Cystitis: significant associations between pathology, histology, and quantitative bacteriology in sows, a cross-sectional study.

BACKGROUND: The importance of cystitis in pig production is controversial and sparse information is available on its frequency and etiology in sows. The aim of this study was to determine the prevalence of bacteriuria, macroscopical and histological lesions of the urinary bladder in culled sows. Urinary bladders and urine samples were obtained from 176 culled sows at slaughter. The urine samples collected by cystocentesis were analyzed to determine bacterial content and pathological findings, macroscopic as well as microscopic, of the bladder were recorded for each sow. RESULTS: The prevalence of bacteriuria, defined by bacterial concentrations ≥ 103 colony forming units per mL of urine, was 34%. Escherichia coli was isolated from 69% of the samples with bacteriuria. Redness of the mucosa was the most frequently observed macroscopic change of the bladder. Intense redness and presence of pus was considered significant pathological changes and occurred in 27% of the urinary bladders. The histopathological examination showed that mononuclear cells were the predominant type of cell infiltration in the bladder mucosa, while neutrophils occurred in very few samples. The criteria for cystitis determined by histopathology were met in 46% of the samples. The criteria were based on presence of hyperemia, edema, and inflammatory cell reaction defined as 40 or more neutrophils or mononuclear cells per high power field. All three indicators of cystitis were significantly associated with each other (p < 0.05) at sow level. CONCLUSION: This study shows that signs of cystitis are common in culled sows. The prevalence of cystitis was 34% based on bacteriological examination, 27% based on macroscopic examination and 46% based on histological examination. Significant associations were found between the three indicators of cystitis: bacteriuria, pathological and histological lesions of the bladder. Based on macroscopic changes and histopathology of the bladder, the cut-off of ≥ 103 colony forming units per mL of urine seems to be appropriate for assessing urine cultures obtained by cystocentesis.

Carprofen-induced depletion of proton motive force reverses TetK-mediated doxycycline resistance in methicillin-resistant Staphylococcus pseudintermedius.

We previously showed that doxycycline (DOX) and carprofen (CPF), a veterinary non-steroidal anti-inflammatory drug, have synergistic antimicrobial activity against methicillin-resistant Staphylococcus pseudintermedius (MRSP) carrying the tetracycline resistance determinant TetK. To elucidate the molecular mechanism of this synergy, we investigated the effects of the two drugs, individually and in combination, using a comprehensive approach including RNA sequencing, two-dimensional differential in-gel electrophoresis, macromolecule biosynthesis assays and fluorescence spectroscopy. Exposure of TetK-positive MRSP to CPF alone resulted in upregulation of pathways that generate ATP and NADH, and promote the proton gradient. We showed that CPF is a proton carrier that dissipates the electrochemical potential of the membrane. In the presence of both CPF and DOX, the energy compensation strategy was attenuated by downregulation of all the processes involved, such as citric acid cycle, oxidative phosphorylation and ATP-providing arginine deiminase pathway. Furthermore, protein biosynthesis inhibition increased from 20% under DOX exposure alone to 75% upon simultaneous exposure to CPF. We conclude that synergistic interaction of the drugs restores DOX susceptibility in MRSP by compromising proton-motive-force-dependent TetK-mediated efflux of the antibiotic. MRSP is unable to counterbalance CPF-mediated PMF depletion by cellular metabolic adaptations, resulting in intracellular accumulation of DOX and inhibition of protein biosynthesis.

Repurposing Azithromycin and Rifampicin Against Gram-Negative Pathogens by Combination With Peptidomimetics.

Synthetic peptidomimetics may be designed to mimic functions of antimicrobial peptides, including potentiation of antibiotics, yet possessing improved pharmacological properties. Pairwise screening of 42 synthetic peptidomimetics combined with the antibiotics azithromycin and rifampicin in multidrug-resistant (MDR) Escherichia coli ST131 and Klebsiella pneumoniae ST258 led to identification of two subclasses of α-peptide/ß-peptoid hybrids that display synergy with azithromycin and rifampicin (fractional inhibitory concentration indexes of 0.03-0.38). Further screening of the best three peptidomimetics in combination with a panel of 21 additional antibiotics led to identification of peptidomimetics that potentiated ticarcillin/clavulanate and erythromycin against E. coli, and clindamycin against K. pneumoniae. The study of six peptidomimetics was extended to Pseudomonas aeruginosa, confirming synergy with antibiotics for five of them. The most promising compound, H-(Lys-ßNPhe)8-NH2, exerted only a minor effect on the viability of mammalian cells (EC50 ≥ 124-210 µM), and thus exhibited the highest selectivity toward bacteria. This compound also synergized with rifampicin and azithromycin at sub-micromolar concentrations (0.25-0.5 µM), thereby inducing susceptibility to these antibiotics at clinically relevant concentrations in clinical MDR isolates. This peptidomimetic lead and its analogs constitute promising candidates for efficient repurposing of rifampicin and azithromycin against Gram-negative pathogens.

International Society for Companion Animal Infectious Diseases (ISCAID) guidelines for the diagnosis and management of bacterial urinary tract infections in dogs and cats.

Urinary tract disease is a common clinical presentation in dogs and cats, and a common reason for antimicrobial prescription. This document is a revision and expansion on the 2011 Antimicrobial Use Guidelines for Treatment of Urinary Tract Disease in Dogs and Cats, providing recommendations for diagnosis and management of sporadic bacterial cystitis, recurrent bacterial cystitis, pyelonephritis, bacterial prostatitis, and subclinical bacteriuria. Issues pertaining to urinary catheters, medical dissolution of uroliths and prophylaxis for urological procedures are also addressed.

Characterization, mechanism of action and optimization of activity of a novel peptide-peptoid hybrid against bacterial pathogens involved in canine skin infections.

Integumentary infections like pyoderma represent the main reason for antimicrobial prescription in dogs. Staphylococcus pseudintermedius and Pseudomonas aeruginosa are frequently identified in these infections, and both bacteria are challenging to combat due to resistance. To avoid use of important human antibiotics for treatment of animal infections there is a pressing need for novel narrow-spectrum antimicrobial agents in veterinary medicine. Herein, we characterize the in vitro activity of the novel peptide-peptoid hybrid B1 against canine isolates of S. pseudintermedius and P. aeruginosa. B1 showed potent minimum inhibitory concentrations (MICs) against canine S. pseudintermedius and P. aeruginosa isolates as well rapid killing kinetics. B1 was found to disrupt the membrane integrity and affect cell-wall synthesis in methicillin-resistant S. pseudintermedius (MRSP). We generated 28 analogues of B1, showing comparable haemolysis and MICs against MRSP and P. aeruginosa. The most active analogues (23, 26) and B1 were tested against a collection of clinical isolates from canine, of which only B1 showed potent activity. Our best compound 26, displayed activity against P. aeruginosa and S. pseudintermedius, but not the closely related S. aureus. This work shows that design of target-specific veterinary antimicrobial agents is possible, even species within a genus, and deserves further exploration.

Repurposing azithromycin and rifampicin against Gram-negative pathogens by combination with peptide potentiators.

Gram-negative bacterial pathogens are intrinsically resistant to several antibiotics that are not able to penetrate the cell envelope barrier. The aim of this study was to identify peptides that at low concentrations induce susceptibility to these antibiotics in multidrug-resistant (MDR) Gram-negative bacterial strains of clinical relevance. Pairwise screening of 34 diverse peptides and four antibiotics (erythromycin, linezolid, rifampicin and vancomycin) with primary activity against Gram-positive bacteria identified 4 peptides that at submicromolar concentrations conferred susceptibility to rifampicin or erythromycin in Escherichia coli ATCC 25922. The identified peptides exhibited synergy with azithromycin and potentiated clindamycin in MDR E. coli ST131 and Klebsiella pneumoniae ST258. The low cytotoxicity toward eukaryotic cells (IC50 > 50 µM) observed for two of these peptides (KLWKKWKKWLK-NH2 and GKWKKILGKLIR-NH2) prompted synthesis and evaluation of the corresponding all-d analogues (D1 and D2), which retained similar synergistic antibacterial profiles. Low concentrations of D1 and D2 in combination with azithromycin and rifampicin inhibited growth of most clinical E. coli, K. pneumoniae and Acinetobacter baumannii strains tested. These data demonstrate that combinatorial screening at low peptide concentrations constitutes an efficient approach to identify clinically relevant peptide-antibiotic combinations. In vivo pharmacokinetic/pharmacodynamic and toxicity studies are needed to further validate the use of the peptides identified in this study for repurposing azithromycin and rifampicin against Gram-negative pathogens.

A High-Throughput Approach To Identify Compounds That Impair Envelope Integrity in Escherichia coli.

The envelope of Gram-negative bacteria constitutes an impenetrable barrier to numerous classes of antimicrobials. This intrinsic resistance, coupled with acquired multidrug resistance, has drastically limited the treatment options against Gram-negative pathogens. The aim of the present study was to develop and validate an assay for identifying compounds that increase envelope permeability, thereby conferring antimicrobial susceptibility by weakening of the cell envelope barrier in Gram-negative bacteria. A high-throughput whole-cell screening platform was developed to measure Escherichia coli envelope permeability to a ß-galactosidase chromogenic substrate. The signal produced by cytoplasmic ß-galactosidase-dependent cleavage of the chromogenic substrate was used to determine the degree of envelope permeabilization. The assay was optimized by using known envelope-permeabilizing compounds and E. coli gene deletion mutants with impaired envelope integrity. As a proof of concept, a compound library comprising 36 peptides and 45 peptidomimetics was screened, leading to identification of two peptides that substantially increased envelope permeability. Compound 79 reduced significantly (from 8- to 125-fold) the MICs of erythromycin, fusidic acid, novobiocin and rifampin and displayed synergy (fractional inhibitory concentration index, <0.2) with these antibiotics by checkerboard assays in two genetically distinct E. coli strains, including the high-risk multidrug-resistant, CTX-M-15-producing sequence type 131 clone. Notably, in the presence of 0.25 µM of this peptide, both strains were susceptible to rifampin according to the resistance breakpoints (R > 0.5 µg/ml) for Gram-positive bacterial pathogens. The high-throughput screening platform developed in this study can be applied to accelerate the discovery of antimicrobial helper drug candidates and targets that enhance the delivery of existing antibiotics by impairing envelope integrity in Gram-negative bacteria.

Large outbreak caused by methicillin resistant Staphylococcus pseudintermedius ST71 in a Finnish Veterinary Teaching Hospital--from outbreak control to outbreak prevention.

INTRODUCTION: The purpose of this study was to describe a nosocomial outbreak caused by methicillin resistant Staphylococcus pseudintermedius (MRSP) ST71 SCCmec II-III in dogs and cats at the Veterinary Teaching Hospital of the University of Helsinki in November 2010 - January 2012, and to determine the risk factors for acquiring MRSP. In addition, measures to control the outbreak and current policy for MRSP prevention are presented. METHODS: Data of patients were collected from the hospital patient record software. MRSP surveillance data were acquired from the laboratory information system. Risk factors for MRSP acquisition were analyzed from 55 cases and 213 controls using multivariable logistic regression in a case-control study design. Forty-seven MRSP isolates were analyzed by pulsed field gel electrophoresis and three were further analyzed with multi-locus sequence and SCCmec typing. RESULTS: Sixty-three MRSP cases were identified, including 27 infections. MRSPs from the cases shared a specific multi-drug resistant antibiogram and PFGE-pattern indicated clonal spread. Four risk factors were identified; skin lesion (OR = 6.2; CI95% 2.3-17.0, P = 0.0003), antimicrobial treatment (OR = 3.8, CI95% 1.0-13.9, P = 0.0442), cumulative number of days in the intensive care unit (OR = 1.3, CI95% 1.1-1.6, P = 0.0007) or in the surgery ward (OR = 1.1, CI95% 1.0-1.3, P = 0.0401). Tracing and screening of contact patients, enhanced hand hygiene, cohorting and barrier nursing, as well as cleaning and disinfection were used to control the outbreak. To avoid future outbreaks and spread of MRSP a search-and-isolate policy was implemented. Currently nearly all new MRSP findings are detected in screening targeted to risk patients on admission. CONCLUSION: Multidrug resistant MRSP is capable of causing a large outbreak difficult to control. Skin lesions, antimicrobial treatment and prolonged hospital stay increase the probability of acquiring MRSP. Rigorous control measures were needed to control the outbreak. We recommend the implementation of a search-and-isolate policy to reduce the burden of MRSP.