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The induction of Staphylococcus aureus biofilm formation or Small Colony Variants is a strain-specific response to host-generated chemical stresses
Microbes and Infection 17 (2015) 77e82 www.elsevier.com/locate/micinf
Short communication
The induction of Staphylococcus aureus biofilm formation or Small Colony Variants is a strain-specific response to host-generated chemical stresses Long M.G. Bui a, John D. Turnidge b, Stephen P. Kidd a,* a
Research Centre for Infectious Disease, School of Molecular and Biomedical Science, The University of Adelaide, Adelaide, South Australia 5005, Australia b SA Pathology, Women's and Children's Hospital, North Adelaide, Adelaide, South Australia 5005, Australia Received 4 August 2014; accepted 24 September 2014 Available online 2 October 2014
Abstract Staphylococcus aureus is extremely versatile. It has a capacity to persist within its host by switching to the alternative lifestyles of biofilm or Small Colony Variants (SCV). The induction of this switch has been presumed to be in response to stressed conditions, however the environmental basis has not been thoroughly investigated. We assessed the response of numerous strains to chemicals that are present in human host. There were some that induced a biofilm or SCV phenotype and indeed some inducing both lifestyles. This result illustrates the diversity within a population and a strain-specific adaptation to the presence of host-generated stresses. © 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.
Keywords: Staphylococcus aureus; Stress response; Biofilm; Small Colony Variants; Oxidative stress
1. Introduction Staphylococcus aureus is an important opportunistic pathogen that can lead to a myriad of diseases via its transit to various sites in the body. These diseases range from the minor infections of superficial skin lesions or deep abscesses, to the major invasive diseases such as pneumonia, meningitis, osteomyelitis, endocarditis, and septicaemia [1]. S. aureus strains isolated from different clinical settings display significant genetic variations and it is the difference that underpins the strain variation in physiology and stress response [2,3]. This includes the emergence of multiple drug resistant S. aureus in hospitals and communities. These drug resistant strains form the basis for Methicillin Resistant S. aureus (MRSA) [4] or more specifically HA-MRSA (Hospital Acquired MRSA) and * Corresponding author. Research Centre for Infectious Disease, School of Molecular and Biomedical Science, Molecular Life Sciences Building, The University of Adelaide, Adelaide, South Australia 5005, Australia. Tel.: þ61 (0)8 83134671; fax: þ61 (0)8 83034362. E-mail address: [email protected] (S.P. Kidd).
CA-MRSA (Community Acquired MRSA) infections. However, in a clinical setting the resistance to antibiotic therapies is not simply based on the presence or absence of a specific gene cassette/s. There are known to be “non-inherited” antibiotic resistance mechanisms that subvert antimicrobial actions via growth pathways, surface structures or processes that are beyond the transport, enzymatic detoxification or target modification mechanisms of antibiotic resistance [5]. These alternative mechanisms include modes of existence that permit S. aureus to survive in a quiescent state and thereby avoid the toxicity of antibiotics or host defences. These specifically are biofilms and Small Colony Variants (SCVs). These lifestyles are highly resistant to antimicrobial agents (therapeutic or host-generated) and are thought to be the basis for S. aureus long-term survival and persistence in the host [6]. Biofilm is characterized by the release of a matrix of polymeric substances (extracellular polymeric substances, EPS); extracellular DNA (eDNA), protein and polysaccharide substances that enclose the colonies and provides protection from host-generated chemicals (stressors) [2,7]. The specific stress response and its coupling to the subsequent production
http://dx.doi.org/10.1016/j.micinf.2014.09.009 1286-4579/© 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.
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of S. aureus biofilm, remains to be defined. The formation of SCVs is characterized by very small, non-pigmented and nonhaemolytic colonies combined with decreased uptake by antibiotics (particularly aminoglycosides). Recent research has clearly shown that the S. aureus persistence within host cells induces SCVs phenotypes and there is some evidence that it is the external environmental stress factors (reactive oxygen species, low pH, cationic peptides, limited nutrition) within host cells that induce this formation of SCVs [6,8]. It is intriguing that amongst the reactive chemicals known to be present, the most stable and equally damaging are the reactive aldehydes that a consequence of rapidly degrading superoxide and nitric oxide being present. These however have been studied very little. The environmental signals triggering the induction of biofilm or SCVs by S. aureus are still unknown. The host-generated chemical stresses as a response to bacterial infection: oxidative stress, nitrosative stress and the presence of reactive aldehydes, are important environmental factors in bacterial pathogenesis. We hypothesized that the role of these stresses could be a part of inducing the formation of biofilm and SCVs for specific strains of S. aureus. 2. Materials and methods 2.1. Sample collection Seventy-two strains of S. aureus were used; nine were accessed from the NARSA (Network of Antimicrobial Resistance in S. aureus): Mu50 (NRS1) Mu3 (NRS2), COL (NRS100), N315 (NRS70), Sanger252 (NRS71), Sanger 476 (NRS72), NCTC8325 (NRS77), MW2 (NRS123) and JE2 (USA300) and 63 from South Australia Pathology, Women's and Children's Hospital (WCH), Adelaide (Australia). This collection represents strains isolated from the blood (BC, 20 samples), lower respiratory (LR, 20 samples), skin and soft tissue infections (SSTI, 20 samples) and cystic fibrosis (CF) patients (3 samples). 2.2. Detection of MRSA and MSSA using oxacillin The susceptibility to oxacillin has been used to classify S. aureus as MRSA or MSSA. Oxacillin and cefoxitin MIC (minimum inhibitory concentration) was performed as recommended by the Clinical Laboratory Standards Institute (CLSI, formerly the National Committee for Clinical Laboratory Standards, NCCLS). Oxacillin MIC was determined by the micro-dilution assay. According to current CLSI guidelines the MRSA strains were identified as MIC 4 mg/mL and MSSA as MIC 2 mg/mL. 2.3. Growth under different conditions of chemical stress There were nine chemicals (stressors) selected to represent the key chemicals present in the host-pathogen environment; S-nitrosoglutathione (GSNO), hydrogen peroxide (H2O2), glyoxal, formaldehyde, methylglyoxal, acrolein, glyco-
laldehyde, glyceraldehyde, and malonaldehyde. All chemicals were purchased from SigmaeAldrich and prepared at a stock concentration (1 M and 1%). The MIC of each chemical stress was determined. Also the growth profile was performed in 96well plates (96 wells, flat bottom, polystyrene, Falcon) containing rich media (Tryptone Soya Broth; TSB). In brief, 5 105 cells (CFU) were inoculated into each well containing 250 mL of TSB and incubated at 37 C. Optical density (OD600 nm, BioTek EL808 Spectrophotometer) was measured after over a period of 24 h. Several concentrations of each stress were assayed and two levels were chosen for further studies; a low level of stress, in which the growth of bacteria in stress was inhibited by 20e30% of the OD630 nm of non stress conditions and high level of stress but sub-lethal; growth inhibition was 70e80% (Table S1). 2.4. Biofilm assay Biofilm formation was assayed in 96-well microtitre plates using a standard, previously described method [9]. In brief, 5 105 cells (CFU) were inoculated in TSB media into the microtitre plates that included added specific concentrations of the different chemicals, and controls of no chemical stress added and then no inoculum. These plates were incubated at 37 C for 24 h, planktonic cells removed by washing and biofilm cells visualized by staining with 0.1% crystal violet. Ethanoleacetone (20:80, v/v) was added and OD630 values were read (Biotek EL808 spectrophotometer). Each sample was performed in three separate assays with eight replicates of each. The values that are presented are an average and error bars are the standard deviation. Included as controls in the assays were strains known to be weak biofilm formers (COL and JE2 (USA300), [10]) and a strong biofilm forming strain (N315, [11]). Scanning Electron Microscopy (SEM) was used to confirm the biofilm formation and true phenotype of selected strains that were seemingly inducing their biofilm. Samples of biofilm cells exactly replicating the above samples, were fixed in SEM fixative solution (4% paraformaldehyde and 1.25% glutaraldehyde in Phosphate Buffer Solution and 4% sucrose, pH 7.2). Following dying in Osmium tetroxide (OsO4 e 2%), the samples were dehydrated using ethanol and then flooded with hexamethyldisilazane (HMDS). All the samples were then mounted on stubs and coated with platinum and visualized using Field Emission Scanning Electron Microscope (Philips XL30, Adelaide Microscopy). As further confirmation, the exact biofilm components were assessed using fluorescent staining. To identify the eDNA, a BacLight kit (Invitrogen) was used according to the manufacturer's instructions. Biofilm was formed on a slide, planktonic cells removed and the biofilm remaining was viewed with an Olympus IX-70 microscope (phase contrast 100 oil immersion objective). A filter of 496e555 nm was used to image SYTO9 fluorescence and 600e700 nm to image PI fluorescence. Fluorescence and phase contrast images were captured and false colour merged with the Metamorph software program (Version 7.7.3.0, Molecular Devices).
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2.5. Detection of SCVs phenotype in the presence of stress Each of the 72 strains (5 105 cells) was separately inoculated into 200 mL TSB in a 96-well microtitre plate with and without chemical stress. The chemical stress with specific concentrations was added and the plates were then incubated at 37 C for 18e24 h. Following the incubation, 100 mL of each culture was diluted and plated onto TSA and grown for 36e72 h at 37 C. Quantitative assessment of SCV was performed by measuring of colony size; SCVs were defined as the diameter of colonies 1 mm (1/5the1/10th the normal size of colonies) and determining the percentage of SCVs per the total number of colonies (CFU/mL). 3. Results 3.1. Identification of MRSA and MSSA We identified 46 MRSA and 17 MSSA in our set of 63 clinical strains. Of 20 BC samples; 10 were MRSA and 10 MSSA. For LR, 16 were MRSA and 4 MSSA. The 20 SSTI samples were all MRSA whereas all 3 CF samples were MSSA. These data were combined with the results generated below for the screening for biofilm and SCVs (Table S2). 3.2. Sensitivity of S. aureus strains in different kinds of stress Two concentrations for nine chemicals were chosen from the growth curves of the reference strain S. aureus JE2 including low level and sub-lethal level (Table S1). These two levels were applied and checked for all clinical isolates and reference strains (data not shown). 3.3. Biofilm formation in non-stress condition Strong biofilm was identified as the mean OD630 is 0.2, weak biofilm was classified as the mean OD630 within 0.1e0.2 and no biofilm as OD630 <0.1. These values are consistent to previous reports for S. aureus biofilm formation [12,13]. Of the clinical strains, 15.9% produced strong biofilm, 54% producing biofilm weakly and 30.1% isolates are biofilm negative (Fig. 1A). Indeed, there were no observed differences in specimen sources for the strong, weak or no biofilm categories. 3.4. Sub-lethal of environmental stress induced biofilm formation A significant induction of biofilm with the addition of chemical stress was defined as greater than two-fold induction compared to that measured with no stress added. With low level of stress there was no significant induction. However at the high concentration of stress some strains induced their biofilm formation. There were 21 strains that significantly induced their biofilm formation (Fig. 1B and Table S2); for 14
Fig. 1. The biofilm formation and biofilm induction by strains of S. aureus. Biofilm was assessed in non-stressed conditions (A) and biofilm formed (strong, weak or no biofilm) by the types (MRSA and MSSA) of strains compared. The induction of biofilm by chemical stress (B) was assayed and strains that showed a 2-fold induction in their biofilm formation in the presence of three chemicals compared to that under non-stress conditions are shown. The chemicals used were: H2O2, hydrogen peroxide; Glycol, glycoladehyde; Formal., formaldehyde; Acrolein; Glyceral., glyceraldehyde; Glox., glyoxal; Malonal., malonaldehyde.
strains the induction was greater than 3-fold. Of these 14 strains, two had been strong biofilm forming strains without stress, while 7 were those producing a weak biofilm and 5 strains had formed no biofilm in non-stressed conditions (Table S2). The biofilm by strain WCH-SK2 was induced by H2O2, GSNO, methylglyoxal and malonaldehyde. For the nine reference strains, we found that some induced biofilm significantly in the presence of chemical stress (Table S2 includes the full data). 3.5. Confirmation of biofilm formation The microtitre plate assay for biofilm formation gives a broad assessment of the potential biofilm that is present. As confirmation the cells that assayed as forming a biofilm were indeed in a biofilm lifestyle, we used SEM and fluorescent staining techniques to identify the key characteristics of a biofilm were present. Under phase contrast microscopy the stress-induced biofilm cells could be seen to aggregate and the presence of a potential EPS matrix could be identified, in particular eDNA (Fig. 2). BacLight staining revealed eDNA as a part of this matrix (Fig. 2AeD). SEM images were taken for strains WCH-SK2 and WCH-SK12. A stress-induced and biofilm-related matrix was clearly seen in SEM (Fig. 2EeF). The EPS in S. aureus biofilm is known to vary in its
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Fig. 2. Confirmation of the biofilm phenotype and biofilm induction by S. aureus strains in the presence of stress. Biofilm factors (an EPS matrix and eDNA) were found, with the presence of eDNA surrounding the biofilm induced S. aureus WCH-SK2 cells when grown in the presence of stress was seen with fluorescent image (A) and phase contrast 100 (B). In contrast, no biofilm factors was seen in a biofilm-negative strain when grown in the presence of stress was seen with fluorescent image (C) and phase contrast (D). SEM images of S. aureus WCH-SK2 showed the image of no biofilm (E) with no stress compared to the biofilm (F) produced by strain WCH-SK2 under stressed conditions.
composition and while previously it was thought to be dominated by polysaccharide (polysaccharide intercellular adhesin, PIA) increasingly there are studies revealing eDNA (or protein) independent of polysaccharide (no PIA is present) [7]. 3.6. Sub-lethal levels of stress formed SCVs The sub-lethal concentration of chemical stresses caused the formation of SCVs by specific strains. With no chemical stress the percentage of SCV was <0.5% for every strain.
Fig. 3 illustrates the SCVs produced by strains in the presence of stress, the full quantification is summarized in Table S2. In total 38 of the 63 clinical strains could form SCVs in the presence of stress in which 18 produced SCVs in 1 chemical and 20 produced SCVs in more than 2 chemicals (Table S2). Of particular note, strain WCH-SK2 possessed the high percentage of SCVs in the presence of five stresses. It can be noted that the distribution of MSSA that changed to SCV phenotype is 5/17 and is not significantly higher than MRSA (15/46). In relation to the original site of isolation, the strains
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Fig. 3. Stress induction of S. aureus SCVs lifestyle. S. aureus strain WCH-SK2 formed normal size colonies when grown in TSA (A). This strain formed SCVs when grown in TSA with methylglyoxal (B) and with glycolaldehyde (C) and acrolein (D).
isolated from BC was slightly higher (8/63) than from LR (6/ 63), SSTI (5/63) or CF (1/63). It is noteworthy that some strains responded to a stress by the induction of SCV and biofilm formation; 6 strains induced biofilm and SCV in the presence of the same stress. 4. Discussion The overarching principle for infection and disease caused by S. aureus is its remarkable resistance to the host defenses, in particular, the innate immune system [14]. This system responds to bacterial infection by producing an array of antimicrobial agents that include reactive oxygen species and reactive nitrogen species and consequently, the more stable reactive aldehydes [15]. Therefore, the surviving S. aureus cells must have mechanisms to counteract the complex mix of toxic chemicals that are present in different host tissues (the exact concentration of these chemical varies between niches, tissues and even within a microenvironment) [16]. There have been several studies into the enzymatic defense against reactive oxygen and nitrogen species but there is very little known about how bacteria defend against reactive aldehydes. Our results indicate that in the presence of relevant reactive aldehydes certain S. aureus strains induced their biofilm formation or switched lifestyle to SCV as a means of survival. A series of investigations have indicated that the presence of defects in the biosynthesis of menadione, hemin and thymidine could be responsible for the formation of SCVs; in menD, hemB and thyA, and resulting in blocked cytochrome
biosynthesis [17]. This affected process subsequently causes a decreased amount of cellular ATP and deficient cell wall biosynthesis, pigmentation and membrane potential. Thus the slow growth, non-pigmentation and decreased uptake of antibiotics by SCV [18]. A recent study has determined that SCVs in both experimental models and human infections are highly resistant to antimicrobial processes [19]. Our work has linked this ability for persistence and SCV lifestyle to the induction and resistance by SCV to the presence of chemical stresses generated by host cells. The data we have presented here show that in a strain-specific manner S. aureus does respond to the chemical stresses it encounters within the host by the induction of its SCV lifestyle. Likewise, several bacteria have clearly been shown to be capable of biofilm formation and these biofilms are clinically relevant [20]. Numerous studies have indicated that there is a high prevalence of biofilm-mediated infections caused by S. aureus. Importantly, its persistence and antibiotic resistance, which are based on biofilm formation of S. aureus, have widely been reported [21e23]. A recent study over a large number of clinical isolates has revealed that the strains with greater multi-resistance to antimicrobial compounds have significantly increased their ability for biofilm formation rather than those with less resistance [24]. However, little is known about the impact of the chemical stress on the induction of biofilm formation. A recent report showed that within the planktonic form, certain ROS and RNS can be produced in the formation of biofilm, and thereby affecting EPS matrix development and repressing biofilm growth [25]. Perhaps as a
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linked corollary to this data, our results showed that the presence of nitrosative (GSNO) and oxidative stress (H2O2) induced the formation of biofilm in a strain-specific manner in S. aureus (Fig. 1). The induction of the biofilm by chemical stress is a definitive switch in bacterial lifestyle by distinct biofilm factors that were seen under microscopy (Fig. 2). It is intriguing to note a possible the link between the biofilm formation and SCV. Previous studies have suggested such parallels. In our study, we found there may be such a link for specific strains which achieve both a high induction in biofilm formation and high percentage of SCV in the presence of the same chemical stress. Within some strains, perhaps the metabolic state and the nature of the surface structures that are associated with SCV act as a precursor to biofilm formation. Likewise highlighting that within a population of cells that exist in a clinical isolate, in some strains especially, there is a diversity in the cell-type and the presence of chemical stresses induces these alternative lifestyle as a means of survival. Conflict of interest All authors declare that there are no conflicts of interest. Acknowledgements The isolates annotated with a NRS numbers were obtained through the Network of Antimicrobial Resistance in Staphylococcus aureus (NARSA) program supported under NIAID/ NIH Contract #HHSN272200700055C. We acknowledge the support of Jan Bell at the Women's and Children's Hospital, for strain isolation.
Appendix A. Supplementary data Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.micinf.2014.09.009. References [1] Mandal S, Berendt AR, Peacock SJ. Staphylococcus aureus bone and joint infection. J Infect 2002;44:143e51. [2] Sabirova JS, Xavier BB, Hernalsteens J-P, De Greve H, Ieven M, Goossens H, et al. Complete genome sequences of two prolific biofilmforming Staphylococcus aureus isolates belonging to USA300 and EMRSA-15 clonal lineages. Gen Ann 2014;2. [3] Stegger M, Wirth T, Andersen PS, Skov RL, De Grassi A, Sim~oes PM, et al. Origin and evolution of European community-acquired methicillinresistant Staphylococcus aureus. mBio 2014;5. [4] Klein E, Smith DL, Laxminarayan R. Hospitalizations and deaths caused by methicillin-resistant Staphylococcus aureus, United States, 1999e2005. Emerg Infect Dis 2007;13:1840e6. [5] Levin BR, Rozen DE. Non-inherited antibiotic resistance. Nat Rev Microbiol 2006;4:556e62. [6] Tuchscherr L, Heitmann V, Hussain M, Viemann D, Roth J, von Eiff C, et al. Staphylococcus aureus small-colony variants are adapted phenotypes for intracellular persistence. J Infect Dis 2010;202:1031e40.
[7] Mann EE, Rice KC, Boles BR, Endres JL, Ranjit D, Chandramohan L, et al. Modulation of eDNA release and degradation affects Staphylococcus aureus biofilm maturation. PLoS One 2009;4:e5822. [8] Tuchscherr L, Medina E, Hussain M, Volker W, Heitmann V, Niemann S, et al. Staphylococcus aureus phenotype switching: an effective bacterial strategy to escape host immune response and establish a chronic infection. EMBO Mol Med 2011;3:129e41. [9] Schembri MA, Klemm P. Biofilm formation in a hydrodynamic environment by novel FimH variants and ramifications for virulence. Infect Immun 2001;69:1322e8. [10] Herbert S, Ziebandt A-K, Ohlsen K, Sch€afer T, Hecker M, Albrecht D, et al. Repair of global regulators in Staphylococcus aureus 8325 and comparative analysis with other clinical isolates. Infect Immun 2010;78:2877e89. [11] Parra-Ruiz J, Vidaillac C, Rose WE, Rybak MJ. Activities of high-dose daptomycin, vancomycin, and moxifloxacin alone or in combination with clarithromycin or rifampin in a novel in vitro model of Staphylococcus aureus biofilm. Antimicrob Agents Chem 2010;54:4329e34. [12] Sanchez C, Mende K, Beckius M, Akers K, Romano D, Wenke J, et al. Biofilm formation by clinical isolates and the implications in chronic infections. BMC Infect Dis 2013;13:47. [13] Tang J, Chen J, Li H, Zeng P, Li J. Characterization of the adhesin genes, Staphylococcal nuclease, hemolysis and biofilm formation among Staphylococcus aureus strains isolated from different sources. Food Path Dis 2013;10:757e63. [14] van Rossum AM, Lysenko ES, Weiser JN. Host and bacterial factors contributing to the clearance of colonization by Streptococcus pneumoniae in a murine model. Infect Immun 2005;73:7718e26. [15] O'Brien PJ, Siraki AG, Shangari N. Aldehyde sources, metabolism, molecular toxicity mechanisms, and possible effects on human health. Crit Rev Toxicol 2005;35:609e62. [16] Perez JM, Arenas FA, Pradenas GA, Sandoval JM, Vasquez CC. Escherichia coli YqhD exhibits aldehyde reductase activity and protects from the harmful effect of lipid peroxidation-derived aldehydes. J Biol Chem 2008;283:7346e53. [17] Singh R, Ray P, Das A, Sharma M. Enhanced production of exopolysaccharide matrix and biofilm by a menadione-auxotrophic Staphylococcus aureus small-colony variant. J Med Microbiol 2010;59:521e7. [18] Proctor RA, von Eiff C, Kahl BC, Becker K, McNamara P, Herrmann M, et al. Small colony variants: a pathogenic form of bacteria that facilitates persistent and recurrent infections. Nat Rev Microbiol 2006;4:295e305. [19] Bates DM, von Eiff C, McNamara PJ, Peters G, Yeaman MR, Bayer AS, et al. Staphylococcus aureus menD and hemB mutants are as infective as the parent strains, but the menadione biosynthetic mutant persists within the kidney. J Infect Dis 2003;187:1654e61. [20] Donlan RM, Costerton JW. Biofilms: survival mechanisms of clinically relevant microorganisms. Clin Microbiol Rev 2002;15:167e93. [21] Singh S, Katiyar R, Kaistha SD. High oxacillin, vancomycin and fluoroquinolone resistance amongst biofilm forming Staphylococcus aureus isolates from ulcerative keratitis infections. Indian J Med Microbiol 2011;29:312e3. [22] Akiyama H, Ueda M, Kanzaki H, Tada J, Arata J. Biofilm formation of Staphylococcus aureus strains isolated from impetigo and furuncle: role of fibrinogen and fibrin. J Dermat Sci 1997;16:2e10. [23] Krismer B, Peschel A. Does Staphylococcus aureus nasal colonization involve biofilm formation? Fut Microbiol 2011;6:489e93. [24] Kwon AS, Park GC, Ryu SY, Lim DH, Lim DY, Choi CH, et al. Higher biofilm formation in multidrug-resistant clinical isolates of Staphylococcus aureus. Int J Antimicrob Agents 2008;32:68e72. [25] Arce Miranda JE, Sotomayor CE, Albesa I, Paraje MG. Oxidative and nitrosative stress in Staphylococcus aureus biofilm. FEMS Microbiol Lett 2011;315:23e9.
Short communication
The induction of Staphylococcus aureus biofilm formation or Small Colony Variants is a strain-specific response to host-generated chemical stresses Long M.G. Bui a, John D. Turnidge b, Stephen P. Kidd a,* a
Research Centre for Infectious Disease, School of Molecular and Biomedical Science, The University of Adelaide, Adelaide, South Australia 5005, Australia b SA Pathology, Women's and Children's Hospital, North Adelaide, Adelaide, South Australia 5005, Australia Received 4 August 2014; accepted 24 September 2014 Available online 2 October 2014
Abstract Staphylococcus aureus is extremely versatile. It has a capacity to persist within its host by switching to the alternative lifestyles of biofilm or Small Colony Variants (SCV). The induction of this switch has been presumed to be in response to stressed conditions, however the environmental basis has not been thoroughly investigated. We assessed the response of numerous strains to chemicals that are present in human host. There were some that induced a biofilm or SCV phenotype and indeed some inducing both lifestyles. This result illustrates the diversity within a population and a strain-specific adaptation to the presence of host-generated stresses. © 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.
Keywords: Staphylococcus aureus; Stress response; Biofilm; Small Colony Variants; Oxidative stress
1. Introduction Staphylococcus aureus is an important opportunistic pathogen that can lead to a myriad of diseases via its transit to various sites in the body. These diseases range from the minor infections of superficial skin lesions or deep abscesses, to the major invasive diseases such as pneumonia, meningitis, osteomyelitis, endocarditis, and septicaemia [1]. S. aureus strains isolated from different clinical settings display significant genetic variations and it is the difference that underpins the strain variation in physiology and stress response [2,3]. This includes the emergence of multiple drug resistant S. aureus in hospitals and communities. These drug resistant strains form the basis for Methicillin Resistant S. aureus (MRSA) [4] or more specifically HA-MRSA (Hospital Acquired MRSA) and * Corresponding author. Research Centre for Infectious Disease, School of Molecular and Biomedical Science, Molecular Life Sciences Building, The University of Adelaide, Adelaide, South Australia 5005, Australia. Tel.: þ61 (0)8 83134671; fax: þ61 (0)8 83034362. E-mail address: [email protected] (S.P. Kidd).
CA-MRSA (Community Acquired MRSA) infections. However, in a clinical setting the resistance to antibiotic therapies is not simply based on the presence or absence of a specific gene cassette/s. There are known to be “non-inherited” antibiotic resistance mechanisms that subvert antimicrobial actions via growth pathways, surface structures or processes that are beyond the transport, enzymatic detoxification or target modification mechanisms of antibiotic resistance [5]. These alternative mechanisms include modes of existence that permit S. aureus to survive in a quiescent state and thereby avoid the toxicity of antibiotics or host defences. These specifically are biofilms and Small Colony Variants (SCVs). These lifestyles are highly resistant to antimicrobial agents (therapeutic or host-generated) and are thought to be the basis for S. aureus long-term survival and persistence in the host [6]. Biofilm is characterized by the release of a matrix of polymeric substances (extracellular polymeric substances, EPS); extracellular DNA (eDNA), protein and polysaccharide substances that enclose the colonies and provides protection from host-generated chemicals (stressors) [2,7]. The specific stress response and its coupling to the subsequent production
http://dx.doi.org/10.1016/j.micinf.2014.09.009 1286-4579/© 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.
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of S. aureus biofilm, remains to be defined. The formation of SCVs is characterized by very small, non-pigmented and nonhaemolytic colonies combined with decreased uptake by antibiotics (particularly aminoglycosides). Recent research has clearly shown that the S. aureus persistence within host cells induces SCVs phenotypes and there is some evidence that it is the external environmental stress factors (reactive oxygen species, low pH, cationic peptides, limited nutrition) within host cells that induce this formation of SCVs [6,8]. It is intriguing that amongst the reactive chemicals known to be present, the most stable and equally damaging are the reactive aldehydes that a consequence of rapidly degrading superoxide and nitric oxide being present. These however have been studied very little. The environmental signals triggering the induction of biofilm or SCVs by S. aureus are still unknown. The host-generated chemical stresses as a response to bacterial infection: oxidative stress, nitrosative stress and the presence of reactive aldehydes, are important environmental factors in bacterial pathogenesis. We hypothesized that the role of these stresses could be a part of inducing the formation of biofilm and SCVs for specific strains of S. aureus. 2. Materials and methods 2.1. Sample collection Seventy-two strains of S. aureus were used; nine were accessed from the NARSA (Network of Antimicrobial Resistance in S. aureus): Mu50 (NRS1) Mu3 (NRS2), COL (NRS100), N315 (NRS70), Sanger252 (NRS71), Sanger 476 (NRS72), NCTC8325 (NRS77), MW2 (NRS123) and JE2 (USA300) and 63 from South Australia Pathology, Women's and Children's Hospital (WCH), Adelaide (Australia). This collection represents strains isolated from the blood (BC, 20 samples), lower respiratory (LR, 20 samples), skin and soft tissue infections (SSTI, 20 samples) and cystic fibrosis (CF) patients (3 samples). 2.2. Detection of MRSA and MSSA using oxacillin The susceptibility to oxacillin has been used to classify S. aureus as MRSA or MSSA. Oxacillin and cefoxitin MIC (minimum inhibitory concentration) was performed as recommended by the Clinical Laboratory Standards Institute (CLSI, formerly the National Committee for Clinical Laboratory Standards, NCCLS). Oxacillin MIC was determined by the micro-dilution assay. According to current CLSI guidelines the MRSA strains were identified as MIC 4 mg/mL and MSSA as MIC 2 mg/mL. 2.3. Growth under different conditions of chemical stress There were nine chemicals (stressors) selected to represent the key chemicals present in the host-pathogen environment; S-nitrosoglutathione (GSNO), hydrogen peroxide (H2O2), glyoxal, formaldehyde, methylglyoxal, acrolein, glyco-
laldehyde, glyceraldehyde, and malonaldehyde. All chemicals were purchased from SigmaeAldrich and prepared at a stock concentration (1 M and 1%). The MIC of each chemical stress was determined. Also the growth profile was performed in 96well plates (96 wells, flat bottom, polystyrene, Falcon) containing rich media (Tryptone Soya Broth; TSB). In brief, 5 105 cells (CFU) were inoculated into each well containing 250 mL of TSB and incubated at 37 C. Optical density (OD600 nm, BioTek EL808 Spectrophotometer) was measured after over a period of 24 h. Several concentrations of each stress were assayed and two levels were chosen for further studies; a low level of stress, in which the growth of bacteria in stress was inhibited by 20e30% of the OD630 nm of non stress conditions and high level of stress but sub-lethal; growth inhibition was 70e80% (Table S1). 2.4. Biofilm assay Biofilm formation was assayed in 96-well microtitre plates using a standard, previously described method [9]. In brief, 5 105 cells (CFU) were inoculated in TSB media into the microtitre plates that included added specific concentrations of the different chemicals, and controls of no chemical stress added and then no inoculum. These plates were incubated at 37 C for 24 h, planktonic cells removed by washing and biofilm cells visualized by staining with 0.1% crystal violet. Ethanoleacetone (20:80, v/v) was added and OD630 values were read (Biotek EL808 spectrophotometer). Each sample was performed in three separate assays with eight replicates of each. The values that are presented are an average and error bars are the standard deviation. Included as controls in the assays were strains known to be weak biofilm formers (COL and JE2 (USA300), [10]) and a strong biofilm forming strain (N315, [11]). Scanning Electron Microscopy (SEM) was used to confirm the biofilm formation and true phenotype of selected strains that were seemingly inducing their biofilm. Samples of biofilm cells exactly replicating the above samples, were fixed in SEM fixative solution (4% paraformaldehyde and 1.25% glutaraldehyde in Phosphate Buffer Solution and 4% sucrose, pH 7.2). Following dying in Osmium tetroxide (OsO4 e 2%), the samples were dehydrated using ethanol and then flooded with hexamethyldisilazane (HMDS). All the samples were then mounted on stubs and coated with platinum and visualized using Field Emission Scanning Electron Microscope (Philips XL30, Adelaide Microscopy). As further confirmation, the exact biofilm components were assessed using fluorescent staining. To identify the eDNA, a BacLight kit (Invitrogen) was used according to the manufacturer's instructions. Biofilm was formed on a slide, planktonic cells removed and the biofilm remaining was viewed with an Olympus IX-70 microscope (phase contrast 100 oil immersion objective). A filter of 496e555 nm was used to image SYTO9 fluorescence and 600e700 nm to image PI fluorescence. Fluorescence and phase contrast images were captured and false colour merged with the Metamorph software program (Version 7.7.3.0, Molecular Devices).
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2.5. Detection of SCVs phenotype in the presence of stress Each of the 72 strains (5 105 cells) was separately inoculated into 200 mL TSB in a 96-well microtitre plate with and without chemical stress. The chemical stress with specific concentrations was added and the plates were then incubated at 37 C for 18e24 h. Following the incubation, 100 mL of each culture was diluted and plated onto TSA and grown for 36e72 h at 37 C. Quantitative assessment of SCV was performed by measuring of colony size; SCVs were defined as the diameter of colonies 1 mm (1/5the1/10th the normal size of colonies) and determining the percentage of SCVs per the total number of colonies (CFU/mL). 3. Results 3.1. Identification of MRSA and MSSA We identified 46 MRSA and 17 MSSA in our set of 63 clinical strains. Of 20 BC samples; 10 were MRSA and 10 MSSA. For LR, 16 were MRSA and 4 MSSA. The 20 SSTI samples were all MRSA whereas all 3 CF samples were MSSA. These data were combined with the results generated below for the screening for biofilm and SCVs (Table S2). 3.2. Sensitivity of S. aureus strains in different kinds of stress Two concentrations for nine chemicals were chosen from the growth curves of the reference strain S. aureus JE2 including low level and sub-lethal level (Table S1). These two levels were applied and checked for all clinical isolates and reference strains (data not shown). 3.3. Biofilm formation in non-stress condition Strong biofilm was identified as the mean OD630 is 0.2, weak biofilm was classified as the mean OD630 within 0.1e0.2 and no biofilm as OD630 <0.1. These values are consistent to previous reports for S. aureus biofilm formation [12,13]. Of the clinical strains, 15.9% produced strong biofilm, 54% producing biofilm weakly and 30.1% isolates are biofilm negative (Fig. 1A). Indeed, there were no observed differences in specimen sources for the strong, weak or no biofilm categories. 3.4. Sub-lethal of environmental stress induced biofilm formation A significant induction of biofilm with the addition of chemical stress was defined as greater than two-fold induction compared to that measured with no stress added. With low level of stress there was no significant induction. However at the high concentration of stress some strains induced their biofilm formation. There were 21 strains that significantly induced their biofilm formation (Fig. 1B and Table S2); for 14
Fig. 1. The biofilm formation and biofilm induction by strains of S. aureus. Biofilm was assessed in non-stressed conditions (A) and biofilm formed (strong, weak or no biofilm) by the types (MRSA and MSSA) of strains compared. The induction of biofilm by chemical stress (B) was assayed and strains that showed a 2-fold induction in their biofilm formation in the presence of three chemicals compared to that under non-stress conditions are shown. The chemicals used were: H2O2, hydrogen peroxide; Glycol, glycoladehyde; Formal., formaldehyde; Acrolein; Glyceral., glyceraldehyde; Glox., glyoxal; Malonal., malonaldehyde.
strains the induction was greater than 3-fold. Of these 14 strains, two had been strong biofilm forming strains without stress, while 7 were those producing a weak biofilm and 5 strains had formed no biofilm in non-stressed conditions (Table S2). The biofilm by strain WCH-SK2 was induced by H2O2, GSNO, methylglyoxal and malonaldehyde. For the nine reference strains, we found that some induced biofilm significantly in the presence of chemical stress (Table S2 includes the full data). 3.5. Confirmation of biofilm formation The microtitre plate assay for biofilm formation gives a broad assessment of the potential biofilm that is present. As confirmation the cells that assayed as forming a biofilm were indeed in a biofilm lifestyle, we used SEM and fluorescent staining techniques to identify the key characteristics of a biofilm were present. Under phase contrast microscopy the stress-induced biofilm cells could be seen to aggregate and the presence of a potential EPS matrix could be identified, in particular eDNA (Fig. 2). BacLight staining revealed eDNA as a part of this matrix (Fig. 2AeD). SEM images were taken for strains WCH-SK2 and WCH-SK12. A stress-induced and biofilm-related matrix was clearly seen in SEM (Fig. 2EeF). The EPS in S. aureus biofilm is known to vary in its
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Fig. 2. Confirmation of the biofilm phenotype and biofilm induction by S. aureus strains in the presence of stress. Biofilm factors (an EPS matrix and eDNA) were found, with the presence of eDNA surrounding the biofilm induced S. aureus WCH-SK2 cells when grown in the presence of stress was seen with fluorescent image (A) and phase contrast 100 (B). In contrast, no biofilm factors was seen in a biofilm-negative strain when grown in the presence of stress was seen with fluorescent image (C) and phase contrast (D). SEM images of S. aureus WCH-SK2 showed the image of no biofilm (E) with no stress compared to the biofilm (F) produced by strain WCH-SK2 under stressed conditions.
composition and while previously it was thought to be dominated by polysaccharide (polysaccharide intercellular adhesin, PIA) increasingly there are studies revealing eDNA (or protein) independent of polysaccharide (no PIA is present) [7]. 3.6. Sub-lethal levels of stress formed SCVs The sub-lethal concentration of chemical stresses caused the formation of SCVs by specific strains. With no chemical stress the percentage of SCV was <0.5% for every strain.
Fig. 3 illustrates the SCVs produced by strains in the presence of stress, the full quantification is summarized in Table S2. In total 38 of the 63 clinical strains could form SCVs in the presence of stress in which 18 produced SCVs in 1 chemical and 20 produced SCVs in more than 2 chemicals (Table S2). Of particular note, strain WCH-SK2 possessed the high percentage of SCVs in the presence of five stresses. It can be noted that the distribution of MSSA that changed to SCV phenotype is 5/17 and is not significantly higher than MRSA (15/46). In relation to the original site of isolation, the strains
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Fig. 3. Stress induction of S. aureus SCVs lifestyle. S. aureus strain WCH-SK2 formed normal size colonies when grown in TSA (A). This strain formed SCVs when grown in TSA with methylglyoxal (B) and with glycolaldehyde (C) and acrolein (D).
isolated from BC was slightly higher (8/63) than from LR (6/ 63), SSTI (5/63) or CF (1/63). It is noteworthy that some strains responded to a stress by the induction of SCV and biofilm formation; 6 strains induced biofilm and SCV in the presence of the same stress. 4. Discussion The overarching principle for infection and disease caused by S. aureus is its remarkable resistance to the host defenses, in particular, the innate immune system [14]. This system responds to bacterial infection by producing an array of antimicrobial agents that include reactive oxygen species and reactive nitrogen species and consequently, the more stable reactive aldehydes [15]. Therefore, the surviving S. aureus cells must have mechanisms to counteract the complex mix of toxic chemicals that are present in different host tissues (the exact concentration of these chemical varies between niches, tissues and even within a microenvironment) [16]. There have been several studies into the enzymatic defense against reactive oxygen and nitrogen species but there is very little known about how bacteria defend against reactive aldehydes. Our results indicate that in the presence of relevant reactive aldehydes certain S. aureus strains induced their biofilm formation or switched lifestyle to SCV as a means of survival. A series of investigations have indicated that the presence of defects in the biosynthesis of menadione, hemin and thymidine could be responsible for the formation of SCVs; in menD, hemB and thyA, and resulting in blocked cytochrome
biosynthesis [17]. This affected process subsequently causes a decreased amount of cellular ATP and deficient cell wall biosynthesis, pigmentation and membrane potential. Thus the slow growth, non-pigmentation and decreased uptake of antibiotics by SCV [18]. A recent study has determined that SCVs in both experimental models and human infections are highly resistant to antimicrobial processes [19]. Our work has linked this ability for persistence and SCV lifestyle to the induction and resistance by SCV to the presence of chemical stresses generated by host cells. The data we have presented here show that in a strain-specific manner S. aureus does respond to the chemical stresses it encounters within the host by the induction of its SCV lifestyle. Likewise, several bacteria have clearly been shown to be capable of biofilm formation and these biofilms are clinically relevant [20]. Numerous studies have indicated that there is a high prevalence of biofilm-mediated infections caused by S. aureus. Importantly, its persistence and antibiotic resistance, which are based on biofilm formation of S. aureus, have widely been reported [21e23]. A recent study over a large number of clinical isolates has revealed that the strains with greater multi-resistance to antimicrobial compounds have significantly increased their ability for biofilm formation rather than those with less resistance [24]. However, little is known about the impact of the chemical stress on the induction of biofilm formation. A recent report showed that within the planktonic form, certain ROS and RNS can be produced in the formation of biofilm, and thereby affecting EPS matrix development and repressing biofilm growth [25]. Perhaps as a
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linked corollary to this data, our results showed that the presence of nitrosative (GSNO) and oxidative stress (H2O2) induced the formation of biofilm in a strain-specific manner in S. aureus (Fig. 1). The induction of the biofilm by chemical stress is a definitive switch in bacterial lifestyle by distinct biofilm factors that were seen under microscopy (Fig. 2). It is intriguing to note a possible the link between the biofilm formation and SCV. Previous studies have suggested such parallels. In our study, we found there may be such a link for specific strains which achieve both a high induction in biofilm formation and high percentage of SCV in the presence of the same chemical stress. Within some strains, perhaps the metabolic state and the nature of the surface structures that are associated with SCV act as a precursor to biofilm formation. Likewise highlighting that within a population of cells that exist in a clinical isolate, in some strains especially, there is a diversity in the cell-type and the presence of chemical stresses induces these alternative lifestyle as a means of survival. Conflict of interest All authors declare that there are no conflicts of interest. Acknowledgements The isolates annotated with a NRS numbers were obtained through the Network of Antimicrobial Resistance in Staphylococcus aureus (NARSA) program supported under NIAID/ NIH Contract #HHSN272200700055C. We acknowledge the support of Jan Bell at the Women's and Children's Hospital, for strain isolation.
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