Nasal Carriage and Molecular Characterization of Methicillin-Resistant Staphylococcus aureus from Healthcare Workers and Community People in Minia City, Upper Egypt

INTRODUCTION

Staphylococcus aureus can be present in the human body as a member of the body flora [1] or as an opportunistic pathogenic bacteria that causes a variety of nosocomial and community diseases [2,3,4]. The nose (anterior nares) is considered the most common biological niche for S. aureus carriage in humans, due to the specific anatomy of this region facilitating the colonization of SA, however, this bacterium can colonize other body regions. These bacteria can live in the nasal cavity in about 20–30% of the human population asymptomatically and persistently [1]. However, ongoing S. aureus nasal colonization may raise the likelihood of subsequent infections [5], moreover, some studies relate S. aureus nasal carriage to some autoimmune diseases[6,7    ].Methicillin-resistant S. aureus (MRSA) infections were initially only found in hospitals, but they have since become a major source of community-acquired (CA) infections worldwide [8]. Extensive and repeated use of broad-spectrum antibiotics is the mandatory contributing factor to the rise in antimicrobial resistance of MRSA. The spread of multi-drug-resistant S. aureus strains, especially MRSA, complicates the treatment of staphylococcal infection and represents a significant economic problem[9].

In addition to antimicrobial resistance, genes involved in virulence are essential for the production of toxins and other substances that exacerbate illness. Increased pathogenicity and the risk of serious infections have been associated with the presence of particular virulence genes, including panton-valentine leucocidin (pvl), alpha-toxin (hla), and toxic shock syndrome toxin-1 (tsst-1) [10].

This study aimed to screen both healthcare workers and community people for nasal carriage of staph aureus, especially MRSA.  In addition, this study was designed to investigate antibiotic resistance profiles and virulence genes (mec A, pvl, hla, seb, fnbA, tsst-1, and icaA) among the isolated strains.

METHODS

Sample Collection:

A total of 233 nasal specimens were taken from community people and healthcare workers of Minia University Hospital during the period between May 2021 to April 2022.  All nasal swabs were streaked onto different media and incubated at 37^oC for 24 hours (11).

Isolation and Identification of S. aureus:

Specimens were identified microscopically and by conventional biochemical tests including Mannitol salt agar culture, catalase production test, coagulase test, and DNase production test [11].

Antibiotic susceptibility testing:

Antibiotic susceptibility was done using the Kirby-Bauer disc susceptibility technique [12], using a standard reference strain, S. aureusATCC (6538), and results were interpreted with the guidance of the Clinical Laboratory Standards Institute (CLSI 2020) [13].  

The following 17 antibiotic discs obtained from (Oxoid, UK) were used: Ampicillin-Sulbactam (SAM:20 µg), Amoxicillin-Clavulanic (AMC:30 µg), Flucloxacillin (FL:5 µg), Oxacillin (OX:1 µg), Piperacillin-Tazobactam (TZP:110 µg), Vancomycin (VA:30 µg),  Azithromycin (AZM:15 µg), Cephalexin (CL:30 µg), Cefoxitin  (FOX:30 µg), Cefepime (FEP:30 µg), Cefaclor (CEC:30 µg), Gentamicin (CN:10 µg), Clindamycin (DA:2 µg), Ciprofloxacin (CIP:5 µg), Levofloxacin (LEV:5 µg), Linezolid (LNZ:30 µg), and Rifampicin (RA:5 µg).

Cefoxitin disc diffusion test:

Using a 30 μg disc, the test was performed on each isolate. The isolate was converted into a 0.5 McFarland standard, and grass culture was performed on the Mueller Hinton agar (MHA) plate. Zone diameters were measured after plates were incubated for eighteen hours at 37⁰C. Oxacillin resistance was defined as an inhibition zone diameterof less than 19 mm, whereas sensitivity to oxacillin was defined as a diameter of more than 20 mm [14].

PCR testing:

DNA Extraction: S. aureus genomic DNA was extracted using the boiling method [15], one to two colonies from overnight cultures were suspended in 500μl of sterile distilled water, then this mixture was boiled at 100 °C for fifteen minutes. After 5 minutes of 14,000 rpm centrifugation, 2μl of the supernatant was used as the genomic template for subsequent testing.

Polymerase Chain Reaction:

Table (1) listed the primers used in that study for the determination of genes encoding methicillin and penicillin-like antibiotics resistance  (mec A),  panton valentine leukocidin (pvl), toxic shock syndrome toxin ( tst-1), staphylococcal enterotoxin b (seb), fibronectin-binding protein A (FnbA), α-hemolysin  (hla), and intracellular adhesion A (icaA). Table(2) illustrates the polymerase chain reaction (PCR) conditions using The DNA thermocycler.

Gel electrophoresis:

 For visualizing the PCR products, 5 μl of the PCR amplicons were loaded in a (1.5) percent agarose gel containing fluorescent ethidium bromide dye (0.5 mg/ml, Medox Biotech India Pvt Ltd) and a molecular weight ladder (1500bp DNA marker; H3 ready to use, Gene Direx). After two hours of electrophoresis at 80 V, different amplified DNA samples were examined using a UV lamp set at 264 nm.

 Statistical analysis:

The data were analyzed using χ2 using SPSS version25 (SPSS, Inc., Chicago, IL, USA). The results were considered significant when P ≤ 0.05.

RESULTS

Ninety-five (40.77%) S. aureus isolates were recovered from 233 collected nasal specimens, including 46 (48.4%) isolates from healthcare workers and 49 (51.6%) from community people. S. aureus was more prevalent in female patients than male ones.

It was observed that S. aureus and MRSA were highly detected in the age group of (21-30) years, while the lowest percentage of their isolation was detected in the age group between (0-10)  as shown in (table 3).

Antibiotic susceptibility test:

Seventeen antibiotics were tested in this study on 95 (40.77%) S. aureus isolates, it was found that the highest resistance percentage of S. aureus isolates was to flucloxacillin for community isolates and to oxacillin for healthcare personnel staph isolates while the least resistance percentage was to clindamycin for all isolates. Table (4) and Fig. (1) showed the resistance pattern of S. aureus isolates, there was no significant difference between S. aureus resistance percentages from healthcare workers and community people for all tested antibiotics except for oxacillin.

Detection of MRSA:

Out of  95 S. aureus isolates, 59 (62.1%) strains were detected as methicillin-resistant staph aureus (MRSA) while 36 (37.9 %) isolates were methicillin-susceptible staph aureus (MSSA), this result was obtained by cefoxitin-resistance testing then confirmed by PCR test for mecA gene. It was detected in healthcare workers (30, 50.8%) more than in community people (29, 49.2%), table (5). In addition, MRSA was found in females (34, 57.6%) more than in males  (20, 42.4%).

Virulence genes:

       It was found that 29 (30.52%) S. aureus strains harbored pvl gene. Panton valentine leucocidin-positive S. aureus isolates were detected more detected in community people, 21(72.4%) than in healthcare workers, 8 (27.6%). Out of 29 community MRSA isolates in this study, there was a high prevalence of pvl (14, 48.27%), however, the prevalence of pvl among 30 MRSA isolated from healthcare workers was 5 (16.6%). The percentage of the pvl-positive MRSA was higher than the pvl-positive MSSA.

It was observed that the distribution of virulence genes among pvl positive strains was as follows: mec A, hla, fnbA, and seb genes were more detected in community people, they were detected in 14 (48.3%), 10 (34.5%), 4 (13.8%) and 2 (6.9%) respectively. Ica A gene was only detected in healthcare workers' pvl positive strains (8, 28%), while the tsst-1 gene wasn't detected among all S. aureus pvl positive strains as shown in Fig (2) and Table (6).

 Table (1). sequences of primers