Evolutionary dynamics and genomic features of the Elizabethkingia anophelis 2015 to 2016 Wisconsin outbreak strain.

An atypically large outbreak of Elizabethkingia anophelis infections occurred in Wisconsin. Here we show that it was caused by a single strain with thirteen characteristic genomic regions. Strikingly, the outbreak isolates show an accelerated evolutionary rate and an atypical mutational spectrum. Six phylogenetic sub-clusters with distinctive temporal and geographic dynamics are revealed, and their last common ancestor existed approximately one year before the first recognized human infection. Unlike other E. anophelis, the outbreak strain had a disrupted DNA repair mutY gene caused by insertion of an integrative and conjugative element. This genomic change probably contributed to the high evolutionary rate of the outbreak strain and may have increased its adaptability, as many mutations in protein-coding genes occurred during the outbreak. This unique discovery of an outbreak caused by a naturally occurring mutator bacterial pathogen provides a dramatic example of the potential impact of pathogen evolutionary dynamics on infectious disease epidemiology.

Community Laboratory Testing for Cryptosporidium: Multicenter Study Retesting Public Health Surveillance Stool Samples Positive for Cryptosporidium by Rapid Cartridge Assay with Direct Fluorescent Antibody Testing.

Cryptosporidium is a common cause of sporadic diarrheal disease and outbreaks in the United States. Increasingly, immunochromatography-based rapid cartridge assays (RCAs) are providing community laboratories with a quick cryptosporidiosis diagnostic method. In the current study, the Centers for Disease Control and Prevention (CDC), the Association of Public Health Laboratories (APHL), and four state health departments evaluated RCA-positive samples obtained during routine Cryptosporidium testing. All samples underwent "head to head" re-testing using both RCA and direct fluorescence assay (DFA). Community level results from three sites indicated that 54.4% (166/305) of Meridian ImmunoCard STAT! positives and 87.0% (67/77) of Remel Xpect positives were confirmed by DFA. When samples were retested by RCA at state laboratories and compared with DFA, 83.3% (155/186) of Meridian ImmunoCard STAT! positives and 95.2% (60/63) of Remel Xpect positives were confirmed. The percentage of confirmed community results varied by site: Minnesota, 39.0%; New York, 63.9%; and Wisconsin, 72.1%. The percentage of confirmed community results decreased with patient age; 12.5% of community positive tests could be confirmed by DFA for patients 60 years of age or older. The percentage of confirmed results did not differ significantly by sex, storage temperature, time between sample collection and testing, or season. Findings from this study demonstrate a lower confirmation rate of community RCA positives when compared to RCA positives identified at state laboratories. Elucidating the causes of decreased test performance in order to improve overall community laboratory performance of these tests is critical for understanding the epidemiology of cryptosporidiosis in the United States (US).

Carbapenem-Resistant Enterobacteriaceae Transmission in Health Care Facilities - Wisconsin, February-May 2015.

Carbapenem-resistant Enterobacteriaceae (CRE) are multidrug-resistant gram-negative bacilli that can cause infections associated with high case fatality rates, and are emerging as epidemiologically important health care-associated pathogens in the United States (1). Prevention of CRE transmission in health care settings is dependent on recognition of cases, isolation of colonized and infected patients, effective use of infection control measures, and the correct use of antibiotics. The use of molecular technologies, including polymerase chain reaction (PCR) testing, pulsed-field gel electrophoresis (PFGE), and whole genome sequencing (WGS), can lead to detection of transmission events and interruption of transmission. In Wisconsin, acute care and critical access hospitals report laboratory-identified CRE to the Wisconsin Division of Public Health (WDPH), and clinical laboratories submit CRE isolates to the Wisconsin State Laboratory of Hygiene (WSLH) for molecular testing. During February-May 2015, a total of 49 CRE isolates from 46 patients were submitted to WSLH. On June 8, WSLH informed WDPH of five carbapenemase-producing CRE isolates with closely related PFGE patterns identified among four inpatients at two hospitals in southeastern Wisconsin. An investigation revealed a high degree of genetic relatedness among the patients' isolates, but did not identify the mechanism of transmission between the two facilities. No breaches in recommended practices were identified; after reviewing respiratory care procedures, no further cases were identified. Routine hospital- and laboratory-based surveillance can detect and prevent health care transmission of CRE.

An investigation of antibiotic susceptibility to empiric therapy for community-associated methicillin-resistant Staphylococcus aureus.

OBJECTIVE: To analyze antibiotic susceptibility patterns of community-associated methicillin-resistant Staphylococcus aureus (MRSA) isolates obtained from skin and soft tissue infections among Wisconsin outpatients. DESIGN: Retrospective genotype testing. SETTING: Isolates were forwarded to the Wisconsin State Laboratory of Hygiene and Marshfield Labs from clinical laboratories throughout Wisconsin. METHODS: MRSA isolates submitted during April, 2010-February, 2012 underwent genotype analysis using pulsed-field gel electrophoresis. Antibiotic susceptibility patterns were determined for all isolates identified by electrophoresis subtyping as strain type USA300, and pattern comparisons were made by public health region. RESULTS: Among 835 MRSA isolates submitted, 217 (26%) were genotyped. Of these, 152 (70%) were USA300 MRSA. Among the 152 USA300 isolates, 95% were susceptible to clindamycin and 99% were susceptible to tetracycline and trimethoprim-sulfamethoxazole. The proportion of clindamycin-susceptible isolates from the southern region was significantly lower when compared to the other 4 regions combined (P = 0.03). One southern region clindamycin-resistant isolate was also resistant to trimethoprim-sulfamethoxazole. CONCLUSIONS: USA300 MRSA was the predominant strain isolated from outpatient skin and soft tissue sites. Antibiotic susceptibility patterns among Wisconsin USA300 MRSA isolates are similar to patterns found in national studies. Local providers should continue to follow national practice guidelines for treatment of outpatient skin infections. A cluster of 4 clindamycin-resistant isolates and 1 trimethoprim-sulfamethoxazole resistant isolate was detected in the southern region, warranting continued surveillance for antibiotic resistance among community-associated MRSA isolates.

Multistate outbreak of Salmonella enterica serotype enteritidis infection associated with pet guinea pigs.

Salmonella causes about one million illnesses annually in the United States. Although most infections result from foodborne exposures, animal contact is an important mode of transmission. We investigated a case of Salmonella enterica serotype Enteritidis (SE) sternal osteomyelitis in a previously healthy child who cared for two recently deceased guinea pigs (GPs). A case was defined as SE pulsed-field gel electrophoresis (PFGE) XbaI pattern JEGX01.0021, BlnI pattern JEGA26.0002 (outbreak strain) infection occurring during 2010 in a patient who reported GP exposure. To locate outbreak strain isolates, PulseNet and the US Department of Agriculture National Veterinary Service Laboratories (NVSL) databases were queried. Outbreak strain isolates underwent multilocus variable-number tandem repeat analysis (MLVA). Traceback and environmental investigations were conducted at homes, stores, and breeder or broker facilities. We detected 10 cases among residents of eight states and four NVSL GP outbreak strain isolates. One patient was hospitalized; none died. The median patient age was 9.5 (range, 1-61) years. Among 10 patients, two purchased GPs at independent stores, and three purchased GPs at different national retail chain (chain A) store locations; three were chain A employees and two reported GP exposures of unknown characterization. MLVA revealed four related patterns. Tracebacks identified four distributors and 92 sources supplying GPs to chain A, including one breeder potentially supplying GPs to all case-associated chain A stores. All environmental samples were Salmonella culture-negative. A definitive SE-contaminated environmental source was not identified. Because GPs can harbor Salmonella, consumers and pet industry personnel should be educated regarding risks.

The role, challenges, and support of pulsenet laboratories in detecting foodborne disease outbreaks.

In recent years, there have been several high-profile nationwide foodborne outbreaks due to enteric organisms in food products, including Salmonella Typhimurium in peanut products, Salmonella Saintpaul in peppers, and Escherichia coli O157:H7 in spinach. PulseNet, the national molecular subtyping network for foodborne disease surveillance, played a key role in detecting each of these outbreaks. PulseNet laboratories use bacterial subtyping methods to rapidly detect clusters of foodborne disease, which are often the first indication that an outbreak is occurring. Rapid outbreak detection reduces ongoing transmission through product recalls, restaurant closures, and other mechanisms. By greatly increasing the sensitivity of outbreak detection, PulseNet allows us to identify and correct problems with our food production and distribution systems that would not otherwise have come to our attention. Annually, millions of potentially preventable cases of foodborne illness result in billions of dollars in lost productivity and health-care expenses. We describe the critical role PulseNet laboratories play in the detection of foodborne outbreaks and discuss current challenges and potential improvements for PulseNet laboratories to more rapidly identify future foodborne outbreaks.

Multistate outbreak of Escherichia coli O157:H7 infection associated with consumption of packaged spinach, August-September 2006: the Wisconsin investigation.

BACKGROUND: Escherichia coli O157:H7 infection often causes hemorrhagic colitis and hemolytic uremic syndrome. METHODS: In 2006, the Wisconsin Division of Public Health and the Wisconsin State Laboratory of Hygiene, in cooperation with other local, state, and federal partners, investigated an outbreak of E. coli O157:H7 infection. RESULTS: In September 2006, the Wisconsin Division of Public Health and the Wisconsin State Laboratory of Hygiene were able to link geographically dispersed E. coli O157:H7 isolates recovered from the stool samples of ill persons, all of which had the same pulsed-field gel electrophoresis pattern (i.e., outbreak pattern). Investigators conducted a case-control study with control subjects (n = 86) matched to case patients (n = 49) by age, sex, and residential location. All case patients' onsets of illness occurred during the period from 20 August through 14 September 2006. Illness was associated with spinach consumption (matched odds ratio, 82.1; 95% confidence interval, 14.7 to >1000). Of the 49 case patients, 26 (53%) recalled eating brand A spinach. On multibrand analysis, only brand A was associated with illness (undefined matched odds ratio; 95% confidence interval, 6.8-infinity). Wisconsin's agriculture laboratory isolated E. coli O157:H7 with the outbreak pattern from spinach in 2 brand A packages, both produced on 15 August 2006. CONCLUSIONS: The rapid multijurisdictional epidemiologic and laboratory response, including timely pulsed-field gel electrophoresis pattern analysis and PulseNet posting, facilitated prompt voluntary recall of brand A spinach.

Comparison of cefoxitin and oxacillin disk diffusion methods for detection of mecA-mediated resistance in Staphylococcus aureus in a large-scale study.

The recommended breakpoints for the cefoxitin disk diffusion test for Staphylococcus aureus were recently modified. In this large-sample study, cefoxitin sensitivity and specificity compared to those of oxacillin were 97.3% and 100%, respectively. This study validated the new cefoxitin breakpoints for the detection of mecA-mediated resistance in S. aureus.

A countywide outbreak of pertussis: initial transmission in a high school weight room with subsequent substantial impact on adolescents and adults.

OBJECTIVE: To describe a large communitywide pertussis outbreak where aggressive diagnostic and treatment measures were used to control the outbreak. DESIGN: Retrospective analysis, May 2003 through February 2004. SETTING: Fond du Lac County, Wisconsin (population 98,882). PARTICIPANTS: Health department personnel conducted case and contact investigations of suspected outbreak-associated illnesses using standard pertussis reporting forms and clinical evaluation and management protocols. Persons with compatible illness were tested for Bordetella pertussis using culture and for B pertussis DNA using polymerase chain reaction. Cases were classified using Council of State and Territorial Epidemiologists definitions. INTERVENTIONS: Health alerts and aggressive testing and treatment of suspected cases of pertussis illness and contact prophylaxis in the community. MAIN OUTCOME MEASURES: Incidences by age, onsets over time, and vaccine coverage in case patients. RESULTS: We identified 261 pertussis cases among county residents; 149 (57%) were laboratory confirmed. Of the first 57 case patients, 47% reported using a particular high school weight room. Pertussis incidence was high in all age groups; 86% of case patients were 10 years or older. Among 156 case patients with reported vaccination histories, 84% had received 5 or more doses of pertussis-containing vaccine. Adults reported significantly more severe pertussis symptoms than adolescents. CONCLUSIONS: Pertussis transmission among adolescents using a school weight room instigated a countywide outbreak with substantial incidence and morbidity among adolescents and adults. Aggressive testing and treatment in the outbreak response likely contributed to a sharp reduction in cases. This labor- and resource-intensive outbreak highlights potential benefits of pertussis booster vaccination among adolescent and adult populations.

Evaluation of polymerase chain reaction and culture for diagnosis of pertussis in the control of a county-wide outbreak focused among adolescents and adults.

During a large pertussis outbreak, culture and polymerase chain reaction (PCR) were used to identify 149 case patients; of these case patients, 79 had positive PCR and culture results, 59 had positive PCR results and negative culture results, 11 had negative PCR results and positive culture results (10 PCR-negative, culture-positive specimens were collected < or = 14 days after illness onset). PCR and culture of samples obtained < or = 2 weeks after illness onset and PCR of samples obtained > 2 weeks after illness onset proved to be most diagnostically useful.

Human multidrug-resistant Salmonella Newport infections, Wisconsin, 2003-2005.

We conducted a retrospective study of Salmonella Newport infections among Wisconsin residents during 2003-2005. Multidrug resistance prevalence was substantially greater in Wisconsin than elsewhere in the United States. Persons with multidrug-resistant infections were more likely than persons with susceptible infections to report exposure to cattle, farms, and unpasteurized milk.

Evidence supporting zoonotic transmission of Cryptosporidium spp. in Wisconsin.

Cryptosporidium hominis and Cryptosporidium parvum are the primary species of Cryptosporidium that infect humans. C. hominis has an anthroponotic transmission cycle, while C. parvum is zoonotic, infecting cattle and other ruminants, in addition to humans. Most cryptosporidiosis outbreaks in the United States have been caused by C. hominis, and this species is often reported as the primary cause of cryptosporidiosis in this country. However, outbreaks account for only 10% of the overall cryptosporidiosis cases, and there are few data on the species that cause sporadic cases. The present study identified the species/genotypes and subgenotypes of Cryptosporidium in 49 cases of sporadic cryptosporidiosis in Wisconsin during the period from 2003 to 2005. The species/genotype of isolates was determined by PCR restriction fragment length polymorphism analysis of the 18S rRNA and Cryptosporidium oocyst wall protein genes. The C. parvum and C. hominis isolates were subgenotyped by sequence analysis of the GP60 gene. Forty-four of 49 isolates were identified as C. parvum, and 1 was identified as C. hominis. Of the remaining isolates, one was identified as being of the cervine genotype, one was identified as being a cervine genotype variant, and two were identified as being of a novel human genotype, previously reported as W17. Nine different subgenotypes were identified within the C. parvum species, and two of these were responsible for 60% of the cases. In this study we found that most sporadic cases of cryptosporidiosis in Wisconsin are caused by zoonotic Cryptosporidium species, indicating that zoonotic transmission could be more frequently associated with sporadic cases in the United States.

Meningococcal disease incidence and mortality in Wisconsin, 1993-2002.

Neisseria meningitidis is a major cause of sepsis and meningitis in children and young adults in the United States. To examine recent epidemiologic features of meningococcal disease in Wisconsin, we evaluated Wisconsin case surveillance data collected during 1993-2002. Surveillance data for cases with onsets during this time were analyzed; statistical trends were assessed. Mortality was examined with regard to age, sex, serogroup, college student status, and young adult status by unadjusted and adjusted analyses. During 1993-2002, 462 cases of meningococcal disease were reported in Wisconsin; 55% of case patients were aged < 19 years. The annual incidence was 0.9 cases per 100,000 persons per year, and incidence was highest among children aged <2 years. Two seasonal peaks in cases were observed during January-April and September-October. The annual mortality rate during the 10-year interval was 0.09 deaths per 100,000 persons per year. Adjusted analysis indicated that serogroup C infection, young adult, and college student status (but not sex) were associated with mortality. Meningococcal disease remains uncommon and sporadic in Wisconsin. Incidence and mortality rates are highest among young children, but young adults who acquire the disease appear to be at an increased mortality risk.

Evaluation of a two-step approach for large-scale, prospective genotyping of Mycobacterium tuberculosis isolates in the United States.

Genotyping of Mycobacterium tuberculosis isolates is useful in tuberculosis control for confirming suspected transmission links, identifying unsuspected transmission, and detecting or confirming possible false-positive cultures. The value is greatly increased by reducing the turnaround time from positive culture to genotyping result and by increasing the proportion of cases for which results are available. Although IS6110 fingerprinting provides the highest discrimination, amplification-based methods allow rapid, high-throughput processing and yield digital results that can be readily analyzed and thus are better suited for large-scale genotyping. M. tuberculosis isolates (n = 259) representing 99% of culture-positive cases of tuberculosis diagnosed in Wisconsin in the years 2000 to 2003 were genotyped by using spoligotyping, mycobacterial interspersed repetitive unit (MIRU) typing, and IS6110 fingerprinting. Spoligotyping clustered 64.1% of the isolates, MIRU typing clustered 46.7% of the isolates, and IS6110 fingerprinting clustered 29.7% of the isolates. The combination of spoligotyping and MIRU typing yielded 184 unique isolates and 26 clusters containing 75 isolates (29.0%). The addition of IS6110 fingerprinting reduced the number of clustered isolates to 30 (11.6%) if an exact pattern match was required or to 44 (17.0%) if the definition of a matching IS6110 fingerprint was expanded to include patterns that differed by the addition of a single band. Regardless of the genotyping method chosen, the addition of a second or third method decreased clustering. Our results indicate that using spoligotyping and MIRU typing together provides adequate discrimination in most cases. IS6110 fingerprinting can then be used as a secondary typing method to type the clustered isolates when additional discrimination is needed.

Transmission of Salmonella enterica serotype typhimurium DT104 to infants through mother's breast milk.

This study documents the first reported transmission of Salmonella enterica serotype Typhimurium definitive type 104 (DT104) to premature fraternal twins via their mother's breast milk. When premature twin neonates developed severe enteritis in the neonatal intensive care unit (NICU), stool samples and the mother's breast milk were cultured for the presence of Salmonella. Antibacterial susceptibility patterns were determined. Semiquantitative organism abundance data were retrospectively gathered on 54 stored breast milk samples collected on 34 different days using a rapid, real-time polymerase chain reaction (PCR) methodology (LightCycler PCR). Fecal samples from other infants in the NICU at that time were also tested. Pulsed-field gel electrophoresis (PFGE) was used to assess the genetic composition of the isolated organisms. The twins' neonatal stools and mother's breast milk cultures revealed a resistance pattern (R-type) to ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline. LightCycler PCR analysis of sequential breast milk samples confirmed this to be the likely source of transmission. In the subsequent outbreak investigation, none of the NICU surveillance fecal samples proved positive for this organism. The genetic composition of organisms isolated from the maternal breast milk was indistinguishable from those isolated from neonatal specimens as determined by PFGE. Antibiotic susceptibility tests coupled with PFGE patterns suggested that these Salmonella isolates were DT104. Because the prevalence of DT104 infections is rising in the United States, neonatologists should be aware of breast milk as a potential mode of transmission.