Recognition of the current role of NTM isolates remains the key step in the management of NTM infections. After recognition, treatment must be guided by attending to the isolated species, the specific syndromes, clinical experience and - for some species - the results of in-vitro susceptibility tests. Surgical therapy is also important for some species (Mycobacterium ulcerans, Mycobacterium scrofulaceum) and for localized infections. The treatment of uncommon species is not yet well defined and recent research on resistance mechanisms has described their importance. The role of biofilms is currently of special concern for various specific infections.

We analyze the effect of amikacin, ciprofloxacin, and clarithromycin, alone and associated with N-acetylcysteine (NAC) and Tween 80, at different times and concentrations in nonpigmented rapidly growing mycobacteria (NPRGM) biofilms. For this purpose, confocal laser scanning microscopy and image analysis were used to study the development and behavior of intrinsic autofluorescence, covered area, thickness, and cell viability in NPRGM biofilms after adding antibiotics alone and associated with antibiofilm agents. In this study, ciprofloxacin is the most active antibiotic against this type of biofilm and thickness is the most affected parameter. NAC and Tween 80 combined with antibiotics exert a synergistic effect in increasing the percentage of dead bacteria and also reducing the percentage of covered surface and thickness of NPRGM biofilms. Tween 80 seems to be an antibiofilm agent more effective than NAC due to its higher reduction in the percentage of cover surface and thickness. In conclusion, the results obtained in this work show that phenotypic parameters (thickness, percentage of covered surface, autofluorescence, percentage of live/dead bacteria) are affected by combining antibiotics and antibiofilm agents, ciprofloxacin and Tween 80 being the most active agents against NPRGM biofilms.

BackgroundThe aim of this study was to analyze the effect of ciprofloxacin at different times on the development and behavior of intrinsic autofluorescence, covered area, thickness and cell viability in a biofilm formed by non-pigmented rapidly growing mycobacteria (NPRGM).Confocal laser scanning microscopy and image analysis were used to study the behavior of ciprofloxacin on biofilms.ResultsThickness was the most affected parameter, although some species showed changes in other parameters. At the same time, we also measured the minimum inhibitory concentration and the minimum biofilm eradication concentration (MBEC). An increase in MBEC was observed in all the strains, M. peregrinum being the species that presented the highest increase.ConclusionsThis study help us to understand better how mycobacterial biofims can be affected by ciprofloxacin.

The structure of biofilms formed by seven nonpigmented rapidly growing mycobacteria, including saprophytes and opportunistic species, was analyzed. Analysis included amount of covered surface, thickness, cell viability, and presence of intrinsic autofluorescence at different times using confocal laser scanning microscopy and image analysis. Autofluorescence was detected inside and outside cells of all mycobacteria. It is known that bacteria grow in nature by forming structured and specialized communities of organisms embedded in a matrix of extrapolymeric substance known as a biofilm (1). Biofilms are also considered to be an important pathogenic factor for many diseases, especially implant-related infections (2).Nonpigmented rapidly growing mycobacteria (NPRGM) (3) are usually considered contaminants or colonizers, although in some patients they are the true cause of the disease (4, 5). The source of human infection is usually the environment (6), with drinking water distribution systems and hospital and household plumbing being the mainly reported sources (4, 7). Findings of recent studies suggest that the biofilm-developing capacity is a property related to the involvement of these bacteria in human pathogenicity (8) and in antimicrobial resistance (9, 10). In a previous report, we observed that NPRGM were able to form biofilms in vitro (5), with differences regarding the importance of biofilms in the pathogenesis of human diseases (11). Other studies have also shown differences among strains within the same species (12). Moreover, there are studies that relate the ability to form biofilms with the presence of cording or rough colonies in the tested strains (12)(13)(14).Intrinsic autofluorescence, including the presence of autofluorescence in the cyan range in Mycobacterium species (16), is a characteristic that has been found previously in several microorganisms (15). In this study, we aimed to analyze the structure of mycobacterial biofilms, with a special focus on detection of autofluorescence.The strains used were Mycobacterium abscessus DSM 44196, Mycobacterium chelonae ATCC 19235, Mycobacterium fortuitum ATCC 6841, Mycobacterium mageritense ATCC 700351, Mycobacterium mucogenicum DSM 44124, Mycobacterium peregrinum ATCC 14467, and Mycobacterium smegmatis ATCC 607.Biofilm development was analyzed at 24, 48, 72, and 96 h using hydrophobic uncoated sterile slide 2-by 4-well plates (ibidy GmbH, Martinsried, Germany), as follows.Mycobacterial colonies were resuspended in sterile phosphatebuffered saline solution (PBS) to achieve a cell density of 1.5 ϫ 10 8 CFU/ml. Three hundred microliters of this suspension was inoculated on each well. Inoculated slides were incubated at 37°C in a 5% CO 2 atmosphere for 30 min. The suspension was then removed, and the wells were washed once with PBS. Three hundred microliters of Middlebrook 7H9 broth was then added to each well, and the slides were placed on an orbital shaker (80 rpm) and incubated at 37°C in normal atmosphere for 4 days. Slides were examined, and the mediu...