Characterization of genotoxin-encoding Escherichia coli isolated from specific-pathogen free cats with impaired fertility.

Escherichia coli strains encoding colibactin (pks), hemolysin-associated cytotoxic necrotizing factor (cnf), and cytolethal distending toxin (cdt) are associated with intestinal inflammation and cancer, urinary tract infection, and septicemia in susceptible hosts. Over a 2-year period, an inbred laboratory colony of specific-pathogen free (SPF) cats (∼25) presented with resorptions, stillbirths, and pyometras in >50 % of pregnancies. Hemolytic E. coli were cultured from vaginal and preputial swabs of clinically normal, intact males, healthy kittens, and placenta and fetal tissues of a dam with reproductive disorders. We hypothesized cats from this colony were colonized with cytotoxin-encoding E. coli. 27 E. coli isolates were cultured from 20 fresh feces representing the majority of cats with and without fertility failures. Two E. coli isolates were also cultured from vaginal swabs from the same cat. 22 isolates (75.9 %) demonstrated hemolysis on blood agar. Twelve isolates (41.4 %) were pks+, 14 (48.3 %) were cnf+, and 10 (34.5 %) were cdt+ by PCR. Serotypes and virulence factor profiles were consistent with the extraintestinal E. coli (ExPEC) pathotype. Antibiotic resistance to cephalothin was exhibited in 13/14 representative isolates. Whole genome sequence analysis of 3 representative isolates confirmed the hemolysin-associated cnf, cdt, and the pks gene island. Representative isolates were cytotoxic to cervical epithelial cells in vitro. This study indicated ExPEC were present in SPF cats with a history of reproductive failure. While causality cannot be established, it is probable ExPEC was associated with impaired reproductive health and breeding success. Since treatment of the colony with cefovecin, reproductive performance has appreciably improved.

Plasmid-Mediated Quinolone Resistance in Shigella flexneri Isolated From Macaques.

Non-human primates (NHPs) for biomedical research are commonly infected with Shigella spp. that can cause acute dysentery or chronic episodic diarrhea. These animals are often prophylactically and clinically treated with quinolone antibiotics to eradicate these possible infections. However, chromosomally- and plasmid-mediated antibiotic resistance has become an emerging concern for species in the family Enterobacteriaceae. In this study, five individual isolates of multi-drug resistant Shigella flexneri were isolated from the feces of three macaques. Antibiotic susceptibility testing confirmed resistance or decreased susceptibility to ampicillin, amoxicillin-clavulanic acid, cephalosporins, gentamicin, tetracycline, ciprofloxacin, enrofloxacin, levofloxacin, and nalidixic acid. S. flexneri isolates were susceptible to trimethoprim-sulfamethoxazole, and this drug was used to eradicate infection in two of the macaques. Plasmid DNA from all isolates was positive for the plasmid-encoded quinolone resistance gene qnrS, but not qnrA and qnrB. Conjugation and transformation of plasmid DNA from several S. flexneri isolates into antibiotic-susceptible Escherichia coli strains conferred the recipients with resistance or decreased susceptibility to quinolones and beta-lactams. Genome sequencing of two representative S. flexneri isolates identified the qnrS gene on a plasmid-like contig. These contigs showed >99% homology to plasmid sequences previously characterized from quinolone-resistant Shigella flexneri 2a and Salmonella enterica strains. Other antibiotic resistance genes and virulence factor genes were also identified in chromosome and plasmid sequences in these genomes. The findings from this study indicate macaques harbor pathogenic S. flexneri strains with chromosomally- and plasmid-encoded antibiotic resistance genes. To our knowledge, this is the first report of plasmid-mediated quinolone resistance in S. flexneri isolated from NHPs and warrants isolation and antibiotic testing of enteric pathogens before treating macaques with quinolones prophylactically or therapeutically.

Role of the AcrAB-TolC efflux pump in determining susceptibility of Haemophilus influenzae to the novel peptide deformylase inhibitor LBM415.

Haemophilus influenzae isolates vary widely in their susceptibilities to the peptide deformylase inhibitor LBM415 (MIC range, 0.06 to 32 microg/ml); however, on average, they are less susceptible than gram-positive organisms, such as Staphylococcus aureus and Streptococcus pneumoniae. Insertional inactivation of the H. influenzae acrB or tolC gene in strain NB65044 (Rd strain KW20) increased susceptibility to LBM415, confirming a role for the AcrAB-TolC pump in determining resistance. Consistent with this, sequencing of a PCR fragment generated with primers flanking the acrRA region from an LBM415-hypersusceptible H. influenzae clinical isolate revealed a genetic deletion of acrA. Inactivation of acrB or tolC in several clinical isolates with atypically reduced susceptibility to LBM415 (MIC of 16 microg/ml or greater) significantly increased susceptibility, confirming that the pump is also a determinant of decreased susceptibility in these clinical isolates. Examination of acrR, encoding the putative repressor of pump gene expression, from several of these strains revealed mutations introducing frameshifts, stop codons, and amino acid changes relative to the published sequence, suggesting that loss of pump repression leads to decreased susceptibility. Supporting this, NB65044 acrR mutants selected by exposure to LBM415 at 8 microg/ml had susceptibilities to LBM415 and other pump substrates comparable to the least sensitive clinical isolates and showed increased expression of pump genes.

Acetate acts as a protonophore and differentially affects bead movement and cell migration of the gliding bacterium Cytophaga johnsonae (Flavobacterium johnsoniae).

Cells of Cytophaga johnsonae (now Flavobacterium johnsoniae) are able to translocate on solid surfaces but are unable to swim in liquid media. Organelles that may be involved in this gliding motility have not been detected, and the mechanism(s) responsible remains unknown. The movement of latex beads attached to the cell surface is considered by some to be a manifestation of the gliding machinery. In this study, acetate (in nutrient-level quantity, 45 mM) was found to inhibit bead movement on cell surfaces, whilst formation and movement of groups of cells (rafts) and typical colony spread were not affected; generation time (in liquid culture) was only slightly increased. Since acetate is a weak acid and is recognized as a protonophore, various electron-transport-associated features were assessed in an effort to understand the differential effects of acetate on bead movement and cell motility. Selected protonophores and electron transport inhibitors were tested to compare their effects on cell translocation and metabolic activities with those of acetate. Although O2 consumption was not significantly affected in the presence of acetate and the protonmotive force decreased only minimally, ATP levels were markedly decreased. Arsenate and cyanide were also shown to inhibit bead movement but did not inhibit either movement of rafts of cells or colony spreading. Cyanide lowered O2 consumption, while arsenate did not; both compounds effected substantial decreases in cellular ATP content, but little or no decrease in protonmotive force. The inhibitory effects of these compounds on bead movement over cell surfaces contrasted with the continued ability of cells to form rafts, to glide and to form spreading colonies and led to the conclusion that bead movement is not a complete correlate of the gliding machinery of C. johnsonae. In addition, it seems likely that bead movement is more affected by the level of cellular ATP than it is by the protonmotive force, which has been assumed to provide the energy (derived from the transmembrane gradients) for the gliding machinery.