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96400.9936Distribution of plasmid-mediated quinolone resistance in Gram-negative bacteria from a tertiary hospital in Nigeria. BACKGROUND: Until recently, mechanisms of resistance to quinolones in Gram-negative bacteria were believed to be only chromosome encoded. However, emergence of plasmid-mediated quinolone resistance (PMQR) has been reported worldwide. AIM: This study investigated distribution of PMQR in Gram-negative bacteria from a tertiary hospital in eastern part of Nigeria. MATERIALS AND METHODS: Seventy-one nonduplicate Gram-negative bacterial isolates of eight species were analyzed for antimicrobial susceptibility, genotypic detection of various PMQRs, typed by random amplified polymorphic DNA (RAPD) and analysis of plasmids present, including replicon typing. RESULTS: The minimum inhibitory concentrations showed MIC90values as high as 256 μg/ml for fluoroquinolones. Carriage of PMQR was found to be 35.2%. Twenty (28.2%) isolates carried various qnr genes, of which seven (9.9%) qnrA1; four (5.6%) qnrB1; eight (11.3%) qnrS1 while one (1.4%) encoded qnrD1. Eighteen (25.4%) isolates were positive for aac(6')-Ib-cr while carriage of multiple genes exists in some strains. Similarly, 13 isolates (18.7%) were found to carry PMQR efflux pump gene, qepA. Conjugation experiments revealed that the plasmids once transferred coded for fluoroquinolone resistance. The transconjugant strains carried a common plasmid estimated to be 65 kb. These plasmids were untypable for replicon/incompatibility. Typing revealed high diversity among all species tested with no identical RAPD pattern seen. CONCLUSION: This study further confirms high level resistance to many antimicrobials in different species of Gram-negative bacteria including fluoroquinolones and spread of PMQR genes in Southern Nigeria.201627510669
244810.9933Emerging coexistence of three PMQR genes on a multiple resistance plasmid with a new surrounding genetic structure of qnrS2 in E. coli in China. BACKGROUND: Quinolones are commonly used for treatment of infections by bacteria of the Enterobacteriaceae family. However, the rising resistance to quinolones worldwide poses a major clinical and public health risk. This study aimed to characterise a novel multiple resistance plasmid carrying three plasmid-mediated quinolone resistance genes in Escherichia coli clinical stain RJ749. METHODS: MICs of ceftriaxone, cefepime, ceftazidime, ciprofloxacin, and levofloxacin for RJ749 and transconjugant c749 were determined by the Etest method. Conjugation was performed using sodium azide-resistant E. coli J53 strain as a recipient. The quinolone resistance-determining regions of gyrA, gyrB, parC, and parE were PCR-amplified. RESULTS: RJ749 was highly resistant to quinolones, while c749 showed low-level resistance. S1-nuclease pulsed-field gel electrophoresis revealed that RJ749 and c749 both harboured a plasmid. PCR presented chromosomal mutation sites of the quinolone resistance-determining region, which mediated quinolone resistance. The c749 genome comprised a single plasmid, pRJ749, with a multiple resistance region, including three plasmid-mediated quinolone resistance (PMQR) genes (aac (6')-Ib-cr, qnrS2, and oqxAB) and ten acquired resistance genes. One of the genes, qnrS2, was shown for the first time to be flanked by two IS26s. Three IS26-mediated circular molecules carrying the PMQR genes were detected. CONCLUSIONS: We revealed the coexistence of three PMQR genes on a multiple resistance plasmid and a new surrounding genetic structure of qnrS2 flanked by IS26 elements. IS26 plays an important role in horizontal spread of quinolone resistance.202032293532
92020.9932Co-existence of bla(IMP), bla(NDM-1), and bla(SHV), genes of Pseudomonas aeruginosa isolated from Quetta: Antimicrobial resistance and clinical significance. OBJECTIVE: Molecular detection and co-presence of carbapenem-resistant genes in the isolates of Pseudomonas aeruginosa are less commonly reported from Quetta. In the present study, we determined to highlight the antibiotic sensitivity profile and genetic mechanism of carbapenem resistance. METHODS: The cross-sectional study was conducted from May to September 2018 at the Hi-tech laboratory, Centre for Advance Studies in Vaccinology and Biotechnology, University of Baluchistan, Quetta. Biochemical and molecular methods were ascertained for the recognition of the isolates and minimum inhibitory concentration was performed using E-test and broth microdilution methods. The molecular basis of carbapenemase activity was determined by identifying carbapenemase genes in the isolates. RESULTS: Of the (n=23) P. aeruginosa isolated from pus aspirates obtained from surgical/burn units, we have detected bla(IMP) (n=7/8) 87.5%, bla(NDM-1) (n=5/8) 62.5%, and bla(SHV) (n=4/8) 50%. The co-existence of multiple antibiotic-resistant genes, bla(IMP), bla(NDM-1) and bla(SHV) was found in (n=2/8) 25% isolates. These isolates displayed resistance against a range of antimicrobials from β-lactams, tetracyclines, cephalosporins, quinolones, monobactams, aminoglycosides, sulphonamides, phosphoric acid, macrolides, and polypeptide groups, suggesting extensive-drug resistance. CONCLUSION: The emergence of MBL and ESBL producers is an alarming threat in the region. It is of great importance to determine the resistance mechanism of bacterial bugs. The lack of new antimicrobials particularly against gram-negative bacteria is quite alarming worldwide.202337680816
116530.9932Isolation, Antimicrobial Susceptibility Profile and Detection of Sul1, blaTEM, and blaSHV in Amoxicillin-Clavulanate-Resistant Bacteria Isolated From Retail Sausages in Kampar, Malaysia. BACKGROUND: Due to the overuse of antibiotics in livestock as a growth-promoting agent, the emergence of multi-antibiotic resistant bacteria is becoming a concern. OBJECTIVES: In this study, we aimed to detect the presence and discover the molecular determinants of foodborne bacteria in retail sausages resistant towards the antibacterial agent amoxicillin-clavulanate. METHODS: Two grams of sausages were chopped into small pieces and transferred into sterile Luria-Bertani (LB) enrichment broths overnight before they were plated on MacConkey agar petri dishes. The bacteria isolated were then screened for amoxicillin-clavulanate resistance, and an antimicrobial susceptibility test of each isolate was performed by using the disc diffusion method. Double synergy and phenotypic tests were carried out to detect the presence of extended spectrum β-lactamase (ESBL). API 20E kit was used to identify the Enterobacteriaceae. All isolates were further examined by polymerase chain reaction (PCR) for resistant genes blaOXA-1, blaOXA-10, plasmid-mediated AmpC (blaCMY and blaDHA), and the chromosome-mediated AmpC, Sul1, blaTEM, and blaSHV genes. RESULTS: A total of 18 amoxicillin-clavulanate resistant isolates were obtained from seven different types of retail sausages. Only half of them were identified as Enterobacteriaceae, but none were ESBL-producers. All the 18 isolated strains demonstrated resistance towards amoxicillin-clavulanate, penicillin and oxacillin (100%), cefotaxime (71.4%), cefpodoxime (66.7%), and ampicillin (83.3%). blaTEM was the most frequently detected β-lactamase gene. Both plasmid- and chromosomal-bound blaTEM genes were detected in all of the isolated Enterobacteriaceae. blaSHV and Sul1 accounted for 22.2% and 11.1% of the amoxicillin-clavulanate resistant isolates, respectively, whereas blaAMPC, blaCMY, blaDHA, blaOXA-1, and blaOXA-10 were not found in any of the isolates. The only one ESBL-producing bacteria detected in this study was Chryseobacterium meningosepticum, which harbored the blaTEM gene. CONCLUSIONS: The multidrug resistant bacteria that carry antibiotic resistant genes from retail sausages may increase the risk of transmission to humans via the consumption of contaminated sausages. Stricter measures must be taken to address the use of antibiotics in animal agriculture and to consider their potential impact on human health.201627942365
89840.9931Characterisation of mobile colistin resistance genes (mcr-3 and mcr-5) in river and storm water in regions of the Western Cape of South Africa. BACKGROUND: Colistin is regarded as a last-resort antimicrobial against multi-drug resistant Gram-negative bacteria (GNB), therefore the dissemination of colistin resistance in the environment is of great concern. Horizontal transfer of mobile colistin resistance (mcr) genes to potential pathogens poses a serious problem. This study aimed to describe the presence of colistin resistant GNB and mcr genes in river and storm water in regions of the Western Cape. METHODS: Water samples were collected from three rivers during May 2019 and January 2020 and two storm water samples were collected in November 2019. Colistin resistant GNB were cultured on MacConkey agar containing colistin and identified by MALDI-TOF. Colistin resistance was confirmed using broth microdilution (BMD). mcr-1-5 genes were detected by PCR performed directly on the water samples and on the colistin resistant isolates. mcr functionality was assessed by BMD after cloning the mcr genes into pET-48b(+) and expression in SHuffle T7 E. coli. RESULTS: mcr-5.1 and various mcr-3 gene variants were detected in the Plankenburg-, Eerste- and Berg rivers and in storm water from Muizenberg, and only mcr-5.1 was detected in storm water from Fish Hoek. Colistin resistant GNB were isolated from all of the water sources. Aeromonas spp. were the most common colistin resistant organisms detected in the water sources; 25% (6/24) of colistin resistant Aeromonas spp. isolated from the Berg river contained novel mcr-3 variants; mcr-3.33 (n = 1), mcr-3.34 (n = 1) mcr-3.35 (n = 1) mcr-3.36 (n = 2) and mcr-3.37 (n = 1), which were confirmed to confer colistin resistance. CONCLUSIONS: The mcr-5.1 and mcr-3 colistin resistance gene variants were present in widely dispersed water sources in regions of the Western Cape. The mcr genes were only detected in water sampled downstream of and alongside communities, suggesting that their presence is driven by human influence/contamination. This is the first documentation of mcr-3 and mcr-5 gene variants in any setting in South Africa. Spill-over of these genes to communities could result in horizontal gene transfer to pathogenic bacteria, exacerbating the challenge of controlling multidrug resistant GNB infections.202134187559
143650.9931Characterisation of carbapenem-resistant Gram-negative organisms from clinical specimens in Yola, Nigeria. OBJECTIVES: This study aimed to identify carbapenem-resistant Gram-negative bacteria from clinical specimens of patients in Yola, Nigeria. METHODS: Routine clinical specimens were screened for the presence of carbapenem-resistant Gram-negative bacteria using chromogenic agar plates. Susceptibility of all presumptive isolates to carbapenems was tested by MIC and disk diffusion methods. Real-time PCR was used to test for the presence of carbapenemase genes. RESULTS: Screening of 1741 clinical specimens yielded 119 (6.8%) presumptive carbapenem-resistant Gram-negative bacteria. Antimicrobial susceptibility testing confirmed carbapenem resistance in 105 of these isolates. New Delhi metallo-β-lactamase (bla(NDM)) gene was detected in 26 isolates and Verona integron-encoded metallo-β-lactamase (bla(VIM)) gene was detected in four. The mechanism of resistance could not be identified in approximately two thirds of the carbapenem-resistant isolates. CONCLUSION: While bla(NDM) and bla(VIM) accounted for 28.6% of the resistance seen, further molecular-based studies are needed to characterise the other mechanisms of carbapenem resistance in these isolates.202031472281
86460.9931Prevalence and molecular characterization of β-lactamase producers and fluoroquinolone resistant clinical isolates from North East India. INTRODUCTION: The rapid emergence and variations of antibiotic resistance among common gram negative bacteria cause a significant concern specially in India and all over the world because of high mortality and morbidity rates. METHODS: In our study, we screened 189 bacterial isolates from Assam Medical College & Hospital, Dibrugarh for antibiotic resistance pattern and tried to identify the resistant genes causing responsible for β-lactam and fluoroquinolones resistance. RESULTS: More than 80% and 45% strains were resistant to all the 3rd generation cephalosporins, fluoroquinolones respectively. Among the 3rd generation cephalosporin resistant strains, 38% and 24% isolates were only ESBL and MBL producers respectively and 11% were reported to have both ESBL and MBL genes. The ESBL positive isolates have shown the dominance of CTX-M3 gene. VIM-1 gene was mostly reported in MBL producers. Our study probably for the first time reporting SIM-1 and SPM-1 MBL gene from India. Mutations in QRDR is found to be the primary cause of fluoroquinolone resistance along with efflux pump and PMQR presence. CONCLUSION: The study represents the first detailed study on antibiotic resistance from NE India this could help to take control measures for the emerging antibiotic resistance in hospital and community based infections in North East India.202133848892
96570.9931Molecular Characterization of Multidrug-Resistant Escherichia coli Isolates from Bovine Clinical Mastitis and Pigs in the Vojvodina Province, Serbia. The aim of the study was to characterize multidrug-resistant (MDR) Escherichia coli isolates collected in Serbia from bovine clinical mastitis cases and diseased pigs, mainly with molecular methods. A total of 48 E. coli isolates was collected during the years 2013-2014, of which 22 were MDR and were included in further analysis. Phylogenetic typing showed that 17 isolates belonged to group A, while two isolates were classified in group B1 and a single one in group D. All isolates showed unique macrorestriction patterns. Phenotypic susceptibility testing revealed resistances of the isolates against up to 13 antimicrobial agents, including resistance to fluoroquinolones. A wide variety of resistance genes was detected by PCR amplification and sequencing of amplicons. Sequence analysis of the quinolone resistance determining regions of topoisomerase genes revealed mutations in gyrA, parC, and/or parE. Plasmid-mediated quinolone resistance genes were detected in two porcine (aac-6'-Ib-cr and qnrS, respectively) isolates and a single bovine (aac-6'-Ib-cr) isolate. Resistance genes were found to be located on conjugative plasmids in 16 cases, many of which conferred a multidrug resistance phenotype. In conclusion, the plentitude of resistance genes located on conjugative plasmids and integrons in E. coli from cows and pigs in Vojvodina, Serbia, pose a high risk for horizontal gene transfer in bacteria from livestock husbandry.201828520501
246380.9931Characterization of Antibiotic-Resistant Stenotrophomonas Isolates from Painted Turtles Living in the Wild. Stenotrophomonas maltophilia is a ubiquitous multidrug-resistant opportunistic pathogen commonly associated with nosocomial infections. The purpose of this study was to isolate and characterize extended-spectrum beta-lactamase (ESBL) producing bacteria from painted turtles (Chrysemys picta) living in the wild and captured in southeastern Wisconsin. Fecal samples from ten turtles were examined for ESBL producing bacteria after incubation on HardyCHROM™ ESBL agar. Two isolates were cultivated and identified by 16S rRNA gene sequencing and whole genome sequencing (WGS) as Stenotrophomonas sp. 9A and S. maltophilia 15A. They were multidrug-resistant, as determined by antibiotic susceptibility testing. Stenotrophomonas sp. 9A was found to produce an extended spectrum beta-lactamase (ESBL) and both isolates were found to be carbapenem-resistant. EDTA-modified carbapenem inactivation method (eCIM) and the modified carbapenem inactivation method (mCIM) tests were used to examine the carbapenemase production and the test results were negative. Through WGS several antimicrobial resistance genes were identified in S. maltophilia 15A. For example a chromosomal L1 β-lactamase gene, which is known to hydrolyze carbapenems, a L2 β-lactamase gene, genes for the efflux systems smeABC and smeDEF and the aminoglycosides resistance genes aac(6')-lz and aph(3')-llc were found. An L2 β-lactamase gene in Stenotrophomonas sp. 9A was identified through WGS.202336729340
151090.9931Fluoroquinolone-resistant and extended-spectrum beta-lactamase producing Escherichia coli isolates from free-living wild animals. During the hunting season 2013-2014, fecal samples collected from hare, roe deer, deer and wild boars were sent to the bacteriology laboratory for the isolation of Escherichia coli and multidrug resistant isolates were characterized phenotypically and genotypically. Out of 106 fecal samples, E. coli was isolated from 101 samples. Although the majority of isolates belonged to phylogenetic groups A and B1, 14 out of 101 isolates were affiliated to group B2. A multidrug resistance phenotype was determined in 7 isolates, all of which had distinguishable genomic macrorestriction profiles. PCR analysis and sequencing revealed a variety of resistance genes, gene cassettes and cassette arrays in these multidrug resistant isolates. Resistance to fluoroquinolones was found in five E. coli isolates (two from a roe deer, one from a deer and two from a wild boar) and multiple mutations in the chromosomal topoisomerase genes were identified. In an E.coli isolate from a hare, the qnrB19 gene was detected. The same isolate carried an aadA23 gene cassette in class 1 integron. In addition, an extended- spectrum beta-lactamase bla(CTX-M-1) gene was detected in an E. coli isolate from a roe deer. The gene was located on a conjugative multi resistance plasmid, which was transferable to a plasmid free E. coli recipient. In conclusion, a number of resistance genes and mobile genetic elements were detected in E. coli isolates from wildlife in Vojvodina, emphasizing the role of environmental pollution in spreading resistant bacteria.201830173743
881100.9931Genetic analysis of multidrug-resistant and AmpC-producing Citrobacter freundii. OBJECTIVE: During the last decade, antimicrobial resistance within pet animals has received worldwide concern owing to their close contact with humans and the possibility of animal-human co-transmission of multidrug-resistant bacteria. This study examined phenotypic as well as molecular mechanisms associated with antimicrobial resistance in a multidrug-resistant, and AmpC-producing Citrobacter freundii recovered from a dog suffering from kennel cough in. MATERIALS AND METHODS: The isolate was recovered from a two-year-old dog suffering from severe respiratory manifestations. Phenotypically, the isolate was resistant to a wide range of antimicrobial agents including, aztreonam, ciprofloxacin, levofloxacin, gentamicin, minocycline, piperacillin, sulfamethoxazole-trimethoprim, and tobramycin. PCR and sequencing confirmed that the isolate harbors multiple antibiotic resistance genes, such as blaCMY-48 and blaTEM-1B which mediate resistance to B-lactams, and qnrB6 which mediate resistance to quinolone antibiotics. RESULTS: Multilocus sequence typing confirmed that the isolate belongs to ST163. Due to the unique characteristics of this pathogen, the whole genome sequencing was performed. In addition to the previously confirmed antibiotic resistance genes by PCR, the isolate was also confirmed to harbor other resistance genes which mediate resistance to aminoglycoside (aac(3)-IId, aac(6')-Ib-cr, aadA16, aph(3'')-Ib, and aph(6)-Id), macrolides [mph(A)), phenicols (floR), rifampicin (ARR-3), sulphonamides (sul1 and sul2), trimethoprim (dfrA27), and tetracycline (tet(A) and tet(B)]. CONCLUSIONS: The results presented in this study confirm that pets are possible sources of highly pathogenic multidrug-resistant microbes with unique genetic characteristics taking into consideration the high potential for their dissemination to humans, which can undoubtedly develop of severe infections in these hosts.202336808363
916110.9931Prospective multicentre study of rectal carriage of multidrug-resistant Enterobacteriaceae among health-care workers in Spain. OBJECTIVES: To investigate the rectal carriage of multidrug-resistant Enterobacteriaceae (colistin-resistant, extended-spectrum β-lactamase (ESBL) -producers and/or carbapenemase-producers) among health-care workers (HCWs) from six Spanish hospitals. METHODS: Rectal swabs from 258 HCWs, employed in intensive care units, haematology wards and clinical microbiology laboratories from six hospitals in northern Spain were studied. They were cultured in selective media for Gram-negative resistant bacteria. Detection of antimicrobial resistance genes and multilocus sequence typing were performed by PCR and further sequencing. A questionnaire including data related to risk factors of colonization/infection by resistant bacteria (age, gender, chronic diseases, immunosuppressive therapies, invasive procedures or antimicrobial treatments) was given to each participant. RESULTS: No carbapenemase-producing Enterobacteriaceae were recovered. However, 8/258 HCWs (3.1%) were positive for ESBL-producing isolates. This rate was not higher than the colonization rate previously reported in Spain for healthy people in the community. Five isolates showed high-level resistance to colistin (MICs ranging from 8 to 128 mg/L) but all of them were negative for the mcr genes tested. No statistically significant risk factors for gut colonization by ESBL-producing or colistin-resistant Enterobacteriaceae were identified among the HCWs participating in the study. CONCLUSIONS: Our data suggest that working in hospitals does not represent a risk for rectal carriage of multidrug-resistant Enterobacteriaceae.202031972320
880120.9931Plasmid-mediated quinolone resistance in non-Typhi serotypes of Salmonella enterica. BACKGROUND: Serious infections with Salmonella species are often treated with fluoroquinolones or extended-spectrum beta-lactams. Increasingly recognized in Enterobacteriaceae, plasmid-mediated quinolone resistance is encoded by qnr genes. Here, we report the presence of qnr variants in human isolates of non-Typhi serotypes of Salmonella enterica (hereafter referred to as non-Typhi Salmonella) from the United States National Antimicrobial Resistance Monitoring System for Enteric Bacteria. METHODS: All non-Typhi Salmonella specimens from the United States National Antimicrobial Resistance Monitoring System for Enteric Bacteria collected from 1996 to 2003 with ciprofloxacin minimum inhibitory concentrations > or = 0.06 microg/mL (233 specimens) and a subset with minimum inhibitory concentrations < or = 0.03 microg/mL (102 specimens) were screened for all known qnr genes (A, B, and S) by polymerase chain reaction. For isolates with positive results, qnr and quinolone resistance-determining region sequences were determined. Plasmids containing qnr genes were characterized by conjugation or transformation. RESULTS: Conjugative plasmids harboring qnrB variants were detected in 7 Salmonella enterica serotype Berta isolates and 1 Salmonella enterica serotype Mbandaka isolate. The S. Mbandaka plasmid also had an extended-spectrum beta -lactamase. Variants of qnrS on nonconjugative plasmids were detected in isolates of Salmonella enterica serotype Anatum and Salmonella enterica serotype Bovismorbificans. CONCLUSIONS: Plasmid-mediated quinolone resistance appears to be widely distributed, though it is still uncommon in non-Typhi Salmonella isolates from the United States, including strains that are quinolone susceptible by the criteria of the Clinical and Laboratory Standards Institute (formerly the National Committee for Clinical Laboratory Standards). The presence of this gene in non-Typhi Salmonella that causes infection in humans suggests potential for spread through the food supply, which is a public health concern.200616804843
1168130.9931Dairy Cattle and the Iconic Autochthonous Cattle in Northern Portugal Are Reservoirs of Multidrug-Resistant Escherichia coli. Background/Objectives: Animals destined for human consumption play a key role in potentially transmitting bacteria carrying antibiotic resistance genes. However, there is limited knowledge about the carriage of antibiotic-resistant bacteria in native breeds. We aimed to characterize the phenotypic profiles and antibiotic resistance genes in Escherichia coli isolated from bovines, including three native Portuguese bovine breeds. Methods: Forty-nine E. coli isolates were selected from 640 fecal samples pooled by age group (eight adult or eight calf samples) from each farm, representing both dairy cattle raised in intensive systems and meat cattle raised in extensive systems in Northern Portugal. The presumptive E. coli colonies plated onto MacConkey agar were confirmed using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). The antibiotic resistance profiles were screened by antimicrobial susceptibility testing (EUCAST/CLSI guidelines), and the antibiotic resistance genes by PCR. Results: Most isolates showed resistance to ampicillin (69%), tetracycline (57%), gentamicin (55%), and trimethoprim + sulfamethoxazole (53%), with no resistance to imipenem. Resistance to at least one antibiotic was found in 92% of isolates, while 59% exhibited multidrug resistance. Most calf isolates, including those from native breeds, showed a multidrug-resistant phenotype. Among the adults, this was only observed in Holstein-Friesian and Barrosã cattle. None of the Holstein-Friesian isolates were susceptible to all the tested antibiotics. ESBL-producing E. coli was identified in 39% of isolates, including those from Holstein-Friesian calves and adults, Cachena calves and Minhota adults. The sul2 gene was detected in 69% of isolates, followed by bla(CTX-M) (45%), aac(3')-IV (41%), and aac(6')-Ib-cr (31%), with a higher prevalence in adults. Conclusions: This pioneering study highlights the concerning presence of multidrug-resistant E. coli in native Portuguese cattle breeds.202439766598
5406140.9931Detection of poxtA- and optrA-carrying E. faecium isolates in air samples of a Spanish swine farm. OBJECTIVE: Two linezolid-resistant Enterococcus faecium isolates, C10004 and C10009, were recovered from air samples of a Spanish swine farm and comprehensively characterized. METHODS: Detection of linezolid resistance mechanisms (mutations and acquisition of resistance genes) was performed by PCR/sequencing. Isolates were characterized by multilocus sequence typing (MLST), antimicrobial susceptibility testing, detection of antimicrobial resistance and virulence genes, and analysis of the genetic environment of the linezolid resistance genes. The characterization of isolate C10009 was performed by Whole-Genome-Sequencing and of isolate C10004 by PCR and amplicon sequencing, where applicable. Conjugation experiments to assess the transferability of the optrA and poxtA genes implicated in linezolid resistance were performed. RESULTS: The linezolid-resistant E. faecium isolates C10004 and C10009, assigned to ST128 and ST437, respectively, harbored the optrA and poxtA genes. Neither mutations in the 23S rRNA nor in the genes for the ribosomal proteins L3, L4 and L22 were detected. C10004 and C10009 carried fourteen and thirteen antimicrobial resistance genes, respectively. The sequence alignment indicated that the genetic environment of the poxtA gene was identical in both isolates, with a downstream-located fexB gene. The poxtA gene was transferred by conjugation together with the fexB gene, and also with tet(M) and tet(L) in the case of isolate C10004. The optrA gene could not be transferred. CONCLUSIONS: This is the first report of the poxtA gene in Spain. The presence of poxtA- and optrA-carrying E. faecium isolates in air samples represents a public health concern, indicating an involvement of swine farms in the spread of linezolid-resistant bacteria.202031884049
1623150.9931Screening for fecal presence of colistin-resistant Escherichia coli and mcr-1 and mcr-2 genes in camel-calves in southern Tunisia. Camels (Camelus dromedarius) are known to harbor multidrug resistant Gram-negative bacteria and to be involved in the transmission of various microorganisms to humans. Data on the occurrence of colistin resistant Escherichia coli as well as mobilized colistin resistance (mcr) genes in camels are lacking. We investigated the presence of colistin resistance and mcr (1-2) genes in E. coli from the feces of camels in Tunisia. Presumptive E. coli isolates from camel-calves in southern Tunisia were qualitatively screened for growth on Mueller-Hinton agar supplemented with 2 mg/L of colistin. The minimal inhibitory concentration of colistin was determined for isolates growing on this medium. All isolates were screened for the presence of the mcr-1 and mcr-2 genes by polymerase chain reaction without detecting any of these genes. However, one isolate was confirmed resistant to colistin and further testing of this isolate revealed it to be Enterobacter cloacae. Our study demonstrated absence of colistin resistance and of the mcr-1 and mcr-2 genes in E. coli isolated from camel feces in southern Tunisia. Thus, there is no evidence that camels represent a major source of mcr genes contamination for the local population or for tourists visiting southern Tunisia.201829866140
1640160.9931First Identification of bla (NDM-1) Producing Escherichia coli ST 9499 Isolated from Musca domestica in the Urban Center of Rio de Janeiro, Brazil. The present study presents phenotypic and molecular characterization of a multidrug-resistant strain of Escherichia coli (Lemef26), belonging to sequence type ST9499 carrying a bla(NDM-1) carbapenem resistance gene. The bacterium was isolated from a specimen of Musca domestica, collected in proximity to a hospital in Rio de Janeiro City, Brazil. The strain was identified as E. coli by matrix-assisted laser desorption-ionization time of flight mass spectrometry (Maldi-TOF-MS) and via genotypic analysis (Whole-Genome Sequencing-WGS), followed by phylogenetic analysis, antibiotic resistance profiling (using phenotypic and genotypic methods) and virulence genotyping. Interestingly, the bla(NDM-1) was the only resistance determinant detected using a panel of common resistance genes, as evaluated by PCR. In contrast, WGS detected genes conferring resistance to aminoglycosides, fluoroquinolones, quinolones, trimethoprim, beta-lactams, chloramphenicol, macrolides, sulfonamide, tetracycline, lincosamide and streptogramin B. Conjugation experiments demonstrated the transfer of carbapenem resistance, via acquisition of the bla(NDM-1) sequence, to a sensitive receptor strain of E. coli, indicating that bla(NDM-1) is located on a conjugative plasmid (most likely of the IncA/C incompatibility group, in association with the transposon Tn3000). Phylogenetic analyses placed Lemef26 within a clade of strains exhibiting allelic and environment diversity, with the greatest level of relatedness recorded with a strain isolated from a human source suggesting a possible anthropogenic origin. Analysis of the virulome revealed the presence of fimbrial and pilus genes, including a CFA/I fimbriae (cfaABCDE), common pilus (ecpABCDER), laminin-bind fimbrae (elfADG), hemorrhagic pilus (hcpABC) and fimbrial adherence determinants (stjC) indicates the ability of strain Lemef26 to colonize animal hosts. To the best of our knowledge, this study represents the first report of bla(NDM-1) carbapenemase gene in an E. coli strain isolated from M. domestica. In concordance with the findings of previous studies on the carriage of MDR bacteria by flies, the data presented herein provide support to the idea that flies may represent a convenient means (as sentinel animals) for the monitoring of environmental contamination with multidrug-resistant bacteria.202337436443
2275170.9931Contribution of β-lactamase and efflux pump overproduction to tazobactam-piperacillin resistance in clinical isolates of Escherichia coli. INTRODUCTION: Tazobactam-piperacillin (TZP) is a mixture of a broad-spectrum penicillin and an irreversible β-lactamase inhibitor. TZP is effective against Gram-negative bacteria that produce extended-spectrum β-lactamases, and it is used as a first-line or second-line drug to treat serious infections. METHODS: This study identified three TZP-resistant and two TZP-intermediate strains among 514 clinical isolates of Escherichia coli. RESULTS: These five isolates possessed one or more β-lactamase genes, bla(TEM-1), bla(CTX-M-2), bla(CTX-M-14), and/or bla(CMY-8). The expression levels of β-lactamase genes and acrAB genes in the strains were examined by using real-time reverse transcription PCR. The total enzymatic piperacillin-degrading activity in cells was determined. Two TZP-resistance mechanisms were identified: hyperproduction of TEM-1 in the two resistant strains; and simultaneous high production of β-lactamase and efflux pump AcrAB in the two TZP-intermediate isolates. The latter are an international high-risk clone O25b:H4-ST131-H30R. CONCLUSION: TZP resistance is still rare in clinical isolates of E. coli. However, resistance can develop on high production and/or combinations of known antimicrobial resistance mechanisms in different ways.202032062000
5418180.9931Detection of optrA in the African continent (Tunisia) within a mosaic Enterococcus faecalis plasmid from urban wastewaters. OBJECTIVES: Oxazolidinone resistance is a serious limitation in the treatment of MDR Enterococcus infections. Plasmid-mediated oxazolidinone resistance has been strongly linked to animals where the use of phenicols might co-select resistance to both antibiotic families. Our goal was to assess the diversity of genes conferring phenicol/oxazolidinone resistance among diverse enterococci and to characterize the optrA genetic environment. METHODS: Chloramphenicol-resistant isolates (>16 mg/L, n = 245) from different sources (hospitals/healthy humans/wastewaters/animals) in Portugal, Angola and Tunisia (1996-2016) were selected. Phenicol (eight cat variants, fexA, fexB) or phenicol + oxazolidinone [cfr, cfr(B), optrA] resistance genes were searched for by PCR. Susceptibility (disc diffusion/microdilution), filter mating, stability of antibiotic resistance (500 bacterial generations), plasmid typing (S1-PFGE/hybridization), MLST and WGS (Illumina-HiSeq) were performed for optrA-positive isolates. RESULTS: Resistance to phenicols (n = 181, 74%) and phenicols + oxazolidinones (n = 2, 1%) was associated with the presence of cat(A-8) (40%, predominant in hospitals and swine), cat(A-7) (29%, predominant in poultry and healthy humans), cat(A-9) (2%), fexB (2%) and fexA + optrA (1%). fexA and optrA genes were co-located in a transferable plasmid (pAF379, 72 918 bp) of two ST86 MDR Tunisian Enterococcus faecalis (wastewaters) carrying several putative virulence genes. MICs of chloramphenicol, linezolid and tedizolid were stably maintained at 64, 4 and 1 mg/L, respectively. The chimeric pAF379 comprised relics of genetic elements from different Gram-positive bacteria and origins (human/porcine). CONCLUSIONS: To the best of our knowledge, we report the first detection of optrA in an African country (Tunisia) within a transferable mosaic plasmid of different origins. Its identification in isolates from environmental sources is worrisome and alerts for the need of a concerted global surveillance on the occurrence and spread of optrA.201729029072
1627190.9930Screening of colistin-resistant bacteria in livestock animals from France. Colistin is frequently used as a growth factor or treatment against infectious bacterial diseases in animals. The Veterinary Division of the European Medicines Agency (EMA) restricted colistin use as a second-line treatment to reduce colistin resistance. In 2020, 282 faecal samples were collected from chickens, cattle, sheep, goats, and pigs in the south of France. In order to track the emergence of mobilized colistin resistant (mcr) genes in pigs, 111 samples were re-collected in 2021 and included pig faeces, food, and water from the same location. All samples were cultured in a selective Lucie Bardet Jean-Marc Rolain (LBJMR) medium and colonies were identified using MALDI-TOF mass spectrometry and then antibiotic susceptibility tests were performed. PCR and Sanger sequencing were performed to screen for the presence of mcr genes. The selective culture revealed the presence of 397 bacteria corresponding to 35 different bacterial species including Gram-negative and Gram-positive. Pigs had the highest prevalence of colistin-resistant bacteria with an abundance of intrinsically colistin-resistant bacteria and from these samples one strain harbouring both mcr-1 and mcr-3 has been isolated. The second collection allowed us to identify 304 bacteria and revealed the spread of mcr-1 and mcr-3 in pigs. In the other samples, naturally, colistin-resistant bacteria were more frequent, nevertheless the mcr-1 variant was the most abundant gene found in chicken, sheep, and goat samples and one cattle sample was positive for the mcr-3 gene. Animals are potential reservoir of colistin-resistant bacteria which varies from one animal to another. Interventions and alternative options are required to reduce the emergence of colistin resistance and to avoid zoonotic transmissions.202236414994