Vol. 9, 1990
References 1. Bryan LE, Bedard J, Wong S, Chamberland S: Quinolone antimicrobial agents: mechanism of action and resistance development. Clinical and Investigative Medicine 1989, 12: 14-19.
2. Shen LL, Kohlbrenner WE, Weigl D, Baranowski J: Mechanism of quinolone inhibition of
3.
4.
5.
6.
7.
DNA gyrase. Appearance of unique norfloxacin binding sites in enzyme-DNA complexes. Journal of Biological Chemistry 1989, 264: 2973-2978. Forsgren A, Schlossman SF, Tedder TF: 4-Quinolone drugs affect cell cycle progression and function of human lymphocytes in vitro. Antimicrobial Agents and Chemotherapy 1987, 31: 768-773, Gollapudi SVS, Prabhala RH, Thadepalli H: Effect of ciprofloxacin on mitogen-stimulated lymphocyte proliferation. Antimicrobial Agents and Chemotherapy 1986, 29: 337-338. Roche Y, Fay M, Gougerot-Poeidalo MA: Interleukin-1 production by antibiotic-treated human monocytes. Journal of Antimicrobial Chemotherapy 1988, 21: 597-607. Bredberg A, Brant M, Riesbeck K, Azou Y, Forsgren A: The 4-quinolone antibiotics: positive genotoxic screening tests despite an apparent lack of mutation induction. Mutation Research 1989, 211: I71-180. Riesbeek K, Bredberg A, Forsgren A: Ciprofloxacin does not inhibit mitochondrial functions but other antibiotics do. Antimicrobial Agents and Chemotherapy 1990, 34: 167-169.
413
16. Kemmerich B, Rossing TH, Pennington JE: Comparative oxidative microbicidal activity of human blood monocytes and alveolar macrophages and activation by recombinant gamma interferon. American Review of Respiratory Disease 1987, 136: 266-270. 17. O'Garra A: Peptide regulatory factors: interleukins and the immune system (1). Lancet 1989, i: 943-947. 18. Ibrahim MS, Maged ZA, Haron A, Khalil RY, Attallah AM: Antibiotics and immunity: effects of antibiotics on mitogen responsiveness of lymphocytes and interleukin-2 production. Chemiotherapia 1988, 7: 369-372. 19. O'Garra, A: Peptide regulatory factors: interleukins and the immune system (2). Lancet 1989, i: 1003-1005. 20. Petit JC, Daguet GL, Richard G, Burghofer B: Influence of ciprofloxacin and piperacillin on interleukin-1 production by murine macrophages. Journal of Antimicrobial Chemotherapy t987, 20: 615-617. 21. Traeey K J, Vlassara H, Cerami A: Peptide regulatory factors: cachectin/tumour necrosis factor. Lancet 1989, i: 1122-1126. 22. Weisbart RH, Gasson JC, Golde DW: Colonystimulating factors and host defense. Annals of Internal Medicine t989,110: 297-303.
8. Forsgren A, Bergh AK, Brandt M, Hansson G: Quinolones affect thymidine incorporation into the DNA of human lymphocytes. Antimierobial Agents and Chemotherapy 1986, 29: 506-508.
9. Forsgren A, Bredberg A, Pardee AB, Sehlossman SF, Tedder TF: Effects of ciprofloxacin on eucaryotic pyrimidine nucleotide biosynthesis and cell growth. Antimicrobial Agents and Chemotherapy 1987, 31: 774-779.
10. Gouenounou M, Ronco E, Kodari E, Vaeheron F, Momrikof V: Modulation de la response proliferative in vitro des cellules spleniques de souris et de la production d'IL-I par les quinolones. Pathologic Biologic 1987, 35: 785-789. 11. De Simone C, Baldinelli L, Ferrazzi M, De Santis S, Pugnaloni L, Sorice F: Influence of ofloxacin, norfloxacin, nalidixic acid, pyromidic acid and pipemidic acid on human gamma-interferon production and blastogenesis. Journal of Antimicrobial Chemotherapy 1986, 17: 811--814. 12. Manzella JP, Clark JK: Effects of quinolones on mitogen-stimulated human mononuclear leukocytes. Journal of Antimicrobial Chemotherapy 1988, 21: 183-186. 13. Pusztai-Markos S, Hauss K: In vitro effect of different antimicrobial agents on IL-2 production and IL-2 receptor expression of human lymphocytes. In: Gillissen G, Opferkuch W, Peters G, Pulverer G (ed): The influence of antibiotics on the host-parasite relationship. Springer Verlag, Berlin, 1989.
14. Riesbeck K, Andersson J, Gullberg M, Forsgren A: Fluorinated 4-quinolones induce hyperproduction of interleukin-2. Proceedings of the National Academy Sciences of the USA 1989, 86: 2809-2813. 15. Roche Y, Fay M, Gnugerot-Poeidalo MA: Enhancement of interleukin-2 production by quinolonetreated human mononuclear leukocytes. International Journal of Immunopharmacology 1988, 10: 161-167.
In Vitro Susceptibility of
Aeromonas caviae, Aeromonas hydrophila and Aeromonas sobria to Fifteen Antibacterial Agents A. Burgos 1'2., G. Quind6s t, R. Martinez 1,2, P. Rojo 2, R. Cisterna t'2 In vitro testing o f tile activity o f 15 antibacterial agents against 522 clinical isolates o f A e r o m o n a s species d e m o n s t r a t e d s o m e speciesassociated trends. A m o x i c i l l i n plus clavulanic acid was effective against approximately 45 % o f Aeromonas caviae and Aeromonas hydrophila, but all Aeroraonas sobria isolates were resistant. A z t r e o n a m , piperacillin and mezlocillin w e r e highly active against all the strains o f A e r o m o n a s tested. Ticarcillin was equally effective against Aeromonas caviae and A e r o monas hydrophila, but m o r e than 50 % o f Aeromonas sobria isolates were resistant. The latter species was m o r e susceptible to cephalosporins than A e r o m o n a s h y d r o p h i l a and 1Departamento de Microbiologia e Immunologia, Facultad de Mediclna y Odontologia, Universidad del Pais Vasco, Apartado 699, 48080 Bilbao, Spain. 2Servicio de Microbiologfa, Hospital Civil de Bilbao, Bilbao, Spain.
414
c a v i a e . Chloramphenicol, tetracycline and trimethoprim-sulfamethoxazole were extremely active against all three A e r o m o n a s species, likewise ofloxacin and ciprofloxacin. Aztreonam, third-generation cephalosporins, chloramphenicol and the quinolones can thus be considered for therapy of infections when A e r o m o n a s is implicated. Aeromonas
Aerornonas are motile microorganisms which are found in aquatic environments and have been implicated in a variety of human infections, including gastroenteritis, superinfection of burns and wounds, and septicaemia in immunocompromised patients. Recent taxonomic studies indicate that these bacteria form a specific group: the family Aeromonadaceae (1). The type genus is Aeromonas, and at least three species produce human disease: A e r o m o n a s caviae, Aeromonas hydrophila and Aeromonas sobria. The majority of Aeromonas hydrophila and Aeromonas sobria strains recovered from gastroenteritis are enterotoxigenic, suggesting a correlation between enterotoxin production and diarrhoeal symptoms. Other indicators of virulence such as the recovery of these microorganisms from blood samples, their lethal dose for laboratory animals and their exoenzymatic activity appear to differ significantly among the there species (2). In previous studies of antibiotic resistance in the genus (2, 3), relatively small numbers of strains, sometimes from different geographical areas, have been tested and there is a need for a definitive study of resistance in a larger number of isolates from different clinical sources which are identified to species level. In this study we tested the susceptibility to 15 antibacterial agents of 522 isolates of Aeromonas spp. isolated from clinical samples, most of which were recovered from patients with a clinical diagnosis of Aeromonas gastroenteritis. Materials and Methods. A total of 522 A e r o monas strains isolated from the same number of hospitalized patients were tested. All isolates were obtained from clinical samples from patients admitted to the Hospital Civil de Bilbao in the last four years. Samples were plated on the following agar media: MacConkey agar (Oxoid, UK), XLD medium (Oxoid), Hektoen enteric agar (Oxoid), selective yersinia agar (Oxoid), selective campylobacter agar (blood agar base No. 2 supplemented with 5 % horse blood, campylobacter growth supplement and Butzler selective antimicrobial supplement (Oxoid)), and DNase-toluidine blue-ampicillin agar (DNase agar (Oxoid), toluidine blue 100 mg/t (Merck, FRG) and ampicillin 30 mg/1).
Eur. J. Clin. Microbiol. Infect Dis.
The identity of the A e r o m o n a s strains was confirmed by standard biochemical tests interpreted according to the criteriaof Popoff (4). The antimicrobial agents tested included Ampicillin, amoxicillin, clavulanic acid and ticarcillin (Beecham, UK), piperacillin (Cyanamid Lederle, Spain), mezlocillin and ciprofloxacin (Bayer, Spain), aztreonam (Squibb, Spain), cefazolin (Antibi6ticos, Spain), cefoxitin and tetracycline (Compafifa Espafiola de la Penicilina y Antibi6ticos, Spain), ceftazidime (Glaxo, Spain), cefotaxime and ofloxacin (Hoechst, Spain), chloramphenicol (Parke Davis, Spain), and trimethoprim and sulfamethoxazole (.Roche, Spain). Antibiotics were dissolved in distilled water, except for ciprofloxacin, ofloxacin and sulfamethoxazole (dissolved in 1 N NaOH), trimethoprim and chloramphenicol (dissolved in 96 % ethanol), aztreonam (dissolved in a saturated solution of Na2CO3), ampicillin (dissolved in PBS of pH 8), and amoxicillin, cefazolin, cefoxitin and ceftazidime (dissolved in PBS of pH 6). MICs were determined by a microdilution method in Mueller-Hinton broth (Oxoid) (5). Microdilution plates contained 50 gl MuellerHinton broth and antibacterial agents at concentrations ranging from 0.062 to 128 gg/ml, except in the case of amoxicillin-clavulanic acid which was incorporated at concentrations ranging from 0.156 to 320 gg/ml. An inoculum of approximately 105 viable cells in 50 p.1 MuellerHinton broth was applied to the plates. Plates were incubated aerobically at 37 °C for 18h. Susceptibility breakpoints for the 15 antibacterial agents were obtained from Table 2 - M2T4 of the NCCLS susceptibility documents (6). Differences in the susceptibility patterns between Aeromonas species were analyzed by the chi-square test with Yates correction where appropriate. Results and Discussion. A total of 504 strains of A e r o m o n a s were isolated from faeces (253 isolates of Aeromonas caviae, 48 of Aeromonas hydrophila and 17 of Aeromonas sobria were isolated from diarrhoeic faeces). Most of the isolates from faecal samples were recovered in mixed culture with other enteric pathogens, with the exception of 124 isolates of Aeromonas caviae, 23 of Aeromonas hydrophila and 7 of Aeromonas sobria which occurred in pure culture. Two bacteraemic isolates and one isolate each of Aeromonas caviae and A e r o monas sobria from wounds were also recovered in pure culture. Another 14 isolates (2 A e r o monas caviae and 12 Aeromonas hydrophila) were recovered in mixed culture from wounds. Almost all of 412 Aeromonas caviae isolates (> 98 %) were susceptible to aztreonam, mezlo-
_Wol. 9, 1990
415
Table 1: In vitro activity (values expressed in I.tg/ml) of 15.antibacterial agents against 412 Aeromonas caviae isolates. Antibacterial agent
MIC 50
Ampicillin Amoxicillin + clavulanic acid Piperacillin Ticarcillin Mezlocillin A z t r e o n am Cefazolin Cefoxitin Ceftazidime Cefotaxime Chloramphenicol Tetracycline Trimethoprim-sutfamethoxazole Ofloxacin Ciprofloxacin
64 10 2 16 4 _ 128 128 1 1 4 2 > 128 _128 1->128 0.25->128 128 _128 _
References 1. Bryan LE, Bedard J, Wong S, Chamberland S: Quinolone antimicrobial agents: mechanism of action and resistance development. Clinical and Investigative Medicine 1989, 12: 14-19.
2. Shen LL, Kohlbrenner WE, Weigl D, Baranowski J: Mechanism of quinolone inhibition of
3.
4.
5.
6.
7.
DNA gyrase. Appearance of unique norfloxacin binding sites in enzyme-DNA complexes. Journal of Biological Chemistry 1989, 264: 2973-2978. Forsgren A, Schlossman SF, Tedder TF: 4-Quinolone drugs affect cell cycle progression and function of human lymphocytes in vitro. Antimicrobial Agents and Chemotherapy 1987, 31: 768-773, Gollapudi SVS, Prabhala RH, Thadepalli H: Effect of ciprofloxacin on mitogen-stimulated lymphocyte proliferation. Antimicrobial Agents and Chemotherapy 1986, 29: 337-338. Roche Y, Fay M, Gougerot-Poeidalo MA: Interleukin-1 production by antibiotic-treated human monocytes. Journal of Antimicrobial Chemotherapy 1988, 21: 597-607. Bredberg A, Brant M, Riesbeck K, Azou Y, Forsgren A: The 4-quinolone antibiotics: positive genotoxic screening tests despite an apparent lack of mutation induction. Mutation Research 1989, 211: I71-180. Riesbeek K, Bredberg A, Forsgren A: Ciprofloxacin does not inhibit mitochondrial functions but other antibiotics do. Antimicrobial Agents and Chemotherapy 1990, 34: 167-169.
413
16. Kemmerich B, Rossing TH, Pennington JE: Comparative oxidative microbicidal activity of human blood monocytes and alveolar macrophages and activation by recombinant gamma interferon. American Review of Respiratory Disease 1987, 136: 266-270. 17. O'Garra A: Peptide regulatory factors: interleukins and the immune system (1). Lancet 1989, i: 943-947. 18. Ibrahim MS, Maged ZA, Haron A, Khalil RY, Attallah AM: Antibiotics and immunity: effects of antibiotics on mitogen responsiveness of lymphocytes and interleukin-2 production. Chemiotherapia 1988, 7: 369-372. 19. O'Garra, A: Peptide regulatory factors: interleukins and the immune system (2). Lancet 1989, i: 1003-1005. 20. Petit JC, Daguet GL, Richard G, Burghofer B: Influence of ciprofloxacin and piperacillin on interleukin-1 production by murine macrophages. Journal of Antimicrobial Chemotherapy t987, 20: 615-617. 21. Traeey K J, Vlassara H, Cerami A: Peptide regulatory factors: cachectin/tumour necrosis factor. Lancet 1989, i: 1122-1126. 22. Weisbart RH, Gasson JC, Golde DW: Colonystimulating factors and host defense. Annals of Internal Medicine t989,110: 297-303.
8. Forsgren A, Bergh AK, Brandt M, Hansson G: Quinolones affect thymidine incorporation into the DNA of human lymphocytes. Antimierobial Agents and Chemotherapy 1986, 29: 506-508.
9. Forsgren A, Bredberg A, Pardee AB, Sehlossman SF, Tedder TF: Effects of ciprofloxacin on eucaryotic pyrimidine nucleotide biosynthesis and cell growth. Antimicrobial Agents and Chemotherapy 1987, 31: 774-779.
10. Gouenounou M, Ronco E, Kodari E, Vaeheron F, Momrikof V: Modulation de la response proliferative in vitro des cellules spleniques de souris et de la production d'IL-I par les quinolones. Pathologic Biologic 1987, 35: 785-789. 11. De Simone C, Baldinelli L, Ferrazzi M, De Santis S, Pugnaloni L, Sorice F: Influence of ofloxacin, norfloxacin, nalidixic acid, pyromidic acid and pipemidic acid on human gamma-interferon production and blastogenesis. Journal of Antimicrobial Chemotherapy 1986, 17: 811--814. 12. Manzella JP, Clark JK: Effects of quinolones on mitogen-stimulated human mononuclear leukocytes. Journal of Antimicrobial Chemotherapy 1988, 21: 183-186. 13. Pusztai-Markos S, Hauss K: In vitro effect of different antimicrobial agents on IL-2 production and IL-2 receptor expression of human lymphocytes. In: Gillissen G, Opferkuch W, Peters G, Pulverer G (ed): The influence of antibiotics on the host-parasite relationship. Springer Verlag, Berlin, 1989.
14. Riesbeck K, Andersson J, Gullberg M, Forsgren A: Fluorinated 4-quinolones induce hyperproduction of interleukin-2. Proceedings of the National Academy Sciences of the USA 1989, 86: 2809-2813. 15. Roche Y, Fay M, Gnugerot-Poeidalo MA: Enhancement of interleukin-2 production by quinolonetreated human mononuclear leukocytes. International Journal of Immunopharmacology 1988, 10: 161-167.
In Vitro Susceptibility of
Aeromonas caviae, Aeromonas hydrophila and Aeromonas sobria to Fifteen Antibacterial Agents A. Burgos 1'2., G. Quind6s t, R. Martinez 1,2, P. Rojo 2, R. Cisterna t'2 In vitro testing o f tile activity o f 15 antibacterial agents against 522 clinical isolates o f A e r o m o n a s species d e m o n s t r a t e d s o m e speciesassociated trends. A m o x i c i l l i n plus clavulanic acid was effective against approximately 45 % o f Aeromonas caviae and Aeromonas hydrophila, but all Aeroraonas sobria isolates were resistant. A z t r e o n a m , piperacillin and mezlocillin w e r e highly active against all the strains o f A e r o m o n a s tested. Ticarcillin was equally effective against Aeromonas caviae and A e r o monas hydrophila, but m o r e than 50 % o f Aeromonas sobria isolates were resistant. The latter species was m o r e susceptible to cephalosporins than A e r o m o n a s h y d r o p h i l a and 1Departamento de Microbiologia e Immunologia, Facultad de Mediclna y Odontologia, Universidad del Pais Vasco, Apartado 699, 48080 Bilbao, Spain. 2Servicio de Microbiologfa, Hospital Civil de Bilbao, Bilbao, Spain.
414
c a v i a e . Chloramphenicol, tetracycline and trimethoprim-sulfamethoxazole were extremely active against all three A e r o m o n a s species, likewise ofloxacin and ciprofloxacin. Aztreonam, third-generation cephalosporins, chloramphenicol and the quinolones can thus be considered for therapy of infections when A e r o m o n a s is implicated. Aeromonas
Aerornonas are motile microorganisms which are found in aquatic environments and have been implicated in a variety of human infections, including gastroenteritis, superinfection of burns and wounds, and septicaemia in immunocompromised patients. Recent taxonomic studies indicate that these bacteria form a specific group: the family Aeromonadaceae (1). The type genus is Aeromonas, and at least three species produce human disease: A e r o m o n a s caviae, Aeromonas hydrophila and Aeromonas sobria. The majority of Aeromonas hydrophila and Aeromonas sobria strains recovered from gastroenteritis are enterotoxigenic, suggesting a correlation between enterotoxin production and diarrhoeal symptoms. Other indicators of virulence such as the recovery of these microorganisms from blood samples, their lethal dose for laboratory animals and their exoenzymatic activity appear to differ significantly among the there species (2). In previous studies of antibiotic resistance in the genus (2, 3), relatively small numbers of strains, sometimes from different geographical areas, have been tested and there is a need for a definitive study of resistance in a larger number of isolates from different clinical sources which are identified to species level. In this study we tested the susceptibility to 15 antibacterial agents of 522 isolates of Aeromonas spp. isolated from clinical samples, most of which were recovered from patients with a clinical diagnosis of Aeromonas gastroenteritis. Materials and Methods. A total of 522 A e r o monas strains isolated from the same number of hospitalized patients were tested. All isolates were obtained from clinical samples from patients admitted to the Hospital Civil de Bilbao in the last four years. Samples were plated on the following agar media: MacConkey agar (Oxoid, UK), XLD medium (Oxoid), Hektoen enteric agar (Oxoid), selective yersinia agar (Oxoid), selective campylobacter agar (blood agar base No. 2 supplemented with 5 % horse blood, campylobacter growth supplement and Butzler selective antimicrobial supplement (Oxoid)), and DNase-toluidine blue-ampicillin agar (DNase agar (Oxoid), toluidine blue 100 mg/t (Merck, FRG) and ampicillin 30 mg/1).
Eur. J. Clin. Microbiol. Infect Dis.
The identity of the A e r o m o n a s strains was confirmed by standard biochemical tests interpreted according to the criteriaof Popoff (4). The antimicrobial agents tested included Ampicillin, amoxicillin, clavulanic acid and ticarcillin (Beecham, UK), piperacillin (Cyanamid Lederle, Spain), mezlocillin and ciprofloxacin (Bayer, Spain), aztreonam (Squibb, Spain), cefazolin (Antibi6ticos, Spain), cefoxitin and tetracycline (Compafifa Espafiola de la Penicilina y Antibi6ticos, Spain), ceftazidime (Glaxo, Spain), cefotaxime and ofloxacin (Hoechst, Spain), chloramphenicol (Parke Davis, Spain), and trimethoprim and sulfamethoxazole (.Roche, Spain). Antibiotics were dissolved in distilled water, except for ciprofloxacin, ofloxacin and sulfamethoxazole (dissolved in 1 N NaOH), trimethoprim and chloramphenicol (dissolved in 96 % ethanol), aztreonam (dissolved in a saturated solution of Na2CO3), ampicillin (dissolved in PBS of pH 8), and amoxicillin, cefazolin, cefoxitin and ceftazidime (dissolved in PBS of pH 6). MICs were determined by a microdilution method in Mueller-Hinton broth (Oxoid) (5). Microdilution plates contained 50 gl MuellerHinton broth and antibacterial agents at concentrations ranging from 0.062 to 128 gg/ml, except in the case of amoxicillin-clavulanic acid which was incorporated at concentrations ranging from 0.156 to 320 gg/ml. An inoculum of approximately 105 viable cells in 50 p.1 MuellerHinton broth was applied to the plates. Plates were incubated aerobically at 37 °C for 18h. Susceptibility breakpoints for the 15 antibacterial agents were obtained from Table 2 - M2T4 of the NCCLS susceptibility documents (6). Differences in the susceptibility patterns between Aeromonas species were analyzed by the chi-square test with Yates correction where appropriate. Results and Discussion. A total of 504 strains of A e r o m o n a s were isolated from faeces (253 isolates of Aeromonas caviae, 48 of Aeromonas hydrophila and 17 of Aeromonas sobria were isolated from diarrhoeic faeces). Most of the isolates from faecal samples were recovered in mixed culture with other enteric pathogens, with the exception of 124 isolates of Aeromonas caviae, 23 of Aeromonas hydrophila and 7 of Aeromonas sobria which occurred in pure culture. Two bacteraemic isolates and one isolate each of Aeromonas caviae and A e r o monas sobria from wounds were also recovered in pure culture. Another 14 isolates (2 A e r o monas caviae and 12 Aeromonas hydrophila) were recovered in mixed culture from wounds. Almost all of 412 Aeromonas caviae isolates (> 98 %) were susceptible to aztreonam, mezlo-
_Wol. 9, 1990
415
Table 1: In vitro activity (values expressed in I.tg/ml) of 15.antibacterial agents against 412 Aeromonas caviae isolates. Antibacterial agent
MIC 50
Ampicillin Amoxicillin + clavulanic acid Piperacillin Ticarcillin Mezlocillin A z t r e o n am Cefazolin Cefoxitin Ceftazidime Cefotaxime Chloramphenicol Tetracycline Trimethoprim-sutfamethoxazole Ofloxacin Ciprofloxacin
64 10 2 16 4 _ 128 128 1 1 4 2 > 128 _128 1->128 0.25->128 128 _128 _
