Analysis of unsuccessful tests and the effect of prolonged clinical sample preprocessing in the GeneXpert MTB/RIF assay.

BACKGROUND: The GeneXpert MTB/RIF (Xpert) assay is a widely used technology for detecting Mycobacterium tuberculosis (MTB) in clinical samples. However, the study on the failure of the Xpert assay during routine implementation and its potential solutions is limited. METHODS: We retrospectively analyzed the records of unsuccessful tests in the Xpert and the GeneXpert MTB/RIF Ultra (Ultra) assays between April 2017 and April 2021 at the Shanghai Public Health Clinical Center. To further investigate the effect of prolonged preprocessing on clinical sputum, an additional 120 sputum samples were collected for Xpert testing after 15 min, 3 h, and 6 h preprocessing. The analysis was performed by SPSS version 19.0 software. RESULTS: A total of 11,314 test records were analyzed, of which 268 (2.37%) had unsuccessful test results. Among these, 221 (1.95%) were reported as "Error", 43 (0.38%) as "Invalid", and 4 (0.04%) as "No result". The most common clinical specimen for Xpert tests was sputum, accounting for 114 (2.17%) unsuccessful tests. The failure rate of urine specimens was lower than that of sputum (OR = 0.12, 95% CI: 0.02-0.88, χ2 = 6.22, p = 0.021). In contrast, the failure rate of stool specimens was approximately twice as high as that of sputum (OR = 1.93, 95% CI: 1.09-3.40, χ2 = 5.35, p = 0.014). In the prolonged preprocessing experiment, 102 cases (85%) yielded consistent results in Xpert tests. Furthermore, 7 cases (5.83%) detected an increase in MTB load, 8 cases (6.67%) detected a decrease in MTB load, and 3 cases (2.5%) yielded incongruent results in MTB and rifampicin resistance detection. CONCLUSIONS: The primary cause of unsuccessful tests in the Xpert assay was reported as "Error". Despite varying failure rates depending on the samples, the Xpert assay can be applied to extrapulmonary samples. For paucibacillary specimens, retesting the remaining preprocessed mixture should be carefully considered.

Analysis of epidemiological characteristics of extrapulmonary tuberculosis from South-Central China.

Objectives: This study aimed to investigate the epidemiological and drug resistance (DR) characteristics of extrapulmonary tuberculosis (EPTB) in South-Central China. Methods: EPTB inpatients who were culture-positive for Mycobacterium tuberculosis were retrospectively included in a study at a provincial TB hospital in Hunan, a province in South-Central China, from January 2013 to December 2021. Demographic, clinical, and drug susceptibility data were retrieved from TB treatment records. Descriptive statistical methods and a Chi-squared test were used to analyze the epidemiological and DR characteristics of EPTB patients. A logistic regression model was used to explore the risk factors of rifampicin-resistant/multidrug-resistant (RR/MDR)-EPTB. Results: A total of 1,324 cases were included. The majority of EPTB patients were in the age range of 20-29 years, were predominantly men (male-to-female ratio: 2.03), and were farmers (65.63%). Most EPTB cases were found in 2013 and 2017 from 2013 to 2021. The most prevalent subtypes of EPTB were lymphatic TB (29.83%, 395/1,324), multiple EPTB (20.85%, 276/1,324), and musculoskeletal TB (14.65%, 194/1,324). Musculoskeletal TB and genitourinary TB predominantly presented as exclusive EPTB forms, while lymphatic TB and pharyngeal/laryngeal TB often co-occurred with pulmonary TB (PTB). Drug susceptibility testing results showed that total DR rates (resistance to any of RFP, isoniazid [INH], streptomycin [STR], and/or ethambutol [EMB]) and RR/MDR rates in EPTB were 25.23% and 12.39%, respectively. Musculoskeletal TB exhibited the highest rates of total DR (31.40%), INH resistance (28.90%), STR resistance (20.10%), EMB resistance (6.20%), MDR (13.90%), and poly-DR (6.70%). The multivariable logistic regression model showed that patients aged from 20 to 59 years (compared to those aged 10 years), workers (compared to retirees), and EPTB patients from the south and west of Hunan (compared to those from the east of Hunan) were at an increased risk of developing RR/MDR EPTB (all OR values > 1). Conclusion: Our study provided a detailed account of the epidemiological and DR characteristics of EPTB in Hunan province, China. The significant DR rates, particularly in musculoskeletal TB cases, highlight the need for timely diagnosis, effective drug susceptibility testing, and the development of more effective treatment regimens for EPTB, especially targeting musculoskeletal TB treatments.

Assembly-Induced Membrane Selectivity of Artificial Model Peptides through Entropy-Enthalpy Competition.

Peptide design and drug development offer a promising solution for combating serious diseases or infections. In this study, using an AI-human negotiation approach, we have designed a class of minimal model peptides against tuberculosis (TB), among which K7W6 exhibits potent efficacy attributed to its assembly-induced function. Comprising lysine and tryptophan with an amphiphilic α-helical structure, the K7W6 sequence exhibits robust activity against various infectious bacteria causing TB (including clinically isolated and drug-resistant strains) both in vitro and in vivo. Moreover, it synergistically enhances the effectiveness of the first-line antibiotic rifampicin while displaying low potential for inducing drug resistance and minimal toxicity toward mammalian cells. Biophysical experiments and simulations elucidate that K7W6's exceptional performance can be ascribed to its highly selective and efficient membrane permeabilization activity induced by its distinctive self-assembly behavior. Additionally, these assemblies regulate the interplay between enthalpy and entropy during K7W6-membrane interaction, leading to the peptide's two-step mechanism of membrane interaction. These findings provide valuable insights into rational design principles for developing advanced peptide-based drugs while uncovering the functional role played by assembly.

EasyNAT MTC Assay: a Rapid Method to Detect Mycobacterium Tuberculosis in Respiratory Specimens.

BACKGROUND: This study aimed to effectively evaluate the diagnostic performance of the EasyNAT Mycobacterium tuberculosis complex (MTC) assay for tuberculosis (TB) detection from sputum. METHODS: The retrospectively analyzed data was collected from September 1, 2021, to November 1, 2023, in our hospital. RESULTS: Forty EasyNAT-positive sputum specimens were simultaneously detected using the GeneXpert MTB/ rifampicin (RIF) assay. The concordance rate between the EasyNAT and GeneXpert MTB/RIF assays was 100%. CONCLUSIONS: Because of the complexity of detecting RIF resistance data information, the rapid EasyNAT system used in conjunction with GeneXpert might be a better choice for the detection of TB in hospitals.

Multi-centric evaluation of Truenat MTB and MTB-RIF Dx assays for diagnosis of extrapulmonary tuberculosis.

Extra-pulmonary TB (EPTB) is difficult to diagnose due to paucibacillary nature of disease. Current study evaluated accuracy of Truenat MTB and MTB-Rif Dx (TN), for detection of Mycobacterium tuberculosis and resistance to rifampicin. Samples were collected from 2103 treatment naive adults with presumptive EPTB, and tested by smear microscopy, liquid culture (LC) (MGIT-960) and GeneXpert MTB/RIF (GX) (Microbiological Reference Standards, MRS). TN results were compared to MRS and Composite Reference Standards (CRS, Microbiology, histopathology, radiology, clinical features prompting decision to treat, response to treatment). CRS grouped patients into 551 confirmed, 1096 unconfirmed, and 409 as unlikely TB. TN sensitivity and specificity was 73.7% and 90.4% against GX. Against LC, Overall sensitivity of GX was 67.6%, while that of TN was 62.3%. Highest sensitivity by TN was observed in pus samples (89%) and highest specificity (92%) in CSF samples, similar to GX. TN sensitivity was better in fluid and biopsy samples and slightly inferior for lymph node aspirates compared to GX. TN sensitivity for RIF resistance detection was slightly superior to GX. TN and GX results were further compared to Clinical Reference Standards. TN detected 170 TB patients initiated on treatment missed by GX, while GX detected 113 such patients missed by TN. Of 124 samples with RIF resistance discordance between GX and TN, GX reported 103/124 as sensitive, 3/124 as indeterminate and 18 as resistant (13/18 samples had low/very low DNA load) while TN reported RIF resistance indeterminate in 103/111 low/very low DNA load samples. Due to paucibacillary nature of EPTB samples, culture yield was poor and phenotypic drug susceptibility testing failed to resolve the discordance. The study establishes TN at par with GX and can be utilized for quick and accurate diagnosis of EPTB.

Pharmacokinetics of anti-Mycobacterium avium-intracellulare disease drugs in silkworms.

Pulmonary Mycobacterium avium-intracellulare complex (MAC) disease is a typical non-tuberculous mycobacterial infection. The incidence of pulmonary MAC is increasing worldwide. This study aimed to clarify the pharmacokinetic parameters of anti-pulmonary MAC disease drugs in silkworms. The pharmacokinetic parameters investigated included maximum concentration, area under the concentration-time curve, total clearance, and volume of distribution at steady-state. In addition, protein-binding rates, fat body transferability, and drug-drug interactions were examined. Antibiotic concentrations were measured using a validated high-performance liquid chromatography-mass spectrometry method. Among the antibiotics investigated, amikacin was not eliminated from silkworms during the 48-h observation period. In contrast, dose-proportional pharmacokinetics were observed in silkworms for all antibiotics tested, except for amikacin. Protein-binding rates in hemolymph for clarithromycin, azithromycin, rifampicin, ethambutol, and amikacin were 39.6 ± 3.0%, 39.5 ± 4.3%, 76.3 ± 3.2%, 20.9 ± 4.2%, and 73.1 ± 4.7%, respectively (mean ± standard deviation). The distribution of antibiotics in the fat bodies of silkworms was related to drug lipophilicity. No drug-drug interactions were observed in the silkworms. The pharmacokinetics of these drugs in silkworms differed significantly from those in humans. Therefore, while it is challenging to predict the pharmacokinetics of these drugs in humans based on silkworm data, the silkworm infection model has facilitated a comprehensive assessment of the relationship between antibiotic exposure and efficacy.

Missed rifampicin and isoniazid resistance by commercial molecular assays.

Drug-resistant tuberculosis (TB) has poor outcomes unless resistance is detected early, ideally by commercially available molecular tests. We present a case of occult multidrug-resistant TB where both rifampicin and isoniazid resistance were missed by molecular testing and were only identified by phenotypic testing.

[A New Method for Determination of Rifampicin and Isoniazid Resistance in Mycobacterium tuberculosis complex Isolates: Capillary Tube Method]. / Mycobacterium tuberculosis Kompleks Izolatlarinda Rifampisin ve Izoniyazid Direncinin Belirlenmesi için Yeni Bir Yöntem: Kapiller Tüp Yöntemi.

Tuberculosis continues to be an important public health problem worldwide. Culture methods are still considered the gold standard in the diagnosis of tuberculosis and the determination of drug resistance. The most important limitation of these methods is their long turnaround time. Commercial culture systems developed to shorten the duration are emerging as an economic problem, especially for developing countries. Therefore, cheap, fast, easy to apply and objectively evaluable tests are needed. In this study, in addition to culture-based methods for determining RIF and INH resistance in Mycobacterium tuberculosis complex isolates, it was aimed to develop the capillary tube method to accelerate the evaluation process. The study included 27 RIF-resistant, 36 RIF -sensitive, 30 INH-resistant, and 33 INH-sensitive isolates obtained from the mycobacteriology laboratory culture collection, for which susceptibility testing to firstline drugs were previously performed using the BACTEC MGIT 960 system (BD, USA) and were stored. H37Rv standard strain and an external quality control strain (IDT3) with known RIF and INH resistance were used as quality control isolates in the study. As a new testing method, the capillary tube method for detecting rifampicin and isoniazid resistance was compared to the standard BACTEC MGIT 960 system. In the determination of RIF and INH resistance, the sensitivity of the capillary tube method compared to the reference method was determined as 85% and 80%, respectively; however, the specificity values (25% and 45.5%, respectively) for both drugs were found to be low in the studies. The time to detect resistance with the capillary tube method varied between 4-9 days. Capillary tube method, which was developed especially for the rapid identification and treatment of multidrug-resistant isolates, is promising in that it detects resistant strains in a short time with a relatively high sensitivity, although its specificity is very low. It is thought that it would be beneficial to continue the study with a larger number of samples and even improve the method with studies conducted directly from clinical samples.

Reassessment of the genetic basis of natural rifampin resistance in the genus Rickettsia.

Rickettsia, a genus of obligate intracellular bacteria, includes species that cause significant human diseases. This study challenges previous claims that the Leucine-973 residue in the RNA polymerase beta subunit is the primary determinant of rifampin resistance in Rickettsia. We investigated a previously untested Rickettsia species, R. lusitaniae, from the Transitional group and found it susceptible to rifampin, despite possessing the Leu-973 residue. Interestingly, we observed the conservation of this residue in several rifampin-susceptible species across most Rickettsia phylogenetic groups. Comparative genomics revealed potential alternative resistance mechanisms, including additional amino acid variants that could hinder rifampin binding and genes that could facilitate rifampin detoxification through efflux pumps. Importantly, the evolutionary history of Rickettsia genomes indicates that the emergence of natural rifampin resistance is phylogenetically constrained within the genus, originating from ancient genetic features shared among a unique set of closely related Rickettsia species. Phylogenetic patterns appear to be the most reliable predictors of natural rifampin resistance, which is confined to a distinct monophyletic subclade known as Massiliae. The distinctive features of the RNA polymerase beta subunit in certain untested Rickettsia species suggest that R. raoultii, R. amblyommatis, R. gravesii, and R. kotlanii may also be naturally rifampin-resistant species.

Isoniazid resistance in Rifampicin sensitive pulmonary tuberculosis in children and adolescents.

BACKGROUND: Isoniazid (INH) and Rifampicin (RIF) are two crucial drugs used in antitubercular therapy. INH is known for its potent bactericidal effects and has a relatively higher prevalence of resistance compared to RIF. However, RIF resistance has been the subject of more extensive research. On the other hand, Ethambutol (EMB) and Streptomycin (STR) resistance have not been thoroughly studied, particularly in the context of children and adolescents. To address this knowledge gap, a study was designed to investigate the resistance patterns of INH, EMB, and STR in RIF-sensitive pulmonary tuberculosis (PTB) cases among children and adolescents. METHODS: Seventy-five newly diagnosed RIF sensitive PTB cases up to 18 years of age were enrolled. Retreatment cases were excluded. Sputum/gastric aspirate sample of these patients were sent for culture in Mycobacterium Growth Indicator Tube (MGIT) followed by drug susceptibility testing and Line Probe Assay. RESULTS: INH, EMB and STR resistance among RIF sensitive PTB cases was found to be 5.7%, 0% and 0.7% respectively. RIF resistance detected by CBNAAT was found to be 8.4%. CONCLUSION: Detection of INH resistance is as important as detecting RIF resistance as prevalence of INH resistance in RIF sensitive PTB among children and adolescents up to 18 years is around 6%.

In-silico transcriptome analysis of antibiotic-treated Mycobacterium tuberculosis identifies novel antibiotic resistance factors.

The emergence of drug resistant Mycobacterium tuberculosis strains increases the burden on the treatment of tuberculosis. In this study, through in-silico transcriptome analysis of drug-treated M. tuberculosis samples, novel drug targets for the treatment of drug resistance in tuberculosis were identified. Gene expression datasets of tuberculosis patients samples treated with different antibiotics (Isoniazid, Rifampicin, Pyrazinamide, Bedaquiline and Linezolid) were considered in this study. DESeq2 was used to identify the differentially regulated genes. Novel genes which were up-regulated during antibiotic treatment were identified which could be antibiotic resistance factors. Further, to understand the resistance mechanism of the novel genes, we performed gene ontology and gene network analysis for the differentially up-regulated genes. Thus, the in-silico transcriptome analysis paves way for a deeper understanding of the antibiotic resistance in M. tuberculosis.

Mutational analysis in drug resistant Mycobacterium tuberculosis in Western Uttar Pradesh.

BACKGROUND: Multi-drug resistant tuberculosis (MDR-TB) results in treatment failure and poor clinical outcomes. This study was carried out with the aim to determine the pattern of drug resistance against Mycobacterium tuberculosis towards first line ATT (anti-tubercular treatment) in sputum smear-positive patients using Line Probe Assay (LPA). METHODS: A cross sectional prospective study was carried out in a tertiary care Hospital of Meerut. A total of 898 sputum samples (on spot and early morning) collected from 449 suspected pulmonary tuberculosis patients as per RNTCP guidelines were screened by microscopy. Decontamination was done by N-acetyl-l-cysteine and sodium hydroxide. Then smear positive samples were subjected to 1st line drug susceptibility testing (DST) using LPA GenoType® MTBDRplus (HAIN Life Science) assay, a molecular method which allows rapid detection of Rifampicin (Rif) and Isoniazid (INH) resistance. RESULTS: The overall burden of MDR TB in this geographical area was 7.9 %. Mono-resistance with Rif alone was around 2.8 %. However, the mono-resistance with INH (inhA gene) and INH (katG gene) was 2.8 % and 1.1 % respectively. Drug resistance of Rif was due to mutations in rpoB gene while resistances to INH were more commonly due to mutation in inhA gene followed by katG gene. TB was more commonly seen in the age group of 30-59 years (43.8 %) and predominantly in males. CONCLUSION: Tuberculosis positivity rate is high in Western Uttar Pradesh. Burden of MDR TB in Western Uttar Pradesh was similar to National data. Line probe assay can be used as a primary method to diagnose multi drug resistant TB as done in present study which can help in earlier initiation of correct therapy.

An open-label study to explore the optimal design of CYP3A drug-drug interaction clinical trials in healthy Chinese people.

A drug-drug interaction (DDI) trial of cytochrome P450 3A (CYP3A) is a necessary part of early-phase trials of drugs mainly metabolized by this enzyme, but CYP3A DDI clinical trials do not have a standard design, especially for Chinese people. We aimed to offer specific recommendations for CYP3A DDI clinical trial design. This was an open, three-cycle, self-controlled study. Healthy subjects were given different administration strategies of CYP3A4 perpetrators. In each cycle, blood samples were collected before and within 24 h after the administration of midazolam, the CYP3A indicator substrate. The plasma concentrations of midazolam and 1-hydroxymidazolam was obtained using liquid chromatography tandem mass spectrometry assay. For CYP3A inhibition, itraconazole exposure with a loading dose could increase the exposure of midazolam by 3.21-fold based on maximum plasma concentration (Cmax), 8.37-fold based on area under the curve Pharmacology Research & Perspectives for review only from zero to the time point (AUC0-t), and 11.22-fold based on area under the curve from zero to infinity (AUC0-∞). The data were similar for itraconazole pretreatment without a loading dose. For CYP3A induction, the exposure of rifampin for 7 days decreased the plasma concentration of midazolam ~0.27-fold based on Cmax, ~0.18-fold based on AUC0-t, and ~0.18-fold based on AUC0-∞. Midazolam exposure did not significantly change when the pretreatment of rifampin increased to 14 days. This study showed that itraconazole pretreatment for 3 days without a loading dose was enough for CYP3A inhibition, and pretreatment with rifampin for 7 days could induce near-maximal CYP3A levels.

Potential of Pan-Tuberculosis Treatment to Drive Emergence of Novel Resistance.

New tuberculosis (TB) drugs with little existing antimicrobial resistance enable a pan-TB treatment regimen, intended for universal use without prior drug-susceptibility testing. However, widespread use of such a regimen could contribute to an increasing prevalence of antimicrobial resistance, potentially rendering the pan-TB regimen ineffective or driving clinically problematic patterns of resistance. We developed a model of multiple sequential TB patient cohorts to compare treatment outcomes between continued use of current standards of care (guided by rifampin-susceptibility testing) and a hypothetical pan-TB approach. A pan-TB regimen that met current target profiles was likely to initially outperform the standard of care; however, a rising prevalence of transmitted resistance to component drugs could make underperformance likely among subsequent cohorts. Although the pan-TB approach led to an increased prevalence of resistance to novel drugs, it was unlikely to cause accumulation of concurrent resistance to novel drugs and current first-line drugs.

A Microtiter Plate Assay at Acidic pH to Identify Potentiators that Enhance Pyrazinamide Activity Against Mycobacterium tuberculosis.

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.

Guiding antibiotics towards their target using bacteriophage proteins.

Novel therapeutic strategies against difficult-to-treat bacterial infections are desperately needed, and the faster and cheaper way to get them might be by repurposing existing antibiotics. Nanodelivery systems enhance the efficacy of antibiotics by guiding them to their targets, increasing the local concentration at the site of infection. While recently described nanodelivery systems are promising, they are generally not easy to adapt to different targets, and lack biocompatibility or specificity. Here, nanodelivery systems are created that source their targeting proteins from bacteriophages. Bacteriophage receptor-binding proteins and cell-wall binding domains are conjugated to nanoparticles, for the targeted delivery of rifampicin, imipenem, and ampicillin against bacterial pathogens. They show excellent specificity against their targets, and accumulate at the site of infection to deliver their antibiotic payload. Moreover, the nanodelivery systems suppress pathogen infections more effectively than 16 to 32-fold higher doses of free antibiotics. This study demonstrates that bacteriophage sourced targeting proteins are promising candidates to guide nanodelivery systems. Their specificity, availability, and biocompatibility make them great options to guide the antibiotic nanodelivery systems that are desperately needed to combat difficult-to-treat infections.

Assessment of polymeric mucin-drug interactions.

Mucosal-delivered drugs have to pass through the mucus layer before absorption through the epithelial cell membrane. Although there has been increasing interest in polymeric mucins, a major structural component of mucus, potentially acting as important physiological regulators of mucosal drug absorption, there are no reports that have systematically evaluated the interaction between mucins and drugs. In this study, we assessed the potential interaction between human polymeric mucins (MUC2, MUC5B, and MUC5AC) and various drugs with different chemical profiles by simple centrifugal method and fluorescence analysis. We found that paclitaxel, rifampicin, and theophylline likely induce the aggregation of MUC5B and/or MUC2. In addition, we showed that the binding affinity of drugs for polymeric mucins varied, not only between individual drugs but also among mucin subtypes. Furthermore, we demonstrated that deletion of MUC5AC and MUC5B in A549 cells increased the cytotoxic effects of cyclosporin A and paclitaxel, likely due to loss of mucin-drug interaction. In conclusion, our results indicate the necessity to determine the binding of drugs to mucins and their potential impact on the mucin network property.

Omeprazole Induces CYP3A4 mRNA Expression but Not CYP3A4 Protein Expression in HepaRG Cells.

Unknown interactions between drugs remain the limiting factor for clinical application of drugs, and the induction and inhibition of drug-metabolizing CYP enzymes are considered the key to examining the drug-drug interaction (DDI). In this study, using human HepaRG cells as an in vitro model system, we analyzed the potential DDI based on the expression levels of CYP3A4 and CYP1A2. Rifampicin and omeprazole, the potent inducers for CYP3A4 and CYP1A2, respectively, induce expression of the corresponding CYP enzymes at both the mRNA and protein levels. We noticed that, in addition to inducing CYP1A2, omeprazole induced CYP3A4 mRNA expression in HepaRG cells. However, unexpectedly, CYP3A4 protein expression levels were not increased after omeprazole treatment. Concurrent administration of rifampicin and omeprazole showed an inhibitory effect of omeprazole on the CYP3A4 protein expression induced by rifampicin, while its mRNA induction remained intact. Cycloheximide chase assay revealed increased CYP3A4 protein degradation in the cells exposed to omeprazole. The data presented here suggest the potential importance of broadening the current DDI examination beyond conventional transcriptional induction and enzyme-activity inhibition tests to include post-translational regulation analysis of CYP enzyme expression.

Efficacy of rifapentine and other rifamycins against Coxiella burnetii in vitro.

Since 1999, doxycycline and hydroxychloroquine have been the recommended treatment for chronic Q fever, a life-threatening disease caused by the bacterial pathogen, Coxiella burnetii. Despite the duration of its use, the treatment is not ideal due to the lengthy treatment time, high mortality rate, resistant strains, and the potential for contraindicated usage. A literature search was conducted to identify studies that screened large panels of drugs against C. burnetii to identify novel targets with potential efficacy against C. burnetii. Twelve candidate antimicrobials approved for use in humans by the US Food and Drug Administration were selected and minimum inhibitory concentrations (MICs) were determined against the low virulence strain Nine Mile phase II. Rifabutin and rifaximin were the best performing antibiotics tested with MICs of ≤0.01 µg mL-1. Further screening of these top candidates was conducted alongside two drugs from the same class, rifampin, well-characterized, and rifapentine, not previously reported against C. burnetii. These were screened against virulent strains of C. burnetii representing three clinically relevant genotypes. Rifapentine was the most effective in the human monocytic leukemia cell line, THP-1, with a MIC ≤0.01 µg mL-1. In the human kidney epithelial cell line, A-498, efficacy of rifapentine, rifampin, and rifabutin varied across C. burnetii strains with MICs between ≤0.001 and 0.01 µg mL-1. Rifampin, rifabutin, and rifapentine were all bactericidal against C. burnetii; however, rifabutin and rifapentine demonstrated impressive bactericidal activity as low as 0.1 µg mL-1 and should be further explored as alternative Q fever treatments given their efficacy in vitro. IMPORTANCE: This work will help inform investigators and physicians about potential alternative antimicrobial therapies targeting the causative agent of Q fever, Coxiella burnetii. Chronic Q fever is difficult to treat, and alternative antimicrobials are needed. This manuscript explores the efficacy of rifamycin antibiotics against virulent strains of C. burnetii representing three clinically relevant genotypes in vitro. Importantly, this study determines the susceptibility of C. burnetii to rifapentine, which has not been previously reported. Evaluation of the bactericidal activity of the rifamycins reveals that rifabutin and rifapentine are bactericidal at low concentrations, which is unusual for antibiotics against C. burnetii.

Lowering mortality risk in CR-HvKP infection in intestinal immunohistological and microbiota restoration.

Gut damage during carbapenem-resistant and hypervirulent Klebsiella pneumoniae (CR-HvKP) infection is associated with a death risk. Understanding the mechanisms by which CR-HvKP causes intestinal damage and gut microbiota alteration, and the impact on immunity, is crucial for developing therapeutic strategies. This study investigated if gastrointestinal tract damage and disruption of gut microbiota induced by CR-HvKP infection undermined host immunity and facilitated multi-organ invasion of CR-HvKP; whether the therapeutic value of the rifampicin (RIF) and zidovudine (ZDV) combination was attributed to their ability to repair damages and restore host immunity was determined. A sepsis model was utilized to assess the intestinal pathological changes. Metagenomic analysis was performed to characterize the alteration of gut microbiota. The effects of the RIF and ZDV on suppressing inflammatory responses and improving immune functions and gut microbiota were evaluated by immunopathological and transcriptomic analyses. Rapid colonic damage occurred upon activation of the inflammation signaling pathways during lethal infections. Gut inflammation compromised host innate immunity and led to a significant decrease in probiotics abundance, including Bifidobacterium and Lactobacillus. Treatment with combination drugs significantly attenuated the inflammatory response, up-regulated immune cell differentiation signaling pathways, and promoted the abundance of Bifidobacterium (33.40 %). Consistently, supplementation of Bifidobacterium alone delayed the death in sepsis model. Gut inflammation and disrupted microbiota are key disease features of CR-HvKP infection but can be reversed by the RIF and ZDV drug combination. The finding that these drugs can restore host immunity through multiple mechanisms is novel and deserves further investigation of their clinical application potential.