Fluoroquinolone Resistance In Clinical Isolates Research Articles

Analysing the Impact of Bedaquiline Resistance in Fluoroquinolone-Resistant Clinical Isolates and Investigating the Molecular Interactions of BDQ with the atpE Gene

The progress of the tuberculosis eradication programme is hindered by the global health issue of drug-resistant tuberculosis, which is a significant threat to the population. Resistance-associated mutations vary geographically, so investigate the prevalence of resistance to fluoroquinolone (anti-TB) drugs and its association with resistance-related mutations in clinical isolates of Tamilnadu (north zone) and Puducherry was carried out. And the resultant fluoroquinolone drug-resistant tuberculosis strains will be subjected to the molecular analysis of bedaquiline resistance to study the mutation in the atpE gene. The study includes 430 specimens received at Intermediate Reference Laboratory, State TB training and Demonstration Centre in the Government Hospital for Chest Diseases for Molecular testing between January 2020 and December 2021. Samples were analysed by GenoType MTBDRslV.2 for the molecular detection of mutations in the gyrA and gyrB genes. FQ resistance was observed in 32 samples (7.4%), most of which were naïve and previous treatment failure cases. Twenty-five isolates had mutations in DNA gyrase subunit -A (gyrA), while mutations in gyrB were observed in only 7 isolates. Mutational analysis revealed that the mutations mainly alter codon 94 (replacing aspartic acid with glycine, D94G), and 90 (replacing alanine with valine, A90V). In Isoniazid Resistance, MDR, and treatment failure cases, the resistance to Fluoroquinolones was most commonly associated with the D94G mutation. A molecular stimulation study evaluates the effect of BDQ on atpE gene by docking, using AutoDock 4.2, showing hydrogen, hydrophobic and electrostatic interactions.

Plasmid-Mediated Fluoroquinolone Resistance in Pseudomonas aeruginosa and Acinetobacter baumannii.

Introduction Pseudomonas aeruginosa and Acinetobacter baumannii are important pathogens in health care–associated infections. Fluoroquinolone resistance has emerged in these pathogens. In this study, we aimed to determine the occurrence of plasmid-mediated quinolone resistance (PMQR) determinants ( qnrA , qnrB , qnrS , aac(6′)-Ib-cr , oqxAB , and qepA ) by polymerase chain reaction (PCR) and the transmissibility of plasmid-borne resistance determinants in clinical isolates of P. aeruginosa and A. baumannii . Materials and Methods The study included P. aeruginosa (85) and A. baumannii (45) which were nonduplicate, clinically significant, and ciprofloxacin resistant. Antibiotic susceptibility testing was done by disk diffusion method for other antimicrobial agents, namely amikacin, ceftazidime, piperacillin/tazobactam, ofloxacin, levofloxacin, and imipenem. Minimum inhibitory concentration of ciprofloxacin was determined. Efflux pump activity was evaluated using carbonyl-cyanide m-chlorophenylhydrazone (CCCP). The presence of PMQR genes was screened by PCR amplification. Transferability of PMQR genes was determined by conjugation experiment, and plasmid-based replicon typing was performed. Results Resistance to other classes of antimicrobial agents was as follows: ceftazidime (86.9%), piperacillin/tazobactam (73.8%), imipenem (69.2%), and amikacin (63.8%). The minimal inhibitory concentration (MIC)50 and MIC90 for ciprofloxacin were 64 and greater than or equal to 256 µg/mL, respectively. There was a reduction in MIC for 37 (28.4%) isolates with CCCP. In P. aeruginosa , 12 (14.1%) isolates harbored qnrB , 12 (14.1%) qnrS , 9 (10.5%) both qnrB and qnrS , 66 (77.6%) aac(6′)-Ib-cr , and 3 (3.5%) oqxAB gene. In A. baumannii , qnrB was detected in 2 (4.4%), 1 (2.2%) harbored both the qnrA and qnrS , 1 isolate harbored qnrB and qnrS , 21 (46.6%) aac(6′)-Ib-cr , and 1 (2.2%) isolate harbored oqxAB gene. Notably, qepA gene was not detected in any of the study isolates. Conjugation experiments revealed that 12 (9.2%) were transferable. Of the transconjugants, seven (58.3%) belonged to IncFII type plasmid replicon, followed by four (33.3%) IncA/C and one (8.3%) IncFIC type. Conclusion The plasmid-mediated resistance aac(6′)-Ib-cr gene is primarily responsible for mediating fluoroquinolone resistance in clinical isolates of P . aeruginosa and A. baumannii . The predominant plasmid type is IncFII.

Burden of Fluoroquinolone Resistance in Clinical Isolates of Escherichia coli at the Ho Teaching Hospital, Ghana.

The growing burden of antibiotic resistance is a threat to the management of infections. Infections by Escherichia coli are routinely treated with fluoroquinolone antimicrobial agents. Due to their frequent use, there has been increasing resistance to these drugs. We set out to determine the burden of fluoroquinolone resistance among clinical E. coli isolates at the Ho Teaching Hospital, Ghana. This was a cross-sectional study conducted from July 2018 to June 2019. One hundred and thirty-five E. coli isolates were cultured from various clinical samples. Antimicrobial susceptibility testing was performed using the Kirby-Bauer disk diffusion method with discs of nalidixic acid (NAL), ciprofloxacin (CIP), norfloxacin (NOR) and levofloxacin (LEV). Deoxyribonucleic acid (DNA) was extracted from the resistant isolates for the detection of fluoroquinolone resistant genes by polymerase chain reaction. Ninety of the 135 isolates (66.7%) were resistant to at least one of the four fluoroquinolone drugs investigated. Resistance to NAL, CIP, NOR, and LEV was 51.0%, 51.1%, 38.8% and 35.7% respectively. Out of the fluoroquinolone resistant isolates, 69 carried one or more fluoroquinolone resistant genes. The predominant resistant genes were aac(6')-Ib-cr (48.9%) and qnrD (25.6%). Seven of the isolates carried both qnrS and aac(6')-Ib-cr genes. Two isolates carried 5 different fluoroquinolone resistant genes. High prevalence of resistance to 4 fluoroquinolone drugs was recorded with associated resistant genes. This is a threat to current efforts to control the spread of antibiotic resistance and calls for concerted efforts to curb the spread of these resistant organisms.

Prevalence and molecular characterization of β-lactamase producers and fluoroquinolone resistant clinical isolates from North East India

IntroductionThe rapid emergence and variations of antibiotic resistance among common gram negative bacteria cause a significant concern specially in India and all over the world because of high mortality and morbidity rates. MethodsIn our study, we screened 189 bacterial isolates from Assam Medical College & Hospital, Dibrugarh for antibiotic resistance pattern and tried to identify the resistant genes causing responsible for β-lactam and fluoroquinolones resistance. ResultsMore than 80% and 45% strains were resistant to all the 3rd generation cephalosporins, fluoroquinolones respectively. Among the 3rd generation cephalosporin resistant strains, 38% and 24% isolates were only ESBL and MBL producers respectively and 11% were reported to have both ESBL and MBL genes. The ESBL positive isolates have shown the dominance of CTX-M3 gene. VIM-1 gene was mostly reported in MBL producers. Our study probably for the first time reporting SIM-1 and SPM-1 MBL gene from India. Mutations in QRDR is found to be the primary cause of fluoroquinolone resistance along with efflux pump and PMQR presence. ConclusionThe study represents the first detailed study on antibiotic resistance from NE India this could help to take control measures for the emerging antibiotic resistance in hospital and community based infections in North East India.

Identification of two novel type II topoisomerase mutations in Enterococcus spp. isolated from a hospital in China

Type II topoisomerases, including DNA gyrase (GyrA) and topoisomerase IV (ParC), contribute to fluoroquinolone resistance in Enterococcus spp. This study investigated the mutational status of the quinolone resistance-determining regions (QRDRs) of GyrA and ParC in the clinical isolates of enterococci from a hospital in Baotou, China. We analyzed 110 enterococcal isolates, including 57 Enterococcus faecalis and 53 Enterococcus faecalis faecium. The resistance rates of E. faecalis and E. faecium to ciprofloxacin were 63.16% and 84.91%, respectively. We found that 32 samples of E. faecalis and 42 of E. faecium had single or combined mutations in gyrA and/or parC, which were all resistant to ciprofloxacin. Only two ciprofloxacin-resistant E. faecalis isolates had no mutation. No mutations in gyrA and parC genes in all ciprofloxacinsusceptible isolates were found. Ciprofloxacin minimal inhibitory concentrations (MICs) in the mutation group were significantly higher than those of the non-mutation group, indicating that mutations in the QRDRs of gyrA and parC were correlated with MIC elevation. Two novel substitutions (GyrA Ser83Phe and ParC Ser80Leu) of E. faecalis were identified herein. Three-dimensional modeling revealed that these novel amino acid substitutions could disrupt the water/metal-ion bridge and decrease the interaction between the enzymes and ciprofloxacin. The data showed a diversity of mutation types in QRDRs of type II topoisomerases whose association with fluoroquinolone resistance in clinical isolates of enterococci warrants further investigation.

Companion Animals Are Spillover Hosts of the Multidrug-Resistant Human Extraintestinal Escherichia coli Pandemic Clones ST131 and ST1193.

Escherichia coli sequence types 131 (ST131) and 1193 are multidrug-resistant extraintestinal pathogens that have recently spread epidemically among humans and are occasionally isolated from companion animals. This study characterized a nationwide collection of fluoroquinolone-resistant (FQR) E. coli isolates from extraintestinal infections in Australian cats and dogs. For this, 59 cat and dog FQR clinical E. coli isolates (representing 6.9% of an 855-isolate collection) underwent PCR-based phylotyping and whole-genome sequencing (WGS). Isolates from commensal-associated phylogenetic groups A (14/59, 24%) and B1 (18/59, 31%) were dominant, with ST224 (10/59, 17%), and ST744 (8/59, 14%) predominating. Less prevalent were phylogenetic groups D (12/59, 20%), with ST38 (8/59, 14%) predominating, and virulence-associated phylogenetic group B2 (7/59, 12%), with ST131 predominating (6/7, 86%) and no ST1193 isolates identified. In a WGS-based comparison of 20 cat and dog-source ST131 isolates with 188 reference human and animal ST131 isolates, the cat and dog-source isolates were phylogenetically diverse. Although cat and dog-source ST131 isolates exhibited some minor sub-clustering, most were closely related to human-source ST131 strains. Furthermore, the prevalence of ST131 as a cause of FQR infections in Australian companion animals was relatively constant between this study and the 5-year-earlier study of Platell et al. (2010) (9/125 isolates, 7.2%). Thus, although the high degree of clonal commonality among FQR clinical isolates from humans vs. companion animals suggests the possibility of bi-directional between-species transmission, the much higher reported prevalence of ST131 and ST1193 among FQR clinical isolates from humans as compared to companion animals suggests that companion animals are spillover hosts rather than being a primary reservoir for these lineages.

Substantial Contribution of SmeDEF, SmeVWX, SmQnr, and Heat Shock Response to Fluoroquinolone Resistance in Clinical Isolates of Stenotrophomonas maltophilia.

Stenotrophomonas maltophilia is an emerging multi-drug resistant opportunistic pathogen. Although fluoroquinolones (FQ) are still clinically valuable for the treatment of S. maltophilia infection, an increasing prevalence in FQ resistance has been reported. Overexpression of SmeDEF, SmeVWX, and SmQnr, and de-repressed expression of heat shock response are reported mechanisms responsible for FQ resistance in S. maltophilia; nevertheless, some of these mechanisms are identified from laboratory-constructed mutants, and it remains unclear whether they occur in clinical setting. In this study, we aimed to assess whether these mechanisms contribute substantially to FQ resistance in clinical isolates. Eighteen ciprofloxacin- and levofloxacin-resistant isolates were selected from 125 clinical isolates of S. maltophilia. The expression of smeE, smeW, and Smqnr genes of these isolates was investigated by RT-qPCR. The de-repressed heat shock response was assessed by rpoE expression at 37°C and bacterial viability at 40°C. The contribution of SmeDEF, SmeVWX, and SmQnr, and heat shock response to FQ resistance was evaluated by mutants construction and susceptibility testing. The results demonstrated that simply assessing the overexpression of SmeDEF, SmeVWX, and SmQnr by RT-qPCR may overestimate their contribution to FQ resistance. Simultaneous overexpression of SmeDEF and SmeVWX did not increase the resistance level to their common substrates, but extended the resistance spectrum. Moreover, the de-repressed expression of heat shock response was not observed to contribute to FQ resistance in the clinical isolates of S. maltophilia.

Investigation of the susceptibility trends in Japan to fluoroquinolones and other antimicrobial agents in a nationwide collection of clinical isolates: A longitudinal analysis from 1994 to 2016

The susceptibilities of clinical isolates to fluoroquinolones and other antimicrobial agents were surveyed to obtain an accurate understanding of trends in incidence and antimicrobial resistance. The samples were collected from across Japan, biennially or triennially, between 1994 and 2016 and a defined level of resistance to fluoroquinolone was determined.Streptococcus pneumoniae, Streptococcus pyogenes and Haemophilus influenzae exhibited stable and high rates of susceptibility to fluoroquinolones over the period examined. For methicillin-resistant Staphylococcus aureus the rate of resistance to levofloxacin and ciprofloxacin was 81.3–93.5% and 83.2–94.2%, respectively, which was markedly higher than that of methicillin-susceptible S. aureus, while sitafloxacin-resistant methicillin-susceptible and methicillin-resistant S. aureus were isolated at 0.3–0.7% and 16.9–36.5%, respectively. The rate of levofloxacin or ciprofloxacin-resistant Escherichia coli increased from around 2–3% between 1994 and 1998 to around 35% in 2016, but the rate of fluoroquinolone-susceptible Klebsiella pneumoniae stayed high at over 94.6% during the study period. Although no fluoroquinolone-resistance in clinical isolates of Salmonella spp. was detected from 1994 to 2002, the resistance rate increased slightly after 2004 and reached to 1.9%–4.7% in 2016. The rate of fluoroquinolone-susceptible Pseudomonas aeruginosa isolated from urinary tract and respiratory tract infections improved during the period examined from 41.8–67.0% to 91.2–94.2%, and from 78.9-88.5% to 90.1–94.6%, respectively. Against Acinetobacter spp., the susceptibility rate of fluoroquinolones was almost constant at around 90%, but one multidrug-resistant isolate was detected in 2013. Overall, the susceptibility to fluoroquinolones was maintained over 20 years against tested bacteria except for MRSA and E. coli.

Prevalence and mechanism of fluoroquinolone resistance in clinical isolates of Proteus mirabilis in Japan

Fluoroquinolone (FQ) and cephalosporin (CEP) resistance among Enterobacteriaceae has been increasingly reported. FQ resistance occurs primarily through mutations in DNA gyrase (gyrA and gyrB) and topoisomerase IV (parC and parE). CEP resistance in Enterobacteriaceae is mainly due to the production of CTX-M type extended-spectrum β-lactamases. Although prevalence and mechanisms of FQ and CEP resistance in Enterobacteriaceae such as Escherichia coli have been well studied, little is known about Proteus mirabilis in Japan. In this study, we assessed the prevalence and mechanism of FQ resistance in Japanese clinical isolates of P. mirabilis. We collected 5845 P. mirabilis isolates from eight hospitals between 2000 and 2013. Prevalence of FQ resistance was calculated as the annual average percentage of all P. mirabilis isolates. We selected 50 isolates exhibiting susceptibility, intermediate resistance, or resistance to levofloxacin (LVX) and identified amino acid substitutions in GyrA, GyrB, ParC, and ParE. The prevalence of FQ-resistant P. mirabilis gradually increased from 2001 to 2004, reaching 16.6% in 2005, and has remained relatively high (13.3–17.5%) since then. Low-level LVX-resistant strains (MIC, 8–16 mg/L) showed significant changes in GyrB (S464Y or -I, or E466D). High-level LVX-resistant strains (MIC, 32–128 mg/L) displayed significant changes in GyrA (E87K) and ParE (D420N). The highest-level LVX-resistant strains (MIC, ≥ 256 mg/L) presented significant changes in GyrA (E87K or -G), GyrB (S464I or -F), and ParE (D420N). Our findings suggest that substitutions in GyrA (E87) and ParE (D420) have played an important role in the emergence of high-level LVX-resistant P. mirabilis isolates (MIC, ≥ 32 mg/L) in Japan.

Genotypic detection of fluoroquinolone resistance in drug-resistant Mycobacterium tuberculosis at a tertiary care centre in south Coastal Karnataka, India.

This study aimed to characterise mutations associated with fluoroquinolone resistance in drug-resistant Mycobacterium tuberculosis clinical isolates at a tertiary care centre in south Coastal Karnataka, India. DNA from 50 M. tuberculosis clinical isolates was extracted and the gyrA and gyrB genes were amplified. Purified amplicons of gyrA and gyrB were sequenced and extended-sequencing PCR products were analysed. Analysis of mutations in gyrA and gyrB was done using the MUBII-TB-DB database. Statistical analysis was performed using SPSS v.22 and data were compared by χ2 test. A P-value of <0.05 was considered statistically significant. Mutations conferring resistance to fluoroquinolones were observed in 9 isolates (18%). The gyrA A281G (D94G) mutation was observed in 3 isolates (6%), whereas mutations G280T (D94Y) and A281C (D94A) were each observed in 1 isolate (2%). Mutation G1498A (D500N) in gyrB alone was observed in 2 isolates (4%). Two isolates (4%) had mutations both in gyrA and gyrB; gyrA mutation T271C (S91P) was observed in one isolate, whereas the other isolate had gyrA C269G (A90G), but both isolates had a common G1498A (D500N) gyrB mutation. G284C mutation conferring S95T polymorphism (not associated with fluoroquinolone resistance) was observed in 39/50 isolates (78%). gyrA mutations at codons 94, 91 and 90 and gyrB mutation G1498A (D500N) were the most common mutations associated with fluoroquinolone resistance in clinical isolates in this study. Future studies including a larger number of samples are desirable to fully explore the true extent of fluoroquinolone resistance and mutations associated with them.

Mutations in the gyrA, parC, and mexR genes provide functional insights into the fluoroquinolone-resistant Pseudomonas aeruginosa isolated in Vietnam.

IntroductionPseudomonas aeruginosa has many mechanisms of resistance to fluoroquinolones. The main mechanism is to change the effect of two enzymes that open the DNA helix – the enzyme DNA gyrase (gyrA) and the topoisomerase IV (parC). In addition, mutations that render the MexAB-oprM pump (mexR) dysfunctional, leading to its overexpression, also enhance resistance to fluoroquinolones. In this study, we aim to detect point mutations of gyrA, parC, and mexR genes that are predicted to be associated with fluoroquinolone resistance in 141 fluoroquinolone-resistant clinical isolates of P. aeruginosa isolated in Vietnam during 2013–2016.MethodsWe tested minimum inhibitory concentrations (MICs) of fluoroquinolone antibiotics in 141 clinical isolates of P. aeruginosa using the VITEK 2 Compact System, followed by PCR assay, to detect and clone the fluoroquinolone resistance-determining region (FRDR) of gyrA, parC, and mexR. Point mutations were analyzed through Sanger sequencing, and the correlation between genetic mutations and phenotypic resistance of 141 clinical isolates was undertaken.ResultsFluoroquinolone-resistant substitution mutations such as Ile for Thr83 and Met for Thr133 in gyrA, Leu for Ser87 in parC, and Val for Glu126 in the repressor of mexR were mainly detected. Comparative analytical data indicated that amino acid alterations within the gyrA and parC genes are highly associated with resistance to ciprofloxacin (CIP) and levofloxacin (LEV) in the isolates, whereas alterations in the efflux regulatory mexR gene are not highly consistent with resistance in these isolates. Moreover, fluoroquinolone-resistant clinical isolates of P. aeruginosa were mainly isolated from pus and sputum specimens.ConclusionIn clinical isolates of P. aeruginosa, a high correlation was observed between MICs of CIP and LEV and alterations in gyrA and parC genes. However, mutations occurring in mexR did not highly correlate with the antibiotic resistance of the bacterium.

Prevalence of Fluoroquinolone-Resistant Clinical Isolates of Escherichia Coli in Urinary Tract Infection

Introduction: The commonest bacterial agent involved in causation of UTIs is Escherichia coli. The emergence of FQ resistant uropathogenic E. coli is of great concern. Aim of the work: to study resistance towards urinary E. coli with various generations of fluoroquinolones. Patients & Methods:: our study was carried out in the Clinical Pathology Department, Sohag University Hospital during the period from June 2016 to May 2017. Our study included 140 participants. Isolates from the specimens were obtained and identified using; Gram staining, colony characteristics on different culture medias. VITEC 2 Compact 15 identification kits were be used to confirm the identification of the isolates Results: E.coli was isolated from 100 patients (71%) of all patients complaining of UTI with positive urinary culture (study or case group). By studying prevalence of Antibiotic resistance of E.coli isolates reveals that fluoroquinolones show sensitivities of 42-46%. Also Nitrofurantoin has the highest sensitivity of 87%. This is followed by meropenem (67%). Ampicillin shows sensitivity in only 6% of cases. Regarding drug sensitivity in out & inpatients, we find that all generations of fluoroquinolones show highly significant resistance ratios among inpatients compared to outpatients. Meropenem show resistance more in inpatients than outpatients, with significant difference, Ampicillin and Nitrofuratoin show non-significant difference. Conclusion: our study show an increased fluoroquinolone resistance among uropathogenic E. coli isolates mainly in hospital admitted patients.

Role of topoisomerase mutations, plasmid mediated resistance (qnr) and acrAB efflux pump in fluoroquinolone resistant clinical isolates of avian Escherichia coli

We investigated the role of topoisomerase mutations, increased level of the multidrug efflux pump AcrAB, and the plasmid-borne genes (qnr) in the fluoroquinolone (FQ) resistant avian Escherichia coli simultaneously. Here, we used four FQs (ciprofloxacin, enrofloxacin, ofloxacin and pefloxacin) and eight clinical isolates of E. coli containing six fluoroquinolone-resistant and two fluoroquinolone- susceptible. PCR and direct sequencing methods were used to detect the role of regulator/ repressor gene (acrR). The objective of this study was to determine the relationship of these resistance mechanisms for fluoroquinolone resistance. The results showed that (i) all four fluoroquinolone- resistant isolates have topoisomerase mutation and plasmid borne genes qnrS and aac(6')-Ib; (ii) three FQ (enrofloxacin, ofloxacin and pefloxacin) resistant isolates harboring qnrS genes; (iii) two FQ (ciprofloxacin and pefloxacin) resistant isolates had topoisomerase mutation and plasmid borne gene qnrS; (iv) all fluoroquinolone susceptible were not harboring qnrS gene and topoisomerase mutation (v) All isolates were negative for qnrA and qnrB. We found that FQs resistance combination was correlated with synergistically contribution of these resistance mechanisms. Plasmid mediated resistance by qnrS was correlated to pefloxacin resistance but did not correlate to ofloxacin, enrofloxacin and ciprofloxacin. This mechanism might be account for the pefloxacin resistance in avian E. coli.

Fitness Cost of Fluoroquinolone Resistance in Clinical Isolates of Pseudomonas aeruginosa Differs by Type III Secretion Genotype.

Fluoroquinolone (FQ) resistance is highly prevalent among clinical strains of Pseudomonas aeruginosa, limiting treatment options. We have reported previously that highly virulent strains containing the exoU gene of the type III secretion system are more likely to be FQ-resistant than strains containing the exoS gene, as well as more likely to acquire resistance-conferring mutations in gyrA/B and parC/E. We hypothesize that FQ-resistance imposes a lower fitness cost on exoU compared to exoS strains, thus allowing for better adaptation to the FQ-rich clinical environment. We created isogenic mutants containing a common FQ-resistance conferring point mutation in parC from three exoU to three exoS clinical isolates and tested fitness in vitro using head-to-head competition assays. The mutation differentially affected fitness in the exoU and exoS strains tested. While the addition of the parC mutation dramatically increased fitness in one of the exoU strains leaving the other two unaffected, all three exoS strains displayed a general decrease in fitness. In addition, we found that exoU strains may be able to compensate for the fitness costs associated with the mutation through better regulation of supercoiling compared to the exoS strains. These results may provide a biological explanation for the observed predominance of the virulent exoU genotype in FQ-resistant clinical subpopulations and represent the first investigation into potential differences in fitness costs of FQ-resistance that are linked to the virulence genotype of P. aeruginosa. Understanding the fitness costs of antibiotic resistance and possibilities of compensation for these costs is essential for the rational development of strategies to combat the problem of antibiotic resistance.

Clinically relevant fluoroquinolone resistance due to constitutive overexpression of the PatAB ABC transporter in Streptococcus pneumoniae is conferred by disruption of a transcriptional attenuator.

ObjectivesConstitutive overexpression of patAB has been observed in several unrelated fluoroquinolone-resistant laboratory mutants and clinical isolates; therefore, we sought to identify the cause of this overexpression.MethodsConstitutive patAB overexpression in two clinical isolates and a laboratory-selected mutant was investigated using a whole-genome transformation approach. To determine the effect of the detected terminator mutations, the WT and mutated patA leader sequences were cloned upstream of a GFP reporter. Finally, mutation of the opposing base in the stem–loop structure was carried out.ResultsWe identified three novel mutations causing up-regulation of patAB. All three of these were located in the upstream region of patA and affected the same Rho-independent transcriptional terminator structure. Each mutation was predicted to destabilize the terminator stem–loop to a different degree, and there was a strong correlation between predicted terminator stability and patAB expression level. Using a GFP reporter of patA transcription, these terminator mutations led to increased transcription of a downstream gene. For one mutant sequence, terminator stability could be restored by mutation of the opposing base in the stem–loop structure, demonstrating that transcriptional suppression of patAB is mediated by the terminator stem–loop structure.ConclusionsThis study showed that a mutation in a Rho-independent transcriptional terminator structure confers overexpression of patAB and fluoroquinolone resistance. Understanding how levels of the PatAB efflux pump are regulated increases our knowledge of pneumococcal biology and how the pneumococcus can respond to various stresses, including antimicrobials.

Molecular analysis of low-level fluoroquinolone resistance in clinical isolates of Moraxella catarrhalis.

We investigated antimicrobial susceptibility and the molecular mechanism underlying low-level resistance to fluoroquinolones in 70 non-duplicate clinical isolates of Moraxella catarrhalis. The isolates were collected in a general hospital in Tokyo, Japan, between January and October 2013 from 38 men and 32 women; most of the isolates (48 out of 70, 68.5%) were obtained from post-nasal drips of children. The antimicrobial susceptibility of M. catarrhalis isolates was determined with an Etest, and low-level fluoroquinolone-resistant isolates were subtyped by PFGE. Mutations in the gyrA and parC genes were determined by PCR and sequencing. PCR products of the gyrA and parC genes from the low-level fluoroquinolone-resistant isolates were transformed into a fluoroquinolone-susceptible strain. Among the 70 isolates, five (7.1%) exhibited elevated fluoroquinolone MICs (levofloxacin, 1.0 mg l(-1); ciprofloxacin, 0.5 mg l(-1)) and different PFGE patterns. The patients from whom these five isolates were isolated had not undergone treatment with fluoroquinolones for the past 6 months. Each of the five low-level fluoroquinolone-resistant isolates had a gyrA gene mutation resulting in a Thr-to-Ile substitution at aa 80 (T80I) in the GyrA protein, while no changes were detected in the parC gene. A transformant carrying the gyrA gene containing the T80I substitution, which corresponded to Ser83 in Escherichia coli, displayed an elevated fluoroquinolone MIC and contained the T80I alteration in GyrA. Thus, our findings reveal that the low-level resistance to fluoroquinolones in M. catarrhalis is due to an amino acid substitution of Thr80 to Ile in GyrA. This is the first evidence of low-level fluoroquinolone resistance in M. catarrhalis.

A novel gyrB gene mutation in fluoroquinolone resistant clinical isolates of Mycobacterium tuberculosis

Fluoroquinolone (FQ) resistance in Mycobacterium tuberculosis can be conferred by mutations in gyrA or gyrB gene. Recent studies suggest that amino acid substitutions in gyrB gene may also play a crucial role in resistance, but genetic studies of these mutations in M. tuberculosis are lacking.

Molecular mechanism of fluoroquinolones resistance in Mycoplasma hominis clinical isolates.

To evaluate the molecular mechanism of fluoroquinolones resistance in Mycoplasma hominis (MH) clinical strains isolated from urogenital specimens. 15 MH clinical isolates with different phenotypes of resistance to fluoroquinolones antibiotics were screened for mutations in the quinolone resistance-determining regions (QRDRs) of DNA gyrase (gyrA and gyrB) and topoisomerase IV (parC and parE) in comparison with the reference strain PG21, which is susceptible to fluoroquinolones antibiotics. 15 MH isolates with three kinds of quinolone resistance phenotypes were obtained. Thirteen out of these quinolone-resistant isolates were found to carry nucleotide substitutions in either gyrA or parC. There were no alterations in gyrB and no mutations were found in the isolates with a phenotype of resistance to Ofloxacin (OFX), intermediate resistant to Levofloxacin (LVX) and Sparfloxacin (SFX), and those susceptible to all three tested antibiotics. The molecular mechanism of fluoroquinolone resistance in clinical isolates of MH was reported in this study. The single amino acid mutation in ParC of MH may relate to the resistance to OFX and LVX and the high-level resistance to fluoroquinolones for MH is likely associated with mutations in both DNA gyrase and the ParC subunit of topoisomerase IV.

Association of norB overexpression and fluoroquinolone resistance in clinical isolates of Staphylococcus aureus from Korea

Although the prevalence of fluoroquinolone resistance among methicillin-resistant Staphylococcus aureus (MRSA) is known to be higher than in methicillin-susceptible S. aureus (MSSA), the reasons have never been identified. We randomly selected 115 isolates of S. aureus collected from 10 different hospitals in Korea between June 2009 and May 2011. To investigate the difference in fluoroquinolone resistance mechanisms between MRSA and MSSA, we evaluated gyrA and parC mutations and the relative expression of the multidrug efflux pump genes norA, norB and norC. All 62 ciprofloxacin-resistant S. aureus had either gyrA or parC mutations. The S84L mutation of gyrA (59/62, 95.2%) and the S80F mutation of parC (61/62, 98.4%) were the most common. Fifty-eight (93.6%) strains had both the S84L mutation of gyrA and the S80F mutation of parC. Among the 115 isolates, norB overexpression was the most common, occurring in 49 (42.6%) strains. There were only two (1.7%) strains with norA overexpression and none with norC overexpression. Strains overexpressing norB were more common among ciprofloxacin-resistant S. aureus (33/62, 53.2%) than ciprofloxacin-susceptible S. aureus (16/53, 30.2%) (P = 0.013). When we analysed 62 ciprofloxacin-resistant S. aureus strains, those overexpressing norB were more common in ciprofloxacin-resistant MRSA (28/46, 60.9%) than in ciprofloxacin-resistant MSSA (5/16, 31.3%) (P = 0.041). Increased expression of norB can be a factor that contributes to ciprofloxacin resistance in MRSA strains.

Abrupt Emergence of a Single Dominant Multidrug-Resistant Strain of Escherichia coli

Fluoroquinolone-resistant Escherichia coli are increasingly prevalent. Their clonal origins--potentially critical for control efforts--remain undefined. Antimicrobial resistance profiles and fine clonal structure were determined for 236 diverse-source historical (1967-2009) E. coli isolates representing sequence type ST131 and 853 recent (2010-2011) consecutive E. coli isolates from 5 clinical laboratories in Seattle, Washington, and Minneapolis, Minnesota. Clonal structure was resolved based on fimH sequence (fimbrial adhesin gene: H subclone assignments), multilocus sequence typing, gyrA and parC sequence (fluoroquinolone resistance-determining loci), and pulsed-field gel electrophoresis. Of the recent fluoroquinolone-resistant clinical isolates, 52% represented a single ST131 subclonal lineage, H30, which expanded abruptly after 2000. This subclone had a unique and conserved gyrA/parC allele combination, supporting its tight clonality. Unlike other ST131 subclones, H30 was significantly associated with fluoroquinolone resistance and was the most prevalent subclone among current E. coli clinical isolates, overall (10.4%) and within every resistance category (11%-52%). Most current fluoroquinolone-resistant E. coli clinical isolates, and the largest share of multidrug-resistant isolates, represent a highly clonal subgroup that likely originated from a single rapidly expanded and disseminated ST131 strain. Focused attention to this strain will be required to control the fluoroquinolone and multidrug-resistant E. coli epidemic.