87
Epidemiol. Infect. (1992), 109, 87-96 Printed in Great Britain
A study into the mechanism of the Crystal Violet reaction in Staphylococcus aureus M. R. BARER, D. BURDESS AND R. FREEMAN Department of Microbiology, University of Newcastle upon Tyne, Framlington Place, Newcastle upon Tyne NE2 4HH, UK
(Accepted 2 March 1992) SUMMARY
The mechanism of the Crystal Violet (CV) reaction, a trait which has been related to biotype, source and pathogenicity in Staphylococcus aureus, was investigated in agar and broth studies. White reactions could be converted to purple and vice versa by altering the incubation conditions on agar. Broth reactions examined macroscopically and by spectrophotometry revealed that both white and purple human biotype strains take up CV but the former then progressively modify the dye more quickly than the latter. A cell-associated product of CV was detected in white and purple strains by reverse-phase thinlayer chromatography of methanol extracts. White strains appear to produce a second additional product from CV. The white reaction was not inhibited by chloramphenicol or azide but did depend on viable cells with a nutrient source. CV MICs and MBCs for 10 white and 10 purple reactors showed no gross differences in susceptibility, while a standardized assay for the rate of CV modifying activity (52 strains) demonstrated that the two categories comprise discrete populations which alter CV at different rates. Although most white strains belong to either or both of phage-typing groups V and II, purple strains with this pattern of susceptibility and white strains without it both occur. The capacity to modify CV slowly or rapidly appears to subdivide human biotype strains independently of their phage group and is associated in the former case with their capacity to produce hospital-acquired and invasive infections. INTRODUCTION
The Crystal Violet (CV) reaction of Staphylococcus aureus has been used to separate biotypes of animal and human origin [1-4] and has been correlated with the capacity to produce enterotoxins [4-7]. We have recently obtained evidence that purple reacting strains (CV type C reactions) are significantly associated with hospital-acquired and invasive infections [8]. Both these and earlier observations suggesting that the CV reaction may provide an indicator for the 'hospital staphylococcus' [9] have stimulated us to examine the nature of the CV reaction. This report documents the effects of varying inoculum size, CV content and incubation conditions on the outcome of the reaction. We also present evidence for a nutritionally dependent CV modifying system which operates at different rates in white and purple reactors.
88
M. R. BARER AND OTHERS MATERIALS AN!) METHODS
Bacterial strains Fifty-two representative strains of S. aureus showing purple, white and yellow CV reactions were selected from the series of clinical isolates reported on previously [8]. Six biotype A strains (see below), two white reactors belonging separately to phage groups II and V, two yellow reactors (both phage group II), and two purple reactors belonging to phage groups II and III, were all used in the initial experiments concerned with reproducibility and inoculum size. From these, strains 10455 (phage group II) and RVI (phage group III) were subsequently used as controls for the white and purple reactions respectively. All strains were examined for fibrinolysin [10], bovine coagulase [8] and fl-haemolysin [8]. These tests showed that, with three exceptions all strains clearly belonged to the human biotype (A) [11]. The three exceptions (one purple and two white reactors), each of which gave negative in all three tests, were typable with the standard human phage set.
Phage typing For the strains listed above, phage typing was done by the Division of Hospital Infection, Central Public Health Laboratory. The remainder were typed as described previously [8]. Media Strains were stored on nutrient agar slopes and subcultured on nutrient agar plates (Lab M, Bury, UK). Standard CV agar and CV broth contained 10 jug/ml Crystal Violet (C.I. 2555. Sigma) in nutrient agar and nutrient broth (Lab M). Preparation of inocula All incubations were at 37 °C in air unless stated otherwise. For the routine CV agar test a visible quantity of growth from separated colonies of overnight growth on nutrient agar was transferred by loop and inoculated in circles of 5-6 mm diameter on CV agar. In some experiments sterile cellulose acetate filters were interposed between the inoculum and the agar surface (13 mm diameter, 0 22 ,um pore size, Nuflow. Oxoid). For the studies requiring more defined inocula t8 24 h broth cultures were prepared in glass Erlenmeyer flasks shaken at approximately 100 cycle/min. Cells were harvested by centrifugation and resuspended in the minimum volume of nutrient broth (approx 15 ml for cells harvested from 1 1 broth) required to allow accurate pipetting. MIC/MBC determinations MICs were determined using a multipoint inoculator to deliver approximately x 6 103 organisms onto nutrient agar plates containing serial twofold dilutions of CV. Inocular were prepared from saline (0-9 %) suspensions of overnight growth on nutrient agar plates by comparison with a no. 1 Brown's turbidity standard. MBCs were determined by the broth macrodilution method [12] (Lab M nutrient broth). For the MBC and time/viability studies. surface viable counts were determined on nutrient agar [13].
Crystal Violet reaction of S. aureus
89
('V reaction in broth Inocula were prepared from overnight broth cultures and the resuspended cell pellets mixed with CVr broth (cell harvest from 10 to 25 ml added to 1 ml CV' broth). (Cells were then pelleted by centrifugation and the colour of the supernatalnt and pellet observed at regular intervals during incubation at 37 0C. Cells were not resuspended between observations.
l1ethan of extracts These were prepared from cells removed from the surface of cellulose acetate filters and cells harvested by centrifugationi from CV reactions done on agar and in broth respectivelv. In each experiment methanol (AnalaR. BDH, l'oole. UK) was added in a fixed ratio to the amount of inoculum present in the sample concerned. This was normally in the range 1 ml methanol added to CV treated cells derived from a test inoculum harvested from 25 to 200 ml overnight broth culture. Extraction was done at room temperature and appeared to be complete after 10 min. The c ells were removed by centrifugation and the methanol extracts stored at 4 'C for up to 48 h prior to analvsis.
Spectrophotonietry The absorption spectra of methanol extracts and C\' broth reaction supernatants were examined over the range 250-700 nm in 0-5 cm quartz cells using a Pve Unicam SP1800 dual beam ultraviolet spectrophotometer.
Thin-layer chrofnatograJphy (Tl,C) Methanol extracts obtained from cells reacted with CV for between 1 and 48 h were applied in 5 1ul samples to 10 x 1O cm. 200 ,um octadecylsilane-bonded, reverse-phase TLC plates (RPTL( KC18, WAhatman, Maidstone, UK). Duplicate plates were run in 90 vol methanol:9 vol distilled water:1 vol 1 m-HCI and 90 vol methanol :9 xvol distilled water: I vol concentrated ammonia (AnalaR, B1)H) until the solvent front reached the top of the plate. Effects of potential, inhibitors and anaerobiosis The effects of sodium azide 25 mg/ml and chloramphenicol 20,ag/ml were examined. Both these agents were shown to be inhibitory but not cidal for the 10455 and RVI strains at the concentrations applied. Both were examined in the standardized broth reaction (see below) by addition to the final incubation medium. Because it was difficult to be sure that sufficient inhibitor was present in this study an additional two-phase experiment was done. Shaken broth cultures (25 ml) were prepared and. after 18 h incubation, sodium azide and chloramphenic ol were added. The cultures were re-incubated for a further 30 min and the cells harvested by centrifugation. washed twice in phosphate buffered saline (PBS) and resuspended by adding 60 1ll PBS to the cell pellet. Aliquots (20 ,l) of each cell suspension were then spotted onto CV agar and CV" agar containing chloramphenicol or azide and the CV reactions observed after overnight incubation. To examine the effects of anaerobiosis on the C\= reaction, strains RVI and
90
M. R. BARER AND OTHERS
10455 were cultured on nutrient agar under anaerobic conditions. Colonial growth was then transferred to pre-reduced CV agar plates as per the standard agar test and the plates re-incubated anaerobically for a further 48 h.
Standardized CV broth assay Shaken overnight broth cultures (25 ml) of each test strain were prepared. Cells were harvested by centrifugation and the pellets resuspended in 2-6 ml nutrient broth and the suspension distributed equally between two sterile pre-weighed 15 ml microcentrifuge tubes. Cells were again pelleted by centrifugation and as much supernatant as possible removed by aspiration. The microcentrifuge tubes were then reweighed and the wet weight of the cell harvest recorded. CV broth and CV broth containing 4 % (wt/vol) formaldehyde were added to the duplicate cell samples at 250 1a per 100 mg of cells, the tubes were vortex-mixed and then incubated at 37 'C. The colour of both tubes was noted at 0, 1, 2, 3, 4, 6, and 24 h. The formaldehyde tube acts as a control indicating the baseline colour of the strain in CV broth. Strains 10455 and RVI were included as CV reaction positive and negative controls in each assay.
REtSULTS The reproducibility of the CVT reaction was examined over ten serial subcultures. All gave the same reaction throughout. Moreover, no variation has occurred in the reactions obtained with our control strains 10455 (white) and RVI (purple) over a period of 2 years. White and yellow strains often show a thin rim of purple around the edge and base of the inoculum after overnight incubation on CV agar. It was thought that cells in this purple region might be acting as a filter retarding the entry of CkV into the centre of the inoculum and facilitating the reaction. To investigate this possibility further, white and purple strains were prepared from overnight broth cultures, and defined inocula applied in 50 ,l drops to the surface of CV' agar. L)ecreasing the inoculum on CV agar was founid to render white and vellow strains purple while purple strains retained their characteristic reaction. Conversely. purple strains could be rendered white by interposing one or more filters between the bacteria an(d the agar surface. Similar reversals of the characteristic reactions could be achieved bv altering the CV! content of the agar. These results are summarized in Table 1 and demonstrated in Fig. 1. The CV' reactions were further investigated by adding cell suspensions to CV' broth. WAAith a sufficiently large inoculum (1010-1011 cells/ml CV broth). the reactions observed on solid media could be reproduced when cells were suspended in CV' broth, mixed, pelleted by centrifugation. and observed over the subsequent 1-4 davs. The pellets were initially purple and the supernatants clear (white and purple strains) indicating that the staining substrate was in excess over the dye. Subsequently. on incubation at 37 'C. the pellets of white strains became white at a rate which could be increased or decreased in relation to the inoculum size. The white reaction was apparently dependent on the nutrient content of the suspending medium since it did not occur when the CV was made up in 0-9 % saline or water.
Crystal Violet reaction of S. aureus
91
Table 1. Effect of inoculum size, Crystal Violet content, and filter interposition on the Crystal Violet reaction Final reaction I'rocedure
White strains*
Purple strains
Standard CV agar, XVhite test I)ecrease inoculumt, Purple < 2-5 x 10tl c.f.u. Change CV contentt, Purple > 20,ug/ml WVhite Change CV content, < I ,ug/ml Interl)ose filters White * Similar results were obtained with yellow t A twofold dilution series was examined.
I 111I I
Purple
Purple Purple
White White strains.
I~~~~~~~~~"11
Fig 1. Reversal of the (X' reaction. Top) row, strain 10455 (w hite) inoculated onto CV agar in a tw~ofold decreasing ser-ies from left to right startinig at 1010 cells/spot. Bottom row. straini R\ I (purple) 5 x 10' (ells inoculated directly onito agar (left). theni from left to i-ight onito 1. 2 and( ~3filters' Note that the R\ I straini was deeply pigmienited ani(l that this is the maini reasoni for its (lal-kel- appearaice in the two inocula oni the right
comp)aied to the tNN-o inoctula oni the left from str-aini 10453. These findirngs suggested that th( (\' reaction depends on aective metabolism in the white strains and that the difference between these and purple strains might result fr-omi different susceptibilities to the inhibitorv effects of CV". However, MWX) determinations on CV" containing agar revealed that 10 white and 10 purple strains as
all showed MICs in the range 10-2-5 pig/ml with no evidence for differential two groups. MBC determinations indicated lethality within a similar range of CV concentrations using a standard inoculum (5 x t0'
senisitivity between the
92
M. R. BARER AND OTHERS
~~~~~~~~~~~~~~~.. ........ .. ... ..
Fig. 2. Reverse-phase thin-layer chromatography of serial methanol extracts from a white and a purple strain. Four plates are shown, two prepared with methanol extracts from the white strain, 10455 (top row), and two from the purple strain, RVI (bottom row). Plates on the left were run with methanol: water: HCI (90:9: 1) and those on the right were run with methanol: water: NH4 (90:9: 1). Samples (5 Iad) were applied (left to right) from serial extracts made after 0, 1. 2. 3, 4. 5. 6, 24. and 48 h incubation in CV broth. The extreme right-hand sample in each plate is a CV' standard in methanol. The arrow indicates the location of the secondary product which appeared in extracts from the white strain.
cells/ml); however, viability studies on preparations containing the inoculum levels used in the CV test indicated that at least 90 % of the inoculum of both white and purple strains remains viable throughout the test period of exposure to 10jug/ml CV. The nature of the white reaction occurring in CV broth was investigated by examining the absorption spectrum (250-700 nm) of the supernatant material and methanol extracts from the cell pellets at defined times after exposure to CV. No changes were observed in the supernatant subsequent to dye uptake, indicating that the products of CV modification were cell-associated. Methanol extracts from serial samples show a decline in the characteristic absorption profile for CV which occurs in both white and purple strains but much faster in the former. It was not possible to identify clearly an absorption spectrum which showed a reciprocal response to the declining CV spectrum; however, new peaks, which were not present in CV broth or extracts from untreated organisms, were observed at 266 and 305 nm. The changes observed in methanol extracts from CV-treated cells were investigated further by reverse-phase thin-layer chromatography. Chromatographs of serial methanol extracts from strain 10566 and RVI are shown in Fig. 2. Both strains show a reciprocal relationship between decline in the primary CV spot and the appearance of a fainter purple spot which ran faster than CV in alkali
93
Crystal Violet reaction of S. aureus
Table 2. Distribution of phage groups and types of the strains examined in the CV broth assay Crystal Violet category (total number of strains examined) Phage-group reactions 1
WAlhite (26)
Purple (26)
10
8
TI
14 6 III 13 15 V 19 4 95 3 0* 81 8 2 0* 7 Non-typable 3 8 I+II+III V alone 7 2 17 No reactions in IT or V' * We have previouslyr reported 5 white strains with Phage 95 reactions. 2 strainis and I purple strain with a group V reaction alone [8].
non-tvpable white
and slower in acid. Dye modification by the RVI (purple) strain proceeds very slowly in comparison to the white strain. In addition, in the white strain only, a second even fainter purple product was apparent in the 24 and 48 h extracts
(Fig. 2). The effects of chloramphenicol, azide, and anaerobict incubation on the CV reaction were examined. Although anaerobic incubation retarded the reaction on agar, none of these agents or conditions inhibited the CV reaction. Finally, in order to determine whether white and purple strains represent a continuous spectrum of CV modifying activity or two discrete populations. a broth assay was established in which the timecourse of colour changes occurring in the cell fraction was observed by comparison with a formaldehyde-fixed control preparation of the same strain. This allowed us to determine the relative rates of the white reactions in cell pellets from white and purple strains. Of the 52 strains tested, all 26 white strains produced the white reaction in under 1 h while comparable reactions for the purple strains all required between 3 and 24 h to become apparent. The phage-group reactions of the test strains are shown in Table 2. While a strong association between phage-group Vr reactions and the white phenotype is apparent, six white strains did not react with phages in this group and four purple strains did. Conversely two white reacting strains were found to react exclusivelxvwith group III phages. a pattern which is commoner amongst purple reacting strains [8]. WVe could find no evidence for differences in the rate of CV' alteration withini white or purple groups which could be correlated with the phage-group reactions. I )18(USSION
The CV" reaction is a reproducible property of strains of S. aureus which appears to be a marker for several significant biological associations of this organism. Although the test has been considered to reflect differential binding properties for CJ1V [14], this view is not supported by our results. The evidence presented here indicates that white an(d purple reactors belonging to the human biotype comprise
94 M. R. BARER AND OTHERS 94 discrete populations on a spectrum of the capacity of strains to convert CV to less coloured products. Yellow strains behaved in all other respects like white strains
CV-containing
media. but produced more pigment on The broth and methanol extract studies show that CV is first bound then processed by bacterial cells. One purple reaction product has been clearly identified by reverse-phase TLC. It travels further than CV on plates run with an alkaline solvent and less than CV in an acidic solvent. A second, fainter product, which travelled further than the primary product in the alkaline solvent, appeared in extracts from the white strain (10455) after prolonged incubation. These findings indicate that the primary product is probably a weak acid which interacts with the hydrophobic phase more readily than CV in an acidic solvent. The CV reaction is dependent on a nutrient source in the medium and appears to be carried out by viable cells yet it cannot be inhibited by anaerobiosis, or by growth inhibitory levels of sodium azide or chloramphenicol. These findings suggest that the reaction is an energy-dependent process which does not require respiratory chain activity or new protein synthesis. The difference between white-of and purple-reacting strains is related to the rate at which they are capable CV. The higher rate of CV modification may be related to their capacity modifying to modify further the primary CV product. A possible biochemical mechanism for this process could be modification of CV by an NAD-dependent enzyme system separate from the respiratory chain. The biological significance of the CV reaction remains unclear. The human biotype of S. aureus was originally described as a purple reactor and the bovine as yellow and white reactors [1]. Our results and canine biotypes, show that both white and purple CV reactors are well represented within the
respectively,
phagetype
and CV human biotype. The lack of an absolute relationship between reaction further supports the notion that the property detected is a determinant which can segregate independently of those which determine phage susceptibility. The strong associations between phage group and CV reaction type which were apparent from our previous report [8], particularly groups and V with theof,white the phenotype, presumably therefore reflect co-selection for, or linkage reaction determinants for these phenotypes rather than dependence of the white on some property central to strains belonging to these groups. Although it is tempting to speculate that strains which modify more rapidly
II
do so
in order to
CV we were unable to demonstrate gross 'detoxify'to CVtheby dre,. MIC/MBC tests. Differential effects of CV on
differences in susceptibility be present but, as hospital infections and enterotoxin growth or death rates with the purple phenotype, even if CV modifying have been associated production capacity is a resistance-related phenomenon then the associations are the inverse of those which conventional wisdom would lead one to It should be emphasized that we have not found any evidence to suggest that reactions confer resistance to noxious agents on strains either white or purple which express these two phenotypes. Antibiograms (eight agents) of the 168 strains examined in our earlier report revealed no significant differences between studies failed to show the white and purple reactors and methicillin evidence of tolerance in either group (Freeman and Burdess, unpublished results). It is also noteworthy that chloramphenicol resistance was not found in either
may
expect.
CV
MIC/MBC
any
Crystal Violet reaction of S. aureus
95
group. This clearly indicates that the CV reaction in S. aureus is not related to the CV staining properties of single colonies of Enterobacteriaceae which contain the CAT (chloramphenicol acetyltransferase) gene [15]. The properties of organisms isolated from a particular environment presumably reflect the selective pressures in that niche. Our experience indicates that purple reacting strains are more likely to be associated with hospital-acquired infections [8] and that this property may be related to the capacity of such strains to colonize human skin in hospital (R. Freeman, S. J. Hudson and D. Burdess, unpublished results). Similarly, others have used the reaction as an indicator of a potential zoonotic source of human infections [16]. The results presented here do not identify a biochemical mechanism for the CV reaction which explains these observations or the pathogenic attributes of purple reactors [14]. Nonetheless, the importance of these associations is such that it is desirable to know whether the reaction itself is directly relevant to these properties or whether it is simply a marker for them. ACKNOWN"LEDGEMENTS
We thank Richard Horobin and Alan Ward for their help with the thin-layer chromatography. This work was supported by a grant from Newcastle Health Authority Research Committee.
1. 2.
3. 4.
5. 6.
REFERENCES Meyer WV. A proposal for subdividing the species Staphylococcus aureus. Int J System Bacteriol 1966; 17: 387-9. Hajek V. Marsalek F. The differentiation of pathogenic staphylococci and a suggestion for their taxonomic classification. Zbl Bakt Hvg 1971: A217: 176-82. Dimitracopoulos G. Kalkani-Boussiakou H, I'apavassiliou J. Animal fecal carriership and biotypes of Staphylococcus aureus. Appl Env Microbiol 1977; 34: 461-4. Gibbs PA, Patterson JT. Harvey J. Bio(hemical characteristics and enterotoxigenicity of Staphylococcus aureus strains isolated from poultry. J Appl Bacteriol 1978; 44: 57-74. Naidu AS. Rathna K. Nirmala P, Yojana Devi D. Rajyalakshmi K. Outbreak of nonmenstrual fatal Toxic Shock Syndrome in India. Lancet 1986: ii: 1452-5. Hirooka EY. Muller EE, Freitas JC. Vicente E. Yoshimoto Y, Bergdoll 'MS. Enterotoxigenicity of Staphylococcus intermedius of canine origin. Int J Food MIicrobiol 1988; 7:
185-91. 7. Naidu AS, Kamme C. Ljungh A, Wadstrorn T. Levels of Toxic Shock Syndrome Toxin-I production among Staphylococcus aureus strains and clinical implications. Zbl Bakt Hyrg 1989; A270: 337-44. 8. Freemani R. Hudson SJ, Burdess 1). (rystal Violet reactions of fresh clinical isolates of Staphylococcu,,s autreus from tw-o British hospitals. Epidemiol Infect 1990: 105: 493-500. 9. Jakonouik P, Jacubicz P. The application of crystal violet ring test to the detection of hospital Staphylococcus aureus strains. In: Jeljaszewicz J. ed. Staphylococci an(l staphylococcal infections. Basel: S. Karger. 1974: 529-33. 10. V ogelsang TM. Wormnes A, Ostervold B. Correlation between staphylococcal phage groups and some staphylococcal enzymes demon4strated by simple methods. Acta Path Microbiol Scand 1962; 54: 218-24. 11. Easnmoni CSF G(oodfellow AM. Staphylococcus and Micrococcus. In: Topley and WVilson's principles and practice of bacteriology, virology and immunity, vol. 2 London: Edward Arnold, 1990: 162-84. 12. Jones RN, Barry AL, Gavan TL, NVashington II. JA. Susceptibility tests: microdilution and macrodilution broth procedures. In: Lennette EH. Balows A, Hausler ,Jr WJ. Shadorny HJ, eds. MaIanual of clinical microbiology. 4th ed. Washington. D.C.: American Society for Microbiology. 1985: 972-7. 4
HY (: 109
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AND OTHERS
13. Miles AA, Misra SS, Irwin JO. The estimation of the bactericidal power of the blood. J Hyg 1938; 38: 732-49. 14. Naidu AS, Jimenez J, Rollof J, et al. Crystal violet binding, cell surface properties and extracellular enzyme profiles of Staphylococcus aureus producing Toxic Shock Syndrome Toxin-1. Zbl Bakt Hyg 1989; A271: 11-21. 15. Proctor GN, Rownd RH. Rosanilins: indicator dyes for chloramphenicol-resistant enterobacteria containing chloramphenicol acetyltransferase. J Bacteriol 1982; 150: 1375-82. 16. Narasimha Rao P, Naidu AS, Ramana Rao P, Rajyalakshmi K. Prevalence of staphylococcal zoonosis in pyogenic skin infection. Zbl Bakt Hyg 1987: A265: 218-2;6.
Epidemiol. Infect. (1992), 109, 87-96 Printed in Great Britain
A study into the mechanism of the Crystal Violet reaction in Staphylococcus aureus M. R. BARER, D. BURDESS AND R. FREEMAN Department of Microbiology, University of Newcastle upon Tyne, Framlington Place, Newcastle upon Tyne NE2 4HH, UK
(Accepted 2 March 1992) SUMMARY
The mechanism of the Crystal Violet (CV) reaction, a trait which has been related to biotype, source and pathogenicity in Staphylococcus aureus, was investigated in agar and broth studies. White reactions could be converted to purple and vice versa by altering the incubation conditions on agar. Broth reactions examined macroscopically and by spectrophotometry revealed that both white and purple human biotype strains take up CV but the former then progressively modify the dye more quickly than the latter. A cell-associated product of CV was detected in white and purple strains by reverse-phase thinlayer chromatography of methanol extracts. White strains appear to produce a second additional product from CV. The white reaction was not inhibited by chloramphenicol or azide but did depend on viable cells with a nutrient source. CV MICs and MBCs for 10 white and 10 purple reactors showed no gross differences in susceptibility, while a standardized assay for the rate of CV modifying activity (52 strains) demonstrated that the two categories comprise discrete populations which alter CV at different rates. Although most white strains belong to either or both of phage-typing groups V and II, purple strains with this pattern of susceptibility and white strains without it both occur. The capacity to modify CV slowly or rapidly appears to subdivide human biotype strains independently of their phage group and is associated in the former case with their capacity to produce hospital-acquired and invasive infections. INTRODUCTION
The Crystal Violet (CV) reaction of Staphylococcus aureus has been used to separate biotypes of animal and human origin [1-4] and has been correlated with the capacity to produce enterotoxins [4-7]. We have recently obtained evidence that purple reacting strains (CV type C reactions) are significantly associated with hospital-acquired and invasive infections [8]. Both these and earlier observations suggesting that the CV reaction may provide an indicator for the 'hospital staphylococcus' [9] have stimulated us to examine the nature of the CV reaction. This report documents the effects of varying inoculum size, CV content and incubation conditions on the outcome of the reaction. We also present evidence for a nutritionally dependent CV modifying system which operates at different rates in white and purple reactors.
88
M. R. BARER AND OTHERS MATERIALS AN!) METHODS
Bacterial strains Fifty-two representative strains of S. aureus showing purple, white and yellow CV reactions were selected from the series of clinical isolates reported on previously [8]. Six biotype A strains (see below), two white reactors belonging separately to phage groups II and V, two yellow reactors (both phage group II), and two purple reactors belonging to phage groups II and III, were all used in the initial experiments concerned with reproducibility and inoculum size. From these, strains 10455 (phage group II) and RVI (phage group III) were subsequently used as controls for the white and purple reactions respectively. All strains were examined for fibrinolysin [10], bovine coagulase [8] and fl-haemolysin [8]. These tests showed that, with three exceptions all strains clearly belonged to the human biotype (A) [11]. The three exceptions (one purple and two white reactors), each of which gave negative in all three tests, were typable with the standard human phage set.
Phage typing For the strains listed above, phage typing was done by the Division of Hospital Infection, Central Public Health Laboratory. The remainder were typed as described previously [8]. Media Strains were stored on nutrient agar slopes and subcultured on nutrient agar plates (Lab M, Bury, UK). Standard CV agar and CV broth contained 10 jug/ml Crystal Violet (C.I. 2555. Sigma) in nutrient agar and nutrient broth (Lab M). Preparation of inocula All incubations were at 37 °C in air unless stated otherwise. For the routine CV agar test a visible quantity of growth from separated colonies of overnight growth on nutrient agar was transferred by loop and inoculated in circles of 5-6 mm diameter on CV agar. In some experiments sterile cellulose acetate filters were interposed between the inoculum and the agar surface (13 mm diameter, 0 22 ,um pore size, Nuflow. Oxoid). For the studies requiring more defined inocula t8 24 h broth cultures were prepared in glass Erlenmeyer flasks shaken at approximately 100 cycle/min. Cells were harvested by centrifugation and resuspended in the minimum volume of nutrient broth (approx 15 ml for cells harvested from 1 1 broth) required to allow accurate pipetting. MIC/MBC determinations MICs were determined using a multipoint inoculator to deliver approximately x 6 103 organisms onto nutrient agar plates containing serial twofold dilutions of CV. Inocular were prepared from saline (0-9 %) suspensions of overnight growth on nutrient agar plates by comparison with a no. 1 Brown's turbidity standard. MBCs were determined by the broth macrodilution method [12] (Lab M nutrient broth). For the MBC and time/viability studies. surface viable counts were determined on nutrient agar [13].
Crystal Violet reaction of S. aureus
89
('V reaction in broth Inocula were prepared from overnight broth cultures and the resuspended cell pellets mixed with CVr broth (cell harvest from 10 to 25 ml added to 1 ml CV' broth). (Cells were then pelleted by centrifugation and the colour of the supernatalnt and pellet observed at regular intervals during incubation at 37 0C. Cells were not resuspended between observations.
l1ethan of extracts These were prepared from cells removed from the surface of cellulose acetate filters and cells harvested by centrifugationi from CV reactions done on agar and in broth respectivelv. In each experiment methanol (AnalaR. BDH, l'oole. UK) was added in a fixed ratio to the amount of inoculum present in the sample concerned. This was normally in the range 1 ml methanol added to CV treated cells derived from a test inoculum harvested from 25 to 200 ml overnight broth culture. Extraction was done at room temperature and appeared to be complete after 10 min. The c ells were removed by centrifugation and the methanol extracts stored at 4 'C for up to 48 h prior to analvsis.
Spectrophotonietry The absorption spectra of methanol extracts and C\' broth reaction supernatants were examined over the range 250-700 nm in 0-5 cm quartz cells using a Pve Unicam SP1800 dual beam ultraviolet spectrophotometer.
Thin-layer chrofnatograJphy (Tl,C) Methanol extracts obtained from cells reacted with CV for between 1 and 48 h were applied in 5 1ul samples to 10 x 1O cm. 200 ,um octadecylsilane-bonded, reverse-phase TLC plates (RPTL( KC18, WAhatman, Maidstone, UK). Duplicate plates were run in 90 vol methanol:9 vol distilled water:1 vol 1 m-HCI and 90 vol methanol :9 xvol distilled water: I vol concentrated ammonia (AnalaR, B1)H) until the solvent front reached the top of the plate. Effects of potential, inhibitors and anaerobiosis The effects of sodium azide 25 mg/ml and chloramphenicol 20,ag/ml were examined. Both these agents were shown to be inhibitory but not cidal for the 10455 and RVI strains at the concentrations applied. Both were examined in the standardized broth reaction (see below) by addition to the final incubation medium. Because it was difficult to be sure that sufficient inhibitor was present in this study an additional two-phase experiment was done. Shaken broth cultures (25 ml) were prepared and. after 18 h incubation, sodium azide and chloramphenic ol were added. The cultures were re-incubated for a further 30 min and the cells harvested by centrifugation. washed twice in phosphate buffered saline (PBS) and resuspended by adding 60 1ll PBS to the cell pellet. Aliquots (20 ,l) of each cell suspension were then spotted onto CV agar and CV" agar containing chloramphenicol or azide and the CV reactions observed after overnight incubation. To examine the effects of anaerobiosis on the C\= reaction, strains RVI and
90
M. R. BARER AND OTHERS
10455 were cultured on nutrient agar under anaerobic conditions. Colonial growth was then transferred to pre-reduced CV agar plates as per the standard agar test and the plates re-incubated anaerobically for a further 48 h.
Standardized CV broth assay Shaken overnight broth cultures (25 ml) of each test strain were prepared. Cells were harvested by centrifugation and the pellets resuspended in 2-6 ml nutrient broth and the suspension distributed equally between two sterile pre-weighed 15 ml microcentrifuge tubes. Cells were again pelleted by centrifugation and as much supernatant as possible removed by aspiration. The microcentrifuge tubes were then reweighed and the wet weight of the cell harvest recorded. CV broth and CV broth containing 4 % (wt/vol) formaldehyde were added to the duplicate cell samples at 250 1a per 100 mg of cells, the tubes were vortex-mixed and then incubated at 37 'C. The colour of both tubes was noted at 0, 1, 2, 3, 4, 6, and 24 h. The formaldehyde tube acts as a control indicating the baseline colour of the strain in CV broth. Strains 10455 and RVI were included as CV reaction positive and negative controls in each assay.
REtSULTS The reproducibility of the CVT reaction was examined over ten serial subcultures. All gave the same reaction throughout. Moreover, no variation has occurred in the reactions obtained with our control strains 10455 (white) and RVI (purple) over a period of 2 years. White and yellow strains often show a thin rim of purple around the edge and base of the inoculum after overnight incubation on CV agar. It was thought that cells in this purple region might be acting as a filter retarding the entry of CkV into the centre of the inoculum and facilitating the reaction. To investigate this possibility further, white and purple strains were prepared from overnight broth cultures, and defined inocula applied in 50 ,l drops to the surface of CV' agar. L)ecreasing the inoculum on CV agar was founid to render white and vellow strains purple while purple strains retained their characteristic reaction. Conversely. purple strains could be rendered white by interposing one or more filters between the bacteria an(d the agar surface. Similar reversals of the characteristic reactions could be achieved bv altering the CV! content of the agar. These results are summarized in Table 1 and demonstrated in Fig. 1. The CV' reactions were further investigated by adding cell suspensions to CV' broth. WAAith a sufficiently large inoculum (1010-1011 cells/ml CV broth). the reactions observed on solid media could be reproduced when cells were suspended in CV' broth, mixed, pelleted by centrifugation. and observed over the subsequent 1-4 davs. The pellets were initially purple and the supernatants clear (white and purple strains) indicating that the staining substrate was in excess over the dye. Subsequently. on incubation at 37 'C. the pellets of white strains became white at a rate which could be increased or decreased in relation to the inoculum size. The white reaction was apparently dependent on the nutrient content of the suspending medium since it did not occur when the CV was made up in 0-9 % saline or water.
Crystal Violet reaction of S. aureus
91
Table 1. Effect of inoculum size, Crystal Violet content, and filter interposition on the Crystal Violet reaction Final reaction I'rocedure
White strains*
Purple strains
Standard CV agar, XVhite test I)ecrease inoculumt, Purple < 2-5 x 10tl c.f.u. Change CV contentt, Purple > 20,ug/ml WVhite Change CV content, < I ,ug/ml Interl)ose filters White * Similar results were obtained with yellow t A twofold dilution series was examined.
I 111I I
Purple
Purple Purple
White White strains.
I~~~~~~~~~"11
Fig 1. Reversal of the (X' reaction. Top) row, strain 10455 (w hite) inoculated onto CV agar in a tw~ofold decreasing ser-ies from left to right startinig at 1010 cells/spot. Bottom row. straini R\ I (purple) 5 x 10' (ells inoculated directly onito agar (left). theni from left to i-ight onito 1. 2 and( ~3filters' Note that the R\ I straini was deeply pigmienited ani(l that this is the maini reasoni for its (lal-kel- appearaice in the two inocula oni the right
comp)aied to the tNN-o inoctula oni the left from str-aini 10453. These findirngs suggested that th( (\' reaction depends on aective metabolism in the white strains and that the difference between these and purple strains might result fr-omi different susceptibilities to the inhibitorv effects of CV". However, MWX) determinations on CV" containing agar revealed that 10 white and 10 purple strains as
all showed MICs in the range 10-2-5 pig/ml with no evidence for differential two groups. MBC determinations indicated lethality within a similar range of CV concentrations using a standard inoculum (5 x t0'
senisitivity between the
92
M. R. BARER AND OTHERS
~~~~~~~~~~~~~~~.. ........ .. ... ..
Fig. 2. Reverse-phase thin-layer chromatography of serial methanol extracts from a white and a purple strain. Four plates are shown, two prepared with methanol extracts from the white strain, 10455 (top row), and two from the purple strain, RVI (bottom row). Plates on the left were run with methanol: water: HCI (90:9: 1) and those on the right were run with methanol: water: NH4 (90:9: 1). Samples (5 Iad) were applied (left to right) from serial extracts made after 0, 1. 2. 3, 4. 5. 6, 24. and 48 h incubation in CV broth. The extreme right-hand sample in each plate is a CV' standard in methanol. The arrow indicates the location of the secondary product which appeared in extracts from the white strain.
cells/ml); however, viability studies on preparations containing the inoculum levels used in the CV test indicated that at least 90 % of the inoculum of both white and purple strains remains viable throughout the test period of exposure to 10jug/ml CV. The nature of the white reaction occurring in CV broth was investigated by examining the absorption spectrum (250-700 nm) of the supernatant material and methanol extracts from the cell pellets at defined times after exposure to CV. No changes were observed in the supernatant subsequent to dye uptake, indicating that the products of CV modification were cell-associated. Methanol extracts from serial samples show a decline in the characteristic absorption profile for CV which occurs in both white and purple strains but much faster in the former. It was not possible to identify clearly an absorption spectrum which showed a reciprocal response to the declining CV spectrum; however, new peaks, which were not present in CV broth or extracts from untreated organisms, were observed at 266 and 305 nm. The changes observed in methanol extracts from CV-treated cells were investigated further by reverse-phase thin-layer chromatography. Chromatographs of serial methanol extracts from strain 10566 and RVI are shown in Fig. 2. Both strains show a reciprocal relationship between decline in the primary CV spot and the appearance of a fainter purple spot which ran faster than CV in alkali
93
Crystal Violet reaction of S. aureus
Table 2. Distribution of phage groups and types of the strains examined in the CV broth assay Crystal Violet category (total number of strains examined) Phage-group reactions 1
WAlhite (26)
Purple (26)
10
8
TI
14 6 III 13 15 V 19 4 95 3 0* 81 8 2 0* 7 Non-typable 3 8 I+II+III V alone 7 2 17 No reactions in IT or V' * We have previouslyr reported 5 white strains with Phage 95 reactions. 2 strainis and I purple strain with a group V reaction alone [8].
non-tvpable white
and slower in acid. Dye modification by the RVI (purple) strain proceeds very slowly in comparison to the white strain. In addition, in the white strain only, a second even fainter purple product was apparent in the 24 and 48 h extracts
(Fig. 2). The effects of chloramphenicol, azide, and anaerobict incubation on the CV reaction were examined. Although anaerobic incubation retarded the reaction on agar, none of these agents or conditions inhibited the CV reaction. Finally, in order to determine whether white and purple strains represent a continuous spectrum of CV modifying activity or two discrete populations. a broth assay was established in which the timecourse of colour changes occurring in the cell fraction was observed by comparison with a formaldehyde-fixed control preparation of the same strain. This allowed us to determine the relative rates of the white reactions in cell pellets from white and purple strains. Of the 52 strains tested, all 26 white strains produced the white reaction in under 1 h while comparable reactions for the purple strains all required between 3 and 24 h to become apparent. The phage-group reactions of the test strains are shown in Table 2. While a strong association between phage-group Vr reactions and the white phenotype is apparent, six white strains did not react with phages in this group and four purple strains did. Conversely two white reacting strains were found to react exclusivelxvwith group III phages. a pattern which is commoner amongst purple reacting strains [8]. WVe could find no evidence for differences in the rate of CV' alteration withini white or purple groups which could be correlated with the phage-group reactions. I )18(USSION
The CV" reaction is a reproducible property of strains of S. aureus which appears to be a marker for several significant biological associations of this organism. Although the test has been considered to reflect differential binding properties for CJ1V [14], this view is not supported by our results. The evidence presented here indicates that white an(d purple reactors belonging to the human biotype comprise
94 M. R. BARER AND OTHERS 94 discrete populations on a spectrum of the capacity of strains to convert CV to less coloured products. Yellow strains behaved in all other respects like white strains
CV-containing
media. but produced more pigment on The broth and methanol extract studies show that CV is first bound then processed by bacterial cells. One purple reaction product has been clearly identified by reverse-phase TLC. It travels further than CV on plates run with an alkaline solvent and less than CV in an acidic solvent. A second, fainter product, which travelled further than the primary product in the alkaline solvent, appeared in extracts from the white strain (10455) after prolonged incubation. These findings indicate that the primary product is probably a weak acid which interacts with the hydrophobic phase more readily than CV in an acidic solvent. The CV reaction is dependent on a nutrient source in the medium and appears to be carried out by viable cells yet it cannot be inhibited by anaerobiosis, or by growth inhibitory levels of sodium azide or chloramphenicol. These findings suggest that the reaction is an energy-dependent process which does not require respiratory chain activity or new protein synthesis. The difference between white-of and purple-reacting strains is related to the rate at which they are capable CV. The higher rate of CV modification may be related to their capacity modifying to modify further the primary CV product. A possible biochemical mechanism for this process could be modification of CV by an NAD-dependent enzyme system separate from the respiratory chain. The biological significance of the CV reaction remains unclear. The human biotype of S. aureus was originally described as a purple reactor and the bovine as yellow and white reactors [1]. Our results and canine biotypes, show that both white and purple CV reactors are well represented within the
respectively,
phagetype
and CV human biotype. The lack of an absolute relationship between reaction further supports the notion that the property detected is a determinant which can segregate independently of those which determine phage susceptibility. The strong associations between phage group and CV reaction type which were apparent from our previous report [8], particularly groups and V with theof,white the phenotype, presumably therefore reflect co-selection for, or linkage reaction determinants for these phenotypes rather than dependence of the white on some property central to strains belonging to these groups. Although it is tempting to speculate that strains which modify more rapidly
II
do so
in order to
CV we were unable to demonstrate gross 'detoxify'to CVtheby dre,. MIC/MBC tests. Differential effects of CV on
differences in susceptibility be present but, as hospital infections and enterotoxin growth or death rates with the purple phenotype, even if CV modifying have been associated production capacity is a resistance-related phenomenon then the associations are the inverse of those which conventional wisdom would lead one to It should be emphasized that we have not found any evidence to suggest that reactions confer resistance to noxious agents on strains either white or purple which express these two phenotypes. Antibiograms (eight agents) of the 168 strains examined in our earlier report revealed no significant differences between studies failed to show the white and purple reactors and methicillin evidence of tolerance in either group (Freeman and Burdess, unpublished results). It is also noteworthy that chloramphenicol resistance was not found in either
may
expect.
CV
MIC/MBC
any
Crystal Violet reaction of S. aureus
95
group. This clearly indicates that the CV reaction in S. aureus is not related to the CV staining properties of single colonies of Enterobacteriaceae which contain the CAT (chloramphenicol acetyltransferase) gene [15]. The properties of organisms isolated from a particular environment presumably reflect the selective pressures in that niche. Our experience indicates that purple reacting strains are more likely to be associated with hospital-acquired infections [8] and that this property may be related to the capacity of such strains to colonize human skin in hospital (R. Freeman, S. J. Hudson and D. Burdess, unpublished results). Similarly, others have used the reaction as an indicator of a potential zoonotic source of human infections [16]. The results presented here do not identify a biochemical mechanism for the CV reaction which explains these observations or the pathogenic attributes of purple reactors [14]. Nonetheless, the importance of these associations is such that it is desirable to know whether the reaction itself is directly relevant to these properties or whether it is simply a marker for them. ACKNOWN"LEDGEMENTS
We thank Richard Horobin and Alan Ward for their help with the thin-layer chromatography. This work was supported by a grant from Newcastle Health Authority Research Committee.
1. 2.
3. 4.
5. 6.
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185-91. 7. Naidu AS, Kamme C. Ljungh A, Wadstrorn T. Levels of Toxic Shock Syndrome Toxin-I production among Staphylococcus aureus strains and clinical implications. Zbl Bakt Hyrg 1989; A270: 337-44. 8. Freemani R. Hudson SJ, Burdess 1). (rystal Violet reactions of fresh clinical isolates of Staphylococcu,,s autreus from tw-o British hospitals. Epidemiol Infect 1990: 105: 493-500. 9. Jakonouik P, Jacubicz P. The application of crystal violet ring test to the detection of hospital Staphylococcus aureus strains. In: Jeljaszewicz J. ed. Staphylococci an(l staphylococcal infections. Basel: S. Karger. 1974: 529-33. 10. V ogelsang TM. Wormnes A, Ostervold B. Correlation between staphylococcal phage groups and some staphylococcal enzymes demon4strated by simple methods. Acta Path Microbiol Scand 1962; 54: 218-24. 11. Easnmoni CSF G(oodfellow AM. Staphylococcus and Micrococcus. In: Topley and WVilson's principles and practice of bacteriology, virology and immunity, vol. 2 London: Edward Arnold, 1990: 162-84. 12. Jones RN, Barry AL, Gavan TL, NVashington II. JA. Susceptibility tests: microdilution and macrodilution broth procedures. In: Lennette EH. Balows A, Hausler ,Jr WJ. Shadorny HJ, eds. MaIanual of clinical microbiology. 4th ed. Washington. D.C.: American Society for Microbiology. 1985: 972-7. 4
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AND OTHERS
13. Miles AA, Misra SS, Irwin JO. The estimation of the bactericidal power of the blood. J Hyg 1938; 38: 732-49. 14. Naidu AS, Jimenez J, Rollof J, et al. Crystal violet binding, cell surface properties and extracellular enzyme profiles of Staphylococcus aureus producing Toxic Shock Syndrome Toxin-1. Zbl Bakt Hyg 1989; A271: 11-21. 15. Proctor GN, Rownd RH. Rosanilins: indicator dyes for chloramphenicol-resistant enterobacteria containing chloramphenicol acetyltransferase. J Bacteriol 1982; 150: 1375-82. 16. Narasimha Rao P, Naidu AS, Ramana Rao P, Rajyalakshmi K. Prevalence of staphylococcal zoonosis in pyogenic skin infection. Zbl Bakt Hyg 1987: A265: 218-2;6.
