The adhesin pertactin (Prn) is one of the major virulence factors of Bordetella pertussis, the etiological agent of whooping cough. However, a significant prevalence of Prn-deficient (Prn−) B. pertussis was observed in Japan. The Prn− isolate was first discovered in 1997, and 33 (27%) Prn− isolates were identified among 121 B. pertussis isolates collected from 1990 to 2009. Sequence analysis revealed that all the Prn− isolates harbor exclusively the vaccine-type prn1 allele and that loss of Prn expression is caused by 2 different mutations: an 84-bp deletion of the prn signal sequence (prn1ΔSS, n = 24) and an IS481 insertion in prn1 (prn1::IS481, n = 9). The frequency of Prn− isolates, notably those harboring prn1ΔSS, significantly increased since the early 2000s, and Prn− isolates were subsequently found nationwide. Multilocus variable-number tandem repeat analysis (MLVA) revealed that 24 (73%) of 33 Prn− isolates belong to MLVA-186, and 6 and 3 Prn− isolates belong to MLVA-194 and MLVA-226, respectively. The 3 MLVA types are phylogenetically closely related, suggesting that the 2 Prn− clinical strains (harboring prn1ΔSS and prn1::IS481) have clonally expanded in Japan. Growth competition assays in vitro also demonstrated that Prn− isolates have a higher growth potential than the Prn+ back-mutants from which they were derived. Our observations suggested that human host factors (genetic factors and immune status) that select for Prn− strains have arisen and that Prn expression is not essential for fitness under these conditions.

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Members of the genus Nocardia are widespread in diverse environments; a wide range of Nocardia species are known to cause nocardiosis in several animals, including cat, dog, fish, and humans. Of the pathogenic Nocardia species, N. seriolae is known to cause disease in cultured fish, resulting in major economic loss. We isolated two N. seriolae strains, CK-14008 and EM15050, from diseased fish and sequenced their genomes using the PacBio sequencing platform. To identify their genomic features, we compared their genomes with those of other Nocardia species. Phylogenetic analysis showed that N. seriolae shares a common ancestor with a putative human pathogenic Nocardia species. Moreover, N. seriolae strains were phylogenetically divided into four clusters according to host fish families. Through genome comparison, we observed that the putative pathogenic Nocardia strains had additional genes for iron acquisition. Dozens of antibiotic resistance genes were detected in the genomes of N. seriolae strains; most of the antibiotics were involved in the inhibition of the biosynthesis of proteins or cell walls. Our results demonstrated the virulence features and antibiotic resistance of fish pathogenic N. seriolae strains at the genomic level. These results may be useful to develop strategies for the prevention of fish nocardiosis.

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The role of collagenase as a mechanism of bacterial pathogenicity in some pathogenic bacteria has been reported. The information on the role of collagenase in Aeromonas spp. pathogenesis is scant. In the present study, a mutant Aeromonas veronii RY001 that is deficient in the putative collagenase gene acg was constructed and compared with the wild-type strain for virulence factors. Bacterial cells and cell-free extracellular products of the mutant had significantly less collagenolytic activity, but there were not significant differences in caseinolytic, gelatinolytic, and elastolytic activities. Adhesion and invasion abilities of the mutant strain on epithelioma papillosum of carp cells was only 56% of that of the wild-type strain, and the cytotoxicity of the mutant strain to epithelioma papillosum of carp cells was only 42% of that of the wild-type strain. The LD50 values of the wild-type strain were determined as 1.6 x 10(6) and 3.5 x 10(5) cfu in goldfish and mice, respectively, whereas the mutant RY001 strain showed slightly higher values (i.e., 2.8 x 10(6) and 1.4 x 10(6) cfu in goldfish and mice, respectively). These results indicated the involvement of the collagenase gene in the pathogenesis of A. veronii.