Mycobacterium Neoaurum Research Articles

Identification and engineering of cholesterol oxidases involved in the initial step of sterols catabolism in Mycobacterium neoaurum

Mycobacteria have been modified to transform sterols to produce valuable steroids. Here, we demonstrated that the oxidation of sterols to sterones is a rate-limiting step in the catabolic pathway of sterols in Mycobacterium neoaurum. Two cholesterol oxidases ChoM1 and ChoM2 involved in the step were identified in M. neoaurum and the ChoM2 shared up to 45% identity with other cholesterol oxidases. We demonstrated that the combination of ChoM1 and ChoM2 plays a significant role in this step. Accordingly, we developed a strategy to overcome this rate-limiting step by augmenting the activity of cholesterol oxidases in M. neoaurum strains to enhance their transformation productivity of sterols to valuable steroids. Our results indicated that the augmentation of ChoM2 achieved 5.57g/l androst-1,4-diene-3,17-dione in M. neoaurum NwIB-01MS and 6.85g/l androst-4-ene-3,17-dione in M. neoaurum NwIB-R10, greatly higher than the original yield, 3.87g/l androst-1,4-diene-3,17-dione and 4.53g/l androst-4-ene-3,17-dione, respectively.

Influence of hydroxypropyl-β-cyclodextrin on phytosterol biotransformation by different strains of Mycobacterium neoaurum

Cyclodextrins (CDs) can improve productivity in the biotransformation of steroids by increasing conversion rate, conversion ratio, or substrate concentration. However, little is known of the proportion of products formed by multi-catabolic enzymes, e.g., via sterol side chain cleavage. Using three strains with different androst-1,4-diene-3,17-dione (ADD) to androst-4-ene-3,17-dione (AD) ratios, Mycobacterium neoaurum TCCC 11028 (MNR), M. neoaurum TCCC 11028 M1 (MNR M1), and M. neoaurum TCCC 11028 M3 (MNR M3), we found that hydroxypropyl-β-cyclodextrin (HP-β-CD) can appreciably increase the ratio of ADD to AD, the reaction rate, and the molar conversion. In the presence of HP-β-CD, conversion of 0.5 g/L of phytosterol (PS) was 2.4, 2.4, and 2.3 times higher in the MNR, MNR M1, and MNR M3 systems, respectively, than in the controls. The ADD proportion increased by 38.4, 61.5, and 5.9 % compared with the control experiment, which resulted in a strong shift in the ADD/AD ratio in the ADD direction. Our results imply that the three PS-biotransforming strains cause efficient side chain degradation of PS, and the increased conversion of PS when using HP-β-CD may be associated with the higher PS concentration in each case. A similar solubilizing effect may not induce a prominent influence on the ADD/AD ratio. However, the different activities of the Δ¹-dehydrogenase of PS-biotransforming strains result in different incremental percentage yields of ADD and ADD/AD ratio in the presence of HP-β-CD.

Two different primary oxidation mechanisms during biotransformation of thymol by gram-positive bacteria of the genera Nocardia and Mycobacterium

Thymol has antibacterial, antifungal, insecticidal, and antioxidative properties which are the basis for the wide use of this compound in the cosmetic, food, and pharmaceutical industries. Although thymol is a ubiquitously occurring substance in the environment, data about its degradation and detoxification by bacteria are sparse. Here, we show the existence of two different pathways for the biotransformation of thymol by Nocardia cyriacigeorgica and Mycobacterium neoaurum which were described for the first time for gram-positive bacteria. The first pathway starts with hydroxylation of thymol to thymohydroquinone (2-isopropyl-5-methylbenzene-1,4-diol) with subsequent oxidation to thymobenzoquinone (2-isopropyl-5-methyl-1,4-benzoquinone). The second pathway involves hydroxylation of the methyl group followed by oxidation to 3-hydroxy-4-isopropylbenzoic acid, possibly via the aldehyde 3-hydroxy-4-isopropylbenzaldehyde. It is noteworthy that the branched side chain of thymol was not oxidized. Similarities and differences of these oxidation processes with those of the gram-negative bacterium Pseudomonas putida, fungi, and plants are discussed and, in addition, the toxicity of thymol towards N. cyriacigeorgica and M. neoaurum was tested. The experiments showed a temporary growth inhibition with 0.025% thymol. This was explained by degradation of thymol and the formation of products which are less toxic than thymol itself.

Cluster of non-tuberculous mycobacteraemia associated with water supply in a haemato-oncology unit

Non-tuberculous mycobacteria (NTM) are ubiquitous environmental organisms but rarely cause infections. Clinical, microbiological and epidemiological investigations and subsequent management of a cluster of NTM bacteraemia on a haemato-oncology unit are reported. From October 2007 to July 2008, five patients being managed for haematological malignancies developed pyrexia and general malaise. Mycobacterium mucogenicum (four patients) and Mycobacterium neoaurum (one patient) were identified from their blood cultures. The environment, in particular the water system, was investigated to identify the source of the infection and multiple water samples were cultured according to established criteria. NTM were also isolated from the hospital water system. Central venous catheters (CVCs) were removed and the patients were successfully treated with antibiotics. Environmental measures and changes in CVC care were introduced to prevent further episodes of NTM bacteraemia in these patients. Despite these measures, NTM continued to be present in the water system, but new clinical cases were not identified. NTM are common environmental organisms and are recognized as being difficult to remove from water systems. CVCs were presumed to be the portal of entry in this cluster of NTM bacteraemia, and the implementation of changes to CVC care protocols was successful in preventing further infections in this immunocompromised patient group.

Identification of phenylalkane derivatives when Mycobacterium neoaurum and Rhodococcus erythropolis were cultured in the presence of various phenylalkanes

Phenylalkanes are ubiquitously found in nature as pollutants originating from oil, gas oil and petrol. Rising commercial demand for mineral oil fractions has led to the increased prevalence of environmental contamination, whereby these particular hydrocarbons are encountered by bacteria which have subsequently developed sophisticated metabolic routes for purposes of degradation. Herein a detailed analysis of these metabolic pathways in the degradation of phenylalkanes by Mycobacterium neoaurum and Rhodococcus erythropolis highlighted preponderance for the formation of certain metabolites of which 17 were identified and whereby striking differences were noticed depending specifically upon the length of the substrate's alkyl chain. Although the degradation of even-numbered phenylalkane substrates was assumed to result in the generation of phenylacetic acid formed due to substrate terminal oxidation and subsequent β-oxidation, cultures of M. neoaurum and R. erythropolis were determined in an extracellular accumulation of odd-numbered acidic metabolites, suggesting a simultaneous presence of sub-terminal degradation mechanisms. However, results obtained from biotransformation assays containing even-chained phenylalkanoic acid intermediates as substrates revealed exclusive β-oxidative mechanisms and no generation of odd-numbered degradation products. R. erythropolis in contrast to M. neoaurum also proved viable for hydroxylation of the aromatic ring of metabolites. Interestingly, the generation of phenylacetic acid and subsequently 2-hydroxyphenyl acetic acid was monitored and entailed the presence of the lactone intermediate 2-coumaranone. These results enhance our understanding of the degradation of phenylalkanes and illustrate the potential application of such species in the bioremediation of these common environmental pollutants and in the strains' diverse abilities to transform mineral oil compounds to new valuable products.

Preparation, Characterization, and Biotransformation of the Inclusion Complex of Phytosterols and Hydroxypropyl-β- cyclodextrin by Mycobacterium neoaurum

The inclusion complex of hydroxypropyl-beta-cyclodextrin (HBbetaCD) and phytosterols (PSs) was prepared and characterized by thermogravimetric analysis (TGA) and infrared (IR) spectroscopy. Biotransformation of the inclusion complex of phytosterols and hydroxypropyl-beta-cyclodextrin (PSs-HBbetaCD) by Mycobacterium neoaurum to 1,4-androstadiene-3,17-dione and 4-androstene-3,17-dione [AD(D)] was studied. The TGA and IR results indicated that the thermal stability of PSs was improved in the complex with HBbetaCD. Biotransformation improved the solubility of PSs in the aqueous medium a lot because the AD(D) production was increased remarkably compared with the control, but growth of the bacteria was inhibited in the presence of HBbetaCD. The optimal inclusion ratio, ultrasonic treating time, dosage, and time of addition of PSs-HBbetaCD complexe were found to be 2:1, 10 min, 1.5 g/30 ml medium, and 48 h after incubation, respectively. This inclusion technique not only increased the availability of the substrates for the microorganisms, but also the capability of these microorganisms to produce AD(D) from PSs.

Conversion of soybean sterols into 3,17-diketosteroids using actinobacteria Mycobacterium neoaurum, Pimelobacter simplex, and Rhodococcus erythropolis

Abstract-Soybean sterols were converted into androst-4-ene-3,17-dione (AD) and 9alpha-hydroxyandrost-4-ene-3,17-dione (9-OH-AD) using three actinobacterium strains. The transformation of a microcrystallic substrate (particle size 5-15 nm) or the transformation in the presence of randomly methylated beta-cyclodextrin (MCD) were carried out by Mycobacterium neoaurum with a phytosterol load of 30 g/l over 144 h with an AD content of 14.5 and 15.2 g/l, respectively. AD obtained in the presence of MCD was transformed into ADD (13.5 g/l) by Pimelobacter simplex cells over 3 h and into 9-OH-AD by Rhodococcus erythropolis cells after 22 h without the isolation of AD from the cultural liquid. The technical product ADD was obtained in 75% yield, based on phytosterol. It contained as impurity 1.25% of AD and 1.5% of 1,2-dehydrotestosterone. In a control experiment-the process of 1,2-dehydrogenation of 20 g/l AD in the water solution of MCD-no by products were isolated. Thus, it is more expedient to introduce the 1,2-double bond into pure AD, whereas R. erythropolis strain with low destructive activity towards steroid nucleus can be used in the mixed culture with M. neoaurum. The crystal product contained, according to HPLC, 80% of 9-OH-AD, and 1.5 AD was combined. The yield of 9-OH-AD (m.p. 218-220 degrees C) based on transformed phytosterol was 56%.

Preparation, Characterization, and Biotransformation of the Inclusion Complex of Phytosterols and Hydroxypropyl-beta-cyclodextrin by Mycobacterium neoaurum

Preparation, Characterization, and Biotransformation of the Inclusion Complex of Phytosterols and Hydroxypropyl-beta-cyclodextrin by Mycobacterium neoaurum

Isolation of rapid growing mycobacteria from soil and water in Iran

A total of 350 soil samples were collected from different part of Uremia city and suburbs. We used 3% sodium lauryl sulfate and 1% NaOH for decontamination of soil samples. Of 350 samples, mycobacteria were isolated from 65 (18.3%) specimens. Mycobacterium fortuitum with 18(5.14) strains yielded the highest frequency of isolation. The other isolates were: Mycobacterium peregrinum 11(3.14%), Mycobacterium flvescens 10 (2.85%), Mycobacterium chelonae 6 (1.71%), Mycobacterium mucogenicum 6(1.71%), Mycobacterium thermoresistable 4(1.14%), Mycobacterium abscessus 3 (0.85%), Mycobacterium neoaurum 2(0.57%), Mycobacterium smegmatis 2 (0.57%) and M. fortuitum third biovalant complex 3 (0.85%). The mean pH of soil was 7.89 ± 0.379 (max 8.5, min 7.5). Our data showed an abundant occurrence of mycobacteria in low pH (P value = 0001). We also collected 120 water samples from rivers, brooks and drinking water. Water samples decontaminated were by adding cetyl pyridinium chloride (CPC) to give final concentration of 0.05%. Mycobacteria isolated from 12 water samples. The predominant isolated species were M. fortuitum and Mycobacterium cheloni. The majority isolates were from brooks and surface waters.

Nontuberculous mycobacterial blood stream and cardiac infections in patients without HIV infection

Nontuberculous mycobacteria rarely cause bacteremia in HIV-negative patients. We describe 16 cases, including the first Mycobacterium neoaurum endocarditis. Nine cases were line related. Most patients were immunocompromised secondary to hematologic malignancy or other comorbid conditions. Amikacin had the most reliable in vitro activity. Combination therapy was frequently used. Mortality was 25%.

A New Steroid-Transforming Strain of Mycobacterium neoaurum and Cloning of 3-Ketosteroid 9α-Hydroxylase in NwIB-01

Using enrichment procedures, five strains that can utilize soybean phytosterols as the sole carbon source were isolated from steroids-contaminated soil samples. Among the isolated strains, the strain NwIB-01 with the highest steroid degradation ability was identified as Mycobacterium neoaurum by morphological, physiological, biochemical tests and 16S rRNA sequence analysis. Meanwhile, the key enzyme gene, which was involved in steroid metabolism and encoding 395-amino acid 3-ketosteroid 9alpha-hydroxylase (KSH), was obtained from M. neoaurum NwIB-01 with the assistance of homology analysis and chromosome walking. To our best knowledge, this is the first report to the gene of key enzyme KSH from M. neoaurum. Strain NwIB-01 exhibited powerful ability of cleaving the side chain specifically from soybean phytosterols to accumulate 4-androstene-3,17-dione (AD) and 1,4-androstadiene-3,17-dione (ADD). It was showed that when cultured in 15 g/l phytosterols, the yield of ADD reached 4.23 g/l while accompanied by 1.76 g/l AD in 96-h-old culture (the molar yield of AD + ADD is 64.7%). The strain NwIB-01 can be applied as excellent phytosterols-transformation strains in potential industrial applications.

Flow injection analysis electrospray ionization mass spectrometry for high-throughput screening of a gene library for amidase activity

In high-throughput screening of gene and mutant libraries, high analysis speeds and short method development times are important factors. Mass spectrometry (MS) is considered to be a generic analytical technique with a relatively short development time. Furthermore, when applying flow injection analysis (FIA) for sample introduction, the requirements for high throughput are met. In this work, the use of a single quadrupole electrospray MS instrument for assaying amidase activity in a gene library is demonstrated. The desired selectivity for measuring the amino acid, the reaction product of the amidase reaction, in the presence of high concentrations of the corresponding amino acid amide substrate was obtained by selective ionization of the amino acid in negative ion mode electrospray. The only sample preparation required was a 200-fold dilution of the reaction mixture. For obtaining quantitative results, a complementary calibration procedure was set up to correct for the change in ionization suppression as a function of conversion. This approach was used to screen a Mycobacterium neoaurum gene library consisting of 11,520 clones with α-methylleucine amide as substrate within 24 h. Conversion was measured on the [M−H] − species of the corresponding α-methylleucine ( m/ z 144). Five positive clones were detected with a conversion ranging from 0.2% to 3.4%.

Infecção por micobactérias de crescimento rápido após procedimentos videocirúrgicos - a hipótese do glutaraldeído

Between August 2006 and February 2007, in the state of Rio de Janeiro, Brazil, a massive outbreak of RGM infections after video laparoscopy was mainly associated to the recently described Mycobacterium massiliense species. All confirmed and probable cases reports described the use of high-level disinfection of medical devices by using 2% glutaraldehyde (2% GA) for 30 min before the surgical procedures. We investigated the susceptibility of the M. massiliense isolates recovered during the outbreak to high-level disinfection after 30 min, 1h, 6h and 10h of exposure to the commercial disinfectants. Reference strains for official mycobactericidal tests such as Mycobacterium abscessus, Mycobacterium bovis, Mycobacterium chelonae, Mycobacterium neoaurum and Mycobacterium smegmatis were included as controls. Although all the reference strains were eliminated in 30 min of exposure to 2% GA, we observed the recovery of all M. massiliense clinical isolates even after 10h of exposure. This study suggests that failures in high-level disinfection and the high tolerance of these M. massiliense clinical strains to the 2% GA were strongly associated to the magnitude of the outbreak.

Phylogenetic analysis of Mycobacterium aurum and Mycobacterium neoaurum with redescription of M. aurum culture collection strains

We examined American Type Culture Collection (ATCC) strains of Mycobacterium aurum and Mycobacterium neoaurum by using multilocus DNA target sequencing. Apart from the type strain, all 10 ATCC M. aurum strains examined were classified incorrectly, with most being reclassified as belonging to the M. neoaurum-'Mycobacterium lacticola' relatedness group. All four M. neoaurum strains were tightly clustered, but heterogeneity was observed within the cluster. As a result of the incorrect annotation of the M. aurum strains, two commonly used methods of identification are compromised and two case reports implicating M. aurum as a human pathogen are probably incorrect, with the isolates probably belonging to the M. neoaurum-'M. lacticola' relatedness group. These findings together with a review of isolates identified at two large reference laboratories suggest that M. aurum is not a clinically significant isolate.

Exochelin Production in Mycobacterium neoaurum

Mycobacterium neoaurum is a soil saprophyte and obligate aerobic bacterium. This group of mycobacterium is relatively fast-growing. They form colonies on nutrient agar at 37 masculineC within 3 - 4 days. In natural soil habitats, bioavailability of iron is limited. To facilitate iron uptake, most mycobacteria produce siderophores. One example is exochelin, which is extracellular and water-soluble. In this report, the production of exochelin in M. neoaurum was induced in iron-deficiency, but repressed under ironsufficiency growth conditions. It is however not induced under zinc-deficiency growth conditions. The growth of this mycobacterium was correlated with exochelin secretion under iron-deficiency culture conditions. When M. neoaurum was grown in defined medium containing 0.04 microg Fe(III)/mL (final concentration), the production of exochelin reached a maximum and the corresponding cell growth was comparable to that under iron-sufficiency conditions. In this study, exochelin was purified from spent supernatant of M. neoaurum by semi-preparative chromatography. When saturated ferric chloride solution was added into the purified exochelin, a ferri-exochelin complex was formed. It is proposed that iron uptake in M. neoaurum is exochelin-mediated.

Mycobacterium neoaurum infection in a patient with renal failure

Mycobacterium neoaurum, a member of the Mycobacterium parafortuitum complex, has only rarely been reported as a pathogen of human infections. We report a case of catheter-related bloodstream infection (CRBSI) due to M. neoaurum in a patient on hemodialysis. The isolate was identified by conventional methods as well as by 16S rRNA gene analysis. The patient was successfully treated with intravenous antibiotics (meropenem and amikacin) for three weeks and the catheter was removed. M. neoaurum should be considered as a possible cause of CRBSI in patients with renal failure. Combination antimicrobial therapy and catheter removal can lead to a favorable clinical outcome.

Degradation of the multiple branched alkane 2,6,10,14-tetramethyl-pentadecane (pristane) in Rhodococcus ruber and Mycobacterium neoaurum

Pristane, a highly branched hydrocarbon that also contains iso-branched termini, was used as a substrate for several alkane-metabolizing bacteria. Rhodococcus ruber and Mycobacterium neoaurum were able to utilize pristane for growth effectively. The intermediates produced by these bacteria during incubation with pristane were analyzed by gas chromatography (GC) and gas chromatography/mass spectra (GC/MS). The products revealed as products of 4-methyl pentanoic acid; methyl butanedioic acid; 2-methyl pentadioic acid; methyl propanedioic acid; 4-methyl heptanedioic acid; and 2,6,10,14-tetramethyl-pentadecan-3-one were detected in M. neoaurum cultures. In R. ruber, methyl butanedioic acid; 2-methyl pentadioic acid; 4,8-dimethylnonanoic acid, 4-methyl heptanedioic acid; 2,6,10-trimethylundecanoic acid; 3,7-dimethyl decanedioic acid; and 2,6,10,14-tetramethyl-pentadecan-3-one were detected. The occurrence of these intermediates showed that pristane could be catabolized not only via mono- but also by a di-terminal oxidation pathway. Furthermore, the presence of 2,6,10,14-tetramethyl-pentadecan-3-one; 3,7-dimethyldecandioate; and 2-methylbutandioate established a third pathway initiated by sub-terminal oxidation at the third carbon atom of pristane. Novel intermediates detected suggest simultaneous sub-terminal and di-terminal oxidation pathways.

Catheter-related bloodstream infections caused by rapidly growing nontuberculous mycobacteria: a case series including rare species

Rapidly growing nontuberculous mycobacteria (RGMs) are responsible for a variety of clinical syndromes in humans including catheter-related blood stream infections (CRBSIs). Recently, we identified a cluster of RGM-associated CRBSI at our institution. We describe the epidemiologic and clinical patterns associated with these infections. We conducted a retrospective single-center review of RGM CRBSI between May 2004 and June 2005. RGMs isolated from blood cultures of 6 patients included Mycobacterium mucogenicum (2), Mycobacterium fortuitum (2), and the rare RGM species, Mycobacterium neoaurum (1) and Mycobacterium septicum (1). All of the patients had a long-term intravascular catheter (mean duration, 6.5 months). Bacteremia was resolved in all patients after catheter removal and appropriate antibiotics. None of the patients suffered a relapse of RGM CRBSI, and all survived to 1 year. RGMs are causative pathogens in both immunosuppressed and immunocompetent individuals with long-term intravascular catheters and blood stream infections. Recent trends at our center suggest that infections with these pathogens are rising.

Mycobacterium neoaurum Bloodstream Infection: Report of 4 Cases and Review of the Literature

We describe a cluster of 4 bloodstream infections with Mycobacterium neoaurum and 5 additional cases from the literature. Infections occurred mainly in immunocompromised hosts who had central venous catheters. Fever was universal at presentation, but local signs of inflammation were rare. Combination antimicrobial therapy and catheter removal resulted in clinical cure.

Cutaneous Mycobacterium neoaurum infection causing scarring alopecia in an immunocompetent host

Cutaneous Mycobacterium neoaurum infection causing scarring alopecia in an immunocompetent host