European Journal of Clinical Microbiology & Infectious Diseases (2020) 39:2235–2246

https://doi.org/10.1007/s10096-020-03984-8

REVIEW

Virulence factors and clonal diversity of Staphylococcus aureus

in colonization and wound infection with emphasis on diabetic foot
infection
Kavitha Shettigar 1,2 & Thokur Sreepathy Murali 1

Received: 28 April 2020 / Accepted: 3 July 2020 / Published online: 18 July 2020
# The Author(s) 2020

Abstract
Foot ulcer is a common complication in diabetic subjects and infection of these wounds contributes to increased rates of
morbidity and mortality. Diabetic foot infections are caused by a multitude of microbes and Staphylococcus aureus, a major
nosocomial and community-associated pathogen, significantly contributes to wound infections as well. Staphylococcus aureus is
also the primary pathogen commonly associated with diabetic foot osteomyelitis and can cause chronic and recurrent bone
infections. The virulence capability of the pathogen and host immune factors can determine the occurrence and progression of S.
aureus infection. Pathogen-related factors include complexity of bacterial structure and functional characteristics that provide
metabolic and adhesive properties to overcome host immune response. Even though, virulence markers and toxins of S. aureus
are broadly similar in different wound models, certain distinguishing features can be observed in diabetic foot infection. Specific
clonal lineages and virulence factors such as TSST-1, leukocidins, enterotoxins, and exfoliatins play a significant role in
determining wound outcomes. In this review, we describe the role of specific virulence determinants and clonal lineages of S.
aureus that influence wound colonization and infection with special reference to diabetic foot infections.

Keywords Clonal diversity . Diabetic foot . Infection . Staphylococcus aureus . Osteomyelitis . Toxins . Virulence

Introduction often colonized by aerobes, anaerobes, and fungi either indi-

vidually or more often as a polymicrobial community.
Foot ulcer is a common complication in diabetic subjects Staphylococcus aureus, a major colonizer of DFU [5–7], pro-
caused due to multitude of underlying risk factors including duces abundant biofilm and thereby inhibits wound healing
neuropathy and vascular insufficiency [1]. These open and exacerbates wound infection [8, 9].
wounds favor colonization by microbes which proliferate in S. aureus with its emerging new clones causes severe
the wound and cause severe infection that can spread to deeper wound infection, skin and soft tissue infections (SSTI), oste-
tissues thereby substantially increasing the risk of hospitaliza- omyelitis, and other unusual infections globally. Most often,
tion and lower limb amputations [2]. Pathophysiology of dia- S. aureus colonizes on skin or mucosal surfaces of children
betic foot infection (DFI) is complex and the wound outcome and HIV or diabetic patients who are more prone to S. aureus
is determinant on both host factors and microbial factors in- colonization [10–13]. Hospital-acquired methicillin-resistant
cluding virulence [3, 4]. Diabetic foot ulcers (DFU) are quite S. aureus (MRSA) strains are largely disseminated in clinical
settings and infect immunosuppressed hosts while
community-associated MRSA strains can cause infections in
* Thokur Sreepathy Murali healthy children and adults [13, 14]. Infection of mucosal
murali.ts@manipal.edu surface or skin is a consequence of initial exposure eventually
triggering upregulation of virulence genes [15]. S. aureus can
1
Department of Biotechnology, Manipal School of Life Sciences, also cause recurrent infections throughout life.
Manipal Academy of Higher Education,
Manipal, Karnataka 576104, India
S. aureus is the predominant bacterial isolate reported from
2
occidental countries in DFI leading to delayed wound healing.
Present address: Department of Medical Laboratory Technology,
Manipal College of Health Professions, Manipal Academy of Higher
Wound adherence, persistence, and infection is enhanced by
Education, Manipal, Karnataka 576104, India virulence factors including wide variety of enzymes and
2236 Eur J Clin Microbiol Infect Dis (2020) 39:2235–2246

toxins elicited by S. aureus such as protease, lipases, nucle- were then collated to understand the role of various S. aureus
ases, hyaluronidases, haemolysins (alpha, beta, gamma, and virulence markers and clonal complexes in wound infections.
delta), and collagenase which make host tissues favorable for
bacterial growth and tissue invasion. Early diagnosis and
proper wound management are critical since spread of S. DFU microbiome and altered
aureus to soft tissue and bone can significantly contribute to physiopathology
amputation of lower extremities [16].
Since DFU is polymicrobial nature, it is essential to con- Studies have reported the polymicrobial nature of DFU and
sider both the microbiological and clinical features to under- the presence of large spectrum of microbes severely limits the
stand microbial virulence potential of diverse microbes that use of traditional culture methods [7]. DFU is commonly col-
cause infection and level of host susceptibility to the microbes onized with aerobic Gram-positive cocci, facultative and ob-
[17, 18]. Each bacterial species differs in its virulence poten- ligate aerobic Gram-negative bacilli, obligate anaerobic bac-
tial in wound environment, and it is important to evaluate the teria [5, 20], and fungi [21]. The widespread occurrence of
intrinsic virulence factors of isolated species to characterize pathogenic and multidrug-resistant strains such as MRSA
and distinguish between pathogens that cause infection and which express several virulence factors negatively influences
colonizers [19]. In addition, it also helps to avoid misuse of treatment outcomes and leads to chronicity of ulcer. Screening
antibiotics since inappropriate antibiotic usage leads to emer- of specific virulence genes and genotyping by multilocus se-
gence of multidrug-resistant pathogens, notably MRSA. Quite quence typing approach have shown that S. aureus isolates
often, differentiation of true infectious pathogens from colo- from monomicrobial and polymicrobial wounds differ in their
nizers is difficult especially in DFU due to the underlying risk clonal diversity and carriage of virulence genes [22]. Though
factors of neuropathy and ischemia. In this regard, studies infection in diabetic subjects by definition can include ab-
have been performed focusing on virulence markers and their scesses, necrotizing fasciitis, and osteomyelitis among many
association in wound adherence and colonization. In this re- others, infected neuropathic diabetic foot ulcers remain the
view, we have focused on S. aureus virulence factors and major problem [18]. Host factors such as neuropathy drasti-
clonal complexes commonly associated with skin and wound cally reduce the sensory functions and pain perception causing
pathogenesis and their role in differentiating colonizing and ulceration which predisposes these wounds to severe bacterial
infecting S. aureus strains in DFI. infections [18]. Furthermore, it has been observed that early
signs of infection can go undetected due to several underlying
risk factors including reduced immunological functions [23],
Search strategy and selection criteria and if left untreated, the infection spreads to deeper tissues
including bones. In diabetic subjects, impaired wound healing
The relevant reference articles were identified through litera- due to an increase in acute inflammatory cells, an absence of
ture search in PubMed and Web of Science databases and cellular growth, and decreased epidermal cell migration have
were restricted to those research articles published between been observed. In addition, the impaired host responses can
January 1980 and March 2020. The following descriptors shift the equilibrium from colonizers to pathogenic species
were used to obtain relevant references: “Staphylococcus leading to chronic non-healing wound ulcers.
aureus,” “ulcer,” “osteomyelitis,” “infection,” “chronic
wound,” “microbiota,” “virulence,” “toxins,” “molecular
methods,” “clonal complexes,” “bacterial colonization,” “an- Diabetic foot osteomyelitis
timicrobial resistance,” “adherence,” “colonization,” “genetic
diversity,” “gene expression,” “host factors,” “pathogenesis,” Osteomyelitis is an inflammatory condition resulting from
and “biofilm” in combination with the term “Staphylococcus infection of bone and is commonly missed or underdiagnosed
aureus” and “diabetic foot” or “diabetic foot osteomyelitis” in patients with underlying diabetic foot ulcer complications.
and the Boolean operators AND, OR, and NOT, in addition to Reports suggest that 60% of DFUs get infected and 10–15%
truncations. We have included cohort studies, cross-sectional of the infected wounds usually develop into osteomyelitis
studies, narrative reviews, and case-control studies. Only full- [24]. S. aureus is the primary pathogen associated with dia-
text articles published in English language were included. The betic foot osteomyelitis (DFOM) and results in substantial
first screening included a review of the titles of the studies. morbidity and mortality. Studies indicate that S. aureus can
The second screening was based on the abstracts and dupli- form biofilms on healthy bones and infect both osteoblasts
cates and articles which did not meet the eligibility criteria and osteoclasts while both in vivo and in vitro studies clearly
were excluded. The final dataset included 140 full-text arti- show that they can also replicate and proliferate inside osteo-
cles, meeting the inclusion criteria. The identified articles were clasts and evade destruction by immune cells [25, 26].
reviewed and then classified based on the study objective and Interestingly, even though antibodies for various S. aureus
Eur J Clin Microbiol Infect Dis (2020) 39:2235–2246 2237

antigens (coagulase, lukD, lukE, fibronectin-binding protein, SSTI [39, 40], recurrent mucocutaneous infections [41], and
etc.) are produced in healthy individuals, S. aureus overcomes necrotizing pneumonia [42]. Further, PVL-carrying strains
protective immune responses and causes recurrent infections can cause chronic SSTI and necrotizing pneumonia in other-
by producing pathogenic antibodies that can drastically over- wise healthy individuals (Table 1). Though PVL-encoding
come adaptive immunity [27, 28]. However, genome se- strains are much less prevalent in community with < 10%
quencing of two S. aureus strains collected longitudinally MSSA clinical isolates found to encode pvl gene, studies in-
from a chronic osteomyelitis patient showed agrC frameshift dicate that isolates carrying gene coding for PVL can result in
mutations over time resulting in reduced virulence and less wound worsening.
tissue damage [29]. Mass-based proteomics approach in a
murine osteomyelitis model demonstrated that mutations in Enterotoxins
exoprotein regulatory protein saeRS and staphylococcal ac-
cessory regulator sarA attenuates virulence by downregulating S. aureus produce several exoproteins including staphylococ-
virulence factor production and degradation of virulence fac- cal enterotoxins (SEA, SEB, SECn, SED, SEE, SEG, SEH,
tors respectively [30]. Víquez-Molina et al. [31] compared the and SEI), exfoliative toxins (ETA and ETB), and leukocidin
prevalence of virulence genes encoding for pvl, etA, etB, and (Fig. 1). Toxic shock syndrome toxin (TSST-1) and staphylo-
tsst in S. aureus strains in SSTI and bone infection and found coccal enterotoxins, collectively termed as pyrogenic toxin
no significant difference in virulence gene profiles except for superantigens (PTSAgs), are known to play a significant role
higher prevalence of pvl+ strains in soft tissue infections. in proliferation of T cells irrespective of antigenic specificity.
Even though several clonal complexes are associated with The majority of S. aureus isolates of DFU produce large num-
DFU colonization and infection, there are limited studies on ber of SAgs [68], while SAg exotoxins have also been shown
virulence genes and clonal complexes associated with DFOM. to contribute significantly to other major illnesses [69]. Higher
Lattar et al. [32] performed molecular fingerprinting of S. number of S. aureus strains isolated from wound grades 2–4
aureus strains from patients with osteomyelitis by pulsed- of Wagner Classification System was shown to harbor genes
field gel electrophoresis and concluded that loss of capsular encoding enterotoxins SEA and SEI than strains from grade 1
polysaccharide production was the major factor associated ulcer [70], making them potent markers to differentiate colo-
with chronic osteomyelitis. They also showed that higher pro- nization from infection. Interestingly, S. aureus strains from
portion of cap5 S. aureus isolates were methicillin-resistant S. DFU share more similarity with strains from atopic dermatitis
aureus (MRSA) and lukS-PV/lukF-PV+ compared with cap8 and normal vaginal mucosa in their distribution and produc-
isolates [32]. Senneville et al. [33] reported bone tropism of tion of more types of SAgs per organism suggesting that DFU
CC398 methicillin-susceptible S. aureus clone and its signif- strains originated and were better adapted to skin compared
icance in DFOM. with mucosae which produce fewer SAgs.

Toxic shock syndrome toxin-1

Virulence factors of S. aureus
Toxic shock syndrome toxin-1 (TSST-1), a 22-kD SAg,
α-Toxin causes toxic shock syndrome. Another new member of the
SAg family, SEI-X, is known to cause necrotizing pneumonia
In skin infections, α-toxin is considered a key virulence factor [58]. Both SEI-X and TSST-1 have potential role in DFU
of S. aureus. This pore-forming toxin consisting primarily of pathogenesis [68]. Even though the carriage of tsst-1 is low
beta sheets is secreted by most of the S. aureus strains as a in DFU isolates, they are significantly more abundant in grade
water-soluble monomer targeting the red blood cells [34–36]. 4 ulcer than in DFOM [33].
The gene coding for alpha toxin hla was present in S. aureus
strains in all the grades of wounds in DFU though some dif- Epidermal cell differentiation factor
ference was observed between MRSA and methicillin-
susceptible S. aureus (MSSA) strains [37, 38]. The epidermal differentiation factor (EDIN) and EDIN-like
factors are a family of exotoxins that specifically inhibit host
Panton-Valentine leukocidin protein RhoA [59], which negatively impacts host tissue by
favoring bacterial dissemination and hindering complement-
Panton-Valentine leukocidin (PVL) is a potent cytotoxin that mediated phagocytosis. Recent findings hypothesize the role
consists of two chromatographically separate protein compo- of EDIN in disseminating between tissues by hematogenous
nents, namely LukS-PV (slow) and LukF-PV (fast). The ac- route through intracellular tunnel formation in endothelial
tive toxin causes lysis of neutrophils by forming a pore on its cells named macroapertures [60, 71]. Edin-positive strains
membrane and is associated with dermonecrosis, chronic were found to be more prevalent in moderate-to-severe grade
2238 Eur J Clin Microbiol Infect Dis (2020) 39:2235–2246

Table 1 S. aureus virulence factors involved in wound progression

Virulence factors Function Role in infection References

MSCRAMMs
Bone sialoprotein-binding Adhesion to extracellular matrix, bone and joint tissue, fibrinogen Osteomyelitis [43, 44]
protein (isoform of SdrE)
(Bbp)
Cap5 and Cap8 Inhibits interaction between C3b, immunoglobulin and receptors; Mastitis, cystic fibrosis, [45]
targets phagocytes; promotes virulence in Caenorhabditis elegans endocarditis
Collagen adhesin (Cna) Collagen-binding adhesin mediates binding to cartilage/ collagen-rich Osteomyelitis, septic arthritis, [46–49]
tissue, blocks complement activation keratitis
Fibronectin-binding proteins FnBPA binds to fibrinogen and elastin; FnBPB binds to fibronectin; Endocarditis, implant orthopaedic [46, 48,
A (FnBPA) and B adhesion to ECM infections, osteomyelits, 50]
(FnBPB) arthritis
Iron-regulated surface Haem uptake and iron acquisition into bacterial cytoplasm SSTI [51]
determinant protein H
(IsdH)
Serine–aspartate Binds desquamated epithelial cells; nasal colonization Bone infection [52–55]
repeat-containing protein
D (SdrD)
SdrE Binds complement factor H; evades immune response; degrades C3b SSTI [56]
Bone sialoprotein-binding SD-rich fibrinogen-binding, bone sialoprotein-binding protein Osteomyelitis, arthritis [57]
protein (isoform of SdrE)
Toxins/superantigens
Epidermal cell Inhibits actin cytoskeleton of epithelial and endothelial barrier; Bacteremia [58–61]
differentiation inhibitor formation of large transcellular tunnels; targets host Rho proteins;
(Edin) inhibits complement-mediated phagocytosis
LukDE Kills leukocytes and macrophages via chemokine receptors Dermonecrosis [62, 63]
PVL Targets complement receptors C5aR and C5L2, apoptosis of Necrotizing pneumonia, [42,
neutrophils, necrosis SSTI, furunculosis 64–67]

DFUs than in low-grade infection. These strains were also several virulence determinants is known to be affected by
associated with agrI cluster and virulence markers including the inhibitory activity of agr groups representing a form of
genes coding for hemolysin, the egc cluster of enterotoxins, bacterial interference [73]. A recent study reported that strains
lukDE, intracellular adhesion proteins (icaA, icaC, and icaD), carrying agr were more pathogenic than those without [74].
cap5, MSCRAMM (clfA, clfB, fib, ebpS, and fnbA), and anti-
biotic resistance (tet and fosB). Edin-positive isolates grouped
to four major clonal complexes, a singleton closely associated Arginine catabolic mobile element
with CC8 (edin-A), a singleton belonging to ST152-MSSA
(edin-B), CC80-MRSA (edin-B), and mostly CC25/28 MSSA Arginine catabolic mobile element (ACME), a genetic island
(edin-A). It is also reported that grade 1 ulcer infected with consisting of clusters of genes, confers S. aureus the ability to
edin-positive strains led to poor wound outcome [72]. While colonize skin. ACME is horizontally transferred from S.
CC25/CC28-MSSA and CC80-MRSA were significantly epidermidis, a skin commensal [75], and encodes multiple
higher in edin-positive isolates, none of them grouped to col- genes, among which arc (arginine deiminase system) and
onizing strains of CC5/CC8 [72]. Association of edin-positive opp-3 (a ABC transporter) are vital in enhanced colonization.
strains with other virulence markers in DFU has also been The arginine deiminase catabolizes L-arginine and by elevat-
reported. Thus, EDIN coding genes can be considered potent ing skin’s pH makes it more amenable for microbial coloni-
markers to categorize S. aureus strains as colonizers or infec- zation [76]. Opp-3 enhances eukaryotic cell adhesion, peptide
tious as well as reliable predictors of the wound outcome. nutrient uptake, and resistance to antimicrobial peptides,
thereby promoting the ability of bacteria to thrive on human
skin. Thus, the acquisition of the mobile element has a poten-
Accessory gene regulator tial role in disruption of skin barrier and bacterial invasion.
Studies with S. aureus USA300 strain highlight the impor-
S. aureus pathogenicity is enhanced by quorum sensing (QS) tance of ACME locus in enhancing pathogenicity and clonal
mechanisms. Virulence factors essential for causing SSTI are dissemination. Even though, ACME has a potent role in suc-
regulated by accessory gene regulator (agr). Expression of cess of USA300, its deletion has shown contradictory effect
Eur J Clin Microbiol Infect Dis (2020) 39:2235–2246 2239

Fig. 1 Schematic diagram illustrating major S. aureus factors associated complex, PSM transporter complex; PSM, phenol-soluble modulins;
with DFI and DFOM. (Adapted from Kong et al. [13]). ACME, arginine PVL, Panton-Valentine leukocidin; SAgs, super antigens; sarA, staphy-
catabolic mobile element; agr, accessory gene regulator; Bbp, bone lococcal accessory regulator; sae, response regulator; SdrD, serine–
sialoprotein-binding protein; CC, clonal complexes; Cna, collagen aspartate repeat-containing protein D; SEs, staphylococcal enterotoxins;
adhesin; FnBP, fibronectin-binding protein; MSCRAMMs, microbial SspA, staphylococcal serine protease; SspB, cysteine protease; TSST-1,
surface components recognizing adhesive matrix molecules; PMT toxic shock syndrome toxin-1

on competitive fitness in skin infection models [77, 78]. inactive small-colony variants, which exhibit significant
ACME speG and ACME arc genes mediate enhanced synthe- phenotypic and metabolic differences from regular S. aureus
sis of polyamines in skin and cause clearance of S. aureus in isolates [82–85]. However, these S. aureus variants are rela-
murine skin abscess model [79]. Survival of USA300 in acidic tively antibiotic resistant and hinder the treatment efficacy
environment is mediated by genes encoded by arc operon [79] [86, 87]. Fibronectin-binding proteins (FnBPs) are the major
and biofilm formation is enhanced by ACME speG-mediated staphylococcal adhesins which help in colonization of hu-
polyamine tolerance [80] by upregulating genes involved in man airway epithelial cells and fibroblasts and thereby es-
biofilm production and by increased adhesion properties, tablish staphylococcal infection [88]. S. aureus FnBPs also
thereby favoring skin colonization, persistence, and play a critical role in orthopaedic implant-associated infec-
transmission. tions, osteomyelitis, and arthritis [82].

Microbial surface components recognizing adhesive Phenol-soluble modulins

matrix molecules
Phenol-soluble modulins (PSMs) also play significant role in
Infection of a host commences with the pathogen binding to S. aureus skin infection [89]. PSMs are pore-forming toxins
host surface components (fibrinogen, fibronectin, and epi- made up of a family of seven amphipathic α-helical peptides.
dermal keratinocytes). A family of staphylococcal cell Most of the S. aureus strains secrete PSMs [14] that provide
wall-anchored adhesins, called MSCRAMMs (microbial them capacity to lyse human neutrophils, monocytes, erythro-
surface components recognizing adhesive matrix mole- cytes, and osteoblasts [89] increasing tissue toxicity. Most
cules), plays a significant role in aiding attachment of S. pathogenic strains of staphylococci elicit different PSMβ pep-
aureus virulence proteins to bone matrix and collagen [81]. tides (PSMβ1 and 2), PSMα peptides (PSMα1–4), and a δ-
In osteoblasts, MSCRAMMs play a significant role by toxin, thus contributing to staphylococcal pathogenesis and
allowing bone invasion and formation of metabolically virulence [89]. PSMs themselves exhibit selective
2240 Eur J Clin Microbiol Infect Dis (2020) 39:2235–2246

antimicrobial function and PSM-inspired peptides are report- Clonal complexes

ed to have considerable bactericidal activity against
multidrug-resistant bacteria [90]. Staphylococci isolated from DFU have been found to be ge-
netically diverse, resistant to many antibiotics and harbor sev-
eral virulence determinants [100]. Using multilocus sequence
Extracellular adherence protein typing, strains of S. aureus could be grouped into clonal line-
ages and the major clonal lineages in humans were found to
Extracellular adherence protein (Eap), a 45–70-kDa protein belong to clonal complex (CC)1, CC5, CC8, CC9, CC12,
that binds to several proteins including fibronectin, is report- CC15, CC22, CC30, CC45, and CC51 [101]. In DFU, CC5
ed to be a significant marker of impaired wound healing in methicillin-sensitive S. aureus (CC5-MSSA), CC8-MSSA,
mouse model [91, 92]. Eap inhibits neovascularization by and CC15-MSSA were considered to be colonizing strains
hindering the inflammatory cell response near the wound with a favorable outcome while CC45-MSSA strains were
area. Studies indicate that Eap interferes in ICAM-1 (inter- shown to cause severe infections [37, 72, 102]. In addition,
cellular adhesion molecule-1)-dependent leukocyte- CC45 and CC30 were also considered as causative clones of
endothelium interactions restricting host leukocyte recruit- severe invasive infections [103, 104]. It is believed that DFU
ment, thereby aiding in persistence of S. aureus in a hostile showing worsening outcome do not colonize with CC5/CC8-
milieu in chronic wounds [93]. In contrast, Eap does not play MSSA strains and clonality of these strains during admission
a major role in virulence of S. aureus in skin wound infection and follow-up visit remain unchanged. CC25/CC28-MSSA
models as well as systemic infection models, since Eap does and CC80-MRSA strains are also considered as infecting
not contribute to bacterial adherence to proteins other than strains in DFU as these CCs were found significantly higher
ICAM-1 [93]. However, Eap does contribute to enhanced in edin-positive strains (Table 2), edin gene being a predictive
adhesion and internalization of staphylococci by risk marker for worsening ulcer [72]. Even though clonal lin-
keratinocytes in a FnBP-independent manner. Eap secreted eages found associated with humans and animals generally are
by S. aureus also significantly contributes to the internaliza- different, livestock-associated CC398 (LA-CC398] strain, as-
tion of other pathogenic bacteria in the wound microenviron- sociated with pigs, has emerged as a major human pathogen
ment [94]. causing severe infections [129–131], ventilator-associated
pneumonia [132], and wound infections [133]. CC398 is sig-
nificantly associated with diabetic foot osteomyelitis (DFOM)
Biofilm factors strains and helps to differentiate DFOM from SSTI—two ma-
jor complications of DFU—both of which are known to carry
Biofilm production is an important strategy adopted by bac- CC45-MSSA [33]. CC398 is distinct with the presence of
teria to colonize and infect skin tissues [95]. Though bacteria hemolysins, genes that code for intracellular adhesion pro-
can be found in planktonic form in chronic wounds, they are teins, cap5, and MSCRAMM genes including bbp, clfA and
most likely observed to form polymicrobial communities in clfB [33], pvl [134], and multiple classes of antimicrobial re-
biofilm matrix [96]. The presence of biofilms in non-healing sistance genes [135] showing potent virulence in SSTI infec-
wounds contributes significantly in hindering the effective- tions in humans.
ness of antimicrobial agents and in overcoming host immu- Association between presence of certain virulence genes
nity. Bioactive compounds from biofilm communities of S. and DFU outcome is reported in many studies. For instance,
aureus and Pseudomonas aeruginosa have been shown to difference in the size of abscess formation in rabbit skin ab-
impair migration and proliferation of keratinocytes in chron- scess model was attributed to different clonal lineages [136].
ic skin wounds and chronic tympanic membrane perforations Different outcomes with difference in abscess diameter rang-
[97]. In vitro studies also have shown that biofilm- ing from 5 to 7 cm (USA300, USA500, and ST80) and from 2
conditioned media (BCM) from these two bacteria could to 4 cm (USA400, USA1000, ST72, USA100) and almost
inhibit cell proliferation while BCM derived from S. aureus complete absence of abscess (USA200, USA1100) were man-
was shown to reduce cell migration in keratinocytes and ifested by different S. aureus clonal lineages. Abscess size
fibroblast cells in wound scratch assays [98]. Proteomic anal- caused by USA300 was found to be comparable with that
ysis of these media revealed several proteins linked to de- caused by USA100, USA200, USA400, USA1100, and
layed wound healing including alpha hemolysin and epider- ST72 strains and different from those carrying USA500,
mal cell differentiation inhibitor [97]. In other studies, loss of USA1000, and ST80 strains. Interestingly, though abscess
HEK cell viability by S. aureus BCM has been reported [98, formation by Panton-Valentine leukocidin (PVL)-positive
99]. HEKa cells treated with BCM showed upregulation of USA300 and PVL-negative USA500 was comparable, the
CXCL2, IL-8, DUSP1, and ATF3 genes which play a major role of PVL in skin infection is thought to be limited in nature.
role in inflammation and apoptosis [99]. Furthermore, neutrophil lysis activity of USA300 was shown
Eur J Clin Microbiol Infect Dis (2020) 39:2235–2246 2241

Table 2 Clonal lineages and associated virulence markers of S. aureus in skin and wound infection

Source of sample Major virulent factors/major findings Prevalent genotype Reference

SSTI pvl ST152, ST121, ST5, ST15, ST1, ST8, and ST88 [105]
SSTI, surgery infection, bone CapH5, capJ5, capK5 CC5, CC8, CC97 [106]
and joint infection, and capH8, capI8, capJ8, and capK8 CC45
others egc cluster CC5, CC45
Absence of fnbB ST228-I
Cna ST239-III and ST45-IV
SSTI hla ST239 [107]
Impetigo eta CC15, CC9, and ST88 (CC88) [108]
eta, etb ST121
Wound, urine, semen egc CC5, CC25, CC30, CC45, CC121 [109]
etd CC25, CC80
edinB CC25, CC80, CC152
Wounds, nares, blood, sputum, egc cluster CC5, CC22, CC30, and CC228 [110]
urine, and others sed, sej, ser CC8
Tst1 CC5, CC30
Wound and respiratory PVL ST80-MRSA-IVc [111]
samples
Bone and joint infections ACME CC8-MSSA [112]
EtD, edinB CC25, CC80
capH8, capI8, capJ8, capK8 CC7, CC12, CC15, CC30, CC45, CC59, ST80,
CC88, ST96, CC101, CC121, ST239 and
ST426
cna CC12, CC22, CC30, CC45, CC96, CC121,
ST239, and ST426
sasG (S.aureus surface protein G) CC5, CC8, CC15, CC22, ST49, CC59, ST80,
CC88, and ST96
Invasive infections Egc CC5, CC25, CC30, and CC45 [113]
Tst CC30
Etd CC25
Invasive infections Tst--1 CC30/CC39 [114]
SSTI, respiratory tract Hla, psmα, RNAIII ST59 [115]
infections, osteomyelitis sasX (cell wall-anchored protein) ST239-MRSA-SCCmecIII-t037
Nasal swabs Increased biofilm production at 0%, 0.1%, CC8 [116]
and 0.25% glucose concentrations
Higher mortality rate; PSMα3 peptide variant with CC30 [117, 118]
reduced immune-stimulatory and cytolytic activity
Osteo-articular infection CC22 [119]
Community settings agr-I CC59 [120]
Community settings PVL ST1153-MSSA [121]
Community, pvl ST1, ST5, ST8, ST22, ST30, ST80, ST772, [122–125]
multiple clinical settings ST452, ST59, ST93, CC121, and ST154
pvl negative ST239 [124]
Hospital settings Tn6072 ST239 [126]
Hospitalized patients at risk of cna CC1, 12, 22, 30, 45, 51, and 239 [127]
MRSA carriage TSST-1 CC30
Multiple clinical samples High level of Hla production CC1, CC5, CC8, CC15, or CC96 [128]
Complete absence of Hla production CC22, CC30, CC45, CC479, CC705

to be significantly higher than that of other strains, and was neutrophils lysis, whereas α-toxin and N-formylated PSMα3
suggested to be a major determinant of MRSA skin infection peptide correlated with neutrophil lysis. Though the role of
pathogenesis [136]. USA300 strains showed correlation be- different S. aureus clonal lineages from blood stream infec-
tween the expression of psmα, hla, and agr (with the excep- tions is available, detailed studies on clonal types on DFI and
tion of lukS-PV) and cause abscess, release cytokine, and lyse their role in wound outcome are relatively less explored.
2242 Eur J Clin Microbiol Infect Dis (2020) 39:2235–2246

ST22 (CC22) is reported as a common type in DFU infec- Pathogenicity was assessed by the survival time of the nema-
tions and all ST22 strains were shown to be positive for viru- tode upon ingestion of S. aureus which was represented by
lence factors clfa and agr I. Several less frequent clones have LT50 and LT100 (time required to kill 50% and 100% of nem-
also been reported suggesting that diabetic patients can be an atodes, respectively). Isolates from ulcer grades 2–4 showed
important route for dissemination of clones between hospital LT50 < 2 days, whereas LT50 was > 3 days for isolates from
and community settings [100]. Sotto et al. [37] reported that grade 1 ulcer. LT50 of strains obtained from healing wounds
foot ulcers with S. aureus strains of CC5 and CC8 showed was higher at the time of entry as well as follow-up while
favorable wound outcome and hypothesized that S. aureus of strains from non-healing ulcers had lower values. Messad
CC5/CC8 clones as colonizing and others as infecting clones. et al. [140] identified genetic elements associated with pro-
phage in S. aureus genome to promote colonization.

Distinguishing colonization from infection

in DFU Conclusion
The Infectious Diseases Society of America and International DFUs are extremely vulnerable to bacterial infections that can
Working Group on the Diabetic Foot together have result in lower limb amputations and even death. Though from a
established specific clinical criteria to distinguish different clinician’s perspective, it is important to differentiate coloniza-
grades of DFI severity [137, 138]. According to this classifi- tion from infection, it might prove cumbersome in DFU due to
cation, grade 1 wound is considered colonized wound while the underlying effects of neuropathy and/or ischemia. The
grade 2 or more is considered infection. Sotto et al. [37] polymicrobial community in DFI further contributes to syner-
screened S. aureus isolates from DFU of varying grades from gistic interaction between wound pathogens and induces various
1 to 4 for various virulence genes and identified several toxins virulence traits and modulates host immunity and overall wound
including leukocidins, enterotoxins, exfoliatins, and toxic deterioration. Prompt recognition of worsening ulcers using pre-
shock syndrome toxin and reported that strains from grade 1 dictive molecular markers will hence considerably help in
foot ulcer to have low prevalence of virulence genes. Further, preventing lower limb amputations. Distribution of isolates into
they extended their study [70] to assess clonality and carriage different clonal complexes allows comparison between coloniz-
of 31 highly prevalent virulence-associated genes to predict ing and infecting strains as well as determining the origin and
the wound outcome. Among the 31 genes screened, 10 genes clonality of the strains infecting wound ulcers. Detection of
(sea, seb, sec, sei, sej, hlb, hlg, hlgv, cap5, and lukE) were specific virulence encoding genes along with clonality in differ-
found to be significantly associated with strains from grade 2– ent grades will help us in identifying S. aureus strains that could
4 ulcers, whereas cap8 gene was associated with strains from cause severe negative wound outcome in DFI and also to avoid
grade 1 ulcers. None of the isolates from worsening wounds misuse of antibiotic therapy in uninfected wounds.
belonged to CC5 and CC8 indicating links between clonality
and wound healing. However, no significant difference was Acknowledgments The authors thank TIFAC-CORE in
found between infected and uninfected ulcers with regard to Pharmacogenomics for funding, DST-FIST, Government of India for
genes coding for PVL and exfoliatins [37]. But contrasting facilities, Manipal Academy of Higher Education for the support and
funding. KS thanks DST – INSPIRE, Government of India for fellow-
observation was found with reference to association of ship. TSM thanks Ms. Apoorva Jnana for help and Dr. Satyamoorthy for
exfoliatins in different grades of DFU. Exfoliatin genes were support and encouragement.
found to be more likely in strains isolated from grade 4 ulcer
compared with lower grades. In addition, their serotype distri- Code availability Not applicable
bution also varied with eta and etb being found very rarely
Authors’ contribution Both the authors contributed to the idea of the
(1.3%) or absent in most samples while etd (3.7%) was found article. Literature search and data analysis were performed by Kavitha.
in higher frequency. However, grade 1 ulcers harboring S. First draft was written by Kavitha and critically revised by Murali. Both
aureus strains carrying etd gene showed worsening wound the authors read and approved the final manuscript.
outcome [72]. Post et al. [139] showed important differences
in the presence of eta and etb gene in diabetic foot infection Funding information Open access funding provided by Manipal
Academy of Higher Education, Manipal.
(eta, 13%; etb, 17%) and osteomyelitis (eta, 22% and etb,
absent). One of the limitations found was the study was con- Availability of data and material Not applicable.
ducted solely on S. aureus isolates of monomicrobial wound
type, while DFU is predominantly polymicrobial in nature.
Compliance with ethical standards
Using a Caenorhabditis elegans model, Sotto et al. [70]
showed that the pathogenicity of S. aureus strains in DFU Conflict of interest The authors declare that they have no conflict of
grades higher than 2 were significantly more than in grade 1. interest.
Eur J Clin Microbiol Infect Dis (2020) 39:2235–2246 2243

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epidemic community-associated methicillin-resistant
Consent to participate Not applicable Staphylococcus aureus. Proc Natl Acad Sci U S A 106:5883–
5888
15. Novick RP (2003) Autoinduction and signal transduction in the
Consent for publication Not applicable
regulation of staphylococcal virulence. Mol Microbiol 48:1429–
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