|Year : 2017 | Volume
| Issue : 1 | Page : 53-60
Prevalence and genetic mechanisms of antimicrobial resistance in Staphylococcus species: A multicentre report of the indian council of medical research antimicrobial resistance surveillance network
Sunanda Rajkumar1, Sujatha Sistla1, Meerabai Manoharan1, Madhan Sugumar1, Niveditha Nagasundaram1, Subhash Chandra Parija1, Pallab Ray2, Yamuna Devi Bakthavatchalam1, Balaji Veeraraghavan3, Arti Kapil4, Kamini Walia1, VC Ohri5
1 Department of Microbiology, Jawaharlal Institute of Postgraduate Medical Education and Research, Puducherry, India
2 Department of Microbiology, Postgraduate Institute of Medical Education and Research, Chandigarh, India
3 Department of Microbiology, Christian Medical College, Vellore, Tamil Nadu, India
4 Department of Microbiology, All India Institute of Medical Sciences, New Delhi, India
5 Division of Epidemiology and Communicable Diseases, Indian Council of Medical Research, New Delhi, India
|Date of Web Publication||16-Mar-2017|
Department of Microbiology, Jawaharlal Institute of Postgraduate Medical Education and Research, Puducherry - 605 006
Source of Support: None, Conflict of Interest: None
Purpose: Routine surveillance of antimicrobial resistance (AMR) is an essential component of measures aimed to tackle the growing threat of resistant microbes in public health. This study presents a 1-year multicentre report on AMR in Staphylococcus species as part of Indian Council of Medical Research-AMR surveillance network. Materials and Methods: Staphylococcus species was routinely collected in the nodal and regional centres of the network and antimicrobial susceptibility testing was performed against a panel of antimicrobials. Minimum inhibitory concentration (MIC) values of vancomycin (VAN), daptomycin, tigecycline and linezolid (LNZ) against selected methicillin-resistant Staphylococcus aureus(MRSA) isolates were determined by E-test and MIC creep, if any, was determined. Resistant genotypes were determined by polymerase chain reaction for those isolates showing phenotypic resistance. Results: The prevalence of MRSA was found to be range from moderate (21%) to high (45%) among the centres with an overall prevalence of 37.3%. High prevalence of resistance was observed with commonly used antimicrobials such as ciprofloxacin and erythromycin in all the centres. Resistance to LNZ was not encountered except for a single case. Full-blown resistance to VAN in S. aureus was not observed; however, a few VAN-intermediate S. aureus isolates were documented. The most common species of coagulase negative staphylococci (CoNS) identified was Staphylococcus haemolyticus and Staphylococcus epidermidis. Resistance among CoNS was relatively higher than S. aureus. Most phenotypically resistant organisms possessed the corresponding resistance genes. Conclusion: There were localised differences in the prevalence of resistance between the centres. The efficacy of the anti-MRSA antimicrobials was very high; however, almost all these antimicrobials showed evidence of creeping MIC.
Keywords: Antimicrobial susceptibility testing, coagulase negative staphylococci, methicillin-resistant Staphylococcus aureus, minimum inhibitory concentration creep, Staphylococcus
|How to cite this article:|
Rajkumar S, Sistla S, Manoharan M, Sugumar M, Nagasundaram N, Parija SC, Ray P, Bakthavatchalam YD, Veeraraghavan B, Kapil A, Walia K, Ohri V C. Prevalence and genetic mechanisms of antimicrobial resistance in Staphylococcus species: A multicentre report of the indian council of medical research antimicrobial resistance surveillance network. Indian J Med Microbiol 2017;35:53-60
|How to cite this URL:|
Rajkumar S, Sistla S, Manoharan M, Sugumar M, Nagasundaram N, Parija SC, Ray P, Bakthavatchalam YD, Veeraraghavan B, Kapil A, Walia K, Ohri V C. Prevalence and genetic mechanisms of antimicrobial resistance in Staphylococcus species: A multicentre report of the indian council of medical research antimicrobial resistance surveillance network. Indian J Med Microbiol [serial online] 2017 [cited 2018 Jan 22];35:53-60. Available from: http://www.ijmm.org/text.asp?2017/35/1/53/202346
| ~ Introduction|| |
Antimicrobial resistance (AMR) is a global public health problem that seriously limits the prevention and treatment of infections and threatens to negate modern advances in medicine. Policies and strategies to tackle the challenges against AMR in India have been envisaged and proposed.,, To this end, the Indian Council of Medical Research (ICMR) in 2013 has initiated the 'antimicrobial resistance surveillance network' (ICMR-AMRSN) as a constituent of the program to bring about national guidelines on rational antimicrobial use for the country. The network's task is to monitor AMR in select pathogen groups of importance to public healthcare system at some of the tertiary care medical institutes and hospitals across the country.
A predominant pathogen group in nosocomial and community-acquired infections are the Gram-positive cocci which are endowed with intrinsic resistance as well as the ability to acquire resistance against antimicrobials, the latter either due to mutations or by horizontal gene transfer., While the diminishing pool of effective antimicrobials against these pathogens is worrisome as exemplified by pervasive inducible resistance against the macrolide-lincosamide-streptogramin B (MLSB), the biggest threat is the emergence and spread of multidrug-resistant staphylococci and enterococci, namely, methicillin-resistant Staphylococcus aureus (MRSA) and glycopeptide-resistant enterococci, especially vancomycin (VAN)-resistant enterococci.,
This study was conducted in Jawaharlal Institute of Postgraduate Medical Education and Research (JIPMER), Puducherry, as the nodal centre for Staphylococcus and Enterococcus species, under the auspices of ICMR-AMRSN, to determine the prevalence and pattern of AMR against a panel of approved antimicrobials in human clinical isolates of Staphylococcus and Enterococcus species and to study the genetic basis of resistance to various antimicrobials by molecular techniques. This report presents a 1-year data of AMR in staphylococci (S. aureus and coagulase negative staphylococci [CoNS]) obtained and analysed in our centre from January to December, 2015 as well as the antimicrobial susceptibility testing (AST) data shared from the regional centres of this pathogen group under the ICMR-AMRSN, namely, All India Institute of Medical Sciences (AIIMS), New Delhi, Postgraduate Institute of Medical Education and Research (PGIMER), Chandigarh and Christian Medical College (CMC), Vellore. The AST data, along with the patient information, that is, sampling data, demographic data, clinical diagnosis and specimen type, were deposited online at ICMR-AMRSN portal (http://bic.icmr.org.in/amr/amrportal/) which serves as the national database for the surveillance network.
| ~ Materials and Methods|| |
Bacterial isolates and antimicrobial susceptibility testing
Non-duplicate isolates of staphylococci were collected on a daily basis from the Department of Microbiology, JIPMER from January to December 2015. All isolates of S. aureus and clinically significant isolates of CoNS were included for analysis. CoNS were distinguished from S. aureus using tube and slide coagulase tests as well as 'S. aureus Test Kit' from Plasmatec, UK. The CoNS were further identified by the automated system, 'Phoenix PID' from BD, USA. Matrix-assisted laser desorption ionisation-time of flight (MALDI TOF) was also used at one of the regional centres.
AST of these isolates was performed against a panel of antimicrobials on Mueller-Hinton agar plates by the standard Kirby-Bauer disk-diffusion method. The antimicrobials tested were penicillin (10 units), erythromycin (ERY) (15 µg), clindamycin (CLI) (2 µg), ciprofloxacin (5 µg), gentamicin (30 µg), cefoxitin (30 µg), tetracycline (30 µg), linezolid (LNZ) (30 µg), teicoplanin (TEC) (30 µg), mupirocin (MUP) (200 µg) and trimethoprim-sulfamethoxazole (SXT) (1.25/23.75 µg). Zone diameters of all antimicrobials were recorded after overnight (16–18 h) incubation at 35°C ± 2°C for S. aureus and 24 h for CoNS and interpreted as per the Clinical and Laboratory Standards Institute (CLSI) guidelines 2014. Quality control for the antimicrobial disks, media and culture conditions was performed once a week with S. aureus (ATCC 25923). Cefoxitin disks were procured from Oxoid Ltd., UK, while all the other antimicrobials and media components were from HiMedia Laboratories, India.
Minimum inhibitory concentration (MIC) values were determined for VAN, LNZ, TEC, daptomycin (DAP) and tigecycline (TIG) against 141 (60 randomly selected MRSA isolates from JIPMER and the rest from the regional centres as part of External Quality Assurance Scheme [EQAS]) by E-test (Biomerieux India Pvt. Ltd., India). The results were compared with data from the previous years to identify any MIC creep which may have occurred.
Detection of resistant phenotypes
MRSA/methicillin-resistant CoNS (MRCoNS) isolates were identified using cefoxitin disk diffusion. A phenotypic 'latex agglutination test (LAT)' (Denka Seiken Co., Ltd., Japan), based on monoclonal antibody to penicillin-binding protein 2a of S. aureus, was carried out to supplement the identification of the phenotype., Isolates with inducible CLI resistance were detected by double disk-diffusion method (D-zone test) with ERY and CLI disks. VAN-intermediate isolates of S. aureus and VAN-intermediate isolates of CoNS (VICoNS) and VAN-resistant isolates of S. aureus (VRSA) and VAN-resistant isolates of CoNS (VRCoNS) were identified using VAN screen agar.
Antimicrobial susceptibility testing data and isolates from regional centres
The AST data of S. aureus and CoNS isolates of the regional centres were provided to JIPMER for this pathogen group. JIPMER also received some isolates of S. aureus and CoNS from the regional centres in two batches during this period under the EQAS program. AST as well as the molecular tests for the resistance phenotypes was performed for these isolates following the same procedures as detailed above.
Approval for this study was obtained from the Human Ethics Committees of the individual institutions.
Chi-square test was employed for the assessment of significant change, if any, among the MIC values of isolates in 3 years in comparison with the median MIC value of the index year (2012). P < 0.05 was considered statistically significant.
Polymerase chain reaction
The resistance phenotypes, namely, MRSA, inducible CLI resistance, high level MUP resistance and LNZ resistance were genotyped and confirmed by polymerase chain reaction (PCR) amplification of a region of the known corresponding resistance gene [Table 1] from the genomic DNA prepared using DNA extraction kit (HiMedia Laboratories, India) according to the manufacturer's recommendations. The PCR amplifications were done with slight modification of the conditions from the original reports ,,,, as follows: For mecA, 94°C for 3 min for the initial denaturation; 94°C for 15 s, 58°C for 15 s and 72°C for 30 s for the next 30 cycles; for the ermA/B/C genes, 94°C for 3 min for the initial denaturation; 94°C for 15 s, 54°C for 15 s and 72°C for 30 s for the next 30 cycles; for mupA, 94°C for 3 min for the initial denaturation; 94°C for 15 s, 58°C for 15 s and 72°C for 30 s for the next 30 cycles; for the cfr gene, 94°C for 3 min for the initial denaturation; 94°C for 15 s, 55°C for 15 s and 72°C for 1 min for the next 30 cycles. All PCR were accompanied with a final extension at 72°C for 5 min. 16S ribosomal RNA PCR was used as an internal control.
|Table 1: Details of the resistance genes, primers and the expected amplicon sizes|
Click here to view
| ~ Results|| |
Isolates of staphylococci
A total of 8032 isolates of staphylococci were analysed in the year 2015, out of which 5403 were S. aureus isolates (67.3%) and 2629 (32.7%) were CoNS. Majority of the S. aureus were from skin and soft tissue infections (SSTI) (73.7%) while those from lower respiratory tract and blood represented 12.4% and 10%, respectively. 3.2% of S. aureus isolates were from sterile body fluids. On the other hand, majority of the CoNS were from blood (71.5%) and pus (21.1%) while only 5.4% were from sterile body fluids. Staphylococcus haemolyticus and Staphylococcus epidermidis were the most common species of CoNS, representing 11.6% and 6.5% of the total CoNS identified. Staphylococcus hominis isolates represented 4.1%. The most common species from sterile body fluids and SSTI was S. haemolyticus (11.9% and 14.9%) followed by S. epidermidis (7.7% and 8.1%). Other rare CoNS species identified included Staphylococcus caprae, Staphylococcus cohnii, Staphylococcus schleiferi, Staphylococcus warneri and Staphylococcus lugdunensis.
Pattern of resistance phenotypes/genotypes in Staphylococcus aureus and coagulase negative staphylococci
The consolidated graph of the resistance pattern of S. aureus isolates against various antimicrobials tested is shown in [Figure 1]. The overall prevalence of MRSA among S. aureus isolates in 2015 was 37.3%. The prevalence of ERY resistance (56.6%) was much higher than CLI resistance (26.6%). Inducible CLI resistance was observed in 18.5% of the isolates. MUP resistance was very low at only 3.4%.
|Figure 1: Overall resistance pattern of Staphylococcus aureus isolates. PEN: Penicillin, ERY: Erythromycin, CLI: Clindamycin, CIP: Ciprofloxacin, GEN: Gentamicin, FOX: Cefoxitin, TET: Tetracycline, LNZ: Linezolid, TEC: Teicoplanin, MUP: Mupirocin, SXT: Trimethoprim-sulfamethoxazole.|
Click here to view
The prevalence of MRSA distribution varied centre wise from as low as 21% at AIIMS to as high as 45% at CMC. MRSA prevalence at PGIMER and JIPMER was 43% and 35%, respectively [Figure 2]. Although resistance rates to other non-beta-lactam antibiotics were mostly similar across the centres, a significant difference was observed with ERY (JIPMER rates being much higher than other centres) and tetracycline (AIIMS rates being 4-fold higher than JIMPER rates).
|Figure 2: Centre-wise distribution of resistance pattern against Staphylococcus aureus isolates. NT: Not tested from respective centre.|
Click here to view
Overall resistance pattern among MRSA and methicillin-sensitive S. aureus (MSSA) isolates. MUP resistance was slightly higher among MRSA (6%) than MSSA (2%). Similarly, MRSA isolates showed a higher resistance to ciprofloxacin, ERY, co-trimoxazole, gentamicin and CLI compared to MSSA isolates [Figure 3]. All MRSA isolates tested were found to be susceptible to VAN, LNZ, DAP and TIG [Figure 1]. MIC50 and MIC90 of all the four antibiotics are shown in [Table 2].
|Figure 3: Overall resistance pattern among methicillin-resistant Staphylococcus aureus and methicillin-sensitive S. aureus isolates.|
Click here to view
|Table 2: MIC50 and MIC90 of the anti-methicillin-resistant Staphylococcus aureus antibiotics against 141 methicillin-resistant Staphylococcus aureus isolates|
Click here to view
The prevalence of methicillin-resistance among CoNS isolates was higher than S. aureus isolates at 66.7% and varied from 44% at AIIMS to 78% at CMC. The MRCoNS prevalence at PGIMER and JIPMER was 74% and 59%, respectively. CoNS which were completely resistant to VAN and TEC were not encountered during the period; however, ten isolates of VICoNS (MIC: >6 µg/ml) were found. Four isolates of LNZ resistant CoNS (LRCoNS) were also encountered. One isolate of TEC resistant was found in VAN sensitive isolate. In the present study, the overall prevalence of resistance encountered was higher in S. haemolyticus than S. epidermidis. In S. haemolyticus, resistance to cefoxitin was 71%, ERY 74%, ciprofloxacin 62% and CLI 54% while in S. epidermidis, cefoxitin resistance was observed in 47%, ERY in 65%, ciprofloxacin in 44% and CLI in 42% of the isolates [Figure 4].
|Figure 4: Overall resistance pattern among Staphylococcus epidermidis and Staphylococcus haemolyticus isolates.|
Click here to view
Pattern of resistance phenotypes according to location
In JIPMER, the overall MRSA prevalence was 26.4% in outpatient department (OPD), 39.9% in wards and 31.5% in the Intensive Care Unit (ICU) in 2015. This pattern of resistance percentage in different healthcare areas, in which the resistance is higher in the overall specimens obtained from wards as compared to those obtained from OPD and ICU, was observed for all the antimicrobials [Table 3]. However, such a resistance pattern in different healthcare areas was not observed with majority of antimicrobials in PGIMER, AIIMS and CMC. In these centres, an increase in the relative MRSA/MRCoNS as well as resistance percentage against most antimicrobials increased from OPD to ward and highest in ICU (data not shown). [Table 3] shows the combined resistance percentages of S. aureus isolates from all specimens for all the centres including the resistance pattern in different healthcare areas during the year 2015.
|Table 3: Combined resistance percentages of S. aureus isolates for all the centres isolated from different healthcare areas from all specimens|
Click here to view
Creeping minimum inhibitory concentration
A comparison of the MIC values of the anti-MRSA antimicrobials, that is, VAN, LNZ, DAP and TIG against sixty randomly selected isolates of MRSA from JIPMER each year showed a significant creep for almost all the antimicrobials. Since the study period between 2014 and 2015 alone was not sufficient enough to understand the trend, we compared the MIC values with the published data of these antimicrobials from our department for the previous years. We reported a creeping MIC for these antimicrobials over a 3 years period (2012–2014) and as revealed with our data, the trend has continued and has become even more marked [Figure 5]. The data were obtained by calculating the number of isolates in each year with an MIC value less than the median MIC value for the index year (2012). Only isolates from JIPMER were included for this calculation. While a steady creep was observed with DAP and TIG, the creeping MIC was much more significant for VAN and LNZ (P < 0.0001).
|Figure 5: Percentage of isolates with minimum inhibitory concentration values equal to or less than the median value from 2013 to 2015 with 2012 data taken as the index year (modified figure used with permission of Editor, Brazilian Journal of Infectious Diseases).|
Click here to view
The MRSA phenotypes were conferred by mecA gene as determined by PCR of 141 randomly selected isolates in 2015. The isolates also included those received at JIPMER from the regional centres under the EQAS program. However, in three of the isolates, which were identified phenotypically as MRSA (by both cefoxitin disc diffusion (CDD) and MRSA LAT), mecA gene was negative by PCR. On the other hand, there were 10 isolates which were identified as MSSA by CDD but were positive by both mecA gene and MRSA latex agglutination.
The inducible CLI resistance was conferred either through ermA or ermC gene only, but ermC gene was more common. None of these was found to be ermB positive [Table 4]. Resistance to the high level MUP (200 µg) conferred by mupA gene, remained very low during the study period (3.4%). A single S. aureus isolate from CMC, Vellore was reported to be resistant to LNZ. PCR analysis of one LNZ-resistant S. haemolyticus isolate from AIIMS (MIC >256) showed the presence of cfr gene. The isolate was also resistant to CLI and chloramphenicol, corroborating the cfr gene-mediated resistance [Figure 6] and [Figure 7]. The only antimicrobial without any observed resistance was VAN as we did not encounter any VRSA during the entire study period; however, one isolate with reduced susceptibility to VAN (MIC: 6 µg/ml) was encountered. Another glycopeptide, TEC, showed a resistance prevalence of 0.1% as determined by disc diffusion.
|Table 4: The resistance phenotypes/genes against non-b-lactam antimicrobials analysed in S. aureus isolates|
Click here to view
|Figure 6: Overall resistance pattern of coagulase negative staphylococci isolates.|
Click here to view
|Figure 7: Centre-wise resistance pattern among coagulase negative staphylococci isolates. NT: Not tested from respective centre.|
Click here to view
India faces unique challenges in tackling AMR due to its geography and vast population, low healthcare spending and inappropriate/overuse of antimicrobials. AMR surveillance constitutes the backbone for programs aimed at tackling AMR by providing in vitro evidence and trend of resistance for further endeavours. The ICMR-AMRSN is a uniform system of AMR surveillance with a network of quality assured laboratories (the nodal and regional centres), which not only give reliable, rapid AMR data but also monitor resistance pattern over a longer period of time.
An Europe-wide survey found the most common infections associated with S aureus to be SSTIs (71% cases), 22.5% of them being MRSA. This finding was reflected in the present study as well.
In our study, the prevalence of MRSA was 37.3%. The observed resistance was lower than what was reported in an earlier study from several centres across India, where rates in 2008 and 2009 were 42% and 40%, respectively. The proportion of MRSA varies among countries ranging from 0.4% in Sweden to 48.4% in Belgium. The differences in the prevalence of resistance phenotypes to almost all the antimicrobials tested between different centres of the surveillance network during the study period, notably, MRSA prevalence which ranged from as low as 21% to as high as 45% between the centres, may be indicative of localised differences in the antibiotic prescription practices and the infection control measures employed. The high and increasing level of resistance to ciprofloxacin, a routine antimicrobial, in all the centres was perhaps expected because of its pervasive use. It is heartening to note that resistance to MUP was very low despite this drug being extensively used both for decolonisation as well as a topical antibiotic. Our study is in agreement with that of Kaur and Narayan where they reported only 1.4% resistance  as well as a multicentre study from France, where resistance of 2.2% was reported. On the other hand, Chaturvedi et al. reported high MUP resistance of 18.8% in their isolates. Studies have reported the association of high level MUP resistance with treatment failure.
MRSA isolates showed a higher resistance to ciprofloxacin, ERY, co-trimoxazole gentamicin and CLI compared to MSSA isolates although comparable rates were observed for MUP resistance. Similar observation was reported in the previous multicentre study from India.
VAN remained very effective against MRSA and MRCoNS as we did not document any VRSA and VRCoNS isolates. VRSA has fortunately remained very rare in India as elsewhere with only few reports so far.,, The relative rarity of VRSA has been explained by the instability of the plasmid carrying vanA gene in S. aureus as well as the fitness burden it imposes on such isolates. There were a few isolates of S. aureus as well as CoNS which showed intermediate susceptibility to VAN as confirmed by broth microdilution.
CoNS are emerging as major nosocomial pathogens with S. epidermidis and S. haemolyticus being the most common species reported. The increasing number of immunocompromised hospitalised patients coupled with increased use of invasive devices and implants has led to the progressive importance of these organisms in health care settings. CoNS possess fewer virulence factors compared to S. aureus and are generally associated with different clinical presentations. However, they are more difficult to treat as they exhibit higher rates of AMR. The same was reflected in the present study with methicillin resistance rates almost double that seen with S. aureus. The identification of CoNS was performed using conventional biochemical reactions, automated methods as well as MALDI-TOF across different centres.
In the present study, the overall prevalence of resistance encountered was higher in S. haemolyticus compared to S. epidermidis. Of all the CoNS species, S. haemolyticus is described as the most resistant phenotype. A study by Cavanagh et al. (2014) noted that 75% of analysed S. haemolyticus isolates displayed multiresistance. This species also plays an important role in the dissemination of resistance genes, contributing to the emergence of epidemic clones of a more virulent nosocomial pathogens.
In any given microbial population, the central MIC tendency can increase over time, and the phenomenon is termed as MIC creep. Categorical representation of data such as percentage resistant, breakpoint MICs, values of MIC50 and MIC90 may fail to detect gradual changes in the MIC values. Conventional broth-dilution methods are incapable of detecting subtle changes while E-test can detect these changes by including intermediate MIC dilutions. Although E-test is a reliable method for MIC determination, caution should be exercised in interpreting the results as it has been observed that E-test consistently gives higher MIC values than that of broth microdilution method. In the current study, a worrisome and significant creep in the MIC values of VAN, LNZ and TIG were observed as compared to previous years. Whether this will culminate in full-blown resistance in the near future remains to be seen.
A recently published study from Brazil reported DAP and TIG resistance in 2 and 10 MRSA isolates, respectively, out of 36 isolates. A point of concern was the finding of higher MIC values for TIG in five of the MRSA isolates (>0.5 µg/ml.) despite this drug not being regularly prescribed in our hospital. Although there is no consensus, the general opinion is that higher VAN MICs would have a deleterious effect on the clinical outcome. Clinical implications of MRSA isolates with higher VAN MIC values (within susceptible range) are controversial. A meta-analysis conducted by Jacob et al. reported higher rates of treatment failure and mortality in such patients while a few others have not documented this finding.
Cefoxitin has been considered as the surrogate marker for the detection of oxacillin resistance as it is a more potent inducer of mecA than penicillins. Most studies have reported excellent results with CDD test. Fernandes et al. reported 100% sensitivity and specificity, whereas Jain et al. reported slightly lower rates of 94.4% sensitivity and 95.8%specificity., With CDD method, there is a scope of misidentification as there is no buffer zone of intermediate susceptibility (≤21 mm is resistant; ≥22 mm is susceptible) more so far isolates with borderline zone diameters. This problem was faced during the EQAS testing in our laboratory with 20 out of 200 (10%) isolates with susceptible zone diameters testing positive for mecA gene as well as in MRSA LAT. On the other hand, there were 4 out of 165 (2.4%) isolates in JIPMER which were resistant by CDD but negative for mecA gene by PCR. Although there are non-mecA gene-mediated mechanisms of MRSA, they are very rarely encountered in clinical practice. A more likely explanation is that there could be some minor variations in the mecA gene near the region of primer annealing leading to negative PCR results although functionally the gene may be unaffected. Such an occurrence can be confirmed using multiple primers targeting different regions of the mecA gene. Till this issue is resolved, it may be prudent to retest those isolates which give borderline zone diameters with another CLSI approved method like oxacillin screen agar.
Molecular analysis of resistance determinants revealed common genes encoding resistance among test MRSA isolates from our centre as well as the other regional centres. The resistance determinants of macrolides vary worldwide. Among our isolates, erm genes were found to be most common determinants than msr genes. In particular, erm C was the predominant gene observed which was contradictory to the studies conducted by Duran et al. and Lim et al., where they documented ermA gene as predominant.,ermB is more common among beta-haemolytic streptococci and enterococci than S. aureus. In inducible MLSB resistant isolates, ermC was the predominant resistance determinant followed by ermA while ermB genes were not detected. This was in agreement with the study by Juda et al. and in contrast to the study conducted by Chavez-Bueno et al., in which ermB was predominant.,
The horizontal transfer of resistance genes from CoNS to S. aureus has been clearly demonstrated for fusidic acid, gentamicin and MUP.
We used high level MUP since high-level resistance to this topical antimicrobial is clinically more significant and likely to be associated with treatment failure, especially in view of the potential spread of mupA. Some of the MUP resistant isolates from JIPMER failed to demonstrate mupA gene. It is possible that these isolates could have carried the other gene responsible for MUP resistance, the plasmid-mediated ileS2 gene. In the previous study, ileS2 was detected in 81 of 82 phenotypically highly MUP-resistant strains. Rapid increase in high-level resistance to MUP and resistance to other antibiotics in S. aureus and CoNS has been associated with an increase in MUP use.
LNZ remains the most effective antimicrobial against S. aureus and CoNS in general, since all but one isolate of S. aureus, and a very small number of cfr-mediated resistant CoNS were encountered. This observation reflects the scenario so far in India as there is still no report of LNZ-resistant S. aureus except few case reports of LRCoNS isolates. Among the older antimicrobials against MRSA in SSTI, namely, SXT, CLI and tetracycline, it is worth mentioning the high level of resistance to SXT. The high level prevalence of ERY as seen in this study as well from other reports in the country is of concern, especially in light of the fact that it can mechanistically confer cross-resistance to CLI.
Although the high in vitro efficacy of anti-MRSA antimicrobials such as the glycopeptides and LNZ among the isolates in JIPMER as well as isolates tested from other centres is continuing, the gradual creeping MIC against these last lines of antimicrobials is of concern underlying the need to continuously monitor MIC levels of these important agents.
Financial support and sponsorship
Indian Council of Medical Research.
Conflicts of interest
There are no conflicts of interest.
| ~ References|| |
Ganguly NK, Arora NK, Chandy SJ, Fairoze MN, Gill JP, Gupta U, et al.
Rationalizing antibiotic use to limit antibiotic resistance in India. Indian J Med Res 2011;134:281-94.
] [Full text]
Kumar SG, Adithan C, Harish BN, Sujatha S, Roy G, Malini A. Antimicrobial resistance in India: A review. J Nat Sci Biol Med 2013;4:286-91.
Chennai Declaration Team. “Chennai Declaration”: 5-year plan to tackle the challenge of anti-microbial resistance. Indian J Med Microbiol 2014;32:221-8.
Giedraitiene A, Vitkauskiene A, Naginiene R, Pavilonis A. Antibiotic resistance mechanisms of clinically important bacteria. Medicina (Kaunas) 2011;47:137-46.
Chancey ST, Zähner D, Stephens DS. Acquired inducible antimicrobial resistance in Gram-positive bacteria. Future Microbiol. 2012;7:959-78.
Indian Network for Surveillance of Antimicrobial Resistance (INSAR) group, India. Methicillin resistant Staphylococcus aureus
(MRSA) in India: Prevalence & susceptibility pattern. Indian J Med Res 2013;137:363-9.
Tarai B, Das P, Kumar D. Recurrent challenges for clinicians: Emergence of methicillin-resistant Staphylococcus aureus
, vancomycin resistance, and current treatment options. J Lab Physicians 2013;5:71-8.
] [Full text]
Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing; Twenty-fourth Informational Supplement. CLSI Document M100-S24. Vol. 33. Wayne, PA, USA: Clinical and Laboratory Standards Institute; 2014.
Cavassini M, Wenger A, Jaton K, Blanc DS, Bille J. Evaluation of MRSA-Screen, a simple anti-PBP 2a slide latex agglutination kit, for rapid detection of methicillin resistance in Staphylococcus aureus
. J Clin Microbiol 1999;37:1591-4.
Hussain Z, Stoakes L, Garrow S, Longo S, Fitzgerald V, Lannigan R. Rapid detection of mecA-positive and mecA-negative coagulase-negative staphylococci by an anti-penicillin binding protein 2a slide latex agglutination test. J Clin Microbiol 2000;38:2051-4.
Fiebelkorn KR, Crawford SA, McElmeel ML, Jorgensen JH. Practical disk diffusion method for detection of inducible clindamycin resistance in Staphylococcus aureus
and coagulase-negative staphylococci. J Clin Microbiol 2003;41:4740-4.
Vannuffel P, Laterre PF, Bouyer M, Gigi J, Vandercam B, Reynaert M, et al.
Rapid and specific molecular identification of methicillin-resistant Staphylococcus aureus
in endotracheal aspirates from mechanically ventilated patients. J Clin Microbiol 1998;36:2366-8.
Martineau F, Picard FJ, Lansac N, Ménard C, Roy PH, Ouellette M, et al.
Correlation between the resistance genotype determined by multiplex PCR assays and the antibiotic susceptibility patterns of Staphylococcus aureus
and Staphylococcus epidermidis
. Antimicrob Agents Chemother 2000;44:231-8.
Strommenger B, Kettlitz C, Werner G, Witte W. Multiplex PCR assay for simultaneous detection of nine clinically relevant antibiotic resistance genes in Staphylococcus aureus
. J Clin Microbiol 2003;41:4089-94.
Rotger M, Trampuz A, Piper KE, Steckelberg JM, Patel R. Phenotypic and genotypic mupirocin resistance among Staphylococci causing prosthetic joint infection. J Clin Microbiol 2005;43:4266-8.
Takaya A, Kimura A, Sato Y, Ishiwada N, Watanabe M, Matsui M, et al.
Molecular characterization of linezolid-resistant CoNS isolates in Japan. J Antimicrob Chemother 2015;70:658-63.
McClure JA, Conly JM, Lau V, Elsayed S, Louie T, Hutchins W, et al.
Novel multiplex PCR assay for detection of the staphylococcal virulence marker Panton-Valentine leukocidin genes and simultaneous discrimination of methicillin-susceptible from -resistant staphylococci. J Clin Microbiol 2006;44:1141-4.
Niveditha N, Sujatha S. Worrisome trends in rising minimum inhibitory concentration values of antibiotics against methicillin resistant Staphylococcus aureus
- Insights from a tertiary care center, South India. Braz J Infect Dis 2015;19:585-9.
Sader HS, Farrell DJ, Jones RN. Antimicrobial susceptibility of Gram-positive cocci isolated from skin and skin-structure infections in European medical centres. Int J Antimicrob Agents 2010;36:28-32.
Kaur DC, Narayan PA. Mupirocin resistance in nasal carriage of Staphylococcus aureus
among healthcare workers of a tertiary care rural hospital. Indian J Crit Care Med 2014;18:716-21. [Full text]
Desroches M, Potier J, Laurent F, Bourrel AS, Doucet-Populaire F, Decousser JW; Microbs Study Group. Prevalence of mupirocin resistance among invasive coagulase-negative staphylococci and methicillin-resistant Staphylococcus aureus
(MRSA) in France: Emergence of a mupirocin-resistant MRSA clone harbouring mupA. J Antimicrob Chemother 2013;68:1714-7.
Chaturvedi P, Singh AK, Singh AK, Shukla S, Agarwal L. Prevalence of mupirocin resistant Staphylococcus aureus
Isolates among patients admitted to a tertiary care hospital. N Am J Med Sci 2014;6:403-7.
Deurenberg RH, Stobberingh EE. The evolution of Staphylococcus aureus
. Infect Genet Evol 2008;8:747-63.
Harris LG, Foster SJ, Richards RG. An introduction to Staphylococcus aureus
, and techniques for identifying and quantifying S. aureus
adhesins in relation to adhesion to biomaterials: Review. Eur Cell Mater 2002;4:39-60.
Somerville G, Proctor RA. The biology of staphylococci. In Staphylococci in Human Disease, editors. Crossley KB, Jefferson KK, Archer GL, Fowler VG. 2nd
ed. Ch. 1. Oxford, UK: Wiley-Blackwell; 2009.
Becker K, Heilmann C, Peters G. Coagulase-negative staphylococci. Clin Microbiol Rev 2014;27:871-925.
Cavanagh JP, Hjerde E, Holden MT, Kahlke T, Klingenberg C, Flægstad T, et al.
Whole-genome sequencing reveals clonal expansion of multiresistant Staphylococcus haemolyticus
in European hospitals. J Antimicrob Chemother 2014;69:2920-7.
van Hal SJ, Fowler VG. Is it time to replace vancomycin in the treatment of methicillin-resistant Staphylococcus aureus
infections? Clin Infect Dis 2013;56:1779-88.
Steinkraus G, White R, Friedrich L. Vancomycin MIC creep in non-vancomycin-intermediate Staphylococcus aureus
(VISA), vancomycin-susceptible clinical methicillin-resistant S. aureus
(MRSA) blood isolates from 2001-05. J Antimicrob Chemother 2007;60:788-94.
Liu C, Bayer A, Cosgrove SE, Daum RS, Fridkin SK, Gorwitz RJ, et al.
Clinical practice guidelines by the infectious diseases society of America for the treatment of methicillin-resistant Staphylococcus aureus
infections in adults and children. Clin Infect Dis 2011;52:e18-55.
Dabul AN, Camargo IL. Molecular characterization of methicillin-resistant Staphylococcus aureus
resistant to tigecycline and daptomycin isolated in a hospital in Brazil. Epidemiol Infect 2014;142:479-83.
Jacob JT, DiazGranados CA. High vancomycin minimum inhibitory concentration and clinical outcomes in adults with methicillin-resistant Staphylococcus aureus
infections: A meta-analysis. Int J Infect Dis 2013;17:e93-100.
Fernandes CJ, Fernandes LA, Collignon P; Australian Group on Antimicrobial Resistance. Cefoxitin resistance as a surrogate marker for the detection of methicillin-resistant Staphylococcus aureus
. J Antimicrob Chemother 2005;55:506-10.
Jain A, Agarwal A, Verma RK. Cefoxitin disc diffusion test for detection of meticillin-resistant staphylococci. J Med Microbiol 2008;57(Pt 8):957-61.
Duran N, Ozer B, Duran GG, Onlen Y, Demir C. Antibiotic resistance genes & susceptibility patterns in staphylococci. Indian J Med Res 2012;135:389-96. [Full text]
Lim KT, Hanifah YA, Yusof M, Thong KL. ermA, ermC, tetM and tetK are essential for erythromycin and tetracycline resistance among methicillin-resistant Staphylococcus aureus
strains isolated from a tertiary hospital in Malaysia. Indian J Med Microbiol 2012;30:203-7. [Full text]
Juda M, Chudzik-Rzad B, Malm A. The prevalence of genotypes that determine resistance to macrolides, lincosamides, and streptogramins B compared with spiramycin susceptibility among erythromycin-resistant Staphylococcus epidermidis
. Mem Inst Oswaldo Cruz 2016;111:155-60.
Chavez-Bueno S, Bozdogan B, Katz K, Bowlware KL, Cushion N, Cavuoti D, et al.
Inducible clindamycin resistance and molecular epidemiologic trends of pediatric community-acquired methicillin-resistant Staphylococcus aureus
in Dallas, Texas. Antimicrob Agents Chemother 2005;49:2283-8.
Archer GL, Scott J. Conjugative transfer genes in staphylococcal isolates from the United States. Antimicrob Agents Chemother 1991;35:2500-4.
Patel JB, Gorwitz RJ, Jernigan JA. Mupirocin resistance. Clin Infect Dis 2009;49:935-41.
Bathoorn E, Hetem DJ, Alphenaar J, Kusters JG, Bonten MJ. Emergence of high-level mupirocin resistance in coagulase-negative staphylococci associated with increased short-term mupirocin use. J Clin Microbiol 2012;50:2947-50.
Kapil A. Need to rationalize linezolid use. Indian J Med Microbiol 2015;33:1-2.
] [Full text]
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]
[Table 1], [Table 2], [Table 3], [Table 4]