gyrA Mutations Research Articles

Molecular testing-guided therapy versus susceptibility testing-guided therapy in first-line and third-line Helicobacter pylori eradication: two multicentre, open-label, randomised controlled, non-inferiority trials

Helicobacter pylori infection is an important causal factor of gastric cancer and peptic ulcer disease and is associated with immune thrombocytopenic purpura and functional dyspepsia. In H pylori strains, point mutations in the 23S rRNA and gyrA genes are associated with clarithromycin resistance and levofloxacin resistance, respectively. Whether the efficacy of molecular testing-guided therapy is non-inferior to that of susceptibility testing-guided therapy for H pylori eradication is unclear. Therefore, we aimed to compare the efficacy and safety of molecular testing-guided therapy and traditional culture-based susceptibility testing-guided therapy in first-line and third-line treatment of H pylori infection. We did two multicentre, open-label randomised trials in Taiwan. In trial 1 (done at seven hospitals), treatment-naive individuals infected with H pylori who were aged 20 years or older were eligible for study inclusion. In trial 2 (done at six hospitals), individuals aged 20 years or older who failed treatment after two or more eradication therapies for H pylori infection were eligible for enrolment. Eligible patients were randomly assigned (1:1) to receive either molecular testing-guided therapy or susceptibility testing-guided therapy. The randomisation sequence was generated by computer using permuted block randomisation with a block size of 4. All investigators were masked to the randomisation sequence. Clarithromycin and levofloxacin resistance were determined by agar dilution test for measuring minimum inhibitory concentrations in the susceptibility testing-guided therapy group, and by PCR and direct sequencing for detection of 23S rRNA and gyrA mutations in the molecular testing-guided therapy group. Study participants received clarithromycin sequential therapy, levofloxacin sequential therapy, or bismuth quadruple therapy according to the resistance status to clarithromycin and levofloxacin. The 13C-urease breath test was used to determine the status of H pylori infection at least 6 weeks after eradication therapy. The primary outcome was the eradication rate by intention-to-treat analysis. The frequency of adverse effects was analysed in patients with available data. The prespecified margins for non-inferiority were 5% for trial 1 and 10% for trial 2. The trials are ongoing for post-eradication follow-up and registered with ClinicalTrials.gov, NCT03556254 for trial 1, and NCT03555526 for trial 2. Between March 28, 2018, and April 23, 2021, 560 eligible treatment-naive patients with H pylori infection were recruited and randomly assigned to the molecular testing-guided therapy group or the susceptibility testing-guided therapy group in trial 1. Between Dec 28, 2017, and Oct 27, 2020, 320 eligible patients with refractory H pylori infection were recruited and randomly assigned to the molecular testing-guided therapy group or the susceptibility testing-guided therapy group in trial 2. 272 men and 288 women were recruited for trial 1, and 98 men and 222 women were recruited for trial 2. In first-line H pylori treatment, infection was eradicated in 241 (86%, 95% CI 82-90) of 280 patients in the molecular testing-guided therapy group and 243 (87%, 83-91) of 280 patients in the susceptibility testing-guided therapy group by intention-to-treat analysis (p=0·81). In third-line H pylori treatment, infection was eradicated in 141 (88%, 83-93) of 160 patients in the molecular testing-guided therapy group and 139 (87%, 82-92) of 160 patients in the susceptibility testing-guided therapy group by intention-to-treat analysis (p=0·74). The difference in the eradication rate between the molecular testing-guided therapy group and the susceptibility testing-guided therapy group was -0·7% (95% CI -6·4 to 5·0; non-inferiority p=0·071) in trial 1 and 1·3% (-6·0 to 8·5; non-inferiority p=0·0018 in trial 2 by intention-to-treat analysis. We found no difference in adverse effects across both treatment groups in trial 1 and trial 2. Molecular testing-guided therapy was similar to susceptibility testing-guided therapy in first-line therapy and non-inferior to susceptibility testing guided therapy in third-line treatment of H pylori infection, supporting the use of molecular testing-guided therapy for H pylori eradication. Ministry of Science and Technology of Taiwan, and Centre of Precision Medicine of the Higher Education Sprout Project by the Ministry of Education of Taiwan.

Evaluation of Microsphere-based xMAP Test for gyrA Mutation Identification in Mycobacterium Tuberculosis

Evaluation of Microsphere-based xMAP Test for gyrA Mutation Identification in Mycobacterium Tuberculosis

High macrolides and fluoroquinolones resistance rate in Mycoplasma genitalium in southern Tenerife

Mycoplasma genitalium (MG) is a recognized sexually transmitted pathogen. Increasing resistance to main lines of treatment (macrolides and quinolones) justifies a genetic study of mutations to improve cure rates. A total of 8,508 samples from April 2018 to July 2022 were processed using AllplexTM STI Essential Assay. In MG positive cases 23S rRNA V domain, gyrA and parC genes were studied. Mutations detected were checked to assess their clinical significance and medical records were reviewed to obtain demographic and treatment information. Resistance study was performed on 92 samples (65 men and 27 women). In relation to the genotypic study, 28 patients presented mutations to macrolides (30.43%). Most common was A2059G (18.48%). For quinolones, 5 patients (5.43%) had clinically relevant mutations in parC gene. Of note was a patient with G295 mutation in gyrA associated with G248T in parC. Thirty subjects underwent a test of cure (TOC). Azithromycin was the most common empirical regimen and moxifloxacin the main alternative. High rate of resistance in our environment evidences the need for targeted therapy by genotypic study of macrolide resistance, supported by the detection of mutations in parC and gyrA to predict quinolones susceptibility and the use of TOC to evaluate treatment response.

A potential role of extracellular DNA in biofilm and ciprofloxacin resistance

Objectives: This study aims to broaden our knowledge of the role of eDNA in bacterial biofilms and antibiotic-resistance gene transfer among isolates.
 Methods: Staphylococcus aureus, E. coli, and Pseudomonas aeruginosa were isolated from different non-repeated 170 specimens. The bacterial isolates were identified using morphological and molecular methods. Different concentrations of genomic DNA were tested for their potential role in biofilms formed by study isolates employing microtiter plate assay. Ciprofloxacin resistance was identified by detecting a mutation in gyrA and parC.
 Results: The biofilm intensity significantly decreased (P < 0.05) concerning S. aureus isolates and insignificantly (P > 0.05) concerning E. coli isolates. Yet, one E. coli isolate's biofilm was significantly decreased (P < 0.05) linearly with increasing eDNA. Of considerable interest, the addition of eDNA led to a significant increase (P < 0.05) in the biofilm of the two-tested P. aeruginosa isolates. Moreover, eDNA participated in transferring Ciprofloxacin resistance to the sensitive isolate when it presents in its biofilm.
 Conclusion: eDNA has a dual effect on bacterial biofilms either supportive or suppressive following bacterial species per se. Also, it seems to play an important role in antibiotic resistance within the biofilm.

Phylogenomics of Globally Spread Clonal Groups 14 and 15 of Klebsiella pneumoniae.

Klebsiella pneumoniae sequence type 14 (ST14) and ST15 caused outbreaks of CTX-M-15 and/or carbapenemase producers worldwide, but their phylogeny and global dynamics remain unclear. We clarified the evolution of K. pneumoniae clonal group 14 (CG14) and CG15 by analyzing the capsular locus (KL), resistome, virulome, and plasmidome of public genomes (n = 481) and de novo sequences (n = 9) representing main sublineages circulating in Portugal. CG14 and CG15 evolved independently within 6 main subclades defined according to the KL and the accessory genome. The CG14 (n = 65) clade was structured in two large monophyletic subclades, CG14-I (KL2, 86%) and CG14-II (KL16, 14%), whose emergences were dated to 1932 and 1911, respectively. Genes encoding extended-spectrum β-lactamase (ESBL), AmpC, and/or carbapenemases were mostly observed in CG14-I (71% versus 22%). CG15 clade (n = 170) was segregated into subclades CG15-IA (KL19/KL106, 9%), CG15-IB (variable KL types, 6%), CG15-IIA (KL24, 43%) and CG15-IIB (KL112, 37%). Most CG15 genomes carried specific GyrA and ParC mutations and emerged from a common ancestor in 1989. CTX-M-15 was especially prevalent in CG15 (68% CG15 versus 38% CG14) and in CG15-IIB (92%). Plasmidome analysis revealed 27 predominant plasmid groups (PG), including particularly pervasive and recombinant F-type (n = 10), Col (n = 10), and new plasmid types. While blaCTX-M-15 was acquired multiple times by a high diversity of F-type mosaic plasmids, other antibiotic resistance genes (ARGs) were dispersed by IncL (blaOXA-48) or IncC (blaCMY/TEM-24) plasmids. We first demonstrate an independent evolutionary trajectory for CG15 and CG14 and how the acquisition of specific KL, quinolone-resistance determining region (QRDR) mutations (CG15), and ARGs in highly recombinant plasmids could have shaped the expansion and diversification of particular subclades (CG14-I and CG15-IIA/IIB). IMPORTANCE Klebsiella pneumoniae represents a major threat in the burden of antibiotic resistance (ABR). Available studies to explain the origin, the diversity, and the evolution of certain ABR K. pneumoniae populations have mainly been focused on a few clonal groups (CGs) using phylogenetic analysis of the core genome, the accessory genome being overlooked. Here, we provide unique insights into the phylogenetic evolution of CG14 and CG15, two poorly characterized CGs which have contributed to the global dissemination of genes responsible for resistance to first-line antibiotics such as β-lactams. Our results point out an independent evolution of these two CGs and highlight the existence of different subclades structured by the capsular type and the accessory genome. Moreover, the contribution of a turbulent flux of plasmids (especially multireplicon F type and Col) and adaptive traits (antibiotic resistance and metal tolerance genes) to the pangenome reflect the exposure and adaptation of K. pneumoniae under different selective pressures.

DETECTION AND CHARACTERIZATION OF MUTATIONS IN GENES RELATED TO SECOND-LINE ANTI-TUBERCULOSIS DRUGS RESISTANCE IN MDR TB

Introduction: Drug-resistant tuberculosis is a major global health issue. Multidrug-resistant TB (MDRTB) and extensively drug-resistant TB (XDR-TB) are particularly concerning. Conventional phenotypic methods being cumbersome and time consuming, molecular assays for the detection of mutations that are associated with resistance to anti-TB drugs have emerged as an important tool for rapid diagnosis. Aims and Objectives: To determine and characterize mutations associated with resistance to second line anti-TB drugs in MDR TB. Methods: First line LPA Conrmed MDR TB and/ or mono rifampicin or mono isoniazid resistant cases were subjected to second line LPA (MTBDRsl ver 2.0 kit)) and banding patterns of resistant cases were obtained. These were then subjected to extended phenotypic DST (Gold Standard) using MGIT 960 for Moxioxacin – 1.0µg/ml, Kanamycin -2.5µg/ml, Capreomycin -2.5µg/ml. Results: The study analysed 1580 samples subjected to SL LPA and found that 37.65% were sensitive to both FQ and SLID. FQ resistance accounted for 60.56% and SLID resistance for 17.02%. Resistance to both seen in 15.15% samples. The most common mutation associated with FQ resistance was gyr A gene at codon D94G, for SLID resistance it was in rrs gene (A1401G) and eis gene (C-12T,G-10A). Conclusion: It was observed that there is a high prevalence (62.34%) of second-line anti-TB drug resistance and mutations in gyrA, gyrB (for FQ), rrs ( for SLID) and eis promoter regions (for low level kanamycin) were signicantly associated with secondline anti-TB drug resistance.

SVR-flaA typing of erythromycin- and ciprofloxacin-resistant Campylobacter jejuni strains isolated from poultry slaughterhouses in southern Brazil.

The emergence of fluoroquinolone and macrolide resistance in C. jejuni, a recognized zoonotic pathogen, has increased worldwide. This study aimed to investigate phenotypic resistance to ciprofloxacin and erythromycin, the molecular mechanisms involved, and the strain of C. jejuni isolated from broiler carcasses. Eighty C. jejuni isolates from broiler carcasses in southern Brazil were investigated for their susceptibility to ciprofloxacin and erythromycin at minimal inhibitory concentrations. Mismatch amplification mutation assay-polymerase chain reaction (MAMA-PCR) was performed to detect substitutions of Thr-86-Ile, A2074C, and A2075G of domain V in the 23S rRNA. The presence of ermB gene and CmeABC operon were investigated by PCR. DNA sequencing was used to detect substitutions in the L4 and L22 proteins of the erythromycin-resistant strains. The Short Variable Region (SVR) of flaA was used to type all the strains resistant to both antimicrobials. Ciprofloxacin and erythromycin resistance were detected in 81.25% and 30.00% of the strains, respectively, and minimal inhibitory concentration values ranged from ≤ 0.125 to 64µg/mL for ciprofloxacin and 0.5 to > 128µg/mL for erythromycin. The Thr-86-Ile mutation in gyrA was observed in 100% of the ciprofloxacin-resistant strains. Mutations in both the A2074C and A2075G positions of 23S rRNA were observed in 62.5% of the erythromycin-resistant strains, while 37.5% had only the mutation A2075G. None of the strains harbored CmeABC operon, and ermB was not detected. Using DNA sequencing, the amino acid substitution T177S was detected in L4, and substitutions I65V, A103V, and S109A were detected in L22. Twelve flaA-SVR alleles were identified among the strains, with the most common SVR-flaA allele, type 287, covering 31.03% of ciprofloxacin- and erythromycin-resistant isolates. The present study revealed a high incidence and high levels of resistance to ciprofloxacin and erythromycin, as well as broad molecular diversity in C. jejuni isolates from broiler carcasses.

Situational analysis on fluoroquinolones use and characterization of high-level ciprofloxacin-resistant Enterococcus faecalis by integrated broiler operations in South Korea.

Fluoroquinolones are classified as "critically important antimicrobials for human medicine"; however, their extensive use in livestock poses a significant health risk to humans as it leads to the rapid spread of antimicrobial resistance. This study confirmed that 40.0%-71.4% of the farms in three of the five integrated broiler operations were administered ciprofloxacin (CIP). Moreover, preventive purposes (60.9%), veterinarian prescriptions (82.6%), drinking water route (100%), and 1 to 3 days (82.6%) of age were significantly highest (P < 0.05). 194 high-level ciprofloxacin-resistant (HLCR) Enterococcus faecalis (E. faecalis) were found in 65 of 74 farms, and of which, the prevalence of qnrA (63.9%), tetM (60.3%), ermB (64.9%), blaz (38.7%), and catA (34.0%) was significantly highest (P < 0.05). 154 (79.4%) isolates showed MDR, and the distribution of MDR was significantly differences among the operations (P < 0.05). All HLCR E. faecalis possessed double mutations in gyrA and parC, and S83I/S80I (90.7%) mutations were most commonly identified. Interestingly, the distribution of isolates with MICs ≥ 512 for both CIP and moxifloxacin was significantly higher in CIP-administered farms (56.5%) than in non-CIP-administered farms (41.4%) (P < 0.05). Also, the prevalence of strong or moderate biofilm formers in HLCR E. faecalis was significantly higher than that of weak and no biofilm formers (P < 0.05). HLCR E. faecalis were heavily distributed in the broiler farms in Korea; therefore, it is necessary to minimize the prevalence of resistant bacteria via structural management regulations such as cleaning and disinfection of farm environments.

Whole genome sequencing reveals novel genetic mutations of Helicobacter pylori associating with resistance to clarithromycin and levofloxacin.

Detection of mutations in one or a couple of genes may not provide enough data or cover all the genomic DNA variance related to antibiotic resistance of Helicobacter pylori to clarithromycin (CLA) and levofloxacin (LVX). We aimed to perform whole genome sequencing to explore novel antibiotic resistance-related genes to increase predictive accuracy for future targeted sequencing tests. Gastric mucosal biopsies were taken during upper endoscopy in 27 H. pylori-infected patients. According to culture-based antibacterial susceptibility test, H. pylori strains were divided into three groups, with nine strains in each group: CLA single-drug resistance (group C), LVX single-drug resistance (group L), and strains sensitive to all antibacterial drugs (group S). Based on whole genome sequencing with group S being the control, group C and group L group-specific single nucleotide variants and amino acid mutations were screened, and potential candidate genes related to CLA and LVX resistance were identified. The median age of study subjects was 35 years (IQR: 31-40), and 17 (63.0%) were male. All nine CLA-resistant strains had A2143G mutations in 23S rRNA, while none of nine sensitive strains had the mutation. Six of nine strains in group L and six of nine strains in group S had 87th or 91st mutation in gyrA. After comparing sequencing data of strains among the three groups, we identified five mutated positions belonging to four genes related to CLA resistance, and 31 mutated positions belonging to 20 genes related to LVX resistance. Novel genetic mutations were detected for CLA resistance (including fliJ and clpX) and LVX resistance (including fliJ, cheA, hemE, Val360Ile, and HP0568). Missense mutations in fliJ and cheA gene were mainly involved in chemotaxis and flagellar motility to facilitate bacterial escape of antibiotics, while the functions of other novel gene mutations underpinning antibiotic resistance remain to be investigated. Whole genome sequencing detected potential novel genetic mutations conferring resistance of H. pylori to CLA and LVX including fliJ and cheA. Further studies to correlate these findings with treatment outcome should be performed.

Differential expression of outer membrane proteins and quinolone resistance determining region mutations can lead to ciprofloxacin resistance in Salmonella Typhi

Multi-drug resistance in Salmonella Typhi remains a public health concern globally. This study aimed to investigate the function of quinolone resistance determining region (QRDR) of gyrA and parC in ciprofloxacin (CIP) resistant isolates and examine the differential expression of outer membrane proteins (OMPs) on exposure to sub-lethal concentrations of CIP in S. Typhi. The CIP-resistant isolates were screened for mutations in the QRDR and analyzed for bacterial growth. Furthermore, major OMPs encoding genes such as ompF, lamB, yaeT, tolC, ompS1, and phoE were examined for differential expression under the sub-lethal concentrations of CIP by real-time PCR and SDS-PAGE. Notably, our study has shown a single-point mutation in gyrA at codon 83 (Ser83-tyrosine and Ser83-phenylalanine), also the rare amino acid substitution in parC gene at codon 80 (Glu80-glycine) in CIP-resistant isolates. Additionally, CIP-resistant isolates showed moderate growth compared to susceptible isolates. Although most of the OMP-encoding genes (tolC, ompS1, and phoE) showed some degree of upregulation, a significant level of upregulation (p < 0.05) was observed only for yaeT. However, ompF and lamB genes were down-regulated compared to CIP-susceptible isolates. Whereas OMPs profiling using SDS-PAGE did not show any changes in the banding pattern. These results provide valuable information on the QRDR mutation, and the difference in the growth, and expression of OMP-encoding genes in resistant and susceptible isolates of S. Typhi. This further provides insight into the involvement of QRDR mutation and OMPs associated with CIP resistance in S. Typhi.

The first evidence of blaCTX-M-55, QnrVC5, and novel insight into the genome of MDR Vibrio vulnificus isolated from Asian sea bass (Lates calcarifer) identified by resistome analysis

Vibrio vulnificus is a bacterium that causes vibriosis and exhibits resistance to many antimicrobials. To comprehensively understand V. vulnificus resistance, four MDRs isolates were selected for resistome analysis. Based on the Average Nucleotide Identity (ANI) and DNA-DNA hybridization (DDH), all isolates showed a high identity score to the V. vulnificus ATCC 33147. Likewise, multilocus sequence typing (MLST) showed that CUVETSS48 isolate was founded closer to two STs (409 and 570), while the others (CUVETCC1, CUVETCC2, and CUVETSS2) were founded closer to a single ST (209, 426, and 372, respectively). MIC determination showed that all isolates were resistant to Amoxicillin and Colistin sulfate, but only two isolates (CUVETCC1 and CUVETSS48) were resistant to all tested antimicrobials. Referring to resistome analysis, multiple antibiotic resistance genes (ARGs) were found, such as QnrVC1, QnrVC7, tetR, tetB, and blaCTX-M-55 (CUVETCC1); tet(59), sul2, and QnrVC5 (CUVETSS48). Moreover, a mutation was founded within the QRDR region of gyrA; S83I (CUVETCC1, CUVETCC2, and CUVETSS48), gyrB; K87N (CUVETCC2, SS2, and SS48), and parC; S80Y (CUVETCC1 and CUVETCC2). In conclusion, this study showed the first evidence for the presence of blaCTX-M-55, QnrVC5 antimicrobial resistance genes, as well as mutations in gyrA, gyrB, and parC genes (at positions 83, 87, and 80) among V. vulnificus isolates. The presence of multiple ARGs may contribute to the MDR phenotype of V. vulnificus.

Prevalence of Mycoplasma genitalium infection with antimicrobial resistance mutations among gay sex workers in China.

Gay sex workers (GSWs) within the population of men who have sex with men in China are known as money boys (MBs). Limited research has been conducted to investigate the infection rate and antimicrobial resistance of Mycoplasma genitalium (M. genitalium) among GSWs in China. This study aimed to evaluate the status of M. genitalium infection among in GSWs. This study was performed among 349 GSWs who were followed up for four years by internet-based sampling collection. The participants were asked to complete an online questionnaire using a mobile app, and trained interviewers took urethral, anorectal, and saliva swab specimens. STIs, including HIV and M. genitalium, were detected. Detection of resistance-associated mutations (RAMs) to macrolides and fluoroquinolones was performed via Sanger sequencing of the 23S rRNA, parC and gyrA genes. GSWs were enrolled by identifying 10 initial "seeds" from the Blued and WeChat apps. Face-to-face interviews were conducted with 349 GSWs from June 2017 to July 2021. The prevalence of M. genitalium and HIV positivity was 92/349 (26.4%, 95% confidence interval [CI] 21.7-31.0) and 71/349 (20.3%, 95% CI 16.3-24.4), respectively. The proportion of GSWs with M. genitalium infection alone in urethral swabs was 16, and the proportion with symptoms was 2/16 (12.5%). The proportion of GSWs with M. genitalium infection alone in anorectal swabs was 36, and the proportion with symptoms was 3/36 (8.3%). Multivariate regression analysis showed that using new types of drugs in the past 3 months and inconsistent condom usage with clients in the past 30 days were associated with M. genitalium infection. Macrolide resistance within the 23S rRNA gene was detected in 73/88 (83.0%) of the M. genitalium-positive GSWs. Moreover, 79.8% (71/89) of parC and 21.1% (19/90) of gyrA genes had mutations responsible for fluoroquinolone resistance. Three cases had no mutations in any of the three genes, 11 cases had mutations in all three genes, five cases had gyrA and parC gene mutations with no mutation in the 23S rRNA gene, and 42 cases had 23S rRNA and parC gene mutations with no mutation in the gyrA gene. M. genitalium infections in our study displayed a high prevalence and very high levels of macrolide and fluoroquinolone resistance among GSWs in China. Asymptomatic M. genitalium infections are quite common among GSWs. Routine resistance testing of M. genitalium-positive specimens and antimicrobial resistance surveillance are crucial.

Genomic analyses of drug-resistant Salmonella enterica serovar Heidelberg strains isolated from meat and related sources between 2013 and 2017 in the south region of Brazil.

Salmonella enterica serovar Heidelberg (S. Heidelberg) is a zoonotic, ubiquitous, and worldwide-distributed pathogen, responsible for gastroenteritis in humans caused by the consumption of contaminated food. In this study, 11 S. Heidelberg strains isolated from chicken and bovine meat, drag swab, and animal feed between 2013 and 2017 in states of the southern region of Brazil were characterized by whole-genome sequencing (WGS) analyses. Antimicrobial resistance against 18 antimicrobials was determined by disk-diffusion and ciprofloxacin's minimum inhibitory concentration by Etest®. The search for resistance and virulence genes, plasmids, Salmonella Pathogenicity Islands (SPIs) plus multi-locus sequence typing (MLST), and single-nucleotide polymorphisms (SNPs) analyses was conducted using WGS data. All strains harbored resistance genes fosA7, aac(6')-Iaa, sul2, tet(A), blaCMY-2, mdsA, and mdsB, and point mutations in gyrA and parC. All strains showed a phenotypic multidrug-resistant profile, with resistant or intermediate resistant profiles against 14 antimicrobials tested. Plasmids ColpVC, IncC, IncX1, and IncI1-I(Alpha) were detected. Virulence genes related to adherence, macrophage induction, magnesium uptake, regulation, and type III secretion systems plus 10 SPIs were detected. All strains were assigned to ST15 and belonged to two SNP clusters showing high similarity to isolates from the United Kingdom, Chile, Germany, the Netherlands, China, South Africa, and South Korea. In conclusion, the presence of multidrug-resistant S. Heidelberg strains in Brazil showing a global genomic relationship may alert for the necessity of stronger surveillance measures by food safety and public health authorities to limit its spread to humans and animals through foods.

Fluoroquinolone Residues in the Environment Rapidly Induce Heritable Fluoroquinolone Resistance in Escherichia coli.

Extensive antibiotic use increases the environmental presence of their residues and may accelerate the development of antibiotic resistance, although this remains poorly understood at environmentally relevant concentrations. Herein, susceptible Escherichia coli K12 was continuously exposed to five antibiotics at such concentrations for 100 days. The de novo-evolved mutants rapidly obtained fluoroquinolone resistance within 10 days, as indicated by the 4- and 16-fold augmentation of minimum inhibitory concentrations against enrofloxacin and ciprofloxacin, respectively. Moreover, the mutants maintained heritable fluoroquinolone resistance after the withdrawal of antibiotics for 30 days. Genomic analysis identified Asp87Gly or Ser83Leu substitutions in the gyrA gene in the mutants. Transcriptomics data showed that the transcriptional response of the mutants to fluoroquinolones was primarily involved in biofilm formation, cellular motility, porin, oxidative stress defense, and energy metabolism. Homologous recombination and molecular docking revealed that mutations of gyrA primarily mainly conferred fluoroquinolone resistance, while mutations at different positions of gyrA likely endowed different fluoroquinolone resistance levels. Collectively, this study revealed that environmentally relevant concentrations of antibiotics could rapidly induce heritable antibiotic resistance; therefore, the discharge of antibiotics into the environment should be rigorously controlled to prevent the development of antibiotic resistance.

Fluoroquinolone resistance among fecal extended spectrum βeta lactamases positive Enterobacterales isolates from children in Dar es Salaam, Tanzania

BackgroundFluoroquinolones have been, and continue to be, routinely used for treatment of many bacterial infections. In recent years, most parts of the world have reported an increasing trend of fluoroquinolone resistant (FQR) Gram-negative bacteria.MethodsA cross-sectional study was conducted between March 2017 and July 2018 among children admitted due to fever to referral hospitals in Dar es Salaam, Tanzania. Rectal swabs were used to screen for carriage of extended-spectrum β-lactamase-producing Enterobacterales (ESBL-PE). ESBL-PE isolates were tested for quinolone resistance by disk diffusion method. Randomly selected fluroquinolone resistant isolates were characterized by using whole genome sequencing.ResultsA total of 142 ESBL-PE archived isolates were tested for fluoroquinolone resistance. Overall phenotypic resistance to ciprofloxacin, levofloxacin and moxifloxacin was found in 68% (97/142). The highest resistance rate was seen among Citrobacterspp. (100%, 5/5), followed by Klebsiella.pneumoniae (76.1%; 35/46), Escherichiacoli (65.6%; 42/64) and Enterobacter spp. (31.9%; 15/47). Whole genome sequencing (WGS) was performed on 42 fluoroquinolone resistant-ESBL producing isolates and revealed that 38/42; or 90.5%, of the isolates carried one or more plasmid mediated quinolone resistance (PMQR) genes. The most frequent PMQR genes were aac(6’)-lb-cr (74%; 31/42), followed by qnrB1 (40%; 17/42), oqx,qnrB6 and qnS1. Chromosomal mutations in gyrA, parC and parE were detected among 19/42 isolates, and all were in E.coli. Most of the E. coli isolates (17/20) had high MIC values of > 32 µg/ml for fluoroquinolones. In these strains, multiple chromosomal mutations were detected, and all except three strains had additional PMQR genes. Sequence types, ST131 and ST617 predominated among E.coli isolates, while ST607 was more common out of 12 sequence types detected among the K.pneumoniae. Fluoroquinolone resistance genes were mostly associated with the IncF plasmids.ConclusionThe ESBL-PE isolates showed high rates of phenotypic resistance towards fluoroquinolones likely mediated by both chromosomal mutations and PMQR genes. Chromosomal mutations with or without the presence of PMQR were associated with high MIC values in these bacteria strains. We also found a diversity of PMQR genes, sequence types, virulence genes, and plasmid located antimicrobial resistance (AMR) genes towards other antimicrobial agents.

Fluoroquinolone-resistance mechanisms and molecular epidemiology of ciprofloxacin-resistant Klebsiella pneumoniae isolates in Iran.

Klebsiella pneumoniae is an important cause of nosocomial infections and displays increasing resistance to fluoroquinolones (FQ). This study surveyed the mechanisms of FQ resistance and molecular typing of K. pneumoniae isolates from intensive care units patients in Tehran, Iran. A total of 48 ciprofloxacin (CIP) resistant K. pneumoniae isolates from urine samples were included in this study. Broth microdilution assays revealed high-level CIP resistance (MIC > 32μg/mL) in 31.25% of the isolates. Plasmid-mediated quinolone resistance genes were detected in 41 (85.4%) isolates. Among which, qnrS (41.67%) was the most prevalent followed by qnrD (35.42%), qnrB (27.1%), qnrA (25%), qepA (22.9%), aac(6')-Ib-cr (20.83%), and qnrC (6.25%). Target site mutations (gyrA and parC) were assessed using PCR and sequencing on all isolates. A single mutation in gyrA (S83I) was found in 13 (27.1%) isolates and two isolates harbored six simultaneous mutations. Fourteen isolates (29.2%) had mutations in parC and S129A and A141V mutations were the most prevalent. Real time PCR showed an increase in the expression level of acrB and oqxB efflux genes in 68.75 and 29.16% isolates, respectively. Enterobacterial repetitive intergenic consensus (ERIC)-PCR revealed 14 genotypes and 11 of them were classified by multilocus sequence typing (MLST) into 11 different sequence types belonging to seven clonal complexes and two singletons, most of them have not been reported in Iran yet. We are concerned about the spread of these clones throughout our country. Most FQ resistance mechanisms were detected among our isolates. However, target site mutation had the greatest effect on CIP resistance among our isolates.

Quinolone-resistant Escherichia coli at the interface between humans, poultry and their shared environment- a potential public health risk

BackgroundCommensal Escherichia coli residing in the guts of humans and animals are reservoirs of multidrug resistance (MDR) genes, including quinolone resistance genes, in humans and poultry. This study aimed to characterize quinolones resistance in E. coli recovered from poultry workers, chickens, and poultry farm/market environments in Abuja, Nigeria.MethodsThis was a cross-sectional study conducted between December 2018 and April 2019 comprising poultry workers, chickens and their poultry farm/market environments. This study characterized E. coli isolates from stool, faecal and environmental samples using antimicrobial susceptibility testing and whole-genome sequencing methods. Core-genome multilocus sequences-based phylogeny was used to determine the relatedness between quinolone-resistant E. coli isolates. Data were analyzed using descriptive statistics.ResultsOf 110 E. coli isolates, quinolone-resistant phenotypes were observed in 68.2% (n = 75) isolates. Whole-genome sequencing detected plasmid-mediated quinolone resistance (PMQR) genes in 63.6% (n = 70) isolates. The most prevalent PMQR gene detected in 56 of these 70 E. coli isolates was qnrS1, followed by qnrB19 in 14 isolates and aac(6’)-lb-cr in two isolates. Fifteen ciprofloxacin and 19 nalidixic acid-resistant isolates respectively showed double mutations in the quinolone-resistance determining regions (QRDRs) of gyrA, with single or double mutations in parC, and a single mutation in parE. The most prevalent amino-acid substitutions observed were S83L + D87N in gyrA (46.5%, n = 20), S80I in parC (51.2%, n = 22) and S458A in parE (14%, n = 6). About 2.9% (2/70) of PMQR isolates were extended-spectrum beta-lactamase (ESBL) producers while 2.9% (2/70) had plasmid-mediated colistin resistance (PMCR) genes.ConclusionsPMQR genes were prevalent in E. coli isolates recovered from healthy humans, chickens and poultry farm/market environments. PMCR genes (mcr-1.1) occurred in PMQR-positive isolates recovered from manure and drinking water originating from poultry farm/market environments. It was found that the gene encoding ESBL coexisted with qnrS-positive isolates of human and avian origin. Horizontal transfer of PMQR genes among E. coli isolates in the human-poultry-environment interface has public health implications for the spread of antimicrobial resistance. Relevant government agencies should enforce regulations to restrict the use of critically important antimicrobials in poultry production.

Whole-genome-based characterization of Campylobacter jejuni from human patients with gastroenteritis collected over an 18 year period reveals increasing prevalence of antimicrobial resistance.

Campylobacteriosis is the most common cause of acute gastrointestinal bacterial infection in Europe, with most infections linked to the consumption of contaminated food. While previous studies found an increasing rate of antimicrobial resistance (AMR) in Campylobacter spp. over the past decades, the investigation of additional clinical isolates is likely to provide novel insights into the population structure and mechanisms of virulence and drug resistance of this important human pathogen. Therefore, we combined whole-genome sequencing and antimicrobial-susceptibility testing of 340 randomly selected Campylobacter jejuni isolates from humans with gastroenteritis, collected in Switzerland over an 18 year period. In our collection, the most common multilocus sequence types (STs) were ST-257 (n=44), ST-21 (n=36) and ST-50 (n=35); the most common clonal complexes (CCs) were CC-21 (n=102), CC-257 (n=49) and CC-48 (n=33). High heterogeneity was observed among STs, with the most abundant STs recurring over the entire study period, while others were observed only sporadically. Source attribution based on ST assigned more than half of the strains to the 'generalist' category (n=188), 25 % as 'poultry specialist' (n=83), and only a few to 'ruminant specialist' (n=11) or 'wild bird' origin (n=9). The isolates displayed an increased frequency of AMR from 2003 to 2020, with the highest rates of resistance observed for ciprofloxacin and nalidixic acid (49.8 %), followed by tetracycline (36.9 %). Quinolone-resistant isolates carried chromosomal gyrA mutations T86I (99.4 %) and T86A (0.6 %), whereas tetracycline-resistant isolates carried tet(O) (79.8 %) or mosaic tetO/32/O (20.2 %) genes. A novel chromosomal cassette carrying several resistance genes, including aph(3')-III, satA and aad(6), and flanked by insertion sequence elements was detected in one isolate. Collectively, our data revealed an increasing prevalence of resistance to quinolones and tetracycline in C. jejuni isolates from Swiss patients over time, linked to clonal expansion of gyrA mutants and acquisition of the tet(O) gene. Investigation of source attribution suggests that infections are most likely related to isolates from poultry or generalist backgrounds. These findings are relevant to guide future infection prevention and control strategies.

Characteristics of rare ST463 carbapenem-resistant Pseudomonas aeruginosa clinical isolates from blood

ObjectivesThis study aimed to elucidate resistance to carbapenems and fluoroquinolones, the transmission mechanism of blaKPC-2, and the virulence characteristics of a Pseudomonas aeruginosa strain (TL3773) isolated in East China. MethodsThe virulence and resistance mechanisms of TL3773 were investigated by whole genome sequencing (WGS), comparative genomic analysis, conjugation experiments, and virulence assays. ResultsThis study isolated carbapenem-resistant P. aeruginosa from blood resistant to carbapenems. The patient's clinical data showed poor prognosis compounded by multiple sites of infection. WGS showed that TL3773 carried aph (3′)-IIb, blaPAO, blaOXA-486, fosA, catB7, and two crpP resistance genes on chromosome, and the carbapenem resistance gene blaKPC-2 on plasmid. We identified a novel crpP gene named TL3773-crpP2. Cloning experiments proved that TL3773-crpP2 was not the primary cause of fluoroquinolone resistance in TL3773. GyrA and ParC mutations may confer fluoroquinolone resistance. The blaKPC-2 genetic environment was IS26-TnpR-ISKpn27-blaKPC-2-ISKpn6-IS26-Tn3-IS26, potentially mediating the transmission of blaKPC-2 in P. aeruginosa. The overall virulence of TL3773 was lower than that of PAO1. However, the pyocyanin and biofilm formation of TL3773 was higher than that of PAO1. WGS further indicated that TL3773 was less virulent than PAO1. Phylogenetic analysis showed that TL3773 was most similar to the P. aeruginosa isolate ZYPA29 from Hangzhou, China. These observations further indicate that ST463 P. aeruginosa is rapidly spreading. ConclusionsThe threat of ST463 P. aeruginosa harbouring blaKPC-2 is emergent and may pose a threat to human health. More extensive surveillance and effective actions are urgently needed to control its further spread.

GyrA Mutations in Mycoplasma genitalium and Their Contribution to Moxifloxacin Failure: Time for the Next Generation of Resistance-Guided Therapy.

Although single nucleotide polymorphisms (SNPs) in Mycoplasma genitalium parC contribute to fluoroquinolone treatment failure, data are limited for the homologous gene, gyrA. This study investigated the prevalence of gyrA SNPs and their contribution to fluoroquinolone failure. Samples from 411 patients (male and female) undergoing treatment for M. genitalium infection (Melbourne Sexual Health Centre, March 2019-February 2020) were analyzed by Sanger sequencing (gyrA and parC). For patients treated with moxifloxacin (n = 194), the association between SNPs and microbiologic treatment outcome was analyzed. The most common parC SNP was G248T/S83I (21.1% of samples), followed by D87N (2.3%). The most common gyrA SNP was G285A/M95I (7.1%). Dual parC/gyrA SNPs were found in 8.6% of cases. One third of infections harboring parC G248T/S83I SNP had a concurrent SNP in gyrA conferring M95I. SNPs in gyrA cooccurred with parC S83I variations. Treatment failure was higher in patients with parC S83I/gyrA dual SNPs when compared with infections with single S83I SNP alone from analysis of (1) 194 cases in this study (81.2% vs 45.8%, P = .047), and (2) pooled analysis of a larger population of 535 cases (80.6% vs 43.2%; P = .0027), indicating a strong additive effect. Compared with parC S83I SNP alone, M. genitalium infections with dual mutations affecting parC/gyrA had twice the likelihood of failing moxifloxacin. Although antimicrobial resistance varies by region globally, these data indicate that gyrA should be considered as a target for future resistance assays in Australasia. We propose a strategy for the next generation of resistance-guided therapy incorporating parC and gyrA testing.