Resistome, mobilome, and virulome explored in clinical isolates derived from acne patients in Egypt: unveiling unique traits of an emerging coagulase-negative Staphylococcus pathogen

Abstract

Coagulase-negative staphylococci (CoNS) are a group of gram-positive staphylococcal species that naturally inhabit the healthy human skin and mucosa. The clinical impact of CoNS-associated infections has recently been regarded as a challenge for diagnosis and therapeutic options. CoNS-associated infections are primarily caused by bacterial resistance to antibiotics and biofilm formation. As antibiotics are still the most used treatment, this problem will likely persist in the future. The present study aimed to investigate the resistance and virulence of CoNS recovered from various acne lesions and explore their genetic basis. Skin swab samples were collected from participants with acne and healthy skin. All samples underwent conventional culture for the isolation of CoNS, MALDI-TOF confirmation, antibiotic susceptibility, and biofilm formation testing. A total of 85 CoNS isolates were recovered from the samples and preliminarily identified as Staphylococcus epidermidis. Isolates from the acne group (n = 60) showed the highest rates of resistance to penicillin (73%), cefoxitin (63%), clindamycin (53.3%), and erythromycin (48%), followed by levofloxacin (36.7%) and gentamycin (31.7%). The lowest rates of resistance were observed against tetracycline (28.3%), doxycycline (11.7%), and minocycline (8.3%). CoNS isolated from mild, moderate acne and healthy isolates did not show strong biofilm formation, whereas the isolates from the severe cases of the acne group showed strong biofilm formation (76.6%). Four extensively drug-resistant and strong biofilm-forming staphylococcal isolates recovered from patients with severe acne were selected for whole-genome sequencing (WGS), and their genomes were investigated using bioinformatics tools. Three of the sequenced genomes were identified as S. epidermidis; however, isolate 29AM was identified as Staphylococcus warneri, which is a newly emerging pathogen that is not commonly associated with acne and was not detected by MALDI-TOF. All the sequenced strains were multidrug-resistant and carried multiple resistance genes, including blaZ, mecA, tet(K), erm(C), lnuA, vgaA, dfrC, fusB, fosBx1, norA, and vanT, which were found to be located on plasmids and chromosomes. Virulence features were detected in all genomes in the presence of genes involved in adherence and biofilm formation (icaA, icaB, icaC, sdrG, sdrH, atl, ebh, and ebp). Only the S. warneri isolate 29AM contained immune evasion genes (capB, capC, acpXL, and manA), an anti-phagocytosis gene (cdsA), and other unique features. As a result of their potential pathogenicity and antibiotic resistance, CoNS must be monitored as an emerging pathogen associated with acne infections. To the best of our knowledge, this is the first report to isolate, identify, and correlate S. warneri with severe acne infections among Egyptian patients using WGS and bioinformatic analysis.

Keywords: CoNS; Staphylococcus epidermidis; Staphylococcus warneri; acne; antibiotic resistance; genome analysis; mobilizable genetic elements; virulence.

Figure 1

Antibiotic resistance among CoNS isolates from acne and healthy groups. Antibiotic susceptibility testing was performed for CoNS isolates using the Kirby–Bauer disk diffusion method, in accordance with the Clinical and Laboratory Standards Institute (CLSI).

Figure 2

Biofilm formation percentage among CoNS isolates from the acne and healthy groups. The biofilm-forming ability of the CoNS isolates was assessed using a crystal violet assay. Adjusted overnight cultures were inoculated in 200 μl of TSB supplemented with 1% (v/v) glucose, in a 96-flat bottom polystyrene microtiter plate. The biofilms were then washed and stained with 0.1% crystal violet. The biofilm biomass was measured colorimetrically at 545 nm and evaluated using the biofilm formation index. Statistical analysis using two-way ANOVA, which was followed by multiple comparisons test with a significance level at ** p < 0.01; *** p < 0.001.

Figure 3

Prevalence of biofilm genes among CoNS isolates. The presence of icaA, icaD, and mecA in the extracted DNA was detected by polymerase chain reaction. The presence of the gene was represented as a percentage of the different acne and healthy groups, and the results are shown as medians with interquartile ranges. Statistical analysis using two-way ANOVA, which was followed by multiple comparisons test with a significance level at * p < 0.05; *** p < 0.001. ns, non-significant.

Figure 4

Heatmap showing the distribution of different virulence determinants among the sequenced isolates in the current study. The red circles denote the presence of virulence genes, whereas the lack of virulence genes is denoted by blue circles. The figure was created using the MORPHEUS online tool (https://software.broadinstitute.org/morpheus).

Figure 5

Whole genome-based phylogenetic tree of S. epidermidis isolates. The S. epidermidis genome sequenced in the current study was compared to the closely related genomes, highlighted in blue. The labels of the S. epidermidis strains sequenced in the current study are shown in red. The figure was created using the iTOL online tool v6.7 (https://itol.embl.de/).

Figure 6

Whole genome-based phylogenetic tree of the S. warneri isolate 29AM. The S. warneri 29AM genome sequenced in the current study was compared to the closely related genomes, highlighted in blue. The label of the S. warneri strain sequenced in this study is shown in red. The figure was created using the iTOL online tool v6.7 (https://itol.embl.de/).

Figure 7

SNP-based phylogenetic tree of Staphylococcus isolates. SNP base phylogeny and heat map displaying the antimicrobial resistance genetic determinants among the sequenced isolates generated and selected genomes: S. epidermidis ATCC^®12228, S. epidermidis ATCC^®35984, S. epidermidis strain 785SEPI, and S. warneri strain SG1. Red and white indicate the presence and absence of ARGs, respectively. The labels of the strains sequenced in this study is shown in red. The figure was created using the iTOL online tool v6.7 (https://itol.embl.de/).