Antimicrobial Agents and Chemotherapy

Testing Plasmodium vivax antimicrobial sensitivity is limited to ex vivo schizont maturation assays, which precludes determining the IC50s of delayed action antimalarials such as doxycycline. Using Plasmodium cynomolgi as a model for P. vivax, we determined the physiologically significant delayed death effect induced by doxycycline (IC50(96h), 1401 {+/-} 607 nM). As expected, IC50(96 h) to chloroquine (20.4 nM), piperaquine (12.6 {micro}M) and tafenoquine (1424 nM) were not affected by extended exposure.

A regimen comprised of bedaquiline, pretomanid and linezolid (BPaL) is the first oral 6-month regimen approved by the US Food and Drug Administration and recommended by the World Health Organization for treatment of extensively drug-resistant tuberculosis. We used a well-established BALB/c mouse model of tuberculosis to evaluate the treatment-shortening potential of replacing bedaquiline with either of two new, more potent diarylquinolines in early clinical trials, TBAJ-587 and TBAJ-876. We also evaluated the effect of replacing linezolid with a new oxazolidinone, TBI-223, exhibiting a larger safety margin with respect to mitochondrial toxicity in preclinical studies. Replacing bedaquiline with TBAJ-587 at the same 25 mg/kg dose significantly reduced the proportion of mice relapsing after 2 months of treatment, while replacing linezolid with TBI-223 at the same 100 mg/kg dose did not significantly change the proportion of mice relapsing. Replacing linezolid or TBI-223 with sutezolid in combination with TBAJ-587 and pretomanid significantly reduced the proportion of mice relapsing. In combination with pretomanid and TBI-223, TBAJ-876 at 6.25 mg/kg was equipotent to TBAJ-587 at 25 mg/kg. We conclude that replacement of bedaquiline with these more efficacious and potentially safer diarylquinolines and replacement of linezolid with potentially safer and at least as efficacious oxazolidinones in the clinically successful BPaL regimen may lead to superior regimens capable of treating both drug-susceptible and drug-resistant TB more effectively and safely.

BackgroundIDSA guideline-based therapy achieves sputum culture conversion rates in 20-34% of patients with Mycobacterium abscessus (MAB) lung disease (LD). Double-{beta}-lactam combinations have been proposed to improve cure, based on time-kill curves. MethodsWe performed minimum inhibitory concentrations (MICs) experiments followed by hollow fiber system model of MAB-LD (HFS-MAB) exposure-effect studies with sulbactam-durlobactam administered every 8h (q8h), q12h, and q24h, to identify target exposures. Next, the sulbactam-durlobactam target exposure plus ceftriaxone was administered in the HFS-MAB inoculated with three different MAB isolates, as was the sulbactam-durlobactam-ceftriaxone combination with epetraborole and omadacycline (SDCEO).{gamma} -slopes (kill-speed) were calculated for all regimens. The minimal sulbactam-durlobactam clinical doses that achieved target exposure were identified using Monte Carlo experiments. ResultsCeftriaxone reduced sulbactam-durlobactam MICs by 8-tube dilutions. In the HFS-MAB, sulbactam-durlobactam microbial kill and antimicrobial resistance were linked to % time concentration persists above MIC (%TMIC), with target exposure of 50%. Sulbactam-durlobactam killed 3.85 log10 CFU/mL below day 0 burden (B0) with regrowth. Sulbactam-durlobactam plus ceftriaxone killed without regrowth and demonstrated Bliss additivity.{gamma} of bacterial population in >95% of virtual subjects were 2.28 (0.97-4.80) log10 CFU/mL/day for sulbactam-durlobactam-ceftriaxone and 2.91 (1.65-4.93) log10 CFU/mL/day for SDCEO. The optimal sulbactam-durlobactam dose co-administered with ceftriaxone was 2G q8h for creatinine clearance >90 mL/min, 2G q12h for 60-90 mL/min, 1G q12h for [&ge;]30 to <60 mL/min, and 1G q24h for <30 mL/min. ConclusionSulbactam-durlobactam-ceftriaxone achieved the highest microbial kill encountered so far in the HFS-MAB. Sulbactam-durlobactam-ceftriaxone should be tested as the backbone for novel treatment shortening regimens.

BackgroundD-Cycloserine (DCS) is a broad-spectrum antibiotic that is currently FDA-approved to treat tuberculosis (TB) disease and urinary tract infection. Despite numerous reports showing good clinical efficacy, DCS fell out of favor as a UTI treatment because of its propensity to cause side effects. NRX-101, a fixed dose combination of DCS and lurasidone, has been awarded Qualified Infectious Disease Product and Fast Track Designation by the US Food and Drug Administration and is being developed for various CNS indications because of its unique synergistic effect; each component mitigates side effects of the other. MethodsIn this study, we tested NRX-101 against the urinary tract pathogens E. coli, P. aeruginosa, K. pneumoniae, and A. baumannii in Mueller Hinton broth (caMHB) and artificial urine media (AUM). Several strains were multidrug resistant. Test compounds were serially diluted in broth/media. Minimum inhibitory concentration (MIC) was defined as the lowest concentration of test compound at which no bacterial growth was observed. ResultsDCS exhibited antibacterial efficacy against all strains tested while lurasidone did not appreciably affect the antibacterial action of DCS in vitro. In AUM, the MICs ranged from 128 to 512 mcg/ml for both DCS and NRX-101. In caMHB, MICs ranged from 8 to 1024 mcg/ml for NRX-101 and 32 to 512 mcg/ml for DCS alone. ConclusionsOur data confirm that DCS as antibacterial activity against reference and drug-resistant urinary pathogens. Furthermore, lurasidone does not interfere with DCSs anti-microbial action in vitro. These results support the clinical development of NRX-101 as a treatment for complicated urinary tract infection.

Indolcarboxamides are a promising series of anti-tubercular agents which target Mycobacterium tuberculosis MmpL3, the exporter of trehalose monomycolate, a key cell wall component. We determined the kill kinetics of the lead indolcarboxamide NITD-349 and determined that while kill was rapid against low density cultures, bactericidal activity was inoculum-dependent. A combination of NITD-349 with isoniazid (which inhibits mycolate synthesis) had an increased kill rate; this combination prevented the appearance of resistant mutants, even at higher inocula.

The global dissemination of Enterobacterales producing both metallo-{beta}-lactamases (MBLs) and serine {beta}-lactamases (SBLs) represents a critical threat to modern medicine, as no currently marketed antibiotics effectively target MBL-mediated resistance. APC148 is a novel, selective zinc-chelating MBL inhibitor designed to restore {beta}-lactam activity in MBL positive isolates, when used in combination with a broad-spectrum carbapenem. In this study, we evaluated the in vitro efficacy of APC148 in triple combinations with either meropenem-avibactam (APC301) or cefepime-avibactam (APC302) against a diverse global collection (JMI collection) of 176 MBL- and SBL-producing Enterobacterales isolates (including NDM, VIM, and IMP variants). Using broth microdilution, the triple combinations were compared against several newly approved and late-stage pipeline antibiotic products. Both APC301 and APC302 demonstrated superior potency, achieving a MIC90 of 0.12 {micro}g/mL. When applying CLSI breakpoint interpretive criteria for the parent {beta}-lactams, 99.4% of the MBL and SBL-containing isolates were susceptible to APC301, while 97.2% were susceptible to APC302. These results indicate that the addition of a selective MBL inhibitor to an SBL-inhibitor/{beta}-lactam antibiotic effectively bypasses complex co-existing {beta}-lactam resistance mechanisms in multidrug-resistant (MDR) pathogens. Given that MDR Enterobacterales frequently harbor multiple {beta}-lactamase classes simultaneously, these triple combinations constitute a highly promising clinical strategy to address the therapeutic void in MBL-mediated resistance

Cefiderocol is a novel siderophore cephalosporin antibiotic exhibiting activities against carbapenem-resistant Gram-negative bacteria including Pseudomonas aeruginosa and Enterobacteriaceae. Drug efflux pumps are reportedly involved in both intrinsic and acquired drug resistance, although their role in bacterial cefiderocol susceptibility remains poorly understood. In this study, we investigated how drug efflux pumps contribute to bacterial cefiderocol susceptibility using the efflux pump(s)-deficient and overexpressing strains of P. aeruginosa, Escherichia coli, and Salmonella enterica. We observed that the mexAB-oprM-deficient P. aeruginosa mutant displayed increased cefiderocol susceptibility compared to the wild-type strain. The overexpression of mexAB-oprM or mexXY-oprM in the mexAB-oprM-deficient mutant increased the MIC value of cefiderocol. Furthermore, the pump inhibitor phenylalanine-arginine {beta}-naphthylamide increased cefiderocol susceptibility in wild-type P. aeruginosa whereas it did not affect the susceptibility of the mexAB-oprM-deficient mutant. These data indicate that the MexAB-OprM drug efflux system contributes to the intrinsic cefiderocol resistance of P. aeruginosa. In addition, MexXY-OprM partially complemented the function of MexAB-OprM in the cefiderocol susceptibility, when expressed.

The standard pharmacodynamic marker in murine tuberculosis drug studies is colony-forming units (CFU). A faster PCR-based marker of bacterial burden is 16S rRNA. For unclear reasons, treatment reduces CFU more than 16S rRNA. We evaluated this CFU-16S gap and estimated the fraction potentially attributable to slow decay of 16S rRNA from dead Mycobacterium tuberculosis (Mtb) versus transition to a viable but not culturable on solid agar (VBNCSA) population. We quantified the CFU-16S gap during and following treatment in six BALB/c mouse studies and one in vitro study. Applying a two-population ordinary differential equation-based model of Mtb death and 16S rRNA decay, we estimated the fraction of the gap potentially attributable to dead Mtb. Using meta-regression, we estimated the association between CFU or 16S rRNA with relapse. For all regimens, CFU fell more than 16S rRNA, ranging from isoniazid-rifampin-pyrazinamide-ethambutol (CFU decreased 39-times more than 16S rRNA at week 4) to bedaquiline-pretomanid-moxifloxacin-pyrazinamide (CFU decreased >500,000-times more). The two-population model suggested that the fraction of the CFU-16S gap attributable to residual 16S rRNA from dead Mtb is modest and decreases over time. After treatment, 16S rRNA often fell while CFU rose. Four-week CFU change explained most variation in relapse (R{superscript 2}=0.90) while four-week 16S rRNA change did not (R{superscript 2}=0.24). CFU-16S gap is only partially explained by slow decay of residual 16S, suggesting development of a VBNCSA population. However, continued decrease in 16S rRNA after treatment cessation and its limited association with relapse suggests VBNCSA may be a transient rather than persistent state.

The ATTACK clinical trial for treatment of carbapenem-resistant Acinetobacter baumannii-calcoaceticus complex (CRAB) isolates determined treatment with sulbactam-durlobactam to be efficacious and safe. However, other newly introduced {beta}-lactam antibiotics, including the novel cephalosporin cefiderocol, have been compromised upon clinical introduction by a type of antibiotic resistance called heteroresistance, in which only a small subpopulation of total cells exhibit phenotypic resistance. Therefore, we sought to test for sulbactam-durlobactam heteroresistance, as well as whether sulbactam-durlobactam was effective against cefiderocol heteroresistant CRAB isolates. We did not observe heteroresistance (or conventional resistance) to sulbactam-durlobactam among the 107 carbapenem-resistant Acinetobacter isolates tested, consistent with the efficacy of this new antibiotic in the ATTACK trial. Further, sulbactam-durlobactam was active against cefiderocol heteroresistant CRAB, highlighting that this antibiotic may be prioritized in relation to cefiderocol in treating CRAB infections.

KBP-7072 is a novel aminomethylcycline with broad-spectrum activity against Gram-positive and Gram-negative multidrug resistant bacterial isolates and strains. Antibacterial activity and the PK/PD relationship were assessed using in vivo infection models. Six to 8-week-old female CD-1 mice were randomized to oral KBP-7072, minocycline and vehicle in a Klebsiella pneumoniae murine model, and KBP-7072, linezolid and vehicle for a Streptococcus pneumoniae murine model. Each animal was inoculated with K. pneumoniae or S. pneumoniae placed on the tip of the nares. KBP-7072 and antibiotics were started 3 hours post inoculation and continued for 3 days for K. pneumoniae, and were started 18 hours post inoculation and continued for 3 days for S. pneumoniae. Animals were euthanized at 0 (control group), 24, 48 or 72 hours post final dose. In vivo efficacy and PK/PD parameters were determined in Staphylococcus aureus isolate (6424MRSA-363), K. pneumoniae isolate (6680kpn-522), and E. coli isolate (6691eco-558) murine thigh infection models. In vivo efficacy and PK/PD parameters (fAUC/MIC, fCmax/MIC and %T>MICfree) were calculated. Respiratory infection occurred in all inoculated mice. KBP-7072 produced a significant (p<0.05 to <0.001) dose-dependent decrease in colony forming units (CFUs) at all doses and a dose-dependent increase in survival rate (p<0.001 vs. vehicle). The median survival in all KBP-7072-treated groups was significantly greater vs. comparators (p<0.001). These results demonstrate potent in vivo efficacy for KBP-7072 and determined that the AUC/MIC parameter was optimal for assessing bacteriostatic and bactericidal effects of KBP-7072.

BackgroundThe nitroimidazoles delamanid and pretomanid play an important role in contemporary tuberculosis treatment. It is unclear whether delamanid and pretomanid have meaningfully different activity since both reduce Mycobacterium tuberculosis colony forming units (CFU) similarly in animal models. The RS ratio is a pharmacodynamic marker of ongoing rRNA synthesis that has been associated with treatment-shortening (i.e., sterilizing) activity. MethodsUsing Mycobacterium tuberculosis Erdman, we conducted dose-ranging studies in aerobic axenic culture and in the conventional BALB/c mouse high-dose aerosol infection model to compare bactericidal and RS ratio activity of delamanid and pretomanid. ResultsIn vitro concentration-response curves showed that delamanid and pretomanid had similar RS ratio effect at maximal concentration but pretomanid was more potent, achieving 90% of the maximal effect (RS-EC90) at a lower concentration (390 ng/mL) than delamanid (810 ng/mL). In mice, delamanid and pretomanid had similar effects on CFU. Human-equivalent doses of delamanid (6 mg/kg) and pretomanid (50 mg/kg) resulted in plasma Cmax concentrations well below (210 ng/mL) and well above (7,825 ng/mL) the RS-EC90, respectively. Delamanid displayed no discernable RS ratio response, even at 16-times the human-equivalent dose. Higher pretomanid doses resulted in significantly greater RS ratio effects. ConclusionsWe found that delamanid and pretomanid have similar bactericidal activity but pretomanid has superior RS ratio activity. Meaningful differences between drugs within the same class were not captured by conventional CFU-based pharmacodynamics, supporting the value of measuring orthogonal drug effects such as the RS ratio. LAY SUMMARYAntibiotics in the nitroimidazole class are used in treatment of drug-resistant tuberculosis. There are two approved nitroimidazole antibiotics: delamanid and pretomanid. For decades, it has been unclear whether delamanid and pretomanid are interchangeable or whether they affect the bacterium M. tuberculosis differently. Most studies of the effect of antibiotics count the number of bacterial colonies that form on a culture plate. "Colony forming units" tell us about change in bacterial burden but does not give information about bacterial health. A new way of thinking about antibiotic effect is the RS ratio. The RS ratio is a test that measures how much ribosomal RNA synthesis is ongoing. Ribosomal RNA synthesis is a "vital sign" of bacterial health and activity. The key finding of this study is that although the two nitroimdazole antibiotics look the same in terms of their effect on bacterial burden, they have different effects on bacterial health. This information deepens understanding of differences between two clinically important antibiotics. It also shows that antibiotics testing should consider not only bacterial burden but also new tests of bacterial health.

Bacteria can adapt to stressful conditions through mutations affecting the RNA polymerase core subunits that lead to beneficial changes in transcription. In response to selection with rifampicin (RIF), mutations arise in the RIF resistance determining region (RRDR) of rpoB that reduce antibiotic binding. These changes can also alter transcription and thereby have pleiotropic effects on bacterial fitness. Here, we studied the evolution of resistance in Bacillus subtilis to the synergistic combination of RIF and the {beta}-lactam cefuroxime (CEF). Two independent evolution experiments led to the recovery of a single rpoB allele (S487L) that was able to confer resistance to RIF and CEF through a single mutation. Two other common RRDR mutations made the cells 32x more sensitive to CEF (H482Y) or led to only modest CEF resistance (Q469R). The diverse effects of these three mutations on CEF resistance are correlated with differences in the expression of peptidoglycan (PG) synthesis genes and in the levels of two metabolites crucial in regulating PG synthesis, glucosamine-6-phosphate (GlcN-6-P) and UDP-N-acetylglucosamine (UDP-GlcNAc). We conclude that RRDR mutations can have widely varying effects on pathways important for cell wall biosynthesis, and this may restrict the spectrum of mutations that arise during combination therapy. ImportanceRifampicin (RIF) is one of the most valued drugs in the treatment of tuberculosis. TB treatment relies on a combination therapy, and for multidrug resistant strains may include {beta}-lactams. Mutations in rpoB present a common route for emergence of resistance to RIF. In this study, using B. subtilis as a model, we evaluate the emergence of resistance for the synergistic combination of RIF and the {beta}-lactam cefuroxime (CEF). One clinically-relevant rpoB mutation conferred resistance to both RIF and CEF, whereas two others increased CEF sensitivity. We were able to link these phenotypes to accumulation of specific PG precursors. Mainly, UDP-GlcNAc through its GlmR mediated influence on GlmS activity has a strong impact on CEF resistance. Since these mutations are clinically relevant, these effects on CEF sensitivity may help refine the use of {beta}-lactams in TB therapy.

The siderophore-cephalosporin cefiderocol(FDC) presents a promising treatment option for carbapenem-resistant (CR) P. aeruginosa (PA). FDC circumvents traditional porin and efflux mediated resistance by utilizing TonB-dependent receptors (TBDRs) to access the periplasmic space. Emerging FDC resistance has been associated with loss of function mutations within TBDR genes or the regulatory genes controlling TBDR expression. Further, difficulties with antimicrobial susceptibility testing (AST) and unexpected negative clinical treatment outcomes have prompted concerns for heteroresistance, where a single lineage isolate contains resistant subpopulations not detectable by standard AST. This study aimed to evaluate the prevalence of TBDR mutations among clinical isolates of P. aeruginosa and the phenotypic effect on FDC susceptibility and heteroresistance. We evaluated the sequence of pirR, pirS, pirA, piuA or piuD from 498 unique isolates collected before the introduction of FDC from 4 clinical sites in Portland, OR (1), Houston, TX (2), and Santiago, Chile (1). At some clinical sites, TBDR mutations were seen in up to 25% of isolates, and insertion, deletion, or frameshift mutations were predicted to impair protein function were seen in 3% of all isolates (n=15). Using population analysis profile testing, we found that P. aeruginosa with major TBDR mutations were enriched for a heteroresistant phenotype and undergo a shift in the susceptibility distribution of the population as compared to susceptible strains with wild type TBDR genes. Our results indicate that mutations in TBDR genes predate the clinical introduction of FDC, and these mutations may predispose to the emergence of FDC resistance.

Piperacillin/tazobactam (TZP) is a widely used penicillin/{beta}-lactamase inhibitor combination with broad antimicrobial activity. Recently, Escherichia coli strains resistant to TZP but susceptible to third generation cephalosporins (TZP-R/3GC-S isolates) have been increasingly identified. Here, we investigated resistance mechanisms underlying the TZP-R/3GC-S phenotype in clinical E. coli isolates. A total of 29 TZP-R/3GC-S E. coli isolates were retrieved from urinary cultures and subjected to whole genome sequencing. Resistance to TZP was confirmed by minimum inhibitory concentration determination. {beta}-lactamase activity in the presence and absence of tazobactam was determined to identify hyperproduction of {beta}-lactamase and assess susceptibility to tazobactam inhibition. A previously unrecognized {beta}-lactamase was identified and cloned to determine its resistance profile. Four different resistance mechanisms underlying the TZP-R/3-GC phenotype were identified: 1) In 18 out of 29 isolates (62%) {beta}-lactamase production was increased and in 16 of these either strong alternative promoters or increased gene copy numbers of blaTEM-1 or blaSHV-1 were identified, 2) seven isolates (24%) produced blaOXA-1, 3) three isolates (10%) produced inhibitor-resistant TEM-{beta}-lactamases, and 4) a single isolate (3%) harboured a blaCTX-M gene as the only {beta}-lactamase present. This {beta}-lactamase, CTX-M-255, only differs from CTX-M-27 by a G239S amino acid substitution. In contrast to CTX-M-27, CTX-M-255 conferred resistance to penicillin/{beta}-lactamase inhibitor combinations but remained susceptible to cephalosporins. In conclusion, hyperproduction of blaTEM was the most prevalent mechanism of TZP-resistance underlying the TZP-R/3GC-S phenotype followed by production of blaOXA-1 and inhibitor-resistant TEM-{beta}-lactamases. Furthermore, we identified a previously unrecognized CTX-M-{beta}-lactamase, CTX-M-255 that was resistant to {beta}-lactamase inhibitors.

Mycobacterium abscessus (Mab) affects patients with immunosuppression, Cystic Fibrosis (CF), or underlying structural lung diseases. Additionally, Mab poses clinical challenges due to its resistance to multiple antibiotics. Herein, we investigated the synergistic effect of dual {beta}-lactams [sulopenem and cefuroxime (CXM)] or the combination of sulopenem and CXM with a {beta}-lactamase inhibitors [BLI; avibactam (AVI) or durlobactam (DUR)]. The sulopenem-CXM combination yielded low minimum inhibitory concentration MIC values for 54 clinical Mab isolates and ATCC19977 (MIC50 and MIC90 [&le;] 0.25 g/mL). Similar synergistic effects were observed in time-kill studies conducted at concentrations achievable in clinical settings. Sulopenem-CXM outperformed monotherapy, yielding [~]1.5 Log10 CFU/mL reduction during 10 days. Addition of BLIs enhanced this antibacterial effect, resulting in additional reduction of CFUs ([~]3 Log10 for sulopenem-CXM and AVI and [~]4 Log10 for sulopenem-DUR). Exploration of the potential mechanisms of the synergy focused on their interactions with L,D-transpeptidases (LDTs; LDTMab1-LDTMab4), Penicillin-Binding-Protein B (PBP-B), and D,D-Carboxypeptidase (DDC). Acyl complexes identified via mass spectrometry analysis, demonstrated the binding of sulopenem with LdtMab2-LdtMab4, DDC, and PBP B, and CXM with LdtMab2 and PBP-B. Molecular docking suggested formation of a covalent adduct between sulopenem and LdtMab2 after the nucleophilic attack of the cysteine residue at the {beta}-lactam carbonyl carbon, leading to the cleavage of the {beta}-lactam ring, and the establishment of a thioester bond linking the LdtMab2 with sulopenem. In conclusion, we demonstrated the biochemical basis of the synergy of sulopenem-CXM with or without BLI. These findings potentially broaden selection of oral therapeutic agents to combat Mab.

Toxoplasmosis is a common protozoan infection that can have severe outcomes in the immunocompromised and during pregnancy, but treatment options are limited. Recently, nucleotide metabolism has received much attention as a target for new antiprotozoal agents and here we focus on pyrimidine salvage by Toxoplasma gondii as a drug target. Whereas uptake of [3H]-cytidine and particularly [3H]-thymidine was at most marginal, [3H]-uracil and [3H]-uridine were readily taken up. Kinetic analysis of uridine uptake was consistent with a single transporter with a Km of 3.3 {+/-} 0.8 {micro}M, which was inhibited by uracil with high affinity (Ki = 1.15 {+/-} 0.07 {micro}M) but not by thymidine or 5-methyluridine, showing that the 5-Me group is incompatible with uptake by T. gondii. Conversely, [3H]-uracil transport displayed a Km of 2.05 {+/-} 0.40 {micro}M, not significantly different from the uracil Ki on uridine transport, and was inhibited by uridine with a Ki 2.44 {+/-} 0.59 {micro}M, also not significantly different from the experimental uridine Km. The reciprocal, complete inhibition, displaying Hill slopes of approximately [~]1, strongly suggest that uridine and uracil share a single transporter with similarly high affinity for both, and we designate it uridine/uracil transporter 1 (TgUUT1). While TgUUT1 excludes 5-methyl substitutions, the smaller 5F substitution was tolerated as 5F-uracil inhibited uptake of [3H]-uracil with a Ki of 6.80 {+/-} 2.12 {micro}M (P > 0.05 compared to uracil Km). Indeed, we found that 5F-Uridine, 5F-uracil and 5F,2-deoxyuridine were all potent antimetabolites against T. gondii with EC50 values well below that of the current first line treatment, sulfadiazine. In vivo evaluation also showed that 5F-uracil and 5F,2-deoxyuridine were similarly effective as sulfadiazine against acute toxoplasmosis. Our preliminary conclusion is that TgUUT1 mediates potential new anti-toxoplasmosis drugs with activity superior to the current treatment.

BackgroundLinezolid (LZD), while effective against Mycobacterium abscessus (MAB), can cause myelosuppression and peripheral neuropathy. Contezolid (CZD) shares a similar antimicrobial profile with improved safety, but biodistribution data remain limited. This study evaluated CZDs biodistribution in MAB-infected mice and humans and its therapeutic potential across infection sites. MethodsMICs of CZD and LZD against 32 clinical MAB isolates and three reference strains were determined. In MAB-infected mice, drug concentrations were quantified in plasma and pulmonary and cerebral tissues at 2, 4, and 8 hours post-administration. In MAB-infected patients, CZD concentrations in bone, plasma, and cerebrospinal fluid were measured at multiple time points and MAB counts in sputum cultures were assessed daily over 14 days. ResultsRespective MIC50 and MIC90 values were 8 and 32 {micro}g/mL (LZD) and 16 and 32 {micro}g/mL (CZD). Pharmacokinetic CZD and LZD level comparisons revealed peak CZD plasma levels within 2 hours, higher systemic CZD levels, comparable pulmonary tissue concentrations, and slightly lower CZD cerebral tissue penetration. In patients, CZD levels in ankle joint effusion samples reached 2.0885 {micro}g/mL at 6 hours, peaking in the posterior malleolus. Plasma CZD concentrations peaked at 8.2349 {micro}g/mL at 3 hours and dropped to 6.1065 {micro}g/mL by 6 hours, while CSF levels were 0.9295 and 0.792 {micro}g/mL at 3 and 6 hours, respectively. Sputum bacterial burden decreased rapidly within 24 hours of CZD treatment, with near-complete clearance by day 4. ConclusionCZD and LZD exhibit comparable tissue distribution but different site-specific penetration, supporting their potential for treating diverse MAB infections.

The rise of multidrug-resistant (MDR) bacteria, particularly carbapenem-resistant Enterobacterales (CRE), poses a significant threat to public health. Infections caused by CRE, such as Escherichia coli and Klebsiella pneumoniae, are associated with high rates of antibiotic treatment failure. {beta}-lactam antibiotics, like meropenem, remain crucial in treating these infections, but their efficacy is undermined by {beta}-lactamase production. This study investigates the potential of APC24-7, a novel broad-spectrum {beta}-lactamase inhibitor (BLi) with dual activity, to restore antimicrobial activity of meropenem against CRE clinical isolates. The in-vitro analysis of a diverse panel of clinically relevant E. coli and K. pneumoniae isolates expressing both serine- and metallo-{beta}-lactamases demonstrated that APC24-7 effectively restored meropenem activity by reducing the minimum inhibitory concentrations (MICs) to below breakpoint. Time-kill assays confirmed that the combination therapy showed dose-dependent bacterial killing, with significant potentiation of meropenem activity against isolates expressing both serine- and metallo-{beta}-lactamases. In-vivo efficacy evaluation in a murine thigh infection model further confirmed APC24-7s potential to restore meropenem efficacy against meropenem resistant strains. These findings suggest that APC24-7offers a promising strategy to combat infections caused by {beta}-lactamase-producing Enterobacterales.

Cefiderocol exhibits excellent in vitro activity against Pseudomonas aeruginosa; however, resistance can emerge. We investigated the molecular mechanisms underlying cefiderocol resistance (MIC >2 mg/L) in 103 clinical strains collected from 61 hospitals (2021-2024). MICs ranged from 4 to >128 mg/L, with 39.8% of strains showing MICs >8 mg/L. Although 37.8% were classified as difficult-to-treat resistant (DTR), acquired {beta}-lactamases were detected in 72.8% of strains, including carbapenemases (39.8%), mainly NDM-1 (29.1%), and Extended Spectrum {beta}-Lactamases (ESBLs) (38.8%). Cloning of 11 {beta}-lactamases into pUCP24, including the acquired cephalosporinase PAC-1 and ESBLs (VEB-1, and VEB-9), resulted in marked increases in cefiderocol MICs (up to 128-fold). Introduction of 6 mutations in the PDC enzyme into a PAO1{Delta}blaPDC-1 background increased MICs up to 4 mg/L and conferred cross-resistance to ceftolozane/tazobactam, notably F121L, G157D, T70I, and E219K. Alterations in siderophore transporters or regulators were identified in 38.8% of strains, most frequently a PirR frameshift (R132fs), consistent with PirR inactivation, which was confirmed in the PAO1 strain to contribute to cefiderocol resistance. Overall, cefiderocol resistance in clinical strains is multifactorial, mainly involving acquired {beta}-lactamases (ESBLs, carbapenemases) and impaired siderophore uptake (PiuA/PiuD, PirA, PiuC), leading to high-level resistance (>8 mg/L). The polyclonal distribution and diversity of mechanisms highlight the need for routine susceptibility testing and surveillance. Detection of NDM producers is critical, as cefiderocol should be used with caution in this context.

Pyrazinamide (PZA) is one of the most important drugs used in combined antituberculous therapy. After the drug enters Mycobacterium tuberculosis it is hydrolyzed by pyrazinamidase (PZAse) to the bactericidal molecule pyrazinoic acid (POA). Ribosomal protein S1 (RpsA) was recently identified as a possible target of PZA based on its binding activity to POA and capacity to inhibit trans-translation. However, its role is not completely understood. It has been proposed that Mycobacterium smegmatis RpsA is not capable of binding POA, unlike M. tuberculosis RpsA. This may be due to the different amino acid sequence in the carboxy-terminal region of the two molecules: in M. smegmatis RpsA it is much closer to the sites that may interact with POA than in M. tuberculosis RpsA. These differences could be contributing, along with the presence of highly active POA efflux, to the natural resistance to PZA in M. smegmatis. To further understand the mechanisms of action of PZA and the role of RpsA in PZA susceptibility, we evaluated the effect of complementing M. tuberculosis RpsA expression in M. smegmatis using pNIT mycobacterial non-integrative expression vector and then performed a PZA susceptibility test determining the minimum inhibitory concentration (MIC) of PZA. It was expected that chimeric ribosomes comprising M. tuberculosis RpsA may be present and may affect PZA susceptibility. Our results showed a reduction in PZA MIC in M. smegmatis complemented with overexpressed M. tuberculosis RpsA compared to non-overexpressed M. smegmatis (468 {micro}g/mL and >7500 {micro}g/mL respectively).