Plasmid-mediated Fluoroquinolone Resistance Research Articles

Distribution and genetic characterization of fluoroquinolone resistance gene qnr among Salmonella strains from chicken in China.

The prevalence and dissemination of the plasmid-mediated fluoroquinolone (FQ) resistance gene qnr in Salmonella are considered serious public health concerns worldwide. So far, no comprehensive large-scale studies have focused on the prevalence and genetic characteristics of the qnr gene in Salmonella isolated from chickens. Herein, this study aimed to investigate the prevalence, antimicrobial resistance (AMR) patterns, and molecular characteristics of chicken-originated qnr-positive Salmonella strains from chicken farms, slaughterhouses, and markets in 12 provinces of China in 2020-2021. The overall prevalence of the qnr gene was 21.13% (56/265), with the highest prevalence in markets (36.11%, 26/72), followed in farms (17.95%, 21/117), and slaughterhouses (10.53%, 9/76). Only the qnrS and qnrB genes were detected, and the prevalence rate of the qnrS gene (19.25%, 51/265) was higher than that of the qnrB gene (1.89%, 5/265). Whole genome sequencing identified 37 distinct AMR genes and 15 plasmid replicons, and the most frequent mutation in quinolone resistance determining regions was parC (T57S; 91.49%, 43/47). Meanwhile, four different qnrS and two qnrB genetic environments were discovered among 47 qnr-positive Salmonella strains. In total, 21.28% (10/47) of the strains were capable of conjugative transfer, and all were qnrS1-positive strains, with the majority of transferable plasmids being IncHI2 types (n = 4). Overall, the prevalence of qnr-positive Salmonella strains from chickens in China and their carriage of multiple resistance and virulence genes and transferable plasmids is a major concern, which calls for continuous surveillance of qnr-positive Salmonella and the development of measures to control its prevalence and transmission.IMPORTANCESalmonella is a common foodborne pathogen responsible for 155,000 deaths annually worldwide. Fluoroquinolones (FQs) are used as first-line drugs for the treatment of Salmonella infections in several countries and regions. However, the emergence and increasing prevalence of the FQ-resistant gene qnr in Salmonella isolated from chickens have been widely reported. Gaining insight into the genetic mechanisms of AMR genes in chicken could lead to the development of preventive measures to control and reduce the risk of drug resistance. In this study, we identified qnr-positive Salmonellae isolated from chickens in different regions of China and their AMR patterns and genome-wide characteristics, providing a theoretical basis for further control of their prevalence and transmission.

The role of the plasmid-mediated fluoroquinolone resistance genes as resistance mechanisms in pediatric infections due to Enterobacterales.

Fluoroquinolones (FQs) are not commonly prescribed in children, yet the increasing incidence of multidrug-resistant (MDR) Enterobacterales (Ent) infections in this population often reveals FQ resistance. We sought to define the role of FQ resistance in the epidemiology of MDR Ent in children, with an overall goal to devise treatment and prevention strategies. A case-control study of children (0-18 years) at three Chicago hospitals was performed. Cases had infections by FQ-susceptible, β-lactamase-producing (bla) Ent harboring a non- or low-level expression of PMFQR genes (PMFQS Ent). Controls had FQR infections due to bla Ent with expressed PMFQR genes (PMFQR Ent). We sought bla genes by PCR or DNA (BD Max Check-Points assay®) and PMFQR genes by PCR. We performed rep-PCR, MLST, and E. coli phylogenetic grouping. Whole genome sequencing was additionally performed on PMFQS Ent positive isolates. Demographics, comorbidities, and device, antibiotic, and healthcare exposures were evaluated. Predictors of infection were assessed. Of 170 β-lactamase-producing Ent isolates, 85 (50%) were FQS; 23 (27%) had PMFQR genes (PMFQS cases). Eighty-five (50%) were FQR; 53 (62%) had PMFQR genes (PMFQR controls). The median age for children with PMFQS Ent and PMFQR Ent was 4.3 and 6.2 years, respectively (p = NS). Of 23 PMFQS Ent, 56% were Klebsiella spp., and of 53 PMFQR Ent, 76% were E. coli. The most common bla and PMFQR genes detected in PMFQS Ent were bla SHV ESBL (44%) and oqxAB (57%), and the corresponding genes detected in PMFQR Ent were bla CTX-M-1-group ESBL (79%) and aac(6')-Ib-cr (83%). Whole genome sequencing of PMFQS Ent revealed the additional presence of mcr-9, a transferable polymyxin resistance gene, in 47% of isolates, along with multiple plasmids and mobile genetic elements propagating drug resistance. Multivariable regression analysis showed that children with PMFQS Ent infections were more likely to have hospital onset infection (OR 5.7, 95% CI 1.6-22) and isolates containing multiple bla genes (OR 3.8, 95% CI 1.1-14.5). The presence of invasive devices mediated the effects of healthcare setting in the final model. Differences in demographics, comorbidities, or antibiotic use were not found. Paradoxically, PMFQS Ent infections were often hospital onset and PMFQR Ent infections were community onset. PMFQS Ent commonly co-harbored multiple bla and PMFQR genes, and additional silent, yet transferrable antibiotic resistance genes such as mcr-9, affecting therapeutic options and suggesting the need to address infection prevention strategies to control spread. Control of PMFQS Ent infections will require validating community and healthcare-based sources and risk factors associated with acquisition.

Genetic and epidemiological analysis of ESBL-producing Klebsiella pneumoniae in three Japanese university hospitals

IntroductionWe aimed to clarify the genetic background and molecular epidemiology of extended-spectrum beta-lactamase (ESBL)-producing Klebsiella pneumoniae (K. pneumoniae) at three geographically separated university hospitals in Japan. MethodsFrom January 2014 to December 2016, 118 ESBL-producing K. pneumoniae (EPKP) strains that were detected and stored at three university hospitals were collected. Molecular epidemiological analysis was performed using enterobacterial repetitive intergenic consensus (ERIC)-polymerase chain reaction (PCR) and multi-locus sequence typing (MLST). The ESBL type was determined using the PCR-sequence method. The presence of plasmid-mediated fluoroquinolone resistance (PMQR) genes was analyzed by PCR. We compared the relationships between PMQR gene possession/quinolone resistance-determining region (QRDR) mutation and levofloxacin (LVFX)/ciprofloxacin (CPFX) susceptibility. ResultsThe detection rate of EPKP was 4.8% (144/2987 patients). MLST analysis revealed 62 distinct sequence types (STs). The distribution of STs was diverse, and only some EPKP strains had the same STs. ERIC-PCR showed discriminatory power similar to that of MLST. The major ESBL genotypes were CTX-M-15-, CTX-M-14-, and SHV-types, which were detected in 47, 30, and 27 strains, respectively. Ninety-one out of 118 strains had PMQR genes and 14 out of 65 strains which were not susceptible to CPFX had QRDR mutations, and the accumulation of PMQR genes and QRDR mutations tended to lead to higher minimum inhibitory concentrations (MICs) of LVFX. ConclusionsAt three geographically separated university hospitals in Japan, the epidemiology of EPKP was quite diverse, and no epidemic strains were found, whereas CTX-M-14 and CTX-M-15 were predominant.

Plasmid-Mediated Fluoroquinolone Resistance Genes in Quinolone-Susceptible Aeromonas spp. Phenotypes Isolated From Recreational Surface Freshwater Reservoir

Aeromonas spp. are recognized as opportunistic pathogens causing diseases. Infections in humans can result mainly in gastrointestinal and wound diseases with or without progression to septicemia. Although Aeromonas spp. are not known uropathogens and they rarely cause urinary tract infection, we hypothesize that the presence of these bacteria in the water and the contact during, e.g., recreational and bathing activity can create the conditions for the colonization of the human body and may result to diseases in various locations, including the urinary tract. Our study presents the occurrence of aeromonad fluoroquinolone-susceptible phenotypes with the presence of plasmid-mediated fluoroquinolone resistance (PMQR) genes in a natural freshwater reservoir occasionally used for recreational activities. Sixty-nine isolates collected during the bathing period were identified by mass spectrometry and screened for the presence of fluoroquinolone-resistant phenotypes and genotypes. Fluoroquinolone susceptibility was determined as minimal inhibitory concentration values. PMQR qnr genes were detected by PCR. Isolates comprising eight species, namely, mainly Aeromonas veronii (50.7% isolates) and Aeromonas media (24.6% isolates) and rarely Aeromonas eucrenophila, Aeromonas caviae, Aeromonas bestiarum, Aeromonas ichthiosmia, and Aeromonas hydrophila, were selected. All isolates were phenotypically susceptible either to ciprofloxacin or levofloxacin. Unexpectedly, at least one to three of the PMQR genes were detected in 42.0% of the fluoroquinolone-susceptible Aeromonas spp. phenotypes. Mainly the qnrS (34.8% isolates) and qnrA (14.5% isolates) determinants were detected. In conclusion, the freshwater reservoir occasionally used for bathing was tainted with aeromonads, with a high occurrence of opportunistic pathogens such as A. veronii and A. media. MALDI‐TOF MS is a powerful technique for aeromonad identification. Our data reveals the mismatch phenomenon between fluoroquinolone-susceptible aeromonad phenotypes and the presence of plasmid-mediated qnr resistance genes. It suggests that phenotypically susceptible bacteria might be a potential source for the storage and transmission of these genes. The exposure during, e.g., a recreational activity may create the potential risk for causing infections, both diagnostically and therapeutically difficult, after expressing the resistance genes and quinolone-resistant strain selection.

Prevalence and Profiles of Antibiotic Resistance Genes mph(A) and qnrB in Extended-Spectrum Beta-Lactamase (ESBL)-Producing Escherichia coli Isolated from Dairy Calf Feces.

The use of antibiotics to treat dairy calves may result in multidrug-resistant extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli. This study investigated fluoroquinolone and macrolide resistance genes among ESBL-producing E. coli isolated from dairy calves. Fresh fecal samples from 147 dairy calves across three age groups were enriched to select for ESBL-producing E. coli. Plasmid-mediated fluoroquinolone (qnrB), macrolide (mph(A)), and beta-lactam (blaCTX-M groups 1 and 9) resistance genes were identified by PCR and gel electrophoresis in ESBL-producing E. coli. Beta-lactamase variants and antibiotic resistance genes were characterized for eight isolates by whole-genome sequencing. Seventy-one (48.3%) samples were positive for ESBL-producing E. coli, with 159 (70.4%) isolates identified as blaCTX-M variant group 1 and 67 (29.6%) isolates as blaCTX-M variant group 9. Resistance gene mph(A) was more commonly associated with blaCTX-M variant group 1, while resistance gene qnrB was more commonly associated with variant group 9. E. coli growth was quantified on antibiotic media for 30 samples: 10 from each age group. Significantly higher quantities of ceftriaxone-resistant E. coli were present in the youngest calves. Results indicate the dominant blaCTX-M groups present in ESBL-producing E. coli may be associated with additional qnrB or mph(A) resistance genes and ESBL-producing E. coli is found in higher abundance in younger calves.

1145. The Role of the Plasmid-Mediated Fluoroquinolone-Resistance (PMFQR) Genes As Resistance Mechanisms in Pediatric Infections due to Enterobacterales (Ent)

BackgroundFluoroquinolones (FQs) are not commonly prescribed in children, yet the increasing incidence of multidrug resistant (MDR) Ent infections in this population often reveals FQ resistance. We sought to define the role of FQ resistance in the epidemiology of MDR Ent in children, with an overall goal to devise treatment and prevention strategies.MethodsA case-control study of children (0-18 years) at 3 Chicago hospitals was performed. Cases had infections by FQ susceptible, 3rd generation cephalosporin-resistant (3GCR) and/or carbapenem-resistant (CR) Ent harboring a non or low level expressed PMFQR gene (PMFQS Ent). Controls had FQR infections due to 3GCR and/or CR Ent with expressed PMFQR genes (PMFQR Ent). We sought bla genes by PCR or DNA (BD Max Check-Points assay®) and PMFQR genes by PCR. We performed Rep-PCR, MLST, and E. coli phylogenetic grouping. Demographics; comorbidities; and device, antibiotic, and healthcare exposures were evaluated. Predictors of infection were assessed.ResultsOf 170 G3CR and/or CR Ent isolates, 85 (50%) were FQS; 23 (27%) had PMFQR genes (PMFQS cases). 85 (50%) were FQR; 53 (62%) had PMFQR genes (PMFQR controls). The median age for children with PMFQS Ent and PMFQR Ent were 4.3 and 6.2 years, respectively (p=NS). Of 23 PMFQS Ent, 53% were Klebsiella and of 53 PMFQR Ent, 76% were E. coli. The most common bla and PMFQR genes in PMFQS Ent were blaSHV ESBL (44%); oqxB (57%) and aac-6’1b-cr (52%) and in PMFQR Ent were blaCTX-M-1 group (76%); aac-6’1b-cr (91%) and oqxA (17%).Multivariable regression analysis showed children with PMFQS Ent infections were more likely to have hospital onset infection (OR 5.7, 95% CI 1.6-22) and isolates with multiple bla genes (OR 3.8, 95% CI 1.1-14.5). The presence of invasive devices mediated the effects of healthcare setting in the final model. Differences in demographics, comorbidities, or antibiotic use were not found.ConclusionParadoxically, PMFQS Ent infections were often hospital onset and PMFQR Ent infections were community onset. PMFQS Ent commonly co-harbored multiple bla and PMFQR genes, affecting therapeutic options and suggesting need for contact precautions. Control of PMFQS Ent infections in children will require validating sources and risk factors.Disclosures Robert A. Bonomo, MD, entasis (Research Grant or Support)Merck (Grant/Research Support)NIH (Grant/Research Support)VA Merit Award (Grant/Research Support)VenatoRx (Grant/Research Support)

230. Nontyphoidal Salmonella from Clinical and Retail Meat Sources Reveal Antimicrobial Resistance Genes for Ceftriaxone and Ciprofloxacin

BackgroundPennsylvania participates in the National Antimicrobial Resistance Monitoring System (NARMS), which includes monitoring of Nontyphoidal Salmonella (NTS), a leading cause of bacterial foodborne illnesses in the United States.MethodsClinical NTS isolates submitted to the Pennsylvania Department of Health (2015-18) were tested for susceptibility to 15 antimicrobial agents and analyzed by whole-genome sequencing (WGS). Concurrently, we conducted a prospective microbiological survey of NTS in retail meat products (chicken breasts, ground turkey, and pork chops) with susceptibility testing and WGS. ResultsOf a sample of 426 clinical Salmonella isolates from humans analyzed for antimicrobial susceptibility, 65 (15.3%) had decreased susceptibility to ciprofloxacin (DSC). Ampicillin resistance was observed in 39 (9.2%) and 15 (3.5%) were ceftriaxone-resistant. Ten ceftriaxone-resistant isolates had genetic elements that confer resistance to third generation extended-spectrum cephalosporins (ESC) [blaCMY−2, n=8 and blaCTX-M-65, n=2]. The blaCTX-M-65- positive isolates had a mutation in gyrA that confers fluoroquinolone resistance. Thirteen clinical isolates carried plasmid-mediated fluoroquinolone resistance genes (PMQR) [qnrB19, qnrS1, qnrA1]. We detected NTS in 131 (3.8%) of 3480 meat samples tested. 7 (5.3%) had DSC, while 38 (29%) and 21 (16%) were resistant to ampicillin and ceftriaxone, respectively. Four S. Infantis isolates had DSC and a blaCTX-M-65 gene plus a mutation in gyrA. Thirteen meat isolates had the blaCMY-2 gene. One additional blaCTX-M-65-positive S. Infantis without gyrA from ground turkey (SRR6351119) differed from four clinical isolates by ≤10 single-nucleotide polymorphisms. Percent of isolates from patients and meat sources that demonstrated resistance to amoxicillin-clavulanate (AMC), ceftriaxone, and decreased susceptibility to ciprofloxacin (DSC) to nine antimicrobial classes tested. Among isolates from patients, resistance to ceftriaxone, a third-generation cephalosporin preferred for severe infections in children, increased from zero in 2015 to 5.8% in 2017. Overall, DSC increased in isolates from human sources while in strains from meat sources, DSC increased from zero in 2015 to over five percent in 2018.ConclusionNTS isolated from human and meat sources were multi-drug resistant. Demonstration of similar resistance genes in meat and in ill humans may be consistent with spread of antibiotic-resistant pathogens from food sources. Dissemination of genes that confer resistance to third generation cephalosporins and fluoroquinolones, including some on mobile plasmids, may undermine recommended treatment for severe NTS infections. These results underscore the need for antimicrobial stewardship efforts in both agriculture and human medicine.Disclosures All Authors: No reported disclosures

The prevalence and mechanism of fluoroquinolone resistance in Escherichia coli isolated from swine farms in China

BackgroundIt has been demonstrated that swine waste is an important reservoir for resistant genes. Moreover, the bacteria carrying resistant genes and originating from swine feces and wastewater could spread to the external environment. Fluoroquinolones (FQs) are widely used in livestock and poultry for the treatment of bacterial infection. However, resistance to FQs has increased markedly.ResultsIn this study, swine feces and wastewater were sampled from 21 swine farms of seven provinces in China to investigate the prevalence of FQ resistance, including plasmid-mediated fluoroquinolone resistance (PMQR) genes and the occurrence of target mutations. All isolates showed moderate rate of resistance to norfloxacin (43.0%), ciprofloxacin (47.6%), ofloxacin (47.0%) and levofloxacin (38.8%). The percentage of strains resistant to the four FQs antimicrobials was positively correlated with the danofloxacin (DANO) MIC. Among the 74 FQ-resistant isolates, 39 (52.70%) had mutations in gyrA (S83L and D87 to N, Y, G, or H), 21 (28.38%) had mutations in parC (S80I and E84K), 2 (2.70%) had mutations in parE (I355T and L416F), 26 (35.14%) had mutations in marR (D67N and G103S), 1 (1.35%) had mutations in acrR (V29G). While, no mutation was found in gyrB. There were 7 (9.46%) strains carried the qnrS gene, 29 (39.19%) strains carried the oqxAB gene, and 9 (12.16%) strains carried the aac (6′)-Ib-cr gene. In addition, the conjugation assays showed that qnrS, oqxAB and aac (6′)-Ib-cr could be successfully transferred to E. coli J53 from 4 (57.1%), 20 (69.0%) and 5 (55.6%) donor strains, respectively. There were no qnrA, qnrB, qnrC, qnrD and qepA genes detected.ConclusionThe present study showed that DANO-resistant E. coli strains isolated from swine farms had significant cross-resistance to other four FQs antimicrobials. Further study revealed that the resistance mechanisms of swine-derived E. coli to FQs may be attributable to the occurrence of chromosomal mutations (gyrA, parC, parE, marR and acrR genes double-site or single-site mutation) and the presence of PMQR genes (qnrS, oqxAB and aac (6′)-Ib-cr). To the best of our knowledge, one novel mutation marR-D67N was found to be associated with FQ resistance, two mutations parE-L416F and acrR-V29G have never been reported in China.

Plasmidic Fluoroquinolone Resistance Genes in Fluoroquinolone-Resistant and/or Extended Spectrum Beta-Lactamase-Producing Escherichia coli Strains Isolated from Pediatric and Adult Patients Diagnosed with Urinary Tract Infection.

Escherichia coli is the leading etiological agent of community-acquired urinary tract infection (UTI). Fluoroquinolones have long been the choice of empirical treatment for UTIs. Plasmid-mediated fluoroquinolone-resistance (PMFR) is important not only for conferring resistance to fluoroquinolones but also because of the presence of PMFR genes on plasmids carrying genes encoding resistance to other antimicrobials. In this study, we aimed at investigating the frequency of PMFR genes in fluoroquinolone-resistant and/or expanded spectrum beta-lactamase (ESBL)-producing E. coli strains isolated from pediatric and adult patients diagnosed with UTI. E. coli strains isolated from urine cultures of 141 adult and 117 pediatric outpatients were evaluated. Antimicrobial susceptibilities were interpreted according to the EUCAST criteria. The presence of PMFR genes (qnrA, qnrB, qnrC, qnrS, qepA, aac(6')-Ib, and aac(6')-Ib-cr) was investigated by multiplex PCR analysis. One hundred-three (73.05%) adult and 92 (78.63%) pediatric isolates were fluoroquinolone resistant and/or ESBL producers. One third (92/258) of all isolates carried at least one PMFR gene, the most prevalent one being qnrS (67.4%). None of the isolates carried qnrC and qepA genes. PMFR determinants were found to be widespread among adult and pediatric isolates. Rational antimicrobial use is crucial for prevention of resistance in both adult and pediatric populations.

The antimicrobial susceptibility, resistance mechanisms and phylogenetic structure of S. Typhi isolated in 2005-2018 in the Russian Federation

Here we present current global epidemiological and microbiological trends for typhoid fever, as well as describe antimicrobial susceptibility and resistance mechanisms of S. Typhi. The data on examining 299 S. Typhi isolates collected in 2005—2018 in the Russian Federation were analyzed from the Russian S. Typhi Reference Center. It was found that S. Typhi population consisted of the isolates with different resistance phenotypes and mechanisms as well as genetic heterogeneity. Moreover, antimicrobial susceptibility was detected in as low as 10.4% S. Typhi strains, whereas 89.6% isolates showed fluoroquinolone resistance (including 7.3% high-level resistance) and 3.0% — multidrug resistance to am-picillin, chloramphenicol, trimethoprim/sulfamethoxazole, tetracycline and fluoroquinolones. All strains preserved susceptibility to extended-spectrum cephalosporins and azithromycin. Fluoroquinolone low-level resistance in S. Typhi was due to single nucleotide substitutions in the gyrA: Asp87Asn (78.7%) Ser83Tyr (5.0%) and Ser83Phe (3.2%). In addition, a plasmid-mediated low-level fluoroquinolone resistance (qnrS) was found in one isolate. In contrast, a fluoroquinolone high-level resistance in S. Typhi was due to accumulation of three single nucleotide substitutions in the genes gyrA (Asp87Asn+Ser83Phe) andparC (Ser80Ile). In multidrug resistant S. Typhi isolates, pHCMl plasmids of incompatibility group IncHI1B(R27) (consisted of blaTEM.1, catA1, dfrA7 and tetB) and single nucleotide substitutions Ser83Tyr or Asp87Asn in gene gyrA were detected. The data of phylogenetic reconstruction based on the analysis of core singlenucleotide variations among examined and previously sequenced S. Typhi genomes, demonstrated that more than 80.0% of S. Typhi isolated in Russia were referred to the Asian genotype as they belonged to subclade 4.3.1 (by Wong et al.) or dominant H58 clade (H58 haplotype by Roumagnac et al.). More than 60.0% isolates in this dominant phylogenetic group possessed a fluoroquinolone low-level resistance due to gyrA Asp87Asn. Less than 20.0% of S. Typhi strains isolated in Russia phylogenetically belonged to the subclades other than 4.3.1 (non-H58) and differed from the major S. Typhi population by lacked antibiotic resistance or exerted fluoroquinolone resistance due to gyrA Ser83Phe. The study data allowed to expand our understanding on genetic diversity in S. Typhi strains isolated recently and pinpoint features of phylogenetic structure for S. Typhi population in the Russian Federation.

Ciprofloxacin-resistant Gram-negative isolates from a tertiary care hospital in Eastern India with novel gyrA and parC gene mutations

BackgroundExpanded-spectrum quinolones (ciprofloxacin) are highly effective against gram-negative bacteria, but significant resistance to quinolones has been increasingly reported. We sought to evaluate the prevalence of gram-negative ciprofloxacin-resistant isolates (CRIs) from our hospital and their mechanism of action. MethodsGram-negative CRIs were identified as per standard procedures and confirmed using the Ezy MICTM Strip (HiMedia). DNA from 67 CRIs was amplified for the quinolone resistance–determining region (QRDR) and plasmid-mediated quinolone resistance genes. Thirty isolates positive for QRDR DNA were sequenced by Sanger's method to detect mutation. ResultsOf the isolates, 42.5% were found to be CRIs, the majority (74.42%) from inpatient departments, and Escherichia coli (64.19%) was the predominant isolate. Among the CRIs, 24.55% were ESBL producers and 35.29% were multidrug resistant. The polymerase chain reaction results showed the majority were amplified by QRDR target regions of gyrA (35.4%) while 4.61% were amplified for the plasmid-mediated fluoroquinolone resistance region of the qnrB gene. Further sequencing of QRDR-positive genes showed point mutations with amino acid changes at codons Ser83 and Asp87 in the gyrA gene and Ser80, Glu84, and Leu88 positions in the parC gene. ConclusionCiprofloxacin resistance observed in our study was mostly due to point mutations. Hence, strategies for rational use of ciprofloxacin and adherence to the dose and duration of treatment could be helpful to prevent selection and spread of mutant CRIs/strains.

Chromosomal and plasmid-mediated fluoroquinolone resistance in human Salmonella enterica infection in Ghana

BackgroundSalmonella infection poses significant public health threat globally, especially in resource-limited countries. Emergence and spread of antibiotic resistant strains to fluoroquinolones have led to treatment failures and increased mortality in Salmonella infection. However, there is dearth of information regarding mechanisms of resistance to fluoroquinolones in Ghana. This study therefore sought to identify chromosomal mutations and plasmid-mediated resistance as possible mechanisms of fluoroquinolone resistance from clinical isolates in Ghana.MethodsThis was a retrospective study of archived isolates biobanked at Kumasi Centre for Collaborative Research in Tropical Medicine, Ghana. Isolates were obtained from blood, stool and oropharynx samples at two hospitals, between May, 2016 and January, 2018. Salmonella identification was done using standard microbiological protocols and antibiotic susceptibility testing performed by Kirby-Bauer disc diffusion method. Isolates with intermediate susceptibility and/or resistance to nalidixic acid and/or ciprofloxacin were selected and examined for chromosomal mutations by Sanger sequencing and plasmid-mediated resistance by PCR.ResultsOf 133 biobanked isolates cultured, 68 (51.1%) and 16 (12%) were identified as Salmonella Typhi and non-typhoidal Salmonella (NTS), respectively. Sequence analysis of gyrA gene revealed the presence of 5 different nonsynonymous mutations, with the most frequent mutation (Ile203Ser) occurring in 12 out of 13 isolates tested. Gyrase B (gyrB) gene had 1 nonsynonymous mutation in 3 out of 13 isolates, substituting phenylalanine with leucine at codon 601 (Phe601Leu). No mutation was observed in parC and parE genes. Two NTS isolates were found to harbour qnrS plasmid-mediated resistant gene of molecular size 550 bp with high ciprofloxacin MIC of 0.5 μg/ml.ConclusionThis study reports for the first time in Ghana plasmid-mediated fluoroquinolone resistant gene qnrS in Salmonella clinical isolates. Nonsynonymous mutations of gyrA and gyrB genes likely to confer Salmonella reduced susceptibility to ciprofloxacin were also reported.

Antibiotic Resistance in Enterobacteriaceae from Surface Waters in Urban Brazil Highlights the Risks of Poor Sanitation.

Surface waters are an unappreciated reservoir of antimicrobial resistance (AMR). Poor sanitation brings different species of environmental bacteria into contact, facilitating horizontal gene transfer. To investigate the role of surface waters as potential reservoirs of AMR, we studied the point prevalence of fecal contamination, AMR genes, and Enterobacteriaceae in an urban lake and rural river system in Northeast Brazil in comparison with a lake and sewer system in Northeast Ohio in the United States. Surface water samples were examined for evidence of human fecal contamination using microbial source tracking and screened for plasmid-mediated fluoroquinolone resistance and carbapenemase genes. Enterobacteriaceae were detected using selective agar followed by antimicrobial susceptibility testing and detection of AMR genes by microarray, and classified by repetitive sequence-based polymerase chain reaction and multilocus sequence typing. Concentrations of human fecal bacteria in the Brazilian urban lake and sewage in Northeast Ohio were similarly high. Filtered water samples from the Brazilian urban lake, however, showed the presence of bla OXA-48, bla KPC, bla VIM-2, qnrS, and aac(6')-lb-cr, whereas only bla VIM-2 was identified in raw sewage from Northeast Ohio. From the Brazilian urban lake, 85% of the Enterobacteriaceae (n = 40) cultured were resistant to at least one clinically important antibiotic, including ST131 Escherichia coli harboring the extended-spectrum beta-lactamase CTX-M. Although two isolates demonstrated polymyxin resistance, mcr-1/2 was not detected. Our findings indicate that surface waters in an urban Brazilian site can serve as an environmental reservoir of AMR and that improving wastewater treatment and sanitation generally may ameliorate AMR dissemination.

Community Origins and Regional Differences Highlight Risk of Plasmid-mediated Fluoroquinolone Resistant Enterobacteriaceae Infections in Children.

Fluoroquinolones are uncommonly prescribed in children, yet pediatric multidrug resistant (MDR) enterobacteriaceae (Ent) infections often reveal fluoroquinolone resistance (FQR). We sought to define the molecular epidemiology of FQR and MDR-Ent in children. A case-control analysis of children with MDR-Ent infections at 3 Chicago hospitals was performed. Cases were children with third-generation cephalosporin-resistant and/or carbapenem-resistant Ent infections. Polymerase chain reaction and DNA analysis assessed bla and plasmid-mediated FQR (PMFQR) genes. Controls were children with third-generation cephalosporin, fluoroquinolone, and carbapenem-susceptible Ent infections matched by age, source and hospital. We assessed clinical-epidemiologic predictors of PMFQR Ent infection. Of 169 third-generation cephalosporin-resistant and/or carbapenem-resistant Ent isolates from children (median age, 4.8 years), 85 were FQR; 56 (66%) contained PMFQR genes. The predominant organism was Escherichia coli, and most common bla gene blaCTX-M-1 group. In FQR isolates, PMFQR gene mutations included aac6'1bcr, oqxA/B, qepA and qnrA/B/D/S in 83%, 15%, 13% and 11% of isolates, respectively. FQR E. coli was often associated with phylogroup B2, ST43/ST131. On multivariable analysis, PMFQR Ent infections occurred mostly in outpatients (odds ratio, 33.1) of non-black-white-Hispanic race (odds ratio, 6.5). Residents of Southwest Chicago were >5 times more likely to have PMFQR Ent infections than those in the reference region, while residence in Central Chicago was associated with a 97% decreased risk. Other demographic, comorbidity, invasive-device, antibiotic use or healthcare differences were not found. The strong association of infection with MDR organisms showing FQR with patient residence rather than with traditional risk factors suggests that the community environment is a major contributor to spread of these pathogens in children.

Evaluation of co-transfer of plasmid-mediated fluoroquinolone resistance genes and blaNDM gene in Enterobacteriaceae causing neonatal septicaemia

BackgroundThe blaNDM-1 (New Delhi Metallo-β-lactamase-1) gene has disseminated around the globe. NDM-1 producers are found to co-harbour resistance genes against many antimicrobials, including fluoroquinolones. The spread of large plasmids, carrying both blaNDM and plasmid-mediated fluoroquinolone resistance (PMQR) markers, is one of the main reasons for the failure of these essential antimicrobials.MethodsEnterobacteriaceae (n = 73) isolated from the blood of septicaemic neonates, admitted at a neonatal intensive care unit (NICU) in Kolkata, India, were identified followed by PFGE, antibiotic susceptibility testing and determination of MIC values for meropenem and ciprofloxacin. Metallo-β-lactamases and PMQRs were identified by PCR. NDM-positive isolates were studied for mutations in GyrA & ParC and for co-transmission of blaNDM and PMQR genes (aac(6′)-Ib-cr, qnrB, qnrS) through conjugation or transformation. Plasmid types, integrons, plasmid addiction systems, and genetic environment of the blaNDM gene in NDM-positive isolates and their transconjugants/ transformants were studied.ResultsIsolated Enterobacteriaceae comprised of Klebsiella pneumoniae (n = 55), Escherichia coli (n = 16), Enterobacter cloacae (n = 1) and Enterobacter aerogenes (n = 1). The rates of ciprofloxacin (90%) and meropenem (49%) non-susceptibility were high. NDM was the only metallo-β-lactamase found in this study. NDM-1 was the predominant metallo-β-lactamase but NDM-5, NDM-7, and NDM-15 were also found. There was no significant difference in ciprofloxacin non-susceptibility (97% vs 85%) and the prevalence of PMQRs (85% vs 77%) between NDM-positive and NDM-negative isolates. Among the PMQRs, aac(6′)-Ib-cr was predominant followed by qnrB1 and qnrS1. Twenty-nine isolates (40%) co-harboured PMQRs and blaNDM, of which 12 co-transferred PMQRs along with blaNDM in large plasmids of IncFIIK, IncA/C, and IncN types. Eighty-two percent of NDM-positive isolates possessed GyrA and/or ParC mutations. Plasmids carrying only blaNDM were of IncHIB-M type predominantly. Most of the isolates had ISAba125 in the upstream region of the blaNDM gene.ConclusionWe hypothesize that the spread of PMQRs was independent of the spread of NDM-1 as their co-transfer was confirmed only in a few isolates. However, the co-occurrence of these genes poses a great threat to the treatment of neonates.

Plasmid-mediated fluoroquinolone resistance associated with extra-intestinal Escherichia coli isolates from hospital samples.

Background & objectives:Infection from fluoroquinolone-resistant extra-intestinal Escherichia coli is a global concern. In this study, isolation and characterization of fluoroquinolone-resistant extra-intestinal E. coli isolates obtained from hospital samples were undertaken to detect plasmid-mediated quinolone resistance (PMQR) genes.Methods:Forty three isolates of E. coli obtained from patients with extra-intestinal infections were subjected to antibiogram to detect fluoroquinolone resistance. The mechanism of fluoroquinolone resistance was determined by the detection of PMQR genes and mutations in quinolone resistance determining region (QRDR).Results:Of the 43 isolates, 36 were resistant to nalidixic acid (83.72%) and 28 to ciprofloxacin (65.11%). Eight E. coli isolates showed total resistance to both the antimicrobials without any minimum inhibitory concentration. The detection of PMQR genes with qnr primers showed the presence of qnrA in two, qnrB in six and qnrS in 21 isolates. The gene coding for quinolone efflux pump (qepA) was not detected in any of the isolates tested. The presence of some unexpressed PMQR genes in fluoroquinolone sensitive isolates was also observed.Interpretation & conclusions:The detection of silent PMQR genes as observed in the present study presents a risk of the transfer of the silent resistance genes to other microorganisms if present in conjugative plasmids, thus posing a therapeutic challenge to the physicians. Hence, frequent monitoring is to be done for all resistance determinants.

Antimicrobial resistance, multilocus sequence types and virulence profiles of ESBL producing and non-ESBL producing uropathogenic Escherichia coli isolated from cats and dogs in Switzerland

Among 64 uropathogenic Escherichia coli (UPEC) isolated from 13 cats and 51 dogs, 35 were extended-spectrum beta-lactamase (ESBL) producers, and 29 were non-ESBL producers. Forty-six (71.9%) of the isolates were multidrug resistant (MDR). Among the ESBL producers, blaCTX-M-15 (n = 17/48.6% of the blaESBLs), blaCTX-M-1 (n = 10/28.6%), blaCTX-M-55 (n = 4/11.4%), blaCTX-M-14 (n = 3/8.6%), and blaCTX-M-27 (n = 1/2.9%) were identified. The plasmid-mediated fluoroquinolone resistance genes aac(6′)-Ib-cr, qnrB and the azithromycin resistance gene mph(A) were detected in 17 (26.6% of all isolates), one (1.6%) and in 13 (20.3%) respectively. The most frequent phylogenetic groups were C (n = 19) and B2 (n = 15). Twenty-six different sequence types (STs) were identified, with two being novel. The most frequent STs were ST410 (n = 16/25%), ST131, and ST73 (both n = 5/7.8%), and ST361 (n = 4/6.3%). Ten (15.6%) of the STs have been associated with urinary tract infection (UTI) in humans, suggesting zoonotic potential. Among seven virulence-associated genes, fyuA was the most prevalent. The overall aggregate virulence factor (VF) score was highest for isolates belonging to phylogenetic group B2 (median aggregate VF score 6, mean score 5,5, range 3–7), and lowest for isolates belonging to phylogenetic group C (0/ 0.5/0-3). The most frequent ST in this study, ST410, harboured the lowest number of VF (0/0,3/0-2).VF scores were higher in NDR (4/3.8/3–4) than in MDR (1/1,9/0–7), and higher in non-ESBL producing isolates (3/3/0–7) than in ESBL producers (1/1,7/0–7). Our data advance our knowledge of the phenotypic and genotypic characteristics of UPEC in companion animals and their potential for infection, zoonotic transmission and dissemination of antimicrobial resistance determinants.

Quinolone Resistance Detection by PCR-RFLP and Multiplex-PCR among Extended- Spectrum β- Lactamase Producing Enterobacteriaceae

Quinolone resistance limits the therapeutic potential for the Extended-Spectrum β- lactamase (ESBL) producing Enterobacteriaceae. The aim of this study was to investigate the most common mechanisms of quinolones resistance in ESBL- producing Enterobacteriaceae using both phenotypic and genotypic methods. Out of 1766 clinical isolates collected between October 2012 and September 2013, 219 Enterobacteriaceae clinical isolates were ESBL producers, nalidixic acid and ciprofloxacin resistant were identified by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDITOF/ MS) and the Minimal Inhibitory Concentration (MIC) values of ciprofloxacin and nalidixic acid wasdetected before and after addition of phenylalanine-arginine β-naphthylamide (PaβN) efflux pump inhibitor. Thirty three isolates were selected for screening of the plasmid-mediated fluoroquinolone resistance (PMQR) genes; (qnr, aac(6’)-Ib-cr and qepA) and the efflux pump genes (oqxAB genes) by multiplex PCR. Whereas GyrA and ParC genes mutations were detected by PCR-RFLP assay. The PaβN changed the MIC values of 28 isolates. The GyrA gene mutation was detected in 24/33 (72.7%), while the par C gene mutation was detected in 3/33 (9.1%). Qnr-genes were detected in 13/33 (39.4%), aac(6')-Ib gene was detected in 24/33 (72.7%). qepA gene was detected in only one Klebsiella pneumoniae isolate. Finally the oqxA gene was detected in 16/33 (48.5%) of the studied isolates. The present study indicated that the PaβN was an effective phenotypic screening method for quinolones resistance efflux pump; moreover PCR-RFLP offered simple and rapid method for detection of ciprofloxacin-resistance that could be useful for clinical diagnosis and epidemiological studies.

Occurrence of the Plasmid-Mediated Fluoroquinolone Resistance qepA1 Gene in Two Clonal Clinical Isolates of CTX-M-15-Producing Escherichia coli from Algeria.

QepA is a plasmid-mediated quinolone resistance determinant of low prevalence described worldwide, mainly in Enterobacteriaceae. This study describes, for the first time in Algeria, two clonally related, QepA-producing Escherichia coli clinical isolates positive for CTX-M-15. The clonal spread of these multidrug-resistant isolates is a major public health concern.

Impact of AAC(6')-Ib-cr in combination with chromosomal-mediated mechanisms on clinical quinolone resistance in Escherichia coli.

aac(6')-Ib-cr is the most prevalent plasmid-mediated fluoroquinolone (FQ) resistance mechanism in Enterobacteriaceae. We aimed to analyse the interplay between this plasmid-mediated gene and chromosomal-mediated quinolone resistance mechanisms on both FQ resistance and bacterial fitness in Escherichia coli. E. coli ATCC 25922 and derived isogenic strains carrying chromosomal-mediated quinolone resistance modifications (Ser83Leu-Asp87Asn in GyrA, Ser80Arg in ParC and/or a marR gene deletion) were electroporated with a pBK-CMV vector encoding AAC(6')-Ib-cr. The MICs of FQs were determined by microdilution and bactericidal activity was determined using time-kill curves. A peritoneal sepsis murine model was used to evaluate the in vivo impact. Bacterial fitness was analysed using growth curves and competition assays. The presence of the aac(6')-Ib-cr gene increased the MICs of ciprofloxacin and norfloxacin 4-8-fold for all E. coli genotypes, independently of the initial resistance level. Combination of the aac(6')-Ib-cr gene with three or four chromosomal mechanisms was necessary to reach MIC values above the susceptible category. Killing curve assays showed a clear selective advantage for survival in strains harbouring the aac(6')-Ib-cr gene (up to 7 log10 cfu/mL after 24 h). AAC(6')-Ib-cr significantly reduced the ciprofloxacin efficacy in vivo. In terms of bacterial fitness cost, maximal OD was significantly lower for all strains harbouring the aac(6')-Ib-cr gene, independently of chromosomal mutations associated. The aac(6')-Ib-cr gene, in spite of producing low-level resistance by itself, plays a relevant role in acquisition of a clinical level of ciprofloxacin and norfloxacin resistance, when combined with three or four chromosomal mutations, both in vitro and in vivo.