Genomic characterisation of nasal isolates of coagulase-negative Staphylococci from healthy medical students reveals novel Staphylococcal cassette chromosome mec elements

Abstract

Coagulase-negative staphylococci (CoNS) are a diverse group of Gram-positive bacteria that are part of the normal human microbiota. Once thought to be non-pathogenic, CoNS has emerged in recent years as opportunistic pathogens of concern particularly in healthcare settings. In this study, the genomes of four methicillin-resistant CoNS isolates obtained from the nasal swabs of healthy university medical students in Malaysia were sequenced using the Illumina short-read platform. Genome sequencing enabled the identification of the four isolates as Staphylococcus warneri UTAR-CoNS1, Staphylococcus cohnii subsp. cohnii UTAR-CoNS6, Staphylococcus capitis subsp. urealyticus UTAR-CoNS20, and Staphylococcus haemolyticus UTAR-CoNS26. The genome of S. cohnnii UTAR-CoNS6 harboured the mecA methicillin-resistance gene on a Staphylococcal cassette chromosome mec (SCCmec) element similar to SCCmec type XIV (5 A) but the SCCmec cassettes identified in the other three CoNS genomes were novel and untypeable. Some of these SCCmec elements also encoded heavy metal resistance genes while the SCCmec type XIV (5 A) variant in S. cohnii UTAR-CoNS6 harboured the complete ica operon, a known virulence factor that functions in biofilm formation. In S. cohnii UTAR-CoNS6, the macrolide resistance genes msrA and mphC along with copper and cadmium resistance genes were located on a 26,630 bp plasmid, pUCNS6. This study showcased the diversity of CoNS in the nasal microbiota of medical students but the discovery of novel SCCmec elements, various antimicrobial and heavy metal resistance along with virulence genes in these isolates is of concern and warrants vigilance due to the likelihood of spread, especially to hospitalised patients.

Keywords: Coagulase-negative Staphylococci (CoNS); Plasmids; SCCmec; Whole-genome sequencing.

Fig. 1

Midpoint-rooted, maximum likelihood core genome phylogenetic tree of the various coagulase-negative staphylococci (CoNS) genomes relevant to this study (n = 204). The four CoNS isolates (i.e., UTAR-CoNS1, UTAR-CoNS6, UTAR-CoNS20, and UTAR-CoNS26) isolated and sequenced in this study were indicated in red star symbol. Innermost ring/coloured strips represent the sub-clades within S. capitis (light to dark orange) and S. cohnii (pale to bright green). Abbreviations used for the sub-clades: CAP represents S. capitis subsp. capitis, URO represents S. capitis subsp. urealyticus, SH represents S. haemolyticus, SW represents S. warneri, SSB represents S. cohnii subsp. barensis, SSC represents S. cohnii subsp. cohnii, and SSU represents S. cohnii subsp. urealyticus. Also shown in the figure are the source of the isolates (middle ring/coloured strips) and their geographical location (outer-most ring/coloured strips) as indicated in the legend on the top left corner.

Fig. 2

Genetic map of the SCCmec elements identified from the assembled CoNS genomes in this study, i.e., S. capitis subsp. urealyticus UTAR-CoNS20, S. cohnii subsp. cohnii UTAR-CoNS6, S. haemolyticus UTAR-CoNS26, and S. warneri UTAR-CoNS1. The signature mec and ccr complexes were bracketed in orange and purple boxes, respectively. The biofilm formation cluster ica, which was found in the SCCmec identified in S. cohnii UTAR-CoNS6, was indicated in a black box with the asterisk (*). The relevant MLST profile and closest SCCmec matches of each genome were indicated in red fonts. NA, not available. Note that the genomes were assembled from Illumina short-read sequences, and hence the SCCmec elements were spread over two to three separate contigs which were stitched together, as indicated with the green needle-and-thread icon, on a scaffold for analysis and as presented here. The SCCmec element from S. cohnii UTAR-CoNS6 was completed by PCR followed by Sanger sequencing to bridge the gap between contigs. However, such a strategy did not yield any results for the remaining three SCCmec elements, inferring that the gaps in between the contigs were possibly larger than could be bridged by conventional PCR.

Fig. 3

Chord diagram presentation of the genotypic AMR profiles of the four CoNS isolates, as identified by AMRFinderPlus and Abricate (see methodology). The upper half of the chord diagram showed all the resistance determinants found, with light grey representing drug-resistance, dark grey representing antiseptic resistance, and black representing heavy-metal resistance. Each coloured ribbon connects the respective strain (labelled on the lower half of the diagram) to the resistance determinants on the upper half of the diagram.

Fig. 4

Linear plasmid map of pUCNS6 from S. cohnii subsp. cohnii UTAR-CoNS6. The plasmid was found to harbour multiple metal resistance determinants, alongside the macrolide resistance determinants msrA and mphC genes.