Antibiotic resistance and mecA characterization of Staphylococcus hominis from filarial lymphedema patients in the Ahanta West District, Ghana: A cross‐sectional study

Abstract Background and Aim Filarial infections affect over 150 million people in the tropics. One of the major forms of filarial pathologies is lymphedema; a condition where the immune response is significantly altered, resulting in changes in the normal flora. Staphylococcus hominis, a human skin commensal, can also be pathogenic in immunocompromised individuals. Therefore, there is the possibility that S. hominis could assume a different behavior in filarial lymphedema patients. To this end, we investigated the levels of antibiotic resistance and extent of mecA gene carriage in S. hominis among individuals presenting with filarial lymphedema in rural Ghana. Method We recruited 160 individuals with stages I–VII lymphedema, in a cross‐sectional study in the Ahanta West District of the Western Region of Ghana. Swabs from lymphedematous limb ulcers, pus, and cutaneous surfaces were cultured using standard culture‐based techniques. The culture isolates were subjected to Matrix‐Assisted Laser Desorption/Ionization Time of Flight (MALDI‐TOF) mass spectrometry for bacterial identification. Antimicrobial susceptibility testing (AST) was performed using the Kirby–Bauer method. mecA genes were targeted by polymerase chain reaction for strains that were cefoxitin resistant. Results In all, 112 S. hominis were isolated. The AST results showed resistance to chloramphenicol (87.5%), tetracycline (83.3%), penicillin (79.2%), and trimethoprim/sulphamethoxazole (45.8%). Of the 112 strains of S. hominis, 51 (45.5%) were resistant to cefoxitin, and 37 (72.5%) of the cefoxitin‐resistant S. hominis haboured the mecA gene. Conclusion This study indicates a heightened level of methicillin‐resistant S. hominis isolated among filarial lymphedema patients. As a result, opportunistic infections of S. hominis among the already burdened filarial lymphedema patients in rural Ghana may have reduced treatment success with antibiotics.


| BACKGROUND
Lymphatic filariasis (LF) caused by lymph-dwelling nematodes (Wuchereria brancrofti, Brugia malayi, and Brugia timori) remains a disease of poverty significantly in Africa, Asia, and some parts of South America. 1 The infection has affected over 120 million people living in 72 countries in the tropics with countries in Africa contributing to the largest burden. 2,3 LF-infected individuals are generally asymptomatic, but most affected patients experience some form of clinical pathologies, including lymphedema and hydrocele, leading to significant disability-adjusted life years (DALYs). 4 According to McPherson et al., 5 over 40 million people suffer from lymphedema and hydrocele with 17 million suffering from chronic lymphedema. 6 Currently, the intervention recommended by the WHO through the GPELF rely on the large-scale administering of mainly microfilaricidal drugs (MDA) to the endemic populations. 7,8 These microfilaricidal drugs such as ivermectin can lower MF loads in infected humans but do not typically target secondary bacterial and fungal pathogens. Bacteria and fungi are believed to be important opportunistic pathogens in patients with filarial lymphedema due to the presence of lesions on their limbs which serve as entry portals. 9 Bacterial products of Staphylococci are known to be responsible for recurrent skin inflammation such as cellulitis. 5 Moreover, previous studies 5,9,10 have also indicted bacterial products (superantigens) in worsening filarial infections, as a result of high cytokine release and mast cell degranulation. These superantigens are highly indicative of Staphylococci aureus coinfection and not necessarily of the coagulase-negative bacteria.
However, some virulent factors such as biofilm formation, that complicate infected wound management have been identified with the coagulase-negative bacteria. 11,12 Staphylococcus hominis is one of the major Staphylococcus species found on the human skin and is mainly found in the axillae, perineal and inguinal areas. 13 S. hominis is ranked third among the coagulasenegative bacteria that are of clinical importance. 14 15 In addition to its occasional pathogenicity, S. hominis may be a reservoir of specific components of the methicillin resistance genetic element, staphylococcal cassette chromosome (SCCmec) that may be transferrable to more pathogenic staphylococcal species. 15 The pathogenicity of S. hominis in immunocompromised individuals have been of grave concern recently. In LF, the pathology of the disease leaves most patients immunocompromised, 5

| MATERIALS AND METHODS
This study employed a cross-sectional design conducted from January 2019 to January 2020. Data collection included interviews using a structured questionnaire and swab sample collection from legs and leg wounds.

| Study area and sampling
The study was conducted in eight (8) 16 All participants between the ages of 18-70 years with filarial lymphedema and who have lived in endemic filarial communities for more than 10 years were included in the study. Children and adults outside the stated age range were excluded. Individuals within the age range presenting with lymphedema of non-filarial origin, existing autoimmune diseases, and debilitating comorbidities were also excluded.

| Ethical considerations
Ethical clearance was obtained from the Committee on Human Research, Publication and Ethics, School of Medicine and Dentistry, KNUST, with approval number (CHRPE/AP/191/18).

| Culture and identification
To ensure the integrity of the samples, they were examined for leakages and correct labeling. Samples were immediately cultured upon arrival in the laboratory. The swabs were plated on Columbia Naladix Acid (CNA) agar and incubated at 35°C-37°C for 18-24 h.
After 24 h, colonies on plates with mixed colonies were subcultured on CNA for pure isolates. The isolates were then stored in a microbank system at −80°C. MALDI-TOF MS was used to identify bacteria. 18

| Antimicrobial susceptibility test
The disc diffusion method of antimicrobial susceptibility testing was used. The isolated S. hominis from the MALDI-TOF analysis were subjected to antimicrobial susceptibility testing (AST), according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines. 19 An inoculum suspension of S. hominis was

| DNA amplification and gel electrophoresis
DNA amplification was done to detect mecA and mecALGA251. Table S1 shows the primer sequences and base pair sizes used.
Multiplex PCR was carried out on each sample, as previously described by Stegger et al., 20 using Applied Biosystem thermal cycler, USA. After DNA amplification, the amplicons were separated by agarose gel electrophoresis as previously described 18

| Statistical analysis
Data analysis was done using International Business Machines version 26. All categorical variables were expressed as frequencies and percentages. Chi-squared and Fischer's Exact test were performed to determine the association between the duration of the wounds and number of S. hominis isolates from the wounds.
Statistical significance was considered at p ≤ 0.05.

| Sociodemographic characteristics of study participants
In this study, 160 participants were recruited across the eight study communities, using the non-probability convenience sampling approach. Most of the participants were in the age category 35-44 years (30.0%), 55-64 (24.4%), and 65 and above (23.8%). Here, 112 (70.0%) of the participants were females. Farming (31.3%) and fish mongering (20.6%) were the major occupations pursued by the participants, however, 30.0% were unemployed. Table 1 shows the sociodemographic data of the study participants.

| DISCUSSION
The potential threat of antibiotic resistance, particularly among commensals, continues to be a major concern for regions where filarial lymphedema is common. 18 The prevalence of methicillinresistant Staphylococcus aureus (MRSA) among filarial lymphedema patients in Ghana has been documented. 18 In that study, Staphylococcus species emerged as the most abundant among the Firmicutes,

CONFLICTS OF INTEREST STATEMENT
The authors declare no conflicts of interest.

DATA AVAILABILITY STATEMENT
The data sets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

TRANSPARENCY STATEMENT
The lead author Alexander Kwarteng affirms that this manuscript is an honest, accurate, and transparent account of the study being reported; that no important aspects of the study have been omitted; and that any discrepancies from the study as planned (and, if relevant, registered) have been explained.