Accumulation of Peptidoglycan O-Acetylation Leads to Altered Cell Wall Biochemistry and Negatively Impacts Pathogenesis Factors of Campylobacter jejuni.

Campylobacter jejuni is a leading cause of bacterial gastroenteritis in the developed world. Despite its prevalence, its mechanisms of pathogenesis are poorly understood. Peptidoglycan (PG) is important for helical shape, colonization, and host-pathogen interactions in C. jejuni Therefore, changes in PG greatly impact the physiology of this organism. O-acetylation of peptidoglycan (OAP) is a bacterial phenomenon proposed to be important for proper cell growth, characterized by acetylation of the C6 hydroxyl group of N-acetylmuramic acid in the PG glycan backbone. The OAP gene cluster consists of a PG O-acetyltransferase A (patA) for translocation of acetate into the periplasm, a PG O-acetyltransferase B (patB) for O-acetylation, and an O-acetylpeptidoglycan esterase (ape1) for de-O-acetylation. In this study, reduced OAP in ΔpatA and ΔpatB had minimal impact on C. jejuni growth and fitness under the conditions tested. However, accumulation of OAP in Δape1 resulted in marked differences in PG biochemistry, including O-acetylation, anhydromuropeptide levels, and changes not expected to result directly from Ape1 activity. This suggests that OAP may be a form of substrate level regulation in PG biosynthesis. Ape1 acetylesterase activity was confirmed in vitro using p-nitrophenyl acetate and O-acetylated PG as substrates. In addition, Δape1 exhibited defects in pathogenesis-associated phenotypes, including cell shape, motility, biofilm formation, cell surface hydrophobicity, and sodium deoxycholate sensitivity. Δape1 was also impaired for chick colonization and adhesion, invasion, intracellular survival, and induction of IL-8 production in INT407 cells in vitro The importance of Ape1 in C. jejuni biology makes it a good candidate as an antimicrobial target.

Prevalence and Risk Factors Associated With Antimicrobial Resistance in Bacteria Related to Bovine Respiratory Disease-A Broad Cross-Sectional Study of Beef Cattle at Entry Into Canadian Feedlots.

A broad, cross-sectional study of beef cattle at entry into Canadian feedlots investigated the prevalence and epidemiology of antimicrobial resistance (AMR) in Mannheimia haemolytica, Pasteurella multocida, Histophilus somni, and Mycoplasma bovis, bacterial members of the bovine respiratory disease (BRD) complex. Upon feedlot arrival and before antimicrobials were administered at the feedlot, deep nasopharyngeal swabs were collected from 2,824 feedlot cattle in southern and central Alberta, Canada. Data on the date of feedlot arrival, cattle type (beef, dairy), sex (heifer, bull, steer), weight (kg), age class (calf, yearling), source (ranch direct, auction barn, backgrounding operations), risk of developing BRD (high, low), and weather conditions at arrival (temperature, precipitation, and estimated wind speed) were obtained. Mannheimia haemolytica, P. multocida, and H. somni isolates with multidrug-resistant (MDR) profiles associated with the presence of integrative and conjugative elements were isolated more often from dairy-type than from beef-type cattle. Our results showed that beef-type cattle from backgrounding operations presented higher odds of AMR bacteria as compared to auction-derived calves. Oxytetracycline resistance was the most frequently observed resistance across all Pasteurellaceae species and cattle types. Mycoplasma bovis exhibited high macrolide minimum inhibitory concentrations in both cattle types. Whether these MDR isolates establish and persist within the feedlot environment, requires further evaluation.

A direct qPCR screening approach to improve the efficiency of Mycoplasma bovis isolation in the frame of a broad surveillance study.

Culturing Mycoplasma bovis is laborious and unpredictable with most laboratories relying on molecular methods for its detection and identification. However, bacterial culture is still necessary to relate phenotypic characteristics to genotypic traits within and between individual strains. Thus, the main objective of this study was to develop a procedure that saved time and consumables during the culturing of M. bovis within the scope of a broad antimicrobial resistance surveillance project. Deep nasopharyngeal swabs (DNPS) collected from feedlot cattle upon arrival at 10 Southern Alberta feedlots were enriched in broth and an aliquot of the culture was directly used in a M. bovis-specific quantitative PCR (qPCR) assay. Only qPCR-positive cultures were plated onto agar media for the isolation of M. bovis. The detection of M. bovis from broth culture by direct-culture-qPCR proved to be more sensitive (1.61 × 102 CFU/mL) than using a commercial kit (1.61 × 103 CFU/mL) to extract DNA from pure cultures of M. bovis. When isolation of M. bovis from broth-enriched DNPS (n = 208 samples) was used as the gold standard for diagnostics, the qPCR screening approach showed 100% sensitivity, 87.27% specificity, and a kappa index = 0.87 (strong agreement). In contrast, qPCR of DNPS samples (n = 58) exhibited 100% sensitivity, 42.86% specificity, and a kappa index = 0.49 (weak agreement). The qPCR protocol described here together with a high throughput direct-culture-qPCR approach for sample testing made it possible to reduce the labor and cost of M. bovis isolation by eliminating the need to process 97.3% of M. bovis-negative samples. This was possible through the use of qPCR Ct values as a predictive tool of the likelihood of M. bovis isolation. This new procedure could be evaluated for its use in antimicrobial resistance surveillance programs that focus on Mycoplasma species.

A Novel aadA Aminoglycoside Resistance Gene in Bovine and Porcine Pathogens.

A novel variant of the AAD(3″) class of aminoglycoside-modifying enzymes was discovered in fatal bovine respiratory disease-associated pathogens Pasteurella multocida and Histophilus somni. The aadA31 gene encodes a spectinomycin/streptomycin adenylyltransferase and was located in a variant of the integrative and conjugative element ICEMh1, a mobile genetic element transmissible among members of the family Pasteurellaceae. The gene was also detected in Mannheimia haemolytica from a case of porcine pneumonia and in Moraxella bovoculi from a case of keratoconjunctivitis. IMPORTANCE Aminoglycosides are important antimicrobials used worldwide for prophylaxis and/or therapy in multiple production animal species. The emergence of new resistance genes jeopardizes current pathogen detection and treatment methods. The risk of resistance gene transfer to other animal and human pathogens is elevated when resistance genes are carried by mobile genetic elements. This study identified a new variant of a spectinomycin/streptomycin resistance gene harbored in a self-transmissible mobile element. The gene was also present in four different bovine pathogen species.

A Bacterial Cell Shape-Determining Inhibitor.

Helicobacter pylori and Campylobacter jejuni are human pathogens and causative agents of gastric ulcers/cancer and gastroenteritis, respectively. Recent studies have uncovered a series of proteases that are responsible for maintaining the helical shape of these organisms. The H. pylori metalloprotease Csd4 and its C. jejuni homologue Pgp1 cleave the amide bond between meso-diaminopimelate and iso-d-glutamic acid in truncated peptidoglycan side chains. Deletion of either csd4 or pgp1 results in bacteria with a straight rod phenotype, a reduced ability to move in viscous media, and reduced pathogenicity. In this work, a phosphinic acid-based pseudodipeptide inhibitor was designed to act as a tetrahedral intermediate analog against the Csd4 enzyme. The phosphinic acid was shown to inhibit the cleavage of the alternate substrate, Ac-l-Ala-iso-d-Glu-meso-Dap, with a Ki value of 1.5 µM. Structural analysis of the Csd4-inhibitor complex shows that the phosphinic acid displaces the zinc-bound water and chelates the metal in a bidentate fashion. The phosphinate oxygens also interact with the key acid/base residue, Glu222, and the oxyanion-stabilizing residue, Arg86. The results are consistent with the "promoted-water pathway" mechanism for carboxypeptidase A catalysis. Studies on cultured bacteria showed that the inhibitor causes significant cell straightening when incubated with H. pylori at millimolar concentrations. A diminished, yet observable, effect on the morphology of C. jejuni was also apparent. Cell straightening was more pronounced with an acapsular C. jejuni mutant strain compared to the wild type, suggesting that the capsule impaired inhibitor accessibility. These studies demonstrate that a highly polar compound is capable of crossing the outer membrane and altering cell shape, presumably by inhibiting cell shape determinant proteases. Peptidoglycan proteases acting as cell shape determinants represent novel targets for the development of antimicrobials against these human pathogens.

Persistence of Escherichia coli in freshwater periphyton: biofilm-forming capacity as a selective advantage.

Recent research has shown that Escherichia coli can persist in aquatic environments, although the characteristics that contribute to their survival remain poorly understood. This study examines periphytic E. coli populations that were continuously present in three temperate freshwater lakes from June to October 2008 in numbers ranging from 2 to 2 × 10(2)  CFU 100 cm(-2) . A crystal violet assay revealed that all tested periphytic E. coli isolates were superior biofilm formers and they formed, on average, 2.5 times as much biofilm as E. coli isolated from humans, 4.5 times as much biofilm as shiga-like toxin-producing E. coli, and 7.5 times as much biofilm as bovine E. coli isolates. Repetitive extragenic palindromic polymerase chain reaction (REP-PCR) DNA fingerprinting analysis demonstrated the genetically diverse nature of the periphytic isolates, with genetic similarity between strains ranging from 40% to 86%. Additionally, the role of curli fibers in biofilm formation was investigated by comparing biofilm formation with curli expression under optimal conditions, although little correlation (R(2)  = 0.095, P = 0.005) was found. The high mean biofilm-forming capacity observed in E. coli isolated from the periphyton suggests that selective pressures may favor E. coli capable of forming biofilms in freshwater environments.