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

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.


1.
Introduction Water is considered an essential natural resource for the maintenance of life. However, the inadequate disposal of large amounts of polluting waste in water bodies has contributed to the scarcity of this resource and has compromised the relationships between living organisms. The urban dejects that are launched in aquatic environments attract decomposing microorganisms, causing ecological imbalances by reducing the oxygen present in the water as well as changes in the dynamics of these ecosystems (Freitas and Gaudio, 2015). The contamination of water bodies by resistant bacteria pathogenic to humans and other animals is another problem that affects these environments (Nascimento and Araújo, 2013;Berglund, 2015). According to the World Health Organization (2016), approximately a half of the population living at the developing world will be affected by infections directly related to water that is out of quality standards and due to inadequate or non-existent sanitation. The Cavouco stream is a tributary of the Capibaribe River, which is one of the main rivers in the Pernambuco state, Brazil. Some works have been carried out in this environment in order to diagnose and monitor the environmental impact. According to Araújo and Oliveira (2013), this stream receives polluting loads of chemical and domestic waste, which compromises the quality of its water. Purificação-Junior et al. (2017), analyzing the microbiota of this environment, detected the presence of pathogenic Gram-positive and Gram-negative isolates, resulting a risk of disease transmission.
One of these isolates previously identified corresponds to the genus Bacillus. Aerobic or facultative anaerobic, catalase-positive and sporeproducing Gram-positive bacteria represent this genus, which may be present in both aquatic and terrestrial environments. It includes species of ecological importance, such as B. thuringiensis (Raymond and Federici, 2017) (Kimouli, 2012).
The present work aimed to investigate the antibiotic susceptibility/resistance profile and biofilm formation ability of isolates belonging to three species of the genus Bacillus collected from the Cavouco stream, an impacted aquatic environment.

Biofilm Formation
To evaluate the potential for biofilm formation, we used the using the crystal violet method described by Stepanovic et al. (2007), performed in microtiter plates. Three culture media were evaluated: Luria Bertani Miller (LB Miller), Tryptose Soy Broth (TSB) and BHI, all supplemented or not with 1% (w/v) glucose. From the readings of optical density (OD), the mean absorbance values of each sample (OD s ) were determined in comparison with the absorbance of the sterility control (OD c ). The isolates were classified as strong (4 × OD c < OD s ), moderate (2 × OD c < OD s ≤ 4 × OD c ) or weak (OD c < OD s ≤ 2 × OD c ) biofilm formers. The isolates presenting values of absorbance equal to or less than the control were classified as non-biofilm producers.

Cellular Hydrophobicity Profile
Cell surface hydrophobicity (CSH) was determined based on the hydrocarbon-binding method described by Tendolkar et al. (2004) with adaptations. The bacterial isolates grown in BHI broth at 37 °C for 18 h were transferred to microtubes and centrifuged for 10 min at 7,000 rpm. The supernatant was discarded and the pellet resuspended in PUM buffer (Trihydrate and Monobasic Potassium Phosphate, Urea and Heptahydrate Magnesium Sulphate), adjusted to 0.5 and the initial reading (OD i ) obtained at 600 nm. Subsequently, the hydrocarbon para-xylene was added to the bacterial suspensions, followed by vortexing for 2 min. After separation of the phases at room temperature, the final reading at 600 nm (OD f ) of the lower phase of each microtube was performed.

Results and Discussion
The antibiotic resistance/susceptibility profiles of the three isolates of the genus Bacillus were evaluated and are shown in Table 1 In the present study, Bacillus cereus isolate stood out because it was resistant to all tested penicillins and cephalosporins, including third and fourth generation. This result is worrying because indicates resistance even to the last generations antibiotics, which are usually prescribed in the case of infections caused by resistant strains Gram-negative. In addition to resistance to two classes of beta-lactam antibiotics, this isolate also showed resistance to trimethoprim, a folic acid antagonist that prevents bacterial replication; thus, it can be considered a multiresistant microorganism (Fernandes et al., 2013; Guimarães; Momesso and Pupo, 2010). However, it is worth mentioning that resistance to different classes of antibiotics depends on the strain obtained and the environment from which the microorganism was obtained (Bottone et al., 2010). Another point that deserves attention is the mechanism of sporulation that the species of the genus Bacillus present. This mechanism, together with other factors such as biofilm formation, may contribute to the permanence and dissemination of these bacteria in different environments, increasing their survival capacity (Logan et al., 2009). The cellular hydrophobicity profile and biofilm formation ability were also evaluated. B. cereus isolate presented a hydrophobicity index equal to 70%, and thus was classified as hydrophobic. The isolates of the other species were moderately hydrophobic except the ATCC, which was hydrophilic ( Figure 1). Hydrophobicity is one of the physicochemical factors that influence the process of microbial adhesion on different substrates. In general, the currently existing methods can measure the interaction between cells and a hydrophobic or hydrophilic material (Wang; Lee and Dykes, 2014). In this study, B. cereus presented a hydrophobic cell surface and strongly produced biofilm, corroborating with other studies showing that as higher the hydrophobicity index of the bacterial cell greater is the capacity for biofilm formation (Rodrigues et al., 2009;Trentin et al., 2014). However, the ATCC was hydrophilic and formed biofilm. Czerwonka et al. (2016), when investigating Proteus mirabilis isolates, demonstrated that those with high biofilm production capacity were hydrophilic. Thus, there seems to be no direct correlation between cell surface hydrophobicity and biofilm formation, being these characteristics specific for each isolate. Concerning biofilm formation, it was possible to verify that all the species formed biofilm in the different media tested, with better results for those supplemented with glucose ( Figure 2). The adhesion of bacteria to a surfacebiotic or abioticis the first step of biofilm formation. Some culture media such as TSB and BHI, supplemented or not, are preferred for evaluation of biofilm formation. Rodrigues et al. (2009) in a study with Salmonella spp evaluated the ability of biofilm formation in TSB medium without and with addition of glucose at 0.5%, 1.0% and 1.5% and observed that the addition of this sugar did not increase the biofilm formation. Similarly, Moliva et al. (2017), evaluating the biofilm formation ability of Streptococcus uberis also in TSB medium supplemented with 0.5% glucose and 0.5% or 5% lactose, also did not notice an increase in the formation. Although supplementation of culture media was apparently not a determining factor for biofilm formation in Salmonella and Streptococcus, this supplementation was favorable in the genus Bacillus. In the present study, all species investigated had a higher biofilm formation capacity in the supplemented media. In the present study, B. pumilus and B. licheniformis were biofilm-forming agents in media supplemented or not with glucose and these species were moderately hydrophobic. These data demonstrate that these species, as well as B. cereus, share common strategies for survival and permanence in impacted aquatic environments despite developing distinct resistance mechanisms.

Conclusion
The present study demonstrates the specificities of the species investigated in relation to the resistance profile and indicates the influence of the mechanisms of sporulation and biofilm formation in the survival and permanence of these microorganisms under adverse conditions.