High-level Fluoroquinolone Resistance Research Articles

Identification of Determinants that Allow Maintenance of High-Level Fluoroquinolone Resistance in Acinetobacter baumannii.

Acinetobacter baumannii is associated with multidrug resistant (MDR) infections in healthcare settings, with fluoroquinolones such as ciprofloxacin being currently ineffective. Clinical isolates largely harbor mutations in the GyrA and TopoIV fluoroquinolone targets, as well as mutations that increase expression of drug resistance-nodulation-division (RND) efflux pumps. Factors critical for maintaining fitness levels of pump overproducers are uncharacterized despite their prevalence in clinical isolates. We here identify proteins that contribute to the fitness of FQR strains overexpressing three known RND systems using high-density insertion mutagenesis. Overexpression of the AdeFGH efflux pump caused hypersensitization to defects in outer membrane homeostatic regulation, including lesions that reduced LOS biosynthesis and blocked production of the major A. baumannii porin. In contrast, AdeAB pump hyperexpression, in the absence of elevated adeC expression (the outer membrane component of the pump), was relatively tolerant to loss of these functions, consistent with the outer membrane protein being the primary disruptive component. Surprisingly, overexpression of proton-transporting efflux pumps had little impact on cytosolic pH, consistent with a compensatory response to pump activity. The most striking transcriptional changes were associated with AdeFGH pump overexpression, including the activation of the phenylacetate (PAA) degradation regulon. Disruption of the PAA pathway resulted in cytosolic acidification and defective expression of genes involved in protection from oxidative stress. These results indicate that RND efflux pump overproduction is compensated by maintenance of outer membrane integrity in A. baumannii to facilitate fitness of FQR isolates.

Interplay between Amino Acid Substitution in GyrA and QnrB19: Elevating Fluoroquinolone Resistance in Salmonella Typhimurium.

Globally, there have been increasing reports of antimicrobial resistance in nontyphoidal Salmonella (NTS), which can develop into severe and potentially life-threatening diarrhea. This study focuses on the synergistic effects of DNA gyrase mutations and plasmid-mediated quinolone resistance (PMQR) genes, specifically qnrB19, on fluoroquinolone (FQ) resistance in Salmonella Typhimurium. By utilizing recombinant mutants, GyrAS83F and GyrAD87N, and QnrB19's, we discovered a significant increase in fluoroquinolones resistance when QnrB19 is present. Specifically, ciprofloxacin and moxifloxacin's inhibitory concentrations rose 10- and 8-fold, respectively. QnrB19 was found to enhance the resistance capacity of mutant DNA gyrases, leading to high-level FQ resistance. Additionally, we observed that the ratio of QnrB19 to DNA gyrase played a critical role in determining whether QnrB19 could protect DNA gyrase against FQ inhibition. Our findings underscore the critical need to understand these resistance mechanisms, as their coexistence enables bacteria to withstand therapeutic FQ levels, posing a significant challenge to treatment efficacy.

Impact of Whole-Genome Sequencing of Mycobacterium tuberculosis on Treatment Outcomes for MDR-TB/XDR-TB: A Systematic Review.

Five electronic databases were searched for studies published in English from the year 2000 onward. Two reviewers independently conducted the article screening, relevant data extraction, and quality assessment. The data of the included studies were synthesized with a narrative method and are presented in a tabular format. The database search identified 949 published articles and 8 studies were included. An unfavorable treatment outcome was reported for 26.6% (488/1834) of TB cases, which ranged from 9.7 to 51.3%. Death was reported in 10.5% (194/1834) of total cases. High-level fluoroquinolone resistance (due to gyrA 94AAC and 94GGC mutations) was correlated as the cause of unfavorable treatment outcomes and reported in three studies. Other drug resistance mutations, like kanamycin high-level resistance mutations (rrs 1401G), rpoB Ile491Phe, and ethA mutations, conferring prothionamide resistance were also reported. The secondary findings from this systematic review involved laboratory aspects of WGS, including correlations with phenotypic DST, cost, and turnaround time, or the impact of WGS results on public health actions, such as determining transmission events within outbreaks. WGS has a significant capacity to provide accurate and comprehensive drug resistance data for MDR/XDR-TB, which can inform personalized drug therapy to optimize treatment outcomes.

Phylogeny, Virulence, and Antimicrobial Resistance Gene Profiles of Enterococcus faecium Isolated from Australian Feedlot Cattle and Their Significance to Public and Environmental Health.

The extent of similarity between E. faecium strains found in healthy feedlot beef cattle and those causing extraintestinal infections in humans is not yet fully understood. This study used whole-genome sequencing to analyse the antimicrobial resistance profile of E. faecium isolated from beef cattle (n = 59) at a single feedlot and compared them to previously reported Australian isolates obtained from pig (n = 60) and meat chicken caecal samples (n = 8), as well as human sepsis cases (n = 302). The E. faecium isolated from beef cattle and other food animal sources neither carried vanA/vanB responsible for vancomycin nor possessed gyrA/parC and liaR/liaS gene mutations associated with high-level fluoroquinolone and daptomycin resistance, respectively. A small proportion (7.6%) of human isolates clustered with beef cattle and pig isolates, including a few isolates belonging to the same sequence types ST22 (one beef cattle, one pig, and two human isolates), ST32 (eight beef cattle and one human isolate), and ST327 (two beef cattle and one human isolate), suggesting common origins. This provides further evidence that these clonal lineages may have broader host range but are unrelated to the typical hospital-adapted human strains belonging to clonal complex 17, significant proportions of which contain vanA/vanB and liaR/liaS. Additionally, none of the human isolates belonging to these STs contained resistance genes to WHO critically important antimicrobials. The results confirm that most E. faecium isolated from beef cattle in this study do not pose a significant risk for resistance to critically important antimicrobials and are not associated with current human septic infections.

Genetic characterization of ESBL-producing and ciprofloxacin-resistant Escherichia coli from Belgian broilers and pigs.

The increasing number of infections caused by Escherichia coli resistant to clinically important antibiotics is a global concern for human and animal health. High overall levels of extended-spectrum beta-lactamase (ESBL)-producing and ciprofloxacin-resistant (ciproR) Escherichia coli in livestock are reported in Belgium. This cross-sectional study aimed to genotypically characterize and trace ESBL-and ciproR-E. coli of Belgian food-producing animals. A total of 798 fecal samples were collected in a stratified-random sampling design from Belgian broilers and sows. Consequently, 77 ESBL-E. coli and 84 ciproR-E. coli were sequenced using Illumina MiSeq. Minimum inhibitory concentration (MIC) for fluoroquinolones and cephalosporins were determined. Molecular in silico typing, resistance and virulence gene determination, and plasmid identification was performed. Scaffolds harboring ESBL or plasmid-mediated quinolone resistance (PMQR) genes were analyzed to detect mobile genetic elements (MGEs) and plasmid origins. Core genome allelic distances were used to determine genetic relationships among isolates. A variety of E. coli sequence types (ST) (n = 63), resistance genes and virulence profiles was detected. ST10 was the most frequently encountered ST (8.1%, n = 13). The pandemic multidrug-resistant clone ST131 was not detected. Most farms harbored more than one ESBL type, with bla CTX-M-1 (41.6% of ESBL-E. coli) being the most prevalent and bla CTX M-15 (n = 3) being the least prevalent. PMQR genes (15.5%, n = 13) played a limited role in the occurrence of ciproR-E. coli. More importantly, sequential acquisition of mutations in quinolone resistance-determining regions (QRDR) of gyrA and parC led to increasing MICs for fluoroquinolones. GyrA S83L, D87N and ParC S80I mutations were strongly associated with high-level fluoroquinolone resistance. Genetically related isolates identified within the farms or among different farms highlight transmission of resistant E. coli or the presence of a common reservoir. IncI1-I(alpha) replicon type plasmids carried different ESBL genes (bla CTX-M-1, bla CTX-M-32 and bla TEM-52C). In addition, the detection of plasmid replicons with associated insertion sequence (IS) elements and ESBL/PMQR genes in different farms and among several STs (e.g., IncI1-I(alpha)/IncX3) underline that plasmid transmission could be another important contributor to transmission of resistance in these farms. Our findings reveal a multifaceted narrative of transmission pathways. These findings could be relevant in understanding and battling the problem of antibiotic resistance in farms.

The international and intercontinental spread and expansion of antimicrobial-resistant Salmonella Typhi: a genomic epidemiology study

SummaryBackgroundThe emergence of increasingly antimicrobial-resistant Salmonella enterica serovar Typhi (S Typhi) threatens to undermine effective treatment and control. Understanding where antimicrobial resistance in S Typhi is emerging and spreading is crucial towards formulating effective control strategies.MethodsIn this genomic epidemiology study, we sequenced the genomes of 3489 S Typhi strains isolated from prospective enteric fever surveillance studies in Nepal, Bangladesh, Pakistan, and India (between 2014 and 2019), and combined these with a global collection of 4169 S Typhi genome sequences isolated between 1905 and 2018 to investigate the temporal and geographical patterns of emergence and spread of antimicrobial-resistant S Typhi. We performed non-parametric phylodynamic analyses to characterise changes in the effective population size of fluoroquinolone-resistant, extensively drug-resistant (XDR), and azithromycin-resistant S Typhi over time. We inferred timed phylogenies for the major S Typhi sublineages and used ancestral state reconstruction methods to estimate the frequency and timing of international and intercontinental transfers.FindingsOur analysis revealed a declining trend of multidrug resistant typhoid in south Asia, except for Pakistan, where XDR S Typhi emerged in 2016 and rapidly replaced less-resistant strains. Mutations in the quinolone-resistance determining region (QRDR) of S Typhi have independently arisen and propagated on at least 94 occasions, nearly all occurring in south Asia. Strains with multiple QRDR mutations, including triple mutants with high-level fluoroquinolone resistance, have been increasing in frequency and displacing strains with fewer mutations. Strains containing acrB mutations, conferring azithromycin resistance, emerged in Bangladesh around 2013 and effective population size of these strains has been steadily increasing. We found evidence of frequent international (n=138) and intercontinental transfers (n=59) of antimicrobial-resistant S Typhi, followed by local expansion and replacement of drug-susceptible clades.InterpretationIndependent acquisition of plasmids and homoplastic mutations conferring antimicrobial resistance have occurred repeatedly in multiple lineages of S Typhi, predominantly arising in south Asia before spreading to other regions.FundingBill & Melinda Gates Foundation.

English

BACKGROUND Urinary tract infection (UTI) is a very important reason for increased hospital stay, increased morbidity and mortality in the hospital setting. Annually, about 150 million people are diagnosed with UTI worldwide.1The fluoroquinolones have assumed an important role in the therapy of UTI. But the prevalence of urinary tract infections caused by fluoroquinolone-resistant bacteria has been increasing over the years. The purpose of this study was to evaluate fluoroquinolone resistance pattern among the bacterial pathogens causing urinary tract infections. METHODS This is a cross-sectional descriptive study. A total of 200 urine samples were collected from clinically suspected UTI patients, attending out-patient department (OPDs) and were admitted to various wards of our hospital. The samples were inoculated using the standard loop, incubated aerobically, isolates were identified by standard biochemical reactions and antibiotic susceptibility testing was performed. RESULTS Out of 200 samples, the most common organism isolated was Escherichia coli (57 %), followed by Klebsiella pneumoniae subsp pneumonia (27 %). 93.9 % of Escherichia coli and 100 % of Klebsiella pneumoniae subsp pneumoniae isolates were resistant to ampicillin. The present study also documented high level of fluoroquinolone resistance among almost all common uropathogens. Among Escherichia coli and Klebsiella pneumoniae subsp pneumoniae; ciprofloxacin resistance was 77.2 % and 85.2 % respectively. Quinolone resistance was found to be more common in hospitalised patients and catheterized patients. According to our study, although levofloxacin is far effective than ciprofloxacin for uropathogens, the resistance to both are increasing. CONCLUSIONS The results presented in this study indicate that it is time to reconsider the frequent use of fluoroquinolones. Use of fluoroquinolones to treat patients before getting the sensitivity pattern of the isolate should be stopped. It is mandatory to formulate antimicrobial policy according to the local resistance pattern. Resistance pattern must be assessed in hospital and laboratory-based surveillance studies. KEY WORDS Urinary Tract Infections, Escherichia coli, Antibiotic Sensitivity, Fluoroquinolones

High prevalence and variable fitness of fluoroquinolone-resistant avian pathogenic Escherichia coli isolated from chickens in Korea

ABSTRACT Colibacillosis caused by avian pathogenic Escherichia coli (APEC) is the most common bacterial disease in poultry, resulting in significant economic losses. Resistance to fluoroquinolones has been found to be high in APEC worldwide, which has increased concerns about risks to human health as well as poultry production. In the present study, we determined the prevalence, genetic traits, and fitness traits of fluoroquinolone-resistant APEC isolated from chickens in Korea using a total of 286 APEC isolates collected between 2014 and 2017. The APEC isolates were highly resistant to nalidixic acid (86.0%), ampicillin (71.7%), tetracycline (69.6%), and sulfisoxazole (61.2%), and 132 (46.2%) of the isolates were resistant to both enrofloxacin and ciprofloxacin. These fluoroquinolone-resistant isolates showed eight mutation combinations including single- or double-point mutations in the gyrA, parC, or parE genes. The isolates with double mutations (codons 83 and 87) in gyrA and additional mutations in parC and parE showed high-level fluoroquinolone resistance (minimum inhibitory concentrations, 16–128 µg/ml). The isolates fell into four phylogenetic groups, and groups A (47/132, 35.6%) and B1 (47/132, 36.4%) were the most predominant. Nine isolates (6.8%) belonged to group B2 and included major lineages of extraintestinal pathogenic E. coli, sequence type (ST) 95 (n = 3) and ST69 (n = 2). The isolates varied in their virulence-associated gene content, biofilm formation, and intramacrophage survival. Overall, fluoroquinolone-resistant APEC in poultry poses a potential risk to public health and represents a highly diverse group of the resistant bacteria that varied in their genetic and fitness traits.

National Prevalence of Salmonella enterica Serotype Kentucky ST198 with High-Level Resistance to Ciprofloxacin and Extended-Spectrum Cephalosporins in China, 2013 to 2017.

Salmonella enterica serotype Kentucky is frequently associated with high-level fluoroquinolone resistance and has gained epidemiological importance globally. A retrospective screening was performed to understand the national prevalence of ciprofloxacin-resistant S Kentucky in China. S. enterica strains (n = 15,405) were collected within the frame of two national surveillance networks between 2013 and 2017. Thirty-three S. Kentucky strains were detected in 5 of 10 provinces, and 27 were assigned to sequence type 198 (ST198). The 27 isolates were multidrug resistant, with high-level resistance to ciprofloxacin, and 21 isolates were further resistant to extended-spectrum cephalosporins (ESCs). Phylogenomic analysis classified ST198 isolates into two clades (198.1 and 198.2), and recent occurrences of inter-/intraregion and interhost transmission were identified. Phylogenetic reconstruction with a global collection showed that one subclade of clade 198.2 was clustered with historical strains from Egypt, and the other one was clustered with strains from Southeast Asia. Isolates of clade 198.1 were clustered with strains isolated from North America. The various patterns of mutations detected in quinolone resistance-determining regions of GyrA and ParC are accordant with the phylogenetic structure. These findings indicate that our isolates may have various origins. SGI1 was exclusively detected in isolates of clade 198.2 with a highly mosaic structure, which were mainly identified as SGI1-K derivatives. Plasmid-mediated quinolone resistance genes qnrS1 and aac(6')-Ib-cr were identified in three isolates, and bla CTX-M-9 and bla CTX-M-27 were detected in 20 of 21 ESC-resistant isolates. This is the first report of the genetic and epidemiological characterization for the S Kentucky epidemic clone ST198 in China, warranting the necessity of surveillance for the high-risk clone.IMPORTANCE Ciprofloxacin and extended-spectrum cephalosporins are the choice for treatment of severe nontyphoidal S. enterica infections in adults. S. enterica serotype Kentucky ST198 has gained epidemiological importance globally, because the clone is frequently resistant to both of these high-level-resistance drug groups. The genetic and epidemiological characterization of S. Kentucky has been well studied in Western countries; however, the information is unclear for China. To fill in the gap, we here did a retrospective screening on a large collection in China, and ST198 isolates were systematically analyzed by whole-genome sequencing. Our study revealed that multidrug-resistant ST198 has spread in five provinces, and the occurrences of interregion and cross-host clonal disseminations were detected. Of note, phylogenomic analysis suggests that the Chinese isolates may have emerged with diverse origins, including Egypt, Southeast Asia, and North America. This study warrants the necessity of surveillance for the high-risk clone to prevent its further dissemination in China.

Molecular mechanisms of fluoroquinolone resistance in Enterobacteriaceae clinical isolates in Azerbaijan, Iran

Fluoroquinolones (FQs) resistance due to their extensive clinical use has been a major problem in clinical settings that lead to limitation of treatment. This study aimed to investigate the resistance genes to FQs in Enterobacteriaceae isolates in Azerbaijan, Iran. A total of 88 Enterobacteriaceae isolated from clinical samples were collected. The presence of mutations in quinolone resistance genes were detected by the PCR and direct sequencing. The expression levels of acrB efflux pump and the ompF porin were evaluated using the Real-Time PCR. Overall, 63 isolates (71.6%) were resistant to FQs. All FQ-resistant Enterobacteriaceae had a mutation in gyrA. We found 100% and 76% mutation at Ser83Leu and Asp87Asn of gyrA, respectively. Mutations of gyrB were including Ser359Ala + Ser367Thr (1.5%) and Ser366Pro + Arg478Trp (1.5%). Also, mutations in Ser80Ile (68%) and Glu84Val (30%) were found in parC gene. The amino-acid substitutions of Ile529Leu, Ser458Ala, and Leu416Phe in parE were detected in 27%, 23.8% and 15.8% of FQ-resistant Enterobacteriaceae, respectively. Forty-eight (92%) of FQ-resistant Enterobacteriaceae were positive for plasmid-mediated quinolone resistance (PMQR) genes. The expression of acrB increased and expression level of ompF decreased by 58.7% and 64.2% FQ-resistant isolates. The high levels of resistance to FQs were observed among Enterobacteriaceae clinical isolates. We found high-level FQ resistance associated with DNA gyrase and topoisomerase IV mutations as a major mechanism, and along with PMQR genes, expression of the acrB efflux pump gene and outer membrane ompF gene in Azerbaijan, Iran.

Genomic trajectories to fluoroquinolone resistance in Francisella tularensis subsp. holarctica live vaccine strain

Objectives: Fluoroquinolone (FQ)-resistant mutants were previously selected from the live vaccine strain (LVS) of Francisella tularensis (F. tularensis) subsp. holarctica. This study further characterised all genetic changes that occurred in these mutants during the evolutionary trajectory toward high-level FQ resistance, and their potential impact on F. tularensis antibiotic resistance and intracellular fitness.Methods: The whole genomes of FQ-resistant mutants were determined and compared with those of their parental strain. All detected mutations were evaluated for their potential impact on FQ resistance and intracellular multiplication of F. tularensis.Results: As compared with the parental LVS genome, 28 mutations were found in the derived FQ-resistant mutants. These mutations involved all genes encoding type II topoisomerases (i.e. gyrA, gyrB, parC, and parE). Interestingly, some of them were not previously associated with FQ resistance, warranting further characterisation. Mutations associated with FQ resistance were also found in other genes, including three encoding proteins involved in transport processes. Most of the detected mutations did not alter multiplication of the corresponding mutants in J774 cells. In contrast, all mutations at locus FTL_0439 encoding FupA/B, a membrane protein involved in iron transport, were associated with FQ resistance and fitness loss.Conclusion: FQ resistance in F. tularensis is complex and may involve single or combined mutations in genes encoding type II topoisomerases, transport systems and FupA/B. In vivo studies are now required to assess the potential role of these mutations in FQ treatment failures.

Initial resistance to companion drugs should not be considered an exclusion criterion for the shorter multidrug-resistant tuberculosis treatment regimen

ObjectivesWe investigated whether companion drug resistance was associated with adverse outcomes of the shorter multidrug-resistant tuberculosis (MDR-TB) treatment regimen in Bangladesh after adjustment for fluoroquinolone resistance. MethodsMDR-TB/rifampicin-resistant tuberculosis patients registered for treatment with a standardized gatifloxacin-based shorter MDR-TB treatment regimen were selected for the study. Drug resistance was determined by the proportion method, gatifloxacin and isoniazid minimum inhibitory concentration testing for selected isolates, and whole-genome sequencing. ResultsLow-level fluoroquinolone resistance and high-level fluoroquinolone resistance were the most important predictors of adverse outcomes, with pyrazinamide resistance having a significant yet lower impact. In patients with fluoroquinolone-/second-line-injectable-susceptible tuberculosis, non-eligibility for the shorter MDR-TB treatment regimen (initial resistance to pyrazinamide, ethionamide, or ethambutol) was not associated with adverse outcome (adjusted odds ratio 1.01; 95% confidence interval 0.4–2.8). Kanamycin resistance was uncommon (1.3%). Increasing levels of resistance to isoniazid predicted treatment failure, also in a subgroup of patients with high-level fluoroquinolone-resistant tuberculosis. ConclusionsOur results suggest that resistance to companion drugs in the shorter MDR-TB treatment regimen, except kanamycin resistance, is of no clinical importance as long as fluoroquinolone susceptibility is preserved. Hence, contrary to current WHO guidelines, exclusions to the standard regimen are justified only in the case of fluoroquinolone resistance. and possibly kanamycin resistance.

Detection of international high-risk clones of food-borne pathogens Salmonella and Escherichia coli in the Russian Federation

The use of whole genome sequencing, standard analyze methods and international databases allows to detect international high-risk clones of food-borne pathogens, to assess their evolution and geographical distribution due to international trade of animal food products and farm animals. In Saint-Petersburg, Russian Federation, Salmonella Newport, Salmonella Kentucky and Escherichia coli O26:H11 of well-known international high-risk clones were first identified from patients with diarrhea. Two S. Kentucky strains isolated in 2015 and 2019 were identical to the international clone ST198 widely distributed in European countries: had multidrug resistance, high-level fluoroquinolone resistance (MIC of ciprofloxacin 32,0 mg/l) due to three single-nucleotide substitutions in the gyrA (Ser83Phe and Asp87Asn) and parC (Ser80Ile). In 2008, a multidrug resistant S. Newport strain resistant to extended-spectrum cephalosporins due to AmpC-cephalosporinase CMY-2 was isolated, belonging to the international clone of S. Newport MDR-AmpC/CMY-2 which caused sporadic cases and outbreaks in the USA and Europe in the 2000s. The plasmid containing the blaCMY-2 had a PstI-restriction profile, one of the most prevalent in the international clone. E. coli O26:H11 strains, isolated in Saint-Petersburg, produced the shiga-like toxin STX1 (stx1a), had additional virulence genes: ehxA (enterohemolysin), katP (catalase peroxidase), espP (serine protease), as well as cba (colicin B), gad (glutamate decarboxylase), cif (type III secreted effector), iss (increased serum survival), belonged to the phylogenetic group B1 and the international high-risk clone E. coli O26:H11 ST21 widely distributed in Europe and the USA. 25% of the strains had multidrug resistance and produced CTX-M extended spectrum beta-lactamases. In the Russian Federation E. coli O26 is consider as the pathogen of diarrheal diseases and registered as Enteropathogenic E. coli in routine bacteriological laboratories without detecting of H-antigen and shiga-like toxins.

Antimicrobial susceptibility profile of Gonococcal isolates obtained from men presenting with urethral discharge in Addis Ababa, Ethiopia: Implications for national syndromic treatment guideline.

Neisseria gonorrhoeae (gonococcus) is the etiologic agent for the sexually transmitted Infection gonorrhea, a disease with a significant global public health impact. The treatment regimen for gonorrhea has been changed frequently over the past few decades due to the organism's propensity for developing antibiotic resistance. This study investigated antimicrobial susceptibility patterns of quinolones, third-generation cephalosporin, and other relevant antimicrobials found in N. gonorrhoeae isolated from men presenting with urethral discharge at selected healthcare facilities in Addis Ababa, Ethiopia, with the aim of revising the national treatment regimen based on the information generated from this study. A total of 599 male patients presenting with urethral discharge were included in the current study. Urethral discharge specimens were cultured on Modified Thayer Martín media and suspected gonococcal colonies were confirmed using Oxidase and Superoxol tests followed by identification through a commercial kit (API-NHR). Antimicrobial susceptibility testing was performed by the Kirby-Bauer disc diffusion method using ciprofloxacin (5μg), ceftriaxone (30μg), cefixime (5μg), cefoxitin (30 μg), penicillin (10μg) and spectinomycin (100 μg) on enriched GC agar. Minimum Inhibitory Concentration (MIC) was also carried out using concentration gradient strips (E-tests) of the same antimicrobial agents. The prevalence of gonococcal isolates in the current study was 69%. Out of the 361 gonococcal isolates, close to 68% were fluoroquinolone non-susceptible, with 60% resistant and 7% having an intermediate status. However, all tested isolates were susceptible to ceftriaxone. In addition, all of the isolates have shown reduced non-susceptibility to spectinomycin and cefoxitin. The prevalence of gonococcal isolates in men presenting with urethral discharge at selected healthcare facilities in Addis Ababa, Ethiopia was found to be high. The high level of fluoroquinolone resistance observed in gonococcal isolates recovered in this study necessitates revision of the national syndromic treatment guideline.

Prediction of treatment outcomes for multidrug-resistant tuberculosis by whole-genome sequencing

Whole-genome sequencing (WGS) has been proposed to be a powerful tool to predict drug resistance for antitubercular drugs. However, the feasibility of WGS in predicting final treatment outcomes for multidrug-resistant tuberculosis (MDR-TB) patients remains unclear PATIENTS AND METHODS: In this prospective observational study conducted from January 2014 to September 2016, MDR-TB patients were enrolled consecutively. Genotypic drug sensitivity testing was performed via WGS using culture isolates. Patients were followed for two years to determine the treatment outcomes. Multivariate analysis was used to identify the association between information provided by WGS and the final treatment outcomes RESULTS: A total of 123 patients with MDR-TB were included in this study. The overall favorable treatment outcome rate was 60.2%. Multivariate analysis showed that independent risk factors associated with unfavorable treatment outcome including high-level moxifloxacin phenotypic resistance (OR, 4.362; 95%CI, 1.364-13.950; p=0.013), cycloserine phenotypic resistance (OR, 7.457; 95%CI, 1.644-33.819; p=0.009), mutations causing high-level fluoroquinolones resistance (OR, 3.947; 95%CI, 1.195-13.034; p=0.024), and ethA mutation (OR, 3.817; 95% CI, 1.154-12.823; p=0.028). WGS costs for each patient are ¥450 ($63), and the average turnaround time was one week CONCLUSIONS: In summary, WGS showed promising feasibility in predicting treatment outcomes for MDR-TB patients within a clinically relevant time frame.

Antimicrobial resistance and clinical significant resistance mechanisms of Salmonella isolated in 2014-2018 in St.Petersburg, Russia.

The article presents the results of antimicrobial resistance monitoring of Salmonella isolated from children and adults with diarrhea in St. Petersburg in 2014-2018. In 746 isolates of 42 serovars more than 90,0% belonged to three: S. enteritidis (79,6%), S. typhimurium (6,8%) and S. infantis (3,8%). The antimicrobial susceptibility testing (according the EUCAST) to 7 classes of antimicrobials revealed the resistance in 78,6% of Salmonella. Low-level quinolone resistance (MIC of ciprofloxacin 0,12-0,5 mg/l) was detected in 63,3% isolates (S. enteritidis -71,0%, S. typhimurium - 15,7%, S. infantis - 89,3%) and was due to five kinds of single nucleotide substitutions in gyrA: Asp87Tyr - 36,1% of studied isolates (only S. infantis); Ser83Phe - 22,2% (only S. enteritidis); Asp87Asn - 19,4% (S. enteritidis, S. typhimurium, S. hadar, S. newport); Ser83Tyr -11,1% (S. enteritidis and S. infantis) and Asp87Gly - 8,3% (only S. enteritidis). Only in one S. kentucky isolate with high-level fluoroquinolone resistance (MIC of ciprofloxacin > 8,0 mg/l) two substitutions (Ser83Phe and Asp87Asn) were detected. Two Salmonella isolates (S. typhimurium and S. corvallis) had plasmid-mediated quinolone resistance (qnrS). Extended-spectrum cephalosporin resistance was found in 6 Salmonella serovars (1,6%). The bla-genes were detected: of genetic group CTX-M1 - in 10 isolates (serovars S. typhimurium, S. enteritidis, S. abony, S. coeln and S. virchow), CTX-M2 - in 2 S. typhimurium isolates, CTX-M9 - in three S. enteritidis isolates. In one S. typhimurium CTX-M1 and CTX-M2 were detected. The gene of CMY-2 (molecular class C cephalosporinase) was revealed in two isolates (S. newport and S. enteritidis). Our study showed that Salmonella (the main bacterial pathogen of acute diarrhea in children and adults) isolated in Saint-Petersburg had antimicrobial resistance to drugs of choice for salmonellosis treatment.

Bacterial resistance to fluoroquinolones and contributing factors in Addis Ababa, Ethiopia: a mixed methods study

Fluoroquinolones are widely used globally and there is a growing trend of resistance to these agents. However, there is scanty information in Ethiopia and this study aimed to assess the level of bacterial resistance to fluoroquinolones and identify contributing factors in Addis Ababa, Ethiopia. Concurrent mixed methods study design was used. Records (January 2013 to December 2016) of the Microbiology Laboratory of International Clinical Laboratories were retrospectively reviewed. In addition, qualitative interviews were conducted with purposively selected 20 prescribers’ and dispensers’ to explore perceived contributing factors to resistance. Descriptive statistics was used to summarize the data and possible association was explored using multivariable logistic regression. Qualitative data was analyzed using thematic analysis. A total of 7,889 bacterial isolates were identified from 4,310 patients. The overall resistance of bacterial isolates to fluoroquinolones was 42.5% and the highest resistance was to nalidixic acid (63.3%). Enterococci and Escherichia coli developed high level of resistance to ciprofloxacin and norfloxacin; while Enterococci had higher resistant to nalidixic acid (77.8%). Resistance to fluoroquinolones was 5-times more likely in patients aged ≥ 60 years than those < 15 years old (AOR = 5.63, 95% CI: 4.71, 6.73). Resistance to fluoroquinolones increased from 40.4% in 2013 to 49.0% in 2015 but declined to 46.3% in 2016. Respondents of the qualitative interviews suggested that injudicious prescribing due to lack of institutional antibiogram and inadequate knowledge, nonprescription sales of antibiotics and/or patient hording and sharing practices contributed for the high level of fluoroquinolone resistance. The study showed a high level of bacterial resistance to fluoroquinolones. Patients’ age and year of testing were significantly associated with resistance. Moreover, inappropriate prescribing practice, illegal over-the-counter sales as well as their unreasonable use by patients were key drivers to the problem. This calls for the strict regulation of non-prescription sales of antibiotics, public awareness creation and development of local antibiogram to guide prescribing. Keywords: fluoroquinolones, bacterial resistance, qualitative interview, enterococci, Escherichia coli

Multiple Benefits of Plasmid-Mediated Quinolone Resistance Determinants in Klebsiella pneumoniae ST11 High-Risk Clone and Recently Emerging ST307 Clone.

International high-risk clones of Klebsiella pneumoniae are among the most common nosocomial pathogens. Increased diversity of plasmid-encoded antimicrobial resistance genes facilitates spread of these clones causing significant therapeutic difficulties. The purpose of our study was to investigate fluoroquinolone resistance in extended-spectrum beta-lactamase (ESBL)-producing strains, including four K. pneumoniae and a single K. oxytoca, isolated from blood cultures in Hungary. Whole-genome sequencing and molecular typing including multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) were performed in selected strains. Gene expression of plasmid-mediated quinolone resistance determinants (PMQR) was investigated by quantitative-PCR. MLST revealed that three K. pneumoniae strains belonged to ST11 and one to ST307 whereas K. oxytoca belonged to ST52. The isolates harbored different β-lactamase genes, however, all K. pneumoniae uniformly carried blaCTX-M-15. The K. pneumoniae isolates exhibited resistance to fluoroquinolones and carried various PMQR genes namely, two ST11 strains harbored qnrB4, the ST307 strain harbored qnrB1 and all K. pneumoniae harbored oqxAB efflux pump. Levofloxacin and moxifloxacin MIC values of K. pneumoniae ST11 and ST307 clones correlated with qnr and oqxAB expression levels. The qnrA1 carrying K. oxytoca ST52 exhibited reduced susceptibility to fluoroquinolones. The maintained expression of qnr genes in parallel with chromosomal mutations indicate an additional protective role of Qnr proteins that can support dissemination of high-risk clones. During development of high-level fluoroquinolone resistance, high-risk clones retain fitness thus, enabling them for dissemination in hospital environment. Based on our knowledge this is the first report of ST307 clone in Hungary, that is emerging as a potential high-risk clone worldwide. High-level fluoroquinolone resistance in parallel with upregulated PMQR gene expression are linked to high-risk K. pneumoniae clones.

Mismatch amplification mutation assay-polymerase chain reaction: A method of detecting fluoroquinolone resistance mechanism in bacterial pathogens.

The mismatch amplification assay is a modified version of polymerase chain reaction (PCR) that permits specific amplification of gene sequences with single base pair change. The basis of the technique relies on primer designing. The single nucleotide mismatch at the 3’ proximity of the reverse oligonucleotide primer makes Taq DNA polymerase unable to carry out extension process. Thus, the primers produce a PCR fragment in the wild type, whereas it is not possible to yield a product with a mutation at the site covered by the mismatch positions on the mismatch amplification mutation assay (MAMA) primer from any gene. The technique offers several advantages over other molecular methods, such as PCR-restriction fragment length polymorphism (RFLP) and oligonucleotide hybridization, which is routinely used in the detection of known point mutations. Since multiple point mutations in the quinolone resistance determining region play a major role in high-level fluoroquinolone resistance in Gram-negative bacteria, the MAMA-PCR technique is preferred for detecting these mutations over PCR-RFLP and sequencing technology.

Frequency of DNA gyrase and topoisomerase IV mutations and plasmid-mediated quinolone resistance genes among Escherichia coli and Klebsiella pneumoniae isolated from urinary tract infections in Azerbaijan, Iran.

This study assessed genetic alterations in gyrA, gyrB, parC and parE and the prevalence of plasmid-mediated quinolone resistance (PMQR) genes among Escherichia coli and Klebsiella pneumoniae isolates from urinary tract infections (UTIs) in Azerbaijan, Iran. A total of 205 clinical isolates of E. coli (n=177) and K. pneumoniae (n=28) were obtained from UTIs. Antimicrobial susceptibility was determined by disk diffusion and agar dilution assays. The presence of PMQR genes was determined by PCR, and sequencing of the gyrA, gyrB, parC and parE was performed. The rate of fluoroquinolone (FQ) resistance among the isolates was 77.1%. The Ser83Leu mutation in gyrA was observed in all 60 FQ-resistant isolates selected for direct sequencing. The second most common mutation in gyrA was Asp87Asn. Frequent mutations in parC were Ser80Ile and Glu84Val. Ser359Ala+Ser367Thr and Gly385Cys mutations in gyrB were identified in one isolate each of K. pneumoniae and E. coli, respectively. The parE gene had mutations at Ile529Leu, Ser458Ala and Leu416Phe. Overall, PMQR determinants were identified in 90% of E. coli and 100% of K. pneumoniae. The prevalence of PMQR genes was as follows: aac(6')-Ib-cr, 71.7%; oqxB, 51.7%; oqxA, 36.7%; qnrB, 28.3%; qnrS, 21.7%; qnrD, 16.7%; qepA, 5.0%; qnrA, 1.7%; and qnrC, 1.7%. FQ resistance rates were high. Mutations in DNA gyrase and topoisomerase IV and the prevalence of PMQR genes in E. coli and K. pneumoniae isolates were alarming. Moreover, the combination of these resistance mechanisms plays an important role in high-level FQ resistance.