Applicability of a multiplex PCR to detect the seven major Shiga toxin-producing Escherichia coli based on genes that code for serogroup-specific O-antigens and major virulence factors in cattle feces

An 11-gene multiplex polymerase chain reaction (mPCR) was developed based on genes that code for serogroup-specific O-antigens and four major virulence factors (intimin, enterohemorrhagic hemolysin, and Shiga toxins [Stx] 1 and 2), to detect O157 and the "top six" non-O157 (O26, O45, O103, O111, O121, and O145) Shiga toxin-producing Escherichia coli (STEC). The assay specificity was validated with pure cultures of seven major STEC (185 strains), 26 other STEC (65 strains), non-STEC (five strains), and 33 strains of other genera and species. Sensitivity of the assay with cattle fecal sample spiked with pooled cultures of seven major STEC was 10(5) colony-forming units (CFU)/g before enrichment and 10(2) CFU/g after enrichment. The applicability of the assay to detect STEC in fecal samples (n=50), before and after enrichment, was evaluated by comparing with culture-based methods for O26, O111, and O157. The mPCR assay of 50 fecal samples showed seven (14%) positive before enrichment and 23 (46%) positive after enrichment for one or more of the seven O-groups. Overall, 17 isolates from 17 fecal samples and 27 isolates (four for O26, three for O45, and 20 for O103) from 19 fecal samples were obtained, by culture-based methods, for O157 and non-O157 serogroups, respectively. None of the 27 non-O157 isolates possessed the stx genes, suggesting that cattle harbor Shiga toxin-negative E. coli belonging to the "top six" non-O157 serogroups. Our data, although based on a limited number of samples, suggest that the sensitivities of the mPCR and culture-based methods in detecting the seven serogroups of STEC in feces differed between O-groups. An obvious limitation of our mPCR is that the concurrent detection of virulence genes and the serogroups in a sample does not necessarily associate the virulence genes with the prevalent serogroups in the same sample. The major application of our 11-gene mPCR assay may be in identifying putative colonies of STEC obtained by culture-based methods.     

Phenotypic and genetic markers for serotype-specific detection of Shiga toxin-producing Escherichia coli O26 strains from North America

Phenotypic and genetic markers of Shiga toxin-producing Escherichia coli (STEC) O26 from North America were used to develop serotype-specific protocols for detection of this pathogen. Carbohydrate fermentation profiles and prevalence of gene sequences associated with STEC O26 (n = 20) were examined. Non-STEC O26 (n = 17), E. coli O157 (n = 20), E. coli O111 (n = 22), and generic E. coli (n = 21) were used as comparison strains. Effects of supplements: cefixime-tellurite, 4-methylumbelliferyl-beta-D-glucuronide (MUG) and chromogenic additives (5-bromo4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal), 5-bromo-4-chloro-3-indolyl-beta-D-glucuronide (X-GlcA) and o-nitrophenyl-beta-D-galactopyranoside (ONPG), added to isolation agar media were examined. Tests for presence of gene sequences encoding beta intimin (eae beta), Shiga toxin 1 and 2 (stx1 and stx2), H7 flagella (flicCh7), enterohemolysin (ehlyA), O26 somatic antigen (wzx), and high pathogenicity island genes (irp2 and fyuA) were conducted using multiplex polymerase chain reaction. Pulsed-field gel electrophoresis (PFGE) of XbaI restriction endonuclease genomic DNA digests was used to establish clonality among E. coli O26 strains. Of the 26 carbohydrates tested, only rhamnose had diagnostic value. Rhamnose non-fermenters included STEC O26 (100%), non-STEC O26 (40%), generic E. coli (29%), E. coli O111 (23%), and E. coli O157 (0%). Rhamnose non-fermenting colonies growing on Rhamnose-McConkey agar supplemented with X-GlcA, X-Gal, or ONPG, respectively, were blue, white, or faint yellow, whereas rhamnose-fermenters were red. Blue colonies from X-GlcA-containing media were the most well-defined and easiest to pick for further tests. All STEC O26 were MUG-fluorescent, while STEC O157 (n = 18) were non-fluorescent. E. coli O111 and generic E. coli strains were either MUG-positive or-negative. Serotype-specific detection of STEC O26 was achieved by selecting cefixime-tellurite-resistant, MUG-fluorescent, rhamnose-nonfermenting colonies, which carried stx1, eae beta, irp2, and wzx gene sequences. STEC O26 prevalence in dairy farm environmental samples determined using the developed isolation and genetic detection protocols was 4%. PFGE indicated the presence of one major cluster of E. coli O26 with 72-100% DNA fragment-length digest similarity among test strains. The serotype-specific detection methods described herein have potential for routine application in STEC O26 diagnosis.     

Importance of culture confirmation of shiga toxin-producing Escherichia coli infection as illustrated by outbreaks of gastroenteritis--New York and North Carolina, 2005

Escherichia coli O157:H7 and other strains of E. coli that produce Shiga toxin are collectively known as Shiga toxin-producing E. coli (STEC). The current outbreak of STEC O157 infections associated with eating fresh spinach illustrates the importance of obtaining isolates to identify the source of the infections. Laboratory methods that do not require bacterial culture of stool specimens to identify STEC are being used increasingly by clinical diagnostic laboratories, sometimes without subsequent confirmation of a strain by isolating it in culture. This report describes findings from outbreaks of gastroenteritis in 2005 in New York and North Carolina in which clinical diagnostic laboratories initially used only non-culture methods to detect Shiga toxin (Stx). The findings highlight the importance of confirmation of Stx-positive stool specimens by bacterial culture for timely and reliable identification of STEC infections, including E. coli O157 and non-O157 STEC, to enable implementation of appropriate public health actions. An important part of that identification is determining the serotype of all STEC isolates and the subtype of STEC O157 strains so that outbreaks can be detected and traced back to sources.     

A long-term study on the prevalence of shiga toxin-producing Escherichia coli (STEC) on four German cattle farms

The occurrence of Shiga toxin-producing Escherichia coli (STEC) was studied on four cattle farms. STEC were detected in 29-82% of the cattle. STEC with additional EHEC markers were detected on all farms. The occurrence of the complete virulence marker pattern (stx1 and/or stx2, eae, EHEC(hlyA), katP, espP) was correlated with the presence of known STEC serotypes. STEC O26:H11 and O165:H25 with the complete pattern of virulence markers were the most prevalent. STEC O157 (H7/H-) STEC O103:H2 and STEC O145:H- were found sporadically. Five clonal subgroups of the STEC O26:H11 isolates were identified by pulsed-field gel electrophoresis. STEC O26:H11 were present in three groups of cattle. This serotype was detected in a single group over the entire fattening period. Most STEC O26:H11 with the complete pattern of potential virulence markers were found in clinically healthy cattle. These animals may represent a risk factor for farmers and consumers.     

Human infections with non-O157 Shiga toxin-producing Escherichia coli, Switzerland, 2000-2009

We characterized 97 non-O157 Shiga toxin (stx)-producing Escherichia coli strains isolated from human patients during 2000-2009 from the national reference laboratory in Switzerland. These strains belonged to 40 O:H serotypes; 4 serotypes (O26:H11/H-, O103:H2, O121:H19, and O145:H28/H-) accounted for 46.4% of the strains. Nonbloody diarrhea was reported by 23.2% of the patients, bloody diarrhea by 56.8%. Hemolytic uremic syndrome developed in 40.0% of patients; serotype O26:H11/H- was most often associated with this syndrome. Forty-five (46.4%) strains carried stx2 genes only, 36 strains (37.1%) carried stx1, and 16 (16.5%) strains carried stx1 and stx2. Genes encoding enterohemolysin and intimin were detected in 75.3% and 70.1% of the strains, respectively. Resistance to ≥1 antimicrobial agent was present in 25 isolates. High genetic diversity within strains indicates that non-O157 stx-producing E. coli infections in Switzerland most often occurred as single cases.     

Detection and characterization of Shiga toxin-producing Escherichia coli from seagulls

Shiga toxin (Stx)-producing Escherichia coli (STEC) strains isolated from a seagull in Japan were examined. A total of 50 faecal samples was collected on a harbour bank in Hokkaido, Japan, in July 1998. Two different STEC strains, whose serotypes were O136:H16 and O153:H-, were isolated from the same individual by PCR screening; both of them were confirmed by ELISA and Vero cell cytotoxicity assay to be producing active Stx2 and Stx1, respectively. They harboured large plasmids, but did not carry the haemolysin or eaeA genes of STEC O157:H7. Based on their plasmid profiles, antibiotic resistance patterns, pulsed-field gel electrophoresis analysis (PFGE), and the stx genes sequences, the isolates were different. Phylogenic analysis of the deduced Stx amino acid sequences demonstrated that the Stx toxins of seagull-origin STEC were closely associated with those of the human-origin, but not those of other animal-origin STEC. In addition, Stx2phi-K7 phage purified from O136 STEC resembled Stx2phi-II from human-origin O157:H7, and was able to convert non-toxigenic E. coli to STEC. These results suggest that birds may be one of the important carriers in terms of the distribution of STEC.     

Characterization and epidemiologic subtyping of Shiga toxin-producing Escherichia coli strains isolated from hemolytic uremic syndrome and diarrhea cases in Argentina

Argentina has a high incidence of hemolytic uremic syndrome (HUS); 12.2 cases per 100,000 children younger than 5 years old were reported in 2002. Shiga toxin (Stx)-producing Escherichia coli (STEC) is the primary etiologic agent of HUS, and STEC O157 is the predominant serogroup isolated. The main objective of the present work was to establish the phenotypic and genotypic characteristics of the STEC strains in general isolated from Argentine children during a prospective study and the clonal relatedness of STEC O157:H7 strains using subtyping techniques. One hundred and three STEC strains isolated from 99 children were included. The phenotypic and genotypic features were established, and a polymerase chain reaction-restriction fragment length polymorphism (PCRRFLP) was performed to determine stx2 variants. The clonal relatedness of E. coli O157 isolates was established by phage typing and pulsed-field gel electrophoresis (PFGE). The 103 STEC strains belonged to 18 different serotypes, and 59% were of serotype O157:H7. Stx2 was identified in 90.3%, and stx1 in 9.7%. Among the 61 STEC O157 strains, 93.4% harbored the stx2/stx2vh-a genes; PT4 (39.3%) and PT2 (29.5%) were the predominant phage types. Using PFGE with the enzyme XbaI, a total of 41 patterns with at least 80% similarity were identified, and seven clusters with identical profiles were established. Some of the clusters were further split by PFGE using BlnI as the second enzyme. Isolates with indistinguishable PFGE patterns were with one exception also indistinguishable by phage typing and stx genotyping. These findings confirmed that some isolates were genetically related. However, no epidemiological linkages were identified. STEC strains with different genotypes and belonging to diverse serotypes were isolated in Argentina. Some STEC O157 strains could not be distinguished by applying subtyping techniques such as PFGE and phage typing.     

大肠埃希菌O157∶H7携带stx2::IS1203v基因研究

目的了解中国部分地区大肠埃希菌O157：H7菌株携带志贺毒素基因变异状况。方法采用聚合酶链反应扩增志贺毒素基因，使用核苷酸序列测定判断是否存在志贺毒素的新变种，用HeLa细胞毒性实验研究其细胞毒性的变化。结果1992—2002年中国部分地区分离到的289株产志贺毒素的大肠埃希菌O157：H7中有3株菌携带的志贺毒素2（stx2）基因有1．3kb的插入序列（IS）插入，且这段IS和IS1203变种（IS1203 variant，IS1203v）有100％的核苷酸序列同源性。IS1203v插入到3株大肠埃希菌O157：H7 stx2基因的位置及开放性读码框（ORF）方向有所不同。除此之外，3株菌原有的stx2基因序列完全一致且为Stx2原型毒素。和Stx2原型毒素相比，这3株携带stx2：：IS1203v基因的菌株对HeLa细胞的毒性明显降低。结论分离到IS1203v插入stx2基因的大肠埃希菌O157：H7菌株；IS1203v的插入可导致对HeLa细胞的细胞毒性降低。     

Duration and magnitude of faecal shedding of Shiga toxin-producing Escherichia coli from naturally infected cattle

To clarify the epidemiological relationship between cattle and human infections of Shiga toxin-producing Escherichia coli (STEC), we studied the duration and magnitude of the excretion of STEC O157 and STEC O26 with rectal faeces from naturally infected cattle at a breeding farm in the Tohoku area of Japan, using microbiological methods. The prevalence of STEC O157 was 3.5% (11/324), whereas that of STEC O26 was 7.9% (14/178). Faecal shedding of STEC O157 persisted for < 1 week to 10 weeks, whereas STEC O26 persisted from < 1 week to < 3 weeks. The magnitude of faecal shedding (per 10 g) ranged from 4 to > 110,000 c.f.u. for STEC O157 and from 3 to 2400 c.f.u. for STEC O26. All isolates of both STEC serotypes contained the stx1 or stx2 genes. Pulsed-field electrophoretic analysis of both STEC serotypes identified predominantly STEC O157 type III and STEC O26 type I in isolates, suggesting that a single STEC strain may be mutated in the intestinal tract of calves. These results indicate that STEC O157 is secreted for longer periods and in higher numbers than STEC O26 from healthy calves with natural infections, suggesting that STEC O157 may have more opportunities than STEC O26 to induce human disease.     

Prevalence and genetic profiling of virulence determinants of non-O157 Shiga toxin-producing Escherichia coli isolated from cattle, beef, and humans, Calcutta, India.

We investigated the prevalence of Shiga toxin-producing Escherichia coli (STEC) in hospitalized diarrhea patients in Calcutta, India, as well as in healthy domestic cattle and raw beef samples collected from the city's abattoir. Multiplex polymerase chain reaction using primers specific for stx1 and stx2 detected STEC in 18% of cow stool samples, 50% of raw beef samples, and 1.4% and 0.6% of bloody and watery stool samples, respectively, from hospitalized diarrhea patients. Various virulence genes in the STEC isolates indicated that stx1 allele predominated. Plasmid-borne markers, namely, hlyA, katP, espP, and etpD, were also identified. Bead enzyme-linked immunosorbent assay and Vero cell assay were performed to detect and evaluate the cytotoxic effect of the Shiga toxins produced by the strains. STEC is not an important cause of diarrhea in India; however, its presence in domestic cattle and beef samples suggests that this enteropathogen may become a major public health problem in the future.     

Shiga Toxin Subtypes Associated with Shiga Toxin-Producing Escherichia coli Strains Isolated from Red Deer, Roe Deer, Chamois, and Ibex

Abstract A total of 52 Shiga toxin-producing Escherichia coli (STEC) strains, isolated from fecal samples of six ibex, 12 chamois, 15 roe deer, and 19 red deer were further characterized by subtyping the stx genes, examining strains for the top nine serogroups and testing for the presence of eae and ehxA. Eleven of the 52 strains belonged to one of the top nine STEC O groups (O26, O45, O91, O103, O111, O113, O121, O145, and O157). Eight STEC strains were of serogroup O145, two strains of serogroup O113, and one strain of serogroup O157. None of the strains harbored stx2a, stx2e, or stx2f. Stx2b (24 strains) and stx1c (21 strains) were the most frequently detected stx subtypes, occurring alone or in combination with another stx subtype. Eight strains harbored stx2g, five strains stx2d, three strains stx1a, two strains stx2c, and one strain stx1d. Stx2g and stx1d were detected in strains not harboring any other stx subtype. The eae and ehxA genes were detected in two and 24 STEC strains, respectively. Considering both, the serogroups and the virulence factors, the majority of the STEC strains isolated from red deer, roe deer, chamois, and ibex do not show the typical patterns of highly pathogenic STEC strains. To assess the potential pathogenicity of STEC for humans, strain isolation and characterization is therefore of central importance.     

不同来源产志贺毒素大肠埃希菌分布特征

目的探讨不同标本中产志贺样毒素大肠埃希菌（STEC）的分布特征。方法采集动物粪便、肉类食品和排污口污泥样品，常规分离大肠埃希菌，血清学分型，PCR鉴定产志贺样毒素（stx1，stx2）菌株。结果293份标本中鉴定出8株STEC，1株为产志贺毒素O157：H7型，2株为不产志贺毒素O157：H7型，5株为产志贺毒素非O157：H7型。结论STEC存在于不同来源的标本中，菌株表型与毒力因子存在一定差异。     

Pathogenic potential to humans of bovine Escherichia coli O26, Scotland

Escherichia coli O26 and O157 have similar overall prevalences in cattle in Scotland, but in humans, Shiga toxin-producing E. coli O26 infections are fewer and clinically less severe than E. coli O157 infections. To investigate this discrepancy, we genotyped E. coli O26 isolates from cattle and humans in Scotland and continental Europe. The genetic background of some strains from Scotland was closely related to that of strains causing severe infections in Europe. Nonmetric multidimensional scaling found an association between hemolytic uremic syndrome (HUS) and multilocus sequence type 21 strains and confirmed the role of stx(2) in severe human disease. Although the prevalences of E. coli O26 and O157 on cattle farms in Scotland are equivalent, prevalence of more virulent strains is low, reducing human infection risk. However, new data on E. coli O26-associated HUS in humans highlight the need for surveillance of non-O157 enterohemorrhagic E. coli and for understanding stx(2) phage acquisition.     

Laboratory-confirmed non-O157 Shiga toxin-producing Escherichia coli--Connecticut, 2000-2005

Shiga toxin-producing Escherichia coli (STEC) infection causes diarrhea that is often bloody and can result in potentially life-threatening hemolytic uremic syndrome (HUS). Escherichia coli O157:H7 is the most common cause of STEC infection in the United States, producing 73,000 illnesses annually, according to the last estimate in 1999. Unlike O157, however, little is known about the incidence of non-O157 strains. Because STEC other than O157 are not commonly identified, the incidence, trends, and epidemiology of non-O157 STEC are not well understood. To assess trends in Shiga toxin enzyme immunoassay (Stx EIA) testing by local clinical laboratories, the Connecticut Department of Public Health (CTDPH) analyzed results of confirmatory testing conducted in the state laboratory during 2000--2005. The findings indicated that a total of 403 STEC infections were reported by clinical laboratories in Connecticut, including 207 identified as STEC by Stx EIA testing alone, and that the use of Stx EIA increased from 2000 to 2005. Use of Stx EIA without prompt culture confirmation can delay or prevent serotyping and subtyping of isolates and detection of both O157 and non-O157 STEC outbreaks. Public health authorities in all states should ensure that clinical laboratories forward Stx EIA-positive specimens to the state laboratory for isolation and identification of STEC, as recommended by the Association of Public Health Laboratories and CDC.     

Prevalence of non-O157:H7 shiga toxin-producing Escherichia coli in diarrheal stool samples from Nebraska

We determined the prevalence of Shiga toxin-producing Escherichia coli (STEC) in diarrheal stool samples from Nebraska by three methods: cefixime-tellurite sorbitol MacConkey (CT- SMAC) culture, enterohemorrhagic E. coli (EHEC) enzyme immunoassay, and stx1,2 polymerase chain reaction (PCR). Fourteen (4.2%) of 335 specimens were positive by at least one method (CT-SMAC culture [6 of 14], EHEC enzyme immunoassay [13 of 14], stx1,2 PCR [14 of 14]). Six contained serogroup O157, while non-O157 were as prevalent as O157 serogroups.     

Prevalence and growth kinetics of Shiga toxin-producing Escherichia coli (STEC) in bovine offal products in Japan

Recent epidemiological data suggest a link between the consumption of bovine offal products and Shiga toxin-producing Escherichia coli (STEC) infection in Japan. This study thus examined the prevalence of STEC in various types of these foods. PCR screened 229 bovine offal products for the presence of Shiga toxin (stx) gene. Thirty-eight (16·6%) samples were stx positive, of which eight were positive for rfbE(O157) and three were positive for wzy(O26). Four O157 and one O26 STEC isolates were finally obtained from small-intestine and omasum products. Notably, homogenates of bovine intestinal products significantly reduced the extent of growth of O157 in the enrichment process compared to homogenates of beef carcass. As co-incubation of O157 with background microbiota complex from bovine intestinal products in buffered peptone water, in the absence of meat samples, tended to reduce the extent of growth of O157, we reasoned that certain microbiota present in offal products played a role. In support of this, inoculation of generic E. coli from bovine intestinal products into the homogenates significantly reduced the extent of growth of O157 in the homogenates of bovine intestinal and loin-beef products, and this effect was markedly increased when these homogenates were heat-treated prior to inoculation. Together, this report provides first evidence of the prevalence of STEC in a variety of bovine offal products in Japan. The prevalence data herein may be useful for risk assessment of those products as a potential source of human STEC infection beyond the epidemiological background. The growth characteristic of STEC O157 in offal products also indicates the importance of being aware when to test these food products.     

Isolation and characterization of Shiga toxin-producing Escherichia coli (STEC) in retail edible beef by-products

The prevalence of Shiga toxin-producing Escherichia coli (STEC) was investigated in 350 edible beef intestinal samples, including omasum (n=110), abomasum (n=120), and large intestines (n=120), collected from traditional beef markets in Seoul, Korea. A total of 23 STEC strains were isolated from 15 samples (four strains from three omasa, 10 from five abomasa, and nine from seven large intestines). The O serotypes and toxin gene types of all STEC isolates were identified, and antimicrobial resistance was assessed using the disk diffusion method. The isolation rates of STEC from edible beef intestines were 2.8% in omasum, 4.2% in abomasums, and 5.9% in large intestines. All STEC isolates harbored either stx1, or both stx1 and stx2 genes simultaneously. Among the 23 isolates, 13 strains were identified as 11 different O serogroups, and 10 strains were untypable. However, enterohemorrhagic Esherichia coli O157, O26, and O111 strains were not isolated. The highest resistance rate observed was against tetracycline (39%), followed by streptomycin (35%) and ampicillin (22%). Of the 23 isolates, 12 isolates (52%) were resistant to at least one antibiotic, nine (39%) isolates were resistant to two or more antibiotics, and one isolate from an abmasum carried resistance against nine antibiotics, including beta-lactam/beta-lactamase inhibitor in combination and cephalosporins. This study shows that edible beef by-products, which are often consumed as raw food in many countries, including Korea, can be potential vehicles for transmission of antimicrobial-resistant pathogenic E. coli to humans.     

Antimicrobial susceptibility of Shiga toxin-producing Escherichia coli isolated from organic dairy farms, conventional dairy farms, and county fairs in Minnesota

This study compared the antimicrobial susceptibility of Shiga toxin-producing Escherichia coli (STEC) isolates from organic dairy farms, conventional dairy farms, and Minnesota county fairs. A total of 83 STEC isolates (43 O157 and 40 non-O157 STEC) were tested for antimicrobial susceptibility as determined by the automated broth microdilution method. Resistance to tetracycline was identified in 19 (23%) isolates and to sulphadimethoxine in 40 (48%) isolates. Half of the STEC isolates were resistant to at least one antimicrobial agent. Resistance to at least one antimicrobial agent was observed in 18 (62%) isolates from conventional farms and in 11 (48%) isolates from organic farms. Resistance to at least one antimicrobial agent was more frequent in isolates from calves (77%) than from cows (39%). Multidrug resistant patterns were more common in non-O157 STEC than O157 STEC. This study provides data to document the degree of STEC antimicrobial resistance from dairy cattle sources in Minnesota. The use of antimicrobial agents on farms, and other environmental influences, may affect resistance patterns in isolates from cattle sources. Systematic surveillance of STEC from cattle could potentially detect emergence of antimicrobial resistance that may be spread to humans through the food chain.     

Antimicrobial resistance and class 1 integrons in pathogenic Escherichia coli from dairy farms

The goal of this study was to assess the prevalence of antimicrobial resistance and class 1 integrons, including integron-associated genes, in 24 Escherichia coli isolates from dairy farms. Escherichia coli isolates (n = 14) from dairy cows with mastitis (ECDM), Shiga toxin-producing (STEC) O157:H7 from cull dairy cow fecal samples (n = 9) and bulk tank milk (n = 1) were evaluated for sensitivity to 19 antimicrobial agents used commonly in human and/or veterinary medicine. Multiplex PCR was used to determine presence of genes associated with class 1 integrons (intI1, qacEDelta1, and sulI1). Class 1 integrons were found only in eight of 10 isolates (one STEC O157:H7 and seven ECDM) that demonstrated antimicrobial resistance, and seven of these were resistant to two or more antimicrobial agents. Eight of 10 STEC O157:H7 and six of 14 ECDM were susceptible to all commonly used antibiotics. Five ECDM demonstrated multiple resistances to four or more antibiotics. Most of the 24 isolates examined exhibited resistance against sulfamethoxazole, followed by streptomycin and tetracycline. STEC O157:H7 strains had less prevalence of antibiotic resistance and integron carriage than ECDM. The multiplex PCR method developed for detection of intI1, qacEDelta1, and sulI1 can be used routinely for monitoring presence of these genes. Class 1 integrons were found in eight of 10 E. coli strains that demonstrated antimicrobial resistance; seven of these were resistant to two or more antibiotics. It appears that integrons played a role in the incidence of antimicrobial resistance of the strains used in this study.     

Survival of Shiga toxin-producing Escherichia coli O157 in marine water and frequent detection of the Shiga toxin gene in marine water samples from an estuary port

Shiga toxin-producing Escherichia coli (STEC) O157 was investigated with respect to its halotolerance and whether it can survive in marine water. STEC O157 could multiply in a medium containing 5% NaCl and in sterilized marine water, and could survive in unsterilized marine water for at least 15 days. On the basis of these results, we postulated that STEC O157 may survive in natural marine water, and attempted to isolate the bacterium and Shiga toxin gene (stx) from marine water in Japan. The stx, comprising stx1 and stx2, was detected from marine water samples by PCR. STEC and other stx-positive bacteria, however, could not be isolated from these samples in this study. These results indicate that stx-positive bacteria may survive in marine water and suggest the necessity of a survey.