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

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-ß-naphthylamide (PAßN) 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 PAßN 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.

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.

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 microg/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 microg/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.

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.

Multiple regulatory pathways associated with high-level ciprofloxacin and multidrug resistance in Salmonella enterica serovar enteritidis: involvement of RamA and other global regulators.

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.

PURPOSE: To examine whether myosin light chain kinase (MLCK) inhibitors can reduce intestinal epithelial permeability increases in vitro. MATERIALS AND METHODS: Isolated 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. RESULTS: Increases 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. CONCLUSIONS: ML-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.

Antibiotic-resistant Campylobacter: could efflux pump inhibitors control infection?

Campylobacter is the most common cause of bacterial gastroenteritis in the world. Poultry is the main reservoir of human infections. The widespread use of antibiotics in agriculture and veterinary medicine has resulted in the emergence of an increasing number of antibiotic-resistant Campylobacter strains that can be transmitted to humans through the food chain. Of particular concern to public health is the prevalence of resistance to macrolides and fluoroquinolones that are used in the treatment of life-threatening campylobacteriosis. The CmeABC efflux system has been shown to contribute to the intrinsic and acquired resistance to these antibiotics. In addition, by mediating resistance to bile, it is essential for colonization of the chicken gut in vivo. Inhibition of CmeABC may provide an effective means of reversing antibiotic resistance and decreasing the transmission of Campylobacter via the food chain. This would positively impact on public health by decreasing the morbidity, mortality and increased healthcare costs associated with the treatment of antibiotic-resistant Campylobacter.

High-level resistance to moxifloxacin and gatifloxacin associated with a novel mutation in gyrB in toxin-A-negative, toxin-B-positive Clostridium difficile.

OBJECTIVES: To determine the mechanism of high-level resistance to fluoroquinolone antimicrobials in toxin-A-negative, toxin-B-positive (A- B+) Clostridium difficile isolates. METHODS: Following culture 16-23S PCR ribotyping was used to determine genomic relationships between A- B+ C. difficile isolates. Antimicrobial susceptibilities were determined using Etests in the presence and absence of the efflux pump inhibitors reserpine (20 microg/mL), L-phenylalanine-L-arginine-beta-naphthylamide (PAbetaN; 20 microg/mL) and verapamil (100 microg/mL). Genomic regions including the quinolone-resistance-determining-region (QRDR) of gyrA and gyrB were amplified and characterized. RESULTS: PCR ribotyping profiles identified one major cluster of A- B+ C. difficile, universally resistant to the fluoroquinolones tested (ofloxacin, ciprofloxacin, levofloxacin, moxifloxacin and gatifloxacin; MICs > 32 mg/L). All isolates with high-level resistance had a transversion mutation (A-->T) resulting in the amino acid substitution Asp-426-->Val in gyrB. Non-clonal isolates were susceptible to moxifloxacin and gatifloxacin (MICs 0.3 and 0.4 mg/L, respectively) with reduced susceptibility to levofloxacin (MIC 3 mg/L) consistent with the wild-type genotype. The MICs for resistant isolates were not significantly affected by the addition of any of the efflux pump inhibitors. No amino acid substitutions were identified in the QRDR of gyrA. CONCLUSIONS: High-level resistance to fluoroquinolones in A- B+ C. difficile is associated with a novel transversion mutation in gyrB. The emergence of universal resistance in different C. difficile strain types may be a factor promoting outbreaks in hospitals.

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.

Role of Rho-kinase in guinea-pig gallbladder smooth muscle contraction.

Guinea-pig gallbladder smooth muscle contractions can be elicited pharmacologically by a range of mechanisms. The involvement of Rho-kinase in contractions mediated by receptor-dependent and receptor-independent mechanisms was investigated using the Rho-kinase inhibitor (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl) cyclohexane carboxamide (Y-27632). In a separate series of experiments, the role of Rho-kinase in the contractile response to Ca2+ entry through store-operated Ca2+ channels and to electrical field stimulation was also examined. Y-27632 (10 microM), which caused a significant decrease (P<0.0005) in basal resting tone, significantly inhibited gallbladder contractions evoked by cumulative additions of the G-protein-coupled agonists, carbachol (1 nM-100 microM; P<0.05) and cholecystokinin (10 nM-1 microM; P<0.005). Y-27632 also inhibited the contractions evoked by a single addition of the sarcoplasmic reticulum ATPase inhibitor, thapsigargin (1 microM; P<0.0005) and cumulative additions of KCl (10-85 mM; P<0.0005). The contractile response to Ca2+ entry through store-operated Ca2+ channels was significantly inhibited by Y-27632 (P<0.05) as were the contractile responses evoked by electrical field stimulation (2-25 Hz; P<0.0005). In contrast, Y-27632 had no significant effect on contractions evoked by phorbol 12,13-dibutyrate (0.1 nM-1 microM; a protein kinase C activator) or by the phosphatase inhibitor, cantharidin (100 microM). In conclusion, Rho-kinase contributes to the contractile response in guinea-pig gallbladder smooth muscle evoked by both G-protein-coupled and non-G-protein-coupled mechanisms in addition to contributing to the maintenance of basal tone. It also contributes to the contractile responses resulting from electrical field stimulation and store-operated Ca2+ channel entry.

An investigation of the molecular mechanisms contributing to high-level erythromycin resistance in Campylobacter.

The molecular mechanisms contributing to high-level erythromycin resistance in Campylobacter jejuni and Campylobacter coli isolates were investigated. The A2075G mutation in the 23S rRNA target genes was identified in all high-level erythromycin-resistant isolates. A number of amino acid substitutions together with insertions and deletions were identified in the corresponding genes encoding L4 and L22 ribosomal proteins both of resistant and susceptible isolates. Amino acid substitutions identified in the resistant strains were located outside regions known to be altered in these proteins. The efflux pump inhibitor L-phenylalanine-L-arginine-beta-naphthylamide (PAbetaN) increased the susceptibility to erythromycin in one of four isolates displaying high-level erythromycin resistance, and reduced the minimal inhibitory concentration displayed by an erythromycin-susceptible C. coli isolate. The A2075G mutation in the 23S rRNA appeared to be the main contributor to high-level erythromycin resistance in Campylobacter. Other mutations/amino acid substitutions found in the 50S ribosomal subunit encoding proteins L4 and L22 do not appear to be linked to the high-level erythromycin-resistant phenotype. Active efflux contributes to the intrinsic resistance to erythromycin in Campylobacter and may contribute to high-level resistance in some isolates.

Antimicrobial resistance in nontyphoidal Salmonella from food sources in Colombia: evidence for an unusual plasmid-localized class 1 integron in serotypes Typhimurium and Anatum.

Seventy-two isolates representing 18 serotypes recovered from various food samples collected in Colombia were tested for antimicrobial susceptibilities. The collection was further characterized for extended-spectrum cephalosporin, aminoglycoside, and tetracycline resistance markers. Multidrug resistant (MDR) isolates were further investigated for class 1 integrons and were evaluated for the presence of conjugative plasmids along with a determination of the incompatibility group by polymerase chain reaction (PCR). Antibiogram analysis showed that the incidence rate of ceftiofur resistance was moderately high (15%). A similar level of resistance to neomycin and oxytetracycline (11% and 10%, respectively) was also observed. There was a high prevalence of gene cassettes as part of one or more class 1 integrons (61%), many of which contained determinants that contributed to the resistance profile. Class 1 integrons identified in MDR Salmonella enterica serotypes Typhimurium and Anatum isolates were characterized. Sequencing identified several incomplete open reading frames (ORFs) as part of a gene cassette (bla-( imp-13 ), dfr7, blr1088, and aac8) along with a complete gene cassette (bla-(oxa2)) in each case. A mosaic of gene cassettes was identical in the two Salmonella serotypes. These integrons were located to a conjugative replicon. Plasmid profiling and incompatibility typing identified three plasmids belonging to Inc groups A/C, P, and W. Our study highlights the role of integrons, contributing to a MDR phenotype that is capable of dissemination to other bacteria.

Characterization of a cmeABC operon in a quinolone-resistant Campylobacter coli isolate of Irish origin.

In this study, a tripartite-operon-encoding efflux system together with its regulatory gene was characterized in an Irish Campylobacter coli isolate CIT-382 showing high-level resistance to nalidixic acid and ciprofloxacin. Sequence comparisons revealed significant homology between C. coli and the cmeABC operon of Campylobacter jejuni. Conservation of functional sequence domains and motifs were noted among C. coli and similar operons in unrelated organisms. A transcriptional regulatory gene cmeR located proximal to cmeABC was also identified. C. coli CIT-382 harbored the Thr-86-Ile amino acid substitution in the gyrA gene. Accumulation studies with ethidium bromide in the presence of known efflux pump inhibitors confirmed the presence of efflux pump activity in C. coli CIT-382. The efflux pump inhibitor PAbetaN had no effect on the MICs to quinolones. Our data suggest that the gyrA gene mutation is the main contributor to the high-level nalidixic acid and ciprofloxacin resistance observed in this Irish C. coli CIT-382 isolate.

Relative contribution of target gene mutation and efflux to varying quinolone resistance in Irish Campylobacter isolates.

The contribution of target gene mutations and active efflux to varying levels of quinolone resistance in Irish Campylobacter isolates was studied. The Thr-86-Ile modification of GyrA did not correlate with the level of quinolone resistance. The efflux pump inhibitor Phe-Arg-beta-Naphthylamide (PAbetaN) had no effect on the MICs to ciprofloxacin. In contrast, a PAbetaN sensitive efflux system contributed to the low-level nalixidic acid resistance phenotype. The lack of effect of PAbetaN in high-level resistant nalidixic isolates may be attributable to mutations identified in the CmeB efflux pump of these isolates. PAbetaN may have limited diagnostic value in the assessment of the contribution of efflux pump activity to ciprofloxacin resistance in Campylobacter.

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

OBJECTIVE: 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. MATERIALS AND METHODS: Rat urinary bladder strips were suspended under 1 g of tension in a physiological buffer at 37 degrees C, gassed with 95% O(2)/5% CO(2). Mechanical activity was recorded isometrically during exposure to neurokinin A and substance P. RESULTS: Both agents produced concentration-dependent contractions of smooth muscle strips which were unaffected by tetrodotoxin (1 micro mol/L), peptidase inhibitors (captopril, thiorphan and bestatin; 1 micro mol/L each) or piroxicam (10 micro mol/L). The rank order of potency of agonists was neurokinin A > substance P > carbachol. Contractile responses to neurokinin A and substance P, like the contractile responses to carbachol, were abolished in a nominally Ca(2+)-free medium and significantly reduced by nifedipine (1 micro mol/L). SKF-96365 (60 micro mol/L), an inhibitor of receptor-mediated Ca(2+) entry, abolished the nifedipine-resistant response to substance P and carbachol, and significantly attenuated the response to neurokinin A. Depleting intracellular Ca(2+) stores with thapsigargin (1 micro mol/L) significantly attenuated neurokinin A-induced contractions but had no effect on substance P- or carbachol- induced contractions. The Rho-kinase inhibitor, Y-27632 (10 micro mol/L), significantly reduced both phasic and tonic components of the contractile responses to neurokinin A, substance P and carbachol. CONCLUSION: The contractile responses induced by tachykinins in rat urinary bladder smooth muscle strips involve a direct action on smooth muscle and are not modulated by peptidases or prostanoids. Neurokinin A and substance P, like carbachol-induced contractions, depend on extracellular Ca(2+) influx largely through voltage-operated and partly through receptor-operated Ca(2+) channels. Intracellular Ca(2+) release contributes to the contractile response to neurokinin A but appears to have no involvement in substance P- and carbachol-induced contractions. Rho-kinase activation contributes to contractions induced by substance P, neurokinin A and carbachol.