Formation of Staphylococcus aureus Biofilm in the Presence of Sublethal Concentrations of Disinfectants Studied via a Transcriptomic Analysis Using Transcriptome Sequencing (RNA-seq).

Staphylococcus aureus is a common biofilm-forming pathogen. Low doses of disinfectants have previously been reported to promote biofilm formation and to increase virulence. The aim of this study was to use transcriptome sequencing (RNA-seq) analysis to investigate global transcriptional changes in S. aureus in response to sublethal concentrations of the commonly used food industry disinfectants ethanol (EtOH) and chloramine T (ChT) and their combination (EtOH_ChT) in order to better understand the effects of these agents on biofilm formation. Treatment with EtOH and EtOH_ChT resulted in more significantly altered expression profiles than treatment with ChT. Our results revealed that EtOH and EtOH_ChT treatments enhanced the expression of genes responsible for regulation of gene expression (sigB), cell surface factors (clfAB), adhesins (sdrDE), and capsular polysaccharides (cap8EFGL), resulting in more intact biofilm. In addition, in this study we were able to identify the pathways involved in the adaptation of S. aureus to the stress of ChT treatment. Further, EtOH suppressed the effect of ChT on gene expression when these agents were used together at sublethal concentrations. These data show that in the presence of sublethal concentrations of tested disinfectants, S. aureus cells trigger protective mechanisms and try to cope with them.IMPORTANCE So far, the effect of disinfectants is not satisfactorily explained. The presented data will allow a better understanding of the mode of disinfectant action with regard to biofilm formation and the ability of bacteria to survive the treatment. Such an understanding could contribute to the effort to eliminate possible sources of bacteria, making disinfectant application as efficient as possible. Biofilm formation plays significant role in the spread and pathogenesis of bacterial species.

A new cultivation-independent tool for fast and reliable detection of Mycobacterium marinum.

The Mycobacterium marinum group (MMG) is a class of mycobacteria that includes M. marinum, the cause of chronic systemic infections in fish. This species occasionally causes granulomatous skin lesions in humans. Other members of MMG are mycolactone-producing mycobacteria (MPM; M. ulcerans, M. shottsii and M. pseudoshottsii). The cultivation-independent approach presented in this study brings a fast and reliable alternative to classically used cultivation methods. The developed triplex erp/IS2404 qPCR assay is based on a primary species-specific erp detection, which allows enumeration of MMG in analysed samples, and secondary IS2404 detection is suitable for the differentiation of M. marinum from MPM. The detection of M. marinum in clinical specimens and in artificially contaminated tissue samples has proven its applicability for diagnostic purposes.

Analysis of sediments and plants from the system of five fishponds in the Czech Republic using culture and PCR methods.

Nontuberculous mycobacteria (NTM) are ubiquitous organisms that have been isolated from a variety of environmental sources. Several NTM species are responsible for diseases in humans and/or animals known as mycobacterioses. The aim of this study was to determine the levels of NTM in the sediments and plants of five fish ponds in the Czech Republic using culture and quantitative real time PCR (qPCR). Additionally, we investigated if there was any link between environmental samples from the fish ponds and the fish occupying them. A total of 8 NTM (14.0%) were cultured from the aquatic environment. qPCR analysis showed that Mycobacterium avium hominissuis was most frequently present (54.4%), followed by Mycobacterium avium paratuberculosis (42.1%). The least frequently isolated NTM was Mycobacterium avium avium (5.3%). Thus, in this study we confirm the presence of mycobacteria in sediments and aquatic plants in fishponds, which are occupied by fish intended for human consumption. We successfully isolated NTM from the tissue of one fish and confirmed a possible transmission of mycobacteria from the aquatic environment to the fish. Consequently, the consumption of such fish represents a possible risk for consumers, particularly immunocompromised individuals.

Relative prevalence of Mycobacterium marinum in fish collected from aquaria and natural freshwaters in central Europe.

A survey was carried out on occurrence of Mycobacterium marinum in fish kept in aquaria and those living in their natural environment. Species-specific qPCR targeting the erp and IS2404 genes together with the conventional culture method were used. The analysis of 72 ornamental fish (n = 216 samples: gills, muscle and intestine) collected from aquaria revealed the presence of M. marinum in 30 individuals (41.7%) of whom 17 (23.6%) were later culture positive. Culture-independent detection revealed the presence of M. marinum in 16 of 83 environmental samples (19.3%) collected in aquaria. The presence of viable M. marinum cells was later confirmed in 5 samples (6.0%). No qPCR or culture positivity was observed when 123 groundwater fish and their corresponding environmental samples (n = 142) were analysed.

[Methods used to detect mycobacterioses in humans--assessment of advantages and disadvantages of mycobacterial identification by sequencing analysis]. / Metody pouzívané pro detekci mykobakterióz u lidí--posouzení výhod a nevýhod identifikace mykobakterií pomocí sekvencní analýzy.

The isolation of potentially pathogenic mycobacteria (PPM) from clinical specimens has become very frequent in the last years. Such organisms are typically environmental and occasionally pathogenic for humans. Standard diagnosis of mycobacterial infections relies on direct examination and culture. Nowadays, molecular tools are available, allowing quicker accurate diagnosis. Detection of PPM can be performed directly from clinical samples, although in most cases identification is carried out after isolation. Sequencing of genomic targets (such as 16S rRNA, rpoB or hsp65) allows accurate and quick identifications but has some technical limitations. Problems concerning sequencing analysis used for PPM identification together with description of available algorithms for PPM identification are the major objectives of this review.

A low prevalence of mycobacteria in freshwater fish from water reservoirs, ponds and farms.

A survey of the occurrence of mycobacteria was conducted from 717 freshwater fish (25 species) in two water reservoirs, five ponds and two farms in the Czech Republic. A total of 2182 tissue samples from these fish were examined using the conventional culture method. Thirteen mycobacterial isolates were obtained from 12 (1.7%) fish belonging to nine species. Isolates were identified using sequence analysis of the 16SrRNA gene as: Mycobacterium algericum, M. fortuitum, M. gordonae, M. insubricum, M. kumamotonense, M. nonchromogenicum, two isolates of M. peregrinum, M. terrae and M. triplex. Mycobacteria were isolated more frequently from fish skin and gills than from internal organs or muscles.

Mycobacterium marinum epididymoorchitis: case report and literature review.

Mycobacterium marinum is the most frequent non-tuberculous Mycobacterium in humans. We report the first ever described case of epididymoorchitis resulting from hematogenous spread of M. marinum from hand oligoarthritis. This was initially mistaken for rheumatoid disease and methylprednisolone-induced immunosuppression led to hematogenous spread of infection to the testis and epididymis.

Morphology and distribution of granulomatous inflammation in freshwater ornamental fish infected with mycobacteria.

Mycobacteriosis in fish is a chronic progressive ubiquitous disease caused by Mycobacterium marinum, M. gordonae and M. fortuitum in most cases. The aim of this study was to describe the morphology and distribution of lesions in 322 freshwater ornamental fish across 36 species. Granulomatous inflammation was diagnosed by gross examination and histopathology testing in 188 fish (58.4%); acid-fast rods (AFR) were determined in only 96 (51.1%) fish from 19 species after Ziehl-Neelsen staining. The most often affected organs with AFR were the kidney (81.2%), digestive tract (54.1%), liver (48.2%), spleen (45.9%) and skin (21.2%); sporadically, AFR were found in the branchiae (9.4%) and gonads (4.7%). In 14 randomly selected fish originating from four different fish tanks, the distribution of mycobacterial infection was studied by culture examination of the skin, gills, muscle tissue, digestive tract, liver, spleen and kidney. In 12 fish, the species M. marinum, M. gordonae, M. fortuitum, M. triviale, and M. avium subsp. hominissuis (serotypes 6 and 8 and genotype IS901- and IS1245+) were detected; mixed infection caused by different mycobacterial species was documented in five of them.

First isolation of a newly described Mycobacterium insubricum from freshwater fish.

Non-tuberculous or potentially pathogenic mycobacteria are ubiquitous in a water environment and are responsible for several diseases in animals and humans known as mycobacterioses. This report describes the first isolation of the novel species Mycobacterium insubricum in clinically healthy freshwater fish (Tinca tinca) in the Czech Republic which is the first to be derived from non-human sources reported in the literature.

Effects of novel 3,4-bisphenylhex(3)enes on cell proliferation in malignant and normal cells.

The growth-inhibitory effect of several newly synthesized alkyl derivatives of 3,4-bisphenylhex(3)ene was studied in four tumor cell lines and three healthy primary cell systems. A marked inhibition of cell proliferation was noted in the neoplastic cells but not in the primary systems. No effect on the cytoplasmic or mitotic microtubule system but an increase in the gross level of 5-methylcytosine in nuclear DNA was observed. It is speculated that the selective growth inhibition of tumor cells is due to DNA-hypermethylation.