Abstract
The in vitro susceptibilities of 170 clinical isolates plus 12 American Type Culture Collection strains of Pasteurella species comprising nine species and three Pasteurella multocida subspecies were studied by an agar dilution method. Garenoxacin (BMS-284756), a new des-fluoro(6) quinolone, was active at ≤0.06 μg/ml against all isolates, including four β-lactamase-producing strains, with >90% of the strains susceptible to ≤0.008 μg/ml. Garenoxacin was generally 1 to 2 dilutions more active than levofloxacin and moxifloxacin and was the most active agent tested. Cefoxitin required 1 μg/ml for inhibition of 51 of 182 (29%) of strains, and 3 strains (also β-lactamase producers) were resistant to doxycycline.
Approximately 245,000 to 725,000 (5 to 15% of the 4.5 million people who suffer animal-bite wounds in the United States annually seek medical attention for infection, including 30,000 patients who visit an emergency department for medical treatment and 10,000 who are hospitalized with a bite wound infection (5, 16). Bite wound infections involve a complex polymicrobial flora (5, 6, 16), among which Pasteurella multocida is considered an important pathogen by clinicians. Recent advances in molecular methods have suggested a more complex taxonomic structure of the genus Pasteurella, with 11 genetically closely related taxa, the establishment of three subspecies of P. multocida, and a reevaluation of 11 taxa with low levels of genetic relatedness (4, 13). Many of the genetically closely related species have different ecological niches, different pathogenic potentials, and different propensities for specific tissue invasion. A recent prospective study (16) demonstrated that Pasteurella species are the most common aerobic isolates from both dog bites (50%) and cat bites (75%). However, P. canis is the predominant isolate present in 26% of dog-bite wounds, followed by P. stomatis (12%) and P. multocida subsp. multocida (12%). In addition, P. dagmatis (4%) and P. multocida subsp. gallicida (2%) and P. multocida subsp. septica (10%) are also encountered in dog-bite wounds. In contrast, P. multocida subsp. multocida and P. multocida subsp. septica are more prevalent in cat-bite wound infections (present in 54 and 28% of such wound infections, respectively). Most studies of the antimicrobial susceptibilities of members of the family Pasteurellaceae are often limited because veterinary and human isolates are combined or the full spectrum of Pasteurella species causing human infections is not tested.
Pasteurella species are associated with a variety of human infections, most but not all of which are associated with animal contact, especially dog and cat bites. While many patients with bite wounds receive a β-lactam agent such as amoxicillin-clavulanate, approximately 20% will report a history of an adverse reaction to penicillin or other β-lactam antibiotics and require an appropriate alternative agent. Fluoroquinolones have been suggested as alternative agents for the treatment of bite wounds. Garenoxacin {BMS-284756; T-3811ME; 1-cyclopro-pyl-8-(difluoromethoxy)-7-[(1R)-1-methyl-2,3-dihydro-1H-5-isoindolyl]-4-oxo-1,4-dihydro-3-quinolinecarboxylic acid methanesulfonate monohydrate} is a new des-fluoro(6) quinolone that lacks the 6-position fluorine that characterizes the previous class of fluoroquinolones. Preliminary data (2, 10, 15) indicate that this drug has a broad spectrum of activity against most gram-positive and gram-negative aerobes including certain strains that are resistant to other fluoroquinolones.
We therefore studied the antimicrobial susceptibilities of 170 clinical isolates and 12 American Type Culture Collection (ATCC) strains of the family Pasteurellaceae consisting of the nine species and three P. multocida subspecies most often associated with human infections.
Almost all of the strains had previously been isolated from bite wounds (almost all of the strains were isolated from 1995 to 2002) and were identified according to standard criteria (3, 12, 13). The sources of the isolates were as follows: dog bites, 60 strains; cat bites, 81 strains; tiger bites, 2 strains; bites caused by other animals, 15 strains; and bovine respiratory sources, 12 strains. The three isolates of P. caballi and six of the eight strains of P. haemolytica were from bovine respiratory sources. For comparative purposes, 12 ATCC strains of 10 species (P. aerogenes ATCC 12192, P. canis ATCC 43326, P. dagmatis ATCC 43325, P. haemolytica ATCC 33396, P. multocida subsp. gallicida ATCC 51689 and ATCC 51696, P. multocida subsp. multocida ATCC 7228 and ATCC 12947, P. multocida subsp. septica ATCC 51688, P. pneumotropica ATCC 35149, P. stomatis ATCC 43327, and P. testudinis ATCC 3368) were also tested.
Standard laboratory powders were supplied, as follows: garenoxacin, Bristol-Myers Squibb Co., Princeton, N.J.; amoxicillin-clavulanate, SmithKline Beecham Pharmaceuticals, Philadelphia, Pa.; ampicillin-sulbactam, Pfizer Inc., New York, N.Y.; levofloxacin, Ortho-McNeil Pharmaceuticals, Raritan, N.J.; moxifloxacin, Bayer Corp., West Haven, Conn.; cefoxitin, Merck & Co., West Point, Pa.; and doxycycline and penicillin G, Sigma Chemical Co., St. Louis, Mo. Antimicrobial agents were reconstituted according to the instructions of the manufacturers. Serial twofold dilutions were added to the media on the day of testing.
Frozen cultures were transferred twice to ensure purity and good growth. Susceptibility testing was performed according to NCCLS standards (14). The basal medium used was Mueller-Hinton agar supplemented with 5% sheep blood. The agar plates were inoculated with a Steers replicator (Craft Machine Inc., Chester, Pa.) with an inoculum of 104 CFU per spot. Control plates without antimicrobial agents were inoculated before and after the inoculation of each set of drug-containing plates. Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 29213 were included as controls. Plates were incubated at 35°C in an aerobic environment for 18 to 20 h and were then examined; the MIC was defined as the lowest concentration of an agent that yielded no growth or a marked change in growth compared to the growth on the control plate.
The results of the present study, in which we tested a large number of clinical isolates of nine Pasteurella species, including the three P. multocida subspecies, are presented in Table 1. Garenoxacin was active at <0.06 μg/ml against all isolates, including four β-lactamase-producing strains (P. aerogenes, n = 1; P. caballi, n = 1; P. haemolytica, n = 2), with >90% of the strains susceptible to <0.008 μg/ml. Three of these four strains, including the two strains of P. haemolytica and the single strain of P. aerogenes, were also resistant to doxycycline. Cefoxitin required 1 to 2 μg/ml for inhibition of 51 of 182 (29%) strains. Garenoxacin was generally 1 to 2 dilutions more active than levofloxacin and moxifloxacin on a weight basis and was the most active agent tested.
TABLE 1.
Comparative in vitro activities of the des-quinolone garenoxacin (BMS-284756) and seven other agents against 182 clinical isolates of Pasteurella species
| Species (no. of isolates) and antimicrobial agent | MIC (μg/ml)a
|
||
|---|---|---|---|
| Range | 50% | 90% | |
| P. canis (24) | |||
| Garenoxacin | 0.001-0.015 | 0.004 | 0.008 |
| Moxifloxacin | 0.008-0.015 | 0.008 | 0.015 |
| Levofloxacin | 0.004-0.015 | 0.015 | 0.015 |
| Penicillin G | 0.02-0.125 | 0.06 | 0.125 |
| Amoxicillin-clavulanate | 0.02-0.25 | 0.125 | 0.25 |
| Ampicillin-sulbactam | 0.02-0.25 | 0.125 | 0.125 |
| Cefoxitin | 0.02-1 | 0.5 | 0.5 |
| Doxycycline | 0.05-0.25 | 0.125 | 0.125 |
| P. dagmatis (12) | |||
| Garenoxacin | 0.004-0.015 | 0.008 | 0.015 |
| Moxifloxacin | 0.008-0.03 | 0.015 | 0.03 |
| Levofloxacin | 0.008-0.015 | 0.015 | 0.015 |
| Penicillin G | 0.02-0.125 | 0.06 | 0.125 |
| Amoxicillin-clavulanate | 0.02-0.25 | 0.125 | 0.25 |
| Ampicillin-sulbactam | 0.06-0.125 | 0.125 | 0.125 |
| Cefoxitin | 0.125-0.5 | 0.25 | 0.5 |
| Doxycycline | 0.125-0.25 | 0.125 | 0.125 |
| P. multocida subsp. multocida (61) | |||
| Garenoxacin | 0.001-0.015 | 0.008 | 0.008 |
| Moxifloxacin | 0.008-0.03 | 0.015 | 0.015 |
| Levofloxacin | 0.008-0.03 | 0.015 | 0.015 |
| Penicillin G | 0.03-0.125 | 0.125 | 0.125 |
| Amoxicillin-clavulanate | 0.125-0.25 | 0.25 | 0.25 |
| Ampicillin-sulbactam | 0.06-0.25 | 0.25 | 0.25 |
| Cefoxitin | 0.25-1 | 0.5 | 1 |
| Doxycycline | 0.05-0.25 | 0.125 | 0.125 |
| P. multocida subsp. septica [45] | |||
| Garenoxacin | 0.001-0.015 | 0.008 | 0.008 |
| Moxifloxacin | 0.008-0.03 | 0.015 | 0.03 |
| Levofloxacin | 0.008-0.03 | 0.015 | 0.03 |
| Penicillin G | 0.02-0.125 | 0.125 | 0.125 |
| Amoxicillin-clavulanate | 0.06-0.25 | 0.25 | 0.25 |
| Ampicillin-sulbactam | 0.06-0.25 | 0.25 | 0.25 |
| Cefoxitin | 0.25-1 | 1 | 1 |
| Doxycycline | 0.06-0.25 | 0.125 | 0.125 |
| P. stomatis (21) | |||
| Garenoxacin | 0.001-0.008 | 0.004 | 0.008 |
| Moxifloxacin | 0.008-0.015 | 0.008 | 0.015 |
| Levofloxacin | 0.008-0.015 | 0.008 | 0.015 |
| Penicillin G | 0.02-0.125 | 0.06 | 0.06 |
| Amoxicillin-clavulanate | 0.02-0.25 | 0.125 | 0.25 |
| Ampicillin-sulbactam | 0.02-0.25 | 0.06 | 0.125 |
| Cefoxitin | 0.25-1 | 0.5 | 0.5 |
| Doxycycline | 0.125-0.25 | 0.125 | 0.25 |
| Other P. speciesb (19) | |||
| Garenoxacin | 0.004-0.06 | 0.015 | 0.06 |
| Moxifloxacin | 0.003-0.125 | 0.015 | 0.06 |
| Levofloxacin | 0.008-0.006 | 0.03 | 0.06 |
| Penicillin G | 0.06->8 | 0.125 | >8 |
| Amoxicillin-clavulanate | 0.02-0.25 | 0.125 | 0.25 |
| Ampicillin-sulbactam | 0.02-2 | 0.125 | 0.25 |
| Cefoxitin | 0.25-2 | 0.5 | 1 |
| Doxycycline | 0.125->8 | 0.5 | >8 |
50% and 90% MICs at which 50 and 90% of isolates tested, respectively, were inhibited.
Includes the following species: P. aerogenes (n =1), P. caballi (n = 3), P. haemolytica (n = 8), P. multocida subsp. gallicida, (n = 4), P. pneumotropica (n = 2), and P. testudinis (n = 1).
Pasteurella species are normal inhabitants of 12 to 92% of dogs and 52 to 99% of cats (9), and most human infections are associated with bites or animal exposure. Many publications do not differentiate or identify the different Pasteurella species, sometimes due to technical difficulties with the interpretation of biochemical tests (3). Our laboratory has recently reported (2) on the use of PCR fingerprinting and α-glucosidase activity as a means of differentiating P. multocida subsp. multocida from P. multocida subsp. septica. Some investigators (1, 11) have suggested different pathogenic potentials and ecological niches for the different species, which increases the clinical importance of differentiating these species in reports of studies. For example, P. multocida subsp. multocida and P. multocida subsp. septica are more frequently recovered from “more serious” infections (11), including bacteremias, and P. multocida subsp. septica is more frequently isolated from cat-bite wounds and has an affinity for the central nervous system (1).
In a prior study from our laboratory (8a), in which the same methodology described here but a limited number of isolates was used, we found that all Pasteurella multocida subsp. multocida and P. multocida subsp. septica isolates were susceptible to ≤0.015 μg of garenoxacin per ml. In general, the quinolones, including gatifloxacin (7), moxifloxacin (7), trovafloxacin (8), and levofloxacin (8, 8a), have consistently been active against P. multocida and other Pasteurella species.
On the basis of our in vitro data, garenoxacin offers an alternative for the treatment of human infections associated with all Pasteurella species.
Acknowledgments
This study was supported, in part, by a grant from Bristol-Myers Squibb Co.
We thank Judee H. Knight and Alice E. Goldstein for assistance.
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