In search of human-associated bacterial pathogens in Antarctic wildlife: report from six penguin colonies regularly visited by tourists.

We investigated the potential role of Antarctic tourism in the introduction of human-associated pathogens into Antarctic wildlife. We collected and analyzed 233 fecal samples from eight bird species. The samples were collected at six localities on the Antarctic Peninsula, which often is visited by tourists. Every sample was investigated for pathogens of potential human origin: Campylobacter jejuni, Salmonella spp., and Yersina spp. None of these bacteria was found. Our data suggest that the tourism industry so far has achieved its goal of not introducing pathogens into the Antarctic region. There is, however, an urgent need to further investigate the situation in areas closer to permanent Antarctic settlements.

Intestinal spirochaetes (genus Brachyspira) colonise wild birds in the southern Atlantic region and Antarctica.

INTRODUCTION: The genus Brachyspira contains well-known enteric pathogens of veterinary significance, suggested agents of colonic disease in humans, and one potentially zoonotic agent. There are recent studies showing that Brachyspira are more widespread in the wildlife community than previously thought. There are no records of this genus in wildlife from the southern Atlantic region and Antarctica. Our aim was therefore, to determine whether intestinal spirochaetes of genus Brachyspira colonise marine and coastal birds in this region. METHOD: Faecal samples were collected from marine and coastal birds in the southern Atlantic region, including sub-Antarctic islands and Antarctica, in 2002, 2009, and 2012, with the aim to isolate and characterise zoonotic agents. In total, 205 samples from 11 bird species were selectively cultured for intestinal spirochaetes of genus Brachyspira. To identify isolates to species level, they were subjected to phenotyping, species-specific polymerase chain reactions, sequencing of partial 16S rRNA, NADH oxidase (nox), and tlyA genes, and phylogenetic analysis. Antimicrobial susceptibility tests were performed. RESULTS: Fourteen unique strains were obtained from 10 birds of three species: four snowy sheathbills (Chionis albus), three kelp geese (Chloephaga hybrida subsp. malvinarum), and three brown skua (Stercorarius antarcticus subsp. lonnbergi) sampled on the Falkland Islands, Tierra del Fuego in Argentina, South Georgia, South Shetland Islands, and the Antarctic Peninsula. Five Brachyspira strains were closely related to potentially enteropathogenic Brachyspira sp. of chickens: B. intermedia (n=2, from snowy sheathbills), and B. alvinipulli (n=3, from a kelp goose and two snowy sheathbills). Three strains from kelp geese were most similar to the presumed non-pathogenic species 'B. pulli' and B. murdochii, whereas the remaining six strains could not be attributed to currently known species. No isolates related to human strains were found. None of the tested strains showed decreased susceptibility to tiamulin, valnemulin, doxycycline, tylvalosin, lincomycin, or tylosin. CONCLUSIONS: This is the first report of intestinal spirochaetes from this region. Despite limitations of current diagnostic methods, our results, together with earlier studies, show that Brachyspira spp., including potentially pathogenic strains, occur globally among free-living avian hosts, and that this genus encompasses a higher degree of biodiversity than previously recognised.

Salmonella Amager, Campylobacter jejuni, and urease-positive thermophilic Campylobacter found in free-flying peregrine falcons (Falco peregrinus) in Sweden.

Rare species with small population sizes are vulnerable to perturbations such as disease, inbreeding, or random events. The threat arising from microbial pathogens could be large and other species could act as reservoirs for pathogens. We report finding three enteric bacterial species, Salmonella Amager, Campylobacter jejuni, and urease-positive thermophilic Campylobacter, in nestling free-flying peregrine falcons (Falco peregrinus) in Sweden in 2000. Campylobacter jejuni isolates exhibited marked genetic similarities to an isolate from a human, providing a possible association between a human-associated strain of this bacterium and peregrine falcons.

The need for high-quality whole-genome sequence databases in microbial forensics.

Microbial forensics is an important part of a strengthened capability to respond to biocrime and bioterrorism incidents to aid in the complex task of distinguishing between natural outbreaks and deliberate acts. The goal of a microbial forensic investigation is to identify and criminally prosecute those responsible for a biological attack, and it involves a detailed analysis of the weapon--that is, the pathogen. The recent development of next-generation sequencing (NGS) technologies has greatly increased the resolution that can be achieved in microbial forensic analyses. It is now possible to identify, quickly and in an unbiased manner, previously undetectable genome differences between closely related isolates. This development is particularly relevant for the most deadly bacterial diseases that are caused by bacterial lineages with extremely low levels of genetic diversity. Whole-genome analysis of pathogens is envisaged to be increasingly essential for this purpose. In a microbial forensic context, whole-genome sequence analysis is the ultimate method for strain comparisons as it is informative during identification, characterization, and attribution--all 3 major stages of the investigation--and at all levels of microbial strain identity resolution (ie, it resolves the full spectrum from family to isolate). Given these capabilities, one bottleneck in microbial forensics investigations is the availability of high-quality reference databases of bacterial whole-genome sequences. To be of high quality, databases need to be curated and accurate in terms of sequences, metadata, and genetic diversity coverage. The development of whole-genome sequence databases will be instrumental in successfully tracing pathogens in the future.

Campylobacter subantarcticus sp. nov., isolated from birds in the sub-Antarctic region.

Six Gram-stain-negative, spiral-shaped, microaerobic isolates were obtained during a sampling from wild birds in the sub-Antarctic region. Based on initial observations, these isolates were classified as Campylobacter lari-like. The isolates were further characterized by whole-cell protein and amplified fragment length polymorphism (AFLP) analysis, which revealed that they were distinct from C. lari and all other known species of the genus Campylobacter. Here, we present comprehensive phylogenetic, genomic and phenotypic evidence that these isolates represent a novel species within the genus Campylobacter, for which the name Campylobacter subantarcticus sp. nov. is proposed. The type strain is R-3023(T) (=LMG 24377(T) =CCUG 38513(T)).

Campylobacter volucris sp. nov., isolated from black-headed gulls (Larus ridibundus).

During a study of the prevalence of Campylobacter jejuni in black-headed gulls (Larus ridibundus) in Sweden, three isolates, strains LMG 24379, LMG 24380T and LMG 24381, were initially identified as Campylobacter lari. Further characterization by both AFLP and whole-cell protein SDS-PAGE analyses revealed that they formed a distinct group in the genus Campylobacter. This unique position was confirmed by phenotypic characterization, 16S rRNA and hsp60 gene sequence analysis and DNA-DNA hybridizations. The combined data confirm that these isolates represent a novel species within the genus Campylobacter, for which the name Campylobacter volucris sp. nov. is proposed. The type strain is LMG 24380T (=CCUG 57498T).

Protozoan Acanthamoeba polyphaga as a potential reservoir for Campylobacter jejuni.

We showed by a laboratory experiment that four different Campylobacter jejuni strains are able to infect the protozoan Acanthamoeba polyphaga. C. jejuni cells survived for longer periods when cocultured with amoebae than when grown in culture alone. The infecting C. jejuni cells aggregated in amoebic vacuoles, in which they were seen to be actively moving. Furthermore, a resuscitation of bacterial cultures that were previously negative in culturability tests was observed after reinoculation into fresh amoeba cultures. After spontaneous rupture of the amoebae, C. jejuni could be detected by microscopy and culturability tests. Our results indicate that amoebae may serve as a nonvertebrate reservoir for C. jejuni in the environment.

Antimicrobial resistance profiles of Campylobacter jejuni isolates from wild birds in Sweden.

In order to determine the occurrence and frequency of resistant strains of the bacterium Campylobacter jejuni and to establish baseline MICs in isolates from an environmental reservoir, the resistance profiles of 10 antimicrobial substances were determined for 137 C. jejuni isolates from wild birds in Sweden. Observed MICs were generally low, with only low to moderate incidence of resistance to the tested compounds. One isolate, however, was resistant to nalidixic acid and ciprofloxacin, indicating that quinolone-resistant genotypes of C. jejuni have the potential to spread to wild bird hosts.

Prevalence of Campylobacter jejuni, Campylobacter lari, and Campylobacter coli in different ecological guilds and taxa of migrating birds.

A total of 1,794 migrating birds trapped at a coastal site in southern Sweden were sampled for detection of Campylobacter spp. All isolates phenotypically identified as Campylobacter jejuni and a subset of those identified as non-C. jejuni were identified to the species level by PCR-based techniques. C. jejuni was found in 5.0% of the birds, Campylobacter lari was found in 5.6%, and Campylobacter coli was found in 0.9%. An additional 10.7% of the tested birds were infected with hippurate hydrolysis-negative Campylobacter spp. that were not identified to the species level. The prevalence of Campylobacter spp. differed significantly between ecological guilds of birds. Shoreline-foraging birds feeding on invertebrates and opportunistic feeders were most commonly infected (76.8 and 50.0%, respectively). High prevalence was also shown in other ground-foraging guilds, i.e., ground-foraging invertebrate feeders (11.0%), ground-foraging insectivores (20.3%), and plant-eating species (18.8%). Almost no Campylobacter spp. were found in ground-foraging granivores (2.3%), arboreal insectivores (0.6%), aerial insectivores (0%), or reed- and herbaceous plant-foraging insectivores (3.5%). During the autumn migration, a high proportion of samples from juveniles were positive (7.1% in passerines, 55.0% in shorebirds), indicating transmission on the breeding grounds or during the early part of migration. Prevalence of Campylobacter spp. was associated with increasing body mass among passerine bird species. Furthermore, prevalence was higher in short-distance migrants wintering in Europe than in long-distance migrants wintering in Africa, the Middle East, or Asia. Among ground-foraging birds of the Muscicapidae, those of the subfamily Turdinae (i.e., Turdus spp.) showed a high prevalence of Campylobacter spp., while the organism was not isolated in any member of the subfamily Muscicapinae (i.e., Erithacus and Luscinia). The prevalence of Campylobacter infection in wild birds thus seems to be linked to various ecological and phylogenetic factors, with great variations in carriership between different taxa and guilds.