Molecular and epidemiological characterization of canine Pseudomonas otitis using a prospective case-control study design.

BACKGROUND: Pseudomonas aeruginosa is an opportunistic pathogen of the canine ear canal and occupies aquatic habitats in the environment. Nosocomial and zoonotic transmission of P. aeruginosa have been documented, including clonal outbreaks. HYPOTHESIS/OBJECTIVES: The primary objective of this study was to assess various environmental exposures as potential risk factors for canine Pseudomonas otitis. It was hypothesized that isolates derived from infected ears would be clonal to isolates derived from household water sources and the mouths of human and animal companions of the study subjects. ANIMALS: Seventy seven privately owned dogs with otitis were enrolled, along with their human and animal household companions, in a case-control design. METHODS: Data on potential risk factors for Pseudomonas otitis were collected. Oral cavities of all study subjects, their human and animal companions, and household water sources were sampled. Pulsed field gel electrophoresis was used to estimate clonal relatedness of P. aeruginosa isolates. RESULTS: In a multivariate model, visiting a dog park was associated with 77% increased odds of case status (P = 0.048). Strains clonal to the infection isolates were obtained from subjects' mouths (n = 18), companion pets' mouths (n = 5), pet owners' mouths (n = 2), water bowls (n = 7) and water taps (n = 2). Clonally related P. aeruginosa isolates were obtained from dogs that had no clear epidemiological link. CONCLUSIONS AND CLINICAL IMPORTANCE: Genetic homology between otic and environmental isolates is consistent with a waterborne source for some dogs, and cross-contamination with other human and animal members within some households.

Genotypic relatedness and antimicrobial resistance of Staphylococcus schleiferi in clinical samples from dogs in different geographic regions of the United States.

BACKGROUND: Staphylococcus schleiferi is a known pathogen that can cause canine skin and ear infections. The aim of this study was to determine the molecular epidemiology and antimicrobial susceptibility of clinical veterinary isolates from different geographic regions in the United States. HYPOTHESIS: It was hypothesized that S. schleiferi would maintain genotypic homogeneity across the different geographic regions and that meticillin-resistant (MR) isolates of S. schleiferi would predominate. METHODS: Isolates were identified as S. schleiferi by a commercial microbiology identification system and confirmed by nuc gene PCR. Antibiotic susceptibility data were collected and PBP2a latex agglutination testing was performed on MR isolates. Pulsed-field gel electrophoresis (PFGE) was performed and clonal clusters were identified with a Dice coefficient similarity of >80%. RESULTS: There were 116 isolates from the Mid-Atlantic region and 101 from across the United States. Of these 217 isolates, 209 (96%) were obtained from cutaneous sites. Of the Mid-Atlantic isolates, 62% (72 of 116) were MR and 16% (18 of 116) were multidrug-resistant (MDR). Of the isolates from the other geographic regions, 73% (74 of 101) were MR and 24% (24 of 101) were MDR. All MR isolates were positive by PBP2a latex agglutination. PFGE identified 155 individual pulsed-field profiles and three major pulsed-field types (PFT) that contained 61% (133 of 217) of the isolates. These pulsed-field types were geographically heterogeneous. CONCLUSIONS: This study demonstrates the dissemination of successful MR pulsed-field types of S. schleiferi across the United States.

Detection of Viable Streptococcus equi equi Using Propidium Monoazide Polymerase Chain Reaction.

There is debate around the clinical significance of Streptococcus equi subsp. equi detection in low numbers using quantitative real-time PCR (qPCR). Propidium monoazide (PMA) qPCR has been used to differentiate DNA from viable and nonviable bacterial cells. The aim of this study was to evaluate the ability of PMA eqbE SEQ2190 triplex qPCR to differentiate DNA from viable and nonviable S. equi in positive and suspect positive clinical specimens. Fifty-seven stored (frozen and refrigerated) positive (36) or suspect positive (21) clinical specimens (determined via SeeI qPCR as the gold standard) were tested using eqbE SEQ2190 triplex qPCR with (+) and without (-) PMA pretreatment. Cycle thresholds were higher when using PMA indicating a mixture of heat killed and viable cells. Number of S. equi positive specimens were as follows: 6/57 eqbE + PMA, 13/57 eqbE -PMA (Chi- squared 3.1, p = .079); 10/57 SEQ2190 +PMA, 53/57 SEQ2190 -PMA (Chi- squared 65.6, p < .0001). The mean cycle thresholds were as follows: 23.88 eqbE -PMA, 29.89 eqbE + PMA (p = .04); 24.9 SEQ2190 -PMA, 31.9 SEQ2190 +PMA (p < .0001). PMA qPCR can be used to determine S. equi viability, but testing should be performed on fresh specimens.

Preparing for the Impact of COVID-19 on the Mental Health of Youth.

The COVID-19 pandemic is continuing to have long-term and global effects that the vaccine may not ease. Children and adolescents endured unprecedented periods of loneliness, social isolation, financial stressors, in-home conflicts, changes in living circumstances, and variable access to healthcare, resulting in increased mental health sequelae. Timely recognition of students' anxiety, depression, and disruptive behaviors will allow appropriate interventions to de-escalate these feelings and prevent suicidal ideations and attempts. As youth return to school, their mental health needs will not subside. School nurses and the multidisciplinary team have a vital role in impacting this population's already surging increase of mental and behavioral health disorders.

Genotypic relatedness and phenotypic characterization of Staphylococcus schleiferi subspecies in clinical samples from dogs.

OBJECTIVE: To assess the degree of biological similarity (on the basis of genotype determined via pulsed-field gel electrophoresis [PFGE]) between isolates of 2 Staphylococcus schleiferi subspecies (S schleiferi subsp coagulans and S schleiferi subsp schleiferi) in clinical samples obtained from dogs. SAMPLE POPULATION: 161 S schleiferi isolates from 160 canine patients. PROCEDURES: A commercial microbiology identification system was used to identify each isolate as S schleiferi. Isolates underwent slide and tube coagulase testing and antimicrobial susceptibility testing. A mecA PCR assay and a latex agglutination test for penicillin-binding protein 2a (PBP2a) were also performed on each isolate. Clonal clusters with a similarity cutoff value of 80% were identified via PFGE. RESULTS: Of the 161 isolates, 61 (38%), 79 (49%), and 21 (13%) were obtained from cutaneous sites, ears, and other sites, respectively; 110 (68%) were coagulase negative, and 51 (32%) were coagulase positive. Among the coagulase-negative and coagulase-positive isolates, 65% (71/110) and 39% (20/51) were oxacillin resistant, respectively. All oxacillin-resistant isolates yielded positive results via mecA PCR assay and PBP2a latex agglutination testing. Via PFGE, 15 major clusters and 108 individual pulsed-field profiles were identified. Oxacillin-resistant and oxacillin-susceptible isolates clustered separately. Clonal clusters were heterogeneous and contained representatives of both subspecies. CONCLUSIONS AND CLINICAL RELEVANCE: Coagulase-positive and coagulase-negative isolates were not genotypically distinct and may represent a single S schleiferi sp with variable coagulase production, rather than 2 biologically distinct subspecies. Further studies are needed to characterize clinical or epidemiological differences associated with infections with coagulase-positive and coagulase-negative S schleiferi in dogs.

Detection of Streptococcus equi subsp. equi in guttural pouch lavage samples using a loop-mediated isothermal nucleic acid amplification microfluidic device.

BACKGROUND: Rapid point-of-care (POC) detection of Streptococcus equi subsp. equi (S. equi) would theoretically reduce the spread of strangles by identifying index and carrier horses. HYPOTHESIS: That the eqbE isothermal amplification (LAMP) assay, and the same eqbE LAMP assay tested in a microfluidic device format, are comparable to a triplex real-time quantitative polymerase chain reaction (qPCR) assay that is commonly used in diagnostic labs. SAMPLES: Sixty-eight guttural pouch lavage (GPL) specimens from horses recovering from strangles. METHODS: Guttural pouch lavage specimens were tested for S. equi retrospectively using the benchtop eqbE LAMP, the eqbE LAMP microfluidic device, and compared to the triplex qPCR, that detects 2 S. equi-specific genes, eqbE and SEQ2190, as the reference standard using the receiver operating characteristic area under the curve (ROC). RESULTS: The 27/68 specimens were positive by benchtop eqbE LAMP, 31/64 by eqbE LAMP microfluidic device, and 12/67 by triplex qPCR. Using the triplex PCR as the reference, the benchtop eqbE LAMP showed excellent discrimination (ROC Area = 0.813, 95% confidence interval [CI] = 0.711-0.915) as did the LAMP microfluidic device (ROC Area = 0.811, 95% CI = 0.529-0.782). There was no significant difference between the benchtop LAMP and LAMP microfluidic device (ROC Area 0.813 ± 0.055 vs 0.811 ± 0.034, P = .97). CONCLUSIONS: The eqbE LAMP microfluidic device detected S. equi in GPL specimens from convalescent horses. This assay shows potential for development as a POC device for rapid, sensitive, accurate, and cost-efficient detection of S. equi.

The prevalence of carriage of meticillin-resistant staphylococci by veterinary dermatology practice staff and their respective pets.

It has been shown that people and pets can harbour identical strains of meticillin-resistant (MR) staphylococci when they share an environment. Veterinary dermatology practitioners are a professional group with a high incidence of exposure to animals infected by Staphylococcus spp. The objective of this study was to assess the prevalence of carriage of MR Staphylococcus aureus (MRSA), MR S. pseudintermedius (MRSP) and MR S. schleiferi (MRSS) by veterinary dermatology practice staff and their personal pets. A swab technique and selective media were used to screen 171 veterinary dermatology practice staff and their respective pets (258 dogs and 160 cats). Samples were shipped by over-night carrier. Human subjects completed a 22-question survey of demographic and epidemiologic data relevant to staphylococcal transmission. The 171 human-source samples yielded six MRSA (3.5%), nine MRSP (5.3%) and four MRSS (2.3%) isolates, while 418 animal-source samples yielded eight MRSA (1.9%) 21 MRSP (5%), and two MRSS (0.5%) isolates. Concordant strains (genetically identical by pulsed-field gel electrophoresis) were isolated from human subjects and their respective pets in four of 171 (2.9%) households: MRSA from one person/two pets and MRSP from three people/three pets. In seven additional households (4.1%), concordant strains were isolated from only the pets: MRSA in two households and MRSP in five households. There were no demographic or epidemiologic factors statistically associated with either human or animal carriage of MR staphylococci, or with concordant carriage by person-pet or pet-pet pairs. Lack of statistical associations may reflect an underpowered study.

Clinical, microbiological, and molecular characterization of methicillin-resistant Staphylococcus aureus infections of cats.

OBJECTIVE: To compare clinical information obtained from medical records of cats with methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-susceptible S aureus (MSSA) infections, evaluate antibiograms of MRSA and MSSA for multiple-drug resistance (MDR), and characterize the strain type and staphylococcal chromosome cassette (SCC)mec type of each MRSA. SAMPLE POPULATION: 70 S aureus isolates obtained from 46 cats. PROCEDURES: Clinical information obtained from medical records, including signalment, clinical signs, histologic examination of affected tissues, and outcomes, was compared between the 2 groups. Composite antibiograms of MRSA and MSSA were compared statistically. The MRSA strains were characterized by use of pulsed-field gel electrophoresis and SCCmec typing. RESULTS: No statistical differences in signalment or subjective differences in clinical signs or outcomes were detected between groups with MRSA or MSSA infection. Significant differences in antimicrobial resistance were detected, with MRSA having complete resistance to fluoroquinolone and macrolide antimicrobials, whereas MSSA maintained a high frequency of susceptibility. Seven pulsed-field patterns were observed in 15 MRSA strains; all but 1 were highly related. All MRSA isolates contained a type II SCCmec element. CONCLUSIONS AND CLINICAL RELEVANCE: Because MDR cannot be predicted in staphylococcal infections in cats on the basis of clinical signalment, culture and susceptibility testing are recommended whenever initial empirical treatment is unsuccessful. Molecular characterization of MRSA strains suggests that there has been reverse-zoonotic transmission from humans. IMPACT FOR HUMAN MEDICINE: The SCCmec type II element is typically associated with nosocomial MRSA infections of people. Cats may serve as reservoirs for MRSA infections in humans.

Distribution of Malassezia organisms on the skin of unaffected psittacine birds and psittacine birds with feather-destructive behavior.

OBJECTIVE: To ascertain whether Malassezia organisms can be detected via cytologic examination and fungal culture of samples from the skin surface of psittacine birds and determine whether the number of those organisms differs between unaffected psittacines and those that have chronic feather-destructive behavior or differs by body region. DESIGN: Prospective study. ANIMALS: 50 unaffected psittacines and 53 psittacines that had feather-destructive behavior. PROCEDURE: Samples were collected by use of acetate tape strips from the skin of the head, neck, proventer, propatagium, inguinal region, and preen gland area of each bird; 0.5-cm(2) sample areas were examined microscopically for yeast, and samples were also incubated on Sabouraud dextrose agar. Polymerase chain reaction assays specific for Malassezia spp, saprophytic fungi, and Candida albicans were performed on DNA prepared from cultured colonies; nested PCR evaluation for Malassezia pachydermatis was then performed. RESULTS: Microscopically, 63 of 618 (10%) tape-strip samples contained yeast. Thirty cultured colonies were assessed via PCR assays, and all yielded negative results for Malassezia spp; C albicans was identified in 2 colony samples. The numbers of yeast identified microscopically in psittacines with feather-destructive behavior and in unaffected birds did not differ significantly, and numbers did not differ by body region. CONCLUSIONS AND CLINICAL RELEVANCE: Yeast were identified infrequently via cytologic examination of samples from the skin surface of unaffected psittacine birds or those that had chronic feather-destructive behavior. If yeast are identified on the skin of birds with feather-destructive behaviors, fungal culture of skin samples should be performed to identify the organism.

A real-time PCR assay to detect the Panton Valentine Leukocidin toxin in staphylococci: screening Staphylococcus schleiferi subspecies coagulans strains from companion animals.

Recent reports suggest that methicillin-resistant strains of Staphylococcus schleiferi subspecies coagulans are now commonly isolated from dogs. Given the association of a potentially mobile SCCmec type IV element with lysogenic phage-encoded Panton Valentine Leukocidin (PVL) toxin genes in community-acquired methicillin-resistant Staphylococcus aureus strains we hypothesized that methicillin-resistant S. schleiferi ssp. coagulans strains may also encode PVL toxin genes. Forty S. schleiferi ssp. coagulans strains isolated from companion animals were studied. Susceptibility to oxacillin was determined by broth microdilution and all isolates were screened by PCR for the presence of the mecA gene. SCCmec typing was performed on 14 isolates. A real-time PCR assay was developed for the detection of the PVL genes using a SmartCycler. Pulsed-field gel electrophoresis (PFGE) was performed to determine whether S. schleiferi ssp. coagulans strains were homogeneous. Twenty-eight of the 40 isolates (70%) were resistant to oxacillin and 26/28 possessed the mecA gene by PCR. SCCmec IV was identified in seven strains; the other seven isolates were not typable by this technique. All 40 strains were negative for the PVL toxin gene. PFGE showed a heterogeneous population and 13 different profiles were determined. In conclusion, this study showed that PVL toxin genes were not detected in a heterogeneous population of methicillin-resistant S. schleiferi ssp. coagulans strains isolated from companion animals.

Panton valentine leukocidin (PVL) toxin positive MRSA strains isolated from companion animals.

Methicillin-resistant Staphylococcus aureus (MRSA) is a highly pathogenic multiple-drug resistant (MDR) microorganism that has recently become more prevalent in the community. It has been found that MRSA strains can also contain genes that encode the panton valentine leukocidin toxin (PVL). The PVL toxin has been shown to be responsible for many of the severe clinical symptoms of infection with MRSA, such as furunculosis, severe necrotizing pneumonia, and necrotic lesions of the skin and soft tissues. The aim of this study was to determine the presence of the S. aureus PVL toxin genes (lukS-PV and lukF-PV) in MRSA strains isolated from companion animals. Eleven MRSA isolates, from a total of 23 tested, were shown to possess the mecA gene and the PVL toxin genes. Pulsed-field gel electrophoresis showed that the 11 PVL toxin positive MRSA strains were highly clonal.

Optimization of an in vitro assay to detect Streptococcus equi subsp. equi.

Streptococcus equi is the etiologic agent of a highly infectious upper respiratory disease of horses known as strangles. Bacterial culture methods and polymerase chain reaction (PCR) of nasopharyngeal washes and guttural pouch lavages are used routinely to test clinical and carrier animals for the presence of S. equi but no definitive or gold standard test method has been shown to be optimal. We hypothesized that (i) a flocked swab submerged in ten-fold serial dilution suspensions of S. equi prepared in 0.9% NaCl would detect more colony forming units (CFU) than a rayon swab when used to inoculate a blood agar plate, (ii) centrifugation of a 1 ml aliquot of each suspension would improve the limit of detection (LOD) by bacterial culture and PCR compared to the culture or PCR of submerged swab samples, (iii) PCR of the centrifuged samples from each suspension would be more sensitive than aerobic culture alone, and (iv) PCR of a 1 ml aliquot directly from a sample would be more sensitive than PCR of a sample following submersion of a flocked swab in 1 ml saline. Using 7 ten-fold serial dilutions of S. equi in 0.9% NaCl, the LOD for 4 bacterial culture methods and 3 PCR methods were compared. The LOD of direct PCR and flocked swab culture was determined at 1 cfu/ml. All PCR methods were equivalent to each other and were more sensitive than any of the culture methods at the lower dilutions. At higher cell densities (>100 cfu/ml) flocked swab culture was not statistically better than rayon swab culture, but it was superior to all other methods tested.

Pseudomonas fluorescens contamination of a feline packed red blood cell unit and studies of canine units.

BACKGROUND: While screening programs have reduced the risk of infectious disease transmission by donors in human and veterinary blood banking, bacterial contamination of blood products has emerged as a major complication in human medicine. OBJECTIVES: To describe a Pseudomonas fluorescens (Pf)-contaminated feline packed RBC (pRBC) unit and experimentally investigate Pf-contaminated canine pRBCs. METHODS: Canine pRBCs were inoculated with Pf-rich pRBCs from the sentinel feline unit and stored at 4 degrees C or 20 degrees C for 72 hours. Aliquots from the pRBCs were serially evaluated by microscopy, culture, and a eubacterial 16S rRNA real-time PCR assay. RESULTS: One Pf-contaminated feline unit turned black after 22 days of storage and was removed from the blood bank; a source was not found, and no other contaminated units were identified. Canine pRBCs spiked with 5 or 25 microL of the sentinel unit became culture- and/or 16S PCR-positive at > or =8 hours at 20 degrees C and 48 hours at 4 degrees C and developed a color change at > or =24 hours. Sensitivity studies indicated that without incubation, inoculation of > or =100 microL Pf-rich pRBCs was necessary for a positive 16S PCR test result. CONCLUSIONS: P. fluorescens grows in stored pRBCs slowly at 4 degrees C and rapidly at 20 degrees C. Screening of blood products for color change, estimating bacterial concentration with microscopy, and 16S PCR testing are simple and fast ways to detect bacteria in stored blood. Aseptic collection, temperature-controlled storage, and regular visual monitoring of stored units is recommended. Discolored units should not be transfused, but examined for bacterial contamination or other blood product quality problems.

Detection of a bla(SHV) extended-spectrum {beta}-lactamase in Salmonella enterica serovar Newport MDR-AmpC.

Salmonella enterica serovar Newport MDR-AmpC expressing TEM-1b and extended-spectrum beta-lactamase SHV-12 was isolated from affected animals during an outbreak of salmonellosis that led to a 3-month closure of one of the largest equine hospitals in the United States.