Characterization of Fosfomycin and Nitrofurantoin Resistance Mechanisms in Escherichia coli Isolated in Clinical Urine Samples. - Related Documents




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523001.0000Characterization of Fosfomycin and Nitrofurantoin Resistance Mechanisms in Escherichia coli Isolated in Clinical Urine Samples. Fosfomycin and nitrofurantoin are antibiotics of choice to orally treat non-complicated urinary tract infections (UTIs) of community origin because they remain active against bacteria resistant to other antibiotics. However, epidemiologic surveillance studies have detected a reduced susceptibility to these drugs. The objective of this study was to determine possible mechanisms of resistance to these antibiotics in clinical isolates of fosfomycin- and/or nitrofurantoin-resistant UTI-producing Escherichia coli. We amplified and sequenced murA, glpT, uhpT, uhpA, ptsI, cyaA, nfsA, nfsB, and ribE genes, and screened plasmid-borne fosfomycin-resistance genes fosA3, fosA4, fosA5, fosA6, and fosC2 and nitrofurantoin-resistance genes oqxA and oqxB by polymerase chain reaction. Among 29 isolates studied, 22 were resistant to fosfomycin due to deletion of uhpT and/or uhpA genes, and 2 also possessed the fosA3 gene. Some modifications detected in sequences of NfsA (His11Tyr, Ser33Arg, Gln67Leu, Cys80Arg, Gly126Arg, Gly154Glu, Arg203Cys), NfsB (Gln44His, Phe84Ser, Arg107Cys, Gly192Ser, Arg207His), and RibE (Pro55His), and the production of truncated NfsA (Gln67 and Gln147) and NfsB (Glu54), were associated with nitrofurantoin resistance in 15/29 isolates; however, the presence of oqxAB plasmid genes was not detected in any isolate. Resistance to fosfomycin was associated with the absence of transporter UhpT expression and/or the presence of antibiotic-modifying enzymes encoded by fosA3 plasmid-mediated gene. Resistance to nitrofurantoin was associated with modifications of NfsA, NfsB, and RibE proteins. The emergence and spread of these resistance mechanisms, including transferable resistance, could compromise the future usefulness of fosfomycin and nitrofurantoin against UTIs. Furthermore, knowledge of the genetic mechanisms underlying resistance may lead to rapid DNA-based testing for resistance.202032847131
121310.9985Fosfomycin resistance determinants in Escherichia coli isolates of human and animal origin from Iran. BACKGROUND: Fosfomycin has regained clinical interest over the last years due to its superior activity against multidrug-resistant bacterial pathogens. In the current study we aimed to characterize genotypic and phenotypic features of fosfomycin resistant (FosR) Escherichia coli isolates originating from human and animal. METHODS AND RESULTS: Five FosR bacteria were selected from a population of 150 E. coli isolates of human and broiler chickens. The sequence types of isolates were determined by multi-locus sequencing typing. Fosfomycin MICs were determined by agar dilution and gradient diffusion methods. Molecular detection of plasmid encoded fosfomycin resistance genes, fosA, fosA3, fosA4, fosA5 and fosC2 was performed by PCR. The modifications of fosfomycin target (MurA), transporters (GlpT, UhpT), and transporter regulator (PtsI) were investigated by gene sequencing. The MICs of fosfomycin were found to be ≥ 128 mg/L according to agar dilution and > 1024 mg/L according to gradient diffusion method. FosR isolates belonged to sequence types ST10 (n = 2), ST361, ST209 and ST1158 (n = 1). While all FosR isolates carried fos genes (fosA3 (n = 2), fosA5 (n = 2) and fosA4(n = 1)), only three isolates revealed amino acid substations in MurA, PtsI and GlpT with MurA P99S change being predicted to have deleterious impact on the function of protein. CONCLUSIONS: Emergence of fosfomycin resistance among studied isolates was mainly attributed to plasmid genes coding for fosfomycin modifying enzymes. The similarity in fosfomycin resistance determinants among clonally diverse E. coli isolates of human and animals indicates a possible cross-sectoral dissemination of fos genes by epidemic plasmids between bacterial isolates of clinical settings and those from animals.202540465084
523120.9981Prediction of nitrofurantoin resistance among Enterobacteriaceae and mutational landscape of in vitro selected resistant Escherichia coli. Nitrofurantoin (NIT) has long been a drug of choice in the treatment of lower urinary tract infections. Recent emergence of NIT resistant Enterobacteriaceae is a global concern. An ordinal logistic regression model based on PCR amplification patterns of five genes associated with NIT resistance (nfsA, nfsB, ribE, oqxA, and oqxB) among 100 clinical Enterobacteriaceae suggested that a combination of oqxB, nfsB, ribE, and oqxA is ideal for NIT resistance prediction. In addition, four Escherichia coli NIT-resistant mutants were in vitro generated by exposing an NIT-susceptible E. coli to varying concentrations of NIT. The in vitro selected NIT resistant mutants (NI2, NI3, NI4 and NI5) were found to have mutations resulting in frameshifts, premature/lost stop codons or failed amplification of nfsA and/or nfsB genes. The in vitro selected NI5 and the transductant colonies with reconstructed NI5 genotype exhibited reduced fitness compared to their parent strain NS30, while growth of a resistant clinical isolate (NR42) was found to be unaffected in the absence of NIT. These results emphasize the importance of strict adherence to prescribed antibiotic treatment regimens and dosage duration. If left unchecked, these resistant bacteria may thrive at sub-therapeutic concentrations of NIT and spread in the community.202234718096
207230.9980Interplay between IncF plasmids and topoisomerase mutations conferring quinolone resistance in the Escherichia coli ST131 clone: stability and resistance evolution. The Escherichia coli ST131 H30-Rx subclone vehicles CTX-M-15 plasmids and mutations in gyrA and parC conferring multidrug resistance successfully in the clinical setting. The aim of this study was (1) to investigate the relationship of specific topoisomerase mutations on the stability of IncF (CTX-M producing) plasmids using isogenic E. coli mutants and (2) to investigate the impact of the IncF-type plasmids present in the E. coli clone ST131 on the evolution of quinolone resistance. E. coli ATCC 25922 (background strain) and derived mutants encoding specific QRDR substitutions were used. Also, NGS-characterized IncFIA and IncFIB plasmids (encoding CTX-M genes) were included. Plasmid stability was evaluated by sequential dilutions into Luria broth medium without antibiotics for 7 days. Mutant frequency to ciprofloxacin was also evaluated. Moderate differences in the IncF plasmids stability were observed among E. coli ATCC 25922 and isogenic mutants. Under our experimental conditions, the fluctuation of bacteria harboring plasmids was less than 0.5-log((10)) in all cases. In the mutant frequency tests, it was observed that the presence of these IncF plasmids increased this value significantly (10-1000-fold). Quinolone resistance substitutions in gyrA or parC genes, frequently found associated with E. coli clone ST131, do not modify the stability of ST131-associated IncFIA and IncFIB plasmids under in vitro conditions. IncF-type plasmids present in E. coli clone ST131 facilitate the selection of resistance to quinolones. These results are consistent with the clinical scenario in which the combination of resistance to quinolones and beta-lactams is highly frequent in the E. coli clone ST131.202134787748
233140.9980Bacteriological and molecular study of fosfomycin resistance in uropathogenic Escherichia coli. The identification of genes associated with resistance has the potential to facilitate the development of novel diagnostic tests and treatment methods. The objective of this study was to examine the antibiotic resistance and Fosfomycin resistance genes in uropathogenic Escherichia coli (UPEC) in patients in Baghdad, Iraq. After analyzing 250 urine samples using various identification methods, including the examination of morphological characteristics, biochemical tests, and genetic detection, it was determined that E. coli was the most common bacteria present, accounting for 63.6% of the samples. Antibiotic susceptibility testing showed a significant prevalence of resistance to various antibiotics, with 99.3% of E. coli isolates exhibiting multiple drug resistance (MDR). Fosfomycin showed antibacterial properties against UPEC. The minimum inhibitory concentration (MIC) ranged from 512 to 1024 μg/mL, while the minimum bactericidal concentration (MBC) was 2048 μg/mL. In the time-kill assay, fosfomycin was effective against fosfomycin-resistant isolates within 8-12 h. The genetic determinants associated with fosfomycin resistance were examined through the utilization of polymerase chain reaction (PCR). The findings indicated that the genes murA, glpT, and cyaA were detected in all the isolates when genomic DNA was used as a template. However, all the tests yielded negative results when plasmid was used as a template. The genes fosA3 and fosA4 were detected in 8.6% and 5% of the isolates when genomic DNA was used as a template. When plasmid was used as a template, the genes fosA3 and fosA4 were found in 5.7% and 2.9% of the isolates, respectively. In conclusion, there is an increasing problem with antibiotic resistance in UPEC, with elevated rates of resistance to several antibiotics. The study also offers novel insights into the genetic foundation of fosfomycin resistance in UPEC.202438367167
88550.9979Emergence of Fosfomycin Resistance by Plasmid-Mediated fos Genes in Uropathogenic ESBL-Producing E. coli Isolates in Mexico. Fosfomycin is currently a viable option against urinary tract infections, particularly against extended-spectrum β-lactamases (ESBL)-producing E. coli, due to its unique mechanism of action and its low resistance among bacteria. The objective of this study was to investigate two of the three most common mechanisms of resistance against this antibiotic among 350 ESBL-producing E. coli strains isolated from the urine of Mexican patients. The prevalence of fosfomycin resistance in our study was 10.9% (38/350). Of all resistant isolates analyzed, 23 (60.5%) were identified as fos-producing organisms, with 14 strains carrying fosA3 and 9, fosA1. Additionally, 11 (28.9%) fosfomycin-resistant isolates presented resistance due to impaired antibiotic transport and 8 (21.0%) both mechanisms. No resistance mechanism investigated in the study was found on 12 strains. All 38 confirmed ESBL-producing isolates carried a bla(CTX-M) subtype, 36 (94.5%) belonged to the O25b-ST131 clone, and all of them were able to transfer the fosfomycin resistance trait to recipient strains horizontally. This is the first study in Mexico demonstrating a plasmid-mediated fosfomycin resistance mechanism among clinical E. coli strains. Since our results suggest a strong association among fos and bla(CTX-M) genes and ST131 clones in uropathogenic E. coli, plasmid-mediated fosfomycin resistance should be closely monitored.202236290041
228460.9979Molecular Mechanisms and Epidemiology of Fosfomycin Resistance in Staphylococcus aureus Isolated From Patients at a Teaching Hospital in China. Staphylococcus aureus is a major cause of hospital- and community-acquired infections placing a significant burden on the healthcare system. With the widespread of multidrug-resistant bacteria and the lack of effective antibacterial drugs, fosfomycin has gradually attracted attention as an "old drug." Thus, investigating the resistance mechanisms and epidemiology of fosfomycin-resistant S. aureus is an urgent requirement. In order to investigate the mechanisms of resistance, 11 fosfomycin-resistant S. aureus isolates were analyzed by PCR and sequencing. The genes, including fosA, fosB, fosC, fosD, fosX, and tet38, as well as mutations in murA, glpT, and uhpT were identified. Quantitative real-time PCR (qRT-PCR) was conducted to evaluate the expression of the target enzyme gene murA and the efflux pump gene tet38 under the selection pressure of fosfomycin. Furthermore, multilocus sequence typing (MLST) identified a novel sequence type (ST 5708) of S. aureus strains. However, none of the resistant strains carried fosA, fosB, fosC, fosD, and fosX genes in the current study, and 12 distinct mutations were detected in the uhpT (3), glpT (4), and murA (5) genes. qRT-PCR revealed an elevated expression of the tet38 gene when exposed to increasing concentration of fosfomycin among 8 fosfomycin-resistant S. aureus strains and reference strain ATCC 29213. MLST analysis categorized the 11 strains into 9 STs. Thus, the mutations in the uhpT, glpT, and murA genes might be the primary mechanisms underlying fosfomycin resistance, and the overexpression of efflux pump gene tet38 may play a major role in the fosfomycin resistance in these isolates.202032670230
179170.9978Complete genome sequence of Enterobacter cloacae R11 reveals multiple genes potentially associated with high-level polymyxin E resistance. Enterobacter cloacae strain R11 is a multidrug-resistant bacterium isolated from sewage water near a swine feedlot in China. Strain R11 can survive in medium containing up to 192 μg/mL polymyxin E, indicating a tolerance for this antibiotic that is significantly higher than that reported for other gram-negative bacteria. In this study, conjugation experiments showed that partial polymyxin E resistance could be transferred from strain R11 to Escherichia coli strain 25922, revealing that some genes related to polymyxin E resistance are plasmid-based. The complete genome sequence of this strain was determined, yielding a total of 4 993 008 bp (G+C content, 53.15%) and 4908 genes for the circular chromosome and 4 circular plasmids. Genome analysis revealed a total of 73 putative antibiotic resistance genes, including several polymyxin E resistance genes and genes potentially involved in multidrug resistance. These data provide insights into the genetic basis of the polymyxin E resistance and multidrug resistance of E. cloacae.201829073359
164580.9978Epidemiological investigation and β-lactam antibiotic resistance of Riemerella anatipestifer isolates with waterfowl origination in Anhui Province, China. Riemerella anatipestifer (R. anatipestifer) is a highly pathogenic and complex serotypes waterfowl pathogen with inherent resistance to multiple antibiotics. This study was aimed to investigate the antibiotic resistance characteristics and genomic features of R. anatipestifer isolates in Anhui Province, China in 2023. A total of 287 cases were analysed from duck farms and goose farms, and the R. anatipestifer isolates were subjected to drug resistance tests for 30 antimicrobials. Whole genome sequencing (WGS) and bioinformatics analysis were performed on the bacterial genomes, targeting the β-lactam resistance genes. The results showed that a total of 74 isolates of R. anatipestifer were isolated from 287 cases, with a prevalence of 25.8%. The antimicrobial susceptibility testing (AST) revealed that all the 74 isolates were resistant to multiple drugs, ranging from 13 to 26 kinds of drugs. Notably, these isolates showed significant resistance to aminoglycosides and macrolides, which are also commonly used in clinical practices. Data revealed the presence of several β-lactamase-related genes among the isolates, including a novel bla(RASA-1) variant (16.2%), the class A extended-spectrum β-lactamase bla(RAA-1) (12.2%), and a bla(OXA-209) variant (98.6%). Functional analysis of the variants bla(RASA-1) and bla(OXA-209) showed that the bla(RASA-1) variant exhibited activity against various β-lactam antibiotics while their occurrence in R. anatipestifer were not common. The bla(OXA-209) variant, on the other hand, did not perform any β-lactam antibiotic resistance. Furthermore, we observed that bla(RAA-1) could undergo horizontal transmission among different bacteria via the insertion sequence IS982. In conclusion, this study delves into the high prevalence of R. anatipestifer infection in waterfowl in Anhui, China. The isolated strains exhibit severe drug resistance issues, closely associated with the prevalence of antibiotic resistance genes (ARG). Additionally, our research investigates the β-lactam antibiotic resistance mechanism in R. anatipestifer.202438387287
206990.9978Two novel CMY-2-type β-lactamases encountered in clinical Escherichia coli isolates. BACKGROUND: Chromosomally encoded AmpC β-lactamases may be acquired by transmissible plasmids which consequently can disseminate into bacteria lacking or poorly expressing a chromosomal bla AmpC gene. Nowadays, these plasmid-mediated AmpC β-lactamases are found in different bacterial species, namely Enterobacteriaceae, which typically do not express these types of β-lactamase such as Klebsiella spp. or Escherichia coli. This study was performed to characterize two E. coli isolates collected in two different Portuguese hospitals, both carrying a novel CMY-2-type β-lactamase-encoding gene. FINDINGS: Both isolates, INSRA1169 and INSRA3413, and their respective transformants, were non-susceptible to amoxicillin, amoxicillin plus clavulanic acid, cephalothin, cefoxitin, ceftazidime and cefotaxime, but susceptible to cefepime and imipenem, and presented evidence of synergy between cloxacilin and cefoxitin and/or ceftazidime. The genetic characterization of both isolates revealed the presence of bla CMY-46 and bla CMY-50 genes, respectively, and the following three resistance-encoding regions: a Citrobacter freundii chromosome-type structure encompassing a blc-sugE-bla CMY-2-type -ampR platform; a sul1-type class 1 integron with two antibiotic resistance gene cassettes (dfrA1 and aadA1); and a truncated mercury resistance operon. CONCLUSIONS: This study describes two new bla CMY-2-type genes in E. coli isolates, located within a C. freundii-derived fragment, which may suggest their mobilization through mobile genetic elements. The presence of the three different resistance regions in these isolates, with diverse genetic determinants of resistance and mobile elements, may further contribute to the emergence and spread of these genes, both at a chromosomal or/and plasmid level.201525885413
5946100.9978Plasmid-encoded fosfomycin resistance in bacteria isolated from the urinary tract in a multicentre survey. Sixty out of 219 fosfomycin-resistant bacteria selected from more than 7400 urinary pathogens in an epidemiological multicentre survey performed in Italy were screened for plasmid genes fosA and fosB conferring fosfomycin resistance. Only five strains, three enterobacteria and two staphylococci, carried plasmids harbouring, respectively, fosA and fosB genes. Fosfomycin resistance in the other isolates was caused by an alteration of the chromosomally encoded GlpT transport system. One strain, Morganella morganii 279, incorporated alpha-glycerolphosphate and its mechanism of fosfomycin resistance needs to be further investigated. Our study showed that PCR amplification is the most accurate, simple and rapid method for epidemiological studies of plasmid-encoded fosfomycin resistance, and that fosfomycin resistance conferred by plasmid genes (both fosA and fosB) accounts for only a low percentage of the fosfomycin-resistant strains.19979338493
2046110.9978QRDR mutations, efflux system & antimicrobial resistance genes in enterotoxigenic Escherichia coli isolated from an outbreak of diarrhoea in Ahmedabad, India. BACKGROUND & OBJECTIVES: Diverse mechanisms have been identified in enteric bacteria for their adaptation and survival against multiple classes of antimicrobial agents. Resistance of bacteria to the most effective fluoroquinolones have increasingly been reported in many countries. We have identified that most of the enterotoxigenic Escherichia coli (ETEC) were resistant to several antimicrobials in a diarrhoea outbreak at Ahmedabad during 2000. The present study was done to identify several genes responsible for antimicrobial resistance and mobile genetic elements in the ETEC strains. METHODS: Seventeen ETEC strains isolated from diarrhoeal patients were included in this study. The antimicrobial resistance was confirmed by conventional disc diffusion method. PCR and DNA sequencing were performed for the identification of mutation in the quinolone resistance-determining regions (QRDRs). Efflux pump was tested by inhibiting the proton-motive force. DNA hybridization assay was made for the detection of integrase genes and the resistance gene cassettes were identified by direct sequencing of the PCR amplicons. RESULTS: Majority of the ETEC had GyrA mutations at codons 83 and 87 and in ParC at codon 80. Six strains had an additional mutation in ParC at codon 108 and two had at position 84. Plasmid-borne qnr gene alleles that encode quinolone resistance were not detected but the newly described aac(6')-Ib-cr gene encoding a fluoroquinolne-modifying enzyme was detected in 64.7 per cent of the ETEC. Class 1 (intI1) and class 2 (intI2) integrons were detected in six (35.3%) and three (17.6%) strains, respectively. Four strains (23.5%) had both the classes of integrons. Sequence analysis revealed presence of dfrA17, aadA1, aadA5 in class 1, and dfrA1, sat1, aadA1 in class 2 integrons. In addition, the other resistance genes such as tet gene alleles (94.1%), catAI (70.6%), strA (58.8%), bla TEM-1 (35.2%), and aphA1-Ia (29.4%) were detected in most of the strains. INTERPRETATION & CONCLUSIONS: Innate gene mutations and acquisition of multidrug resistance genes through mobile genetic elements might have contributed to the emergence of multidrug resistance (MDR) in ETEC. This study reinforces the necessity of utilizing molecular techniques in the epidemiological studies to understand the nature of resistance responsible for antimicrobial resistance in different species of pathogenic bacteria.201121911975
974120.9978First report on class 1 integrons and Trimethoprim-resistance genes from dfrA group in uropathogenic E. coli (UPEC) from the Aleppo area in Syria. Horizontal gene transfer (HGT) introduces advantageous genetic elements into pathogenic bacteria using tools such as class1 integrons. This study aimed at investigating the distribution of these integrons among uropathogenic E. coli (UPEC) isolated from patients in Aleppo, Syria. It also set to uncover the frequencies of the clinically relevant DfrA1 and DfrA17,7, as well as various associations leading to reduced susceptibility. This study involved 75 Trimethoprim-resistant E. coli isolates from in- and outpatients with urinary tract infections (UTIs) from 3 major hospitals in Aleppo. Bacterial identification, resistance and extended-spectrum-β-lactamase (ESBL) production testing were performed according to Clinical Laboratory Standards Institute guidelines. Detection of integrons and DfrA genes was done using PCR and statistical significance was inferred through χ2 (Fisher's) test. Class1 integrons were detected in 54.6% of isolates while DfrA1 and DfrA17,7 were found in 16% and 70.6% of tested samples respectively. Furthermore, only DfrA17,7 were strongly associated with class1 integrons, as were reduced susceptibility to the majority of individual antibiotics, multidrug resistance and ESBL production. This study demonstrated the high prevalence of class1 integrons among UPEC strains in Aleppo, Syria, as well as their significant associations with MDR. This data give information for local healthcare provision using antibiotic chemotherapy.201323956949
2913130.9978Distribution of resistance genetic determinants among Vibrio cholerae isolates of 2012 and 2013 outbreaks in IR Iran. The objective of this study was to characterize antimicrobial resistance determinants in relation to antimicrobial susceptibility and genotyping profile in 20 clinical isolates of Vibrio cholerae. All of the isolates were resistant to streptomycin. The second most prevalent resistance was observed to trimethoprim (75%), co-trimoxazole (60%), tetracycline (50%), and minocycline (45%). About 50% of the isolates fulfilled the criteria of Multi Drug Resistance (MDR) phenotype. None of the isolates carried tet A, B, C, and, D determinants. This finding shows that tetracycline resistance determinants recognized so far, does not satisfactorily describe the 50% tetracycline resistance phenotype in this study, suggesting the possible contribution of other not yet characterized resistance mechanisms involved. Class 1 integron, widely distributed among enteric bacteria, was not detected among V. cholerae strains under study. Conversely, 100% of the isolates harbored SXT constin((int)), among which 70% were positive for dfrA1, strA, and strB genes. The sul1gene was present in 60% of the isolates while none of them contained floR gene. All the isolates uniformly appeared to be identical in fingerprinting profiles expected from outbreak strains. In conclusion, SXT element with its mosaic structure was the exclusive antimicrobial resistance determinant of clonal V. cholerae isolates taken from outbreaks of 2012 and 2013 in Iran.201728062293
1520140.9977Colistin resistance in Salmonella and Escherichia coli isolates from a pig farm in Great Britain. OBJECTIVES: The objective of this study was to characterize colistin-resistant bacteria isolated from pigs on a farm in Great Britain following identification of a plasmid-borne colistin resistance mechanism in Escherichia coli from China. METHODS: Phenotypic antimicrobial susceptibility testing was undertaken by broth dilution and WGS was performed to detect the presence of genes encoding resistance and virulence. Transferable colistin resistance was investigated by conjugation. RESULTS: Two E. coli and one Salmonella Typhimurium variant Copenhagen were shown to be MDR, including resistance to colistin, with one E. coli and the Salmonella carrying the mcr-1 gene; all three harboured chromosomal mutations in genes conferring colistin resistance and both E. coli harboured β-lactamase resistance. The Salmonella mcr-1 plasmid was highly similar to pHNSHP45, from China, while the E. coli mcr-1 plasmid only had the ISApII and mcr-1 genes in common. The frequency of mcr-1 plasmid transfer by conjugation to recipient Enterobacteriaceae from Salmonella was low, lying between 10(-7) and 10(-9) cfu/recipient cfu. We were unable to demonstrate mcr-1 plasmid transfer from the E. coli. Plasmid profiling indicated transfer of multiple plasmids from the Salmonella resulting in some MDR transconjugants. CONCLUSIONS: Identification of the mcr-1 gene in Enterobacteriaceae from pigs confirms its presence in livestock in Great Britain. The results suggest dissemination of resistance through different horizontally transferable elements. The in vitro transfer of multiple plasmids carrying colistin and other resistances from the Salmonella isolate underlines the potential for wider dissemination and recombination.201627147305
1609150.9977Analysis of Salmonella enterica with reduced susceptibility to the third-generation cephalosporin ceftriaxone isolated from U.S. cattle during 2000-2004. Over the past decade enteric bacteria in Europe, Africa, and Asia have become increasingly resistant to cephalosporin antimicrobial agents. This is largely due to the spread of genes encoding extended-spectrum beta-lactamase (ESBL) enzymes that can inactivate many cephalosporins. Recently, these resistance mechanisms have been identified in Salmonella isolated from humans in the United States. Due to the potential for transmission of resistant bacteria to humans via food animals, Salmonella animal isolates were monitored for ESBL production. During 2000-2004, Salmonella cattle slaughter isolates (n = 3,984) were tested, and 97 (2.4%) of these were found to have decreased susceptibility (minimum inhibitory concentration [MIC] >32 microg/ml) to the third-generation cephalosporin ceftriaxone. The majority of these were serotypes Newport (58) and Agona (14), some of which were genetically indistinguishable by pulsed field gel electrophoresis (PFGE) analysis. None of the isolates had an ESBL phenotype; all were susceptible to the fourth-generation cephalosporins cefepime and cefquinome. PCR and sequence analysis for resistance genes detected the bla(CMY-2) gene in 93 isolates and the bla(TEM-1) gene in 12 isolates; however, neither gene encodes an ESBL. These data indicate that bovine Salmonella isolates from the United States with decreased susceptibility or resistance to ceftriaxone do not exhibit an ESBL phenotype and most contain the bla(CMY-2) gene.200819025468
1580160.9977Polyclonal Spread of Fosfomycin Resistance among Carbapenemase-Producing Members of the Enterobacterales in the Czech Republic. Fosfomycin (FOS) has been recently reintroduced into clinical practice, but its effectiveness against multidrug-resistant (MDR) Enterobacterales is reduced due to the emergence of FOS resistance. The copresence of carbapenemases and FOS resistance could drastically limit antibiotic treatment. The aims of this study were (i) to investigate fosfomycin susceptibility profiles among carbapenem-resistant Enterobacterales (CRE) in the Czech Republic, (ii) to characterize the genetic environment of fosA genes among the collection, and (iii) to evaluate the presence of amino acid mutations in proteins involved in FOS resistance mechanisms. During the period from December 2018 to February 2022, 293 CRE isolates were collected from different hospitals in the Czech Republic. FOS MICs were assessed by the agar dilution method (ADM), FosA and FosC2 production was detected by the sodium phosphonoformate (PPF) test, and the presence of fosA-like genes was confirmed by PCR. Whole-genome sequencing was conducted with an Illumina NovaSeq 6000 system on selected strains, and the effect of point mutations in the FOS pathway was predicted using PROVEAN. Of these strains, 29% showed low susceptibility to fosfomycin (MIC, ≥16 μg/mL) by ADM. An NDM-producing Escherichia coli sequence type 648 (ST648) strain harbored a fosA10 gene on an IncK plasmid, while a VIM-producing Citrobacter freundii ST673 strain harbored a new fosA7 variant, designated fosA7.9. Analysis of mutations in the FOS pathway revealed several deleterious mutations occurring in GlpT, UhpT, UhpC, CyaA, and GlpR. Results regarding single substitutions in amino acid sequences highlighted a relationship between ST and specific mutations and an enhanced predisposition for certain STs to develop resistance. This study highlights the occurrence of several FOS resistance mechanisms in different clones spreading in the Czech Republic. IMPORTANCE Antimicrobial resistance (AMR) currently represents a concern for human health, and the reintroduction of antibiotics such as fosfomycin into clinical practice can provide further option in treatment of multidrug-resistant (MDR) bacterial infections. However, there is a global increase of fosfomycin-resistant bacteria, reducing its effectiveness. Considering this increase, it is crucial to monitor the spread of fosfomycin resistance in MDR bacteria in clinical settings and to investigate the resistance mechanism at the molecular level. Our study reports a large variety of fosfomycin resistance mechanisms among carbapenemase-producing Enterobacterales (CRE) in the Czech Republic. Our study summarizes the main achievements of our research on the use of molecular technologies, such as next-generation sequencing (NGS), to describe the heterogeneous mechanisms that reduce fosfomycin effectiveness in CRE. The results suggest that a program for widespread monitoring of fosfomycin resistance and epidemiology fosfomycin-resistant organisms can aide timely implementation of countermeasures to maintain the effectiveness of fosfomycin.202337098942
2063170.9977Nalidixic acid-a good marker of fluoroquinolone resistance mechanisms in Escherichia coli. The purpose of this study was to evaluate how ciprofloxacin, pefloxacin, and nalidixic acid disks perform in screening fluoroquinolone resistance mechanisms in 278 Escherichia coli isolates collected from a prospective clinical material. Antimicrobial susceptibility testing of ciprofloxacin, pefloxacin, and nalidixic acid was performed with the disk diffusion method. PCR-based and sequencing methods were used to detect chromosomal mutations in the gyrA and parC genes and the presence of plasmid-mediated qnr and aac(6')-1b-cr genes. In addition, whole-genome sequencing was used to confirm these results. Our results show that fluoroquinolone resistance mechanisms were discovered, even in ciprofloxacin-susceptible isolates, and plasmid-mediated low-level fluoroquinolone resistance is easily missed if only ciprofloxacin disk is used. E. coli strains with chromosomal gyrA and/or parC mutations were well detected with pefloxacin disk. However, nalidixic acid was a superior tool to detect and differentiate between low- (plasmid-mediated) and high-level (chromosomal mutations) fluoroquinolone resistance in E. coli. Thus, more clinical studies are needed to evaluate the clinical relevance of fluoroquinolone resistance mechanisms in enteric bacteria and pathogens that show potential but are not yet phenotypically fluoroquinolone-resistant. IMPORTANCE: We show in our clinical setting that fluoroquinolone resistance mechanisms are discovered, even among phenotypically fluoroquinolone-susceptible Escherichia coli isolates. When plasmid-mediated quinolone-resistance determinants are present, they are a potential risk for treatment failures due to accumulation of resistance mechanisms during the antimicrobial treatment. Therefore, when it is clinically relevant, fluoroquinolone resistance mechanisms in E. coli should be monitored more closely, and we also recommend testing nalidixic acid susceptibility.202540401973
2330180.9977Antimicrobial and disinfectant resistance of Escherichia coli isolated from giant pandas. AIMS: The study aims to demonstrate the antimicrobial and disinfectant resistance phenotypes and genotypes of Escherichia coli isolates obtained from giant pandas (Ailuropoda melanoleuca). METHODS AND RESULTS: Antimicrobial testing was performed according to the standard disk diffusion method. The minimal inhibitory concentrations (MICs) of disinfectants were determined using the agar dilution method. All isolates were screened for the presence of antimicrobial and disinfectant resistance genes and further analysed for genetic relatedness by pulse-field gel electrophoresis (PFGE). Results showed that 46·6% of the isolates were resistant to at least one antimicrobial. Escherichia coli isolates showed resistance to fewer antimicrobials as panda age increased. Among antimicrobial-resistant E. coli isolates, the antimicrobial resistance genes blaCTX-M (88·2%) and sul1 (92·3%) were most prevalent. The disinfectant resistance genes emrE, ydgE/ydgF, mdfA and sugE(c) were commonly present (68·2-98·9%), whereas qac and sugE(p) were relatively less prevalent (0-21·3%). The frequencies of resistance genes tended to be higher in E. coli isolated in December than in July, and PFGE profiles were also more diverse in isolates in December. The qacEΔ1 and sugE(p) genes were higher in adolescent pandas than in any other age groups. PFGE revealed that antimicrobial resistance correlated well with sampling time and habitat. CONCLUSIONS: This study demonstrated that antimicrobial and disinfectant resistance was common in giant panda-derived E. coli, and the antimicrobial resistance was associated with sampling time and habitat. Escherichia coli could serve as a critical vector in spreading disinfectant and antimicrobial resistance. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study that demonstrated the phenotypic and genetic characterizations of antimicrobial and disinfectant resistance in E. coli isolates from more than 60 giant pandas. Frequent transfer of pandas to other cages may lead to the dissemination of antimicrobial resistance. The study highlights the need for regularly monitoring the antimicrobial and disinfectant resistance in bacteria from giant pandas.201525846200
2064190.9977Co-spread of metal and antibiotic resistance within ST3-IncHI2 plasmids from E. coli isolates of food-producing animals. Concerns have been raised in recent years regarding co-selection for antibiotic resistance among bacteria exposed to heavy metals, particularly copper and zinc, used as growth promoters for some livestock species. In this study, 25 IncHI2 plasmids harboring oqxAB (20/25)/blaCTX-M (18/25) were found with sizes ranging from ∼260 to ∼350 kb and 22 belonged to the ST3-IncHI2 group. In addition to blaCTX-M and oqxAB, pcoA-E (5/25) and silE-P (5/25), as well as aac(6')-Ib-cr (18/25), floR (16/25), rmtB (6/25), qnrS1(3/25) and fosA3 (2/25), were also identified on these IncHI2 plasmids. The plasmids carried pco and sil contributed to increasing in the MICs of CuSO4 and AgNO3. The genetic context surrounding the two operons was well conserved except some variations within the pco operon. The ~32 kb region containing the two operons identified in the IncHI2 plasmids was also found in chromosomes of different Enterobacteriaceae species. Further, phylogenetic analysis of this structure showed that Tn7-like transposon might play an important role in cross-genus transfer of the sil and pco operons among Enterobacteriaceae. In conclusion, co-existence of the pco and sil operons, and oqxAB/blaCTX-M as well as other antibiotic resistance genes on IncHI2 plasmids may promote the development of multidrug-resistant bacteria.201627143648