Discovery of 2-phenylquinazolines as potent Mycobacterium avium efflux pump inhibitors able to synergize with clarithromycin against clinical isolate.

Nontuberculous mycobacteria (NTM), which include the Mycobacterium avium complex, are classified as difficult-to-treat pathogens due to their ability to quickly develop drug resistance against the most common antibiotics used to treat NTM infections. The overexpression of efflux pumps (EPs) was demonstrated to be a key mechanism of clarithromycin (CLA) resistance in NTM. Therefore, in this work, 24 compounds from an in-house library, characterized by chemical diversity, were tested as potential NTM EP inhibitors (EPIs) against Mycobacterium smegmatis mc2 155 and M. avium clinical isolates. Based on the acquired results, 12 novel analogs of the best derivatives 1b and 7b were designed and synthesized to improve the NTM EP inhibition activity. Among the second set of compounds, 13b emerged as the most potent NTM EPI. At a concentration of 4 µg/mL, it reduced the CLA minimum inhibitory concentration by 16-fold against the clinical isolate M. avium 2373 overexpressing EPs as primary mechanism of CLA resistance.

Specific Cationic Antimicrobial Peptides Enhance the Recovery of Low-Load Quiescent Mycobacterium tuberculosis in Routine Diagnostics.

The culture confirmation of Mycobacterium tuberculosis (MTB) remains the gold standard for the diagnosis of Tuberculosis (TB) with culture conversion representing proof of cure. However, over 40% of TB samples fail to isolate MTB even though many patients remain infectious due to the presence of viable non-culturable forms. Previously, we have shown that two short cationic peptides, T14D and TB08L, induce a hormetic response at low concentrations, leading to a stimulation of growth in MTB and the related animal pathogen Mycobacterium bovis (bTB). Here, we examine these peptides showing they can influence the mycobacterial membrane integrity and function through membrane potential reduction. We also show this disruption is associated with an abnormal reduction in transcriptomic signalling from specific mycobacterial membrane sensors that normally monitor the immediate cellular environment and maintain the non-growing phenotype. We observe that exposing MTB or bTB to these peptides at optimal concentrations rapidly represses signalling mechanisms maintaining dormancy phenotypes, which leads to the promotion of aerobic metabolism and conversion into a replicative phenotype. We further show a practical application of these peptides as reagents able to enhance conventional routine culture methods by stimulating mycobacterial growth. We evaluated the ability of a peptide-supplemented sample preparation and culture protocol to isolate the MTB against a gold standard routine method tested in parallel on 255 samples from 155 patients with suspected TB. The peptide enhancement increased the sample positivity rate by 46% and decreased the average time to sample positivity of respiratory/faecal sampling by seven days. The most significant improvements in isolation rates were from sputum smear-negative low-load samples and faeces. The peptide enhancement increased sampling test sensitivity by 19%, recovery in samples from patients with a previously culture-confirmed TB by 20%, and those empirically treated for TB by 21%. We conclude that sample decontamination and culture enhancement with D-enantiomer peptides offer good potential for the much-needed improvement of the culture confirmation of TB.

High genetic heterogeneity of Mycobacterium intracellulare isolated from respiratory specimens.

BACKGROUND: M. intracellulare is a frequent causative pathogen of nontuberculous mycobacteria infection that causes infections in the respiratory tract, whose incidence is increasing in many countries. This study aimed at determining the VNTR-based genetic diversity of a collection of 39 M. intracellulare human strains isolated from respiratory specimens over the last 5 years. RESULTS: The VNTR analysis showed that M. intracellulare strains displayed a high genetic diversity, indicating that the M. intracellulare genotypes are quite heterogeneous in our geographical area. Moreover, a comparison with VNTR profiles of strains from other countries confirmed that genotypes of clinical strains of M. intracellulare are not related to geographical origin. CONCLUSIONS: VNTR typing has proved to be a highly discriminatory method for better understanding the molecular epidemiology of M. intracellulare.

LysX2 is a Mycobacterium tuberculosis membrane protein with an extracytoplasmic MprF-like domain.

BACKGROUND: Aminoacyl-phosphatidylglycerol (aaPG) synthases are bacterial enzymes that usually catalyze transfer of aminoacyl residues to the plasma membrane phospholipid phosphatidylglycerol (PG). The result is introduction of positive charges onto the cytoplasmic membrane, yielding reduced affinity towards cationic antimicrobial peptides, and increased resistance to acidic environments. Therefore, these enzymes represent an important defense mechanism for many pathogens, including Staphylococcus aureus and Mycobacterium tuberculosis (Mtb), which are known to encode for lysyl-(Lys)-PG synthase MprF and LysX, respectively. Here, we used a combination of bioinformatic, genetic and bacteriological methods to characterize a protein encoded by the Mtb genome, Rv1619, carrying a domain with high similarity to MprF-like domains, suggesting that this protein could be a new aaPG synthase family member. However, unlike homologous domains of MprF and LysX that are positioned in the cytoplasm, we predicted that the MprF-like domain in LysX2 is in the extracytoplasmic region. RESULTS: Using genetic fusions to the Escherichia coli proteins PhoA and LacZ of LysX2, we confirmed this unique membrane topology, as well as LysX and MprF as benchmarks. Expression of lysX2 in Mycobacterium smegmatis increased cell resistance to human ß-defensin 2 and sodium nitrite, enhanced cell viability and delayed biofilm formation in acidic pH environment. Remarkably, MtLysX2 significantly reduced the negative charge on the bacterial surface upon exposure to an acidic environment. Additionally, we found LysX2 orthologues in major human pathogens and in rapid-growing mycobacteria frequently associated with human infections, but not in environmental and non-pathogenic mycobacteria. CONCLUSIONS: Overall, our data suggest that LysX2 is a prototype of a new class within the MprF-like protein family that likely enhances survival of the pathogenic species through its catalytic domain which is exposed to the extracytoplasmic side of the cell membrane and is required to decrease the negative charge on the bacterial surface through a yet uncharacterized mechanism.

Efflux Pump Inhibitors Against Nontuberculous Mycobacteria.

Over the last years, nontuberculous mycobacteria (NTM) have emerged as important human pathogens. Infections caused by NTM are often difficult to treat due to an intrinsic multidrug resistance for the presence of a lipid-rich outer membrane, thus encouraging an urgent need for the development of new drugs for the treatment of mycobacterial infections. Efflux pumps (EPs) are important elements that are involved in drug resistance by preventing intracellular accumulation of antibiotics. A promising strategy to decrease drug resistance is the inhibition of EP activity by EP inhibitors (EPIs), compounds that are able to increase the intracellular concentration of antimicrobials. Recently, attention has been focused on identifying EPIs in mycobacteria that could be used in combination with drugs. The aim of the present review is to provide an overview of the current knowledge on EPs and EPIs in NTM and also, the effect of potential EPIs as well as their combined use with antimycobacterial drugs in various NTM species are described.

Increase in non-tuberculous mycobacteria isolated from humans in Tuscany, Italy, from 2004 to 2014.

BACKGROUND: In Italy, the prevalence of non-tuberculous mycobacteria (NTM) in human infections is largely unknown. Herein, we report the epidemiology of NTM infections in a region of central Italy, Tuscany, over the last 11 years, and provide a review of the recent literature on NTM isolation rates in different geographic regions. METHODS: The complete collection of NTM strains isolated from a total of 42,055 clinical specimens at the Laboratory of Clinical Mycobacteriology of Pisa University Hospital, Italy, from 1 January 2004 to 31 December 2014 was included. RESULTS: In our setting, in the period 2004-2014 a total of 147 patients had cultures positive for NTM. The number of NTM isolates increased considerably from five isolates in 2004 to 29 in 2014; a sharp increase occurred in the last 3 years. Overall, 16 NTM species were isolated; the most common were M. avium, M. intracellulare and M. gordonae detected in respectively in 41.5, 14.3 and 11.6% of NTM patients. In general, NTM isolates were largely prevalent in people older than 60 (57.8%); patients aged 1-10 year-old almost exclusively yielded M. avium and M. intracellulare. Of the 147 NTM clinical isolates, 76.2% were from respiratory specimens, 10.9% from lymph nodes, 2.7% from blood (yielding exclusively M. avium), and the remaining 10.2% from other clinical specimens. CONCLUSIONS: The observed increase in NTM isolation rate in our setting is in keeping with the general increase in NTM infections reported worldwide in the past two decades, although the distribution of the NTM prevalent species differs by geographic region.

Intracellular cytokine detection by fluorescence-activated flow cytometry: basic principles and recent advances.

Intracellular cytokine staining is a flow cytometric technique consisting of culturing stimulated cytokine-producing cells in the presence of a protein secretion inhibitor, followed by fixation, permeabilization and staining of intracellular cytokines and cell markers (surface or cytoplasmic) with fluorescent antibodies. Up to 18 different colors can be detected by modern flow cytometers, making it the only immunological technique allowing simultaneous determination of antigen-specific T cell function and phenotype. In addition, cell proliferation and viability can be also measured. For this reason, it is probably the most popular method to measure antigenicity during vaccine trials and in the study of infectious diseases, along with ELISPOT. In this review, we will summarize its features, provide the protocol used by most laboratories and review its most recent applications.

Fatal disseminated Mycobacterium avium infection with bone marrow infiltration and hemophagocytic syndrome.

Disseminated non-tuberculous mycobacterial (NTM) infection can affect patients with underlying immunosuppressive conditions. Despite being rare, delay in diagnosis can lead to life-threatening uncontrolled immune response and hemophagocytic syndrome (HPS). We report a case of a 63-year-old female with suspected autoimmune disease, in whom HPS was diagnosed according to HLH-2004 criteria and H-score. Mycobacterium avium (M. avium) was isolated from blood culture, bronchoalveolar lavage (BAL) and bone marrow biopsy. In immunosuppressed patients, early clinical suspicion and prompt microbiological diagnosis of mycobacterial infection together with drug susceptibility tests (DST)-based treatment, as well as HPS, are pivotal to increase the likelihood of treatment success.

A rare case of primary cutaneous Nocardia vinacea in an immunocompetent patient: A case report and a review of the literature.

The nocardiae are a complex group of bacteria belonging to the aerobic saprophytes actinomycetes. Although nocardiosis typically occurs in immunocompromised patients, infection may occasionally develop in immunocompetent patients as well. Here we describe a rare case of primary cutaneous nocardiosis due to Nocardia vinacea in an immunocompetent 79-year-old patient. Since cutaneous nocardiosis presents variably and mimics other cutaneous infections, acid-fast and Gram stainings on clinical samples are significant to obtain a rapid and presumptive diagnosis.

Evaluation of antibiofilm activity of cefiderocol alone and in combination with imipenem against Pseudomonas aeruginosa.

OBJECTIVES: The main aim of this study was to evaluate the antibiofilm activity of cefiderocol alone and in combination with imipenem vs. sessile cells of Pseudomonas aeruginosa, assessing a potential synergistic bactericidal effect. METHODS: Ten P. aeruginosa clinical isolates from infected implants and bloodstream were included in the study. Cefiderocol was tested alone and in combination with imipenem on 24-h-old P. aeruginosa biofilm formed on porous glass beads. For each antibiotic formulation, minimum bactericidal biofilm concentration (MBBC), defined as the lowest concentration that determined a reduction of at least 3 log10 CFU/mL compared with the untreated control, was evaluated. Scanning electron microscopy (SEM) was used to investigate the biofilm of P. aeruginosa treated with cefiderocol, imipenem, or their combination. RESULTS: Cefiderocol and imipenem were tested alone on P. aeruginosa biofilm and a reasonable reduction in the number of viable cells was observed, especially at high drug concentrations tested. The synergistic effect of cefiderocol in combination with imipenem was evaluated for five selected isolates. Cotreatment with the two drugs led to a remarkable reduction of cell viability by resulting in synergistic bactericidal activity in all tested strains and in synergistic eradicating activity in only one isolate. SEM analysis revealed that, in cefiderocol-treated biofilm, bacterial cells became more elongated than in the untreated control, forming filaments in which bacterial division seems to be inhibited. CONCLUSIONS: Cefiderocol exhibited an encouraging antibiofilm activity against tested strains, representing a valid option for the treatment of P. aeruginosa biofilm-associated infections, especially when administered in combination with imipenem.

Evaluation of underreporting tuberculosis in Central Italy by means of record linkage.

BACKGROUND: Tuberculosis (TB) surveillance systems have some pitfalls outside of a National Tuberculosis Program and lack of efficient surveillance hampers accurate epidemiological quantification of TB burden.In the present study we assessed the quality of surveillance at the University Hospital in Pisa (UHP), Italy, and TB incidence rates over a ten year period (1999-2008). METHODS: Assessment of underreporting was done by record-linkage from two sources: databases of TB diagnoses performed in the UHP and the Italian Infectious Disease Surveillance (IIDS) system. Two different databases were examined: a) TB diagnoses reported in the Hospital Discharge Records (HDR) from three Units of UHP (Respiratory Pathophysiology, Pulmonology and Infectious Diseases Units) (TB database A); b) TB diagnoses reported in HDR of all Units of UHP plus TB positive cases obtained by the Laboratory Register (LR) of UHP (TB database B). For the TB database A, the accuracy of TB diagnosis in HDR was assessed by direct examination of the Clinical Record Forms of the cases. For the TB database B, clinical and population data were described, as well as the trend of incidence and underreporting over 10 yrs. RESULTS: In the first study 293 patients were found: 80 patients (27%) with a confirmed TB diagnosis were underreported, 39 of them were microbiologically confirmed. Underreporting was related to age (Reported vs Non Reported, mean age: 49.27 ± 20 vs 55 ± 19, p < 0.005 ), diagnosis (smear positive vs negative cases 18.7 vs 81.2%, p = 0.001), microbiological confirmation (49% vs 51%, p < 0.05), X-ray findings (cavitary vs non-cavitary cases: 12.5 vs 87.5%, p = 0.001) but not to nationality.In the second study, 666 patients were found. Mean underreporting rate was 69.4% and decreased over time (68% in 1999, 48% in 2008). Newly diagnosed TB cases were also found to decrease in number whereas immigration rate increased. Underreporting was related to nationality (Immigrants vs Italians: 18% vs 68%, p < 0.001), diagnosis (microbiological confirmation: 25% vs 75%, p < 0.01), kind of hospital regimen (hospitalized patients vs Day Hospital: 70% vs 16%, p < 0.001), and position of TB code in the HDR (TB code in first position vs in the following position: 39,5% vs 45% p < 0.001). CONCLUSIONS: TB is underreported in Pisa, particularly in older patients and those without microbiological confirmation. The TB code in first position of HDR seems fairly accurate in confirming TB diagnosis.

Rapid Molecular Diagnosis of Extra-Pulmonary Tuberculosis by Xpert/RIF Ultra.

Rapid detection of Mycobacterium tuberculosis complex and determination of drug resistance are essential for early diagnosis and treatment of tuberculosis (TB). Xpert MTB/RIF Ultra (Xpert Ultra), a molecular test that can simultaneously identify M. tuberculosis complex and resistance to rifampicin directly on clinical samples, is currently used. Xpert Ultra represents a helpful tool for rapid pulmonary TB diagnosis, especially in patients with paucibacillary infection. The aim of this review is to provide an overview of the diagnostic performance of Xpert Ultra in detection of extra-pulmonary tuberculosis.

Rapid and Accurate Identification of Nontuberculous Mycobacteria Directly from Positive Primary MGIT Cultures by MALDI-TOF MS.

Over the last years, nontuberculous mycobacteria (NTM) have emerged as important human pathogens. Accurate and rapid mycobacterial species identification is needed to successfully diagnose, treat, and manage infections caused by NTM. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry, MALDI-TOF MS, was demonstrated to effectively identify mycobacteria isolates subcultured from solid or liquid media rather than new positive cultures. The present study aims to develop a new extraction protocol to yield rapid and accurate identification of NTM from primary MGIT cultures by MALDI-TOF MS. A total of 60 positive MGIT broths were examined by the Bruker Biotyper system with Mycobacteria Library v. 2.0 (Bruker Daltonics GmbH & Co. KG., Bremen, Germany). The results were compared with those obtained by the molecular method, line probe assay GenoType Mycobacterium CM/AS/NTM-DR. All samples were concordantly identified by MALDI-TOF MS and the molecular test for all the tested mycobacteria. Fifty-seven (95%) MGIT positive cultures for NTM from clinical samples had a MALDI-TOF MS analysis score S ≥ 1.8. Although a small number of strains and a limited diversity of mycobacterial species were analysed, our results suggest that MALDI-TOF MS could represent a promising routine diagnostic tool for identifying mycobacterial species directly from primary liquid culture.

Pretomanid for tuberculosis treatment: an update for clinical purposes.

Coronavirus disease (COVID-19) pandemic determined a 10 years-set back in tuberculosis (TB) control programs. Recent advances in available therapies may help recover the time lost. While Linezolid (LZD) and Bedaquiline (BDQ), previously Group D second line drugs (SLDs) for TB, have been relocated to Group A, other drugs are currently being studied in regimens for drug resistant TB (DR-TB). Among these, Pretomanid (PA), a recently introduced antimycobacterial drug derived from nitroimidazole with both solid bactericidal and bacteriostatic effect, and with an excellent effectiveness and tolerability profile, is in the spotlight. Following promising data obtained from recently published and ongoing randomized controlled trials (RCTs), the World Health Organization (WHO) determined to include PA in its guidelines for the treatment of rifampicin-resistant (RR), multi drug resistant (MDR) and pre-extensively drug resistant TB (pre-XDR-TB) with BDQ, LZD and Moxifloxacine (MFX) in a 6-month regimen. Although further studies on the subject are needed, PA may also represent a treatment option for drug-susceptible TB (DS-TB), latent TB infection (LTBI) and non tuberculous mycobacteria (NTM). This narrative review aims to examine current implementation options and future possibilities for PA in the never-ending fight against TB.

Identification of a human immunodominant T-cell epitope of mycobacterium tuberculosis antigen PPE44.

BACKGROUND: Recently our group has identified a novel antigen of Mycobacterium tuberculosis, protein PPE44, belonging to the "PPE protein" family. Although its role in infection is largely unknown, PPE44-specific immune responses were detected in mice infected with M. tuberculosis; moreover, immunization of mice with PPE44 subunit vaccines resulted in protective efficacy comparable to the one afforded by BCG against M. tuberculosis (Romano et al., Vaccine 26, 6053-6063, 2008). RESULTS: In the present paper, we investigated anti-PPE44 T-lymphocyte responses during human infection by evaluating the frequency of PPE44-specific interferon (IFN)-γ-secreting cells by ELISpot and flow cytometry in a small cohort of healthy subjects that had proven positive to PPD (PPD+) in vitro, in patients with active tuberculosis, in subjects vaccinated with BCG and in unvaccinated, PPD- healthy controls. We showed IFN-γ+ T cell immune responses to recombinant PPE44 in at least a very high proportion of PPD+ individuals tested and, to a lower extent, in subjects vaccinated with BCG. By the use of a panel of overlapping synthetic 20-mer peptides spanning the PPE44 primary amino acid sequence, we identified a strong CD4+ T-cell epitope, encompassed by peptide p1L (VDFGALPPEVNSARMYGGAG), in the NH2-terminus of the PPE44 molecule at the amino acid position 1-20. Conversely, our experiments did not provide evidence of a significant IFN-γ+ CD4+ T cell response to PPE44 or its immunodominant peptide p1L in most (7 out of 8) patients with active TB. CONCLUSIONS: Our data suggest an important immunological role of PPE44 and its immunodominant epitope p1L that could be useful in the design of anti-tuberculosis vaccines and in the immunological diagnosis of M. tuberculosis infection.

Effect of efflux pump inhibitors on the susceptibility of Mycobacterium avium complex to clarithromycin.

In this study we aimed to evaluate the effect of the combination of clarithromycin and four inhibitors of efflux pumps (EPIs), including berberine (BER), carbonyl cyanide m-chlorophenylhydrazone (CCCP), piperine (PIP) and tetrandrine (TET), against 12 Mycobacterium avium complex clinical isolates. The minimum inhibitory concentration (MIC) of clarithromycin showed at least a fourfold reduction in presence of BER (83% of total isolates), CCCP (67%), PIP (25%) and TET (75%). Our results showed that the EPIs tested are active against both clarithromycin susceptible and resistant isolates. In particular, among the six resistant isolates, TET reversed the resistance phenotype of three strains, BER of two strains, and CCCP and PIP of one strain. Overall, our findings show the importance of these compounds in increasing the efficacy of clarithromycin in MAC clinical isolates.

Evolutionary pathway of the Beijing lineage of Mycobacterium tuberculosis based on genomic deletions and mutT genes polymorphisms.

Among the genotypes that prevail in the modern spectrum of Mycobacterium tuberculosis strains, the Beijing genotype is the one that causes major concern, as it is geographically widespread and it is considered hypervirulent. Comparative genomic studies have shown that Beijing strains have principally evolved through mechanisms of deletion of chromosomal regions, designated regions of difference (RD), and mutations. In this paper, we aimed to determine the evolutionary history of Beijing strains through the analysis of polymorphisms generated by deletions of large specific sequences, i.e., RD105, RD181, RD150, and RD142, and by single nucleotide substitutions in genes mutT4 and mutT2, coding for DNA repair enzymes. Based on the molecular characteristics of a collection of Beijing strains recently isolated in Tuscany, Italy, we propose a phylogenetic reconstruction of the Beijing family. According to our model, the Beijing family evolved from a M. tuberculosis progenitor following deletion of the RD207 region, an event responsible for the loss of spacers 1-34 in the direct repeat (DR) locus. The major lineages of the Beijing family then evolved via subsequent deletions of regions RD105, RD181 and RD150. In the most ancient evolutionary lineages genes mutT4 and mutT2 were in wild type configuration; the mutT4 mutation was acquired subsequent to the RD181 deletion in a progenitor strain that, in turn, gave rise to a sublineage bearing the mutT2 mutation. Within the major branches of the Beijing family, deletion of additional spacers in the DR locus led to evolution of sublineages characterized by different spoligotypes. Our evolutionary model of the Beijing family provides a deeper framework than previously proposed for epidemiologic and phylogenetic studies of circulating M. tuberculosis Beijing strains, thus allowing a more systematic and comprehensive evaluation of the relevance of Beijing strain variability.

Virulence of Mycobacterium avium Subsp. hominissuis Human Isolates in an in vitro Macrophage Infection Model.

Background: Mycobacterium avium subsp. hominissuis (MAH) is an environmental opportunistic pathogen for humans and swine worldwide; in humans, the vast majority of MAH infections is due to strains belonging to specific genotypes, such as the internal transcribed spacer (ITS)-sequevars Mav-A and Mav-B that mostly cause pulmonary infections in elderly patients and severe disseminated infections in acquired immunodeficiency syndrome patients, respectively. To test whether the different types of infections in distinct patients' populations might reflect a different virulence of the infecting genotypes, MAH human isolates, genotyped by ITS sequencing and MIRU-VNTR minisatellite analysis, were studied for the capacity to infect and replicate in human macrophages in vitro. Methods: Cultures of human peripheral blood mononuclear cells and phagocytic human leukemic cell line THP-1 cells were infected with each MAH isolate and intracellular colony-forming units (CFU) were determined. Results: At 2 h after infection, i.e., immediately after cell entry, the numbers of intracellular bacteria did not differ between Mav-A and Mav-B organisms in both phagocytic cell types. At 5 days, Mav-A organisms, sharing highly related VNTR-MIRU genotypes, yielded numbers of intracellular CFUs significantly higher than Mav-B organisms in both phagocytic cell types. MIRU-VNTR-based minimum spanning tree analysis of the MAH isolates showed a divergent phylogenetic pathway of Mav-A and Mav-B organisms. Conclusion: Mav-A and Mav-B sequevars might have evolved different pathogenetic properties that might account for their association with different human infections.