Genetic Polymorphism of agr Locus and Antibiotic Resistance of Staphylococcus aureus at two hospitals in Pakistan

Objective: The accessory gene regulator (agr) locus in Staphylococcus aureus (S. aureus) is a global regulator of quorum sensing and controls the production of virulence factors. This study was carried out to investigate the agr specific groups both in methicillin resistant and sensitive Staphylococcus aureus (MRSA and MSSA) and their relation with antibiotic resistance. Methods: A total of 90 clinical S. aureus isolates were studied from two tertiary care hospitals. The isolates were identified by standard biochemical tests. Methicillin resistance was confirmed by oxacillin and cefoxitin resistance. Multiplex PCR was used to determine the agr groups. Results: MRSA prevalence was found to be 53.3%.The agr groups’ distribution in MRSA was as follows: 22 (45.8%) belonged to group I, 14 (29.1%) belonged to group III and 2 (4.1%) belonged to group II. agrIV was not detected in MRSA. For 17 isolates, the agr group was not detected.agr III isolates showed higher antibiotic resistance than agrI isolates except in case of oxacillin and linezolid. Conclusions: Strict infection control policy and antibiotic guidelines should be adopted to control the problem of MRSA. Higher prevalence of agr I and agr III shows that they are dominant agr groups of our area.


INTRODUCTION
Staphylococcus aureus is an extracellular Gram positive pathogen, which causes a number of infections such as pneumonia, endocarditis, and septic arthritis. 1 In many cases, the infection originates from hospital derived antibiotic resistant bacteria, among which the most common is MRSA whose prevalence varies markedly between different regions and hospitals. 2 Within hospitals, MRSA accounts for 40-70% of infections in Intensive care units 3 and overall it is responsible for 50% or more of hospital acquired infections in many countries. Normal nasal carriage of S. aureus is 25-30% whereas less than 2% of normal individuals are colonized with MRSA. 4 In Pakistan, the prevalence of MRSA has increased tremendously over the years. It was reported as 5% in 1989 and since then has increased up to 51%. It is reported to range from 42 to 51%, increasing from the 1990 to 2000 5,6 and from 19.5% in 2001 to 40% in 2008. 7 S. aureus produces many virulence factors comprising of toxins and enzymes, regulated by agr and sar systems. 8 The accessory gene regulatory (agr) system down regulates the expression of surface proteins while up regulates the expression of expoproteins. It encodes two transcripts, RNAII and RNAIII where RNAII encodes for agrA, agrB, agrC and agrD. S. aureus isolates can be divided into four agr groups on the basis of the specificity of the auto-inducing peptide (AgrC). 9 Further, S. aureus strains belong to specific agr groups 10 implicating the importance of the knowledge of agr gene groups.
The current study was designed to analyze the genetic polymorphism of agr locus among S. aureus isolates and to assess its relationship with antibiotic resistance profile.

Bacterial Isolates:
The study includes a total of 90 S. aureus clinical isolates, out of which 35 were collected from Holy Family Hospital, Rawalpindi whereas, 55 isolates were collected from Microbiology Laboratory, Pakistan Institute of Medical Sciences, Islamabad during the months of April to Oct 2011. These isolates were taken from different sources where 18 were from nasal swab, 50 from pus, 3 from peri rectal swab, 10 from blood, 4 from tracheal secretion, and one each from the following sources: tissue, prostatic secretion, throat swab, semen and CVP tip.The culture media for isolation of S. aureus were blood agar, mannitol salt agar and brain heart infusion (BHI) broth/agar. Identification of S. aureus Isolates: Identification of isolates was performed by Gram staining and routine biochemical tests including catalase, coagulase, mannitol salt fermentation, and DNase tests. Antibiotic Susceptibility Testing: Susceptibility testing was conducted by disk diffusion method according to the guidelines of Clinical and Laboratory Standards Institute (CLSI). 11 Fusidic acid (susceptibility and resistance were ≥22 mm and <22 mm) and tigecycline susceptible breakpoints≥ 19 mm zone size were interpreted according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) and US FDA clinical breakpoints respectively. S. aureus ATCC 25923 was used as quality control strain. DNA Extraction: Extraction procedure was followed according to manufacturer's instructions using Wizard Genomic DNA Extraction Kit (Promega Inc., Madison, USA).

PCR Amplification and Detection of agr Groups:
Primers provided by Integrated DNA Technologies (California, USA) were chosen from published sequences. 10 The PCR assay was performed using green master mix (Promega Inc., Madison, USA). Amplified samples were analyzed by electrophoresis on a 1% agarose gel and stained with ethidium bromide. Statistical Analysis: Statistical analysis was performed using the software SPSS 17.0 (SPSS Inc, Chicago, USA). Differences among different groups were analyzed using χ 2 test. p value less than 0.05 was considered as significant.

RESULTS
Ninety clinical isolates were confirmed as S. aureus by Gram staining and standard biochemical tests. All isolates were mannitol fermenters and positive for catalase, DNase and coagulase. Resistance to oxacillin and cefoxitin or both according to CLSI presented the prevalence of MRSA to be 53.3%.   Table-I shows the antibiotic resistance of MRSA and MSSA isolates. Majority of MRSA isolates exhibited high level of resistance to penicillin, cefoxitin and fusidic acid and comparatively low resistance to vancomycin. MSSA isolates were also highly resistant to penicillin. Tigecycline against MRSA and vancomycin and cefoxitin against MSSA clinical isolates were found to be the most effective antibiotics. Prevalence of agr Specific Groups in MRSA and MSSA: Using multiplex PCR, MRSA and MSSA clinical isolates were grouped in four agr specific groups (Fig.1). Among all groups, agrI was the most prevalent followed by agrIII and agrII. agrIV was absent in MRSA while 4.7% MSSA isolates were positive for it (Table-II). Non-type able agr group among MRSA and MSSA were 20.8% and 16.6%, respectively. Association between Antibiotic Resistance and agr Specific Groups: MRSA and MSSA isolates showed high resistance against penicillin with statistically significant difference (p = 0.002) in all agr groups as shown in Table-III. However, all agr containing MRSA and MSSA isolates were sensitive to tigecycline.

DISCUSSION
In this study, prevalence of MRSA was found to be 53.3% which is consistent with the results reported by other researchers. 5,12 Although higher rate of MRSA was observed in males with nonsignificant difference that is similar to the earlier reports. 2,3 Higher prevalence of MRSA was observed in case of patients aged 44+ years (68.4%) and 1-18 years (60.9%) which is close to the data, 3 where it was 61.4% in 41-80 years age group. Similar results were reported in studies conducted in Malaysia and India. 13,14 Previously, it has been shown that more cases of MRSA are reported from patients staying in intensive care units 2,3 and the same was observed in Sadia Khan et al.    this study. Since the patients in ICUs are acutely ill and immune-compromised, it generates more risk for the infections. A higher percentage (94%) of MRSA was found in nasal swabs than other specimens in contrast to the pus and sputum samples reported by others. 3,15 This is due to the reason that nasal swabs constitute the major portion of samples coming from ICU.

Antibiotic agrI n (%) agr II n (%) agrIII n (%) agrIV n (%) Non-typeable n (%) Total n (%) p value
For all the antibiotics tested, MRSA isolates showed great resistance than MSSA. Most of MRSA isolates presented multiple drug resistance. All isolates indicated 21.1% resistance to linezolid while MRSA presented 20.8% resistance. These results are in contradiction with other reports 3,16 and up to 10% has been reported from Iran. 17 This high level of resistance observed in isolates could be due to mutations of multiple gene copies, chloramphenicolflorfenicol resistance (cfr) gene carriage or misuse of this effective drug. Levofloxacin and tetracycline presented the resistance in the range of 16%-19% in MRSA isolates while 14.6% isolates were resistant to chloramphenicol which is similar to other studies. 17,18 This study also identified 3% MRSA isolates which were resistant to vancomycin. This finding is in contrast with other studies from the region. 3,14,19 Currently the drug of choice for treating life threatening infection caused by multidrug resistant MRSA is vancomycin so the emerging resistance against it would be a serious concern for the clinicians in Pakistan.
In this study, tigecycline was the only drug with 100% sensitivity showing similarity with the earlier findings. 3 Reports from other studies suggest that tigecycline is a good choice and has not yet been influenced by any of the resistance mechanisms which are involved in other antimicrobials. 19,20 By amplification of the hyper-variable domain of the agr locus, we assigned agr groups to our clinical isolates. Agr group I was most prevalent group in both MRSA and MSSA clinical isolates followed by agr III which is similar with other studies. 10,21,22 agr group I and III are closely related having 80% sequence homology that would propose an exclusive genetic characteristics of our isolates and selection for the coexistence of S. aureus strains in the population.
No agr group was identified for 17 isolates which is in accordance with a previous report. 21 Only 6 isolates were typed as agr II and 2 isolates as agr IV which represents reduced prevalence of these groups in our locality that is opposite to the earlier findings. 22 agr IV was only detected in case of MSSA while it was absent for MRSA isolates which is similar to results of few other studies. 10,21 Their absence shows that competition does not favor these strains.
Although agr I was dominant in all sources, hospital wards and age groups, it was higher in nasal swabs (50%), OPD (50%) and 44+ years age group (57.8%) respectively. agrIII was more prevalent in sources other than pus or nasal swabs (31.8%), in 44+ years age group (10.4%) as compared to other age groups and in outpatients (28.2%) as compared to hospitalized patients. Resistance profile suggests that agr III isolates are more resistant than agr I. Resistance to oxacillin is almost similar in all agr groups except agr IV and all agr IV isolates were resistant to penicillin in this study similar to the report by other study. 22 In conclusion, agrI was the most prevalent group in all the hospital departments, all type of sources and age groups followed by agrIII. The uniform fitness of S. aureusagr groups in some cases suggests that they also have comparable competitive ability within the host. The allocation of agr groups in this study perhaps reflects ecological and geographical structuring or sampling bias.