Disc diffusion susceptibility testing of Haemophilus influenzae by NCCLS methodology using low-strength ampicillin and co-amoxiclav discs

Pauliina Kärpänoja1,*, Antti Nissinen2, Pentti Huovinen3 and Hannu Sarkkinen1 on behalf of the Finnish Study Group for Antimicrobial Resistance (FiRe network)§

1 Päijät-Häme Central Hospital, Department of Clinical Microbiology, Keskussairaalankatu 7, FIN–15850 Lahti; 2 Keski-Suomi Central Hospital, Jyväskylä; 3 National Public Health Institute, Turku, Finland

Received 16 September 2003; returned 29 October 2003; revised 7 January 2003; accepted 8 January 2004


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Objectives: The objective of this multicentre study was to define the accuracy and reproducibility of the NCCLS disc diffusion method for Haemophilus influenzae against ampicillin and co-amoxiclav in Finnish clinical microbiology laboratories. Special attention was paid to the ability of the laboratories to detect ß-lactamase-negative ampicillin-resistant (BLNAR) strains.

Methods: Three BLNAR and two ß-lactamase-negative ampicillin-susceptible isolates (BLNAS)—originating from the American Type Culture Collection (ATCC) and UK National External Quality Assessment (UKNEQAS) schemes—were included in this study. Susceptibility tests for these isolates were performed in 26 clinical microbiology laboratories, in accordance with NCCLS guidelines. Additionally, low-strength discs for ampicillin (2 µg) and co-amoxiclav (3 µg) were tested.

Results: The low-strength discs for ampicillin and co-amoxiclav categorized more accurately BLNAR and BLNAS H. influenzae isolates than did the high-strength discs recommended by the NCCLS. In addition, the high-strength discs produced more major errors than the low-strength discs (22 versus six for ampicillin and 40 versus seven for co-amoxiclav). Great variation occurred in the method regardless of the antibiotic concentration of the discs.

Conclusions: The use of low-content ampicillin and co-amoxiclav discs is recommended for the susceptibility testing of H. influenzae. Interpretative criteria of S>=17 mm and R<=13 mm for both discs are suggested.

Keywords: H. influenzae, BLNAR, susceptibility testing


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
The first reports of ampicillin-resistant H. influenzae were published in 1974 by several authors. The plasmid-mediated production of TEM or ROB ß-lactamases is the prevalent mechanism of this resistance, exceeding 30% in the USA, and increasing in Finland over 5 years (from 8% in 1988–1990 to 24.4% in 1995 in middle ear isolates of children).13 Non-ß-lactamase-mediated resistance to ampicillin in H. influenzae was reported in the early 1980s by Markowitz.4 These ß-lactamase-negative, ampicillin-resistant (BLNAR) or intermediate strains remained rare in the 1990s in Finland (0.2%), western European countries (0.3%) and the USA (2.5%).2,5,6 In more recent surveillance studies from Spain and Japan, however, higher proportions (9.3% and up to 8.9%) have been reported.7,8 Altered penicillin binding proteins (PBPs) 3, 4 and 5 are associated with this type of resistance.9 Strains carrying two mechanisms for ampicillin resistance (the TEM-1 ß-lactamase gene and a mutation in the ftsI gene affecting PBP3) have also been described, but are unusual worldwide.10

Problems detecting BLNAR strains using the NCCLS disc diffusion method11 have been reported previously by several groups and different solutions have been proposed to correct this methodological difficulty. The use of a low-strength disc (oxacillin 1 µg) is acknowledged as a screening method to identify penicillin non-susceptible pneumococci. In cases of H. influenzae and ampicillin, the ability to discriminate BLNAR strains from ß-lactamase-negative, ampicillin-susceptible (BLNAS) strains was better using a 2 µg disc rather than the 10 µg disc recommended by the NCCLS.12

Here we report the results and conclusions of an evaluation of the NCCLS disc diffusion method for the susceptibility testing of H. influenzae in Finland. The main focus was to assess the method used to identify the BLNAR strains, and therefore discs with lower contents of ampicillin and co-amoxiclav were included. H. influenzae isolates from the ATCC collection and the UK NEQAS schemes with reference susceptibilities were used as challenge strains.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Disc diffusion susceptibility tests for ampicillin and co-amoxiclav were performed for five H. influenzae control strains in 26 Finnish clinical microbiology laboratories (Finnish Study Group for Antimicrobial Resistance, FiRe laboratories). The challenge strains and their reference MIC values given by the suppliers are presented in Table 1 (isolates 1–5). Isolates 2, 3 and 5 were regarded as BLNAR strains on the basis of decreased MIC values for ampicillin, co-amoxiclav and/or cephalosporins. Unfortunately, the information concerning altered PBPs 3, 4 and 5 associated with this type of resistance9 was not available from ATCC or UK NEQAS. The technical procedure for the susceptibility testing was that outlined by the NCCLS.11 Briefly: haemophilus test medium (HTM) agar plates (Oxoid, Basingstoke, Hampshire, UK) were prepared for the laboratories centrally in the Antimicrobial Research Laboratory, National Public Health Institute, Turku, Finland. Standard, 6 mm paper discs (Oxoid, Basingstoke, Hampshire, UK) were used. The inoculum was adjusted to 0.5 on the McFarland scale, and the inoculated plates were incubated for 16–18 h at 35 ± 2°C, 5% CO2. The following antibiotics and concentrations were used: ampicillin 10 and 2 µg and co-amoxiclav 30 (10 + 20) and 3 (1 + 2) µg.


View this table:
[in this window]
[in a new window]
 
Table 1. Haemophilus influenzae strains used for the validation and combined results of the disc diffusion tests in 26 laboratories for these five control strains. The reference MIC values of ampicillin, co-amoxiclav, cefaclor and cefuroxime are from the NCCLS standard 2003 (ATCC strains) or from the UK NEQAS distributions feedback information
 
The results were analysed using the WHONET 5.3 computer program and SPSS for Windows statistical software, version 11.5


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
A total of 128 measured results were reported for the ampicillin 10 µg disc and 129 for each of the ampicillin 2 µg, co-amoxiclav 30 µg and co-amoxiclav 3 µg discs.

Isolates 2, 3 and 5 were considered BLNAR strains resistant to ampicillin, co-amoxiclav and first and/or second-generation cephalosporins. The S/I/R classification is based on the interpretative criteria given by the NCCLS for ampicillin 10 µg and co-amoxiclav 30 µg discs. Interpretative criteria suggested by Zerva et al. (S>=17 mm, R<=13 mm)12 were applied for both low-strength discs. Resistant and intermediate categories were combined for the analysis.

Both low-strength discs categorized more accurately the BLNAR isolates (Table 1). Sensitivities and specificities estimated for each test (disc) were as follows: ampicillin 10 µg: 71%/88%, ampicillin 2 µg: 92%/90%, co-amoxiclav 30 µg: 44%/96%, co-amoxiclav 3 µg: 91%/87%. Thus, the methods using low-strength discs were more sensitive for both antimicrobial agents. The specificity was acceptable for all tests (discs). Altogether, the NCCLS susceptibility method produced 22 major errors (false-susceptible results) for ampicillin and 40 for co-amoxiclav. The corresponding figures for the low-strength discs were six for ampicillin and seven for co-amoxiclav. In a three-centre, collaborative study in the USA, major errors—according to the NCCLS—were produced for two ß-lactamase-inhibitor combination discs (co-amoxiclav and ampicillin/sulbactam).13 However, in the same study, contrary to our results, ampicillin testing—according to the NCCLS—did not produce major errors.

The variation in the zone diameters of the inhibition zones was considerable, as the ranges and standard deviations reveal (Table 1). Several of the 26 laboratories reported difficulties in determining the edge of the inhibition zones because of hazy limits of growth. This could, in theory, be due to two methodological factors: the medium or the inoculum density. Several reports have been published concerning the use of HTM in general, but we have found no evaluation of the Oxoid HTM. However, the composition of HTM by different manufacturers is identical and therefore we do not believe that the medium we used would be the main reason for the lack of good reproducibility in this study. Rather, the inoculum density may explain these difficulties. The inoculum density recommended by the NCCLS11 is McFarland 0.5, which corresponds to 1–4 x 108 cfu/mL. According to our unpublished data, however, the actual cfu/mL varies from 1–7 x 108, even though the density is adjusted to McFarland 0.5 using a photometric device. Thus, the use of too dense inocula may, in part, explain the great variability in the zone diameters. The worst results were seen for isolate 2, where the variation of zone diameters and misclassifications were greatest even with the low-strength discs. This is somewhat surprising, because the reference MIC values given by the supplier (UK NEQAS) were, out of all the isolates, highest for ampicillin (8 mg/L) and cefuroxime (64 mg/L) and one might assume that this strain would be the easiest to classify correctly. Most comments of hazy growth within the inhibition zones were for this isolate.

Although BLNAR H. influenzae isolates are uncommon in most western countries, their detection is important for two reasons. First, these isolates are resistant to first- and second-generation cephalosporins, and treatment failures have been reported with these antibiotics.4 Second, the BLNAR strains have become increasingly prevalent in certain countries. The widespread use of oral cephalosporins has been suggested as a reason for this increase, for instance in Japan.10 In Finland, the consumption of cephalosporins, all in all, has been considerably higher than in other Nordic countries (in 1995, three versus less than one DDD/1000 inhabitants per day). In outpatient care, oral cephalosporins have been particularly popular for the treatment of skin and middle ear infections.14

To conclude, in our opinion, low-strength ampicillin and co-amoxiclav discs should be added to the routine susceptibility testing of H. influenzae to detect BLNAR isolates. The sensitivity of the NCCLS method is not satisfactory. The high-strength discs do not seem to add value to the detection of BLNAR H. influenzae. However, ß-lactamase-positive isolates were not included in this study, and we do not know, at the moment, whether the detection of co-amoxiclav resistance among these isolates is accurate with the low-strength disc. The interpretative criteria used in this study are applicable for both low-strength discs, but require further validation. The suggested detection of BLNAR H. influenzae by the disc diffusion method should always be confirmed by MIC testing.


    Acknowledgements
 
Members of the FiRe network (in 1999): Anja Kostiala-Thompson and Merja Rautio (Jorvi Hospital); Risto Renkonen and Anna Muotiala (Medix-Diacor, laboratories); Martti Vaara and Petteri Carlson (HYKS, laboratories); Hannele Jousimies-Somer (Mehiläinen Hospital); Jukka Korpela and Ritva Heikkilä (Kanta-Häme Central Hospital); Suvi-Sirkku Kaukoranta (Pohjois-Karjala Central Hospital); Antti Nissinen (Keski-Suomi Central Hospital); Pekka Ruuska (Kainuu Central Hospital); Henrik Jägerroos (Lappi Central Hospital); Martti Larikka (Länsi-Pohja Central Hospital); Simo Räisänen (Keski-Pohjanmaa Central Hospital); Ulla Larinkari and Benita Forsblom (Kymenlaakso Central Hospital); Marja-Leena Katila and Ulla Kärkkäinen (Kuopio University Hospital); Hannu Sarkkinen and Pauliina Kärpänoja (Päijät-Häme Central Hospital); Maritta Kauppinen and Seppo Paltemaa (Etelä-Karjala Central Hospital); Päivi Kärkkäinen (Mikkeli Central Hospital, Savonlinna Central Hospital); Markku Koskela (Oulu University Hospital); Sini Pajarre (Satakunta Central Hospital); Sinikka Oinonen and Virpi Ratia (Seinäjoki Central Hospital), Paul Grönroos (Health Center Koskiklinikka); Risto Vuento and Oili Liimatainen (Tampere University Hospital); Maj-Rita Siro (Health Center Pulssi); Erkki Eerola and Raija Manninen (Turku University); Olli Meurman (Turku University Hospital); Heikki Hiekkaniemi (Vaasa Central Hospital); Pentti Huovinen and Katrina Lager (National Public Health Institute).


    Footnotes
 
* Corresponding author. Tel: +358-3-819-2459; Fax: +358-3-819-2875; E-mail: pauliina.karpanoja{at}phks.fi Back

§ The members of the FiRe network are listed in the Acknowledgements. Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
1 . Scriver, S. R., Walmsley, S. L., Kau, C. L. et al. (1994). Determination of antimicrobial susceptibilities of Canadian isolates of Haemophilus influenzae and characterization of their ß-lactamases. Antimicrobial Agents and Chemotherapy 38, 1678–80.[Abstract]

2 . Doern, G. V., Brueggeman, A. B., Pierce, G. et al. (1997). Antibiotic resistance among clinical isolates of Haemophilus influenzae in the United States in 1994 and 1995 and detection of ß-lactamase-positive strains resistant to amoxicillin-clavulanate: results of a national multicenter surveillance study. Antimicrobial Agents and Chemotherapy 41, 292–7.[Abstract]

3 . Manninen, R., Huovinen, P. & Nissinen, A. (1997). Increasing antimicrobial resistance in Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis in Finland. Journal of Antimicrobial Chemotherapy 40, 387–92.[Abstract]

4 . Markowitz, S. M. (1980). Isolation of an ampicillin-resistant, non-ß-lactamase-producing strain of Haemophilus influenzae. Antimicrobial Agents and Chemotherapy 17, 80–3.[ISI][Medline]

5 . Nissinen, A., Herva, E., Katila M. L. et al. (1995). Antimicrobial resistance in Haemophilus influenzae isolated from blood, cerebrospinal fluid, middle ear fluid and throat samples of children. A nationwide study in Finland 1988–1990. Scandinavian Journal of Infectious Diseases 27, 57–61.[ISI][Medline]

6 . Kayser, F. H., Morenzoni, G. & Santanam, P. (1990). The second European collaborative study on the frequency of antimicrobial resistance in Haemophilus influenzae. European Journal of Clinical Microbiology and Infectious Diseases 9, 810–17.[ISI][Medline]

7 . Marco, F., Garcia-de-Lomas, J., Garcia-Rey, C. et al. (2001). Antimicrobial susceptibilities of 1,730 Haemophilus influenzae respiratory tract isolates in Spain 1998–1999. Antimicrobial Agents and Chemotherapy 45, 3226–8.[Abstract/Free Full Text]

8 . Ohkusu, K., Nakamura, A. & Sawada, K. (2000). Antibiotic resistance among clinical isolates of Haemophilus influenzae in Japanese children. Diagnostic Microbiology and Infectious Disease 36, 249–54.[CrossRef][ISI][Medline]

9 . Mendelman, P. M., Chaffin, D. O. & Kalaitzoglou G. (1990). Penicillin binding proteins and ampicillin resistance in Haemophilus influenzae. Journal of Antimicrobial Chemotherapy 25, 525–34.[Abstract]

10 . Ubukata, K., Chiba, N., Hasewaga, K. et al. (2002). Differentiation of ß-lactamase-negative ampicillin-resistant Haemophilus influenzae from other H. influenzae strains by a disc method. Journal of Infection and Chemotherapy 8, 50–8.[Medline]

11 . National Committee for Clinical Laboratory Standards. (2003). Performance Standards for Antimicrobial Disk Susceptibility Tests—Eighth Edition: Approved Standard M2-A8. NCCLS, Villanova, PA, USA.

12 . Zerva, L., Biedenbach, D. J. & Jones R. N. (1996). Reevaluation of interpretative criteria for Haemophilus influenzae by using meropenem (10-microgram), imipenem (10-microgram) and ampicillin (2- and 10-microgram) disks. Journal of Clinical Microbiology 34, 1970–4.[Abstract]

13 . Doern, G. V., Jorgensen, J. H., Thornsberry, C. et al. (1990). Disk diffusion susceptibility testing of Haemophilus influenzae using Haemophilus test medium. European Journal of Clinical Microbiology and Infectious Diseases 9, 329–36.[ISI][Medline]

14 . Bergan, T. (2001). Antibiotic usage in Nordic countries. International Journal of Antimicrobial Agents 18, 279–82.[CrossRef][ISI][Medline]