Use of critically important antimicrobial classes early in life may adversely impact bacterial resistance profiles during adult years: potential co-selection for plasmid-borne fluoroquinolone and macrolide resistance via extended-spectrum beta-lactam use in dairy cattle.

The transfer of antimicrobial resistance genes commonly occurs via vertical and horizontal gene transfer, as such genes are often found on the same mobile genetic element. This occurrence can lead to the co-selection of resistance to antimicrobials without their application. Dairy cattle located in the south-western United States were enrolled in a matched-pair longitudinal study to evaluate the effects of a two-dose ceftiofur treatment for metritis on levels of third-generation cephalosporin resistance among faecal Escherichia coli temporally. Escherichia coli chosen for further investigation were isolated on selective media, harboured extended-spectrum beta-lactam, fluoroquinolone and macrolide resistance genes. This combination has previously been unreported; importantly, it included genes encoding for resistance to antibiotics that can only be used in dairy cattle less than 20 months of age. Fluoroquinolones, macrolides and third and higher generation cephalosporins are considered critically important and highest priority for human medicine by the World Health Organization.

Culture-independent and dependent evaluation of the equine paranasal sinus microbiota in health and disease.

BACKGROUND: Horses with bacterial sinusitis frequently undergo empirical treatment with antimicrobials, however, in some cases bacterial culture of the affected sinus is used to direct therapy. Data regarding which organisms are part of the commensal microbiota of the equine sinus are lacking making it difficult to interpret culture results and guide empiric antimicrobial selection. OBJECTIVES: Our objectives were to describe the bacterial and fungal microbiota of the paranasal sinuses in clinically normal horses using culture-dependent and independent approaches and to compare the bacterial culture and susceptibility patterns of normal horses with those from horses affected with primary and secondary sinusitis. STUDY DESIGN: Experimental study and descriptive retrospective review of case records. METHODS: Sinus washes were collected from 23 healthy horses. Washes were submitted for routine culture and susceptibility testing and DNA was isolated for next generation sequencing of bacterial and fungal marker genes. For clinical cases of sinusitis, medical records from 2010 to 2017 were reviewed and horses diagnosed with primary and/or secondary sinusitis were included. RESULTS: The paranasal sinus cavity hosts multiple bacterial and fungal organisms. The bacterial microbiota of healthy horses consists largely of uncultivable, aerobic bacteria. While few anaerobes were isolated from normal horses, the majority of clinical cases resulted in growth of anaerobic organisms with no difference in the proportion of aerobic and anaerobic bacteria isolated from clinical cases. MAIN LIMITATIONS: Small sample size in both populations of horses and heterogeneity of the population prevent a more in-depth analysis. CONCLUSIONS: The microbiota of the paranasal sinuses of horses consists primarily of aerobic bacteria and fungal organisms, the majority of which are uncultivable via common clinical methods. Anaerobic bacteria are found in the majority of horses with clinical sinusitis. These findings suggest anaerobic bacteria are associated with sinusitis and their presence should be considered when treating horses with sinusitis.

Macrolide-susceptible probiotic Enterococcus faecium ST296 exhibits faecal-environmental-oral microbial community cycling among beef cattle in feedlots.

Enterococci are included in the United States National Antimicrobial Resistance Monitoring System to track antibiotic resistance among commensal Gram-positive enteric bacteria, largely due to their high abundance in food animals and in retail meat. In the U.S. cattle industry, macrolides are used to prevent and control liver abscesses, which cause significant economic losses. Previous studies have suggested that feeding tylosin and the intensity of the pen environment, both expand and sustain respectively the prevalence of multidrug resistance among enterococci in feedlot cattle. This has led to research into alternative feed supplements and improved stewardship practices. In a randomized controlled trial, we measured the impact of a probiotic and an altered pen environment on antimicrobial resistance among faecal Enterococcus spp. in cattle fed tylosin. Supplementing cattle with an Enterococcus faecium and Saccharomyces cerevisiae-based probiotic yielded the isolation of E. faecium of the probiotic sequence type (ST296) from faecal and environmental samples in treatment groups, as well as from cattle and the manure pack in nearby pens. Of importance, the probiotic strain also was found in a desiccated and milled manure pack sample taken 120 days after the initial trial ended. Phylogenetic and SNP analyses revealed clonal survival and spread compatible with faecal-environmental-oral recycling of the probiotic strain within and among cattle and pens. The increase in prevalence of the ST296 strain occurred concomitant with a decrease in ST240, the dominant sequence type associated with ermB and tet(M) resistance genes in this trial. SIGNIFICANCE AND IMPACT OF THE STUDY: We demonstrate that a macrolide-susceptible probiotic Enterococcus faecium ST296 strain fed to beef cattle becomes fully embedded in the microbial community cycling of bacteria via faecal-environmental-oral transmission within and among feedlot pens. An initial investment in feeding the probiotic is thereby leveraged into expanding numbers of susceptible bacteria in cattle and their environment, even among those cattle fed tylosin.

The effects of antibiotic type and extender storage method on sperm quality and antibacterial effectiveness in fresh and cooled-stored stallion semen.

Two experiments were conducted to evaluate the effects of antibiotic-containing extender of on sperm quality and control of bacterial growth. In Experiment 1, ejaculates were diluted in extender containing no antibiotics, potassium penicillin G-amikacin disulfate (PEN-AMIK), ticarcillin disodium-potassium clavulanate (TICAR-CLAV), piperacillin sodium/tazobactam sodium (PIP-TAZ), or meropenem (MERO). In freshly extended semen, only slight differences were detected among some antibiotic treatments for total sperm motility, curvilinear velocity, and viable acrosome-intact sperm (P < 0.05). In cool-stored semen, slight differences were also detected among certain antibiotic treatments for curvilinear velocity and chromatin integrity (P < 0.05). In Experiment 2, ejaculates were diluted in extender and subjected to no bacterial spiking, or inoculated with lower or higher doses of K. pneumoniae or P. aeruginosa. Following cooled storage of semen, colony forming units/ml (CFU/mL) were less in PEN-AMIK (706 ±â€¯244) and MERO (1576 ±â€¯1076) treatment groups than in TICAR-CLAV (4678 ±â€¯1388) or PIP-TAZ (8108 ±â€¯3198) treatment groups (P < 0.05). The CFU/mL were lower in all antibiotic-containing treatment groups than the control group (18478 ±â€¯4374; P < 0.05). The percentage of culture plates containing no bacterial growth in unspiked semen was greater in PEN-AMIK (75%) than PIP-TAZ (15%) or TICAR-CLAV (20%; P < 0.05). The percentages of culture plates containing no bacterial growth in semen spiked with a lower doses of K. pneumoniae or P. aeruginosa were higher in PEN-AMIK (70% and 50%, respectively) then in all other treatment groups (0-40% and 0-15% for K. pneumonia and P. aeruginosa, respectively; P < 0.05); however, complete control of bacterial load was only modest even with PEN-AMIK. In both experiments, freezing and thawing extender prior to use did not have any appreciable detrimental effect on sperm quality or antibiotic efficacy. In summary, all antibiotics tested had minimal effects on measures of sperm quality in fresh or cool-stored semen extenders; however, PEN-AMIK, followed by MERO, yielded the best results in terms of antimicrobial efficacy. None of the antibiotic types controlled bacterial growth, in comparison with the antibiotic-free control group, when extended semen was spiked with a high concentration of Pseudomonas aeruginosa. Cooled storage of extended semen reduced bacterial growth in comparison with freshly extended semen.

Serotype Diversity and Antimicrobial Resistance among Salmonella enterica Isolates from Patients at an Equine Referral Hospital.

Although Salmonella enterica can produce life-threatening colitis in horses, certain serotypes are more commonly associated with clinical disease. Our aim was to evaluate the proportional morbidity attributed to different serotypes, as well as the phenotypic and genotypic antimicrobial resistance (AMR) of Salmonella isolates from patients at an equine referral hospital in the southern United States. A total of 255 Salmonella isolates was obtained from clinical samples of patients admitted to the hospital between 2007 and 2015. Phenotypic resistance to 14 antibiotics surveilled by the U.S. National Antimicrobial Resistance Monitoring System was determined using a commercially available panel. Whole-genome sequencing was used to identify serotypes and genotypic AMR. The most common serotypes were Salmonella enterica serotype Newport (18%), Salmonella enterica serotype Anatum (15.2%), and Salmonella enterica serotype Braenderup (11.8%). Most (n = 219) of the isolates were pansusceptible, while 25 were multidrug resistant (≥3 antimicrobial classes). Genes encoding beta-lactam resistance, such as blaCMY-2, blaSHV-12, blaCTX-M-27, and blaTEM-1B, were detected. The qnrB2 and aac(6')-Ib-cr genes were present in isolates with reduced susceptibility to ciprofloxacin. Genes encoding resistance to gentamicin (aph(3')-Ia, aac(6')-IIc), streptomycin (strA and strB), sulfonamides (sul1), trimethoprim (dfrA), phenicols (catA), tetracyclines [tet(A) and tet(E)], and macrolides [ere(A)] were also identified. The main predicted incompatibility plasmid type was I1 (10%). Core genome-based analyses revealed phylogenetic associations between isolates of common serotypes. The presence of AMR Salmonella in equine patients increases the risk of unsuccessful treatment and causes concern for potential zoonotic transmission to attending veterinary personnel, animal caretakers, and horse owners. Understanding the epidemiology of Salmonella in horses admitted to referral hospitals is important for the prevention, control, and treatment of salmonellosis.IMPORTANCE In horses, salmonellosis is a leading cause of life-threatening colitis. At veterinary teaching hospitals, nosocomial outbreaks can increase the risk of zoonotic transmission, lead to restrictions on admissions, impact hospital reputation, and interrupt educational activities. The antimicrobials most often used in horses are included in the 5th revision of the World Health Organization's list of critically important antimicrobials for human medicine. Recent studies have demonstrated a trend of increasing bacterial resistance to drugs commonly used to treat Salmonella infections. In this study, we identify temporal trends in the distribution of Salmonella serotypes and their mechanisms of antimicrobial resistance; furthermore, we are able to determine the likely origin of several temporal clusters of infection by using whole-genome sequencing. These data can be used to focus strategies to better contain the dissemination and enhance the mitigation of Salmonella infections and to provide evidence-based policies and guidelines to steward antimicrobial use in veterinary medicine.

Evaluation of Oxacillin and Cefoxitin Disk Diffusion and MIC Breakpoints Established by the Clinical and Laboratory Standards Institute for Detection of mecA-Mediated Oxacillin Resistance in Staphylococcus schleiferi.

Staphylococcus schleiferi is a beta-hemolytic, coagulase-variable colonizer of small animals that can cause opportunistic infections in humans. In veterinary isolates, the rate of mecA-mediated oxacillin resistance is significant, with reported resistance rates of >39%. The goal of this study was to evaluate oxacillin and cefoxitin disk diffusion (DD) and MIC breakpoints for detection of mecA-mediated oxacillin resistance in 52 human and 38 veterinary isolates of S. schleiferi Isolates were tested on multiple brands of commercial media and according to Clinical and Laboratory Standards Institute (CLSI) methods. Zone diameters and MIC values were interpreted using CLSI breakpoints (CLSI, Performance Standards for Antimicrobial Susceptibility Testing. M100-S27, 2017) for Staphylococcus aureus/Staphylococcus lugdunensis, coagulase-negative staphylococci (CoNS), and Staphylococcus pseudintermedius Results were compared to those of mecA PCR. Twenty-nine of 90 (32%) isolates were mecA positive. Oxacillin inhibition zone sizes and MICs interpreted by S. pseudintermedius breakpoints reliably differentiated mecA-positive and mecA-negative isolates, with a categorical agreement (CA) of 100% and no very major errors (VMEs) or major errors (MEs) for all media. For cefoxitin DD results interpreted using S. aureus/S. lugdunensis and CoNS breakpoints, CA values were 85% and 75%, respectively, and there were 72% and 64% VMEs, respectively, and 0 MEs. For cefoxitin MICs interpreted using S. aureus/S. lugdunensis breakpoints, CA was 81%, and there were 60% VMEs and no MEs. Our data demonstrate that oxacillin DD or MIC testing methods using the current S. pseudintermedius breakpoints reliably identify mecA-mediated oxacillin resistance in S. schleiferi, while cefoxitin DD and MIC testing methods perform poorly.

Faecal Campylobacter shedding among dogs in animal shelters across Texas.

Epidemiologic studies on faecal Campylobacter shedding among dogs in the United States have been limited, despite evidence that the incidence of human campylobacteriosis has increased over the last decade. Our objectives were to estimate the prevalence of faecal Campylobacter shedding among shelter dogs in Texas, to estimate the specific prevalence of Campylobacter jejuni and Campylobacter coli shedding, and to identify risk factors for Campylobacter-positive status. Using a cross-sectional study design, we collected faecal samples from dogs in six animal shelters across Texas between May and December, 2014. Quantitative PCR protocols were used to detect Campylobacter in samples and to specifically identify C. jejuni and C. coli. The prevalence of faecal Campylobacter shedding among sampled dogs was 75.7% (140/185). Prevalence varied significantly by shelter (p = .03), ranging from 57% to 93%. There was a marginal association (p = .06) between abnormal faecal consistency and positive Campylobacter status, after controlling for shelter as a random effect. However, approximately 70% of Campylobacter-positive dogs had grossly normal faeces. Campylobacter prevalence did not vary significantly by age group or sex. The prevalence of C. jejuni-positive samples was 5.4% (10/185), but C. coli was not detected in any samples. Dogs are a potential source of zoonotic Campylobacter transmission.

Rhodococcus equi Infections in Dogs.

Five cases of Rhodococcus equi infection in dogs were identified from 2003 to 2014. Three of the dogs had severe, internal lesions attributable to R. equi that have not been previously described: endophthalmitis, endocarditis, and suppurative pleuropneumonia. Isolates from 4 of the dogs were analyzed by polymerase chain reaction for Rhodococcus virulence-associated plasmid (vap) genes. One isolate was vapA-positive, 2 lacked a virulence plasmid, and 1 carried the novel vapN-associated plasmid (pVAPN) recently characterized in bovine isolates. The pVAPN plasmid has not been described in isolates cultured from companion animals. Four of the dogs either were receiving immunosuppressive drugs or had endocrinopathies. R. equi has the potential to cause significant infections in dogs, and immunocompromised animals should be considered at risk for infection.

Evaluation of an Immunochromatographic Assay for Rapid Detection of Penicillin-Binding Protein 2a in Human and Animal Staphylococcus intermedius Group, Staphylococcus lugdunensis, and Staphylococcus schleiferi Clinical Isolates.

The performance of a rapid penicillin-binding protein 2a (PBP2a) detection assay, the Alere PBP2a culture colony test, was evaluated for identification of PBP2a-mediated beta-lactam resistance in human and animal clinical isolates of Staphylococcus intermedius group, Staphylococcus lugdunensis, and Staphylococcus schleiferi. The assay was sensitive and specific, with all PBP2a-negative and PBP2a-positive strains testing negative and positive, respectively.

Evaluation of Oxacillin and Cefoxitin Disk and MIC Breakpoints for Prediction of Methicillin Resistance in Human and Veterinary Isolates of Staphylococcus intermedius Group.

Staphylococcus pseudintermedius is a coagulase-positive species that colonizes the nares and anal mucosa of healthy dogs and cats. Human infections with S. pseudintermedius range in severity from bite wounds and rhinosinusitis to endocarditis; historically, these infections were thought to be uncommon, but new laboratory methods suggest that their true incidence is underreported. Oxacillin and cefoxitin disk and MIC tests were evaluated for the detection of mecA- or mecC-mediated methicillin resistance in 115 human and animal isolates of the Staphylococcus intermedius group (SIG), including 111 Staphylococcus pseudintermediusand 4 Staphylococcus delphini isolates, 37 of which were mecA positive. The disk and MIC breakpoints evaluated included the Clinical and Laboratory Standards Institute (CLSI) M100-S25 Staphylococcus aureus/Staphylococcus lugdunensis oxacillin MIC breakpoints and cefoxitin disk and MIC breakpoints, the CLSI M100-S25 coagulase-negative Staphylococcus (CoNS) oxacillin MIC breakpoint and cefoxitin disk breakpoint, the CLSI VET01-S2 S. pseudintermedius oxacillin MIC and disk breakpoints, and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) S. pseudintermedius cefoxitin disk breakpoint. The oxacillin results interpreted by the VET01-S2 (disk and MIC) and M100-S25 CoNS (MIC) breakpoints agreed with the results of mecA/mecC PCR for all isolates, with the exception of one false-resistant result (1.3% of mecA/mecC PCR-negative isolates). In contrast, cefoxitin tests performed poorly, ranging from 3 to 89% false susceptibility (very major errors) and 0 to 48% false resistance (major errors). BD Phoenix, bioMérieux Vitek 2, and Beckman Coulter MicroScan commercial automated susceptibility test panel oxacillin MIC results were also evaluated and demonstrated >95% categorical agreement with mecA/mecC PCR results if interpreted by using the M100-S25 CoNS breakpoint. The Alere penicillin-binding protein 2a test accurately detected all mecA-positive isolates, although for four isolates, cefoxitin induction was required prior to testing. These data demonstrate that the cefoxitin surrogate test does not reliably detect the presence of mecA in S. pseudintermedius isolates and that laboratories should perform oxacillin disk or MIC tests of these isolates when they are encountered.

Plane of nutrition influences the performance, innate leukocyte responses, and resistance to an oral Salmonella enterica serotype Typhimurium challenge in Jersey calves.

Two experiments investigated how plane of nutrition influences performance, leukocyte responses, and resistance to an oral Salmonella enterica serotype Typhimurium challenge. In experiment 1, 46 (2±1 d of age) calves were randomly assigned to 2 diets: a low (LPN; n=23) and high plane of nutrition (HPN; n=23). The LPN calves were fed 409 g/d of dry matter (DM) of a 20% crude protein and 20% fat milk replacer, whereas HPN calves were fed 610 and 735 g/d of DM of a 28% crude protein and 25% fat milk replacer during wk 1 and 2 to 6, respectively. In experiment 2, 20 bull calves (LPN; n=11 and HPN; n=9) were orally challenged on d 80 with 1.5×10(7) cfu of Salmonella Typhimurium (ATCC #14028). The HPN calves had a greater incidence (87.5 vs. 45.5%) and duration of days with high fecal scores (5.5 vs. 3.5 d). The LPN calves had greater neutrophil surface expression of L-selectin on d 7, 21, and 42. Following the Salmonella Typhimurium challenge, calf starter DM intake was greater among the HPN calves. The percentage of neutrophils producing an oxidative burst was also greater among HPN calves on d 1 to 5 after the challenge. Similarly, the intensity of the oxidative burst tended to be greater among the HPN calves on d 2 and 3 postchallenge. The secretion of tumor necrosis factor-α from whole-blood cultures stimulated with lipopolysaccharide tended to be greater on d 1 and was greater on d 5 and 6 among HPN calves. The median ranks of haptoglobin concentrations were greater and plasma zinc concentrations tended to be decreased among LPN calves. These data indicate that feeding a HPN to Jersey calves improved average daily gain and feed efficiency, but increased the incidence of high fecal scores during the first few weeks of life; however, the HPN Jersey calves may be more resistant to Salmonella Typhimurium after weaning.

Amikacin resistance in Staphylococcus pseudintermedius isolated from dogs.

Staphylococcus pseudintermedius is the most common microorganism isolated from canine pyoderma and postoperative wound infections. The prevalence of methicillin-resistant S. pseudintermedius (MRSP) has increased, and recently, isolates that are resistant not only to methicillin but also to other classes of antibiotic drugs, including aminoglycosides, have become common. A total of 422 S. pseudintermedius isolates collected from 413 dogs were analyzed for amikacin and methicillin resistance using broth microdilution and disk diffusion testing. Methicillin-resistant isolates were significantly (P < 0.0001) more likely to be resistant to amikacin (37%, 31/84) than were methicillin-susceptible isolates (7%, 22/338). Additionally, resistance to non-ß-lactam antibiotics was significantly associated with resistance to amikacin irrespective of methicillin resistance. Among the 422 isolates, 32 that tested positive for amikacin resistance by broth microdilution or disk diffusion testing were investigated further for the presence of aminoglycoside-modifying enzyme genes using multiplex PCR. Of these isolates, 66% (21/32) were methicillin resistant. In contrast to previous studies of Staphylococcus aureus, the most prevalent gene detected was aph(3')-IIIa found in 75% (24/32) of isolates followed by aac(6')/aph(2") and ant(4')-Ia in 12% (4/32) and 3% (1/32), respectively. Understanding the differences in antimicrobial resistance gene carriage between different species of Staphylococcus may improve antimicrobial drug selection for clinical therapy and provide insights into how resistance develops in S. pseudintermedius.

Meningoencephalitis in a dog due to Trichosporon montevideense.

Meningoencephalitis due to infection with Trichosporon montevideense was diagnosed in a 4-year-old dog with a brief clinical history of rapidly progressing neurological signs that culminated in a comatose state. No significant gross lesions were found at post-mortem examination. Microscopically, a few scattered areas of pyogranulomatous inflammation with a few small, non-pigmented fungal hyphae were found within the cerebrum surrounding the lateral ventricles. A Trichosporon sp. was identified through culture of the brain and species was determined via sequence analysis of the internal transcribed spacer region of the Trichosporon rRNA gene. DNA in-situ hybridization confirmed the diagnosis. This is the first reported case of Trichosporon-associated meningoencephalitis in a dog.

In vitro susceptibility of equine-obtained isolates of Corynebacterium pseudotuberculosis to gallium maltolate and 20 other antimicrobial agents.

This study's objective was to determine the in vitro antimicrobial activities of gallium maltolate (GaM) and 20 other antimicrobial agents against clinical equine isolates of Corynebacterium pseudotuberculosis. The growth of cultured isolates was not inhibited by any concentration of GaM. MIC data revealed susceptibility to commonly used antimicrobials.

Evaluation of a catalase-based urine test for the detection of urinary tract infection in dogs and cats.

BACKGROUND: Bacterial infection of the urinary tract is a common disorder in dogs and cats. Although microscopic examination of urine sediment is routinely used to screen for infection, this test can lack sensitivity or require expertise. A reliable in-clinic screening test would be a useful adjunct for the identification of dogs and cats with bacterial urinary tract infection (UTI). HYPOTHESIS: That a catalase-based urine test (Accutest Uriscreen™) is a more sensitive screening test for UTI in dogs and cats than urine microscopic sediment examination. ANIMALS: One hundred and sixty client-owned dogs and cats. METHODS: Surplus urine from animals presented to a veterinary teaching hospital was used in this prospective observational study. A routine urinalysis, aerobic bacterial culture, and the Uriscreen test were performed on cystocentesis samples. Sensitivity and specificity with 95% confidence intervals and positive and negative likelihood ratios were calculated for Uriscreen and microscopic sediment examination using culture results as the gold standard. RESULTS: Bacterial culture was positive in 27/165 (16.4%) samples. The sensitivity, specificity, and positive and negative likelihood ratios for the Uriscreen were 89%, 71%, 3.0, and 0.15, respectively. Sensitivity, specificity, and positive and negative likelihood ratios for urine sediment microscopic examination were 78%, 90%, 7.8, and 0.24, respectively. CONCLUSIONS AND CLINICAL IMPORTANCE: The Uriscreen is a more sensitive screening test for UTI in dogs and cats than sediment examination; however, the urine sediment examination was more specific. A negative Uriscreen result helps exclude UTI; however, urine bacterial culture is still necessary to exclude or confirm UTI in all cases.

Caprine abscess model of tulathromycin concentrations in interstitial fluid from tissue chambers inoculated with Corynebacterium pseudotuberculosis following subcutaneous or intrachamber administration.

Corynebacterium pseudotuberculosis causes chronic, suppurative, abscessing conditions in livestock and humans. We used an in vivo model to evaluate antimicrobial efficacy for focal abscesses caused by C. pseudotuberculosis. Tissue chambers were surgically implanted in the subcutaneous tissues of the right and left paralumbar fossa of 12 goats to serve as a model for isolated, focal abscesses. For each goat, one tissue chamber was inoculated with C. pseudotuberculosis, while the contralateral chamber served as an uninoculated control. Six goats were administered a single dose of tulathromycin at 2.5 mg/kg of body weight subcutaneously, while the other six received the same dose by injection directly into the inoculated chambers. Our objective was to compare the effects and tulathromycin concentrations in interstitial fluid (IF) samples collected from C. pseudotuberculosis-infected and control chambers following subcutaneous or intrachamber injection of tulathromycin. In addition, the effects of tulathromycin on the quantity of C. pseudotuberculosis reisolated from inoculated chambers were assessed over time. Tulathromycin IF concentrations from C. pseudotuberculosis-infected and control tissue chambers were similar to those in plasma following subcutaneous administration. Following intrachamber administration, tulathromycin IF concentrations in infected chambers were continuously above the MIC for the C. pseudotuberculosis isolate for 15 days. There were no significant differences for plasma area under the curve and elimination half-lives between subcutaneous and intrachamber administration. Six of the 12 infected chambers had no growth of C. pseudotuberculosis 15 days postadministration. Results of this study indicate that tulathromycin may be beneficial in the treatment of focal infections such as those caused by C. pseudotuberculosis.

Frequency of resistance in obligate anaerobic bacteria isolated from dogs, cats, and horses to antimicrobial agents.

Clinical specimens from dogs, cats, and horses were examined for the presence of obligate anaerobic bacteria. Of 4,018 specimens cultured, 368 yielded 606 isolates of obligate anaerobic bacteria (248 from dogs, 50 from cats, and 308 from horses). There were 100 specimens from 94 animals from which only anaerobes were isolated (25 dogs, 8 cats, and 61 horses). The most common sites tested were abdominal fluid (dogs and cats) and intestinal contents (horses). The most common microorganism isolated from dogs, cats, and horses was Clostridium perfringens (75, 13, and101 isolates, respectively). The MICs of amoxicillin with clavulanate, ampicillin, chloramphenicol, metronidazole, and penicillin were determined using a gradient endpoint method for anaerobes. Isolates collected at necropsy were not tested for antimicrobial susceptibility unless so requested by the clinician. There were 1/145 isolates tested that were resistant to amoxicillin-clavulanate (resistance breakpoint ≥ 16/8 µg/ml), 7/77 isolates tested were resistant to ampicillin (resistance breakpoint ≥ 2 µg/ml), 4/242 isolates tested were resistant to chloramphenicol (resistance breakpoint ≥ 32 µg/ml), 12/158 isolates tested were resistant to clindamycin (resistance breakpoint ≥ 8 µg/ml), 10/247 isolates tested were resistant to metronidazole (resistance breakpoint ≥ 32 µg/ml), and 54/243 isolates tested were resistant to penicillin (resistance breakpoint ≥ 2 µg/ml). These data suggest that anaerobes are generally susceptible to antimicrobial drugs in vitro.

Morphologic and cytokine profile characterization of Salmonella enterica serovar typhimurium infection in calves with bovine leukocyte adhesion deficiency.

The role of neutrophils in the pathogenesis of Salmonella enterica Typhimurium-induced ruminant and human enteritis and diarrhea has yet to be characterized with in vivo models. To address this question, the in vivo bovine ligated ileal loop model of nontyphoidal salmonellosis was used in calves with the naturally occurring bovine leukocyte adhesion deficiency (BLAD) mutation whose neutrophils are unable to extravasate and infiltrate the extravascular matrix. Data obtained from 4 BLAD Holstein calves homozygous for BLAD (CD18-), 1 to 5 weeks of age, were compared with 4 controls, age-matched Holstein calves negative for BLAD (CD18+). Morphologic studies revealed that infection of CD18- calves with S Typhimurium resulted in no significant tissue infiltration by neutrophils, less tissue damage, reduced luminal fluid accumulation, and increased bacterial invasion, when compared with CD18+ calves. Ultrastructurally, lesions in enterocytes induced by S Typhimurium infection in CD18- calves--including attachment and disruption of the brush border, apical membrane ruffling formation, and cellular degeneration--were similar to the ones reported in the literature for CD18- calves. Study of cytokine gene expression by quantitative real-time polymerase chain reaction revealed that early stages of acute infection (4-8 hours postinfection) were associated with increased interleukin 8 gene expression in the absence of tissue influx of neutrophils in CD18- calves, whereas later stages of infection (12 hours postinfection) were associated with increased expression of growth-related oncogene alpha in the presence of neutrophil influx in CD18+ calves. In contrast, the proinflammatory cytokines interleukin 1beta and tumor necrosis factor alpha were poorly correlated with the presence or absence of tissue neutrophils.

Early phase morphological lesions and transcriptional responses of bovine ileum infected with Mycobacterium avium subsp. paratuberculosis.

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of chronic enteritis in ruminants (Johne's disease) and a possible etiopathologic agent in human Crohn's disease. The host-pathogen interaction in this chronic disease has largely depended on the randomly collected static lesions studied in subclinically or clinically infected animals. We have established and utilized the neonatal calf ligated ileal loop model to study the early temporal host changes during MAP infection. After inoculation of ligated ileal loop with MAP, samples were analyzed for bacterial invasion, histologic and ultrastructural morphologic changes, and gene expression at several times (0.5-12 hours) postinfection. Our results indicate that MAP invades the intestinal mucosa as early as 0.5 hour postinoculation. Distribution and migration of neutrophils, monocytes/macrophages, and goblet cells were confirmed by histopathology, scanning and transmission electron microscopy. Coincident with the morphologic analysis, we measured by real-time polymerase chain reaction gene expression of various cytokines/chemokines that are involved in the recruitment of mononuclear and polymorphonuclear leukocytes to the site of infection. We also detected expression of several other genes, including intestinal-trefoil factor, profilin, lactoferrin, and enteric ss-defensin, which may play significant roles in the early MAP infection. Thus, the calf ligated intestinal loop model may be used as a human disease model to understand the role of MAP in the pathogenesis of Crohn's disease.

Regulation of Salmonella enterica serovar typhimurium invasion genes by csrA.

Penetration of intestinal epithelial cells by Salmonella enterica serovar Typhimurium requires the expression of invasion genes, found in Salmonella pathogenicity island 1 (SPI1), that encode components of a type III secretion apparatus. These genes are controlled in a complex manner by regulators within SPI1, including HilA and InvF, and those outside SPI1, such as the two-component regulators PhoP/PhoQ and BarA/SirA. We report here that epithelial cell invasion requires the serovar Typhimurium homologue of Escherichia coli csrA, which encodes a regulator that alters the stability of specific mRNA targets. A deletion mutant of csrA was unable to efficiently invade cultured epithelial cells and showed reduced expression of four tested SPI1 genes, hilA, invF, sipC, and prgH. Overexpression of csrA from an induced araBAD promoter also negatively affected the expression of these genes, indicating that CsrA can act as both a positive and a negative regulator of SPI1 genes and suggesting that the bacterium must tightly control the level or activity of CsrA to achieve maximal invasion. We found that CsrA affected hilA, a regulator of the other three genes we tested, probably by controlling one or more genetic elements that regulate hilA. We also found that both the loss and the overexpression of csrA reduced the expression of two regulators of hilA, hilC and hilD, suggesting that csrA exerts its control of hilA through one or both of these regulators. We further found, however, that CsrA could affect the expression of both invF and sipC independent of its effects on hilA. One additional striking phenotype of the csrA mutant, not observed in a comparable E. coli mutant, was its slow growth. Phenotypic revertants that had normal growth rates, while maintaining the csrA mutation, were common. These suppressed strains, however, did not recover the ability to invade cultured cells, indicating that the csrA-mediated loss of invasion cannot be attributed simply to poor growth and that the growth and invasion deficits of the csrA mutant arise from effects of CsrA on different targets.