Brucella and Its Hidden Flagellar System

Brucella, a Gram-negative bacterium with a high infective capacity and a wide spectrum of hosts in the animal world, is found in terrestrial and marine mammals, as well as amphibians. This broad spectrum of hosts is closely related to the non-classical virulence factors that allow this pathogen to establish its replicative niche, colonizing epithelial and immune system cells, evading the host’s defenses and defensive response. While motility is the primary role of the flagellum in most bacteria, in Brucella, the flagellum is involved in virulence, infectivity, cell growth, and biofilm formation, all of which are very important facts in a bacterium that to date has been described as a non-motile organism. Evidence of the expression of these flagellar proteins that are present in Brucella makes it possible to hypothesize certain evolutionary aspects as to where a free-living bacterium eventually acquired genetic material from environmental microorganisms, including flagellar genes, conferring on it the ability to reach other hosts (mammals), and, under selective pressure from the environment, can express these genes, helping it to evade the immune response. This review summarizes relevant aspects of the presence of flagellar proteins and puts into context their relevance in certain functions associated with the infective process. The study of these flagellar genes gives the genus Brucella a very high infectious versatility, placing it among the main organisms in urgent need of study, as it is linked to human health by direct contact with farm animals and by eventual transmission to the general population, where flagellar genes and proteins are of great relevance.

Antibiotic sensitivity of Pseudomonas aeruginosa strains isolated from milk of highly productive dairy cows

Pseudomonas aeruginosa is a free-living bacterium that is conditionally pathogenic in natural conditions, but when ingested by an animal can cause severe infectious diseases, depending on the location and stage of infection, increasing the time of convalescence. It is naturally resistant to a number of widely used antibacterial drugs: fluoroquinolones, tetracyclines, chloramphenicol; it is capable of forming a biofilm. The aim of this work was to assess the level of sensitivity of strains isolated from cow's milk to the main groups of antibiotics with clinically significant anti-pseudomonasal activities. The work provides microbiological monitoring data for 2020-2021. During this period, 350 milk samples were taken from Holstinized black-and-white cows of the dairy direction of various lactation ages. All isolates were characterized by generally accepted phenotypic methods, with confirmation of biochemical properties. A number of antibiotics selected in this work comply with the recommendations of EUCAST. Sensitivity testing of the isolated strains was carried out by the disco-diffuse method. Pseudomonas aeruginosa was detected in 69 milk samples, which accounted for 19.7% of the total number of milk samples. Pseudomonas Aeruginosa was isolated as a monoculture in 42.03% of cases, in association with gram (-) bacteria in 20.29% and gram (+) in 37.68%. The percentage of pigmented strains in our work was 98.55% of all isolated strains, and 1.45% of poorly pigmented. During the research work, it was found that more than 90% of the strains were resistant to cefepime, the rest showed partial resistance. Therefore, it can be recommended for use only after a correction for sensitivity to this drug. The aminoglycoside group drugs, amikacin and gentamicin, had the highest activity - over 90% against the isolated Ps.a. strains. The results obtained indicate that the isolated strains of Ps.a. they showed high resistance to representatives of the cephalosporin group, which is also increasing with respect to fluoroquinolones, which is a serious problem in the spread of antibiotic resistance.

Multiple concurrent and convergent stages of genome reduction in bacterial symbionts across a stink bug family

Nutritional symbioses between bacteria and insects are prevalent and diverse, allowing insects to expand their feeding strategies and niches. A common consequence of long-term associations is a considerable reduction in symbiont genome size likely influenced by the radical shift in selective pressures as a result of the less variable environment within the host. While several of these cases can be found across distinct insect species, most examples provide a limited view of a single or few stages of the process of genome reduction. Stink bugs (Pentatomidae) contain inherited gamma-proteobacterial symbionts in a modified organ in their midgut and are an example of a long-term nutritional symbiosis, but multiple cases of new symbiont acquisition throughout the history of the family have been described. We sequenced the genomes of 11 symbionts of stink bugs with sizes that ranged from equal to those of their free-living relatives to less than 20%. Comparative genomics of these and previously sequenced symbionts revealed initial stages of genome reduction including an initial pseudogenization before genome reduction, followed by multiple stages of progressive degeneration of existing metabolic pathways likely to impact host interactions such as cell wall component biosynthesis. Amino acid biosynthesis pathways were retained in a similar manner as in other nutritional symbionts. Stink bug symbionts display convergent genome reduction events showing progressive changes from a free-living bacterium to a host-dependent symbiont. This system can therefore be used to study convergent genome evolution of symbiosis at a scale not previously available.

Vibrio natriegens as a pET-Compatible Expression Host Complementary to Escherichia coli

Efficient and novel recombinant protein expression systems can further reduce the production cost of enzymes. Vibrio natriegens is the fastest growing free-living bacterium with a doubling time of less than 10 min, which makes it highly attractive as a protein expression host. Here, 196 pET plasmids with different genes of interest (GOIs) were electroporated into the V. natriegens strain VnDX, which carries an integrated T7 RNA polymerase expression cassette. As a result, 65 and 75% of the tested GOIs obtained soluble expression in V. natriegens and Escherichia coli, respectively, 20 GOIs of which showed better expression in the former. Furthermore, we have adapted a consensus “what to try first” protocol for V. natriegens based on Terrific Broth medium. Six sampled GOIs encoding biocatalysts enzymes thus achieved 50–128% higher catalytic efficiency under the optimized expression conditions. Our study demonstrated V. natriegens as a pET-compatible expression host with a spectrum of highly expressed GOIs distinct from E. coli and an easy-to-use consensus protocol, solving the problem that some GOIs cannot be expressed well in E. coli.

Msh Pilus Mutations Increase the Ability of a Free-Living Bacterium to Colonize a Piscine Host

Symbioses between animals and bacteria are ubiquitous. To better understand these relationships, it is essential to unravel how bacteria evolve to colonize hosts. Previously, we serially passaged the free-living bacterium, Shewanella oneidensis, through the digestive tracts of germ-free larval zebrafish (Danio rerio) to uncover the evolutionary changes involved in the initiation of a novel symbiosis with a vertebrate host. After 20 passages, we discovered an adaptive missense mutation in the mshL gene of the msh pilus operon, which improved host colonization, increased swimming motility, and reduced surface adhesion. In the present study, we determined that this mutation was a loss-of-function mutation and found that it improved zebrafish colonization by augmenting S. oneidensis representation in the water column outside larvae through a reduced association with environmental surfaces. Additionally, we found that strains containing the mshL mutation were able to immigrate into host digestive tracts at higher rates per capita. However, mutant and evolved strains exhibited no evidence of a competitive advantage after colonizing hosts. Our results demonstrate that bacterial behaviors outside the host can play a dominant role in facilitating the onset of novel host associations.

Vector competence of the African argasid tick Ornithodoros moubata for the Q fever agent Coxiella burnetii

Q fever is a widespread zoonotic disease caused by the intracellular bacterium Coxiella burnetii. While transmission is primarily but not exclusively airborne, ticks are usually thought to act as vectors on the basis of early microscopy studies. However, recent observations revealed that endosymbionts of ticks have been commonly misidentified as C. burnetii, calling the importance of tick-borne transmission into question. In this study, we re-evaluated the vector competence of the African soft tick Ornithodoros moubata for an avirulent strain of C. burnetii. To this end, we used an artificial feeding system to initiate infection of ticks, specific molecular tools to monitor further infections, and culture assays in axenic and cell media to check for the viability of C. burnetii excreted by ticks. We observed typical traits associated with vector competence: The exposure to an infected blood meal resulted in viable and persistent infections in ticks, trans-stadial transmissions of infection from nymphs to adults and the ability of adult ticks to transmit infectious C. burnetii. However, in contrast to early studies, we found that infection differed substantially between tick organs. In addition, while adult female ticks were infected, we did not observe C. burnetii in eggs, suggesting that transovarial transmission is not effective. Finally, we detected only a sporadic presence of C. burnetii DNA in tick faeces, but no living bacterium was further isolated in culture assays, suggesting that excretion in faeces is not a common mode of transmission in O. moubata.

Msh Pilus Mutations Increase the Ability of a Free-Living Bacterium to Colonize a Piscine Host

Symbioses between animals and bacteria are ubiquitous. To better understand these relationships, it is essential to unravel how bacteria evolve to colonize hosts. Previously, we serially passaged the free-living bacterium, Shewanella oneidensis, through the digestive tracts of germ-free larval zebrafish (Danio rerio) to uncover the evolutionary changes involved in the initiation of a novel symbiosis with a vertebrate host. After 20 passages, we discovered an adaptive missense mutation in the mshL gene of the msh pilus operon, which improved host colonization, increased swimming motility, and reduced surface adhesion. In the present study, we have determined that this mutation was a loss-of-function mutation and found that it improved zebrafish colonization by augmenting S. oneidensis representation in the water column outside larvae through a reduced association with environmental surfaces. Additionally, we found that strains containing the mshL mutation were able to immigrate into host digestive tracts at higher rates per capita. However, mutant and evolved strains exhibited no evidence of a competitive advantage after colonizing hosts. Our results demonstrate that bacterial behaviors outside the host can play a dominant role in facilitating the onset of novel host associations.

Phenotypic Parallelism during Experimental Adaptation of a Free-Living Bacterium to the Zebrafish Gut

ABSTRACT Although animals encounter a plethora of bacterial species throughout their lives, only a subset colonize vertebrate digestive tracts, and these bacteria can profoundly influence the health and development of their animal hosts. However, our understanding of how bacteria initiate symbioses with animal hosts remains underexplored, and this process is central to the assembly and function of gut bacterial communities. Therefore, we used experimental evolution to study a free-living bacterium as it adapts to a novel vertebrate host by serially passaging replicate populations of Shewanella oneidensis through the intestines of larval zebrafish (Danio rerio). After approximately 200 bacterial generations, isolates from evolved populations improved their ability to colonize larval zebrafish during competition against their unpassaged ancestor. Genome sequencing revealed unique sets of mutations in the two evolved isolates exhibiting the highest mean competitive fitness. One isolate exhibited increased swimming motility and decreased biofilm formation compared to the ancestor, and we identified a missense mutation in the mannose-sensitive hemagglutinin pilus operon that is sufficient to increase fitness and reproduce these phenotypes. The second isolate exhibited enhanced swimming motility but unchanged biofilm formation, and here the genetic basis for adaptation is less clear. These parallel enhancements in motility and fitness resemble the behavior of a closely related Shewanella strain previously isolated from larval zebrafish and suggest phenotypic convergence with this isolate. Our results demonstrate that adaptation to the zebrafish gut is complex, with multiple evolutionary pathways capable of improving colonization, but that motility plays an important role during the onset of host association. IMPORTANCE Although animals encounter many bacterial species throughout their lives, only a subset colonize vertebrate digestive tracts, and these bacteria can profoundly influence the health and development of their animal hosts. We used experimental evolution to study a free-living bacterium as it adapts to a novel vertebrate host by serially passaging replicate populations of Shewanella oneidensis through the intestines of larval zebrafish (Danio rerio). Our results demonstrate that adaptation to the zebrafish gut is complex, with multiple evolutionary pathways capable of improving colonization, but that motility plays an important role during the onset of host association.

Apparent Intracellular Helicobacter pylori Detected by Immunohistochemistry: The Missing Link in Eradication Failure

Background Helicobacter pylori is primarily an extracellularly living bacterium. However, seemingly intracellular occurrence can often be detected by immunohistochemical stains. Considering antimicrobial resistance, we investigated the impact of the apparent intracellular H. pylori (aiHp) on treatment failure of first-line triple therapies. Methods Gastric biopsies of 814 patients infected with H. pylori naive to treatment were analyzed before and after eradication therapy by immunohistochemistry. Of these, 373 received treatment consisting of amoxicillin, clarithromycin, and proton pump inhibitor (AC/PPI). Availability of polymerase chain reaction-based clarithromycin susceptibility test results from pretreatment gastric biopsies was a precondition for matching 52 aiHp to 52 non-aiHp cases within the AC/PPI group. Results AiHp were detected mostly in low counts predominantly in corpus biopsies, rarely in antrum biopsies (95.2% vs 24.6%); they were found in 497 (61%) of all patients and in 192 of 373 patients (51.5%) in the AC/PPI group. The eradication rate in aiHp versus non-aiHp cases was 44.4% versus 72.9% in the entire sample and 45.3% versus 66.8% in the AC/PPI group. Among the 104 paired patients, respective values were 46.2% versus 78.8%; in clarithromycin-susceptible cases, 60.6% versus 91.9%. Both aiHp and resistance to clarithromycin proved to be highly significant (P ≤ .001) and independent predictors of eradication failure. Twelve of 13 aiHp cases with a clarithromycin-sensitive strain who failed eradication developed resistance to the antibiotic. Conclusions AiHp found by immunohistochemical staining especially in corpus biopsies proved to be a risk factor for failure of first-line triple therapies; occurrence of aiHp should be considered with regard to therapy options.

Phenotypic parallelism during experimental adaptation of a free-living bacterium to the zebrafish gut

Despite the fact that animals encounter a plethora of bacterial species throughout their lives, only a subset are capable of colonizing vertebrate digestive tracts, and these bacteria can profoundly influence the health and development of their animal hosts. However, it is still unknown how bacteria evolve symbioses with animal hosts, and this process is central to both the assembly and function of gut bacterial communities. Therefore, we used experimental evolution to study a free-living bacterium as it adapts to a novel vertebrate host. We serially passaged replicate populations of Shewanella oneidensis, through the digestive tracts of larval zebrafish (Danio rerio). After only 20 passages, representing approximately 200 bacterial generations, isolates from replicate evolved populations displayed an improved ability to colonize larval zebrafish digestive tracts during competition against their unpassaged ancestor. Upon sequencing the genomes of these evolved isolates, we discovered that the two isolates with the highest mean competitive fitness accumulated unique sets of mutations. We characterized the swimming motility and aggregation behavior of these isolates, as these phenotypes have previously been shown to alter host-microbe interactions. Despite exhibiting different biofilm characteristics, both isolates evolved augmented swimming motility. These enhancements are consistent with expectations based on the behavior of a closely related Shewanella strain previously isolated from the zebrafish digestive tract and suggest that our evolved isolates are pursuing a convergent adaptive trajectory with this zebrafish isolate. In addition, parallel enhancements in swimming motility among isolates from independently adapted populations implicates increased dispersal as an important factor in facilitating the onset of host association. Our results demonstrate that free-living bacteria can rapidly improve their associations with vertebrate hosts.