ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, May 1975, P. 693-697
Copyright O 1975 American Society for Microbiology
Vol. 7, No. 5
Printed in U.S.A.
Assessment of Susceptibility of Ampicillin-Resistant Enterobacteria to Cephalosporins D. GREENWOOD,'* CHING HAAN TEOH,2ICHAN AND F. O'GRADY' Department of Medical Microbiology, St. Bartholomew's Hospital, London EClA 7RE, England Received for publication 22 October 1974
The susceptibility of 103 ampicillin-resistant strains of enterobacteria to two cephalosporins was assessed by turbidimetric monitoring and disk diffusion tests. The results obtained suggest that in most cases a correlation exists between the suppression of growth of a dense bacterial culture for a therapeutically acceptable period by a fixed, high concentration of cephalosporin and a large zone of inhibition (>24 mm) using a high potency disk. This correlation was found to hold good when a moderate, subconfluent inoculum and commercially obtained blood-agar plates were used for the disk test, but not when the stringent conditions required by the Kirby-Bauer test (which demands a dense inoculum on Mueller-Hinton agar) were employed. Better correlation was obtained with cephaloridine than with cephalothin. This approach to the interpretation of disk diffusion tests gives a grossly different picture of the susceptibility of ampicillinresistant enterobacteria to cephalosporins than do conventional minimal inhibitory concentration-based correlations.
Turbidimetric monitoring shows that concentrations of cephalosporins well above the conventionally determined minimal inhibitory concentrations (MIC) exert only a transient effect on dense populations of enterobacteria (5). This marked inoculum effect is particularly well shown by ampicillin-resistant strains (Greenwood and O'Grady, manuscript in preparation). This raises the question whether the conventional MIC is a useful guide to the likely performance of the agent against the high concentration of organisms commonly present in infected lesions, and therefore to what extent conventional disk diffusion sensitivity tests are a valid indication of susceptibility of the organism in the therapeutic sense. In an attempt to explore this problem further, we have compared the susceptibility of a variety of ampicillin-resistant enterobacteria to cephalosporins as judged by both disk tests and by turbidimetric monitoring of dense cultures and discuss the discrepancies observed. MATERIALS AND METHODS The 103 strains consisted of 46 Escherichia coli, 28 Klebsiella sp., four Enterobacter sp., eight Proteus mirabilis, two indole-positive Proteus, five Hatnia sp., six Serratia marcescens, three Proteus inconstans, and one Citrobacter. I Present address: Department of Microbiology, University of Nottingham, City Hospital, Nottingham NG5 1PH. 2Present address: Department of Microbiology, University of Hong Kong, Queen Mary Hospital, Hong Kong.
The strains were isolated from infected urine in the diagnostic bacteriology department of St. Bartholomew's Hospital. All the strains failed to show a zone of inhibition when tested against a 10-,ug ampicillin disk on blood agar plates using a subconfluent inoculum, with the exception of eight Klebsiella strains which gave inhibition zones of > 14 mm when tested in this manner. Antibiotic disks containing cephaloridine (5 and 25
Mg) and cephalothin (5 and 30 Mg)
were
freshly
obtained from Mast Laboratories Ltd. Disk diffusion tests were performed under two sets of conditions. For the first the stringent conditions recommended by Bauer et al. (1) were used. Plates of Mueller-Hinton agar (Difco) were poured on a horizontal surface to a depth of 5 to 6 mm (30 ml per 8.5-cm diameter petri dish). The plates were stored at 4 C for not longer than 4 days; before use they were dried in a 37 C incubator for about 30 min. Organisms were grown for 4 h in Trypticase soy broth (Difco) and diluted to the density of a freshly prepared barium sulphate standard (0.5 ml of 1% BaCl, added to 99.5 ml of 1% H,S04) with sterile distilled water. Plates were then immediately inoculated by streaking with sterile cotton-wool swabs (dipped in the bacterial suspension and drained) evenly in three directions. The four antibiotic-containing disks were firmly applied to the surface of the plate after the inoculum had been allowed to dry (about 5 min). The inoculum used gives rise to a confluent growth on overnight incubation. The second method employed the considerably lower inoculum recommended by the International Collaborative Study on Antibiotic Senstivity Testing (3). Blood agar plates (ready-poured, Oxoid Ltd.) were inoculated with 0.001 ml of a 1/1,000 dilution of an
693
694
GREENWOOD, TEOH, AND O'GRADY
ANTIMICROB. AGENTS CHEMOTHER.
turbidimetric monitoring, is shown in Table 1. The numbers of strains of which regrowth was suppressed for longer than 8 h are shown in Table 2 in relation to their zone sizes when tested by the two disk methods at two disk potencies. The disk zones obtained with those strains judged to be very susceptible to cephaloridine (13 strains) and cephalothin (16 strains) in that growth was suppressed for 20 h or more in the turbidimetric system are shown in Table 3. According to the criteria of the Bauer-Kirby method, strains which show a zone of inhibition of 18 or more mm around a high potency cephalothin disk are fully susceptible. Zones of 15 to 17 mm are considered to indicate intermediate susceptibility (1) and this has generally been thought to imply that infections due to these organisms should respond to treatment in situations where high antibiotic concentrations are obtainable. All 16 strains judged in this way to be of intermediate susceptiblity and 12 of 27 strains judged to be fully susceptible were resistant according to the turbidimetric criterion of regrowth in less than 8 h after a dose of cephaloridine sufficient to produce an initial concentration of 250 ,ug/ml. Cephalothin gave comparable results: 11 of 15 strains of intermediate susceptiblity and 11 of 34 strains fully susceptible according to the Bauer-Kirby tests were judged resistant according to our turbidimetric criteria. If a low content disk was used with the high inoculum of the Bauer-Kirby test, cephaloridine and cephalothin gave widely different results. When the disk contained only 5 ,ug of cephaloridine and 14 mm was adopted as the RESULTS dividing line between susceptible and resistant, The susceptibility of the 103 ampicillin- agreement between the disk and turbidimetric resistant strains of enterobacteria, as judged by assesssments was good except that six of the 90
overnight broth culture using a 2-mm diameter wire loop. This inoculum yields a dense, but not confluent, growth on overnight incubation. The inoculum was spread with a glass rod and allowed to dry for about 5 min before application of the four antibiotic disks. Both sets of plates were incubated at 37 C overnight and inhibition zones were measured with dividers and a millimeter rule. Turbidimetric system. Turbidimetric studies of the response of dense bacterial populations to cephalosporins were made in the manner previously described (5) using a development of the simple turbidity cell of Watson et al. (7) in which the opacity of 12 independent bacterial cultures can be continuously monitored photometrically (6). Growth medium was the complete broth (osmolality ca. 325 osmol per kg) described previously (5). At a standard point in the logarithmic growth phase when the opacity had reached 30% of maximum (equivalent to a viable count of ca. 5 x 107 organisms per ml), sufficient antibiotic was added to produce a concentration of 250 Ag/ml. This concentration was chosen because it is readily achieved in urine and had previously been shown to produce rapid lysis of the majority of strains. Criteria of susceptibility or resistance. Cultures of susceptible organisms exposed to 3-lactam agents exhibit a fall in opacity due to lysis of the organisms (5). Highly resistant organisms show no deviation from the normal growth curve. Other organisms show an initial fall in opacity but through the interaction of several factors (4, 5) ultimately regrow. Organisms affected by the agents to such a limited extent that their original numbers are restored before the next dose of antibiotic would be due on a normal therapeutic regimen may reasonably be regarded as resistant. Strains which regrew in less than 8 h were consequently regarded as resistant and those that did not as susceptible. Strains that failed to regrow over the 20-h period for which the observations were continued were considered very susceptible.
TABLE 1. Susceptibility of 103 ampicillin-resistant coliform bacilli as judged by turbidimetric criteria Number in which regrowth occurred in: 9 to 19 h (susceptible)
8 h or less
Species and number tested
(resistant)
20 h or more (very susceptible)
CERa
CTN"
CER
CTN
CER
CTN
42 18
31
1 2 0 0
14 5 2 4
3 8 0 1 0 0 0 1 0
1 12 1 1
Escherichia coli (46) Klebsiella sp. (28) Enterobacter sp. (4) P. mirabilis (8) Indole-positive Proteus (2) Hatnia sp. (5) Serratia marcescens (6) Proteus inconstans A & B (3) Citrobacter sp. (l) a CER, Cephaloridine; CTN, cephalothin.
4 7 2 5 5 2 1
11 1 3 2 5 6 2 1
-
0 0 1
0 0
0 0 0 0 0
0 0 0 1 0
695
ENTEROBACTERIAL SUSCEPTIBILITY TO CEPHALOSPORINS
VOL. 7, 1975
TABLE 2. Comparison of susceptibility by turbidimetric and disk diffusion methods Inoculum for disk test
Agent
No. of strains suppressed in the turbidity system which gave a zone diameter (mm) of:a
Disk content
(Mg) High (Bauer-Kirby)
Cephaloridine Cephalothin
Moderate (ICS)b
Cephaloridine Cephalothin
24to28
Copyright O 1975 American Society for Microbiology
Vol. 7, No. 5
Printed in U.S.A.
Assessment of Susceptibility of Ampicillin-Resistant Enterobacteria to Cephalosporins D. GREENWOOD,'* CHING HAAN TEOH,2ICHAN AND F. O'GRADY' Department of Medical Microbiology, St. Bartholomew's Hospital, London EClA 7RE, England Received for publication 22 October 1974
The susceptibility of 103 ampicillin-resistant strains of enterobacteria to two cephalosporins was assessed by turbidimetric monitoring and disk diffusion tests. The results obtained suggest that in most cases a correlation exists between the suppression of growth of a dense bacterial culture for a therapeutically acceptable period by a fixed, high concentration of cephalosporin and a large zone of inhibition (>24 mm) using a high potency disk. This correlation was found to hold good when a moderate, subconfluent inoculum and commercially obtained blood-agar plates were used for the disk test, but not when the stringent conditions required by the Kirby-Bauer test (which demands a dense inoculum on Mueller-Hinton agar) were employed. Better correlation was obtained with cephaloridine than with cephalothin. This approach to the interpretation of disk diffusion tests gives a grossly different picture of the susceptibility of ampicillinresistant enterobacteria to cephalosporins than do conventional minimal inhibitory concentration-based correlations.
Turbidimetric monitoring shows that concentrations of cephalosporins well above the conventionally determined minimal inhibitory concentrations (MIC) exert only a transient effect on dense populations of enterobacteria (5). This marked inoculum effect is particularly well shown by ampicillin-resistant strains (Greenwood and O'Grady, manuscript in preparation). This raises the question whether the conventional MIC is a useful guide to the likely performance of the agent against the high concentration of organisms commonly present in infected lesions, and therefore to what extent conventional disk diffusion sensitivity tests are a valid indication of susceptibility of the organism in the therapeutic sense. In an attempt to explore this problem further, we have compared the susceptibility of a variety of ampicillin-resistant enterobacteria to cephalosporins as judged by both disk tests and by turbidimetric monitoring of dense cultures and discuss the discrepancies observed. MATERIALS AND METHODS The 103 strains consisted of 46 Escherichia coli, 28 Klebsiella sp., four Enterobacter sp., eight Proteus mirabilis, two indole-positive Proteus, five Hatnia sp., six Serratia marcescens, three Proteus inconstans, and one Citrobacter. I Present address: Department of Microbiology, University of Nottingham, City Hospital, Nottingham NG5 1PH. 2Present address: Department of Microbiology, University of Hong Kong, Queen Mary Hospital, Hong Kong.
The strains were isolated from infected urine in the diagnostic bacteriology department of St. Bartholomew's Hospital. All the strains failed to show a zone of inhibition when tested against a 10-,ug ampicillin disk on blood agar plates using a subconfluent inoculum, with the exception of eight Klebsiella strains which gave inhibition zones of > 14 mm when tested in this manner. Antibiotic disks containing cephaloridine (5 and 25
Mg) and cephalothin (5 and 30 Mg)
were
freshly
obtained from Mast Laboratories Ltd. Disk diffusion tests were performed under two sets of conditions. For the first the stringent conditions recommended by Bauer et al. (1) were used. Plates of Mueller-Hinton agar (Difco) were poured on a horizontal surface to a depth of 5 to 6 mm (30 ml per 8.5-cm diameter petri dish). The plates were stored at 4 C for not longer than 4 days; before use they were dried in a 37 C incubator for about 30 min. Organisms were grown for 4 h in Trypticase soy broth (Difco) and diluted to the density of a freshly prepared barium sulphate standard (0.5 ml of 1% BaCl, added to 99.5 ml of 1% H,S04) with sterile distilled water. Plates were then immediately inoculated by streaking with sterile cotton-wool swabs (dipped in the bacterial suspension and drained) evenly in three directions. The four antibiotic-containing disks were firmly applied to the surface of the plate after the inoculum had been allowed to dry (about 5 min). The inoculum used gives rise to a confluent growth on overnight incubation. The second method employed the considerably lower inoculum recommended by the International Collaborative Study on Antibiotic Senstivity Testing (3). Blood agar plates (ready-poured, Oxoid Ltd.) were inoculated with 0.001 ml of a 1/1,000 dilution of an
693
694
GREENWOOD, TEOH, AND O'GRADY
ANTIMICROB. AGENTS CHEMOTHER.
turbidimetric monitoring, is shown in Table 1. The numbers of strains of which regrowth was suppressed for longer than 8 h are shown in Table 2 in relation to their zone sizes when tested by the two disk methods at two disk potencies. The disk zones obtained with those strains judged to be very susceptible to cephaloridine (13 strains) and cephalothin (16 strains) in that growth was suppressed for 20 h or more in the turbidimetric system are shown in Table 3. According to the criteria of the Bauer-Kirby method, strains which show a zone of inhibition of 18 or more mm around a high potency cephalothin disk are fully susceptible. Zones of 15 to 17 mm are considered to indicate intermediate susceptibility (1) and this has generally been thought to imply that infections due to these organisms should respond to treatment in situations where high antibiotic concentrations are obtainable. All 16 strains judged in this way to be of intermediate susceptiblity and 12 of 27 strains judged to be fully susceptible were resistant according to the turbidimetric criterion of regrowth in less than 8 h after a dose of cephaloridine sufficient to produce an initial concentration of 250 ,ug/ml. Cephalothin gave comparable results: 11 of 15 strains of intermediate susceptiblity and 11 of 34 strains fully susceptible according to the Bauer-Kirby tests were judged resistant according to our turbidimetric criteria. If a low content disk was used with the high inoculum of the Bauer-Kirby test, cephaloridine and cephalothin gave widely different results. When the disk contained only 5 ,ug of cephaloridine and 14 mm was adopted as the RESULTS dividing line between susceptible and resistant, The susceptibility of the 103 ampicillin- agreement between the disk and turbidimetric resistant strains of enterobacteria, as judged by assesssments was good except that six of the 90
overnight broth culture using a 2-mm diameter wire loop. This inoculum yields a dense, but not confluent, growth on overnight incubation. The inoculum was spread with a glass rod and allowed to dry for about 5 min before application of the four antibiotic disks. Both sets of plates were incubated at 37 C overnight and inhibition zones were measured with dividers and a millimeter rule. Turbidimetric system. Turbidimetric studies of the response of dense bacterial populations to cephalosporins were made in the manner previously described (5) using a development of the simple turbidity cell of Watson et al. (7) in which the opacity of 12 independent bacterial cultures can be continuously monitored photometrically (6). Growth medium was the complete broth (osmolality ca. 325 osmol per kg) described previously (5). At a standard point in the logarithmic growth phase when the opacity had reached 30% of maximum (equivalent to a viable count of ca. 5 x 107 organisms per ml), sufficient antibiotic was added to produce a concentration of 250 Ag/ml. This concentration was chosen because it is readily achieved in urine and had previously been shown to produce rapid lysis of the majority of strains. Criteria of susceptibility or resistance. Cultures of susceptible organisms exposed to 3-lactam agents exhibit a fall in opacity due to lysis of the organisms (5). Highly resistant organisms show no deviation from the normal growth curve. Other organisms show an initial fall in opacity but through the interaction of several factors (4, 5) ultimately regrow. Organisms affected by the agents to such a limited extent that their original numbers are restored before the next dose of antibiotic would be due on a normal therapeutic regimen may reasonably be regarded as resistant. Strains which regrew in less than 8 h were consequently regarded as resistant and those that did not as susceptible. Strains that failed to regrow over the 20-h period for which the observations were continued were considered very susceptible.
TABLE 1. Susceptibility of 103 ampicillin-resistant coliform bacilli as judged by turbidimetric criteria Number in which regrowth occurred in: 9 to 19 h (susceptible)
8 h or less
Species and number tested
(resistant)
20 h or more (very susceptible)
CERa
CTN"
CER
CTN
CER
CTN
42 18
31
1 2 0 0
14 5 2 4
3 8 0 1 0 0 0 1 0
1 12 1 1
Escherichia coli (46) Klebsiella sp. (28) Enterobacter sp. (4) P. mirabilis (8) Indole-positive Proteus (2) Hatnia sp. (5) Serratia marcescens (6) Proteus inconstans A & B (3) Citrobacter sp. (l) a CER, Cephaloridine; CTN, cephalothin.
4 7 2 5 5 2 1
11 1 3 2 5 6 2 1
-
0 0 1
0 0
0 0 0 0 0
0 0 0 1 0
695
ENTEROBACTERIAL SUSCEPTIBILITY TO CEPHALOSPORINS
VOL. 7, 1975
TABLE 2. Comparison of susceptibility by turbidimetric and disk diffusion methods Inoculum for disk test
Agent
No. of strains suppressed in the turbidity system which gave a zone diameter (mm) of:a
Disk content
(Mg) High (Bauer-Kirby)
Cephaloridine Cephalothin
Moderate (ICS)b
Cephaloridine Cephalothin
24to28
