Nocardia species distribution and antimicrobial susceptibility within Australia.

BACKGROUND: Nocardia is a ubiquitous saprophyte capable of causing human disease. Disease is primarily respiratory or cutaneous, usually acquired via inhalation or inoculation. Under the influence of environmental and host factors, Nocardia incidence and species distribution demonstrate geographical variation. AIMS: To examine for differences in Nocardia incidence within Western Australia (WA) and analyse species distribution in the context of prior published studies. To analyse antibiogram data from a nationwide passive antimicrobial resistance surveillance program. METHODS: Retrospective extraction of laboratory data for Western Australian Nocardia isolates over a 21-year period. Analysis of Nocardia antimicrobial susceptibility testing data submitted to the Australian Passive Antimicrobial Resistance Surveillance (APAS) program between 2005 and 2022. RESULTS: Nine hundred sixty WA isolates were identified, giving an annual incidence of 3.03 per 100 000 population with apparent latitudinal variation. The four most common species identified within WA and amongst APAS isolates were N. nova, N. cyriacigeorgica, N. brasiliensis and N. farcinica. APAS data demonstrated that all species exhibited high rates of susceptibility to linezolid (100%) and trimethoprim-sulfamethoxazole (98%). Amikacin (>90% susceptibility for all species except N. transvalensis) was the next most active parenteral agent, superior to both carbapenems and third-generation cephalosporins. Susceptibility to oral antimicrobials (other than linezolid) demonstrated significant interspecies variation. CONCLUSIONS: We demonstrate geographical variation in the distribution of Nocardia incidence. Four species predominate in the Australian setting, and nationwide data confirm a high in vitro susceptibility to trimethoprim-sulphamethoxazole and linezolid, justifying their ongoing role as part of first-line empiric therapy.

Antimicrobial susceptibility of ceftolozane-tazobactam against multidrug-resistant Pseudomonas aeruginosa isolates from Melbourne, Australia.

We collected 163 clinical Pseudomonas aeruginosa isolates at a tertiary hospital specialising in adult cystic fibrosis (CF) and lung transplantation (LTx) in Melbourne, Australia, to explore the activity of ceftolozane-tazobactam (C/T) in populations at high-risk for antimicrobial resistance. Of these, 144 (88.3%) were collected from sputum, and 19 (11.7%) from bronchoalveolar lavage. Most (85.3%) were derived from patients with cystic fibrosis and included a subset of patients that had undergone LTx. These isolates were tested against 11 antibiotics, including C/T, using Sensititre plates for broth microdilution (BMD) testing. Sixty (36.8%) isolates were classified as multidrug resistant (MDR) and 32 (19.6%) were extensively drug resistant (XDR). Overall, 133/163 (81.6%) isolates were susceptible to C/T. For MDR and XDR isolates, 88.3% and 28.1% were C/T susceptible, respectively. Among the non-MDR/XDR isolates, 100% remained susceptible to C/T. Comparisons of C/T susceptibility were made using BioMérieux Etests and Liofilchem MIC test strips (MTS). Categorical agreement to BMD was >93% for both test strips, but essential agreement to BMD was slightly higher with Etest (89.0%) compared to Liofilchem (74.8%). In conclusion, C/T retained activity against most MDR and over a quarter of XDR P. aeruginosa isolates from complex patients with CF and post-LTx.

Therapeutics for Vancomycin-Resistant Enterococcal Bloodstream Infections.

Vancomycin-resistant enterococci (VRE) are common causes of bloodstream infections (BSIs) with high morbidity and mortality rates. They are pathogens of global concern with a limited treatment pipeline. Significant challenges exist in the management of VRE BSI, including drug dosing, the emergence of resistance, and the optimal treatment for persistent bacteremia and infective endocarditis. Therapeutic drug monitoring (TDM) for antimicrobial therapy is evolving for VRE-active agents; however, there are significant gaps in the literature for predicting antimicrobial efficacy for VRE BSIs. To date, TDM has the greatest evidence for predicting drug toxicity for the three main VRE-active antimicrobial agents daptomycin, linezolid, and teicoplanin. This article presents an overview of the treatment options for VRE BSIs, the role of antimicrobial dose optimization through TDM in supporting clinical infection management, and challenges and perspectives for the future.

The impact of daptomycin therapeutic drug monitoring on clinical outcomes: a systematic review.

AIM: Daptomycin therapeutic drug monitoring (TDM) is a potentially valuable intervention for a relatively new drug. The aim of this study was to determine whether daptomycin TDM, including dose adjustment where necessary, improves the clinical outcomes of adult patients with Gram-positive infections. METHODS: A systematic review of English-language studies in MEDLINE (Ovid MEDLINE and Epub Ahead of Print, In-process, In-Data-Review & Other Non-Indexed Citations, Daily and Versions), EMBASE via OVID, Cochrane Central Register of Controlled Trials via the OVID platform, Scopus and Web of Science online databases was performed and conducted in accordance with Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. There was no discrimination on study type or time of publication. STUDY SELECTION: Adults (age ≥18 years) with a Gram-positive infection requiring treatment with daptomycin who received TDM, with subsequent reporting of serum concentrations and dose adjustment where necessary, were included. RESULTS: In total, 2869 studies were identified, of which nine met the inclusion criteria. No studies of daptomycin TDM including a relevant control arm have been published to date. All of the included studies were single-arm observational cohort studies. Broad heterogeneity was observed between the studies in terms of included pathogens, infection types, daptomycin TDM practices, reported clinical outcomes, and reporting of potential confounders. CONCLUSIONS: No studies exploring the efficacy of routine daptomycin TDM on patient-centred outcomes in comparison with fixed dosing regimens have been published to date. This represents a key knowledge gap as opposed to an inherent lack of efficacy. Further well-designed, comparative studies are required to determine the role of daptomycin TDM in patients with Gram-positive infections.

Antibiotic management of urinary tract infections in the post-antibiotic era: a narrative review highlighting diagnostic and antimicrobial stewardship.

BACKGROUND: As one of the most common indications for antimicrobial prescription in the community, the management of urinary tract infections (UTIs) is both complicated by, and a driver of, antimicrobial resistance. OBJECTIVES: To highlight the key clinical decisions involved in the diagnosis and treatment of UTIs in adult women, focusing on clinical effectiveness and both diagnostic and antimicrobial stewardship as we approach the post-antimicrobial era. SOURCES: Literature reviewed via directed PubMed searches and manual searching of the reference list for included studies to identify key references to respond to the objectives. A strict time limit was not applied. We prioritised recent publications, randomised trials, and systematic reviews (with or without meta-analyses) where available. Searches were limited to English language articles. A formal quality assessment was not performed; however, the strengths and limitations of each paper were reviewed by the authors throughout the preparation of this manuscript. CONTENT: We discuss the management of UTIs in ambulatory adult women, with particular focus on uncomplicated infections. We address the diagnosis of UTIs, including the following: definition and categorisation; bedside assessments and point-of-care tests; and the indications for, and use of, laboratory tests. We then discuss the treatment of UTIs, including the following: indications for treatment, antimicrobial sparing approaches, key considerations when selecting a specific antimicrobial agent, specific treatment scenarios, and duration of treatment. We finally outline emerging areas of interest in this field. IMPLICATIONS: The steady increase in antimicrobial resistance among common uropathogens has had a substantial affect on the management of UTIs. Regarding both diagnosis and treatment, the clinician must consider both the patient (clinical effectiveness and adverse effects, including collateral damage) and the community more broadly (population-level antimicrobial selection pressure).

Oral ciprofloxacin activity against ceftriaxone-resistant Escherichia coli in an in vitro bladder infection model.

OBJECTIVES: Pharmacodynamic profiling of oral ciprofloxacin dosing for urinary tract infections caused by ceftriaxone-resistant Escherichia coli isolates with ciprofloxacin MIC ≥ 0.25 mg/L. BACKGROUND: Urine-specific breakpoints for ciprofloxacin do not exist. However, high urinary concentrations may promote efficacy in isolates with low-level resistance. METHODS: Ceftriaxone-resistant E. coli urinary isolates were screened for ciprofloxacin susceptibility. Fifteen representative strains were selected and tested using a dynamic bladder infection model. Oral ciprofloxacin dosing was simulated over 3 days (250 mg daily, 500 mg daily, 250 mg 12 hourly, 500 mg 12 hourly and 750 mg 12 hourly). The model was run for 96 h. Primary endpoint was change in bacterial density at 72 h. Secondary endpoints were follow-up change in bacterial density at 96 h and area-under-bacterial-kill-curve. Bacterial response was related to exposure (AUC0-24/MIC; Cmax/MIC). PTA was determined using Monte-Carlo simulation. RESULTS: Ninety-three clinical isolates demonstrated a trimodal ciprofloxacin MIC distribution (modal MICs at 0.016, 0.25 and 32 mg/L). Fifteen selected clinical isolates (ciprofloxacin MIC 0.25-512 mg/L) had a broad range of quinolone-resistance genes. Following ciprofloxacin exposure, E. coli ATCC 25922 (MIC 0.008 mg/L) was killed in all dosing experiments. Six isolates (MIC ≥ 16 mg/L) regrew in all experiments. Remaining isolates (MIC 0.25-8 mg/L) regrew variably after an initial period of killing, depending on simulated ciprofloxacin dose. A >95% PTA, using AUC0-24/MIC targets, supported 250 mg 12 hourly for susceptible isolates (MIC ≤ 0.25 mg/L). For isolates with MIC ≤ 1 mg/L, 750 mg 12 hourly promoted 3 log10 kill at the end of treatment (72 h), 1 log10 kill at follow-up (96 h) and 90% maximal activity (AUBKC0-96). CONCLUSIONS: Bladder infection modelling supports oral ciprofloxacin activity against E. coli with low-level resistance (ciprofloxacin MIC ≤ 1 mg/L) when using high dose therapy (750 mg 12 hourly).

Genomic dissection of Klebsiella pneumoniae infections in hospital patients reveals insights into an opportunistic pathogen.

Klebsiella pneumoniae is a major cause of opportunistic healthcare-associated infections, which are increasingly complicated by the presence of extended-spectrum beta-lactamases (ESBLs) and carbapenem resistance. We conducted a year-long prospective surveillance study of K. pneumoniae clinical isolates in hospital patients. Whole-genome sequence (WGS) data reveals a diverse pathogen population, including other species within the K. pneumoniae species complex (18%). Several infections were caused by K. variicola/K. pneumoniae hybrids, one of which shows evidence of nosocomial transmission. A wide range of antimicrobial resistance (AMR) phenotypes are observed, and diverse genetic mechanisms identified (mainly plasmid-borne genes). ESBLs are correlated with presence of other acquired AMR genes (median n = 10). Bacterial genomic features associated with nosocomial onset are ESBLs (OR 2.34, p = 0.015) and rhamnose-positive capsules (OR 3.12, p < 0.001). Virulence plasmid-encoded features (aerobactin, hypermucoidy) are observed at low-prevalence (<3%), mostly in community-onset cases. WGS-confirmed nosocomial transmission is implicated in just 10% of cases, but strongly associated with ESBLs (OR 21, p < 1 × 10-11). We estimate 28% risk of onward nosocomial transmission for ESBL-positive strains vs 1.7% for ESBL-negative strains. These data indicate that K. pneumoniae infections in hospitalised patients are due largely to opportunistic infections with diverse strains, with an additional burden from nosocomially-transmitted AMR strains and community-acquired hypervirulent strains.

Oral fosfomycin activity against Klebsiella pneumoniae in a dynamic bladder infection in vitro model.

INTRODUCTION: The use of oral fosfomycin for urinary tract infections (UTIs) caused by non-Escherichia coli uropathogens is uncertain, including Klebsiella pneumoniae, the second most common uropathogen. METHODS: A multicompartment bladder infection in vitro model was used with standard media and synthetic human urine (SHU) to simulate urinary fosfomycin exposure after a single 3 g oral dose (fAUC0-72 16884 mg·h/L, t½ 5.5 h) against 15 K. pneumoniae isolates including ATCC 13883 (MIC 2 to >1024 mg/L) with a constant media inflow (20 mL/h) and 4-hourly voiding of each bladder. The impact of the media (CAMHB + G6P versus SHU) on fosfomycin MIC measurements, drug-free growth kinetics and regrowth after fosfomycin administration was assessed. A low and high starting inoculum (5.5 versus 7.5 log10 cfu/mL) was assessed in the bladder infection model. RESULTS: Compared with CAMHB, isolates in SHU had a slower growth rate doubling time (37.7 versus 24.1 min) and reduced growth capacity (9.0 ± 0.3 versus 9.4 ± 0.3 log10 cfu/mL), which was further restricted with increased inflow rate (40 mL/h) and more frequent voids (2-hourly). Regrowth was commonly observed in both media with emergence of fosfomycin resistance promoted by a high starting inoculum in CAMHB (MIC rise to ≥1024 mg/L in 13/14 isolates). Resistance was rarely detected in SHU, even with a high starting inoculum (MIC rise to ≥1024 mg/L in 2/14 isolates). CONCLUSIONS: Simulated in an in vitro UTI model, the regrowth of K. pneumoniae urinary isolates was inadequately suppressed following oral fosfomycin therapy. Efficacy was further reduced by a high starting inoculum.

Pharmacokinetic/pharmacodynamic analysis of oral fosfomycin against Enterobacterales, Pseudomonas aeruginosa and Enterococcus spp. in an in vitro bladder infection model: impact on clinical breakpoints.

OBJECTIVES: Fosfomycin is an established treatment for uncomplicated urinary tract infections (UTIs), yet evidence supporting susceptibility breakpoints is limited. We examine the UTI susceptibility criteria. METHODS: Fosfomycin susceptibility, heteroresistance and in vitro growth in a bladder infection model, after a single 3 g dose of oral fosfomycin, were bridged to human pharmacokinetics with pharmacokinetic/pharmacodynamic and Monte Carlo analyses. Data from common uropathogens (24 Escherichia coli, 20 Klebsiella pneumoniae, 4 Enterobacter cloacae, 14 Pseudomonas aeruginosa, 8 Enterococcus faecalis and 8 Enterococcus faecium) were compared and analysed to ascertain species-specific PTA. RESULTS: Glucose-6-phosphate (G6P) increased MICs of E. coli, K. pneumoniae and E. cloacae (median 2-fold dilutions 3-5), but not of P. aeruginosa and Enterococcus. Atypical E. coli lacking G6P potentiation were killed in the bladder infection model despite high MICs (32-128 mg/L). Fosfomycin heteroresistance was uncommon in E. coli (MIC > 2 mg/L) but was detected in the majority of K. pneumoniae (MIC > 1 mg/L) and P. aeruginosa (MIC >8 mg/L). For these species, baseline heteroresistance was a strong predictor for treatment failure in the model. No heteroresistance was found in Enterococcus. The fAUC/MIC targets for stasis were 1935, 3393, 9968, 2738 and 283 for typical E. coli, K. pneumoniae, E. cloacae, P. aeruginosa and E. faecalis, respectively (synthetic human urine medium alone promoted a 1 log10 kill in E. faecium). A >95% PTA for stasis was only found at MIC ≤ epidemiological cut-off (ECOFF) for E. coli (4 mg/L). For other species, PTAs were low for WT populations. CONCLUSIONS: With the exception of E. coli, fosfomycin is a poor target for other uropathogen species. A reduction in oral fosfomycin UTI breakpoints is supported.

Genomic surveillance of antimicrobial resistant bacterial colonisation and infection in intensive care patients.

BACKGROUND: Third-generation cephalosporin-resistant Gram-negatives (3GCR-GN) and vancomycin-resistant enterococci (VRE) are common causes of multi-drug resistant healthcare-associated infections, for which gut colonisation is considered a prerequisite. However, there remains a key knowledge gap about colonisation and infection dynamics in high-risk settings such as the intensive care unit (ICU), thus hampering infection prevention efforts. METHODS: We performed a three-month prospective genomic survey of infecting and gut-colonising 3GCR-GN and VRE among patients admitted to an Australian ICU. Bacteria were isolated from rectal swabs (n = 287 and n = 103 patients ≤2 and > 2 days from admission, respectively) and diagnostic clinical specimens between Dec 2013 and March 2014. Isolates were subjected to Illumina whole-genome sequencing (n = 127 3GCR-GN, n = 41 VRE). Multi-locus sequence types (STs) and antimicrobial resistance determinants were identified from de novo assemblies. Twenty-three isolates were selected for sequencing on the Oxford Nanopore MinION device to generate completed reference genomes (one for each ST isolated from ≥2 patients). Single nucleotide variants (SNVs) were identified by read mapping and variant calling against these references. RESULTS: Among 287 patients screened on admission, 17.4 and 8.4% were colonised by 3GCR-GN and VRE, respectively. Escherichia coli was the most common species (n = 36 episodes, 58.1%) and the most common cause of 3GCR-GN infection. Only two VRE infections were identified. The rate of infection among patients colonised with E. coli was low, but higher than those who were not colonised on admission (n = 2/33, 6% vs n = 4/254, 2%, respectively, p = 0.3). While few patients were colonised with 3GCR- Klebsiella pneumoniae or Pseudomonas aeruginosa on admission (n = 4), all such patients developed infections with the colonising strain. Genomic analyses revealed 10 putative nosocomial transmission clusters (≤20 SNVs for 3GCR-GN, ≤3 SNVs for VRE): four VRE, six 3GCR-GN, with epidemiologically linked clusters accounting for 21 and 6% of episodes, respectively (OR 4.3, p = 0.02). CONCLUSIONS: 3GCR-E. coli and VRE were the most common gut colonisers. E. coli was the most common cause of 3GCR-GN infection, but other 3GCR-GN species showed greater risk for infection in colonised patients. Larger studies are warranted to elucidate the relative risks of different colonisers and guide the use of screening in ICU infection control.

Antimicrobial pharmacokinetics and preclinical in vitro models to support optimized treatment approaches for uncomplicated lower urinary tract infections.

INTRODUCTION: Urinary tract infections (UTIs) are extremely common. Millions of people, particularly healthy women, are affected worldwide every year. One-in-two women will have a recurrence within 12-months of an initial UTI. Inadequate treatment risks worsening infection leading to acute pyelonephritis, bacteremia and sepsis. In an era of increasing antimicrobial resistance, it is critical to provide optimized antimicrobial treatment. AREAS COVERED: Literature was searched using PubMed and Google Scholar (up to 06/2020), examining the etiology, diagnosis and oral antimicrobial therapy for uncomplicated UTIs, with emphasis on urinary antimicrobial pharmacokinetics (PK) and the application of dynamic in vitro models for the pharmacodynamic (PD) profiling of pathogen response. EXPERT OPINION: The majority of antimicrobial agents included in international guidelines were developed decades ago without well-described dose-response relationships. Microbiology laboratories still apply standard diagnostic methodology that has essentially remained unchanged for decades. Furthermore, it is uncertain how relevant standard in vitro susceptibility is for predicting antimicrobial efficacy in urine. In order to optimize UTI treatments, clinicians must exploit the urine-specific PK of antimicrobial agents. Dynamic in vitro models are valuable tools to examine the PK/PD and urodynamic variables associated with UTIs, while informing uropathogen susceptibility reporting, optimized dosing schedules, clinical trials and treatment guidelines.

Treatment of invasive IMP-4 Enterobacter cloacae infection in transplant recipients using ceftazidime/avibactam with aztreonam: A case series and literature review.

Infections caused by carbapenemase-producing Enterobacteriaceae (CPE) are an emerging threat in both solid organ and stem cell transplant recipients. Invasive CPE infections in transplant recipients are associated with a high mortality, often due to limited therapeutic options and antibacterial toxicities. One of the most therapeutically challenging group of CPE are the metallo-ß-lactamase (MBL)-producing Gram-negative bacteria, which are now found worldwide, and often need treatment with older, highly toxic antimicrobial regimens. Newer ß-lactamase inhibitors such as avibactam have well-established activity against certain carbapenemases such as Klebsiella pneumoniae carbapenemases (KPC), but have no activity against MBL-producing organisms. Conversely, aztreonam has activity against MBL-producing organisms but is often inactivated by other co-existing ß-lactamases. Here, we report four cases of invasive MBL-CPE infections in transplant recipients caused by IMP-4-producing Enterobacter cloacae who were successfully treated with a new, mechanism-driven antimicrobial combination of ceftazidime/avibactam with aztreonam. This novel antimicrobial combination offers a useful treatment option for high-risk patients with CPE infection, with reduced drug interactions and toxicity.

Efficacy of single and multiple oral doses of fosfomycin against Pseudomonas aeruginosa urinary tract infections in a dynamic in vitro bladder infection model.

OBJECTIVES: We used a dynamic bladder infection in vitro model with synthetic human urine (SHU) to examine fosfomycin exposures to effectively kill, or prevent emergence of resistance, among Pseudomonas aeruginosa isolates. METHODS: Dynamic urinary fosfomycin concentrations after 3 g oral fosfomycin were simulated, comparing single and multiple (daily for 7 days) doses. Pharmacodynamic response of 16 P. aeruginosa (MIC range 1 to >1024 mg/L) were examined. Baseline disc diffusion susceptibility, broth microdilution MIC and detection of heteroresistance were assessed. Pathogen kill and emergence of resistance over 72 h following a single dose, and over 216 h following daily dosing for 7 days, were investigated. The fAUC0-24/MIC associated with stasis and 1, 2 and 3 log10 kill were determined. RESULTS: Pre-exposure high-level resistant (HLR) subpopulations were detected in 11/16 isolates after drug-free incubation in the bladder infection model. Five of 16 isolates had >2 log10 kill after single dose, reducing to 2/16 after seven doses. Post-exposure HLR amplification occurred in 8/16 isolates following a single dose and in 11/16 isolates after seven doses. Baseline MIC ≥8 mg/L with an HLR subpopulation predicted post-exposure emergence of resistance following the multiple doses. A PK/PD target of fAUC0-24/MIC >5000 was associated with 3 log10 kill at 72 h and 7 day-stasis. CONCLUSIONS: Simulated treatment of P. aeruginosa urinary tract infections with oral fosfomycin was ineffective, despite exposure to high urinary concentrations and repeated daily doses for 7 days. Emergence of resistance was observed in the majority of isolates and worsened following prolonged therapy. Detection of a baseline resistant subpopulation predicted treatment failure.

Oral Fosfomycin Treatment for Enterococcal Urinary Tract Infections in a Dynamic In Vitro Model.

There are limited treatment options for enterococcal urinary tract infections, especially vancomycin-resistant Enterococcus (VRE). Oral fosfomycin is a potential option, although limited data are available guiding dosing and susceptibility. We undertook pharmacodynamic profiling of fosfomycin against E. faecalis and E. faecium isolates using a dynamic in vitro bladder infection model. Eighty-four isolates underwent fosfomycin agar dilution susceptibility testing (E. faecalis MIC50/90 32/64 µg/ml; E. faecium MIC50/90 64/128 µg/ml). Sixteen isolates (including E. faecalis ATCC 29212 and E. faecium ATCC 35667) were chosen to reflect the MIC range and tested in the bladder infection model with synthetic human urine (SHU). Under drug-free conditions, E. faecium demonstrated greater growth restriction in SHU compared to E. faecalis (E. faecium maximal growth 5.8 ± 0.6 log10 CFU/ml; E. faecalis 8.0 ± 1.0 log10 CFU/ml). Isolates were exposed to high and low fosfomycin urinary concentrations after a single dose, and after two doses given over two days with low urinary concentration exposure. Simulated concentrations closely matched the target (bias 2.3%). E. faecalis isolates required greater fosfomycin exposure for 3 log10 kill from the starting inoculum compared with E. faecium The ƒAUC0-72/MIC and ƒ%T > MIC0-72 for E. faecalis were 672 and 70%, compared to 216 and 51% for E. faecium, respectively. There was no rise in fosfomycin MIC postexposure. Two doses of fosfomycin with low urinary concentrations resulted in equivalent growth inhibition to a single dose with high urinary concentrations. With this urinary exposure, fosfomycin was effective in promoting suppression of regrowth (>3 log10 kill) in the majority of isolates.

Evaluation of pooled human urine and synthetic alternatives in a dynamic bladder infection in vitro model simulating oral fosfomycin therapy.

The impact of the bladder environment on fosfomycin activity and treatment response is uncertain. Standard laboratory media does not reflect the biomatrix of urine, where limited nutritional factors are important for growth and antimicrobial kill rates. We compared fosfomycin activity against Enterobacteriaceae in laboratory media, human urine and synthetic alternatives. Sixteen clinical isolates (8-Escherichia coli, 4-Enterobacter cloacae, 4-Klebsiella pneumoniae) were studied with broth microdilution (BMD) susceptibility, static time-kill assays and dynamic testing in a bladder infection model simulating a 3 g oral fosfomycin dose. Mueller-Hinton broth (MHB) with and without 25 mg/L glucose-6-phosphate (G6P), pooled midstream urine (MSU), pooled 24 h urine collection (24 U), artificial urine medium (AUM) and synthetic human urine (SHU) were compared. BMD susceptibility, bacterial growth and response to static fosfomycin concentrations in urine were best matched with SHU and were distinctly different when tested in MHB with G6P. Fosfomycin exposure in the bladder infection model was accurately reproduced (bias 4.7 ± 6.2%). Under all media conditions, 8 isolates (2-E. coli, 2-E. cloacae, 4-K. pneumoniae) re-grew and 4 isolates (4-E. coli) were killed. The remaining isolates (2-E. coli, 2-E. cloacae) re-grew variably in urine and synthetic media. Agar dilution MIC failed to predict re-growth, whereas BMD MIC in media without G6P performed better. Emergence of resistance was restricted in synthetic media. Overall, SHU provided the best substitute for urine for in vitro modelling of antimicrobial treatment of uropathogens, and these data have broader utility for improved preclinical testing of antimicrobials for urinary tract infections.

Oral Fosfomycin Efficacy with Variable Urinary Exposures following Single and Multiple Doses against Enterobacterales: the Importance of Heteroresistance for Growth Outcome.

Oral fosfomycin trometamol is licensed as a single oral dose for the treatment of uncomplicated urinary tract infections, with activity against multidrug-resistant uropathogens. The impact of interindividual variability in urinary concentrations on antimicrobial efficacy, and any benefit of giving multiple doses, is uncertain. We therefore performed pharmacodynamic profiling of oral fosfomycin, using a dynamic bladder infection in vitro model, to assess high and low urinary exposures following a single oral dose and three repeat doses given every 72 h, 48 h, and 24 h against 16 clinical isolates with various MICs of fosfomycin (8 Escherichia coli, 4 Enterobacter cloacae, and 4 Klebsiella pneumoniae isolates). Baseline fosfomycin high-level-resistant (HLR) subpopulations were detected prior to drug exposure in half of the isolates (2 E. coli, 2 E. cloacae, and 4 K. pneumoniae isolates; proportion, 1 × 10-5 to 5 × 10-4% of the total population). Fosfomycin exposures were accurately reproduced compared to mathematical modeling (linear regression slope, 1.1; R2, 0.99), with a bias of 3.8% ± 5.7%. All 5/5 isolates with MICs of ≤1 µg/ml had no HLR and were killed, whereas 8/11 isolates with higher MICs regrew regardless of exposure to high or low urinary concentrations. A disk diffusion zone of <24 mm was a better predictor for baseline HLR and regrowth. Administering 3 doses with average exposures provided very limited additional kill. These results suggest that baseline heteroresistance is important for treatment response, while increased drug exposure and administering multiple doses may not be better than standard single-dose fosfomycin therapy.

Impact of bacterial species and baseline resistance on fosfomycin efficacy in urinary tract infections.

OBJECTIVES: To assess the antibacterial effects of a single 3 g oral fosfomycin dose on Escherichia coli and Klebsiella pneumoniae clinical isolates within a dynamic bladder infection model. METHODS: An in vitro model simulating dynamic urinary fosfomycin concentrations was used. Target fosfomycin exposure (Cmax = 1984 mg/L and Tmax = 7.5 h) was validated by LC-MS/MS. Pharmacodynamic responses of 24 E. coli and 20 K. pneumoniae clinical isolates were examined (fosfomycin MIC ≤0.25-128 mg/L). Mutant prevention concentration (MPC), fosfomycin heteroresistance, fosfomycin resistance genes and fosA expression were examined. Pathogen kill and emergence of high-level resistance (HLR; MIC >1024 mg/L) were quantified. RESULTS: Following fosfomycin exposure, 20 of 24 E. coli exhibited reductions in bacterial counts below the lower limit of quantification without regrowth, despite baseline fosfomycin MICs up to 128 mg/L. Four E. coli regrew (MIC = 4-32 mg/L) with HLR population replacement. At baseline, these isolates had detectable HLR subpopulations and MPC >1024 mg/L. All E. coli isolates were fosA negative. In contrast, 17 of 20 K. pneumoniae regrew post exposure, 6 with emergence of HLR (proportion = 0.01%-100%). The three isolates without regrowth did not have a detectable HLR subpopulation after dynamic drug-free incubation. All K. pneumoniae had MPC >1024 mg/L and were fosA positive. WGS analysis and fosA expression failed to predict fosfomycin efficacy. CONCLUSIONS: E. coli and K. pneumoniae isolates demonstrate discrepant responses to a single fosfomycin dose in a dynamic bladder infection in vitro model. Treatment failure against E. coli was related to an HLR subpopulation, not identified by standard MIC testing. Activity against K. pneumoniae appeared limited, regardless of MIC testing, due to universal baseline heteroresistance.

Urinary antibacterial activity of fosfomycin and nitrofurantoin at registered dosages in healthy volunteers.

Given emerging uropathogen resistance to more recent antibiotics, old antibiotics used for uncomplicated urinary tract infection (UTI) warrant re-examination. In this study, the urinary antibacterial activities of fosfomycin and nitrofurantoin were investigated by determining the urinary inhibitory titre and urinary bactericidal titre against uropathogens in urine samples from female volunteers following administration of single-dose fosfomycin (3 g) or nitrofurantoin (50 mg q6h or 100 mg q8h). Urine samples were collected over 48 h (fosfomycin) or 6 or 8 h (nitrofurantoin), with drug levels quantified with every void. Fosfomycin concentrations ranged from <0.75 mg/L [lower limit of quantification (LLOQ)] to 5729.9 mg/L and nitrofurantoin concentrations ranged from <4 mg/L (LLOQ) to 176.3 mg/L (50 mg q6h) or 209.4 mg/L (100 mg q8h). There was discrepancy in the response to fosfomycin between Escherichia coli and Klebsiella pneumoniae, with fosfomycin displaying strong bactericidal activity for 48 h against E. coli but moderate bactericidal activity for 18 h against K. pneumoniae. This effect was not related to the strain's baseline minimum inhibitory concentration but rather to the presence of a resistant subpopulation. Maximum titres of nitrofurantoin were obtained during the first 2 h, but no antibacterial effect was found in most samples regardless of the dose. In the rare samples in which antibacterial activity was detectable, titres were comparable for both species tested. These findings confirm doubts regarding fosfomycin administration in UTIs caused by K. pneumoniae and reveal a discrepancy between nitrofurantoin's measurable ex vivo activity and its clinical effect over multiple dosing intervals.