Journal of Antimicrobial Chemotherapy (1992) 29, 383-393
In-vitro antimicrobial susceptibility of Rhodococcus equi P. Nordmam* and E. Ronco
Rhodococcus equi is an intraccllular facultative, Gram-positive cocco-batiHary organism of increasing importance as a pulmonary pathogen in HIV-positive patients. This study was carried out to evaluate the optimal antibiotic combinations for treating such infections. Four human R. equi isolates and one reference strain were tested for their susceptibilities to 36 antibiotics. In-vitro the most active antibiotics were amikacin, gentamicin, netilmicin, erythromycin, clarithromydn, roxithromytin, ciprofloxacin, sparfloxacin, rifampicin, vancomycin, teicoplanin, doxycycUne, minocycline, imipenem, meropenem and trimethoprim/sulphamethoxazole. The only bactericidal antibiotics were the aminoglycosides, ciprofloxacin, sparfloxacin and vancomycin. As determined by FIC indices, four combinations were synergistic: rifampidn-erythromycin, rifampicin-minocycline, erythromytin-minocycline and imipenem-amikacin. However, no antibiotic combinations were synergistic with the time-kill kinetic method at achievable serum concentrations or at ten-fold and halffokl the MICs. Frequencies of selection of antibiotic-resistant mutants determined at concentrations of five- and ten-fold the MICs ranged from < 1 x 10~* for erythromycin and trimethoprim/sulphamethoxazole to 5 x 10~4 for amikacin. These results may be of help in selecting the antibiotics for treating infected HIV-positive patients.
Introduction
Rhodococcus equi, (formerly known as Corynebacterhon equi) has been described as an opportunistic pathogen in immunocompromised patients (Golub, Falk & Spink, 1967; Van Etta et al., 1983; Sane & Durack, 1986; Allen et al., 1989; Prescott, 1991). Since the spread of AIDS, an increasing number of R. equi cases has been reported (Sarnies et al., 1986; Sane & Durack, 1986; Bishopric et al., 1988; Jones et al., 1989; Emmons, Reichwein & Winslow, 1991; Harvey & Sunstrum, 1991). Consequently, this pathogen is becoming a significant cause of pneumonia in HIV-positive patients. R. equi is a Gram-positive cocco-bacillary bacterium, classified as an Actinomycetales (Goodfellow, 1987). Well known to veterinary microbiologists since its first isolation by Magnusson in 1923 (Magnusson, 1923), R. equi is involved in 10% of pneumonia in foals (Hillidgc, 198S; Prescott, 1987). Both animal and human infections are thought to be acquired via the respiratory route in farms and in stables where R. equi is found not only in the soil but also in the bowels of horses and of other farm animals (Yager, 1987; Prescott, 1991). R. equi is a facultative intracellular pathogen infecting both macrophages and polymorphonuclear leucocytes but persisting in macrophages which may explain its •Corrapondence to: Dr P. Nordmann, Laboratoire de Microbiologtt Medicate, Hdpital Raymond Poincare, 104 Bd Raymond Poincare, 92380 Garches, France. O305-74S3/92/O4O383 +11 S02.00/0
383 © 1992 The British Society for Antimicrobial Chemotherapy
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Service de Microbiologie, Hdpital Raymond Poincari, Faculti de Midecine Paris-Quest, Garches, France
384
P- Nonbnann and E. Rooco
Materials and methods Bacterial strains Five distinct R. equi strains were used; four clinical isolates and a human reference strain (52T2, Institut Pasteur collection, Paris, France). The clinical strains were isolated from blood culture and bronchoalveolar brush samples from four HIV-positive patients with pneumonia. Their identification was confirmed by API Corynebacteria tests (La Balme-les-Grottes, France) and the identification centre of the Institut Pasteur. Antibiotics The antimicrobial agents used in this study were obtained from laboratory standard powders and were used immediately after their dilution. The agents and their sources were: amikarin, oxacillin, penicillin G, and netilmicin (Bristol), amoxycillin, clavulanic acid1, and ticarcillin (Beecham), latamoxef and vancomycin (Eli Lilly & Co), cepha-
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ability to escape the normal pulmonary defence mechanisms (Zink et al., 1987). In humans, pneumonia and lung abscesses are the most frequent infections (accounting for 70% of cases), occurring mainly in immunocompromised patients (Marsh & Von Gracvenitz, 1973; Berg et al., 1977; Lcbar & Pensler, 1986; Hillerdal et al., 1988; Emmons et al., 1991) and rarely in the immunocompetent (Harvey & Sunstrum, 1991). Contact with horses is often reported in infected patients (Prescott, 1991). Non-pulmonary infections have also been described, such as infectious diarrhoea, endophthalmitis, osteomyelitis, brain or pelvic abscesses (Fierer et al., 1987; Ebersole & Paturzo, 1988; Novak et al., 1988; Garretson & Fiscella, 1989). Based on experimental treatments of R. equi pneumonia in foals, rifampicin combined with erythromycin is known to be successful (Hillidge, 1987; Sweeney, Sweeney & Divers, 1987). Nevertheless, the optimal antimicrobial treatment for human infections is not yet known and the overall mortality of AIDS patients with R. equi infections remains around 50% whatever treatment is given (Harvey & Sunstrum, 1991). Various antibiotic combinations have been tried. However, the course of the disease is characterized by frequent relapses, occurrence of antibiotic-resistant mutants during treatment, and often a need for complementary surgery. Results based on the bactericidal activity of drugs alone or in combination on R. equi are few and contradictory. Prescott & Nicholson (1984) reported in-vitro synergic activity of erythromycin combined with rifampicin, as well as penicillin combined with gentamicin or erythromycin, though Novak et al. (1988) failed to detect any antibiotic synergy. Therefore, the goals of this study were to determine MICs of 36 antibiotics, including new drugs such as third generation cephalosporins, carbapenems, fluoroquinolones and new macrolides. Thus, providing information not only on the antibacterial activities of drugs used in veterinary medicine but also those recently introduced for the treatment of human infections. Furthermore, to determine MBCs which should be helpful in the choice of bactericidal antibiotics, the in-vitro antibiotic activities of drugs alone and in combination and in addition to calculate the frequencies of in-vitro selection of antibioticresistant mutants with antibiotics alone or in combination to estimate the risk of clinical selection of antibiotic-resistant mutants (since several weeks treatment is necessary for such infections), was determined.
In-Tttro susceptibility of R. eqtd
385
lothin, ceftazidime (Glaxo), ceftriaxone and trimethoprim/sulphamethoxazole (Roche), clarithromycdn and erythromycin (Abbott), cefoxitin and imipenem (Merck Sharp & Dohme), gentamicin (Schering Corp.), pristinamycin, sparfloxacin and spiramycin (Rhone-Poulenc), fusidic acid (Leo), cefotaxime, chloramphenicol and roxitnromycin (Roussel), ciprofloxacin (Bayer-Pharma), pefloxacin (Roger & Bellon), ofloxacin (Diamant), teicoplanin (Merrell Dow) rifampicin (Gruppo Lepetit), doxycycline and tetracycline base (Pfizer), lomcfloxacin (Searle), minocycline (Lederlfc), fosfomycin (Sanofi) and meropenem (ICI Pharma). In-vitro susceptibility to antibiotics
Study of combined antimicrobial activity Combinations of antibiotics were studied by two techniques (Schoenknecht, Sabath & Thornsberry, 1985; Leclercq et al., 1991). All experiments were performed in triplicate with four out of the five strains (one strain was more resistant to rifampicin and fluoroquinolones than the others). Firstly, the strains were studied by the microdilution chequerboard technique. Trays were prepared using a plastic microdilution technique (Dynatec Laboratories, Inc., Alexandria, VA) as follows. Seven antibiotics were selected from the various antibiotic groups according to some or all of the following criteria: low MIC, low MBC, high Cn^JMIC, availability of oral and intravenous dosages; these included amikacin, ciprofloxacin, erythromycin, imipenem, minocycline, rifampicin, and vancomycin. Serial two-fold dilutions of the antibiotics (concentrations from 16- to 0-12-fold the MICJQ) were performed to study antibiotic combinations. The inoculum of strains tested was adjusted to 10* cfu/mL. After 24 h incubation FIC indices were calculated using the following formula: FIC index = FIC A + FIC B = MIC of A in combination/MIC of A alone+MIC of B in combination/MIC of B alone. The FIC indices were calculated as the antibiotic combinations that were both most effective and comprised the lowest concentrations of antibiotics. An FIC index < 0-5 was considered as showing synergy, an FIC index of 0-5-4 indifference and a FIC index > 4 as showing antagonism to the combination of antibiotics.
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MICs and MBCs were determined by a macrodilution broth dilution technique in glass tubes (Jones et al., 1985) containing 5 mL of Mueller-Hinton broth (Diagnostics Pasteur, Paris). Bacterial inocula were prepared by appropriate dilutions of overnight shaked cultures of organisms in Mueller-Hinton broth. A standard inoculum of 106 cfu/mL was used in all cases. For the determination of trimethoprim/sulphamethoxazole (1:5) susceptibility, a standard agar dilution technique on Mueller-Hinton agar supplemented with 5% of lysed horse blood (Diagnostics Pasteur, Paris) was used. All these experiments were performed at 37°C. The MICs were determined as the first tube with no visible growth after 24 h incubation. MBCs were calculated as the least concentrations of drug to achieve a 99-9% reduction of the original inoculum. For antibiotics with low MBCs, MBCs and MICs were also determined after 24 h incubation at 37°C in a shaking water-bath or at 30cC in an unshaken waterbath. These latter MICs and MBCs were determined in order to take into account the aerobic requirement for maximal growth of R. equi and its optimal growth temperature of 30°C (Hughes & Sulaiman, 1987). All MIC and MBC determinations were performed in triplicate for each strain. Thefivestrains were also screened for /Mactamase production using nitrocefin (Glaxo Laboratories, Middlesex, England).
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P. Nordnuum and E. Rooco
Frequencies of in-vitro selection of antibiotic-resistant mutants Mutation rates were determined by counting the number of colonies arising from a large inoculum (10* cfu) on antibiotic containing plates at concentrations five- and ten-fold the MICs using four different strains. The same eight antibiotics were used as in the time-kill kinetics. The mutation rates were also calculated with antibiotics combined two by two at five- and ten-fold their MIC. The MICs of the mutants were subsequently determined as previously described. Results Antibiotic susceptibilities of the strains The MICs and MBCs of the antibiotics against the five strains are shown in Table I. The five strains possessed the same pattern of antibiotic susceptibilities except for one which was less susceptible to rifampicin and the fluoroquinolones. This strain had been isolated from a patient treated with rifampicin alone and then ofloxacin. The /Mactam MICs were high with the exception of imipenem (0-25 mg/L), and meropenem (0-50 mg/L). No /Mactamases were found in any strains using the nitrocefin test The MBCs for all 0-lactams were > 256 mg/L with a MBC/MIC ratio > 32. The antimicrobial activities of the aminoglycosides were similar among each other with low
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Secondly, time-kill kinetics were performed to test the bactericidal activity of antibiotic combinations with the same seven antibiotics as those studied in the FIC index test plus, trimethoprim/sulphamethoxazole. The drugs were tested at concentrations clinically achievable in serum and ten- and 0-5-fold the MICs. The antibiotic concentrations (mg/L) used weie: ciprofloxacin 3-5, 2-5, 0-12; minocycline 5, 1-2, 0-06; erythromycin 6, 2-5, 012; amikacin 24, 20, 1; imipenem 30, 2-5, 0-12; rifampicin 15, 0-6, 0-03; vancomycin 40, 2-5, 0-12; trimethoprim/sulphamethoxazole 8/32, 32/128, 1-6/6-4. Time-kill kinetic studies were determined for each antibiotic alone and in combination. All experiments were performed in glass tubes containing 5 mL of Mueller-Hinton broth in a shaking water-bath at 37°C. An inoculum of 106 cfu/mL was obtained from a dilution of fresh overnight cultures. After 0, 3, 6, 24 h incubation, water-diluted samples were plated on Mueller-Hinton agar. After 24 h incubation, the organisms isolated were tested for their antibiotic susceptibility in order to ensure that no antibiotic-resistant mutants had been selected. For trimethoprim/sulphamethoxazole time-kill determinations, the same procedure was used with supplementation of Mueller-Hinton broth with 5% lysed horse blood. Synergy was defined as > 2 logl0 decrease in cfu/mL between the combination and its most active component after 24 h incubation. Antagonism was defined as a > 2 log,0 increase in cfu/mL between the combination and either drug alone. Intermediate results were considered as indifference. Bactericidal activity was defined as > 3 log,0 reduction in growth at 24 h incubation. To ensure that antibiotic carry-over could not affect the result of the timekill kinetics, preliminary experiments were performed as follows (Pearson et al., 1980). Low numbers of bacteria (103 cfu/mL) were resuspended in liquid medium in the presence or absence of each antibiotic alone or in combination. Thereafter the same serial dilutions used for the time-kill kinetics studies were plated immediately on Mueller-Hinton agar. All plates were incubated at 37CC for 48 h before cfu were determined.
In-ritro susceptibility of R. eqtd
387
Table L MICs and MBCs of 36 antibiotics against five R. equi strains
Antibiotic Penicillin G Amoxydllin Amoxydllin/davulanic
add
ITTlipCTldH
Meropcnem Latamoxef Gentamidn Amikadn Netihnidn Tetracycline DoxycycHne Minocydrae Erythromydn Spiramydn Qarithromydn Roxithromydn Pristinamycin Gprofloxadn Ofloxadn Pefloxadn Sparfloxadn Lomefloxadn Chloramphenicol Vancomydn Teicoplanin Fusidic add Rifampidn Fosfomydn Trimethoprim/ sulphamethoxazole
2-16 4-8 4-8 64-128 256-512 128-512 8-16 32-64 1-4 >256 0-12-0-5 0-25-0-50 16-32 0-5-1 0-5-4 1-2 1-4 0-5-1 0-12-0-5 0O6-0-25 4-8 0-12-0-25 0-12-0-25 4-8 0-12-4 4-16 8-64 0-12-4 4-32 8-32 0-12-0-25 0O6-0-12 4-16 003-25 >256 1-6-^4/12-8-25^
50% 4 4 4 64 256 128 8 32 2 >256 0-25 0-50 16 0-5 2 2 2 1 0-12 0-25 8 0-12 0-12 4 0-25 4 16 0-25 4 8 0-12 006 8 006 >256 3-2/12-8
MBC (mg/L) range 50% >256 >256
>256 >256
>256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 1-2 4-8 2-4 >256 >256 >256 >256 >256 >256 >256 >256 1-16 16-256 16-> 256 1-16 16-32 >256 2-4 4-16 64-128 >256 >256 ND
>256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 2 8 4 >256 >256 >256 >256 >256 >256 >256 >256 1 32 32 1 32 >256 2 8 128 >256 >256 ND
ND, Not detennined
MIC and low MBC. The MBCs of fluoroquinoloncs were four-fold higher than those of the MICs. Among these drugs, dprofloxadn and sparfloxadn were the most active. Among the tetracycline analogues tested, the MIC of minocycline was the lowest but none was bacteriddal. Among the macrolides and pristinamycin, the MICs of erythromydn, darithromydn, and roxithromydn were the lowest No macrolides were bacteriddal at clinically achievable serum concentrations. Glycopcptidc MICs were low but
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OxadlHn Ticardllin Cephalothin Ccfoxitin Cefotaxime Ceftriaxone Ceftazidime
MIC (mg/L) range
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Table IL Values (range) of FIC indices of combinations of eight antibiotics against four R. equi strains
AMI AMI
CIP 1-8-2-2
CIP
ERY 4-5-6
2-3 ERY
IMP 0-25-0-40 0-5-1
2-3 IMP
MIN 2-4 1-2-2-5 0O3-O06 0-75-1
MIN
RIF 1-3 1-3 0-01-004 0-90-1-20 0-03-0O6
RIF
VAN 0-75-1 0-75-1
1-3 0-75-1 1-1-5 1-5-2-5
VAN
only vancomycin was bactericidal. Rifampicin showed high antibacterial activity without being bactericidal. Among the remaining antibiotics tested, the MICs of trimethoprim/sulphamethoxazole were low but MBCs were high. The M B C (mg/L) of the antibiotics found to be bactericidal were amikacin 32, gentamicin 8, netilmidn 16, ciprofloxacin 8, sparfloxacin 8, vancomycin 64, whether shaken or incubated at 30°C. Therefore, in these modified conditions, no antibiotic was bactericidal and the MIC values for these antibiotics increased in the same order of magnitude as the MBC values.
Table HI. Frequency of selection of antibiotic-resistant mutants to eight antibiotics. Antibiotic selection concentrations were five- and ten-fold the MICs. Experiments were performed with four different R. equi strains. The average increase in MICs of the antibiotic-resistant mutants are also indicated. Concentration of selection (mg-L)
Mutation frequency
Fold increase in MIC of the mutants
10 20
3-5 x10" 4 1-4 xlO" 5
8 8
Ciprofloxacin
1-25 2-50
5-8 xlO" 7 < 1 x10"'
1000
Erythromycin
1-25 2-50
< 1 xlO" 1 < 1 x10"'
Imipenem
1-25 2-50
2-9 x10"' 1-2 xlO- 6
Minocycline
0-60 1-20
4-5 x 1 0 - 7 1-2 xlO" 7
Rifampicin
0-30 O60
1-2 xlO" 7 0-5-1 x lO"7
Trimethoprim/ sulphamethoxazole
16/64 32/128
Amikacin
Vancomycin
0-6 1-2
8 8 OS OO
Antibiotic
10,000 10,000
< 1 x10"' < 1 x10"' 0-5-2x10-' < 1 x 10"'
4
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AMI, Amikacin; CIP, tiprofloxacin; ERY, erythrotnycin; IMP, imipenem; MIN, minocycHne; RIF, rifampidn; VAN, vancomycin.
In-Tttro UKeptibffity of X. eqtd
389
I09 I08 I07
J I0 6 "5 I0 5
I0 4
1
1
1
1
1
1
1
1
24
Time ( h ) Hgmc. Bactericidal effects of achievable serum concentrations of •milr»rin (AMI), ciproflozacin (CIP), erythromycin (ERY), imipcnem (IMP), minocydine (MTN),rifampicin(RTF), trirnethoprim/sulphamethoxaxole (TMS) and vancomycin (VAN) alone or in combination against four K. equi strains. The curves rcprumt mean values of bacterial counts over 24 h incubation; standard deviations are indicated by vertical bars. Control without any antibiotics (C); antibiotic concentrations alone or in combination as follows; (1): ERY, IMP, MIN, RIF, TMS, ERY-IMP, ERY-MIN, ERY-RIF, ERY-TMS, IMP-IMP, ERY-MIN, IMP-TMS, MIN-RIF, MIN-TMS, RIF-TMS, or ERY-AMI; (2): AMI, CIP, VAN, AMI-OP, AMI-VAN, OP-VAN, AMI-IMP, AMI-MIN, AMI-RIF, AMI-TMS, OP-IMP, OP-ERY, OP-RIF, OP-MIN, OP-TMS, VAN-IMP, VAN-MIN, VAN-RIF or VAN-TMS.
Combined antimicrobial activities Values of the FIC for the combinations of antibiotics are shown in Table II for four strains. The synergistic combinations were rifampicin-erythromycin, rifampicinminocycline, erythromycin-minocycline, and imipenem-amikacin. Erythromycin combined with amikacin was the only antagonistic combination. All other antibiotic combinations were indifferent In time-kill studies, the results of the bactericidal activity of the drugs are shown in the Figure. These results are obtained with antibiotic concentrations equivalent to achievable serum values (similar results were obtained when concentrations of ten-fold and half-fold the MICs were used). Ciprofloxacin, amikacin, vancomycin alone or combined with any other antibiotic (with the exception of amikacin-erythromycin) showed an average of only 2-5 log,0 cfu/mL decrease after 24 h incubation as compared to the original inoculum. The other antibiotics (rifampicin, erythromycin, imipenem, minocycline and trimethoprim/sulphamethoxazole) alone or in combination as well as ntnilracin plug erythromycin led to an average of a 1-5 log10 cfu/mL decrease compared to the original inoculum. The antibiotic combination in which one or both of the antibiotics was bactericidal, according to the above mentioned MBCs, were more bactericidal than the others. Subinhibitory concentrations of antibiotics in combination did not show any synergic effect Frequencies of in-vitro selection of antibiotic-resistant mutants Antibiotic concentrations at five- and ten-fold the MICs were used to screen for antibiotic-resistant mutants. The results of this selection are indicated in Table III.
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I0 3
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P- Nonhnaim and El Ronco
Combinations of antibiotics (even at the concentration offive-foldtheir MIC) reduced the mutation frequencies for all antibiotics to < 1 x 10"'. All antibiotic-resistant mutants showed a similar increase in their MIC values with antibiotics of the same group.
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Discussion Since only a few strains were studied, the results of MICs, MBCs, combined antimicrobial activities, and mutation frequencies were similar, with the exception of susceptibility to rifampicin and fluoroquinolones of one strain. To our knowledge, this is the first report of such extensive susceptibility testing of human R. equi isolates. The MICs are in general accordance with those previously reported for animal or human strains (Woolcock & Mutimer, 1980; Prescott, 1981; Harvey & Sunstrum, 1991; Deere et al., 1991). The isolates had intermediate susceptibility or were resistant to all /Mactam agents as previously described for clinical isolates but not for soil or animal strains (Prescott, 1991). Carbapenems showed low MICs. Since the MIC of ceftriaxone was 2 mg/L, it should be mentioned that when susceptibility was determined by the disc diffusion method, colonies occurred around the ceftriaxone disc (data not shown). This suggests an inducible mechanism of resistance to ceftriaxone. The /Mactam resistance of human isolates is not explained by the presence of /Mactamase. Only one R. equi isolate has been described previously as /Mactamase-positive but neither extended /Mactam susceptibilities nor characterization of this enzyme has been reported (Fierer et al., 1987). As with other Gram-positive bacteria, low affinity of /Mactams for penicillin binding proteins may be an explanation. Whichever /Mactam agent was studied MBCs were always > 256 mg/L. It is suggested therefore that tolerance may be a widespread mechanism of resistance in R. equi. This possibility, associated with a high in-vitro mutation frequency towards imipenem underlines the fact that /Mactams should never be used as monotherapy to treat infections caused by R. equi. Differences in susceptibilities occurred among antibiotics of the same group. Strains were susceptible to ciprofloxacin and sparfloxacin but less so to lomefloxacin, ofloxacin, and pefloxacin. The MICs of erythromycin, roxithromycin and clarithromycin were low, but those of pristinamycin and spiramycin were relatively high. Strains were susceptible to minocycline and doxycycline but less so to tetracycline. Therefore, testing one antibiotic from each class may not allow one to deduce the overall susceptibility to other antibiotics of the same group. This may be clinically relevant. Among the remaining antibiotics, R. equi strains were fully susceptible to aminoglycosides, rifampicin and glycopeptides as has been reported previously by Emmons el al. (1991). However, fosfomycin was totally inactive (Woolcock & Mutimer, 1980). Few data are available on the bactericidal activity of antibiotics against R. equi. Novak et al. (1988) did not find any antibiotic to be bactericidal though Emmons et al. (1991) showed that vancomycin, erythromycin, imipenem were bactericidal. In our study, the aminoglycosides, ciprofloxacin, sparfloxacin and vancomycin were bactericidal when the same MBC procedures were used. However, no antibiotics were bactericidal in shake cultures. Such differences in bactericidal activity may have arisen from the aerobic requirements for the maximal growth of R. equi. Thus, incubation temperature as well as shaking conditions may be critical for the MBC determinations of these slow growing organisms. Of the antibiotic combinations tested, only four were synergic as determined by the FIC indices: erythromycin-rifampicin, erythromycin-minocycline, rifampicin-
In-ritro •accyUbfltty of R. eqtd
391
Antibiotic-resistant mutants were selected in vitro at detectable frequencies with single antibiotics, except for erythromycin and trimethoprim/sulphamethoxazole. Such results correlate well with the high frequency of in-vivo selection of R. equi antibioticresistant mutants. The selection frequency of amikacin-resistant mutants was very high as it is for aminoglycosides and other Actinomycetales such as Mycobacterium tuberculosis. Rifampicin- and ciprofloxacin-resistant mutants possessed MICs which were greater than clinically achievable serum concentrations which may explain treatment failures (Nordmann et al., 1992). However, antibiotics in combination provided undetectable in-vitro mutation frequencies. This result justifies the clinical use of antibiotics in combination. Imipenem-resistant mutants are interesting as they show cross-resistance for carbapenems and cephamycins only. High susceptibility to imipenem may be explained by the existence of a special penicillin binding protein of high affinity for carbapenems and cephamycins (P. Nordmann, M. H. Nicolas & L. Gutmann, unpublished). In addition it should be mentioned that the range of selection frequencies was reduced for the four strains tested, which may indicate a similar risk of in-vivo selection of antibiotic-resistant mutants, whichever R. equi strain is isolated. For treatment of R. equi infections in foals, rifampicin combined with erythromycin is successful, whereas penicillin combined with gentamicin is not (Hillidge et al., 1987). The efficiency of combined erythromycin and rifampicin may result from high macrophage concentrations of these antibiotics, in relation to the intracdlular persistence of R. equi. Nevertheless, a review of the literature shows that optimal treatment has not yet been denned for humans (Harvey & Sunstrum, 1991; Prescott, 1991). Some infections have been successfully treated with antibiotics with low macrophage uptake, such as vancomycin, imipenem and aminoglycosides (Van Etta et al., 1983; Emmons et al., 1991; Harvey & Sunstrum, 1991; Rouquet et al., 1991), relapses have been described
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minocyclinc and imipenem-amikacin. The crythromycin-amikacin combination was antagonistic. Similar results were obtained when erythromycin was replaced by clarithromycin or roxithromycin; when amikacin was replaced by gentamicin or when minocydine was replaced by doxycycline (data not shown). In time-kill studies ciprofloxacin, vancomycin and amikacin were more bactericidal than erythromycin, imipenem, rifampicin, trimethoprim/sulphamethoxazole and minocyclinc The decrease in cfu/mL was always less than 3 logl0. Antibiotic combinations in time-kill kinetics at any of the concentrations tested failed to identify any synergic effect The apparent discrepancy between the FIC index, MIC and MBC, and the time-kill kinetic results may have arisen from the shake culture conditions used in the time-kill kinetics which provide for maximal growth. In time-kill kinetics, the absence of antibiotic synergy is in agreement with the study of Novak et al. (1988) which reported no synergy between rifampicin and erythromycin. However, Prescott & Nicholson (1984) reported synergy when erythromycin was combined with penicillin (not tested in the present study) or rifampicin, or when penicillin (but not ampicillin) was combined with gentamicin. Such differences may arise from differences in methodology. Technical differences between the study of Prescott & Nicholson (1984) and the present report were, absence of shaking versus presence of shaking cultures; criteria of synergy ten-fold vs 100-fold increase in killing; bacterial inoculum, 107 vs 106 cfu/mL. Our decision to shake the tubes in the time-kill kinetic studies was to provide maximal growth. Therefore, the relative inefficiency of antibiotics to cure human infections may be partially explained by the poor correlation between in-vitro testing techniques and in-vivo growth of R. equi.
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P- Nordmann and E. Rooco
for antibiotics with high intramacrophage uptake such as rifampicin, erythromycin, doxycycline and trimetboprim/sulphamethoxazole (Emmons et al., 1991; Harvey & Sunstrum, 1991; Nordmann et al., 1991). Though these reports could affect the choice of antibiotic combinations, the development of an animal model is needed. Together with the in-vitro antibacterial properties of drugs, it is important to consider other criteria for the choice of antibiotics in vivo such as lung and intracellular drug concentrations, and the availability of intravenous and oral preparations since this infection often requires treatment of several weeks. Acknowledgements
References Allen, V. D., Nice, A., Kercm, E., Greenberg, M. & Gold, R. (1989). Rhodococcus equi pneumonia in a child with leukemia. Pediatric Infectious Disease Journal 8, 656-8. Berg, R., Chmel, H., Mayo, J. & Armstrong, D. (1977). Corynebacterium equi infection complicating neoplastic disease. American Journal of Clinical Pathology 68, 73-7. Bishopric, G. A., D'Agay, M. F., Schlemmer, B., Sarfati, E. & Brocheriou, C. (1988). Pulmonary pseudotumour due to Corynebacterium equi in a patient with the acquired immunodeficiency syndrome. Thorax 43, 486-7. Deere, D., Bure, A., Pangon, B., Philippon, A. & Bergogne-Berezin, E. (1991). In-vitro susceptibility of Rhodococcus equi to 27 antibiotics. Journal of Antimicrobial Chemotherapy 28,311-3. Ebersole, L. L. & Paturzo, J. L. (1988). Endophthalmitis caused by Rhodococcus equi Prescott serotype 4. Journal of Clinical Microbiology 26, 1221-2. Emmons, W., Reichwein, B. & Winslow, D. L. (1991). Rhodococcus equi infection in the patient with AIDS: literature review and report of an unusual case. Reviews of Infectious Diseases 13, 91-6. Fiercr, J., Wolf, P., Seed, L., Gay, T., Noonan, K. & Haghighi, P. (1987). Non-pulmonary Rhodococcus equi infections in patients with acquired immune deficiency syndrome (AIDS). Journal of Clinical Pathology 40, 556-8. Gohib, B., Falk, G. & Spink, W. W. (1967). Lung abscess due to Corynebacterium equi. Report of first human infection. Annals of Internal Medicine 66, 1174-7. Goodfcllow, M. (1987). The tazonomic status of Rhodococcus equi. Veterinary Microbiology 14, 205-9. Harvey, R. L. & Sunstrum, J. C. (1991). Rhodococcus equi infection in patients with and without human immunodeficiency virus infection. Reviews of Infectious Diseases 13, 139-45. HiHerdal, G., Riesenfeldt-Orn, I., Pedersen, A. & Ivanicova, E. (1988). Infection with Rhodococcus equi in a patient with sarcoidosis treated with corticosteroids. Scandinavian Journal of Infectious Diseases 20, 673-7. Hillidge, C. J. (1986). Review of Corynebacterium (Rhodococcus equi) lung abscesses in foals: pathogenesis, diagnosis and treatment. Veterinary Record 119, 261-4. Hillidge, C. J. (1987). Use of erythromycin-rifampin combination in treatment of Rhodococcus equi pneumonia. Veterinary Microbiology 14, 337-42. Hillman, D., Garretson, B. & FisceHa, R. (1989). Rhodococcus equi endophthalmitis. Case report Archives of Ophthalmology 107, 20. Hughes, K. L. & Sulaiman, I. (1987). The ecology of Rhodococcus equi and physicocbemical influences on growth. Veterinary Microbiology 14, 241-50. Jones, M. R., Neale, T. J., Say, P. J. & Home, J. G. (1989). Rhodococcus equt an emerging opportunistic pathogen? Australian and New Zealand Journal of Medicine 19, 103-7.
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We thank L. Gutmann for helpful advice, C. Wilkerson for critical reading of the manuscript, J. Caillon, S. Francoual, and M. H. Nicolas for donating the strains and C. Nauciel for constant interest in this work. This work was funded by a Paris-Ouest Medical School grant.
In-vllro gusccptlMItty of R. equi
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{Received 18 July 1991; revised version accepted 16 October 1991)
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Jones, R. N., Barry, A. L., Gavan, T. L. & Washington, J. A. (1985). Susceptibility test* microdilution and macrodihition broth procedures. In Manual of Clinical Microbiology, 4th cdn (Lennette, E. H., Balows, A., Hauster, W. J. & Shadomy, H. J., Eds), pp. 972-7. American Society for Microbiology, Washington, DC. Lebar, W. D. & Pensler, M. I. (1986). Pleural effusion due to Rhodococcus eqtd. Journal of Infectious Diseases 154, 919-20. Ledercq, R., Bingen, E., Su, Q. H., Lambert-Zechovski, N., CourvaHn, P. & Duval, J. (1991). Effects of combinations of /Mactams, daptomycin, gentamicin and glycopeptides against grycopeptide-resistant enterococci. Antimicrobial Agents and Chemotherapy 35, 92-8. Magnusson, H. (1923). Spezifische infektiose Pneumonic bcim Fohlen. Ein neuer Eitererreger beim Pferde. Archivfur Wissenschqftliche und Praktische Tierheilkunde 50, 22-38. Marsh, J. C. & Von Graevenitz, A. (1973). Recurrent Corynebacterium equi infection with lymphoma. Cancer 32, 147-9. Nordmann, P., Caumes, E., Grosset, J. & Gentilini, M. (1991). Pneumopathie ahveolaire recidivante a Rhodococcus equi chez un malade atteint de SID A. Medecine et Maladies Infectieuses 21, 319-21. Nordmann, P, Chavanet, P., Caillon, J., Duez, J. M. & Portier, H. (1992). Recurrent pneumonia due to rifampicin-resistant Rhodococcus equi in a patient infected with HIV. Journal of Infection, 24, 104-7 Novak, R. M., PoKsky, E. L., Janda, W. M. & Libcrtin, C. R. (1988). Osteomyelitis caused by Rhodococcus equi in a renal transplant recipient Infection 16, 186-8. Pearson, R. D., Steigbiel, R. T., Davis, H. T. & Chapman, S. W. (1980). Method of reliable determination of minimal lethal antibiotic concentrations. Antimicrobial Agents and Chemotherapy 18, 699-708. Prescott, J. F. (1981). The susceptibility of isolates of Corynebacterium equi to antimicrobial drugs. Journal of Veterinary Pharmacology and Therapeutics 4, 27-31. Prescott, J. F. (1987). Epidemiology of Rhodococcus equi infection in horses. Veterinary Microbiology 14, 211-4. Prescott, J. F. (1991). Rhodococcus equi: an animal and human pathogen. Clinical Microbiological Reviews 4, 20-34. Prescott, J. F. & Nicholson, V. M. (1984). The effects of combinations of selected antibiotics on the growth of Corynebacterium equi. Journal of Veterinary Pharmacology and Therapeutics 7, 61-4. Rouquet, R. M., Clave, D., Massip, P., Moatti, N. & Leophonte, P. (1991). Imipencm/vancomycin for Rhodococcus equi pulmonary infection in HIV-positive patient. Lancet 337, 375. Sarnies, J. H., Hathaway, B. N., Echols, R. M., Veazey J. M. & Pilon, V. A. (1986). Lung abscess due to Corynebacterium equi. Report of the first case in a patient with acquired immune deficiency syndrome. American Journal of Medicine 80, 685-8. Sane, D. C. & Durack, D. T. (1986). Infection with Rhodococcus equi in AIDS. New England Journal of Medicine 314, 56-7. Schoenknecht, F. D., Sabath, L. D. & Thornsberry, C. (1985). Susceptibility tests: special tests. In Manual of Clinical Microbiology, 4th edn (Lennette, E. H., Balows, A., Hauslcr W. J. & Shadomy, H. J., Eds), pp. 1000-8. American Society for Microbiology, Washington, DC. Sweeney, C. R., Sweeney, R. W. & Divers, T. J. (1987). Rhodococcus equi pneumonia in 48 foals: response to antimicrobial therapy. Veterinary Microbiology 14, 329-36. Van Etta, L. L., FiHce, G. A., Ferguson, R. M. & Gerding, D. N. (1983). Corynebacterium equi: a review of 12 cases of human infection. Reviews of Infectious Diseases 5, 1012-8. Wookock, J. B. & Mutimer, M. D. (1980). Corynebacterium equi: in vitro susceptibility to twenty-six antimicrobial agents. Antimicrobial Agents and Chemotherapy 18, 976-7. Yager, J. A. (1987). The pathogenesis of Rhodococcus equi pneumonia in foals. Veterinary Microbiology 14, 225-32. Zink, M. C , Yager, J. A., Prescott, J. F. & Fernando, M. A. (1987). Electron microscopic investigation of intracellular events after ingestion of Rhodococcus equi by foal alveolar macrophages. Veterinary Microbiology 14, 295-305.
In-vitro antimicrobial susceptibility of Rhodococcus equi P. Nordmam* and E. Ronco
Rhodococcus equi is an intraccllular facultative, Gram-positive cocco-batiHary organism of increasing importance as a pulmonary pathogen in HIV-positive patients. This study was carried out to evaluate the optimal antibiotic combinations for treating such infections. Four human R. equi isolates and one reference strain were tested for their susceptibilities to 36 antibiotics. In-vitro the most active antibiotics were amikacin, gentamicin, netilmicin, erythromycin, clarithromydn, roxithromytin, ciprofloxacin, sparfloxacin, rifampicin, vancomycin, teicoplanin, doxycycUne, minocycline, imipenem, meropenem and trimethoprim/sulphamethoxazole. The only bactericidal antibiotics were the aminoglycosides, ciprofloxacin, sparfloxacin and vancomycin. As determined by FIC indices, four combinations were synergistic: rifampidn-erythromycin, rifampicin-minocycline, erythromytin-minocycline and imipenem-amikacin. However, no antibiotic combinations were synergistic with the time-kill kinetic method at achievable serum concentrations or at ten-fold and halffokl the MICs. Frequencies of selection of antibiotic-resistant mutants determined at concentrations of five- and ten-fold the MICs ranged from < 1 x 10~* for erythromycin and trimethoprim/sulphamethoxazole to 5 x 10~4 for amikacin. These results may be of help in selecting the antibiotics for treating infected HIV-positive patients.
Introduction
Rhodococcus equi, (formerly known as Corynebacterhon equi) has been described as an opportunistic pathogen in immunocompromised patients (Golub, Falk & Spink, 1967; Van Etta et al., 1983; Sane & Durack, 1986; Allen et al., 1989; Prescott, 1991). Since the spread of AIDS, an increasing number of R. equi cases has been reported (Sarnies et al., 1986; Sane & Durack, 1986; Bishopric et al., 1988; Jones et al., 1989; Emmons, Reichwein & Winslow, 1991; Harvey & Sunstrum, 1991). Consequently, this pathogen is becoming a significant cause of pneumonia in HIV-positive patients. R. equi is a Gram-positive cocco-bacillary bacterium, classified as an Actinomycetales (Goodfellow, 1987). Well known to veterinary microbiologists since its first isolation by Magnusson in 1923 (Magnusson, 1923), R. equi is involved in 10% of pneumonia in foals (Hillidgc, 198S; Prescott, 1987). Both animal and human infections are thought to be acquired via the respiratory route in farms and in stables where R. equi is found not only in the soil but also in the bowels of horses and of other farm animals (Yager, 1987; Prescott, 1991). R. equi is a facultative intracellular pathogen infecting both macrophages and polymorphonuclear leucocytes but persisting in macrophages which may explain its •Corrapondence to: Dr P. Nordmann, Laboratoire de Microbiologtt Medicate, Hdpital Raymond Poincare, 104 Bd Raymond Poincare, 92380 Garches, France. O305-74S3/92/O4O383 +11 S02.00/0
383 © 1992 The British Society for Antimicrobial Chemotherapy
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Service de Microbiologie, Hdpital Raymond Poincari, Faculti de Midecine Paris-Quest, Garches, France
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P- Nonbnann and E. Rooco
Materials and methods Bacterial strains Five distinct R. equi strains were used; four clinical isolates and a human reference strain (52T2, Institut Pasteur collection, Paris, France). The clinical strains were isolated from blood culture and bronchoalveolar brush samples from four HIV-positive patients with pneumonia. Their identification was confirmed by API Corynebacteria tests (La Balme-les-Grottes, France) and the identification centre of the Institut Pasteur. Antibiotics The antimicrobial agents used in this study were obtained from laboratory standard powders and were used immediately after their dilution. The agents and their sources were: amikarin, oxacillin, penicillin G, and netilmicin (Bristol), amoxycillin, clavulanic acid1, and ticarcillin (Beecham), latamoxef and vancomycin (Eli Lilly & Co), cepha-
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ability to escape the normal pulmonary defence mechanisms (Zink et al., 1987). In humans, pneumonia and lung abscesses are the most frequent infections (accounting for 70% of cases), occurring mainly in immunocompromised patients (Marsh & Von Gracvenitz, 1973; Berg et al., 1977; Lcbar & Pensler, 1986; Hillerdal et al., 1988; Emmons et al., 1991) and rarely in the immunocompetent (Harvey & Sunstrum, 1991). Contact with horses is often reported in infected patients (Prescott, 1991). Non-pulmonary infections have also been described, such as infectious diarrhoea, endophthalmitis, osteomyelitis, brain or pelvic abscesses (Fierer et al., 1987; Ebersole & Paturzo, 1988; Novak et al., 1988; Garretson & Fiscella, 1989). Based on experimental treatments of R. equi pneumonia in foals, rifampicin combined with erythromycin is known to be successful (Hillidge, 1987; Sweeney, Sweeney & Divers, 1987). Nevertheless, the optimal antimicrobial treatment for human infections is not yet known and the overall mortality of AIDS patients with R. equi infections remains around 50% whatever treatment is given (Harvey & Sunstrum, 1991). Various antibiotic combinations have been tried. However, the course of the disease is characterized by frequent relapses, occurrence of antibiotic-resistant mutants during treatment, and often a need for complementary surgery. Results based on the bactericidal activity of drugs alone or in combination on R. equi are few and contradictory. Prescott & Nicholson (1984) reported in-vitro synergic activity of erythromycin combined with rifampicin, as well as penicillin combined with gentamicin or erythromycin, though Novak et al. (1988) failed to detect any antibiotic synergy. Therefore, the goals of this study were to determine MICs of 36 antibiotics, including new drugs such as third generation cephalosporins, carbapenems, fluoroquinolones and new macrolides. Thus, providing information not only on the antibacterial activities of drugs used in veterinary medicine but also those recently introduced for the treatment of human infections. Furthermore, to determine MBCs which should be helpful in the choice of bactericidal antibiotics, the in-vitro antibiotic activities of drugs alone and in combination and in addition to calculate the frequencies of in-vitro selection of antibioticresistant mutants with antibiotics alone or in combination to estimate the risk of clinical selection of antibiotic-resistant mutants (since several weeks treatment is necessary for such infections), was determined.
In-Tttro susceptibility of R. eqtd
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lothin, ceftazidime (Glaxo), ceftriaxone and trimethoprim/sulphamethoxazole (Roche), clarithromycdn and erythromycin (Abbott), cefoxitin and imipenem (Merck Sharp & Dohme), gentamicin (Schering Corp.), pristinamycin, sparfloxacin and spiramycin (Rhone-Poulenc), fusidic acid (Leo), cefotaxime, chloramphenicol and roxitnromycin (Roussel), ciprofloxacin (Bayer-Pharma), pefloxacin (Roger & Bellon), ofloxacin (Diamant), teicoplanin (Merrell Dow) rifampicin (Gruppo Lepetit), doxycycline and tetracycline base (Pfizer), lomcfloxacin (Searle), minocycline (Lederlfc), fosfomycin (Sanofi) and meropenem (ICI Pharma). In-vitro susceptibility to antibiotics
Study of combined antimicrobial activity Combinations of antibiotics were studied by two techniques (Schoenknecht, Sabath & Thornsberry, 1985; Leclercq et al., 1991). All experiments were performed in triplicate with four out of the five strains (one strain was more resistant to rifampicin and fluoroquinolones than the others). Firstly, the strains were studied by the microdilution chequerboard technique. Trays were prepared using a plastic microdilution technique (Dynatec Laboratories, Inc., Alexandria, VA) as follows. Seven antibiotics were selected from the various antibiotic groups according to some or all of the following criteria: low MIC, low MBC, high Cn^JMIC, availability of oral and intravenous dosages; these included amikacin, ciprofloxacin, erythromycin, imipenem, minocycline, rifampicin, and vancomycin. Serial two-fold dilutions of the antibiotics (concentrations from 16- to 0-12-fold the MICJQ) were performed to study antibiotic combinations. The inoculum of strains tested was adjusted to 10* cfu/mL. After 24 h incubation FIC indices were calculated using the following formula: FIC index = FIC A + FIC B = MIC of A in combination/MIC of A alone+MIC of B in combination/MIC of B alone. The FIC indices were calculated as the antibiotic combinations that were both most effective and comprised the lowest concentrations of antibiotics. An FIC index < 0-5 was considered as showing synergy, an FIC index of 0-5-4 indifference and a FIC index > 4 as showing antagonism to the combination of antibiotics.
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MICs and MBCs were determined by a macrodilution broth dilution technique in glass tubes (Jones et al., 1985) containing 5 mL of Mueller-Hinton broth (Diagnostics Pasteur, Paris). Bacterial inocula were prepared by appropriate dilutions of overnight shaked cultures of organisms in Mueller-Hinton broth. A standard inoculum of 106 cfu/mL was used in all cases. For the determination of trimethoprim/sulphamethoxazole (1:5) susceptibility, a standard agar dilution technique on Mueller-Hinton agar supplemented with 5% of lysed horse blood (Diagnostics Pasteur, Paris) was used. All these experiments were performed at 37°C. The MICs were determined as the first tube with no visible growth after 24 h incubation. MBCs were calculated as the least concentrations of drug to achieve a 99-9% reduction of the original inoculum. For antibiotics with low MBCs, MBCs and MICs were also determined after 24 h incubation at 37°C in a shaking water-bath or at 30cC in an unshaken waterbath. These latter MICs and MBCs were determined in order to take into account the aerobic requirement for maximal growth of R. equi and its optimal growth temperature of 30°C (Hughes & Sulaiman, 1987). All MIC and MBC determinations were performed in triplicate for each strain. Thefivestrains were also screened for /Mactamase production using nitrocefin (Glaxo Laboratories, Middlesex, England).
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P. Nordnuum and E. Rooco
Frequencies of in-vitro selection of antibiotic-resistant mutants Mutation rates were determined by counting the number of colonies arising from a large inoculum (10* cfu) on antibiotic containing plates at concentrations five- and ten-fold the MICs using four different strains. The same eight antibiotics were used as in the time-kill kinetics. The mutation rates were also calculated with antibiotics combined two by two at five- and ten-fold their MIC. The MICs of the mutants were subsequently determined as previously described. Results Antibiotic susceptibilities of the strains The MICs and MBCs of the antibiotics against the five strains are shown in Table I. The five strains possessed the same pattern of antibiotic susceptibilities except for one which was less susceptible to rifampicin and the fluoroquinolones. This strain had been isolated from a patient treated with rifampicin alone and then ofloxacin. The /Mactam MICs were high with the exception of imipenem (0-25 mg/L), and meropenem (0-50 mg/L). No /Mactamases were found in any strains using the nitrocefin test The MBCs for all 0-lactams were > 256 mg/L with a MBC/MIC ratio > 32. The antimicrobial activities of the aminoglycosides were similar among each other with low
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Secondly, time-kill kinetics were performed to test the bactericidal activity of antibiotic combinations with the same seven antibiotics as those studied in the FIC index test plus, trimethoprim/sulphamethoxazole. The drugs were tested at concentrations clinically achievable in serum and ten- and 0-5-fold the MICs. The antibiotic concentrations (mg/L) used weie: ciprofloxacin 3-5, 2-5, 0-12; minocycline 5, 1-2, 0-06; erythromycin 6, 2-5, 012; amikacin 24, 20, 1; imipenem 30, 2-5, 0-12; rifampicin 15, 0-6, 0-03; vancomycin 40, 2-5, 0-12; trimethoprim/sulphamethoxazole 8/32, 32/128, 1-6/6-4. Time-kill kinetic studies were determined for each antibiotic alone and in combination. All experiments were performed in glass tubes containing 5 mL of Mueller-Hinton broth in a shaking water-bath at 37°C. An inoculum of 106 cfu/mL was obtained from a dilution of fresh overnight cultures. After 0, 3, 6, 24 h incubation, water-diluted samples were plated on Mueller-Hinton agar. After 24 h incubation, the organisms isolated were tested for their antibiotic susceptibility in order to ensure that no antibiotic-resistant mutants had been selected. For trimethoprim/sulphamethoxazole time-kill determinations, the same procedure was used with supplementation of Mueller-Hinton broth with 5% lysed horse blood. Synergy was defined as > 2 logl0 decrease in cfu/mL between the combination and its most active component after 24 h incubation. Antagonism was defined as a > 2 log,0 increase in cfu/mL between the combination and either drug alone. Intermediate results were considered as indifference. Bactericidal activity was defined as > 3 log,0 reduction in growth at 24 h incubation. To ensure that antibiotic carry-over could not affect the result of the timekill kinetics, preliminary experiments were performed as follows (Pearson et al., 1980). Low numbers of bacteria (103 cfu/mL) were resuspended in liquid medium in the presence or absence of each antibiotic alone or in combination. Thereafter the same serial dilutions used for the time-kill kinetics studies were plated immediately on Mueller-Hinton agar. All plates were incubated at 37CC for 48 h before cfu were determined.
In-ritro susceptibility of R. eqtd
387
Table L MICs and MBCs of 36 antibiotics against five R. equi strains
Antibiotic Penicillin G Amoxydllin Amoxydllin/davulanic
add
ITTlipCTldH
Meropcnem Latamoxef Gentamidn Amikadn Netihnidn Tetracycline DoxycycHne Minocydrae Erythromydn Spiramydn Qarithromydn Roxithromydn Pristinamycin Gprofloxadn Ofloxadn Pefloxadn Sparfloxadn Lomefloxadn Chloramphenicol Vancomydn Teicoplanin Fusidic add Rifampidn Fosfomydn Trimethoprim/ sulphamethoxazole
2-16 4-8 4-8 64-128 256-512 128-512 8-16 32-64 1-4 >256 0-12-0-5 0-25-0-50 16-32 0-5-1 0-5-4 1-2 1-4 0-5-1 0-12-0-5 0O6-0-25 4-8 0-12-0-25 0-12-0-25 4-8 0-12-4 4-16 8-64 0-12-4 4-32 8-32 0-12-0-25 0O6-0-12 4-16 003-25 >256 1-6-^4/12-8-25^
50% 4 4 4 64 256 128 8 32 2 >256 0-25 0-50 16 0-5 2 2 2 1 0-12 0-25 8 0-12 0-12 4 0-25 4 16 0-25 4 8 0-12 006 8 006 >256 3-2/12-8
MBC (mg/L) range 50% >256 >256
>256 >256
>256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 1-2 4-8 2-4 >256 >256 >256 >256 >256 >256 >256 >256 1-16 16-256 16-> 256 1-16 16-32 >256 2-4 4-16 64-128 >256 >256 ND
>256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 2 8 4 >256 >256 >256 >256 >256 >256 >256 >256 1 32 32 1 32 >256 2 8 128 >256 >256 ND
ND, Not detennined
MIC and low MBC. The MBCs of fluoroquinoloncs were four-fold higher than those of the MICs. Among these drugs, dprofloxadn and sparfloxadn were the most active. Among the tetracycline analogues tested, the MIC of minocycline was the lowest but none was bacteriddal. Among the macrolides and pristinamycin, the MICs of erythromydn, darithromydn, and roxithromydn were the lowest No macrolides were bacteriddal at clinically achievable serum concentrations. Glycopcptidc MICs were low but
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OxadlHn Ticardllin Cephalothin Ccfoxitin Cefotaxime Ceftriaxone Ceftazidime
MIC (mg/L) range
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Table IL Values (range) of FIC indices of combinations of eight antibiotics against four R. equi strains
AMI AMI
CIP 1-8-2-2
CIP
ERY 4-5-6
2-3 ERY
IMP 0-25-0-40 0-5-1
2-3 IMP
MIN 2-4 1-2-2-5 0O3-O06 0-75-1
MIN
RIF 1-3 1-3 0-01-004 0-90-1-20 0-03-0O6
RIF
VAN 0-75-1 0-75-1
1-3 0-75-1 1-1-5 1-5-2-5
VAN
only vancomycin was bactericidal. Rifampicin showed high antibacterial activity without being bactericidal. Among the remaining antibiotics tested, the MICs of trimethoprim/sulphamethoxazole were low but MBCs were high. The M B C (mg/L) of the antibiotics found to be bactericidal were amikacin 32, gentamicin 8, netilmidn 16, ciprofloxacin 8, sparfloxacin 8, vancomycin 64, whether shaken or incubated at 30°C. Therefore, in these modified conditions, no antibiotic was bactericidal and the MIC values for these antibiotics increased in the same order of magnitude as the MBC values.
Table HI. Frequency of selection of antibiotic-resistant mutants to eight antibiotics. Antibiotic selection concentrations were five- and ten-fold the MICs. Experiments were performed with four different R. equi strains. The average increase in MICs of the antibiotic-resistant mutants are also indicated. Concentration of selection (mg-L)
Mutation frequency
Fold increase in MIC of the mutants
10 20
3-5 x10" 4 1-4 xlO" 5
8 8
Ciprofloxacin
1-25 2-50
5-8 xlO" 7 < 1 x10"'
1000
Erythromycin
1-25 2-50
< 1 xlO" 1 < 1 x10"'
Imipenem
1-25 2-50
2-9 x10"' 1-2 xlO- 6
Minocycline
0-60 1-20
4-5 x 1 0 - 7 1-2 xlO" 7
Rifampicin
0-30 O60
1-2 xlO" 7 0-5-1 x lO"7
Trimethoprim/ sulphamethoxazole
16/64 32/128
Amikacin
Vancomycin
0-6 1-2
8 8 OS OO
Antibiotic
10,000 10,000
< 1 x10"' < 1 x10"' 0-5-2x10-' < 1 x 10"'
4
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AMI, Amikacin; CIP, tiprofloxacin; ERY, erythrotnycin; IMP, imipenem; MIN, minocycHne; RIF, rifampidn; VAN, vancomycin.
In-Tttro UKeptibffity of X. eqtd
389
I09 I08 I07
J I0 6 "5 I0 5
I0 4
1
1
1
1
1
1
1
1
24
Time ( h ) Hgmc. Bactericidal effects of achievable serum concentrations of •milr»rin (AMI), ciproflozacin (CIP), erythromycin (ERY), imipcnem (IMP), minocydine (MTN),rifampicin(RTF), trirnethoprim/sulphamethoxaxole (TMS) and vancomycin (VAN) alone or in combination against four K. equi strains. The curves rcprumt mean values of bacterial counts over 24 h incubation; standard deviations are indicated by vertical bars. Control without any antibiotics (C); antibiotic concentrations alone or in combination as follows; (1): ERY, IMP, MIN, RIF, TMS, ERY-IMP, ERY-MIN, ERY-RIF, ERY-TMS, IMP-IMP, ERY-MIN, IMP-TMS, MIN-RIF, MIN-TMS, RIF-TMS, or ERY-AMI; (2): AMI, CIP, VAN, AMI-OP, AMI-VAN, OP-VAN, AMI-IMP, AMI-MIN, AMI-RIF, AMI-TMS, OP-IMP, OP-ERY, OP-RIF, OP-MIN, OP-TMS, VAN-IMP, VAN-MIN, VAN-RIF or VAN-TMS.
Combined antimicrobial activities Values of the FIC for the combinations of antibiotics are shown in Table II for four strains. The synergistic combinations were rifampicin-erythromycin, rifampicinminocycline, erythromycin-minocycline, and imipenem-amikacin. Erythromycin combined with amikacin was the only antagonistic combination. All other antibiotic combinations were indifferent In time-kill studies, the results of the bactericidal activity of the drugs are shown in the Figure. These results are obtained with antibiotic concentrations equivalent to achievable serum values (similar results were obtained when concentrations of ten-fold and half-fold the MICs were used). Ciprofloxacin, amikacin, vancomycin alone or combined with any other antibiotic (with the exception of amikacin-erythromycin) showed an average of only 2-5 log,0 cfu/mL decrease after 24 h incubation as compared to the original inoculum. The other antibiotics (rifampicin, erythromycin, imipenem, minocycline and trimethoprim/sulphamethoxazole) alone or in combination as well as ntnilracin plug erythromycin led to an average of a 1-5 log10 cfu/mL decrease compared to the original inoculum. The antibiotic combination in which one or both of the antibiotics was bactericidal, according to the above mentioned MBCs, were more bactericidal than the others. Subinhibitory concentrations of antibiotics in combination did not show any synergic effect Frequencies of in-vitro selection of antibiotic-resistant mutants Antibiotic concentrations at five- and ten-fold the MICs were used to screen for antibiotic-resistant mutants. The results of this selection are indicated in Table III.
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I0 3
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Combinations of antibiotics (even at the concentration offive-foldtheir MIC) reduced the mutation frequencies for all antibiotics to < 1 x 10"'. All antibiotic-resistant mutants showed a similar increase in their MIC values with antibiotics of the same group.
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Discussion Since only a few strains were studied, the results of MICs, MBCs, combined antimicrobial activities, and mutation frequencies were similar, with the exception of susceptibility to rifampicin and fluoroquinolones of one strain. To our knowledge, this is the first report of such extensive susceptibility testing of human R. equi isolates. The MICs are in general accordance with those previously reported for animal or human strains (Woolcock & Mutimer, 1980; Prescott, 1981; Harvey & Sunstrum, 1991; Deere et al., 1991). The isolates had intermediate susceptibility or were resistant to all /Mactam agents as previously described for clinical isolates but not for soil or animal strains (Prescott, 1991). Carbapenems showed low MICs. Since the MIC of ceftriaxone was 2 mg/L, it should be mentioned that when susceptibility was determined by the disc diffusion method, colonies occurred around the ceftriaxone disc (data not shown). This suggests an inducible mechanism of resistance to ceftriaxone. The /Mactam resistance of human isolates is not explained by the presence of /Mactamase. Only one R. equi isolate has been described previously as /Mactamase-positive but neither extended /Mactam susceptibilities nor characterization of this enzyme has been reported (Fierer et al., 1987). As with other Gram-positive bacteria, low affinity of /Mactams for penicillin binding proteins may be an explanation. Whichever /Mactam agent was studied MBCs were always > 256 mg/L. It is suggested therefore that tolerance may be a widespread mechanism of resistance in R. equi. This possibility, associated with a high in-vitro mutation frequency towards imipenem underlines the fact that /Mactams should never be used as monotherapy to treat infections caused by R. equi. Differences in susceptibilities occurred among antibiotics of the same group. Strains were susceptible to ciprofloxacin and sparfloxacin but less so to lomefloxacin, ofloxacin, and pefloxacin. The MICs of erythromycin, roxithromycin and clarithromycin were low, but those of pristinamycin and spiramycin were relatively high. Strains were susceptible to minocycline and doxycycline but less so to tetracycline. Therefore, testing one antibiotic from each class may not allow one to deduce the overall susceptibility to other antibiotics of the same group. This may be clinically relevant. Among the remaining antibiotics, R. equi strains were fully susceptible to aminoglycosides, rifampicin and glycopeptides as has been reported previously by Emmons el al. (1991). However, fosfomycin was totally inactive (Woolcock & Mutimer, 1980). Few data are available on the bactericidal activity of antibiotics against R. equi. Novak et al. (1988) did not find any antibiotic to be bactericidal though Emmons et al. (1991) showed that vancomycin, erythromycin, imipenem were bactericidal. In our study, the aminoglycosides, ciprofloxacin, sparfloxacin and vancomycin were bactericidal when the same MBC procedures were used. However, no antibiotics were bactericidal in shake cultures. Such differences in bactericidal activity may have arisen from the aerobic requirements for the maximal growth of R. equi. Thus, incubation temperature as well as shaking conditions may be critical for the MBC determinations of these slow growing organisms. Of the antibiotic combinations tested, only four were synergic as determined by the FIC indices: erythromycin-rifampicin, erythromycin-minocycline, rifampicin-
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Antibiotic-resistant mutants were selected in vitro at detectable frequencies with single antibiotics, except for erythromycin and trimethoprim/sulphamethoxazole. Such results correlate well with the high frequency of in-vivo selection of R. equi antibioticresistant mutants. The selection frequency of amikacin-resistant mutants was very high as it is for aminoglycosides and other Actinomycetales such as Mycobacterium tuberculosis. Rifampicin- and ciprofloxacin-resistant mutants possessed MICs which were greater than clinically achievable serum concentrations which may explain treatment failures (Nordmann et al., 1992). However, antibiotics in combination provided undetectable in-vitro mutation frequencies. This result justifies the clinical use of antibiotics in combination. Imipenem-resistant mutants are interesting as they show cross-resistance for carbapenems and cephamycins only. High susceptibility to imipenem may be explained by the existence of a special penicillin binding protein of high affinity for carbapenems and cephamycins (P. Nordmann, M. H. Nicolas & L. Gutmann, unpublished). In addition it should be mentioned that the range of selection frequencies was reduced for the four strains tested, which may indicate a similar risk of in-vivo selection of antibiotic-resistant mutants, whichever R. equi strain is isolated. For treatment of R. equi infections in foals, rifampicin combined with erythromycin is successful, whereas penicillin combined with gentamicin is not (Hillidge et al., 1987). The efficiency of combined erythromycin and rifampicin may result from high macrophage concentrations of these antibiotics, in relation to the intracdlular persistence of R. equi. Nevertheless, a review of the literature shows that optimal treatment has not yet been denned for humans (Harvey & Sunstrum, 1991; Prescott, 1991). Some infections have been successfully treated with antibiotics with low macrophage uptake, such as vancomycin, imipenem and aminoglycosides (Van Etta et al., 1983; Emmons et al., 1991; Harvey & Sunstrum, 1991; Rouquet et al., 1991), relapses have been described
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minocyclinc and imipenem-amikacin. The crythromycin-amikacin combination was antagonistic. Similar results were obtained when erythromycin was replaced by clarithromycin or roxithromycin; when amikacin was replaced by gentamicin or when minocydine was replaced by doxycycline (data not shown). In time-kill studies ciprofloxacin, vancomycin and amikacin were more bactericidal than erythromycin, imipenem, rifampicin, trimethoprim/sulphamethoxazole and minocyclinc The decrease in cfu/mL was always less than 3 logl0. Antibiotic combinations in time-kill kinetics at any of the concentrations tested failed to identify any synergic effect The apparent discrepancy between the FIC index, MIC and MBC, and the time-kill kinetic results may have arisen from the shake culture conditions used in the time-kill kinetics which provide for maximal growth. In time-kill kinetics, the absence of antibiotic synergy is in agreement with the study of Novak et al. (1988) which reported no synergy between rifampicin and erythromycin. However, Prescott & Nicholson (1984) reported synergy when erythromycin was combined with penicillin (not tested in the present study) or rifampicin, or when penicillin (but not ampicillin) was combined with gentamicin. Such differences may arise from differences in methodology. Technical differences between the study of Prescott & Nicholson (1984) and the present report were, absence of shaking versus presence of shaking cultures; criteria of synergy ten-fold vs 100-fold increase in killing; bacterial inoculum, 107 vs 106 cfu/mL. Our decision to shake the tubes in the time-kill kinetic studies was to provide maximal growth. Therefore, the relative inefficiency of antibiotics to cure human infections may be partially explained by the poor correlation between in-vitro testing techniques and in-vivo growth of R. equi.
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for antibiotics with high intramacrophage uptake such as rifampicin, erythromycin, doxycycline and trimetboprim/sulphamethoxazole (Emmons et al., 1991; Harvey & Sunstrum, 1991; Nordmann et al., 1991). Though these reports could affect the choice of antibiotic combinations, the development of an animal model is needed. Together with the in-vitro antibacterial properties of drugs, it is important to consider other criteria for the choice of antibiotics in vivo such as lung and intracellular drug concentrations, and the availability of intravenous and oral preparations since this infection often requires treatment of several weeks. Acknowledgements
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We thank L. Gutmann for helpful advice, C. Wilkerson for critical reading of the manuscript, J. Caillon, S. Francoual, and M. H. Nicolas for donating the strains and C. Nauciel for constant interest in this work. This work was funded by a Paris-Ouest Medical School grant.
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{Received 18 July 1991; revised version accepted 16 October 1991)
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