|Year : 2016 | Volume
| Issue : 4 | Page : 500-505
Coagulase-negative staphylococci causing blood stream infection at an Indian tertiary care hospital: Prevalence, antimicrobial resistance and molecular characterisation
S Singh1, B Dhawan2, A Kapil2, SK Kabra3, A Suri4, V Sreenivas5, BK Das2
1 Department of Medical Microbiology, Post Graduate Institute of Medical Education and Research, Chandigarh, India
2 Department of Microbiology, All Institute of Medical Sciences, New Delhi, India
3 Department of Pediatrics, All Institute of Medical Sciences, New Delhi, India
4 Department of Neurosurgery, All Institute of Medical Sciences, New Delhi, India
5 Department of Biostatistics, All Institute of Medical Sciences, New Delhi, India
|Date of Submission||29-Jul-2016|
|Date of Acceptance||13-Oct-2016|
|Date of Web Publication||8-Dec-2016|
Department of Medical Microbiology, Post Graduate Institute of Medical Education and Research, Chandigarh
Source of Support: None, Conflict of Interest: None
Introduction: Recent years have seen a rise of coagulase-negative staphylococci (CoNS) from common contaminants to agents of nosocomial blood stream infections (BSI's). Molecular typing and establishing a correlation with antibiotic resistance is essential particularly in countries like India where genotyping studies for drug-resistant CoNS are sparse. Methods: A prospective study was done over 18 months, wherein 42,693 blood samples were received, and 59 patients with BSI due to CoNS were evaluated. The isolates recovered were identified by a biochemical test panel and matrix-assisted laser desorption ionization – time of flight mass spectrometry followed by antimicrobial susceptibility testing by Kirby–Baur disc diffusion method and E-test strips. Staphylococcal chromosomal cassette mec (SCCmec) element was characterised by multiplex polymerase chain reaction for all methicillin-resistant (MR) isolates. Results: The majority of CoNS isolated were constituted by Staphylococcus haemolyticus (47.5%) followed by Staphylococcus epidermidis (33.9%), Staphylococcus hominis (11.86%), Staphylococcus cohnii (5.08%) and Staphylococcus warneri (1.69%). Among all isolates 57.6% were MR with statistically significant higher resistance versus methicillin sensitive-CoNS. This difference was significant for erythromycin (76% vs. 44%, P = 0.011), rifampicin (50% vs. 12%,P= 0.002) and amikacin (26.5% vs. 4%, P = 0.023), ciprofloxacin (64.7% vs. 20%, P = 0.001) and cotrimoxazole (55.9% vs. 20%, P = 0.006). SCCmec type I was predominant (61.8%, P = 0.028) and exhibited multidrug resistance (76.2%). Coexistence of SCCmec type I and III was seen in 8.82% MR isolates. Conclusion: CoNS exhibit high antimicrobial resistance thereby limiting treatment options. The presence of new variants of SCCmec type in hospital-acquired CoNS may predict the antibiotic resistance pattern. This is the first evaluation of the molecular epidemiology of CoNS causing BSI from India and can serve as a guide in the formulation of hospital infection control and treatment guidelines.
Keywords: Antimicrobial resistance, bloodstream infection, coagulase negative staphylococci, mec A, staphylococcal chromosomal cassette mec typing
|How to cite this article:|
Singh S, Dhawan B, Kapil A, Kabra S K, Suri A, Sreenivas V, Das B K. Coagulase-negative staphylococci causing blood stream infection at an Indian tertiary care hospital: Prevalence, antimicrobial resistance and molecular characterisation. Indian J Med Microbiol 2016;34:500-5
|How to cite this URL:|
Singh S, Dhawan B, Kapil A, Kabra S K, Suri A, Sreenivas V, Das B K. Coagulase-negative staphylococci causing blood stream infection at an Indian tertiary care hospital: Prevalence, antimicrobial resistance and molecular characterisation. Indian J Med Microbiol [serial online] 2016 [cited 2020 Feb 20];34:500-5. Available from: http://www.ijmm.org/text.asp?2016/34/4/500/195374
| ~ Introduction|| |
Recent years have seen an increased recognition of coagulase-negative staphylococci (CoNS) as agents of hospital and community acquired infections. Previously dismissed as contaminants, these pathogens have now established their role in infections of the bloodstream, urinary tract, surgical sites, prosthetic devices and shunts. Another concern is the rising incidence of methicillin-resistant (MR)-CoNS in hospitalised patients. Resistance to β-lactams is determined by the mec A gene harboured on a mobile genetic element, i.e., staphylococcal chromosomal cassette mec (SCCmec). This not only limits treatment options but also enables transfer of these resistance elements to other Staphylococci. It is imperative that the molecular characteristics of nosocomial MRCoNS isolates from India be elucidated to understand the mobilisation and evolution of these genetic elements in our health-care settings. The objective of our study was to understand the epidemiology of CoNS causing blood stream infection (BSI) at our institute. The identification, antimicrobial susceptibility testing and SCCmec typing of the clinical isolates were done to guide us in the establishment of institutional guidelines regarding diagnosis and treatment of such infections.
| ~ Methods|| |
A hospital-based, prospective study was planned over an 18 months period.
2200 bedded, tertiary, teaching and referral hospital in North India.
Significant CoNS bacteraemia was defined by a modified clinical and microbiological algorithm, derived from that described by Beekmann et al. As per our protocol, the isolation of the same CoNS species from 2 or more blood culture samples within a 5-day period was considered significant. In cases where only one blood sample was available, in addition to the positive blood culture, the presence of at least two clinical parameters constituted significance. The clinical parameters included were: body temperature >38°C or <36°C, systolic blood pressure of <90 mmHg, total leucocyte count of >12,000/µL or <2000/µL and presence of >10% immature neutrophil granulocytes. Patients with significant bacteraemia due to CoNS were included in this study.
Ethical clearance was obtained from the Institute ethical review board (reference no. IESC/T-34) before commencement.
Identification of coagulase-negative staphylococci
All clinical isolates were identified by a biochemical test panel adapted from the Kloos and Schleifer scheme  and any ambiguous results were resolved by matrix-assisted laser desorption ionization – time of flight mass spectrometry (MALDI-TOF MS)-Bruker Biotyper Real Time Classification 3.1.
Antimicrobial susceptibility testing
The procedure and interpretation of antimicrobial susceptibility of the isolates were as per Clinical and Laboratory Standards Institute 2014 guidelines. Kirby–Bauer disc diffusion test was performed for cefoxitin, penicillin, amoxicillin-clavulanic acid, gentamicin amikacin, doxycycline, ciprofloxacin, trimethoprim-sulfamethoxazole, erythromycin, clindamycin, linezolid, netilmicin and teicoplanin. The estimation of the minimum inhibitory concentration of vancomycin was done using E-test strips. Confirmation of MR was done by polymerase chain reaction (PCR)-based amplification and detection of mec A gene. The resistance to three or more non-β-lactam group of drugs was considered as multidrug resistance (MDR).
Staphylococcal chromosomal cassette mec typing
The mec A PCR confirmed MRCoNS were subjected to SCCmec typing. The detection of SCCmec types I–IV was performed by multiplex PCR strategy for SCCmec typing as described previously for MR-Staphylococcus aureus. The S. aureus strains used as controls for SCCmec typing were: 665U (type I), UK EMRSA-16 (type II), UK EMRSA-1 (type III) and UK EMRSA-15 (type IV). Isolates which were non-typeable (NT) by this PCR were further subjected to singleplex PCR for SCCmec type V  using WIS (type V) as control.
The analysis was done using SPSS 18.0 for Windows (SPSS, Inc., Chicago, IL, USA). Categorical variables were compared using Chi-square test or Fisher's exact test, as appropriate. All tests for significance were two-tailed, and a P ≤ 0.05 was considered as statistically significant.
| ~ Results|| |
A total blood culture positivity of 4.9% was seen and 59 cases of CoNS BSI were identified, comprising 2.8% of all the positive blood cultures. The overall prevalence of Staphylococcal BSI was 2.3/1000 blood cultures while that of CoNS causing BSI was 1.4/1000 blood cultures. CoNS accounted for 61.5% of the staphylococcal isolates. The majority of the CoNS BSI cases were admitted to Intensive Care Units (ICUs) (n = 12) followed by oncology (n = 11) among all medical and surgical wards.
Staphylococcus haemolyticus (47.5%) formed the majority of the cases followed by Staphylococcus epidermidis (33.9%), S. hominis (11.86%), Staphylococcus cohnii (5.08%) and Staphylococcus warneri (1.69%). The majority of S. haemolyticus isolates were recovered from patients CoNS BSI admitted to medicine wards (80%), oncology (54.5%) and ICU's (50%). Isolation of S. epidermidis was higher among patients admitted to paediatric (70%) and surgical wards (55%) [Figure 1].
|Figure 1: Species distribution of coagulase-negative staphylococci among various wards|
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Antimicrobial susceptibility testing
Among the 59 isolates, a 57.6% prevalence of MRCoNS was seen. An overall high prevalence of resistance to the non-β-lactam antimicrobials was observed including clindamycin (69.4%), erythromycin (62.7%), ciprofloxacin (45.7%) and cotrimoxazole (40.7%). All the isolates were uniformly susceptible to vancomycin, teicoplanin, netilmicin and linezolid. The MRCoNS showed a higher level of resistance to all non-β-lactam antimicrobials tested as compared to Methicillin-sensitive (MS)-CoNS [Table 1]. MRCoNS showed higher level of resistance to all non-β-lactam antimicrobials compared to MSCoNS with difference being statistically significant for erythromycin (76% vs. 44%, P = 0.011), rifampicin (50% vs. 12%, P = 0.002) and amikacin (26.5% vs. 4%, P = 0.023), ciprofloxacin (64.7% vs. 20%, P = 0.001) and cotrimoxazole (55.9% vs. 20%, P = 0.006).
|Table 1: Resistance profile of methicillin resistant coagulase negative staphylococci and methicillin sensitive coagulase negative staphylococci isolates to non-β lactam antimicrobials|
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The prevalence of MR was highest among isolates of S. haemolyticus (67.9%, P = 0.273). There was no statistically significant association between CoNS species and resistance to non-β-lactams.
MDR was present in 49.2% of the CoNS isolates (n = 29). Among the MRCoNS, MDR was found in 85.3%. The maximum number of MDRCoNS isolates were recovered from patients admitted to ICU's (91.67%) and minimum from those admitted to oncology (18.2%, P = 0.006.) All the isolates of S. cohnii were MDR followed by isolates of S. haemolyticus (57.1%), S. epidermidis (40%) and S. hominis (28.6%) (P = 0.485).
SCCmec typing of the 34 MRCoNS isolates identified 21 (61.8%) isolates with SCCmec type I, 3 (8.82%) with SCCmec type III, 1 (2.9%) with SCCmec type II, 1 (2.9%) with SCCmec type IV and 3 (8.82%) with coexisting type I + III. Of all the isolates tested 5 (14.7%) were NT [Figure 2].
|Figure 2: Multiplex polymerase chain reaction for SCCmec typing (I–IV) of methicillin resistant coagulase negative staphylococci|
Click here to view
SCCmec type I was the predominant type among all isolates with 68.4% of S. haemolyticus isolates harbouring it, followed by S. hominis (66.7%), S. epidermidis (55.5%) and S. cohnii (33.3%) isolates (P = 0.028). All NT isolates were that of S. haemolytics [Table 2].
|Table 2: Distribution of Staphylococcal chromosomal cassette mec type among methicillin- resistant Coagulase negative Staphylococci species|
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An analysis of the susceptibility profile of different SCCmec types among the MRCoNS to non-β-lactam antimicrobials was done revealed that isolates with SCCmec type I, the most frequently identified SCCmec type, also exhibited high rates of resistance to erythromycin (76.2%), clindamycin (76.2%), ciprofloxacin (57.1%), cotrimoxazole (47.6%) and rifampicin (47.6%) [Table 3].
|Table 3: Distribution of Staphylococcal chromosomal cassette mec types in methicillin- resistant Coagulase negative Staphylococci according to resistance to nonβ-lactams|
Click here to view
Among isolates harbouring SCCmec type I, 76.2% were MDR, while in those harbouring both SCCmec type I + III, 66.7% were MDR (P = 0.720). All the three SCCmec type III isolates and the single isolates each of type II and IV were MDR.
| ~ Discussion|| |
Novel therapeutic interventions and increased use of foreign medical devices have been paralleled by the emergence of CoNS as formidable pathogens in health-care settings. We observed that CoNS are emerging primarily among Gram-positive cocci as agents of BSI. In some Indian studies evaluating the ecology of BSI in neonatal ICU's, a higher prevalence has been found. However, clinical significance of the CoNS isolated was not defined in these studies and may have resulted in an overestimation of prevalence. Western literature suggests that CoNS account for 25%–57% of the cases of BSI. There is a multifactorial cause for this difference between western and Indian literature, perhaps due to variations in the use of invasive medical devices, ambient humidity, prevalent flora in the hospital environment or on the health-care workers and patients.
The majority of CoNS isolated in our study were among ICU inpatients followed by those in oncology units. The presence of intravascular catheters, immunosuppression with severe neutropenia, mucosal breakdown caused by cytotoxic chemotherapy may precipitate BSIs in these groups. Since ours is a tertiary care, referral centre catering to the entire country this ecology could be representative of most Indian centres.
The species distribution of CoNS in our study is in concordance with previous studies from India where S. haemolyticus was the most common followed by S. epidermidis. In developed countries, S. epidermidis is the most frequent CoNS causing BSI followed by S. haemolyticus. Different species distribution in developed versus developing countries could be due to the difference in colonisation characteristics of patients and the varying adaptability of different species to selective pressures such as biocides and antimicrobials in the environment.
Prevalence of MRCoNS ranging from 48.2% to 60% has been previously reported., We found the highest MR in S. haemolyticus, supporting the findings of other centres wherein resistance rates as high as 90% have been reported.
An overall high prevalence of resistance to all antibiotics was seen with MRCoNS showing higher resistance to non-β-lactam antimicrobials as compared to MSCoNS, difference being statistically significant for erythromycin, rifampicin, amikacin, ciprofloxacin and cotrimoxazole. Hence, these antibiotics no longer remain options for empirical treatment of CoNS infections. The non-β-lactam agents most active against MRCoNS were doxycycline and rifampicin probably due infrequent use at our centre, resulting in low selection pressure.
All CoNS isolates were susceptible to vancomycin, teicoplanin, linezolid and netilmicin. These drugs may be included in therapy for patients with MRCoNS infections; however, empirical use must be avoided before recording results of susceptibility tests as overuse of these antibiotics can promote glycopeptide and oxazolidinone resistance.
Our study highlights a high prevalence of MDRCoNS primarily in S. haemolyticus. Studies analysing the whole genome of S. haemolyticus have revealed as many as 82 insertion sequences. These may represent hotspots for the acquisition of antibiotic resistance genes accounting for the high prevalence of MDR in these CoNS species.,
The multiplex PCR used in this study detects the most prevalent SCCmec types in CoNS isolates causing health-care associated infections and hence can be used as a cost-effective mode of SCCmec typing in resource-limited laboratories since a majority of MRCoNS isolates harbour a single or a combination of SCCmec I–V types.
SCCmec type I was predominant among our isolates and the low proportion of SCCmec IV can be explained since our strains were from hospitalised patients, while SCCmec IV is predominantly detected in community-acquired MRCoNS strains.
A study from South India on the SCCmec typing of CoNS isolated from various clinical samples of HIV-infected patients has also revealed type I (50%) to be predominant followed by type V (19%), type III (14%), type IV (7%).
It had been proposed that difference in the distribution of various SCCmec types among MRCoNS depends on the geographical locations and the host species involved as varying SCCmec types have been documented in different parts of the world.,
In this study, 14.7% of the MRCoNS were NT and coexistence of SCCmec type I + III were observed. In a study from South India, 6% of the isolates were NT by PCR for SCCmec I–V and 4% harboured both type I and V. The presence of novel structures, rearrangements and recombination of the mec element may explain the variability in the SCCmec types. SCCmec element variants such as SCCmec type II without kpd operon, SCCmec type IV without locus D of the dcs gene have been described previously. Since these loci are being detected in the multiplex PCR used in our study, the presence of such mutants could account for the rather large number of NT isolates in our study and merits further evaluation.
The frequent coexistence of SCCmec types and the presence of NT elements in MRCoNS is a challenge for SCCmec typing. Coexisting SCCmec elements may constitute a composite rather than two distinct elements. One of the limitations of the PCR-based schemes we used is the inability to differentiate separate elements from such composites.
Studies on the distribution of SCCmec types among the CoNS species suggest that type III and IV are preferentially associated with S. epidermidis while type I and IV predominate in S. haemolyticus. We describe a statistically significant association of both S. epidermidis and S. haemolyticus with SCCmec type I. All isolates with NT SCCmec type were identified as S. haemolyticus and this could be due to structural rearrangements in the SCCmec element or may indicate clonality which needs to be elucidated further.
Molecular characterisation of CoNS is gaining importance since some SCCmec types are associated with higher rates of antimicrobial resistance. In this study, CoNS isolates with SCCmec types I and III showed higher rates of MDR.
Interestingly, a majority of MRSA clinical isolates previously characterised in our hospital possessed type I and were significantly more resistant to antibiotics. Horizontal transfer of SCCmec between S. aureus and CoNS has been contemplated in literature. Thus, we can speculate that SCCmec type I found widely in MRSA strains in our hospital might have originated from MRCoNS.
| ~ Conclusion|| |
To the best of our knowledge, this is the first evaluation of the molecular epidemiology of CoNS causing BSI from India. Molecular typing and establishing a correlation with antibiotic resistance is essential particularly in countries like India where genotyping studies for drug-resistant CoNS are scant. Since CoNS are capable of transmitting drug-resistance genes to organisms like S. aureus, typing a large number of CoNS isolates and prospectively monitoring of their drug resistance is paramount in designing antibiotic policies and hospital infection control strategies.
Dr. Arunaloke Chakrabarti, Dr. Pallab Ray and Dr. Vikas Gautam of Post Graduate Institute of Medical Education and Research, Chandigarh for granting access to MALDI-TOF MS equipment.
Financial support and sponsorship
The study was conducted as part of the MD thesis of Dr. Shreya Singh.
Conflicts of interest
There are no conflicts of interest.
| ~ References|| |
Latif M, Usman J, Gilani M, Munir T, Mushtaq M, Anjum R. Coagulase negative staphylococci – A fast emerging threat. J Pak Med Assoc 2015;65:283-6.
Becker K, Heilmann C, Peters G. Coagulase-negative staphylococci. Clin Microbiol Rev 2014;27:870-926.
Beekmann SE, Diekema DJ, Doern GV. Determining the clinical significance of coagulase-negative staphylococci isolated from blood cultures. Infect Control Hosp Epidemiol 2005;26:559-66.
Kloos WE, Schleifer KH. Isolation and characterization of staphylococci from human skin II. Descriptions of four new species: Staphylococcus warneri
, Staphylococcus capitis
, Staphylococcus hominis
, and Staphylococcus simulans
1. Int J Syst Evol Microbiol 1975;25:62-79.
CLSI. Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing. Twenty-Fourth Informational Supplement CLSI Document M100-S24, Wayne, PA.; 2014.
Pérez-Roth E, Claverie-Martín F, Villar J, Méndez-Alvarez S. Multiplex PCR for simultaneous identification of Staphylococcus aureus
and detection of methicillin and mupirocin resistance. J Clin Microbiol 2001;39:4037-41.
Agvald-Ohman C, Lund B, Edlund C. Multiresistant coagulase-negative staphylococci disseminate frequently between intubated patients in a multidisciplinary intensive care unit. Crit Care 2004;8:R42-7.
Oliveira DC, de Lencastre H. Multiplex PCR strategy for rapid identification of structural types and variants of the mec element in methicillin-resistant Staphylococcus aureus
. Antimicrob Agents Chemother 2002;46:2155-61.
Milheiriço C, Oliveira DC, de Lencastre H. Update to the multiplex PCR strategy for assignment of mec element types in Staphylococcus aureus
. Antimicrob Agents Chemother 2007;51:3374-7.
Wattal C, Raveendran R, Goel N, Oberoi JK, Rao BK. Ecology of blood stream infection and antibiotic resistance in intensive care unit at a tertiary care hospital in North India. Braz J Infect Dis 2014;18:245-51.
Keim LS, Torres-Filho SR, Silva PV, Teixeira LA. Prevalence, aetiology and antibiotic resistance profiles of coagulase negative staphylococci isolated in a teaching hospital. Braz J Microbiol 2011;42:248-55.
Jain A, Agarwal A, Verma RK, Awasthi S, Singh KP. Intravenous device associated blood stream staphylococcal infection in paediatric patients. Indian J Med Res 2011;134:193-9.
Pereira VC, Cunha Mde L. Coagulase-negative staphylococci strains resistant to oxacillin isolated from neonatal blood cultures. Mem Inst Oswaldo Cruz 2013;108:939-42.
Barros EM, Ceotto H, Bastos MC, Dos Santos KR, Giambiagi-Demarval M. Staphylococcus haemolyticus
as an important hospital pathogen and carrier of methicillin resistance genes. J Clin Microbiol 2012;50:166-8.
Takeuchi F, Watanabe S, Baba T, Yuzawa H, Ito T, Morimoto Y, et al.
Whole-genome sequencing of Staphylococcus haemolyticus
uncovers the extreme plasticity of its genome and the evolution of human-colonizing staphylococcal species. J Bacteriol 2005;187:7292-308.
Saravanan M, Dass SB, Abirami SB, Suriakumar JK, Krishnan P. Prevalence of SCCmec types among methicillin resistant coagulase negative staphylococci isolated from HIV patients in Chennai, South India. BMC Infect Dis 2014;14 Suppl 3:51.
Mombach Pinheiro Machado AB, Reiter KC, Paiva RM, Barth AL. Distribution of staphylococcal cassette chromosome mec (SCCmec) types I, II, III and IV in coagulase-negative staphylococci from patients attending a tertiary hospital in southern Brazil. J Med Microbiol 2007;56(Pt 10):1328-33.
Gürkan ME, Kiliç A, Orhan BE, Başustaoğlu AC. Clinical signifi cance and staphylococcal cassette chromosome mec (SCCmec) characterization of coagulase-negative staphylococci isolated from blood cultures. Turk J Med Sci 2011;41:859-65.
Shore A, Rossney AS, Keane CT, Enright MC, Coleman DC. Seven novel variants of the staphylococcal chromosomal cassette mec in methicillin-resistant Staphylococcus aureus
isolates from Ireland. Antimicrob Agents Chemother 2005;49:2070-83.
Ruppé E, Barbier F, Mesli Y, Maiga A, Cojocaru R, Benkhalfat M, et al.
Diversity of staphylococcal cassette chromosome mec structures in methicillin-resistant Staphylococcus epidermidis
and Staphylococcus haemolyticus
strains among outpatients from four countries. Antimicrob Agents Chemother 2009;53:442-9.
Gadepalli R, Dhawan B, Kapil A, Sreenivas V, Jais M, Gaind R, et al.
Clinical and molecular characteristics of nosocomial meticillin-resistant Staphylococcus aureus
skin and soft tissue isolates from three Indian hospitals. J Hosp Infect 2009;73:253-63.
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3]