Pathogen invasion-dependent tissue reservoirs and plasmid-encoded antibiotic degradation boost plasmid spread in the gut

Many plasmids encode antibiotic resistance genes. Through conjugation, plasmids can be rapidly disseminated. Previous work identified gut luminal donor/recipient blooms and tissue-lodged plasmid-bearing persister cells of the enteric pathogen Salmonella enterica serovar Typhimurium (S.Tm) that survive antibiotic therapy in host tissues, as factors promoting plasmid dissemination among Enterobacteriaceae. However, the buildup of tissue reservoirs and their contribution to plasmid spread await experimental demonstration. Here, we asked if re-seeding-plasmid acquisition-invasion cycles by S.Tm could serve to diversify tissue-lodged plasmid reservoirs, and thereby promote plasmid spread. Starting with intraperitoneal mouse infections, we demonstrate that S.Tm cells re-seeding the gut lumen initiate clonal expansion. Extended spectrum beta-lactamase (ESBL) plasmid-encoded gut luminal antibiotic degradation by donors can foster recipient survival under beta-lactam antibiotic treatment, enhancing transconjugant formation upon re-seeding. S.Tm transconjugants can subsequently re-enter host tissues introducing the new plasmid into the tissue-lodged reservoir. Population dynamics analyses pinpoint recipient migration into the gut lumen as rate-limiting for plasmid transfer dynamics in our model. Priority effects may be a limiting factor for reservoir formation in host tissues. Overall, our proof-of-principle data indicates that luminal antibiotic degradation and shuttling between the gut lumen and tissue-resident reservoirs can promote the accumulation and spread of plasmids within a host over time.

The utilization of Blaptica dubia cockroaches as an in vivo model to test antibiotic efficacy

Insects are now well recognized as biologically relevant alternative hosts for dozens of mammalian pathogens and they are routinely used in microbial pathogenesis studies. Unfortunately, these models have yet to be incorporated into the drug development pipeline. The purpose of this work was to begin to evaluate the utility of orange spotted (Blaptica dubia) cockroaches in early antibiotic characterization. To determine whether these model hosts could exhibit mortality when infected with bacteria that are pathogenic to humans, we subjected B. dubia roaches to a range of infectious doses of Klebsiella pneumoniae, Escherichia coli, Staphylococcus aureus, and Acinetobacter baumannii to identify the medial lethal dose. These results showed that lethal disease did not develop following infection of high doses of S. aureus, and A. baumannii. However, cockroaches infected with E. coli and K. pneumoniae succumbed to infection (LD50s of 5.82 × 106 and 2.58 × 106 respectively) suggesting that this model may have limitations based on pathogen specificity. However, because these cockroaches were susceptible to infection from E. coli and K. pneumoniae, we used these bacterial strains for subsequent antibiotic characterization studies. These studies suggested that β-lactam antibiotic persistence and dose was associated with reduction of hemolymph bacterial burden. Moreover, our data indicated that the reduction of bacterial CFU was directly due to the drug activity. Altogether, this work suggests that the orange-spotted cockroach infection model provides an alternative in vivo setting from which antibiotic efficacy can be evaluated.

Cephalexin removal by persulfate activation using simulated sunlight and ferrous ions

This study presents the main results related to the use of activated persulfate (PS) in the elimination of the beta-lactam antibiotic cephalexin (CPX). Experiments were done using K2S2O8 and simulated sunlight. A face-centered central composite experimental design was used to analyze the effects of the solution pH and the PS concentration on the reaction, and to determine the optimized conditions that favor the CPX elimination. The results indicated that the removal of CPX is promoted by an acidic pH and under the higher evaluated PS dose (7.5 mg L−1). CPX total removal was achieved in 30 min. The analysis of the effect of the pollutant initial concentration indicated that a pseudo-first order kinetic model can be used to describe the reaction. Likewise, the use of Fe2+ ions for PS activation (in the dark) was evaluated and allowed to establish that a higher concentration of ions favors the pollutant removal. Control tests and under the presence of scavenger agents indicated that both HO• and SO4−• radicals would be present in the solution and promote the CPX elimination. The assessment of the solution dissolved organic carbon, nitrates and sulfates was also carried out, and indicated that a portion of the organic matter was mineralized.

656. Sulbactam-Durlobactam MIC Determination: Comparative Evaluation of the New ETEST® SUD to the CLSI 2021 Broth Microdilution Method

Background Species belonging to the Acinetobacter baumannii-calcoaceticus (ABC) complex, such as A. baumannii, A. pittii and A. nosocomialis, are a major cause of hospital acquired infections and outbreaks with increasing occurrence of multidrug-resistance. Sulbactam-durlobactam (SUD), a combination of one active β-lactam antibiotic (sulbactam) with a new β-lactamase inhibitor (durlobactam), is currently being tested in a phase 3 clinical trial by Entasis Therapeutics for the treatment of serious infections caused by ABC, including multidrug-resistant strains. At the same time, an ETEST® SUD (sulbactam-durlobactam - MIC range 0.004/4-64/4 µg/mL) has been developed and calibrated versus the broth microdilution reference method (BMD) as described by the Clinical and Laboratory Standards Institute (CLSI). This test is intended to determine the MIC of sulbactam-durlobactam for species of the ABC complex. The aim of this study was to perform a first comparative study of ETEST SUD with the CLSI BMD method on a panel of 263 isolates. Methods The panel consisted of 204 A. baumannii, 29 A. pittii, 30 A. nosocomialis, including 24 SUD-resistant strains, and one CLSI QC strain. BMD was performed using the 2021 CLSI guidelines. ETEST SUD was evaluated using the standard ETEST procedure for Acinetobacter spp. (inoculum 0.5 McFarland, Mueller Hinton medium, incubation at 35°C for 20-24h). For each method, the MIC was read at complete inhibition of visible growth. To determine category agreement (CA) and error rates, the sulbactam-durlobactam provisional breakpoint of 4 µg/mL was applied. Results The QC strain MICs were in the expected range with reproducible results. The essential MIC agreement [EA, ±1 dilution] was 97.7% without any tendency to over- or underestimate the MIC when compared to BMD. The CA was 98.5%. Two Very Major Errors, both within the EA, and two Major Errors, one within the EA, were observed. Conclusion In this study, the ETEST SUD was found to be equivalent to the CLSI reference method. MIC end points were easy to read. With a 15-dilution range and simplicity of use, ETEST SUD could represent a valuable tool for MIC determination and could be an alternative to BMD. For Research Use Only. The performance characteristics of this product have not been established yet. Disclosures All Authors: No reported disclosures

1043. Activity of Mecillinam Against Enterobacterales Isolates Collected From Patients With Urinary Tract Infections (UTIs) in the USA During 2019

Background Mecillinam is a β-lactam antibiotic that exerts its antibacterial activity by binding to penicillin-binding protein 2. In the USA, intravenous (IV) mecillinam is in development for the treatment of complicated UTIs in the hospital setting and as step-down therapy transitioning from IV mecillinam to oral pivmecillinam so that patients can continue treatment at home. To support the clinical development of mecillinam in the USA for the treatment of both complicated and uncomplicated UTI, this observational study investigated the activity of mecillinam against Enterobacterales isolates from patients with UTI in the USA, collected during 2019. Methods This study evaluated the activity of mecillinam and other antimicrobial agents against 1075 selected Enterobacterales clinical isolates collected from patients with UTI in the USA during 2019. Antibiotic activity (minimum inhibitory concentration [MIC]) was determined by Clinical & Laboratory Standards Institute (CLSI) agar dilution methodology, and susceptibility was interpreted according to CLSI guidelines. Results Among the selected 1075 isolates, producers of extended-spectrum beta-lactamase (ESBL) represented 9.6% of Escherichia coli and 50% of Klebsiella pneumoniae. Ninety-five percent of the isolates tested were susceptible to mecillinam (Table 1). The MIC50 and MIC90 values for mecillinam were 0.25 and 2 µg/mL, respectively. Fosfomycin MIC50 and MIC90 were 1 and 32 µg/mL, respectively (97.6% of isolates were susceptible). Mecillinam showed the lowest MIC90 value of all single antibiotics tested. The highest MIC90 was 128 µg/mL for both nitrofurantoin and cefotaxime. The lowest percentage of resistance was obtained with fosfomycin (1.7%), followed by mecillinam (4%). Table 1: Summary MIC and susceptibility data for all isolates tested (n=1075) Conclusion Overall, mecillinam showed excellent activity and a comparable resistance profile to fosfomycin. Resistance rates to ceftazidime, cefotaxime, ciprofloxacin and trimethoprim/sulfamethoxazole of greater than 20% are concerning due to the frequent use of these antibiotics in clinical practice to treat UTIs. Taken together, these data demonstrate that mecillinam has promising activity, with low resistance observed in Enterobacterales species causing UTIs in the USA. Clinical development of mecillinam in the USA is ongoing. Disclosures Stephen Hawser, PhD, Utility Therapeutics (Grant/Research Support) Ian Morrissey, Utility Therapeutics (Grant/Research Support) Anne Santerre Henriksen, MS, Advanz (Consultant)Shionogi BV (Consultant)UTILITY Therapeutics (Consultant)