Pulsed-field Gel Electrophoresis Subtypes Research Articles

Pulsed-field gel electrophoresis (PFGE) analysis of Listeria monocytogenes isolates from different sources and geographical origins and representative of the twelve serovars

Multiplex-PCR (MPCR) serogrouping and pulsed-field gel electrophoresis (PFGE) subtyping analysis are currently used by several public and private laboratories for the characterization of Listeria monocytogenes. In this study a set of 80 L. monocytogenes isolates belonging to the twelve serovars was used to investigate (i) the typeability of the rare serovars, (ii) the ability of PFGE analysis with ApaI and AscI to differentiate serovars within MPCR serogroups and (iii) the association of molecular types with the specific source or geographical origin of the isolates. With the exception of three isolates (rare serovars 4a and 4c) that were not amenable to restriction with ApaI, all the other analyzed isolates were subtyped by both enzymes. PFGE discriminated the 80 isolates into 62 combined ApaI and AscI PFGE patterns (pulsotypes), but could not differentiate serovars within MPCR serogroups, in which isolates from different serovars displaying the same pulsotype were found. Clustering analysis suggests that for some pulsotypes grouping according to Portuguese origin or source can be suggested. On the other hand, some L. monocytogenes clones are widely distributed. Two pulsotypes from Portuguese human isolates were identical to the ones displayed by human outbreak clones in the UK and in the USA and Switzerland, respectively, although they were not temporally matched. Computer-assisted data analysis of large and diverse PFGE type databases will improve the correct interpretation of subtyping data in epidemiological studies and in tracing routes and sources of contamination in the food industry.

Outbreaks of Salmonellosis in Minnesota (1998 through 2006) Associated with Frozen, Microwaveable, Breaded, Stuffed Chicken Products

From 1998 through 2006, four outbreaks of salmonellosis associated with raw, frozen, microwaveable, breaded, pre-browned, stuffed chicken products were identified in Minnesota. In 1998, 33 Salmonella Typhimurium cases were associated with a single brand of Chicken Kiev. In 2005, four Salmonella Heidelberg cases were associated with a different brand and variety (Chicken Broccoli and Cheese). From 2005 to 2006, 27 Salmonella Enteritidis cases were associated with multiple varieties of product, predominately of the same brand involved in the 1998 outbreak. In 2006, three Salmonella Typhimurium cases were associated with the same brand of product involved in the 2005 Salmonella Heidelberg outbreak. The outbreak serotype and pulsed-field gel electrophoresis subtype of Salmonella were isolated from product in each outbreak. In these outbreaks, most individuals affected thought that the product was precooked due to its breaded and prebrowned nature, most used a microwave oven, most did not follow package cooking instructions, and none took the internal temperature of the cooked product. Similar to previous salmonellosis outbreaks associated with raw, breaded chicken nuggets or strips in Canada and Australia, inadequate labeling, consumer responses to labeling, and microwave cooking were the key factors in the occurrence of these outbreaks. Modification of labels, verification of cooking instructions by the manufacturer, and notifications to alert the public that these products contain raw poultry, implemented because of the first two outbreaks, did not prevent the other outbreaks. Microwave cooking is not recommended as a preparation method for these types of products, unless they are precooked or irradiated prior to sale.

Investigation and Characterization of the Frozen Feeder Rodent Industry in Texas Following a Multi‐StateSalmonellaTyphimurium Outbreak Associated with Frozen Vacuum‐Packed Rodents

A multi-state outbreak investigation of Salmonella Typhimurim cases associated with pet snakes and the frozen vacuum-packed rodents used to feed them identified a Texas frozen feeder rodent facility (Supplier A) as the source of the Salmonella-infected frozen rodents. Texas authorities collected samples directly from Supplier A. Seven Salmonella-positive samples out of 49 environmental swabs were found and one adult mouse out of 88 frozen feeder rodents was Salmonella-positive by culture. No Salmonella strains were isolated from rodent feeds. The pulsed-field gel electrophoresis (PFGE) subtype patterns of S. Typhimurium isolates from feeder rodent and environment samples were indistinguishable from the outbreak strain isolated from humans. A follow-up investigation was performed on all additional feeder rodent facilities identified in Texas. Salmonella was isolated at one of four facilities; seven of 100 rodent samples were positive for Salmonella at this facility. The serotype S. I 4,[5],12:i:- was isolated from seven feeder rodent samples, and PFGE patterns of the seven isolates were indistinguishable. As observed in the initial outbreak investigation, no Salmonella were cultured from rodent feeds at any of the facilities. The feeder rodent industry is an insufficiently recognized industry in the United States. Outbreak investigation and testing of additional feeder rodent facilities in Texas indicate that further evaluation of feeder rodent facilities as a source of Salmonella for pet snakes and humans is warranted.

A Multi‐stateSalmonellaTyphimurium Outbreak Associated with Frozen Vacuum‐packed Rodents used to Feed Snakes

From December 2005 through January 2006, the Minnesota Department of Health (MDH) identified four human clinical isolates of Salmonella Typhimurium that were indistinguishable by pulsed-field gel electrophoresis (PFGE). During routine interviews, three of the cases reported attending the same junior high school and two handled snakes in the science classroom. MDH collected environmental samples from the school's science classroom for Salmonella culturing; these included environmental samples and frozen vacuum-packed mice purchased over the internet to feed the classroom snakes. Through PulseNet, a national molecular subtyping surveillance network for enteric bacteria, 21 human S. Typhimurium isolates with indistinguishable PFGE patterns were identified in the United States since December 2005. Each state determined whether these human cases had recent exposure to snakes fed vacuum-packed rodents. Texas state officials conducted tracebacks of the vacuum-packed mice and collected samples at the breeding facility. Nineteen of 21 cases were interviewed, and seven reported contact with frozen vacuum-packed rodents from the same internet-based supplier in Texas. In Minnesota, the outbreak PFGE subtype of S. Typhimurium was isolated from the snakes, frozen feed rodents, and the classroom environment. Three human cases were identified in Michigan, Pennsylvania, and Wyoming. The outbreak PFGE subtype of S. Typhimurium was isolated from the Pennsylvania case's frozen rodents and the Michigan case's pet snake. The outbreak PFGE subtype of S. Typhimurium was also isolated from the supplier's rodent facility. This was a S. Typhimurium outbreak associated with frozen rodents. Human transmission likely occurred through direct contact with snakes and contaminated environmental surfaces. This report represents the second recent multi-state salmonellosis outbreak associated with commercially distributed rodents. Stronger oversight of the commercial rodent industry is warranted.

The Effect of Chilling in Cold Air or Ice Water on the Microbiological Quality of Broiler Carcasses and the Population of Campylobacter

Cold air or ice water can be used to chill poultry carcasses after slaughter. The objective of this study was to compare the effect of 2 chill methods on broiler carcass bacteria. Broiler carcasses were cut in half along the dorsal-ventral midline; one half was subjected to an ice-water immersion chill in an agitated bath for 50 min, whereas the reciprocal half was subjected to an air chill in a 1 degrees C cold room for 150 min. Total aerobic bacteria, coliforms, Escherichia coli, and Campylobacter were enumerated from half-carcass rinses. Species of Campylobacter isolates was determined by a commercial PCR method, which was followed by molecular subtyping with pulsed-field gel electrophoresis and determination of antimicrobial susceptibility to 9 drugs. Although significantly fewer of each bacterial type were detected per milliliter from immersion-chilled carcasses than from air-chilled carcasses, in each case the difference was less than 1 log(10) cfu/mL. Chilling method did not affect species; both Campylobacter jejuni and Campylobacter coli were recovered. Results of pulsed-field gel electrophoresis subtyping did not suggest that either chilling method selected for any specific subtypes; most subtypes were found on carcass halves used for both the air chill and water immersion chill. Resistance to 2 antimicrobial drugs was noted in 9 C. coli isolates, 6 from air-chilled carcass halves and 3 from immersion-chilled carcass halves. These data showed that immersion-chilled carcasses had lower numbers of bacteria; however, the difference was not large and may have been due to simple dilution. Both methods were effective for lowering carcass temperature, and neither chilling method seemed to select for specific species, subtypes, or antimicrobial-resistant Campylobacter.

Comparison of multiple-locus variable-number tandem repeat analysis and pulsed-field gel electrophoresis in a setting of polyclonal endemicity of vancomycin-resistant Enterococcus faecium

In order to assess whether multiple-locus-variable number tandem repeat analysis (MLVA) could replace pulsed-field gel electrophoresis (PFGE) for genotyping vancomycin-resistant isolates of Enterococcus faecium (VREF), this study compared the typeability, discriminatory power, concordance and costs of these methods for VREF isolates obtained from patients, environmental samples and the hands of healthcare workers (HCWs) in a medical intensive care unit (ICU) where VREF was endemic. Over a 58-day period, 393 VREF isolates (373 vanA, one vanA/B, 19 vanB) were cultured from patient rectal swabs (n = 76), the environment (n = 270) and the hands of HCWs (n = 47). PFGE was able to divide 358 (91.1%) isolates into 19 PFGE types (>six bands different) and 24 subtypes (one to three bands different). MLVA was able to type 391 (99.5%) isolates into 11 genotypes. The discriminatory power of PFGE subtypes was 83%, as compared to 68% for MLVA. Concordance between the two methods, based on matched or mismatched MLVA types and PFGE types or subtypes, was 67.5% and 82.8%, respectively. Using PFGE, 13 isolates could be genotyped in 3 days; MLVA genotyped 94 isolates in 2 days. For both methods, the estimated costs were Euro 7 ($10)/isolate. PFGE and MLVA produced highly concordant results when assigning genotypes to nosocomial VREF isolates. MLVA was faster, but PFGE subtyping was more discriminatory.

Identification of the First Vancomycin Intermediate-ResistantStaphylococcus aureus(VISA) Isolate from a Hospital in Portugal

A clinical isolate of methicillin-resistant Staphylococcus aureus (MRSA) with intermediate resistance to vancomycin (minimal inhibitory concentration [MIC] of 4 mug/ml) was isolated in 2006 from a surgical wound of a patient hospitalized at the orthopedics ward of Hospital de São Marcos--Braga, in the town of Braga. A combination of molecular typing methods, including pulsed-field gel electrophoresis (PFGE), spa typing, multilocus sequence typing, and staphylococcal chromosomal cassette mec typing, identified the vancomycin intermediate-resistant S. aureus VISA-BRAGA as a derivative of the epidemic MRSA (EMRSA)-15 clone, which has been isolated with increasing frequency from several Portuguese hospitals recently. Compared to another EMRSA-15 isolate with the same genetic background (including PFGE subtype) the VISA-BRAGA isolate exhibited relatively high oxacillin MIC, slow growth, loss of hemolytic activity, and increased resistance to vancomycin and to daptomycin although neither of these two antibiotics was used in therapy. The VISA-BRAGA isolate described here appears to represent the first S. aureus with decreased susceptibility to vancomycin identified in a Portuguese hospital.

Epidemiology of European community-associated methicillin-resistant Staphylococcus aureus clonal complex 80 type IV strains isolated in Denmark from 1993 to 2004.

In Europe, community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) infections have been caused predominantly by isolates belonging to the "European CA-MRSA" clone (sequence type 80, staphylococcal cassette chromosome mec type IV). In this study, the epidemiology of European CA-MRSA was investigated on a nationwide scale, covering the period from 1993 to 2004. Denmark has been a low-prevalence country regarding MRSA since the mid-1970s but has experienced an increase in the number of new MRSA cases in recent years. Our results show that European CA-MRSA contributed to this increase. The isolates primarily caused skin and soft tissue infections (SSTIs) in patients outside hospitals, and transmission between household members was the predominant mode of spread. Although some of the isolates were found in hospitalized patients, nosocomial transmission seemed likely in only one instance, pointing to endogenous infections as an important factor. Compared to the CA-MRSA clone most common in the United States (USA300), the European CA-MRSA clone seems less well adapted to persist in hospital environments. Patients with a recent history of travel or family relation to the Mediterranean or Middle East were highly overrepresented. The epidemiological data indicated that the European CA-MRSA isolates were introduced into Denmark on multiple occasions, paralleled by an increasing level of genetic diversity of the isolates found during the study period. European CA-MRSA has previously been described as a rather uniform clone. However, we found pronounced, diverse pulsed-field gel electrophoresis subtypes, staphylococcal protein A gene (spa) types, and susceptibility patterns.

Multilocus sequence typing of Candida tropicalis shows clonal cluster enriched in isolates with resistance or trailing growth of fluconazole

Multilocus sequence typing (MLST) was used to characterize the genetic profiles of 52 Candida tropicalis isolates collected from 10 hospitals in Taiwan. A total of 33 diploid sequence types (DSTs) were differentiated among the 52 isolates and all were novel to the Internet C. tropicalis DST database ( http://pubmlst.org/ctropicalis/). Eleven (33.3%) of the 33 DSTs could be assigned to 3 major clonal clusters (1–3) based on eBURST calculation. Only clonal cluster 1 co-clustered isolates from UK and the United States. Clonal cluster 2 was enriched with isolates with resistance or trailing growth of fluconazole (14/20, 70%). Furthermore, there are a number of pulsed-field gel electrophoresis subtypes associated with the isolates in this cluster, demonstrating that possibly more than one clone with resistant or trailing property have emerged and spread in Taiwan in 1999.

Isolation of Salmonella Typhimurium from Outbreak-Associated Cake Mix

During May and June of 2005, 26 persons in several states were infected by a single strain (isolates indistinguishable by pulsed-field gel electrophoresis) of Salmonella enterica serotype Typhimurium after eating cake batter ice cream. The cake mix used to prepare the cake batter in the ice cream was implicated by epidemiologic investigation as the source of Salmonella contamination. Initial tests did not detect Salmonella in cake mix collected during the outbreak investigation. The objective of this study was to evaluate different procedures to isolate Salmonella from the implicated cake mix, cake, and ice cream. All outbreak-associated food samples (14 samples) were collected during the outbreak investigation by health departments of several of the states involved. Different combinations of Salmonella isolation procedures, including sample size, preenrichment broth, enrichment broth, enrichment temperature, and isolation medium, were used. Salmonella Typhimurium was isolated from two cake mix samples; the food isolates were indistinguishable from the outbreak pattern by pulsed-field gel electrophoresis subtyping. Universal preenrichment broth was substantially better than was lactose broth for preenrichment, and tetrathionate broth was better than was Rappaport-Vassiliadis broth for isolating Salmonella from the two positive cake mix samples. Although more typical Salmonella colonies were observed on plates from enrichment cultures grown at 35°C, more confirmed Salmonella isolates were obtained from plates of enrichment cultures grown at 42°C. Brilliant green agar, xylose lysine tergitol 4 agar, xylose lysine desoxycholate agar, Hektoen enteric agar, and bismuth sulfite agar plates were equally effective in isolating Salmonella from cake mix. The best combination of preenrichment-enrichment conditions for isolating the outbreak strain of Salmonella was preenrichment of cake mix samples in universal preenrichment broth at 35oC for 24 h, followed by enrichment in tetrathionate broth at 42°C for 24 h.

Genotypic analysis of invasive Streptococcus pneumoniae from Mali, Africa, by semiautomated repetitive-element PCR and pulsed-field gel electrophoresis.

As part of a large, ongoing study of invasive infections in pediatric patients in Bamako, Mali, 106 cases of invasive pneumococcal disease were identified from June 2002 to July 2003 (J. D. Campbell et al., Pediatr. Infect. Dis. J. 23:642-649, 2004). Of the 12 serotypes present, the majority of isolates were not contained in PCV7 (the 7-valent pneumococcal conjugate vaccine), including 1 isolate that was serotype 1, 12 isolates that were serotype 2, 58 isolates that were serotype 5, 7 isolates that were serotype 7F, and 1 isolate that was serotype 12F. To determine whether clonal dissemination of the predominant serotypes had taken place, genotyping was performed on 100 S. pneumoniae isolates by using two methods: pulsed-field gel electrophoresis (PFGE) of SmaI-digested genomic DNA, and the Bacterial Barcodes repetitive-element PCR (rep-PCR) method. Criteria for delineating rep-PCR genotypes were established such that isolates of different serotypes were generally not grouped together. The two methods were equally discriminatory within a given pneumococcal serotype. PFGE separated the isolates into 15 genotypes and 7 subtypes; rep-PCR separated isolates into 15 genotypes and 6 subtypes. Using either method, isolates within serotypes 2, 5, and 7 formed three large, separate clusters containing 1 genotype each. Both methods further distinguished related subtypes within serotypes 2 and 5. Interestingly, one of the PFGE subtypes of serotype 5 is indistinguishable from the Columbia(5)-19 clone circulating in Latin America since 1994. The data support that serotypes 2 and 5 were likely to be the result of dissemination of particular clones, some of which are responsible for invasive disease over a broad population range.

Phage types, virulence genes and PFGE profiles of Shiga toxin-producing Escherichia coli O157:H7 isolated from raw beef, soft cheese and vegetables in Lima (Peru)

The present study was conducted in Lima Metropolitana to evaluate the prevalence of Shiga toxin-producing Escherichia coli (STEC) O157:H7 in raw beef, raw ground beef, soft cheese and fresh vegetables, sampled at different markets in the city. Between October 2000 and February 2001, 407 food samples were collected from different markets in the 42 districts of Lima Metropolitana. Samples were assayed for E. coli O157 by selective enrichment in modified Tryptic Soy Broth containing novobiocin, followed by immunomagnetic separation (IMS) and plating onto sorbitol MacConkey agar supplemented with cefixime and potassium tellurite. Fifty (12.3%) of 407 food samples resulted positive for E. coli O157 isolation (23 of 102 ground beef; 15 of 102 beef meat; eight of 102 soft cheese and four of 101 fresh vegetables). Thirty-five E. coli O157 isolates were further analysed for the presence of virulence genes. All 35 were positive by PCR for O157 rfbE, fliCh7, eae-γ1 and ehxA genes. In addition, genes encoding Shiga toxins were detected in 33 of 35 isolates, five isolates (14%) encoded stx 1, stx 2, and 28 (80%) stx2 only. The isolates were of seven different phage types (PT4, PT8, PT14, PT21, PT34, PT54, and PT87) with three phage types accounting for 80% of isolates: PT4 (15 isolates), PT14 (8 isolates), and PT21 (5 isolates). Interestingly, the majority (31 of 35; 89%) of E. coli O157:H7 isolates characterized in this study belonged mainly to the phage types previously found in STEC O157:H7 strains associated with severe human disease in Europe and Canada. Pulsed-field gel electrophoresis (PFGE) of 32 isolates revealed 14 XbaI-PFGE groups (I to XIV) of similarity > 85%, with 23 (72%) isolates grouped in five clusters. Some isolates from different districts presented a high clonal relatedness. Thus, PFGE group VIII clustered eleven strains from nine different districts. The broad range of PFGE subtypes found in this study demonstrates the natural occurrence of many genetic variants among STEC O157:H7 spread in Lima.

Use of Pulsed-Field Gel Electrophoresis of Conserved XbaI Fragments for Identification of Swine Salmonella Serotypes

Swine Salmonella isolates (n=674) from various locations throughout the United States and Canada were analyzed via pulsed-field gel electrophoresis (PFGE) with XbaI. PFGE subtypes were analyzed by cluster analysis and compared to conventional serotyping results. The analysis showed a correlation of serotype to PFGE subtype. In addition, conserved fragments were identified within the restriction patterns that were unique to each serotype. PFGE using XbaI restriction provided a possible alternative method for screening and identifying swine Salmonella serotypes.

Detection of genetic diversity by pulsed-field gel electrophoresis among Escherichia coli O157 isolated from bovine faecal samples by immunomagnetic separation technique

Escherichia coli O157 is considered to be one of most important human pathogens of animal origin which causes serious clinical complications. One of the most common methods to isolate E. coli O157 is the immunomagnetic separation (IMS) technique which employs specific antibodies coupled to magnetic beads to bind and extract cells from enrichment broths followed by plating onto sorbitol MacConkey agar supplemented with cefixime and potassium tellurite (CT-SMAC) plates. The aim of this study was to determine strain variation by pulsed-field gel electrophoresis (PFGE) among E. coli O157 on IMS/CT-SMAC plates. Every suspect colony of E. coli O157 was tested following isolation by the IMS/CT-SMAC technique. From 124 colonies detected; six XbaI-PFGE profiles were identified. Our results demonstrate that mixed populations of E. coli O157 with distinguishable PFGE profiles that are simultaneously present in bovine faeces can be isolated with IMS/CT-SMAC technique. If the aim of the study was to analyse diversity of PFGE profiles of E. coli O157 in a faecal sample following isolation by the IMS/CT-SMAC technique, at least five colonies per sample should be analysed to detect different PFGE subtypes if present.

Effects of concentrate level of diet and pen configuration on prevalence of Escherichia coli O157 in finishing goats

Boer-sired goat kids ( n = 181) from native pastures were transported to outdoor pens in a beef cattle research facility to evaluate the effects of dietary concentrate level and pen configuration on prevalence of Escherichia coli O157 and to determine if horizontal transmission of E. coli O157 occurs between goats and cattle. Goats were blocked into heavy and light BW groups and randomly assigned to 12 pens, with each containing 15 or 16 animals. Treatments were randomly assigned to pens in a 3 × 2 factorial arrangement. Dietary treatments consisted of diets containing 50, 70, or 90% of the total diet (DM basis) as concentrate, with the balance of the diet as alfalfa hay. Goats were housed in pens with concrete floors, and a large concrete block (45 cm × 75 cm × 150 cm) was placed in the center of 6 of the 12 pens, thus providing two different pen configurations. Fecal samples were collected on day 0, 20, 34, 55, 83, and 105, and cultured for E. coli O157. Feces from cattle housed in adjacent pens were also sampled on day 1, 20, and 34. Isolation and identification of E. coli O157 from fecal samples were by selective enrichment in a broth, followed by immunomagnetic separation and plating on cefixime, tellurite, sorbitol MacConkey agar. Sorbitol-negative colonies that were positive for indole production, positive for O157 antigen agglutination, and speciated by API test, were considered as E. coli O157. Pulsed-field gel electrophoresis (PFGE) of chromosomal DNA was performed on all E. coli O157 isolates to characterize the genetic types and to determine if horizontal transfer of strains occurred between cattle and goats. Detection of the attaching and effacing gene ( eae), shiga toxin-1 and -2 genes ( stx1 and stx2) was done by polymerase chain reaction (PCR). Mean prevalence of E. coli O157 across all treatments and sampling days was 4.6% and no statistical differences ( P > 0.10) were observed for the different pen configurations or diet concentrate levels, but prevalence was numerically higher in the 70% concentrate treatment on 4 of the 6 sampling days. The eae and stx2 genes were present in 95% of the isolates, but the stx1 gene was present in only 51% of the isolates. One E. coli O157 isolate from a goat shared a common PFGE subtype with cattle isolates. Results suggest that in goats with low prevalence of E. coli O157 the level of concentrate in the diet did not influence the shedding pattern.

PFGE and PCR/RFLP typing of Campylobacter jejuni strains from poultry

1. Pulsed-field gel electrophoresis (PFGE) and PCR-restriction fragment length polymorphisms of the flagellin gene (fla-RFLP) were used to analyse 92 poultry and 110 human strains of Campylobacter jejuni. 2. Among poultry strains, 11 fla-RFLP and 11 PFGE subtypes were found, while human strains could be divided into 23 fla-RFLP and 32 PFGE subtypes. Altogether, 31 fla-RFLP and 32 PFGE subtypes were found. 3. The results show that individual flocks in farms are mostly infected with a single C. jejuni clone, while during subsequent colonisation their genotypes altered. fla-RFLP and PFGE profiles in poultry and humans were identical in less than 6% of cases. The results found so far confirm previous findings that chicken meat does not represent as important a source of campylobacteriosis as was previously believed. 4. The typing of Campylobacter sp. forms the basis for an evaluation of the current state and risk assessment of various Campylobacter sp. sources in relation to humans. Examination of samples with only one method is insufficient for epidemiology studies, because apparently different clones identified with one method could originate from a single clone, which could be proved with the other method.

Salmonella Outbreaks in Restaurants in Minnesota, 1995 through 2003: Evaluation of the Role of Infected Foodworkers

The 23 restaurant-associated salmonellosis outbreaks that occurred in Minnesota from 1995 through 2003 were reviewed to characterize the role of infected foodworkers. The median duration of the outbreaks was 21 days (range, 1 to 517 days). The median number of culture-confirmed patron cases per outbreak was seven (range, 1 to 36 cases). The median incubation for patron cases ranged from9hto5.9 days. A specific food vehicle was implicated in four outbreaks and suspected in five. Salmonella of the same serotype and pulsed-field gel electrophoresis subtype as that found in patrons was recovered from foodworkers in 19 outbreaks. Overall, 12% (129 of 1,033) of foodworkers tested positive for Salmonella. Sixty-four (53%) of 121 Salmonella-positive foodworkers reported not having had a recent gastrointestinal illness. Overall, the median duration of Salmonella shedding was 16 days. Among foodworkers who reported gastrointestinal illness, the median shedding duration was 30 days as compared with 3 days for asymptomatic foodworkers. Positive environmental samples were recovered in 4 (33%) of 12 outbreaks. No specific food vehicle was identified in any outbreaks associated with Salmonella-positive environmental samples. The median duration of outbreaks with positive environmental samples (187 days) was significantly longer than the median duration of outbreaks with negative environmental results (26 days, P = 0.03). A higher proportion of Salmonella-positive foodworkers (22 versus 8%) was identified in outbreaks with positive environmental samples. Salmonella outbreaks in restaurants are frequently prolonged yet produce a small number of confirmed patron cases. Prolonged outbreak durations suggest a persistent reservoir of contamination. Infected foodworkers likely serve as an important source for Salmonella transmission. Therefore, assessment of foodworker infection is essential for controlling restaurant outbreaks.

Intra-specific composition and succession of Bifidobacterium longum in human feces

Intra-species analysis of pulsed field gel electrophoresis (PFGE) on human fecal Bifidobacterium longum isolates revealed that a majority of 12 Japanese subjects harbored strains of unique PFGE types or subtypes over a 68-week period, suggesting that "indigenous"Bifidobacterium strains remain stable for a considerable time in each individual intestinal microbiota.

Development of a rapid strain differentiation method for methicillin-resistant Staphylococcus aureus isolated in Japan by detecting phage-derived open-reading frames

To develop a rapid genotyping method for investigating outbreaks of methicillin-resistant strains of Staphylococcus aureus (MRSA) isolated in Japan. Isolates were genotyped by detecting the keeping pattern of 16 open-reading frames (ORFs), a process we call phage ORF typing (POT). Thirteen of the ORFs were selected from phage genomes and one from a genomic island SaGIm in the genome of strain Mu50. The other two ORFs, one from Tn554 and one from staphylococcal cassette chromosome mec (SCCmec) type II, were used as strain markers. Three hundred and sixty-eight isolates from five hospitals were classified into 133 types by POT, whereas they were classified into 139 types by pulsed-field gel electrophoresis (PFGE) subtyping. The discriminatory power of POT (D=0.989) was equal to that of PFGE subtyping (D=0.986). MRSA isolates collected in Japan can be genotyped by detecting the keeping pattern of phage-derived ORFs with a discriminatory power equal to that of PFGE subtyping. MRSA isolates can be genotyped rapidly by detecting phage-derived ORFs. As particular pandemic clones can be found in a specific region, a typing method localized to a pandemic clone may be effective for the rapid genotyping of MRSA during outbreaks.

Molecular Epidemiology of Ceftiofur-Resistant Escherichia coli Isolates from Dairy Calves

Healthy calves (n = 96, 1 to 9 weeks old) from a dairy herd in central Pennsylvania were examined each month over a five-month period for fecal shedding of ceftiofur-resistant gram-negative bacteria. Ceftiofur-resistant Escherichia coli isolates (n = 122) were characterized by antimicrobial resistance (disk diffusion and MIC), serotype, pulsed-field gel electrophoresis subtypes, beta-lactamase genes, and virulence genes. Antibiotic disk diffusion assays showed that the isolates were resistant to ampicillin (100%), ceftiofur (100%), chloramphenicol (94%), florfenicol (93%), gentamicin (89%), spectinomycin (72%), tetracycline (98%), ticarcillin (99%), and ticarcillin-clavulanic acid (99%). All isolates were multidrug resistant and displayed elevated MICs. The E. coli isolates belonged to 42 serotypes, of which O8:H25 was the predominant serotype (49.2%). Pulsed-field gel electrophoresis classified the E. coli isolates into 27 profiles. Cluster analysis showed that 77 isolates (63.1%) belonged to one unique group. The prevalence of pathogenic E. coli was low (8%). A total of 117 ceftiofur-resistant E. coli isolates (96%) possessed the bla(CMY2) gene. Based on phenotypic and genotypic characterization, the ceftiofur-resistant E. coli isolates belonged to 59 clonal types. There was no significant relationship between calf age and clonal type. The findings of this study revealed that healthy dairy calves were rapidly colonized by antibiotic-resistant strains of E. coli shortly after birth. The high prevalence of multidrug-resistant nonpathogenic E. coli in calves could be a significant source of resistance genes to other bacteria that share the same environment.