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Antimicrobial resistance among invasive isolates of Streptococcus pneumoniae collected across Canada
Diagnostic Microbiology and Infectious Disease 59 (2007) 75 – 80 www.elsevier.com/locate/diagmicrobio
Antimicrobial Susceptibility Studies
Antimicrobial resistance among invasive isolates of Streptococcus pneumoniae collected across Canada☆ Ross J. Davidsona,b,c,d,⁎, Roberto Melanoa , Canadian Invasive Pneumococcal Disease Surveillance Group 1 , Kevin R. Forwarda,b,c,d a Queen Elizabeth II Health Sciences Centre, Dalhousie University, Halifax, Nova Scotia, Canada Department of Microbiology and Immunology, Dalhousie University, Halifax, Nova Scotia, Canada c Department of Pathology and Laboratory Medicine, Dalhousie University, Halifax, Nova Scotia, Canada d Department of Medicine, Dalhousie University, Halifax, Nova Scotia, Canada Received 6 December 2006; accepted 25 March 2007 b
Abstract Between 2002 and 2003, 736 nonduplicate Streptococcus pneumoniae isolated from blood cultures were collected from 7 of 10 Canadian provinces (10 tertiary care centers). Microdilution broth susceptibility testing was performed using the method prescribed by the Clinical Laboratory Standards Institute. Of the isolates, 16.85% were nonsusceptible to penicillin and 5.4% were highly resistant. Of the S. pneumoniae, 14.1% had reduced susceptibility to erythromycin and 47% had been accounted for by the M phenotype. No isolates were recovered that were resistant to telithromycin. Only 6 isolates were resistant to levofloxacin and gatifloxacin. Of these, 5 strains had intermediate susceptibility to moxifloxacin and 1 was considered susceptible. The rates observed in this study are in keeping with previous surveillance studies among noninvasive isolates. © 2007 Elsevier Inc. All rights reserved. Keywords: Invasive S. pneumoniae; Antibiotic resistance; Surveillance
1. Introduction Streptococcus pneumoniae remains the leading bacterial cause of upper and lower community-acquired respiratory infections in both children and adults (File and Tan 1997; Georges et al., 1999; Mandel et al., 2007). S. pneumoniae is also the leading cause of bacterial meningitis and is associated with significant morbidity and mortality (Sinner ☆ This study was supported by an unrestricted grant from Aventis Pharmaceuticals, Canada. ⁎ Corresponding author. Queen Elizabeth II Health Sciences Centre, Halifax, Nova Scotia, Canada B3H-1V8. Tel.: +1-902-473-5520; fax: +1902-473-4432. E-mail address: [email protected] (R.J. Davidson). 1 Members of the Canadian Invasive Pneumococcal Disease Surveillance Group: Daryl Hoban, Winnipeg, Manitoba; Zafar Hussain, London, Ontario; Magdalena Kuhn, Moncton, NB; Christine Lee, Hamilton, Ontario; Allison McGeer, Toronto, Ontario; Robert Rennie, Edmonton, Alberta; Diane Roscoe, Vancouver, British Columbia; Andrew Simor, Toronto, Ontario; Baldwin Toye, Ottawa, Ontario; Karl Weiss, Montreal, Quebec.
0732-8893/$ – see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.diagmicrobio.2007.03.024
and Tunkel, 2004). The annual incidence of invasive disease due to S. pneumoniae is estimated to be between 12 and 17 cases per 100 000 population (NACI, 2000). Timely and appropriate antimicrobial therapy has been shown to reduce the morbidity and mortality of pneumococcal disease, particularly among patients with invasive disease (Feikin et al., 2000). However, the increasing rates of resistance to antimicrobial agents among S. pneumoniae documented in Canada and worldwide may threaten our ability to adequately manage patients with pneumococcal disease (Ball, 1999; Davidson et al., 2002; Lonks et al., 2002; Low, 1998; Low et al., 2002). Surveillance studies performed over the last decade have demonstrated alarming rates of resistance, particularly among traditional 1st-line agents such as penicillin, trimethoprim–sulfamethoxazole, tetracyclines, and macrolides (Doern et al., 1998, 2001; Felmingham and Gruneberg, 2000; Low et al., 2002; Low 2004; NACI, 2000; Powis et al., 2004; Whitney et al., 2000). For patients managed in the community setting, current evidence suggests that the use of a macrolide or ketolide as
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R.J. Davidson et al. / Diagnostic Microbiology and Infectious Disease 59 (2007) 75–80
monotherapy is suitable (File and Tan, 1997; Mandel et al., 2007); however, there is increasing concern about the frequency and role of macrolide resistance. Seriously ill patients, those with comorbidities, and patients admitted to hospital should be treated with combination therapy (β-lactam + macrolide or ketolide) or with newer agents such as the fluoroquinolones (Mandel et al., 2007). Ample evidence exists demonstrating that treatment with discordant empirical therapy at time of presentation results in increased morbidity and mortality in patients with serious bacterial infections (Gleason et al., 1999; Heffelfinger et al., 2000; Mufson and Stanek, 1999; Waterer et al., 2000). Because physicians rarely have information on the specific etiology of the infection when a patient presents, initial management is almost always empiric. The obvious benefit of administering adequate antimicrobial therapy dictates that current data on the epidemiology of antimicrobial resistance should be readily available to physicians so that appropriate therapy can be initiated. This study examines the regional differences in antimicrobial susceptibility of invasive S. pneumoniae (blood isolates), collected from across Canada over a 2-year period. 2. Materials and methods 2.1. Bacterial strains A total of 736 nonduplicate isolates of S. pneumoniae, collected at 10 tertiary care centers from 7 Canadian provinces, were included in this study. All were blood culture isolates collected between 2002 and 2003. S. pneumoniae were identified at each local site using standard criteria and confirmed at the Queen Elizabeth II Health Sciences Centre, Nova Scotia, Canada, using several criteria including Gram stain, optochin susceptibility, and bile solubility. Strains were stored in at −86 °C and subcultured at least twice before further testing. Subcultures were performed on Mueller–Hinton agar plates supplemented with 5% (vol/vol) defibrinated sheep's blood. S. pneumoniae ATCC 49619 was used as a control in susceptibility testing.
2.2. Susceptibility testing Microdilution broth susceptibility testing was performed at the Queen Elizabeth II Health Sciences Centre using the method prescribed by the Clinical Laboratory Standards Institute (CLSI) and appropriate controls suggested by the CLSI included in each run (CLSI, 2005). Susceptibility to the following antimicrobial agents were tested: levofloxacin, gatifloxacin, moxifloxacin, penicillin, amoxicillin, cefuroxime, ceftriaxone, tetracycline, erythromycin, and telithromycin. Current CLSI interpretive criteria were used to define antimicrobial resistance (CLSI, 2005). To distinguish between M and MLSB phenotypes for erythromycin-resistant strains, a double-disk approximation technique was performed using erythromycin (15 μg) and clindamycin (2 μg) disks. Both disks were placed 14 mm apart, edge to edge, and the plates were incubated overnight at 35 °C in 5% CO2. Interpretation of the results was performed using criteria described previously (de Azavedo et al., 1999). 2.3. Identification of resistance genes The erythromycin resistance genes, ermB and mefA/E, were amplified using a polymerase chain reaction technique previously published (Reinert et al., 2005; Sutcliffe et al., 1996). The amplicons were labeled with the Gene Images AlkPhos Direct labeling system (Amersham Pharmacia Biotech, Buckinghamshire, UK) for use as probes. DNA of each ERY-resistant strain was prepared as follows: 500 μL of a cellular suspension (3.0 McFarland in water) was lysed by boiling for 10 min and cellular debris removed by centrifugation. The supernatant was transferred to nylon membranes using the Minifold I Dot-Blot Systems (Schleicher and Schuell BioScience, Keene, NH), and the hybridization was performed under high-stringency conditions. Chemiluminescent detection was performed with CDP-Star reagent according to the manufacturer's instructions (Amersham Pharmacia Biotech). The quinolone resistance-determining region (QRDR) of the gyrA, gyrB, parC, and parE genes amplified from strains exhibiting reduced susceptibility to fluoroquinolones were
Table 1 In vitro susceptibility for 736 invasive S. pneumoniae isolates collected across Canada Antimicrobial agent
No. (%) of isolates Resistant
Intermediate
Susceptible
MIC50
MIC90
Range
Levofloxacin Moxifloxacin Gatifloxacin Cefuroxime Ceftriaxone Amoxicillin Penicillin Tetracycline Erythromycin Telithromycin
6 0 6 66 1 8 40 66 98 0
1 (0.14%) 5 (0.68%) 0 (0%) 14 (1.9%) 23 (3.12%) 1 (0.13%) 84 (11.42%) 6 (0.82%) 6 (0.82%) 0 (0%)
729 (99.04%) 731 (99.32%) 730 (99.18%) 656 (89.13%) 712 (96.74% 727 (98.78%) 612 (83.15%) 664 (90.22%) 632 (85.87%) 736 (100%)
1 0.12 0.5 b0.12 b0.03 b0.03 0.03 0.25 0.06 0.015
1 0.25 0.5 1 0.5 0.25 0.25 2 4 0.06
0.25 to 16 b0.06 to 2 b0.12 to 8 b0.12 to 32 b0.03 to 4 b0.03 to 8 b0.015 to N4 b0.12 to N32 b0.06 to N32 b0.008 to 1
(0.81%) (0%) (0.81%) (8.97%) (0.14%) (1.1%) (5.43%) (8.97%) (13.31%) (0%)
MIC (mg/L)
R.J. Davidson et al. / Diagnostic Microbiology and Infectious Disease 59 (2007) 75–80
amplified using primers previously described (Pan et al., 1996) and sequenced using the BigDye terminator methodology (Applied Biosystems/Perkin Elmer, Foster City, CA). Multiple nucleotide sequence alignments were performed with the Clustal X program.
3. Results 3.1. β-Lactam resistance The susceptibility profile to penicillin is displayed in Table 1. Greater than 80% of the isolates were susceptible (612, 83.15%) (MIC, ≤0.06 μg/mL); 84 isolates (11.42%) displayed an intermediate susceptibility (MIC, 0.12– 1 μg/mL), and 40 (5.43%) were resistant (MIC, ≥2 μg/mL). 3.2. Macrolide resistance
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Table 3 Fluoroquinolone resistance Strains
T164-57 V157-32 W155-53 W155-38 E162-77 T164-56
MIC (mg/L)
QRDR mutations
LEV
GAT
MOX
gyrA
parC
16 16 16 8 8 8
8 4 4 4 4 4
2 2 2 2 2 1
Ser81Phe Ser81Phe Ser81Phe Ser81Phe Ser81Phe Ser81Phe
Ser79Phe Ser79Phe Ser79Tyr Ser79Phe Asp83Asn Ser79Phe
Phenotypes of resistant strains and mutations found in QRDR regions. E = Edmonton; T = Toronto; V = Vancouver; W = Winnipeg; LEV = levofloxacin; GAT = gatifloxacin; MOX = moxifloxacin.
for both genes displayed MICs between 4 and 16 μg/mL. Phenotypically, these isolates displayed a cMLSB phenotype. 3.3. Fluoroquinolone resistance
In total, 104 strains (14.1%) had reduced susceptibility to erythromycin: 98 resistant strains (13.3%) and 6 with intermediate susceptibility (0.8%). Among the 98 erythromycin-resistant strains, the M and cMLSB phenotypes were observed in 41 (41.8%) and 50 (51%) strains, respectively, using the double-disk approximation test. Only 1 strain (from Winnipeg) with the iMLSB phenotype (ermB+ mefA−) was detected. A bimodal distribution was observed between erythromycin-resistant strains. This distribution had a good correlation with the presence of ermB or mefA genes (Table 2). The ermB gene was detected in 52% (n = 51) of the macrolide-resistant strains (MLSB phenotype), including 3 with both ermB and mefA genes. The erythromycin MICs for these strains ranged from 2 to N32 μg/mL, 44 of these had erythromycin MICs of N32 μg/mL. Forty-two percent (n = 41) were positive for mefA gene (M phenotype) alone. The MIC range of these strains was 1 to 16 μg/mL; however, most of these isolates had an MIC to erythromycin of 2 to 8 μg/mL. The 6 strains (6.1%) negative
In total, only 6 strains (0.8%) were resistant to levofloxacin and gatifloxacin (MICs values between 8–16 and 4–8 μg/mL, respectively). Among these, 5 strains had intermediate susceptibility to moxifloxacin (2 μg/mL), and 1 was considered susceptible (1 μg/mL). One additional strain displayed intermediate susceptibility to levofloxacin (4 μg/mL) (Table 1). Sequencing of QRDR regions revealed mutations in the gyrA and parC genes consistent with fluoroquinolone resistance (Table 3), that is, amino acids 81 in GyrA (Ser→Phe), and 79 (Ser→Phe or Ser→Tyr) and 83 (Asp→Asn) in ParC. QRDR from gyrB and parE were sequenced, but no mutations were detected. 3.4. Cross-resistance A correlation between the macrolide and tetracycline resistance and the degree of penicillin resistance was observed (Fig. 1). Resistance to erythromycin and tetracycline was more common among penicillinR N penicillinI N penicillinS.
Table 2 Molecular characterization of macrolide resistance in S. pneumoniae City
No. of No. of No. of genotypes Isolates erythromycinermB+ ermB− resistant mefA− mefA+ isolates (%) a
Montreal 88 Moncton 28 London 57 Vancouver 43 Toronto 123 Ottawa 74 Halifax 29 Winnipeg 98 Hamilton 100 Edmonton 96 Total 736 a
26 (29.5) 5 (17.9) 9 (15.8) 6 (14) 17 (13.8) 9 (12.2) 3 (10.3) 8 (8.2) 8 (8) 7 (7.3) 98 (100)
18 4 4 2 5 5 2 5 2 1 48 (49)
7 1 5 4 6 3 – 3 6 6 41 (41.8)
ermB+ ermB− mefA+ mefA− – – – – 2 – 1 – – – 3 (3.1)
1 – – – 4 1 – – – – 6 (6.1)
Percentage of erythromycin-resistant isolates collected from each city.
Fig. 1. Distribution (%) of erythromycin (ERY) and tetracycline (TET) resistance among penicillin-susceptible, penicillin-intermediate, and penicillin-resistant S. pneumoniae.
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A correlation between erythromycin and tetracycline resistance was observed: 80.3% (53 isolates) of tetracyclineresistant strains (n = 66) were also erythromycin resistant. Of 44 erythromycin-resistant strains with MICs of N32 μg/mL, 41 were tetracycline resistant (62.1% of the tetracyclineresistant strains). We failed to detect an isolate resistant to telithromycin. The MIC90 to this antibiotic was 0.06 μg/mL. No strain had an MIC above 1 μg/mL. Five hundred eighty-seven strains had MICs of ≤0.015 μg/mL.
4. Discussion In this study, 16.85% of isolates were nonsusceptible to penicillin, and 5.4% were highly resistant. These rates are in keeping with previous surveillance studies performed in Canada among noninvasive isolates (Zhanel et al., 2003). There have been suggestions that bacteria, having acquired resistance, may be less virulent because of the biologic cost of maintaining their resistant phenotype (Andersson and Levin, 1999; Cirz et al., 2005). Because the rates of penicillin resistance among our invasive isolates are similar to those observed in noninvasive isolates from other investigators, our results suggest that penicillin resistance does not appear to hinder the virulence or the ability of S. pneumoniae to cause invasive disease. Only 11 isolates in this study had an MIC to penicillin of ≥4 μg/mL. It is possible that isolates displaying this level of resistance may be less virulent, but that observation is beyond the scope of this study. With the recent adjustment in the breakpoints for amoxicillin and ceftriaxone by the CLSI, the rates of resistance to these 2 agents remain very low. In patients infected with strains exhibiting penicillin MICs between 2 and 4 μg/mL, some evidence exist to suggest that these patients may have an increased risk of mortality (Dowell et al., 1999; Feikin et al., 2000; Turret et al., 1999). Feikin et al. (2000) reported no increased mortality in patients infected with strains exhibiting penicillin MICs between 0.12 and 2 μg/mL compared with susceptible strains, but they found that mortality increased 7-fold in patients infected with pneumococci with penicillin MICs ≥4 μg/mL. The low rate of highly resistant strains to penicillin suggests that the use of a β-lactam agent, with the possible exception of cefuroxime, continues to be a good choice for the empiric management of nonmeningeal pneumococcal disease in Canada. However, the finding that N5% of invasive isolates were highly resistant to penicillin suggests that vancomycin be added to the empiric management of patients with suspected bacterial meningitis until culture and antimicrobial susceptibility results are available. The overall rate of resistance to the macrolides was 14.1%. Efflux, mediated by the mef gene, accounted for approximately 42% of the resistance. Similar to the results
observed for penicillin, the rates of macrolide resistance among invasive strains approximate the rates observed among noninvasive isolates (Whitney et al., 2000). As well, previous studies have demonstrated significant regional variation in macrolide resistance between the provinces. The one interesting observation was the lower rate of macrolide resistance observed among invasive isolates from the east coast of Canada compared with the rates seen among noninvasive isolates (Davidson et al., 2003). Despite the rate of macrolide resistance, we failed to isolate a single organism resistant to the ketolide, telithromycin. In part, because of relatively low serum concentrations, most guidelines suggest that seriously ill patients and patients admitted to the hospital should not receive a macrolide as monotherapy but rather in combination with a β-lactam agent (File and Tan, 1997; Georges et al., 1999; Mandel et al., 2007). Daneman et al. (2006) recently reported that macrolide failures were significantly more common among cases of pneumococcal bacteremia with isolates exhibiting an erythromycin MIC of 1 μg/mL. Fluoroquinolone resistance among invasive S. pneumoniae is exceeding low, ≤1% overall. Although fluoroquinolone resistance among all strains of S. pneumoniae is low, our study suggests the rate is somewhat lower than is observed among noninvasive isolates. The rates of resistance to levofloxacin and gatifloxacin were identical in this study; no isolates were found to be fully resistant to moxifloxacin. Among the 6 levofloxacin-resistant isolates, 5 were highly resistant and 1 was intermediately resistant. Previous surveillance studies have also demonstrated that intermediate resistance to levofloxacin is rarely observed. The concern here, and not specifically addressed in this study, is the proportion of isolates containing a single topoisomerase IV mutation, yet remaining susceptible in vitro. Several studies have recently demonstrated that a significant percentage of levofloxacin-susceptible isolates harbor a single mutation. As well, additional studies have shown that this is a significant risk factor for the development fluoroquinolone resistance and/or clinical failure in patients repeatedly exposed to the fluoroquinolones (Davidson et al., 2002; Low 2004). For patients with severe disease, particularly those with a bacteremic component, recent evidence has emerged to suggest that monotherapy may provide suboptimal coverage (Martinez et al., 2003; Stahl et al., 1999; Waterer et al., 2001). Three retrospective studies have suggested that combination therapy that included a macrolide as part of the regimen reduced mortality in patients with bacteremic pneumococcal pneumonia (Martinez et al., 2003; Stahl et al., 1999; Waterer et al., 2001). Waterer et al. (2001) observed that patients with bacteremic pneumococcal pneumonia who received at least 2 effective antibiotics within the first 24 h after presentation to a hospital had significantly lower rates of mortality than patients receiving only one effective antibiotic. In severely ill patients (pneumonia severity index class IV or V), mortality was
R.J. Davidson et al. / Diagnostic Microbiology and Infectious Disease 59 (2007) 75–80
5-fold greater in the group receiving only one antibiotic compared with patients receiving 2 antibiotics. Martinez et al. (2003) observed that treating bacteremic pneumococcal pneumonia patients with a regimen that did not include a macrolide was an independent predictor of inhospital mortality. Stahl et al. (1999) demonstrated that inclusion of a macrolide to the antibiotic regimen also significantly reduced patient length of stay in hospital. All of the abovementioned studies evaluated combination therapy empirically before obtaining blood culture results. That is, none of the studies directed examined the effects of pathogen-directed therapy, and no evidence has been provided to contradict the principles of pathogen-directed therapy once culture and susceptibility information has been obtained. This study examined the susceptibility patterns of a large number of S. pneumoniae enhanced for demonstrated virulence by virtue of their ability to cause invasive disease. Among this collection, the fluoroquinolones and telithromycin were the most reliably active in vitro. References Andersson DI, Levin BR (1999) The biological cost of antibiotic resistance. Curr Opin Microbiol 2:489–493. Ball P (1999) Therapy for pneumococcal infection at the millennium: doubts and certainties. Am J Med 107(Suppl 1A):77S–85S. Cirz RT, Chin JK, Andes DR, de Crecy-Lagard V, Craig WA, Romesberg FE (2005) Inhibition of mutation and combating the evolution of antibiotic resistance. PLOS Biol 3:1–10. Clinical Laboratory Standards Institute. (2005). Performance Standards for Antimicrobial Susceptibility Testing: 15th Informational Supplement. Wayne (PA): Clinical Laboratory Standards Institute. Daneman N, McGeer A, Green K, Low DE, and the Toronto Invasive Bacterial Diseases Network (2006) Macrolide resistance in bacteremic pneumococcal disease: implications for patient management. Clin Infect Dis 43:432–438. Davidson R, Cavalcanti R, Brunton JL, et al (2002) Resistance to levofloxacin and failure of treatment of pneumococcal pneumonia. N Engl J Med 346:747–750. Davidson RJ, Chan CK, Doern G, Zhanel GG. Abstr. 13th Eur. Congress Clin. Microbiol. Infect. Dis, Abstr 1736, 2003. de Azavedo J, Yeung RH, bast DJ, Duncan CL, Borgia SB, Low DE (1999) Prevalence and mechanisms of macrolide resistance in clinical isolates of group A streptococci from Ontario, Canada. Antimicrob Agents Chemother 43:2144–2147. Doern GV, Pfaller MA, Kugler K, Freeman J, Jones RN (1998) Prevalence of antimicrobial resistance among respiratory tract isolates of Streptococcus pneumoniae in North America: 1997 results from the SENTRY antimicrobial surveillance program. Clin Infect Dis 27:764–770. Doern GV, Heilmann KP, Huynh HK, et al (2001) Antimicrobial resistance among clinical isolates of Streptococcus pneumoniae in the United States during 1999–2000, including a comparison of resistance rates since 1994–1995. Antimicrob Agents Chemother 45:1721–1729. Dowell SF, Smith T, Leversedge K, Snitzer J (1999) Pneumonia treatment failure associated with highly resistant pneumococci. Clin Infect Dis 29:462–463. Feikin DR, Schuchat A, Kolczak N, Berrett L, Harrison H, Lefkowitz L, et al (2000) Mortality from invasive pneumococcal pneumonia in the era of antibiotic resistance, 1995–1997. Am J Public Health 90: 223–229.
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Felmingham D, Gruneberg RN (2000) The Alexander project 1996–1997: latest susceptibility data from this international study of bacterial pathogens from community-acquired lower respiratory tract infections. J Antimicrob Chemother 45:191–203. File Jr TM, Tan JS (1997) Incidence, etiologic pathogens, and diagnostic testing of community-acquired pneumonia. Curr Opin Pulm Med 3:89–97. Georges H, Leroy O, Vandenbussche C, et al (1999) Epidemiological features and prognosis of severe community-acquired pneumococcal pneumonia. Intensive Care Med 25:198–206. Gleason PP, Meehan TP, Fine JM, Galusha DH, Fine MJ (1999) Associations between initial antimicrobial therapy and medical outcomes for hospitalized elderly patients with pneumonia. Arch Intern Med 159:2562–2572. Heffelfinger JD, Dowell SF, Jorgensen JH, et al (2000) Management of community-acquired pneumonia in the era of pneumococcal resistance. Arch Intern Med 160:1399–1408. Lonks JR, Garau J, Gomez L, Xercavins M, Ochoa DE, Gareen F, et al (2002) Failure of macrolide antibiotic treatment in patients with bacteremia due to erythromycin resistant Streptococcus pneumoniae. Clin Infect Dis 35:556–564. Low DE (1998) Resistance and treatment implications: pneumococcus, Staphylococcus aureus and gram-negative rods. Infect Dis Clin North Am 3:613–630. Low DE (2004) Quinolone resistance among pneumococci: therapeutic and diagnostic implications. Clin Infect Dis 38(Suppl 4):S357–S362. Low DE, de Azavedo J, Weiss K, et al (2002) Antimicrobial resistance among clinical isolates of Streptococcus pneumoniae in Canada during 2000. Antimicrob Agents Chemother 46:1295–1301. Mandel LA, Wunderink RG, Anzueto A, Bartlett JG, Campbell D, Dean NC, et al (2007) Infectious diseases society of America/American thoracic society consensus guidelines on the management of community acquired pneumonia in adults. Clin Infect Dis 44: S27–S72. Martinez JA, Horcajada JP, Almeda M, et al (2003) Addition of a macrolide to a β-lactam–based empirical antibiotic regimen is associated with lower in-hospital mortality for patients with bacteremic pneumococcal pneumonia. Clin Infect Dis 36:389–395. Mufson MA, Stanek RJ (1999) Bacteremic pneumococcal pneumonia in one American city: a 20-year longitudinal study, 1978–1997. Am J Med 107(Suppl):34S–43S. National Advisory Committee on Immunization (NACI) (2000) Statement on recommended use of pneumococcal conjugate vaccine. Can Commun Dis Rep 28:1–32. Pan XS, Ambler J, Mehtar S, Fisher LM (1996) Involvement of topoisomerase IV and DNA gyrase as ciprofloxacin targets in Streptococcus pneumoniae. Antimicrob Agents Chemother 40: 2321–2326. Powis J, McGeer A, Green K, Vanderkooi O, Weiss K, Zhanel G, et al (2004) In vitro antimicrobial susceptibilities of Streptococcus pneumoniae clinical isolates obtained in Canada in 2002. Antimicrob Agents Chemother 48:3305–3311. Reinert RR, Reinert S, van der Linden M, Cil MY, Al-Lahham A, Appelbaum P (2005) Antimicrobial Susceptibility of Streptococcus pneumoniae in Eight European Countries from 2001 to 2003. Antimicrob Agents Chemother 49:2903–2913. Sinner SW, Tunkel AR (2004) Antimicrobial agents in the treatment of bacterial meningitis. Infect Dis Clin North Am 18:581–602. Stahl JE, Barza M, DesJardin J, Martin R, Eckman MH (1999) Effect of macrolides as part of initial empiric therapy on length of stay in patients hospitalized with community-acquired pneumonia. Arch Intern Med 159:2576–2580. Sutcliffe J, Grebe T, Tait-Kamradt A, Wondrack L (1996) Detection of erythromycin-resistant determinants by PCR. Antimicrob Agents Chemother 40:2562–2566. Turret GS, Blum S, Fazal BA, Justman JE, Telzak EE (1999) Penicillin resistance and other predictors of mortality in pneumococcal bacteremia
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in a population with high HIV seroprevalence. Clin Infect Dis 29:321–327. Waterer GW, Wunderink RG, Jones CB (2000) Fatal pneumococcal pneumonia attributed to macrolide resistance and azithromycin monotherapy. Chest 118:1839–1840. Waterer GW, Somes GW, Wunderink RG (2001) Monotherapy may be suboptimal for severe bacteremic pneumococcal pneumonia. Arch Intern Med 161:1837–1842.
Whitney CG, Farley MM, Hadler J, et al (2000) Increasing prevalence of multidrug resistant Streptococcus pneumoniae in the United States. N Engl J Med 343:1917–1924. Zhanel GG, Palatnick L, Nichol K, Bellyou T, Low DE, Hoban DJ (2003) Antimicrobial resistance in respiratory tract Streptococcus pneumoniae isolates: results of the Canadian Respiratory Organism Susceptibility Study, 1997 to 2002. Antimicrob Agents Chemother 47:1867–1874.
Antimicrobial Susceptibility Studies
Antimicrobial resistance among invasive isolates of Streptococcus pneumoniae collected across Canada☆ Ross J. Davidsona,b,c,d,⁎, Roberto Melanoa , Canadian Invasive Pneumococcal Disease Surveillance Group 1 , Kevin R. Forwarda,b,c,d a Queen Elizabeth II Health Sciences Centre, Dalhousie University, Halifax, Nova Scotia, Canada Department of Microbiology and Immunology, Dalhousie University, Halifax, Nova Scotia, Canada c Department of Pathology and Laboratory Medicine, Dalhousie University, Halifax, Nova Scotia, Canada d Department of Medicine, Dalhousie University, Halifax, Nova Scotia, Canada Received 6 December 2006; accepted 25 March 2007 b
Abstract Between 2002 and 2003, 736 nonduplicate Streptococcus pneumoniae isolated from blood cultures were collected from 7 of 10 Canadian provinces (10 tertiary care centers). Microdilution broth susceptibility testing was performed using the method prescribed by the Clinical Laboratory Standards Institute. Of the isolates, 16.85% were nonsusceptible to penicillin and 5.4% were highly resistant. Of the S. pneumoniae, 14.1% had reduced susceptibility to erythromycin and 47% had been accounted for by the M phenotype. No isolates were recovered that were resistant to telithromycin. Only 6 isolates were resistant to levofloxacin and gatifloxacin. Of these, 5 strains had intermediate susceptibility to moxifloxacin and 1 was considered susceptible. The rates observed in this study are in keeping with previous surveillance studies among noninvasive isolates. © 2007 Elsevier Inc. All rights reserved. Keywords: Invasive S. pneumoniae; Antibiotic resistance; Surveillance
1. Introduction Streptococcus pneumoniae remains the leading bacterial cause of upper and lower community-acquired respiratory infections in both children and adults (File and Tan 1997; Georges et al., 1999; Mandel et al., 2007). S. pneumoniae is also the leading cause of bacterial meningitis and is associated with significant morbidity and mortality (Sinner ☆ This study was supported by an unrestricted grant from Aventis Pharmaceuticals, Canada. ⁎ Corresponding author. Queen Elizabeth II Health Sciences Centre, Halifax, Nova Scotia, Canada B3H-1V8. Tel.: +1-902-473-5520; fax: +1902-473-4432. E-mail address: [email protected] (R.J. Davidson). 1 Members of the Canadian Invasive Pneumococcal Disease Surveillance Group: Daryl Hoban, Winnipeg, Manitoba; Zafar Hussain, London, Ontario; Magdalena Kuhn, Moncton, NB; Christine Lee, Hamilton, Ontario; Allison McGeer, Toronto, Ontario; Robert Rennie, Edmonton, Alberta; Diane Roscoe, Vancouver, British Columbia; Andrew Simor, Toronto, Ontario; Baldwin Toye, Ottawa, Ontario; Karl Weiss, Montreal, Quebec.
0732-8893/$ – see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.diagmicrobio.2007.03.024
and Tunkel, 2004). The annual incidence of invasive disease due to S. pneumoniae is estimated to be between 12 and 17 cases per 100 000 population (NACI, 2000). Timely and appropriate antimicrobial therapy has been shown to reduce the morbidity and mortality of pneumococcal disease, particularly among patients with invasive disease (Feikin et al., 2000). However, the increasing rates of resistance to antimicrobial agents among S. pneumoniae documented in Canada and worldwide may threaten our ability to adequately manage patients with pneumococcal disease (Ball, 1999; Davidson et al., 2002; Lonks et al., 2002; Low, 1998; Low et al., 2002). Surveillance studies performed over the last decade have demonstrated alarming rates of resistance, particularly among traditional 1st-line agents such as penicillin, trimethoprim–sulfamethoxazole, tetracyclines, and macrolides (Doern et al., 1998, 2001; Felmingham and Gruneberg, 2000; Low et al., 2002; Low 2004; NACI, 2000; Powis et al., 2004; Whitney et al., 2000). For patients managed in the community setting, current evidence suggests that the use of a macrolide or ketolide as
76
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monotherapy is suitable (File and Tan, 1997; Mandel et al., 2007); however, there is increasing concern about the frequency and role of macrolide resistance. Seriously ill patients, those with comorbidities, and patients admitted to hospital should be treated with combination therapy (β-lactam + macrolide or ketolide) or with newer agents such as the fluoroquinolones (Mandel et al., 2007). Ample evidence exists demonstrating that treatment with discordant empirical therapy at time of presentation results in increased morbidity and mortality in patients with serious bacterial infections (Gleason et al., 1999; Heffelfinger et al., 2000; Mufson and Stanek, 1999; Waterer et al., 2000). Because physicians rarely have information on the specific etiology of the infection when a patient presents, initial management is almost always empiric. The obvious benefit of administering adequate antimicrobial therapy dictates that current data on the epidemiology of antimicrobial resistance should be readily available to physicians so that appropriate therapy can be initiated. This study examines the regional differences in antimicrobial susceptibility of invasive S. pneumoniae (blood isolates), collected from across Canada over a 2-year period. 2. Materials and methods 2.1. Bacterial strains A total of 736 nonduplicate isolates of S. pneumoniae, collected at 10 tertiary care centers from 7 Canadian provinces, were included in this study. All were blood culture isolates collected between 2002 and 2003. S. pneumoniae were identified at each local site using standard criteria and confirmed at the Queen Elizabeth II Health Sciences Centre, Nova Scotia, Canada, using several criteria including Gram stain, optochin susceptibility, and bile solubility. Strains were stored in at −86 °C and subcultured at least twice before further testing. Subcultures were performed on Mueller–Hinton agar plates supplemented with 5% (vol/vol) defibrinated sheep's blood. S. pneumoniae ATCC 49619 was used as a control in susceptibility testing.
2.2. Susceptibility testing Microdilution broth susceptibility testing was performed at the Queen Elizabeth II Health Sciences Centre using the method prescribed by the Clinical Laboratory Standards Institute (CLSI) and appropriate controls suggested by the CLSI included in each run (CLSI, 2005). Susceptibility to the following antimicrobial agents were tested: levofloxacin, gatifloxacin, moxifloxacin, penicillin, amoxicillin, cefuroxime, ceftriaxone, tetracycline, erythromycin, and telithromycin. Current CLSI interpretive criteria were used to define antimicrobial resistance (CLSI, 2005). To distinguish between M and MLSB phenotypes for erythromycin-resistant strains, a double-disk approximation technique was performed using erythromycin (15 μg) and clindamycin (2 μg) disks. Both disks were placed 14 mm apart, edge to edge, and the plates were incubated overnight at 35 °C in 5% CO2. Interpretation of the results was performed using criteria described previously (de Azavedo et al., 1999). 2.3. Identification of resistance genes The erythromycin resistance genes, ermB and mefA/E, were amplified using a polymerase chain reaction technique previously published (Reinert et al., 2005; Sutcliffe et al., 1996). The amplicons were labeled with the Gene Images AlkPhos Direct labeling system (Amersham Pharmacia Biotech, Buckinghamshire, UK) for use as probes. DNA of each ERY-resistant strain was prepared as follows: 500 μL of a cellular suspension (3.0 McFarland in water) was lysed by boiling for 10 min and cellular debris removed by centrifugation. The supernatant was transferred to nylon membranes using the Minifold I Dot-Blot Systems (Schleicher and Schuell BioScience, Keene, NH), and the hybridization was performed under high-stringency conditions. Chemiluminescent detection was performed with CDP-Star reagent according to the manufacturer's instructions (Amersham Pharmacia Biotech). The quinolone resistance-determining region (QRDR) of the gyrA, gyrB, parC, and parE genes amplified from strains exhibiting reduced susceptibility to fluoroquinolones were
Table 1 In vitro susceptibility for 736 invasive S. pneumoniae isolates collected across Canada Antimicrobial agent
No. (%) of isolates Resistant
Intermediate
Susceptible
MIC50
MIC90
Range
Levofloxacin Moxifloxacin Gatifloxacin Cefuroxime Ceftriaxone Amoxicillin Penicillin Tetracycline Erythromycin Telithromycin
6 0 6 66 1 8 40 66 98 0
1 (0.14%) 5 (0.68%) 0 (0%) 14 (1.9%) 23 (3.12%) 1 (0.13%) 84 (11.42%) 6 (0.82%) 6 (0.82%) 0 (0%)
729 (99.04%) 731 (99.32%) 730 (99.18%) 656 (89.13%) 712 (96.74% 727 (98.78%) 612 (83.15%) 664 (90.22%) 632 (85.87%) 736 (100%)
1 0.12 0.5 b0.12 b0.03 b0.03 0.03 0.25 0.06 0.015
1 0.25 0.5 1 0.5 0.25 0.25 2 4 0.06
0.25 to 16 b0.06 to 2 b0.12 to 8 b0.12 to 32 b0.03 to 4 b0.03 to 8 b0.015 to N4 b0.12 to N32 b0.06 to N32 b0.008 to 1
(0.81%) (0%) (0.81%) (8.97%) (0.14%) (1.1%) (5.43%) (8.97%) (13.31%) (0%)
MIC (mg/L)
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amplified using primers previously described (Pan et al., 1996) and sequenced using the BigDye terminator methodology (Applied Biosystems/Perkin Elmer, Foster City, CA). Multiple nucleotide sequence alignments were performed with the Clustal X program.
3. Results 3.1. β-Lactam resistance The susceptibility profile to penicillin is displayed in Table 1. Greater than 80% of the isolates were susceptible (612, 83.15%) (MIC, ≤0.06 μg/mL); 84 isolates (11.42%) displayed an intermediate susceptibility (MIC, 0.12– 1 μg/mL), and 40 (5.43%) were resistant (MIC, ≥2 μg/mL). 3.2. Macrolide resistance
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Table 3 Fluoroquinolone resistance Strains
T164-57 V157-32 W155-53 W155-38 E162-77 T164-56
MIC (mg/L)
QRDR mutations
LEV
GAT
MOX
gyrA
parC
16 16 16 8 8 8
8 4 4 4 4 4
2 2 2 2 2 1
Ser81Phe Ser81Phe Ser81Phe Ser81Phe Ser81Phe Ser81Phe
Ser79Phe Ser79Phe Ser79Tyr Ser79Phe Asp83Asn Ser79Phe
Phenotypes of resistant strains and mutations found in QRDR regions. E = Edmonton; T = Toronto; V = Vancouver; W = Winnipeg; LEV = levofloxacin; GAT = gatifloxacin; MOX = moxifloxacin.
for both genes displayed MICs between 4 and 16 μg/mL. Phenotypically, these isolates displayed a cMLSB phenotype. 3.3. Fluoroquinolone resistance
In total, 104 strains (14.1%) had reduced susceptibility to erythromycin: 98 resistant strains (13.3%) and 6 with intermediate susceptibility (0.8%). Among the 98 erythromycin-resistant strains, the M and cMLSB phenotypes were observed in 41 (41.8%) and 50 (51%) strains, respectively, using the double-disk approximation test. Only 1 strain (from Winnipeg) with the iMLSB phenotype (ermB+ mefA−) was detected. A bimodal distribution was observed between erythromycin-resistant strains. This distribution had a good correlation with the presence of ermB or mefA genes (Table 2). The ermB gene was detected in 52% (n = 51) of the macrolide-resistant strains (MLSB phenotype), including 3 with both ermB and mefA genes. The erythromycin MICs for these strains ranged from 2 to N32 μg/mL, 44 of these had erythromycin MICs of N32 μg/mL. Forty-two percent (n = 41) were positive for mefA gene (M phenotype) alone. The MIC range of these strains was 1 to 16 μg/mL; however, most of these isolates had an MIC to erythromycin of 2 to 8 μg/mL. The 6 strains (6.1%) negative
In total, only 6 strains (0.8%) were resistant to levofloxacin and gatifloxacin (MICs values between 8–16 and 4–8 μg/mL, respectively). Among these, 5 strains had intermediate susceptibility to moxifloxacin (2 μg/mL), and 1 was considered susceptible (1 μg/mL). One additional strain displayed intermediate susceptibility to levofloxacin (4 μg/mL) (Table 1). Sequencing of QRDR regions revealed mutations in the gyrA and parC genes consistent with fluoroquinolone resistance (Table 3), that is, amino acids 81 in GyrA (Ser→Phe), and 79 (Ser→Phe or Ser→Tyr) and 83 (Asp→Asn) in ParC. QRDR from gyrB and parE were sequenced, but no mutations were detected. 3.4. Cross-resistance A correlation between the macrolide and tetracycline resistance and the degree of penicillin resistance was observed (Fig. 1). Resistance to erythromycin and tetracycline was more common among penicillinR N penicillinI N penicillinS.
Table 2 Molecular characterization of macrolide resistance in S. pneumoniae City
No. of No. of No. of genotypes Isolates erythromycinermB+ ermB− resistant mefA− mefA+ isolates (%) a
Montreal 88 Moncton 28 London 57 Vancouver 43 Toronto 123 Ottawa 74 Halifax 29 Winnipeg 98 Hamilton 100 Edmonton 96 Total 736 a
26 (29.5) 5 (17.9) 9 (15.8) 6 (14) 17 (13.8) 9 (12.2) 3 (10.3) 8 (8.2) 8 (8) 7 (7.3) 98 (100)
18 4 4 2 5 5 2 5 2 1 48 (49)
7 1 5 4 6 3 – 3 6 6 41 (41.8)
ermB+ ermB− mefA+ mefA− – – – – 2 – 1 – – – 3 (3.1)
1 – – – 4 1 – – – – 6 (6.1)
Percentage of erythromycin-resistant isolates collected from each city.
Fig. 1. Distribution (%) of erythromycin (ERY) and tetracycline (TET) resistance among penicillin-susceptible, penicillin-intermediate, and penicillin-resistant S. pneumoniae.
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A correlation between erythromycin and tetracycline resistance was observed: 80.3% (53 isolates) of tetracyclineresistant strains (n = 66) were also erythromycin resistant. Of 44 erythromycin-resistant strains with MICs of N32 μg/mL, 41 were tetracycline resistant (62.1% of the tetracyclineresistant strains). We failed to detect an isolate resistant to telithromycin. The MIC90 to this antibiotic was 0.06 μg/mL. No strain had an MIC above 1 μg/mL. Five hundred eighty-seven strains had MICs of ≤0.015 μg/mL.
4. Discussion In this study, 16.85% of isolates were nonsusceptible to penicillin, and 5.4% were highly resistant. These rates are in keeping with previous surveillance studies performed in Canada among noninvasive isolates (Zhanel et al., 2003). There have been suggestions that bacteria, having acquired resistance, may be less virulent because of the biologic cost of maintaining their resistant phenotype (Andersson and Levin, 1999; Cirz et al., 2005). Because the rates of penicillin resistance among our invasive isolates are similar to those observed in noninvasive isolates from other investigators, our results suggest that penicillin resistance does not appear to hinder the virulence or the ability of S. pneumoniae to cause invasive disease. Only 11 isolates in this study had an MIC to penicillin of ≥4 μg/mL. It is possible that isolates displaying this level of resistance may be less virulent, but that observation is beyond the scope of this study. With the recent adjustment in the breakpoints for amoxicillin and ceftriaxone by the CLSI, the rates of resistance to these 2 agents remain very low. In patients infected with strains exhibiting penicillin MICs between 2 and 4 μg/mL, some evidence exist to suggest that these patients may have an increased risk of mortality (Dowell et al., 1999; Feikin et al., 2000; Turret et al., 1999). Feikin et al. (2000) reported no increased mortality in patients infected with strains exhibiting penicillin MICs between 0.12 and 2 μg/mL compared with susceptible strains, but they found that mortality increased 7-fold in patients infected with pneumococci with penicillin MICs ≥4 μg/mL. The low rate of highly resistant strains to penicillin suggests that the use of a β-lactam agent, with the possible exception of cefuroxime, continues to be a good choice for the empiric management of nonmeningeal pneumococcal disease in Canada. However, the finding that N5% of invasive isolates were highly resistant to penicillin suggests that vancomycin be added to the empiric management of patients with suspected bacterial meningitis until culture and antimicrobial susceptibility results are available. The overall rate of resistance to the macrolides was 14.1%. Efflux, mediated by the mef gene, accounted for approximately 42% of the resistance. Similar to the results
observed for penicillin, the rates of macrolide resistance among invasive strains approximate the rates observed among noninvasive isolates (Whitney et al., 2000). As well, previous studies have demonstrated significant regional variation in macrolide resistance between the provinces. The one interesting observation was the lower rate of macrolide resistance observed among invasive isolates from the east coast of Canada compared with the rates seen among noninvasive isolates (Davidson et al., 2003). Despite the rate of macrolide resistance, we failed to isolate a single organism resistant to the ketolide, telithromycin. In part, because of relatively low serum concentrations, most guidelines suggest that seriously ill patients and patients admitted to the hospital should not receive a macrolide as monotherapy but rather in combination with a β-lactam agent (File and Tan, 1997; Georges et al., 1999; Mandel et al., 2007). Daneman et al. (2006) recently reported that macrolide failures were significantly more common among cases of pneumococcal bacteremia with isolates exhibiting an erythromycin MIC of 1 μg/mL. Fluoroquinolone resistance among invasive S. pneumoniae is exceeding low, ≤1% overall. Although fluoroquinolone resistance among all strains of S. pneumoniae is low, our study suggests the rate is somewhat lower than is observed among noninvasive isolates. The rates of resistance to levofloxacin and gatifloxacin were identical in this study; no isolates were found to be fully resistant to moxifloxacin. Among the 6 levofloxacin-resistant isolates, 5 were highly resistant and 1 was intermediately resistant. Previous surveillance studies have also demonstrated that intermediate resistance to levofloxacin is rarely observed. The concern here, and not specifically addressed in this study, is the proportion of isolates containing a single topoisomerase IV mutation, yet remaining susceptible in vitro. Several studies have recently demonstrated that a significant percentage of levofloxacin-susceptible isolates harbor a single mutation. As well, additional studies have shown that this is a significant risk factor for the development fluoroquinolone resistance and/or clinical failure in patients repeatedly exposed to the fluoroquinolones (Davidson et al., 2002; Low 2004). For patients with severe disease, particularly those with a bacteremic component, recent evidence has emerged to suggest that monotherapy may provide suboptimal coverage (Martinez et al., 2003; Stahl et al., 1999; Waterer et al., 2001). Three retrospective studies have suggested that combination therapy that included a macrolide as part of the regimen reduced mortality in patients with bacteremic pneumococcal pneumonia (Martinez et al., 2003; Stahl et al., 1999; Waterer et al., 2001). Waterer et al. (2001) observed that patients with bacteremic pneumococcal pneumonia who received at least 2 effective antibiotics within the first 24 h after presentation to a hospital had significantly lower rates of mortality than patients receiving only one effective antibiotic. In severely ill patients (pneumonia severity index class IV or V), mortality was
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5-fold greater in the group receiving only one antibiotic compared with patients receiving 2 antibiotics. Martinez et al. (2003) observed that treating bacteremic pneumococcal pneumonia patients with a regimen that did not include a macrolide was an independent predictor of inhospital mortality. Stahl et al. (1999) demonstrated that inclusion of a macrolide to the antibiotic regimen also significantly reduced patient length of stay in hospital. All of the abovementioned studies evaluated combination therapy empirically before obtaining blood culture results. That is, none of the studies directed examined the effects of pathogen-directed therapy, and no evidence has been provided to contradict the principles of pathogen-directed therapy once culture and susceptibility information has been obtained. This study examined the susceptibility patterns of a large number of S. pneumoniae enhanced for demonstrated virulence by virtue of their ability to cause invasive disease. Among this collection, the fluoroquinolones and telithromycin were the most reliably active in vitro. References Andersson DI, Levin BR (1999) The biological cost of antibiotic resistance. Curr Opin Microbiol 2:489–493. Ball P (1999) Therapy for pneumococcal infection at the millennium: doubts and certainties. Am J Med 107(Suppl 1A):77S–85S. Cirz RT, Chin JK, Andes DR, de Crecy-Lagard V, Craig WA, Romesberg FE (2005) Inhibition of mutation and combating the evolution of antibiotic resistance. PLOS Biol 3:1–10. Clinical Laboratory Standards Institute. (2005). Performance Standards for Antimicrobial Susceptibility Testing: 15th Informational Supplement. Wayne (PA): Clinical Laboratory Standards Institute. Daneman N, McGeer A, Green K, Low DE, and the Toronto Invasive Bacterial Diseases Network (2006) Macrolide resistance in bacteremic pneumococcal disease: implications for patient management. Clin Infect Dis 43:432–438. Davidson R, Cavalcanti R, Brunton JL, et al (2002) Resistance to levofloxacin and failure of treatment of pneumococcal pneumonia. N Engl J Med 346:747–750. Davidson RJ, Chan CK, Doern G, Zhanel GG. Abstr. 13th Eur. Congress Clin. Microbiol. Infect. Dis, Abstr 1736, 2003. de Azavedo J, Yeung RH, bast DJ, Duncan CL, Borgia SB, Low DE (1999) Prevalence and mechanisms of macrolide resistance in clinical isolates of group A streptococci from Ontario, Canada. Antimicrob Agents Chemother 43:2144–2147. Doern GV, Pfaller MA, Kugler K, Freeman J, Jones RN (1998) Prevalence of antimicrobial resistance among respiratory tract isolates of Streptococcus pneumoniae in North America: 1997 results from the SENTRY antimicrobial surveillance program. Clin Infect Dis 27:764–770. Doern GV, Heilmann KP, Huynh HK, et al (2001) Antimicrobial resistance among clinical isolates of Streptococcus pneumoniae in the United States during 1999–2000, including a comparison of resistance rates since 1994–1995. Antimicrob Agents Chemother 45:1721–1729. Dowell SF, Smith T, Leversedge K, Snitzer J (1999) Pneumonia treatment failure associated with highly resistant pneumococci. Clin Infect Dis 29:462–463. Feikin DR, Schuchat A, Kolczak N, Berrett L, Harrison H, Lefkowitz L, et al (2000) Mortality from invasive pneumococcal pneumonia in the era of antibiotic resistance, 1995–1997. Am J Public Health 90: 223–229.
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