Ultrastructural Pathology, Early Online, 1–11, 2014 ! Informa Healthcare USA, Inc. ISSN: 0191-3123 print / 1521-0758 online DOI: 10.3109/01913123.2014.950780
ORIGINAL ARTICLE
Ultrastructural Morphologic Changes in Mycobacterial Biofilm in Different Extreme Condition Virendra Kumar, PhD*, Tarun Kumar Sachan, MSc*, Pragya Sharma, PhD, and Krishna Dutta Rawat, MSc
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National JALMA Institute for Leprosy and Other Mycobacterial Diseases, Agra, Uttar Pradesh, India
ABSTRACT The aim of this study was to investigate the morphologic and ultrastructural features of biofilms of slow and fast-growing mycobacteria in different stress conditions, presence and absence of oleic acid albumin dextrose catalase (OADC) enrichment and at different temperatures: 30, 37 and 42 C. Four hundred mycobacterial isolates were taken. The biomass of each biofilm was quantified using a modified microtiter plate assay method. Isolates were divided into those that formed fully established biofilms, moderately attached biofilms and weakly adherent biofilms by comparison with a known biofilm-forming strain. The large quantity of biofilm was produced by Mycobacterium smegmatis at temperature 37 and 42 C as compared to 30 C. Mycobacterium fortuitum and M. avium developed large amount of biofilm at 30 C as compared to 37 and 42 C. Mycobacterium tuberculosis developed strong biofilm at 37 C and no biofilm at 30 and 42 C in Sauton’s media. The selected non-tuberculous mycobacteria and H37Rv developed strong biofilm in the presence of OADC enrichment in Sauton’s medium. Microscopic examination of biofilms by scanning electron microscopy revealed that poorly adherent biofilm formers failed to colonize the entire surface of the microtiter well. While moderately adherent biofilm formers grew in uniform monolayers but failed to develop a mature three-dimensional structure. SEM analysis of an isolate representative of the group formed fully established biofilms with a textured, multilayered, three-dimensional structure. Keywords: ECM, M. avium, M. fortuitum, M. smegmatis, M. tuberculosis, SEM
environment in which it develops such as pH, iron, oxygen, ionic strength, temperature and nutrient level [5]. Excess available carbon and the limitation of nitrogen, potassium or phosphate promote EPS and thickness of biofilm [6]. Colonies can vary widely in morphology, and there is a clear correlation between highly structured morphologies and the ability of a cell to produce an ECM [7]. Biofilms can form on almost any abiotic or biologic surface and it is estimated that 65% of all human bacterial infections involve biofilms. Biofilm formation provide protection from a wide range of environmental challenges, such as UV exposure [8], metal toxicity [9], acid exposure [10], dehydration and salinity phagocytosis, and several antibiotics and antimicrobial agents [11]. Mycobacterium tuberculosis causes deadly infectious diseases tuberculosis (TB) and during the last 10–15
Biofilm is a microbially derived sessile community characterized by cells that are irreversibly attached to a substratum or interface or to each other, are embedded in a matrix of extracellular polymeric substances (EPS) that they have produced, and exhibit an altered phenotype with respect to growth rate and gene transcription [1]. Biofilms are composed primarily of microbial cells and EPS. EPS may account for 50–90% of the total organic carbon of biofilms [2] and can be considered as the primary matrix material of the biofilm. The ECM (extracellular matrix) synthesized by microbial cells vary greatly in their composition and hence in their chemical and physical properties. The electron micrograph of structured colonies showed that they are composed of cells surrounded by an ECM [3,4]. The exact structure of any biofilm is probably a unique feature of the
Received 18 July 2014; Accepted 24 July 2014; Published online 5 September 2014 *These authors contributed equally for the paper. Correspondence: Dr Virendra Kumar, PhD, Scientist ‘C’ and Head, Department of Electron Microscopy, National JALMA Institute for Leprosy and Other Mycobacterial Diseases, Tajganj, Agra, Uttar Pradesh 282001, India. Tel: +91-562-2331751-54 (Extn: 208). Fax: +91-562-2331755. E-mail: [email protected]
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years, several cases of non-tuberculous mycobacteria (NTM) which are involved in various opportunistic infections as well as wound infection exist in biofilm formation such as M. avium, M. intracellulare and M. fortuitum [12]. In addition, Roberts et al. [13] observed biofilm formation in M. gordonae, M. fortuitum, M. abscessus, M. septicum and M. gilvum [14] while Ojha and Hatfull [15] reported biofilm formation in M. tuberculosis H37Rv. Furthermore, Low et al. 2011 [16] found biofilm-like structure in vitro and in vivo in M. ulcerans displaying an abundant ECM. Very little is known about the ultrastructure of mycobacterial biofilm or about biofilm architecture in different conditions. The present study describes the development of biofilm in different environmental conditions and highlights the ultrastructural features, characteristics, distribution and morphology of mycobacterial biofilms that develop at liquid–air interface in presence or absence of oleic acid albumin dextrose catalase (OADC) enrichment and at different temperatures.
MATERIALS AND METHODS Four hundred mycobacterial isolates were taken for this study. Mycobacterium fortuitum, M. smegmatis and M. tuberculosis H37Rv were obtained from Mycobacterial Repository Centre of National JALMA Institute for Leprosy and Other Mycobacterial Diseases, Agra, Uttar Pradesh, India, and fourth characterize isolate, M. avium was obtained from National Institute of Tuberculosis, Chennai, Tamilnadu, India. Additionally, control strain of M. smegmatis MC2 155 was obtained from Molecular Biophysics Unit, Indian Institute of Science, Bangalore, Karnataka, India.
Molecular identification of mycobacterial isolates DNA extraction DNA from selected mycobacterial isolates was extracted from growth on Lowenstein–Jensen (LJ) slants. It included lysis with lysozyme, proteinase K/sodium dodecyl sulfate (SDS) and extraction with N-cetyl-N,N,N-trimethyl ammonium bromide/ sodium chloride (CTAB/NaCl) as described elsewhere van Soolingen et al. [17]. Polymerase chain reaction–restriction fragment length polymorphism and spoligotyping Polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) analysis of hsp65 gene [18] was applied to characterize four mycobacterial isolates, M. smegmatis, M. fortuitum and M. tuberculosis
FIGURE 1. PCR-restriction enzyme pattern analysis (PRA) of selected mycobacteria: (Lane 1) 50 bp ladder, (Lane 2) M. tuberculosis, (Lane 3) M. smegmatis and (Lane 4 and 5) M. fortuitum.
(Figure 1). Additionally, M. tuberculosis H37Rv was confirmed by spoligotyping which was done with the help of a commercially available kit (Ocimum Biosolutions, Hyderabad, Telangana, India) and as described previously by Kamerbeek et al. [19]. Results were compared with the international database Spol DB 4.0 (Unite´ de la Tuberculose et des Mycobacte´ries, Institut Pasteur de Guadeloupe, Guadeloupe, France) [20] (Figure 2). Culture of biofilm by fast-growing mycobacteria Developed planktonic cultures of M. smegmatis and M. fortuitum in Middlebrook 7H9 broth (Difco, Middlebrook, France) supplemented with 0.05% Tween-80 and 2% glucose. For assessment of biofilm formation, M. smegmatis and M. fortuitum were grown in modified Sauton’s medium (containing, g L 1: ferric ammonium citrate 0.0167, L-aspargine 1.33, citric acid 0.66, magnesium sulfate 0.166, dipotassium hydrogen phosphate 0.287, sodium dihydrogen phosphate 0.633, sodium chloride 0.4, polysorbate-80 0.833, and glycerol 2%, w/v) incubated at 7 d. In addition, Middlebrook 7H9 agar medium was supplemented with 2% glucose and used as the solid medium for mycobacterial growth. Initial observations of biofilm formation were carried out in 25 mL polystyrene tissue culture flasks (NUNC, Roskilde, Denmark). Five milliliter of Sauton’s medium was added into the flasks and they were inoculated with 1:100 dilutions from a stationary-phase culture. The flasks were incubated in a humidified incubator and observed at different time of intervals. For observing the surface pellicles, cultures were similarly inoculated in 30 mL Sauton’s medium in 90-mm diameter polystyrene petri dishes. Culture of biofilm by slow grower mycobacteria Single-isolated colony of M. tuberculosis and M. avium from Middlebrook’s 7H10 agar medium containing 0.5% (vol/vol) glycerol and 10% (vol/vol) OADC was Ultrastructural Pathology
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FIGURE 2. Spoligotypig of M. tuberculosis H37Rv.
used to inoculate 5 mL of Middlebrook’s 7H9 broth medium containing 0.5% (vol/vol) glycerol, 0.05% Tween-80 and 10% (vol/vol) OADC and incubated at 37 C for 7 d. However after 7 d, 1 mL primary culture was used to inoculate 9 mL of Middlebrook’s 7H9 media to prepare secondary culture. These secondary cultures were incubated for 7 d at 37 C. The culture from these bottles was centrifuged at 1467 RCF for 5 min at 4 C in Eppendorf tubes. Subsequently, the pellet was washed in PBS to remove the media and suspended in 200 mL MB7H9 media containing 10% OADC and without OADC. The 200 mL cultures were inoculated with 10 mL Sauton’s Media in 25 mL polystyrene flask. Quantification of biofilm of slow grower and fast grower mycobacteria in different stress conditions Biofilm formation in the presence and absence of OADC as well as different temperature, 30, 37 and 42 C was adopted as described by O’toole et al. [5]. The plates were incubated at different temperatures for 1 and 2 weeks for fast-growing mycobacteria and 2 and 4 weeks for slow-growing mycobacteria. To observe the effect of OADC enrichment (with and without OADC) 1 107 cells were poured in Sauton’s media in the wells of left and rightmost row of the microtiter plate. The effects of OADC and different temperatures on the development of biofilm of selected mycobacteria were recorded based upon their optical density at 570 nm. Biofilm biomass was quantified using a modification of a methodology described by Monsenego [21]. After incubation, the microtiter plate was rinsed twice in PBS to remove loosely attached planktonic cells and dried for 30 min at 37 C. Each well of the microtiter plate was stained with filtered 0.5% (w/v) crystal violet for 5 min. Excess crystal violet was removed by gently washing twice with distilled water. Subsequently, 250 mL 70% ethanol was added to leach the crystal violet from the stained biofilms. The A570 was measured using a microtiter plate reader (SpectraMax M2, Sunnyvale, CA). The wells of the polystyrene microtiter plate, contained biomass that remained bound to the surface, followed by washing steps for viewing as a genuine biofilm. The sample of the biofilm was not taken from the wells of the microtiter plate because the biomass may not have a biofilm rather a deposit of planktonic cells. !
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Analysis of biofilm formation. The capacity of each mycobacterial isolate to form a biofilm was compared with that of the confluent biofilm-forming M. smegmatis MC2 155 control by analyzing the absorbance of the crystal violet stain obtained for each biofilm. Each isolate to be assigned a percentage value depending on the proportion of biofilm and biomass which was able to establish after 7 d in comparison with the control (taken as 100%). Eight replicate wells were included for each isolate of each biofilm assay which was carried out three times. However, each isolate were also divided into three groups depending on observation whether they formed fully established biofilms with 475% of the biomass of the positive control, moderately adherent biofilms with 25–75% biomass or weak biofilms with525% of the biomass of the positive control. Scanning electron microscopy. The mycobacterial biofilm developed in microtiter dish as described above were fixed in a solution of 2.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4, overnight at 4 C. The samples were rinsed once in the same buffer and dehydrated by increasing concentrations of ethanol (30, 50, 70, 90 and 100%). The samples were dried in a fume hood and fixed on to stubs with conductive selfadhesive carbon tapes, coated with gold film sputtering and used for analysis with scanning electron microscopy (SEM) (S3000-N; Hitachi, Tokyo, Japan). The ultrastructural picture shown by SEM described as thin, thick and thicker of the biofilm developed in the microtiter plate and described as weak, moderate and strong, respectively. Ruthenium red staining. A two loopful growth were taken on glass slide and a smear was prepared. One milliliter of 0.15% ruthenium red 0.5% glutaraldehyde dissolved in 100 mM cacodylate buffer was poured on the glass slide and kept for 1 h at room temperature. This stain was removed, 0.05% ruthenium red 5% glutaraldehyde in a 100-mM cacodylate buffer solution was added, and the preparation was incubated for 2 h at room temperature. This stain was removed, and the slides were washed five times for 10 min in 100 mM cacodylate buffer. The slides were viewed by light microscopy for exopolysaccharide (EPS) staining, see Prouty and Gunn [22].
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RESULTS One well of each isolate was characterized as weakly adherent, moderately adherent and fully established biofilm which was confirmed by SEM, after incubation. The weak biofilm former failed to colonize the majority of the surface of the polystyrene peg. Small clusters of cells were observed, but these did not aggregate to form a monolayer or a more mature biofilm structure. The representative moderately adherent isolate grew in a uniform monolayer, but did not form a mature multi-layered biofilm. The SEM analysis of the isolates that formed 475% of the biofilm biomass of the control confirmed that this isolate developed a dense, mature biofilm on the polystyrene peg, with a multi-layered threedimensional structure. The SEM images supported
the results achieved by crude crystal violet staining of biofilm biomass and highlight the value of the 96-peg plate format for high-throughput analysis of biofilm formation in these isolates.
Analysis of biofilm formation in M. smegmatis The SEM analysis of single rod-shaped cells of biofilm of M. smegmatis showed that EPS adhere to the wall of cells (Figure 3). In Sauton’s media at first week, M. smegmatis developed moderate biofilm at 30 and 42 C and weak biofilm at 37 C (Figures 4 and 5). At second week in Sauton’s media, these bacteria developed moderate biofilm at 30 C and strong biofilm at 42 and 37 C (Figure 6). However, addition of OADC into Sauton’s media induced more biofilm development at
FIGURE 3. EPS secreted by single mycobacterial cell (in bracket).
FIGURE 4. Thickness of biofilm of M. smegmatis [(A) weak, (B) moderate and (C) strong].
FIGURE 5. Thickness of biofilm of M. smegmatis in Sauton’s media at first week at 30, 37 and 42 C [(A) moderate, (B) weak and (C) moderate]. Ultrastructural Pathology
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FIGURE 6. Thickness of biofilm of M. smegmatis in Sauton’s media at second week in 30, 37 and 42 C [(A) moderate, (B) strong and (C) strong].
FIGURE 7. Thickness of biofilm of M. smegmatis in presence [(A) weak and (B) moderate] and absence of OADC condition [(C) moderate and (D) strong].
second week as compared to first week. At first week these mycobacteria developed weak biofilm without OADC and became moderate with OADC addition in Sauton’s media. At second week these bacteria developed moderate amount of biofilm without OADC and strong biofilm with OADC in Sauton’s media. The strong biofilm revealed irregular smooth colony and bacteria encased in a thick matrix of EPS. The biofilm appeared to have more abundant ECM holding the rods together, interspersed with channels and the bacilli appeared to be arranged together into linear cord-like formations. The cells of M. smegmatis replicate in this biofilm community and the quantity of the ECM increases with time (Figure 7). The ruthenium red staining also showed the amount of ECM justifying the thickness of biofilm (Figure 8; Tables 1 and 2).
High-resolution micrographs indicated that cell clusters exhibited heterogeneous morphology with a mycelial-like texture. The bacilli of different sizes were seen ranging from curved and rod shaped. At second week, these bacteria developed strong biofilm at 30 C and weak biofilm at 37 C and moderate biofilm at 42 C in Sauton’s media but at first week no significant differences were observed (Figure 9). Significant differences were observed in Sauton’s media at second week in which moderate amount of biofilm was developed without OADC and strong biofilm with OADC. At first week in Sauton’s media, weak amount of biofilm was developed without OADC and moderate biofilm with OADC (Figure 10). The ruthenium red staining also showing the thickness of biofilm (Figure 11; Tables 1 and 2).
Analysis of biofilm formation in M. fortuitum
Analysis of M. avium biofilm formation
The ultrastructure of M. fortuitum showed dense aggregation of cells, composed of large cell clusters.
SEM analysis revealed irregular and smooth crystalline structures, appeared to be calcifications of biofilm
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FIGURE 8. Ruthenium red staining of M. smegmatis biofilm developed in different temperature and OADC condition [(A) weak, (B) moderate and (C) strong].
TABLE 1. Quantification of biofilm at different temperatures in Sauton’s medium. Mycobacterium smegmatis Temperature ( C) 30 37 42
Mycobacterium fortuitum
Mycobacterium avium
Mycobacterium tuberculosis
Week I
Week II
Week I
Week II
Week II
Week IV
Week II
Week IV
M W M
M S S
S M M
S W M
M W M
M W S
W S W
No S No
M, moderate; S, strong; W, weak.
TABLE 2. Quantification of biofilm in presence (+) and absence ( ) of OADC in Sauton’s media.
OADC – +
Mycobacterium smegmatis
Mycobacterium fortuitum
Mycobacterium avium
Mycobacterium tuberculosis
Week I
Week II
Week I
Week II
Week II
Week IV
Week II
Week IV
W M
M S
W M
M S
No W
M S
W M
M S
M, moderate; S, strong; W, weak.
FIGURE 9. Thickness of biofilm of M. fortuitum in culture [(A) weak, (B) moderate and (C) strong].
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FIGURE 10. Effect of OADC and temperature on M. fortuitum biofilm formation [(A) weak, (B) moderate and (C) strong].
FIGURE 11. Ruthenium red staining of M. fortuitum biofilm developed in different temperature and OADC condition [(A) weak, (B) moderate and (C) strong].
FIGURE 12. Effect of OADC and temperature on M. avium biofilm formation [(A) weak, (B) moderate and (C) strong].
material or the formation of microcrystalline structures. At higher magnification, the surfaces were not completely smooth but exhibited microabrasions and irregularities, which may contribute to bacterial adhesion. The overall surface configuration, with its furrowed corrugated facade would appear to make the appliance more conducive to bacterial and biofilm accumulation. In Sauton’s media at fourth week, these mycobacteria developed strong biofilm with OADC and moderate biofilm without OADC significantly but at second week no significant differences were found. For development of biofilm, we selected temperature point i.e. 30 and 37 C, 42 C in Sauton’s media. In Sauton’s media in second week, these bacteria develop moderate amount of biofilm at 30 and 42 C and weak biofilm at 37 C but at fourth week in Sauton’s media moderate biofilm was !
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developed at 30 C, weak at 37 C and strong at 42 C (Figures 12 and 13). The ruthenium red staining also showing the thickness of biofilm in Figure 14 and Tables 1 and 2.
Analysis of M. tuberculosis H37Rv biofilm formation This is the first report of the ultrastructural observation of the biofilm of M. tuberculosis H37Rv. They depicted distinctly different morphologic features as compared to NTM. The globular deposits were evident and distributed across the entire surface but seemed to be concentrated around the raised edges of the furrowed surface. The furrows and groove were also observed in these biofilm and the rod-shaped cell
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FIGURE 13. Effect of OADC and temperature on M. avium [(A) weak, (B) moderate and (C) strong].
FIGURE 14. Ruthenium red staining of M. avium biofilm developed in different temperature and OADC condition [(A) weak, (B) moderate and (C) strong].
FIGURE 15. Effect of OADC and temperature on M. tuberculosis biofilm formation [(A) weak, (B) moderate and (C) strong].
was inserted into the ECM and dense amount of ECM was found in this biofilm architecture. Mycobacterium tuberculosis H37Rv does not develop biofilm in Sauton’s media at second week at 30 and 42 C while strong biofilm was developed at 37 C. In Sauton’s media, these mycobacteria developed weak biofilm at 30 and 42 C and strong biofilm at 37 C at second week. In Sauton’s media at fourth week, these bacteria developed no biofilm at 30 and 42 C and strong biofilm at 37 C. In Sauton’s media, at fourth week, these mycobacteria significantly developed strong biofilm in the presence of OADC as compared to without OADC and at second week significantly moderate biofilm developed with OADC and weak amount of biofilm developed without OADC
(Figures 15 and 16). The ruthenium red staining also showing the thickness of biofilm in Figure 17 and Tables 1 and 2.
DISCUSSION Mycobacterium tuberculosis, commonly called MTB or simply the tubercle bacillus, causes TB. TB remains a major global health problem. In 2011, there were 8.7 million new cases of active TB worldwide and in India 2.0–2.5 illion approximately. It has been estimated that there were 310,000 incident cases of multidrugresistant TB (MDR) and 63,000 were in India. M. tuberculosis related to M. leprae, the causative agent of Ultrastructural Pathology
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FIGURE 16. Effect of OADC and temperature on M. tuberculosis biofilm formation [(A) no biofilm, (B) weak, (C) moderate and (D) strong biofilm].
FIGURE 17. Ruthenium red staining of M. tuberculosis biofilm developed in different temperature and OADC condition [(A) weak, (B) moderate and (C) strong].
leprosy, as well as to numerous other mycobacterial species, which are referred to collectively as NTM are widely distributed in the environment, particularly in wet soil, marshland, streams, rivers and estuaries. The global incidence of NTM pulmonary disease averages one case per 100,000 person years. The presence and development of biofilm have now been reported increasingly to play a major role in pathogenesis of disease as it aids bacterial survival within the host shielded in an EPS. Various factors which include composition of media, types of sugars available in the media, temperature have been suggested to play a role in the development and survival of these biofilms in the host. The quality and quantity of nutrients, ions and several other substances present in the media are known to influence bacterial behavior and would certainly be expected to have a regulatory role in the formation of biofilm. For example, Pseudomonas aeruginosa uses organic acids as a source of carbon to form biofilm, see O’toole et al. [5]. Further, they have suggested that pH, nutrient levels, iron, oxygen, ionic strength and temperature, may also play a role in the rate of microbial attachment to a substratum. The optimal temperature for most cultures of NTM is between 28 and 37 C. Mycobacterium avium and M. fortuitum develop strong biofilm at 30 C and weak biofilm at 37 and 42 C while Johansen et al. [23] reported that M. avium developed more amount of biofilm in MB7H9 media in presence of OADC at 28 C and at 3 week of !
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incubation period. Our study is similar to Johansen et al. [23] and showed that highest amount of biofilm formation in Sauton’s media and in the presence of OADC enrichment and low amount of biofilm without OADC enrichment. In the present study, it was observed that Middlebrook OADC enrichment provides the essential nutrients for the cultivation of mycobacteria. However, oleic acid is a long chain of fatty acid utilized by mycobacterium species in their metabolism while dextrose used as a source of carbon or energy. The albumin binds toxins to protect the bacilli from toxic agents and the catalase catalyzes the reaction of iron with molecular oxygen to stimulate revival of damaged cells. Therefore, OADC enrichment are recommended for the isolation, cultivation and susceptibility testing of mycobacterium especially M. tuberculosis. Therefore, our study also suggested that OADC is the best supplement for biofilm growth as agreed by Nyvad and Fejerskov [24]. Mycobacterium smegmatis developed moderate biofilm at 30 C and strong biofilm at 42 and 37 C. Mycobacterium smegmatis demonstrates rapid growth on solid media over the temperature range of 24–45 C [7]. SEM was employed in the present study to visualize the general morphology and the detailed ultrastructural features of biofilm formed on a hydrophobic surface such as polystyrene surface in presence and absence of OADC. The use of SEM to determine the morphology and ultrastructure of bacteria and biofilms has been utilized before in
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10 V. Kumar et al. several other studies [25–27]. When visualized at high magnification, the surfaces were not completely smooth but exhibited irregularities which may contribute to bacterial adhesion. The overall surface configuration with its furrowed corrugated fac¸ade would appear to make the appliance more conductive to bacterial biofilm accumulation. The fact that biofilm structure were observed in fully hydrated living specimen using a compound microscope is strongly suggestive that the biofilm structure observed here are real, and not the result of either sample preparation or specimen handling. In the present investigation, results suggested that there are specialized zone within the biofilm consisting of bacteria associated with extracellular proteins. The extracellular ultrastructure of multispecies studied here by forming a more heterogenous matrix in other species. Therefore, it is possible that the observed high degree of matrix organization could be the result of growing pure culture under constant condition and may not be as pronounced in the environment. In the present study, techniques were used to investigate the detailed surface structure and configuration of the material, which may lead to an understanding the patterns of biofilm formation in mycobacteria. Observations represent a rapid and convenient means of assessing the pattern of colonization as well as surface structure of the biofilm. In case of biofilm formation, ECM has a pre-eminent role with its presence being demonstrated in all the biofilms studied so far. The chemical components of the ECM can be polysaccharides, nucleic acids, proteins, extracellular DNA and possibly even the debris left behind by the dead cells are also reported Sutherland [28] and Webb et al. [29]. However, the composition of mycobacterial biofilms is significantly different from that of other bacteria, containing an ECM rich in lipids rather than polysaccharides, see Ojha et al. [30]. To confirm that does mycobacteria produce an ECM, ruthenium red stain, a specific stain used in EPS detection, and reacts with the surface of mycobacteria was used and different amount of biofilm was found as also observed by O’Toole et al. [5]. The ultrastructure of biofilm showed different three-dimensional structure such as corncob appearance, mycelia like appearance, honeycomb and mold-like appearance. Mycobacterium fortuitum biofilm revealed the cell clusters exhibited heterogeneous morphology with a mycelial like and pleiomorphic cell structures. At higher magnification, mycobacteria of different sizes ranging from short curved rods to longer branching rods were evident. These observations are in agreement with earlier finding of Lie [31]. However, September et al. [12] observed microcolony branching cell, void and channel between microcolony and develops patches of aggregate cells formed biofilm with heterogeneous morphology in M. chelonae.
The SEM image of biofilm of M. chubuensie, M. gilvum, M. obuense, M. parafortuitum and M. vaccae showed curved structures arranged in a definite order and voids were clearly visible with long fiber and short fiber [32]. In our ultrastructural analysis, biofilm of M. smegmatis showed more abundant ECM holding the rods together, interspersed with water channels. It was observed that tendency of the bacilli to become arranged together into linear cord-like formations. On the other hand M. fortuitum exhibited heterogeneous morphology with a mycelial-like texture while M. avium and M. tuberculosis showed crystalline and globular structure. However, we have clearly observed ultrastructurally that OADC enrichment is most important for thickness of biofilm. Probably, this is the first report so far, demonstrating the ultrastructure of biofilm developed in different selected temperature as well as in presence and absence of OADC.
CONCLUSION The present work provides the information regarding the ultrastructure details of biofilm of M. fortuitum, M. smegmatis, M. avium and M. tuberculosis, developed in presence and absence of OADC enrichment. It is evident that bacterial cells have the ability to aggregate into particular three-dimensional assemblages. But little is known about the three-dimensional ultrastructural feature of mycobacterial biofilm developed in various conditions. SEM is helpful in employing a rapid and convenient technique for assessing the pattern of colonization as well as screening samples for major bacterial morph types which may lead to an understanding of the patterns of biofilm formation and the designing of new drug target. This improved and more comprehensive understanding of process connected with biofilm will lead to new knowledge that would help in developing novel, effective control strategies for prevention of biofilm and a resulting improvement in patient management.
ACKNOWLEDGEMENTS Authors are thankful to Dr. V.M. Katoch, Dr. R.M. Agarwal, Dr. K. Venkatesan, Dr. K.K. Mohanty, Dr. Beenu Joshi, Dr. V.D. Sharma and Dr. D.S. Chauhan for support and to prepare this manuscript.
DECLARATION OF INTEREST The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article. Ultrastructural Pathology
Morphologic Changes in Mycobacterial Biofilm We are highly thankful for funding from the Department of Biotechnology Govt. of India Grant No. BT/PR10435/Med/30/85/2008.
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REFERENCES 1. Donlan RM, Costerton, JW. Biofilms: Survival mechanisms of clinically relevant microorganisms. J Clin Microbiol Rev 2002;15: 167–93. 2. Flemming HC, Mayer WJ, Korstgens C, Borchard W. Cohesiveness in biofilm matrix polymers. Community structure and co-operation in biofilms, SGM Symposium Series, Vol. 59. Cambridge: Cambridge University Press; 2000:87–105. 3. Friedman L, Kolter R. Genes involved in matrix formation in Pseudomonas aeruginosa PA14 biofilm. Mol Microbiol 2004; 51: 675–90. 4. Yarwood JM, Bartels DJ, Volper EM, Greenberg EP. Quorum sensing in Staphylococcus aureus biofilms. J Bacteriol 2004;186: 1838–50. 5. O’Toole G, Kaplan HB, Kolter R. Biofilm formation as microbial development. Ann Rev Microbiol 2000;54: 49–79. 6. Sukontapatipark W, El-Agroudi MA, Selliseth NJ, et al. Bacterial colonization associated with fixed orthodontic appliances. A scanning electron microscopy study. Eur J Orthod 2001;23: 475–84. 7. Rastogi P, Saini H, Dixit J, Singhal R. Probiotics and oral health. Natl J Maxillofac Surg 2011;2: 6–9. 8. Espeland EM, Wetzel RG. Complexation, stabilization, and UV photolysis of extracellular and surface-bound glucosidase and alkaline phosphatase: Implications for biofilm microbiota. Microb Ecol 2001;42: 572–85. 9. Teitzel GM, Parsek MR. Heavy metal resistance of biofilm and planktonic Pseudomonas aeruginosa. Appl Environ Microbiol 2003;69: 2313–20. 10. McNeill K, Hamilton IR. Acid tolerance response of biofilm cells of Streptococcus mutans. FEMS Microbiol Lett 2003;221: 25–30. 11. Mah TF, O’Toole GA. Mechanisms of biofilm resistance to antimicrobial agents. Trends Microbiol 2001;9: 34–9. 12. September SM, Brozel VS, Venter SN. Diversity of non tuberculoid mycobacterium species in biofilms of urban and semi urban drinking water distribution systems. Appl Environ Microbiol 2004;70: 7571–3. 13. Roberts GD, Koneman EW, Kim YK. In: Balows A, Hausler WJ, Hermann KL, et al., eds. Mycobacterium in manual of clinical microbiology, 5 eds. Washington: American Society for Microbiol Press; 1991:304–39. 14. Wolfaardt GM, Lawrence JR, Robarts RD, et al. Multicellular organization in a degradative biofilm community. Appl Environ Microbiol 1994;60: 434–46. 15. Ojha A, Hatfull GF. The role of iron in Mycobacterium smegmatis biofilm formation: The exochelin siderophore is essential in limiting iron conditions for biofilm formation but not for planktonic growth. Mol Microbiol 2007;66: 468–83. 16. Low B, Lee W, Seneviratne CJ, et al. Ultrastructure and morphology of biofilms on thermoplastic orthodontic appliances in ‘fast’ and ‘slow’ plaque formers. Eur J Orthod 2011;33: 577–83.
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17. van Soolingen D, Qian L, de Haas PE, et al. Predominance of a single genotype of Mycobacterium tuberculosis in countries of east Asia. J Clin Microbiol 1995;33: 3234–8. 18. Telenti A, Marchesi F, Balz M, et al. Rapid identification of mycobacteria to the species level by polymerase chain reaction and restriction enzyme analysis. J Clin Microbiol 1993;31: 175–8. 19. Kamerbeek J, Schouls LM, Kolk A, et al. Simultaneous detection and strain differentiation of Mycobacterium tuberculosis for diagnosis and epidemiology. J Clin Microbiol 1997;35: 907–14. 20. Brudey K, Driscoll JR, Rigout L, et al. Mycobacterium tuberculosis complex genetic diversity: Mining the fourth international database (SpolDB 4) for classification, population genetics and epidemiology. BMC Microbiol 2006;6: 23. doi:10.1186/1471-2180-6-23. 21. Monsenego P. The presence of microorganisms on the fitting denture complete surface: A study in vivo. J Oral Rehabil 2000;27: 708–13. 22. Prouty AM, Gunn. Comparative analysis of Salmonella enterica Serovar Typhimurium biofilm formation on gallstones and on glass december. Infect Immun 2003;71:7154–8. 23. Johansen BT, Agdestein A, Oslen I, et al. Biofilm formation by Mycobacterium avium isolates originating from human, swine and birds. BMC Microbiol 2009;9: 159. doi:10.1186/ 1471-2180-9-159. 24. Nyvad B, Fejerskov O. Scanning electron microscopy of early microbial colonization of human enamel and root surfaces in vivo. Scand J Dent Res 1987;95: 287–96. 25. Yildiz FH, Schoolnik GK. Vibrio cholerae O1 El Tor: Identification of a gene cluster required for the rugose colony type, exopolysaccharide production, chlorine resistance, and biofilm formation. Proc Natl Assoc Sci USA 1999; 96: 4028–33. 26. Marsollier L, Brodin P, Jackson M, et al. Impact of Mycobacterium ulcerans biofilm on transmissibility to ecological niches buruli ulcer pathogenesis. PLoS Pathog 2007;3: 62. 27. Stoodley P, Dodds I, Boyle JD, Lappin-Scott HM. Influence of hydrodynamics and nutrients on biofilm structure. J Appl Microbiol 1998;85: 19s–28s. 28. Sutherland IW. The biofilm matrix-an immobilized but dynamic microbial environment. Trends Microbiol 2001;9: 222–7. 29. Webb JS, Thompson LS, James S, et al. Cell death in Pseudomonas aeruginosa biofilm development. J Bacteriol 2003;185: 4585–92. 30. Ojha AK, Baughn AD, Sambandan D. Growth of Mycobacterium tuberculosis biofilms containing free mycolic acids and harbouring drug-tolerant bacteria. Mol Microbiol 2008;69: 164–74. 31. Lie T. Morphological studies on dental plaque formation. Acta Odontol Scand 1979;37: 73–85. 32. Agusti G, Astola O, Guell ER, et al. Surface spreading motility shown by a group of phylogenetically related, rapidly growing pigmented mycobacteria suggests that motility is a common property of mycobacterial species but is restricted to smooth colonies. J Bacteriol 2008;190: 6894–902.
ORIGINAL ARTICLE
Ultrastructural Morphologic Changes in Mycobacterial Biofilm in Different Extreme Condition Virendra Kumar, PhD*, Tarun Kumar Sachan, MSc*, Pragya Sharma, PhD, and Krishna Dutta Rawat, MSc
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National JALMA Institute for Leprosy and Other Mycobacterial Diseases, Agra, Uttar Pradesh, India
ABSTRACT The aim of this study was to investigate the morphologic and ultrastructural features of biofilms of slow and fast-growing mycobacteria in different stress conditions, presence and absence of oleic acid albumin dextrose catalase (OADC) enrichment and at different temperatures: 30, 37 and 42 C. Four hundred mycobacterial isolates were taken. The biomass of each biofilm was quantified using a modified microtiter plate assay method. Isolates were divided into those that formed fully established biofilms, moderately attached biofilms and weakly adherent biofilms by comparison with a known biofilm-forming strain. The large quantity of biofilm was produced by Mycobacterium smegmatis at temperature 37 and 42 C as compared to 30 C. Mycobacterium fortuitum and M. avium developed large amount of biofilm at 30 C as compared to 37 and 42 C. Mycobacterium tuberculosis developed strong biofilm at 37 C and no biofilm at 30 and 42 C in Sauton’s media. The selected non-tuberculous mycobacteria and H37Rv developed strong biofilm in the presence of OADC enrichment in Sauton’s medium. Microscopic examination of biofilms by scanning electron microscopy revealed that poorly adherent biofilm formers failed to colonize the entire surface of the microtiter well. While moderately adherent biofilm formers grew in uniform monolayers but failed to develop a mature three-dimensional structure. SEM analysis of an isolate representative of the group formed fully established biofilms with a textured, multilayered, three-dimensional structure. Keywords: ECM, M. avium, M. fortuitum, M. smegmatis, M. tuberculosis, SEM
environment in which it develops such as pH, iron, oxygen, ionic strength, temperature and nutrient level [5]. Excess available carbon and the limitation of nitrogen, potassium or phosphate promote EPS and thickness of biofilm [6]. Colonies can vary widely in morphology, and there is a clear correlation between highly structured morphologies and the ability of a cell to produce an ECM [7]. Biofilms can form on almost any abiotic or biologic surface and it is estimated that 65% of all human bacterial infections involve biofilms. Biofilm formation provide protection from a wide range of environmental challenges, such as UV exposure [8], metal toxicity [9], acid exposure [10], dehydration and salinity phagocytosis, and several antibiotics and antimicrobial agents [11]. Mycobacterium tuberculosis causes deadly infectious diseases tuberculosis (TB) and during the last 10–15
Biofilm is a microbially derived sessile community characterized by cells that are irreversibly attached to a substratum or interface or to each other, are embedded in a matrix of extracellular polymeric substances (EPS) that they have produced, and exhibit an altered phenotype with respect to growth rate and gene transcription [1]. Biofilms are composed primarily of microbial cells and EPS. EPS may account for 50–90% of the total organic carbon of biofilms [2] and can be considered as the primary matrix material of the biofilm. The ECM (extracellular matrix) synthesized by microbial cells vary greatly in their composition and hence in their chemical and physical properties. The electron micrograph of structured colonies showed that they are composed of cells surrounded by an ECM [3,4]. The exact structure of any biofilm is probably a unique feature of the
Received 18 July 2014; Accepted 24 July 2014; Published online 5 September 2014 *These authors contributed equally for the paper. Correspondence: Dr Virendra Kumar, PhD, Scientist ‘C’ and Head, Department of Electron Microscopy, National JALMA Institute for Leprosy and Other Mycobacterial Diseases, Tajganj, Agra, Uttar Pradesh 282001, India. Tel: +91-562-2331751-54 (Extn: 208). Fax: +91-562-2331755. E-mail: [email protected]
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years, several cases of non-tuberculous mycobacteria (NTM) which are involved in various opportunistic infections as well as wound infection exist in biofilm formation such as M. avium, M. intracellulare and M. fortuitum [12]. In addition, Roberts et al. [13] observed biofilm formation in M. gordonae, M. fortuitum, M. abscessus, M. septicum and M. gilvum [14] while Ojha and Hatfull [15] reported biofilm formation in M. tuberculosis H37Rv. Furthermore, Low et al. 2011 [16] found biofilm-like structure in vitro and in vivo in M. ulcerans displaying an abundant ECM. Very little is known about the ultrastructure of mycobacterial biofilm or about biofilm architecture in different conditions. The present study describes the development of biofilm in different environmental conditions and highlights the ultrastructural features, characteristics, distribution and morphology of mycobacterial biofilms that develop at liquid–air interface in presence or absence of oleic acid albumin dextrose catalase (OADC) enrichment and at different temperatures.
MATERIALS AND METHODS Four hundred mycobacterial isolates were taken for this study. Mycobacterium fortuitum, M. smegmatis and M. tuberculosis H37Rv were obtained from Mycobacterial Repository Centre of National JALMA Institute for Leprosy and Other Mycobacterial Diseases, Agra, Uttar Pradesh, India, and fourth characterize isolate, M. avium was obtained from National Institute of Tuberculosis, Chennai, Tamilnadu, India. Additionally, control strain of M. smegmatis MC2 155 was obtained from Molecular Biophysics Unit, Indian Institute of Science, Bangalore, Karnataka, India.
Molecular identification of mycobacterial isolates DNA extraction DNA from selected mycobacterial isolates was extracted from growth on Lowenstein–Jensen (LJ) slants. It included lysis with lysozyme, proteinase K/sodium dodecyl sulfate (SDS) and extraction with N-cetyl-N,N,N-trimethyl ammonium bromide/ sodium chloride (CTAB/NaCl) as described elsewhere van Soolingen et al. [17]. Polymerase chain reaction–restriction fragment length polymorphism and spoligotyping Polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) analysis of hsp65 gene [18] was applied to characterize four mycobacterial isolates, M. smegmatis, M. fortuitum and M. tuberculosis
FIGURE 1. PCR-restriction enzyme pattern analysis (PRA) of selected mycobacteria: (Lane 1) 50 bp ladder, (Lane 2) M. tuberculosis, (Lane 3) M. smegmatis and (Lane 4 and 5) M. fortuitum.
(Figure 1). Additionally, M. tuberculosis H37Rv was confirmed by spoligotyping which was done with the help of a commercially available kit (Ocimum Biosolutions, Hyderabad, Telangana, India) and as described previously by Kamerbeek et al. [19]. Results were compared with the international database Spol DB 4.0 (Unite´ de la Tuberculose et des Mycobacte´ries, Institut Pasteur de Guadeloupe, Guadeloupe, France) [20] (Figure 2). Culture of biofilm by fast-growing mycobacteria Developed planktonic cultures of M. smegmatis and M. fortuitum in Middlebrook 7H9 broth (Difco, Middlebrook, France) supplemented with 0.05% Tween-80 and 2% glucose. For assessment of biofilm formation, M. smegmatis and M. fortuitum were grown in modified Sauton’s medium (containing, g L 1: ferric ammonium citrate 0.0167, L-aspargine 1.33, citric acid 0.66, magnesium sulfate 0.166, dipotassium hydrogen phosphate 0.287, sodium dihydrogen phosphate 0.633, sodium chloride 0.4, polysorbate-80 0.833, and glycerol 2%, w/v) incubated at 7 d. In addition, Middlebrook 7H9 agar medium was supplemented with 2% glucose and used as the solid medium for mycobacterial growth. Initial observations of biofilm formation were carried out in 25 mL polystyrene tissue culture flasks (NUNC, Roskilde, Denmark). Five milliliter of Sauton’s medium was added into the flasks and they were inoculated with 1:100 dilutions from a stationary-phase culture. The flasks were incubated in a humidified incubator and observed at different time of intervals. For observing the surface pellicles, cultures were similarly inoculated in 30 mL Sauton’s medium in 90-mm diameter polystyrene petri dishes. Culture of biofilm by slow grower mycobacteria Single-isolated colony of M. tuberculosis and M. avium from Middlebrook’s 7H10 agar medium containing 0.5% (vol/vol) glycerol and 10% (vol/vol) OADC was Ultrastructural Pathology
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FIGURE 2. Spoligotypig of M. tuberculosis H37Rv.
used to inoculate 5 mL of Middlebrook’s 7H9 broth medium containing 0.5% (vol/vol) glycerol, 0.05% Tween-80 and 10% (vol/vol) OADC and incubated at 37 C for 7 d. However after 7 d, 1 mL primary culture was used to inoculate 9 mL of Middlebrook’s 7H9 media to prepare secondary culture. These secondary cultures were incubated for 7 d at 37 C. The culture from these bottles was centrifuged at 1467 RCF for 5 min at 4 C in Eppendorf tubes. Subsequently, the pellet was washed in PBS to remove the media and suspended in 200 mL MB7H9 media containing 10% OADC and without OADC. The 200 mL cultures were inoculated with 10 mL Sauton’s Media in 25 mL polystyrene flask. Quantification of biofilm of slow grower and fast grower mycobacteria in different stress conditions Biofilm formation in the presence and absence of OADC as well as different temperature, 30, 37 and 42 C was adopted as described by O’toole et al. [5]. The plates were incubated at different temperatures for 1 and 2 weeks for fast-growing mycobacteria and 2 and 4 weeks for slow-growing mycobacteria. To observe the effect of OADC enrichment (with and without OADC) 1 107 cells were poured in Sauton’s media in the wells of left and rightmost row of the microtiter plate. The effects of OADC and different temperatures on the development of biofilm of selected mycobacteria were recorded based upon their optical density at 570 nm. Biofilm biomass was quantified using a modification of a methodology described by Monsenego [21]. After incubation, the microtiter plate was rinsed twice in PBS to remove loosely attached planktonic cells and dried for 30 min at 37 C. Each well of the microtiter plate was stained with filtered 0.5% (w/v) crystal violet for 5 min. Excess crystal violet was removed by gently washing twice with distilled water. Subsequently, 250 mL 70% ethanol was added to leach the crystal violet from the stained biofilms. The A570 was measured using a microtiter plate reader (SpectraMax M2, Sunnyvale, CA). The wells of the polystyrene microtiter plate, contained biomass that remained bound to the surface, followed by washing steps for viewing as a genuine biofilm. The sample of the biofilm was not taken from the wells of the microtiter plate because the biomass may not have a biofilm rather a deposit of planktonic cells. !
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Analysis of biofilm formation. The capacity of each mycobacterial isolate to form a biofilm was compared with that of the confluent biofilm-forming M. smegmatis MC2 155 control by analyzing the absorbance of the crystal violet stain obtained for each biofilm. Each isolate to be assigned a percentage value depending on the proportion of biofilm and biomass which was able to establish after 7 d in comparison with the control (taken as 100%). Eight replicate wells were included for each isolate of each biofilm assay which was carried out three times. However, each isolate were also divided into three groups depending on observation whether they formed fully established biofilms with 475% of the biomass of the positive control, moderately adherent biofilms with 25–75% biomass or weak biofilms with525% of the biomass of the positive control. Scanning electron microscopy. The mycobacterial biofilm developed in microtiter dish as described above were fixed in a solution of 2.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4, overnight at 4 C. The samples were rinsed once in the same buffer and dehydrated by increasing concentrations of ethanol (30, 50, 70, 90 and 100%). The samples were dried in a fume hood and fixed on to stubs with conductive selfadhesive carbon tapes, coated with gold film sputtering and used for analysis with scanning electron microscopy (SEM) (S3000-N; Hitachi, Tokyo, Japan). The ultrastructural picture shown by SEM described as thin, thick and thicker of the biofilm developed in the microtiter plate and described as weak, moderate and strong, respectively. Ruthenium red staining. A two loopful growth were taken on glass slide and a smear was prepared. One milliliter of 0.15% ruthenium red 0.5% glutaraldehyde dissolved in 100 mM cacodylate buffer was poured on the glass slide and kept for 1 h at room temperature. This stain was removed, 0.05% ruthenium red 5% glutaraldehyde in a 100-mM cacodylate buffer solution was added, and the preparation was incubated for 2 h at room temperature. This stain was removed, and the slides were washed five times for 10 min in 100 mM cacodylate buffer. The slides were viewed by light microscopy for exopolysaccharide (EPS) staining, see Prouty and Gunn [22].
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RESULTS One well of each isolate was characterized as weakly adherent, moderately adherent and fully established biofilm which was confirmed by SEM, after incubation. The weak biofilm former failed to colonize the majority of the surface of the polystyrene peg. Small clusters of cells were observed, but these did not aggregate to form a monolayer or a more mature biofilm structure. The representative moderately adherent isolate grew in a uniform monolayer, but did not form a mature multi-layered biofilm. The SEM analysis of the isolates that formed 475% of the biofilm biomass of the control confirmed that this isolate developed a dense, mature biofilm on the polystyrene peg, with a multi-layered threedimensional structure. The SEM images supported
the results achieved by crude crystal violet staining of biofilm biomass and highlight the value of the 96-peg plate format for high-throughput analysis of biofilm formation in these isolates.
Analysis of biofilm formation in M. smegmatis The SEM analysis of single rod-shaped cells of biofilm of M. smegmatis showed that EPS adhere to the wall of cells (Figure 3). In Sauton’s media at first week, M. smegmatis developed moderate biofilm at 30 and 42 C and weak biofilm at 37 C (Figures 4 and 5). At second week in Sauton’s media, these bacteria developed moderate biofilm at 30 C and strong biofilm at 42 and 37 C (Figure 6). However, addition of OADC into Sauton’s media induced more biofilm development at
FIGURE 3. EPS secreted by single mycobacterial cell (in bracket).
FIGURE 4. Thickness of biofilm of M. smegmatis [(A) weak, (B) moderate and (C) strong].
FIGURE 5. Thickness of biofilm of M. smegmatis in Sauton’s media at first week at 30, 37 and 42 C [(A) moderate, (B) weak and (C) moderate]. Ultrastructural Pathology
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FIGURE 6. Thickness of biofilm of M. smegmatis in Sauton’s media at second week in 30, 37 and 42 C [(A) moderate, (B) strong and (C) strong].
FIGURE 7. Thickness of biofilm of M. smegmatis in presence [(A) weak and (B) moderate] and absence of OADC condition [(C) moderate and (D) strong].
second week as compared to first week. At first week these mycobacteria developed weak biofilm without OADC and became moderate with OADC addition in Sauton’s media. At second week these bacteria developed moderate amount of biofilm without OADC and strong biofilm with OADC in Sauton’s media. The strong biofilm revealed irregular smooth colony and bacteria encased in a thick matrix of EPS. The biofilm appeared to have more abundant ECM holding the rods together, interspersed with channels and the bacilli appeared to be arranged together into linear cord-like formations. The cells of M. smegmatis replicate in this biofilm community and the quantity of the ECM increases with time (Figure 7). The ruthenium red staining also showed the amount of ECM justifying the thickness of biofilm (Figure 8; Tables 1 and 2).
High-resolution micrographs indicated that cell clusters exhibited heterogeneous morphology with a mycelial-like texture. The bacilli of different sizes were seen ranging from curved and rod shaped. At second week, these bacteria developed strong biofilm at 30 C and weak biofilm at 37 C and moderate biofilm at 42 C in Sauton’s media but at first week no significant differences were observed (Figure 9). Significant differences were observed in Sauton’s media at second week in which moderate amount of biofilm was developed without OADC and strong biofilm with OADC. At first week in Sauton’s media, weak amount of biofilm was developed without OADC and moderate biofilm with OADC (Figure 10). The ruthenium red staining also showing the thickness of biofilm (Figure 11; Tables 1 and 2).
Analysis of biofilm formation in M. fortuitum
Analysis of M. avium biofilm formation
The ultrastructure of M. fortuitum showed dense aggregation of cells, composed of large cell clusters.
SEM analysis revealed irregular and smooth crystalline structures, appeared to be calcifications of biofilm
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FIGURE 8. Ruthenium red staining of M. smegmatis biofilm developed in different temperature and OADC condition [(A) weak, (B) moderate and (C) strong].
TABLE 1. Quantification of biofilm at different temperatures in Sauton’s medium. Mycobacterium smegmatis Temperature ( C) 30 37 42
Mycobacterium fortuitum
Mycobacterium avium
Mycobacterium tuberculosis
Week I
Week II
Week I
Week II
Week II
Week IV
Week II
Week IV
M W M
M S S
S M M
S W M
M W M
M W S
W S W
No S No
M, moderate; S, strong; W, weak.
TABLE 2. Quantification of biofilm in presence (+) and absence ( ) of OADC in Sauton’s media.
OADC – +
Mycobacterium smegmatis
Mycobacterium fortuitum
Mycobacterium avium
Mycobacterium tuberculosis
Week I
Week II
Week I
Week II
Week II
Week IV
Week II
Week IV
W M
M S
W M
M S
No W
M S
W M
M S
M, moderate; S, strong; W, weak.
FIGURE 9. Thickness of biofilm of M. fortuitum in culture [(A) weak, (B) moderate and (C) strong].
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FIGURE 10. Effect of OADC and temperature on M. fortuitum biofilm formation [(A) weak, (B) moderate and (C) strong].
FIGURE 11. Ruthenium red staining of M. fortuitum biofilm developed in different temperature and OADC condition [(A) weak, (B) moderate and (C) strong].
FIGURE 12. Effect of OADC and temperature on M. avium biofilm formation [(A) weak, (B) moderate and (C) strong].
material or the formation of microcrystalline structures. At higher magnification, the surfaces were not completely smooth but exhibited microabrasions and irregularities, which may contribute to bacterial adhesion. The overall surface configuration, with its furrowed corrugated facade would appear to make the appliance more conducive to bacterial and biofilm accumulation. In Sauton’s media at fourth week, these mycobacteria developed strong biofilm with OADC and moderate biofilm without OADC significantly but at second week no significant differences were found. For development of biofilm, we selected temperature point i.e. 30 and 37 C, 42 C in Sauton’s media. In Sauton’s media in second week, these bacteria develop moderate amount of biofilm at 30 and 42 C and weak biofilm at 37 C but at fourth week in Sauton’s media moderate biofilm was !
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developed at 30 C, weak at 37 C and strong at 42 C (Figures 12 and 13). The ruthenium red staining also showing the thickness of biofilm in Figure 14 and Tables 1 and 2.
Analysis of M. tuberculosis H37Rv biofilm formation This is the first report of the ultrastructural observation of the biofilm of M. tuberculosis H37Rv. They depicted distinctly different morphologic features as compared to NTM. The globular deposits were evident and distributed across the entire surface but seemed to be concentrated around the raised edges of the furrowed surface. The furrows and groove were also observed in these biofilm and the rod-shaped cell
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FIGURE 13. Effect of OADC and temperature on M. avium [(A) weak, (B) moderate and (C) strong].
FIGURE 14. Ruthenium red staining of M. avium biofilm developed in different temperature and OADC condition [(A) weak, (B) moderate and (C) strong].
FIGURE 15. Effect of OADC and temperature on M. tuberculosis biofilm formation [(A) weak, (B) moderate and (C) strong].
was inserted into the ECM and dense amount of ECM was found in this biofilm architecture. Mycobacterium tuberculosis H37Rv does not develop biofilm in Sauton’s media at second week at 30 and 42 C while strong biofilm was developed at 37 C. In Sauton’s media, these mycobacteria developed weak biofilm at 30 and 42 C and strong biofilm at 37 C at second week. In Sauton’s media at fourth week, these bacteria developed no biofilm at 30 and 42 C and strong biofilm at 37 C. In Sauton’s media, at fourth week, these mycobacteria significantly developed strong biofilm in the presence of OADC as compared to without OADC and at second week significantly moderate biofilm developed with OADC and weak amount of biofilm developed without OADC
(Figures 15 and 16). The ruthenium red staining also showing the thickness of biofilm in Figure 17 and Tables 1 and 2.
DISCUSSION Mycobacterium tuberculosis, commonly called MTB or simply the tubercle bacillus, causes TB. TB remains a major global health problem. In 2011, there were 8.7 million new cases of active TB worldwide and in India 2.0–2.5 illion approximately. It has been estimated that there were 310,000 incident cases of multidrugresistant TB (MDR) and 63,000 were in India. M. tuberculosis related to M. leprae, the causative agent of Ultrastructural Pathology
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FIGURE 16. Effect of OADC and temperature on M. tuberculosis biofilm formation [(A) no biofilm, (B) weak, (C) moderate and (D) strong biofilm].
FIGURE 17. Ruthenium red staining of M. tuberculosis biofilm developed in different temperature and OADC condition [(A) weak, (B) moderate and (C) strong].
leprosy, as well as to numerous other mycobacterial species, which are referred to collectively as NTM are widely distributed in the environment, particularly in wet soil, marshland, streams, rivers and estuaries. The global incidence of NTM pulmonary disease averages one case per 100,000 person years. The presence and development of biofilm have now been reported increasingly to play a major role in pathogenesis of disease as it aids bacterial survival within the host shielded in an EPS. Various factors which include composition of media, types of sugars available in the media, temperature have been suggested to play a role in the development and survival of these biofilms in the host. The quality and quantity of nutrients, ions and several other substances present in the media are known to influence bacterial behavior and would certainly be expected to have a regulatory role in the formation of biofilm. For example, Pseudomonas aeruginosa uses organic acids as a source of carbon to form biofilm, see O’toole et al. [5]. Further, they have suggested that pH, nutrient levels, iron, oxygen, ionic strength and temperature, may also play a role in the rate of microbial attachment to a substratum. The optimal temperature for most cultures of NTM is between 28 and 37 C. Mycobacterium avium and M. fortuitum develop strong biofilm at 30 C and weak biofilm at 37 and 42 C while Johansen et al. [23] reported that M. avium developed more amount of biofilm in MB7H9 media in presence of OADC at 28 C and at 3 week of !
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incubation period. Our study is similar to Johansen et al. [23] and showed that highest amount of biofilm formation in Sauton’s media and in the presence of OADC enrichment and low amount of biofilm without OADC enrichment. In the present study, it was observed that Middlebrook OADC enrichment provides the essential nutrients for the cultivation of mycobacteria. However, oleic acid is a long chain of fatty acid utilized by mycobacterium species in their metabolism while dextrose used as a source of carbon or energy. The albumin binds toxins to protect the bacilli from toxic agents and the catalase catalyzes the reaction of iron with molecular oxygen to stimulate revival of damaged cells. Therefore, OADC enrichment are recommended for the isolation, cultivation and susceptibility testing of mycobacterium especially M. tuberculosis. Therefore, our study also suggested that OADC is the best supplement for biofilm growth as agreed by Nyvad and Fejerskov [24]. Mycobacterium smegmatis developed moderate biofilm at 30 C and strong biofilm at 42 and 37 C. Mycobacterium smegmatis demonstrates rapid growth on solid media over the temperature range of 24–45 C [7]. SEM was employed in the present study to visualize the general morphology and the detailed ultrastructural features of biofilm formed on a hydrophobic surface such as polystyrene surface in presence and absence of OADC. The use of SEM to determine the morphology and ultrastructure of bacteria and biofilms has been utilized before in
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10 V. Kumar et al. several other studies [25–27]. When visualized at high magnification, the surfaces were not completely smooth but exhibited irregularities which may contribute to bacterial adhesion. The overall surface configuration with its furrowed corrugated fac¸ade would appear to make the appliance more conductive to bacterial biofilm accumulation. The fact that biofilm structure were observed in fully hydrated living specimen using a compound microscope is strongly suggestive that the biofilm structure observed here are real, and not the result of either sample preparation or specimen handling. In the present investigation, results suggested that there are specialized zone within the biofilm consisting of bacteria associated with extracellular proteins. The extracellular ultrastructure of multispecies studied here by forming a more heterogenous matrix in other species. Therefore, it is possible that the observed high degree of matrix organization could be the result of growing pure culture under constant condition and may not be as pronounced in the environment. In the present study, techniques were used to investigate the detailed surface structure and configuration of the material, which may lead to an understanding the patterns of biofilm formation in mycobacteria. Observations represent a rapid and convenient means of assessing the pattern of colonization as well as surface structure of the biofilm. In case of biofilm formation, ECM has a pre-eminent role with its presence being demonstrated in all the biofilms studied so far. The chemical components of the ECM can be polysaccharides, nucleic acids, proteins, extracellular DNA and possibly even the debris left behind by the dead cells are also reported Sutherland [28] and Webb et al. [29]. However, the composition of mycobacterial biofilms is significantly different from that of other bacteria, containing an ECM rich in lipids rather than polysaccharides, see Ojha et al. [30]. To confirm that does mycobacteria produce an ECM, ruthenium red stain, a specific stain used in EPS detection, and reacts with the surface of mycobacteria was used and different amount of biofilm was found as also observed by O’Toole et al. [5]. The ultrastructure of biofilm showed different three-dimensional structure such as corncob appearance, mycelia like appearance, honeycomb and mold-like appearance. Mycobacterium fortuitum biofilm revealed the cell clusters exhibited heterogeneous morphology with a mycelial like and pleiomorphic cell structures. At higher magnification, mycobacteria of different sizes ranging from short curved rods to longer branching rods were evident. These observations are in agreement with earlier finding of Lie [31]. However, September et al. [12] observed microcolony branching cell, void and channel between microcolony and develops patches of aggregate cells formed biofilm with heterogeneous morphology in M. chelonae.
The SEM image of biofilm of M. chubuensie, M. gilvum, M. obuense, M. parafortuitum and M. vaccae showed curved structures arranged in a definite order and voids were clearly visible with long fiber and short fiber [32]. In our ultrastructural analysis, biofilm of M. smegmatis showed more abundant ECM holding the rods together, interspersed with water channels. It was observed that tendency of the bacilli to become arranged together into linear cord-like formations. On the other hand M. fortuitum exhibited heterogeneous morphology with a mycelial-like texture while M. avium and M. tuberculosis showed crystalline and globular structure. However, we have clearly observed ultrastructurally that OADC enrichment is most important for thickness of biofilm. Probably, this is the first report so far, demonstrating the ultrastructure of biofilm developed in different selected temperature as well as in presence and absence of OADC.
CONCLUSION The present work provides the information regarding the ultrastructure details of biofilm of M. fortuitum, M. smegmatis, M. avium and M. tuberculosis, developed in presence and absence of OADC enrichment. It is evident that bacterial cells have the ability to aggregate into particular three-dimensional assemblages. But little is known about the three-dimensional ultrastructural feature of mycobacterial biofilm developed in various conditions. SEM is helpful in employing a rapid and convenient technique for assessing the pattern of colonization as well as screening samples for major bacterial morph types which may lead to an understanding of the patterns of biofilm formation and the designing of new drug target. This improved and more comprehensive understanding of process connected with biofilm will lead to new knowledge that would help in developing novel, effective control strategies for prevention of biofilm and a resulting improvement in patient management.
ACKNOWLEDGEMENTS Authors are thankful to Dr. V.M. Katoch, Dr. R.M. Agarwal, Dr. K. Venkatesan, Dr. K.K. Mohanty, Dr. Beenu Joshi, Dr. V.D. Sharma and Dr. D.S. Chauhan for support and to prepare this manuscript.
DECLARATION OF INTEREST The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article. Ultrastructural Pathology
Morphologic Changes in Mycobacterial Biofilm We are highly thankful for funding from the Department of Biotechnology Govt. of India Grant No. BT/PR10435/Med/30/85/2008.
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