Neisseria arctica sp. nov., isolated from nonviable eggs of greater white-fronted geese (Anser albifrons) in Arctic Alaska.

During the summers of 2013 and 2014, isolates of a novel Gram-stain-negative coccus in the genus Neisseriawere obtained from the contents of nonviable greater white-fronted goose (Anseralbifrons) eggs on the Arctic Coastal Plain of Alaska. We used a polyphasic approach to determine whether these isolates represent a novel species. 16S rRNA gene sequences, 23S rRNA gene sequences, and chaperonin 60 gene sequences suggested that these Alaskan isolates are members of a distinct species that is most closely related to Neisseria canis, Neisseriaanimaloris and Neisseriashayeganii. Analysis of the rplF gene additionally showed that the isolates are unique and most closely related to Neisseriaweaveri. Average nucleotide identity of the whole genome sequence of the type strain was between 71.5 and 74.6 % compared to close relatives, further supporting designation as a novel species. Fatty acid methyl ester analysis showed a predominance of C14 : 0, C16 : 0 and C16 : 1ω7c fatty acids. Finally, biochemical characteristics distinguished the isolates from other species of the genus Neisseria. On the basis of these combined data, the isolates are proposed to represent a novel species of the genus Neisseria, with the name Neisseria arctica sp. nov. The type strain is KH1503T (=ATCC TSD-57T=DSM 103136T).

Microbial Infections Are Associated with Embryo Mortality in Arctic-Nesting Geese.

To address the role of bacterial infection in hatching failure of wild geese, we monitored embryo development in a breeding population of Greater white-fronted geese (Anser albifrons) on the Arctic Coastal Plain of Alaska. During 2013, we observed mortality of normally developing embryos and collected 36 addled eggs for analysis. We also collected 17 infertile eggs for comparison. Using standard culture methods and gene sequencing to identify bacteria within collected eggs, we identified a potentially novel species of Neisseria in 33 eggs, Macrococcus caseolyticus in 6 eggs, and Streptococcus uberis and Rothia nasimurium in 4 eggs each. We detected seven other bacterial species at lower frequencies. Sequences of the 16S rRNA genes from the Neisseria isolates most closely matched sequences from N. animaloris and N. canis (96 to 97% identity), but phylogenetic analysis suggested substantial genetic differentiation between egg isolates and known Neisseria species. Although definitive sources of the bacteria remain unknown, we detected Neisseria DNA from swabs of eggshells, nest contents, and cloacae of nesting females. To assess the pathogenicity of bacteria identified in contents of addled eggs, we inoculated isolates of Neisseria, Macrococcus, Streptococcus, and Rothia at various concentrations into developing chicken eggs. Seven-day mortality rates varied from 70 to 100%, depending on the bacterial species and inoculation dose. Our results suggest that bacterial infections are a source of embryo mortality in wild geese in the Arctic.

Francisella novicida pathogenicity island encoded proteins were secreted during infection of macrophage-like cells.

Intracellular pathogens and other organisms have evolved mechanisms to exploit host cells for their life cycles. Virulence genes of some intracellular bacteria responsible for these mechanisms are located in pathogenicity islands, such as secretion systems that secrete effector proteins. The Francisella pathogenicity island is required for phagosomal escape, intracellular replication, evasion of host immune responses, virulence, and encodes a type 6 secretion system. We hypothesize that some Francisella novicida pathogenicity island proteins are secreted during infection of host cells. To test this hypothesis, expression plasmids for all Francisella novicida FPI-encoded proteins with C-terminal and N-terminal epitope FLAG tags were developed. These plasmids expressed their respective epitope FLAG-tagged proteins at their predicted molecular weights. J774 murine macrophage-like cells were infected with Francisella novicida containing these plasmids. The FPI proteins expressed from these plasmids successfully restored the intramacrophage growth phenotype in mutants of the respective genes that were deficient for intramacrophage growth. Using these expression plasmids, the localization of the Francisella pathogenicity island proteins were examined via immuno-fluorescence microscopy within infected macrophage-like cells. Several Francisella pathogenicity island encoded proteins (IglABCDEFGHIJ, PdpACE, DotU and VgrG) were detected extracellularly and they were co-localized with the bacteria, while PdpBD and Anmk were not detected and thus remained inside bacteria. Proteins that were co-localized with bacteria had different patterns of localization. The localization of IglC was dependent on the type 6 secretion system. This suggests that some Francisella pathogenicity island proteins were secreted while others remain within the bacterium during infection of host cells as structural components of the secretion system and were necessary for secretion.

The biochemical properties of the Francisella pathogenicity island (FPI)-encoded proteins IglA, IglB, IglC, PdpB and DotU suggest roles in type VI secretion.

The Francisella pathogenicity island (FPI) encodes proteins thought to compose a type VI secretion system (T6SS) that is required for the intracellular growth of Francisella novicida. In this work we used deletion mutagenesis and genetic complementation to determine that the intracellular growth of F. novicida was dependent on 14 of the 18 genes in the FPI. The products of the iglABCD operon were localized by the biochemical fractionation of F. novicida, and Francisella tularensis LVS. Sucrose gradient separation of water-insoluble material showed that the FPI-encoded proteins IglA, IglB and IglC were found in multiple fractions, especially in a fraction that did not correspond to a known membrane fraction. We interpreted these data to suggest that IglA, IglB and IglC are part of a macromolecular structure. Analysis of published structural data suggested that IglC is an analogue of Hcp, which is thought to form long nano-tubes. Thus the fractionation properties of IglA, IglB and IglC are consistent with the current model of the T6SS apparatus, which supposes that IglA and IglB homologues form an outer tube structure that surrounds an inner tube composed of Hcp (IglC) subunits. Fractionation of F. novicida expressing FLAG-tagged DotU (IcmH homologue) and PdpB (IcmF homologue) showed that these proteins localize to the inner membrane. Deletion of dotU led to the cleavage of PdpB, suggesting an interaction of these two proteins that is consistent with results obtained with other T6SSs. Our results may provide a mechanistic basis for many of the studies that have examined the virulence properties of Francisella mutants in FPI genes, namely that the observed phenotypes of the mutants are the result of the disruption of the FPI-encoded T6SS structure.

Streptococcus phocae isolated from a spotted seal (Phoca largha) with pyometra in Alaska.

A spotted seal harvested by subsistence hunters in Kotzebue Sound, Alaska (USA), showed a grossly enlarged uterus and associated lymph nodes. Streptococcus phocae was isolated from the purulent uterine discharge. Histopathologic examination revealed inflammation that was limited to the uterine mucosa. Lymph nodes draining the affected organ were reactive but no evidence of active infection was found in the lymph nodes. This report is the first Streptococcus phocae isolated from spotted seals as well as the first report of pyometra as the main pathologic finding associated with this pathogen. Isolation of this pathogen from Alaska expands the reported range to arctic pinnipeds. Zoonotic potential remains unknown.