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190600.8941Spread of Antimicrobial Resistance by Salmonella enterica Serovar Choleraesuis between Close Domestic and Wild Environments. The Salmonellaenterica serovar Choleraesuis affects domestic pig and wild boar (WB), causing clinical salmonellosis. Iberian swine production is based on a free-range production system where WB and Iberian pig (IP) share ecosystems. This study focuses on the negative impact on the pork industry of infections due to this serotype, its role in the spread of antibiotic resistance, and its zoonotic potential. Antibiotic resistance (AR) and genetic relationships were analyzed among 20 strains of S. Choleraesuis isolated from diseased WB and IP sampled in the southwest region of the Iberian Peninsula. AR was studied using the Kirby-Bauer method with the exception of colistin resistance, which was measured using the broth microdilution reference method. Resistance and Class 1 integrase genes were measured using PCR, and the genetic relationship between isolates and plasmid content by pulsed field gel electrophoresis. The results show a higher incidence of AR in isolates from IP. Phylogenetic analysis revealed seven profiles with two groups containing isolates from IP and WB, which indicates circulation of the same clone between species. Most pulsotypes presented with one plasmid of the same size, indicating vertical transmission. AR determinants bla(TEM) and tetA were routinely found in IP and WB, respectively. One isolate from IP expressed colistin resistance and presented the mcr-1 gene carried by a plasmid. This study suggests that S. Choleraesuis circulates between WB and IP living in proximity, and also that the mobilization of AR genes by plasmids is low. Furthermore, the detection of plasmid-mediated colistin resistance in bacteria from IP is alarming and should be monitored.202033137987
80410.8871Cloning, mutagenesis, and characterization of the microalga Parietochloris incisa acetohydroxyacid synthase, and its possible use as an endogenous selection marker. Parietochloris incisa is an oleaginous fresh water green microalga that accumulates an unusually high content of the valuable long-chain polyunsaturated fatty acid (LC-PUFA) arachidonic acid within triacylglycerols in cytoplasmic lipid bodies. Here, we describe cloning and mutagenesis of the P. incisa acetohydroxyacid synthase (PiAHAS) gene for use as an herbicide resistance selection marker for transformation. Use of an endogenous gene circumvents the risks and regulatory difficulties of cultivating antibiotic-resistant organisms. AHAS is present in plants and microorganisms where it catalyzes the first essential step in the synthesis of branched-chain amino acids. It is the target enzyme of the herbicide sulfometuron methyl (SMM), which effectively inhibits growth of bacteria and plants. Several point mutations of AHAS are known to confer herbicide resistance. We cloned the cDNA that encodes PiAHAS and introduced a W605S point mutation (PimAHAS). Catalytic activity and herbicide resistance of the wild-type and mutant proteins were characterized in the AHAS-deficient E. coli, BUM1 strain. Cloned PiAHAS wild-type and mutant genes complemented AHAS-deficient bacterial growth. Furthermore, bacteria expressing the mutant PiAHAS exhibited high resistance to SMM. Purified PiAHAS wild-type and mutant proteins were assayed for enzymatic activity and herbicide resistance. The W605S mutation was shown to cause a twofold decrease in enzymatic activity and in affinity for the Pyruvate substrate. However, the mutant exhibited 7 orders of magnitude higher resistance to the SMM herbicide than that of the wild type.201222488216
301720.8859The ancient small mobilizable plasmid pALWED1.8 harboring a new variant of the non-cassette streptomycin/spectinomycin resistance gene aadA27. The small mobilizable plasmid pALWED1.8 containing a novel variant of the streptomycin/spectinomycin resistance gene aadA27 was isolated from the permafrost strains of Acinetobacter lwoffii. The 4135bp plasmid carries mobА and mobC genes that mediate its mobilization by conjugative plasmids. The nucleotide sequences of mobА and mobC are similar to those of mobilization genes of the modern plasmid pRAY* and its variants, which contain aadB gene, and are widespread among the pathogenic strains of Acinetobacter baumannii. Almost identical pALWED1.8 variants were detected in modern environmental Аcinetobacter strains. A highly similar plasmid was revealed in a strain of Acinetobacter parvus isolated from mouse intestine. Furthermore, we discovered six previously unidentified variants of plasmids related to pALWED1.8 and pRAY* in public databases. In contrast to most known variants of aadA which are cassette genes associated with integrons, the aadA27 variant harbored by pALWED1.8 is a non-cassette, autonomously transcribed gene. Non-cassette aadA genes with 96% sequence identity to aadA27 were detected in the chromosomes of Acinetobacter gyllenbergii and several uncharacterized strains of Аcinetobacter sp. Moreover, we revealed that the autonomous aadA-like genes are present in the chromosomes of many gram-positive and gram-negative bacteria. The phylogenetic analysis of amino acid sequences of all identified AadA proteins showed the following: (i) cassette aadA genes form a separate monophyletic group and mainly reside on plasmids and (ii) chromosomal non-cassette aadA genes are extremely diverse and can be inherited both vertical and via horizontal gene transfer.201626896789
106730.8854Virulence and plasmidic resistance determinants of Escherichia coli isolated from municipal and hospital wastewater treatment plants. Escherichia coli is simultaneously an indicator of water contamination and a human pathogen. This study aimed to characterize the virulence and resistance of E. coli from municipal and hospital wastewater treatment plants (WWTPs) in central Portugal. From a total of 193 isolates showing reduced susceptibility to cefotaxime and/or nalidixic acid, 20 E. coli with genetically distinct fingerprint profiles were selected and characterized. Resistance to antimicrobials was determined using the disc diffusion method. Extended spectrum β-lactamase and plasmid-mediated quinolone resistance genes, phylogroups, pathogenicity islands (PAIs) and virulence genes were screened by polymerase chain reaction (PCR). CTX-M producers were typed by multilocus sequence typing. Resistance to beta-lactams was associated with the presence of bla(TEM), bla(SHV), bla(CTX-M-15) and bla(CTX-M-32). Plasmid-mediated quinolone resistance was associated with qnrA, qnrS and aac(6')-Ib-cr. Aminoglycoside resistance and multidrug-resistant phenotypes were also detected. PAI IV(536), PAI II(CFT073), PAI II(536) and PAI I(CFT073), and uropathogenic genes iutA, papAH and sfa/foc were detected. With regard to the clinical ST131 clone, it carried bla(CTX-M-15), blaTEM-type, qnrS and aac(6')-lb-cr; IncF and IncP plasmids, and virulence factors PAI IV(536), PAI I(CFT073), PAI II(CFT073), iutA, sfa/foc and papAH were identified in the effluent of a hospital plant. WWTPs contribute to the dissemination of virulent and resistant bacteria in water ecosystems, constituting an environmental and public health risk.201526042965
33540.8851Construction and characterization of a replication-competent retroviral shuttle vector plasmid. We constructed two versions of an RCASBP-based retroviral shuttle vector, RSVP (RCASBP shuttle vector plasmid), containing either the zeocin or blasticidin resistance gene. In this vector, the drug resistance gene is expressed in avian cells from the long terminal repeat (LTR) promoter, whereas in bacteria the resistance gene is expressed from a bacterial promoter. The vector contains a bacterial origin of replication (ColE1) to allow circular viral DNA to replicate as a plasmid in bacteria. The vector also contains the lac operator sequence, which binds to the lac repressor protein, providing a simple and rapid way to purify the vector DNA. The RSVP plasmid contains the following sequence starting with the 5" end: LTR, gag, pol, env, drug resistance gene, lac operator, ColE1, LTR. After this plasmid was transfected into DF-1 cells, we were able to rescue the circularized unintegrated viral DNA from RSVP simply by transforming the Hirt DNA into Escherichia coli. Furthermore, we were able to rescue the integrated provirus. DNA from infected cells was digested with an appropriate restriction enzyme (ClaI) and the vector-containing segments were enriched using lac repressor protein and then self-ligated. These enriched fractions were used to transform E. coli. The transformation was successful and we did recover integration sites, but higher-efficiency rescue was obtained with electroporation. The vector is relatively stable upon passage in avian cells. Southern blot analyses of genomic DNAs derived from successive viral passages under nonselective conditions showed that the cassette (drug resistance gene-lac operator-ColE1) insert was present in the vector up to the third viral passage for both resistance genes, which suggests that the RSVP vectors are stable for approximately three viral passages. Together, these results showed that RSVP vectors are useful tools for cloning unintegrated or integrated viral DNAs.200211799171
264650.8848Detection of Antimicrobial Resistance Genes in Escherichia coli Isolated from Black Howler Monkeys (Alouatta pigra) and Domestic Animals in Fragmented Rain-Forest Areas in Tabasco, Mexico. The appearance and spread of antimicrobial resistance (AMR) in bacteria in natural environments and wildlife are related to agricultural and livestock activities and are a global health and conservation problem. We assessed the presence of AMR genes in Escherichia coli isolated from black howler monkeys (Alouatta pigra), sheep (Ovis aries), cattle (Bos taurus), and horses (Equus caballus) from a highly fragmented forest in southern Mexico. Fresh fecal samples were collected using swabs, seeded on eosin-methylene blue agar, and E. coli colonies identified by PCR; multiplex-PCR was performed on E. coli DNA for the detection of 10 AMR genes from four families (sulfonamides, tetracycline, β-lactamase, and chloramphenicol). We detected E. coli in 94% (48/51) of fecal samples, of which 33% (16/48) tested positive for at least one AMR gene. We detected AMR genes in at least one individual from each sampled animal species, with the most prevalent genes being tet(B) 18% (9/48), sul2 14% (7/48), sul1, and blaTEM 12% (6/48). Sheep samples contained AMR genes from the four families of antibiotics detected in this study and 50% (5/10) tested positive for the presence of at least one gene. A total of 12% (2/16) of fecal samples from black howler monkeys tested positive for AMR genes. The presence of AMR genes in A. pigra and domestic animals has not been reported in the Balancán area of Tabasco, Mexico. Transmission of AMR bacteria from domestic animals to monkeys is rare; however, this is a potential health risk for wildlife and species conservation.202032402234
208460.8846Characterization of Four Multidrug Resistance Plasmids Captured from the Sediments of an Urban Coastal Wetland. Self-transmissible and mobilizable plasmids contribute to the emergence and spread of multidrug-resistant bacteria by enabling the horizontal transfer of acquired antibiotic resistance. The objective of this study was to capture and characterize self-transmissible and mobilizable resistance plasmids from a coastal wetland impacted by urban stormwater runoff and human wastewater during the rainy season. Four plasmids were captured, two self-transmissible and two mobilizable, using both mating and enrichment approaches. Plasmid genomes, sequenced with either Illumina or PacBio platforms, revealed representatives of incompatibility groups IncP-6, IncR, IncN3, and IncF. The plasmids ranged in size from 36 to 144 kb and encoded known resistance genes for most of the major classes of antibiotics used to treat Gram-negative infections (tetracyclines, sulfonamides, β-lactams, fluoroquinolones, aminoglycosides, and amphenicols). The mobilizable IncP-6 plasmid pLNU-11 was discovered in a strain of Citrobacter freundii enriched from the wetland sediments with tetracycline and nalidixic acid, and encodes a novel AmpC-like β-lactamase (bla(WDC-1)), which shares less than 62% amino acid sequence identity with the PDC class of β-lactamases found in Pseudomonas aeruginosa. Although the IncR plasmid pTRE-1611 was captured by mating wetland bacteria with P. putida KT2440 as recipient, it was found to be mobilizable rather than self-transmissible. Two self-transmissible multidrug-resistance plasmids were also captured: the small (48 kb) IncN3 plasmid pTRE-131 was captured by mating wetland bacteria with Escherichia coli HY842 where it is seemed to be maintained at nearly 240 copies per cell, while the large (144 kb) IncF plasmid pTRE-2011, which was isolated from a cefotaxime-resistant environmental strain of E. coli ST744, exists at just a single copy per cell. Furthermore, pTRE-2011 bears the globally epidemic bla(CTX-M-55) extended-spectrum β-lactamase downstream of ISEcp1. Our results indicate that urban coastal wetlands are reservoirs of diverse self-transmissible and mobilizable plasmids of relevance to human health.201729067005
122970.8846Detection of multi-drug resistance and AmpC β-lactamase/extended-spectrum β-lactamase genes in bacterial isolates of loggerhead sea turtles (Caretta caretta) from the Mediterranean Sea. Sea turtles are useful sentinels to monitor the dissemination of antimicrobial resistance (AMR) in the marine coastal ecosystems. Forty Gram negative bacteria were isolated from wounds of 52 injured Caretta caretta, living in the Mediterranean Sea. Bacteria were identified using 16S rRNA gene sequencing and tested for susceptibility to 15 antibiotics. In addition, NGS amplicon sequencing was performed to detect the presence of AmpC β-lactamase genes (bla(AmpC)) and extended-spectrum β-lactamase (ESBL) genes (bla(CTX-M,)bla(SHV,)bla(TEM)). Seventy-five percent of the isolates (30/40 isolates) exhibited multidrug resistance (MDR) phenotypes and 32.5% (13/40 isolates) were confirmed to be positive for at least one gene. The variants of ESBLs genes were bla(CTX-M-3,)bla(TEM-236) and bla(SHV-12). Variants of the bla(AmpC)β-lactamase gene i.e., bla(ACT-24), bla(ACT-2), bla(ACT-17), bla(DHA-4) and bla(CMY-37), were also detected. In addition, 4 isolates were found simultaneously harboring CTX and AmpC genes while 2 strains harbored 3 genes (bla(ACT-2+TEM-236+SHV-12), and bla(CTX-M-3+ACT-24+TEM-236)).202133513540
528180.8845Bacteria populating freshly appeared supraglacial lake possess metals and antibiotic-resistant genes. Antibiotic resistance (AR) has been extensively studied in natural habitats and clinical applications. AR is mainly reported with the use and misuse of antibiotics; however, little is known about its presence in antibiotic-free remote supraglacial lake environments. This study evaluated bacterial strains isolated from supraglacial lake debris and meltwater in Dook Pal Glacier, northern Pakistan, for antibiotic-resistant genes (ARGs) and metal-tolerant genes (MTGs) using conventional PCR. Several distinct ARGs were reported in the bacterial strains isolated from lake debris (92.5%) and meltwater (100%). In lake debris, 57.5% of isolates harbored the bla(TEM) gene, whereas 58.3% of isolates in meltwater possessed bla(TEM) and qnrA each. Among the ARGs, qnrA was dominant in debris isolates (19%), whereas in meltwater isolates, qnrA (15.2%) and bla(TEM) (15.2%) were dominant. ARGs were widely distributed among the bacterial isolates and different bacteria shared similar types of ARGs. Relatively greater number of ARGs were reported in Gram-negative bacterial strains. In addition, 92.5% of bacterial isolates from lake debris and 83.3% of isolates from meltwater harbored MTGs. Gene copA was dominant in meltwater isolates (50%), whereas czcA was greater in debris bacterial isolates (45%). Among the MTGs, czcA (18.75%) was dominant in debris strains, whereas copA (26.0%) was greater in meltwater isolates. This presents the co-occurrence and co-selection of MTGs and ARGs in a freshly appeared supraglacial lake. The same ARGs and MTGs were present in different bacteria, exhibiting horizontal gene transfer (HGT). Both positive and negative correlations were determined between ARGs and MTGs. The research provides insights into the existence of MTGs and ARGs in bacterial strains isolated from remote supraglacial lake environments, signifying the need for a more detailed study of bacteria harboring ARGs and MTGs in supraglacial lakes.202438262510
150890.8844First Detection and Genomic Insight into mcr-1 Encoding Plasmid-Mediated Colistin-Resistance Gene in Escherichia coli ST101 Isolated from the Migratory Bird Species Hirundo rustica in Thailand. Background: This study aimed to investigate the occurrence of mcr-1 encoding plasmid-mediated colistin-resistance gene in Escherichia coli isolated from migratory birds in Thailand. Materials and Methods: A total of 178 cloacal swabs from migratory birds was sampled and isolated from 2016 to 2017 in Nan, Trang, and Bangkok, Thailand. The multiplex polymerase chain reaction was used to screen the resistance genes. After screening, a disk diffusion assay and the minimum inhibitory concentration were investigated. The draft genome sequence of isolate 2A85589 was obtained using an Illumina HiSeq X-Ten platform. The genome was assembled using SPAdes 3.0.0. Antimicrobial resistance genes were identified using ResFinder 3.1. Results: We reported E. coli ST101 of isolate 2A85589, an mcr-1-carrying resistance gene isolated from the migratory bird species Hirundo rustica in Thailand. The draft genome of 2A85589 was 4,621,016 bp in size. IncHI1A plasmid was identified using PlasmidFinder with high coverage. In silico analysis detected the presence of eight putative acquired resistance genes, namely blaTEM-1B, mcr-1, mef(A), mef(B), QnrS1, sul3, tet(A), and tet(B), which conferred resistance to β-lactam, colistin, macrolide, quinolone, sulfonamide, and tetracycline. Conclusion: This study underlines the potential risk of the environmental contamination of mcr-1-carrying E. coli isolated from the migratory bird. The long range migration of birds can result in dissemination of mcr-1-carrying bacteria globally. Therefore, plasmid-mediated colistin is an urgent need to be addressed in both human and veterinary medicine for disease control and prevention.201931334682
1382100.8843Surveillance of antimicrobial-resistant Escherichia coli in Sheltered dogs in the Kanto Region of Japan. There is a lack of an established antimicrobial resistance (AMR) surveillance system in animal welfare centers. Therefore, the AMR prevalence in shelter dogs is rarely known. Herein, we conducted a survey in animal shelters in Chiba and Kanagawa prefectures, in the Kanto Region, Japan, to ascertain the AMR status of Escherichia coli  (E. coli) prevalent in shelter dogs. E. coli was detected in the fecal samples of all 61 and 77 shelter dogs tested in Chiba and Kanagawa, respectively. The AMR was tested against 20 antibiotics. E. coli isolates derived from 16.4% and 26.0% of samples from Chiba and Kanagawa exhibited resistance to at least one antibiotic, respectively. E. coli in samples from Chiba and Kanagawa prefectures were commonly resistant to ampicillin, piperacillin, streptomycin, kanamycin, tetracycline, and nalidixic acid; that from the Kanagawa Prefecture to cefazolin, cefotaxime, aztreonam, ciprofloxacin, and levofloxacin and that from Chiba Prefecture to chloramphenicol and imipenem. Multidrug-resistant bacteria were detected in 18 dogs from both regions; β-lactamase genes (blaTEM, blaDHA-1, blaCTX-M-9 group CTX-M-14), quinolone-resistance protein genes (qnrB and qnrS), and mutations in quinolone-resistance-determining regions (gyrA and parC) were detected. These results could partially represent the AMR data in shelter dogs in the Kanto Region of Japan.202235031646
1384110.8842Antimicrobial resistance in wildlife: detection of antimicrobial resistance genes in Apennine wolves (Canis lupus italicus Altobello, 1921) from Central Italy. The aim of this study was to molecularly investigate the presence of antimicrobial resistance genes (ARGs) in organ samples from 11 Apennine wolves (Canis lupus italicus) collected in Central Italy. Samples from lung, liver, spleen, kidney, tongue and intestine were investigated by PCRs targeting the following genes: tet(A), tet(B), tet(C), tet(D), tet(E), tet(G), tet(K), tet(L), tet(M), tet(O), tetA(P), tet(Q), tet(S), tet(X), sul1, sul2, sul3, bla(CTX-M), bla(SHV), bla(TEM) and mcr-1. A PCR positivity was highlighted for 13 out of the 21 tested genes; no positive results were obtained for tet(C), tet(D), tet(E), tet(G), sul3, bla(CTX), bla(SHV) and mcr-1 genes. All 11 animals sampled showed positivity for one or more resistance genes. The results confirm the potential role of the wolf as an indicator and/or vector of antimicrobial-resistant bacteria or ARGs.202438499909
5872120.8839Characterization of the plasmids harbouring the florfenicol resistance gene floR in Glaesserella parasuis and Actinobacillus indolicus. OBJECTIVES: The aim of this study was to characterize the floR-carrying plasmids originating from Glaesserella parasuis and Actinobacillus indolicus isolated from pigs with respiratory disease in China. METHODS: A total of 125 G. parasuis and 28 A. indolicus strains collected between 2009 and 2022 were screened for florfenicol resistance. Characterization of floR-positive isolates and plasmids were determined by antimicrobial susceptibility testing, serotyping, multilocus sequence typing (MLST), conjugation and transformation assays, whole-genome sequencing (WGS), and phylogenetic analysis. RESULTS: One A. indolicus and six G. parasuis were identified as positive for floR. The six G. parasuis were assigned to four different serovars, including serovars 6, 7, 9, and unknown. In addition to strain XP11, six floR genes were located on plasmids. The six floR-bearing plasmids could be transformed into Pasteurella multocida and divided into two different types, including ∼5000 bp and ∼6000 bp plasmids. The ∼5000 bp plasmids consisting of rep, lysR, mobB, and floR genes, exhibited high similarity among Pasteurellaceae bacteria. Furthermore, the ∼6000 bp plasmids, consisting of rep, lysR, mobC, mobA/L, and floR genes, showed high similarity between G. parasuis and Actinobacillus Spp. Notably, WGS results showed that the floR modules of the two types of plasmids could be transferred and integrated into the diverse Pasteurellaceae- origined plasmids. CONCLUSION: This study firstly reported the characterization of floR-carrying plasmids from A. indolicus and a non-virulent serovar of G. parasuis in pigs in China and elucidated the transmission mechanism of the floR resistance gene among the Pasteurellaceae family.202337726088
1169130.8839Determination and molecular analysis of antibiotic resistance in Gram-negative enteric bacteria isolated from Pelophylax sp. in the Eastern Black Sea Region. The aim of this study was to evaluate the prevalence and types of antimicrobial resistance among Gram-negative enteric bacteria isolated from Pelophylax sp. Fifty-four frogs were collected from six provinces in the Eastern Black Sea Region of Turkey. In the cloacal swab cultures, bacteria from 160 different colonies were identified by biochemical tests, automated systems, and matrix-assisted laser desorption ionisation-time of flight mass spectrometry. The antimicrobial susceptibility tests were performed by the disk diffusion method. The observed drug resistance rate was the highest to ampicillin and cefazolin, while the lowest against ciprofloxacin and tetracycline. In the molecular assays, bla TEM (8 Citrobacter spp.), bla SHV (2 Escherichia coli, 1 Hafnia alvei, and a Serratia liquefaciens), tetA genes (E. coli and Klebsiella spp.) and a class 1 integron without any gene cassette (E. coli) were detected. Among the strains, no plasmid-mediated quinolone resistance [qnrA, qnrB, qnrS, qepA and aac (6 ')-Ib-cr] was found. However, two of three quinolone-resistant Klebsiella strains showed the novel amino acid substitution in the gyrA gene resulting in Ser83Asp and Asp87Glu.The clonality between E. coli isolates was also examined by pulsed-field gel electrophoresis. We consider that multidrug-resistant Gram-negative enteric bacteria in the intestinal microbiota of a cosmopolitan frog species might be a reservoir for antibiotic resistance genes.202134570716
1510140.8838Fluoroquinolone-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
2647150.8837Antibiotic Susceptibility and Virulence Factors in Escherichia coli from Sympatric Wildlife of the Apuan Alps Regional Park (Tuscany, Italy). Today a growing number of studies are focusing on antibiotic resistance in wildlife. This is due to the potential role of wild animals as reservoirs and spreaders of pathogenic and resistant bacteria. This study focused on isolating and identifying Escherichia coli from the feces of wild animals living in the Apuan Alps Regional Park (Tuscany, Italy) and evaluating some of their antibiotic resistance and pathogenicity traits. Eighty-five fecal samples from different species were studied. Seventy-one E. coli were identified by matrix assisted laser desorption ionization-time of flight mass spectrometry analysis, subjected to antibiograms and polymerase chain reaction for the detection of antibiotic resistance genes and pathogenicity factors. The highest resistance rates were found against cephalothin (39.4%) and ampicillin (33.8%), followed by amoxicillin/clavulanic acid (15.5%), streptomycin (12.7%), and tetracycline (5.6%). Regarding resistance genes, 39.4% of the isolates were negative for all tested genes. The remaining isolates were positive for bla(CMY)(-2), sul2, strA-strB and aadA1, tet(B), and tet(A), encoding resistance to beta-lactams, trimethoprim/sulfamethoxazole, streptomycin, and tetracycline, respectively. With regard to virulence factors, 63.4% of the isolates were negative for all genes; 21.1% carried astA alone, which is associated with different pathotypes, 9.9% carried both escV and eaeA (aEPEC); single isolates (1.4%) harbored escV (aEPEC), escV associated with astA and eaeA (aEPEC), astA with stx2 and hlyA (EHEC) or astA and stx1, stx2, and hlyA (EHEC). These results show that wildlife from nonanthropized environments can be a reservoir for antibiotic-resistant microorganisms and suggest the need for a deeper knowledge on their origin and diffusion mechanisms through different ecological niches.201930676273
1088160.8837Detection and Molecular Characterization of Escherichia coli Strains Producers of Extended-Spectrum and CMY-2 Type Beta-Lactamases, Isolated from Turtles in Mexico. Multidrug-resistant bacteria are a growing problem in different environments and hosts, but scarce information exists about their prevalence in reptiles. The aim of this study was to analyze the resistance mechanisms, molecular typing, and plasmid content of cefotaxime-resistant (CTX(R)) Escherichia coli isolates recovered from cloacal samples of 71 turtles sheltered in a herpetarium in Mexico. CTX(R)-E. coli were recovered in 11 of 71 samples (15.5%), and one isolate/sample was characterized. Extended-spectrum β-lactamase (ESBL)-producing E. coli isolates were detected in four samples (5.6%): two strains carried the blaCTX-M-2 gene (phylogroup D and ST2732) and two contained the blaCTX-M-15 gene (phylogroup B1 and lineages ST58 and ST156). The blaCMY-2 gene was detected by PCR in E. coli isolates of eight samples (9.8%) (one of them also carried blaCTX-M-2); these isolates were distributed into phylogroups A (n = 1), B1 (n = 6), and D (n = 1) and typed as ST155, ST156, ST2329, and ST2732. Plasmid-mediated quinolone resistance (PMQR) genes were detected in five isolates [aac(6')Ib-cr, qnrA, qnrB19, and oqxB]. From three to five replicon plasmids were detected among the strains, being IncFIB, IncI1, IncFrep, and IncK the most prevalent. ESBL or pAmpC genes were transferred by conjugation in four strains, and the blaCTX-M-15 and blaCMY-2 genes were localized in IncFIB or IncI1 plasmids by Southern blot hybridization assays. Class 1 and/or class 2 integrons were detected in eight strains with six different structures of gene cassette arrays. Nine pulsed-field gel electrophoresis patterns were found among the 11 studied strains. To our knowledge, this is the first detection of ESBL, CMY-2, PMQR, and mobile determinants of antimicrobial resistance in E. coli of turtle origin, highlighting the potential dissemination of multidrug-resistant bacteria from these animals to other environments and hosts, including humans.201627482752
1089170.8837Diversity of plasmids harboring bla(CMY-2) in multidrug-resistant Escherichia coli isolated from poultry in Brazil. Multidrug-resistance (MDR) has been increasingly reported in Gram-negative bacteria from the intestinal microbiota, environment and food-producing animals. Resistance plasmids able to harbor different transposable elements are capable to mobilize antimicrobial resistance genes and transfer to other bacterial hosts. Plasmids carrying bla(CMY) are frequently associated with MDR. The present study assessed the presence of plasmid-encoded ampC genes (bla(cmy), bla(mox), bla(fox), bla(lat), bla(act), bla(mir), bla(dha), bla(mor)) in commensal E. coli isolated from apparently healthy broiler chickens. Furthermore, we characterized the plasmids and identified those harboring the resistance genes. We isolated 144/200 (72%) of E. coli isolates with resistance to cefotaxime and the resistance gene identified was bla(CMY-2). The pulsed-field gel electrophoresis (PFGE) analysis showed high diversity of the genetic profiles. The phylogenetic groups A, B1, B2, and D were identified among E. coli isolates and group D was the most prevalent. The PCR-based replicon typing (PBRT) analysis identified four distinct plasmid incompatibility groups (Inc) in MDR isolates. Moreover, plasmids harboring bla(CMY-2), ranged in size from 50kb to 150kb and 51/144 (35%) belonged to IncK, 21/144 (14.5%) to IncB/O, 8/144 (5.5%) to IncA/C, 1/144 (0.5%) to IncI, while 63/144 (44.5%) were not typeable by PBRT. Overall, a high prevalence of bla(CMY-2) genes was found in a diverse population of commensal MDR E. coli from poultry in Brazil, harbored into different plasmids.201728602519
1078180.8837Prevalence of integrons, blaCTX-M and blaTEM resistance markers among ESBL-producing uropathogenic Escherichia coli isolates: first report of genomic blaCTX-M from India. Integrons have been observed to be frequently associated with uropathogenic bacteria. This study aimed at 1) determining the prevalence of class 1 integrons among ESBLl-producing uropathogenic Escherichia coli, and 2) analyzing resistance genes associated with different phylogenetic groups of the integron-positive isolates with special reference to bla(CTX-M) and bla(TEM). Twenty-three ESBL-producing E. coli were studied. Enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR) displayed 14 major patterns. Pulse field Gel electrophoresis-typing of 8 randomly selected integron-positive strains ruled out any correlation between genotype and antibiotype. Genomic DNA from 14 strains was PCR-positive for class 1 integrons, bla(CTX-M-15) and bla(TEM-1)-like genes. Integron-sequencing revealed "aadA5-dfrA17-dfrA7" as the most prevalent gene cassette. Our findings unveil the increasing role of the bla(CTX-M) genes in antibiotic resistance and emphasize on the significance of appropriate empirical treatment for Urinary tract infections. Moreover, this is the first study which reports bla(CTX-M) located on genomic DNA of bacteria from India.201121742580
2480190.8835GLO1 Contributes to the Drug Resistance of Escherichia coli Through Inducing PER Type of Extended-Spectrum β-Lactamases. BACKGROUND: Escherichia coli-associated antimicrobial resistance (AMR) issue so far needs urgent considerations. This study aims to screen the potent genes associated with extended-spectrum β-lactamases (ESBLs) in drug-resistant Escherichia coli and elucidate the specific drug-resistant mechanism. METHODS: Clinical ESBLs-EC samples were obtained based on the microbial identification, and the whole genome was sequenced. In combination with the significantly enriched pathways, several differently expressed genes were screened and verified by RT-PCR. Furthermore, through knocking out glyoxalase 1 (GLO1) gene and transfecting overexpressed plasmids, the potential relationship between GLO1 and ESBLs was then investigated. Lastly, the concentrations of β-lactamases in bacteria and supernatant from different groups were examined by enzyme-linked immunosorbent assay (ELISA). RESULTS: After successful isolation and identification of ESBLs-EC, the whole genome and eighteen differential metabolic pathways were analyzed to select differently expressed genes, including add, deoD, guaD, speG, GLO1, VNN1, etc. RT-PCR results showed that there were no differences in these genes between the standard bacteria and susceptible Escherichia coli. Remarkably, the relative levels of four genes including speG, Hdac10, GLO1 and Ppcdc were significantly increased in ESBLs-EC in comparison with susceptible strains, whereas other gene expression was decreased. Further experiments utilizing gene knockout and overexpression strains confirmed the role of GLO1. At last, a total of 10 subtypes of β-lactamases were studied using ELISA, including BES-, CTX-M1-, CTX-M2-, OXA1-, OXA2-, OXA10-, PER-, SHV-, TEM-, and VEB-ESBLs, and results demonstrated that GLO1 gene expression only affected PER-β-lactamases but had no effects on other β-lactamases. CONCLUSION: SpeG, Hdac10, GLO1 and Ppcdc might be associated with the drug-resistant mechanism of Escherichia coli. Of note, this study firstly addressed the role of GLO1 in the drug resistance of ESBLs-EC, and this effect may be mediated by increasing PER-β-lactamases.202235414749