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523000.9727Characterization 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
121310.9678Fosfomycin 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
228420.9672Molecular 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
517130.9664Adaptive laboratory-evolved MRSA with PPEF manifests cross-susceptibility to oxacillin and hypersensitivity to ciprofloxacin. Emerging resistance to current antibiotics is a global threat to human health. Therefore, comprehending the mechanism behind antibiotic resistance holds paramount importance. In the pursuit of finding new antibacterial agents, our group has developed a small molecule, PPEF (2'-(4-ethoxyphenyl)-5-(4-propylpiperazin-1-yl)-1H,1'H-2,5'-bibenzo(d)imidazole), having bisbenzimidazole as a pharmacophore, targeting bacterial type IA topoisomerase, a novel drug target in bacteria. We examined the emergence of mutations leading to PPEF resistance in laboratory-evolved Staphylococcus aureus strains. The growth curve revealed that S. aureus 25923 PPEF-resistant (SA-PR) and methicillin-resistant S. aureus 43300 PPEF-resistant (MRSA-PR) attained stationary phase earlier than their respective reference strains. RNA sequencing analysis revealed that atpD (ATP synthase gene) was downregulated by 2 log(2)-fold in both SA-PR and MRSA-PR strains, whereas there was 10 to 13 log(2)-fold downregulation of mecR1 (methicillin resistance-inducing gene), ble (bleomycin resistance-inducing gene), blaZ (beta-lactamase), pbp (penicillin-binding protein gene), ermA (rRNA adenine methyltransferase gene), and kdpB (potassium-transporting ATPase) in the MRSA-PR strain. Quantitative reverse-transcriptase PCR data confirmed these results. Additionally, MRSA-PR showed a 5 log(2)-fold upregulation of comG and a 9 log(2)-fold downregulation of topB, indicating increased genomic variability and stress adaptation contributing to resistance. Genomic sequencing revealed deletions of resistance genes, including aac(6')-aph(2''), aadD, mecA, and blaZ in MRSA-PR, resulting in a gain in resistance and a diminishing returns epistasis pattern in PPEF-evolved S. aureus strains. This led to the development of an evolved MRSA-PR strain susceptible to oxacillin, ciprofloxacin, gentamicin, and imipenem. Our findings indicate that adaptation to PPEF has increased antibiotic susceptibility, thereby changing the clinical outcomes of infections.IMPORTANCEThis study investigates how Staphylococcus aureus bacteria, including methicillin-resistant Staphylococcus aureus (MRSA) strain, develop resistance to a new candidate antibacterial compound, PPEF (2'-(4-ethoxyphenyl)-5-(4-propylpiperazin-1-yl)-1H,1'H-2,5'-bibenzo(d)imidazole). The research found that resistant strains grew slower and showed significant changes in the activity of genes related to antibiotic resistance. Some resistance genes were deleted in the resistant MRSA strain, making it more sensitive to other antibiotics like oxacillin and ciprofloxacin. These findings highlight how resistance to PPEF leads to increased sensitivity to conventional antibiotics. This suggests that developing combination therapies of PPEF with other antibiotics could optimize treatment regimens and slow resistance evolution. This study also indicates that the antibiotic regimens could be designed to force resistant bacteria into evolutionary trade-offs, where they lose resistance to widely used antibiotics while gaining resistance to a new compound like PPEF.202540662666
523140.9661Prediction 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
140050.9659Comparative genomic analysis of Escherichia coli strains obtained from continuous imipenem stress evolution. The carbapenem-resistant Escherichia coli has aroused increasing attention worldwide, especially in terms of imipenem (IMP) resistance. The molecular mechanism of IMP resistance remains unclear. This study aimed to explore the resistance mechanisms of IMP in E. coli. Susceptible Sx181-0-1 strain was induced into resistance strains by adaptive laboratory evolution. The drug resistance spectrum was measured using the disk diffusion and microbroth dilution methods. Whole-genome sequencing and resequencing were used to analyze the nonsynonymous single-nucleotide polymorphisms (nsSNPs) between the primary susceptible strain and resistant strains. The expression levels of these genes with nsSNPs were identified by real-time quantitative PCR (RT-qPCR). Resistance phenotype appeared in the induced 15th generation (induction time = 183 h). Sx181-32 and Sx181-256, which had the minimum inhibitory concentrations of IMP of 8 and 64 µg ml-1, were isolated during continuous subculture exposed to increasing concentrations of IMP, respectively. A total of 19 nsSNPs were observed both in Sx181-32 and Sx181-256, distributed in rpsU, sdaC, zwf, ttuC, araJ, dacC, mrdA, secF, dacD, lpxD, mrcB, ftsI, envZ, and two unknown function genes (orf01892 and orf01933). Among these 15 genes, five genes (dacC, mrdA, lpxD, mrcB, and ftsI) were mainly involved in cell wall synthesis. The mrdA (V338A, L378P, and M574I) and mrcB (P784L, A736V, and T708A) had three amino acid substitutions, respectively. The expression levels of rpsU, ttuC, and orf01933 were elevated in both Sx181-32 and Sx181-256 compared to Sx181-0-1. The expression levels of these genes were elevated in Sx181-256, except for araJ. Bacteria developed resistance to antimicrobials by regulating various biological processes, among which the most involved is the cell wall synthesis (dacC, mrdA, lpxD, mrcB, and ftsI). The combination mutations of mrdA, envZ, and ftsI genes may increase the resistance to IMP. Our study could improve the understanding of the molecular mechanism of IMP resistance in E. coli.202235147175
121160.9658Molecular characterization of multidrug-resistant Escherichia coli of the phylogroups A and C in dairy calves with meningitis and septicemia. Escherichia coli is an important cause of septicemia (SEPEC) and neonatal meningitis (NMEC) in dairy calves. However, the diversity of virulence profiles, phylogroups, antimicrobial resistance patterns, carriage of integron structures, and fluoroquinolone (FQ) resistance mechanisms have not been fully investigated. Also, there is a paucity of knowledge about the virulence profiles and frequency of potential SEPEC in feces from calves with or without diarrhea. This study aimed to characterize the virulence potential, phylogroups, antimicrobial susceptibility, integron content, and FQ-resistance mechanisms in Escherichia coli isolated from calves with meningitis and septicemia. Additionally, the virulence genes (VGs) and profiles of E. coli isolated from diarrheic and non-diarrheic calves were compared between them and together with NMEC and SEPEC in order to identify shared profiles. Tissue and fluid samples from eight dairy calves with septicemia, four of which had concurrent meningitis, were processed for bacteriology and histopathology. Typing of VGs was assessed in 166 isolates from diverse samples of each calf. Selected isolates were evaluated for antimicrobial susceptibility by the disk diffusion test. Phylogroups, integron gene cassettes cartography, and FQ-resistance determinants were analyzed by PCR, sequencing, and bioinformatic tools. Furthermore, 109 fecal samples and 700 fecal isolates from dairy calves with or without diarrhea were evaluated to detect 19 VGs by uniplex PCR. Highly diverse VG profiles were characterized among NMEC and SEPEC isolates, but iucD was the predominant virulence marker. Histologic lesions in all calves supported their pathogenicity. Selected isolates mainly belonged to phylogroups A and C and showed multidrug resistance. Classic (dfrA17 and arr3-dfrA27) and complex (dfrA17-aadA5::ISCR1::bla(CTX-M-2)) class 1 integrons were identified. Target-site mutations in GyrA (S83L and D87N) and ParC (S80I) encoding genes were associated with FQ resistance. The VGs detected more frequently in fecal samples included f17G (50%), papC (30%), iucD (20%), clpG (19%), eae (16%), and afaE-8 (13%). Fecal isolates displaying the profiles of f17 or potential SEPEC were found in 25% of calves with and without diarrhea. The frequency of E. coli VGs and profiles did not differ between both groups (p > 0.05) and were identical or similar to those found in NMEC and SEPEC. Overall, multidrug-resistant E. coli isolates with diverse VG profiles and belonging to phylogroups A and C can be implicated in natural cases of meningitis and septicemia. Their resistance phenotypes can be partially explained by class 1 integron gene cassettes and target-site mutations in gyrA and parC. These results highlight the value of antimicrobial resistance surveillance in pathogenic bacteria isolated from food-producing animals. Besides, calves frequently shed potential SEPEC in their feces as commensals ("Trojan horse"). Thus, these bacteria may be disseminated in the farm environment, causing septicemia and meningitis under predisposing factors.202234982979
627270.9657Inactivation of ackA and pta Genes Reduces GlpT Expression and Susceptibility to Fosfomycin in Escherichia coli. Fosfomycin is used to treat a variety of bacterial infections, including urinary tract infections caused by Escherichia coli. In recent years, quinolone-resistant and extended-spectrum β-lactamase (ESBL)-producing bacteria have been increasing. Because fosfomycin is effective against many of these drug-resistant bacteria, the clinical importance of fosfomycin is increasing. Against this background, information on the mechanisms of resistance and the antimicrobial activity of this drug is desired to enhance the usefulness of fosfomycin therapy. In this study, we aimed to explore novel factors affecting the antimicrobial activity of fosfomycin. Here, we found that ackA and pta contribute to fosfomycin activity against E. coli. ackA and pta mutant E. coli had reduced fosfomycin uptake capacity and became less sensitive to this drug. In addition, ackA and pta mutants had decreased expression of glpT that encodes one of the fosfomycin transporters. Expression of glpT is enhanced by a nucleoid-associated protein, Fis. We found that mutations in ackA and pta also caused a decrease in fis expression. Thus, we interpret the decrease in glpT expression in ackA and pta defective strains to be due to a decrease in Fis levels in these mutants. Furthermore, ackA and pta are conserved in multidrug-resistant E. coli isolated from patients with pyelonephritis and enterohemorrhagic E. coli, and deletion of ackA and pta from these strains resulted in decreased susceptibility to fosfomycin. These results suggest that ackA and pta in E. coli contribute to fosfomycin activity and that mutation of these genes may pose a risk of reducing the effect of fosfomycin. IMPORTANCE The spread of drug-resistant bacteria is a major threat in the field of medicine. Although fosfomycin is an old type of antimicrobial agent, it has recently come back into the limelight because of its effectiveness against many drug-resistant bacteria, including quinolone-resistant and ESBL-producing bacteria. Since fosfomycin is taken up into the bacteria by GlpT and UhpT transporters, its antimicrobial activity fluctuates with changes in GlpT and UhpT function and expression. In this study, we found that inactivation of the ackA and pta genes responsible for the acetic acid metabolism system reduced GlpT expression and fosfomycin activity. In other words, this study shows a new genetic mutation that leads to fosfomycin resistance in bacteria. The results of this study will lead to further understanding of the mechanism of fosfomycin resistance and the creation of new ideas to enhance fosfomycin therapy.202337199605
158080.9656Polyclonal 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
517090.9656Synergistic effect of imp/ostA and msbA in hydrophobic drug resistance of Helicobacter pylori. BACKGROUND: Contamination of endoscopy equipment by Helicobacter pylori (H. pylori) frequently occurs after endoscopic examination of H. pylori-infected patients. In the hospital, manual pre-cleaning and soaking in glutaraldehyde is an important process to disinfect endoscopes. However, this might not be sufficient to remove H. pylori completely, and some glutaraldehyde-resistant bacteria might survive and be passed to the next patient undergoing endoscopic examination through unidentified mechanisms. We identified an Imp/OstA protein associated with glutaraldehyde resistance in a clinical strain, NTUH-C1, from our previous study. To better understand and manage the problem of glutaraldehyde resistance, we further investigated its mechanism. RESULTS: The minimal inhibitory concentrations (MICs) of glutaraldehyde andexpression of imp/ostA RNA in 11 clinical isolates from the National Taiwan University Hospital were determined. After glutaraldehyde treatment, RNA expression in the strains with the MICs of 4-10 microg/ml was higher than that in strains with the MICs of 1-3 microg/ml. We examined the full-genome expression of strain NTUH-S1 after glutaraldehyde treatment using a microarray and found that 40 genes were upregulated and 31 genes were downregulated. Among the upregulated genes, imp/ostA and msbA, two putative lipopolysaccharide biogenesis genes, were selected for further characterization. The sensitivity to glutaraldehyde or hydrophobic drugs increased in both of imp/ostA and msbA single mutants. The imp/ostA and msbA double mutant was also hypersensitive to these chemicals. The lipopolysaccharide contents decreased in individual imp/ostA and msbA mutants and dramatically reduced in the imp/ostA and msbA double mutant. Outer membrane permeability assay demonstrated that the imp/ostA and msbA double mutation resulted in the increase of outer membrane permeability. Ethidium bromide accumulation assay demonstrated that MsbA was involved in efflux of hydrophobic drugs. CONCLUSION: The expression levels of imp/ostA and msbA were correlated with glutaraldehyde resistance in clinical isolates after glutaraldehyde treatment. Imp/OstA and MsbA play a synergistic role in hydrophobic drugs resistance and lipopolysaccharide biogenesis in H. pylori.200919594901
1793100.9656Comparative Genome Analysis of an Extensively Drug-Resistant Isolate of Avian Sequence Type 167 Escherichia coli Strain Sanji with Novel In Silico Serotype O89b:H9. Extensive drug resistance (XDR) is an escalating global problem. Escherichia coli strain Sanji was isolated from an outbreak of pheasant colibacillosis in Fujian province, China, in 2011. This strain has XDR properties, exhibiting sensitivity to carbapenems but no other classes of known antibiotics. Whole-genome sequencing revealed a total of 32 known antibiotic resistance genes, many associated with insertion sequence 26 (IS26) elements. These were found on the Sanji chromosome and 2 of its 6 plasmids, pSJ_255 and pSJ_82. The Sanji chromosome also harbors a type 2 secretion system (T2SS), a type 3 secretion system (T3SS), a type 6 secretion system (T6SS), and several putative prophages. Sanji and other ST167 strains have a previously uncharacterized O-antigen (O89b) that is most closely related to serotype O89 as determined on the basis of analysis of the wzm-wzt genes and in silico serotyping. This O89b-antigen gene cluster was also found in the genomes of a few other pathogenic sequence type 617 (ST617) and ST10 complex strains. A time-scaled phylogeny inferred from comparative single nucleotide variant analysis indicated that development of these O89b-containing lineages emerged about 30 years ago. Comparative sequence analysis revealed that the core genome of Sanji is nearly identical to that of several recently sequenced strains of pathogenic XDR E. coli belonging to the ST167 group. Comparison of the mobile elements among the different ST167 genomes revealed that each genome carries a distinct set of multidrug resistance genes on different types of plasmids, indicating that there are multiple paths toward the emergence of XDR in E. coli. IMPORTANCE E. coli strain Sanji is the first sequenced and analyzed genome of the recently emerged pathogenic XDR strains with sequence type ST167 and novel in silico serotype O89b:H9. Comparison of the genomes of Sanji with other ST167 strains revealed distinct sets of different plasmids, mobile IS elements, and antibiotic resistance genes in each genome, indicating that there exist multiple paths toward achieving XDR. The emergence of these pathogenic ST167 E. coli strains with diverse XDR capabilities highlights the difficulty of preventing or mitigating the development of XDR properties in bacteria and points to the importance of better understanding of the shared underlying virulence mechanisms and physiology of pathogenic bacteria.201930834329
5168110.9655Bacteriophage Resistance Affects Flavobacterium columnare Virulence Partly via Mutations in Genes Related to Gliding Motility and the Type IX Secretion System. Increasing problems with antibiotic resistance have directed interest toward phage therapy in the aquaculture industry. However, phage resistance evolving in target bacteria is considered a challenge. To investigate how phage resistance influences the fish pathogen Flavobacterium columnare, two wild-type bacterial isolates, FCO-F2 and FCO-F9, were exposed to phages (FCO-F2 to FCOV-F2, FCOV-F5, and FCOV-F25, and FCO-F9 to FCL-2, FCOV-F13, and FCOV-F45), and resulting phenotypic and genetic changes in bacteria were analyzed. Bacterial viability first decreased in the exposure cultures but started to increase after 1 to 2 days, along with a change in colony morphology from original rhizoid to rough, leading to 98% prevalence of the rough morphotype. Twenty-four isolates (including four isolates from no-phage treatments) were further characterized for phage resistance, antibiotic susceptibility, motility, adhesion, and biofilm formation, protease activity, whole-genome sequencing, and virulence in rainbow trout fry. The rough isolates arising in phage exposure were phage resistant with low virulence, whereas rhizoid isolates maintained phage susceptibility and high virulence. Gliding motility and protease activity were also related to the phage susceptibility. Observed mutations in phage-resistant isolates were mostly located in genes encoding the type IX secretion system, a component of the Bacteroidetes gliding motility machinery. However, not all phage-resistant isolates had mutations, indicating that phage resistance in F. columnare is a multifactorial process, including both genetic mutations and changes in gene expression. Phage resistance may not, however, be a challenge for development of phage therapy against F. columnare infections since phage resistance is associated with decreases in bacterial virulence. IMPORTANCE Phage resistance of infectious bacteria is a common phenomenon posing challenges for the development of phage therapy. Along with a growing world population and the need for increased food production, constantly intensifying animal farming has to face increasing problems of infectious diseases. Columnaris disease, caused by Flavobacterium columnare, is a worldwide threat for salmonid fry and juvenile farming. Without antibiotic treatments, infections can lead to 100% mortality in a fish stock. Phage therapy of columnaris disease would reduce the development of antibiotic-resistant bacteria and antibiotic loads by the aquaculture industry, but phage-resistant bacterial isolates may become a risk. However, phenotypic and genetic characterization of phage-resistant F. columnare isolates in this study revealed that they are less virulent than phage-susceptible isolates and thus not a challenge for phage therapy against columnaris disease. This is valuable information for the fish farming industry globally when considering phage-based prevention and curing methods for F. columnare infections.202134106011
5065120.9654Locus of Heat Resistance (LHR) in Meat-Borne Escherichia coli: Screening and Genetic Characterization. Microbial resistance to processing treatments poses a food safety concern, as treatment tolerant pathogens can emerge. Occasional foodborne outbreaks caused by pathogenic Escherichia coli have led to human and economic losses. Therefore, this study screened for the extreme heat resistance (XHR) phenotype as well as one known genetic marker, the locus of heat resistance (LHR), in 4,123 E. coli isolates from diverse meat animals at different processing stages. The prevalences of XHR and LHR among the meat-borne E. coli were found to be 10.3% and 11.4%, respectively, with 19% agreement between the two. Finished meat products showed the highest LHR prevalence (24.3%) compared to other processing stages (0 to 0.6%). None of the LHR(+)E. coli in this study would be considered pathogens based on screening for virulence genes. Four high-quality genomes were generated by whole-genome sequencing of representative LHR(+) isolates. Nine horizontally acquired LHRs were identified and characterized, four plasmid-borne and five chromosomal. Nine newly identified LHRs belong to ClpK1 LHR or ClpK2 LHR variants sharing 61 to 68% nucleotide sequence identity, while one LHR appears to be a hybrid. Our observations suggest positive correlation between the number of LHR regions present in isolates and the extent of heat resistance. The isolate exhibiting the highest degree of heat resistance possessed four LHRs belonging to three different variant groups. Maintenance of as many as four LHRs in a single genome emphasizes the benefits of the LHR in bacterial physiology and stress response.IMPORTANCE Currently, a "multiple-hurdle" approach based on a combination of different antimicrobial interventions, including heat, is being utilized during meat processing to control the burden of spoilage and pathogenic bacteria. Our recent study (M. Guragain, G. E. Smith, D. A. King, and J. M. Bosilevac, J Food Prot 83:1438-1443, 2020, https://doi.org/10.4315/JFP-20-103) suggests that U.S. beef cattle harbor Escherichia coli that possess the locus of heat resistance (LHR). LHR seemingly contributes to the global stress tolerance in bacteria and hence poses a food safety concern. Therefore, it is important to understand the distribution of the LHRs among meat-borne bacteria identified at different stages of different meat processing systems. Complete genome sequencing and comparative analysis of selected heat-resistant bacteria provide a clearer understanding of stress and heat resistance mechanisms. Further, sequencing data may offer a platform to gain further insights into the genetic background that provides optimal bacterial tolerance against heat and other processing treatments.202133483306
449130.9653Oxygen-insensitive nitroreductases: analysis of the roles of nfsA and nfsB in development of resistance to 5-nitrofuran derivatives in Escherichia coli. Nitroheterocyclic and nitroaromatic compounds constitute an enormous range of chemicals whose potent biological activity has significant human health and environmental implications. The biological activity of nitro-substituted compounds is derived from reductive metabolism of the nitro moiety, a process catalyzed by a variety of nitroreductase activities. Resistance of bacteria to nitro-substituted compounds is believed to result primarily from mutations in genes encoding oxygen-insensitive nitroreductases. We have characterized the nfsA and nfsB genes of a large number of nitrofuran-resistant mutants of Escherichia coli and have correlated mutation with cell extract nitroreductase activity. Our studies demonstrate that first-step resistance to furazolidone or nitrofurazone results from an nfsA mutation, while the increased resistance associated with second-step mutants is a consequence of an nfsB mutation. Inferences made from mutation about the structure-function relationships of NfsA and NfsB are discussed, especially with regard to the identification of flavin mononucleotide binding sites. We show that expression of plasmid-carried nfsA and nfsB genes in resistant mutants restores sensitivity to nitrofurans. Among the 20 first-step and 53 second-step mutants isolated in this study, 65 and 49%, respectively, contained insertion sequence elements in nfsA and nfsB. IS1 integrated in both genes, while IS30 and IS186 were found only in nfsA and IS2 and IS5 were observed only in nfsB. Insertion hot spots for IS30 and IS186 are indicated in nfsA, and a hot spot for IS5 insertion is evident in nfsB. We discuss potential regional and sequence-specific determinants for insertion sequence element integration in nfsA and nfsB.19989791100
1212140.9651Virulence Factors and Antimicrobial Resistance of Uropathogenic Escherichia coli EQ101 UPEC Isolated from UTI Patient in Quetta, Balochistan, Pakistan. Infectious diseases have been tremendously increasing as the organisms of even normal flora become opportunistic and cause an infection, and Escherichia coli (E. coli EQ101) is one of them. Urinary tract infections are caused by various microorganisms, but Escherichia coli is the primary cause of almost 70%-90% of all UTIs. It has multiple strains, possessing diverse virulence factors, contributing to its pathogenicity. Furthermore, these virulent strains also can cause overlapping pathogenesis by sharing resistance and virulence factors among each other. The current study is aimed at analyzing the genetic variants associated with multi-drug-resistant (MDR) E. coli using the whole genome sequencing platform. The study includes 100 uropathogenic Escherichia coli (UPEC) microorganisms obtained from urine samples out of which 44% were multi-drug-resistant (MDR) E. coli. Bacteria have been isolated and antimicrobial susceptibility test (AST) was determined by disk diffusion method on the Mueller-Hinton agar plate as recommended by the Clinical and Laboratory Standards Institute (CLSI) 2020, and one isolate has been selected which shows resistance to most of the antibiotics, and that isolate has been analyzed by whole genome sequencing (WGS), accompanied by data and phylogenetic analysis, respectively. Organisms were showing resistance against ampicillin (10 μg), cefixime (5 μg), ceftriaxone (30 μg), nalidixic acid (30 μg), ciprofloxacin (5 μg), and ofloxacin (5 μg) on antimicrobial susceptibility test. WGS were done on selected isolate which identified 25 virulence genes (air, astA, chuA, fyuA, gad, hra, iha, irp2, iss, iucC, iutA, kpsE, kpsMII_K1, lpfA, mchF, ompT, papA_F43, sat, senB, sitA, terC, traT, usp, vat, and yfcV) and seven housekeeping genes (adk, fumC, gyrB, icd, mdh, purA, and recA). Among resistance genes, seven genes (TolC, emrR, evgA, qacEdelta1, H-NS, cpxA, and mdtM) were identified to be involved in antibiotic efflux, three AMR genes (aadA5, mphA, and CTX-M-15) were involved in antibiotic inactivation, and two genes (sul1 and dfrA14) were found to be involved in antibiotic drug replacement. Our data identified antibiotic resistance and virulence genes of the isolate. We suggest further research work to establish region-based resistance profile in comparison with the global resistance pattern.202337727279
2336150.9651Distribution of disinfectant resistant genes in mcr-1-carrying Escherichia coli isolated from children in southern China. BACKGROUND: Colistin, a polymyxin antibiotic, serves as a crucial defense against multidrug-resistant gram-negative bacteria, despite its nephrotoxicity. However, the plasmid-mediated mobilization of the polymyxin resistance gene, mcr-1, presents a significant public health threat. The widespread use of disinfectants has resulted in Escherichia coli (E. coli) carrying mcr-1 also showing disinfectant resistance. The aim of this study is to investigate the distribution of disinfectant genes and resistance to disinfectants in mcr-1-carring E coli from children in the South China. METHODS: We evaluated the distribution of twelve disinfectant-resistance genes by PCR. Evaluated the correlation between disinfectant-resistance genes and resistance to disinfectants and antibiotics. We also examined the correlation between the strains' biofilm formation and the presence of disinfectant-resistance genes. Bioinformatic tools were employed to analyze resistance genes, virulence genes, and insertion sequences. Five strains were randomly selected to examine the effects of sub-inhibitory concentration (sub-MIC) of 8 disinfectants on the expression of the mcr-1 gene by qRT-PCR. RESULTS: The most prevalent of the nine biocide resistance genes were mdfA, sugE(c), ydgE, and ydgF (n = 21; all 100 %). The qacG, qacF, sugE(p) and tehA gene was not detected. Furthermore, benzalkonium chloride (BC) and potassium hydrogen persulfate (PMPS)-based disinfectants were effective against all mcr-1-carrying E. coli strains. The majority of mcr-1 were distributed among the InHI2 plasmid types, although three strains lacked mcr-1 on their plasmids. Biofilm formation was observed in 48 % of the strains. emrD and sitABCD showed significant associations with the susceptibility of the strains to 84 disinfectants (P of 0.0351 and 0.0300). In addition, sitABCD was significantly associated with susceptibility to povidone-iodine (PVP-I) (P value of 0.0062). Compared to the untreated group, stimulation with sub-MIC of peracetic acid (PAA) and PVP-I resulted in decreased or increased mcr-1 expression in five E. coli strains, respectively (P of 0.0011 for PAA and P of 0.0476 for PVP-I). CONCLUSION: BC and PMPS based disinfectants were effective against all mcr-1 carrying E. coli strains. Most of the mcr-1 genes were distributed among the InHI2 plasmid types. The emrD and sitABCD genes are highly associated with resistance to 84 disinfectants, and the sitABCD gene was highly associated with resistance to PVP-I. PVP-I selective pressure may encourage the maintenance of mcr-1 gene in E. coli.202539551109
6364160.9650Characterization of clumpy adhesion of Escherichia coli to human cells and associated factors influencing antibiotic sensitivity. Escherichia coli intestinal infection pathotypes are characterized by distinct adhesion patterns, including the recently described clumpy adhesion phenotype. Here, we identify and characterize the genetic factors contributing to the clumpy adhesion of E. coli strain 4972. In this strain, the transcriptome and proteome of adhered bacteria were found to be distinct from planktonic bacteria in the supernatant. A total of 622 genes in the transcriptome were differentially expressed in bacteria present in clumps relative to the planktonic bacteria. Seven genes targeted for disruption had variable distribution in different pathotypes and nonpathogenic E. coli, with the pilV and spnT genes being the least frequent or absent from most groups. Deletion (Δ) of five differentially expressed genes, flgH, ffp, pilV, spnT, and yggT, affected motility, adhesion, or antibiotic stress. ΔflgH exhibited 80% decrease and ΔyggT depicted 184% increase in adhesion, and upon complementation, adhesion was significantly reduced to 13%. ΔflgH lost motility and was regenerated when complemented, whereas Δffp had significantly increased motility, and reintroduction of the same gene reduced it to the wild-type level. The clumps produced by Δffp and ΔspnT were more resistant and protected the bacteria, with ΔspnT showing the best clump formation in terms of ampicillin stress protection. ΔyggT had the lowest tolerance to gentamicin, where the antibiotic stress completely eliminated the bacteria. Overall, we were able to investigate the influence of clump formation on cell surface adhesion and antimicrobial tolerance, with the contribution of several factors crucial to clump formation on susceptibility to the selected antibiotics. IMPORTANCE: The study explores a biofilm-like clumpy adhesion phenotype in Escherichia coli, along with various factors and implications for antibiotic susceptibility. The phenotype permitted the bacteria to survive the onslaught of high antibiotic concentrations. Profiles of the transcriptome and proteome allowed the differentiation between adhered bacteria in clumps and planktonic bacteria in the supernatant. The deletion mutants of genes differentially expressed between adhered and planktonic bacteria, i.e., flgH, ffp, pilV, spnT, and yggT, and respective complementations in trans cemented their roles in multiple capacities. ffp, an uncharacterized gene, is involved in motility and resistance to ampicillin in a clumpy state. The work also affirms for the first time the role of the yggT gene in adhesion and its involvement in susceptibility against another aminoglycoside antibiotic, i.e., gentamicin. Overall, the study contributes to the mechanisms of biofilm-like adhesion phenotype and understanding of the antimicrobial therapy failures and infections of E. coli.202438530058
5066170.9650Genetic Alterations Associated with Colistin Resistance Development in Escherichia coli. Background: The increased incidence of infections due to multidrug-resistant Gram-negative bacteria has led to the renewed interest in the use of 'forgotten' antibiotics such as colistin. In this work, we studied the chromosomal colistin resistance mechanisms among laboratory-induced colistin-resistant Escherichia coli isolates. Methods: Three colistin-susceptible (ColS) clinical isolates of E. coli assigning to ST131, ST405, and ST361 were exposed to successively increasing concentrations of colistin. The nucleotide sequences of pmrA, pmrB, pmrD, phoP, phoQ, and mgrB genes were determined. The fitness burden associated with colistin resistance acquisition was determined by measuring the in vitro growth rate. Results: Colistin resistance induction resulted in 16-64 times increase in colistin MICs in mutants (n = 8) compared with parental isolates. Analysis of chromosomal genes in colistin-resistant mutants compared with those of ColS ancestors revealed genetic alterations confined to PmrAB two-component system and included PmrA G53R/R81S/L105P and PmrB E121K/E121A/A159P/A159V/G302E changes. The PmrB E121 was found as a critical position for colistin resistance development being altered in three mutants with different ancestors. The acquired colistin-resistance phenotype was stable following 10 consecutive passages in the absence of selective pressure of colistin and it did not alter the susceptibility of mutants to other antimicrobial agents. All mutants exhibited growth rates similar to their respective ColS ancestors, except for one isolate, which revealed a significant growth defect. Conclusion: Our results revealed that colistin resistance in E. coli was more related to PmrAB alterations, which did not impose a fitness cost in most cases.202438905152
5032180.9650Hijacking a small plasmid to confer high-level resistance to aztreonam-avibactam and ceftazidime-avibactam. Acquired β-lactamase-encoding genes are typically carried by large plasmids in Gram-negative bacteria, which also commonly carry multi-copy small plasmids. This study found that mobile genetic elements carrying antimicrobial resistance genes are capable of hijacking small plasmids. This study focused on aztreonam-avibactam (ATM-AVI) as this combination can be used to effectively counter almost all β-lactamases produced by bacteria, and has been recommended against carbapenem-resistant Enterobacterales. A clinical strain (085003) of carbapenem-resistant Escherichia coli was investigated, and mutants (085003R32 and 085003R512) able to grow under 32/4 and 512/4 mg/L of ATM-AVI were obtained as representatives of low- and high-level resistance, respectively, by induction. Comparative genomics showed that 085003R32 and 085003R512 had a single nucleotide mutation of β-lactamase gene bla(CMY-2), encoding a novel CMY with a Thr319Ile substitution, assigned 'CMY-2R'. Cloning and enzyme kinetics were used to verify that CMY-2R conferred ATM-AVI resistance by compromising binding of AVI and subsequent protection of ATM. Mechanisms for the discrepant resistance between 085003R32 and 085003R512 were investigated. Three tandem copies of bla(CMY-2R) were identified on a self-transmissible IncP1 plasmid of 085003R32 due to IS1294 misrecognizing its end terIS and rolling-circle replication. 085003R512 had only a single copy of bla(CMY-2R) on the IncP1 plasmid, but possessed anther bla(CMY-2R) on an already present 4-kb small plasmid. IS1294-mediated mobilization on to this multi-copy small plasmid increased the copy number of bla(CMY-2R) significantly, rendering higher resistance. This study shows that bacteria can employ multiple approaches to accommodate selection pressures imposed by exposure to varied concentrations of antimicrobial agents.202337769749
9042190.9650Resistance of Francisella novicida to fosmidomycin associated with mutations in the glycerol-3-phosphate transporter. The methylerythritol phosphate (MEP) pathway is essential in most prokaryotes and some lower eukaryotes but absent from human cells, and is a validated target for antimicrobial drug development. The formation of MEP is catalyzed by 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR). MEP pathway genes have been identified in many category A and B biothreat agents, including Francisella tularensis, which causes the zoonosis tularemia. Fosmidomycin (Fos) inhibits purified Francisella DXR. This compound also inhibits the growth of F. tularensis NIH B38, F. novicida and F. tularensis subsp. holarctica LVS bacteria. Related compounds such as FR900098 and the lipophilic prodrug of FR900098 (compound 1) have been developed to improve the bioavailability of these DXR inhibitors. In performing disk-inhibition assays with these compounds, we observed breakthrough colonies of F. novicida in the presence of Fos, suggesting spontaneous development of Fos resistance (Fos(R)). Fos(R) bacteria had decreased sensitivity to both Fos and FR900098. The two most likely targets for the development of mutants would be the DXR enzyme itself or the glycerol-3-phosphate transporter (GlpT) that allows entry of Fos into the bacteria. Sensitivity of Fos(R)F. novicida bacteria to compound 1 was not abated suggesting that spontaneous resistance is not due to mutation of DXR. We thus predicted that the glpT transporter may be mutated leading to this resistant phenotype. Supporting this, transposon insertion mutants at the glpT locus were also found to be resistant to Fos. DNA sequencing of four different spontaneous Fos(R) colonies demonstrated a variety of deletions in the glpT coding region. The overall frequency of Fos(R) mutations in F. novicida was determined to be 6.3 × 10(-8). Thus we conclude that one mechanism of resistance of F. novicida to Fos is caused by mutations in GlpT. This is the first description of spontaneous mutations in Francisella leading to Fos(R).201222905031