Ib-cr Genes Research Articles

Occurrence of ESBL-Producing Escherichia coli ST131, Including the H30-Rx and C1-M27 Subclones, Among Urban Seagulls from the United Kingdom.

Antimicrobial resistance is a public health concern. Understanding any role that urban seagulls may have as a reservoir of resistant bacteria could be important for reducing transmission. This study investigated fecal Escherichia coli isolates from seagulls (herring gulls and lesser black-backed gulls) to determine the prevalence of extended-spectrum cephalosporin-resistant (ESC-R) and fluoroquinolone-resistant E. coli among gull species from two cities (Taunton and Birmingham) in the United Kingdom (UK). We characterized the genetic background and carriage of plasmid-mediated resistance genes in extended-spectrum β-lactamase (ESBL)-producing E. coli obtained from these birds. Sixty ESC-R E. coli isolates were obtained from 39 seagulls (39/78, 50%), of which 28 (28/60, 46.7%) were positive for plasmid-mediated CTX-M and/or AmpC β-lactamase resistance genes. Among these, blaCTX-M-15, blaCTX-M-14, and blaCMY-2 predominated. Three isolates belonging to the B2-ST131 clone were detected, of which two harbored blaCTX-M-15 (typed to C2/H30Rx) and one harbored blaCTX-M-27 and was typed to C1/H30-R (recently described as the C1-M27 sublineage). The plasmid-mediated quinolone resistance (PMQR) gene carriage prevalence (11.7%) consisted of aac(6')-Ib-cr and qnrB genes. No carbapenem or colistin resistance genes were detected. Urban seagulls in the UK are colonized and can spread major antimicrobial-resistant E. coli isolates harboring ESBL and PMQR determinants, including clinically important strains such as the pandemic clone B2-ST131 and the C1-M27 subclade. This is the first report of ST131-C1-M27 subclade in wildlife in the UK and in seagulls worldwide.

Whole genome sequence of an Escherichia coli ST410 isolate co-harbouring blaNDM-5, blaOXA-1, blaCTX-M-15, blaCMY-2, aac(3)-IIa and aac(6')-Ib-cr genes isolated from a patient with bloodstream infection in China.

Mortality associated with carbapenemase-producing Enterobacteriaceae is high as there are few therapeutic options. Escherichia coli sequence type 410 (ST410) is currently an international high-risk clone and is responsible for a large number of clinical infections. Here we report the draft genome sequence of a ST410 clinical E. coli isolate (ECS9) co-harbouring blaNDM-5, blaOXA-1, blaCTX-M-15, blaCMY-2, aac(3)-IIa and aac(6')-Ib-cr genes, obtained from a patient with bloodstream infection in China. The genome of E. coli ECS9 was sequenced using an Illumina HiSeqTM 4000 instrument with a 150-bp paired-end approach. Generated sequence reads were assembled using Velvet 1.2.10. Contigs were annotated using Rapid Annotation using Subsystem Technology (RAST), and further whole-genome sequence data analyses were performed. Escherichia coli ECS9 belongs to multilocus sequence typing (MLST) ST410. The total number of assembled bases was 4 935 145 bp, with 5077 protein-coding sequences. The presence of the blaNDM-5, blaOXA-1, blaCTX-M-15 and blaCMY-2 genes was detected in addition to other antimicrobial resistance genes conferring resistance to fluoroquinolones, aminoglycosides, trimethoprim, sulfonamides and tetracyclines. To our knowledge, this is the first report of anE. coli ST410 strain co-harbouring blaNDM-5, blaOXA-1, blaCTX-M-15, blaCMY-2, aac(3)-IIa and aac(6')-Ib-cr, obtained from a bloodstream infection in China. The presented genome sequence of carbapenemase-producing E. coli strain ST410 could provide further insight into the acquisition of multiple resistance genes by this successful lineage.

Plasmid-mediated quinolone resistance in Escherichia coli isolates from commercial broiler chickens and selection of fluoroquinolone-resistant mutants

Plasmid-mediated quinolone resistance (PMQR) is a potential concern for animal husbandry and public health. Escherichia coli isolates from a total of 109 fecal samples collected from 6 commercial broiler farms between 2007 and 2011 were examined for PMQR genes, and transfer of these genes was tested by conjugation analysis to elucidate the prevalence and spread of PMQR in broiler chickens. Two isolates from 2 farms harbored the aac(6')-Ib-cr gene that was not detected in plasmids using Southern blot analysis of S1 nuclease-digested genomic DNA separated by pulsed-field gel electrophoresis. In these 2 isolates, nucleotide mutations in the gyrA and parC genes that result in amino acid substitutions were detected. Additionally, a total of 6 isolates originating from 6 chickens from the 2 farms were positive for the qnrS1 gene. In 2 of the 6 isolates, the qnrS1 gene was transferred to a recipient strain. Two transconjugants harboring the qnrS1 gene were cultured on media supplemented with successively higher concentrations of enrofloxacin (ERFX). After a 5-time subcultivation, the ERFX MICs reached 8 and 16 μg/mL, and no nucleotide mutations were detected in the gyrA, gyrB, parC, and parE genes. Our results suggest that the prevalence of PMQR was relatively low in broiler chickens and that exposure of bacteria carrying PMQR genes to the selective pressure of fluoroquinolones can result in resistance to fluoroquinolone, which is not caused by mutations in genes encoding topoisomerases.

Exploring the profile of antimicrobial resistance genes harboring by bacteriophage in chicken feces

Bacteriophage may play an important role in antimicrobial resistance genes (ARGs) transmission. However, the contribution of bacteriophage to the spread of ARGs in environment, especially in poultry farm environment, is rarely known. In this study, the prevalence of ARGs in bacteriophage DNA was investigated in chicken feces from 30 different poultry farms in China. Then the abundance of the aac(6′)-Ib-cr, blaCTX-M, ermB, floR, mcr-1, sul1, tetM and intI1 genes was determined by qPCR in bacteriophage and compared with certain representative plasmid DNA samples. The results showed that 12 ARGs (aac(6′)-Ib-cr, aph(3′)-IIIa, blaCTX-M, ermB, ermF, floR, mcr-1, qnrS, sul1, sul2, vanA, tetM genes) and class 1 integron gene intI1 were detected in bacteriophage DNA fraction. The sul1, tetM and aac(6′)-Ib-cr genes were most prevalent with high detection rates of 77%, 61% and 55%, respectively. To our best knowledge, this study firstly reported the presence of the mcr-1 gene in bacteriophage DNA derived from farms environments. We found that the gene copy (GC) numbers of the aac(6′)-Ib-cr, ermB and sul1 genes were as high as 5.47, 5.22 and 5.54 log10 GC/g, respectively. Both the prevalence and abundance of ARGs in broiler fecal wastes were also generally higher than in laying hens. In addition, although the GC numbers of the aac(6′)-Ib-cr, floR and tetM genes in plasmid DNA was higher than that in phage DNA fraction by 4.68, 3.59 and 3.9 orders of magnitude, respectively, the absolute abundances of the blaCTX-M and mcr-1 genes in phage DNA were close to or even higher than that in plasmid DNA at farm SIL2, SIL4 and SIB1. As potential vessels for ARGs, bacteriophage could not be ignored due to their unique extracellular persistence in environments. Overall, this is the first comprehensive survey about bacteriophage carried ARGs from farms in different regions in China.

Prevalence and Characteristics of Quinolone Resistance in Salmonella Isolated from Retail Foods in Lanzhou, China

The aim of this study was to determinate the prevalence of Salmonella in retail foods and its resistance to quinolones in retail foods in Lanzhou, People's Republic of China. In this work, 2,182 food samples, collected from March 2015 to December 2018, were analyzed to detect Salmonella and then analyzed for serotype distribution, quinolone resistance, and quinolone-resistant gene detection. The findings demonstrate that the overall prevalence of Salmonella in these food categories was low. A total of 41 (1.9%) of 2,182 food samples were found to be positive for Salmonella. Ten distinct serovars were identified, and Salmonella Derby, Salmonella Anatum, and Salmonella Enteritidis were the most prevalent serovars. According to the broth microdilution test, the resistance percentages were 90.2% to nalidixic acid, 39.0% to enrofloxacin, 41.5% to ciprofloxacin, 29.3% to ofloxacin, and 26.8% to levofloxacin. Among the quinolone-resistant isolates, 12 strains had a single mutation in gyrA at codon 83 (Ser→Phe) or codon 87 (Asp→Asn or Asp→Gly). Five isolates had one parC mutation (Ser80→Arg) and one or two gyrA hot spot mutations. qnr genes were found in seven isolates (five qnrB and two qnrD), and the aac(6')-Ib gene in seven isolates. Two isolates carry both qnrB and aac(6')-Ib-cr genes. Based on these results, a low prevalence of Salmonella contamination in retail foods was found, but it might play a potential risk factor in the spread of quinolone-resistant Salmonella strains in the Lanzhou region.

Molecular epidemiological of extended-spectrum β-lactamase producing Escherichia coli isolated in Djibouti.

While the molecular epidemiology of extended-spectrum-b-lactamase (ESBL)-producing E. coli is well known in Europe due to effective surveillance networks and substantial literature, data for Africa are less available, especially in Djibouti. We studied 31 isolates of ESBL-producing E. coli from Djibouti and compared these molecular results with data available in Africa. Susceptibility rates were 3.2% for ceftazidim, 48.4% for piperacillin-tazobactam, 90.3% for amikacine and 16.1% for ofloxacin. No isolate showed resistance to carbapenems or colistin. 30 E. coli (96.8%) were positive to blaCTX-M-15, 1 (3.2%) to blaCTX-M-14 and 10 (32.3%) to narrow-broad-spectrum blaTEM. No blaSHV were detected. Fluoroquinolone resistance analysis showed that 30 ofloxacin-resistant E. coli had the mutation Ser-83->Leu on the gyrA gene. 24 E. coli (77.4%) harboured the plasmid-borne aac(6 ')-Ib-cr gene. No E. coli carried the genes qnrA, qnrB and qepA. 10 isolates (32.3%) belonging to the ST131 clone. The plasmid incompatibility group most widely represented in our collection was IncFIA/IB/II. There is no major difference with African epidemiology. In particular, we notice the international diffusion of specific clonal group ST131.

A study on prevalence of plasmid mediated quinolone resistant genes in Klebsiella pneumoniae

Introduction: Fluoroquinolone resistance is increasingly been reported in Enterobacteriaceae. Resistance to quinolones among Enterobacteriaceae is mediated by point mutations in chromosomal genes that encode the subunits of DNA-gyrase and topoisomerase IV. Objectives: The present study was done to identify quinolone resistant Klebsiella pneumoniae isolates from clinical samples and to detect the prevalence of plasmid mediated quinolone resistance genes. Materials & Methods: A total of 63 isolates from various clinical samples were identified by standard biochemical methods. Antimicrobial resistance pattern of these isolates were performed by disk diffusion method as per guidelines of CLSI. Minimum Inhibitory Concentration for Ciprofloxacin was determined by E test strips with concentration gradient of 0.002-32 µg/ml. Detection of plasmid mediated quinolone resistance genes was performed by PCR. Results: By disk diffusion, 90.99% isolates were found to be resistant to Ciprofloxacin. The sensitivity of isolates to Imipenem was found to be 99.10%. About 50.74% isolates were resistant to Aminoglycosides and 61.19% were resistant to third generation Cephalosporins. By E test, 80.95% isolates were resistant for Ciprofloxacin with MIC of >1µg/ml. By PCR, out of 63 isolates, 6 (9.52%) showed aac (6’) – Ib - cr gene, 7 (11.11%) were positive for qnr A gene, 29 (46.03%) for both qnr B and aac (6’) - Ib-cr gene and 8 (12.70%) for qnr B gene. Conclusion: The transferrable nature of Plasmid mediated fluoroquinolone resistant genes favours the dissemination of the resistant genes between different bacterial genera and limit therapeutic options available. The prevalence of plasmid mediated quinolone resistance mediated by qnr A, qnrB and aac (6’) - Ib-cr gene results in multidrug resistance among Klebsiella pneumoniae.

Multidrug resistant Aeromonas spp. isolated from zebrafish (Danio rerio): antibiogram, antimicrobial resistance genes and class 1 integron gene cassettes.

Aeromonas spp. are Gram-negative opportunistic bacteria which have been commonly associated with fish diseases. In this study, antibiogram, antimicrobial resistance genes and integrons of 43 zebrafish-borne Aeromonas spp. were studied. The isolates were identified as six Aeromonas species (A. veronii biovar veronii (n=26), A. veronii biovar sobria (n=3), A. hydrophila (n=8), A. caviae (n=3), A. enteropelogenes (n=2) and A. dhakensis (n=1)). Antibiogram of the isolates indicated that most of them were resistant to amoxicillin (100·00%), nalidixic acid (100·00%), oxytetracycline (100·00%), ampicillin (93·02%), tetracycline (74·42%), rifampicin (67·44%) and imipenem (65·15%). Multiple antimicrobial resistance (MAR) index values ranged from 0·19-0·44 to 90·70% isolates showed multidrug resistance. PCR of antimicrobial resistance genes revealed that the tetracycline resistance gene (tetA) was the most predominant (67·44%) among the isolates. The qnrS (53·49%), tetB (30·23%), tetE (30·23%), qnrB (23·26%) and aac(6')-Ib-cr (4·65%) genes were also detected. Class 1 integrase (IntI1) gene was found in 46·51% of the isolates. Two types of class 1 integron gene cassette profiles (qacG-aadA6-qacG and drfA1) were identified. The results showed that zebrafish-borne aeromonads can harbour different types of antimicrobial resistance genes and class 1 integrons. SIGNIFICANCE AND IMPACT OF THE STUDY: Aeromonas spp. are important pathogens found in diverse environments. Antimicrobial resistance genes and integrons of ornamental fish-borne Aeromonas spp. are not well studied. The antibiogram, antimicrobial resistance genes and class 1 integrons of Aeromonas spp. isolated from zebrafish were characterized for the first time in Korea. The prevalence of tetracycline resistance genes, plasmid-mediated quinolone resistance genes and class 1 integron gene cassettes were observed among the isolates. The qacG-aadA6-qacG gene cassette was identified for the first time in Aeromonas spp. The results suggest that the wise use of antimicrobials is necessary for the better management of the ornamental fish.

Characterization of uropathogenic ESBL-producing Escherichia coli isolated from hospitalized patients in western Algeria.

The aim of this study is to assess the prevalence and molecular characterization of uropathogenic Extended spectrum β-lactamases (ESBLs) producing Escherichia coli. During 3 years, all hospitalized patients at the University-affiliated hospital of Tlemcen and presenting urinary tract infections caused by E. coli were considered as potential study participants. These E. coli isolates were examined phenotypically for ESBL production. ESBL strains were subjected to antimicrobial susceptibility testing and were investigated for the presence of plasmid mediated quinolone resistance genes, 16SrRNA methylase genes and virulence genes by using conventional PCR and DNA sequencing. The molecular characterization of ESBL strains was established by phylogenetic grouping method and ERIC-PCR. The overall prevalence of ESBL was 32.5%. The blaCTX-M-15 was the most frequently detected in ESBL isolates, followed by blaCTX-M-14, blaCTX-M-28, blaCTX-M-1 and blaSHV-12 respectively. The plasmid-mediated quinolone resistance genes were detected in the 15 ESBL strains with the aac(6')-Ib-cr gene was the most detected followed by qnrB1 and qnrA1 gene respectively. Among the 22 ESBL isolates resistant to gentamicin and amikacin, the 16SrRNA methylase genes were detected in 4 isolates. The sfa and pap virulent genes were founds in 26% and 22% of isolates receptively. The genotyping analysis of all strains revealed that almost were belonged to phylogenetic groups A1 and A0 and fourteen distinct clones. The emergence of uropathogenic ESBL isolates and the high rate of blaCTX-M are alarming in Algeria. Strict measure must be required to control the further spread of these strains in Algerian hospitals.

Meta-transcriptomics reveals a diverse antibiotic resistance gene pool in avian microbiomes

BackgroundAntibiotic resistance is rendering common bacterial infections untreatable. Wildlife can incorporate and disperse antibiotic-resistant bacteria in the environment, such as water systems, which in turn serve as reservoirs of resistance genes for human pathogens. Anthropogenic activity may contribute to the spread of bacterial resistance cycling through natural environments, including through the release of human waste, as sewage treatment only partially removes antibiotic-resistant bacteria. However, empirical data supporting these effects are currently limited. Here we used bulk RNA-sequencing (meta-transcriptomics) to assess the diversity and expression levels of functionally viable resistance genes in the gut microbiome of birds with aquatic habits in diverse locations.ResultsWe found antibiotic resistance genes in birds from all localities, from penguins in Antarctica to ducks in a wastewater treatment plant in Australia. Comparative analysis revealed that birds feeding at the wastewater treatment plant carried the greatest resistance gene burden, including genes typically associated with multidrug resistance plasmids as the aac(6)-Ib-cr gene. Differences in resistance gene burden also reflected aspects of bird ecology, taxonomy, and microbial function. Notably, ducks, which feed by dabbling, carried a higher abundance and diversity of resistance genes than turnstones, avocets, and penguins, which usually prey on more pristine waters.ConclusionsThese transcriptome data suggest that human waste, even if it undergoes treatment, might contribute to the spread of antibiotic resistance genes to the wild. Differences in microbiome functioning across different bird lineages may also play a role in the antibiotic resistance burden carried by wild birds. In summary, we reveal the complex factors explaining the distribution of resistance genes and their exchange routes between humans and wildlife, and show that meta-transcriptomics is a valuable tool to access functional resistance genes in whole microbial communities.

Residual waters vs. multiresistant bacteria

Introduction: Water is used daily in numerous domestic and industrial activities, and when discarded is considered wastewater. The self-medication, antibiotics inappropriate prescription, and the use of antibiotics in animal feed, leads to the environment being a reservoir of bacteria carrying resistance genes, allowing their dissemination among bacterial communities of water and soil. Bacteria can have several associated resistance mechanisms, giving them the ability to be resistant to different classes of antibiotics. The 3rd generation cephalosporins have an extended action spectrum and are hydrolyzed by the extended spectrum β-lactamases (ESBLs). The same bacterium can express these enzymes associated with an acetyltransferase encoded by the aac (6’)-Ib-cr gene, which confers resistance to aminoglycosides (amikacin and tobramycin) and simultaneously to Fluorquinolones. Objective: Identification of resistant genes in wastewater isolates. Materials and methods: 164 isolates from the residual water, were studied for antibiotic susceptibility and 144 were selected for identification of resistance genes by PCR reaction and sequencing. Results: The antimicrobial study of 144 strains, showed that 78% (112/144) were resistant to ciprofloxacin. The resistance to ciprofloxacin was studied by the detection of aac (6’)-Ib-cr gene, found in 26% (37/144). The remained resistant isolates could have different mechanisms, such as qnr genes plasmid-mediated quinolone resistance and/or mutations on chromosomal genes (gyrA and parC). Among the 3rd generation cephalosporin resistance, were found 35% (50/114) were found resistant to cefotaxime and 26% (37/144) to ceftazidime. After sequencing, the following enzymes were predominant: CTX-M-15 (17/55); CTX-M-14 (6/55). The enzymes CTX-M-1, CTX-M-2, CTX-M-98 were found one by each isolate. Conclusion: The existence of bacteria with resistance genes to 3rd generation cephalosporins simultaneously with fluoroquinolones and aminoglycosides constitutes a serious public health problem, with the aggravation of having the same susceptibility profile and the same genetic identity of the hospital strains.

Genotypic Characterization of Antimicrobial Resistance-Associated Genes in Citrobacter Freundii Isolated from Patients with Urinary Tract Infection in Al-Najaf Governorate-Iraq

Citrobacter freundii is one of the most important pathogens that cause urinary tract infection in human because it harbors many types of antimicrobials resistance-associated genes. Therefore, this study aimed to evaluate the prevalence of antimicrobial resistance-associated genes citrobacter freundii isolated from patients with urinary tract infection in Al-Najaf governorate-Iraq. A total of 30 strains of citrobacter freundii isolated from urine samples of patients with urinary tract infection in Al-Najaf central hospital in Al-Najaf City, Iraq, during April to December 2018. Polymers chain reaction technique was used to detect the presence of antimicrobial resistance-associated genes. From the 30 strains of citrobacter freundii, 15 strains (50%) were positive for bla-tem gene, 16 strains (53.3%) were positive for bla-shv gene, 21 strains (70%) were positive for bla-ctxm gene, 3 strains (10%) were positive for bla-ctxm-1 gene, 6 strains (20%) were positive for bla-ctxm-2 gene. Interestingly, it should be noted that no any strain form the 30 we tested was carrying pse1, bla-ctxm-8, bla-ctxm-9 or bla-ctxm-25 genes. Our data showed that 19 strains (63.3%) were positive for cat1 gene, 9 strains (30%) were positive for cat2 gene and 4 strains (13.3%) were positive for cat3 gene. On the other hand, 17 strains (56.6%) were carrying qnrA gene, 11 strains (36.6%) were carrying qnrB gene and aac(6')-Ib-cr gene. Also, the results proved that there were 7 strains (23.3%) being positive for sul1 gene and 11 strains (36.6%) were positive for sul2 gene. We also found 22 strains (73.3%) being positive for strA-strB gene and 4 isolates (13.3%) were positive for aacC1 genes. No strain carrying aacC2, tetA and tetB genes was identified. Citrobacter freundii harbor large numbers of different antimicrobial resistance-associated genes that enable this pathogen to escape the effect of antibiotics which make this bacteria highly resistant causing urinary tract infection.

Dissemination of Escherichia coli carrying plasmid-mediated quinolone resistance (PMQR) genes from swine farms to surroundings

Different samples were collected from three swine farms in China to investigate the spread of antibiotic-resistant Escherichia coli. A total of 130 E. coli isolates were obtained from feces, air, river water, silt, and soil samples and characterized. The susceptibility of the E. coli isolates to 19 antibiotics was tested. The results revealed that the resistance rates of the E. coli isolates against 9 antibiotics were high. The minimum inhibitory concentration (MIC) values of ciprofloxacin, ofloxacin, and nalidixic acid were mainly in the ranges of 2–64, 8–64, and 8–64 μg/ml. The plasmid-mediated quinolone resistance (PMQR) genes qnr, aac(6′)-Ib-cr, qepA, and oqxAB were detected by polymerase chain reaction (PCR), and the similarity of E. coli from different samples was identified by pulsed-field gel electrophoresis (PFGE). The detection rates of the qnrA, qnrB, qnrS, aac(6′)-Ib-cr, qepA, and oqxAB genes in the E. coli isolates from three swine farms were in the range of 10.87–23.08%, 13.04–20.51%, 40.00–43.48%, 30.43–38.46%, 6.52–12.82%, and 7.69–17.39%, respectively. The PFGE result showed that 49% (49/100) of isolates originating from air, river water, soil, and silt samples had ≥85% similarity to fecal-obtained isolates, and 40.82% (20/49) of them shared the same PMQR genes with fecal-obtained isolates. This indicated that E. coli carrying PMQR genes and originating from feces in swine farms could spread to the external environment, which could be a potential threat to the public environment and human health.

Emergence of bla NDM-1 associated with the aac(6')-Ib-cr, acrB, cps, and mrkD genes in a clinical isolate of multi-drug resistant Klebsiella pneumoniae from Recife-PE, Brazil.

The emergence of New Delhi metallo-β-lactamase (NDM) is concernig because it reduces the antibiotic therapy options for bacterial infections. Resistant and virulent genes from an isolate of Klebsiella pneumoniae derived from a patient with sepsis in a hospital in Recife-PE, Brazil, were investigated using PCR and DNA sequencing. bla NDM-1, aac(6')-Ib-cr and acrB resistance genes, and cps and mrkD virulence genes were detected. To our knowledge, this is the first report on bla NDM-1 in Recife-PE. This detection alerts researchers to the need to control the spread of bla NDM-1 resistance gene by this bacterium in Brazil.

Draft genome sequence of an Escherichia coli ST410 isolate co-harbouring blaCTX-M-15, blaCMY-42, blaOXA-1, aac(3)-IIa and aac(6')-Ib-cr genes with gyrA and parC mutations isolated from a paediatric patient in Poland.

Escherichia coli is one of the major causative agents of nosocomial infections. Here we report the first draft genome sequence of an E. coli strain (no. 158) isolated in Poland carrying blaCTX-M-15, blaCMY-42, blaOXA-1, aac(3)-IIa and aac(6')-Ib-cr genes together with mutations in the gyrA and parC genes. Total DNA was sequenced using an Illumina NextSeq 500 platform. The draft genome of E. coli strain 158 was assembled using SPAdes 3.9 assembler. Contigs were annotated using the Prokka v.1.12 algorithm. Species confirmation, multilocus sequence typing (MLST), serotyping, molecular virulence and resistance traits, and plasmid replicons were analysed using appropriate bioinformatics tools available at the Centre for Genomic Epidemiology website. Additional in silico analyses were also conducted. The genome size was estimated at 4883487bp, with 4601 predicted coding sequences. The presence of blaCTX-M-15, blaCMY-42, blaOXA-1, aac(3)-IIa and aac(6')-Ib-cr genes was detected in addition to other antimicrobial resistance genes as well as mutations in the gyrA (Ser83Leu and Asp87Asn) and parC (Ser80Ile) genes. The investigated strain E. coli 158 belongs to ST410. To our knowledge, this is the first draft genome of an E. coli strain co-harbouring blaCTX-M-15, blaCMY-42, blaOXA-1, aac(3)-IIa and aac(6')-Ib-cr genes with mutations in gyrA and parC reported in Poland. The reported genome sequence contains valuable information on genetic features of antimicrobial resistance mechanisms of E. coli in Poland.

Genomic features of a multidrug-resistant Enterobacter cloacae ST279 producing CTX-M-15 and AAC(6')-Ib-cr isolated from fatal infectious stomatitis in a crossed pit viper (Bothrops alternatus).

The widespread dissemination of extended-spectrum β-lactamase-producing Enterobacteriaceae has become a major issue in veterinary medicine. However, until now, there has been no report of bacteria with such a phenotype in infected snakes. The aim of this study was to report the first draft genome sequence of an Enterobacter cloacae isolate (SERP1) recovered from a snake with infectious stomatitis. The whole genome of E. cloacae strain SERP1 was sequenced on an Illumina NextSeq platform and was de novo assembled using CLC NGS Cell v.10. Data analysis was performed using online tools from the Center of Genomic Epidemiology. The genome size was calculated at 4966856bp, containing a total of 4796 protein-coding sequences. The strain was assigned to sequence type 279 (ST279) and, besides the clinically relevant blaCTX-M-15 and aac(6')-Ib-cr genes, it also presented resistance genes to β-lactams, aminoglycosides, phenicols, sulphonamides, tetracyclines, trimethoprim, quinolones and fosfomycin. These data offer novel information regarding multidrug-resistant E. cloacae dissemination in wild animals and might contribute to further comparative genomic analysis.

Characterization of Quinolone-Resistant Determinants in Tribe Proteeae Isolated from Pet Turtles with High Prevalence of qnrD and Novel gyrB Mutations.

Development of antibiotic resistance in bacteria has challenged significantly in both veterinary and human medicine. In this study, we analyzed the potential risk of pet turtles harboring tribe Proteeae as a source of quinolone-resistant determinants, including plasmid-mediated quinolone resistance (PMQR) genes and target gene alterations in the quinolone resistance-determining region (QRDR). Antimicrobial susceptibility of 54 Proteeae isolates against ciprofloxacin, ofloxacin, levofloxacin, and nalidixic acid was examined. The PMQR genes and QRDR alterations were identified using conventional PCR assays and sequencing. Four isolates were resistant to all quinolones tested in this study. Nine isolates showed resistance to nalidixic acid and showed either intermediate resistance or susceptibility to other tested quinolones. All isolates resistant to one or more tested quinolones harbored mutations in gyrB and some also had gyrA and parC mutations. Of 54, 12 Proteeae isolates displayed the novel E466D, N440T, Q411S, and F417L mutations in gyrB. Among the PMQR genes, 41 (76%) isolates harbored the qnrD gene with the highest prevalence, whereas aac(6')Ib-cr, qnrS, qnrA, and qnrB genes were detected in 28 (52%), 9 (17.0%), 7 (13.0%), and 1 (1.9%) study isolates, respectively. The QRDR analysis of selected mutants revealed that increasing quinolone selective pressure led to a predominance of gyrA mutants. All results indicate that a healthy pet turtle can play as a potential reservoir for quinolone-resistant Proteeae, which it might cause public health risk on pet owners.

First report of blaOXA-24 carbapenemase gene, armA methyltransferase and aac(6′)-Ib-cr among multidrug-resistant clinical isolates of Proteus mirabilis in Algeria

Carbapenemase-producing, or carbapenem-resistant, Enterobacteriaceae are an emerging threat to human and animal health because they are resistant to many of the last-line antimicrobials available for treatment of infection. The aim of this study was to analyse the antimicrobial resistance patterns and their encoding genes of Proteus mirabilis isolated in Constantine, Algeria. A total of 108 Proteus, Morganella and Providencia (PMP) strains were isolated from a large variety of clinical specimens at University Hospital of Constantine in Algeria. Isolates were identified using the API 20E system and matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF/MS). Diagnostic accuracy was determined by independent comparison of each method to phylogenetic analysis based on 16S rRNA gene sequencing. Antimicrobial susceptibility was determined by the standard disk diffusion and Etest methods. The presence of antimicrobial resistance genes was screened for by PCR amplification and sequencing. A total of 72 PMP strains were multidrug-resistant (MDR). Among them, one P. mirabilis isolate was resistant to imipenem with a minimum inhibitory concentration (MIC) of ≥12μg/mL. PCR and sequencing showed the presence of various antimicrobial resistance genes, including blaCTX-M-15, blaTEM-1, blaTEM-2, blaPER-1, blaSHV-11, aadA1, aadA2, armA, aac(6')-Ib, aac(6')-Ib-cr, aac(3)-Ia and ant(2″)-I, forming different resistance profiles. Moreover, the blaOXA-24 gene was detected in the imipenem-resistant P. mirabilis strain. In this study, a MDR P. mirabilis isolate harbouring the blaOXA-24, armA 16S rRNA methylase and aac(6)-Ib-cr genes was found for the first time in Algeria.

Fluoroquinolone-resistant Achromobacter xylosoxidans clinical isolates from Serbia: high prevalence of the aac-(6')-Ib-cr gene among resistant isolates.

The aim of this study was to evaluate the contribution of plasmid-mediated genes and efflux to fluoroquinolone resistance in collection of Achromobacter spp. gathered during a 3-year period. Susceptibility to ciprofloxacin and levofloxacin was tested by disk diffusion and microdilution tests for a collection of 98 Achromobacter spp. clinical isolates. Identification of fluoroquinolone-resistant isolates was performed by sequencing and phylogenetic analyses of the nrdA gene. Genetic relatedness among resistant isolates was determined by pulsed-field gel electrophoresis (PFGE) analysis. The influence of an H+ conductor cyanide m-chlorophenyl hydrazone (CCCP) and a resistance-nodulation-division-type efflux pump inhibitor phenylalanine-arginine beta-naphthylamide (PAβN) on minimal inhibitory concentration (MIC) value was evaluated by broth microdilution. The presence of the plasmid-mediated qnrA, qnrB, qnrC, qnrS, and aac-(6')-Ib-cr genes was investigated by PCR and sequencing. Achromobacter spp. isolates that were resistant or intermediately resistant to fluoroquinolones in disk diffusion tests (44/98) were subjected to microdilution. As a result, 20/98 isolates were confirmed to be resistant to ciprofloxacin while 10/98 was resistant to levofloxacin. CCCP decreased twofold MIC value for ciprofloxacin in six isolates and more than 16 times in one isolate, while MIC value for levofloxacin was decreased in all isolates (twofold to more than eightfold). Fluoroquinolone-resistant isolates were identified as A. xylosoxidans with the nrdA gene sequencing. PFGE revealed that resistant isolates belonged to seven different genotypes. Ten isolates belonging to four genotypes were positive for the aac-(6')-Ib-cr gene. Although resistance to fluoroquinolones was not widespread among analyzed isolates, detected contribution of efflux pumps and the presence of the aac-(6')-Ib-cr gene present a platform for emergence of more resistant strains.

PREVALENCE AND CHARACTERIZATION OF QUINOLONE RESISTANCE GENES IN PROTEUS SPECIES ISOLATED FROM PET TURTLES

Proteus spp. are widely recognized as opportunistic pathogens causing urinary tract and septic infections in humans and animals. The aim of this study was to investigate the prevalence of plasmid-mediated quinolone resistance genes and mutations in the quinolone resistance determining region in association with the detection of quinolone susceptibility of 24 strains of pet turtle-borne Proteus spp. Susceptibility of 4 antimicrobials including nalidixic acid, ciprofloxacin, ofloxacin, and levofloxacin was examined by disk diffusion and minimum inhibitory concentration test. Six isolates were resistant to nalidixic acid showing either intermediate resistance or resistance to other quinolones. All nalidixic acid, resistant isolates harbored mutations in gyrB (N440T/A401G/Q411S). Two of the isolates had both gyrA (S83I) and parC (S80I) mutations. Twenty-one isolates were positive for the presence of plasmid-mediated quinolone resistance genes; the qnrD gene had the highest prevalence with 19 (79.2%), while qnrS, qnrA, qnrB, and aac(6′), Ib-cr genes were present in 9 (37.5%), 2 (8.3%), 1 (4.2%), and 11 (45.8%) isolates, respectively. These results suggest that pet turtle-associated Proteus spp. should be considered a potential source of antimicrobial resistance determinants.