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July 1989, Volume 39, Issue 7

Original Article

NEISSERIA MENINGITIDIS STORAGE IDENTIFICATION AND MIC DETERMINATION

Salim Hafiz  ( Department of Microbiology, Aga Khan University Hospital, Karachi. )
Neville Lyall  ( Department of Microbiology, Aga Khan University Hospital, Karachi. )
Mehboob Alam  ( Department of Microbiology, Aga Khan University Hospital, Karachi. )

Abstract

The significance Minimum Inhibitory Concentration (MIC) of determination for Meningococci is described and according to the present study a shift in the sensitivity of Meningococcus is occurring, local isolates show higher Minimum Inhibitory Concentration for Chloramphenicol and Azactam. Strains of Meningococcus can successfully be stored in a domestic freezer (JPMA 39: 177, 1989).

INTRODUCTION

Neisseria nieningitidis is an extremely intri­guing organism. On one hand it inhibits the upper respiratory tract and other mucosal surfaces of humans, the only known reservoir, without causing any ill effects. On the other hand it causes invasive infection, killing the patient in few hours. Menin­gococcal disease tends to occur in clusters and usually in epidemic forms. Epidemics due to N. Meningococcus group A tend to occur in cyclic waves approximately every decade and have been documented since the beginning of 20th century1. In Karachi the first documented outbreak of N. ineningococcus occurred during 1966-67. Unfor­tunately the group causing the outbreak is not known while the recently concluded epidemic was caused by sero group A and interestingly it fol­lowed identical steps in its spread. The isolates were sensitive to the routine drugs2. N meningococcus is closely related to N. gonorrhoeae and the difference is very subtle3. Both members of the family were highly sensitive to Penicillin but with passage of time one member N. gonorrhoeae become gradually resistant to Penicillin4 and according to one report, N. gonor­rhoeae had the ability to inactivate Penicillin even in 19455; the time Penicillin was commercially available yet N. meningitidis a sister member has failed to acquire this ability. It is universally ac­cepted that N. ineningitidis is sensitive to Penicillin and Chioramphenicol and as the isolates in the recently concluded epidemic were all found to be thus sensitive tested by disc sensitivity method, we decided to compare the MIC of local isolates be­cause of considerable abuse of antibiotics com­pared to foreign isolate where use of antibiotic is restricted.

MATERIALS AND METHODS

Organisms:
A total of 22 clinical isolates of N. ineizin­ gitidis were used for the determination of Mini­mum Inhibitory Concentration (MIC). They in­cluded 5 strains isolated in UK and provided by Dr Abbott, 9 strains were isolated by our labora­tory and 8 strains were provided by “The Lab” and “Sind Lab”, Karachi. AU the local isolates belon­ged to Sero-group A. The UK strains were freeze dried. They were grown on chocolate agar identified and confirmed by gram’s staining, oxidase and carbohydrate fer­mentation reaction. All local strains, as and when isolated, were confirmed, sensitivity test carried out by Kirby Bauer method and 10 colonies were emulsified in 1 ml of sterile tryptone soya broth (oxoid) containing 10% glycerol, stored in the freezer compartment of refrigerator at —15 to — 20°C. When needed, the tubes containing the or­ganisms were thawed, subcultured onto chocolate agar, incubated at 36-37°C in a carbon dioxide incubator with enhanced humidity and 24 hour growth was used for determination of MIC.
Determination of Minimum Inhibitory
Concentrations:
Agar dilution methods were used for the determination of MIC. Media: The basal media used was Difco G.C. agar base with defined supplement6.
Carbohydrate Fermentation Reaction:
Carbohydrate fermentation reactions were determined by incorporating 1% carbohydrate in the medium and 1 ml of 0.02% Phenol red in­dicator sterilized by autoclaving at 10 lbs/sq inch (115°C) for 15 minutes and plates were poured.
Determination of Minimum Inhibitory
Concentrations:
Agar dilution method was used for the de­termination of MIC. A range of concentration of Penicillin, Chioramphenicol, Azactam and Ami­kacin were prepared according to the recommen­dations7to give the required final concentration as given in the Table.

Plates were poured and used within a week of preparation.
Inoculation of Plates:
A multipoint inoculator (Denley Instru­mcnts Ltd, Billinghurst, Sussex, United Kingdom) was used to deliver 0.001 ml inocula (106CFU) to the surface of antibiotic containing plates, car­bohydrate containing plates and control plates without any antibiotics or carbohydrate were also inoculated as growth control. After inoculation, the carbohydrate plates were cut by using sterile scalpel blade in order to avoid smudging of fer­mentation results. All plates were incubated at 36°C in an atmosphere of 5% carbon dioxide with enhanced humidity. Results were recorded after 24 and 48 hours and the MIC’s were taken as the lowest concentrations of antibiotic preventing the growth. Carbohydrate fermentation were recor­ded by observing for growth and change in colour from red to yellow.

RESULTS

Storage Results:
All the strains of N. ineningitidis stored in freezer survived for over 6 months. This provides a simple, convenient and economic method for preserving cultures where sophisticated facilities are not available.
Carbohydrate Fermentation:
All the strains tested fermented Glucose and Maltose while Sucrose and Lactose were negative and the results fully matched with fermentation reactions compared with serum free sugars.
MIC Determination:
On disc sensitivity all strains were sensitive to Chloramphenicol, Penicillin and Azactam. The results of MIC are recorded in the Table and it is interesting to note that there is a difference be­tween the United Kingdom isolates and local iso­lates as far as MIC are concerned. In all cases, local isolates show an increased resistance, with Penicillin it is only marginal 5.8% (1 strain) has MIC of 0.05ug/ml, with chloramphenicol 17% iso­lates shows MIC greater than I ug/ml, with Azac­tam it amounts to 23% and in the case of Amikacin more than 41% local isolate has MIC > 8 ug/ml as opposed to 20% of UK isolates.

DISCUSSION

Inspite of the close relationship enjoyed by M. meningitidis with N. gonorrhocae, N. menin­gitidis has failed to acquire the ability to produce Beta lactamase but if this ever happens it would be devastating. One way would be to carry out MIC on meningococcal isolates specially in the parts of the world where antibiotic abuse is a common practice and all laboratories in the deve­loping world carry out antibiotic sensitivity tests by Disc diffusion method. The study involves a limi­ted number of isolates but reveals that the sen­sitivity pattern of a Meningococcal isolates is ex­hibiting a gradual shift towards resistance to the commonly used antibiotics, Penicillin and Chlo­ramphenicol. the increase in MIC for Chloram­phenicol is higher as compared to Penicillin. Organisms can be stored conveniently in do­mestic refrigerators for appreciable length of time and carbohydrate fermentation reactions carried out using a multipoint inoculator. The abuse of antibiotics, specially underdos­ing, may have the clue for the gradual shift of MIC in Meningoeocci.

ACKNOWLEDGEMENT

We thank Drs. J.D. Abbott of Manchester, UK, Iftikhar Ahmad, Sind Lab, Karachi, Iqbal Ahmad, the Lab, Karachi for supplying strains of meningococci and Mrs Fatima Nadir for her sec­retarial help.

REFERENCES

1. Lapeyssonnie, L La meningite cerebrospinale en AM­que. Bull. WHO 28 (supp.): 3.
2. Hafiz, A. Acute meningitis in Karachi—Past and present. Med. Spectrum, 1988; 9: 25.
3. Odugbemi, t.O. and Hafiz, S. Identification of Neisseria gonorrhoeae by carbohydrate disc reactions on a modi­fied fermentation medium. J. Trop. Med. Hyg., 81: 106.
4. Jamil, J.J., Hafiz, S., and McEntegart, M.G. The effect of Benzylpenicillin on strains of Neisseria gonorrhoeae in liquid and solid media. Antimicrob. Chemotherapy, 1981; 210.
5. Hatiz, S., McEntegart, M.G. and Gooch, H. Did Neis­ seria gonorrhoeae acquire the ability to produce B-lac­tmase in 1976? Lancet, 1982; 1: 558.
6. Flynn, J. and Waitkins, S.A. A serum-free medium for testing fermentation reactions in Neisseria gonorrhoeae. Clin. Pathol., 1972; 25: 525.
7. Anahalt, J.P. and Washington, J.A. Preparation and stor­age of antimicrobial solution in ASMs, manual of clinical microbiology 4th ed. Bethesda, 1985, p. 1019.

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