Distribution of thermostable direct hemolysin (TDH)- and TDH-related hemolysin (TRH)-producing Vibrio parahaemolyticus in coastal Shimane Prefecture and TDH and TRH V parahaemolyticus contamination of retail shellfish

We studied distribution of thermostable direct hemolysin (TDH)- and TDH-related hemolysin (TRH)-producing Vibrio parahaemolyticus in coastal sea water, sediment, and shellfish and related retail shellfish contamination in Shimane Prefecture, Japan, between 2002 and 2004. V. parahaemolyticus was isolated from > 80% of sea water, sediment, and shellfish. The detection of TDH gene (tdh) and TRH gene (trh)-positive V parahaemolyticus in sea water was 11%, in sediment 16%, and in shellfish 26%. The number of genes and gene-related in seawater was 23 MPN/L, in sediment 29 MPN/100 g, and in shellfish 460 MPN/10 g. TDH- and TRH-producing V. parahaemolyticus detected in seawater was 5%, in sediment 11% and in shellfish 14%. The continuous distribution of TDH-producing O2:K28, O4:K88, O4:K37, and O4:KUT organisms on the western coast and TRH2-producing O5:k30, O5:K43, O10:K19, O10:KUT, O11:K40, O11:KUT, and OUT:KUT organisms on the Oki Island coast suggested the settlement of these organisms in these coastal environments. From 7 (12%) of 59 retail short-necked clam samples, we isolated TDH-producing O 1:KUT, O3:K6 (2 strains from 2 samples imported from Korea), O4:K12, OUT:K8, and TRH2-producing OUT:K40 and OUT:K51 organisms. These findings suggested that TDH- and TRH-producing V. parahaemolyticus are widely distributed along the coast of this prefecture and are transported by contaminated retail shellfish from other areas.     

Isolation of thermostable direct hemolysin producing Vibrio parahaemolyticus from food using screening by PCR in food-borne outbreaks

The producibility of thermostable direct hemolysin (TDH) is the most important pathogenic factor in Vibrio parahaemolyticus. TDH (+) V. parahaemolyticus is usually isolated from patients having V. parahaemolyticus food-borne disease. TDH (+) V. parahaemolyticus is, however, very difficult to isolate from food and environmental samples. In the 5 years from 2000 to 2004 in Tokyo, V. parahaemolyticus was isolated from food samples related to 67 of 227 V parahaemolyticus food-borne outbreaks. In these outbreaks, TDH (+) strains were also tried to isolate using PCR as the screening methods. TDH (+) V. parahaemolyticus strains were able to isolate from enrichment broth in which toxR and tdh genes become positive in PCR. TDH (+) strains of the same serotype with patients were able to be isolated from 23 food samples related to 11 outbreaks (16.4%); 3 outbreaks in 2000, 2 in 2001, 2 in 2002, 1 in 2003, and 3 in 2004. The serotypes of V. parahaemolyticus isolated from food were O3 : K6 (10 samples), O3 : K5 (6 samples), O1 : K25 (4 samples), O3 : K29 (2 samples), O4 : K 8 (1 sample), and O4 : K11 (1 sample). The isolation rate of the TDH (+) strain from enrichment broth differed with samples. In several samples TDH (+) strains were isolated easily only by examining 3 colonies, hence no TDH (+) strains were isolated in spite of the examination of 250 colonies. No correlation was seen between the number of V. parahaemolyticus and the isolation rate of TDH (+) strains in food samples. Screening using PCR is very effective method for isolating TDH (+) V. parahaemolyticus from food samples.     

O3:K6 serotype of Vibrio parahaemolyticus identical to the global pandemic clone associated with diarrhea in Peru.

OBJECTIVES: To determine if the Vibrio parahaemolyticus O3:K6 global pandemic clone has spread into Peru. METHODS: A collection of 100 V. parahaemolyticus strains isolated from diarrhea cases in Peru were serotyped for O:K antigens and genotyped for the presence of the species-specific toxR gene and for the tdh and trh genes. In addition, the group-specific PCR (GS-PCR) and PCR for the presence of the open reading frame ORF8 of the filamentous phage f237 was performed to determine the pandemic status of the strains. RESULTS: Fifty strains of V. parahaemolyticus in this collection were identified as pandemic strains. Forty-six ORF8 and GS-PCR positive strains were identical to the global pandemic clone O3:K6, while four strains that also possessed the pandemic genotype and were ORF8 and GS-PCR positive belonged to serotypes O3:K68, O3:K58 and OUT (untypable):K6. One of the O3:K6 strains was isolated in 1996, indicating that the pandemic strain was present in Peru at about the same time that it caused the first outbreak in Calcutta in February 1996. CONCLUSIONS: Based on this first report in Peru of such strains, we recommend including V. parahaemolyticus in the differential diagnosis of the etiologic agents for diarrhea in this part of the world.     

The trends of Vibrio parahaemolyticus foodborne outbreaks in Tokyo: 1989-2000

The total number of foodborne outbreaks due to Vibrio parahaemolyticus in Tokyo during the last 12 years between 1989 and 2000 were 710. The number of outbreaks in a year was 55 in 1989, 75 in 1990, and there was a gradual decrease to 24 outbreaks in 1993 which was the smallest number during those 12 years. After 1994, the number of outbreaks increased dramatically year by year until 1998 (107 outbreaks). Then they had decreased slightly to 74 in 1999, 65 in 2000. The monthly incidence of V. parahaemolyticus foodborne outbreaks showed a peak in August (44.2%) each year. In the last 12 years, 88.7% of V. parahaemolyticus foodborne outbreaks occurred during the 3 months between July and September, while 99.9% occurred between June and October. The most prevalent serotype of V. parahaemolyticus also changed, the most prevalent was O4:K4 in 1989, O4:K8 in both 1990 and 1991, O1:K56 in 1992, and O4:K8 from 1993 through 1995. Serotype O3:K6 became the most prevalent in 1996 and has remained so to date. In addition, the new serotype O4:K68 had also appeared in 1998. The number of outbreaks due to serotype O4:K68 followed that of O3:K6. Thus, the trends of V. parahaemolyticus foodborne outbreaks during the last 12 years in Tokyo showed various characteristics and dramatic changes in causal organisms.     

Comparison of epidemiological markers for Vibrio parahaemolyticus isolated from food poisoning

Vibrio parahaemolyticus food poisoning, is the most prevalent among bacterial food poisoning in Japan. Study of epidemiologic markers is important in an attempt to trace the source of contamination. The purpose of this study was to compare seven different typing methods (serotyping, plasmid profile, antibiogram, phage susceptibility. TDH production, tdh and trh gene and pulsed-field gel electrophoresis [PFGE]) for V. parahaemolyticus. Outbreaks of V. parahaemolyticus food poisoning which occurred during the 13 years from 1981 to 1993 numbered 43 including 481 cases in Nagano Prefecture. Serovar O4:K8 was the most prlevalent serovar isolated, serovar O2:K3, O4:K63 and O3:K5 followed. Forty one strains of V. parahaemolyticus were used in this study. All of the strains were isolated from 12 food poisoning cases at Nagano Prefectual Research Institute for Health and Pollution. Of the 41 strains, twenty two strains (O4:K8, O4:K63) were sensitive to both phi VP 253 and phi VP 143 phages, six strains (O3:K5) to phage phi VP 143. Thirteen strains (O3:K29, O4:K11, O4:K12 and O5:KUT) were insensitive to both phages. CBPC, CBPC.CEZ and CBPC.CEZ.KM.SM resistant strains was determined in 22 strains out of 41 strains. Five strains of V. parahaemolyticus carried plasmid. Of the 41 strains, thirty nine strains were possessive to tdh gene and productive to TDH. Chromosomal DNA of the isolates from 12 different outbreaks was analysed by PFGE after Not I digestion. PFGE analysis of the digested DNA yielded 11 to 21 DNA fragments. Twelve distinctive fragment patterns were identified in 41 V. parahaemolyticus isolates from 12 different food poisonings. These results showed that the PFGE method is an useful tool to analyse an epidemiological survey for isolates of Vibrio parahaemolyticus food poisoning.     

Epidemiological study of outbreaks and sporadic cases due to Vibrio parahaemolyticus--serotype O3:K6 in Aichi Prefecture, Japan, during 1988 and 2001

Percentage of the outbreaks by O3:K6 Vibrio parahaemolyticus (V. p) in Aichi Prefecture Japan increased from 3% (3/86) for 1988-95 to 75% (33/44) for 1996-2001. The percentage of the sporadic diarrhea cases caused by O3:K6 V. p in a general hospital in Aichi Prefecture also increased from 0% (0/253) to 61% (135/221) during the same periods. Thermostable direct hemolysin (TDH)-positive O3:K6 were isolated from 95% (19/20) of the outbreak incidents and 100% (135/135) of the sporadic cases. Only one TRH (TDH-related hemolysin)-positive O3:K6 was isolated from one outbreak incident. Percentage of the outbreaks by O3:K6 V. p associated with the consumption of boiled shellfishes increased from 5% (6/117) for 1988-95 to 25% (15/59) for 1996-2001, in particular, boiled crabs and squillas associated outbreaks increased from 2% (2/117) to 17% (10/59) and from 2% (2/117) to 10% (6/59), respectively. From 1,548 raw sea foods sampled in the Nagoya Central Wholesale Market in Aichi Prefecture in 1995-99, one TDH-positive O3:K6 was isolated from one live squilla (1/30). Increase in the percentage of outbreaks associated with TDH-positive O3:K6 V. p after 1996 in Aichi Prefecture was revealed to correlate with the increase in the outbreaks associated with consumption of boiled sea foods, especially boiled crabs as well as squillas. Accordingly, it becomes clear that sanitary handling of these boiled foods is important to prevent outbreaks and sporadic cases of diarrhea caused by O3:K6 V. p infection.     

Review and guidelines for treatment of diarrhea caused by Vibrio parahaemolyticus

In Chile Vibrio parahaemolyticus has been detected in 3 gastroenteritis outbreaks since 1998. The most recent outbreak occurred during the summer of 2005, affecting over 10,000 people of whom one died. Affected individuals presented with one or more of the following symptoms: diarrhea, nausea, vomiting, abdominal pain and/or fever. Fecal white blood cells were detected in only 6% of patients. The predominant serotype in the 3 outbreaks was the pandemic O3:K6 strain. Diagnosis was confirmed by isolation and identification of V. parahaemolyticus in stool cultures and/or by establishing an epidemiological link. V. parahaemolyticus isolates were 100% susceptible to tetracycline, ciprofloxacin and chloramphenicol, and universally resistant to ampicillin. Due to the public health impact of the 2005 outbreak, the Ministry of Health called for a National Task Force mandated to review epidemiological, clinical and microbiological features of the outbreak and to propose management guidelines.     

广东省2003～2008年副溶血性弧菌血清学分型研究

目的了解广东省副溶血性弧菌食物中毒患者和水产品分离株的血清型分布。方法采用血清玻片凝集试验对365株从食物中毒患者和水产品中分离的副溶血性弧菌进行血清分型。结果365株副溶血性弧菌中,205株食物中毒患者分离株以O3：K6型为主,血清型中排前3位的是O3：K6（71.71%）、O4：K8（10.24%）、O2：K3（4.39%）;160株水产品分离株共分50个血清型,排前3位的是O5：K17（8.13%）、O2：K28（6.25%）、O2：K3（5.63%）,无优势菌型。结论2003～2008年广东省副溶血性弧菌食物中毒分离株血清型以O3：K6型为主,水产品分离株血清型呈多样性。     

浙江省副溶血性弧菌03：K6血清型菌株多位点序列分型研究

目的掌握浙江省不同来源的副溶血性弧菌O3：K6血清型菌株的分子分型特征,为副溶血性弧菌食源性疾病的预防控制提供技术支持。方法选择副溶血性弧菌的7个管家基因dnaE、gyrB、recA、dtdS、pntA、pyrC及tnaA,对62株不同来源的副溶血性弧菌03：K6血清型菌株样本进行PCR扩增、测序,Chromas软件和DNAStar软件分析,核酸序列上传至MLST数据库进行比对,获得每株菌的序列型,绘制多位点序列分型遗传进化树并进行亲缘性分析。结果62株副溶血性弧菌03：K6血清型菌株中,59株菌为ST-3型（3,4,19,4,29,4,22）,1株菌为ST-121型（3,2,82,52,4,78,66）,1株菌为新的ST型（5,10,34,27,77,49,23）,1株菌仅gyrB基因第562位点由C突变为T,其余基因序列与ST4型（3,5,22,12,20,22,25）一致。结论浙江省副溶血性弧菌03：K6血清型菌株的主要MLST型别为ST-3型。     

Bacteriological studies of traveller's diarrhoea (6). Analysis of enteropathogenic bacteria at Kansai Airport Quarantine Station from September 4th, 1994 through December 1996

During the period of investigation from Sept. 4, 1994 to Dec, 1996, a total of 11,446,534 overseas travellers were quarantined at Kansai Airport Quarantine Station, and 22,187 voluntarily reported of episodes suffering from diarrhoea. Bacteriological examination of the stools a total of 9,299 individuals' was performed, and the following results were obtained. 1) Various enteropathogenic bacteria were isolated from 33.3% of the stools examined. Bacterial species isolated were as follows: Plesiomonas shigelloides, 2,066 cases (66.7%); Vibrio parahaemolyticus, 358 cases (11.6%); Aeromonas sobria, 360 cases (11.6%); Shigella spp., 291 cases (9.4%); Salmonella spp., 183 cases (5.9%); A. hydrophila, 126 cases (4.1%); and V. cholerae non-O1, 121 cases (3.9%). However, ETEC was not done with an object of test. 2) In 502 cases (16.2%), plural enteropathogenic bacteria were isolated from single patient, suggesting high frequency of a mixed infections. 3) From Feb. to Mar. 1995, thirteen cases cholera were found from patients who had travelled to Bali, Indonesia. Cases with enteropathogenic bacteria other than V. cholerae O1 were found without any seasonal variation. 4) The major regions where the travellers were infected with the pathogens are as follows: Vibrio spp., were from only Asia; Shigella, widely distributed but especially in India and Indonesia; P. shigelloides and Salmonella, widely distributed. 5) Among the Shigella strains, S. sonnei were isolated the most, followed by S. flexneri, S. boydii and S. dysenteriae. A strain of S. boydii provisional serovar E 16553 was isolated from a patient infected in India. 6) Among the Salmonella serovars, Salmonella Enteritidis was isolated the most frequently (49 cases, 25.7%). 7) 265 (89.2%) of 297 Shigella strains, 52 (27.2%) of 19] Salmonella strains, and 19 (95.0%) of 20 V. cholerae O1 were resistant to one or more drugs tested (SM. CP. TC. KM. ABPC. NA. OFLX). 8) All of the 20 V. cholerae O1 strains were Ogawa, E1 Tor. All of them were toxigenic strains. 9) The most frequently isolated serovar of V. parahaemolyticus was O3: K6. 89.8% of all V. parahaemolyticus strains were positive for thermostable direct hemolysin (TDH) gene, and 14.6% of them were positive for TDH-related hemolysin (TRH) gene by DNA-probe or PCR method.     

丽水市贝类产品中副溶血性弧菌的血清分型及耐药性研究

目的了解丽水市贝类产品中副溶血性弧菌的血清型分布及耐药性,为防治副溶血性弧菌引起的食源性疾病提供依据。方法从丽水市区农贸市场等采集贝壳类水产品,常规方法分离副溶血性弧菌,参照GB/T 4789.7—2003方法,用标准血清进行分型,用纸片扩散法进行药敏试验。结果检出阳性标本28份,检出率为26.67%(28/105)。分离得到的28株副溶血性弧菌分属于7个血清群,分别为O:4群占39.29%(11/28)、O:3群占14.29%(4/28)、O:2群占14.29%(4/28)、O:11群占14.29%(4/28)、O:1群占7.14%(2/28)、O:7群占7.14%(2/28)、O:10群占3.57%(1/28)。28株副溶血性弧菌中有25株对氨苄西林耐药,1株对四环素耐药。结论从丽水市贝类产品分离的副溶血性弧菌具有血清分群多样化和耐药性简单的特点。     

Bacteriological study of traveller's diarrhoea. 4) Isolation of enteropathogenic bacteria from patients with traveller's diarrhoea at Osaka Airport Quarantine Station during 1984-1991]

During the last 8 years (1984 to 1991), 16,639,233 overseas travellers were quarantined at Osaka Airport Quarantine Station and 38,326 travellers reported that they were (or had been) suffering from diarrhoea. Bacteriological examination of stools from 12,573 persons revealed the following results. 1) Various enteropathogenic bacteria were isolated from 3,669 cases (29.2%) examined. The predominant species of bacteria isolated were as follows: Salmonella, 1049 cases; Plesiomonas shigelloides, 1030 cases; Vibrio parahaemolyticus, 789 cases; Shigella, 607 cases; enterotoxigenic Escherichia coli, 422 cases; Vibrio cholerae non-O1, 212 cases. 2) There were no apparent seasonal variations in the isolation rate of these pathogens. 3) The suspected regions for infection with these pathogens were as follows: a) Salmonella, Enterotoxigenic E. coli and Plesiomonas, mainly South-East and South-West Asia. b) Shigella, South-West Asia, especially India (59.8%). c) V. parahaemolyticus and V. fluvialis, mainly South-East and East Asia. d) V. cholerae non-O1, V. mimicus, almost restricted to Asia, mainly South-East Asia. 4) 22 strains of V. cholerae O1 were isolated and 19 were Ogawa, E1 Tor. Of these strains, 13 were cholera toxin-producing strains and 9 were non-toxigenic strains. 5) Several pathogens (mixed infection) were isolated simultaneously from 670 cases. 6) The 1247 Salmonella strains were identified into 98 serovars. 7) Of 624 Shigella strains isolated, 57.9% were S. sonnei, 29.2% were S. flexneri, 8.6% were S. boydii, 4.3% were S. dysenteriae. 8) The most predominant serovar of V. parahaemolyticus was O4:K8. Of 1,247 strains isolated, 9.8% were not producing thermostable direct hemolysin (TDH). 9) 570 (91.3%) of 624 Shigella strains and 409 (32.8%) of 1,247 Salmonella strains isolated were resistant to any one of the drugs tested (SM. CP. TC. KM. ABPC. NA. OFLX). The resistance rate and the number of multiple drug-resistance strains increased year by year. 10) Enterotoxigenic E. coli was isolated from 422 cases (10.7%) of 3,939 cases. Cases with enterotoxigenic E. coli strains producing ST (heat-stable), LT (heat-labile) or both ST and LT were 53.8%, 24.2% and 14.2% respectively. The others were cases with mixed types of enterotoxin production.     

副溶血性弧菌Vop T蛋白的表达及其抗体的毒力菌株特异性研究

目的 研究副溶血性弧菌VopT蛋白的菌群特异性,为建立致病性副溶血性弧菌的快速检测方法以及探讨VopT的作用机制奠定基础。方法 根据副溶血性弧菌VopT蛋白基因(VPA1327)序列设计合成引物,通过PCR从副溶血性弧菌致病株WX06152(血清型O3∶K6)中扩增出目的基因,构建原核表达载体pET-30a(+)-VPA1327并转化大肠杆菌BL21(DE3)。经IPTG诱导表达,重组蛋白应用MALDI-TOF-MS/MS鉴定和His镍柱纯化,并通过免疫BALB/c小鼠制备抗血清,建立副溶血性弧菌VopT蛋白的间接ELISA检测方法,并分析其敏感性和毒力菌株特异性。结果 成功表达了大小为33 kDa融合蛋白,肽指纹图谱与副溶血性弧菌VopT蛋白一致。重组VopT抗血清能够特异性检测副溶血性弧菌毒力株的全菌细胞及其裂解蛋白,与环境株及其它种属菌株无明显交叉反应,灵敏度达到10⁵CFU·mL^-1。结论 重组VopT的抗血清具有良好特异性,为探讨VopT致病机理和分泌机制研究提供了一定的参考依据。     

Human gastroenteritis associated with Vibrio parahaemolyticus in Recife, Brazil]

A study was carried out on the occurrence of Vibrio parahaemolyticus in 1,100 diarrheal feces, routinely sent to a private clinical laboratory for microbiologic diagnosis, in Recife. V. parahaemolyticus was isolated from 14 (1.3%) fecal samples. However, if we considered only the specimens from adult patients, the isolation rate of V. parahaemolyticus rose to 7.1%. In most cases (92.86%), V. parahaemolyticus was the only enteropathogen recognized. Among the isolates, seven K antigen serovars were demonstrated, and three were untypable. Only two human isolates, both ureolytic, did not produce the thermostable direct hemolysin. We concluded that V. parahaemolyticus is an important cause of sea food linked diarrhea among adults in Recife.     

引起食物中毒副溶血性弧菌的病原学鉴定

目的分离食物中毒患者粪便及食物加工工具样本中的病原菌,对分离菌株进行表型和毒力基因鉴定。方法采用TCBS平板法分离食物中毒样本中的病原菌。采用细菌系统鉴定方法,确定所分离的副溶血性弧菌(Vibrio parahaemolyticus,Vp)生化反应特性和血清型。采用PCR检测Vp分离株的种属特异基因、直接耐热溶血素毒力基因(tdh)和直接耐热相关溶血素毒力基因(trh)。采用K-B纸片法检测Vp分离株对14种抗生素的敏感性。结果从1例携带者和3例病人粪便标本中分离出4株Vp,从2份食物加工工具样本中分离出2株Vp。6株Vp分离株均属于含Vp种属特异基因的O1∶K56血清型,tdh基因阳性但trh基因阴性。6株Vp分离株生物学性状和药敏试验结果一致。结论tdh+/trh-O1∶K56血清型Vp是引起本次食物中毒的病原菌。     

A CAMP phenomenon between Vibrio cholerae biotype El Tor and staphylococcal B-hemolysin

A CAMP phenomenon was demonstrated by Vibrio cholerae biotype El Tor and B-lysin producing Staphylococcus aureus in 5% sheep red blood cells-tryptic soy agar medium. All 394 El Tor vibrio strains tested, all showed a crescent-shaped hemolysis (positive CAMP) when the cultures were incubated in a candle jar whereas 67% were CAMP positive when incubated aerobically. Only 9% of the isolates produced detectable hemolysin in a standard tube test using heart infusion broth and 72% in a tube test using heart infusion broth containing 1% glycerol. Seven classical V. cholerae tested were CAMP negative. The CAMP reaction is easy to perform and may be useful for routine use in the differentiation of V. cholerae biotype El Tor from classical V. cholerae.     

Non-cholera vibrios in enterobacteriologic routine]

Of 3250 diarrheal stools received for microbiologic diagnosis at a private clinical laboratory in Recife, Brazil, strains of Vibrio were isolated from 55 (1.7%). The study was carried out from May 1989 through May 1991. For recovering Vibrio, fecal samples were enriched in alkaline peptone water supplemented with 2% NaCl and subcultured on thiosulfate-citrate-bile salts-sucrose agar (TCBS). Of the recovered species, V. parahaemolyticus was most commonly found (24 strains), followed by V. furnissii (15 strains), V. cholerae non-01 (6 strains), V. alginolyticus (4 strains), V. fluvialis (2 strains), and Vibrio sp. (1 strain). The low isolation rate of Vibrio raises doubts about the cost-effectiveness of the use of TCBS in the routine enterobacteriologic workup of clinical laboratories.     

Characterization of the pili isolated from Vibrio parahaemolyticus O3:K6

Pilus of Vibrio parahaemolyticus O3:K6 strain LVP9 belonging to the newly identified clone was purified and characterized. The molecular mass of the pilin was estimated to be about 18 kDa by SDS-PAGE, and the isoelectric point of the pilin was 5.0 +/- 0.2. The LVP9 pili were antigenically different from the other V. parahaemolyticus Na2 pili and Ha7 pili as previously reported, nevertheless all three had indistinguishable morphology and shared a high degree of homology in their N-terminal amino acid sequences. Strain LVP9 and its purified pili did not agglutinate human and rabbit erythrocytes. The LVP9 organisms and the purified pili were adhesive to the rabbit intestine. The adhesion was inhibited by pretreatment of the rabbit intestine with the purified pili or by pretreatment of the organisms with the Fab fractions of anti-pilus antibody. These results indicate that the LVP9 pilus is an adherent factor to the rabbit intestine.     

Direct identification of mycobacteria from the positive cultures in mycobacteria growth indicator tube (MGIT)

The ability of the AccuProbe (Kyokuto) to identify mycobacteria directly from the positive cultures in an automatic detector for mycobacteria (BACTEC MGIT960, Becton Dickinson) was evaluated. Sputum samples were collected from patients with suspected mycobacteriosis between February and April 1999 and conventionally incubated in MGIT960 (37 degrees C for 42 days). The MGIT-positive cultures were successively incubated for several days and were directly identified with the AccuProbe. Monomycobacterial strains were detected from 93 (93.9%) of the 99 sputum samples and polymycobacterial strains were detected from 6 sputum samples (6.1%). Viable cell counts in the positive cultures in MGIT960 were determined using Middlebrook 7H10 agar. The cell counts of Mycobacterium tuberculosis and Mycobacterium avium-intracellulare complex (MAC) varied greatly among the individual samples; the former, 3.8 x 10(2)-2.5 x 10(6) cfu/ml and the latter 1.5 x 10(3)-1.9 x 10(8) cfu/ml. The great differences in the cell counts were observed among these samples. Although the cultures in MGIT were estimated as positive, the early stage of bacterial growth might be used as the samples for the cell counting. By this method, 96 of the total 101 cases were successfully identified as follows: M. tuberculosis 57 cases (56.4%) and MAC 39 cases (38.6%). In conclusion, the present results indicate that the direct identification of mycobacteria from the positive cultures in MGIT960 using AccuProbe is useful for a rapid diagnosis in microbiological testing for mycobacteriosis which has tended to increase in recent years.     

Enzymatic characterization of Vibrio alginolyticus strains isolated from bivalves harvested at Venice Lagoon (Italy) and Guanabara Bay (Brazil)

The aquatic ecosystem is the natural habitat of microorganisms including Vibrio and Aeromonas genus which are pathogenic to human and animals. In the present investigation the frequency of these bacteria and the enzymatic characteristics of 34 Vibrio alginolyticus strains isolated from bivalves harvested in Venice Lagoon (Italy) and Guanabara Bay (Brazil) were carried out from November 2003 to February 2004. The mussels' samples were submitted to enrichment in Alkaline Peptone Water (APW) added with 1% of sodium chloride (NaCl) and APW plus 3% NaCl incubated at 37 degrees C for 18-24 h. Following the samples were streaked onto TCBS Agar (Thiossulfate Citrate Bile Sucrose Agar) and the suspected colonies were submitted to biochemical characterization. Also, the Vibrio alginolyticus strains were evaluated to collagenase, elastase and chondroitinase production. The results showed the isolation of 127 microorganisms distributed as follows: 105 Vibrio strains such as V. alginolyticus (32.4%), V. harveyi (19%) and V. parahaemolyticus (7.6%), 20 Aeromonas strains and two Plesiomonas shigelloides were the main pathogens isolated. We observed the production of the three enzymes from V. alginolyticus strains considered as the main virulence factors of the bacteria, especially in cases of human dermatological infection.