Improved Microbiological Techniques Using the Polymerase Chain Reaction and Pulsed-Field Gel Electrophoresis for Diagnosis and Follow-Up of Enterohaemorrhagic Escherichia coli Infection

 The aims of the present investigation were to evaluate the microbiological diagnostic procedures, especially polymerase chain reaction (PCR) versus culture and seroagglutination, in relation to the clinical features of enterohaemorrhagic Escherichia coli (EHEC) infection and to study the status of EHEC in the western part of Sweden. During 1997 and 1998, stool specimens from 3,948 patients were analysed by PCR for the presence of EHEC with verotoxin (VT)1- and/or VT2-producing DNA sequences. The stool specimens were also cultured for Escherichia coli O157 : H7, Salmonella, Campylobacter, Shigella and Yersinia. Fifty-five patients were positive by PCR. Thirty-nine patients were positive for EHEC by PCR and culture. Of these, 29 were infected with EHEC serogroup O157 : H7 strains. All EHEC isolates were analysed by pulsed-field gel electrophoresis (PFGE); 17 different clones were identified. Studies on the duration of the presence of EHEC in the gut showed that EHEC often disappears rather quickly, i.e. within 2 weeks. In one patient, however, EHEC remained for several months. In conclusion, PCR, rather than culture and agglutination, should be the method of choice for microbiological diagnosis of EHEC infection. PCR is more sensitive than culture for detecting EHEC in the gut.     

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.     

Laboratory investigation of a multistate food-borne outbreak of Escherichia coli O157:H7 by using pulsed-field gel electrophoresis and phage typing.

Two hundred thirty-three isolates of Escherichia coli O157:H7 were analyzed by both pulsed-field gel electrophoresis (PFGE) and bacteriophage typing. All 26 isolates from persons whose illness was associated with a recent multistate outbreak of E. coli O157:H7 infections linked to the consumption of undercooked hamburgers and all 27 isolates from incriminated lots of hamburger meat had the same phage type and the same PFGE pattern. Twenty-five of 74 E. coli O157:H7 isolates from Washington State and 10 of 27 isolates from other states obtained during the 6 months before the outbreak had the same phage type as the outbreak strain, but only 1 isolate had the same PFGE pattern. PFGE thus appeared to be a more sensitive method than bacteriophage typing for distinguishing outbreak and non-outbreak-related strains. The PFGE patterns of seven preoutbreak sporadic isolates and five sporadic isolates from the outbreak period differed from that of the outbreak strain by a single band, making it difficult to identify these isolates as outbreak or non-outbreak related. Phage typing and PFGE with additional enzymes were helpful in resolving this problem. While not as sensitive as PFGE, phage typing was helpful in interpreting PFGE data and could have been used as a simple, rapid screen to eliminate the need for performing PFGE on unrelated isolates.     

Genomic Comparisons and Shiga Toxin Production among Escherichia coli O157:H7 Isolates from a Day Care Center Outbreak and Sporadic Cases in Southeastern Wisconsin

Contour-clamped homogeneous electric field pulsed-field gel electrophoresis (CHEF-PFGE) was used to compare Wisconsin isolates of Escherichia coli O157:H7, including 39 isolates from a 1994 day care center outbreak, 28 isolates from 18 individuals from the surrounding geographic area with sporadic cases occurring during the 3 months before the outbreak, and 3 isolates, collected in 1995, from patients with hemolytic-uremic syndrome (HUS) who were from eastern Wisconsin counties other than those inhabited by the day care center and sporadic-case individuals. The technique of CHEF-PFGE using XbaI identified seven highly related restriction endonuclease digestion profiles (REDPs) (93 to 98% similarity) among the 39 day care center isolates and nine XbaI REDPs (63 to 93% similarity) among the 28 isolates from sporadic-case individuals, including REDP 33, which was exhibited by both day care and sporadic-case isolates. PFGE analyses of sequential E. coli O157:H7 isolates from symptomatic day care center attendees revealed that the REDPs of 25 isolates from eight patients were indistinguishable whereas the REDPs of 2 of 6 isolates from two patients differed slightly (93 to 95% similarity). The REDPs of the three isolates from 1995 HUS patients were 78 to 83% similar, with REDP 26 being exhibited by one HUS-associated isolate and an isolate from one day care attendee who did not develop HUS. The genes for both Shiga toxins I and II (stx₁ and stx₂, respectively) were detected in all but one isolate (sporadic case), and Shiga toxin production by the day care center isolates was not significantly different from that of the other isolates, including the three HUS-associated isolates. Analyses of E. coli O157:H7 isolates from both the day care center outbreak and sporadic cases by CHEF-PFGE permitted us to define the REDP variability of an outbreak and geographic region and demonstrated that the day care center outbreak and a HUS case in 1995 were caused by E. coli O157:H7 strains endemic to eastern Wisconsin.     

Isolation and Characterization of Verocytotoxin-Producing Escherichia coli O157 Strains from Dutch Cattle and Sheep

In the periods from July to November 1995 and 1996, fecal samples from Dutch cattle and sheep were collected at the main slaughterhouses of The Netherlands, located at different geographic sites. The samples were examined for the presence of verocytotoxin (VT)-producing Escherichia coli (VTEC) of serogroup O157. E. coli O157 strains could be isolated from 57 (10.6%) of 540 adult cattle, 2 (0.5%) of 397 veal calves, 2 (3.8%) of 52 ewes, and 2 (4.1%) of 49 lambs. Immunomagnetic separation with O157-specific-antibody-coated beads appeared to be significantly more sensitive than conventional plating for detection of the organism in feces. With the exception of two isolates from adult cattle which appeared to be negative for VT genes, all animal isolates were positive for both VT (VT1 and/or VT2) and E. coli attaching-and-effacing gene sequences, and therefore, they were regarded as potential human pathogens. Although genomic typing by pulsed-field gel electrophoresis revealed a wide variety of distinct restriction patterns, comparison of the 63 animal isolates with 33 fecal O157 VTEC strains previously isolated from humans with the diarrhea-associated form of the hemolytic-uremic syndrome by their phage types and VT genotypes showed a marked similarity between animal and human isolates: 30 (90.9%) of the 33 human isolates appeared to be of E. coli O157 strain types also isolated from cattle and sheep. It was concluded that Dutch cattle and sheep are an important reservoir of E. coli O157 strains that are potentially pathogenic for humans.     

Use of Phenotyping and Genotyping to Verify Transmission of <Emphasis Type="Italic">Escherichia coli</Emphasis> O157:H7 from Dairy Farms

A total of 80 human infections by Escherichia coli O157:H7 were documented in Finland in 1997 and 1998. Most were sporadic and their sources undetermined. Five cases not associated with one another, one of which led to secondary transmission within a family, could be traced to five different dairy farms. These five case patients (age range 2–17 years, median age 3 years) were hospitalised with bloody diarrhoea; two of them developed haemolytic uraemic syndrome. All nine human isolates obtained were sorbitol negative, carried the verocytotoxin 2 and eae genes, and produced verocytotoxin and enterohaemolysin. The phage and pulsed-field gel electrophoresis types of the human and bovine isolates from the corresponding farms were indistinguishable. The cattle (20–70 animals per farm) were monitored for up to 2 years after the human cases. The proportion of cattle excreting the type that caused the human infections varied from 3.2 to 66.7% when sampled soon after the human cases, and from 0.0 to 5.3% about a year or so later. On most of the farms, the animals excreted the pathogen intermittently. On one farm, Escherichia coli O157 isolates with other characteristics were also occasionally isolated. Although the infections were traced back to the farms, it could not be established whether the source was unpasteurised milk or direct or indirect contact with cattle. The results of this study emphasise the need for special recommendations for children visiting or living on a farm to prevent these infections. Electronic Publication     

Stability of multiple-locus variable-number tandem repeats in Salmonella enterica serovar typhimurium

Variable-number tandem repeats (VNTRs) may evolve so rapidly that multiple profiles emerge during an outbreak. A total of 190 isolates from eight Salmonella enterica serovar Typhimurium outbreaks and 15 isolates from seven patients were analyzed by pulsed-field gel electrophoresis and VNTR typing. Small changes in loci were noted; otherwise, the VNTR profiles were stable during the course of the outbreaks.     

Evaluation of ribotyping as epidemiologic tool for typing Escherichia coli serogroup O157 isolates.

A total of 121 representative Escherichia coli O157:H7 and O157:NM (nonmotile) isolates were characterized by ribotype, phage type, verotoxin genotype, and genomic fingerprints generated by pulsed-field gel electrophoresis. Ribotyping was not able to discriminate between O157:H7 isolates, and phage typing and pulsed-field gel electrophoresis were the most valuable and discriminatory techniques.     

Pseudo-outbreak of listeriosis elucidated by pulsed-field gel electrophoresis

Listeria monocytogenes was isolated from three patients hospitalized in two departments of the same hospital over a two-week period. A nosocomial outbreak of listeriosis was suspected. Patients presented with tenosynovitis, central venous catheter infection, and bacteremia. The first patient had a community-acquired infection, the second a nosocomial infection, and the source of the third patient's illness was uncertain. Epidemiological investigations failed to identify a common source of contamination within the hospital. The three strains were nontypeable by phage typing, but Smal macrorestriction analysis and pulsed-field gel electrophoresis yielded three distinct profiles. Therefore, the three cases seemed to represent a cluster of sporadic cases as opposed to an outbreak of listeriosis. Rapid typing of isolates is essential in the early investigation of potential outbreaks of listeriosis and may prevent the initiation of expensive and time-consuming epidemiological investigations.     

Molecular Epidemiology of Methicillin-Resistant Staphylococcus aureus in Finland

 This study reports the recent trends in the occurrence of methicillin-resistant Staphylococcus aureus in Finland, with special focus on characterization of the strains linked to interhospital epidemics and local outbreaks. Between 1981 and 1997, the annual number of methicillin-resistant Staphylococcus aureus isolations ranged from 89 to 272. Of all blood isolates of Staphylococcus aureus reported to the National Infectious Disease Register during the period 1995–97 (n=2049), only six were resistant to methicillin. Between 1992 and 1997, typing analysis by various methods (i.e., antibiogram, phage typing, ribotyping, and pulsed-field gel electrophoresis) identified 18 different strains capable of causing intrahospital outbreaks or interhospital epidemics. These 18 strains were separated into 13 different ribotypes and 14 major pulsed-field gel electrophoresis types. Multiresistance was investigated as a possible marker for epidemicity. Eight of the ten interhospitally spread strains were multiresistant compared to only three of the eight intrahospitally spread outbreak strains. More than one-third of the epidemic and local outbreak strains were suspected to be of foreign origin. The majority (6 of 10) of the epidemics were localized in southern and western Finland, and the largest epidemic, which occurred in the Helsinki metropolitan area, involved over 200 persons. Thus far, the epidemics have remained primarily intracity problems, and only two strains have become endemic.     

Importance of environmental transmission in cases of EHEC O157 causing hemolytic uremic syndrome

A local outbreak of Shiga toxin (Stx)-producing enterohemorrhagic Escherichia coli (EHEC) O157:H7 causing severe hemolytic-uremic syndrome (HUS) was found to be caused by environmental transmission. Automated ribotyping and pulsed-field gel electrophoresis revealed that four stx₂-positive EHEC isolates obtained from two unrelated children, one mother and one cow were identical. Results of an epidemiological investigation strongly suggest that both children were infected via a meadow strewn with manure containing EHEC-positive feces from the infected cow a few days prior to the onset of illness. The cow belonged to a cattle farm neighboring the meadow. This report highlights the risk of acquiring EHEC O157 through indirect contact with a farm environment.     

Molecular epidemiology of Escherichia coli O157:H7 by pulsed-field gel electrophoresis and comparison with that by bacteriophage typing.

One hundred twenty-four Escherichia coli O157:H7 isolates were characterized by pulse-field gel electrophoresis, bacteriophage typing, and PCR of verotoxin genes. Diversity indices obtained--0.786 for phage types and 0.987 for pulsed-field gel electrophoresis types--demonstrated that phage typing falls below the critical value (0.9) required for confident interpretation of results.     

Genetic and Antigenic Characteristics of Neisseria meningitidis Strains Isolated in the Czech Republic in 1997–1998

 Strains of Neisseria meningitidis isolated from patients and asymptomatic individuals who had been in contact with patients were investigated using four typing methods with the aim of identifying any heterogeneity and/or homogeneity among the strains. In 1993, a dramatic change in the incidence and severity of invasive meningococcal disease in the Czech Republic occurred as a consequence of the appearance of a Neisseria meningitidis strain of phenotype C : 2a : P1.5,2 and electrophoretic type ET-15, belonging to the ET-37 complex, which had not previously been identified in this country. Presented here are the results of a study of the relationships between 58 Neisseria meningitidis isolates collected between January 1997 and June 1998. Forty-nine isolates originating from patients with invasive meningococcal disease and nine from healthy contacts were analyzed using the following four methods: whole-cell enzyme immunoassay, multilocus enzyme electrophoresis, pulsed-field gel electrophoresis, and randomly amplified polymorphic DNA analysis. A high prevalence of phenotype C : 2a : P1.5,2 electrophoretic type ET-15 was confirmed among the patients' strains. Nevertheless, during the study period, they became heterogeneous. Strains isolated from healthy contacts showed greater heterogeneity in serological phenotypes and electrophoretic types from the beginning of the study, and electrophoretic type ET-15 strains were less frequent. Within the electrophoretic type ET-15 clone, strains showing the identical serological phenotype (with the exception of one isolate) were indistinguishable using randomly amplified polymorphic DNA analysis, while pulsed-field gel electrophoresis revealed heterogeneity with 12 pulsed-field gel electrophoretic types identified. The strains from the same cluster displaying the same serological phenotype were indistinguishable with any of the methods used.     

Genotyping of verocytotoxin-producing Escherichia coli O157: comparison of isolates of a prevalent phage type by fluorescent amplified-fragment length polymorphism and pulsed-field gel electrophoresis analyses

We applied the high-resolution genotyping technique fluorescent amplified-fragment length polymorphism (FAFLP) analysis to 71 isolates of a single phage type (PT8) of pulsed-field gel electrophoresis (PFGE)-characterized verocytotoxin-producing Escherichia coli O157. Twenty-seven similar, but not identical, groupings were defined by both FAFLP analysis and the PFGE profiles. Given the FAFLP analysis conditions described here, these two methods exhibited equivalent discriminatory powers.     

Genomic diversity and molecular differentiation of Riemerella anatipestifer associated with eight outbreaks in five farms

Riemerella anatipestifer causes infectious serositis of ducks and geese. The genomic diversity of R. anatipestifer associated with outbreaks in waterfowls was studied using 24 multidrug-resistant R. anatipestifer isolates collected from the visceral organs of ducks and geese from seven outbreaks in four goose farms and one outbreak in one duck farm. Seven methods were used to differentiate these isolates. Plasmid patterns differed in plasmid number and size, ranging from 2.9 kb to 20 kb, and provided seven profiles. Divergent nucleotide sequences (predominant in 670 to 830 base pairs) of the ompA gene categorized the 24 isolates into three groups based on cluster analysis and polymerase chain reaction–restriction fragment length polymorphism. Repetitive-sequence polymerase chain reaction and pulsed-field gel electrophoresis analysis revealed the highest genotypic variations among the isolates. Genotypes and serotypes differed among farms and within the same farm and even within a single goose. In conclusion, a difference in R. anatipestifer genotypes and serotypes was observed for multiple outbreaks in waterfowls.     

Fluorescent amplified-fragment length polymorphism subtyping of the Salmonella enterica serovar enteritidis phage type 4 clone complex

Fluorescent amplified-fragment length polymorphism (FAFLP) analysis, a high-resolution PCR-based genome fingerprinting method, was used to subtype Salmonella enterica serovar Enteritidis phage type 4. This single phage type is responsible for the majority of salmonellosis in Europe. Twenty strains isolated from nine outbreaks, five isolates from sporadic cases of human infection, four strains of poultry origin, and one laboratory-derived strain were comparatively studied by pulsed-field gel electrophoresis (PFGE) and FAFLP analysis. Following macrorestriction with XbaI, PFGE classified 73% of PT4 strains as a single type. FAFLP analysis was carried out with the primer pair EcoRI+0 and MseI+C, by simultaneously sampling 170 to 190 loci throughout the PT4 genome. Twenty-three FAFLP profiles, with 1 to 61 amplified-fragment differences, were found among the 30 strains. The index of discriminatory power of FAFLP analysis was 0.98, compared to 0.47 for PFGE. FAFLP analysis assigned genotypes to each PT4 outbreak, as well as sporadic PT4 infections, a significant development for the epidemiology and control of this zoonotic enteric pathogen.     

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.     

Evaluation of the molecular epidemiology of an outbreak of multiply resistant Shigella sonnei in a day-care center by using pulsed-field gel electrophoresis and plasmid DNA analysis.

Outbreaks of diarrhea in child day-care centers (DCC) are common. This study was undertaken to evaluate the molecular epidemiology of an outbreak of diarrhea due to Shigella sonnei. This outbreak involved 25 of 52 (48%) DCC children and 14 of 132 (11%) teachers and household contacts. S. sonnei isolates from nine children and five contacts were characterized by antimicrobial susceptibility, plasmid content, plasmid DNA restriction fragment pattern, and pulsed-field gel electrophoresis (PFGE) of total genomic DNA; 33 isolates from Houston, Tex., Chicago, Ill., and Mexico City, Mexico, also were studied. All outbreak isolates were resistant to ampicillin and trimethoprim-sulfamethoxazole and shared five to six plasmids ranging from 3.3 to 70 MDa. A total of 8 of 12 temporally associated nonoutbreak Houston isolates had plasmid profiles and restriction fragment patterns similar to those of the outbreak strain, despite possessing different antibiotic susceptibility patterns. PFGE demonstrated identical DNA patterns among outbreak isolates and similar or identical patterns among temporally associated sporadic Houston isolates with plasmid profiles similar to that of the outbreak strain. All other nonoutbreak strains from Houston, Chicago, and Mexico had plasmid profiles, restriction fragment patterns, and PFGE patterns different from those of the outbreak strain. DCC outbreak isolates could be distinguished from most sporadic isolates by antimicrobial susceptibility testing, but plasmid analysis and PFGE could not differentiate common-source isolates from sporadic isolates in the same location during the same time period, indicating that isolates present in the community were genetically similar to those producing outbreaks in the DCC.     

DNA fingerprinting of Salmonella enterica subsp. enterica serovar typhimurium with emphasis on phage type DT104 based on variable number of tandem repeat loci

Seventy-eight human and environmental strains of Salmonella enterica subsp. enterica serovar Typhimurium, as well as 18 isolates of other Salmonella serovars and 6 isolates of Escherichia coli, were subjected to a novel variable number of tandem repeats (VNTR)-based fingerprinting method that showed high discrimination and reproducibility for typing serovar Typhimurium isolates. The method is based on capillary separation of PCR products from fluorescence-labeled VNTR in the serovar Typhimurium genome. The serovar Typhimurium isolates displayed 54 VNTR patterns, and the VNTR assay correctly identified strains from a well-characterized outbreak. Among 37 serovar Typhimurium phage type DT104 isolates, 28 distinct VNTR patterns were found. This VNTR-based method is fast and suitable for complete automation. Our VNTR-based method was capable of high discrimination within the homogeneous serovar Typhimurium DT104 phage type and can be used to trace outbreaks and to monitor DT104 as well as other phage types. The VNTR assay was compared to XbaI pulsed-field gel electrophoresis, amplified fragment length polymorphism analysis, integron-cassette profiles and gene PCR of intI1, qacEDelta1, sulI1, and floR. The VNTR assay showed greatly improved resolution compared to all other tested methods in this study.     

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.