Accuracy of different methods for identification of Staphylococcus haemolyticus

Abstract

Objectives: Staphylococcus haemolyticus is associated with device-related infections in immunocompromised individuals and acts as a reservoir for antibiotic resistance genes. It is also the species with the highest antibiotic resistance rates. However, identification is still difficult in most clinical laboratories. Simplified biochemical tests give variable results while newer methods such as MALDI-TOF MS and automated systems may not be readily available. Aim: To compare the performance of the simplified biochemical scheme, BD-Phoenix automated system, and PCR for nuc gene for the identification of S. haemolyticus with MALDI-TOF MS as the gold standard. Methods: This study included 427 coagulase-negative staphylococci (CoNS) isolates of which 356 were identified as S. haemolyticus and 71 as other species by MALDI-TOF MS. These isolates were subjected to a simplified biochemical scheme using tests like the fermentation of maltose, sucrose, trehalose, mannose, urease, xylose, ornithine, and susceptibility to novobiocin. Conventional PCR targeting the nuc gene and BD-Phoenix were also used for identification. The accuracy of these methods was assessed in comparison with MALDI-TOF MS. Results: The sensitivity and specificity of biochemical tests, BD- Phoenix and nuc PCR were 97.5% and 97.2%: 97.8% and 100%: 100% and 100% respectively. Inaccurate identification was observed for some of the isolates (2.2% by BD- Phoenix and 2.5% by biochemical tests). These isolates were identified as S. haemolyticus by the other methods. Conclusion: Identification of S. haemolyticus by biochemical tests and BD-Phoenix had good accuracy comparable to PCR as well as MALDI-TOF MS. This simplified biochemical scheme can be easily implemented even in laboratories with limited resources. J Microbiol Infect Dis 2021; 11(1):8-14.

Keywords

• S. haemolyticus
• coagulase negative staphylococci
• MALDI-TOF MS
• nuc gene

References

1. 1. Bouchami O, de Lencastre H, Miragaia M. Impact of insertion sequences and recombination on the population structure of Staphylococcus haemolyticus. PloS One. 2016; 11(6):e0156653-e0156653.
2. 2. Czekaj T, Ciszewski M, Szewczyk EM. Staphylococcus haemolyticus - an emerging threat in the twilight of the antibiotics age. Microbiology 2015; 161(11):2061-8.
3. 3. Cavanagh JP, Hjerde E, Holden MTG, et al. Whole-genome sequencing reveals clonal expansion of multi-resistant Staphylococcus haemolyticus in European hospitals. J Antimicrob Chemother 2014; 69(11):2920-7.
4. 4. Cunha M de LR, Sinzato YK, Silveira LV. Comparison of methods for the identification of coagulase-negative staphylococci. Mem Inst Oswaldo Cruz 2004; 99(8):855-60.
5. 5. Becker K, Heilmann C, Peters G. Coagulase-negative Staphylococci. Clin Microbiol Rev 2014; 27(4):870-926.
6. 6. Kosecka-Strojek M, Sabat AJ, Akkerboom V, et al. Development and Validation of a Reference Data Set for Assigning Staphylococcus Species Based on Next-Generation Sequencing of the 16S-23S rRNA Region. Front Cell Infect Microbiol 2019; 9:278-278.
7. 7. Kloos WE, Schleifer KH. Simplified scheme for routine identification of human Staphylococcus species. J Clin Microbiol 1975; 1(1):82-8.
8. 8. Koneman E.W, Allen S.D, Janda W.M, et al. Staphylococci and related organisms. Color Atlas and Textbook of Diagnostic Microbiology 1997; 539-576.

9. 9. Rossi CC, Pereira MF, Giambiagi-deMarval M. Underrated Staphylococcus species and their role in antimicrobial resistance spreading. Genet Mol Biol 2020; 43(1 suppl 2):e20190065.
10. 10. Kleinschmidt S, Huygens F, Faoagali J, et al. Staphylococcus epidermidis as a cause of bacteremia. Future Microbiol 2015; 10(11):1859-79.
11. 11. Manoharan M, Sistla S, Ray P. Prevalence and molecular determinants of antimicrobial resistance in clinical isolates of Staphylococcus haemolyticus from India. Microb Drug Resist [Internet] 2020 Mar 19 [cited 2020 Jul 14].
12. 12. Gautam V, Sethuraman N, Kaur R, et al. Changing epidemiology of coagulase-negative staphylococci in normal flora of skin. Indian J Med Microbiol 2017; 35(2):277.
13. 13. Sah S, Bordoloi P, Vijaya D, et al. Simple and economical method for identification and speciation of Staphylococcus epidermidis and other coagulase negative Staphylococci and its validation by molecular methods. J Microbiol Methods 2018; 149:106-19.
14. 14. Pinheiro L, Brito CI, Pereira VC, et al. Susceptibility profile of Staphylococcus epidermidis and Staphylococcus haemolyticus isolated from Blood cultures to vancomycin and novel antimicrobial drugs over a period of 12 years. Microb Drug Resist 2016; 22(4):283-93.
15. 15. de Oliveira A, Cataneli Pereira V, Pinheiro L, et al. Antimicrobial resistance profile of planktonic and biofilm cells of Staphylococcus aureus and coagulase-negative staphylococci. Int J Mol Sci 2016; 17(9):1423.
16. 16. de Freitas Guimarães F, Nóbrega DB, Richini-Pereira VB, et al. Enterotoxin genes in coagulase-negative and coagulase-positive staphylococci isolated from bovine milk. J Dairy Sci 2013; 96(5):2866-72.
17. 17. Goyal R, Singh NP, Kumar A, et al. Simple and economical method for speciation and resistotyping of clinically significant coagulase negative staphylococci. Indian J Med Microbiol 2006; 24(3):201-4.
18. 18. Spanu T, De Carolis E, Fiori B, et al. Evaluation of matrix-assisted laser desorption ionization-time-of-flight mass spectrometry in comparison to rpoB gene sequencing for species identification of bloodstream infection staphylococcal isolates. Clin Microbiol Infect 2011; 17(1):44-9.
19. 19. Trevisoli LE, Bail L, Rodrigues LS, et al. Matrix-assisted laser desorption ionization-time of flight: a promising alternative method of identifying the major coagulase-negative staphylococci species. Rev Soc Bras Med Trop 2018; 51(1):85-7.
20. 20. Loonen AJM, Jansz AR, Bergland JNB, et al. Comparative study using phenotypic, genotypic, and proteomics methods for identification of coagulase-negative staphylococci. J Clin Microbiol 2012; 50(4):1437-9.
21. 21. Tomazi T, Gonçalves JL, Barreiro JR, et al. Identification of coagulase-negative staphylococci from bovine intramammary infection by Matrix-assisted laser desorption ionization–time of flight mass spectrometry. J Clin Microbiol 2014; 52(5):1658-63.
22. 22. Layer F, Ghebremedhin B, Moder K-A, et al. Comparative study using various methods for identification of Staphylococcus species in clinical specimens. J Clin Microbiol 2006; 44(8):2824-30.
23. 23. Chatzigeorgiou K-S, Sergentanis TN, Tsiodras S, et al. Phoenix 100 versus Vitek 2 in the identification of gram-positive and gram-negative bacteria: a comprehensive meta-analysis. J Clin Microbiol 2011; 49(9):3284-91.
24. 24. Hirotaki S, Sasaki T, Kuwahara-Arai K, et al. Rapid and accurate identification of human-associated staphylococci by use of multiplex PCR. J Clin Microbiol 2011; 49(10):3627-31.