Differences between the activity of penicillin, amoxycillin, and co-amoxyclav against 5,252 Streptococcus pneumoniae isolates tested in the Alexander Project 1992- 1996

D. L. Butlera,*, R. C. Gagnonb, L. A. Millera, J. A. Pouparda, D. Felminghamc and R. N. Grünebergc

a Anti-Infectives , SmithKline Beecham Pharmaceuticals, Collegeville, PA, USA; b Department of Biostatistics and Data Sciences, SmithKline Beecham Pharmaceuticals, Collegeville, PA, USA; c GR Micro Ltd., London, UK


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
A number of published studies have shown that the MICs of amoxycillin and/or co-amoxyclav are lower than those of ampicillin and/or penicillin for Streptococcus pneumoniae. Other published studies have concluded that the activities of amoxycillin and co-amoxyclav are comparable with that of penicillin for S. pneumoniae. A collection of 5252 S. pneumoniae isolates obtained during a 5 year period (1992- 1996) was analysed to determine differences between the MICs of penicillin, amoxycillin and co-amoxyclav. Among the isolates analysed, 3788 (72%) were penicillin-susceptible, 615 (12%) were penicillin-intermediate and 849 (16%) were penicillin-resistant. Differences between the agents were assessed by examination of MIC distribution functions and simultaneous 95% CIs. In addition, penicillin-intermediate and -resistant isolates were analysed to determine the number and percentage of isolates which had an amoxycillin and co-amoxyclav MIC less than, equal to, or greater than the penicillin MIC. Results showed that the amoxycillin and co-amoxyclav MIC 90s were one two-fold dilution lower than those of penicillin for all isolates collected between 1992- 1993 and 1994- 1996. Simultaneous 95% CIs showed that the mean differences between MICs of amoxycillin and penicillin, and between MICs of co-amoxyclav and penicillin, were less than zero. The majority of the penicillin-intermediate and penicillin-resistant isolates had an amoxycillin and co-amoxyclav MIC less than the penicillin MIC. In conclusion, amoxycillin and co-amoxyclav MICs were shown to be lower than the penicillin MICs for the S. pneumoniae isolates analysed in this study.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Prior to January 1995, NCCLS guidelines stated that the susceptibility of Streptococcus pneumoniae to amoxycillin and co-amoxyclav was predicted by testing penicillin since there were no breakpoints established for those agents. In January 1995, the NCCLS established breakpoints for amoxycillin and co-amoxyclav distinct from those of penicillin. 1 Breakpoints were granted in part because the data presented to the NCCLS showed that amoxycillin and amoxycillin/clavulanic acid had greater activity than penicillin and ampicillin for S. pneumoniae.The breakpoints established were based on in-vitro S. pneumoniae data, pharmacokinetics, animal studies and outcome data from two major clinical studies.

In a surveillance study of 1527 clinical isolates of S. pneumoniae the activity of amoxycillin and co-amoxyclav appeared to be essentially comparable with that of penicillin. 2 A recent surveillance study of 898 S. pneumoniae isolates reported that the MIC90 for co-amoxyclav was one dilution higher than the penicillin MIC90.3 Another recently published surveillance study grouped ampicillin and amoxycillin together in a S. pneumoniae results table.4 These results differ from most other published in-vitro studies on S. pneumoniae which have shown that amoxycillin and/or co-amoxyclav MICs are lower than the MICs of ampicillin and/or penicillin. 5,6,7,8,9,10,11

The purpose of our analysis was to compare the MICs of amoxycillin and co-amoxyclav to those of penicillin. A collection of 5252 isolates of S. pneumoniae collected during a 5 year period (1992- 1996) were analysed. These isolates were collected as part of the Alexander Project, a multicentre, international surveillance study of community-acquired lower respiratory tract pathogens. For this study, isolates were categorized by year of collection and by penicillin susceptibility group (e.g. penicillin-susceptible, penicillin-intermediate and penicillin-resistant). Isolates were analysed to determine differences between the amoxycillin, co-amoxyclav and penicillin MICs based on examination of MIC distribution functions and simultaneous 95% CIs. In addition, all penicillin-intermediate and penicillin-resistant isolates were analysed to determine the number and percentage of isolates which had an amoxycillin and co-amoxyclav MIC less than, equal to, or greater than the penicillin MIC.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Isolates

Ten centres in Europe and five in the USA participated in the Alexander Project between 1992- 1995. 12,13 In 1996, twenty centres from eighteen countries participated. During this 5 year period, a total of 5252 S. pneumoniae isolates were tested. Outpatient sputum isolates and those collected during the first 48 h of admission were included in the collection. Blood isolates cultured during the same period were also included. The isolates were sent to the central Alexander Project Laboratory, GR Micro Ltd., London, UK, on heavily inoculated swabs maintained in Amies transport medium (Bibby Sterilin Ltd., Stone, UK).

Identification

Identification was performed at GR Micro Ltd. according to standard methodology using the following tests: colonial morphology, Gram's stain morphology, catalase reaction, optochin susceptibility and bile solubility.

Susceptibility testing

MICs were determined for all isolates using microbroth dilution methodology. The microtitre trays were commercially prepared by Sensititre (Accumed International Ltd., East Grinstead, UK). Each inoculum was approximately 104 cfu in 50 µL of medium. Mueller- Hinton medium was supplemented with saponin-lysed horse blood (final concentration 2% v/v) and nicotinamide adenine dinucleotide (final concentration 10 mg/L) for susceptibility testing of S. pneumoniae.

S. pneumoniae ATCC 49619 was used as quality control. Results were accepted only when the control strain MICs were within ranges established by the NCCLS.

Different concentrations of penicillin, amoxycillin and co-amoxyclav in doubling dilutions were tested between 1992- 1993 and 1994- 1996. Following review of MIC distribution data collected between 1992- 1993, concentration ranges were adjusted in 1994. For 1992- 1993, the concentration of penicillin tested was 0.03- 16 mg/L. For the same time period, the concentrations of amoxycillin and co-amoxyclav tested were 0.06- 32 mg/L. For 1994- 1996, 0.004- 8 mg/L was the concentration range tested for all three antimicrobials. Co-amoxyclav was tested in a 2:1 ratio. The concentrations listed for co-amoxyclav refer to the amoxycillin component.

Data analysis

Data were analysed using version 6.12 of the Statistical Analysis System (SAS). MIC distributions for penicillin, amoxycillin and co-amoxyclav were categorized by year of collection (1992- 1993 and 1994- 1996), and summarized by their respective distribution functions.14 Distribution functions were also utilized to summarize MIC distributions for isolates categorized by penicillin susceptibility group (penicillin-susceptible, penicillin-intermediate and penicillin-resistant). From these distribution functions, which are step functions, the modes (longest length of a vertical line), and MICxs (where x is any quantile), were determined. For example, the median (MIC50), was determined by drawing a horizontal line through 0.5 on the vertical axis of the distribution function; the MIC value where this line intersects the distribution function was the median. Statistical tests comparing the mean of the differences between penicillin and amoxycillin MICs, and between penicillin and co-amoxyclav MICs, were conducted using simultaneous 95% CIs.15 If these intervals did not include zero, a statistically significant difference existed between MIC means. Tabular reports summarizing the difference in MICs of amoxycillin and co-amoxyclav relative to penicillin were generated for penicillin susceptibility groups (penicillin-intermediate and penicillin-resistant).


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
A total of 5252 S. pneumoniae isolates collected between 1992- 1996 were analysed. There were 3788 (72%) penicillin-susceptible, 615 (12%) penicillin-intermediate and 849 (16%) penicillin-resistant isolates. The distribution of MICs is displayed in Figure 1 for isolates categorized by year of collection, 1992- 1993 (Figure 1a) and 1994- 1996 (Figure 1b). The data were categorized by these year groups because concentrations of penicillin, amoxycillin and co-amoxyclav below 0.03 and 0.06 mg/L, respectively, were not tested in 1992- 1993. Furthermore, the MICs of penicillin, amoxycillin and co-amoxyclav at and below 0.06 mg/L were categorized as <=0.06 mg/L for isolates in 1992- 1993.



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Figure 1. Distributions of penicillin (······), amoxycillin (- - - ) and co-amoxyclav (— - ) MICs for S. pneumoniae: (a) 1992- 1993, 1856 isolates; (b) 1994- 1996, 3396 isolates.

 
The MIC90s of amoxycillin and co-amoxyclav were one two-fold dilution lower than penicillin among 1856 S. pneumoniae and 3396 S. pneumoniae isolates collected between 1992- 1993 and 1994- 1996, respectively (Table I). In fact, distribution functions in Figure 1a and 1b show that the MICs of amoxycillin and co-amoxyclav are consistently lower than the MICs of penicillin for concentrations of >0.06 mg/L (1992- 1993) and >0.03 mg/L (1994- 1996).


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Table I. In-vitro activities of penicillin and amoxycillin with and without clavulanate for 5252 isolates of S. pneumoniaea
 
A similar analysis was performed for all isolates, except those susceptible to penicillin for 1992 and 1993, categorized by penicillin susceptibility. Distributions of MICs are displayed in Figure 2 for isolates categorized as penicillin-susceptible, penicillin-intermediate and penicillin-resistant. The MIC90s of amoxycillin and co-amoxyclav were one two-fold dilution lower than penicillin for the penicillin-susceptible isolates (Table II and Figure 2a). The MICs of amoxycillin and co-amoxyclav were consistently one two-fold dilution lower than the MICs of penicillin for the penicillin-intermediate isolates (Figure 2b). For most of the penicillin-resistant strains, the MICs of amoxycillin and co-amoxyclav were a two-fold dilution lower than the penicillin MIC. However, the MIC 90s of all three agents were the same (4 mg/L) (Table II and Figure 2c).



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Figure 2. Distributions of penicillin (······), amoxycillin (- - - ) and co-amoxyclav (— - ) MICs for S. pneumoniae: (a) in penicillin-susceptible isolates; (b) in isolates of intermediate susceptibility; (c) in penicillin-resistant isolates.

 

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Table II. In-vitro activities of penicillin and amoxycillin with or without clavulanate for 3846 isolates of S. pneumoniaea
 
Table III lists the number and percentage of penicillin-intermediate and penicillin-resistant isolates having amoxycillin and co-amoxyclav MICs which are less than, equal to, or greater than the penicillin MICs. Of 615 penicillin-intermediate isolates, 69% and 73% had an amoxycillin and co-amoxyclav MIC less than the penicillin MIC, respectively. Furthermore, 29% of amoxycillin and 34% of co-amoxyclav MICs were two or more dilutions lower than the penicillin MIC. Of 849 penicillin-resistant isolates, 64% and 71% had an amoxycillin and co-amoxyclav MIC less than penicillin, respectively, with 7% of amoxycillin and 11% of co-amoxyclav MICs two or more doubling- dilutions lower than the penicillin MIC.


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Table III. Difference in MICs for amoxycillin with or without clavulanate relative to penicillin for the penicillin-intermediate and penicillin-resistant strains of S. pneumoniae collected between 1992– 1996
 
For penicillin-intermediate isolates, the lower amoxycillin and co-amoxyclav MICs resulted in different category interpretations between penicillin, amoxycillin and co-amoxyclav. Using current NCCLS breakpoints, of the 615 penicillin-intermediate isolates, 89% and 91% were amoxycillin and co-amoxyclav-susceptible, respectively. Of the 849 penicillin-resistant isolates, 3% and 4% were amoxycillin and co-amoxyclav-susceptible, respectively.

Applying a pharmacodynamic breakpoint of 2 mg/L, 99.5% of the 615 penicillin-intermediate isolates are amoxycillin and co-amoxyclav-susceptible.16 Seventy-nine percent and 80% of the 849 penicillin-resistant isolates are amoxycillin and co-amoxyclav-susceptible, respectively.

The penicillin-intermediate and penicillin-resistant isolates were also analysed to determine if there were differences between the MICs of amoxycillin and co-amoxyclav. For the penicillin-intermediate isolates, 10% had an co-amoxyclav MIC less than the amoxycillin MIC, while 3% had an amoxycillin MIC less than the co-amoxyclav MIC. For the penicillin-resistant isolates, 18% had an co-amoxyclav MIC less than the amoxycillin MIC, while 7% had an amoxycillin MIC less than the co-amoxyclav MIC. The majority of these differences were within one doubling-dilution.

To determine statistical significance among the MICs of penicillin, amoxycillin and co-amoxyclav, simultaneous 95% CIs were calculated for the MICs of all isolates categorized similarly to Figures 1 and 2. The MICs of amoxycillin and co-amoxyclav were significantly lower than the penicillin MIC for S. pneumoniae isolates categorized by year groups, 1992- 1993 and 1994- 1996, and by penicillin susceptibility (Table IV).


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Table IV. Simultaneous 95% CIs for the MIC differences between amoxycillin– penicillin and amoxycillin/clavulanate– penicillin
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The increased activity, based on MIC differences, of amoxycillin and/or co-amoxyclav compared to ampicillin and/or penicillin among penicillin-intermediate and penicillin-resistant isolates has been described previously in the literature.5,6,7,8,9,10,11 However, the differences shown here for the penicillin-intermediate and penicillin-resistant groups were not observed in another published surveillance study. 2 In addition to MIC differences according to penicillin susceptibility group, differences were also observed between the three agents for S. pneumoniae isolates categorized by year of collection.

There were statistically significant differences between the MICs of amoxycillin and co-amoxyclav compared with those of penicillin for a large group of S. pneumoniae strains isolated between 1992 and 1996. Differences between the three agents were assessed by comparing the MIC distribution functions and simultaneous 95% CIs. Differences were also assessed by comparing the percentage of isolates which had a lower amoxycillin or co-amoxyclav MIC than penicillin. The results of this analysis show that amoxycillin and co-amoxyclav MICs were significantly lower than the penicillin MICs for the S. pneumoniae isolates analysed in this study.

The significance of these lower MICs may have clinical relevance for ß-lactams. The length of time that serum levels of an antibiotic are above the MIC (T>MIC) of an organism is the pharmacokinetic/pharmacodynamic parameter that correlates best with in-vivo efficacy of ß-lactam antibiotics.17 Animal models and clinical studies have identified T>MIC as the key parameter that correlates most closely with clinical and bacteriological outcomes.18 Lower S. pneumoniae MICs of a ß-lactam, such as amoxycillin, will result in a longer T>MIC which correlates to better clinical efficacy.

The results of this study clearly show that the activity of amoxycillin and co-amoxyclav cannot be predicted by testing penicillin. Additionally, amoxycillin and co-amoxyclav have distinct S. pneumoniae breakpoints from several organizations including the NCCLS, and need to be tested independently. In many cases, isolates that are intermediate or resistant to penicillin, are susceptible to amoxycillin and/or co-amoxyclav. Using appropriate breakpoints will allow clinicians the choice of selecting another more potent anti-pneumococcal penicillin antibiotic, such as amoxycillin or co-amoxyclav, instead of a macrolide, cephalosporin, or fluoroquinolone for treatment of infections caused by penicillin-resistant S. pneumoniae.


    Acknowledgments
 
We gratefully acknowledge the members of The Alexander Project Group and the laboratory staff at GR Micro Ltd.


    Notes
 
* Corresponding address. SmithKline Beecham Pharmaceuticals, 1250 S. Collegeville Road, UP1340, PO Box 5089, Collegeville, PA 19426-0989, USA. Tel: +1-610-917-6278; Fax: +1-610-917-4617 Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . National Committee for Clinical Laboratory Standards. (1995). Performance Standards for Antimicrobial Susceptibility Testing— Sixth Information Supplement M100-S6. NCCLS, Villanova, PA.

2 . Doern, G. V., Brueggemann, A., Holley, H. P. & Rauch, A. M. (1996). Antimicrobial resistance of Streptococcus pneumoniae recovered from outpatients in the United States during the winter months of 1994 to 1995: results of a 30-centre national surveillance study. Antimicrobial Agents and Chemotherapy 40, 1208– 13.[Abstract]

3 . Jones, R. N., Pfaller, M. A., Doern, G. V., Verhoff, J., Jones, M., Sader, H. S.et al . ( 1997). Initial report of a longitudinal, international antimicrobial surveillance study (SENTRY): alarming resistance rates in monitored sites (68 medical centres) in the USA, Canada, South America, and Europe. In Program and Abstracts of the Thirty-Seventh Interscience Conference on Antimicrobial Agents and Chemotherapy, Toronto, Canada, 1997. Abstract E-109, p. 133. American Society for Microbiology, Washington, DC.

4 . Pfaller, M. A., Jones, R. N., Doern, G. V., Kugler, K. & the SENTRY Participants Group. (1998). Bacterial pathogens isolated from patients with bloodstream infection: frequencies of occurrence and antimicrobial susceptibility patterns from the SENTRY antimicrobial surveillance program (United States and Canada, 1997). Antimicrobial Agents and Chemotherapy 42, 1762– 70.[Abstract/Free Full Text]

5 . Goldstein, F. W., Emirian, M. F., Guerrier, M. L. & Acar, J. F. (1992). Comparative activity of amoxycillin and cefotaxime against 392 penicillin-resistant and susceptible Streptococcus pneumoniae. In Program and Abstracts of the Thirty-Second Interscience Conference on Antimicrobial Agents and Chemotherapy, Anaheim, CA, 1992. Abstract 1025, p. 281. American Society for Microbiology, Washington, DC.

6 . Jorgensen, J. H., Doern, G. V., Maher, L. A., Howell, A. W. & Redding, J. S. (1990). Antimicrobial resistance among respiratory isolates of Haemophilus influenzae, Moraxella catarrhalis, and Streptococcus pneumoniae in the United States. Antimicrobial Agents and Chemotherapy 34 , 2075– 80.[ISI][Medline]

7 . Korgenski, E. K., Christenson, J. C. & Daly, J. A. (1997). Comparison of amoxycillin/clavulanate versus penicillin MICs for drug-resistant Streptococcus pneumoniae. In Abstracts of the Ninety-Seventh General Meeting of the American Society for Microbiology, Miami Beach, FL, 1997. Abstract C-295, p. 171. American Society for Microbiology, Washington, DC.

8 . Spangler, S. K., Jacobs, M. R. & Appelbaum, P. C. (1994). In vitro susceptibilities of 185 penicillin-susceptible and resistant pneumococci to WY-49605 (SUN/SY 5555), a new oral penem, compared with those to penicillin G, amoxycillin, amoxycillin-clavulanate, cefixime, cefaclor, cefpodoxime, cefuroxime, and cefdinir. Antimicrobial Agents and Chemotherapy 38, 2902– 4.[Abstract]

9 . Thornsberry, C., Ogilvie, P., Kahn, J., Mauriz, Y. & the Laboratory Investigator Group. (1997). Surveillance of antimicrobial resistance in Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis in the United States in 1996- 1997 respiratory season. Diagnostic Microbiology and Infectious Disease 29, 249– 57.[ISI][Medline]

10 . Trujillo, H., Callejas, R., Mejia, G. I. & Castrillon, L. (1989). Bacteriology of middle ear fluid specimens obtained by tympanocentesis from 111 Colombian children with acute otitis media. Pediatric Infectious Disease Journal 8, 361– 3.[ISI][Medline]

11 . Pankuch, G. A., Visalli, M. A., Jacobs, M. R. & Appelbaum, P. C. (1995). Activities of oral and parenteral agents against penicillin- susceptible and -resistant pneumococci. Antimicrobial Agents and Chemotherapy 39, 1499– 504.[Abstract]

12 . Gruneberg, R. N., Felmingham, D. & the Alexander Project Group. (1996). Results of the Alexander Project: A continuing, multicentre study of the antimicrobial susceptibility of community-acquired lower respiratory tract bacterial pathogens. Diagnostic Microbiology and Infectious Disease 25, 169– 81.[ISI][Medline]

13 . Schito, G. C., Mannelli, S., Pesce, A. & the Alexander Project Group. (1997). Trends in the activity of macrolide and ß -lactam antibiotics and resistance development. Journal of Chemotherapy 9, 18– 28.[ISI][Medline]

14 . Larson, H. J. (1982). Probability Theory and Statistical Inference. John Wiley and Sons.

15 . Wright, S. W. (1992). Adjusted P -values for simultaneous inference. Journal of the American Statistical Association 48, 1005– 13.

16 . Woodnutt, G. & Berry, V. (1999). Two pharmacodynamic models for assessing the efficacy of amoxicillin/clavulanate against experimental respiratory tract infections caused by strains of Streptococcus pneumoniae. Antimicrobial Agents and Chemotherapy 43, 29– 34.[Abstract/Free Full Text]

17 . Craig, W. A. (1998). Pharmacokinetic/pharmacodynamic parameters: rationale for antibacterial dosing of mice and men. Clinical Infectious Diseases 26, 1– 12.[ISI][Medline]

18 . Drusano, G. L. & Craig, W. A. (1997). Relevance of pharmacokinetics and pharmacodynamics in the selection of antibiotics for respiratory tract infections. Journal of Chemotherapy 9, Suppl. 3, 38– 44.[ISI][Medline]

Received 3 August 1998; returned 6 October 1998; revised 30 November 1998; accepted 10 February 1999