In vitro susceptibility of the Bacteroides fragilis group in community hospitals

DIAGNMICROBIOLINFECT DIS 1986;5:317-322

317

ANTIBIOTIC SUSCEPTIBILITYTESTING

In Vitro Susceptibility of the Bacteroides fragilis Group in Community Hospitals Thomas M. File Jr, Richard B. Thomson Jr, James S. Tan, Sara-Jane Salstrom, George A. Jacobs, Lynette Johnson, and Lulu Tan

The antimicrobial susceptibility of clinical isolates of the Bacteroides ~ragilis group was determined at six community hospitals (one large, >600 beds; and five smaller, 96-325 beds). Imipenem was the most active/3-1actam with 100% of isolates being sensitive at 4 pg/ml. The percentage of isolates inhibited at 16 pg/ml (and 32 vLg/ml)for the 7-alpha-methoxy antibiotics was: cefoxitin 66 (901; moxalactam 73 (851; cefotetan 68 (721; cefmetazole 40 (61). Metronidazole, chloremphenicol, and clindamycin were active against 100%, 100%, and 89% at breakpoints of 8 pg/ml, 8 pg/m], and 4 pg/ml, respectively. The activity of several [3-Iactams in our report differed slightly from that reported from university teaching hospitals. There were differences at many of the breakpoints for activity of some of the #-lactam antibiotics for isolates from the large community hospital as compared with the combined isolates from the smaller community hospitals. Interestingly, the pattern was one of more resistance at the smaller community hospitals. INTRODUCTION It is our experience that m a n y hospitals do not r o u t i n e l y perform susceptibility testing of anaerobic bacteria. Therefore, most p h y s i c i a n s rely on p r e v i o u s l y p u b l i s h e d information c o n c e r n i n g s u s c e p t i b i l i t y patterns. The majority of p r e v i o u s l y p u b l i s h e d information comes from large u n i v e r s i t y referral centers. It is quite possible that the patterns of s u s c e p t i b i l i t y to a n t i m i c r o b i a l agents in such centers m a y differ from smaller c o m m u n i t y hospitals. This s t u d y provides a d d i t i o n a l information concerning the s u s c e p t i b i l i t y patterns of the Bocteroides frogilis group w i t h i n c o m m u n i t y hospitals and evaluates the s u s c e p t i b i l i t y patterns of these organisms to some n e w l y available antimicrobials.

From the service of Infectious Disease and Microbiology, Laboratory Akron City Hospital, Akron, OH and the Northeastern Ohio Universities College of Medicine, Rootstown, OH. This paper was presented in part at the 24th Interscience Conference on Antimicrobial Agents and Chemotherapy. Address reprint requests to: Thomas M. File, Jr., M.D., 75 Arch Street, Suite 204, Akron, OH 44304. Received October 31, 1985; revised and accepted April 7, 1986. © 1986 Elsevier Science Publishing Co., Inc. 52 Vanderbilt Avenue, New York, NY 10017

0732-8893/86/$03.50

318

T.M. File et al.

MATERIALS AND METHODS

Bacteroides fragilis group strains were collected from nonduplicate clinical samples in six community hospitals. Akron City Hospital (ACH) is a 618-bed teaching hospital. The five remaining smaller hospitals ranged from 96 to 325 beds and included the following: Cuyahoga Falls General Hospital, Cuyahoga Falls, OH; Robinson Memorial Hospital, Ravenna, OH; Wadsworth Rittman Hospital, Wadsworth, OH; Wooster Community Hospital, Wooster, OH and Barberton Citizen Hospital, Barberton, OH. Isolates were collected from January 1983 through December 1983. All isolates were identified by API 20A (Analytab Products, Plainview, NY) supplemented with methods developed by Finegold and Martin (1982) and by Holdeman et al. (1977). Isolates were stored in chopped meat broth without glucose at room temperature for a maximum of 3 months before determination of susceptibility. Standards of antimicrobial drugs were supplied by the manufacturers. Minimum inhibitory concentrations (MICs) were determined in the bacteriology laboratory of ACH by an agar dilution method as previously described (Sutter and Washington, 1980). In brief, portions of five colonies were inoculated into thioglycollate medium without indicator, supplemented with hemin (5 ~g/ml) and vitamin K1 (0.1 ~g/ml), and incubated for 18-20 hr. Just prior to the test, the turbidity was adjusted to a 0.5 McFarland standard by adding the thioglycollate broth culture to brucella broth medium. Plates were prepared containing Wilkins-Chalgren agar (Difco Laboratories, Detroit, MI) and serial twofold dilutions of the antimicrobial in concentrations ranging from 128 to 0.125 ~.g/ml. Plates were inoculated using a Steers replicator. Approximately 105 colony forming units (CFU) were inoculated per spot. Plates were incubated in an anaerobic chamber for 48 hr at 35-37°C. The minimal inhibitory concentration of each strain is the lowest concentration of drug yielding no growth, one discrete colony, or a fine, barely visible haze, Control organisms included Bacteroides fragilis ATCC 25285, B. thetaiotaomicron ATCC 29741, and Clostridium perfringens ATCC 13124. The MIC of the control organisms were within the accepted standards (Sutter and Washington, 1980). Breakpoints for susceptibility were selected on the basis of achievable blood levels as used by Cuchural et al. (1984). Statistical analysis was performed by using ×2 analysis with the Yates correction (Guilford and Fruchter, 1970). RESULTS There was a total of 123 strains of the B. fragilis group evaluated: 82 from ACH and 41 from the other hospitals. The species tested and the number of isolates tested for each species were: B. fragilis 82, B. thetaiotaomicron 21, B. distasonis 8, B. vulgatus 23, B. ovatus 3, B. uniformis 1. The percentage of the species of the B. fragilis group isolated from ACH and from the other hospitals was similar (B. fragilis: ACH 67%, others 61%; B. thetaiotaomicron: ACH 15%, others 12%; B. distasonis: ACH 6%, others 5%; B. vulgatus: ACH 11%, others 20%; B. ovatus: ACH 0%, others 2%; B. uniformis: ACH 1%, others 0%). The susceptibility rates (MICs for 50% and 90% of isolates, respectively and percent of isolates susceptible at two breakpoints) of the B. fragilis group to 15 antimicrobial agents are shown in Table 1. The ~-lactam antibiotics had variable activity with imipenem showing the greatest activity. Of the cephalosporin type antimicrobials, moxalactam had the lowest MICso (4.0 p.g/ml) whereas cefoxitin had the lowest MICgo (32 p.g/ml). Metronidazole and chloramphenicol were also consistently active. Clindamycin was also very active but 6% of isolates had an MIC ~8 t~g/ml. Figure 1 indicates the cumulative activity of the antimicrobial agents against the 123 strains isolated. A comparison of the susceptibility of the B. fragilis group strains isolated from

B. fragilis Susceptibility in C o m m u n i t y Hospitals

319

TABLE 1. Susceptibility of B. fragi|is Group (Combined Data of Six Northeastern Ohio Community Hospitals--123 Isolates) Percent susceptible at breakpoint

MIC (ug/ml) Antimicrobial agent

50%o

Imipenem Moxalactam Cefoxitin Cefotetan Cefmetazole Ceftizoxime Cefotaxime Cefmenoxime Ceftriaxone Cefoperazone Metronidazole Chloramphenicol Clindamycin Mezlocillin

0.5 4.0 16 16 32 32 32 32 32 64 1.0 4.0 1.0 32

90%

Range

1.0 64 32 128 128 >128 >128 128 >128 >128 1.0 8.0 8.0 128

Breakpoint (ug/ml)

(%0)

8 16(32) ° 16(32) 16(32) 16(32) 16(32) 16(32) 16(32) 16(32) 16(32) 8(16) 8(16) 4( 8) 64(128)

100 73(85) b 66(90) 68(72) 40(61) 34(63) 33(67) 18(50) 26(51) 4(14) 100 100 89(94) 87(92)

<0.1254 0.25->128 4 - 128 4 - 128 4->128 0.25->128 0.5->128 1->128 1->128 2->128 0.252 28 <0.125->128 2->128

°Higher breakpoint bPercent susceptible at higher breakpoint.

-

Z:S'"

. COMBIN123 ED strai/ rl~iili~i / "/ f#+"" 3~/ "'''/''" 8O

b"

_z

/.::,I~"Y l 14;

40

20

I

t

/

/ !

/..'"

.-'"

.,'/.., /k /I/,

/'

/

0

.

: .

.

.

.

-

I

___

J

...

i

i

/./.,/lx" ~"

i

......:,'~

.~ ...~

I

... ~

~//

I::

......~;.,

10

I-

I

I

!.S

,

/-

'

.... ,,,....

~10.1250.25 0.5 1 2 4

8

16

32

64

128

M i c (m~i.)

FIGURE 1. Cumulative susceptibility o f B. fmgilis group isolates from community hospitals. A, cefoperazoue (CPZ); B, cefmenoxime (MEN); C, cefrnetazole (MET); D, cefotaxime (CTX); E, cefotetan (TET); F, cefoxitin (FOX); G, ceftizoxime (CZX); H, ceftriaxone (RO); O, chloramphenical (CHL); J, clindamycin (CLD); K, metronidazole (MTN); L, mezlocillin (MZ); M, moxalactam (MOX); N, imipenem (IPM).

320

T.M, F i l e e t a l .

TABLE 2. C o m p a r i s o n of In Vitro S u s c e p t i b i l i t i e s of B. f r a g i l i s G r o u p i n Large a n d Small Hospitals Percent susceptible at Antimicrobial agent

8 ug/ml

16 ug/ml

32 ug/ml

ACH a

Other b

ACH

Other

ACH

Other

Imipenem Moxalactam Cefoxitin Cefotetan

100 68 16 35

100 73 17 15c

100 73 66 71

100 73 66 63

100 83 91 73

100 88 88 71

Cefmetazole Ceftizoxime

9 28

2 2

46 40

27 22

60 73

63 41

Cefotaxime

17

5

40

17

77

49

7

5

20

15

57

34

20

2

33

12

61

32

4

0

6

0

34

7

100 100 93 16

100 100 98 12

100 100 95 57

100 100 98 32

100 100 96 80

100 100 98 66

Cefmenoxime ~ Ceftriaxone Cefoperazone Metronidazole Chloramphenicol Clindamycin Mezlociliin

°Strains (82) from ACH (618 beds). bStrains (41) from five small hospitals (average 228 beds, range 96-325 beds). CRules indicate significant (p < 0.05) difference between underline values.

TABLE 3. S u s c e p t i b i l i t y of S p e c i e s of B. f r a g i l i s G r o u p a ( C o m b i n e d Data of Six N o r t h e a s t e r n O h i o C o m m u n i t y H o s p i t a l s ) Percent susceptible to antimicrobial agent for Antimicrobial agent Imipenem Moxalactam Cefoxitin Cefotetan Cefmetazole Cetizoxime Cefotaxime Cefmenoxirne Ceftriaxone Cefoperazone

Breakpoint (ug/ml)

B. fragilis (80 isolates)

B. thetaiotaomicon (17 isolates)

B. distasonis B. vulgatus (7 isolates) (17 isolates)

8 16 16 16 16 16 16 16 16 16

100 95 78 89 49 37 37 24 30 5

100 6 12 0 0 24 18 0 6 0

(53) (88) (0) (0) (35) (23) (6) (6) (18)

100 29 29 29 14 29 29 14 29 14

100 100 59 (94) 100

100 100 100 71

(32) b (32) (32) (32) (32) (32) (32) (32) (32)

Metronidazole 8 (16) Chloramphenicol 8 (16) Clindamycin 4 (8) Mezlocillin 64

100 100 97 82

(99) c (93) (93) (78) (66) (80) (63) (59) (26)

(29] (71) (29) (29) (71) (43) (43) (57) (14)

100 100 88 65 12 35 29 12 29 0

(94) (71) (24) (71) (71) (58) (59) (24)

100 100 100 76

°There was one isolate each of B. ovatus and B uniformis not included separately because of insignificant numbers. Although there were only seven isolate of B. distosonis, the results are presented separately to show their specific findings. bHigher breakpoint. CPercent Susceptible at higher breakpoint.

B. fragilis Susceptibility in Community Hospitals

321

ACH as compared with those strains isolated from the combined group of smaller hospitals is indicated in Table 2. In all cases in which there was a statistically significant difference the result indicated a decreased susceptibility at the smaller hospitals. An evaluation of the isolates from each hospital indicated that this difference was not due to strains from any one hospital alone. The susceptibility of the specific species of B. fragilis group for the antimicrobial tested is indicated in Table 3. B. thetaiotaomicron and B. distasonis were more resistant compared with B. fragi|is and B. vulgatus.

DISCUSSION

Imipenem was the most active of the ~-lactam antibiotics evaluated demonstrating effective inhibition of 100% of the Bacteroides fragilis group isolates from our community hospitals. This agrees with previously published data from large medical centers (Aldridge eta]., 1984; Owens and Finegold, 1983). Of the other ~-lactams tested the 7-alpha-methoxy agents (moxalactam, cefoxitin, cefotetan, cefmetazole) had the greatest activity, particularly moxalactam and cefoxitin. Most of the isolates had one of two breakpoints for cefoxitin (16 or 32 p.g/ml). On the other hand the susceptibility of moxalactam was far more variable with 65% having MIC <4 ~Lg/ml but 15% being resistant to 32 p~g/ml. The activity of cefotetan was comparable to cefoxitin at the 16 ~g/ml breakpoint level (68% vs. 66%, respectively): however, cefmetazole was active against only 40% at 16 ~g/ml. The other cephalosporin type antibiotics had rather high rates of resistance. The activity of metronidazole, chloramphenicol, and clindamycin in the present study was very similar to that found by others at large university hospitals (Cuchural et al., 1984). All isolates were susceptible in low concentrations to metronidazole and chloramphenicol whereas 94% were susceptible to clindamycin (breakpoint of 8 p~g/ml). The activity of many of the established ~-lactam antimicrobial agents was less in our study than that reported in the multicenter university hospital study by Cuchural et el. (1984). The MICso and MIC9oof cefoxitin was 8 and 16 p,g/ml, respectively in their study as compared with 16 and 32 p,g/ml in the present study. This may be due to differences in inoculum s i z e - - - 1 0 s CFU per inoculum spot for the present study versus -104 CFU per inoculum spot used in the study by Cuchural et al. (1984) and by Tally et al. (1975). On the other hand Aldridge et el. (1984) in a different study found very similar results to ours for cefoxitin but showed greater activity for ceftizoxime (MICgoof 16 p,g/ml compared with >128 p,g/ml in the present study). Such differences may likely reflect resistant patterns that vary geographically. There were differences at many of the breakpoints for activity of some of the ~lactam antibiotics for isolates from ACH as compared with the combined isolates from the smaller community hospitals. Interestingly, the pattern was one of more resistance at the smaller community hospitals. The reason for this difference was not directly discernable from information obtained in the study. A previous report comparing the susceptibility of B. fragilis isolates from four large teaching hospitals in Detroit found that isolates from two of the hospitals were more resistant to clindamycin and speculated that this was due to increased use of clindamycin in these specific hospitals (Bawdon et al., 1979). Increased use of the antimicrobials that had decreased activity could not be the explanation for our results since those antibiotics were not used at the community hospitals (indeed none of them were marketed at the time of this study). With the exception of cefotetan at a break point of 8 p.g/ml, the decreased activity was not observed with the 7-alpha-methoxy [3-1actams which appear to have the greatest stability among the cephalosporinlike antibiotics to the

322

T.M. File et al.

c o m m o n l y f o u n d ~-lactamases (Jones a n d Wilson, 1983). These results of the susceptibility of B. fragilis isolates s u p p o r t previous r e c o m m e n d a t i o n s (Cuchural et al., 1984; A l d r i d g e et al., 1983) that s u s c e p t i b i l i t y testing be performed on significant isolates in each hospital in order to d e t e r m i n e local susceptibility patterns. Certainly our results indicate that this s h o u l d a p p l y to smaller c o m m u n i t y hospitals as well. As p r e v i o u s l y reported (Cuchural et al., 1984; Eley and Greenwood, 1984) there were significant differences among susceptibility rates of the i n d i v i d u a l species of the B. fragilis group thus e m p h a s i z i n g the need for performing identification of species in order to judge e m p i r i c t h e r a p y prior to k n o w l e d g e of susceptibility results. The authors thank Winnie Kelleher and Leslie Harth for their secretarial assistance.

REFERENCES Aldridge KE, Sanders CV, Janney A, Sebastian F, Marier RL (1984} Comparison of the activities of penicillin G and new D-lactam antibiotics against clinical isolates of bacteroides species. Antimicrob Agents Chemother 26:410. Bawdon RE, Rozmiej E, Palchaudhuri S, Krakowiak J (1979) Variability in the susceptibility pattern of bacteroides fragilis in four Detroit area hospitals. Antimicrob Agents Chemother 16:664. Cuchural GJ Jr, Tally FP, Jacobus NV, Gorbach SL, Aldridge K, Cleary T, Finegold SM, Hill G, Iannini P, O'Keefe JP, Pierson C (1984) Antimicrobial susceptibilities of 1,292 isolates of the Bacteroides fragilis group in the United States: comparison of 1981 with 1982. Antimicrob Agents Chemother 26:145. Eley A, Greenwood D (1984) Variations in susceptibility to latamoxef (moxalactam) and cefoxitin within the Bacteroides fragilis group. J Antimicrob Chemother 13:245. Finegold SM, Martin J (1982) Diagnostic Microbiology. St. Louis: CV Mosby Co., pp 138-139; 309-318. Guilford JT, Fruchter B (1970) Fundamental Statistics in Psychology and Education. New York: McGraw-Hill, pp 193-211. Holdeman LV, Cato EP, Moore WEC (1977) Anaerobic Laboratory Manual, 4th ed. Blacksburg, VA: Virginia Polytechnic Institute. Jones RN, Wilson HW (1983) Antimicrobial activity, beta-lactamase stability and beta-lactamase inhibition of cefotetan and other 7-alpha-methoxy beta-lactam antimicrobials. Diagn Microbiol Infect Dis 1:71. Owens WE, Finegold SM (1983) Comparative in vitro susceptibilities of anaerobic bacteria to cefmenoxime, cefotetan, and N-formimidoyl thienamycin. Antimicrob Agents Chemother 23:626. Sutter V, Washington J (1980) Susceptibility testing of Anaerobes. In Manual of Clinical Microbiology, 3rd ed. Eds., EH Lennette et al. Washington, D.C,: American Society for Microbiology, pp 475-477. Tally FP, Jacobus NV, Bartlett JG, Gorbach SL (1975) Susceptibility of anaerobes to cefoxitin and other cephalosporins. Antimicrob Agents Chemother 7:128.