Enterococci as a One Health indicator of antimicrobial resistance.

The rapid increase of antimicrobial-resistant bacteria in humans and livestock is concerning. Antimicrobials are essential for the treatment of disease in modern day medicine, and their misuse in humans and food animals has contributed to an increase in the prevalence of antimicrobial-resistant bacteria. Globally, antimicrobial resistance is recognized as a One Health problem affecting humans, animals, and environment. Enterococcal species are Gram-positive bacteria that are widely distributed in nature. Their occurrence, prevalence, and persistence across the One Health continuum make them an ideal candidate to study antimicrobial resistance from a One Health perspective. The objective of this review was to summarize the role of enterococci as an indicator of antimicrobial resistance across One Health sectors. We also briefly address the prevalence of enterococci in human, animal, and environmental settings. In addition, a 16S RNA gene-based phylogenetic tree was constructed to visualize the evolutionary relationship among enterococcal species and whether they segregate based on host environment. We also review the genomic basis of antimicrobial resistance in enterococcal species across the One Health continuum.

Genomic Characterization of Carbapenem-Resistant Bacteria from Beef Cattle Feedlots.

Carbapenems are considered a last resort for the treatment of multi-drug-resistant bacterial infections in humans. In this study, we investigated the occurrence of carbapenem-resistant bacteria in feedlots in Alberta, Canada. The presumptive carbapenem-resistant isolates (n = 116) recovered after ertapenem enrichment were subjected to antimicrobial susceptibility testing against 12 different antibiotics, including four carbapenems. Of these, 72% of the isolates (n = 84) showed resistance to ertapenem, while 27% of the isolates (n = 31) were resistant to at least one other carbapenem, with all except one isolate being resistant to at least two other drug classes. Of these 31 isolates, 90% were carbapenemase positive, while a subset of 36 ertapenem-only resistant isolates were carbapenemase negative. The positive isolates belonged to three genera; Pseudomonas, Acinetobacter, and Stenotrophomonas, with the majority being Pseudomonas aeruginosa (n = 20) as identified by 16S rRNA gene sequencing. Whole genome sequencing identified intrinsic carbapenem resistance genes, including blaOXA-50 and its variants (P. aeruginosa), blaOXA-265 (A. haemolyticus), blaOXA-648 (A. lwoffii), blaOXA-278 (A. junii), and blaL1 and blaL2 (S. maltophilia). The acquired carbapenem resistance gene (blaPST-2) was identified in P. saudiphocaensis and P. stutzeri. In a comparative genomic analysis, clinical P. aeruginosa clustered separately from those recovered from bovine feces. In conclusion, despite the use of selective enrichment methods, finding carbapenem-resistant bacteria within a feedlot environment was a rarity.

Comparative Genomic Analysis of Enterococci across Sectors of the One Health Continuum.

Enterococci are Gram-positive bacteria that can be isolated from a variety of environments including soil, water, plants, and the intestinal tract of humans and animals. Although they are considered commensals in humans, Enterococcus spp. are important opportunistic pathogens. Due to their presence and persistence in diverse environments, Enterococcus spp. are ideal for studying antimicrobial resistance (AMR) from the One Health perspective. We undertook a comparative genomic analysis of the virulome, resistome, mobilome, and the association between the resistome and mobilome of 246 E. faecium and 376 E. faecalis recovered from livestock (swine, beef cattle, poultry, dairy cattle), human clinical samples, municipal wastewater, and environmental sources. Comparative genomics of E. faecium and E. faecalis identified 31 and 34 different antimicrobial resistance genes (ARGs), with 62% and 68% of the isolates having plasmid-associated ARGs, respectively. Across the One Health continuum, tetracycline (tetL and tetM) and macrolide resistance (ermB) were commonly identified in E. faecium and E. faecalis. These ARGs were frequently associated with mobile genetic elements along with other ARGs conferring resistance against aminoglycosides [ant(6)-la, aph(3')-IIIa], lincosamides [lnuG, lsaE], and streptogramins (sat4). Study of the core E. faecium genome identified two main clades, clade 'A' and 'B', with clade A isolates primarily originating from humans and municipal wastewater and carrying more virulence genes and ARGs related to category I antimicrobials. Overall, despite differences in antimicrobial usage across the continuum, tetracycline and macrolide resistance genes persisted in all sectors.

Germline Mutation Analysis in Sporadic Breast Cancer Cases With Clinical Correlations.

Demographics for breast cancers vary widely among nations. The frequency of germline mutations in breast cancers, which reflects the hereditary cases, has not been investigated adequately and accurately in highly-consanguineous Pakistani population. In the present discovery case series, germ-line mutations in twenty-seven breast cancer candidate genes were investigated in eighty-four sporadic breast cancer patients along with the clinical correlations. The germ-line variants were also assessed in two healthy gender-matched controls. The clinico-pathological features were evaluated by descriptive analysis and Pearson χ2 test (with significant p-value <0.05). The most frequent parameters associated with hereditary cancer cases are age and ethnicity. Therefore, the analyses were stratified on the basis of age (≤40 years vs. >40 years) and ethnicity. The breast cancer gene panel assay was carried out by BROCA, which is a genomic capture, massively parallel next generation sequencing assay on Illumina Hiseq2000 with 100bp read lengths. Copy number variations were determined by partially-mapped read algorithm. Once the mutation was identified, it was validated by Sanger sequencing. The ethnic analysis stratified on the basis of age showed that the frequency of breast cancer at young age (≤40 years) was higher in Sindhis (n = 12/19; 64%) in contrast to patients in other ethnic groups. Majority of the patients had stage III (38.1%), grade III (50%), tumor size 2-5 cm (54.8%), and invasive ductal carcinoma (81%). Overall, the analysis revealed germ-line mutations in 11.9% of the patients, which was not significantly associated with younger age or any particular ethnicity. The mutational spectrum was restricted to three genes: BRCA1, BRCA2, and TP53. The identified mutations consist of seven novel germ-line mutations, while three mutations have been reported previously. All the mutations are predicted to result in protein truncation. No mutations were identified in the remaining twenty-four candidate breast cancer genes. The present study provides the framework for the development of hereditary-based preventive and treatment strategies against breast cancers in Pakistani population.

Genomic Characterization of Enterococcus hirae From Beef Cattle Feedlots and Associated Environmental Continuum.

Enterococci are commensal bacteria of the gastrointestinal tract of humans, animals, and insects. They are also found in soil, water, and plant ecosystems. The presence of enterococci in human, animal, and environmental settings makes these bacteria ideal candidates to study antimicrobial resistance in the One-Health continuum. This study focused on Enterococcus hirae isolates (n = 4,601) predominantly isolated from beef production systems including bovine feces (n = 4,117, 89.5%), catch-basin water (n = 306, 66.5%), stockpiled bovine manure (n = 24, 0.5%), and natural water sources near feedlots (n = 145, 32%), and a few isolates from urban wastewater (n = 9, 0.2%) denoted as human-associated environmental samples. Antimicrobial susceptibility profiling of a subset (n = 1,319) of E. hirae isolates originating from beef production systems (n = 1,308) showed high resistance to tetracycline (65%) and erythromycin (57%) with 50.4% isolates harboring multi-drug resistance, whereas urban wastewater isolates (n = 9) were resistant to nitrofurantoin (44.5%) and tigecycline (44.5%) followed by linezolid (33.3%). Genes for tetracycline (tetL, M, S/M, and O/32/O) and macrolide resistance erm(B) were frequently found in beef production isolates. Antimicrobial resistance profiles of E. hirae isolates recovered from different environmental settings appeared to reflect the kind of antimicrobial usage in beef and human sectors. Comparative genomic analysis of E. hirae isolates showed an open pan-genome that consisted of 1,427 core genes, 358 soft core genes, 1701 shell genes, and 7,969 cloud genes. Across species comparative genomic analysis conducted on E. hirae, Enterococcus faecalis and Enterococcus faecium genomes revealed that E. hirae had unique genes associated with vitamin production, cellulose, and pectin degradation, traits which may support its adaptation to the bovine digestive tract. E. faecium and E. faecalis more frequently harbored virulence genes associated with biofilm formation, iron transport, and cell adhesion, suggesting niche specificity within these species.

Absence of Glutathione S-Transferase Theta 1 Gene Is Significantly Associated With Breast Cancer Susceptibility in Pakistani Population and Poor Overall Survival in Breast Cancer Patients: A Case-Control and Case Series Analysis.

PURPOSE: Deletion of Glutathione S-Transferase Theta 1 (GSTT1) encoding gene is implicated in breast cancer susceptibility, clinical outcomes, and survival. Contradictory results have been reported in different studies. The present investigation based on a representative Pakistani population evaluated the GSTT1-absent genotype in breast cancer risk and prognosis. METHODS: A prospective study comprising case-control analysis and case series analysis components was designed. Peripheral blood samples were collected from enrolled participants. After DNA extraction, GSTT1 genotyping was carried out by a multiplex PCR with ß-globin as an amplification control. Association evaluation of GSTT1 genotypes with breast cancer risk, specific tumor characteristics, and survival were the primary endpoints. RESULTS: A total of 264 participants were enrolled in the molecular investigation (3 institutions). The study included 121 primary breast cancer patients as cases and 143 age-matched female subjects, with no history of any cancer, as controls. A significant genetic association between GSTT1-absent genotype and breast cancer susceptibility (p-value: 0.03; OR: 2.13; 95% CI: 1.08-4.29) was reported. The case-series analysis showed lack of association of GSTT1 genotypes with menopause (p-value: 0.86), tumor stage (p-value: 0.12), grade (p-value: 0.32), and size (p-value: 0.07). The survival analysis revealed that GSTT1-absent genotype cases had a statistically significant shorter overall survival (OS) than those with the GSTT1-present genotype cases (mean OS: 23 months vs 33 months). The HR (95% CI) for OS in patients carrying GSTT1-absent genotype was 8.13 (2.91-22.96) when compared with the GSTT1-present genotype. CONCLUSIONS: The present study is the first report of an independent significant genetic association between GSTT1-absent genotype and breast cancer susceptibility in a Pakistani population. It is also the foremost report of the association of this genotype with OS in breast cancer cases. Upon further validation, GSTT1 variation may serve as a marker for devising better population-specific strategies. The information may have translational implications in the screening and treatment of breast cancers.