ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, May 1975, p. 622-628
Vol. 7, No. 5 Printed in U.S.A.
Copyright 0 1975 American Society for Microbiology
Epidemiology of Antibiotic and Heavy Metal Resistance in Bacteria: Resistance Patterns in Staphylococci Isolated from Populations in Iraq Exposed and Not Exposed to Heavy Metals or Antibiotics DAVID J. GROVES, H. SHORT, A. J. THEWAINI,
AND
FRANK E. YOUNG*
Department of Microbiology, The University of Rochester School of Medicine and Dentistry,* Strong Memorial Hospital, Rochester, New York 14642 and Medical City Hospital, University of Baghdad, Baghdad, Iraq Received for publication 26 November 1974
Staphylococci were isolated from rural and urban populations in Iraq, which not known to be exposed to either heavy metals or antibiotics. The antibiotic and heavy metal resistance patterns of these strains were analyzed in both mannitol-fermenting and nonfermenting strains. Over 90% of the strains were resistant to at least one of the following antibiotics: penicillin, chloramphenicol, erythromycin, tetracycline, cephalothin, lincomycin, or methicillin. In general, mannitol-fermenting strains were resistant to penicillin and cupric ions. Mannitol-negative strains were more frequently associated with mercuric ion and tetracycline resistance. Although resistance to penicillin and tetracycline can coexist, the combination of penicillin resistance and tetracycline resistance usually occurred in mannitol-negative strains. The possibility of selection of heavy metal-resistant strains due to exposure to toxic levels of methylmercury was examined. No significant increase in mercuric ion-resistant strains of staphylococci or Escherichia coli were detected in exposed populations as compared to control groups. The possible reasons for this result are discussed.
were
The current interest in subtle effects of environmental contaminants on ecosystems has resulted in a renewed emphasis on the interactions of pollutants with microorganisms. As man further contaminates his own environment, he alters the milieu of those organisms for whom he is the host. For example, selection of antibiotic-resistant strains in human patients (4, 8, 19) and live stock (18) is a well recognized phenomenon. There is also evidence to indicate that there may be a correlation between the emergence of resistance to antibiotics and heavy metals. Thus the exposure of industrial workers to metallic mercury or inorganic mercury has been shown to be associated with an increased colonization by antibiotic- or mercury-resistant staphylococci in the nasal passages (7). Even in populations that had not been known to have been exposed to mercury, there is a high correlation between certain types of antibiotic resistance patterns and resistance to heavy metals. Recent studies have demonstrated an association between resistance to penicillin, copper ions, and the production of coagulase, whereas resistance to mercury was more commonly noted in strains that were resistant to tetracycline and coagulase negative (6; D. Groves and F.. Young, manuscript in preparation).
Because there was an increased incidence of intercurrent infections leading to death in patients poisoned with methylmercury at Minimata (14), it was considered particularly relevant to determine the effects of exposure to toxic amounts of heavy metals on resistance patterns in microorganisms. In the period from September 1971 to mid-January 1972, a severe epidemic of methylmercury poisoning of farmers and their families occurred in Iraq, due to the consumption of home-made bread prepared from seed grain treated with a methyl mercurial fungicide. A total of 6,530 cases were admitted to hospitals, and there were 459 hospital deaths attributed to methylmercury poisoning (3). As part of an inter-university collaboration between the University of Baghdad (Iraq) and the University of Rochester (U.S.A.) we explored the effect of methylmercury poisoning on the microbial flora of man. The results of this study indicate that over 90%7o of the strains of staphylococci isolated from rural and urban populations in Iraq, that were obtained from individuals who were not known to be exposed to either heavy metals or antibiotics, were resistant to one of the following antibiotics: penicillin, chloramphenicol, erythromycin, tetracycline, cephalothin, lincomycin, or methicillin. Al-
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HEAVY METAL RESISTANCE IN STAPHYLOCOCCI
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though associations could be made between otic resistance for urban and rural populations types of resistance patterns, there was no signif- are shown in Table 1. The urban populations icant increase in mercuric-resistant strains of were derived from patients which were hospital-
staphylococci or Escherichia coli in populations ized or visited the out-patient clinic. None of exposed to methylmercury as compared with these were known to be treated with antibiotics. The rural populations were obtained from pathe population selected as the control. tients visiting the popular clinic of the Mesaib MATERIALS AND METHODS al-Kabir Irrigation Project (columns 5 and 6) or Bacterial isolates. Strains were isolated from from rural populations in the same area (colrectal swabs and anterior nasal swabs using Fisher umns 7 and 8). In these samples there were very Handi swabs containing modified Stuart transport few strains that were susceptible to all antibiotmedium (Fisher Scientific Co., Pittsburgh, Pa.). Out- ics tested (35 out of 474). There was no signifiside the hospital supplies and specimens were trans- cant difference between mannitol-positive and ported in an ice chest at 4 to 10 C to minimize thermal mannitol-negative strains. With the exception damage. Staphylococcal strains were isolated by streaking nasal swabs on mannitol salt agar. Rectal of fewer multiply resistant strains, the patterns swabs were streaked on McConkey's agar for isolation were similar in urban and rural populations. Note that penicillin resistance occurs predomiof lactose-positive enteric organisms. Antibiotic and heavy metal resistance. The assay nantly in mannitol-positive strains. The heavy metal resistance patterns of these for susceptibility and resistance to antibiotics and heavy metals were performed as described elsewhere staphylococcal strains are shown in Table 2. (6; D. Groves and F. Young, manuscript in prepara- Resistance to metals is not as common as tion). resistance to antibiotics in the same strains. Data manipulation. Data storage, reducttn, and Although a total of only 35 strains were susceppattern recognition were carried out with a program tible to all of the antibiotics tested, 160 strains designed specifically for analysis of resistance markwere susceptible to mercuric, cupric, and caders (6). Analysis of mercury levels. Blood samples were mium ions. These susceptible strains were collected in heparinized Vacutainer tubes. The blood largely mannitol negative. All of the strains resistant only to mercuric ions were mannitol was analyzed for total and inorganic mercury by the atomic absorption method (9). nonfermentors, whereas strains resistant only to Cu2+ (or Cd2+) were predominantly able to RESULTS ferment mannitol. Even when mercury resistAntibiotic and heavy metal resistance pat- ance was present with resistance to other metterns in microorganisms isolated from indi- als, the strain was usually unable to ferment viduals who were not known to be exposed to mannitol. This association can be more clearly antibiotics or heavy metals. To study the visualized by analyzing the distribution of the effect of toxicity to mercury on the plasmids in five predominant markers: resistance to penicilmicrobes which inhabit man, we attempted to lin, resistance to tetracycline, resistance to obtain populations which were not known to be Hg2+, resistance to Cu2+, and resistance to Cd2+ exposed to mercury from rural, urban, and independent of the others (Table 3). Because of hospital environments. The patterns of antibi- the existence of multiply resistant strains, each TABLE 1. Distribution of mannitol fermentation and antibiotic resistance patterns Mannitol reaction
Rural populations
Urban populations
Antibiotic resistance pattemsa
Non-clinic
Clinic
Outpatients
Hospital
P
C
E
T
Cf
L
Dp
+
-
+
-
+
-
+
-
S R S R R R
S S S S R R
S S S S S R
S S R R R R
S S S S S S
S S S S S S
S S S S S S
5 48 2 17 5 3
7 23 5 38 38 9
5 28 1 10 3 3
7 17 1 19 16 4
3 41 0 3 0 1
6 18 1 7 3 0
0 19 0 1 0 0
2 6 1 1 0 0
14
8
8
12
1
3
0
1
Others
a Abbreviations: P, penicillin; C, chloramphenicol; E, erythromycin; T, tetracycline; Cf, cephalothin; L, lincomycin; Dp, methicillin; S, susceptible; R, resistant.
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ANTIMICROB. AGENTS CHEMOTHER.
GROVES ET AL.
TABLE 2. Distribution of mannitol fermentation and heavy metal resistance patterns Mannitol reaction
Urban
Heavy metal resistancea
Rural populations
populations OutHosl italpatients
Hoptal
Hg2+ Cu2+ Cd2+ + S
R S S
R R S R a
S S R S
S S S R
R
S R R R
S R R
-
+
-
Clinic
Nonclinic
+
+
-
-
18 64 10 38 5 16 1 8 0 23 0 5 0 1 0 0 35 17 30 20 28 18 16 3 3 1 1 0 0 0 1 0 3 8 1 2 1 2 0 0 3 4 0 2 0 0 0 0 27 5 15 7 15 0 2 0 5 6 1 2 0 1 0 0
S, susceptible; R, resistant.
TABLE 3. Analysis of independent resistance traits in mannitol fermenting and nonfermenting strains Mannitol reaction Independent
resistancea
Urban
Rural
populations
populations
OutHospital patients +
Penicillin
.
86 115 96 41 36 38 16
Tetracycline .. 35 Mercuric ion ... 11 Cupric ion . 70 Cadmium ion
51 21 2 47 17
Clinic
Nonclinic
+
+
66 46 30 20 8 46 4 11 1 2 11 1 4 0 0 31 44 21 18 3 11 15 1 3 0
aThe strains are resistant to each agent independently. Thus the penicillin-resistant strains may or may not be resistant to any of the other agents tested.
total population is less than the sum of the values in the column above it. The strains resistant to penicillin were distributed between mannitol-positive and mannitol-negative strains in ratios very similar to those of the total populations, as in each case the penicillin resistant strains comprise the majority of the population. In strains resistant to tetracyclines or to mercury, there is a disproportionate decrease in the fraction of mannitol-positive strains. Copper and cadmium resistance, on the other hand, causes a disproportionate increase in the mannitol-positive strains. These results (Table 3) outline the general association of resistance to penicillin and copper with mannitol fermentation as opposed to the association between resistance to tetracycline, resistance to
mercury, and the lack of mannitol fermentation. Therefore, the general association between resistance markers and the mannitol reaction would appear to be the same as described for other populations (D. Groves and F. E. Young, manuscript in preparation). One might expect a three-vector comparison of antibiotic resistance, metal resistance, and mannitol fermentation to show the two predominant classes to be (i) those with the mannitolpositive trait and copper as well as penicillin resistance; and (ii) those with resistance to tetracycline, resistance to mercury, and the lack of mannitol fermentation. In Table 4 we have listed, for each population, the distribution of the penicillin, tetracycline, mercury, and copper resistance patterns in fermenting and nonfermenting organisms. We have eliminated the subsets which contained less than six organisms in any pattern. In the populations resistant to only two of the test compounds (penicillin, tetracycline, mercury, and copper), the major patterns are PenRTetRHgsCus and PenRTetsCuRHgs. Thus penicillin resistance and tetracycline resistance can coexist. The classes of microorganisms with resistance only to the following pairs of agents are seldom found: penicillin and mercury; tetracycline and mercury; tetracycline and copper; or mercury and copper. Most of the strains resistant to tetracycline are also resistant to penicillin. The strains resistant only to these two antibiotics are predominantly mannitol negative. Furthermore, strains resistant to mercuric ions are usually multiply resistant strains and predominantly mannitol negative. Effect of exposure to heavy metals or antibiotics on patterns of heavy metal resistance in microorganisms. Two populations exposed to methylmercury and one population exposed to antibiotics were studied. The first population exposed to methylmercury consisted of 40 patients, largely families with children, who were kept in Medical City Hospital for treatment and research. The specimens taken from these patients were compared with control specimens taken from approximately 400 hospital patients, a subpopulation of whom had received antibiotic therapy. The second population consisted of residents of the Mesaib-al-Kabir Irrigation Project. This particular area was severely affected by methylmercury poisoning, with approximately 800 people exhibiting clinical symptoms of mercury poisoning out of a total population of 20,000. Control populations were selected from nonexposed isolated families, and from patients attending the medical clinic at Mesaib village. Both exposed and nonexposed
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TABLE 4. Major patterns of resistance to antibiotics and heavy metals Mannitol reaction
T
S R S R R R R R
S S S R S R R R
Hg2+| Cu2+ S S S S S R S R
S S R S R S R R
Others
Non-clinic
Clinic
Outpatients
Hospital
P
Rural populations
Urban populations
Resistance patterna
+
-
+
-
+
-
+
3 9 3 8 42 3 16 6
5 15 2
40 8 24 12 14
2 5 3 4 26 0 15 1
4 13 4 21 7 5 16 3
2 1 1 2 40 0 2 0
3 7 4 6 11 0 3 1
0 2 0 0 17 0 1 0
1 6 1 1 1 0 0 0
4
8
2
3
1
2
0
1
aAbbreviations: Penicillin, P; tetracycline, T; susceptible, S; resistant, R. populations were validated by analysis of blood samples for total and inorganic mercury content.
TABLE 5. Mannitol-positive staphylococci isolated from patients in Medical City Hospital Metal resistance
Exposure The resistance patterns of staphylococci to patterna heavy metals and antibiotics were compared in populations of patients that were exposed to None Mercury bitic Hg2+| Cu2+ Cd2+ methylmercury and antibiotics to ascertain whether exposure influenced the types of resist11 6 18 S S S ance patterns in these strains. The distribution 0 S 0 0 R S of resistance to copper, cadmium, and mercuric 28 S 13 35 R S 4 3 ions in mannitol-fermenting strains is shown in 3 S R S 1 1 3 R S Table 5. Exposure to methylmercury did not .R 1 S 0 3 R R influence the incidence of mercury-resistant 12 2 S 27 R R strains ip the population, whereas exposure to 22 0 5 R R R antibiotics significantly increased the incidence of straing resistant to mercuric ions. This was a S, Susceptible; R, resistant. most evident by an increase in strains that were multiply resistant to mercuric, copper, and TABLE 6. Mannitol negative staphylococci isolated from patients in Medical City Hospital cadmium ions (5 and 28% for nonexposed and antibiotic exposed, respectively). The highest Metal resistance Exposure incidence of mercury resistance in staphylococci patteMa occurred in the nonexposed population (Table 6). There was no significant difference in the None Mercury biotic Hg2+ Cu2+ Cd2+ incidence of mercury-resistant strains isolated from patients that were exposed to antibiotics 48 7 64 S S S or methylmercury as compared to the control 10 S S 23 5 R population. S S 5 21 R 17 1 2 S S A similar analysis of the heavy metal resist1 R 9 8 R R 1 S ance patterns was performed on strains of S R 1 3 4 R staphylococci isolated from rural populations 1 S R 1 R 5 (Tables 7 and 8). The size of the nonexposed 6 0 R R 6 R population precludes a detailed analysis. However, summation of mannitol-positive and man5, Susceptible; R, resistant. nitol-negative staphylococci gives zero out of 29 mercury-resistant strains from the mercury This increase is not quite significant. A larger nonexposed population, compared to 12 out of control population might well still have had no 185 from the exposed population. This gives mercury-resistant strains. Forty-five straihs 1 and P 0.25 to 0.1. with no mercury resistance in the control group an x2 of 1.87 with d.f. =
=
626
ANTIMICROB. AGENTS CHEMOTrHER.
GROVES ET AL.
would have made the increase in the exposed group significant with a P = 0.1. There is a possibility that the resistance of E. coli isolates might be more affected by exposure to organic mercury, as the feces contain a large portion of the eliminated mercury, thus exposing fecal microorganisms to elevated levels. Table 9 shows the data for mercury resistance and susceptibility of the E. coli isolated from fecal samples and rectal swabs of both exposed and nonexposed populations. As observed with staphylococci there was a higher incidence of TABLE 7. Heavy metal resistance of mannitol-positive staphylococci isolated from rural subjects Metal resistance patterna
Hg2+
CU2+
S
S
S R S R S S R R S R R S R R a R, Resistant; S,
Cd2+
S S S R S R R R
susceptible.
TABLE 8. Heavy metal resistance of mannitol-negative staphylococci isolated from rural subjects Metal resistance patterna
Exposure
Hg2+
Cu2+
Cd2+
None
Mercury
S R S S R R S R
S S R S R S R R
S S S R S R R R
8 0 3 0 0 0 0 0
28 1 54 0 6 0 0 2
aR, Resistant; S,
susceptible.
TABLE 9. Effect of exposure to methylmercury on resistance of mercuric ions in strains of Escherichia coli Resistance to mercuric ion
Rb
Sb
Non-clinic Mesaib project
7 69
1 14
Hospital (no
antibiotic
exposure) 10 35
40 71
Exposure to mercury indicated by + and -. b R, Resistant; S, susceptible. a
resistance to mercury in the organisms isolated from individuals in the urban environment. Even with the small sample studied it is unlikely that poisoning with methylmercury shifted significantly the levels of resistance to mercuric ions at the time of sampling of the population.
DISCUSSION Microorganisms frequently contain extrachromosomal elements that determine resistance to toxic compounds such as antibiotics and heavy metals, or the determinants for genes that regulate complex pathways of biodegradation such as the metabolism of camphor in Pseudomonas putida (15). In staphylococci a single genetic determinant, coagulase, has been utilized traditionally to separate Staphylococcus aureus from Staphylococcus epidermidis. Other traits such as mannitol fermentation (13), phage sensitivity, and biotyping (2) have reinforced this grouping. The observation that genetic markers can be exchanged between the S. aureus and S. epidermidis (20) indicates that this grouping may be quite artificial. Nevertheless, we utilized mannitol fermentation as an additional trait in the analysis of the distribution of heavy metal resistance among staphylococci. If the possession of resistance to mercury produced a selective advantage to microorganisms inhabiting poisoned individuals, it would not be unreasonable to expect an increased incidence of mercury-resistant strains either in the staphylococci which usually inhabit the nasopharynx (S. aureus) or those which usually inhabit the skin (S. epidermidis). However, no significant increase in the incidence of mercuryresistant strains occurred in individuals that were severely poisoned with mercury. A number of explanations should be considered in evaluating this result. First, the exposure to mercury was limited in duration. Within 2 months of the onset of ingestion of contaminated bread, symptoms appeared and the treated grain was recovered. Thus the blood levels rose to a maximum, then decayed with a half-life of approximately 70 days (3). Second, there was an interval of at least 6 months between the consumption of the contaminated bread and the sampling of the population for analysis of the microbial flora. Nevertheless, the total mercury levels in the blood of exposed subjects were still elevated, with a range of 100 ng/ml to 1,350 ng/ml. Even when a limited population was studied in which the blood levels were greater than 500 ng/ml, there was no significant increase in the mercury-resistant fraction of E.
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HEAVY METAL RESISTANCE IN STAPHYLOCOCCI
coli or staphylococci. Third, the blood level values used are total mercury levels, with the inorganic mercury composing less than 10% of this value (3). Fourth, although strains of staphylococci which are resistant to mercury ions are also resistant to phenyl mercuric ions, there is no relationship to their resistance to methylmercury (G. D. Groves and F. E. Young, manuscript in preparation). Thus, poisoning with alkyl mercurial compounds may not provide selective pressure for resistance to mercuric ions even though the elevated circulating inorganic mercuric levels in the blood show that significant degradation to inorganic mercury occurs. Finally, the high frequency of antibiotic-resistant staphylococci, even among those not known to have been treated with antibiotics, would obscure subtle changes in the microbial flora. For instance, antibiotic-resistant staphylococci occurred with a frequency of greater than 0.90 with an incidence of 0.86 for penicillin resistance. The only trend that indicated a selective effect of mercury was in the rural population; however, the low numbers of isolates precluded a detailed analysis (Tables 7 and 8). In addition, the infrequent occurrence of mercuryresistant organisms in the rural environment where the original exposure to organo-mercurials occurred would result in a limited gene pool from which mercury-resistant organisms might be selected. The ideal population for study would be carriers of mercury-susceptible staphylococci and E. coli who become exposed to high levels of inorganic or organic mercury with sampling prior to, during, and after exposure. It is also necessary to have a pool of mercuryresistant strains in the environment for selection by the elevated mercury levels. Such ideal human populations are not possible, but model animal systems are under consideration. Studies are also under way to sample populations with chronic exposure to organo-mercurials, as well as chronic and transitory exposure to inorganic mercury. Many of the genes which regulate antibiotic and heavy metal resistance in staphylococci reside on plasmids. Although there appears to be no incompatibility between plasmid-borne resistance to penicillin, tetracycline, chloramphenicol, and erythromycin in S. aureus (5, 17), little is known about the interactions between markers that reside on different plasmids and how these plasmids interact to form multiple resistant strains. The most extensively studied plasmids in S. aureus are the penicillinase-containing plasmids of phage groups I and II. Resistance to heavy metals such as mercuric and cadmium ions are usually linked to penicil-
627
linase on these plasmids (11, 12, 16). Tetracycline resistance can be determined by a plasmid which is lost or transduced independently of the penicillinase-containing plasmids (1, 10). The data in this study demonstrate that patterns of antibiotic and heavy metal resistance vary between coagulase-positive or mannitol-fermenting and coagulase-negative or mannitol-noniermenting strains. For instance, resistance to penicillin and copper ions are most frequently associated with the presence of mannitol fermentation, whereas the resistance to tetracycline and mercuric ions is most frequently found in mannitol nonfermenting strains. Although it is possible to have resistance to penicillin, tetracycline, mercuric ions and copper ions in the same strain, this most frequently occurs when the organism cannot ferment mannitol. It may be of interest to note that similar association between the resistance to mercury and tetracycline in coagulase-negative strains in contrast to the resistance to penicillin and copper in coagulase-positive strains was observed in the microbiology laboratory at the University of Rochester. Detailed studies are in progress to determine whether the insights from the epidemiological survey may aid in obtaining a more detailed understanding of the incompatibility and interaction of plasmids in pathogenic organisms. ACKNOWLEDGMENTS We are grateful to the Scientific Committee on Mercury Poisoning in the Baghdad Medical College for allowing us to publish these results. The analytical data on mercury were supplied by the Mercury Research Laboratory under the direction of Hashim I. Dhahir. We wish to acknowledge P. Dhahir and M. R. Greenwood for supervising the analytical team and for data processing. The analytical determinations were made by Ilham M. Al-Jubouri, Amir Khayat, Selma M. Matook, and Mansour Al-Muntasir. We wish to also extend our thanks to Ammar Abdul Razzak and Muhammed Ali Majeed for their invaluable help in collecting the samples and in other aspects of the work. We also wish to express our appreciation to Buthaina Al-Nakash, Agnes Haik, Suhaila Saadalla, Rasoul Al-Dabagh, Hashim Al-Mosawi, and other workers in the Microbiology Laboratory in the Medical City Hospital for their indispensable help. David L. Hollis contributed to the technical aspects of the study at the University of Rochester. This research was supported in part by National Science Foundation (RANN) grant GI 30097 and in part by funds supplied by Baghdad University. We are grateful to Bioquest Division of Baltimore Biological Laboratory and Difco Laboratories for their kind donation of supplies and media. LITERATURE CITED 1. Asheshov, E. H. 1966. Chromosomal location of the genetic elements controlling penicillinase production in a strain of Staphylococcus aureus. Ntature (London) 210:804-806. 2. Baird-Parker, A. C. 1965. Staphylococci and their classification. Ann. N. Y. Acad. Sci. 128:4-25. 3. Bakir, F., S. F. Damluji, L. Amin-zaki, M. Murtadha, A.
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4.
5. 6.
7. 8.
9. 10.
11.
12.
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Khalidi, N. Y. Al-Rawi, S. Tikriti, H. I. Dhahir, T. W. Clarkson, J. C. Smith, and R. A. Doherty. 1973. Methylmercury poisoning in Iraq: an interuniversity report. Science 181:230-241. Bentley, D. W., J. J. Hahn, and M. H. Lepper. 1970. Transmission of chloramphenicol-resistant Staphylococcus epidermidis: epidemiologic and laboratory studies. J. Infect. Dis. 122:365-375. Dyke, K. G. H., and M. H. Richmond. 1967. Occurrence of various types of penicillinase plasmids among hospital staphylococci. J. Clin. Pathol. 20:75-79. Groves, D. J., L. Maroglio, C. W. Merriam, and F. E. Young. 1974. Epidemiology of antibiotic and heavy metal resistance in bacteria: a computer-based data system. Comput. Programs Biomed. 3:123-134. Hall, B. M. 1970. Mercury resistance of Staphylococcus aureus. J. Hyg. 68:121-129. Lepper, M. H., P. Tillman, and R. Devetsky. 1965. Patterns of transmission of staphylococci. Ann. N. Y. Acad. Sci. 128:404-427. Magos, L., and T. W. Clarkson. 1972. Atomic-absorption determination of total, inorganic, and organic mercury in blood. J. Assoc. Off. Anal. Chem. 55:966-971. May, J. W., R. H. Houghton, and C. J. Perret. 1964. The effect of growth at elevated temperatures on some heritable properties of Staphylococcus aureus. J. Gen. Microbiol. 37:157-169. Moore, B. 1960. A new screen test and selective medium for the rapid detection of epidemic strains of Staphylococcus aureus. Lancet 2:453-458. Novick, R. P., and C. Roth. 1968. Plasmid-linked resistance to inorganic salts in Staphylococcus aureus. J.
ANTIMICROB. AGENTS CHEMOTHER. Bacteriol. 95:1335-1342. 13. Raymond, E. A., and W. H. Traub. 1970. Identification of staphylococci isolated from clinical material. Appl. Microbiol. 19:919-922. 14. Report from an expert group. 1971. Methylmercury in fish. A toxicologic-epidemiologic evaluation of risks, p. 126. Nordisk Hygienisk Tidskrift Supplement 4, Stockholm. 15. Rheinwald, J. G., A. M. Chakrabarty, and I. C. Gunsalus. 1973. A transmissible plasmid controlling camphor oxidation in Pseudomonas putida. Proc. Natl. Acad. Sci. U.S.A. 70:885-889. 16. Richmond, M. H., and M. John. 1964. Cotransduction by a staphylococcal phage of the genes responsible for penicillinase synthesis and resistance to mercury salts. Nature (London) 202:1360-1361. 17. Richmond, M. H., M. T. Parker, M. P. Jevons, and M. John. 1964. High penicillinase production correlated with multiple antibiotic resistance in Staphylococcus aureus. Lancet 1:293-296. 18. Smith, W. H. 1969. Veterinary implications of transfer activity, p. 213-223. In G. E. W. Wolstenholme and M. O'Conner (ed.), Ciba foundation symposium on bacterial episomes and plasmids. Little, Brown and Co., Boston. 19. Watanabe, T. 1963. Infective heredity of multiple drug resistance. Bacteriol. Rev. 27:87-115. 20. Yu, L., and J. N. Baldwin. 1971. Intraspecific transduction in Staphylococcus epidermidis and interspecific transduction between Staphylococcus aureus and Staphylococcus epidermidis. Can. J. Microbiol. 17:767-773.
Vol. 7, No. 5 Printed in U.S.A.
Copyright 0 1975 American Society for Microbiology
Epidemiology of Antibiotic and Heavy Metal Resistance in Bacteria: Resistance Patterns in Staphylococci Isolated from Populations in Iraq Exposed and Not Exposed to Heavy Metals or Antibiotics DAVID J. GROVES, H. SHORT, A. J. THEWAINI,
AND
FRANK E. YOUNG*
Department of Microbiology, The University of Rochester School of Medicine and Dentistry,* Strong Memorial Hospital, Rochester, New York 14642 and Medical City Hospital, University of Baghdad, Baghdad, Iraq Received for publication 26 November 1974
Staphylococci were isolated from rural and urban populations in Iraq, which not known to be exposed to either heavy metals or antibiotics. The antibiotic and heavy metal resistance patterns of these strains were analyzed in both mannitol-fermenting and nonfermenting strains. Over 90% of the strains were resistant to at least one of the following antibiotics: penicillin, chloramphenicol, erythromycin, tetracycline, cephalothin, lincomycin, or methicillin. In general, mannitol-fermenting strains were resistant to penicillin and cupric ions. Mannitol-negative strains were more frequently associated with mercuric ion and tetracycline resistance. Although resistance to penicillin and tetracycline can coexist, the combination of penicillin resistance and tetracycline resistance usually occurred in mannitol-negative strains. The possibility of selection of heavy metal-resistant strains due to exposure to toxic levels of methylmercury was examined. No significant increase in mercuric ion-resistant strains of staphylococci or Escherichia coli were detected in exposed populations as compared to control groups. The possible reasons for this result are discussed.
were
The current interest in subtle effects of environmental contaminants on ecosystems has resulted in a renewed emphasis on the interactions of pollutants with microorganisms. As man further contaminates his own environment, he alters the milieu of those organisms for whom he is the host. For example, selection of antibiotic-resistant strains in human patients (4, 8, 19) and live stock (18) is a well recognized phenomenon. There is also evidence to indicate that there may be a correlation between the emergence of resistance to antibiotics and heavy metals. Thus the exposure of industrial workers to metallic mercury or inorganic mercury has been shown to be associated with an increased colonization by antibiotic- or mercury-resistant staphylococci in the nasal passages (7). Even in populations that had not been known to have been exposed to mercury, there is a high correlation between certain types of antibiotic resistance patterns and resistance to heavy metals. Recent studies have demonstrated an association between resistance to penicillin, copper ions, and the production of coagulase, whereas resistance to mercury was more commonly noted in strains that were resistant to tetracycline and coagulase negative (6; D. Groves and F.. Young, manuscript in preparation).
Because there was an increased incidence of intercurrent infections leading to death in patients poisoned with methylmercury at Minimata (14), it was considered particularly relevant to determine the effects of exposure to toxic amounts of heavy metals on resistance patterns in microorganisms. In the period from September 1971 to mid-January 1972, a severe epidemic of methylmercury poisoning of farmers and their families occurred in Iraq, due to the consumption of home-made bread prepared from seed grain treated with a methyl mercurial fungicide. A total of 6,530 cases were admitted to hospitals, and there were 459 hospital deaths attributed to methylmercury poisoning (3). As part of an inter-university collaboration between the University of Baghdad (Iraq) and the University of Rochester (U.S.A.) we explored the effect of methylmercury poisoning on the microbial flora of man. The results of this study indicate that over 90%7o of the strains of staphylococci isolated from rural and urban populations in Iraq, that were obtained from individuals who were not known to be exposed to either heavy metals or antibiotics, were resistant to one of the following antibiotics: penicillin, chloramphenicol, erythromycin, tetracycline, cephalothin, lincomycin, or methicillin. Al-
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HEAVY METAL RESISTANCE IN STAPHYLOCOCCI
VOL. 7, 1975
623
though associations could be made between otic resistance for urban and rural populations types of resistance patterns, there was no signif- are shown in Table 1. The urban populations icant increase in mercuric-resistant strains of were derived from patients which were hospital-
staphylococci or Escherichia coli in populations ized or visited the out-patient clinic. None of exposed to methylmercury as compared with these were known to be treated with antibiotics. The rural populations were obtained from pathe population selected as the control. tients visiting the popular clinic of the Mesaib MATERIALS AND METHODS al-Kabir Irrigation Project (columns 5 and 6) or Bacterial isolates. Strains were isolated from from rural populations in the same area (colrectal swabs and anterior nasal swabs using Fisher umns 7 and 8). In these samples there were very Handi swabs containing modified Stuart transport few strains that were susceptible to all antibiotmedium (Fisher Scientific Co., Pittsburgh, Pa.). Out- ics tested (35 out of 474). There was no signifiside the hospital supplies and specimens were trans- cant difference between mannitol-positive and ported in an ice chest at 4 to 10 C to minimize thermal mannitol-negative strains. With the exception damage. Staphylococcal strains were isolated by streaking nasal swabs on mannitol salt agar. Rectal of fewer multiply resistant strains, the patterns swabs were streaked on McConkey's agar for isolation were similar in urban and rural populations. Note that penicillin resistance occurs predomiof lactose-positive enteric organisms. Antibiotic and heavy metal resistance. The assay nantly in mannitol-positive strains. The heavy metal resistance patterns of these for susceptibility and resistance to antibiotics and heavy metals were performed as described elsewhere staphylococcal strains are shown in Table 2. (6; D. Groves and F. Young, manuscript in prepara- Resistance to metals is not as common as tion). resistance to antibiotics in the same strains. Data manipulation. Data storage, reducttn, and Although a total of only 35 strains were susceppattern recognition were carried out with a program tible to all of the antibiotics tested, 160 strains designed specifically for analysis of resistance markwere susceptible to mercuric, cupric, and caders (6). Analysis of mercury levels. Blood samples were mium ions. These susceptible strains were collected in heparinized Vacutainer tubes. The blood largely mannitol negative. All of the strains resistant only to mercuric ions were mannitol was analyzed for total and inorganic mercury by the atomic absorption method (9). nonfermentors, whereas strains resistant only to Cu2+ (or Cd2+) were predominantly able to RESULTS ferment mannitol. Even when mercury resistAntibiotic and heavy metal resistance pat- ance was present with resistance to other metterns in microorganisms isolated from indi- als, the strain was usually unable to ferment viduals who were not known to be exposed to mannitol. This association can be more clearly antibiotics or heavy metals. To study the visualized by analyzing the distribution of the effect of toxicity to mercury on the plasmids in five predominant markers: resistance to penicilmicrobes which inhabit man, we attempted to lin, resistance to tetracycline, resistance to obtain populations which were not known to be Hg2+, resistance to Cu2+, and resistance to Cd2+ exposed to mercury from rural, urban, and independent of the others (Table 3). Because of hospital environments. The patterns of antibi- the existence of multiply resistant strains, each TABLE 1. Distribution of mannitol fermentation and antibiotic resistance patterns Mannitol reaction
Rural populations
Urban populations
Antibiotic resistance pattemsa
Non-clinic
Clinic
Outpatients
Hospital
P
C
E
T
Cf
L
Dp
+
-
+
-
+
-
+
-
S R S R R R
S S S S R R
S S S S S R
S S R R R R
S S S S S S
S S S S S S
S S S S S S
5 48 2 17 5 3
7 23 5 38 38 9
5 28 1 10 3 3
7 17 1 19 16 4
3 41 0 3 0 1
6 18 1 7 3 0
0 19 0 1 0 0
2 6 1 1 0 0
14
8
8
12
1
3
0
1
Others
a Abbreviations: P, penicillin; C, chloramphenicol; E, erythromycin; T, tetracycline; Cf, cephalothin; L, lincomycin; Dp, methicillin; S, susceptible; R, resistant.
624
ANTIMICROB. AGENTS CHEMOTHER.
GROVES ET AL.
TABLE 2. Distribution of mannitol fermentation and heavy metal resistance patterns Mannitol reaction
Urban
Heavy metal resistancea
Rural populations
populations OutHosl italpatients
Hoptal
Hg2+ Cu2+ Cd2+ + S
R S S
R R S R a
S S R S
S S S R
R
S R R R
S R R
-
+
-
Clinic
Nonclinic
+
+
-
-
18 64 10 38 5 16 1 8 0 23 0 5 0 1 0 0 35 17 30 20 28 18 16 3 3 1 1 0 0 0 1 0 3 8 1 2 1 2 0 0 3 4 0 2 0 0 0 0 27 5 15 7 15 0 2 0 5 6 1 2 0 1 0 0
S, susceptible; R, resistant.
TABLE 3. Analysis of independent resistance traits in mannitol fermenting and nonfermenting strains Mannitol reaction Independent
resistancea
Urban
Rural
populations
populations
OutHospital patients +
Penicillin
.
86 115 96 41 36 38 16
Tetracycline .. 35 Mercuric ion ... 11 Cupric ion . 70 Cadmium ion
51 21 2 47 17
Clinic
Nonclinic
+
+
66 46 30 20 8 46 4 11 1 2 11 1 4 0 0 31 44 21 18 3 11 15 1 3 0
aThe strains are resistant to each agent independently. Thus the penicillin-resistant strains may or may not be resistant to any of the other agents tested.
total population is less than the sum of the values in the column above it. The strains resistant to penicillin were distributed between mannitol-positive and mannitol-negative strains in ratios very similar to those of the total populations, as in each case the penicillin resistant strains comprise the majority of the population. In strains resistant to tetracyclines or to mercury, there is a disproportionate decrease in the fraction of mannitol-positive strains. Copper and cadmium resistance, on the other hand, causes a disproportionate increase in the mannitol-positive strains. These results (Table 3) outline the general association of resistance to penicillin and copper with mannitol fermentation as opposed to the association between resistance to tetracycline, resistance to
mercury, and the lack of mannitol fermentation. Therefore, the general association between resistance markers and the mannitol reaction would appear to be the same as described for other populations (D. Groves and F. E. Young, manuscript in preparation). One might expect a three-vector comparison of antibiotic resistance, metal resistance, and mannitol fermentation to show the two predominant classes to be (i) those with the mannitolpositive trait and copper as well as penicillin resistance; and (ii) those with resistance to tetracycline, resistance to mercury, and the lack of mannitol fermentation. In Table 4 we have listed, for each population, the distribution of the penicillin, tetracycline, mercury, and copper resistance patterns in fermenting and nonfermenting organisms. We have eliminated the subsets which contained less than six organisms in any pattern. In the populations resistant to only two of the test compounds (penicillin, tetracycline, mercury, and copper), the major patterns are PenRTetRHgsCus and PenRTetsCuRHgs. Thus penicillin resistance and tetracycline resistance can coexist. The classes of microorganisms with resistance only to the following pairs of agents are seldom found: penicillin and mercury; tetracycline and mercury; tetracycline and copper; or mercury and copper. Most of the strains resistant to tetracycline are also resistant to penicillin. The strains resistant only to these two antibiotics are predominantly mannitol negative. Furthermore, strains resistant to mercuric ions are usually multiply resistant strains and predominantly mannitol negative. Effect of exposure to heavy metals or antibiotics on patterns of heavy metal resistance in microorganisms. Two populations exposed to methylmercury and one population exposed to antibiotics were studied. The first population exposed to methylmercury consisted of 40 patients, largely families with children, who were kept in Medical City Hospital for treatment and research. The specimens taken from these patients were compared with control specimens taken from approximately 400 hospital patients, a subpopulation of whom had received antibiotic therapy. The second population consisted of residents of the Mesaib-al-Kabir Irrigation Project. This particular area was severely affected by methylmercury poisoning, with approximately 800 people exhibiting clinical symptoms of mercury poisoning out of a total population of 20,000. Control populations were selected from nonexposed isolated families, and from patients attending the medical clinic at Mesaib village. Both exposed and nonexposed
HEAVY METAL RESISTANCE IN STAPHYLOCOCCI
VOL. 7, 1975
625
TABLE 4. Major patterns of resistance to antibiotics and heavy metals Mannitol reaction
T
S R S R R R R R
S S S R S R R R
Hg2+| Cu2+ S S S S S R S R
S S R S R S R R
Others
Non-clinic
Clinic
Outpatients
Hospital
P
Rural populations
Urban populations
Resistance patterna
+
-
+
-
+
-
+
3 9 3 8 42 3 16 6
5 15 2
40 8 24 12 14
2 5 3 4 26 0 15 1
4 13 4 21 7 5 16 3
2 1 1 2 40 0 2 0
3 7 4 6 11 0 3 1
0 2 0 0 17 0 1 0
1 6 1 1 1 0 0 0
4
8
2
3
1
2
0
1
aAbbreviations: Penicillin, P; tetracycline, T; susceptible, S; resistant, R. populations were validated by analysis of blood samples for total and inorganic mercury content.
TABLE 5. Mannitol-positive staphylococci isolated from patients in Medical City Hospital Metal resistance
Exposure The resistance patterns of staphylococci to patterna heavy metals and antibiotics were compared in populations of patients that were exposed to None Mercury bitic Hg2+| Cu2+ Cd2+ methylmercury and antibiotics to ascertain whether exposure influenced the types of resist11 6 18 S S S ance patterns in these strains. The distribution 0 S 0 0 R S of resistance to copper, cadmium, and mercuric 28 S 13 35 R S 4 3 ions in mannitol-fermenting strains is shown in 3 S R S 1 1 3 R S Table 5. Exposure to methylmercury did not .R 1 S 0 3 R R influence the incidence of mercury-resistant 12 2 S 27 R R strains ip the population, whereas exposure to 22 0 5 R R R antibiotics significantly increased the incidence of straing resistant to mercuric ions. This was a S, Susceptible; R, resistant. most evident by an increase in strains that were multiply resistant to mercuric, copper, and TABLE 6. Mannitol negative staphylococci isolated from patients in Medical City Hospital cadmium ions (5 and 28% for nonexposed and antibiotic exposed, respectively). The highest Metal resistance Exposure incidence of mercury resistance in staphylococci patteMa occurred in the nonexposed population (Table 6). There was no significant difference in the None Mercury biotic Hg2+ Cu2+ Cd2+ incidence of mercury-resistant strains isolated from patients that were exposed to antibiotics 48 7 64 S S S or methylmercury as compared to the control 10 S S 23 5 R population. S S 5 21 R 17 1 2 S S A similar analysis of the heavy metal resist1 R 9 8 R R 1 S ance patterns was performed on strains of S R 1 3 4 R staphylococci isolated from rural populations 1 S R 1 R 5 (Tables 7 and 8). The size of the nonexposed 6 0 R R 6 R population precludes a detailed analysis. However, summation of mannitol-positive and man5, Susceptible; R, resistant. nitol-negative staphylococci gives zero out of 29 mercury-resistant strains from the mercury This increase is not quite significant. A larger nonexposed population, compared to 12 out of control population might well still have had no 185 from the exposed population. This gives mercury-resistant strains. Forty-five straihs 1 and P 0.25 to 0.1. with no mercury resistance in the control group an x2 of 1.87 with d.f. =
=
626
ANTIMICROB. AGENTS CHEMOTrHER.
GROVES ET AL.
would have made the increase in the exposed group significant with a P = 0.1. There is a possibility that the resistance of E. coli isolates might be more affected by exposure to organic mercury, as the feces contain a large portion of the eliminated mercury, thus exposing fecal microorganisms to elevated levels. Table 9 shows the data for mercury resistance and susceptibility of the E. coli isolated from fecal samples and rectal swabs of both exposed and nonexposed populations. As observed with staphylococci there was a higher incidence of TABLE 7. Heavy metal resistance of mannitol-positive staphylococci isolated from rural subjects Metal resistance patterna
Hg2+
CU2+
S
S
S R S R S S R R S R R S R R a R, Resistant; S,
Cd2+
S S S R S R R R
susceptible.
TABLE 8. Heavy metal resistance of mannitol-negative staphylococci isolated from rural subjects Metal resistance patterna
Exposure
Hg2+
Cu2+
Cd2+
None
Mercury
S R S S R R S R
S S R S R S R R
S S S R S R R R
8 0 3 0 0 0 0 0
28 1 54 0 6 0 0 2
aR, Resistant; S,
susceptible.
TABLE 9. Effect of exposure to methylmercury on resistance of mercuric ions in strains of Escherichia coli Resistance to mercuric ion
Rb
Sb
Non-clinic Mesaib project
7 69
1 14
Hospital (no
antibiotic
exposure) 10 35
40 71
Exposure to mercury indicated by + and -. b R, Resistant; S, susceptible. a
resistance to mercury in the organisms isolated from individuals in the urban environment. Even with the small sample studied it is unlikely that poisoning with methylmercury shifted significantly the levels of resistance to mercuric ions at the time of sampling of the population.
DISCUSSION Microorganisms frequently contain extrachromosomal elements that determine resistance to toxic compounds such as antibiotics and heavy metals, or the determinants for genes that regulate complex pathways of biodegradation such as the metabolism of camphor in Pseudomonas putida (15). In staphylococci a single genetic determinant, coagulase, has been utilized traditionally to separate Staphylococcus aureus from Staphylococcus epidermidis. Other traits such as mannitol fermentation (13), phage sensitivity, and biotyping (2) have reinforced this grouping. The observation that genetic markers can be exchanged between the S. aureus and S. epidermidis (20) indicates that this grouping may be quite artificial. Nevertheless, we utilized mannitol fermentation as an additional trait in the analysis of the distribution of heavy metal resistance among staphylococci. If the possession of resistance to mercury produced a selective advantage to microorganisms inhabiting poisoned individuals, it would not be unreasonable to expect an increased incidence of mercury-resistant strains either in the staphylococci which usually inhabit the nasopharynx (S. aureus) or those which usually inhabit the skin (S. epidermidis). However, no significant increase in the incidence of mercuryresistant strains occurred in individuals that were severely poisoned with mercury. A number of explanations should be considered in evaluating this result. First, the exposure to mercury was limited in duration. Within 2 months of the onset of ingestion of contaminated bread, symptoms appeared and the treated grain was recovered. Thus the blood levels rose to a maximum, then decayed with a half-life of approximately 70 days (3). Second, there was an interval of at least 6 months between the consumption of the contaminated bread and the sampling of the population for analysis of the microbial flora. Nevertheless, the total mercury levels in the blood of exposed subjects were still elevated, with a range of 100 ng/ml to 1,350 ng/ml. Even when a limited population was studied in which the blood levels were greater than 500 ng/ml, there was no significant increase in the mercury-resistant fraction of E.
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HEAVY METAL RESISTANCE IN STAPHYLOCOCCI
coli or staphylococci. Third, the blood level values used are total mercury levels, with the inorganic mercury composing less than 10% of this value (3). Fourth, although strains of staphylococci which are resistant to mercury ions are also resistant to phenyl mercuric ions, there is no relationship to their resistance to methylmercury (G. D. Groves and F. E. Young, manuscript in preparation). Thus, poisoning with alkyl mercurial compounds may not provide selective pressure for resistance to mercuric ions even though the elevated circulating inorganic mercuric levels in the blood show that significant degradation to inorganic mercury occurs. Finally, the high frequency of antibiotic-resistant staphylococci, even among those not known to have been treated with antibiotics, would obscure subtle changes in the microbial flora. For instance, antibiotic-resistant staphylococci occurred with a frequency of greater than 0.90 with an incidence of 0.86 for penicillin resistance. The only trend that indicated a selective effect of mercury was in the rural population; however, the low numbers of isolates precluded a detailed analysis (Tables 7 and 8). In addition, the infrequent occurrence of mercuryresistant organisms in the rural environment where the original exposure to organo-mercurials occurred would result in a limited gene pool from which mercury-resistant organisms might be selected. The ideal population for study would be carriers of mercury-susceptible staphylococci and E. coli who become exposed to high levels of inorganic or organic mercury with sampling prior to, during, and after exposure. It is also necessary to have a pool of mercuryresistant strains in the environment for selection by the elevated mercury levels. Such ideal human populations are not possible, but model animal systems are under consideration. Studies are also under way to sample populations with chronic exposure to organo-mercurials, as well as chronic and transitory exposure to inorganic mercury. Many of the genes which regulate antibiotic and heavy metal resistance in staphylococci reside on plasmids. Although there appears to be no incompatibility between plasmid-borne resistance to penicillin, tetracycline, chloramphenicol, and erythromycin in S. aureus (5, 17), little is known about the interactions between markers that reside on different plasmids and how these plasmids interact to form multiple resistant strains. The most extensively studied plasmids in S. aureus are the penicillinase-containing plasmids of phage groups I and II. Resistance to heavy metals such as mercuric and cadmium ions are usually linked to penicil-
627
linase on these plasmids (11, 12, 16). Tetracycline resistance can be determined by a plasmid which is lost or transduced independently of the penicillinase-containing plasmids (1, 10). The data in this study demonstrate that patterns of antibiotic and heavy metal resistance vary between coagulase-positive or mannitol-fermenting and coagulase-negative or mannitol-noniermenting strains. For instance, resistance to penicillin and copper ions are most frequently associated with the presence of mannitol fermentation, whereas the resistance to tetracycline and mercuric ions is most frequently found in mannitol nonfermenting strains. Although it is possible to have resistance to penicillin, tetracycline, mercuric ions and copper ions in the same strain, this most frequently occurs when the organism cannot ferment mannitol. It may be of interest to note that similar association between the resistance to mercury and tetracycline in coagulase-negative strains in contrast to the resistance to penicillin and copper in coagulase-positive strains was observed in the microbiology laboratory at the University of Rochester. Detailed studies are in progress to determine whether the insights from the epidemiological survey may aid in obtaining a more detailed understanding of the incompatibility and interaction of plasmids in pathogenic organisms. ACKNOWLEDGMENTS We are grateful to the Scientific Committee on Mercury Poisoning in the Baghdad Medical College for allowing us to publish these results. The analytical data on mercury were supplied by the Mercury Research Laboratory under the direction of Hashim I. Dhahir. We wish to acknowledge P. Dhahir and M. R. Greenwood for supervising the analytical team and for data processing. The analytical determinations were made by Ilham M. Al-Jubouri, Amir Khayat, Selma M. Matook, and Mansour Al-Muntasir. We wish to also extend our thanks to Ammar Abdul Razzak and Muhammed Ali Majeed for their invaluable help in collecting the samples and in other aspects of the work. We also wish to express our appreciation to Buthaina Al-Nakash, Agnes Haik, Suhaila Saadalla, Rasoul Al-Dabagh, Hashim Al-Mosawi, and other workers in the Microbiology Laboratory in the Medical City Hospital for their indispensable help. David L. Hollis contributed to the technical aspects of the study at the University of Rochester. This research was supported in part by National Science Foundation (RANN) grant GI 30097 and in part by funds supplied by Baghdad University. We are grateful to Bioquest Division of Baltimore Biological Laboratory and Difco Laboratories for their kind donation of supplies and media. LITERATURE CITED 1. Asheshov, E. H. 1966. Chromosomal location of the genetic elements controlling penicillinase production in a strain of Staphylococcus aureus. Ntature (London) 210:804-806. 2. Baird-Parker, A. C. 1965. Staphylococci and their classification. Ann. N. Y. Acad. Sci. 128:4-25. 3. Bakir, F., S. F. Damluji, L. Amin-zaki, M. Murtadha, A.
628
4.
5. 6.
7. 8.
9. 10.
11.
12.
GROVES ET AL.
Khalidi, N. Y. Al-Rawi, S. Tikriti, H. I. Dhahir, T. W. Clarkson, J. C. Smith, and R. A. Doherty. 1973. Methylmercury poisoning in Iraq: an interuniversity report. Science 181:230-241. Bentley, D. W., J. J. Hahn, and M. H. Lepper. 1970. Transmission of chloramphenicol-resistant Staphylococcus epidermidis: epidemiologic and laboratory studies. J. Infect. Dis. 122:365-375. Dyke, K. G. H., and M. H. Richmond. 1967. Occurrence of various types of penicillinase plasmids among hospital staphylococci. J. Clin. Pathol. 20:75-79. Groves, D. J., L. Maroglio, C. W. Merriam, and F. E. Young. 1974. Epidemiology of antibiotic and heavy metal resistance in bacteria: a computer-based data system. Comput. Programs Biomed. 3:123-134. Hall, B. M. 1970. Mercury resistance of Staphylococcus aureus. J. Hyg. 68:121-129. Lepper, M. H., P. Tillman, and R. Devetsky. 1965. Patterns of transmission of staphylococci. Ann. N. Y. Acad. Sci. 128:404-427. Magos, L., and T. W. Clarkson. 1972. Atomic-absorption determination of total, inorganic, and organic mercury in blood. J. Assoc. Off. Anal. Chem. 55:966-971. May, J. W., R. H. Houghton, and C. J. Perret. 1964. The effect of growth at elevated temperatures on some heritable properties of Staphylococcus aureus. J. Gen. Microbiol. 37:157-169. Moore, B. 1960. A new screen test and selective medium for the rapid detection of epidemic strains of Staphylococcus aureus. Lancet 2:453-458. Novick, R. P., and C. Roth. 1968. Plasmid-linked resistance to inorganic salts in Staphylococcus aureus. J.
ANTIMICROB. AGENTS CHEMOTHER. Bacteriol. 95:1335-1342. 13. Raymond, E. A., and W. H. Traub. 1970. Identification of staphylococci isolated from clinical material. Appl. Microbiol. 19:919-922. 14. Report from an expert group. 1971. Methylmercury in fish. A toxicologic-epidemiologic evaluation of risks, p. 126. Nordisk Hygienisk Tidskrift Supplement 4, Stockholm. 15. Rheinwald, J. G., A. M. Chakrabarty, and I. C. Gunsalus. 1973. A transmissible plasmid controlling camphor oxidation in Pseudomonas putida. Proc. Natl. Acad. Sci. U.S.A. 70:885-889. 16. Richmond, M. H., and M. John. 1964. Cotransduction by a staphylococcal phage of the genes responsible for penicillinase synthesis and resistance to mercury salts. Nature (London) 202:1360-1361. 17. Richmond, M. H., M. T. Parker, M. P. Jevons, and M. John. 1964. High penicillinase production correlated with multiple antibiotic resistance in Staphylococcus aureus. Lancet 1:293-296. 18. Smith, W. H. 1969. Veterinary implications of transfer activity, p. 213-223. In G. E. W. Wolstenholme and M. O'Conner (ed.), Ciba foundation symposium on bacterial episomes and plasmids. Little, Brown and Co., Boston. 19. Watanabe, T. 1963. Infective heredity of multiple drug resistance. Bacteriol. Rev. 27:87-115. 20. Yu, L., and J. N. Baldwin. 1971. Intraspecific transduction in Staphylococcus epidermidis and interspecific transduction between Staphylococcus aureus and Staphylococcus epidermidis. Can. J. Microbiol. 17:767-773.
