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Antimicrobial susceptibilities of Haemophilus species in Hong Kong
Journalof Infection(I989) I9, I35-I42 Antimicrobial
susceptibilities of Haemophilus species in Hong Kong
Julia M. Ling, H a z e l K h i n - T h i - O o , Y.-W. H u i a n d G. L. F r e n c h
Department of Microbiology, The Chinese University of Hong Kong, The Prince of Wales Hospital, Shatin, New Territories, Hong Kong Accepted for publication 9 March I989 Summary Altogether 4o3 Haemophilus spp. were isolated in seven hospital laboratories in Hong Kong during June I986, mostly from sputum. Of these 73 % were Haemophilus influenzae and 27 % Haemophilusparainfluenzae. All the isolates of H. influenzae were non-capsulated; Haemophilus spp. were not isolated from blood or cerebrospinal fluid (CSF) during the period of the study. Antimicrobial resistance, including multiple resistance, was common. Of all the strains of H. influenzae, 2o % were resistant to I mg/1 ampicillin, (all except one by production of T E M - I fl-lactamase), 65 % were resistant to 0"5 mg/1 erythromycin, 25 % to I mg/1 tetracycline, I4% to I mg/1 chloramphenicol (mediated by the production of a chloramphenicol-destroying enzyme) and less than 1% to 8 rag/1 cefaclor and 0'5 mg/1 trimethoprim. All isolates were susceptible to cephamandole and cefuroxime. Haemophilusparainfluenzae showed similar susceptibilities, except that a greater proportion of strains was sensitive to erythromycin and chloramphenicol. Only 50 % of the ampicillin-resistant strains of H. influenzae and H. parainfluenzae contained detectable plasmid s of 2-55 Mdal arranged in six to nine different plasmid profiles. Resistance to ampicillin and chloramphenicol has increased markedly in isolates of H. influenzae in Hong Kong over the last 5 years. This resistance may be associated with transposable genes.
Introduction T h e incidence of antimicrobiat resistance in Haemophilus spp. has increased t h r o u g h o u t the world in recent years. 1-~ In a previous study of strains of/-/. influenzae isolated in H o n g K o n g in I 9 8 i , 6 3 % of isolates were resistant to ampicillin and 6 % to chloramphenicol, both at a concentration of I mg/1. Anecdotal evidence suggested that resistance in these organisms has been increasing. We therefore c o n d u c t e d a new study of I-Iaemophilus spp. isolated in I986. All strains of Haemophilus spp. isolated from routine cultures of clinical specimens during June I986 in seven hospitals in H o n g K o n g were saved. T h e strains were reidentified, tested for susceptibility to nine antimicrobial agents by an agar dilution m e t h o d and examined for possession of plasmids and the p r o d u c t i o n of fl-lactamase.
Materials and methods Organisms All strains of Haemophilus spp. isolated in seven hospital laboratories during June I986 were saved on ' c h o c o l a t e ' agar slants and stored as stock cultures o613-4453/89/o5oi35+o8 $o2.oo/o
© I989 The British Societyfor the Study of Infection
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j.M. LING ET AL.
in 5% glycerol broth at --7o °C. Staphylococcus aureus N C T C 6 5 7 I and Escherichia coli ATCC25922 were used as controls. Identification
Organisms were submitted to the Department of Microbiology, Chinese University of Hong Kong for confirmation of their identity and for susceptibility testing. T h e y were identified as H. influenzae if they required both X and V factors and did not produce porphyrinogen; as H. parainfluenzae if they required only V factor and produced porphyrinogen. 7,s The few isolates of other Haemophilus spp. were not further identified. Organisms were grown on Fildes' agar for examination of irridescence by the method of T u r k and May. 9 T h e y were serotyped by slide agglutination with antiserum of types a to f (Wellcome, Beckenham, England) and by latex agglutination for type b (Wellcome). A n t i m i c r o b i a l susceptibility tests
Minimal inhibitory concentrations (MICs) of nine antimicrobial agents were determined by an agar dilution method. Stock cultures were grown on chocolate agar, by incubation overnight at 37 °C in an atmosphere of 5 ~o CO2. T h e inoculum in the tests was prepared by emulsifying a few colonies in i ml brain heart infusion broth so as to give a turbidity corresponding to that of a MacFarland o'5 standard. A ten-fold dilution was made from this in brainheart infusion broth. T h e medium used for susceptibility tests was MuellerHinton agar (Oxoid) containing 5 % lysed horse blood and I ~o vitox (Oxoid). Plates containing serial two-fold dilution of the following antibiotics were prepared the day before use and stored at 4 °C: ampicillin, erythromycin, chloramphenicol, tetracycline, cefaclor, cephamandole, cefuroxime, trimethoprim and co-trimoxazole (which contained trimethoprim and sulfamethoxazole in the ratio of I : 19). These plates were inoculated by means of an MIC2ooo inoculator (Dynatech, U.S.A.) with a final inoculum of approximately IO~ colony forming units per spot. Except for plates containing erythromycin, which were incubated in air, plates were incubated at 37 °C in an atmosphere of 5 % CO2 and read after 18 h. T h e M I C was taken to be the lowest concentration of drug that inhibited visible growth. Breakpoints
Except for chloramphenicol, resistance breakpoints were those recommended by the British Society for Antimicrobial Chemotherapy (BSAC). 1° A chloramphenicol breakpoint of I mg/1 was used instead of 2 rag/1 as recommended by the BSAC for Haemophilus spp. since we found that some of our strains which produced chloramphenicol-inactivating enzymes had MICs of 2 mg/1. D e t e c t i o n and characterisation o f [I-lactamase
All isolates were tested for the production of fl-lactamase by means of fllactamase detection papers (Oxoid). fl-lactamase was extracted from fllactamase-producing strains by scraping overnight cultures on 'chocolate' agar into 600/,1 phosphate-buffered saline (pH 7"3) which was then centrifuged
Susceptibilities of Haemophilus species
I37
in a microcentrifuge (Microfuge, MSE). Cell pellets were washed, resuspended in IOO #1 distilled water and then sonicated twice at an amplitude of Io microns for 30 s (Soniprep I5O, MSE). Cell debris was removed by centrifugation and the supernatant stored at - 70 °C or used immediately, fl-lactamases were then characterised by isoelectric focussing according to the method of Vecoli and colleagues. 11 Detection o f chloramphenieol-inactivation Chloramphenicol-resistant isolates were tested for chloramphenicol-inactivation by the method of Howard and colleagues. 12 T h e nature of the enzyme detected was not determined but was presumed to be chloramphenicol acetyltransferase. Plasmid profile analysis by agarose gel electrophoresis Plasmid D N A was extracted from ampicillin-resistant H. influenzae and H. parainfluenzae isolates by the method of Kado and Liu 13 and electrophoresed in a 0"7% agarose gel (Sigma, U.S.A.) with T r i s - b o r a t e - E D T A buffer pH 8"314 at 45 mA for 2 h, and use of a vertical Protean slab cell (Bio-Rad, U.S.A.). Gels were stained with ethidium bromide (Sigma) and then photographed through a red filter with Polaroid 665 film under short-wave ultraviolet transillumination. Standard sized markers used were: T P I I 6 ; T P I 2 9 ; T P I 2 5 ; T P I 2 o ; T P I 9 3 ; S-a; N T P 2 and ColE1 (from D r B. Rowe, Colindale, U.K.); pWG615; p W G I I 5 ; pWG3 and pEI94 (from D r W-B. Grubb, Perth, Western Australia). Results Altogether 403 strains of Haemophilus spp. were isolated during the study, all from sputum except for four from eye swabs and three from nasal swabs. None was isolated from blood or cerebrospinal fluid (CSF). Of the total, 292 (72"5 %) were H. influenzae, Io 7 (26"6%) were H. parainfluenzae, one was H. parahemolyticus and three were Haemophilus spp. which were not further identified. Irridescent colonies of the strains of Haemophilus spp isolated on Fildes' agar were not detected and many of the H. influenzae isolates autoagglutinated with both serological methods. We were thus unable to identify any capsulated strains of H. influenzae during the study. Review of laboratory records from the seven hospitals taking part in the study revealed that during the i2 months of i985, Haemophilus spp. (usually H. influenzae type b) were recovered from blood and/or CSF of only I9 patients, thereby indicating that capsulated, invasive H. influenzae strains are u n c o m m o n in Hong Kong. Of the 393 isolates which survived for further study, 288 were H. influenzae and Io2 were H. parainfluenzae. Table I shows the susceptibility of these isolates to four fl-lactam and five other antimicrobial agents. Fifty-seven (I9"8%) strains of H. influenzae were resistant to I mg/1 ampicillin and, of these, 94"7% had MICs > = 8 mg/1. All ampicillinresistant isolates except one with an M I C of 2 mg/1 produced fl-lactamase detectable by the Oxoid papers. Except for two fl-lactamase-positive isolates,
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Susceptibilities of H a e m o p h i l u s species
139
T a b l e II. Distribution of resistance patterns of H a e m o p h i l u s spp. isolated in
Hong Kong Percentage of strains Number of resistances o I 2 3 4 5
Resistance patterns Sensitive E Others AT ET Others ATC AET Others AETC AETTmCr
H. influenzae
H. parainfluenzae
(288)
(1o2)
23.3 49'3 3"4 1"4 2.8 1"7 5'6 4"9 1'6 5"6 0"3
47"I 12'7 17"6 7"8 4'9 3"9 2"o o'o 3"9 o.o o'o
A = ampicillin, E = erythromycin, T = tetracycline, C = chloramphenicol, Cr = cefaclor, Tm = trimethoprim. which were resistant to 8 mg/1 cefaclor, all were susceptible to cefaclor (8 mg/1), cephamandole and cefuroxime (4 mg/1). F o u r t e e n per cent were resistant to I mg/1 chloramphenicol and were shown by the m e t h o d of H o w a r d and colleagues, 12 to inactivate chloramphenicol, probably by the p r o d u c t i o n of chloramphenicol acetyltransferase. Sixty-five per cent were resistant to 0"5 rag/1 e r y t h r o m y c i n and 25 % tO I mg/1 tetracycline but less than z % were resistant to t r i m e t h o p r i m or co-trimoxazole. T h e proportion of H. parainfluenzae isolates resistant to ampicillin and tetracycline was similar to that of H. influenzae (Table I). T h e ampicillinresistance was also m e d i a t e d by the p r o d u c t i o n of fi-lactamase. All isolates were susceptible to cefaclor (8 mg/1), cephamandole and cefuroxime (4 mg/1). F e w e r H. parainfluenzae isolates, were resistant to e r y t h r o m y c i n (I9 % had M I C s > 0"5 mg/1) and chloramphenicol (4% had M I C s > I mg/1) and the chloramphenicol-resistance was due to a chloramphenicol-inactivating enzyme. In contrast, more isolates were resistant to t r i m e t h o p r i m and cotrimoxazole (5 and 3 % respectively had M I C s > o'5 mg/1). Only 23 % of H. influenzae isolates were susceptible to all the agents tested; 53 % were resistant to one drug, and 24 % to two or more drugs (Table II). A lower percentage of H. parainfluenzae isolates exhibited multiple resistance, 47 % being susceptible to all drugs tested and 23 % resistant to two or more. F o r t y - o n e ampicillin-resistant, fl-lactamase-positive H. influenzae isolates were analysed for possession of plasmids. T w e n t y (48"8 %) did not contain detectable plasmids b u t there were nine different plasmid profiles (plasmid types i-6) among the remaining 21 isolates (Table III). T h e s e contained plasmids ranging in size f r o m 2I to 55 M d a l except for one isolate which had only one small plasmid of 4-5 Mdal. fl-lactamases extracted from 25 of these isolates were all of the T E M - I type with an isoelectric point of 5"4.
14o
J.M.
LING
ET AL.
Table III. Plasmid profiles of ampicillin-resistant fl-lactamase-positive strains of H. influenzae and H. parainfluenzae Sizes of p l a s m i d s in Mdal
I
IA
2
2A
2B
3
Plasmid type 4 5 6
54'8 46 43 40 34 28 23 20"6 4"8 4"0 3"6 2- 7 2"52 No. of isolates :
H. influenzae H. parainfluenzae Total
7
8
9
3"6
-2. 7
Io
Nil
2'52
--
o 2 2
2o 13 33
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46
43 40
40 34 28 23
23
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23 20"6
4'8 4' 0
I o I
4 3 7
I o I
I o I
I o I
6 I 7
4 o 4
2 o 2
I o I
o 4 4
o I I
o I I
Of 25 ampicillin-resistant fl-lactamase-positive H. parainfluenzae tested, I3 (52 %) did not contain plasmids while the remaining I2 exhibited six plasmid profiles (plasmid types IA, 3, 7-Io), three of which were similar to those found in H. influenzae (Table III). Of the four plasmid profiles unique to H. parainfluenzae, one contained a 46 Mdal plasmid while the others contained plasmids of 2-4 Mdal. T w e n t y H. parainfluenzae strains were analysed for fllactamase by isoelectric focussing and all produced the T E M - I enzyme. Discussion
Invasive H. influenzae infections are uncommon in Hong Kong. T h e seven laboratories which participated in this study isolated capsulated H. influenzae type b from the blood or CSF of only I9 patients during I985. These hospitals (which included the infectious diseases hospital) served a population of about four million. T h e y had approximately 6000 beds and iooooo admissions per annum. Standard investigations, including culture and antigen detection, were used in their laboratories. Thus the infrequent isolation of capsulated H. influenzae from blood and CSF is probably a true reflection of a low incidence of invasive H. influenzae infections in Hong Kong despite the large number of children in the population. In contrast, H. influenzae and H. parainfluenzae were often isolated from respiratory tract specimens. Haemophilus parainfluenzae comprised about a quarter of the isolates although their clinical significance was not investigated. Capsulated strains of H. influenzae were not detected. Single and multiple antibiotic resistances were common among strains of both H. influenzae and H. parainfluenzae with I9 % or more of isolates being
Susceptibilities of H a e m o p h i l u s species
I4I
resistant to ampicillin and tetracycline (I mg/1). Ampicillin-resistance in b o t h species was mediated by the p r o d u c t i o n of T E M - I fl-lactamase. F o u r t e e n per cent of H. influenzae and 4 % of H. parainfluenzae were resistant to i rag/1 chloramphenicol as a result of their producing a d r u g - d e s t r o y i n g enzyme. Since I 9 8 I , resistance to ampicillin and chloramphenicol has increased significantly in H o n g K o n g among isolates of H. influenzae. 6 In contrast, less than 1 % of Haemophilus spp. were resistant to trimethoprim, co-trimoxazole or cefaclor and all isolates were susceptible to cephamandole and cefuroxime. In all b u t one strain, ampicillin-resistance was mediated b y T E M - I fllactamase. While this is the usual m e c h a n i s m of ampicillin-resistance in H. influenzae,15.1~ in several recent studies some strains have been observed that were resistant b y non-enzymatic mechanisms. 5'1G'17 T h i s p h e n o m e n o n was seen in only one of our isolates. In the present study, about 50 % of fl-lactamase-producing strains of H. influenzae and H. parainfluenzae did not contain detectable plasmids. Other workers have noted that resistance factors in H. influenzae may exist extrachromosomally or b e c o m e integrated into the chromosome. ~8-2° In particular, ampicillin-resistance has been f o u n d to be associated with a transposon, T n A . 2~ T h u s multiply-resistant strains m a y lack detectable plasmids. It is likely that determinants of ampicillin-resistance in H o n g K o n g strains of Haemophilus spp. have been transposed on to the chromosome. This p h e n o m e n o n warrants further study. While invasive, capsulated strains of H. influenzae are relatively u n c o m m o n in H o n g K o n g , and anecdotal evidence suggests that such virulent organisms are usually susceptible to ampicillin and chloramphenicol, clinicians should be alert for the appearance of resistant and multiply-resistant strains of H. influenzae type b in H o n g Kong. Ampicillin-resistant strains of non-capsulated H. influenzae remain susceptible to the second generation cephalosporins. T h e s e drugs m a y therefore be considered for use in the treatment of serious infections caused by these organisms. (We thank the following colleagues who collected strains of Haemophilus spp. in their laboratories for this study: Drs E. Gwi, M. Ho, K. M. Kam, T. Lat, R. Lycette, W. P. Mak, T. K. Ng, D. Tsang, and Mr H. T. Yeung. We also thank Mr Thomas Ling for the results of isoelectric focussing, Mr W. S. Au, Mr K. K. Mong and Mr E. Ng for technical assistance and Miss A. Cheung and Miss C. Ng for computer data entry.) References
i. Simasathien S, Duangmani C, Echeverria P. Haemophilus influenzae type b resistant to ampicillin and chloramphenicol in an orphanage in Thailand. Lancet I98o; ii: I2 I4-I2I 7. 2. Schlech NF, Band JD, Hightower AW, Broome CV. Bacterial meningitis in the United States (Abstr no. 3II). In: Program and abstracts of the 22nd Interscienee Conference on Antimicrobial Agents and Chemotherapy. Washington, DC: American Society for Microbiology, i982. 3. Campos J, Garcia-Tornel S, Sanfeliu I. Susceptibility studies of multiple resistant Haemophilus influenzae isolated from paediatric patients and contacts. Antimicrob Agents Chemother I984; 25: 7o6-709. 4. Doern GV, Jorgensen JH, Thornsberry C, Preston DA. Prevalence of antimicrobial
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resistance among clinical isolates of Haemophilus influenzae: a collaborative study. Diagn Microbiol Infect Dis 1986; 4 : 95-1o7. 5. Powell M, Koutsia-Carouzou C, Voutsinas D, Seymour A, Williams JD. Resistance of clinical isolates of Haemophilus influenzae in United Kingdom I986. Br M e d J 1987; 295: 176-179. 6. Ling J, Chau PY, Leung YK, Ng WS, So SY. Antibiotic susceptibility of pneumococci and Haemophilus influenzae isolated from patients with acute exacerbations of chronic bronchitis: prevalence of tetracycline-resistant strains in Hong Kong. J Infect 1983; 6: 33-37. 7. Biberstein EL, Mini PD, Gills MG. Action of Haemophilus cultures on gammaaminolevulinic acid. J Bacteriol 1973 ; 86 : 814-819. 8. Kilian M. A taxonomic study of the genus Haemophilus, with the proposal of a new species. J Gen Microbiol 1976; 93: 9-62. 9. Turk DC, May JR. Isolation and identification of H. influenzae. In: Haemophilus influenzae : clinical importance. The English Universities Press, London, 1967: I17-2o. io. Report by a Working Party of the British Society for Antimicrobial Chemotherapy. Breakpoints in in-vitro antibiotic sensitivity testing. J Antimicrob Chemother I988; 2 I : 7oi-7IO. II. Vecoli C, Prevost FE, Ververis JJ, Medeiros AA, O'Leary Jr GP. Comparison of polyacrylamide and agarose gel thin-layer isoelectric focussing for the characterization of fl-lactamases. Antimicrob Agents Chemother I983 ; 24: I86-189. 12. Howard AJ, Williams HM, Poole MC. Chloramphenicol resistance in Haemophilus influenzae. Lancet 1986; ii: 745-74613. Kado CI, Liu S.-T. Rapid procedure for detection and isolation of large and small plasmids. J Bacteriol 198I; I45: 1365-1373. 14. Meyers JA, Sanchez D, Elwell LP, Falkow S. Simple agarose gel electrophoretic method for the identification and characterization of plasmid deoxyribonucleic acid. J Bacteriol 1976; 127: 1529-1537. 15. Brunton J, Clare D~ Meier MA. Molecular epidemiology of antibiotic resistance plasmids of Haemophilus species and Neisseria gonorrhoeae. Rev Infect Dis 1986; 8:713-724. 16. Mendelman PM, Chaffin DO, Clausen C, et al. Failure to detect ampicillin-resistant, nonfl-lactamase-producing Haemophilus influenzae by standard disk susceptibility testing. Antimicrob Agents Chemother 1986; 3o: 274-280. I7. Mendelman PM, Chaffin DO, Stull TL, Rubens CE, Mack KD, Smith AL. Characterization of non-fl-lactamase-rnediated ampicillin resistance in Haemophilus influenzae. Antimicrob Agents Chemother 1984; 26: 235-244. 18. Willard JE, Johnson EJ, Daum RS. Interrelationships between physical state, phenotypic stability and transferability offl-lactamase genes in Haemophilus influenzae. J Gen Microbiol 1982; 128: 2353-2360. 19. Mendelman PM, Doroshow CA, Gandy SL, Syriopoulou V, Weigen CP, Smith AL. Plasmid-mediated resistance in multiply resistant Haemophilus influenzae type b causing meningitis; molecular characterization of one strain and review of the literature. J Infect Dis 1984; 15o: 30-39 . 20. Murphy-Corb M, Nolan-Willard M, Daum RS. Integration of plasmid DNA coding for fl-lactamase production in the Haemophilus influenzae chromosome. J Bacteriol 1984; 16o: 815-817 . 21. Laufs R, Kaulfers P.-M. Molecular characterization of a plasmid specifying ampicillin resistance and relationship to other R factors from Haemophilus influenzae. J Gen Microbiol 1977; lO3: 277-286.
susceptibilities of Haemophilus species in Hong Kong
Julia M. Ling, H a z e l K h i n - T h i - O o , Y.-W. H u i a n d G. L. F r e n c h
Department of Microbiology, The Chinese University of Hong Kong, The Prince of Wales Hospital, Shatin, New Territories, Hong Kong Accepted for publication 9 March I989 Summary Altogether 4o3 Haemophilus spp. were isolated in seven hospital laboratories in Hong Kong during June I986, mostly from sputum. Of these 73 % were Haemophilus influenzae and 27 % Haemophilusparainfluenzae. All the isolates of H. influenzae were non-capsulated; Haemophilus spp. were not isolated from blood or cerebrospinal fluid (CSF) during the period of the study. Antimicrobial resistance, including multiple resistance, was common. Of all the strains of H. influenzae, 2o % were resistant to I mg/1 ampicillin, (all except one by production of T E M - I fl-lactamase), 65 % were resistant to 0"5 mg/1 erythromycin, 25 % to I mg/1 tetracycline, I4% to I mg/1 chloramphenicol (mediated by the production of a chloramphenicol-destroying enzyme) and less than 1% to 8 rag/1 cefaclor and 0'5 mg/1 trimethoprim. All isolates were susceptible to cephamandole and cefuroxime. Haemophilusparainfluenzae showed similar susceptibilities, except that a greater proportion of strains was sensitive to erythromycin and chloramphenicol. Only 50 % of the ampicillin-resistant strains of H. influenzae and H. parainfluenzae contained detectable plasmid s of 2-55 Mdal arranged in six to nine different plasmid profiles. Resistance to ampicillin and chloramphenicol has increased markedly in isolates of H. influenzae in Hong Kong over the last 5 years. This resistance may be associated with transposable genes.
Introduction T h e incidence of antimicrobiat resistance in Haemophilus spp. has increased t h r o u g h o u t the world in recent years. 1-~ In a previous study of strains of/-/. influenzae isolated in H o n g K o n g in I 9 8 i , 6 3 % of isolates were resistant to ampicillin and 6 % to chloramphenicol, both at a concentration of I mg/1. Anecdotal evidence suggested that resistance in these organisms has been increasing. We therefore c o n d u c t e d a new study of I-Iaemophilus spp. isolated in I986. All strains of Haemophilus spp. isolated from routine cultures of clinical specimens during June I986 in seven hospitals in H o n g K o n g were saved. T h e strains were reidentified, tested for susceptibility to nine antimicrobial agents by an agar dilution m e t h o d and examined for possession of plasmids and the p r o d u c t i o n of fl-lactamase.
Materials and methods Organisms All strains of Haemophilus spp. isolated in seven hospital laboratories during June I986 were saved on ' c h o c o l a t e ' agar slants and stored as stock cultures o613-4453/89/o5oi35+o8 $o2.oo/o
© I989 The British Societyfor the Study of Infection
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j.M. LING ET AL.
in 5% glycerol broth at --7o °C. Staphylococcus aureus N C T C 6 5 7 I and Escherichia coli ATCC25922 were used as controls. Identification
Organisms were submitted to the Department of Microbiology, Chinese University of Hong Kong for confirmation of their identity and for susceptibility testing. T h e y were identified as H. influenzae if they required both X and V factors and did not produce porphyrinogen; as H. parainfluenzae if they required only V factor and produced porphyrinogen. 7,s The few isolates of other Haemophilus spp. were not further identified. Organisms were grown on Fildes' agar for examination of irridescence by the method of T u r k and May. 9 T h e y were serotyped by slide agglutination with antiserum of types a to f (Wellcome, Beckenham, England) and by latex agglutination for type b (Wellcome). A n t i m i c r o b i a l susceptibility tests
Minimal inhibitory concentrations (MICs) of nine antimicrobial agents were determined by an agar dilution method. Stock cultures were grown on chocolate agar, by incubation overnight at 37 °C in an atmosphere of 5 ~o CO2. T h e inoculum in the tests was prepared by emulsifying a few colonies in i ml brain heart infusion broth so as to give a turbidity corresponding to that of a MacFarland o'5 standard. A ten-fold dilution was made from this in brainheart infusion broth. T h e medium used for susceptibility tests was MuellerHinton agar (Oxoid) containing 5 % lysed horse blood and I ~o vitox (Oxoid). Plates containing serial two-fold dilution of the following antibiotics were prepared the day before use and stored at 4 °C: ampicillin, erythromycin, chloramphenicol, tetracycline, cefaclor, cephamandole, cefuroxime, trimethoprim and co-trimoxazole (which contained trimethoprim and sulfamethoxazole in the ratio of I : 19). These plates were inoculated by means of an MIC2ooo inoculator (Dynatech, U.S.A.) with a final inoculum of approximately IO~ colony forming units per spot. Except for plates containing erythromycin, which were incubated in air, plates were incubated at 37 °C in an atmosphere of 5 % CO2 and read after 18 h. T h e M I C was taken to be the lowest concentration of drug that inhibited visible growth. Breakpoints
Except for chloramphenicol, resistance breakpoints were those recommended by the British Society for Antimicrobial Chemotherapy (BSAC). 1° A chloramphenicol breakpoint of I mg/1 was used instead of 2 rag/1 as recommended by the BSAC for Haemophilus spp. since we found that some of our strains which produced chloramphenicol-inactivating enzymes had MICs of 2 mg/1. D e t e c t i o n and characterisation o f [I-lactamase
All isolates were tested for the production of fl-lactamase by means of fllactamase detection papers (Oxoid). fl-lactamase was extracted from fllactamase-producing strains by scraping overnight cultures on 'chocolate' agar into 600/,1 phosphate-buffered saline (pH 7"3) which was then centrifuged
Susceptibilities of Haemophilus species
I37
in a microcentrifuge (Microfuge, MSE). Cell pellets were washed, resuspended in IOO #1 distilled water and then sonicated twice at an amplitude of Io microns for 30 s (Soniprep I5O, MSE). Cell debris was removed by centrifugation and the supernatant stored at - 70 °C or used immediately, fl-lactamases were then characterised by isoelectric focussing according to the method of Vecoli and colleagues. 11 Detection o f chloramphenieol-inactivation Chloramphenicol-resistant isolates were tested for chloramphenicol-inactivation by the method of Howard and colleagues. 12 T h e nature of the enzyme detected was not determined but was presumed to be chloramphenicol acetyltransferase. Plasmid profile analysis by agarose gel electrophoresis Plasmid D N A was extracted from ampicillin-resistant H. influenzae and H. parainfluenzae isolates by the method of Kado and Liu 13 and electrophoresed in a 0"7% agarose gel (Sigma, U.S.A.) with T r i s - b o r a t e - E D T A buffer pH 8"314 at 45 mA for 2 h, and use of a vertical Protean slab cell (Bio-Rad, U.S.A.). Gels were stained with ethidium bromide (Sigma) and then photographed through a red filter with Polaroid 665 film under short-wave ultraviolet transillumination. Standard sized markers used were: T P I I 6 ; T P I 2 9 ; T P I 2 5 ; T P I 2 o ; T P I 9 3 ; S-a; N T P 2 and ColE1 (from D r B. Rowe, Colindale, U.K.); pWG615; p W G I I 5 ; pWG3 and pEI94 (from D r W-B. Grubb, Perth, Western Australia). Results Altogether 403 strains of Haemophilus spp. were isolated during the study, all from sputum except for four from eye swabs and three from nasal swabs. None was isolated from blood or cerebrospinal fluid (CSF). Of the total, 292 (72"5 %) were H. influenzae, Io 7 (26"6%) were H. parainfluenzae, one was H. parahemolyticus and three were Haemophilus spp. which were not further identified. Irridescent colonies of the strains of Haemophilus spp isolated on Fildes' agar were not detected and many of the H. influenzae isolates autoagglutinated with both serological methods. We were thus unable to identify any capsulated strains of H. influenzae during the study. Review of laboratory records from the seven hospitals taking part in the study revealed that during the i2 months of i985, Haemophilus spp. (usually H. influenzae type b) were recovered from blood and/or CSF of only I9 patients, thereby indicating that capsulated, invasive H. influenzae strains are u n c o m m o n in Hong Kong. Of the 393 isolates which survived for further study, 288 were H. influenzae and Io2 were H. parainfluenzae. Table I shows the susceptibility of these isolates to four fl-lactam and five other antimicrobial agents. Fifty-seven (I9"8%) strains of H. influenzae were resistant to I mg/1 ampicillin and, of these, 94"7% had MICs > = 8 mg/1. All ampicillinresistant isolates except one with an M I C of 2 mg/1 produced fl-lactamase detectable by the Oxoid papers. Except for two fl-lactamase-positive isolates,
138
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Susceptibilities of H a e m o p h i l u s species
139
T a b l e II. Distribution of resistance patterns of H a e m o p h i l u s spp. isolated in
Hong Kong Percentage of strains Number of resistances o I 2 3 4 5
Resistance patterns Sensitive E Others AT ET Others ATC AET Others AETC AETTmCr
H. influenzae
H. parainfluenzae
(288)
(1o2)
23.3 49'3 3"4 1"4 2.8 1"7 5'6 4"9 1'6 5"6 0"3
47"I 12'7 17"6 7"8 4'9 3"9 2"o o'o 3"9 o.o o'o
A = ampicillin, E = erythromycin, T = tetracycline, C = chloramphenicol, Cr = cefaclor, Tm = trimethoprim. which were resistant to 8 mg/1 cefaclor, all were susceptible to cefaclor (8 mg/1), cephamandole and cefuroxime (4 mg/1). F o u r t e e n per cent were resistant to I mg/1 chloramphenicol and were shown by the m e t h o d of H o w a r d and colleagues, 12 to inactivate chloramphenicol, probably by the p r o d u c t i o n of chloramphenicol acetyltransferase. Sixty-five per cent were resistant to 0"5 rag/1 e r y t h r o m y c i n and 25 % tO I mg/1 tetracycline but less than z % were resistant to t r i m e t h o p r i m or co-trimoxazole. T h e proportion of H. parainfluenzae isolates resistant to ampicillin and tetracycline was similar to that of H. influenzae (Table I). T h e ampicillinresistance was also m e d i a t e d by the p r o d u c t i o n of fi-lactamase. All isolates were susceptible to cefaclor (8 mg/1), cephamandole and cefuroxime (4 mg/1). F e w e r H. parainfluenzae isolates, were resistant to e r y t h r o m y c i n (I9 % had M I C s > 0"5 mg/1) and chloramphenicol (4% had M I C s > I mg/1) and the chloramphenicol-resistance was due to a chloramphenicol-inactivating enzyme. In contrast, more isolates were resistant to t r i m e t h o p r i m and cotrimoxazole (5 and 3 % respectively had M I C s > o'5 mg/1). Only 23 % of H. influenzae isolates were susceptible to all the agents tested; 53 % were resistant to one drug, and 24 % to two or more drugs (Table II). A lower percentage of H. parainfluenzae isolates exhibited multiple resistance, 47 % being susceptible to all drugs tested and 23 % resistant to two or more. F o r t y - o n e ampicillin-resistant, fl-lactamase-positive H. influenzae isolates were analysed for possession of plasmids. T w e n t y (48"8 %) did not contain detectable plasmids b u t there were nine different plasmid profiles (plasmid types i-6) among the remaining 21 isolates (Table III). T h e s e contained plasmids ranging in size f r o m 2I to 55 M d a l except for one isolate which had only one small plasmid of 4-5 Mdal. fl-lactamases extracted from 25 of these isolates were all of the T E M - I type with an isoelectric point of 5"4.
14o
J.M.
LING
ET AL.
Table III. Plasmid profiles of ampicillin-resistant fl-lactamase-positive strains of H. influenzae and H. parainfluenzae Sizes of p l a s m i d s in Mdal
I
IA
2
2A
2B
3
Plasmid type 4 5 6
54'8 46 43 40 34 28 23 20"6 4"8 4"0 3"6 2- 7 2"52 No. of isolates :
H. influenzae H. parainfluenzae Total
7
8
9
3"6
-2. 7
Io
Nil
2'52
--
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2o 13 33
54"8 --
46
43 40
40 34 28 23
23
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23 20"6
4'8 4' 0
I o I
4 3 7
I o I
I o I
I o I
6 I 7
4 o 4
2 o 2
I o I
o 4 4
o I I
o I I
Of 25 ampicillin-resistant fl-lactamase-positive H. parainfluenzae tested, I3 (52 %) did not contain plasmids while the remaining I2 exhibited six plasmid profiles (plasmid types IA, 3, 7-Io), three of which were similar to those found in H. influenzae (Table III). Of the four plasmid profiles unique to H. parainfluenzae, one contained a 46 Mdal plasmid while the others contained plasmids of 2-4 Mdal. T w e n t y H. parainfluenzae strains were analysed for fllactamase by isoelectric focussing and all produced the T E M - I enzyme. Discussion
Invasive H. influenzae infections are uncommon in Hong Kong. T h e seven laboratories which participated in this study isolated capsulated H. influenzae type b from the blood or CSF of only I9 patients during I985. These hospitals (which included the infectious diseases hospital) served a population of about four million. T h e y had approximately 6000 beds and iooooo admissions per annum. Standard investigations, including culture and antigen detection, were used in their laboratories. Thus the infrequent isolation of capsulated H. influenzae from blood and CSF is probably a true reflection of a low incidence of invasive H. influenzae infections in Hong Kong despite the large number of children in the population. In contrast, H. influenzae and H. parainfluenzae were often isolated from respiratory tract specimens. Haemophilus parainfluenzae comprised about a quarter of the isolates although their clinical significance was not investigated. Capsulated strains of H. influenzae were not detected. Single and multiple antibiotic resistances were common among strains of both H. influenzae and H. parainfluenzae with I9 % or more of isolates being
Susceptibilities of H a e m o p h i l u s species
I4I
resistant to ampicillin and tetracycline (I mg/1). Ampicillin-resistance in b o t h species was mediated by the p r o d u c t i o n of T E M - I fl-lactamase. F o u r t e e n per cent of H. influenzae and 4 % of H. parainfluenzae were resistant to i rag/1 chloramphenicol as a result of their producing a d r u g - d e s t r o y i n g enzyme. Since I 9 8 I , resistance to ampicillin and chloramphenicol has increased significantly in H o n g K o n g among isolates of H. influenzae. 6 In contrast, less than 1 % of Haemophilus spp. were resistant to trimethoprim, co-trimoxazole or cefaclor and all isolates were susceptible to cephamandole and cefuroxime. In all b u t one strain, ampicillin-resistance was mediated b y T E M - I fllactamase. While this is the usual m e c h a n i s m of ampicillin-resistance in H. influenzae,15.1~ in several recent studies some strains have been observed that were resistant b y non-enzymatic mechanisms. 5'1G'17 T h i s p h e n o m e n o n was seen in only one of our isolates. In the present study, about 50 % of fl-lactamase-producing strains of H. influenzae and H. parainfluenzae did not contain detectable plasmids. Other workers have noted that resistance factors in H. influenzae may exist extrachromosomally or b e c o m e integrated into the chromosome. ~8-2° In particular, ampicillin-resistance has been f o u n d to be associated with a transposon, T n A . 2~ T h u s multiply-resistant strains m a y lack detectable plasmids. It is likely that determinants of ampicillin-resistance in H o n g K o n g strains of Haemophilus spp. have been transposed on to the chromosome. This p h e n o m e n o n warrants further study. While invasive, capsulated strains of H. influenzae are relatively u n c o m m o n in H o n g K o n g , and anecdotal evidence suggests that such virulent organisms are usually susceptible to ampicillin and chloramphenicol, clinicians should be alert for the appearance of resistant and multiply-resistant strains of H. influenzae type b in H o n g Kong. Ampicillin-resistant strains of non-capsulated H. influenzae remain susceptible to the second generation cephalosporins. T h e s e drugs m a y therefore be considered for use in the treatment of serious infections caused by these organisms. (We thank the following colleagues who collected strains of Haemophilus spp. in their laboratories for this study: Drs E. Gwi, M. Ho, K. M. Kam, T. Lat, R. Lycette, W. P. Mak, T. K. Ng, D. Tsang, and Mr H. T. Yeung. We also thank Mr Thomas Ling for the results of isoelectric focussing, Mr W. S. Au, Mr K. K. Mong and Mr E. Ng for technical assistance and Miss A. Cheung and Miss C. Ng for computer data entry.) References
i. Simasathien S, Duangmani C, Echeverria P. Haemophilus influenzae type b resistant to ampicillin and chloramphenicol in an orphanage in Thailand. Lancet I98o; ii: I2 I4-I2I 7. 2. Schlech NF, Band JD, Hightower AW, Broome CV. Bacterial meningitis in the United States (Abstr no. 3II). In: Program and abstracts of the 22nd Interscienee Conference on Antimicrobial Agents and Chemotherapy. Washington, DC: American Society for Microbiology, i982. 3. Campos J, Garcia-Tornel S, Sanfeliu I. Susceptibility studies of multiple resistant Haemophilus influenzae isolated from paediatric patients and contacts. Antimicrob Agents Chemother I984; 25: 7o6-709. 4. Doern GV, Jorgensen JH, Thornsberry C, Preston DA. Prevalence of antimicrobial
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resistance among clinical isolates of Haemophilus influenzae: a collaborative study. Diagn Microbiol Infect Dis 1986; 4 : 95-1o7. 5. Powell M, Koutsia-Carouzou C, Voutsinas D, Seymour A, Williams JD. Resistance of clinical isolates of Haemophilus influenzae in United Kingdom I986. Br M e d J 1987; 295: 176-179. 6. Ling J, Chau PY, Leung YK, Ng WS, So SY. Antibiotic susceptibility of pneumococci and Haemophilus influenzae isolated from patients with acute exacerbations of chronic bronchitis: prevalence of tetracycline-resistant strains in Hong Kong. J Infect 1983; 6: 33-37. 7. Biberstein EL, Mini PD, Gills MG. Action of Haemophilus cultures on gammaaminolevulinic acid. J Bacteriol 1973 ; 86 : 814-819. 8. Kilian M. A taxonomic study of the genus Haemophilus, with the proposal of a new species. J Gen Microbiol 1976; 93: 9-62. 9. Turk DC, May JR. Isolation and identification of H. influenzae. In: Haemophilus influenzae : clinical importance. The English Universities Press, London, 1967: I17-2o. io. Report by a Working Party of the British Society for Antimicrobial Chemotherapy. Breakpoints in in-vitro antibiotic sensitivity testing. J Antimicrob Chemother I988; 2 I : 7oi-7IO. II. Vecoli C, Prevost FE, Ververis JJ, Medeiros AA, O'Leary Jr GP. Comparison of polyacrylamide and agarose gel thin-layer isoelectric focussing for the characterization of fl-lactamases. Antimicrob Agents Chemother I983 ; 24: I86-189. 12. Howard AJ, Williams HM, Poole MC. Chloramphenicol resistance in Haemophilus influenzae. Lancet 1986; ii: 745-74613. Kado CI, Liu S.-T. Rapid procedure for detection and isolation of large and small plasmids. J Bacteriol 198I; I45: 1365-1373. 14. Meyers JA, Sanchez D, Elwell LP, Falkow S. Simple agarose gel electrophoretic method for the identification and characterization of plasmid deoxyribonucleic acid. J Bacteriol 1976; 127: 1529-1537. 15. Brunton J, Clare D~ Meier MA. Molecular epidemiology of antibiotic resistance plasmids of Haemophilus species and Neisseria gonorrhoeae. Rev Infect Dis 1986; 8:713-724. 16. Mendelman PM, Chaffin DO, Clausen C, et al. Failure to detect ampicillin-resistant, nonfl-lactamase-producing Haemophilus influenzae by standard disk susceptibility testing. Antimicrob Agents Chemother 1986; 3o: 274-280. I7. Mendelman PM, Chaffin DO, Stull TL, Rubens CE, Mack KD, Smith AL. Characterization of non-fl-lactamase-rnediated ampicillin resistance in Haemophilus influenzae. Antimicrob Agents Chemother 1984; 26: 235-244. 18. Willard JE, Johnson EJ, Daum RS. Interrelationships between physical state, phenotypic stability and transferability offl-lactamase genes in Haemophilus influenzae. J Gen Microbiol 1982; 128: 2353-2360. 19. Mendelman PM, Doroshow CA, Gandy SL, Syriopoulou V, Weigen CP, Smith AL. Plasmid-mediated resistance in multiply resistant Haemophilus influenzae type b causing meningitis; molecular characterization of one strain and review of the literature. J Infect Dis 1984; 15o: 30-39 . 20. Murphy-Corb M, Nolan-Willard M, Daum RS. Integration of plasmid DNA coding for fl-lactamase production in the Haemophilus influenzae chromosome. J Bacteriol 1984; 16o: 815-817 . 21. Laufs R, Kaulfers P.-M. Molecular characterization of a plasmid specifying ampicillin resistance and relationship to other R factors from Haemophilus influenzae. J Gen Microbiol 1977; lO3: 277-286.