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ECPB 2018, 84(4): 68–73
https://doi.org/10.25040/ecpb2018.04.068
Research articles

Changes of Cultural and Morphological Features and the Formation of the Antibiotic Resistance in Corynebacterium Diphtheriae Induced by Subinhibitory Concentrations of Penicillin

O. MOTYKA1, K. KAPUSTIAK2, O. KORNIYCHUK1
Abstract

The influence of subminimal inhibitory concentrations (sub-MIC) of benzylpenicillin on antibiotic susceptibility levels, the cultural-morphological and biochemical properties of C. diphtheriae have been investigated. Two C. diphtheriae strains (biovariants gravis and belfanti) were used. The MICs and the minimal inhibitory concentrations (MBCs) of penicillin have been determined by using the standard broth dilution method (macromethod). The MICs were registered after 24 and 48 years of growth. Initially, both strains were susceptible to the bacteriostatic and bactericidal action of penicillin: MICs were 0.05 and 0.13 mg/L, MBCs - 1.0 and 2.0 mg/L. After passing the broth with sub-MIC penicillin, the increase of MICs and MBCs was observed in both strains. The maximum lin- tolerance was developed (MBC/MIC = 128). Corynebacteria that had lost penicillin-susceptibility exhibited typical phenotypic properties which are required for the bacteriological identification of this pathogen. The penicillin-resistance culture of corynebacteria began growing slowly, demonstrating the delayed fermentation or did not show the ability to utilize saccharides. At the same time, the ability of toxin production was maintained. The ability of penicillin-susceptibility and penicillin-tolerance of C. diphtheriae to the viability in the presence of high antibiotic concentrations (128.0 - > 256.0 mg/L) was tested. After 48-hour presence in high concentrations of penicillin in cultures of susceptible strains, the viability has maintained in no more than 0.001 % of cells. In cultures of corynebacteria with acquired tolerance, the percentage of living cells was more than 10 % of the total number before the end of the exposure period; the ability to colonize in the blood agar was retained in about 0.1 % of cells, forming, however, atypical small colonies. Also the formation of sub-populations of non-culturable persister cells was observed in cultures of penicillin-tolerant C. diphtheriae. Significant changes in the cell wall and the intracellular structure were observed in these cells. The recirculation of the C. diphtheriae from persister-cells occurred after their transfer to a fresh serum broth, holding at a temperature of 2-6 °C and the subsequent incubation at 37 °C during 48-96 hours. Corynebacteria, being recultivated from non-culturable forms retained the acquired level of penicillin-resistance (including tolerance), atypical culture-morphological properties and slowed down (during 24-72 h) the manifestation of the basic biochemical properties. Atypical manifestations of biological properties of C. diphtheriae can lead to the complication of indication and identification of the diphtheria causative agent according to the standard pattern, during the examination of patients after antibiotic therapy, in particular. The persister formation is important for C. diphtheriae population strategy in adverse conditions. Recieved: 14.11.2018

Keywords: Corynebacterium diphtheriae, benzylpenicillin, subminimal concentration, cultural and morphological changes, persister

Full text: PDF (Ukr) 259K

References
  1. 1. Efsratiou A, Maple PAC. Manual for the laboratory diagnosis of diphtheria. The expanded programme of immunization in the European region of WHO. ICP/EPI038(C). World Health Organization. Regional Office for Europe. 1994.
  2. 2. Nakaz MOZ Ukrainy No 192 vid 03.08.99 r. "Pro zakhody shchodo pokrashchennia bakteriolohichnoi diahnostyky dyfterii v Ukraini».
  3. 3. Dos Santos LS, Antunes CA, de Oliveira DM, Sant'Anna Lde O, Pereira JA, Hirata Júnior R et al. Tellurite resistance: a putative pitfall in Corynebacterium diphtheriae diagnosis? Antonie van Leeuwenhoek. 2015;108(5):1275-9. doi.org/10.1007/s10482-015-0558-4
  4. 4. Neal SE, Efstratiou A. International external quality assurance for laboratory diagnosis of diphtheria. J Clin Microbiol. 2009 Dec;47(12):4037-42. doi.org/10.1128/JCM.00473-09
  5. 5. Lázaro-Díez M, Remuzgo-Martínez S, Rodríguez-Mirones C, Acosta F, Icardo JM, Martínez- Martínez L et al. Effect of subinhibitory concentrations of ceftaroline on meticillin-resistant Staphylococcus aureus (MRSA) biofilms. PloS One. 2016 Jan;11(1). doi.org/10.1371/journal. pone.0147569
  6. 6. Gomes DL, Peixoto RS, Barbosa EA, Napoleão F, Sabbadini PS, dos Santos KR et al. SubMICs of penicillin and erythromycin enhance biofilm formation and hydrophobicity of Corynebacterium diphtheriae strains. J Med Microbiol. 2013 May; 62(Pt5):754-60.
  7. 7. EUCAST Breakpoints Tables for interpretation of MICs and zone diameters, version 8.0, valid from 2018-01-01.
  8. 8. Von Hunolstein C, Alfarone G, Scopetti F, Pataracchia M, La Valle R, Franchi F et al. Molecular epidemiology and characteristics of Corynebacterium diphtheriae and Corynebacterium ulcerans strains isolated in Italy during the 1990 s. J Med Microbiol. 2003;52(Pt 2):181-8. doi.org/10.1099/jmm.0.04864-0
  9. 9. Reinolds ES. The use of lead citrate at high pH as an electropaque stain in electron microskory. Jorn Cell Biol. 1963;17:208-12. doi.org/10.1083/jcb.17.1.208
  10. 10. Zakikhany K, Efstratiou A. Diphtheria in Europe: current problems and new challenges. Future Microbiol. 2012 May;7(5):595-607. doi.org/10.2217/fmb.12.24
  11. 11. Wagner KS, White JM, Lucenko I, Mercer D, Crowcroft NS, Neal S, et al. Diphtheria in the postepidemic period, Europe, 2000-2009. Emerg Infect Dis. 2012;18(2):217-25.
  12. 12. Rousseau C, Belchior E, Broche B, Badell E, Guiso N, Laharie I et al. Diphtheria in the south of France, March 2011; Euro Surveill. 2011;16(19).
  13. 13. Zasada AA., Baczewska-Rej A, Wardak S. An increase in non-toxigenic Corynebacterium diphtheriae infections in Poland – molecular epidemiology and antimicrobial susceptibility of strains isolated from past outbreaks and those currently circulating in Poland. Int J Infect Dis. 2010 Oct;14(10). doi.org/10.1016/j.ijid.2010.05.013
  14. 14. Lewis K. Persister cells. Ann Rev Microbiol. 2010;64:357-72.
  15. 15. Kwan BW, Valenta JA, Benedik MJ, Wood TK. Arrested protein synthesis increases persister-lice cell formation. Antimicrob Agents Chemother. 2013 Mar;57(3):1468-73. doi.org/10.1128/ AAC.02135-12


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