- Email: [email protected]
METHICILLIN-RESISTANT STAPHYLOCOCCI 1965-75
650
Occasional
Our recent experience of the epidemiology of methicillin-resistance (Mth-R) has revealed a changed picture, Having made a continuous survey of staphylococcal resistance since January, 1965, in Zurich and in parts of adjacent districts (1-1million inhabitants)," we were able to obtain information about the relative frequency of Mth-R in Staph. aureus in a circumscribed geographical area and about the clinical significance of this resis-
Survey
METHICILLIN-RESISTANT STAPHYLOCOCCI 1965-75 F. H. KAYSER
in our population. This epidemiological survey has shown a significant decrease in the number of staphylococcal infections caused by Mth-R strains, although the use of -lactam antibiotics has not been restricted in the past few years. This report summarises our data on the epidemiology of methicillin-resistance in Staph. aureus and compares the findings with results obtained since 1965. tance
Institute of Medical Microbiology, CH-8028 Zürich, Switzerland
Methicillin resistance in Staphylococcus has been one of the major problems of gram positive infections in hospitals in the Zurich area. Up to 1971, about 20% of staphylococcal disease was caused by these peculiar organisms. Since 1972, however, a gradual decrease in the number of methicillin-resistant organisms has been observed, with an unprecedented low of 3% in 1975. The nearly 700 methicillin-resistant cultures that have isolated since 1965 exhibited, with rare exceptions, conventional group-III patterns of lysis in phage-typing and similar antibiotypes. It is suggested that all these isolates are derivatives of a strain which has long existed in the staphylococcal population. The reasons for the changes in the frequency of this strain as an agent causing staphylococcal disease are unclear. The use of penicillinase-resistant &bgr;-lactam antibiotics in hospitals does not seem to play a major role in the distribution and spread or in the disappearance of this strain.
Summary
aureus
Methods Cultures
Staphylococci were isolated from clinical samples and classiaccording to the criteria proposed by
fied as Staph. aureus Baird-Parker. 12
Staph. epidermidis isolates were not included in the study, An isolate or culture was defined as the first isolate obtained from a patient during his staphylococcal disease. A strain was defined as a collection of isolates exhibiting similar patterns of phage lysis and similar antibiotic-resistance characters. Bacteriological Methods Methicillin resistance was detected by inoculating approximately 104 cells, grown overnight in brain-heart infusion (BHI) broth, on to the surface of oxacillin-containing or methicillincontaining Meuller-Hinton (MH) agar plates with a modified Steers replicator. The plates were incubated at 300C for 24 and 48 hours. A minimal inhibitory concentration (Mtc) of 12-$g of the drugs per ml of medium was taken as evidence of resistance. In the early part of the study, Mth-R was detected in a broth (BHi) dilution test using oxacillin. The tubes were incubated at 37°C for 48 hours and resistance was defined according to the criteria just described. Disc susceptibility testing was performed by means of the et al. and the slightly modified single-disc method of Bauer U.S. Food and Drug Administration, 14 15 using MH-agar (BBL), supplemented with 5% old human blood from the blood bank. Penicillin-&bgr;-Iactamase was detected according to Perret’s slightly modified iodide method.16 "
Introduction The extensive
of antimicrobial agents such as benzylpenicillin erythromycin in staphylococcal disease has often been followed by the appearance of large numbers of Staphylococcus aureus resistant to these agents.’ Such a situation seemed also to arise with methicillin-resistant staphylococci. With the introduction of penicillinase-resistant -lactam antibiotics into therapy, increasing numbers of isolations of these staphylococci were reported throughout the world. 2-8 Clinical data also showed that such strains may cause serious infections in the compromised host.9 10 use
or
TABLE
I.-FREQUENCY
OF ANTIBIOTIC RESISTANCE IN
I
*)’nor admission
to or recent
Staph.
I
antibiotic
aureus, ZURICH
I
1965-75 .
i
therapy not determined
651 TAAI I].--
!BNTlRtOTIC-RFS1STANCE I’A’Cf1 RNS OF .lr’fFil<:11 LIN°RFCICTANT
Staph.
aureus
r:{1I.TIJRI’I
I
TARLE HI.—PHAGE-TYPtNG PATTHRNS OF MFTH;(:H ) LIN-RESISTANT
Heterogeneity of Mth-R was detected by streaking O’lml of different dilutions of overnight broth cultures on to the surface of agar plates containing increasing concentrations of oxacillin. In some experiments heterogeneity was examined by inoculating antibiotic plates with different dilutions of cultures with the help of a modified Steers replicator. The phage-type of the isolates was determined according to Blair and Williams. 19 From the beginning of this survey in 1965, all cultures were preserved as lyophilysed samples. Results Disease due
to
Methicillin-resistant Staphylococci
Table t shows the frequency of methicillin resistance and further antibiotic resistance from 1965. to 1975. In the years 1965, 1966, and 1967 all staphylococcal cultures isolated during the year were examined for Mth-R. Because of the large increase in staphylococcal isolations in the following years, a 3-month period in the middle of 1968 and 1969 was taken as representing the whole year. In the following years staphylococci isolated in December, 1970, and January/February, 1971, were included in the study. From 1972 up to 1975, staphylococcal isolates in the first 3 months of the year were collected and examined. From table i it can be concluded that, from 1966 to 1971, a high proportion of staphylococcal cultures exhibited Mth-R. From 1972, however, the proportion dec-
lined, reaching significantly an unprecedented low of 3% in 1975. All the isolates showed the typical heteroresistancethat is, cells of pure cultures tolerated widely different amounts of 3-lactam antibiotics. All isolates expressed their resistance best on media containing sodium chloride or at an incubation temperature of 30°C. Despite the frequent occurrence of Mth-R up to 1973, the percentage of this resistance in staphylococci was always
Sraph.
aureus
CB1I.TliRES
lower than that to other standard antibiotics. Mth-R staphylococci in the Zurich area did not exhibit resistance to vancomycin and to the gentamycin antibiotics.
Methicillin-resistant
Staph.
aureus was
isolated
ex-
clusively from hospital inpatients. The problem was observed for the most part in large hospitals, whereas in small hospitals methicillin-resistance did not play any major role. From 1969 to 1975, 80% and more of all the isolated resistant staphylococci came from two hospitals: the large university hospital in Zurich and a mediumsize general hospital in Chur. The kinds of infections observed in hospital inpatients did not change significantly from 1965 to 1975. They were the expected hospital-acquired infections and consisted of respiratory infections (20-38%), surgical wound infections (22-30%), skin and muscle infections (9-16%), urinary-tract infections (8-12%), bone and joint infections (9-11%), septicaemia of unknown origin (3-10%), and miscellaneous infections (4-8%).
Antibiotypes of Methicillin-resistantStaphylococci Table ii summarises the patterns of resistance to standard antimicrobials found in Mth-R staphylococci from 1965 to 1975. All cultures were resistant to the sulphonamides and to streptomycin and produced penicillinase. With few exceptions, the isolates exhibited resistance to tetracycline and to erythromycin. 85% had the "dissociated" type of resistance with cross-resistance to oleandomycin after induction with erythromycin, whereas 15% had the "undissociated" resistance to all macrolide antibiotics. Resistance to kanamycin/neomycin and to chloramphenicol was not constantly observed. The genetic markers of these resistances, however, were found to be part of relatively unstable staphylococcal plasmids.19
652
Phage-types of Methicillin-resistant Staphylococci. Table in summarises the phage-typing patterns of Mth-R staphylococci isolated in Zurich. Most of the isolates exhibited conventional group iit patterns of lysis at routine test dilution (RTD), or 1000 times RTD. Phage-types 80/81 and 52A/80 were encountered only once, type 29 three times, and type 29/80 twice. The susceptibility of the cultures to the typing phages did not change significantly throughout the observation period. It was observed, however, that since 1969 the number of trains typable only by 1000 times the RTD and since 1972 the frequency of cultures untypable increased. Discussion The similar antibiotypes and similar phage-typing patterns of the 687 methicillin-resistant Staph. aureus cultures isolated since 1965 in the Zurich area reveal that the problem of methicillin resistance results mainly from the spread of one epidemiologically distinct strain. In addition to Mth-R, the "Zurich strain" is constantly resistant to the sulphonamides (Sul-R), erythromycin (Ery-R), tetracycline (Tet-R), streptomycin (Str-R), and penicillin (Pen-R). Molecular studies have shown that the genetic markers of these resistances-with the exception of Pen-R-are very probably part of the bacterial chromosome.19 On the other hand, it has been found that the determinants of resistance to chloramphenicol (Cml-R) and neomycin/kanamycin (Neo-R) as well as of penicillinase production are located on different, mutually compatible plasmids. 19 20 Plasmids usually are responsible for a high degree of genetic variability in bacteria. Isolates which lack Cml-R or Neo-R owe this fact to the instability of their respective genetic markers. Penicillinase production, on the other hand, is thought to be absolutely necessary for a staphylococcus strain to survive for a long period in the hospital environment. Hence, all isolates were observed to produce this enzyme, although the markers are part of the penicillinase plasmid. The "one-strain hypothesis" of methicillin resistance in the Zurich area is further supported by the similar phage-typing patterns of cultures isolated between 1965 and 1975. A trend toward restriction in phage susceptibility of our cultures is detectable. This, however, is an overall trend also noted in other staphylococci,and has presumably resulted from alterations in prophage carriage. Further evidence of the "single-clone hypothesis" is mentioned by Lacey2l who even argues that all Mth-R staphylococci isolated so far in the world are derivatives of one single strain. The question, then, remains as to the causes of the increased prominence of this strain in our hospitals up to 1971 and the rapid decrease in the frequency of infections caused by it since 1972. We suggested that the growing number of Mth-R isolates might be connected with the increasing use of &bgr;-lacantibiotics since 1964 in our hospitals.l1 Our recent experience indicates that this suggestion may be incorrect, because such antibiotics have not been used less in the years in which the isolations of this strain have diminished. Furthermore, in hospitals of the United States Mth-R staphylococci, although present in low numbers, never played a major role22 despite the frequent use of penicillins and cephalosporins.23 tam
Possibly resistance in vitro is not always equivalent to resistance in vivo. The specific properties of Mth-R staphylococci suggest that resistance per se may not be clinically important in all patients. At normal body temperatures, only few cells in populations can express their resistance to high levels of &bgr;-Iactam antibiotics. These high-resistance minorities grow slowly and have ontv low virulence, especially in the presence of -)actam in vivo antibiotic therapy. 24 Thus selective exerted by antimicrobial therapy may not have resulted in the varying frequencies of isolations observed from 1965 to 1975. That does not mean, however, that infections caused by Mth-R staphylococci should be treated with p-Iactam antibiotics. In patients with severe underlying disease, even the minorities with low virulence might cause serious staphylococcal infections. Alternative antimicrobials (e.g. gentamycin, tobramycin, vancomycin) can be applied in the treatment of methicillin-resistant infections. The increase in methicillin-resistant staphylococcal infections in our hospitals from 1965 may be better explained by other characters, besides Mth-R, which enabled this strain to survive in the hospital-e.g., resistance to many other antimicrobials, including production of large amounts of penicillinase. The reduction in the number of Mth-R isolations since 1972 is more difficult to understand. Possibly the genes for Mth-R became mobilised bB plasmids or bacteriophage, and spontaneous loss of these genomes, resulting in methicillin-susceptible and more virulent variants, occurred. The presence of additional extrachromosomal DNA, which has to be maintained and replicated in a bacterial host, can result in decreased virulence. Plasmid-negative variants thus may be better equipped to survive in the hospital environment. Experience in our laboratory20 and in others,25 26 however, has shown that Mth-R seems to be a very stable genetic character. A connection of this resistance with extrachromosomal elements was not observed until now in the Zurich strain. Change of the patient population could have resulted in a decreased capacity of Mth-R staphylococci to survive and multiply in the human host. We have no indication at all that this happened in our hospitals. Unknown extrachromosomal virulence factors could have become lost from Mth-R cultures, with consequently reduced capacity of this strain to compete with other staphylococcal organisms.
pressures
A
new
methicillin-susceptible .Staph.
aureus
strain,
highly virulent and resistant to many other antimicrobials, could have appeared in our hospitals. Unfortunately we lack data on phage-types of methicillin-susceptible staphylococci from 1965 to 1975. A conclusive explanation of the changing ecology of Mth-R staphylococci in the Zurich area cannot be advanced at present. For the sake of our patients, however, we hope that the incidence of infections by Mth-R staphylococci will decrease further or at least remain at the present low level. REFERENCES 1. Finland, M. J. infect.Dis. 1970, 122, 419. 2. Parker, M. T., Hewitt, J. H.Lancet, 1970, i, 800 3. Jessen, O., Rosendal, K., Bülow, P., Faber, V., J.Med. 1969, 281, 627.
Eriksen,
K R. New Es
653 trees. The surroundings are rather barren and dry. On the morning of July 14, 1973, an unknown stray dog entered the colony at about 0800 hours. It attacked various persons along the road and in houses till it came to the school house. Here, it entered the classroom and bit several of the children before they could run away. It was chased out and ran through the compounds of some of the houses, where it was finally located and killed at about 1030. In all 19 persons had been bitten by the dog, some with severe injuries. The doctor at the dispensary sent an urgent message to the Medical College Hospital at Aurangabad for antirabic vaccine. Since it appeared from the report that more comprehensive measures would be required, a personal visit was made to the colony to see the cases and outline the necessary treatment.
few, recently planted,
Public Health FOLLOW-UP OF RABID DOG BITE VICTIMS USHA SHAH*
G. S.
JASWAL†
Department of Preventive and Social Medicine, Medical College, Aurangabad, India. INTRODUCTION
Rabies is endemic in India and bites by stray dogs are the commonest cause of human disease. Case studies or detailed investigations on victims of bites by rabid animals are extremely rare, and a study of publications reveals no report where all the victims of a single rabid dog have been followed up. In July, 1973, an opportunity occurred for detailed investigation of an incident where a rabid dog entered a community and attacked 19 persons, children and adults, before it was killed. Subsequent laboratory studies showed that the dog was highly infective-i.e., rabies virus was present in high titre in the salivary glands. The details of the incident and follow-up of all the victims thus contribute to make a unique account. HISTORY OF ATTACK
Nath Nagar (North) is a small colony which has been recently built to house workers constructing the Jayakwadi Dam Project, in Aurangabad District of Maharashtra State. It is about 42 km from Aurangabad along the Paithan road. The colony consists of barrack-like buildings to house the workers, some bungalows for engineers and other staff, as well as offices, warehouses, &c. A dispensary for the colony and a primary school for the workers’ children are maintained by the project authority of the irrigation department. There are a
* Present address: Department of Preventive and Social Medicine, B. J. Medical College, Poona, India. †Dr Jaswal died on July 10, 1974.
DR KAYSER AND OTHERS: REFERENCES -
4 5
continued
Barrett, F. F., McGhee, R. P., Finland, M. ibid. 1968, 279, 441. O’Toole, R. D., Drew, W. L., Dahlgren, B. J., Beatty, H. N. J. Am. med.
Ass. 1970, 213, 257. 6 Rountree, P. M., Beard, M. A. Med. J. Aust. 1968, i, 1163 7 Courtieu, A. L., Guillermet, F. N., Longeray, C., Maka, G., Chabbert, Y.-A. Ann. Inst. Past. 1964, 107, 691. 8 Borowski, J., Kamienska, K., Rutecka, J. Br. med. J. 1964, i, 983 9 Benner, E. J., Kayser, F. H. Lancet, 1968, ii, 741. 10 Acar, J. F., Courvalin, P., Chabbert, Y.-A. Antimicrob. Agents Chemother.
INVESTIGATION AND TREATMENT
patients were seen on the afternoon of July 14 in the dispensary at Nath Nagar (North). The patients wounds had been dressed and, in some cases, stitches applied. All had been given an injection of tetanus toxoid. All the
Wound Treatment The wounds were
reopened and stitches removed. They by application of a quaternary ammonium compound (’Cetavlon’ liquid, undiluted) and dressed again were
cauterised
without stitches.
Serum Antirabic serum was given to patients 6, 8, 10, and 12 (tables I and n) who had severe lacerations and face wounds. After an intradermal test, to exclude sensitization against horse serum, half the quantity of serum was infiltrated around the wound, and half injected intramuscularly. The total dose was 750 i.u. for an adult, and proportionate dose for a child. Vaccine Antirabic vaccine (A.R.V.) treatment was started immediately, all patients receiving the first injection on the same day. Further treatment (booster injections, &c.) was outlined for each case and was carried out at the Rural Health Unit, Paithan, which is attached to the Medical College. The details of these cases, their injuries, and treatment given are shown in table I. The Semple-type antirabic vaccine (A.R.V.), consisting of 5% suspension of sheep brain, and the hyperimmune antirabies serum of equine origin (A.R.S.) were supplied by the Haffkine Institute, Bombay. Blood Samples To study the response to treatment, blood-samples were drawn for antibody studies on days 2, 10, 30, and 90 after the bite. 7 patients cooperated in this study. The sera were examined, by the method of plaque reduction in tissue culture, for neutralising antibodies against rabies, by Dr T. J. Wiktor, at the Wistar Institute, Philadelphia, U.S.A. The carcass of the dog, which had been buried, was exhumed and carried to the Medical College, Aurangabad for nuropsy. To establish infec-
1970, p. 280. 11 Kayser, F. H., Mak, T. M. Am. J. med. Sci. 1972, 264, 197. 12 Baird-Parker, A. C. J. gen. Microbiol. 1965, 38, 363. 13 Bauer, A. W., Kirby, M. M., Sherris, J. C., Turck, M. Am. J. clin. Pathl. 1966, 45, 493. 14 Federal Register, 1972, 37, 20525. 15 ibid 1973, 38, 2576. 16 Perret, C J Nature, 1954, 174, 1012. 17 Kayser, F. H., Wiemer, U. Zeitschr. Hyg. 1964, 150, 308. 1S Blair, J E., Williams, R. E. O. Bull. Wld Hlth Org. 1961, 24, 771. 19 Kayser, F. H., Wüst, J., Corrodi, P. Antimicrob. Agents Chemother. 1972,
TABLE II-NEUTRALISING-ANTIBODY LEVELS IN SEVEN CASES TREATED FOR RABID DOG BITE
2, 217. 217. 20 Kayser, F H., Santanam, P., Wüst, J. J. med. Microbiol. (in the press). 21 Lacey, R. W. Bact. Rev. 1975, 39, 1. 22 Finland, M Am J. med. Sci. 1972, 264, 207. 23 Craig. W. A. Abstracts Ninth International Congress of Chemotherapy, Lon-
don, 1975. 24 Kayser, F H. Adv. antimicrob. antineopl. Chemother. 1972, 1, 657. 25 Suffler. P W., Sweeney, H. M., Cohen, S. J. Bact. 1973, 116, 771. 26 Lacey, R W., Grinsted, J. J. med. Microbiol. 1973, 6, 511.
are anomalous and may be ignored. They may transposition of samples during labelling and transport.
* these figures
be
explained by
Occasional
Our recent experience of the epidemiology of methicillin-resistance (Mth-R) has revealed a changed picture, Having made a continuous survey of staphylococcal resistance since January, 1965, in Zurich and in parts of adjacent districts (1-1million inhabitants)," we were able to obtain information about the relative frequency of Mth-R in Staph. aureus in a circumscribed geographical area and about the clinical significance of this resis-
Survey
METHICILLIN-RESISTANT STAPHYLOCOCCI 1965-75 F. H. KAYSER
in our population. This epidemiological survey has shown a significant decrease in the number of staphylococcal infections caused by Mth-R strains, although the use of -lactam antibiotics has not been restricted in the past few years. This report summarises our data on the epidemiology of methicillin-resistance in Staph. aureus and compares the findings with results obtained since 1965. tance
Institute of Medical Microbiology, CH-8028 Zürich, Switzerland
Methicillin resistance in Staphylococcus has been one of the major problems of gram positive infections in hospitals in the Zurich area. Up to 1971, about 20% of staphylococcal disease was caused by these peculiar organisms. Since 1972, however, a gradual decrease in the number of methicillin-resistant organisms has been observed, with an unprecedented low of 3% in 1975. The nearly 700 methicillin-resistant cultures that have isolated since 1965 exhibited, with rare exceptions, conventional group-III patterns of lysis in phage-typing and similar antibiotypes. It is suggested that all these isolates are derivatives of a strain which has long existed in the staphylococcal population. The reasons for the changes in the frequency of this strain as an agent causing staphylococcal disease are unclear. The use of penicillinase-resistant &bgr;-lactam antibiotics in hospitals does not seem to play a major role in the distribution and spread or in the disappearance of this strain.
Summary
aureus
Methods Cultures
Staphylococci were isolated from clinical samples and classiaccording to the criteria proposed by
fied as Staph. aureus Baird-Parker. 12
Staph. epidermidis isolates were not included in the study, An isolate or culture was defined as the first isolate obtained from a patient during his staphylococcal disease. A strain was defined as a collection of isolates exhibiting similar patterns of phage lysis and similar antibiotic-resistance characters. Bacteriological Methods Methicillin resistance was detected by inoculating approximately 104 cells, grown overnight in brain-heart infusion (BHI) broth, on to the surface of oxacillin-containing or methicillincontaining Meuller-Hinton (MH) agar plates with a modified Steers replicator. The plates were incubated at 300C for 24 and 48 hours. A minimal inhibitory concentration (Mtc) of 12-$g of the drugs per ml of medium was taken as evidence of resistance. In the early part of the study, Mth-R was detected in a broth (BHi) dilution test using oxacillin. The tubes were incubated at 37°C for 48 hours and resistance was defined according to the criteria just described. Disc susceptibility testing was performed by means of the et al. and the slightly modified single-disc method of Bauer U.S. Food and Drug Administration, 14 15 using MH-agar (BBL), supplemented with 5% old human blood from the blood bank. Penicillin-&bgr;-Iactamase was detected according to Perret’s slightly modified iodide method.16 "
Introduction The extensive
of antimicrobial agents such as benzylpenicillin erythromycin in staphylococcal disease has often been followed by the appearance of large numbers of Staphylococcus aureus resistant to these agents.’ Such a situation seemed also to arise with methicillin-resistant staphylococci. With the introduction of penicillinase-resistant -lactam antibiotics into therapy, increasing numbers of isolations of these staphylococci were reported throughout the world. 2-8 Clinical data also showed that such strains may cause serious infections in the compromised host.9 10 use
or
TABLE
I.-FREQUENCY
OF ANTIBIOTIC RESISTANCE IN
I
*)’nor admission
to or recent
Staph.
I
antibiotic
aureus, ZURICH
I
1965-75 .
i
therapy not determined
651 TAAI I].--
!BNTlRtOTIC-RFS1STANCE I’A’Cf1 RNS OF .lr’fFil<:11 LIN°RFCICTANT
Staph.
aureus
r:{1I.TIJRI’I
I
TARLE HI.—PHAGE-TYPtNG PATTHRNS OF MFTH;(:H ) LIN-RESISTANT
Heterogeneity of Mth-R was detected by streaking O’lml of different dilutions of overnight broth cultures on to the surface of agar plates containing increasing concentrations of oxacillin. In some experiments heterogeneity was examined by inoculating antibiotic plates with different dilutions of cultures with the help of a modified Steers replicator. The phage-type of the isolates was determined according to Blair and Williams. 19 From the beginning of this survey in 1965, all cultures were preserved as lyophilysed samples. Results Disease due
to
Methicillin-resistant Staphylococci
Table t shows the frequency of methicillin resistance and further antibiotic resistance from 1965. to 1975. In the years 1965, 1966, and 1967 all staphylococcal cultures isolated during the year were examined for Mth-R. Because of the large increase in staphylococcal isolations in the following years, a 3-month period in the middle of 1968 and 1969 was taken as representing the whole year. In the following years staphylococci isolated in December, 1970, and January/February, 1971, were included in the study. From 1972 up to 1975, staphylococcal isolates in the first 3 months of the year were collected and examined. From table i it can be concluded that, from 1966 to 1971, a high proportion of staphylococcal cultures exhibited Mth-R. From 1972, however, the proportion dec-
lined, reaching significantly an unprecedented low of 3% in 1975. All the isolates showed the typical heteroresistancethat is, cells of pure cultures tolerated widely different amounts of 3-lactam antibiotics. All isolates expressed their resistance best on media containing sodium chloride or at an incubation temperature of 30°C. Despite the frequent occurrence of Mth-R up to 1973, the percentage of this resistance in staphylococci was always
Sraph.
aureus
CB1I.TliRES
lower than that to other standard antibiotics. Mth-R staphylococci in the Zurich area did not exhibit resistance to vancomycin and to the gentamycin antibiotics.
Methicillin-resistant
Staph.
aureus was
isolated
ex-
clusively from hospital inpatients. The problem was observed for the most part in large hospitals, whereas in small hospitals methicillin-resistance did not play any major role. From 1969 to 1975, 80% and more of all the isolated resistant staphylococci came from two hospitals: the large university hospital in Zurich and a mediumsize general hospital in Chur. The kinds of infections observed in hospital inpatients did not change significantly from 1965 to 1975. They were the expected hospital-acquired infections and consisted of respiratory infections (20-38%), surgical wound infections (22-30%), skin and muscle infections (9-16%), urinary-tract infections (8-12%), bone and joint infections (9-11%), septicaemia of unknown origin (3-10%), and miscellaneous infections (4-8%).
Antibiotypes of Methicillin-resistantStaphylococci Table ii summarises the patterns of resistance to standard antimicrobials found in Mth-R staphylococci from 1965 to 1975. All cultures were resistant to the sulphonamides and to streptomycin and produced penicillinase. With few exceptions, the isolates exhibited resistance to tetracycline and to erythromycin. 85% had the "dissociated" type of resistance with cross-resistance to oleandomycin after induction with erythromycin, whereas 15% had the "undissociated" resistance to all macrolide antibiotics. Resistance to kanamycin/neomycin and to chloramphenicol was not constantly observed. The genetic markers of these resistances, however, were found to be part of relatively unstable staphylococcal plasmids.19
652
Phage-types of Methicillin-resistant Staphylococci. Table in summarises the phage-typing patterns of Mth-R staphylococci isolated in Zurich. Most of the isolates exhibited conventional group iit patterns of lysis at routine test dilution (RTD), or 1000 times RTD. Phage-types 80/81 and 52A/80 were encountered only once, type 29 three times, and type 29/80 twice. The susceptibility of the cultures to the typing phages did not change significantly throughout the observation period. It was observed, however, that since 1969 the number of trains typable only by 1000 times the RTD and since 1972 the frequency of cultures untypable increased. Discussion The similar antibiotypes and similar phage-typing patterns of the 687 methicillin-resistant Staph. aureus cultures isolated since 1965 in the Zurich area reveal that the problem of methicillin resistance results mainly from the spread of one epidemiologically distinct strain. In addition to Mth-R, the "Zurich strain" is constantly resistant to the sulphonamides (Sul-R), erythromycin (Ery-R), tetracycline (Tet-R), streptomycin (Str-R), and penicillin (Pen-R). Molecular studies have shown that the genetic markers of these resistances-with the exception of Pen-R-are very probably part of the bacterial chromosome.19 On the other hand, it has been found that the determinants of resistance to chloramphenicol (Cml-R) and neomycin/kanamycin (Neo-R) as well as of penicillinase production are located on different, mutually compatible plasmids. 19 20 Plasmids usually are responsible for a high degree of genetic variability in bacteria. Isolates which lack Cml-R or Neo-R owe this fact to the instability of their respective genetic markers. Penicillinase production, on the other hand, is thought to be absolutely necessary for a staphylococcus strain to survive for a long period in the hospital environment. Hence, all isolates were observed to produce this enzyme, although the markers are part of the penicillinase plasmid. The "one-strain hypothesis" of methicillin resistance in the Zurich area is further supported by the similar phage-typing patterns of cultures isolated between 1965 and 1975. A trend toward restriction in phage susceptibility of our cultures is detectable. This, however, is an overall trend also noted in other staphylococci,and has presumably resulted from alterations in prophage carriage. Further evidence of the "single-clone hypothesis" is mentioned by Lacey2l who even argues that all Mth-R staphylococci isolated so far in the world are derivatives of one single strain. The question, then, remains as to the causes of the increased prominence of this strain in our hospitals up to 1971 and the rapid decrease in the frequency of infections caused by it since 1972. We suggested that the growing number of Mth-R isolates might be connected with the increasing use of &bgr;-lacantibiotics since 1964 in our hospitals.l1 Our recent experience indicates that this suggestion may be incorrect, because such antibiotics have not been used less in the years in which the isolations of this strain have diminished. Furthermore, in hospitals of the United States Mth-R staphylococci, although present in low numbers, never played a major role22 despite the frequent use of penicillins and cephalosporins.23 tam
Possibly resistance in vitro is not always equivalent to resistance in vivo. The specific properties of Mth-R staphylococci suggest that resistance per se may not be clinically important in all patients. At normal body temperatures, only few cells in populations can express their resistance to high levels of &bgr;-Iactam antibiotics. These high-resistance minorities grow slowly and have ontv low virulence, especially in the presence of -)actam in vivo antibiotic therapy. 24 Thus selective exerted by antimicrobial therapy may not have resulted in the varying frequencies of isolations observed from 1965 to 1975. That does not mean, however, that infections caused by Mth-R staphylococci should be treated with p-Iactam antibiotics. In patients with severe underlying disease, even the minorities with low virulence might cause serious staphylococcal infections. Alternative antimicrobials (e.g. gentamycin, tobramycin, vancomycin) can be applied in the treatment of methicillin-resistant infections. The increase in methicillin-resistant staphylococcal infections in our hospitals from 1965 may be better explained by other characters, besides Mth-R, which enabled this strain to survive in the hospital-e.g., resistance to many other antimicrobials, including production of large amounts of penicillinase. The reduction in the number of Mth-R isolations since 1972 is more difficult to understand. Possibly the genes for Mth-R became mobilised bB plasmids or bacteriophage, and spontaneous loss of these genomes, resulting in methicillin-susceptible and more virulent variants, occurred. The presence of additional extrachromosomal DNA, which has to be maintained and replicated in a bacterial host, can result in decreased virulence. Plasmid-negative variants thus may be better equipped to survive in the hospital environment. Experience in our laboratory20 and in others,25 26 however, has shown that Mth-R seems to be a very stable genetic character. A connection of this resistance with extrachromosomal elements was not observed until now in the Zurich strain. Change of the patient population could have resulted in a decreased capacity of Mth-R staphylococci to survive and multiply in the human host. We have no indication at all that this happened in our hospitals. Unknown extrachromosomal virulence factors could have become lost from Mth-R cultures, with consequently reduced capacity of this strain to compete with other staphylococcal organisms.
pressures
A
new
methicillin-susceptible .Staph.
aureus
strain,
highly virulent and resistant to many other antimicrobials, could have appeared in our hospitals. Unfortunately we lack data on phage-types of methicillin-susceptible staphylococci from 1965 to 1975. A conclusive explanation of the changing ecology of Mth-R staphylococci in the Zurich area cannot be advanced at present. For the sake of our patients, however, we hope that the incidence of infections by Mth-R staphylococci will decrease further or at least remain at the present low level. REFERENCES 1. Finland, M. J. infect.Dis. 1970, 122, 419. 2. Parker, M. T., Hewitt, J. H.Lancet, 1970, i, 800 3. Jessen, O., Rosendal, K., Bülow, P., Faber, V., J.Med. 1969, 281, 627.
Eriksen,
K R. New Es
653 trees. The surroundings are rather barren and dry. On the morning of July 14, 1973, an unknown stray dog entered the colony at about 0800 hours. It attacked various persons along the road and in houses till it came to the school house. Here, it entered the classroom and bit several of the children before they could run away. It was chased out and ran through the compounds of some of the houses, where it was finally located and killed at about 1030. In all 19 persons had been bitten by the dog, some with severe injuries. The doctor at the dispensary sent an urgent message to the Medical College Hospital at Aurangabad for antirabic vaccine. Since it appeared from the report that more comprehensive measures would be required, a personal visit was made to the colony to see the cases and outline the necessary treatment.
few, recently planted,
Public Health FOLLOW-UP OF RABID DOG BITE VICTIMS USHA SHAH*
G. S.
JASWAL†
Department of Preventive and Social Medicine, Medical College, Aurangabad, India. INTRODUCTION
Rabies is endemic in India and bites by stray dogs are the commonest cause of human disease. Case studies or detailed investigations on victims of bites by rabid animals are extremely rare, and a study of publications reveals no report where all the victims of a single rabid dog have been followed up. In July, 1973, an opportunity occurred for detailed investigation of an incident where a rabid dog entered a community and attacked 19 persons, children and adults, before it was killed. Subsequent laboratory studies showed that the dog was highly infective-i.e., rabies virus was present in high titre in the salivary glands. The details of the incident and follow-up of all the victims thus contribute to make a unique account. HISTORY OF ATTACK
Nath Nagar (North) is a small colony which has been recently built to house workers constructing the Jayakwadi Dam Project, in Aurangabad District of Maharashtra State. It is about 42 km from Aurangabad along the Paithan road. The colony consists of barrack-like buildings to house the workers, some bungalows for engineers and other staff, as well as offices, warehouses, &c. A dispensary for the colony and a primary school for the workers’ children are maintained by the project authority of the irrigation department. There are a
* Present address: Department of Preventive and Social Medicine, B. J. Medical College, Poona, India. †Dr Jaswal died on July 10, 1974.
DR KAYSER AND OTHERS: REFERENCES -
4 5
continued
Barrett, F. F., McGhee, R. P., Finland, M. ibid. 1968, 279, 441. O’Toole, R. D., Drew, W. L., Dahlgren, B. J., Beatty, H. N. J. Am. med.
Ass. 1970, 213, 257. 6 Rountree, P. M., Beard, M. A. Med. J. Aust. 1968, i, 1163 7 Courtieu, A. L., Guillermet, F. N., Longeray, C., Maka, G., Chabbert, Y.-A. Ann. Inst. Past. 1964, 107, 691. 8 Borowski, J., Kamienska, K., Rutecka, J. Br. med. J. 1964, i, 983 9 Benner, E. J., Kayser, F. H. Lancet, 1968, ii, 741. 10 Acar, J. F., Courvalin, P., Chabbert, Y.-A. Antimicrob. Agents Chemother.
INVESTIGATION AND TREATMENT
patients were seen on the afternoon of July 14 in the dispensary at Nath Nagar (North). The patients wounds had been dressed and, in some cases, stitches applied. All had been given an injection of tetanus toxoid. All the
Wound Treatment The wounds were
reopened and stitches removed. They by application of a quaternary ammonium compound (’Cetavlon’ liquid, undiluted) and dressed again were
cauterised
without stitches.
Serum Antirabic serum was given to patients 6, 8, 10, and 12 (tables I and n) who had severe lacerations and face wounds. After an intradermal test, to exclude sensitization against horse serum, half the quantity of serum was infiltrated around the wound, and half injected intramuscularly. The total dose was 750 i.u. for an adult, and proportionate dose for a child. Vaccine Antirabic vaccine (A.R.V.) treatment was started immediately, all patients receiving the first injection on the same day. Further treatment (booster injections, &c.) was outlined for each case and was carried out at the Rural Health Unit, Paithan, which is attached to the Medical College. The details of these cases, their injuries, and treatment given are shown in table I. The Semple-type antirabic vaccine (A.R.V.), consisting of 5% suspension of sheep brain, and the hyperimmune antirabies serum of equine origin (A.R.S.) were supplied by the Haffkine Institute, Bombay. Blood Samples To study the response to treatment, blood-samples were drawn for antibody studies on days 2, 10, 30, and 90 after the bite. 7 patients cooperated in this study. The sera were examined, by the method of plaque reduction in tissue culture, for neutralising antibodies against rabies, by Dr T. J. Wiktor, at the Wistar Institute, Philadelphia, U.S.A. The carcass of the dog, which had been buried, was exhumed and carried to the Medical College, Aurangabad for nuropsy. To establish infec-
1970, p. 280. 11 Kayser, F. H., Mak, T. M. Am. J. med. Sci. 1972, 264, 197. 12 Baird-Parker, A. C. J. gen. Microbiol. 1965, 38, 363. 13 Bauer, A. W., Kirby, M. M., Sherris, J. C., Turck, M. Am. J. clin. Pathl. 1966, 45, 493. 14 Federal Register, 1972, 37, 20525. 15 ibid 1973, 38, 2576. 16 Perret, C J Nature, 1954, 174, 1012. 17 Kayser, F. H., Wiemer, U. Zeitschr. Hyg. 1964, 150, 308. 1S Blair, J E., Williams, R. E. O. Bull. Wld Hlth Org. 1961, 24, 771. 19 Kayser, F. H., Wüst, J., Corrodi, P. Antimicrob. Agents Chemother. 1972,
TABLE II-NEUTRALISING-ANTIBODY LEVELS IN SEVEN CASES TREATED FOR RABID DOG BITE
2, 217. 217. 20 Kayser, F H., Santanam, P., Wüst, J. J. med. Microbiol. (in the press). 21 Lacey, R. W. Bact. Rev. 1975, 39, 1. 22 Finland, M Am J. med. Sci. 1972, 264, 207. 23 Craig. W. A. Abstracts Ninth International Congress of Chemotherapy, Lon-
don, 1975. 24 Kayser, F H. Adv. antimicrob. antineopl. Chemother. 1972, 1, 657. 25 Suffler. P W., Sweeney, H. M., Cohen, S. J. Bact. 1973, 116, 771. 26 Lacey, R W., Grinsted, J. J. med. Microbiol. 1973, 6, 511.
are anomalous and may be ignored. They may transposition of samples during labelling and transport.
* these figures
be
explained by