Biofilm Formation in Bacillus cereus, B. licheniformis and B. pumilus: An Alternative for Survival in Impacted Environments

Author(s): Cícero José Luiz dos Ramos Almeida, Sivoneide Maria da SILVA, Thiago Henrique NAPOLEÃO, Márcia Vanusa da SILVA, Maria Betânia Melo de OLIVEIRA

Bacillus is a genus of Gram-positive and rod-shaped bacteria that may be facultative anaerobes and resistant to stress conditions in the environment. In view of the physiological diversity of the vegetative forms of this genus, the species are considered as ubiquitous microorganisms, being isolated from soil, freshwater and seawater, as well as food. In the present study, isolates of three species (B. cereus, B. licheniformis and B. pumilus), obtained from an impacted environment (Cavouco stream, Recife, Brazil), were investigated for the susceptibility/resistance profile toward antibiotics by antibiogram (disk diffusion), hydrophobicity by the hydrocarbon-binding method, and ability to form biofilm in different culture media by the crystal violet method. B. cereus was the species with the highest resistance (nine antimicrobials), followed by B. pumilus (two) and B. licheniformis (one). The isolates were biofilm formers, with higher formation in media supplemented with 1% glucose, and all were hydrophobic. This work is an indication that B. cereus, B. licheniformis and B. pumilus appear to possess distinct resistance mechanisms that are not directly related to biofilm formation ability. Further studies are needed to understand better the dynamics of survival of these species in impacted environments.

susceptibility, cellular hydrophobicity, Gram-positive

1. Adesida SA, Ezenta CC, Adagbada AO, Aladesokan AA, Coker AO. (2017). Carriage of multidrug resistant enterococcus faecium and enterococcus faecalis among apparently healthy humans. African Journal of Infectious Diseases, 11: 83–89. http://doi.org/10.21010/ajid.v11i2.11
2. Araújo MC, Oliveira, MBM. (2013). Monitoring of the water quality of a stream at the Federal University of Pernambuco, Brazil. Rev. Environment & Water. 8: 3. http://dx.doi.org/10.4136/ambi-agua.1192.
3. Bae YM, Zheng L, Hyun JE, Jung KS, Heu S, Lee SY. (2014). Growth characteristics and biofilm formation of various spoilage bacteria isolated from fresh produce. J Food Sci. 79: 2072–80. Doi: 10.1111/1750-3841.12644.
4. Banada PP, Deshpande S, Russo R, Singleton E, Shah D, Patel B, Burday M, Koshy R, Wang Q, Jones M, Gall A, Lokhov S, Kwiatkowski R, Persing D, Connell N, Alland D. (2017). Rapid Detection of Bacillus anthracis Blood Stream Infections Using a Novel Assay in the GeneXpert System. J. Clin. Microbiol. JCM.00466-17. Doi: 10.1128/JCM.00466-17.
5. Berglund B. (2015). Environmental dissemination of antibiotic resistance genes and correlation to anthropogenic contamination with antibiotics. Infection Ecology & Epidemiology. 5: 28564. http://dx.doi.org/10.3402/iee.v5.28564
6. Bottone EJ. (2010). Bacillus cereus, a volatile human pathogen. Clinical microbiology reviews. 23: 382-398. Doi:10.1128/CMR.00073-09.
7. Celandroni F, Salvetti S, Gueye SA, Mazzantini D, Lupetti A, Senesi S. (2016). Identification and pathogenic potential of clinical Bacillus and Paenibacillus isolates. Plos One. 11: 0152831. https://doi.org/10.1371/journal.pone.0152831.
8. Chong YP, Park SJ, Kim ES, Bang KM, Kim MN, Kim SH, Lee SO, Choi SH, Jeong JY, Woo JH, Kim YS. (2015). Prevalence of blaZ gene types and the cefazolin inoculum effect among methicillin-susceptible Staphylococcus aureus blood isolates and their association with multilocus sequence types and clinical outcome. Eur J Clin Microbiol Infect Dis. 34: 349-355. Doi: 10.1007/s10096-014-2241-5.
9. CLSI. (2015). Performance Standards for Antimicrobial Susceptibility Testing; Twenty-Fourth Informat. Supplement - M100-S24. Clinical and Laboratory Standards Institute.
10. Czerwonka G, Guzy A, Kaluz K, Grosicka M, Danczuk M, Lechowicz L, Gmiter D, Kowalczyk P, Kaca W. 2016. The role of Proteus mirabilis cell wall features in biofilm formation. Arch Microbiol. 198:877-84. Doi: 10.1007/s00203-016-1249-x.
11. Fernandes R, Amador P, Prudêncio C. (2013). β-Lactams: chemical structure, mode of action and mechanisms of resistance. Reviews in Medical Microbiology. 24:7–17. DOI:10.1097/MRM.0b013e3283587727.
12. Freitas ESM, Gaudio RSD. (2015). Ecological crisis, water shortage and ideologies: a critical analysis of the 2070 Letter. Rev. Society & Nature 27: 439-452. http://dx.doi.org/10.1590/1982-451320150306
13. Ghafourian S, Sadeghifard N, Soheili S, Zamberi Sekawi Z. (2015). Extended Spectrum Beta-lactamases: Definition, Classification and Epidemiology. Curr. Issues Mol. Biol. 17: 11-22.
14. Guimarães DO, Momesso LS, Pupo MT. (2010). Antibiotics: therapeutic importance and prospects for the discovery and development of new agents. New Quimica. 33: 667-679.
15. Kimouli M, Vrioni G, Papadopoulou M, Koumaki V,Petropoulou D, Gounaris A, Friedrich AW,Tsakris S. (2012). Two cases of severe sepsis caused by Bacillus pumilus in neonatal infants. Journal of Medical Microbiology. 61: 596–599. DOI 10.1099/jmm.0.033175-0.
16. Kurinčič M, Jeršek B, Klančnik A, Možina SS, Fink R, Dražić G, Bohinc, K. (2016). Effects of natural antimicrobials on bacterial cell hydrophobicity, adhesion, and zeta potential/Vpliv naravnih protimikrobnih snovi na bakterijsko hidrofobnost, adhezijo in zeta potencial. Archives of Industrial Hygiene and Toxicology, 67: 39-45. Doi: 10.1515/aiht-2016-67-2720.
17. Lobova TI, Yemelyanova E, Andreeva IS, Puchkova LI, Repin VY. (2015) Antimicrobial resistance and plasmid profile of bacterial strains isolated from the Urbanized Eltsovka-1 river (Russia). Microbial Drug Resistance. 21: 477–490. DOI:10.1089/mdr.2014.0203
18. Logan NA, Popovic T, Hoffmaster A. (2009) Bacillus and other aerobic endospore-forming bacteria. In Manual of Clinical Microbiology. 9: 455–473.
19. Majed R, Faille C, Kallassy M, Gohar M. (2016). Bacillus cereus Biofilms - Same, Only Different. Frontiers in microbiology. 7: 1054. https://doi.org/10.3389/fmicb.2016.01054
20. Mohapatra BR, La Duc MT. (2012). Rapid detection of viable Bacillus pumilus SAFR-032 encapsulated spores using novel propidium monoazide-linked fluorescence in situ hybridization. Journal of Microbiological Methods. 90: 15-19. Doi: 10.1016/j.mimet.2012.04.006.
21. Moliva VM, Cerioli F, Reinoso LB. (2017). Evaluation of environmental and nutritional factors and sua gene on in vitro biofilm formation of Streptococcus uberis isolates. Microbial pathogenesis. 107: 144-148. Doi: 10.1016/j.micpath.2017.03.028.
22. Nascimento VFS, Araújo MFF. (2013). Occurrence of opportunistic pathogenic bacteria in a semiarid reservoir in Rio Grande do Norte, Brazil. Rev. Environmental Sciences. 7: 91-104.
23. Nicolau DP, Silberg BN. (2017). Cefazolin potency against methicillin-resistant Staphylococcus aureus: a microbiologic assessment in support of a novel drug delivery system for skin and skin structure infections. Infection and drug resistance. 10: 227–230. Doi: 10.2147/IDR.S134497.
24. Owusu-Kwarteng J, Wuni,A, Akabanda F, Tano-Debrah K, Jespersen L. (2017). Prevalence, virulence factor genes and antibiotic resistance of Bacillus cereus sensu lato isolated from dairy farms and traditional dairy products. BMC Microbiology. 17: 2-8. DOI 10.1186/s12866-017-0975-9.
25. Purificação-Júnior AF, Araújo LCA, Lopes ACS, Sobral MA, Lima GMS, Silva MV, Correia MTS, Oliveira MBM. 2017. Microbiota sampled from a polluted stream in Recife-PE, Brazil and its importance to public health. African Journal of Microbiology Research.11: 1142-1149. DOI: 10.5897/AJMR2017.8577
26. Raymond B, Federici BA. (2017). In defence of Bacillus thuringiensis, the safest and most successful microbial insecticide available to humanity - a response to EFSA. FEMS Microbiology Ecology, 93: 7. https://doi.org/10.1093/femsec/fix084
27. Rodrigues LB, Santos LR, Rizzo NN, Tagliari VZ, Oliveira AP, Trenhago G, Rodegheri SC, Taglieti RM, Dickel EL, Nascimento, VP. (2009). Hydrophobicity and biofilm formation on polystyrene by Salmonella Heidelberg isolated from a poultry slaughterhouseActa Scientiae Veterinaria. 37: 225-230.
28. Shivamurthy VM, Gantt S, Reilly C, Tilley P, Guzman J, Tucker L. (2016). Bacillus pumilus Septic Arthritis in a Healthy Child. Canadian Journal of Infectious Diseases and Medical Microbiology. 2016: 3265037. http://dx.doi.org/10.1155/2016/3265037
29. Stepanovic S, Vukovic D, Hola V, Bonaventura G, Djukic S, Cirkovic I, Ruzicka F. (2017). Quantification of biofilm in microtiter plates: overview of testing conditions and practical recommendations for assessment of biofilm production by staphylococci. Journal Compilation APMIS. 115: 891-8. DOI: 10.1111/j.1600-0463.2007.apm_630.x.
30. Tendolkar PM, Baghdayan AS, Gilmore MS, Shankar N. (2014). Enterococcal surface protein, Esp, enhances biofilm formation by Enterococcus faecalis. Infect Immun. 72: 6032–6039. DOI: 10.1128/IAI.72.10.6032–6039.2004.
31. Trentin DS, Bonatto F, Zimmer KR, Ribeiro VB, Antunes ALS, Bart AL, Soares GV, Krug C, Baumvol IJR, Macedo AJ. (2014). N2/ H2 plasma surface modifications of polystyrene inibit the adhesion of multidrug resistant bacteria. Surface & Coatings Technology. 245: 84–91. https://doi.org/10.1016/j.surfcoat.2014.02.046
32. Wang Y, Lee SM, Dykes G. (2014). The physicochemical process of bacterial attachment to abiotic surfaces: Challenges for mechanistic studies, predictability and the development of control strategies. Crit Rev Microbiol. 1-13. DOI: 10.3109/1040841X.2013.866072.
33. WHO. (2016). Sanitation safety planning: manual for safe use and disposal of wastewater, greywater and excreta. World Health Organization, 20 Avenue Appia, Geneva.
34. Zain SN, Bennett R, Flint S. (2017). The Potential Source of B. licheniformis Contamination During Whey Protein Concentrate 80 Manufacture. Journal of Food Science. 82: 751-756. Doi: 10.1111/1750-3841.13633.