A descriptive analysis of PVL-positive multidrug-resistant Staphylococcus aureus in hospital-associated infections in Saudi Arabia

Methicillin resistant Staphylococcus aureus infections impose a huge risk to public health in healthcare and community settings worldwide. Therefore, it is of interest to document data on the anti-biogramas and genotypes of isolates from Saudi Arabia. We assessed the antimicrobial susceptibility, determined spa (protein A gene) and analyzed multilocus MLST genotypes, and detected PVL gene in these isolates. We collected 28 clinical MRSA isolates, cultured and determined the minimum inhibitory concentrations of 17 antimicrobial agents using Vitek2 system (BioMerieux, USA) from 3 hospitals in Saudi Arabia during the year 2012. Polymorphic region of the spa and seven housekeeping genes were amplified and sequenced. BioNumerics v.5.1 (Applied Maths) was used for spa typing and MLST. Samples were screened for the presence of PVL and mecA genes using polymerase chain reaction (PCR). Analysis shows that all isolates were susceptible to chloramphenicol, rifampicin, nitrofurantoin, teicoplanin, daptomycin and vancomycin. The T4573/ST22 strains are found to be prevalent in the Saudi Arabia (N=6, 21%). We further noted that three isolates (t363/ST240 strain) were resistant to eight antimicrobial agents. Most of t4573/ST22 strains were PVL positive, resistant to ciprofloxacin and linked to HA-MRSA infections. We document data for the presence of emerging multi drug resistant S. aureus strains carrying the PVL gene circulating within hospitals. This highlights the urgent need for continuous active surveillance and implementation of prevention measures.


Background:
Staphylococcus aureus infections are a major cause of communityacquired (CA-) and hospital-acquired (HA-) infections and are associated with significant morbidity and mortality rates. In 1999, National Healthcare Safety Network-USA (NHSN) reported that 52.3% of nosocomial infections occurring in ICUs were due to methicillin resistant Staphylococcus aureus (MRSA) [1]. HAIs caused by multidrug-resistance have become a serious public health problem, especially in intensive care units (ICUs) due to patients' existing condition, impaired immunity and exposure to many invasive devices and antibiotic treatments, leading to increasing cost, morbidity and mortality [2][3][4]. Infections with CA-MRSA in patients with no contact with the hospital environment have been described worldwide [5][6][7][8]. Exposure of CA-MRSA strains to the selective pressure of antibiotics used in hospitals has the potential for selection of expanded antibiotic resistance profiles in the pathogen. Consequently, constant re-introduction of MRSA into hospitals from the community reservoir could significantly hamper infection control efforts [7,9]. CA-MRSA strains are polyclonal, with an epidemiological association between clone type and geographical origin, whereas HA-MRSA infections are caused by a limited number of clones [7, 10, 11]. The rate of MRSA infection ranges from 12% to 49%, with variations in susceptibility pattern profiles, but there are very limited data on the prevalence of different genotypes in Saudi Arabia [12][13][14][15]. A systemic review and meta-analysis of the prevalence of MRSA in the Kingdom of Saudi Arabia, based on 26 articles published between 2002 and 2012, revealed variation in the prevalence and antimicrobial susceptibility in patients, but also very limited data on genotyping and virulence gene detection [16]. Panton-valentine leukocidine-positive Staphylococcus aureus causes recurring skin and soft tissue infections. Recently, a molecular characterization study on MRSA infections in Saudi Arabia showed a high diversity clonal complex encoding for Panton Valentine Leukocidine (PVL) and carrying resistance markers in CA-MRSA strains [17]. It is of interest to document data on the anti-biogramas and genotypes of multi drug resistance PLV gene positive S. aureus strains from Saudi Arabia.

Methodology: Bacterial isolates:
Twenty-eight clinical MRSA isolates were collected randomly from three referral hospitals in Saudi. Isolates were cultured on sheep blood agar and incubated at 37°C for 24 h. S. aureus isolates were identified as round white colonies positive to Gram stain, catalase and slide agglutination tests [18].

Detection of MRSA isolates:
Methicillin-resistant phenotype was confirmed according to British Society for Antimicrobial Chemotherapy (BSAC) standards using the Vitek2 system (BioMerieux, USA). An isolate is considered methicillin resistant when the minimum inhibitory concentration (MIC) breakpoint of oxacillin is >2 mg/L and of cefoxitin >4 mg/L [19].

Staphylococcal protein A (Spa) and Multi locus sequence typing (MLST-typing):
The polymorphic regions of the spa gene and seven housekeeping genes (arc, aroE, glp, gmk, pta, tpi and yqiL) were amplified by PCR using the primers (listed in Table 1), as described previously [20,21]. All sequencing reactions were carried out using the ABI PRISM BigDye Terminator cycle sequencing ready reaction kit (Applied Biosystems, Foster City, California, USA). The sequence data were analysed in BioNumerics v.5.1 (Applied Maths).

Detection of PVL and mecA genes:
A PCR was carried out for PVL and mecA gene amplification individually in two runs using a thermo cycler (Eppendorf, Hamburg, Germany) as described previously with modifications [22]. In brief, 20 µL of final reaction mixture of each run containing 10 µL of 1X Taq master-mix reaction, 1 µL of each primer (100µM) (listed in table 1) and 2 µL of the DNA (20 ng) template and mecA-F: 5`-mecA-R: 5`-were used. PCR conditions were as follows: initial denaturation at 95°C for 10 min, followed by 25 cycles of denaturation at 95°C for 1 min, annealing at 74°C for 1 min and extension at 72 °C for 1 min and a final extension step at 72°C for 10 min. MRSA ATCC 35591 and DNase/RNase-free distilled water were included as positive and negative controls, respectively. For visualisation of the product, 10 µL of PCR amplicons were mixed with 1 µL of EzVision One loading dye (Amresco, Solon, OH, USA) and loaded into a 1.5% (wt/vol) agarose gel (Agarose I™). Electrophoresis was carried out in 1X TAE buffer at 80v for 50 minutes. A molecular weight marker 100-bp ladder (Promega, Madison, WI, USA) was included on each gel. Bands were visualised using an Alpha Innotech UV imager (FluorChem™).

Discussion:
MRSA infections are prevalent in healthcare and community settings and more prevention efforts are needed to overcome newly emerging multidrug resistance. The incidence rates and susceptibility patterns to antimicrobial agents are variable across geographical regions, due to many factors such as surveillance methods, economic status and use of antimicrobials. Here, we have conducted an investigation on MRSA strains from Taif hospitals in Saudi Arabia. MRSA isolates were collected randomly January-August 2012 from three governmental hospitals serving 1400000 populations in Taif and Hail city; King Abdul Aziz Specialist Hospital (KAASH), King Khalid Hospital (KFH) and Children Hospital (CH) to assess HA-MRSA and CA-MRSA infections including antimicrobial susceptibility pattern, MLST and spatyping, PVL gene detection. All MRSA isolates were susceptible to chloramphenicol, daptomycin, nitrofurantoin, rifampicin, teicoplanin and vancomycin. There is wide variation in the antimicrobial susceptibility pattern in Saudi Arabia. Rifampicin resistance has previously been observed in Al Khubar (76%), Qassem (60%), Taif and Riyadh (33%), Aseer (11% and Makkah (6%), but not in Najran city [24][25][26][27][28][29][30]. Chloramphenicol resistance has been recorded in Taif (30%) and Asser (1.5%) [25][26]. Only one case of daptomycin resistance has been reported in Hail, Saudi Arabia, in Najran (9.4%) [28]. This variation can be assumed to be a reflection of differences in study design and surveillance method. Multidrug-resistant S. aureus has been reported in many hospitals world-wide but, to the best of my knowledge, this thesis is the first study to report multi-drug resistant MRSA with resistance to ciprofloxacin, clindamycin, erythromycin, fusidic acid, gentamicin, mupirocin, tetracycline and trimethoprim in Saudi Arabia.
A diversity of spa and MLST types were identified in Hail, with t4573/ST-22 being the most prevalent in the hospital (21%). Similar diversity has been reported in Riyadh [30,31]. Interestingly, although t4573, harbouring PVL gene and resistant to ciprofloxacin, is common in Taif city (n=6, 21%), the strain has a very rare distribution worldwide and is not well characterised. One isolate has been reported in Saudi Arabia (Riyadh) [30], one in New Zealand [32] and two in Sweden (www.spaserver.ridom.de). One isolate of t037 was detected in Taif, but t037 is common in Riyadh (35%) and has a wide global distribution. This variation probably reflects the low sample collection in the present study. Clonal complex (CC) 22 is the most common MLST type (N= 9, 32%) followed by CC97, CC80, CC241, CC2882, CC6, CC5 and . It is possible that CA-MRSA category infections (SCCmecA types I, II or III) are transferred to, and colonise, patients or hospital environments and may cause hospital infections, thus becoming HA-MRSA category. We state limitations on the epidemiology of MRSA infections in Taif city due to (1) lack of SCCmecA-typing; (2) small sample size; and (3) dataset is through hospital-based surveillance.
Association between MRSA ST-80 and PVL positive is found without susceptibility to antimicrobial agents. An in-depth study using a larger number of clinical MRSA ST-80 samples is required to establish such association. Thus, we document data for the presence of emerging multi drug resistant S. aureus strains carrying the PVL gene circulating within hospitals in Saudi Arabia. This highlights the urgent need for continuous active surveillance and implementation of prevention measures.

Conclusion:
The presence of multi drug resistance PLV gene positive S. aureus strains circulating within hospitals in Saudi Arabia warrants the need for continuous active surveillance and implementation of prevention measures.