Journal of Antimicrobial Chemotherapy (1978) 4, 47-54
/3-Lactamases of subspecies of Bacteroides fragilis
T. Leung and J. D. Williams
The /Mactamase activities of 64 strains of Bacteroides fragilis were studied. The subspecies of Bacteroides fragilis were found to have /Mactamases which focussed at distinctive isoelectric points. Five strains which showed a high degree of resistance to /J-lactam antibiotics showed three separate types of resistance, two of which were enzymic. Cephalosporins showed variable degrees of susceptibility to hydrolysis by the Bacteroides fragilis enzymes but cefoxitin was unaffected. Introduction With increasing awareness of the role of anaerobic organisms in abdominal sepsis Bacteroides fragilis has received increasing study. Only a small number of antibiotics are active against Bacteroides fragilis and /J-lactam antibiotics are generally regarded as inactive against most strains of Bacteroides other than those which are isolated from the oral cavity and related sites. The resistance is often associated with /Mactamase production but may also be caused by inability of the antibiotic to penetrate the bacterial cell in order to reach the site of action of the antibiotic (Derland & Birnbaum, 1977). The /Mactamases produced by Bacteroides fragilis usually only hydrolyse cephalosporins resembling type lb of Richmond & Jack (1971) and confer increased resistance to cephalosporin antibiotics. We have examined 64 strains of Bacteroides species, including some with very high resistance to /Mactam antibiotics, and the hydrolysis patterns of the enzymes for several cephalosporins were determined. Methods and materials Sixty-four strains of Bacteroides fragilis were obtained from clinical lesions and from faecal samples from normal individuals. The 32 faecal strains were kindly given by Dr B. S. Drasar, Central Public Health Laboratory, Colindale. The strains were subspeciated by the methods outlined in the Virginia Polytechnic Institute (V.P.I.) Manual (1975) and comprised 27 ss. fragilis, 2 ss. ovatus, 10 ss. vulgatus, 11 ss. thetaiotamicron, 8 distasonis and 6 untyped strains. Susceptibility testing Minimum inhibitory concentrations (MICs) were determined using Oxoid DST agar with 8% lysed horse blood and an inoculum of 103 organisms. Serial dilutions of the 03O5-7453/78/O701-BO47 $01.00/0
47 © (1978) The British Society for Antimicrobial Chemotherapy
Downloaded from http://jac.oxfordjournals.org/ at University of Birmingham on July 15, 2015
The London Hospital Medical College, London El 2 A D, England
48
T. Leung and J. D. Williams
following antibiotics were incorporated into the agar before pouring the plates— cephaloridine, cephalothin, cephazolin, cephamandole, cephalexin, cephradine, cefuroxime and cefoxitin. Two penicillins—ampicillin and carbenicillin—were also examined. ^-Lactamase activity of all strains was screened by the chromogenic cephalosporin 87/312 (O'Callaghan, Morris, Kirby & Shingler, 1972). Twelve of the strains which produced /9-lactamase were selected for further study. These included 7 strains with the mode MICs comprising 3 subspecies fragilis, one strain each of thetaiotamicron, vulgatus, ovatus and distasonis and also 5 strains which were highly resistant to most cephalosporins, of which were subspecies/raf His and one was a vulgatus strain.
Results Subspecies of Bacteroides fragilis The subspecies of B. fragilis examined are listed in Table I with the mode MICs of cephalosporins for these strains. Strains of subspecies fragilis, ovatus and distasonis were slightly more resistant than the other two subspecies but the interspecies differences were Table I. Mode MICs of penicillins and cephalosporins for subspecies of Bacteroides fragilis; the numbers of strains tested are shown in parentheses
Ampicillin Carbenicillin Cephaloridine Cephalothin Cefazolin Cefamandole Cephalexin Cephradine Cefuroxime Cefoxitin
B. fragilis (27)
B. ovatus (2)
B. vulgatus (10)
16 16 16 64 16 32 64 64 16 4
32 64 32 128 32 128 32 64 32 16
4 2 16 32 16 64 8 16 4 4
B. thetaiotaomicron B. distasonis (8) (ID 8 8 8 32 32 8 16 8 1 1
32 64 64 128 32 64 64 64 16 4
Downloaded from http://jac.oxfordjournals.org/ at University of Birmingham on July 15, 2015
Enzyme extraction study The organisms were grown in peptone-yeast extract-glucose medium supplemented with haemin and menadione (VPI Manual p. 124). The cell pellets were washed twice with phosphate buffer pH 7-2 and disrupted using a MSE ultrasonicator. The cell debris was removed by centrifuging at 4°C at 20,000 £ for 30 min and the crude extract used for hydrolysis and isoelectric focussing experiments. The rate of hydrolysis of the cephalosporins was determined at 35°C on a Unicam SP1800 spectrophotometer with controlled temperature cell carrier by measuring the rate of drop in absorbance at wavelengths specific for each cephalosporin in the u.v. region (O'Callaghan, Muggleton & Ross, 1968). The protein content of these extracts was determined by the Folin reagent (Lowry, 1951). Isoelectric focussing of the /Mactamase was performed by the technique described by Matthew (1977). To obtain better resolution of the isoelectric points, the broad spectrum pH 3-10 carrier ampholine was supplemented with a narrow range ampholine pH 4 to 4-5.
/3 -Lactaraases of B. fragUb
49
marginal. Cefoxitin was the most active agent followed by cefuroxime. The activities of ampicillin and carbenicillin were similar to that of cephaloridine. There were some differences in susceptibility between strains isolated from clinical sources and those from faeces. The faecal strains included more strains which were relatively sensitive to cephalosporins (Table II). Cefoxitin at 16^g/ml inhibited 100% of the faecal strains and 87% of the clinical isolates.
Cumulative % susceptible to (/ig/ml) Antibiotic
Strains (No) 0-5
Cephaloridine Cephalothin Cephazolin Cephamandole Cephalexin/ Cephradine Cefuroxime Cefoxitin
F (32) C (32) F C F C F C F C F C F C
1
2
9
4
8
16
32
64
128
>128
27
42
57 21 30
75 63 54 24 72 66 51 45 75 53 84 69
93 84 66 72 81 90 63 69 84 75
96
87
100 100 100 100 100 100 100 100 100 100 100 100
97
100
9 9 12 6 12
21
12 6 15
33 9 20
6 3
30
9 9 3
30
18 2
3 30 6
57 9
63 24
69 36 87 72
54 30 30 15 39 7 75 58 100 87
84 84 96 78 96
F = Strains isolated from faeces. C = Strains isolated from clinical sources. Screening tests showed that /Mactamase was produced by the majority of the strains in all 5 subspecies although in most of these strains, the amount produced was small. Isoelectric focussing showed characteristic pi values for the enzymes derived from the several subspecies. Thirteen B. fragilis var fragilis strains focussed at pH 4-9, three vulgalus strains at pH 4-6, four thetaiotaomicron strains at pH 4-6, with subsidiary weaker bands at pH 4-3 and 4-4, three distasonis strains at pH 5-1 and the two ovatus strains at pH 6-7. The two ovatus strains produced very weak bands (Figure 1). The hydrolysis profile of the subspecies is shown in Table III. Specific activity of the enzymes in general was low, less than 0-2 fimo\ of antibiotics hydrolysed per minute per mg protein. There was considerable variation in the rates of hydrolysis of the 8 antibiotics. Most variation was seen with cefazolin. Cefoxitin was not hydrolysed by any of the subspecies tested but cefuroxime was hydrolysed by all strains to a varying extent. Cephalexin and cephradine were also relatively resistant to hydrolysis.
Strains highly resistant to cephalosporins The 5 strains showing particularly high resistance to cephalosporins were also examined by these methods and the results are summarised in Table IV. Three separate patterns of resistance were seen. The first pattern of resistance was seen in three strains. These all showed at least tenfold increase in activity compared to the standard strains having the
Downloaded from http://jac.oxfordjournals.org/ at University of Birmingham on July 15, 2015
Table II. MICs of 7 cephalosporins and cefoxitin for faecal and clinical isolates of Bacteroides fragilis
T. Leung and J. D. Williams
50
* *
PH
-
• ! . " ) - •
'
\•
t
' *
5-
in Figure 1. Isoeledric focussing of ^-lactamascs produced by subspecies of Bacteroides fragilis. 1, ss. fragilis pH 4-9. 2, ss. dislasonis pH 5 1 . 3, ss. thetaiotaomicron pH4-5. 4, ss. vulgatus p H 4 6 . 5,ss. ovatus
pH 6-7.
mode resistance. They produced an intense j3-lactamase band at pH 4-9 and one weak subsidiary band at pH 5-1 (Figure 2). The second resistance pattern was seen in one strain which had a very high specific activity to most substrates apart from cefoxitin and it exhibited a complex enzyme pattern comprising of at least 4 intense bands with weaker bands in between with pi values from 4-8-5-2. One further strain (B. fragilis var vulgatus) was resistant to both cefoxitin and cefuroxime as well as to other cephalosporins despite having very low specific activity (Tables III and IV). It did not produce a discernible ^-lactamase band on the gel.
Downloaded from http://jac.oxfordjournals.org/ at University of Birmingham on July 15, 2015
6-
- =Not
tested.
Cephaloridine Cephalothin Cefazolin Cephamandole Cephalexin Cephradine Cefuroxime Cefoxitin
ss. fragilis b
c
ss. ooatus SS. uulgatus
omicron
ss. thetaiotass. diastonis
Table 111. Hydrolysis by B. fragilis subspecies b-lactamases of 7 cephalosporins, and cefoxitin. The specific activity is expressed in pmoles hydrolysed per min per mg protein (and relative % hydrolysis compared to cephaloridine)
rom http://jac.oxfordjournals.org/ at University of Birmingham on July 15, 2015
?!
?
T. kmg and J. D. William
52
Table IV. Activities of plactamase from resistant strains of Bacferoides/ragilis and the standard NCTC strain against 8 cephalosporins. Specific activity in pmoles per min per mg protien (relative % hydrolysis compared to cephaloridine). -= Prominent band
NCTC 9343
a
Resistance patterns I b c
I1
I11
Isoelcctric points
Multiple 4.8 to 5.2
Not detectable
Figure 2. lsoelectric focussing of ~lactamasesproduced by 5 Bae~eroidcs/ragilisspecies strains which to B. /rogilis NCTC 9343 strain. 1 a, b, c, 11, Ill-see
m r e highly resistant to cephalosporins compared t u t . S = NCTC 9343.
Downloaded from http://jac.oxfordjournals.org/ at University of Birmingham on July 15, 2015
Cephaloridine Cephalothin Cephalexin Cephradine Cephamandole Cefazolin Ccfuroxime Cefoxitin
Table IV summarised the relationship between MIC of these highly resistant Bacteroides fragilis and susceptibility to hydrolysis by p-lactamax. For the first two patterns, resistance correlated with plactamase production but the third type which showed the highest resistance to all cephalosporins had the same hydrolytic activity to that of the standard strain.
Dbmsion
Downloaded from http://jac.oxfordjournals.org/ at University of Birmingham on July 15, 2015
Resistance of bacteria to j?-lactam antibiotics is due to a combination of factorsthe affinity of the antibiotic for the peptidoglycan synthesising enzymes, the extent of the barrier to penetration of the antibiotic to the cell wall, and the activity of the varying amounts of /I-lactamase present inside the bacterial cell. Bacteroides fragilis of intestinal origin are moderately resistant to &lactam antibiotics. The role of & lactamase in resistance has come under increasing study and the development of new j?-lactam agents such as the cephamycin compounds with good activity against Bacteroides had added stimulus to these investigations. There have been several recent studies on Bacteroides fragilis p-lactamases (Del Bene & Farrar, 1973; Weinrich & Del Bene, 1976; Olsson, Nord & Wadstrom, 1976; Darland & Birnbaum, 1977). Our study was undertaken after marked differences were noted in susceptibility of faecal and clinical isolates of B. fragilis to p-lactam antibiotics. We had differentiated the enzymes present in all five subspecies of B. fragilis and had related the activity of these enzymes to the activity of wphalosporin antibiotics against those organisms. There was a difference in susceptibility to cephalosporins against B. fragilis from clinical isolates and faeces of normal individuals (Table II). Those from faecal origin were more sensitive than those isolated from clinical wound specimens. This might reflect the selection pressure exerted by antibiotic use in hospital, the most commonly used drugs being the &lactam agents. We also found that B. fragilis ss. fragilis was the most common subspecies isolated from wound infections. The differences between strains isolated from sepsis and from normal faecal specimens may be due to differences in pathogenicity of the subspecies or selection of the more resistant pathogens in a polymicrobial anaerobic infection or simply due to the relative ease of recovery of some Bacteroides subspecies in routine culture methods. The plactamases present in B. fragilis have been reported to be mainly non-inducible cephalosporinases (Andersen & Sykes, 1973; Del Bene & Farrar, 1973) and they have little activity against penicillins. We found that there was in most strains only a low level of Blactamase activity and this might not be the predominant factor in causing the mode resistance they exhibited. The rates at which the enzymes hydrolyse different cephalosporins vary but cefoxitin was completely stable to all the enzymes examined. The value of isoelectric focussing of 8-lactamases in differentiation of microorganisms was pointed out by Matthew & Harris (1976). The isoelectric points of & lactamases of the subspecies of B. fragilis also appear to be of some guidance in subspeciation. The strains of B. fragilis we examined which showed a marked increase of resistance to cephalosporins fell into three patterns. Two types of resistance associated with increased Slactamase production were seen (Table V). The commonest type was characterised by an enzyme focussing at pH 4-9 and 5.1. The second type which occurred in one strain gave a complex enzyme pattern and possessed very high hydrolytic activity. Attempts to transfer this Blactamase have so far been unsuccessful and growth at a higher temperature of incubation did not cure the ability to produce this enzyme.
54
T.Leung and J. D.Williams
Table V. Relationship between MIC and susceptibility to hydrolysis by the resistant strains of Bacteroides fragilis. Hydrolysis was expressed in specific activity-/moles hydrolysed per min per mg protein and MIC in pglml Cephaloridine MIC Hydrolysis Standard strain Resistant pattern I Resistant pattern I1 Resistant pattern 111
16 256 >256 >256
0.14 1.6 20 0.16
Cefoxitin
Cefuroxime
MIC Hydrolysis 4 8 8 64
0 0 0 0
MIC Hydrolysis 8 256 16 >256
0.03 1 1 0.03
References
Downloaded from http://jac.oxfordjournals.org/ at University of Birmingham on July 15, 2015
Cefoxitin was stable t o all types of enzymes found in these resistant strains. I n the third type of resistance enzyme activity was very low and it is probable that there is an increased barrier t o the penetration of antibiotics in these cells which resulted in increased resistance t o all cephalosporins and to the cephamycin cefoxitin.
Anderson, J. D. & Sykes, R. B. Characterisation of a beta-lactamase obtained from a strain of B. frcgilis resistant to beta-lactam antibiotics. Journal of Medical Microbiology 6 : 201-6 (1973): Darland, G. & Birnbaum, J. Cefoxitin resistance to beta-lactamases: a major factor for susceptibility of Bacteroides fragilis to the antibiotic. Antimicrobial Agents and Chemotherapy 11 : 725-34 (1977). Del Bene, V. & Farrar, W. E. Cephalosporinase activity in Bacteroides fragilis. Antimicrobial Agents and Chemotherapy 3: 369-72 (1973). Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193: 265-75 (1951). Matthew, M. & Harris, A. M. Identification of beta-lactamases by analytical isoelectric focussing: correlation with bacterial taxonomy. Journal of General Microbiology 94,5547 (1976). Matthew, M. Isoelectric focussing studies of beta-lactamases. In Zsoelectric Focussing (Arbuthnott, J. P. & Beeley, J. A., Eds). Butterworth, London (1977) pp. 248-53. O'Callaghan, C. H., Muggleton, P. W. & Ross, G. W. Effects of beta-lactamase from gram negative organisms on cephalosporins and penicillins. Antimicrobial Agents and Chemotherapy-1968: 57-63 (1968). O'Callaghan, C. H., Morris, A., Kirby, S. & Shingler, A. H. Novel method for detection of beta-lactamases by using a chromogenic cephalosporin substance. Antimicrobial Agents and Chemotherapy 1: 283-8 (1972). Olsson, B., Nord, C. E. & Wadstrom, T. Formation of beta-lactamase in B. fragilis: cell bound and extracellular activity. Antimicrobial Agents and Chemotherapy 9, 727-35 (1976). Richmond, M. H. & Sykes, R. B. The beta-lactamases of gram negative bacteria and their possible physiological role. Advances in Microbial Physiology 9: 31-88 (1973). Virginia Polytechnic Institute Anaerobic Laboratory Manual 3rd Edition. (Holdeman, L. V. & Moore, W. E. C., Eds.) (1975). Weinrich, A. E. & Del Bene, V. Beta-lactamases activity in anaerobic bacteria. Antimicrobial Agents and Chemotherapy 10: 106-1 11 (1976).
/3-Lactamases of subspecies of Bacteroides fragilis
T. Leung and J. D. Williams
The /Mactamase activities of 64 strains of Bacteroides fragilis were studied. The subspecies of Bacteroides fragilis were found to have /Mactamases which focussed at distinctive isoelectric points. Five strains which showed a high degree of resistance to /J-lactam antibiotics showed three separate types of resistance, two of which were enzymic. Cephalosporins showed variable degrees of susceptibility to hydrolysis by the Bacteroides fragilis enzymes but cefoxitin was unaffected. Introduction With increasing awareness of the role of anaerobic organisms in abdominal sepsis Bacteroides fragilis has received increasing study. Only a small number of antibiotics are active against Bacteroides fragilis and /J-lactam antibiotics are generally regarded as inactive against most strains of Bacteroides other than those which are isolated from the oral cavity and related sites. The resistance is often associated with /Mactamase production but may also be caused by inability of the antibiotic to penetrate the bacterial cell in order to reach the site of action of the antibiotic (Derland & Birnbaum, 1977). The /Mactamases produced by Bacteroides fragilis usually only hydrolyse cephalosporins resembling type lb of Richmond & Jack (1971) and confer increased resistance to cephalosporin antibiotics. We have examined 64 strains of Bacteroides species, including some with very high resistance to /Mactam antibiotics, and the hydrolysis patterns of the enzymes for several cephalosporins were determined. Methods and materials Sixty-four strains of Bacteroides fragilis were obtained from clinical lesions and from faecal samples from normal individuals. The 32 faecal strains were kindly given by Dr B. S. Drasar, Central Public Health Laboratory, Colindale. The strains were subspeciated by the methods outlined in the Virginia Polytechnic Institute (V.P.I.) Manual (1975) and comprised 27 ss. fragilis, 2 ss. ovatus, 10 ss. vulgatus, 11 ss. thetaiotamicron, 8 distasonis and 6 untyped strains. Susceptibility testing Minimum inhibitory concentrations (MICs) were determined using Oxoid DST agar with 8% lysed horse blood and an inoculum of 103 organisms. Serial dilutions of the 03O5-7453/78/O701-BO47 $01.00/0
47 © (1978) The British Society for Antimicrobial Chemotherapy
Downloaded from http://jac.oxfordjournals.org/ at University of Birmingham on July 15, 2015
The London Hospital Medical College, London El 2 A D, England
48
T. Leung and J. D. Williams
following antibiotics were incorporated into the agar before pouring the plates— cephaloridine, cephalothin, cephazolin, cephamandole, cephalexin, cephradine, cefuroxime and cefoxitin. Two penicillins—ampicillin and carbenicillin—were also examined. ^-Lactamase activity of all strains was screened by the chromogenic cephalosporin 87/312 (O'Callaghan, Morris, Kirby & Shingler, 1972). Twelve of the strains which produced /9-lactamase were selected for further study. These included 7 strains with the mode MICs comprising 3 subspecies fragilis, one strain each of thetaiotamicron, vulgatus, ovatus and distasonis and also 5 strains which were highly resistant to most cephalosporins, of which were subspecies/raf His and one was a vulgatus strain.
Results Subspecies of Bacteroides fragilis The subspecies of B. fragilis examined are listed in Table I with the mode MICs of cephalosporins for these strains. Strains of subspecies fragilis, ovatus and distasonis were slightly more resistant than the other two subspecies but the interspecies differences were Table I. Mode MICs of penicillins and cephalosporins for subspecies of Bacteroides fragilis; the numbers of strains tested are shown in parentheses
Ampicillin Carbenicillin Cephaloridine Cephalothin Cefazolin Cefamandole Cephalexin Cephradine Cefuroxime Cefoxitin
B. fragilis (27)
B. ovatus (2)
B. vulgatus (10)
16 16 16 64 16 32 64 64 16 4
32 64 32 128 32 128 32 64 32 16
4 2 16 32 16 64 8 16 4 4
B. thetaiotaomicron B. distasonis (8) (ID 8 8 8 32 32 8 16 8 1 1
32 64 64 128 32 64 64 64 16 4
Downloaded from http://jac.oxfordjournals.org/ at University of Birmingham on July 15, 2015
Enzyme extraction study The organisms were grown in peptone-yeast extract-glucose medium supplemented with haemin and menadione (VPI Manual p. 124). The cell pellets were washed twice with phosphate buffer pH 7-2 and disrupted using a MSE ultrasonicator. The cell debris was removed by centrifuging at 4°C at 20,000 £ for 30 min and the crude extract used for hydrolysis and isoelectric focussing experiments. The rate of hydrolysis of the cephalosporins was determined at 35°C on a Unicam SP1800 spectrophotometer with controlled temperature cell carrier by measuring the rate of drop in absorbance at wavelengths specific for each cephalosporin in the u.v. region (O'Callaghan, Muggleton & Ross, 1968). The protein content of these extracts was determined by the Folin reagent (Lowry, 1951). Isoelectric focussing of the /Mactamase was performed by the technique described by Matthew (1977). To obtain better resolution of the isoelectric points, the broad spectrum pH 3-10 carrier ampholine was supplemented with a narrow range ampholine pH 4 to 4-5.
/3 -Lactaraases of B. fragUb
49
marginal. Cefoxitin was the most active agent followed by cefuroxime. The activities of ampicillin and carbenicillin were similar to that of cephaloridine. There were some differences in susceptibility between strains isolated from clinical sources and those from faeces. The faecal strains included more strains which were relatively sensitive to cephalosporins (Table II). Cefoxitin at 16^g/ml inhibited 100% of the faecal strains and 87% of the clinical isolates.
Cumulative % susceptible to (/ig/ml) Antibiotic
Strains (No) 0-5
Cephaloridine Cephalothin Cephazolin Cephamandole Cephalexin/ Cephradine Cefuroxime Cefoxitin
F (32) C (32) F C F C F C F C F C F C
1
2
9
4
8
16
32
64
128
>128
27
42
57 21 30
75 63 54 24 72 66 51 45 75 53 84 69
93 84 66 72 81 90 63 69 84 75
96
87
100 100 100 100 100 100 100 100 100 100 100 100
97
100
9 9 12 6 12
21
12 6 15
33 9 20
6 3
30
9 9 3
30
18 2
3 30 6
57 9
63 24
69 36 87 72
54 30 30 15 39 7 75 58 100 87
84 84 96 78 96
F = Strains isolated from faeces. C = Strains isolated from clinical sources. Screening tests showed that /Mactamase was produced by the majority of the strains in all 5 subspecies although in most of these strains, the amount produced was small. Isoelectric focussing showed characteristic pi values for the enzymes derived from the several subspecies. Thirteen B. fragilis var fragilis strains focussed at pH 4-9, three vulgalus strains at pH 4-6, four thetaiotaomicron strains at pH 4-6, with subsidiary weaker bands at pH 4-3 and 4-4, three distasonis strains at pH 5-1 and the two ovatus strains at pH 6-7. The two ovatus strains produced very weak bands (Figure 1). The hydrolysis profile of the subspecies is shown in Table III. Specific activity of the enzymes in general was low, less than 0-2 fimo\ of antibiotics hydrolysed per minute per mg protein. There was considerable variation in the rates of hydrolysis of the 8 antibiotics. Most variation was seen with cefazolin. Cefoxitin was not hydrolysed by any of the subspecies tested but cefuroxime was hydrolysed by all strains to a varying extent. Cephalexin and cephradine were also relatively resistant to hydrolysis.
Strains highly resistant to cephalosporins The 5 strains showing particularly high resistance to cephalosporins were also examined by these methods and the results are summarised in Table IV. Three separate patterns of resistance were seen. The first pattern of resistance was seen in three strains. These all showed at least tenfold increase in activity compared to the standard strains having the
Downloaded from http://jac.oxfordjournals.org/ at University of Birmingham on July 15, 2015
Table II. MICs of 7 cephalosporins and cefoxitin for faecal and clinical isolates of Bacteroides fragilis
T. Leung and J. D. Williams
50
* *
PH
-
• ! . " ) - •
'
\•
t
' *
5-
in Figure 1. Isoeledric focussing of ^-lactamascs produced by subspecies of Bacteroides fragilis. 1, ss. fragilis pH 4-9. 2, ss. dislasonis pH 5 1 . 3, ss. thetaiotaomicron pH4-5. 4, ss. vulgatus p H 4 6 . 5,ss. ovatus
pH 6-7.
mode resistance. They produced an intense j3-lactamase band at pH 4-9 and one weak subsidiary band at pH 5-1 (Figure 2). The second resistance pattern was seen in one strain which had a very high specific activity to most substrates apart from cefoxitin and it exhibited a complex enzyme pattern comprising of at least 4 intense bands with weaker bands in between with pi values from 4-8-5-2. One further strain (B. fragilis var vulgatus) was resistant to both cefoxitin and cefuroxime as well as to other cephalosporins despite having very low specific activity (Tables III and IV). It did not produce a discernible ^-lactamase band on the gel.
Downloaded from http://jac.oxfordjournals.org/ at University of Birmingham on July 15, 2015
6-
- =Not
tested.
Cephaloridine Cephalothin Cefazolin Cephamandole Cephalexin Cephradine Cefuroxime Cefoxitin
ss. fragilis b
c
ss. ooatus SS. uulgatus
omicron
ss. thetaiotass. diastonis
Table 111. Hydrolysis by B. fragilis subspecies b-lactamases of 7 cephalosporins, and cefoxitin. The specific activity is expressed in pmoles hydrolysed per min per mg protein (and relative % hydrolysis compared to cephaloridine)
rom http://jac.oxfordjournals.org/ at University of Birmingham on July 15, 2015
?!
?
T. kmg and J. D. William
52
Table IV. Activities of plactamase from resistant strains of Bacferoides/ragilis and the standard NCTC strain against 8 cephalosporins. Specific activity in pmoles per min per mg protien (relative % hydrolysis compared to cephaloridine). -= Prominent band
NCTC 9343
a
Resistance patterns I b c
I1
I11
Isoelcctric points
Multiple 4.8 to 5.2
Not detectable
Figure 2. lsoelectric focussing of ~lactamasesproduced by 5 Bae~eroidcs/ragilisspecies strains which to B. /rogilis NCTC 9343 strain. 1 a, b, c, 11, Ill-see
m r e highly resistant to cephalosporins compared t u t . S = NCTC 9343.
Downloaded from http://jac.oxfordjournals.org/ at University of Birmingham on July 15, 2015
Cephaloridine Cephalothin Cephalexin Cephradine Cephamandole Cefazolin Ccfuroxime Cefoxitin
Table IV summarised the relationship between MIC of these highly resistant Bacteroides fragilis and susceptibility to hydrolysis by p-lactamax. For the first two patterns, resistance correlated with plactamase production but the third type which showed the highest resistance to all cephalosporins had the same hydrolytic activity to that of the standard strain.
Dbmsion
Downloaded from http://jac.oxfordjournals.org/ at University of Birmingham on July 15, 2015
Resistance of bacteria to j?-lactam antibiotics is due to a combination of factorsthe affinity of the antibiotic for the peptidoglycan synthesising enzymes, the extent of the barrier to penetration of the antibiotic to the cell wall, and the activity of the varying amounts of /I-lactamase present inside the bacterial cell. Bacteroides fragilis of intestinal origin are moderately resistant to &lactam antibiotics. The role of & lactamase in resistance has come under increasing study and the development of new j?-lactam agents such as the cephamycin compounds with good activity against Bacteroides had added stimulus to these investigations. There have been several recent studies on Bacteroides fragilis p-lactamases (Del Bene & Farrar, 1973; Weinrich & Del Bene, 1976; Olsson, Nord & Wadstrom, 1976; Darland & Birnbaum, 1977). Our study was undertaken after marked differences were noted in susceptibility of faecal and clinical isolates of B. fragilis to p-lactam antibiotics. We had differentiated the enzymes present in all five subspecies of B. fragilis and had related the activity of these enzymes to the activity of wphalosporin antibiotics against those organisms. There was a difference in susceptibility to cephalosporins against B. fragilis from clinical isolates and faeces of normal individuals (Table II). Those from faecal origin were more sensitive than those isolated from clinical wound specimens. This might reflect the selection pressure exerted by antibiotic use in hospital, the most commonly used drugs being the &lactam agents. We also found that B. fragilis ss. fragilis was the most common subspecies isolated from wound infections. The differences between strains isolated from sepsis and from normal faecal specimens may be due to differences in pathogenicity of the subspecies or selection of the more resistant pathogens in a polymicrobial anaerobic infection or simply due to the relative ease of recovery of some Bacteroides subspecies in routine culture methods. The plactamases present in B. fragilis have been reported to be mainly non-inducible cephalosporinases (Andersen & Sykes, 1973; Del Bene & Farrar, 1973) and they have little activity against penicillins. We found that there was in most strains only a low level of Blactamase activity and this might not be the predominant factor in causing the mode resistance they exhibited. The rates at which the enzymes hydrolyse different cephalosporins vary but cefoxitin was completely stable to all the enzymes examined. The value of isoelectric focussing of 8-lactamases in differentiation of microorganisms was pointed out by Matthew & Harris (1976). The isoelectric points of & lactamases of the subspecies of B. fragilis also appear to be of some guidance in subspeciation. The strains of B. fragilis we examined which showed a marked increase of resistance to cephalosporins fell into three patterns. Two types of resistance associated with increased Slactamase production were seen (Table V). The commonest type was characterised by an enzyme focussing at pH 4-9 and 5.1. The second type which occurred in one strain gave a complex enzyme pattern and possessed very high hydrolytic activity. Attempts to transfer this Blactamase have so far been unsuccessful and growth at a higher temperature of incubation did not cure the ability to produce this enzyme.
54
T.Leung and J. D.Williams
Table V. Relationship between MIC and susceptibility to hydrolysis by the resistant strains of Bacteroides fragilis. Hydrolysis was expressed in specific activity-/moles hydrolysed per min per mg protein and MIC in pglml Cephaloridine MIC Hydrolysis Standard strain Resistant pattern I Resistant pattern I1 Resistant pattern 111
16 256 >256 >256
0.14 1.6 20 0.16
Cefoxitin
Cefuroxime
MIC Hydrolysis 4 8 8 64
0 0 0 0
MIC Hydrolysis 8 256 16 >256
0.03 1 1 0.03
References
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Cefoxitin was stable t o all types of enzymes found in these resistant strains. I n the third type of resistance enzyme activity was very low and it is probable that there is an increased barrier t o the penetration of antibiotics in these cells which resulted in increased resistance t o all cephalosporins and to the cephamycin cefoxitin.
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