Alessandro Mustazzolu

Prevention of False Resistance Results Obtained in Testing the Susceptibility of Mycobacterium tuberculosis to Pyrazinamide with the Bactec MGIT 960 System Using a Reduced Inoculum

ABSTRACT The susceptibility of 211 clinical isolates of Mycobacterium tuberculosis complex (201 M. tuberculosis and 10 Mycobacterium bovis isolates) to pyrazinamide (PZA) was assessed by the nonradiometric Bactec MGIT 960 system (M960). Detection of PZA resistance was followed by a repeat testing using a reduced inoculum (RI) of 0.25 ml instead of 0.5 ml. According to the first M960 analysis, resistance was observed in 55 samples. In the RI assay, 32 samples turned out to be susceptible and 23 proved to be resistant (58.2% false positivity). The Bactec 460 assay confirmed as resistant those strains detected by the RI assay, while discrepant results were found susceptible. Mutation analysis performed on 13 M. tuberculosis isolates detected pncA mutations in 11 samples. On the basis of our data, we suggest using the RI assay to confirm all PZA resistance results obtained with the standard M960 assay. Further studies are required to confirm our findings.

Activities of Drug Combinations against Mycobacterium tuberculosis Grown in Aerobic and Hypoxic Acidic Conditions

ABSTRACT Mycobacterium tuberculosis is exposed to hypoxia and acidity within granulomatous lesions. In this study, an acidic culture model of M. tuberculosis was used to test drug activity against aerobic 5-day-old (A5) and hypoxic 5-, 12-, and 19-day-old (H5, H12, and H19, respectively) bacilli after 7, 14, and 21 days of exposure. In A cultures, CFU and pH rapidly increased, while in H cultures growth stopped and pH increased slightly. Ten drugs were tested: rifampin (R), isoniazid (I), pyrazinamide (Z), ethambutol (E), moxifloxacin (MX), amikacin (AK), metronidazole (MZ), nitazoxanide (NZ), niclosamide (NC), and PA-824 (PA). Rifampin was the most active against A5, H5, H12, and H19 bacilli. Moxifloxacin and AK efficiently killed A5 and H5 cells, I was active mostly against A5 cells, Z was most active against H12 and H19 cells, and E showed low activity. Among nitrocompounds, NZ, NC, and PA were effective against A5, H5, H12, and H19 cells, while MZ was active against H12 and H19 cells. To kill all A and H cells, A5- and H5-active agents R, MX, and AK were used in combination with MZ, NZ, NC, or PA, in comparison with R-I-Z-E, currently used for human therapy. Mycobacterial viability was determined by CFU and a sensitive test in broth (day to positivity, MGIT 960 system). As shown by lack of regrowth in MGIT, the most potent combination was R-MX-AK-PA, which killed all A5, H5, H12, and H19 cells in 14 days. These observations demonstrate the sterilizing effect of drug combinations against cells of different M. tuberculosis stages grown in aerobic and hypoxic acidic conditions.

Rifampin Induces Hydroxyl Radical Formation in Mycobacterium tuberculosis

ABSTRACT The antituberculosis (anti-TB) drug rifampin (RIF) binds to the beta subunit of the RNA polymerase (RpoB) of Mycobacterium tuberculosis, but the bactericidal responses triggered after target interaction are not known. To evaluate whether RIF induced an oxidative burst, lysates of RIF-treated M. tuberculosis were tested for determination of reactive oxygen species (ROS) by the electron paramagnetic resonance (EPR) technique using 1-hydroxy-3-carboxy-pyrrolidine (CPH) and 5,5-dimethyl-1-pyrrolidine-N-oxide (DMPO) as spin traps. M. tuberculosis killing by RIF stimulated an increase in the rate of formation of the CPH radical (CP·). Lysate pretreatment with the O2·− and ·OH scavengers superoxide dismutase (SOD) and thiourea (THIO), respectively, or with the metal chelator diethylene triamine pentaacetic acid (DTPA) inhibited CP· formation, arguing in favor of a metal-catalyzed ROS response. Formation of CP· did not increase following treatment of RIF-resistant strains with RIF, indicating that the ROS were induced after RpoB binding. To identify the ROS formed, lysates of RIF-treated bacilli were incubated with DMPO, a spin trap specific for ·OH and O2·−, with or without pretreatment with SOD, catalase, THIO, or DTPA. Superoxide dismutase, catalase, and THIO decreased formation of the DMPO-OH adduct, and SOD plus DTPA completely suppressed it, suggesting that RIF activated metal-dependent O2·−-mediated mechanisms producing ·OH inside tubercle bacilli. The finding that the metal chelator DTPA reduced the bactericidal activity of RIF supported the possibility that ·OH was generated through these mechanisms and that it participated at least in part in M. tuberculosis killing by the drug.

Drug-resistant tuberculosis among foreign-born persons in Italy

To the Editors: Over the last few years, drug-resistant tuberculosis (TB) has emerged as an important threat to public health in industrialised countries. In Italy, the most recent data on resistance to the first-line drugs (FLDs) streptomycin (S), isoniazid (H), rifampicin (R) and ethambutol (E) were reported for the period 1998–2001 [1]. These studies determined the prevalence of resistance among new cases and previously treated cases, but no information was available on the contribution of immigration, which plays an important role on TB epidemiology in low-incidence countries [2]. In the last decade, while the notified incidence of TB in Italy was stable at approximately seven cases per 100,000 people annually, the proportion of foreign-born persons (FBPs) with TB increased from 22% in 1999 to 46% in 2008 [3]. In the same period, the proportion of African-born persons with TB decreased from 51% to 30%, whereas the proportion of European cases increased from 16% to 33%, most of them being born in Eastern Europe, including Former Soviet Union (FSU) countries. Eastern European countries are among those with the highest TB rates caused by multidrug-resistant (MDR) Mycobacterium tuberculosis strains ( i.e. resistant to at least H and R) and extensively drug-resistant (XDR) strains ( i.e. MDR strains resistant to any fluoroquinolone and to at least one injectable second-line drug (SLD): kanamycin (KM), capreomycin (CM), amikacin (AK)) [4]. Reliable drug susceptibility testing (DST) is essential to diagnose TB caused by drug-resistant strains. In Italy, a network of laboratories coordinated by the World Health Organization (WHO) Supranational Reference Laboratory (SRL) in Rome performs drug susceptibility proficiency testing for S, H, R, E (five rounds from 1997 to 2010) and SLD (KM, AK, CM and ofloxacin (OFL)) (one round in 2010) [5]. In order to understand …

Tuberculosis in migrants from 106 countries to Italy, 2008-2014

Tuberculosis (TB) is a major infectious disease worldwide. Over recent years, TB caused by multidrug-resistant (MDR) Mycobacterium tuberculosis strains (resistant to at least isoniazid and rifampicin) and extensively drug-resistant (XDR) strains (MDR strains resistant to any fluoroquinolone and to at least one injectable second-line drug (SLD), i.e. kanamycin, capreomycin or amikacin) has emerged as a public health concern in industrialised countries, due to increasing migration from regions where TB is endemic. In migrants coming to Italy from 106 countries, MDR-TB was high from the former Soviet Union and low from Africa http://ow.ly/WZDbo

Proficiency testing of first- and second-line anti-tuberculosis drugs in Italy

To the Editors: The emergence of drug-resistant tuberculosis (TB) is an increasing threat to public health in industrialised countries; thus, it is important to supervise mycobacteriology laboratories by performing periodic proficiency of anti-TB drug susceptibility testing (DST). In 1994, the World Health Organization (WHO) and the International Union against Tuberculosis and Lung Diseases developed a global project of anti-TB drug resistance surveillance to assist countries via a network of supranational reference laboratories (SRLs). Proficiency test (PT) results of first-line anti-TB drugs have been reported for the SRL network [1] and for some individual countries [2, 3]. The SRL in Rome, Italy, coordinated two PTs of first-line drugs in endemic countries in 2002–2006 [4] and two PTs of first-line drugs in Italy in 1998–2000 [5, 6]. The present study aims to verify whether the quality of DST in Italy changed after that time; to this end, a comprehensive survey of five PTs during a 13-yr period (1998–2010) is reported here, together with a pilot round of second-line drug PTs in 2010. Laboratories covering 18 out of 20 Italian regions participated in the PT exercise: 22 laboratories in 1998, 20 in 2000, 28 in 2003, 29 in 2007 and 30 in 2010. To maintain knowledge and skills, the laboratories were selected by the SRL on the basis of the number of patient samples analysed for DST. For instance, a mean of 88 first-line DSTs per laboratory (range 21–357) were performed in 2009. In 2010, 13 laboratories with a mean of 113 first-line and six second-line DSTs per laboratory in 2009 also performed the second-line drug PT. A mean of nine second-line DSTs per laboratory was performed in 2010. The Mycobacterium tuberculosis panels for first- and second-line drug PTs distributed by the Rome …

Mycobacterium tuberculosis gene expression at different stages of hypoxia-induced dormancy and upon resuscitation

The physiology of dormant Mycobacterium tuberculosis was studied in detail by examining the gene expression of 51 genes using quantitative Reverse-Transcription Polymerase Chain Reaction. A forty-day period of dormancy in the Wayne culture model depicted four major transcription patterns. Some sigma factors and many metabolic genes were constant, whereas genes belonging to the dormancy regulon were activated on day 9. In particular, alpha-crystallin mRNA showed more than a 1,000-fold increase compared to replicating bacilli. Genes belonging to the enduring hypoxic response were up-regulated at day 16, notably, transcription factors sigma B and E. Early genes typical of log-phase bacilli, esat-6 and fbpB, were uniformly down-regulated during dormancy. Late stages of dormancy showed a drop in gene expression likely due to a lack of substrates in anaerobic respiration as demonstrated by the transcriptional activation observed following nitrates addition. Among genes involved in nitrate metabolism, narG was strongly up-regulated by nitrates addition. Dormant bacilli responded very rapidly when exposed to oxygen and fresh medium, showing a transcriptional activation of many genes, including resuscitation-promoting factors, within one hour. Our observations extend the current knowledge on dormant M. tuberculosis gene expression and its response to nutrients and to aerobic and anaerobic respiration.

Mycobacterium tuberculosis is selectively killed by rifampin and rifapentine in hypoxia at neutral pH.

ABSTRACT The activities of rifampin, rifapentine, bedaquiline, PA-824, clofazimine, nitazoxanide, isoniazid, amikacin, moxifloxacin, niclosamide, thioridazine, and pyrazinamide were tested against nonreplicating (dormant) Mycobacterium tuberculosis H37Rv under conditions of hypoxia at pHs 5.8 and 7.3, mimicking environments of cellular granulomas and caseous granulomas, respectively. At pH 5.8, several drugs killed dormant bacilli, with the best being rifampin and rifapentine. At pH 7.3, only rifampin and rifapentine efficiently killed dormant bacilli, while all other drugs showed little activity.

Fighting tuberculosis by drugs targeting nonreplicating Mycobacterium tuberculosis bacilli

Current tuberculosis (TB) treatment requires 6 months of combination therapy with isoniazid (INH), rifampin (RIF), pyrazinamide (PZA), and ethambutol for active TB and 9 months of INH or 3 months of rifapentine (RFP) + INH for latent TB. The lungs of patients with active and latent TB contain heterogeneous mixtures of cellular and caseous granulomas harboring Mycobacterium tuberculosis bacilli ranging from actively replicating (AR) to nonreplicating (NR), phenotypically drug-resistant stages. Several in vitro models to obtain NR cells were reported, including exposure to hypoxia, nutrient starvation, acid + nitric oxide, and stationary phase. Overall, these models showed that RIF, RFP, PA-824 (PA), metronidazole (MZ), bedaquiline (BQ), and fluoroquinolones were the most active drugs against NR M. tuberculosis. In hypoxia at pH 5.8, some combinations killed AR plus NR cells, as shown by lack of regrowth in liquid media, whereas in hypoxia at pH 7.3 (the pH of the caseum), only RIF and RFP efficiently killed NR bacilli while several other drugs showed little effect. In conventional mouse models, combinations containing RFP, BQ, PA, PZA, moxifloxacin, sutezolid, linezolid, and clofazimine sterilized animals in ≤2 months, as shown by lack of viable bacilli in lung homogenates after 3 months without therapy. Drugs were less effective in C3HeB/FeJ mice forming caseous granulomas. Overall, in vitro observations and in vivo studies suggest that the search for new TB drugs could be addressed to low lipophilic molecules (e.g., new rpoB inhibitors with clogP < 3) killing NR M. tuberculosis in hypoxia at neutral pH and reaching high rates of unbound drug in the caseum.

Activity of drugs against dormant Mycobacterium tuberculosis

Objective/background: Heterogeneous mixtures of cellular and caseous granulomas coexist in the lungs of tuberculosis (TB) patients, with Mycobacterium tuberculosis (Mtb) existing from actively replicating (AR) to dormant, nonreplicating (NR) stages. Within cellular granulomas, the pH is estimated to be less than 6, whereas in the necrotic centres of hypoxic, cholesterol/triacylglycerol-rich, caseous granulomas, the pH varies between 7.2 and 7.4. To combat TB, we should kill both AR and NR stages of Mtb. Dormant Mtb remodels lipids of its cell wall, and so lipophilic drugs may be active against NR Mtb living in caseous, lipid-rich, granulomas. Lipophilicity is expressed as logP, that is, the logarithm of the partition coefficient (P) ratio P octanol/P water. In this study, the activity of lipophilic drugs (logP>0) and hydrophilic drugs (logP ≤0) against AR and NR Mtb was measured in hypoxic conditions under acidic and slightly alkaline pHs. Methods: The activity of drugs was determined against AR Mtb (5-day-old aerobic cells: A5) and NR Mtb (12- and 19-day-old hypoxic cells: H12 and H19) in a Wayne dormancy model of Mtb H37Rv at pH 5.8, to mimic the environment of cellular granulomas. Furthermore, AR and NR bacilli were grown for 40 days in Wayne models at pH 6.6, 7.0, 7.4, and 7.6, to set up conditions mimicking the caseous granulomas (hypoxia+slightly alkaline pH), to measure drug activity against NR cells. Mtb viability was determined by colony-forming unit (CFU) counts. Results: At pH 5.8, lipophilic drugs (rifampin, rifapentine, bedaquiline, PA-824, clofazimine, nitazoxanide: logP ≥2.14) reduced CFU of all cells (H12, H19, and A5) by ≥2log10. Among hydrophilic drugs (isoniazid, pyrazinamide, ethambutol, amikacin, moxifloxacin, metronidazole: logP ≤0.01), none reduced H12 and H19 CFUs by ≥2log10, with the exception of metronidazole. When Mtb was grown at different pHs the following Mtb growth was noted: at pH 6.6, AR cells grew fluently while NR cells grew less, with a CFU increase up to Day 15, followed by a drop to Day 40. AR and NR Mtb grown at pH 7.0, 7.4, and 7.6 showed up to 1 log10 CFU lower than their growth at pH 6.6. The pHs of all AR cultures tended to reach pH 7.2–7.4 on Day 40. The pHs of all NR cultures remained stable at their initial values (6.6, 7.0, 7.4, and 7.6) up to Day 40. The activity of drugs against H12 and H19 cells was tested in hypoxic conditions at a slightly alkaline pH. Under these conditions, some lipophilic drugs were more active (>5 log CFU decrease after 21 days of exposure) against H12 and H19 cells than clofazimine, nitazoxanide, isoniazid, pyrazinamide, amikacin (<1 log CFU decrease after 21 days of exposure). Testing of other drugs is in progress. Conclusion: Lipophilic drugs were more active than hydrophilic agents against dormant Mtb in hypoxic conditions at pH 5.8. The Wayne model under slightly alkaline conditions was set up, and in hypoxic conditions at a slightly alkaline pH some lipophilic drugs were more active than other drugs against NR Mtb. Overall, these models can be useful for testing drug activity against dormant Mtb under conditions mimicking the environments of cellular and caseous granulomas.