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.
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.
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.
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.
Significance and Impact of the Study: Risks of infection associated with tattoo and permanent makeup (PMU) inks contaminated with microorganisms continues to be a public health concern. This study confirms the results from our previous study showing that a high portion of tattoo and PMU inks were contaminated with micro‐organisms, including pathogenic bacteria. The results of this survey highlight the safety concerns associated with intradermal injection of microbially contaminated tattoo inks. Furthermore, this study shows the importance of continuously monitoring the levels of microbial contamination of tattoo and PMU inks marketed in the US.
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.
We present 2 cases of lateral incomplete impending fracture of the femoral neck without trauma in elderly patients taking long-term bisphosphonate (BP) treatment, and we defined it as atypical ...femoral neck fracture (AFNF). To the best of our knowledge, this is the first report on the follow-up results of AFNF.
Patients in both cases had been taking BP drugs for a long time with osteoporosis. The duration of BP treatment was 6 years, and there was no history of repeated stresses.
All fractures were linear at the lateral aspect of the mid portion of the femoral neck, and the BMD of the femoral neck was -0.9, and -1.8, respectively.
Internal fixation was performed in both cases (73 years, 68 years) using cannulated screws.
In both patients who underwent screw fixation, the fracture line started to extend distally at 4 weeks and 2 weeks following surgery. In the 3-month follow-up image, the length of the fracture increased by 20.1 mm and 9.9 mm, respectively. There was a problem with active rehabilitation, and the possibility of revision was also found to be a burden in terms of mortality and cost in older patients.
In the case of AFNF, guidelines for treatment should be set in consideration of the decreased bone healing, even when the fracture pattern is simple. Arthroplasty based on a wider indication may be worth considering.
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.
Halocynthia roretzi (phylum Cordata), also called 'sea pineapples', live in shallow coastal waters and typically feed on plankton and detritus that they filter from seawater. It has been reported ...that symbiotic microflora associated with H. roretzi act as protective agents that strengthen its immune system or control energy metabolism. This study analyzed the culturable microflora from the coelomic fluid of the sea pineapple using MALDI-biotyping and 16S rRNA sequencing, combining a recent technology with the conventional method of bacteria identification. The MALDI-biotyper enabled the classification of the symbiotic microflora into 5 groups based on the specific patterns of their mass spectrum. The 16S rRNA sequencing was then used to establish the identity of the dominant bacteria in 4 groups, later revealed as 2 groups of Vibrio spp., Shwanella spp. and Bacillus spp. MALDI-biotyping was applied for the identification of microorganisms directly from cultured agar, and, coupled with numerical taxonomic analyses, we determined the major microflora associated with H. roretzi.
We report the isolation of activating transcription factor of chaperone (ATFC), a novel cDNA from
Bombyx mori BM5 cells that encodes a putative transducer of endoplasmic reticulum (ER) stress. The ...236 amino acids of ATFC include both a basic region and a leucine zipper at the C-terminus, in contrast to Hac1p of yeast which features these structures at its N-terminus. ATFC expression was strongly up-regulated by ER stress. ATFC could specifically bind to the unfolded protein response element. BM5 cells transfected with ATFC cDNA displayed enhanced ER chaperone expression in response to ER stress. These results indicate that ATFC encodes a putative transducer of ER stress.
To elucidate the molecular mechanisms associated with metamorphic phenomenon relating to
Bombyx mori, an important organism in the sericulture industry, we identified genes that are expressed in the ...different developmental stages, specifically the embryonic (ES) and larval (LS) stages of
B. mori. Of 8230 high-quality ESTs from two full-length enriched cDNA libraries, 3442 of the ES ESTs were coalesced into 1325 clusters, while 4788 were coalesced into 927 clusters. The functional classification of these ESTs based on Gene Ontology showed that the types of genes that are associated with oxidoreductase activity, enzyme inhibition, and larval development were highly observed in LS, whereas the types of genes that are involved in nucleotide binding, enzyme activity, and protein transport activity were highly observed in ES. In addition, when the gene expression profile between ES and LS was examined by counting the EST frequencies in each library, 69 genes were identified as being either up- or down-regulated in the larval stage compared to the embryonic stage (
P
>
0.99) and this was confirmed by semi-quantitative RT-PCR. The results show that genes involved in proteolysis and peptidolysis, and lipid and carbohydrate metabolism were dramatically up-regulated in LS, while those related to protein metabolism, DNA/RNA, and coenzymes were highly down-expressed. In particular, a GO analysis of these genes revealed that genes that are involved in hydrolase activity were observed to be highly expressed in amount as well as diversity in LS, while those involved in nucleic acid binding were highly expressed in ES. These data may contribute to elucidating genetic events that distinguish the developmental stage and to our understanding of the metamorphosis of
B. mori.
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