Quinolone Resistance Genes qnrS Research Articles

Analysis of Antimicrobial Resistance in Bacterial Pathogens Recovered from Food and Human Sources: Insights from 639,087 Bacterial Whole-Genome Sequences in the NCBI Pathogen Detection Database.

Understanding the role of foods in the emergence and spread of antimicrobial resistance necessitates the initial documentation of antibiotic resistance genes within bacterial species found in foods. Here, the NCBI Pathogen Detection database was used to query antimicrobial resistance gene prevalence in foodborne and human clinical bacterial isolates. Of the 1,843,630 sequence entries, 639,087 (34.7%) were assigned to foodborne or human clinical sources with 147,788 (23.14%) from food and 427,614 (76.88%) from humans. The majority of foodborne isolates were either Salmonella (47.88%), Campylobacter (23.03%), Escherichia (11.79%), or Listeria (11.3%), and the remaining 6% belonged to 20 other genera. Most foodborne isolates were from meat/poultry (95,251 or 64.45%), followed by multi-product mixed food sources (29,892 or 20.23%) and fish/seafood (6503 or 4.4%); however, the most prominent isolation source varied depending on the genus/species. Resistance gene carriage also varied depending on isolation source and genus/species. Of note, Klebsiella pneumoniae and Enterobacter spp. carried larger proportions of the quinolone resistance gene qnrS and some clinically relevant beta-lactam resistance genes in comparison to Salmonella and Escherichia coli. The prevalence of mec in S. aureus did not significantly differ between meat/poultry and multi-product sources relative to clinical sources, whereas this resistance was rare in isolates from dairy sources. The proportion of biocide resistance in Bacillus and Escherichia was significantly higher in clinical isolates compared to many foodborne sources but significantly lower in clinical Listeria compared to foodborne Listeria. This work exposes the gaps in current publicly available sequence data repositories, which are largely composed of clinical isolates and are biased towards specific highly abundant pathogenic species. We also highlight the importance of requiring and curating metadata on sequence submission to not only ensure correct information and data interpretation but also foster efficient analysis, sharing, and collaboration. To effectively monitor resistance carriage in food production, additional work on sequencing and characterizing AMR carriage in common commensal foodborne bacteria is critical.

Phenotypic and Genotypic Characterization of a Hypervirulent Carbapenem-Resistant Klebsiella pneumoniae ST17-KL38 Clinical Isolate Harboring the Carbapenemase IMP-4.

ABSTRACTCarbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP) is a threat to global public health. We characterized a sequence type 17 (ST17) K. pneumoniae clinical isolate that was resistant to carbapenems and belonged to serotype KL38/O2. Its complete genome is comprised of a 5.1-Mb chromosome and two conjugative plasmids. The 52,578-bp N-type plasmid pXH210-IMP contains the blaIMP-4 carbapenemase gene and the quinolone resistance gene qnrS1. The 272,742-bp FII(K)-9:FIB(K)-10 plasmid pXH210-AMV carries an array of genes that confer resistance to aminoglycosides, chloramphenicol, quinolones, tetracycline, sulfonamides, trimethoprim, arsenic, copper, and silver. However, the XH210 genome otherwise lacks the genes that are considered characteristic markers of hypervirulence in K. pneumoniae. The virulence potential of XH210 was assessed using a random forest algorithm predictive model, as well as Galleria mellonella and mouse infection models. The results of these were concordant and suggested that XH210 is hypervirulent and therefore a CR-hvKP strain. This worrying convergence of virulence and clinically significant antibiotic resistance is particularly concerning given the absence of typical hypervirulence markers. Further investigations are required to understand the virulence mechanisms of XH210 and to improve the diagnostics of hypervirulent K. pneumoniae.IMPORTANCE The combination of drug resistance and hypervirulence significantly limits the available treatment options for life-threatening infections caused by multidrug-resistant hvKP, especially CR-hvKP. To date, research on IMP-producing CR-hvKP is extremely scarce, and the virulence mechanisms of CR-hvKP are far more complicated and diverse than has been described in the literature so far. In this study, we characterized the tigecycline-resistant and IMP-4 carbapenemase-producing ST17 K. pneumoniae isolate XH210 from a human blood sample. Importantly, XH210 exhibits hypervirulence but does not possess traits that are frequently associated with the phenotype, highlighting the urgent need to improve identification of potentially hypervirulent isolates and enhance active surveillance of CR-hvKP strains to prevent their dissemination.

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.

Characterization of a carbapenem-resistant Citrobacter amalonaticus coharbouring bla IMP-4 and qnrs1 genes

Introduction. Members of the genus Citrobacter are facultative anaerobic Gram-negative bacilli belonging to the Enterobacterales [Janda J Clin Microbiol 1994; 32(8):1850-1854; Arens Clin Microbiol Infect 1997;3(1):53-57]. Formerly, Citrobacter species were occasionally reported as nosocomial pathogens with low virulence [Pepperell Antimicrob Agents Chemother 2002;46(11):3555-60]. Now, they are consistently reported to cause nosocomial infections of the urinary tract, respiratory tract, bone, peritoneum, endocardium, meninges, intestines, bloodstream and central nervous system. Among Citrobacter species, the most common isolates are C. koseri and C. freundii, while C. amalonaticus has seldom been isolated [Janda J Clin Microbiol 1994; 32(8):1850-1854; Marak Infect Dis (Lond) 2017;49(7):532-9]. Further, Citrobacter spp. are usually susceptible to carbapenems, aminoglycosides, tetracyclines and colistin [Marak Infect Dis (Lond) 2017;49(7):532-9].Hypothesis/Gap Statement. As C. amalonaticus is rare, only one clinical isolate, coharbouring carbapenem resistance gene bla IMP-4 and quinolone resistance gene qnrs1, has been reported.Aim. To characterize a carbapenem-resistant C. amalonaticus strain from PR China coharbouring bla IMP-4 and qnrs1.Methodology. Three hundred and forty nonrepetitive carbapenem-resistant Enterobacterales (CRE) strains were collected during 2011-2018. A carbapenem-resistant C. amalonaticus strain was detected and confirmed using a VITEK mass spectrometry-based microbial identification system and 16S rRNA sequencing. Minimum inhibitory concentrations (MICs) for clinical antimicrobials were obtained by the broth microdilution method. Whole-genome sequencing (WGS) was performed for antibiotic resistance gene analysis, and a phylogenetic tree of C. amalonaticus strains was constructed using the Bacterial Pan Genome Analysis (BPGA) tool. The transferability of the resistance plasmid was verified by conjugal transfer.Results. A rare carbapenem-resistant C. amalonaticus strain (CA71) was recovered from a patient with cerebral obstruction and the sequences of 16S rRNA gene shared more than 99 % similarity with C. amalonaticus CITRO86, FDAARGOS 165. CA71 is resistant to β-lactam, quinolone and aminoglycoside antibiotics, and even imipenem and meropenem (MICs of 2 and 4 mg l-1 respectively), and is only sensitive to polymyxin B and tigecycline. Six antibiotic resistance genes were detected via WGS, including the β-lactam genes bla IMP-4, bla CTX-M-18 and bla Sed1, the quinolone gene qnrs1, and the aminoglycoside genes AAC(3)-VIIIa, AadA24. Interestingly, bla IMP-4 and qnrs1 coexist on an IncN1-type plasmid (pCA71-IMP) and successfully transferred to Escherichia coli J53 via conjugal transfer. Phylogenetic analysis showed that CA71 is most similar to C. amalonaticus strain CJ25 and belongs to the same evolutionary cluster along with seven other strains.Conclusion. To the best of our knowledge, this is the first report of a carbapenem-resistant C. amalonaticus isolate coharbouring bla IMP-4 and qnrs1.

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.

Antimicrobial resistance patterns among different Escherichia coli isolates in the Kingdom of Saudi Arabia.

Antimicrobial resistance patterns among different Escherichia coli isolates in the Kingdom of Saudi Arabia. This study aimed to investigate the patterns of antimicrobial resistance in E. coli isolated from different samples, and to identify potential pathogenic isolates in Riyadh, Kingdom of Saudi Arabia (KSA). In total, 51 bacterial isolates were recovered from 113 samples of human urine, food (raw meat, raw chicken, raw egg surface, and fresh vegetables), water, and air. Twenty-four E. coli isolates were tested for susceptibility to 26 antibiotics. The air sample isolates were most resistant to amoxicillin, ampicillin, amoxicillin/clavulanic acid, amoxicillin/sulbactam, piperacillin/tazobactam, cefalotin, cefuroxime, cefoxitin, cefixime, nitrofurantoin, and trimethoprim/sulfamethoxazol. The isolates from vegetable samples were resistant to amoxicillin, ampicillin, amoxicillin/clavulanic acid, amoxicillin/sulbactam, cefalotin, cefuroxime, cefoxitin, and cefixime. By contrast, the isolates from the water samples were resistant only to amoxicillin and ampicillin. The isolates from the human urine samples were most frequently resistant to norfloxacin (80%) followed by amoxicillin and ampicillin (70%), trimethoprim/sulfamethoxazole (55%), ciprofloxacin and ofloxacin (50%), cefalotin (30%), cefuroxime, cefixime and cefotaxime (25%), ceftazidime, ceftriaxone, cefepime and aztreonam (20%), amoxicillin/clavulanic acid, piperacillin/tazobactam and gentamicin (10%), and amoxicillin/sulbactam and cefoxitin (5%). Almost all (23/25, 95.8%) (n = 23) of the isolates were multi-drug resistant (MDR) (i.e., resistant to 3 or more classes of antibiotics), and 16.7% (n = 4) of those were positive for extended spectrum β-lactamase (ESBL). Of the 4 ESBL-producers, 3 were positive for blaCTX-M-15 and blaCTX-M1group, 2 were positive for blaCMY-2, and 1 each was positive for blaCTX-M-2 group, blaSHV, and blaOXA-47. The quinolone resistance gene qnrS was detected in 25% (n = 6) of the E. coli strains isolated from urine (N = 5) and air (N = 1) samples. The considerable number of antimicrobial resistance genes detected among E. coli isolates tested here is alarming and should raise public health concern.

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

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

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.

Characterization of an IMP-4-Producing Klebsiella pneumoniae ST1873 Strain Recovered from an Infant with a Bloodstream Infection in China.

PurposeImipenemase (IMP), an Ambler class B metallo-β-lactamase, is an important carbapenemase that confers resistance to almost all β-lactams. In this study, we characterized the genomic feature of an IMP-4-producing Klebsiella pneumoniae ST1873 strain, a rare sequence type (ST) isolated from an infant with a bloodstream infection in China.Patients and MethodsK. pneumoniae strain, BKP19, was collected from a bloodstream infection in an infant who was hospitalized at the department of paediatrics. The whole genome sequence of the strain was sequenced using the Illumina NovaSeq 6000 platform and long-read MinION sequencer. Multilocus sequence typing, antimicrobial resistance gene identification, plasmid and phylogenetic relationship analysis of the strain were analysed by various bioinformatics approaches.ResultsK. pneumoniae BKP19 was resistant to multiple antimicrobials, including carbapenems. Eleven antimicrobial resistance genes corresponding to beta-lactam resistance, quinolone resistance, phenicol resistance and fosfomycin resistance could be identified in the genome. The carbapenem resistance gene blaIMP-4 was located in an IS26-associated class 1 integron of an IncN-type plasmid with 39,033 bp (pIMP-4-BKP19). Sequence alignment revealed that pIMP-4-BKP19 is closely related to the common plasmid carrying IMP-4 in K. pneumoniae (pIMP-HZ1-like plasmid) but is smaller, lacking the quinolone resistance gene qnrS1 and multiple tra gene orthologs. Conjugation experiment revealed that pIMP-4-BKP19 is a non-conjugative plasmid. According to in silico MLST analysis, K. pneumoniae strain BKP19 belongs to a sporadic clone ST1873.ConclusionIn summary, our study reports the first genome sequence of a K. pneumoniae ST1873 strain harbouring the class B β-lactamase blaIMP-4 in an IncN-type plasmid recovered from an infant with a bloodstream infection in China. Considering the global emergence of IMP-4 carbapenemase, more attention must be paid to prevent its future prevalence.

Whole genome sequencing snapshot of multi-drug resistant Klebsiella pneumoniae strains from hospitals and receiving wastewater treatment plants in Southern Romania.

We report on the genomic characterization of 47 multi-drug resistant, carbapenem resistant and ESBL-producing K. pneumoniae isolates from the influent (I) and effluent (E) of three wastewater treatment plants (WWTPs) and from Romanian hospital units which are discharging the wastewater in the sampled WWTPs. The K. pneumoniae whole genome sequences were analyzed for antibiotic resistance genes (ARGs), virulence genes and sequence types (STs) in order to compare their distribution in C, I and E samples. Both clinical and environmental samples harbored prevalent and widely distributed ESBL genes, i.e. blaSHV, blaOXA, blaTEM and blaCTX M. The most prevalent carbapenemase genes were blaNDM-1, blaOXA-48 and blaKPC-2. They were found in all types of isolates, while blaOXA-162, a rare blaOXA-48 variant, was found exclusively in water samples. A higher diversity of carbapenemases genes was seen in wastewater isolates. The aminoglycoside modifying enzymes (AME) genes found in all types of samples were aac(6’), ant(2'')Ia, aph(3'), aaD, aac(3) and aph(6). Quinolone resistance gene qnrS1 and the multi-drug resistance oqxA/B pump gene were found in all samples, while qnrD and qnrB were associated to aquatic isolates. The antiseptics resistance gene qacEdelta1 was found in all samples, while qacE was detected exclusively in the clinical ones. Trimethroprim-sulfamethoxazole (dfrA, sul1 and sul2), tetracyclines (tetA and tetD) and fosfomycin (fosA6, known to be located on a transpozon) resistance genes were found in all samples, while for choramphenicol and macrolides some ARGs were detected in all samples (catA1 and catB3 / mphA), while other (catA2, cmIA5 and aac(6’)Ib / mphE and msrE) only in wastewater samples. The rifampin resistance genes arr2 and 3 (both carried by class I integrons) were detected only in water samples. The highly prevalent ARGs preferentially associating with aquatic versus clinical samples could ascribe potential markers for the aquatic (blaSHV-145, qacEdelta1, sul1, aadA1, aadA2) and clinical (blaOXA-1, blaSHV-106,blaTEM-150, aac(3)Iia, dfrA14, oqxA10; oqxB17,catB3, tetD) reservoirs of AR. Moreover, some ARGs (oqxA10; blaSHV-145; blaSHV-100, aac(6')Il, aph(3')VI, armA, arr2, cmlA5, blaCMY-4, mphE, msrE, oqxB13, blaOXA-10) showing decreased prevalence in influent versus effluent wastewater samples could be used as markers for the efficiency of the WWTPs in eliminating AR bacteria and ARGs. The highest number of virulence genes (75) was recorded for the I samples, while for E and C samples it was reduced to half. The most prevalent belong to three functional groups: adherence (fim genes), iron acquisition (ent, fep, fyu, irp and ybt genes) and the secretion system (omp genes). However, none of the genes associated with hypervirulent K. pneumoniae have been found. A total of 14 STs were identified. The most prevalent clones were ST101, ST219 in clinical samples and ST258, ST395 in aquatic isolates. These STs were also the most frequently associated with integrons. ST45 and ST485 were exclusively associated with I samples, ST11, ST35, ST364 with E and ST1564 with C samples. The less frequent ST17 and ST307 aquatic isolates harbored blaOXA-162, which was co-expressed in our strains with blaCTX-M-15 and blaOXA-1.

Genomic Characterization of Antibiotic Resistant Escherichia coli Isolated From Domestic Chickens in Pakistan.

Poultry husbandry is important for the economic health of Pakistan, but the Pakistani poultry industry is negatively impacted by infections from Escherichia coli. We performed Illumina whole genome sequencing on 92 E. coli isolates obtained from the livers of deceased chickens originating in five Pakistani geographical regions. Our analysis indicates that the isolates are predominantly from the B1 and A clade and harbor a diverse number of antibiotic resistance and virulence genes, with no linkage between phylogeny and antibiotic resistance gene presence but some association between phylogeny and virulence gene and SNP presence for the B1 and E phylogroups. The colistin resistance gene mcr-1 and the quinolone resistance gene qnrS1 were both found in 13/92 isolates. Alarmingly, 82/92 of the E. coli strains characterized in this study are multidrug resistant with 100% (92/92) resistance to lincomycin, 81.5% (75/92) to streptomycin, 79.3% (73/92) to ampicillin and 66.3% (61/92) to ciprofloxacin. These results provide a high-resolution analysis of poultry-associated E. coli isolates in an area with a high endemic burden of antibiotic resistance. Surveillance of antibiotic resistance in poultry associated E. coli isolates is an important pillar of the One Health concept to integrate analysis of potential pathogens in human, animal, and environmental niches.

Early emergence of OXA-181-producing Escherichia coli ST410 in China

The aim of this study was to investigate the prevalence and characterisation of OXA-48-like-producing Enterobacteriaceae in Henan Province, China. A total of 339 carbapenem-non-susceptible clinical Enterobacteriaceae isolates [imipenem or meropenem minimum inhibitory concentration (MIC) of >1μg/mL] obtained between January 2013 and December 2016 were screened for the presence of the blaOXA-48-like gene by PCR and sequencing. Antimicrobial susceptibility to various antimicrobials agents was examined by MIC testing. Multilocus sequence typing (MLST) was performed for bacterial genotyping. The plasmid pEC21-OXA-181 was transformed into Escherichia coli DH5α by electroporation and was sequenced using an Illumina MiSeq platform, followed by subsequent annotation and genetic analysis. Among the 339 carbapenem-non-susceptible Enterobacteriaceae isolates, only one (0.3%) E. coli strain EC21, belonging to ST410, was positive for blaOXA-181, a variant of blaOXA-48. This OXA-181-producing E. coli, recovered from a patient without a history of foreign travel, was obtained earlier than the first reported blaOXA-181-positive E. coli (WCHEC14828) in Sichuan Province, China. Plasmid analysis revealed that blaOXA-181 together with the quinolone resistance gene qnrS1 was carried by an IS26-flanked composite transposon on a 51-kb IncX3-type plasmid. These findings indicate the emergence of OXA-181-producing E. coli in China earlier than previously thought. The blaOXA-181 gene is associated with the widely disseminated potentially endemic E. coli ST410 clone and is carried by an IncX3 plasmid, a common vehicle for spreading NDM-type carbapenemases, which might promote the further dissemination of blaOXA-181 among the Enterobacteriaceae in China.

Occurrence and characterisation of ESBL-encoding plasmids among Escherichia coli isolates from fresh vegetables

Extended-spectrum β-lactamase (ESBL)-producing Escherichia coli isolates have been increasingly reported in different reservoirs. The aims of this study were to investigate the presence of ESBL-producing E. coli in fresh vegetables and to characterise their ESBL gene-carrying plasmids. Among the 245 samples from vegetables investigated during 2011–2013, seven putative ESBL-producing E. coli (salad n = 2, sprouts n = 5) were found. They were subjected to ESBL phenotypic confirmatory tests, detection/sequencing of ESBL genes, antimicrobial susceptibility testing (AST), phylotyping, XbaI-macrorestriction analysis, multilocus sequence typing and transformation. Transformants were characterised by AST, S1-nuclease PFGE, replicon typing, conjugation and investigated for co-located antimicrobial resistance genes. Two ESBL gene-carrying plasmids were sequenced using a HiSeq 2500 system. The seven isolates were confirmed as ESBL producers, displayed unrelated XbaI-patterns and unique sequence types (STs) and belonged to the phylogroups A, B1 or D. The ESBL genes were located on plasmids. Two plasmids carrying blaCTX-M-14 genes (incompatibility group IncK or IncHI2) were seen in isolates from salad (ST973) and sprout (ST527). Two blaCTX-M-15- (IncFIB; non-typeable) and the IncN blaCTX-M-65- and IncHI2 blaCTX-M-125-carrying plasmids were found in isolates from sprouts (ST410, ST847, ST10, ST542). All plasmids were conjugative, except for the IncFIA-FIB blaCTX-M-2-carrying plasmid. Sequence analysis of two plasmids identified the ESBL genes in close location to other resistance genes: sulfonamide resistance gene sul2, streptomycin resistance genes strA and strB, the plasmid-mediated quinolone resistance gene qnrS1 and blaTEM-1 (sul2-strA-strB-IS66-blaTEM-1-tnpR-ΔtnpA-ISEcp1-blaCTX-M-15-Δorf477-ΔtnpA-qnrS1) or the fosfomycin resistance gene fosA3 (ΔISEcp1-blaCTX-M-125-ΔIS903B-fosA3). These observations underline the importance of vegetables as reservoirs for multidrug resistant ESBL-producing E. coli.

Distribution of extended-spectrum β-lactam, quinolone and carbapenem resistance genes, and genetic diversity among uropathogenic Escherichia coli isolates in Tehran, Iran.

Antimicrobial resistance among uropathogenic Escherichia coli (UPEC) is increasing. The aim of this study was to evaluate antimicrobial resistance in UPEC isolates and its correlation with virulence factors. A total of 120 E. coli isolates were collected from patients with urinary tract infection in Tehran, Iran. Biofilm formation and antimicrobial susceptibility were determined by phenotypic tests. The combination disk diffusion test and modified Hodge test (MHT) were performed for phenotypic detection of extended-spectrum β-lactamases (ESBLs) and carbapenemases, respectively. PCR was used for the detection of virulence genes, ESBL-encoding genes, and quinolone (qnr) and carbapenem resistance genes. Pulsed-field gel electrophoresis (PFGE) was performed to explore the genetic relatedness among isolates. Most isolates exhibited biofilm formation, and different frequencies of virulence genes were observed. There was a high rate of resistance, especially multidrug resistance, to most of the antimicrobial agents tested. Phenotypically, 67.5% of the isolates produced an ESBL and were resistant to different antimicrobial classes. In total, 83.3% of the isolates harboured ESBL genes, especially blaTEM and blaCTX-M, and 32.5% were positive for the quinolone resistance genes qnrS and qnrB. All isolates were negative for carbapenemases by the MHT and PCR. These results indicate the association between the presence of various virulence genes and antimicrobial susceptibility. PFGE analysis showed that there was great clonal diversity among the selected isolates, with 17 isolates divided into five clusters. These results indicate that the high antimicrobial resistance among UPEC isolates is alarming and requires urgent attention.

Plasmid-Mediated Quinolone Resistance in Shigella flexneri Isolated From Macaques.

Non-human primates (NHPs) for biomedical research are commonly infected with Shigella spp. that can cause acute dysentery or chronic episodic diarrhea. These animals are often prophylactically and clinically treated with quinolone antibiotics to eradicate these possible infections. However, chromosomally- and plasmid-mediated antibiotic resistance has become an emerging concern for species in the family Enterobacteriaceae. In this study, five individual isolates of multi-drug resistant Shigella flexneri were isolated from the feces of three macaques. Antibiotic susceptibility testing confirmed resistance or decreased susceptibility to ampicillin, amoxicillin-clavulanic acid, cephalosporins, gentamicin, tetracycline, ciprofloxacin, enrofloxacin, levofloxacin, and nalidixic acid. S. flexneri isolates were susceptible to trimethoprim-sulfamethoxazole, and this drug was used to eradicate infection in two of the macaques. Plasmid DNA from all isolates was positive for the plasmid-encoded quinolone resistance gene qnrS, but not qnrA and qnrB. Conjugation and transformation of plasmid DNA from several S. flexneri isolates into antibiotic-susceptible Escherichia coli strains conferred the recipients with resistance or decreased susceptibility to quinolones and beta-lactams. Genome sequencing of two representative S. flexneri isolates identified the qnrS gene on a plasmid-like contig. These contigs showed >99% homology to plasmid sequences previously characterized from quinolone-resistant Shigella flexneri 2a and Salmonella enterica strains. Other antibiotic resistance genes and virulence factor genes were also identified in chromosome and plasmid sequences in these genomes. The findings from this study indicate macaques harbor pathogenic S. flexneri strains with chromosomally- and plasmid-encoded antibiotic resistance genes. To our knowledge, this is the first report of plasmid-mediated quinolone resistance in S. flexneri isolated from NHPs and warrants isolation and antibiotic testing of enteric pathogens before treating macaques with quinolones prophylactically or therapeutically.

Dissemination of IMP-4-encoding pIMP-HZ1-related plasmids among Klebsiella pneumoniae and Pseudomonas aeruginosa in a Chinese teaching hospital.

A total of 26 blaIMP-4-carrying strains of Pseudomonas aeruginosa and Klebsiella pneumoniae were isolated from 2009 to 2013 in a Chinese teaching hospital, and these strains can be assigned into multiple sequence types or allelic profiles as determined by multilocus sequence typing. Of these strains, P. aeruginosa P378 and K. pneumoniae 1220 harbor the IMP-4-encoding plasmids pP378-IMP and p1220-IMP, respectively, whose complete nucleotide sequences are determined to be genetically closely related to the IncN1-type plasmid pIMP-HZ1. pP378-IMP/p1220-IMP-like plasmids are hinted to be present in all the other blaIMP-4-carrying strains, indicating the dissemination of pIMP-HZ1-related plasmids among K. pneumoniae or P. aeruginosa of different genotypes in this hospital. pP378-IMP carries two distinct accessory resistance regions, a blaIMP-4-carrying class 1 integron In823b, and a truncated Tn3-family unit transposon ΔTn6292-3′ harboring the quinolone resistance gene qnrS1. Massive fragmentation and rearrangement of these accessory genetic contents occur among p1220-IMP and IMP-HZ1 relative to pP378-IMP. blaIMP-4 is also present in the In823b remnants from p1220-IMP and IMP-HZ1, while qnrS1 is located in a Tn6292-derive fragment from pIMP-HZ1 but not found in p1220-IMP. pP378-IMP represents the first fully sequenced IncN-type plasmid from P. aeruginosa.

Dissemination of the transmissible quinolone-resistance gene qnrS1 by IncX plasmids in Nigeria.

The plasmid-encoded quinolone resistance gene qnrS1 was recently found to be commonly associated with ciprofloxacin resistance in Nigeria. We mapped the qnrS1 gene from an Escherichia coli isolate obtained in Nigeria to a 43.5 Kb IncX2 plasmid. The plasmid, pEBG1, was sufficient to confer ciprofloxacin non-susceptibility, as well as tetracycline and trimethoprim resistance, on E. coli K-12. Deletion analysis confirmed that qnrS1 accounted for all the ciprofloxacin non-suceptibility conferred by pEBG1 and tetracycline and trimethoprim resistance could be attributed to tetAR and dfrA14 genes respectively. While it contained a complete IncX conjugation system, pEBG1 was not self-transmissible likely due to an IS3 element inserted between the pilX5 and pilX6 genes. The plasmid was however efficiently mobilizable. pEBG1 was most similar to another qnrS1-bearing IncX2 plasmid from Nigeria, but both plasmids acquired qnrS1 independently and differ in their content of other resistance genes. Screening qnrS1–positive isolates from other individuals in Nigeria revealed that they carried neither pEBG1 nor pNGX2-QnrS1 but that IncX plasmids were prevalent. This study demonstrates that the IncX backbone is a flexible platform that has contributed to qnrS1 dissemination in Nigeria.

Prevalence and characterization of Salmonella enterica in dried milk-related infant foods in Shaanxi, China

The aim of this study was to investigate the existence and characteristics of Salmonella enterica in dried milk-related infant foods. Twenty-four (3.4%) of 705 samples, including 5 (2.0%) of 246 powdered infant formula, 18 (4.0%) of 445 infant rice cereal, and 1 (7.1%) of 14 other infant foods, were positive for Salmonella. Fifteen serotypes were identified in 40 Salmonella isolates; Salmonella Duesseldorf (15.0%) and Salmonella Indiana (15.0%) were more frequently detected than other serotypes. Resistance to chloramphenicol (82.5%) was most common, followed by tetracycline (57.5%), ceftiofur (52.5%), kanamycin (52.5%), streptomycin (50.0%), gentamycin (45.0%), nalidixic acid (35.0%), ceftriaxone (32.5%), ciprofloxacin (25.0%), amikacin (20.0%), and cefoxitin (15.0%). Twenty-eight (70.0%) isolates were resistant to ≥8 antimicrobials, with 5 (12.5%) being resistant to 14 antimicrobials. Amino acid substitutions in gyrase A (GyrA) were most frequently detected as Ser83Arg/Asp87Glu and in p53-associated Parkin-like cytoplasmic protein (ParC), they were all Ser80Arg; the quinolone resistance gene qnrS (47.5%) was commonly detected as well as aminoglycoside acetyltransferase [aac(6′)-Ib; 25.0%], qnrA (17.5%), and qnrB (15.0%) genes. Thirty distinct pulsed-field gel electrophoresis patterns were identified among 40 isolates; no identical pulsed-field gel electrophoresis pattern was detected among Salmonella isolates with the same serovar that was recovered in 2010 and 2012. Our results suggest that dried milk-related infant foods could be contaminated with Salmonella and highlight that the dangers to infant health should not be neglected.

Complete sequences of IncHI1 plasmids carrying blaCTX-M-1 and qnrS1 in equine Escherichia coli provide new insights into plasmid evolution.

To determine the structure of two multidrug-resistant IncHI1 plasmids carrying blaCTX-M-1 in Escherichia coli isolates disseminated in an equine clinic in the Czech Republic. A complete nucleotide sequencing of 239 kb IncHI1 (pEQ1) and 287 kb IncHI1/X1 (pEQ2) plasmids was performed using the 454-Genome Sequencer FLXsystem. The sequences were compared using bioinformatic tools with other sequenced IncHI1 plasmids. A comparative analysis of pEQ1 and pEQ2 identified high nucleotide identity with the IncHI1 type 2 plasmids. A novel 24 kb module containing an operon involved in short-chain fructooligosaccharide uptake and metabolism was found in the pEQ backbones. The role of the pEQ plasmids in the metabolism of short-chain fructooligosaccharides was demonstrated by studying the growth of E. coli cells in the presence of these sugars. The module containing the blaCTX-M-1 gene was formed by a truncated macrolide resistance cluster and flanked by IS26 as previously observed in IncI1 and IncN plasmids. The IncHI1 plasmid changed size and gained the quinolone resistance gene qnrS1 as a result of IS26-mediated fusion with an IncX1 plasmid. Our data highlight the structure and evolution of IncHI1 from equine E. coli. A plasmid-mediated sugar metabolic element could play a key role in strain fitness, contributing to the successful dissemination and maintenance of these plasmids in the intestinal microflora of horses.

Complete Sequence of a KPC-Producing IncN Multidrug-Resistant Plasmid from an Epidemic Escherichia coli Sequence Type 131 Strain in China

We report here the nucleotide sequence of a novel blaKPC-2-harboring incompatibility group N (IncN) plasmid, pECN580, from a multidrug-resistant Escherichia coli sequence type 131 (ST131) isolate recovered from Beijing, China. pECN580 harbors β-lactam resistance genes blaKPC-2, blaCTX-M-3, and blaTEM-1; aminoglycoside acetyltransferase gene aac(6')-Ib-cr; quinolone resistance gene qnrS1; rifampin resistance gene arr-3; and trimethoprim resistance gene dfrA14. The emergence of a blaKPC-2-harboring multidrug-resistant plasmid in an epidemic E. coli ST131 clone poses a significant potential threat in community and hospital settings.