Kasus Vibrio parahaemolyticus di Dalam Seafood

January 26, 2011 at 6:07 am | Posted in Uncategorized | Leave a comment

Disalin dari Blog Personal milik ELVIRA SYAMSIR. Staf Pengajar Dept. Ilmu & Teknologi Pangan, Fateta, Institut Pertanian Bogor (elvira_tpg@yahoo.com)

RINGKASAN

Vibrio parahaemolyticus (Vp) merupakan bakteri halofilik Gram negatif. Bakteri ini tumbuh pada kadar NaCl optimum 3%, kisaran suhu 5 – 43°C, pH 4.8 – 11 dan aw 0.94 – 0.99. Pertumbuhan berlangsung cepat pada kondisi suhu optimum (37°C) dengan waktu generasi hanya 9–10 menit. Seafood yang merupakan produk hasil laut, memberikan semua kondisi yang dibutuhkan oleh Vp untuk tumbuh dan berkembang biak: keberadaan garam, nutrien yang baik serta pH dan aw yang cocok sehingga Vp sering terdapat sebagai flora normal di dalam seafood. Mereka terkonsentrasi dalam saluran pencernaan moluska, seperti kerang, tiram dan mussel yang mendapatkan makanannya dengan cara mengambil dan menyaring air laut.

Strain Vp patogen merupakan penyebab penyakit gastroenteritis yang disebabkan oleh produk hasil laut (seafood), terutama yang dimakan mentah, dimasak tidak sempurna atau terkontaminasi dengan seafood mentah setelah pemasakan. Gastroenteritis berlangsung akut, diare tiba-tiba dan kejang perut yang berlangsung selama 48 – 72 jam dengan masa inkubasi 8 – 72 jam. Gejala lain adalah mual, muntah, sakit kepala, badan agak panas dan dingin. Pada sebagian kecil kasus, bakteri juga menyebabkan septisemia.

Sejak tahun 1997, jumlah kasus Kejadian Luar Biasa (KLB) yang disebabkan oleh Vp meningkat secara tajam di berbagai kawasan dunia. Terjadinya KLB ini telah teridentifikasi disebabkan oleh konsumsi seafood terutama tiram (oyster) mentah yang terkontaminasi oleh Vp. Sejak tahun 1997 tersebut, maka seafood terutama tiram dianggap sebagai jenis pangan yang penting diwaspadai dari aspek keamanan pangan. Strain Vp patogen penyebab gastroenteritis sangat beragam. Strain Vp patogen dengan serotype O3:K6 sejak tahun 1996 muncul menjadi sumber patogen baru penyebab keracunan pangan.

Kasus keracunan karena Vp lebih banyak terjadi pada musim panas. Kondisi ini berkorelasi positif dengan prevalensi dan jumlah kontaminasi Vp pada sampel seafood lingkungan yang juga meningkat dengan meningkatnya suhu perairan. Tingkat salinitas air laut juga berpengaruh pada tingkat kontaminasi.

Teknik analisis berpengaruh pada tingkat prevalensi dan tingkat isolasi Vp dari seafood. Untuk pengendalian tingkat kontaminasi didalam seafood, diperlukan pemilihan metode analisis yang lebih sensitifitas dengan waktu deteksi yang lebih cepat. Teknik analisis berdasarkan deteksi gen (tlh, tdh dan/atau trh) memberikan hasil yang lebih akurat untuk mendeteksi strain patogen dibandingkan dengan teknik MPN-konvensional yang berdasarkan pada reaksi biokimiawi.

Pada sampel seafood dari lingkungan dan pasar ritel, Vp patogen hanya terdeteksi dalam jumlah rendah (<100 sel per-gram). Prevalensi dan tingkat kontaminasi Vp dalam sampel seafood lingkungan dan pasar ritel juga seringkali jauh lebih kecil dari batas maksimum Vp yang diijinkan FDA didalam seafood yang akan dijual (10^4 sel per-gram). Kondisi ini juga terjadi pada sampel yang diambil selama terjadinya KLB. Sehingga, praktek penilaian seafood yang hanya didasarkan pada penghitungan total Vp sebagai deteksi keberadaan Vp patogen, hendaknya diperbaiki dengan juga mempertimbangkan faktor virulen tdh dan/atau trh. Tingkat kontaminasi maksimal yang diijinkan dalam seafood yang dijual juga perlu dikaji ulang.

Terjadinya kasus epidemik yang besar dengan mengkonsumsi seafood yang terdeteksi hanya mengandung Vp dalam jumlah kecil dapat disebabkan oleh beberapa faktor. Beberapa faktor penyebab diduga adalah kemampuan Vp patogen memproduksi tdh dapat diperkaya oleh adanya asam empedu terkonjugasi, asam glikokolat dan asam taurokolat dan atau dosis infeksi dari strain patogen yang sangat rendah.

Selama distribusi dan pemasaran, diperlukan praktek-praktek penanganan yang dapat menekan pertumbuhan mikroba. Praktek penyimpanan di suhu rendah (<4°C) dapat menekan dan menurunkan tingkat pertumbuhan Vp. Selain itu, proses pasteurisasi minimal (50°C, 15 menit) dan iradiasi sinar gamma 3 kGy dapat meningkatkan keamanan pangan dari produk-produk seafood.

PENDAHULUAN

Vibrio parahaemolyticus (Vp) adalah bakteri halofilik Gram negatif yang merupakan flora normal dari daerah estuaria dan pantai. Bakteri ini muncul secara musiman. Biasanya, pada musim panas Vp relatif mudah dideteksi pada air laut, sedimen, plankton, ikan, krustasea dan moluska yang merupakan tempat hidupnya di ekosistem. Mereka terkonsentrasi dalam saluran pencernaan moluska, seperti kerang, tiram dan mussel yang mendapatkan makanannya dengan cara mengambil dan menyaring air laut (Charles-Hernández et al., 2006).

Beberapa strain dari bakteri Vp, merupakan penyebab utama dari penyakit gastroenteritis yang disebabkan oleh produk hasil laut (seafood), terutama yang dimakan mentah, dimasak tidak sempurna atau terkontaminasi dengan seafood mentah setelah pemasakan. Gastroenteritis yang disebabkan oleh Vp berlangsung akut, diare yang tiba-tiba dan kejang perut yang berlangsung selama 48 – 72 jam. Masa inkubasi berkisar antara 8 – 72 jam dengan rata-rata sekitar 18 jam. Gejala lain yang dilaporkan dengan frekuensi yang berturut-turut menurun adalah mual, muntah, sakit kepala dan badan panas dingin. Pada sebagian kecil kasus, bakteri menyebabkan kerusakan (luka) pada mukosa usus sehingga tinja dari beberapa penderita selain mengandung bakteri, juga berdarah dan mengandung leukosit serta memicu terjadinya septisemia (Kaysner, 2000).

Kasus keracunan karena mengkonsumsi pangan tercemar Vp, biasanya berlangsung secara musiman. Karena Vp biasanya muncul pada saat suhu lingkungan perairan di atas 15°C, maka kasus keracunan karena Vp biasa terjadi pada musim panas dimana suhu permukaan laut naik hingga mencapai di atas 15°C (McLaughlin et al, 2005).

Sejak tahun 1997, jumlah kasus Kejadian Luar Biasa (KLB) yang disebabkan oleh Vp meningkat secara tajam di berbagai kawasan dunia. Terjadinya KLB ini telah teridentifikasi disebabkan oleh konsumsi seafood terutama tiram (oyster) mentah yang terkontaminasi oleh Vp. Sejak tahun 1997 tersebut, maka seafood terutama tiram dianggap sebagai jenis pangan yang penting diwaspadai dari aspek keamanan pangan.

Tulisan ini akan mencoba menjelaskan mengenai frekuensi isolasi Vp dari seafood, dan melihat faktor-faktor apa saja yang berpengaruh terhadap tingkat isolasi Vp dari seafood tersebut. Beberapa faktor yang akan dilihat adalah faktor lingkungan, teknik analisis yang digunakan serta aspek penyimpanan dan penanganan seafood. Diharapkan, kajian ini dapat menjelaskan keterkaitan antara frekuensi isolasi Vp dari dalam seafood dengan beberapa faktor yang mempengaruhinya dan dapat menjadi bahan masukan untuk pengembangan metode atau teknik pengendalian yang efisien untuk mengurangi resiko kontaminasi Vp dan menjamin keamanan pangan.

KARAKTERISTIK KEJADIAN LUAR BIASA Vp TERKAIT SEAFOOD

Vp teridentifikasi sebagai patogen pangan pertama kali di Jepang, pada tahun 1950. Infeksi disebabkan oleh konsumsi sarden, dengan 272 orang sakit dan 20 meninggal. Sejak itu, Vp dikenal sebagai penyebab penyakit karena seafood mentah atau setengah matang di Jepang dan beberapa negara Asia lainnya (Daniels, 2000).

Kejadian luar biasa keracunan pangan karena Vp (KLB Vp) didefinisikan sebagai kejadian dua atau lebih kasus penyakit dengan gejala klinis yang mirip, yang terjadi setelah mengkonsumsi suatu jenis seafood. Pada kasus infeksi Vp 1988 – 1997 di Florida, Alabama, Louisiana dan Texas, 59%-nya merupakan penyakit gastroenteritis, 8% dengan septisemia dan 34% dengan infeksi kulit. Sebanyak 88% dari penderita gastroenteritis tercatat mengkonsumsi tiram mentah sebelum sakit, sementara 91% penderita septisemia juga mengkonsumsi makanan yang sama sebelum sakit. Dari total 345 kasus, 45% di antaranya dirawat dan 4% meninggal dunia (Daniels et al., 2000).

1. Kasus-kasus KLB Vp Karena Konsumsi Seafood

Beberapa kasus KLB Vp di Chile karena konsumsi seafood mentah ditampilkan pada Tabel 1. Sebelum 2004, kasus keracunan karena Vp jarang terjadi di Chile dan hal ini terkait dengan kondisi suhu perairan yang rendah (11-16°C). Pada saat KLB terjadi, suhu permukaan air laut mencapai 18.3–19.2°C (Puerto Montt, KLB 2004 dan 2005) dan 20°C (Antofagasta, KLB 1997–1998).

Tabel 1. KLB keracunan pangan di Chile dan Spanyol, 1997 – 2005
——————————————————————————–
Tahun Jumlah kasus (daerah epidemi) Pembawa
——————————————————————————–
Chile (Fuenzalida et al, 2005) :
1997-1998 300 (Antofagasta) Ikan kulit keras (shellfish) mentah
2004 1500 (Puerto Montt) Ikan kulit keras (shellfish) mentah
2005 3600 (Puerto Mont)
11000 (seluruh Chile)

Spanyol (Martinez-Urtaza et al, 2004 dan 2005) :
1999 64 (Galicia) Tiram mentah
2004 80 (A Coruña) Kepiting rebus
——————————————————————————

Di Spanyol, rekaman medis dari beberapa rumah sakit menunjukkan bahwa infeksi Vp lebih sering dari yang diduga. Dari rekaman medis, Vp teridentifikasi dari sampel klinis pasien gastroenteritis di Barcelona (1986, 1987 dan 1999), Zaragoza (1993) dan Madrid (1998 dan 2000). Kasus KLB terjadi pada tahun 1999 dan 2004, disebabkan oleh konsumsi seafood mentah dan yang telah dimasak (Tabel 1). Kepiting rebus yang merupakan penyebab KLB Juli 2004, dimasak pada kondisi higiene dan sanitasi yang buruk dan kemudian disimpan di suhu ruang selama beberapa jam sebelum dikonsumsi (Martinez-Urtaza et al, 2004 dan 2005).

Pada periode 1986 – 1995, setiap tahun rata-rata terjadi 85 KLB karena Vp. Insiden KLB Vp melonjak drastis pada 1996 dan bertahan hingga sekarang. Pada 1996–1999, 61-71% dari total KLB keracunan pangan di Taiwan disebabkan oleh Vp (Chiou et al, 2000).

Di Amerika Serikat, sepanjang 1973 – 1998 terjadi 40 kasus KLB Vp di 15 negara bagian dan wilayah Guam dengan 1064 penderita dan median tingkat serangan 56% (3 – 100%) dimana sebagian besar kasus terjadi di bulan Juli. Pembawa adalah seafood atau yang terkontaminasi dengan seafood, terutama yang dikonsumsi mentah (38% kasus) atau setengah matang. Penyebab utama adalah tiram dan kerang. Pada periode ini, 30% KLB terjadi pada 1997 – 1998 dan tiga diantaranya cukup besar. Pada Juli–Agustus 1997, keracunan disebabkan oleh konsumsi tiram mentah dari Puget Sound, Washington; dua kasus KLB gastroenteritis Vp pada Mei–Juni 1998 terjadi karena mengkonsumsi tiram mentah yang berasal dari Galveston Bay, Texas dan di akhir Juli 1998, KLB Vp terkait dengan konsumsi tiram dan kerang mentah yang berasal dari Teluk Oyster, Long Island, New York (Daniels et al, 2000).

Kasus KLB juga terjadi di Alaska pada Juli 2004, yang disebabkan oleh tiram Alaska dengan tingkat serangan 29%. Suhu air laut di daerah pemanenan pada Juli 2006 tercatat di atas 15°C (McLaughlin et al, 2005).

Kasus KLB Vp di New York, Oregon dan Washington, kembali terjadi pada 20 Mei – 31 Juli 2006 setelah mengkonsumsi tiram dan remis dalam bentuk mentah atau masak yang dimakan di restoran. Dari 177 kasus yang secara epidemiologis terhubung dengan infeksi Vp, 41% positif disebabkan oleh Vp berdasarkan analisis sampel klinis penderita. Tiram dan remis berasal dari daerah pantai Washington dan British Columbia, Canada; yang didistribusikan secara nasional ke pasar ikan dan restoran. Luasnya daerah pemasaran berdampak pada meluasnya daerah sebaran penyakit. Pada KLB 2006, 122 kasus berasal dari 17 sumber seafood yang sama. Kasus ini berimplikasi pada penutupan perusahaan pemanenan tiram yang merupakan pemasok utama tiram penyebab KLB (Balter et al, 2006).

Dari beberapa kasus KLB yang terjadi dapat disimpulkan bahwa keracunan karena Vp merupakan kasus musiman yang kemunculannya sangat terkait dengan meningkatnya suhu perairan. Dari data yang ditampilkan terlihat bahwa keracunan biasanya terjadi pada bulan-bulan yang suhunya hangat (musim panas), dimana suhu permukaan laut lebih besar dari 15°C. Seafood yang paling sering dilaporkan sebagai penyebab keracunan adalah tiram dan kerang.

Besarnya frekuensi keracunan karena Vp terkait juga dengan cara mengkonsumsi seafood. Besarnya prevalensi kasus keracunan Vp di Taiwan disebabkan oleh kebiasaan masyarakatnya untuk mengkonsumsi seafood dalam kondisi mentah. Kondisi yang sama tampaknya juga terjadi di beberapa negara Asia lainnya yang mempunyai kebiasaan mengkonsumsi seafood mentah, seperti Jepang dan Thailand.

Pada kasus keracunan yang terjadi setelah mengkonsumsi seafood yang dimasak, faktor penyebab adalah proses pemasakan yang tidak sempurna sehingga tidak membunuh semua Vp yang ada, atau proses penanganan yang buruk (kondisi higiene dan sanitasi tidak terjaga, seafood disimpan disuhu ruang selama beberapa jam sebelum diolah/dikonsumsi, atau terjadinya kontaminasi silang antara produk yang telah dimasak dengan produk mentah).

2. Karakteristik Vp patogen pada Sampel Klinis

Dilihat dari serotypenya, maka strain Vp patogen penyebab setiap kejadian KLB sangat beragam seperti ditampilkan pada Tabel 2. Walaupun demikian, menurut Chowdhury et al (2000) yang disitasi oleh Martinez-Urtaza et al (2004), pandemik Vp dalam beberapa tahun terakhir ini terutama disebabkan oleh tiga serotype utama yaitu O3:K6, O4:K68 dan O1:K untypeable (KUT).

Tabel 2. Serotype dominan pada beberapa sampel klinis KLB Vp
——————————————————————————
Daerah Tahun Strain penyebab dominan (serotype) Referensi

——————————————————————————
Spanyol 1999 O4:K11
2004 O3:K6 Martinez-Urtaza et al, 2005

Chile 1997-1998, O3:K6 Fuenzalida et al, 2005
2004, 2005

USA 1997 O4:K12 dan O1:K56 Daniels et al, 2000
1998 O3:K6
2004 O6:K18 McLaughlin et al, 2005
2006 O4:K12 Balter et al, 2006

Taiwan 1995–1999 O3:K6 Chiou et al, 2000
—————————————————————————-

Vp dari strain O3:K6 pertama kali diketahui sebagai penyebab pandemik pada 1996 di Asia Tenggara (Fuenzalida, et al, 2005). Isolat memiliki gen toxR, tlh dan tdh, tidak memiliki trh dan positif sebagai strain pandemik dari klon pandemic O3:K6 (Martinez-Urtaza et al, 2005).

Tabel 3. Data serotype Vp yang diisolasi dari sampel klinis penderita di daerah Asia, Amerika dan Eropa
—————————————————————
Serotype Tahun isolasi Asal Strain
—————————————————————
1 O1:K1 1965 Jepang ATCC17802
2 O1:K25 1999 Thailand VPHY191
3 O1:KUT 1998 Bangladesh AV-16000
1999 Thailand VPHY123
4 O3:K4 1970 UK ATCC43996
5 O3:K6 1985 Int. traveler AQ4037
1996 India VP81
1997 Laos 97LVP2
1997 Taiwan DOH-958 15
1998 Bangladesh AN-8373
1998 Jepang JKY-VP6
1998 Korea VP2
1998 Thailand VP47
1998 USA BE98-2062
1999 Thailand VPHY67
6 O3:K7 1978 UK NCTC11344
7 O4:K11 1998 Spanyol 428/00
1999 Spanyol 30824
1999 Spanyol 30825
1999 Spanyol 447/00
8 O4:K68 1998 Bangladesh AN-5034
1999 Thailand VPHY145
Sumber: Martinez-Urtaza et al (2004)

Dari data yang ditampilkan pada Tabel 3 terlihat bahwa serotype O3:K6 selalu muncul pada kasus KLB pada akhir tahun 1990-an. Di Spanyol, 2/3 isolat klinis KLB 2004, memiliki strain O3:K6 dan sisanya (1/3) O3:K untypeable. Strain O3:K6 juga ditemukan sebagai strain dominan dari isolat klinis pasien kasus KLB Chile (19997-1998, 2004 dan 2005) dan pertama kali muncul di Amerika Serikat sebagai penyebab utama KLB pada 1998. Di Taiwan, uji serotyping terhadap 3743 isolat Vp sepanjang 1995 – 1999, teridentifikasi 40 serotype dengan O3:K6 sebagai serotype dominan. Di Taiwan, serotype O3:K6 ini terdeteksi pertamakali pada Oktober 1995 dan levelnya hanya 0.6% dari total isolat Vp. Jumlah ini menjadi 50.1% pada 1996 dan mencapai puncaknya (83.8%) pada 1997 (Chiou et al, 2000).

Di Amerika Serikat, walaupun muncul pada 1998, serotype O3:K6 tidak menjadi penyebab utama kasus tahun 1997, 2004 dan 2004. Vp dominan dari sampel klinis KLB 1997 memiliki serotype O4:K12 dan O1:K56. Pada KLB 2004 di Alaska, serotype mayoritas dalam sampel klinis adalah O6:K18 sementara 18 dari 23 isolat Vp kasus 2006 merupakan serotype O4:K12 (McLaughlin et al, 2005; Balter et al, 2006).

Seperti di Amerika Serikat, data serotype Vp yang diisolasi dari sampel klinis di dareah Asia, Amerika dan Eropa (Tabel 3) menunjukkan bahwa strain Vp patogen yang diisolasi di daerah Asia sangat beragam tetapi dari 1985-1999, sebagian besar didominasi oleh strain dengan serotype O3:K6 (Martinez-Urtaza et al, 2004). Dari paparan diatas bisa dikatakan bahwa sejak 1996, Vp strain O3:K6 merupakan bakteri patogen baru yang harus diwaspadai.

PENGARUH LINGKUNGAN TERHADAP PREVALENSI DAN TINGKAT CEMARAN Vp DI DALAM SEAFOOD

Dari kasus KLB di atas, diketahui bahwa keracunan karena Vp biasanya terjadi pada musim panas, dan strain yang diperoleh dari isolat klinis adalah strain patogen yang memiliki tlh dan/atau trh. Besarnya jumlah kasus keracunan Vp yang terjadi setelah mengkonsumsi tiram mentah, menyebabkan seafood terbanyak yang diteliti terkait dengan keberadaan Vp adalah tiram (oyster).

Beberapa penelitian pada sampel seafood dari lingkungan menunjukkan prevalensi dan tingkat kontaminasi Vp yang sangat beragam (Tabel 4). Di daerah dengan empat musim prevalensi Vp biasanya meningkat selama musim panas, sementara di daerah beriklim tropis seperti India, prevalensi Vp di dalam sampel seafood dari lingkungan mencapai hampir 100% (Deepanjali et al, 2005). Secara umum, tingkat kontaminasi yang terjadi 10^4 koloni/gram (batas maksimum total Vp di dalam seafood yang akan dijual, yang direkomendasikan FDA) seperti dilaporkan oleh DePaole et al (2000) dan DePaola et al (2003).

Beberapa hasil penelitian secara jelas menunjukkan bahwa terdapat korelasi yang kuat antara prevalensi Vp dan tingkat kontaminasinya di dalam seafood dengan kondisi lingkungan perairan tempat seafood tersebut berasal. Dari beberapa penelitian (Tabel 4) disimpulkan bahwa suhu air berkorelasi positif dengan prevalensi Vp dan tingkat kontaminasinya di dalam air. Prevalensi dan tingkat kontaminasi Vp pada seafood lebih sering terjadi di musim panas atau pada daerah perairan yang suhunya diatas 15°C.

Tabel 4. Pengaruh ekologis lingkungan perairan terhadap prevalensi dan tingkat kontaminasi Vp di dalam seafood
——————————————————————————————-
No Sampel Hasil Referensi
——————————————————————————————-
No.1
Sampel.1 Tiram dari berbagai daerah pesisir USA, beberapa minggu setelah KLB Vp (1997 – 1998)
Hasil.1.1 Prevalensi Vp ditemukan pada tiram dari Puget Sound, Washington; Galveston Bay, Texas dan Long Island, New York.
Hasil.1.2 Hanya dua sampel yang jumlah Vp-nya diatas batas yang diijinkan (>10000/g)
Hasil.1.3 Strain patogen (tdh dan/atau trh positif) hanya terdeteksi pada sedikit sampel, sebagian besar pada tiram dari Puget Sound, jumlahnya <10/g
Hasil.1.4 Kondisi Galveston Bay selama musim panas (setelah KLB): suhu air 27.8 – 31.7C; salinitas 14.9 – 29.3 ppt dan tk cemaran Vp pada tiram 100 – 1000/g;
Hasil.1.5 Terdapat korelasi negatif antara jumlah Vp didalam tiram dengan salinitas air
Referensi.1 DePaola et al, 2000

No.2
Sampel.2 Tiram (Crassostrea virginica) dari Mobile Bay, Alabama (Mei 1998 – April 1999)
Hasil.2.1 Jumlah Vp dalam tiram dipengaruhi oleh suhu air
Hasil.2.2 T air > 20C, Vp rata-rata 130 CFU/g;
Hasil.2.3 T air < 20C, Vp rata-rata 15 CFU/g
Referensi.2 Gooch et al, 2002

No.3
Sampel.3 Moluska, 671 sampel dari pantai negara bagian Gulf dan Atlantic (1999 – 2000)
Hasil.3.1 Kandungan Vp dalam sampel berkore-lasi positif dengan suhu perairan
Hasil.3.2 Kandungan Vp sampel dari pantai Gulf lebih tinggi dari pantai Atlantic
Hasil.3.3 Vp pada 5% sampel > 1000 CFU/g
Hasil.3.4 6% sampel mengandung Vp patogen (tdh+)
Hasil.3.5 Frekuensi deteksi Vp patogen dan to-tal Vp berkorelasi positif dengan suhu air dan
Hasil.3.6 Kegagalan deteksi Vp didalam seafood lebih dikarenakan oleh jumlah yang terlalu kecil dan distribusi Vp yang tidak homogen
Referensi.3 Cook et al, 2002

No. 4
Sampel.4 Tiram, dari Mobile Bay Alabama (Juni, Juli, September 2001@ 20 perbulan
Hasil.4.1 Kandungan Vp sampel pada Juni, Juli dan September berturut-turut log 2.90±0.91, 2.88±0.36; 2.47±0.26 CFU/g;
Hasil.4.2 40% sampel bulan Juni & Juli mengan-dung Vp patogen (10 – 20 CFU/g), Vp patogen negatif pada sampel bulan September 2001
Referensi.4 Kaufman et al, 2003

No.5
Sampel.5 Sampel tiram, dari Mobile Bay Alabama sampling dua-mingguan (Maret 1999 – September 2000)
Hasil.5.1 Semua sampel mengandung Vp, jumlah <10 – 12000 CFU/g.
Hasil.5.2 Jumlah Vp dalam sampel berbanding lurus dengan suhu air laut. Tetapi jumlah Vp patogen berbanding terbalik dengan suhu air laut
Hasil.5.3 Vp patogen terdeteksi dalam 34 (21.8%) dari 156 sampel
46 dari 6018 isolat (dari enrichment plate) dan 31 dari 6992 isolat (direct plate) positif memiliki tdh
Hasil.5.4 Serotype strain sangat beragam, 97% punya gen trh dan memproduksi urease
Hasil.5.5 Tidak terdeteksi strain pandemik O3:K6
Referensi.5 DePaola et al, 2003

No.6
Sampel.6 Air laut dan bahan organik dari pantai Laut Seto-Inlad Jepang; pada musim dingin
Hasil.6.1 Teknik MPN-PCR:
95% sampel positif Vp;
jumlah 3 – >1400 /100 ml air atau 10 /g sampel organik
gen tdh dan trh positif berturut-turut pada 55% dan 20% sampel
Hasil.6.2 Teknik MPN-kultur konvensional:
40% sampel positif Vp;
jumlah 3 – 240 /100 ml air atau 10 /g sampel organik
gen tdh & trh negatif pd semua sampel
Referensi.6 Alam et al, 2002

No.7
Sampel.7 Air laut dan bahan organik dari pantai Laut Seto-Inlad Jepang; pada musim panas
Hasil.7.1 Teknik MPN-PCR:
99% sampel positif Vp;
jumlah 3 – >1400 /100 ml air atau 10 /g sampel organik
gen tdh dan trh positif berturut-turut pada 41.5% dan 8.5% sampel

Hasil.7.2 Teknik MPN-kultur konvensional:
76.6% sampel positif Vp;
jumlah 3 – >1400 /100 ml air atau 10 /g sampel organik
gen tdh & trh negatif pd semua sampel
Referensi.7 Alam et al, 2003

No.8
Sampel.8 800 sampel (12 batch) tiram dari sebuah muara sungai di daerah timur laut Brazil
Hasil.8.1 Salinitas air laut sampling 3 – 27‰.
Hasil.8.2 hanya satu dari 12 batch yang positif mengandung Vp dan bersifat Kanagawa negatif (non patogen)
Referensi.8 De Sousa et al, 2004

No.9
Sampel.9.1 309 sampel moluska & sampel klinis dr Chile barat daya (Januari 2004 – Maret 2005)
Hasil.9.1 Hampir semua sampel positif Vp terjadi pada musim panas (Januari – Maret)
Sampel.9.2 204 sampel moluska (Januari – Maret 2004 dan 2005 (KLB Vp))
Hasil.9.2.1 53% mengandung Vp.
Hasil.9.2.2 Hanya 3 dr 50 sampel dengan Vp + yg mengandung Vp patogen klon pandemik
Sampel.9.3 25 dari 48 sampel positif Vp pada musim panas 2005
Hasil.9.3.1 Suhu air laut bulanan rata-rata 18.3 – 19.2°C
Hasil.9.3.2 Seafood mengandung Vp rata-rata 9.4 g-1 MPN (kisaran 3 – 93 g-1, MPN).
Referensi.9 Fuenzalida et al, 2005

No.10
Sampel.10.1 2 sampel tiram dari Alaska (saat kejadian KLB, Juni 2004)
Hasil.10.1.1 Mengandung Vp 2.1 dan 3.5 MPN/gram
Hasil.10.1.2 Strain dominan adalah O6:K18 (5 dari 10 isolat yang diidentifikasi), sisanya O1:K9 (3/10), O5:K17 (1/10), dan O10:K68 (1/10). Semua isolat positif mengandung gen tdh
Hasil.10.1.3 ada korelasi yang tinggi antara strain pada sampel klinis dengan sampel lingkungan (tiram) yang diperoleh dari Teluk Alaska (analisis PFGE)
Hasil.10.1.4 Vp patogen pada sampel bulan Juli lebih besar dari sampel bulan Agustus. T air lebih tinggi pada bulan Agustus
Sampel.10.2 29 sampel tiram Alaska (21 Juli – 15 September 2004)
Hasil.10.2.1 13/29 sampel mengandung Vp dalam kisaran 0.3 – 430 MPN/gram (nilai median 3.5 MPN/gram)
Hasil.10.2.2 Isolat Vp patogen (tdh positif) 85% dari isolat bulan Juli dan 20% pada isolat dari bulan Agustus. Rasio prevalensi (tdh positif) bulan Juli dibandingkan dengan Agustus = 4.6. Suhu perairan bulan Juli = 16.6°C, bulan Agustus = 17.4°C.
Hasil.10.2.3 Vp dengan tdh tidak terdeteksi pada isolat bulan September.
Referensi.10 McLaughlin et al, 2005

No.11
Sampel.11 Tiram, dari estuaria sepanjang pantai India barat daya
Hasil.11.1 Vp terdeteksi pada 93.87% sampel; jumlah <10 – 104 CFU/gram.
Hasil.11.2 Vp patogen terdeteksi pada 5 dari 49 sampel (10.2%) dengan menggunakan tdh-probe; dan 3 dari 49 sampel (6.1%) dengan PCR. Isolat dari satu sampel memiliki serotype pandemik: O3:K6.
Hasil.11.3 59.3% sampel memiliki gen trh
Referensi.11 Deepanjali et al, 2005

Salinitas air juga berpengaruh terhadap prevalensi dan tingkat kontaminasi Vp kedalam seafood. Cook et al. (2002) menduga salinitas optimal untuk Vp adalah 19 ppt, dan jumlah Vp akan lebih rendah jika salinitas berada di luar salinitas optimal tersebut. Kondisi tingkat salinitas air yang berfluktuasi sangat lebar dan jauh dari kisaran optimal pertumbuhan Vp, diduga sebagai salah satu penyebab rendahnya prevalensi dan tingkat cemaran Vp yang terdeteksi pada seafood (de Sousa, 2004). Daerah atau habitat asal seafood berpengaruh terhadap prevalensi dan tingkat cemaran Vp didalam seafood dan hal ini mungkin disebabkan oleh kondisi suhu air dan kadar salinitasnya.

Beberapa penelitian dengan data terbatas menyebutkan prevalensi dan tingkat isolasi Vp patogen di dalam seafood selama musim panas meningkat dengan menurunnya suhu perairan (DePaola et al, 2003 dan McLaughlin et al, 2005). Diduga, strain Vp yang patogen lebih tahan terhadap suhu perairan yang lebih dingin, dan secara perlahan digantikan oleh strain yang non patogen dengan meningkatnya suhu perairan (McLaughlin et al, 2005). Strain Vp patogen yang terdeteksi sangat beragam dan strain Vp patogen yang pandemik hanya terdeteksi pada sedikit sampel, bahkan pada sampel seafood selama terjadinya KLB.

PENGARUH TEKNIK ANALISIS TERHADAP PREVALENSI DAN TINGKAT CEMARAN Vp DI DALAM SEAFOOD

Fuenzalida et al (2005) yang mensitasi dari beberapa literatur menyebutkan bahwa rendahnya tingkat cemaran Vp (termasuk Vp patogen) dapat disebab-kan oleh rendahnya recovery dari Vp dengan menggunakan prosedur analisis yang digunakan. Rendahnya tingkat recovery ini bisa disebabkan oleh prevalensi dari strain yang hidup tetapi tidak bisa tumbuh di dalam media uji (viable but non-culturable). Beberapa penelitian yang melihat prevalensi dan tingkat kontaminasi Vp didalam seafood terkait dengan teknik analisis, dirangkum pada Tabel 5.

Penentuan total Vp dapat dilakukan dengan metode MPN konvensional yang dikembangkan oleh BAM (menggunakan konfirmasi biokimia) atau dengan pemupukan pada media non selektif yang dilanjutkan dengan deteksi menggunakan pelacak (probe) gen tlh (thermolabile hemolysin). Sementara untuk identifikasi strain Vp patogen dapat dilakukan dengan uji kanagawa atau menggunakan pelacak DNA dengan atau tanpa kombinasi dengan PCR (Polymerase Chain Reaction-perbanyakan kopi sekuens DNA) untuk mendeteksi gen tdh dan trh didalam Vp (BAM, 2004).

Teknik analisis sangat berpengaruh pada tingkat isolasi bakteri dan waktu analisis. Dari beberapa penelitian (Tabel 5) dikatakan bahwa metode pelacak DNA berkorelasi sangat baik dengan teknik penghitungan konvensional menggunakan konfirmasi biokimia, dengan waktu analisis yang lebih cepat.

Untuk strain patogen, analisis dengan pelacak gen jauh lebih sensitif dibandingkan dengan teknik analisis konvensional. Pada hasil penelitian yang dilaporkan oleh Alam et al (2002), diketahui bahwa teknik PCR dengan menggunakan isolat DNA yang berasal dari media pengkayaan memberikan hasil yang jauh lebih baik dari metode MPN konvensional (MPN-BAM). Prevalensi Vp mencapai 95% dengan deteksi gen tdh dan trh mencapai 20% dari sampel, sementara prevalensi dengan teknik konvensional hanya 40% dan tidak bisa mendeteksi keberadaan gen tdh dan trh (Tabel 5).

Media yang digunakan untuk deteksi vibrio dalam pangan dan air dikembangkan berdasarkan pertimbangan kemampuan bakteri ini untuk tumbuh cepat pada pH alkali, tahan terhadap efek penghambatan yang diberikan oleh garam empedu dan natrium tellurite, dan toleran terhadap garam (NaCl). Media pengkayaan yang umum digunakan untuk Vibrio adalah APW (broth alkaline peptone water), NTSB (salt trypticase soy broth) dan SPB (salt polimiksin broth). Penelitian Hara-Kudo et al (2001) menyebutkan bahwa pengkayaan dua tahap memberikan hasil recovery Vp yang lebih baik dibandingkan pengkayaan 1 tahap.

Sebagai media selektif, TCBS (thiosulfate-citrate-bile-saccharose) adalah yang paling umum digunakan. Kelemahan media ini adalah tidak terlalu spesifik membedakan Vp dari V. hollisae, V. mimicus dan V. vulnificus yang sama-sama membentuk koloni berwarna hijau. Hara-Kudo et al (2001) mengembangkan media selektif CV (chromogenic agar) yang mengandung substrat untuk ß-galaktosidase (CV) pada CV agar, yang bisa membedakan Vp dari koloni peng-ganggu sebagai koloni berwarna violet (Gambar 1). Prevalensi Vp didalam sea-food lebih tinggi jika menggunakan media selektif CV (65% pada pengkayaan 1 tahap dan 80% pada pengkayaan 2 tahap) daripada media TCBS (50 pada pengka-yaan 1 tahap dan 70% dengan pengkayaan 2 tahap) dengan tingkat isolasi yang juga lebih baik.


Gambar 1. Koloni Vp pada agar CV (a, warna ungu) dan TCBS (b, warna hijau)

PENGARUH PENANGANAN PASCA PANEN TERHADAP PREVALENSI DAN TINGKAT CEMARAN Vp DI DALAM SEAFOOD

Vp menyukai kisaran suhu 5 – 43°C untuk pertumbuhannya, dengan suhu pertumbuhan optimum 37°C. Pertumbuhan berlangsung cepat pada kondisi suhu optimum, dengan waktu generasi hanya 9–10 menit. Nilai pH optimum pertumbuhan Vp adalah 4.8–11, dan ketahanan terhadap keasaman akan meningkat jika suhu lingkungan mendekati kondisi suhu pertumbuhan optimum. Walaupun lebih menyukai kondisi lingkungan anaerob untuk pertumbuhannya, Vp juga dapat tumbuh pada kondisi aerob. Bakteri ini tergolong halofilik dengan kadar NaCl optimum 3% dan membutuhkan aw 0.94-0.99 dengan optimum 0.98 untuk pertumbuhannya (Kaysner, 2000).

Seafood yang berasal dari daerah laut, memberikan semua kondisi yang dibutuhkan oleh Vp untuk tumbuh dan berkembang biak: keberadaan garam, nutrien yang baik serta pH dan aw yang cocok. Guna mencegah terjadinya bahaya karena mengkonsumsi seafood, perlu dilihat prevalensi dan tingkat kontaminasi Vp pada seafood yang dijual di tingkat ritel serta pengaruh kondisi penanganan pasca panen seafood terhadap prevalensi dan tingkat kontaminasi Vp didalamnya. Beberapa hasil penelitian terkait dengan kondisi penanganan pasca panen ini dapat dilihat pada Tabel 6.

Tabel 5. Evaluasi beberapa teknik analisis Vp
——————————————-
No Sampel Metode Hasil Referensi
——————————————-
No.1
Sampel.1 Tiram
Metode.1 MPN-VPAP labeled DNA probe dengan Direct-VPAP labeled DNA-probe (tlh-Vp)
Hasil.1.1 Ada korelasi (r = 0.78) anta-ra metode MPN_BAM de-ngan direct-VPAP & DNA probe (gen tlh, MPN Vp).
Hasil.1.2 Waktu analisis dengan metode direct lebih cepat
Referensi.1 Ellison et al, 2001

No.2
Sampel.2 Tiram
Metode.2 MPN-VPAP -vs- di-rect-VPAP & direct-VPDig (digoxigenin-labeled probe)
Hasil.2.1 Waktu analisis dengan metode direct lebih cepat
Hasil.2.2 Teknik MPN-VPAP berkore-lasi sangat baik (r > 0.85) dengan Direct-VPAP & Direct-VPDig
Referensi.2 Gooch et al, 2001

No.3
Sampel.3 Seafood
Metode.3.1 Media pengkayaan: 2 tahap (salt trypticase soy broth/NTSB & salt polimiksin broth (SPB) / 1 tahap (SPB)
Hasil.3.1.1 Media pengkayaan 2 tahap lebih efektif
Hasil.3.1.2 Pada TCBS, koloni Vp (juga V. hollisae, V. mimicus dan V. vulnificus) berwarna hijau sementara pada CV agar, Vp berwarna violet & berbeda dari spesies Vibrio lainnya
Media.3.2 Media selektif: thio-sulfate citrate bile salts sucrose (TCBS) atau chromogenic agar yg mengandung substrat untuk ß-ga-laktosidase (CV)
Hasil.3.2.1 Prevalensi Vp dengan media selektif CV (65-80%) lebih baik dari media TCBS (50-70%)
Referensi.3 Hara-Kudo et al, 2001

No.4
Sampel.4 Air laut dan bahan organik
Metode.4 Teknik MPN-PCR (menggunakan isolat DNA dari media pengkayaan) dan Teknik MPN-kultur konvensional
Hasil.4.1 Teknik MPN-PCR: 95% sampel positif Vp; jumlah 3 – >1400 /100 ml air atau 10 /g sampel organik; gen tdh & trh positif berturut-turut 55% & 20%
Hasil.4.2 Teknik MPN-kultur: 40% sampel positif Vp; jumlah 3 – 240 /100 ml air atau 10 /g sampel organik; gen tdh & trh negatif pada semua sampel
Referensi.4 Alam, et al, 2002

No.5
Sampel.5 Seafood
Metode.5 MPN-PCR (deteksi gen tlh) dan MPN-TCBS; pengkayaan pada APW (Alkalin Peptone Water)
Hasil.5.1 Pada sampel spike: MPN-PCR mendeteksi Vp ≥ jum-lah spike sel sementara MPN-TCBS mendeteksi Vp < jumlah spike sampel
Hasil.5.2 Pada sampel seafood: MPN-PCR mendeteksi Vp > dari metode MPN-TCBS
Referensi.5 Miwa et al, 2003

No.6
Sampel.6 Tiram, 131 ekor
Metode.6 Real time-PCR dan streak plate APW-probe DNA utk tdh real time-PCR: sampel + tdh 46.6%; streak plate-probe tdh: sampel + tdh 11.5%
Referensi.6 Blacstone, et al, 2003

Dari hasil penelitian ini disimpulkan bahwa prevalensi Vp dalam seafood di tingkat ritel bervariasi dari 6.32% (Franco-Monsreal dan Flores-Abuxapqui, 1989) sampai 90% (Kaufman et al, 2003). Di Amerika Serikat, prevalensi Vp dalam seafood ritel dalam jumlah besar (90%) ditemukan pada sampel musim panas (Kaufman et al, 2003).

Penelitian Miwa et al (2006) terhadap sampel seafood dari pasar ritel Jepang menyebutkan bahwa prevalensi Vp dalam sampel moluska lebih besar dari sampel udang. Tingkat prevalensi juga ditemukan jauh lebih tinggi pada seafood yang akan dimasak daripada yang akan dimakan mentah.

Tabel 6. Tingkat cemaran Vp di dalam seafood pasca panen
——————————————————————-
No Sampel Waktu Hasil Referensi
——————————————————————-
No.1
Sampel.1 Seafood dari restoran, kota Yucatan, Mexico; 190 sampel: mentah, masak tidak sempurna, masak parsial dengan perebusan
Waktu.1 Maret-Agustus 1987
Hasil.1 Prevalensi Vp: 6.32%
Referensi.1 Franco-Monsreal dan Flores-Abuxapqui, 1989

No.2
Sampel.2 Hancuran daging tiram,
T 4°C, 0°C, -18°C, -24°C
Hasil.2.1 Jumlah Vp merupakan fungsi log dari log waktu.
Referensi.2 Muntada-Garriga et al, 1995

Hasil.2&3 Penyimpanan suhu rendah signifikan menurunkan Vp

No.3
Sampel.3 Tiram, pasteurisasi 50°C, 15 menit
Hasil.3 Proses min. 10 menit efektif menurunkan jumlah Vp dari >10000 mjd tdk terdeteksi
Referensi.3 Andrew et al, 2000

No.4
Sampel.4 Tiram dari 4 restoran, 3 pasar induk di Gainesville, Fla. (@ dua sampel), bulanan
Waktu.4 September 1997 – Mei 1998
Hasil.4 Jumlah rata-rata Vp sampel tertinggi di bulan Oktober 1997 (3000 CFU/g); Vp ting-gi sepanjang September-November 1997
Referensi.4 Ellison, et al, 2001

No.5
Sampel.5 Tiram (Crassostrea virgin-ica) dari Mobile Bay, Alabama
Waktu.5 Mei 1998 – April 1999
Hasil.5.1 Jam ke-0: jumlah Vp oleh suhu air pada saat panen: T air > 20C, nilai Vp rata-rata 130 CFU/g; T air < 20C, Vp rata-rata 15 CFU/g
Hasil.5.2 10 jam penyimpanan di 26C, Vp dalam tiram hidup naik 50 kali lipat (log 1.7 CFU/g)
Hasil.5.3 24 jam penyimp. di 26C, Vp dalam tiram hidup naik 790 kali lipat (log 2.9 CFU/g)
Hasil.5.4 penyimpanan 14 hari di 3C (setelah disimpan di 26C selama 24 jam), Vp turun mjd 1/6 jumlah sblm disimpan dingin (log 0.8 CFU/g)
Referensi.5 Cook, et al, 2002

No.6
Sampel.6 Tiram 370 lot, pasar ritel USA (71% dari restoran); disuplay dari Gulf Coast Pasifik, Mid-Atlantic, North Atlantic dan Canada
Waktu.6 Juni 1998 – Juli 1999
Hasil.6.1 Jumlah Vp seafood pada musim panas >10000 MPN/g
Hasil.6.2 Tingkat cemaran dipengaruhi oleh lokasi asal panen
Hasil.6.3 Tingkat cemaran menurun (7%/hari) selama penyimpanan di pasar ritel
Hasil.6.4 tdh terdeteksi pd 9/3429 (0.3%) kultur Vp atau 4% lot tiram
Hasil.6.5 Desember 1997 – Mei 1998 Vp didalam sampel < 100/g
Referensi.6 Cook, et al, 2002

No.7
Sampel.7 Seafood (tiram, kerang), 329 sampel, Jepang
Hasil.7 Prevalensi gen tdh 10%; jumlah < 3 – 93 per-10 g
Referensi.7 Harakudo et al, 2003

No.8
Sampel.8 Tiram hidup, di iradiasi sinar gamma (60Co)
Hasil.8 Dosis 1 kGy menurunkan tk kontaminasi Vp sebesar 6-10 log. Sampai dosis 3 kGy, tidak membunuh tiram & tidak menyebabkan penyimpangan bau, flavor & penampakan
Referensi.8 Jakabi et al, 20003

No.9
Sampel.9 Tiram, diambil perbulan @ 20 sampel, dari Mobile Bay Alabama
Waktu.9 , Juli, September 2001
Hasil.9.1 saat panen, Vp pd 90% sam-pel (dari 10) 200–2000CFU/ 00 lm nsi l, t;0.91, 2.88±0.36;alam litian onal, en meman pelacak DNA akan memnpa kombinasi dilanjutkan dengan deteksi menggung; (Juni, juli, September berturut-turut log 2.90±0.91, 2.88±0.36; 2.47±0.26 CFU/g
Hasil.9.2 40% sampel bulan Juni dan Juli 2001mengandung Vp patogen (10 – 20 CFU/g), Vp patogen negatif
Hasil.9.3 Setelah penyimpanan 24 jam di suhu ruang, Vp dalam tiram hidup meningkat 13 – 26 kali lipat. Vp terdeteksi pada beberapa tiram dengan jumlah lebih dari 100 CFU/g
Referensi.9 Kaufman et al, 2003

No.10
Sampel.10 Seafood dari pasar retail, Jepang
Hasil.10.1 Prevalensi Vp dalam seafood untuk konsumsi mentah: 55% (11/20) pada udang; 96.7% (29/30) pd moluska
Hasil.10.2 Jumlah Vp dlm seafood un-tuk konsumsi mentah 10^4 MPN/100g dalam 36.7% sampel moluska
Hasil.10.3 Prevalensi Vp dalam seafood untuk konsumsi masak: 45% (9/20) pada udang; 100% (20/20) pada moluska
Hasil.10.4 Jumlah Vp dlm seafood un-tuk konsumsi masak > 10^4 MPN/100g dalam 5% sampel udang & > 10^4 MPN/100g dalam 90% sampel moluska
Hasil.10.5 Jumlah Vp patogen pd sam-pel seafood yang akan dimakan mentah dibawah limit deteksi (<30MPN/100g)
Hasil.10.6 Prevalensi Vp patogen pada sampel moluska untuk kon-sumsi masak 35%; jumlah 3.6×10 – 1.1×10^3MPN/100g
Hasil.10.7 Insiden Vp patogen cenderung lebih besar jk cemaran Vp dalam sampel tinggi
Hasil.10.8 Serotype sebag. besar strain patogen dlm 11/33 sampel dgn tdh+ adl O3:K6 dan merupakan klon pandemik
Referensi.10 Miwa et al, 2006

No.11
Sampel.11 Sampel 300 seafood yang dipanen di daerah Cina Timur
Hasil.11 Prevalensi Vp 26% dengan teknik konvensional & 32.3% dengan teknik TaqMan PCR
Referensi.11 Cai, et al, 2006

No.12
Sampel.12 Tiram
Hasil.12 Prevalensi Vp 51.5%; Vp patogen 12.1%
Referensi.12 Ward dan Bej, 2006

Pada beberapa kasus yang meneliti prevalensi dan tingkat kontaminasi Vp didalam seafood selama terjadinya KLB (Tabel 6) diketahui bahwa jumlah Vp di dalam sampel selama periode kejadian tersebut jauh lebih kecil dari batas maksimum yang diijinkan oleh FDA, yaitu 104 sel per-gram. Rendahnya tingkat kontaminasi Vp (<104 sel per-gram) di dalam sampel seafood yang diambil dari tempat asal seafood penyebab kontaminasi ini menyebabkan perlunya peninjauan kembali terhadap batas maksimal tingkat cemaran Vp yang diijinkan di dalam seafood terutama yang akan dikonsumsi mentah. Rendahnya prevalensi dan tingkat cemaran Vp patogen (10000 koloni menjadi tidak terdeteksi. Proses iradiasi sinar gamma (Co60) juga dapat menurunkan tingkat kontaminasi Vp (Jakabi et al, 2003). Dosis 1 kGy bisa menurunkan tingkat kontaminasi Vp sebesar 6-10 log. Sampai dosis 3 kGy, proses tidak membunuh tiram dan juga tidak menyebabkan penyimpangan bau, flavor dan penampakan tiram.

KESIMPULAN

* Seafood terutama tiram yang dimakan mentah merupakan jenis pangan yang paling sering membawa Vp penyebab gastroenteritis. Kasus keracunan karena Vp lebih banyak terjadi pada musim panas. Strain Vp patogen penyebab gastroenteritis sangat beragam. Strain Vp patogen dengan serotype O3:K6 sejak tahun 1996 muncul menjadi sumber patogen baru penyebab keracunan pangan.
* Prevalensi dan jumlah kontaminasi Vp pada sampel seafood lingkungan meningkat dengan meningkatnya suhu perairan. Tingkat salinitas air laut juga berpengaruh pada tingkat kontaminasi.
* Teknik analisis berpengaruh pada tingkat prevalensi dan tingkat isolasi Vp dari seafood. Untuk pengendalian tingkat kontaminasi didalam seafood, diperlukan pemilihan metode analisis yang lebih sensitifitas dengan waktu deteksi yang lebih cepat.
* Prevalensi dan tingkat kontaminasi Vp dalam sampel seafood lingkungan dan pasar ritel seringkali jauh lebih kecil dari batas maksimum yang diijinkan FDA (10^4 sel per-gram) termasuk pada beberapa kasus KLB.
* Pada sampel seafood dari lingkungan dan pasar ritel, Vp patogen hanya terdeteksi dalam jumlah rendah (< 100 sel per-gram), bahkan pada sampel yang berasal dari lokasi KLB.
Praktek penilaian seafood yang hanya didasarkan pada penghitungan total Vp sebagai deteksi keberadaan Vp patogen, hendaknya diperbaiki dengan juga mempertimbangkan faktor virulen tdh dan/atau trh. Tingkat kontaminasi maksimal yang diijinkan ada didalam seafood yang dijual juga perlu dikaji ulang.
* Praktek penyimpanan di suhu rendah (< 4°C) dapat menekan dan menurunkan tingkat pertumbuhan Vp. Selain itu, proses pasteurisasi minimal (50°C, 15 menit) dan iradiasi sinar gamma 3 kGy dapat meningkatkan keamanan pangan dari produk-produk seafood.

DAFTAR PUSTAKA

- Alam, M.J., K.I. Tomochika, S.I. Miyoshi, S. Shinoda. 2002. Environmental investigation of potentially pathogenic Vibrio parahaemolyticus in the Seto-Inland Sea, Japan. FEMS Microbiol Lett. Feb 19;208(1):83-7.

- Alam, M.J., S. Miyoshi, S. Shinoda. 2003. Studies on pathogenic Vibrio parahaemolyticus during a warm weather season in the Seto Inland Sea, Japan. Environ Microbiol. Aug;5(8):706-10.

- Andrews, L.S., D.L. Park, Y.P. Chen. 2000. Low temperature pasteurization to reduce the risk of vibrio infections from raw shell-stock oysters. Food Addit Contam. Sep;17(9):787-91

- Balter et al. 2006. Vibrio parahaemolyticus Infections Associated with Consumption of Raw Shellfish – Three States, 2006. http://www.cdc.gov/epo/dphsi/phs/infdis.htm. Diakses 20 Desember 2006.

- Blackstone, G.M., J.L. Nordstrom JL, M.C. Vickery, M.D. Bowen, R.F. Meyer, A. DePaola. 2003. Detection of pathogenic Vibrio parahaemolyticus in oyster enrichments by real time PCR. J Microbiol Methods. May;53(2):149-55.

- Cai T, L. Jiang, C. Yang, K. Huang. 2006. Application of real-time PCR for quantitative detection of Vibrio parahaemolyticus from seafood in eastern China. FEMS Immunol Med Microbiol. Mar;46(2):180-6.

- Charles-Hernández, G.L., E. Cifuentes, S.J. Rothenberg. 2006. Environmental factors associated with the presence of Vibrio parahaemolyticus in sea products and the risk of food poisoning in communities bordering the Gulf of Mexico. Journal of Environmental Health Research, Vol. 5 issue 2

- Chiou, C-S., S-Y. Hsu, S-I. CHIU, T-K WANG dan C-S. CHAO. 2000. Vibrio parahaemolyticus Serovar O3:K6 as Cause of Unusually High Incidence of Food-Borne Disease Outbreaks in Taiwan from 1996 to 1999. J. of Clinical Microbiol. Dec., p. 4621–4625 Vol. 38, No. 12

- Cook, D.W, P. Oleary, J.C. Hunsucker, E.M. Sloan, J.C. Bowers, R.J. Blodgett, A. Depaola. 2002. Vibrio vulnificus and Vibrio parahaemolyticus in U.S. retail shell oysters: a national survey from June 1998 to July 1999. J Food Prot. Jan;65(1):79-87.

- Cook, D.W., J.C. Bowers, A. DePaola. 2002. Density of total and pathogenic (tdh+) Vibrio parahaemolyticus in Atlantic and Gulf coast molluscan shellfish at harvest. J. Food Prot. Dec;65(12):1873-80

- Daniels, N.A., L. MacKinnon, R. Bishop, S. Altekruse, B. Ray, R.M. Hammond, S. Thompson, S. Wilson, N.H. Bean, P.M. Griffin and L. Slutsker. 2000. Vibrio parahaemolyticus Infections in the United States, 1973–1998. The Journal of Infectious Diseases;181:1661–6.

- de Sousa, O.V., R.H. Vieira, F.G. de Menezes, C.M. dos Reis, E. Hofer. 2004. Detection of Vibrio parahaemolyticus and Vibrio cholerae in oyster, Crassostrea rhizophorae, collected from a natural nursery in the Coco river estuary, Fortaleza, Ceara, Brazil. Rev Inst Med Trop Sao Paulo. Mar-Apr;46(2):59-62. Epub 2004 May 5.

- Deepanjali, A., H.S. Kumar, I. Karunasagar, I. Karunasagar. 2005. Seasonal variation in abundance of total and pathogenic Vibrio parahaemolyticus bacteria in oysters along the southwest coast of India. Appl Environ Microbiol. Jul;71(7):3575-80.

- DePaola, A., C.A. Kaysner, J. Bowers, D.W. Cook. 2000. Environmental investigations of Vibrio parahaemolyticus in oysters after outbreaks in Washington, Texas, and New York (1997 and 1998). Appl Environ Microbiol. Nov;66(11):4649-54.

- DePaola, A., J. Ulaszek, C.A. Kaysner, B.J. Tenge, J.L. Nordstrom, J. Wells, N. Puhr, S.M. Gendel. 2003. Molecular, serological, and virulence characteristics of Vibrio parahaemolyticus isolated from environmental, food, and clinical sources in North America and Asia. Appl Environ Microbiol. Jul;69(7):3999-4005.

- Ellison, R.K., E. Malnati, A. Depaola, J. Bowers, G.E. Rodrick. 2001. Populations of Vibrio parahaemolyticus in retail oysters from Florida using two methods. J Food Prot. May;64(5):682-6.

- Franco-Monsreal, J. dan J. Flores-Abuxapqui J. 1989. Prevalence of Vibrio parahaemolyticus in seafood from restaurants in the city of Merida, Yucatan. Salud Publica Mex. May-Jun;31(3):314-25.

- Fuenzalida, L., C. Hernández, J. Toro, M.L. Rioseco, J. Romero dan R.T. Espejo. 2006. Vibrio parahaemolyticus in shellfish and clinical samples during two large epidemics of diarrhoea in southern Chile. Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology.

- Gooch, J.A., A. DePaola A, C.A. Kaysner, D.L. Marshall. 2001. Evaluation of two direct plating methods using nonradioactive probes for enumeration of Vibrio parahaemolyticus in oysters. Appl Environ Microbiol. Feb;67(2):721-4

- Gooch, J.A., A. DePaola, J. Bowers, D.L. Marshall. 2002. Growth and survival of Vibrio parahaemolyticus in postharvest American oysters. J Food Prot. Jun;65(6):970-4.

- Hara-Kudo, Y., K. Sugiyama, M. Nishibuchi, A. Chowdhury, J. Yatsuyanagi, Y. Ohtomo, A. Saito, H. Nagano, T. Nishina, H. Nakagawa, H. Konuma, M. Miyahara, S. Kumagai. 2003. Prevalence of pandemic thermostable direct hemolysin-producing Vibrio parahaemolyticus O3:K6 in seafood and the coastal environment in Japan. Appl Environ Microbiol. Jul;69(7):3883-91.

- Hara-Kudo, Y., T. Nishina, H. Nakagawa, H. Konuma, J. Hasegawa, S. Kumagai. 2001. Improved method for detection of Vibrio parahaemolyticus in seafood. Appl Environ Microbiol. Dec;67(12):5819-23.

- Jakabi, M, D.S. Gelli, J.C. Torre, M.A. Rodas, B.D. Franco, M.T. Destro, M. Landgrafi. 2003. Inactivation by ionizing radiation of Salmonella enteritidis, Salmonella infantis, and Vibrio parahaemolyticus in oysters (Crassostrea brasiliana). J Food Prot. 2003 Jun;66(6):1025-9.

- Kaufman, G.E., A.K. Bej, J. Bowers, A. DePaola. 2003. Oyster-to-oyster variability in levels of Vibrio parahaemolyticus. J Food Prot. Jan;66(1):125-9

- DePaola, A., J.L. Nordstrom, J.C. Bowers, J.G. Wells, D.W. Cook. 2003. Seasonal abundance of total and pathogenic Vibrio parahaemolyticus in Alabama oysters. Appl Environ Microbiol. 2003 Mar;69(3):1521-6.

- Kaysner, C.A. 2000. Vibrio species. Didalam The Microbilogical Safety and Quality of Food (Vol. II). Lund., B.M., T.C. Baird-Parker dan G.W. Gould (Ed). Aspen Publisher, Inc. Gaithersburg, Maryland.

- Martinez-Urtaza, J., A. Lozano-Leon, A. DePaola, M. Ishibashi, K. Shimada, M. Nishibuchi dan E. Liebana. 2004. Characterization of Pathogenic Vibrio parahaemolyticus Isolates from Clinical Sources in Spain and Comparison with Asian and North American Pandemic Isolates. Journal of Clinical Microbiology, Oct 2004, Vol. 42, No. 10 (p. 4672–4678). American Society for Microbiology.

- Martinez-Urtaza, J., L. Simental, D. Velasco, A. DePaola, M. Ishibashi, Y. Nakaguchi, M. Nishibuchi, D. Carrera-Flores, C. Rey-Alvarez dan A. Pousa. 2005. Pandemic Vibrio parahaemolyticus O3:K6, Europe. Emerging Infectious Diseases. http://www.cdc.gov/eid. Vol. 11, No. 8, August 2005.

- McLaughlin, J.B., A. DePaola, C.A. Bopp, K.A. Martinek, N.P. Napolilli, C.G. Allison, S.L. Murray, E.C. Thompson, M.M. Bird, and J.P. Middaugh. 2005. Outbreak of Vibrio parahaemolyticus Gastroenteritis Associated with Alaskan Oysters. The new england journal of medicine 353;14 http://www.nejm.org October 6

- Miwa N, M. Kashiwagi, F. Kawamori, T. Masuda, Y. Sano, M. Hiroi, H. Kurashige. 2006. Levels of Vibrio parahaemolyticus and thermostable direct hemolysin gene-positive organisms in retail seafood determined by the most probable number-polymerase chain reaction (MPN-PCR) method. Shokuhin Eiseigaku Zasshi. Apr;47(2):41-5.

- Miwa N, Nishio T, Arita Y, Kawamori F, Masuda T, Akiyama M. 2003. Evaluation of MPN method combined with PCR procedure for detection and enumeration of Vibrio parahaemolyticus in seafood. Shokuhin Eiseigaku Zasshi. Dec;44(6):289-93.

- Muntada-Garriga, JM, J.J. Rodriguez-Jerez, El. Lopez-Sabater, M.T. Mora-Ventura. 1995. Effect of chill and freezing temperatures on survival of Vibrio parahaemolyticus inoculated in homogenates of oyster meat. Lett Appl Microbiol. Apr;20(4):225-7.

- Osawa, R dan S. Yamai. 1996. Production of thermostable direct hemolysin by Vibrio parahaemolyticus enhanced by conjugated bile acids. Appl Environ Microbiol. Aug;62(8):3023-5.

- Sharp, A.N. 2000. Detection of microorganisms in foods: principles of physical method for separation and associated chemical and enzymological methods of detection. Didalam The Microbilogical Safety and Quality of Food (Vol. II). Lund., B.M., T.C. Baird-Parker dan G.W. Gould (Ed). Aspen Publisher, Inc. Gaithersburg, Maryland.

- Vuddhakul, V., S. Soboon, W. Sunghiran, S. Kaewpiboon, A. Chowdhury, M. Ishibashi, Y. Nakaguchi, M. Nishibuchi. 2006. Distribution of virulent and pandemic strains of Vibrio parahaemolyticus in three molluscan shellfish species (Meretrix meretrix, Perna viridis, and Anadara granosa) and their association with foodborne disease in southern Thailand. J Food Prot. Nov;69(11):2615-20

- Ward, LN dan A.K. Bej. 2006. Detection of Vibrio parahaemolyticus in shellfish by use of multiplexed real-time PCR with TaqMan fluorescent probes. Appl Environ Microbiol. Mar;72(3):2031-42.

Link tentang Vibrio Parahaemolyticus

January 25, 2011 at 8:27 am | Posted in Uncategorized | Leave a comment

1. Kasus Vibrio parahaemolyticus di Dalam Seafood (Oleh: Elvira Syamsir)
2. More information about Vibrio parahaemolyticus
3. Vibrio parahaemolyticus – General Information
4. ….coming soon

Cholera, Vibrio cholerae O1 and O139, and Other Pathogenic Vibrios

January 25, 2011 at 5:24 am | Posted in Uncategorized | 2 Comments

Chapter 24. Cholera, Vibrio cholerae O1 and O139, and Other Pathogenic Vibrios

Richard A. Finkelstein.


Medical Microbiology. 4th edition.
Baron S, editor.
Galveston (TX): University of Texas Medical Branch at Galveston; 1996.
[Table of Contents Page]

General Concepts

Cholera and Vibrio cholerae
Clinical Manifestations: Cholera is a potentially epidemic and life-threatening secretory diarrhea characterized by numerous, voluminous watery stools, often accompanied by vomiting, and resulting in hypovolemic shock and acidosis. It is caused by certain members of the species Vibrio cholerae which can also cause mild or inapparent infections. Other members of the species may occasionally cause isolated outbreaks of milder diarrhea whereas others—the vast majority—are free-living and not associated with disease.

Structure, Classification, and Antigenic Types: Vibrios are Gram-negative, highly motile curved rods with a single polar flagellum. They tolerate alkaline media that kill most intestinal commensals, but they are sensitive to acid. Numerous free-living vibrios are known, some potentially pathogenic. Until 1992, cholera was caused by only two serotypes, Inaba (AC) and Ogawa (AB), and two biotypes, classical and El Tor, of toxigenic O group 1 V cholerae. These organisms may be identified by agglutination in O group 1-specific antiserum directed against the lipopolysaccharide component of the cell wall and by demonstration of their enterotoxigenicity. In 1992, cholera caused by serogroup O139 (synonym “Bengal” the 139th and latest serogroup of V cholerae to be identified) emerged in epidemic proportions in India and Bangladesh. This serovar is identified by 1) absence of agglutination in O group 1 specific antiserum; 2) by agglutination in O group 139 specific antiserum; and 3) by the presence of a capsule.

Pathogenesis: Cholera is transmitted by the fecal-oral route. Vibrios are sensitive to acid, and most die in the stomach. Surviving virulent organisms may adhere to and colonize the small bowel, where they secrete the potent cholera enterotoxin (CT, also called “choleragen”). This toxin binds to the plasma membrane of intestinal epithelial cells and releases an enzymatically active subunit that causes a rise in cyclic adenosine 51-monophosphate (cAMP) production. The resulting high intracellular cAMP level causes massive secretion of electrolytes and water into the intestinal lumen.

Host Defenses: Gastric acid, mucus secretion, and intestinal motility are the prime nonspecific defenses against V cholerae. Breastfeeding in endemic areas is important in protecting infants from disease. Disease results in effective specific immunity, involving primarily secretory immunoglobulin (IgA), as well as IgG antibodies, against vibrios, somatic antigen, outer membrane protein, and/or the enterotoxin and other products.

Epidemiology: Cholera is endemic or epidemic in areas with poor sanitation; it occurs sporadically or as limited outbreaks in developed countries. In coastal regions it may persist in shellfish and plankton. Long-term convalescent carriers are rare. Enteritis caused by the halophile V parahaemolyticus is associated with raw or improperly cooked seafood.

Diagnosis: The diagnosis is suggested by strikingly severe, watery diarrhea. For rapid diagnosis, a wet mount of liquid stool is examined microscopically. The characteristic motility of vibrios is stopped by specific antisomatic antibody. Other methods are culture of stool or rectal swab samples on TCBS agar and other selective and nonselective media; the slide agglutination test of colonies with specific antiserum; fermentation tests (oxidase positive); and enrichment in peptone broth followed by fluorescent antibody tests, culture, or retrospective serologic diagnosis. More recently the polymerase chain reaction (PCR) and additional genetically-based rapid techniques have been recommended for use in specialized laboratories.

Control: Control by sanitation is effective but not feasible in endemic areas. A good vaccine has not yet been developed. A parenteral vaccine of whole killed bacteria has been used widely, but is relatively ineffective and is not generally recommended. An experimental oral vaccine of killed whole cells and toxin B-subunit protein is less than ideal. Living attenuated genetically engineered mutants are promising, but such strains can cause limited diarrhea as a side effect. Antibiotic prophylaxis is feasible for small groups over short periods.

Other Vibrio Infections:

Other serogroups of V cholerae may cause diarrheal disease and other infections but are not associated with epidemic cholera. Vibrio parahaemolyticus is an important cause of enteritis associated with the ingestion of raw or improperly prepared seafood. Other Vibrio species, including V vulnificus, can cause infections of humans and other animals including fish. Campylobacter species (formerly included with vibrios) can cause enteritis. C pylori, now known as Helicobacter pylori, is associated with gastric and duodenal ulcers (see Ch. 23).

Introduction

Vibrios are highly motile, gram-negative, curved or comma-shaped rods with a single polar flagellum. Of the vibrios that are clinically significant to humans, Vibrio cholerae O group 1, the agent of cholera, is the most important. Vibrio cholerae was first isolated in pure culture by Robert Koch in 1883, although it had been seen by other investigators, including Pacini, who is credited with describing it first in Florence, Italy, in 1854.

Cholera is a life-threatening secretory diarrhea induced by an enterotoxin secreted by V cholerae. Cholera and the cholera enterotoxin are increasingly recognized as the prototypes for a wide variety of non-invasive diarrheal diseases, collectively known as the enterotoxic enteropathies; of these, diarrhea due to enterotoxigenic strains of Escherichia coli (see Ch. 26) is the most important. Cholera remains a major epidemic disease. There have been seven great pandemics. The latest, which started in 1961, invaded the Western Hemisphere (for the first time this century) with a massive outbreak in Peru in 1991. There have since been more than a million cases in Central and South America as well as a few imported cases in the U.S. and Canada. V cholerae serogroup O139, which arose in October of 1992 in India and Bangladesh, may become the cause of the 8th great pandemic of cholera.

Other vibrios may also be clinically significant in humans, and some are known to cause diseases in domestic animals. Nonpathogenic vibrios are widely distributed in the environment, particularly in estuarine waters and seafoods. For this reason, isolation of a vibrio from a patient with diarrheal disease does not necessarily indicate an etiologic relationship.

Vibrio Cholerae

Clinical Manifestations

Following an incubation period of 6 to 48 hours, cholera begins with the abrupt onset of watery diarrhea (Fig. 24-1). The initial stool may exceed 1 L, and several liters of fluid may be secreted within hours, leading to hypovolemic shock. Vomiting usually accompanies the diarrheal episodes. Muscle cramps may occur as water and electrolytes are lost from body tissues. Loss of skin turgor, scaphoid abdomen, and weak pulse are characteristic of cholera. Various degrees of fluid and electrolyte loss are observed, including mild and subclinical cases. The disease runs its course in 2 to 7 days; the outcome depends upon the extent of water and electrolyte loss and the adequacy of water and electrolyte repletion therapy. Death can occur from hypovolemic shock, metabolic acidosis, and uremia resulting from acute tubular necrosis.


Figure 24-1
Pathophysiology of cholera.

Structure, Classification, and Antigenic Types

The cholera vibrios are Gram-negative, slightly curved rods whose motility depends on a single polar flagellum. Their nutritional requirements are simple. Fresh isolates are prototrophic (i.e., they grow in media containing an inorganic nitrogen source, a utilizable carbohydrate, and appropriate minerals). In adequate media, they grow rapidly with a generation time of less than 30 minutes. Although they reach higher population densities when grown with vigorous aeration, they can also grow anaerobically. Vibrios are sensitive to low pH and die rapidly in solutions below pH 6; however, they are quite tolerant of alkaline conditions. This tolerance has been exploited in the choice of media used for their isolation and diagnosis.

Until 1992, the vibrios that caused epidemic cholera were subdivided into two biotypes: classical and El Tor. Classical V cholerae was first isolated by Koch in 1883. Subsequently, in the early 1900s, some vibrios resembling V cholerae were isolated from Mecca-bound pilgrims at the quarantine station at El Tor, in the Sinai peninsula, that had been established to try to control cholera associated with pilgrimages to Mecca. These vibrios resembled classical V cholerae in many ways but caused lysis of goat or sheep erythrocytes in a test known as the Greig test. Because the pilgrims from whom they were isolated did not have cholera, these hemolytic El Tor vibrios were regarded as relatively insignificant except for the possibility of confusion with true cholera vibrios. In the 1930s, similar hemolytic vibrios were associated with relatively restricted outbreaks of diarrheal disease, called paracholera, in the Celebes. In 1961, cholera caused by El Tor vibrios erupted in Hong Kong and spread virtually worldwide. Although in the course of this pandemic most V cholerae biotype El Tor strains lost their hemolytic activity, a number of ancillary tests differentiate them from vibrios of the classical biotype.

The operational serology of the cholera vibrios which belong in O antigen group 1 is relatively simple. Both biotypes (El Tor and classical) contain two major serotypes, Inaba and Ogawa (Fig. 24-2). These serotypes are differentiated in agglutination and
vibriocidal antibody tests on the basis of their dominant heat-stable lipopolysaccharide somatic antigens. The cholera group has a common antigen, A, and the serotypes are differentiated by the type-specific antigens, B (Ogawa) and C (Inaba). An additional serotype, Hikojima, which has both specific antigens, is rare. V cholerae O139 appears to have been derived from the pandemic El Tor biotype but has lost the characteristic O1 somatic antigen; it has gained the ability to produce a polysaccharide capsule; it produces the same cholera enterotoxin; and it seems to have retained the epidemic potential of O1 strains.


Figure 24-2
Vibrio cholerae (O group 1 antigen).

Other antigenic components of the vibrios, such as outer membrane protein antigens, have not been extensively studied. The cholera vibrios also have common flagellar antigens. Cross-reactions with Brucella and Citrobacter species have been reported. Because of DNA relatedness and other similarities, other vibrios formerly called “nonagglutinable” are now classified as V cholerae. The term nonagglutinable is a misnomer because it implies that these vibrios are not agglutinable; in fact, they are not agglutinable in antisera against the O antigen group 1 cholera vibrios, but they are agglutinable in their own specific antisera. More than 139 serotypes are now recognized. Some strains of non-O group 1 V cholerae cause diarrheal disease by means of an enterotoxin related to the cholera enterotoxin and, perhaps, by other mechanisms, but these strains have not been associated with devastating outbreaks like those caused by the true cholera vibrios. Recently, vibrio strains that agglutinate in some O group 1 cholera diagnostic antisera but not in others have been isolated from environmental sources. Volunteer feeding experiments have shown that these atypical O group 1 vibrios are not enteropathogenic in humans. Recent studies using specific toxin gene probes indicate that these environmental isolates not only are nontoxigenic, but also do not possess any of the genetic information encoding cholera toxin, although some isolates from diarrheal stools do.

The cholera vibrios cause many distinctive reactions. They are oxidase positive. The O group 1 cholera vibrios almost always fall into the Heiberg I fermentation pattern; that is, they ferment sucrose and mannose but not arabinose, and they produce acid but not gas. Vibrio cholerae also possesses lysine and ornithine decarboxylase, but not arginine dihydrolase. Freshly isolated agar-grown vibrios of the El Tor biotype, in contrast to classical V cholerae, produce a cell-associated mannose-sensitive hemagglutinin active on chicken erythrocytes. This activity is readily detected in a rapid slide test. In addition to hemagglutination, numerous tests have been proposed to differentiate the classical and El Tor biotypes, including production of a hemolysin, sensitivity to selected bacteriophages, sensitivity to polymyxin, and the Voges-Proskauer test for acetoin. El Tor vibrios originally were defined as hemolytic. They differed in this characteristic from classical cholera vibrios; however, during the most recent pandemic, most El Tor vibrios (except for the recent isolates from Texas and Louisiana) had lost the capacity to express the hemolysin. Most El Tor vibrios are Voges-Proskauer positive and resistant to polymyxin and to bacteriophage IV, whereas classical vibrios are sensitive to them. As both biotypes cause the same disease, these characteristics have only epidemiologic significance. Strains of the El Tor biotype, however, produce less cholera enterotoxin, but appear to colonize intestinal epithelium better than vibrios of the classical variety. Also, they seem some what more resistant to environmental factors. Thus, El Tor strains have a higher tendency to become endemic and exhibit a higher infection-to-case ratio than the classical biotype.

Pathogenesis

Recent studies with laboratory animal models and human volunteers have provided a detailed understanding of the pathogenesis of cholera. Initial attempts to infect healthy American volunteers with cholera vibrios revealed that the oral administration of up to 1011 living cholera vibrios rarely had an effect; in fact, the organisms usually could not be recovered from stools of the volunteers. After the administration of bicarbonate to neutralize gastric acidity, however, cholera diarrhea developed in most volunteers given 104 cholera vibrios. Therefore, gastric acidity itself is a powerful natural resistance mechanism. It also has been demonstrated that vibrios administered with food are much more likely to cause infection.

Cholera is exclusively a disease of the small bowel. To establish residence and multiply in the human small bowel (normally relatively free of bacteria because of the effective clearance mechanisms of peristalsis and mucus secretion), the cholera vibrios have one or more adherence factors that enable them to adhere to the microvilli (Fig. 24-3). Several hemagglutinins and the toxin-coregulated pili have been suggested to be involved in adherence but the actual mechanism has not been defined. In fact, there may be multiple mechanisms. The motility of the vibrios may affect virulence by enabling them to penetrate the mucus layer. They also produce mucinolytic enzymes, neuraminidase, and proteases. The growing cholera vibrios elaborate the cholera enterotoxin (CT or choleragen), a polymeric protein (Mr 84,000) consisting of two major domains or regions. The A region (Mr 28,000), responsible for biologic activity of the enterotoxin, is linked by noncovalent interactions with the B region (Mr 56,000), which is composed of five identical noncovalently associated peptide chains of Mr 11,500. The B region, also known as choleragenoid, binds the toxin to its receptors on host cell membranes. It is also the immunologically dominant portion of the holotoxin. The structural genes that encode the synthesis of CT reside on a transposon-like element in the V cholerae chromosome, in contrast to those for the heat-labile enterotoxins (LTs) of E coli (Ch. 25), which are encoded by plasmids. The amino acid sequences of these structurally, functionally, and immunologically related enterotoxins are very similar. Their differences account for the differences in physicochemical behavior and the antigenic distinctions that have been noted. There are at least two antigenically related but distinct forms of cholera enterotoxin, called CT-1 and CT-2. Classical O1 V cholerae and the Gulf Coast El Tor strains produce CT-1 whereas most other El Tor strains and O139 produce CT-2. Vibrio cholerae exports its enterotoxin, whereas the E coli LTs occur primarily in the periplasmic space. This may account for the reported differences in severity of the diarrheas caused by these organisms.


Figure 24-3
Vibrio cholerae attachment and colonization in experimental rabbits. The events are assumed to be similar in human cholera. (A) Scanning electron microscopy during early infection. Curved (more…)

Studies in adult American volunteers have shown that 5µ g of CT, administered orally with bicarbonate, causes 1 to 6 L of diarrhea; 25µg causes more than 20 L.

Synthesis of CT and other virulence-associated factors such as toxin-coregulated pili are believed to be regulated by a transcriptional activator, Tox R, a transmembrane DNA-binding protein.

The molecular events in these diarrheal diseases involve an interaction between the enterotoxins and intestinal epithelial cell membranes (Fig. 24-4). The toxins bind through region B to a glycolipid, the GM1 ganglioside, which is practically ubiquitous in eukaryotic cell membranes. Following this binding, the A region, or a major portion of it known as the A1 peptide (Mr 21,000), penetrates the host cell and enzymatically transfers ADP-ribose from nicotinamide adenine dinucleotide (NAD) to a target protein, the guanosine 5′-triphosphate (GTP)-binding regulatory protein associated with membrane-bound adenylate cyclase. Thus, CT (and LT) resembles diphtheria toxin in causing transfer of ADP-ribose to a substrate. With diphtheria toxin, however, the substrate is elongation factor 2 and the result is cessation of host cell protein synthesis. With CT, the ADP-ribosylation reaction essentially locks adenylate cyclase in its “on mode” and leads to excessive production of cyclic adenosine 51-monophosphate (cAMP). Pertussis toxin, another ADP-ribosyl transferase, also increases cAMP levels, but by its effect on another G-protein, Gi (Fig. 24-5).

The subsequent cAMP-mediated cascade of events has not yet been delineated, but the final effect is hypersecretion of chloride and bicarbonate followed by water, resulting in the characteristic isotonic voluminous cholera stool. In hospitalized patients, this can result in losses of 20 L or more of fluid per day. The stool of an actively purging, severely ill cholera patient can resemble rice water—the supernatant of boiled rice. Because the stool can contain 108 viable vibrios per ml, such a patient could shed 2 × 1012 cholera vibrios per day into the environment. Perhaps by production of CT, the cholera vibrios thus ensure their survival by increasing the likelihood of finding another human host. Recent evidence suggests that prostaglandins may also play a role in the secretory effects of cholera enterotoxin. Recent studies in volunteers using genetically-engineered Tox strains of V cholerae have revealed that the vibrios have putative mechanisms in addition to CT for causing (milder) diarrheal disease. These include Zot (for Zonula occludens toxin) and Ace (for accessory cholera enterotoxin), and perhaps others, but their role has not been established conclusively. Certainly CT is the major virulence factor and the act of colonization of the small bowel may itself elicit an altered host response (e.g., mild diarrhea), perhaps by a trans-membrane signaling mechanism.


Figure 24-4
Mechanism of action of cholera enterotoxin. Cholera toxin approaches target cell surface. B subunits bind to oligosaccharide of GM1 ganglioside. Conformational alteration of holotoxin occurs, allowing (more…)


Figure 24-5
Comparison of activities of cholera enterotoxin (CT) with pertussis toxin (PT). The α-subunits of Gs and Gi, with GTP-binding sites, are ADP-ribosylated, respectively, by A1 (more…)

Various animal models have been used to investigate pathogenic mechanisms, virulence, and immunity. Ten-day-old suckling rabbits develop a fulminating diarrheal disease after intraintestinal inoculation with virulent V cholerae or CT. Adult rabbits are relatively resistant to colonization by cholera vibrios; however, they do respond, with characteristic out pouring of fluid, to the intraluminal inoculation of live vibrios or enterotoxin in surgically isolated ileal loops. Suckling mice are susceptible to intragastric inoculation of vibrios and to orally administered toxin. Adult conventional mice are also susceptible to orally administered toxin, but resist colonization except in isolated intestinal loops. Interestingly, however, germ-free mice can be colonized for months with cholera vibrios. They rarely show adverse effects, although they are susceptible to cholera enterotoxin. Dogs have been used experimentally, although they are relatively refractory and require enormous inocula to elicit choleraic manifestations. Chinchillas also are susceptible to diarrhea following intraintestinal inoculation with moderate numbers of cholera vibrios. Infections initiated by extraintestinal routes of inoculation (e.g., intraperitoneal) largely reflect the toxicity of the lipopolysaccharide endotoxin. The intraperitoneal infection in mice has been used to assay the protective effect of conventional killed vibrio vaccines (no longer widely used).

Various animals, including humans, rabbits, and guinea pigs, also respond to intradermal inoculation of relatively minute amounts of CT with a characteristic delayed (maximum response at 24 hours), sustained (visible up to 1 week or more), erythematous, edematous induration associated with a localized alteration of vascular permeability. In laboratory animals, this response can be measured after injecting a protein-binding dye, such as trypan blue, that extravasates to produce a zone of bluing at the site of intracutaneous inoculation of toxin. This observation has been exploited in the assay of CT and its antibody and in the detection of other enterotoxins.

In addition, because of the broad spectrum of activity of CT on cells and tissues that it never contacts in nature, various in vitro systems can be used to assay the enterotoxin and its antibody. In each, the toxin causes a characteristically delayed, but sustained, activation of adenylate cyclase and increased production of cAMP, and it may cause additional, readily recognizable, morphologic alterations of certain cultured cell lines. The cells most widely used for this purpose are Chinese hamster ovary (CHO) cells, which elongate in response to picogram doses of the toxin, and mouse Y-l adrenal tumor cells, which round up. Cholera toxin has become an extremely valuable experimental probe to identify other cAMP-mediated responses. It also activates adenylate cyclase in pigeon erythrocytes, a procedure that was used by D. Michael Gill to define its mode of action.

These assays and models also have been applied in the study of an expanding number of CT-related and unrelated enterotoxins. These include the LTs of E coli, which are structurally and immunologically similar to it and are effective in any model that is responsive to CT. The family of small molecular weight heat-stable enterotoxins (ST) of E coli, which activate guanylate cyclase, and which are rapidly active in the infant mouse and certain other intestinal models, are clearly unrelated to CT. CT-related enterotoxins have been reported from certain nonagglutinable (non-O group I) Vibrio strains and a Salmonella enterotoxin was shown to be related immunologically to CT. CT-like factors from Shigella and V parahaemolyticus have thus far been demonstrated only in sensitive cell culture systems. Other enterotoxins and enterocytotoxins, which elicit cytotoxic effects on intestinal epithelial cells, also have been described from Escherichia, Klebsiella, Enterobacter, Citrobacter, Aeromonas, Pseudomonas, Shigella, V parahaemolyticus, Campylobacter, Yersinia enterocolitica, Bacillus cereus, Clostridium perfringens, C difficile, and staphylococci. Escherichia coli, some vibrio strains, and some other enteric bacteria produce cytotoxins that, like Shiga toxin of Shigella dysenteriae, act on Vero (African green monkey kidney) cells in vitro. These toxins have been called Shiga-like toxins, Shiga toxin-like toxins, Vero toxins, and Vero cytotoxins. The classic staphylococcal enterotoxins perhaps should more properly be called neurotoxins, as they seem to affect the central nervous system rather than the gut directly to cause fluid secretion or histopathologic effects.

Host Defenses

Infection with cholera vibrios results in a spectrum of responses. These range from no observed manifestations except perhaps a serologic response ( the most common) to acute purging, which must be treated by hospitalization and fluid replacement therapy; this is the classic response. The reasons for these differences are not entirely clear, although it is known that individuals differ in gastric acidity and that hypochlorhydric individuals are most prone to cholera. Whether individuals differ in the availability of intestinal receptors for cholera vibrios or for their toxin has not been established. Prior immunologic experience of subjects at risk is certainly a major factor. For example, in heavily endemic regions such as Bangladesh, the attack rate is relatively low among adults in comparison with children. In neoepidemic areas, cholera is more frequent among the working adult population. Resistance is related to the presence of circulating antibody and, perhaps more importantly, local immunoglobulin A (IgA) antibody against the cholera bacteria or the cholera enterotoxin or both. Intestinal IgA antibody can prevent attachment of the vibrios to the mucosal surface and neutralize or prevent binding of the cholera enterotoxin. For reasons that are not clear, individuals of blood group O are slightly more susceptible to cholera. Breastfeeding is highly recommended as a means of increasing immunity of infants to this and other diarrheal disease agents.

Recovery from cholera probably depends on two factors: elimination of the vibrios by antibiotics or the patient’s own immune response, and regeneration of the poisoned intestinal epithelial cells. Treatment with a single 200-mg dose of doxycycline has been recommended. As studies in volunteers demonstrated conclusively, the disease is an immunizing process. Patients who have recovered from cholera are solidly immune for at least 3 years.

Cholera vaccines consisting of killed cholera bacteria administered parenterally have been used since the turn of the century. However, recent controlled field studies indicate that little, if any, effective immunity is induced in immunologically virgin populations by such vaccines, although they do stimulate preexisting immunity in the adult population in heavily endemic regions. Controlled studies have likewise shown that a cholera toxoid administered parenterally was ineffective in preventing cholera. Probably the natural disease should be simulated to induce truly effective immunity although a parenterally administered conjugate vaccine consisting of the polysaccharide of the vibrio LPS covalently linked to cholera toxin has given promising results in preliminary studies. Studies in volunteers have shown that orally administered, chemically mutagenized or genetically engineered mutants which do not produce CT or produce only its B subunit protein can induce immunity against subsequent challenge. However, most of these candidate vaccines also produce unacceptable side effects—primarily mild to moderate diarrhea. An exception is strain CVD103-HgR (a mercury resistant AB+ derivative of classical biotype Inaba serotype strain 569B). This strain has minimal reactogenicity but does not colonize well and therefore has to be given in higher doses. Field studies with this strain are in progress. Combined preparations of bacterial somatic antigen and toxin antigen have been reported to act synergistically in stimulating immunity in laboratory animals; that is, the combined protective effect is closer to the product than to the sum of the individual protective effects. However, a large field study evaluating such nonviable oral vaccines in Bangladesh revealed that neither the whole-cell bacterin nor the killed vibrios supplemented with the B-subunit protein of the cholera enterotoxin induced sufficient long term protection, especially in children, to justify their recommendation for public health use. No clear-cut advantage of the inclusion of the B-subunit was demonstrated.

In any case, even if these vaccines were effective, the requirement for large and repeated doses would make them too expensive for use in the developing areas that are usually afflicted with epidemic cholera. Moreover, they were clearly less effective in children—the primary target population in heavily endemic areas. Neither the killed whole cell vaccine nor strain CVD103-HgR could be expected to protect against the new O139 serovar.

Epidemiology

Humans apparently are the only natural host for the cholera vibrios. Cholera is acquired by the ingestion of water or food contaminated with the feces of an infected individual. Previously, the disease swept the world in six great pandemics and later receded into its ancestral home in the Indo-Pakistani subcontinent. In 1961, the El Tor biotype (a subset distinguished by physiologic characteristics) of V cholerae, not previously implicated in widespread epidemics, emerged from the Celebes (now Sulawesi), causing the seventh great cholera pandemic. In the course of their migration, the El Tor biotype cholera vibrios virtually replaced V cholerae of the classic biotype that formerly was responsible for the annual cholera epidemics in India and East Pakistan (now Bangladesh). The pandemic that began in 1961 is now heavily seeded in Southeast Asia and in Africa. It has also invaded Europe, North America, and Japan, where the outbreaks have been relatively restricted and self-limited because of more highly developed sanitation. Several new cases were reported in Texas in 1981 and sporadic cases have since been reported in Louisiana and other Gulf Coast areas. This now endemic focus appears to be due to a clone which is unique from the pandemic strain. In 1991, the pandemic strain hit Peru with massive force and has since spread through most of the Western Hemisphere, causing more than a million cases. Fortunately, mortality has been less than 1 percent because of the effectiveness of oral rehydration therapy. The vibrios surprised us again, in 1992, with the emergence of O139 in India and Bangladesh. For a while it appeared that O139 would replace O1 (both classical and El Tor) but it has exhibited quiescent periods when O1 reemerges.

Cholera appears to exhibit three major epidemiologic patterns: heavily endemic, neoepidemic (newly invaded, cholera-receptive areas), and, in developed countries with good sanitation, occasional limited outbreaks. These patterns probably depend largely on environmental factors (including sanitary and cultural aspects), the prior immune status or antigenic experience of the population at risk, and the inherent properties of the vibrios themselves, such as their resistance to gastric acidity, ability to colonize, and toxigenicity. In the heavily endemic region of the Indian subcontinent, cholera exhibits some periodicity; this may vary from year to year and seasonally, depending partly on the amount of rain and degree of flooding. Because humans are the only reservoirs, survival of the cholera vibrios during interepidemic periods probably depends on a relatively constant availability of low-level undiagnosed cases and transiently infected, asymptomatic individuals. Long-term carriers have been reported but are extremely rare. The classic case occurred in the Philippines, where “cholera Dolores” harbored cholera vibrios in her gallbladder for 12 years after her initial attack in 1962. Her carrier state resolved spontaneously in 1973; no secondary cases had been associated with her well-marked strain. Recent studies, however, have suggested that cholera vibrios can persist for some time in shellfish, algae or plankton in coastal regions of infected areas and it has been claimed that they can exist in “a viable but nonculturable state.”

During epidemic periods, the incidence of infection in communities with poor sanitation is high enough to frustrate the most vigorous epidemiologic control efforts. Although transmission occurs primarily through water contaminated with human feces, infection also may be spread within households and by contaminated foods. Thus, in heavily endemic regions, adequate supplies of pure water may reduce but not eliminate the threat of cholera.

In neoepidemic cholera-receptive areas, vigorous epidemiologic measures, including rapid identification and treatment of symptomatic cases and asymptomatically infected individuals, education in sanitary practices, and interruption of vehicles of transmission (e.g., by water chlorination), may be most effective in containing the disease. In such situations, spread of cholera usually depends on traffic of infected human beings, although spread between adjacent communities can occur through bodies of water contaminated by human feces. John Snow was credited with stopping an epidemic in London, England, by the simple expedient of removing the handle of the “Broad Street pump” (a contaminated water supply) in 1854, before acceptance of the “germ theory” and before the first isolation of the “Kommabacillus” by Robert Koch.

In such developed areas as Japan, Northern Europe, and North America, cholera has been introduced repeatedly in recent years, but has not caused devastating outbreaks; however, Japan has reported secondary cases and, in 1978, the United State experienced an outbreak of about 12 cases in Louisiana. In that outbreak, sewage was infected, and infected shellfish apparently were involved. Interestingly, the hemolytic vibrio strain implicated was identical to one that caused an unexplained isolated case in Texas in 1973.

Diagnosis

Rapid bacteriologic diagnosis offers relatively little clinical advantage to the patient with secretory diarrhea, because essentially the same treatment (fluid and electrolyte replacement) is employed regardless of etiology. Nevertheless, rapid identification of the agent can profoundly affect the subsequent course of a potential epidemic outbreak. Because of their rapid growth and characteristic colonial morphology, V cholerae can be easily isolated and identified in the bacteriology laboratory, provided, first, that the presence of cholera is suspected and, second, that suitable specific diagnostic antisera are available. The vibrios are completely inhibited or grow somewhat poorly on usual enteric diagnostic media (MacConkey agar or eosin-methylene blue agar). An effective selective medium is thiosulfate-citrate-bile salts-sucrose (TCBS) agar, on which the sucrose-fermenting cholera vibrios produce a distinctive yellow colony. However, the usefulness of this medium is limited because serologic testing of colonies grown on it occasionally proves difficult, and different lots vary in their productivity. This medium is also useful in isolating V parahaemolyticus. They can also be isolated from stool samples or rectal swabs from cholera cases on simple meat extract (nutrient) agar or bile salts agar at slightly alkaline pH values. Following observation of characteristic colonial morphology with a stereoscopic microscope using transmitted oblique illumination, microorganisms can be confirmed as cholera vibrios by a rapid slide agglutination test with specific antiserum. Classic and El Tor biotypes can be differentiated at the same time by performing a direct slide hemagglutination test with chicken erythrocytes: all freshly isolated agar-grown El Tor vibrios exhibit hemagglutination; all freshly isolated classic vibrios do not. In practice, this can be accomplished with material from patients as early as 6 hours after streaking the specimen in which the cholera vibrios usually predominate. However, to detect carriers (asymptomatically infected individuals) and to isolate cholera vibrios from food and water, enrichment procedures and selective media are recommended. Enrichment can be accomplished by inoculating alkaline (pH 8.5) peptone broth with the specimen and then streaking for isolation after an approximate 6-hour incubation period; this process both enables the rapidly growing vibrios to multiply and suppresses much of the commensal microflora.

The classic case of cholera, which includes profound secretory diarrhea and should evoke clinical suspicion, can be diagnosed within a few minutes in the prepared laboratory by finding rapidly motile bacteria on direct, bright-field, or dark-field microscopic examination of the liquid stool. The technician can then make a second preparation to which a droplet of specific anti-V cholerae O group 1 antiserum is added. This quickly stops vibrio motility. Another rapid technique is the use of fluorescein isothiocyanate-labeled specific antiserum (fluorescent antibody technique) directly on the stool or rectal swab smear or on the culture after enrichment in alkaline peptone broth. For cultural diagnosis, both nonselective and selective (TCBS) media may be used. Although demonstration of typical agglutination essentially confirms the diagnosis, additional conventional tests such as oxidase reaction, indole reaction, sugar fermentation reactions, gelatinase, lysine, arginine, and ornithine decarboxylase reactions may be helpful. Tests for chicken cell hemagglutination, hemolysis, polymyxin sensitivity, and susceptibility to phage IV are useful in differentiating the El Tor biotype from classic V cholerae. Tests for toxigenesis may be indicated.

Diagnosis can be made retrospectively by confirming significant rises in specific serum antibody titers in convalescents. For this purpose, conventional agglutination tests, tests for rises in complement-dependent vibriocidal antibody, or tests for rises in antitoxic antibody can be employed. Convenient microversions of these tests have been developed. Passive hemagglutination tests and enzyme-linked immunosorption assays (ELISAs) have also been proposed.

Cultures that resemble V cholerae but fail to agglutinate in diagnostic antisera (nonagglutinable or non-O group 1 vibrios) present more of a problem and require additional tests such as oxidase, decarboxylases, inhibition by the vibriostatic pteridine compound 0/129, and the “string test.” The string test demonstrates the property, shared by most vibrios and relatively few other genera, of forming a mucus-like string when colony material is emulsified in 0.5 percent aqueous sodium deoxycholate solution. Additional tests for enteropathogenicity and toxigenesis may be useful. Genetically based tests such as PCR are increasingly being used in specialized laboratories.

Control

Treatment of cholera consists essentially of replacing fluid and electrolytes. Formerly, this was accomplished intravenously, using costly sterile pyrogen-free intravenous solutions. The patient’s fluid losses were conveniently measured by the use of buckets, graduated in half-liter volumes, kept underneath an appropriate hole in an army-type cot on which the patient was resting. Antibiotics such as tetracycline, to which the vibrios are generally sensitive, are useful adjuncts in treatment. They shorten the period of infection with the cholera vibrios, thus reducing the continuous source of cholera enterotoxin; this results in a substantial saving of replacement fluids and a markedly briefer hospitalization. Note, however, that fluid and electrolyte replacement is all-important; patients who are adequately rehydrated and maintained will virtually always survive, and antibiotic treatment alone is not sufficient.

Recently it has been recognized that almost all cholera patients and others with similar severe secretory diarrheal disease can be maintained by fluids given orally if the solutions contain a usable energy source such as glucose. Because of this discovery, packets containing appropriate salts are distributed by such organizations as WHO and UNICEF to cholera-afflicted areas, where they are dissolved in water as needed. One such formulation, called ORS for oral rehydration salts, contains NaCl, 3.5 g; KCl,1.5 g; NaHCO3, 2.5 g (or trisodium citrate, 2.9 g); and glucose, 20.0 g. This mixture is dissolved in 1 L of water and taken orally in increments. Flavoring may be added. Improved versions of ORS, including rice-based formulations that reduce stool output and can be made at home, have been recommended. Unfortunately, this technique, which will save countless millions of lives in developing countries, has not yet been widely accepted by practicing physicians in developed countries.

The possibility of pharmacologic intervention (e.g., a pill that will stop choleraic diarrhea after it has started), has been considered. Two drugs, chlorpromazine and nicotinic acid, have been effective in experimental animals, although the precise mechanism of action has yet to be defined.

Like smallpox and typhoid, cholera—under natural circumstances—appears to affect only humans; therefore, V cholerae as an etiologic entity could conceivably disappear with the last human infection. Nevertheless, the spectrum of cholera-like diarrheal diseases probably will persist for some time.

Cholera is essentially a disease associated with poor sanitation. The simple application of sanitary principles—protecting drinking water and food from contamination with human feces—would go a long way toward controlling the disease. However, at present, this is not feasible in the underdeveloped areas that are afflicted with epidemic cholera or are considered to be cholera receptive. Meanwhile, development of a vaccine that would effectively prevent colonization and manifestations of cholera would be extremely helpful. As indicated above, such vaccines are presently being tested. Antibiotic or chemotherapeutic prophylaxis is feasible and may be indicated under certain circumstances. It also should be mentioned that the incidence of cholera is significantly higher in formula-fed than in breast-fed babies.

Present information indicates that V parahaemolyticus enteritis could be almost completely prevented by applying appropriate procedures to prevent multiplication of the organisms in contaminated seafood, such as keeping it refrigerated continually.

Other Vibrio Infections

Other vibrios may be clinically significant also. These include non-O group 1 V cholerae. Vibrio parahaemolyticus, a halophilic (salt-loving) vibrio associated with enteritis is acquired by ingestion of raw or improperly cooked seafoods. Another halophilic vibrio, which ferments lactose and for this reason was called the L + vibrio, has recently been identified as V vulnificus. It has been associated with wound infections as well as fatal septicemias. Other groups of vibrios, previously referred to as group F and EF-6, have recently been classified into species: V fluvialis, V hollisae, V furnissia, and V damsela. Vibrio mimicus is a recently described sucrose-negative species. Vibrio fetus, a group of anaerobic to microaerophilic spirally curved rods associated with venereally transmitted infertility and abortion in domestic animals, is now called Campylobacter jejuni and is considered to belong in the family Spirillaceae rather than in the family Vibrionaceae. Campylobacter jejuni has been associated with dysentery-like gastroenteritis, duodenal and gastric ulcers, as well as with other types of infection, including bacteremic and central nervous system infections in humans (see Ch. 23). Another vibrio-like organism, Helicobacter pylori (formerly known as C pylori) causes gastritis and predisposes to duodenal ulcers and gastric cancer. Although some similarities in habitat and other properties occur, members of the family Vibrionaceae are separated taxonomically from members of the family Enterobacteriaceae. The oxidase test (vibrios are usually oxidase positive) is particularly useful. Other vibrios exist, and some of these may be responsible for diseases in fish and other lower animals. As vibrios are widely distributed in the environment, particularly in estuarine waters and in seafoods, reports of their isolation from patients with diarrheal disease do not necessarily always imply an etiologic relationship.

Cholera-like vibrios have been reported in Maryland’s Chesapeake Bay but have not been associated with any human cases despite more than 15 years of extensive surveillance. These vibrios are probably nonpathogenic nonagglutinable (non-O group 1) vibrios, or the atypical O group 1 vibrios mentioned above, which do not contain the genes for toxin production, do not colonize, and are avirulent.

Relatively little is known about the epidemiology of nonagglutinable vibrios. When sought, these vibrios have been found widely in brackish surface waters (sewers, marshes, bogs, and coastal areas), and are generally more numerous in warmer months. They appear to be free-living aquatic organisms; whether particular subsets are potential pathogens is not yet clear. Strains isolated from humans with diarrheal disease more frequently give positive responses in assays for enterotoxins or enteropathogenicity, but the pathogenic mechanism of other isolates associated with shellfish remains undefined. An epidemiologic pattern is more evident with V parahaemolyticus, which is clearly part of the normal flora of coastal and estuarine waters throughout the world. Although originally recognized in Japan, V parahaemolyticus enteritis has been reported virtually worldwide within the last decade. Its reported frequency varies widely, partly because of inherent differences in distribution and partly because many laboratories do not use the appropriate culture medium (TCBS) to isolate these organisms. Two types of clinical syndromes, both usually self-limited, have been observed. The most common is a watery diarrhea, perhaps with associated abdominal cramps, nausea, vomiting, and fever, with a modal incubation period of 15 hours. A dysenteric syndrome with a short incubation period of 2 1/2 hours also has been described. In Japan, about 24 percent of reported cases of food poisoning are attributed to V parahaemolyticus. The disease occurs primarily during summer, possibly reflecting the increased presence of the organism in the marine environment during those months, as well as the enhanced opportunity for it to multiply in unrefrigerated foods. It appears to be transmitted exclusively by food, primarily raw or improperly prepared seafood. As growth of this organism is inhibited at temperatures below 15° C, rapid cooling and refrigeration of seafoods that are eaten raw would vastly reduce the incidence of disease. The organisms are killed by heating to 65° C for 10 minutes; therefore, properly handled cooked seafood should present no problem. The role played in virulence and pathogenesis by the thermostable direct hemolysin, which is responsible for the positive Kanagawa phenomenon (a hemolytic reaction around colonies growing on a particular blood agar medium), is not yet fully defined. This hemolysin is clearly associated with pathogenicity, but whether it is merely an associated marker or intimately involved in the disease process awaits further research. Be this as it may, only strains that possess the Kanagawa hemolysin are considered pathogenic. In laboratory studies, the isolated hemolysin has been reported to be cytotoxic, cardiotoxic, and lethal.

References

1. Albert MJ. Vibrio cholerae O139 Bengal. J Clin Microbiol. 1994;32:2345. [PubMed]
2. Barua D, Greenough III WB: Cholera. Plenum Book Company, New York and London, 1992 .
3. Blake JD, Weaver RE, Hollis DG. Diseases of humans (other than cholera) caused by vibrios. Annu Rev Microbiol. 1980;34:341. [PubMed]
4. Clemens JD. et al. Field trial of cholera vaccines in Bangladesh: results from three-year follow-up. Lancet. 1990;335:270. [PubMed]
5. Finkelstein RA. Cholera. Crit Rev Microbiol. 1973;2:553.
6. Finkelstein RA: Cholera. In Germanier R (ed): Bacterial vaccines. Academic Press, San Diego, 1984 .
7. Finkelstein RA: Cholera, the cholera enterotoxins, and the cholera enterotoxin-related enterotoxin family. p. 85. In Owen P, Foster TS (eds): Immuno-chemical and Molecular Genetic Analysis of Bacterial Pathogens. Elsevier, Amsterdam, 1988 .
8. Finkelstein RA, Burks MF, Zupan A. et al. Epitopes of the cholera family of enterotoxins. Rev Infect Dis. 1987;9:544. [PubMed]
9. Gill DM: Seven toxic peptides that cross cell membranes. p. 291. In Jeljaszewicz I,
10. Wadstrom T (eds): Bacterial Toxins and Cell Membranes. Academic Press, San Diego, 1978 .
11. Hoge CW, Watsky D, Peeler RN. et al. Epidemiology and spectrum of Vibrio infections in a Chesapeake Bay community. J Infect Dis. 1989;160:985. [PubMed]
12. Kaper JB, Morris JG, Jr, Levine MM. Cholera. Clin Microbiol Rev. 1995;8:48. [PubMed] [Free Full text in PMC]
13. Kaper JB, Moseley SL, Falkow S. Molecular characterization of environmental and nontoxigenic strains of Vibrio cholerae. Infect Immun. 1981;32:661. [PubMed]
14. Levine MM, Kaper JBV, Black RE, Clements ML. New knowledge on pathogenesis of bacterial enteric infections as applied to vaccine development. Microbiol Rev. 1983;47:510. [PubMed]
15. Levine MM, Kaper JP, Herrington D. et al. Volunteer studies of deletion mutants of Vibrio cholerae O1 prepared by recombinant techniques. Infect Immun. 1988;56:161. [PubMed]
16. Marchlewicz BA, Finkelstein RA. Immunologic differences among the cholera/coli family of enterotoxins. Diagn Microbiol Infect Dis. 1983;1:129. [PubMed]
17. Mekalanos JJ, Swartz DJ, Pearson GDN. et al. Cholera toxin Miller VL, Taylor RK, Mekalanos JJ: Cholera toxin transcriptional activator Tox R is a transmembrane DNA binding protein. Cell. 1987;48:271. [PubMed]
18. Morris JG, Jr, Black RE. Cholera and other vibrioses in the United States. N Engl J Med. 1985;312:343. [PubMed]
19. Moss J, Vaughn M. Activation of adenylate cyclase by choleragen. Annu Rev Biochem. 1979;48:581. [PubMed]
20. Ouchterlony O, Holmgren J (eds): Cholera and related diarrheas; molecular aspects of a global health problem. 43rd Nobel Symposium, co-sponsored by the World Health Organization. S Karger, Basel, 1980 .
21. Peterson JW, Ochoa LG. Role of prostaglandins and cAMP in the secretory effects of cholera toxin. Science. 1989;245:857. [PubMed]
22. Wachsmuth IK, Blake PA, Olsvik O. Vibrio cholerae and Cholera: Molecular to Global Perspectives. ASM Press, Washington, DC,1994 .
23. World Health Organization. Diarrheal diseases control programme. Report of the tenth meeting of the technical advisory group (Geneva, March 13—17, 1989). WHO/D/89. 1989;32:1.
24. van Heyningen WE, Seal JR: Cholera: The American Scientific Experience, 1947-1980. Westview Press, Boulder CO, 1983 .

Copyright © 1996, The University of Texas Medical Branch at Galveston.

Vibrio parahaemolyticus

January 25, 2011 at 1:33 am | Posted in Uncategorized | Leave a comment

Vibrio parahaemolyticus
From Wikipedia, the free encyclopedia Vibrio parahaemolyticus


SEM image of V. parahaemolyticus
Scientific classification
Kingdom: Bacteria
Phylum: Proteobacteria
Class: Gamma Proteobacteria
Order: Vibrionales
Family: Vibrionaceae
Genus: Vibrio
Species: V. parahaemolyticus
Binomial name
Vibrio parahaemolyticus
(Fujino et al. 1951)
Sakazaki et al. 1963

Vibrio parahaemolyticus is a curved, rod-shaped, Gram-negative bacterium found in brackish[1] saltwater, which, when ingested, causes gastrointestinal illness in humans.[1] V. parahaemolyticus is oxidase positive, facultatively aerobic, and does not form spores. Like other members of the genus Vibrio, this species is motile, with a single, polar flagellum.[2]

Contents:
1 Pathogenesis
2 Epidemiology
3 Hosts
4 References
5 External links

Pathogenesis

While infection can occur via the fecal-oral route, ingestion of bacteria in raw or undercooked seafood, usually oysters, is the predominant cause the acute gastroenteritis caused by V. parahaemolyticus.[3] Wound infections also occur, but are less common than seafood-borne disease. The disease mechanism of V. parahaemolyticus infections has not been fully elucidated.[4]

Clincal isolates usually possess two pathogenicity islands (PAI), which are acquired via horizontal gene transfer. Although the pathogenicity islands have ben sequenced, the functions of many of the PAI genes have not been elucidated. Each pathogenicity island contains a genetically-distinct Type III Secretion System, which is capable of injecting virulence proteins into host cells to cause disease. Additionally, two well-characterized virulence proteins are typically found in the pathogenicity islands, the thermostable direct hemolysin gene (tdh) or the tdh-related hemolysin gene (trh). Strains possessing the hemolysins exhibit beta-hemolysis on blood agar plates.

Epidemiology

Outbreaks tend to be concentrated along coastal regions during the summer and early fall when higher water temperatures favor higher levels of bacteria. Seafood most often implicated includes squid, mackerel, tuna, sardines, crab, shrimp, and bivalves like oysters and clams. The incubation period of ~24 hours is followed by explosive, watery diarrhea accompanied by nausea, vomiting, abdominal cramps, and sometimes fever. Vibrio parahaemolyticus symptoms typically resolve with-in 72 hours, but can persist for up to 10 days in immunocompromised individuals. As the vast majority of cases of V. parahaemolyticus food infection are self-limiting, treatment is not typically necessary. In severe cases, fluid and electrolyte replacement is indicated.

Additionally, swimming or working in affected areas can lead to infections of the eyes or ears[5] and open cuts and wounds. Following Hurricane Katrina, there were 22 vibrio wound infections 3 of which were caused by V. parahaemolyticus and 2 of these led to death.

Hosts

Hosts of Vibrio parahaemolyticus include:
Clithon retropictus[6]
Nerita albicilla[6]

References
^ a b CDC Disease Info vibrioparahaemolyticus_g
^ a b Ryan KJ; Ray CG (editors) (2004). Sherris Medical Microbiology (4th ed.). McGraw Hill. ISBN 0-8385-8529-9.
^ Finkelstein RA (1996). Cholera, Vibrio cholerae O1 and O139, and Other Pathogenic Vibrios. In: Barron’s Medical Microbiology (Barron S et al., eds.) (4th ed.). Univ of Texas Medical Branch. (via NCBI Bookshelf) ISBN 0-9631172-1-1.
^ Baffone W, Casaroli A, Campana R, Citterio B, Vittoria E, Pierfelici L, Donelli G (2005). “‘In vivo’ studies on the pathophysiological mechanism of Vibrio parahaemolyticus TDH(+)-induced secretion”. Microb Pathog 38 (2-3): 133–7. doi:10.1016/j.micpath.2004.11.001. PMID 15748815.
^ Penland RL, Boniuk M, Wilhelmus KR (2000). “Vibrio ocular infections on the U.S. Gulf Coast”. Cornea 19 (1): 26–9. doi:10.1097/00003226-200001000-00006. PMID 10632004.
^ a b Kumazawa NH, Kato E, Takaba T, Yokota T. (August) 1988. Survival of Vibrio parahaemolyticus in two gastropod molluscs, Clithon retropictus and Nerita albicilla. Nippon Juigaku Zasshi. 50(4): 918-24.

External links
CDC Disease Info vibrioparahaemolyticus_g
FDA Bad Bug Book entry on V. parahaemolyticus

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Infectious diseases · Bacterial diseases: Proteobacterial G− (primarily A00–A79, 001–041, 080–109)

Categories: Vibrionales

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