Molecular epidemiologic analysis of Vibrio cholerae O1 isolates by pulsed-field gel electrophoresis.

Isolates of Vibrio cholerae O1 El Tor from two well-defined cholera outbreaks in Malaysia were analyzed by using pulsed-field gel electrophoresis (PFGE). Isolates from sporadic cases occurring during the same time period were also studied. Digestion of chromosomal DNA from these isolates of V. cholerae O1 with restriction endonucleases NotI (5'-GCGGCCGC-3') and SfiI (5'-GGCCNNNN-3'), followed by PFGE, produced restriction endonuclease analysis (REA) patterns consisting of 13 to 24 bands (ranging in size from 46 to 398 kbp). Analysis of the REA patterns generated by PFGE after digestion with NotI and SfiI suggested the clonal nature and close genetic identity of the isolates obtained during each of the two outbreaks (Dice coefficient, 0.93 to 1.0). Although they had very similar REA patterns, the two outbreak clones were not identical. Isolates of V. cholerae O1 from sporadic cases, on the other hand, appeared to be much more heterogeneous (five different REA patterns detected in the five isolates tested; Dice coefficient, 0.31 to 0.81) than those obtained during the two outbreaks. We conclude that PFGE of V. cholerae O1 chromosomal DNA digested with infrequently cutting restriction endonucleases is a useful method for molecular typing of V. cholerae isolates for epidemiological purposes.     

Concomitant infection of enterotoxigenic Escherichia coli in an outbreak of cholera caused by Vibrio cholerae O1 and O139 in Ahmedabad, India

In Ahmedabad, a major city in the state of Gujarat, India, an outbreak of acute secretory diarrhea caused by Vibrio cholerae O1 Ogawa El Tor, V. cholerae O139, and multiple serotypes of enterotoxigenic Escherichia coli (ETEC) occurred in January 2000. All of the representative V. cholerae O1 and O139 isolates examined harbored the ctxA gene (encoding the A subunit of cholera toxin) and the El Tor variant of the tcpA gene (encoding toxin-coregulated pilus). ETEC isolates of different serotypes were positive for the elt gene, encoding heat-labile enterotoxin. To further understand the molecular characteristics of the pathogens, representative isolates were examined by ribotyping and pulsed-field gel electrophoresis (PFGE). Ribotyping showed that the isolates of V. cholerae O1 Ogawa exhibited a pattern identical to that of the prevailing clone of O1 in areas where cholera is endemic in India, and all of the O139 isolates were identical to the BII clone of V. cholerae O139. PFGE of the representative O1 Ogawa isolates exhibited an identical pattern, comparable to the H pattern of the new clone of O1 reported in Calcutta, India. PFGE analysis of the V. cholerae O139 isolates showed identical patterns, but these differed from the PFGE patterns of O139 isolates reported during 1992 to 1997 in Calcutta. ETEC isolates showed genetic heterogeneity among isolates belonging to the same serotype, although the identical PFGE pattern was also observed among ETEC isolates of different serotypes. Antibiograms of the isolates were unusual, because all of the O139 isolates were resistant to nalidixic acid. Likewise, all of the E. coli isolates showed resistance to ciprofloxacin, norfloxacin, and nalidixic acid. This is a unique outbreak, and we believe that it is the first in which V. cholerae and ETEC were concomitantly involved.     

Molecular evolution of Vibrio cholerae O1 strains isolated in Lima, Peru, from 1991 to 1995.

Following the emergence of cholera in Lima, Peru, in 1991, isolates of Vibrio cholerae O1 biotype El Tor recovered from patients in various parts of Lima were selected and characterized. Ribotyping and pulsed-field gel electrophoresis (PFGE) revealed four BglI ribotypes and eight NotI PFGE types among 50 V. cholerae O1 strains recovered from patients with cholera in Lima from 1991 to 1995, with certain genotypes appearing to cluster geographically. While differences in ribotype and PFGE type patterns suggest that genetic changes are occurring in the strain responsible for the Latin American cholera epidemic, more frequently than previously reported, 40 (80%) O1 strains showed an identical ribotype pattern and 41 (82%) strains showed closely related PFGE types, types 1, 2, or 3, that differed by less than three restriction fragments. All strains were susceptible to nine antibacterial agents studied. In 1991, more than 95% of the clinical V. cholerae O1 strains were serotype Inaba, whereas from 1992, serotype Ogawa began to predominate, with more than 90% of the isolates being of the Ogawa serotype in 1995. The small differences in genotypes of V. cholerae O1 is remarkable because cholera is highly seasonal in coastal areas of Peru and support the hypothesis that the epidemic strain reemerges from an environmental source. However, the relative high rate of genetic changes within V. cholerae O1 as shown by ribotyping and PFGE should be taken into consideration when typing patterns of V. cholerae O1 associated with cholera in Latin America are evaluated.     

Molecular characterization of Vibrio cholerae O1 strains by pulsed-field gel electrophoresis.

Pulsed-field gel electrophoresis (PFGE) was performed on 180 isolates of Vibrio cholerae serogroup O1 representing 6 different multilocus enzyme electrophoresis (MEE) types and 27 rRNA restriction fragment length polymorphism types (ribotypes). Isolates were digested with the restriction enzyme NotI and were separated into 63 patterns on the basis of differences in band arrangements. In general, strains which were different by MEE or ribotyping also had different PGFE patterns. PFGE identified individual strains within a single MEE type or ribotype; isolates with one PFGE pattern were less frequently distinguished by ribotyping. All V. cholerae O1 isolates tested from the Latin American epidemic were indistinguishable by their MEE, ribotype, or PFGE patterns. PFGE could further distinguish strains of this same ribotype isolated in Africa, Europe, the South Pacific, or Southeast Asia. Although both MEE and PFGE could identify the strain from the Latin American epidemic, PFGE was more rapid and less labor intensive. PFGE also distinguished nontoxigenic isolates endemic to the U.S. Gulf Coast from unrelated nontoxigenic isolates. In the present study PFGE was more discriminating than other previously described subtyping assays for V. cholerae O1 and appears to be a useful epidemiologic tool.     

A molecular and phenotypic study of Vibrio cholerae in Iran

Vibrio cholerae is again the subject of attention on account of the current increase in the world-wide incidence of cholera. In this study, 200 clinical isolates of V. cholerae serotypes O1 and non-O1, non-O139, were collected from different provinces in Iran. The isolates were subjected to biochemical analysis, antibiogram, PCR of toxin genes, plasmid profile, ribotyping and pulsed-field gel electrophoresis (PFGE). The analysis of plasmid content showed that 33-96% of V. cholerae isolated from different provinces carry a large plasmid. PCR analysis of V. cholerae O1 showed that the genes encoding cholera toxin (ctx), toxin co-regulated pilus (tcp), accessory cholera enterotoxin (ace) and zonula occludens toxin (zot) were present in 55-97% of isolates in different provinces. Restriction fragment length polymorphism (RFLP) of BglI-digested DNA probed with five oligonucleotides revealed three different ribotype patterns in isolates of V. cholerae O1. The ribotype pattern B21 of V. cholerae O1 El Tor was found to be the predominant pattern in the isolates studied. V. cholerae non-O1, non-O139 isolates showed a single ribotype pattern. PFGE analysis also showed 10 different patterns amongst the isolates, 9 of which were in V. cholerae O1. Overall, the analysis of polymorphism of ribotypes and PFGE patterns of the isolates showed that the provinces in Iran were affected by a limited number of clones of V. cholerae O1 and non-O1, non-O139 strains.     

Phenotypic and genotypic changes in Vibrio cholerae O139 Bengal.

To find reasons for the recent decline of Vibrio cholerae O139 Bengal cholera in Bangladesh, phenotypic and genotypic changes in O139 isolates obtained from patients with cholera from 1993 to 1996 were studied. The isolates were tested for the presence of ctx and tcpA genes, hemagglutinin/protease (HA/P), capsule, D-mannose-sensitive hemagglutinin (MSHA), L-fucose-sensitive hemagglutinin (FSHA), tube test (tube) and CAMP test (CAMP) hemolytic activities, resistance to 2,4-diamino-6,7-diisopropyl pteridine (O/129) and trimethoprim-sulfamethoxazole (TMP-SMX), and genotype by pulsed-field gel electrophoresis (PFGE). All isolates possessed ctx and tcpA genes, HA/P, and a capsule. Most isolates were negative for FSHA, but although the majority of the isolates were positive for MSHA, no discernible trend in the activity was found during the study period. All early isolates were CAMP hemolysin positive and resistant to the vibriostatic compound O/129 and TMP-SMX, the two properties that could be used for the presumptive diagnosis of O139 cholera. However, subsequently, isolates that were CAMP hemolysin negative and susceptible to TMP-SMX and O/129 were increasingly encountered, with all the 1996 isolates being so, which suggested that these properties can no longer be used for the presumptive diagnosis of O139 cholera. V. cholerae O139 isolates that were CAMP hemolysin positive and resistant to O/129 and TMP-SMX produced a disease of greater severity than that caused by the CAMP hemolysin-negative and susceptible isolates on the basis of the lengths of stay of the hospitalized patients. The study period witnessed the evolution of four different genotypes by PFGE. All of these data suggested that the V. cholerae O139 isolates have undergone changes in some properties. However, how these changes influenced their prevalence relative to that of V. cholerae O1 in human infection is not clear. Studies of the environmental factors will provide the key for an understanding of the relative abundance of these vibrios.     

Epidemiologic subtyping of Escherichia coli serogroup O157 strains isolated in Ontario by phage typing and pulsed-field gel electrophoresis

Phage typing and DNA macrorestriction fragment analysis by pulsed-field gel electrophoresis (PFGE) were evaluated for use in the epidemiological subtyping of Escherichia coli serogroup O157 strains isolated in Ontario, Canada. Among 30 strains isolated from patients with sporadic cases of infection, 22 distinct XbaI macrorestriction patterns were identified and 17 strains exhibited unique PFGE patterns. In contrast, phage typing identified only seven different phage types and 17 strains belonged to the same phage type. A total of 25 phage type-macrorestriction pattern combinations were identified among the strains from patients with sporadic cases of infection. PFGE subtyping differentiated between unrelated strains that exhibited the same phage type, and in one group of strains, phage typing differentiated between strains of the same PFGE subtype. Both typing procedures correctly identified outbreak-related isolates as belonging to the same type in four separate outbreaks. Each outbreak strain was characterized by a distinct macrorestriction pattern, while phage typing subdivided the outbreak strains into only three different types. A small percentage of outbreak-related isolates had PFGE patterns that differed slightly (one or two DNA fragment differences) from that of the outbreak strain. On the other hand, each isolate from the same outbreak belonged to the same phage type as that of the outbreak strain. We conclude that phage typing and PFGE fingerprinting represent complementary procedures for the subtyping of E. coli serogroup O157 and that the combined use of these procedures provides optimal discrimination.     

Genetic diversity of Vibrio cholerae O1 in Argentina and emergence of a new variant

The genetic diversity of Vibrio cholerae O1 strains from Argentina was estimated by random amplified polymorphic DNA (RAPD) analysis and pulsed-field gel electrophoresis (PFGE). Twenty-nine isolates carrying the virulence genes ctxA, zot, ace, and tcpA appeared to represent a single clone by both typing methods; while 11 strains lacking these virulence genes exhibited several heterogeneous RAPD and PFGE patterns. Among the last group, a set of isolates from the province Tucumán showed a single RAPD pattern and four closely related PFGE profiles. These strains, isolated from patients with diarrhea, did not produce the major V. cholerae O1 virulence determinants, yet cell supernatants of these isolates caused a heat-labile cytotoxic effect on Vero and Y-1 cells and elicited significant variations on the water flux and short-circuit current in human small intestine mounted in an Ussing chamber. All these effects were completely abolished by incubation with a specific antiserum against El Tor hemolysin, suggesting that this virulence factor was responsible for the toxic activity on both the epithelial cells and the small intestine specimens and may hence be involved in the development of diarrhea. We propose "Tucumán variant" as the designation for this new cluster of cholera toxin-negative V. cholerae O1 strains.     

应用PFGE分析广州稻叶型霍乱弧菌的同源性

目的分析广州40株稻叶型霍乱弧菌的毒力基因和PFGE特征,探讨菌株的同源性和广州稻叶型霍乱弧菌流行的特点。方法应用PCR方法检测稻叶型菌株的4种毒力基因,采用限制性内切酶NotI对菌株进行PFGE分型,使用BioNumerics Version4.0软件（复选Dice相关系数和UPGMA方法）对菌株进行聚类和同源分析。结果绝大多数水型稻叶霍乱疫情相关菌株为genotypeA型菌,而绝大多数环境分离稻叶型霍乱弧菌为genotypeB型和C型菌。40株霍乱菌株分布于18种pulsotype,水型霍乱疫情相关菌株的pulsotype极为相近（相似性系数98%以上）,而环境分离的稻叶型霍乱弧菌呈现明显的pulsotype多样性。结论是否携带ctxA基因是稻叶型霍乱弧菌人源分离株与环境分离株的主要区别之一,绝大多数环境分离的稻叶型霍乱弧菌与人源分离株的同源性较低,但仍需加强对不同来源菌株的同源性分析和溯源追踪。     

Polymorphic amplified typing sequences provide a novel approach to Escherichia coli O157:H7 strain typing

Escherichia coli O157:H7 (O157) strains are commonly typed by pulsed-field gel electrophoresis (PFGE) following digestion of genomic DNA with the restriction enzyme XbaI. We have shown that O157 strains differ from each other by a series of discrete insertions or deletions, some of which contain recognition sites for XbaI, suggesting that these insertions and deletions are responsible for the differences in PFGE patterns. We have devised a new O157 strain typing protocol, polymorphic amplified typing sequences (PATS), based on this information. We designed PCR primer pairs to amplify genomic DNA flanking each of 40 individual XbaI sites in the genomes of two O157 reference strains. These primer pairs were tested with 44 O157 isolates, 2 each from 22 different outbreaks of infection. Thirty-two primer pairs amplified identical fragments from all 44 isolates, while eight primer pairs amplified regions that were polymorphic between isolates. The isolates could be differentiated solely on the basis of which of the eight polymorphic amplicons was detected. PATS correctly identified 21 of 22 outbreak pairs as being identical or highly related, whereas PFGE correctly identified 14 of the 22 outbreak pairs as being identical or highly related; PATS was also able to type isolates from three outbreaks that were untypeable by PFGE. However, PATS was less sensitive than PFGE in discriminating between outbreaks. These data suggest that typing by PATS may provide a simple procedure for strain typing of O157 and other bacteria and that further evaluation of the utility of this method for epidemiologic investigations is warranted.     

Escalating association of Vibrio cholerae O139 with cholera outbreaks in India

Between December 1999 and December 2000, teams from the National Institute of Cholera and Enteric Diseases, Calcutta, India, examined eight outbreaks of cholera, which occurred in different parts of the country distant from each other. In two of these outbreaks each, only V. cholerae O1 biotype ElTor or V. cholerae O139 could be isolated, while in the remaining four outbreaks, both O1 and O139 were isolated. The interesting feature is the escalating association of V. cholerae O139 with outbreaks of cholera; two of the most recent outbreaks, one in Calcutta and one in Orissa, were caused exclusively by O139. The O139 strains from the six different outbreaks were genotypically closely related. These trends indicate a shift in the outbreak propensity of V. cholerae O139.     

Multilocus sequence typing has better discriminatory ability for typing Vibrio cholerae than does pulsed-field gel electrophoresis and provides a measure of phylogenetic relatedness

Twenty-two Vibrio cholerae isolates, including some from "epidemic" (O1 and O139) and "nonepidemic" serogroups, were characterized by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) by using three housekeeping genes, gyrB, pgm, and recA; sequence data were also obtained for the virulence-associated genes tcpA, ctxA, and ctxB. Even with the small number of loci used, MLST had better discriminatory ability than did PFGE. On MLST analysis, there was clear clustering of epidemic serogroups; much greater diversity was seen among tcpA- and ctxAB-positive V. cholerae strains from other, nonepidemic serogroups, with a number of tcpA and ctxAB alleles identified.     

Evaluating the usefulness of spa typing,in comparison with pulsed-field gel electrophoresis,for epidemiological typing of methicillin-resistant Staphylococcus aureus in a low-prevalence region in Sweden 2000–2004

The usefulness of spa typing was evaluated in relation to pulsed-field gel electrophoresis (PFGE), as a tool for epidemiological typing of methicillin-resistant Staphylococcus aureus (MRSA) in a low-prevalence region in southern Sweden. Bacterial isolates from 216 MRSA cases, newly identified in 2000–2004, were studied. The isolates were obtained from infected patients (31%), and from colonized individuals found by screening (69%). In total, 49 spa types and 73 PFGE patterns were identified. The discriminatory power of spa typing was lower (94.9 ± 1.8%) than that of PFGE (97.3 ± 1.2%). For two spa types (t002 and t008) the Panton–Valentine leukocidin results added useful discriminatory information. The most common spa types were t044 (n = 31; four PFGE patterns), t002 (n = 24; 10 PFGE patterns), t067 (n = 12; four PFGE patterns), t050 (n = 12; one PFGE pattern), and t324 (n = 11; one PFGE pattern). Epidemiological investigations identified 91 single cases and 39 transmission chains, each involving two to 13 cases. All the transmission chains were held together both by spa and PFGE typing. Among the 91 single-case isolates, 33 spa types and 50 PFGE patterns were unique (matchless) at the time of identification. The low prevalence of MRSA, the low number of outbreaks, and the wide spectrum of strains due to frequent acquisitions abroad (49% of the cases), makes spa typing a useful complement to epidemiological investigations in our setting. However, we still recommend the continued use of PFGE for further discrimination of isolates with identical spa types when epidemiological data can not exclude possible transmission.     

Clonal diversity of Shiga toxin-producing Escherichia coli O157:H7/H- in Germany--a ten-year study

Two hundred and ten E. coli O157:H7/H- strains isolated from single cases and outbreaks of diarrhoea and haemolytic uraemic syndrome (HUS) in Germany between 1988 and 1998 were characterised by a range of molecular subtyping methods and phage typing in order to analyse their clonal nature. A high clonal heterogeneity, together with a considerable clonal stability, has been identified among the bacterial isolates and no single clonal type appeared to be geographically dominant. It is recommended to apply pulsed-field gel electrophoresis (PFGE) together with P gene profile determination (number and genomic positions of lambdoid bacteriophages) as laboratory tools for an extended epidemiological surveillance of E. coli OOFF phage typing will remain helpful as a first line of analysis, particularly in outbreak situations.     

Lack of polymorphism in a Vibrio cholerae O139-specific DNA region encoding the somatic antigen in strains isolated during 1993-1998

Vibrio cholerae O139 Bengal emerged as a second aetiologic agent of cholera in South Asia in late 1992. This new serogroup arose from a Vibrio cholerae O1 strain by deletion of the chromosomal region encoding O1 specificity and acquisition of a novel 35-kb region encoding the O139 specificity. Previous studies indicated significant phenotypic and genotypic changes in O139 isolates over the years since its first appearance. This prompted us to study possible polymorphism in the 35-kb novel region encoding the O139 specificity. A total of 17 V. cholerae O139 isolates originating from different countries and years in South Asia and China, and a single unrelated V. cholerae O139 isolate from Argentina were studied. The 35-kb chromosomal region was amplified as two fragments of 12 and 23 kb in an extended PCR from all isolates. These amplicons were then treated separately with seven different restriction enzymes and separated by agarose gel electrophoresis. The South Asian and Chinese isolates gave identical patterns for the same enzymes, but different patterns for different enzymes, thus exhibiting no polymorphism in the 35-kb region. However, the Argentine isolate gave distinct patterns for most of the enzymes confirming its different origin. This data indicated that the portion of the chromosome encoding the O139 antigen specificity is highly conserved. As found in previous studies, the early O139 isolates were resistant to trimethoprim-sulfamethoxazole (TMP-SMX) and vibriostatic compound, O/129, and CAMP- haemolysin positive. The isolates of later years diverged exhibiting different patterns by pulsed-field gel electrophoresis (PFGE), and becoming susceptible to TMP-SMX and O/129, and CAMP-haemolysin negative.     

Evaluation of pulsed-field gel electrophoresis in epidemiological investigations of meningococcal disease outbreaks caused by Neisseria meningitidis serogroup C

Since 1990, the frequency of Neisseria meningitidis serogroup C (NMSC) outbreaks in the United States has increased. Based on multilocus enzyme electrophoresis (MEE), the current molecular subtyping standard, most of the NMSC outbreaks have been caused by isolates of several closely related electrophoretic types (ETs) within the ET-37 complex. We chose 66 isolates from four well-described NMSC outbreaks that occurred in the United States from 1993 to 1995 to evaluate the potential of pulsed-field gel electrophoresis (PFGE) to identify outbreak-related isolates specific for each of the four outbreaks and to differentiate between them and 50 sporadic isolates collected during the outbreak investigations or through active laboratory-based surveillance from 1989 to 1996. We tested all isolates collected during the outbreak investigations by four other molecular subtyping methods: MEE, ribotyping (ClaI), random amplified polymorphic DNA assay (two primers), and serotyping and serosubtyping. Among the 116 isolates, we observed 11 clusters of 39 NheI PFGE patterns. Excellent correlation between the PFGE and the epidemiological data was observed, with an overall sensitivity of 85% and specificity of 71% at the 95% pattern relatedness breakpoint using either 1.5 or 1.0% tolerance. For all four analyzed outbreaks, PFGE would have given public health officials additional support in declaring an outbreak and making appropriate public health decisions.     

Multilocus variable-number tandem repeat analysis distinguishes outbreak and sporadic Escherichia coli O157:H7 isolates

Escherichia coli O157:H7 is a major cause of food-borne illness in the United States. Outbreak detection involves traditional epidemiological methods and routine molecular subtyping by pulsed-field gel electrophoresis (PFGE). PFGE is labor-intensive, and the results are difficult to analyze and not easily transferable between laboratories. Multilocus variable-number tandem repeat (VNTR) analysis (MLVA) is a fast, portable method that analyzes multiple VNTR loci, which are areas of the bacterial genome that evolve quickly. Eighty isolates, including 21 isolates from five epidemiologically well-characterized outbreaks from Pennsylvania and Minnesota, were analyzed by PFGE and MLVA. Strains in PFGE clusters were defined as strains that differed by less than or equal to one band by using XbaI and the confirmatory enzyme SpeI. MLVA was performed by comparing the number of tandem repeats at seven loci. From 6 to 30 alleles were found at the seven loci, resulting in 64 MLVA types among the 80 isolates. MLVA correctly identified the isolates from all five outbreaks if only a single-locus variant was allowed. MLVA differentiated strains with unique PFGE types. Additionally, MLVA discriminated strains within PFGE-defined clusters that were not known to be part of an outbreak. In addition to being a simple and validated method for E. coli O157:H7 outbreak detection, MLVA appears to have a sensitivity equal to that of PFGE and a specificity superior to that of PFGE.     

Presence of CTX gene cluster in environmental non-O1/O139 Vibrio cholerae and its potential clinical significance

Purpose: The aim of this study was to understand the epidemiological linkage of clinical and environmental isolates of Vibrio cholerae and to determine their genotypes and virulence genes content. Materials and Methods: A total of 60 V. cholerae strains obtained from clinical specimens (n = 40) and surface waters (n = 20) were subjected to genotyping using PFGE and determination of their virulence-associated gene clusters. Result: PCR analysis showed the presence of chromosomally located hly and RTX genetic elements in 100% and 90% of the environmental isolates, respectively. The phage-mediated genetic elements such as CTX, TLC and VPI were detected in 5% of the environmental isolates suggesting that the environmental isolates cannot acquire certain mobile gene clusters. A total of 4 and 18 pulsotypes were obtained among the clinical and environmental V. cholerae isolates, respectively. Non-pathogenic environmentally isolated V. cholerae constituted a distinct cluster with one single non-O1, non-O139 strain (EP6) carrying the virulence genes similar to the epidemic strains. This may suggest the possible potential of conversion of non-pathogenic to a pathogenic environmental strain. Conclusions: The emergence of a single environmental isolate in our study containing the pathogenicity genes amongst the diverse non-pathogenic environmental isolates needs to be further studied in the context of V. cholerae pathogenicity sero-coversion.     

Vitek system antimicrobial susceptibility testing of O1, O139, and non-O1 Vibrio cholerae.

Vibrio cholerae causes epidemic diarrhea throughout the world. Fluid replacement is the primary therapy for cholera; however, high mortality rates often necessitate the use of antibiotics. V. cholerae, like most bacteria, has developed resistance to some antibiotics. In the early 1990s a new serotype strain, Bengal 0139, began a new wave of cholera epidemics. Bengal isolates showed unique trends in antimicrobial resistance. Many clinical laboratories use automated antibiotic susceptibility testing for V. cholerae. It is important to know if automated susceptibility test results for V. cholerae coincide with reported trends in antibiotic susceptibility. In the present study, we used the Vitek automated susceptibility system to determine the susceptibilities of 79 V. cholerae O1 isolates, 100 O139 isolates, and 112 non-O1 isolates. Vitek susceptibilities for V. cholerae showed a good correlation with preestablished epidemiological data. Although the new O139 serogroup showed a trend of increased resistance to trimethoprim-sulfamethoxazole and nitrofurantoin, it was more susceptible to ampicillin than previous serogroup O1 and non-O1 strains. Regardless of serogroup, > or = 98% of the V. cholerae isolates tested were susceptible to most antibiotics tested by us. It is important to continue susceptibility testing of all new isolates of V. cholerae because of emerging resistant strains. However, V. cholerae remains susceptible to most of the available antibiotics.     

Use of polymerase chain reaction for detection of toxigenic Vibrio cholerae O1 strains from the Latin American cholera epidemic.

In January 1991, an outbreak of cholera started in Peru and spread throughout most of Latin America within 8 months. As of March 1992, over 450,000 cases and approximately 4,000 deaths have been reported to the Pan American Health Organization. The causative organism is toxigenic Vibrio cholerae O1 of the El Tor biotype and is distinct from the U.S. Gulf Coast strains. A polymerase chain reaction (PCR) that amplifies a 564-bp fragment of the cholera toxin A subunit gene (ctxA) was used to identify toxigenic V. cholerae O1 strains. A total of 150 V. cholerae O1 isolates were tested. They were of unknown toxin status, were associated with recent outbreaks, and were isolated from patients, food, and water. One hundred forty isolates were found to be toxigenic both by PCR and the routine diagnostic enzyme-linked immunosorbent assay. Thirty-eight known toxigenic strains isolated worldwide from 1921 to 1991 were also positive in the PCR. A collection of 18 nontoxigenic V. cholerae O1 strains, 35 Escherichia coli heat-labile-enterotoxin-I-producing strains, 26 Campylobacter strains, and 8 strains of Aeromonas hydrophila, previously reported to produce cholera toxin-like toxin, were all negative in the ctxA PCR. We conclude that this PCR is a diagnostic method that specifically detects toxin genes in V. cholerae O1 strains in a reference laboratory. It is more rapid and less cumbersome than other diagnostic methods for detection of toxicity in these strains.