Identification of Clinical Isolate CCGC 19/16 as Bacillus cytotoxicus

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Resumo

Bacillus cereus sensu lato s.l. comprises genetically, morphologically and physiologically similar gram-positive spore-forming bacterial species with high pathogenic potential, such as B. anthracis, B. cereus and B. thuringiensis. Toxin-producing strains of B. cereus s.l. pose a major threat to human health. The high degree of similarity between these species makes it very difficult to identify them and to take adequate measures to treat the diseases they cause. Previously, we characterized a clinical isolate CCGC 19/16 belonging to B. cereus s.l. that exhibited features of both B. cereus and B. cytotoxicus. In the present work, CCGC 19/16 was identified as B. cytotoxicus using multilocus sequence typing (MLST) and mass spectrometric analysis. It was also shown that, unlike other representatives of the B. cytotoxicus species, strain CCGC 19/16 is not thermotolerant. Unlike B. cereus, strain CCGC 19/16 is sensitive to most antibiotics and shows increased motility. Like B. cereus strain CCGC 19/16 forms β-hemolysis zones in blood agar. In addition, it has been shown that prolonged storage of samples prior to analysis can lead to misidentification of the isolate. Our results indicate that “rapid methods” of analysis using single genes have insufficient resolving power in the identification of B. cereus s.l. species. The combination of MLST analysis with MALDI-TOF MS provides sufficient resolution.

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Sobre autores

N. Polyakov

Gamaleya National Research Centre for Epidemiology and Microbiology; Vernadsky Institute of Geochemistry and Analytical Chemistry of the Russian Academy of Sciences

Email: mzubasheva@mail.ru
Rússia, Moscow, 123098; Moscow, 119334

D. Karpov

Engelhardt Institute of Molecular Biology of the Russian Academy of Sciences

Email: mzubasheva@mail.ru
Rússia, Moscow, 119991

M. Zubasheva

Gamaleya National Research Centre for Epidemiology and Microbiology

Autor responsável pela correspondência
Email: mzubasheva@mail.ru
Rússia, Moscow, 123098

A. Polyakova

Lomonosov Moscow State University

Email: mzubasheva@mail.ru
Rússia, Moscow, 119234

D. Shcherbinin

Gamaleya National Research Centre for Epidemiology and Microbiology

Email: mzubasheva@mail.ru
Rússia, Moscow, 123098

A. Solovyev

Gamaleya National Research Centre for Epidemiology and Microbiology

Email: mzubasheva@mail.ru
Rússia, Moscow, 123098

M. Lavrentyev

Gamaleya National Research Centre for Epidemiology and Microbiology

Email: mzubasheva@mail.ru
Rússia, Moscow, 123098

T. Smirnova

Gamaleya National Research Centre for Epidemiology and Microbiology

Email: mzubasheva@mail.ru
Rússia, Moscow, 123098

M. Sukhina

Ryzhikh State Research Center of Coloproctology

Email: mzubasheva@mail.ru
Rússia, Moscow, 123423

V. Zhukhovitsky

Gamaleya National Research Centre for Epidemiology and Microbiology; Russian Medical Academy of Continuing Professional Education

Email: mzubasheva@mail.ru
Rússia, Moscow, 123098; Moscow, 125993

Bibliografia

  1. Liu Y., Du J., Lai Q., Zeng R., Ye D., Xu J., Shao Z. (2017) Proposal of nine novel species of the Bacillus cereus group. Int. J. Syst. Evol. Microbiol. 67(8), 2499–2508.
  2. Miyata J., Tasaka S., Miyazaki M., Yoshida S., Naoki K., Sayama K., Asano K., Fujiwara H., Ohkusu K., Hasegawa N., Betsuyaku T. (2013) Bacillus cereus necrotizing pneumonia in a patient with nephrotic syndrome. Int. Med. 52(1), 101–104.
  3. Worapongsatitaya P.T., Pupaibool J. (2022) Bacillus cereus meningoencephalitis in an immunocompetent patient. IDCases. 29, e01577.
  4. Inoue D., Nagai Y., Mori M., Nagano S., Takiuchi Y., Arima H., Kimura T., Shimoji S., Togami K., Tabata S., Yanagita S., Matsushita A., Nagai K., Imai Y., Takegawa H., Takahashi T. (2010) Fulminant sepsis caused by Bacillus cereus in patients with hematologic malignancies: analysis of its prognosis and risk factors. Leuk. Lymphoma. 51(5), 860–869.
  5. Ikeda M., Yagihara Y., Tatsuno K., Okazaki M., Okugawa S., Moriya K. (2015) Clinical characteristics and antimicrobial susceptibility of Bacillus cereus blood stream infections. Ann. Clin. Microbiol. Antimicrob. 14, 43.
  6. Guinebretiere M.H., Auger S., Galleron N., Contzen M., De Sarrau B., De Buyser M. L., Lamberet G., Fagerlund A., Granum P. E., Lereclus D., De Vos P., Nguyen-The C., Sorokin A. (2013) Bacillus cytotoxicus sp. nov. is a novel thermotolerant species of the Bacillus cereus group occasionally associated with food poisoning. Int. J. Syst. Evol. Microbiol. 63(Pt 1), 31–40.
  7. Liu Y., Lai Q., Goker M., Meier-Kolthoff J.P., Wang M., Sun Y., Wang L., Shao Z. (2015) Genomic insights into the taxonomic status of the Bacillus cereus group. Sci. Rep. 5, 14082.
  8. Carroll L.M., Cheng R. A., Wiedmann M., Kovac J. (2022) Keeping up with the Bacillus cereus group: taxonomy through the genomics era and beyond. Crit. Rev. Food Sci. Nutr. 62(28), 7677–7702.
  9. Schoch C.L., Ciufo S., Domrachev M., Hotton C. L., Kannan S., Khovanskaya R., Leipe D., McVeigh R., O’Neill K., Robbertse B., Sharma S., Soussov V., Sullivan J. P., Sun L., Turner S., Karsch-Mizrachi I. (2020) NCBI Taxonomy: a comprehensive update on curation, resources and tools. Database (Oxford). 2020, baaa062.
  10. Daffonchio D., Raddadi N., Merabishvili M., Cherif A., Carmagnola L., Brusetti L., Rizzi A., Chanishvili N., Visca P., Sharp R., Borin S. (2006) Strategy for identification of Bacillus cereus and Bacillus thuringiensis strains closely related to Bacillus anthracis. Appl. Environ. Microbiol. 72(2), 1295–1301.
  11. Chelliah R., Wei S., Park B. J., Kim S. H., Park D. S., Kim S. H., Hwan K. S., Oh D. H. (2017) Novel motB as a potential predictive tool for identification of B. cereus, B. thuringiensis and differentiation from other Bacillus species by triplex real-time PCR. Microb. Pathog. 111, 22–27.
  12. Olsen J.S., Skogan G., Fykse E. M., Rawlinson E. L., Tomaso H., Granum P. E., Blatny J. M. (2007) Genetic distribution of 295 Bacillus cereus group members based on adk-screening in combination with MLST (Multilocus Sequence Typing) used for validating a primer targeting a chromosomal locus in B. anthracis. J. Microbiol. Meth. 71(3), 265–274.
  13. Clark A.E., Kaleta E. J., Arora A., Wolk D. M. (2013) Matrix-assisted laser desorption ionization-time of flight mass spectrometry: a fundamental shift in the routine practice of clinical microbiology. Clin. Microbiol. Rev. 26(3), 547–603.
  14. Cairo J., Gherman I., Day A., Cook P. E. (2022) Bacillus cytotoxicus — a potentially virulent food-associated microbe. J. Appl. Microbiol. 132(1), 31–40.
  15. Morgulis A., Coulouris G., Raytselis Y., Madden T. L., Agarwala R., Schaffer A. A. (2008) Database indexing for production MegaBLAST searches. Bioinformatics. 24(16), 1757–1764.
  16. Jolley K.A., Bray J. E., Maiden M. C.J. (2018) Open-access bacterial population genomics: BIGSdb software, the PubMLST.org website and their applications. Wellcome Open Res. 3, 124.
  17. Liang Q., Liu C., Xu R., Song M., Zhou Z., Li H., Dai W., Yang M., Yu Y., Chen H. (2021) fIDBAC: a platform for fast bacterial genome identification and typing. Front. Microbiol. 12, 723577.
  18. Beaman T.C., Gerhardt P. (1986) Heat resistance of bacterial spores correlated with protoplast dehydration, mineralization, and thermal adaptation. Appl. Environ. Microbiol. 52(6), 1242–1246.
  19. Sauer S., Freiwald A., Maier T., Kube M., Reinhardt R., Kostrzewa M., Geider K. (2008) Classification and identification of bacteria by mass spectrometry and computational analysis. PLoS One. 3(7), e2843.
  20. Смирнова Т. А., Сухина М. А., Гречников А.А., Поддубко С.В., Зубашева М.В., Грумов Д.А., Богданов И.А., Переборова А. А., Козлова В.А., Плиева З.С., Щербинин Д. Н., Андреевская С.Г., Шевлягина Н.В., Соловьев А.И., Карпов Д.С., Поляков Н.Б., Жуховицкий В.Г. (2023) Идентификация клинических изолятов группы Bacillus cereus и их характеристика методами масс-спектрометрии и электронной микроскопии. Молекуляр. биология. 57(4), 609–622.
  21. Shu L.J., Yang Y.L. (2017) Bacillus classification based on matrix-assisted laser desorption ionization time-of-flight mass spectrometry — effects of culture сonditions. Sci. Rep. 7(1), 15546.
  22. Kim K., Seo J., Wheeler K., Park C., Kim D., Park S., Kim W., Chung S. I., Leighton T. (2005) Rapid genotypic detection of Bacillus anthracis and the Bacillus cereus group by multiplex real-time PCR melting curve analysis. FEMS Immunol. Med. Microbiol. 43(2), 301–310.
  23. Hsieh Y.M., Sheu S. J., Chen Y.L., Tsen H.Y. (1999) Enterotoxigenic profiles and polymerase chain reaction detection of Bacillus cereus group cells and B. cereus strains from foods and food-borne outbreaks. J. Appl. Microbiol. 87(4), 481–490.
  24. Oliwa-Stasiak K., Molnar C.I., Arshak K., Bartoszcze M., Adley C.C. (2010) Development of a PCR assay for identification of the Bacillus cereus group species. J. Appl. Microbiol. 108(1), 266–273.
  25. Ehling-Schulz M., Svensson B., Guinebretiere M.H., Lindback T., Andersson M., Schulz A., Fricker M., Christiansson A., Granum P.E., Martlbauer E., Nguyen-The C., Salkinoja-Salonen M., Scherer S. (2005) Emetic toxin formation of Bacillus cereus is restricted to a single evolutionary lineage of closely related strains. Microbiology (Reading). 151(1), 183–197.
  26. Parte A.C., Sarda Carbasse J., Meier-Kolthoff J.P., Reimer L.C., Goker M. (2020) List of prokaryotic names with standing in nomenclature (LPSN) moves to the DSMZ. Int. J. Syst. Evol. Microbiol. 70(11), 5607–5612.
  27. Riley E.P., Schwarz C., Derman A.I., Lopez-Garrido J. (2020) Milestones in Bacillus subtilis sporulation research. Microb. Cell. 8(1), 1–16.
  28. Klee S.R., Brzuszkiewicz E.B., Nattermann H., Bruggemann H., Dupke S., Wollherr A., Franz T., Pauli G., Appel B., Liebl W., Couacy-Hymann E., Boesch C., Meyer F.D., Leendertz F.H., Ellerbrok H., Gottschalk G., Grunow R., Liesegang H. (2010) The genome of a Bacillus isolate causing anthrax in chimpanzees combines chromosomal properties of B. cereus with B. anthracis virulence plasmids. PLoS One. 5(7), e10986.
  29. Wilson M.K., Vergis J.M., Alem F., Palmer J.R., Keane-Myers A.M., Brahmbhatt T.N., Ventura C.L., O’Brien A.D. (2011) Bacillus cereus G9241 makes anthrax toxin and capsule like highly virulent B. anthracis Ames but behaves like attenuated toxigenic nonencapsulated B. anthracis Sterne in rabbits and mice. Infect. Immun. 79(8), 3012–3019.
  30. Apriliana U., Wibawa H., Ruhiat E., Untari T., Indarjulianto S. (2021) Isolation and identification of avirulent strains of Bacillus anthracis from environmental samples in Central Java, Indonesia. Int. J. One Hlth. 7(2), 204–211.
  31. Okinaka R.T., Price E.P., Wolken S.R., Gruendike J.M., Chung W.K., Pearson T., Xie G., Munk C., Hill K.K., Challacombe J., Ivins B.E., Schupp J.M., Beckstrom-Sternberg S.M., Friedlander A., Keim P. (2011) An attenuated strain of Bacillus anthracis (CDC684) has a large chromosomal inversion and altered growth kinetics. BMC Genomics. 12, 477.

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2. Fig. 1. Dendrograms of similarity of mass spectra of clinical isolates with the spectra of B. cereus and B. cytotoxicus in the MALDI biotyper reference database depending on the duration of incubation of cultures of clinical isolates in a liquid nutrient medium (strain B. cereus CCGC1208 (clinical isolate) and collector strain B. cereus ATCC10876 were described in detail earlier in [20]).

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3. Fig. 2. Strain similarity tree based on pairwise alignments of the search results for the combined gene sequence glpF-gmk-ilvD-pta-pur-pycA-tpi in the NCBI database. The tree was built using the Fast Minimum Evolution method with default parameters.

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4. Fig. 3. Assessment of the thermal stability of clinical isolates and reference strains from the B. cereus group. a is the relative survival curve of the spores. The number of viable spores at the starting point is assumed to be 100%. b — The values of D100 calculated from the data presented in panel A. The D100 value is the spore processing time (minutes) at 100°C, during which the number of viable spores is reduced by 10 times. The data spread is represented by the standard deviation for the three biological repeats. Statistical significance: NS is an insignificant difference, *0.05 > p > 0.01 in accordance with the Student's t-test.

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5. Fig. 4. Assessment of the mobility of B. cereus ATCC 10702 and CCGC19/16. a — The diameter of colonies of strains after 24 hours of incubation; b — the diameter of colonies after 48 hours of incubation.

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6. Fig. 5. Estimation of the size of hemolysis zones in B. cereus group strains. a is Strain B. cereus ATCC10702, b is Strain CCGC19/16.

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