Vibrio cholerae O1 Inhabit Intestines and Spleens of Fish in Aquaculture Ponds.

Vibrio cholerae is the causative agent of cholera, an acute diarrheal disease that spreads locally and globally in epidemics and pandemics. Although it was discovered that fish harbor V. cholerae strains in their intestines, most investigations revealed non-toxic V. cholerae serogroups in fish. Due to the rarity of toxigenic V. cholerae serogroups, it is difficult to cultivate these strains from environmental samples. Hence, here we aimed to uncover evidence of the occurrence of toxigenic V. cholerae in the intestines and spleens of various fish species. By using molecular detection tools, we show that V. cholerae O1 and strains positive for the cholera toxin inhabit both healthy and diseased fish intestines and spleens, suggesting that fish may serve as intermediate vectors of toxigenic V. cholerae. No significant differences were found between the abundance of toxigenic V. cholerae (either O1 or cholera toxin positive strains) in the healthy and the diseased fish intestines or spleens. In conclusion, a variety of fish species may serve as potential vectors and reservoirs of toxigenic V. cholerae as they form a link between the other reservoirs of V. cholerae (chironomids, copepods, and waterbirds). Similarly, they may aid in the spread of this bacterium between water bodies.

Aeromonashydrophila infection in tilapia triggers changes in the microbiota composition of fish internal organs.

Aeromonas hydrophila is a major pathogenic species that causes mass mortality in various freshwater fish species including hybrid tilapia, the main fish species in Israeli aquaculture. Our hypothesis was that A. hydrophila infection may cause changes in the microbiota composition of fish internal organs, and therefore we aimed to study the effect of A. hydrophila infection by injection or by net handling on the microbiota compositions of fish intestine, spleen, and liver. Significant differences in the microbiota composition were found between the internal organs of the diseased and the healthy fish in both experimental setups. Fusobacteriota was the most dominant phylum in the microbiota of healthy fish (∼70%, liver). Cetobacterium was the most abundant genus and relatively more abundant in healthy, compared to diseased fish. When A. hydrophila was inoculated by injection, it was the only pathogenic genus in the spleen and liver of the diseased fish. However, in the handling experiment, Vibrio was also detected in the diseased fish, demonstrating coinfection interactions. Based on these experiments, we conclude that indeed, A. hydrophila infection in tilapia causes changes in the microbiota composition of fish internal organs, and that fish net handling may trigger bacterial infection in freshwater aquaculture.

Intestine and spleen microbiota composition in healthy and diseased tilapia.

Symbiotic bacteria within the gut microbiome of various organisms, including fish, provide the host with several functions that improve the immune system. Although the spleen plays an important role in the modulation of immune responses, the role of spleen microbiota in shaping the immune system is unclear. Our study aimed at understanding the relationship between fish health and microbiota composition in the intestine and spleen. Our model organism was the hybrid tilapia (Oreochromis aureus × Oreochromis niloticus). We sampled intestine and spleen from healthy and diseased adult tilapia and determined their microbiota composition by sequencing the 16S rRNA gene. Significant differences were found between the intestine and the spleen microbiota composition of healthy compared to diseased fish as well as between intestines and spleens of fish with the same health condition. The microbiota diversity of healthy fish compared to diseased fish was significantly different as well. In the intestine of healthy fish, Cetobacterium was the most abundant genus while Mycoplasma was the most abundant genus in the spleen. Vibrio was the most abundant genus in the intestine and spleen of diseased fish. Moreover, it seems that there is a co-infection interaction between Vibrio and Aeromonas, which was reflected in the spleen of diseased fish. While Vibrio, Aeromonas and Streptococcus were the probable pathogens in the diseased fish, the role of Mycoplasma as a pathogen of cultured hybrid tilapia remains uncertain. We conclude that the intestine and spleen microbiota composition is strongly related to the health condition of the fish.

Comparative Microbiota Composition Across Developmental Stages of Natural and Laboratory-Reared Chironomus circumdatus Populations From India.

Chironomids are aquatic insects that undergo a complete metamorphosis of four life stages. Here we studied, for the first time, the microbiota composition of Chironomus circumdatus, a tropical midge species, both from the Mula and Mutha Rivers in Pune, India and as a laboratory-reared culture. We generated a comparative microbial profile of the eggs, larvae and pupae, the three aquatic life stages of C. circumdatus. Non-metric multidimensional scaling analysis (NMDS) demonstrated that the developmental stage had a more prominent effect on the microbiota composition compared to the sampling location. Notably, the microbiota composition of the egg masses from the different sampling points clustered together and differed from laboratory culture larvae. Proteobacteria was the dominant phylum in all the environmental and laboratory-reared egg masses and pupal samples, and in the laboratory-reared larvae, while Fusobacteria was the dominant phylum in the larvae collected from the field environment. The most abundant genera were Cetobacterium, Aeromonas, Dysgonomonas, Vibrio, and Flavobacterium. The ten amplicon sequence variants (ASVs) that most significantly contributed to differences in microbiota composition between the three sampled locations were: Burkholderiaceae (ASVs 04 and 37), C39 (Rhodocyclaceae, ASV 14), Vibrio (ASV 07), Arcobacter (ASV 21), Sphaerotilus (ASV 22), Bacteroidia (ASVs 12 and 28), Flavobacterium (ASV 29), and Gottschalkia (ASV 10). No significant differences were found in the microbial richness (Chao1) or diversity (Shannon H') of the three sampled locations. In contrast, significant differences were found between the microbial richness of the three life stages. Studying the microbiota of this Chironomus species may contribute to a better understanding of the association of C. circumdatus and its microbial inhabitants.

Comparative Study of Intestinal Microbiota Composition of Six Edible Fish Species.

Intensive freshwater aquaculture in the Spring Valley, Israel, is implemented mainly in earthen fishponds and reservoirs that are stocked with a variety of edible fish species. Here we sampled six different healthy fish species from these intensive aquacultures. The fish were hybrid striped bass, European bass, red drum (all carnivores), hybrid tilapia, flathead grey mullet (both herbivores), and common carp (an omnivore). Significant differences were found among the intestinal microbiota of the six studied fish species. The microbiota composition diversity was strongly related to the trophic level of the fish, such that there was a significant difference between the carnivore and the herbivore species, while the omnivore species was not significantly different from either group. The most abundant genus in the majority of the fishes' intestinal microbiota was Cetobacterium. Furthermore, we found that beside Cetobacterium, a unique combination of taxa with relative abundance >10% characterized the intestine microbiota of each fish species: unclassified Mycoplasmataceae, Aeromonas, and Vibrio (hybrid striped bass); Turicibacter and Clostridiaceae 1 (European bass); Vibrio (red drum); ZOR0006-Firmicutes (hybrid tilapia); unclassified Mycoplasmataceae and unclassified Vibrionaceae (flathead grey mullet); and Aeromonas (common carp). We conclude that each fish species has a specific bacterial genera combination that characterizes it. Moreover, diet and the trophic level of the fish have a major influence on the gut microbiota of healthy fish that grow in intensive freshwater aquaculture.

Chironomus ramosus Larval Microbiome Composition Provides Evidence for the Presence of Detoxifying Enzymes.

Chironomids (Diptera; Chironomidae) are aquatic insects that are abundant in freshwater. We aimed to study the endogenous microbiota composition of Chironomus ramosus larvae that were sampled from the Mutha River and a laboratory culture in India. Furthermore, we performed a metagenomic analysis of the larval microbiome, sampled from the Mutha River. Significant differences were found between the bacterial community composition of C. ramosus larvae that were sampled from the Mutha River and the laboratory culture. A total of 54.7% of the amplicon sequence variants (ASVs) that were identified in the larvae from the Mutha River were unique, compared to only 12.9% of unique ASVs that were identified from the laboratory-reared larvae. The four most abundant phyla across all samples were: Proteobacteria, Fusobacteria, Firmicutes, and Bacteroidetes, while the nine most abundant genera were: Aeromonas, Alkanindiges, Breznakia, Cetobacterium, Chryseobacterium, Desulfovibrio, Dysgonomonas, Thiothrix, and Vibrio. Moreover, in the metagenomic analysis, we detected bacterial genes and bacterial pathways that demonstrated the ability to degrade different toxic compounds, detoxify metal, and confer resistance to antibiotics and UV radiation, amongst other functions. The results illuminate the fact that there are detoxifying enzymes in the C. ramosus larval microbiome that possibly play a role in protecting the insect in polluted environments.

Tsukamurella pulmonis conjunctivitis in patients with an underlying nasolacrimal duct obstruction - report of two cases.

Tsukamurella pulmonis ( Actinobacteria ), a Gram-positive, obligate aerobic and weakly or variably acid-fast bacterium, is an opportunistic pathogen. Here we report two cases of conjunctivitis caused by T. pulmonis . Both patients had a previous history of nasolacrimal duct obstruction (NLDO). Isolation of T. pulmonis was performed on chocolate, tryptic soy blood and Columbia nalidixic agars. After 24 h of incubation, odourless, white-greyish, membrane-like colonies were observed. The VITEK-2 bacterial identifier system failed to identify the species, while Vitek-MS matrix-assisted laser desorption ionization time-of-flight technology, successfully identified the isolate from case 2 but not from case 1. Final identification was verified using 16S rRNA gene sequencing. An antibiogram was performed and according to the results cefazoline in addition to vancomycin eye drops for 5 days, were suggested as a treatment in case 1. In case 2 the infection was ended without treatment. This is the first report of Tsukamurella as a pathogen that causes conjunctivitis in patients with NLDO.

Direct Evidence That Sunbirds' Gut Microbiota Degrades Floral Nectar's Toxic Alkaloids.

Orange-tufted sunbirds (Cinnyris osea) feed on the nectar of the tobacco tree (Nicotiana glauca) which contains toxic pyridine alkaloids characterized by high concentrations of anabasine and much lower concentrations of nicotine. We aimed at determining whether the gut microbiota of sunbirds harbors bacterial species that enable the birds to cope with these toxic alkaloids. An in vivo experiment that included 12 birds showed that inducing dysbiosis in sunbirds' guts by the addition of sulfamethoxazole and trimethoprim, significantly reduced the birds' ability to degrade anabasine (n = 3) compared to control birds (n = 3) with undisturbed microbiota. Sunbirds whose gut bacterial communities were altered by the antibacterial agents and who were fed with added nicotine, also showed a lower percentage of nicotine degradation (n = 3) in their excreta compared to the sunbirds with undisturbed microbiota (n = 3), though this difference was not significant. In an in vitro experiment, we studied the ability of Lactococcus lactis, Enterobacter hormaechei, Chryseobacterium gleum, Kocuria palustris, and Methylorubrum populi that were isolated from sunbirds' excreta, to degrade anabasine and nicotine. By using gas chromatography-mass spectrometry (GC-MS) analysis, we successfully demonstrated, for the first time, the ability of these species to degrade the focal secondary metabolites. Our findings demonstrate the role of gut bacteria in detoxifying toxic secondary metabolites found in the N. glauca nectar. The degradation products may supply the birds with nitrogen which is scarce in nectar-rich diets. These findings support another role of bacteria in mediating the interactions between plants and their pollinators.

Copper and chromium exposure affect chironomid larval microbiota composition.

Chironomids are aquatic insects that are known to be pollution tolerant. We have recently demonstrated that endogenous chironomid microbiota protects its host from toxic metals. Following these findings, we hypothesized that under different environmental conditions, a different bacterial consortium will evolve. Our aim was to explore the change in chironomid larval microbiota composition triggered by exposure to toxic copper and hexavalent chromium. Chironomid larvae were collected from the environment and treated in the laboratory with copper, hexavalent chromium, and no metal (control). After six days, the microbial composition of the surviving larvae was examined. We found a significant change in larval microbiota composition between the three treatments and for different copper concentrations. The abundance of specific taxa varied significantly between the treatments. At the genus level, the abundance of some genera (e.g. Yersinia, Acinetobacter) increased in the presence of copper, and some genera (e.g. Yersinia, Dysgonomonas, Delftia, Enterococcus) increased in the presence of hexavalent chromium, compared to the control. The change in the larval microbiota composition was rapid and metal-specific. We suggest that each larva hosts a consortium of bacterial species that can proliferate under a specific environmental change and thus, protect the insect under unstable environmental conditions.

Quorum-sensing signaling by chironomid egg masses' microbiota, affects haemagglutinin/protease (HAP) production by Vibrio cholerae.

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.

Geographic partitioning or environmental selection: What governs the global distribution of bacterial communities inhabiting floral nectar?

Floral nectar harbors microbial communities which have significant impacts on its chemistry, volatiles, nutritional contents, and attractiveness for pollinators. Yet, fundamental knowledge regarding the structure and composition of nectar-associated microbiomes remains largely unknown. Especially elusive are the environmental factors and spatial effects that shape nectar-inhabiting microbial communities. The aim of this study was to explore and analyze the role of geographical and environmental factors affecting the composition and global distribution of floral nectar microbiota. We explored and compared the structure of bacterial communities inhabiting the floral nectar of the widely spread and invasive tobacco tree (Nicotiana glauca) in six continents: South and North America, Australia, Europe, Africa, and Asia, using 16S rRNA gene sequencing. Environmental abiotic data for each sampled plant was obtained from the Worldclim database and applied for inferring the effects of environmental conditions on bacterial community structure and diversity. Most abundant in the nectar were the Proteobacteria, Firmicutes, and Actinobacteria phyla, with Acinetobacter and Rosenbergiella (Proteobacteria) being the dominant bacterial genera that contributed most to the dissimilarities between sites. Acinetobacter and Rosenbergiella abundances were negatively correlated and significantly higher in the Mediterranean regions (Greece, Israel, and the Canary Islands) compared to Argentina and Australia. Temperature, site-elevation, rainfall, and density of vegetation were found to have significant effects on the structure and diversity of these bacterial communities in the nectar. Vegetation density was positively correlated with microbial diversity, while increased temperatures and elevation reduced the diversity and evenness of bacterial communities. Mantel's test showed that the similarity between the bacterial communities' composition significantly decreased as distances between them increased. We conclude that both geographical distance and local environmental abiotic conditions affect and shape the composition and diversity of nectar inhabiting bacterial communities.

Identification of chironomid species as natural reservoirs of toxigenic Vibrio cholerae strains with pandemic potential.

Vibrio cholerae causes the fatal cholera diarrhea. Chironomids (Diptera; Chironomidae) are abundant in freshwater aquatic habitats and estuaries and are natural reservoirs of V. cholerae. Until now, only the non-O1/O139 serogroups of V. cholerae were identified in chironomids. Here, we explored whether chironomids are natural reservoirs of V. cholerae O1/O139 serogroups, which are associated with cholera endemics and pandemics. All four life stages of chironomids were sampled from two rivers, and a laboratory culture in Pune, India, and from a pond in Israel. In total, we analyzed 223 chironomid samples. The presence of V. cholerae O1/O139 serogroups was verified using molecular tools. Nine chironomid species were identified; of them, Chironomus circumdatus was the most abundant. The presence of V. cholerae serogroup O1 and the cholera toxin genes were detected in samples from all chironomid species. However, serogroup O139 was detected in only two chironomid species. Besides PCR to detect specific genes, a metagenomic analysis that was performed in three selected C. ramosus larvae, identified a list of virulence genes associated with V. cholerae. The findings provide evidence that chironomids are natural reservoirs of toxigenic V. cholerae O1/O139. Chironomid populations and V. cholerae show biannual peak patterns. A similar pattern is found for cholera epidemics in the Bengal Delta region. Thus, we hypothesize that monitoring chironomids in endemic areas of the disease may provide a novel tool for predicting and preventing cholera epidemics. Moreover, serogroup O139 was detected only in two chironomid species that have a restricted distribution in the Indian subcontinent, possibly explaining why the distribution of the O139 serogroup is limited.

Changes in Microbiota Composition Along the Metamorphosis Developmental Stages of Chironomus transvaalensis.

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.

The effect of toxic pyridine-alkaloid secondary metabolites on the sunbird gut microbiome.

Sunbirds feed on tobacco tree nectar which contains toxic nicotine and anabasine secondary metabolites. Our aim was to understand the effect of nicotine and anabasine on the gut microbiota composition of sunbirds. Sixteen captive sunbirds were randomly assigned to two diets: artificial nectar either with (treatment) or without (control) added nicotine and anabasine. Excreta were collected at 0, 2, 4 and 7 weeks of treatment and samples were processed for bacterial culture and high-throughput amplicon sequencing of the 16S rRNA gene. The gut microbiome diversity of the treated and control birds changed differently along the seven-week experiment. While the diversity decreased in the control group along the first three samplings (0, 2 and 4 weeks), it increased in the treatment group. The microbiota composition analyses demonstrated that a diet with nicotine and anabasine, significantly changed the birds' gut microbiota composition compared to the control birds. The abundance of nicotine- and anabasine- degrading bacteria in the excreta of the treated birds, was significantly higher after four and seven weeks compared to the control group. Furthermore, analysis of culturable isolates, including Lactococcus, showed that sunbirds' gut-associated bacteria were capable of degrading nicotine and anabasine, consistent with their hypothesised role as detoxifying and nutritional symbionts.

Draft genome of Rosenbergiella nectarea strain 8N4T provides insights into the potential role of this species in its plant host.

BACKGROUND: Rosenbergiella nectarea strain 8N4T, the type species of the genus Rosenbergiella, was isolated from Amygdalus communis (almond) floral nectar. Other strains of this species were isolated from the floral nectar of Citrus paradisi (grapefruit), Nicotiana glauca (tobacco tree) and from Asphodelus aestivus. R. nectarea strain 8N4T is a Gram-negative, oxidase-negative, facultatively anaerobic bacterium in the family Enterobacteriaceae. RESULTS: Here we describe features of this organism, together with its genome sequence and annotation. The DNA GC content is 47.38%, the assembly size is 3,294,717 bp, and the total number of genes are 3,346. The genome discloses the possible role that this species may play in the plant. The genome contains both virulence genes, like pectin lyase and hemolysin, that may harm plant cells and genes that are predicted to produce volatile compounds that may impact the visitation rates by nectar consumers, such as pollinators and nectar thieves. CONCLUSIONS: The genome of R. nectarea strain 8N4T reveals a mutualistic interaction with the plant host and a possible effect on plant pollination and fitness.

Comparative Analysis of Intestine Microbiota of Four Wild Waterbird Species.

Waterbirds are ubiquitous and globally distributed. Yet, studies on wild waterbirds' gut microbiota are still rare. Our aim was to explore and compare the gut microbial community composition of wild waterbird species. Four wild waterbird species that are either wintering or all-year residents in Israel were studied: great cormorants, little egrets, black-crowned night herons and black-headed gulls. For each bird, three intestinal sections were sampled; anterior, middle and posterior. No significant differences were found among the microbiota compositions in the three intestine sections of each individual bird. Each waterbird species had a unique microbial composition. The gut microbiota of the black-headed gulls' fundamentally deviated from that of the other bird species, probably due to a very high abundance (58.8%) of the genus Catellicoccus (Firmicutes). Our results suggest a correlation between the waterbird species' phylogeny and their intestine microbial community hierarchical tree, which evinced phylosymbiosis. This recent coinage stands for eco-evolutionary patterns between the host phylogeny and its microbiota composition. We conclude that eco-evolutionary processes termed phylosymbiosis may occur between wild waterbird species and their gut microbial community composition.

Accumulating evidence suggests that some waterbird species are potential vectors of Vibrio cholerae.

Vibrio cholerae is the causative agent of cholera, a life-threatening diarrheal disease. Cholera causes epidemics and pandemics, but the ways this disease spreads worldwide is still unclear. This review highlights a relatively new hypothesis regarding the way V. cholerae can be globally dispersed. Copepods and chironomids are natural reservoirs of V. cholerae and are part of different fish species' diet. Furthermore, V. cholerae inhabits marine and freshwater fish species. Waterbird species feed on fish or on small invertebrates such as copepods and chironomids. Waterbirds have also been found to carry living copepods and/or chironomids internally or externally from one waterbody to another. All of the above points to the fact that some waterbird species might be vectors of V. cholerae. Indeed, we and others have found evidence for the presence of V. cholerae non-O1 as well as O1 in waterbird cloacal swabs, feces, and intestine samples. Moreover, hand-reared cormorants that were fed on tilapia, a fish that naturally carries V. cholerae, became infected with this bacterial species, demonstrating that V. cholerae can be transferred to cormorants from their fish prey. Great cormorants as well as other waterbird species can cover distances of up to 1,000 km/day and thus may potentially transfer V. cholerae in a short time across and between continents. We hope this review will inspire further studies regarding the understanding of the waterbirds' role in the global dissemination of V. cholerae.

Extended phenotype in action. Two possible roles for silica needles in plants: not just injuring herbivores but also inserting pathogens into their tissues.

Phytoliths are silica bodies of various shapes including in the shape of sharp needles formed by many land plants. Defense from herbivory is one of the several known functions of phytoliths, especially the mechanical defense by abrasion of the mouthparts of arthropods and the teeth of mammalian herbivores. Another, although somewhat lesser-known, anti-herbivory defensive mechanism of phytoliths is wounding by sharp silica needles. We discuss and illuminate an even much less known defensive mechanism by phytoliths, i.e., the ability of needle-like phytoliths to insert microscopic pathogens (bacteria, fungi, viruses) into herbivores' tissues. We do it by comparison and by showing analogy with the better-known insertion of microbial pathogens into the body of herbivores by thorns, spines, and prickles. This largely overlooked and understudied defensive mechanism is a special case of a double extended phenotype; plants' defense, and the multiplication and dispersal of microorganisms, and is thus a case of mutualism.

Antimicrobial agent susceptibilities of Legionella pneumophila MLVA-8 genotypes.

Legionella pneumophila causes human lung infections resulting in severe pneumonia. High-resolution genotyping of L. pneumophila isolates can be achieved by multiple-locus variable-number tandem-repeat analysis (MLVA-8). Legionella infections in humans occur as a result of inhalation of bacteria-containing aerosols, thus, our aim was to study the antimicrobial susceptibilities of different MLVA-8 genotypes to ten commonly used antimicrobial agents in legionellosis therapy. Epidemiological cut-off values were determined for all antibiotics. Significant differences were found between the antimicrobial agents' susceptibilities of the three studied environmental genotypes (Gt4, Gt6, and Gt15). Each genotype exhibited a significantly different susceptibility profile, with Gt4 strains (Sequence Type 1) significantly more resistant towards most studied antimicrobial agents. In contrast, Gt6 strains (also Sequence Type 1) were more susceptible to six of the ten studied antimicrobial agents compared to the other genotypes. Our findings show that environmental strains isolated from adjacent points of the same water system, exhibit distinct antimicrobial resistance profiles. These differences highlight the importance of susceptibility testing of Legionella strains. In Israel, the most extensively used macrolide for pneumonia is azithromycin. Our results point at the fact that clarithromycin (another macrolide) and trimethoprim with sulfamethoxazole (SXT) were the most effective antimicrobial agents towards L. pneumophila strains. Moreover, legionellosis can be caused by multiple L. pneumophila genotypes, thus, the treatment approach should be the use of combined antibiotic therapy. Further studies are needed to evaluate specific antimicrobial combinations for legionellosis therapy.