Acta path. microbiol. scand. Sect. B, 84: 359-368, 1976
STAPHYLOCOCCUS AUREUS STRAINS ISOLATED IN DANISH HOSPITALS FROM JANUARY lst, 1966, TO DECEMBER 31st, 1974 KIRSTEN ROSENDAL, PERBULOW,MICHAEL WEISBENTZON and KNUDRIEWERTS ERIKSEN Statens Seruminstitut, Department of HospitaI Infections, Copenhagen, Department of Medical Bacteriology, Arhus, Biostatistical Department, Copenhagen, and Department of Medical Bacteriology, Blegdamshospitalet, Copenhagen, Denmark
Rosendal, K., Biilav, P., Bentzon, M. W. & Eriksen, K. R. Staphylococcus aureus strains isolated in Danish hospitals from January lst, 1966, to December 31st, 1974. Acta path. microbiol. scand. Sect. B, 84: 359-368, 1976. During the years 1966-1974, 167,297 strains isolated from 167,297 patients or staff members in Danish hospitals were registered at Statens Seruminstitut. All the strains were phage-typed and examined for production of a ‘Tween’-80-splitting enzyme and resistance to mercuric chloride. 158,236 strains were examined for resistance to antibiotics. Since 1968, a steep decrease in the number of strains resistant to three or more antibiotics (multiple-resistant) and in strains of the 83A complex was noticed. I n recent years an increase in strains belonging to phage-group I and in those referred to as miscellaneous and non-identified was registered. The increase in the non-typable strains might be explained by the shift of the concentration of the typing phages from 1000 x RTD to 100 x RTD. I t is concluded that a t least two factors may have contributed to the reduction of the multiple-resistant strains: an altered antibiotic policy, restricting the use of streptomycin and tetracyclines, and an improved hospital hygiene, diminishing the spread of identical strains within the various departments. However, it is emphasized that the consumption of methicillin is still increasing. Kefr words: Staphylococcus aureus strains; antibiotic resistance; phage-typing ; Danish hospitals.
K. Rosendal, Department of Hospital Infections, Statens Seruminstitut, Amager Boulevard 80, DK-2300 Copenhagen S, Denmark.
Received 14.vi.76
Accepted 23.vi.76
At Statens Seruminstitut, Copenhagen, the registration of phage-typed Staphylococcus aureus strains from hospitals all over the country is still performed as described in publications from 1963 (20) and 1967 (16), which deal with the material from the beginning of the registration in April, 1960 until the end of December, 1965. In these surveys, a steep decrease in the
number of strains belonging to the 52, 52A, 80, 81 complex was noticed, whereas strains belonging to the 83A, 84, 85, 93, 89 complex ( = 83A complex) and especially those resistant to three or more antibiotics were registered with increasing frequency. The present survey deals with the material collected over a nine-year period ending December 31, 1974. I t was found that the changes described previously continued dur-
359
TABLE 1. T h e Materiul Year Number Examined for resistance to antibiotics % TWHg+ %
1966 15682
1967 16044
1968 16235
1969 18626
1970 19305
1971 18393
1972 20002
1973 21303
1974 21707
Total 167297
14712
14990
15111
17604
18393
17478
19024
20254
20670
158236
34 36
36 35
36 32
35 29
33 26
29 21
27 18
22 13
19 11
~~~
TWHg+
= do not produce the ‘Tween’-8O-splitting enzyme. = resistant to mercuric chloride.
ing the first three years and that from then on a decrease in the multiple-resistant strains was observed. Since 1966 it has been possible to record the occurrence of the methicillin-resistant strains. Furthermore, evolutionary events within the 83A complex have been followed during recent years, as it has been possible to perform a subdivision of the complex by using the new international typing phages 84, 85 and 89.
89) (17) and in 1973 (phage 94) ( 3 ) . The strains typed by phage 94 have been recorded as “ m i s cellaneous”. The phage previously referred to as “6557” ( 5 ) has been internationally designated as phage 93. From June Ist, 1971, the strains non-typable at RTD (routine test dilution) have been retested with the phages in a concentration of 100 x R T D instead of 1000 x RTD. In order to examine the effect of this change, freeze dried bacteraemia strains, non-typable at 100 x RTD, were retyped, using the concentration of 1000 x RTD (Table 2 ) .
RESULTS MATERIALS AND METHODS
T h e Material A “Total material”, (Table 1) comprises all phage-typed Staphylococcus aureus strains isolated from persons connected with Danish hospitals, patients and staff members. Only one strain per person is included, selected as previously described (20). B Bacteraemia strains. For comparison, some figures from a material consisting of bacteraemia strains only (19) have been made use of. The methods are mainly those described in papers from 1963 (20) and 1967 (16). The alterations are as follows: Registration The information from the material from 1966 was extracted from the original cards. The results from 1967-1974 were transferred to punch-cards, using special cipher-codes for the registration of phage-types/groups, sources, resistance to antibiotics and mercuric chloride and production of a ‘Tween’-80-splitting enzyme. Bacteriophage-typing New typing phages have been added to the basic set ( 2 ) in 1966 (phage 84 and 85), 1969 (phage
360
From Table 1 it is seen that the material consists of 167,297 strains (isolated from the same number of persons) most of which have been examined for resistance to antibiotics. During the whole period the number of strains has been steadily increasing. Changes in the Properties of the Strains Resistance to antibiotics (Fig. 1 ) . The percentage of strains sensitive to all antibiotics and resistant to penicillin (P) has been fairly constant, but strains resistant to the other antibiotics tested (streptomycin (S), tetracyclines (T), chloramphenicol ( C ) , erythromycin (E) and methicillin ( M ) ) have since 1968 become less frequent. I t is remarkable that the declining parts of the curves representing S and T resistance run parallel. The changes of resistance are further analysed in Fig. l B which shows a decrease in multiple-resistant strains (resistant to three or more antibiotics) from 25 per cent in 1967 to 7 in 1974 and an increase in strains resistant
A 80’60
-
40,
::p O
I
93, 89 (83A) complex reached a peak in 1969-1970, whereafter the percentage fell from 25 to 14 in 1974, and a considerable increase of non-typable strains was noticed from 1971. Further investigation of the 83A complex (Fig. 3) showed that strains lysed by all five typing phages have almost disappeared, whereas strains sensitive to phage 83A plus some of the other phages and types 84 and 85 are increasing. Types 89 and 84/85/93/89 have decreased since 1969.
*
0
2ot c
“1 E
NT
0 64
68
72
907
B
%
6.*fo“-l ‘0
-
% 100
,..* .”, ,..a’
,----
”.‘.‘.
0
68
64
72
80, 70- 8 3 A COMPLEX
1
Fig. 1. Antibiotic sensitivity of the strains (percentage). T h e antibiotic sensitivity of strains from a previously published material (16) from the years 1964-1965 is also shown in this figure.
A. Strains resistant to: P = penicillin; T = tetracyclines ; C = chloramphenicol;
B.
0- -0
0
-0
0-0
S
= streptomycin; M = methicillin; E = erythromycin.
Strains resistant to P and sensitive to other antibiotics investigated. Strains resistant to P + S + T f other antibiotics as well. Strains sensitive to all antibiotics.
20
% 52,52A,80,81 o COMPLEX
j
to P only, from 36 per cent to 60 over the same period. Phage-typeslgroups. The percentage occurrences of the various phage-groups are shown in Figs. 2 and 3. The major changes are: strains of the 52, 52A, 80, 81 complex a.re still decreasing, strains of the 83A, 84, 85,
0
66
68
70
72
b
74
0
Fig. 2. Percentage contribution of the various phage-groups/complexes. Cumulative curves. NI = Non-identified. Misc. = Miscellaneous. N T = Non-typable.
36 1
% 100
-
84/85 + 93
90 80
70
60 50
40 30
20 10
0 1
71
73
Fig. 3. 83A complex. Cumulative curves. 84/(89) = Strains lysed by phage 84 whether or not in combination with phage 89. “83A” = Strains lysed by phage 83A either solely or in combination with some, but never all, of the phages 84, 85, 89 and 93.
/////// = Strains lysed by all five typing phages in question.
As the increase in the non-typable strains coincided with the change of phage concentration from 1000 x R T D to 100 x RTD, bacteraemia strains from recent years were tested with concentrations at 1000 x RTD, which led to a reduction of non-typable 362
strains to a percentage of 6, equal to that of previous materials (Table 2 ) . Resistance t o mercuric chloride and production of the ‘Tween’-80-splitting enzyme. The percentages of strains resistant to mercuric chloride ( H g + ) and those not producing the ‘Tween’-80-splitting enzyme (TW-) have decreased since 1968 and 1971, respectively (Table 1) .
Correlation of the Various Properties of the Strains Resistance to antibiotics and phage-type/ group. In Table 3 the material from 1974 is compared with that from 1969, in which a more detailed subdivision of the 83A complex became possible. The previously described prevalence of certain antibiotic patterns within certain phage-groups (16) is also found in the present material. The highest percentage of strains resistant to P + S is still seen within the 52, 52A, 80, 81 complex, and that of multiple-resistant strains among strains of the 83A complex. However, the changes in antibiotic resistance found in the material as a whole are also found within the various groups: a higher proportion of strains are resistant to P only, and the multiple-resistant strains have become fewer; among strains of the 83A complex, the multiple-resistant strains have decreased from 70 per cent in 1969 to 38 in 1974. These changes are to a certain degree caused by variations in the proportion of the TABLE 2. Percentages of Non-typable Strains among Bacteraemia Strains ~
Total
N T at 100 x RTD
N T at 1000 x RTD
1971 1972 1973 1974
21 18 13 12
10 5 5 4
329 577 61 1 516
Total
16
6
2033
n
~
~
16 4
Per cent among strains of the antibiotic pattern TWHg +
15 3
40 13 40 44 32 26 14 8 9 11 3 9
1974
For an explanation of the nomenclature, see Fig. 3.
28 4 29 39 42 33 18 4 15 11 2 6
Phage-type/group/complex 52,52A, 80,81 complex Type 80/8 1 Type 52/52A/80/81 Other types of the complex Group I Group I1 Group I11 83A complex Type “83A” Type 84/ ( 89 ) Type 89 Other types of the complex
1969
Per cent sensitive to antibiotics
7
20
24 21 23 30 51 60 60 20 68 45 3 37
1969
P
16 4
32 25 32 33 63 67 71 46 70 16 13 63
1974
61
27
24 39 27 9 2 1 2 1 1 2 1 1
1969
19 34
1 1 2 1 1 1
1 1
12 39 13 4
1974
83 91
16 25 15 12 2 1 11 70 8 37 91 49
1969
56 80
1 4 38 14 67 76 16
1
7 13 7 6
1974
Resistant to (per cent) : PST + PSTE + PSTEM + PSTM PS
TABLE 3. Correlation of Various Properties of the Strains
-
30 25 16 29
5 3 56 36
-
-
5 73 22
-
-
1974
15 57 28 -
1969
Per cent of the complex in question
2490 382 1407 701 2095 2802 3212 4412 223 121 2468 1600
1969
1373 73 1004 296 3776 3189 3327 3005 889 747 487 882
1974
Total number
TABLE 4. Correlation of Various Properties of Multiple-resistant* Strains Per cent
Phage-type/group Group I11 83A complex Type “83A” Type 84/ ( 89 ) Type 89 Other strains Total
Per cent of multiple-resistant
Hg +
TW1969 1974
1969
1974
1969
1974
73 94 (44) 73 97 35
44 65 16 66 74 29
81 96 (83) 89 97 73
72 90 87 90 93 47
9 76 1 1 55 15
8 72 8 32 24 20
83
56
91
80 4086
1491
Number of strains
*
Multiple-resistant = resistant to PST, PSTE, PSTEM and PSTM.
The brackets indicate that the figures are too small to permit a calculation. For an explanation of the nomenclature, see Fig. 3.
various phage-types (Table 3, column 4 ) , whose antibiotic resistance is fairly stable. For instance, the resistance of the 83A complex is influenced by the increase of type “83A”, of which 70 per cent is resistant to P only. Yet, strains resistant to P + S have become less frequent in type 52/52A/80/81, and the percentage of multiple-resistant strains has increased in type 84 and decreased in type 89. Resistance to antibiotics and occurrence of H g + and TW- strains. The highest proportion of H g + and TW- strains is found among multiple-resistant strains, even though the percentage of the TW- strains shows a decreasing tendency (Table 3 ) . Phage-pattern and occurrence of H g + and TW- strains. The properties of the multipleresistant strains are further examined in Table 4. The percentage of TW- strains has decreased in all phage-groups. The percentage of H g + strains does not vary much for the two years registered, except among strains referred to as “other”, the percentage composition of which does not differ for the years in question. As a rule, resistance to mercuric chloride and ‘Tween’ negativity cannot be correlated to phage-pattern. However, there are a few
364
exceptions: the percentage of TW- strains is higher among strains of phage-type 80 and 7 1 and lower in strains of phage-type “83A” than among other strains of the same antibiotic pattern within the same phage-group. Furthermore, type 80181 includes a higher proportion of H g + strains than do other strains of the 52, 52A, 80, 81 complex.
Clinical Sources and Distribution of Phage-groups f Complexes As the contribution of strains from most sources was found not to vary much from year to year, only the percentages from 1974 are given in the upper part of Table 5. However, there is an increase in strains isolated from cicatrices and wounds (Table 5, lower part). The percentage Occurrence of some phagegroupsfcomplexes among strains from some sources is given in Table 6. I n nearly all instances, strains of the 52, 52A, 80, 81 and 83A complexes have decreased since 1967. Group I11 strains were more frequently isolated in 1970 than in 1967, but have also decreased during the last four-year period.
TABLE 5. Clinical Sources of Strains. Percentage of Total Material
1974 Source
Per cent
Abscesses Pustuli Eye Ear Nose Blood Fistula Urine Bums
8 3 1 7 5 3 2 10 2
pita1 strains can be separated from other strains. Table 7 shows that strains from the “Total material” occupy an intermediate position between strains of known hospital origin and strains originated outside hospitals, which indicates that the “Total material”-as might be expected-contains strains from hospital cases as well as from outside cases. DISCUSSION
Source
1967 Per cent
1974 Per cent
Cicatrices and wounds Not specified
12 32
21 28
in strains from wounds group ‘I1 is now the most frequently found phage-group. The percentages of the M-resisbnt strains, given in 6, have decreased since 1967; for some of the sources, however, the highest percentages are found in 1970. Comparison with Bacteraemia Strains I t is not possible to state whether the strains of the present material were isolated from processes originating before or after hospitalization. However, it is possible to compare properties of these strains with those of strains from bacteraemia cases, where hos-
The changes in antibiotic resistance described here are similar to those observed by authors from various countries (15, 22, 13, 14), dating back to 1959 (4). These results may give rise to the questions of why certain strains become endemic, and what properties are able to give them advantages over other strains under certain circumstances. I n the Danish materia], muItiple-resistant strains of the 83A complex are being replaced by strains resistant to p only, which do not belong to the genetically related group 111 but mainly to unrelated groups. It is possible that strains resistant to a limited number of antibiotics may in certain respects be superior to multiple-resistant strains; resuh recently published by Lacey & Chopra (10) showed that loss of resistant markers gave the strain an advantage over the multiple-resistant parent strain. If this example can be generally applied, it supports the theory that multiple-resistant strains are superior to
TABLE 6. Percentage Occurrence of Methicillin Resistant Strains and of some Phage Groups/Complexes among Strains from Various Clinical Sources, from the Years 1967, 1970 and 1974
Source
Abscesses Cicatrices Wounds Trachea Sputum Urine
M-resistant strains 1967 1970 1974
2 16 7 21 14 23
3 19 11
18 22 20
1 4 2 4 8 11
52,52A, 80,81 complex 1967 1970 1974 31 13 11 9 9 7
23 12 10 9 9 9
11
7 5 5 5 5
Group I11
1967 1970 1974 9 16 19 14 16 19
16 20 25 21 21 21
10 13 18 15 16 21
83A complex 1967 1970 1974 9 34 28 31 33 45
11 32 25 28 32 43
9 15 15 15 19 26
Non-typable strains 1967 1970 1974 6
7 7 13 8 10
5 5 5 5 5
14 9 14 12 12
5
10
365
TABLE 7. Various Properties of Strains o f Different Origin. Strains f r o m Total Material Compared with Bacteraemia Strains Res. to P. only per cent
Res. to T per cent
83A complex per cent
Res. to M per cent
Year
Hos
TM
OH
Hos
TM
OH
Hos
TM
OH
Hos
TM
OH
1967 1970 1974
23 31 56
36 46 60
47 47 62
60 50 22
30 25 13
17 18 9
46 40 15
16 17 6
5 8 3
53 46 26
23 24 14
10 18 14
Hos = Bacteraemia strains from cases of hospital origin. OH = Bacteraemia strains from cases originated outside hospitals. T M = Total Material.
less resistant strains only when given protection by an antibiotic pressure. However, a correlation between the decline of M resistant strains and the diminished use of methicillin has not been demonstra’ted, neither in four large Danish hospitals (19) nor in any other country (11, 23, 8 ) . O n the other hand, a correlation between the proportion of resistant strains and the reduced use of streptomycin and tetracyclines has been found both here and elsewhere (19, 14). Yet, even if it is accepted that the altered antibiotic policy at least partly explains the increase in strains resistant to P only, the shift to strains of group I and miscellaneous strains is not accounted for. These strains must possess properties enabling them to outgrow other strains under the present circumstances. Several authors (4, 11, 23) have suggested that improved hygienic measures may also contribute to the reduction of the multipleresistant strains through a decrease of the cross-infection rate. The present authors investigated the spread of identical strains within larger hospital departments in the years 1969 and 1974 (19). As expected, the multiple-resistant strains from 1969 had been replaced by strains resistant to P only. Furthermore, the number of cases caused by one strain was modest in 1974 compared to the number in 1969; but experience, rather than statistical evidence, told that the P resistant strains actually began to spread when they 366
had been introduced into the hospitals. Thus, an improved hospital hygiene cannot alone account for the changes observed, and it is probable that the explanation is more complex. The suggestion forwarded by Pulverer & D a m e n (15) and Otaya ( 1 2 ) that “newer” antibiotics, e.g. the cephalosporins, might be able to eradicate the multiple-resistant strains, cannot explain the changes found in the Danish material, as in Denmark the consumption of these drugs as compared to that of methicillin is insignificant. The mechanisms causing changes of resistance patterns in staphylococci are known to be mutation and transduction (see 9 ) , whereas lysogenization, which does influence the phage pattern, has not previously been shown to affect antibiotic resistance. Recently, however, Blackwell & Feingold ( 1 ) found that loss of the prophage ( s ) blocking lipase production was combined with loss of certain resistance determinants as well. I n the Danish material more of the multiple-resistant strains have become T W + ; thus showing loss of the prophage ( s ) blocking the ‘Tween’-80-splitting enzyme. These strains are assumed to be genetically unstable, owing to their abundant content of DNA elements, and the possibility cannot be excluded that the T W + variants are the more stable descendants lacking some of the original resistance markers. Furthermore, the S and T determinants seem to be lost simultaneously (Fig. 1) . These observa-
tions seem to be in agreement with the experimental data obtained by Biilow ( 6 ) suggesting that resistance markers to S and T are linked and can be transduced by a phage blocking the above mentioned enzyme. The changes within the 52, 52A, 80, 81 complex with shift from type 80181 to type 52/52A/80/81 also indicate phage activity influencing the phage type (21). A preliminary investigation of the strains of the 83A complex has indicated that similar events may take place here; these findings will be published separately ( 18). The strains non-typable at 100 x RTD do not represent a homogeneous group. Retyping at 1000 x R T D disclosed that their distribution corresponded exactly to that of the whole material, and that their increase was due to the change in concentration of the phage suspensions. However, it is noteworthy that the percentage of strains non-typable at RTD has been constant throughout the whole period investigated. The changes of the properties registered here are presumed to influence the outcome of staphylococcal septicaemia cases from the same years ( 7 ) . During this period, a slight decrease in mortality was seen, which was most probably due to better facilities for adequate antibiotic treatment. At the same time, another improvement was noticed: the spread of identical strains within hospitals was diminished. Thus in Denmark at present two factors may have contributed to the more favourable situation: a more restricted use of streptomycin and tetracyclines, and an improved hospital hygiene. But the equilibrium may easily be disturbed, and it is to be hoped that future developments will continue along the same lines.
3.
4.
5.
6.
7. 8. 9.
10.
11.
12.
13.
14.
15.
REFERENCES
1. Blackwell, C. C . & Feingold, D . S.: Frequency and same properties of clinical isolates of methicillin-resistant Staphylococcus aureus. Amer. J. Clin. Path. 64: 372-377, 1975. 2. Blair, J . E. & Williams, R . E. 0.:Phage
16.
17.
typing of staphylococci. Bull. W.H.O. 24: 771-784, 1961. Blouse, L. E., Stringfield, W . B., Marraro, R . V . & Dupuy, H . J . : Activity and characteristics of a new staphylococcus phage 94. Exper. Biol. and Med. 142: 572-576, 1973. Bulger, R . J . & Sherris, J . C.: Decreased incidence of antibiotic resistance among Staphylococcus aureus. A study in a university hospital over a 9-year period. Ann. Intern. Med. 69: 1099-1108, 1968. Biilow, P.: A new epidemic phage type of Staphylococcus aureus. 1. The experimental typing phage 6557’. Acta path. microbiol. scand. 72: 147-159, 1968. Biilow, P.: Staphylococci in Danish hospitals during the last decade: factors influencing some properties of predominant epidemic strains. Ann. New York Acad. Sci. 182: 2139, 1971. Jessen, O., Rosendal, K . , Biilow, P. & Faber, V . : Unpublished results. Kayser, F. H . : Methicillin-resistant staphylococci 1965-1975. Lancet ii: 650-653, 1975. Lacey, R . W.: Genetic basis, epidemiology, and future significance of antibiotic resistance in Staphylococcus aurew: A review. J. Clin. Path. 26: 899-913, 1973. Lacey, R . W. & Chopra, I.: Effect of plasmid carriage on the virulence of Staphylococcus aureus. J. med. Microbiol. 8: 137-147, 1975. Noone, P. & Shufi, M . S.: Controlling infection in a district general hospital. J. Clin. Path. 26: 140-145, 1973. Otaya, H.: Sensitivity of Staphylococcus aureus and Escherichia coli to antibiotics. V . Differentiation of drug sensitive- and resistant-strains by minimal inhibitory concentration of the drug and annual change of sensitivity of the bacteria to various drugs. J. Antibiotics (Tokyo) 27: 686-695, 1974. Parker, M . T., Asheshov, E . A., Hewitt, 1.H., Nakhla, L. S . & Brock, B. M . : Endemic staphylococcal infections in hospitals. Ann. New York Acad. Sci. 236: 466-484, 1974. Plorde, I. J . & Sherris, J . C.: Staphylococcal resistance to antibiotics: origin, measurement, and epidemiology. Ann. New York Acad. Sci. 236: 413-434, 1974. Pulverer, G. & Damen, G.: Aktuelle Resistenzsituation von Staphylococcus aureus. Med. KIin. 67: 608-614, 1972. Rosendal, K. & Biilow, P.: Staphylococcus aureus strains isolated in Danish hospitals. 2. From January lst, 1961, to December 31st, 1965. Acta path. microbiol. scand. 71: 422438, 1967. Rosendal, K . & Biilow, P.: A subdivision of Staphylococcus aureus strains belonging to the
367
83A, 84, 85, 6557, 592 complex with special reference to antibiotic resistance. Acta path. microbiol. scand. Sect. B, 79: 377-389, 1971. 18. Rosendal, K. & Biilow, P.: Unpublished results. 19. Rosendal, K., Jessen, O., Bentzon, M . W . & Biilow, P . : Unpublished results. 20. Rosendal, K., Stenderup, A . , Helms, P . & Eriksen, K . R.: Staphylococcus aureus strains isolated in Danish hospitals from April 1st to December 31st, 1960. Acta path. microbiol. scand. 58: 72-84, 1963.
368
21. Rountree, P. M . & Asheshov, E. A.: Further observations on changes in the phage-pattern of phage type 80/81 staphylococci. J. gen. Microbiol. 26: 111-122, 1961. 22. Rountree, P. M . & Vickery, A . M.: Further observations on methicillin-resistant staphylococci. Med. J. Austral. I: 1030-1034, 1973. 23. Williams, R . E. 0 . & Dean, B. A.: Phage types of Staphylococcus aureus in one hospital 1961-72. J. Hyg. (Lond.) 73: 375-382, 1974.
STAPHYLOCOCCUS AUREUS STRAINS ISOLATED IN DANISH HOSPITALS FROM JANUARY lst, 1966, TO DECEMBER 31st, 1974 KIRSTEN ROSENDAL, PERBULOW,MICHAEL WEISBENTZON and KNUDRIEWERTS ERIKSEN Statens Seruminstitut, Department of HospitaI Infections, Copenhagen, Department of Medical Bacteriology, Arhus, Biostatistical Department, Copenhagen, and Department of Medical Bacteriology, Blegdamshospitalet, Copenhagen, Denmark
Rosendal, K., Biilav, P., Bentzon, M. W. & Eriksen, K. R. Staphylococcus aureus strains isolated in Danish hospitals from January lst, 1966, to December 31st, 1974. Acta path. microbiol. scand. Sect. B, 84: 359-368, 1976. During the years 1966-1974, 167,297 strains isolated from 167,297 patients or staff members in Danish hospitals were registered at Statens Seruminstitut. All the strains were phage-typed and examined for production of a ‘Tween’-80-splitting enzyme and resistance to mercuric chloride. 158,236 strains were examined for resistance to antibiotics. Since 1968, a steep decrease in the number of strains resistant to three or more antibiotics (multiple-resistant) and in strains of the 83A complex was noticed. I n recent years an increase in strains belonging to phage-group I and in those referred to as miscellaneous and non-identified was registered. The increase in the non-typable strains might be explained by the shift of the concentration of the typing phages from 1000 x RTD to 100 x RTD. I t is concluded that a t least two factors may have contributed to the reduction of the multiple-resistant strains: an altered antibiotic policy, restricting the use of streptomycin and tetracyclines, and an improved hospital hygiene, diminishing the spread of identical strains within the various departments. However, it is emphasized that the consumption of methicillin is still increasing. Kefr words: Staphylococcus aureus strains; antibiotic resistance; phage-typing ; Danish hospitals.
K. Rosendal, Department of Hospital Infections, Statens Seruminstitut, Amager Boulevard 80, DK-2300 Copenhagen S, Denmark.
Received 14.vi.76
Accepted 23.vi.76
At Statens Seruminstitut, Copenhagen, the registration of phage-typed Staphylococcus aureus strains from hospitals all over the country is still performed as described in publications from 1963 (20) and 1967 (16), which deal with the material from the beginning of the registration in April, 1960 until the end of December, 1965. In these surveys, a steep decrease in the
number of strains belonging to the 52, 52A, 80, 81 complex was noticed, whereas strains belonging to the 83A, 84, 85, 93, 89 complex ( = 83A complex) and especially those resistant to three or more antibiotics were registered with increasing frequency. The present survey deals with the material collected over a nine-year period ending December 31, 1974. I t was found that the changes described previously continued dur-
359
TABLE 1. T h e Materiul Year Number Examined for resistance to antibiotics % TWHg+ %
1966 15682
1967 16044
1968 16235
1969 18626
1970 19305
1971 18393
1972 20002
1973 21303
1974 21707
Total 167297
14712
14990
15111
17604
18393
17478
19024
20254
20670
158236
34 36
36 35
36 32
35 29
33 26
29 21
27 18
22 13
19 11
~~~
TWHg+
= do not produce the ‘Tween’-8O-splitting enzyme. = resistant to mercuric chloride.
ing the first three years and that from then on a decrease in the multiple-resistant strains was observed. Since 1966 it has been possible to record the occurrence of the methicillin-resistant strains. Furthermore, evolutionary events within the 83A complex have been followed during recent years, as it has been possible to perform a subdivision of the complex by using the new international typing phages 84, 85 and 89.
89) (17) and in 1973 (phage 94) ( 3 ) . The strains typed by phage 94 have been recorded as “ m i s cellaneous”. The phage previously referred to as “6557” ( 5 ) has been internationally designated as phage 93. From June Ist, 1971, the strains non-typable at RTD (routine test dilution) have been retested with the phages in a concentration of 100 x R T D instead of 1000 x RTD. In order to examine the effect of this change, freeze dried bacteraemia strains, non-typable at 100 x RTD, were retyped, using the concentration of 1000 x RTD (Table 2 ) .
RESULTS MATERIALS AND METHODS
T h e Material A “Total material”, (Table 1) comprises all phage-typed Staphylococcus aureus strains isolated from persons connected with Danish hospitals, patients and staff members. Only one strain per person is included, selected as previously described (20). B Bacteraemia strains. For comparison, some figures from a material consisting of bacteraemia strains only (19) have been made use of. The methods are mainly those described in papers from 1963 (20) and 1967 (16). The alterations are as follows: Registration The information from the material from 1966 was extracted from the original cards. The results from 1967-1974 were transferred to punch-cards, using special cipher-codes for the registration of phage-types/groups, sources, resistance to antibiotics and mercuric chloride and production of a ‘Tween’-80-splitting enzyme. Bacteriophage-typing New typing phages have been added to the basic set ( 2 ) in 1966 (phage 84 and 85), 1969 (phage
360
From Table 1 it is seen that the material consists of 167,297 strains (isolated from the same number of persons) most of which have been examined for resistance to antibiotics. During the whole period the number of strains has been steadily increasing. Changes in the Properties of the Strains Resistance to antibiotics (Fig. 1 ) . The percentage of strains sensitive to all antibiotics and resistant to penicillin (P) has been fairly constant, but strains resistant to the other antibiotics tested (streptomycin (S), tetracyclines (T), chloramphenicol ( C ) , erythromycin (E) and methicillin ( M ) ) have since 1968 become less frequent. I t is remarkable that the declining parts of the curves representing S and T resistance run parallel. The changes of resistance are further analysed in Fig. l B which shows a decrease in multiple-resistant strains (resistant to three or more antibiotics) from 25 per cent in 1967 to 7 in 1974 and an increase in strains resistant
A 80’60
-
40,
::p O
I
93, 89 (83A) complex reached a peak in 1969-1970, whereafter the percentage fell from 25 to 14 in 1974, and a considerable increase of non-typable strains was noticed from 1971. Further investigation of the 83A complex (Fig. 3) showed that strains lysed by all five typing phages have almost disappeared, whereas strains sensitive to phage 83A plus some of the other phages and types 84 and 85 are increasing. Types 89 and 84/85/93/89 have decreased since 1969.
*
0
2ot c
“1 E
NT
0 64
68
72
907
B
%
6.*fo“-l ‘0
-
% 100
,..* .”, ,..a’
,----
”.‘.‘.
0
68
64
72
80, 70- 8 3 A COMPLEX
1
Fig. 1. Antibiotic sensitivity of the strains (percentage). T h e antibiotic sensitivity of strains from a previously published material (16) from the years 1964-1965 is also shown in this figure.
A. Strains resistant to: P = penicillin; T = tetracyclines ; C = chloramphenicol;
B.
0- -0
0
-0
0-0
S
= streptomycin; M = methicillin; E = erythromycin.
Strains resistant to P and sensitive to other antibiotics investigated. Strains resistant to P + S + T f other antibiotics as well. Strains sensitive to all antibiotics.
20
% 52,52A,80,81 o COMPLEX
j
to P only, from 36 per cent to 60 over the same period. Phage-typeslgroups. The percentage occurrences of the various phage-groups are shown in Figs. 2 and 3. The major changes are: strains of the 52, 52A, 80, 81 complex a.re still decreasing, strains of the 83A, 84, 85,
0
66
68
70
72
b
74
0
Fig. 2. Percentage contribution of the various phage-groups/complexes. Cumulative curves. NI = Non-identified. Misc. = Miscellaneous. N T = Non-typable.
36 1
% 100
-
84/85 + 93
90 80
70
60 50
40 30
20 10
0 1
71
73
Fig. 3. 83A complex. Cumulative curves. 84/(89) = Strains lysed by phage 84 whether or not in combination with phage 89. “83A” = Strains lysed by phage 83A either solely or in combination with some, but never all, of the phages 84, 85, 89 and 93.
/////// = Strains lysed by all five typing phages in question.
As the increase in the non-typable strains coincided with the change of phage concentration from 1000 x R T D to 100 x RTD, bacteraemia strains from recent years were tested with concentrations at 1000 x RTD, which led to a reduction of non-typable 362
strains to a percentage of 6, equal to that of previous materials (Table 2 ) . Resistance t o mercuric chloride and production of the ‘Tween’-80-splitting enzyme. The percentages of strains resistant to mercuric chloride ( H g + ) and those not producing the ‘Tween’-80-splitting enzyme (TW-) have decreased since 1968 and 1971, respectively (Table 1) .
Correlation of the Various Properties of the Strains Resistance to antibiotics and phage-type/ group. In Table 3 the material from 1974 is compared with that from 1969, in which a more detailed subdivision of the 83A complex became possible. The previously described prevalence of certain antibiotic patterns within certain phage-groups (16) is also found in the present material. The highest percentage of strains resistant to P + S is still seen within the 52, 52A, 80, 81 complex, and that of multiple-resistant strains among strains of the 83A complex. However, the changes in antibiotic resistance found in the material as a whole are also found within the various groups: a higher proportion of strains are resistant to P only, and the multiple-resistant strains have become fewer; among strains of the 83A complex, the multiple-resistant strains have decreased from 70 per cent in 1969 to 38 in 1974. These changes are to a certain degree caused by variations in the proportion of the TABLE 2. Percentages of Non-typable Strains among Bacteraemia Strains ~
Total
N T at 100 x RTD
N T at 1000 x RTD
1971 1972 1973 1974
21 18 13 12
10 5 5 4
329 577 61 1 516
Total
16
6
2033
n
~
~
16 4
Per cent among strains of the antibiotic pattern TWHg +
15 3
40 13 40 44 32 26 14 8 9 11 3 9
1974
For an explanation of the nomenclature, see Fig. 3.
28 4 29 39 42 33 18 4 15 11 2 6
Phage-type/group/complex 52,52A, 80,81 complex Type 80/8 1 Type 52/52A/80/81 Other types of the complex Group I Group I1 Group I11 83A complex Type “83A” Type 84/ ( 89 ) Type 89 Other types of the complex
1969
Per cent sensitive to antibiotics
7
20
24 21 23 30 51 60 60 20 68 45 3 37
1969
P
16 4
32 25 32 33 63 67 71 46 70 16 13 63
1974
61
27
24 39 27 9 2 1 2 1 1 2 1 1
1969
19 34
1 1 2 1 1 1
1 1
12 39 13 4
1974
83 91
16 25 15 12 2 1 11 70 8 37 91 49
1969
56 80
1 4 38 14 67 76 16
1
7 13 7 6
1974
Resistant to (per cent) : PST + PSTE + PSTEM + PSTM PS
TABLE 3. Correlation of Various Properties of the Strains
-
30 25 16 29
5 3 56 36
-
-
5 73 22
-
-
1974
15 57 28 -
1969
Per cent of the complex in question
2490 382 1407 701 2095 2802 3212 4412 223 121 2468 1600
1969
1373 73 1004 296 3776 3189 3327 3005 889 747 487 882
1974
Total number
TABLE 4. Correlation of Various Properties of Multiple-resistant* Strains Per cent
Phage-type/group Group I11 83A complex Type “83A” Type 84/ ( 89 ) Type 89 Other strains Total
Per cent of multiple-resistant
Hg +
TW1969 1974
1969
1974
1969
1974
73 94 (44) 73 97 35
44 65 16 66 74 29
81 96 (83) 89 97 73
72 90 87 90 93 47
9 76 1 1 55 15
8 72 8 32 24 20
83
56
91
80 4086
1491
Number of strains
*
Multiple-resistant = resistant to PST, PSTE, PSTEM and PSTM.
The brackets indicate that the figures are too small to permit a calculation. For an explanation of the nomenclature, see Fig. 3.
various phage-types (Table 3, column 4 ) , whose antibiotic resistance is fairly stable. For instance, the resistance of the 83A complex is influenced by the increase of type “83A”, of which 70 per cent is resistant to P only. Yet, strains resistant to P + S have become less frequent in type 52/52A/80/81, and the percentage of multiple-resistant strains has increased in type 84 and decreased in type 89. Resistance to antibiotics and occurrence of H g + and TW- strains. The highest proportion of H g + and TW- strains is found among multiple-resistant strains, even though the percentage of the TW- strains shows a decreasing tendency (Table 3 ) . Phage-pattern and occurrence of H g + and TW- strains. The properties of the multipleresistant strains are further examined in Table 4. The percentage of TW- strains has decreased in all phage-groups. The percentage of H g + strains does not vary much for the two years registered, except among strains referred to as “other”, the percentage composition of which does not differ for the years in question. As a rule, resistance to mercuric chloride and ‘Tween’ negativity cannot be correlated to phage-pattern. However, there are a few
364
exceptions: the percentage of TW- strains is higher among strains of phage-type 80 and 7 1 and lower in strains of phage-type “83A” than among other strains of the same antibiotic pattern within the same phage-group. Furthermore, type 80181 includes a higher proportion of H g + strains than do other strains of the 52, 52A, 80, 81 complex.
Clinical Sources and Distribution of Phage-groups f Complexes As the contribution of strains from most sources was found not to vary much from year to year, only the percentages from 1974 are given in the upper part of Table 5. However, there is an increase in strains isolated from cicatrices and wounds (Table 5, lower part). The percentage Occurrence of some phagegroupsfcomplexes among strains from some sources is given in Table 6. I n nearly all instances, strains of the 52, 52A, 80, 81 and 83A complexes have decreased since 1967. Group I11 strains were more frequently isolated in 1970 than in 1967, but have also decreased during the last four-year period.
TABLE 5. Clinical Sources of Strains. Percentage of Total Material
1974 Source
Per cent
Abscesses Pustuli Eye Ear Nose Blood Fistula Urine Bums
8 3 1 7 5 3 2 10 2
pita1 strains can be separated from other strains. Table 7 shows that strains from the “Total material” occupy an intermediate position between strains of known hospital origin and strains originated outside hospitals, which indicates that the “Total material”-as might be expected-contains strains from hospital cases as well as from outside cases. DISCUSSION
Source
1967 Per cent
1974 Per cent
Cicatrices and wounds Not specified
12 32
21 28
in strains from wounds group ‘I1 is now the most frequently found phage-group. The percentages of the M-resisbnt strains, given in 6, have decreased since 1967; for some of the sources, however, the highest percentages are found in 1970. Comparison with Bacteraemia Strains I t is not possible to state whether the strains of the present material were isolated from processes originating before or after hospitalization. However, it is possible to compare properties of these strains with those of strains from bacteraemia cases, where hos-
The changes in antibiotic resistance described here are similar to those observed by authors from various countries (15, 22, 13, 14), dating back to 1959 (4). These results may give rise to the questions of why certain strains become endemic, and what properties are able to give them advantages over other strains under certain circumstances. I n the Danish materia], muItiple-resistant strains of the 83A complex are being replaced by strains resistant to p only, which do not belong to the genetically related group 111 but mainly to unrelated groups. It is possible that strains resistant to a limited number of antibiotics may in certain respects be superior to multiple-resistant strains; resuh recently published by Lacey & Chopra (10) showed that loss of resistant markers gave the strain an advantage over the multiple-resistant parent strain. If this example can be generally applied, it supports the theory that multiple-resistant strains are superior to
TABLE 6. Percentage Occurrence of Methicillin Resistant Strains and of some Phage Groups/Complexes among Strains from Various Clinical Sources, from the Years 1967, 1970 and 1974
Source
Abscesses Cicatrices Wounds Trachea Sputum Urine
M-resistant strains 1967 1970 1974
2 16 7 21 14 23
3 19 11
18 22 20
1 4 2 4 8 11
52,52A, 80,81 complex 1967 1970 1974 31 13 11 9 9 7
23 12 10 9 9 9
11
7 5 5 5 5
Group I11
1967 1970 1974 9 16 19 14 16 19
16 20 25 21 21 21
10 13 18 15 16 21
83A complex 1967 1970 1974 9 34 28 31 33 45
11 32 25 28 32 43
9 15 15 15 19 26
Non-typable strains 1967 1970 1974 6
7 7 13 8 10
5 5 5 5 5
14 9 14 12 12
5
10
365
TABLE 7. Various Properties of Strains o f Different Origin. Strains f r o m Total Material Compared with Bacteraemia Strains Res. to P. only per cent
Res. to T per cent
83A complex per cent
Res. to M per cent
Year
Hos
TM
OH
Hos
TM
OH
Hos
TM
OH
Hos
TM
OH
1967 1970 1974
23 31 56
36 46 60
47 47 62
60 50 22
30 25 13
17 18 9
46 40 15
16 17 6
5 8 3
53 46 26
23 24 14
10 18 14
Hos = Bacteraemia strains from cases of hospital origin. OH = Bacteraemia strains from cases originated outside hospitals. T M = Total Material.
less resistant strains only when given protection by an antibiotic pressure. However, a correlation between the decline of M resistant strains and the diminished use of methicillin has not been demonstra’ted, neither in four large Danish hospitals (19) nor in any other country (11, 23, 8 ) . O n the other hand, a correlation between the proportion of resistant strains and the reduced use of streptomycin and tetracyclines has been found both here and elsewhere (19, 14). Yet, even if it is accepted that the altered antibiotic policy at least partly explains the increase in strains resistant to P only, the shift to strains of group I and miscellaneous strains is not accounted for. These strains must possess properties enabling them to outgrow other strains under the present circumstances. Several authors (4, 11, 23) have suggested that improved hygienic measures may also contribute to the reduction of the multipleresistant strains through a decrease of the cross-infection rate. The present authors investigated the spread of identical strains within larger hospital departments in the years 1969 and 1974 (19). As expected, the multiple-resistant strains from 1969 had been replaced by strains resistant to P only. Furthermore, the number of cases caused by one strain was modest in 1974 compared to the number in 1969; but experience, rather than statistical evidence, told that the P resistant strains actually began to spread when they 366
had been introduced into the hospitals. Thus, an improved hospital hygiene cannot alone account for the changes observed, and it is probable that the explanation is more complex. The suggestion forwarded by Pulverer & D a m e n (15) and Otaya ( 1 2 ) that “newer” antibiotics, e.g. the cephalosporins, might be able to eradicate the multiple-resistant strains, cannot explain the changes found in the Danish material, as in Denmark the consumption of these drugs as compared to that of methicillin is insignificant. The mechanisms causing changes of resistance patterns in staphylococci are known to be mutation and transduction (see 9 ) , whereas lysogenization, which does influence the phage pattern, has not previously been shown to affect antibiotic resistance. Recently, however, Blackwell & Feingold ( 1 ) found that loss of the prophage ( s ) blocking lipase production was combined with loss of certain resistance determinants as well. I n the Danish material more of the multiple-resistant strains have become T W + ; thus showing loss of the prophage ( s ) blocking the ‘Tween’-80-splitting enzyme. These strains are assumed to be genetically unstable, owing to their abundant content of DNA elements, and the possibility cannot be excluded that the T W + variants are the more stable descendants lacking some of the original resistance markers. Furthermore, the S and T determinants seem to be lost simultaneously (Fig. 1) . These observa-
tions seem to be in agreement with the experimental data obtained by Biilow ( 6 ) suggesting that resistance markers to S and T are linked and can be transduced by a phage blocking the above mentioned enzyme. The changes within the 52, 52A, 80, 81 complex with shift from type 80181 to type 52/52A/80/81 also indicate phage activity influencing the phage type (21). A preliminary investigation of the strains of the 83A complex has indicated that similar events may take place here; these findings will be published separately ( 18). The strains non-typable at 100 x RTD do not represent a homogeneous group. Retyping at 1000 x R T D disclosed that their distribution corresponded exactly to that of the whole material, and that their increase was due to the change in concentration of the phage suspensions. However, it is noteworthy that the percentage of strains non-typable at RTD has been constant throughout the whole period investigated. The changes of the properties registered here are presumed to influence the outcome of staphylococcal septicaemia cases from the same years ( 7 ) . During this period, a slight decrease in mortality was seen, which was most probably due to better facilities for adequate antibiotic treatment. At the same time, another improvement was noticed: the spread of identical strains within hospitals was diminished. Thus in Denmark at present two factors may have contributed to the more favourable situation: a more restricted use of streptomycin and tetracyclines, and an improved hospital hygiene. But the equilibrium may easily be disturbed, and it is to be hoped that future developments will continue along the same lines.
3.
4.
5.
6.
7. 8. 9.
10.
11.
12.
13.
14.
15.
REFERENCES
1. Blackwell, C. C . & Feingold, D . S.: Frequency and same properties of clinical isolates of methicillin-resistant Staphylococcus aureus. Amer. J. Clin. Path. 64: 372-377, 1975. 2. Blair, J . E. & Williams, R . E. 0.:Phage
16.
17.
typing of staphylococci. Bull. W.H.O. 24: 771-784, 1961. Blouse, L. E., Stringfield, W . B., Marraro, R . V . & Dupuy, H . J . : Activity and characteristics of a new staphylococcus phage 94. Exper. Biol. and Med. 142: 572-576, 1973. Bulger, R . J . & Sherris, J . C.: Decreased incidence of antibiotic resistance among Staphylococcus aureus. A study in a university hospital over a 9-year period. Ann. Intern. Med. 69: 1099-1108, 1968. Biilow, P.: A new epidemic phage type of Staphylococcus aureus. 1. The experimental typing phage 6557’. Acta path. microbiol. scand. 72: 147-159, 1968. Biilow, P.: Staphylococci in Danish hospitals during the last decade: factors influencing some properties of predominant epidemic strains. Ann. New York Acad. Sci. 182: 2139, 1971. Jessen, O., Rosendal, K . , Biilow, P. & Faber, V . : Unpublished results. Kayser, F. H . : Methicillin-resistant staphylococci 1965-1975. Lancet ii: 650-653, 1975. Lacey, R . W.: Genetic basis, epidemiology, and future significance of antibiotic resistance in Staphylococcus aurew: A review. J. Clin. Path. 26: 899-913, 1973. Lacey, R . W. & Chopra, I.: Effect of plasmid carriage on the virulence of Staphylococcus aureus. J. med. Microbiol. 8: 137-147, 1975. Noone, P. & Shufi, M . S.: Controlling infection in a district general hospital. J. Clin. Path. 26: 140-145, 1973. Otaya, H.: Sensitivity of Staphylococcus aureus and Escherichia coli to antibiotics. V . Differentiation of drug sensitive- and resistant-strains by minimal inhibitory concentration of the drug and annual change of sensitivity of the bacteria to various drugs. J. Antibiotics (Tokyo) 27: 686-695, 1974. Parker, M . T., Asheshov, E . A., Hewitt, 1.H., Nakhla, L. S . & Brock, B. M . : Endemic staphylococcal infections in hospitals. Ann. New York Acad. Sci. 236: 466-484, 1974. Plorde, I. J . & Sherris, J . C.: Staphylococcal resistance to antibiotics: origin, measurement, and epidemiology. Ann. New York Acad. Sci. 236: 413-434, 1974. Pulverer, G. & Damen, G.: Aktuelle Resistenzsituation von Staphylococcus aureus. Med. KIin. 67: 608-614, 1972. Rosendal, K. & Biilow, P.: Staphylococcus aureus strains isolated in Danish hospitals. 2. From January lst, 1961, to December 31st, 1965. Acta path. microbiol. scand. 71: 422438, 1967. Rosendal, K . & Biilow, P.: A subdivision of Staphylococcus aureus strains belonging to the
367
83A, 84, 85, 6557, 592 complex with special reference to antibiotic resistance. Acta path. microbiol. scand. Sect. B, 79: 377-389, 1971. 18. Rosendal, K. & Biilow, P.: Unpublished results. 19. Rosendal, K., Jessen, O., Bentzon, M . W . & Biilow, P . : Unpublished results. 20. Rosendal, K., Stenderup, A . , Helms, P . & Eriksen, K . R.: Staphylococcus aureus strains isolated in Danish hospitals from April 1st to December 31st, 1960. Acta path. microbiol. scand. 58: 72-84, 1963.
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21. Rountree, P. M . & Asheshov, E. A.: Further observations on changes in the phage-pattern of phage type 80/81 staphylococci. J. gen. Microbiol. 26: 111-122, 1961. 22. Rountree, P. M . & Vickery, A . M.: Further observations on methicillin-resistant staphylococci. Med. J. Austral. I: 1030-1034, 1973. 23. Williams, R . E. 0 . & Dean, B. A.: Phage types of Staphylococcus aureus in one hospital 1961-72. J. Hyg. (Lond.) 73: 375-382, 1974.
