ABSTRACT: Staphylococcus aureus is a major cause of nosocomial and community acquired infections. The increasing prevalence of multiple drug resistant strains causes failure of treatment of patients resulting into prolonged hospital stay.
Phagetyping is a successful method of strain characterization in S. aureus and can be applied when studying the spread of staphylococcal infection and its origin in an outbreak.
The present study was conducted to identify the currently existing phagegroups of S. aureus, their prevalence and resistance to antibiotics.
A total of 99 isolates of S. aureus were phage typed and their antibiotic resistance was determined using standard microbiological procedures. Of these,72.72% isolates were typable.
Phage group III was the most predominant (54.54%)out of which 66.66% were typed by phage type 42 E and38.88% were typed by phage type 47. MSSA strains were more sensitive as compared to MRSA strains. Phage group II isolates were resistant to penicillin.
Keywords: Staphylococcus aureus, MRSA, MSSA, Phage-grouping, Phage-typing.
INTRODUCTION : -
Staphylococcus aureus is a major cause of nosocomial and community acquired infections around the world. These strains cause a high rate of morbidity and mortality and are the cause of grave concern to clinicians. The increasing prevalence of multiple drug resistant strains result into failure of treatment and prolonged hospital stay. It is an additional problem to the hospital performing infection control and prevention program.
Phagetyping has been successful as a method of strain characterization in S. aureus and it can be applied when studying the spread of staphylococcal infection and its origin in an outbreak. These phagetypes responsible for infections may vary from time to time and hospital to hospital in their antibiotic resistance pattern. Typing of these strains is important in epidemiology to analyze the spread of infection.
The present study was undertaken to study the epidemiology of S. aureus isolates with reference to currently existing phage-groups, their prevalence in the hospital and community and the correlation of various phage-groups with antibiotic resistance
MATERIALS AND METHODS:-
A total of 100 S. aureus isolates including 75 isolates recovered from clinical samples and25 isolates from nasal swabs of healthcare workers were included in the study. All the isolates were identified using standard microbiological procedures as per the guidelines of Kloos and Schleiffer's scheme (1975) and Coneman's dichotomous key (2006). They were subjected to antibiotic susceptibility test by Kirby Bauer's disc diffusion method (1966) on Mueller Hinton (MH) agar and the zones were interpreted as per CLSI guidelines(2007). Antibiotic discs with their respective potencies were as under
Antibiotic
Symbol
Potency
Penicillin G
P
10 units
Ampicillin+Sulbactam
AS
10/10 mcg
Amoxicillin+Clavulanic acid
AMC
20/10 mcg
Erythromycin
E
15mcg
Ciprofloxacin
CF
15mcg
Cefotaxime
CE
30mcg
Gentamicin
G
10mcg
Co-trimoxazole
BA
1.25+23.75mcg
Tetracycline
TE
30mcg
Vancomycin
VA
30mcg
Methicillin resistance was checked using oxacillin discs (1mcg) on Mueller Hinton Agar (MHA) with 4% salt and cefoxitin discs (30mcg)on MHA as per CLSI guidelines and the strains were identified as Methicillin resistant S. aureus (MRSA) and Methicillin sensitive S. aureus (MSSA).
Phage typing:-
A total of 100 S. aureus strains comprising of 75 clinical and 25 from healthcare workers were sent to National Phage-typing centre, Maulana Azad Medical College, New Delhi, for phage-typing.
Seventy five clinical isolates were designated as community acquired and hospital acquired strains as per following criterion:
The isolate was labeled as hospital acquired when it was obtained after 48 hours of admission or was directly related to the hospital intervention. The community acquired isolates are obtained within 48 hours of admission, were unrelated to the hospital intervention and also the strains isolated from OPD patients.
At the staphylococci phage typing centre the phage typing was done by standard method at 100 X RTD using International basic set of 23 typing phages.
Phage Group I 29 52 52A 79 80
Phage Group II 3A 3C 55 71
Phage Group III 6 42E 47 53 54 75 77 83A 84 85
Phage Group V 94 96
Phage Group NA 81 95
Statistical Analysis - Chi square was used to compare the antibiotic susceptibility between the MSSA and MRSA strains and between the phage groups.
RESULTS :
Of the total 100 strains, 42were isolated from the community acquired infections (CAI),33 from the hospital acquired infections (HAI), and 25 from the nasal swabs (NS) of healthy hospital staff. Distribution of MRSA, MSSA and Phage groups in the strains isolated from community, hospital and nasal isolates is as shown inTable -1.
Table 1: Distribution of Phage Groups amongst MRSA and MSSA from various sources
Phage Groups
I
II
III
V
NA
Mix
NT
Source
Community (42)
MRSA (4)
2
1
2
1
1
3
1
%
50.0%
25.0%
50.0%
25.0%
25.0%
75.0%
25.0%
MSSA (38)
18
4
24
5
2
22
6
%
47.4%
10.5%
63.2%
13.2%
5.3%
57.9%
15.8%
Hospital (33)
MRSA (16)
4
1
9
3
3
6
5
%
25.0%
6.3%
56.3%
18.8%
18.8%
37.5%
31.3%
MSSA (17)
7
1
8
2
2
6
5
%
41.2%
5.9%
47.1%
11.8%
11.8%
35.3%
29.4%
Nasal (24)
MRSA (7)
2
0
2
0
0
0
3
%
28.6%
0.0%
28.6%
0.0%
0.0%
0.0%
42.9%
MSSA (17)
5
1
9
4
3
7
7
%
29.4%
5.9%
52.9%
23.5%
17.6%
41.2%
41.2%
Total MRSA (27)
8
2
13
4
4
9
9
%
29.6%
7.4%
48.1%
14.8%
14.8%
33.3%
33.3%
Total MSSA (72)
30
6
41
11
7
35
18
%
41.7%
8.3%
56.9%
15.3%
9.7%
48.6%
25.0%
Out of total 100 S. aureus isolates, one nasal isolate was lost during transport. Out of 99 isolates,27 were nontypable by the current set of phages which could type 72.72% of total isolates. Percentage typeability among MSSA and MRSA was found to be 75% and 66.66% respectively. Of the total 99 clinical isolates, 48.61% of MSSA and 33.33 % of MRSA strains were typable by more than one phage group and were considered as mixed group.
Of the community isolates, 16.7%were nontypable and 59.52% belonged to mixed group. The most predominant was phage-group III (63.2% MSSA and 50% MRSA) followed by phage-group I (47.4% MSSA and 50% MRSA).
The most predominant phage group in our hospital was group III (47.1% MSSA & 56.3% MRSA) followed by phage group I (41.2% MSSA & 25% MRSA). Phage-group II was the least common (5.9% MSSA and 6.3% MRSA).
Amongst nasal isolates, 37% were nontypable. Phage-group II and V were not detected in nasal MRSAs. Phage-group III (52.9% MSSA and28.6% MRSA) was the most predominant followed by phage-group I (29.5 MSSA and 28.6% MRSA).
Phage-group distribution amongst MSSA and MRSA strains is shown in Table-1 and Figure 1, Figure 2 and Figure 3. It was found as Group I (41.7% MSSA and 29.6% MRSA), Group II (8.3% MSSA and 7.4% MRSA), Group III (56.9% MSSA and 48.1% MRSA), Group V (15.3% MSSA and 14.8% MRSA), Mix Group (48.6% MSSA and 33.3% MRSA) and NA Group (9.7% MRSA and 14.8% MRSA).
Predominant phage types in specific phage groups are shown inTable-2.
Table 2: Predominance of phagetypes in specific phagegroups of Staphylococci
Phage group & Types
Community
MRSA
MSSA
Hospital
MRSA
MSSA
Nasal
MRSA
MSSA
Phage group III
Phage Type 42 E (36)
16
2
14
13
6
7
7
0
7
Phage Type 47 (21)
10
1
9
9
4
5
0
0
2
Phage group I
Phage Type 52 (22)
10
2
8
9
2
7
3
0
3
Phage Type 52 A (17)
10
2
8
6
1
5
1
0
1
Phage group NA
Phage Type 81 (18)
10
1
9
5
3
2
3
0
3
Phage group II
Phage Type 55 (7)
5
1
4
1
1
0
1
0
1
Phage Type 71 (6)
5
1
4
1
1
0
0
0
0
Amongst phage group III, phage type 42E was the commonest followed by phage type 47. The predominant phage types in phage group I were 52 and 52A, in phage group NA, type 81 and in phage group II, type 55 and 71.
Correlation of different phage groups of MRSA and MSSA with their antibiotic susceptibility is shown in Table 3 and Table 4 respectively.
Table 3: Prevalence of various Phage Groups and their antimicrobial resistance pattern amongst MRSA isolated from clinical specimens
Antibiotics
P
AS
AMC
E
CF
CE
G
BA
TE
VA
Phage Group
Phage Group I (6)
6
0
0
4
3
4
1
4
2
0
%
100.0%
0.0%
0.0%
66.7%
50.0%
66.7%
16.7%
66.7%
33.3%
0.0%
Phage Group II (2)
2
0
0
0
0
1
0
1
0
0
%
100.0%
0.0%
0.0%
0.0%
0.0%
50.0%
0.0%
50.0%
0.0%
0.0%
Phage Group III (11)
9
0
0
7
5
8
2
7
4
0
%
81.8%
0.0%
0.0%
63.6%
45.5%
72.7%
18.2%
63.6%
36.4%
0.0%
Phage Group V (4)
4
0
0
3
1
4
2
3
2
0
%
100.0%
0.0%
0.0%
75.0%
25.0%
100.0%
50.0%
75.0%
50.0%
0.0%
Phage Group NA (4)
4
0
0
3
2
2
1
4
3
0
%
100.0%
0.0%
0.0%
75.0%
50.0%
50.0%
25.0%
100.0%
75.0%
0.0%
Phage Group Mix (9)
9
0
0
6
3
6
2
7
4
0
%
100.0%
0.0%
0.0%
66.7%
33.3%
66.7%
22.2%
77.8%
44.4%
0.0%
Phage Group NT (6)
4
2
2
5
2
6
4
4
4
0
%
66.7%
33.3%
33.3%
83.3%
33.3%
100.0%
66.7%
66.7%
66.7%
0.0%
Table 4: Prevalence of various Phage Groups and their antimicrobial resistance pattern amongst MSSA isolated from clinical specimens
Antibiotic
P
AS
AMC
E
CF
CE
G
BA
TE
VA
Phage Group
Phage Group I (25)
18
0
0
3
14
7
0
4
3
0
%
72.0%
0.0%
0.0%
12.0%
56.0%
28.0%
0.0%
16.0%
12.0%
0.0%
Phage Group II (5)
5
0
0
2
1
2
0
2
1
0
%
100.0%
0.0%
0.0%
40.0%
20.0%
40.0%
0.0%
40.0%
20.0%
0.0%
Phage Group III (32)
23
0
0
6
18
12
1
4
4
0
%
71.9%
0.0%
0.0%
18.8%
56.3%
37.5%
3.1%
12.5%
12.5%
0.0%
Phage Group V (7)
5
0
0
1
4
3
0
0
0
0
%
71.4%
0.0%
0.0%
14.3%
57.1%
42.9%
0.0%
0.0%
0.0%
0.0%
Phage Group NA (13)
10
0
0
4
7
8
1
3
2
0
%
76.9%
0.0%
0.0%
30.8%
53.8%
61.5%
7.7%
23.1%
15.4%
0.0%
Phage Group Mix (28)
21
0
0
5
16
10
1
5
3
0
%
75.0%
0.0%
0.0%
17.9%
57.1%
35.7%
3.6%
17.9%
10.7%
0.0%
Phage Group NT (11)
7
0
0
4
6
3
1
2
0
0
%
63.6%
0.0%
0.0%
36.4%
54.5%
27.3%
9.1%
18.2%
0.0%
0.0%
DISCUSSION:
The epidemiology of S. aureus has continued to change during the past few years. It is important to know the epidemiological relatedness between the organisms to know the spread of these strains. Bacteriophagetyping is an established method for epidemiological typing of staphylococci.
Different workers have reported predominance of different phage-groups and phagetypes in their studies. On analysis of our results of phagetyping, we found that phage group III was the most predominant in all the three groups i.e. hospital isolates, community isolates and nasal isolates followed by phage group I in both MSSA as well as MRSA strains.
Mehndiratta et al (2010) have reported predominance of phage group III amongst the MRSA isolates from HAI and phage group NA amongst MSSA from community. Vidhani et al (2001) have also reported that group III was predominant in MRSA. In the mid nineties phage group III was reported predominantly in MRSA and group II in MSSA by Usman et al(1996) and Samba & Gadba(1993). Mixed phage group was reported to be the most predominant phage group in the late nineties as reported by Gupta et al. in 1999. Computer analysis of Staphylococcus aureus phage typing data from 1957 to 1975 published by Zierdt et al. (1980) showed that 54% of the strains belonged to mixed group of phages. This indicates that there is cyclic fluctuation in the appearance of specific phage groups.
Prevalence of phagetypes in different phage groups was also studied. In our study the most predominant phagetypes in phage group III were 42E followed by type 47 whereas in phage group I, the commonest phage type was 52 followed by 52A. Predominant phage pattern amongst individuals group seams to vary in different studies. Phagetype 80 was reported to be the commonest by Rountree and Freeman (1955). Predominance of phagetypes 53 / 75 / 77 have been reported by Barber (1951) and Barber &Duttan (1958). Pahujani et al (1965) have reported that phage group I with 80 / 81 complex is the most common phage-type.
All the staphylococci were sensitive to vancomycin. More than 50% MRSA isolates belonging to phage group NA, V and I showed resistance to more than 5 antibiotics tested out of ten whereas more than 50% isolates of group three were resistant to more than 4 antibiotics. MSSA isolates belonging to phage group NA were slightly more resistant as compared to other phage groups. On comparing antibiotic resistance of MRSA and MSSA strains, it was observed that MRSA strains were significantly more resistant to majority of antibiotics as compared to MSSA strains. (P value< 0.001 for E, CE, G, BA and T). This may be because majority of our MSSA strains were isolated from community. Similar finding is also documented by Mehndiratta et al.(2010) and Suntharam et al.(2001). Multiple drug resistance is lower in community isolates due to less antibiotic selective pressure within the community than in the hospital. All phage group II isolates were found resistant to penicillin.
CONCLUSION:
Bacteriophage typing is an established method of epidemiological typing for S. aureus. Our study showed predominance of phage group III in all the three study groups. Among phage-group III, the most common phagetypes were 42 E followed by47. All phage group II isolates were resistant to penicillin. This study brings to light the importance of phagetyping, as certain phagetypes may be associated higher drug resistance.Of the 99 isolates under study, 26.26% isolates were nontypable. There is increasing problem of non-typeability amongst S. aureus and need for newer set of phages. It also implies the need to develop a local set of phages pertaining to a particular area so as to increase the typeability by phagetyping.
Limitations of the study
1) A larger number of MRSA isolates need to be studied, to determine the significance of certain phage-types in relation to community or hospital acquired infections but due to limited number of isolates were accepted at the National Phagetyping centre, we had to limit our study to 100 isolates only.
2) Only 72.72% S. aureus strains could be typed with current set of phages. There is a need to develop local set of phages.
Scope for further research
There is a need to develop a local set of phages to increase the typeability by phage typing
Phage typing can be compared with other typing methods of staphylococci.
Figure 1 : Distribution of Phage Groups amongst MRSA and MSSA from community
Figure 2 : Distribution of Phage Groups amongst MRSA and MSSA from Hospital
Figure - 3 Distribution of Phage Groups amongst MRSA and MSSA from Nasal Isolates
