- Open Access
Molecular epidemiology of clinical and carrier strains of methicillin resistant Staphylococcus aureus (MRSA) in the hospital settings of north India
© Dar et al; licensee BioMed Central Ltd. 2006
- Received: 12 July 2006
- Accepted: 14 September 2006
- Published: 14 September 2006
The study was conducted between 2000 and 2003 on 750 human subjects, yielding 850 strains of staphylococci from clinical specimens (575), nasal cultures of hospitalized patients (100) and eye & nasal sources of hospital workers (50 & 125 respectively) in order to determine their epidemiology, acquisition and dissemination of resistance genes.
Organisms from clinical samples were isolated, cultured and identified as per the standard routine procedures. Susceptibility was measured by the agar diffusion method, as recommended by the Nat ional Committee for Clinical Laboratory Standards (NCCLS). The modified method of Birnboin and Takahashi was used for isolation of plasmids from staphylococci. Pulsed-field gel electrophoresis (PFGE) typing of clinical and carrier Methicillin resistant Staphylococcus aureus (MRSA) strains isolated during our study was performed as described previously.
It was shown that 35.1% of Staphylococcus aureus and 22.5% of coagulase-negative staphylococcal isolates were resistant to methicillin. Highest percentage of MRSA (35.5%) was found in pus specimens (n = 151). The multiple drug resistance of all MRSA (n = 180) and Methicillin resistant Coagulase-negative Staphylococcus aureus (MRCNS) (n = 76) isolates was detected. In case of both methicillin-resistant as well as methicillin-sensitive Saphylococcal isolates zero resistance was found to vancomycin where as highest resistance was found to penicillin G followed by ampicillin. It was shown that the major reservoir of methicillin resistant staphylococci in hospitals are colonized/infected inpatients and colonized hospital workers, with carriers at risk for developing endogenous infection or transmitting infection to health care workers and patients. The results were confirmed by molecular typing using PFGE by Sma I-digestion.
It was shown that the resistant markers G and T got transferred from clinical S. aureus (JS-105) to carrier S. aureus (JN-49) and the ciprofloxacin (Cf) and erythromycin (E) resistance seemed to be chromosomal mediated. In one of the experiments, plasmid pJMR1O from Staphylococcus aureus coding for ampicillin (A), gentamicin (G) and amikacin (Ak) resistance was transformed into Escherichia coli. The minimal inhibitory concentrations (MICs) for A and G were lower in E. coli than in S. aureus. However, the MIC for Ak was higher in E. coli transformants than in S. aureus.
There is a progressive increase in MRSA prevalence and multi-drug resistance in staphylococci. Vancomycin is still the drug of choice for MRSA infections. The major reservoir of methicillin resistant staphylococci in hospitals is colonized/infected inpatients and colonized hospital workers. Resistance transfer from staphylococci to E. coli as well as from clinical to carrier staphylococci due to antibiotic stress seemed to be an alarming threat to antimicrobial chemotherapy.
- Nasal Swab
- Hospital Worker
- PFGE Pattern
- Staphylococcal Strain
- Colonization Pressure
Staphylococci are one of the important causes of human infections but are also found as non-pathogenic microorganisms in human samples [1–5]. The spectrum of S. aureus infections includes toxic shock syndrome , food poisoning, meningitis [6, 7] as well as dermatological disorders ranging from minor infections and eczema to blisters and scalded skin syndrome . Recent reports have begun to document infections caused by Staphylococcus epidermidis, such as bacterial endocarditis  prosthetic heart valve endocarditis , bacteraemia, surgical wound infections , intravascular catheters , post-operative endophathalmitis , conjunctivitis and keratitis . Several other coagulase negative staphylococci (CNS) species have been implicated at low incidence in a variety of infections. The CNS species Staphylococcus saprophyticus was often regarded as a more important opportunistic pathogen than S. epidermidis in human urinary tract infections (UTIs), especially in young sexually active females. It was considered to be the second most common cause of acute cystatitis or pyelonephritis in these patients [15, 16]. The major reservoir of Staphylococci in hospitals are colonized/infected in-patients and colonized hospital workers, with carriers at risk for developing endogenous infection or transmitting infection to health care workers and patients [3, 17–23], while transient hand carriage of the organism on the hands of health care workers account for the major mechanism for patient to patient transmission .
Methicillin-resistant strains of staphylococci were identified immediately upon the introduction of methicillin into clinical practice. Methicillin-resistant S. aureus (MRSA) was initially identified for the first time in 1961 by Jevons [25, 26]. Since then strains of methicillin-resistant Staphylococcus aureus and methicillin-resistant coagulase-negative staphylococci have spread worldwide [27, 28] and have become established inside and outside of the hospital environment . Already multiresistant to different classes of antibiotics, MRSA had been reported to acquire resistance to gentamicin and related aminoglycosides  therefore the treatment of infections due to these organisms and their eradication is very difficult. Constant monitoring of these strains is essential in order to control their spread in the hospital environment and transmission to the community.
The present study was undertaken with the aim of determining epidemiology of clinical and carrier staphylococci and molecular studies of their acquisition and dissemination of resistance in a hospital setting in northern India.
The study population (n = 750) was divided into healthy personnel (n = 175) and patients (n = 575) (including 50 medical personnel attending wound infections). For the healthy personnel, 125 hospital workers contributed nasal swabs and 50 hospital works contributed ocular swabs. None of the healthy personnel had taken any kind of antibiotics 7 days before the time of specimen collection. For the patients, 50 patients with wound infections admitted in Orthopedic Surgical Ward of J.N. Medical College, Aligarh Muslim University, Aligarh India, contributing nasal swabs besides pus culture and 525 subjects contributing different clinical sources were included. Informed consent was obtained from all the subjects before sample collection. The Ethical committees of Sher-e-Kashmir Institute of Medical Sciences, Srinagar and J.N. Medical College, Aligarh Muslim University, Aligarh India approved the study.
Organisms from clinical samples were cultured as per the routine procedures. The anterior nares were sampled as follows. A sterile cotton-tipped swab was moistened in a culture tube containing 2 ml of 0.1% buffered Tween 80. The swab was wrung out within the tube, swirled inside the anterior nares for five clockwise, and five counter clockwise rotations, reintroduced into the culture tube and wrung out. The ocular swabs were obtained as described previously . Swabs were deposited in tubes containing 2 ml of detergent fluid (0.1% buffered Tween 80) serially diluted in 10 fold steps. 40 μl per dilution was then drop plated onto the 5% sheep blood agar plates and mannitol salt agar plates (Hi-Media Mumbai, India), and the plates were incubated for 24 h at 35°C and observed or the growth of suspected Staphylococci colonies. After the identity of the cultures was confirmed according to Bergey's manual including Gram's staining, catalase and coagulase test (slide and tube methods) [32, 33], they were stored at -70°C in freezer vials containing 15% glycerol for further analysis.
Antimicrobial susceptibility test
Susceptibility was measured by the agar diffusion method, as recommended by the National Committee for Clinical Laboratory Standards (NCCLS) , with the following discs; amikacin (Ak), 30 μg; ampicillin (A), 10 μg; cefazolin (Cz), 30 μg; cephalexin (Ce), 30 μg; cephotaxime (Cp), 30 μg; chloramphenicol (C), 30 μg; ciprofloxacin (Cf), 5 μg; clinadamycin (Cd), 2 μg; Co-trimoxazole (Co), 25 μg; fusidic acid (Fc), 10 μg; gentamicin (G), 10 μg; Imipenem (I), 10 μg; methicillin (M), 5 μg; penicillin G (P), 10 U; rifampin (R), 5 μg; roxithromycin (Ro), 30 μg; Tetracycline (T), 30 μg; and vancomycin, 30 μg; (Hi Media Mumbai, India). Methicillin-resistant Staphylococci were also tested for oxacillin resistance using the oxacillin-salt screening test performed according to NCCLS guidelines . S. aureus ATCC® 29213, S. aureus ATCC® 43300; (American Type Culture Collection Manassas, VA USA) were used as the positive and negative controls respectively.
Plasmid isolation, transformation and conjugation
Antibiotic resistance profiles of 61 strains of MRSA isolated in an orthopaedic surgical ward
Antibiotic resistance profiles of MRSA
No. of resistance markers
JMR-3, 55, 33, 59
JMR-6, 38, 51
JMR-24, 46, 48, 39
JMR-37, 45, 50, 54
For resistance transfer to be studied through cell – to – cell contacts between clinical and carrier S. aureus strains, the conjugation was performed through mixed culture tests as studied previously [33, 39]. Here S. aureus strains were isolated from a patient having postoperative ocular infection admitted in the postoperative ward of the Institute of Ophthalmology. JN-49 strain (AR, PR, CfR, ER, GS, TS) was isolated from the nose of the same patient having postoperative ocular infection and JS-105 (TR, ER, GR, AS, PS, CfS) was isolated from ocular swab.
Chromosomal DNA from MRSA isolates was prepared in agarose blocks and was cleaved with Sma I (Bangalore Genei Pvt. Ltd. India) as described by Bannerman et al . PFGE typing of clinical and carrier MRSA strains isolated during our study was performed as described previously [39, 41]. For restriction endonuclease digestion, approximately 1 to 1.5 mm of a plug was cut and incubated with 250 μl of restriction buffer containing 20 U of Sma I at 25°C for 4 h. After DNA digestion, the agarose plugs were incubated with 1 ml of TE buffer at 37°C for 1 h. The plugs were then inserted into 1% agorose gel in 0.5× TBE buffer, and restriction fragments were separated using a contour-clamped homogeneous electric field system (CHEF-DRII; Bio-Rad, Laboratories). Electrophoresis was performed using the following conditions: Block 1: initial switch time 5 sec; final switch time 15 sec; run time 10 h; voltage 200 V or 6 V/cm. Block 2: initial switch time 15 sec, final switch time 60 sec. run time 13 h, voltage 20 V or 6 V/cm.
Age and gender distribution
Age and gender distribution of staphylococcal infected/colonized subjects (n = 750) according to clinical diagnosis
Clinical Diagnosis n = 750 (100%)
5–14 n = 63 (%)
15–24 n = 133 (%)
25–44 n = 224 (%)
45–64 n = 182 (%)
>65 n = 96 (%)
Unknowna n = 52 (%)
Male n = 31(49.2)
Female n = 32 (50.8)
Male n = 67(50.4)
Female n = 66(49.6)
Male n = 116(51.8)
Female n = 108(48.2)
Male n = 101(55.5)
Female n = 81(44.5)
Male n = 56(58.3)
Female n = 40(41.7)
Male n = 23(44.2)
Female n = 29(55.8)
Wound infection n = 122 (16.2)
Postoperative infection n = 35 (4.7)
Bacteraemia n = 25 (3.3)
Pneumonia n = 21 (2.8)
Septicaemia n = 15 (2.0)
Urinary tract infections n = 89 (11.9)
Mastatitis n = 12 (1.6)
Prostatitis n = 18 (2.4)
Conjunctivitis n = 52 (6.9)
Corneal ulcer n = 20 (2.7)
Endophthalmitis n = 11 (1.5)
Otherb n = 110 (14.7)
Normalc n = 175 (23.3)
Unknownd n = 45 (6.0)
Frequency of staphylococcal isolates among clinical sources
Resistance profilesa of staphylococcal isolates (n = 850) from eyes and other clinical sources in health and disease
Antimicrobial Agent (n = 18)b
Percentage of Resistancec Displayed by
S. aureus n = 513 (%)
Clinical S. arueus n = 338(%)
Carrier S. arueus n = 175 (%)
Coagulase-negative Staphylococci (CNS) n = 337 (%)
Clinical CNS n = 237 (%)
Carrier CNS n = 100 (%)
Resistance Profiles of Staphylococcal Isolates
The resistance patterns of staphylococcal isolates (n = 850) to 18 antimicrobial agents are shown in Table 1. Vancomycin appeared to be the most effective drug. In case of Staphylococcus aureus (n = 513), the highest resistance was observed to penicillin G (92.0%) followed by ampicillin (80.5%) and co-trimoxazole (50.0%). Though the highest resistance in coagulase-negative staphylococci (n = 337) was shown to penicillin G (89.9%) but it was lesser by (2.1%) as compared to its resistance in Staphylococcus aureus, followed by the same pattern as to ampicillin (71.5%) and co-trimoxazole (40.3%).
As far as the clinical versus carrier staphylococcal islates are concerned the general observation was that the resistance in carrier isolates was lesser than clinical strains in case of all antibiotics. In clinical (n = 338) and carrier (n = 175) isolates of S. aureus the highest resistance was found to penicillin G (92.6% versus 90.60%) followed by ampicillin (81.4% versus 78.8%) and co-trimoxazole (51.8% versus 46.3%) respectively. The least resistance was shown to fusidic acid (4.1% versus 2.3) followed by chloramphenicol (4.7% versus 3.4%), tetracycline (12.7% versus 7.1%) and amikacin (13.9% versus 10.3%) respectively in clinical and carrier isolates of S. aureus. Almost similar trend was observed in clinical (n = 237) and carrier (n = 100) isolates of coagulase-negative staphylococci, with the exception that in carrier isolates zero resistance to amikacin was seen.
Comparative multi-drug resistance patterns of methicillin-resistant and methicillin-sensitive clinical and carrier isolates
Antibiotic resistance profiles of MRSA isolates in an orthopedic surgical ward
PFGE patterns of 61 MRSA isolates
Number of isolates
JMR-1, 2, 3, 4, 6, 38, 55
JMR-5, 14, 18
JMR-9, 13, 19, 26, 52
JMR-16, 25, 29
JMR-24, 46, 48, 39
JMR-37, 50, 54, 15, 58
JMR-21, 40, 42, 44, 59, 61
JMR-17, 20, 23, 27, 30, 32,
JMR-11, 12, 28, 41, 53 60,
JMR-22, 33, 34, 35, 36
JMR-43, 45, 51
JMR-7, 10, 47, 49, 57
PFGE profiles of MRSA
Minimal inhibitory concentrations of A, G, Ak for S. aureus strain JMR10 and one E. coli transformant
S. aureus strain JMR10
E. coli transformants
Furthermore the bacteria that normally colonize the human body (the resident microflora) could act as reservoirs for resistance genes, which could then be transferred to pathogens during their temporary colonization of the same site, and need to be focused while treating infections.
Plasmid-determined resistance transfer
There are reports of emergence and high occurrence of strains resistant to methicillin from various parts of the world . Recent studies have documented the increased costs associated with MRSA infection, as well as the importance of colonization pressure [42, 43]. Already multiresistant to different classes of antibiotics, MRSA had been reported to acquire resistance to gentamicin and related aminoglycosides , therefore the treatment of infections due to these organisms and their eradication is very difficult. Constant monitoring of these strains is essential in order to control their spread in the hospital environment and transmission to the community. Of the 513 clinical strains of S. aureus, 180 (65.1%) were methicillin-resistant S. aureus (MRSA) and out of 337 coagulase-negative staphylococcal isolates 76 (22.5%) were methicillin-resistant (MRCNS). Of the 180 MRSA strains the highest number was found in pus followed by urine and blood. Of the 76 MRCNS studied the highest number was obtained from urine followed by conjunctiva and nasal swabs of patients having ocular infections. The number of MRSA isolates being drastically high in wound infections, this might be due to the fact that orthopedic unit is a fertile environment for MRSA. The open wounds and the frequent dressing changes often necessitate a dressing team or multiple persons plus the inherent immunosupression of the wound patients might lead to MRSA colonization.
The present study suggests that MRSA most likely remains a hospital-acquired infection, but a significant proportion may be acquired in community facilities like nursing and residential homes . The major reservoir of staphylococci in hospitals are colonized/infected in-patients and colonized hospital workers, with carriers at risk for developing endogenous infection or transmitting infection to health care workers and patients [2, 3, 17–19, 44], while transient hand carriage of the organism on the hands of health care workers account for the major mechanism for patient to patient transmission . Low prevalence of MRSA colonization in an adult outpatient population indicated that MRSA carriers most likely acquired the organism through contact with healthcare facilities rather than in the community . These data show that care must be taken when attributing MRSA colonization to the community if detected in outpatients or during the first 24 to 48 hours of hospitalization. The risk to patients in terms of transmission of MRSA seems to be influenced strongly by the proportion of patients with colonization at intensive care unit admission and is associated with severity of illness, length of stay, and exposures to antibiotics and medical devices .
Colonization with S. aureus can occur soon after birth and at any given time, the nasal carriage rate in adults is estimated at between 20% and 40% . Healthcare workers have a higher incidence of colonization. The carrier state is clinically important because carriers undergoing surgery will experience more infections than non-carriers. Fierobe et al  has shown a relationship between MRSA postoperative intra-abdominal sepsis and nasal colonization of MRSA. MRSA strains are usually introduced into an institution by an infected or colonized patient or by a colonized healthcare worker, and transfer from one patient to another has led to major epidemics in tertiary care hospitals as well as chronic care facilities . Colonization of anterior nares with MRSA carries a significantly higher risk for infection than does colonization by sensitive strains [18, 47].
It is imperative we continue with basic infection control principles like hand washing and contact isolation and barrier nursing. Intranasal 2% mupirocin applied twice daily for five days appears to be the most effective topical agent against MRSA [48–50] and eliminates 91% of stable carrier states. By confining the investigation to nasal carriage in healthy state, colonization at other body sites may have remained undetected and the 'true' prevalence in this study may have been underestimated. There is, however, overall agreement that sensitivity of nose swabs in detecting MRSA carriage is reasonably high (>70%) [51–53], and it was decided to confine the investigation to nasal specimens for reasons of accessibility, compliance and consistency with other investigations. The design of the study does not allow one to distinguish between patients who acquired MRSA during their current episode of hospital stay or patients already colonized on arrival to the hospital. Despite this, most of the carriers appeared to have acquired the organisms in hospital settings, as most of the isolates seem to be of the same types of strains as isolated from nose of the hospital workers.
From this study we suggest that all patients having history of previous hospital admission and patients admitted directly from nursing homes should be screened for MRSA prior to any elective surgical or orthopedic operative procedures. We believe that patients for routine elective procedures should have a negative result before undergoing the procedure. If surgery cannot be delayed due to medical reason then prophylaxis against MRSA should be given prior to the operation, though we appreciate this may not have much benefit in case of emergency admissions. However we would suggest that in patients with any of the above risk factors admitted for emergency surgery which may involve prosthetic implant, should be screened as well as given MRSA prophylaxis prior to their operations.
Broad-spectrum insusceptibility of all 180 MRSA and 76 MRCNS isolates to common antimicrobial agents were observed. Among all 17 antimicrobial agents used only vancomycin was shown to be consistently effective against all MRSA and MRCNS. We did not find any glycopeptide resistant S. aureus in our study, and vancomycin and teicoplanin remain the drugs of choice, although decreased susceptibility as well as resistance to vancomycin has been reported recently [54, 55].
Of the 850 stephylococcal isolates, surprisingly about 90%, 80% and 50% strains were resistant to Pencillin G, ampicillin and co-trimoxazole, respectively. This might be due to the fact that at present time these agents are tremendously used in the treatment of general infections. On the contrary to the multiple resistance of MRSA, MSSA seemed to be much more susceptible to all tested antimicrobial agents except for pencillin, ampicillin and co-trimoxozable. Our observation was that resistance in carrier isolates was lesser than clinical strains in case of all antibiotics tested. In the present study it is documented that resistance to chloramphenicol and ciprofloxacin was comparatively higher in ocular isolates as compared to staphylococcal strains isolated from other clinical sources. This might be due to more pronounced use of these antibiotics for treatment of ocular infections. This study underscores the need for hospital clinicians to be aware of the common bacterial isolates in their unit and their usual antibiotic susceptibility. This is imperative in order to make rational decisions for the prudent use of antibiotics, particularly for empirical therapy. Another important cause of resistance is excessive or inappropriate use of antibiotics in hospitals . The magnitude of the problem of multi-resistance is such that clinicians must be familiar with the causes of antibiotic resistance and the measures for preventing or minimizing the emergence of resistance.
PFGE, because of its great discriminatory power and high degree of specimen typeability is accepted as the gold standard for the molecular typing of S. aureus isolates. It has successfully been used to study the epidemiology of S. aureus nosocomial infection and methicillin resistance [40, 57–59]. Nevertheless, PFGE is time-consuming and labor intensive, in this study PFGE exhibited superiority as a technique for analyzing epidemiology of S. aureus. In the present study it was observed that the 61 strains of MRSA isolated in an Orthopaedic Surgical Ward displayed 42 patterns of antibiogram. These 61 MRSA strains were subjected to PFGE analysis whereby 14 PFGE patterns were observed. These results suggest that most of the MRSA appeared to have been acquired by patients during their current episode of hospital stay. Moreover similarity of PFGE patterns of MRSA isolates from pus, skin and nasal cavity suggest that most MRSA types isolated from pus were derived from the nasal cavity but some types were derived from the nearby skin and that these microorganisms occasionally cause wound infections.
MRSA acquisition depends on 2 major and independent determinants: colonization pressure and antimicrobial selective pressure. In case of colonization with distinct multiple clones of MRSA, antimicrobial pressure plays a major role; in the case of colonization with a single dominant clone of MRSA, colonization pressure plays a major role. McGown  proposed a biological model to explain the relationship between antimocrobial use and the emergence of resistance. At the level of individual patient, antimicrobial treatment leads to a large modification in the endogenous flora. The usual result is that susceptible strains are replaced by resistant ones. At the collective level, antimicrobial use in a hospital unit tends to maintain the presence of multidrug – resistant organisms in inpatients, healthcare workers, and the environment. In cases in which basic infection-control practices are inconsistently applied, these pathogens are implicated in the majority of infections. Antimicrobials such as β-lactams and fluoroquinolones, which are ineffective against MRSA and have excellent tissue diffusion, could promote the acquisition of MRSA by increasing the 'receptiveness' of the patients and thereby allowing the progression towards colonization and infection.
The spread of resistance to antimicrobial agents in S. aureus is largely due to the acquisition of plasmids and/or transposons . Although transfer of resistance between staphylococcal strains in the laboratory has been shown to occur via transformation, transduction, and conjugation  only conjugative transfer appears to be significant in vivo . In staphylococci, the conjugative transfer of resistant determinants is usually mediated by conjugative plasmids  but has also been shown to occur in the absence of detectable conjugative plasmids . Conjugative plasmids, usually 35 to 50 kb, spread resistance determinants between species and genera [66, 67]. Besides transferring the resistance determinants, they can mobilize non-conjugative plasmids  recombine with nonconjugative plasmids to form new plasmids or acquire and transfer resistance transposons [61, 63]. Studies with human staphylococcal strains indicate that Staphylococcus epidermidis is a reservoir of antibiotic resistance genes that can be transferred to S. aureus under in vitro and in vivo conditions . In vitro studies of drug resistance transfer between clinical and carrier staphylococcal strains, was done in the present study. The results indicated that tetracycline resistance was transferred from clinical to carrier isolates. This reflects that the use of antibiotics in humans to treat infections can promote resistance in normal flora.
The widespread occurrence and dissemination of resistance markers leading to multiple antibiotics ineffective, thus increasing the cost of health care, needs to be tackled logistically by wise and judicious use of existing antibiotics and by developing ideal and cost effective antibiotics having least chances of acquiring resistance. Furthermore the hospital acquired MRSA infections through colonization of patients and hospital workers demands appropriate and timely measure to counteract this health problem.
We appreciate the support from technical staff of Microbial Division, Institute of Ophthalmology, Division of Bacteriology and Department of Microbiology J.N. Medical College, Aligarh Muslim University Aligarh, India; Department of Microbiology, Sher-e-Kashmir Institute of Medical Sciences, Srinagar, India and Lab of Molecular and Cell Biology (LMCB), Center for DNA Fingerprinting and Diagnostics, Hyderabad, India.
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