The role of denitrification genes in anaerobic growth and virulence of Flavobacterium columnare.

AIMS: Comparative genomics analyses indicated that the Flavobacterium columnare genome has unique denitrification genes relative to Flavobacterium psychrophilum and Flavobacterium johnsoniae, including nasA (nitrate reductase), nirS (nitrite reductase), norB (nitric oxide reductase) and nosZ (nitrous oxide reductase). The current study determines the roles of nasA, nirS, norB and nosZ in anaerobic growth, nitrate reduction, biofilm formation and virulence. METHODS AND RESULTS: Four in-frame deletion mutants in virulent F. columnare strain 94-081 were constructed by allelic exchange using pCP29 plasmid. Compared with parent strain 94-081, FcΔnasA,FcΔnirS and FcΔnosZ mutants did not grow as well anaerobically, whereas the growth of FcΔnorB strain was similar to the parent strain (FcWT). Exogenous nitrate was not significantly consumed under anaerobic conditions in FcΔnasA, FcΔnirS and FcΔnosZ compared to parent strain 94-081. Under anaerobic conditions, Fc∆nasA, Fc∆norB and Fc∆nosZ formed significantly less biofilm than the wild type strain at 24 and 96 h, but FcΔnirS was not significantly affected. The nitrite reductase mutant FcΔnirS was highly attenuated in catfish, whereas FcΔnasA, FcΔnorB and FcΔnosZ had similar virulence to FcWT. CONCLUSIONS: These results show, for the first time, that denitrification genes enable F. columnare to grow anaerobically using nitrate as an electron acceptor, and nitrite reductase contributes to F. columnare virulence. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings indicate potential for F. columnare to grow in nitrate-rich anaerobic zones in catfish production ponds, and they suggest that a Fc∆nirS strain could be useful as a safe live vaccine if it protects catfish against columnaris disease.

Microbial contamination of tattoo and permanent makeup inks marketed in the US: a follow-up study.

In a 2018 survey, U.S. Food and Drug Administration (FDA) identified microbial contamination in 42 (49%) of 85 unopened tattoo and permanent makeup (PMU) inks purchased from 13 manufacturers in the US between November 2015 and April 2016. To confirm the results of our previous survey, we evaluated the level of microbial contamination in an additional 27 samples from 10 manufacturers from September 2017 to December 2017, including 21 unopened tattoo and PMU inks which were selected based on our previous survey results and 6 ink diluents that were not previously analysed. Aerobic plate count and enrichment culture methods from the FDA's Bacteriological Analytical Manual revealed 11 (52%) out of 21 inks, from six manufacturers, were contaminated with micro-organisms, with contamination levels up to 3·6 × 108  CFU per gram, consistent with our previous survey results. We identified 25 bacterial strains belonging to nine genera and 19 species. Strains of Bacillus sp. (11 strains, 44%) were dominant, followed by Paenibacillus sp. (5 strains, 20%). Clinically relevant strains, such as Kocuria rhizophila and Oligella ureolytica, were also identified, as similar to the findings in our previous survey. No microbial contamination was detected in any of the six ink diluents.

Construction and evaluation of type III secretion system mutants of the catfish pathogen Edwardsiella piscicida.

Catfish is the largest aquaculture industry in the United States. Edwardsiellosis is considered one of the most significant problems affecting this industry. Edwardsiella piscicida is a newly described species within the genus Edwardsiella, and it was previously classified as Edwardsiella tarda. It causes gastrointestinal septicaemia, primarily in summer months, in farmed channel catfish in the south-eastern United States. In the current study, we adapted gene deletion methods used for Edwardsiella to E. piscicida strain C07-087, which was isolated from a disease outbreak in a catfish production pond. Four genes encoding structural proteins in the type III secretion system (T3SS) apparatus of E. piscicida were deleted by homologous recombination and allelic exchange to produce in-frame deletion mutants (EpΔssaV, EpΔesaM, EpΔyscR and EpΔescT). The mutants were phenotypically characterized, and virulence and vaccine efficacy were evaluated. Three of the mutants, EpΔssaV, EpΔyscR and EpΔesaM, were significantly attenuated compared to the parent strain (p < .05), but EpΔescT strain was not. Vaccination of catfish with the four mutant strains (EpΔssaV, EpΔesaM, EpΔyscR and EpΔescT) provided significant protection when subsequently challenged with wild-type strain. In conclusion, we report methods for gene deletion in E. piscicida and development of vaccine candidates derived from a virulent catfish isolate.

Microbiological survey of commercial tattoo and permanent makeup inks available in the United States.

AIMS: Tattooing and use of permanent makeup (PMU) has dramatically increased over the last decade, with a concomitant increase in ink-related infections. The aim of this study was to determine whether micro-organisms are present, and if so, the number and their identification in the commercial tattoo and PMU inks available in the United States. METHODS AND RESULTS: We surveyed 85 unopened tattoo and PMU inks, purchased from 13 companies. We incubated 100 µl of ink samples on trypticase soy agar plates for bacterial growth, 7H10 Middlebrook medium for mycobacterial growth, and Sabouraud dextrose medium for fungal growth. In total, 42 inks were contaminated with micro-organisms (49%). Thirty-three inks were contaminated with bacteria, 2 inks with fungi, and 7 inks had both bacterial and fungal growth. Mycobacteria were not detected in any of the examined tattoo and PMU inks. In 26 inks, microbial concentrations ranged between 101 and 103 CFU per ml, but higher counts (>103 CFU per ml) were recorded in 16 inks. We identified 83 bacteria by their 16S rDNA sequences, including 20 genera and 49 species. Strains of Bacillus spp. (53%) were dominant, followed by Lysinibacillus fusiformis (7%) and Pseudomonas aeruginosa (5%). Thirty-four (41%) possibly clinically relevant strains were identified, including P. aeruginosa, Dermacoccus barathri and Roseomonas mucosa, some of which have been previously reported to be associated with human skin infections. CONCLUSIONS: The results indicate that commercial tattoo and PMU inks on the US market surveyed in this study contain a wide range of micro-organisms, including pathogenic bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: Microbial contaminants in tattoo and PMU inks are an emerging safety concern for public health. This study provides evidence that microbial contamination of tattoo and PMU inks available in the United States is more common than previously thought and highlights the importance of monitoring these products for potentially pathogenic micro-organisms.

Identification of high-risk Listeria monocytogenes serotypes in lineage I (serotype 1/2a, 1/2c, 3a and 3c) using multiplex PCR.

AIMS: Using molecular subtyping techniques, Listeria monocytogenes is divided into three major phylogenetic lineages, and a multiplex PCR method can differentiate five L. monocytogenes subgroups: 1/2a-3a, 1/2c-3c, 1/2b-3b-7, 4b-4d-4e and 4a-4c. In this study, we conducted genome comparisons and evaluated serotype-associated genes for their utility as a multiplex PCR-based method for distinguishing high-risk serotypes 1/2a and 1/2c in lineage I from low-risk serotypes 3a and 3c. METHODS AND RESULTS: Primer sets were developed that are specific for serotype 1/2c (LMOSLCC2372_0308) and serotype 3a (LMLG_0742). These primers were then tested in a multiplex format with primers specific for serotype 1/2a (flaA) to separate serotypes 1/2a, 1/2c, 3a and 3c using 25 strains of lineage I L. monocytogenes. CONCLUSIONS: Here, for the first time, we report primers specific for L. monocytogenes serotype 1/2c and serotype 3a, and we demonstrate a multiplex PCR method for separating the four serotypes of lineage I L. monocytogenes. SIGNIFICANCE AND IMPACT OF THE STUDY: The described multiplex PCR assay consistently showed successful separation of 1/2a and 1/2c strains from 3a and 3c strains. PCR is routinely performed in many diagnostic and epidemiologic investigations for L. monocytogenes, and these primers should increase the feasibility and accessibility of L. monocytogenes serotyping.

Immunoproteomic analysis of capsulate and non-capsulate strains of Lactococcus garvieae.

A comparative immunoproteomic study was carried out to investigate the immunogenicity of capsulate (KG9408) and non-capsulate (NSS9310) strains of Lactococcus garvieae. Immunoblot assays, following two-dimensional gel electrophoresis (2-DE) for L. garvieae strains, revealed a significant difference between anti-capsulate and anti-non-capsulate rabbit sera with respect to the number and antigenicity of antigenic spots. Anti-capsulate and anti-non-capsulate rabbit sera reacted with an average of 72 and 127 antigenic spots, respectively. The strong reaction of anti-non-capsulate sera with elongation factor (EF)-G and -Tu, and GMP synthase, of the L. garvieae strains identifies these as specific major antigens. This study clearly demonstrates the differences in 2-DE immunoblot profiles between the capsulate and non-capsulate strains of L. garvieae. These differences may be the reason for variations in immunogenicity between capsulate and non-capsulate strains. Glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, arginine deaminase and ornithine carbamoyltransferase were identified from the 2-DE immunoblot profiles of both strains. Therefore, these common antigens are potential markers for the development of vaccines against L. garvieae, irrespective of strain. Immunoproteomics, a powerful tool for studying antigens at the proteomic level, allowed a comparative investigation of the immunogenicity of capsulate and non-capsulate strains of L. garvieae for vaccine development.