Fosfomycin Resistance Research Articles

Identification and characterisation of fosfomycin resistance in Escherichia coli urinary tract infection isolates from Australia

Of 1033 Escherichia coli urinary tract infection isolates collected from females >12 years of age in Australia in 2019, only 2 isolates were resistant to fosfomycin with a minimum inhibitory concentration (MIC) of >256 mg/L. Despite having different multilocus sequence types, the two isolates harboured an identical plasmid-encoded fosA4 gene. The fosA4 gene has previously been identified in a single clinical E. coli isolate cultured in Japan in 2014. Each fosfomycin-resistant isolate harboured two conjugative plasmids that possessed an array of genes conferring resistance to aminoglycosides, β-lactams, macrolides, quinolones, sulfonamides and/or trimethoprim.

Characterization of a fosA3 Carrying IncC-IncN Plasmid From a Multidrug-Resistant ST17 Salmonella Indiana Isolate.

The aim of this study was to investigate the characteristics of a fosA3 carrying IncC–IncN plasmid from a multidrug-resistant Salmonella isolate HNK130. HNK130 was isolated from a chicken and identified as ST17 Salmonella enterica serovar Indiana and exhibited resistance to 13 antibiotics including the cephalosporins and fosfomycin. S1 nuclease pulsed-field gel electrophoresis and Southern blot assays revealed that HNK130 harbored only one ∼180-kb plasmid carrying fosA3 and blaCTX–M–14, which was not transferable via conjugation. We further examined 107 Escherichia coli electro-transformants and identified 3 different plasmid variants, pT-HNK130-1 (69), pT-HNK130-2 (15), and pT-HNK130-3 (23), in which pT-HNK130-1 seemed to be the same as the plasmid harbored in HNK130. We completely sequenced an example of each of these variants, and all three variants were IncC–IncN multi-incompatible plasmid and showed a mosaic structure. The fosA3 gene was present in all three and bounded by IS26 elements in the same orientation (IS26-322bp-fosA3-1758bp-IS26) that could form a minicircle containing fosA3. The blaCTX–M–14 gene was located within an IS15DI-ΔIS15DI-iroN-IS903B-blaCTX–M–14-ΔISEcp1-IS26 structure separated from the fosA3 gene in pT-HNK130-1, but was adjacent to fosA3 in pT-HNK130-3 in an inverted orientation. Linear comparison of the three variants showed that pT-HNK130-2 and pT-HNK130-3 resulted from the sequence deletion and inversion of pT-HNK130-1. Stability tests demonstrated that pT-HNK130-1 and pT-HNK130-3 could be stably maintained in the transformants without antibiotic selection but pT-HNK130-2 was unstable. This is the first description of an IncC–IncN hybrid plasmid from an ST17 S. Indiana strain and indicates that this plasmid may further facilitate dissemination of fosfomycin and cephalosporin resistance in Salmonella.

Multiple Plasmid Vectors Mediate the Spread of fosA3 in Extended-Spectrum-β-Lactamase-Producing Enterobacterales Isolates from Retail Vegetables in China.

The plasmid-mediated fosfomycin resistance gene fosA3 has been detected in Enterobacterales from various sources but has rarely been reported in vegetables. In this study, the aim was to investigate the prevalence of and, subsequently, to characterize fosA3-positive Enterobacterales isolates from retail vegetables. Seventeen (7.3%) fosA3-carrying strains were identified from 233 extended-spectrum-β-lactamase-producing Enterobacterales isolates from vegetables. All 17 isolates, including six Escherichia coli, seven Klebsiella pneumoniae, two Raoultella ornithinolytica, and two Citrobacter freundii isolates, carried blaCTX-M S1-nuclease pulsed-field gel electrophoresis (S1-PFGE) and hybridization confirmed that the fosA3 genes in 16 isolates were located on plasmids ranging in size from ∼40 kb to ∼250 kb, except one located on chromosome of C. freundii All the fosA3-carrying plasmids from 16 fosA3-positive isolates were successfully transferred into the recipient bacteria by transformation or conjugation. In agreement with data determined with isolates from food animals, the IncHI2/ST3 and IncN-F33:A-:B-/F33:A-:B plasmids were the main vectors of fosA3 in E. coli Additionally, F24:A-:B1, IncFIIK-IncR, IncFIIS, IncR, and two untypeable plasmids were found for the first time to be vectors for fosA3 in Enterobacterales The genetic contexts of fosA3 in 15 Enterobacterales isolates differed due to insertion and/or loss of molecular modules mediated by mobile elements. However, all fosA3 genes were flanked by IS26, as commonly observed in other fosA3-carrying plasmids. Here, we report a high rate of detection of fosA3 genes, mediated by multiple plasmid vectors, in ESBL-producing Enterobacterales from retail vegetables. FosA3-producing Enterobacterales could be transmitted to the human body by direct contact or consumption of vegetables, which might pose a potential threat to public health.IMPORTANCE This report provides important information on the transmission and epidemiology of fosA3 among Enterobacterales isolates from vegetables. The rate of occurrence of fosA3 in ESBL-producing Enterobacterales from retail vegetables is high, and fosA3 was found to be carried by diverse plasmids. Some novel genetic contexts of fosA3 and novel fosA3-carrying plasmids, including several plasmid types common in K. pneumoniae, were identified, increasing the number of known transfer vectors for the fosA3 gene and reflecting the complexity of fosA3 transmission in Enterobacterales The capture of fosA3 by the resident plasmid of K. pneumoniae will accelerate the spread of fosA3 in K. pneumoniae, one of the most pathogenic species in clinical medicine. Considering the clinical importance of fosfomycin, and the fact that vegetables are directly consumed, the fosfomycin resistance genes present a risk of transmission to the human body through the food chain and thus pose a threat to public health.

Fosfomycin, from susceptibility to resistance: Impact of the new guidelines on breakpoints

The fosfomycin breakpoint using the disc diffusion method (DDM) changed in the 2019 CA-SFM/EUCAST guidelines v2 (24mm versus 19mm). We assessed its impact on categorization of Enterobacterales recovered from urine samples in emergency departments. A total of 7749 and 2348 strains were tested using the DDM and the broth microdilution method (BMD), respectively. The DDM with the 19-mm breakpoint was in accordance with the BMD. Using the 24-mm breakpoint, the overall rate of fosfomycin resistance in Enterobacterales increased by three-fold (5.6% vs 18.1%, P<0.01) and reached 2.8% and 86.5% in E. coli and K. pneumoniae, respectively. French guidelines for the management of community-acquired UTI remain appropriate. The accuracy of the methods for routine fosfomycin susceptibility testing should be assessed. The role of fosfomycin in the treatment of documented CA-UTI due to Enterobacterales other than E. coli should be evaluated considering its rate of resistance and recent data reporting low accuracy.

Analysis of β-lactam, azithromycin and fosfomycin resistance in non-typhoidal Salmonella: Characterisation of an S. Infantis plasmid

Non-typhoidal Salmonella (NTS)infections are associated with high morbidity and mortality. β-lactams are used as first-line treatment but resistance to these has increased considerably in recent years. Azithromycin and fosfomycin are used as alternatives; however, the incidence of resistance in these drugs is also increasing. Epidemiological surveillance on 35,372 NTS received by Public Health England was conducted for analysis of demographics, including global travel. Genomic typing and antimicrobial resistance data for Salmonellaisolates were used to determine the prevalence of β-lactam, azithromycin and fosfomycin resistance in NTSover a four year period. No isolates were resistant to β-lactams, azithromycin or fosfomycin alone but all isolates were resistant to multiple antimicrobial classes. IncHI2, IncY and IncN plasmids were predominantly found in the most multi-drug resistant isolates. Multi-drug resistance (MDR) was particularly a concern in the S. Infantis population. Therefore, long read sequencing was used to characterise an MDR S. Infantis isolate. Three drug regions were identified in a IncFIB, a mega plasmid identified in this isolate. The resistance determinants fosA, arsA, arsD and blaCTXM65,were discovered on the same drug region. Analysis of IncFIB in this S.Infantis isolate revealed 99% similarity to a IncFIB plasmid in S. Infantis isolated from chickens in the USA. Thishas not been reported before, warranting efforts for enhanced surveillance programmes to identify sources of emerging resistance, which will aid in establishing control measures for prevention of spread of resistance.

Sequence analysis of Salmonella enterica isolates obtained from shelter dogs throughout Texas.

Dogs are a potential source of zoonotic Salmonella transmission. We had previously estimated the prevalence of Salmonella shedding among shelter dogs throughout Texas using a repeated cross‐sectional study design. Our current objectives were to fully characterize the Salmonella isolates and to assess their relatedness, using whole‐genome sequencing. Antimicrobial resistance (AMR) genes were detected in 4/27 (15%) of the isolates. The fosfomycin resistance gene fosA7 was identified in two isolates; to our knowledge, there are no published reports of this gene in canine Salmonella isolates. The biocide resistance gene qacEdelta1, conferring resistance to quaternary ammonium compounds, was detected in an isolate that had four additional AMR genes. The most frequently identified serotypes were Newport (6/27, 22%) and Javiana (4/27, 15%), both of which were widespread among animal shelters. For these serotypes, there was evidence of both transmission of Salmonella within the shelter environment and separate introductions of Salmonella into a shelter. Several canine Salmonella isolates were closely related to human clinical isolates (four canine isolates within 10 SNPs and six more within 20 SNPs), suggesting a shared pathogen population. Educational outreach programmes targeting animal shelter workers would be useful for optimizing knowledge of Salmonella and other canine‐associated zoonotic pathogens.

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.

Draft Genome Sequence of Staphylococcus epidermidis UMB8493, Isolated from the Female Urinary Tract.

Staphylococcus epidermidis is a native member of the human microbiota. Here, we report the draft genome sequence of S. epidermidis UMB8493, an isolate from a catheterized urine sample from a female with overactive bladder symptoms. The 2.54-Mbp draft genome encodes genes associated with beta-lactam and fosfomycin resistance.

The Inactivation of Enzymes Belonging to the Central Carbon Metabolism Is a Novel Mechanism of Developing Antibiotic Resistance.

ABSTRACTFosfomycin is a bactericidal antibiotic, analogous to phosphoenolpyruvate, that exerts its activity by inhibiting the activity of MurA. This enzyme catalyzes the first step of peptidoglycan biosynthesis, the transfer of enolpyruvate from phosphoenolpyruvate to uridine-diphosphate-N-acetylglucosamine. Fosfomycin is increasingly being used, mainly for treating infections caused by Gram-negative multidrug-resistant bacteria. The mechanisms of mutational resistance to fosfomycin in Stenotrophomonas maltophilia, an opportunistic pathogen characterized by its low susceptibility to commonly used antibiotics, were studied in the current work. None of the mechanisms reported so far for other organisms, which include the production of fosfomycin-inactivating enzymes, target modification, induction of an alternative peptidoglycan biosynthesis pathway, and the impaired entry of the antibiotic, are involved in the acquisition of such resistance by this bacterial species. Instead, the unique cause of resistance in the mutants studied is the mutational inactivation of different enzymes belonging to the Embden-Meyerhof-Parnas central metabolism pathway. The amount of intracellular fosfomycin accumulation did not change in any of these mutants, showing that neither inactivation nor transport of the antibiotic is involved. Transcriptomic analysis also showed that the mutants did not present changes in the expression level of putative alternative peptidoglycan biosynthesis pathway genes or any related enzyme. Finally, the mutants did not present an increased phosphoenolpyruvate concentration that might compete with fosfomycin for its binding to MurA. On the basis of these results, we describe a completely novel mechanism of antibiotic resistance based on mutations of genes encoding metabolic enzymes.IMPORTANCE Antibiotic resistance has been largely considered a specific bacterial response to an antibiotic challenge. Indeed, its study has been mainly concentrated on mechanisms that affect the antibiotics (mutations in transporters, efflux pumps, and antibiotic-modifying enzymes, or their regulators) or their targets (i.e., target mutations, protection, or bypass). Usually, antibiotic resistance-associated metabolic changes were considered a consequence (fitness costs) and not a cause of antibiotic resistance. Herein, we show that alterations in the central carbon bacterial metabolism can also be the cause of antibiotic resistance. In the study presented here, Stenotrophomonas maltophilia acquires fosfomycin resistance through the inactivation of glycolytic enzymes belonging to the Embden-Meyerhof-Parnas pathway. Besides resistance to fosfomycin, this inactivation also impairs the bacterial gluconeogenic pathway. Together with previous work showing that antibiotic resistance can be under metabolic control, our results provide evidence that antibiotic resistance is intertwined with the bacterial metabolism.

Prevalence of O25b-ST131 clone and fosfomycin resistance in urinary Escherichia coli isolates and their relation to CTX-M determinant

Escherichia coli ST131 clone and H30-R/H30-Rx subclones are the most common multidrug-resistant high-risk clones in UTIs. Antimicrobial susceptibility of fosfomycin was compared to five other agents in consecutively collected 299 urinary isolates using the agar dilution method. Prevalence of the ST131 clone and the occurrence of blaCTX-M were also investigated. Overall resistance to fosfomycin, cefuroxime, and ceftriaxone were 2.7%, 35.4%, and 30.1% respectively. fosA, fosA3, and fosC2 genes were not detected. In isolates resistant to ciprofloxacin (34.7%), the prevalence of ST131 clone was 31.7%, of which 81.8% belonged to H30-R and 66.7% to H30-Rx subclones. None of the isolates of the ST131 clone were resistant to fosfomycin. However, blaCTX-M occurred in 57.6% of the isolates among this clone, 62.9% in H30-R and 68.2% in H30-Rx subclones. The results of this study suggest that fosfomycin resistance is not prevalent in urinary isolates, however, blaCTX-Mpositive ST131 clone is quite common.

Contribution of hypermutation to fosfomycin heteroresistance in Escherichia coli.

To explore the effect of combining defects in DNA repair systems with the presence of fosfomycin-resistant mechanisms to explain the mechanisms underlying fosfomycin heteroresistance phenotypes in Enterobacteriaceae. We used 11 clinical Escherichia coli isolates together with isogenic single-gene deletion mutants in the E. coli DNA repair system or associated with fosfomycin resistance, combined with double-gene deletion mutants. Fosfomycin MICs were determined by gradient strip assay (GSA) and broth microdilution (BMD). Mutant frequencies for rifampicin (100 mg/L) and fosfomycin (50 and 200 mg/L) were determined. Using two starting inocula, in vitro fosfomycin activity was assessed over 24 h in growth (0.5-512 mg/L) and time-kill assays (64 and 307 mg/L). Strong and weak mutator clinical isolates and single-gene deletion mutants, except for ΔuhpT and ΔdnaQ, were susceptible by GSA. By BMD, the percentage of resistant clinical isolates reached 36%. Single-gene deletion mutants showed BMD MICs similar to those for subpopulations by GSA. Strong mutators showed a higher probability of selecting fosfomycin mutants at higher concentrations. By combining the two mechanisms of mutation, MICs and ranges of resistant subpopulations increased, enabling strains to survive at higher fosfomycin concentrations in growth monitoring assays. In time-kill assays, high inocula increased survival by 37.5% at 64 mg/L fosfomycin, compared with low starting inocula. The origin and variability of the fosfomycin heteroresistance phenotype can be partially explained by high mutation frequencies together with mechanisms of fosfomycin resistance. Subpopulations should be considered until clinical meaning is established.

Antimicrobial resistance and extended spectrum beta-lactamases in urinary tract infections: A serious problem in Northern Mexico

Objective: To describe the causal agents, prevalence of antimicrobial resistance, and risk factors associated with extended spectrum beta-lactamase (ESBL)-producing agents in urinary tract infections (UTIs).&#x0D; Materials and methods: A retrospective study was conducted at a tertiary care hospital in Monterrey, Mexico. Inclusion criteria were patients that clinically presented with a UTI and had a positive urine culture, within the time frame of March to October 2017. The association with ESBL-producing agents was determined through the Χ2 test for categorical variables. Statistical significance was set at a p &lt;0.05, utilizing SPSS version 20.0 software.&#x0D; Results: A total of 353 positive urine cultures were confirmed. ESBL production was found in 21.5% of the strains. There was a high level of resistance (&gt;50%) to amoxicillin-clavulanate, ciprofloxacin, levofloxacin, fosfomycin, and trimethoprim-sulfamethoxazole and moderate resistance (10-50%) to gentamicin and ceftriaxone. Amikacin, ertapenem, nitrofurantoin, and colistin had the lowest resistance rates (&lt;10%). The ESBL-producing agents were associated with complicated UTI (p≤0.0001). The comorbidities associated with ESBL-positive UTIs were diabetes mellitus (p=0.02) and immunodeficiency (p=0.008), as was having undergone radiotherapy (p=0.025) and previous antibiotic use (p≤0.001).&#x0D; Limitations: The clonal relationship of isolates, especially of E. coli, was not analyzed. We could not establish whether there was a high level of genetic diversity between the isolates or whether independent acquisition or cross-transmission occurred.&#x0D; Value: We evaluated the epidemiologic characteristics of the ESBL-producing agents in UTIs at a Mexican tertiary care hospital.&#x0D; Conclusions: One out of every five UTIs was caused by ESBLs in our study population. There was a high level of resistance to the antibiotics used as first-line empiric therapy in the patients studied.

Resistencia antibiótica y agentes beta-lactamasa de espectro extendido en las infecciones del tracto urinario: un problema grave en el norte de México

Objective: To describe the causal agents, prevalence of antimicrobial resistance, and risk factors associated with extended spectrum beta-lactamase (ESBL)-producing agents in urinary tract infections (UTIs).&#x0D; Materials and methods: A retrospective study was conducted at a tertiary care hospital in Monterrey, Mexico. Inclusion criteria were patients that clinically presented with a UTI and had a positive urine culture, within the time frame of March to October 2017. The association with ESBL-producing agents was determined through the Χ2 test for categorical variables. Statistical significance was set at a p &lt;0.05, utilizing SPSS version 20.0 software.&#x0D; Results: A total of 353 positive urine cultures were confirmed. ESBL production was found in 21.5% of the strains. There was a high level of resistance (&gt;50%) to amoxicillin-clavulanate, ciprofloxacin, levofloxacin, fosfomycin, and trimethoprim-sulfamethoxazole and moderate resistance (10-50%) to gentamicin and ceftriaxone. Amikacin, ertapenem, nitrofurantoin, and colistin had the lowest resistance rates (&lt;10%). The ESBL-producing agents were associated with complicated UTI (p≤0.0001). The comorbidities associated with ESBL-positive UTIs were diabetes mellitus (p=0.02) and immunodeficiency (p=0.008), as was having undergone radiotherapy (p=0.025) and previous antibiotic use (p≤0.001).&#x0D; Limitations: The clonal relationship of isolates, especially of E. coli, was not analyzed. We could not establish whether there was a high level of genetic diversity between the isolates or whether independent acquisition or cross-transmission occurred.&#x0D; Value: We evaluated the epidemiologic characteristics of the ESBL-producing agents in UTIs at a Mexican tertiary care hospital.&#x0D; Conclusions: One out of every five UTIs was caused by ESBLs in our study population. There was a high level of resistance to the antibiotics used as first-line empiric therapy in the patients studied.

Identification of fosA10, a Novel Plasmid-Mediated Fosfomycin Resistance Gene of Klebsiella pneumoniae Origin, in Escherichia coli.

PurposeSeveral subtypes of plasmid-mediated fosfomycin resistance gene fosA in Enterobacteriaceae have been reported worldwide and have caused concern. The present study characterized a novel member of fosA gene located on a plasmid from Escherichia coli.Materials and MethodsA fosfomycin-resistant E. coli isolate PK9 was recovered from a chicken meat sample in 2018. The presence of fosA genes was detected by PCR and sequencing. Whole-genome sequencing (WGS), conjugation, and cloning were performed to identify the mechanism responsible for fosfomycin resistance. Oxford Nanopore MinION sequencing was carried out to characterize the plasmid carrying fosfomycin resistance gene and the genetic context of the novel fosA variant.ResultsA novel fosA gene with significant homology (>98%) with fosA6 and fosA5 genes was identified by WGS and was named fosA10. FosA10 shared 56.1% to 98.6% amino acid sequence identity with other reported plasmid-mediated FosA enzymes. Fosfomycin resistance and fosA10 gene were successfully transferred to E. coli C600 by conjugation. Cloning confirmed that FosA10 could confer fosfomycin resistance (MIC > 128 μg/mL). The fosA10 gene was localized on a 53kb IncFII (F35:A-:B-) plasmid. The ∆lysR-fosA10-∆hp fragment (4328 bp), located between two copies of IS10R, showed 100% identity with the chromosomal sequences of 17 Klebsiella pneumoniae strains of ST664 and one of ST3821 in GenBank.ConclusionOur findings indicated that the fosA10 gene of E. coli might be captured from the chromosome of K. pneumoniae by IS10, which further demonstrated that K. pneumoniae might act as a reservoir of fosA-like genes acquired by E. coli.

In vitro susceptibility of OXA-48, NDM, VIM and IMP enzyme- producing Klebsiella spp. and Escherichia coli to fosfomycin.

Infections caused by Carbapenemase-producing Enterobacterales (CPE) are an important public health issue. Intravenous fosfomycin can be considered as an alternative for the treatment of serious infections caused by CPE. In this study, in vitro activity of fosfomycin was investigated among CPE isolates. Overall, 158 clinically relevant isolates obtained from 18 hospitals of 13 cities in Turkey with predetermined carbapenemase types were evaluated in the study, including Escherichia coli (n = 19) and Klebsiella spp. (n = 139). In vitro activity of fosfomycin was determined with agar dilution method. Among Klebsiella spp., 104 harbored blaOXA-48, 15 isolates carried both blaOXA-48 and blaNDM; three had both blaOXA-48 and blaVIM and nine isolates had blaNDM alone. Four isolates carried only blaVIM and two isolates harbored blaIMP alone. One isolate co-harbored blaVIM and blaNDM. Among E. coli isolates, blaOXA-48 and blaNDM were carried by 18 and one isolates, respectively. Resistance to fosfomycin was detected in 43.7% of the isolates. Among Klebsiella spp. and E. coli, these rates were 46.8% and 21.1%, respectively. In Klebsiella spp. resistance to fosfomycin was 49.5% in blaOXA-48 carriers; 26.7% in isolates co-harbouring blaOXA-48 and blaNDM and 66.7% in blaNDM carriers. In E. coli, fosfomycin resistance was detected among 16.7% of the blaOXA-48 carriers. High level of fosfomycin resistance in these isolates may be attributable to the fact that these isolates are multidrug resistant. The genetic background of resistance should also be investigated in order to understand the co-occurrence and transfer of resistance among the CPE.

Whole genome sequencing (WGS) fails to detect antimicrobial resistance (AMR) from heteroresistant subpopulation of Salmonella enterica

Due to rapidly falling costs, whole genome sequencing (WGS) is becoming an essential tool in the surveillance of antimicrobial resistance (AMR) in Salmonella enterica. Although there have been many recent works evaluating the accuracy of WGS in predicting AMR from a large number of Salmonella isolates, little attention has been devoted to deciphering the underlying causes of disagreement between the WGS genotype and experimentally determined AMR phenotype. This study analyzed the genomes of six S. enterica isolates previously obtained from raw chicken which exhibited disagreements between WGS genotype and AMR phenotype. A total of five WGS false negative predictions toward ampicillin, amoxicillin/clavulanate, colistin, and fosfomycin resistance were presented in conjunction with their corresponding empirical phenotypic and/or genetic evidence of heteroresistance. A further case study highlighting the inherent limitations of WGS to detect the underlying genetic mechanisms of colistin heteroresistance was presented. These findings implicate heteroresistance as an underlying cause for false negative WGS-based AMR predictions in S. enterica and suggest that widespread use of WGS in the surveillance of AMR in food isolates might severely underestimate true resistance rates.

ISEcp1-Mediated Transposition Leads to Fosfomycin and Broad-Spectrum Cephalosporin Resistance in Klebsiella pneumoniae.

A fosfomycin-resistant and carbapenemase (OXA-48)-producing Klebsiella pneumoniae isolate was recovered, and whole-genome sequencing revealed ISEcp1-blaCTX-M-14b tandemly inserted upstream of the chromosomally encoded lysR-fosA locus. Quantitative evaluation of the expression of lysR and fosA genes showed that this insertion brought a strong hybrid promoter leading to overexpression of the fosA gene, resulting in fosfomycin resistance. This work showed the concomitant acquisition of resistance to broad-spectrum cephalosporins and fosfomycin due to a single genetic event.

Oral fosfomycin use for pyelonephritis and complicated urinary tract infections: a 1 year review of outcomes and prescribing habits in a large municipal healthcare system.

The rising incidence of MDR uropathogens has driven increased use of oral fosfomycin for treatment of complicated urinary tract infections (cUTIs). However, there are limited data to support its use for cUTI, especially pyelonephritis. We performed a retrospective review of all oral fosfomycin prescriptions between 1 January and 31 December 2017 in the Los Angeles County Department of Health Service system, the second largest US municipal health system. We examined demographics, clinical characteristics, adverse events and 30 day treatment success for patients with cUTI. Follow-up urine cultures till 31 December 2018 were examined for emergence of fosfomycin resistance. Of 154 patients prescribed fosfomycin, 99 (64%) had cUTI. Of these, 39 (39%) had lower tract, 37 (37%) pyelonephritis and 23 (23%) non-pyelonephritis upper tract cUTI. Escherichia coli ESBL producers were the predominant pathogens (73%). Of the 63 patients with 30 day follow-up, 49 (78%) had clinical success, including 16/20 (80%) treated for pyelonephritis. Treatment failure was associated with male sex (P < 0.01), urological abnormalities (P = 0.05), non-E. coli cUTI (P = 0.03) and receipt of <25% IV therapy prior to fosfomycin switch (P = 0.03). Of patients prescribed fosfomycin (n = 154), fosfomycin-resistant E. coli were found in 9/64 (14%) of the patients with follow-up urine cultures >30 days after initial treatment. Despite the lack of data supporting its use, we found that most patients receiving oral fosfomycin off-label for cUTI, including pyelonephritis, had clinical success. However, emergence of subsequent resistance warrants caution. Prospective comparative studies should be done to better evaluate oral fosfomycin use for cUTI.

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.

Characterization of FosL1, a Plasmid-Encoded Fosfomycin Resistance Protein Identified in Escherichia coli.

Fosfomycin is gaining renewed interest for treating urinary tract infections. Monitoring fosfomycin resistance is therefore important in order to detect the emergence of novel resistance mechanisms. Here, we used the Rapid Fosfomycin NP test to screen a collection of extended-spectrum-β-lactamase-producing Escherichia coli isolates from Switzerland and found a fosfomycin-resistant isolate in which a novel plasmid-mediated fosfomycin resistance gene, named fosL1, was identified. The FosL1 protein is a putative glutathione S-transferase enzyme conferring high-level resistance to fosfomycin and sharing between 57% to 63% amino acid identity with other FosA-like family members. Genetic analyses showed that the fosL1 gene was embedded in a mobile insertion cassette and had likely been acquired by transposition through a Tn7-related mechanism. In silico analysis over GenBank databases identified the FosL1-encoding gene in addition to another variant (fosL1 and fosL2, respectively) in two Salmonella enterica isolates from the United States. Our study further highlights the necessity of monitoring fosfomycin resistance in Enterobacteriaceae to identify the emergence of novel mechanisms of resistance.