Targeting 16S rRNA Genes Research Articles

A Molecular Approach to Understand the Involvement of Mycoplasma in Suppurative Otitis Cases of Cattle in Chittoor District of Andhra Pradesh

In bovines the economic losses mainly due to the outbreak of diseases, leads to mortality, morbidity, treatment cost and reduced production. Recently in August month of 2024, received 13 bovine suppurativeotitis samples from outbreak in KuppamMandal of Chittoor district. The history having the purulent discharges with foul smell from ear infections of cattle that to crossbreed cattle of all age groups are affected. After 5 days of appearing aural discharges the animals were also shown the nervous signs, head tilting and other clinical findings includes unilateral or bilateral ear droop, epiphora, ptosis, abnormal nystagmus, strabismus, regurgitation, stiff neck, opisthotonus, facial hyperesthesia, purulent aural discharge, nasopharyngeal collapse, recumbency and finally death were noticed in severely affected animals. All the samples were processed for cultural tests by inoculating in PPLO selective broth and selective media PPLO agar then incubated anaerobically at 370C and observed the color development in broth pink to yellow color after 3- 5 days of incubation indicative of positive growth and fried egg micro colonies on selective PPLO agar media after 8-14 days of incubation observed under low power and 40X. The DNA was isolated from all the samples and screened for presence of Mycoplasma by targeting 16s rRNA gene and found that out of 13 samples 10 were positive for genus Mycoplasma and produced the predicted 280bp size product in all positive samples. The suppurativeotitis in cattle usually is caused by Actinomyces spp., Corynebacterium pseudotuberculosis, E. coli, Heamophilus, P.multocida, Pseudomonas spp, Streptococcus spp., and Mycoplasma bovis. This study was targeted only the emerging pathogen i.e the cellwall deficient bacteria Mycoplasma because of infections is hampered by a lack of effective vaccines and specific treatments, leads to increasing trends in antimicrobial resistance. Concluded that isolation and identification of the pathogenic ........

Characterizing the urobiome in geriatric males with chronic indwelling urinary catheters: an exploratory longitudinal study.

The impact of chronic indwelling urinary catheters (IUCs) on the composition and stability of the urinary microbiota remains unknown. The primary aim of this study was to describe the urinary microbiomes of geriatric males with chronic IUCs. A secondary aim was to explore clinical catheter-associated urinary tract infection (CAUTI) courses of the participants. Geriatric male patients with chronic IUCs were followed longitudinally. Catheterized urine, catheter tips, and both urethral and periurethral swabs were collected from participants at monthly intervals. Microbes were isolated and identified from each specimen using an enhanced culture method called expanded quantitative urine culture (EQUC) and targeted 16S rRNA gene DNA sequencing. Microbial outcomes were examined both in the absence of urinary symptoms and in the context of clinical diagnosis of CAUTI. Ten male participants (mean age 86 years) were enrolled. Urinary microbiomes differed for each participant. However, within each individual, microbiomes were similar over time and across niches (bladder, catheter, urethra, and periurethra). Within-niche microbiomes differed across individuals, and this was observed over time. The most abundant bacteria isolated from all niches were known uropathogens. Six of 10 individuals met diagnostic criteria for CAUTI at least once during the 12-month observation period, but no evidence of this or antibiotic treatment/response was discernable in our monthly samples. The microbiomes of each participant were unique and remained similar over time and across niches. Longitudinal EQUC or 16S rRNA gene sequencing data could be useful to clinicians when diagnosing or treating possible CAUTI.IMPORTANCECatheter-associated urinary tract infections (CAUTIs) are serious but preventable nosocomial infections. The most common risk factor for developing CAUTI is prolonged use of indwelling urinary catheters (IUCs). This study provides the first longitudinal description of the urinary microbiomes of geriatric males with chronic IUCs, in the absence of urinary signs and symptoms, as a first step toward enhancing our knowledge of the impact of chronic IUCs on the composition and stability of the urinary microbiota. This is an understudied area, particularly for males.

Viability discrimination of bacterial microbiomes in home kitchen dish sponges using propidium monoazide treatment.

Dish sponges are known to support the proliferation of human bacterial pathogens, yet they are commonly used by consumers. Exposure to foodborne pathogens via sponge use may lead to illness, a serious concern among susceptible populations. The extent of exposure risks from sponge use has been limited by constraints associated with culture-independent or dependent methods for bacterial community characterization. Therefore, five used dish sponges were characterized to evaluate the presence of viable bacterial foodborne pathogens using the novel application of propidium monoazide (PMA) treatment and targeted 16S rRNA gene amplicon sequencing. Select pathogen viability was confirmed using targeted selective enrichment. The taxonomic abundance profiles of total and viable sponge microbiomes did not vary significantly. The numbers of unique bacterial species (p = 0.0465) and foodborne pathogens (p = 0.0102) identified were significantly lower in viable sponge microbiomes. Twenty unique bacterial foodborne pathogens were detected across total and viable sponge microbiomes, and three to six viable foodborne pathogens were identified in each sponge. Escherichia coli and Staphylococcus aureus were identified in each viable sponge microbiome, and viable E. coli were recovered from two sponges via targeted selective enrichment. These findings suggest that sponge-associated bacterial communities are primarily viable and contain multiple viable bacterial foodborne pathogens.

Microbial community profiling in intestinal tract of indigenous chickens from different villages

Village chickens (Gallus gallus domesticus) are commonly reared in rural households of South Africa and other developing countries. They play a vital role as a primary source of protein through the provision of meat and eggs. The chicken gut microbiota plays an important role in chicken’s immune system, its health, physiological development of the gut, digestion of food, nutrient absorption and productivity. Thus, it is imperative to critically investigate the chicken microbial composition in order to develop effective disease control measures and increase production. In this present study, microbial DNA was isolated from 34 non-descript mixed gender matured village chickens’ intestinal contents followed by high throughput Illumina sequencing targeting 16S rRNA gene. Senwamokgope village had the largest microbiota composition as compared to Itieleni and Thakgalang villages. Overall, Firmicutes (74%) was the most abundant phylum observed, followed by Proteobacteria (8%), Actinobateria (5%), and Bacteroidota (3%). At the genus level, Lactobacillus was the dominant bacteria. Other genera found included Sphingomonas (7%), Cutibacterium (4%), and Clostridium_sensu_stricto_1 (2%). The richness of female intestinal microbiota was higher compared to the male microbiota. The findings of this study provide baseline information that can assist to better understand the chicken gut microbiota and its interaction with diseases and parasites.

Seasonal variation in airborne microbial communities of the Akiyoshido Cave: Lampenflora dispersed by phototrophic bioaerosols

Lampenflora, such as phototrophic microorganisms, multiply on speleothem surfaces under the show-cave lighting, causing deterioration of the natural and cultural heritages of the caves. Speleothems change color from white to green through microbial photosynthesis and are destroyed by microbial metabolisms. Aerosol transmission are suspected to disperses photosynthetic microorganisms. However, the mechanism underlying the process remains unclear, because the complexity of air ventilation affects the cave atmosphere. In the present study, we collected aerosol samples from the Akiyoshido Cave, which is a special natural monument in Japan and possesses various types of speleothems, to analyze microbial concentrations and communities in the cave atmosphere. Under a fluorescence microscope, phototrophic and heterotrophic microorganisms were observed in the aerosols with in the cave. The aerosol concentrations showed seasonal changes depending on air-flow variations in cave ventilation. High-throughput DNA sequencing targeting 16S rRNA genes revealed that the airborne bacterial communities inside the cave were dominated by members of the phyla Actinomycetota, Bacillota, and Pseudomonadota, originating from the external terrestrial, phyllosphere or freshwater environments, as well as its interior (guano). Cyanobacteria showed small relative abundances (from 1.0 to 10%) randomly in the aerosol samples at several locations in the cave, suggesting that the lampenflora-derived cyanobacteria were dispersed throughout the cave by the ventilation. Additionally, phototrophic microorganisms closely related to the relatives of the genera Leptolyngbya, Calothrix, and Chroococcidiopsis from the phylum Cyanobacteria were isolated from the aerosol samples. These results confirm that Cyanobacteria are one of the candidate microorganisms responsible for lampenflora dispersion and are “alive and airborne” in caves.

The impacts of film mulching and ridging on N2O emissions, relevant functional genes, and microbial communities in rain-fed potato fields

Rain-fed potato (Solanum tuberosum) fields in drylands significantly contribute to nitrous oxide (N2O) emissions, making them an important focus of agricultural greenhouse gas research. Film mulching and ridging are key agricultural methods in potato cultivation. Investigating the impact of these methods on N2O emissions, nitrifying/denitrifying functional genes, and microbial communities can provide a theoretical basis for soil emission reduction and more sustainable dryland agriculture. We examine the effects of flat tillage with mulching, ridge tillage with mulching, flat tillage without mulching, and ridge tillage without mulching, on potato fields under natural rainfall conditions in Wuchuan County, China. N2O emission fluxes were monitored using a static (dark) chamber and gas chromatography. Real-time quantitative PCR (q-PCR) was used to quantify abundances of nitrifying and denitrifying bacteria related to N2O emissions at various potato-growth stages. Illumina high-throughput sequencing was used to investigate microbial community structure by targeting 16S rRNA genes; related soil elements (soil temperatures and moisture) are analyzed. Mulching and ridging indirectly influence N2O emissions, nitrifying/denitrifying functional gene copy numbers, and microbial community structure by altering soil temperature and moisture. Cumulative N2O emissions and emission intensity were both consistently higher in ridge tillage with mulching during the potato-growing period. Ammonia-oxidizing archaea are the main microorganisms that control N2O emissions, with nitrification-coupled denitrification also being an important mechanism contributing to high N2O emissions during soil dry–wet cycles. Increased soil temperature and moisture elevated N2O emissions and functional gene copy numbers. The combination of mulching and ridging effectively uses the characteristics of both practices, making Nitrospira the dominant genus, and significantly increases N2O emissions.

Clostridium perfringens in central Colombia: frequency, toxin genes, and risk factors

Clostridium perfringens is an opportunistic bacterium that causes intestinal diseases in both humans and animals. This study aimed to assess the frequency of C. perfringens and the presence of toxin-encoding genes in fecal samples from individuals with or without gastrointestinal symptoms in the Department of Boyacá, Colombia. Additionally, risk factors associated with carriage and disease development were analyzed. A total of 114 stool samples were analyzed using a molecular test based on specific polymerase chain reaction (PCR) targeting 16S-rRNA and alpha toxin (cpa) genes. For individuals with a positive result for the PCR test, stool samples were cultured on Tryptose Sulfite Cycloserine (TSC) agar. Two to five colonies forming units were selected based on phenotypic characteristics, resulting in 56 bacterial isolates. These isolates were then analyzed for toxin-coding genes associated with gastrointestinal diseases. In addition, sociodemographic and clinical data from 77 individuals were also analyzed. The overall frequency of C. perfringens was 19.3% (n = 22/114). The detection frequency in 77 individuals with clinical data was 16.6% (n = 5/30) among symptomatic individuals and 21.2% (n = 10/47) among asymptomatic individuals. All 56 isolates obtained carried the cpa gene, while cpb2 was present in 10.7% (n = 6/56); cpe and cpb genes were not detected. Notably, diabetes and autoimmune diseases are significantly associated with an increased risk of C. perfringens detection (adjusted OR 8.41: 95% CI 1.32–35.89). This study highlights an elevated frequency of C. perfringens and the presence of the cpb2 gene in asymptomatic individuals compared with their symptomatic counterparts. These findings offer insights into the distribution and virulence factors of C. perfringens at a micro-geographical level. This information supports the need for developing tailored prevention strategies based on local characteristics to promote active surveillance programs based on molecular epidemiology.

Prevalence and predictors of NTM in presumed/confirmed drug-resistant TB.

Non-tuberculous mycobacteria (NTM) are increasingly isolated in individuals with presumed/confirmed pulmonary TB. We aimed to estimate the prevalence and species distribution of NTM among presumed/confirmed drug-resistant TB (DR-TB) individuals and determine NTM isolation predictors. Sputum samples collected for DR-TB diagnosis and follow-up from 2012 to 2021 in Ghana were retrospectively analysed. Samples were subjected to sputum smear microscopy (SSM) and mycobacterial culture. The MPT64 assay was performed on positive cultures to distinguish between Mycobacterium tuberculosis complex MTBc and NTM. NTM isolates were re-cultured for species identification using GenoType® Mycobacterium CM/AS line-probe assay, polymerase chain reaction, and Sanger sequencing targeting 16S rRNA and rpoB genes. MTBc isolates identified by GenoType underwent spoligotyping. A logistic regression model was used to identify the predictors of NTM isolation. Of the 2,492 samples, 839 (33.7%) tested culture-positive for mycobacteria, with 257 (30.6%) presumed to be NTM. Of these, 53 (23.6%) were identified at the species level, with a predominance of M. intracellulare (66.0%). MPT64 testing missed 18 (3%) MTBc isolates. Logistic regression showed increased odds of NTM isolation in follow-up samples (aOR 2.41, 95% CI 1.46-3.99). NTM species were isolated from 46 patients, with four classified as NTM pulmonary disease. Enhancing our understanding of local NTM epidemiology and improving local diagnostic capabilities can optimise patient management strategies and outcomes.

Unraveling changes in the duck microbiome and inflammatory processes due to allithiamine-enriched feed

The gastrointestinal tract of poultry harbors a diverse and intricate microbiome that plays a crucial role in nutrient digestion and absorption, immune system development, and enhances resistance against pathogens. Maintaining a healthy state and proper production is fundamentally determined by the symbiosis between the host and microbes. Due to genetic and technological improvements, intensive growth rate can be associated with many pathological conditions, such as increased susceptibility to infections. Intestinal inflammation in poultry industries detrimentally affects productivity by hindering nutrient absorption and the efficient allocation of nutrients for growth. The host releases different biomarkers in response to inflammation. Hence, there is an utmost interest of reliable, precise, sensitive and robust biomarkers to evaluate both the gastrointestinal health status and inflammation in poultry. The aim of this study was to determine how the developed feed prototype (allithiamine) affects the community diversity in raised duck, and the relationship between gut microbiome composition and inflammatory factor as calprotectin, using targeted 16S rRNA gene amplicon sequencing and Chicken Calprotectin ELISA Kit.

Low-cost conventional PCR techniques enable simultaneous detection of bacterial sexually transmitted infections with enhanced sensitivity and specificity

PurposeNeisseria gonorrhoeae (NG), Chlamydia trachomatis (CT) and Mycoplasma hominis (MH), the three most common treatable bacterial sexually transmitted infections (STIs) worldwide can lead to many complications if remain untreated. Screening of high-risk population with highly sensitive methods will lead to significant improvement in patient outcomes and will prevent downward transmission. The advantages of Polymerase chain reaction (PCR) based assay are not only high sensitivity and specificity, but also detection of multiple organisms in a single reaction which reduce the result turn-around time. The aim of the present study was to evaluate the feasibility of a multiplex PCR assay method targeting 16S rRNA gene for simultaneous detection of NG, CT and MH infection along with their trend and occurrence among high-risk population in Assam, Northeast India. MethodsA cross-sectional study was undertaken, where a total of 200 randomly selected patients from high-risk population were included. After validation of singleplex PCR, Multiplex PCR (M-PCR) was performed along with the traditional culture method for NG. Results & conclusionThe overall agreement of M-PCR with singleplex PCR was very high (100%). The occurrence of STI was found to be very high (101/200; 50.5%). Furthermore, co-infection was detected in 10/200; 5%) individuals. Infection was more common among young individuals (p < 0.05) and males out-numbered females (p < 0.05). The most common organism detected was CT (42/200; 21%) followed by NG (41/200; 20.5%) and MH (20/200; 10%). The M-PCR assay workflow is simple, cost effective and can be used in routine diagnostic laboratories with basic molecular facilities.

Heterosis of endophytic microbiomes in hybrid rice varieties improves seed germination.

Seed endophytic microbiomes are shaped by host and environmental factors and play a crucial role in their host growth and health. Studies have demonstrated that host genotype, including hybridization, affects seed microbiomes. Heterosis features are also observed in root-associated microbiomes. It remains unclear, however, whether heterosis exists in seed endophytic microbiomes and whether hybrid microbiota provide noticeable advantages to host plant growth, especially to seed germination. Here, we investigated the structure of seed endophytic bacterial and fungal communities from three hybrid rice varieties and their respective parents using amplicon sequencing targeting 16S rRNA and ITS2 genes. Heterosis was found in diversity and composition of seed endophytic microbiomes in hybrids, which hosted more diverse communities and significantly higher abundances of plant growth-promoting taxa, such as Pseudomonas and Rhizobium genera compared with their parental lines. Co-occurrence network analysis revealed that there are potentially tighter microbial interactions in the hybrid seeds compared with their parent seeds. Finally, inoculation of seed-cultivable endophytes, isolated from hybrids, resulted in a greater promotion of seed germination compared with those isolated from parent lines. These findings suggest that heterosis exists not only in plant traits but also in seed endophytic microbiota, the latter in turn promotes seed germination, which offers valuable guidance for microbiome-assisted rice breeding.IMPORTANCEGenetic and physiological changes associated with plant hybridization have been studied for many crop species. Still, little is known about the impact of hybridization on the seed microbiota. In this study, we indicate that hybridization has a significant impact on the endophytic bacterial and fungal communities in rice seeds. The seed endophytic microbiomes of hybrids displayed distinct characteristics from those of their parental lines and exhibited potential heterosis features. Furthermore, the inoculation of seed-cultivable endophytes isolated from hybrids exhibited a greater promotion effect on seed germination compared with those isolated from the parents. Our findings make a valuable contribution to the emerging field of microbiome-assisted plant breeding, highlighting the potential for a targeted approach that aims to achieve not only desired plant traits but also plant-beneficial microbial communities on the seeds.

Clinico-diagnostic and Therapeutic Management of Ehrlichiosis in Dog

Background: The research work was undertaken to investigate the efficacy of various diagnostic tests by using conventional microscopic examination, rapid Ab detection test and molecular technique, haemato-biochemical changes, Abdominal organs changes using ultrasonography, and therapeutic efficacy of different treatment protocols on Canine Monocytic Ehrlichiosis (CME) infected dogs from in and around Navsari, Gujarat, India. Methods: 69 dogs suspected of ehrlichiosis based on clinical signs and hematological values and then subjected to blood and buffy coat smear examination, rapid Ab detection test and PCR based assay targeting 16S rRNA gene. The pre and post-treatment haematology and biochemical analysis of treatment groups along with all the positive dogs were performed. Further, all the treatment group dogs were subjected to ultrasound for evaluating organ abnormalities. Modified Clinical Sign Grading (MCSG) score for CME-infected dogs was carried out to evaluate therapeutic responses based on minimizing the clinical score. Descriptive statistics along with Student t-test and One-way analysis of variance (ANOVA) followed by DMRT was performed to compare differences between means wherever applicable. Result: CME incidence among the 69 suspected cases was 49.28%. Out of 69 suspected dogs, Ehrlichial inclusions or morulae in the monocytes could be identified in only 4.35% of blood smears and 10.14% of buffy coat smears examined by Giemsa and or Field-stained and 15.94% of dogs were found positive for Ehrlichia genus by PCR. 72.5 per cent of dogs were found positive for antibodies to E. canis, out of 40 dogs. The sensitivity of blood smear examination, buffy coat smear examination, and thrombocytopenia was 10.34%, 24.14% and 89.66%, respectively. The specificity of all the diagnostic tests was 100%. The anemia along with leucopenia, were the most significant hematological alteration recorded while ALP, creatinine, and BUN were elevated and albumin was significantly decreased in CME-infected dogs. Significant improvement of haemato-biochemical parameters towards normalcy in both treatment groups. However, a higher percentage of reduction was observed in the clinical score of imidocarb treated CME affected dogs than in the oxy-doxycycline therapy after 14 days.

Surveillance and screening of Stomoxyinae flies from Mallorca Island (Spain) reveal the absence of selected pathogens but confirm the presence of the endosymbiotic bacterium Wolbachia pipientis

Adult brachycera biting flies can significantly impact livestock through both direct effects (reduction of food intake, disturbance, painful bites, and blood loss) and indirect effects (pathogen transmission), leading to substantial economic losses and production damage. This study aimed to assess the presence of blood-sucking flies in six mixed-animal farm environments on the island of Mallorca (Balearic Islands, Spain) by employing multiple trapping methods. Additionally, distribution maps of brachycera biting fly species recorded in Spain were created, based on data extracted thorough review of scientific literature and citizen digital databases. Investigation of several pathogens, including equine infectious anemia virus (EIAV), Anaplasmataceae bacteria, and piroplasm protozoa, was carried out using different PCR targets (18S rRNA, 16S rRNA, groESL, and tat genes). Citizen science databases and literature review corroborated the consistent distribution trend for two Stomoxyinae species, underscoring the importance of citizen collaboration as a complement to traditional entomological surveillance. Our study confirmed the presence of two biting Stomoxyinae species: the prevalent stable fly Stomoxys calcitrans across all sampled farms, and the horn fly Haematobia irritans, which turned out to be less abundant. DNA barcoding techniques validated the identification of the two species. Neither EIAV nor bacterial/protozoan pathogens were detected using the selected PCR targets in either fly species. However, Wolbachia pipientis (clustered in the supergroup A together with the only sequence of W. pipientis from the USA) was identified through PCR targeting 16S rRNA, groESL and wsp genes in all pools of H. irritans (n = 13) collected from two of the examined farms. This study represents the first attempt to investigate pathogens in Stomoxyinae biting flies in Spain. The discovery of the endosymbiotic Wolbachia organism in H. irritans represents the first record in Spain and the second from Europe. This finding holds significant implications for future research on the applications of this bacterium in biocontrol programs.

Characterization of the Effects of a Novel Probiotic on Salmonella Colonization of a Piglet-Derived Intestinal Microbiota Using Improved Bioreactor.

The carriage of Salmonella in pigs is a major concern for the agri-food industry and for global healthcare systems. Humans could develop salmonellosis when consuming contaminated pig products. On the other hand, some Salmonella serotypes could cause disease in swine, leading to economic losses on farms. The purpose of the present study was to characterize the anti-Salmonella activity of a novel Bacillus-based probiotic using a bioreactor containing a piglet-derived intestinal microbiota. Two methods of probiotic administration were tested: a single daily and a continuous dose. Salmonella enumeration was performed using selective agar at T24h, T48h, T72h, T96h and T120h. The DNA was extracted from bioreactor samples to perform microbiome profiling by targeted 16S rRNA gene sequencing on Illumina Miseq. The quantification of short-chain fatty acids (SCFAs) was also assessed at T120h. The probiotic decreased Salmonella counts at T96 for the daily dose and at T120 for the continuous one. Both probiotic doses affected the alpha and beta diversity of the piglet-derived microbiota (p < 0.05). A decrease in acetate concentration and an increase in propionate proportion were observed in the continuous condition. In conclusion, the tested Bacillus-based product showed a potential to modulate microbiota and reduce Salmonella colonization in a piglet-derived intestinal microbiota and could therefore be used in vivo.

Genotyping of Methicillin Resistant Staphylococcus aureus Clinical Isolates in Sulaimani City using Pulsed -Field Gel Electrophoresis

Methicillin-resistant Staphylococcus aureus (MRSA) strains cause serious nosocomial infections. The objective of this study was to detect and find the epidemiological relatedness among MRSA isolates which was collected in Emergency burn-and-plastic surgery hospital in Sulaimani city using Pulsed-filed gel electrophoresis (PFGE). Routine methods were used for detection of Staphylococcus aureus, further molecular confirmation was done by PCR assays targeting 16S rRNA gene. The cefoxitin-disc diffusion assay was used for detection MRSA, which is subsequently confirmed by the presence of the mecA gene. PFGE was chosen to observe the genetic relatedness of 10 MRSA strains using SmaI restriction endonuclease. All the isolates (100%, 50/50) carried 16S rRNA gene specific for S.aureus and (42%, 21/50) exhibited resistance to cefoxitin and harboured the mecA gene, thereby being classified as MRSA. Among 10 MRSA isolates, the PFGE pattern were grouped into four clusters. Three isolates belonging to cluster (C3) were indistinguishable, 4 isolates belonging to cluster (C4) were closely related, 2 isolates of the second cluster (C2) were possibly related and the first cluster (C1) with 1 MRSA isolate was unrelated. This finding shows 30% of the samples had indistinguishable fingerprints with no changes in banding profiles, while 40% of the samples had closely related patterns. PFGE is a good discriminatory tool for typing MRSA strains.

Phylogenetic analysis of Klebsiella pneumoniae isolates of respiratory tract infections in humans and sheep

Background: Klebsiella pneumoniae is an important opportunistic pathogen, which is capable to colonizing the respiratory system in both human and animals causing mild to severe infections. Aim: This study aims to isolate K. pneumoniae from the nasal discharges of human and sheep as well as identifying the antibiotic resistance and molecular phylogeny of local isolates. Methods: A total of 100; 50 humans and 50 sheep, positive nasal swab isolates were selected, confirmed biochemically and by the VITEK-2 system. Molecular testing using the polymerase chain reaction (PCR) and phylogeny was conducted. Results: On MacConkey agar, Klebsiella isolates were appeared as large, pinkish, and mucoid colonies; while microscopically, it appeared as Gram-negative rods. Traditional biochemical tests revealed that 62% and 78% of human and sheep isolates were positive Klebsiella isolates; whereas respectively, 54.84% and 71.8% of these isolates were positive by VITEK-2. Antibiotic susceptibility test showed that the human isolates were sensitive to aztreonam, piperacillin-tazobactam, ciprofloxacin and cefuroxime. Subsequently, sheep isolates were sensitive to cefuroxime, ciprofloxacin, piperacillin-tazobactam, ampicillin, cefoxitin and tetracycline. Targeting 16S rRNA gene, a total of 17 human and 28 sheep isolates were molecularly positive K. pneumoniae. Phylogenetic analysis of study human and sheep isolates showed its identity to NCBI Indian (LC747146.1) and Iraqi (LC711141.1) isolates, respectively. Comparative analysis between the local human and sheep isolates revealed a significant identity that ranged from 99.82% to 99.88% with a percentage of mutation ranged from 0.008% to 0.002%. Conclusion: Klebsiella pneumoniae is highly prevalent bacterium in both human and sheep with an observable resistance to antibiotics. Molecular phylogeny of study isolates demonstrated their closely relation, suggesting the possible direct or indirect transmission of the bacterium from sheep to human or vice versa. Moreover studies are greatly important to estimate the routes of bacterial transmission. Also, extensive hygiene practices could be lowered the spreading of K. pnuemoniae to farm workers.

Non-Tuberculous Mycobacteria Infections based on 16S rRNA detections in patients with Suspected Chronic Tuberculosis at a Tertiary Referral Hospital in Indonesia

This study identified non-tuberculous mycobacteria (NTM) isolates from the clinical respiratory specimen of patients suspected of chronic tuberculosis at a tertiary referral hospital in Indonesia. In this study, 20 clinical non-tuberculous mycobacteria (NTM) isolates were obtained from respiratory samples examined in the tuberculosis section of the Clinical Microbiology laboratory at Dr Soetomo General Hospital. The isolates were identified using TB Ag MPT 64 (SD Bioline) and targeting 16S rRNA genes sequence for analysis at the species level. Data on clinical features were collected and chest radiographs were evaluated. The highest bacterium found in the isolates was Mycobacterium kansasii (12 strains, 60%.), followed by Mycobacterium gordonae (2 strains, 5%), Mycobacterium parascofulaceum (2 strains, 5%), Mycobacterium avium (1 strain, 2.5%), Mycobacterium simiae (1 strain, 2.5%). Mycobacterium abcessus (1 strain, 2.5%), and Mycobacterium paraterrae (1 strain, 2.5%). Males dominated patients with Mycobacterium kansasii. Clinical symptoms include cough, dyspneu, hemoptysis, chest pain, and sweats. Half of the patients had a history of previous tuberculosis and other comorbid diabetes mellitus. Non-tuberculous mycobacteria (NTM) species identified were dominated by Mycobacterium kansasii. The predominance of non-tuberculous mycobacteria (NTM) species from clinical respiratory can be a reference to determine prudent treatment for patients in high-burden TB countries.

Genomic characterization of Mycobacterium lepromatosis from ENL patients from India

BackgroundLeprosy is caused by Mycobacterium leprae and Mycobacterium lepromatosis. Both organisms cannot be cultured in vitro. M. lepromatosis was found to be associated mainly with diffuse lepromatous leprosy and with Lucio's phenomena initially. Later, M. lepromatosis was observed in borderline leprosy cases (BL), lepromatous leprosy cases (LL) and leprosy reactional cases (T1R and ENL). Although many cases are being reported with similar clinical features like Lucio phenomenon in India but M. lepromatosis was not isolated from these cases. The aim of this study was to screen MB patients and patients with type 2 reaction for the presence of M. lepromatosis. MethodologyWe recruited a total of 75 multibacillary leprosy cases (45 MB cases without reaction and 30 type 2 reaction (ENL) cases) from TLM hospitals Purulia (West Bengal), Barabanki (Uttar Pradesh), Shahdara (Delhi) and PGIMER (Chandigarh), India. Punch biopsies of 5 mm were collected in 70% ethanol from all the study subjects. DNA was extracted followed by Hemi-nested PCR targeting 16S rRNA gene specific for M. lepromatosis. Further, PCR products were processed for Sanger sequencing for an absolute confirmation of M. lepromatosis. Whole genome sequencing was done to confirm the presence of M. lepromatosis. ResultWe observed presence of M. lepromatosis in 4 necrotic ENL patients by heminested PCR. There was 100% 16S rRNA sequence similarity with M. lepromatosis FJ924 in one case, 98.96% in two cases and in one case it was 90.9% similarity by nucleotide BLAST (BLASTn) by using the NCBI website. On the basis of Sanger sequencing, we noted presence of M. lepromatosis in 3 necrotic ENL patients as one sample only gave 90.9% similarity by BLASTn. On the basis of de novo assembly and genome obtained, only one sample S4 with a 2.9 mb genome size was qualified for downstream analysis. Sixteen M. lepromatosis- specific proteins were identified in this case and the closest species was M. lepromatosis strain FJ924 based on whole genome level phylogeny. ConclusionThese results provide valuable insights into the prevalence of M. lepromatosis in ENL patients in different regions of India and contribute to our understanding of the genetic characteristics of this pathogen in the context of leprosy.

Genetic Characterization of Hyalomma Anatolicum (Ixodoidea: Ixodidae) in Babylon Province Middle Iraq

This study was included molecular study of Hyalomma anatolicum (Ixodoidea: Ixodidae) based on 16S rRNA gene collected from three regions' in Babylon province. The results of gel electrophoresis image (1.5 % agarose) shows the amplicons of a partial region within large subunit RNA gene (size= 450 bp). The sequencing data has reported species of H. anatolicum 16S rRNA gene (OQ162293) to OQ162298 these species revealed close matching on the phylogenetic tree to an isolate of H. anatolicum for different countries. This study represents first in Babylon Governorate to diagnose H. anatolicum by targeting 16S rRNA gene.

Molecular and serological detection of Anaplasma spp. in small ruminants in an area of Cerrado Biome in northeastern Brazil

Anaplasmosis, caused by bacteria of the genus Anaplasma, is an important tick-borne disease that causes economic losses to livestock farms in many countries. Even though Anaplasma spp. have been detected in goats and sheep worldwide, few studies investigate the occurrence and genetic identity of these agents in small ruminants from Brazil. Thus, this work aimed to detect and determine the genetic identity of Anaplasma spp. in small ruminants from the Baixo Parnaíba region, state of Maranhão, northeastern Brazil. For this purpose, blood samples were collected from 161 animals (91 goats; 70 sheep) from 4 municipalities in the Baixo Parnaíba region. Sheep and goat serum samples were subjected to recombinant membrane surface protein (MSP5)-based iELISA. Whole blood samples were subject to DNA extraction and molecular diagnosis using PCR assays for Anaplasma spp. targeting msp1β, msp1α, 16S rRNA and msp4 genes. Positive samples were sequenced and then subjected to Anaplasma marginale msp1α genetic diversity analysis and phylogenetic inferences based on the 16S rRNA and msp4 genes. The serological survey detected the presence of anti-A. marginale IgG antibodies in 18 animals (11.1%): 2.9% (2/70) sheep and 17.4% (16/91) goats. Anaplasma marginale DNA was detected in 2 goats (1.2%) using qPCR based on the msp1β gene. Two distinct A. marginale msp1α strains, namely α β and α β ΓγΓγΓγΓγ were found in the infected goats, each one found in a different animal, both belonging to the H genotype. Phylogenetic analysis based on the 16S rRNA gene showed the sequences positioned in three different clades and grouped with sequences from ‘Candidatus Anaplasma boleense’, A. platys and A. marginale. Phylogenetic inferences based on the msp4 gene positioned the sequence variants in the A. marginale clade. The present work represents the first molecular detection of sequence variants phylogenetic associated to ‘Candidatus Anaplasma boleense’ and A. platys and α β and α β ΓγΓγΓγΓγ in goats from Brazil.