Mycobacterium lentiflavum in Drinking Water Supplies, Australia

Mycobacterium lentiflavum, a slow-growing nontuberculous mycobacterium, is a rare cause of human disease. It has been isolated from environmental samples worldwide. To assess the clinical significance of M. lentiflavum isolates reported to the Queensland Tuberculosis Control Centre, Australia, during 2001-2008, we explored the genotypic similarity and geographic relationship between isolates from humans and potable water in the Brisbane metropolitan area. A total of 47 isolates from 36 patients were reported; 4 patients had clinically significant disease. M. lentiflavum was cultured from 13 of 206 drinking water sites. These sites overlapped geographically with home addresses of the patients who had clinically significant disease. Automated repetitive sequence-based PCR genotyping showed a dominant environmental clone closely related to clinical strains. This finding suggests potable water as a possible source of M. lentiflavum infection in humans.

Characterization of Actinobacteria Degrading and Tolerating Organic Pollutants

Species of the genera Rhodococcus, Gordonia and Mycobacterium are known as degraders of recalcitrant pollutants. These bacteria are good survivors in harsh environments. Due to such properties these organisms are able to occupy a wide range of environmental niches. The members of these taxa have been suggested as tools for biotechnical applications such as bioremediation and biosynthesis. At the same time several of the species are known as opportunistic human pathogens. Therefore, the detailed characterization of any isolate that has potential for biotechnological applications is very important. This thesis deals with several corynebacterial strains originating from different polluted environments: soil, water-damaged indoor walls, and drinking water distribution systems. A polyphasic taxonomic approach was applied for characterization of the isolates. We found that the strains degrading monoaromatic compounds belonged to Rhodococcus opacus, a species that has not been associated with any health problem. The taxonomic position of strain B293, used for many years in degradation research under different names, was clarified. We assigned it to the species Gordonia polyisoprenivorans. This species is classified under European Biohazard grouping 1, meaning that it is not considered a health hazard for humans. However, there are reports of catheter-associated bacteraemia caused by G. polyisoprenivorans. Our results suggested that the ability of the organism to grow on phthalate esters, used as softeners in medical plastics, may be associated with the colonization of catheters and other devices. In this thesis Mycobacterium lentiflavum, a new emerging opportunistic human pathogen, was isolated from biofilms growing in public drinking water distribution systems. Our report on isolation of M. lentiflavum from water supplies is the second report on this species from drinking water systems, which may thus constitute a reservoir of M. lentiflavum. Automated riboprinting was evaluated for its applicability in rapidly identifying environmental mycobacteria. The technique was found useful in the characterization of several species of rapidly and slowly growing environmental mycobacteria. The second aspect of this thesis refers to characterization of the degradation and tolerance power of several R. opacus, M. murale and G. polyisoprenivorans strains. R. opacus GM-14 utilizes a wide range of aromatic substrates, including benzene, 15 different halobenzenes, 18 phenols and 7 benzoates. This study revealed the high tolerance of R. opacus strains toward toxic hydrophobic compounds. R. opacus GM-14 grew in mineral medium to which benzene or monochlorobenzene was added in amounts of 13 or 3 g l-1, respectively. R. opacus GM-29 utilized toluene and benzene for growth. Strain GM-29 grew in mineral medium with 7 g l-1 of liquid toluene or benzene as the sole carbon source, corresponding to aqueous concentrations of 470 and 650 mg l-1, respectively. Most organic solvents, such as toluene and benzene, due to their high level of hydrophobicity, pass through the bacterial membrane, causing its disintegration. In this thesis the mechanisms of adaptation of rhodococci to toxic hydrophobic compounds were investigated. The rhodococcal strains increased the level of saturation of their cellular fatty acids in response to challenge with phenol, chlorophenol, benzene, chlorobenzene or toluene. The results indicated that increase in the saturation level of cellular fatty acids, particularly that in tuberculostearic acid, is part of the adaptation mechanism of strains GM-14 and GM-29 to the presence of toxic hydrophobic compounds.

Detection of nontuberculous mycobacteria from water buffalo raw milk in Brazil

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Occurrence of mycobacteria in bovine milk samples from both individual and collective bulk tanks at farms and informal markets in the southeast region of Sao Paulo, Brazil

Background: Mycobacterium spp. is one of the most important species of zoonotic pathogens that can be transmitted from cattle to humans. The presence of these opportunistic, pathogenic bacteria in bovine milk has emerged as a public-health concern, especially among individuals who consume raw milk and related dairy products. To address this concern, the Brazilian control and eradication program focusing on bovine tuberculosis, was established in 2001. However, bovine tuberculosis continues to afflict approximately 1,3 percent of the cattle in Brazil. In the present study, 300 samples of milk from bovine herds, obtained from both individual and collective bulk tanks and informal points of sale, were cultured on Löwenstein-Jensen and Stonebrink media. Polymerase chain reaction (PCR)-based tests and restriction-enzyme pattern analysis were then performed on the colonies exhibiting phenotypes suggestive of Mycobacterium spp., which were characterized as acid-fast bacilli.Results: Of the 300 bovine milk samples that were processed, 24 were positively identified as Mycobacterium spp.Molecular identification detected 15 unique mycobacterial species: Mycobacterium bovis, M. gordonae, M. fortuitum, M. intracellulare, M. flavescens, M. duvalii, M. haemophilum, M. immunogenum, M. lentiflavum, M. mucogenicum, M. novocastrense, M. parafortuitum, M. smegmatis, M. terrae and M. vaccae. The isolation of bacteria from the various locations occurred in the following proportions: 9 percent of the individual bulk-tank samples, 7 percent of the collective bulk-tank samples and 8 percent of the informal-trade samples. No statistically significant difference was observed between the presence of Mycobacterium spp. in the three types of samples collected, the milk production profiles, the presence of veterinary assistance and the reported concerns about bovine tuberculosis prevention in the herds.Conclusion: The microbiological cultures associated with PCR-based identification tests are possible tools for the investigation of the presence of Mycobacterium spp. in milk samples. Using these methods, we found that the Brazilian population may be regularly exposed to mycobacteria by consuming raw bovine milk and related dairy products. These evidences reinforces the need to optimize quality programs of dairy products, to intensify the sanitary inspection of these products and the necessity of further studies on the presence of Mycobacterium spp. in milk and milk-based products. © 2013 Franco et al.; licensee BioMed Central Ltd.

Isolation and Identification of Environmental Mycobacteria in the Waters of a Hemodialysis Center

The use of poorly treated water during hemodialysis may lead to contamination with nontuberculous mycobacteria (NTM). This study aimed to isolate and identify NTM species in the water of a Brazilian hemodialysis center. We collected 210 samples of water from the hydric system of the unit (post-osmosis system, hemodialysis rooms, reuse system, and hemodialysis equipment) and from the municipal supply network; we isolated the NTM by a classic microbiological technique and identified them by the PCR restriction enzyme pattern of the hsp65 gene (PRA). Fifty-one (24.3 %) of the collected samples tested positive for NTM; both the municipal supply network (2 samples, 3.2 %) and the hydric system of the hemodialysis center (49 samples, 96.1 %) contained NTM. We isolated and identified potentially pathogenic bacteria such as Mycobacterium lentiflavum (59.0 %) and M. kansasii (5.0 %), as well as rarely pathogenic bacteria like M. gordonae (24.0 %), M. gastri (8.0 %), and M. szulgai (4.0 %). The ability of NTM to cause diseases is well documented in the literature. Therefore, the identification of NTM in the water of a Brazilian hemodialysis center calls for more effective water disinfection procedures in this unit.

Clinical significance of Mycobacterium asiaticum isolates in Queensland, Australia

Mycobacterium asiaticum was first reported as a cause of human disease in 1982, with only a few cases in the literature to date. This study aims to review the clinical significance of M. asiaticum isolates in Queensland, Australia. A retrospective review (1989 to 2008) of patients with M. asiaticum isolates was conducted. Data were collected through the Queensland TB Control Centre database. Disease was defined in accordance with the American Thoracic Society criteria. Twenty-four patients (13 female) had a positive culture of M. asiaticum, many residing around the Tropic of Capricorn. M. asiaticum was responsible for pulmonary disease (n = 2), childhood lymphadenitis (n = 1), olecranon bursitis (n = 1), 6 cases of possible pulmonary disease, and 2 possible wound infections. Chronic lung disease was a risk factor for pulmonary infection, and wounds/lacerations were a risk factor for extrapulmonary disease. Extrapulmonary disease responded to local measures. Pulmonary disease responded to ethambutol-isoniazid-rifampin plus pyrazinamide for the first 2 months in one patient, and amikacin-azithromycin-minocycline in another patient. While M. asiaticum is rare in Queensland, there appears to be an environmental niche. Although often a colonizer, it can be a cause of pulmonary and extrapulmonary disease. Treatment of pulmonary disease remains challenging. Extrapulmonary disease does not mandate specific nontuberculous mycobacterium (NTM) treatment.

Risk factors for Mycobacterium tuberculosis infection among children in Greenland

Objective To examine the risk factors for Mycobacterium tuberculosis infection (MTI) among Greenlandic children for the purpose of identifying those at highest risk of infection. Methods Between 2005 and 2007, 1797 Greenlandic schoolchildren in five different areas were tested for MTI with an interferon gamma release assay (IGRA) and a tuberculin skin test (TST). Parents or guardians were surveyed using a standardized self-administered questionnaire to obtain data on crowding in the household, parents’ educational level and the child’s health status. Demographic data for each child – i.e. parents’ place of birth, number of siblings, distance between siblings (next younger and next older), birth order and mother’s age when the child was born – were also extracted from a public registry. Logistic regression was used to check for associations between these variables and MTI, and all results were expressed as odds ratios (ORs) and 95% confidence intervals (CIs). Children were considered to have MTI if they tested positive on both the IGRA assay and the TST. Findings The overall prevalence of MTI was 8.5% (152/1797). MTI was diagnosed in 26.7% of the children with a known TB contact, as opposed to 6.4% of the children without such contact. Overall, the MTI rate was higher among Inuit children (OR: 4.22; 95% CI: 1.55–11.5) and among children born less than one year after the birth of the next older sibling (OR: 2.48; 95% CI: 1.33–4.63). Self-reported TB contact modified the profile to include household crowding and low mother’s education. Children who had an older MTI-positive sibling were much more likely to test positive for MTI themselves (OR: 14.2; 95% CI: 5.75–35.0) than children without an infected older sibling. Conclusion Ethnicity, sibling relations, number of household residents and maternal level of education are factors associated with the risk of TB infection among children in Greenland. The strong household clustering of MTI suggests that family sources of exposure are important.

Mycobacterium abscessus infection and potable water

Mycobacterium abscessus is a rapidly growing mycobacteria responsible for progressive pulmonary disease, soft tissue and wound infections, and can contaminate clinical specimens. Nontuberculous mycobacteria (NTM) are generally considered environmental organisms though M. abscessus has not featured frequently in environmental studies, particularly those examining potable water. In a study of Brisbane potable water, M. abscessus was isolate from ten different locations. The incidence of disease due to M. abscessus has been increasing in Queensland. Aim: To compare genotypically the M. abscessus isolates obtained from water to those obtained from human clinical specimens. Methods: From a study of Brisbane potable water between 2007 and 2009, ten isolates confirmed as M. abscessus were recovered. In addition, one strain was isolated from a rainwater tank of a patient with disease due to M. avium, and another from the swimming pool of a patient with M. intracellulare disease. A random sample of 74 clinical isolates referred to the QLD Mycobacterial reference laboratory during the same time period was available for comparison using repPCR strain typing (Diversilab). Results: The drinking water isolates formed two distinct strain patterns (A and B) that shared >90% similarity. The tankwater isolate (pattern C) shared >85% similarity with the potable water isolates, but the pool isolate (D) was distinctly different. Fifty-three clinical isolates clustered tightly (>95% similarity) with the Group A potable water isolates, 4 patients with Group B. Thirteen patient isolates clustered with the Rainwater tank isolate. One patient matched the pool isolate. There were a further 3 patient isolates that were unrelated to the water isolates. No differences were found between strain types in terms of geographic origin, gender, age, or site/type of infection. Conclusion: The high degree of similarity between strains of M. abscessus from potable water and strains causing infection in humans from the same area, strengthens the possibility that drinking water may be a source of infection in these patients.

SRL 172 (killed Mycobacterium vaccae) in addition to standard chemotherapy improves quality of life without affecting survival, in patients with advanced non-small-cell lung cancer : phase III results

Background: This open-label, randomised phase III study was designed to further investigate the clinical activity and safety of SRL172 (killed Mycobacterium vaccae suspension) with chemotherapy in the treatment of non-small-cell lung cancer (NSCLC). Patients and methods: Patients were randomised to receive platinum-based chemotherapy, consisting of up to six cycles of MVP (mitomycin, vinblastine and cisplatin or carboplatin) with (210 patients) or without (209 patients) monthly SRL172. Results: There was no statistical difference between the two groups in overall survival (primary efficacy end point) over the course of the study (median overall survival of 223 days versus 225 days; P = 0.65). However, a higher proportion of patients were alive at the end of the 15-week treatment phase in the chemotherapy plus SRL172 group (90%), than in the chemotherapy alone group (83%) (P = 0.061). At the end of the treatment phase, the response rate was 37% in the combined group and 33% in the chemotherapy alone group. Patients in the chemotherapy alone group had greater deterioration in their Global Health Status score (-14.3) than patients in the chemotherapy plus SRL172 group (-6.6) (P = 0.02). Conclusion: In this non-placebo controlled trial, SRL172 when added to standard cancer chemotherapy significantly improved patient quality of life without affecting overall survival times. © 2004 European Society for Medical Oncology.

Mycobacterium abscessus isolated from municipal water : a potential source of human infection

Background Mycobacterium abscessus is a rapidly growing mycobacterium responsible for progressive pulmonary disease, soft tissue and wound infections. The incidence of disease due to M. abscessus has been increasing in Queensland. In a study of Brisbane drinking water, M. abscessus was isolated from ten different locations. The aim of this study was to compare genotypically the M. abscessus isolates obtained from water to those obtained from human clinical specimens. Methods Between 2007 and 2009, eleven isolates confirmed as M. abscessus were recovered from potable water, one strain was isolated from a rainwater tank and another from a swimming pool and two from domestic taps. Seventy-four clinical isolates referred during the same time period were available for comparison using rep-PCR strain typing (Diversilab). Results The drinking water isolates formed two clusters with ≥97% genetic similarity (Water patterns 1 and 2). The tankwater isolate (WP4), one municipal water isolate (WP3) and the pool isolate (WP5) were distinctly different. Patient isolates formed clusters with all of the water isolates except for WP3. Further patient isolates were unrelated to the water isolates. Conclusion The high degree of similarity between strains of M. abscessus from potable water and strains causing infection in humans from the same geographical area, strengthens the possibility that drinking water may be the source of infection in these patients.

Heterogeneity of clinical and environmental isolates of Mycobacterium fortuitum using repetitive element sequence-based PCR: municipal water an unlikely source of community-acquired infections

M. fortuitum is a rapidly growing mycobacterium associated with community-acquired and nosocomial wound, soft tissue, and pulmonary infections. It has been postulated that water has been the source of infection especially in the hospital setting. The aim of this study was to determine if municipal water may be the source of community-acquired or nosocomial infections in the Brisbane area. Between 2007 and 2009, 20 strains of M. fortuitum were recovered from municipal water and 53 patients’ isolates were submitted to the reference laboratory. A wide variation in strain types was identified using repetitive element sequence-based PCR, with 13 clusters of ≥2 indistinguishable isolates, and 28 patterns consisting of individual isolates. The clusters could be grouped into seven similar groups (>95% similarity). Municipal water and clinical isolates collected during the same time period and from the same geographical area consisted of different strain types, making municipal water an unlikely source of sporadic human infection.

Mycobacterium tuberculosis groE promoter controls the expression of the bicistronic groESL1 operon and shows differential regulation under stress conditions

Heat shock promoters of mycobacteria are strong promoters that become rapidly upregulated during macrophage infection and thus serve as valuable candidates for expressing foreign antigens in recombinant BCG vaccine. In the present study, a new heat shock promoter controlling the expression of the groESL1 operon was identified and characterized. Mycobacterium tuberculosis groESL1 operon codes for the immunodominant 10 kDa (Rv3418c, GroES/Cpn10/Hsp10) and 60 kDa (Rv3417c, GroEL1/Cpn60.1/Hsp60) heat shock proteins. The basal promoter region was 115 bp, while enhanced activity was seen only with a 277-bp fragment. No promoter element was seen in the groES-groEL1 intergenic region. This operon codes for a bicistronic mRNA transcript as determined by reverse transcriptase-PCR and Northern blot analysis. Primer extension analysis identified two transcriptional start sites (TSSs) TSS1 (-236) and TSS2 (-171), out of which one (TSS2) was heat inducible. The groE promoter was more active than the groEL2 promoter in Mycobacterium smegmatis. Further, it was found to be differentially regulated under stress conditions, while the groEL2 promoter was constitutive.

Sequence specific interaction of Mycobacterium smegmatis topoisomerase I with duplex DNA

We have identified strong topoisomerase sites (STS) for Mycobacteruim smegmatis topoisomerase I in double-stranded DNA context using electrophoretic mobility shift assay of enzyme-DNA covalent complexes; Mg2+, an essential component for DNA relaxation activity of the enzyme, is not required for binding to DNA, The enzyme makes single-stranded nicks, with transient covalent interaction at the 5'-end of the broken DNA strand, a characteristic akin to prokaryotic topoisomerases. More importantly, the enzyme binds to duplex DNA having a preferred site with high affinity, a. property similar to the eukaryotic type I topoisomerases, The preferred cleavage site is mapped on a 65 bp duplex DNA and found to be CG/TCTT. Thus, the enzyme resembles other prokaryotic type I topoisomerases in mechanistics of the reaction, but is similar to eukaryotic enzymes in DNA recognition properties.