Plasmid-mediated Quinolone Resistance Genes qnrS Research Articles

The scope of antimicrobial resistance in residential aged care facilities determined through analysis of Escherichia coli and the total wastewater resistome.

High and often inappropriate antibiotic use has been documented for residential aged care facilities (RACFs). As a result, RACFs represent selective environments favoring the development of antimicrobial resistance (AMR). However, surveillance of the prevalence and scope of AMR in RACFs is limited. Here, wastewater-based epidemiology encompassing culture-based methods in combination with whole genome sequencing and metagenomics allowed for the in-depth analysis of the prevalence of antimicrobial-resistant Escherichia coli in two RACFs and one retirement facility. Wastewater was collected at five different time points over 18 months. From these, E. coli were isolated and assessed for phenotypic and genotypic resistance. The antimicrobial resistome of each wastewater sample was also determined. A comparison of facilities revealed a higher prevalence of AMR and multidrug-resistant E. coli observed in one RACF. The international high-risk E. coli clone, ST131, carrying CTX-M-like extended-spectrum beta-lactamases was exclusively isolated from this facility. A high proportion of these isolates were classified as belonging to phylogroups B2 and D, associated with virulent extra-intestinal E. coli infections. The resistome of this facility also revealed a higher prevalence of mobile resistance genes such as sul1 and sul2, conferring sulfamethoxazole resistance, the plasmid-mediated quinolone-resistance gene qnrS, and qacEdelta1 that confers biocide resistance. High fluoroquinolone resistance rates were observed in all three sample sites despite measures in place limiting the use of this class of antibiotics. The findings of this study illustrate that RACFs are highly selective environments that require measures to limit AMR development, potentially through antimicrobial stewardship. IMPORTANCE Antimicrobial resistance (AMR) is a global threat that imposes a heavy burden on our health and economy. Residential aged care facilities (RACFs), where frequent inappropriate antibiotic use creates a selective environment that promotes the development of bacterial resistance, significantly contribute to this problem. We used wastewater-based epidemiology to provide a holistic whole-facility assessment and comparison of antimicrobial resistance in two RACFs and a retirement village. Resistant Escherichia coli, a common and oftentimes problematic pathogen within RACFs, was isolated from the wastewater, and the phenotypic and genotypic AMR was determined for all isolates. We observed a high prevalence of an international high-risk clone, carrying an extended-spectrum beta-lactamase in one facility. Analysis of the entire resistome also revealed a greater number of mobile resistance genes in this facility. Finally, both facilities displayed high fluoroquinolone resistance rates-a worrying trend seen globally despite measures in place aimed at limiting their use.

High β-Lactam and Quinolone Resistance of Enterobacteriaceae from the Respiratory Tract of Sheep and Goat with Respiratory Disease.

Simple Summaryβ-lactams and quinolones are major groups of antibiotics that are commonly used for the treatment of severe infection both in animals and humans. Little is known about their resistance mechanisms in animals. Our results revealed high resistance rates against both groups in Gram-negative bacteria recovered from small ruminants suffering from respiratory disease. Phenotypically, 9.2% of the isolates were multidrug-resistant, and 11.8% and 6.6% of the isolates were positive for AmpC and ESBL production, respectively. Genetic characterization identified different β-lactamase-encoding genes such as blaTEM, blaSHV, and blaCTX-M which are responsible for β-lactam resistance. Furthermore, the plasmid-mediated quinolone resistance gene, qnrS, was identified to be associated with quinolone resistance. Our results regenerate interest in the wise use of antimicrobials in animal fields as well as to apply a One Health approach to prevent and/or mitigate their dissemination to the human environment.During the last decade’s increase of antimicrobial resistance (AMR) in animals, animal-human transmission has become a major threat. Therefore, the present study aimed to evaluate the genetic basis of AMR in Gram-negative bacteria recovered from sheep and goats with respiratory disease. Nasal and ocular swabs were collected from 69 diseased animals, and 76 Gram-negative bacterial isolates were identified from 59 animals. All isolates were checked phenotypically for resistance and genotypically for different resistance mechanisms, including β-lactam, quinolone, and aminoglycoside resistance. Our results demonstrated that 9.2% (95% CI 4.5–17.8%) of the isolates were multidrug-resistant, with high resistance rates to β-lactams and quinolones, and 11.8% (95% CI 6.4–21%) and 6.6% (95% CI 2.8–14.5%) of the isolates were phenotypically positive for AmpC and ESBL, respectively. Genotypically, blaTEM was the most identified β-lactamase encoding gene in 29% (95% CI 20–40%) of the isolates, followed by blaSHV (14.5%, 95% CI 8.3–24.1%) and blaCTX-M (4%, 95% CI 1.4–11%). Furthermore, 7.9% (95% CI 3.7–16.2%) of the isolates harbored plasmid-mediated quinolone resistance gene qnrS. Our study revealed for the first time to our knowledge high β-lactam and quinolone resistance associated with the bacteria recovered from sheep and one goat with respiratory disease. Furthermore, different antimicrobial resistant determinants were identified for the first time from animals in Africa, such as blaLEN-13/55, blaTEM-176 and blaTEM-198/214. This study highlights the potential role of sheep and goats in disseminating AMR determinants and/or resistant bacteria to humans. The study regenerates interest for the development of a One Health approach to combat this formidable problem.

Molecular characterisation of antimicrobial resistance determinants and class 1 integrons of Salmonella enterica subsp. enterica serotype Enteritidis strains from retail food in China

Salmonella is a genus responsible for a majority of foodborne disease outbreaks affecting public health worldwide, and Salmonella enterica subsp. enterica serotype Enteritidis (S. Enteritidis) is the most common pathogen that presents alarming rates of antimicrobial resistance. The aim of this study was to analyse the antimicrobial resistance of 124 S. Enteritidis strains collected from retail foods in 39 cities across China and to conduct a comprehensive molecular characterisation of their antimicrobial resistance genes (ARGs) and class 1 integrons through PCR amplification, antibiotic susceptibility testing, and conjugation assays. The strains exhibited a multidrug resistance rate as high as 68.5% and were frequently resistant to nalidixic acid (97.6%) and ampicillin (74.2%). Correspondingly, the analysis of quinolone genes revealed that 97.5% of the strains harboured single mutations in gyrA; some possessed the plasmid-mediated quinolone resistance genes qnrS (0.8%), oqxAB (2.4%), as well as the blaTEM-1 (67.7%); and the extended spectrum beta-lactamase (ESBL) genes blaCTX-M-55 were detected in 2.4% of the strains. Class 1 integrons were observed in 11.3% (14/124) of the strains and integrated three different gene cassettes, namely, aadA22 (1/14), dfrA1-aadA1 (5/14), and dfrA17-aadA5 (8/14). Conjugation experiments and antimicrobial susceptibility testing showed that the integrons including antimicrobial resistance genes could be transferred to the recipient strain via mobile plasmids, rendering the recipient strain resistant. Both multilocus variable-number tandem-repeat analysis (MLVA) and core genome multilocus sequence typing (cgMLST) show a better discrimination in this study, hence a combination of MLVA and cgMLST analyses would improve the accuracy and cost-effectiveness. This study provides a systematic and comprehensive analysis of the antimicrobial resistance profiles of S. Enteritidis strains isolated from retail food in China. The present data will help to gain insight into the public health impact of S. Enteritidis and guide the development of control strategies.

Extensively-Drug Resistant Klebsiella pneumoniae Recovered From Neonatal Sepsis Cases From a Major NICU in Egypt.

BackgroundNeonatal sepsis is a nuisance to clinicians and medical microbiologists, particularly those cases caused by Klebsiella pneumoniae. Thus, we aimed at investigating the profile and mechanisms of antibiotic resistance and the clonal relationships between K. pneumoniae isolated from neonates at the largest tertiary care hospital’s neonatal intensive care units (NICUs) in Minia, Egypt.MethodsThis study comprised 156 neonates diagnosed with culture-proven sepsis from February 2019 to September 2019, at a major NICU of Minia City. All K. pneumoniae isolates were collected and characterized by antimicrobial profile, resistance genotype, and pulsed-field gel electrophoresis typing.ResultsTwenty-four K. pneumoniae isolates (15.3%) were collected out of the 156 sepsis diagnosed neonates. These samples showed extensive drug resistance (XDR) to most of the tested antimicrobials, except fluoroquinolones. All the K. pneumoniae isolates possessed blaVIM and blaNDM carbapenemase genes, while blaKPC gene was detected in 95.8%. Considering extended-spectrum β-lactamases genes, blaCTX–M was found in all the isolates and blaOXA–1 gene in 75% of them. The plasmid-mediated quinolone resistance gene qnrS, was predominantly found among our isolates in comparison to qnrB or qnrA. A moderate degree of clonal relatedness was observed between the isolates.ConclusionTo the best of our knowledge, this the first report of an alarming occurrence of XDR among K. penumoniae isolates recovered from neonatal sepsis in Egypt. Our data necessitate proper antimicrobial stewardship as the choices will be very limited.

Molecular Characterization of Extended-Spectrum β-Lactamase-Producing Multidrug Resistant Escherichia coli From Swine in Northwest China.

Objectives: The aim of the present study was to explore the prevalence and molecular characterization of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli collected from pig farms in Northwest China.Methods: Between May 2015 and June 2017, a total of 456 E. coli isolates were collected from fecal samples of healthy and diarrheal pigs in Northwest China to screen the ESBL producers. The β-lactamases, plasmid-mediated quinolone resistance (PMQR) genes and virulence genes among ESBL producers were corroborated by PCR and sequencing. Finally, ESBL producers were further grouped according to phylogenetic background and genetic relatedness.Results: Forty-four (9.6%) out of the 456 E. coli isolates were identified as ESBL-producing isolates. All ESBL producers exhibited multidrug resistance (MDR) phenotype, and more than 90% of the ESBL producers were resistant to amoxicillin, amoxicillin-clavulanic acid, oxytetracycline, enrofloxacin and sulfamethoxazole/trimethoprim. All ESBL producers harbored at least one type of β-lactamase, with blaCTX−M, blaTEM, blaSHV, blaOXA−48, and blaKPC−2 being detected in forty, thirty, seven, four, two and one isolates, respectively. Sequencing revealed the most common blaCTX−M subtype was blaCTX−M−14 (n = 24), followed by blaCTX−M−15 (n = 14), blaCTX−M−64 (n = 11), blaCTX−M−9 (n = 10) and blaCTX−M−123 (n = 9). qnrS (n = 23) was the predominant PMQR gene, and all PMQR genes were detected in co-existence with β-lactamase genes. estA (n = 18) and F4 (n = 18) were the most prevalent enterotoxin and fimbrial adhesin, respectively, and 27 different virotypes were found with respect to the association of enterotoxins and fimbrial adhesins. Twenty-four different sequence types (STs) were identified among 44 ESBL producers, and clones ST405, ST10 and ST648 were strongly present in more than one-third (34.1%) of ESBL producers.Conclusion: All ESBL-producing E. coli isolates exhibited MDR phenotype, and showed high prevalence of β-lactamase and PMQR genes. Especially, one isolate harbored ESBL genes blaTEM, blaSHV, blaCTX−M−9, blaCTX−M−14, blaCTX−M−64, and carbapenemase gene blaOXA−48 and blaKPC−2, as well as PMQR genes qnrS, qnrB, qnrD, qepA and aac(6')-Ib-cr.

Genomic Identity of Fluoroquinolone-Resistant blaCTX-M-15-Type ESBL and pMAmpC β-Lactamase Producing Klebsiella pneumoniae from Buffalo Milk, India

We investigated the occurrence of extended-spectrum β-lactamase (ESBL) and AmpC-type β-lactamase (ACBL) producing quinolone-resistant Klebsiella pneumoniae (KP) in milk samples of apparently healthy buffaloes (n = 348) and buffaloes (n = 19) with evidence of subclinical mastitis from seven districts of West Bengal, India. In total, 12 ESBL producing KP were isolated with blaCTX-M-15 gene and 7 of them were ACBL producers, as well. The blaCTX-M-15 genes were carried by transposable element ISEcp1. The plasmid-mediated quinolone resistance genes-qnrS, qnrA, qnrB, qepA, and aac(6')-Ib-cr were detected in five, one, three, four, and one isolate (s), respectively. In addition, eight isolates carried mutation in gyrase (gyrA) and six in topoisomerase IV (parC). Resistance markers/genes for sulfonamide (sul1), tetracycline [tet(A) and tet(B)], and aminoglycoside (aacC2) were also detected in eight, four, and one isolate(s), respectively. The class I integrons identified in five isolates carried aad2/aad5 and dfrA12/dfrA17 gene cassettes. The enterobacterial repetitive intergenic consensus-PCR revealed that all the isolates were genetically diverse and comprised a heterogeneous population. Isolation of multidrug-resistant KP, a typical nosocomial pathogen from buffalo milk, reiterates the need to monitor farm animals for ESBL producing Enterobacteriaceae and emphasizes on judicious use of antibiotics in animal husbandry sector.

High diversity of genes and plasmids encoding resistance to third-generation cephalosporins and quinolones in clinical Escherichia coli from commercial poultry flocks in Italy

The aim was to investigate occurrence and diversity of plasmid-mediated resistance to third-generation cephalosporins (3GC) and quinolones in clinical Escherichia coli from 200 industrial poultry farms across Italy. E. coli was isolated from colibacillosis lesions in turkeys (n = 109), broilers (n = 98) and layers (n = 22) between 2008 and 2012. 3GC-resistant isolates were screened for extended-spectrum and AmpC β-lactamase (ESBL/AmpC), while all isolates were tested for plasmid-mediated quinolone resistance (PMQR) genes. ESBL/AmpC- and PMQR-positive isolates were typed by pulsed-field gel electrophoresis and antimicrobial susceptibility testing, and their plasmids were characterised by replicon typing, multilocus sequence typing, restriction fragment length polymorphism and conjugation. EBSL/AmpC genes (blaCTX-M-1, blaCTX-M-14, blaCTX-M-2, blaSHV-12 and blaCMY-2) were detected in 7%, 9% and 4% of isolates from turkeys, broilers and layers, respectively. We identified seven ESBL/AmpC-encoding plasmid types, usually conjugative (78%), with a marked prevalence of IncI1/pST3 plasmids carrying blaCTX-M-1. PMQR occurred less frequently among isolates from turkeys (0.9%) compared to those from broilers (5%) and layers (4%). The PMQR genes qnrS, qnrB19 and oqxA/B were located on three plasmid types and two non-typeable plasmids, mostly (85%) conjugative. ESBL/AmpC- and PMQR-positive isolates were genetically unrelated and 64% of them were additionally resistant to aminoglycosides, sulfonamides and tetracyclines. Our data show that 3GC- and quinolone-resistant clinical E. coli in Italian poultry production represent a highly diverse population often resistant to most antimicrobials available for poultry. These findings underline the crucial need to develop new strategies for prevention and control of colibacillosis.

NDM-1 carbapenemase-producing Salmonella enterica subsp. enterica serovar Corvallis isolated from a wild bird in Germany

Sir, Thirdand fourth-generation cephalosporins, as well as carbapenems, are b-lactam antimicrobial agents with a broad in vitro spectrum against many human pathogens. They are considered critically important antimicrobials for human treatment. During the last decade, the prevalence of antimicrobial-resistant carbapenemase-producing Enterobacteriaceae among humans has increased and is considered a public health concern worldwide. However, the isolation of carbapenemase-producing enteric bacteria from animals has rarely been reported. – 6 One of the most widespread carbapenemases is the New Delhi metallo-b-lactamase (NDM). The genes encoding this enzyme have been found in different bacterial species (mostly Acinetobacter spp., Escherichia coli and Klebsiella spp.) and are located on highly efficient mobile genetic elements. NDM-1-producing bacteria, mostly isolated from human patients, are frequent in some environmental niches (i.e. water and sewage in India) and have recently been isolated from food-producing animals in China. The Indian subcontinent and the Balkan states seem to be the main reservoirs for NDM-1-producing strains. Among Salmonella, the blaNDM-1 gene has rarely been reported, and in all cases only in patients who had travelled to India. The German Salmonella Reference Laboratory (NRL-Salm Federal Institute of Risk Assessment, BfR) collection contains about 67000 isolates gathered since 1997, mainly from foodproducing animals and foods, but also from non-food-producing animals, the environment, feed and humans. Among this collection, 184 Salmonella spp. isolates that showed clinical resistance to third-generation cephalosporins (MICs ≥4 mg/L for cefotaxime, collected since 2006) were further characterized for their susceptibility to a panel of b-lactams/b-lactamase inhibitors and the presence of extended-spectrum b-lactamase/AmpC genes as previously described. One Salmonella enterica subsp. enterica serovar Corvallis (NRL-Salm-12-1738), multilocus sequence type ST1541 (http:// mlst.ucc.ie/mlst/dbs/Senterica) isolated from a wild bird (black kite, Milvus migrans) showed carbapenem susceptibility values that suggested the presence of a carbapenemase (zone diameters of 24, 20 and 24 mm for, respectively, imipenem, ertapenem and meropenem 10 mg discs, Oxoid-Thermo Fisher Scientific, Wesel, Germany). When Salmonella Corvallis 12-1738 was tested for the MICs of these compounds (Etest, bioMerieux, Craponne, France), it showed reduced susceptibility to them all (0.25, 0.5 and 0.125 mg/L for imipenem, ertapenem and meropenem, respectively; non-wild-type by the EUCAST cut-off values, but susceptible or intermediate according to the CLSI clinical breakpoints). When the isolate was grown in liquid medium with imipenem (Luria-Bertani broth with 16 mg/L imipenem inoculated with 1:1000 overnight culture), it showed full resistance (clinical breakpoints CLSI versus EUCAST, imipenem≥4 versus.8 mg/L). This isolate also showed resistance to chloramphenicol (floR), kanamycin [accA4, also named aac(6)-Ib], tetracycline[tet(A)], trimethoprim(dfrA17), streptomycin (strA/B), sulphonamides (sul1, sul2) and fosfomycin, carried the plasmid-mediated quinolone resistance geneqnrS, andwas susceptible to tigecycline and nitrofurantoin. The resistance determinants (genes cited above, integrons and plasmids) responsible for these resistance phenotypes were analysed by PCR amplification/sequencing and plasmid typing as described before. In this way, the presence of a blaNDM-1 carbapenemase gene was determined (European Nucleotide Archive accession number HG007972; sequence of a 960 bp PCR fragment obtained using the ISAb125-F1/bleBML-B1: TTGAAACTGTCGCACC TCA/TCCAACTCGTCGCAAAGC primers designed for the present work), together with a blaCMY-16 AmpC gene. Both genes were co-localized on pRH-1738, an 180 kb IncA/C conjugative plasmid, which also harboured two class 1 integrons (carrying dfrA1-aadA5 or aacA4 in their variable regions) as well as all resistance genes detected except qnrS. The blaNDM-1 genes have been found located on plasmids of different sizes and incompatibility groups, with IncA/C and IncHI1 being the most frequent. The low number of Salmonella Corvallis in the NRL-Salm collection (62 isolates from different countries isolated from food, livestock, the environment and non-food animals) reflects the low prevalence of Salmonella Corvallis in human infection in Germany (579 cases of salmonellosis, 0.09%, since 2001; Robert Koch-Institut: SurvStat, http://www3.rki.de/SurvStat, data status: 21 May 2013) and Europe (http://www.efsa.europa.eu/en/ efsajournal/doc/3129.pdf). However, Salmonella Corvallis has been detected with noticeably higher prevalence in humans and food products in non-European countries, is endemic in SouthEast Asia (associated with pig and pork) and is emerging in North Africa and Nigeria, where it is widely found in different animal species and the environment (R. S. Hendriksen, Technical University of Denmark, personal communication). The intake of water polluted with faeces or human waste seems to be the most important route for wild birds to acquire antimicrobial-resistant bacteria. The black kite is a migratory bird of prey that lives close to water and spends the summer in Europe

Plasmid-mediated quinolone resistance determinants qnr and aac(6′)-Ib-cr in Escherichia coli and Klebsiella spp. from Norway and Sweden

The prevalence of the plasmid-mediated quinolone resistance genes qnr and aac(6′)-Ib-cr was investigated among clinical isolates of Escherichia coli and Klebsiella spp. selected from 2 collections of consecutive isolates collected in 2004 to 2005 in Norway ( n = 2479) and Sweden ( n = 2980) and 1 group of extended-spectrum β-lactamase (ESBL)-producing isolates collected in 2003 in Norway ( n = 71). A total of 414 isolates was selected for screening based on resistance to nalidixic acid and/or reduced susceptibility/resistance to ciprofloxacin. The prevalence of both qnr and aac(6′)-Ib-cr was higher among the ESBL producers (9.1% and 52.3%, respectively) than in the consecutive isolates (1.1% and 3.2%, respectively). qnrS1 was detected in 6 isolates, whereas qnrB1 and qnrB7 were detected in 2 isolates. The genetic structure surrounding qnrS1 was similar to previously described structures. In 2 isolates, qnrS1 was located on conjugative IncN-type plasmids of approximately 140 kb.

Mechanisms of resistance and mobility among multidrug-resistant CTX-M–producing Escherichia coli from Canadian intensive care units: the 1st report of QepA in North America

Abstract We studied the molecular mechanisms of resistance and mobility of 18 multidrug-resistant CTX-M–producing Escherichia coli isolates isolated from patients in Canadian intensive care units. Fluoroquinolone-resistant isolates (83.3%) had mutations in gyrA and parC. Plasmid-mediated quinolone resistance genes qnr ( A, B, and S), qepA, and aac(6′)-Ib-cr were detected in 0%, 5.6%, and 44.4%, respectively. Sulfamethoxazole/trimethoprim-resistant isolates (61.1%) carried a dfr gene, and 10 (90.9%) of the 11 carried 1 or more sul genes. Gentamicin-resistant isolates (27.8%) carried the aac(3′)-II gene, and doxycycline-resistant isolates (33.3%) carried 1 or more tet efflux genes. Both genetically related and unrelated groups of E. coli harboring extended-spectrum β-lactamases were observed. The bla CTX-M genes were primarily located on diverse IncF plasmids of multiple replicon types downstream of the IS Ecp1 element. The spread of the bla CTX-M genes among E. coli in Canada occurs through a diversity of different mechanisms and does not correspond to a single CTX-M determinant, or a single clone, or a single plasmid but rather through the combination of clonal spread of virulent strains and acquisition of diverse CTX-M–bearing plasmids. We report the 1st qepA-producing E. coli in North America.