Biofilm Formation and Biocide Susceptibility Testing of Mycobacterium fortuitum and Mycobacterium marinum

The ability of non-tuberculous mycobacteria to form biofilms may allow for their increased resistance to currently used biocides in medical and industrial settings. This study examines the biofilm growth of Mycobacterium fortuitum and Mycobacterium marinum, using the MBEC™ assay system, and compares the susceptibility of planktonic and biofilm cells to commercially available biocides. With scanning electron microscopy, both M. fortuitum and M. marinum form biofilms that are morphologically distinct. Biocide susceptibility testing suggested that M. fortuitum biofilms displayed increased resistance over their planktonic state. This is contrasted with M. marinum biofilms, which were generally as or more susceptible over their planktonic state. Received: 15 February 2002 / Accepted: 28 March 2002     

Characterization of biofilm growth and biocide susceptibility testing of Mycobacterium phlei using the MBEC assay system

The importance of non-tuberculosis mycobacterial biofilm species in medicine, industry and the environment has recently gained attention. Our objectives were to characterize biofilm growth of Mycobacterium phlei M4, as a model of rapidly growing mycobacteria using the minimal biofilm eradication concentration (MBEC) and to compare biocide susceptibility of planktonic and biofilm organisms. Scanning electron microscopy was also carried out to observe biofilm morphology. With the exception of Sporicidin and Virkon the minimum bactericidal concentration values for all biocides tested were lower than the MBEC values. The MBEC assay system was seen to produce multiple and reproducible biofilms of M. phlei and to be a useful tool for susceptibility studies.     

Mycobacterium marinum biofilm formation reveals cording morphology

Abstract The emergence of the nontuberculosis mycobacteria (NTM) as clinically relevant pathogens has warranted the study of these ubiquitous organisms in the context of their likely environmental niche, the biofilm. We assayed the NTM bacterium Mycobacterium marinum strain 1218R, a fish outbreak isolate, for biofilm formation on different surfaces over time using three different methods. Using the MBEC system, biofilm development occurred continually over the 14-day culture period reaching a mature or stable biofilm state after 7 days postinoculation. Quantification of M. marinum biofilm formation on high-density polyethylene (HDPE), polycarbonate (PC) and silicon (Si) coupons over a 14-day period was evaluated using a continuous flow reactor system. M. marinum developed biofilms on all of the surfaces tested. However, substantially more biofilm accumulated on the silicon than on the other substrates (Si>HDPE>PC) under the same growth conditions indicating that silicon was the most effective substratum studied for the generation of M. marinum biofilms and suggesting a correlation between surface hydrophobicity and attachment. Finally, confocal laser scanning microscopy was used to visualize M. marinum biofilm development in situ over time and revealed an unusual biofilm ultrastructure. Large cell clusters attached to the surface grew in parallel sinuous arrays of cells that formed large cords.     

Mycobacterium fortuitum and Mycobacterium chelonae biofilm formation under high and low nutrient conditions

The rapidly growing mycobacteria (RGM) are broadly disbursed in the environment. They have been recovered from freshwater, seawater, wastewater and even potable water samples and are increasingly associated with non-tuberculous mycobacterial disease. There is scant evidence that non-tuberculous mycobacteria (NTM) and RGM form biofilms. Therefore, an experimental system was designed to assess the ability of RGM to form biofilms under controlled laboratory conditions. A flat plate reactor flow cell was attached to either a high or low nutrient reservoir and monitored by image analysis over time. Two surfaces were chosen for assessment of biofilm growth: silastic which is commonly used in medical settings and high density polyethylene (HDPE) which is prevalent in water distribution systems. The results show that Mycobacterium fortuitum and M. chelonae formed biofilms under both high and low nutrient conditions on both surfaces studied. These results suggest that RGM may form biofilms under a variety of conditions in industrial and medical environments.     

Mycobacterium fortuitum subspecies acetamidolyticum, a new subspecies of Mycobacterium fortuitum

Mycobacterium fortuitum subspecies acetamidolyticum is a new subspecies of M. fortuitum and has an intermediate growth rate. It is a nonphotochromogenic mycobacterium. It does not utilize glutamate but utilizes acetamide as a simultaneous nitrogen and carbon source. It is able to utilize acetate, malate, pyruvate, fumarate, glucose, fructose, and n-propanol as the sole sources of carbon in the presence of ammoniacal nitrogen, but does not utilize them in the presence of glutamate-nitrogen. It is easily differentiated from all rapidly growing mycobacteria by its inability to utilize glutamate as a simultaneous nitrogen and carbon source, and from all slowly growing mycobacteria by its capacity to utilize acetamide as a simultaneous nitrogen and carbon source. Its mycolic acid pattern is different from that of M. fortuitum. However, its deoxyribonucleic acid showed 94% relatedness with that of M. fortuitum. In view of the above findings, it has been designated as a new subspecies of M. fortuitum. The organism was isolated from sputum of a 56-year-old patient with lung disease and is considered to be a lung pathogen. The type strain is ATCC 35931 (NCH E11620).     

Differential Identification of Mycobacterium kansasii and Mycobacterium marinum

This report deals with the differential diagnosis between Mycobacterium marinum and M. kansasii. We found that the two species could be differentiated by using six main tests, namely, the nitrate reduction test, the arylsulfatase test, the ability to grow in the presence of 10.0 mug of amithiazone per ml, the ability to grow in the presence of 5.0 mug of kanamycin per ml, the temperature-ratio test, and the rate of growth on solid medium. In contrast to M. kansasii, considerable variation was observed among strains of M. marinum. However, the evidence obtained was not considered sufficient to justify the conclusion that more than one species was represented among the strains identified as M. marinum.     

Comparative pathogenesis of Mycobacterium marinum and Mycobacterium tuberculosis

A thorough understanding of Mycobacterium tuberculosis pathogenesis in humans has been elusive in part because of imperfect surrogate laboratory hosts, each with its own idiosyncrasies. Mycobacterium marinum is the closest genetic relative of the M. tuberculosis complex and is a natural pathogen of ectotherms. In this review, we present evidence that the similar genetic programmes of M. marinum and M. tuberculosis and the corresponding host immune responses reveal a conserved skeleton of Mycobacterium host–pathogen interactions. While both species have made niche-specific refinements, an essential framework has persisted. We highlight genetic comparisons of the two organisms and studies of M. marinum in the developing zebrafish. By pairing M. marinum with the simplified immune system of zebrafish embryos, many of the defining mechanisms of mycobacterial pathogenesis can be distilled and investigated in a tractable host/pathogen pair.     

Biofilm formation on copper and its control by inhibitor/biocide in cooling water environment

The present study has successfully identified the nitrate reducing bacteria present in the cooling water system and also investigated the performance of industrially applied biocide and inhibitor on the bacterial inhibition. In order to carry out the objective of this study, facilities and methods such as 16S rRNA gene sequencing, Lowry assay, SEM, EIS, ICP-MS and weight loss analysis were being utilized. In this study, two out of the five morphologically dis- similar colonies identified through 16S rRNA gene sequencing, namely the Massilia timonae and the Pseudomonas, were being utilized in the biocorrosion study on copper metal. From the surface analysis using SEM demonstrated the phenomenon of biofilm formation on the copper surface. 2-methylbenzimidazole has the addition of methyl group in the diazole ring position of benzimidazole it has create basicity environment and inhibit the metal deterioration. Meanwhile, it is also deducible from the EIS and protein analysis that com- bination of biocide with either of the inhibitors gives rise to better biocorrosion suppression (0.00178 mpy and 0.00171mpy) as compared to the sole effect of either biocide or inhibitor (0.00219 mpy, 0.00162 and 0.00143). Biocorrosion system biocide with MBM was found to exhibit 65% corrosion inhibition efficiency. Moreover, adoption of 2-Methylbenzimidazole seems to display better performance as compared to Multionic 8151, which is adopted in cooling water system.     

Equivalence of mycobactins from Mycobacterium senegalense, Mycobacterium farcinogenes and Mycobacterium fortuitum

Mycobactins were isolated from five strains designated Mycobacterium farcinogenes and a similar number designated Mycobacterium senegalense following growth under conditions of iron-limitation. These lipid-soluble iron-chelating compounds were characterized by a combination of thin-layer and high-performance liquid chromatography. The mycobactins from both the slow-growing M. farcinogenes and the rapidly-growing M. senegalense strains proved impossible to differentiate both from each other and from those produced by strains of Mycobacterium fortuitum, indicating a close relationship between all three species. However, Nocardia farcinica, previously implicated with the bovine farcy strains, produced a different mycobactin which was easily distinguished by thin-layer chromatography alone.     

Biofilm formation by and antifungal susceptibility of Candida isolates from urine

Biofilm formation (BF) in the setting of candiduria has not been well studied. We determined BF and MIC to antifungals in Candida spp. isolates grown from urine samples of patients and performed a retrospective chart review to examine the correlation with risk factors. A total of 67 Candida spp. isolates were grown from urine samples from 55 patients. The species distribution was C. albicans (54%), C. glabrata (36%), and C. tropicalis (10%). BF varied greatly among individual Candida isolates but was stable in sequential isolates during chronic infection. BF also depended on the growth medium and especially in C. albicans was significantly enhanced in artificial urine (AU) compared to RPMI medium. In nine of the C. albicans strains BF was 4- to 10-fold higher in AU, whereas in three of the C. albicans strains and two of the C. glabrata strains higher BF was measured in RPMI medium than in AU. Determination of the MICs showed that planktonic cells of all strains were susceptible to amphotericin B (AMB) and caspofungin (CASPO) and that three of the C. glabrata strains and two of the C. albicans strains were resistant to fluconazole (FLU). In contrast, all biofilm-associated adherent cells were resistant to CASPO and FLU. The biofilms of 14 strains (28%) were sensitive to AMB (MIC(50) of <1 mug/ml). Correlation between degree of BF and MIC of AMB was not seen in RPMI grown biofilms but was present when grown in AU. A retrospective chart review demonstrated no correlation of known risk factors of candiduria with BF in AU or RPMI. We conclude that BF is a stable characteristic of Candida strains that varies greatly among clinical strains and is dependent on the growth medium. Resistance to AMB is associated with higher BF in AU, which may represent the more physiologic medium to test BF. Future studies should address whether in vitro BF can predict treatment failure in vivo.     

Random insertion transposon mutagenesis of Mycobacterium fortuitum identified mutant defective in biofilm formation

Mycobacterium fortuitum has emerged as a nosocomial infectious agent and biofilm formation attributed for the presence of this bacterium in hospital environment. Transposon random mutagenesis was used to identify membrane-proteins for biofilm formation in M. fortuitum. Ten mutants were shortlisted from a library of 450 mutants for examine their biofilm forming ability. Comparative biofilm ability with respect to wild type M. fortuitum ATCC 6841 showed an altered and delayed biofilm formation in one mutant namely, MT721. Sequence analysis revealed mutation in anthranilate phosphoribosyl transferase (MftrpD), which is associated with tryptophan operon. Functional interaction study of TrpD protein through STRING showed its interaction with chorismate utilizing proteins, majorly involved in synthesis of aromatic amino acid and folic acid, suggesting that biofilm establishment and maintenance requires components of central metabolism. Our study indicates important role of MftrpD in establishment and maintenance of biofilm by M. fortuitum, which may further be explored for drug discovery studies against mycobacterial infections.     

Microbial sensor for drug susceptibility testing of Mycobacterium tuberculosis

Aims

Drug susceptibility testing (DST) of clinical isolates of Mycobacterium tuberculosis is critical in treating tuberculosis. We demonstrate the possibility of using a microbial sensor to perform DST of M. tuberculosis and shorten the time required for DST.

Methods and Results

The sensor is made of an oxygen electrode with M. tuberculosis cells attached to its surface. This sensor monitors the residual oxygen consumption of M. tuberculosis cells after treatment with anti‐TB drugs with glycerine as a carbon source. In principle, after drug pretreatment for 4–5 days, the response differences between the sensors made of drug‐sensitive isolates are distinguishable from the sensors made of drug‐resistant isolates. The susceptibility of the M. tuberculosis H37Ra strain, its mutants and 35 clinical isolates to six common anti‐TB drugs: rifampicin, isoniazid, streptomycin, ethambutol, levofloxacin and para‐aminosalicylic acid were tested using the proposed method. The results agreed well with the gold standard method (LJ) and were determined in significantly less time. The whole procedure takes approximately 11 days and therefore has the potential to inform clinical decisions.

Conclusions

To our knowledge, this is the first study that demonstrates the possible application of a dissolved oxygen electrode‐based microbial sensor in M. tuberculosis drug resistance testing. This study used the microbial sensor to perform DST of M. tuberculosis and shorten the time required for DST.

Significance and Impact of the Study

The overall detection result of the microbial sensor agreed well with that of the conventional LJ proportion method and takes less time than the existing phenotypic methods. In future studies, we will build an O₂ electrode array microbial sensor reactor to enable a high‐throughput drug resistance analysis.     

Biofilm formation and antifungal susceptibility of Trichosporon asahii isolates from Mexican patients

Background

Trichosporon asahii is a yeast-like fungus that has recently gained importance as a cause of opportunistic systemic infections. The pathogenicity and virulence factors of T. asahii remain largely unknown. Because of the association between invasive infections and the use of catheters and related devices, the ability of the microorganism to adhere and form biofilms may play an important role in the pathogenicity during a trichosporonosis.

海分枝杆菌mas基因的功能研究

目的研究海分枝杆菌结核蜡酸合酶(mycocerosic acid synthase,mas)基因在致病中的机制。方法在海分枝杆菌转座子随机突变库中,以菌落形态和抗酸染色的索状形态为标志筛选出索状结构改变的突变株;检测细菌在巨噬细胞内的增殖能力以及斑马鱼感染后的生存率及病理改变。结果获得插入位点位于mas基因不同位置的3个索状结构突变株。海分枝杆菌mas突变株在巨噬细胞内增殖能力减弱;其对斑马鱼的致死能力急剧下降,在斑马鱼体内不能形成肉芽肿并很快被清除。结论海分枝杆菌mas基因与其索状结构形成密切相关,并且对其在巨噬细胞内及宿主体内的生存和繁殖十分重要。     

Characterisation of phenolic glycolipids from Mycobacterium marinum

The phenolic glycolipids from two strains of Mycobacterium marinum have been isolated and characterised. The glycolipids from M. marinum MNC 170 were principally glycosides of diacyl C37, C39 and C41 phenolphthiocerols A, but in M. marinum MNC 842, these lipids were accompanied by glycosides of diacyl phenolphthiodiolones A and novel phthiotriols A with the same overall chain-lengths. The main acyl components of the phenolic glycolipids from M. marinum MNC 170 were C26 dimethyl and C27 and C29 trimethyl-branched fatty acids, but in the lipids of M. marinum MNC 842, the C27 trimethyl acid was the only principal component. The sugar composition of all these glycolipids had been previously shown to correspond to 3-O-methylrhamnose.     

Comparison of the Pathogenicity for Mice of Mycobacterium fortuitum and Mycobacterium abscessus

The pathogenicity for mice of 12 strains of Mycobacterium abscessus was compared with that for 8 strains of M. fortuitum. Both species caused lesions in kidneys and produced "spinning disease" resulting from inner ear infections. No major differences in pathogenicity of these two species were demonstrated. Strain to strain variation was marked, especially with M. abscessus. For example, 1.6 x 10(6) organisms of strain 11188 of M. abscessus produced death in four of five animals within 42 days, whereas strain 380 of M. abscessus failed to produce any deaths within 42 days. In the case of M. fortuitum, the greatest mortality observed was one of five animals, yet the incidence of spinning disease and kidney disease occurred earlier postinfection than in mice infected with M. abscessus. Histologically, abscess formation by a strain of M. abscessus was greater than by a strain of M. fortuitum, but this difference cannot be interpreted as a species difference.