Bacteria in aquatic environments are a principal food source for predatory protists. Whereas interactions between bacteria and protists are recognized to determine the pathogenesis and epidemiology of several human pathogens, few studies have systematically characterized the interactions between specific aquatic bacteria and protists beyond the prey-predator relation. We, therefore, surveyed individual co-cultures between 18 different genome-sequenced marine bacteria with known virulence gene repertoires and three model protist species widely used for assessing bacteria-protist interactions. Strikingly, 10, 5, and 3 bacterial isolates were capable of lysing the protists Acanthamoeba polyphaga, Tetrahymena pyriformis, and Euglena gracilis, respectively. A majority of the bacteria were able to grow and/or maintain viable populations in the presence of viable protists. Some bacteria survived longer with viable protists but not heat-killed protists and were observed in protist vacuoles. In this respect, marine bacteria are similar to several protist-dependent human pathogens, including Legionella. Analyses of growth patterns in low-nutrient media showed that co-cultivation with A. polyphaga allowed one bacterial strain to overcome nutritional stress and obtain active growth. Five isolates depended on viable amoebae to grow, notwithstanding nutrient media status. The remarkable capability of these marine bacteria to survive encounters with, and even actively kill, model predatory protists under laboratory conditions suggests that diverse bacterial defense strategies and virulence mechanisms to access nutrients may be important in shaping microbial interactions. If verified with native marine and freshwater populations, the diversity of interactions uncovered here has implications for understanding ecological and evolutionary consequences of population dynamics in bacteria and protists.IMPORTANCEThe microbiome constitutes the base of food webs in aquatic environments. Its composition partly reflects biotic interactions, where bacteria primarily are considered prey of predatory protists. However, studies that focus on one or a few species have shown that some bacteria have abilities to escape grazing and may even be capable of lysing their protist predators. In this study, we substantially extend these findings by systematically investigating interactions among multiple taxa of both bacteria and protists. Our results show that marine bacteria display a wider and more complex range of interactions with their predators than generally recognized-from growth dependency to protist lysis. Given that such interactions play key roles in the pathogenesis and epidemiology of several human pathogens, our findings imply that bacterial virulence traits can contribute to defining the structure and ecology of aquatic microbiomes.

Association of the water- and foodborne pathogen Campylobacter jejuni with free-living Acanthamoeba spp. trophozoites enhances C. jejuni survival and resistance to biocides and starvation. When facing less than optimal environmental conditions, however, the Acanthamoeba spp. host can temporarily transform from trophozoite to cyst and back to trophozoite, calling the survival of the internalized symbiont and resulting public health risk into question. Studies investigating internalized C. jejuni survival after A. castellanii trophozoite transformation have neither been able to detect its presence inside the Acanthamoeba cyst after encystation nor to confirm its presence upon excystation of trophozoites through culture-based techniques. The purpose of this study was to detect C. jejuni and Mycobacterium avium recovered from A. polyphaga trophozoites after co-culture and induction of trophozoite encystation using three different encystation methods (Neff's medium, McMillen's medium and refrigeration), as well as after cyst excystation. Internalized M. avium was used as a positive control, since studies have consistently detected the organism after co-culture and after host excystation. Concentrations of C. jejuni in A. polyphaga trophozoites were 4.5 × 10⁵ CFU/ml, but it was not detected by PCR or culture post-encystation. This supports the hypothesis that C. jejuni may be digested during encystation of the amoebae. M. avium was recovered at a mean concentration of 1.9 × 10⁴ from co-cultured trophozoites and 4.4 × 10¹ CFU/ml after excystation. The results also suggest that M. avium recovery post-excystation was statistically significantly different based on which encystation method was used, ranging from 1.3 × 10¹ for Neff's medium to 5.4 × 10¹ CFU/ml for refrigeration. No M. avium was recovered from A. polyphaga cysts when trophozoites were encysted by McMillen's medium. Since C. jejuni internalized in cysts would be more likely to survive harsh environmental conditions and disinfection, a better understanding of potential symbioses between free-living amoebae and campylobacters in drinking water distribution systems and food processing environments is needed to protect public health. Future co-culture experiments examining survival of internalized C. jejuni should carefully consider the encystation media used, and include molecular detection tools to falsify the hypothesis that C. jejuni may be present in a viable but not culturable state.

Sammanfattning: Föreliggande rapport syftar till att vara en kartläggning över nuvarande situation med identifierade problem och behov gällande tillgång till information mellan olika huvudmän för att vara ett underlag till strategi och handlingsplan. Arbetet i denna rapport har genomförts i två steg; dels en enkätundersökning och dels genomförande av två workshops.Kartläggningen visade att det finns ett stort antal personer som är behöriga till olika systemstöd men som inte använder dem. Det framkom även att en stor del upplever brister i befintliga rutiner och att rutiner inte följs. Brister inom området med rutiner kan hanteras genom utbildning och revidering av befintliga dokument. Resultatet pekar också på att det brister i förståelse för varandra, mellan huvudmännen, hur rutinerna ser ut och hur man arbetar på olika sätt inom olika verksamheter. Man efterfrågar utbildning och mer specifikt en gemensam utbildning för landsting och kommuner för att få en ökad förståelse och insyn i varandras verksamheter eftersom det i praktiken påverkar och berör det egna dagliga arbetet. En stor del av deltagarna anser att systemen saknar viss typ av informationsmängd eller funktionalitet. Vidare är det även en stor del som upplever dubbelarbete som följd av användningen av systemstöden. Enkätsvaren visar att det finns ett behov från landstingets personal att digitalt kunna ta del av information såsom hälsostatus från kommunerna. Dessutom säger enkätsvaren att båda huvudmännen (men främst kommunernas Socialtjänst) efterfrågar möjligheten att kommunicera på ett säkert och digitalt sätt med andra myndigheter.

BACKGROUND: Campylobacter jejuni is a common cause of human bacterial diarrhea in most parts of the world. Most C. jejuni infections are acquired from contaminated poultry, milk, and water. Due to health care costs and human suffering, it is important to identify all possible sources of infection. Unpasteurized milk has been associated with several outbreaks of C. jejuni infection. Campylobacter has been identified on fresh fruit, and other gastrointestinal pathogens such as Salmonella, E. coli O157:H7 and Cryptosporidium have been involved in fruit juice outbreaks. C. jejuni is sensitive to the acidic environment of fruit juice, but co-cultures with the amoeba, Acanthamoeba polyphaga, have previously been shown to protect C. jejuni at low pH.

METHODS: To study the influence of A. polyphaga on the survival of C. jejuni in milk and juice, the bacteria were incubated in the two products at room temperature and at 4°C with the following treatments: A) C. jejuni preincubated with A. polyphaga before the addition of product, B) C. jejuni mixed with A. polyphaga after the addition of product, and C) C. jejuni in product without A. polyphaga. Bacterial survival was assessed by colony counts on blood agar plates.

RESULTS: Co-culture with A. polyphaga prolonged the C. jejuni survival both in milk and juice. The effect of co-culture was most pronounced in juice stored at room temperature. On the other hand, A. polyphaga did not have any effect on C. jejuni survival during pasteurization of milk or orange juice, indicating that this is a good method for eliminating C. jejuni in these products.

CONCLUSION: Amoebae-associated C. jejuni in milk and juice might cause C. jejuni infections.

The Gram-negative bacterium Campylobacter jejuni is able to enter, survive and multiply within the free living amoeba Acanthamoeba polyphaga, but the molecular mechanisms behind these events are still unclear. We have studied the uptake and intracellular trafficking of viable and heat killed bacterial cells of the C. jejuni strain 81-176 in A. polyphaga. We found that viable bacteria associated with a substantially higher proportion of Acanthamoeba trophozoites than heat killed bacteria. Furthermore, the kinetics of internalization, the total number of internalized bacteria as well as the intracellular localization of internalized C. jejuni were dramatically influenced by bacterial viability. Viable bacteria were internalized at a high rate already after 1 h of co-incubation and were observed in small vacuoles tightly surrounding the bacteria. In contrast, internalization of heat killed C. jejuni was low at early time points and did not peak until 96 h. These cells were gathered in large spacious vacuoles that were part of the degradative pathway as determined by the uptake of fluorescently labeled dextran. The amount of heat killed bacteria internalized by A. polyphaga did never reach the maximal amount of internalized viable bacteria. These results suggest that the uptake and intracellular survival of C. jejuni in A. polyphaga is bacterially induced.

Low concentrations of Campylobacter jejuni cells in environmental samples make them difficult to study with conventional culture methods. Here, we show that enrichment by amoeba cocultures works well with low-concentration samples and that this method can be combined with molecular techniques without loss of genetic specificity.

Several species of free-living amoebae can cause disease in humans. However, in addition to the direct pathogenicity of e.g. Acanthamoebae and Naegleria species, they are recognized as environmental hosts, indirectly involved in the epidemiology of many pathogenic bacteria. Although several studies have demonstrated intracellular survival of many different bacteria in these species, the extent of such interactions as well as the implications for the epidemiology of the bacterial species involved, are largely unknown and probably underestimated. In this study, we evaluated eight different unicellular eukaryotic organisms, for their potential to serve as environmental hosts for Campylobacter species. These organisms include four amoebozoas (Acanthamoeba polyphaga, Acanthamoeba castellanii, Acanthamoeba rhysodes and Hartmanella vermiformis), one alveolate (Tetrahymena pyriformis), one stramenopile (Dinobryon sertularia), one eugoenozoa (Euglena gracilis) and one heterolobosea (Naegleria americana). Campylobacter spp. including Campylobacter jejuni, Campylobacter coli and Campylobacter lari are the most common cause of gastroenteritis in the western world. Survival and replication of these three species as well as Campylobacter hyointestinalis were assessed in co-cultures with the eukaryotic organisms. Campylobacter spp. generally survived longer in co-cultures, compared to when incubated in the corresponding growth media. The eukaryotic species that best promoted bacterial survival was the golden algae D. sertularia. Three species of amoebozoas, of the genus Acanthamoeba promoted both prolonged survival and replication of Campylobacter spp. The high abundance in lakes, ponds and water distribution networks of these organisms indicate that they might have a role in the epidemiology of campylobacteriosis, possibly contributing to survival and dissemination of these intestinal pathogens to humans and other animals. The results suggest that not only C. jejuni, but a variety of Campylobacter spp. can interact with different eukaryotic unicellular organisms.

Campylobacter jejuni is a common cause of bacterial gastroenteritis in most parts of the world. The bacterium has a broad host range and has been isolated from many animals and environments. To investigate shedding patterns and putative effects on an avian host, we developed a colonization model in which a wild bird species, the European Robin Erithacus rubecula, was inoculated orally with C. jejuni from either a human patient or from another wild bird species, the Song Thrush Turdus philomelos. These two isolates were genetically distinct from each other and provoked very different host responses. The Song Thrush isolate colonized all challenged birds and colonization lasted 6.8 days on average. Birds infected with this isolate also showed a transient but significant decrease in body mass. The human isolate did not colonize the birds and could be detected only in the feces of the birds shortly after inoculation. European Robins infected with the wild bird isolate generated a specific antibody response to C. jejuni membrane proteins from the avian isolate, which also was cross-reactive to membrane proteins of the human isolate. In contrast, European Robins infected with the human isolate did not mount a significant response to bacterial membrane proteins from either of the two isolates. The difference in colonization ability could indicate host adaptations.