Functional characterization of Francisella tularensis subspecies holarctica genotypes during tick cell and macrophage infections using a proteogenomic approach.

Tularemia is a vector-borne disease caused by the Gram-negative bacterium Francisella tularensis. Known hosts and vectors in Europe are hare and ticks. F. tularensis is transmitted from ticks and animals, but also from the hydrotelluric environment and the consumption of contaminated water or food. A changing climate expands the range in which ticks can live and consequently might contribute to increasing case numbers of tularemia. Two subspecies of F. tularensis are human pathogenic. Francisella tularensis tularensis (Ftt) is endemic in North America, while Francisella tularensis holarctica (Fth) is the only subspecies causing tularemia in Europe. Ft is classified as a category A bioterrorism agent due to its low infectious dose, multiple modes of transmission, high infectivity and potential for airborne transmission and has become a global public health concern. In line with the European survey and previous phylogenetic studies, Switzerland shows the co-distribution of B.6 and B.12 strains with different geographical distribution and prevalence within the country. To establish itself in different host environments of ticks and mammals, F. tularensis presumably undergoes substantial changes on the transcriptomics and proteomic level. Here we investigate the transcriptomic and proteomic differences of five strains of Fth upon infection of rabbit macrophages and tick cells.

Mutation of wbtJ, a N-formyltransferase involved in O-antigen synthesis, results in biofilm formation, phase variation and attenuation in Francisella tularensis.

Two clinically important subspecies, Francisella tularensis subsp. tularensis (type A) and F. tularensis subsp. holarctica (type B) are responsible for most tularaemia cases, but these isolates typically form a weak biofilm under in vitro conditions. Phase variation of the F. tularensis lipopolysaccharide (LPS) has been reported in these subspecies, but the role of variation is unclear as LPS is crucial for virulence. We previously demonstrated that a subpopulation of LPS variants can constitutively form a robust biofilm in vitro, but it is unclear whether virulence was affected. In this study, we show that biofilm-forming variants of both fully virulent F. tularensis subspecies were highly attenuated in the murine tularaemia model by multiple challenge routes. Genomic sequencing was performed on these strains, which revealed that all biofilm-forming variants contained a lesion within the wbtJ gene, a formyltransferase involved in O-antigen synthesis. A ΔwbtJ deletion mutant recapitulated the biofilm, O-antigen and virulence phenotypes observed in natural variants and could be rescued through complementation with a functional wbtJ gene. Since the spontaneously derived biofilm-forming isolates in this study were a subpopulation of natural variants, reversion events to the wbtJ gene were detected that eliminated the phenotypes associated with biofilm variants and restored virulence. These results demonstrate a role for WbtJ in biofilm formation, LPS variation and virulence of F. tularensis.

Treatment Outcome of Severe Respiratory Type B Tularemia Using Fluoroquinolones.

BACKGROUND: Fluoroquinolones lack approval for treatment of tularemia but have been used extensively for milder illness. Here, we evaluated fluoroquinolones for severe illness. METHODS: In an observational study, we identified case-patients with respiratory tularemia from July to November 2010 in Jämtland County, Sweden. We defined severe tularemia by hospitalization for >24 hours and severe bacteremic tularemia by Francisella tularensis subsp. holarctica growth in blood or pleural fluid. Clinical data and drug dosing were retrieved from electronic medical records. Chest images were reexamined. We used Kaplan-Meier curves to evaluate time to defervescence and hospital discharge. RESULTS: Among 67 case-patients (median age, 66 years; 81% males) 30-day mortality was 1.5% (1 of 67). Among 33 hospitalized persons (median age, 71 years; 82% males), 23 had nonbacteremic and 10 had bacteremic severe tularemia. Subpleural round consolidations, mediastinal lymphadenopathy, and unilateral pleural fluid were common on chest computed tomography. Among 29 hospitalized persons with complete outcome data, ciprofloxacin/levofloxacin (n = 12), ciprofloxacin/levofloxacin combinations with doxycycline and/or gentamicin (n = 11), or doxycycline as the single drug (n = 6) was used for treatment. One disease relapse occurred with doxycycline treatment. Treatment responses were rapid, with median fever duration 41.0 hours in nonbacteremic and 115.0 hours in bacteremic tularemia. Increased age-adjusted Charlson comorbidity index predicted severe bacteremic tularemia (odds ratio, 2.7 per score-point; 95% confidence interval, 1.35-5.41). A 78-year-old male with comorbidities and delayed ciprofloxacin/gentamicin treatment died. CONCLUSIONS: Fluoroquinolone treatment is effective for severe tularemia. Subpleural round consolidations and mediastinal lymphadenopathy were typical findings on computed tomography among case-patients in this study.

Identification of zoonotic pathogenic bacteria from blood and ticks obtained from hares and long-eared hedgehogs (Hemiechinus megalofis) in eastern Iran.

The role of wildlife in the complex balance of tick-borne diseases within ecosystems is crucial, as they serve as hosts for tick carriers and reservoirs for the pathogens carried by these ticks. This study aimed to investigate the presence of zoonotic pathogenic bacteria in wildlife, specifically in hares and long-eared hedgehogs (Hemiechinus megalofis), in the eastern region of Iran. The focus was on the detection of Borrelia spp., Coxiella burnetii, Anaplasma spp., Francisella spp., and Leptospira spp., using the Nested-PCR method. We analyzed a total of 124 blood samples, and 196 ticks collected from hares and long-eared hedgehogs were analyzed. The Nested-PCR method was employed to identify the presence of zoonotic pathogenic bacteria DNA. Our study revealed the presence of these zoonotic pathogenic bacteria in both wildlife species, indicating their potential role as hosts and reservoirs for the ticks carrying these pathogens. The specific presence and prevalence of Borrelia spp., Coxiella burnetii, Anaplasma spp., Francisella spp., and Leptospira spp. were determined through the Nested-PCR method. This study contributes to the limited knowledge about the involvement of wild animals in the transmission of tick-borne diseases. By using the Nested-PCR method, we successfully identified the presence of zoonotic pathogenic bacteria in hares and long-eared hedgehogs. This study emphasizes the need for further research to better understand the ecological process of tick-borne diseases, particularly the role of wildlife in their spread. Such knowledge is crucial for wildlife conservation efforts and the management of tick-borne diseases, ultimately benefiting both animal and human health.

Diagnosis of piscine francisellosis in Largemouth Bass from a public display exhibit in north-central Florida, USA.

OBJECTIVE: The Largemouth Bass Micropterus salmoides is an important freshwater fish that is native to the southeastern United States and is cultured for conservation, food, and for the sports fishing industry. Francisella orientalis is a globally distributed bacterial pathogen of warmwater fish species and is associated with granulomatous inflammation and high mortalities. Outbreaks of piscine francisellosis in the United States have been reported in only a few fish species. This study describes three case presentations of francisellosis in Largemouth Bass from a public display system in north-central Florida. Additionally, laboratory-controlled immersion challenges using an F. orientalis isolate from tilapia Oreochromis spp. evaluate susceptibility of Largemouth Bass fingerlings to F. orientalis infection and mortality through this exposure route. METHODS: Necropsy, histologic examination, immunohistochemistry, bacterial recovery and culture, and quantitative polymerase chain reaction were used as diagnostic tools to evaluate both the affected display fish and the immersion-challenged fingerlings. RESULT: Although the display fish and immersion-challenged fingerlings presented with nonspecific clinical signs, gross and histological changes were indicative of granulomatous disease. Immunohistochemical and molecular testing methods confirmed F. orientalis infection in affected fish. CONCLUSION: The three case presentations described here mark the first reporting of naturally occurring piscine francisellosis in Largemouth Bass that were held in a public display exhibit. Additionally, causality was proven in the Largemouth Bass fingerlings through the immersion challenges. These findings demonstrate susceptibility through immersion-based exposure and assert that francisellosis should be considered among the list of differential diagnoses for Largemouth Bass with granulomatous disease.

Case report: Francisella philomiragia bacteremia in a patient with acute lymphoblastic leukemia.

Francisella philomiragia is a Gram-negative coccobacillus, which is a very rare human opportunistic pathogen causing pneumonia and systemic infection. It is difficult to identify this bacterium through conventional Gram-staining and biochemical methods due to an amorphous Gram stain appearance after 24 h culture and its relatively fastidious and slow growth giving weak and/or delayed reactions in biochemical tests. It is often misidentified as other bacteria including Haemophilus spp., Pseudomonas aeruginosa, or Sphingomonas paucimobilis. False identification may delay the therapy of the patients and even endanger the patient's life. Here, we report a case of a 34-year-old man with acute lymphoblastic leukemia infected by F. philomiragia, which was almost misdiagnosed. This case describes our identification of a patient with a systemic F. philomiragia infection. To our knowledge, this is the first such case reported in China.

Nasal responses to elevated temperature and Francisella noatunensis infection in Atlantic cod (Gadus morhua).

We report the histological and transcriptomic changes in the olfactory organ of Atlantic cod exposed to Francisella noatunensis. Experimental infection was performed at either 12 °C or 17 °C. Infected fish presented the classic gross pathologies of francisellosis. Nasal morpho-phenotypic parameters were not significantly affected by elevated temperature and infection, except for the number of mucus cells in the 12 °C group seven weeks after the challenge. A higher number of genes were altered through time in the group reared at 17 °C. At termination, the nasal transcriptome of infected fish in both groups was similar to the control. When both infected groups were compared, 754 DEGs were identified, many of which were involved in signalling, defence, transmembrane and enzymatic processes. In conclusion, the study reveals that elevated temperature could trigger responses in the olfactory organ of Atlantic cod and shape the nasal response to F. noatunensis infection.

Tolfenpyrad displays Francisella-targeted antibiotic activity that requires an oxidative stress response regulator for sensitivity.

IMPORTANCE: Francisella species are highly pathogenic bacteria that pose a threat to global health security. These bacteria can be made resistant to antibiotics through facile methods, and we lack a safe and protective vaccine. Given their history of development as bioweapons, new treatment options must be developed to bolster public health preparedness. Here, we report that tolfenpyrad, a pesticide that is currently in use worldwide, effectively inhibits the growth of Francisella. This drug has an extensive history of use and a plethora of safety and toxicity data, making it a good candidate for development as an antibiotic. We identified mutations in Francisella novicida that confer resistance to tolfenpyrad and characterized a transcriptional regulator that is required for sensitivity to both tolfenpyrad and reactive oxygen species.

Impact of endosymbionts on tick physiology and fitness.

Ticks transmit pathogens and harbour non-pathogenic, vertically transmitted intracellular bacteria termed endosymbionts. Almost all ticks studied to date contain 1 or more of Coxiella, Francisella, Rickettsia or Candidatus Midichloria mitochondrii endosymbionts, indicative of their importance to tick physiology. Genomic and experimental data suggest that endosymbionts promote tick development and reproductive success. Here, we review the limited information currently available on the potential roles endosymbionts play in enhancing tick metabolism and fitness. Future studies that expand on these findings are needed to better understand endosymbionts' contributions to tick biology. This knowledge could potentially be applied to design novel strategies that target endosymbiont function to control the spread of ticks and pathogens they vector.

Bipolar Biogeographical Distribution of Parafrancisella Bacteria Carried by the Ciliate Euplotes.

Parafrancisella adeliensis, a Francisella-like endosymbiont, was found to reside in the cytoplasm of an Antarctic strain of the bipolar ciliate species, Euplotes petzi. To inquire whether Euplotes cells collected from distant Arctic and peri-Antarctic sites host Parafrancisella bacteria, wild-type strains of the congeneric bipolar species, E. nobilii, were screened for Parafrancisella by in situ hybridization and 16S gene amplification and sequencing. Results indicate that all Euplotes strains analyzed contained endosymbiotic bacteria with 16S nucleotide sequences closely similar to the P. adeliensis 16S gene sequence. This finding suggests that Parafrancisella/Euplotes associations are not endemic to Antarctica, but are common in both the Antarctic and Arctic regions.

Discovery of a glutathione utilization pathway in Francisella that shows functional divergence between environmental and pathogenic species.

Glutathione (GSH) is an abundant metabolite within eukaryotic cells that can act as a signal, a nutrient source, or serve in a redox capacity for intracellular bacterial pathogens. For Francisella, GSH is thought to be a critical in vivo source of cysteine; however, the cellular pathways permitting GSH utilization by Francisella differ between strains and have remained poorly understood. Using genetic screening, we discovered a unique pathway for GSH utilization in Francisella. Whereas prior work suggested GSH catabolism initiates in the periplasm, the pathway we define consists of a major facilitator superfamily (MFS) member that transports intact GSH and a previously unrecognized bacterial cytoplasmic enzyme that catalyzes the first step of GSH degradation. Interestingly, we find that the transporter gene for this pathway is pseudogenized in pathogenic Francisella, explaining phenotypic discrepancies in GSH utilization among Francisella spp. and revealing a critical role for GSH in the environmental niche of these bacteria.

Disparate dynamics of pathogen prevalence in Ixodes ricinus and Dermacentor reticulatus ticks occurring sympatrically in diverse habitats.

Ixodes ricinus and Dermacentor reticulatus ticks are important reservoirs and vectors of pathogens. The aim of the present study was to investigate the dynamic of the prevalence and genetic diversity of microorganisms detected in these tick species collected from two ecologically diverse biotopes undergoing disparate long-term climate condition. High-throughput real time PCR confirmed high prevalence of microorganisms detected in sympatrically occurring ticks species. D. reticulatus specimens were the most often infected with Francisella-like endosymbiont (FLE) (up to 100.0%) and Rickettsia spp. (up to 91.7%), while in case of I. ricinus the prevalence of Borreliaceae spirochetes reached up to 25.0%. Moreover, pathogens belonging to genera of Bartonella, Anaplasma, Ehrlichia and Babesia were detected in both tick species regardless the biotope. On the other hand, Neoehrlichia mikurensis was conformed only in I. ricinus in the forest biotope, while genetic material of Theileria spp. was found only in D. reticulatus collected from the meadow. Our study confirmed significant impact of biotope type on prevalence of representatives of Borreliaceae and Rickettsiaceae families. The most common co-infection detected in D. reticulatus was Rickettsia spp. + FLE, while Borreliaceae + R. helvetica was the most common in I. ricinus. Additionally, we found significant genetic diversity of R. raoultii gltA gene across studied years, however such relationship was not observed in ticks from studied biotopes. Our results suggest that ecological type of biotope undergoing disparate long-term climate conditions have an impact on prevalence of tick-borne pathogens in adult D. reticulatus and I. ricinus.

Applying next generation sequencing to detect tick-pathogens in Dermacentor nuttalli, Ixodes persulcatus, and Hyalomma asiaticum collected from Mongolia.

Ticks and tick-borne diseases represent major threats to the public health of the Mongolian population, of which an estimated 26% live a traditional nomadic pastoralist lifestyle that puts them at increased risk for exposure. Ticks were collected by dragging and removal from livestock in Khentii, Selenge, Tuv, and Umnugovi aimags (provinces) during March-May 2020. Using next-generation sequencing (NGS) with confirmatory PCR and DNA sequencing, we sought to characterize the microbial species present in Dermacentor nuttalli (n = 98), Hyalomma asiaticum (n = 38), and Ixodes persulcatus (n = 72) tick pools. Rickettsia spp. were detected in 90.4% of tick pools, with Khentii, Selenge, and Tuv tick pools all having 100% pool positivity. Coxiella spp. were detected at an overall pool positivity rate of 60%, while Francisella spp. were detected in 20% of pools and Borrelia spp. detected in 13% of pools. Additional confirmatory testing for Rickettsia-positive pools demonstrated Rickettsia raoultii (n = 105), Candidatus Rickettsia tarasevichiae (n = 65) and R. slovaca/R. sibirica (n = 2), as well as the first report of Candidatus Rickettsia jingxinensis (n = 1) in Mongolia. For Coxiella spp. reads, most samples were identified as a Coxiella endosymbiont (n = 117), although Coxiella burnetii was detected in eight pools collected in Umnugovi. Borrelia species that were identified include Borrelia burgdorferi sensu lato (n = 3), B. garinii (n = 2), B. miyamotoi (n = 16), and B. afzelii (n = 3). All Francisella spp. reads were identified as Francisella endosymbiont species. Our findings emphasize the utility of NGS to provide baseline data across multiple tick-borne pathogen groups, which in turn can be used to inform health policy, determine regions for expanded surveillance, and guide risk mitigation strategies.

Therapeutic efficacy of enrofloxacin in treatment of Francisella orientalis infections in juvenile Nile tilapia (Oreochromis niloticus L.).

Outbreaks of infections by Francisella orientalis represent one of the main obstacles to Nile tilapia (Oreochromis niloticus L.) farming. It is responsible for acute mortality in fingerlings and juveniles. The main control measure available is oral antibiotic therapy. This study compared the therapeutic efficacy of the antibiotics enrofloxacin and oxytetracycline, the most commonly used antimicrobial, against francisellosis in juvenile Nile tilapia (O. niloticus). Fish were challenged with a virulent isolate of F. orientalis and treated with medicated feed containing one of two doses of oxytetracycline (100 or 300 mg/kg of live weight (LW)) or 10 mg/kg of LW of enrofloxacin. The positive and negative control groups received feed without antibiotics; the negative control group was unchallenged. The results showed that enrofloxacin at a dose of 10 mg/kg of LW is effective against francisellosis in juvenile Nile tilapia (O. niloticus). Treatment with oxytetracycline did not eliminate the pathogen from the infected host, and the surviving fish became carriers. Enrofloxacin was able to cure the fish of infection with F. orientalis. This study suggests that enrofloxacin is a better option for treating francisellosis in Nile tilapia (O. niloticus L.). It controls mortality and avoids the carrier state in the fish, thus reducing the possibility of recurrence in the affected batches.

Molecular detection of Coxiella-like endosymbionts in Rhipicephalus microplus from north India.

Apart from the tick-borne pathogens affecting human and animal health, ticks also harbor various non-pathogenic endosymbionts with dynamic ecological interactions. These endosymbionts are unexplored from the Indian ticks; hence this pilot study was conducted. Seventy-nine ticks were collected from Nainital district of Uttarakhand state of north India and were identified as Rhipicephalus microplus morphologically and by molecular analysis. PCR and sequence analysis were carried out to detect the presence of Rickettsia-like, Coxiella-like and Francisella-like endosymbionts in these ticks. Based on the partial 16S rRNA gene sequence, Coxiella-like endosymbiont (CLE) was detected in the adult and other life-cycle stages of ticks with 96.6-97.7% nucleotide sequence identity with the published CLE sequences from GenBank. The phylogenetic analysis revealed that the CLE from R. microplus were clustered with the CLE from other Rhipicephalus species. All these CLE formed distinct clades from the pathogenic Coxiella burnetii. None of the tick samples was found positive for Rickettsia-like and Francisella-like endosymbionts in the present study. We also demonstrated the vertical transmission of CLE from surface sterilized and laboratory reared fully engorged adult females to the eggs and the larvae. However, large scale studies are to be conducted to detect various endosymbionts and endosymbiont-tick associations in the Indian tick species and to explore these associations for tick and tick-borne disease control.

Arginine 58 is indispensable for proper function of the Francisella tularensis subsp. holarctica FSC200 HU protein, and its substitution alters virulence and mediates immunity against wild-type strain.

HU protein, a member of the nucleoid-associated group of proteins, is an important transcription factor in bacteria, including in the dangerous human pathogen Francisella tularensis. Generally, HU protein acts as a DNA sequence non-specific binding protein and it is responsible for winding of the DNA chain that leads to the separation of transcription units. Here, we identified potential HU protein binding sites using the ChIP-seq method and two possible binding motifs in F. tularensis subsp. holarctica FSC200 depending upon growth conditions. We also confirmed that FSC200 HU protein is able to introduce negative supercoiling of DNA in the presence of topoisomerase I. Next, we showed interaction of the HU protein with a DNA region upstream of the pigR gene and inside the clpB gene, suggesting possible regulation of PigR and ClpB expression. Moreover, we showed that arginine 58 and partially arginine 61 are important for HU protein's DNA binding capacity, negative supercoiling induction by HU, and the length and winding of FSC200 chromosomal DNA. Finally, in order to verify biological function of the HU protein, we demonstrated that mutations in arginine 58, arginine 61, and serine 74 of the HU protein decrease virulence of FSC200 both in vitro and in vivo and that immunization using these mutant strains is able to protect as many as 100% of mice against wild-type challenge. Taken together, our findings deepen knowledge about the role of the HU protein in tularaemia pathogenesis and suggest that HU protein should be addressed in the context of tularaemia vaccine development.

Evaluation of Francisella orientalis ΔpdpA as a Live Attenuated Vaccine against Piscine Francisellosis in Nile Tilapia.

Francisella orientalis is an important bacterial pathogen of marine and freshwater fish with worldwide distribution. Fish francisellosis is a severe subacute to chronic granulomatous disease, with high mortalities and high infectivity rates in cultured and wild fish. To date, there is no approved vaccine for this disease. In this study, we evaluated the efficacy of a defined F. orientalis pathogenicity determinant protein A (pdpA) mutant (ΔpdpA) as a live attenuated immersion vaccine against subsequent immersion challenge with the wild-type organism. Immunized Nile tilapia Oreochromis niloticus were protected (45% relative percent survival) from the lethal challenges and presented significantly lower mortality than nonvaccinated and challenged treatments. Although serum IgM was significantly higher in immunized fish, similar bacterial loads were detected in vaccinated and nonvaccinated survivors. In conclusion, although the F. orientalis ΔpdpA is attenuated and effectively stimulated an adaptive immune response, the low relative percent survival and high bacterial persistence in survivors of immunized and challenged treatments indicates low suitability of ΔpdpA as a mucosal vaccine for tilapia under conditions used in this study.

Novel development of cationic surfactant-based mucoadhesive nanovaccine for direct immersion vaccination against Francisella noatunensis subsp. orientalis in red tilapia (Oreochromis sp.).

Francisella noatunensis subsp. orientalis (Fno) is one of the infectious diseases that causes economic losses associated with tilapia mortality. Even though direct immersion administration of vaccines is more practicable for small fish and fry compared with oral and injection vaccination in the fields, the efficacy is still insufficient due to lower potency of antigen uptake. Herein, we accomplished the development of a mucoadhesive nanovaccine platform using cetyltrimethylammonium bromide (CTAB), a cationic surfactant, to improve the efficiency of immersion vaccination against Fno in tilapia. Cationic Fno nanovaccine (CAT-Fno-NV) was prepared though emulsification using an ultrasonic method. In our investigation, the CAT-Fno-NV increased the opportunity of Fno vaccine uptake by extending the contact time between vaccine and mucosal surface of fish gills and enhancing the protective efficacy against Fno infection. Fish were vaccinated with the CAT-Fno-NV by a direct immersion protocol. The challenge trial by Fno injection revealed that CAT-Fno-NV at the concentration 1:100 ratio (approximately 1 × 106 cfu/mL) had the highest efficacy to protect fish from Fno infection at day 30 after post challenge period according to the total number of Fno detected in head kidney, spleen and liver. A significant upregulation of IgM gene was observed in gills, skin, head kidney, serum and peripheral blood lymphocytes (PBLs) and spleen tissues treated with WC and CAT-Fno-NV (1:100) vaccines, while IgT gene was highly expressed in only gills and skin tissues for treated WC and CAT-Fno-NV (1:100) groups. We anticipate that the cationic surfactant-based nanovaccine developed in this study could become an efficient alternative for direct immersion vaccination to induce humoral immune responses against Fno in vaccinated tilapia.

Efficient multiplex CRISPR/Cpf1 (Cas12a) genome editing system in Aspergillus aculeatus TBRC 277.

CRISPR/Cas technology is a versatile tool for genome engineering in many organisms, including filamentous fungi. Cpf1 is a multi-domain protein of class 2 (type V) RNA-guided CRISPR/Cas endonuclease, and is an alternative platform with distinct features when compared to Cas9. However, application of this technology in filamentous fungi is limited. Here, we present a single CRISPR/Cpf1 plasmid system in Aspergillus aculeatus strain TBRC 277, an industrially relevant cell factory. We first evaluated the functionality of three Cpf1 orthologs from Acidaminococcus sp. BV3L6 (AsCpf1), Francisella tularensis subsp. novicida U112 (FnCpf1), and Lachnospiraceae bacterium (LbCpf1), in RNA-guided site-specific DNA cleavage at the pksP locus. FnCpf1 showed the highest editing efficiency (93 %) among the three Cpf1s. It was further investigated for its ability to delete a 1.7 kb and a 0.5 kb from pksP and pyrG genes, respectively, using two protospacers targeting these gene loci in a single crRNA array. Lastly, simultaneous editing of three sites within TBRC 277 genome was performed using three guide sequences targeting these two genes as well as an additional gene, kusA, which resulted in combined editing efficiency of 40 %. The editing of the NHEJ pathway by targeting kusA to generate a NHEJ-deficient strain of A. aculeatus TBRC 277 improved gene targeting efficiency and yielded more precise gene-editing than that of using wild-type strain. This promising genome-editing system can be used for strain improvement in industrial applications such as production of valuable bioproducts.

Influence of tick sex and geographic region on the microbiome of Dermacentor variabilis collected from dogs and cats across the United States.

As tick-borne diseases continue to increase across North America, current research strives to understand how the tick microbiome may affect pathogen acquisition, maintenance, and transmission. Prior high throughput amplicon-based microbial diversity surveys of the widespread tick Dermacentor variabilis have suggested that life stage, sex, and geographic region may influence the composition of the tick microbiome. Here, adult D. variabilis ticks (n = 145) were collected from dogs and cats from 32 states with specimens originating from all four regions of the United States (West, Midwest, South, and Northeast), and the tick microbiome was examined via V4-16S rRNA gene amplification and Illumina sequencing. A total of 481,246 bacterial sequences were obtained (median 2924 per sample, range 399-11,990). Fifty genera represented the majority (>80%) of the sequences detected, with the genera Allofrancisella and Francisella being the most abundant. Further, 97%, 23%, and 5.5% of the ticks contained sequences belonging to Francisella spp., Rickettsia spp., and Coxiella spp., respectively. No Ehrlichia spp. or Anaplasma spp. were identified. Co-occurrence analysis, by way of correlation coefficients, between the top 50 most abundant genera demonstrated five strong positive and no strong negative correlation relationships. Geographic region had a consistent effect on species richness with ticks from the Northeast having a significantly greater level of richness. Alpha diversity patterns were dependent on tick sex, with males exhibiting higher levels of diversity, and geographical region, with higher level of diversity observed in ticks obtained from the Northeast, but not on tick host. Community structure, or beta diversity, of tick microbiome was impacted by tick sex and geographic location, with microbiomes of ticks from the western US exhibiting a distinct community structure when compared to those from the other three regions (Northeast, South, and Midwest). In total, LEfSe (Linear discriminant analysis Effect Size) identified 18 specific genera driving these observed patterns of diversity and community structure. Collectively, these findings highlight the differences in bacterial diversity of D. variabilis across the US and supports the interpretation that tick sex and geographic region affects microbiome composition across a broad sampling distribution.