Microbes and Infectious Diseases Phenotypic and genotypic detection of antimicrobial resistance and virulence factors among Staphylococcus aureus clinical isolates

Background: Staphylococcus aureus (S. aureus) is a world-wide nosocomial and community-acquired infectious agent. This study aimed to determine the prevalence rate of S. aureus infections with assessment of their antibiotic susceptibility patterns and virulence profiles using available phenotypic and genotypic methods. Methodology: Staphylococcus aureus isolates were collected and identified by conventional methods. Antimicrobial susceptibility testing was done and interpreted according to Clinical and Laboratory Standards Institute guidelines (2022) followed by macrolide lincosamide streptogramin B (MLS B ) phenotyping by D test. Detection of staphylococcal virulence (hla and etb) and antibiotic resistance (ermB and msrA) genes were also done. Results:


Introduction
Staphylococcus aureus (S. aureus) is one of the leading causes of morbidity and mortality in both hospital and community-acquired infections especially those with high antimicrobial resistance rates [1]. About 500,000 S. aureus-related infections occur annually in the United States [2]. In Egypt, methicillin resistant S. aureus (MRSA) contributes to about 40-80% of S. aureus healthcare-associated infections [3].
Many reports described MRSA general prevalence rate of ≥ 20% among hospitalized patients in different Arabian countries. Staphylococcus aureus has the ability to survive for days or even weeks on environmental surfaces in healthcare facilities. It can withstand a wide range of temperatures, humidity and exposure to sunlight. These characteristics enable S. aureus to contaminate a wide range of hospital items with high infectivity [4,5].
The World Health Organization (WHO) has considered MRSA as important antibiotic resistant bacteria and put them on their priority list. All organisms on that list require novel therapeutic approaches and substantiate an urgent need for new antibiotics options [6].
Vancomycin is the drug of choice for treating severe MRSA infections. However, its use has several limitations like: poor tissue penetration, narrow therapeutic index, slow activity potential nephrotoxicity and ototoxicity [7]. Few alternatives are available for treatment of MRSA infections. Ceftaroline (CPT) is a fifth-generation cephalosporin approved by the Food and Drug Administration for the treatment of complicated staphylococcal infections with increased affinity to penicillin binding proteins [8].

Streptogramins
(Dalfopristin / Quinupristin) represent one of the few potential protein interrupting anti-staphylococcal agents that interfere with protein synthesis by binding to the 50S subunit of the bacterial ribosome like macrolides and lincosamides explaining the cross resistance to macrolide, lincosamide and streptogramins (MLS) antibiotics mediated by many resistance genes including; erm & msrA genes [9]. Different S. aureus strains carry different virulence factors with varying pathogenic outcomes [10]. Haemolysins and exfoliative toxins are characterized virulence factors in S. aureus. Haemolysins make holes in the host cell membrane, facilitating toxins entry and cell damage [8]. Also, exfoliative toxins enhance host colonization and invasion of skin and injured mucosa as they are proteases which can recognize and hydrolyze skin proteins [11].
The alarming high levels of virulent MRSA in Egyptian hospitals made awareness of MRSA control measures among the medical staff at different health centers a must; emphasizing the need for well-organized antibiotic stewardship programs with proper hand hygiene and strict disinfection measures to keep the prevalence of MRSA carriage and infections as low as possible [7]. The aim of this work is to determine the prevalence rate of S. aureus infections in Menoufia University Hospitals and to assess their virulence profiles & antibiotic susceptibility patterns (with the evaluation of alternative treatment options) using available phenotypic and genotypic methods focusing on MLSB phenotypes.

Methods
This cross-sectional study was performed in Medical Microbiology and Immunology Department and Clinical Pathology Department, Faculty of Medicine, Menoufia University during the period from February 2021 to May 2022. Written consents and full patient history (name, age, sex, duration of hospital stay, presence of associated co-morbidities and exposure to invasive procedures) were obtained from all patients who were admitted at different departments and ICUs of Menoufia University Hospitals. This study protocol was approved by the local Ethics Committee of the Menoufia University.

Processing of the samples:
Different clinical samples (blood, pus, urine, sputum plus surgical wound swabs) were collected from patients. These collected specimens were immediately delivered to Microbiology Laboratory within two hours to be processed and examined. Each sample was inoculated on nutrient, sheep blood and mannitol salt agar. All were incubated aerobically at 37°C for 24-48h [12].

Identification of Staphylococcus aureus:
Staphylococcus aureus isolates were identified according to conventional methods by colonial appearance (golden yellow colonies on nutrient agar, white to yellow, creamy opaque beta haemolytic colonies on sheep blood agar, mannitol fermenting colonies on mannitol salt agar); Gram's staining (Gram positive cocci arranged in in grape like clusters) and biochemical reactions (catalase positive & tube coagulase positive) [12].

Detection of methicillin resistance
Detection of methicillin resistance was done using cefoxitin disc (30 µg); the diameter of inhibition zone was measured, categorized as resistant if zone diameter ≤ 21mm after 16-18 incubation hours at 34 o C considered as MRSA [13].

Macrolide lincosamide streptogramin B (MLSB) phenotyping
Staphylococcus aureus isolates were investigated for macrolide lincosamide streptogramin B (MLSB) phenotyping by erythromycin-clindamycin double disk diffusion test (D-Zone test). The test results were interpreted as different phenotypes as follows [13,14]: • Constitutive cMLS phenotype: if isolate was resistant to both erythromycin and clindamycin.
• Inducible iMLSB phenotype: if isolate was resistant to erythromycin and susceptible to clindamycin with positive D-zone test (flattening of clindamycin growth inhibition zone adjacent to erythromycin disc or even hazy growth).

Statistical analysis
The collected data were tabulated and analyzed by SPSS (statistical package for the social science; SPSS Inc., Chicago, IL, USA) version 15 for Microsoft Windows. Descriptive statistics were used and expressed as number and approximated percentages.
According to methicillin susceptibility, S. aureus isolates were classified into methicillin resistant S. aureus (MRSA) (84/152-55.3%) and methicillin susceptible S.aureus (MSSA) (68/152-44.7%). There was no statistically significant difference between MRSA and MSSA regarding type of sample but there was statistically significant difference between them regarding patient gender (53.6% of MRSA were isolated from female patients) as shown in table (2). Also, there was statistically significant difference regarding age, duration of hospital stay, associated co-morbidities and exposure to invasive procedures as approximately 52.4%, 73.8%, 41.7% and 36.9% of MRSA strains were isolated from patients aged more than 50 years, stayed for more than 7 days in hospitals, with associated co-morbidities and invasive procedures respectively.
Antimicrobial susceptibility patterns of S. aureus isolates obtained by disk diffusion method are illustrated in table (3). MRSA isolates were highly susceptible to linezolid, streptogramins (100% for each), vancomycin (95.2%) and ceftaroline (90.5%). There was a high statistically significant difference between MRSA and MSSA regarding penicillin, erythromycin and ciprofloxacin susceptibility and a statistically significant difference regarding ceftaroline, clindamycin, nitrofurantoin and trimethoprimsulfamethoxazole susceptibility.
According to macrolide lincosamide streptogramin B (MLSB) phenotyping by D test, statistically significant difference was shown between MRSA and MSSA regarding cMLSB, iMLSB, MSB and S phenotypes but without statistically significant difference regarding L phenotypes as demonstrated in table (4).
Regarding vancomycin susceptibility by broth dilution method, 95.2% of MRSA strains were VSSA and 4.8% were VISA. While all MSSA were VSSA as shown in table (5).
By multiplex PCR, haemolysin hla gene was detected in 77 MRSA isolates (91.7%). Regarding antibiotic resistance genes, erm B resistance gene was detected in 9 (10.7%) MRSA isolates. While msrA resistance gene was detected only in one MRSA isolate (1.2%). On the other hand, etb gene was not detected in any isolate under previously recommended PCR thermos-cycling conditions. Antimicrobial susceptibility profile regarding erm B gene presence in MRSA isolates was illustrated in table (6).

Discussion
The center for disease prevention & control (CDC) has labeled MRSA as a serious threat to human health. It is implicated in numerous diseases ranging from superficial skin infections to sepsis. Accurate numbers describing the prevalence and characteristics of these infections and the added burden they cause to developing countries in the Eastern Mediterranean and African regions are lacking [5,7] [30,31]. Prospective studies with larger sample size are warranted to support or to verify our findings.

Conclusion
Methicillin and MLSB resistance among S. aureus are concerning. Therefore; great significant efforts should be made for their regular surveillance and accurate detection in hospital settings with strict compliance to infection prevention and control measures to limit their spread.
Linezolid, streptogramins, vancomycin and ceftaroline have acceptable susceptibility patterns. Proper antibiotic stewardship program is strongly recommended to keep the benefit of these antibiotics in treatment of MRSA infections. A great diversity in virulence patterns was determined; haemolysin production was detected in almost all MRSA phenotypically and genotypically. While, exfoliative (etb) gene was not detected at all. Further analytical studies with larger sample size are recommended to overcome any fundal or numerical limitation

Limitations of the study
We identified S.aureus by manual biochemical reactions and assessed vancomycin susceptibility by broth dilution method (homeprepared). Using of more accurate diagnostic methods for proper identification of S.aureus and proper differentiation of vancomycin nonsusceptible S.aureus isolates into VRSA, VISA and hVISA with detection of responsible resistance genes are the subject of further studies by the authors.

Acknowledgments:
Thanks to Medical Microbiology and Immunology Department and Clinical Pathology Department, Faculty of Medicine, Menoufia University for their cooperation and continuous support.

Conflict of interest:
The authors declare that there is no conflict of interest.

Antimicrobial Resistance in the WHO African
Region: a systematic literature review.
Brazzaville: WHO Regional Office for Africa