The Prevalence of Resistance to Methicillin in Staphylococcus aureus Strains Isolated from Patients by PCR Method for Detec-tion of mecA and nuc Genes.

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) is the main cause of hospital infection emerged over the last decades. Rapid detection of MRSA is important for patient care and proper usage of infection control. Detection of mecA genes (encoding resistance to methicillin and other similar antibiotics) and nuc genes (encoding staphylococcal thermostable nuclease) by PCR method is now considered for rapid identification of MRSA strain. The aim of this study was to determine the prevalence of MRSA isolated from patients in Tehran, Iran by PCR method for detection of mecA and nuc genes.
METHOD: Phenotypic method such as microscopic and colony morphology and catalase and coagulase tests were used for identification of S. aureus isolates. DNA was extracted from all isolates and the presence of nuc and mecA gene was detected by PCR method. For determination of MRSA by phenotypic methods, oxacillin disk diffusion test were used. Data were analyzed by SPSS software.
RESULTS: Out of 126 clinical sample identified by phenotypic method, 101 isolates had nuc gene. In disk diffusion tests by oxacillin disk, 78.2% of isolates were considered to be MRSA, but in PCR method for mecA gene, 69% isolates were positive.
CONCLUSIONS: The results showed a high prevalence of methicillin-resistance among S. aureus isolates. Identifying MRSA strains, isolating MRSA-positive patients and carrier's treatment in a hospital to prevent MRSA infection is important in limiting the spread of MRSA. The PCR method for detection of nuc and mecA genes has potential for rapid and accurate diagnosis of MRSA strains.

Introduction

Staphylococcus aureus is one of the most important human pathogens, causing a wide range of nosocomial and community acquired infections, from mild skin and soft tissue infections to wound infections and bacteriemia (1). Currently several methods are used to distinguish S. aureus from other bacteria, which include culture methods, followed Gram's staining, grow in mannitol salt agar and fermentation of mannitol and catalase and coagulase tests; however these routine methods are time consuming and cumbersome and the accuracy of most of them has constantly been questioned the course of over time (2). PCR-based method is used as the single most reliable and sensitive test for accurate and rapid identification of S. aureus (3). S. aureus produces an extracellular thermostable nuclease, encoded by nuc gene, which is one of the most distinguishing and successful characteristics that might be used for distinguishing S. aureus from other staphylococcus spp. This suggests that nuc gene is a specific marker gene and PCR is a useful method for identifying this gene in S. aureus (4).

Methicillin-resistant S. aureus (MRSA) is a variant of S. aureus bacteria that has evolved resistance to methicillin and other antibiotics including all other beta lactams, aminoglycosides and macrolides (5). Chromosome and plasmids mediated resistance to antimicrobial agents in S. aureus strains (6). Methicillin resistance in MRSA is mediated by the presence of 78-kDa penicillin binding protein PBP2′ (or PBP2a) which has a very low affinity for beta-lactam antibiotics. PBP2a is encoded by the mecA gene (7). mecA gene is located on a mobile genetic element (from 21-to 67-kb), that is called staphylococcal cassette chromosome mec elements (SCCmec) (8). The first strain of MRSA was reported in the UK and Europe in 1961, just less than 1 year after the introduction of methicillin for clinical use. Since that time, MRSA has become a major public health problem worldwide and the prevalence of MRSA has been dramatically rising in recent years (9). Thus, rapid and exact methods for identification of MRSA in clinical specimens are essential for accurate diagnosis and antimicrobial therapy (10).

Disk diffusion method and agar dilution method are the most commonly used methods in routine clinical diagnostic laboratories to determine methicillin resistance; but these methods have low specificity in detection of methicillin resistance and by the mere results of disk diffusion methods or agar dilution methods, the true prevalence of MRSA cannot be determined (11). Using polymerase chain reaction (PCR) method for detection of mecA gene is considered as the gold standard method for the detection and identification of prevalence of MRSA and has been described in recently published reports (12).

The aim of this study was to determine the prevalence of resistance to methicillin in S. aureus isolated from patients in Tehran, Iran, by combination of phenotypic and genotypic (PCR method for detection of mecA and nuc genes) methods.

Materials and Methods

Identification of S. aureus isolates

A total of 126 Staphylococcusaureus isolates were collected from January 2008 to June 2008 from various clinical specimens of patients admitted to four university hospitals (Imam Khomeini, Mostafa Khomeini, Shariati, Motahari) in Tehran, Iran. These isolates were transferred to Microbiology Department of Medical School of Shahed University.

Multiple isolates from the same patients, even if the site of infection was different, were excluded. After prepare the new cultures of isolates, Gram's staining were done on isolates smear to ensure the absence of contamination and study of bacteria morphology. All isolates were reconfirmed and identified as S. aureus based on colony and microscopic morphology (grape-like Gram-positive cocci) and positivity in catalase and coagulase tests. Altogether, 126 isolates were collected and stored at -70°C in freezing medium (nutrient broth containing 15% glycerol). Growth on mannitol salt agar medium and fermentation of mannitol were also checked for all isolates.

Detection of methicillin resistance by phenotypic method

Disk diffusion test was performed by oxacillin (1µg) disk (MAST Diagnostics, UK) on Mueller Hinton agar (Merck, Germany) plate inoculated by each isolate according to the Clinical Laboratory Standards Institute (CLSI) guidelines (13). Control strain was S. aureus ATCC25923. Intermediate-resistant strains were considered resistant.

DNA extraction

For rapid DNA extraction, five colonies from overnight growth on brain heart infusion (BHI) agar plates were picked up and suspended in 500 µl of sterile distilled water. The suspension was then heated at 100°C for 15 min. After centrifugation for 5 min at 14,000 rpm to sediment the debris, the clear supernatant was used as template DNA in PCR method (14).

Primers for PCR

All primers used in this study were synthesized and purchased from Cinnagen Company (Tehran, Iran),according to sequences obtained from GenBank and compared with the sequence of nuc and mecA genes published by Zhang et al. (4). The primer sequences were as follows: Nuc1 (5'-GCG ATT GAT GGT GAT ACG GTT-3') and Nuc2 (5′-AGC CAA GCC TTG ACG AAC TAA AGC-3′), for nuc gene, and MecA1 (5'-CCA ATT CCA CAT TGT TTC GGT CAT A- 3') and MecA2 (5'-GTA GAA ATG ACT GAA CGT CCG ATA A -3') ′), for mecA gene.

Molecular detection of nuc gene by PCR method

PCR method were performed in a final volume of 20µl, containing 2 µl of template DNA, 2 µl of PCR buffer (10x), 1 µl of MgCl2 (50mM), 4 µl of dNTPs (1mM), 1 µl of each Nuc1 and Nuc2 primers (10 Pmol), 0.25 µl of Taq DNA polymerase (5u/µl) and 8.75 µl of double distilled water. All materials were purchased from Cinnagen Company (Tehran, Iran). PCR amplifications were performed with Techne thermocycler (Touchgene Gradient, UK), using the following cycle conditions: an initial denaturation step at 94°C for 5 min; 30 cycles of 94°C for 1 min, 50°C for 1 min, and 72°C for 2 min, and a final extension step at 72°C for 10 min. The PCR products were electrophoresed in 2% agarose gels with 1X Tris-acetate-EDTA (TAE) buffer at 100V for 100 min and then gels were stained with ethidium bromide (Cinnagen Co., Tehran, Iran) to see the amplified DNA fragments (279 bp) under UV light box by comparison with a molecular size marker (100 bp ladders, eurobio, UK). Positive and negative controls were S. aureus ATCC29213 and molecular grade water, respectively (4).

Detection of mecA gene by PCRmethod

The PCR reactions were carried out in a 20 µl reaction volume, containing 2 µl of template DNA, 2 µl of PCR buffer (10x), 1 µl of MgCl2 (50mM), 4 µl of dNTPs (1mM), 4 µl of each MecA1 and MecA2 primers (10 Pmol), 0.25 µl of Taq DNA polymerase (5u/µl), and 2.75 µl of double distilled water. All materials were purchased from Cinnagen Company (Tehran, Iran). Thermocycling conditions in a Techne thermocycler (Touchgene Gradient,UK),were as follows: an initial denaturation step at 94°C for 5 min, followed by 30 cycles of 94°C for 30 sec, 52°C for 30 sec, and 72°C for 30 sec, with a final extension at 72°C for 5 min. PCR products were run on 2% agarose gel and analyzed as described above for amplified DNA (310 bp). Methicillin resistant S. aureus clinical isolate containing mecA gene and molecular grade water were used as positive and negative controls, respectively (4). All the personal information of the patients was remained private during all steps of the research.

Results

A total of 126 isolates were identified as S. aureus by routine phenotypic methods including Gram's staining, colony morphology and catalase and coagulase test. The results of culture on mannitol salt agar have shown that, 95 (75.9%) of isolates showed positive results with grown on mannitol salt agar and fermentation of mannitol. Thus culture on mannitol salt agar to identify S. aureus, is not able to make a definite identification alone.

Figure 1 show image of agarose gel electrophoresis after amplification of nuc gene (279 bp) for some isolates by PCR method. By applying PCR method, among the 126 clinical samples that were identified as S. aureus with phenotypic methods, 101 (80.2%) isolates were found to be nuc positive (Fig. 1). The presence of some discrepancies between the results of phenotypic and genotypic methods for detection of S. aureus strain, make it clear that, the method for identification of nuc genes is not sufficient alone. So, phenotypic and genotypic methods together were used for identification of S. aureus strain. Table 1 to 3 shows the frequency of S. aureus isolates, accor-ding to hospitals, admitted wards and type of clinical specimens.

Fig. 1

Image of agarose gel electrophoresis after amplification of nuc gene (270 bp) for some isolates by PCR method. Lane M is 100 bp DNA ladder, lanes 1 to 5 and 7 to 11 is nuc gene, NC: negative control, PC: positive control (S. aureus ATCC29213)

Table 1

Frequency of S. aureus isolates according to hospitals

Hospital	Number of S. aureus isolates	Frequency distribution of strains (%)
Imam Khomeini	72	57
Mostafa Khomeini	28	22
Motahari	14	11
Shariati	12	10
Total	126	100

Table 3

Frequency of S. aureus isolates according to type of clinical specimens

Origin	Number of S. aureus isolates	Frequency distribution of strains (%)
Respiratory	73	57.6
Wound	31	25
Blood	11	8.7
Urine	1	1.1
Others	10	7.6
Total	126	100

Figure 2 show image of agarose gel electrophoresis after amplification of mecA gene (310 bp) for some isolates by PCR method. From 126 S. aureus isolates, 87 (69%) isolates harbor the mecA gene and identified as methicillin-resistant S. aureus (MRSA) and the remaining 39 (31%) isolates were methicillin-susceptible (MSSA).

By disk diffusion test, among 126 isolates of S. aureus, 98 (78.2%) of isolates were determined MRSA and 21.8% MSSA. The results of comparison between the phenotypic and genotypic methods are displayed in Table 2. Frequency distribution (percentage) of MRSA and MSSA isolates according to different hospital, admitted wards and clinical specimens were compared and significant differences were observed (Fig. 3 to 5).

Table 2

Frequency of S. aureus isolates according to admitted wards/Departments

Wards/Departments	Number of S. aureus isolates	Frequency distribution of strains (%)
Operation	11	9
Intensive care unit	33	26
Emergency	4	3
Respiratory	28	22
Burn	14	11
Internal	36	29
Total	126	100

Fig. 3

Frequency distribution of MRSA and MSSA isolates in hospitals. Most of MRSA strains were obtained from Imam Khomeini, Mostafa Khomeini and Motahari Hospitals, while most of MSSA strains were obtained from Shariati Hospital

Fig. 5

Frequency distribution of MRSA and MSSA isolates in clinical specimens. The prevalence of MRSA strains in all clinical specimens was significantly higher than MSSA, except for wounds specimen.

Image of agarose gel electrophoresis after amplification of mecA gene (310 bp) for some isolates by PCR method. Lane M is molecular size marker (100 bp DNA ladder), lanes 3 to 6, 8 and 11 is mecA gene, PC: Positive control (Methicillin resistant S. aureus clinical isolate containing mecA gene), NC: Negative control

Frequency distribution of MRSA and MSSA isolates in wards. The majority of the strains obtained from different hospital wards were identified as MRSA strains, except the respiratory ward that the prevalence of MRSA and MSSA were almost same

In this study, there was no significant difference between MRSA and MSSA isolates regarding the prevalence of presence of nuc gene; the nuc genes were observed in 69 (79.3%) of the MRSA isolates (out of 87 isolates) and in 32 (82.1%) of the MSSA isolates (out of 39 isolates).

Discussion

MRSA is type of S. aureus that has developed resistance to beta-lactam antibiotics and is responsible for community-associated and nosocomial infections (8,15). Therefore, finding the rapid and accurate techniques for identification of these bacteria, can contribute to the rapid diagnosis and timely treatment of infections. Currently, various laboratory methods for detecting S. aureus and MRSA strains have been reported (16). The conventional phenotypic methods have a high ability to detect S. aureus and MRSA strains and they are still widely used in clinical laboratories as routine identifications tests. But phenotypic methods have some disadvantages. So many studies are being conducted to improve and increase the sensitivity and specificity of methods for determination of S. aureus. On the other hand, there is still no consensus on the best rapid and accurate detection method with high sensitivity; therefore, many articles are published about the comparison of different rapid methods for the detection of these bacteria and advantages of each technique (3).

Our study showed that the mannitol salt agar test was negative for 5% of S. aureus isolates; as this shown in other studies (17, 18).S. aureus can be easily identified by PCR amplification of nuc gene; therefore, nuc gene has been used for the detection of S. aureus by some researchers (15, 19). The diagnostic values for detection of nuc gene by PCR based method were 93.3% sensitivity and 89.6% specificity (1). Brakstad et al. used the nuc gene as target DNA to identify S. aureus strains and recorded amplification product of nuc gene at 279 bp region for all 90 of 90 reference clinical isolates of S. aureus (20). Therefore, we used primers of their study in our project. A high percentage (80.2%) of S. aureus isolates were positive for the presence of nuc gene, that can confirm the ability of PCR method as fast and reliable method for detection of the nuc gene to identify S. aureus strains. However, some phenotypically S. aureus isolates were shownnegative result probably due to non-optimal experimental conditions for PCR method, the differences in the nucleotide sequence among the nuc genes caused by some mutation or the absence of nuc gene in some S. aureus strains. So it just seems that, a negative PCR method result for nuc gene cannot prove the absence of S. aureus among clinical isolates.

In this study, we used phenotypic (oxacillin disk diffusion test) and genotypic (PCR method for mecA gene) methods for detection of MRSA. The results of our study showed that 78.2% and 69% of S. aureus isolates were recognized as MRSA by disc diffusion test and PCR method, respectively. Whenever 107 (84.9%) of isolates had shown similar results in phenotypic and genotypic assays, 10 (8.3%) of isolates were mecA-positive in PCR but methicillinsensitive in disk diffusion test. This could be attributed to not consistently expression of mecA gene. Besides, 19 (15.1%) of the phenotypically methicillin-resistant strains were negative for mecA gene. This resistance can be due to the presence of other resistance mechanisms, such as large amounts of produced beta-lactamase or the lack of optimal PCR conditions or change in mecA gene due to the mutations. Phenotypically methicillin resistant strains without mecA gene and methicillin sensitive strains harboring mecA gene are also shown in other studies (21, 22).

Considering that detection of the mecA gene by PCR method is gold standard method for identifying methicillin resistance in S. aureus isolates, the prevalence of MRSA in this study have determined 69%. This prevalence of MRSA is high and comparable to results of other studies in Tehran, Iran; 53% by Rahbar et al. (23) and 88% by Rahimi et al. (24). This prevalence is also similar to other international studies performed in other areas, such as United States, France, Canada, Australia, European countries (25-28).

This study also demonstrates that the distribution of MRSA isolates vary in different hospital wards. This de?nitely shows that some patients are more likely to catch infection (26). In the present study, the maximum and minimum isolation of MRSA was from ICU and emergency ward respectively; as CDC (Centers for Disease Control and Prevention) reported that in the United States, more than 50% of S. aureus isolates in ICU and 40% of S. aureus infections in other hospital units are methicillin resistant (29). In this study, the prevalence of MRSA was significantly different among various clinical specimens and MRSA isolates showed higher prevalence in most clinical specimens (except wounded samples). The variation in prevalence of MRSA among S. aureus isolated from various specimens might be due to the variation in antibiotics usage, infection control practices in different hospitals and prolonged antibiotic treatment of severely sick patients, who generally stay longer in hospital (28). Finally, this study has demonstrated a high prevalence of MRSA, regarding this problem in community, the identification of MRSA in isolates by PCR method, offers a very specific, sensitive and relatively rapid alternative to conventional assays. Moreover, due to the absence of mecA gene in some isolates of MRSA, the use of both phenotypic and genotypic tests combined may provide the best information for obtaining general resultfor detection of MRSA strains.

Conclusion

Considering a significant increase in the prevalence of methicillin-resistance in S. aureus strains caused by the indiscriminate and excessive use of antibiotics during the last decade, our study emphasize on identifying MRSA isolates, isolating MRSA-positive patients and carrier's treatment in a hospital to establish effective measures to prevent MRSA infection. This study shows that PCR method is a useful method for detection of nuc and mecA genes which leads to rapid detection and identification of MRSA cultured from patient's specimens (in less than 6 h) and may provide substantial benefits for infection control byallowing prompt and cost-effective implementation of contact precautions. Here, it is suggested that in order to obtain more reliable results, further studies about the distribution of isolates according to different variablesare required.

Ethical considerations

Ethical issues (Including plagiarism, Informed Consent, misconduct, data fabrication and/or falsification, double publication and/or submission, redundancy, etc.) have been completely observed by the authors.

Table 4

Number of MRSA and MSSAstrains detected byoxacillin disk diffusion test and PCR method for comparison of used tests for 126 strains

Result in		Disk diffusion test
		MRSA	MSSA
	MRSA	107	10
PCR method
	MSSA	19	116