Assessment of three antibiotic combination regimens against Gram-negative bacteria causing neonatal sepsis in low- and middle-income countries.

Gram-negative bacteria (GNB) are a major cause of neonatal sepsis in low- and middle-income countries (LMICs). Although the World Health Organization (WHO) reports that over 80% of these sepsis deaths could be prevented through improved treatment, the efficacy of the currently recommended first- and second-line treatment regimens for this condition is increasingly affected by high rates of drug resistance. Here we assess three well known antibiotics, fosfomycin, flomoxef and amikacin, in combination as potential antibiotic treatment regimens by investigating the drug resistance and genetic profiles of commonly isolated GNB causing neonatal sepsis in LMICs. The five most prevalent bacterial isolates in the NeoOBS study (NCT03721302) are Klebsiella pneumoniae, Acinetobacter baumannii, E. coli, Serratia marcescens and Enterobacter cloacae complex. Among these isolates, high levels of ESBL and carbapenemase encoding genes are detected along with resistance to ampicillin, gentamicin and cefotaxime, the current WHO recommended empiric regimens. The three new combinations show excellent in vitro activity against ESBL-producing K. pneumoniae and E. coli isolates. Our data should further inform and support the clinical evaluation of these three antibiotic combinations for the treatment of neonatal sepsis in areas with high rates of multidrug-resistant Gram-negative bacteria.

Selective bacteriophages reduce the emergence of resistant bacteria in bacteriophage-antibiotic combination therapy.

Escherichia coli O157:H7 is a globally important foodborne pathogen with implications for food safety. Antibiotic treatment for O157 may potentially contribute to the exacerbation of hemolytic uremic syndrome, and the increasing prevalence of antibiotic-resistant strains necessitates the development of new treatment strategies. In this study, the bactericidal effects and resistance development of antibiotic and bacteriophage monotherapy were compared with those of combination therapy against O157. Experiments involving continuous exposure of O157 to phages and antibiotics, along with genetic deletion studies, revealed that the deletion of glpT and uhpT significantly increased resistance to fosfomycin. Furthermore, we found that OmpC functions as a receptor for the PP01 phage, which infects O157, and FhuA functions as a receptor for the newly isolated SP15 phage, targeting O157. In the glpT and uhpT deletion mutants, additional deletion in ompC, the receptor for the PP01 phage, increased resistance to fosfomycin. These findings suggest that specific phages may contribute to antibiotic resistance by selecting the emergence of gene mutations responsible for both phage and antibiotic resistance. While combination therapy with phages and antibiotics holds promise for the treatment of bacterial infections, careful consideration of phage selection is necessary.IMPORTANCEThe combination treatment of fosfomycin and bacteriophages against Escherichia coli O157 demonstrated superior bactericidal efficacy compared to monotherapy, effectively suppressing the emergence of resistance. However, mutations selected by phage PP01 led to enhanced resistance not only to the phage but also to fosfomycin. These findings underscore the importance of exercising caution in selecting phages for combination therapy, as resistance selected by specific phages may increase the risk of developing antibiotic resistance.

FosA8-producing E. coli ST131: clinical cases in Italy, February 2023.

Fosfomycin-resistant FosA8-producing Enterobacterales are uncommon strains with extremely low incidence in Europe, based on only three reports in the literature. We detected FosA8-producing Escherichia coli ST131 in clinical isolates from two patients admitted in February 2023 to a rehabilitation unit in Italy. The occurrence of rare fosA-like genes in the high-risk clone ST131 is of clinical relevance. The dissemination of FosA-producing E. coli, although still at low levels, should be continuously monitored.

Effect of ceftazidime-avibactam combined with different antimicrobials against carbapenem-resistant Klebsiella pneumoniae.

This study aimed to assess the in vitro efficacy of ceftazidime-avibactam (CZA) in combination with various antimicrobial agents against carbapenem-resistant Klebsiella pneumoniae (CRKP). We selected 59 clinical CRKP isolates containing distinct drug resistance mechanisms. The minimum inhibitory concentrations (MICs) of meropenem (MEM), colistin (COL), eravacycline (ERA), amikacin (AK), fosfomycin (FOS), and aztreonam (ATM), both individually and in combination with CZA, were tested using the checkerboard method. The interactions of antimicrobial agent combinations were assessed by fractional inhibitory concentration index (FICI) and susceptible breakpoint index (SBPI). The time-kill curve assay was employed to dynamically evaluate the effects of these drugs alone and in combination format. In the checkerboard assay, the combination of CZA+MEM showed the highest level of synergistic effect against both KPC-producing and carbapenemase-non-producing isolates, with synergy rates of 91.3% and 100%, respectively. Following closely was the combination of FOS+CZA . For metallo-beta-lactamases (MBLs) producing strains, ATM+CZA displayed complete synergy, while the combination of MEM+CZA showed a synergy rate of only 57.14% for NDM-producing strains and 91.67% for IMP-producing strains. In the time-kill assay, MEM+CZA also demonstrated significant synergistic effects against the two KPC-2-producing isolates (Y070 and L70), the two carbapenemase-non-producing isolates (Y083 and L093), and the NDM-1-producing strain L13, with reductions in log10 CFU/mL exceeding 10 compared to the control. Against the IMP-producing strain Y047, ATM+CZA exhibited the highest synergistic effect, resulting in a log10 CFU/mL reduction of 10.43 compared to the control. The combination of CZA and MEM exhibited good synergistic effects against KPC-producing and non-enzyme-producing strains, followed by the FOS+CZA combination. Among MBL-producing strains, ATM+CZA demonstrated the most pronounced synergistic effect. However, the combinations of CZA with ERA, AK, and COL show irrelevant effects against the tested clinical isolates. IMPORTANCE: Our study confirmed the efficacy of the combination CZA+MEM against KPC-producing and non-carbapenemase-producing strains. For metalloenzyme-producing strains, CZA+ATM demonstrated the most significant synergy. Additionally, CZA exhibited a notable synergy effect when combined with FOS. These combination therapies present promising new options for the treatment of CRKP infection.

Reverse N-Substituted Hydroxamic Acid Derivatives of Fosmidomycin Target a Previously Unknown Subpocket of 1-Deoxy-d-xylulose 5-Phosphate Reductoisomerase (DXR).

Reverse analogs of the phosphonohydroxamic acid antibiotic fosmidomycin are potent inhibitors of the nonmevalonate isoprenoid biosynthesis enzyme 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR, IspC) of Plasmodium falciparum. Some novel analogs with large phenylalkyl substituents at the hydroxamic acid nitrogen exhibit nanomolar PfDXR inhibition and potent in vitro growth inhibition of P. falciparum parasites coupled with good parasite selectivity. X-ray crystallographic studies demonstrated that the N-phenylpropyl substituent of the newly developed lead compound 13e is accommodated in a subpocket within the DXR catalytic domain but does not reach the NADPH binding pocket of the N-terminal domain. As shown for reverse carba and thia analogs, PfDXR selectively binds the S-enantiomer of the new lead compound. In addition, some representatives of the novel inhibitor subclass are nanomolar Escherichia coli DXR inhibitors, whereas the inhibition of Mycobacterium tuberculosis DXR is considerably weaker.

Small Molecule IITR08367 Potentiates Antibacterial Efficacy of Fosfomycin against Acinetobacter baumannii by Efflux Pump Inhibition.

Fosfomycin is a broad-spectrum single-dose therapy approved for treating lower urinary tract infections. Acinetobacter baumannii, one of the five major UTI-causing pathogens, is intrinsically resistant to fosfomycin. Reduced uptake and active efflux are major reasons for this intrinsic resistance. AbaF, a major facilitator superfamily class of transporter in A. baumannii, is responsible for fosfomycin efflux and biofilm formation. This study describes the identification and validation of a novel small-molecule efflux pump inhibitor that potentiates fosfomycin efficacy against A. baumannii. An AbaF inhibitor screening was performed against Escherichia coli KAM32/pUC18_abaF, using the noninhibitory concentration of 24 putative efflux pump inhibitors. The inhibitory activity of IITR08367 [bis(4-methylbenzyl) disufide] against fosfomycin/H+ antiport was validated using ethidium bromide efflux, quinacrine-based proton-sensitive fluorescence, and membrane depolarization assays. IITR08367 inhibits fosfomycin/H+ antiport activity by perturbing the transmembrane proton gradient. IITR08367 is a nontoxic molecule that potentiates fosfomycin activity against clinical strains of A. baumannii and prevents biofilm formation by inhibiting efflux pump (AbaF). The IITR08367-fosfomycin combination reduced bacterial burden by > 3 log10 in kidney and bladder tissue in the murine UTI model. Overall, fosfomycin, in combination with IITR08367, holds the potential to treat urinary tract infections caused by A. baumannii.

Change in Tissue Microbiome and Related Human Beta Defensin Levels Induced by Antibiotic Use in Bladder Carcinoma.

It is now generally accepted that the success of antitumor therapy can be impaired by concurrent antibiotic therapy, the presence of certain bacteria, and elevated defensin levels around the tumor tissue. The aim of our current investigation was to identify the underlying changes in microbiome and defensin levels in the tumor tissue induced by different antibiotics, as well as the duration of this modification. The microbiome of the tumor tissues was significantly different from that of healthy volunteers. Comparing only the tumor samples, no significant difference was confirmed between the untreated group and the group treated with antibiotics more than 3 months earlier. However, antibiotic treatment within 3 months of analysis resulted in a significantly modified microbiome composition. Irrespective of whether Fosfomycin, Fluoroquinolone or Beta-lactam treatment was used, the abundance of Bacteroides decreased, and Staphylococcus abundance increased. Large amounts of the genus Acinetobacter were observed in the Fluoroquinolone-treated group. Regardless of the antibiotic treatment, hBD1 expression of the tumor cells consistently doubled. The increase in hBD2 and hBD3 expression was the highest in the Beta-lactam treated group. Apparently, antibiotic treatment within 3 months of sample analysis induced microbiome changes and defensin expression levels, depending on the identity of the applied antibiotic.

Population pharmacokinetic/pharmacodynamic target attainment analysis of IV fosfomycin for the treatment of MDR Gram-negative bacterial infections.

BACKGROUND: IV fosfomycin is used against MDR Gram-negative bacilli (GNB) but has dose-limiting side effects, especially in patients with impaired kidney function. OBJECTIVES: To determine the optimal dosage of IV fosfomycin for patients with varying degrees of kidney function. METHODS: Adult patients receiving IV fosfomycin for treatment of GNB were eligible. Five serial blood samples were collected after at least three doses of fosfomycin; plasma was assayed by LC-MS/MS and modelled by population pharmacokinetic analysis. The PTA for AUC24/MIC of 98.9 for Escherichia coli and Klebsiella pneumoniae, and 40.8 for Pseudomonas aeruginosa were computed by Monte Carlo simulations. Cumulative fractions of response (CFR) were analysed for each pathogen using EUCAST MIC distributions. RESULTS: A total of 24 patients were included. Creatinine clearance (CLCR) and gender significantly influenced fosfomycin clearance. The kidney function-adjusted dosing regimens are proposed by using the lowest dose that can achieve ≥90% PTA for AUC24/MIC of 98.9 at an MIC of ≤32 mg/L (EUCAST v.13 susceptibility breakpoint for Enterobacterales). For patients with normal kidney function (CLCR 91-120 mL/min), a dosage of 15 g/day is suggested. This regimen achieved 97.1% CFR against E. coli, whereas CFR was 72.9% for K. pneumoniae and 76.7% for P. aeruginosa. CONCLUSIONS: A fosfomycin dosage of 15 g/day with adjustment according to kidney function provided high PTA and CFR when treating E. coli. This dosage is lower than that used in current practice and may improve tolerability. Higher dosages may be needed for P. aeruginosa; however, safety data are limited.

Treatment of infections caused by multidrug-resistant Gram-negative bacilli: A practical approach by the Italian (SIMIT) and French (SPILF) Societies of Infectious Diseases.

INTRODUCTION: The emergence of multidrug-resistant Gram-negative bacilli and the development of new antibiotics have complicated the selection of optimal regimens. International guidelines are valuable tools, but are limited by the scarcity of high-quality randomized trials in many situations. METHODS: A panel of experts from the French and Italian Societies of Infectious Diseases aimed to address unresolved issues in clinical practice based on their experience, an updated literature review and open discussions. RESULTS: The panel reached consensus for the following 'first choices': (i) cefepime for ventilator-acquired pneumonia due to AmpC ß-lactamase-producing Enterobacterales; (ii) the ß-lactam/ß-lactamase inhibitor combination most active in vitro, or cefiderocol combined with fosfomycin, and aerosolized colistin or aminoglycosides, for severe pneumonia due to Pseudomonas aeruginosa resistant to ceftolozane-tazobactam; (iii) high-dose piperacillin-tazobactam (including loading dose and continuous infusion) for complicated urinary tract infections (cUTIs) caused by extended-spectrum ß-lactamase-producing Enterobacterales with piperacillin-tazobactam minimum inhibitory concentration (MIC) ≤8 mg/L; (iv) high-dose cefepime for cUTIs due to AmpC ß-lactamase-producing Enterobacterales other than Enterobacter spp. if cefepime MIC ≤2 mg/L; (v) ceftolozane-tazobactam or ceftazidime-avibactam plus metronidazole for intra-abdominal infections (IAIs) due to third-generation cephalosporin-resistant Enterobacterales; (vi) ceftazidime-avibactam plus aztreonam plus metronidazole for IAIs due to metallo-ß-lactamase-producing Enterobacterales; (vii) ampicillin-sulbactam plus colistin for bloodstream infections (BSIs) caused by carbapenem-resistant Acinetobacter baumannii; (viii) meropenem-vaborbactam for BSIs caused by Klebsiella pneumoniae carbapenemase-producing Enterobacterales; and (ix) ceftazidime-avibactam plus fosfomycin for neurological infections caused by carbapenem-resistant P. aeruginosa. CONCLUSIONS: These expert choices were based on the necessary balance between antimicrobial stewardship principles and the need to provide optimal treatment for individual patients in each situation.

Identification of the novel fosfomycin resistance gene fosSC in Staphylococcus capitis.

OBJECTIVES: Fosfomycin has regained attention for treating infections caused by methicillin-resistant Staphylococcus aureus and multidrug-resistant coagulase-negative staphylococci. In this research, our objective was to investigate the mechanisms underlying fosfomycin resistance in Staphylococcus capitis. METHODS: The minimum inhibitory concentrations (MICs) of fosfomycin were assessed in 109 clinical S. capitis isolates by the agar dilution method. By cloning the fos-like genes into the shuttle vector, pTSSCm-Pcap, and observing the change in fosfomycin MICs, the gene function was verified. Core genome multilocus sequence typing and comparative genomics analysis were conducted to determine the population characteristics of S. capitis isolates and analyse the genetic environment of the fos-like genes. RESULTS: We identified a novel fosfomycin resistance gene, fosSC, on the chromosome in 58 out of 109 (53.2%) S. capitis isolates. The deduced products of the fosSC genes shared 67.15-67.88% amino acid sequence identity with FosB. The RN-pT-fosSC transformants carrying fosSC showed a 512-fold increase in the fosfomycin MICs. The fosSC gene was embedded in a conserved genetic context, but IS431mec was located to the left of the fosSC gene in cluster L due to the insertion of staphylococcal cassette chromosome mec. CONCLUSIONS: The chromosomal fosSC genes in some lineages of S. capitis explained their high-level fosfomycin resistance. Ongoing surveillance is crucial for monitoring the potential threat of horizontal transfer, which could be facilitated by the presence of mobile genetic elements surrounding the fosSC gene.

[Investigation of the Antibacterial Synergistic Activity of Fosfomycin-Teicoplanin Combination in Methicillin-Resistant Staphylococcus aureus Strains Isolated from Various Clinical Sample]. / Çesitli Klinik Örneklerden Elde Edilen Metisiline Dirençli Staphylococcus aureus Izolatlarinda Fosfomisin-Teikoplanin Kombinasyonunun Antibakteriyel Sinerjistik Etkinliginin Arastirilmasi.

The aim of this study was to investigate the detection of teicoplanin and fosfomycin antibiotic susceptibility of methicillin-resistant Staphylococcus aureus (MRSA) strains by different methods and to evaluate the antibacterial synergistic effect of teicoplanin-fosfomycin combination by using checkerboard assay and time kill curve assay. Forty-five MRSA strains isolated from clinical samples in routine medical microbiology laboratory of Göztepe Prof. Dr. Süleyman Yalçin City Hospital were included in the study. In the first stage of the combination study, minimum inhibitory concentrations (MIC) were investigated by broth microdilution for teicoplanin and by both broth microdilution and agar dilution methods for fosfomycin. The combination of teicoplanin and fosfomycin was tested by the checkerboard method in 45 MRSA strains and combination effect was determined according to fractional inhibitory concentration index (ΣFIC) calculation. The synergistic effect and bactericidal activity of antibiotic combination were studied against a randomly selected strain from the strains used in the study by using time-kill method for 24 hours. As a result of teicoplanin and fosfomycin antibiotic susceptibility studies, all isolates were found to be susceptible to both antibiotics according to the susceptibility breakpoints determined by the European Committee on Antimicrobial Susceptibility Testing (EUCAST). A synergistic effect was found in 22 (49%), additive effect in 22 (49%) and indifferent effect in one (2%) of the 45 strains studied with the checkerboard method. The mean ΣFIC of 45 isolates was found to be 0.5. In the combination study of the antibiotics of the isolate that was studied with time-kill method, synergism was detected for 1/8 MIC concentrations at 12th hour and 24th hour and synergism at 1/4 MIC concentration at sixth hour, 12th hour and 24th hour. In the combination study of 1/4 MIC concentrations of antibiotics, bactericidal effect was detected at sixth hour and this effect was observed to disappear at 12th and 24th hours. High rate of synergistic antibacterial effect of teicoplanin-fosfomycin combination on MRSA isolates was demonstrated as a result of in vitro tests. Such studies conducted on antibiotic-resistant bacterial infections will provide clinicians different treatment options and will contribute to increasing survival. As a result of this study, provided that it is supported by future clinical studies, it can be stated that the teicoplanin-fosfomycin combination may be an effective treatment option in community and hospital-acquired infections caused by MRSA.

Hydroxamate-Containing Bisphosphonates as Fosmidomycin Analogues: Design, Synthesis, and Proherbicide Activity.

Fosmidomycin (FOS) is a natural product inhibiting the DXR enzyme in the MEP pathway and has stimulated interest for finding more suitable FOS analogues. Herein, two series of FOS analogue hydroxamate-containing bisphosphonates as proherbicides were designed, with bisphosphonate replacing the phosphonic unit in FOS while retaining the hydroxamate (BPF series) or replacing it with retro-hydroxamate (BPRF series). The BPF series were synthesized through a three-step reaction sequence including Michael addition of vinylidenebisphosphonate, N-acylation, and deprotection, and the BPRF series were synthesized with a retro-Claisen condensation incorporated into the reaction sequence. Evaluation on model plants demonstrated several compounds having considerable herbicidal activities, and in particular, compound 8m exhibited multifold activity enhancement as compared to the control FOS. The proherbicide properties were comparatively validated. Furthermore, DXR enzyme assay, dimethylallyl pyrophosphate rescue, and molecular docking verified 8m to be a promising proherbicide candidate targeting the DXR enzyme. In addition, 8m also displayed good antimalarial activities.

Bacterial Persistence in Urinary Tract Infection Among Postmenopausal Population.

IMPORTANCE: Urinary tract infections (UTIs) are common in older-aged women. Our study examined bacterial persistence with commonly prescribed antibiotics. Bacterial growth was demonstrated despite antibiotic treatment. OBJECTIVES: The aims of this study were to quantify the bacterial persister phenotype in urine collected from postmenopausal women with acute and recurrent UTI and to determine the capabilities of first-line antibiotics to effectively treat persister cells. STUDY DESIGN: This was an institutional review board-approved cross-sectional analysis within a large academic referral center. Uropathogens were cultured from postmenopausal women with acute or recurrent UTI and screened for persister cells using persistence assays. Demographic and clinical variables were collected and analyzed. The entire experimental process was repeated in triplicate. Data were analyzed for significance (P < 0.05) between the persister culture and antibiotic treatments using a 1-way analysis of variance with multiple comparisons in Prism 9.3.0. RESULTS: Forty participants were included: 62.5% White, 22.5% Black, 3% Asian, and 2% Hispanic with a mean age of 72.3 ± 11.62 years. The persister phenotype was demonstrated in all of Escherichia coli isolates. Treatment with fosfomycin demonstrated reduced colony-forming units per milliliter compared with control (P < 0.01). Among recurrent isolates, there was a statistically significant decrease in colony-forming units per milliliter after antibiotic treatment with all 4 antibiotics (P < 0.05). CONCLUSIONS: This study demonstrated in vitro bacterial persistence in uropathogens from urogynecology patients despite treatment with commonly prescribed antibiotics. Fosfomycin generated the least amount of persister cells. Results suggest that persistence may be one bacterial defense mechanism involved in UTIs. Further research is needed to understand the clinical implications.

In vitro and in silico studies of enterobactin-inspired Ciprofloxacin and Fosfomycin first generation conjugates on the antibiotic resistant E. coli OQ866153.

BACKGROUND: The emergence of antimicrobial resistance in bacterial pathogens is a growing concern worldwide due to its impact on the treatment of bacterial infections. The "Trojan Horse" strategy has been proposed as a potential solution to overcome drug resistance caused by permeability issues. OBJECTIVE: The objective of our research was to investigate the bactericidal activity and mechanism of action of the "Trojan Horse" strategy using enterobactin conjugated with Ciprofloxacin and Fosfomycin against the antibiotic-resistant Escherichia coli strain OQ866153. METHODOLOGY: Enterobactin, a mixed ligand of E. coli OQ866153, was conjugated with Ciprofloxacin and Fosfomycin individually to aid active absorption via specific enterobactin binding proteins (FepABCDG). The effectiveness of the conjugates was assessed by measuring their bactericidal activity against E. coli OQ866153, as well as their ability to inhibit DNA gyrase enzyme and biofilm formation. RESULTS: The Fe+3-enterobactin-Ciprofloxacin conjugate effectively inhibited the DNA gyrase enzyme (Docking score = -8.597 kcal/mol) and resulted in a lower concentration (25 µg/ml) required to eliminate supercoiled DNA plasmids compared to the parent drug (35 µg/ml; Docking score = -6.264 kcal/mol). The Fe+3-Enterobactin-Fosfomycin conjugate showed a higher inhibition percentage (100%) of biofilm formation compared to Fosfomycin (21.58%) at a concentration of 2 mg/ml, with docking scores of -5.481 and -3.756 kcal/mol against UDP-N acetylglucosamine 1-carboxyvinyltransferase MurA. CONCLUSION: The findings of this study suggest that the "Trojan Horse" strategy using enterobactin conjugated with Ciprofloxacin and Fosfomycin can effectively overcome permeability issues caused by efflux proteins and enhance the bactericidal activity of these drugs against antibiotic-resistant strains of E. coli.

International guidelines for the treatment of carbapenem-resistant Gram-negative Bacilli infections: A comparison and evaluation.

OBJECTIVES: This study aimed to appraise clinical practice guidelines (CPGs) for the treatment of carbapenem-resistant Gram-negative Bacilli (CRGNB) infections and to summarise the recommendations. METHODS: A systematic search of the literature published from January 2012 to March 2023 was undertaken to identify CPGs related to CRGNB infections treatment. The methodological and reporting quality of eligible CPGs were assessed using six domains of the Appraisal of Guidelines for Research and Evaluation (AGREE) II tool and seven domains of the Reporting Items for practice Guidelines in HealThcare (RIGHT) checklist. Basic information and recommendations of included CPGs were extracted and compared. RESULTS: A total of 21 CPGs from 7953 relevant articles were included. The mean overall AGREE II score was 62.7%, and was highest for "clarity of presentation" (90.2%) and lowest for "stakeholder involvement" (44.8%). The overall reporting quality of all of the CPGs was suboptimal, with the proportion of eligible items ranging from 45.7 to 85.7%. The treatment of CRGNB infections is related to the type of pathogen, the sensitivity of antimicrobial agents, and the site of infection. In general, the recommended options mainly included novel ß-lactam/ ß-lactamase inhibitors, cefiderocol, ampicillin-sulbactam (mainly for carbapenem-resistant Acinetobacter baumannii [CRAB]), and combination therapy, involving polymyxin B/colistin, tigecycline (except for carbapenem-resistant Pseudomonas aeruginosa), aminoglycosides, carbapenems, fosfomycin, and sulbactam (mainly for CRAB). CONCLUSIONS: The methodological and reporting quality of CPGs for the treatment of CRGNB infections are generally suboptimal and need further improvement. Both monotherapy with novel drugs and combination therapy play important roles in the treatment.

Bacteriological and molecular study of fosfomycin resistance in uropathogenic Escherichia coli.

The identification of genes associated with resistance has the potential to facilitate the development of novel diagnostic tests and treatment methods. The objective of this study was to examine the antibiotic resistance and Fosfomycin resistance genes in uropathogenic Escherichia coli (UPEC) in patients in Baghdad, Iraq. After analyzing 250 urine samples using various identification methods, including the examination of morphological characteristics, biochemical tests, and genetic detection, it was determined that E. coli was the most common bacteria present, accounting for 63.6% of the samples. Antibiotic susceptibility testing showed a significant prevalence of resistance to various antibiotics, with 99.3% of E. coli isolates exhibiting multiple drug resistance (MDR). Fosfomycin showed antibacterial properties against UPEC. The minimum inhibitory concentration (MIC) ranged from 512 to 1024 µg/mL, while the minimum bactericidal concentration (MBC) was 2048 µg/mL. In the time-kill assay, fosfomycin was effective against fosfomycin-resistant isolates within 8-12 h. The genetic determinants associated with fosfomycin resistance were examined through the utilization of polymerase chain reaction (PCR). The findings indicated that the genes murA, glpT, and cyaA were detected in all the isolates when genomic DNA was used as a template. However, all the tests yielded negative results when plasmid was used as a template. The genes fosA3 and fosA4 were detected in 8.6% and 5% of the isolates when genomic DNA was used as a template. When plasmid was used as a template, the genes fosA3 and fosA4 were found in 5.7% and 2.9% of the isolates, respectively. In conclusion, there is an increasing problem with antibiotic resistance in UPEC, with elevated rates of resistance to several antibiotics. The study also offers novel insights into the genetic foundation of fosfomycin resistance in UPEC.

Pharmacokinetics/pharmacodynamics cut-off determination for fosfomycin using Monte Carlo simulation in healthy horses.

Fosfomycin (FOM) is an approved veterinary medicinal product for large animals in Japan, but Clinical breakpoint (CBP) for antimicrobial susceptibility test (AST) is not defined for animals. This study aimed at conducting a pharmacokinetics/pharmacodynamics (PK/PD) analysis to determine the PK/PD cutoff for the CBP in horses. Drug concentrations following single intravenous administration (IV) of 20 mg/kg body weight (BW) FOM in nine horses were measured using liquid chromatography/mass spectrometry. The data were modelled using a nonlinear mixed-effects model, followed by Monte Carlo simulations. A 90% probability of target attainment for a PK/PD target of the ratio of Area Under the free plasma concentration-time curve divided by the minimal inhibitory concentration (MIC) >24 hr was set as PK/PD cut-off. The PK/PD cutoff for FOM 20 mg/kg BW q12 hr IV was estimated with the MIC value of ≤16.0 mg/L, and this regimen was considered effective against E. coli (MIC90; 16.0 mg/L) in healthy horses based on the MIC90 values of the wild population. Owing to the relevance of FOM to human health, veterinarians should use q 12 hr FOM 20 mg /kg against E. coli infections with an MIC <16 µg/mL, as suggested by our PK/PD cutoff after AST.

In vitro susceptibility to fosfomycin in clinical and environmental extended-spectrum beta-lactamase producing and/or ciprofloxacin-non-susceptible Escherichia coli isolates.

Extended-spectrum beta-lactamase producing and ciprofloxacin-non-susceptible Escherichia coli are clinical and environmental issues. We evaluated the susceptibility profile of fosfomycin in non-susceptible E. coli isolated from urine and the environment. We measured the activity of fosfomycin against 319 and 36 E. coli strains from urine and environmental isolates, respectively, collected from rivers. Fosfomycin resistance profiles were investigated using the minimal inhibitory concentration (MIC), according to the Clinical and Laboratory Standards Institute (CLSI) and the European Committee for Antimicrobial Susceptibility Testing (EUCAST) guidelines. Antibiotic susceptibility testing revealed that 5% and 6.6% of urine samples were non-susceptible to fosfomycin according to CLSI and EUCAST guidelines, respectively. The fosfomycin MIC50/90 was 0.5/4 mg/L. Of the 36 E. coli isolates from river water, 11.1% and 13,8% were non-susceptible to fosfomycin according to CLSI and EUCAST, respectively (range ≤0.25 ≥512 mg/L). All the isolates with MIC ≥512 mg/L for fosfomycin showed the fosA3 gene. Fosfomycin resistance was more frequent in the environment than in clinical samples.

Raising the alarm: fosfomycin resistance associated with non-susceptible inner colonies imparts no fitness cost to the primary bacterial uropathogen.

IMPORTANCE: While fosfomycin resistance is rare, the observation of non-susceptible subpopulations among clinical Escherichia coli isolates is a common phenomenon during antimicrobial susceptibility testing (AST) in American and European clinical labs. Previous evidence suggests that mutations eliciting this phenotype are of high biological cost to the pathogen during infection, leading to current recommendations of neglecting non-susceptible colonies during AST. Here, we report that the most common route to fosfomycin resistance, as well as novel routes described in this work, does not impair virulence in uropathogenic E. coli, the major cause of urinary tract infections, suggesting a re-evaluation of current susceptibility guidelines is warranted.