Quinolone-resistant Mutants Research Articles

Co-Occurrence of Two Plasmids Encoding Transferable blaNDM-1 and tet(Y) Genes in Carbapenem-Resistant Acinetobacter bereziniae.

Acinetobacter bereziniae has emerged as a significant human pathogen, acquiring multiple antibiotic resistance genes, including carbapenemases. This study focuses on characterizing the plasmids harboring the blaNDM-1 and tet(Y) genes in two carbapenem-resistant A. bereziniae isolates (UCO-553 and UCO-554) obtained in Chile during the COVID-19 pandemic. Methods: Antibiotic susceptibility testing was conducted on UCO-553 and UCO-554. Both isolates underwent whole-genome sequencing to ascertain their sequence type (ST), core genome multilocus sequence-typing (cgMLST) profile, antibiotic resistance genes, plasmids, and mobile genetic elements. Conjugation experiments were performed for both isolates. Results: Both isolates exhibited broad resistance, including resistance to carbapenems, third-generation cephalosporins, fluoroquinolones, tetracycline, cotrimoxazole, and aminoglycosides. Both isolates belong to sequence type STPAS1761, with a difference of 17 out of 2984 alleles. Each isolate carried a 47,274 bp plasmid with blaNDM-1 and aph(3')-VI genes and two highly similar plasmids: a 35,184 bp plasmid with tet(Y), sul2, aph(6)-Id, and aph(3″)-Ib genes, and a 6078 bp plasmid containing the ant(2″)-Ia gene. Quinolone-resistance mutations were identified in the gyrA and parC genes of both isolates. Importantly, blaNDM-1 was located within a Tn125 transposon, and tet(Y) was embedded in a Tn5393 transposon. Conjugation experiments successfully transferred blaNDM-1 and tet(Y) into the A. baumannii ATCC 19606 strain, indicating the potential for horizontal gene transfer. Conclusions: This study highlights the critical role of plasmids in disseminating resistance genes in A. bereziniae and underscores the need for the continued genomic surveillance of this emerging pathogen. The findings emphasize the importance of monitoring A. bereziniae for its potential to cause difficult-to-treat infections and its capacity to spread resistance determinants against clinically significant antibiotics.

Pathogen diversity and antimicrobial resistance transmission of Salmonella enterica serovars Typhi and Paratyphi A in Bangladesh, Nepal, and Malawi: a genomic epidemiological study

Enteric fever is a serious public health concern. The causative agents, Salmonella enterica serovars Typhi and Paratyphi A, frequently have antimicrobial resistance (AMR), leading to limited treatment options and poorer clinical outcomes. We investigated the genomic epidemiology, resistance mechanisms, and transmission dynamics of these pathogens at three urban sites in Africa and Asia. S Typhi and SParatyphi Abacteria isolated from blood cultures of febrile children and adults at study sites in Dhaka (Bangladesh), Kathmandu (Nepal), and Blantyre (Malawi) during STRATAA surveillance were sequenced. Isolates were charactered in terms of their serotypes, genotypes (according to GenoTyphi and Paratype), molecular determinants of AMR, and population structure. We used phylogenomic analyses incorporating globally representative genomic data from previously published surveillance studies and ancestral state reconstruction to differentiatelocally circulating from imported pathogen AMR variants. Clusters of sequences without any single-nucleotide variants in their core genome were identified and used to explore spatiotemporal patterns and transmission dynamics. We sequenced 731genomes from isolates obtained during surveillance across the three sites between Oct 1, 2016, and Aug 31, 2019 (24 months in Dhaka and Kathmandu and 34months in Blantyre). SParatyphi Awas present in Dhaka and Kathmandu but not Blantyre. STyphi genotype 4.3.1 (H58) was common in all sites, but with different dominant variants (4.3.1.1.EA1 in Blantyre, 4.3.1.1in Dhaka, and 4.3.1.2in Kathmandu). Multidrug resistance (ie,resistance to chloramphenicol, co-trimoxazole, and ampicillin) was common in Blantyre (138 [98%] of 141cases) and Dhaka (143 [32%] of 452), but absent from Kathmandu. Quinolone-resistance mutations were common in Dhaka (451 [>99%] of 452) and Kathmandu (123 [89%] of 138), but not in Blantyre (three [2%] of 141). Azithromycin-resistance mutations in acrB were rare, appearing only in Dhaka (five [1%] of 452). Phylogenetic analyses showed that most cases derived from pre-existing, locally established pathogen variants; 702(98%) of 713 drug-resistant infections resulted from local circulation of AMR variants, not imported variants or recent de novo emergence; and pathogen variants circulated across age groups. 479(66%) of 731cases clustered with others that were indistinguishable by point mutations; individual clusters included multiple age groups and persisted for up to 2·3years, and AMR determinants were invariant within clusters. Enteric fever was associated with locally established pathogen variants that circulate across age groups. AMR infections resulted from local transmission of resistant strains. These results form a baseline against which to monitor the impacts of control measures. Wellcome Trust, Bill & Melinda Gates Foundation, EU Horizon 2020, and UK National Institute for Health and Care Research.

Mycoplasma genitalium's Antibiotic Resistance in Sexually Transmitted Infections Clinics in Israel.

To determine the prevalence of antibiotic resistance rate in Mycoplasma genitalium, and distribution of mutations associated with this resistance, among patients that attended sexually transmitted infections (STI) investigation clinics. This cross-sectional study included M. genitalium-positive samples (urine, vaginal, rectal, and pharyngeal swabs) collected from 170 patients attending two STI investigation clinics, which were subjected to macrolide and quinolone resistance mutations analyses. Data regarding patient age, sex, and material/anatomical site of testing were collected. Macrolide-resistance mutations were identified in 48.8% of samples and were more common among males (p < .0001) and in rectal samples (p < .05). A2059C was the most prevalent macrolide-resistance mutation (18.2%). Quinolone resistance was detected in 23% of the samples, with S83I being the most common (17.1%) mutation. Rate of co-resistance to macrolides and quinolones was 21.2%. The high rate of antibiotic resistance found in the current study, especially to macrolides, underscores the importance of antibiotic resistance monitoring in M. genitalium isolates in cases of persistent or recurrent urethritis/cervicitis, in cases of treatment failure and among specific populations. Such surveillance will improve treatment regimens and cure rates.

Epidemiological and genetic characterization of multidrug-resistant non-O1 and non-O139 Vibrio cholerae from food in southern China

This study reports a comprehensive epidemiological and genetic analysis of V. cholerae strains, specifically non-O1/non-O139 serogroups, isolated from animal-derived food samples in Guangdong province from 2015 to 2019. A total of 21 V. cholerae strains were obtained, which exhibited high resistance rates for nalidixic acid (57.14 %, 12/21), ampicillin (33.33 %, 7/21), and ciprofloxacin (19.05 %, 4/21). The quinolone resistance-related gene, qnrVC, was prevalent in 80.95 % (17/21) of the isolates. Additionally, chromosomally mediated quinolone-resistance mutations, including mutations in GyrA at position 83 (S83I) and ParC at position 85 (S85L), were detected in 47.62 % of the isolates. The combination of target mutation and qnrVC genes was shown to mediate resistance or intermediate resistance to ciprofloxacin in V. cholerae. Furthermore, an IncC-type conjugative plasmid carrying thirteen antibiotic resistance genes, including genes conferring resistance to two clinically important antibiotics, cephalosporins and fluoroquinolones, was identified in the shrimp-derived strain Vc516. While none of our food isolates harbored the toxigenic CTX- and TCP-encoding genes, they did possess genes encoding toxins such as HlyA and Autoinducer-2. Notably, some V. cholerae strains from this study exhibited a close genetic relationship with clinical strains, suggesting their potential to cause human infections. Taken together, this study provides a comprehensive view of the epidemiological features and genetic basis of antimicrobial resistance and virulence potential of V. cholerae strains isolated from food in southern China, thereby advancing our understanding of this important pathogen.

Dissemination of ESBL-producing E. coli ST131 through wastewater and environmental water in Switzerland

The E. coli lineage ST131 is a major cause of multidrug-resistant urinary tract and bloodstream infections worldwide. Recently emerged ST131 sublineages spread globally within few years, but their dissemination routes are incompletely understood. In this study, we investigate the potential role of wastewater and surface water in the spread of extended-spectrum β-lactamase (ESBL)-producing ST131. Streams, lakes, and two wastewater treatment plants (WWTPs) in the canton of Zug, Switzerland, were consecutively sampled over 1.5 years. ST131 was detected in 38% of the samples taken downstream (1–5 km) of WWTP discharge sites, but usually absent in water bodies distant from urban areas or WWTP discharge. Specific strains were repeatedly isolated (≤5 pairwise cgSNP distance) from wastewater or river sites downstream of effluent discharge, indicating their repeated entry or persistence in WWTPs in large concentrations. Genetic characterization of the ESBL-producing water isolates revealed a predominance of clades A and C1 and an emerging ciprofloxacin-resistant sublineage with mutations in quinolone resistance determining regions (QRDR) within clade A. Multiple isolates belonged to internationally circulating sublineages, including C1-M27 and papGII + sublineages with chromosomally encoded ESBLs. This study demonstrates that the clinically relevant E. coli lineage ST131 pollutes river ecosystems, representing a significant challenge to public health and to technologies to minimize their entry into the water environment.

Transcriptome analysis reveals the important roles of a two-component system, flagellar assembly, active efflux system and outer membrane proteins in the anti-quinolone ability of Vibrio harveyi from orange-spotted grouper (Epinephelus coioides)

The drug resistance of Vibrio harveyi in aquaculture became more severe because the control of Vibriosis depends majorly on the current antibiotics. Transcriptomes of a wild-type strain (VS) and its quinolone-resistant mutants (VR) of V. harveyi were respectively sequenced by RNA-seq technology. A total of 2,082 unigenes were obtained after de novo splicing and assembly. 129 genes were identified with significant differential expression in strain VR compared to strain VS, among which 65 were up-regulated and 64 down-regulated. Then, functional annotation and enrichment analysis of these differentially expressed genes (DEGs) were performed. GO enrichment results showed that DEGs focused mainly on cell structure, substance metabolism, and transporter. COG classification of the DEGs mainly focused on amino acid transport and metabolism, cell wall/membrane biosynthesis, carbohydrate transport and metabolism, ribosomal structure, and biosynthesis. KEGG pathways related to a two-component system, ABC transport system and flagellar assembly (ko02040) were enriched significantly, and 9 genes associated with quinolone-resistance ability, including genes for resistance-related transport proteins, outer membrane proteins, and DNA repair-related proteins were discovered through analysis of the drug-resistance related genes. Ten DEGs (including the above part genes of 9 drug resistance-related genes) in the transcriptome data were taken to analyze their expression with real-time qPCR. The results were the same as the changes of the above transcriptome analysis, further confirming the reliability of the transcriptome sequencing and data analysis. In a word, genes from a two-component system, flagellar assembly, active efflux system and outer membrane proteins take great roles in the quinolone-resistance of V. harveyi. These results provide enough information for further study on the molecular mechanism of quinolone-resistance and give a helpful transcriptomic resource to unravel the contact between quinolone-resistance and metabolic pathways in Vibrios.

Dissecting the genotypic features of a fluoroquinolone-resistant Pseudomonas aeruginosa ST316 sublineage causing ear infections in Shanghai, China.

Limited information is available regarding the genomic characteristics of P. aeruginosa causing ear infections. Our aim is to characterize the genotypic features of an emerging ST316 sublineage causing aural infections in Shanghai. A total of 199 ear swab isolates were subjected to whole genome sequencing (WGS). Complete genomes for two isolates were resolved. We showed this recently emerged sublineage exhibited high-level resistance to fluoroquinolones (FQs) primarily by accumulation of known mutations in quinolone resistance determining regions (QRDRs). Loss-of-function mutations in mexR and mexCD were frequently detected. Mutations in fusA1 (P166S) and parE (S492F) were resident in this sublinage about 2 years after its emergence. Recombination events might be a key driver of genomic diversity in this sublineage. Convergent evolution events on Multidrug-resistant (MDR) determinants were also observed. We generated predictive machine models and identified biomarkers of resistance to gentamicin, fosfomycin, and cefoperazone-sulbactam in this sublineage. This sublineage tended to be less virulent by loss of a series virulence genes represented by ppkA, rhlI, and iron uptake- and antimicrobial activity-related genes. Specific mutations were detected in pilU and lpxB genes that related to surface structures. Moreover, this sublineage differed from non-ST316 isolates in several ways, including virulence genes related to cell surface structure. Our analysis suggested acquisition of a roughly 390 kbp MDR plasmid carrying qnrVC1 might play an important role in the success of this sublinage. Clonal expansion of this sublineage exhibiting enhanced adaptation to cause ear infections is concerning, which requires urgent control measures to be implemented.

Hotspots sequences of gyrA, gyrB, parC, and parE genes encoded for fluoroquinolones resistance from local Salmonella Typhi strains in Jakarta

BackgroundInfection of Salmonella enterica subsp. enterica serovar Typhi is the primary etiology of typhoid fever globally and is common in many developing countries, especially those with dense populations and poor environmental sanitation. Antibiotic fluoroquinolones were used for the treatment in the 1980s due to the resistance to the first-line antibiotics. However, many cases of treatment failure of fluoroquinolones in typhoidal patients have been reported from numerous countries in Asia, Europe, Africa, and America. Mutations in quinolone resistance determining regions (QRDR) genes, gyrA, gyrB, parC, and parE, are found in fluoroquinolone-resistant Salmonella Typhi. Contrast reports came from the S. Typhi isolates in Indonesia, mainly Jakarta and the surroundings, obtained from patients with typhoid fever, with good sensitivity to the fluoroquinolones, i.e., nalidixic acid, ciprofloxacin, moxifloxacin, and levofloxacin. The present study, therefore, aimed to identify the hotspot sequences of gyrA, gyrB, parC, and parE genes of the local S. Typhi strains based on their susceptibility to fluoroquinolones from patients with typhoid fever in Jakarta and its satellite cities.ResultsA total of 28 isolates were identified as S. Typhi. All isolates were susceptible to nalidixic acid, levofloxacin, and moxifloxacin. Twenty-seven isolates (96.4%) were susceptible to ciprofloxacin, with one isolate (3.6%) being intermediate. The hotspot sequences of gyrA, gyrB, parC, and parE genes from all isolates were identical to the fluoroquinolone-sensitive reference sequence Salmonella enterica subsp. enterica serovar Typhi Ty2 (NCBI GenBank AE014613.1), including the isolate with intermediate susceptibility. The mutation was not found, and amino acid deduced from all hotspots in susceptible and intermediate isolates showed no replacement in all reported codons.ConclusionsThis study showed that the local S. Typhi strains from Jakarta and surroundings were susceptible to fluoroquinolones (nalidixic acid, ciprofloxacin, levofloxacin, and moxifloxacin), and the hotspot sequences of the gyrA, gyrB, parC, and parE genes were all identical to the reference sequence. Thus, the hotspot sequences of the gyrA, gyrB, parC, and parE genes seemingly were conserved in Jakarta’s local S. Typhi strains and could be considered wild type. The phenotypic susceptibility was consistent with the genotypic characteristic without non-synonymous mutations associated with drug resistance.

Molecular characteristics of fluoroquinolone-resistant Escherichia coli isolated from suckling piglets with colibacillosis

ObjectivesColibacillosis is a frequent enteric disease in the pig industry that causes significant economic losses. The objective of this study was to investigate the molecular characteristics of fluoroquinolone (FQ)-resistant E. coli isolates from suckling piglets with colibacillosis.ResultsA total of 43 FQ-resistant E. coli isolates were tested in this study and all isolates showed multi-drug resistance (MDR) and mutations in quinolone resistance determining regions (gyrA or parC). Especially, FQ-resistant E. coli isolates with double mutations in both gyrA and parC were shown a high FQs minimum inhibitory concentration (≥ 64 mg/L for ciprofloxacin, ≥ 128 mg/L for enrofloxacin, and ≥ 256 mg/L for norfloxacin). Among 43 FQ-resistant E. coli isolates, 12 (27.9%) were showed plasmid-mediated quinolone resistance (PMQR) positive E. coli. Prevalence of PMQR gene, aac(6’)-Ib-cr, qnrS, and qepA, were identified in 7, 3, and 2 E. coli isolates, respectively. We identified the following in PMQR-positive E. coli isolates: the tetracycline resistance genes tetD (12 isolates, 100.0%), tetE (12 isolates, 100.0%), tetA (11 isolates, 91.7%), and tetB (1 isolate, 8.3%); β-lactamases–encoding blaCMY-2 (10 isolates, 83.3%), blaTEM-1 (7 isolates, 58.3%), blaOXA-1 (7 isolates, 58.3%), blaSHV-1 (3 isolates, 16.7%), and blaAAC-2 (1 isolate, 8.3%); and the chloramphenicol resistance genes (10 isolates, 83.3%); the sulfonamide resistance genes sul1 (9 isolates, 75.0%) and sul2 (10 isolates, 83.3%); the aminoglycoside modifying enzyme gene aac(3)-II (2 isolates, 16.7%). The F4 (7 isolates, 58.3%), LT:STb:EAST1 (5 isolates, 41.7%), and paa (3 isolates, 25.0%) were most common fimbrial antigen, combinations of toxin genes, and non-fimbrial adhesins genes, respectively. All PMQR-positive E. coli carried class I integrons but only 4 isolates carried the gene cassette. The most prevalent plasmid replicon was FIB (9 isolates, 75.0%), followed by FIC, HI1, and N (7 isolates, 58.3%), respectively.ConclusionsBecause FQ-resistant E. coli can serve as a reservoir of FQ resistant genetic determinants that can be transferred to pathogenic bacteria in humans or pigs, this represents a public health hazard.

In vitro antibacterial activity of OPS-2071 against Gram-positive and Gram-negative enteropathogenic bacteria.

Enteric infections are a major public health issue in developing countries. Antimicrobial resistance is also a problem for enteric infection. OPS-2071 is a novel quinolone antibiotic with low oral absorption and potent antibacterial activity against Clostridioides difficile. This study was conducted to confirm the antimicrobial activity of OPS-2071 against major enteropathogenic bacteria and to evaluate the risk of emergence of drug resistance. The antibacterial activity was evaluated by the agar dilution method. The inhibitory activity against DNA gyrase and topoisomerase IV was determined by supercoiling assay and decatenation assay, respectively. The mutant prevention concentration and frequency of spontaneous resistance were determined by inoculation on drug-containing agar. Compared with the reference drugs, the antibacterial activity of OPS-2071 was more potent against Gram-positive bacteria and Campylobacter jejuni, including quinolone-resistant strains. Against other Gram-negative bacteria, OPS-2071 was comparable to existing quinolones. The inhibitory activities against DNA gyrase with quinolone-resistant mutations closely correlated with the antibacterial activity. Spontaneous resistance to OPS-2071 was not observed in Staphylococcus aureus and Escherichia coli and was lower than that of existing quinolones and higher than that of azithromycin in C. jejuni. The mutant prevention concentration of OPS-2071 was lower than that of tested compounds in S. aureus and C. jejuni and slightly higher than that of existing quinolones in E. coli. The broad and potent in vitro antibacterial activity and lower risk of drug resistance suggested that OPS-2071 may be useful for enteric infections caused by major pathogens including quinolone-resistant Campylobacter.

Sublethal concentrations of heavy metals Cu2+ and Zn2+ can induce the emergence of bacterial multidrug resistance

The emergence of multidrug resistance has become an environmental safety issue of worldwide concern. Numerous studies have confirmed that heavy metals can promote the spread of bacterial resistance. However, in addition to co-resistance plasmids, the role and mechanism of metals at sublethal levels (< minimum inhibitory concentration [MIC]) on antibiotic resistance remain poorly defined. Here we investigated the alteration of resistance phenotype under Cu 2 + and Zn 2 + exposure and explored the molecular mechanism by which heavy metals affect antibiotic resistance. The results showed that the MIC of Escherichia coli and Staphylococcus aureus on antibiotics, especially norfloxacin and tetracyclines, was significantly increased after seven consecutive days of exposure to Cu 2 + and Zn 2 + and exhibited a higher cross-resistance rate, which meant that heavy metals can induce bacteria to develop antibiotic resistances that were not previously available. It was observed the downregulation of outer membrane protein gene omp C after heavy metal exposure, while the expression of other efflux pump and resistance genes in E. coli was upregulated, especially tet B, tol C and arc AB genes, suggesting that heavy metals might enhance antibiotic resistance by altering the expression of efflux pump genes. Moreover, many point mutations were found in the efflux system of resistant mutant strains. In particular, quinolone-resistant mutant strains showed a point mutation in the conversion of serine (Ser) to phenylalanine (Phe) at residue 464 of the gyr B gene, which may also be an important cause of increased quinolone resistance. This study provides an explanation for the effect of heavy metals on bacterial resistance. • The MIC of E. coli and S. aureus on antibiotics increased after Cu 2+ and Zn 2+ exposure. • After exposure to heavy metals, the cross resistance rates also showed an increase. • Heavy metals may enhance antibiotic resistance by altering efflux pump expression. • Many point mutations were found in the efflux system of resistant mutant strains.

Impact on quinolone resistance of plasmid-mediated quinolone resistance gene and mutations in quinolone resistance-determining regions in extended spectrum beta lactamase-producing Klebsiella pneumoniae isolated from urinary tract infection patients.

Klebsiella pneumoniae is a typical pathogen in urinary tract infections (UTI), and the emergence of extended spectrum beta-lactamase (ESBL)-producing strains has been frequently reported, accompanied by higher quinolone resistance rates. There are two major mechanisms of quinolone resistance, mutations in quinolone resistance-determining regions (QRDR) and the presence of the plasmid-mediated quinolone resistance (PMQR) genes. This study aimed to investigate quinolone resistance among 105 ESBL-producing K. pneumoniae specimens isolated from UTI patients in Indonesia. These were characterized for antimicrobial resistance to nalidixic acid, ciprofloxacin, and levofloxacin, QRDR mutations in gyrA and parC and the presence of PMQR genes. We found that 84.8% of the collected isolates were resistant to at least one of the quinolones. QRDR mutation in gyrA was observed in 49.5% of these strains and parC mutations in 61.0%. PMQR genes were identified in 84.8% of strains. The QRDR mutations clearly had a greater effect on resistance than the PMQR genes. In conclusion, we found high quinolone resistance rates in Indonesian ESBL-producing K. pneumoniae, in which QRDR mutation played a major role.

Prevalence, species diversity, and antimicrobial susceptibility of Campylobacter strains in patients with diarrhea and poultry meat samples: one-year prospective study.

Source tracking of antimicrobial resistance in Campylobacter is useful for control measures. In this study, Campylobacter-associated diarrhea and homology in antimicrobial resistance of humans and poultry meat isolates were investigated. A total of 400 stools of patients and 100 poultry meat samples were analyzed. Susceptibility of the isolates was detected by disk diffusion, Etest, and agar dilution methods. Mismatch amplification mutation assay was used for the detection of mutations in the gyrA quinolone resistance determining region (QRDR). Campylobacter spp., including C. jejuni, C. coli, and C. lari, were detected in 35% of the chicken meat and 6.75% of the stool samples, respectively. The QRDR mutation was detected in most of the stool and chicken meat samples. Although the frequency of resistance to tetracycline (53.5% and 62.8%), erythromycin (39.2% and 37.1%), and gentamicin (32.1% and 31.4%) was relatively similar, higher frequency of resistance to ciprofloxacin (51.4% vs 28.6%) and nalidixic acid (42.15% vs 28.6%) among the chicken meat, and ampicillin (50% and 17.1%) among the human stool was detected. High percentage of poultry meat samples is contaminated with different Campylobacter species, which shows homology with the patients' isolates in Tehran.

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.

Genotypic Diversity of Ciprofloxacin Nonsusceptibility and Its Relationship with Minimum Inhibitory Concentrations in Nontyphoidal Salmonella Clinical Isolates in Taiwan.

This study analyzed the genetic diversity of ciprofloxacin (CIP) nonsusceptibility and the relationship between two major mechanisms and minimum inhibitory concentrations (MICs) of CIP in nontyphoidal Salmonella (NTS). Chromosomal mutations in quinolone resistance-determining regions (QRDRs) and plasmid-mediated quinolone resistance (PMQR) genes were searched from ResFinder, ARG-ANNOT, and PubMed for designing the sequencing regions in gyrA, gyrB, parC, and parE, and the 13 polymerase chain reactions for PMQR genes. We found that QRDR mutations were detected in gyrA (82.1%), parC (59.0%), and parE (20.5%) but not in gyrB among the 39 isolates. Five of the 13 PMQR genes were identified, including oqxA (28.2%), oqxB (28.2%), qnrS (18.0%), aac(6′)-Ib-cr (10.3%), and qnrB (5.1%), which correlated with the MICs of CIP within 0.25–2 μg/mL, and it was found that oxqAB contributed more than qnr genes to increase the MICs. All the isolates contained either QRDR mutations (53.8%), PMQR genes (15.4%), or both (30.8%). QRDR mutations (84.6%) were more commonly detected than PMQR genes (46.2%). QRDR mutation numbers were significantly associated with MICs (p < 0.001). Double mutations in gyrA and parC determined high CIP resistance (MICs ≥ 4 μg/mL). PMQR genes contributed to intermediate to low CIP resistance (MICs 0.25–2 μg/mL), thus providing insights into mechanisms underlying CIP resistance.

Molecular characterization of fluoroquinolone-resistant Escherichia coli from broiler breeder farms

Fluoroquinolones (FQs) have been used effectively antimicrobial agents of choice for treatment of various infections caused by E. coli and FQs-resistance of E. coli from broiler breeders has been implicated in its vertical transmission to their offspring. The objective of this study investigated the phenotypic and genotypic characteristics of FQ-resistant E. coli isolates from broiler breeder farms in Korea. A total of 106 FQ-resistant E. coli isolates were tested in this study and all isolates had mutations in quinolone resistance determining regions; all (100%) had mutations in gyrA, 89 (84.0%) had mutations in parE, 8 (7.5%) isolates showed the mutations with parC and parE, and none had mutations in gyrB. The predominant mutation type was double mutation in gyrA (S83L and D87N), and all FQ-resistant E. coli isolates that had mutations in parC or parE also had double mutations in gyrA. Especially, FQ-resistant E. coli isolates which possessed double mutations in gyrA in combination with double mutations in parC or single mutations in both parC and parE were shown high levels of minimum inhibitory concentrations rage. Of the 23 plasmid-mediated quinolone resistance (PMQR)-positive E. coli isolates, qnrS was detected in 10 (9.4%) isolates, and followed by qnrA (7 isolates, 6.6%), qnrB (4 isolates, 3.8%), and aac(6′)-Ib-cr (2 isolates, 1.9%). Sixteen (69.6%) of the 23 PMQR-positive E. coli isolates harbored class 1 integrons with four different gene cassette arrangements and total of 9 plasmid replicon types were also identified in 23 PMQR-positive E. coli isolates. This is the first study to investigate the prevalence and characteristics of FQ-resistant and PMQR-positive E. coli isolated from the broiler breeder in Korea; it supports that constant monitoring and studies at the broiler breeder level are required to prevent the pyramidal transmission of FQ-resistant E. coli.

Molecular testing for H. pylori clarithromycin and quinolone resistance: a prospective Chinese study.

In China, there is a high prevalence of antibiotic-resistant Helicobacter pylori infections in the population. The aim of the study was to assess a new ARMS-PCR test for detection of H. pylori clarithromycin resistance (CR) and quinolone resistance (QR) mutations and evaluate the spectrum of antibiotic resistance in patients from three Chinese provinces. Sanger sequencing and multiplex ARMS-PCR were used to detect H. pylori CR and QR bacteria in gastric biopsy samples. Among the 1,182 patients enrolled with gastritis, 643 (54.4%) were positive for H. pylori. Of these, 371 (57.7%) had antibiotic-resistant strains, comprising 236 (63.6%) with a single drug antibiotic-resistant strain and 135 (36.4%) with multiple drug-resistant strains. Following Sanger sequencing analysis of 23S rRNA and gyrA gene for mutations (antibiotic resistance markers), rates of CR, QR, and multidrug resistance (CR and QR) were 19.9, 12.0, and 25.8%, respectively. The 23S rRNA CR mutation A2143G (286, 96.9%) and the gyrA QR mutations C261A (85, 31.5%) and G271A (71, 26.3%) were common. Benchmarking against Sanger sequencing results, multiplex ARMS-PCR test had a high diagnostic sensitivity and specificity for detection of CR (96 and 93%), QR (95 and 92%) and multidrug resistance (95 and 95%). Based on our findings, the high incidence of single and multiple antibiotic resistance requires the routine checking of antibiotic resistance in all patients with suspected H. pylori infections. Multiplex ARMS-PCR is a simple and rapid test that can be now used for more efficient treatment of H. pylori infections and reduces the misuse of antibiotics.

Analysis of Genetic Mutations in Quinolone Resistance and Virulence Factor Gene Profile of Enterococcus faecalis

Analysis of Genetic Mutations in Quinolone Resistance and Virulence Factor Gene Profile of Enterococcus faecalis

Molecular characterization of fluoroquinolone resistant Escherichia coli from Ghana

Background: The indiscriminate use of antibiotics in humans and animals has led to an increase in drug resistant bacteria. This has become a global health threat and a limitation to first- and second-line antibiotic treatment options with consequences including treatment failure and higher medical cost. Using Escherichia coli (E. coli) as an indicator of resistant bacteria, the study sought to determine the distribution of antibiotic resistant isolates from clinical samples and characterize quinolone-resistant genes. Methods and materials: Methods: An experimental quantitative approach was adopted to obtain clinical samples followed by strain identification and antimicrobial susceptibility testing. E-tests were used to determine the Minimum Inhibitory Concentration (MIC) for ciprofloxacin resistant isolates after which chromosomal DNA and plasmids were extracted and probed for the genes (gyr A, parC, aac(6′)1bcr gyr B, Qnr A, Qnr B, Qnr C, Qnr S, qep A and oqx B) known to confer ciprofloxacin–resistance. The amplicons were sequenced and analyzed for mutational hot spots. Results: In total, 47/83 (56.6%) E. coli isolates were resistant to ciprofloxacin and 29/47 (61.7%) showed MIC > 32 μg/mL. Majority 21/29 (72.4%) of the ciprofloxacin resistant E. coli were positive for Gyr A and mutations were found within codon 83 and 87. In parC, the mutations were at codons 71 and 80. Conclusion: The study revealed a high prevalence of fluoroquinolone resistance and found chromosomal and plasmid mediated genes to be widespread and linked to ciprofloxacin-resistance in Ghana. The putative mutations in Quinolone Resistance Determining Regions (QRDR) and the new mutations identified in parC requires further investigations into its association with ciprofloxacin-resistant E. coli. However the preliminary data justifies the need for intensified surveillance in Ghana.

Changing trends of antimicrobial susceptibility and resistance mechanisms to quinolones in typhoidal salmonellae isolated from India in last 5 years

Background: Antimicrobial resistance in enteric fever is a major therapeutic challenge and there is a need to monitor the pattern of resistance to the antityphoidal agents and detection of molecular mechanisms of resistance.The study was designed to monitor the antimicrobial susceptibility of Salmonella enterica serovars Typhi and Paratyphi A isolated from patients of enteric fever during 2014 to 2019 and detection of antimicrobial resistance mechanisms to quinolones. Methods and materials: Total 281 strains isolated from January 2014 to September 2019 were selected for the study. Antimicrobial susceptibility was done as per CLSI guidelines (2019) for amoxicillin, chloramphenicol, co-trimoxazole, ciprofloxacin, ceftriaxone and azithromycin. Minimum inhibitory concentration (MIC) determination for ciprofloxacin, ceftriaxone and azithromycin were done by E-Test method according to manufacturer's (ABs Biodisk, Sweden) instructions. DNA sequencing was done for gyrA, gyrB, parC and parE genes to find mutations in quinolone resistance determining region (QRDR). Results: Out of total 281 strains included in the study 214 (76.2%) were S. Typhi and 67 (23.8%) were S. Paratyphi A. As per CLSI 2019, antimicrobial susceptibility for chloramphenicol, ampicillin and cotrimoxazole was 0.7% (2/281), 1.42% (4/281) and 1.06% (3/281) respectively. Only 6/214 (2.8%) were susceptible for S. Typhi and no strain was found to be susceptible in S. Paratyphi A. Azithromycin susceptibility was 100% in S. Typhi, for S. Paratyphi there is no breakpoint available. Ciprofloxacin MIC50 and MIC90 values found to be 0.25 μg/mL and 1 μg/mL in S. Typhi and 0.5 μg/mL and 6 μg/mL in S. Paratyphi A. Ceftriaxone MIC50 and MIC90 values found to be 0.023 μg/mL and 0.5 μg/mL in both S. Typhi and S. Paratyphi A. For azithromycin MIC50 and MIC90 were 2 μg/mL and 6 μg/mL for S. Typhi and 4 μg/mL and 12 μg/mL for S. Paratyphi A respectively. All quinolone resistance strains had mutations in QRDR of gyrA and parC genes. Conclusion: Ciprofloxacin is no longer a drug of choice for treatment of enteric fever in India. Although susceptibility to ceftriaxone is 100% but MIC is creeping towards resistance. Azithromycin can be a promising treatment option for uncomplicated enteric fever.