Bronchopneumonia in sheep associated with Providencia stuartii

Bacteria of the genus Providencia are opportunistic pathogens in humans, widely distributed in the environment and associated with greater resistance to antibiotics, and this being uncommon the association with clinical diseases. This study reports the isolation of P. stuartii in two sheep that presented clinical signs of pneumonia. At necropsy there was severe and acute fibrinopurulent bronchopneumonia. Histologically, there were infiltrated neutrophils and fibrin in the alveolar lumen, and the alveolar septa presented multifocal thickening with moderate proliferation of pneumocytes and mononuclear interstitial infiltrate. Providencia sp. was isolated in the microbiological tests of the lung and tracheal secretions. The isolate was subjected to DNA extraction, polymerase chain reaction (PCR) for the 16SrRNA gene and sequencing of genomic DNA, which demonstrated 100% homology with P. stuartii. This is the first report of the presence of this microorganism as a cause of interstitial and fibrinopurulent bronchopneumonia in sheep. Therefore, it is suggested that epidemiological surveillance strategies should be carried out in animals to better understand their role in the dissemination of this pathogen.

Genome analysis of Lysinibacillus sphaericus isolate 6.2 pathogenic to Culex quinquefasciatus Say, 1823 (Diptera: Culicidae)

Viersanova A, Purwanto H. 2021. Genome analysis of Lysinibacillus sphaericus isolate 6.2 pathogenic to Culex quinquefasciatus Say, 1823 (Diptera: Culicidae). Biodiversitas 22: 5211-5222. Lysinibacillus sphaericus is an entomopathogenic bacteria that is specific to vector mosquitoes, especially Culex spp., and Anopheles spp., so it has been widely used as a bioinsecticide. L. sphaericus has a wide variation of toxicity efficiencies, which have led to continuous exploration of new isolates with higher toxicity and a new toxin to deal with resistance problems. This study aimed to identify the genomic characteristics and toxin characteristics of isolate 6.2 based on whole genome analysis and analyze the identification of isolate 6.2. Isolate 6.2 was previously obtained from rhizosphere in Yogyakarta. To analyze the genome and toxins, the NGS technique was used and then the analysis was carried out using a couple of freely available bioinformatics tools. Molecular identification was carried out with the 16SrRNA gene and the relationship was analyzed by reconstructing the phylogenetic tree using Neighbours-Joining. The genomic analysis of isolate 6.2 showed good results with G+C content and genome size that matched the reference genome of L. sphaericus. The result of the 16SrRNA gene blasting showed that the closest related gene of isolate 6.2 is L. fusiformis (NR_042072.1). However, the reconstructed phylogenetic tree did not show the formation of clusters according to the species. Toxin analysis indicates that isolate 6.2 has Mtx, s-layer protein, hemolysin, and chitin-binding protein genes. All of which are known to be associated with the toxicity of L. sphaericus to binary toxin resistant population of Culex quinquefasciatus.

Heavy metal (As, Cd, Cr, Hg, Pb) analysis and identification of heavy metal resistant bacteria in sediments from Manado Bay

Environmental pollution from heavy metals is becoming a growing concern due to the adverse effects it is causing throughout the world. This study aims to analyze heavy metal concentrations and identify heavy metal resistant bacteria in the bay of Manado. Sediment samples were collected from five bays in Manado. The concentrations of heavy metals As, Cd, Cr, Hg and Pb were analyzed using ICP-OES, and Hg using CV-AFS. Bacteria from the sediment were grown in nutrient broth media containing heavy metals As, Cd, Cr, Hg and Pb respectively. Microbiology and 16SrRNA gene analysis were used to identify the bacteria that grown on media containing varying concentrations of heavy metals. The results showed that the sediments from the five bays in Manado contained heavy metals with an average concentration of As <1mg/kg, Cd 1.8mg/kg), Cr 6.2mg/kg, Hg <0.07mg/kg). and Pb 11.2mg/kg. The results of microbiological and molecular analysis showed that 5 species of heavy metal resistant bacteria were Pseudomonas aeruginosa, Bacillus cereus, Staphylococcus arlettae, Acinobacter sp., and Brevibacterium sp. The five bacteria found to be resistant to heavy metals can be used to detoxify As, Cd, Cr, Hg, and Pb.

Intestinal Dominance by Serratia marcescens and Serratia ureilytica among Neonates in the Setting of an Outbreak

(1) Background: We determined the relevance of intestinal dominance by Serratia spp. during a neonatal outbreak over 13 weeks. (2) Methods: Rectal swabs (n = 110) were obtained from 42 neonates. Serratia spp. was cultured from swabs obtained from 13 neonates (Group 1), while the other 29 neonates were culture-negative (Group 2). Total DNA was extracted from rectal swabs, and quantitative PCRs (qPCRs) using Serratia- and 16SrRNA-gene-specific primers were performed. relative intestinal loads (RLs) were determined using ΔΔCt. Clonality was investigated by random amplified polymorphic DNA analysis and whole-genome sequencing. (3) Results: The outbreak was caused by Serratia marcescens during the first eight weeks and Serratia ureilytica during the remaining five weeks. Serratia spp. were detected by qPCR in all Group 1 neonates and eleven Group 2 neonates. RLs of Serratia spp. were higher in Group 1 as compared to Group 2 (6.31% vs. 0.09%, p < 0.05) and in the first swab compared to the last (26.9% vs. 4.37%, p < 0.05). Nine neonates had extraintestinal detection of Serratia spp.; eight of them were infected. RLs of the patients with extraintestinal spread were higher than the rest (2.79% vs. 0.29%, p < 0.05). (4) Conclusions: Intestinal dominance by Serratia spp. plays a role in outbreaks and extraintestinal spread.

Efflux Might Participate in Decreased Susceptibility to Oxytetracycline in Contagious Agalactia-Causative Mycoplasma spp.

Contagious agalactia is associated with mastitis, keratoconjunctivitis, arthritis, pneumonia, and septicemia in small ruminants in countries with large dairy industries worldwide. The causative agents belong to four (sub)species of the Mycoplasma genus that have remained essentially susceptible to antimicrobials, including to the widely-used tetracycline family. However, some clinical isolates have been detected that show increased minimum inhibitory concentrations of tetracyclines, although they do not harbor the mutation in the 16SrRNA gene usually associated with resistance. The present work aimed to assess whether efflux pumps, infrequently described in mycoplasmas, could participate in the observed moderate loss of susceptibility. General efflux mechanisms were measured (i) using the fluorescence property of ethidium bromide when accumulated intracellularly and intercalated in the mycoplasma genomes, its active extrusion resulting in a temperature-dependent decrease in fluorescence and (ii) monitoring the growth inhibition of mycoplasmas by subinhibitory concentrations of tetracycline with or without reserpine, a known inhibitor of efflux in other bacteria. Both methods revealed non-specific efflux phenomena in most of the isolates tested, although their efficacy was difficult to quantify. This property could contribute to the acquisition of mutations conferring resistance by maintaining intracellular concentrations of tetracyclines at subinhibitory levels.

Correlation of Streptococcus Mutans by 16SrRNA Gene Sequencing in Children with Different Caries Experience- A PCR Based Study

Background and Objectives: Early Childhood Caries is considered as a common chronic disease in children. Since some research had concluded that children with higher levels of Streptococcus mutans are associated with a higher incidence of decayed, missing, and filled teeth, the present study was undertaken to analyze the salivary Streptococcus mutans of children with different caries status using species specific 16S rRNA gene sequencing technology to evaluate the quantity of Streptococcus mutans with respect to different caries status. Materials and Methods: Children between 3-6 years were selected and divided into 3 groups, Group I- Caries free, Group II-Early Childhood caries and Group III- Severe Early childhood caries. The caries status was assessed using dmfs and the severity of caries was assessed using pufa index. Salivary samples were collected to isolate DNA and Real time PCR was done with 16SrRNA primer specific to Streptococcus mutans to estimate the quantity of Streptococcus mutans in children with different caries status. Results: There was a significant difference in the mean CT values among the study groups (p < 0.001). Post hoc Tukey test revealed that, Group I had a significantly higher mean CT values than that of Groups II and III. Pearson's correlation analysis was carried out to correlate dmfs value with CT values and to correlate the pufa value with the fold change. There was a negative correlation between dmfs score and CT values and positive correlation between pufa score and fold change. Conclusion: There was a higher expression of 16SrRNA gene in Severe Early childhood caries group, followed by Early childhood caries and caries free group, indicating a high level of Streptococcus mutans in Severe Early childhood caries group and there was a negative correlation between dmfs score and CT values and a positive correlation between pufa score and fold change.

Molecular techniques for the assessment of Cr (VI) reduction by Bacillus thuringiensis

Effluent pollution with Cr (VI) is a worldwide environmental problem. In the Pasto River (southeastern, Colombia), previous studies reported contamination with this metal at points near tanneries. To establish the role of Bacillus thuringiensis in Cr (VI) reduction in water from Pasto River, experiments were carried out with untreated Pasto River water (treatment 1), sterile Pasto River water inoculated with B. thuringiensis (treatment 2), and unsterilized Pasto River water inoculated with B. thuringiensis (treatment 3). All experiments were conducted in bioreactors with a controlled temperature of 20 °C and constant agitation for 156 h. Samples of 20 mL were taken every 12 h from each treatment to track Cr (VI) reduction levels and to confirm microorganism identity via molecular methods involving denaturing gradient gel electrophoresis (DGGE), restriction enzyme digestion profiles (RFLP), and bioinformatic analysis. Cr (VI) reduction was higher in treatment 3 (99:42 %) as opposed to treatment 2 (76:12 %) and treatment 1 (74:46 %). The molecular identity of B. thuringiensis was determined via sequencing of the 16SrRNA gene, and RFLP assessments in all three treatments revealed B. thuringiensis profiles. Since B. thuringiensis was present in all three treatments trough time, Cr (VI) reduction can be attributed to this bacterium.

Molecular detection of Theileria species, Anaplasma species, Candidatus Mycoplasma haemobos, Trypanosoma evansi and first evidence of Theileria sinensis-associated bovine anaemia in crossbred Kedah-Kelantan x Brahman cattle

Background Serious disease outbreaks in cattle are usually associated with blood pathogens. This study aims to detect blood pathogens namely Theileria species, Anaplasma species, Candidatus Mycoplasma haemobos and Trypanosoma evansi, and determine their phylogenetic relationships and haemato-biochemical abnormalities in naturally infected cattle. Methods Molecular analysis was achieved by PCR amplification and sequencing of PCR amplicons of 18SrRNA gene of Theileria species, 16SrRNA genes of Anaplasma and Mycoplasma species, MPSP genes of T. orientalis and T. sinensis, MSP4 gene of A. marginale, 16SrRNA gene of Candidatus Mycoplasma haemobos, and RoTat1.2 VSG gene of Trypanosoma evansi, in sixty-one (61) clinically ill Kedah-Kelantan x Brahman cattle in Pahang, Malaysia. Results A total of 44 (72.13%) cattle were infected with more than one blood pathogen. Theileria species was the blood pathogen with the highest molecular detection rate (72.13, 95% CI 59.83–81.81%). Nucleotide blast analyses of all sequences demonstrated high degree of molecular similarity (98–100%) in comparison with their respective reference sequences. Analysis of 18SrRNA gene sequences of Theileria species and 16SrRNA gene sequences of Anaplasma species revealed Theileria sinensis and Anaplasma platys respectively as additional species detected in these cattle. MPSP-PCR analysis was conducted for further confirmation of T. sinensis. The blood picture of eight infected cattle groups revealed poikilocytosis, anisocytosis, rouleaux formation and degenerative left shift. High mean erythrocyte fragility values were common in infected cattle groups. Anaemia of the macrocytic normochromic type and spherocytes were observed in the T. evansi and Anaplasma platys + Theileria sinensis double species co-infected cattle group. Normocytic normochromic anaemia was observed in the T. sinensis infected cattle group. Significant (p < 0.05) increases in serum liver and kidney parameters, total protein, globulin, total and unconjugated bilirubin and decreased albumin values were observed in the T. evansi infected cattle when compared to clinically healthy cattle. Conclusion We present the first evidence of Theileria sinensis-associated bovine anaemia (TSABA) in Malaysian cattle. Because of the high occurrence of bovine theileriosis and detection of A. platys, there is an urgent need for appropriate preventive and control measures against these blood pathogens.

P677 Fungal abundance is related to inflammatory status in Ulcerative Colitis

Background Fungi account for approximately 0.1% of the total microorganisms in the gut. Despite the vast body of literature on the bacterial component of the gut microbiota, little has been published on the fungal microbiota. Fungal-bacterial interactions may be significant in inflammatory bowel disease (IBD), with several lines of evidence linking fungi and IBD. Our study aimed to explore and compare the fungal and bacterial loads in fecal samples from different ulcerative colitis (UC) patients’ groups. Methods Using two qPCR systems to amplify the ITS2 sequence from fungi and the 16srRNA gene from bacteria, we characterized and compared fungal and bacterial loads in 3 groups: 1) UClr: UC patients in long-term remission (≥5 years of flare-free disease, clinical, endoscopic and histological remission at inclusion); 2) UCsr: UC patients in short-term remission (3 months in clinical remission at inclusion and previously more than one relapse/year); 3) UCfl: UC patients with active disease (CAI &gt;4 at inclusion). We obtained two frozen fecal samples from all subjects, except for the UCfl group from which we obtained 1 sample at flare onset. Results were expressed in copies/g of feces. Results We included 87 UC patients, 29 in UClr, 20 in UCsr, and 38 in UCfl groups. Median age was 39 years, women comprised 52%. Fecal samples contained a significantly lower number of ITS2 gene copies (median 9.27E+05) than 16srRNA gene copies (median 4.28E+11). 16s rRNA gene copies were similar among the different groups with a median 3.88E+11 for UClr, median 5.29E+11 for UCsr, and median 4.315E+11 for UCfl patients (FDR p=0.65). In contrast, copies of ITS2 gene were increased in UCfl (median 1.95E+06) when compared to UClr (3.68E+04, FDR p=0.0036) but not significantly different to UCsr (5.24E+05 copies, FDR p=0.20). We analyzed the variation between samples over time; we compared the number of copies of ITS2 and 16s rRNA genes in fecal samples collected at two different time points: basal versus 8-weeks. We calculated overall fold changes that reflect stability over time. We found a trend for a more significant variation in the quantity of the ITS2 gene than the 16srRNA gene, but this was not significant. Analysis of the ITS2/16S rRNA ratio assessing the frequency of fungi compared to bacteria showed that this ratio was higher in UCfl (median 6.94E-06) when compared to UClr (median 2.63E-07, FDR p=0.0005) and UCsr patients (median 5.60E-07, FDR p=0.0072), there were no differences between UClr and UCsr patients. Conclusion Fungal abundance was increased in UC flare patients compared to UC patients in long remission, while there were no differences in bacterial abundance. This high number of fungi could be involved in the inflammatory response of UC patients.

Genetic Diversity of Staphylococcus Saprophyticus Strains Causing Urinary Tract Infections in Lagos Metropolis

ObjectiveStaphylococcus saprophyticus is a Gram-positive bacterium implicated in urinary tract infections in sexually active women along with other bacteria. There is a special need to accurately isolate and identify this organism in clinical specimens in order to prevent misidentification in the laboratory. The present study was carried out to evaluate the genetic diversity of one hundred different Staphylococcus saprophyticus strains isolated from women presenting with urinary tract infections reporting in four government hospitals in Lagos Metropolis using 16SrRNA gene sequence analysis. ResultsThe PCR-amplified and sequenced 16SrRNA gene of 100 isolates and confirmed 88 strains of S. saprophyticus to subspecies level, while 8 belong to other genera and 4 could not be ascertained due to low significant similarity. Phylogenetic analysis showed the interrelationship between the isolates from different hospitals. The results showed the distribution of the isolates into two broad clusters and twenty-two sub-clusters. The results revealed the genetic diversity of Staphylococcus saprophyticus isolated from women with urinary tract infections in Lagos Metropolis using 16SrRNA gene sequence.