Identification of plasmids in avian-associated Escherichia coli using nanopore and illumina sequencing.

BACKGROUND: Avian pathogenic Escherichia coli (APEC) are the causative agents of colibacillosis in chickens, a disease which has significant economic impact on the poultry industry. Large plasmids detected in APEC are known to contribute to strain diversity for pathogenicity and antimicrobial resistance, but there could be other plasmids that are missed in standard analysis. In this study, we determined the impact of sequencing and assembly factors for the detection of plasmids in an E. coli whole genome sequencing project. RESULTS: Hybrid assembly (Illumina and Nanopore) combined with plasmid DNA extractions allowed for detection of the greatest number of plasmids in E. coli, as detected by MOB-suite software. In total, 79 plasmids were identified in 19 E. coli isolates. Hybrid assemblies were robust and consistent in quality regardless of sequencing kit used or if long reads were filtered or not. In contrast, long read only assemblies were more variable and influenced by sequencing and assembly parameters. Plasmid DNA extractions allowed for the detection of physically smaller plasmids, but when averaged over 19 isolates did not significantly change the overall number of plasmids detected. CONCLUSIONS: Hybrid assembly can be reliably used to detect plasmids in E. coli, especially if researchers are focused on large plasmids containing antimicrobial resistance genes and virulence factors. If the goal is comprehensive detection of all plasmids, particularly if smaller sized vectors are desired for biotechnology applications, the addition of plasmid DNA extractions to hybrid assemblies is prudent. Long read sequencing is sufficient to detect many plasmids in E. coli, however, it is more prone to errors when expanded to analyze a large number of isolates.

Comparative genomics of multidrug-resistant Enterococcus spp. isolated from wastewater treatment plants.

BACKGROUND: Wastewater treatment plants (WWTPs) are considered hotspots for the environmental dissemination of antimicrobial resistance (AMR) determinants. Vancomycin-Resistant Enterococcus (VRE) are candidates for gauging the degree of AMR bacteria in wastewater. Enterococcus faecalis and Enterococcus faecium are recognized indicators of fecal contamination in water. Comparative genomics of enterococci isolated from conventional activated sludge (CAS) and biological aerated filter (BAF) WWTPs was conducted. RESULTS: VRE isolates, including E. faecalis (n = 24), E. faecium (n = 11), E. casseliflavus (n = 2) and E. gallinarum (n = 2) were selected for sequencing based on WWTP source, species and AMR phenotype. The pangenomes of E. faecium and E. faecalis were both open. The genomic fraction related to the mobilome was positively correlated with genome size in E. faecium (p < 0.001) and E. faecalis (p < 0.001) and with the number of AMR genes in E. faecium (p = 0.005). Genes conferring vancomycin resistance, including vanA and vanM (E. faecium), vanG (E. faecalis), and vanC (E. casseliflavus/E. gallinarum), were detected in 20 genomes. The most prominent functional AMR genes were efflux pumps and transporters. A minimum of 16, 6, 5 and 3 virulence genes were detected in E. faecium, E. faecalis, E. casseliflavus and E. gallinarum, respectively. Virulence genes were more common in E. faecalis and E. faecium, than E. casseliflavus and E. gallinarum. A number of mobile genetic elements were shared among species. Functional CRISPR/Cas arrays were detected in 13 E. faecalis genomes, with all but one also containing a prophage. The lack of a functional CRISPR/Cas arrays was associated with multi-drug resistance in E. faecium. Phylogenetic analysis demonstrated differential clustering of isolates based on original source but not WWTP. Genes related to phage and CRISPR/Cas arrays could potentially serve as environmental biomarkers. CONCLUSIONS: There was no discernible difference between enterococcal genomes from the CAS and BAF WWTPs. E. faecalis and E. faecium have smaller genomes and harbor more virulence, AMR, and mobile genetic elements than other Enterococcus spp.

Methods for Genomic Epidemiology of Bacterial Pathogens: Example Salmonella.

Genomics has revolutionized how we characterize and monitor infectious diseases for public health. The surveillance and characterization of Salmonella has improved drastically within the past decade. In this chapter, we discuss the prerequisites for good bacterial genomics studies and make note of advantages and disadvantages of this research approach. We discuss methods for outbreak detection and the evolutionary and epidemiological characterization of Salmonella spp. We provide an outline for determining the sequence type and serotype of isolates, building a core genome phylogenetic tree, and detecting antimicrobial resistance genes, virulence factors, and mobile genetic elements. These methods can be used to study other pathogenic bacterial species.

Hybrid Genome Assemblies of 245 Avian and Broiler Barn Environment-Associated Escherichia coli Strains Isolated from Saskatchewan Broiler Farms.

Escherichia coli infections in poultry cause significant morbidity and economic losses for producers each year. In a 3-year period, we collected and sequenced the whole genomes of E. coli disease isolates (n = 91), isolates from presumed healthy birds (n = 61), and isolates from 8 barn sites (n = 93) on broiler farms in Saskatchewan.

Exploring the mobilome and resistome of Enterococcus faecium in a One Health context across two continents.

Enterococcus faecium is a ubiquitous opportunistic pathogen that is exhibiting increasing levels of antimicrobial resistance (AMR). Many of the genes that confer resistance and pathogenic functions are localized on mobile genetic elements (MGEs), which facilitate their transfer between lineages. Here, features including resistance determinants, virulence factors and MGEs were profiled in a set of 1273 E. faecium genomes from two disparate geographic locations (in the UK and Canada) from a range of agricultural, clinical and associated habitats. Neither lineages of E. faecium, type A and B, nor MGEs are constrained by geographic proximity, but our results show evidence of a strong association of many profiled genes and MGEs with habitat. Many features were associated with a group of clinical and municipal wastewater genomes that are likely forming a new human-associated ecotype within type A. The evolutionary dynamics of E. faecium make it a highly versatile emerging pathogen, and its ability to acquire, transmit and lose features presents a high risk for the emergence of new pathogenic variants and novel resistance combinations. This study provides a workflow for MGE-centric surveillance of AMR in Enterococcus that can be adapted to other pathogens.

Effect of combinations of feed-grade urea and slow-release urea in a finishing beef diet on fermentation in an artificial rumen system.

This study evaluated the effect of combinations of feed-grade urea and slow-release urea (SRU) on fermentation and microbial protein synthesis within two artificial rumens (Rusitec) fed a finishing concentrate diet. The experiment was a completely randomized, dose-response design with SRU substituted at levels of 0% (control), 0.5%, 1%, or 1.75% of dry matter (DM) in place of feed-grade urea, with four replicate fermenters per dosage. The diet consisted of 90% concentrate and 10% forage (DM basis). The experiment was conducted over 15 d, with 8 d of adaptation and 7 d of sampling. Dry matter and organic matter disappearances were determined after 48 h of incubation from day 9 to 12, and daily ammonia (NH3) and volatile fatty acid (VFA) production were measured from day 9 to 12. Microbial protein synthesis was determined on days 13-15. Increasing the level of SRU quadratically affected total VFA (Q, P = 0.031) and ammonia (Q, P = 0.034), with a linear increment in acetate (L, P = 0.01) and isovalerate (L, P = 0.05) and reduction in butyrate (L, P = 0.05). Disappearance of neutral detergent fiber (NDF) and acid detergent fiber (ADF) was quadratically affected by levels of SRU, plateauing at 1% SRU. Inclusion of 1% SRU resulted in the highest amount of microbial nitrogen associated with feed particles (Q, P = 0.037). Responses in the efficiency of microbial protein synthesis fluctuated (L, P = 0.002; Q, P = 0.001) and were the highest for 1% SRU. In general, the result of this study showed that 1% SRU in combination with 0.6% urea increased NDF and ADF digestibility and total volatile fatty acid (TVFA) production.

Investigation of a Reduction in Tylosin on the Prevalence of Liver Abscesses and Antimicrobial Resistance in Enterococci in Feedlot Cattle.

Recent concerns over linkages between antimicrobial resistance in human pathogens and antimicrobial use in livestock have prompted researchers to investigate management strategies that reduce the current reliance on in-feed tylosin to control liver abscesses in feedlot cattle. A total of 7,576 crossbred yearlings were allocated to the study (~253 animals/pen, 10 replicate pens per treatment) and individually randomized to one of three treatments. Tylosin phosphate (11 ppm) was included in-feed (1) for the first 125 days on feed (DOF) (FIRST-78%), (2) for DOF 41 to 161 (LAST-75%), or (3) for the entire feeding period (CON; day 0-161). Fecal composites were collected from the pen floor on days 0, 81, and 160 of the finishing period. Serial dilutions were spread plated for enumeration of enterococci on Bile Esculin Azide (BEA) agar and BEA amended with 8 µg/ml erythromycin. Results indicated that although the proportion of EryR enterococci increased with DOF (P < 0.01), neither treatment (P = 0.34) or treatment × DOF (P = 0.37) affected antimicrobial resistance. Of the 538 isolates, 97% were enterococci, with mixed species isolated early in the feeding period and only Enterococcus hirae isolated at the end. Isolates were most frequently resistant to tylosin (86%), erythromycin (84%), and doxycycline (31%). Macrolide and tetracycline resistant isolates harbored erm(B), msrC, and tet(L), tet(M), tet(O) genes, respectively. Overall, the proportion of EryR enterococci increased (P < 0.05) in all three treatments over the feeding period. Compared to the control cattle, FIRST-78% cattle had more severe (P < 0.05) liver abscesses, while there was a trend (P < 0.08) for this response in LAST-75% cattle. There was no difference (P > 0.05) in total liver abscesses, growth performance, carcass traits, morbidity, or mortality among treatments. These results support the potential to reduce the duration and therefore quantity of tylosin administered to feedlot cattle during the feeding period without impacting animal productivity.

Comparison of biochemical and genotypic speciation methods for vancomycin-resistant enterococci isolated from urban wastewater treatment plants.

Enterococci species in wastewater including Enterococcus faecalis, Enterococcus faecium, Enterococcus casseliflavus and Enterococcus gallinarum isolates (n = 308) with low or high level vancomycin resistance were determined and compared using a phenotypic method (RapID™ STR system), 16S rRNA sequencing, and multi-locus (atpA, groESL, and pheS) sequence analysis (MLSA). Error rates for the RapID™ STR system were E. faecalis (15.9%), E. faecium (21.5%), and E. casseliflavus/E. gallinarum (56.9%) when referenced to the consensus of all methods tested. Comparison of single nucleotide polymorphism (SNP) distances and phylogenetic trees suggested that the groESL locus delineated species more effectively than other loci. The groESL locus was the most reliable loci for the correct identification of Enterococcus spp., including E. casseliflavus and E. gallinarum, with high congruence compared to the consensus (Adjusted Rand Index = 0.954; Adjusted Wallace Co-efficient = 0.941). All of the methods were compared to whole genome sequencing, which acted as a gold standard, for the isolates from this study and those downloaded from NCBI.

Quantification and Multidrug Resistance Profiles of Vancomycin-Resistant Enterococci Isolated from Two Wastewater Treatment Plants in the Same Municipality.

Wastewater treatment plants (WWTPs) are points of control for the environmental dissemination of antimicrobial resistant bacteria. Vancomycin-resistant enterococci (VRE) were used as indicators of antimicrobial resistance (AMR) in two WWTPs (biologically aerated filter (BAF) and conventional activated sludge (CAS)) in the same municipality. The removal and abundance of enterococci and VRE as well as the species and antimicrobial resistance profiles of VRE were assessed. Enterococci and VRE from the primary and final effluents were enumerated. Results were assessed from an ecological context. VRE was not selected for by either WWTP but the BAF system outperformed the CAS system for the removal of enterococci/VRE. Enterococcus faecalis (n = 151), E. faecium (n = 94) and E. casseliflavus/E. gallinarum (n = 59) were the dominant VRE species isolated. A decrease in levofloxacin resistance in enterococci was observed in the BAF WWTP. An increase in nitrofurantoin resistant (p < 0.001) and a decrease in quinupristin/dalfopristin (p = 0.003) and streptomycin (p = 0.022) resistant enterococci were observed in the CAS WWTP, corresponding to a shift of VRE from E. faecalis to E. faecium. Wastewater treatment processes can be managed to limit the dissemination of antimicrobial resistance determinants into the surrounding environment.

Serotyping and antimicrobial resistance of Mannheimia haemolytica strains from European cattle with bovine respiratory disease.

The objective of this study was to assess the prevalence of three serotypes, A1, A2, and A6 in 98 M. haemolytica isolates collected from clinical BRD cases in European cattle and assess their antimicrobial resistance profiles. Isolates were characterized by serotyping (plate agglutination and serotype specific PCR) and antimicrobial susceptibility testing. The study identified a predominance of serotypes A1 (59%) and A6 (22%) in European M. haemolytica isolates exhibiting a relatively low level of antimicrobial resistance. A comprehensive understanding of the relative prevalence of different M. haemolytica serotypes in Europe informs a targeted approach for vaccine design against BRD.

Effect of exogenous fibrolytic enzymes and ammonia fiber expansion on the fermentation of wheat straw in an artificial rumen system (RUSITEC)1.

This study investigated the effect of treatment of wheat straw using ammonia fiber expansion (AFEX) and exogenous fibrolytic enzymes (Viscozyme) on fiber digestibility, rumen fermentation, microbial protein synthesis, and microbial populations in an artificial rumen system [Rumen Simulation Technique (RUSITEC)]. Four treatments were assigned to 16 vessels (4 per treatment) in 2 RUSITEC apparatuses in a randomized block design. Treatments were arranged as a 2 × 2 factorial using untreated or AFEX-treated wheat straw with or without exogenous fibrolytic enzymes [0 or 500 µg of protein/g straw dry matter (DM)]. Fibrolytic enzymes were applied to straw, prior to sealing in nylon bags. The concentrate mixture was provided in a separate bag within each fermentation vessel. The RUSITECs were adapted for 8 d and disappearance of DM, neutral detergent fiber (NDF), acid detergent fiber (ADF), and crude protein (CP) was measured after 48 h of incubation. Ammonia fiber expansion increased (P < 0.01) the disappearance of wheat straw DM (69.6 vs. 38.3%), NDF (65.6 vs. 36.8%), ADF (61.4 vs. 36.0%), and CP (68.3 vs. 24.0%). Total dietary DM, organic matter (OM), and NDF disappearance was also increased (P ≤ 0.05) by enzymes. Total microbial protein production was greater (P < 0.01) for AFEX-treated (72.9 mg/d) than untreated straw (63.1 mg/d). Total gas and methane (CH4) production (P < 0.01) were also greater for AFEX-treated wheat straw than untreated straw, with a tendency for total gas to increase (P = 0.06) with enzymes. Ammonia fiber expansion increased (P < 0.01) total volatile fatty acid (VFA) production and the molar proportion of propionate, while it decreased (P < 0.01) acetate and the acetate-to-propionate ratio. The AFEX-treated straw had lower relative quantities of fungi, methanogens, and Fibrobacter succinogenes (P < 0.01) and fewer protozoa (P < 0.01) compared to untreated straw. The pH of fermenters fed AFEX-treated straw was lower (P < 0.01) than those fed untreated straw. Both AFEX (P < 0.01) and enzymes (P = 0.02) decreased xylanase activity. There was an enzyme × straw interaction (P = 0.02) for endoglucanase activity. Enzymes increased endoglucanase activity of AFEX-treated wheat straw, but had no effect on untreated straw. The addition of enzymes lowered the relative abundance of Ruminococcus flavefaciens, but increased F. succinogenes. These results indicate that AFEX increased the ruminal disappearance of wheat straw and improved fermentation and microbial protein synthesis in the RUSITEC.