How to survive pig farming: Mechanism of SCC mec element deletion and metabolic stress adaptation in livestock-associated MRSA

Abstract

Previous research on methicillin susceptible Staphylococcus aureus (MSSA) belonging to livestock-associated (LA-) sequence type (ST) 398, isolated from pigs and their local surroundings, indicated that differences between these MSSA and their methicillin resistant predecessors (MRSA) are often limited to the absence of the staphylococcal cassette chromosome mec (SCCmec) and few single nucleotide polymorphisms. So far, our understanding on how LA-MRSA endure the environmental conditions associated with pig-farming as well as the putative impact of this particular environment on the mobilisation of SCCmec elements is limited. Thus, we performed in-depth genomic and transcriptomic analyses using the LA-MRSA ST398 strain IMT38951 and its methicillin susceptible descendant. We identified a mosaic-structured SCCmec region including a putative replicative SCCmecVc which is absent from the MSSA chromosome through homologous recombination. Based on our data, such events occur between short repetitive sequences identified within and adjacent to two distinct alleles of the large cassette recombinase genes C (ccrC). We further evaluated the global transcriptomic response of MRSA ST398 to particular pig-farm associated conditions, i.e., contact with host proteins (porcine serum) and a high ammonia concentration. Differential expression of global regulators involved in stress response control were identified, i.e., ammonia-induced alternative sigma factor B-depending activation of genes for the alkaline shock protein 23, the heat shock response and the accessory gene regulator (agr)-controlled transcription of virulence factors. Exposure to serum transiently induced the transcription of distinct virulence factor encoding genes. Transcription of genes reported for mediating the loss of methicillin resistance, especially ccrC, was not significantly different compared to the unchallenged controls. We concluded that, from an evolutionary perspective, bacteria may save energy by incidentally dismissing a fully replicative SCCmec element in contrast to the induction of ccr genes on a population scale. Since the genomic SCCmec integration site is a hot-spot of recombination, occasional losses of elements of 16 kb size may restore capacities for the uptake of foreign genetic material. Subsequent spread of resistance, on the other hand, might depend on the autonomous replication machinery of the deleted SCCmec elements that probably enhance chances for reintegration of SCCmec into susceptible genomes by mere multiplication.

Keywords: SCCmec; ammonium; deletion; livestock associated; manure (litter); methicillin resistant Staphylococcus aureus; recombination; transcriptome analysis.

Figure 1

Genome-wide comparison of MRSA ST398 (IMT38951) and its descendant MSSA (IMT38951_42). A combinatorial approach including long reads (PacBio) and short reads (Illumina sequencing) allowed precise reconstruction of both genomes. Five regions harbouring phages or phage-associated genes identified using PHASTER are indicated in green. Location of resistance genes identified using ResFinder 2.1 and the SCCmec intergration site are indicated in blue. Comparative genomics determined a difference of 10 single nucleotide polymorphisms in total (excluding phage regions).

Figure 2

Sequence comparison of IMT38951 and IMT38951_42 from rlmH (previously: orfX) to dus (previously orfY). Repetitive elements and homologues regions challenged the precise reconstruction of MRSA IMT38951 and its isogenic descendant lacking SCCmecVc (region B). Since sequence identities of genes and intergenic spacer regions present in this region range from 98/99% (light blue-green) to 100% (blue-green), including sequences and genes that flank (regions A and C) the missing SCCmec element in IMT38951_42. DR, direct repeat of 15 bp length; CCPol-MP-Cch2, operon containing an A-family DNA polymerase (CCPol) together with a small protein lacking conserved domains (“middle protein,” MP) as well as a putative helicase (Cch2) (Bebel et al., 2020); ccrC, chromosomal cassette recombinase C; tpn, transposase associated with insertion element (IS) 431; ISR, intergenic spacer region of 224 bp length.

Figure 3

Tracking the loss of SCCmecVc. MRSA ST398 IMT38951: Illustration of the SCCmec integration site downstream of rlmH. (A) Comparison of the terminal sequences of the intergenic sequences spacer region 1 (I) and 2 (III) and the initial basepairs of ccrC8 (II) and ccrC1 (IV) (start codon: green) revealed two possibilities of homologues recombination after deletion of SCCmec: A & C, intergenic regions 1 and 2; and B & D, ccrC8 and ccrC1. (B) The region between rlmH (previously “orfX“) and dus (previously orfY) carries various repetitive DNA sequences. (C) Both recombination events would allow deletion of an identical and complete SCCmecVc element. (D) Circular SCCmec element with putative functional recombinase ccrC8 and a CCPol-MP-Cch2 II complex (Bebel et al., 2020) conferring its autonomous replication capabilities. ccr, chromosomal cassette recombinase; IS, insertion sequence; DR, directs repeats associated with ccr-mediated integration and excision of SCCmec elements (Semmler et al., 2016).

Figure 4

Global overview on differentially expressed genes for samples and conditions after 10 and 60 min of exposure. Pattern analysis of differentially expressed genes (DEGs) are visualized based on their log2 fold-change (log2FC) compared to the corresponding control samples, ranging from up- (yellow) to downregulated (blue). Each column corresponds to a distinct growth condition (S = serum, A = ammonia, AS = ammonia & serum) and time point (10 min and 60 min of exposure). Genes are clustered based on their respective expression across the tested conditions (left). Expression ranges from-10 log2FC to +10 log2FC.

Figure 5

Heatmap of the top differentially expressed genes across all sample conditions. Top differentially expressed genes (DEGs) (n = 41) are visualized based on their respective log2 fold-change (log2FC) compared to the corresponding control samples. Each column corresponds to a stress condition (S = serum, A = ammonia, AS = ammonia & serum) and time point (10 min and 60 min). Genes are either strongly upregulated with log2FC > 5 (yellow) or downregulated with log2FC < −4 (blue). Genes are furthermore annotated with corresponding COG ontologies (left side).

Figure 6

Heatmap illustrating transcription of genes at the chromosomal SCCmec integration site from rlmH (“orfX”) to dus (“orfY”). Gene transcripts are visualized based on their log2 fold-change (log2FC) with either red (upregulated), blue (downregulated) or white (no changes) compared to the corresponding control samples. Each column corresponds to a growth condition (S = serum, A = ammonia, AS = ammonia & serum) and time point (10 min and 60 min). Overall expression ranges from-2 log2FC to +2 log2FC.