htrA Gene Research Articles

Foodborne bacteria in milk and milk products along the water buffalo milk chain in Bangladesh

Controlling foodborne pathogens in buffalo milk is crucial for ensuring food safety. This study estimated the prevalence of nine target genes representing seven critical foodborne bacteria in milk and milk products, and identified factors associated with their presence in buffalo milk chain nodes in Bangladesh. One hundred and forty-three milk samples from bulk tank milk (n = 34), middlemen (n = 37), milk collection centers (n = 37), and milk product shops (n = 35) were collected and analyzed using RT-PCR. Escherichia (E.) coli, represented through yccT genes, was the most prevalent throughout the milk chain (81–97%). Chi-squared tests were performed to identify the potential risk factors associated with the presence of foodborne bacteria encoded for different genes. At the middleman level, the prevalence of E. coli was associated with the Mymensingh, Noakhali, and Bhola districts (P = 0.01). The prevalence of Listeria monocytogenes, represented through inlA genes, and Yersinia (Y.) enterocolitica, represented through yst genes, were the highest at the farm level (65–79%). The prevalence of both bacteria in bulk milk was associated with the Noakhali and Bhola districts (P < 0.05). The prevalence of Y. enterocolitica in bulk milk was also associated with late autumn and spring (P = 0.01) and was higher in buffalo-cow mixed milk than in pure buffalo milk at the milk collection center level (P < 0.01). The gene stx2 encoding for Shiga toxin-producing (STEC) E. coli was detected in 74% of the milk products. At the middleman level, the prevalence of STEC E. coli was associated with the use of cloths or tissues when drying milk containers (P = 0.01). Salmonella enterica, represented through the presence of invA gene, was most commonly detected (14%) at the milk collection center. The use of plastic milk containers was associated with a higher prevalence of Staphylococcus aureus, represented through htrA genes, at milk product shops (P < 0.05). These results suggest that raw milk consumers in Bangladesh are at risk if they purchase and consume unpasteurized milk.

A cross sectional study on molecular prevalence of Orientia tsutsugamushi in household rat population of South India

This study aimed to assess the molecular prevalence of mite-borne zoonotic pathogen O. tsutsugamushi in household rats of South India through nested polymerase chain reaction amplification of O. tsutsugamushi 47-kDa htrA gene and to determine the most suitable sample type for screening of O. tsutsugamushi in rats. Out of 85 rats trapped in Tamil Nadu, Karnataka, and Puducherry regions, 47 rats were found positive for the O. tsutsugamushi genome with prevalence of 55.29 %. Among different sample types screened, faecal samples exhibited the highest positivity rate, followed by liver, spleen, kidney, and blood samples. Agreement between faecal and spleen samples of rats for the presence of O. tsutsugamushi was the highest. Principal component analysis revealed a positive correlation between the spleen, liver, and faeces and a negative correlation between blood and faeces for the presence of O. tsutsugamushi genome. These findings underscore the varied distribution of O. tsutsugamushi among different samples and indicate that the faecal and liver samples of rats are an ideal choice of samples for epidemiological studies. This is the first study to report a high level of presence of O. tsutsugamushi in faecal samples of rats.

Effect of environmental variables on the abundance of Amblyomma ticks, potential vectors of Rickettsia parkeri in central Brazil.

Amblyomma ticks are vectors of both Rickettsia rickettsii and R. parkeri in the Americas, where capybaras (Hydrochoerus hydrochaeris) are the main hosts in urban areas, thus contributing to the transmission of spotted fever. Herein, we studied: (i) the seasonal dynamics and abundance of ticks in areas where capybaras live, (ii) the effect of environmental variables on tick abundance, and (iii) the presence of Rickettsia-infected ticks. Between September 2021 and September 2022, we sampled ticks using cloth-dragging at 194 sites on the shore of Lake Paranoá in Brasília, Brazil. We measured environmental data (season, vegetation type, canopy density, temperature, humidity, and presence or vestige of capybara) at each site. Nymphs and adults were morphologically identified to the species level, and a selected tick sample including larvae was subjected to genotypic identification. We investigated Rickettsia-infected ticks by PCR (gltA, htrA, ompB, and ompA genes) and associations between tick abundance and environmental variables using Generalized Linear Models. A total of 30,334 ticks (96% larvae) were captured. Ticks were identified as Amblyomma, with A. sculptum comprising 97% of the adult/nymphs. Genotype identification of a larval sample confirmed that 95% belonged to A. dubitatum. Seasonal variables showed significant effects on tick abundance. Most larvae and nymphs were captured during the early dry season, while the adults were more abundant during the wet season. Vegetation variables and the presence of capybaras showed no association with tick abundance. Rickettsia parkeri group and R. bellii were identified in A. dubitatum, while A. sculptum presented R. bellii. We conclude that: (i) Amblyomma ticks are widely distributed in Lake Paranoá throughout the year, especially larvae at the dry season, (ii) the abundance of Amblyomma ticks is explained more by climatic factors than by vegetation or presence of capybaras, and (iii) A. dubitatum ticks are potential vectors of R. parkeri in Brasília.

Microbiome and mitogenomics of the chigger mite Pentidionis agamae: potential role as an Orientia vector and associations with divergent clades of Wolbachia and Borrelia.

Trombiculid mites are globally distributed, highly diverse arachnids that largely lack molecular resources such as whole mitogenomes for the elucidation of taxonomic relationships. Trombiculid larvae (chiggers) parasitise vertebrates and can transmit bacteria (Orientia spp.) responsible for scrub typhus, a zoonotic febrile illness. Orientia tsutsugamushi causes most cases of scrub typhus and is endemic to the Asia-Pacific Region, where it is transmitted by Leptotrombidium spp. chiggers. However, in Dubai, Candidatus Orientia chuto was isolated from a case of scrub typhus and is also known to circulate among rodents in Saudi Arabia and Kenya, although its vectors remain poorly defined. In addition to Orientia, chiggers are often infected with other potential pathogens or arthropod-specific endosymbionts, but their significance for trombiculid biology and public health is unclear. Ten chigger species were collected from rodents in southwestern Saudi Arabia. Chiggers were pooled according to species and screened for Orientia DNA by PCR. Two species (Microtrombicula muhaylensis and Pentidionis agamae) produced positive results for the htrA gene, although Ca. Orientia chuto DNA was confirmed by Sanger sequencing only in P. agamae. Metagenomic sequencing of three pools of P. agamae provided evidence for two other bacterial associates: a spirochaete and a Wolbachia symbiont. Phylogenetic analysis of 16S rRNA and multi-locus sequence typing genes placed the spirochaete in a clade of micromammal-associated Borrelia spp. that are widely-distributed globally with no known vector. For the Wolbachia symbiont, a genome assembly was obtained that allowed phylogenetic localisation in a novel, divergent clade. Cytochrome c oxidase I (COI) barcodes for Saudi Arabian chiggers enabled comparisons with global chigger diversity, revealing several cases of discordance with classical taxonomy. Complete mitogenome assemblies were obtained for the three P. agamae pools and almost 50 SNPs were identified, despite a common geographic origin. P. agamae was identified as a potential vector of Ca. Orientia chuto on the Arabian Peninsula. The detection of an unusual Borrelia sp. and a divergent Wolbachia symbiont in P. agamae indicated links with chigger microbiomes in other parts of the world, while COI barcoding and mitogenomic analyses greatly extended our understanding of inter- and intraspecific relationships in trombiculid mites.

Molecular detection of virulence genes for multi drug resistance Klebseilla pneumoniae in patients with urinary tract infections, Thi-Qar Provenance, Iraq

An agent that contributes to hospital acquired illnesses is Klebsiella pneumoniae. In community-acquired infections and illnesses of the urinary tract, it is the primary and most frequent causal agent. The strains were detected by using Analytical Profile Index (Api-20 E) and 16s rRNA techniques, fifteen trains of K. pneumoniae were obtained from eighteen urine samples collected from patients in Al-Hussein Hospital, Thi-Qar Governorate, Iraq. This research sought to identify the antibiotic resistance of K. pneumoniae to Aztreonam, Ceftriaxone, Trimethoprim, Amikacin, Ciprofloxacin, Meropenem, and Piperacillin, the resistance was high and ranged between (86.6-100)%. The multidrug resistance percentage was 100%, in addition the investigation aimed to detect some virulence genes, (i.e. HtrA and MrkA gene) in K. pneumoniae. The HtrA and MrkA genes were found using the PCR method, which revealed that 6 (40%) of the isolates had the HtrA gene and that all strains under study lacked the MrkA gene.

Emergent spotted fever group Rickettsiae infections among hard ticks in Islamic Republic of Iran.

Tick-borne rickettsioses have become a health concern worldwide following the increasing incidence in recent decades. However, there is limited information about these diseases in Islamic Republic of Iran. This cross-sectional study was conducted to estimate the Rickettsia infection among ixodid ticks collected from cattle, sheep and goats in Islamic Republic of Iran. The DNA of ixodid ticks collected from cattle, sheep and goats in 54 villages of Zanjan Province, Islamic Republic of Iran, were collected and analysed using a spectrophotometer. Rickettsial-positive samples were screened by targeting the htrA gene and fragments of gltA gene were analysed. The variables were analysed using descriptive statistics and the χ2 test was used to compare the variables. A total of 528 ticks were tested. Overall, Rickettsia infection rate was 6.44%. Nine of the 12 tick species were infected. Rickettsial positive rates in Hyalomma marginatum and Dermacentor marginatus were 21.33% and 12.77%, respectively. R. aeschlimannii, the predominant rickettsia, was detected only in Hy. marginatum. R. raoultii, R. sibirica and R. slovaca comprised about half of the positive ticks and were recovered from more than one tick species. Considering the discovery of infected ticks in the Islamic Republic of Iran, there is a need to establish a tick control programme in the country, paying attention to populations at high-risk.

Genetic heterogeneity of &lt;i&gt;Rickettsia helvetica&lt;/i&gt; population

Background. To date, the genetic variability of Rickettsia helvetica has not been sufficiently studied.The aim. To study the prevalence and genetic variability of R. helvetica in Ixodes spp. collected in Western Siberia and the Russian Far East.Materials and methods. Ixodes spp. collected from rodents in the Omsk province, Western Siberia (n = 280) and collected by flagging on Putyatin and Russky Islands in Primorsky Krai, Russian Far East (n = 482) were analyzed for the presence of Rickettsia spp. All positive samples were genotyped for the gltA gene fragment. For a number of R. helvetica samples, fragments of the 16S rRNA, ompA, ompB, sca4, htrA, and groEL genes and 23S–5S intergenic spacer were additionally sequenced.Results. Four Rickettsia species (R. helvetica, “Candidatus Rickettsia tarasevichiae”, “Candidatus Rickettsia uralica”, and “Candidatus Rickettsia mendelii”) were found. Of them, R. helvetica was identified in 72.2 % of Ixodes apronophorus and 18.8 % of Ixodes trianguliceps from the Omsk province and in single Ixodes persulcatus from the Omsk province and Putyatin Island. This is the first finding of Rickettsia spp. in I. apronophorus. All known R. helvetica sequences from this study and the GenBank database belonged to four well supported monopheletic groups forming genetic lineages I–IV. Lineage I included European isolates from Ixodes ricinus, Western Siberian isolates from I. persulcatus, and some sequences from I. apronophorus. All R. helvetica sequences from I. trianguliceps from the Omsk province and I. persulcatus from the Komi Republic and one sequence from I. apronophorus were assigned to lineage II. Most sequences from I. apronophorus formed lineage III; all known R. helvetica sequences from I. persulcatus from the Far East formed genetic lineage IV.Conclusion. The genetic heterogeneity of R. helvetica population was first demonstrated. Known isolates of R. helvetica are reliably assigned to four genetic lineages, but not in all cases the association of different lineages with a specific tick species or specific territory was observed.

Epidemiology of feline bartonellosis and molecular characteristics of Bartonella henselae in Bangladesh

Bartonellosis, a neglected vector-borne zoonotic disease transmitted from animals to humans, continues to threaten human and animal health significantly. This study aims to determine the epidemiology of feline bartonellosis and the molecular characteristics of Bartonella spp. in cats. From June 2018 to June 2020, 304 oral swabs were randomly collected from Bangladesh's Dhaka, Mymensingh, and Rajshahi districts. A pre-tested questionnaire was administered to collect data. Oral swabs were subjected to PCR targeting htrA gene to confirm Bartonella spp., which was subsequently validated through sequencing. Risk factors were identified using multivariable logistic regression analysis. The overall prevalence of feline bartonellosis was found to be 15.1 %. The following factors were significantly (p < 0.05) associated with Bartonella infection in risk factor analysis: cats aged ≥ 1 year (OR: 3.23, 95 % CI: 1.38–24.40), local breed cats (OR: 3.37, 95 % CI: 1.05–10.81), cats carrying fleas (OR: 2.33, 95 % CI: 1.93–13.45), antifleacidal drugs inconsistently administered cats (OR: 6.74, 95 % CI: 3.17–14.31), outdoor access cats (OR: 2.54, 95 % CI: 1.16–5.57). Notably, zoonotic B. henselae was confirmed through sequencing, establishing it as the causal agent of cat scratch disease. Phylogenetic analysis showed homology with B. henselae sequences from Brazil, Saint Kitts, and Nevis. We recommend consistent and appropriate flea control measures to curb its spread among Bangladeshi cats. Moreover, limiting outdoor exposure or implementing preventive measures for outdoor cats could reduce the disease burden. The associated human health risk can be decreased by effectively controlling this disease within the cat population.

Analysis of protein secretion in Bacillus subtilis by combining a secretion stress biosensor strain with an in vivo split GFP assay

BackgroundBacillus subtilis is one of the workhorses in industrial biotechnology and well known for its secretion potential. Efficient secretion of recombinant proteins still requires extensive optimization campaigns and screening with activity-based methods. However, not every protein can be detected by activity-based screening. We therefore developed a combined online monitoring system, consisting of an in vivo split GFP assay for activity-independent target detection and an mCherry-based secretion stress biosensor. The split GFP assay is based on the fusion of a target protein to the eleventh β-sheet of sfGFP, which can complement a truncated sfGFP that lacks this β-sheet named GFP1-10. The secretion stress biosensor makes use of the CssRS two component quality control system, which upregulates expression of mCherry in the htrA locus thereby allowing a fluorescence readout of secretion stress.ResultsThe biosensor strain B. subtilis PAL5 was successfully constructed by exchanging the protease encoding gene htrA with mCherry via CRISPR/Cas9. The Fusarium solani pisi cutinase Cut fused to the GFP11 tag (Cut11) was used as a model enzyme to determine the stress response upon secretion mediated by signal peptides SPPel, SPEpr and SPBsn obtained from naturally secreted proteins of B. subtilis. An in vivo split GFP assay was developed, where purified GFP1-10 is added to the culture broth. By combining both methods, an activity-independent high-throughput method was created, that allowed optimization of Cut11 secretion. Using the split GFP-based detection assay, we demonstrated a good correlation between the amount of secreted cutinase and the enzymatic activity. Additionally, we screened a signal peptide library and identified new signal peptide variants that led to improved secretion while maintaining low stress levels.ConclusionOur results demonstrate that the combination of a split GFP-based detection assay for secreted proteins with a secretion stress biosensor strain enables both, online detection of extracellular target proteins and identification of bottlenecks during protein secretion in B. subtilis. In general, the system described here will also enable to monitor the secretion stress response provoked by using inducible promoters governing the expression of different enzymes.

First isolation of Rickettsia amblyommatis from Amblyomma mixtum in Colombia

BackgroundRickettsiae are obligate intracellular Gram-negative bacteria that are the causative agent of rickettsioses and are spread to vertebrate hosts by arthropods. There are no previous reports of isolation of Rickettsia amblyommatis for Colombia.MethodsA convenience sampling was executed in three departments in Colombia for direct collection of adult ticks on domestic animals or over vegetation. Ticks were screened for the presence of Rickettsia spp. by real-time polymerase chain reaction (qPCR) amplifying the citrate synthase gene (gltA), and the positive sample was processed for isolation and further molecular characterization by conventional PCR. The absolute and relative frequencies were calculated for several tick species variables. All products from conventional PCR were further purified and sequenced by the Sanger technique. Representative sequences of 18 Rickettsia species were downloaded from GenBank. Consensus phylogenetic trees were constructed for the gltA, ompB, ompA, and htrA genes with 1000 replicates, calculating bootstrap values through the maximum likelihood method and the generalized time reversible substitution model in the MEGA 7.0 software program.ResultsOne female Amblyomma mixtum collected on vegetation was amplified by qPCR (gltA), indicating a frequency of 1.6% (1/61) for Rickettsia spp. infection. Sequence analysis of a rickettsial isolate from this tick in BLASTn showed 100% identity with gltA (340 base pairs [bp]), 99.87% for ompB (782 bp), 98.99% for htrA (497 bp), and 100% for ompA (488 bp) to R. amblyommatis. Concatenated phylogenetic analysis confirmed these findings indicating that the isolate is grouped with other sequences of Amblyomma cajennense complex from Panama and Brazil within the R. amblyommatis clade.ConclusionsThis paper describes the isolation and early molecular identification of a R. amblyommatis strain from A. mixtum in Colombia.Graphical

Pathogenic Rickettsia, Anaplasma, and Ehrlichia in Rhipicephalus microplus ticks collected from cattle and laboratory hatched tick larvae.

The order Rickettsiales contains a group of vector-borne gram-negative obligate intracellular bacteria, which often cause human emerging infectious diseases and economic losses for dairy and meat industries. The purpose of this study is to investigate the distribution of the pathogens including Rickettsia spp., Anaplasma spp., and Ehrlichia spp. in the order Rickettsiales in ticks from Yueyang, a prefecture-level city of Hunan Province in Sothern China, and assess the potentiality of transovarial transmission of these rickettsial organisms. Ticks were collected from cattle in a farm in Yueyang City and the tick DNA was used as template to amplify the htrA, rrs, gltA, ompA and ompB genes of Rickettsia as well as rrs and groEL genes of Anaplasma and Ehrlichia. All ticks (465) collected were the cattle tick, Rhipicephalus microplus. PCR showed the minimum infection rate (MIR) was 1.5% (7/465) for Candidatus Rickettsia xinyangensis, 1.9% (9/465) for C. Anaplasma boleense, 1.3% (6/465) for Anaplasma platys, 0.6% (3/465) for A. marginale, and 1.17% (2/465) for each of A. bovis, Ehrlichia minasensis, and a non-classified Ehrlichia sp. A human pathogen, C. Rickettsia xinyangensis and A. platys were detected in 100% (3/3) and 33.3% (2/6) laboratory-hatched larval pools from infected females respectively. Our study revealed a diversity of pathogenic rickettsial species in R. microplus ticks from Hunan Province suggesting a threat to people and animals in China. This study also provided the first molecular evidence for the potential transovarial transmission of C. Rickettsia xinyangensis and A. platys in R. microplus, indicating that R. microplus may act as the host of these two pathogens.

HtrA is involved in stress response and adhesion in Glaesserella parasuis serovar 5 strain Nagasaki

Glaesserella parasuis is an important pathogen that causes fibrinous polyserositis, peritonitis and meningitis in pigs, leading to considerable economic losses to the swine industry worldwide. It is well established that the serine protease HtrA is closely associated with bacterial virulence, but the role of HtrA in G. parasuis pathogenesis remains largely unknown. To characterize the function of the htrA gene in G. parasuis, a ΔhtrA mutant was constructed. We found that the ΔhtrA mutant showed significant growth inhibition under heat shock and alkaline stress conditions, indicating HtrA is involved in stress tolerance and survival of G. parasuis. In addition, deletion of htrA gene resulted in decreased adherence to PIEC and PK-15 cells and increased phagocytic resistance to 3D4/2 macrophages, suggesting that htrA is essential for adherence of G. parasuis. Scanning electron microscopy revealed morphological surface changes of the ΔhtrA mutant, and transcription analysis confirmed that a number of adhesion-associated genes are downregulated, which corroborated the aforementioned phenomenon. Furthermore, G. parasuis HtrA induced a potent antibody response in piglets with Glässer’s disease. These observations confirmed that the htrA gene is related to the survival and pathogenicity of G. parasuis.

A novel Rickettsia species closely related to Rickettsia felis in Anopheles mosquitoes from Yingkou City, Northeast China.

Mosquitoes are generally recognized as the most important vector of many zoonotic pathogens. In this study, seven mosquitoes species were identified (Anopheles pullus, Anopheles sinensis, Anopheles lesteri, Anopheles kleini, Ochlerotatus dorsalis, Aedes koreicus and Culex inatomii) in samples collected from Yingkou City, Liaoning Province, Northeastern China. A novel Rickettsia species was detected in Anopheles sinensis (two of 71, 2.82%) and Anopheles pullus (one of 106, 0.94%) mosquitoes. Genetic analysis indicated that the rrs and ompB genes have highest 99.60% and 97.88%-98.14% identities to Rickettsia felis, an emerging human pathogen of global concern mainly harboured by fleas, mosquitoes and booklice. The gltA sequences of these strains have 99.72% of nucleotide similarity with Rickettsia endosymbiont of Medetera jacula. The groEL sequences have 98.37% similarity to both Rickettsia tillamookensis and Rickettsia australis. The htrA sequences have 98.77% similarity to Rickettsia lusitaniae. In the phylogenetic tree based on concatenated nucleotide sequences of rrs, gltA, groEL, ompB and htrA genes, these strains are closely related to R. felis. Herein, we name it 'Candidatus Rickettsia yingkouensis'. Its human pathogenicity to humans and animals is still to be determined.

Multilocus genetic analysis indicates taxonomic status of “Candidatus Rickettsia mendelii” as a separate basal group

To date, the phylogeny of Rickettsia spp. from basal groups is based on the small number of identified species. Thus, the finding of “Candidatus Rickettsia mendelii” in 2016 is of great interest. In this study, “Ca. R. mendelii” was first identified in the Asian region in a new carrier, Ixodes pavlovskyi. “Candidatus R. mendelii”, along with “Candidatus Rickettsia tarasevichiae”, were found in Ixodes ticks collected on Russky Island (the Far East), where I. pavlovskyi coexists with I. persulcatus. To establish the taxonomic position of “Ca. R. mendelii”, a detailed genetic study was carried out. “Candidatus R. mendelii” was genotyped by five genetic fragments (16S rRNA, gltA, and ompB genes, groESL operon, and 23S–5S IGS region); among them, the ompB gene, groESL operon and 23S–5S IGS region were sequenced for the first time. In addition, “Ca. R. tarasevichiae” was genetically characterized by eight genetic loci (16S rRNA, gltA, ompA, ompB, sca4, htrA genes, groESL operon, and 23S–5S IGS region), of which the sca4 gene was first determined. Phylogenetic analysis indicated that regardless of analyzed genetic loci, “Ca. R. mendelii” formed a separate well-supported cluster on each phylogenetic tree. Phylogenetic analysis based on concatenated sequences of gltA, ompB, and groEL gene fragments (total length of 3191 bp) demonstrated that “Ca. R. mendelii”, like Rickettsia bellii, is a basal group of Rickettsia.

Detection of Rickettsia spp. in ticks of wildlife fauna from Costa Rica: First report of Rickettsia rhipicephali in Central America

In the past two decades, new species of Rickettsia have been detected and described worldwide, some of them considered pathogenic for humans. Although Costa Rica is considered a biodiversity hotspot, the knowledge about rickettsiae in sylvatic ecosystems and wild animals is scarce. The aim of this preliminary study was to detect and identify species of Rickettsia in ticks collected from wild animals in Costa Rica. A total 119 ticks were collected from 16 animal host species belonging to diverse vertebrate families (Didelphidae, Procyonidae, Felidae, Choloepodidae, Bradypodidae, Myrmecophagidae, Tayassuidae, Tapiridae, Phyllostomidae, Bufonidae, Geoemydidae, Boidae, Colubridae), and they were grouped into 43 pools to detect the presence of Rickettsia spp. DNA by PCR targeting the gltA gene. In positive pools, amplicons of the ompA, sca5 (ompB), and/or htrA genes were also amplified to identify the species present. The identification of some ticks was also confirmed by molecular methods. Four species of Rickettsia were detected in eight (19%) tick pools: Rickettsia amblyommatis in four pools of Amblyomma geayi (host: Caluromys derbianus) and one pool of Amblyomma cf. parvum (host: Nasua narica), Rickettsia rhipicephali in one pool of Dermacentor latus (host: Tayassu pecari), ‘Candidatus Rickettsia colombianensi’ in one pool of Amblyomma sp. nymphs (host: Boa constrictor), and Rickettsia sp. genotype IbR/CRC in one pool of Ixodes cf. boliviensis (host: Puma concolor). This is the first molecular detection of R. rhipicephali in Central America, and of ‘Candidatus R. colombianensi’ in Costa Rica. Results show that diverse wild animals and their ticks are associated with several species of rickettsiae in Costa Rica, which may come in contact with humans and other domestic animals in sylvatic environments.

Identification of genes associated with environmental persistence in Campylobacter jejuni and Campylobacter coli isolates from processing in a broiler abattoir

The aim of this study was to determine the prevalence of the htrA, htrB and ppk1 genes -all of which are related to environmental persistence- in C. jejuni and C. coli isolates obtained from abattoir samples at the arrival of broilers (initial stage) and in meat products after processing (final stage). A total of 119 DNA extracts (55 C. jejuni and 64 C. coli) were included in the study. Identification of genes was performed by conventional PCR (one for each gene). The overall prevalence was 40.3%, 93.3% and 68.9% for the htrA, htrB and ppk1 genes, respectively. Statistically significant differences were found (p < 0.05) between prevalence of C. jejuni and C. coli for all three genes. In C. coli the prevalence was significantly higher for the htrA (p = 0.007) and htrB (p = 0.015) genes, while ppk1 gene prevalence was significantly higher in C. jejuni (p < 0.001). In addition, statistically significant increase in the frequency of htrA (p = 0.007) and htrB (p = 0.013) genes in the final product compared to broilers on arrival at the abattoir was observed in C. jejuni, but not in C. coli. These results suggest that htrA and htrB genes are involved in environmental persistence of Campylobacter jejuni.

Transcriptome analysis of heat resistance regulated by quorum sensing system in Glaesserella parasuis.

The ability of bacteria to resist heat shock allows them to adapt to different environments. In addition, heat shock resistance is known for their virulence. Our previous study showed that the AI-2/luxS quorum sensing system affects the growth characteristics, biofilm formation, and virulence of Glaesserella parasuis. The resistance of quorum sensing system deficient G. parasuis to heat shock was obviously weaker than that of wild type strain. However, the regulatory mechanism of this phenotype remains unclear. To illustrate the regulatory mechanism by which the quorum sensing system provides resistance to heat shock, the transcriptomes of wild type (GPS2), ΔluxS, and luxS complemented (C-luxS) strains were analyzed. Four hundred forty-four differentially expressed genes were identified in quorum sensing system deficient G. parasuis, which participated in multiple regulatory pathways. Furthermore, we found that G. parasuis regulates the expression of rseA, rpoE, rseB, degS, clpP, and htrA genes to resist heat shock via the quorum sensing system. We further confirmed that rseA and rpoE genes exerted an opposite regulatory effect on heat shock resistance. In conclusion, the findings of this study provide a novel insight into how the quorum sensing system affects the transcriptome of G. parasuis and regulates its heat shock resistance property.

Phenotypicand Genotypic Characterization of Clinical Isolates of Intracellular Adherent-Invasive Escherichia coli Among Different Stages, Family History, and Treated Colorectal Cancer Patients in Iran.

There is increasing evidence showing that microbial dysbiosis impacts the health and cancer risk of the host. An association between adherent–invasive Escherichia coli (AIEC) and colorectal cancer (CRC) has been revealed. Cyclomodulins (CMs) have been receiving increasing attention for carcinogenic changes. In this study, the incidence and features of intracellular AIEC and cyclomodulin-encoding genes were investigated and the phylogenetic grouping and genetic relatedness were evaluated. E. coli strains were isolated from the colorectal biopsies. Adhesion and invasion assays and intramacrophage cell survival test were performed to separate the AIEC isolates. Virulence genotyping for the genes htrA, dsbA, chuA, and lpfA and the cyclomodulin toxins was also conducted. In addition, phylogenetic grouping of the isolates was determined. Subsequently, repetitive element sequence-based PCR (rep-PCR) fingerprinting was performed. A total of 24 AIEC pathovars were isolated from 150 patients. The prevalence rates of htr, dsbA, and lpfA were 70.83% and that of chuA was 91.66%. The frequencies of the cyclomodulin toxins were as follows: cnf1, 29.2%; cnf2, 25%; colibactin, 29.2%; and cdt, 4.2%; cif was not found. Among the AIEC isolates, 4.2%, 4.2%, 54.2%, 29.2%, and 8.3% with phylotypes A or C, B1, B2, D, and E were identified, respectively. Left-sided colon carcinoma and adenocarcinoma T≥1 stage (CRC2) were colonized by B2 phylogroup AIEC-producing CMs more often than the samples from the other groups. Close genetic relatedness was observed in AIEC isolates with rep-PCR.

Development of paper-based DNA sensor for detection of O. tsutsugamushi using sustainable GQDs@AuNPs nanocomposite

The graphene quantum dots (GQDs) was synthesized using potato starch and water by hydrothermal method and further used for reduction of tetracholoroauric acid to form graphene quantum dots-gold (GQDs@AuNPs) nanocomposite. The GQDs/GQDs@AuNPs were analyzed using FTIR, UV–Vis, Flourometry and HR-TEM. The synthesized GQDs@AuNPs were further used for fabrication of cost-effective screen-printed paper electrode (SPPE) based DNA sensor for the detection of O. tsutsugamushi using htrA gene specific 5′NH2 linked DNA probe. Modification of SPPE using GQDs@AuNPs nanocomposite and ssDNA probe was monitored using EIS, FTIR, FE-SEM and AFM. The sensor detection limit (LOD) was assessed as 0.002 ng/μl from the standard calibration curve with the correlation coefficient, R2 = 0.993. The sensitivity of the DNA sensor was calculated as 7700 μA/cm2/ng for ssGDNA of O. tsutsugamushi using cyclic voltammetry. The sensor validation was done using scrub typhus patient's blood DNA samples. The sensor showed good storage stability at 4 °C for six months with just a loss of 12% of the initial current values. The SPPE/DNA sensor developed is very specific, sensitive, stable and detects O. tsutsugamushi in less time.

“Candidatus Rickettsia uralica” and “Candidatus Rickettsia thierseensis” are genetic variants of one species

The spotted fever group is the largest group in the Rickettsia genus, and new candidate species of this group are found throughout the world. "Candidatus Rickettsia thierseensis" was observed in a single Ixodes ricinus in Austria in 2020. However, sequences of five genes (total length: 5305 bp) of the "Candidatus R. thierseensis" were nearly identical to those available for "Candidatus Rickettsia uralica" identified in Ixodes trianguliceps and small mammals in Russia in 2015. To confirm the identity of these species, appropriate fragments of seven genetic loci from four isolates of "Candidatus R. uralica" were sequenced and compared with the "Candidatus R. thierseensis" sequences. Additionally, the groEL gene fragment was first sequenced for "Candidatus R. uralica". In this analysis, "Candidatus R. uralica" and "Candidatus R. thierseensis" demonstrated 100% identity in the 23S-5S IGS region (354 bp) and in fragments of the 16S rRNA (991 bp), gltA (1119 bp), and htrA (463 bp) genes, differing by only one substitution in each of the ompA (3696 bp), ompB (4871 bp), and sca4 (2971 bp) gene fragments. Thus, "Candidatus R. uralica" and "Candidatus R. thierseensis" are likely the same species based on comparative sequence analysis.