BACTERIOPHAGE PATTERN AND METHICILLIN RESISTANCE OF STAPHYLOCOCCUS AUREUS ISOLATED FROM HOSPITALISED PATIENTS

BACTERIOPHAGE PATTERN AND METHICILLIN RESISTANCE OF STAPHYLOCOCCUSAUREUS ISOLATED FROM HOSPITALISED PATIENTS •

Surg Cdr RN MISRA, Surg Cdr YO SINGH

+

(INHS Sanjivani, Cochin)

ABSTRACT Ninety two strains of Staphylococcus aureus isolated from hospitalized patients were phage grouped and phage typed and their resistance pattern to methicillin were studied. Forty five (48.9%) strains were nontypable, followed by mixed group in 23 (25%). The phage groups I, II. III. V and miscellaneous groups were 3.3 per cent. 7.6 per cent, 8.7 per cent, t per cent and 3.3 per cent respectively. Majority of the strains were methicillin resistant. Out of 45 nontypable strains 33 (73.3%) were methicillin resistant whereas in mixed phage group methicillin resistance was found in 52 per cent. The inadequacy of available phages for typing. rising incidence of methicillin resistance in untypable strains and variance of phage group and phage types at various places has been highlighted. MJAFI 1997; 53 : 191-194 KEYWORDS: Drug resistance; Methicillin; Phage typing; Staphylococcus aureus.

Introduction

S

taphylococcus aureus is a common isolate from localized and systemic infections in hospitalized patients [IJ. It is responsible for nearly 10 per cent of nosocomial infections [2]. Bacteriophage typing is helpful in precise typing of bacteria and it provides valuable information in epidemiological studies of infection in the community. The temperate phages of Staph. aureus have a narrow host range and are suitable for use as typing phages. The phage typing system was originally introduced by Wilson and Atkins [3] and later it was reclassified and used internationally with 23 standard typing phages in 5 groups. The phage typing of a staphylococcus is usually stable though it may occasionally change by the loss or gain of a carried type-determining prophage as the bacteria pass from person to person in the community (2]. In 1950's a particularly virulent bacteriophage type 80/81 caused outbreaks of serious nosocomial

staphylococcal infections in the hospitals throughout the world and was also found to colonise hospital workers. The introduction of anti-staphylococcal antibiotics like penicillins and other betalactams in the 1960's coincided with the eclipse of strain 80/81 which was replaced by diverse phage types responsible for nosocomial infections [4]. However emergence of resistance to methicillin by alteration of penicillin binding proteins (PBP) is a growing cause for concern [5,6] and prompted us to study the prevalence of methicillin resistant Staph. aureus (MRSA) in the hospital environment. An effort was made to type these strains and correlate their typability with methicillin resis. tance.

Material and Methods Ninety two strains of Staph. aureus from pus and other material from hospitalized patients were obtained. The clinical sources were 51 patients with pyoderma. 18 with surgical wound infections, and 23 with abscess and other infections. The specimens were cultured on fresh 10 per cent

'Classified Specialist (Pathology), Dept of Microbiology, "Classiflcd Specialist (Medicine). Dept of Medicine, Armcd Forces Medical College, Pune ·411040.

Misra and Singh

192

blood agar, incubated aerobically at 37°C. Staph aureus was identified hy standard methods [2]. The strains were subjected to bacteriophage typing both by routine test dilution (RTD) and RTDxlOO at the Staphylococcal Phage Typing Centre (ICMR), Maulana Azad Medical College, New Delhi. The standard phage groups are as under [7] Group I· 29, 52, S2A, 79, 80 Group II· 3A, 3C, 55, 71 Group III· 6, 42E, 47, 53, 54, 75, 77, 83A, 84. 85 Group IV· (420) Group V· 94. 96 Miscellaneous Group ~ 81,95 The strains were tested for their antibiotic sensitivity pattern to methicillin 5 }lg disc against NCTC Staph. aureus 6571 at 30°C for 24 h in Mueller Hinton agar. The interpretation was done as recommended by Stokes [8]. The resistance was con finned by break point minimum inhibitory concentration method to 4 J1g/mL of methicillin

[8].

TABLE I

Phage pattern of Stop/iytocOCCU!1 aureus isolated frolll hospitalised patients Phage group

Number of isolates

Phage typt.'S and no of strains

RTD-

RTlh 100

I II

30.3)

2Qfi9

:i9179; 80

717.61

3'\/55171.3U55 3N71

3A(21.55.71

III

8(8.7)

54.75.83A.

6/47/54/84/85. 53n5/114,42EI 54184

47/54/8JI/84/85. 47/53/85(3)

IV

Nil

V

1(1.1)

Mise

3(3.3)

Mixed

25 (27.1)

NT

96 81 (3) 81/83A(2) 47154/75!85/81 ( 4) 801JA!75(2),55/80(3 ) 71/75/79(2).80/95196 53/71/96(2)

80195(3) 80/54/52(2)

47180184(2) 81/96(2)

45(48.9\

1'(1(31

92

-Number in brackets indicate percentage of total strains studied

: NT - Not typable; RTD - Routine test dilutions

Results The phage pattern of 92 Staph. aureus strains showed that 45 strains were untypable (Table I). Maximum number (25%) belonged to the mixed phage group. Phage group III were 8.7 per cent and those belonging to phage group V were the least (1%). In phage group II (7.6%), phage type 3A occurred more frequently and in the miscellaneous group phage type 81 contributed the max i~ III um, Phage types 47/54175/85/81 occurred more frequently in mixed phage group. None of the stains belonged to group IV. Fifty seven (62%) Staph. aureus strains were methicillin resistant. Thirty three (35.9%) MRSA strains were nontypable whereas 13.6 per cent were in the mixed phage group. Discussion The non-typability of 45 (48.9%) strains show that the staphylococci are not fully typable with the present set of bacteriophages. This has also been found by others [4,9,10]. The mixed phage pattern was commonest in this study where the organisms were lysed by phages belonging to more than one phage group. Similar observations have been made in earlier studies [9,12].

TABLE 2

Methidllin resistant pattern in relalion to phage types MR(%)

MS(%)

Total

Phage Gp I

2

I

PhageGp II Phage Gp III PhageGp IV

2

5

7

4

4

8

Phage Gp V Misc Gp

3

Nil

I

I

J

MixedGp

2 13 (52)

1 12 (48)

25

Not typable

33 (733)

12 (26fi)

45

57 (62)

35 (38\

92

MR ~ Methicillin resistant; MS = Methicillin sensitive

The commonest phage group reported in different studies in India [10,12] was Group III and these were 8.7 per cent in this study and is commoner than other phage groups. 47/53/85 occurred more frequently among the phage types. The miscellaneous phage group consisted of 3.3 per cent, which is comparatively lower than the findings reported by Basalas et al [II]. This could be due to inclusion of phage types 94, 96 in phage group V, .\LIM1- 1'01.. 53. NO. 3. /997

193

Phage Typing of Staph, aureus TABLE 3 Pha~e

paltern or Staphylococcus aureus reported by different workers Total

Worker

Strains

Bhujawala (1972) New Delhi

350

Bhaskran (1979)

80

I'irupati Ramani (1980)

76

Visnkhapatnarn

202

Basales (1988)

Group I

Group II

87 (24.9)

27 (7.7)

7 (8.7)

(113)

9

92

rrselll study Cochin

phage groups Mise

Mixed

NT

(12.8)

88 (25.2)

\03 (294)

15 1187)

4

(50)

45 (563)

45

5

5

\7

15

(0.6)

(6.6)

(4)

(197)

32 (42.1)

16

5 (25) 7 (7J»)

(·Hll

(7.9)

Gorakhpur

No of strains in dlffer~nl Group III

3

(3.3)

25

21

(12.5)

(10.4)

48 (23.11)

8

3 13.3)

(1)

(8,7)

25

87 45 (59,8)

Number in brackets indicate percentage of total isolates

as well as a possible real reduction in occurrence. The variance in phage groups at different places (Table 3) is possible due to nonspecific lysis by phage suspensions. activation of latent prophages and spontaneous variation of phage susceptibility [15]. Sixty two per cent methicillin resistance among the 92 strains is considerably higher than 33.6 per cent MRSA strains reported in Pune [16]. Majority (35.9%) of the methicillin resistant strains were in the non-typable group followed by mixed phage group (13%) which is comparable with the tindings of Alex et al [6] where majority of the 48 MRSA strains belong to non-typable group MRSA. Strains which produce unique PBP2' also produce penicillinase and full armamentarium of pathogenic factors including protein A. coagulase and DNAse L17]. Numerous in vitro studies have shown that MRSA strains are fully virulent and show intraphagocytic survival potential and lethality in animal studies [18]. Most of these strains are resistant to multitude of antibiotics including fluroquinolones but they are sensitive to vancomycin and teicoplanin which are the drugs of choice [19]. In phage group II. majority of strains (517). showed methicilline resistance where as MRSA and MSSA were equally distributed in phage group III and miscellaneous phage group. The phage types 52. 52A. 80 and 81 of phage group I were prevalent in hospitals between 1954-84 and were gradually replaced by other phage types after advent of penicillins [20J. In this study the phage HIAH, ''01. 53.

f,'()

3. /99 7

type 29/79 was cOlMft'lonly seen among the phage group I. Phage group III strains 83.'\. 84. 85 are characterised by resistance to neomycin. bacitracin and b-lysin production and failure to produce staphylokinase was responsible tor wide-spread post-operative sepsis in the 1960's [13J. In this study they accounted tor 8.7 per cent of which 47/53/85 phage type was found more frequently. Resistance to methicilline is seen in 50 per cent of strains but the number of strains are not adequate to substantiate this point. It seems that nontypable strains are more often methicillin resistant and do not belong to any classical phage groups. More number of bacteriophages are required to type staphylococci or Dutch MRSA specific phages may be utilised to type them. Other more advanced criteria like polymerase chain reaction may be utilised to characterise them as has been done by Alex et al [6]. Changing pattern of phage types demand a continuation of study in these areas to control the ever increasing nontypable MRSA strains in the hospital set-up. ACKNOWLEDGEMENT We thank Dr OS Agarwal and Prof R Natarajan of Maulana Azad Medical College, New Delhi lor their valuable support in conducting phage typing.

RHERENCES I. Ghosh B, Gupta 1\1. Bhattacharya SR. Clmico-bactenological study of pyoderma.fndian J Dermatol 1974; 19: 35-8. 2, Cnllee JG. Duguid JI' Practical Medical Microbiology. /Jlh Edillon. Churchill Livingstone 19R9,

Misra and Singh

194 3. Williams GS, Atkins JD, Phage types of Staphylococcus aureus. Lancet 1945; 1: 647-8, 4. Paschricha A, Bhujawala RA, Shriniwas. Bacteriology of pyoderma. Indian J Pathology Bacteriology 1972; 15: 131-

8. 5. Khalifa IK. Anwar AH, Gary H. Nontypable bacteriophage pattern of methicillin resistant Staphylococcus aureus involved in a hospital outbreak. J Clin Microbiol 1989: 27: 2249-51. 6. Alex von B, Bak R. Broom PI', Gossen WHF. Lewen N, Quint WGV, Comparison of phage typing and DNA finger printing by polymerase chain reaction for discrimination of methicillin resistant Staphylococcus aureus. J Clin Microbioi 1993; 31: 798. 7. Easman CSF, Goodfellow M. Staphylococcusand Micrococcus. In: Parker MT, Collicr LH. editors. Topley Wilson's Principles of Bacteriology, Virology and Immunity, 8th cd. London: Edward Arnold. 1990; 161-86. 8. Stokes EJ. Ridgway OL. Wren MWD. Clinical Microbiology. 7th ed. Boston: Edward Arnold 1993; 251-2. 9. Bhujawala RA. Mohapatro LN. Bacteriophage pattern and antibiotic resistance pettern of Staphylococcusaureus Indian Journal Pathology Bacteriology 1972; IS: 66-72. 10. Bhaskaran CG, Ran PS, Krishnamurthy T. Tarachand P. Bacteriological study of pyoderma Indian Journal Dermatology Venerology Leprology 1979; 45: 162-70. 11. Ramani TV, Jayakar PA. Bacteriological study of 100 cases of pyoderma with special reference to StaphylOCOCCI. their antibiotic sensitivity and phage pattern. Indian Journal Dermatology Venerology Leprology 1980: 46: 282-6. 12. Basalas RO. Arora SK. Mukhija RD, Singh UK, Mohan L. Changing pattern of Staphylococci in pyoderma cases. In-

dian J Pathology and Microbiol 1990; 33: 299-303.

13, Khare AK, Bansal NK, Dhruva AK. A Clinical and Bacteriological study of pyoderma. Indian Journal Dermatology Venerology Leprology 1988: 54: 192-5. 14. Cohen LS, Fever FR, Cluff LD. Studies of the epidemiology of staphylococcal infection. New Engl J Med 1982;

266: 367-72. 15. Waldvogel FA. Staphylococcus aureus (including toxic shock syndrome). In: Mandell OL. Bennett JE, Dolin R, editors. Principles and practrcc of Infectious Diseases, 4th ed, New York: Churchill Livingstone, 1995; 1754-77. 16. Roo MR, Ohri VC, Kher SK, Raghunath D. Emergence of methicillin resistance Staphylococcus aureus (MRSA) in Command Hospital (SC). Pune. Medical Journal Armed Forces India 1990; 46: 235-7. 17. Crossly K. Landsman B, Zaske D. An outbreak of infection caused by strains of Staphylococcusaureus resistant to methicillin and aminoglycosides. J Infect Dis 1979; 139: 280-7. 18. Peacock JE, Moorman DR, Wenzel RP. Methicillin resistant Staphylococcus aureus: microbiologic characteristics, antimicrobial susceptibilities and assessment of virulence of epidemic strains. J Infect Dis 1981; 144: 575-9. 19. Kinsman 0, Naidoo J, Noble We. Some effects of plasmid coding for antibiotic resistance of the virulence of Staphylococcus aureus. Pathology 1985; 66: 325-30. 20. Vaudause P, Weldvogcl FA. Methicillin resistant Staphylococcus aureus: relation between expression of resistance and phagocytosis by polymorphonuclear leukocytes. J Infect Dis 1979: 139: 547-5 I.