A geospatial approach to identify patterns of antibiotic susceptibility at a neighborhood level in Wisconsin, United States.

The global threat of antimicrobial resistance (AMR) varies regionally. This study explores whether geospatial analysis and data visualization methods detect both clinically and statistically significant variations in antibiotic susceptibility rates at a neighborhood level. This observational multicenter geospatial study collected 10 years of patient-level antibiotic susceptibility data and patient addresses from three regionally distinct Wisconsin health systems (UW Health, Fort HealthCare, Marshfield Clinic Health System [MCHS]). We included the initial Escherichia coli isolate per patient per year per sample source with a patient address in Wisconsin (N = 100,176). Isolates from U.S. Census Block Groups with less than 30 isolates were excluded (n = 13,709), resulting in 86,467 E. coli isolates. The primary study outcomes were the results of Moran's I spatial autocorrelation analyses to quantify antibiotic susceptibility as spatially dispersed, randomly distributed, or clustered by a range of - 1 to + 1, and the detection of statistically significant local hot (high susceptibility) and cold spots (low susceptibility) for variations in antibiotic susceptibility by U.S. Census Block Group. UW Health isolates collected represented greater isolate geographic density (n = 36,279 E. coli, 389 = blocks, 2009-2018), compared to Fort HealthCare (n = 5110 isolates, 48 = blocks, 2012-2018) and MCHS (45,078 isolates, 480 blocks, 2009-2018). Choropleth maps enabled a spatial AMR data visualization. A positive spatially-clustered pattern was identified from the UW Health data for ciprofloxacin (Moran's I = 0.096, p = 0.005) and trimethoprim/sulfamethoxazole susceptibility (Moran's I = 0.180, p < 0.001). Fort HealthCare and MCHS distributions were likely random. At the local level, we identified hot and cold spots at all three health systems (90%, 95%, and 99% CIs). AMR spatial clustering was observed in urban areas but not rural areas. Unique identification of AMR hot spots at the Block Group level provides a foundation for future analyses and hypotheses. Clinically meaningful differences in AMR could inform clinical decision support tools and warrants further investigation for informing therapy options.

Direct Tissue PCR and Genotyping for Species Identification in a Case of Laryngeal Blastomycosis.

Otolaryngologic manifestations of infection with Blastomyces species are extremely rare and restricted geographically to recognized endemic regions. Here, we describe a case of laryngeal blastomycosis that presented as slowly progressive dysphonia. While a preliminary diagnosis was made using routine histopathology, a species identification of Blastomyces dermatitidis was made using polymerase chain reaction amplification and rapid genotyping without the need for fungal culture. All symptoms resolved following 1 month of antifungal therapy. Rapid molecular differentiation of B dermatitidis from Blastomyces gilchristii provides important insights into pathogenesis given recent recognition of differences in clinical spectra.

Surveillance for multidrug resistant Escherichia coli carriage in cattle, dogs and humans reveals predominance of CMY-2, CTX-M-15 and CTX-M-9 groups of ß-lactamases.

Global spread of antimicrobial multidrug resistance (MDR) in human and veterinary medicine relies upon diagnostics, surveillance and stewardship to guide mitigation. Utilizing surveillance of fecal samples from our service area for detecting MDR Escherichia coli carriage in humans (2143), dogs (627), and cattle (130), we found isolates resistant to third/fourth generation cephems present in 3.7 %, 13.1 %, and 51.5 %, respectively. CMY-2, CTX-M-15-like and CTX-M9 group genes in descending order were predominant in all hosts and accounted for 83.3 % of non-wild-type gene targets. MDR carriage mirrored cephem non-susceptibility rates as published in annual antibiograms for humans and dogs; notably, no carbapenem-resistant carriage isolates were detected. Given the scale of MDR E. coli carriage in cattle (14X) and dogs (3.5X) compared to humans, bench-marking of the resistance gene pool by host species utilizing regional One Health surveillance may aid in assessing occupational and geographic risks for acquiring resistance and for monitoring of mitigation strategies.

Multicenter Clinical Evaluation of Modified Two-Tiered Testing Algorithms for Lyme Disease Using Zeus Scientific Commercial Assays.

Modified two-tiered testing (MTTT) algorithms for Lyme disease (LD), which involve the sequential use of orthogonal enzyme immunoassays (EIAs) without immunoblotting, are acceptable alternatives to standard two-tiered testing (STTT; EIA followed by immunoblots) provided the EIAs have been FDA-cleared for this intended use. We evaluated four Zeus Scientific LD EIAs used in two distinct MTTT algorithms for FDA review. MTTT 1 used a VlsE1/pepC10 polyvalent EIA followed by a whole-cell sonicate (WCS) polyvalent EIA. MTTT 2 used the same first-tier EIA followed by separate IgM and IgG WCS EIAs. In a retrospective phase, we compared each MTTT algorithm to STTT using archived samples from LD patients or control subjects. In a prospective phase, we used the same algorithms to analyze consecutive excess samples submitted for routine LD serology to three clinical laboratories. For the retrospective phase, MTTTs 1 and 2 were more sensitive (56% and 74%) than STTT (41%; P ≤ 0.03) among 61 patients with acute erythema migrans (EM). In LD patients with neuroborreliosis, carditis, or arthritis (n = 75), sensitivity was comparable between algorithms (96 to 100%; P = 1.0). Among 190 control subjects without past LD, all algorithms were highly and comparably specific (≥99%, P = 0.48). For the prospective phase, (n = 2,932), positive percent-agreement (PPA), negative percent-agreement (NPA), and overall agreement of MTTT 1 with STTT were 93%, 97.7% and 97.4% (kappa 0.80). MTTT 2 yielded higher PPA (98%) but lower NPA (96.1%) and overall agreement (96.2%, kappa 0.74; all P < 0.05). Compared with STTT, both MTTT algorithms provided increased sensitivity in EM patients, comparable sensitivity in later disease and non-inferior specificity.

Comparison of Two Commercial Matrix-Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) Systems for Identification of Nontuberculous Mycobacteria.

OBJECTIVES: This multicenter study's aim was to assess the performance of two commercially available matrix-assisted laser desorption/ionization time of flight mass spectrometry systems in identifying a challenge collection of clinically relevant nontuberculous mycobacteria (NTM). METHODS: NTM clinical isolates (n = 244) belonging to 23 species/subspecies were identified by gene sequencing and analyzed using Bruker Biotyper with Mycobacterial Library v5.0.0 and bioMérieux VITEK MS with v3.0 database. RESULTS: Using the Bruker or bioMérieux systems, 92% and 95% of NTM strains, respectively, were identified at least to the complex/group level; 62% and 57%, respectively, were identified to the highest taxonomic level. Differentiation between members of Mycobacterium abscessus, M fortuitum, M mucogenicum, M avium, and M terrae complexes/groups was problematic for both systems, as was identification of M chelonae for the Bruker system. CONCLUSIONS: Both systems identified most NTM isolates to the group/complex level, and many to the highest taxonomic level. Performance was comparable.

Beta-Hemolytic Nongroup A Streptococcal Pharyngitis in Children.

OBJECTIVE: To evaluate the epidemiology, clinical features, and antibiotic prescribing patterns for nongroup A streptococci (NGAS) in children. STUDY DESIGN: Throat cultures obtained for pharyngitis were assessed at a large community-based health system over 10 years. Epidemiologic and clinical features of children with NGAS were compared with children with group A Streptococcus (GAS) and negative cultures. Antibiotic prescribing patterns were evaluated. RESULTS: A total of 224 328 rapid streptococcal antigen tests and 116 578 throat cultures were performed. Clinical analysis was completed for 602 GAS-positive patients, 535 NGAS-positive patients, and 480 patients with negative cultures. Incidence of NGAS did not vary annually or by season but increased with age from 2% at ≤5 years to 7% at 18 years of age. Patients with NGAS were more likely than those with negative cultures to have tonsillar exudate (20.3% vs 13.1%, P = .003) and enlarged tonsils (28.6% vs 19.3%, P < .001). Modified Centor scores did not differ between groups (score ≥2, P = 1.0; score ≥3, P = .50). Patients with GAS were more likely than those with NGAS to have fever (32.6% vs 24.5%, P = .003), palatal petechiae (14.0% vs 3.1%, P < .001), and modified Centor score ≥2 (47.8% vs 27.1%; P < .001). Of patients with NGAS, 65% were prescribed antibiotics. CONCLUSIONS: NGAS likely exist in both carriage and infectious states and incidence increases with age. Infections associated with NGAS are milder than with GAS, and complications are rare. Laboratory reporting of NGAS results in high antibiotic use, despite current recommendations against treatment.

Infectious Mononucleosis and Lyme Disease as Confounding Diagnoses: A Report of 2 Cases.

Lyme disease and infectious mononucleosis are common illnesses that share similar clinical presentations. Significant cross-reactivity is known to occur between Lyme and EBV serologic assays complicating the diagnosis. To date, no prior cases of concurrent acute Lyme and EBV infections have been reported. We describe the clinical presentation of two children with confirmed early Lyme disease and features suggestive of infectious mononucleosis, including one case of probable Lyme and EBV co-infection.

Development and Validation of a Serologic Test Panel for Detection of Powassan Virus Infection in U.S. Patients Residing in Regions Where Lyme Disease Is Endemic.

Powassan virus (POWV) is an emerging tick-borne arbovirus presenting a public health threat in North America. POWV lineage II, also known as deer tick virus, is the strain of the virus most frequently found in Ixodes scapularis ticks and is implicated in most cases of POWV encephalitis in the United States. Currently, no commercial tests are available to detect POWV exposure in tick-borne disease (TBD) patients. We describe here the development and analytical validation of a serologic test panel to detect POWV infections. The panel uses an indirect enzyme immunoassay (EIA) to screen. EIA-positive samples reflex to a laboratory-developed, POWV-specific immunofluorescence assay (IFA). The analytical sensitivity of the test panel was 89%, and the limit of detection was a plaque reduction neutralization test (PRNT) titer of 1:20. The analytical specificity was 100% for the IgM assay and 65% for the IgG assay when heterologous-flavivirus-positive samples were tested. On samples collected from regions where Lyme disease is endemic, seroprevalence for POWV in TBD samples was 9.4% (10 of 106) versus 2% when tested with non-TBD samples (2 of 100, P = 0.034). No evidence of POWV infection was seen in samples collected from a region where Lyme disease was not endemic (0 of 22). This test panel provides a sensitive and specific platform for detecting a serologic response to POWV early in the course of infection when neutralizing antibodies may not be detectable. Combined with clinical history, the panel is an effective tool for identifying acute POWV infection. IMPORTANCE Approximately 100 cases of POWV disease were reported in the United States over the past 10 years. Most cases have occurred in the Northeast (52) and Great Lakes (45) regions (https://www.cdc.gov/powassan/statistics.html). The prevalence of POWV in ticks and mammals is increasing, and POWV poses an increasing threat in a greater geographical range. In areas of the Northeast and Midwest where Lyme disease is endemic, POWV testing is recommended for patients with a recent tick bite, patients with Lyme disease who have been treated with antibiotics, or patients with a tick exposure who have tested negative for Lyme disease or other tick-borne illnesses and have persistent symptoms consistent with posttreatment Lyme disease. Testing could also benefit patients with tick exposure and unexplained neurologic symptoms and chronic fatigue syndrome (CFS) patients with known tick exposure. Until now, diagnostic testing for Powassan virus has not been commercially available and has been limited to patients presenting with severe, neurologic complications. The lack of routine testing for Powassan virus in patients with suspected tick-borne disease means that little information is available regarding the overall prevalence of the virus and the full spectrum of clinical symptoms associated with infection. As Ixodes scapularis is the tick vector for Powassan virus and multiple other tick-borne pathogens, including the Lyme disease bacterium, Borrelia burgdorferi, the clinical presentations and long-term outcomes of Powassan virus infection and concurrent infection with other tick-borne disease pathogens remain unknown.

The nasal microbiota of dairy farmers is more complex than oral microbiota, reflects occupational exposure, and provides competition for staphylococci.

Allergic and autoimmune diseases had been attributed to lack of exposure to biodiversity, an important factor in regulating immune homeostasis in a healthy host. We posit that the microbiome of healthy dairy farmers (DF) will be richer than non-farmers (NF) living in urban settings due to exposure to a greater biodiversity in the dairy environment. However, no studies have investigated the relationships between microbiota of dairy farmers (DF) compared with urban non-farmers (NF). We compared the nasal and oral microbiota of dairy farmers (N_DF, O_DF, respectively) with nasal and oral microbiota of NF in the same geographical area. The N_DF showed high microbial diversity with hundreds of unique genera that reflected environmental/occupational exposures. The nasal and oral microbiomes clustered separately from each other using Principal Coordinate Analysis, and with DF harboring two-fold and 1.5-fold greater exclusive genera in their nose and mouth respectively, than did non-farmers. Additionally, the N_DF group had a lower burden of Staphylococcus spp. suggesting a correlation between higher microbial diversity and competition for colonization by staphylococci. The N_DF samples were negative for the mecA gene, a marker of methicillin-resistance in staphylococci. The lower burden of staphylococci was found to be independent of the abundance of Corynebacterium spp. Exposure to greater biodiversity could enhance microbial competition, thereby reducing colonization with opportunistic pathogens. Future studies will analyze whether exposure to livestock microbiomes offers protection from acute and chronic diseases.

Risk Factors for Severe Infection, Hospitalization, and Prolonged Antimicrobial Therapy in Patients with Babesiosis.

Babesiosis is an emerging tick-borne disease transmitted by the hard tick Ixodes scapularis, which also transmits Lyme disease. Better gradation of prognostic indicators are needed to determine which patients may develop serious complications requiring hospitalization, and to provide early guidance on appropriate therapy. In this study, we evaluated 128 patients with smear or real time polymerase chain reaction-confirmed Babesia microti infections over a period of 16 years. Patients with asplenia or immunocompromising conditions were more likely to have severe infection (P < 0.01), require hospitalization (P < 0.01), or receive prolonged courses of antimicrobials (P < 0.01). Nausea or vomiting (P < 0.01) and diarrhea (P < 0.01) along with hyperbilirubinemia (P < 0.01) were predictive of severe infection, hospitalization, and prolonged antimicrobial therapy. Patients with concurrent Lyme disease were less likely to require hospitalization and had similar severity of disease and length of antibiotic treatment compared with those without Lyme disease.

Serologic Evidence of Powassan Virus Infection in Patients with Suspected Lyme Disease1.

Powassan virus (POWV) lineage II is an emerging tickborne flavivirus with an unknown seroprevalence in humans. In a Lyme disease-endemic area, we examined the seroreactivity to POWV in 2 patient cohorts and described the clinical features of the POWV-seroreactive patients. POWV disease might be less neuroinvasive than previously thought.

T2 Magnetic Resonance Assay-Based Direct Detection of Three Lyme Disease-Related Borrelia Species in Whole-Blood Samples.

In early Lyme disease (LD), serologic testing is insensitive and seroreactivity may reflect active or past infection. In this study, we evaluated a novel assay for the direct detection of three species of Borrelia spirochetes in whole blood. The T2 magnetic resonance (T2MR) assay platform was used to amplify Borrelia DNA released from intact spirochetes and to detect amplicon. Analytical sensitivity was determined from blood spiked with known concentrations of spirochetes, and the assay's limit of detection was found to be in the single-cell-per-milliliter range: 5 cells/ml for B. afzelii and 8 cells/ml for Borrelia burgdorferi and Borrelia garinii Clinical samples (n = 66) from confirmed or suspected early LD patients were also analyzed. B. burgdorferi was detected using T2MR in 2/2 (100%) of blood samples from patients with confirmed early LD, based on the presence of erythema migrans and documentation of seroconversion or a positive real-time blood PCR. T2MR detected B. burgdorferi in blood samples from 17/54 (31%) of patients with probable LD, based on the presence of erythema migrans without documented seroconversion or of documented seroconversion in patients with a compatible clinical syndrome but without erythema migrans. Out of 21 clinical samples tested by real-time PCR, only 1 was positive and 13 were negative with agreement with T2MR. An additional 7 samples that were negative by real-time PCR were positive with T2MR. Therefore, T2MR enables a low limit of detection (LoD) for Borrelia spp. in whole blood samples and is able to detect B. burgdorferi in clinical samples.

Pro-inflammatory immune responses are associated with clinical signs and symptoms of human anaplasmosis.

Human anaplasmosis (HA) is an emerging tick-borne disease that may present as a mild flu-like illness or a life threatening, sepsis-like condition. Although disease severity is hypothesized to relate to immunopathology and immune dysfunction in humans, studies to directly measure immune responses in infected humans have been very limited. We quantified cytokines in 80 confirmed HA patients using a multiplex chemiluminescence immunoassay system and compared similarly measured responses in 1000 control subjects. Pro-inflammatory cytokines were significantly elevated in HA patients (all seven p<0.0001). Interferon gamma (IFN-γ) concentrations were particularly high, with average concentrations 7.8 times higher in the HA patients than the controls. A subset of cytokines consisting of IL-1ß, IL-8, IL-6, TNF-α, and IL-10 was also coordinately high and significantly associated with severity of thrombocytopenia in HA patients. Patients with infections in the very acute stage (≤ 4 days ill) tended to have the highest IFN-γ, IL-12p70, and IL-2 levels. Higher concentrations of IL-13 and IL-5 were associated with diarrhea and vomiting. Our findings support a pathophysiological role for a pro-inflammatory response in HA, especially with regard to the modulation of hematopoiesis and subsequent hematopoietic complications.

Serologic screening of United States blood donors for Babesia microti using an investigational enzyme immunoassay.

BACKGROUND: The tick-borne pathogen Babesia microti has become recognized as the leading infectious risk associated with blood transfusion in the United States, yet no Food and Drug Administration-licensed screening tests are currently available to mitigate this risk. The aim of this study was to evaluate the performance of an investigational enzyme immunoassay (EIA) for B. microti as a screening test applied to endemic and nonendemic blood donor populations. STUDY DESIGN AND METHODS: The study aimed to test 20,000 blood donors from areas of the United States considered endemic for B. microti and 10,000 donors from a nonendemic area with the investigational B. microti EIA. Repeat-reactive samples were retested by polymerase chain reaction (PCR), blood smear, immunofluorescent assay (IFA), and immunoblot assay. In parallel, serum samples from symptomatic patients with confirmed babesiosis were tested by EIA, IFA, and immunoblot assays. RESULTS: A total of 38 of 13,757 (0.28%) of the donors from New York, 7 of 4583 (0.15%) from Minnesota, and 11 of 8363 (0.13%) from New Mexico were found repeat reactive by EIA. Nine of the 56 EIA repeat-reactive donors (eight from New York and one from Minnesota) were positive by PCR. The specificity of the assay in a nonendemic population was 99.93%. Among IFA-positive clinical babesiosis patients, the sensitivity of the assay was 91.1%. CONCLUSION: The B. microti EIA detected PCR-positive, potentially infectious blood donors in an endemic population and exhibited high specificity among uninfected and unexposed individuals. The EIA promises to provide an effective tool for blood donor screening for B. microti in a format amenable to high-throughput and cost-effective screening.

Surveillance of Wisconsin Antibacterial Susceptibility Patterns.

BACKGROUND: Antimicrobial resistance presents a threat to quality patient care. Knowledge of localantibacterial susceptibility patterns can guide clinicians in empiric antibacterial administration andassist pharmacists and infectious disease physicians in development of appropriate therapeutic pathways. METHODS: To characterize Wisconsin antibacterial susceptibility patterns and elucidate geographicor temporal variation in antibacterial resistance, a retrospective, observational analysis of antibiogram data was performed. Seventy-two members of the Wisconsin Clinical Laboratory Network(WCLN) submitted antibiograms describing clinically significant isolates tested in calendar year 2013 to the WCLN Laboratory Technical Advisory Group. RESULTS: In the context of commonly reported antibacterial agents, data were compiled for approximately 75,800 isolates of Escherichia coi; 13,300 Klebsiella pneumoniae; 6300 Proteus mirobilis;2800 Enterobacter cloacae; 8400 Pseudomonas aeruginosa; 30,000 S aureus; 11,200 coagulase-negative Staphylococcus spp; and 13,800 Enterococcus spp. P mirobilis isolates from northern Wisconsin were more likely to demonstrate resistance than those in the southern region. In contrast, P aeruginosa isolates from southern Wisconsin had decreased susceptibility to a number ofagents when compared to other regions. Temporal trending in decreased E coli and P mirabilis susceptibility to fluoroquinolones and trimethoprimsulfamethoxazole was observed. Increased methicillin-resistant Staphylococcus oureus (MRSA) rates were observed in northwest and southeastWisconsin. In general, northeast Wisconsin exhibited less frequency of antibacterial resistance. CONCLUSIONS: Geographic variation exists with respect to antibacterial resistance, particularly inareas of Wisconsin adjacent to large population centers of neighboring states. Antibacterial surveillance in Wisconsin is indicated on a regular basis to assess emerging trends in antibacterial resistance. Existing WCLN infrastructure allows for such investigations.

Human Infection with Ehrlichia muris-like Pathogen, United States, 2007-2013(1).

An Ehrlichia muris-like (EML) pathogen was detected among 4 patients in Minnesota and Wisconsin during 2009. We characterized additional cases clinically and epidemiologically. During 2004-2013, blood samples from 75,077 patients from all 50 United States were tested by PCR from the groEL gene for Ehrlichia spp. and Anaplasma phagocytophilum. During 2007-2013, samples from 69 (0.1%) patients were positive for the EML pathogen; patients were from 5 states: Indiana (1), Michigan (1), Minnesota (33), North Dakota (3), and Wisconsin (31). Most (64%) patients were male; median age was 63 (range 15-94) years; and all 69 patients reported likely tick exposure in Minnesota or Wisconsin. Fever, malaise, thrombocytopenia, and lymphopenia were the most common symptoms. Sixteen (23%) patients were hospitalized (median 4 days); all recovered, and 96% received doxycycline. Infection with the EML pathogen should be considered for persons reporting tick exposure in Minnesota or Wisconsin.

Appearances are Deceptive: Staphylococcus Superinfection of Clavicular Tuberculous Osteomyelitis.

A man, aged 25 years, presented with pain, swelling, and drainage from the right clavicular area. He had a past history of abscess at the sternoclavicular joint. The cultures from the drainage site grew methicillin-sensitive Staphylococcus aureus, and he was placed on appropriate antibiotics. As S. aureus infection of the clavicle is often secondary in nature, particularly in adults, a thorough workup was done to identify the underlying cause. Quantiferon gold, done as a part of the workup, came back positive, while the bone cultures grew S. aureus and Mycobacterium tuberculosis. He was placed on 9 months of combination therapy for tuberculosis osteomyelitis with a good clinical outcome.

A clinical, diagnostic, and ecologic perspective on human anaplasmosis in the Upper Midwest.

INTRODUCTION: Human anaplasmosis caused by the bacterial pathogen Anaplasma phagocytophilum was first discovered in the Upper Midwest in 1990. Since that time the number of cases in the region has steadily increased, such that today, the pathogen rivals that of Lyme disease in causing human tick-borne-related illness. OBJECTIVE: We provide an overview of the biology, clinical characteristics, and epidemiology of the disease in the Upper Midwest and discuss currently available diagnostic methods. FINDINGS: Rapid differentiation of anaplasmosis from other acute febrile illnesses and targeted treatment are important for preventing severe disease and potentially fatal outcomes in infected individuals. Beyond blood smear analysis and serology, the development of real-time polymerase chain reaction (PCR) assays for clinical use holds promise in improving our ability to make rapid diagnoses and to differentiate A phagocytophilum infections from those produced by closely related Ehrlichia pathogens, which are also present in the region. CONCLUSION: Continuing expansion of the range of the black-legged tick (Ixodes scapuloris), the principal vector of the disease, into areas heavily populated or visited by humans in the region likely will result in this pathogen becoming an even greater burden on human health. Efforts are needed to better characterize the current geographic distribution of human Anaplasma and Ehrlichia cases to identify emerging foci and to better understand the enzootic cycles that maintain the pathogens in the region. Improved diagnostics may assist with such efforts.

High diversity of plasmids harbouring blaCMY-2 among clinical Escherichia coli isolates from humans and companion animals in the upper Midwestern USA.

OBJECTIVES: To determine the population structure and genetic relatedness of plasmids encoding CMY-2 ß-lactamase in clinical Escherichia coli from humans and companion animals within a defined geographical area. METHODS: In total, 42 human and 73 companion animal isolates displaying an AmpC phenotype were isolated at a regional diagnostic reference laboratory in the upper Midwestern USA during 2009-11. Following PCR screening for transferable AmpC genes and plasmid transformation, blaCMY-2-positive plasmids were characterized by S1 nuclease PFGE, PCR-based replicon typing, antimicrobial susceptibility testing of transformants, conjugation experiments, plasmid multilocus sequence typing and restriction fragment length polymorphism. RESULTS: blaCMY-2 occurred in 6 (14%), 56 (86%) and 6 (75%) isolates from humans, dogs and cats, respectively. Usually plasmids carrying blaCMY-2 were conjugative (78%) and did not contain additional resistance genes (82%). The replicon types were IncI1 (52%), IncA/C (13%), IncFII (10%), IncI2 (5%), IncL/M (3%), IncB/O (2%) or non-typeable (15%). Related IncI1/ST12 plasmids were detected in one human and five canine isolates, while the remaining plasmids did not show similarity across host species. A novel epidemiological linkage of blaCMY-2 with IncL/M plasmids and a new CMY gene variant (blaCMY-108) were found in human isolates. CONCLUSIONS: This study is one of the first One Health attempts to compare plasmids encoding CMY-2 ß-lactamase among clinical isolates from humans and companion animals in the same region. The results indicate an unforeseen heterogeneity of plasmid backgrounds and suggest limited exchange between the two populations, in which blaCMY-2 occurred at very different frequencies and was harboured by distinct plasmid types.

Evidence of multiple virulence subtypes in nosocomial and community-associated MRSA genotypes in companion animals from the upper midwestern and northeastern United States.

OBJECTIVE: Not much is known about the zoonotic transmission of methicillin-resistant Staphylococcus aureus (MRSA) in companion animals in the United States. We report the rate of prevalence of S. aureus and MRSA recovered from clinical samples of animals requiring treatment at veterinary clinics throughout the upper midwestern and northeastern United States. DESIGN: We compared phenotypes, genotypes, and virulence profiles of the MRSA isolates identified in companion animals, such as cats, dogs, horses, and pigs, with typical human nosocomial and community-associated MRSA (CA-MRSA) genotypes to assess implied zoonotic transmission or zooanthroponosis. Five hundred thirty-three coagulase-positive staphylococci (CPS) isolates recovered between 2006 and 2008 from a variety of animal-source samples were screened for S. aureus by S. aureus-specific 16S rDNA primers and were screened for methicillin-resistance. All MRSA isolates were genotyped by pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and spa typing. They were also screened for common staphylococcal enterotoxin and adhesion genes by multiplex and singleplex PCR. RESULTS: Among the 533 CPS isolates recovered, 66 (12.4%) were determined to be S. aureus and 24 (4.5%) were MRSA. The percent of animals that were positive for S. aureus were as follows: 6.6% (32 of 487) dogs, 39.6% (19 of 48) cats, 83.3% (10 of 12) horses, and 100% of pigs, rabbits, hamsters and rats. Notably, 36.4% of all S. aureus identified were MRSA. Methicillin-resistant S. aureus was present in clinical samples from 12 of 487 dogs (2.5%), 6 of 48 cats (12.5%), 5 of 12 horses (42%), and 1 of 2 pigs (50%). The 24 MRSA isolates resolved into 4 PFGE clones: USA100 (50%), USA300 (16.7%), USA500 (20.8%) and USA800 (12.5%) and 6 sequence types (ST5, ST8, ST105, ST830, and ST986) or 2 clonal complexes, CC5 and CC8. Five major virulence profiles (clusters A to E) were observed in these MRSA isolates. Genotypic and virulence profiles of cats and dogs were more similar to each other than to those of horses. A Panton-Valentine leukocidin positive isolate with ST8:USA300 background was identified in a pig causing skin and soft infection. CONCLUSION: The presence of human MRSA clones in these animals suggests possible reverse zoonotic transmission. This study reports the first case of a USA300 genotype in a pig. Presence of multiple virulence profiles within a MRSA genotype in these animals suggests the potential of emergence of new MRSA clones by gaining or losing additional virulence genes.