Evaluation of mycobacteria infection prevalence and optimization of the identification process in North Sardinia, Italy.

IMPORTANCE: Mycobacterial infection is a major threat to public health worldwide. Accurate identification of infected species and drug resistance detection are critical factors in treatment. We focused on shortening the turn-around time of identifying mycobacteria species and antibiotic resistance tests.

Drug resistance and the genotypic characteristics of rpoB and katG in rifampicin- and/or isoniazid-resistant Mycobacterium tuberculosis isolates in central Vietnam.

BACKGROUND: Tuberculosis (TB) and drug-resistant TB (DR-TB) are national health burdens in Vietnam. In this study, we investigated the prevalence of rifampicin (RIF) and/or isoniazid (isonicotinic acid hydrazide, INH) resistance in patients with suspected TB, and applied appropriate techniques to help rapidly target DR-TB. METHODS: In total, 1,547 clinical specimens were collected and cultured using the BACTEC MGIT system (Becton Dickinson and Co.). A resazurin microtiter assay (REMA) was used to determine the proportions of RIF and/or INH resistance. A real-time polymerase chain reaction panel with TaqMan probes was employed to identify the mutations of rpoB and katG associated with DR-TB in clinical isolates. Genotyping of the identified mutations was also performed. RESULTS: A total of 468 Mycobacterium tuberculosis isolates were identified using the REMA. Of these isolates, 106 (22.6%) were found to be resistant to 1 or both antibiotics. Of the resistant isolates, 74 isolates (69.8%) were resistant to isoniazid (INH) only, while 1 isolate (0.94%) was resistant to RIF only. Notably, 31 isolates (29.24%) were resistant to both antibiotics. Of the 41 phenotypically INH-resistant isolates, 19 (46.3%) had the Ser315Thr mutation. There were 8 different rpoB mutations in 22 (68.8%) of the RIF-resistant isolates. The most frequently detected mutations were at codons 531 (37.5%), 526 (18.8%), and 516 (6.3%). CONCLUSION: To help prevent new cases of DR-TB in Vietnam, it is crucial to gain a comprehensive understanding of the genotypic DR-TB isolates.

A rare presentation of tubercular osteomyelitis of the foot.

Tuberculosis is a communicable disease that is a major cause of ill health. It is one of the top ten causes of death worldwide and the leading cause of death from a single infectious agent. Its most common clinical presentation is pulmonary involvement. However, approximately 23-30% of tuberculosis patients have extrapulmonary symptoms. A rare (1%) clinical presentation of tuberculosis is foot and ankle infection. This is complicated by the fact that the diagnosis of osteoarticular tuberculosis is difficult. Our case was a 66-year-old multi-pathological pensioner, who, while working in the countryside, had an ankle sprain on the left foot, with a lacerated wound of about 2 cm diameter. The non-endemic area and the negative chest X-ray made the diagnosis extremely complex. However, a multidisciplinary approach with the radiologists and the infectious disease department led to clinical stabilization of the patient. Therefore, awareness and high index of suspicion of the disease is essential and referral to experts should be made if diagnosis is indeterminate despite extensive investigations. The knowledge allows early identification of the disease and prompt therapy in order to avoid long-standing untreated infections which typically cause bone destruction and loss of function. The knowledge is also mandatory for western physicians due to increasing international travel, immigration from less developed countries and increased use of immunosuppressive medications. We believe that this article can bring awareness around osteoarticular tuberculosis and help with improving outcome and eradication of the infection. Level of clinical evidence: 4.

Use of bedaquiline in spinal osteomyelitis and soft tissue abscess caused by multidrug-resistant Mycobacterium tuberculosis: A case report.

INTRODUCTION: Spinal Tuberculosis (STB) represents between 1% and 2% of total tuberculosis cases. STB management remains challenging; the first-line approach consists of medical treatment, while surgery is reserved for patients with complications. No data regarding STB treatment with bedaquiline-containing regimens are available in the literature. CASE DESCRIPTION: Herein, we report the case of a 21-year-old man from Côte d'Ivoire with a multidrug resistance STB with subcutaneous abscess. After approval of the hospital off-label drug committee, we started bedaquiline 400 mg daily for two weeks, followed by 200 mg three times per week, for 22 weeks, associated with linezolid 600 mg daily, rifabutin 450 mg daily, and amikacin 750 mg daily (interrupted after eight weeks). During treatment, we performed a weekly EKG. No QT prolongation was shown, but inverted T waves appeared, requiring several cardiological consultations and cardiac MRI, but no cardiac dysfunction was found. After 24 weeks, bedaquiline was replaced with moxifloxacin 400 mg daily. The patient continued treatment for another year. We performed another computer tomography at the end of treatment, confirming the cure. DISCUSSION: A salvage regimen containing bedaquiline proved effective in treating multidrug-resistance tuberculosis spinal infection without causing severe adverse effects. However, further studies are needed to evaluate better bedaquiline bone penetration and the correct duration of treatment with bedaquiline in MDR spinal tuberculosis.

Quinoxaline-based efflux pump inhibitors restore drug susceptibility in drug-resistant nontuberculous mycobacteria.

Nontuberculous mycobacteria (NTM) comprise several ubiquitous, environmentally localized bacteria that may be responsible for serious human diseases. NTM-associated pulmonary infections largely affect individuals with underlying respiratory disease or chronic disease and immunosuppressed patients. Mycobacterium simiae and M. abscessus are two NTMs responsible for lung disease in immunocompetent and immunocompromised individuals. In this study, two NTM strains were isolated from two patients admitted to an Italian hospital and were identified as M. simiae and M. abscessus. The two NTMs were tested for drug susceptibility against different antibiotics. To restore drug susceptibility, a new series of 2-aryl-3-phenoxymethyl-quinoxaline derivatives (QXs) was designed, synthesized, and investigated as efflux pump inhibitors (EPIs) against two clinical isolates of the above-cited NTMs, evaluating how EPIs can influence the drug minimal inhibitory concentration values and, therefore, the activity. The different\ resistance levels tracked in the clinical strains were reduced by EPIs, and in several cases, the susceptibility was completely restored. QXs also resulted as potential chemical probes to be used in drug susceptibility tests to identify the resistance origin when detected.

Use of bedaquiline in spinal osteomyelitis and soft tissue abscess caused by multidrug-resistant Mycobacterium tuberculosis: A case report

Abstract Introduction Spinal Tuberculosis (STB) represents between 1% and 2% of total tuberculosis cases. STB management remains challenging; the first-line approach consists of medical treatment, while surgery is reserved for patients with complications. No data regarding STB treatment with bedaquiline-containing regimens are available in the literature. Case description Herein, we report the case of a 21-year-old man from Côte d'Ivoire with a multidrug resistance STB with subcutaneous abscess. After approval of the hospital off-label drug committee, we started bedaquiline 400 mg daily for two weeks, followed by 200 mg three times per week, for 22 weeks, associated with linezolid 600 mg daily, rifabutin 450 mg daily, and amikacin 750 mg daily (interrupted after eight weeks). During treatment, we performed a weekly EKG. No QT prolongation was shown, but inverted T waves appeared, requiring several cardiological consultations and cardiac MRI, but no cardiac dysfunction was found. After 24 weeks, bedaquiline was replaced with moxifloxacin 400 mg daily. The patient continued treatment for another year. We performed another computer tomography at the end of treatment, confirming the cure. Discussion A salvage regimen containing bedaquiline proved effective in treating multidrug-resistance tuberculosis spinal infection without causing severe adverse effects. However, further studies are needed to evaluate better bedaquiline bone penetration and the correct duration of treatment with bedaquiline in MDR spinal tuberculosis.

Solid Lipid Nanoparticles as Formulative Strategy to Increase Oral Permeation of a Molecule Active in Multidrug-Resistant Tuberculosis Management.

The role of mycobacterial efflux pumps in drug-resistant tuberculosis has been widely reported. Recently, a new compound, named SS13, has been synthesized, and its activity as a potential efflux inhibitor has been demonstrated. In this work, the chemical-physical properties of the SS13 were investigated; furthermore, a formulative study aimed to develop a formulation suitable for oral administration was performed. SS13 shows nonintrinsic antitubercular activity, but it increases the antitubercular activity of all the tested drugs on several strains. SS13 is insoluble in different simulated gastrointestinal media; thus, its oral absorption could be limited. Solid lipid nanoparticles (SLNs) were, therefore, developed by using two different lipids, Witepsol and/or Gelucire. Nanoparticles, having a particle size (range of 200-450 nm with regards to the formulation composition) suitable for intestinal absorption, are able to load SS13 and to improve its permeation through the intestinal mucosa compared to the pure compound. The cytotoxicity is influenced by the concentration of nanoparticles administered. These promising results support the potential application of these nanocarriers for increasing the oral permeation of SS13 in multidrug-resistant tuberculosis management.

Phytochemical Compositions and Biological Activities of Essential Oils from the Leaves, Rhizomes and Whole Plant of Hornstedtia bella Skornick.

The rapid emergence of drug-resistant strains and novel viruses have motivated the search for new anti-infectious agents. In this study, the chemical compositions and cytotoxicity, as well as the antibacterial, antifungal, antitrichomonas, and antiviral activities of essential oils from the leaves, rhizomes, and whole plant of Hornstedtia bella were investigated. The GC/MS analysis showed that ß-pinene, E-ß-caryophyllene, and α-humulene were found at high concentrations in the essential oils. The essential oils exhibited (i) inhibition against Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Staphylococcus epidermidis with minimum inhibitory concentrations (MIC) and minimum lethal concentration (MLC) values from 1 to 4% (v/v); (ii) MIC and MLC values from 2 to 16% (v/v) in Candida tropicalis and Candida parapsilosis; (iii) MIC and MLC values from 4 to 16% in Enterococcus faecalis; and (iv) MIC and MLC values from 8 to greater than or equal to 16% (v/v) in the remaining strains, including Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Candida albicans, and Candida glabrata. In antitrichomonas activity, the leaves and whole-plant oils of Hornstedtia bella possessed IC50, IC90, and MLC values of 0.008%, 0.016%, and 0.03% (v/v), respectively, whilst those of rhizomes oil had in turn, 0.004%, 0.008%, and 0.016% (v/v).Besides, the leaf oil showed a weak cytotoxicity against Vero 76 and MRC-5; meanwhile, rhizomes and whole-plant oils did not exert any toxic effects on cell monolayers. Finally, these oils were not active against EV-A71.

"In vitro" activity of Melaleuca cajuputi against mycobacterial species.

The increasing incidence of resistance in tuberculosis and in atypical mycobacterial infections has prompted the search for alternative agents. We explored the antimycobacterial activity of Melaleuca cajuputi essential oil against tubercular and non tubercular mycobacterials isolates. The good activity observed towards M. cajuputi indicated that this essential oil might represent a promising antimicrobial agents, particularly in the management of microbial resistance.

The bifunctional protein GlmU is a key factor in biofilm formation induced by alkylating stress in Mycobacterium smegmatis.

Living organisms have developed specific defence mechanisms to counteract hostile environmental conditions. Alkylation stress response mechanisms also occur in Mycobacterium tuberculosis (MTB) the pathogen responsible for tuberculosis. The effect of alkylating agents on the cellular growth of MTB was investigated using methyl methanesulfonate (MMS) as methyl donor demonstrating that limited doses of alkylating agents might affect MTB cell viability. A global investigation of Mycobacterium smegmatis response to alkylating stress was then pursued by differential proteomics to identify the most affected cellular pathways. Quantitative analysis of proteomic profiles demonstrated that most of the proteins upregulated in the presence of alkylating agents are involved in biofilm formation and/or cell wall biosynthesis. Tailored experiments confirmed that under stress conditions M. smegmatis elicits physical defence mechanisms by increasing biofilm formation. Among the upregulated proteins, we identified the GlmU bifunctional enzyme as a possible factor involved in biofilm production. Experiments with both conditional deletion and overexpressing glmU mutants demonstrated that down regulation of GlmU decreased M. smegmatis capabilities to produce biofilm whereas overexpression of the enzyme increased biofilm formation. These results were supported by inhibition of GlmU acetyltransferase activity with two different inhibitors, suggesting the involvement of this enzyme in the M. smegmatis defence mechanisms.

Design, synthesis and antitubercular activity of 4-alkoxy-triazoloquinolones able to inhibit the M. tuberculosis DNA gyrase.

A number of new F-triazolequinolones (FTQs) and alkoxy-triazolequinolones (ATQs) were designed, synthesized and evaluated for their activity against Mycobacterium tuberculosis H37Rv. Five out of 21 compounds exhibited interesting minimum inhibitory concentration (MIC) values (6.6-57.9 µM), ATQs generally being more potent than FTQs. Two ATQs, 21a and 30a, were endowed with the best anti-Mtb potency (MIC = 6.9 and 6.6 µM, respectively), and were not cytotoxic in a Vero cell line. Tested for activity against M. tuberculosis DNA gyrase in a DNA supercoiling activity assay, 21a and 30a showed IC50 values (27-28 µM) comparable to that of ciprofloxacin (10.6 µM). 21a was next selected for screening against several Mtb strains obtained from clinical isolates, including multi-drug-resistant (MDR) variants. Importantly, this compound was effective in all cases, with very promising MIC values (4 µM) in the case of some isoniazid/rifampicin-resistant Mtb strains. Finally, computer-based simulations revealed that the binding mode of 21a in the Mtb gyrase cleavage core complexed with DNA and the relevant network of intermolecular interactions are utterly similar to those described for ciprofloxacin, yielding a molecular rationale for the comparable anti-mycobacterial and DNA gyrase inhibition activity of this quinolone.

Enhancement of antimicrobial activity of pump inhibitors associating drugs.

INTRODUCTION: with the continuous emergence of pathogenic resistance to conventional drugs through efflux pumps, increasing efforts are directed toward discovering efflux inhibitory molecules. METHODOLOGY: in this study three P-glycoprotein (P13CP, P22CP, P34CP) efflux-inhibitors (EIs), belonging to the series of phenoxymethylquinoxalines capable to restore/potentiate the antiproliferative activity of doxorubicin and vincristine against human tumor cell lines and different antibiotics against clinical isolates, were investigated on 10 clinical strains of Candida and 12 clinical and ATCC strains of Gram positive and Gram-negative bacteria. RESULTS: MFC values of FLC were reduced in all Candida strains by the P22CP and P34CP inhibitors, and in 5/10 fungal strains by the P13CP inhibitor. CONCLUSION: novel antibiotics with new modes of action are urgently required to suppress the rise of MDR bacteria. An alternative approach would be to identify molecules that can interfere with the process of efflux.

Levels of different cytokines in women and men with asymptomatic genital infection caused by Chlamydia.

INTRODUCTION: Immune response to genital Chlamydia trachomatis infection is involved in both immunity and pathology. The cytokine profile during infection has been implicated in the disease outcome, either resolution or severe sequelae. METHODOLOGY: In total, 3900 patients were analyzed for presence of genital infections caused by Chlamydia using molecular assays. Interleukins (IL) IL-10, IL-17, IL-6, IL-2 and chemokine IP-10 were estimated by ELISA in urine, cervical swabs and semen samples. Statistical analysis was performed using the T student test. RESULTS: A total of 47 out of 3900 samples (1.2%) were found to be positive for Chlamydia trachomatis based on the Real Time (RT) PCR results. Statistical analysis revealed that the differences between Chlamydia trachomatis positive and negative samples regarding levels of cytokines were not significant. CONCLUSIONS: Our results demonstrated that no significant difference in cytokine concentrations exists in Chlamydia trachomatis infected patients when compared to healthy controls. In further study, we aim to test on a greater number of positive samples a greater number of cytokines involved in the immune response to Chlamydia trachomatis infections.

Potential of Borojoa patinoi Cuatrecasas water extract to inhibit nosocomial antibiotic resistant bacteria and cancer cell proliferation in vitro.

Borojó (Borojoa patinoi Cuatrec.) is a fruit used in Colombian traditional medicine with supposed antihypertensive, antitumoral, diuretic, healing, immunological, anti-inflammatory and aphrodisiac effects. To explore the relative merits in terms of biological activities of borojó aqueous extract (BAE), we investigated in vitro its antimicrobial activity on nosocomial pathogenic and multidrug resistant (MDR) strains of Pseudomonas aeruginosa (6), Staphylococcus aureus (1) and Candida species (6), as well as its cytotoxicity on human conjunctive Wong-Kilbourne derivative (WKD) cells and Caco-2 cells from heterogeneous human epithelial colorectal adenocarcinoma. The bacteriostatic activity was observed overall on P. aeruginosa strains, as evidenced by the increase of the lag phase (43 hours) and reduction of the maximum growth rate detected using 187.5 mg BAE per mL. The bactericidal activity, instead, was observed at 375 mg BAE per mL. On the other hand, BAE showed an anti-proliferative effect against the Caco-2 cell line and was shown to be toxic on the WKD cell line at concentrations ranging from 0.05 to 187.5 µg mL-1. The analysis of the phenolic fraction of the fruit aqueous extract (BAE) using UHPLC-MS/MS showed the presence of 26 compounds, with vanillic, syringic and o-coumaric acids as the most abundant. Among these molecules, 7.81 ng mL-1 luteolin and myricetin, singly tested, were able to reduce bacterial growth. To the best of our knowledge, we are unaware of any previous studies demonstrating the anti-bacterial activity of borojó aqueous extract against antibiotic resistant strains of P. aeruginosa, and its anti-proliferative effect against WKD and Caco-2 cell lines. The latter result offers a potential base for new interest and investigations in relation to colon carcinoma models and borojó fruit consumption, since in Colombia this fruit is consumed also for its supposed antitumoral effects.

In vitro activity of essential oils against Pseudomonas aeruginosa isolated from infected hip implants.

INTRODUCTION: Essential oils have been used since ancient times and are known for their anti-inflammatory, anti-depressive, antiseptic, antifungal and antimicrobial properties. METHODOLOGY: in this study the antimicrobial activity of two essential oils from Melaleuca alternifolia and Thymus vulgaris-red thyme geraniol was tested against 16 multidrug-resistant P. aeruginosa strains from infected hip implants as well as the "in vitro" cytotoxic activity on normal human Wong-Kilbourne derivative (WKD) cells. RESULTS: Thymus vulgaris-red thyme geraniol showed lower antimicrobial activity when compared to Melaleuca alternifolia. All tested oils were cytotoxic at concentrations lower than 0.12%. CONCLUSION: Increase in drug resistance and lack of new antibiotics may encourage the development of natural treatments together with higher concern on environmental issues and natural lifestyle.

In vitro activity of hybrid lavender essential oils against multidrug resistant strains of Pseudomonas aeruginosa.

INTRODUCTION: Lavender is an evergreen shrub native to Northern Africa and other mountainous Mediterranean regions. It grows throughout Southern Europe, the United States, and Australia. Lavender essential oil has been used since ancient times and is known for its anti-inflammatory, antidepressant, antiseptic, antifungal and antimicrobial properties. METHODOLOGY: in this study, the antimicrobial activity of two Lavender essential oils (Lavanda sumian and Lavanda grosso) against 16 multidrug-resistant P. aeruginosa strains from clinical ocular samples taken from migrant patients has been investigated. The in vitro cytotoxic activity on human Wong-Kilbourne derivative (WKD) conjunctiva cells from healthy patients and nitric oxide synthase (NOS) activity on murine macrophage (J774.1A) were also evaluated. RESULTS: L. sumian showed lower antimicrobial activity when compared to L. grosso. Both lavender oils tested had no cytotoxic effect at very low concentrations, mostly L. grosso. The essential oils extracted from L. sumian and L. grosso significantly reduced NOS in a cell model. CONCLUSION: Increase in drug resistance and lack of new antibiotics may encourage the development of natural antimicrobial treatments.

Beyond pulmonary nontuberculous mycobacteria disease: do extra-pulmonary forms represent an emerging clinical and public health threat?

Pulmonary and extra-pulmonary diseases caused by nontuberculous mycobacteria: new clinical and public health threats http://ow.ly/87Dm30eMFd9.

Pediatric Tuberculosis in Northern Sardinia.

BACKGROUND AND OBJECTIVES: Migration flux is an increasing phenomenon in Italy, and it raises several public health issues and concerns in pediatric infectious diseases. This study investigated the clinical characteristics and outcomes of a pediatric population at high-risk for tuberculosis (TB) and the potential role of immigration as a risk factor. DESIGN: We performed an observational retrospective study of children referred to the only Pediatric Infectious Diseases Unit for Northern Sardinia over a 6-year-period (2009-2014). Main variables assessed included TB skin test (TST), confirmed by quantiFERON Gold in Tube test, thorax X-ray (TX), microbiological culture, direct microscopy for acid-fast bacilli and molecular assays. RESULTS: Of the 246 children (mean age = 5.8 ± 3.9 years) identified, 222 (90.2%) were native to Sardinia and 24 (9.8%) were immigrants. The majority of children (n=205; 83%) were TB-exposed but not infected based on a negative TST and TX. Among the TST positive group (n= 39; 16%), 19 (49%) had latent TB (TX negative), while 20 (51%) had active TB (TX positive). The percent of TST positive children was significantly higher in the immigrant than the native group (42.5% versus 14%, p<0.001). Clinical presentations included pulmonary involvement with hilar lymphadenopathy (72%), pleurisy (13,5%), lateral-cervical lymphadenopathy (9%), pneumonia with calcifications (4.5%) and disseminated TB (4.5%). One child had multidrug-resistant tuberculosis. CONCLUSIONS: Pediatric TB represents a relevant and potentially worsening public health problem in Northern Sardinia. A strict surveillance system and appropriate treatment can prevent the most severe forms and reduce TB transmission.