Research Note: Epidemiological cutoff values and acquired resistance mechanisms of three veterinary antibiotics against Escherichia coli from chicken respiratory tract infections.

Florfenicol, apramycin, and danofloxacin are antibiotics approved only for veterinary use and that have good therapeutic effects on chicken respiratory infections caused by Escherichia coli. We established epidemiological cutoff values (ECV) for these antibiotics using 363 E. coli isolates from tracheal samples of chickens in 5 veterinary clinics in Guangdong Province, China. The minimum inhibitory concentrations (MIC) were determined using the agar dilution method as per Clinical and Laboratory Standards Institution guidelines. The ECV were then calculated using the statistical method and verified by normalized resistance interpretation and ECOFFinder software programs. The ECV of florfenicol, apramycin, and danofloxacin against E. coli were 16, 16, and 0.125 µg/mL, respectively. Susceptibility tests indicated that these isolates were resistant to florfenicol (66.7%), apramycin (22.3%), and danofloxacin (92.3%). Strains carrying floR were distributed in the range of MIC ≥32 µg/mL for florfenicol. Apramycin resistance was found in 77 strains (77/363, 21.1%), and isolates that carried aac(3)-IV were all in the range of MIC ≥512 µg/mL. Danofloxacin resistance was found in the range of MIC ≤0.125 µg/mL, but there were no mutations in the quinolone resistance-determining regions and plasmid-mediated quinolone resistance genes qnrA, qnrB, qnrC, qnrD, aac-(6')-Ib-cr, qep, and oqxB. The presence of the qnrS gene was verified in a few of the strains with an MIC of 0.06 µg/mL. The establishment of ECV was significant for monitoring of resistance development and therapy guidance.

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.

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.

Characterization of a P1-like bacteriophage carrying CTX-M-27 in Salmonella spp. resistant to third generation cephalosporins isolated from pork in China.

The aim of this study was to elucidate the epidemiology of third generation cephalosporin resistant Samonella isolates from pork of a slaughterhouse in China and the features of transferable elements carrying blaCTX-M genes. One hundred and twenty-six (7.3%) Salmonella isolates were identified; S. Derby and S. Rissen were the most two prevalent serotypes. Among these isolates 20 (15.8%) were resistant to third generation cephalosporins and nine of them carried blaCTX-M-27. S1-PFGE and replicon typing of blaCTX-M-27-carrying plasmids showed that seven were untypeable plasmids of about 104 Kb and two were IncP plasmids of about 300 Kb. Complete sequence analysis of one PBRT-untypeable plasmid showed it was a P1-like bateriophage, named SJ46, which contained a non-phage-associated region with several mobile elements, including Tn1721, ISEcp1B and IS903D. The other six 104 Kb PBRT-untypeable blaCTX-M-27-carrying plasmids also harboured the same phage-insertion region of SJ46 suggesting that they were the same P1-like bacteriophage. PFGE profiles of the parental strains revealed both potential vertical and horizontal spread of this P1-like blaCTX-M-27-containing element. Additionally, the representative gene of the P1 family bacteriophage, repL, was detected in 19.0% (24/126) of the isolates. This study indicated a potential role of P1-family bacteriophage in capture and spread of antimicrobial resistance in pathogens.

Over-expression of atf4 in Xenopus embryos interferes with neurogenesis and eye formation.

Accumulated evidence indicates that the activating transcription factor 4 (atf4) is a developmentally relevant gene. Here, we report on the characterization of atf4 in Xenopus embryos, which is differentially expressed in the central nervous system, eyes, blood, and the pronephros, as well as in developing endodermal organs such as the stomach, duodenum, liver, and pancreas. Ectopic expression of atf4 in the animal hemisphere of Xenopus embryos had no obvious effects on the induction of neural progenitors, but suppressed neurogenesis and eye formation without promoting apoptosis. Our data suggest that tightly controlled atf4 activities may be crucial for normal neurogenesis and early eye patterning.