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Whole genome sequencing and pan-genome analysis of Staphylococcus/Mammaliicoccus spp. isolated from diabetic foot ulcers and contralateral healthy skin of Algerian patients

BMC Microbiology volume 23, Article number: 342 (2023) Cite this article

Abstract

Background

Diabetic foot infections (DFIs) are the most common complications of diabetic foot ulcers (DFUs), and a significant cause of lower extremity amputation. In this study we used whole genome sequencing to characterize the clonal composition, virulence and resistance genetic determinants of 58 Staphylococcus/Mammaliicoccus spp. isolates from contralateral healthy skin and DFU from 44 hospitalized patients.

Results

S. aureus (n = 32) and S. epidermidis (n = 10) isolates were recovered from both DFUs and healthy skin, whereas, S. haemolyticus (n = 8), M. sciuri (n = 1), S. hominis (n = 1) and S. simulans (n = 3) were recovered exclusively from healthy skin. In contrast, S. caprae (n = 2) and S. saprophyticus (n = 1) were recovered only from DFUs. Among S. aureus isolates, MRSA were present with high prevalence (27/32, 84.4%), 18 of which (66.7%) were from DFUs and 9 (33.3%) from healthy skin. In contrast, the coagulase-negative Staphylococcus (CoNS)/Mammaliicoccus isolates (n = 26), in particular S. epidermidis and S. haemolyticus were more prevalent in healthy skin, (10/26, 38.5%) and (8/26, 30.8%), respectively. MLST, spa and SCCmec typing classified the 32 S. aureus isolates into 6 STs, ST672, ST80, ST241, ST1, ST97, ST291 and 4 unknown STs (STNF); 8 spa types, t044, t037, t3841, t1247, t127, t639, t937 and t9432 and 2 SCCmec types, type IV and type III(A). Among CoNS, the S. epidermidis isolates belonged to ST54, ST35 and ST640. S. haemolyticus belonged to ST3, ST25, ST29, ST1 and ST56. The sole M. sciuri isolate was found to carry an SCCmec type III(A). A wide range of virulence genes and antimicrobial resistance genes were found among our isolates, with varying distribution between species or STs. The pan-genome analysis revealed a highly clonal population of Staphylococcus isolates, particularly among S. aureus isolates. Interestingly, the majority of S. aureus isolates including MRSA, recovered from the healthy skin and DFUs of the same patient belonged to the same clone and exhibited similar virulence/resistance genotype.

Conclusions

Our study provides clinically relevant information on the population profile, virulence and antibiotic resistance of Staphylococcus/Mammaliicoccus spp. in DFIs, which could serve as a basis for further studies on these as well as other groups of pathogens associated with DFIs.

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Background

Diabetes is a fast-growing global problem with huge social, health, and economic consequences [1]. The prevalence of diabetes in 2021 was estimated to be 10.5% (536.6 million people), and it is expected to raise to 12.2% (783.2 million) in 2045 [2].

People with diabetes are at increased risk of long-term complications such as coronary heart disease, cerebrovascular and peripheral vascular diseases and diabetic foot ulcers (DFUs) [3]. It was estimated that 15% of diabetics will develop DFUs during their existence [4].

Microbial infections of the DFUs, termed diabetic foot infections (DFIs) are key contributors to the amputation risk [5]. Limb loss associated with DFUs have a significant negative impact on mobility, psychosocial well-being, and quality of life of the patients and increase healthcare costs [6, 7].

Bacterial species belonging to the genus Staphylococcus are common colonizers of skin and mucous membranes of humans and animals, but also opportunistic pathogens capable of causing a wide range of infections. The staphylococci can be differentiated into Coagulase-Positive (CoPS) and Coagulase-Negative (CoNS), based on their ability to produce coagulase. Recently, five species among the CoNS (S. sciuri, S. fleurettii, S. lentus, S. stepanovicii and S. vitulinus), and belonging to S. sciuri group, were reclassified into the novel genus Mammaliicoccus, with Mammaliicoccus (M.) sciuri as the type species [8].

The CoPS, which are considered as more pathogenic than the CoNS, include the notorious pathogen, S. aureus, which is the main causative agent of both community acquired and nosocomial infections in humans as well as in animals [9,10,11,12], including DFIs [12, 13]. S. aureus can deploy numerous virulence factors which are implicated in DFI and delayed wound healing process [14, 15]. However, the CoNS and the Mammaliicoccus are now known to be also frequently associated with clinical infections, including diabetic foot osteomyelitis [16], particularly in immune-compromised and hospitalized patients [17].

Both Staphylococcus and Mammaliicoccus species are becoming increasingly resistant to several antibiotics, as a result of the acquisition of resistance genes [18], limiting further the therapeutic options against the infections caused by these bacteria, and leading to worse clinical outcomes [19].

Thus, understanding the genetic characteristics of Staphylococcus and related bacteria in DFIs can be exploited for both therapeutic and diagnostic purposes.

Apart from one study by Djahmi et al. (2013) [20], data on the staphylococci associated with DFIs in Algeria are lacking. Therefore, in the present study we used whole genome sequencing (WGS) to characterize the genetic diversity, antibiotic resistance and virulence genetic determinants of Staphylococcus and Mammaliicoccus spp. isolates recovered from DFIs; and to assess the potential association between clones/species and/or virulence/resistance genes with PEDIS grades and the source of isolates.

Results

Species and source of Staphylococcus/Mammaliicoccus isolates

Eight Staphylococcus/Mammaliicoccus species were identified among the 58 isolates, including S. aureus (32/58, 55.2%), S. epidermidis (10/58, 17.2%), S. haemolyticus (8/58, 13.8%), S. simulans (3/58, 5.2%), S. caprae (2/58, 3.5%), S. hominis, S. saprophyticus and M. sciuri (1/58 each, 1.7%). The characteristics of the 58 isolates included in this study are shown in Table 1.

Table 1 Clinical, epidemiological and molecular characteristics of Staphylococcus/Mammaliicoccus species recovered from DFUs and Healthy skin (n = 58)
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Among the 32 S. aureus isolates, 23/32 (71.9%) were recovered from DFUs and 9/32 (28.1%) from healthy skin. The 10 S. epidermidis isolates were recovered from 8 patients, including 6/10 (60%) from healthy skin and 4/10 (40%) from DFUs. All the S. haemolyticus (n = 8), M. sciuri (n = 1), S. hominis (n = 1) and S. simulans (n = 3) isolates were recovered exclusively from healthy skin, but S. caprae (n = 2) and S. saprophyticus (n = 1) were recovered only from DFUs.

Among the 32 S. aureus isolates, 27/32 (84.4%) carried the mecA gene, and were therefore MRSA; 18/27 of which (66.7%) were from DFUs and 9/27 (33.3%) from healthy skin.

Statistically, S. aureus including MRSA isolates and CoNS were significantly associated with the source of isolation (p-value = 0.004).

Molecular typing of the Staphylococcus/Mammaliicoccus isolates

Clones of S. aureus were characterized based on the combination of MLST, SCCmec and spa typing. In silico determination of MLST revealed that S. aureus isolates belonged to 6 known STs including ST80 (16/32, 50%), ST241 (5/32, 15.6%), ST672 (3/32, 9.4%), ST1 (2/32, 6.3%), ST97 and ST291 (1/32 each, 3.1%), and 4 STNF (4/32, 12.5%).

Eight spa types were identified among S. aureus isolates. The dominant one was t044 (13/32, 40.6%), followed by t037 (9/32, 28.1%), t3841 (3/32, 9.4%), t1247 and t127 (2/32 each, 6.3%), t639, t9432 and t937 (1/32 each, 3.1%).

Two SCCmec types were identified among MRSA isolates, type IV (19/27, 70.4%) and type III(A) (8/27, 29.6%). The type IV isolates were assigned to subtype IVc(2B) (16/27, 59.3%) and IVd(2B) (3/27, 11.11%).

Among CoNS/Mammaliicoccus isolates, SCCmec type III(A) (1/26, 3.8%) was detected in the sole M. sciuri isolate.

The dominant MRSA clone (13/27, 48.1%) was ST80- t044- IVc(2B), followed by ST241-t037- III(3 A)) (5/27,18.5%); whereas, ST672- t3841- IVd(2B) and STNF- t037- III(3 A) were each represented by 3/27 (11.1%) isolates. In addition, 2 other spa types were detected among ST80- IVc(2B) isolates, t1247 (2/27, 7.4%) and t639 (1/27, 3.7%).

2/5 (40%) of MSSA isolates belonged to ST1-t127, while ST97-t9432, ST291-t937 and STNF-t037 were each represented by one isolate (1/5, 20%).

Among S. epidermidis isolates, 6/10 (60%) belonged to ST54, 2/10 (20%) to ST35 and 1/10 (10%) to ST640. 3/8 (37.5%) S. haemolyticus belonged to ST3 and 2/8 (25%) to ST25, while ST29, ST1 and ST56 were each represented by 1/8 (12.5%) isolate.

Virulence genes

The presence and distribution of the virulence genes are summarized in Tables 2,3 and S2.

A total of 116 virulence genes were detected among S. aureus isolates including 42 adhesion genes and a large number of type 8 capsular polysaccharide, immune evasion and exoenzyme genes.

Table 2 Virulence gene profiles in S. aureus isolates recovered from DFUs and Healthy skin (n = 32)
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Thirty-five toxin-encoding genes were found among MRSA/MSSA isolates including 6 hemolysins (hlgA, hlgB, hlgC, hlb, hld and hla/hly), 13 staphylococcal enterotoxins (se) and staphylococcal enterotoxin-like toxins (sel) (sea, seb, seh, sek, seq, seg, sei, sem, sen, seo, seu selk and selq), with different carriage proportions ranging from 3.1 to 100%. ST1 isolates carried the highest number (n = 7) of se/sel genes, sea, seb, seh, sek, seq, selk and selq. In contrast, none of the se/sel genes were detected among the ST80 isolates.

Remarkably, none of the Staphylococcus/Mammaliicoccus isolates harbored an exfoliatine toxin gene (eta or etb) or a toxic shock syndrome toxin (tst) gene.

The leukocidins genes (lukD, lukE) were detected in all S. aureus isolates (32/32,100%), while lukF/lukS-PV were detected only in ST80 isolates (16/32, 50%). In addition, edinB gene encoding epidermal cell differentiation inhibitors was also detected only in ST80 isolates (16/32, 50%) and in ST291- t937 (1/32, 3.1%).

On the other hand, the sole virulence factor found among the 26 CoNS/Mammaliicoccus isolates was the arginine catabolic mobile element (ACME), which was exclusively present in S. epidermidis isolates (9/26, 34.6%).

Table 3 Virulence gene profiles in CoNS/M. sciuri isolates recovered from DFUs and Healthy skin (n = 26)
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The association of virulence genes with S. aureus ST or CoNS/Mammaliicoccus species was statistically significant. In contrast, no statistically significant association was found between the presence/absence of virulence genes and PEDIS grades or the source of isolates (p-value > 0.05).

Resistance genes

S. aureus

The distribution of the genetic determinants of antibiotic resistance among the S. aureus is shown in Tables 4 and S2. Genes encoding β-lactam resistance, mecA and blaZ, were detected among S. aureus isolates at frequencies of 84.4% (27/32) and 50% (16/32), respectively.

Table 4 Resistance gene profiles in S. aureus isolates recovered from DFUs and Healthy skin (n = 32)
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The genes encoding aminoglycoside-modifying enzymes (AME), aph(3’)- III/ aph(3’)- IIIa, were the most prevalent among S. aureus isolates (25/32, 78.1%). All ST80 isolates (16/32, 50%) were positive for ant(6)-Ia and aph(3’)-III/ aph(3’)-IIIa genes, and all ST241 isolates (5/32, 15.6%) were positive for 3 AME genes, ant(9)-Ia, aac(6’)- aph(2’’) and aph(3’)- III.

Four genes encoding resistance to macrolide-lincosamide-streptogramin B (MLSB) were detected in S. aureus isolates. The msr(A) and mph(C) genes were detected in all the ST672 isolates (3/32, 9.4%), and the erm(A) in all the ST241 isolates (5/32, 15.6%).

Three genes encoding resistance to tetracycline were detected among S. aureus isolates, tet(M) (9/32, 28.1%), tet(K) (2/32, 6.3%) and tet(38) (6/32, 18.8%). Remarkably, only isolates belonging to t037 and harboring SCCmec-III (ST241 and STNF) carried tet(M).

The fusB and fusC genes coding for fusidic acid resistance were detected in all the ST80-t1247 (2/32, 6.3%) and ST1-t127 (2/32, 6.3%), respectively. The dfrG gene coding for trimethoprim-sulfamethoxazole resistance was detected only in ST241 isolates (5/32, 15.6%).

CoNS/ M. sciuri

As presented in Table 5, various resistance genes were also identified among the twenty-six CoNS/ M. sciuri isolates. 18/26 (69.2%) carried both mecA and blaZ genes. Remarkably, the single M. sciuri isolate harbored both mecA and mecA1 (1/1, 100%). 9/10 (90%) S. epidermidis isolates carried both fusB and fosB genes. In addition, msr(A) and mph(C) were detected in S. epidermidis (2/10, 20%) and S. haemolyticus (8/8,100%) isolates. Moreover, ermC was found in S. haemolyticus (5/8, 62.5%) and S. epidermidis (1/10, 10%) isolates. aac(6’)- aph(2’’)/ aac(6’)-Ie/aph(2’’)-Ia genes were detected in S .haemolyticus (7/8, 87.5%) and M. sciuri (1/1, 100%).

Table 5 Resistance gene profiles in CoNS species and M. sciuri recovered from DFUs and Healthy skin (n = 26)
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The aph(3’)-III/ aph(3’)-IIIa (6/8, 75%) and tet(K) (7/10, 70%) genes were found exclusively in S. haemolyticus and in S. epidermidis, respectively. Genes conferring resistance to streptogramin (vat(B), vat(C) and vgb(B)), macrolides (vga(B) and vga(A)LC), kanamycin/neomycin (aadD), tetracycline (tet(L)), streptomycin (str) and to quaternary ammonium compounds (qacB) were detected only in S. haemolyticus isolates, at a frequency of one gene per isolate(1/8, 12.5%).

Pan-genome analysis

The pan-genome of each species was determined and phylogenetic trees were built based on gene presence/absence matrix. Interestingly, isolates from DFUs are intermingled among the healthy skin isolates throughout the trees. A high number of accessory genes was observed, particularly, in S. epidermidis (4383, 72.3%), S. aureus (3997, 67.1%), S. haemolyticus (3874, 65.9%) and M. sciuri (3813, 65.7%) (Fig. 1).

Fig. 1
figure 1

Pan-genome representation of Staphylococcus/Mammaliicoccus spp. The pan-genome pie charts show gene content of Staphylococcus/Mammaliicoccus spp., determined by the Roary software. The pan-genome can be classified into core genes (the combination of core and soft core genes) and accessory genes (the combination of shell and cloud genes)

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The pan-genome analysis separated the ST80 S. aureus isolates into three subgroups (Fig. 2), one comprised of 10 closely related t044 isolates, the second comprised of 2 t1247 and 1 t639 related isolates, and the third contained 3 t044 isolates, which were more distantly related to ST80 strains from the other countries. S. aureus ST80, ST1, ST672, ST241 and STNF were found to be closely related to each other and were more distantly related to the reference strains.

Fig. 2
figure 2

Phylogenetic analysis of S. aureus based on the pan-genomes with Roary. On the right, the heatmap was generated using the presence and absence of core and accessory genes produced by Roary, genes are represented by white and blue bar for absence and presence, respectively. The phylogenetic tree was visualized in the online interactive viewer Phandango using the absence and presence matrix of genes and the tree file in the standard Newick tree format generated by Roary. Meta data were shown in the middle, reference strains (11819-97, S1475, S0924, S0924, AA45, MW2, LK34, AA51, MSSA476, 3688STDY6125016, AA6, GR1, VB12268, 3688STDY6124880, 3688STDY6124954, CM17, TW20 VB1490, 3688STDY6124945) were highlighted by black in the strain name

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The phylogenetic tree of S. epidermidis revealed two major clusters, the first included ST54 isolates, which were distinct from the reference S. epidermidis strains, and the second included the reference ST54 strains (Fig. 3). The ST35 isolates clustered together, but separate from the ST35 strains from other countries.

Fig. 3
figure 3

Phylogenetic analysis of S. epidermidis based on the pan-genomes with Roary. On the right of each panel, the heatmap was generated using the presence and absence of core and accessory genes produced by Roary, genes are represented by white and blue bar for absence and presence, respectively. Phylogenetic tree was visualized in the online interactive viewer Phandango using the absence and presence matrix of genes and the tree files in the standard Newick tree format generated by Roary. Meta data were shown in the middle, reference strains (SE68, BB424986M, BB403186T, C36, C40, 14.1.R1, C146, AK-612) were highlighted by black in the strain name

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The phylogenetic tree of S. haemolyticus revealed a clear distinction between our isolates and the reference strains (Fig. 4).

Fig. 4
figure 4

Phylogenetic analysis of S. haemolyticus based on the pan-genomes with Roary. On the right of each panel, the heatmap was generated using the presence and absence of core and accessory genes produced by Roary, genes are represented by white and blue bar for absence and presence, respectively. Phylogenetic tree was visualized in the online interactive viewer Phandango using the absence and presence matrix of genes and the tree files in the standard Newick tree format generated by Roary. Meta data were shown in the middle, reference strains (VB5326, OG2, SH_16, DE0439, CN1219, AA12, AA83, AA48) were highlighted by black in the strain name

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Similarly, S. simulans and S. caprae isolates (Figs. 5 and 6) from our study were closely related and more distantly related to the clinical isolates from China (CJ16) and Japan (JMUB145, JMUB590 and JMUB898), respectively.

Fig. 5
figure 5

Phylogenetic analysis of S. simulans based on the pan-genomes with Roary. On the right of each panel, the heatmap was generated using the presence and absence of core and accessory genes produced by Roary, genes are represented by white and blue bar for absence and presence, respectively. Phylogenetic tree was visualized in the online interactive viewer Phandango using the absence and presence matrix of genes and the tree files in the standard Newick tree format generated by Roary. Meta data were shown in the middle, reference strains (SNUC 1336, UMC-CNS-990, NCTC 11,046, 1H5, HAA294, MR1, CJ16) were highlighted by black in the strain name

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Fig. 6
figure 6

Phylogenetic analysis of S. caprae based on the pan-genomes with Roary. On the right of each panel, the heatmap was generated using the presence and absence of core and accessory genes produced by Roary, genes are represented by white and blue bar for absence and presence, respectively. Phylogenetic tree was visualized in the online interactive viewer Phandango using the absence and presence matrix of genes and the tree files in the standard Newick tree format generated by Roary. Meta data were shown in the middle, reference strains (JMUB145, JMUB590, JMUB898, M23864:W1, 1H22, 9557, SY333) were highlighted by black in the strain name

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S. saprophyticus and S. hominis isolates clustered with clinical isolates from India and the Netherlands, respectively (Figs. 7 and 8).

Fig. 7
figure 7

Phylogenetic analysis of S. hominis based on the pan-genomes with Roary. On the right of each panel, the heatmap was generated using the presence and absence of core and accessory genes produced by Roary, genes are represented by white and blue bar for absence and presence, respectively. Phylogenetic tree was visualized in the online interactive viewer Phandango using the absence and presence matrix of genes and the tree files in the standard Newick tree format generated by Roary. Meta data were shown in the middle, reference strains (HAB38, 2842STDY5753564, 1H9, C5, acrll, NC15, 384, ZBW5, UFMG-H7B) were highlighted by black in the strain name

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Fig. 8
figure 8

Phylogenetic analysis of S. saprophyticus based on the pan-genomes with Roary. On the right of each panel, the heatmap was generated using the presence and absence of core and accessory genes produced by Roary, genes are represented by white and blue bar for absence and presence, respectively. Phylogenetic tree was visualized in the online interactive viewer Phandango using the absence and presence matrix of genes and the tree files in the standard Newick tree format generated by Roary. Meta data were shown in the middle, reference strains (F2AH2Ly, HAF121, FDAARGOS_168, 63, SS536, SNUC 2120, CHK146-2161, VjHHoM0pEl_bin.12.MAG, SW396) were highlighted by black in the strain name

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However, our single M. sciuri isolate formed an outgroup, which was distinct from the rest of the reference strains (Fig. 9).

Fig. 9
figure 9

Phylogenetic analysis of M. sciuri based on the pan-genomes with Roary. On the right, the heatmap was generated using the presence and absence of core and accessory genes produced by Roary, genes are represented by white and blue bar for absence and presence, respectively. The phylogenetic tree was visualized in the online interactive viewer Phandango using the absence and presence matrix of genes and the tree file in the standard Newick tree format generated by Roary. Meta data were shown in the middle, reference strains (MIN-176, NS202, SS02, UFMG-H6, GDQ20D70P, SNUC 1353, FDAARGOS_285, MGBC107897, NCTC12103, 82,104, WHA07, BL01) were highlighted by black in the strain name

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Discussion

In this study, we investigated clonal composition, virulence and resistance determinants of Staphylococcus/Mammaliicoccus species isolated from DFUs and healthy skin. S. aureus was recovered from the same sampling site alone or in combination with other CoNS/Mammaliicoccus species.

S. aureus isolates, including MRSA recovered from the healthy skin and DFUs of each patient, belonged mostly to the same clone and had similar genotype (P1, P3, P9, P15 and P32). This is consistent with earlier findings that S. aureus isolates recovered from 4 distinct anatomical sites (oro-nasal cavity, periodontal pockets, skin and ulcer) of patients with type 2 diabetes were highly related in the same patient [21]. However, MRSA isolates belonging to different clones were also recovered from the same sampling site (P2).

S. aureus isolates, in particular MRSA, were more prevalent in DFIs than the healthy skin. This finding led to suggest that S. aureus, especially MRSA play a significant role in the development and the chronicity of DFIs as described in previous studies [22, 23].

In contrast, CoNS species were mostly recovered from healthy skin, with the exception of S. caprae and S. saprophyticus which were isolated only from DFIs.

Diabetic patients are thought to be major vehicles for clonal dissemination of staphylococci between hospitals and the community [24]; this could explain the relatively high genetic diversity of our staphylococci. Indeed, our MRSA isolates carried SCCmec III or IV, which are commonly associated with healthcare- and community-acquired infections, respectively.

This study revealed that the dominant MRSA clone (13/27, 48.1%) had the characteristics of the European clone (ST80- t044- IVc(2B)) [25]. Only isolates belonging to this clone carried lukF/lukS-PV.

Despite the known community origin of PVL-positive ST80- IV [26], this clone was highly prevalent in our investigation, confirming the results of other studies from Algeria, which reported the widespread occurrence of PVL-positive ST80- IV in Algerian hospitals [27, 28]. Similarly, several studies have also reported that ST80 CA-MRSA was spreading in healthcare settings in Tunisia [29], Jordan [30] and Kuwait [31, 32].

In addition to PVL, isolates belonging to the European clone (ST80- t044- IVc(2B)) carried edinB and SCCmec type IV considered as stable genetic markers for CA-MRSA [26, 33].These properties suggest the community origin of this clone.

The EDIN coding genes are powerful molecular markers associated with poor wound outcomes, that could differentiate colonization from infection in DFUs [34, 35]. In our study, edinB-positive ST80- IVc(2B) isolates were recovered from different grades of severity (2–4), from both DFUs and the healthy skin, which indicates the lack of association between these virulence markers and the severity of DFUs. These results contrast with data obtained in France [34], where edin were found to be highly prevalent in S. aureus isolates from high-grade foot ulcers.

The Brazilian clone (ST241-t037- III(3 A)) was the second most prevalent clone in our study. This clone includes ST239, ST240 and ST241 harboring SCCmec-III(A), which differ in mutations in pta or yqiL genes [36].

Given that isolates belonging to the Brazilian clone (ST241-t037- III(3 A)) carried genes that confer resistance to several classes of antibiotics, corroborates a recent Algerian study performed in the province of Constantine that reported a high prevalence (72.5%) of a worrisome emerging multidrug resistant Brazilian clone (PVL-negative ST239/241 SCCmec-III mercury) [37]. Furthermore, this clone has been reported to be the major HA-MRSA clone in hospitals in another Algerian province [38].

Interestingly, another study from Algeria reported that 82.2% of the MRSA isolated from DFIs belonged to ST239 [20]. Furthermore, studies from India suggest that the Brazilian clone has been found to be associated with high biofilm production in DFUs, and positive for luk-DE and icaA-B [39].

ST672-t3841-IVd(2B) is another MRSA clone found in this study, it was detected among healthy skin and DFU isolates. ST672 is an emerging MRSA clone in India and Australia [40, 41] and commonly associated with CA-MRSA [42]. In addition, this clone has been reported in DFU patients in India [39]. To the best of our knowledge, this is the first report of this clone in Algeria.

The 5 MSSA isolates belonged to 4 different clones, ST1-t127 (2/5, 40%), ST97-t9432, ST291-t937 and STNF-t037 (1/5 each, 20%). The finding of ST1 among MSSA isolates was consistent with previous European studies [26, 43]. However, in the USA, this clone was reported as CA-MRSA, and was also found associated with DFIs across all healing categories [44].

Interestingly, two of our MSSA clones, ST291-t937 and ST97-t9432, were previously identified as livestock-associated [45]. ST291 was also reported in DFU patients in India [39]. We noted that the ST291-t937 isolate lacked the human innate immune evasion cluster (IEC) (sak, chp, scn and sea) which confirms its animal origin [46]. In contrast, the ST97-t9432 carried the IEC, harboring the sak and scn genes, which could suggest a human origin of this clone [47].

The phylogenetic analysis revealed that Staphylococcus/Mammaliicoccus spp. carried a high number of accessory genes which have features characteristic of transferred elements (presence of mobility genes) and may provide selective advantages under particular conditions such as antibiotic resistance, adaptation, colonization and pathogenicity [48].

Despite that certain strains clustered with refence strains, there was a clear distinction between our isolates and those from other countries. The phylogenetic comparison of ST80 with the European (11819-97 and S1475), Egyptian (AA45) and the USA (S0924) strains revealed that the Algerian ST80 strains were quite diverse from all the reference strains and mostly clonal, indicated by the extremely short branches at the tip of each clonal branch.

Both the MRSA- and MSSA-STNF exhibited a close clonality and slight variation in gene content, suggesting that the STNF-MRSA clone emerged following the acquisition of SCCmec [26]. In addition, the pan-genome analysis confirmed also that the STNF shared a common ancestor with ST241 and ST239.

S. haemolyticus isolates belonging to the same ST were not clustered together throughout the phylogenetic tree. Hence, STs that are intermingled with another may be a result of recent divergence or recombination of the MLST genes [49].

No statistically significant association was found between the presence of virulence genetic determinants and the severity of DFUs. This result contrasts with the findings of a previous report that suggested that infected DFU markers sea, sei, lukE and hlgγ, were strongly associated with strains from grades 2–4 DFUs, and that cap8 was associated with strains from grade 1 ulcers and MSSA strains [50].

The higher frequency of adhesin-encoding genes among our S. aureus isolates suggests that they have a potential to form biofilms, which could contribute to their persistence and chronicity in DFU [39, 51].

Likewise, the higher prevalence of γ-hemolysins, lukE-lukD and cap8 cluster genes among our S. aureus isolates was consistent with a previous study which reported high frequency of γ -hemolysin genes in MRSA isolates recovered from DFU specimens and patients nares [30].

The absence of se/sel genes in the ST80 clone, is in agreement with previous studies which found that ST80 CA-MRSA did not harbor any enterotoxin genes [29, 52]. However, a study in Kuwait hospitals reported that PVL- positive ST80 CA-MRSA carried sed, sei, seg, seb, seh and sea, suggesting that ST80 isolates arose from SE negative isolates due to the acquisition of SE-carrying bacteriophages [32].

In contrast, a high number of se/sel genes was found among ST672 and ST1 isolates. The production of a large number of superantigen exotoxins (Sag) might contribute to the worsening of DFUs by the activation of T cells and the production of proinflammatory cytokines [53].

The ACME was detected only in S. epidermidis isolates, which is in agreement with a previous study that reported a higher prevalence of ACME amongst S. epidermidis [54]. ACME contributes to the success of bacteria in acidic environments as the acid environment on the skin [55]. In fact, the extensive success of certain S. aureus strains, such as USA300 (ST8-MRSA-IVa), the US epidemic CA-MRSA strain, has been partially attributed to the presence of ACME which is thought to originate from S. epidermidis [56].

We have identified a wide range of antimicrobial resistance genes among our isolates, with varying distribution between species or ST. Genes coffering resistance to tetracycline, fusidic acid and fosfomycin were prevalent only in S. epidermidis. The macrolide resistance genes erm(A) and erm(C) were predominant in S. aureus ST241 clone and S. haemolyticus, respectively. Aouati et al. (2021) reported that ermA and ermC were responsible for erythromycin-resistance in multidrug resistant HA-MRSA ST239/241 strains in Algeria [37], which is in perfect agreement with our finding. Noteworthily, the erm(A) gene was previously reported as the most prevalent gene in MRSA strains in DFIs in Algeria [20].

Carriage of AME genes was mostly associated with S. haemolyticus and S. aureus, particularly ST80 and ST241 isolates, which proves that the monotherapy with AME fails to eradicate DFIs due to these bacteria.

Our data suggest a widespread distribution of resistant genes among S. epidermidis and S. haemolyticus isolates, the opportunistic pathogens which form part of the normal commensal flora of humans, whilst it is difficult to eradicate because of their resistance to multiple antibiotics [17]. In addition, S. epidermidis and other CoNS can provide a reservoir of genes facilitating MRSA infection such as antibiotic resistance determinants [11]. Thus, it has been suggested that S. epidermidis may play an essential role in DFI etiology [21].

The tet(K) and fusB genes were less abundant among ST80 isolates, which was in contrast with previous studies that demonstrated that Algerian PVL-positive ST80- IV strains were resistant to multiple antibiotics, in particular to these drugs [57,58,59].

Although our study provided some important information on the population and genetic profile of Staphylococcus/Mammaliicoccus spp. isolated from DFIs, it suffers from few limitations; (i) small sample size; (ii) lack of phenotypic antibiotic resistance data; and (iii) non-inclusion of a control group of patients who had not received antibiotics.

Conclusions

In conclusion, our pan-genome analysis demonstrated that the Algerian S. aureus and CoNS/Mammaliicoccus isolates were closely related to each other, and presented novel genetic features, with a widespread distribution of virulence factors and antibiotic resistance genes, rendering this pathology more difficult to manage. The detection of the same S. aureus/CoNS clone in both DFIs and the healthy skin suggests that the autochthonous skin staphylococci can act as a reservoir for DFIs. To the best of our knowledge, this study represents the first investigation in Algeria, employing WGS and pan-genome analysis to get an insight on the underlying diversity and pathogenicity of Staphylococcus/Mammaliicoccus in DFIs. Most importantly, this study highlights the importance of WGS in disease surveillance and outbreak investigation, as it allows fine typing and detailed gene profiling of bacterial isolates.

Methods

Study group

Patients aged over 18 years who were hospitalized with infected DFUs at the University Regional Military Hospital and the University Hospital Ben Badis, in the province of Constantine, Algeria, from October to December 2019, were included in this study. 32 patients had a single ulcer and were sampled from both healthy foot skin (contralateral site to the chronic wound) and DFU. Patients who underwent surgical procedure including amputations were excluded from the study.

In addition, 14 strains obtained from the routine diagnostic recovered from 12 hospitalized DFU patients were added, including 2 patients who presented with 2 ulcers on the same foot.

DFUs were classified by clinicians using PEDIS classification (grade 2–4) proposed by the Diseases Society of America (IDSA) and the International Working Group on the Diabetic Foot Classifications of Diabetic Foot Infection (IWGDF) [60]. Patient demographics including age, gender, PEDIS grade, ulcer location and antibiotics taken during the 15 previous days were recorded.

Sample collection

After wound debridement and cleansing with sterile saline solution, pus samples were collected in deep wounds from infected tissues. The healthy skin samples were obtained by swabbing of an intact skin area measuring 50 cm2. Swabs were immediately transported to the laboratory of microbiology in 1 ml of sterile saline 0.9% for culture. The strains were isolated on mannitol salt agar after incubation at 37 °C for 24 h.

Whole genome sequencing analysis

Genomic DNA was extracted from 58 non duplicate Staphylococcus/Mammaliicoccus isolates by lytic treatment using achromopeptidase (Wako Pure Chemical Industries, Osaka, Japan) and then Sodium Dodecyl Sulfate (10%). DNA was purified using Zymo Research kit (Zymoresearch, Irvine, Ca, USA), according to the manufacturer’s instructions.

A DNA sequencing library (insert size, 300 to 500 bp) was prepared using a QIAseq FX DNA Library Kits (Qiagen, Germany). WGS was performed using the Illumina NextSeq 500 platform with the 300-cycle NextSeq 500 paired-end read sequencing (2 × 150-mer).

Bioinformatic analysis

Annotation of the genomes was performed with Prokka [61]. Putative bacterial species were determined using Krona [62].

To characterize isolates, sequencing reads were analyzed in silico by multi locus sequence typing (MLST) [63]. spa types and SCCmec were identified in silico with the online tools spaTyper and SCCmecFinder [64]. Antimicrobial resistance genes were identified by homology searching against the ResFinder database [65].

For pan-genome analysis, 58 isolates from this study and publicly available sequences either at draft or complete genome sequences (a total of 78 strains) [see Additional file 3] was performed using Roary [66]. Tree construction was performed using FastTree and visualized in the online interactive viewer Phandango [67] using the absence and presence matrix of genes and the tree file in the standard Newick tree format generated by Roary.

Statistical analysis

Data were analyzed using the Statistical Package for Social Sciences (SPSS; ver. 26.0). Contingency tables were constructed and Chi-squared tests were used to calculate p-value for describing possible associations between species/ST and virulence/resistance genes with PEDIS grades and the source of isolates. p-values < 0.05 were considered statistically significant.

Data Availability

All data generated or analyzed in this study are included within the article and its additional files. The Short Read Archives (SRA) were deposited in NCBI database, [https://0-www-ncbi-nlm-nih-gov.brum.beds.ac.uk/bioproject?LinkName=sra_bioproject&from_uid=26401144] with the accession number PRJDB13730. All complete sequences in this study are available, as shown in the additional file 1.

Abbreviations

CA-MRSA:

Community-acquired MRSA

CoNS:

Coagulase-negative Staphylococcus

CoPS:

Coagulase-positive Staphylococcus

DFI:

Diabetic foot infection

DFU:

Diabetic foot ulcer

HA-MRSA:

Healthcare-acquired MRSA

MRSA:

Methicillin-resistant S. aureus

MSSA:

Methicillin-sensitive S. aureus

MSCoNS:

Methicillin-sensitive coagulase negative staphylococci

WGS:

Whole genome sequencing

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Acknowledgements

We gratefully acknowledge the National Institute of Infectious Diseases of Japan, for providing chemical products for experiments and supported experimental facilities to carry out experiments.

We thank also the clinical and support teams at the University Regional Military Hospital and the University Hospital Ben Badis of Constantine, Algeria, for their support in undertaking this study.

Funding

This study was funded by the Algerian Ministry of Higher Education and Scientific Research (DGRSDT/MESRS). This work was supported by the Research Program on Emerging and Reemerging Infectious Diseases of the Japan Agency for Medical Research and Development (grant number: JP23fk0108666j0401).

Author information

Authors and Affiliations

  1. Laboratory of Molecular Biology, Genomics and Bioinformatics, Department of Biology, Faculty of Nature and Life Sciences, University Hassiba Benbouali, Chlef, Algeria

    Nerdjes Ferhaoui & Mohammed Sebaihia

  2. Pathogen Genomics Center, National Institute of Infectious Diseases (NIID), Tokyo, Japan

    Rina Tanaka, Tsuyoshi Sekizuka & Makoto Kuroda

Authors
  1. Nerdjes Ferhaoui
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  2. Rina Tanaka
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  3. Tsuyoshi Sekizuka
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  4. Makoto Kuroda
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  5. Mohammed Sebaihia
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Contributions

MS and MK designed the study and reviewed the manuscript. RT and NF performed the whole genome sequencing. TS and MK performed the bioinformatic analysis. NF isolated bacterial strains, performed in silico typing, pan-genome analysis, statistical analysis and wrote the draft manuscript. MS supervised the research and revised the manuscript. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Mohammed Sebaihia.

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Competing interests

The authors declare no competing interests.

Ethics approval and consent to participate

All methods were carried out in accordance with relevant guidelines and regulations, all rules of confidentiality and ethics as prescribed in the Helsinki Declaration have been respected and all experimental protocols were approved by ethics committee of the University Regional Military Hospital and University Hospital Ben Badis of Constantine, Algeria. Informed consent was obtained from all subjects and/or their legal guardians.

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Laboratory of Molecular Biology, Genomics and Bioinformatics, Department of Biology, Faculty of Nature and Life Sciences, University Hassiba Benbouali, Chlef, Algeria

Nerdjes Ferhaoui and Mohammed Sebaihia.

Pathogen Genomics Center, National Institute of Infectious Diseases (NIID), Tokyo, Japan

Makoto Kuroda, Tsuyoshi Sekizuka and Rina Tanaka.

Nerdjes Ferhaoui

n.ferhaoui@univ-chlef.dz.

Rina Tanaka: r-tanaka@niid.go.jp.

Tsuyoshi Sekizuka

tsuyoshi.sekizuka@gmail.com.

Makoto Kuroda

makokuro@niid.go.jp.

Additional information

Corresponding authors.

Correspondence to Mohammed Sebaihia: m.sebaihia@univ-chlef.dz.

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Ferhaoui, N., Tanaka, R., Sekizuka, T. et al. Whole genome sequencing and pan-genome analysis of Staphylococcus/Mammaliicoccus spp. isolated from diabetic foot ulcers and contralateral healthy skin of Algerian patients. BMC Microbiol 23, 342 (2023). https://0-doi-org.brum.beds.ac.uk/10.1186/s12866-023-03087-2

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Keywords

BMC Microbiology

ISSN: 1471-2180

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