Antibiotic sensitivity analysis of clinical coagulase-negative staphylococci

Background. The increasing role of coagulase-negative staphylococci in the occurrence of staphylococcal infections leads to the need for close attention to them. Special control is required over the sensitivity of bacteria to antibiotics and the spread of methicillin resistance, as a sign of multiple resistance to antibacterial drugs. It is also important to identify the virulence factors of coagulase-negative staphylococci, which determine their behavior in the environment.
The aim. To evaluate the sensitivity of strains of coagulase-negative staphylococci to clinically significant antibiotics daptomycin, vancomycin, linezolid and oxacillin and lantibiotic warnerin.
Methods. Determination of the minimal inhibitory concentrations of antibacterial compounds for clinical coagulase-negative staphylococci by standard methods of serial dilutions and disc diffusion. Identification of the phenomenon of decreased susceptibility of bacteria to vancomycin by population analysis and concentration gradient. Lipid analysis by thin layer chromatography.
Results. High antibacterial activity of vancomycin, daptomycin and linezolid against clinical strains of coagulase-negative staphylococci was shown. The upper limit of the minimum inhibitory concentrations of vancomycin within the sensitive phenotype and the expansion of the ranges of the minimum inhibitory concentrations of daptomycin and warnerin towards an increase in oxacillin-resistant isolates were revealed. The heterogeneous nature of sensitivity to vancomycin of the cultures of the studied strains and the possibility of their rapid enrichment with subpopulations with reduced sensitivity to this antibiotic have been established. The selection of resistance of coagulase-negative staphylococci to vancomycin was accompanied by an increase in the synthesis of lysylphosphatidylglycerol and a decrease in their sensitivity to cationic peptide compounds.
Conclusion. The revealed prevalence of the methicillin-resistant phenotype of clinical strains of coagulase-negative staphylococci, along with the presence in the lipid spectrum of the universal factor of resistance to cationic antibacterial compounds, lysylphosphatidylglycerol, entails the need for new methodological solutions for diagnosing infections caused by coagulase-negative staphylococci.

1. Dekhnich AV, Nikulin AA, Rjabkova EL, Krechikova OI, Suhorukova MV, Kozlov RS, et al. Epidemiology of antimicrobial resistance of S. aureus isolated from ICU patients in Russia: Results of prospective multicenter study. Clinical Microbiology and Antimicrobial Chemotherapy. 2008; 10(4): 333-344. (In Russ.).

2. Romanov АV, Dekhnich АV, Sukhorukova МV, Skleenova ЕYu, Ivanchik NV, Edelstein МV, et al. Antimicrobial resistance of nosocomial Staphylococcus aureus isolates in Russia: Results of multicenter epidemiological study «MARATHON» 2013-2014. Clinical Microbiology and Antimicrobial Chemotherapy. 2017; 19(1): 57-62. (In Russ.).

3. Zaitsev AA, Karpov OI, Sidorenko SV. Staphylococci and vancomycin: Trends in confrontation. Antibiotics and Chemotherapy. 2003; 48(6): 20-26. (In Russ.).

4. Hiramatsu K, Aritaka N, Hanaki H, Kawasaki S, Hosoda Y, Hori S, et al. Dissemination in Japanese hospitals of strains of Staphylococcus aureus heterogeneously resistant to vancomycin. Lancet. 1997; 350(9092): 1670-1673. doi: 10.1016/S0140-6736(97)07324-8

5. Center KJ, Reboli AC, Hubler R, Rodgers GL, Long SS. Decreased vancomycin susceptibility of coagulase-negative staphylococci in a neonatal intensive care unit: evidence of spread of Staphylococcus warneri. J Clin Microbiol. 2003; 41(10): 4660-4665. doi: 10.1128/JCM.41.10.4660-4665.2003

6. Cremniter J, Slassi A, Quincampoix JC, Sivadon-Tardy V, Bauer T, Porcher R, et al. Decreased susceptibility to teicoplanin and vancomycin in coagulase-negative staphylococci isolated from orthopedic-device-associated infections. J Clin Microbiol. 2010; 48(4): 1428-14231. doi: 10.1128/JCM.02098-09

7. Jones T, Yeaman MR, Sakoulas G, Yang SJ, Proctor RA, Sahl HG, et al. Failures in clinical treatment of Staphylococcus aureus infection with daptomycin are associated with alterations in surface charge, membrane phospholipid asymmetry, and drug binding. Antimicrob Agents Chemother. 2008; 52(1): 269-278. doi: 10.1128/AAC.00719-07

8. Oku Y, Kurokawa K, Ichihashi N, Sekimizu K. Characterization of the Staphylococcus aureus mprF gene, involved in lysinylation of phosphatidylglycerol. Microbiology (Reading). 2004; 150(1): 45-51. doi: 10.1099/mic.0.26706-0

9. World Health Organization, Antimicrobial Resistance Division. 2020 antibacterial agents in clinical and preclinical development: An overview and analysis. Geneva, Switzerland; 2021. URL: https://www.who.int/publications/i/item/9789240021303 [date of access: 11.05.2022].

10. Sabirova EV, Gordinskaya NA, Abramova NV, Nekaeva ES. Antimicrobial resistance of nosocomial staphylococci isolated from burn center patients in 2002-2008. Clinical Microbiology and Antimicrobial Chemotherapy. 2010; 12(1): 77-81. (In Russ.).

11. Becker K, Heilmann C, Peters G. Coagulase-negative staphylococci. Clin Microbiol Rev. 2014; 27(4): 870-926. doi: 10.1128/CMR.00109-13

12. Clinical and laboratory standards institute (CLSI). Performance standards for antimicrobial susceptibility testing; twentyfourth informational supplement. 2014: 34(1).

13. Polyudova TV, Lemkina LM, Korobov VP, Likhatskaya GN. Optimization of production conditions and 3D-structure modeling of novel antibacterial peptide of lantibiotic family. Prikladnaya biokhimiya i mikrobiologiya. 2017; 53(1): 40-46. (In Russ.). doi: 10.1134/S0003683817010148

14. Kononova LI, Korobov VP. Physiological properties of the vancomycin-resistant strain Staphylococcus epidermidis 33 GISK VANR. Microbiology (Mikrobiologiya). 2015; 84(1): 41-48. (In Russ.). doi: 10.7868/S0026365615010061

15. Korobov VP, Lemkina LM, Polyudova TV, Akimenko VK. Isolation and characterization of a new low-molecular antibacterial peptide of the lantibiotics family. Microbiology (Mikrobiologiya). 2010; 79(2): 206-215. (In Russ.).

16. Korobov VP, Lemkina LM, Polyudova TV. Broad spectrum antibacterial peptide hominin KLP-1: Patent N 2528055 of the Russian Federation. 2014; (25). (In Russ.).

17. Clinical and laboratory standards institute (CLSI). Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. Approved standard; 9th edition. 2012: 32(2).

18. Wootton M, Howe RA, Hillman R, Walsh TR, Bennett PM, MacGowan AP. A modified population analysis profile (PAP) method to detect hetero-resistance to vancomycin in Staphylococcus aureus in a UK hospital. J Antimicrob Chemother. 2001; 47(4): 399-403. doi: 10.1093/jac/47.4.399

19. Hanaki H, Hiramatsu K. Detection methods of glycopeptideresistant Staphylococcus aureus I: Susceptibility testing. Antibiotic Resistance. Methods in Molecular Medicine™. Ed. SH Gillespie. Humana Press; 2001; 48: 85-92. doi: 10.1385/1-59259-077-2:85

20. Sieradzki K, Roberts RB, Haber SW, Tomasz A. The development of vancomycin resistance in a patient with methicillinresistant Staphylococcus aureus infection. New Engl J Med. 1999; 340(7): 517-523. doi: 10.1056/NEJM199902183400704

21. Bligh EG, Dyer WJ. A rapid method of total lipid extraction and purification. Can J Biochem Physiol. 1959; 37(8): 911-917. doi: 10.1139/o59-099

22. Fewster ME, Burns BJ, Mead JF. Quantitative densitometric thin-layer chromatography of lipids using copper acetate reagent. J Chromatogr. 1969; 43(1): 120-126. doi: 10.1016/s0021-9673(00)99173-8

23. Kates M. Techniques of lipidology. Isolation, analysis and identification of lipids. Moscow: Mir Piblishing House; 1975. (In Russ.).

24. Granichnaya NV, Zaytseva EA, Perelomova OV. Resistance of coagulase negative staphylococci recovered from different biomaterials in cardiac patients. Pacific Medical Journal. 2019; 2(76): 38-42. (In Russ.). doi: 10.17238/PmJ1609-1175.2019.2.28-42

25. Kelley PG, Gao W, Ward PB, Howden BP. Daptomycin nonsusceptibility in vancomycin-intermediate Staphylococcus aureus (VISA) and heterogeneous-VISA (hVISA): Implications for therapy after vancomycin treatment failure. J Antimicrob Chemother. 2011; 66(5): 1057-1060. doi: 10.1093/jac/dkr066

26. Howden BP, Johnson PDR, Ward PB, Stinear TP, Davies JK. Isolates with low-level vancomycin resistance associated with persistent methicillin-resistant Staphylococcus aureus bacteremia. Antimicrob Agents Chemother. 2006; 50(9): 3039-3047. doi: 10.1128/AAC.00422-06

27. Pinheiro L, Brito CI, Pereira VC, de Oliveira A, Camargo CH, Cunha M de L. Reduced susceptibility to vancomycin and biofilm formation in methicillin-resistant Staphylococcus epidermidis isolated from blood cultures. Mem Inst Oswaldo Cruz. 2014; 109(7): 871-878. doi: 10.1590/0074-0276140120

28. Barber KE, Werth BJ, Ireland CE, Stone NE, Nonejuie P, Sakoulas G, et al. Potent synergy of ceftobiprole plus daptomycin against multiple strains of Staphylococcus aureus with various resistance phenotypes. J Antimicrob Chemother. 2014; 69(11): 3006-3010. doi: 10.1093/jac/dku236

29. Chopra L, Singh G, Taggar R, Dwivedi A, Nandal J, Kumar P, et al. Antimicrobial peptides from bacterial origin: Potential alternative to conventional antibiotics. High Value Fermentation Products; ed. by S. Saran, V. Babu, A. Chuabey. Scrivener; 2019: 193-204. doi: 10.1002/9781119460053.ch8

30. Werth BJ, Vidaillac C, Murray KP, Newton KL, Sakoulas G, Nonejuie P, et al. Novel combinations of vancomycin plus ceftaroline or oxacillin against methicillin-resistant vancomycin-intermediate Staphylococcus aureus (VISA) and heterogeneous VISA. Antimicrob Agents Chemother. 2013; 57(5): 2376-2379. doi: 10.1128/AAC.02354-12

31. Mun S-H, Kang O-H, Joung D-K, Kim S-B, Choi J-G, Shin D-W, et al. In vitro anti-MRSA activity of carvone with gentamicin. Exp Ther Med. 2014; 7(4): 891-896. doi: 10.3892/etm.2014.1498

32. Ernst CM, Peschel A. Broad-spectrum antimicrobial peptide resistance by MprF-mediated aminoacylation and flipping of phospholipids. Mol Microbiol. 2011; 80(2): 290-299. doi: 10.1111/j.1365-2958.2011.07576.x

33. Geiger O, González-Silva N, López-Lara IM, Sohlenkamp C. Amino acid-containing membrane lipids in bacteria. Prog Lipid Res. 2010; 49(1): 46-60. doi: 10.1016/j.plipres.2009.08.002

34. Peschel A, Jack RW, Otto M, Collins LV, Staubitz P, Nicholson G, et al. Staphylococcus aureus resistance to human defensins and evasion of neutrophil killing via the novel virulence factor MprF is based on modification of membrane lipids with l-lysine. J Exp Med. 2001; 193(9): 1067-1076. doi: 10.1084/jem.193.9.1067

35. Satola SW, Farley MM, Anderson KF, Patel JB. Comparison of detection methods for heteroresistant vancomycin-intermediate Staphylococcus aureus, with the population analysis profile method as the reference method. J Clin Microbiol. 2011; 49(1): 177-183. doi: 10.1128/JCM.01128-10

36. Sieradzki K, Villari P, Tomasz A. Decreased susceptibilities to teicoplanin and vancomycin among coagulase-negative methicillin-resistant clinical isolates of staphylococci. Antimicrob Agents Chemother. 1998; 42(1): 100-107. doi: 10.1128/AAC.42.1.100

37. Walsh TR, Bolmstrom A, Qwarnstrom A, Ho P, Wootton M, Howe RA, et al. Evaluation of current methods for detection of staphylococci with reduced susceptibility to glycopeptides. J Clin Microbiol. 2001; 39(7): 2439-2444. doi: 10.1128/JCM.39.7.2439-2444.2001

38. Lulitanond A, Chanawong A, Sribenjalux P, Kaewkes W, Vorachit M, Chongtrakool P, et al. Detection of heterogeneous, intermediate-vancomycin-resistant Staphylococcus aureus (hVISA) using low-concentration vancomycin disks. Southeast Asian J Trop Med Public Health. 2006; 37(4): 761-767.

39. Cafiso V, Bertuccio T, Spina D, Purrello S, Campanile F, Di Pietro C, et al. Modulating activity of vancomycin and daptomycin on the expression of autolysis cell-wall turnover and membrane charge genes in hVISA and VISA strains. PLoS One. 2012; 7(1): e29573. doi: 10.1371/journal.pone.0029573