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 ~ Introduction
 ~  Materials and Me...
 ~ Results
 ~ Discussion
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  Table of Contents  
ORIGINAL ARTICLE
Year : 2018  |  Volume : 36  |  Issue : 4  |  Page : 522-525
 

Occurrence of II and V staphylococcal cassette chromosome mec types among coagulase-negative staphylococci from Northeastern part of India


Department of Microbiology, Assam University, Silchar, Assam, India

Date of Web Publication18-Mar-2019

Correspondence Address:
Dr. Amitabha Bhattacharjee
Department of Microbiology, Assam University, Silchar - 788 011, Assam
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijmm.IJMM_18_285

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 ~ Abstract 


Introduction: Coagulase Negative Staphylococci, the most commonly isolated pathogen are becoming emerging threats to the community as well as to the nosocomial environment. The present study underscores the distribution of Staphylococcal cassette chromosome mec (SCCmec) types among Methicillin resistant Coagulase Negative Staphylococci from the environmental origin. Methods and Materials: Environmental and food sample (n = 460) from different location of northeastern region of India were collected for a period of one year and were phenotypically and genotypically screened using cefoxitin disc and PCR techniques for mecA and mecC gene detection. All the MR-CoNS isolates possessing mecA gene were subjected to 16srDNA sequencing for species identification. SCCmec typing was determined by evaluating using primer sets from type I to type V. Antibiotic susceptibility testing was performed for all the isolates. Statistical analysis with chi-square test using SPSS-21 statistical software. Results: Methicillin resistance shown by one hundred forty three isolates were carried out for molecular analysis, among them 53.84% serves as mecA carrier. Distribution of Staphylococcus haemolyticus was more frequent and was found that SCCmec types II and V were predominant among the study isolates. Linezolid was the drug of choice for the CoNS isolates. Statistical analysis showed an insignificant result for the tested antibiotics and SCCmec types. Conclusion: This study therefore interprets the relative importance of SCCmec types among MR-CoNS isolates.


Keywords: Methicillin-resistant coagulase-negative staphylococci, mec genes, staphylococcal cassette chromosome mec, SPSS-21


How to cite this article:
Bhowmik D, Chakraborty P, Bhowmick R, Sarkar R, Chetri S, Bhattacharjee A. Occurrence of II and V staphylococcal cassette chromosome mec types among coagulase-negative staphylococci from Northeastern part of India. Indian J Med Microbiol 2018;36:522-5

How to cite this URL:
Bhowmik D, Chakraborty P, Bhowmick R, Sarkar R, Chetri S, Bhattacharjee A. Occurrence of II and V staphylococcal cassette chromosome mec types among coagulase-negative staphylococci from Northeastern part of India. Indian J Med Microbiol [serial online] 2018 [cited 2019 May 21];36:522-5. Available from: http://www.ijmm.org/text.asp?2018/36/4/522/254391



 ~ Introduction Top


Methicillin-resistant coagulase-negative staphylococci (MR-CoNS) are now an emerging threat to the society, i.e., community as well as nosocomial environment; as an opportunistic pathogen, it persists and proliferates in diverse environmental sources.[1] Infections caused by CoNS are the most commonly isolated pathogens from the neonatal intensive care unit (NICU), and hence, Staphylococcus haemolyticus and Staphylococcus epidermidis became the most frequent causative agents of leading NICU infections over the past decades.[2],[3] Staphylococcus sciuri also being the important human pathogens are responsible for endocarditis, peritonitis, septic shock, urinary tract infection, pelvic inflammatory disease and wound infections, with an exception for Staphylococcus saprophyticus and Staphylococcus lugdunensis where they rarely attack a healthy host, due to their nonaggressive virulence properties.[2]

MR-CoNS in the environment serve as a reservoir of genetically diverse staphylococcal cassette chromosome mec (SCCmec) types, which function as a vehicle for the transfer of resistance genes horizontally to other organisms.[4] Till date, 12 different SCCmec types (I–XII) have been discovered in Staphylococcus aureus, and it was also reported that many composite SCCmec elements have been found in MR-CoNS.[5] Based on such diversity, five SCCmec types (I–V) have also been described in CoNS isolates.[6]

Genetic diversity of SCCmec types varies with environmental locations as well as with their introduction of newly evolved CoNS isolates. SCCmec typing of MR-CoNS is very elemental stage for studying the evolution of mobile genetic elements and also for providing information regarding the antibiotic resistance prototype in staphylococci.[4]

As there is a paucity of data regarding the status of SCCmec types in the MR-CoNS from this country, the present study detects the SCCmec types among MR-CoNS isolated from environment and food sources.


 ~ Materials and Methods Top


Isolation and identification of the bacterial isolates

A total of 460 samples were collected from the environmental and food samples of various location of southern part of Assam, India, for a period of 1 year from January 2017 to December 2017. The environmental sites of the collected isolates include river water (21), pond water (33), raw milk (181), faecal matter of poultry (23), chicken meat (163) and soil sample from paddy fields (39). All the samples were inoculated onto mannitol salt broth and incubated overnight. Then, the isolates were streaked onto mannitol salt agar (Himedia, India), Chromo agar (Himedia, India), Baird Parker Agar (Himedia, India) and Hi Chrome Rapid MRSA Agar Base (Himedia, India) incubated aerobically at 37°C for 24–72 h.

The isolates were initially identified by Gram staining, catalase and coagulase test. Only the CoNS isolates that showed different staphylococcal characteristics were selected for further study.

Phenotypic and genotypic screening of the isolates

All the CoNS isolates were then screened with cefoxitin disc diffusion method and oxacillin agar dilution method according to the Clinical and Laboratory Standards Institute (CLSI) guidelines.[7] Screening for MR gene, namely, mecA and mecC carriage, was performed by polymerase chain reaction assay.[8],[7]

16s rDNA sequencing

Those MR-CoNS isolates that possess mecA gene were prepared for DNA fragment amplification for 16s rDNA (27F-5'-AGAGTTTGATCMTGGCTCAG-3' and 1492R-5'-TACGGYTACCTTGTTACGACTT-3') sequencing for species identification, and the result has been interpreted by sequence similarity search with BLAST, Chromas software and the DDBJ/EMBL/GeneBank databases.

Determination of staphylococcal cassette chromosome mec types

For all mecA-positive isolates, SCCmec types were determined by evaluating Type I, II, III, IV (a), IV (b), IV (c), IV (d) and Type V. The primers for the designated Types I–V were described previously.[9]

Antibiotic susceptibility testing

Antibiotic susceptibility testing was performed by disc diffusion method on Mueller–Hinton Agar according to the recommendation of CLSI guidelines [7] with the following antibiotics: ciprofloxacin (5 μg), gentamicin (10 μg), tetracycline (30 μg), clindamycin (2 μg), erythromycin (15 μg), tigecycline (15 μg), co-trimoxazole (25 μg) and linezolid (15 μg). S. aureus ATCC 25923 was used as a quality control.

Statistical analysis

The data were generated using descriptive statistics (frequency, percentage and mean ± standard deviation) and Chi-square test with statistical package for the social sciences SPSS-21 statistical software (IBM, Armonk, NY, USA). This study was performed to evaluate the statistical significant impact of antibiotics on SCCmec types. The Chi-square test with P < 0.05 significance level was used for statistical analysis.


 ~ Results Top


A total of 238 isolates were found to be CoNS of which 143 showed methicillin resistance, established by cefoxitin disc diffusion and oxacillin agar dilution method. Molecular analysis showed that 79 (53.84%) isolates serve as a carrier for mecA gene which encodes for its resistance [Table 1].
Table 1: Dissemination of staphylococcal isolates in environmental zone

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Among all the mec A harbouring MR-CoNS, it was found that S. haemolyticus (52.72%) was predominantly carrying mecA gene followed by S. sciuri (30.90%) and Staphylococcus xylosus (16.36%). The distribution of SCCmec types in S. haemolyticus is more frequent, 31.03% isolates bear SCCmec Type V, 24.13% isolates harboured SCCmec Type II, followed by 13.79% Type III and 20.69% Type IV (a), whereas SCCmec Type IV (b), IV (c) and IV (d) could not be typed. However, in S. sciuri, SCCmec Type II and Type V were more predominant followed by Type IV (b) and (c) whereas S. xylosus harboured both SCCmec Type II and Type III [Table 2]. It was also found from the current study that 20 (36.36%) isolates were identified as SCCmec Type II, 14 (25.45%) belongs to SCCmec Type V, 6 (10.90%) were SCCmec Type IV (a) while 12.72% isolates were in SCCmec Type III and 0.36% fits in SCCmec Type I and Type IV (c). Twenty-four (43.63%) isolates of MR-CoNS could not be typed using this method.
Table 2: Distribution of staphylococcal cassette chromosome mec types among methicillin-resistant coagulase-negative staphylococci isolates

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Susceptibility profile of MR-CoNS isolates, with non-beta-lactam antibiotics among SCCmec types showed that S. haemolyticus with SCCmec Types II and V were found to be more susceptible towards linezolid (79.3%) followed by ciprofloxacin (53.3%) and gentamicin (46.6%). While for S. sciuri, linezolid (66.6%) and tetracycline (55.6%) were more efficient followed by co-trimoxazole (44.4%) and gentamicin (33.3%), and in S. xylosus, gentamicin (66.6%) showed higher susceptibility pattern [Table 3].
Table 3: Antibiotic susceptibility testing of methicillin-resistant coagulase-negative staphylococci isolates

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According to the result of statistical analysis, i.e., Chi-square test for the resistance profile among the SCCmec types, significance index (P = 0.05) and degree of freedom (P = 5), all the data were statistically insignificant to almost all the tested antibiotics with different SCCmec types: ciprofloxacin, (P = 0.99); gentamicin, (P = 0.96); tetracycline, (P = 1.00); clindamycin, (P = 0.77); erythromycin, (P = 0.77); linezolid, (P = 0.99); tigecycline, (P = 0.34) and co-trimoxazole, (P = 1.00) [Table 4].
Table 4: Statistical analysis of resistance non-beta-lactam antibiotics among staphylococcal cassette chromosome mec type

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 ~ Discussion Top


CoNS are becoming an increasing concern in both clinical and community environment as because of the fact that they harbour SCCmec elements.[10] SCCmec as the mobile genetic element of the chromosome has been delineated by the carriage of mecA gene complex, ccr gene complex, integration at a specific nucleotide region (att Bscc) and demarcation by a pair of direct and inverted repeats.[11],[12]

Dissemination of mecA gene in CoNS species has been already established in previous studies [6],[13] which was further evident in our study too where three environmental isolates, namely S. haemolyticus, S. sciuri and S. xylosus were found to carry SCCmec. This study also focused on the distribution of SCCmec Types I, II, III, IV (a), IV (b), IV (c), IV (d) and V among the environmental CoNS isolates obtained from different sites of southern Assam.

Predominance of SCCmec Type II and Type V was quite similar to the findings of a study from Beijing where they showed the prevalence of SCCmec Types II, III and V [10] whereas in the study of Zong et al., SCCmec Types III, IV and V were found to be the most predominant types.[14]

In the study conducted by Schoenfelder et al., it was found that 64% of the 158 S. sciuri isolates were resistant to six different antibiotics and 16% showed multidrug-resistant phenotype.[15] Earlier study also reported that S. epidermis had higher resistance towards erythromycin, penicillin and cefoxitin, and other half of the strains were found to bear multidrug resistance.[16] The present study showed S. sciuri with higher resistance towards ciprofloxacin, which was followed by moderate resistance towards erythromycin, tigecycline and clindamycin. In case of S. haemolyticus, it was also found that tigecycline displayed highest resistance rate followed by clindamycin and erythromycin, while S. xylosus was resistant towards four groups of antibiotics. A previous study also exhibited a similar pattern.[16]

The statistical analysis of our study explained the resistance profile of MR-CoNS isolates to the non-beta-lactam antibiotics among SCCmec types. This depicts that SCCmec Type I is found to confer more resistance to all the tested antimicrobials which was followed by SCCmec type II, III, IV (a–d) and V, showing no statistical significance. In contrary to this finding, another report showed statistically significant result towards rifampicin (P = 0.010), levofloxacin (P = 0.005) and clindamycin (P = 0.012) among SCCmec Type III.[12]


 ~ Conclusion Top


This study underscores environment as a reservoir of the MR-CoNS isolates and has also revealed that MR-CoNS isolates are predominantly associated with S. haemolyticus. Since CoNS are capable of transmitting multidrug resistance genes, molecular typing can be a prospective towards monitoring the drug resistance and infection control management.

Financial support and sponsorship

The study was financially supported by the Department of Biotechnology, Government of India programme (BT/PR22757/MED/29/1172/2016).

Conflicts of interest

There are no conflicts of interest.

 
 ~ References Top

1.
Seng R, Kitti T, Thummeepak R, Kongthai P, Leungtongkam U, Wannalerdsakun S, et al. Biofilm formation of methicillin-resistant coagulase negative staphylococci (MR-coNS) isolated from community and hospital environments. PLoS One 2017;12:e0184172.  Back to cited text no. 1
    
2.
Becker K, Heilmann C, Peters G. Coagulase-negative staphylococci. Clin Microbiol Rev 2014;27:870-926. [doi.org/10.1128/CMR.00109-13].  Back to cited text no. 2
    
3.
Katayama Y, Ito T, Hiramatsu K. A new class of genetic element, Staphylococcus cassette chromosome mec, encodes methicillin resistance in Staphylococcus aureus. Antimicrob Agents Chemother 2000;44:1549-55.  Back to cited text no. 3
    
4.
Ghosh A, Singh Y, Kapil A, Dhawan B. Staphylococcal cassette chromosome mec (SCCmec) typing of clinical isolates of coagulase-negative staphylocci (CoNS) from a tertiary care hospital in New Delhi, India. Indian J Med Res 2016;143:365-70.  Back to cited text no. 4
[PUBMED]  [Full text]  
5.
Available from: http://www.sccmec.org/Pages/SCC_aboutIWG-SCCEN.html. [Last accessed on 2018 May 24].  Back to cited text no. 5
    
6.
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;56:1328-33.  Back to cited text no. 6
    
7.
Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing. CLSI Supplement M100. 27th ed. Wayne, Pennsylvania, USA: Clinical and Laboratory Standards Institute; 2017.  Back to cited text no. 7
    
8.
Becker K, Ballhausen B, Köck R, Kriegeskorte A. Methicillin resistance in Staphylococcus isolates: The “mec alphabet” with specific consideration of mecC, a mec homolog associated with zoonotic S. Aureus lineages. Int J Med Microbiol 2014;304:794-804.  Back to cited text no. 8
    
9.
Ghaznavi-Rad E, Nor Shamsudin M, Sekawi Z, van Belkum A, Neela V. A simplified multiplex PCR assay for fast and easy discrimination of globally distributed staphylococcal cassette chromosome mec types in meticillin-resistant Staphylococcus aureus. J Med Microbiol 2010;59:1135-9.  Back to cited text no. 9
    
10.
Chen XP, Li WG, Zheng H, Du HY, Zhang L, Zhang L, et al. Extreme diversity and multiple SCCmec elements in coagulase-negative Staphylococcus found in the clinic and community in Beijing, China. Ann Clin Microbiol Antimicrob 2017;16:57.  Back to cited text no. 10
    
11.
Zhang K, McClure JA, Elsayed S, Conly JM. Novel staphylococcal cassette chromosome mec type, tentatively designated type VIII, harboring class A mec and type 4 ccr gene complexes in a Canadian epidemic strain of methicillin-resistant Staphylococcus aureus. Antimicrob Agents Chemother 2009;53:531-40.  Back to cited text no. 11
    
12.
Ito T, Katayama Y, Asada K, Mori N, Tsutsumimoto K, Tiensasitorn C, et al. Structural comparison of three types of staphylococcal cassette chromosome mec integrated in the chromosome in methicillin-resistant Staphylococcus aureus. Antimicrob Agents Chemother 2001;45:1323-36.  Back to cited text no. 12
    
13.
Pierre J, Williamson R, Bornet M, Gutmann L. Presence of an additional penicillin-binding protein in methicillin-resistant Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus hominis, and Staphylococcus simulans with a low affinity for methicillin, cephalothin, and cefamandole. Antimicrob Agents Chemother 1990;34:1691-4.  Back to cited text no. 13
    
14.
Zong Z, Peng C, Lü X. Diversity of SCCmec elements in methicillin-resistant coagulase-negative staphylococci clinical isolates. PLoS One 2011;6:e20191.  Back to cited text no. 14
    
15.
Schoenfelder SM, Dong Y, Feßler AT, Schwarz S, Schoen C, Köck R, et al. Antibiotic resistance profiles of coagulase-negative staphylococci in livestock environments. Veterinary Microbiol 2017;200:79-87. [doi.org/10.1016/j.vetmic 2016.04.019].  Back to cited text no. 15
    
16.
Sani NA, Sapri HF, Neoh HM, Hussin S. First report on the molecular epidemiology of Malaysian Staphylococcus epidermidis isolated from a university teaching hospital. BMC Res Notes 2014;7:597.  Back to cited text no. 16
    



 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]



 

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