REVIEWS OF INFECTIOUS DISEASES. VOL. 1, NO.5 .• SEPTEMBER-OCTOBER 1979 © 1979 by The University of Chicago. 0162-0886/79/0105-0006$00.81
Effect of Serum and Blood on Enterobacteriaceae Grown in the Presence of Subminimal Inhibitory Concentrations of Ampicillin and Mecillinam Victor Lorian and Barbara A. Atkinson
From the Section of Microbiology, Department of Pathology, The Bronx-Lebanon Hospital Center; and the Department of Laboratory Medicine, Albert Einstein College of Medicine, Bronx, New York
It was of interest to know whether the filamentous or round cells of Enterobacteriaceae that formed in the presence of sub-MICs of f3-lactam antibiotics differ from bacteria not exposed to these drugs in their susceptibility to the bactericidal effect of serum and to phagocytosis.
Gram-negative bacilli exposed to low concentrations of penicillin [1-3] and of some other agents [4-6] elongate and eventually become long filaments. However, exposure to three antibacterial agents-mecillinam [7-9], 6-aminopenicillanic acid [8-11], or fosfomycin [12, B]-resulted predominantly in the formation of oval and round cells. Some bacteria exposed to subminimal inhibitory concentrations (sub-MICs) of antibiotics have shown increased vulnerability to immunodefense mechanisms. Serum in combination with antibiotics is known to be bactericidal for some species of Enterobacteriaceae [14, 15]. Polymyxin sensitized Proteus mirabilis to the bactericidal effect of serum [16], as did streptomycin and tetracycline with Escherichia coli [17]. Staphylococcus aureus grown in the presence of sub-MICs of nafcillin showed an increased susceptibility to phagocytosis [19].
Materials and Methods
Strains. The following strains of Enterobacteriaceae were tested for their susceptibility to the bactericidal effect of serum and blood: four strains of E. coli, one serum sensitive (KI2), one serum resistant (10), and two moderately sensitive to serum (14, Elliott); two strains of P. mirabilis (6, 7), and one strain each of Serratia marcescens, Salmonella group B, Salmonella typhimurium (710), and Klebsiella pneumoniae. Antibiotics. Ampicillin (Ayerst Laboratories, New York, N.Y.) was used to induce the formation of filamentous bacteria (figure 1, bottom left). Mecillinam (Hoffman-LaRoche, Nutley, N.J.) was used to induce the formation of oval and round bacteria (figure 1, right). Preparation of serum and blood. Blood was drawn by venipuncture with a 20-ml plastic syringe from five healthy volunteers who claimed
This research was funded by a grant from HoffmanLaRoche, Nutley, N. J. Please address requests for reprints to Dr. Victor Lorian, Division of Microbiology and Epidemiology, The BronxLebanon Hospital Center, Albert Einstein College of Medicine, 1276 Fulton Avenue, Bronx, New York 10456.
797
Downloaded from http://cid.oxfordjournals.org/ at University of Glasgow on June 28, 2015
Bacteria from strains of five species of Enterobacteriaceae grown in the presence of subminimal inhibitory concentrations of ampicillin or mecillinam formed into filamelltous or round cells. These filamentous and round cells as well as normal control bacteria were incubated with either fresh human serum or blood, the bac!er~cidaleff~(;t.s of which were then determined. In most. c~ses, the bactericidal effect of either serum or blood on filamentous or round cells was },ess that} the effect on control cells. In some cases, the effect on thes~ drug-exposed cells was similar to that on control cells, but in no instance was the effect greater for the drug-expo~ed cells than for the control cells. However, in all cases in which the bactericidal effect of either serum or blood on the control cells was ~996Jo, the bactericidal effect on the drug-exposed cells was close to 900(0. Although drug-exposed cells were not much more resistant to the bactericidal effect of serum or blood than were normal bacteria, they clearly were not more susceptible to these effects.
798
Lorian and Atkinson
not to be taking any medication. A 10-ml volume was shaken for a few minutes with sterile glass beads in a sterile 50-ml flask. Another 5 ml of the blood was placed in a sterile glass tube, allowed to clot for 1 hr, and centrifuged at 150..g for 10 min. The serum was then re'moved and diluted 1:5 with Hanks' balanced salt solution (HijSS) plus 0.1 % gelatin (Hanks' gel) (both from Difco, Detroit, Mich.). From two to 10 sets of serum and blood were used to test the sensitivity of each strain. Determination of MICs and sub-MICs to be used in preparing test organisms. The MIC for each strain of bacteria was determined by a twofold agar dilution method [20]. The morphology of each strain exposed to antibiotic at the MIC and at dilutions down to 1/128 of the MIC was observed by interference phase contrast microscopy. The concentration of antibiotic that induced the greatest change in cell morphology without de-
stroying the cell (i.e., filaments 10-40 /Am long, or oval or round cells 4-12/Am in diameter) was used fOIJh~Jest. Table 1 shows the MICs for ampicillin and mecillinam for each strain of bacteria tested. jJreparation of test organisms. Filter membranes with a pore size of 0.3 /Am (no. PHW047S0; Millipore, Bedford, Mass.) were placed on drug-free trypticase soy agar (TSA) and warmed for 1 hr at 37 C before inoculation with 0.1 ml of a 1: 10 dilution of a culture of the organism to be tested that had been grown 20 hr in trypticase soy broth (TSB). The inoculated membranes were incubated for 90 min at 37,C, transferred either to TSA containing the antibiotic at one-half to one-eighth the MIC or to drug-free TSA, and then incubated for an additional 3.5 hr. The control organisms, which were grown on drug-free TSA (six membranes) were eluted with 15 ml of Hanks' gel; the organisms grown on antibiotic-
Downloaded from http://cid.oxfordjournals.org/ at University of Glasgow on June 28, 2015
Figure 1. Normal, untreated cells of Proteus mirabilis (top, left), filaments of P. mirabilis grown in ampicillin at one-fourth the MIC (bottom, le!f), and oval and round cells of P. mirabilis grown in mecillinam at one-fourth the MIC (right). Cell morphology was observed in each case by interference phase contrast microscopy at a magnification of x2,500.
Effect of Serum and Blood on Enterobacteria
799
+
Table 1. The MIC of ampicillin and mecillinam for all strains of Enterobacteriaceae tested. MIC of
ampicillin (I-lg/ml)
mecillinam (I-lg/ml)
6.3 4.0 5 4,000
0.4 0.25 0.19 7.8
Salmonella typhimurium 710 Salmonella group B
2.5 1.0
0.8 0.8
Serratia marcescens
50
200
Klebsiella pneumoniae 10
3.0
0.78
Proteus mirabilis 6 Proteus mirabilis 7
3.0 2.0
50 50
Organism
0
A
~
M
U Q)
Escherichia coli Escherichia coli Escherichia coli Escherichia coli
K12
10
14 Elliott
....... .......
~ ~
90
~\ \\
C "'0
.u
\\
~
Q) ~
U C
99
\\ \\
en
\\ \\ \\
0~
99.9
\ containing TSA (40 membranes) were eluted in 50ml plastic screw-capped tubes (Falcon Plastics, Oxnard, Calif.) with 15 ml of Hanks' gel containing the same concentration of antibiotic as was present on the TSA during incubation. The suspensions were shaken, and the volume of each was adjusted to give the approximate turbidity of the 0.5 McFarland standard (5 x 107 organisms/ml). These bacterial suspensions were used for the studies with serum or blood described below. This technique has been described in detail previously [8].
Test procedure. One 15-ml tube contained 0.1 ml of bacteria grown on drug-free TSA combined with 1.0 ml of Hanks' gel (control normal bac-
1/ 2 Figure 2.
The
1/ 2
Hours
bactericidal
effect
of serum on
Escherichia coli K12 exposed to one-half the MIC of ampicillin (A) and to one-half the MIC of mecillinam (M). - - - = control organisms; - - = drugexposed organisms.
teria). A pair of tubes contained 0.1 ml of normal bacteria, 0.1 ml of Hanks' gel, and either 0.9 ml of fresh or heat-inactivated serum diluted 1:5 in Hanks' gel or 0.9 ml of defibrinated blood. Another set of tubes contained 0.1 ml of the suspension of bacteria grown on antibiotic-contain-
+ o
u
u
~
a>
Q)
......
a>
90
"0 '1:l
'1:l
U
U
~
~
Q)
Q)
U
90
0
99
U 99
0
0
CD 0~
CD 0~
99.9
99.9
1/2
2
Hours
3
1/ 2
2
3
Hours
Figure 3. The bactericidal effect of serum on Escherichia coli 14 exposed to one-fourth the MIC of ampicillin (left) and to one-fourth the MIC of mecillinam (right) . . - - -e = control organisms; • • = drug-exposed organisms.
Downloaded from http://cid.oxfordjournals.org/ at University of Glasgow on June 28, 2015
MIC of
800
Lorian and Atkinson
+
o
---
....
a
90
-= u
Q)
Q)
+
b
0
o
"0
t-
(1)
u
0
o
u
~
't
90
CO
+
~
0
o
c
90
0 "0
u ~
Q)
90
U 99 0
m ~ 0
99.9 2
I
3
2
Hours
Hours
Figure 4. The bactericidal effect of serum on Escherichia coli 10 exposed to ampicillin at one-fourth the MIC (left) and to mecillinam (right) at the MIC (a), at one-half the MIC (b), and at one-fourth the MIC (c). e- - -e = control organisms; • • = drug-exposed organisms.
ing TSA plus 0.1 ml of antibiotic (at the concentration used to produce the filaments or round forms), and either 0.9 ml of Hanks' gel (antibiotic control), 0.9 ml of serum diluted 1:5 with Hanks' gel, or 0.9 ml of defibrinated blood. The bactericidal activity of the serum or blood was determined by mixing the suspensions on a vortex mixer and incubating the tubes at 37 C on a multipurpose rotator (Scientific Products, Chamblee, Ga.) at 20 rpm. After incubation for 0, 0.5, I, 2, and 3 hr, O.I-ml aliquots were removed from the suspensions and placed in 9.9 ml of saline at 10 C (to prevent growth). The number of cfu in the suspension was determined by plating 0.2 ml of 10-fold serial dilutions of the suspension on TSA plates and then incubating the plates. for 24 hr. The morphology of the bacteria eluted from the membranes as well as those in the suspensions
+ 0
u
~ Qj
90
0 "0 U ~
Q)
U 99 0
m 0~
99.9 112
2
3
Hours
Figure 5. The bactericidal effect of serum on Serratia marcescens exposed to mecillinam at one-fourth the MIC. e- - -e = control organisms; • • = drug-exposed organisms.
Downloaded from http://cid.oxfordjournals.org/ at University of Glasgow on June 28, 2015
U
+
Effect ofSerum and Blood on Enterobacteria
801
+ 0
-
+
(J
-
am
Cl)
0
1/4 M1C
'+Cl)
U
~
't
c
90
"'0
90
u
"0
~
"0
Cl)
u
~
U 99
C (l)
CO
0~
Q)
0
1/2 MIC
+
0 ~iiiiiiFiil~C===f==:::::::'----e 1/4 MIC ---------
90
99.9
Y2
2
1
3
2
Hours
Hours
Figure 6. The bactericidal effect of serum on Proteus mirabilis 7. The graph at left shows P. mirabilis exposed to ampicillin at one-fourth the MIC and incubated with 10 sera. The graph at right shows P. mirabilis exposed to various concentrations of ampicillin (am) and of mecillinam (mec) and incubated with serum. __ - - . =. control organisms; • • = drug-exposed organisms.
used for testing were studied by interference phase contrast microscopy during each test. The percentage of antibiotic-exposed bacteria (filaments or oval and round cells) that were killed by either serum or blood was calculated by dividing the number of cfu present in the suspension after exposure to serum or blood by the number of cfu present in the antibiotic control suspension (incubated in HBSS for the appropriate length of time).
in which drug-exposed organisms were slightly more sensitive to serum than were control organisms was in E. coli 14 that was exposed to mecillinam (figure 3, right). Figure 4 (left) shows the effect of serum on E. coli 10 (serum resistant). Whereas the effect of serum on the control bacteria of this strain was small to minimal, depending on the sample of serum used, the filaments of E. coli 10 that re-
Results
With the exception of E. coli K12 (serum sensitive), E. coli 14 and E. coli Elliott (both moderately sensitive to serum), and S. marcescens, all the species and strains tested showed little sensitivity to serum as either normal organisms, filaments, or oval and round cells. Figure 2 shows the effect of serum on E. coli K12 exposed to ampicillin or to mecillinam. In 99070 bactericidal effect both on control organisms and on drug-exposed organisms. Serum also had a >99.9% bactericidal effect on the control cells of E. coli 14 (moderately sensitive to serum), but after 2 hr, and especially after 3 hr, of incubation in serum, the filaments of this strain showed a small but increasing number of live organisms (figure 3, left). The only instance
a ----~:::-
-
o 90 "0
:~ .... Q.l
o
CD U
+ 0
;t.
r~~~~~ ---..-b -----
90 2
3
Hours
Figure 7. The bactericidal effect of serum on Salmonella typhimurium 710 exposed to ampicillin at one-half the MIC (a) and at the MIC (b) . . - - -e = control organisms;. • = drug-exposed organisms.
Downloaded from http://cid.oxfordjournals.org/ at University of Glasgow on June 28, 2015
mec
0~
1/16 M1C
Control
Lorian and Atkinson
802
== ...
a
=======::
b
+ 0
""" : : : :::a=--=-:::' =~----------""'"
....=-,=-:
90
+ 0
ua> 90 a>
0 "0
u
...
+
C
0
a>
U 0
•
co 0~
Figure 8. The bactericidal effect of serum on various Enterobacteriaceae exposed to mecillinam at onefourth the MIC. (a) Salmonella group B; (b) Klebsiella pneumoniae; (c) Proteus mirabilis 6; (d) Escherichia coli Elliott. e---e = control organisms; • = drug-exposed organisms.
90
+ 0
90
\,\ \,
\ '\\
d
\,\
\~
~\, '\,
1/ 2
2
3
Hours
suIted from exposure to ampicillin showed a continuing and significant increase in the number of live organisms (number of cfu) after 1 hr of contact with serum. The oval and round cells of E. coli 10 that formed in the presence of mecillinam at various concentrations, including the MIC, appeared to be completely resistant to the bactericidal effect of serum (figure 4, right). In S. marcescens, both the control cells and round cells that resulted from exposure to mecillinam showed equal sensitivity to serum, which had a 900/0-990/0 bactericidal effect on the organism after 3 hr of incubation (figure 5). S. marcescens is highly resistant to the effects of ampicillin, so no filaments were available for study. In P. mirabilis 7, both control cells and the filaments produced with ampicillin showed little or no susceptibility to the bactericidal effect of
serum, depending on the serum, for up to 2 hr of incubation. However, after 3 hr of incubation serum showed a 90%-99% bactericidal effect on control cells of P. mirabilis, whereas it showed little or no bactericidal effect on its filaments (figure 6, left). Even though P. mirabi/is 7 was exposed to ampicillin at concentrations of one-third or even one-half the MIC, its filaments remained resistant . to the effects of serum. The same phenomenon occurred with round cells of P. mirabilis produced by exposure to mecillinam (figure 6, right). Control cells of S. typhimurium 710 as well as filaments and round cells produced by exposure to ampicillin and mecillinam, respectively, showed complete resistance to the bactericidal effect of serum after 3 hr of incubation (figure 7). Round cells formed by bacteria of a strain of Salmonella group B and by K. pneumoniae, P. mirabilis 6,
Downloaded from http://cid.oxfordjournals.org/ at University of Glasgow on June 28, 2015
-
Effect ofSerum and Blood on Enterobacteria
803
+ 0
M
A
..... u
Q)
'+'+-
(J)
90
0 "0
.-U ~
.....Q) 99 u 0
en ~ 0
99.9
Hours Figure 9. The bactericidal effects of blood on Escherichia coli K12 exposed to one-half the MIC of ampicillin (A) and of mecillinam (M). . - - -e = control organisms; • • = drug-exposed organisms.
o + U
0
~
--
~,
\,\ ,
u
\,
~
Q;
90
u ~
u
~
u
,, \
99
\
0
\\
"0
~ ........
"0
\
"0
\', \
"0 ~
\
90
Q)
....
\\ \\ \\ \\
99
Q)
....
--,
U 0
CD "-
"-
~
0
:--
\\
99.9
\\
CD
\\ \\
~ 0
\\
99.99
99.9
1/2
3
Hours
0
2
3
Hours
Figure 10. The bactericidal effect of blood on Escherichia coli 14 exposed to one-fourth the MIC of ampicillin (left) and to one-fourth the MIC of mecillinam (right) . . - - -e = control organisms; • • = drug-exposed organisms.
Downloaded from http://cid.oxfordjournals.org/ at University of Glasgow on June 28, 2015
and E. coli Elliott (moderately sensitive to serum) by exposure to mecillinam were simultaneously incubated with serum for 3 hr. With the exception of E. coli Elliott, both the control cells and the round cells of these species showed total resistance to the bactericidal effect of serum. Whereas the round cells of E. coli Elliott showed some susceptibility to serum, they were much more resistant to its effects than were the control organisms of this strain (figure 8). Heat-inactivated serum showed no antibacterial effect for any of these organisms. Effect of blood. The bactericidal effect of blood on either the control cells, filaments, or round cells of E. coli K12 (figure 9) and E. coli 14 (figure 10) was very similar to that of serum (99070-99.9070). Whereas 99070 of the control cells of E. coli 10 were killed by incubation with blood, a greater proportion of its filaments and round cells survived; incubation with blood killed only 50070-90070 of these cells (figure 11). Both control cells and round cells of S. marcescens were slightly more susceptible to the bactericidal effect of blood than they were to that of serum (figure 12). Control cells and filaments of P. mirabilis 7 were equally susceptible to the bactericidal effect of blood, with a bactericidal rate of between 90070 and 99.9070 (figure 13). Finally, the control cells of S. typhimurium 710 were slightly affected by the bactericidal activity of blood, whereas the filaments were unaffected (figure 14).
804
Lorian and Atkinson
a u
~
+
99
Q)
+
o
0
"0 U
'0
Qi "0
"0
+ o
U ~
cu
'0 99
c
0
CD ~ 0
99.9 99 '----
1/2
2
.1.--
---'-
3
Hours
2
----'--
3
Hours
Figure 11.
The bactericidal effect of blood on Escherichia coli 10 exposed to ampicillin at one-fourth the MIC (left) and to mecillinam (right) at the MIC (a), at one-half the MIC (b), and at one-fourth the MIC (c). e- - __ = controlorganisms; • • = drug-exposed organisms. Discussion
Complement is the agent that is most often the source of the bactericidal effect of serum [21-23]. In this study, only E. coli and S. marcescens showed significant sensitivity to the bactericidal + 0
U 2
Qi 90 0 "0 () ~
2 u
99
0
CD ~
........
0
- ..... ......
99.9 112
2
3
Hours
Figure 12. The bactericidal effect of blood on Serratia marcescens exposed to mecillinam at one-fourth the MIC. e- - __ = control organisms; • • =
drug-exposed organisms.
effect of serum. Blood was bactericidal for all of the species and strains tested, with the exception of-Salmonella group Band S. fyphimurium 710. Even E. coli 10 and P. mirabilis, which were practically unaffected by serum, were killed at a rate of 90070-99.9070 by blood. These gifferences between the effect of serum and that of blood are probably attributable to phagocytosis. In most cases, the bactericidal effect of either serum or blood on filaments or on oval and round cells was less than the effect on control cells. In some instances, the bactericidal effect on drug-exposed cells was similar to the effect on control cells, but in only one instance was the effect greater than that on the control cells. It is possible that a few filaments that were not killed by either serum or blood separated into many normal-sized bacilli, thereby producing a larger number of cfu than were generated by the surviving normal bacilli. The round cells produced by exposure to mecillinam have a significantly thinner cell wall than do the control cells [8], and thus a greater-than-normal susceptibility of these cells to the action of complement was expected.
Downloaded from http://cid.oxfordjournals.org/ at University of Glasgow on June 28, 2015
b
~
Effect ofSerum and Blood on Enterobacteria
805
+ 0
...u Q)
....... .......
Q)
Figure 13. The bactericidal effect of blood on Proteus mirabilis 7 exposed to ampicillin at one-fourth the' MIC. e-- - -e = control organisms; • • = drug-exposed organisms.
--
90
-----
0 \:l U
--
~
Q)
...... u
I
99
0
m ~
0
1/ 2
Whereas this larger size could account for the lower rate of killing of these cells by blood (p~~gocytQ~is), the lower bactericidal rate caused by'seruin remains unexplained. Although the bactericidal rate for filaments and round cells was usually lower than for control organisms, it was only slightly so. In all cases in which the bactericidal effect on control organisms was ~99070, the filaments or round cells were killed in a proportion close to 90070. Therefore, it appears that, although the oval, round, and filamentous morphologies did not present a significant obstacle to the bactericidal effect of either serum or blood, neither did these changes in morphology enhance the effects. Conclusion
The conclusion of this study is at variance with the
u
2 .....
+
3
majority of data found in the literature [14-17]. Possible explanations for this include the following. (1) Different ~riteria were used to evaluate the effects of sub-MICs of antibiotics on bacteria before the organisms were exposed to serum or blood. In our study, cell morphology and the number of cfu were strict criteria used to demonstrate nonlethal antibacterial activity. (2) Other studies used different antibacterial agents to induce changes in cell morphology. (3) Different concentrations of antibacterial agents were used. (4) Most important, different techniques were used to prepare organisms. The filaments and round cells used in this study were prepared on membranes. In other studies the cells were prepared in liquid media, in which osmotic effects could have altered the integrity of the cells. Therefore, these latter cells could have been more susceptible to the effects of serum and of blood than were cells grown on membranes and thus less exposed to osmotic forces.
0 ~~::::::::::::::--"""'---::::::::--:::::::""--4~----"
References
Q)
o
1. Duguid, J. P. The sensitivity of bacteria to the action of penicillin. Edinburgh Med. J. 53:401-412,
-0
U
2 Hours
90
~
Q)
1946.
uo
,.-
(l)
,.-
./ ./
t!2
3
Hours
Figure 14. The bactericidal effect of blood on Salmonella typhimurium 710 exposed to ampicillin at one-half the MIC. e-- - -e = control organisms; • • = drug-exposed organisms.
2. Gardner, A. D. Morphological effects of penicillin on bacteria. Nature 146:837-838, 1940. 3. Lorian, V., Sabath, L. D. Penicillins and cephalosporins: differences in morphologic effects on Proteus mirabilis. J. Infect. Dis. 125:560-564, 1972. 4. Brzin, B. Unusual cell form of Bacterium anUratum produced by sulfonamides. Experientia 22: 149-150, 1966. 5. Goss, W. A., Deitz, W. H., Cook, T. M. Mechanism of action of nalidixic acid on Escherichia coli. J. Bacteriol. 88: 1112-1118, 1964.
Downloaded from http://cid.oxfordjournals.org/ at University of Glasgow on June 28, 2015
99,9
806
15. Wardla\V. A. C. The complement-dependent bacteriolytic activity of normal ~)Jman serum. J. Exp. Med. 115: 1231-1249, 1962. 16. Sud, I. J., Feingold, D. S. Detection of agents that alter the bacterial cell surface. Antimicrob. Agents Chemother. 8:34-37, 1975. 17. Dutcher, B. S., Reynard, A. M., Beck, M. E., Cunningham, R. K. Potentiation of antibiotic bactericidal activity by normai human serum. Antimicrob. Agents Chemother. 13:820-826, 1978. 18. Nishida, M., Mine, Y., Nonoyama, S., Yokota, Y. Effect of antibiotics on the phagocytosis and killing of Pseudomonas aeruginosa by rabbit polymorphonuclear leukocytes. Chemotherapy 22:203-210, 1976. 19. Friedman, H., Warren, G. H. Enhanced susceptibility of penicillin resistant staphylococci to phagocytosis after in vitro incubation with low doses of nafcillin (38177). Proc. Soc. Exp. BioI. Med. 146:707-711, 1974. 20. Steers, E., Foltz, E. L., Graves,.B. S. An inocula replicating apparatus for routine testing of bacterial susceptibility to antibiotics. Antibiot. Chemother. 9:307-311, 1959. 21. Feingold, D. S., Goldman, J. N., Kuritz, H. M. Locus of the action of serum and the role of lysozyme in the serum bactericidal reaction. J. Bacteriol. 96:2118-2126 j 1968. 22. Rowley, D., Wardlaw, A. C. Lysis of gram-negative bacteria by serum. J. Gen. Microbiol. 18:529-533, 1958. 23. Glynn, A. A., Milne, M. A kinetic study of the bacteriolytic and bactericidal action of human serum. Immunology 12:639-653, 1967.
Downloaded from http://cid.oxfordjournals.org/ at University of Glasgow on June 28, 2015
6. Lorian, V., Popoola, B. The effect of nitrofurantoin on the morphology of gram-negative bacilli. J. Infect. Dis. 125:187-189, 1972. 7. Greenwood, D., O'Grady, F. FL 1060: a new beta-Iactam antibiotic with novel properties. J. Clin. Pathol. 26: 1-6, 1973. 8. Lorian, V., Atkinson, B. Comparison of the effects of mecillinam and 6-aminopenicillanic acid on Proteus mirabilis, Escherichia coli, and Staphylococcus aureus. Antimicrob. Agents Chemother. 11:541-552, 1977. 9. Melchior, N. H., BJorn, J., Tybring, L., Birch-Andersen, A. Light and electron microscopy of the early response of Escherichia coli to a 6 {3-amidinopenicillanic acid (FL 1060). Acta Pathol. MicrobioI. Scand. [B] 81:393-407, 1973. 10. De Rosnay-Dulong, C., Latrille, J. Proprietes metaboliques des spheroplastes d'Enterobacteries induits par I'acide 6-aminopenicillanique. C. R. Soc. BioI. (Paris) 159:1663-1668. 11. Sprat, B. G., Pardee, A. G. Penicillin binding proteins and cell shape in E. coli. Nature 254:516-517, 1975. 12. Lorian, V., Atkinsoll, B. Effects of subinhibitory concentrations of fosfomycin on bacteria. J. Ital. de Chemoterapia 23:65-74, 1976. 13. Stapley, E. 0., Hendlin, D., Mata, J. M., Jackson, M., Wallick, H., Hernandez, S., Mochales, S., Currie, S. A., Miller, R. M. Phosphonomycin. I. Discovery and in vitro biological characterization. Antimicrob. Agents Chemother. 1969:284-290, 1970. 14. Traub, M. H., Sherris, J. C. Studies on the interaction between serum bactericidal activity and antibiotics in vitro. Chemotherapy 15:70-83, 1970.
Lorian and Atkinson
Effect of Serum and Blood on Enterobacteriaceae Grown in the Presence of Subminimal Inhibitory Concentrations of Ampicillin and Mecillinam Victor Lorian and Barbara A. Atkinson
From the Section of Microbiology, Department of Pathology, The Bronx-Lebanon Hospital Center; and the Department of Laboratory Medicine, Albert Einstein College of Medicine, Bronx, New York
It was of interest to know whether the filamentous or round cells of Enterobacteriaceae that formed in the presence of sub-MICs of f3-lactam antibiotics differ from bacteria not exposed to these drugs in their susceptibility to the bactericidal effect of serum and to phagocytosis.
Gram-negative bacilli exposed to low concentrations of penicillin [1-3] and of some other agents [4-6] elongate and eventually become long filaments. However, exposure to three antibacterial agents-mecillinam [7-9], 6-aminopenicillanic acid [8-11], or fosfomycin [12, B]-resulted predominantly in the formation of oval and round cells. Some bacteria exposed to subminimal inhibitory concentrations (sub-MICs) of antibiotics have shown increased vulnerability to immunodefense mechanisms. Serum in combination with antibiotics is known to be bactericidal for some species of Enterobacteriaceae [14, 15]. Polymyxin sensitized Proteus mirabilis to the bactericidal effect of serum [16], as did streptomycin and tetracycline with Escherichia coli [17]. Staphylococcus aureus grown in the presence of sub-MICs of nafcillin showed an increased susceptibility to phagocytosis [19].
Materials and Methods
Strains. The following strains of Enterobacteriaceae were tested for their susceptibility to the bactericidal effect of serum and blood: four strains of E. coli, one serum sensitive (KI2), one serum resistant (10), and two moderately sensitive to serum (14, Elliott); two strains of P. mirabilis (6, 7), and one strain each of Serratia marcescens, Salmonella group B, Salmonella typhimurium (710), and Klebsiella pneumoniae. Antibiotics. Ampicillin (Ayerst Laboratories, New York, N.Y.) was used to induce the formation of filamentous bacteria (figure 1, bottom left). Mecillinam (Hoffman-LaRoche, Nutley, N.J.) was used to induce the formation of oval and round bacteria (figure 1, right). Preparation of serum and blood. Blood was drawn by venipuncture with a 20-ml plastic syringe from five healthy volunteers who claimed
This research was funded by a grant from HoffmanLaRoche, Nutley, N. J. Please address requests for reprints to Dr. Victor Lorian, Division of Microbiology and Epidemiology, The BronxLebanon Hospital Center, Albert Einstein College of Medicine, 1276 Fulton Avenue, Bronx, New York 10456.
797
Downloaded from http://cid.oxfordjournals.org/ at University of Glasgow on June 28, 2015
Bacteria from strains of five species of Enterobacteriaceae grown in the presence of subminimal inhibitory concentrations of ampicillin or mecillinam formed into filamelltous or round cells. These filamentous and round cells as well as normal control bacteria were incubated with either fresh human serum or blood, the bac!er~cidaleff~(;t.s of which were then determined. In most. c~ses, the bactericidal effect of either serum or blood on filamentous or round cells was },ess that} the effect on control cells. In some cases, the effect on thes~ drug-exposed cells was similar to that on control cells, but in no instance was the effect greater for the drug-expo~ed cells than for the control cells. However, in all cases in which the bactericidal effect of either serum or blood on the control cells was ~996Jo, the bactericidal effect on the drug-exposed cells was close to 900(0. Although drug-exposed cells were not much more resistant to the bactericidal effect of serum or blood than were normal bacteria, they clearly were not more susceptible to these effects.
798
Lorian and Atkinson
not to be taking any medication. A 10-ml volume was shaken for a few minutes with sterile glass beads in a sterile 50-ml flask. Another 5 ml of the blood was placed in a sterile glass tube, allowed to clot for 1 hr, and centrifuged at 150..g for 10 min. The serum was then re'moved and diluted 1:5 with Hanks' balanced salt solution (HijSS) plus 0.1 % gelatin (Hanks' gel) (both from Difco, Detroit, Mich.). From two to 10 sets of serum and blood were used to test the sensitivity of each strain. Determination of MICs and sub-MICs to be used in preparing test organisms. The MIC for each strain of bacteria was determined by a twofold agar dilution method [20]. The morphology of each strain exposed to antibiotic at the MIC and at dilutions down to 1/128 of the MIC was observed by interference phase contrast microscopy. The concentration of antibiotic that induced the greatest change in cell morphology without de-
stroying the cell (i.e., filaments 10-40 /Am long, or oval or round cells 4-12/Am in diameter) was used fOIJh~Jest. Table 1 shows the MICs for ampicillin and mecillinam for each strain of bacteria tested. jJreparation of test organisms. Filter membranes with a pore size of 0.3 /Am (no. PHW047S0; Millipore, Bedford, Mass.) were placed on drug-free trypticase soy agar (TSA) and warmed for 1 hr at 37 C before inoculation with 0.1 ml of a 1: 10 dilution of a culture of the organism to be tested that had been grown 20 hr in trypticase soy broth (TSB). The inoculated membranes were incubated for 90 min at 37,C, transferred either to TSA containing the antibiotic at one-half to one-eighth the MIC or to drug-free TSA, and then incubated for an additional 3.5 hr. The control organisms, which were grown on drug-free TSA (six membranes) were eluted with 15 ml of Hanks' gel; the organisms grown on antibiotic-
Downloaded from http://cid.oxfordjournals.org/ at University of Glasgow on June 28, 2015
Figure 1. Normal, untreated cells of Proteus mirabilis (top, left), filaments of P. mirabilis grown in ampicillin at one-fourth the MIC (bottom, le!f), and oval and round cells of P. mirabilis grown in mecillinam at one-fourth the MIC (right). Cell morphology was observed in each case by interference phase contrast microscopy at a magnification of x2,500.
Effect of Serum and Blood on Enterobacteria
799
+
Table 1. The MIC of ampicillin and mecillinam for all strains of Enterobacteriaceae tested. MIC of
ampicillin (I-lg/ml)
mecillinam (I-lg/ml)
6.3 4.0 5 4,000
0.4 0.25 0.19 7.8
Salmonella typhimurium 710 Salmonella group B
2.5 1.0
0.8 0.8
Serratia marcescens
50
200
Klebsiella pneumoniae 10
3.0
0.78
Proteus mirabilis 6 Proteus mirabilis 7
3.0 2.0
50 50
Organism
0
A
~
M
U Q)
Escherichia coli Escherichia coli Escherichia coli Escherichia coli
K12
10
14 Elliott
....... .......
~ ~
90
~\ \\
C "'0
.u
\\
~
Q) ~
U C
99
\\ \\
en
\\ \\ \\
0~
99.9
\ containing TSA (40 membranes) were eluted in 50ml plastic screw-capped tubes (Falcon Plastics, Oxnard, Calif.) with 15 ml of Hanks' gel containing the same concentration of antibiotic as was present on the TSA during incubation. The suspensions were shaken, and the volume of each was adjusted to give the approximate turbidity of the 0.5 McFarland standard (5 x 107 organisms/ml). These bacterial suspensions were used for the studies with serum or blood described below. This technique has been described in detail previously [8].
Test procedure. One 15-ml tube contained 0.1 ml of bacteria grown on drug-free TSA combined with 1.0 ml of Hanks' gel (control normal bac-
1/ 2 Figure 2.
The
1/ 2
Hours
bactericidal
effect
of serum on
Escherichia coli K12 exposed to one-half the MIC of ampicillin (A) and to one-half the MIC of mecillinam (M). - - - = control organisms; - - = drugexposed organisms.
teria). A pair of tubes contained 0.1 ml of normal bacteria, 0.1 ml of Hanks' gel, and either 0.9 ml of fresh or heat-inactivated serum diluted 1:5 in Hanks' gel or 0.9 ml of defibrinated blood. Another set of tubes contained 0.1 ml of the suspension of bacteria grown on antibiotic-contain-
+ o
u
u
~
a>
Q)
......
a>
90
"0 '1:l
'1:l
U
U
~
~
Q)
Q)
U
90
0
99
U 99
0
0
CD 0~
CD 0~
99.9
99.9
1/2
2
Hours
3
1/ 2
2
3
Hours
Figure 3. The bactericidal effect of serum on Escherichia coli 14 exposed to one-fourth the MIC of ampicillin (left) and to one-fourth the MIC of mecillinam (right) . . - - -e = control organisms; • • = drug-exposed organisms.
Downloaded from http://cid.oxfordjournals.org/ at University of Glasgow on June 28, 2015
MIC of
800
Lorian and Atkinson
+
o
---
....
a
90
-= u
Q)
Q)
+
b
0
o
"0
t-
(1)
u
0
o
u
~
't
90
CO
+
~
0
o
c
90
0 "0
u ~
Q)
90
U 99 0
m ~ 0
99.9 2
I
3
2
Hours
Hours
Figure 4. The bactericidal effect of serum on Escherichia coli 10 exposed to ampicillin at one-fourth the MIC (left) and to mecillinam (right) at the MIC (a), at one-half the MIC (b), and at one-fourth the MIC (c). e- - -e = control organisms; • • = drug-exposed organisms.
ing TSA plus 0.1 ml of antibiotic (at the concentration used to produce the filaments or round forms), and either 0.9 ml of Hanks' gel (antibiotic control), 0.9 ml of serum diluted 1:5 with Hanks' gel, or 0.9 ml of defibrinated blood. The bactericidal activity of the serum or blood was determined by mixing the suspensions on a vortex mixer and incubating the tubes at 37 C on a multipurpose rotator (Scientific Products, Chamblee, Ga.) at 20 rpm. After incubation for 0, 0.5, I, 2, and 3 hr, O.I-ml aliquots were removed from the suspensions and placed in 9.9 ml of saline at 10 C (to prevent growth). The number of cfu in the suspension was determined by plating 0.2 ml of 10-fold serial dilutions of the suspension on TSA plates and then incubating the plates. for 24 hr. The morphology of the bacteria eluted from the membranes as well as those in the suspensions
+ 0
u
~ Qj
90
0 "0 U ~
Q)
U 99 0
m 0~
99.9 112
2
3
Hours
Figure 5. The bactericidal effect of serum on Serratia marcescens exposed to mecillinam at one-fourth the MIC. e- - -e = control organisms; • • = drug-exposed organisms.
Downloaded from http://cid.oxfordjournals.org/ at University of Glasgow on June 28, 2015
U
+
Effect ofSerum and Blood on Enterobacteria
801
+ 0
-
+
(J
-
am
Cl)
0
1/4 M1C
'+Cl)
U
~
't
c
90
"'0
90
u
"0
~
"0
Cl)
u
~
U 99
C (l)
CO
0~
Q)
0
1/2 MIC
+
0 ~iiiiiiFiil~C===f==:::::::'----e 1/4 MIC ---------
90
99.9
Y2
2
1
3
2
Hours
Hours
Figure 6. The bactericidal effect of serum on Proteus mirabilis 7. The graph at left shows P. mirabilis exposed to ampicillin at one-fourth the MIC and incubated with 10 sera. The graph at right shows P. mirabilis exposed to various concentrations of ampicillin (am) and of mecillinam (mec) and incubated with serum. __ - - . =. control organisms; • • = drug-exposed organisms.
used for testing were studied by interference phase contrast microscopy during each test. The percentage of antibiotic-exposed bacteria (filaments or oval and round cells) that were killed by either serum or blood was calculated by dividing the number of cfu present in the suspension after exposure to serum or blood by the number of cfu present in the antibiotic control suspension (incubated in HBSS for the appropriate length of time).
in which drug-exposed organisms were slightly more sensitive to serum than were control organisms was in E. coli 14 that was exposed to mecillinam (figure 3, right). Figure 4 (left) shows the effect of serum on E. coli 10 (serum resistant). Whereas the effect of serum on the control bacteria of this strain was small to minimal, depending on the sample of serum used, the filaments of E. coli 10 that re-
Results
With the exception of E. coli K12 (serum sensitive), E. coli 14 and E. coli Elliott (both moderately sensitive to serum), and S. marcescens, all the species and strains tested showed little sensitivity to serum as either normal organisms, filaments, or oval and round cells. Figure 2 shows the effect of serum on E. coli K12 exposed to ampicillin or to mecillinam. In 99070 bactericidal effect both on control organisms and on drug-exposed organisms. Serum also had a >99.9% bactericidal effect on the control cells of E. coli 14 (moderately sensitive to serum), but after 2 hr, and especially after 3 hr, of incubation in serum, the filaments of this strain showed a small but increasing number of live organisms (figure 3, left). The only instance
a ----~:::-
-
o 90 "0
:~ .... Q.l
o
CD U
+ 0
;t.
r~~~~~ ---..-b -----
90 2
3
Hours
Figure 7. The bactericidal effect of serum on Salmonella typhimurium 710 exposed to ampicillin at one-half the MIC (a) and at the MIC (b) . . - - -e = control organisms;. • = drug-exposed organisms.
Downloaded from http://cid.oxfordjournals.org/ at University of Glasgow on June 28, 2015
mec
0~
1/16 M1C
Control
Lorian and Atkinson
802
== ...
a
=======::
b
+ 0
""" : : : :::a=--=-:::' =~----------""'"
....=-,=-:
90
+ 0
ua> 90 a>
0 "0
u
...
+
C
0
a>
U 0
•
co 0~
Figure 8. The bactericidal effect of serum on various Enterobacteriaceae exposed to mecillinam at onefourth the MIC. (a) Salmonella group B; (b) Klebsiella pneumoniae; (c) Proteus mirabilis 6; (d) Escherichia coli Elliott. e---e = control organisms; • = drug-exposed organisms.
90
+ 0
90
\,\ \,
\ '\\
d
\,\
\~
~\, '\,
1/ 2
2
3
Hours
suIted from exposure to ampicillin showed a continuing and significant increase in the number of live organisms (number of cfu) after 1 hr of contact with serum. The oval and round cells of E. coli 10 that formed in the presence of mecillinam at various concentrations, including the MIC, appeared to be completely resistant to the bactericidal effect of serum (figure 4, right). In S. marcescens, both the control cells and round cells that resulted from exposure to mecillinam showed equal sensitivity to serum, which had a 900/0-990/0 bactericidal effect on the organism after 3 hr of incubation (figure 5). S. marcescens is highly resistant to the effects of ampicillin, so no filaments were available for study. In P. mirabilis 7, both control cells and the filaments produced with ampicillin showed little or no susceptibility to the bactericidal effect of
serum, depending on the serum, for up to 2 hr of incubation. However, after 3 hr of incubation serum showed a 90%-99% bactericidal effect on control cells of P. mirabilis, whereas it showed little or no bactericidal effect on its filaments (figure 6, left). Even though P. mirabi/is 7 was exposed to ampicillin at concentrations of one-third or even one-half the MIC, its filaments remained resistant . to the effects of serum. The same phenomenon occurred with round cells of P. mirabilis produced by exposure to mecillinam (figure 6, right). Control cells of S. typhimurium 710 as well as filaments and round cells produced by exposure to ampicillin and mecillinam, respectively, showed complete resistance to the bactericidal effect of serum after 3 hr of incubation (figure 7). Round cells formed by bacteria of a strain of Salmonella group B and by K. pneumoniae, P. mirabilis 6,
Downloaded from http://cid.oxfordjournals.org/ at University of Glasgow on June 28, 2015
-
Effect ofSerum and Blood on Enterobacteria
803
+ 0
M
A
..... u
Q)
'+'+-
(J)
90
0 "0
.-U ~
.....Q) 99 u 0
en ~ 0
99.9
Hours Figure 9. The bactericidal effects of blood on Escherichia coli K12 exposed to one-half the MIC of ampicillin (A) and of mecillinam (M). . - - -e = control organisms; • • = drug-exposed organisms.
o + U
0
~
--
~,
\,\ ,
u
\,
~
Q;
90
u ~
u
~
u
,, \
99
\
0
\\
"0
~ ........
"0
\
"0
\', \
"0 ~
\
90
Q)
....
\\ \\ \\ \\
99
Q)
....
--,
U 0
CD "-
"-
~
0
:--
\\
99.9
\\
CD
\\ \\
~ 0
\\
99.99
99.9
1/2
3
Hours
0
2
3
Hours
Figure 10. The bactericidal effect of blood on Escherichia coli 14 exposed to one-fourth the MIC of ampicillin (left) and to one-fourth the MIC of mecillinam (right) . . - - -e = control organisms; • • = drug-exposed organisms.
Downloaded from http://cid.oxfordjournals.org/ at University of Glasgow on June 28, 2015
and E. coli Elliott (moderately sensitive to serum) by exposure to mecillinam were simultaneously incubated with serum for 3 hr. With the exception of E. coli Elliott, both the control cells and the round cells of these species showed total resistance to the bactericidal effect of serum. Whereas the round cells of E. coli Elliott showed some susceptibility to serum, they were much more resistant to its effects than were the control organisms of this strain (figure 8). Heat-inactivated serum showed no antibacterial effect for any of these organisms. Effect of blood. The bactericidal effect of blood on either the control cells, filaments, or round cells of E. coli K12 (figure 9) and E. coli 14 (figure 10) was very similar to that of serum (99070-99.9070). Whereas 99070 of the control cells of E. coli 10 were killed by incubation with blood, a greater proportion of its filaments and round cells survived; incubation with blood killed only 50070-90070 of these cells (figure 11). Both control cells and round cells of S. marcescens were slightly more susceptible to the bactericidal effect of blood than they were to that of serum (figure 12). Control cells and filaments of P. mirabilis 7 were equally susceptible to the bactericidal effect of blood, with a bactericidal rate of between 90070 and 99.9070 (figure 13). Finally, the control cells of S. typhimurium 710 were slightly affected by the bactericidal activity of blood, whereas the filaments were unaffected (figure 14).
804
Lorian and Atkinson
a u
~
+
99
Q)
+
o
0
"0 U
'0
Qi "0
"0
+ o
U ~
cu
'0 99
c
0
CD ~ 0
99.9 99 '----
1/2
2
.1.--
---'-
3
Hours
2
----'--
3
Hours
Figure 11.
The bactericidal effect of blood on Escherichia coli 10 exposed to ampicillin at one-fourth the MIC (left) and to mecillinam (right) at the MIC (a), at one-half the MIC (b), and at one-fourth the MIC (c). e- - __ = controlorganisms; • • = drug-exposed organisms. Discussion
Complement is the agent that is most often the source of the bactericidal effect of serum [21-23]. In this study, only E. coli and S. marcescens showed significant sensitivity to the bactericidal + 0
U 2
Qi 90 0 "0 () ~
2 u
99
0
CD ~
........
0
- ..... ......
99.9 112
2
3
Hours
Figure 12. The bactericidal effect of blood on Serratia marcescens exposed to mecillinam at one-fourth the MIC. e- - __ = control organisms; • • =
drug-exposed organisms.
effect of serum. Blood was bactericidal for all of the species and strains tested, with the exception of-Salmonella group Band S. fyphimurium 710. Even E. coli 10 and P. mirabilis, which were practically unaffected by serum, were killed at a rate of 90070-99.9070 by blood. These gifferences between the effect of serum and that of blood are probably attributable to phagocytosis. In most cases, the bactericidal effect of either serum or blood on filaments or on oval and round cells was less than the effect on control cells. In some instances, the bactericidal effect on drug-exposed cells was similar to the effect on control cells, but in only one instance was the effect greater than that on the control cells. It is possible that a few filaments that were not killed by either serum or blood separated into many normal-sized bacilli, thereby producing a larger number of cfu than were generated by the surviving normal bacilli. The round cells produced by exposure to mecillinam have a significantly thinner cell wall than do the control cells [8], and thus a greater-than-normal susceptibility of these cells to the action of complement was expected.
Downloaded from http://cid.oxfordjournals.org/ at University of Glasgow on June 28, 2015
b
~
Effect ofSerum and Blood on Enterobacteria
805
+ 0
...u Q)
....... .......
Q)
Figure 13. The bactericidal effect of blood on Proteus mirabilis 7 exposed to ampicillin at one-fourth the' MIC. e-- - -e = control organisms; • • = drug-exposed organisms.
--
90
-----
0 \:l U
--
~
Q)
...... u
I
99
0
m ~
0
1/ 2
Whereas this larger size could account for the lower rate of killing of these cells by blood (p~~gocytQ~is), the lower bactericidal rate caused by'seruin remains unexplained. Although the bactericidal rate for filaments and round cells was usually lower than for control organisms, it was only slightly so. In all cases in which the bactericidal effect on control organisms was ~99070, the filaments or round cells were killed in a proportion close to 90070. Therefore, it appears that, although the oval, round, and filamentous morphologies did not present a significant obstacle to the bactericidal effect of either serum or blood, neither did these changes in morphology enhance the effects. Conclusion
The conclusion of this study is at variance with the
u
2 .....
+
3
majority of data found in the literature [14-17]. Possible explanations for this include the following. (1) Different ~riteria were used to evaluate the effects of sub-MICs of antibiotics on bacteria before the organisms were exposed to serum or blood. In our study, cell morphology and the number of cfu were strict criteria used to demonstrate nonlethal antibacterial activity. (2) Other studies used different antibacterial agents to induce changes in cell morphology. (3) Different concentrations of antibacterial agents were used. (4) Most important, different techniques were used to prepare organisms. The filaments and round cells used in this study were prepared on membranes. In other studies the cells were prepared in liquid media, in which osmotic effects could have altered the integrity of the cells. Therefore, these latter cells could have been more susceptible to the effects of serum and of blood than were cells grown on membranes and thus less exposed to osmotic forces.
0 ~~::::::::::::::--"""'---::::::::--:::::::""--4~----"
References
Q)
o
1. Duguid, J. P. The sensitivity of bacteria to the action of penicillin. Edinburgh Med. J. 53:401-412,
-0
U
2 Hours
90
~
Q)
1946.
uo
,.-
(l)
,.-
./ ./
t!2
3
Hours
Figure 14. The bactericidal effect of blood on Salmonella typhimurium 710 exposed to ampicillin at one-half the MIC. e-- - -e = control organisms; • • = drug-exposed organisms.
2. Gardner, A. D. Morphological effects of penicillin on bacteria. Nature 146:837-838, 1940. 3. Lorian, V., Sabath, L. D. Penicillins and cephalosporins: differences in morphologic effects on Proteus mirabilis. J. Infect. Dis. 125:560-564, 1972. 4. Brzin, B. Unusual cell form of Bacterium anUratum produced by sulfonamides. Experientia 22: 149-150, 1966. 5. Goss, W. A., Deitz, W. H., Cook, T. M. Mechanism of action of nalidixic acid on Escherichia coli. J. Bacteriol. 88: 1112-1118, 1964.
Downloaded from http://cid.oxfordjournals.org/ at University of Glasgow on June 28, 2015
99,9
806
15. Wardla\V. A. C. The complement-dependent bacteriolytic activity of normal ~)Jman serum. J. Exp. Med. 115: 1231-1249, 1962. 16. Sud, I. J., Feingold, D. S. Detection of agents that alter the bacterial cell surface. Antimicrob. Agents Chemother. 8:34-37, 1975. 17. Dutcher, B. S., Reynard, A. M., Beck, M. E., Cunningham, R. K. Potentiation of antibiotic bactericidal activity by normai human serum. Antimicrob. Agents Chemother. 13:820-826, 1978. 18. Nishida, M., Mine, Y., Nonoyama, S., Yokota, Y. Effect of antibiotics on the phagocytosis and killing of Pseudomonas aeruginosa by rabbit polymorphonuclear leukocytes. Chemotherapy 22:203-210, 1976. 19. Friedman, H., Warren, G. H. Enhanced susceptibility of penicillin resistant staphylococci to phagocytosis after in vitro incubation with low doses of nafcillin (38177). Proc. Soc. Exp. BioI. Med. 146:707-711, 1974. 20. Steers, E., Foltz, E. L., Graves,.B. S. An inocula replicating apparatus for routine testing of bacterial susceptibility to antibiotics. Antibiot. Chemother. 9:307-311, 1959. 21. Feingold, D. S., Goldman, J. N., Kuritz, H. M. Locus of the action of serum and the role of lysozyme in the serum bactericidal reaction. J. Bacteriol. 96:2118-2126 j 1968. 22. Rowley, D., Wardlaw, A. C. Lysis of gram-negative bacteria by serum. J. Gen. Microbiol. 18:529-533, 1958. 23. Glynn, A. A., Milne, M. A kinetic study of the bacteriolytic and bactericidal action of human serum. Immunology 12:639-653, 1967.
Downloaded from http://cid.oxfordjournals.org/ at University of Glasgow on June 28, 2015
6. Lorian, V., Popoola, B. The effect of nitrofurantoin on the morphology of gram-negative bacilli. J. Infect. Dis. 125:187-189, 1972. 7. Greenwood, D., O'Grady, F. FL 1060: a new beta-Iactam antibiotic with novel properties. J. Clin. Pathol. 26: 1-6, 1973. 8. Lorian, V., Atkinson, B. Comparison of the effects of mecillinam and 6-aminopenicillanic acid on Proteus mirabilis, Escherichia coli, and Staphylococcus aureus. Antimicrob. Agents Chemother. 11:541-552, 1977. 9. Melchior, N. H., BJorn, J., Tybring, L., Birch-Andersen, A. Light and electron microscopy of the early response of Escherichia coli to a 6 {3-amidinopenicillanic acid (FL 1060). Acta Pathol. MicrobioI. Scand. [B] 81:393-407, 1973. 10. De Rosnay-Dulong, C., Latrille, J. Proprietes metaboliques des spheroplastes d'Enterobacteries induits par I'acide 6-aminopenicillanique. C. R. Soc. BioI. (Paris) 159:1663-1668. 11. Sprat, B. G., Pardee, A. G. Penicillin binding proteins and cell shape in E. coli. Nature 254:516-517, 1975. 12. Lorian, V., Atkinsoll, B. Effects of subinhibitory concentrations of fosfomycin on bacteria. J. Ital. de Chemoterapia 23:65-74, 1976. 13. Stapley, E. 0., Hendlin, D., Mata, J. M., Jackson, M., Wallick, H., Hernandez, S., Mochales, S., Currie, S. A., Miller, R. M. Phosphonomycin. I. Discovery and in vitro biological characterization. Antimicrob. Agents Chemother. 1969:284-290, 1970. 14. Traub, M. H., Sherris, J. C. Studies on the interaction between serum bactericidal activity and antibiotics in vitro. Chemotherapy 15:70-83, 1970.
Lorian and Atkinson
