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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Genotyping and clarithromycin susceptibility testing of Mycobacterium avium subsp. hominissuis isolated in Tuscany, Italy.

Mycobacterium avium subsp. hominissuis (MAH) is a major cause of nontuberculous mycobacteria infection and the incidence of MAH infections is increasing in many countries. This study aimed at determining the VNTR-based genetic diversity and the susceptibility to clarithromycin of a collection of 71 MAH human strains isolated in the last seven years. The VNTR analysis, revealing 16 unique patterns and 8 clusters including a total of 55 isolates, showed that most MAH isolates displayed a close genetic relationship, indicating that the MAH genotypes are quite homogeneous in our geographical area. Clarithromycin showed strong antimicrobial activity against MAH isolates, as indicated by the high proportion (94.4%) of susceptible strains. No association between specific VNTR patterns and the clinical features or the MIC of clarithromycin was found.

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.

Detection of Mycobacterium tuberculosis from paraffin-embedded tissues by GeneXpert MTB/RIF.

GeneXpert MTB/RIF (Xpert) assay, a rapid and automated system based on real-time PCR and molecular beacon technology, proved to be a sensitive and specific tool capable of detecting Mycobacterium tuberculosis and rifampin resistance in clinical specimens. In this study we provide a Xpert-dedicated successful protocol for processing paraffin-embedded tissue and assess the feasibility of the Xpert assay-based tuberculosis (TB) diagnosis on these specimens, thus proving the Xpert assay as a valuable TB diagnostic tool in supporting conventional histopathological methods.

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.

Genomic diversity of Mycobacterium tuberculosis Beijing strains isolated in Tuscany, Italy, based on large sequence deletions, SNPs in putative DNA repair genes and MIRU-VNTR polymorphisms.

The Beijing genotype of Mycobacterium tuberculosis is cause of global concern as it is rapidly spreading worldwide, is considered hypervirulent, and is most often associated to massive spread of MDR/XDR TB, although these epidemiological or pathological properties have not been confirmed for all strains and in all geographic settings. In this paper, to gain new insights into the biogeographical heterogeneity of the Beijing family, we investigated a global sample of Beijing strains (22% from Italian-born, 78% from foreign-born patients) by determining large sequence polymorphism of regions RD105, RD181, RD150 and RD142, single nucleotide polymorphism of putative DNA repair genes mutT4 and mutT2 and MIRU-VNTR profiles based on 11 discriminative loci. We found that, although our sample of Beijing strains showed a considerable genomic heterogeneity, yielding both ancient and recent phylogenetic strains, the prevalent successful Beijing subsets were characterized by deletions of RD105 and RD181 and by one nucleotide substitution in one or both mutT genes. MIRU-VNTR analysis revealed 47 unique patterns and 9 clusters including a total of 33 isolates (41% of total isolates); the relatively high proportion of Italian-born Beijing TB patients, often occurring in mixed clusters, supports the possibility of an ongoing cross-transmission of the Beijing genotype to autochthonous population. High rates of extra-pulmonary localization and drug-resistance, particularly MDR, frequently reported for Beijing strains in other settings, were not observed in our survey.

Genomic variability of Mycobacterium tuberculosis strains of the Euro-American lineage based on large sequence deletions and 15-locus MIRU-VNTR polymorphism.

A sample of 260 Mycobacterium tuberculosis strains assigned to the Euro-American family was studied to identify phylogenetically informative genomic regions of difference (RD). Mutually exclusive deletions of regions RD115, RD122, RD174, RD182, RD183, RD193, RD219, RD726 and RD761 were found in 202 strains; the RD(Rio) deletion was detected exclusively among the RD174-deleted strains. Although certain deletions were found more frequently in certain spoligotype families (i.e., deletion RD115 in T and LAM, RD174 in LAM, RD182 in Haarlem, RD219 in T and RD726 in the "Cameroon" family), the RD-defined sublineages did not specifically match with spoligotype-defined families, thus arguing against the use of spoligotyping for establishing exact phylogenetic relationships between strains. Notably, when tested for katG463/gyrA95 polymorphism, all the RD-defined sublineages belonged to Principal Genotypic Group (PGG) 2, except sublineage RD219 exclusively belonging to PGG3; the 58 Euro-American strains with no deletion were of either PGG2 or 3. A representative sample of 197 isolates was then analyzed by standard 15-locus MIRU-VNTR typing, a suitable approach to independently assess genetic relationships among the strains. Analysis of the MIRU-VNTR typing results by using a minimum spanning tree (MST) and a classical dendrogram showed groupings that were largely concordant with those obtained by RD-based analysis. Isolates of a given RD profile show, in addition to closely related MIRU-VNTR profiles, related spoligotype profiles that can serve as a basis for better spoligotype-based classification.

Genetic diversity and phylogeny of Mycobacterium avium.

Mycobacterium avium, one of the species of the M. avium complex (MAC), includes 4 subspecies, i.e., M. avium subsp. hominissuis (MAH), M. avium subsp. avium (MAA), M. avium subsp. silvaticum (MAS) and M. avium subsp. paratuberculosis (MAP), in turn classified into the S (sheep) and C (cattle) types. These subspecies, although closely related, represent distinct organisms, each endowed with specific pathogenetic and host range characteristics, ranging from environmental opportunistic bacteria that cause infections in swine and immunocompromised patients to pathogens of birds and ruminants. The present review summarizes the basic epidemiological and pathological features of the M. avium subspecies, describes the major genomic events responsible of M. avium subspecies diversity (insertion sequences, sequence variations in specific chromosome loci or genes, deletions, duplications and insertions of large genomic regions) and then reconstructs the phylogenetic relationships among the M. avium subspecies.