Multidrug-Resistant Staphylococcus aureus in Diabetic Foot Infections (DFI) from Beira, Mozambique: Prevalence and Virulence Profile

Introduction

Diabetic foot infection (DFI) is a common complication resulting from soft tissue or bone infection, often associated with neuropathy or peripheral arterial disease in patients with type 2 diabetic mellitus.¹ DFI is associated with considerable morbidity, an increased risk of lower limb amputation, and a high mortality.² DFI is mainly polymicrobial, caused by different bacterial communities, and the presence of this wide variety of species is a factor that complicates diagnosis and treatment.^3,4 Staphylococcus aureus is one of the most frequently isolated pathogens of DFI, often exhibiting worrying trends of high virulence and antibiotic resistance.⁵

Staphylococcus is one of the most common genera on the skin and nasal mucosa of humans and animals.^5,6 The presence of Staphylococcus in wounds can cause increased inflammation and delay the healing process.⁶ This scenario is aggravated by the combination of the presence of different virulent genes and high antibiotic resistance, which may compromise the success of the treatment. The presence of virulence genes, such as those encoding the protein adhesin A (SpA), which facilitates the attachment of bacteria to host cells and tissues, fibronectin (FnBP), which binds to molecules on the surface of the host cells, and α-and-β-hemolysins (hlA and hlB) that can cause damage to host cells and induce cell death, contributes to the successful invasion and pathogenicity of S. aureus.^7,8 Exfoliative toxins, expressed by the ET genes, are exotoxins produced by several staphylococcal species, capable of causing blistering in human and animal infections.⁹ Three of these toxins, encoded by the etA, etB, and etD genes, are directly linked to infections in humans.¹⁰ Toxic shock syndrome-associated toxin (TSST, or TSST-1), which encodes a protein considered a superantigen, and enterotoxins, such as those of the SE family (seA, seB, seD, seI and seH), are other examples of S. aureus virulence genes.^11,12

The occurrence of antibiotic-resistant strains, such as methicillin-resistant S. aureus (MRSA), is another factor that increases the severity of S. aureus infections in diabetic patients, making treatment of these infections challenging to say the least.^13,14 MRSA strains thus play a significant role in DFI and have become a public health concern due to their high virulence and resistance to an increasingly broad spectrum of antibiotics.¹⁵ MRSA infections are associated with prolonged hospital stay, increased morbidity and mortality rates, and higher healthcare costs compared with infections caused by methicillin-sensitive strains.¹⁶ Furthermore, MRSA strains pose a significant threat to public health due to their propensity for nosocomial transmission and their ability to form antibiotic-resistant biofilms on medical devices.¹⁷ Biofilm formation involves several steps, such as adhesion, accumulation, maturation and biofilm dispersion, determined by several genes. In this process, the icaABCD operon, which encodes Polysaccharide Intercellular Adhesion (PIA), the main component of biofilms, plays a very relevant role.^18,19 The regulation of the genes of this operon is complex and affected by several factors, including the presence of antimicrobials, the presence of cell wall-binding proteins and the presence of other bacterial virulence genes.²⁰

S. aureus is a major opportunistic pathogen in DFIs and is frequently associated with persistent and severe infections in diabetic patients, often leading to poor healing outcomes, increased risk of amputation, and prolonged hospitalization.^5,21 The evaluation of surveillance of this opportunistic pathogen is critically important for guiding effective clinical management and controlling the spread of antimicrobial resistance.^21,22 Regular monitoring of its antibiotic resistance patterns and virulence profiles enables early detection of multidrug-resistant and highly virulent strains, informing targeted antimicrobial therapy and infection control measures.²² Moreover, surveillance data are essential for developing local and regional treatment guidelines, optimizing antibiotic stewardship programs, and shaping public health interventions aimed at reducing the clinical and economic burden of DFIs, particularly in resource-limited settings.^23,24

In Africa, the increasing incidence and inadequate management of type 2 diabetes represent crucial public health challenges, requiring active surveillance.²⁵ In Mozambique, there is a progressive increase in the prevalence of type 2 diabetes. The lack of awareness among the population about the disease and its control strategies is alarming.²⁶ In view of this, there is an urgent need for immediate adaptation in health service delivery, especially in the diagnosis and characterization of opportunistic pathogens associated with type 2 diabetes, with a view to improving diabetes management. In this context, this study aimed at the molecular characterization of S. aureus isolates associated with DFI from patients assisted at the Matacuane military health center in the city of Beira, Mozambique.

Material and Methods

Study Area

The Matacuane Military Health Center is a military health facility integrated into the national health system of Mozambique, dedicated to providing healthcare services to military and paramilitary forces. However, as part of the national health system, this facility also provides primary healthcare services to civilians living in the vicinity of the health center. Located in the urban area of the municipality of Beira, on average, the center provides primary healthcare services to 1900 to 2500 patients per month, from various neighborhoods, with the majority of patients coming from the Matacuane neighborhood.²⁷

The most important services offered include general medicine consultations, pediatric consultations, monitoring of patients on antiretroviral therapy and nursing services. Nursing services involve the administration of intramuscular or intravenous medications and the treatment of ulcers, wounds or surgical dressings, in patients referred by the Beira Central Hospital.

Inclusion and Exclusion Criteria

All civilian patients with a confirmed diagnosis of type 2 diabetes mellitus, who presented clinical signs of infected foot ulcers and provided informed consent, as stated under ethical considerations, were included in the study. On other way, military or paramilitary personnel, civilian patients who refused or were unable to provide informed consent, and those who had not initiated antibiotic treatment were excluded from the study.

Sampling and Characteristics of Patients and Lesions

Sampling was conducted between September 2023 and January 2024, in 45 civilian patients with type 2 diabetes and DFI ulcers. Ethical considerations were strictly observed. All patients were taking antibiotics to treat the infection. Demographic and lesion information, including age, sex, duration of DFI, occurrence of diabetes medication, lesion characteristics, and lesion location were collected.

Isolation and Identification of Staphylococcus aureus

A sample was collected from each patient by swab, after debridement of the ulcer. A total of 45 swabs were sampled and, respectively, inoculated into Brain Heart Infusion (BHI) broth (Oxoid Ltd., Basingstoke, UK) and incubated overnight at 37°C. From this culture broth, Mannitol Salt Agar (MSA) (Oxoid Ltd., Basingstoke, UK) and Columbia agar (supplemented with 5% sheep blood) plates were inoculated and incubated at 37°C for 24 to 48 h. The plates were inspected for characteristic staphylococcal morphology and presumptive Staphylococcus aureus colonies were identified using conventional methods (Gram staining, catalase and coagulase reactions) according to standard microbiological methods. Identification as S. aureus was performed by PCR targeting the 447-bp fragment of nuc gene, with the forward primer 5′-GCGATTGATGGTGATACGGTI-3′ and reverse primer 5′-AGCCAAGCCTTGACGAACTAAAGC-3′.^28,29 The primers were commercially synthesized by STAB VIDA (STAB VIDA, Caparica, Portugal). S. aureus (ATCC 25923) and S. epidermidis (ATCC 12228) were used as positive and negative controls, respectively.

DNA extraction was performed using the QIAamp DNA Mini® Kit (Qiagen, Hilden, North Rhine-Westphalia, Germany) according to the manufacturer’s instructions. Briefly, bacterial pellets were resuspended in lysis buffer containing proteinase K and incubated at 56°C to ensure complete cell lysis. Ethanol was then added to the lysate, and the mixture was transferred to a QIAamp spin column. DNA was bound to the silica membrane, followed by successive washing steps to remove contaminants. Finally, purified DNA was eluted in the provided elution buffer and stored at –20°C until use.

The mix for PCR was prepared with 12.5 µL of Taq DNA Polymerase NZYTaq II2x Colourless Master Mix (MZYTech, Lisboa, Portugal), 1 µL of each forward and reverse primer (final concentration 0.3 µM), 2 µL of template DNA and sterile ultrapure water to fill 25 µL of total volume. The reaction mixtures were subjected to the following amplification conditions: initial denaturation, 94°C for 5 min, followed by 30 cycles of denaturation 94°C for 30 s, annealing 52°C for 30 s, elongation 72°C for 30 s, and a final elongation at 72°C for 5 min. PCR reactions were run in a thermocycler GeneAmp® PCR System 9700, Bio-Rad (Bio-Rad Laboratories, Segrate, Milan, Italy). The resulting PCR products were resolved on 2% (m/v) agarose gels in 1× TAE buffer, in an EC330 Thermo Fisher Scientific tank (Atlanta, GA, USA) at 8 V/cm for 60 min. The gels were stained with GelRed (Frilabo, Maia, Portugal) and analyzed using a Gel Doc™ EZ System (Bio-Rad Laboratories, Segrate, Milan, Italy). For calculating the size of the PCR products, the molecular marker 100 bp DNA Ladder (Invitrogen, CA, USA) was used.

Antibiotic Resistance Profile

Antimicrobial resistance profiling was performed by the disk diffusion method on Mueller–Hinton (MH) agar plates (Biokar Diagnostics, Beauvais, France) with antibiotic disks (Liofilchem, Roseto degli Abruzzi, Italy), according to the Clinical Laboratory Standards Institute (CLSI, 2021).³⁰ Colonies grown on Trypto-Casein-Soy agar (Biokar Diagnostics, Beauvais, France) for 22 ± 2 h at 37°C were suspended in sterile saline until the turbidity was equivalent to the McFarland 0.5 standard (ca. 10⁶ CFU/mL). Of the resulting bacterial suspensions, 100 µL was used to inoculate MH plates under the conditions described by CLSI.³⁰ After deposition of the antibiotic disks, the plates were incubated for 18 ± 2 h at 37°C. Eight antibiotics were tested: cefoxitin (FOX) 30 μg; penicillin G 10U; vancomycin (VAN) 5 μg; chloramphenicol (CHL) 30 μg; tetracycline (TET) 30 μg; gentamicin (GEN) 10 μg; trimethoprim/sulfamethoxazole (SXT) 1.25/23.75 μg; erythromycin (ERY) 16 μg. On each 90 mm diameter plate, four different antibiotic disks were placed. For each isolate, two replicates were performed.

To assess the antimicrobial resistance profile of the S. aureus isolates, inhibition zones diameters were measured (millimeter) and compared to those described in the CLSI (2021).³⁰ Isolates were considered non-susceptible to a given antibiotic when they showed intermediate or full resistance to that antibiotic, according to the CLSI clinical breakpoints. Multidrug resistance was considered as non-susceptibility to at least one agent, in three or more antimicrobial categories and/or resistance to methicillin, cefoxitin or oxacillin.³¹

Table 1 Primers Used for the Detection of Virulent Genes and Respective Sizes of Amplified Product

Identification of Virulence and Antibiotic Resistance Genes

Multiplex PCR (MPCR) investigated the presence of 32 virulence genes (Table 1). Namely: Genes encoding hemolysin (hlA, hlB and hlgCB); genes associated with colonization, such as fibronectins (fnbA and fnbB); genes encoding adhesins (clfA and clfB) and gene sasX related to nasal colonization; accessory gene regulators (agr I, Agr II, agr III, agr IV and arcA); biofilm regulator genes (icaA, icaB, icaC and icaD); exotoxins, such as exfoliative toxin genes (etA, etC and etD) and toxic shock syndrome (TSST); enterotoxins (sea, seb, sec, sed, see, seI and seH); capsular polysaccharides (cap5 and cap8); and genes encoding pore-forming leukotoxins (lukED and lukS/F-PV). Based on the annealing temperature and PCR product size of each primer, the reactions were grouped into different MPCRs. Namely: MPCR for seH, icaD, TSST, etD, hlgCB and fnbB genes; MPCR for fnbB, icaA and agr III genes; MPCR for hlB, Agr I, agr II and agr IV genes; MPCR for icaC, icaB, etB and clfA genes; MPCR for clfB, lukED, cap5 and cap8 genes; MPCR for seA, seB, seC and seE genes; MPCR for seD, ACME-arcA and lukS/F-PV genes; MPCR for etA, sasX and hlA genes. The gene seI was screened as a single PCR.

The MPCR master mixes were respectively prepared with 12.5 µL of Taq DNA Polymerase NZYTaq II2x Colourless Master Mix (MZYTech, Lisboa, Portugal), 1 µL of each forward and reverse primers (Table 2) (final concentration 0.3 µM), 2 µL of template DNA and sterile ultrapure water up to 25 µL of total volume. All primers commercially synthesized by STAB VIDA (STAB VIDA, Caparica, Portugal).

Table 2 Primers Used for the Detection of Antimicrobial Resistance Genes and Respective Sizes of Amplified Products

Genes encoding resistance to methicillin (mecA), penicillin (blaZ), erythromycin (ermA, ermB and ermC), and vancomycin (vancA and vancB) were screened by MPCR (Table 2). The MPCR master mix was prepared with 12.5 µL of Taq DNA Polymerase NZYTaq II2x Colourless Master Mix (MZTech, Lisboa, Portugal), 1 µL of each forward and reverse primers (Table 2) (final concentration 0.3 µM), 2 µL of template DNA, and sterile ultrapure water to complete 25 µL of total volume. MPCR conditions were performed as previous described.^17,29,43 All primers commercially synthesized by STAB VIDA (STAB VIDA, Caparica, Portugal).

All PCR reactions were performed in a GeneAmp® PCR System 9700 thermocycler (Applied Biosystems, Bio-Rad Laboratories, Segrate, Milan, Italy). PCR products were resolved on 2% (m/v) agarose gels in 1×TAE buffer, in an EC330 Thermo Fisher Scientific tank (Georgia, USA) at 8 V/cm for 60 min. Gels were stained with GelRed (Frilabo, Maia, Portugal) and analysed using a Gel Doc™ EZ System (Bio-Rad Laboratories, Segrate, Milan, Italy). To calculate the size of PCR products, the molecular marker 100 bp DNA Ladder (InVitrogen, California, USA) was used.

Results

Demographic Data

Demographic and lesion information, including age, sex, duration of DFI, occurrence of diabetes medication, lesion characteristics, and lesion location are shown in Table 3. The age of the patients ranged from 36 to 75 years (mean 65.8 ± 13.76 years) and 64.4% were female (Table 3). Most patients (93%) were taking medications to control diabetes. Most patients (55.6%) had DIF for more than three months (Table 3). The types of lesions among the patients were mainly deep ulcers (86.7%) with chronic wounds (77.8%). Most lesions were located on the right toe (37.8%) and plantar region (28.8%) (Table 3).

Table 3 Characterization of DFI Patients and Lesions

The diabetic foot ulcers of the 45 patients evaluated in this study resulted in the identification of 45 isolates of S. aureus. These S. aureus isolates were subjected to analysis of the antibiotic resistance profile and the presence of virulence and antibiotic resistance genes.

Table 4 Distribution of Virulent Genes by Function

Virulent Genes

To facilitate the recognition of the most relevant virulence genes in the 45S. aureus isolates, the genes were grouped according to their respective functions (Table 4). Thus, the most frequent virulence genes were those encoding exotoxins (TSST, 57.8% and etA, 35.6%), followed by genes encoding colonization (clfB, 37.8%) and regulators factors (arcA, 37.8%) (Table 4). Regarding leukocidin genes, LukPV was found in 26.7% and LukED in 15.6 of the isolates. The cap5 (20%), icaA (17.8%) and icaD (11.1%) genes were the most representative adhesion genes. Among the seven genes investigated that encode staphylococcal enterotoxins, only the seI gene was found (13.3%) (Table 4). Among the 32 genes accessed, 15 (46.9%) were detected in at least one isolate (Table 5). Seventeen (clfA, etB, etD, fnbA, sasX, hlA, seA, seB, seC, seD, seE, seh, icaB, icaC, agr III, agr IV and cap8) of the 32 target genes were not found in any isolate.

Table 5 Virulence Genes Detected in the 45 Staphylococcus aureus Isolates

Most isolates that present virulent genes contain more than one gene, related or not (Tables 4 and 5). Regarding similar virulence genes, the most predominant co-occurrences were observed with the exotoxin gene TSST and etA (31.1%), and the adhesion factors icaA/icaD and cap5 (13.3%). The co-occurrence of multiples (three or more) virulence genes involved in different functions was found in 42.2% of the isolates. On the other hand, 31.1% of the isolates presented two genes encoding different types of virulence. In five of the 45 isolates, none of the 32 virulent genes under analysis were detected (Table 5).

Table 6 Frequencies of Antibiotic Resistance Profile and Associated Genes

Antibiotic Resistance

The most prevalent antimicrobial resistance profiles were penicillin (100%) and cefoxitin (53.3%) (Table 6). Among non-β-lactam antibiotics, the most frequent resistance profiles were to trimethoprim/sulfamethoxazole (40%), erythromycin (33.3%) and vancomycin (22.2%). Very few isolates showed resistance to chloramphenicol or gentamicin (8.9 and 4.4%, respectively) (Table 6). Among the 45 S. aureus isolates from DFI, 31 (68.9%) were multidrug-resistant as they showed resistance to cefoxitin and/or more than three groups of unrelated antibiotics (Table 7).

Table 7 Antibiotic Resistance Profile and Presence of Genes Encoding Antibiotic Resistance in the 45 Staphylococcus aureus Isolates from DFI

Regard the frequency of antibiotic resistance genes, the blaZ gene, which encodes penicillin resistance, was found in all 45 isolates analyzed (Table 6). The mecA gene, encoding methicillin/penicillin resistance, was found in 53.3% of the isolates. Two types of genes encoding vancomycin resistance were found (vancA, in 28.9% and vancB, 15.6%) (Table 6). Among the genes encoding erythromycin resistance, the ermB gene (15.6%) was the most common gene, followed by ermC (11.1%). Gene encoding resistance to trimethoprim/sulfamethoxazole dfrA (31.1%) and dfrG (17.8%) was found among 40% of isolates that showed resistance profile.

Co-occurrence of resistance genes was observed in 84.4% of the isolates (Table 7). The most frequent co-occurrence was of blaZ and mecA (48.8%). In three of the 45 (6.7%) S. aureus isolates, all types of antibiotic resistance genes evaluated in this study were detected (blaZ, mecA, vanc, dfr and erm) (Table 7).

Beyond the high overall resistance to penicillin and cefoxitin, Table 7 shows a concerning trend of convergence between multidrug resistance and the presence of multiple virulence determinants in S. aureus isolates from DFI. Strains harboring both mecA and blaZ genes frequently co-expressed virulence genes such as TSST, etA, and leukocidin genes (lukPV, lukED), highlighting the potential for these strains to cause severe and hard-to-treat infections. Particularly notable were isolates like FDB33 and FDB34, which combined resistance to five or more antibiotics with genes associated with toxin production and biofilm formation (icaA, icaD, cap5), suggesting enhanced persistence and immune evasion capabilities.

Discussion

Methicillin-resistant S. aureus (MRSA) has been documented as the most dominant healthcare-associated Staphylococcus worldwide.^49,50 However, in Mozambique, there is no information on the characterization of S. aureus isolated from DIF. Hopefully, this study will contribute to fill this gap. In general, low genetic diversity was found among the isolates, demonstrated by the similarity of the virulent genes found. The results of this study highlight high resistance to penicillin (100%), cefoxitin (53.3%) and trimethoprim/sulfamethoxazole (40%).

Among diabetic patients with foot ulcers, MRSA has emerged as an important and commonly opportunistic pathogen, often community- and hospital-associated. MRSA can infect ulcers, skin, and soft tissues, making treatment very difficult.⁵¹ Among the 53.3% (24/45) MRSA isolates identified in this study, 45.8% were resistant to trimethoprim/sulfamethoxazole, 41.7% to erythromycin and 29.2% to vancomycin. Similar results were found by other authors in Poland,¹⁰ Egypt¹⁶ and Brazil.⁵² S. aureus recovered from DFI in a Tunisian hospital showed 100% resistance to methicillin and 33.3% to trimethoprim/sulfamethoxazole.⁵⁰ In Egypt, a high prevalence of MRSA strains (100%)¹⁶ exhibiting resistance to erythromycin and vancomycin was noted, possibly reflecting different prescription habits and infection control policies. In Brazil, high resistance rates (61%) of MRSA were also observed.⁵² However, the socioeconomic factors, such as better access to healthcare and structured diabetes care programs, might partially mitigate the burden compared to the Mozambican context. In Mozambique, co-trimoxazole (trimethoprim/sulfamethoxazole (SXT)) plays a very important role in the treatment of opportunistic diseases in immunocompromised patients and, together with amoxicillin and clavamox (another penicillin antibiotics), is the most prescribed and recommended antibiotic by health professionals.⁵³

The mecA gene was found in all isolates that showed resistance to cefoxitin. These isolates also carried the blaZ gene encoding penicillin resistance and 29.2% had the vanc and or erm gene. Several studies have found a high prevalence of antibiotic resistance and genes associated with antibiotic resistance in S. aureus recovered from clinical and food samples in Mozambique.^43,54,55 In food samples, Salamandane et al⁴³ reported the occurrence of the blaZ gene in 42.1% of S. aureus isolates from ready-to-eat foods, as well as the occurrence of the mecA gene in 36.8% and vancA in 31.6%. On the other hand, in clinical isolates, blaZ was found in 79.2% of samples and mecA in 100%.⁵⁵ Co-occurrence of multiple resistance genes further suggests potential for treatment failure and nosocomial spread.

In South Africa, MRSA prevalence among DFI patients has been reported at levels similar to or slightly higher than those observed in Mozambique, while studies from Nigeria and Kenya show even higher rates of multidrug resistance in S. aureus isolates.⁵⁶ Several factors as limitations in healthcare infrastructure, with reduced laboratory capacity for culture and sensitivity testing, leading to widespread empirical antibiotic use without proper microbiological diagnosis in different African countries could explain these differences.^57,58

Regarding the genes encoding virulence factors, the most frequent were exotoxins TSST (57.8%), colonization factor clfB (40%), regulator factor arcA (37.8%) and exfoliative toxin etA (33.3%). A total of 53.8% of the isolates that presented TSST also presented the etA gene. Several studies found a low prevalence of TSST in S. aureus isolates from DFI.^50,59,60 In Costa Rica, Víquez-Molina et al detected the TSST gene in 5.2% of the samples,⁵⁹ and in India, Shettigar et al⁶⁰ found 13.9% of TSST in S. aureus recovered from individuals with foot ulcers. Combination of several factors, including high glucose, low oxygen levels resulting from the combination of neuropathy and poor circulation and indiscriminate use of a variety of antibiotics,⁶¹ may have contributed to the high frequency of TSST genes in this work. Exfoliative toxins cause scalded skin syndrome, most commonly in newborns, young children and immunosuppressed individuals. Scalded skin syndrome is a staphylococcal infection that causes redness, blisters and peeling, causing the top layer of skin to peel off.⁶²

The gene encoding Panton-Valentine Leucocidin (PVL) was found in 40% of the isolates. Of these, 11 isolates were positive for lukPV, six isolates for lukED and one isolate showed both genes (lukPV and lukED). Detection of lukED in MRSA strains associated with DFI has also been reported in Iran.⁶³ In Iran, lukED gene was found in 85% of S. aureus recovered from DIF. PVL is one of the main virulence factors associated with hospital-acquired pneumonia and skin infections. This toxin damages white blood cells, which are essential in fighting infection.⁶⁴ Meeren et al found 37.5% and 90.5% of the gene encoding this toxin PVL in hospital- and community-acquired S. aureus in Beira city, Mozambique.⁵⁴ Other relevant genes found in the present study are related to fibrinogen-binding adhesin of the host cell, such as clfB (37.8%) and fnbB (4.4%).

These genes encode proteins known as clumping factors, fibronectin-/fibrinogen-binding proteins, and adhesins,⁶⁵ which play an important role in biofilm formation. A recent study reported high biofilm formation in MRSA isolated from street food in Mozambique.¹⁷ Regarding enterotoxins, only the seI gene was detected in 13.3% of the isolates. Although they are considered enterotoxins, seI and seH encode proteins that are also factors associated with the colonization of host tissues by S. aureus, due to their presumed participation in the infectious process.^66,67

Some strains are of particular concern, as they are multidrug-resistant or multidrug-resistant MRSA, carry several virulent genes and isolated from high-risk patients. Namely, the MRSA isolates FDB03, that presented the clfB, TSST, etA, lukPV, lukED and agr I genes, and FDB04, with virulence genes clfB, TSST, etA and arcA, recovered from obese patients with ulcers for more than 90 days. Also, the MRSA isolates FDB11 and FDB39 recovered from ulcers older than 90 or 60 days, from elderly or obese patients, respectively, carried six virulence genes each. Although they were isolated from non-obese individuals with recent ulcers (less than 30 days), the multidrug-resistant isolate FDB33 carrying the hlB, TSST, etA, arcA, IcaA, icaD, cap5 and seI; and the MRSA isolate FDB34, carrying the genes TSST, etA, arcA, IcaA, icaD and cap5, may be considered high risk, as they present genes related to biofilm formation. In fact, in the biofilm state, bacteria are more resistant to antibiotics than their planktonic counterparts due to the multilevel protection conferred by the extracellular matrix (which hinders the penetration of antibiotics), altered metabolic states and lower growth rate.^17,28,68

We did not find data on the prevalence of DFI in Mozambique. However, in South Africa, a country bordering Mozambique and sharing similarities in terms of prevalence of non-transmissible diseases, DIF is estimated to affect 28% of diabetic patients.⁶⁹ The prevalence of DFI in Zimbabwe was estimated to be 53%. Longer duration of diabetes, absence of pedal pulses, and peripheral neuropathy were considered risk factors for foot ulceration in these populations.^25,70–73

Conclusions

This study aimed to investigate antibiotic resistance and the presence of virulence genes in S. aureus isolated from DFI. To the best of our knowledge, this is the first study addressing antibiotic resistance and virulence genes in S. aureus isolated from DFI patients in Mozambique. The results revealed alarming levels of antibiotic resistance, particularly to penicillin, cefoxitin, co-trimoxazole (trimethoprim/sulfamethoxazole) and vancomycin, as well as high percentages of multidrug-resistant strains. Furthermore, the study identified a high presence of virulent genes. The presence of multiple virulence genes, including TSST, clfB, and lukPV, underscores the pathogenic potential of these strains and their capacity to cause severe infections. The presence of virulent genes in multidrug-resistant strains represents a worrying scenario, as it indicates the potential for greater severity of infections caused by these bacteria, making treatment difficult and increasing the risk of serious complications.