In Vitro Susceptibilities of the Bacteroides fragilis Group Species: Change in Isolation Rates Significantly Affects Overall Susceptibility Data

Abstract

A comparison of antimicrobial susceptibility data of species of the Bacteroides fragilis group for 1989-1990 and 1998-1999 studies showed statistically significant increases or decreases in in vitro activity. Overall significant increases in resistance were noted for ampicillin-sulbactam and clindamycin, while significant decreases in resistance were noted for ertapenem and cefoxitin. Susceptibilities to piperacillin-tazobactam, imipenem, meropenem, and trovafloxacin remained virtually the same for the two studies. Importantly, a change in the rates of isolation of the various species showed the B. fragilis species comprised 58% of the isolates in 1989 to 1990 and 45% of the isolates in 1998 to 1999. This change in rates of isolation of B. fragilis versus non-B. fragilis species had an overall effect on susceptibility data.

The Bacteroides fragilis group is a predominant component of the normal bacterial flora of the gastrointestinal tract. Moreover, these organisms are frequent isolates from mixed aerobic-anaerobic infections, such as intra-abdominal, diabetic foot, and soft tissue infections and contribute to the severity of the infections due to virulence factors (2). Several reports have indicated that in patients with mixed infections (including bacteremia), the B. fragilis group organisms can serve as a marker for increased morbidity and mortality and that patients treated with inappropriate antibiotics based on in vitro susceptibility data had a poorer clinical outcome than patients receiving appropriate antibiotic therapy (9, 11, 13).

The B. fragilis group is a heterogeneous group of species that vary in their susceptibility to antimicrobial agents, particularly β-lactams and clindamycin (1, 12). The B. fragilis species unexplainably remains more susceptible to many antimicrobial agents than the other species within the group (1, 7, 17). It is therefore important, especially in the choice of empirical therapy, that sufficient susceptibility data from sufficient numbers of isolates be available for local or regional isolates. The Anaerobe Working Group for the National Committee for Clinical Laboratory Standards (NCCLS) recommends that susceptibility testing of the B. fragilis group be done with a distribution of test isolates to approximate the species distribution normally isolated from infections (7). Several multicenter susceptibility studies have shown that the B. fragilis species comprises 58 to 63% of isolates (1, 5, 6, 17). Such distribution rates have been relatively evident in studies reported during the 1980s and early 1990s. Snydman et al. (15) reported that during 1995 to 1996, 53% of 961 isolates were of the B. fragilis species. However, it was recently reported from an ongoing national susceptibility study that the distribution of isolates has changed (Y. Golan, N. V. Jacobus, L. A. McDermott, S. Supran, D. W. Hecht, E. Goldstein, R. Venezia, L. Harrell, S. Jenkins, J. Rihs, C. Pierson, S. L. Gorbach, S. Finegold, and D. R. Snydman, Abstr. 41st Intersci. Conf. Antimicrob. Agents Chemother., abstr. K1209, p. 412, 2001). They found that the incidence of the B. fragilis species decreased from 63% in 1984 to 1985 to 50% in 1999 to 2000. Here we report a similar comparison of changing isolation rates from 1989 and 1990 to 1998 and 1999 and the effect on antimicrobial susceptibility results.

During 1989 to 1990 and 1998 to 1999, respectively, 1,240 and 401 nonduplicate clinical isolates of species of the B. fragilis group were collected from intra-abdominal, wound, blood, tissue, and abscess specimens. Isolates were identified with selective media, biochemical profiles, and gas-liquid chromatography (8, 16). Each antimicrobial agent listed in Table 2 was provided by the manufacturer. Susceptibility testing in both studies was performed by a broth microdilution method as recommended by the NCCLS (10) with Anaerobe MIC broth (Difco) to prepare twofold dilutions of each agent within a dilution range of 0.03 to 256 μg/ml. Ampicillin was combined with sulbactam in a 2:1 ratio, while twofold dilutions of piperacillin were combined with tazobactam at a constant concentration of 4 μg/ml. The inoculum for each isolate was prepared by suspending growth from a 24- to 48-h sheep blood agar plate to a suspension equal to a no. 1 McFarland standard. The suspension was further diluted to give a final inoculum size of 10⁵ CFU per well (10⁶ CFU/ml). All susceptibility plates were incubated at 35°C anaerobically for 48 h and read. The MIC was defined as the lowest concentration of each antimicrobial agent that inhibited the visible growth of the test isolate. With each susceptibility run, quality control was performed with B. fragilis ATCC 25285 and Bacteroides thetaiotaomicron ATCC 2974.

TABLE 2.

Comparison of effects of the change in isolation rates of B. fragilis versus non-B. fragilis species^a during 1989 to 1990 and 1998 to 1999

Antimicrobial and organism	Results for 1989-1990								Results for 1998-1999
	No. tested	MIC (μg/ml)b				% of MICs			No. tested	MIC (μg/ml)				% of MICs
		Range	Mode	50	90	S	I	R		Range	Mode	50	90	S	I	R
Piperacillin-tazobactam
B. fragilis group	496	0.25-64	4	4	8	99.6	0.4	0	401	0.06-32	0.12	0.12	1	100	0	0
B. fragilis	274	0.25-32	2	2	8	100	0	0	180	0.06-32	0.12	0.12	1	100	0	0
Other species	222	0.25-64	8	4	16	99.1	0.9	0	221	0.06-32	2	0.5	4	100	0	0
Ampicillin-sulbactam
B. fragilis group	1,240	0.03-128	0.5	1	4	96.9	1.9	1.2	401	0.03-64	1	2	8	89.1	8.5	2.4
B. fragilis	714	0.12-64	0.5	0.5	4	98.3	0.8	0.9	180	0.5-64	1	2	8	91.6	6.1	2.3
Other species	526	0.03-128	1	1	8	95.1	3.2	1.7	221	0.03-64	1	2	16	86.9	10.4	2.7
Imipenem
B. fragilis group	742	0.03-16	0.06	0.06	0.5	99.6	0	0.4	401	0.015-8	0.03	0.06	0.25	99.8	0.2	0
B. fragilis	438	0.03-16	0.03	0.06	0.5	99.3	0	0.7	180	0.015-8	0.03	0.06	0.25	99.4	0.6	0
Other species	304	0.03-4	0.12	0.12	0.5	100.0	0	0	221	0.015-2	0.12	0.06	0.25	100	0	0
Ertapenem
B. fragilis group	496	0.25-64	0.25	0.25	4	91.7	4	4.3	401	0.015-32	0.12	0.12	1	98.8	0.5	0.7
B. fragilis	274	0.25-32	0.25	0.25	2	94.5	3.6	1.9	180	0.03-32	0.12	0.12	1	97.8	1.1	1.1
Other species	222	0.25-64	0.25	1	8	88.3	4.5	7.2	221	0.015-32	0.5	0.25	1	99.5	0	0.5
Meropenem
B. fragilis group	742	0.03-64	0.12	0.12	0.5	98.9	0.7	0.4	401	0.015-64	0.06	0.12	0.5	98.8	0.5	0.7
B. fragilis	438	0.03-64	0.12	0.12	0.5	98.7	0.9	0.4	180	0.03-32	0.06	0.12	0.5	98.3	0.6	1.1
Other species	304	0.03-64	0.12	0.12	0.5	99.3	0.7	0	221	0.015-8	0.12	0.12	0.5	99.1	0.9	0
Cefoxitin
B. fragilis group	1,240	0.06-128	8	8	32	82.4	12.2	5.4	401	0.25-128	4	4	16	91.5	4.3	4.2
B. fragilis	714	0.12-128	8	8	32	89.5	7	3.5	180	0.25-32	4	4	16	92.4	3.8	3.8
Other species	526	0.06-128	16	16	32	72.8	19.2	8	221	0.015-32	4	4	16	90.5	5.0	4.5
Clindamycin
B. fragilis group	1,240	0.03-64	0.5	0.5	32	76.4	8.3	15.3	401	0.03-16	0.25	0.25	16	71.3	5	23.7
B. fragilis	714	0.03-64	0.5	0.25	4	89.5	1.0	9.5	180	0.015-16	0.25	0.25	16	77.2	0	22.8
Other species	526	0.03-64	4	2	32	58.6	18.3	23.1	221	0.015-16	16	1	16	66.5	9	24.5
Trovafloxacin
B. fragilis group	496	0.25-32	0.25	0.25	2	91.9	4.6	3.5	401	0.015-4	0.25	0.25	2	92.8	6	1.2
B. fragilis	274	0.25-32	0.25	0.25	1	96.3	2.6	1.1	180	0.015-4	0.25	0.25	2	93.3	6.7	0
Other species	222	0.25-32	1	1	4	86.5	7.2	6.3	221	0.015-16	0.5	0.5	2	92.3	5.4	2.3

^aOther species included isolates of B. thetaiotaomicron, B. ovatus, B. distasonis, B. vulgatus, B. uniformis, B. caccae, B. stercoris, and B. eggerthii.

^b50 and 90, MIC₅₀ and MIC₉₀, respectively.

Individual MICs for each antimicrobial were collated to establish MIC ranges, MICs at which 50 and 90% of the isolates tested are inhibited(MIC₅₀s and MIC₉₀s, respectively), and the percentage of isolates categorized as susceptible (S), intermediate (I), and resistant (R) based on NCCLS recommendations (10). Breakpoints for ertapenem were ≤4 as S, 8 as I, and ≥16 as R (provisional breakpoints from the NCCLS summary minutes, Meeting of the Subcommittee on Antimicrobial Susceptibility Testing, Reston, Va., 7 to 9 June 1998, p. 15-16).

Table 1 compares the temporal distributions of the test isolates. In the 1989-1990 study, the B. fragilis species accounted for 57.6% of the isolates, whereas in 1998 to 1999, B. fragilis species isolation rates decreased by 12.6 to 45% of the isolates (P < 0.0001). Concomitantly, the isolation rates of the non-B. fragilis species rose from 42.4% to 55% (P < 0.0001), with a notable increase in the percentage of B. uniformis and B. caccae isolates. These percent changes are similar to those recently reported by Golan et al. (41st ICAAC). They found a decrease of 13% (P < 0.0001) in B. fragilis species isolates in an ongoing antimicrobial resistance survey between 1984 to 1985 and 1999 to 2000.

TABLE 1.

Distribution of test isolates for susceptibility studies during 1989 to 1990 and 1998 to 1999

Isolate	No. of isolates
	1989-1990	1998-1999
B. fragilis group	1,240	401
B. fragilis	714	180
Non-B. fragilis
B. thetaiotaomicron	234	73
B. ovatus	117	41
B. distasonis	81	27
B. vulgatus	76	33
B. uniformis	14	21
B. caccae	3	22
B. stercoris	0	4
B. eggerthii	1	0

^aThe 714 1989-1990 and 180 1998-1999 B. fragilis isolates represent 57.6 and 45%, respectively, of the B. fragilis group isolates. The 526 1989-1990 and 221 1998-1999 non-B. fragilis isolates represent 42.8 and 55%, respectively, of the total number of isolates tested.

The changes in antimicrobial susceptibility parameters of the B. fragilis group varied by type of antimicrobial agent and percentage of B. fragilis isolates within the test pool for the two studies (Table 2). Table 3 compares the statistical significance of these differences for the susceptibility rates. For piperacillin-tazobactam, a single B. ovatus isolate had an intermediate MIC in the 1989-1990 study, with all other isolates susceptible, while 100% of the isolates were susceptible during 1998 to 1999. In contrast, susceptibility to ampicillin-sulbactam decreased overall from 96.9% in 1989 and 1990 to 89% in 1998 and 1999. This decrease in ampicillin-sulbactam susceptibility was seen among both B. fragilis and non-B. fragilis species, but a greater contribution was made by a greater percentage of non-B. fragilis species. Overall, susceptibility to the carbapenems remained the same for imipenem and meropenem from 1989 and 1990 to 1998 and 1999. Among the members of the B. fragilis group, resistance to imipenem is primarily among the B. fragilis species (1, 3, 4, 13, 15), whereas resistance to meropenem is found among other species as well (3). Our data presented here support these findings. Ertapenem, a newly approved carbapenem, however, showed a significant increase in susceptibility between 1989 to 1990 and 1998 to 1999. In 1989 to 1990, the highest rates of resistance to ertapenem were noted among non-B. fragilis species; however, in 1998 to 1999, even though the percentage of non-B. fragilis isolates tested increased, the resistance rate decreased appreciably, was lower than that among the B. fragilis species, and was similar to those of imipenem and meropenem. Interestingly, cefoxitin susceptibility increased in the latter study for both the B. fragilis and non-B. fragilis species. Clindamycin resistance increased from 1989 and 1990 to 1998 and 1999 and was higher among the non-B. fragilis species in both studies. Similarly, overall resistance to trovafloxacin decreased slightly in the later study, including a decrease in non-B. fragilis species resistance rates compared to those in the 1989-1990 data.

TABLE 3.

Comparison of antimicrobial susceptibilities of the B. fragilis group, B. fragilis species, and other species^a or 1989 to 1990 and 1998 to 1999

Antimicrobial and organism	Result for:				P valueb
	1989-1990		1998-1999
	No. tested	% Susceptible	No. tested	% Susceptible
Piperacillin-tazobactam
B. fragilis group	496	99.6	401	100	0.505
B. fragilis	274	100	180	100	NAc
Other species	222	99.1	221	100	0.499
Ampicillin-sulbactam
B. fragilis group	1,240	96.9	401	89.1	0.001*
B. fragilis	714	98.3	180	91.6	<0.001*
Other species	526	95.1	221	86.9	<0.001*
Imipenem
B. fragilis group	742	99.6	401	99.8	1.000
B. fragilis	438	99.3	180	99.4	1.000
Other species	304	100	221	100	NA
Ertapenem
B. fragilis group	496	91.7	401	98.8	0.001*
B. fragilis	274	94.5	180	97.8	0.090
Other species	222	88.3	221	99.5	<0.001*
Meropenem
B. fragilis group	742	98.9	401	98.8	0.777
B. fragilis	438	98.7	180	98.3	0.724
Other species	304	99.3	221	99.1	1.000
Cefoxitin
B. fragilis group	1,240	82.4	401	91.5	0.001*
B. fragilis	714	89.5	180	92.4	0.187
Other species	526	72.8	221	90.5	<0.001*
Clindamycin
B. fragilis group	1,240	76.4	401	71.3	0.042*
B. fragilis	714	89.5	180	77.2	<0.001*
Other species	526	58.6	221	66.5	0.042*
Trovafloxacin
B. fragilis group	496	91.9	401	92.8	0.642
B. fragilis	274	96.3	180	93.3	0.143
Other species	222	86.5	221	92.3	0.047*

^aOther species included isolates of B. thetaiotaomicron, B. ovatus, B. distasonis, B. vulgatus, B. uniformis, B. caccae, B. stercoris, and B. eggerthii.

^bP values are derived from Fisher's exact test or Pearson's chi-square test, as appropriate. ∗, P is statistically significant at an alpha level of 0.05.

^cNA, data not available.

Table 4 compares the susceptibility rates of the antimicrobials among the B. fragilis group species with >10 isolates in each study. For piperacillin-tazobactam, only a single strain of B. ovatus with an intermediate MIC was noted in 1989 to 1990, all other strains in both studies being susceptible. Imipenem resistance was noted only in B. fragilis isolates compared to the other species. For ertapenem and meropenem, resistance was detected among B. fragilis isolates in both studies, but varied among the non-B. fragilis species in each study, with the exception that B. uniformis isolates for which MICs were >4 μg/ml were isolated in both studies. For ampicillin-sulbactam and clindamycin, all B. fragilis group species showed a decrease in susceptibility from 1989 and 1990 to 1998 and 1999.

TABLE 4.

Comparison of the susceptibility rates of the B. fragilis group species tested during 1989 to 1990 and 1998 to 1999

Antimicrobial agent	% Susceptibilitya
	B. fragilis		B. thetaiotaomicron		B. ovatus		B. distasonis		B. vulgatus		B. uniformis
	1989-1990	1998-1999	1989-1990	1998-1999	1989-1990	1998-1999	1989-1990	1998-1999	1989-1990	1998-1999	1998-1990	1998-1999
Piperacillin-tazobactam	100	100	100	97	100	100	100	100	100	100	100
Ampicillin-sulbactam	98	92*	96	90	99	88*	85	67*	99	88*	100	86
Imipenem	99.3	99.4	100	100	100	100	100	100	100	100	100	100
Ertapenem	95	98	95	100	88	100*	59	100*	100	97	100	100
Meropenem	98	98	99	100	100	100	100	100	100	97	88	95
Cefoxitin	90	93	69	90*	73	95*	70	89	86	91	79	81
Clindamycin	90	77*	50	77*	60	59	52	59	78	70	79	76
Trovafloxacin	96	93	86	99*	82	93	82	96	97	76*	100	91

^aP values were derived from Fisher's exact test or Pearson's chi-square test, as appropriate. ∗, P is statistically significant at an alpha level of 0.05 and indicates a significant change between the 1989-1990 and 1998-1999 studies.

A limitation of our analysis was that during the 1989-1990 study, there was a modification of the antimicrobial test panel, and not all isolates were tested against all antimicrobials. The percentage of B. fragilis species isolates tested against piperacillin-tazobactam, imipenem, ertapenem, meropenem, and trovafloxacin varied slightly from the overall average, but not enough to influence the overall susceptibility data.

Anaerobic bacteriology by nature is time-consuming compared with aerobe bacteriology, even for the most-rapidly-growing anaerobes, such as species in the B. fragilis group. This often delays identification and susceptibility data for use in individual patients. Therefore, the empirical choice of antimicrobial therapy by clinicians for mixed aerobic-anaerobic infections is important. Multicenter susceptibility studies are effective tools to monitor changing antimicrobial susceptibilities on a local, regional, or national basis. Today antimicrobial resistance among the species in the B. fragilis group spans all classes of approved antimicrobials, including recent reports of metronidazole resistance responsible for clinical failures (14; R. Chaudry, P. Mathur, B. Dhawan, and L. Kumar, Letter, Emerg. Infect. Dis. 7:485-486, 2001). This study has shown the following. (i) The distribution of species of the B. fragilis group has changed from 10 years previously in favor of greater numbers of non-B. fragilis isolates. (ii) The decrease in B. fragilis isolates, usually the most susceptible species, resulted in increasing resistance rates for some antimicrobials (clindamycin and ampicillin-sulbactam) and decreasing resistance rates for other agents (carbapenems). (iii) Complete species identification and antimicrobial susceptibilities are important because they most accurately delineate any change in antimicrobial resistance. Thus, future surveys like this one will be important in monitoring new and significant changes in antimicrobial susceptibilities among anaerobes.