Teresa Quinn

Enterobacter sakazakii: An Emerging Pathogen in Powdered Infant Formula

Enterobacter sakazakii represents a significant risk to the health of neonates. This bacterium is an emerging opportunistic pathogen that is associated with rare but life-threatening cases of meningitis, necrotizing enterocolitis, and sepsis in premature and full-term infants. Infants aged <28 days are considered to be most at risk. Feeding with powdered infant formula (PIF) has been epidemiologically implicated in several clinical cases. Infants should be exclusively breast-fed for the first 6 months of life, and those who are not should be provided with a suitable breast-milk substitute. PIF is not a sterile product; to reduce the risk of infection, the reconstitution of powdered formula should be undertaken by caregivers using good hygienic measures and in accordance with the product manufacturers food safety guidelines.

Multiple Regulatory Pathways Associated with High-Level Ciprofloxacin and Multidrug Resistance in Salmonella enterica Serovar Enteritidis: Involvement of ramA and Other Global Regulators

ABSTRACT Mechanisms of antibiotic resistance were examined in nalidixic acid-resistant Salmonella enterica serovar Enteritidis field isolates displaying decreased susceptibility to ciprofloxacin and in in vitro-derived ciprofloxacin-resistant mutants (104-cip and 5408-cip). All field isolates harbored a single gyrA mutation (D87Y). Deletion of acrB and complementation with wild-type gyrA increased quinolone susceptibility. Selection for ciprofloxacin resistance was associated with the development of an additional gyrA (S83F) mutation in 104-cip, novel gyrB (E466D) and parE (V461G) mutations in 5408-cip, overexpression of acrB and decreased susceptibility to nonquinolone antibiotics in both mutants, and decreased OmpF production and altered lipopolysaccharide in 104-cip. Complementation of mutated gyrA and gyrB with wild-type alleles restored susceptibility to quinolones in 104-cip and significantly decreased the ciprofloxacin MIC in 5408-cip. Complementation of parE had no effect on quinolone MICs. Deletion of acrB restored susceptibility to ciprofloxacin and other antibiotics tested. Both soxS and marA were overexpressed in 104-cip, and ramA was overexpressed in 5408-cip. Inactivation of each of these global regulators lowered ciprofloxacin MICs, decreased expression of acrB, and restored susceptibility to other antibiotics. Mutations were found in soxR (R20H) and in soxS (E52K) in 104-cip and in ramR (G25A) in 5408-cip. In conclusion, both efflux activity and a single gyrA mutation contribute to nalidixic acid resistance and reduced ciprofloxacin sensitivity. Ciprofloxacin resistance and decreased susceptibility to multiple antibiotics can result from different genetic events leading to development of target gene mutations, increased efflux activity resulting from differential expression of global regulators associated with mutations in their regulatory genes, and possible altered membrane permeability.

Myosin Light Chain Kinase Inhibition: Correction of Increased Intestinal Epithelial Permeability In Vitro

PurposeTo examine whether myosin light chain kinase (MLCK) inhibitors can reduce intestinal epithelial permeability increases in vitro.Materials and MethodsIsolated rat, mouse and human colonic tissue mucosae and Caco-2 monolayers were exposed to cytochalasin D (cD) and sodium caprate (C10), in the absence and presence of the MLCK inhibitors, ML-9 and D PIK. Transepithelial electrical resistance (TEER) and Papp of [14C]-mannitol or FITC-dextran 4000 (FD-4) were measured. Western blots were used to measure MLC phosphorylation.ResultsIncreases in Papp of [14C]-mannitol and decreases in TEER were induced by tight junction openers. These changes were attenuated by ML-9. D-PIK offset the FD-4 Papp increase induced by C10 in Caco-2 only, while ML-9 and PIK inhibited MLC directly. cD induced constriction of peri-junctional actin in Caco-2 monolayers, but this was prevented by ML-9. Although mannitol fluxes across colonic mucosae from dextran-sulphate (DSS)-treated mice were higher than control, they were not ameliorated by either ML-9 or PIK in vitro.ConclusionsML-9 inhibits paracellular permeability increases in several intestinal epithelial models. D-PIK reduced stimulated paracellular fluxes in Caco-2 monolayers, but not in tissue. Pre-established increases were not modified by two MLCK inhibitors in a mouse model of IBD.

Fitness Costs and Stability of a High-Level Ciprofloxacin Resistance Phenotype in Salmonella enterica Serotype Enteritidis: Reduced Infectivity Associated with Decreased Expression of Salmonella Pathogenicity Island 1 Genes

ABSTRACT The fitness costs associated with high-level fluoroquinolone resistance were examined for phenotypically and genotypically characterized ciprofloxacin-resistant Salmonella enterica serotype Enteritidis mutants (104-cip and 5408-cip; MIC, >32 μg/ml). The stability of the fluoroquinolone resistance phenotype in both mutants was investigated to assess whether clones with better fitness could emerge in the absence of antibiotic selective pressure. Mutants 104-cip and 5408-cip displayed altered morphology on agar and by electron microscopy, reduced growth rates, motility and invasiveness in Caco-2 cells, and increased sensitivity to environmental stresses. Microarray data revealed decreased expression of virulence and motility genes in both mutants. Two clones, 104-revert and 1A-revertC2, with ciprofloxacin MICs of 3 and 2 μg/ml, respectively, were recovered from separate lineages of 104-cip after 20 and 70 passages, respectively, on antibiotic-free agar. All fitness costs, except motility, were reversed in 104-revert. Potential mechanisms associated with reversal of the resistance phenotype were examined. Compared to 104-cip, both 104-revert and 1A-revertC2 showed decreased expression of acrB and soxS but still overexpressed marA. Both acquired additional mutations in SoxR and ParC, and 1A-revertC2 acquired two mutations in MarA. The altered porin and lipopolysaccharide (LPS) profiles observed in 104-cip were reversed. In contrast, 5408-cip showed no reversal in fitness costs and maintained its high-level ciprofloxacin resistance for 200 passages on antibiotic-free agar. In conclusion, high-level ciprofloxacin resistance in S. Enteritidis is associated with fitness costs. In the absence of antibiotic selection pressure, isolates may acquire mutations enabling reversion to an intermediate-level ciprofloxacin resistance phenotype associated with less significant fitness costs.

Mechanisms of fluoroquinolone resistance in Escherichia coli isolates from food-producing animals.

ABSTRACT Eleven multidrug-resistant Escherichia coli isolates (comprising 6 porcine and 5 bovine field isolates) displaying fluoroquinolone (FQ) resistance were selected from a collection obtained from the University Veterinary Hospital (Dublin, Ireland). MICs of nalidixic acid and ciprofloxacin were determined by Etest. All showed MICs of nalidixic acid of >256 μg/ml and MICs of ciprofloxacin ranging from 4 to >32 μg/ml. DNA sequencing was used to identify mutations within the quinolone resistance-determining regions of target genes, and quantitative real-time PCR (qRT-PCR) was used to evaluate the expression of the major porin, OmpF, and component genes of the AcrAB-TolC efflux pump and its associated regulatory loci. Decreased MIC values to nalidixic acid and/or ciprofloxacin were observed in the presence of the efflux pump inhibitor phenylalanine-arginine-β-naphthylamide (PAβN) in some but not all isolates. Several mutations were identified in genes coding for quinolone target enzymes (3 to 5 mutations per strain). All isolates harbored GyrA amino acid substitutions at positions 83 and 87. Novel GyrA (Asp87 → Ala), ParC (Ser80 → Trp), and ParE (Glu460 → Val) substitutions were observed. The efflux activity of these isolates was evaluated using a semiautomated ethidium bromide (EB) uptake assay. Compared to wild-type E. coli K-12 AG100, isolates accumulated less EB, and in the presence of PAβN the accumulation of EB increased. Upregulation of the acrB gene, encoding the pump component of the AcrAB-TolC efflux pump, was observed in 5 of 11 isolates, while 10 isolates showed decreased expression of OmpF. This study identified multiple mechanisms that likely contribute to resistance to quinolone-based drugs in the field isolates studied.

Multi-drug resistance in Salmonella enterica: efflux mechanisms and their relationships with the development of chromosomal resistance gene clusters.

Bacterial drug resistance represents one of the most crucial problems in present day antibacterial chemotherapy. Of particular concern to public health is the continuing worldwide epidemic spread of Salmonella enterica serovar Typhimurium phage type DT104 harbouring a genomic island called Salmonella genomic island I (SGI-1). This island contains an antibiotic gene cluster conferring resistance to ampicillin, chloramphenicol, florfenicol, streptomycin, sulfonamides and tetracyclines. These resistance genes are assembled in a mosaic pattern, indicative of several independent recombinational events. The mobility of SGI-1 coupled with the ability of various antibiotic resistance genes to be integrated and lost from the chromosomal resistance locus allows for the transfer of stable antibiotic resistance to most of the commonly used antibiotics and adaptation to new antibiotic challenges. This, coupled with the incidence of increasing fluoroquinolone resistance in these strains increases the risk of therapeutic failure in cases of life-threatening salmonellosis. Fluoroquinolone resistance has largely been attributed to mutations occurring in the genes coding for intracellular targets of these drugs. However, efflux by the AcrAB-TolC multi-drug efflux pump has recently been shown to directly contribute to fluoroquinolone resistance. Furthermore, the resistance to chloramphenicol-florfenicol and tetracyclines in DT104 isolates, is due to interaction between specific transporters for these antibiotics encoded by genes mapping to the SGI-1 and the AcrAB-TolC tripartite efflux pump. The potential for the use of efflux pump inhibitors to restore therapeutic efficacy to fluoroquinolones and other antibiotics offers an exciting developmental area for drug discovery.

Development of Multiple-Locus Variable-Number Tandem-Repeat Analysis for the Molecular Subtyping of Enterobacter sakazakii

ABSTRACT The genomic content of Enterobacter sakazakii strain ATCC BAA-894 was analyzed for variable-number tandem repeats (VNTRs). In this study we report the development of a multiple-locus VNTR analysis (MLVA) strategy for the subtyping of E. sakazakii. The method is based on a GeneScan analysis of four VNTR loci labeled with multiple fluorescent dyes. This approach was applied to a collection of 112 isolates representing all 16 of the currently defined E. sakazakii biogroups. MLVA successfully discriminated among these isolates and compared favorably with pulsed-field gel electrophoresis. The method was relatively fast and easy to perform. The potential value of MLVA as an epidemiological tool is discussed.

Antimicrobial resistance profiling and fla-typing of Irish thermophillic Campylobacter spp. of human and poultry origin.

Aims:  To determine the genetic relationship between a random collection of Irish human and poultry Campylobacter isolates and to determine the frequency of antibiotic resistance.

Mechanisms of neurokinin A- and substance P-induced contractions in rat detrusor smooth muscle in vitro

To investigate the mechanisms of neurokinin A‐ and substance P‐induced contractions of rat urinary bladder smooth muscle, and to compare them with those of the muscarinic agonist carbachol.

Antimicrobial resistance profiles and mechanisms of resistance in Campylobacter jejuni isolates from pets.

The presence of antimicrobial resistance in 51 Campylobacter jejuni isolates obtained from cats and dogs was determined by E-testing. Resistance to nalidixic acid (37.3% of isolates), ciprofloxacin (19.6%), tetracycline (13.7%), ampicillin (13.7%), erythromycin (11.8%), and chloramphenicol (5.9%) was detected. Resistance to two antimicrobials or more was present in 31.4% of isolates, and one isolate was resistant to all six antimicrobials. Of the isolates with ciprofloxacin and/or nalidixic acid resistance, 54.5% had the gyrA substitution Thr-86-Ile on sequencing. The tet o gene was detected in 75.0% isolates with high-level resistance to tetracycline. With the observed antimicrobial resistance in C. jejuni isolates from pets in this study, and the detection of identical mechanisms for quinolone and tetracycline resistance in pets and humans, pets should be considered a potential source of (multi)resistant C. jejuni infections in humans.