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The evaluation of novel chromogenic substrates for the detection of lipolytic activity in clinical isolates of Staphylococcus aureus and MRSA from two European study groups

Simon W.J. Gould, Maureen Chadwick, Paul Cuschieri, Susan Easmon, Anthony C. Richardson, Robert G. Price, Mark D. Fielder
DOI: http://dx.doi.org/10.1111/j.1574-6968.2009.01654.x 10-16 First published online: 1 August 2009

Abstract

Eight novel chromogenic substrates were evaluated for their efficacy in detecting lipase activity in clinical isolates of Staphylococcus aureus from the United Kingdom and Malta. All isolates metabolized the chromogenic lipase substrates 5-(4-hydroxy-3,5-dimethoxyphenylmethylene)-2-thioxothia-zolidin-4-one-3-ethanoic acid (SRA)-propionate, SRA-butyrate, SRA-octanoate and 2-[2-(4-hydroxy-3,5-dimethoxyphenyl)-vinyl]-3-methy-benzothiazolium salt (SBZTM)-acetate. Over 90% of the isolates metabolized the lipase substrates SRA-decanoate and SRA-laurate. However, only 0.6% of UK isolates and 2% of Maltese isolates metabolized the lipase substrate SRA-myristate; none of the isolates tested metabolized SBZTM-butyrate. Traditional Tween 80 assays showed that over 73% of the UK methicillin-resistant Staphylococcus aureus (MRSA) isolates and 83% of the UK methicillin-sensitive Staphylococcus aureus (MSSA) isolates demonstrated lipolytic activity. In contrast, Maltese isolates showed lipase activity in 94% and 88% of the MRSA and MSSA strains, respectively. Lipases in MRSA and MSSA demonstrated substrate specificity whose activity appeared dependent upon hydrocarbon chain length of the chromogen. These novel chromogens can be used for lipase enzyme detection and have application for full characterization of numerous S. aureus lipases.

Keywords
  • Staphylococcus aureus
  • lipase
  • novel chromogenic substrates
  • Tween 80

Introduction

Methicillin-sensitive and methicillin-resistant Staphylococcus aureus (MSSA and MRSA, respectively) are important nosocomial pathogens (Apfalter et al., 2002). As a group of organisms, the staphylococci produce many different extracellular enzymes including DNAse, proteinase and lipase (Arvidson, 2000). In the recent past there has been an increase in the number of studies carried out on these extracellular enzymes, some focusing on lipase activity (Nostro et al., 2001). The lipase enzyme is active against a number of natural and synthetic lipid substrates and also water-soluble triglycerides and Tweens (Arvidson, 2000). These substrates are broken down to their constituent alcohol and fatty acids by ester linkage hydrolysis (Easmon & Adlam, 1983). To date, five different staphylococcal lipase genes from different species have been cloned and sequenced, two from S. aureus, two from Staphylococcus epidermidis and one from Staphylococcus hyicus (Nikoleit et al., 1995). Of the two lipases produced by S. aureus, only one is described as a true lipase, an enzyme that is able to hydrolyse water-insoluble long-chain trigylycerols as well as water-soluble trigylycerols (Arvidson, 2000). The second enzyme can only hydrolyse water-soluble short-chain trigylycerols, and is referred to as a short-chain glycerol ester hydrolase (Arvidson, 2000). The pathogenic role played by lipase during an S. aureus infection remains unclear; however, it has been shown that the enzymes are produced during infection and it is suggested that they play a role in the interference of phagocytosis (Easmon & Adlam, 1983).

Currently, there are two established approaches to detecting lipase activity. The methods involve the use of nutrient agar supplemented with 1% Tween 80 or nutrient agar supplemented with egg yolk (Cooke et al., 1999). Tween 80 degradation is specific for lipase activity, whereas egg yolk medium demonstrates both lipase and phospholipase activity (Cooke et al., 1999). Lipolysis of Tween 80 is characterized by the appearance of a zone of opacity, due to precipitation of liberated fatty acids that combine with calcium to form salts (Nostro et al., 2001). Although the Tween 80 assay is normally used for qualitative detection of S. aureus lipase activity, the results can be difficult to interpret (Nostro et al., 2001).

Chromogenic media are increasingly recognized as useful tools in diagnostic laboratories for the rapid detection of bacterial species and strain characterization. Current successful examples of these media include CHROMagar for the detection of Candida spp. (Cooke et al., 2002), oxacillin resistance screening agar base for the detection of MRSA (Perry et al., 2004) and chromogenic Salmonella esterase for the detection of salmonellae (Cooke et al., 1999).

The aim of the current study was to evaluate a range of novel chromogens against a variety of clinical strains in terms of lipase activity and to establish whether any variance could be exploited in a diagnostic or analytical context. The clarity and rapidity of resolution of these chromogens was also compared with traditional Tween 80-based tests.

Materials and methods

Bacterial strains

Genome type strains as well as clinical isolates were tested in this study. Genome type strains were obtained from the Network of Antibicrobial Resistance on Staphylococcus aureus (NARSA) and include S. aureus N315, Mu50, NCTC 08325, EMRSA-16 (strain 252), MSSA 476 (strain 476), COL and MW2.

Clinical isolates of MRSA and MSSA were collected from three hospitals in the United Kingdom (Kingston Hospital, Surrey, kindly provided by Mr M. Smith; the Royal Brompton Hospital, London; and the Royal Marsden Hospital, London) and from St Luke's Hospital, Malta. A total of 378 isolates were collected from the UK hospitals (260 MRSA and 118 MSSA) and 309 isolates were collected from the Maltese hospital (216 MRSA and 93 MSSA).

Tween 80 assay

Nutrient agar (Oxoid Ltd) plates were supplemented with 1% Tween 80 (Fisher Scientific Ltd). Overnight cultures were suspended in Ringers solution to the equivalent of 0.5 McFarland's standard (equivalent to 1.5 × 108 CFU mL−1). These cultures were dispensed in 10-μL aliquots onto predried plates and incubated at 35 °C for 48 h; tests were carried out in triplicate. After incubation, the presence or absence of opalescent zones around the colonies were recorded.

Chromogenic substrates

All chromogenic substrates were provided by PPR Diagnostics Ltd, London, UK. The test substrates were 5-(4-hydroxy-3,5-dimethoxyphenylmethylene)-2-thioxothia-zolidin-4-one-3-ethanoic acid (SRA)-propionate (C3), SRA-butyrate (C4), SRA-octanoate (C8), SRA-decanoate (C10), SRA-laurate (C12), SRA-myristate (C14), 2-[2-(4-hydroxy-3,5-dimethoxyphenyl)-vinyl]-3-methy-benzothiazolium salt (SBZTM)-acetate (C2) and SBZTM-butyrate (C4). An example of the chemical structure of the chromogenic substrate can be seen in Fig. 1.

1

Structure of representative chromogenic substrates. The left-hand panel shows SRA; R=propionate (C3), butyrate (C4), octanoate (C8), decanoate (C10), laurate (C12) or myristate (C14). The right-hand panel shows SBzTM-tosylate where R is either acetate (C2) or butyrate (C4). (Structural diagrams provided by Dr A.C. Richardson of PPR Diagnostics.)

Chromogenic medium

The chromogenic substrates were added to nutrient agar (Oxoid Ltd) to a final concentration of 0.3 g L−1. The substrates were dissolved in 4-mL aliquots of a methanol–water mix (2–10% v/v). The substrates were then added to cooled (55 °C), presterilized nutrient agar (Oxoid Ltd) and poured into 90-mm Petri dishes.

Inoculation of chromogenic media

Overnight cultures were streaked onto plates as a single line, in a radiating pattern from the centre of the plates, using six isolates per plate. All plates were incubated at 35 °C for 48 h and all tests were carried out in triplicate. Culture control plates were incubated under similar conditions. After incubation, they were observed for any change in colour.

Pulse-field gel electrophoresis (PFGE)

PFGE was performed as described previously by Gould et al. (2008).

Results

Lipolytic activity against Tween 80

The seven genome type strains tested demonstrated lipolytic activity on Tween 80 plates. Analysis of the UK isolates demonstrated that 73% (189/260) of MRSA and 83% (98/118) of MSSA showed lipolytic activity on Tween 80 plates. A higher number of the Maltese isolates – 94% (203/216) of the MRSA and 88% (82/93) of the MSSA – were observed to be Tween 80 positive.

Lipolytic activity against chromogenic substrates

Following incubation for 48 h, colour changes were observed with all but one of the substrates, namely SBZTM-butyrate. This information is summarized in Table 1 (for the genome type strains) and Table 2 (clinical isolates). The colour changes varied with the different substrates as did the size of the coloured zone produced around the isolates. The coloured zones ranged from 3 to 4 mm for isolates, when SRA-myristate was used, to total colouration of the agar medium when SRA-propionate was used as a substrate. All isolates produced colour changes with SRA-propionate and SRA-butyrate both of which resulted in colouration of the agar, with the colour radiating across the plate with the greatest intensity of colouration adjacent to the colony. Isolates on both SRA-propionate and SRA-butyrate agar showed activity that diffused away from the colony producing a red colouration in the medium within 24 h (Fig. 2).

View this table:
1

Detection of lipolytic activity of the genome type strains against the novel chromogenic substrates

SubstrateStaphylococcus aureus genome type strain
N315Mu5008325MRSA-16 (252)MSSA (476)ColMW2
SRA-propionate
SRA-butyrate
SRA-octanoate
SRA-decanoate
SRA-laurate
SRA-myristate
SBzTM-acetate
SBzTM-butyrate
  • Grey shading represents lipolytic activity, no shading represents no detectable lipolytic activity against the indicated substrate.

View this table:
2

Data representing the number of clinical isolates of Staphylococcus aureus with the ability to metabolize the novel chromogenic lipase or esterase substrates

SubstrateOriginal media colourationColour observed inTime (h)UK isolates (%)Maltese isolates (%)
ColonyWhole plate
SRA-propionateTranslucent pale yellowRedRed24100100
SRA-butyrateTranslucent pale yellowRedRed24100100
SRA-octanoateTranslucent pale yellowPurpleYellow48100100
SRA-decanoateTranslucent pale yellowPinkPink zone489798
SRA-laurateTranslucent pale yellowPinkPink zone489290
SRA-myristateTranslucent pale yellowPinkPink zone480.52
SBzTM-acetateTranslucent redDark purplePurple48100100
SBzTM-butyrateTranslucent redNo changeNo change48100100
  • The results detail the observed colouration for both the isolate and the agar plate, the time taken for the observed colour changes and the percentage of isolate group that were scored positive for lipase or esterase activity (using the SBzTM-acetate, SBzTM-butyrate substrates) against the given novel chromogenic substrates.

2

Examples of lipolytic activity of the same clinical isolate of Staphylococcus aureus against the chromogenic substrates SRA-butyrate (b), SRA-propionate (c) and the traditional Tween 80 lipase detection method (a) after a 24-h aerobic incubation at 37°C. The arrow shown in (a) indicates the presence of the opalescent zone observed with lipase activity on Tween 80 assays, demonstrating the comparative ease with which lipolytic activity can be observed with the sample chromogenic substrates illustrated. The inset image in (a) shows a backlit view of the Tween 80 lipolysis zone.

All of the clinical isolates produced colour changes on SRA-octanoate with the colonies turning a deep purple hue; however, one of the genome type strains, MSSA-476, did not produce any colour change. These colour changes were observed after 24 h of incubation. No colour change was observed in the agar.

The use of both SRA-decanoate and SRA-laurate media gave rise to growth with a dark pink zone around the resultant colonies. These zones of colour were consistently larger with the isolates grown on the SRA-decanoate plates when compared with those grown on the SRA-laurate plates; the rest of the plate retained the original pale yellow colouration. All except one of the genome type strain (MSSA 476) metabolized the SRA-decanoate substrate with a subsequent colour change evident on the plate. Most of the isolates changed colour on these two different plates, 97% (362/372) of the UK isolates and 98% (301/308) of the Maltese isolates, demonstrating colour changes on the SRA-decanoate plates. Three of the genome type strains (08325, MSSA 476, COL) showed no colour change on SRA-laurate plates. Similarly, 92% of UK isolates (345/372) and 91% of Maltese isolates (279/308) demonstrated metabolism of the SRA-laurate substrate. The UK isolates showed similar levels of substrate metabolism in both the MRSA and MSSA group 97% (253/257) and 98% (109/115), respectively, as did the Maltese isolates, with both MRSA and MSSA showing 98% (215/216, 90/92, respectively) of isolate metabolism of the substrate. The SRA-laurate substrate was metabolized by 92% (345/372) of the UK isolates and 91% (279/308) of the Maltese isolates. The UK isolates showed similar activity levels when compared with SRA-laurate, the MRSA and MSSA group showing 91% (235/257) and 94% (108/115) of the isolates, respectively, with both groups demonstrating ability to metabolize the substrate. A similar trend was also observed with the Maltese isolates, with similar results produced on comparing MRSA and MSSA, 90% (194/216) and 92% (85/92), respectively.

SRA-myristate was metabolized by the lowest number of isolates in the study groups. A small zone of pale pink colour could be seen around the isolates, but the colonies did not change in colour. None of the genome strains were able to metabolize SRA-myristate, only 0.5% (2/372) of UK isolates and 2% (5/308) of the Maltese isolates produced any colour change, with 0.4% (1/372) of MRSA and 0.9% (1/115) of MSSA of the UK isolates and 0.9% (2/216) of MRSA and 3.2% (3/92) of MSSA groups of the Maltese isolates showing any metabolism of this substrate.

All genome type strains and clinical isolates produced colour changes on SBZTM-acetate with the plates showing a deep purple colouration dispersed across the whole plate.

None of the genome type strains or the clinical isolates produced any notable colour change on SBZTM-butyrate supplemented agar plates. Some slight colour changes were observed following incubation; however, when compared with controls no colour difference was evident, suggesting that this slight change was the result of substrate degradation upon incubation. In addition, no pigment uptake was observed in any of the isolates with this substrate.

Comparison of chromogenic substrates and Tween 80 tests

Both Tween 80 and chromogenic agar methods detected lipase activity in the isolates panel to varying degrees. The most notable difference between the two media was the ease with which the result on the chromogenic plate could be read due to the formation of colour on the chromogenic plate. Results from the Tween 80 assay can be difficult to determine, possibly due to low-level lipase activity (Fig. 2). In these cases, there is very little precipitation observed in the medium and this can lead to a false negative result being reported. Figure 2 demonstrates the necessity, in some cases, to view the Tween 80 test plate illuminated with back light to reveal faint zone of lipolysis. However, the use of the novel chromogenic substrates described here eliminate any ambiguity in the detection of lipase activity (Fig. 2). When comparing the results obtained using the two different assay types four of the chromogenic media, SRA-propionate, SRA-butyrate (Fig. 2), SRA-octanoate and SBZTM-acetate were hydrolysed by all of the clinical isolates in the study. This outcome contrasts with the results of the Tween 80 assay where positive results ranged from 73% (of the UK MRSA isolates) to 94% (of the Maltese MRSA isolates) for the same isolate panel.

Tween 80 medium requires an incubation time of 48 h before results can be read. However, both SRA-propionate and SRA-butyrate results can be read clearly within 24 h, representing a reduction in the required time to read the completed test.

Strain typing by PFGE

PFGE banding patterns were determined for 86.2% (586/680) of the clinical isolates within the study. PFGE profiles could not be determined for the remaining 13.8% of isolates (Gould et al., 2008). A total of 47 different strain types were present in the culture collection. Some strain groups also contained a varying number of subtypes, which ranged from three to 39 subtypes. A total of four main strain types were evident, which consisted of c. 68% of the isolates tested. The largest concentration of isolates (43% of total isolates) was found in a strain type that appeared to be similar in banding pattern to epidemic-MRSA-15 (EMRSA-15); this strain is one of the most common strains of EMRSA in the United Kingdom. The second largest strain type contained 12% of the total number of isolates and was found to be similar to EMRSA-16. The final two type strains contained 6% and 7%, respectively, the first of these types was determined to be similar to EMRSA-1; however, the isolates in this group were predominantly isolates of MSSA. The second strain type contained predominantly Maltese isolates (except for the two UK isolates) and therefore may be a local strain. The remaining 32% of isolates were found in the remaining 43 strain type; the frequency of isolates in these strain types ranged from 0.2% to 2%.

Discussion

In the current study, two different agar-based assays were examined for their efficacy in detecting lipase activity from clinical isolates of MRSA and MSSA from two study countries (UK and Malta). The study compared nutrient agar supplemented with either Tween 80 (1%) or each one of eight novel chromogenic substrates. When comparing these two types of media, the action of lipase was most apparent when using the chromogenic agar plates due to the production of colouration, compared with the precipitate observed with the Tween 80-based assay. A high percentage of isolates demonstrated lipase activity on both media. All the clinical isolates and seven of the eight genomic type strains (MSSA 476 did not metabolize SRA-octanoate) demonstrated metabolism of the chromogenic substrate with fatty chain length up to and including octanoate (C=8). However, some of these isolates were recorded as having no lipase activity when the Tween 80 assay was used. This observation demonstrated that these short-chain fatty acid chromogenic substrates have a higher sensitivity than the traditional Tween 80 agar plates in the detection of lipase activity. In the cases of MRSA isolates from the United Kingdom, 27% (71/260) were recorded as lipase negative on Tween 80 plates, but all isolates were positive when tested on the short-chain fatty acid substrates.

Of the eight different chromogenic substrates tested in the study, two substrates were either not metabolized or produced a colour change in a small number of isolates. SBZTM-butyrate gave no colour change at all while SRA-myristate produced a colour change with only a small number of isolates (≤2%) in the test groups. The result with the SRA-myristate might indicate that few clinical isolates of S. aureus are capable of metabolizing such a large lipase substrate (C14). With the SBZTM-butyrate substrate colouration was observed of equal intensity pre- and postincubation on both test and control plates.

The intensity of colour that developed on the remaining plates appears to be linked to the size of the fatty acid side chain. Observing the plates that contained either the SRA-propionate substrate (C=3), or the SRA-butyrate (C=4) or the SBZTM-acetate (C=2) substrate, the colour change radiated across the plate. However, on the plates containing SRA-octanoate (C=8) the colour change was only seen within the colony, while the SRA-decanoate (C=10) and SRA-laurate (C=12) substrates both produced colour changes that migrated from the colonies, suggesting extracellular action of the lipase. The size of this zone of colour was observed to be greater with the SRA-decanoate substrate than that noted with the SRA-laurate substrate. The changes seen between SRA-decanoate and SRA-laurate may be explained by the fact that SRA-decanoate is smaller than SRA-laurate by two hydrocarbons, suggesting a variation in enzyme activity due to substrate side-chain length. It has been proposed that free fatty acids may play a role in inhibiting phagocytosis (Easmon & Adlam, 1983), and it has also been shown that the long-chain fatty acids have bactericidal properties (Easmon & Adlam, 1983). However, 80% of S. aureus strains produce an enzyme called fatty acid-modifying enzymes (FAME), which catalyses the esterification of the lipid to alcohols or cholesterol (Arvidson, 2000). It should be noted that this enzyme is most effective on saturated fatty acids composed of 15–19 carbons. It is, therefore, tempting to speculate that the reduction in hydrolysis of the substrates of 10–12 hydrocarbons such as SRA-decanoate and SRA-laurate may be a mechanism to prevent the production of potentially bacteriocidal, free long-chain fatty acids not modified by FAME.

The differences in the amount of colour produced may possibly be related to the different action of two types of lipase enzymes. It has been reported that S. aureus has two lipase genes: geh (isolated from S. aureus strain PS54) and lip, geh (isolated from S. aureus strain NCTC 8530) (Arvidson, 2000). These genes encode for two different lipase activities, with geh encoding for the true lipase and lip, geh encoding for the short-chain glycerol ester hydrolase. Therefore, it is possible that the zone of the colour seen may be due to the action of these two enzymes possibly working synergistically.

Colour changes seen with SRA-propionate, SRA-butyrate, SRA-octanoate and SBZTM-acetate may be due to the action of the short-chain glycerol ester hydrolase. Other groups have reported similar findings, although they referred to these enzymes as SAL-1 (Kloos et al., 1991). These authors reported that SAL-1 has a strong preferential action against short-chain fatty acids, with maximal activity towards butyric acid esterified to glycerol, p-nitrophenol or umbelliferone. This activity decreased with acyl chain length of one methyl group and hardly hydrolyses the larger chain trioctanyloglyercol (Kloos et al., 1991). Zones of colour were seen following metabolism of the small fatty acid chain molecules, SRA-propionate, SRA-butyrate and SBZTM-butyrate, as the hydrocarbon side chain length fell within the active range of short-chain glycerol ester hydrolase. True lipase can also hydrolyse short-chain water-soluble triacylglycerols; therefore, the hydrolysis of these substrates may also be due to the true lipases, as well as the activity of the short-chain glycerol ester hydrolase.

Colour changes with SRA-decanoate, SRA-laurate and SRA-myristate might possibly be due to the action of the true lipase, as these can hydrolyse long-chain triacylglycerols. Interestingly, short-chain glycerol ester hydrolases are believed to be produced by most S. aureus isolates, whereas true lipases are less common (Arvidson, 2000). If the hydrolysis of SRA-decanoate and SRA-laurate are due to true lipase activity, these results suggest that in the clinical isolates used in this study there is a higher level of true lipase activity at 90% and above. This may contrast with other reports (Arvidson, 2000) and could represent a salient feature of hospital strains of MRSA and MSSA.

The colour changes seen with SRA-octanoate pose a challenge, as the change in colour was only seen within the colony and did not radiate out from the colony as seen with other substrates used in this study. It is tempting to speculate that the hydrolysis of the substrate was due to the action of short-chain glycerol ester hydrolase. This may explain the poor hydrolysis and lack of radiating colour around the colony. However, true lipases have a wide range of activity. It has already been suggested that the hydrolysis of the short-chain triacylglycerols may be due to either true lipase or short-chain glycerol ester hydrolyase; therefore, we can speculate that SRA-octanoate might be hydrolysed by a true lipase.

In conclusion, seven of the eight chromogenic lipase and esterase substrates in the test panel demonstrated improved detection enzyme activity in clinical isolates of S. aureus when compared with the standard Tween 80 assay. The development of a coloured product as a result of metabolism yielded an unambiguous test result not previously possible with the Tween 80 assay (Fig. 2). The use of novel chromogenic substrates with varied chain length also allowed for more detailed analysis of the range of lipid metabolism present in S. aureus isolates. Following the observations reported in this paper, we suggest that these chromogenic substrates may have potential use in enzymatic and physiological research involving staphylococci as well as other microorganisms. Currently, there is limited knowledge in the area of S. aureus lipase activity and its role in pathology; the main focus of the published work has been directed towards S. hyicus (Rosenstein & Götz, 2000). Hence, it is possible that these novel substrates may act as a key in future S. aureus lipase research acting as a metabolism marker in molecular studies concerned with the control of lipase production. Lipase activity in staphylococci is regulated by two genes, agr and the sar genes, which, respectively, positively and negatively regulate lipase production. It is unknown which lipase is regulated by these genes as the substrates previously used did not discriminate been the action of these lipases (Arvidson, 2000). Novel, modified chromogenic substrates such as those detailed in here may play a role in the elucidation of lipase activity controlled by the agr and the sar genes to provide a better understanding of the metabolism of these important organisms.

Acknowledgements

We would like to thank the network of antimicrobial resistance in S. aureus (NARSA) for providing us with the genome type strains N315, Mu50, EMRSA-16 (strain 252), MSSA 476, Col and MW2. S.W.J.G. is in receipt of a Kingston University studentship. We are also grateful to Dr Gary Forster-Wilkins for useful discussions during the preparation of this manuscript.

Footnotes

  • Editor: Mark Enright

References

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