Fosfomycin Resistance Research Articles

Aliidiomarina shirensis as Possible Source of the Integron- and Plasmid-Mediated Fosfomycin Resistance Gene fosC2.

In-silico analysis and cloning experiments identified a fosC2-like fosfomycin resistance gene in the chromosome of Aliidiomarina shirensis, with our data suggesting that this bacterium might be added to the list of species identified as reservoirs of fos-like genes that were subsequently acquired by other Gram-negative species. Indeed, the fosC2 gene was identified as acquired in Providencia huaxinensis and Aeromonas hydrophila isolates, with this gene being located in class 1 integron structures in the latter cases. Biochemical characterization and site-directed mutagenesis showed a higher catalytic efficiency for the intrinsic FosC2AS (from A. shirensis) than for the acquired FosC2 (from P. huaxinensis) enzyme due to a single substitution in the amino acid sequence (Gly43Glu). Notably, this study constitutes the first identification of the likely natural reservoir of a complete gene cassette (including its attC site).

The Genomic Characterization of KPC-Producing Klebsiella pneumoniae from the ICU of a Teaching Hospital in Shanghai, China.

PurposeThis study retrospectively analyzed the genome characteristics of blaKPC-2 in multidrug-resistant Klebsiella pneumoniae collected from the ICU of a teaching hospital in Shanghai, China.MethodsFrom February 2018 to December 2019, 36 strains of multidrug-resistant Klebsiella pneumoniae were collected from the bronchoalveolar lavage fluid of critically ill patients. The genome of all isolates was obtained through the Illumina sequence, and single nucleotide polymorphisms of the blaKPC-2 gene were analyzed to explore blaKPC-2’s evolutionary characteristics. Different strains’ genetic relationships and homology were studied by constructing an evolutionary tree on a single copy orthologue. Pacbio combined Illumina sequence was conducted to evaluate the structure and potential mobility of drug-resistant plasmids of the strain KP-s26.ResultsThe distribution of resistance and virulence genes had little difference, but most strains had significant differences in the plasmid-encoded region. Most strains (31/36) carried the carbapenemase gene blaKPC-2, with no single nucleotide polymorphism in different strains. Extended-spectrum β-lactamase resistance genes, such as blaCTX-M and blaSHV, were found in the isolates, but no metallo-β-lactamases were detected. All strains with blaKPC-2 coexisted with chromosomal-associated fosfomycin resistance genes fosA6, and the coexistence of blaKPC-2 and blaCTX variants (blaCTX-M-15, blaCTX-M-65, and blaCTX-M-27) was also detected in 29/31 strains. The isolate KP-s26 carried five circular plasmids. pA and pB were conjugate plasmids, as they carried drug resistance genes and contained a complete IV secretion system.ConclusionThe blaKPC-2 carbapenemase gene is relatively conservative in the process of evolution; drug-resistant plasmids containing conjugated transfer elements contribute to the spreading of drug resistance. The coexistence of blaKPC-2 with fosA6 or blaCTX-M variants was associated with increased fosfomycin resistance and broad-spectrum β-lactam resistance, respectively.Clinical Trials RegistrationClinical Trials.gov Identifier: NCT03950544

Whole-genome analysis of carbapenem-resistant Acinetobacter baumannii from clinical isolates in Southern Thailand

The worldwide spread of carbapenem-resistant Acinetobacter baumannii (CRAB) has become a healthcare challenge for some decades. To understand its molecular epidemiology in Southern Thailand, we conducted whole-genome sequencing (WGS) of 221 CRAB clinical isolates. A comprehensive bioinformatics analysis was performed using several tools to assemble, annotate, and identify sequence types (STs), antimicrobial resistance (AMR) genes, mobile genetic elements (MGEs), and virulence genes. ST2 was the most prevalent ST in the CRAB isolates. For the detection of AMR genes, almost all CRAB isolates carried the blaOXA-23 gene, while certain isolates harbored the blaNDM-1 or blaIMP-14 genes. Also, various AMR genes were observed in these CRAB isolates, particularly aminoglycoside resistance genes (e.g., armA, aph(6)-Id, and aph(3″)-Ib), fosfomycin resistance gene (abaF), and tetracycline resistance genes (tet(B) and tet(39)). For plasmid replicon typing, RepAci1 and RepAci7 were the predominant replicons found in the CRAB isolates. Many genes encoding for virulence factors such as the ompA, adeF, pgaA, lpxA, and bfmR genes were also identified in all CRAB isolates. In conclusion, most CRAB isolates contained a mixture of AMR genes, MGEs, and virulence genes. This study provides significant information about the genetic determinants of CRAB clinical isolates that could assist the development of strategies for improved control and treatment of these infections.

Chromosomally Located fosA7 in Salmonella Isolates From China.

This study aimed to investigate the prevalence of fosfomycin fosA7 in Salmonella enterica isolates from food animals and retail meat products in China and the impact of fosA7 on bacterial fitness. A total of 360 Salmonella isolates collected from 11 provinces and cities in China were detected for fosA7. All fosA7-positive Salmonella isolates were determined minimum inhibitory concentrations (MICs) and sequenced by Illumina Hiseq. The fosA7 gene of S. Derby isolate HA2-WA5 was knocked out. The full length of fosA7 was cloned into vector pBR322 and then transformed into various hosts. MICs of fosfomycin, growth curves, stability, and fitness of fosA7 were evaluated. The fosA7 gene was identified in S. Derby (ST40, n = 30) and S. Reading (ST1628, n = 5). MICs to fosfomycin of 35 fosA7-positive isolates were 1 to 32 mg/L. All fosA7 were located on chromosomes of Salmonella. The deletion of fosA7 in HA2-WA5 decreased fosfomycin MIC by 16-fold and slightly affected its fitness. The acquisition of plasmid-borne fosA7 enhanced MICs of fosfomycin in Salmonella (1,024-fold) and Escherichia coli (16-fold). The recombinant plasmid pBR322-fosA7 was stable in Salmonella Typhimurium, S. Pullorum, S. Derby, and E. coli, except for Salmonella Enteritidis, and barely affected on the growth of them but significantly increased biological fitness in Salmonella. The spread of specific Salmonella serovars such as S. Derby ST40 will facilitate the dissemination of fosA7. fosA7 can confer high-level fosfomycin resistance and enhance bacterial fitness in Salmonella if transferred on plasmids; thus, it has the potential to be a reservoir of the mobilized fosfomycin resistance gene.

The Antibiotic Fosfomycin Mimics the Effects of the Intermediate Metabolites Phosphoenolpyruvate and Glyceraldehyde-3-Phosphate on the Stenotrophomonas maltophilia Transcriptome.

Stenotrophomonas maltophilia is an opportunistic pathogen with an environmental origin, which presents a characteristically low susceptibility to antibiotics and is capable of acquiring increased levels of resistance to antimicrobials. Among these, fosfomycin resistance seems particularly intriguing; resistance to this antibiotic is generally due to the activity of fosfomycin-inactivating enzymes, or to defects in the expression or the activity of fosfomycin transporters. In contrast, we previously described that the cause of fosfomycin resistance in S. maltophilia was the inactivation of enzymes belonging to its central carbon metabolism. To go one step further, here we studied the effects of fosfomycin on the transcriptome of S. maltophilia compared to those of phosphoenolpyruvate—its structural homolog—and glyceraldehyde-3-phosphate—an intermediate metabolite of the mutated route in fosfomycin-resistant mutants. Our results show that transcriptomic changes present a large degree of overlap, including the activation of the cell-wall-stress stimulon. These results indicate that fosfomycin activity and resistance are interlinked with bacterial metabolism. Furthermore, we found that the studied compounds inhibit the expression of the smeYZ efflux pump, which confers intrinsic resistance to aminoglycosides. This is the first description of efflux pump inhibitors that can be used as antibiotic adjuvants to counteract antibiotic resistance in S. maltophilia.

Structural and functional characterization of fosfomycin resistance conferred by FosB from Enterococcus faecium.

The Gram-positive pathogen Enterococcus faecium is one of the leading causes of hospital-acquired vancomycin resistant enterococci (VRE) infections. E. faecium has extensive multidrug resistance and accounts for more than two million infections in the United States each year. FosB is a fosfomycin resistance enzyme found in Gram-positive pathogens like E. faecium. Typically, the FosB enzymes are Mn2+ -dependent bacillithiol (BSH) transferases that inactivate fosfomycin through nucleophilic addition of the thiol to the antibiotic. However, our kinetic analysis of FosBEf shows that the enzyme does not utilize BSH as a thiol substrate, unlike the other well characterized FosB enzymes. Here we report that FosBEf is a Mn2+ -dependent L-cys transferase. In addition, we have determined the three-dimensional X-ray crystal structure of FosBEf in complex with fosfomycin at a resolution of 2.0Å. A sequence similarity network (SSN) was generated for the FosB family to investigate the unexpected substrate selectivity. Three non-conserved residues were identified in the SSN that may contribute to the substrate selectivity differences in the family of enzymes. Our structural and functional characterization of FosBEf establishes the enzyme as a potential target and may prove useful for future structure-based development of FosB inhibitors to increase the efficacy of fosfomycin.

Clonal Dissemination of Clinical Carbapenem-Resistant Klebsiella pneumoniae Isolates Carrying fosA3 and bla KPC-2 Coharboring Plasmids in Shandong, China.

Treatment strategies of infection by carbapenem-resistant Klebsiella pneumoniae (CRKP) are limited. Fosfomycin, a broad-spectrum antibiotic, has attracted renewed interest in combination therapy to fight K. pneumoniae infections. However, reports on fosfomycin-resistant K. pneumoniae are increasing. Among the 57 CRKP strains, 40 (70.2%) were resistant to fosfomycin. Thus, whole-genome sequencing and bioinformatics analysis were conducted to reveal molecular characteristics of fosfomycin-resistant K. pneumoniae. Twenty-three isolates coharbored fosAkp and fosA3, with K. pneumoniae carbapenemase (KPC)-producing ST11-KL64-wzi64-O2 (n = 13) and ST11-KL47-wzi209-OL101 (n = 8), the predominating clonal groups, while fosA3 was not detected in isolates carrying class B carbapenemase genes. Twenty-two (out of 26) ST11-KL64 strains were positive for rmpA2, of which 12 carried fosA3. Four of the 23 fosA3-positive isolates could successfully transfer their fosfomycin-resistant determinants to Escherichia coli J53AziR. All four strains belonged to ST11-KL47 with the same pulsed-field gel electrophoresis profile, and their transconjugants acquired fosfomycin, carbapenem, and aminoglycoside resistance. A 127-kb conjugative pCT-KPC-like hybrid plasmid (pJNKPN52_KPC_fosA) coharboring fosA3, blaKPC–2, blaCTX–M–65, blaSHV–12, rmtB, and blaTEM–1 was identified. ST11-KL64 and ST11-KL47 K. pneumoniae, with higher resistance and virulence, should be critically monitored to prevent the future dissemination of resistance.

Pharmacodynamics of Linezolid Plus Fosfomycin Against Vancomycin–Resistant Enterococcus faecium in a Hollow Fiber Infection Model

The optimal therapy for severe infections caused by vancomycin-resistant Enterococcus faecium (VREfm) remains unclear, but the combination of linezolid and fosfomycin may be a good choice. The 24-h static-concentration time-kill study (SCTK) was used to preliminarily explore the pharmacodynamics of linezolid combined with fosfomycin against three clinical isolates. Subsequently, a hollow-fibre infection model (HFIM) was used for the first time to further investigate the pharmacodynamic activity of the co-administration regimen against selected isolates over 72 h. To further quantify the relationship between fosfomycin resistance and bacterial virulence in VREfm, the Galleria mellonella infection model and virulence genes expression experiments were also performed. The results of SCTK showed that the combination of linezolid and fosfomycin had additive effect on all strains. In the HFIM, the dosage regimen of linezolid (12 mg/L, steady-state concentration) combined with fosfomycin (8 g administered intravenously every 8 h as a 1 h infusion) not only produced a sustained bactericidal effect of 3∼4 log10 CFU/mL over 72 h, but also completely eradicated the resistant subpopulations. The expression of virulence genes was down-regulated to at least 0.222-fold in fosfomycin-resistant strains compared with baseline isolate, while survival rates of G. mellonella was increased (G. mellonella survival ≥45% at 72 h). For severe infections caused by VREfm, neither linezolid nor fosfomycin monotherapy regimens inhibited amplification of the resistant subpopulations, and the development of fosfomycin resistance was at the expense of the virulence of VREfm. The combination of linezolid with fosfomycin produced a sustained bactericidal effect and completely eradicated the resistant subpopulations. Linezolid plus Fosfomycin is a promising combination for therapy of severe infections caused by VREfm.

647. Investigation of Heteroresistance Among Klebsiella pneumoniae (KP) Inner Colonies (IC) Observed During Fosfomycin Disk Diffusion (DD) Testing

Abstract Background During fosfomycin DD testing, the frequent occurrence of non-susceptible IC within the zone of inhibition of susceptible isolates has been noted. The Clinical & Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility Testing (EUCAST) have contradicting recommendations on how IC should be interpreted; CLSI recommends considering IC when interpreting DD results whereas EUCAST recommends ignoring them. This study sought to identify the susceptibility of these IC and to understand whether heteroresistance contributes to the appearance of IC during fosfomycin DD. Methods This study included a convenience sample of 71 KP clinical isolates from 3 United States locations. During DD testing, 58 (81.7%) of these isolates displayed at least one IC. Broth microdilution (BMD) minimal inhibitory concentration (MIC) testing, using extrapolated CLSI Escherichia coli breakpoints, was performed on a subset (n=32) of the IC in duplicate for comparison to the corresponding parent MIC values. This was followed by a modified disk elution screening test for heteroresistance to compare the frequency of low level resistance (LLR) and high level resistance (HLR) between the susceptible isolates that produced resistant IC (n=6) and those that did not produce any IC (n=3). Results The MIC range for the IC isolates (128 to > 1024 μg/mL) increased as compared to the parent isolates (< 2 to > 256 μg/mL) and MIC50/90 increased from the parent (128/ > 256 μg/mL) to IC (1024/ > 1024 μg/mL) isolates. All IC isolates had a resistant MIC value vs. 46.5% of parent isolates, and over 90% of IC isolates had an MIC at least 2 dilutions higher than their corresponding parent isolate. Heteroresistance screening found all tested isolates to be positive for LLR, and 8 of 9 positive for HLR, while the one HLR-negative isolate was IC-producing. Conclusion IC were frequent during fosfomycin DD testing and were commonly more resistant than their corresponding KP parent isolates. A small subset of these isolates tested via a modified disk elution test displayed either LLR or HLR regardless of the absence of IC. These results call for further investigation among a larger isolate set to understand what mechanisms are responsible for the frequency of IC and their increased fosfomycin resistance. Disclosures All Authors: No reported disclosures

First Detection of Klebsiella pneumoniae Isolate Co-Harboring Fosfomycin Resistance Gene fosA3 and blactx-m Among Gram Negative Urine Isolates in a Turkish Hospital.

The most common gram-negative pathogens in urinary tract infections are Escherichia coli and Klebsiella pneumoniae. Therapy that is often empirical relies on local antibiotic resistance data, hence monitorization of antimicrobial resistance periodically in each hospital is a requirement. In this study, antibiotic susceptibility profiles of consecutive urinary isolates (E. coli [n = 235] and K. pneumoniae [n = 56]) of adult patients collected between February 2018 and February 2019 from inpatients in Hacettepe University Hospital were assessed. Isolates resistant to fosfomycin (minimum inhibitory concentration >32 mg/L) were further investigated for the presence of fosA, fosA3, and fosC2. Fosfomycin susceptibility was determined by agar dilution method. Broth microdilution method was performed for amikacin, gentamicin, ceftazidime, ceftriaxone, meropenem, ciprofloxacin, tigecycline, trimethoprim/sulfamethoxazole (TMP/SMX), colistin, and piperacillin/tazobactam (PIP/TAZ). PCR method was employed to investigate fosA, fosA3, fosC2, and blaCTX-M. Existence of fosA3 gene was confirmed by sequencing. Resistance rates to amikacin, gentamicin, ceftazidime, ceftriaxone, meropenem, ciprofloxacin, tigecycline, TMP/SMX, colistin, and PIP/TAZ were 2.7%, 18.5%, 25.4%, 33.0%, 3.4%, 45.4%, 2.4%, 43.6%, 6.2%, and 23.7%, respectively. Sixteen isolates (5.5%) were resistant to fosfomycin. Resistance was most frequently observed in K. pneumoniae (n = 9). fosA3 gene was detected in one fosfomycin-resistant K. pneumoniae isolate. This isolate also carried blaCTX-M. fosC2 and fosA genes could not be detected in any of the isolates. In this study, we report for the first time the existence of fosA3 in Turkey and its association with the blaCTX-M gene. As a result of increasing blaCTX-M producing Enterobacterales isolates globally, increase in fosfomycin resistance may be expected in near future.

Genomic analysis of antibiotic-resistant Enterobacter spp. from wastewater sources in South Africa: The first report of the mobilisable colistin resistance mcr-10 gene in Africa

This study delineated the resistome, virulome, mobile genetic elements, clonal lineages, phylogenetic and epidemiological insights of antibiotic-resistant Enterobacter cloacae complex (ECC) isolates obtained from a wastewater treatment plant (WWTP) and its associated waters in KwaZulu Natal, South Africa. The ECC isolates obtained from the final effluent of a (WWTP) and the receiving river upstream and downstream of the WWTP were sequenced on an Illumina MiSeq machine. Genomic analysis revealed that E. asburiae and E. kobei both harboured the fosfomycin resistance gene ( fosA ) and E. kobei was positive for the colistin resistance gene mcr-10 . The genetic environment of the fosA gene was not associated with any MGE and was found on the chromosome in both E. asburiae and E. kobei. The mcr-10 cassette was found associated with a tyrosine integrase and the insertion sequence IS3 (tyrosine-type recombinase/integrase: mcr-10 :IS3) genetic context located on an E. kobei plasmid pSTW0522-51-1 (with a 100% homology). All three isolates carried different bla ACT genes that were not associated with any MGE but had the sugE gene (encodes the quaternary ammonium compound resistance protein) in their genetic environment suggesting co-resistance to biocides and antibiotics. E. kobei harboured the virulence gene terC which confers resistance to tellurium. The E. kobei isolate was phylogenetically related to clinical isolates whereas E. asburiae and E. hormaechei isolates were closely related to environmental isolates. The occurrence of an E. kobei isolate carrying a mobilisable colistin resistance mcr-10 gene in the river water upstream of a major WWTP is the first report of the mcr-10 gene in Africa and is a public health concern warranting monitoring to gain genomic insights into the full extent of ABR in river systems in South Africa and globally.

Whole-genome sequencing analysis of highly prevalent Salmonella serovars in wild boars from a national park in Sardinia

The study investigated Salmonella prevalence in slaughtered wild boars from Asinara island National Park in Sardinia (Italy). Salmonella isolates were serotyped and the antimicrobial resistance profile was evaluated. Isolates were furthermore characterized with Whole Genome Sequencing. Additional analysis were performed in order to evaluate the microbial hygiene of wild boars carcasses. A total of 306 samples were tested for Salmonella presence, of which n. 270 were from n. 90 slaughtered wild boars and n. 36 were from slaughterhouse environment. Salmonella was detected in 32/90 (35.6%) colon content and 16/90 (17.8%) mesenteric lymph nodes samples. The overall prevalence of Salmonella carriers wild boars, either in colon content and/or lymph nodes, was 42/90 (46.7%) but only one animal was found positive at carcass level. Prevalence was not influenced ( P > 0.05) by the age and the gender of the animals. Eight different Salmonella serotypes were identified between 50 isolates, with the most common ones being Abony and Newport. The sequence analysis showed that S. Abony and S. Newport strains clustered separately from public genomes. This finding refects the origin of the animals from the geographical closed environment of Asinara island. Moreover, sequence analysis showed high similarity between strains belonging to the same serovar. S . Derby and S . Agona strains exhibited phenotypic resistance to fosfomycin and harboured the fosA7 gene. The mean aerobic colony count and Enterobacteriaceae values showed a good hygiene level of the carcasses. This study demonstrated that Salmonella enterica is widespread in wild boar population of Asinara island and could play an important role in transmission to humans. The finding of Salmonella strains resistant to fosfomycin is of primary importance as this drug is used as an alternative treatment for infections caused by multi-drug resistant microorganisms. • Salmonella in wild boar play a role in transmission to humans. • High variability of Salmonella serovars. • High similarity of Salmonella serovars with whole genome sequencing. • Extended sensitivity to antimicrobials. • Fosfomycin resistance encoded by the gene fosA 7 in S. Agona and S. Derby isolates.

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.

Investigation of In-vitro Efficacy of Intravenous Fosfomycin in Extensively Drug-Resistant Klebsiella pneumoniae Isolates and Effect of Glucose 6-Phosphate on Sensitivity Results

BackgroundThe aim of this study was to determine the in vitro efficacy of intravenous (IV) fosfomycin against extensively drug-resistant Enterobacterales strains and the effect of glucose 6-phosphate (G6-P) on sensitivity results. Material methodThirty-two extensively drug-resistant Klebsiella pneumonia strains were included in the study. Detection of the carbapenemase genes was performed using the Gene-Xpert® System Carba R® kit. Susceptibility of IV fosfomycin was assessed using the agar dilution method. The agar dilution method was repeated using Muller-Hinton Agar medium without G6-P to assess the effect of G6-P on sensitivity results. ResultsAll strains in the study produced carbapenemases and were resistant to all drugs tested, including carbapenems, piperacillin-tazobactam, ceftriaxone, and ceftazidime. Fosfomycin resistance was detected in 3 (9.3%) strains. When the sensitivity test was repeated without G6-P, fosfomycin resistance was detected in 82.7% of the fosfomycin-susceptible strains. The Gene-Xpert® System showed NDM-1 in 46.8%, OXA-48 in 18.7%, KPC in 3.1%, and NDM-1 + OXA-48 in 21.8% of the strains. OXA-48 was detected in one of the resistant strains, and none of the viable genes were detected in two of the resistant strains. ConclusionThis study shows that IV fosfomycin is a potentially important treatment alternative for infections caused by common resistant strains. Accurate results may not be obtained unless G6-P is used in the agar dilution method for in vitro susceptibility studies of fosfomycin.

Differences in Fosfomycin Resistance Mechanisms between Pseudomonas aeruginosa and Enterobacterales.

Multidrug-resistant (MDR) Pseudomonas aeruginosa presents a serious threat to public health due to its widespread resistance to numerous antibiotics. P. aeruginosa commonly causes nosocomial infections including urinary tract infections (UTI) which have become increasingly difficult to treat. The lack of effective therapeutic agents has renewed interest in fosfomycin, an old drug discovered in the 1960s and approved prior to the rigorous standards now required for drug approval. Fosfomycin has a unique structure and mechanism of action, making it a favorable therapeutic alternative for MDR pathogens that are resistant to other classes of antibiotics. The absence of susceptibility breakpoints for fosfomycin against P. aeruginosa limits its clinical use and interpretation due to extrapolation of breakpoints established for Escherichia coli or Enterobacterales without supporting evidence. Furthermore, fosfomycin use and efficacy for treatment of P. aeruginosa are also limited by both inherent and acquired resistance mechanisms. This narrative review provides an update on currently identified mechanisms of resistance to fosfomycin, with a focus on those mediated by P. aeruginosa such as peptidoglycan recycling enzymes, chromosomal Fos enzymes, and transporter mutation. Additional fosfomycin resistance mechanisms exhibited by Enterobacterales, including mutations in transporters and associated regulators, plasmid-mediated Fos enzymes, kinases, and murA modification, are also summarized and contrasted. These data highlight that different fosfomycin resistance mechanisms may be associated with elevated MIC values in P. aeruginosa compared to Enterobacterales, emphasizing that extrapolation of E. coli breakpoints to P. aeruginosa should be avoided.

First Report of the Plasmid-mediated fosB Gene in Enterococcus faecalis from Pigs.

Plasmid-mediated fosfomycin determinants is a global public health concern due to the increasing dissemination of fosfomycin resistance and limited clinical treatment options. Information about the fosfomycin resistant and molecular genetic among Enterococcus spp. is still lacking. In this study, we found the first plasmid-medieted fosB in Enterococcus faecalis from pigs, and all the fosfomycin resistant Enterococcus spp. (FRE) isolates were multi-drug resistant. S1-PFGE, Southern blot and conjugation experiments indicated that the fosB gene located on ~54.7 kb transferable plasmids. Relative competition assay confirmed that the fosB-carrying plasmid impaired fitness in recipient E. faecalis JH2-2. Illumina and the MinION sequencing data revealed that both E. faecalis ES-1 and ES-2 isolates belonged to novel ST (ST964), and had 71 SNPs difference. WGS showed that the genetic environments of fosB were diverse among different species, and the linezolid resistance gene optrA was found in the fosB-carrying strains. To summarize, for the first time, we reported plasmid-mediated fosB in E. faecalis from pigs. And, the co-occurrence of fosB and optrA pose a serious threat to public health.

Investigation of Fosfomycin Susceptibility in Extended Spectrum Beta-Lactamase Producing Enteric Bacteria by Disk Diffusion, Automated System and Agar Dilütion Kit

Due to the increase of bacteria that develop resistance to classical antibiotics in recent years, fosfomycin has become one of the antibiotics gaining importance again. Fosfomycin exerts its bactericidal effect on many bacteria by binding to a cytoplasmic enzyme, MurA, and inhibiting the enzyme enopurivil transferase, which is involved in peptidoglycan synthesis. Bacteria producing ESBL are common pathogens in nosocomial infections and resistance is frequently encountered in their treatment. Fosfomycin is considered as an alternative drug in these infections. With its reintroduction, it is important to establish reliable and reproducible susceptibility testing methods for fosfomycin. In this study, it was aimed to evaluate the utility of disk diffusion, agar dilution kit and automated MicroScan methods (Beckman Coulter, Brea, CA, ABD) for fosfomycin susceptibility of ESBL-producing enteric bacteria. The study included 120 ESBL positive isolates of enteric bacteria (89 Escherichia coli, 31 Klebsiella pneumoniae) cultivated from the samples sent to the Microbiology Laboratory of the Mersin University Medical Faculty of Medicine Hospital between May 2019 and November 2019. Fosfomycin susceptibilities of the isolates included in the study were tested by disk diffusion, automated MicroScan and the commercial agar dilution kit. In the study, 37 (30.8%) of 120 isolates were determined as resistant to fosfomycin by disk diffusion method and six (5%) of the isolates were found to be resistant to fosfomycin with the MicroScan automatized system. With the agar dilution kit, 11 (9.1%) of 120 isolates were found to be resistant as the minimum inhibitory concentration (MIC) values were > 32 µg/ml. Fosfomycin resistance was significantly higher in K.pneumoniae isolates than E.coli isolates by agar dilution method (p= 0.006). Ertapenem resistance was detected in seven of 120 isolates. All seven isolates that were resistant to ertapenem were susceptible to fosfomycin by agar dilution method. All three isolates, which were found resistant to colistin, were susceptible to fosfomycin by agar dilution method. In the study, the categorical agreement of the disk diffusion and MicroScan methods in E.coli isolates with the agar dilution was high and the error rates were low. For K.pneumoniae, categorical concordance was calculated as 22.5% for disk diffusion test and 74.1% for MicroScan, while major and minor error rates were found to be quite high. As a result; fosfomycin is thought to be an alternative drug option in the treatment of infections caused by especially carbapenem and colistin resistant bacteria, with its high activity on ESBL-producing enteric bacteria. It has been determined that there is a great agreement between the agar dilution, which is the reference method for the determination of fosfomycin resistance for E.coli, and the MicroScan and disk diffusion methods, and that the disk diffusion method which provides ease of use in terms of cost and labor compared to the agar dilution method, can be used as a reliable method. However, it has been demonstrated that the agar dilution method should be used in K.pneumoniae isolates. Nevertheless; It was concluded that if the automatized system is used in laboratories where agar dilution cannot be performed, the results should be interpreted carefully, since the error rates are very high.

502: Combating fosfomycin resistance in CF-related bacterial lung infections

502: Combating fosfomycin resistance in CF-related bacterial lung infections

Interplay between Phenotypic Resistance to Relevant Antibiotics in Gram-Negative Urinary Pathogens: A Data-Driven Analysis of 10 Years’ Worth of Antibiogram Data

The global emergence of antimicrobial resistance (AMR) has become a critical issue for clinicians, as it puts the decades of developments in the medical field in jeopardy, by severely limiting the useful therapeutic arsenal of drugs, both in nosocomial and community-acquired infections. In the present study, a secondary analysis of taxonomic and resistance data was performed, corresponding to urinary tract infections (UTIs) caused by Gram-negative bacteria, detected between 1 January 2008 to 31 December 2017 at the Albert Szent-Györgyi Health Center, University of Szeged. The following were identifiable from the data collected: year of isolation; outpatient (OP)/inpatient (IP) origin of the isolate; taxonomy; and susceptibility/resistance to selected indicator antibiotics. Principal component analysis (PCA) and a correlation matrix were used to determine the association between the presences of resistance against indicator antibiotics in each taxonomic group. Overall, data from n = 16,240 outpatient and n = 13,964 inpatient Gram-negative UTI isolates were included in the data analyses. In E. coli, strong positive correlations were seen between resistance to ciprofloxacin (CIP) and gentamicin (GEN) resistance (OP: r = 0.6342, p = 0.049; IP: r = 0.9602, p < 0.001), whereas strong negative correlations were shown for fosfomycin (FOS) and nitrofurantoin (NIT) resistance (OP: r = −0.7183, p = 0.019; IP: r = −0.7437; p = 0.014). For Klebsiella spp. isolates, CIP resistance showed strong positive correlation with resistance to third-generation cephalosporins (3GC) and GEN (r = 0.7976, p = 0.006 and r = 0.7428, p = 0.014, respectively) in OP isolates, and with resistance to trimethoprim-sulfamethoxazole (SXT) and FOS (r = 0.8144, p = 0.004 and r = 0.7758, p < 0.001, respectively) in IP isolates. For members of the Citrobacter-Enterobacter-Serratia group, the resistance among indicator antibiotics showed a strong positive correlation, with the exception of FOS resistance. In the Proteus-Providencia-Morganella group, the strongest association was noted between CIP and SXT resistance (OP: r = 0.9251, p < 0.001; IP: r = 0.8007; p = 0.005). In the case of OP Acinetobacter spp., CIP showed strong and significant positive correlations with most indicator antibiotics, whereas for IP isolates, strong negative correlations arose among imipenem (IMI) resistance and resistance to other drugs. For Pseudomonas spp., strong and positive correlations were noted among resistance to β-lactam antibiotics and aminoglycosides, with the exception of ceftazidime (CEFT), showing strong, but negative correlations. Though molecular tests and sequencing-based platforms are now considered as the gold-standard for AMR surveillance, standardized collection of phenotypic resistance data and the introduction of Big Data analytic methods may be a viable alternative for molecular surveillance, especially in low-resource settings.

Retrospective evaluation of intravenous fosfomycin in multi-drug resistant infections at a tertiary care hospital in Lebanon.

Fosfomycin has re-emerged as a possible therapeutic alternative for the treatment of resistant bacterial pathogens. Its main mechanism of action is the inhibition of the initial step of cell wall synthesis and is active against both Gram-positive and Gram-negative bacteria. However, its clinical effectiveness against multidrug resistant bacteria remains largely unknown. Therefore, we aim to evaluate the clinical and microbiological effectiveness of intravenous fosfomycin as well as its safety in a tertiary care teaching hospital in Lebanon. This is a retrospective chart review of adult patients who had presented to the hospital and were treated with intravenous fosfomycin for at least 24 hours for any type of infection between 2014 and 2019. Among 31 episodes treated with intravenous fosfomycin, 68% had an overall favorable clinical response. In 84% of the episodes, fosfomycin was administered in combination with other antibiotics, commonly tigecycline. Of those with available cultures at end of therapy, 73% achieved microbiological success. No relapse was documented within 30 days of completion of therapy. In the episodes secondary to resistant pathogens, the rates of favorable clinical outcome and microbiological success at the end of therapy were 71% and 73%, respectively. Fosfomycin resistance developed in two cases and mild adverse events occurred in 65% of the episodes during the course of treatment. Fosfomycin is a safe and effective option in the treatment of multi-drug resistant infections. Nevertheless, careful stewardship is important to maintain its efficacy and to reduce the risk of selection of antimicrobial resistance.