Joseph E. Rubin

Antimicrobial resistance and genetic characterization of fluoroquinolone resistance of Pseudomonas aeruginosa isolated from canine infections

Infections with antimicrobial-resistant bacteria are a great challenge in both human and veterinary medicine. The purpose of this study was to determine antimicrobial susceptibility of 106 strains of Pseudomonas aeruginosa isolated from dogs with otitis and pyoderma from 2003 to 2006 in the United States. Three antimicrobial panels, including 6 classes and 32 antimicrobial agents, were used. A wide range of susceptibility patterns were noted with some isolates being resistant to between 8 and 28 (mean 16) of the antimicrobials tested. Among the beta-lactams, all isolates were resistant to ampicillin, cefoxitin, cefpodoxime, cephalothin and cefazolin followed by amoxicillin/clavulanic acid (99%), ceftiofur (97%), ceftriaxone (39%), cefotaxime (26%), and cefotaxime/clavulanic acid (20%), whereas less than 7% of isolates were resistant to ceftazidime/clavulanic acid, ceftazidime, piperacillin/tazobactam or cefepime. Two isolates were resistant to the carbapenems. Among the quinolones and fluoroquinolones, the most isolates were resistant to naladixic acid (96%), followed by orbifloxacin (52%), difloxacin (43%), enrofloxacin (31%), marbofloxacin (27%), gatifloxacin (23%), levofloxacin (21%), and ciprofloxacin (16%). Among the aminoglycosides, the most resistance was seen to kanamycin (90%), followed by streptomycin (69%), gentamicin (7%), and amikacin (3%). Of the remaining antimicrobials 100% of the isolates were resistant to chloramphenicol followed by tetracycline (98%), trimethoprim/sulfamethoxazole (57%), and sulfisoxazole (51%). Point mutations were present in gyrA, gyrB, parC, and/or parE genes among 34 of the 102 naladixic acid-resistant isolates. Two isolates contained class 1 integrons carrying aadA gene conferring streptomycin and spectinomycin resistance. The findings suggest that many antimicrobial agents commonly used in companion animals may not constitute appropriate therapy for canine pseudomonas infections.

Reproduction of Mucohaemorrhagic Diarrhea and Colitis Indistinguishable from Swine Dysentery following Experimental Inoculation with “Brachyspira hampsonii” Strain 30446

Background Mucohaemorrhagic diarrhea caused by Brachyspira hyodysenteriae, swine dysentery, is a severe production limiting disease of swine. Recently, pigs in western Canada with clinical signs indistinguishable from swine dysentery were observed. Despite the presence of spirochetes on fecal smears, recognized Brachyspira spp. including B. hyodysenteriae could not be identified. A phylogenetically distinct Brachyspira, called “B. hampsonii” strain 30446, however was isolated. The purpose of this study was to experimentally reproduce mucohaemorrhagic colitis and characterize strain 30446 shedding following inoculation. Methods and Findings Eighteen 13-week-old pigs were randomly assigned to inoculation (n = 12) or control (n = 6) groups in each of two trials. In trial 1, pigs were inoculated with a tissue homogenate collected from clinically affected field cases. In trial 2, pigs were inoculated with a pure broth culture of strain 30446. In both trials, mucohaemorrhagic diarrhea was significantly more common in inoculated pigs than controls, all of which remained healthy. In animals with mucohaemorrhagic diarrhea, significantly more spirochetes were observed on Gram stained fecal smears, and higher numbers of strain 30446 genome equivalents were detected by quantitative PCR (qPCR). Strain 30446 was cultured from colon and/or feces of all affected but no control animals at necropsy. Conclusions “Brachyspira hampsonii” strain 30446 causes mucohaemorrhagic diarrhea in pigs following a 4–9 day incubation period. Fecal shedding was detectable by day 4 post inoculation, and rarely preceded the onset of mucoid or haemorrhagic diarrhea by more than 2 days. Culture and 30446-specific qPCR are reliable methods of detection of this organism in feces and tissues of diarrheic pigs. The emergence of a novel Brachyspira spp., such as “B. hampsonii”, creates diagnostic challenges including higher risk of false negative diagnostic tests. We therefore recommend diagnostic laboratories routinely use Brachyspira culture, nox-based and species-specific PCR, and DNA sequencing to diagnose Brachyspira-associated colitis in pigs.

Prevalence, Sites of Colonization, and Antimicrobial Resistance Among Staphylococcus Pseudintermedius Isolated from Healthy Dogs in Saskatoon, Canada

In dogs, Staphylococcus pseudintermedius is a common colonizer and is associated with pyoderma, otitis externa, and urinary tract infections. In the current study, nasal, pharyngeal, and rectal swabs were taken from 175 healthy dogs and cultured for S. pseudintermedius. The organism was found in 153 dogs (87.4%), including individuals in which it exclusively colonized in the nares (n = 1), pharynx (n = 16), and rectum (n = 17). Antimicrobial susceptibility testing revealed that a remarkably susceptible population, 46.4% of isolates, was susceptible to all drugs tested, and resistance to penicillin (39.9%) and tetracycline (23.5%) were most common. No methicillin (oxacillin)-resistant isolates were identified. Although 3.3% of isolates were erythromycin resistant, no inducible clindamycin resistance was found. The data provide a baseline for future resistance surveillance and indicate that multiple body sites, including at least the pharynx and rectum, should be tested.

Inducibly Cefoxitin-Resistant Macrococcus-Like Organism Falsely Identified as Methicillin-Resistant Staphylococcus aureus on CHROMagar with Oxacillin

In a screening study for methicillin-resistant Staphylococcus aureus (MRSA), nasal, pharyngeal, and rectal swabs were collected from healthy dogs at the University of Saskatchewan. Multiple solid media, including CHROMagar Staph aureus (CHROMagar, Paris, France), were inoculated; S. aureus grows as

Letter to editor-colony morphology is insufficient to identify methicillin-resistant Staphylococcus aureus isolated from stethoscopes.

To the Editor:—Dr. Merlin and colleagues examined the prevalence of methicillin-resistant Staphylococcus aureus (MRSA) contamination of stethoscopes. They found that 16 (32%) of 50 stethoscopes sampled were MRSA-positive, which was higher than previously reported.1 In this study, stethoscopes were swabbed and samples were inoculated onto mannitol salt agar (MSA) containing 4 μg/mL oxacillin. Samples were considered to be MRSA-positive if at least one suspicious colony was found after 72 hours of incubation. However, biochemical tests (Gram stain, coagulase and catalase test as a minimum database) to positively identify these bacteria were not done, calling these findings into question. The identification of colonies as MRSA from MSA + oxacillin without confirmatory testing is inappropriate, as the specificity of MSA + 6 μg/mL oxacillin is only 79%.2 Similarly, MRSA false positives (coagulase-negative or methicillin-susceptible) were found among 6.5% of samples inoculated onto MSA + cefoxitin.2,3 Another study reported that among nasal swabs, Bacillus species and coagulase-negative staphylococci grown on MSA can be misidentified as S. aureus.4 In my experience, colonies with typical S. aureus morphology on MSA with or without oxacillin are not infrequently bacteria other than S. aureus, particularly after prolonged incubation periods. The previously reported specificity of MSA + oxacillin (79%) included only patient swabs and may underestimate the false-positive rate of fomite samples where the prevalence of Bacillus species is unknown and coagulase-negative staphylococci are common.2,5 Further confounding these results, media were incubated at 37◦C as opposed to 33◦C–35◦C as recommended by Clinical and Laboratory Standards Institute (CLSI) guidelines.6 While

Comparison of dog and rabbit plasmas in the tube coagulase test for Staphylococcus aureus.

The tube coagulase test, an invaluable laboratory tool for identifying Staphylococcus aureus, is most often done using rabbit plasma. However, there is evidence that depending on the origin of the isolates, other plasmas may be superior. The current study sought to compare the utility of dog and rabbit plasma in the coagulase test for S. aureus isolated from canine (n = 28), bovine (n = 29), and human (n = 30) hosts. Overall, coagulation times were significantly faster for dog (2.38 hr) than rabbit (3.19 hr) plasma. When coagulation times were compared by isolate origin, no significant differences were found for rabbit plasma, whereas bovine isolates clotted dog plasma significantly faster (1.86 hr) than canine (2.79 hr) or human (2.38 hr) isolates. Investigators should be aware that rabbit plasma may not be the ideal coagulase-testing medium for S. aureus from all sources.