causes water-based infections resulting in severe pneumonia. Recently, we showed that different MLVA-8 (multilocus variable-number tandem-repeat analysis using 8 loci) genotypes dominated different sites of a drinking-water distribution system. Each genotype displayed a unique temperature-dependent growth behavior. Here we compared the pathogenicity potentials of different MLVA-8 genotypes of environmental and clinical strains. The virulence traits studied were hemolytic activity and cytotoxicity toward amoebae and macrophages. Clinical strains were significantly more hemolytic than environmental strains, while their cytotoxicity toward amoebae was significantly lower at 30°C. No significant differences were detected between clinical and environmental strains in cytotoxicity toward macrophages. Significant differences in virulence were observed between the environmental genotypes (Gt). Gt15 strains showed a significantly higher hemolytic activity. In contrast, Gt4 and Gt6 strains were more infective toward Moreover, Gt4 strains exhibited increased cytotoxicity toward macrophages and demonstrated a broader temperature range of amoebal lysis than Gt6 and Gt15 strains. Understanding the virulence traits of genotypes may improve the assessment of public health risks of in drinking water. is the causative agent of a severe form of pneumonia. Here we demonstrated that clinical strains were significantly more cytotoxic toward red blood cells than environmental strains, while their cytotoxicity toward macrophages was similar. Genotype 4 (Gt4) strains were highly cytotoxic toward amoebae and macrophages and lysed amoebae in a broader temperature range than to the other studied genotypes. The results can explain the relatively high success of Gt4 in the environment and in clinical samples; thus, Gt4 strains should be considered a main factor for the assessment of public health risks of in drinking water. Our findings shed light on the ecology, virulence, and pathogenicity potential of different genotypes, which can be a valuable parameter for future modeling and quantitative microbial risk assessment of in drinking-water systems.

Chironomids (Diptera; Chironomidae), also known as non-biting midges, are one of the most abundant insects in freshwater habitats. Our aim was to understand whether the metamorphosis developmental stages affect the endogenous microbiota composition of Chironomus transvaalensis. Toward our objective, we analyzed the endogenous microbiota composition of C. transvaalensis' four life stages: egg masses, larvae, pupae, and adults. Significant differences were found between the microbiota compositions of the different developmental stages of this Chironomus species. We observed a decline in bacterial diversity as the insect evolved from egg mass to adult, while the highest richness was observed in the pupal stage. Although there were significant differences between the microbiota compositions of each life stage, a bacterial core, which included 27 Amplicon Sequence Variants (ASVs), was found in all the developmental life stages (in ≥75% of samples). Chironomids are natural reservoirs of Vibrio cholerae and Aeromonas species, and the Vibrio and Aeromonas ASVs were part of the core bacteria. The presence of the ompW gene, which is specific to V. cholerae, confirmed the presence of this species in all the chironomid's life stages. Thus, the results provide important insights about the host-microbe interactions in chironomids with a specific understanding of chironomids-Vibrio-Aeromonas-microbiota interactions.

No abstract

Chironomids (Diptera; Chironomidae) are the most abundant insects in freshwater environments and are considered natural reservoirs of Vibrio cholerae. We monitored the annual dynamics of chironomid populations along with their microbiota in order to better understand host–microbiota interactions. Chironomus transvaalensis populations peaked biannually in August and May–June. The composition of the endogenous bacterial communities of their egg masses clustered in two groups according to the sampling periods August–November and May–July. Nevertheless, a core bacterial community (43%) was present in all egg-mass samples. The most abundant phyla were: Proteobacteria, Firmicutes, Cyanobacteria and Bacteroidetes. The abundance of several genera (e.g. Rheinheimera and Pseudomonas) was positively correlated with C. transvaalensis population dynamics, while a predator–prey interaction was observed between the relative abundance of Vibrio OTUs and C. transvaalensis population size. Chironomids are known to tolerate toxic and stress conditions, and our results demonstrated that bacterial genera that may protect the insect under these conditions are present in the egg masses. After hatching, the first larval meal is the gelatinous matrix that surrounds the eggs. This meal contains a probiotic consortium that may protect the larva during its metamorphosis. The results provide important insights into the host–microbe interactions of chironomids.

Vibrio cholerae, the causative agent of cholera, is commonly isolated, along with other bacterial species, from chironomid insects (Diptera: Chironomidae). Nevertheless, its prevalence in the chironomid egg masses’ microbiota is less than 0.5%. V. cholerae secretes haemagglutinin/protease (HAP) that degrades the gelatinous matrix of chironomid egg masses and prevents hatching. Quorum sensing (QS) activates HAP production in response to accumulation of bacterial autoinducers (AIs). Our aim was to define the impact of chironomid microbiota on HAP production by V. cholerae. To study QS signaling, we used V. cholerae bioluminescence reporter strains (QS‐proficient O1 El‐Tor wild‐type and QS‐deficient mutants) and different bacterial species that we isolated from chironomid egg masses. These egg mass isolates, as well as a synthetic AI‐2, caused an enhancement in lux expression by a V. cholerae QS‐deficient mutant. The addition of the egg mass bacterial isolate supernatant to the QS‐deficient mutant also enhanced HAP production and egg mass degradation activities. Moreover, the V. cholerae wild‐type strain was able to proliferate using egg masses as their sole carbon source, while the QS‐deficient was not. The results demonstrate that members of the chironomid bacterial consortium produce external chemical cues that, like AI‐2, induce expression of the hapA gene in V. cholerae. Understanding the interactions between V. cholerae and the insects’ microbiota may help uncover the interactions between this pathogen and the human gut microbiota.