The Alexander Project 1998–2000: susceptibility of pathogens isolated from community-acquired respiratory tract infection to commonly used antimicrobial agents

Michael R. Jacobs1,*, David Felmingham2, Peter C. Appelbaum3, Reuben N. Grüneberg2 and the Alexander Project Group§

1 Department of Pathology, Case Western Reserve University and University Hospitals of Cleveland, 11100 Euclid Ave, Cleveland, OH 44106; 3 Hershey Medical Center, Hershey, PA, USA; 2 GR Micro, London, UK

Received 24 May 2002; returned 1 July 2002; revised 30 April 2003; accepted 7 May 2003


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objectives: The Alexander Project is a continuing surveillance study, begun in 1992, examining the susceptibility of pathogens involved in adult community-acquired respiratory tract infections (CARTI) to a range of antimicrobial agents.

Materials and methods: This study tested the susceptibility of isolates of Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis collected between 1998 and 2000 to 23 antimicrobials. Minimum inhibitory concentrations of agents were determined using the broth microdilution method and interpreted according to NCCLS and pharmacokinetic/pharmacodynamic (PK/PD) breakpoints.

Results: In total, 8882 isolates of S. pneumoniae, 8523 isolates of H. influenzae and 874 isolates of M. catarrhalis were collected during 1998–2000 from centres in 26 countries. The world-wide prevalence of penicillin resistance (penicillin MICs >= 2 mg/L) in isolates of S. pneumoniae was 18.2% over the study period, and the prevalence of macrolide resistance (erythromycin MICs >= 1 mg/L) in this pathogen was 24.6%. Over the study period, macrolide resistance exceeded penicillin resistance in 19 of the 26 countries included in the study. Of the non-fluoroquinolone agents, the only oral agents to which over 90% of S. pneumoniae isolates were susceptible at both NCCLS and PK/PD breakpoints were amoxicillin (95.1%) and co-amoxiclav (95.5–97.9%). The prevalence of fluoroquinolone-resistant S. pneumoniae (ofloxacin MICs >= 8 mg/L) was 1.1%. Gemifloxacin was the most potent fluoroquinolone tested against S. pneumoniae (99.9% susceptible). In isolates of H. influenzae, ß-lactamase production was 16.9%, whereas the prevalence of ß-lactamase-negative, ampicillin-resistant strains was low (0.2%). ß-Lactamase production in M. catarrhalis world-wide remained high over the period studied (92.1%). Using PK/PD breakpoints, the most active non-fluoroquinolone agents against H. influenzae were ceftriaxone (100% susceptible), cefixime (99.8%) and co-amoxiclav (98.1–99.6%). Co-amoxiclav, cefdinir and cefixime (100%) were the most active ß-lactams against M. catarrhalis. Both H. influenzae and M. catarrhalis were highly susceptible to the fluoroquinolones.

Conclusions: These data demonstrate the continued evolution of and geographical variation in bacterial resistance and highlight the need for appropriate prescribing of antimicrobials in CARTI, using agents with adequate activity, based on local susceptibility profiles and PK/PD parameters.

Keywords: surveillance, antimicrobial resistance, community-acquired respiratory tract infection, Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis are the major bacterial pathogens involved in community-acquired respiratory tract infections (CARTI).13 Choice of antimicrobial therapy in CARTI is usually empirical. However, this choice is complicated by the increasing prevalence of resistance amongst these three major bacterial pathogens. In Haemophilus and Moraxella species, ß-lactamase production is the principal resistance mechanism to penicillins. S. pneumoniae has acquired resistance to several classes of antimicrobial agent, including penicillins, macrolides, co-trimoxazole and fluoroquinolones, by a variety of mechanisms.4 It is, therefore, important to monitor the emergence and spread of antimicrobial resistance both geographically and over time.

Established in 1992, the Alexander Project is a continuing surveillance study examining the antimicrobial susceptibility of bacterial CARTI pathogens to a range of compounds, with testing undertaken in three central laboratories using standardized procedures.5 From 1992 to 1995, isolates were collected from centres in a number of European Union countries and states in the USA. In 1996, the study extended to centres in Mexico, Brazil, South Africa, Saudi Arabia, Hong Kong and further European countries,6,7 with centres in Israel, Japan, Kenya, Russia and Singapore added in 1998 and 1999. Antimicrobial susceptibility data for 1998–2000 for the three major CARTI pathogens are now presented.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Collaborating centres

In 1998–2000, the following centres took part in the study. Africa: Kenya, Nairobi; South Africa, Johannesburg. Eastern Europe: the Czech Republic, Prague; Poland, Warsaw; Russia, Moscow; the Slovak Republic, Bratislava. Western Europe: Austria, Vienna; Belgium, Leuven; France, Toulouse, Paris, Nancy, Lille, Strasbourg; Germany, Weingarten, Berlin; Greece, Athens; Italy, Genoa; The Netherlands, Maastricht; Portugal, Lisbon; the Republic of Ireland, Dublin; Spain, Barcelona; Switzerland, Basel, Berne, Geneva, La Chaux des Fonds, Lausanne, Lugano, Sion, St Gallen, Zurich; UK, London. Far East: Hong Kong, Pokfulam; Japan, Tokyo, Nagasaki, Kanagawa, Sendai City; Singapore. Middle East: Israel, Ramat-Gan; Saudi Arabia, Riyadh. Latin America: Brazil, Sao Paulo; Mexico, Mexico City, Baja California, Monterrey, Durango. USA: 30 centres in 23 states were included in 1998, 16 centres in 11 states in 1999 and 23 centres in 20 states in 2000. The investigators for each of these regions are listed in the Acknowledgements. Some centres contributed isolates from more than one hospital and region, and there was no restriction as to the type of hospital or laboratory that could contribute strains.

Bacterial isolates and antimicrobial susceptibility testing

Inclusion criteria for bacterial isolates, and methods used for transportation, storage and confirmation of identification of isolates, as well as testing methods have been described in detail, previously.57 Briefly, strains isolated from the sputum or blood of adult patients with CARTI were included. Duplicate isolates from the same patient were not accepted. Isolates were accepted from hospitalized patients only if they were cultured from samples obtained in the first 48 h after admission. Centres were requested to collect up to 400 isolates. After isolation, identification and susceptibility testing at the participating laboratory, stored isolates were sent to one of three central testing centres for confirmation of identity and standardized susceptibility testing. Susceptibility testing was carried out by the NCCLS broth microdilution method,8 using dried commercial plates (Sensititre, Trek Diagnostics, East Grinstead, UK) reconstituted with Mueller–Hinton broth with 5% lysed horse blood for S. pneumoniae and Haemophilus Test Medium for H. influenzae and M. catarrhalis. The antimicrobials tested against S. pneumoniae included (concentration ranges tested in mg/L): penicillin (0.015–8), amoxicillin (0.015–16), co-amoxiclav (0.015–16), cefaclor (0.5–64), cefuroxime (0.015–16), cefixime (0.12–16), ceftriaxone (0.015–4), cefprozil (0.12–64), cefdinir (0.016–16), erythromycin (0.015–32), clarithromycin (0.015–32), azithromycin (0.03–32), clindamycin (0.015–2), doxycycline (0.06–8), chloramphenicol (1–16), co-trimoxazole (0.06–8, trimethoprim component 1:19 ratio with sulfamethoxazole), ciprofloxacin (0.25–16), ofloxacin (0.25–16), gemifloxacin (0.004–0.5), levofloxacin (0.12–2), gatifloxacin (0.03–1) and moxifloxacin (0.03–1). The antimicrobials tested against H. influenzae and M. catarrhalis included (concentration ranges tested in mg/L): ampicillin (0.12–16), amoxicillin (0.12–16), co-amoxiclav (0.12–16), cefaclor (0.5–64), cefuroxime (0.12–32), cefixime (0.008–16), ceftriaxone (0.004–2), cefprozil (0.5–64), cefdinir (0.06–16), erythromycin (0.5–64), clarithromycin (0.5–64), azithromycin (0.12–64), doxycycline (0.06–8), chloramphenicol (0.12–32), co-trimoxazole (0.015–4, trimethoprim component 1:19 ratio with sulfamethoxazole), ciprofloxacin (0.002–1), ofloxacin (0.008–2), gemifloxacin (0.001–0.5), levofloxacin (0.004–0.5), gatifloxacin (0.0002–0.5) and moxifloxacin (0.001–0.5). Breakpoint concentrations used to interpret MIC data qualitatively were those published by NCCLS8 as well as pharmacokinetic/pharmacodynamic (PK/PD) breakpoints,915 and are indicated in Table 1.


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Table 1.  Breakpoints (mg/L) used to determine susceptible (S), intermediate (I) and resistant (R) categories, based on PK/PD and NCCLS 2002 interpretative breakpoints8–15
 
Quality control and quality assurance

Quality control strains recommended by the NCCLS were used on each day of testing, and results of testing carried out were accepted if results of control strains were within published limits. In addition, each lot of dried panels was tested with a battery of 50 isolates of S. pneumoniae and H. influenzae. These results were compared with those obtained with reference frozen panels, and lots of dried trays were accepted if cumulative MIC plots were comparable to those obtained in the frozen trays. Additionally, growth properties of media used to reconstitute microdilution trays were monitored and used only if performance was satisfactory. ß-Lactamase-negative, ampicillin-resistant (BLNAR, ampicillin MIC >= 4 mg/L) and ß-lactamase-positive, co-amoxiclav-resistant (BLPACR, co-amoxiclav MIC >= 2 mg/L) isolates were retested.16


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Streptococcus pneumoniae

Antimicrobial susceptibility: A total of 8882 isolates of S. pneumoniae submitted from centres in 26 countries were tested. The antimicrobial susceptibility of world-wide isolates of S. pneumoniae and MIC50/90s against this pathogen are shown in Table 2. Based on NCCLS breakpoints, the most active non-fluoroquinolone antimicrobials against S. pneumoniae were co-amoxiclav, amoxicillin and ceftriaxone (>95% susceptibility). Cefaclor was the least active ß-lactam with only 60.2% of isolates susceptible to this agent. Susceptibility to erythromycin, clarithromycin and azithromycin was similar (75.3–75.5%). Ofloxacin was the least active fluoroquinolone and gemifloxacin the most active in this class (92.7% and 99.9% susceptibility, respectively). Chloramphenicol was more active than doxycycline which was more active than co-trimoxazole (88.1%, 71.3% and 63.3% susceptibility, respectively).


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Table 2.  Susceptibility of world-wide isolates of S. pneumoniae, H. influenzae and M. catarrhalis to 23 antimicrobials and MIC50s and MIC90s, Alexander Project 1998–2000
 
Table 3 shows antimicrobial MIC50/90s from centres in each country. There was considerable variation in MIC50/90s for all of the ß-lactams. For the macrolides, MIC90s were >=16 mg/L in 16 of the 26 countries included. Chloramphenicol MIC90s were >=8 mg/L for 12 countries and doxycycline MIC90s were >=4 mg/L except for centres in Germany, The Netherlands and the UK (<=0.5 mg/L). Co-trimoxazole MIC90s were >=1 mg/L in all countries. Ofloxacin MIC90s were <=2 mg/L in most countries, but were 4 mg/L in Italy, Spain, Japan and Israel, and >8 mg/L in Hong Kong.


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Table 3.  MIC50/MIC90 (mg/L) for antimicrobials tested against S. pneumoniae, Alexander Project 1998–2000
 
The susceptibility of S. pneumoniae to the antimicrobials tested for centres in each country is shown in Table 4. The prevalence of penicillin resistance exceeded 10% in centres in 14 of the 26 countries included and was highest in Hong Kong (69.9%) and lowest in The Netherlands and Brazil (1.1%) (Figure 1). The susceptibility of S. pneumoniae to penicillin was >=90% for centres in the Czech Republic, Russia, Belgium, Germany, Italy and The Netherlands, with Hong Kong recording the lowest susceptibility (26.4%). The most active oral ß-lactam agents overall were amoxicillin (95.1% susceptible) and co-amoxiclav (95.5% for current formulations and 97.9% for 2000/125 mg twice daily formulation). The difference between the coverage of the two formulations of co-amoxiclav was particularly prominent in Hong Kong (81.9% for current formulations versus 100% for 2000/125 mg twice daily formulation). The prevalence of erythromycin resistance exceeded that of penicillin resistance in 19 of the 26 participating countries (Table 4 and Figure 1). The susceptibility of S. pneumoniae to erythromycin was >=90% for centres in Kenya, the Czech Republic, Poland, Russia, Austria, Germany, The Netherlands and Brazil, with Hong Kong recording the lowest susceptibility (19.7%) (Table 4 and Figure 1). Centres in 13 of the 26 countries reported S. pneumoniae susceptibilities of >=90% for chloramphenicol, compared with only The Netherlands and UK for doxycycline and no countries for co-trimoxazole. Susceptibility to ofloxacin was <90% in Italy, Spain Japan, Israel, and lowest in Hong Kong (77.7%) (Table 4).


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Table 4.  Susceptibility (%) of S. pneumoniae to antimicrobials (susceptible [S], intermediate [I] and resistant [R] shown for penicillin) using NCCLS breakpoints except for co-amoxiclav 2000/125 mg, doxycycline, co-trimoxazole and gemifloxacin for which PK/PD breakpoints were used (see Table 1), Alexander Project 1998–2000
 


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Figure 1. Comparative prevalences of erythromycin (filled bars) and penicillin resistance (open bars) in S. pneumoniae (MICs >=0.5 and >=2 mg/L, respectively). Alexander Project 1998–2000.

 
Cross-resistance and co-resistance: Overall, 11.5% of penicillin-resistant strains were also resistant to amoxicillin, though this varied from 0% in Belgium, the Republic of Ireland, UK, Hong Kong and Saudi Arabia to 21.3% in the USA (Table 5). The proportion of penicillin-resistant S. pneumoniae that were also erythromycin-resistant varied from 7.7% in the Republic of Ireland to 97.0% in Hong Kong and Singapore (Table 5). Cross-resistance between clindamycin (MIC > 0.5 mg/L) and erythromycin can be used as an approximation of the prevalence of the erm(B)-mediated methylation mechanism (MLSB-phenotype resistance: resistant to macrolides and clindamycin) versus the mef(A)-mediated efflux mechanism (M-phenotype resistance: resistant to macrolides but susceptible to clindamycin) of S. pneumoniae macrolide resistance.1719 Cross-resistance between erythromycin and clindamycin was at least 50% in all countries except the UK (48.4%), Hong Kong (31.0%), Singapore (38.8%), Saudi Arabia (34.4%) and the USA (32.2%), suggesting that a greater proportion of isolates from centres in these countries expressed the mef(A) resistance mechanism (Table 5). The proportion of penicillin-resistant strains that were also resistant to chloramphenicol was 39.2% overall, ranging from 9.1% in Israel to 85.9% in Hong Kong (Table 5). Co-resistance between penicillin and doxycycline was 61.2% overall (range 22.7% in Israel to 97.0% in Singapore) and between penicillin and co-trimoxazole, 89.6% of isolates displayed co-resistance (range 52.2% in Japan to 100% in the Slovak Republic, Italy and the Republic of Ireland). A total of 98/8882 S. pneumoniae isolates (1.1%) were resistant to ofloxacin (MIC >= 8 mg/L) between 1998 and 2000. The majority of these isolates came from centres in the Far East (44), the USA (30) and Western Europe (15) (Table 5). In the Far East, 72.2% of ofloxacin-resistant strains were also resistant to penicillin and 93.1% to erythromycin compared with 20.0% and 40.0% for the USA, respectively. The MIC distributions for the fluoroquinolones tested against ofloxacin-resistant isolates are shown in Table 6 and indicate that the majority of these strains (>=77.1%) were non-susceptible to all the fluoroquinolones tested except for gemifloxacin (10.2% non-susceptible).


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Table 5. Cross-resistance (%) between amoxicillin and penicillin, and erythromycin and clindamycin; and co-resistance (%) between penicillin and erythromycin, doxycycline or co-trimoxazole in S. pneumoniae. Country of isolation for ofloxacin-resistant S. pneumoniae is also shown, Alexander Project 1998–2000
 

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Table 6.  MIC distribution (number of isolates inhibited) and comparative in vitro activity of fluoroquinolone antimicrobials against S. pneumoniae ofloxacin-resistant isolates (MICs >= 8 mg/L)
 
Multidrug resistance: Six different drug classes were tested against S. pneumoniae: ß-lactams, macrolides, a tetracycline, a phenicol, a folate pathway inhibitor and fluoroquinolones (Table 7). The prevalence of strains multidrug-resistant to any three of erythromycin (>=0.5 mg/L), doxycycline (>=0.5 mg/L), chloramphenicol (>=8 mg/L), co-trimoxazole (>=1 mg/L) and ofloxacin (>=8 mg/L) was 17.5% overall, ranging from 0% in centres in The Netherlands to 76.2% for Hong Kong centres. When this was expanded to resistance to any three classes of the six tested, including penicillin (MIC >= 0.12 mg/L), then 23.7% of isolates were multidrug-resistant (range The Netherlands and the Czech Republic 1.1% to Hong Kong 79.3%). The prevalence of multidrug resistance defined by resistance to any four of the six classes was 14.6% overall, and of resistance to five or more of the six classes was 5.9% overall (Table 7).


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Table 7. Prevalence of S. pneumoniae non-susceptible to three or more drug classes, Alexander Project 1998–2000 
 
Haemophilus influenzae

Antimicrobial susceptibility: A total of 8523 isolates of H. influenzae were tested. Using PK/PD breakpoints, ceftriaxone, cefixime, co-amoxiclav and chloramphenicol were the most active of the non-fluoroquinolone agents tested (100%, 99.8%, 98.1–99.6% and 98.1% susceptibility, respectively) (Table 2). Although NCCLS breakpoints for cefaclor, clarithromycin and azithromycin show 89.7%, 79.6% and 99.5% of H. influenzae susceptible to these agents, respectively, based on PK/PD breakpoints, the susceptibility of H. influenzae to cefaclor, the macrolides and azithromycin was <=1.4%. H. influenzae was highly susceptible (>=99.8%) to all the fluoroquinolones (Table 2). Table 8 provides the MIC50/90s for the antimicrobials tested against H. influenzae for centres in each country.


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Table 8.  MIC50/MIC90 (mg/L) for antimicrobials tested against H. influenzae, Alexander Project 1998–2000
 
The principal mechanism of aminopenicillin resistance observed in H. influenzae was ß-lactamase production (Figure 2). Overall, 16.9% of H. influenzae isolates were ß-lactamase positive. Fourteen of the 26 countries included in the study had prevalences of ß-lactamase-positive H. influenzae in excess of 10%, with centres in Russia having the lowest (4.2%) and centres in the USA the highest (29.6%) prevalences. The effect of ß-lactamase production can be seen in Table 2, with 81.6% of isolates susceptible to amoxicillin versus 98.1–99.6% susceptible to co-amoxiclav, and this enzyme is responsible for most of the variations in susceptibility between countries for the penicillins (Table 9). Twenty-one BLNAR isolates were identified in 1998–2000. Twelve were isolated from centres in Japan, three were from the USA and there was one each isolated from The Netherlands, Spain, UK, Hong Kong, Singapore and Saudi Arabia. There were six BLPACR strains isolated over the study period, five from Japan and one from Hong Kong.



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Figure 2. Prevalence of co-trimoxazole resistance (MIC >= 1 mg/L) (filled bars) and ß-lactamase production (open bars) in H. influenzae, Alexander Project 1998–2000.

 

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Table 9.  Susceptibility (%) of H. influenzae to antimicrobials tested based on PK/PD breakpoints (refer to Table 1 for further information), Alexander Project 1998–2000 
 
There was considerable variation in the prevalence of co-trimoxazole resistance between countries, from 8.5% for centres in Belgium to 55.2% in Kenya (Table 9, Figure 2). Co-trimoxazole resistance exceeded ß-lactamase production in 19 of the 26 participating countries, with centres in Russia, Poland, South Africa, Brazil and Kenya having prevalences of co-trimoxazole resistance more than four times that of ß-lactamase production (Figure 2). The prevalence of chloramphenicol resistance in H. influenzae was the highest for centres in Hong Kong at 9.2%.

Moraxella catarrhalis

ß-Lactamase production was the primary aminopenicillin resistance mechanism in M. catarrhalis isolates, with 92.1% of 874 isolates tested being ß-lactamase positive. There was little variation between countries in the susceptibility of M. catarrhalis to the antimicrobials tested or in the prevalence of ß-lactamase production, so results are not presented for each country. All M. catarrhalis isolated were susceptible to co-amoxiclav, cefixime, cefdinir, chloramphenicol, gatifloxacin and moxifloxacin (Table 2). Cefaclor and cefprozil were the least active agents tested against this pathogen (10.9% and 16.0% susceptibility, respectively) (Table 2). Although 72.0% of isolates were susceptible to co-trimoxazole, MICs clustered around the breakpoint.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The Alexander Project data presented here add to those reported from previous years and show the trend in many countries towards high prevalences of antimicrobial resistance in the major RTI pathogens from adults with lower RTIs.57,20,21 Major changes since 1997 are highlighted in this discussion. However, as the results for 1998–2000 have been combined in this paper, significant changes that have occurred during the 1998–2000 period are also noted.

Over the 3 year study period, the susceptibility of the pathogens tested has been generally stable with the following exceptions. (1) Penicillin resistance in S. pneumoniae increased significantly in Poland between 1998 (2.2%) and 1999/2000 (10.7%) (P = 0.001), Portugal between 1998/1999 (8.8%) and 2000 (16.7%) (P = 0.04), South Africa between 1998 (7.3%) and 1999/2000 (20.7%) (P = 0.013), Spain between 1998 (22.3%) and 1999 (37.4%) (P = 0.016) and the USA between 1998 (22.2%) and 1999 (31.0%) (P < 0.001); significant decreases were seen in Spain between 1999 (37.4%) and 2000 (20.8%) (P = 0.009), the UK between 1998 (17.6%) and 1999/2000 (7.2%) (P = 0.013) and the USA between 1999 (31.0%) and 2000 (22.7%) (P < 0.001). (2) Macrolide resistance in S. pneumoniae increased significantly in Israel between 1998 (9.3%) and 1999 (20.5%) (P = 0.045), Mexico between 1999 (18.3%) and 2000 (28.9%) (P = 0.04), Spain between 1998 (19.4%) and 1999/2000 (28.6%) (P = 0.016) and the USA between 1998 (25.1%) and 1999 (34.6%) (P < 0.001); significant decreases were seen in Switzerland between 1998 (19.2%) and 1999/2000 (9.4%) (P = 0.008) and the USA between 1999 (34.6%) and 2000 (26.8%) (P < 0.001). (3) Co-trimoxazole resistance in S. pneumoniae increased significantly in the USA between 1998 (35.2%) and 1999 (43.4%), but then decreased to 34.1% in 2000 (P < 0.001). (4) ß-Lactamase production in H. influenzae increased significantly in France between 1998 (20.3%) and 1999/2000 (30.8%) (P = 0.018); but decreased significantly in Spain between 1998 (28.2%) and 1999/2000 (16.4%) (P = 0.014) and the USA between 1998/1999 (31.0%) and 2000 (25.9%) (P = 0.012). (5) Chloramphenicol resistance in H. influenzae increased in Italy between 1998/1999 (0%) and 2000 (4.2%) (P = 0.01) and South Africa between 1998 (0%) and 1999/2000 (5.0%) (P = 0.023). (6) Co-trimoxazole resistance in H. influenzae increased significantly in Brazil between 1998 (47.5%) and 1999 (71.1%) (P = 0.013), Mexico between 1999 (33.3%) and 2000 (47.1%) (P = 0.05) and South Africa between 1998/1999 (35.0%) and 2000 (47.2%) (P = 0.019); significant decreases were seen in Brazil between 1999 (71.1%) and 2000 (46.8%) (P = 0.007), Germany between 1998/1999 (27.2%) and 2000 (12.9%) (P = 0.001), Poland between 1998 (28.0%) and 1999 (19.5%) (P = 0.0027), the Slovak Republic between 1998 (17.8%) and 1999/2000 (9.3%) (P = 0.03), Spain between 1998/1999 (44.7%) and 2000 (28.0%) (P = 0.004) and Switzerland between 1998 (23.4%) and 1999/2000 (13.1%) (P = 0.02).

Centres in nine of the 18 countries also included in the Alexander Project in 19976 have had increases of at least 5% in the prevalence of penicillin-resistant S. pneumoniae (based on the proportion of penicillin-resistant isolates to that of -susceptible plus -intermediate strains), and five of those had significant increases of 10% or more (South Africa, P = 0.003; Saudi Arabia, P = 0.03; Mexico, P = 0.016; France, P = 0.005; Hong Kong, P = 0.04). For centres in France and Hong Kong, these increases add to already high penicillin resistance prevalences and are part of an established trend.6,21 For centres in Mexico, Saudi Arabia and South Africa, trend data are only available from 1996, but nevertheless show significant increases in penicillin resistance.21 Of note is that, in 1996 and 1997, centres in South Africa had a low (<=4.5%) prevalence of penicillin resistance, which remained low in 1998 (7.3%), but increased thereafter (24.8% in 1999 and 15.4% in 2000).6,21 A similar trend has been seen in pneumococci isolated from blood and cerebrospinal fluid (CSF) in this country.22

The prevalence of erythromycin-resistant S. pneumoniae increased by at least 5% in six of 18 countries compared with 1997, with two countries showing increases of more than 10% (South Africa, P < 0.001; USA, P < 0.001).6 In the USA, the significant increase in erythromycin resistance from 16.9% in 1997 to 28.6% in 1998–2000 is confirmed by the SENTRY data set, which reported a significant increase in erythromycin resistance between 1997–1998 (9.8–15.9%) and 1999 (23.7%).23 In South Africa, between 1991 and 1998, erythromycin resistance in pneumococci from blood and CSF increased from 10.6% to 28.7%,22 consistent with the approximate three-fold increase observed in this study between 1996 and 1998–2000. In contrast, the prevalence of erythromycin resistance was similar between 1997 and 1998–2000 for centres in the Czech Republic (2.2% and 1.5%, respectively) and Germany (6.5% and 6.9%, respectively).6

Penicillin resistance in S. pneumoniae is a pharmacokinetic issue and can be overcome with appropriate dosing regimens.9,11,13,14,24 The majority of isolates were found to be susceptible to ceftriaxone (95.1% for non-meningeal infections), amoxicillin (95.1%), current formulations of co-amoxiclav (95.5%) and the new formulation of co-amoxiclav (2000/125 mg twice daily) (97.9%). In particular, the new formulation of co-amoxiclav may be clinically useful in countries where the proportion of strains susceptible to the new formulation compared to current formulations increased, with a 3.3–4.5% increase found in South Africa, France, Republic of Ireland, Spain, Israel, Saudi Arabia and Mexico, and an 18.1% increase in Hong Kong (Table 4). In contrast, resistance to other drug classes is generally absolute, and the overall prevalences of both erythromycin resistance and co-trimoxazole resistance were high (24.6% and 24.8%, respectively) in S. pneumoniae. In addition, the prevalence of erythromycin resistance exceeded that of penicillin resistance in the majority of countries included in the study. Erythromycin resistance was also seen in penicillin-susceptible isolates, particularly in Hong Kong, Japan and Italy. Overall, the prevalence of both penicillin- and erythromycin-resistant S. pneumoniae was highest in Hong Kong and Singapore, and this is consistent with findings from other surveillance studies.25,26 Forty-three percent of all erythromycin-resistant S. pneumoniae in this study were of the M phenotype (erythromycin resistant, clindamycin susceptible) with Hong Kong, Singapore, Saudi Arabia and the USA having higher prevalences of M than MLS phenotypes, with similar findings reported in other studies.23,27

The prevalence of S. pneumoniae isolates with elevated ofloxacin MICs (>=8 mg/L) increased in the Alexander Project from 11 isolates (0.54%) in 1997 to 98 isolates (1.1%) in 1998–2000 (P = 0.01).6 Although the number of isolates is small, these data are consistent with other reports indicating a trend for decreasing susceptibility of S. pneumoniae to fluoroquinolones.23,28 As the majority of strains with decreased ofloxacin susceptibility were isolated from centres in the USA, Western Europe, Japan and Hong Kong, the emergence of fluoroquinolone resistance should be monitored closely in these locations. In the Far East, the observed association of ofloxacin resistance with penicillin and erythromycin resistance in S. pneumoniae is a cause for concern.29 In the current study, some ofloxacin-resistant S. pneumoniae from the USA also demonstrated co-resistance with penicillin (20.0%) and erythromycin (40.0%).

ß-Lactamase production remains the most important mechanism of resistance to aminopenicillins expressed by isolates of H. influenzae, with considerable geographical variation.25 Compared with 1997 Alexander Project data, the prevalence of ß-lactamase-positive H. influenzae was stable (<5% change) for the majority (14/18) of countries.6 The prevalence of ß-lactamase-positive isolates fluctuated considerably in the UK, being between 6% and 17% from 1992 to 1996, falling to 6.3% in 1997, then rising to between 13% and 22% from 1998 to 2000.6,21 Centres in France (8.5% increase, P < 0.001) and the USA (6.3% increase, P = 0.03) also showed increases of more than 5%, and this is in line with previous trends.6,21 Centres in Spain had the only major decrease, falling from 31.7% to 20.5% (P = 0.001) and continuing the downward trend seen in this country since 1992.6,21 The evolution of ß-lactamase production in M. catarrhalis has continued with overall prevalence increasing from 75.7% in 1992 to 92.1% in 1998–2000.57,21

BLNAR strains of H. influenzae remain rare (0.2% in 1998–2000). This type of resistance occurs as a result of changes in the affinities of penicillin-binding proteins (PBPs), particularly PBP 3A and PBP 3B.30 However, some isolates of H. influenzae with ampicillin MICs of 0.5–2 mg/L also exhibit changes in these PBPs, indicating a step-wise acquisition of resistance analogous to that seen in S. pneumoniae.31 A small number of BLPACR strains were also isolated (six strains [0.1%] in 1998–2000), five from Japan and one from Hong Kong.

Overall, the data presented here are consistent with other multi-national and national surveillance projects over approximately the same time period. Additional factors, such as adaptive or ‘fitness’ costs to the bacteria, the development of herd immunity in the human population, cross-infection and differences in the epidemiology across regions and between settings (e.g. daycare centres) will also influence resistance.32

One application of surveillance data is in attempting to define the relationship between antimicrobial usage and resistance. A comparison of resistance prevalences reported here and antimicrobial usage data from 11 European countries showed a generally consistent relationship between macrolide use and erythromycin resistance in S. pneumoniae.33 However, in Portugal, Italy and Belgium there was a dissociation between high broad-spectrum penicillin usage and penicillin resistance in this pathogen.33 Baquero & Negri examined why, despite high levels of both penicillin and cephalosporin usage in Italy, the prevalence of ß-lactam (penicillin)-resistant S. pneumoniae was relatively low.34 These authors hypothesized that the low prevalence of penicillin-resistant S. pneumoniae in Italy was due to the widespread use of parenteral rather than oral cephalosporins in this country.34 Oral cephalosporins have been found to be more effective at selecting for penicillin resistance in S. pneumoniae than aminopenicillins in France.35 France had the highest prevalence of penicillin-resistant and erythromycin-resistant S. pneumoniae as well as the highest total antimicrobial usage, broad-spectrum penicillin and macrolide usage, and the second highest cephalosporin usage of the 11 European countries studied (based on 1997 data).33 In the current data set, Italy has a prevalence of erythromycin-resistant S. pneumoniae (34.9%), that is more than eight times that of penicillin-resistant strains (Figure 1), consistent with a 1997–1999 national surveillance study.36 Italy was ranked third highest in macrolide consumption and this corresponds with a ranking of second highest for macrolide resistance in the Alexander Project.33

Past data from the Alexander Project have indicated a strong correlation (R = 0.896) between the use of long-acting macrolides and erythromycin resistance in S. pneumoniae, whereas the relationship between short-acting macrolides and resistance prevalence was very weak (R = –0.099).37 Differences in the pharmacokinetic properties of the agents are thought to play a role in this; long-acting macrolides, with their increased half-lives, provide a significant ‘selection window’ where sub-inhibitory concentrations allow the selection of resistant clones.9

Empirical prescribing of antimicrobial therapy requires the use of agents that have adequate coverage of all of the three major respiratory pathogens.38 However, as the prevalence of antimicrobial resistance has increased, the therapeutic options available to physicians have become restricted. For example, of the non-fluoroquinolone agents included in this study, amoxicillin and co-amoxiclav were the only oral agents to which >90% of S. pneumoniae strains were susceptible. The lowest susceptibility was seen for cefaclor, co-trimoxazole and cefixime (all <70%).

The impact of antimicrobial resistance on clinical efficacy is debated.3941 For agents such as parenteral penicillin G, doses can be increased to overcome resistance in non-meningeal infections, and there is little evidence that resistance has had a negative impact on clinical outcome of pneumonia and bacteraemia where dosing is sufficient.40,41 For oral ß-lactams, the application of PK/PD principles has greatly assisted our interpretation of MICs in relation to clinical outcomes and has also been used to optimize doses to maintain bacteriological efficacy against strains with resistance mechanisms and increased MICs.38 For the macrolides, however, there is emerging evidence that macrolide resistance in S. pneumoniae, due to both erm and mef mechanisms, results in clinical failure both in otitis media and in pneumonia.24,39,40,42 The susceptibility of S. pneumoniae to erythromycin was above 90% in only eight of the 26 countries included in the study. Given these data, for the majority of countries in the Alexander Project, macrolide antimicrobials would not be appropriate for the empirical therapy of infections for which S. pneumoniae is a major pathogen. Of the fluoroquinolones, gemifloxacin was the most potent agent in vitro against S. pneumoniae. The fluoroquinolones ciprofloxacin and ofloxacin have marginal activity against Gram-positive pathogens, such as S. pneumoniae, due to pharmacokinetic limitations, and treatment failures with these older quinolones are well documented.40 More recently, treatment failures in community-acquired pneumonia and the emergence of resistance on-therapy have been described for levofloxacin.43 With the more widespread use of fluoroquinolones, both the prevalence of resistance and the frequency of treatment failures are likely to increase.43

Based on NCCLS and PK/PD breakpoints, ceftriaxone, cefixime and co-amoxiclav were the most active ß-lactams against H. influenzae, with susceptibility consistently >99% for the 3 years reported. Using breakpoints based on PK/PD parameters, cefprozil and particularly cefaclor were the least active ß-lactams against H. influenzae. In addition, the use of PK/PD breakpoints indicates that very few H. influenzae isolates are susceptible to the macrolides (<=1.2%) or doxycycline (28.9%). This finding is consistent with clinical data indicating bacteriological failures associated with macrolide therapy in otitis media.39,44 Fluoroquinolone MICs for H. influenzae were low, and resistance to this class of agent was found very infrequently.

As with most other multi-national surveillance projects, the Alexander Project has a limited number of centres contributing isolates from each country. For the USA, a large number of centres distributed across the country were included, but for most of the other countries, only one or very few centres were involved and local and regional differences may not be adequately described. This is an issue even with national surveillance studies where regional differences have been found,27 and locally generated data should be used wherever possible.38

By the end of 2001, the Alexander Project will have collected antimicrobial susceptibility data for 10 years. The data collected demonstrate increasing antimicrobial resistance in many countries and highlight geographical and temporal changes in resistance phenotypes. This surveillance study is part of the international effort to monitor and track changes in antimicrobial susceptibility in order to maintain the clinical utility of current agents, suggest targets for new therapeutic strategies, highlight the increasing problem of antimicrobial resistance and promote the application of surveillance data to clinical decision making.38


    Acknowledgements
 
We acknowledge Laura Koeth for coordinating collecting sites and transport of isolates; Saralee Bajaksouzian, Christine Dencer, Gengrong Lin, Glenn Pankuch, Marion J. Robbins and Anne Windau for expert technical assistance; Ian Harding and Vaughan Reed for data analysis; GlaxoSmithKline for support of the project; and Naomi Richardson for assistance with manuscript preparation. The Alexander Project is supported by an unrestricted educational grant from GlaxoSmithKline.

The contributions of the following Alexander Group investigators are acknowledged. Africa: S. Kariuki, K. Klugman. Eastern Europe: P. Urbáková, W. Hryniewicz, S.V. Sidorenko, H. Upková. Western Europe: U. Theuretzbacher, J. Verhaegen, J. Lemozy, H. Dabernat, F. Goldstein, M. Weber, M. Roussel-Delvallez, F. Jehl, H. Grimm, M. Seewald, H. Mauch, H. Giamarellou, G.C. Schito, E.E. Stobberingh, J.-M. Cristino, E. Smyth, A. Gilleece, L. Fenelon, J. Liñares, J. Bille, D. Felmingham. Far East: S. Wing Hong, K. Yamaguchi, S. Kono, M. Inoue, M. Kaku, M. Yeo, R. Lin. Middle East: N. Keller, A.M. Shibl. Latin America: C. Mendes, J. Sifuentes-Osornio, F. Quiñones, L.E. Espinosa, G. Alvarez, R. Ma. Hinojosa, J.C. Tinoco, F. Ojeda. USA: B. Grover, S. Gamble, M. Bay, D. Lamb, S. Munroe, G. Teskie, P. Wong, S. Cyprian, T. Cleary, M. Rivera, C. Watkins, H. Phillips, D. Prince, S. Walker, M. Beard, R. Carey, B. Droege, J. Tjhio, G. Denys, R. Cheek, G. Munier, B. Cato, W. Eppling, D. Cosmidis, D. DeMarco, L. McDermott, D. Schwartz, M. Welty, R. VanEnk, J. Loomis, L. McClure, L. Temme, S. Matthey, M. Hostetter, L. Buck, G. Overturf, R. Cammarata, S. Jenkins, L. Rosenstein, J.R. DiPersio, M. Jacobs, C. Hogan, B. Rourke, L. Kaufmann, J. Griffin, B. Smith, L. Brown, B. Cavagnolo, N. Lee, L. Mann, K. Korgenski, K. Hazen, W. Winn, J. Claridge, M. Coyle, M. Patera, J. Quick, M. Schmitz.


    Footnotes
 
* Corresponding author. Tel: +1-216-844-3484; Fax: +1-216-844-5601; E-mail: mrj6{at}po.cwru.edu Back

§ The Alexander Study group participants are listed in the Acknowledgements. Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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