Legionella septentrionalis sp. nov., isolated from aquatic environments in the northern PR China.

Four strains (km711T, km714, km542 and km524), representing a novel Legionella species, were isolated from aquatic environments in northern PR China. Cells were Gram-stain-negative, rod-shaped, microaerobic, motile and growth depended on l-cysteine. They grew at 25‒42 °C (optimum, 35‒37 °C) and could tolerate up to 1.5 % (w/v) NaCl (optimum, 0.5 %). The major fatty acids (>5 %) of the type strain km711T were C17 : 0 anteiso, C15 : 0 anteiso, iso-C16 : 0 and C16 : 1 ω7c and/or iso-C15 : 0 2OH. The pairwise comparison values were <96.1 % for 16S rRNA gene sequences, 23.3‒28.7 % interspecies variation for mip gene sequences, <93.6 % average nucleotide identity and <72.8 % average amino acid identity between these four strains and related type strains within the genus Legionella. The phylogenetic tree based on the four concatenated genes (16S rRNA, mip, rpoB and rnpB) and protein-concatamer tree based on concatenation of 21 protein markers both revealed that these four strains formed a separate phylogenetic branch cluster within the genus Legionella. The results of phenotypic and genotypic features suggest that these four strains represent a novel species of the genus Legionella, for which the name Legionella septentrionalis sp. nov. is proposed (type strain km711T=KCTC 15655T=NBRC 113219T).

Legionella qingyii sp. nov., isolated from water samples in China.

Three Legionella-like strains, designed km488T, km489 and km521, were isolated from freshwater samples in China. Cells were Gram-stain-negative, rod-shaped and non-spore-forming. Growth was observed on BCYEα agar, but not on BCYEα agar without l-cysteine, chocolate agar with PolyViteX or Columbia blood agar. The major fatty acids (>5 %) of strains km488T, km489 and km521 were C16 : 0, anteiso-C15 : 0, iso-C16 : 0 and anteiso-C17 : 0. The mip gene sequences (574 nt) showed the isolates were almost identical with more than 99.7 % sequence similarities, and closely matched to L. gormanii ATCC 33297T with 95.4-95.6 % sequence similarities. Phylogenetic analyses based on concatenated gene (16S rRNA, mip, rpoB and rnpB) sequences indicated that the isolates formed a distinct cluster along with L. gormanii within the genus Legionella. Matrix-assisted laser desorption ionization time-of-flight analyses also demonstrated a clear separation between the isolates and other closely and distantly related Legionella species. DNA-DNA hybridization studies demonstrated that the isolates were closely related (92.0 -95.0 % DNA-DNA relatedness) but differentiated from their phylogenetic neighbours (<70 % DNA-DNA relatedness). The whole genome of km488T was sequenced, and showed a G+C content of 37.8 mol%. Based on the findings from this polyphasic taxonomic study, the isolates are considered to represent a single novel species, for which the name Legionella qingyii sp. nov. is proposed. The type strain is km488T (KCTC 15636T=CCTCC AB 2018025T=NRBC 113223T).

Targeting single-nucleotide polymorphisms in the 16S rRNA gene to detect and differentiate Legionella pneumophila and non-Legionella pneumophila species.

A PCR-based method targeting single-nucleotide polymorphisms (SNPs) in the 16S rRNA gene was developed for differential identification of Legionella pneumophila and non-Legionella pneumophila. Based on the bioinformatics analysis for 176 Legionella 16S rRNA gene fragments of 56 different Legionella species, a set of SNPs, A(628)C(629) was found to be highly specific to L. pneumophila strains. A multiplex assay was designed that was able to distinguish sites with limited sequence heterogeneity between L. pneumophila and non-L. pneumophila in the targeted 16S rRNA gene. The assay amplified a 261-bp amplicon for Legionella spp. and a set of 203- and 97-bp amplicons only specific to L. pneumophila species. Among 49 ATCC strains and 284 Legionella isolates from environmental water and clinical samples, 100 % of L. pneumophila and non-L. pneumophila strains were correctly identified and differentiated by this assay. The assay presents a more rapid, sensitive and alternative method to the currently available PCR-sequencing detection and differentiation method.

Different distribution patterns of ten virulence genes in Legionella reference strains and strains isolated from environmental water and patients.

Virulence genes are distinct regions of DNA which are present in the genome of pathogenic bacteria and absent in nonpathogenic strains of the same or related species. Virulence genes are frequently associated with bacterial pathogenicity in genus Legionella. In the present study, an assay was performed to detect ten virulence genes, including iraA, iraB, lvrA, lvrB, lvhD, cpxR, cpxA, dotA, icmC and icmD in different pathogenicity islands of 47 Legionella reference strains, 235 environmental strains isolated from water, and 4 clinical strains isolated from the lung tissue of pneumonia patients. The distribution frequencies of these genes in reference or/and environmental L. pneumophila strains were much higher than those in reference non-L. pneumophila or/and environmental non-L. pneumophila strains, respectively. L. pneumophila clinical strains also maintained higher frequencies of these genes compared to four other types of Legionella strains. Distribution frequencies of these genes in reference L. pneumophila strains were similar to those in environmental L. pneumophila strains. In contrast, environmental non-L. pneumophila maintained higher frequencies of these genes compared to those found in reference non-L. pneumophila strains. This study illustrates the association of virulence genes with Legionella pathogenicity and reveals the possible virulence evolution of non-L. pneumophia strains isolated from environmental water.

Two-step scheme for rapid identification and differentiation of Legionella pneumophila and non-Legionella pneumophila species.

A rapid two-step scheme based on PCR amplification and enzymatic digestion analysis of a 226-bp fragment of the 16S rRNA gene was developed to identify the Legionella genus by PCR amplification and to differentiate the Legionella pneumophila and non-Legionella pneumophila species by enzymatic digestion analysis. Among 42 ATCC strains (16 strains of L. pneumophila and 26 strains of non-L. pneumophila) and 200 Legionella isolates from environmental water samples, including pools, rivers, lakes, and cooling towers in Guangdong province, 99.59% of L. pneumophila and non-L. pneumophila strains were correctly identified and differentiated by this scheme. The procedure of this two-step identification and differentiation scheme is simple and takes only about 4 h. These results suggest that this two-step scheme provides a simple and convenient method for the rapid identification and differentiation of L. pneumophila and non-L. pneumophila species.

[Association study on the mitochondrial genome region np16181-16193 variation with type 2 diabetes mellitus].

OBJECTIVE: To investigate the association of the mitochondrial DNA region np16181-16193 variations with type 2 diabetes mellitus (T2DM). METHODS: Blood samples of 199 unrelated T2DM patients and 205 normal controls were collected to detect the mitochondrial DNA region np16181-16193 variations by PCR and sequencing, and to analyze the association of the variations with the major clinical symptoms. RESULTS: The mitochondrial DNA np16181-16193 region is a hypervariable area, with several polymorphisms. Four types of np16181-16193 region variations were found only in T2DM. The 1-hour postprandial blood glucose (P1BG) in the T2DM individuals with np16181-16193 region variations was significantly higher than those without variations (P<0.05), while there was no significant difference in other biochemical parameters (P>0.05). CONCLUSION: The mitochondrial DNA np16181-16193 variations could not be regarded as a risk factor for T2DM.

[Detection of fungi in liquor workers with tinea corporis and tinea cruris using arbitrarily primed polymerase chain reaction].

OBJECTIVE: To explore the method of rapid detection of skin fungi and the significance of conventional diagnosis liquor worker tinea corporis and tinea cruris using arbitrarily primed polymerase chain reaction AP-PCR. METHODS: Among liquor workers who were 50 tinea corporis patients, 58 tinea cruris patients and 50 health persons, we amplified the DNAs of the dermatophytes were amplified using AP-PCR and random primers OPD18 5'-GAGAGCCAAC-3' and OPAA11 5'-ACCCGACCTG-3', at the same time, the dermatophytes with microscope were detected and cultured. RESULTS: AP-PCR analysis detected fungal DNA in 45 patients(90.00%) among 50 liquor worker patients with tinea corporis, 31 patients(62.00%) had the positive results of microscope detection, and 41 patients(82.00%) had the positive results of standard culture. Among these workers who suffered from tinea corporis, T.rubrum, T.mentagrophyte, M. canis and E.floccosum were detected by AP-PCR. T.rubrum, T.mentagrophyte and M.canis were detected by standard culture. AP-PCR analysis detected fungal DNA in 53 patients(91.38%) among 58 liquor worker patients with tinea cruris, 37 patients(63.79%) had the positive results of microscope detection, and 48(82.76%) had the positive results of standard culture. Among the 58 workers who had tinea cruris, T.rubrum, E.floccosum and T.mentagrophyte were detected by AP-PCR and standard culture. Among 50 health persons, AP-PCR analysis detected fungal DNA in 3 persons(6.00%). The detection result with AP-PCR indicated that the kinds of fungi were T.rubrum and T.mentagrophyte. No one health person had the positive result in detection of fungi using microscope detection. Only one(2.00%) health person was detected to be infected by fungus with cultural way. The kind of fungus was T.rubrum. CONCLUSION: AP-PCR is a rapid, sensitive and specific detection method for occupational dermatophyte patients. It can be used to detect and diagnose professional dermatophytosis.

Effects of antiarrhythmic peptide 10 on acute ventricular arrhythmia.

OBJECTIVE: To observe the effects antiarrhythmic peptide 10 (AAP10) aon acute ventricular arrhythmia and the phosphorylation state of ischemic myocardium connexin. METHODS: Acute total ischemia and partial ischemia models were established by ceasing perfusion and ligating the left anterior descending coronary artery in SD rats. The effects of AAP10 (1 mg/L) on the incidence rate of ischemia-induced ventricular arrhythmia were observed. The ischemic myocardium was sampled to detect total-Cx43 and NP-Cx43 by immunofluorescent staining and western blotting. the total-Cx43 expression was detected through image analysis system by semi-quantitative analysis. RESULTS: AAP10 could significantly decrease the incidence of ischemia-induced ventricular tachycardia and ventricular fibrillation. During ischemic stage, total ischemia (TI) and AAP10 total ischemia (ATI) groups were compared with partial ischemia (PI) and AAP10 partial ischemia (API) groups. The rates of incidence for arrhythmia in the ATI and API groups (10% and 0%) were lower than those in the TI and PI groups (60% and 45%). The difference between the two groups was statistically significant (P=0.019, P=0.020). The semi-quantitative analysis results of the ischemic myocardium showed that the total-Cx43 protein expression distribution areas for TI, ATI, PI and API groups were significantly decreased compared with the control group. On the other hand, the NP-Cx43 distribution areas of TI, ATI, PI and API groups were significantly increased compared with the control group (P>0.05). AAP10 could increase the total-Cx43 expression in the ischemic area and decrease the NP-Cx43 expression. Western blot results were consistent with the results of immunofluorescence staining. CONCLUSIONS: AAP10 can significantly decrease the rate of incidence of acute ischemia-induced ventricular tachycardia and ventricular fibrillation. Acute ischemic ventricular arrhythmias may have a relationship with the decreased phosphorylation of Cx43 induced by ischemia. AAP10 may stimulate the phosphorylation of Cx43 by increasing the total-Cx43 expression and decreasing the NP-Cx43 expression in the ischemic area, so as to decrease ventricular arrhythmia.