Molecular characterisation of methicillin-resistant Staphylococcus aureus (MRSA) from keratitis patients: a microbiological analysis
- 1Institute of Ophthalmology, Jawaharlal Nehru Medical College, Faculty of Medicine, Aligarh Muslim University, Aligarh, India
- 2Department of Biochemistry, Faculty of Life Sciences, Aligarh Muslim University, Aligarh, India
- Correspondence to Professor Naheed Banu, Department of Biochemistry, Faculty of Life Sciences, Aligarh Muslim University, Aligarh 202001, India;
- Accepted 24 January 2010
- Published Online First 14 May 2010
Background In order to diagnose and characterise a major corneal infection, ‘keratitis,’ which can lead to blindness, molecular studies were conducted to assess the prevalence of methicillin-resistant Staphylococcus aureus (MRSA) strains in patients suffering from keratitis
Methods The selected culture positive for strains of S aureus were subjected to 18 antimicrobial drugs for their sensitivity behaviour, and the minimum inhibitory concentration (MIC) of oxacillin was determined by the microdilution method as recommended by the National Committee for Clinical Laboratory Standards. Further, S strains were characterised using molecular tools.
Results Among the 202 cases included in the present study, 64 (31.2%) cases were found to be S aureus-positive, of which only six strains were MRSA. The highest resistance was observed for penicillin (65.6%) which was followed by a significant number of bacterial isolates showing resistance to methicillin (9.3%), while all organisms were susceptible to vancomycin. The minimum inhibitory concentrations of oxacillin for MRSA strains ranged between 16 and 128 μg/ml. A multiplex PCR assay for Staphylococcal Cassette Chromosome mec complex (SCCmec) of MRSA strains (N=6) showed that five MRSA strains had a Type III cassette, and one had a Type II cassette. The pulsed field gel electrophoresis of three MRSA strains showed closely related band patterns.
Conclusion The close relatedness among bacterial strains as observed by employing different typing techniques suggests that the clonal characteristics of multiresistant MRSA isolates could be deciphered. The presence of Type II SCCmec in keratitis subjects is probably the first report from India, as it has not been reported earlier.
- SCCmec type II cassette
- keratitis PFGE
Infectious keratitis is a sight-threatening clinical problem that develops as a result of complication of ocular surgery or penetrating ocular injury. Of the various pathogenic organisms, Staphylococcus aureus, a versatile and dangerous bacterial pathogen, has been reported as the second most common pathogen of bacterial keratitis across the world.1 2
This organism infects compromised corneas—for example, bulbous keratopathy, chronic herpetic keratits, keratoconjuctivitis sicca, ocular rosacea or atopic kerato-conjunctivitis. The frequent and sometimes indiscriminate use of antibiotics has led to the development of resistance in many bacterial pathogens including S aureus. Therefore, the occurrence of MRSA in infectious keratitis has previously been reported by numerous workers across the world.3 4
Of special concern to ophthalmic surgeons are increasing reports of postoperative MRSA infection. For example, MRSA wound infections have been reported with clear corneal phacoemulsification wounds,5 6 whereas MRSA keratitis, a serious and increasing complication following ophthalmic surgery, has been found after laser in situ keratomileusis7 8 and photorefractive keratoctomy (PRK)9 including a bilateral PRK in medical residents.10 11 Furthermore, Sotozono et al12 in a study reported three cases of MRSA keratitis following penetrating keratopathy, one case after lamellar keratopathy and four cases following epithelial transplantation for Stevens–Johnson syndrome. These and other associated data suggest that MRSA has become increasingly prevalent worldwide, as is evident from many surveillance studies.13–15 Epidemiology and the phenotypic and genotypic characterisation of MRSA strains16 have been widely discussed after its evolution from single clone and intercontinental spread.17 18 The first report on MRSA genotype in India appeared a few years ago.19 We have, however, reported earlier the prevalence of MRSA from our hospital,20 even though the geographic distribution and evolutionary pattern of a few MRSA strains of Indian origin were known.21
Still, to the best of our knowledge, there is a distinct lack of information or no information regarding molecular characteristics of MRSA causing ocular infections. Considering these, the present study was designed to characterise the ocular MRSA strains genotypically in order to identify the type of SCCmec cassette and also to establish their clonal relationship with international spread clones.
Materials and methods
Sample collection and isolation
Bacterial cultures were obtained from a total of 202 patients suffering from bacterial keratitis, referred to the culture laboratory at the Institute of Ophthalmology, Jawaharlal Nehru Medical College, AMU, Aligarh, India, between September 2004 and June 2005. The cultures were taken from infected eyes, by corneal scraping with a sterile blade and then inoculated onto a 5% sheep blood agar and mannitol salt agar (Hi Media, Mumbai, India) to obtain the growth of suspected S aureus. Plates were incubated for 24 h at 32°C. Cultures were characterised using Gram staining, catalase reaction, coagulase (tube coagulase) and DNAase test,22 23 and the identity of the cultures was confirmed by comparing the properties as observed in this study with those given in Bergey's Manual of Determinative Bacteriology. The clinical details of the patients with MRSA culture positive are presented in table 1.
Antibiotic susceptibility test
The susceptibility of 18 antimicrobial agents was measured by the agar diffusion method as recommended by the National Committee for Clinical Laboratory Standards (NCCLS)24 using the following commercially available antibiotic discs: penicllin-G (P) 10 units, ampicillin (A) 10 μg, amoxicillin (Ac) 30 μg, gentamicin (G) 10 μg, tetracycline (T) 30 μg, co-trimoxazole (Co) 25 μg, cefazoline (Cz) 30 μg, ciprofloxacin (Cf) 5 μg, chloramphenicol (C) 30 μg, ceftazidime (Ca) 30 μg, clindamycin (Cd) 2 μg, roxithromycin (Ro) 30 μg, methicillin (M) 5 μg, vancomycin (V) 30 μg, erythromycin (E) 30 μg, fusidic acid (Fc) 10 μg, imipenem (I) 10 μg and rifampicin (R) 5 μg (Hi Media, Mumbai, India). Strains of MRSA were also subjected to oxacillin to see if there were any strain resistants to oxacillin or not, using the oxacillin salt screening test as per NCCLS guidelines.25 Oxacillin was used instead of methicillin, as it is a more stable antibiotic for in vitro testing and simulates methicillin results.
The overnight grown cell cultures were centrifuged to collect pellet. Pellets were suspended in lysis buffer (phosphate-buffered saline containing 0.5% sodium dodecyl sulfate and 100μg/ml proteinase K) and incubated at 37°C for 1 h. An equal volume of phenol:chloroform (1:1) mixture was added to the cell suspension and vortexed. The suspension was centrifuged for 5 min at 10 000 rpm, and the aqueous phase was transferred to a fresh tube. The DNA was precipitated by adding 60 μl of 3 M sodium chloride and three volumes of absolute alcohol. After overnight incubation at −20°C, the DNA pellet was washed twice with 99% cold ethanol, air-dried and suspended in 500 μl of TE buffer (10 mM Tris-HCI (pH 8) and 1 mM EDTA (pH 8)).
Multiplex PCR for SCCmec cassette typing
Multiplex PCR was performed, according to the protocol and conditions described by Oliveira et al.26 For PCR amplification, the reaction was carried out in Gene Amp9700 (Applied Biosystems, Foster City, California). The four loci (A, B, D, E) along with cassette type and mecA gene were chosen to characterise the SCCmec (table 2).
PCR for typing ccrAB
PCR was performed for detecting the types of recombinase systems for type II and type III cassette in MRSA strains with primer 5′-ATTGCCTTGATAATAGCCITC-3′ (forward) and 5′-TAAAGGCATCAATGCACAAACACT-3′ (reverse for type 2ccr) and 5′-AGCTCAAAAGCAAGCAATAGAAT-3′ (reverse for type3 ccr) using the conditions as suggested by Kunyan et al.27
Pulsed field gel electrophoresis
Pulsed field gel electrophoresis (PFGE) typing of MRSA strains isolated in this study was performed as described by McDoughal et al.28 The bacterial pellets were processed for lysostaphin treatment, and the agarose plugs were prepared. The plugs were digested with EC lysis buffer for 4 h, washed with TE buffer and then digested with restriction enzyme SmaI (Promega, Madison, Wisconsin) for 3 h. The plugs were then inserted into 1.5% agarose gel in 0.5 TBE buffer, and restriction fragments were separated using CHEF-DRIll device (BioRad, Richmond, California). Electrophoresis was performed using the following conditions; initial switch time 5 s and final switch time 35 s; voltage 6 V/cm, including angle 120°, run for 21 h. The criteria to analyse the DNA fingerprints generated by PFGE was followed as proposed by Tenover et al.29
Clinical details of patients with MRSA infection
Except for the postoperative cases, the other five preoperative patients had consulted a local ophthalmologist prior to visiting us, of which four were treated with antifungal and antibacterial antibiotics (medical details shown in table 1). Based on susceptibility results, the antibiotic treatment was modified for all the patients. One patient with ciprofloxacin-resistant MRSA was treated with the combination of cefazolin and gentamicin solution, and the rest were treated with fluoroquinolones.
Resistance profiles of staphylococci isolates
The resistance pattern of S aureus obtained from a total of 202 cases to 18 antimicrobial agents is shown in figure 1. Of these, 31.2% (N=64) cases were found to be positive for S aureus. Among the antibiotics tested, vancomycin was most susceptible to all bacterial isolates included in this study. The highest resistance was observed against penicillin-G (65.5%; N=42), followed by co-trimoxazole (53%; N=34). However, a significant number (9.3%; N=6) of bacterial isolates displayed resistance to methicillin. Interestingly, a total of 17.2% (N=l1) S aureus isolates were sensitive to all antimicrobial agents. The resistance profile of MRSA isolates is presented in table 3. All the MRSA isolates were found to exhibit multiple drug-resistant properties and showed resistance to five or more antimicrobial drugs belonging to different classes at a time. These MRSA strains were found to be resistant to aminoglycoside and β-lactam antibiotics. The minimum inhibitory concentration of oxacillin for MRSA ranged between 16 (strain OC-94) and 128 μg/ml (strain OC-7, strain OC-73 and strain OC-143) (table 3).
Multiplex PCR assay
The first round of Multiplex PCR assay for determining the SCCmec types revealed that five strains had SCCmec type III, and one strain (OC-94) harboured the SCCmec type II cassette. All the MRSA strains were mecA-positive (figure 2). To further analyse the presence of any variant in type III cassette, another Multiplex PCR assay was run and showed that two strains (OC-7 and OC-73) were found to be SCCmec type III-A variant (303 bp band is absent and is a product of plasmid pT181) (figure 3). Further, to consolidate the fact that the MRSA strain indeed possessed the type III and type II cassette, a PCR was performed for the detection of ccrAB gene, as this gene is specific for each type of cassette. By amplifying different sized products, PCR generated a 1600 bp product for the type III cassette for strain OC-7 and OC-73 and a 1300 bp product for the type II cassette in the OC-94 strain (data not shown). The primer sequence and product size of the corresponding gene are given in table 2.
Pulsed field gel electrophoresis
The PFGE patterns of the SmaI digest of selected strains of MRSA are shown in figure 4. PFGE was performed using Brazilian strain HSJ216 and NCTC 8325 as a DNA marker. The interpretation was done as described by Tenover et al.29 Both staphylococcal strains, OC-7 and OC-73, differed from the Brazilian clone HSJ216 by four and three bands, respectively. Between the two strains used in this study, the degree of similarity differed by three bands only suggesting that both strains are subtypes of each other and closely related to Brazilian clone HSJ216. In contrast to type III-A isolates, the type II strain OC-94 showed a unique banding pattern.
Discussion and conclusion
The purpose of the present study was to characterise the MRSA isolates, which are still considered to be an infrequent cause of external ocular infections. The cases of keratitis observed here consisted of a minimal superficial defect with an associated subepithelial infiltrate and were non-destructive. We observed in this study that five out of six cases were treated with flouroquinolones, a most commonly used and highly effective drug in this geographical area. The discrepancy in the antibiogram of MRSA, isolated from a corneal ulcer, is very poor, since these strains are resistant to almost all antibiotics including major β-lactam antibiotics. At least five types of SCCmec elements numbered I to V30 31 have been identified, SCCmec Type I contains the mecA gene as the sole resistance determinant, whereas SCCmec types II and III also have determinants for resistance to non-β-lactam antibiotics.32
Because of their larger size, horizontal transfer of SCCmec types II and III occurs less easily than with type IV.33 34 Thus, the spread of MRSA strains with type II and III mainly occurs due to selective pressure of antibiotic exposure over time.30 SCCmec types II and III are commonly found in multidrug-resistant nosocomial MRSA isolates.32 Upon SCCmec cassette genotyping using primers mentioned in table 2 and as shown in figures 2, 3, we identified two distinct genotypes of MRSA strains, type III-A and type II. The type III-A cassette corresponds to the major clones found in Asian countries, 21 whereas the type II cassette is more often found in Korean and Japanese strains.21 Therefore, the presence of the type II cassette in keratitis probably is the first report of its kind in India, as it is not prevalent here.
PFGE, due to its great discriminatory power and high degree of specimen typeability, is accepted as the gold standard for molecular typing of S aureus isolates. It has been successfully used to study the epidemiology of S aureus nosocomial infection and methicillin resistance.35 36 Nevertheless, PFGE is time-consuming and labour-intensive. In this study, PFGE exhibited superiority as a technique for analysing the epidemiology of S aureus strains. In the present investigation, when three MRSA strains were subjected to PFGE analysis, three pulsotypes or PFGE patterns were observed. Upon interpretation of PFGE banding patterns, MRSA strains OC-7 and OC-73 were found to be closely related to each other and showed clonal similarity to Brazilian clone HSJ216. This suggests that keratitis MRSA isolates share a common lineage to international clones. However, MRSA strain OC-94 showed a distinct PFGE pattern.
We are thankful to G Arakari, D Tata Centre for Research in Tropical Diseases, Indian Institute of Sciences, Bangalore, for extending the laboratory facilities for carrying out PCR and PFGE based studies, and K Hiramatshu, Department of Microbiology and Infection Control Science, Juntendo University, Tokyo, Japan, for providing control MRSA strains. Technical assistance provided by the laboratory staff, especially by S Ahmad and K Fakhan, Microbiology Section, Institute of Ophthalmology, AMU, Aligarh, is thankfully acknowledged. We are also grateful for the cooperation and help from the many ophthalmologists including R Maheshwari (Director), RR Sukul, H Ashraf, SN Askari and M Ashraf of the Institute of Ophthalmology, AMU, Aligarh. Thanks are due to M Zaidi, Institute of Ophthalmology, for editing the manuscript.
Competing interests None.
Provenance and peer review Not commissioned; externally peer reviewed.