Whole-Genome Sequencing to Predict Antimicrobial Susceptibility Profiles in Neisseria gonorrhoeae.

BACKGROUND: Neisseria gonorrhoeae is a major public health problem due to increasing incidence and antimicrobial resistance. Genetic markers of reduced susceptibility have been identified; the extent to which those are representative of global antimicrobial resistance is unknown. We evaluated the performance of whole-genome sequencing (WGS) used to predict susceptibility to ciprofloxacin and other antimicrobials using a global collection of N. gonorrhoeae isolates. METHODS: Susceptibility testing of common antimicrobials and the recently developed zolifodacin was performed using agar dilution to determine minimum inhibitory concentrations (MICs). We identified resistance alleles at loci known to contribute to antimicrobial resistance in N. gonorrhoeae from WGS data. We tested the ability of each locus to predict antimicrobial susceptibility. RESULTS: A total of 481 N. gonorrhoeae isolates, collected between 2004 and 2019 and making up 457 unique genomes, were sourced from 5 countries. All isolates with demonstrated susceptibility to ciprofloxacin (MIC ≤0.06 µg/mL) had a wild-type gyrA codon 91. Multilocus approaches were needed to predict susceptibility to other antimicrobials. All isolates were susceptible to zoliflodacin, defined by an MIC ≤0.25 µg/mL. CONCLUSIONS: Single marker prediction can be used to inform ciprofloxacin treatment of N. gonorrhoeae infection. A combination of molecular markers may be needed to determine susceptibility for other antimicrobials.

High Prevalence of Macrolide and Fluoroquinolone Resistance-Mediating Mutations in Mycoplasma genitalium-Positive Urine Specimens From Saskatchewan.

BACKGROUND: Mycoplasma genitalium is an emerging, sexually transmitted infection, which is more prevalent than Chlamydia trachomatis in some regions. An increase in antibiotic resistance, that is, azithromycin and moxifloxacin, recommended for treating M. genitalium infections has been noted. This is the first detailed report on the prevalence of M. genitalium and its antimicrobial resistance in Saskatchewan, Canada. METHODS: Aptima urine specimens (n = 1977), collected for the diagnosis of C. trachomatis/Neisseria gonorrhoeae, were tested for M. genitalium using the Aptima M. genitalium assay (MG-TMA). Antimicrobial resistance was ascertained using polymerase chain reaction and DNA sequencing of 23S rRNA (azithromycin) and parC (moxifloxacin) from Aptima M. genitalium assay-positive specimens; mutations predictive of resistance were noted. RESULTS: The prevalence of M. genitalium was 9.6% (189/1977). Predicted resistance to azithromycin (substitutions at positions 2058/2059 in 23S rRNA) was observed in 63.6% (70/110) of the specimens tested, whereas resistance to moxifloxacin (S83I in ParC) was observed in 10.6% (9/85) of the specimens. Mutations in both 23S rRNA and ParC were observed in 2.12% (4/189) of the specimens. Women aged 20 to 24 years had the highest prevalence (18.3%, P < 0.001), and in females, M. genitalium was significantly associated with C. trachomatis or N. gonorrhoeae/C. trachomatis (P < 0.001) coinfection. The prevalence of M. genitalium (9.6%) in the province of Saskatchewan was higher than that of the other 2 bacterial sexually transmitted infections (N. gonorrhoeae (3.09%) and C. trachomatis (6.85%). CONCLUSIONS: The prevalence of M. genitalium (9.6%) and associated resistance to azithromycin (63.6%) in Saskatchewan high, suggesting that empiric azithromycin therapy may not be adequate for treating M. genitalium infections.

Genomic Analysis Reveals Antibiotic-Susceptible Clones and Emerging Resistance in Neisseria gonorrhoeae in Saskatchewan, Canada.

Whole-genome sequencing was used to identify mutations in antibiotic resistance-conferring genes to compare susceptibility predictions with MICs and to ascertain strain types in 99 isolates of Neisseria gonorrhoeae Genotypes associated with susceptibility, as well as MIC creep or emerging resistance, were noted. Phylogenomic analysis revealed three distinctive clades and putative gonococcal transmission linkages involving a tetracycline-resistant N. gonorrhoeae outbreak and the clonal spread of susceptible isolates in men.

Functional gene profiling through metaRNAseq approach reveals diet-dependent variation in rumen microbiota of buffalo (Bubalus bubalis).

Recent advances in next generation sequencing technology have enabled analysis of complex microbial community from genome to transcriptome level. In the present study, metatranscriptomic approach was applied to elucidate functionally active bacteria and their biological processes in rumen of buffalo (Bubalus bubalis) adapted to different dietary treatments. Buffaloes were adapted to a diet containing 50:50, 75:25 and 100:0 forage to concentrate ratio, each for 6 weeks, before ruminal content sample collection. Metatranscriptomes from rumen fiber adherent and fiber-free active bacteria were sequenced using Ion Torrent PGM platform followed by annotation using MG-RAST server and CAZYmes (Carbohydrate active enzymes) analysis toolkit. In all the samples Bacteroidetes was the most abundant phylum followed by Firmicutes. Functional analysis using KEGG Orthology database revealed Metabolism as the most abundant category at level 1 within which Carbohydrate metabolism was dominating. Diet treatments also exerted significant differences in proportion of enzymes involved in metabolic pathways for VFA production. Carbohydrate Active Enzyme(CAZy) analysis revealed the abundance of genes encoding glycoside hydrolases with the highest representation of GH13 CAZy family in all the samples. The findings provide an overview of the activities occurring in the rumen as well as active bacterial population and the changes occurring through different dietary treatments.

Influence of Diet Composition on Cattle Rumen Methanogenesis: A Comparative Metagenomic Analysis in Indian and Exotic Cattle.

Comparative metagenomics approach has been used in this study to discriminate colonization of methanogenic population in different breeds of cattle. We compared two Indian cattle breeds (Gir and Kankrej) and two exotic cattle (Holstein and Jersey) breeds. Using a defined dietary plan for selected Indian varieties, the diet dependent shifts in microbial community and abundance of the enzymes associated with methanogenesis were studied. This data has been compared with the available rumen metagenome data from Holstein and Jersey dairy cattle. The abundance of genes for methanogenesis in Holstein and Jersey cattle came from Methanobacteriales order whereas, majority of the enzymes for methanogenesis in Gir and Kankrej cattle came from Methanomicrobiales order. The study suggested that by using slow/less digestible feed, the propionate levels could be controlled in rumen; and in turn, this would also help in further reducing the hydrogenotrophic production of methane. The study proposes that with the designed diet plan the overall methanogenic microbial pool or the individual methanogens could be targeted for development of methane mitigation strategies.

Characterization of the rumen microbiome of Indian Kankrej cattle (Bos indicus) adapted to different forage diet.

Present study described rumen microbiome of Indian cattle (Kankrej breed) to better understand the microbial diversity and largely unknown functional capacity of the rumen microbiome under different dietary treatments. Kankrej cattle were gradually adapted to a high-forage diet (four animals with dry forage and four with green forage) containing 50 % (K1), 75 % (K2) to 100 % (K3) forage, and remaining concentrate diet, each for 6 weeks followed by analysis of rumen fiber adherent and fiber-free metagenomic community by shotgun sequencing using ion torrent PGM platform and EBI-metagenomics annotation pipeline. Taxonomic analysis indicated that rumen microbiome was dominated by Bacteroidetes followed by Firmicutes, Fibrobacter, Proteobacteria, and Tenericutes. Functional analysis based on gene ontology classified all reads in total 157 categories based on their functional role in biological, molecular, and cellular component with abundance of genes associated with hydrolase activity, membrane, transport, transferase, and different metabolism (such as carbohydrate and protein). Statistical analysis using STAMP revealed significant differences (P < 0.05) between solid and liquid fraction of rumen (in 65 categories), between all three treatments (in 56 categories), and between green and dry roughage (17 categories). Diet treatment also exerted significant difference in environmental gene tags (EGTs) involved in metabolic pathways for production of volatile fatty acids. EGTs for butyrate production were abundant in K2, whereas EGTs for propionate production was abundant during K1. Principal component analysis also demonstrated that diet proportion, fraction of rumen, and type of forage affected rumen microbiome at taxonomic as well as functional level.

Characterization of antimicrobial resistance genes from Neisseria gonorrhoeae positive remnant Aptima urine specimens.

Aim: To ascertain the antimicrobial resistance and strain types (STs) of Neisseria gonorrhoeae from 50 remnant Aptima urine specimens using molecular methods. Methods: Mutations predictive of resistance to six antibiotics were identified in eight genes. STs were determined using NG-MAST and NG-STAR. Results: All eight antimicrobial resistance genes could be characterized in 36 specimens. A total of 17 specimens were predicted to be susceptible to all antibiotics, including ceftriaxone. Decreased susceptibility to cefixime and ciprofloxacin resistance was predicted in 11 specimens (PBP2 type 34.001). Overall, 38/50 specimens were predicted to be ciprofloxacin susceptible; three were azithromycin resistant. Nineteen NG-MAST and 21 NG-STAR STs were noted. Conclusion: Molecular analysis of remnant Aptima specimens enabled the prediction of emerging gonococcal cefixime and azithromycin resistance which would otherwise have been undetected.

Deep insights into carbohydrate metabolism in the rumen of Mehsani buffalo at different diet treatments.

Ruminants are known to harbour a vast and diverse microbial community that functions in utilizing the fibrous and starchy feedstuffs. The microbial fermentation of fibrous and starchy feed is carried out by different groups of microbiota, which function in synergistic mechanism. The exploration of the shift in carbohydrate utilizing microbial community with the change in diet will reveal the efficient role of that group of microbial community in particular carbohydrate utilization. The present study explains the shifts in microbial enzymes for carbohydrate utilization with the change in the feed proportions and its correlation with the microbial community abundance at that particular treatment. The sequencing data of the present study is submitted to NCBI SRA with experiment accession IDs (ERX162128, ERX162129, ERX162130, ERX162131, ERX162139, ERX162134, ERX162140, ERX162141, ERX197218, ERX197219, ERX197220, ERX197221, ERX162158, ERX162159, ERX162160, ERX162161, ERX162176, ERX162164, ERX162165, ERX162166, ERX162167, ERX162168, ERX162169, ERX162177).

Metagenome of Mehsani buffalo rumen microbiota: an assessment of variation in feed-dependent phylogenetic and functional classification.

AIM: To gain a greater understanding of the ecology and metabolic potential of the rumen microbiome with the changes in the animal diet. METHODS: Diet composed of varying proportion of green and dry roughages along with grains was given to 8 Mehsani buffaloes, and rumen metagenome was sketched using shotgun semiconductor sequencing. RESULTS: In the present study, the Bacteroidetes were found to be dominant at the phyla level and Prevotella at the genus level. The ratio of Firmicutes to Bacteroidetes was found to be higher in the solid fraction as compared to the liquid fraction. In the solid fraction of the dry roughage group, the significant increment (p < 0.05) in Bacteroidetes abundance was observed with increment of roughage concentration. At the genus level, Clostridium significantly increased with the increment in roughage concentration. A comparison of glycoside hydrolase and cellulosome functional genes revealed more glycoside hydrolase 3 encoding genes with higher fiber diet and significant difference in carbohydrate-active enzymes family composition between green and dry roughage groups of the liquid fraction. CONCLUSION: The present study provides a base to understand the modulating behavior of microbiota which can be manipulated to improve livestock nutrient utilization efficiency and for targeting the efficient catabolism of complex carbohydrate molecules as well.

Microbial and Carbohydrate Active Enzyme profile of buffalo rumen metagenome and their alteration in response to variation in the diet.

Rumen microbiome represents rich source of enzymes degrading complex plant polysaccharides. We describe here analysis of Carbohydrate Active Enzymes (CAZymes) from 3.5 gigabase sequences of metagenomic data from rumen samples of Mehsani buffaloes fed on different proportions of green or dry roughages to concentrate ration. A total of 2597 contigs encoding putative CAZymes were identified by CAZyme Analysis Toolkit (CAT). The phylogenetic analysis of these contigs by MG-RAST revealed predominance of Bacteroidetes, followed by Firmicutes, Proteobacteria, and Actinobacteria phyla. Moreover, a higher abundance of oligosaccharide degrading and debranching enzymes in buffalo rumen metagenome and that of cellulases and hemicellulases in termite hindgut was observed when we compared glycoside hydrolase (GH) profile of buffalo rumen metagenome with cow rumen, termite hindgut and chicken caecum metagenome. Further, comparison of microbial profile of green or dry roughage fed animals showed significantly higher abundance (p-value<0.05) of various polysaccharide degrading bacterial genera like Fibrobacter, Prevotella, Bacteroides, Clostridium and Ruminococcus in green roughage fed animals. In addition, we found a significantly higher abundance (p-value<0.05) of enzymes associated with pectin digestion such as pectin lyase (PL) 1, PL10 and GH28 in green roughage fed animals. Our study outlines CAZyme profile of buffalo rumen metagenome and provides a scope to study the role of abundant enzyme families (oligosaccharide degrading and debranching enzymes) in digestion of coarse feed.