Emergence of ciprofloxacin heteroresistance in foodborne Salmonella enterica serovar Agona.

BACKGROUND: Bacterial heteroresistance has been increasingly identified as an important phenomenon for many antibiotic/bacterium combinations. OBJECTIVES: To investigate ciprofloxacin heteroresistance in Salmonella and characterize mechanisms contributing to ciprofloxacin heteroresistance. METHODS: Ciprofloxacin-heteroresistant Salmonella were identified by population analysis profiling (PAP). Target mutations and the presence of PMQR genes were detected using PCR and sequencing. Expression of acrB, acrF and qnrS was conducted by quantitative RT-PCR. Competition ability and virulence were also compared using pyrosequencing, blue/white screening, adhesion and invasion assays and a Galleria model. Two subpopulations were whole-genome sequenced using Oxford Nanopore and Illumina platforms. RESULTS: PAP identified one Salmonella from food that yielded a subpopulation demonstrating heteroresistance to ciprofloxacin at a low frequency (10-9 to 10-7). WGS and PFGE analyses confirmed that the two subpopulations were isogenic, with six SNPs and two small deletions distinguishing the resistant from the susceptible. Both subpopulations possessed a T57S substitution in ParC and carried qnrS. The resistant subpopulation was distinguished by overexpression of acrB and acrF, a deletion within rsxC and altered expression of soxS. The resistant population had a competitive advantage against the parental population when grown in the presence of bile salts but was attenuated in the adhesion and invasion of human intestinal cells. CONCLUSIONS: We determined that heteroresistance resulted from a combination of mutations in fluoroquinolone target genes and overexpression of efflux pumps associated with a deletion in rsxC. This study warns that ciprofloxacin heteroresistance exists in Salmonella in the food chain and highlights the necessity for careful interpretation of antibiotic susceptibility.

The Emergence of Chromosomally Located bla CTX-M-55 in Salmonella From Foodborne Animals in China.

The emergence and increase in prevalence of resistance to cephalosporins amongst isolates of Salmonella from food animals imposes a public health threat. The aim of the present study was to investigate the prevalence and characteristics of CTX-M-producing Salmonella isolates from raw meat and food animals. 27 of 152 (17.76%) Salmonella isolates were ESBL-positive including 21/70 (30%) from food animals and 6/82 (7.32%) from raw meat. CTX-M-55 was the most prevalent ESBL type observed (12/27, 44.44%). 7 of 12 CTX-M-55-positive Salmonella isolates were Salmonella Indiana, 2 were Salmonella Typhimurium, 2 were Salmonella Chester, and the remaining isolate was not typeable. Eight CTX-M-55-positive Salmonella isolates were highly resistant to fluoroquinolones (MICCIP = 64 ug/mL) and co-harbored aac(6')-Ib-cr and oqxAB. Most of the CTX-M-55 positive isolates (11/12) carried bla CTX-M-55 genes on the chromosome, with the remaining isolate carrying this gene on a transferable 280 kb IncHI2 plasmid. A chromosomal bla CTX-M-55 gene from one isolate transferred onto a 250 kb IncHI2 plasmid which was subsequently conjugated into recipient strain J53. PFGE and MLST profiles showed a wide range of strain types were carrying bla CTX-M-55. Our study demonstrates the emergence and prevalence of foodborne Salmonella harboring a chromosomally located bla CTX-M-55 in China. The co-existence of PMQR genes with bla CTX-M-55 in Salmonella isolates suggests co-selection and dissemination of resistance to both fluoroquinolones and cephalosporins in Salmonella via the food chain in China represents a public health concern.

Upregulation of AcrEF in Quinolone Resistance Development in Escherichia coli When AcrAB-TolC Function Is Impaired.

We studied mechanisms of drug resistance development in Escherichia coli strains lacking efflux pump components. E. coli K12 deletion mutants were subjected to increasing concentrations of ciprofloxacin (CIP) to determine the frequency of target gene mutations. We generated a series of mutants that were selected based on their minimum inhibitory concentrations (MICs) to CIP, as well as their corresponding point mutations in target genes. The mutants displayed a number of target modifications and, in particular, gyrA mutations altering codons Ser83Leu, Asp87Gly, and Asp87His as well as a change in parC at 78 (substitution of Gly for Asp). All these mutations were related to drug resistance. When exposed to CIP, mutants lacking efflux pump genes acrA and acrB demonstrated a low level of resistance that was because of point mutations in the target genes. High-level resistance was achieved with a 100- to 500-fold increase in expression of efflux pump genes acrE and acrF that compensated for the loss of AcrA and AcrB, and thus resulted in an obvious increase of CIP MIC. We demonstrate that an intact AcrAB-TolC efflux pump is crucial to the development of bacterial resistance. Its activity is complemented by expression of the alternative AcrEF efflux pump.

Coordinated Expression of acrAB-tolC and Eight Other Functional Efflux Pumps Through Activating ramA and marA in Salmonella enterica serovar Typhimurium.

The aim of this study was to determine the expression of eight other functional transporter genes upon acrAB inactivation and also the expression of acrAB when the function of eight other transporters are impaired in Salmonella enterica. We used single- or multigene deletion mutants (i.e., ΔacrA, ΔacrB, ΔtolC, ΔacrAB, ΔacrEF, ΔacrD, ΔmdsABC, ΔmdtABC, ΔemrAB, ΔmacAB, ΔmdfA, ΔmdtK, ΔacrABramA, ΔacrABmarA, and ΔacrABsoxS) and real time (RT)-PCR to quantify the expression of different pump and regulator genes; infection ability was characterized by adhesion and invasion assays. The expression of acrAB operon was increased upon acrB inactivation. Single deletion of acrA or tolC also increased expression of acrB. The deletion of acrAB increased expression of eight other functional efflux pumps genes and vice versa, in which increased expression of ramA and marA was also detected. Mutants containing single deletions of functional pump genes were attenuated in cells. In conclusion, there is a feedback mechanism that coordinates regulation of AcrAB-TolC and eight other functional efflux pumps through the global transcriptional regulators ramA and marA in S. enterica serovar Typhimurium.

Resistance mechanisms and fitness of Salmonella Typhimurium and Salmonella Enteritidis mutants evolved under selection with ciprofloxacin in vitro.

The aim of this study was to investigate the difference in resistance mechanisms and fitness of Salmonella Typhimurium (ST) and Salmonella Enteritidis (SE) mutants selected during the evolution of resistance under exposure to increasing ciprofloxacin concentrations in vitro. Mutations in quinolone target genes were screened by PCR. Phenotypic characterization included susceptibility testing by the broth dilution method, investigation of efflux activity and growth rate, and determination of the invasion of human intestinal epithelium cells in vitro. The two Salmonella serotypes exhibited differences in target gene mutations and efflux pump gene expression during the development of resistance. In the parental strains, ST had a competitive advantage over SE. During the development of resistance, initially, the SE strain was more competitive. However, once ciprofloxacin resistance was acquired, ST once again became the more competitive strain. In the absence of bile salts or at 0.1% bile, the growth rate of SE was initially greater than that of ST, but once ciprofloxacin resistance was acquired, ST had higher growth rates. ST strains showed decreased invasion of epithelial cells in 0.1% bile. These data indicate that ciprofloxacin-resistant ST strains are more competitive than ciprofloxacin-resistant SE strains.

In Vitro Development of Ciprofloxacin Resistance of Salmonella enterica Serovars Typhimurium, Enteritidis, and Indiana Isolates from Food Animals.

Difference in the development of resistance may be associated with the epidemiological spread and drug resistance of different Salmonella enterica serovar strains. In the present study, three susceptible S. enterica serovars, Typhimurium (ST), Enteritidis (SE), and Indiana (SI) strains, were subjected to stepwise selection with increasing ciprofloxacin concentrations. The results indicated that the mutation frequencies of the SI group were 101-104 higher and developed resistance to ciprofloxacin more rapidly compared with the ST and SE groups. Ciprofloxacin accumulation in the SI strain was also higher than the other two strains in the presence of an efflux pump inhibitor. The development of ciprofloxacin resistance was quite different among the three serovar strains. In SI, increasing AcrAB-TolC efflux pump expression and single or double mutations in gyrA with or without a single parC mutation (T57S) were found in the development of ciprofloxacin resistance. In SE, an increase in the AcrAB-TolC efflux pump regulatory gene ramA gradually decreased as resistant bacteria developed; then resistance resulted from gyrA D87G and gyrB E466D mutations and/or in other active efflux pumps besides AcrAB-TolC. For ST, ramA expression increased rapidly along with gyrA D87 N and/or gyrB S464F mutations. In conclusion, persistent use of ciprofloxacin may aggravate the resistance of different S. enterica serovars and prudent use of the fluoroquinolones is needed. The quicker resistance and higher mutation frequency of the SI isolates present a potential public health threat.

Equivocal association of RAD51 polymorphisms with risk of esophageal squamous cell carcinoma in a Chinese population.

AIM: To study the contribution of genetic variation in RAD51 to risk of esophageal squamous cell carcinoma (ESCC). METHODS: Three single nucleotide polymorphisms (SNPs) in RAD51 (rs1801320, rs4144242 and rs4417527) were genotyped in 316 ESCC patients and 316 healthy controls in Anyang area of China using PCR- RFLP (polymerase chain reaction-restriction fragment length polymorphism). Demographic variables between cases and controls were statistically compared by T test and Chi-square test. Hardy-Weinberg equilibrium was evaluated by the Chi-square test. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated to measure any association with ESCC. Haplotype frequencies were estimated by Phase 2.1. RESULT: The genotype frequencies of rs1801320, rs4144242 and rs4417527 in patients with ESCC demonstrated no significant differences from those in control group (P>0.05). When the haplotypes of these three SNPs were constructed and their relationships with ESCC risk investigated, however, CGG was observed to increase the risk (P=0.020, OR=2. 289). CONCLUSIONS: There was no association between the three SNPs of RAD51 and ESCC susceptibility in our Chinese population. However, the CGG haplotype might be a risk factor.

[Correlation of XPD gene with susceptibility to gastric cancer].

BACKGROUND AND OBJECTIVE: Mutations in DNA repair system are related to carcinogenesis. This study was to evaluate the correlations of polymorphisms and haplotypes of XPD gene with individual susceptibility to gastric cancer. METHODS: Genomic DNA were extracted from peripheral blood leukocytes of 207 gastric cancer patients and 212 healthy controls. Genotypes at codon 312 and codon 751 polymorphic sites were identified by amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) or polymerase chain reaction-restriction fragment length polymorphism (PCR-PFLP), respectively. RESULTS: At codon 312, the frequency of GA or AA genotype was higher in the gastric cancer patients than in the healthy controls (P<0.01, OR=3.41, 95% CI: 2.06-4.79; P<0.01, OR=3.47, 95% CI: 1.39-8.68). No significant difference was found in the distribution of the polymorphism at codon 751 between the two groups (P>0.05). By the haplotype AA (codon 312A-codon 751A) analysis, the frequency of heterozygote (-/AA) or homozygote (AA/AA) was higher in the patients than in the controls (P<0.01,OR=2.81, 95% CI:1.82-4.34;P=0.02,OR=3.92, 95% CI:1.31-11.70, respectively). Whereas there were no significant differences of the other three haplotypes between the patients and the controls (P>0.05). CONCLUSIONS: The polymorphism of XPD at codon 312 might contribute to the etiology of gastric cancer. The haplotype AA (codon 312A-codon 751A) would be a critical risk factor of the susceptibility to gastric cancer.