Lara Brunori

Evaluation of a New Line Probe Assay for Rapid Identification of gyrA Mutations in Mycobacterium tuberculosis

ABSTRACT Resistance of Mycobacterium tuberculosis to fluoroquinolones (FQ) results mostly from mutations in the gyrA gene. We developed a reverse hybridization-based line probe assay in which oligonucleotide probes carrying the wild-type gyrA sequence, a serine-to-threonine (S95T) polymorphism, and gyrA mutations (A90V, A90V-S95T, S91P, S91P-S95T, D94A, D94N, D94G-S95T, D94H-S95T) were immobilized on nitrocellulose strips and hybridized with digoxigenin-labeled PCR products obtained from M. tuberculosis strains. When a mutated PCR product was used, hybridization occurred to the corresponding mutated probe but not to the wild-type probe. A panel of M. tuberculosis complex strains including 19 ofloxacin-resistant (OFL-R) and 9 ofloxacin-susceptible (OFL-S) M. tuberculosis strains was studied for detection and identification of gyrA mutations by the line probe assay and nucleotide sequencing, in comparison with testing of in vitro susceptibility to FQ. Results were 100% concordant with those of nucleotide sequencing. The S95T polymorphism, which is not related to FQ resistance, was found in 5 OFL-S and 2 OFL-R strains; the other 17 OFL-R strains harbored single mutations associated with serine or threonine at codon 95. No mutations were found in the other OFL-S strains. Overall, on the basis of the MICs on solid medium, the new line probe assay correctly identified all OFL-S and 17 out of 19 (89.5%) OFL-R strains. A nested-PCR protocol was also evaluated for the assay to amplify PCR products from M. tuberculosis-spiked sputa, with a good specificity and a sensitivity of 2 × 103M. tuberculosis CFU per ml of sputum.

Mycobacterium bovis Bacillus Calmette-Guérin infects DC-SIGN– dendritic cell and causes the inhibition of IL-12 and the enhancement of IL-10 production

The only available vaccine against tuberculosis is Mycobacterium bovis Bacillus Calmette Guérin (BCG), although its efficacy in preventing pulmonary tuberculosis is controversial. Early interactions between dendritic cells (DC) and BCG or Mycobacterium tuberculosis (Mtb) are thought to be critical for mounting a protective antimycobacterial immune response. Recent studies have shown that BCG and Mtb target the DC‐specific C‐type lectin intercellular adhesion molecule‐3‐grabbing nonintegrin (DC‐SIGN) to infect DC and inhibit their immunostimulatory function. This would occur through the interaction of the mycobacterial mannosylated lipoarabinomannan to DC‐SIGN, which would prevent DC maturation and induce the immunosuppressive cytokine interleukin (IL)‐10 synthesis. Here, we confirm that DC‐SIGN is expressed in DC derived from monocytes cultured in granulocyte macrophage‐colony stimulating factor (GM‐CSF) and IL‐4 and show that it is not expressed in DC derived from monocytes cultured in GM‐CSF and interferon‐α (IFN‐α). We also demonstrate that DC‐SIGN– DC cultured in GM‐CSF and IFN‐α are able to phagocytose BCG and to undergo a maturation program as well as DC‐SIGN+ DC cultured in IL‐4 and GM‐CSF. We also show that BCG causes the impairment of IL‐12 and the induction of IL‐10 secretion by DC, irrespective of DC‐SIGN expression. Finally, we demonstrate that the capacity to stimulate a mixed leukocyte reaction of naïve T lymphocytes is not altered by the treatment of both DC populations with BCG. These data suggest that DC‐SIGN cannot be considered as the unique DC receptor for BCG internalization, and it is more interesting that the mycobacteria‐induced immunosuppression cannot be attributed to the engagement of a single receptor.

In vitro activity of protegrin-1 and beta-defensin-1, alone and in combination with isoniazid, against Mycobacterium tuberculosis

The antimicrobial peptide protegrin-1 (PG-1) inhibited the growth in vitro of drug-susceptible and multidrug-resistant Mycobacterium tuberculosis; a lower activity was shown by human beta-defensin-1 (HBD-1) against both strains. The combination of PG-1 or HBD-1 with isoniazid significantly reduced M. tuberculosis growth in comparison with the peptides or isoniazid alone.

Activities of moxifloxacin alone and in combination with other antimicrobial agents against multidrug-resistant Mycobacterium tuberculosis infection in BALB/c mice

ABSTRACT The activity of moxifloxacin was enhanced by the addition of ethionamide but not by that of cycloserine, thiacetazone, capreomycin, para-aminosalicylic acid, or linezolid in BALB/c mice infected with a strain of Mycobacterium tuberculosis resistant to isoniazid, rifampin, and six other drugs. These observations are important for the therapy of multidrug-resistant tuberculosis.

The LTK63 adjuvant improves protection conferred by Ag85B DNA-protein prime-boosting vaccination against Mycobacterium tuberculosis infection by dampening IFN-γ response

T helper type-1 response is essential to control Mycobacterium tuberculosis (MTB) infection but excessive antigen-mediated inflammation concurs to pathology. In mice challenged with MTB, the protection elicited by an Ag85B-encoding DNA vaccine, was lost when mice were boosted with Ag85B-protein in the absence of adjuvant. This effect was due to the expansion of a set of IFN-gamma secreting-CD4+ T cells highly responsive to Ag85B-protein but which lost the ability to interact with MTB-infected macrophages and control MTB growth. Ag85B-protein co-administration with the adjuvant LTK63 reduced the expansion of Ag85B-protein-responding CD4+ T cells and allowed the survival of those protective Ag85B-specific CD4+ T cells induced by the Ag85B-encoding DNA vaccine. Consequently, the protection against MTB-infection was restored. LTK63 caused also a marked augmentation of Ag85B-specific antibodies, in particular those belonging to the IgG2b isotype. The recovery of protection through a down-modulation of antigen-specific IFN-gamma response by an adjuvant is a novel finding which could be of relevance in tuberculosis vaccination.

The Ag85B protein of Mycobacterium tuberculosis may turn a protective immune response induced by Ag85B‐DNA vaccine into a potent but non‐protective Th1 immune response in mice

Clarifying how an initial protective immune response to tuberculosis may later loose its efficacy is essential to understand tuberculosis pathology and to develop novel vaccines. In mice, a primary vaccination with Ag85B‐encoding plasmid DNA (DNA‐85B) was protective against Mycobacterium tuberculosis (MTB) infection and associated with Ag85B‐specific CD4+ T cells producing IFN‐γ and controlling intramacrophagic MTB growth. Surprisingly, this protection was eliminated by Ag85B protein boosting. Loss of protection was associated with a overwhelming CD4+ T cell proliferation and IFN‐γ production in response to Ag85B protein, despite restraint of Th1 response by CD8+ T cell‐dependent mechanisms and activation of CD4+ T cell‐dependent IL‐10 secretion. Importantly, these Ag85B‐responding CD4+ T cells lost the ability to produce IFN‐γ and control MTB intramacrophagic growth in coculture with MTB‐infected macrophages, suggesting that the protein‐dependent expansion of non‐protective CD4+ T cells determined dilution or loss of the protective Ag85B‐specific CD4+ induced by DNA‐85B vaccination. These data emphasize the need of exerting some caution in adopting aggressive DNA‐priming, protein‐booster schedules for MTB vaccines. They also suggest that Ag85B protein secreted during MTB infection could be involved in the instability of protective anti‐tuberculosis immune response, and actually concur to disease progression.

Metronidazole plus Rifampin Sterilizes Long-Term Dormant Mycobacterium tuberculosis

ABSTRACT Long-term nonreplicating (dormant) Mycobacterium tuberculosis populations (26-day-old cells) were sterilized by metronidazole plus rifampin, but not by metronidazole or rifampin alone, after 7 and 11 days of exposure to the drugs. Lower or no drug activity was observed against 19- or 12-day-old dormant or 5-day-old actively replicating populations.

Mycobacterium tuberculosis Drug Resistance, Abkhazia

To the Editor: Drug-resistant tuberculosis (TB) has been identified as a major problem in the former Soviet Union, and was recently surveyed in the Aral Sea regions of Dashoguz (Turkmenistan) and Karakalpakstan (Uzbekistan) (1). However, few data are available for the Caucasian region and published reports have focused mainly on prisons (2,3). We report a drug resistance survey for first- and second-line anti-TB drugs conducted in Abkhazia, a Caucasian region of 8,600 km2 with approximately 250,000 inhabitants, at the western end of Georgia on the Black Sea. The collapse of the Soviet Union lead to disruption of TB control activities in all Eastern bloc regions (4). In Abkhazia, the shortage and poor quality of drugs, self-medication, and poor adherence to the therapy became even more evident during the war with Georgia in 1993 and the international embargo that followed. A TB program based on the World Health Organization/International Union against Tuberculosis and Lung Disease (WHO/IUATLD) recommendations was initiated in Abkhazia with the support of Medecins Sans Frontieres (MSF) in 1999. In 2000, monitoring of drug resistance was started for new cases and previously treated case-patients. The study was performed in collaboration with the Guliripchi TB Hospital, MSF, and the Istituto Superiore di Sanita (ISS), a WHO/IUATLD Supranational Reference Laboratory for anti-TB drug resistance. Sputa were collected from all patients attending Guliripchi TB Hospital in Sukhumi, the capital of Abkhazia, from September 2000 to April 2004. Patients were either referred by their practitioners or came spontaneously because TB was suspected. Diagnosis, treatment, and hospitalization were provided free. Samples were treated as previously described (5). Of 489 sputa collected from individual patients, 447 were culture positive (246 from new case-patients and 201 from previously treated case-patients) and 42 were culture negative; of these, >90% showed a negative, doubtful, or 1+ smear result. Susceptibility to first-line (streptomycin, isoniazid, rifampin, and ethambutol) and second-line (kanamycin, ethionamide, capreomycin, cycloserine, p-aminosalicylic acid, and ofloxacin) drugs was determined by the proportion method on Middlebrook 7H10 agar. The critical concentrations used were streptomycin, 2 µg/mL; isoniazid, 0.2 µg/mL; rifampin, 1 µg/mL; ethambutol, 5 µg/mL; kanamycin, 5 µg/mL; ethionamide, 5 µg/mL; capreomycin, 10 µg/mL; p-aminosalicylic acid, 2 µg/mL; and ofloxacin, 2 µg/mL (6–8). Cycloserine was used at a concentration of 30 µg/mL (9). If a strain was resistant to >1 first-line drugs, the susceptibility to all second-line drugs was determined. Data on resistance to the first- and second-line drugs are given in the Table. The strains isolated from 35.8% of the new cases and 57.2% of the previously treated case-patients were resistant to >1 first-line drugs. The highest monoresistance was seen for isoniazid and streptomycin in both new and previously treated case-patients, while monoresistance to rifampin and ethambutol was low ( 1 second-line drugs. Table First-line and second line antituberculosis drug resistance in 447 Mycobacterium tuberculosis strains collected in Abkhazia from September 2000 to April 2004* Few data have been reported on drug resistance to first- and second-line drugs in the former Soviet Union and in the Caucasian region (1–4). Overall, in Abkhazia, monoresistance to isoniazid was higher than in Karakalpakstan and Dashoguz (1), while monoresistance to streptomycin was lower. MDR-TB in new and previously treated case-patients showed levels intermediate between these 2 regions. Resistance to kanamycin and ethionamide was 14.3% and 12.8%, respectively, while resistance to ofloxacin was low (1.5%). Fluoroquinolones have not been commonly used in Abkhazia and former regions of the Soviet Union. Currently, regimens for the treatment of MDR-TB in Abkhazia combine an intensive phase for a minimum of 6 months with at least 4 drugs to which the MTB strain is susceptible, including 1 parenteral agent and 1 fluoroquinolone (ofloxacin), followed by a continuation phase of at least 15 months with >3 drugs. This is the first survey reporting drug susceptibility data for MTB within the Caucasus. It indicates that the prevalence of MDR strains is similar to that in other central Asia regions (1). Our results are representative of the present situation in Abkhazia since sampling systematically covered all TB cases for the period examined. The Guliripchi TB Hospital of Sukhumi is the only TB treatment center in the region, and all cases were included in the study. Overall, our data show that second-line drug resistance is present in Abkhazia, particularly among cases with MDR, and suggest the adoption of strategies for access and correct use of second-line drugs (10).

Isolation of Nocardia asiatica from Cutaneous Ulcers of a Human Immunodeficiency Virus-Infected Patient in Italy

ABSTRACT A strain of Nocardia was isolated from cutaneous ulcers of a human immunodeficiency virus-infected patient in Italy. Comparative 16S rRNA gene sequence analysis revealed that the isolate represented a strain of Nocardia asiatica. Antimicrobial susceptibility testing was essential to guide the clinicians to successfully treat this infection.

Infection of human THP-1 cells with dormant Mycobacterium tuberculosis.

Dormant, non-replicating Mycobacterium tuberculosis H37Rv strain cultured in hypoxic conditions was used to infect THP-1 cells. CFUs counting, Kinyoun staining and electron microscopy showed that dormant bacilli infected THP-1 cells at a rate similar to replicating M. tuberculosis, but failed to grow during the first 6 days of infection. The absence of growth was specific to the intracellular compartment, as demonstrated by efficient growth in liquid medium. Quantification of β-actin mRNA recovered from infected cells showed that, in contrast with log-phase bacteria, infection with dormant bacilli determined a reduced THP-1 cell death. Gene expression of intracellular non-replicating bacteria showed a pattern typical of a dormant state. Intracellular dormant bacteria induced the activation of genes associated to a proinflammatory response in THP-1 cells. Though, higher levels of TNFα, IL-1β and IL-8 mRNAs compared to aerobic H37Rv infected cells were not paralleled by increased cytokine accumulation in the supernatants. Moreover, dormant bacilli induced a higher expression of inducible cox-2 gene, accompanied by increased PGE2 secretion. Overall, our data describe a new model of in vitro infection using dormant M. tuberculosis that could provide the basis for understanding how non-replicating bacilli survive intracellularly and influence the maintenance of the hypoxic granuloma.