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The Isolation of Certain Types of Micrococci from
Dental Students and Related Antibiotic Studies
IVAN GARDNER and DONALD E. SHAY
Department of Microbiology, Baltimore College of Dental Surgery, University of
Maryland, Baltimore
The recent rise in the incidence of antibiotic-resistant strains of pathogenic micrococci
and in the percentage of surgical postoperative infections attributable to these organisms
suggests the need for a specific investigation of this field as it relates to dentistry.'-6
Many implications have arisen in considering the treatment of postoperative micrococcal
oral infections resulting from dental surgery. With reports from Benham et al.
and Landy et al., the epidemiological concept of oral infections has changed greatly;
the thought that the mucosal membranes of the surgeon himself might be the source
of the infective agent had not been widespread.7'8
No work has been reported interrelating the dentist and patient to the transfer of
etiologic agents in postoperative infections. Considering the proximity of dentist and
patient in the normal course of operative and surgical proceedings, an investigation
into this area should prove most valuable in determining the course of such microbiologic
transfer and resulting infections.
Basing their work on preliminary studies, the authors have selected the following
objectives for consideration: (1) the incidence of micrococcal organisms in the nasal
mucosa, with special emphasis on those pathogens demonstrating hemolytic and serum
coagulase activity; (2) a study of antibiotic-resistance patterns of isolates; and (3)
studies of acquired resistance. Experimental results obtained by utilizing these objectives
as a guide should provide some insight into the problem of postoperative infection.
HISTORICAL REVIEW
Since the initiation of antibiotic therapy subsequent to the discovery of penicillin,
micrococcal resistance patterns have changed markedly and swiftly. Increased antibiotic
resistance of all micrococci has presented a serious surgical problem to the
general surgeon and dental surgeon alike.
The penicillin-resistant pathogenic micrococci encountered in hospital laboratories
have mounted steadily since 1943. Spink reported only 12 per cent of all cultures encountered
as resistant9 Gallardo confirmed this.10 Blair and Barber and Dorozenko
reported steadily increasing percentages of penicillin-resistant strains of 21.5, 41.0,
and 59, respectively, for the years 1945, 1946, and 1948.1" 12 More recently Nichols
and Needham in 1949 and 1953, Biegelman and Rantz in 1950, and Tamblyn and
This investigation was supported by Grant D-765 from the N.I.H., U. S. Public Health Service.
Received for publication July 20, 1959; revised by authors October 2, 1959.
99
100 GARDNER AND SHAY J. D. Res. January-February 1960
Moyer in 1954 reported, respectively, 68, 56, and 82 per cent of hospital isolates as
being penicillin-resistant in the years of publication listed.6' 13-15
Based on the severe problem presented by postoperative micrococcal infection,
Benham et al. studied antibiotic sensitivity patterns of Micrococcus pyogenes from
infected surgical wounds and compared these with patterns of those organisms isolated
from the respiratory tracts of surgery personnel.7 In vitro sensitivity assays of penicillin,
chlortetracycline, oxytetracycline, chloramphenicol, bacitracin, and streptomycin
were studied. Of the strains isolated from surgical wounds following treatment with
penicillin and streptomycin, 94 per cent were resistant to penicillin, and 79 per cent
were resistant to streptomycin in vitro. Of the strains isolated from surgery personnel,
19 per cent were sensitive to penicillin and 81 per cent resistant. The predominant
pattern among strains isolated from wounds indicated resistance to penicillin, streptomycin,
chlortetracycline, oxytetracycline, and tetracycline. The general pattern (36
per cent) among surgery personnel was one of resistance to penicillin only. Where
infection of surgical wounds with M. pyogenes occurred, a definite correlation between
the sensitivity of the organisms recovered from such wounds and that of those from
respiratory tracts of surgery personnel was indicated.
Further investigations in the field, opened by the work of Benham et al.,7 led researchers
at the University of Chicago under Landy to study surgical wound infections
in seven clinical cases occurring within 13 days.8 These micrococcal infections were
traced to contamination by organisms from the nasal mucosa of the first assistant
surgeon. The etiologic agent was described as "positive, plasma clumping" M. pyogenes
var. aureus. This organism displayed the following antibiotic sensitivity pattern: resistant
to penicillin, streptomycin, aureomycin, terramycin, and tetracycline; sensitive
to chloramephenicol, bacitracin, erythromycin, and magnamycin. All seven wounds
demonstrated the same organism with identical sensitivity patterns. Direct correlation
of the antibiotic tube-dilution assay with the impregnated-disk method was definitely
established through comparative studies. Control cultures of the nasal mucosa of patients,
surgeon, and second assistant disclosed no pathogenic micrococci. Proof of
patient-operator transfer of etiologic agent was demonstrated by the occurrence of
fewer infections when the first assistant surgeon donned two masks and extended the
scrubbing time. However, this first assistant retained identically resistant micrococci,
described above, as normal nasal flora for 2 years. This work established the pattern
for the etiology and epidemiology of many postoperative infections.
Numerous and conflicting results have been reported concerning the resistance of
various organisms toward many of the antibiotics. Baron reported no increased resistance
by M. pyogenes var. aureus toward aureomycin when cultured with increasing
concentrations of the antibiotic. Lawrence and Francis have described the development
of drug resistance to aureomycin to be "infrequent and generally of a low degree.'
7 Pratt and Dufrenoy also report the development of bacterial resistance to
erythromycin.'8 Lubash, Van der Meulen, Berntsen, and Tompsett have reported on
the production of a strain of M. pyogenes var. aureus resistant to novobiocin.19 In a
similar manner Jones, Nichols, and Finland have noted that "the rate of development
of resistance in strains of M. aureus during repeated subcultures on antibiotic containing
agar was rapid."720
Basic knowledge, provided by a variety of investigators, as to the swift increases in
Vol. 39, No. 1 ISOLATION OF MICROCOCCI FROM DENTAL STUDENTS 101
antibiotic-resistant pathogenic micrococci over a period of years, coupled with recent
correlation of surgeon-to-patient transmission of etiologic agents, has indicated the
importance of a greater knowledge of the distribution and activity of micrococcal flora
of the dentist, dental surgeon, and patient. The proximity of dentist and patient in
operative procedures has prompted the desire for further investigation into the pathogenic
micrococcal flora of the nasal mucosa of dental students, in an attempt to survey
both the distribution of the organisms mentioned above and the antibiotic sensitivity
patterns of such organisms.
A discrete knowledge of the pathogenic micrococcal population of the nasal mucosa
of dental students and antibiotic resistance patterns of this group may further aid the
application of postoperative therapy. Clarification of dentist-patient transfer of an
etiologic agent may result from studies directed along these lines. Investigations into
the antibiotic resistance patterns and resistance acquisition, as modified by mutational
variation, are reported with the hope of providing a better understanding of the phenomenon
and treatment of postoperative infection.
MATERIALS AND METHODS
Cultures of micrococci were isolated from nasal swabbings of 347 dental students
and were maintained on brain-heart infusion agar slants. All cultures were collected
during the months of March and December.
All media and glassware, except the dehydrated human plasma (supplied sterile),
were sterilized by autoclaving; antimicrobial "sensi-discs"* for sensitivity tests were
supplied sterile, and whole human blood was supplied from a hospital blood bank.
Specimens were obtained by introducing sterile cotton swabs, moistened with 0.85
NaCl solution, I inch into the inferior meatus of the nose. The specimens were streaked
directly onto blood agar and incubated at 370 C. for 24 hours. Micrococcal strains
showing beta hemolysis were transferred to brain-heart infusion agar slants for subsequent
analysis. Coagulase activity tests were performed using desiccated plasma.
Only organisms demonstrating beta hemolytic action and coagulase activity were considered
as pathogens.22'23 As an adjunct for further separation of pathogenic from
non-pathogenic micrococci mannitol fermentation tests were performed on the isolates.
24
Sensitivity tests were performed on trypticase agar, utilizing the "sensi-disc" technique.
Two concentrations of each antibiotic were used. No attempt to correlate zone
size of different antibiotics was attempted, owing to unknown variation in the rates of
diffusion and efficiency of antibiotics in vitro.25
To determine the rapidity with which these micrococcal isolates become drug tolerant,
certain isolates were selected to be subjected to an environment containing
increasing concentrations of certain antibiotics. Nine isolates were selected on the basis
of a marked susceptibility toward aureomycin, erythromycin, and novobiocin. Seven
of the nine strains were pathogenic is determined by their ability to ferment mannitol,
clot normal human plasma, and produce zones of hemolysis on blood agar plates. The
remaining tow strains were non-pathogenic.
A tube-culture titration was performed to ascertain the baseline tolerance of each
organism toward each antibiotic. By consecutive culturing of the organisms in increas-
* Sensi-disc," registered trade mark, Baltimore Biological Laboratory, Baltimore, Maryland
102 GARDNER AND SHAY J. D. Res. January-February 1960
ing concentrations of each antibiotic, an attempt was made to raise the level of tolerance
of each organism to the individual antibiotics. Ten consecutive transfers were
made over a period of 12 calendar days. The incubation increments ranged from 24
to 72 hours, depending upon the time required to produce visible turbidity in the
culture tube.
Crystalline aureomycin HCl,* erythromycin glucoheptonatet and novobiocin,4 were
the antibiotics utilized.
The tube-dilution method was utilized to obtain the desired concentrations of the
antibiotics.
RESULTS AND DISCUSSION
Patient history.-A survey of the patient's general health was conducted, primarily
to determine the therapeutic usage of antibiotics by the patient in recent years.
Besides revealing factors influencing the sensitivities of the nasal flora of the individual,
the history also provides basic information concerning the patient's general health
and past micrococcal infections. The completed chart gives a concise record of the
historical and experimental data. Nasal swabs were taken from 347 dental students.
The incidence of micrococci isolated from such swabbings was 98 per cent.
The incidence of pathogenic micrococci for the four groups was 23.5 per cent. The
incidence per group varied from 12 to 34 per cent.
No apparent pattern was established for numbers of non-pathogenic or pathogenic
micrococci between the various clinical and preclinical groups.
The majority of microorganisms recovered were of the M. pyogenes var. albus type,
while the majority of pathogens recovered were of the M. pyogenes var. aureus type.
It was routine to isolate two or more different morphologic and hemolytic types of
micrococci from a single swabbing (Table 1).
Antibiotic resistance patterns.-Organisms when subjected to the activity of aureomycin,
carbomycin, chloromycetin, neomycin, streptomycin, matromycin, novobiocin,
terramycin, tetracycline, and erythromycin demonstrated little or no resistance to the
concentrations utilized. Strains tested in vitro against polymyxin showed a general
resistance to the antibiotic. Only in the case of penicillin was a definite pattern established.
Resistance was apparent in approximately 33 per cent of all strains tested.
Penicillin-susceptible strains showed extreme sensitivity to high and low concentrations
of the drug.
Four M. pyogenes var. aureus strains that demonstrated no coagulase activity were
selected for sensitivity studies, in speculation that some detectable relationship between
coagulase activity and antibiotic resistance might be demonstrated. Results
indicated that the general pattern of the coagulase-negative strains shows a greater
degree of resistance to many of the antibiotics than those patterns of the coagulasepositive
strains.
Acquired resistance studies.-Table 2 discloses the variations in antibiotic tolerance.
It is interesting to note that there is no apparent difference between the non-pathogens
(organisms H and I) and the pathogens in regard to their ability to become drugtolerant.
* Lederle Laboratories Division, American Cyanamid Company.
t Eli Lilly and Company. $ The Upjohn Company.
Vol. 39, No. 1 ISOLATION OF MICROCOCCI FROM DENTAL STUDENTS 103
The experimental results demonstrate, to varying degrees, the ability of each of
these nine isolates to become more tolerant toward certain antibiotics. The ratio of
initial to final concentration varied from 2 to 82 with aureomycin, 3 to 115 with novobiocin,
and 0 to 240 with erythromycin. The figures thus obtained with aureomycin
are inconsistent with those reported by Baron, who found no increased resistance in
the ratio of initial to final concentration of aureomycin after fourteen transfers on
media containing increasing concentrations of aureomycin.'6 These data, however, are
in accord with work reported by Lubash, Van der Meulen, Bernsten, and Tompsett,
as well as by Jones, Nichols, and Finland.'9'20
The results obtained from this study serve to illustrate that the acquisition of drug
TABLE 1
HEMOLYTIC CHARACTER, COAGULASE ACTIVITY, AND ACTION IN 5 PER CENT
MANNITOL BROTH OF MICROCOCCAL ISOLATES
GROUP
DESCRIPTION
See Chart in complete study
* Minimum and maximum figures represent micrograms of antibiotic per
t Fold-increase figures are approximated.
104 GARDNER AND SHAY J. D. Res. January-February 1960
tolerance by micrococci is fairly rapid and that the levels of tolerance achieved, within
the limits of this experiment, can be multifold.
SUMMARY AND CONCLUSIONS
An investigation into the micrococcal flora of the nasal mucosa of 347 dental students
is presented.
No significant pattern was established in the recovery of pathogenic micrococci
isolated from preclinical and clinical students. The percentage recovery of micrococci
in all groups approached 100, while 23.5 per cent of this total represented pathogens.
All pathogenic isolates were subjected to sensitivity testing with a variety of proprietary
antibiotics. The organisms demonstrated little or no resistance to the majority
of drugs tested. There was a general pattern of resistance to the action of polymyxin.
Only extremes were noted with penicillin-organisms being generally sensitive or
resistant to the concentrations of antibiotic utilized.
The possible interrelationships of acquired antibiotic resistance and the importance
of the determination of micrococcal populations in the treatment of postoperative infection
are presented. Multifold increases in antibiotic tolerance and the rapid rate
of the acquisition of such tolerances are reported for selected micrococci.
The import of greater understanding of the phenomenon of postoperative infection
as related to dentist-patient proximity under operative and surgical technics is implicated.
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