Epidemiology of Enterotoxigenic Escherichia coli infection in Minnesota, 2016-2017.

Enterotoxigenic Escherichia coli (ETEC) is a well-established cause of traveller's diarrhoea and occasional domestic foodborne illness outbreaks in the USA. Although ETEC are not detected by conventional stool culture methods used in clinical laboratories, syndromic culture-independent diagnostic tests (CIDTs) capable of detecting ETEC have become increasingly prevalent in the last decade. This study describes the epidemiology of ETEC infections reported to the Minnesota Department of Health (MDH) during 2016-2017. ETEC-positive stool specimens were submitted to MDH to confirm the presence of ETEC DNA by polymerase chain reaction (PCR). Cases were interviewed to ascertain illness and exposures. Contemporaneous Salmonella cases were used as a comparison group in a case-case comparison analysis of risk factors. Of 222 ETEC-positive specimens received by MDH, 108 (49%) were concordant by PCR. ETEC was the sixth most frequently reported bacterial enteric pathogen among a subset of CIDT-positive specimens. Sixty-nine (64%) laboratory-confirmed cases had an additional pathogen codetected with ETEC, including enteroaggregative E. coli (n = 40) and enteropathogenic E. coli (n = 39). Although travel is a risk factor for ETEC infection, only 43% of cases travelled internationally, providing evidence for ETEC as an underestimated source of domestically acquired enteric illness in the USA.

Prospective Salmonella Enteritidis surveillance and outbreak detection using whole genome sequencing, Minnesota 2015-2017.

Clusters of Salmonella Enteritidis cases were identified by the Minnesota Department of Health using both pulsed-field gel electrophoresis (PFGE) and whole genome sequencing (WGS) single nucleotide polymorphism analysis from 1 January 2015 through 31 December 2017. The median turnaround time for obtaining WGS results was 11 days longer than for PFGE (12 vs. 1 day). WGS analysis more than doubled the number of clusters compared to PFGE analysis, but reduced the total number of cases included in clusters by 34%. The median cluster size was two cases for WGS compared to four for PFGE, and the median duration of WGS clusters was 27 days shorter than PFGE clusters. While the percentage of PFGE clusters with a confirmed source (46%) was higher than WGS clusters (32%), a higher percentage of cases in clusters that were confirmed as outbreaks reported the vehicle or exposure of interest for WGS (78%) than PFGE (46%). WGS cluster size was a significant predictor of an outbreak source being confirmed. WGS data have enhanced S. Enteritidis cluster investigations in Minnesota by improving the specificity of cluster case definitions and has become an integral part of the S. Enteritidis surveillance process.

Prevalence and characterization of Staphylococcus aureus, including methicillin-resistant Staphylococcus aureus, isolated from bulk tank milk from Minnesota dairy farms.

Staphylococcus aureus is a common causative agent of bovine mastitis in dairy herds. The emergence of methicillin-resistant Staphylococcus aureus (MRSA) in hospitals as well as the community is a significant and costly public health concern. S. aureus-related bovine mastitis is a common reason for therapeutic and/or prophylactic use of antibiotics on dairy farms. In this study, herd prevalence of S. aureus, including MRSA, was estimated from bulk tank milk (BTM) from Minnesota farms. A total of 150 pooled BTM samples from 50 farms, collected over 3 seasons (spring, summer, and fall of 2009), were assessed. Herd prevalence of methicillin-susceptible S. aureus (MSSA) was 84%, while MRSA herd prevalence was 4%. A total of 93 MSSA isolates and 2 MRSA isolates were recovered from 150 BTM samples. Antibiotic susceptibility testing of S. aureus isolates showed pansusceptibility in 54 isolates, resistance to a single antibiotic class in 21 isolates, resistance to two antibiotic classes in 13 isolates, and resistance to ≥3 antibiotics classes and thus multidrug resistance in 5 isolates. The two MRSA isolates displayed resistance to ß-lactams, cephalosporins, and lincosamides and were multiresistant. Staphylococcal protein A gene (spa) typing identified spa types t529 and t034 most frequently among methicillin-susceptible isolates, while t121 was observed in MRSA isolates. Seven isolates, including the two MRSA isolates, produced staphylococcal enterotoxins B, C, D, and E on overnight culture. MRSA isolates were further genotyped using multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). Of the 2 MRSA isolates, one had a composite genotype profile of MLST ST 5-PFGE USA100-unknown spa type, which has been reported among hospital-associated MRSA isolates, while the second isolate carried the MLST ST 8-PFGE USA300-spa type t121 genotype, commonly identified among community-associated MRSA isolates. These results suggest that MRSA genotypes associated with hospitals and community can be isolated from milk at very low rates.

Dynamics of Escherichia coli O157:H7 outbreak detection and investigation, Minnesota 2000-2008.

We determined characteristics of Escherichia coli O157:H7 pulsed-field gel electrophoresis clusters that predict their being solved (i.e. that result in identification of a confirmed outbreak). Clusters were investigated by the Minnesota Department of Health (MDH) using a dynamic iterative model. During 2000-2008, 19 (23%) of 84 clusters were solved. Clusters of ≥3 isolates were more likely to be solved than clusters of two isolates. Clusters in which the first two case isolates were received at MDH on the same day were more likely to be solved than were clusters in which the first two case isolates were received over ≥8 days. Investigation of clusters of ≥3 E. coli O157:H7 cases increased the success of cluster investigations.

Methicillin-resistant Staphylococcus aureus in resident animals of a long-term care facility.

UNLABELLED: Animals provide benefits to elderly and chronically ill people by decreasing loneliness, increasing social interactions, and improving mental health. As a result, many hospitals and long-term care facilities allow family pets to visit ill or convalescing patients or support animal-assisted therapy programs. These include programs that have resident animals in long-term care facilities. Despite the benefits, there are concerns about disease transmission between pets and patients. Antibiotic-resistant bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA), are a recognized problem in healthcare settings leading to refractory infections and potentially life-threatening illnesses. MRSA has been isolated from numerous animal species, yet few studies are available on the carriage of this pathogen in animals residing in long-term care facilities. Our objective was to characterize MRSA carriage among resident animals in a long-term care facility. METHODS: To document MRSA colonization, nasal swabs from 12 resident animals (one dogs and 11 cats) of a long-term care facility were collected weekly for 8 weeks. Staphylococcus isolates were characterized by antimicrobial susceptibility and MRSA isolates were further characterized by pulsed-field gel electrophoresis (PFGE). PFGE isolate patterns were compared with an existing database of MRSA isolate patterns at the Minnesota Department of Health. RESULTS: Two of 11 cats were colonized with MRSA. MRSA was recovered from five of eight weekly samples in one cat and two of eight weekly samples in the other cat. All isolates were classified as USA100 (healthcare-associated strains). DISCUSSION: Long-term care resident animals may acquire MRSA. Clonally related strains were identified over the 8-week sampling period. It is unclear if pets serve as an on-going source of infection to their human companions in long-term care facilities.

Use of multiple-locus variable number tandem repeat analysis and phage typing for subtyping of Salmonella Enteritidis from sporadic human cases in the United States.

AIMS: To investigate the genetic diversity among S. Enteritidis isolates from different geographic regions to evaluate the relationship between phage types (PTs) and variable number tandem repeat analysis (VNTR) loci. METHODS AND RESULTS: We performed multiple-locus variable number tandem repeat analysis (MLVA) and phage typing on 245 S. Enteritidis isolates collected from sporadic human clinical cases in Michigan, Minnesota, New York, and Washington states between 2000 and 2007. Ninety-four MLVA types and 22 different PTs were identified. Specific PTs were associated with a predominant allele for certain VNTR loci. Cluster analysis using a minimum-spanning tree demonstrated two major clusters (I, II) and one minor cluster of isolates. PTs 8, 13a, 13 and 34 were significantly associated with MLVA cluster I. Phage types 1, 4, 6a, and 18 were significantly associated with MLVA cluster II. CONCLUSIONS: We found significant association between MLVA-based clusters and PTs. Certain VNTR loci were associated with specific PTs and could serve as useful molecular markers for S. Enteritidis in epidemiological investigations. SIGNIFICANCE AND IMPACT OF THE STUDY: MLVA genotyping in combination with phage typing can be used for effective characterization of S. Enteritidis isolates. It can also be useful for tracing possible sources during investigations of sporadic and outbreak cases of S. Enteritidis.

International foodborne outbreak of Shigella sonnei infection in airline passengers.

During 22-24 August 2004, an outbreak of Shigella sonnei infection affected air travellers who departed from Hawaii. Forty-seven passengers with culture-confirmed shigellosis and 116 probable cases who travelled on 12 flights dispersed to Japan, Australia, 22 US states, and American Samoa. All flights were served by one caterer. Pulsed-field gel electrophoresis of all 29 S. sonnei isolates yielded patterns that matched within one band. Food histories and menu reviews identified raw carrot served onboard as the likely vehicle of infection. Attack rates for diarrhoea on three surveyed flights with confirmed cases were 54% (110/204), 32% (20/63), and 12% (8/67). A total of 2700 meals were served on flights with confirmed cases; using attack rates observed on surveyed flights, we estimated that 300-1500 passengers were infected. This outbreak illustrates the risk of rapid, global spread of illness from a point-source at a major airline hub.

Comparison of multiple-locus variable-number tandem repeat analysis, pulsed-field gel electrophoresis, and phage typing for subtype analysis of Salmonella enterica serotype Enteritidis.

Strain subtyping is an important tool for detection of outbreaks caused by Salmonella enterica serotype Enteritidis. Current subtyping methods, however, yield less than optimal subtype discrimination. In this study, we describe the development and evaluation of a multiple-locus variable-number tandem repeat analysis (MLVA) method for subtyping Salmonella serotype Enteritidis. The discrimination ability and epidemiological concordance of MLVA were compared with those of pulsed-field gel electrophoresis (PFGE) and phage typing. MLVA provided greater discrimination among non-epidemiologically linked isolates than did PFGE or phage typing. Epidemiologic concordance was evaluated by typing 40 isolates from four food-borne disease outbreaks. MLVA, PFGE, and, to a lesser extent, phage typing exhibited consistent subtypes within an outbreak. MLVA was better able to differentiate isolates between the individual outbreaks than either PFGE or phage typing. The reproducibility of MLVA was evaluated by subtyping sequential isolates from an infected individual and by testing isolates following multiple passages and freeze-thaw cycles. PFGE and MLVA patterns were reproducible for isolates that were frozen and passaged multiple times. However, 2 of 12 sequential isolates obtained from an individual over the course of 36 days had an MLVA type that differed at one locus and one isolate had a different phage type. Overall, MLVA typing of Salmonella serotype Enteritidis had enhanced resolution, good reproducibility, and good epidemiological concordance. These results indicate that MLVA may be a useful tool for detection and investigation of outbreaks caused by Salmonella serotype Enteritidis.

Comparative molecular analysis of community- or hospital-acquired methicillin-resistant Staphylococcus aureus.

Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) is a growing public health concern that has been associated with pediatric fatalities. It is hypothesized that the evolution of CA-MRSA is a recent event due to the acquisition of mec DNA by previously methicillin-susceptible strains that circulated in the community. This study investigated the genetic relatedness between CA-MRSA, hospital-associated MRSA (HA-MRSA), and nonmenstrual toxic shock syndrome (nmTSS) isolates. Thirty-one of 32 CA-MRSA isolates were highly related as determined by pulsed-field gel electrophoresis and spa typing yet were distinguishable from 32 HA-MRSA strains. The 31 related CA-MRSA isolates produced either staphylococcal enterotoxin B (n = 5) or C (n = 26), and none made TSS toxin 1. All CA-MRSA isolates tested contained a type IV staphylococcal cassette chromosome mec (SCCmec) element. In comparison, none of the HA-MRSA isolates (n = 32) expressed the three superantigens. Antibiotic susceptibility patterns were different between the CA-MRSA and HA-MRSA isolates; CA-MRSA was typically resistant only to beta-lactam antibiotics. Six of twenty-one nmTSS isolates were indistinguishable or highly related to the CA-MRSA isolates. MnCop, an nmTSS isolate obtained in Alabama in 1986, was highly related to the CA-MRSA isolates except that it did not contain an SCCmec element. These data suggest that CA-MRSA strains may represent a new acquisition of SCCmec DNA in a previously susceptible genetic background that was capable of causing nmTSS. CA-MRSA poses a serious health risk not only because it is resistant to the antibiotics of choice for community-acquired staphylococcal infections but also because of its ability to cause nmTSS via superantigen production.

Molecular analysis of a hospital cafeteria-associated salmonellosis outbreak using modified repetitive element PCR fingerprinting.

A hospital cafeteria-associated outbreak of gastroenteritis due to Salmonella enterica serotype Infantis was retrospectively evaluated using modified repetitive element PCR (rep-PCR) fingerprinting with the ERIC2 and BOXA1R primers and computer-assisted gel analysis and dendrogram construction. Rep-PCR yielded objective between-cycler, same-strain similarity values of from 92% (composite fingerprints) to 96% (ERIC2 fingerprints). The 70 Salmonella isolates (which included 19 serotype Infantis isolates from the hospital outbreak, 10 other serotype Infantis isolates, and 41 isolates representing 14 other serotypes) were resolved well to the serotype level with each of the three fingerprint types (ERIC2, BOXA1R, and composite). Rep-PCR typing uncovered several historical serotyping errors and provided presumptive serotype assignments for other isolates with incomplete or undetermined serotypes. Analysis of replicate fingerprints for each isolate, as generated on two different thermal cyclers, indicated that most of the seeming subserotype discrimination noted in single-cycler dendrograms actually represented assay variability, since it was not reproducible in combined-cycler dendrograms. Rep-PCR typing, which would have been able to identify the presence of the hospital-associated serotype Infantis outbreak after the second outbreak isolate, could be used as a simple surrogate for serotyping by clinical microbiology laboratories that are equipped for diagnostic PCR.

Community-acquired methicillin-resistant Staphylococcus aureus in a rural American Indian community.

CONTEXT: Until recently, methicillin-resistant Staphylococcus aureus (MRSA) infections have been acquired primarily in nosocomial settings. Four recent deaths due to MRSA infection in previously healthy children in the Midwest suggest that serious MRSA infections can be acquired in the community in rural as well as urban locations. OBJECTIVES: To document the occurrence of community-acquired MRSA infections and evaluate risk factors for community-acquired MRSA infection compared with methicillin-susceptible S aureus (MSSA) infection. DESIGN: Retrospective cohort study with medical record review. SETTING: Indian Health Service facility in a rural midwestern American Indian community. PATIENTS: Patients whose medical records indicated laboratory-confirmed S aureus infection diagnosed during 1997. MAIN OUTCOME MEASURES: Proportion of MRSA infections classified as community acquired based on standardized criteria; risk factors for community-acquired MRSA infection compared with those for community-acquired MSSA infection; and relatedness of MRSA strains, determined by pulsed-field gel electrophoresis (PFGE). RESULTS: Of 112 S aureus isolates, 62 (55%) were MRSA and 50 (45%) were MSSA. Forty-six (74%) of the 62 MRSA infections were classified as community acquired. Risk factors for community-acquired MRSA infections were not significantly different from those for community-acquired MSSA. Pulsed-field gel electrophoresis subtyping indicated that 34 (89%) of 38 community-acquired MRSA isolates were clonally related and distinct from nosocomial MRSA isolates found in the region. CONCLUSIONS: Community-acquired MRSA may have replaced community-acquired MSSA as the dominant strain in this community. Antimicrobial susceptibility patterns and PFGE subtyping support the finding that MRSA is circulating beyond nosocomial settings in this and possibly other rural US communities.

Epidemiology and clonality of community-acquired methicillin-resistant Staphylococcus aureus in Minnesota, 1996-1998.

Methicillin-resistant Staphylococcus aureus (MRSA) has emerged among patients in the general population who do not have established risk factors for MRSA. Records from 10 Minnesota health facilities were reviewed to identify cases of MRSA infection that occurred during 1996-1998 and to identify which cases were community acquired. Susceptibility testing and pulsed-field gel electrophoresis (PFGE) subtyping were performed on available isolates. A total of 354 patients (median age, 16 years) with community-acquired MRSA (CAMRSA) infection were identified. Most case patients (299 [84%]) had skin infections, and 103 (29%) were hospitalized. More than 90% of isolates were susceptible to all antimicrobial agents tested, with the exception of beta-lactams and erythromycin. Of 334 patients treated with antimicrobial agents, 282 (84%) initially were treated with agents to which their isolates were nonsusceptible. Of 174 Minnesota isolates tested, 150 (86%) belonged to 1 PFGE clonal group. CAMRSA infections were identified throughout Minnesota; although most isolates were genetically related and susceptible to multiple antimicrobials, they were generally nonsusceptible to initial empirical therapy.

Use of molecular subtyping in surveillance for Salmonella enterica serotype typhimurium.

BACKGROUND: Because Salmonella enterica serotype typhimurium is the most common serotype isolated from persons with salmonellosis in the United States, it is difficult to detect unusual clusters or outbreaks. To determine whether molecular subtyping could be useful in public health surveillance for S. enterica serotype typhimurium, the Minnesota Department of Health initiated the routine use of pulsed-field gel electrophoresis (PFGE) of isolates. METHODS: Beginning in 1994, all S. enterica serotype typhimurium isolates submitted by clinical laboratories to the Department of Health were subtyped by PFGE. A standard questionnaire was used to interview patients about possible sources of infection. RESULTS: From 1994 through 1998, 998 cases of infection with S. enterica serotype typhimurium were reported to the Minnesota Department of Health (4.4 cases per 100,000 person-years). PFGE was performed on 958 of the isolates (96 percent), and 174 different patterns were identified. Sixteen outbreaks with a common source were identified, accounting for 154 cases. PFGE subtyping made it possible to confirm 10 outbreaks that involved small numbers of cases in institutional settings. Of six larger, community-based outbreaks, four would probably not have been recognized without PFGE subtyping. These four outbreaks accounted for 96 of the 154 culture-confirmed outbreak cases (62 percent). Fifty-six of 209 isolates tested for antimicrobial susceptibility (27 percent) were resistant to at least five antimicrobial agents. The multidrug-resistant isolates identified had unique PFGE patterns. CONCLUSIONS: Routine molecular subtyping of S. enterica serotype typhimurium by PFGE can improve the detection of outbreaks and aid in the identification of multidrug-resistant strains. Combining routine molecular subtyping with a method of rapid communication among public health authorities can improve surveillance for S. enterica serotype typhimurium infections.

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.

Surveillance for Escherichia coli O157:H7 infections in Minnesota by molecular subtyping.

BACKGROUND: Escherichia coli O157:H7 is a leading cause of diarrhea and the hemolytic-uremic syndrome. Current public health surveillance for E. coli O157:H7 requires considerable resources; traditional methods lack the sensitivity and specificity to detect outbreaks effectively. METHODS: During 1994 and 1995, the Minnesota Department of Health requested that all clinical isolates of E. coli O157:H7 be submitted to our laboratory. Isolates were subtyped by pulsed-field gel electrophoresis (PFGE), and patients were interviewed about potential sources of infection. RESULTS: In 1994 and 1995, 344 cases of E. coli O157:H7 infection were reported to the Minnesota Department of Health; 317 (92 percent) were subtyped by PFGE, and 143 distinct PFGE patterns were identified. Ten outbreaks of E. coli O157:H7 were identified; these accounted for 56 (18 percent) of the 317 subtyped cases. Four outbreaks were detected solely as a result of subtype-specific surveillance. In 11 two-week periods, the number of reported cases of E. coli O157:H7 doubled from the previous two weeks. In eight of these instances, the patterns identified were dissimilar and there were no outbreaks. Two of the remaining three increases resulted from multiple simultaneous outbreaks. CONCLUSIONS: Subtype-specific surveillance for E. coli O157:H7 can identify outbreaks that are not detected by traditional methods and can ascertain whether sudden increases in reported cases are due to sporadic isolated cases or to one or more outbreaks.