Cover Page

Cite item


Abstract. Currently, a search for augmenting antibiotics activity is still crucial due to elevated frequency of detecting carbapenem-resistant Gran-positive bacterial isolates. To resolve this, it might be reasonable to combine carbapenems metal-â-lactamase (MâL) inhibitors. Unfortunately, no MâL inhibitors approved for treatment of carbapenem-resistant infections are currently available. Pathogenic bacteria may survive antibiotic attack, exert tolerance and persistence accompanied with the ongoing infectious process. In connection with this, determining dependence between antimicrobialrelated bactericidal effect and exposure time on microbes at 4, 8, 12 and 24 hours after the onset, a so called time-kill assay, is necessary. A synergy between both agents was noted upon reduced microbial population by ≥ 3 log10. A checkerboard array followed by seeding the microplate well contents onto a dense nutrient medium at various time points were used to assess a synergistic efficacy of carbapenems applied together with clodronic acid against MâL-producing VIMgenotype P. aeruginosa 532/14 clinical isolate obtained from patients with infectious complications (minimal inhibitory concentrations [MIC] for imipenem or meropenem were 512 μg/ml), microbial burden 106 CFU/ml. Optical density was measured at two wavelengths (490 and 630 nm) in ELx800 reader, within 4–24 hour exposure time to determine time of logarithmic growth phase emerging in test culture. It is noteworthy that magnitude of optical density is a difference between two bichromatic measurements resulting in remarkably reduced inaccuracy due to scratches or fingerprints left on the plate. It was found that clodronic acid exhibited a synergic bactericidal effect with carbapenems against a clinically resistant MâL-producing VIM-genotype P. aeruginosa 532/14 strain. Upon that, imipenem-related antimicrobial activity was evident as early as 8 hours after the onset decreasing cell growth down to 1.4 log10 compared to control, whereas 12 hours later it resulted in total inhibition of test strain by decreasing growth of the test strain by 6 log10. Meropenem in combination with clodronic acid showed a more pronounced activity: complete absence of P. aeruginosa 532/14 growth by 8 hours of incubation, growth suppression by 3.2 log10, which reached 6 log10 12–24 hours after the onset. Time-kill assay allows to identify efficient combinations of carbapenems and MâL inhibitors, which is of great importance for increasing therapeutic efficacy of patients with severe purulent-septic complications.

About the authors

A. G. Afinogenova

St. Petersburg Pasteur Institute, St. Petersburg; St. Petersburg State University, St. Petersburg

Author for correspondence.
Email: spbtestcenter@mail.ru

PhD, MD (Biology), Head of Laboratory Testing Centre;

Professor of Surgical Dentistry Department 

197101, Russian Federation, St. Petersburg, Mira str., 14
Phone: +7 (812) 232-86-31 (office); +7 921 557-88-94 (mobile)

Russian Federation

T. M. Voroshilova

Nikiforov Russian Center of Emergency and Radiation Medicine, St. Petersburg

Email: fake@neicon.ru
Bacteriologist, Head of Bacterial Laboratory Russian Federation

G. E. Afinogenov

St. Petersburg State University, St. Petersburg

Email: fake@neicon.ru
PhD, MD (Medicine), Professor of Surgical Dentistry Department Russian Federation

D. Yu. Maday

St. Petersburg State University, St. Petersburg

Email: fake@neicon.ru
PhD, MD (Medicine), Professor, Head of Surgical Dentistry Department Russian Federation

A. A. Spiridonova

Pavlov First St. Petersburg State Medical University, St. Petersburg

Email: fake@neicon.ru
Head of Clinical Microbiology Department Russian Federation


  1. Афиногенова А.Г., Ворошилова Т.М., Афиногенов Г.Е., Мадай Д.Ю. Оценка эффективности нового ингибитора металло-бета-лактамазы в условиях модельной системы in vitro // Инфекция и иммунитет. 2016. Т. 6, № 4. С. 335– 344. [Afinogenova A.G., Voroshilova T.M., Afinogenov G.E., Maday D.Y. The new metall-beta-lactamase’s inhibitor efficacy in a model system in vitro. Infektsiya i immunitet = Russian Journal of Infection and Immunity, 2016, vol. 6, no. 4, pp. 335–344. doi: 10.15789/2220-7619-2016-4-335-344 (In Russ.)]
  2. Афиногенова А.Г., Ворошилова Т.М., Афиногенов Г.Е., Родионов Г.Г. «Метод шахматной доски» как тест для оценки снижения уровня резистентности грамотрицательных микроорганизмов к карбапенемам в присутствии бисфосфоната // Клиническая микробиология и антимикробная химиотерапия. 2015. Т. 17, № 1. С. 24–32. [Afinogenova A.G., Voroshilova T.M., Afinogenov G.E., Rodionov G.G. «Checkerboard array» as a test for evaluation of decrease of microbial resistance decrease to capbapenems in the presence of bisphosphonate. Klinicheskaya mikrobiologiya i antimikrobnaya khimio terapiya = Clinical Microbiology and Antimicrobial Chemotherapy, 2015, vol. 17, no. 1, pp. 24–32. (In Russ.)]
  3. Борисова М.И., Лазакович Д.Н., Сидорова Н.А., Савушкин А.И. Биопленкообразующая активность и феномен персистенции микроорганизмов // Journal of Biomedical Technologies. 2015. № 2. С. 28–35. [Borisova M.I., Lazakovich D.N., Sidorova N.A., Savushkin A.I. Biofilm-forming activity and the phenomenon of persistense in microorganisms. Journal of Biomedical Technologies, 2015, no. 2, pp. 28–35. doi: 10.15393/j6.art.2015.3382 (In Russ.)]
  4. Бухарин О.В. Персистенция бактериальных патогенов как физиологический феномен // Вестник Московского Университета. Серия 16. Биология. 2008. № 1. С. 6–13. [Bukharin O.V. Persistence of bacterial pathogens as a physiological phenomenon. Vestnik Moskovskogo universiteta. Seriya 16. Biologiya = Herald of the Moscow State University. Series 16. Biology, 2008, no. 1, pp. 6–13. (In Russ.)]
  5. Определение чувствительности микроорганизмов к антимикробным препаратам: методические указания. М.: Федеральный центр госсанэпиднадзора Минздрава России, 2004. 91 с. [Determination of the sensitivity of microorganisms to antimicrobial drugs: methodological guidelines. Moscow: Federal Center for State Sanitary and Epidemiological Supervision of the Russian Ministry of Health, 2004, 91 p.]. URL: http://fcgie.ru/download/elektronnaya_baza_metod_dokum/muk_1890-04.pdf (19.10.2018)]
  6. Поляк М.С. Лабораторное обеспечение антибиотикотерапии. СПб.: ООО «Анатолия», 2012. 256 с. [Polyak M.S. Laboratornoe obespechenie antibiotikoterapii [Laboratory provision of antibiotic therapy]. SPb: LLC “Anatoliya”, 2012. 256 p.]
  7. Aiyegoro O.A., Afolayan A.J., Okoh A.I. In vitro time kill assessment of crude methanol extract of Helichrysum pedunculatum leaves. African J. Biotechnol., 2008, vol. 7, no. 11, pp. 1684–1688. doi: 10.5897/AJB08.055
  8. Basri D.F., Khairon R. Pharmacodynamic interaction of Quercus infectoria galls extract in combination with vancomycin against MRSA using microdilution checkerboard and time-kill assay. Evid. Based Complement. Alternat. Med., 2012, vol. 2012: 493156. doi: 10.1155/2012/493156
  9. Bedenic B., Vraneš J., Sviben M., Beader N., Kalenić S. Postantibiotic and post-beta-lactamase inhibitor effect of carbapenems combined with EDTA against Pseudomonas aeruginosa strains producing VIM-metallo beta-lactamases. Chemotherapy, 2008, vol. 54, pp. 188–193. doi: 10.1159/000140461
  10. Bhardwaj M., Singh B.R., Sinha D.K., Kumar V., Prasanna Vadhana O.R., Varan Singh S., Nirupama K.R., Pruthvishree and Archana Saraf B.S. Potential of herbal drug and antibiotic combination therapy: a new approach to treat multidrug resistant bacteria. Pharm. Anal. Acta, 2016, vol. 7, no. 11. doi: 10.4172/2153-2435.1000523
  11. Drawz S.M., Bonomo R.A. Three decades of β-lactamase inhibitors. Clin. Microb. Rev., 2010, vol. 23, no. 1, pp. 160–201. doi: 10.1128/CMR.00037-09
  12. Eliopoulos G.M., Moellering R.C. Antimicrobial combinations. In: Antibiotics in Laboratory Medicine; 4th edition. Ed. V. Lorian. Baltimore, MD, USA: Williams & Wilkins, 1996, pp. 330–396.
  13. Leber A. Synergism testing: broth microdilution checkerboard and broth macrodilution methods. In: Clinical Microbiology Procedures Handbook; 4th edition. Washington, DC: ASM Press, 2016, pp. 5.16.1-5.16.23. doi: 10.1128/9781555818814.ch5.16
  14. Rani R., Sharma D., Chaturvedi M., Yadav J.P. Antibacterial activity of twenty different endophytic fungi isolated from Calotropis procera and time kill assay. Clin. Microbiol., 2017, vol. 6, no. 3. doi: 10.4172/2327-5073.1000280

Copyright (c) 2018 Afinogenova A.G., Voroshilova T.M., Afinogenov G.E., Maday D.Y., Spiridonova A.A.

Creative Commons License
This work is licensed under a Creative Commons Attribution 4.0 International License.

СМИ зарегистрировано Федеральной службой по надзору в сфере связи, информационных технологий и массовых коммуникаций (Роскомнадзор).
Регистрационный номер и дата принятия решения о регистрации СМИ: серия ПИ № ФС 77 - 64788 от 02.02.2016.

This website uses cookies

You consent to our cookies if you continue to use our website.

About Cookies