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Current Medical Research and Opinion
Vol. 12, No. 9,1992
Comparative in vitro activity of enoxacin and other fluoroquinolones against multi-resistant strains of Salmonella typhi
Abdul Hannan, M.B., B.S.,
Curr. Med. Res. Opin., (1992), 12,560.
-
D.C.P.,Dip.Bact.,F.R.C.Path. Armed Forces Institute of Pathology, Rawalpindi, Pakistan
Received: 6th December 1991
Summary The in vitro activities of enoxacin, lomefloxacin, norfloxacin, ofloxacin, and pefloxacin against 274 strains of Salmonella typhi isolated from suspected typhoid fever patients (137 multi-resistant strains and 137 strains sensitive to chloramphenicol, ampicillin and/or co-trimoxazole) were determined using disk diffusion and agar dilution techniques. In vitro, enoxacin was active against all tested strains with a MIC,, and inhibition zone size against multi-resistant strains of 0.12 mg/l and 34 m m diamete6 respectively. Similar results were found with the other fluoroquinolones. Enoxacin and other fluoroquinolones may be the therapy of choice in cases of typhoid fever caused by organisms resistant to the standard therapy, chloramphenicol. Key words: Enoxacin - anti-infective agents, quinolone - Salmonella typhi microbial sensitivity tests
Introduction Chloramphenicol therapy has been the treatment of choice for typhoid fever, followed by either ampicillin or co-trimoxazole therapy.' Recently, however, strains of Salmonella typhi have been identified which are resistant to chloramphenicol, ampicillin and/or co-trimoxazole.* Enoxacin, a newly developed member of the quinolone-azaquinolone class of antibiotics, has been shown to have a broad spectrum of in vitro activity against Gram-negative and Grampositive pathogen^.^ The present study was designed to determine the in vitro activity of enoxacin and other fluorinated quinolones against chloramphenicol-, ampicillin-, and/or co-trimoxazole-resistant strains of Salmonella typhi.
Material and methods Bacterial isolates Strains of Salmonella typhi were isolated from blood cultures obtained from patients with signs/symptoms compatible with a diagnosis of typhoid fever. Freeze-dried cultures were reconstituted with 0.5 ml of 7.5% glucose broth and incubated overnight at 37OC. Isolates were then sub-cultured on MacConkey agar and blood agar.4 In addition to the clinical blood isolates, 5 Salmonella typhi strains with known minimum inhibitory concentrations (MICs) were also tested for control purposes. The control strains were designated: AFIP-22103 S. typhi (chloramphenicol MIC:256 mg/l), AFIP-45673 S. tvphi (chloramphenicol MIC: 560
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Abdul Hannan
4 mg/l), AFIP-25625 S. typhi (ofloxacin MIC:0.03 mg/l), 'wild'-African (chloramphenicol MIC:lmg/l), and JV-1052 (chloramphenicol MIC:128 mg/l). AFIP strains represented blood culture isolates. The 'wild' and JV strains were obtained as nutrient agar sub-cultures courtesy of Prof. J. Vandepitte (Universitaire Ziekenhuis St. Rafael, Leuven, Belgium). Clinical and control strains were stored freeze dried until sub-cultured for sensitivity testing. The identity of each strain was serologically confirmed using specific antisera (Wellcome Reagents Ltd., U.K.). Biochemical profiles of each strain were constructed using 'sugar sets' (AFIP Rawalpindi, Pakistan) and API-20E galleries (API-System, Montalieu-Vercieu, France).
Disk diffusion sensitivity testing The base materials for the various antibiotics were obtained from the suppliers as follows: enoxacin - Abbott Laboratories, Karachi; norfloxacin - Merck Sharp & Dohme, Karachi; ofloxacin - Hoechst, Pakistan; and pefloxacin RhBne-Poulenc, Pakistan. Filter paper disks (6 mm) were prepared by soaking them in the appropriate concentration of stock solution for each agent for 30 minutes and then drying each disk at 5OOC. The disk concentrations of antibiotic agent (pg/disk) were as follows: ampicillin 10; chloramphenicol 10; co-trimoxazole 25; enoxacin 10; lomefloxacin 10; norfloxacin 5 ; ofloxacin 5; and pefloxacin 5. Lomefloxacin disks (10 pg/disk) were supplied commercially by Searle, Pakistan. Disk diffusion was performed using the Kirby-Bauer method. Inoculated plates with antibiotic disks applied were incubated for 16 to 18 hours at 37OC and inhibition zones were measured using a millimeter ruler (Fowler Ultra Cal 11; Sylvac, U.S.A.).
MIC determinations MICs were determined using the agar dilution technique (ICS) with Mueller Hinton agar and Denley's multipoint applicator (Mast Laboratories Ltd., Liverpool, U.K.). Plates were inoculated with a suspension of organism containing lo5 to 10" colony forming units (CFUs) per ml and incubated for 16 to 20 hours at 37OC. The lowest concentration of an antibiotic that inhibited the growth of a particular strain was recorded as the MIC.
Results Two hundred and seventy-four strains of Salmonella typhi were isolated. Of these isolates, 137 were resistant and 137 were sensitive to chloramphenicol, ampicillin and/or co-trimoxazole. The sensitive strains were identified as follows: 75 S. typhi, 45 S. paratyphi A, and 17 S. parutyphi B. The sensitivities of all isolates studied to the various antibiotic agents are summarized in Table I. The mean zones of inhibition and MIC of the various antibiotic agents against resistant and sensitive strains of S. typhi are summarized in Tables I1 and 111, respectively. 56 1
Comparative in vitro activity of enoxacin and other fluoroquinolones against multi-resistant strains of Salmonella tvphi
Table I.
Susceptibility of 274 bacterial isolates studied to the various antibiotics
Susceptibility
Antibiotic agent
Multi-resistant
Chloramphenicol Ampicillin Co-trimoxazole Chlorarnphenicol Co-trimoxazole Ampicillin Co-trimoxazole Ampicillin Co-trimoxazole
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Resistant Resistant Resistant Resistant Sensitive
No. strains 91
6 24 15
1 137
Chloramphenicol Ampicillin Co-trimoxazole
Table 11. Zone diameters of inhibition of various antibiotics against resistant and sensitive strains of typhoid Salmonellae Disk concentration if%)
No. strains
Zone of inhibition (mmt
10
10 10 10 1.25/23.75 1.25/23.75
97 40 130 7 122 15
0 20 0 0 20
Enoxacin Lomefloxacin Norfloxacin Ofloxacin Pefloxacin
10 10 5 5 5
137 137 137 137 137
34 34 36 35 35
I r i = 137) Chloramphenicol Ampicillin Co-trimoxazole
10 10 1.25/23.75
137 137 137
20 20 20
Enoxacin Lomefloxacin Norfloxacin Ofloxacin Pefloxacin
10 10 5 5 5
137 137 137 137 137
34 34 36 35 35
Antibiotic
Resistant ( n= 1.171 Chloramphenicol
Ampicillin Co-trimoxazole
18
Serisitive
Note: Resistant = resistant to one, two or all the conventional anti-typhoid antibiotics: sensitive =sensitive to all the three conventional anti-typhoid antibiotics
Enoxacin was active against all tested strains of 5’.typhi, sensitive and resistant to chloramphenicol, ampicillin and/or co-trimoxazole. Only 1 of 137 resistant strains was not inhibited by an enoxacin concentration of 0.12 mg/ml. The MIC9,,: 562
Abdul Hannan
maximum serum concentration ratio was approximately 25, which would be in the therapeutic range. The MIC and/or inhibition zone diameters obtained with norfloxacin and lomefloxacin were similar to those obtained with enoxacin (Tables I1 and 111). Ofloxacin and pefloxacin demonstrated marginally superier MICs and better M1C:serum concentration ratios when compared to enoxacin.
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Table 111. Minimum inhibitory concentration (MIC)of quinolones against resistant and sensitive strains of typhoid Salmonellae An tibiotic
Minimum inhibitory concentration (mg/l)
Serum concentration (mg/l)
Range
MGO
MGOO
Resistant (n = 137) Enoxacin Norfloxacin Ofloxacin Pefloxacin
0.06 to 0.25 0.06 to 0.25 0.03 to0.12 0.03 to0.12
0.12 0.12 0.03 0.06
0.25 0.25 0.12 0.12
2.0 to 3.0* (X25) 1.5 (X12.5) 3.8 (X125) 4.0 to 5.8 (X96)
Sensitive In = 137) Enoxacin Norfloxacin Ofloxacin Pefloxacin
0.06 to0.12 0.06 to 0.25 0.03 to 0.12 0.03 to0.12
0.06 0.12 0.03 0.06
0.12 0.25 0.12 0.12
2.0 to 3.0 (X25) 1.5 (X12.5) 3.8 (X125) 4.0 to 5.8 (X96)
Note: See footnote to Table 11. *3.5 to 4.5 mg/l on achieving a steady state in 2 to 3 days with scheduled dosing
Discussion In the present study, the results demonstrated that enoxacin had good in vitro activity against all strains of Salmonella typhi tested, irrespective of the sensitivity/ resistance of the strain to standard anti-typhoid drugs. The activity of enoxacin compared well with that of other fluoroquinolones, i.e. lomefloxacin, norfloxacin, ofloxacin, and pefloxacin. The MIC,,, MICloo,and serum concentrations for each of the quinolones tested in this study indicate that therapeutic concentrations of these antibiotics are easily obtained. In conclusion, fluorinated quinolones may prove to be the therapy of choice in cases of typhoid fever caused by strains of Salmonella typhi resistant to current standard antibiotics. References 1. Hannan, A., (1986). In vitvo activity of ofloxacin against 210 clinical isolates of typhoid salmonella. Infection, 14, Suppl. 4, S243-S244. 2. Hannan, A., Ahmed, S. A,, Akhtar, M. A., Answar, C. M., Mahmood, S . , Kaleem, M., and Iqbal, M., (1988). A possible break-through in the treatment of typhoid fever caused by chloramphenicol resistant Salmonelfa typhi. In: “Proceedings 6th Medical Congress of Chemotherapy, Taormina-Giardini Naxos, Italy”, pp. 383-385. Eds.: G. Nicoletti and G. Cocuzza. Edizioni Riviste Scientifiche, Florence. 3. Siporin, C., and Towse, G., (1984). Enoxacin: world-wide in vitro activity against 22,451 clinical isolates. J. Antimicrob. Chernother:. 14, Suppl. C, 47-55. 4. Stoke, E. J., and Ridgeway, G. L.. (1980). Antibacterial drugs: In: “Clinical Bacteriology”, 5th edit., pp. 200-254. Arnold, London.
563
Current Medical Research and Opinion
Vol. 12, No. 9,1992
Comparative in vitro activity of enoxacin and other fluoroquinolones against multi-resistant strains of Salmonella typhi
Abdul Hannan, M.B., B.S.,
Curr. Med. Res. Opin., (1992), 12,560.
-
D.C.P.,Dip.Bact.,F.R.C.Path. Armed Forces Institute of Pathology, Rawalpindi, Pakistan
Received: 6th December 1991
Summary The in vitro activities of enoxacin, lomefloxacin, norfloxacin, ofloxacin, and pefloxacin against 274 strains of Salmonella typhi isolated from suspected typhoid fever patients (137 multi-resistant strains and 137 strains sensitive to chloramphenicol, ampicillin and/or co-trimoxazole) were determined using disk diffusion and agar dilution techniques. In vitro, enoxacin was active against all tested strains with a MIC,, and inhibition zone size against multi-resistant strains of 0.12 mg/l and 34 m m diamete6 respectively. Similar results were found with the other fluoroquinolones. Enoxacin and other fluoroquinolones may be the therapy of choice in cases of typhoid fever caused by organisms resistant to the standard therapy, chloramphenicol. Key words: Enoxacin - anti-infective agents, quinolone - Salmonella typhi microbial sensitivity tests
Introduction Chloramphenicol therapy has been the treatment of choice for typhoid fever, followed by either ampicillin or co-trimoxazole therapy.' Recently, however, strains of Salmonella typhi have been identified which are resistant to chloramphenicol, ampicillin and/or co-trimoxazole.* Enoxacin, a newly developed member of the quinolone-azaquinolone class of antibiotics, has been shown to have a broad spectrum of in vitro activity against Gram-negative and Grampositive pathogen^.^ The present study was designed to determine the in vitro activity of enoxacin and other fluorinated quinolones against chloramphenicol-, ampicillin-, and/or co-trimoxazole-resistant strains of Salmonella typhi.
Material and methods Bacterial isolates Strains of Salmonella typhi were isolated from blood cultures obtained from patients with signs/symptoms compatible with a diagnosis of typhoid fever. Freeze-dried cultures were reconstituted with 0.5 ml of 7.5% glucose broth and incubated overnight at 37OC. Isolates were then sub-cultured on MacConkey agar and blood agar.4 In addition to the clinical blood isolates, 5 Salmonella typhi strains with known minimum inhibitory concentrations (MICs) were also tested for control purposes. The control strains were designated: AFIP-22103 S. typhi (chloramphenicol MIC:256 mg/l), AFIP-45673 S. tvphi (chloramphenicol MIC: 560
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Abdul Hannan
4 mg/l), AFIP-25625 S. typhi (ofloxacin MIC:0.03 mg/l), 'wild'-African (chloramphenicol MIC:lmg/l), and JV-1052 (chloramphenicol MIC:128 mg/l). AFIP strains represented blood culture isolates. The 'wild' and JV strains were obtained as nutrient agar sub-cultures courtesy of Prof. J. Vandepitte (Universitaire Ziekenhuis St. Rafael, Leuven, Belgium). Clinical and control strains were stored freeze dried until sub-cultured for sensitivity testing. The identity of each strain was serologically confirmed using specific antisera (Wellcome Reagents Ltd., U.K.). Biochemical profiles of each strain were constructed using 'sugar sets' (AFIP Rawalpindi, Pakistan) and API-20E galleries (API-System, Montalieu-Vercieu, France).
Disk diffusion sensitivity testing The base materials for the various antibiotics were obtained from the suppliers as follows: enoxacin - Abbott Laboratories, Karachi; norfloxacin - Merck Sharp & Dohme, Karachi; ofloxacin - Hoechst, Pakistan; and pefloxacin RhBne-Poulenc, Pakistan. Filter paper disks (6 mm) were prepared by soaking them in the appropriate concentration of stock solution for each agent for 30 minutes and then drying each disk at 5OOC. The disk concentrations of antibiotic agent (pg/disk) were as follows: ampicillin 10; chloramphenicol 10; co-trimoxazole 25; enoxacin 10; lomefloxacin 10; norfloxacin 5 ; ofloxacin 5; and pefloxacin 5. Lomefloxacin disks (10 pg/disk) were supplied commercially by Searle, Pakistan. Disk diffusion was performed using the Kirby-Bauer method. Inoculated plates with antibiotic disks applied were incubated for 16 to 18 hours at 37OC and inhibition zones were measured using a millimeter ruler (Fowler Ultra Cal 11; Sylvac, U.S.A.).
MIC determinations MICs were determined using the agar dilution technique (ICS) with Mueller Hinton agar and Denley's multipoint applicator (Mast Laboratories Ltd., Liverpool, U.K.). Plates were inoculated with a suspension of organism containing lo5 to 10" colony forming units (CFUs) per ml and incubated for 16 to 20 hours at 37OC. The lowest concentration of an antibiotic that inhibited the growth of a particular strain was recorded as the MIC.
Results Two hundred and seventy-four strains of Salmonella typhi were isolated. Of these isolates, 137 were resistant and 137 were sensitive to chloramphenicol, ampicillin and/or co-trimoxazole. The sensitive strains were identified as follows: 75 S. typhi, 45 S. paratyphi A, and 17 S. parutyphi B. The sensitivities of all isolates studied to the various antibiotic agents are summarized in Table I. The mean zones of inhibition and MIC of the various antibiotic agents against resistant and sensitive strains of S. typhi are summarized in Tables I1 and 111, respectively. 56 1
Comparative in vitro activity of enoxacin and other fluoroquinolones against multi-resistant strains of Salmonella tvphi
Table I.
Susceptibility of 274 bacterial isolates studied to the various antibiotics
Susceptibility
Antibiotic agent
Multi-resistant
Chloramphenicol Ampicillin Co-trimoxazole Chlorarnphenicol Co-trimoxazole Ampicillin Co-trimoxazole Ampicillin Co-trimoxazole
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Resistant Resistant Resistant Resistant Sensitive
No. strains 91
6 24 15
1 137
Chloramphenicol Ampicillin Co-trimoxazole
Table 11. Zone diameters of inhibition of various antibiotics against resistant and sensitive strains of typhoid Salmonellae Disk concentration if%)
No. strains
Zone of inhibition (mmt
10
10 10 10 1.25/23.75 1.25/23.75
97 40 130 7 122 15
0 20 0 0 20
Enoxacin Lomefloxacin Norfloxacin Ofloxacin Pefloxacin
10 10 5 5 5
137 137 137 137 137
34 34 36 35 35
I r i = 137) Chloramphenicol Ampicillin Co-trimoxazole
10 10 1.25/23.75
137 137 137
20 20 20
Enoxacin Lomefloxacin Norfloxacin Ofloxacin Pefloxacin
10 10 5 5 5
137 137 137 137 137
34 34 36 35 35
Antibiotic
Resistant ( n= 1.171 Chloramphenicol
Ampicillin Co-trimoxazole
18
Serisitive
Note: Resistant = resistant to one, two or all the conventional anti-typhoid antibiotics: sensitive =sensitive to all the three conventional anti-typhoid antibiotics
Enoxacin was active against all tested strains of 5’.typhi, sensitive and resistant to chloramphenicol, ampicillin and/or co-trimoxazole. Only 1 of 137 resistant strains was not inhibited by an enoxacin concentration of 0.12 mg/ml. The MIC9,,: 562
Abdul Hannan
maximum serum concentration ratio was approximately 25, which would be in the therapeutic range. The MIC and/or inhibition zone diameters obtained with norfloxacin and lomefloxacin were similar to those obtained with enoxacin (Tables I1 and 111). Ofloxacin and pefloxacin demonstrated marginally superier MICs and better M1C:serum concentration ratios when compared to enoxacin.
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Table 111. Minimum inhibitory concentration (MIC)of quinolones against resistant and sensitive strains of typhoid Salmonellae An tibiotic
Minimum inhibitory concentration (mg/l)
Serum concentration (mg/l)
Range
MGO
MGOO
Resistant (n = 137) Enoxacin Norfloxacin Ofloxacin Pefloxacin
0.06 to 0.25 0.06 to 0.25 0.03 to0.12 0.03 to0.12
0.12 0.12 0.03 0.06
0.25 0.25 0.12 0.12
2.0 to 3.0* (X25) 1.5 (X12.5) 3.8 (X125) 4.0 to 5.8 (X96)
Sensitive In = 137) Enoxacin Norfloxacin Ofloxacin Pefloxacin
0.06 to0.12 0.06 to 0.25 0.03 to 0.12 0.03 to0.12
0.06 0.12 0.03 0.06
0.12 0.25 0.12 0.12
2.0 to 3.0 (X25) 1.5 (X12.5) 3.8 (X125) 4.0 to 5.8 (X96)
Note: See footnote to Table 11. *3.5 to 4.5 mg/l on achieving a steady state in 2 to 3 days with scheduled dosing
Discussion In the present study, the results demonstrated that enoxacin had good in vitro activity against all strains of Salmonella typhi tested, irrespective of the sensitivity/ resistance of the strain to standard anti-typhoid drugs. The activity of enoxacin compared well with that of other fluoroquinolones, i.e. lomefloxacin, norfloxacin, ofloxacin, and pefloxacin. The MIC,,, MICloo,and serum concentrations for each of the quinolones tested in this study indicate that therapeutic concentrations of these antibiotics are easily obtained. In conclusion, fluorinated quinolones may prove to be the therapy of choice in cases of typhoid fever caused by strains of Salmonella typhi resistant to current standard antibiotics. References 1. Hannan, A., (1986). In vitvo activity of ofloxacin against 210 clinical isolates of typhoid salmonella. Infection, 14, Suppl. 4, S243-S244. 2. Hannan, A., Ahmed, S. A,, Akhtar, M. A., Answar, C. M., Mahmood, S . , Kaleem, M., and Iqbal, M., (1988). A possible break-through in the treatment of typhoid fever caused by chloramphenicol resistant Salmonelfa typhi. In: “Proceedings 6th Medical Congress of Chemotherapy, Taormina-Giardini Naxos, Italy”, pp. 383-385. Eds.: G. Nicoletti and G. Cocuzza. Edizioni Riviste Scientifiche, Florence. 3. Siporin, C., and Towse, G., (1984). Enoxacin: world-wide in vitro activity against 22,451 clinical isolates. J. Antimicrob. Chernother:. 14, Suppl. C, 47-55. 4. Stoke, E. J., and Ridgeway, G. L.. (1980). Antibacterial drugs: In: “Clinical Bacteriology”, 5th edit., pp. 200-254. Arnold, London.
563
