Staphylococcus haemolyticus is a reservoir of antibiotic resistance genes in the preterm infant gut

Abstract

Staphylococcus haemolyticus is an important cause of sepsis in preterm infants, with gut colonization being recognized as a risk factor for infection. To better understand the diversity of S. haemolyticus among preterm infants, we generated genome sequences of S. haemolyticus strains (n = 140) from 44 stool samples of 22 preterm infants from four hospitals in England. Core genome phylogenetic analyses, incorporating 126 publicly available S. haemolyticus genome sequences, showed that 85/140 (60.1%) of the isolates, from three different hospitals, formed a clonal group with 78/85 (91.7%) strains having Multi-Locus Sequence Type (ST) 49. Antibiotic resistance genes were prevalent in the genomes. There was a strong association between the presence of mecA and phenotypic resistance to oxacillin, and the aacA-aphD gene and phenotypic resistance to gentamicin. While mecA was near-ubiquitous, none of the strains from the preterm infant cohort had a complete Staphylococcal Cassette Chromosome mec (SCCmec) element. The aacA-aphD gene was associated with the transposon Tn4001 in multiple chromosomal and plasmid contexts. Our data suggest the existence of a distinct sub-population of S. haemolyticus that has adapted to colonize the gut of preterm infants, and widespread horizontal gene transfer and recombination among this frequent colonizer of the preterm infant gut.

Keywords: Staphylococcus; antibiotic resistance; gut microbiota; microbial evolution; microbial genomics; neonates.

Figure 1.

Estimation of Staphylococcus haemolyticus abundance in the preterm infant gut microbiota based on 16S rRNA gene sequencing data. The figure visualizes previously analyzed 16S rRNA gene sequencing data of 497 stool samples from 192 preterm infants. bubble plots show the relative abundance of OTUs identified as Staphylococcus haemolyticus.

Figure 2.

Midpoint-rooted phylogenetic tree illustrating core genome comparisons among strains sequenced in this study and publicly available strains (n = 266). The concentric rings display metadata related to stool isolates collected from preterm infants at various hospitals across the UK. Ring A indicates the hospital of origin for each isolate. Ring B denotes individual infants from whom the strains were isolated. Ring C shows multilocus sequence typing (MLST) designations for each strain. The central ring (ring D) uses alternating dark and light gray blocks to represent fastbaps clusters, numbered for reference. Sequence types (STs) with three or more isolates are labeled individually in the legend, while STs with fewer than three isolates are grouped under “other.” strains for which sequence types could not be determined are labeled as “ud” (undetermined).

Figure 3.

Antibiotic resistance gene (ARG) profiles in Staphylococcus haemolyticus isolates. This figure displays the presence or absence of specific ARGs and their associated antibiotic classes across isolates from preterm infants, grouped by their hospitals of origin. A core genome phylogenetic tree, with fastbaps clusters indicated, is shown on the left, together with information on the hospitals each infant was hospitalized in. Abbreviations: QAC – quaternary ammonium compounds.

Figure 4.

Presence of aminoglycoside and β-lactam resistance genes and outcome of antibiotic susceptibility testing. Coloured purple circles represent the presence of aminoglycoside resistance genes aacA-aphD, ant(4’)-Ib, aph(3’)-IIIa, and ant(6)-iIa. Coloured pink circles represent the presence of β-lactam resistance genes blaZ and mecA. Filled circles indicate that the gene is present, open circles indicate that the gene is absent. Filled triangles indicate phenotypic resistance to an antibiotic, open triangles indicate phenotypic sensitivity to antibiotics. The half-circle labeled “I” represents individual infants, and the half-circle labeled “H” indicates the hospitals from which these infants’ isolates were obtained, with the same color coding as in Figures 2 and 3. The midpoint-rooted phylogenetic tree is based on a core genome alignment of S. haemolyticus genomes.

Figure 5.

Genetic context of the Tn4001 region found in S. haemolyticus isolates from the preterm infant cohort. Coloured purple circles represent the presence (filled circle) or absence (open circle) of aacA-aphD gene. Different genetic contexts of Tn4001 are indicated: squares represent the configuration of Tn4001 in plasmids (pBAMBI1 – pBAMBI4) and circles represent the configuration of Tn4001 in the chromosome (chr1_config1 and chr_config 2) (Figure S3). The half-circle labeled “C” represents the Tn4001 genetic context, the half-circle labeled “I” represents individual infants, and the half-circle labeled “H” indicates the hospitals from which these infants’ isolates were obtained and these use the same colors as in Figure 1 and 2. The midpoint-rooted phylogenetic tree is based on a core genome alignment of S. haemolyticus genomes.