Surveillance of antimicrobial-resistant Escherichia coli in Sheltered dogs in the Kanto Region of Japan

There is a lack of an established antimicrobial resistance (AMR) surveillance system in animal welfare centers. Therefore, the AMR prevalence in shelter dogs is rarely known. Herein, we conducted a survey in animal shelters in Chiba and Kanagawa prefectures, in the Kanto Region, Japan, to ascertain the AMR status of Escherichia coli (E. coli) prevalent in shelter dogs. E. coli was detected in the fecal samples of all 61 and 77 shelter dogs tested in Chiba and Kanagawa, respectively. The AMR was tested against 20 antibiotics. E. coli isolates derived from 16.4% and 26.0% of samples from Chiba and Kanagawa exhibited resistance to at least one antibiotic, respectively. E. coli in samples from Chiba and Kanagawa prefectures were commonly resistant to ampicillin, piperacillin, streptomycin, kanamycin, tetracycline, and nalidixic acid; that from the Kanagawa Prefecture to cefazolin, cefotaxime, aztreonam, ciprofloxacin, and levofloxacin and that from Chiba Prefecture to chloramphenicol and imipenem. Multidrug-resistant bacteria were detected in 18 dogs from both regions; β-lactamase genes (blaTEM, blaDHA-1, blaCTX-M-9 group CTX-M-14), quinolone-resistance protein genes (qnrB and qnrS), and mutations in quinolone-resistance-determining regions (gyrA and parC) were detected. These results could partially represent the AMR data in shelter dogs in the Kanto Region of Japan.

Rapid Detection of Quinolone Resistance Mutations in gyrA of Helicobacter pylori by Real-Time PCR

The treatment of infections by the gastric pathogen Helicobacter pylori (H. pylori) has become more difficult due to increased rates of resistances against various antibiotics. Typically, atriple therapy, employing a combination of at least two antibiotics and a proton pump inhibitor, is used to cure H. pylori infections. In case of first-line therapy failure, quinolones are commonly applied in a second-line therapy. To prevent second-line treatment failures, we developed an improved method to detect the most common quinolone-resistance mutations located in the quinolone-resistance-determining region (QRDR) of the bacterial gyrA gene. Biopsy material from the gastric mucosa of infected patients was used to identify quinolone-resistant strains before the onset of drug administration. Two different wild-type and six mutant QRDR sequences were included. Melting curve analyses were performed with corresponding gyrA plasmid DNAs using a real-time polymerase chain reaction (RT-PCR) assay. By applying a combination of only two different fluorescent probes, this assay allows wild-type sequences to be unambiguously distinguished from all known mutant QRDR sequences of H. pylori. Next, the Tm values of patient DNAs were established, and the genotypes were confirmed by sequencing. Thus, quinolone-resistant H. pylori strains can be easily and quickly diagnosed before treatment, which will help to avoid the administration of ineffective drug regimes.

High prevalence of plasmid-mediated quinolone resistance (PMQR) among E. coli from aquatic environments in Bangladesh

Fluro(quinolones) is an important class of antibiotic used widely in both human and veterinary medicine. Resistance to fluro(quinolones) can be acquired by either chromosomal point mutations or plasmid-mediated quinolone resistance (PMQR). There is a lack of studies on the prevalence of PMQR in organisms from environmental sources in Bangladesh. In this study, we investigated the occurrence of PMQR genes in E. coli from various water sources and analysed associations between multi-drug resistance (MDR) and resistance to extended spectrum β-lactam antibiotics. We analysed 300 E. coli isolates from wastewaters of urban live-bird markets (n = 74) and rural households (n = 80), rural ponds (n = 71) and river water samples (n = 75) during 2017–2018. We isolated E. coli by filtering 100 ml of water samples through a 0.2μm cellulose membrane and incubating on mTEC agar media followed by identification of isolated colonies using biochemical tests. We selected one isolate per sample for detection of PMQR genes by multiplex PCR and tested for antibiotic susceptibility by disc diffusion. Clonal relatedness of PMQR-positive isolates was evaluated by enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR). About 66% (n = 199) of E. coli isolates harbored PMQR-genes, predominantly qnrS (82%, n = 164) followed by aac(6’)-lb-cr (9%, n = 17), oqxAB (7%, n = 13), qnrB (6%, n = 11) and qepA (4%, n = 8). Around 68% (n = 135) of PMQR-positive isolates were MDR and 92% (n = 183) were extended spectrum β-lactamase (ESBL)-producing of which the proportion of positive samples was 87% (n = 159) for blaCTX-M-1’ 34% (n = 62) for blaTEM, 9% (n = 16) for blaOXA-1, blaOXA-47 and blaCMY-2, and 2% (n = 4) for blaSHV. Further, 16% (n = 32) of PMQR-positive isolates were resistant to carbapenems of which 20 isolates carried blaNDM-1. Class 1 integron (int1) was found in 36% (n = 72) of PMQR-positive E. coli isolates. PMQR genes were significantly associated with ESBL phenotypes (p≤0.001). The presence of several PMQR genes were positively associated with ESBL and carbapenemase encoding genes such as qnrS with blaCTXM-1 (p<0.001), qnrB with blaTEM (p<0.001) and blaOXA-1 (p = 0.005), oqxAB and aac(6’)-lb-cr with blaSHV and blaOXA-1 (p<0.001), qnrB with blaNDM-1 (p<0.001), aac(6’)-lb-cr with blaOXA-47 (p<0.001) and blaNDM-1 (p = 0.002). Further, int1 was found to correlate with qnrB (p<0.001) and qepA (p = 0.011). ERIC-PCR profiles allowed identification of 84 of 199 isolates with 85% matching profiles which were further grouped into 33 clusters. Only 5 clusters had isolates (n = 11) with identical ERIC-PCR profiles suggesting that PMQR-positive E. coli isolates are genetically heterogeneous. Overall, PMQR-positive MDR E. coli were widely distributed in aquatic environments of Bangladesh indicating poor wastewater treatment and highlighting the risk of transmission to humans and animals.

High Carriage Rate of the Multiple Resistant Plasmids Harboring Quinolone Resistance Genes in Enterobacter spp. Isolated from Healthy Individuals

Antimicrobial-resistant bacteria causing intractable and even fatal infections are a major health concern. Resistant bacteria residing in the intestinal tract of healthy individuals present a silent threat because of frequent transmission via conjugation and transposition. Plasmids harboring quinolone resistance genes are increasingly detected in clinical isolates worldwide. Here, we investigated the molecular epidemiology of plasmid-mediated quinolone resistance (PMQR) in Gram-negative bacteria from healthy service trade workers. From 157 rectal swab samples, 125 ciprofloxacin-resistant strains, including 112 Escherichia coli, 10 Klebsiella pneumoniae, two Proteus mirabilis, and one Citrobacter braakii, were isolated. Multiplex PCR screening identified 39 strains harboring the PMQR genes (including 17 qnr,19 aac(6′)-Ib-cr, and 22 oqxA/oqxB). The genome and plasmid sequences of 39 and 31 strains, respectively, were obtained by short- and long-read sequencing. PMQR genes mainly resided in the IncFIB, IncFII, and IncR plasmids, and coexisted with 3–11 other resistance genes. The high PMQR gene carriage rate among Gram-negative bacteria isolated from healthy individuals suggests the high-frequency transmission of these genes via plasmids, along with other resistance genes. Thus, healthy individuals may spread antibiotic-resistant bacterial, highlighting the need for improved monitoring and control of the spread of antibiotic-resistant bacteria and genes in healthy individuals.

Quinolone resistance is transferred horizontally via uptake signal sequence recognition in Haemophilus influenzae

The presence of Haemophilus influenzae strains with low susceptibility to quinolones has been reported worldwide. However, the emergence and dissemination mechanisms remain unclear. In this study, a total of 14 quinolone-low-susceptible H. influenzae isolates were investigated phylogenetically and in vitro resistance transfer assay in order to elucidate the emergence and dissemination mechanisms. The phylogenetic analysis based on gyrA sequences showed that strains with the same sequence type determined by multilocus sequence typing were classified into different clusters, suggesting that H. influenzae quinolone resistance emerges not only by point mutation, but also by the horizontal transfer of mutated gyrA . Moreover, the in vitro resistance transfer assay confirmed the horizontal transfer of quinolone resistance and indicated an active role of extracellular DNA in the resistance transfer. Interestingly, the horizontal transfer of parC only occurred in those cells that harbored a GyrA with amino acid substitutions, suggesting a possible mechanism of quinolone resistance in clinical settings. Moreover, the uptake signal and uptake-signal-like sequences located downstream of the quinolone resistant-determining regions of gyrA and parC , respectively, contributed to the horizontal transfer of resistance in H. influenzae . Our study demonstrates that the quinolone resistance of H. influenzae could emerge due to the horizontal transfer of gyrA and parC via recognition of an uptake signal sequence or uptake-signal-like sequence. Since the presence of quinolone-low-susceptible H. influenzae with amino acid substitutions in GyrA have been increasing in recent years, it is necessary to focus our attention to the acquisition of further drug resistance in these isolates.

The molecular mechanisms of fluoroquinolone resistance found in rectal swab isolates of Enterobacterales from men undergoing a transrectal prostate biopsy: the rationale for targeted prophylaxis

Background Transrectal ultrasound-guided prostate biopsy (TRUS-Bx) is considered an essential urological procedure for the histological diagnosis of prostate cancer. It is, however, considered a “contaminated” procedure which may lead to infectious complications. Recent studies suggest a significant share of fluoroquinolone-resistant rectal flora in post-biopsy infections. Methods The molecular mechanisms of fluoroquinolone resistance, including PMQR (plasmid-mediated quinolone resistance) as well as mutation in the QRDRs (quinolone-resistance determining regions) of gyrA, gyrB, parC and parE, among Enterobacterales isolated from 32 of 48 men undergoing a prostate biopsy between November 2015 and April 2016 were investigated. Before the TRUS-Bx procedure, all the patients received an oral antibiotic containing fluoroquinolones. Results In total, 41 Enterobacterales isolates were obtained from rectal swabs. The MIC of ciprofloxacin and the presence of common PMQR determinants were investigated in all the isolates. Nine (21.9%) isolates carried PMQR with qnrS as the only PMQR agent detected. DNA sequencing of the QRDRs in 18 Enterobacterales (E. coli n = 17 and E. cloacae n = 1) isolates with ciprofloxacin MIC ≥ 0.25 mg/l were performed. Substitutions in the following codons were found: GyrA—83 [Ser → Leu, Phe] and 87 [Asp → Asn]; GyrB codon—605 [Met → Leu], ParC codons—80 [Ser → Ile, Arg] and 84 [Glu → Gly, Met, Val, Lys], ParE codons—458 [Ser → Ala], 461 [Glu → Ala] and 512 [Ala → Thr]. Six isolates with ciprofloxacin MIC ≥ 2 mg/l had at least one mutation in GyrA together with qnrS. Conclusions This study provides information on the common presence of PMQRs among Enterobacterales isolates with ciprofloxacin MIC ≥ 0.25 mg/l, obtained from men undergoing TRUS-Bx. This fact may partially explain why some men develop post-TRUS-Bx infections despite ciprofloxacin prophylaxis.

Mutational Diversity in the Quinolone Resistance-Determining Regions of Type-II Topoisomerases of Salmonella Serovars

Quinolone resistance in bacterial pathogens has primarily been associated with mutations in the quinolone resistance-determining regions (QRDRs) of bacterial type-II topoisomerases, which are DNA gyrase and topoisomerase IV. Depending on the position and type of the mutation (s) in the QRDRs, bacteria either become partially or completely resistant to quinolone. QRDR mutations have been identified and characterized in Salmonella enterica isolates from around the globe, particularly during the last decade, and efforts have been made to understand the propensity of different serovars to carry such mutations. Because there is currently no thorough analysis of the available literature on QRDR mutations in different Salmonella serovars, this review aims to provide a comprehensive picture of the mutational diversity in QRDRs of Salmonella serovars, summarizing the literature related to both typhoidal and non-typhoidal Salmonella serovars with a special emphasis on recent findings. This review will also discuss plasmid-mediated quinolone-resistance determinants with respect to their additive or synergistic contributions with QRDR mutations in imparting elevated quinolone resistance. Finally, the review will assess the contribution of membrane transporter-mediated quinolone efflux to quinolone resistance in strains carrying QRDR mutations. This information should be helpful to guide the routine surveillance of foodborne Salmonella serovars, especially with respect to their spread across countries, as well as to improve laboratory diagnosis of quinolone-resistant Salmonella strains.

Prevalence and Characterization of Quinolone Resistance in Campylobacter spp. Isolates in Chicken Livers from Retail Stores in Georgia, USA

Campylobacter is the leading bacterial pathogen that causes human foodborne illnesses worldwide and outbreaks have been associated with consumption of under-cooked chicken livers. The objectives of this study were to compare two PCR assays for speciation of 250 Campylobacter isolates, to assess antibiotic resistance of the isolates, and to analyze genetic diversity of the quinolone resistance determining regions (QRDR) of the isolates. Speciation was performed in a double-blind manner, and results showed that 181 (72%) isolates were identified as Campylobacter jejuni and 69 (28%) isolates were identified as Campylobacter coli by both PCR assays. A total of 93 (37.2%) isolates were determined to be resistant to at least one antibiotic. Among 88 C. jejuni isolates, 33 isolates (18%) were resistant to nalidixic acid (NAL) and ciprofloxacin (CIP), followed by 25 (14%) isolates resistant to tetracycline (TET) and 18 (10%) isolates resistant to NAL and TET. Two isolates were resistant to four antibiotics tested. One isolate was resistant to five antibiotics tested. For C. coli, two isolates were resistant to TET, and two were resistant to NAL, CIP and TET. The amino acid sequences of the QRDR regions among the isolates revealed eight point mutations and could be classified into 12 groups. Thirty-eight C. jejuni isolates resistant to NAL and CIP had a point mutation at residue 86 (Thr to Ile substitution). However, six isolates without the substitution at the same position were resistant to NAL and/or CIP. In addition, 10 isolates with a point mutation at residue 86 were susceptible to NAL and CIP. This observation suggests that besides the substitution at 86, other mechanisms may confer resistance to quinolones. Further studies are needed to elucidate mechanisms for quinolone resistance in Campylobacter. Based on our results, the Campylobacter spp. isolated from chicken livers were found to be resistant to the quinolones and other classes of antibiotics.

Molecular mechanisms of macrolide and fluoroquinolone resistance among Korean isolates of Mycoplasma genitalium over a period of five years 2014–2019

Antimicrobial resistance in Mycoplasma genitalium has become a global issue, and certain groups have a higher probability of acquiring resistant strains. Little is known about the genetic diversity and characteristics of the antimicrobial resistance-determining sites (ARDSs) of M. genitalium in the Korean population. Therefore, we examined the genetic diversity of the ARDSs of M. genitalium-positive urogenital samples obtained from Korean females (G1) and males (G2) visiting primary care clinics and DNA samples from referred males (G3) with persistent urethritis. From 2014 to 2019, 54 patients from G1, 86 patients from G2, and 68 patients from G3 were included in the study. Sanger sequencing was performed on the 2058/2059 sites in the 23S rRNA gene and quinolone resistance-determining regions (QRDRs) of M. genitalium . The rates of mutation in G1, G2, and G3 were 1.85, 5.81, and 48.53 %, respectively, for A2059G in the 23S rRNA gene (P<0.001); 1.85, 0, and 17.78 %, respectively, for M95R or I in gyrA (P<0.001); 0, 0, and 31.11 %, respectively, for D99N or G in gyrA (P<0.001); and 7.41, 16.28, and 30 %, respectively, for S83R or N or I in parC (P=0.015). A2059G significantly increased the risk of mutations at the gyrA95, gyrA99, and parC83 sites (all P<0.01). In conclusion, although the genetic diversity of the ARDSs of M. genitalium was variable among the groups, it was generally lower in isolates with macrolide resistance and higher in isolates with quinolone resistance in Korea compared with the isolates in other countries. The G3 group demonstrated increased genetic diversity at the A2059G, gyrA95, gyrA99, and parC83 sites.