The modification of metabolic pathways to allow for a dormant lifestyle appears to be an important feature for the survival of pathogenic bacteria within their host. One regulatory mechanism for persistent Mycobacterium tuberculosis infections is the stringent response. In this study, we analyze the stringent response of a nonpathogenic, saprophytic mycobacterial species, Mycobacterium smegmatis. The use of M. smegmatis as a tool for studying the mycobacterial stringent response was demonstrated by measuring the expression of two M. tuberculosis genes, hspX and eis, in M. smegmatis in the presence and absence of rel Msm . The stringent response plays a role in M. smegmatis cellular and colony formation that is suggestive of changes in the bacterial cell wall structure.The ability of Mycobacterium tuberculosis to persist in the human host is a major challenge for both vaccine-and drugbased strategies for controlling the spread of tuberculosis (TB) (17). Persistent M. tuberculosis cells are capable of initiating active growth in the host, a condition known as reactivation TB. Although it is somewhat controversial, it is generally believed that the site of viable M. tuberculosis in the host is inside caseous, necrotic, granulomatous lesions in the lungs. It has been reported that M. tuberculosis cells persisting inside granulomas lack the acid-fast staining characteristic of bacteria recovered from sputa and lesions of patents with active disease, indicating that changes occur in the bacterial cell wall during metabolic adaptation to a state of dormancy in the host (18,22). Various models have been developed to create in vitro growth conditions that mimic the presumed state of M. tuberculosis inside granulomas, including oxygen limitation (32, 33) and nutrient starvation (1,20). For example, when M. tuberculosis cultures are suspended in distilled water, they appear to lose their acid-fast staining ability, but the cells can remain viable for over 2 years in this extreme nutrient-deprived environment (22). This implies that in vitro observations of mycobacterial physiology may provide important insights into understanding how pathogenic mycobacteria survive in hosts. In addition, several mutants of M. tuberculosis that are impaired in de novo biosynthesis of various amino acids and vitamins (14) are attenuated in mouse models of tuberculosis infection, suggesting that the organism resides in a nutrient-poor environment. This supports the significance of in vitro nutrient starvation models for understanding in vivo persistence.Recently, the stringent response of M. tuberculosis has been shown to play an important role in the in vitro and in vivo survival of this bacterium (9, 29). Escherichia coli has two homologous genes that are responsible for initiating the stringent response, namely, relA and spoT. Collectively, RelA and SpoT can sense nutrient deprivation and respond by synthesizing guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp), which can alter the promoter specificity of RNA polymeras...

Histoplasmosis is one of the most common and deadly opportunistic infections among persons living with human immunodeficiency virus (HIV)/acquired immune deficiency syndrome in Latin America, but due to limited diagnostic capacity in this region, few data on the burden and clinical characteristics of this disease exist. Between 2005 and 2009, we enrolled patients ≥ 18 years of age with suspected histoplasmosis at a hospital-based HIV clinic in Guatemala City. A case of suspected histoplasmosis was defined as a person presenting with at least three of five clinical or radiologic criteria. A confirmed case of histoplasmosis was defined as a person with a positive culture or urine antigen test for . Demographic and clinical data were also collected and analyzed. Of 263 enrolled as suspected cases of histoplasmosis, 101 (38.4%) were confirmed cases. Median time to diagnosis was 15 days after presentation (interquartile range [IQR] = 5-23). Crude overall mortality was 43.6%; median survival time was 19 days (IQR = 4-69). Mycobacterial infection was diagnosed in 70 (26.6%) cases; 26 (25.7%) histoplasmosis cases were coinfected with mycobacteria. High mortality and short survival time after initial symptoms were observed in patients with histoplasmosis. Mycobacterial coinfection diagnoses were frequent, highlighting the importance of pursuing diagnoses for both diseases.

Mycobacterial species are characterized by the presence of lipid-rich, hydrophobic cell envelopes. These cell envelopes contribute to properties such as roughness of colonies, aggregation of cells in liquid culture without detergent, and biofilm formation. We describe here a mutant strain of Mycobacterium smegmatis, called DL1215, which demonstrates marked deviations from the above-mentioned phenotypes. DL1215 arose spontaneously from a strain deficient for the stringent response (M. smegmatis ⌬rel Msm strain) and is not a reversion to a wild-type phenotype. The nature of the spontaneous mutation was a single base-pair deletion in the lsr2 gene, leading to the formation of a truncated protein product. The DL1215 strain was complicated by having both inactivated rel Msm and lsr2 genes, and so a single lsr2 mutant was created to analyze the gene's function. The lsr2 gene was inactivated in the wild-type M. smegmatis mc 2 155 strain by allelic replacement to create strain DL2008. Strain DL2008 shows characteristics unique from those of both the wild-type and ⌬rel Msm strains, some of which include a greatly enhanced ability to slide over agar surfaces (referred to here as "hypermotility"), greater resistance to phage infection and to the antibiotic kanamycin, and an inability to form biofilms. Complementation of the DL2008 mutant with a plasmid containing lsr2 (pLSR2) reverts the strain to the mc 2 155 phenotype. Although these phenotypic differences allude to changes in cell surface lipids, no difference is observed in glycopeptidolipids, polar lipids, apolar lipids, or mycolic acids of the cell wall.Mycobacterium smegmatis is a fast-growing, saprophytic mycobacterial species. Although considered nonpathogenic, M. smegmatis provides a popular model for studying virulence mechanisms of slow-growing, pathogenic relatives such as Mycobacterium tuberculosis (9,16,37,41) and Mycobacterium leprae (35,42). An important aspect of mycobacterial pathogenesis is the ability of the pathogen to establish latent infections in hosts lasting for several years. Persistent M. tuberculosis bacilli in the host manifest drastic changes in gene expression that set the cells apart from actively growing tubercle bacilli (36,40). One bacterial regulatory network that coordinates nutrient deprivation with adaptive metabolism is the stringent response. In mycobacteria this global regulatory system is controlled by a single gene called rel, and deletion of this gene in M. tuberculosis results in a severe defect in both long-term in vitro and in vivo survival (10, 30). We recently reported that the rel gene of M. smegmatis (rel Msm ) is involved in the regulation of cellular and colony morphologies (9). As seen with M. tuberculosis, the stringent response is required for long-term survival of M. smegmatis in culture, since the rel Msm mutant readily dies over a month-long period while in stationary phase.Here we report the appearance of a mutant strain, called DL1215, that arose spontaneously from the parental M. smegmatis ⌬rel Msm strain. We...

Background A Diagnostic Laboratory Hub (DLH) was set up in Guatemala to provide opportunistic infection (OI) diagnosis for people with HIV (PWH). Methods Patients newly presenting for HIV, PWH not receiving antiretrovirals (ARVs) for >90 days but returned to care (Return/Restart), and PWH on ARVs with symptoms of OIs (ARV treatment) were prospectively included. Screening for tuberculosis, nontuberculous mycobacteria (NTM), histoplasmosis, and cryptococcosis was done. Samples were couriered to the DLH, and results were transmitted electronically. Demographic, diagnostic results, disease burden, treatment, and follow-up to 180 days were analyzed. Results In 2017, 1953 patients were included, 923 new HIV infections (an estimated 44% of all new HIV infections in Guatemala), 701 on ARV treatment, and 315 Return/Restart. Three hundred seventeen (16.2%) had an OI: 35.9% tuberculosis, 31.2% histoplasmosis, 18.6% cryptococcosis, 4.4% NTM, and 9.8% coinfections. Histoplasmosis was the most frequent AIDS-defining illness; 51.2% of new patients had <200 CD4 cells/mm3 with a 29.4% OI incidence; 14.3% of OIs in new HIV infections occurred with CD4 counts of 200–350 cells/mm3. OIs were the main risk factor for premature death for new HIV infections. At 180 days, patients with OIs and advanced HIV had 73-fold greater risk of death than those without advanced disease who were OI-free. Conclusions The DLH OI screening approach provides adequate diagnostic services and obtains relevant data. We propose a CD4 screening threshold of <350 cells/mm3. Mortality remains high, and improved interventions are required, including expansion of the DLH and access to antifungal drugs, especially liposomal amphotericin B and flucytosine.

Guatemala is a developing country in Central America with a high burden of HIV and endemic fungal infections; we attempted to estimate the burden of serious fungal infections for the country. A full literature search was done to identify epidemiology papers reporting fungal infections from Guatemala. We used specific populations at risk and fungal infection frequencies in the population to estimate national rates. The population of Guatemala in 2013 was 15.4 million; 40% were younger than 15 and 6.2% older than 60. There are an estimated 53,000 adults with HIV infection, in 2015, most presenting late. The estimated cases of opportunistic fungal infections were: 705 cases of disseminated histoplasmosis, 408 cases of cryptococcal meningitis, 816 cases of Pneumocystis pneumonia, 16,695 cases of oral candidiasis, and 4,505 cases of esophageal candidiasis. In the general population, an estimated 5,568 adult asthmatics have allergic bronchopulmonary aspergillosis (ABPA) based on a 2.42% prevalence of asthma and a 2.5% ABPA proportion. Amongst 2,452 pulmonary tuberculosis patients, we estimated a prevalence of 495 for chronic pulmonary aspergillosis in this group, and 1,484 for all conditions. An estimated 232,357 cases of recurrent vulvovaginal candidiasis is likely. Overall, 1.7% of the population are affected by these conditions. The true fungal infection burden in Guatemala is unknown. Tools and training for improved diagnosis are needed. Additional research on prevalence is needed to employ public health measures towards treatment and improving the reported data of fungal diseases.