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.

Molecular characterization of cephalosporin-resistant Salmonella enterica serotype Newport isolates from animals in Pennsylvania.

Multidrug-resistant (MDR) strains of Salmonella enterica serotype Newport have been described for many years. However, the recognition of Newport strains with resistance to cephalosporin antibiotics is more recent. Plasmid-mediated CMY-2 AmpC beta-lactamases have been identified in Salmonella in the United States, and the bla(CMY-2) gene has been shown to be present in Salmonella serotype Newport. This organism is currently undergoing epidemic spread in both animals and humans in the United States, and this is to our knowledge the first description of the molecular epidemiology of this Salmonella strain in animals. Forty-two isolates were included in this study. All isolates were characterized by pulsed-field gel electrophoresis, plasmid analysis, and antibiogram. Four pulsed-field profiles with XbaI were observed. Plasmid analyses showed that although the majority of isolates harbored a single plasmid of 140 kb, this plasmid was not identical in all strains. All isolates showed the presence of the bla(CMY) gene by PCR. Integrons were detected in 16 of the 42 isolates; a fragment of approximately 1,000 bp, amplified with the intI-F and aadAI-R primers, confirmed the presence of the aadAI gene cassette within an integron in these 16 isolates. The potential for coselection of the bla(CMY) gene, if located on an MDR replicon, may not be dependent on any particular antibiotic but rather may be the result of more general antimicrobial use. If this replicon is mobile, it is to be expected that similar MDR strains of additional Salmonella serotypes will be recognized in due course.