Rhodococcus Spp Research Articles

The Detection of Propionate Utilization by Bacteria Isolated from a Plastic Recycling Site

(1) The study aims to utilize a reported approach for culturing mesophilic bacteria from a plastic waste environment; (2) The work revived mesophilic microbial population from an aged PET recycling site using a culture-based approach, and determined the purified isolates in genus level in 16S identification; (3) A total of 59 bacterial isolates were obtained, in which microbial species, including Pseudomonas spp, Rhodococcus spp, and Burkholderia spp were identified as abundance. It was observed that the surviving microbes favoured sodium propionate as a short-chain carbon source for growth, rather than the intended plastic substrate, PET. The preference of sodium propionate utilization by several bacterial isolates, including 5601W (detected as Rhodococcus spp.), 5601Y, 7801, and 7802 (detected as Burkholderia spp.), was confirmed through growth curve analysis and cell enumeration conducted in a medium where sodium propionate served as the sole carbon source.; (4) The microbial demonstration revealed the metabolic complex of microbial communities in the environment and indicated the challenges associated with bacterial isolation from environments with accumulated plastic waste.

PECULIARITIES OF THE GUT MICROBIOTA IN PATIENTS WITH MIGRAINE COMPARING TO HEALTHY INDIVIDUALS.

The aim: Analyze the gut microbiome state in patients with migraine (M) and healthy individuals, to assess possible correlations between the detected changes in patients with migraine and the frequency, intensity of headaches, psycho-emotional state of the patients, and their quality of life. Materials and methods: 100 objects were enrolled, divided into 2 groups: main - patients with M and control - healthy volunteers. Investigation of the intestinal microbiome was performed by chromato-mass spectrometry. For M patients the following scales were used: Visual Analogue Scale (VAS), Migraine Disability Assessment (MIDAS), Back Depression Inventory (BDI). Results: In main group increased amount of Alcaligenes spp (p = 0.0061), Clostridium coccoides (p = 0.0021), Clostridium propionicum (p = 0.0287), Eggerthella lenta (p = 0.0138), Pseudonocardia spp (p = 0.0210), Rhodococcus spp (p = 0.0164), Candida spp (p = 0.0079), Micromycetes spp (campesterol) (p = 0.0011) were found. Patients with M had a raised amount of Herpes simplex (p = 0.0305) and endotoxin level (p = 0.0459). Differences in gut microorganisms in both groups were significant. In patients with M negative correlations were observed between Alcaligenes spp ammount and BDI score (r = -0.6226, p =0.007), VAS score (r = -0.489, p = 0.046), headache frequency (r = -0.487, p = 0.046); between the levels of Clostridium coccoides and MIDAS score (r =-0.51, p = 0.035), BDI score (r = -0.54, p = 0.025) and positive correlation between Eggerthella lenta level and VAS score (r =0.4830, p=0.049). Conclusions: Correlations between changes of gut microbiome and M are promising for further research.

Effect of native microorganism Rhodococcus spp. SL-9 for dibenzothiophene degradation and its application towards clean coal approach

Abstract Combustion of coal emits many harmful gases, causing huge environmental problems. Harmful gases are, sulfur dioxide, nitrogen oxides and carbon dioxide, which disturbing eco-condition of environment. Apart from all other emissions sulfur emissions has significant effect on environment as well as on human health. To remove sulfur from coal various methods had been proposed as well as investigated by various researchers. It is necessary to utilize economic as well as ecofriendly technique, which is nothing but the microbial technique. Current study focus on removal of organic sulfur from coal using novel Rhodococcus spp. (SL-9). From literature and previous studies, it was proving that the amount of higher content of type of sulfur present in coal is organic sulfur, which can be found in the form of dibenzothiophene (DBT). Rhodococcus spp. (SL-9). was utilized as bio-catalyst for degradation of DBT via 4S-Pathway. Rhodococcus spp. (SL-9) degrade 0.26 mM DBT within 6 days via 4S pathway. Impact of various process parameters on bio-desulfurization were studied, which are temperature, pH, agitation intensity and different carbon source, which have considerable effect on degradation of DBT. The efficiency of Rhodococcus spp. (SL-9) for maximum degradation and conversion of DBT into 2-HBP (2-hydroxybiphenyl) at optimized parameters are 30 °C, 160 rpm, and glucose as carbon source. This could suggest that Rhodococcus spp. (SL-9) had ability to degrade DBT compound from coal.

Bio-Guided Isolation of Antimalarial Metabolites from the Coculture of Two Red Sea Sponge-Derived Actinokineospora and Rhodococcus spp.

Coculture is a productive technique to trigger microbes’ biosynthetic capacity by mimicking the natural habitats’ features principally by competition for food and space and interspecies cross-talks. Mixed cultivation of two Red Sea-derived actinobacteria, Actinokineospora spheciospongiae strain EG49 and Rhodococcus sp. UR59, resulted in the induction of several non-traced metabolites in their axenic cultures, which were detected using LC–HRMS metabolomics analysis. Antimalarial guided isolation of the cocultured fermentation led to the isolation of the angucyclines actinosporins E (1), H (2), G (3), tetragulol (5) and the anthraquinone capillasterquinone B (6), which were not reported under axenic conditions. Interestingly, actinosporins were previously induced when the axenic culture of the Actinokineospora spheciospongiae strain EG49 was treated with signalling molecule N-acetyl-d-glucosamine (GluNAc); this finding confirmed the effectiveness of coculture in the discovery of microbial metabolites yet to be discovered in the axenic fermentation with the potential that could be comparable to adding chemical signalling molecules in the fermentation flask. The isolated angucycline and anthraquinone compounds exhibited in vitro antimalarial activity and good biding affinity against lysyl-tRNA synthetase (PfKRS1), highlighting their potential developability as new antimalarial structural motif.

Genomic deletions in Rhodococcus based on transformation of linear heterologous DNA.

Several genome engineering methods have been developed for Rhodococcus. However, they suffer from limitations such as extensive cloning, multiple steps, successful expression of heterologous genes via plasmid etc. Here, we report a rapid method for performing genomic deletions/disruptions in Rhodococcus spp. using heterologous linear DNA. The method is cost effective and less labour intensive. The applicability of the method was demonstrated by successful disruption of rodA and orphan parA. None of the disrupted genes were found to be essential for the viability of the cell. Disruption of orphan parA and rodA resulted in elongated cells and short rods, respectively. This is the first report demonstrating disruption of rodA and orphan parA genes by electroporation of heterologous linear DNA in Rhodococcus spp.

Study of duplicated galU genes in Rhodococcus jostii and a putative new metabolic node for glucosamine-1P in rhodococci

BackgoundStudying enzymes that determine glucose-1P fate in carbohydrate metabolism is important to better understand microorganisms as biotechnological tools. One example ripe for discovery is the UDP-glucose pyrophosphorylase enzyme from Rhodococcus spp. In the R. jostii genome, this gene is duplicated, whereas R. fascians contains only one copy. MethodsWe report the molecular cloning of galU genes from R. jostii and R. fascians to produce recombinant proteins RjoGalU1, RjoGalU2, and RfaGalU. Substrate saturation curves were conducted, kinetic parameters were obtained and the catalytic efficiency (kcat/Km) was used to analyze enzyme promiscuity. We also investigated the response of R. jostii GlmU pyrophosphorylase activity with different sugar-1Ps, which may compete for substrates with RjoGalU2. ResultsAll enzymes were active as pyrophosphorylases and exhibited substrate promiscuity toward sugar-1Ps. Remarkably, RjoGalU2 exhibited one order of magnitude higher activity with glucosamine-1P than glucose-1P, the canonical substrate. Glucosamine-1P activity was also significant in RfaGalU. The efficient use of the phospho-amino-sugar suggests the feasibility of the reaction to occur in vivo. Also, RjoGalU2 and RfaGalU represent enzymatic tools for the production of (amino)glucosyl precursors for the putative synthesis of novel molecules. ConclusionsResults support the hypothesis that partitioning of glucosamine-1P includes an uncharacterized metabolic node in Rhodococcus spp., which could be important for producing diverse alternatives for carbohydrate metabolism in biotechnological applications. General significanceResults presented here provide a model to study evolutionary enzyme promiscuity, which could be used as a tool to expand an organism's metabolic repertoire by incorporating non-canonical substrates into novel metabolic pathways.

The cell organization underlying structural colour is involved in Flavobacterium IR1 predation

Flavobacterium IR1 is a gliding bacterium with a high degree of colonial organization as a 2D photonic crystal, resulting in vivid structural coloration when illuminated. Enterobacter cloacae B12, an unrelated bacterium, was isolated from the brown macroalga Fucus vesiculosus from the same location as IR1. IR1 was found to be a predator of B12. A process of surrounding, infiltration, undercutting and killing of B12 supported improved growth of IR1. A combination of motility and capillarity facilitated the engulfment of B12 colonies by IR1. Predation was independent of illumination. Mutants of IR1 that formed photonic crystals less effectively than the wild type were reduced in predation. Conversely, formation of a photonic crystal was not advantageous in resisting predation by Rhodococcus spp. PIR4. These observations suggest that the organization required to create structural colour has a biological function (facilitating predation) but one that is not directly related to the photonic properties of the colony. This work is the first experimental evidence supporting a role for this widespread type of cell organization in the Flavobacteriia.

Construction of Genetic Logic Gates Based on the T7 RNA Polymerase Expression System in Rhodococcus opacus PD630.

Rhodococcus opacus PD630 (R. opacus) is a nonmodel, Gram-positive bacterium that holds promise as a biological catalyst for the conversion of lignocellulosic biomass to value-added products. In particular, it demonstrates both a high tolerance for and an ability to consume inhibitory lignin-derived aromatics, generates large quantities of lipids, exhibits a relatively rapid growth rate, and has a growing genetic toolbox for engineering. However, the availability of genetic parts for tunable, high-activity gene expression is still limited in R. opacus. Furthermore, genetic logic circuits for sophisticated gene regulation have never been demonstrated in Rhodococcus spp. To address these shortcomings, two inducible T7 RNA polymerase-based expression systems were implemented for the first time in R. opacus and applied to the construction of AND and NAND genetic logic gates. Additionally, three isopropyl β-d-1-thiogalactopyranoside (IPTG)-inducible promoters were created by inserting LacI binding sites into newly characterized constitutive promoters. Furthermore, four novel aromatic sensors for 4-hydroxybenzoic acid, vanillic acid, sodium benzoate, and guaiacol were developed, expanding the gene expression toolbox. Finally, the T7 RNA polymerase platform was combined with a synthetic IPTG-inducible promoter to create an IMPLY logic gate. Overall, this work represents the first demonstration of a heterologous RNA polymerase system and synthetic genetic logic in R. opacus, enabling complex and tunable gene regulation in this promising nonmodel host for bioproduction.

A Novel Approach for Bioleaching of Sulfur, Iron, and Silica Impurities from Coal by Growing and Resting Cells of Rhodococcus spp

Coal is one of the most important sources of fossil energy on earth. However, direct combustion of coal with a high sulfur content can cause various environmental problems. Other constituents of coal that can cause environmental problems include iron oxide (hematite), iron hydroxide, and silica. In this study, growing and resting cells of Rhodococcus erythropolis strains PD1, R1, and FMF, and R. qingshengii were used in heterotrophic removal of sulfur and bioleaching of iron and silica from coal. All of the mentioned strains have an ability of dibenzothiophene (DBT) desulfurization via 4-S pathway. 2-hydroxybiphenyl, sulfate, and ferric ions (Fe3+) were assayed by Gibb’s test, barium chloride (BaCl2), and thiocyanate ions (SCN−), respectively. FTIR and XRF analyzer were used for detection of the coal bioleaching process by the selected strain of R. erythropolis (PD1). Results indicated that all strains have the ability to grow on coal as the sulfur source. Among them, strain PD1 produced the highest optical density and continued to grow even after 150-h incubation. In both growing- and resting-cells experiments, strain PD1 desulfurized coal most readily compared to other strains. Results of XRF showed that growing cells of strain PD1 had high desulfurizing ability of coal (46%) compared to resting cells in the absence of any carbon sources (24%). Growing cells of strain PD1 also leached 46% of the iron and 14% of the silicate after 7 days of incubation. Resting cells of PD1 leached 32% of the iron as determined by XRF analysis. Also, growing cells of PD1 removed most SiO2 from coal as detected and confirmed by FTIR and XRF. To the best of our knowledge, this is the first report on bioleaching of iron and silica from coal by R. erythropolis strain PD1, making it a suitable candidate for coal bioremediation.

Assessment of Indoor Microbial Quality of Library’s Premise: Case of Central Library of the University of Yaoundé I

Background: Good indoor air quality is important for human health and comfort, because people spend a most of their time within buildings. Microbial pollution is a key element of indoor air pollution. Bacteria and fungi growing indoors when sufficient moisture is available usually cause indoor air pollution. Methods: This study was conducted to assess the microbial concentration and to identify the main bacteria and fungi in the indoor environment of Central Library of the University of Yaounde I. A total of 76 samples were taken from indoor air, surfaces and mouldy books. Bioaerosol sampling and air concentration were made by passive air sampling technique using petri dishes containing different culture media and exposed for 30, 60 and 90 min in the morning and afternoon. Sampling of surfaces and mouldy books were made by rubbing using sterile swab. The identification of the isolated microorganisms was based on macroscopic, microscopic and biochemical characters. Results: The concentrations of bacteria and fungi in the indoor environment of Central Library of the University of Yaounde I ranged between 747 and 2324 CFU/m for the air and 40 and 500 CFU/cm2 for surfaces. In the examined area, the predominant culturable species of microflora were members of the following bacteria genera; Bacillus spp, Staphylococcus spp, Micrococcus spp, Pseudomonas spp, Rhodococcus spp, Enterobacter spp, Klebsiella spp and Escherichia spp and fungi; Aspergillus spp, Penicillium spp, Curvularia spp, Mucor spp, Cladosporium spp, Candida spp Rhodotorula spp, Fusarium spp, Trichophyton spp, Acremonium spp, Aureobasidium spp, Rhizopus spp and Chrysonilia spp. Conclusion: High concentrations of bacteria and fungi were observed in the central library of the University of Yaounde I. Precautions and safety measures should be taken to reduce microbial pollution at universities libraries by improving libraries ventilation and disinfection.

Biodegradation and chemotaxis of polychlorinated biphenyls, biphenyls, and their metabolites by Rhodococcus spp.

Two biphenyl-degrading bacterial strains, SS1 and SS2, were isolated from polychlorinated biphenyl (PCB)-contaminated soil. They were identified as Rhodococcus ruber and Rhodococcus pyridinivorans based on the 16S rRNA gene sequence, as well as morphological, physiological and biochemical characteristics. SS1 and SS2 exhibited tolerance to 2000 and 3000mg/L of biphenyl. And they could degrade 83.2 and 71.5% of 1300mg/L biphenyl within 84h, respectively. In the case of low-chlorinated PCB congeners, benzoate and 3-chlorobenzoate, the degradation activities of SS1 and SS2 were also significant. In addition, these two strains exhibited chemotactic response toward TCA-cycle intermediates, benzoate, biphenyl and 2-chlorobenzoate. This study indicated that, like the flagellated bacteria, non-flagellated Rhodococcus spp. might actively seek substrates through the process of chemotaxis once the substrates are depleted in their surroundings. Together, these data provide supporting evidence that SS1 and SS2 might be good candidates for restoring biphenyl/PCB-polluted environments.

Evaluation of Heavy Metals Resistance in Biofilm Cells of Native Rhodococcus spp. Isolated from Soil

Evaluation of Heavy Metals Resistance in Biofilm Cells of Native Rhodococcus spp. Isolated from Soil

Biodegradation of the organic disulfide 4,4'-dithiodibutyric acid by Rhodococcus spp.

Four Rhodococcus spp. exhibited the ability to use 4,4'-dithiodibutyric acid (DTDB) as a sole carbon source for growth. The most important step for the production of a novel polythioester (PTE) using DTDB as a precursor substrate is the initial cleavage of DTDB. Thus, identification of the enzyme responsible for this step was mandatory. Because Rhodococcus erythropolis strain MI2 serves as a model organism for elucidation of the biodegradation of DTDB, it was used to identify the genes encoding the enzymes involved in DTDB utilization. To identify these genes, transposon mutagenesis of R. erythropolis MI2 was carried out using transposon pTNR-TA. Among 3,261 mutants screened, 8 showed no growth with DTDB as the sole carbon source. In five mutants, the insertion locus was mapped either within a gene coding for a polysaccharide deacetyltransferase, a putative ATPase, or an acetyl coenzyme A transferase, 1 bp upstream of a gene coding for a putative methylase, or 176 bp downstream of a gene coding for a putative kinase. In another mutant, the insertion was localized between genes encoding a putative transcriptional regulator of the TetR family (noxR) and an NADH:flavin oxidoreductase (nox). Moreover, in two other mutants, the insertion loci were mapped within a gene encoding a hypothetical protein in the vicinity of noxR and nox. The interruption mutant generated, R. erythropolis MI2 noxΩtsr, was unable to grow with DTDB as the sole carbon source. Subsequently, nox was overexpressed and purified, and its activity with DTDB was measured. The specific enzyme activity of Nox amounted to 1.2 ± 0.15 U/mg. Therefore, we propose that Nox is responsible for the initial cleavage of DTDB into 2 molecules of 4-mercaptobutyric acid (4MB).

Isolation and characterization of actinobacteria from Yalujiang coastal wetland, North China

ObjectiveTo evaluate various types of samples from the different marine environments as sources of actinomycetes from the Yalujiang coastal wetland, North China, and to screen their antimicrobial properties. Further, the identified actinomycetes were characterized based on morphological, biochemical, and physiological characteristics. MethodsEight different production media were used to isolate actinomycetes from different stations of marine soil sediments in Yalujiang coastal wetland and the genotypic positions were established by 16S rDNA. ResultsA total of 172 actinomycetal isolates were obtained from 13 samples using five media. The most effective culture media in the isolation of actinobacteria were Gause's Synthetic agar and Starch-casein agar. Among 172 isolates, 46 isolates (26.74%) showed antibacterial activity, 70.93% belonged to the genus Streptomyces, others were Micromonospora spp. and Rhodococcus spp. Out of the 46 isolates, two cultures were further supported by morphological characterization analysis. ConclusionsThis is the first report about actinomycetes isolated from Yalujiang coastal wetland and it seems that the promising isolates from the unusual/unexplored wetland may prove to be an important step in the development of microbial natural product research.

Comparative integrated omics: identification of key functionalities in microbial community-wide metabolic networks.

Background:Mixed microbial communities underpin important biotechnological processes such as biological wastewater treatment (BWWT). A detailed knowledge of community structure and function relationships is essential for ultimately driving these systems towards desired outcomes, e.g., the enrichment in organisms capable of accumulating valuable resources during BWWT.Methods:A comparative integrated omic analysis including metagenomics, metatranscriptomics and metaproteomics was carried out to elucidate functional differences between seasonally distinct oleaginous mixed microbial communities (OMMCs) sampled from an anoxic BWWT tank. A computational framework for the reconstruction of community-wide metabolic networks from multi-omic data was developed. These provide an overview of the functional capabilities by incorporating gene copy, transcript and protein abundances. To identify functional genes, which have a disproportionately important role in community function, we define a high relative gene expression and a high betweenness centrality relative to node degree as gene-centric and network topological features, respectively.Results:Genes exhibiting high expression relative to gene copy abundance include genes involved in glycerolipid metabolism, particularly triacylglycerol lipase, encoded by known lipid accumulating populations, e.g., Candidatus Microthrix parvicella. Genes with a high relative gene expression and topologically important positions in the network include genes involved in nitrogen metabolism and fatty acid biosynthesis, encoded by Nitrosomonas spp. and Rhodococcus spp. Such genes may be regarded as ‘keystone genes’ as they are likely to be encoded by keystone species.Conclusion:The linking of key functionalities to community members through integrated omics opens up exciting possibilities for devising prediction and control strategies for microbial communities in the future.

Selective isolation of Actinomycetes from mangrove sediment of Tanjung Lumpur, Kuantan, Malaysia.

Aims: Mangroves of Tanjung Lumpur, Kuantan, Pahang is considered as a relatively underexplored resource of actinomycetes. Based on the above perspective, a study was conducted on mangrove sediments of Tanjung Lumpur, Kuantan to isolate potential actinomycetes using several pretreatments and various selective media. Methodology and results: Sediments from five different sites at Tanjung Lumpur mangrove were collected and selectively pre-treated. The pretreated sediments were diluted and plated onto eight different selective media. A total of 172 potential actinomycetes were isolated from all the media. Antimicrobial activities of 61 selected strains were checked against 8 test microorganisms using cross streak method. Pretreatment of wet heat with seawater was the most effective method for the isolation of actinomycetes as it yielded a maximum of 105 actinomycete isolates and IM7 was the most suitable medium for actinomycete isolation with highest percentage of recovery (31 %). Forty three isolates (70.5 %) showed antimicrobial activities against one or more test microorganisms. Isolates IIUM B21 and IIUM B31 showed antimicrobial activity against all test microorganisms. Seven isolates showed antifungal activity as they inhibited only C. albicans. Ten isolates were randomly selected for identification based on partial sequences of 16S rRNA gene. Six isolates were found belong to the genus Streptomyces, two isolates belong to the genus Micromonospora and two isolates were identified as Rhodococcus spp. Conclusion, significance and impact of study: These findings revealed the potential of mangrove sediment of Tanjung Lumpur as an important source of actinomycetes with biosynthetic capabilities which might be beneficial to pharmaceutical industries.

A Case of Recurrent Meningitis Caused by Rhodococcus species Successfully Treated with Antibiotic Treatment and Intrathecal Injection of Vancomycin through an Ommaya Reservoir.

Human infection by Rhodococcus species is rare and mostly limited to immunocompromised hosts such as patients infected with the human immunodeficiency virus (HIV) or organ transplant recipients. The most common strain is R. equi, and the most common clinical presentation is pulmonary infection, reported in 80% of Rhodococcus spp. infections. The central nervous system is an uncommon infection site. We report a case of a patient with pneumonia, brain abscess, and recurrent meningitis caused by Rhodococcus spp. He initially presented with pneumonia with necrosis, which progressed to brain abscess and recurrent meningitis. Rhodococcus spp. was identified from the cerobrospinal fluid (CSF) collected during his fourth hospital admission. Despite prolonged treatment with appropriate antibiotics, meningitis recurred three times. Finally, in order to administer antibiotics directly into the CSF and bypass the blood-brain barrier, an Ommaya reservoir was inserted for administration of 90 days of intrathecal vancomycin and amikacin in conjunction with intravenous and oral antibiotics; the patient was finally cured with this treatment regimen.

Chloromuconolactone dehalogenase ClcF of actinobacteria

This work investigated the distribution of the clcF gene in actinobacteria isolated from different ecotopes. The gene encodes chloromuconolactone dehalogenase (CMLD) ClcF, the enzyme found to date in only one representative of Gram‐positive bacteria, Rhodococcus opacus 1CP, adapted to 2‐chlorophenol (2CP). Using primers specific to the clcF gene, from the DNA matrix of rhodococcal strains closely related to species Rhodococcus wratislaviensis (P1, P12, P13, P20, G10, KT112, KT723, BO1) we obtained PCR products whose nucleotide sequences were 100% identical to that of the clcF gene from strain R. opacus 1CP. CMLDs isolated from the biomass of strains Rhodococcus spp. G10 and P1 grown on 2CP did not differ by their subunit molecular mass deduced from the known amino acid sequence of the clcF gene from the ClcF of strain R. opacus 1CP. Matrix‐assisted laser dissociation/ionization time‐of‐flight mass spectrometry showed the presence of a peak with m/z 11,194–11,196 Da both in whole cells and in protein solutions with a ClcF activity. Thus, we have first time shown the distribution of ClcF among actinobacteria isolated from geographically distant habitats.

Sputum Smear Concentration May Misidentify Acid-Fast Bacilli As Mycobacterium Tuberculosis in HIV-Infected Patients

Tuberculosis (TB) diagnosis in most resource-limited settings still depends on smear microscopy for identification of acid-fast bacilli (AFB). However, recently developed molecular diagnostics that test for the presence of Mycobacterium tuberculosis (Mtb) DNA have been shown to be superior for confirmation of TB diagnosis. At regular clinical visits over a 12-month period, we collected sputa from HIV-infected patients presenting with signs or symptoms of TB at 2 Nigerian clinics. Sputa were stained for AFB and tested using the Genotype MTBDRplus to confirm the presence of Mtb. Other species were identified using 16S rRNA sequence. In 56% (233/415) of AFB-positive patients, Mtb was confirmed. The patients on antiretroviral therapy were less likely than those not on antiretroviral therapy to be infected with Mtb [odds ratio (OR) = 0.25, P = 0.003]. In a multivariate logistic regression model using clinical features and diagnostic results, abnormal respiratory findings on auscultation (OR = 3.28, P = 0.03) and a direct sputum smear grade >3/100 (OR = 6.4, 4.6, P < 0.02) were significant predictors of Mtb infection. Concentrated sputum smear was predictive of Mtb infection only at the highest grades (2+, 3+). Interestingly, among 65 samples that could not be confirmed for Mtb, 32 (49%) were found to contain other, possibly novel, actinomycetes, including atypical Mycobacteria, Rhodococcus spp, Nocardia spp, and Corynebacterium spp. We conclude that concentrated sputum smears may misidentify other bacteria as Mtb in HIV-infected patients. The use of molecular diagnostics could reduce unnecessary or inappropriate treatment and improve identification of pathogens in resource-limited settings with high HIV burden.

Gene Cluster Encoding Cholate Catabolism in Rhodococcus spp

Bile acids are highly abundant steroids with important functions in vertebrate digestion. Their catabolism by bacteria is an important component of the carbon cycle, contributes to gut ecology, and has potential commercial applications. We found that Rhodococcus jostii RHA1 grows well on cholate, as well as on its conjugates, taurocholate and glycocholate. The transcriptome of RHA1 growing on cholate revealed 39 genes upregulated on cholate, occurring in a single gene cluster. Reverse transcriptase quantitative PCR confirmed that selected genes in the cluster were upregulated 10-fold on cholate versus on cholesterol. One of these genes, kshA3, encoding a putative 3-ketosteroid-9α-hydroxylase, was deleted and found essential for growth on cholate. Two coenzyme A (CoA) synthetases encoded in the cluster, CasG and CasI, were heterologously expressed. CasG was shown to transform cholate to cholyl-CoA, thus initiating side chain degradation. CasI was shown to form CoA derivatives of steroids with isopropanoyl side chains, likely occurring as degradation intermediates. Orthologous gene clusters were identified in all available Rhodococcus genomes, as well as that of Thermomonospora curvata. Moreover, Rhodococcus equi 103S, Rhodococcus ruber Chol-4 and Rhodococcus erythropolis SQ1 each grew on cholate. In contrast, several mycolic acid bacteria lacking the gene cluster were unable to grow on cholate. Our results demonstrate that the above-mentioned gene cluster encodes cholate catabolism and is distinct from a more widely occurring gene cluster encoding cholesterol catabolism.