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Multidrug resistant (MDR) tuberculosis (TB) is defined by the resistance of Mycobacterium tuberculosis, the causative organism, to the first-line antibiotics rifampicin and isoniazid. Mitigating or reversing resistance to these drugs offers a means of preserving and extending their use in TB treatment. R-loops are RNA/DNA hybrids that are formed in the genome during transcription, and can be lethal to the cell if not resolved. RNase HI is an enzyme that removes R-loops, and this activity is essential in M. tuberculosis: knockouts of rnhC, the gene encoding RNase HI, are non-viable. This essentiality supports it as a candidate target for the development of new antibiotics. In the model organism Mycolicibacterium smegmatis, RNase HI activity is provided by two RNase HI enzymes, RnhA and RnhC. We show that the partial depletion of RNase HI activity in M. smegmatis, by knocking out either of the genes encoding RnhA or RnhC, led to the accumulation of R-loops. The sensitivity of the knockout strains to the antibiotics moxifloxacin, streptomycin and rifampicin was increased, with sensitivity to the transcriptional inhibitor rifampicin strikingly increased by nearly 100-fold. We also show that R-loop accumulation accompanies partial transcriptional inhibition, suggesting a mechanistic basis for the synergy between RNase HI depletion and transcriptional inhibition. A model of how transcriptional inhibition can potentiate R-loop accumulation is presented. Finally, we identified four small molecules that inhibit recombinant RnhC activity and that also potentiated rifampicin activity in whole-cell assays against M. tuberculosis, supporting an on-target mode of action, and providing the first step in developing a new class of anti-mycobacterial drug.ImportanceThis study validates mycobacterial RNase HI as a druggable, vulnerable candidate for a new therapeutic treatment of M. tuberculosis with a novel mode of action. RNase HI depletion shows synergistic bacterial killing with some current first- and second-line antibiotics, suggesting that RNase HI inhibitors would combine well with these regimens, and could potentially accelerate the clearance of drug-sensitive strains. RNase HI inhibitors also have the potential to reduce the effective dose of rifampicin, with the comcommitant reduction in side effects. The potentiation of rifampicin efficacy conferred by RNase HI deficiency suggests that RNase HI inhibitors may be able to mitigate against development of rifampicin resistance. The synergy may also be able to reverse rifampicin resistance, rescuing this antibiotic for therapy. The surprising finding that low levels of transcriptional inhibition potentiate R-loop formation provides a key new insight into R-loop metabolism.

Pseudomonas aeruginosa is a common pathogen that is associated with multidrug-resistant (MDR) and carbapenem-resistant (CR) phenotypes; therefore, we investigated its resistance patterns and mechanisms by using data from the Antimicrobial Testing Leadership and Surveillance (ATLAS) program in the Asia-Pacific region from 2015 to 2019. MICs were determined using the broth microdilution method. Genes encoding major extended-spectrum β-lactamases and carbapenemases were investigated by multiplex PCR assays. Susceptibility was interpreted using the Clinical and Laboratory Standards Institute (CLSI) breakpoints. A total of 6,349 P. aeruginosa isolates were collected in the ATLAS program between 2015 and 2019 from 14 countries. According to the CLSI definitions, the numbers (and rates) of CR and MDR P. aeruginosa isolates were 1,198 (18.9%) and 1,303 (20.5%), respectively. For 747 of the CR P. aeruginosa strains that were available for gene screening, 253 β-lactamase genes were detected in 245 (32.8%) isolates. The most common gene was bla(VIM) (29.0%, 71/245), followed by bla(NDM) (24.9%, 61/245) and bla(VEB) (20.8%, 51/245). The resistance patterns and associated genes varied significantly between the countries in the Asia-Pacific region. India had the highest rates of carbapenem resistance (29.3%, 154/525) and gene detection (17.7%, 93/525). Compared to those harboring either class A or B β-lactamase genes, the CR P. aeruginosa isolates without detected β-lactamase genes had lower MICs for most of the antimicrobial agents, including ceftazidime-avibactam and ceftolozane-tazobactam. In conclusion, MDR and CR P. aeruginosa infections pose a major threat, particularly those with detected carbapenemase genes. Continuous surveillance is important for improving antimicrobial stewardship and antibiotic prescriptions.

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

Cyclohexyl-griselimycin is a preclinical candidate for use against tuberculosis (TB). Here, we show that this oral cyclodepsipeptide is also active against the intrinsically drug-resistant nontuberculous mycobacterium Mycobacterium abscessus in vitro and in a mouse model of infection. This adds a novel advanced lead compound to the M. abscessus drug pipeline and supports a strategy of screening chemical matter generated in TB drug discovery efforts to fast-track the discovery of novel antibiotics against M. abscessus.

The failure of antibiotic therapy in respiratory tract infections in cystic fibrosis is partly due to the high tolerance observed in Pseudomonas aeruginosa biofilms. This tolerance is mediated by changes in bacterial metabolism linked to growth in biofilms, opening up potential avenues for novel treatment approaches based on modulating metabolism. The goal of the present study was to identify carbon sources that increase the inhibiting and/or eradicating activity of tobramycin, ciprofloxacin, and ceftazidime against P. aeruginosa PAO1 biofilms grown in a synthetic cystic fibrosis sputum medium (SCFM2) and to elucidate their mode of action. After screening 69 carbon sources, several combinations of antibiotics 1 carbon sources that showed markedly higher anti-biofilm activity than antibiotics alone were identified. D,L-malic acid and sodium acetate could potentiate both biofilm inhibiting and eradicating activity of ciprofloxacin and ceftazidime, respectively, while citric acid could only potentiate biofilm inhibitory activity of tobramycin. The mechanisms underlying the increased biofilm eradicating activity of combinations ciprofloxacin/D,L-malic acid and ceftazidime/sodium acetate are similar but not identical. Potentiation of ceftazidime activity by sodium acetate was linked to increased metabolic activity, a functional TCA cycle, increased ROS production, and high intracellular pH, whereas the latter was not required for D,L-malic acid potentiation of ciprofloxacin. Finally, our results indicate that the potentiation of antibiotic activity by carbon sources is strain dependent.

Identified in the 1970s as the leading cause of invasive bacterial disease in neonates and young infants, group B Streptococcus (GBS) is now also recognized as a significant cause of morbidity and mortality among adults with underlying medical conditions and the elderly. Concomitant with the increasing incidence of GBS invasive disease in adults is the rise of resistance among GBS isolates to second line antibiotics. Previous research shows that among serotype V GBS, one of the most common capsular types causing adult invasive disease, sequence type 1 (ST1), accounts for an overwhelming majority of adult invasive disease isolates and frequently harbors macrolide resistance. In this study, using whole-genome sequencing data from strains isolated in the United States and Canada over a 45-year period, we examined the association of antimicrobial resistance with the emergence of invasive serotype V ST1 GBS. Our findings show a strong temporal association between increased macrolide resistance and the emergence of serotype V ST1 GBS subpopulations that currently co-circulate to cause invasive disease in adults and young infants. ST1 GBS subpopulations are defined, in part, by the presence of macrolide resistance genes in mobile genetic elements. Increased frequency of macrolide resistance-encoding mobile genetic elements among invasive GBS ST1 strains suggests the presence of such elements contributes to GBS virulence. Our work provides a foundation for the investigation of genetic features contributing to the increasing prevalence and pathogenesis of serotype V GBS in adult invasive disease.

Pseudomonas aeruginosa is among the highest priority pathogens for drug development, because of its resistance to antibiotics, extraordinary adaptability, and persistence. Anti-pseudomonal research is strongly encouraged to address the acute scarcity of innovative antimicrobial lead structures. In an effort to understand the physiological response of P. aeruginosa to clinically relevant antibiotics, we investigated the proteome after exposure to ciprofloxacin, levofloxacin, rifampicin, gentamicin, tobramycin, azithromycin, tigecycline, polymyxin B, colistin, ceftazidime, meropenem, and piperacillin/tazobactam. We further investigated the response to CHIR-90, which represents a promising class of lipopolysaccharide biosynthesis inhibitors currently under evaluation. Radioactive pulse-labeling of newly synthesized proteins followed by 2D-PAGE was used to monitor the acute response of P. aeruginosa to antibiotic treatment. The proteomic profiles provide insights into the cellular defense strategies for each antibiotic. A mathematical comparison of these response profiles based on upregulated marker proteins revealed similarities of responses to antibiotics acting on the same target area. This study provides insights into the effects of commonly used antibiotics on P. aeruginosa and lays the foundation for the comparative analysis of the impact of novel compounds with precedented and unprecedented modes of action.

The emergence of daptomycin-resistant (DAP-R) Staphylococcus aureus strains has become a global problem. Point mutations in mprF are the main cause of daptomycin (DAP) treatment failure. However, the impact of these specific point-mutations in methicillin-resistant S. aureus (MRSA) strains associated with DAP resistance and the "see-saw effect" of distinct beta-lactams remains unclear. In this study, we used three series of clinical MRSA strains with three distinct mutated mprF alleles from clone complexes (CC) 5 and 59 to explore the "see-saw effect" and the combination effect of DAP plus beta-lactams. Through construction of mprF deletion and complementation strains of SA268, we determined that mprF-S295A, mprF-S337L and one novel mutation of mprF-I348del within the bifunctional domain lead to DAP resistance. Compared with wild-type mprF cloned from a DAP-susceptible (DAP-S) strain, these three mprF mutations conferred the "see-saw effect" to distinct beta-lactams in the SA268ΔmprF strains and mutated-mprF (I348del and S337L) did not alter the cell surface positive charge (P > 0.05). The susceptibility to beta-lactams increased significantly in DAP-R CC59 strains and the "see-saw effect" was found to be associated with distinct mutated mprF alleles and the category of beta-lactams. The synergistic activity of DAP plus oxacillin was detected in all DAP-R MRSA strains. Continued progress in understanding the mechanism of restoring susceptibility to beta-lactam antibiotics mediated by the mprF mutation and its impact on beta-lactam combination therapy will provide fundamental insights into treatment of MRSA infections.

Contains fulltext : 248649.pdf (Publisher’s version ) (Open Access) Mycobacterium abscessus is an opportunistic pathogen notorious for its resistance to most classes of antibiotics and low cure rates. M. abscessus carries an array of mostly unexplored defense mechanisms. A deeper understanding of antibiotic resistance and tolerance mechanisms is pivotal in development of targeted therapeutic regimens. We provide the first description of all major transcriptional mechanisms of tolerance to all antibiotics recommended in current guidelines, using RNA sequencing-guided experiments. M. abscessus ATCC 19977 bacteria were subjected to subinhibitory concentrations of clarithromycin (CLR), amikacin (AMK), tigecycline (TIG), cefoxitin (FOX), and clofazimine (CFZ) for 4 and 24 h, followed by RNA sequencing. To confirm key mechanisms of tolerance suggested by transcriptomic responses, we performed time-kill kinetic analysis using bacteria after preexposure to CLR, AMK, or TIG for 24 h and constructed isogenic knockout and knockdown strains. To assess strain specificity, pan-genome analysis of 35 strains from all three subspecies was performed. Mycobacterium abscessus shows both drug-specific and common transcriptomic responses to antibiotic exposure. Ribosome-targeting antibiotics CLR, AMK, and TIG elicit a common response characterized by upregulation of ribosome structural genes, the WhiB7 regulon and transferases, accompanied by downregulation of respiration through NuoA-N. Exposure to any of these drugs decreases susceptibility to ribosome-targeting drugs from multiple classes. The cytochrome bd-type quinol oxidase contributes to CFZ tolerance in M. abscessus, and the sigma factor sigH but not antisigma factor MAB_3542c is involved in TIG resistance. The observed transcriptomic responses are not strain-specific, as all genes involved in tolerance, except erm(41), are found in all included strains.

Although intra-articular vancomycin powder (VP) is sometimes applied before the closure of the incision to prevent periprosthetic joint infection (PJI) after joint replacement, the dosage, efficacy, and safety remain controversial. This study aimed to explore the dosage, efficacy, and safety of intra-articular VP in the prophylaxis of infection after total knee arthroplasty (TKA) in a rat model. Sixty male rats were randomly divided into five groups after receiving TKA: control (no antibiotics); systemic vancomycin (SV) (intraperitoneal injection, 88 mg/kg of body weight, equal to 1 g in a patient weighing 70 kg); and VP0.5, VP1.0, and VP2.0 (44 mg/kg, 88 mg/kg and 176 mg/kg, respectively; intra-articular). All animals were inoculated in the knee with methicillin-resistant S. aureus (MRSA). General status, serum biomarkers, radiology, microbiological assay, and histopathological tests were assessed within 14 days postoperation. Compared with the control and SV groups, bacterial counts, knee width, tissue inflammation, and osteolysis were reduced in the VP0.5, VP1.0, and VP2.0 groups, without notable body weight loss and incision complications. Among all the VP groups, VP1.0 and VP2.0 groups presented superior outcomes with regard to knee width and tissue inflammation than the VP0.5 group. Microbial culture indicated that no MRSA survived in the knee of VP1.0 and VP2.0 groups, while bacteria growth was observed in the VP0.5 group. No obvious changes in the structure and functional biomarkers of liver and kidney were observed in either the SV or VP groups. Therefore, intra-articular vancomycin powder at a dosage from 88 mg/kg to 176 mg/kg may be effective and safe in preventing PJI induced by methicillin-resistant S. aureus in the rat TKA model.

Uropathogenic Escherichia coli (UPEC), the major causative agent of urinary tract infections, can invade different types of host cells. To compare the pharmacodynamic properties of antibiotics against intra- and extracellular UPEC, an in vitro model of intracellular infection was established in J774 mouse macrophages infected by the UPEC strain CFT073. We tested antibiotics commonly prescribed against urinary tract infections (gentamicin, ampicillin, nitrofurantoin, trimethoprim, sulfamethoxazole, and ciprofloxacin) and the investigational fluoroquinolone finafloxacin. The metabolic activity of individual bacteria was assessed by expressing the fluorescent reporter protein TIMERbac within CFT073. Concentration-response experiments revealed that all tested antibiotics were much less effective against intracellular bacteria than extracellular ones. Most antibiotics, except fluoroquinolones, were unable to reach a bactericidal effect intracellularly at clinically achievable concentrations. Ciprofloxacin and finafloxacin killed 99.9% of extracellular bacteria at concentrations around the MIC, while for intracellular bacteria, concentrations more than 100× over the MIC were required to achieve a bactericidal effect. Time-kill curves showed that finafloxacin was more rapidly bactericidal in acidic medium than at neutral pH, while the reverse observation was made for ciprofloxacin. Intracellularly, kill curves showed biphasic kinetics for both fluoroquinolones, suggesting the presence of drug-tolerant subpopulations. Flow cytometry analysis of TIMERbac fluorescence revealed a marked heterogeneity in intracellular growth of individual bacteria, suggesting that the presence of subpopulations reaching a state of metabolic dormancy was the main reason for increased antibiotic tolerance of intracellular UPEC.

Staphylococcus aureus is a serious threat to public health due to the rise of antibiotic resistance in this organism, which can prolong or exacerbate skin and soft tissue infections (SSTIs). Methicillin-resistant S. aureus is a Gram-positive bacterium and a leading cause of SSTIs. As such, many efforts are under way to develop therapies that target essential biological processes in S. aureus. Antimicrobial photodynamic therapy is an effective alternative to antibiotics; therefore we developed an approach to simultaneously expose S. aureus to intracellular and extracellular photosensitizers. A near infrared photosensitizer was conjugated to human monoclonal antibodies (MAbs) that target the S. aureus iron-regulated surface determinant (Isd) heme acquisition proteins. In addition, the compound VU0038882 was developed to increase photoactivatable porphyrins within the cell. Combinatorial photodynamic treatment of drug-resistant S. aureus exposed to VU0038882 and conjugated anti-Isd MAbs proved to be an effective antibacterial strategy in vitro and in a murine model of SSTIs.

Gepotidacin is a novel, first-in-class triazaacenaphthylene antibiotic that inhibits bacterial DNA replication by a distinct mechanism of action with an in vitro spectrum of activity that includes Escherichia coli. Our objectives herein were the following: (i) to identify the pharmacokinetic-pharmacodynamic (PK-PD) index associated with the efficacy of gepotidacin against E. coli; (ii) to determine the magnitude of the above-described PK-PD index associated with various bacterial reduction endpoints for E. coli; and (iii) to characterize the relationship between gepotidacin exposure and on-therapy E. coli resistance amplification. A 24-h one-compartment in vitro infection model was used to investigate the first two study objectives, and a 10-day hollow-fiber in vitro infection model was used to evaluate the third objective. For the dose-fractionation studies (objective i) in which E. coli NCTC 13441 (gepotidacin MIC, 2 mg/liter) was evaluated, gepotidacin free-drug area under the concentration-time curve (AUC) from 0 to 24 h to the MIC (AUC/MIC ratio) was identified as the PK-PD index most closely associated with change in bacterial burden (r2 = 0.925). For the dose-ranging studies (objective ii), in which four E. coli isolates (gepotidacin MIC range, 1 to 4 mg/liter) were studied, the magnitude of the median gepotidacin free-drug AUC/MIC ratio associated with net bacterial stasis and 1- and 2-log10 CFU reductions for the pooled data set was 33.9, 43.7, and 60.7, respectively. For the hollow-fiber in vitro infection model studies (objective iii), in which one isolate (E. coli NCTC 13441; gepotidacin MIC, 2 mg/liter) was evaluated, gepotidacin free-drug AUC/MIC ratios of 275 and greater were sufficient to suppress on-therapy resistance amplification. Together, the data generated from these studies will be useful to support discrimination among candidate dosing regimens for future clinical study.

We recently reported the successful treatment of a case of periprosthetic joint infection (PJI) with phage. Phage activity against bacteria causing PJI has not been systematically evaluated. Here, we examined the in vitro activity of seven phages against 122 clinical isolates of Staphylococcus aureus recovered between April 1999 and February 2018 from subjects with PJI. Phages were assessed against planktonic and biofilm phenotypes. Activity of individual phages was demonstrated against up to 73% of bacterial isolates in the planktonic state and up to 100% of biofilms formed by isolates that were planktonically phage susceptible. Susceptibility to phage was not correlated with small-colony-variant phenotype for planktonic or biofilm bacteria; correlation between antibiotic susceptibility and planktonic phage susceptibility and between biofilm phage susceptibility and strength of biofilm formation were noted under select conditions. These results demonstrate that phages can infect S. aureus causing PJI in both planktonic and biofilm phenotypes, and thus are worthy of investigation as an alternative or addition to antibiotics in this setting.

Telavancin, a lipoglycopeptide antibiotic, is traditionally dosed at 10 mg/kg based on total body weight but is associated with toxicities that limit its use. This study supports the use of a capped dosing regimen of 750 mg in obese patients, which is associated with equal efficacy and fewer adverse effects compared to traditional dosing.

Biofilm has recently been highlighted as a complicating feature of necrotizing soft tissue infections (NSTI) caused by Streptococcus pyogenes (i.e., group A Streptococcus [GAS]) contributing to a persistence of bacteria in tissue despite prolonged antibiotic therapy. Here, we assessed the standard treatment of benzylpenicillin and clindamycin with or without rifampin in a tissue-like setting. Antibiotic efficacy was evaluated by CFU determination in a human organotypic skin model infected for 24 or 48 h with GAS strains isolated from NSTI patients. Antibiotic effect was also evaluated by microcalorimetric metabolic assessment in in vitro infections of cellular monolayers providing continuous measurements over time. Adjunctive rifampin resulted in enhanced antibiotic efficacy of bacterial clearance in an organotypic skin tissue model, 97.5% versus 93.9% (P = 0.006). Through microcalorimetric measurements, adjunctive rifampin resulted in decreased metabolic activity and extended lag phase for all clinical GAS strains tested (P

Contezolid (MRX-I), a novel oxazolidinone antibiotic, was recently approved for the treatment of serious Gram-positive infections. The pharmacokinetics and disposition of [(14)C]contezolid were investigated in a single-dose human mass balance study. Cross-species comparison of plasma exposure for contezolid and metabolites was performed, and the safety of the disproportionate metabolite in human was evaluated with additional nonclinical studies. After an oral administration of 99.1 μCi/602-mg dose of [(14)C]contezolid, approximately 91.5% of the radioactivity was recovered in 0 to 168 h postdose, mainly in urine followed by that in feces. The principal metabolic pathway of contezolid in human comprised an oxidative ring opening of the 2,3-dihydropyridin-4-one fragment into polar metabolites MRX445-1 and MRX459, with recovery of approximately 48% and 15% of the dose, respectively, in urine and feces. Contezolid, MRX445-1, and MRX459 accounted for 68.0%, 19.5%, and 4.84% of the plasma exposure of the total radioactivity, respectively. Metabolites MRX445-1 and MRX459 were observed in disproportionately larger amounts in human plasma than in samples from rat or dog, the rodent and nonrodent species, respectively, used for the general nonclinical safety assessment of this molecule. This discrepancy was resolved with additional nonclinical studies, wherein the primary metabolite, MRX445-1, was further characterized. The no-observed-adverse-effect level (NOAEL) of MRX445-1 was determined as 360 mg/kg body weight/day in a 14-day repeat-dose test in pregnant and nonpregnant Sprague Dawley rats. Furthermore, MRX445-1 exhibited no antibacterial activity in vitro. Thus, MRX445-1 is not expected to exert clinically relevant pharmacology and toxicity.

Shiga toxin-producing Escherichia coli (STEC) is an important foodborne pathogen that contributes to over 250,000 infections in the United States each year. Because antibiotics are not recommended for STEC infections, resistance in STEC has not been widely researched despite an increased likelihood for the transfer of resistance genes from STEC to opportunistic pathogens residing within the same microbial community. From 2001 to 2014, 969 STEC isolates were collected from Michigan patients. Antibiotic susceptibility profiles to clinically relevant antibiotics were determined using disc diffusion, while epidemiological data were used to identify factors associated with resistance. Whole-genome sequencing was used for serotyping, examining genetic relatedness, and identifying genetic determinants and mechanisms of resistance in the non-O157 isolates. Increasing frequencies of resistance to at least one antibiotic were observed over the 14 years (P = 0.01). While the non-O157 serogroups were more commonly resistant than O157 (odds ratio, 2.4; 95% confidence interval,1.43 to 4.05), the frequency of ampicillin resistance among O157 isolates was significantly higher in Michigan than the national average (P = 0.03). Genomic analysis of 321 non-O157 isolates uncovered 32 distinct antibiotic resistance genes (ARGs). Although mutations in genes encoding resistance to ciprofloxacin and ampicillin were detected in four isolates, most of the horizontally acquired ARGs conferred resistance to aminoglycosides, β-lactams, sulfonamides, and/or tetracycline. This study provides insight into the mechanisms of resistance in a large collection of clinical non-O157 STEC isolates and demonstrates that antibiotic resistance among all STEC serogroups has increased over time, prompting the need for enhanced surveillance.

Neisseria gonorrhoeae is an increasing public health threat due to its rapidly rising incidence and antibiotic resistance. There are an estimated 106 million cases per year worldwide, there is no vaccine available to prevent infection, and N. gonorrhoeae strains that are resistant to all antibiotics routinely used to treat the infection have emerged. In many strains, antibiotic resistance is mediated by overexpression of the MtrCDE efflux pump, which enables the bacteria to transport toxic antibiotics out of the cell. Genetic mutations that inactivate MtrCDE have previously been shown to render resistant strains susceptible to certain antibiotics. Here, we show that peptides rationally designed to target and disrupt the activity of each of the three protein components of MtrCDE were able to increase the susceptibility of N. gonorrhoeae strains to antibiotics in a dose-dependent manner and with no toxicity to human cells. Cotreatment of bacteria with subinhibitory concentrations of the peptide led to 2- to 64-fold increases in susceptibility to erythromycin, azithromycin, ciprofloxacin, and/or ceftriaxone in N. gonorrhoeae strains FA1090, WHO K, WHO P, and WHO X. The cotreatment experiments with peptides P-MtrC1 and P-MtrE1 resulted in increased susceptibilities of WHO P and WHO X to azithromycin, ciprofloxacin, and ceftriaxone that were of the same magnitude seen in MtrCDE mutants. P-MtrE1 was able to change the azithromycin resistance profile of WHO P from resistant to susceptible. Data presented here demonstrate that these peptides may be developed for use as a dual treatment with existing antibiotics to treat multidrug-resistant gonococcal infections.

Hospitalized patients with SARS-CoV-2 infection (COVID-19) often receive antibiotics for suspected bacterial coinfection. We estimated the incidence of bacterial coinfection and secondary infection in COVID-19 using clinical diagnoses to determine how frequently antibiotics are administered when bacterial infection is absent. We performed a retrospective cohort study of inpatients with COVID-19 present on admission to hospitals in the Premier Healthcare Database between April and June 2020. Bacterial infections were defined using ICD-10-CM diagnosis codes and associated “present on admission” coding. Coinfections were defined by bacterial infection present on admission, while secondary infections were defined by bacterial infection that developed after admission. Coinfection and secondary infection were not mutually exclusive. A total of 18.5% of 64,961 COVID-19 patients (n = 12,040) presented with bacterial infection at admission, 3.8% (n = 2,506) developed secondary infection after admission, and 0.9% (n = 574) had both; 76.3% (n = 49,551) received an antibiotic while hospitalized, including 71% of patients who had no diagnosis of bacterial infection. Secondary bacterial infection occurred in 5.7% of patients receiving steroids in the first 2 days of hospitalization, 9.9% receiving tocilizumab in the first 2 days of hospitalization, and 10.3% of patients receiving both. After adjusting for patient and hospital characteristics, bacterial coinfection (adjusted relative risk [aRR], 1.15; 95% confidence interval [CI], 1.11 to 1.20) and secondary infection (aRR 1.93; 95% CI, 1.82 to 2.04) were both independently associated with increased mortality. Although 1 in 5 inpatients with COVID-19 presents with bacterial infection, secondary infections in the hospital are uncommon. Most inpatients with COVID-19 receive antibiotic therapy, including 71% of those not diagnosed with bacterial infection.

Q fever, caused by the intracellular pathogen Coxiella burnetii, is traditionally treated using tetracycline antibiotics, such as doxycycline. Doxycycline is often poorly tolerated, and antibiotic-resistant strains have been isolated. In this study, we have evaluated a panel of antibiotics (doxycycline, ciprofloxacin, levofloxacin, and co-trimoxazole) against C. burnetii using in vitro methods (determination of MIC using liquid and solid media; efficacy assessment in a THP cell infection model) and in vivo methods (wax moth larvae and mouse models of infection). In addition, the schedule for antibiotic treatment has been evaluated, with therapy initiated at 24 h pre- or postchallenge. Both doxycycline and levofloxacin limited overt clinical signs during treatment in the AJ mouse model of aerosol infection, but further studies are required to investigate the possibility of disease relapse or incomplete bacterial clearance after the antibiotics are stopped. Levofloxacin was well tolerated and therefore warrants further investigation as an alternative to the current recommended treatment with doxycycline.

Antibiotics inhibit essential bacterial processes, resulting in arrest of growth and, in some cases, cell death. Many antibiotics are also reported to trigger endogenous production of reactive oxygen species (ROS), which damage DNA, leading to induction of the mutagenic SOS response associated with the emergence of drug resistance. However, the type of DNA damage that arises and how this triggers the SOS response are largely unclear. We found that several different classes of antibiotic triggered dose-dependent induction of the SOS response in Staphylococcus aureus, indicative of DNA damage, including some bacteriostatic drugs. The SOS response was heterogenous and varied in magnitude between strains and antibiotics. However, in many cases, full induction of the SOS response was dependent upon the RexAB helicase/nuclease complex, which processes DNA double-strand breaks to produce single-stranded DNA and facilitate RecA nucleoprotein filament formation. The importance of RexAB in repair of DNA was confirmed by measuring bacterial survival during antibiotic exposure, with most drugs having significantly greater bactericidal activity against rexB mutants than against wild-type strains. For some, but not all, antibiotics there was no difference in bactericidal activity between wild type and rexB mutant under anaerobic conditions, indicative of a role for reactive oxygen species in mediating DNA damage. Taken together, this work confirms previous observations that several classes of antibiotics cause DNA damage in S. aureus and extends them by showing that processing of DNA double-strand breaks by RexAB is a major trigger of the mutagenic SOS response and promotes bacterial survival.

Cefiderocol (FDC) is a recently developed siderophore cephalosporin showing excellent antibacterial activity against Gram-negative bacteria, including Acinetobacter baumannii . By investigating a series of A. baumannii clinical isolates with elevated MICs of FDC, we showed that PER-like β-lactamases and, to a lesser extent, NDM-like β-lactamases, significantly contributed to reduced susceptibility to that antibiotic.

Linezolid is an important last-resort antibiotic for the treatment of multidrug-resistant enterococci. The aim of this study was to further characterize the genetic context of optrA and poxtA in 10 florfenicol-resistant enterococci isolated from flowing surface water. In most genomes, optrA and poxtA were embedded in transposition units integrated into plasmids or into the chromosomal radC . For the first time, a chromosomally integrated optrA in an Enterococcus raffinosus isolate is described.

Exebacase (CF-301) is a novel antistaphylococcal lysin (cell wall hydrolase) in phase 3 of clinical development for the treatment of Staphylococcus aureus bacteremia, including right-sided endocarditis, used in addition to standard-of-care antibiotics. In the current study, the potential for exebacase to treat S. aureus pneumonia was explored in vitro using bovine pulmonary surfactant (Survanta) and in vivo using a lethal murine pneumonia model. Exebacase was active against a set of methicillin-sensitive S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA) strains, with an MIC90 of 2 μg/ml (n = 18 strains), in the presence of a surfactant concentration (7.5%) inhibitory to the antistaphylococcal antibiotic daptomycin, which is inactive in pulmonary environments due to specific inhibition by surfactant. In a rigorous test of the ability of exebacase to synergize with antistaphylococcal antibiotics, exebacase synergized with daptomycin in the presence of surfactant in vitro, resulting in daptomycin MIC reductions of up to 64-fold against 9 MRSA and 9 MSSA strains. Exebacase was also observed to facilitate the binding of daptomycin to S. aureus and the elimination of biofilm-like structures formed in the presence of surfactant. Exebacase (5 mg/kg of body weight 1 time every 24 h [q24h], administered intravenously for 3 days) was efficacious in a murine model of staphylococcal pneumonia, resulting in 50% survival, compared to 0% survival with the vehicle control; exebacase in addition to daptomycin (50 mg/kg q24h for 3 days) resulted in 70% survival, compared to 0% survival in the daptomycin-alone control group. Overall, exebacase is active in pulmonary environments and may be appropriate for development as a treatment for staphylococcal pneumonia.

Carbapenem-resistant Enterobacterales, such as Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae, represent a major threat to public health due to their rapid spread. Novel drug combinations such as ceftazidime-avibactam (CZA), combining a broad-spectrum cephalosporin along with a broad-spectrum β-lactamase inhibitor, have recently been introduced and have been shown to exhibit excellent activity toward multidrug-resistant KPC-producing Enterobacterales strains. However, CZA-resistant K. pneumoniae isolates are now being increasingly reported, mostly corresponding to producers of KPC variants. In this study, we evaluated in vitro the nature of the mutations in the KPC-2 and KPC-3 β-lactamase sequences (the most frequent KPC-type enzymes) that lead to CZA resistance and the subsequent effects of these mutations on susceptibility to other β-lactam antibiotics. Single-step in vitro selection assays were conducted, resulting in the identification of a series of mutations in the KPC sequence which conferred the ability of those mutated enzymes to confer resistance to CZA. Hence, 16 KPC-2 variants and 10 KPC-3 variants were obtained. Production of the KPC variants in an Escherichia coli recombinant strain resulted in a concomitant increased susceptibility to broad-spectrum cephalosporins and carbapenems, with the exceptions of ceftazidime and piperacillin-tazobactam, compared to wild-type KPC enzymes. Enzymatic assays showed that all of the KPC variants identified exhibited an increased affinity toward ceftazidime and a slightly decreased sensitivity to avibactam, sustaining their impact on CZA resistance. However, their respective carbapenemase activities were concurrently negatively impacted.

Aliarcobacter butzleri is an emergent enteropathogen for which resistance to several classes of antimicrobial agents has been described, although the underlying mechanisms have been poorly addressed. We aimed to evaluate the contribution of the resistance-nodulation-division-type (RND) efflux system, AreABC, to drug resistance in A. butzleri. A. butzleri strains were first tested against several antimicrobials with and without an efflux pump inhibitor. Then, erythromycin-resistant strains were screened for the presence of a premature stop codon in a putative transcriptional regulator of the AreABC system, areR. Lastly, antimicrobial susceptibility and ethidium bromide (EtBr) accumulation were evaluated using an areB knockout strain and a strain overexpressing the AreABC system through areR truncation. The presence of the efflux pump inhibitor resulted in increased susceptibility to most of the antimicrobials tested. A correlation between erythromycin resistance and the presence of premature stop codons in areR was observed. The truncation of areR resulted in increased expression of the AreABC system and decreased susceptibility to various antimicrobials. In contrast, areB inactivation resulted in increased susceptibility and a higher intracellular accumulation of EtBr. In conclusion, the AreABC efflux pump plays a role in the resistance of A. butzleri to multiple drugs and is regulated by a putative transcriptional repressor, areR. Our results support the importance of efflux pumps in this bacterium's resistance to major classes of antibiotics and other antimicrobials. Susana Ferreira acknowledges the Universidade da Beira Interior (UBI) and the Foundation for Science and Technology (FCT), both involved in the scientific employment contract according to DL57/2016. Cristiana Mateus acknowledges the fellowship reference BID/ICI-FCS/Santander Universidades-UBI/2019 within the scope of the protocol signed between the UBI and Santander Universidades. Sanger sequencing was performed at the Unidade de Tecnologia e Inovação (Departamento de Genética Humana, Instituto Nacional de Saúde Ricardo Jorge, Lisbon, Portugal). This work was supported by the Foundation for Science and Technology (FCT) through funds from the state budget and by the European Regional Development Fund (ERDF) under the Portugal 2020 Program through the Regional Operational Program of the Center (Centro2020) and through the project with the reference UIDB/00709/2020. info:eu-repo/semantics/publishedVersion

Methicillin-resistant Staphylococcus aureus (MRSA) bacteremia has a high case-fatality rate, but currently recommended antimicrobial therapies have many shortcomings. The efficacy and safety of lincosamide therapy for MRSA bacteremia is incompletely defined. A retrospective audit was done of the management of all adults with MRSA bacteremia at an Australian tertiary referral hospital between 1 January 2007 and 31 December 2020. A total of 176 patients were included. The case-fatality rate declined from 14/57 (25%) in the first half of the study to 12/119 (10%) in the second half (P = 0.01). Of the 172 patients receiving antibiotics, 62 (36%) received a lincosamide-predominant regimen (lincosamide monotherapy for >50% of the intravenous course). The patients receiving lincosamide-predominant intravenous therapy had lower in-hospital mortality (odds ratio [OR], 0.07; 95% confidence interval [CI], 0.01 to 0.53; P = 0.01) and a lower incidence of renal complications (OR [95% CI], 0.34 [0.15−0.75]; P = 0.008) than patients receiving an alternative regimen. In multivariate analysis that also considered age, disease severity, comorbidity, infectious diseases consultation, source control, and the year of admission, patients receiving a lincosamide-predominant regimen were still less likely to die in the hospital than those receiving an alternative regimen (OR [95% CI], 0.05 [0.00 to 0.65]; P = 0.02). Lincosamides appear to have utility, at least as stepdown therapy, in the treatment of MRSA bacteremia, particularly in young, clinically stable patients with few comorbidities in whom endocarditis has been excluded. Prospective studies will help define their optimal role.

Extremely drug-resistant (XDR) Acinetobacter baumannii causes challenging nosocomial infections. We report the case of a patient with XDR A. baumannii pneumonia and septic shock successfully treated with cefiderocol and a novel antibiotic obtained via expanded access protocol. With focused research and drug development efforts, the poor outcomes associated with these infections may be mitigated.

BackgroundVancomycin is a first line antibiotic for many common infectious diseases and is the most commonly prescribed antibiotic in the United States hospital setting. Vancomycin is also well known to cause kidney injury; two recent prospective studies have identified that increasing vancomycin area under the concentration curve predicts vancomycin induced kidney injury (VIKI). However, outside of clinical trials, it is unclear if pre-clinical data can quantitatively describe VIKI in patients.MethodsData were simultaneously analyzed from a pre-clinical rat model and two prospective clinical studies. Logged vancomycin area under the concentration curve (AUC) data for rats (n=48) and patients from PROVIDE (n=263) and CAMERA2 (n=291) were included. VIKI was defined as urinary KIM-1 concentrations ≥9.42 ng/mL in the rat and according to KDIGO stage 1 kidney injury for all human patients. Multiple generalized linear models were explored, and the order of magnitude was calculated between the probability of acute kidney injury (AKI) from the average obtained in the clinical studies (i.e. CAMERA2 and PROVIDE) and the rat for 0.1 increments in Log10AUC bounded common concentrations obtained in the therapeutic range (i.e. ~200 −800 mg*24h/L).ResultsA logit link model best fit the data. When calculating the multiplicative factors between the studies therapeutic range AUCs, the rat was an average 2.7 to 4.2 times more sensitive to AKI between AUCs of 199.5 (i.e. log 10 AUC=2.3) and 794.3 mg*h/L (i.e. log 10 AUC=2.9), respectively.ConclusionsA pre-clinical rat model was quantitatively linked to toxicity data from two large human studies. The rat is an attractive pre-clinical model to explore exposure toxicity relationships with vancomycin. External validation is required.

The poxtA gene encodes a protein belonging to the F lineage of the ATP-binding cassette superfamily, which can protect the bacterial ribosome from some anti-ribosomal antibiotics, including oxazolidinones (1).….