JOURNAL OF BIOLUMINESCENCE AND CHEMILUMINESCENCE VOL 4 267-271 (1989)

Opsonophagocytosis of Bacteria Studied by Chemiluminescence in Microtitre Plates

J. G. M. Hastings*, L. A. Jewes’ and K. M. Oxley Departments of Medical Microbiology and ’Bacteriology, University of Sheffield Medical School, Beech Hill Road, Sheffield, S10 2RX. UK

A protocol for polymorphonuclear leukocyte chemiluminescence (PMN CL) assays of opsonophagocytosis was developed for a microtitre-plate luminometer. The complete procedure was performed in a single microtitre plate and was simpler and more efficient than previous protocols. The kinetics of the PMN CL response were best when microtitre plates were incubated on a shaking incubator between readings. The new protocol was used in a study of the pathogenicity of Corynebacterium jeikeium, an organism found in association with infection in the immunocompromised. No differences were found when PMN CL induction by 15 strains of C. jeikeium were compared with 15 isolates of other corynebacteria. Both groups of organisms required complement for efficient opsonophagocytosis; C. jeikeium strains showed no requirement for specific antibody. Resistance to opsonophagocy- tosis does not appear to be an explanati~n for the increased pathogenicity of C. jeikeium. Microtitre-plate luminometers are particularly well suited to bacterial opsonization studies where large numbers of strains often need to be assessed.

Keywords: Chemiluminescence; microtitre-plate lurninometer; opsonophagocytosis

INTRODUCTION

The chemiluminescence (CL) response of phago- cytes has been widely used to study cell function, opsonic potential of serum and microbial pathogenicity in relation to resistance to opso- nophagocytosis. Initially most CL work was performed on scintillation counters but purpose- built luminometers are now more commonly used. These are generally single-chamber instru- ments, either manually operated or fully auto- mated with loading via a carousel to allow handling of multiple samples. Recently lumino- meters which work to a microtitre-plate format have become available. The Amerlite Analyser

(Amersham International, Little Chalfont, UK) was originally developed for automated signal detection in luminescence immunoassays but is equally suited to the measurement of cellular CL.

Strains of Corynebacterium jeikeium (‘JK coryneforms’) are isolated more frequently from infected, immunocompromised patients than other corynebacteria (Young et al., 1981; Gill et al., 1981). This may be related to the increased resistance to antimicrobial agents shown by many of these isolates but the possibility they are more resistant to host defences has not been investi- gated.

We describe a rapid, streamlined protocol for studying opsonization of bacteria by phagocytic

*Author for correspondence.

0884-39!96/89/030267-05$05.00 @ 1989 by John Wiley & Sons. Ltd.

268 J. G. M. HASTINGS, L. A. JEWES AND K. M. OXLEY

CL assayed on the Amerlite Analyser. An assessment of the opsonization of C . jeikeiurn in comparison to other corynebacteria is presented as an illustration of the use of this protocol.

METHODS

Bacteria

Fifteen strains of C. jeikeiurn isolated from a variety of clinical conditions and 15 skin isolates of other corynebacteria were selected for study. The latter included strains of C. bovis, C. xerosis, C. ulcerans and C. 0 2 .

Serum

Pooled human sera was obtained from five healthy donors and stored at -70°C. Comple- ment was inactivated by heating to 56°C for 30 min and the classical pathway was blocked using 10 mmol magnesium chloride/ethylene gly- col tetraacetate (MgEGTA). Antibody to C. jeikeiurn was absorbed out using the type strain (NCTC 11913) at 4°C in the presence of ethylenediaminetetraacetate.

Polymorphonuclear leukocyte (PMN) preparation

Fresh heparinized blood was collected from healthy human volunteers and sedimented over dextran. Residual red cells were lysed with distilled water and the remaining leukocyte-rich suspension washed twice in HBSS and resus- pended to a concentration o f 10" PMNlml in HBSS supplemented with 1% foetal calf serum.

Opsonization and PMN CL (Fig. 1)

Serum (lo%), HBSS and bacteria (1501.11; lo8 cfu/ ml) were added in duplicate to wells of a microtitre tray and incubated for 30 min at 37 "C in a shaking incubator (Amersham Internation- al). Trays were then centrifuged in a standard bench centrifuge fitted with a microtitre-plate head. Supernates were discarded and the bacteria washed twice with saline before resuspension in 150pl of HBSS. As a control, opsonized zymosan

37°C 1 30rnlrl

Ceritr tfuynd

Washed lwice

I

Figure 1. Protocol for bacterial opsonization and PMN CL assay in microtitre trays

(OZ; 20mg/ml, 10% serum) was prepared in two wells of each tray.

PMN (100 PI; 1O'lml) and luminol (200 pi; 1 0 - 4 ~ ) were added to each well and light emission measured in the Amerlite Analyser. Readings were taken at 3-rnin intervals over a period of 30min. Between readings, trays were left in the instrument or incubated in either a standard microtitre-tray incubator or a horizontal shaking model (see 'Results'). For the corynebac- teria work, CL results were expressed as a percentage of CL peaks obtained with opsonized zymosan.

RESULTS

Assay conditions

The Amerlite Analyser has no built-in heating mechanism but when operating has an internal temperature of about 30 "C under normal ambient temperatures. Peak OZ-induced CL readings were slightly reduced and the time-to-peak increased if assays were performed with the microtitre tray left in the instrument between readings rather than removed and placed in an incubator at 37°C. Kinetics of PMN CL produc- tion were improved further if a shaking incubator rather than a static incubator was used (Fig. 2). This procedure was adopted for all the opso- nophagocytosis work.

CHEMILUMINESCENCE IN MICROTITRE PLATES

C Er l l CL tF . r la t i r l i A t , t t u r t i t I

269

Figure 2. Peak CL production and time-to-peak for PMN stimulated with opsonized zymosan (0%. 1 Yo, 5%. 10% or 20% serum). Between readings microtitre trays were incubated in either a static (plain) or a shaking incubator (hatched) at 37°C

Opsonization of corynebacteria rent. Typical patterns of opsonic requirement are shown in Fig. 4. All strains of corynebacteria

There was a wide strain-to-strain variation in were poorly opsonized by heated serum. How- PMN CL induction by both opsonized and ever, PMN CL induction was only slightly unopsonized corynebacteria (Fig. 3). Mean PMN reduced when the same isolates were opsonized CL peaks induced by C. jeikeiurn strains and with absorbed and MgEGTA-chelated serum. other corynebacteria were not significantly diffe- Other corynebacteria were not tested against

270 J. G. M. HASTINGS, L. A. JEWES AND K. M. OXLEY

I JK NJK JK NJK

UNOPSONISED 10% SERUM

Figure 3. PMN CL (relative to OZ) induced by 15 C. jeikeium isolates (JK) and 15 other corynebacterium (NJK)

1 v UN WS HS MgE l " w s H s A b ~

Strain C34 Strain C36

Figure 4. PMN CL (relative to OZ) induced by a non-C. jeikeiurn corynebacteria (strain C34, C. ulcerans) and a C. jeikeiurn isolate (strain C36). unopsonized (UN). or opsonized with whole untreated serum (WS), heated serum (HS). absorbed serum (Ab, C. jeikeiurn only) and MgEGTA-chelated serum (MgE)

absorbed serum but otherwise behaved in a similar manner to the C. jeikeium strains.

DISCUSSION

In CL opsonophagocytosis studies it is often necessary to assess a range of bacterial isolates against a variety of different serum preparations. The traditional approach has been to perform

opsonization and subsequent centrifugation/ washing steps in tubes, and then transfer aliquots to a luminometer cuvette containing PMN and luminol. With large numbers of tubes involved this can be a laborious and time-consuming process. The microtitre-tray-based protocol we have developed has a number of advantages. The complete procedure, opsonization through to PMN CL measurement, is performed in a single microtitre tray. Addition and removal of fluids is simplified by the use of multichannel pipettes which makes serial washing steps rapid and efficient. Furthermore, the microvolumes used allow a significant reduction in the amount of PMN and human serum required for each assay run.

We used a study of the pathogenicity of C. jeikeium to help develop and assess this new protocol. Epidemiologically C. jeikeium appear to have a greater potential to infect immuncom- promised patients than other corynebacteria. The PMNL results suggest that increased resistance of C. jeikeium isolates to opsonophagocytosis is not an explanation for this increased pathogenicity; mean peak CL values induced by the two groups of organisms were not significantly different. All the corynebacteria we tested, including the 15 C. jeikeium isolates, required complement for effi- cient opsonophagocytosis. In contrast, the absorption studies with C. jeikeium suggested that specific antibody to this organism did not play an important role. Opsonization also appeared to proceed normally in the absence of the classical pathway. The association of C. jeikeium with infections of the immunocompromised may be related to other factors such as resistance to antibiotics or an increased ability to adhere to and colonize intravenous devices which are commonly a feature of the management of such patients.

Over the last five years we have utilized a number of different luminometers for PMN CL studies. After our recent experiences with CL measurement in microtitre trays we feel this format is ideally suited to the evaluation of large numbers of bacterial strains and serum samples in opsonophagocytosis studies. The Arnerlite Ana- lyser will read all 96 wells in under 90 seconds, which is a major improvement over single- chamber luminometers. The lack of an internal heating element did not appear to be critical and, although the shaking incubator improved the kinetics of CL production, results with the static incubator were satisfactory for this type of work.

CHEMILUMINESCENCE IN MICROTITRE PLATES 27 1

REFERENCES

Gill, V. J . , Manning, C. , Lamson, M. , Wollering, P. and Pizzo, P. (1981). Antibiotic resistant Group JK bacteria in hospitals. Journal of Clinical Microbiology, 13, 47247l.

Young, V. M., Meyers, W. E., Moody, M. R. and Schimpff, S. C. (1981). The emergence of coryneform bacteria as a cause of nosocomial infections in compromised hosts. American Journal of Medicine, 70,646-650.