JOURNAL OF BACTERIOLOGY, Oct. 1968, p.

1441-1442
Copyright © 1968 American Society for Microbiology
3-Lactamase of R Factors
JACQUELINE EVANS, E. GALINDO, J. OLARTE, AND STANLEY FALKOW
Department ofMicrobiology, Georgetown University Schools of Medicine and Dentist
ry, Washington, D.C. 20007,
and Laboratoria de Bacteriologia Intestinal, Hospital Infantil de Mexico, Mexico
7, D. F.
Received for publication 12 June 1968
The widespread use of antibiotics in Mexico
City has been associated with the appearance of
a large proportion of drug-resistant enteric species
(5). It did not seem surprising, therefore, that of
13 different multiple-drug-resistant strains of
enteropathogenic Escherichia coli and Shigella
serotypes isolated from 1956 to 1959, 8 were found
to harbor resistance transfer factors (R factors;
11). The R factors were primarily of the fi+
genotype (12) and mediated resistance to three
or more of the following: sulfonamides (SU),
tetracycline (TC), streptomycin (SM), chloramphenicol
(CM), kanamycin (KM), and
ampicillin (AM). Two R factors conferring
centrifugation, washing in 0.1 M P04 buffer (pH
7.2) and disrupting the cells in a Branson sonic
oscillator. After centrifugation at 30,000 x g for
20 min, the supernatant fluids were employed
directly without further purification. It has been
our experience that the commonly employed
iodometric assay for ,B-lactamase (6) is not suitable
for use with the ,B-lactamases of enteric
bacteria because the enzyme is markedly inhibited
by iodine. Consequently, we employed
the acidimetric method (8) and a spectrophotometric
assay (4).
The R-associated ,B-lactamase in W1485(R7)
and in W1485 (RI6) has a rather unusual sub-
TABLE 1. Relative rates of hydrolysis of various substrates by ,3-lactamase
Enzyme aScpteivciitfyi"c' Benzylb Pmheetnhoyxly- Ampicillin 6-APAe Cloxacillin M
ethicillin
B. cereus 2.02 1.00 1.29 1.22 0.07 0.01 0.12
S. aureus 0.918 1.00 0.60 1.12 0.06 0.01 0.01
E. coli r' 0.329 1.00 0.47 1.09 0.94 0.05 0.01
E. coli r7 0.020 1.00 3.04 2.74 1.35 0.22 0.79
E. coli r"l 0.024 1.00 5.30 3.75 2.35 0.31 0.84
a Units per milligram of crude extract of protein
I Rate of hydrolysis of benzylpenicillin = 1.00.
c 6-Aminopenicillanic acid.
resistance to AM were identified in strains of E.
coli isolated in 1956: R7-SU, SM, TC, AM
carried by strain 086:B7, and RI6-SU, SM, TC,
CM, AM carried by strain 0126:B16. The
isolation of these strains seemed unusual, since
R-associated AM resistance was not reported
until late in 1962 (1) nor was ampicillin (first
synthesized in 1961) employed in Mexico prior
to 1962. Datta and Richmond (2) reported that
resistance to ampicillin is associated with a /-
lactamase and we were prompted to examine the
mechanism of resistance in our early isolates.
For comparison, we included another R factor
(R2-SU, SM, TC, CM, AM) known to carry a
,B-lactamase (3) similar to that described by
Datta and Richmond.
Each of the R factors was transferred to the
E. coli K-12 strain W1485F- to provide an
identical genetic background. Enzyme samples
were prepared from nutrient broth cultures by
on benzylpenicillin.
strate profile (Table 1). The enzyme from the E.
coli strains isolated in 1956 hydrolyzes semisynthetic
penicillins such as phenoxymethylpenicillin
(penicillin V) and a-aminobenzylpenicillin
(ampicillin) at a rate three to five
times faster than benzylpenicillin (penicillin G).
The enzyme produced by W1485 (R2), like that
reported by Datta and Richmond (2), has almost
equal activity on penicillin G and ampicillin and
a reduced rate on penicillin V. Compared to R2,
the f-lactamase of R7 and RI6 is also capable of
hydrolyzing significantly more cloxacillin [5-
methyl-3-(o-chlorophenyl)-4-isoxazolyl penicillin],
6-aminopenicillanic acid, and methicillin
(dimethoxyphenyl penicillin). The ,B-lactamase of
Bacillus cereus 569 and Staphylococcus aureus
was included and gives results comparable to
those reported by Citri and Pollock (7).
Both types of R-associated ,B-lactamase were
constitutive and not apparently inducible, nor
1441
Vol. 96, No 4
Printed in UI.S.A.
Downloaded from jb.asm.org by on April 4, 2010
J. BACTERIOL.
was there any evidence of concomitant Rassociated
penicillin amidase activity. The very
low specific activity of R7- and R1I-penicillinase
was not changed by varying growth conditions,
or medium, or by continued growth in the presence
of ampicillin. Subsequent studies have shown
that the f3-lactamase associated with R7 and RI6
is not unique but rather is found frequently in
R factors isolated in a wide variety of strains
from many locales. It constitutes, therefore, a
second general class of R-factor associated
/3-lactamase.
The finding that a 3-lactamase with a high
degree of specificity for semisynthetic penicillins
was already present in 1956 suggests that it was
previously evolved to fit a naturally occurring
substrate. The nature of such a substratewhether
an antibiotic or natural cell constituentis
not clear. These data complement the recent
findings of Smith (9, 10) that suggest that R
factors are not a recent phenomenon and that
drug use and the selection of R-factor genes need
not be as direct as originally presumed.
This study was supported by contract C-7061 from
the U.S. Army Research and Development Command.
LITERATURE CITED
1. Anderson, E. S., and N. Datta. 1965. Resistance
to penicillins and its transfer in Enterobacteriaceae.
Lancet. 1:407-409.
2. Datta, N., and M. Richmond. 1966. The purification
and properties of a penicillinase whose synthesis
is mediated by an R-factor in Escherichia
coli. Biochem. J. 98:204-209.
3. Farrar, W. E., Jr., and L. Dekle. 1967. Transferable
antibiotic resistance associated with an outbreak
of Shigellosis. Ann. Int. Med. 67:1208-1215.
4. Jansson, J. A. T. 1965. A direct spectrophotometric
assay for penicillin ,B-lactamase (penicillinase).
Biochim. Biophys. Acta. 99:171-172.
5. Olarte, J., E. Galindo, and A. Joachin. 1963.
Sensitivity of Salmonella, Shigella, and enteropathogenic
Escherichia coli species to
cephalothin, ampicillin, chloramphenicol, and
tetracycline. Antimicrobial Agents and Chemotherapy-
1962, p. 787-793.
6. Perret, C. J. 1954. lodometric assay of penicillinase.
Nature 174:1012-1013.
7. Citri, N., and Pollock, M. R. 1966. The biochemistry
and function of ,-lactamase (penicillinase).
Advan. Enzymol. 28:237-323.
8. Saz, A. K., D. L. Lowery, and L. J. Jackson.
1961. Staphylococcal penicillinase. I. Inhibition
and stimulation of activity. J. Bacteriol.
82:298-304.
9. Smith, D. H. 1967. R-factor mediate resistance to
mercury, nickel, and cobalt. Science 156:1114-
1116.
10. Smith, D. H. 1967. R factor infection of
Escherichia coli lyophilized in 1946. J. Bacteriol.
94:2071-2072.
11. Watanabe, T. 1963. Infective heredity of multiple
drug resistance in bacteria. Bacteriol. Rev.
27:87-115.
12. Watanabe, T., H. Nishida, C. Ogata, T. Arai, and
S. Sato. 1964. Episome-mediated transfer of
drug resistance in enterobacteriaceae. VII.
Two types of naturally occurring R factors. J.
Bacteriol. 88:716-726.
1442 NOTES
Downloaded from jb.asm.org by on April 4, 2010