(p)ppGpp modifies RNAP function to confer ß-lactam resistance in a peptidoglycan-independent manner.

(p)ppGpp is a nucleotide alarmone that controls bacterial response to nutrient deprivation. Since elevated (p)ppGpp levels confer mecillinam resistance and are essential for broad-spectrum ß-lactam resistance as mediated by the ß-lactam-insensitive transpeptidase YcbB (LdtD), we hypothesized that (p)ppGpp might affect cell wall peptidoglycan metabolism. Here we report that (p)ppGpp-dependent ß-lactam resistance does not rely on any modification of peptidoglycan metabolism, as established by analysis of Escherichia coli peptidoglycan structure using high-resolution mass spectrometry. Amino acid substitutions in the ß or ß' RNA polymerase (RNAP) subunits, alone or in combination with the CRISPR interference-mediated downregulation of three of seven ribosomal RNA operons, were sufficient for resistance, although ß-lactams have no known impact on the RNAP or ribosomes. This implies that modifications of RNAP and ribosome functions are critical to prevent downstream effects of the inactivation of peptidoglycan transpeptidases by ß-lactams.

CTX-M-127 with I176F mutations found in bacteria isolates from Bangladeshi circulating banknotes.

Extended-spectrum beta-lactamase (ESBL)-producing organisms are widely recognized as clinically relevant causes of difficult-to-treat infections. CTX-M has formed a rapidly growing family distributed worldwide among a wide range of clinical bacteria, particularly members of Enterobacteriaceae. Circulating banknotes, exchanged daily among people, pose a potential vehicle for transmitting multidrug resistance. We screened for ESBL-carrying bacteria in the present study and reported CTX-M mutations in Bangladesh's banknotes. We sequenced the genes and performed homology modeling using the Swiss model with CTX-M-15 (4HBT) as a template. Then, we performed molecular docking of mecillinam with the template and the generated model using Autodock 4.2 (Release 4.2.6). After docking, we visually inspected the complexes built using Autodock tools for polar contacts and pi-pi interactions in PyMOL 2.5.4. Our partially sequenced blaCTX-M was related to blaCTX-M-10 and blaCTX-M-15. We observed multiple single-nucleotide substitution mutations, i.e., G613T (silent mutation), A626T (I176F), and A503G (N135D). Homology modeling showed high similarity when the model was superimposed over the template. The orientation of Asn (135) in the template and Asp (135) in the model does not show a significant difference. Likewise, Ile (176) in the template and Phe (176) in the model offer the same orientation. Our generated model could bind to Lys237, Ser240, and Asp135 residues with the lowest binding energy on docking. Our predicted binding of the mecillinam to the mutated D-135 residue in the model indicates contributions and supports previous reports proposing CTX-M-15 to CTX-M-127 mutational conversion on the mecillinum resistance phenotype.

Phage-induced bacterial morphological changes reveal a phage-derived antimicrobial affecting cell wall integrity.

In a looming post-antibiotic era, antibiotic alternatives have become key players in the combat against pathogens. Although recent advances in genomic research allow scientists to fully explore an organism's genome in the search for novel antibacterial molecules, laborious work is still needed in order to dissect each individual gene product for its antibacterial activity. Here, we exploited phage-induced bacterial morphological changes as anchors to explore and discover a potential phage-derived antimicrobial embedded in the phage genome. We found that, upon vibriophage KVP40 infection, Vibrio parahaemolyticus exhibited morphological changes similar to those observed when treated with mecillinam, a cell wall synthesis inhibitor, suggesting the mechanism of pre-killing that KVP40 exerts inside the bacterial cell upon sieging the host. Genome analysis revealed that, of all the annotated gene products in the KVP40 genome that are involved in cell wall degradation, lytic transglycosylase (LT) is of particular interest for subsequent functional studies. A single-cell morphological analysis revealed that heterologous expression of wild-type KVP40-LT induced similar bacterial morphological changes to those treated with the whole phage or mecillinam, prior to cell burst. On the contrary, neither the morphology nor the viability of the bacteria expressing signal-peptide truncated- or catalytic mutant E80A- KVP40-LT was affected, suggesting the necessity of these domains for the antibacterial activities. Altogether, this research paves the way for the future development of the discovery of phage-derived antimicrobials that is guided through phage-induced morphological changes.

PBP Target Profiling by ß-Lactam and ß-Lactamase Inhibitors in Intact Pseudomonas aeruginosa: Effects of the Intrinsic and Acquired Resistance Determinants on the Periplasmic Drug Availability.

The lack of effective treatment options against Pseudomonas aeruginosa is one of the main contributors to the silent pandemic. Many antibiotics are ineffective against resistant isolates due to poor target site penetration, efflux, or ß-lactamase hydrolysis. Critical insights to design optimized antimicrobial therapies and support translational drug development are needed. In the present work, we analyzed the periplasmic drug uptake and binding to PBPs of 11 structurally different ß-lactams and 4 ß-lactamase inhibitors (BLIs) in P. aeruginosa PAO1. The contribution of the most prevalent ß-lactam resistance mechanisms to MIC and periplasmic target attainment was also assessed. Bacterial cultures (6.5 log10 CFU/mL) were exposed to 1/2× PAO1 MIC of each antibiotic for 30 min. Unbound PBPs were labeled with Bocillin FL and analyzed using a FluorImager. Imipenem extensively inactivated all targets. Cephalosporins preferentially targeted PBP1a and PBP3. Aztreonam and amdinocillin bound exclusively to PBP3 and to PBP2 and PBP4, respectively. Penicillins bound preferentially to PBP1a, PBP1b, and PBP3. BLIs displayed poor PBP occupancy. Inactivation of oprD elicited a notable reduction of imipenem target attainment, and it was to a lesser extent in the other carbapenems. Improved PBP occupancy was observed for the main targets of the widely used antipseudomonal penicillins, cephalosporins, meropenem, aztreonam, and amdinocillin upon oprM inactivation, in line with MIC changes. AmpC constitutive hyperexpression caused a substantial PBP occupancy reduction for the penicillins, cephalosporins, and aztreonam. Data obtained in this work will support the rational design of optimized ß-lactam-based combination therapies against resistant P. aeruginosa infections. IMPORTANCE The growing problem of antibiotic resistance in Gram-negative pathogens is linked to three key aspects, (i) the progressive worldwide epidemic spread of multidrug-resistant (MDR), extensively drug-resistant (XDR), and pandrug-resistant (PDR) Gram-negative strains, (ii) a decrease in the number of effective new antibiotics against multiresistant isolates, and (iii) the lack of mechanistically informed combinations and dosing strategies. Our combined efforts should focus not only on the development of new antimicrobial agents but the adequate administration of these in combination with other agents currently available in the clinic. Our work determined the effectiveness of these compounds in the clinically relevant bacteria Pseudomonas aeruginosa at the molecular level, assessing the net influx rate and their ability to access their targets and achieve bacterial killing without generating resistance. The data generated in this work will be helpful for translational drug development.

Pivmecillinam, the paradigm of an antibiotic with low resistance rates in Escherichia coli urine isolates despite high consumption.

OBJECTIVES: Pivmecillinam, the oral version of mecillinam, represents one of the major recommended and used antibiotics for empiric and targeted treatment of urinary tract infections in primary care in Denmark, Norway and Sweden. Mecillinam resistant mutants in Escherichia coli develop easily in vitro, but their fitness cost has been shown to be high. METHODS: We revisited the resistance and consumption data from the monitoring programmes in the three countries and compared pivmecillinam with ciprofloxacin from 2010 to 2020. RESULTS: Mecillinam resistance rates in Escherichia coli remained around 6% in Denmark and Norway relative to a constant consumption in Norway of 1.6-1.8 DID (defined daily doses per 1000 inhabitants per day), and even increasing in Denmark from 1.6 to 2.3 DID. In Sweden resistance was significantly lower at 4% related to the lower consumption of 0.5 DID. For ciprofloxacin, resistance rates fluctuated around 6%-12%, highest in Sweden with the highest consumption (0.8-0.6 DID) and lowest in Denmark (0.55-0.35 DID) and Norway (0.7-0.3 DID), although consumption declined significantly in all three countries. CONCLUSIONS: Pivmecillinam is an example of an antibiotic, which easily develops resistance in vitro, but apparently can be used broadly in primary care without increase in resistance rates.

Intracellular uropathogenic Escherichia coli are undetectable in urinary bladders after oral mecillinam treatment: An experimental study in a pig model of cystitis.

OBJECTIVES: Experiments in murine models of urinary tract infection (UTI) show that uropathogenic Escherichia coli (UPEC) form bacterial reservoirs in the bladder tissue that can survive beta lactam antibiotics and give rise to reinfection. The observed reinfection cascade suggests intracellular bacterial persistence as a possible explanation for recurrent UTI in humans. To test this hypothesis in an animal model closer to humans, we here investigated whether UPEC infecting the bladders of experimentally inoculated pigs are able to survive standard oral mecillinam treatment. Moreover, we analyzed the infected pig bladders by microscopy for the presence of intracellular UPEC colonies. METHODS: Seven pigs were experimentally inoculated with the UPEC cystitis strain, UTI89, to induce cystitis. After 5 days of infections, a 3-day oral treatment with the extracellularly active ß-lactam, mecillinam, was initiated. The infection was monitored with regular urine and blood samples. When terminated, whole bladders were removed and homogenized to quantify viable intracellular bacteria. In addition, two pigs were inoculated with UTI89pMAN01 constitutively expressing green fluorescent protein and the bladders subsequently analyzed by microscopy for bacterial location and morphology. RESULTS: Experimental inoculation resulted in cystitis in all animals. After 3-day treatment with mecillinam, no viable UPEC were detectable in urine or bladder homogenates. Microscopy analysis of pig bladders at 12 h post infection, revealed no detectable intracellular bacterial colonies and no filamentous UPEC phenotypes were identified. CONCLUSIONS: Pigs experimentally infected with UPEC completely clear their infection upon mecillinam treatment, which contrasts earlier findings from similar experiments in mice. Moreover, the hallmarks of induced UTI in mice, i.e. intracellular bacterial communities and bacterial filamentation, could not be identically reproduced in a pig model of acute UTI. This result suggests that significant differences might exist between UTI in mice and larger mammals, and therefore perhaps also between mice and humans. Additional studies are needed to reveal details on the Escherichia coli acute UTI pathogenesis cascade in larger mammals to assess to which extent observations in mice can be transferred to humans.

Activity of mecillinam against carbapenem-resistant Enterobacterales.

BACKGROUND: Despite the fact that carbapenem-resistant Enterobacterales (CRE) mostly cause urinary tract infections (UTIs), only few studies have focused on the efficacity of mecillinam against these CRE. OBJECTIVES: To evaluate the mecillinam susceptibility of a huge collection of CRE, including carbapenemase-producing Enterobacterales (CPE) and non-CPE (ESBL and AmpC producers with decreased permeability of the outer membrane). METHODS: A total of 8310 non-duplicate clinical CRE, including 4042 OXA-48-like producers, 1094 NDM producers, 411 VIM producers, 174 KPC producers, 42 IMI producers, 153 multiple-carbapenemase producers and 45 isolates producing other types of carbapenemases (such as IMP-like enzymes or GES-5), were included in the study. WGS was performed on all CPE using Illumina technology. Categorization of susceptibility to mecillinam was performed using disc diffusion (mecillinam discs at 10 µg; I2A, France) according to EUCAST recommendations. The results were interpreted according to EUCAST guidelines (S ≥15 mm). RESULTS: Significantly higher susceptibility rates were observed for carbapenem-resistant Proteus spp. (85%) and carbapenem-resistant Escherichia coli (84%), which are the two most common species responsible for UTIs, than for Klebsiella pneumoniae (67%), Enterobacter cloacae complex (75%), Citrobacter spp. (65%), Serratia spp. (34%) and Morganella morganii (12%). Susceptibility rates were 84%, 71% and 91% for OXA-48-like, NDM and IMI producers and 70% for non-CPE CRE. Mecillinam was less active against VIM and KPC producers (14% and 0%, respectively). CONCLUSIONS: Mecillinam might be an alternative for the treatment of infections due to CRE, particularly UTIs, except for VIM and KPC producers and for M. morganii and Serratia spp species.

LC-MS/MS methods for determination of unstable pivmecillinam and mecillinam in acidified human plasma: Application to a pharmacokinetic study.

Pivmecillinam, the ester of biologically active antibiotic mecillinam, is an effective oral preparation to treat urinary tract infections. To study pharmacokinetics in humans, LC-MS/MS methods were developed to quantify pivmecillinam and mecillinam in human plasma, respectively. Considering cephalexin as internal standard, analytes were separated on UltimateXB-C18 columns after protein precipitation by acetonitrile. The mobile phase was composed of water containing 0.1% formic acid and methanol. The multiple reactions monitoring transitions of m/z 440.2→167.1, 326.1→167.1, and 348.1→158.1 were selected to inspect pivmecillinam, mecillinam, and the internal standard in positive ion mode. No apparent matrix effect was perceived. Linearities were obtained over calibration ranges of 0.0500-12.0 and 10.0-15,000 ng/mL, respectively. The intraday precisions were below 5.5%, the interday precisions were below 6.1%, and accuracies were within -8.1 to 13.0%. Stability tests were conducted and an acidification step was explored to enhance the stability of pivmecillinam and mecillinam. Further stability was validated under various storage and processing conditions. Both methods were applied to a pharmacokinetic study of pivmecillinam and mecillinam after oral administration of 400 mg pivmecillinam hydrochloride tablets in healthy Chinese subjects.

SUsceptibility and Resistance to Fosfomycin and other antimicrobial agents among pathogens causing lower urinary tract infections: findings of the SURF study.

Urinary tract infections (UTIs) are prevalent worldwide, particularly among women. Their incidence increases with age, and treatment is increasingly challenging owing to antibiotic resistance and the lack of new agents. We investigated the susceptibility of current urinary isolates to fosfomycin and other antibiotics across Europe. This cross-sectional study collected consecutive urinary isolates from non-hospitalised women at 20 centres in Belgium, the UK, Italy, Spain and Russia. Bacteria were tested by disk diffusion with relevant antibiotics. As a quality control, a central laboratory re-tested, by agar dilution, (i) isolates found resistant to fosfomycin and (ii) every tenth isolate; all non-Russian sites were included. A total of 2848 isolates were analysed, principally Escherichia coli (2064; 72.5%), Klebsiella spp. (275; 9.7%) and Proteus spp. (103; 3.6%). For E. coli, agents active against >90% of isolates were nitrofurantoin (98.5%), fosfomycin (96.4%) and mecillinam (91.8%). Fosfomycin and nitrofurantoin remained active against >90% of cephalosporin-resistant E. coli. Among 143 E. coli recorded as susceptible locally by disk tests, 138 (96.5%) were confirmed susceptible by minimum inhibitory concentration (MIC) tests, however resistance was only confirmed in 29/58 (50.0%) of those reported resistant by local disk tests. Escherichia coli was found to be the most common uropathogen isolated and was highly susceptible to fosfomycin, nitrofurantoin and mecillinam, all used effectively for more than 30 years. Guidelines advocating fosfomycin for uncomplicated UTIs in women remain microbiologically valid.

Increased energy demand from anabolic-catabolic processes drives ß-lactam antibiotic lethality.

ß-Lactam antibiotics disrupt the assembly of peptidoglycan (PG) within the bacterial cell wall by inhibiting the enzymatic activity of penicillin-binding proteins (PBPs). It was recently shown that ß-lactam treatment initializes a futile cycle of PG synthesis and degradation, highlighting major gaps in our understanding of the lethal effects of PBP inhibition by ß-lactam antibiotics. Here, we assess the downstream metabolic consequences of treatment of Escherichia coli with the ß-lactam mecillinam and show that lethality from PBP2 inhibition is a specific consequence of toxic metabolic shifts induced by energy demand from multiple catabolic and anabolic processes, including accelerated protein synthesis downstream of PG futile cycling. Resource allocation into these processes is coincident with alterations in ATP synthesis and utilization, as well as a broadly dysregulated cellular redox environment. These results indicate that the disruption of normal anabolic-catabolic homeostasis by PBP inhibition is an essential factor for ß-lactam antibiotic lethality.

Clavulanate combinations with mecillinam, cefixime or cefpodoxime against ESBL-producing Enterobacterales frequently associated with blaOXA-1 in a paediatric population with febrile urinary tract infections.

OBJECTIVES: Oral treatment of febrile urinary tract infections (FUTIs) can be impaired by MDR Enterobacterales often combining ESBL and inhibitor-resistant genes. We studied the impact of ß-lactamases and Enterobacterales' genotypes on the cefixime, cefpodoxime and mecillinam ± amoxicillin/clavulanate MICs. MATERIALS AND METHODS: In this multicentric study, we included 251 previously whole-genome-sequenced ESBL-producing Enterobacterales, isolated in French children with FUTIs. The MICs of cefixime, cefpodoxime, mecillinam alone and combined with amoxicillin/clavulanate were determined and analysed with respect to genomic data. We focused especially on the isolates' ST and their type of ß-lactamases. Clinical outcomes of patients who received cefixime + amoxicillin/clavulanate were also analysed. RESULTS: All isolates were cefixime and cefpodoxime resistant. Disparities depending on blaCTX-M variants were observed for cefixime. The addition of amoxicillin/clavulanate restored susceptibility for cefixime and cefpodoxime in 97.2% (MIC50/90 of 0.38/0.75 mg/L) and 55.4% (MIC50/90 of 1/2 mg/L) of isolates, respectively, whatever the ST, the blaCTX-M variants or the association with inhibitor-resistant ß-lactamases (34.2%). All isolates were susceptible to mecillinam + amoxicillin/clavulanate with MIC50/90 of 0.19/0.25 mg/L, respectively. Neither therapeutic failure nor any subsequent positive control urine culture were reported for patients who received cefixime + amoxicillin/clavulanate as an oral relay therapy (n = 54). CONCLUSIONS: Despite the frequent association of ESBL genes with inhibitor-resistant ß-lactamases, the cefixime + amoxicillin/clavulanate MICs remain low. The in vivo efficacy of this combination was satisfying even when first-line treatment was ineffective. Considering the MIC distributions and pharmacokinetic parameters, mecillinam + amoxicillin/clavulanate should also be an alternative to consider when treating FUTIs in children.

Chemical-genetic interrogation of RNA polymerase mutants reveals structure-function relationships and physiological tradeoffs.

The multi-subunit bacterial RNA polymerase (RNAP) and its associated regulators carry out transcription and integrate myriad regulatory signals. Numerous studies have interrogated RNAP mechanism, and RNAP mutations drive Escherichia coli adaptation to many health- and industry-relevant environments, yet a paucity of systematic analyses hampers our understanding of the fitness trade-offs from altering RNAP function. Here, we conduct a chemical-genetic analysis of a library of RNAP mutants. We discover phenotypes for non-essential insertions, show that clustering mutant phenotypes increases their predictive power for drawing functional inferences, and demonstrate that some RNA polymerase mutants both decrease average cell length and prevent killing by cell-wall targeting antibiotics. Our findings demonstrate that RNAP chemical-genetic interactions provide a general platform for interrogating structure-function relationships in vivo and for identifying physiological trade-offs of mutations, including those relevant for disease and biotechnology. This strategy should have broad utility for illuminating the role of other important protein complexes.

Intravenous mecillinam compared with other ß-lactams as targeted treatment for Escherichia coli or Klebsiella spp. bacteraemia with urinary tract focus.

BACKGROUND: Mecillinam (amdinocillin) is active against Gram-negative bacteria. Clinical data on the efficacy of IV mecillinam for severe urinary tract infections is sparse. OBJECTIVES: To assess the effectiveness of targeted IV mecillinam compared with other ß-lactams for bacteraemia with Escherichia coli and Klebsiella spp. and a urinary tract focus. PATIENTS AND METHODS: We performed a retrospective cohort study at five university hospitals in the Capital Region of Denmark from 1 January 2012 to 31 December 2017. We used Cox proportional hazard regression to compare the primary composite endpoint (all-cause mortality or bacteraemia recurrence within 30 days) between patients treated with mecillinam versus ampicillin, cefuroxime, piperacillin/tazobactam and meropenem. RESULTS: We included 1129 patients in the primary analysis, of which 146 were given IV mecillinam as targeted treatment. We found no significant difference in the primary endpoint between patients treated with mecillinam versus ampicillin and cefuroxime, but found a higher risk for the primary endpoint in the piperacillin/tazobactam and meropenem groups, with adjusted HRs of 2.22 (95% CI 1.24-3.97, P < 0.01) and 2.48 (95% CI 1.04-5.93, P = 0.04), respectively, compared with mecillinam. CONCLUSIONS: The results of this study suggest that IV mecillinam may be a suitable targeted treatment for bacteraemia with a urinary tract focus. However, these results need confirmation by randomized controlled studies.

CombiANT: Antibiotic interaction testing made easy.

Antibiotic combination therapies are important for the efficient treatment of many types of infections, including those caused by antibiotic-resistant pathogens. Combination treatment strategies are typically used under the assumption that synergies are conserved across species and strains, even though recent results show that the combined treatment effect is determined by specific drug-strain interactions that can vary extensively and unpredictably, both between and within bacterial species. To address this problem, we present a new method in which antibiotic synergy is rapidly quantified on a case-by-case basis, allowing for improved combination therapy. The novel CombiANT methodology consists of a 3D-printed agar plate insert that produces defined diffusion landscapes of 3 antibiotics, permitting synergy quantification between all 3 antibiotic pairs with a single test. Automated image analysis yields fractional inhibitory concentration indices (FICis) with high accuracy and precision. A technical validation with 3 major pathogens, Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus, showed equivalent performance to checkerboard methodology, with the advantage of strongly reduced assay complexity and costs for CombiANT. A synergy screening of 10 antibiotic combinations for 12 E. coli urinary tract infection (UTI) clinical isolates illustrates the need for refined combination treatment strategies. For example, combinations of trimethoprim (TMP) + nitrofurantoin (NIT) and TMP + mecillinam (MEC) showed synergy, but only for certain individual isolates, whereas MEC + NIT combinations showed antagonistic interactions across all tested strains. These data suggest that the CombiANT methodology could allow personalized clinical synergy testing and large-scale screening. We anticipate that CombiANT will greatly facilitate clinical and basic research of antibiotic synergy.

In Vivo Evolution of CTX-M-215, a Novel Narrow-Spectrum ß-Lactamase in an Escherichia coli Clinical Isolate Conferring Resistance to Mecillinam.

Here, we report a novel narrow-spectrum ß-lactamase CTX-M-215 identified in an Escherichia coli clinical isolate in China and conferring high-level resistance to mecillinam but not to cefotaxime. CTX-M-215 differed from CTX-M-125, a CTX-M extended-spectrum ß-lactamase (ESBL), by an N132D substitution, which decreased hydrolytic activities toward penicillins and cephalosporins except for mecillinam. High similarity was observed between CTX-M-215- and CTX-M-125-bearing plasmids, carried by different isolates in the same patient, indicating in vivo evolution of CTX-M-215 from CTX-M-125.

Stability of ß-lactam antibiotics in bacterial growth media.

Laboratory assays such as MIC tests assume that antibiotic molecules are stable in the chosen growth medium-but rapid degradation has been observed for antibiotics including ß-lactams under some conditions in aqueous solution. Degradation rates in bacterial growth medium are less well known. Here, we develop a 'delay time bioassay' that provides a simple way to estimate antibiotic stability in bacterial growth media, using only a plate reader and without the need to measure the antibiotic concentration directly. We use the bioassay to measure degradation half-lives of the ß-lactam antibiotics mecillinam, aztreonam and cefotaxime in widely-used bacterial growth media based on MOPS and Luria-Bertani (LB) broth. We find that mecillinam degradation can occur rapidly, with a half-life as short as 2 hours in MOPS medium at 37°C and pH 7.4, and 4-5 hours in LB, but that adjusting the pH and temperature can increase its stability to a half-life around 6 hours without excessively perturbing growth. Aztreonam and cefotaxime were found to have half-lives longer than 6 hours in MOPS medium at 37°C and pH 7.4, but still shorter than the timescale of a typical minimum inhibitory concentration (MIC) assay. Taken together, our results suggest that care is needed in interpreting MIC tests and other laboratory growth assays for ß-lactam antibiotics, since there may be significant degradation of the antibiotic during the assay.