Improvements on colony morphology identification towards bacterial profiling
Introduction
Isolation of specimens by bacteriological techniques such as the culturing in selective or differential media is a routine procedure, especially in clinical laboratories. Growing on agar surfaces, microorganisms form colonies whose appearance helps the clinicians and researchers to identify genera or even species. One of the most intriguing aspects of this approach is the observation of similar colony patterns in different systems and the existence of distinct patterns when culturing a sole strain in analogous conditions. The large number of reckonable patterns turns the identification of colony morphologies a real challenge for microbiologists, clinicians and technicians.
Rapid technological advances in bacterial identification methods have occurred providing a formidable wide range of techniques to detect, identify and differentiate bacteria. Molecular methods such as ELISA, PCR and MALDI-TOF MS have introduced great improvements in bacterial identification as they contributed to speed up the analysis and the reduction of handling (Weile and Knabbe, 2009). Despite the technological advances, culture-based strategies are still necessary to obtain information about the microbiological effect of the antibiotic courses, by determining the number of bacteria before and after antibiotic administration, as well as to determine whether phenotypic selection is occurring. Antibiotic treatments may exert a pressure selection over bacteria that may not be detected using molecular methods. Atypical colony morphologies can often exhibit unusual biochemical and metabolic features turning clinical identification into a challenge. For instance, Staphylococcus aureus small colony variants (SCV) have altered metabolic activity, thus interfering with the results of biochemical tests as the negative results of coagulase tests (Hilmi et al., 2013). Therefore, colony morphology characterization often complements conventional microbial identification detecting intra-strain diversity (Qamer et al., 2003).
Intra-population diversity generated by bacterial phenotypic and genetic adaptation may be beneficial since it allows both evolution and adaptation to new and changing environments increasing the chances of bacteria survival (Boles et al., 2004, Goerke et al., 2007, Yachi and Loreau, 1999). Alterations in colony morphological traits can be a macroscopic manifestation of the several biological strategies adopted by microorganisms to face stress conditions, as starvation, depletion of oxygen, antibiotics and host defences (Sousa et al., 2011). Furthermore, the different aspect of colonies may reflect differences in virulence (Davies et al., 2007, Martin et al., 1993, Rossignol et al., 2009, Tannaes et al., 2000), antimicrobial resistance (Lewis, 2005, Massey et al., 2001, Sousa et al., 2011) and persistence (Balaban et al., 2004, Singh et al., 2009, Spoering and Lewis, 2001). Therefore, and despite being described by several authors as old-fashioned (Braga et al., 2013, Weile and Knabbe, 2009), colony morphology characterization can provide valuable insights into individual microbial diversity, both derived from genetic changes or reversible changes (Sousa et al., 2011).
In chronic infections, including cystic fibrosis (CF), the existence of multiple colony morphology variants is recurrent. One of the most important clinical features in CF is the Pseudomonas aeruginosa conversion from non- to mucoid form, being the later phenotype more difficult to eradicate (Hogardt and Heesemann, 2010, Lyczak et al., 2002). Mucoid variants are markedly more resistant to antibiotics, such as gentamicin, aminoglycosides, ciprofloxacin and imipenem, or can be even multi-resistant (Agarwal et al., 2005, Manno et al., 2005). Several other morphotypes have been identified in bacteria related to chronic and acute infections. The most common and best studied are SCV (Haussler, 2004, Haussler et al., 1999, Haussler et al., 2003, Hoffman et al., 2006, Massey et al., 2001, Proctor et al., 2006, Wellinghausen et al., 2009), the rough (small) colonies (Drenkard and Ausubel, 2002) and the hyperpiliated colonies (Deziel et al., 2001, Haussler et al., 2003).
Biofilm formation, the microbial organization in multicellular communities, is another relevant strategy used by microorganisms to face stress conditions and to introduce microbial diversity (Costerton et al., 1987, Donlan, 2002, Drenkard, 2003, Lewis, 2001, Stewart, 2002). Bacteria may develop some phenotypic changes to facilitate the growth as a biofilm (Sauer et al., 2002), being these changes observed when bacteria are recovered from biofilms, plated on agar media and form colonies with distinct morphological patterns (Sousa et al., 2011).
Apart from obvious morphological differences that have been reported, the conditions in which evaluation has been performed were different among published works. This variety is especially significant in what the medium used and the growth time for colony development is concerned. To obtain new insights on colony morphology identification, this study used two P. aeruginosa strains (a reference strain and a clinical isolate) to perform a detailed and broad evaluation of colony morphologies during their development experiencing different growth times, plate densities, culture media and mode of growth, including planktonic and biofilm lifestyle. This study aimed at determining the impact of the experimental conditions on each colony morphological features, including form, margin, texture, size and colour, in a systematic way.
Section snippets
Bacterial strains and culture conditions
P. aeruginosa ATCC 10145 and a clinical isolated from medical equipment (from now on referred as clinical strain) were used throughout this study. Bacteria were routinely cultured on tryptic soy broth (TSB) or agar (TSA) medium at 37 °C. All strains were preserved in cryovials (Nalgene) at − 80 ± 2 °C. Prior to each experiment, bacterial cells were grown on TSA plates for 24 h at 37 °C. The use of a reference strain and a clinical isolate ensured the different genetic background.
Planktonic cultures
Planktonic bacteria
Results
To describe each colony regarding the distinct morphological characteristics, the following terms were used as synonymous: colony morphology, colony type, colony variant and morphotype, all meaning a group of bacteria grown from a single cell on agar surface, exhibiting a typical colonial pattern. It must be remarked for further studies that this definition does not exclude the possibility that different strains or species exhibit the same morphotype or that a strain or species exhibits more
Discussion
The recognition of typical colony morphologies is crucial, among others, for clinical diagnosis. Scientific and clinical laboratories frequently use the colony morphology displayed by bacteria on agar media as an auxiliary means to identify bacterial species because of their different and specific growth patterns.
Changes in colony morphology are gaining attention because they are thought to be the expression of the adaptation to different environments, thus hampering the pathogen identification
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