Scand J Infect Dis 23: 337-340, 1991
Antibiotic Susceptibility of lnvasive Haemophilus influenzae Type b Isolates in Denmark in 1988 and 1989 KIM KRISTENSEN' and IDA MORTENSEN2
Scand J Infect Dis Downloaded from informahealthcare.com by V U L Periodicals Rec on 12/29/14 For personal use only.
From the 'Paediatric Departmenl GGK, Rigshospitalet and the 'Departmenl of Diagnostic Baclerioiogy and Antibiotics, Statens Serumrnstitur, Copenhagen. Denmark
A system for surveillance of invasive Haemophilus influenzae type b infections in Denmark yielded 135 strains isolated from b l o d or cerebrospinal fluid from August 1988 through December 1989. The susceptibility of the strains to 7 antibiotics was investigated by an agar dilution method. Ceftriaxone was found to be the most active drug followed by cefotaxime, ceftazidime, rifampicin, ampicillin, cefuroxime, and chloramphenicol. Except for ampicillin the MICs for the individual antibiotics were similar. Seven strains (5.2 %), which all produced beta-lactamase, were resistant to ampicillin. Since H. influenzae type b meningitis in otherwise healthy individuals almost exclusively occurs in children aged 2 months to 4 years, a third generation cephalosporin such as ceftriaxone or cefotaxime may be considered the drug of choice for initial therapy of meningitis in this age group. K. Kristensen, MD, Paediatric Department GGK, Rigshospitalet, DK-2100 Copenhagen 0, Denmark
INTRODUCTION Since Haemophilus influenzae type b (Hib) is a major cause of life-threatening invasive infections such as meningitis and epiglottitis in children. precise knowledge of antibiotic susceptibility of invasive Hib isolates is of vital importance. Two recent Danish studies on this subject showed rates of resistance to ampicillin of only 5 and 8 Yo (1, 2), which is much lower than those reported from some other countries, in which the prevalence of ampicillin resistance ranges from 27 to 57 Yo (3-5). However, none of the Danish studies were based on recent data, and because the prevalence of ampicillin resistance among Hib isolates has increased substantially during the last decade elsewhere (3-5), there is a need for new Danish data on this subject. Resistance to chloramphenicol is still uncommon in the UK and USA (3,4), but is present in 65.7% of invasive Hib isolates in Spain (5). Until now, resistance to chloramphenicol has only been described twice in Denmark (in strains also resistant to ampicillin, 6). Resistance to second or third generation cephalosporins has been reported from Canada (7), but is generally rare. Since August 1988 we have collected all Hib strains isolated from blood or cerebrospinal fluid (CSF) in Denmark. We report here on their susceptibility to 7 antibiotics relevant in the treatment or prophylaxis of invasive Hib infection. MATERIAL AND METHODS Collection of strains From August 1988 through December 1989 all departments of clinical microbiology in Denmark sent all Hib strains isolated from blood or CSF to Statens Seruminstitut. A total of 135 strains were collected. All strains were serotyped in our laboratory using rabbit antiserum and kept at -70°C until tested. Antibiotics The antibiotics studied were ampicillin, cefuroxime, cefotaxime, ceftazidime, ceftriaxone, chloramphenicol. and rifampicin. All antibiotics were kindly provided by their manufacturers.
338 K. Kristensen and I. Mortensen
Scand J Infect Dis 23 (1991)
Scand J Infect Dis Downloaded from informahealthcare.com by V U L Periodicals Rec on 12/29/14 For personal use only.
dY
Fig. 1. MICs of the antibiotics tested. 0,00075
0,003
0,O 125
0.05
0.8
OV2
3'2
12.5
50
CONCENTRATDN moll
Medium Agar dilution method was performed on 5% blood agar containing beef 509'0, Orthana pepton (NS Orthana) 1 I NaCl , Ph. Nord. (Bie & Berntsen) 0.3%,Na2HP04*12H 2 0 0.29'0, 5 N NaOH 10.5 mM, SoBigel agar type USA (Hasche) 1.39/0, CaCI2.2 H 2 0 0.00929'0, MgS04.7 H 2 0 0.0068Y0, and defibrinated horse blood 5%. The plates were supplemented with nicotine amide adenine dinucleotide (Nad 0.015 Yo).
Agar dilution Antibiotic solutions were prepared to obtain final concentrations in 2-fold dilutions ranging from 100 to 0.00075 mg/l. An agar dilution-multiple inoculator method was used. Colonies from overnight cultures on chocolate agar plates were picked and diluted in sterile saline to a density of lo9 colony forming units (cfu)/ml as measured by colorimetry (model 252 Coming Colorimeter) from which appropriate dilutions in sterile saline were prepared for inoculation (Denley A400, Sussex, England). Drops containing 103-104 cfu were delivered onto the antibiotic-containing agar plates. A control plate without antibiotics was inoculated before and after each series. Plates were incubated overnight at 35°C in an atmosphere of 59'0 carbon dioxide and 95% air. Minimum inhibitory concentration (MIC) was defined as the lowest concentration of an antibiotic which completely inhibited growth disregarding g 3 colonies or a faint haze caused by inoculum.
Table I. Etiology in 393 cases of bacterial meningitis in children aged 2 months to 4 years notified in Denmark I986 to 1988" Data: T. Renne, Department of Epidemiology, Statens Seruminstitut, Copenhagen ~
~~~
~
Bacteria
No. of cases reported
Hib Meningococci Pneumococci Listeria monocytogenes Others
158 203 26 I 5
Another 38 cases were notified in which no information about the causative organism was available.
Scand J Infect Dis 23
Antibiotic susceptibility of H. injluenzae type b 339
Detection of beta-lactamase production The chromogenic cephalosporin substrate (Glaxo compound) was used for detection of beta-lactamase production (8).
Scand J Infect Dis Downloaded from informahealthcare.com by V U L Periodicals Rec on 12/29/14 For personal use only.
RESULTS Assuming that a MIC < 1 mg/l for all antibiotics tested indicates clinical susceptibility, all strains were found susceptible to all antibiotics tested, except for 7 strains (5.2%), which were not susceptible to ampicillin (Fig. 1). All these strains produced beta-lactamase, but showed the same susceptibility to the cephalosporins as those that did not produce beta-lactamase. Ceftriaxone was found to be the most active drug with a MIC as low as 0.003 mg/l. DISCUSSION This study confirms the finding of Cordtz et al. (2) that still only about 5 'Yo of invasive Hib isolates in Denmark are resistant to ampicillin, which emphasizes that a restrictive policy on the use of antibiotics such as that pursued in Denmark probably is of value in this respect. In patients with meningitis no delay in effective treatment should be accepted, and it is therefore important to assess the risk of ampicillin resistance in a case of meningitis of unknown etiology. Of the organisms causing meningitis in otherwise healthy individuals Hib is the only one, which may be resistant to ampicillin, and because Hib meningitis in previously healthy individuals almost exclusively occurs in children aged 2 months to 4 years (9), the risk that the infecting organism should be resistant to ampicillin in a case of meningitis of unknown etiology should be calculated for this age group solely. Table I shows the distribution by etiology of cases of bacterial meningitis notified 1986 to 1988 in Denmark in children aged 2 months to 4 years. On the assumption that the rate of notification is the same for all kinds of meningitis, 40% of the cases were caused by Hib. Thus, at present the risk of ampicillin resistance in a case of meningitis of unknown etiology in Denmark in this age group is about 2 'Yo. No strains were found to be resistant to chloramphenicol or to the cephalosporins tested, and therefore we possess several possibilities for treatment in case of ampicillin resistance. The third generation cephalosporins ceftriaxone and cefotaxime seem to be the best because: (i) They show the highest activity of the antibiotics tested; (ii) Clinical trials have shown that they are as effective as ampicillin in the treatment not only of Hib meningitis in children, but also of meningitis caused by meningococci and pneumococci (10-13); (iii) They have in clinical studies proven superior to chloramphenicol and cefuroxime in the treatment of meningitis in children (11-13); (iiii) No serious side effects or interactions with other drugs that may be used in patients with meningitis like those occurring with chloramphenicol (14, 15) have until now been reported. The risk of ampicillin resistance in patients with meningitis in Denmark is modest. The prognosis in patients with Hib meningitis may, however, depend on how fast the CSF is sterilized (1 I , 13, 16), and therefore, a third generation cephalosporin such as ceftriaxone or cefotaxime should probably be chosen as initial therapy for patients aged 2 months to 4 years with meningitis of unknown etiology or meningitis proved to be caused by Hib (by microscopy, culture or antigen detection). REFERENCES 1. Arpi M, Hsnberg PZ, Frimodt-Msller N. Antibiotic susceptibility of Haemophilus influenzae isolated
from cerebrospinal fluid and blood. Acta Pathol Microbiol Immunol Scand [B] 94: 167-171, 1986. 2. Cordtz T, Jepsen OB, Arpi M, Henberg P. Antibiotic therapy of meningitis caused by ampicillin22-918553
340 K.Kristensen and I. Mortensen
3. 4.
5. 6.
Scand J Infect Dis Downloaded from informahealthcare.com by V U L Periodicals Rec on 12/29/14 For personal use only.
7.
8. 9.
10. 11.
12.
Scand J Infect Dis 23 (1991)
resistant Haemophilus influenzae in Denmark and Greenland 1981 to 1987. Eur J Clin Microbiol Infect Dis 7: 646-650, 1988. Powell M, Koutsia-Carouzou C, Voutsinas D, Seymour A, Williams JD. Resistance of clinical isolates of Haemophilus influenzae in United Kingdom 1986. Br Med J 295: 176-179, 1987. Doern GV, Jorgensen JH, Thornsberry C, Preston DA, Tubert T, Redding JS, Maher LA. National collaborative study of the prevalence of antimicrobial resistance among clinical isolates of Haemophilus influenzae. Antimicrob Agents Chemother 32: 180-185, 1988. Campos J, Santiago G, Gairi JM, Bregues I. Multiply resistant Haemophilus influenzae type b causing meningitis: Comparative clinical and laboratory study. J Pediatr 108: 897-902, 1986. Pers C. Fatalt tilfzlde af meningitis forhaget af ampicillin- og kloramfenikolresistent Haemophilus influenzae type b. Nyt om Mikrobiologi, Oct. 1987. (In Danish.) Bergeron MG, Lavoie GY, Boucher FDW. Comparative bactericidal activity of cefixime, carumonam, enoxacin and roxithromycin with those of other antibiotics against resistant Haemophilus influenzae including beta-lactam tolerant strains. J Antimicrob Chemother 20: 663-669, 1987. OCallaghan CH, Morris A, Kirby SM, Shingler AH. Novel method for detection ofbeta-lactamases by using a chromogenic cephalosporin substrate. Antimicrob Agents Chemother 1: 283-288, 1972. Kristensen K, Kaaber K, Rernne T, Larsen SO, Henrichsen J. Epidemiology of Haemophilus influenzae type b infections among children in Denmark 1985 and 1986. Acta Paediatr Scand 79: 587-592, 1990. Jacobs RF, Wells TG, Steele RW, Yamauchi TA. Prospective randomized comparison of cefotaxime vs. ampicillin and chloramphenicol for bacterial meningitis in children. J Pediatr 107: 129-133, 1985. Lebel MH, Hoyt J, McCracken GH. Comparative efficacy of ceftriaxone and cefuroxime for treatment of bacterial meningitis. J Pediatr 114: 1049-1054, 1989. Peltola K, Anttila M, Renkonen 0, The Finnish Study Group. Randomized comparison of chloramphenicol, ampicillin, cefotaxime, and ceftriaxone for childhood bacterial meningitis. Lancet 2
1281-1287, 1989. 13. Schaad UB, Suter S, Gianelli-Borradori A, Phenninger J, Auckenthaler R, Bernath 0, Cheseaux J,
Wedgwood J. A comparison of ceftriaxone and cefuroxime for the treatment of bacterial meningitis in children. N Engl J Med 322: 141-147, 1990. 14. Lafferriere CI, Marks MI. Chloramphenicol: properties and clinical use. Pediatr Infect Dis 1: 257-264, 1982. 15. Powell DA, Nahata MC, Durrell DC, Glazer JP, Hilty MD. Interactions among chloramphenicol, phenytoin, and phenobarbital in a pediatric patient. J Pediatr 98: 1001-1003, 1981. 16. Lebel MH, McCracken GH. Delayed cerebrospinal fluid sterilization and adverse outcome of bacterial meningitis in infants and children. Pediatrics 83: 161-167, 1989.
Antibiotic Susceptibility of lnvasive Haemophilus influenzae Type b Isolates in Denmark in 1988 and 1989 KIM KRISTENSEN' and IDA MORTENSEN2
Scand J Infect Dis Downloaded from informahealthcare.com by V U L Periodicals Rec on 12/29/14 For personal use only.
From the 'Paediatric Departmenl GGK, Rigshospitalet and the 'Departmenl of Diagnostic Baclerioiogy and Antibiotics, Statens Serumrnstitur, Copenhagen. Denmark
A system for surveillance of invasive Haemophilus influenzae type b infections in Denmark yielded 135 strains isolated from b l o d or cerebrospinal fluid from August 1988 through December 1989. The susceptibility of the strains to 7 antibiotics was investigated by an agar dilution method. Ceftriaxone was found to be the most active drug followed by cefotaxime, ceftazidime, rifampicin, ampicillin, cefuroxime, and chloramphenicol. Except for ampicillin the MICs for the individual antibiotics were similar. Seven strains (5.2 %), which all produced beta-lactamase, were resistant to ampicillin. Since H. influenzae type b meningitis in otherwise healthy individuals almost exclusively occurs in children aged 2 months to 4 years, a third generation cephalosporin such as ceftriaxone or cefotaxime may be considered the drug of choice for initial therapy of meningitis in this age group. K. Kristensen, MD, Paediatric Department GGK, Rigshospitalet, DK-2100 Copenhagen 0, Denmark
INTRODUCTION Since Haemophilus influenzae type b (Hib) is a major cause of life-threatening invasive infections such as meningitis and epiglottitis in children. precise knowledge of antibiotic susceptibility of invasive Hib isolates is of vital importance. Two recent Danish studies on this subject showed rates of resistance to ampicillin of only 5 and 8 Yo (1, 2), which is much lower than those reported from some other countries, in which the prevalence of ampicillin resistance ranges from 27 to 57 Yo (3-5). However, none of the Danish studies were based on recent data, and because the prevalence of ampicillin resistance among Hib isolates has increased substantially during the last decade elsewhere (3-5), there is a need for new Danish data on this subject. Resistance to chloramphenicol is still uncommon in the UK and USA (3,4), but is present in 65.7% of invasive Hib isolates in Spain (5). Until now, resistance to chloramphenicol has only been described twice in Denmark (in strains also resistant to ampicillin, 6). Resistance to second or third generation cephalosporins has been reported from Canada (7), but is generally rare. Since August 1988 we have collected all Hib strains isolated from blood or cerebrospinal fluid (CSF) in Denmark. We report here on their susceptibility to 7 antibiotics relevant in the treatment or prophylaxis of invasive Hib infection. MATERIAL AND METHODS Collection of strains From August 1988 through December 1989 all departments of clinical microbiology in Denmark sent all Hib strains isolated from blood or CSF to Statens Seruminstitut. A total of 135 strains were collected. All strains were serotyped in our laboratory using rabbit antiserum and kept at -70°C until tested. Antibiotics The antibiotics studied were ampicillin, cefuroxime, cefotaxime, ceftazidime, ceftriaxone, chloramphenicol. and rifampicin. All antibiotics were kindly provided by their manufacturers.
338 K. Kristensen and I. Mortensen
Scand J Infect Dis 23 (1991)
Scand J Infect Dis Downloaded from informahealthcare.com by V U L Periodicals Rec on 12/29/14 For personal use only.
dY
Fig. 1. MICs of the antibiotics tested. 0,00075
0,003
0,O 125
0.05
0.8
OV2
3'2
12.5
50
CONCENTRATDN moll
Medium Agar dilution method was performed on 5% blood agar containing beef 509'0, Orthana pepton (NS Orthana) 1 I NaCl , Ph. Nord. (Bie & Berntsen) 0.3%,Na2HP04*12H 2 0 0.29'0, 5 N NaOH 10.5 mM, SoBigel agar type USA (Hasche) 1.39/0, CaCI2.2 H 2 0 0.00929'0, MgS04.7 H 2 0 0.0068Y0, and defibrinated horse blood 5%. The plates were supplemented with nicotine amide adenine dinucleotide (Nad 0.015 Yo).
Agar dilution Antibiotic solutions were prepared to obtain final concentrations in 2-fold dilutions ranging from 100 to 0.00075 mg/l. An agar dilution-multiple inoculator method was used. Colonies from overnight cultures on chocolate agar plates were picked and diluted in sterile saline to a density of lo9 colony forming units (cfu)/ml as measured by colorimetry (model 252 Coming Colorimeter) from which appropriate dilutions in sterile saline were prepared for inoculation (Denley A400, Sussex, England). Drops containing 103-104 cfu were delivered onto the antibiotic-containing agar plates. A control plate without antibiotics was inoculated before and after each series. Plates were incubated overnight at 35°C in an atmosphere of 59'0 carbon dioxide and 95% air. Minimum inhibitory concentration (MIC) was defined as the lowest concentration of an antibiotic which completely inhibited growth disregarding g 3 colonies or a faint haze caused by inoculum.
Table I. Etiology in 393 cases of bacterial meningitis in children aged 2 months to 4 years notified in Denmark I986 to 1988" Data: T. Renne, Department of Epidemiology, Statens Seruminstitut, Copenhagen ~
~~~
~
Bacteria
No. of cases reported
Hib Meningococci Pneumococci Listeria monocytogenes Others
158 203 26 I 5
Another 38 cases were notified in which no information about the causative organism was available.
Scand J Infect Dis 23
Antibiotic susceptibility of H. injluenzae type b 339
Detection of beta-lactamase production The chromogenic cephalosporin substrate (Glaxo compound) was used for detection of beta-lactamase production (8).
Scand J Infect Dis Downloaded from informahealthcare.com by V U L Periodicals Rec on 12/29/14 For personal use only.
RESULTS Assuming that a MIC < 1 mg/l for all antibiotics tested indicates clinical susceptibility, all strains were found susceptible to all antibiotics tested, except for 7 strains (5.2%), which were not susceptible to ampicillin (Fig. 1). All these strains produced beta-lactamase, but showed the same susceptibility to the cephalosporins as those that did not produce beta-lactamase. Ceftriaxone was found to be the most active drug with a MIC as low as 0.003 mg/l. DISCUSSION This study confirms the finding of Cordtz et al. (2) that still only about 5 'Yo of invasive Hib isolates in Denmark are resistant to ampicillin, which emphasizes that a restrictive policy on the use of antibiotics such as that pursued in Denmark probably is of value in this respect. In patients with meningitis no delay in effective treatment should be accepted, and it is therefore important to assess the risk of ampicillin resistance in a case of meningitis of unknown etiology. Of the organisms causing meningitis in otherwise healthy individuals Hib is the only one, which may be resistant to ampicillin, and because Hib meningitis in previously healthy individuals almost exclusively occurs in children aged 2 months to 4 years (9), the risk that the infecting organism should be resistant to ampicillin in a case of meningitis of unknown etiology should be calculated for this age group solely. Table I shows the distribution by etiology of cases of bacterial meningitis notified 1986 to 1988 in Denmark in children aged 2 months to 4 years. On the assumption that the rate of notification is the same for all kinds of meningitis, 40% of the cases were caused by Hib. Thus, at present the risk of ampicillin resistance in a case of meningitis of unknown etiology in Denmark in this age group is about 2 'Yo. No strains were found to be resistant to chloramphenicol or to the cephalosporins tested, and therefore we possess several possibilities for treatment in case of ampicillin resistance. The third generation cephalosporins ceftriaxone and cefotaxime seem to be the best because: (i) They show the highest activity of the antibiotics tested; (ii) Clinical trials have shown that they are as effective as ampicillin in the treatment not only of Hib meningitis in children, but also of meningitis caused by meningococci and pneumococci (10-13); (iii) They have in clinical studies proven superior to chloramphenicol and cefuroxime in the treatment of meningitis in children (11-13); (iiii) No serious side effects or interactions with other drugs that may be used in patients with meningitis like those occurring with chloramphenicol (14, 15) have until now been reported. The risk of ampicillin resistance in patients with meningitis in Denmark is modest. The prognosis in patients with Hib meningitis may, however, depend on how fast the CSF is sterilized (1 I , 13, 16), and therefore, a third generation cephalosporin such as ceftriaxone or cefotaxime should probably be chosen as initial therapy for patients aged 2 months to 4 years with meningitis of unknown etiology or meningitis proved to be caused by Hib (by microscopy, culture or antigen detection). REFERENCES 1. Arpi M, Hsnberg PZ, Frimodt-Msller N. Antibiotic susceptibility of Haemophilus influenzae isolated
from cerebrospinal fluid and blood. Acta Pathol Microbiol Immunol Scand [B] 94: 167-171, 1986. 2. Cordtz T, Jepsen OB, Arpi M, Henberg P. Antibiotic therapy of meningitis caused by ampicillin22-918553
340 K.Kristensen and I. Mortensen
3. 4.
5. 6.
Scand J Infect Dis Downloaded from informahealthcare.com by V U L Periodicals Rec on 12/29/14 For personal use only.
7.
8. 9.
10. 11.
12.
Scand J Infect Dis 23 (1991)
resistant Haemophilus influenzae in Denmark and Greenland 1981 to 1987. Eur J Clin Microbiol Infect Dis 7: 646-650, 1988. Powell M, Koutsia-Carouzou C, Voutsinas D, Seymour A, Williams JD. Resistance of clinical isolates of Haemophilus influenzae in United Kingdom 1986. Br Med J 295: 176-179, 1987. Doern GV, Jorgensen JH, Thornsberry C, Preston DA, Tubert T, Redding JS, Maher LA. National collaborative study of the prevalence of antimicrobial resistance among clinical isolates of Haemophilus influenzae. Antimicrob Agents Chemother 32: 180-185, 1988. Campos J, Santiago G, Gairi JM, Bregues I. Multiply resistant Haemophilus influenzae type b causing meningitis: Comparative clinical and laboratory study. J Pediatr 108: 897-902, 1986. Pers C. Fatalt tilfzlde af meningitis forhaget af ampicillin- og kloramfenikolresistent Haemophilus influenzae type b. Nyt om Mikrobiologi, Oct. 1987. (In Danish.) Bergeron MG, Lavoie GY, Boucher FDW. Comparative bactericidal activity of cefixime, carumonam, enoxacin and roxithromycin with those of other antibiotics against resistant Haemophilus influenzae including beta-lactam tolerant strains. J Antimicrob Chemother 20: 663-669, 1987. OCallaghan CH, Morris A, Kirby SM, Shingler AH. Novel method for detection ofbeta-lactamases by using a chromogenic cephalosporin substrate. Antimicrob Agents Chemother 1: 283-288, 1972. Kristensen K, Kaaber K, Rernne T, Larsen SO, Henrichsen J. Epidemiology of Haemophilus influenzae type b infections among children in Denmark 1985 and 1986. Acta Paediatr Scand 79: 587-592, 1990. Jacobs RF, Wells TG, Steele RW, Yamauchi TA. Prospective randomized comparison of cefotaxime vs. ampicillin and chloramphenicol for bacterial meningitis in children. J Pediatr 107: 129-133, 1985. Lebel MH, Hoyt J, McCracken GH. Comparative efficacy of ceftriaxone and cefuroxime for treatment of bacterial meningitis. J Pediatr 114: 1049-1054, 1989. Peltola K, Anttila M, Renkonen 0, The Finnish Study Group. Randomized comparison of chloramphenicol, ampicillin, cefotaxime, and ceftriaxone for childhood bacterial meningitis. Lancet 2
1281-1287, 1989. 13. Schaad UB, Suter S, Gianelli-Borradori A, Phenninger J, Auckenthaler R, Bernath 0, Cheseaux J,
Wedgwood J. A comparison of ceftriaxone and cefuroxime for the treatment of bacterial meningitis in children. N Engl J Med 322: 141-147, 1990. 14. Lafferriere CI, Marks MI. Chloramphenicol: properties and clinical use. Pediatr Infect Dis 1: 257-264, 1982. 15. Powell DA, Nahata MC, Durrell DC, Glazer JP, Hilty MD. Interactions among chloramphenicol, phenytoin, and phenobarbital in a pediatric patient. J Pediatr 98: 1001-1003, 1981. 16. Lebel MH, McCracken GH. Delayed cerebrospinal fluid sterilization and adverse outcome of bacterial meningitis in infants and children. Pediatrics 83: 161-167, 1989.
