Mycobacterium Species Research Articles

Insight into characteristics and pathogenicity of five rapidly growing non-tuberculous Mycobacterium species isolated from the Siamese fighting fish, Betta splendens

Siamese fighting fish (Betta splendens) play an important role in the global aquarium trade, but their susceptibility to mycobacteriosis could challenge the long-term sustainability of the industry. Thus, this study aimed to characterize rapidly growing non-tuberculous mycobacteria (RGM) isolated from the fish and to investigate their pathogenicity. Five RGM species were investigated, namely Mycobacterium chelonae, M. cosmeticum, M. farcinogenes, M. mucogenicum, and M. senegalense. The isolates were phenotypically and biochemically characterized and assessed for susceptibility to 18 antibiotics and five disinfectants. The pathogenicity of the isolated bacteria was evaluated through experimental infection of the fish by intraperitoneal injection. The results of phenotypic and biochemical typing allowed the identification of some of the isolates and provided preliminary information to distinguish between the bacteria. All isolates were resistant to at least four antibiotics, with multiple antibiotic resistance indexes ranging from 0.22 to 0.61, with M. chelonae having the highest value. Four disinfectants (i.e., ethanol, formalin, chlorine, and povidone‑iodine) showed strong antibacterial activity, whereas potassium permanganate proved less effective. An LD50 value within 7 days showed that M. chelonae had the highest virulence at 7.12 × 105 CFU/fish, followed by M. mucogenicum at 4.25 × 106 CFU/fish and a range of 1.01–1.65 × 107 CFU/fish for the other isolates. Depending on the dose and the isolate administered, some fish displayed acute disease symptoms, while others developed a chronic condition. The acute disease was characterized by short median survival, severe peritonitis, and tissue necrosis. Fish with a chronic infection survived the 42-day trial but were emaciated and had systemic granulomas within their viscera. The findings of this study have not only improved our understanding of the nature of these RGM species but also promoted the development of control strategies to mitigate the negative impact of mycobacteriosis on the Siamese fighting fish industry.

Canine leproid granuloma caused by a member of the Mycobacterium tuberculosis complex.

Canine leproid granuloma (CLG) is a chronic form of dermatitis that has been associated with nontuberculous mycobacterial infections in Africa, Oceania, the Americas, and Europe. We report here a case of CLG associated with a member of the Mycobacterium tuberculosis complex (MTBC), which could be of public health concern. An 8-y-old pet dog developed 0.5-1-cm diameter, raised, firm, nonpruritic, alopecic, painless skin nodules on the external aspects of both pinnae. Histologic examination revealed severe pyogranulomatous dermatitis with intracellular Ziehl-Neelsen-positive bacilli that were immunoreactive by immunohistochemistry using a polyclonal primary antibody that recognizes tuberculous and nontuberculous Mycobacterium species. DNA extracted from formalin-fixed, paraffin-embedded skin sections was tested by a Mycobacterium genus-specific nested PCR assay targeting the 16S rRNA gene. BLAST sequence analysis of 214-bp and 178-bp amplicons showed 99.5% identity with members of the MTBC; however, the agent could not be identified at the species level. Although CLG has been associated traditionally with nontuberculous mycobacterial infections, the role of Mycobacterium spp. within the MTBC as a cause of this condition, and the role of dogs with CLG as possible sources of MTBC to other animals and humans, should not be disregarded given its zoonotic potential.

Functional annotation of rhizospheric phageome of the wild plant species Moringa oleifera.

The study aims to describe phageome of soil rhizosphere of M.oleifera in terms of the genes encoding CAZymes and other KEGG enzymes. Genes of the rhizospheric virome of the wild plant species Moringa oleifera were investigated for their ability to encode useful CAZymes and other KEGG (Kyoto Encyclopedia of Genes and Genomes) enzymes and to resist antibiotic resistance genes (ARGs) in the soil. Abundance of these genes was higher in the rhizospheric microbiome than in the bulk soil. Detected viral families include the plant viral family Potyviridae as well as the tailed bacteriophages of class Caudoviricetes that are mainly associated with bacterial genera Pseudomonas, Streptomyces and Mycobacterium. Viral CAZymes in this soil mainly belong to glycoside hydrolase (GH) families GH43 and GH23. Some of these CAZymes participate in a KEGG pathway with actions included debranching and degradation of hemicellulose. Other actions include biosynthesizing biopolymer of the bacterial cell wall and the layered cell wall structure of peptidoglycan. Other CAZymes promote plant physiological activities such as cell-cell recognition, embryogenesis and programmed cell death (PCD). Enzymes of other pathways help reduce the level of soil H2O2 and participate in the biosynthesis of glycine, malate, isoprenoids, as well as isoprene that protects plant from heat stress. Other enzymes act in promoting both the permeability of bacterial peroxisome membrane and carbon fixation in plants. Some enzymes participate in a balanced supply of dNTPs, successful DNA replication and mismatch repair during bacterial cell division. They also catalyze the release of signal peptides from bacterial membrane prolipoproteins. Phages with the most highly abundant antibiotic resistance genes (ARGs) transduce species of bacterial genera Pseudomonas, Streptomyces, and Mycobacterium. Abundant mechanisms of antibiotic resistance in the rhizosphere include "antibiotic efflux pump" for ARGs soxR, OleC, and MuxB, "antibiotic target alteration" for parY mutant, and "antibiotic inactivation" for arr-1. These ARGs can act synergistically to inhibit several antibiotics including tetracycline, penam, cephalosporin, rifamycins, aminocoumarin, and oleandomycin. The study highlighted the issue of horizontal transfer of ARGs to clinical isolates and human gut microbiome.

N-terminal PPE domain plays an integral role in extracellular transportation and stability of the immunomodulatory Rv3539 protein of the Mycobacterium tuberculosis

Multigene PE/PPE family is exclusively present in mycobacterium species. Only few selected genes of this family have been characterized till date. Rv3539 was annotated as PPE63 with conserved PPE domain at N-terminal and PE-PPE at C-terminal. An α/β hydrolase structural fold, characteristic of lipase/esterase, was present in the PE-PPE domain. To assign the biochemical function to Rv3539, the corresponding gene was cloned in pET-32a (+) as full-length, PPE, and PE-PPE domains individually, followed by expression in E. Coli C41 (DE3). All three proteins demonstrated esterase activity. However, the enzyme activity in the N-terminal PPE domain was very low. The enzyme activity of Rv3539 and PE-PPE proteins was approximately same with the pNP-C4 as optimum substrate at 40 °C and pH 8.0. The loss of enzyme activity after mutating the predicted catalytic triad (Ser296Ala, Asp369Ala, and His395Ala) found only in the PE-PPE domain, confirmed the candidature of the bioinformatically predicted active site residue. The optimal activity and thermostability of the Rv3539 protein was altered by removing the PPE domain. CD-spectroscopy analysis confirmed the role of PPE domain to the thermostability of Rv3539 by maintaining the structural integrity at higher temperatures. The presence of the N-terminal PPE domain directed the Rv3539 protein to the cell membrane/wall and the extracellular compartment. The Rv3539 protein could generate humoral response in TB patients. Therefore, results demonstrated that Rv3539 demonstrated esterase activity. PE-PPE domain of Rv3539 is functionally automated, however, N-terminus domain played a role in protein stabilization and its transportation. Both domains participated in immunomodulation.

Multiple mutations of Mycobacterium intracellulare subsp. chimaera causing false-negative reaction to the transcription-reverse transcription concerted method for pathogen detection

ObjectivesTo report an isolate of Mycobacterium intracellulare subsp. chimaera with multiple mutations in 16S ribosomal RNA (rRNA) gene, resulting in the false-negative reaction to the transcription-reverse transcription concerted (TRC) method for Mycobacterium avium-intracellulare complex. MethodsWe used TRC, polymerase chain reaction (PCR), and Matrix-assisted laser desorption/ionization Time-of-Flight/Mass Spectrometry (MALDI-TOF/MS) methods to identify a clinical isolate in 2021. Due to the discordant results between TRC and PCR or MALDI-TOF MS methods, 16S rRNA sequencing, whole-genome shotgun (WGS) sequencing, and average nucleotide identity (ANI) analysis were employed to identify the isolate. ResultsA mycobacterial isolate from a sputum sample gave negative results for the detection of Mycobacterium tuberculosis complex or M. avium-intracellulare complex by the TRC method. However, the isolate was identified as M. intracellulare by both PCR method and MALDI-TOF MS method. WGS sequencing of 16S rRNA genome revealed eight substitution mutations and one insertion mutation within the region, which could hamper the correct reaction to TRC method. Subsequent ANI analysis between the isolate and various species of nontuberculosis mycobacteria revealed that the isolate could be identified as M. intracellulare subsp. chimaera. ConclusionRare mutations within the 16S rRNA genome resulted in the false-negative identification of Mycobacterium chimaera by the TRC method. WGS sequencing and ANI analysis was necessary to identify the isolate.

Exploring the role of wastewater-based epidemiology in understanding tuberculosis burdens in Africa

Tuberculosis (TB) remains a persistent challenge to public health and presents a substantial menace, especially in developing nations of sub-Saharan Africa. It exerts a considerable strain on healthcare systems in these regions. Effective control requires reliable surveillance, which can be improved by incorporating environmental data alongside clinical data. Molecular advances have led to the development of alternative surveillance methods, such as wastewater-based epidemiology. This studyinvestigated the presence, concentration, and diversity of Mycobacterium tuberculosis complex, the cause of TB, in from six African countries: Ghana, Nigeria, Kenya, Uganda, Cameroon, and South Africa. Samples were collected from wastewater treatment plants. All samples were found to contain Mycobacterium species that have been linked to TB in both humans and animals, including Mycobacterium tuberculosis complex, Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium africanum, and Mycobacterium caprae, at varying concentrations. The highest median concentration was found in Ghana, reaching up to 4.7 Log copies/ml for MTBC, 4.6 Log copies/ml for M. bovis, and 3.4 Log copies/ml for M. africanum. The presence of M. africanum outside of West Africa was found in South Africa, Kenya, and Uganda and could indicate the spread of the pathogen. The study underscores the usefulness of wastewater-based epidemiology for tracking TB and shows that even treated wastewater may contain these pathogens, posing potential public health risks.

Heterologous Expression, Purification and Structural Characterization of Ribonuclease E from Mycobacterium smegmatis

Rapid adaptations to various environmental changes are key to the survival of bacterial cells, which is mediated at the level of transcription initiation and RNA degradation/processing. Ribonuclease E (RNase E), an essential endoribonuclease gene in Mycobacterium species, is a key mediator in most reactions of RNA metabolism such as messenger RNA (mRNA), ribosomal RNA (rRNA) and transfer RNA (tRNA) processing and their degradation qualifying it as an important drug target. Here, we first report the cloning of full-length Mycobacterium smegmatis mc2155 (M. smegmatis) RNase E [MSMEG_4626] and its expression with isopropyl β-D-1-thiogalactopyranoside (IPTG) in Escherichia coli (E. coli) as a glutathione S-transferase (GST) fusion protein. The best expression of the recombinant protein was optimized with respect to different parameters, such as E. coli strains, temperature, IPTG concentrations and time points of induction. Despite the use of protease inhibitors, we observed low-molecular-weight degradation products along with full-length fusion proteins. Maximum expression of soluble and least degraded full-length RNase E was obtained at 32 °C/0.1-mM IPTG, further purified by affinity chromatography using glutathione agarose beads and confirmed by immunoblotting technique using anti-GST antibody. In silico characterization using multiple sequence alignment, homology modeling and docking studies revealed four amino acid residues, Asp613, Phe657, Ile662 and Glu688, in the catalytic domain of RNase E that are completely conserved in both E. coli and Mycobacterium species, also involved in RNA binding and might act as candidate drug targets to combat antibiotic-resistant mycobacterial diseases. Additionally, recombinant GST-RNase E protein can unravel the mechanism of RNA degradation/processing through protein–protein interaction studies.

Mixed infection of three nontuberculous mycobacteria species identified by metagenomic next-generation sequencing in a patient with peritoneal dialysis-associated peritonitis: a rare case report and literature review

BackgroundPeritonitis caused by nontuberculous mycobacteria (NTM) is an infrequent but important complication in patients undergoing peritoneal dialysis (PD). There has been no report of mixed infections with multiple NTM. Peritoneal dialysis-associated peritonitis (PDAP) caused by Mycobacterium abscessus is more common than that caused by M. smegmatis and M. goodii.Case presentationThis case concerns a patient with PDAP caused by gram-positive bacilli, which could not be identified at the species level in successive detections of initial peritoneal effluent. Later, M. smegmatis was detected with no sensitivity results in bacterial culture. However, metagenomic next-generation sequencing (mNGS) and first whole-genome sequences indicated that there were three species coexisting in the culture, including M. smegmatis (24,708 reads), M. abscessus (9224 reads), and M. goodii (8305 reads). This is the first case of PDAP with specific evidence that conventional detection methods isolated a poorly pathogenic NTM, whereas mNGS and first whole-genome sequences identified multiple NTM. Pathogenic bacteria might not be detected using conventional methods due to their lower abundance. This case report is the first description of mixed infections with more than two species of NTM during PDAP.ConclusionsPDAP caused by multiple NTM is rare, and the diagnosis is difficult. When NTM are isolated by conventional tests in patients who are suspected of infection, clinicians should be vigilant, and further tests should be performed to determine the presence of rare or even previously unknown bacteria, for which the quantity is relatively low, but the pathogenicity is high. The rare pathogen may be a primary agent in causing such complications.

Transcriptional Self-Regulation of the Master Nitrogen Regulator GlnR in Mycobacteria.

As a master nitrogen regulator in most actinomycetes, GlnR both governs central nitrogen metabolism and regulates many carbon, phosphate, and secondary metabolic pathways. To date, most studies have been focused on the GlnR regulon, while little is known about the transcriptional regulator for glnR itself. It has been observed that glnR transcription can be upregulated in Mycobacterium smegmatis under nitrogen-limited growth conditions; however, the detailed regulatory mechanism is still unclear. Here, we demonstrate that the glnR gene in M. smegmatis is transcriptionally activated by its product GlnR in response to nitrogen limitation. The precise GlnR binding site was successfully characterized in its promoter region using the electrophoretic mobility shift assay and the DNase I footprinting assay. Site mutagenesis and genetic analyses confirmed that the binding site was essential for in vivo self-activation of glnR transcription. Moreover, based on bioinformatic analyses, we discovered that most of the mycobacterial glnR promoter regions (144 out of 147) contain potential GlnR binding sites, and we subsequently proved that the purified M. smegmatis GlnR protein could specifically bind 16 promoter regions that represent 119 mycobacterial species, including Mycobacterium tuberculosis. Together, our findings not only elucidate the transcriptional self-regulation mechanism of glnR transcription in M. smegmatis but also indicate the ubiquity of the mechanism in other mycobacterial species. IMPORTANCE In actinomycetes, the nitrogen metabolism not only is essential for the construction of life macromolecules but also affects the biosynthesis of secondary metabolites and even virulence (e.g., Mycobacterium tuberculosis). The transcriptional regulation of genes involved in nitrogen metabolism has been thoroughly studied and involves the master nitrogen regulator GlnR. However, the transcriptional regulation of glnR itself remains elusive. Here, we demonstrated that GlnR functions as a transcriptional self-activator in response to nitrogen starvation in the fast-growing model Mycobacterium species Mycobacterium smegmatis. We further showed that this self-regulation mechanism could be widespread in other mycobacteria, which might be beneficial for those slow-growing mycobacteria to adapt to the nitrogen-starvation environments such as within human macrophages for M. tuberculosis.

Mycobacterium avium Subspecies paratuberculosis in Asymptomatic Zoo Herbivores in Poland

Mycobacterial infections are significant issues in zoo animals, influencing animal welfare, conservation efforts, and the zoonotic potential of pathogens. Although tuberculosis is recognised to be highly dangerous, paratuberculosis can also lead to animal losses and is potentially dangerous for humans. The aim of the current study was to confirm whether Mycobacterium avium spp. paratuberculosis (MAP) infections are currently present in zoos in Poland. Faeces samples (n = 131) were collected from different animal species from eight zoos in Poland. The faeces were decontaminated and inoculated into Herrold's Egg Yolk Media. The species was determined using commercial DNA testing. The IS900 was checked using RT-PCR. The culture was positive in seven samples: five with M. avium, one with Mycobacterium fortiatum, and one without any identified Mycobacterium species. RT-PCR confirmed MAP genetic material in nine animals. Our findings represent the first confirmation of MAP in bongo (Tragelaphus eurycerus), indicating that it is present in Polish zoological gardens. Fortunately, the disease can be monitored more easily due to recent legislation (the Animal Health Law).

Ocular Mycobacterium haemophilum infection originating in the cornea: a case report

BackgroundMycobacterium haemophilum is a slow-growing non-chromogenic nontuberculous Mycobacterium species that can cause skin infection or arthritis in an immunocompromised population or in children. Primary infection of the healthy adult cornea is rare. The special requirements for culture make this pathogen difficult to diagnose. The study aims to report the clinical manifestation and treatment process of corneal infection and notify the awareness of M. Haemophilus keratitis among clinicians. This is the first case report of primary M. haemophilum infection in the cornea of healthy adults reported in the literature.Case presentationA 53-year-old healthy goldminer presented with left eye redness and a history of vision loss for four months. The patient was misdiagnosed with herpes simplex keratitis until M. haemophilum was detected using high-throughput sequencing. Penetrating keratoplasty was performed, and a large number of mycobacteria were detected by Ziehl-Neelsen staining of the infected tissue. Three months later, the patient developed conjunctival and eyelid skin infections that manifested as caseous necrosis of the conjunctiva and skin nodules. After excision and debridement of the conjunctival lesions and systemic antituberculosis drug treatment for 10 months, the patient was cured.ConclusionM. haemophilum could cause primary corneal infection in healthy adults, which is an infrequent or rare infection. Owing to the need for special bacterial culture conditions, conventional culture methods do not provide positive results. High-throughput sequencing can rapidly identify the presence of bacteria, which aids in early diagnosis and timely treatment. Prompt surgical intervention is an effective treatment option for severe keratitis. Long-term systemic antimicrobial therapy is crucial.

First identification of mycobacteriosis in Atlantic mackerel (Scomber scombrus).

Mycobacterium infection in fish is a well-known disease problem globally, mainly in the farming of ornamental fish or fish for food. Less is known about the prevalence, distribution and the effects such infections have on wild fish species. Presumptive mycobacteriosis has previously been observed in Atlantic mackerel (Scomber scombrus). Since 2018, there has been an increase in reports of granulomatous kidney disease in Atlantic mackerel with the suspicion of this being mycobacteriosis. A total of six individuals were sent to the Institute of Marine Research for further examination. They were caught in the Nordic Sea by either commercial fishing vessels or during the International Ecosystem Summer Survey in the Nordic Seas (IESSNS research cruise) between 2018 and 2020. Samples for both histological and molecular analysis were collected. Here, we detect a likely novel Mycobacterium species in tissue samples from Atlantic mackerel with this condition, on the basis of rDNA and protein gene sequences. The same unnamed bacterium seems to have been found in some Pacific marine fishes. The macroscopic and histological manifestation of the disease is described. Over the past years, there has been an increase in reports of mycobacteriosis worldwide and climate change has been suggested as one of the driving forces as these bacteria prefer warm water.

Mycobacterium biofilms

The genus Mycobacterium includes some of the deadliest pathogens of History (Mycobacterium tuberculosis, Mycobacterium leprae), but most of the species within the genus are environmental microorganisms. Because some of these nontuberculous mycobacteria (NTM) species can be human pathogens, the study of these mycobacterial biofilms has increased during the last decades, and the interest in this issue increased as well as the growing number of patients with diseases caused by NTM. Different molecular mechanisms have been described, being especially well known the importance of glycopeptidolipids. Moreover, the knowledge of the extracellular matrix has shown important differences with other microorganisms, especially because of the presence of lipidic molecules as a key component of this structure. The clinical importance of mycobacterial biofilms has been described for many chronic diseases, especially lung diseases and implant-related ones, both in vitro and in vivo, and even in patients. Moreover, the biofilm-producing capacity has been proven also in M. tuberculosis, while its importance is not well understood. Biofilm studies have also shown the increasing resistance of mycobacteria in sessile form, and the importance of this resistance in the management of the patients is beyond doubt, being surgery necessary in some cases to cure the patients. Diagnosis of mycobacterial diseases is still based on culture-based techniques designed for the detection of M. tuberculosis. Molecular biology-based methods are also broadly used but again designed for tuberculosis diagnosis. Antimicrobial susceptibility testing is also well developed for tuberculosis, but only some species of NTM have standardized techniques for this purpose. New tools or approaches are necessary to treat these patients, whose importance is increasing, as the number of potential hosts is also increasing throughout the world.

A nontuberculous mycobacterium could solve the mystery of the lady from the Franciscan church in Basel, Switzerland

BackgroundIn 1975, the mummified body of a female has been found in the Franciscan church in Basel, Switzerland. Molecular and genealogic analyses unveiled her identity as Anna Catharina Bischoff (ACB), a member of the upper class of post-reformed Basel, who died at the age of 68 years, in 1787. The reason behind her death is still a mystery, especially that toxicological analyses revealed high levels of mercury, a common treatment against infections at that time, in different body organs. The computed tomography (CT) and histological analysis showed bone lesions in the femurs, the rib cage, and the skull, which refers to a potential syphilis case.ResultsAlthough we could not detect any molecular signs of the syphilis-causing pathogen Treponema pallidum subsp. pallidum, we realized high prevalence of a nontuberculous mycobacterium (NTM) species in brain tissue sample. The genome analysis of this NTM displayed richness of virulence genes and toxins, and similarity to other infectious NTM, known to infect immunocompromised patients. In addition, it displayed potential resistance to mercury compounds, which might indicate a selective advantage against the applied treatment. This suggests that ACB might have suffered from an atypical mycobacteriosis during her life, which could explain the mummy’s bone lesion and high mercury concentrations.ConclusionsThe study of this mummy exemplifies the importance of employing differential diagnostic approaches in paleopathological analysis, by combining classical anthropological, radiological, histological, and toxicological observations with molecular analysis. It represents a proof-of-concept for the discovery of not-yet-described ancient pathogens in well-preserved specimens, using de novo metagenomic assembly.

Bovine brucellosis and tuberculosis at a livestock-wildlife interface in Zimbabwe: A nexus for amplification of a zoonosis or a myth?

Bovine brucellosis and tuberculosis are important zoonoses affecting both livestock and wildlife. The study aimed to investigate seroprevalence of brucellosis and to isolate Brucella spp. and Mycobacterium bovis in cattle from livestock-wildlife interface areas. Three sites were selected from high, medium and low interface. The high interface was adjacent to the park and separated by a broken fence, while the medium and low interface were 15-20 and 50km from the perimeter fence, respectively. Cattle aged ≥2 years were randomly selected and bled for serology. Culture for brucellae and Mycobacterium species was attempted on lymph nodes collected at the slaughter floor. Sera were screened for Brucella antibodies using the Rose Bengal test and confirmed by the Complement fixation test. Data were analysed using a multivariable logistic regression model. Overall, seroprevalence was 11.7% (125/1068; 95% CI: 9.8-13.6%). High interface areas recorded significantly higher (p < 0.05) seroprevalence of 20.9% (85/406; 95% CI: 17.0-24.9%), compared to low 8.9% (31/350; 95% CI: 5.9-11.8%) and medium interface 2.9% (9/312; 95% CI: 1.0-4.8%). Brucella seropositivity was approximately three times higher (OR = 3.3, 95% CI: 2.1-5.3) for Malipati compared to Chiredzi. Similarly, the odds were twice (OR = 2.0, 95% CI: 1.2-3.5) in cows with history of abortion compared to those without. Brucella and Mycobacterium species were not isolated from all samples. The study highlighted the significance of high interface as a nexus for amplification of brucellosis in cattle. Thus, a brucellosis control programme that takes into consideration limiting livestock-wildlife interaction should be considered.

Impact of Pipe Material and Temperature on Drinking Water Microbiome and Prevalence of Legionella, Mycobacterium, and Pseudomonas Species

In drinking water distribution systems (DWDSs), pipe material and water temperature are some of the critical factors affecting the microbial flora of water. Six model DWDSs consisting of three pipe materials (galvanized steel, copper, and PEX) were constructed. The temperature in three systems was maintained at 22 °C and the other 3 at 32 °C to study microbial and elemental contaminants in a 6-week survey using 16S rRNA next-generation sequencing (NGS) and inductively coupled plasma-optical emission spectrometry (ICP-OES). Pipe material and temperature were preferentially linked with the composition of trace elements and the microbiome of the DWDSs, respectively. Proteobacteria was the most dominant phylum across all water samples ranging from 60.9% to 91.1%. Species richness (alpha diversity) ranking was PEX < steel ≤ copper system and elevated temperature resulted in decreased alpha diversity. Legionellaceae were omni-prevalent, while Mycobacteriaceae were more prevalent at 32 °C (100% vs. 58.6%) and Pseudomonadaceae at 22 °C (53.3% vs. 62.9%). Heterogeneity between communities was disproportionately driven by the pipe material and water temperature. The elevated temperature resulted in well-defined microbial clusters (high pseudo-F index) in all systems, with the highest impact in PEX (10.928) followed by copper (9.696) and steel (5.448). Legionellaceae and Mycobacteriaceae are preferentially prevalent in warmer waters. The results suggest that the water temperature has a higher magnitude of impact on the microbiome than the pipe material.

Non-tuberculous mycobacterial pulmonary disease: Awareness survey of front-desk healthcare workers in Northern Tanzania.

Over the past decade, there have been increasing reports of non-tuberculous mycobacteria (NTM) species being implicated in tuberculosis (TB) treatment failure or misdiagnosed as TB. Inadequate awareness of NTM pulmonary disease among healthcare workers (HCWs) may contribute to a low index of suspicion for patients presenting to their hospitals. In this study, we assessed the awareness of NTM pulmonary disease (NTM-PD) among front desk HCWs in Northern Tanzania. A cross-sectional descriptive survey was carried out among front desk HCWs in four administrative regions of Northern Tanzania. A standardized questionnaire was administered to consented participants from four clusters; clinicians, laboratory scientists, nurses, and pharmacists serving TB patients from Regional and District Health Facilities. Each participant was asked a set of questions, scored and the total score for each participant was determined. An awareness score was used to measure the level of awareness. The average score for all participants was estimated including the 95% confidence interval (CI). The overall awareness score was 24.1%, 95% CI 22.0-26.2%. History of training, experience in TB care, level of health facilities, age group, and setting were found to be statistically associated with the level of awareness of study participants. More than two-thirds (67%) of participants believe that pulmonary NTM and TB are clinically similar and 60% are not aware that AFB Microscopy cannot distinguish between the two. Only 13% of participants could mention at least one risk factor for NTM pulmonary disease. The level of awareness of NTM pulmonary disease was poor among HCWs in the surveyed TB clinics. National TB Programs are advised to include a topic on NTM in various on-job TB training packages for HCWs.

Specificity of the innate immune responses to different classes of non-tuberculous mycobacteria.

Mycobacterium avium is the most common nontuberculous mycobacterium (NTM) species causing infectious disease. Here, we characterized a M. avium infection model in zebrafish larvae, and compared it to M. marinum infection, a model of tuberculosis. M. avium bacteria are efficiently phagocytosed and frequently induce granuloma-like structures in zebrafish larvae. Although macrophages can respond to both mycobacterial infections, their migration speed is faster in infections caused by M. marinum. Tlr2 is conservatively involved in most aspects of the defense against both mycobacterial infections. However, Tlr2 has a function in the migration speed of macrophages and neutrophils to infection sites with M. marinum that is not observed with M. avium. Using RNAseq analysis, we found a distinct transcriptome response in cytokine-cytokine receptor interaction for M. avium and M. marinum infection. In addition, we found differences in gene expression in metabolic pathways, phagosome formation, matrix remodeling, and apoptosis in response to these mycobacterial infections. In conclusion, we characterized a new M. avium infection model in zebrafish that can be further used in studying pathological mechanisms for NTM-caused diseases.

Clinical features of rare disseminated Mycobacterium colombiense infection in nine patients who are HIV-negative in Guangxi, China

ObjectivesLocalized or disseminated infection caused by different nontuberculous mycobacteria (NTM) species has been increasingly reported in recent years, but reports of Mycobacterium colombiense infection are extremely rare. Herein, we analyzed the clinical features of patients with disseminated M. colombiense infection. MethodsPatients diagnosed with disseminated M. colombiense infection between February 4, 2016 and August 25, 2021 at the First Affiliated Hospital of Guangxi Medical University were retrospectively analyzed. ResultsNTM infection was diagnosed in 248 HIV-negative patients. Of these, nine patients with disseminated M. colombiense infection were enrolled. Five of these patients were positive for anti-interferon-γ autoantibodies. The lung, lymph nodes, bones, and joints were the most commonly involved organs. Anemia, fever, lymphadenopathy, cough and expectoration, and ostealgia were the most common symptoms. The levels of white blood cells and neutrophils were increased in eight patients. M. colombiense was detected by both metagenomic next-generation sequencing (mNGS) and culture in four patients and only by mNGS in the remaining five patients. All patients received combination anti-NTM therapy; five underwent surgery. The condition of eight patients improved, and one died during the treatment. ConclusionPatients infected with M. colombiense can present as disseminated infections, easily involving multiple organs, such as the lung, lymph nodes, bone, and joints, with fever, lymphadenopathy, and increased white blood cell and neutrophil counts. mNGS plays a crucial role in the early diagnosis of M. colombiense infection. Once diagnosed, timely and effective anti-NTM therapy, combined with local surgery if necessary, can improve the prognosis of patients with this condition.

Clinical pharmacists’ interventions and therapeutic drug monitoring in patients with mycobacterial infections

PurposeInfections caused by non-tuberculous mycobacterium (NTM) are increasing, less well-known by health care clinicians, and usually require long term treatment with multiple antimicrobials. There is no existing evidence on clinical pharmacists’ involvement in the care of patients with NTM infections. We sought to characterize pharmacists’ interventions in providing medication management for patients with NTM infections. MethodsA retrospective review of patients aged 18 years or older seen by a pharmacist specializing in NTM from January 1, 2018 through June 1, 2020 was performed. Charts were reviewed for drug therapy problems identified by a pharmacist. Details regarding therapeutic drug monitoring (TDM) and subsequent dose adjustments were obtained. ResultsSeventy-seven patients were included. Median age was 68.5 years, and most patients were female. The most common mycobacterium species treated was Mycobacterium avium/intracellulare complex. Majority of pharmacist consults (63.6%) were referred by Pulmonology physicians, with remainder by Infectious Diseases clinicians. Identified drug therapy problems included: needs additional therapy (23%), unnecessary therapy (24.3%), different drug needed (6.8%), dose too low (75.7%), dose too high (20.3%), adverse drug reaction (31.1%), and adherence (8.1%). Fifteen patients had TDM performed during treatment. A clinical pharmacist was involved in evaluation of all TDM results. Over half of patients with TDM levels had at least 1 dose change made. A minority of patients (16.9%) experienced clinical failure. ConclusionClinical pharmacists should be involved in this complex care to optimize medication management through identification of drug interactions, tailoring antimicrobial dosing, managing TDM results, and providing adherence counseling.