HAM-ART: An optimised culture-free Hi-C metagenomics pipeline for tracking antimicrobial resistance genes in complex microbial communities.

Shotgun metagenomics is a powerful tool to identify antimicrobial resistance (AMR) genes in microbiomes but has the limitation that extrachromosomal DNA, such as plasmids, cannot be linked with the host bacterial chromosome. Here we present a comprehensive laboratory and bioinformatics pipeline HAM-ART (Hi-C Assisted Metagenomics for Antimicrobial Resistance Tracking) optimised for the generation of metagenome-assembled genomes including both chromosomal and extrachromosomal AMR genes. We demonstrate the performance of the pipeline in a study comparing 100 pig faecal microbiomes from low- and high-antimicrobial use pig farms (organic and conventional farms). We found significant differences in the distribution of AMR genes between low- and high-antimicrobial use farms including a plasmid-borne lincosamide resistance gene exclusive to high-antimicrobial use farms in three species of Lactobacilli. The bioinformatics pipeline code is available at https://github.com/lkalmar/HAM-ART.

Mechanisms of ß-lactam resistance of Streptococcus uberis isolated from bovine mastitis cases.

A number of veterinary clinical pathology laboratories in New Zealand have been reporting emergence of increased minimum in inhibitory concentrations for ß-lactams in the common clinical bovine mastitis pathogen Streptococcus uberis. The objective of this study was to determine the genetic basis of this increase in MIC for ß-lactams amongst S. uberis. Illumina sequencing and determination of oxacillin MIC was performed on 265 clinical isolates. Published sequences of the five penicillin binding proteins pbp1a, pbp1b, pbp2a, pbp2b, and pbp2x were used to identify, extract and align these sequences from the study isolates. Amino acid substitutions resulting from single nucleotide polymorphisms (SNP) within these genes were analysed for associations with elevated (≥ 0.5 mg/L) oxacillin MIC together with a genome wide association study. The population structure of the study isolates was approximated using a phylogenetic tree generated from an alignment of the core genome. A total of 53 % of isolates had MIC ≥ 0.5 mg/L for oxacillin. A total of 101 substitutions within the five pbp were identified, of which 11 were statistically associated with an MIC ≥ 0.5 mg/L. All 140 isolates which exhibited an increased ß-lactam MIC had SNPs leading to pbp2x E381K and Q554E substitutions. The phylogenetic tree indicated that the genotype and phenotype associated with the increased MIC for oxacillin were present in several different lineages suggesting that acquisition of this increased ß-lactam MIC had occurred in multiple geographically distinct regions. Reanalysis of the data from the intervention studies from which the isolates were originally drawn found a tendency for the pbp2x E381K substitution to be associated with lower cure rates. It is concluded that there is geographically and genetically widespread presence of pbp substitutions associated with reduced susceptibility to ß-lactam antimicrobials. Additionally, presence of pbp substitutions tended to be associated with poorer cure rate outcomes following antimicrobial therapy for clinical mastitis.

A novel and sensitive functional assay for complement Factor I based on the third proteolytic clip of C3b.

A sensitive assay for the functional activity of complement Factor I is described. This is based on its third proteolytic clip whereby Factor I cleaves cell-bound iC3b to cell-bound C3dg and soluble C3c, thereby abolishing conglutination of the cells. Factor H is required as a co-factor for Factor I activity. Because of the low affinity of iC3b for Factor H, the assay needs to be performed at low ionic strength. This assay is easier to perform than those based on the conversion of C3b to iC3b (the first two Factor I clips), there being no need for the unstable intermediate EAC142 or for purified C3.

Streptococcus bovimastitidis sp. nov., isolated from a dairy cow with mastitis.

Here we describe a new species of the genus Streptococcus that was isolated from a dairy cow with mastitis in New Zealand. Strain NZ1587T was Gram-positive, coccus-shaped and arranged as chains, catalase and coagulase negative, γ-haemolytic and negative for Lancefield carbohydrates (A-D, F and G). The 16S rRNA sequence did not match sequences in the NCBI 16S rRNA or GreenGenes databases. Taxonomic classification of strain NZ1587T was investigated using 16S rRNA and core genome phylogeny, genome-wide average nucleotide identity (ANI) and predicted DNA-DNA hybridisation (DDH) analyses. Phylogeny based on 16S rRNA was unable to resolve the taxonomic position of strain NZ1587T, however NZ1587T shared 99.4 % identity at the 16S rRNA level with a distinct branch of S. pseudoporcinus. Importantly, core genome phylogeny demonstrated that NZ1587T grouped amongst the 'pyogenic' streptococcal species and formed a distinct branch supported by a 100 % bootstrap value. In addition, average nucleotide identity and inferred DNA-DNA hybridisation analyses showed that NZ1587T represents a novel species. Biochemical profiling using the rapid ID 32 strep identification test enabled differentiation of strain NZ1587T from closely related streptococcal species. In conclusion, strain NZ1587T can be classified as a novel species, and we propose a novel taxon named Streptococcus bovimastitidis sp. nov.; the type strain is NZ1587T. NZ1587T has been deposited in the Culture Collection University of Gothenburg (CCUG 69277T) and the Belgian Co-ordinated Collections of Micro-organisms/LMG (LMG 29747).

Experimental confirmation of the C3 tickover hypothesis by studies with an Ab (S77) that inhibits tickover in whole serum.

The complement component 3 (C3) tickover hypothesis was put forward in the early 1970s to account for the spontaneous activation of the alternative complement pathway that occurs after the genetic absence or in vitro depletion of Factor I, the enzyme that is essential for the breakdown of C3b. The hypothesis was widely accepted, but experimental demonstration of the tickover was elusive. A phage Ab against C3b that inhibited the alternative complement pathway, but not the classical pathway, was described in 2009. Studies using this Ab in a variety of assays have now demonstrated that it acts primarily by inhibiting tickover, thereby confirming that tickover really exists.-Lachmann, P. J., Lay, E., Seilly, D. J. Experimental confirmation of the C3 tickover hypothesis by studies with an Ab (S77) that inhibits tickover in whole serum.

Baseline sensitivity of HSV-1 and HSV-2 clinical isolates and defined acyclovir-resistant strains to the helicase-primase inhibitor pritelivir.

Fifty-nine US isolates of HSV-1 and HSV-2 obtained between 1998 and 2004 were tested for sensitivity to the helicase-primase inhibitor, pritelivir (AIC316, BAY 57-1293) by plaque-reduction assay. All isolates, which were collected prior to any clinical use of primase-helicase inhibitors, were sensitive and showed mean EC50 values of 0.026 and 0.029µM for HSV-1 and HSV-2, respectively. Furthermore, several laboratory-selected acyclovir-resistant HSV mutants were also sensitive to pritelivir. These data provide a baseline for HSV sensitivity to pritelivir in general population before it is introduced and broadly used to treat HSV infection. The data also validate pritelivir as an appropriate therapy for nucleoside-resistant HSV infections.