Opportunistic Bacterial Pathogen Research Articles

Citrobacter freundii-associated lesions in stranded loggerhead sea turtles (Caretta caretta).

Citrobacter freundii, an opportunistic bacterial pathogen belonging to the family Enterobacteriaceae, has been sporadically reported in sea turtles. Here, the authors describe 3 unusual lesions associated with C. freundii infection in 3 loggerhead sea turtles stranded on the coast of Gran Canaria Island, Spain. It is possible that these 3 distinct lesions played a major role in the death of these turtles. The first turtle had caseous cholecystitis, a lesion not previously described in sea turtles. The second turtle had large intestinal diverticulitis, a rare condition in loggerheads. The third turtle had bilateral caseous salt gland adenitis. Histologically, numerous gram-negative bacilli were observed at the deepest edge of inflammation in all cases. Pure cultures of C. freundii were obtained from these 3 lesions. Molecular detection of C. freundii DNA in formalin-fixed paraffin-embedded samples from the lesions of the 3 turtles confirmed the microbiological isolation. These cases, in addition to expanding the limited body of knowledge on bacterial infections in sea turtles, highlight the potential pathogenic role of C. freundii in loggerhead turtles.

Bacterial contamination of water used as thermal transfer fluid in fluid-warming devices

Recent reports implied heater-cooler units (HCU), which are used for warming of infusions, blood or in ECMO devices, as possible origin of healthcare-associated infections (HAIs) with potentially pathogenic bacteria like nontuberculous mycobacteria [1]. This represents a source of contamination in a usually sterile setting. Aim of this study is to analyse water from infusion heating devices (IHDs) for bacterial contamination and to raise the question if IHDs act as a possible source in the transmission of HAIs. 300-500 ml of thermal transfer fluid (TTF) derived from the reservoirs of 22 independent IHDs were collected and processed on different selective and non-selective media for colony count and identification of bacteria. Strains of Mycobacterium species (spp.) were further analysed by whole genome sequencing. Bacterial growth was observed in each of the 22 collected TTFs after cultivation at 22°C and 36°C. Pseudomonas aeruginosa was the most frequent pathogen identified, being present in 13.64% (3/22) at > 100 CFU/100mL. Colonization with Mycobacterium chimaera, Ralstonia pickettii and Ralstonia mannitolilytica was detectable in 9.09% (2/22) of the isolates. Primary sequencing of the detected M. chimaera suggests a close relationship to a M. chimaera strain detected in an outbreak in Switzerland, which led to the death of two patients. A contamination of the TTF represents a germ reservoir in a sensitive setting. Handling errors of the IHD may lead to distribution of opportunistic or facultative bacterial pathogens, increasing the risk of nosocomial infection transmission.

Parasitological, Bacteriological and Virological Research of Carp (Cyprinus carpio) in a Case in Hirfanlı Dam Lake in Türkiye

The aim of the present study was shedding light on the disease factors underlying mortality event among common carps inhabiting Hirfanlı Dam Lake in Türkiye in spring of the year 2021. For this purpose various parasitological, bacteriological and virological research techniques were employed. Five parasite genera, Dactylogyrus spp., Myxobolus spp., Eimeria spp., Trichodina spp., Diplostomum spp. were isolated from 13 common carps. The opportunistic bacterial pathogen of fresh water fishes Aeromonas hydrophila was also isolated from wounded fish. No viruses were detected. Fungal infections were encountered in the skin lesions of carps. The source of the encountered morbidity and mortality picture among the studied carps points to the combined action of parasitic, bacterial and fungal agents in these fish. Further investigations on seasonal variations of parasitofauna on the lake are needed for better understanding the dynamics in the ecology of the parasites which are hosted by carps in the Hirfanlı Dam Lake.

Delivery mechanism can enhance probiotic activity against honey bee pathogens

Managed honey bee (Apis mellifera) populations play a crucial role in supporting pollination of food crops but are facing unsustainable colony losses, largely due to rampant disease spread within agricultural environments. While mounting evidence suggests that select lactobacilli strains (some being natural symbionts of honey bees) can protect against multiple infections, there has been limited validation at the field-level and few methods exist for applying viable microorganisms to the hive. Here, we compare how two different delivery systems—standard pollen patty infusion and a novel spray-based formulation—affect supplementation of a three-strain lactobacilli consortium (LX3). Hives in a pathogen-dense region of California are supplemented for 4 weeks and then monitored over a 20-week period for health outcomes. Results show both delivery methods facilitate viable uptake of LX3 in adult bees, although the strains do not colonize long-term. Despite this, LX3 treatments induce transcriptional immune responses leading to sustained decreases in many opportunistic bacterial and fungal pathogens, as well as selective enrichment of core symbionts including Bombilactobacillus, Bifidobacterium, Lactobacillus, and Bartonella spp. These changes are ultimately associated with greater brood production and colony growth relative to vehicle controls, and with no apparent trade-offs in ectoparasitic Varroa mite burdens. Furthermore, spray-LX3 exerts potent activities against Ascosphaera apis (a deadly brood pathogen) likely stemming from in-hive dispersal differences, whereas patty-LX3 promotes synergistic brood development via unique nutritional benefits. These findings provide a foundational basis for spray-based probiotic application in apiculture and collectively highlight the importance of considering delivery method in disease management strategies.

WS07.03 A long-term study of Pseudomonas aeruginosa early patient isolate adaptation to the hypoxic environment

Pseudomonas aeruginosa is an opportunistic bacterial pathogen which causes chronic lung infections in people with cystic fibrosis (CF) and is a major contributor to morbidity and mortality. Despite this, the mechanism driving its adaptation towards chronic colonisation in the CF lung is not yet fully understood. This work focuses on the adaptations of P. aeruginosa to hypoxia, one of the important environmental pressures present in the lung. Long term hypoxia has yet to be examined as a driver of adaptation towards the persistence of this bacterium in CF patients. We have studied the effect of long term adaptation to 6% oxygen for up to 28 days. Interestingly, distinctive colony morphotypes developed throughout the experiments, including small colony variants (SCV). One of the observed morphotypes appeared exclusively under low-oxygen pressure. Importantly, the colonies with visibly different morphologies were more common in hypoxia adapted cultures, comprising up to 98% of the population, while they never exceeded 35% in normoxia adapted cultures. Proteomic analysis showed changes in the abundance of over 700 proteins within 5 days of adaptation, including those involved in respiration and carbon metabolism, quorum sensing and phenazine biosynthesis. Interestingly, comparable changes in several pathways were consistent across both conditions while phenotype differences in motility, pyocyanin production, and protease production between the different colony morphotypes in samples taken over time were observed. Both the SCV and the normal colony variants (NCV) from the hypoxia adapted strains showed decreases in motility, pyocyanin production and protease production. A Congo red assay showed increased exopolysaccharide production in both hypoxia adapted SCV and NCVs. Overall, these results confirm that hypoxia indeed promotes the appearance of many phenotypes in P. aeruginosa that are associated with persistence in the lung and with poor patient outcomes.

An Intestinal Bacillus velezensis Isolate Displays Broad-Spectrum Antibacterial Activity and Prevents Infection of Both Gram-Positive and Gram-Negative Pathogens In Vivo.

The increasing prevalence of drug-resistant bacteria has significantly diminished the effectiveness of antibiotics in clinical settings, leading to the emergence of untreatable bacterial infections. To address this public health challenge, the gut microbiome represents a promising source of novel antimicrobial therapeutics. In this study, we screened mouse intestinal isolates for growth inhibitory activity against the human enteric pathogen Vibrio cholerae and identified a strain of spore-forming Bacillus velezensis, named BVM7, that produced a potent antibiotic with activity against V. cholerae and a broad spectrum of enteric and opportunistic pathogens. Characterization of the antimicrobial compounds produced by BVM7 revealed that they were primarily secreted antimicrobial peptides (AMPs) produced during stationary-phase growth. Furthermore, our results showed that introducing either BVM7 vegetative cells or spores into mice precolonized with V. cholerae or Enterococcus faecalis significantly reduced the burden of infection. Interestingly, we also observed that BVM7 was sensitive to a group of Lactobacillus probiotic strains and that inoculation of Lactobacilli could eliminate BVM7 and potentially restore the native gut microbiome. These findings highlight the potential of bacteria from the gut microbiome as a source for novel antimicrobial compounds and a tool for managing bacterial infections by in situ bio-delivery of multiple AMPs. IMPORTANCE The rise of antibiotic-resistant pathogens poses a challenge to public health. The gut microbiome presents a promising source of new antimicrobials and treatments. By screening murine gut commensals, we found a spore-forming Bacillus velezensis strain, BVM7, that exhibited antimicrobial activity toward a wide array of enteric and opportunistic bacterial pathogens. In addition to showing that this killing effect occurred through the action of secreted antimicrobial peptides (AMPs), we demonstrate that BVM7 vegetative cells and spores can be used to treat infections of both Gram-positive and Gram-negative pathogens in vivo. By expanding our knowledge of the antimicrobial properties of bacteria in the gut microbiome, we hope to contribute insights for developing novel drugs and therapeutic interventions.

A unique AA5 alcohol oxidase fused with a catalytically inactive CE3 domain from the bacterium Burkholderia pseudomallei.

Copper radical oxidases (CROs) are redox enzymes able to oxidize alcohols or aldehydes, while only requiring a single copper atom as cofactor. Studied CROs are found in one of two subfamilies within the Auxiliary Activities family 5 (AA5) in the carbohydrate-active enzymes database. We here characterize an AA5 enzyme outside the subfamily classification from the opportunistic bacterial pathogen Burkholderia pseudomallei, which curiously was fused to a carbohydrate esterase family 3 domain. The enzyme was shown to be a promiscuous primary alcohol oxidase, with an activity profile similar to enzymes from subfamily 2. The esterase domain was inactive on all tested substrates, and structural predictions revealed this being an effect of crippling substitutions in the expected active site residues.

Mechanisms of Antibiotic and Biocide Resistance That Contribute to Pseudomonas aeruginosa Persistence in the Hospital Environment.

Pseudomonas aeruginosa is an opportunistic bacterial pathogen responsible for multiple hospital- and community-acquired infections, both in human and veterinary medicine. P. aeruginosa persistence in clinical settings is worrisome and is a result of its remarkable flexibility and adaptability. This species exhibits several characteristics that allow it to thrive under different environmental conditions, including the ability to colonize inert materials such as medical equipment and hospital surfaces. P. aeruginosa presents several intrinsic mechanisms of defense that allow it to survive external aggressions, but it is also able to develop strategies and evolve into multiple phenotypes to persevere, which include antimicrobial-tolerant strains, persister cells, and biofilms. Currently, these emergent pathogenic strains are a worldwide problem and a major concern. Biocides are frequently used as a complementary/combination strategy to control the dissemination of P. aeruginosa-resistant strains; however, tolerance to commonly used biocides has also already been reported, representing an impediment to the effective elimination of this important pathogen from clinical settings. This review focuses on the characteristics of P. aeruginosa responsible for its persistence in hospital environments, including those associated with its antibiotic and biocide resistance ability.

A Novel Zinc (II) Porphyrin Is Synergistic with PEV2 Bacteriophage against Pseudomonas aeruginosa Infections.

Pseudomonas aeruginosa (PsA) is an opportunistic bacterial pathogen that causes life-threatening infections in individuals with compromised immune systems and exacerbates health concerns for those with cystic fibrosis (CF). PsA rapidly develops antibiotic resistance; thus, novel therapeutics are urgently needed to effectively combat this pathogen. Previously, we have shown that a novel cationic Zinc (II) porphyrin (ZnPor) has potent bactericidal activity against planktonic and biofilm-associated PsA cells, and disassembles the biofilm matrix via interactions with eDNA In the present study, we report that ZnPor caused a significant decrease in PsA populations in mouse lungs within an in vivo model of PsA pulmonary infection. Additionally, when combined with an obligately lytic phage PEV2, ZnPor at its minimum inhibitory concentration (MIC) displayed synergy against PsA in an established in vitro lung model resulting in greater protection of H441 lung cells versus either treatment alone. Concentrations above the minimum bactericidal concentration (MBC) of ZnPor were not toxic to H441 cells; however, no synergy was observed. This dose-dependent response is likely due to ZnPor's antiviral activity, reported herein. Together, these findings show the utility of ZnPor alone, and its synergy with PEV2, which could be a tunable combination used in the treatment of antibiotic-resistant infections.

Evaluation of the Clinical Impact and In Vitro Antibacterial Activities of Two Bioactive Restoratives against S. mutans ATCC 25175 in Class II Carious Restorations

Streptococcus mutans is a Gram-positive opportunistic bacterial pathogen and that causes dental caries and then restorative treatment remains the best clinical practice approach to repair and prevent dental caries. This study compared the antimicrobial performance of resin modified glass ionomer cement (RM-GIC) and ACTIVA restoratives by evaluating the S. mutans count, pH levels, and plaque index (PI) scores before and on the 7th day of restoration, and then determined the antimicrobial activities against S. mutans ATCC 25175 in both restoratives in vitro. Seventy-eight eligible Saudi female participants, with class II carious lesions, were randomly distributed into RM-GIC and ACTIVA restorative groups. We evaluated the S. mutans count by the serial dilution technique and salivary pH by using a portable pH meter. The PI scores were determined by Silness-Löe method and the antibacterial activity by the agar well diffusion method. Statistical analysis of normality distribution was performed with the Kolmogorov-Smirnov and the difference between groups was an analysis by paired t-test. In addition, the independent sample was compared with the independent samples t-test. Both groups reduced the S. mutans count, pH acidity, and PI scores, and this reduction was statistically significant on the 7th day of restoration (P < 0.05), preference for ACTIVA. The in vitro antibacterial activity against S. mutans ATCC 25175 showed a non-significant difference between both bioactive restorative materials (P < 0.05). The novel application of ACTIVA restorative material is a promising option for patients at risk of caries.

Climate warming and increasing Vibrio vulnificus infections in North America

Vibrio vulnificus is an opportunistic bacterial pathogen, occurring in warm low-salinity waters. V. vulnificus wound infections due to seawater exposure are infrequent but mortality rates are high (~ 18%). Seawater bacterial concentrations are increasing but changing disease pattern assessments or climate change projections are rare. Here, using a 30-year database of V. vulnificus cases for the Eastern USA, changing disease distribution was assessed. An ecological niche model was developed, trained and validated to identify links to oceanographic and climate data. This model was used to predict future disease distribution using data simulated by seven Global Climate Models (GCMs) which belong to the newest Coupled Model Intercomparison Project (CMIP6). Risk was estimated by calculating the total population within 200 km of the disease distribution. Predictions were generated for different “pathways” of global socioeconomic development which incorporate projections of greenhouse gas emissions and demographic change. In Eastern USA between 1988 and 2018, V. vulnificus wound infections increased eightfold (10–80 cases p.a.) and the northern case limit shifted northwards 48 km p.a. By 2041–2060, V. vulnificus infections may expand their current range to encompass major population centres around New York (40.7°N). Combined with a growing and increasingly elderly population, annual case numbers may double. By 2081–2100 V. vulnificus infections may be present in every Eastern USA State under medium-to-high future emissions and warming. The projected expansion of V. vulnificus wound infections stresses the need for increased individual and public health awareness in these areas.

The Antimicrobial Peptide Octopromycin Suppresses Biofilm Formation and Quorum Sensing in Acinetobacter baumannii

Acinetobacter baumannii is an opportunistic bacterial pathogen that causes severe infections in immunocompromised individuals. A. baumannii forms biofilm and produces extracellular matrix, which supports bacteria to survive under harsh conditions and be resistant to antibacterial treatments. In the present study, we investigated the biofilm and quorum-sensing inhibitory effects of antimicrobial peptide, octopromycin in A. baumannii. Field emission-scanning electron microscopy results clearly showed significantly reduced biofilm mass and caused a collapse in biofilm architecture at the minimum inhibitory concentration (50 µg/mL) and minimum bactericidal concentration (200 µg/mL) of octopromycin. Antibiotic-resistant persister cells of A. baumannii were successfully killed by octopromycin treatment, and it inhibited violacein production in Chromobacterium violaceum in a concentration-dependent manner. Octopromycin also inhibited alginate production, surface movements (swarming and swimming), and twitching motility of A. baumannnii, confirming its anti-quorum-sensing activity. Multiple metabolic pathways, two-component regulation systems, quorum-sensing, and antibiotic synthesis-related pathways in A. baumannii biofilms were strongly affected by octopromycin treatment. The collective findings indicate that the antibacterial peptide octopromycin may control A. baumannii biofilms through multi-target interactions. Octopromycin could be a desirable therapeutic option for the prevention and control of A. baumannii infections.

Cryo-EM structure of the RuvAB-Holliday junction intermediate complex from Pseudomonas aeruginosa.

Holliday junction (HJ) is a four-way structured DNA intermediate in homologous recombination. In bacteria, the HJ-specific binding protein RuvA and the motor protein RuvB together form the RuvAB complex to catalyze HJ branch migration. Pseudomonas aeruginosa (P. aeruginosa, Pa) is a ubiquitous opportunistic bacterial pathogen that can cause serious infection in a variety of host species, including vertebrate animals, insects and plants. Here, we describe the cryo-Electron Microscopy (cryo-EM) structure of the RuvAB-HJ intermediate complex from P. aeruginosa. The structure shows that two RuvA tetramers sandwich HJ at the junction center and disrupt base pairs at the branch points of RuvB-free HJ arms. Eight RuvB subunits are recruited by the RuvA octameric core and form two open-rings to encircle two opposite HJ arms. Each RuvB subunit individually binds a RuvA domain III. The four RuvB subunits within the ring display distinct subdomain conformations, and two of them engage the central DNA duplex at both strands with their C-terminal β-hairpins. Together with the biochemical analyses, our structure implicates a potential mechanism of RuvB motor assembly onto HJ DNA.

Computational Modelling, Functional Characterization and Molecular Docking to Lead Compounds of Bordetella pertussis Diaminopimelate Epimerase.

Bordetella pertussis, the causative agent of whooping cough, is an opportunistic virulent bacterial pathogen that is resistant to a wide range of antibiotics due to a variety of resistance mechanisms. Looking at the increasing number of infections caused by B. pertussis and its resistance to diverse antibiotics, it is essential to develop alternative strategies to fight against B. pertussis. Diaminopimelate epimerase (DapF) is an important enzyme of the lysine biosynthesis pathway in B. pertussis that catalyzes the formation of meso-2, 6-diaminoheptanedioate (meso-DAP), which is an important step in lysine metabolism. Therefore, Bordetella pertussis diaminopimelate epimerase (DapF) becomes an ideal target for antimicrobial drug development. In the present study, computational modelling, functional characterization, binding studies, and docking studies of BpDapF with lead compounds were carried out using different in silico tools. In silico prediction results in the secondary structure, 3-D structure analysis, and protein-protein interaction analysis of BpDapF. Docking studies further showed the respective amino acid residues for ligands in the phosphate‑binding loop of BpDapF play a vital role in the formation of H‑bonds with these ligands. The site where the ligand was bound is a deep groove, which is regarded as the binding cavity of the protein. Biochemical studies indicated that Limonin (binding energy - 8.8kcal/mol), Ajmalicine (binding energy - 8.7kcal/mol), Clinafloxacin (binding energy - 8.3kcal/mol), Dexamethasone (binding energy - 8.2kcal/mol), and Tetracycline (binding energy - 8.1kcal/mol) exhibited promising binding towards the drug target DapF of B. pertussis in comparison with the binding between other drugs and act as the potential inhibitors of BpDapF that eventually can reduce the catalytic activity of BpDapF.

Soil Inoculation and Blocker-Mediated Sequencing Show Effects of the Antibacterial T6SS on Agrobacterial Tumorigenesis and Gallobiome

The type VI secretion system (T6SS) is deployed by many proteobacteria to secrete effector proteins into bacterial competitors for competition or eukaryotic cells for pathogenesis. Agrobacteria, a group of soilborne phytopathogens causing crown gall disease on various plant species, deploy the T6SS to attack closely and distantly related bacterial species in vitro and in planta. Current evidence suggests that the T6SS is not essential for pathogenesis under direct inoculation, but it remains unknown whether the T6SS influences natural disease incidence or the microbial community within crown galls (i.e., the gallobiome). To address these two key questions, we established a soil inoculation method on wounded tomato seedlings that mimics natural infections and developed a bacterial 16S rRNA gene amplicon enrichment sequencing platform. By comparing the Agrobacterium wild-type strain C58 with two T6SS mutants, we demonstrate that the T6SS influences both disease occurrence and gallobiome composition. Based on multiple inoculation trials across seasons, all three strains induced tumors, but the mutants had significantly lower disease incidences. The season of inoculation played a more important role than the T6SS in shaping the gallobiome. The influence of the T6SS was evident in summer, during which two Sphingomonadaceae species and the family Burkholderiaceae were enriched in the gallobiome induced by the mutants. Further in vitro competition and colonization assays demonstrated the T6SS-mediated antagonism to a Sphingomonas sp. R1 strain isolated from tomato rhizosphere in this study. In conclusion, this work demonstrates that the Agrobacterium T6SS promotes tumorigenesis in infection processes and provides competitive advantages in gall-associated microbiota. IMPORTANCE The T6SS is widespread among proteobacteria and used for interbacterial competition by agrobacteria, which are soil inhabitants and opportunistic bacterial pathogens causing crown gall disease in a wide range of plants. Current evidence indicates that the T6SS is not required for gall formation when agrobacteria are inoculated directly on plant wounding sites. However, in natural settings, agrobacteria may need to compete with other bacteria in bulk soil to gain access to plant wounds and influence the microbial community inside crown galls. The role of the T6SS in these critical aspects of disease ecology have remained largely unknown. In this study, we successfully developed a soil inoculation method coupled with blocker-mediated enrichment of bacterial 16S rRNA gene amplicon sequencing, named SI-BBacSeq, to address these two important questions. We provided evidence that the T6SS promotes disease occurrence and influences crown gall microbiota composition by interbacterial competition.

Legionella longbeachae Regulates the Association of Polyubiquitinated Proteins on Bacterial Phagosome with Multiple Deubiquitinases.

Legionella spp. are the causative agents of a severe pneumonia known as Legionnaires' disease. Upon being engulfed by host cells, these environmental bacteria replicate intracellularly in a plasma membrane-derived niche termed Legionella-containing vacuole (LCV) in a way that requires the defective in organelle trafficking/intracellular multiplication (Dot/Icm) protein transporter. Our understanding of interactions between Legionella and its hosts was mostly based on studies of Legionella pneumophila. In this study, we found that the LCVs created by virulent Legionella longbeachae are similarly decorated by polyubiquitinated proteins to those formed by L. pneumophila and that the ubiquitin-proteasome system (UPS) is required for optimal intracellular growth of L. longbeachae. Furthermore, we utilized bioinformatics methods and the ubiquitin-vinylmethyl ester probe to obtain potential deubiquitinases (DUBs) encoded by L. longbeachae. These efforts led to the identification of 9 L. longbeachae DUBs that displayed distinct specificity toward ubiquitin chain types. Among these, LLO_1014 and LLO_2238 are associated with the LCVs and impact the accumulation of polyubiquitinated species on the bacterial phagosome. Moreover, LLO_1014 and LLO_2238 could fully restore the phenotypes associated with Δceg23 (lotB) and Δlem27 (lotC) mutants of L. pneumophila, indicating that these DUBs have similar functions. Together, these results reveal that L. longbeachae uses multiple DUBs to construct an intracellular niche for its replication. IMPORTANCE Legionella spp. are opportunistic intracellular bacterial pathogens that cause Legionnaires' disease. Legionella utilizes the Dot/Icm type IV secretion system to deliver effector protein into host cells to modulate various cellular functions. At least 26 L. pneumophila effectors are known to hijack the host ubiquitin system via diverse mechanisms. L. longbeachae is the second leading cause of Legionnaires' disease worldwide. However, our knowledge about the interactions between L. longbeachae and its hosts is very limited. Here, we found that, similar to L. pneumophila infection, the host ubiquitin proteasome system is also important for the intracellular replication of L. longbeachae. In addition, the bacterial phagosomes harboring L. longbeachae are enriched with polyubiquitinated proteins in a Dot/Icm system-dependent manner. We further identified 9 L. longbeachae proteins that function as DUBs with distinct ubiquitin chain specificity. Of note, several of the phagosome-associated L. longbeachae DUBs regulate the recruitment of polyubiquitinated proteins to the LCV.

Whole-genome sequencing of Histophilus somni strains isolated in Russia.

Histophilus somni is a Gram-negative bacterium belonging to the Pasteurellaceae family that can cause bovine histophilosis. Histophilus may act as a commensal or opportunistic bacterial cattle pathogen. Comparing genomes of the pathogenic strain 2336 with the non-pathogenic preputial 129Pt isolate revealed some putative virulence factors. The study of the complete genomes of H. somni strains circulating in Russia has never been conducted before. This study aimed to identify genetic features of the H. somni strains isolated in Russia and evaluate the possibility of using strains for vaccine development. Three strains of H. somni were isolated from different sources. Strain 188-VIEV was isolated from a vaginal swab sample of cattle with endometritis. 532-VIEV and 551-VIEV were cultured from the cryopreserved bull semen samples imported from Canada. Histophilus somni strain ATCC 700025 provided by ATCC (American Type Culture Collection) was also used in the study. DNA extraction was performed using QIAamp DNA Mini Kit (QIAGEN, USA). The whole-genome sequencing of the four strains was performed using Illumina Miseq. The comparison of the resulting sequences with the complete genomes of H. somni 2336 and 129Pt, and detection of the resistance genes and virulence factors, was performed using the ResFinder and Virulence Factor Database web services. The genome size of the samples varied from 1.9 to 2.3 Mb. The number of coding sequences varied from 1795 to 2256. The average sequence density was 90%. The total guanine-cytosine (GC) content was 36.8%-37.2%, which coincided with data previously obtained for H. somni. Three out of four studied strains encoded putative virulence factors such as filamentous hemagglutinin homologs, lipooligosaccharide biosynthesis proteins, and proteins involved in iron transport and utilization. The Ser83Ile substitution was identified in the DNA topoisomerase II (gyrA) in H. somni strains 532-VIEV and 551-VIEV cultured from bull semen which led to resistance to fluoroquinolones. The gene (AAC-6-Ia + APH-2'') encoding a bifunctional aminoglycoside modification enzyme was detected in strain 551-VIEV. Strains with virulence genes identified could be candidates for designing vaccines and potentially represent antigen sources. The results show that antibiotic-resistant H. somni can be spread with semen used for artificial insemination.

Dietary Stimuli, Intestinal Bacteria and Peptide Hormones Regulate Female Drosophila Defecation Rate

Peptide hormones control Drosophila gut motility, but the intestinal stimuli and the gene networks coordinating this trait remain poorly defined. Here, we customized an assay to quantify female Drosophila defecation rate as a proxy of intestinal motility. We found that bacterial infection with the human opportunistic bacterial pathogen Pseudomonas aeruginosa (strain PA14) increases defecation rate in wild-type female flies, and we identified specific bacteria of the fly microbiota able to increase defecation rate. In contrast, dietary stress, imposed by either water-only feeding or high ethanol consumption, decreased defecation rate and the expression of enteroendocrine-produced hormones in the fly midgut, such as Diuretic hormone 31 (Dh31). The decrease in defecation due to dietary stress was proportional to the impact of each stressor on fly survival. Furthermore, we exploited the Drosophila Genetic Reference Panel wild type strain collection and identified strains displaying high and low defecation rates. We calculated the narrow-sense heritability of defecation rate to be 91%, indicating that the genetic variance observed using our assay is mostly additive and polygenic in nature. Accordingly, we performed a genome-wide association (GWA) analysis revealing 17 candidate genes linked to defecation rate. Downregulation of four of them (Pmp70, CG11307, meso18E and mub) in either the midgut enteroendocrine cells or in neurons reduced defecation rate and altered the midgut expression of Dh31, that in turn regulates defecation rate via signaling to the visceral muscle. Hence, microbial and dietary stimuli, and Dh31-controlling genes, regulate defecation rate involving signaling within and among neuronal, enteroendocrine, and visceral muscle cells.

The Oligosaccharide Region of LPS Governs Predation of E. coli by the Bacterivorous Protist, Acanthamoeba castellanii.

Protozoan predation is a major cause of bacterial mortality. The first step of predation for phagocytic amoebae is the recognition of their prey. Lipopolysaccharide (LPS) is a major component of Gram-negative bacteria and is only present on the outer leaflet of the outer membrane lipid bilayer. LPS consists of three distinct regions: lipid A, an oligosaccharide core, and O-polysaccharide. Previous research in our lab determined that the oligosaccharide (OS) region of LPS mediates the recognition and internalization of Escherichia coli by Acanthamoeba castellanii. The oligosaccharide region is conceptually divided into the inner core and outer core. The LPS of any given E. coli strain contains only one of five different OS structures: K-12 and R1 to R4. All OSs contain the same inner core sugars but different outer core sugars. Here, we show that the Kdo2 moiety of the inner core is necessary and sufficient for E. coli recognition and internalization by A. castellanii. We also show that the precise composition of the variable outer core OS region modulates the efficiency with which A. castellanii consumes bacteria. The latter finding indicates that outer core OS composition plays a role in bacterial defense against phagocytic predators. IMPORTANCE Rather than being transmitted from host to host, most opportunistic bacterial pathogens reside in the environment for significant amounts of time. Protist predation is a major cause of bacterial mortality. To enhance their survival in the environment, bacteria have evolved various defense strategies such as filamentation, increased motility, biofilm formation, toxin release, and modification of cell wall structure; strategies which also enhance their virulence to humans. This work shows that the major component of the bacterial cell wall, LPS, also known as bacterial endotoxin, is a "dual use" factor, regulating amoeba predation of bacteria in addition to its well-known role as a human virulence factor. Both these functions are governed by the same parts of LPS. Thus, the structure and composition of this "dual use" factor likely evolved as a response to constant voracious protist grazing pressure in the environment, rather than during short-term infections of human and animals.

Bacterial topography of the upper and lower respiratory tract in pigs

BackgroundUnderstanding the complex structures and interactions of the bacterial communities inhabiting the upper (URT) and lower (LRT) respiratory tract of pigs is at an early stage. The objective of this study was to characterize the bacterial topography of three URT (nostrils, choana, and tonsils) and LRT (proximal trachea, left caudal lobe and secondary bronchi) sites in pigs. Thirty-six post-mortem samples from six pigs were analysed by 16S rRNA gene quantification and sequencing, and the microbiota in nostrils and trachea was additionally profiled by shotgun sequencing.ResultsThe bacterial composition obtained by the two methods was congruent, although metagenomics recovered only a fraction of the diversity (32 metagenome-assembled genomes) due to the high proportion (85–98%) of host DNA. The highest abundance of 16S rRNA copies was observed in nostrils, followed by tonsils, trachea, bronchi, choana and lung. Bacterial richness and diversity were lower in the LRT compared to the URT. Overall, Firmicutes and Proteobacteria were identified as predominant taxa in all sample types. Glasserella (15.7%), Streptococcus (14.6%) and Clostridium (10.1%) were the most abundant genera but differences in microbiota composition were observed between the two tracts as well as between sampling sites within the same tract. Clear-cut differences were observed between nasal and tonsillar microbiomes (R-values 0.85–0.93), whereas bacterial communities inhabiting trachea and lung were similar (R-values 0.10–0.17). Moraxella and Streptococcus were more common in bronchial mucosal scraping than in lavage, probably because of mucosal adherence. The bacterial microbiota of the choana was less diverse than that of the nostrils and similar to the tracheal microbiota (R-value 0.24), suggesting that the posterior nasal cavity serves as the primary source of bacteria for the LRT.ConclusionWe provide new knowledge on microbiota composition and species abundance in distinct ecological niches of the pig respiratory tract. Our results shed light on the distribution of opportunistic bacterial pathogens across the respiratory tract and support the hypothesis that bacteria present in the lungs originate from the posterior nasal cavity. Due to the high abundance of host DNA, high-resolution profiling of the pig respiratory microbiota by shotgun sequencing requires methods for host DNA depletion.