Biofilm released cells can easily be obtained in a fed-batch system using ica+ but not with ica- isolates

Biofilm released cells can easily be obtained in a fed-batch system using ica+ but not with ica- isolates Vânia Gaio and Nuno Cerca Centre of Biological Engineering (CEB), Laboratory of Research in Biofilms Rosário Oliveira (LIBRO), University of Minho, Braga, Portugal ABSTRACT Submitted 20 April 2020 Accepted 24 June 2020 Published 15 July 2020 Corresponding author Nuno Cerca, Staphylococcus epidermidis is one of the major opportunistic bacterial pathogens in healthcare facilities, mainly due to its strong ability to form biofilms in the surface of indwelling medical devices. To study biofilms under in vitro conditions, both fed-batch and flow systems are widely used, with the first being the most frequent due to their low cost and ease of use. Aim. To assess if a fed-batch system previously developed to obtain biofilm released cells (Brc) from strong biofilm producing S. epidermidis isolates could also be used to obtain and characterize Brc from isolates with lower abilities to form biofilms. Methodology. The applicability of a fed-batch system to obtain Brc from biofilms of 3 ica+ and 3 ica− isolates was assessed by quantifying the biofilm and Brc biomass by optical density (OD) and colony-forming units (CFU) measurements. The effect of media replacement procedures of fed-batch systems on the amount of biofilm was determined by quantifying the biofilm and biofilm bulk fluid, by CFU, after consecutive washing steps. Results. The fed-batch model was appropriate to obtain Brc from ica+ isolates, that presented a greater ability to form biofilms and release cells. However, the same was not true for ica− isolates, mainly because the washing procedure would physically remove a significant number of cells from the biofilm. Conclusions. This study demonstrates that a fed-batch system is only feasible to be used to obtain Brc from S. epidermidis when studying strong and cohesive biofilm-forming isolates. Subjects Biotechnology, Microbiology, Infectious Diseases Keywords Biofilm-released cells, Fed-batch systems, Biofilm disassembly, S. epidermidis Academic editor Jonathan Thomas Additional Information and Declarations can be found on page 10 DOI 10.7717/peerj.9549 Copyright 2020 Gaio and Cerca Distributed under Creative Commons CC-BY 4.0 OPEN ACCESS INTRODUCTION Staphylococcus epidermidis is a well-known nosocomial pathogenic associated with recurrent biofilm-infections, acknowledged as the major agent involved in biofilmassociated medical devices infections (Becker, Heilmann & Peters, 2014). Importantly, this bacterium, which was previously seen as a commensal microorganism due to its benign relationship with the host (Cogen, Nizet & Gallo, 2008; Gardiner et al., 2017), is nowadays accepted as an important opportunistic pathogen, of particular concern in ill and immunocompromised patients (Otto, 2009). S. epidermidis infections are more likely to happen upon invasive procedures involving indwelling medical devices, in which the How to cite this article Gaio V, Cerca N. 2020. Biofilm released cells can easily be obtained in a fed-batch system using ica+ but not with ica- isolates. PeerJ 8:e9549 http://doi.org/10.7717/peerj.9549 physiological barriers are compromised, since this bacterium is a ubiquitous inhabitant of the skin and mucosae in humans (Ziebuhr et al., 2006) and has a strong ability to form biofilms on the surface of medical devices (Cerca et al., 2005c; Laverty, Gorman & Gilmore, 2013). Bacteria within biofilms are undoubtedly more resistant to antibiotics (Albano et al., 2019; Cerca et al., 2005a; Dias et al., 2018) and to the host immune defense (Cerca et al., 2006; Gray et al., 1984; Yao, Sturdevant & Otto, 2005), contributing to the persistence and recurrence of infections (Mah, 2012; Schommer et al., 2011; Singh & Ray, 2014). For all these reasons, biofilms have been a major research target and extensive studies allowed to characterize the biofilm lifecycle and divide it into three main stages: attachment, maturation and disassembly (as reviewed in Boles & Horswill, 2011; Otto, 2013). The importance of a better characterization of the disassembly process in biofilms has been pointed out, since cells released from the biofilm can enter the systemic circulation and contribute to the spreading of the infection (Boles & Horswill, 2011; Kaplan, 2010) and cause severe systemic diseases, as bacteraemia (Cervera et al., 2009; Wang et al., 2011) which are associated with high levels of morbidity and mortality among immunocompromised patients (Kleinschmidt et al., 2015; Rogers, Fey & Rupp, 2009). Both fed-batch and dynamic systems have been used to study and characterize initial adhesion (Cerca et al., 2005b; Isberg & Barnes, 2002) and maturation of the biofilm (Moormeier & Bayles, 2014; Periasamy et al., 2012). However, both present advantages and drawbacks, depending on the main focus of the study (Bahamondez-Canas, Heersema & Smyth, 2019). The few studies addressing disassembly rely almost entirely on dynamic systems, which is not surprising, as these systems present key advantages such as a controlled flow, allowing a continuous diffusion of oxygen, nutrients and waste, and are thought to be a more accurate representation of the conditions in which biofilms are formed in various diseases, as previously reviewed (Azeredo et al., 2017; Bahamondez-Canas, Heersema & Smyth, 2019). However, these systems are significantly more expensive and are often more difficult to assemble, being essential to have good background knowledge on hydrodynamics to study biofilms in such conditions (Yawata et al., 2016). Hence, it is no wonder that fed-batch systems are more frequently used on biofilm research, since they are easier to implement and already widely used under in vitro conditions (Azeredo et al., 2017; Bahamondez-Canas, Heersema & Smyth, 2019). Thus, the ability to implement fed-batch systems to high-throughput research in biofilms disassembly would be beneficial, as it would allow more studies to be undertaken on this research topic. Earlier, we demonstrated the feasibility to use a fed-batch system to obtain S. epidermidis cells released from biofilms (Brc) (França et al., 2016a; Gaio & Cerca, 2019). However, we failed to include low biofilm-forming isolates on those studies and, as a consequence, the applicability of this model on such isolates could be questioned. Hence, the aim of the current study was to better understand the limitations of a fed-batch system to obtain Brc from S. epidermidis biofilms, by testing its potential to characterize Brc from ica+ and ica− isolates with distinct abilities to form biofilms. Gaio and Cerca (2020), PeerJ, DOI 10.7717/peerj.9549 2/15 MATERIALS & METHODS Bacterial isolates and growth conditions Six isolates of S. epidermidis, with different abilities to form biofilms and characterized by the presence (+) or absence (-. ) of the intercellular adhesion gene (ica), generally involved in S. epidermidis biofilm formatio (...truncated)