uhpT Genes Research Articles

Generation and Characterization of Stable Small Colony Variants of USA300 Staphylococcus aureus in RAW 264.7 Murine Macrophages.

Intracellular survival and immune evasion are typical features of staphylococcal infections. USA300 is a major clone of methicillin-resistant S. aureus (MRSA), a community- and hospital-acquired pathogen capable of disseminating throughout the body and evading the immune system. Carnosine is an endogenous dipeptide characterized by antioxidant and anti-inflammatory properties acting on the peripheral (macrophages) and tissue-resident (microglia) immune system. In this work, RAW 264.7 murine macrophages were infected with the USA300 ATCC BAA-1556 S. aureus strain and treated with 20 mM carnosine and/or 32 mg/L erythromycin. Stable small colony variant (SCV) formation on blood agar medium was obtained after 48 h of combined treatment. Whole genome sequencing of the BAA-1556 strain and its stable derivative SCVs when combining Illumina and nanopore technologies revealed three single nucleotide differences, including a nonsense mutation in the shikimate kinase gene aroK. Gene expression analysis showed a significant up-regulation of the uhpt and sdrE genes in the stable SCVs compared with the wild-type, likely involved in adaptation to the intracellular milieu.

An insight into genes responsible for fosfomycin resistance among uropathogens of asymptomatic bacteriuria during pregnancy: A North Indian study.

Asymptomatic bacteriuria (ASB) is a common finding during pregnancy. Effective antibiotic treatment could reduce its adverse effects on both mother and fetus. However, emerging antimicrobial resistance limits the treatment options. Fosfomycin might be a promising drug in this regard, as its resistance is still low. The aim of the study was to determine the antimicrobial susceptibility pattern of fosfomycin in isolates causing ASB by disc diffusion and agar dilution (in selected isolates), determine minimum inhibitory contribution (MIC) by agar dilution in isolates resistant by disc diffusion and detect the genes responsible for fosfomycin resistance. This was a 2-year study carried in the Department of Microbiology, Jawaharlal Nehru Medical College and Hospital (JNMCH), Aligarh Muslim University (AMU), Aligarh. A total of 10 252 urine samples from asymptomatic pregnant females (18-45 years) attending the antenatal care (ANC) outpatient department (OPD) were submitted. Identification of pathogen and antimicrobial susceptibility testing (AST) was carried out as per standard methods of CLSI. There was phenotypic detection of methicillin-resistant Staphylococcus aureus (MRSA) and other Staphylococcus species (MRSS), high-level aminoglycoside resistance (HLAR), vancomycin resistant Enterococci (VRE) and S. aureus (VRSA), extended spectrum β-lactamase (ESBL) and carbapenem-resistant Enterobacterales (CRE). All the fosfomycin-resistant isolates (by disk diffusion) were tested by agar dilution. Conventional PCR was performed for murA, fosA, uhpT and glpT genes on all resistant isolates. In this study, the prevalence of ASB among pregnant females was 1173(11.4 %), in which Escherichia coli 495(42 %) was the predominant organism. The overall sensitivity of fosfomycin among Gram-positive cocci (GPC) and Gram-negative bacilli (GNB) was 99 % and 97.6 %, respectively. MRSA and MRSS accounted for 50 (66.6 %) and 71 (76 %), respectively. The highest rates of MIC >2048 µg ml-1 were shown by most isolates (mainly E. coli ) on agar dilution. PCR studies revealed four E. coli strains possessed both murA (also present in one K. pneumoniae strain) and glpT genes. While only one isolate ( E. faecalis ) was positive for fosA gene. But none of the strain possessed the uhpT gene. According to this study, murA and glpT genes were more frequent than fosA. We cannot comment on the prevalence and regional distribution of fosfomycin-resistant genes based on this preliminary study. Therefore, more Indian studies should be carried out to create awareness about the presence of genes in a particular area.

HptA Mutation May Mediate Fosfomycin Resistance in Methicillin-Resistant Staphylococcus aureus Clinical Isolates.

Fosfomycin can be used alone or in combination to treat methicillin-resistant Staphylococcus aureus (MRSA) infection. However, fosfomycin resistance has been observed in MRSA. In S. aureus, fosfomycin resistance is mediated by the fosfomycin-modifying enzyme FosB, or mutations in the target enzyme MurA. Mutations in the chromosomal glpT and uhpT genes, which encode fosfomycin transporters, also result in fosfomycin resistance. The three-component regulatory system HptRSA mediates the expression of uhpT and glpT in S. aureus. This study aimed to investigate the role of hptRSA mutation in fosfomycin resistance in MRSA clinical isolates. We found that hptRSA mutations were common in MRSA strains isolated from our hospital. Most mutations were amino acid substitutions and widely distributed in fosfomycin-sensitive and fosfomycin-resistant strains. However, HptA-truncated mutations were only found in fosB-negative fosfomycin-resistant strains with wild-type uhpT and glpT genes. Quantitative real-time PCR results showed that the transcription level of uhpT decreased by 13.7-25.6-fold in the HptA-truncated strains. Concordantly, the fosfomycin minimum inhibitory concentration (MIC) of HptA-truncated strains was 64-128 μg/mL, while SA240 was 2 μg/mL. The low transcription level of uhpT and high increase in MIC suggest that hptA mutation may lead to fosfomycin resistance in MRSA. We complemented hptA in one of the HptA-truncated clinical strains (SA179), showing reversal of fosfomycin resistance (from 128 to 32 μg/mL). Then we knocked out hptA in S. aureus Newman; fosfomycin MIC increased from 4 to 64 μg/mL, suggesting that HptA mutation may play an important role in fosfomycin resistance.

Molecular Analysis of Fosfomycin Resistance Among Escherichia coli Isolates from Urinary Tract Infections in Kidney Transplant Patients During 2019 - 2020

Background: This study aimed to estimate the prevalence of fosfomycin resistance and the frequency of extended-spectrum beta-lactamase (ESBL) production in Escherichia coli isolates from three kidney transplant patients (KTPs) in Tehran. Methods: Sixty clinical isolates of uropathogenic E. coli were collected from three kidney transplant centers in Tehran between April and May 2019. Antimicrobial susceptibility testing (AST), minimum inhibitory concentration (MIC) of fosfomycin, and screening for ESBL production were conducted following the protocols established by the Clinical and Laboratory Standards Institute (CLSI). The presence of the blaTEM, blaSHV, blaCTX-M, fosA3, and fosC2 genes was evaluated using polymerase chain reaction (PCR) and sequencing. Additionally, mutations in the murA, glpT, uhpT, and cya genes were assessed. The activity of the carbohydrate phosphate transporter was measured using the real-time PCR assay. Results: According to the AST results, ampicillin showed the highest resistance rate (86%), while ertapenem and doripenem exhibited complete susceptibility (100%). According to the E-test, 1.6% of E. coli isolates were resistant to fosfomycin. Furthermore, 33.4% of E. coli isolates in KTPs were ESBL producers, with the most frequent occurrence of the blaTEM gene (55%). Additionally, mutations were identified in the murA, uhpT, and glpT genes of resistant samples. No plasmid genes for fosA3 and fosC2 were detected. The expression of the uhpT gene increased 32-fold in a susceptible isolate, as determined by qPCR. Conclusions: The high resistance of E. coli isolates from urinary tract infections (UTIs) of KTPs to β-lactam antibiotics remains a significant clinical challenge. However, no correlation was found between ESBL production and resistance to fosfomycin. The resistance rate to fosfomycin was low, and the primary cause of resistance was mutations in chromosomal genes.

MurA, uhpT, glpT and fosA Genes of Fosfomycin Resistance in Multi-drug Resistant E. coli Isolated from Hematological Malignancies' Patients with Blood Stream Infection

Aim: To estimate the prevalence of murA, uhpT, glpT and fosA resistance genes of fosfomycin in multidrug resistant Escherichia coli isolated from patients suffering from hematological malignancies with on top blood stream infection together with correlating this distribution to the rate of expression of AmpC, ESBLs and MBLs in such isolates.
 Methods: 205 blood samples collected from patients with underlying hematological malignancies were cultured to isolate E. coli strains. Multidrug resistance was detected. PCR was done to determine fosfomycin resistance genes; murA, uhpT, glpT and fosA.
 Results: A total of 83 (40.5%) E. coli strains were isolated from blood samples. 58 (69.9%) were found to be multidrug resistant. AmpC beta-lactamase production was deduced in 15 (25.8%) isolates. 43 (74.1%) isolates were ESBLs producers whereas 9 (15.5%) were MBL class A carbapene-mases producers. Depending on PCR results, murA gene was detected in one isolate (1.7%), uhpT gene in 3 (5.2%), glpT gene in 4 (6.9%) whereas fosA gene was found in 7 E. coli strains (12.1%).
 Conclusion: Fosfomycin is a promising antibiotic with limited degree of prevalence of its resistant genes; however disseminating resistance might increase in multidrug resistant E. coli

Molecular detection of glpT and uhpT genes as fosfomycin pathways in UTI infection patients

Escherichia coli in immunocompetent hosts is the causative agent of uncomplicated urinary tract infections and opportunistic infections in immunocompromised hosts and causes urinary infections. The minimum inhibitory MIC concentration among UPEC isolates ranged from 128 μg/ml to 512 μg/ml under this study. Molecular pattern of glpT and uhpT resistance factors genes toward fosfomycin in E. coli isolates of Iraqi UTI patients were determined in this review to clarify their role in fosfomycin uptake resistance system. MethodsFrom the hospitals in Baghdad-Iraq, 50 urinary collected samples were obtained from fifty patients. The diagnosis was made using housekeeping gene 16sDNA gene. Minimum fosfomycin inhibitory concentrations (MICs) were tested the method of agar dilution with a concentrations between 16 and 1024 μg/mL. In addition, the investigation of glpT and uhpT genes was determined with specific primers using polymerase chain reaction PCR. ResultsThe genetic identifying of Escherichia coli isolates with 16sDNA recorded all isolates gave positive results with amplified size of 544 bp for 16sDNA were recorded as specific gene detection for all isolates. In comparison to the break point, the highest MIC value was recorded about 256 μg/mL, 512 μg/mL and 1024 μg/mL which appeared resistance in fosfomycin ≤128 μg/ml. Moreover, the results showed that the presence of glpT and uhpT in all UPEC tested isolates which contributing in elevating the fosfomycin resistance by inducing the efflux pump system. ConclusionOur results showed highly susceptibility of UPEC isolates under this study toward the fosfomycin. We concluded, that the prevalence of glpT and uhpT It is considerably related to the progress of fosfomycin resistant by inducing efflux pump system to eliminate the drug out of the bacterial cells.

Novel Chromosomal Mutations Responsible for Fosfomycin Resistance in Escherichia coli.

Fosfomycin resistance in Escherichia coli results from chromosomal mutations or acquisition of plasmid-mediated genes. Because these mechanisms may be absent in some resistant isolates, we aimed at decipher the genetic basis of fosfomycin resistance in E. coli. Different groups of isolates were studied: fosfomycin-resistant mutants selected in vitro from E. coli CFT073 (MIC = 1 mg/L) and two groups (wildtype and non-wildtype) of E. coli clinical isolates. Single-nucleotide allelic replacement was performed to confirm the implication of novel mutations into resistance. Induction of uhpT expression by glucose-6-phosphate (G6P) was assessed by RT-qPCR. The genome of all clinical isolates was sequenced by MiSeq (Illumina). Two first-step mutants were obtained in vitro from CFT073 (MICs, 128 mg/L) with single mutations: G469R in uhpB (M3); F384L in uhpC (M4). Second-step mutants (MICs, 256 mg/L) presented additional mutations: R282V in galU (M7 from M3); Q558∗ in lon (M8 from M4). Introduction of uhpB or uhpC mutations by site-directed mutagenesis conferred a 128-fold increase in fosfomycin MICs, whereas single mutations in galU or lon were only responsible for a 2-fold increase. Also, these mutations abolished the induction of uhpT expression by G6P. All 14 fosfomycin-susceptible clinical isolates (MICs, 0.5–8 mg/L) were devoid of any mutation. At least one genetic change was detected in all but one fosfomycin-resistant clinical isolates (MICs, 32 – >256 mg/L) including 8, 17, 18, 5, and 8 in uhpA, uhpB, uhpC, uhpT, and glpT genes, respectively. In conclusion, novel mutations in uhpB and uhpC are associated with fosfomycin resistance in E. coli clinical isolates.

Surveillance of murA and the plasmid-mediated fosfomycin resistance fosA gene in uropathogenic E. coli isolates from UTI patients

BackgroundUrinary tract infection, is a common nosocomial infection which became difficult to treat recently as multidrug-resistant strains have increased. The goal of this review was to determine phenotypic and molecular pattern of fosfomycin resistance represented in modifying enzyme FosA in Iraqi Escherichia coli isolates from patients hospitalised with UTI. MethodsUrine samples were got from 50 patients in the hospitals of Baghdad/Iraq. After isolation diagnosis was done using 16sDNA and murA genes. Minimum inhibitory concentrations (MICs) of isolates to fosfomycin were determined using the agar dilution method over a concentration range of 0.5–1024 μg/mL. Also, the presence of chromosomal and plasmid extracted modifying enzyme (fosA gene) investigated using polymerase chain reaction with specific primers. ResultsGenotypic identification of E. coli by using 16sDNA revealed that all the isolated 100% were gave appositive results with amplified size of 544 bp and 984 bp for murA as genes detection. The gene sequencing for murA showed a mutation (conversion in base 503 T→G), thus only one amino acid alteration when Histiden (H) residue change to Glutamine. The highest rate of MIC was 256mg/ml regarding fosfomycin as compared with break point 128 μg/mL. In this study, PCR assays for glpT and uhpT genes in 100% of uropathogenic E. coli. The high frequency of fosfomycin modifying-enzyme fosA is associated with its origin on chromosome or transferred by mobile elements like plasmid. ConclusionOur results indicated the bactericidal activity of fosfomycin against uropathogenic E. coli isolates. In agreement with similar studies, we concluded that the presence of murA is significantly associated with the development of resistance to fosfomycin. This type of resistance is in correlation with co-resistant by other classes of antimicrobial. Moreover, fosA gene on chromosomal genome and mediated-plasmid being the most prevalent fosfomycin-resistant determinant in UPEC. Therefore the results indicate that both fosA is contributed in genetic fosfomycin resistance.

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.

Prevalence of fosfomycin resistance and gene mutations in clinical isolates of methicillin-resistant Staphylococcus aureus

BackgroundFosfomycin exhibits excellent in vitro activity against multidrug-resistant pathogens, including methicillin-resistant Staphylococcus aureus (MRSA). Increasing fosfomycin resistance among clinical MRSA isolates was reported previously, but little is known about the relative abundance of Fosfomycin resistance genes in MRSA isolates circulating in Taiwan.MethodsAll MRSA isolates, collected in 2002 and 2012 by the Taiwan Surveillance of Antimicrobial Resistance (TSAR) program, were used in this study. Susceptibility to various antimicrobial agents, including fosfomycin, was determined by broth microdilution. Genetic determinants of fosfomycin resistance, including fosB carriage and murA, glpT and uhpT mutations, were investigated using PCR and sequencing of amplicons. Staphylococcal protein A (spa) typing was also performed to determine the genetic relatedness of MRSA isolates.ResultsA total of 969 MRSA strains, 495 in the year 2002 and 474 in the year 2012, were analyzed. The overall in vitro susceptibility was 8.2% to erythromycin, 18.0% to clindamycin, 29.0% to tetracycline, 44.6% to ciprofloxacin, 57.5% to trimethoprim/sulfamethoxazole, 86.9% to rifampicin, 92.9% to fosfomycin and 100% to linezolid and vancomycin. A significant increase in the fosfomycin resistance rate was observed from 3.4% in 2002 to 11.0% in 2012. Of 68 fosfomycin-resistant MRSA isolates, several genetic backgrounds probably contributing to fosfomycin resistance were identified. Twelve isolates harbored the fosB gene, and various mutations in murA, uhpT, and glpT genes were noted in 11, 59, and 66 isolates, respectively. The most prevalent gene mutations were found in the combination of uhpT and glpT genes (58 isolates). The vast majority of the fosfomycin-resistant MRSA isolates belonged to spa type t002.ConclusionsAn increased fosfomycin resistance rate of MRSA isolates was observed in our present study, mostly due to mutations in the glpT and uhpT genes. Clonal spread probably contributed to the increased fosfomycin resistance.

Molecular 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.

Mechanisms of fosfomycin resistance in clinical isolates of carbapenem-resistant Klebsiella pneumoniae

ObjectivesOlder antimicrobials such as fosfomycin are being considered as alternative agents in the treatment of drug-resistant organisms. However, there are limited data on the usefulness of fosfomycin against carbapenem-resistant Klebsiella pneumoniae (CRKP). This study aimed to investigate the prevalence of fosfomycin resistance and associated mechanisms in CRKP. MethodsA total of 99 clinical CRKP isolates were collected in the Second Affiliated Hospital of Wenzhou Medical University (Wenzhou, China) between January 2017 and June 2018. Fosfomycin susceptibility testing was performed by the agar dilution method. Carbapenemase and fosfomycin resistance genes were detected by PCR. Analysis of the murA, glpT, uhpT, uhpA, ptsI and cyaA genes was performed by PCR and sequencing of four fosfomycin-resistant fosA3-negative CRKP strains. Conjugation experiments were employed to determine the mobility of the fosA3 gene. ResultsOf the 99 CRKP isolates, fosfomycin-non-susceptibility was detected in 48 (48.5%) isolates, among which the fosA3 gene was detected in 44. Among the four fosfomycin-resistant fosA3-negative CRKP isolates, one isolate possessed a single nucleotide insertion and deletion mutations as well as 219 nucleotide substitution mutations in murA, two isolates possessed deletion or mutation of large DNA fragments in glpT, and one isolate possessed a fragment insertion sequence in glpT. Transfection into Escherichia coli J53 via plasmid conjugation was successful for 19 (43.2%) of the 44 fosA3-positive CRKP isolates. ConclusionThe fosA3 gene is the primary mechanism of fosfomycin resistance in CRKP and can be transmitted widely by plasmid in hospitals. Mutations in murA and glpT were found in fosfomycin-resistant fosA3-negative CRKP.

Mutations of the Transporter Proteins GlpT and UhpT Confer Fosfomycin Resistance in Staphylococcus aureus.

With the increasing spread of methicillin-resistant Staphylococcus aureus worldwide, fosfomycin has begun to be used more often, either alone or in combination with other antibiotics, for treating methicillin-resistant S. aureus infections, resulting in the emergence of fosfomycin-resistant strains. Fosfomycin resistance is reported to be mediated by fosfomycin-modifying enzymes (FosA, FosB, FosC, and FosX) and mutations of the target enzyme MurA or the membrane transporter proteins UhpT and GlpT. Our previous studies indicated that the fos genes might not the major fosfomycin resistance mechanism in S. aureus, whereas mutations of glpT and uhpT seemed to be more related to fosfomycin resistance. However, the precise role of these two genes in S. aureus fosfomycin resistance remains unclear. The aim of the present study was to investigate the role of glpT and uhpT in S. aureus fosfomycin resistance. Homologous recombination was used to knockout the uhpT and glpT genes in S. aureus Newman. Gene complementation was generated by the plasmid pRB473 carrying these two genes. The fosfomycin minimal inhibitory concentration (MIC) of the strains was measured by the E-test to observe the influence of gene deletion on antibiotic susceptibility. In addition, growth curves were constructed to determine whether the mutations have a significant influence on bacterial growth. Deletion of uhpT, glpT, and both of them led to increased fosfomycin MIC 0.5 μg/ml to 32 μg/ml, 4 μg/ml, and >1024 μg/ml, respectively. By complementing uhpT and glpT into the deletion mutants, the fosfomycin MIC decreased from 32 to 0.5 μg/ml and from 4 to 0.25 μg/ml, respectively. Moreover, the transporter gene-deleted strains showed no obvious difference in growth curves compared to the parental strain. In summary, our study strongly suggests that mutations of uhpT and glpT lead to fosfomycin resistance in S. aureus, and that uhpT mutation may play a more important role. The high resistance and low biological fitness cost resulting from uhpT and glpT deletion suggest that these strains might have an evolutionary advantage in a fosfomycin-rich clinical situation, which should be closely monitored.

Molecular insights into fosfomycin resistance in Escherichia coli.

Fosfomycin activity in Escherichia coli depends on several genes of unknown importance for fosfomycin resistance. The objective was to characterize the role of uhpT , glpT , cyaA and ptsI genes in fosfomycin resistance in E. coli. WT E. coli BW25113 and null mutants, Δ uhpT , Δ glpT , Δ cyaA , Δ ptsI , Δ glpT-uhpT , Δ glpT-cyaA , Δ glpT-ptsI , Δ uhpT-cyaA , Δ uhpT-ptsI and Δ ptsI-cyaA , were studied. Susceptibility to fosfomycin was tested using CLSI guidelines. Fosfomycin mutant frequencies were determined at concentrations of 64 and 256 mg/L. Fosfomycin in vitro activity was tested using time-kill assays at concentrations of 64 and 307 mg/L (human C max ). Fosfomycin MICs were: WT E. coli BW25113 (2 mg/L), Δ glpT (2 mg/L), Δ uhpT (64 mg/L), Δ cyaA (8 mg/L), Δ ptsI (2 mg/L), Δ glpT-uhpT (256 mg/L), Δ glpT-cyaA (8 mg/L), Δ glpT-ptsI (2 mg/L), Δ uhpT-cyaA (512 mg/L), Δ uhpT-ptsI (64 mg/L) and Δ ptsI-cyaA (32 mg/L). In the mutant frequency assays, no mutants were recovered from BW25113. Mutants appeared in Δ glpT , Δ uhpT , Δ cyaA and Δ ptsI at 64 mg/L and in Δ uhpT and Δ cyaA at 256 mg/L. Δ glpT-ptsI , but not Δ glpT-cyaA , Δ uhpT-cyaA or Δ uhpT-ptsI , increased the mutant frequency compared with the highest frequency found in each single mutant. In time-kill assays, all mutants regrew at 64 mg/L. Initial bacterial reductions of 2-4 log 10 cfu/mL were observed for all strains, except for Δ uhpT-ptsI , Δ glpT-uhpT and Δ uhpT-cyaA . Only Δ glpT and Δ ptsI mutants were cleared using 307 mg/L. Fosfomycin MIC may not be a good efficacy predictor, as highly resistant mutants may appear, depending on other pre-existing mutations with no impact on MIC.

Prevalence of Fosfomycin Resistance and Mutations in murA, glpT, and uhpT in Methicillin-Resistant Staphylococcus aureus Strains Isolated from Blood and Cerebrospinal Fluid Samples.

In China, fosfomycin alone or in combination with other antibiotics is commonly used in the treatment of infections caused by Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA). Although fosfomycin-resistant S. aureus strains have continued to emerge and increase, the research on them is rare. In order to determine the prevalence and mechanisms of fosfomycin resistance in MRSA clinical isolates, a total of 96 non-duplicate MRSA isolates were collected from blood and cerebrospinal fluid samples at Huashan Hospital in Shanghai, China between 2004 and 2014. Antimicrobial susceptibility testing was performed by agar dilution. Meanwhile, the fosfomycin-resistance-related genes, fosB, murA, glpT, and uhpT, were amplified by PCR and subjected to sequencing analysis. Multilocus sequence typing (MLST) was conducted to assess strain types. The minimum inhibitory concentration (MIC) of fosfomycin for the 96 MRSA strains ranged from 1.0 to >1,024 mg/L, and approximately 70% (67/96) of the isolates were resistant to fosfomycin (MIC ≥ 64.0 mg/L). Nine isolates with MICs ≥ 128 mg/L carried fosB gene. Twenty-five distinct mutations were detected in the murA (7), glpT (10), and uhpT (8) genes. While five of the murA mutations and five of the glpT mutations were observed only in fosfomycin-sensitive isolates and one of the murA mutation was found both in fosfomycin-resistant and fosfomycin-sensitive isolates, the remaining 14 mutations (1 murA, 5 glpT, and all uhpT mutations) were present only in fosfomycin-resistant isolates. MLST analysis demonstrated that the majority (46/67) of the glpT and/or uhpT mutants belong to ST5, the predominant sequence type among the fosfomycin-resistant MRSA isolates. In conclusion, there is a high rate of fosfomycin resistance in MRSA strains. The mutations in the murA, glpT, and uhpT genes are common in fosfomycin-resistant MRSA strains, and may play a greater role in the development of fosfomycin resistance than the presence of the fosB gene in these organisms.

Antimicrobial Susceptibility and Molecular Mechanisms of Fosfomycin Resistance in Clinical Escherichia coli Isolates in Mainland China.

Escherichia coli is one of the most common pathogens in nosocomial and community-acquired infections in humans. Fosfomycin is a broad-spectrum antibiotic which inhibits peptidoglycan synthesis responsible for bacterial cell wall formation. Although low, the exact E. coli susceptibility to fosfomycin as well as the mechanisms of resistance in the population from Mainland China are mostly unknown. 1109 non-duplicate clinical E. coli strains isolated from urine, sputum, blood and pus samples in 20 widely dispersed tertiary hospitals from Mainland China were collected from July 2009 to June 2010, followed by determination of minimum inhibitory concentrations of fosfomycin. Detection of the murA, glpT, uhpT, fosA, fosA 3 and fosC genes was performed in fosfomycin non-susceptible E. coli strains and conjugation experiments were employed to determine the mobility of fosA 3 gene. In this study, 7.8% (86/1109) E. coli strains were fosfomycin non-susceptible. Amino acid substitutions in GlpT and MurA were found in six and four E.coli strains, respectively, while the uhpT gene was absent in eighteen E.coli strains. Twenty-nine isolates carried the transferable plasmid with the fosA 3 gene at high frequencies of around 10−6 to 10−7 per donor cell in broth mating. The majority of isolates were susceptible to fosfomycin, showing that the drug is still viable in clinical applications. Also, the main mechanism of E. coli resistance in Mainland China was found to be due to the presence of the fosA 3 gene.

Characterization of a novel two-component regulatory system, HptRS, the regulator for the hexose phosphate transport system in Staphylococcus aureus.

Hexose phosphate is an important carbon source within the cytoplasm of host cells. Bacterial pathogens that invade, survive, and multiply within various host epithelial cells exploit hexose phosphates from the host cytoplasm through the hexose phosphate transport (HPT) system to gain energy and synthesize cellular components. In Escherichia coli, the HPT system consists of a two-component regulatory system (UhpAB) and a phosphate sensor protein (UhpC) that tightly regulate expression of a hexose phosphate transporter (UhpT). Although growing evidence suggests that Staphylococcus aureus also can invade, survive, and multiply within various host epithelial cells, the genetic elements involved in the HPT system in S. aureus have not been characterized yet. In this study, we identified and characterized the HPT system in S. aureus that includes the hptRS (a novel two-component regulatory system), the hptA (a putative phosphate sensor), and the uhpT (a hexose phosphate transporter) genes. The hptA, hptRS, and uhpT markerless deletion mutants were generated by an allelic replacement method using a modified pMAD-CM-GFPuv vector system. We demonstrated that both hptA and hptRS are required to positively regulate transcription of uhpT in response to extracellular phosphates, such as glycerol-3-phosphate (G3P), glucose-6-phosphate (G6P), and fosfomycin. Mutational studies revealed that disruption of the hptA, hptRS, or uhpT gene impaired the growth of bacteria when the available carbon source was limited to G6P, impaired survival/multiplication within various types of host cells, and increased resistance to fosfomycin. The results of this study suggest that the HPT system plays an important role in adaptation of S. aureus within the host cells and could be an important target for developing novel antistaphylococcal therapies.

CTX-M-15-producing urinary Escherichia coli O25b-ST131-phylogroup B2 has acquired resistance to fosfomycin

To describe trends in fosfomycin resistance in urinary isolates of Escherichia coli producing extended-spectrum beta-lactamases (ESBLs) in relation to fosfomycin consumption and to characterize representative fosfomycin-resistant isolates. In 2007-08, an unexpected increase in fosfomycin resistance in ESBL-producing urinary E. coli was observed. Laboratory records were reviewed and a prospective surveillance study was initiated on all urinary tract infections caused by ESBL-producing, fosfomycin-resistant E. coli. bla(ESBL) types, phylogroups, genetic environment and afa/dra operon were determined by PCR and sequencing. Molecular epidemiology was analysed by PFGE and multilocus sequence typing. To elucidate possible mechanisms of fosfomycin resistance, uhpT, glpT, uhpA, ptsI, cyaA and murA genes were analysed. Fosfomycin consumption was determined as recommended by WHO. From 2004 to 2008, fosfomycin consumption increased by 50%, while fosfomycin resistance in ESBL producers increased from 2.2% to 21.7%. Of 26 isolates studied, 24 produced CTX-M-15 and belonged to the O25b-ST131-phylogroup B2 clonal strain. PFGE revealed two clusters. Cluster I included 18 isolates, 16 of them indistinguishable from strains producing CTX-M-15 previously described in Madrid. The five isolates of Cluster II had the IS26 linked to bla(CTX-M-15) and the afa/dra operon. In Cluster I isolates, no mutations in glpT, uhpT, uhpA, ptsI, cyaA and murA were detected. Cluster II isolates showed a 15 bp deletion (A(169)-C(183)) in uhpA. Fosfomycin resistance in urinary E. coli has increased due to the acquisition of this resistance by a previously circulating CTX-M-15-producing E. coli O25b-ST131-phylogroup B2 strain. This happened during a period when the use of fosfomycin increased by 50%.

Identification of chromosomal Shigella flexneri genes induced by the eukaryotic intracellular environment.

Upon entry into the eukaryotic cytosol, the facultative intracellular bacterium Shigella flexneri is exposed to an environment that may necessitate the expression of particular genes for it to survive and grow intracellularly. To identify genes that are induced in response to the intracellular environment, we screened a library containing fragments of the S. flexneri chromosome fused to a promoterless green fluorescent protein gene (gfp). Bacteria containing promoter fusions that had a higher level of gfp expression when S. flexneri was intracellular (in Henle cells) than when S. flexneri was extracellular (in Luria-Bertani broth) were isolated by using fluorescence-activated cell sorting. Nine different genes with increased expression in Henle cells were identified. Several genes (uhpT, bioA, and lysA) were involved in metabolic processes. The uhpT gene, which encoded a sugar phosphate transporter, was the most frequently isolated gene and was induced by glucose-6-phosphate in vitro. Two of the intracellularly induced genes (pstS and phoA) encode proteins involved in phosphate acquisition and were induced by phosphate limitation in vitro. Additionally, three iron-regulated genes (sufA, sitA, and fhuA) were identified. The sufA promoter was derepressed in iron-limiting media and was also induced by oxidative stress. To determine whether intracellularly induced genes are required for survival or growth in the intracellular environment, we constructed mutations in the S. flexneri uhpT and pstS genes by allelic exchange. The uhpT mutant could not use glucose-6-phosphate as a sole carbon source in vitro but exhibited normal plaque formation on Henle cell monolayers. The pstS mutant had no apparent growth defect in low-phosphate media in vitro but formed smaller plaques on Henle cell monolayers than the parent strain. Both mutants were as effective as the parent strain in inducing apoptosis in a macrophage cell line.