Studies On Mechanisms Of Fosfomycin Resistance In Extended-spectrum β-lactamase-producing Enterobacteriaceae

Enterobacteriaceae can cause various community and hospital-acquired infections, including urinary tract infections (UTIs). About80%of uncomplicated UTIs and40%of complicated UTIs was caused by Enterobacteriaceae infection. In recent years, the increasing rate of extended-spectrum β-lactamases (ESBLs)-producing Enterobacteriaceae results in fewer choices of antimicrobial drugs during clinical treatment.Fosfomycin has a broad spectrum of activity against a wide range of Gram-positive and Gram-negative bacteria. It provides a synergistic effect to other antibiotics and no significant cross-resistance. Fosfomycin was often used in treatment of infections caused by methicillin-resistant Staphylococcus aureus (MRSA) and other multi-drug resistant bacteria combined with vancomycin. Meanwhile, fosfomycin has been used for treatment of uncomplicated lower urinary tract infection for more than40years internationally. Recently, several studies found that fosfomycin was still highly effective in the treatment of UTIs. Infectious Diseases Society of America (IDSA) and European Society for Microbiology and Infectious Diseases (ESCMID) recommended fosfomycin orally for the treatment of uncomplicated UTIs and intravenously for UTIs caused by ESBLs-producing Enterobacteriaceae. Fosfomycin was also used in the treatment of complicated UTIs domestically due to high incidence of ESBLs-producing Escherichia collThe fosfomycin breakpiont for E. coli isolated from the urinary tract only was proposed by the Clinical and Laboratory Standards Institute (CLSI) with an MIC<64μg/ml considered susceptible in January,1999. The majority of studies applied this breakpoint to all species of Enterobacteriaceae. The European Committee on Antimicrobial Susceptibility Testing (EUCAST) has adopted a breakpoint of susceptibility of Enterobacteriaceae to fosfomycin of32μg/ml or less, irrespective of the site of infections, which was used rarely.Mechanisms of resistance to fosfomycin in Enterobacteriaceae mainly includes decreased drug uptake, modification of the target site and inactivation of the antibiotic.A total of257ESBLs-producing Enterobacteriaceae were collected at Huashan Hospital, Fudan University in2008, including144strains of E. coli and113Klebsiella pneumoniae. Our object is to determinate the susceptibility rate of ESBLs-producing Enterobacteriaceae to fosfomycin and other antimicrobials, to investigate the prevalence and surrounding sequences of fos gene by PCR amplifying and sequencing, and to study the resistant mechanism mediated by transport systems and target enzyme in fosfomycin-resistant bacteria which did not carry fos gene. The study included the following three parts.Part One Fosfomycin and other antimicrobial susceptibility testing of ESBLs-producing EnterobacteriaceaeMethods and Results:The minimum inhibit concentration (MIC) of18kinds of antimicrobial drugs to all of the257ESBLs-producing Enterobacteriaceae strains was detected using the agar dilution method. The18antimicrobials included fosfomycin, piperacillin, piperacillin/tazobactam, cefazolin, cefotaxime, ceftazidime, ceftriaxone, cefepime, cefoxitin, aztreonam, imipenem, meropenem, gentamicin, amikacin, ciprofloxacin, trimethoprim/sulfamethoxazole, tetracycline and chloramphenicol.25μg/ml glucose-6-phosphate (G-6-P) were added to Mueller-Hinton agar while fosfomycin susceptibility testing. Interpretive criteria for fosfomycin were applied using EUCAST interpretive criteria for all isolates of Enterobacteriaceae. Other antimicrobials breakpoints follow the CLSI interpretive criteria. E. coli ATCC25922was used as quality control strain.According to the EUCAST criteria, the susceptibility to fosfomycin was76.4%in ESBLs-producing E. coli and77.9%in K.pneumonia. Meanwhile, using the CLSI criteria for urinary tract isolates of E. coli, the susceptibility was82.6%and84.1%, respectively.All of the ESBLs-producing E. coli and K.pneumonia strains were resistant to cefazolin, cefotaxime and ceftriaxone, and susceptibility rates to piperacillin were below5%. Only part of strains was sensitive to ceftazidime, cefepime and aztreonam. The susceptibility rate was>96%for imipenem and meropenem and59%to63%for cefoxitin. The susceptibility rates to piperacillin/tazobactam was83.3%and46.9%in E. coli and K. pneumoniae, respectively.The susceptibility rate of E. coli isolates remains below50%for gentamicin, ciprofloxacin, trimethoprim/sulfa isoxazole, tetracycline, chloramphenicol, except for amikacin (88.9%).61.0%and below50%of K. pneumoniae isolates were sensitive to tetracycline and other antimicrobials, respectively.Amikacin-non-sensitive strains of E. coli and K. pneumoniae showed susceptibility rate of31.2%and71.6%to fosfomycin. The susceptibility rates of non-sensitive strains to other antimicrobial were>68%to fosfomycin according to the EUCAST criteria, apart from stains nonsensitive to amikacin, meropenem and imipenem.CTX-M type ESBLs genes were found in98.6%of E. coli and86.7%K.pneumonia. There was no significant relationship between different CTX-M gene types and fosfomycin resistance rates (CTX-M-1group and CTX-M-9group compared, P=0.882,0.664).23.0%strains of K. pneumoniae carried SHV type ESBLs alone or in combination of CTX-M.Conclusion:Fosfomycin has good antibacterial activity not only for ESBLs-producing E. coli and K. pneumoniae, but also for non-sensitive strains to other antimicrobial.Part Two Resistant mechanisms of Enterobacteriaceae to fosfomycin related with fos genesBackground:Resistance to fosfomycin can be associated with the presence of enzymes that inactivate the antibiotic. Four main types have been described as FosA, FosB, FosC, FosX and subtypes. Only fosA, fosA2, fosA3and fosC2gene has been reported in fosfomycin-resistant Enterobacteriaceae. Plasmid-encoded fosA gene has been found among other Enterobacteriaceae, Pseudomonas spp and Acinetobacter spp besides Serratia marcescens. One study found that10%of fosfomycin-resistant Enterobacteriaceae carried fosA gene. Wachino etc found two new plasmid-mediated fosfomycin-resistant genes, named fosA3and fosC2in CTX-M-producing E. coli in Japan. Several studies have shown1.0%-9.0%of E. coli and1.1%of K. pneumoniae carried fosA3gene. Meanwhile, some studies found fosA3gene is closely associated with blaCTX-M, rmtB gene and often located on the same plasmid in ESBL-producing Enterobacteriaceae. Plasmids carrying fosA3gene may accelerate the spread of fosfomycin resistance and may cause common propagation of multidrug resistance. We investigate the prevalence and surrounding sequences of fos gene by PCR amplifying and sequencing. Methods and Results:13.2%of ESBLs-producing E. coli and3.5%of K. pneumoniae carried fosA3gene. fosA3gene-positive strains are highly resistant to fosfomycin.55.9%and16%fosfomycin resistant strains harbored fosA3gene. We defined strains with MIC≥256μg/ml as highly resistant ones. And the proportion of fosA3gene-positive strains in highly resistant strains was76.0%and28.6%. One K. pneumoniae K117harboring fosA gene was susceptible to fosfomycin (MIC=32μg/ml). No fosA2, fosC2, fosB, fosB2, fosC or fosX gene was detected among these isolates.The complete sequence of fosA3genes in all of23fosA3-carring strains were100%identical to the sequence of fosA3in E. coli originally found in Japan. fosAK117had95%nucleotide identity with fosATn2921. The deduced amino acid sequences of FosAK117showed97.9%identities to FosATn2921. fosATn2921and fosA3was cloned into Top10and cloning strains were all highly resistant to fosfomycin (MIC1024μg/ml,≥1024μg/ml).The fosA3genes were successfully transferred to the recipients from20donors by conjugation. blaCTX-M and blaTEM-1genes were cotransferred to the recipients with fos A3from16and12donors, fos A gene-positive K. pneumoniae K117failed to conjugate to the recipient.We investigated the genetic environment of the fosA3gene of by primer walking in five transconjugants. The5’end of fosA3was insertion sequence IS26. The length between IS26and fosA3was322bp. The downstream sequences of fosA3were not exactly the same. The3’end of fosA3was IS26and the space between fosA3and IS26was536bp or1758bp in3strains. In the other2strains, IS1294was found located at the3’end of fosA3. We sequenced three plasmids for long distance to investigate the positional relationship between fosA3and ESBLs gene. blaCTX-M was located on the upstream in all three plasmids. Two plasmids carried four resistance genes:fosA3, blaCTX-M, blaTEM-1, rmtB.A fosfomycin resistant E. coli E265, which was negative foR fOs gene by PCR amplification, was successfully conjugated. We extracted the conjugative plasmid of E265and digested with Psti. A novel plasmid mediated fOs gene subtype (named fosA4) was found to confer fosfomycin resistance. fosA4had70%,69%,73%identity with fosATn2921,fosA2, fosA3respectively. The amino acid sequence of FosA4was69%-80%identity to FosATn2921, FosA2and FosA3, and14%-31%identity to FosB, FosC and FosX. The sequence types (STs) of E. coli and K. pneumoniae isolates harboring fosA3gene were determined.19E. coli and4K. pneumoniae isolates were assigned to11and3distinct STs respectively.Conclusion:fosA3gene is the most popular type of fos genes in ESBLs-producing Enterobacteriaceae. The main mechanism of highly resistance to fosfomycin in E. coli was the prevalence of fosA3gene.fosA3genes often cotransfer with the ESBLs gene. IS26might be involved in the horizontal spread of fosA3.Part Three Resistant mechanisms of Enterobacteriaceae to fosfomycin related with the transport system and target enzymeBackground:Fosfomycin specifically binds to the enzyme UDP-N-acetylglucosamine enolpyruvyl transferase (MurA), inhibiting the formation of peptidoglycan and thus playing a bactericidal effect. Mutation in or overexpression of MurA confer the bacterial resistance to fosfomycin.Fosfomycin enters cells by means of two different transport uptake systems:the glycerol-3-phosphate transporter (GlpT) and hexose phosphate transporter (UhpT). GlpT, which transport glycerol-3-phosphate (G-3-P) is constitutively expressed whereas UhpT, which transported G-6-P, was induced by extracellular G-6-P. Strains with defect in GlpT or UhpT function could not grow on M9minimal medium with G-3-P or G-6-P as sole carbon source (G-3-P-M9or G-6-P-M9). Defect in one of the two transporters caused by mutations in the structural gene can lead to reduced fosfomycin uptake. Mutations in the uhpA, ptsl and cyaA gene can also down-regulate UhpT or GlpT expression which confers resistance.Methods and Results:15isolates, including5strains of E. coli and10K. pneumoniae, were highly resistant to fosfomycin and negative for fos gene by PCR amplification. The murA genes were amplified by PCR and sequenced. The amino acid sequence of MurA had no mutations in5strains of E. coli. An amino acid substitution Gln7Pro was found in MurA of one K. pneumoniae isolate K672. Amplification of murA was failed in K2103, K2610, K190and K2304. The other five K. pneumoniae isolates had no mutations in MurA.15isolates and ATCC25922was inoculated on M9minimal medium agar with G-3-P or G-6-P as single carbon source.5strains of E. coli and1of K. pneumonia did not grow on neither G-3-P-M9nor G-6-P-M9, suggesting that both the two transporter systems, GlpT and UhpT, have lost function in these six strains. The other9strains of K. pneumoniae grew on the G-6-P-M9, but not on the G-3-P-M9, suggesting that UhpT transporter can work normally, while there are dysfunctions in the GlpT System in these nine strains.The glpT, uhpT, uhpA, pstl and cyaA genes of the15resistant strains and ATCC25922were amplified by PCR and sequencing. The complete sequences of glpT gene in5strains of E. coli were failed to amplify. But PCR amplification for internal conserved sequence was positive. This result suggested that there might be large segment deletions or mutations on the edge sequence of the glpT gene in these5strains. For10strains of K. pneumoniae, the complete sequence and conserved sequence of glpT gene were amplified failed, suggesting that there might be an entire gene lost of glpT in the10K. pneumoniae. Deletions or mutations of glpT gene are likely to be a direct result of GlpT transport system dysfunction.The six strains which did not grow on G-6-P-M9had no mutations of UhpT, suggesting the dysfunction of UhpT might be related to decrease in transcription or expression levels. The remaining nine strains could grow on G-6-P-M9, of which UhpT gene was also analysised. No mutations in UhpT were found in four strains. Three strains had an amino acid substitution Ile127Val. The whole sequence of UhpT could not be successfully amplified in two strains.Sequencing of UhpA in two E. coli E2393and E2967and amplification of UhpA in a K. pneumoniae K672were failed. No mutations were found in UhpA of the other strains. Several amino acid substitutions were found in PstI and CyaA of five strains of E. coli. It was pretty difficult to amplify or sequence for PstI and CyaA of K. pneumoniae. PstI was successfully amplified in only two strains of K. pneumoniae and had amino acid substitutions Val229Ile and Asn241Ser. Whether the UhpA, PstI and CyaA can confer resistance by affecting expression of GlpT or UhpT needs further study.Conclusion:Dysfunction of transport systems may be main mechanism of fosfomycin resistance in fos-negative Enterobacteriaceae. ESBLs-producing K. pneumoniae showed highly resistant to fosfomycin mainly because of deletion in glpT sequence. The fos-negative E. coli, which were highly resistant to fosfomycin, present dysfunction of both two transporter systems. Target enzyme mutations existed in few strains of K. pneumoniae. Whether the mutations confer resistance needed further studies.