Fosfomycin Resistance Gene fosA3 Research Articles

Investigation of Fosfomycin Resistance and Plasmid-mediated Fosfomycin Resistance Genes in Enterobacterales Isolates

The study aimed to investigate the fosfomycin resistance in Enterobacterales isolates and the presence of plasmid-mediated fosfomycin resistance genes fosA3 and fosC2 in fosfomycin-resistant Enterobacterales isolates. A total of 2095 Enterobacterales isolates obtained from urine samples were included in the study. Identification of isolates was performed in the Vitek MS. The antimicrobial susceptibility of the isolates and detection of ESBL was determined using the Vitek 2 Compact. In 185 fosfomycin-resistant isolates, fosA3 and fosC2 genes were investigated by PCR. In addition, carbapenemase genes were investigated in carbapenem-resistant isolates. In 2095 Enterobacterales, 65.8% of the isolates were E. coli, and fosfomycin resistance was determined as 22%. ESBL production was defined as 36.5%, 58.1%, and 32.5% in all isolates, K. pneumoniae, and E. coli isolates, respectively. K. pneumoniae (56.2%) were the most frequently isolated bacteria among 185 fosfomycin-resistant Enterobacterales isolates. In these isolates, 66.95% were ESBL-producing Enterobacterales, and 35.7% were carbapenem-resistant isolates. According to the PCR results, two of the 185 Enterobacterales isolates were positive for the fosA3 gene, fosC2 gene was not detected. According to PCR for carbapenemase genes results with these isolates, blaOXA–48 positivity was 66.7%, and blaNDM positivity was 3%. blaKPC and blaVIM positivity could not be detected. In our study, the prevalence of fosA3 was 1.1%. The occurrence of fosA3 gene positivity in Enterobacterales isolates is important for the future treatment of UTIs.

Investigation of O25b-ST131 Clone Frequency and Presence of Carbapenem and Fosfomycin Resistance Genes in Escherichia coli Isolates: First Detection of fosA3 from Escherichia coli O25bSTf131 Clone from Türkiye

Escherichia coli ST131 clone was first reported in 2008 and defined as a 'high-risk pandemic clone'. This clone plays a critical role in the spread of antimicrobial resistance worldwide. It was reported in many studies that the E.coli ST131 clone is widespread worldwide and can carry virulence and antibiotic resistance genes. E.coli ST131 clone is associated with a fluoroquinolone and broad-spectrum cephalosporin resistance. The agents used in the treatment of infections caused by the E.coli ST131 clone are limited. For this reason, monitoring the resistance status of these limited agents is crucial. In this study, we aimed to investigate the prevalence of the O25b-ST131 clone, and the presence of carbapenem and fosfomycin resistance genes in fluoroquinolone-resistant E.coli isolates isolated from mid-stream urine samples. For the detection of the O25b-ST131 clone in fluoroquinolone-resistant E.coli isolates, amplification was performed with primers O25pabBspe-F and O25pabBspe-R. For the determination of resistance genes, carbapenem resistance genes blaNDM, blaVIM, blaKPC, blaIMP, and blaOXA-48 in carbapenem resistant isolates and plasmid-mediated fosfomycin resistance genes fosA3 and fosC2 in fosfomycin resistant isolates were investigated by multiplex PCR method. According to PCR results, the prevalence of E.coli O25b-ST131 isolates was 51.2%. Carbapenem resistance rate was 3.41%, and fosfomycin resistance rate was 3.41% in fluoroquinolone-resistant E.coli isolates. Carbapenem and fosfomycin resistance rates in E.coli O25bST131 isolates were determined as 0.83% and 2.5%, respectively. At least one of the carbapenem-resistance genes (blaNDM and/or blaOXA-48) was detected in six of the eight carbapenem-resistant isolates. Fosfomycin resistance gene fosA3 was seen in four of eight fosfomycin-resistant isolates. fosC2 gene was not detected in any of the isolates. In addition, the plasmid-mediated fosfomycin resistance gene fosA3 was detected in an E.coli O25b-ST131 isolate, and this result was confirmed by sequence analysis. To the best of our knowledge, this is the second report about fosA3 positivity in E.coli ST131 isolates from the world and the first reported from Europe and Türkiye. As a result, approximately half of the fluoroquinolone-resistant E.coli isolates were identified as E.coli ST131 clones. Despite the high rate of this clone, the carbapenem and fosfomycin resistance rates are still relatively low, which is pleasing for the future of treatment. However, it should not be forgotten that resistance rates and the prevalence of resistance genes should be constantly monitored.

Emergence of fosfomycin resistance among isolates of multidrug-resistant Enterobacter species at a tertiary care hospital in Dhaka, Bangladesh

Background: The emergence of multidrug-resistant Enterobacter species as a worrying resistant pathogen seriously threatened human health. The rising rate of resistance to commonly used antibiotics limit the choice hence it is urgent to evaluate the antimicrobial activity of older drug like Fosfomycin. Objective: The study aimed to seek the frequency of fosfomycin resistance in the clinical isolates of Enterobacter species and to detect the fosfomycin resistance gene along with antibiotic resistance pattern. Methods: This cross-sectional study was conducted in the Department of Microbiology of a tertiary care hospital in Bangladesh from July 2018 to June 2019. Enterobacter spp. was isolated from a total of 350 samples by a standard microbiological method. Antibiotic susceptibility was performed by the disk diffusion technique. Antibiotic susceptibility and minimum inhibitory concentration (MIC) of fosfomycin were determined by the agar dilution method. Fosfomycin resistance gene fosA, fosA3, fosA4, fosA5, fosB, fosB2, fosC, fosC2 and fosX among fosfomycin‑resistant Enterobacter spp detected by polymerase chain reaction (PCR) using specific primer. Sequencing of fosA and fosA₅ was performed by capillary method, and the nucleotide sequence of fosA₅ has been deposited to GenBank. Results: Out of 28 Enterobacter spp. 7 (25%) fosfomycin resistant Enterobacter spp. were detected by agar dilution method. Out of 7 fosfomycin-resistant strains, 4 (57.14%) were isolated from urine samples. Fifteen (53.57%) isolates of Enterobacter spp. were multidrug-resistant detected by disc diffusion technique. All of the fosfomycin-resistant isolates were MDR. A significant rise in the MIC was found between 256µg/ml- ≥4096 µg/ml to fosfomycin. PCR revealed that 100% of fosfomycin resistant isolates are positive for fosA, 71.43% and 28.57% were positive for fosA₅ and fosB₂ respectively. Sequencing of fosA₅ gene established the FosA family fosfomycin resistance glutathione transferase gene. Conclusion: The results of this study showed a high proportion of fosfomycin resistance among multidrug-resistant Enterobacter spp. irrespective of fosfomycin usage in Bangladesh. FosA family fosfomycin resistance glutathione transferase gene emerging in Bangladesh. Bangladesh Medical Res Counc Bull 2022; 48(3): 203-210

The Co-occurrence of mcr-3 and fosA3 in IncP plasmid in ST131 Escherichia coli: A novel case.

Plasmid-mediated colistin resistance genes, especially mcr-3 combined with the fosfomycin resistance gene fosA3, are a grave health concern. Our study was designed to determine the epidemiological characteristics of the combination of mcr-3 and fosA3 in Anhui province, China. A total of 127 multi-drug-resistant (MDR) E. coli strains were assessed for antibiotic resistance/sensitivity to detect mcr-3 and fosA3 using polymerase chain reaction (PCR) and sequencing. The genes of interest were conjugated using EC600, and replicon and sequence types (STs) were identified by PCR-based replicon typing (PBRT) and multilocus sequence typing (MLST). Cluster similarity and genomic relatedness among the positive isolates were confirmed by Xbal PFGE. The processed E. coli isolates were highly resistant to the tested antibiotics; the prevalence of mcr-3 was 0.78% in the transferable IncP-type plasmid in ST131, whereas fosA3 prevalence was 38.58% among different transferable plasmids, including IncFIIK, IncFII and IncA/C, and in various STs including ST69, ST1193, ST12, ST46, ST57, ST1196, ST38, ST95, ST131, ST7584 and ST10184. Both were successfully transferred to EC600. The Xbal PFGE cluster exposed similarities among the STs. Our results show that to control the spread of colistin and fosfomycin resistance genes in human pathogens, the ban on colistin must be continued in animal feeding farms not only in China but around the world; additionally, awareness platforms on the use of colistin must be implemented and strict policies in poultry and pig farms must be maintained. Furthermore, fosfomycin misuse by patients and overuse by physicians must be strictly managed to stop the spread of fosfomycin resistance.

Occurrence and molecular epidemiology of fosA3-bearing Escherichia coli from ducks in Shandong province of China

The plasmid-borne fosfomycin resistance gene fosA3 has been identified in Escherichia coli (E. coli) from various animals but has rarely been reported in ducks. In this study, we investigated the fosA3 prevalence and molecular characteristics of fosA3-harboring E. coli strains from ducks in Shandong province of China. In 416 E. coli isolates, 91 (21.88%) were identified as fosA3-bearing strains, and the fosfomycin-resistant phenotype of 88 of the 91 fosA3-harboring strains was successfully transferred to the recipient strains. Seven different genetic structures surrounding the fosA3 gene were detected and 2 new contexts were discovered among the fosA3-carrying E. coli. Twenty fosA3-harboring isolates and their trans-conjugants were randomly selected for pulsed-field gel electrophoresis (PFGE) typing and S1-nuclease PFGE, respectively. The PFGE patterns revealed that the 20 randomly selected fosA3-bearing isolates were not a result of clonal dissemination. S1-PFGE showed that 15 of the 20 randomly selected trans-conjugants carried a single plasmid, and these 15 plasmids that harbored fosA3 (55–190 kb) were distributed into the following replicon types: IncF (n = 11), IncI1 (n = 1), IncN (n = 1), untypable (n = 1), and W-FIC (n = 1). Additionally, as vectors for fosA3 in E. coli, F-:A1:B6, N/ST1, IncI1/ST2, W-FIC, and one untypable plasmid had never been reported before. These observations highlighted the importance of ducks as a reservoir for multidrug-resistant fosA3-carrying E. coli.

Prevalence and Antibiotic Resistance Characteristics of Extraintestinal Pathogenic Escherichia coli among Healthy Chickens from Farms and Live Poultry Markets in China.

Simple SummaryChicken meat has been proved to be a suspected source of extraintestinal pathogenic Escherichia coli (ExPEC), causing several diseases in humans, and bacteria in healthy chickens can contaminate chicken carcasses at the slaughter; however, reports about the prevalence and molecular characteristics of ExPEC in healthy chickens are still rare. In this study, among 926 E. coli isolates from healthy chickens in China, 22 (2.4%) were qualified as ExPEC and these ExPEC isolates were clonally unrelated. A total of six serogroups were identified in this study, with O78 being the most predominant type, and all the six serogroups had been frequently reported in human ExPEC isolates in many countries. All the 22 ExPEC isolates were multidrug-resistant and most isolates carried both blaCTX-M and fosA3 resistance genes. Notably, plasmid-borne colistin resistance gene mcr-1 was identified in six ExPEC isolates, among which two carried additional carbapenemase gene blaNDM, compromising both the efficacies of the two critically important drugs for humans, carbapenems and colistin. These results highlight that healthy chickens can serve as a potential reservoir for multidrug resistant ExPEC isolates, including mcr-1-containing ExPEC.Chicken products and chickens with colibacillosis are often reported to be a suspected source of extraintestinal pathogenic Escherichia coli (ExPEC) causing several diseases in humans. Such pathogens in healthy chickens can also contaminate chicken carcasses at the slaughter and then are transmitted to humans via food supply; however, reports about the ExPEC in healthy chickens are still rare. In this study, we determined the prevalence and characteristics of ExPEC isolates in healthy chickens in China. A total of 926 E. coli isolates from seven layer farms (371 isolates), one white-feather broiler farm (78 isolates) and 17 live poultry markets (477 isolates from yellow-feather broilers) in 10 cities in China, were isolated and analyzed for antibiotic resistance phenotypes and genotypes. The molecular detection of ExPEC among these healthy chicken E. coli isolates was performed by PCRs, and the serogroups and antibiotic resistance characteristics of ExPEC were also analyzed. Pulsed-field gel electrophoresis (PFGE) and Multilocus sequence typing (MLST) were used to analyze the genetic relatedness of these ExPEC isolates. We found that the resistance rate for each of the 15 antimicrobials tested among E. coli from white-feather broilers was significantly higher than that from brown-egg layers and that from yellow-feather broilers in live poultry markets (p < 0.05). A total of 22 of the 926 E. coli isolates (2.4%) from healthy chickens were qualified as ExPEC, and the detection rate (7.7%, 6/78) of ExPEC among white-feather broilers was significantly higher than that (1.6%, 6/371) from brown-egg layers and that (2.1%, 10/477) from yellow-feather broilers (p < 0.05). PFGE and MLST analysis indicated that clonal dissemination of these ExPEC isolates was unlikely. Serogroup O78 was the most predominant type among the six serogroups identified in this study, and all the six serogroups had been frequently reported in human ExPEC isolates in many countries. All the 22 ExPEC isolates were multidrug-resistant (MDR) and the resistance rates to ampicillin (100%) and sulfamethoxazole-trimethoprim (100%) were the highest, followed by tetracycline (95.5%) and doxycycline (90.9%). blaCTX-M was found in 15 of the 22 ExPEC isolates including 10 harboring additional fosfomycin resistance gene fosA3. Notably, plasmid-borne colistin resistance gene mcr-1 was identified in six ExPEC isolates in this study. Worryingly, two ExPEC isolates were found to carry both mcr-1 and blaNDM, compromising both the efficacies of carbapenems and colistin. The presence of ExPEC isolates in healthy chickens, especially those carrying mcr-1 and/or blaNDM, is alarming and will pose a threat to the health of consumers. To our knowledge, this is the first report of mcr-1-positive ExPEC isolates harboring blaNDM from healthy chickens.

Prevalence and mechanisms of fosfomycin resistance among KPC-producing Klebsiella pneumoniae clinical isolates in China

The threat of antibiotic resistance has increased dramatically in recent years. Fosfomycin, an old antibiotic agent, has been re-introduced to fight infections caused by Klebsiella pneumoniae carbapenemase-producing K. pneumoniae (KPC-KP). However, the trend of fosfomycin resistance among KPC-KP strains is increasing. In this study, 80 KPC-KP clinical isolates were collected from three teaching hospitals during 2014-2017 in China and were subjected to whole-genome sequencing (WGS). The fosfomycin resistance phenotype and resistance mechanisms were investigated by antimicrobial susceptibility testing and carbon source growth test, respectively. Among all KPC-KP strains, 80.0% (64/80) were resistant to fosfomycin and 36.3% (29/80) were positive for the mobile fosfomycin resistance gene fosA3. Among the 63 strains that were unable to grow in M9 basic medium with glycerol-3-phosphate (G3P) as the sole carbon source (mediated by mutation of the target gene glpT), there was no significant difference regarding the MIC distribution of fosfomycin between fosA3-positive and fosA3-negative strains (P=0.577). Among the 50 strains that were negative for fosA3 but positive for fosA, the fosfomycin MICs of strains unable to grow in M9 basic medium with G3P as the sole carbon source were significantly higher (P < 0.001) than in strains that were able to grow in M9 basic medium with G3P as the sole carbon source. Our findings indicate that fosfomycin resistance among KPC-KP in China is an emerging problem and the two major mechanisms of resistance identified were plasmid-mediated fosfomycin resistance gene fosA3 and mutation of the target gene glpT.

Characterization 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.

Emergence of mcr- and fosA3-mediated colistin and fosfomycin resistance among carbapenem-resistant Acinetobacter baumannii in Iraq

IntroductionAcinetobacter baumannii infections have spread in healthcare and community settings with extensive drug-resistance in various locations. Considering importance of source tracking, our aim was molecular characterization and genetic analysis of fosfomycin- and colistin-resistant A. baumannii. MethodsIn this study, 205 A. baumannii were identified from hospital settings during 2014–2018. Their drug resistance pattern was assessed using disk diffusion method as per clinical and laboratory standards institute (CLSI). The colistin, imipenem and meropenem minimum inhibitory concentrations (MICs) were determined using broth microdilution method. Among multidrug-resistant strains, colistin and fosfomycin resistance genes (mcr1-mcr4 and fosA3, respectively) were screened by polymerase chain reaction (PCR) technique. Genetic relation of isolates was evaluated using pulsed field gel electrophoresis (PFGE) method. ResultsOf 205 A. baumannii, the resistance rate against carbapenems, fosfomycin and colistin included 95%, 30% and 32%, respectively. The colistin MIC≥4 μg/ml was observed among 26.34% of them. The fosA3 and mcr-1 genes were detected in 7% (n = 15) and 11% (n = 22) of them, but mcr-2, mcr-3 and mcr-4 were not detected. The strains exhibited 13 pulsotypes (A-M). Hence, the genotypic patterns were different in each hospital of Baghdad city. ConclusionIn this study, we observed an increase in the rate of fosfomycin- and colistin-resistant A. baumannii in Baghdad, Iraq during 2014–2018. The fosA3 and mcr-1 participated in fosfomycin and colistin resistance, respectively. Major risk factors for isolation of colistin-resistant A. baumannii included prior six months consumption of cephalosporins and carbapenems and previous two months of hospitalization. PFGE results unraveled 13 pulsotypes (A-M) without genetic relation.

Evaluation of Fosfomycin Activity Against Extended Spectrum Beta Lactamase (ESBL) Producing Enterobacteriaceae Isolated from Three Centers of Tehran, Iran.

Rising rates of antimicrobial resistance among Enterobacteriaceae limit the use of reliably active forms of available drugs. The aim of this study was to investigate the prevalence of fosfomycin (US6794490B2) resistance gene among ESBL producing isolates in Iran. We tested 355 isolates of Enterobacteriacea collected from various clinical samples including urine, wounds, blood and other sources during June 2016 to July 2017. Antibiotic sensitivity and Extended Spectrum Beta Lactamase (ESBL) production were tested using agar dilution method according to the Clinical and Laboratory Standards Institute (CLSI) guidelines. ESBL genes (blaTEM, bla SHV,bla CTX-M), plasmid-encoded fosfomycin resistance genes (fosA, fosB, fosA3 and fosC2) and chromosomal mutations (murA, glpT, uhpT) were detected by Polymerase Chain Reaction (PCR). In this study, 151 of the 355 isolates were ESBL-positive. blaCTX-M (77%) was the most common gene followed by blaSHV (70%) and blaTEM (58%), either alone or in combination. Eighty nine percent (132/151) of the ESBL-positive isolates were MDR. Antimicrobial susceptibility rates were higher for fosfomycin (92.8%) and imipenem (35.5%) among ESBL-positive isolates. None of the ESBL- positive isolates harbored any mutations or plasmid-mediated fosfomycin resistance determinants. In conclusion, fosfomycin showed good antimicrobial activity against multidrug resistance ESBL- positive Enterobacteriaceae.

Characterization of a Novel SXT/R391 Integrative and Conjugative Element Carrying cfr, blaCTX-M-65, fosA3, and aac(6')-Ib-cr in Proteus mirabilis.

A novel 139,487-bp SXT/R391 integrative and conjugative element, ICEPmiChnBCP11, was characterized in Proteus mirabilis of swine origin in China. ICEPmiChnBCP11 harbors 20 different antimicrobial resistance genes, including the clinically important rRNA methyltransferase gene cfr, the extended-spectrum β-lactamase gene blaCTX-M-65, fosfomycin resistance gene fosA3, and fluoroquinolone resistance gene aac(6')-Ib-cr An ISPpu12-mediated composite transposon containing various resistance genes and 10 copies of IS26 is inserted in hot spot 4. ICEPmiChnBCP11 was successfully transferred to Escherichia coli.

Molecular Epidemiology of Plasmid-Mediated Fosfomycin Resistance Gene Determinants in Klebsiella pneumoniae Carbapenemase-Producing Klebsiella pneumoniae Isolates in China.

The Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae has become a serious problem because the species is wide ranging and there are few treatment options. Fosfomycin has attracted renewed interest in combination therapy for infections caused by KPC-producing K. pneumoniae isolates. Because of the increasing use of fosfomycin, resistant isolates have been continually reported in carbapenem-resistant K. pneumoniae (CRKP). At present, multiple mechanisms can result in fosfomycin resistance. However, there is limited knowledge with respect to plasmid-mediated fosfomycin resistance gene (fosA3) determinants in KPC-producing K. pneumoniae isolates. In this study, a total of 101 CRKP strains were collected from four hospitals in Zhejiang province from January 2013 to August 2014; 28.7% (29/101) of CRKP isolates were resistant to fosfomycin. Gene fosA3 was detected in 29 fosfomycin-resistant KPC-producing K. pneumoniae isolates, whereas genes fosA, fosB, fosB2, fosC, fosC2, and fosX were all negative among the resistant isolates. In addition, among 29 fosfomycin-resistant KPC-producing K. pneumoniae isolates, pulsed-field gel electrophoresis (PFGE) analysis revealed five pulsotypes. S1-PFGE and Southern blot showed that the fosA3 gene was located on an approximately 140-kb plasmid in all isolates. Eight of the 29 isolates (27.6%) tested could successfully transfer their fosfomycin-resistant phenotype to Escherichia coli strain J53. All fosA3-positive isolates were determined to have an identical genetic background, IS26-tetR-cadC-orf1-fosA3-IS26, which is the same as that of the fosA3-positive plasmid pFOS18 in China. The primary resistance mechanism to fosfomycin was caused by a plasmid-mediated fosA3. Furthermore, it is noteworthy that the plasmid genetically carrying a combination of the fosA3 and blaKPC-2 genes could accelerate the spread of antibiotic resistance. Effective and persistent monitoring and surveillance will be vital to prevent further dissemination of these resistance genes.

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

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

Antimicrobial susceptibility and mechanisms of fosfomycin resistance in extended-spectrum β-lactamase-producing Escherichia coli strains from urinary tract infections in Wenzhou, China

Fosfomycin in combination with various antibiotics represents an excellent clinically efficacious regimen for the treatment of urinary tract infections (UTIs) caused by extended-spectrum β-lactamase (ESBL)-producing Escherichia coli. Underlying mechanisms of fosfomycin resistance remain largely uncharacterised. To investigate the antibacterial efficacy of fosfomycin against ESBL-producing E. coli, 356 non-repetitive ESBL-producing E. coli clinical isolates were collected from urine specimens from patients with UTI in Wenzhou, China, from January 2011 to December 2015. Antimicrobial sensitivity testing indicated that 6.7% (24/356) of the ESBL-producing E. coli strains were resistant to fosfomycin. The fosA3 gene encoding a fosfomycin-modifying enzyme was detected in 20 isolates by PCR and sequencing, alone or in combination with other ESBL determinants. Conjugation experiments and Southern blotting demonstrated that 70% (14/20) of the fosA3-positive isolates possessed transferable plasmids (ca. 54.2 kb) co-harbouring the ESBL resistance gene blaCTX-M and the fosfomycin resistance gene fosA3. Among the four fosfomycin-resistant fosA3-negative E. coli isolates, three contained amino acid substitutions (Ile28Asn and Phe30Leu in MurA and Leu297Phe in GlpT). The results indicate that presence of the fosA3 gene is the primary mechanism of fosfomycin resistance in ESBL-producing E. coli isolates in Wenzhou, China. In addition, a plasmid (ca. 54.2 kb) co-harbouring fosA3 and blaCTX-M genes is horizontally transferable. Furthermore, a low degree of homology in the fosfomycin-resistant E. coli was confirmed using multilocus sequence typing (MLST), suggesting that there is no obvious phenomenon of clonal dissemination.

Prevalence of Plasmid-Mediated Fosfomycin Resistance Gene fosA3 Among CTX-M-Producing Escherichia coli Isolates from Chickens in China.

The aim of this study was to investigate the prevalence of fosfomycin resistance gene fosA3 and characterize plasmids harboring fosA3 among CTX-M-producing Escherichia coli from chickens in China. A total of 234 CTX-M-producing E. coli isolates collected from chickens from 2014 to 2016 were screened for the presence of plasmid-mediated fosfomycin resistance genes (fosA, fosA3, and fosC2). Clonal relatedness of fosA3-positive isolates was determined by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). The genetic environment of fosA3 was analyzed by polymerase chain reaction (PCR) mapping. Plasmids were studied by using conjugation experiments, PCR-based replicon typing and plasmid MLST. Sixty-four (27.4%) fosA3-positive E. coli isolates were identified in this study. The gene blaCTX-M-55 (31/64) was predominant among these strains, followed by blaCTX-M-14 (18/64) and blaCTX-M-65 (14/64). Various PFGE patterns and sequence types (STs) indicated that these isolates were clonally unrelated. Seven different genetic environments of fosA3 were identified and two new combinations (ISEcp1-blaCTX-M-65-ΔIS903D-IS26-fosA3-orf1-orf2-Δorf3-IS26 and IS26-ISEcp1-blaCTX-M-3-orf477-blaTEM-1-IS26-fosA3-orf1-orf2-Δorf3-IS26) were discovered for the first time. Conjugation experiments were successful for 47 isolates and 33 transconjugants harbored a single plasmid. Plasmids carrying fosA3 belonged to incompatibility group IncFII (17/33), IncI1 (2/33), IncHI2 (3/33), and IncB/O (1/33). F33:A-:B- plasmids carrying blaCTX-M-55, IncHI2/ST3 plasmids carrying blaCTX-M-65, and F2:A-:B-plasmids carrying blaCTX-M-55 were found in E. coli isolates from different provinces. Our results revealed a considerable prevalence of fosA3 gene among CTX-M-producing E. coli with clonal diversity from chickens in China. The transmission of different kinds of plasmids is responsible for the dissemination of fosA3 in chicken farms in China.

Dissemination of a clone carrying a fosA3-harbouring plasmid mediates high fosfomycin resistance rate of KPC-producing Klebsiella pneumoniae in China

Fosfomycin has been proposed as an adjunct to other active agents for treating KPC-producing Klebsiella pneumoniae infections. This study aimed to investigate the prevalence of fosfomycin resistance and plasmid-mediated resistance determinants among KPC-producing K. pneumoniae isolates from clinical samples in China. In total, 278 KPC-producing and 80 extended-spectrum β-lactamase (ESBL)-producing (non-KPC-producing) clinical K. pneumoniae isolates were collected in 12 hospitals from 2010 to 2013. Fosfomycin susceptibility testing was carried out using the agar dilution method. Phylogenetic clonal patterns were revealed by pulsed-field gel electrophoresis (PFGE). Isolates were screened for plasmid-mediated fosfomycin resistance genes (fosA, fosA3 and fosC2) by PCR amplification. A plasmid was completely sequenced by next-generation sequencing. The fosfomycin resistance rate in KPC-producers (60.8%; 169/278) was significantly higher than in ESBL-producers (12.5%; 10/80). In addition, 94 KPC-producing isolates were positive for fosA3 and most of them were clonally related. A 23939-bp plasmid (pFOS18) co-harbouring fosA3 and blaKPC-2 was completely sequenced, revealing that the fosA3 gene was flanked by two copies of IS26; however, blaKPC-2 was located on a Tn3–Tn4401 integration structure. Although the fosA3 and blaKPC-2 genes are located on different transposon systems, they are able to spread together worldwide through plasmid transfer. Dissemination of the clone carrying the fosA3-harbouring plasmid mediates the high fosfomycin resistance rate of KPC-producing K. pneumoniae in China. Fosfomycin as an alternative option for treating infections caused by KPC-producing K. pneumoniae should not be recommended in hospitals in which fosfomycin-resistant clonal dissemination is emerging.

P43 First detection of acquired fosfomycin resistance gene fosA3 among CTX-M-producing Escherichia coli isolates from healthy Japanese people

P43 First detection of acquired fosfomycin resistance gene fosA3 among CTX-M-producing Escherichia coli isolates from healthy Japanese people

Detection of the plasmid-encoded fosfomycin resistance gene fosA3 in Escherichia coli of food-animal origin

To investigate the occurrence of plasmid-mediated fosfomycin resistance genes among Escherichia coli from food animals in China. A total of 892 E. coli isolates collected from individual pigs (n=368), chickens (n=196), ducks (n=261), geese (n=35), pigeons (n=20) and partridges (n=12) in Guangdong Province during 2002-08 were screened for the presence of fosA3, fosA and fosC2 by PCR amplification and sequencing. The clonal relationship of fosA3-positive isolates, plasmid content and other associated resistance genes were also characterized. Twelve (1.3%) E. coli isolates showed resistance to fosfomycin and 10 (1.1%) isolates (4 from pigs, 2 from chickens, 2 from ducks, 1 from a goose and 1 from a pigeon) were positive for fosA3. None of the E. coli isolates was positive for fosA or fosC2. All of the isolates carrying fosA3 were CTX-M producers, and three of them carried rmtB. Most of the fosA3-harbouring isolates were found to be clonally unrelated. The fosA3 genes were flanked by IS26. Two fosA3 genes co-localized with rmtB and blaCTX-M-65 on indistinguishable F33:A-:B- plasmids that carried three addiction systems (pemI/pemK, hok/mok/sok and srnB). Four, one and one fosA3 genes were found to be associated with IncN (ST8 type), IncI1 and F2:A-:B- plasmids, respectively. We discovered that fosA3 is always associated with blaCTX-M, which facilitates its quick dispersal. The emergence of fosA3 in food animals could impact on human medicine by the potential transfer of resistance through the food chain.

Prevalence of acquired fosfomycin resistance among extended-spectrum -lactamase-producing Escherichia coli and Klebsiella pneumoniae clinical isolates in Korea and IS26-composite transposon surrounding fosA3

To investigate the prevalence of plasmid-mediated fosfomycin resistance determinants among extended-spectrum β-lactamase (ESBL)-producing Escherichia coli and Klebsiella pneumoniae and their genetic environments. A total of 347 non-duplicate ESBL-producing E. coli (165) and K. pneumoniae (182) were collected. The fosfomycin MICs were determined by the agar dilution method according to CLSI guidelines. PCR was used to detect the plasmid-encoded fosfomycin resistance genes (fosA, fosA3, fosB and fosC2). For isolates harbouring plasmid-encoded fosfomycin resistance genes, sequence types (STs) were determined. The transformation experiment was performed using E. coli TOPO10 (Invitrogen, USA) as a recipient strain. With the plasmids from the transformants, plasmid replicon typing was performed and the nucleotide sequences adjacent to fosA3 were determined. The susceptibility to fosfomycin was 92.9% in E. coli and 95.2% in K. pneumoniae. Of the 21 isolates non-susceptible to fosfomycin (8 E. coli and 13 K. pneumoniae), 7 (5 E. coli and 2 K. pneumoniae) isolates harboured fosA3 and all of them co-harboured bla(CTX-M-1group) or bla(CTX-M-9group). The STs of the isolates harbouring fosA3 were diverse (E. coli: ST1, ST1, ST533, ST2 and ST86; K. pneumoniae: ST11 and ST101). The plasmid replicon types of transformants co-harbouring bla(CTX-M-1group) and bla(CTX-M-9group) were IncF and IncN, respectively. By sequence analysis, we found the common feature that the fosA3 gene, connected to bla(CTX-M) via insertion sequences, was located between two IS26 elements oriented in the opposite direction, composing an IS26-composite transposon. An IS26-composite transposon appears to be the main vehicle for dissemination of fosA3 in E. coli and K. pneumoniae of diverse clones.