Phenotypic and genotypic assessment of iron acquisition in diverse bovine-associated non-aureus staphylococcal strains

Abstract

Although the role of iron in bacterial infections has been well described for Staphylococcus (S.) aureus, iron acquisition in (bovine-associated) non-aureus staphylococci and mammaliicocci (NASM) remains insufficiently mapped. This study aimed at elucidating differences between four diverse bovine NASM field strains from two species, namely S. chromogenes and S. equorum, in regards to iron uptake (with ferritin and lactoferrin as an iron source) and siderophore production (staphyloferrin A and staphyloferrin B) by investigating the relationship between the genetic basis of iron acquisition through whole genome sequencing (WGS) with their observed phenotypic behavior. The four field strains were isolated in a previous study from composite cow milk (CCM) and bulk tank milk (BTM) in a Flemish dairy herd. Additionally, two well-studied S. chromogenes isolates originating from a persistent intramammary infection and from a teat apex were included for comparative purpose in all assays. Significant differences between species and strains were identified. In our phenotypical iron acquisition assay, while lactoferrin had no effect on growth recovery for all strains in iron deficient media, we found that ferritin served as an effective source for growth recovery in iron-deficient media for S. chromogenes CCM and BTM strains. This finding was further corroborated by analyzing potential ferritin iron acquisition genes using whole-genome sequencing data, which showed that all S. chromogenes strains contained hits for all three proposed ferritin reductive pathway genes. Furthermore, a qualitative assay indicated siderophore production by all strains, except for S. equorum. This lack of siderophore production in S. equorum was supported by a quantitative assay, which revealed significantly lower or negligible siderophore amounts compared to S. aureus and S. chromogenes. The WGS analysis showed that all tested strains, except for S. equorum, possessed complete staphyloferrin A (SA)-synthesis and export operons, which likely explains the phenotypic absence of siderophore production in S. equorum strains. While analyzing the staphyloferrin A and staphyloferrin B operon landscapes for all strains, we noticed some differences in the proteins responsible for iron acquisition between different species. However, within strains of the same species, the siderophore-related proteins remained conserved. Our findings contribute valuable insights into the genetic elements associated with bovine NASM pathogenesis.

Keywords: Dairy cows; Staphylococcus chromogenes; Staphylococcus equorum; WGS; ferritin; iron-acquisition; lactoferrin; mastitis; non-aureus staphylococci; siderophore.

Figure 1

Overview of strain growth (optical density, OD600) over 24 h in different growth media. The four field strains, Staphylococcus chromogenes from composite cow milk (CCM) and from bulk tank milk (BTM) (A, B) and S. equorum from CCM and BTM (C, D), and the two comparative strains, Staphylococcus chromogenes isolates from a persistent intramammary infection (IM) and from the teat apex of a dairy heifer (TA) (E, F), are grown in tryptic soy broth (TSB, solid line), deferrated tryptic soy broth (dTSB, dotted line), deferrated tryptic soy broth with ferritin from equine spleen (dTSBF, short-dash line), and deferrated tryptic soy broth with human recombinant lactoferrin (dTSBL, long-dash line). All experiments were performed in duplicate. Different letters within each figure (A–C) indicate significant differences when applying the Bonferroni correction between growth media within strains (P ≤ 0.05).

Figure 2

Siderophore-associated protein homology in diverse NASM isolates. Presence of siderophore-associated proteins within S. chromogenes isolates [from a persistent intramammary infection (IM), the teat apex of a dairy heifer (TA), composite cow milk (CCM) and bulk tank milk (BTM)] and S. equorum isolates (from CCM and BTM) highlighted in pink, including 100 isolates (S. chromogenes = 83, S. equorum = 17) from the Mastitis Pathogen Collection of the Canadian Bovine Mastitis and Milk Quality Research Network (CBMQRN) [35], and quality control reference strain S. aureus ATCC 25923. An ML tree (1000 ultrafast bootstraps) representing phylogenetic relationship of S. chromogenes, S. equorum, and quality control strain S. aureus ATCC 25923 on whole genome SNP level with the S. aureus ATCC 25923 (CP009361) as reference. The final phylogenetic tree was annotated with the presence of siderophore- and ferritin-related protein hits across genomes. Color coded (blue-red) represents amino acid homology of the identified proteins as compared to the NCBI siderophore- and ferritin-related protein hits. Only hits with amino acid homology above 30% and 50% query coverage are shown.

Figure 3

Siderophore-related sfa-hts (A) and sbn-sir(B) operon landscapes. Operon landscapes for the four field strains, Staphylococcus chromogenes from composite cow milk (CCM) and from bulk tank milk (BTM) and S. equorum from CCM and BTM, the two comparative strains, S. chromogenes isolated from a persistent intramammary infection (IM) and from the teat apex of a dairy heifer (TA), and positive control, Staphylococcus aureus ATCC 25923.