Stephen A. Kania

Evaluation of susceptibility test breakpoints used to predict mecA-mediated resistance in Staphylococcus pseudintermedius isolated from dogs.

Clinical and Laboratory Standards Institute interpretive breakpoints for in vitro susceptibility tests that predict mecA-mediated oxacillin resistance in Staphylococcus pseudintermedius isolates from animals have been changed twice in the past decade. Moreover, there are no counterpart recommendations for human isolates of S. pseudintermedius. Individual medical and veterinary laboratories variably use interpretive breakpoints identical to those recommended for use with Staphylococcus aureus or identical to those recommended for use with coagulase-negative staphylococci. The purpose of the current study was to examine correlations between oxacillin disk diffusion, oxacillin gradient diffusion, oxacillin microbroth dilution, and cefoxitin disk diffusion tests used to predict mecA-mediated resistance in S. pseudintermedius and to retrospectively estimate, from disk diffusion zone diameter measurements, the prevalence and rate of increase of oxacillin resistance among canine S. pseudintermedius isolates submitted to a veterinary teaching hospital laboratory. Oxacillin disk diffusion zone diameters of ≤17 mm and oxacillin minimum inhibitory concentrations of ≥0.5 μg/ml were highly correlated with detection of mecA in canine S. pseudintermedius isolates by polymerase chain reaction. MecA-mediated resistance among S. pseudintermedius isolates from dogs increased from less than 5% in 2001 to near 30% in 2007. More than 90% of the methicillin-resistant S. pseudintermedius isolates in 2006 and 2007 were also resistant to representatives of ≥4 additional antimicrobial drug classes. Cefoxitin disk diffusion with the resistance breakpoint set at ≤24 mm significantly underestimated the presence of mecA in S. pseudintermedius.

Deletions in the 7a ORF of feline coronavirus associated with an epidemic of feline infectious peritonitis

Abstract A population of Persian cats experienced an epidemic of feline infectious peritonitis (FIP) over 2 years. Twelve cases of FIP occurred in litters born during this period. Cats contracting FIP were all genetically related through the sire. Feline coronavirus (FCoV) genomic RNA was detected consistently in this study in biologic samples from adult cats, kittens suffering from FIP, and their siblings. Analysis of viral 7a/7b open reading frame (ORFs) were analyzed and revealed two distinct virus variants circulating in the population, one with an intact 7a ORF and one with two major deletions in the 7a ORF. The 7b ORFs were intact and similar among all virus isolates, although point mutations resulting in amino acid changes were present. The sire was determined to be infected with both variants, and was persistently virus-infected. We speculate the deletion variant arose from the non-deletion variant during viral replication in this population, possibly in the sire.

Identification of a predominant multilocus sequence type, pulsed-field gel electrophoresis cluster, and novel staphylococcal chromosomal cassette in clinical isolates of mecA-containing, methicillin-resistant Staphylococcus pseudintermedius

Methicillin resistance encoded by the mecA gene is increasingly observed in Staphylococcus pseudintermedius. Little is known about the population genetics of veterinary staphylococci bearing methicillin resistance. The aim of this study was to determine the relatedness of resistant bacteria and to compare them with methicillin-susceptible isolates. Multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) fragment profiling were performed on methicillin-resistant S. pseudintermedius (MRSP) and methicillin-susceptible S. pseudintermedius (MSSP) isolates obtained from canine samples submitted to the veterinary teaching hospital bacteriology service between 2006 and 2008. Multilocus sequence typing detected 20 different sequence types, 16 of which were not previously described. Methicillin-resistant isolates were predominantly ST 68, possessed the Staphylococcus aureus-associated staphylococcal chromosomal cassette mec (SCCmec) type V(T) and fell within the largest PFGE cluster; whereas methicillin-susceptible strains were more genetically diverse. This suggests that most methicillin resistance within the population of isolates tested originated from a single source which has persisted and expanded for several years.

Role of chromodomain helicase DNA-binding protein 2 in DNA damage response signaling and tumorigenesis

The chromodomain helicase DNA-binding proteins (CHDs) are known to affect transcription through their ability to remodel chromatin and modulate histone deacetylation. In an effort to understand the functional role of the CHD2 in mammals, we have generated a Chd2 mutant mouse model. Remarkably, the Chd2 protein appears to play a critical role in the development, hematopoiesis and tumor suppression. The Chd2 heterozygous mutant mice exhibit increased extramedullary hematopoiesis and susceptibility to lymphomas. At the cellular level, Chd2 mutants are defective in hematopoietic stem cell differentiation, accumulate higher levels of the chromatin-associated DNA damage response mediator, γH2AX, and exhibit an aberrant DNA damage response after X-ray irradiation. Our data suggest a direct role for the chromatin remodeling protein in DNA damage signaling and genome stability maintenance.

Multilocus Sequence Typing for Characterization of Staphylococcus pseudintermedius

ABSTRACT Staphylococcus pseudintermedius is an opportunistic pathogen in dogs. Four housekeeping genes with allelic polymorphisms were identified and used to develop an expanded multilocus sequence typing (MLST) scheme. The new seven-locus technique shows S. pseudintermedius to have greater genetic diversity than previous methods and discriminates more isolates based upon host origin.

Risk of colonization or gene transfer to owners of dogs with meticillin‐resistant Staphylococcus pseudintermedius

To determine the zoonotic risk from meticillin-resistant staphylococcal species or transfer of resistance genes between dogs with pyoderma and their owners, 25 dog-owner pairs were studied. Cultures were obtained from the dogs lesions and the owners nasal cavity on the initial visit. Staphylococcus isolates were identified and antimicrobial susceptibility tests were performed by the Kirby-Bauer disk diffusion method. Presence of the mecA gene was determined by PCR. Staphylococcal Cassette Chromosome (SCCmec) typing was performed by multiplex PCR. Eighteen dogs had a meticillin-resistant staphylococcal species, with meticillin-resistant S. pseudintermedius (MRSP) isolated from 15 dogs. MRSP was isolated from two owners of dogs with MRSP skin infections. Both organisms had the same susceptibility pattern and SCCmec type. MRSP was not isolated from the owners after treating both dogs for 1 month. At least one coagulase-negative Staphylococcus spp. (CoNS) was isolated from each owner, with meticillin resistance found in 16 (64%) of the isolates. The mecA gene was identified in all but two of the meticillin-resistant Staphylococcus spp. Multiplex PCR identified SCCmec type V in all MRSP. The mecA gene-possessing CoNS isolates from owners contained either SCCmec type IVa or IVc. In conclusion, MRSP colonization of owners appeared to be uncommon and transient. Human nasal carriage of meticillin-resistant CoNS was common, but the SCCmec types were different from those in the canine MRSP isolates. Owners do not appear to be at great risk of zoonotic transfer of organisms or antimicrobial resistance genes from dogs with MRSP infections, but the findings should be confirmed with a much larger cohort.

Comparison of Tests To Detect Oxacillin Resistance in Staphylococcus intermedius, Staphylococcus schleiferi, and Staphylococcus aureus Isolates from Canine Hosts

ABSTRACT Multiple tests were compared to the reference standard PBP2a latex agglutination test for detection of mecA-mediated oxacillin resistance in canine staphylococci. Cefoxitin disk diffusion, using breakpoints for human isolates of coagulase-negative Staphylococcus spp., had low sensitivity for detection of oxacillin resistance in members of the Staphylococcus intermedius group.

Binding and neutralization of bacterial lipopolysaccharide by colistin nonapeptide.

Polymyxin nonapeptides, proteolytic derivatives of polymyxin antibiotics, are less toxic than their parent compounds but retain some of their antibacterial activities. To confirm and expand observations that polymyxin nonapeptides have anti-endotoxin activity, we studied the ability of colistin nonapeptide to bind to bacterial lipopolysaccharide (LPS) and to inhibit the effects of LPS on Limulus amoebocyte lysate and lymphocyte mitogenicity. Colistin nonapeptide was purified by high-pressure liquid chromatography and was demonstrated to bind to LPS by equilibrium dialysis. The ability of colistin nonapeptide to render E. coli ATCC 25922 cells sensitive to erythromycin was abrogated by 50% after incubation with E. coli O18 LPS in a ratio by weight of LPS to colistin nonapeptide of 3.9:1. The presence of 4 micrograms of colistin nonapeptide or colistin per ml increased by 130- and 800-fold, respectively, the concentration of E. coli O113 LPS required to produce 50% gelation of Limulus amoebocyte lysate as measured by a spectrophotometric assay. Neutralization of LPS by colistin nonapeptide was time and concentration dependent. In contrast to the neutralization seen with LPS derived from a colistin-sensitive organism, colistin nonapeptide neutralized very little LPS extracted from a strain of Serratia marcescens that was resistant to colistin. Colistin nonapeptide also inhibited LPS-induced [3H]thymidine uptake by splenic lymphocytes, but its activity was less than 1/10 that of colistin. We conclude that colistin nonapeptide binds to LPS and possesses antiendotoxin activity. However, the anti-endotoxin activity of the nonapeptide is considerably less than that of its parent compound, colistin.

Seroprevalence of Ehrlichia canis, Ehrlichia chaffeensis and Ehrlichia ewingii in dogs in North America

BackgroundThis study evaluated the exposure of dogs to three different Ehrlichia spp. in the south and central regions of the United States where vector-borne disease prevalence has been previously difficult to ascertain, particularly beyond the metropolitan areas.MethodsDog blood samples (n = 8,662) were submitted from 14 veterinary colleges, 6 private veterinary practices and 4 diagnostic laboratories across this region. Samples were tested for E. canis, E. chaffeensis and E. ewingii specific antibodies using peptide microtiter ELISAs.ResultsOverall, E. canis, E. chaffeensis and E. ewingii seroprevalence was 0.8%, 2.8%, and 5.1%, respectively. The highest E. canis seroprevalence (2.3%) was found in a region encompassing Arkansas, Louisiana, Oklahoma, Tennessee and Texas. E. chaffeensis seroreactivity was 6.6% in the central region (Arkansas, Kansas, Missouri, and Oklahoma) and 4.6% in the southeast region (Georgia, Maryland, North Carolina, South Carolina, Tennessee and Virginia). Seroreactivity to E. ewingii was also highest in the central region (14.6%) followed by the southeast region (5.9%). The geospatial pattern derived from E. chaffeensis and E. ewingii seropositive samples was similar to previous reports based on E. chaffeensis seroreactivity in white-tailed deer and the distribution of human monocytic ehrlichiosis (HME) cases reported by the CDC.ConclusionsThe results of this study provide the first large scale regional documentation of exposure to E. canis, E. chaffeensis and E. ewingii in pet dogs, highlighting regional differences in seroprevalence and providing the basis for heightened awareness of these emerging vector-borne pathogens by veterinarians and public health agencies.

NEOSPORA CANINUM AND TOXOPLASMA GONDII ANTIBODY PREVALENCE IN ALASKA WILDLIFE

Free-ranging caribou and moose populations in some regions of Alaska undergo periodic declines in numbers. Caribou and moose are managed by the state as valuable resources for not only sustenance and subsistence, but also for cultural heritage. Incidence and prevalence of diseases that may impact herd health and recruitment from year to year are relevant to management decisions aimed to protect the long-term viability of these herds. Neospora caninum and Toxoplasma gondii are two apicomplexan parasites that can cause neurologic disease and abortions in their intermediate hosts and less frequently cause disease in their definitive hosts. The definitive hosts of N. caninum and T. gondii are canids and felids, respectively, and prevalence in the environment is in part dependent on maintenance of the life cycle through the definitive hosts. Serum samples from caribou (Rangifer tarandus, n=453), wolf (Canis lupus, n=324), moose (Alces alces, n=201), black-tailed deer (Odocoileus hemionus, n=55), coyote (Canis latrans, n=12), and fox (Vulpes vulpes, n=9) collected in Alaska were assayed for N. caninum– and T. gondii–reactive antibodies with an immunofluorescent antibody test (IFAT) and a modified agglutination test (MAT), respectively. Seroprevalence of N. caninum was greater in caribou (11.5%) than in wolves (9.0%), moose (0.5%), or black-tailed deer (0%). Seroprevalence of T. gondii was greater in wolves (17.8%) than in caribou (0.4%), moose (0%), or black-tailed deer (0%). Seroprevalence of N. caninum and T. gondii were 16.7% and 0.0% in coyotes and 0.0% and 12.5% in fox, but small sample sizes prevented further analysis. Antibodies to N. caninum in young caribou compared to adult caribou suggest that vertical transmission may be an important component of new infections in Alaskan caribou. The spatial distribution of antibody-positive individuals across Alaska may reflect differences in frequency of definitive hosts and alteration of predation patterns among regions.