Pyrazinamide (PZA) is a key component of chemotherapy for the treatment of drug-susceptible tuberculosis (TB) and is likely to continue to be included in new drug combinations. Potentiation of PZA could be used to reduce the emergence of resistance, shorten treatment times, and lead to a reduction in the quantity of PZA consumed by patients, thereby reducing the toxic effects. Acidified medium is required for the activity of PZA against Mycobacterium tuberculosis. In vitro assessments of pyrazinamide activity are often avoided because of the lack of standardization, which has led to a lack of effective in vitro tools for assessing and/or enhancing PZA activity.We have developed and optimized a novel, robust, and reproducible, microtiter plate assay, that centers around acidity levels that are low enough for PZA activity. The assay can be applied to the evaluation of novel compounds for the identification of potentiators that enhance PZA activity. In this assay, potentiation of PZA is demonstrated to be statistically significant with the addition of rifampicin (RIF), which can, therefore, be used as a positive control. Conversely, norfloxacin demonstrates no potentiating activity with PZA and can be used as a negative control. The method, and the associated considerations, described here, can be adapted in the search for potentiators of other antimicrobials.

Although the incidence of infections in orthopedic surgeries, including periprosthetic surgeries, remains low at approximately 1-2%, the number of surgeries and the incidence of drug-resistant bacteria is increasing. The cost and morbidity associated with revision surgeries are huge. More effective drug combinations and delivery methods are urgently needed. In this paper, three anti-infective drugs (vancomycin, rifampicin, and silver sulfadiazine) have been jointly and effectively electrospun in thin (0.1 mm) flexible nanofiber mats of either poly (methyl methacrylate) (PMMA) or poly (lactic-co-glycolic acid) (PLGA). The inclusion of poly (ethylene glycol) (PEG) enabled optimal drug release with a reduced water contact angle for wetting. The controlled release of these three agents from 20% PEG (w/w to polymer)-blended PMMA or PLGA nanofiber mats may allow for the prophylactical prevention of implant-related infections or provide methods to treat orthopedic infections at the time of revision surgeries. These combinations of drugs provide excellent additive or synergistic antibiotic action against a broader spectrum of bacteria than each drug alone.

Multidrug-resistant tuberculosis (TB) (MDR-TB), or TB that is simultaneously resistant to both isoniazid (INH) and rifampicin (RIF), is a barrier to successful TB control and treatment. Stratified data on MDR-TB, particularly in the high-burden western Kenya region, remain unknown. This data is important to monitor the efficacy of TB control and treatment efforts. Herein, we determined the molecular epidemiology of drug-resistant TB and associated risk factors in western Kenya. This was a non-experimental, population-based, cross-sectional study conducted between January and August 2018. Morning sputum samples of individuals suspected of pulmonary TB were collected, processed, and screened for Mycobacterium tuberculosis (Mtb) and drug resistance using line probe assay (LPA) and Mycobacterium growth indicator tubes (MGIT) culture. MGIT-positive samples were cultured on brain heart infusion (BHII) agar media, and the presence of Mtb was validated using Immunochromatographic assay (ICA). Drug sensitivity was performed on MGIT and ICA-positive but BHI-negative samples. Statistical significance was set at P < 0.05. Of the 622 Mtb isolates, 536 (86.2%) were susceptible to RIF and INH. The rest, 86 (13.83%), were resistant to either drugs or both. A two-sample proportional equality test revealed that the MDR-TB prevalence in western Kenya (5%) did not vary significantly from the global MDR-TB estimate (3.9%) (P = 0.196). Men comprised the majority of susceptible and resistant TB (75.9% and 77.4%%, respectively). Also, compared with healthy individuals, the prevalence of HIV was significantly higher in MDR-TB patients (35.9% vs 5.6%). Finally, TB prevalence was highest in individuals aged 25-44 years, who accounted for 58.4% of the total TB cases. Evidently, the prevalence of MDRTB in western Kenya is high. Particular attention should be paid to men, young adults, and those with HIV, who bear the greatest burden of resistant TB. Overall, there is a need to refine TB control and treatment programs in the region to yield better outcomes.

Methicillin-resistant Staphylococcus aureus (MRSA) bacteremia is a serious clinical challenge with high mortality rates. Antibiotic combination therapy is currently used in cases of persistent infection; however, the limited development of new antibiotics will likely increase the need for combination therapy, and better methods are needed for identifying effective combinations for treating persistent bacteremia. To identify pairwise combinations with the most consistent potential for benefit compared to monotherapy with a primary anti-MRSA agent, we conducted a systematic study with an in vitro high-throughput methodology. We tested daptomycin and vancomycin each in combination with gentamicin, rifampicin, cefazolin, and oxacillin, and ceftaroline with daptomycin, gentamicin, and rifampicin. Combining cefazolin with daptomycin lowered the daptomycin concentration required to reach 95% growth inhibition (IC95) for all isolates tested and lowered daptomycin IC95 below the sensitivity breakpoint for five out of six isolates that had daptomycin minimum inhibitory concentrations at or above the sensitivity breakpoint. Similarly, vancomycin IC95s were decreased when vancomycin was combined with cefazolin for 86.7% of the isolates tested. This was a higher percentage than was achieved by adding any other secondary antibiotic to vancomycin. Adding rifampicin to daptomycin or vancomycin did not always reduce IC95s and failed to produce synergistic interaction in any of the isolates tested; the addition of rifampicin to ceftaroline was frequently synergistic and always lowered the amount of ceftaroline required to reach the IC95. These analyses rationalize further in vivo evaluation of three drug pairs for MRSA bacteremia: daptomycin+cefazolin, vancomycin+cefazolin, and ceftaroline+rifampicin.IMPORTANCEBloodstream infections caused by methicillin-resistant Staphylococcus aureus (MRSA) have a high mortality rate despite the availability of vancomycin, daptomycin, and newer antibiotics including ceftaroline. With the slow output of the antibiotic pipeline and the serious clinical challenge posed by persistent MRSA infections, better strategies for utilizing combination therapy are becoming increasingly necessary. We demonstrated the value of a systematic high-throughput approach, adapted from prior work testing antibiotic combinations against tuberculosis and other mycobacteria, by using this approach to test antibiotic pairs against a panel of MRSA isolates with diverse patterns of antibiotic susceptibility. We identified three antibiotic pairs-daptomycin+cefazolin, vancomycin+cefazolin, and ceftaroline+rifampicin-where the addition of the second antibiotic improved the potency of the first antibiotic across all or most isolates tested. Our results indicate that these pairs warrant further evaluation in the clinical setting.

Staphylococcus aureus is a major causative pathogen of osteomyelitis. Intracellular infections of resident bone cells including osteocytes can persist despite gold-standard clinical intervention. The mechanisms by which intracellular S. aureus evades antibiotic therapy are unknown. In this study, we utilised an in vitro S. aureus infection model of human osteocytes to investigate whether antibiotic-mediated dysregulation of autophagy contributes to this phenomenon. Infected or non-infected osteocyte-like cells were exposed to combinations of rifampicin, vancomycin, and modulators of autophagy. Intracellular bacterial growth characteristics were assessed using colony-forming unit (CFU) analysis, viable bacterial DNA abundance, and the rate of escape into antibiotic-free medium, together with measures of autophagic flux. Rifampicin, alone or in combination with vancomycin, caused a rapid decrease in the culturability of intracellular bacteria, concomitant with stable or increased absolute bacterial DNA levels. Both antibiotics significantly inhibited autophagic flux. However, modulation of autophagic flux did not affect viable bacterial DNA levels. In summary, autophagy was shown to be a factor in the host-pathogen relationship in this model, as its modulation affected the growth state of intracellular S. aureus with respect to both their culturability and propensity to escape the intracellular niche. While rifampicin and vancomycin treatments moderately suppressed autophagic flux acutely, this did not explain the paradoxical response of antibiotic treatment in decreasing S. aureus culturability whilst failing to clear bacterial DNA and hence intracellular bacterial load. Thus, off-target effects of rifampicin and vancomycin on autophagic flux in osteocyte-like cells could not explain the persistent S. aureus infection in these cells.

OBJECTIVE: We aimed to assess the frequency, management, and burden of enterococcal-related vascular graft infection.
METHODS: From 2008 to 2021, data regarding all episodes of vascular graft infections initially managed or secondarily referred to our referral center were prospectively collected. We described the history and management of the infection, depending on the type of prosthesis used.
RESULTS: The frequency of enterococcal-related vascular graft infections was 29/249 (12 %). Most of them were early infections (22/29, 76 %). Infections were polymicrobial (26/29, 90 %), mostly associated with Enterobacterales. Among patients with positive blood cultures, 7/8 (88 %) involved enterococci. Patients with enterococcal-related vascular graft infections were mainly (22/29, 76 %) treated with an association of antibiotics. Mortality and relapse occurred in 28 % and 7 % respectively of the cases.
CONCLUSIONS: Enterococcal-related vascular graft infections occurred in patients with comorbidities, during the early period following surgery and were more frequent in cases of intra-cavitary prosthesis. Their potential virulence needs to be considered, especially in polymicrobial infections.

Folliculitis decalvans (FD) is a rare type of inflammatory scalp disorder that leads to scarring alopecia. It is classified as primary neutrophilic cicatricial alopecia. FD presents a challenging scenario in clinical dermatology due to its rarity, resistance to treatment, and potential for scarring alopecia. This inflammatory scalp disorder primarily affects middle-aged adults, predominantly males. While its exact pathogenesis remains uncertain, a deficient host immune response to Staphylococcus aureus infection is hypothesized. Therapeutic interventions for FD pose difficulties, with limited treatment options available A 58-year-old female patient presented with a history of follicular papules that gradually progressed to form clusters of pustules, crusting, and hemorrhagic lesions with tufting of hairs on the crown area of the scalp, and was diagnosed with FD. Considering isotretinoin\'s role in inhibiting abnormal keratinization and inflammation, and rifampicin\'s ability to eradicate S. aureus, the combination of both provides a comprehensive approach to tackling the underlying factors contributing to FD. Despite previous unsuccessful treatments, combination therapy with isotretinoin and rifampicin yielded a remarkable outcome, prompting further exploration of this approach.

This study assessed the presence of the genotoxic impurity 1-methyl-4-nitrosopiperazine (MNP) in 27 batches of rifampicin capsules obtained from 11 manufacturers in China. While they were below the temporary limit of 5 ppm set by the US Food and Drug Administration, the observed levels (0.33-2.36 ppm) exceeded the acceptable threshold of 0.16 ppm. Building upon preliminary findings and degradation experiments, we concluded that MNP is a by-product of the oxidative degradation of rifampicin or is introduced via oxidation or nitrosation during the synthesis process involving 1-methyl-4-aminopiperazine. The pathways of MNP formation were confirmed in this study. Furthermore, we observed that the addition of antioxidants, sealed storage, and selection of dominant crystal forms can aid in controlling MNP levels.

Persister cells are transiently tolerant to antibiotics and are associated with recalcitrant chronic infections due to recolonization of host cells after antibiotic removal. Brucella spp. are facultative pathogens that establish intracellular infection cycles in host cells which results in chronic persistent infections. Brucella abortus forms multi-drug persister cells which are promoted by the (p)ppGpp synthetase Rsh during rifampicin exposure. Here, we confirmed that Rsh promoted persister cells formation in B. abortus stationary phase treated with rifampicin and enrofloxacin. Deletion of the gene for Rsh decreased persister cells level in the presence of these drugs in different growth phases. However, persister cells formation by deletion strain varied in different growth phases in the presence of other antibiotics. Rsh also was involved in persister cells formation during rifampicin treatment under certain stress conditions, including acidic conditions, exposure to PBS, and heat stress. Moreover, Rsh impacted persister cell levels during rifampicin or enrofloxacin treatment in RAW264.7 macrophages. Certain typeIItoxin-antitoxin modules were upregulated under various stress conditions in B. abortus. We established that Rsh positively regulated the type II toxin-antitoxin mbcTA. Moreover, rifampicin-tolerant persister cells formation was elevated and ATP levels were decreased when mbcTA promoter was overexpressed in Rsh deletion background in stationary phase. Our results establish that (p)ppGpp synthetase Rsh plays a key role in B. abortus persistence and may serve as a potent novel target in combination with rifampicin in the development of new therapeutic approaches and prevention strategies to treat chronic infections of Brucella.

BACKGROUND: The widespread use and abuse of antibiotics has resulted in the pollution of water sources with antibiotic residues, posing a threat to human health, the environment, and the economy. Therefore, a highly sensitive and selective method is required for their detection in water samples. Herein, advanced ultrasensitive electrochemical sensor platform was developed by integrating gold-silver alloy nanocoral clusters (Au-Ag-ANCCs) with functionalized multi-walled carbon nanotube-carbon paste electrode (f-MWCNT-CPE) and choline chloride (ChCl) nanocomposites for simultaneously determining the residues of antimicrobial drugs, rifampicin (RAMP) and norfloxacin (NFX), in water samples.
RESULTS: The developed sensor (Au-Ag-ANCCs/f-MWCNTs-CPE/ChCl) was extensively characterized using several analytical (UV-Vis, FT-IR, XRD, SEM, and EDX) and electrochemical (EIS, CV, and SWV) techniques. It exhibited outstanding performance in a wide linear range, from 14 pM to 115 μM for RAMP, and from 0.9 nM to 200 μM for NFX, with a limit of detection (LOD, 3σ/m, S/N = 3, n = 5) and a limit of quantification (LOQ, 10σ/m, S/N = 3, n = 5) values of 2.7 pM and 8.85 pM for RAMP, and 0.14 nM and 0.47 nM for NFX, respectively. The sensor also exhibited exceptional reproducibility, stability, and resistance to interference.
CONCLUSIONS: The developed sensor was effectively utilized to determine RAMP and NFX residues in hospital wastewater, river, and tap water samples, yielding recoveries within the range of 96.8-103 % and relative standard deviations below 5 %. Generally, the proposed sensor demonstrated remarkable performance in detecting the target analytes, making it an ideal tool and the first of its kind for addressing global antibiotic residue pollutants in water sources.