Background Antimicrobial therapy is considered an important component in the medical management of most patients with acute exacerbation of chronic bronchitis (AECB). The three predominant bacterial species isolated are nontypeable  Haemophilus influenzae ,  Moraxella catarrhalis , and  Streptococcus pneumoniae .  Staphylococcus aureus  is also frequently isolated while atypical bacteria are thought to cause up to 10% of exacerbations. Antibacterial resistance is increasing worldwide and little surveillance data exist concerning pathogens isolated from patients with AECB.
Background
Antimicrobial therapy is considered an important component in the medical management of most patients with acute exacerbation of chronic bronchitis (AECB). The three predominant bacterial species isolated are nontypeable  Haemophilus influenzae ,  Moraxella catarrhalis , and  Streptococcus pneumoniae .  Staphylococcus aureus  is also frequently isolated while atypical bacteria are thought to cause up to 10% of exacerbations. Antibacterial resistance is increasing worldwide and little surveillance data exist concerning pathogens isolated from patients with AECB.Haemophilus influenzaeMoraxella catarrhalisStreptococcus pneumoniaeStaphylococcus aureusMethods This study examines the prevalence of antibacterial resistance in isolates obtained from patients with clinically diagnosed AECB. A total of 3043 isolates were obtained from 85 centres in 29 countries, between 1999–2003, and were tested against the new ketolide telithromycin and a panel of commonly used antibiotics.
Methods
This study examines the prevalence of antibacterial resistance in isolates obtained from patients with clinically diagnosed AECB. A total of 3043 isolates were obtained from 85 centres in 29 countries, between 1999–2003, and were tested against the new ketolide telithromycin and a panel of commonly used antibiotics.Results and Discussion Of the  S. pneumoniae  isolates, 99.9% were susceptible to telithromycin, but only 71% were susceptible to erythromycin and 75.3% to penicillin. Of the  H. influenzae  isolates, 99.6% were susceptible to telithromycin. 11.7% of these isolates produced β-lactamase. Almost 10% of  S. pneumoniae  were multidrug-resistant; 99.0% of these isolates were susceptible to telithromycin. Telithromycin also demonstrated good  in vitro  activity against  M. catarrhalis  (MIC 90  = 0.12 mg/L) and was the most active compound against methicillin-susceptible  S. aureus  (98.9% susceptible).
Results and Discussion
Of the  S. pneumoniae  isolates, 99.9% were susceptible to telithromycin, but only 71% were susceptible to erythromycin and 75.3% to penicillin. Of the  H. influenzae  isolates, 99.6% were susceptible to telithromycin. 11.7% of these isolates produced β-lactamase. Almost 10% of  S. pneumoniae  were multidrug-resistant; 99.0% of these isolates were susceptible to telithromycin. Telithromycin also demonstrated good  in vitro  activity against  M. catarrhalis  (MIC 90  = 0.12 mg/L) and was the most active compound against methicillin-susceptible  S. aureus  (98.9% susceptible).S. pneumoniaeH. influenzaeS. pneumoniaein vitroM. catarrhalisS. aureusConclusion Telithromycin demonstrated similar or better activity against the bacterial species investigated than the other agents, with the most complete coverage overall. These species are the predominant causative bacterial pathogens in AECB and thus the spectrum of activity of telithromycin makes it a potential alternative for the empirical treatment of AECB.
Conclusion
Telithromycin demonstrated similar or better activity against the bacterial species investigated than the other agents, with the most complete coverage overall. These species are the predominant causative bacterial pathogens in AECB and thus the spectrum of activity of telithromycin makes it a potential alternative for the empirical treatment of AECB.

Introduction The World Health Organization (WHO) estimates that chronic obstructive pulmonary disease (COPD) is the fourth leading cause of death worldwide. In the year 2000, it was estimated that 2.74 million people died from COPD worldwide [ 1 ]. COPD is defined by the presence of irreversible or partially irreversible airway obstruction in patients with chronic bronchitis or emphysema [ 2 , 3 ]. The disease is characterized by recurrent (1–4 per year) acute exacerbations of chronic bronchitis (AECB), defined by a subjective increase from baseline of one or more symptoms including shortness of breath, cough, sputum production, and sputum purulence [ 4 ]. The precipitating factors for AECB have been extensively researched and determined to be heterogeneous with complex aetiology [ 5 - 10 ]. Results from a number of placebo-controlled clinical investigations have demonstrated that antibacterial agents are of significant clinical benefit in the treatment of AECB, particularly for those patients with at least two of the three cardinal symptoms of AECB (worsening dyspnoea, increased sputum volume, and increased sputum purulence) and/or severe airway obstruction [ 11 - 13 ]. Other clinical trials measuring non-traditional endpoints have shown that antibiotic therapy reduces the time to symptom resolution and has long-term benefits including greater intervals between episodes of exacerbation [ 14 , 15 ]. Consequently, antibiotic therapy is considered an important component in the medical management of patients with AECB. Bacteria can be isolated from 40–60% of sputum samples of patients experiencing AECB [ 16 ]. The three predominant bacterial species isolated are non-typeable  Haemophilus influenzae ,  Moraxella catarrhalis , and  Streptococcus pneumoniae . Other less frequently isolated potential pathogens are Gram-negative enterobacteria,  Haemophilus parainfluenzae ,  Staphylococcus aureus , and  Pseudomonas aeruginosa . Gram negative enterobacteria and  Pseudomonas aeruginosa  are more frequently isolated in patients with severe underlying disease [ 10 ]. Viral infections are present in approximately 30% of exacerbations,  Mycoplasma pneumoniae  in 1–10%, and  Chlamydophila pneumoniae  in 4–5% (serologically identified) [ 6 - 10 ]. Amoxycillin, ampicillin, sulfamethoxazole-trimethoprim (trimethoprim-sulphamethoxazole), tetracyclines, and erythromycin are considered first-line antimicrobial therapy for AECB [ 17 ]. The clinical utility of these agents is, however, being hampered by the increasing global spread of pathogens with resistance to one or more of these agents. Up to 40% of  H. influenzae  isolates and more than 90% of  M. catarrhalis  isolates produce β-lactamase and this limits the value of penicillins and some other β-lactams [ 18 ]. Furthermore, resistance to penicillin and macrolides has spread rapidly among isolates of  S. pneumoniae  [ 19 ]. Other agents used include extended spectrum cephalosporins, amoxycillin/clavulanate, azithromycin, clarithromycin, and levofloxacin. Telithromycin is the first ketolide available for clinical use. Derivatives of erythromycin-A, the ketolides, like the macrolides, exert their antimicrobial action by binding to the bacterial ribosome. Although both macrolides and ketolides bind strongly to a region of domain V in the 23S rRNA of the ribosome, telithromycin has additional strong binding to a region in domain II to which the macrolides bind weakly [ 20 ]. Ketolides are also poor substrates for the efflux pump (mefA) responsible for macrolide resistance in  S. pneumoniae  [ 21 ]. Consequently, telithromycin has been found to have potent activity against macrolide resistant  S. pneumoniae  with methylase, efflux or ribosomal mutations as the mechanisms of resistance [ 22 , 23 ]. There is a need for alterative therapeutic options for the treatment of AECB and surveillance data are needed to help determine the suitability of new agents. The PROTEKT (Prospective Resistant Organism Tracking and Epidemiology for the Ketolide Telithromycin) study is an international, longitudinal, antibacterial resistance surveillance study, which was initiated in 1999 to monitor the spread of resistance among respiratory tract pathogens worldwide. Here we analyze the  in vitro  antimicrobial activity of bacterial isolates obtained from patients clinically diagnosed with AECB in 3 consecutive years of the PROTEKT study. Using these data, and previously published clinical data, the potential role of telithromycin in the treatment of AECB will be discussed.
Introduction
The World Health Organization (WHO) estimates that chronic obstructive pulmonary disease (COPD) is the fourth leading cause of death worldwide. In the year 2000, it was estimated that 2.74 million people died from COPD worldwide [ 1 ]. COPD is defined by the presence of irreversible or partially irreversible airway obstruction in patients with chronic bronchitis or emphysema [ 2 , 3 ]. The disease is characterized by recurrent (1–4 per year) acute exacerbations of chronic bronchitis (AECB), defined by a subjective increase from baseline of one or more symptoms including shortness of breath, cough, sputum production, and sputum purulence [ 4 ]. The precipitating factors for AECB have been extensively researched and determined to be heterogeneous with complex aetiology [ 5 - 10 ].Results from a number of placebo-controlled clinical investigations have demonstrated that antibacterial agents are of significant clinical benefit in the treatment of AECB, particularly for those patients with at least two of the three cardinal symptoms of AECB (worsening dyspnoea, increased sputum volume, and increased sputum purulence) and/or severe airway obstruction [ 11 - 13 ]. Other clinical trials measuring non-traditional endpoints have shown that antibiotic therapy reduces the time to symptom resolution and has long-term benefits including greater intervals between episodes of exacerbation [ 14 , 15 ]. Consequently, antibiotic therapy is considered an important component in the medical management of patients with AECB.Bacteria can be isolated from 40–60% of sputum samples of patients experiencing AECB [ 16 ]. The three predominant bacterial species isolated are non-typeable  Haemophilus influenzae ,  Moraxella catarrhalis , and  Streptococcus pneumoniae . Other less frequently isolated potential pathogens are Gram-negative enterobacteria,  Haemophilus parainfluenzae ,  Staphylococcus aureus , and  Pseudomonas aeruginosa . Gram negative enterobacteria and  Pseudomonas aeruginosa  are more frequently isolated in patients with severe underlying disease [ 10 ]. Viral infections are present in approximately 30% of exacerbations,  Mycoplasma pneumoniae  in 1–10%, and  Chlamydophila pneumoniae  in 4–5% (serologically identified) [ 6 - 10 ].Haemophilus influenzaeMoraxella catarrhalisStreptococcus pneumoniaeHaemophilus parainfluenzaeStaphylococcus aureusPseudomonas aeruginosaPseudomonas aeruginosaMycoplasma pneumoniaeChlamydophila pneumoniaeAmoxycillin, ampicillin, sulfamethoxazole-trimethoprim (trimethoprim-sulphamethoxazole), tetracyclines, and erythromycin are considered first-line antimicrobial therapy for AECB [ 17 ]. The clinical utility of these agents is, however, being hampered by the increasing global spread of pathogens with resistance to one or more of these agents. Up to 40% of  H. influenzae  isolates and more than 90% of  M. catarrhalis  isolates produce β-lactamase and this limits the value of penicillins and some other β-lactams [ 18 ]. Furthermore, resistance to penicillin and macrolides has spread rapidly among isolates of  S. pneumoniae  [ 19 ]. Other agents used include extended spectrum cephalosporins, amoxycillin/clavulanate, azithromycin, clarithromycin, and levofloxacin.H. influenzaeM. catarrhalisS. pneumoniaeTelithromycin is the first ketolide available for clinical use. Derivatives of erythromycin-A, the ketolides, like the macrolides, exert their antimicrobial action by binding to the bacterial ribosome. Although both macrolides and ketolides bind strongly to a region of domain V in the 23S rRNA of the ribosome, telithromycin has additional strong binding to a region in domain II to which the macrolides bind weakly [ 20 ]. Ketolides are also poor substrates for the efflux pump (mefA) responsible for macrolide resistance in  S. pneumoniae  [ 21 ]. Consequently, telithromycin has been found to have potent activity against macrolide resistant  S. pneumoniae  with methylase, efflux or ribosomal mutations as the mechanisms of resistance [ 22 , 23 ].S. pneumoniaeS. pneumoniaeThere is a need for alterative therapeutic options for the treatment of AECB and surveillance data are needed to help determine the suitability of new agents. The PROTEKT (Prospective Resistant Organism Tracking and Epidemiology for the Ketolide Telithromycin) study is an international, longitudinal, antibacterial resistance surveillance study, which was initiated in 1999 to monitor the spread of resistance among respiratory tract pathogens worldwide. Here we analyze the  in vitro  antimicrobial activity of bacterial isolates obtained from patients clinically diagnosed with AECB in 3 consecutive years of the PROTEKT study. Using these data, and previously published clinical data, the potential role of telithromycin in the treatment of AECB will be discussed.in vitroMaterials and Methods Patients and bacterial isolates Details of the study design, including the selection of patients and the methodology for the identification of isolates and their storage in the PROTEKT study has been described previously [ 24 ]. Isolates in this study were obtained from patients diagnosed with AECB from in 85 centres in 29 countries (Table  1 ). To be included in this analysis, an isolate was deemed pathogenic in AECB by clinical and laboratory findings. Isolates were only acceptable if the patient was ≥ 30 years old and the specimen was obtained from blood, bronchoalveolar lavage (BAL), or sputum. Isolates from patients diagnosed with AECB obtained from other sites (e.g., ear, throat, nasopharynx) and isolates obtained from patients <30 years of age were excluded from this analysis because AECB is more likely to be present in patients ≥ 30 years of age and the responsible bacterial pathogen is more likely to be correctly isolated from the blood, BAL, or sputum. Table 1 Geographical distribution of isolates from AECB patients used in this study Area Countries Centres Isolates North America 2 4 319 South America 6 14 427 Europe 13 41 1847 Australasia 6 19 437 South Africa 1 6 13 Totals 29 85 3043 In Year 1 (1999–2000), each centre had a quota of 60 isolates of  S. pneumoniae , 40  H. influenzae , 15  H. parainfluenzae , 20  M. catarrhalis , 25  Streptococcus pyogenes  and 20  S. aureus  to collect. In years 2 (2000–2001) and 3 (2001–2002),  H. parainfluenzae  were not collected and 15 extra isolates of  S. pneumoniae  were collected instead. Antimicrobial testing The comparator agents used were four β-lactams; penicillin (for  S. pneumoniae  and  S. aureus ), ampicillin (for  H. influenzae ,  H. parainfluenzae  and  M. catarrhalis ), amoxycillin/clavulanate, and cefuroxime, three macrolides/azalides; erythromycin, clarithromycin, and azithromycin, the folate synthesis inhibitor; trimethoprim-sulphamethoxazole, the tetracycline; tetracycline and a fluoroquinolone, levofloxacin. Minimum inhibitory concentrations (MIC) of each antibacterial were determined using the National Committee for Clinical and Laboratory Standards (NCCLS) broth microdilution methodology and lyophilised microtitre plates (Sensititre, Trek Diagnostics) at a central laboratory (GR Micro Ltd., London, UK) [ 26 ]. NCCLS breakpoints [ 25 , 26 ] were used to interpret the MIC data and to determine susceptibility status. The NCCLS breakpoints for telithromycin for  S. pneumoniae  and for  S. aureus  are ≤ 1 mg/l is susceptible, 2 mg/l is intermediate, and ≥ 4 mg/l is resistant, and for  H. influenzae  ≤ 4 mg/l is susceptible, 8 mg/l is intermediate, and ≥ 16 mg/l is resistant [ 27 ].
Materials and Methods
Patients and bacterial isolates Details of the study design, including the selection of patients and the methodology for the identification of isolates and their storage in the PROTEKT study has been described previously [ 24 ]. Isolates in this study were obtained from patients diagnosed with AECB from in 85 centres in 29 countries (Table  1 ). To be included in this analysis, an isolate was deemed pathogenic in AECB by clinical and laboratory findings. Isolates were only acceptable if the patient was ≥ 30 years old and the specimen was obtained from blood, bronchoalveolar lavage (BAL), or sputum. Isolates from patients diagnosed with AECB obtained from other sites (e.g., ear, throat, nasopharynx) and isolates obtained from patients <30 years of age were excluded from this analysis because AECB is more likely to be present in patients ≥ 30 years of age and the responsible bacterial pathogen is more likely to be correctly isolated from the blood, BAL, or sputum. Table 1 Geographical distribution of isolates from AECB patients used in this study Area Countries Centres Isolates North America 2 4 319 South America 6 14 427 Europe 13 41 1847 Australasia 6 19 437 South Africa 1 6 13 Totals 29 85 3043 In Year 1 (1999–2000), each centre had a quota of 60 isolates of  S. pneumoniae , 40  H. influenzae , 15  H. parainfluenzae , 20  M. catarrhalis , 25  Streptococcus pyogenes  and 20  S. aureus  to collect. In years 2 (2000–2001) and 3 (2001–2002),  H. parainfluenzae  were not collected and 15 extra isolates of  S. pneumoniae  were collected instead.
Patients and bacterial isolates
Details of the study design, including the selection of patients and the methodology for the identification of isolates and their storage in the PROTEKT study has been described previously [ 24 ]. Isolates in this study were obtained from patients diagnosed with AECB from in 85 centres in 29 countries (Table  1 ). To be included in this analysis, an isolate was deemed pathogenic in AECB by clinical and laboratory findings. Isolates were only acceptable if the patient was ≥ 30 years old and the specimen was obtained from blood, bronchoalveolar lavage (BAL), or sputum. Isolates from patients diagnosed with AECB obtained from other sites (e.g., ear, throat, nasopharynx) and isolates obtained from patients <30 years of age were excluded from this analysis because AECB is more likely to be present in patients ≥ 30 years of age and the responsible bacterial pathogen is more likely to be correctly isolated from the blood, BAL, or sputum.Geographical distribution of isolates from AECB patients used in this studyIn Year 1 (1999–2000), each centre had a quota of 60 isolates of  S. pneumoniae , 40  H. influenzae , 15  H. parainfluenzae , 20  M. catarrhalis , 25  Streptococcus pyogenes  and 20  S. aureus  to collect. In years 2 (2000–2001) and 3 (2001–2002),  H. parainfluenzae  were not collected and 15 extra isolates of  S. pneumoniae  were collected instead.S. pneumoniaeH. influenzaeH. parainfluenzaeM. catarrhalisStreptococcus pyogenesS. aureusH. parainfluenzaeS. pneumoniaeAntimicrobial testing The comparator agents used were four β-lactams; penicillin (for  S. pneumoniae  and  S. aureus ), ampicillin (for  H. influenzae ,  H. parainfluenzae  and  M. catarrhalis ), amoxycillin/clavulanate, and cefuroxime, three macrolides/azalides; erythromycin, clarithromycin, and azithromycin, the folate synthesis inhibitor; trimethoprim-sulphamethoxazole, the tetracycline; tetracycline and a fluoroquinolone, levofloxacin. Minimum inhibitory concentrations (MIC) of each antibacterial were determined using the National Committee for Clinical and Laboratory Standards (NCCLS) broth microdilution methodology and lyophilised microtitre plates (Sensititre, Trek Diagnostics) at a central laboratory (GR Micro Ltd., London, UK) [ 26 ]. NCCLS breakpoints [ 25 , 26 ] were used to interpret the MIC data and to determine susceptibility status. The NCCLS breakpoints for telithromycin for  S. pneumoniae  and for  S. aureus  are ≤ 1 mg/l is susceptible, 2 mg/l is intermediate, and ≥ 4 mg/l is resistant, and for  H. influenzae  ≤ 4 mg/l is susceptible, 8 mg/l is intermediate, and ≥ 16 mg/l is resistant [ 27 ].
Antimicrobial testing
The comparator agents used were four β-lactams; penicillin (for  S. pneumoniae  and  S. aureus ), ampicillin (for  H. influenzae ,  H. parainfluenzae  and  M. catarrhalis ), amoxycillin/clavulanate, and cefuroxime, three macrolides/azalides; erythromycin, clarithromycin, and azithromycin, the folate synthesis inhibitor; trimethoprim-sulphamethoxazole, the tetracycline; tetracycline and a fluoroquinolone, levofloxacin.S. pneumoniaeS. aureusH. influenzaeH. parainfluenzaeM. catarrhalisMinimum inhibitory concentrations (MIC) of each antibacterial were determined using the National Committee for Clinical and Laboratory Standards (NCCLS) broth microdilution methodology and lyophilised microtitre plates (Sensititre, Trek Diagnostics) at a central laboratory (GR Micro Ltd., London, UK) [ 26 ]. NCCLS breakpoints [ 25 , 26 ] were used to interpret the MIC data and to determine susceptibility status. The NCCLS breakpoints for telithromycin for  S. pneumoniae  and for  S. aureus  are ≤ 1 mg/l is susceptible, 2 mg/l is intermediate, and ≥ 4 mg/l is resistant, and for  H. influenzae  ≤ 4 mg/l is susceptible, 8 mg/l is intermediate, and ≥ 16 mg/l is resistant [ 27 ].S. pneumoniaeS. aureusH. influenzaeResults A total of 3043 bacterial pathogens were isolated from patients in 29 countries around the world, with by far the largest number of specimens (1841, 60.5%) coming from Europe (Table  1 ). Percentage of isolates by country were as follows: Argentina 8.0%, Australia 1.1%, Austria 0.6%, Brazil 4.0%, Canada 9.1%, China 1.7%, Colombia 0.1%, Ecuador 0.6%, Eire 0.03%, France 3.4%, Germany 14.3%, Hungary 1.2%, Indonesia 0.03%, Italy 18.9%, Japan 10.0%, Mexico 1.3%, Poland 10.2%, Portugal 2.8%, Russia 0.2%, South Africa 0.4%, South Korea 0.9%, Spain 5.3%, Sweden 0.4%, Switzerland 0.6%, Taiwan 0.7%, Turkey 0.3%, United Kingdom 2.4%, United States 1.4%, Venezuela 0.1%. Of these isolates identified as causative pathogens for bacterial AECB,  S. pneumoniae  and  H. influenzae  formed the majority (1075 and 1037 respectively), followed by  M. catarrhalis  (536) (Table  2 ). Patients were predominantly male (63.5%), with 47.5% of patients belonging to the (30–64) year age group and 52.5% in the >64 year old age group. No difference in the distribution of pathogens by age group was observed (data not shown). Table 2 Distribution of specimen types by species for the 3043 bacterial pathogens described in this study Specimen S. pneumoniae H. influenzae M. catarrhalis S. aureus H. parainfluenzae 1 Total  [n (%)] Sputum 832 895 492 219 43 2481 (81.5) BAL 2 144 135 44 66 17 406 (13.4) Blood 99 7 0 50 0 156 (5.1) Total  [n (%)] 1075 (35.3) 1037 (34.1) 536 (17.6) 335 (11.0) 60 (2.0) 3043 (100) 1 Only isolated in the first year of the study 2 Bronchoalveolar lavage Table  3  shows the range of MIC values, the MIC 50  and MIC 90  of the various agents against the five species. Where breakpoints were available the percentage of isolates to the various agents is also included. Telithromycin had similar or better  in vitro  susceptibility than the comparator agents against all of these species. Activity against  S. pneumoniae  was particularly good, with telithromycin being the most active agent; 99.9% of isolates were classified as susceptible and the MIC 90  (0.12 mg/L) was substantially lower than all other compounds tested. Table 3 In vitro  activity of antibacterial agents against 3043 bacterial pathogens isolated from patients with AECB and % susceptibilities to antibacterial agents. Organism Antibacterial MIC mg/l % susceptible range 50 90 total MSSA MRSA S. pneumoniae  N = 1075 Telithromycin 0.004–2 0.015 0.12 99.9 Azithromycin 0.03->64 0.12 >64 71.2 Clarithromycin 0.015->32 0.03 >32 71 Erythromycin 0.03->64 0.06 >64 71 Penicillin 0.008–8 0.03 2 75.3 Amox/clavulanate 1 0.015–8 0.03 2 96.1 Cefuroxime 0.015–16 0.03 2 82.2 Trimethoprim-sulphamethoxazole 0.12–32 0.25 8 62 Tetracycline 0.12–32 0.25 32 69.6 Levofloxacin 0.5->32 1 1 98.9 H. influenzae  N = 1037 Telithromycin 0.002–16 1 2 99.6 Azithromycin 0.06–32 1 2 99.7 Clarithromycin 0.25->64 8 16 82.4 Erythromycin 0.25->64 4 8 - 2 Ampicillin 0.12–32 0.25 16 87.3 Amox/clavulanate 1 0.12–4 0.5 1 100 Cefuroxime 0.12–16 1 2 99.5 Trimethoprim-sulphamethoxazole 0.03–32 0.06 4 80.7 Tetracycline 0.12–32 0.5 1 97.4 Levofloxacin 0.008–8 0.015 0.015 99.8 M. catarrhalis  N = 536 Telithromycin 0.004–0.5 0.06 0.12 Azithromycin 0.06–0.25 0.06 0.06 Clarithromycin 0.25–0.5 0.25 0.25 Erythromycin 0.25–1 0.25 0.25 Ampicillin 0.12–32 8 16 Amox/clavulanate 1 0.12–0.5 0.12 0.25 Cefuroxime 0.12–16 1 2 Trimethoprim-sulphamethoxazole 0.06–4 0.25 0.5 Tetracycline 0.12–32 0.25 0.5 Levofloxacin 0.008–0.06 0.03 0.03 S. aureus  N = 335 Telithromycin 0.03->32 0.06 >32 85.1 98.9 24.2 Azithromycin 0.12->64 1 >64 70.4 84.2 9.7 Clarithromycin 0.03->32 0.25 >32 70.4 84.2 9.7 Erythromycin 0.12->64 0.25 >64 70.4 84.6 9.7 Penicillin 0.008–8 4 8 23.6 28.9 0 Amox/clavulanate 1 0.06–8 0.5 8 83.1 100 0 Cefuroxime 0.12–16 1 16 81.2 100 0 Trimethoprim-sulphamethoxazole 0.12–32 0.12 0.25 94.9 97.4 83.9 Tetracycline 0.12–32 0.5 32 84.8 92.7 50 Levofloxacin 0.5–64 0.5 8 76.7 81.7 6.5 H. parainfluenzae  N = 60 Telithromycin 0.06–4 1 2 100 Azithromycin 0.06–2 0.5 1 100 Clarithromycin 0.25–16 4 8 93.3 Erythromycin 0.5–8 2 4 - 2 Ampicillin 0.12–32 0.25 1 90 Amox/clavulanate 1 0.12–2 0.5 1 100 Cefuroxime 0.12–4 0.25 0.5 100 Trimethoprim-sulphamethoxazole 0.03–32 0.03 1 88.3 Tetracycline 0.12–16 0.5 4 88.3 Levofloxacin 0.008–8 0.015 0.06 98.4 1 Amox/clavulanate = Amoxycillin/clavulanate 2 No CLSI interpretive criteria for erythromycin and  Haemophilus  spp. One hundred and three (9.6%)  S. pneumoniae  isolates (from 51 and 53 patients in the 30–64 and >64 year old age groups respectively)) were resistant to both penicillin (MIC ≥ 2 mg/L) and erythromycin (MIC ≥ 1 mg/L) and this was reflected in resistance to amoxycillin, cefuroxime, clarithromycin and azithromycin also (Table  4 ). These isolates were found in 35 centres in 16 countries. Sixty of these resistant isolates were also resistant to both trimethoprim-sulphamethoxazole and tetracycline. Both telithromycin and levofloxacin had good activity against these isolates, 99% susceptibility to telithromycin and 98.1% to levofloxacin. The MIC 50  and MIC 90  values for telithromycin in this population were 0.06 mg/L and 0.5 mg/L, respectively. Table 4 Antibacterial activity against 103  Streptococcus pneumoniae  isolates with combined macrolide and penicillin resistance Antibacterial % susceptible % intermediate % resistant Telithromycin 99.0 1.0 0.0 Azithromycin 0 0 100 Clarithromycin 0 0 100 Erythromycin 0 0 100 Penicillin 0 0 100 Amoxycillin 0 0 100 Amoxycillin-clavulanate 72.8 13.6 13.6 Cefuroxime 1.0 1.0 98.0 Trimethoprim-sulphamethoxazole 19.4 16.5 64.1 Tetracycline 11.7 0.0 88.3 Levofloxacin 98.1 0.0 1.9 Over 99% of  H. influenzae  isolates were susceptible to amoxycillin-clavulanate, cefuroxime, telithromycin, azithromycin, and levofloxacin. Tetracycline also had good activity with 97.4% of isolates susceptible. Only 11.7% of  H. influenzae  isolates produced β-lactamase. There were only 60 isolates of  H. parainfluenzae  and 100% of these were susceptible to four of the eight compounds tested, telithromycin, amoxycillin/clavulanate, cefuroxime and azithromycin. Trimethoprim-sulphamethoxazole and tetracycline were the least active compounds. In terms of MICs, levofloxacin, azithromycin and telithromycin were the most potent compounds against  M. catarrhalis  with MIC 90  values of 0.03 mg/l, 0.06 mg/l and 0.12 mg/l respectively. There are currently no interpretative NCCLS guidelines available for  M. catarrhalis  to allow classification into susceptible or resistant categories. The total number of isolates of  S. aureu s was 335 and of these only 62 were resistant to methicillin (MRSA). Trimethoprim-sulphamethoxazole was the most active compound overall, with 94.9% of all isolates being susceptible. Telithromycin and tetracycline were the next most active with 85.1% and 84.8% of all isolates susceptible. Telithromycin was the most active compound against the MSSA isolates, with 98.9% being susceptible. The susceptibility of MSSA to tetracycline and trimethoprim-sulphamethoxazole was 92.7% and 97.4% susceptible respectively. These three compounds were the only ones to have activity against the MRSA isolates (trimethoprim-sulphamethoxazole 83.9%, tetracycline 50% and telithromycin 24.2%). Less than 10% of the MRSA isolates were susceptible to the remainder of the compounds.
Results
A total of 3043 bacterial pathogens were isolated from patients in 29 countries around the world, with by far the largest number of specimens (1841, 60.5%) coming from Europe (Table  1 ). Percentage of isolates by country were as follows: Argentina 8.0%, Australia 1.1%, Austria 0.6%, Brazil 4.0%, Canada 9.1%, China 1.7%, Colombia 0.1%, Ecuador 0.6%, Eire 0.03%, France 3.4%, Germany 14.3%, Hungary 1.2%, Indonesia 0.03%, Italy 18.9%, Japan 10.0%, Mexico 1.3%, Poland 10.2%, Portugal 2.8%, Russia 0.2%, South Africa 0.4%, South Korea 0.9%, Spain 5.3%, Sweden 0.4%, Switzerland 0.6%, Taiwan 0.7%, Turkey 0.3%, United Kingdom 2.4%, United States 1.4%, Venezuela 0.1%.Of these isolates identified as causative pathogens for bacterial AECB,  S. pneumoniae  and  H. influenzae  formed the majority (1075 and 1037 respectively), followed by  M. catarrhalis  (536) (Table  2 ). Patients were predominantly male (63.5%), with 47.5% of patients belonging to the (30–64) year age group and 52.5% in the >64 year old age group. No difference in the distribution of pathogens by age group was observed (data not shown).S. pneumoniaeH. influenzaeM. catarrhalisDistribution of specimen types by species for the 3043 bacterial pathogens described in this studySpecimenS. pneumoniaeH. influenzaeM. catarrhalisS. aureusH. parainfluenzaeTotalSputumBAL 2BloodTotal1 Only isolated in the first year of the study2 Bronchoalveolar lavageTable  3  shows the range of MIC values, the MIC 50  and MIC 90  of the various agents against the five species. Where breakpoints were available the percentage of isolates to the various agents is also included. Telithromycin had similar or better  in vitro  susceptibility than the comparator agents against all of these species. Activity against  S. pneumoniae  was particularly good, with telithromycin being the most active agent; 99.9% of isolates were classified as susceptible and the MIC 90  (0.12 mg/L) was substantially lower than all other compounds tested.in vitroS. pneumoniaeIn vitro  activity of antibacterial agents against 3043 bacterial pathogens isolated from patients with AECB and % susceptibilities to antibacterial agents.In vitroOrganismAntibacterialMIC mg/l% susceptiblerange5090totalMSSAMRSAS. pneumoniaeH. influenzaeM. catarrhalisS. aureusH. parainfluenzae1 Amox/clavulanate = Amoxycillin/clavulanate2 No CLSI interpretive criteria for erythromycin and  Haemophilus  spp.HaemophilusOne hundred and three (9.6%)  S. pneumoniae  isolates (from 51 and 53 patients in the 30–64 and >64 year old age groups respectively)) were resistant to both penicillin (MIC ≥ 2 mg/L) and erythromycin (MIC ≥ 1 mg/L) and this was reflected in resistance to amoxycillin, cefuroxime, clarithromycin and azithromycin also (Table  4 ). These isolates were found in 35 centres in 16 countries. Sixty of these resistant isolates were also resistant to both trimethoprim-sulphamethoxazole and tetracycline. Both telithromycin and levofloxacin had good activity against these isolates, 99% susceptibility to telithromycin and 98.1% to levofloxacin. The MIC 50  and MIC 90  values for telithromycin in this population were 0.06 mg/L and 0.5 mg/L, respectively.S. pneumoniaeAntibacterial activity against 103  Streptococcus pneumoniae  isolates with combined macrolide and penicillin resistanceStreptococcus pneumoniaeAntibacterial% susceptible% intermediate% resistantOver 99% of  H. influenzae  isolates were susceptible to amoxycillin-clavulanate, cefuroxime, telithromycin, azithromycin, and levofloxacin. Tetracycline also had good activity with 97.4% of isolates susceptible. Only 11.7% of  H. influenzae  isolates produced β-lactamase. There were only 60 isolates of  H. parainfluenzae  and 100% of these were susceptible to four of the eight compounds tested, telithromycin, amoxycillin/clavulanate, cefuroxime and azithromycin. Trimethoprim-sulphamethoxazole and tetracycline were the least active compounds. In terms of MICs, levofloxacin, azithromycin and telithromycin were the most potent compounds against  M. catarrhalis  with MIC 90  values of 0.03 mg/l, 0.06 mg/l and 0.12 mg/l respectively.H. influenzaeH. influenzaeH. parainfluenzaeM. catarrhalisThere are currently no interpretative NCCLS guidelines available for  M. catarrhalis  to allow classification into susceptible or resistant categories.M. catarrhalisThe total number of isolates of  S. aureu s was 335 and of these only 62 were resistant to methicillin (MRSA). Trimethoprim-sulphamethoxazole was the most active compound overall, with 94.9% of all isolates being susceptible. Telithromycin and tetracycline were the next most active with 85.1% and 84.8% of all isolates susceptible. Telithromycin was the most active compound against the MSSA isolates, with 98.9% being susceptible. The susceptibility of MSSA to tetracycline and trimethoprim-sulphamethoxazole was 92.7% and 97.4% susceptible respectively. These three compounds were the only ones to have activity against the MRSA isolates (trimethoprim-sulphamethoxazole 83.9%, tetracycline 50% and telithromycin 24.2%). Less than 10% of the MRSA isolates were susceptible to the remainder of the compounds.S. aureuDiscussion The primary cause of COPD is exposure to tobacco smoke, the major risk factor being cigarette smoking. The demography of the disease in this study and others reflects this, as the majority of patients in this analysis were male and half were elderly (>64 yrs of age) (2).  S. pneumoniae  is most frequently isolated in the least severe cases of AECB, whereas  H. influenzae  is more commonly isolated from moderate to severe cases, with  P. aeruginosa  occurring in severe hospitalised cases [ 28 ]. Telithromycin does not have good activity against  Pseudomonas  spp. (GR Micro Limited, data on file, internal report number 141-02-99) and hence may not be an appropriate empirical therapeutic option for AECB patients with severe underlying disease who are hospitalized for an acute exacerbation. Whether the isolation of a pathogen during AECB represents an infection responsible for the exacerbation has been debated for many years [ 29 - 31 ]. Bacteria have been isolated almost as frequently from patients with stable COPD as those with an AECB, and clinical trials of antibiotic therapy in AECB show contradictory and sometimes unconvincing results [ 30 ]. The presence of bacteria in the lower airways is, however, regarded as abnormal since these airways are sterile in healthy adults, and it has been hypothesized that the presence of bacteria in stable COPD represents a low-grade smouldering infection. In addition, a recent study has shown that infection with different strains of pathogens that are new to the patient is associated with development of exacerbation [ 32 , 33 ]. Amoxycillin-clavulanate, azithromycin, and levofloxacin have been shown to be effective in the treatment of AECB, however, there is concern regarding their long-term usefulness, because of the development of resistance to these agents among the causative pathogens [ 34 , 35 ]. Telithromycin has a more focused spectrum of activity than the β-lactams and the fluoroquinolones; it is specifically targeted against pathogens causing community-acquired respiratory disease, including those most commonly associated with AECB. In addition, it is active against penicillin- and macrolide-resistant strains of  S. pneumoniae  and hence offers a viable potential option for the empiric treatment of AECB in non-hospitalised patients [ 36 ]. The data in this study demonstrate that telithromycin has high  in vitro  activity against the commonest bacterial pathogens causing AECB. These data also show that telithromycin has the highest overall activity against bacterial isolates from patients with AECB, regardless of species. Almost 10% of  S. pneumoniae  isolated were resistant to penicillin, macrolides, and at least one of the other antibiotics tested, with only telithromycin and levofloxacin retaining high activity against these isolates (99.0% and 98.1%, respectively). The validity of this finding is strengthened as the isolates were obtained from a large number of patients over a wide geographical distribution. Although atypical pathogens were not examined in the PROTEKT study, telithromycin has been shown to have superior activity  in vitro  against  Chlamydophila pneumoniae  to the other macrolides with the exception of clarithromycin and has similar activity to the fluoroquinolones [ 37 ]. In guinea pig models, telithromycin had better activity than erythromycin against  Legionella pneumophila  infections [ 38 ].  In vitro , the activity of telithromycin against  L. pneumophila  was similar to levofloxacin but better than erythromycin [ 38 ]. β-lactams and cephalosporins have no activity against  Mycoplasma pneumoniae  as this species lacks a typical bacterial cell wall, the site of activity for these drugs. Telithromycin has been found to have higher activity than doxycycline and levofloxacin against  M. pneumoniae  [ 39 ]. As the atypical pathogens can represent up to 10% of infections associated with AECB, the efficacy of telithromycin against these pathogens could be a consideration in the selection of empiric therapy for AECB. Telithromycin has been shown to penetrate into respiratory tissues well [ 40 ]. The concentration of telithromycin in alveolar macrophages and epithelial lining fluid exceeds that of plasma markedly and remains at therapeutic levels for 24 hours after dosing. Bactericidal levels are also maintained in plasma. A good post-antibiotic effect has also been observed [ 41 ]. Telithromycin causes only moderate ecological disturbance to oral and intestinal flora comparable to that associated with clarithromycin and it does not significantly increase the development of resistance in the normal flora, although the MIC of oral streptococci can be slightly raised [ 42 ]. Telithromycin can be administered once a day for AECB. Clinical studies have demonstrated that 800 mg administered once daily for 5 days was as effective and well tolerated as a 10-day course of amoxycillin/clavulanate (500/125 mg 3 times daily for 10 days), cefuroxime axetil (500 mg twice daily for 10 days) or clarithromycin (500 mg twice daily for 10 days) [ 43 ]. Other clinical studies have also confirmed the safety and tolerability of telithromycin 800 mg administered for 5 – 10 days [ 44 ]. Once a day dosing schedules and shorter courses may promote patient adherence to therapy, and this in turn could delay the development of resistance. Although this study provides valuable information on the overall antimicrobial profile of bacteria causing AECB, care should be taken when interpreting data related to specific demographics. The prevalence of species could not be calculated in this study as a major limitation, inherent to most surveillance studies, is the requirement for collecting centres to fulfil a specified quota of isolates over a defined time period (1 year). If, for instance, a centre managed to fulfil the quota for  S. pneumoniae  isolates from patients with community-acquired pneumonia, it could then only send  H. influenzae  from patients with AECB to fulfil the quota for this organism. In addition, atypical pathogens were not sampled and they can represent up to 10% of the causative pathogens [ 28 ]. In summary, the data presented here demonstrate that telithromycin has good  in vitro  activity against  H. influenzae ,  S. pneumoniae , and  M. catarrhalis , respiratory pathogens commonly isolated in AECB. It is as active as or more active than agents that are currently used in this clinical setting. Additionally, although not shown here, telithromycin has better  in vitro  activity against atypical pathogens than other agents; an important advantage in this clinical setting as these pathogens may represent 10% of AECB associated infections. The development of resistance will always be a threat to the usefulness of antibacterial compounds, however surveillance studies such as PROTEKT allow the rapid detection and characterization of resistance mechanisms and highlight the need for and examine the  in vitro  efficacy of newer antibacterial agents. Providing careful surveillance for the development of resistance is maintained, telithromycin currently offers a useful agent in the treatment of AECB.
Discussion
The primary cause of COPD is exposure to tobacco smoke, the major risk factor being cigarette smoking. The demography of the disease in this study and others reflects this, as the majority of patients in this analysis were male and half were elderly (>64 yrs of age) (2).  S. pneumoniae  is most frequently isolated in the least severe cases of AECB, whereas  H. influenzae  is more commonly isolated from moderate to severe cases, with  P. aeruginosa  occurring in severe hospitalised cases [ 28 ]. Telithromycin does not have good activity against  Pseudomonas  spp. (GR Micro Limited, data on file, internal report number 141-02-99) and hence may not be an appropriate empirical therapeutic option for AECB patients with severe underlying disease who are hospitalized for an acute exacerbation.S. pneumoniaeH. influenzaeP. aeruginosaPseudomonasWhether the isolation of a pathogen during AECB represents an infection responsible for the exacerbation has been debated for many years [ 29 - 31 ]. Bacteria have been isolated almost as frequently from patients with stable COPD as those with an AECB, and clinical trials of antibiotic therapy in AECB show contradictory and sometimes unconvincing results [ 30 ]. The presence of bacteria in the lower airways is, however, regarded as abnormal since these airways are sterile in healthy adults, and it has been hypothesized that the presence of bacteria in stable COPD represents a low-grade smouldering infection. In addition, a recent study has shown that infection with different strains of pathogens that are new to the patient is associated with development of exacerbation [ 32 , 33 ].Amoxycillin-clavulanate, azithromycin, and levofloxacin have been shown to be effective in the treatment of AECB, however, there is concern regarding their long-term usefulness, because of the development of resistance to these agents among the causative pathogens [ 34 , 35 ]. Telithromycin has a more focused spectrum of activity than the β-lactams and the fluoroquinolones; it is specifically targeted against pathogens causing community-acquired respiratory disease, including those most commonly associated with AECB. In addition, it is active against penicillin- and macrolide-resistant strains of  S. pneumoniae  and hence offers a viable potential option for the empiric treatment of AECB in non-hospitalised patients [ 36 ].S. pneumoniaeThe data in this study demonstrate that telithromycin has high  in vitro  activity against the commonest bacterial pathogens causing AECB. These data also show that telithromycin has the highest overall activity against bacterial isolates from patients with AECB, regardless of species. Almost 10% of  S. pneumoniae  isolated were resistant to penicillin, macrolides, and at least one of the other antibiotics tested, with only telithromycin and levofloxacin retaining high activity against these isolates (99.0% and 98.1%, respectively). The validity of this finding is strengthened as the isolates were obtained from a large number of patients over a wide geographical distribution.in vitroS. pneumoniaeAlthough atypical pathogens were not examined in the PROTEKT study, telithromycin has been shown to have superior activity  in vitro  against  Chlamydophila pneumoniae  to the other macrolides with the exception of clarithromycin and has similar activity to the fluoroquinolones [ 37 ]. In guinea pig models, telithromycin had better activity than erythromycin against  Legionella pneumophila  infections [ 38 ].  In vitro , the activity of telithromycin against  L. pneumophila  was similar to levofloxacin but better than erythromycin [ 38 ]. β-lactams and cephalosporins have no activity against  Mycoplasma pneumoniae  as this species lacks a typical bacterial cell wall, the site of activity for these drugs. Telithromycin has been found to have higher activity than doxycycline and levofloxacin against  M. pneumoniae  [ 39 ]. As the atypical pathogens can represent up to 10% of infections associated with AECB, the efficacy of telithromycin against these pathogens could be a consideration in the selection of empiric therapy for AECB.in vitroChlamydophila pneumoniaeLegionella pneumophilaIn vitroL. pneumophilaMycoplasma pneumoniaeM. pneumoniaeTelithromycin has been shown to penetrate into respiratory tissues well [ 40 ]. The concentration of telithromycin in alveolar macrophages and epithelial lining fluid exceeds that of plasma markedly and remains at therapeutic levels for 24 hours after dosing. Bactericidal levels are also maintained in plasma. A good post-antibiotic effect has also been observed [ 41 ]. Telithromycin causes only moderate ecological disturbance to oral and intestinal flora comparable to that associated with clarithromycin and it does not significantly increase the development of resistance in the normal flora, although the MIC of oral streptococci can be slightly raised [ 42 ].Telithromycin can be administered once a day for AECB. Clinical studies have demonstrated that 800 mg administered once daily for 5 days was as effective and well tolerated as a 10-day course of amoxycillin/clavulanate (500/125 mg 3 times daily for 10 days), cefuroxime axetil (500 mg twice daily for 10 days) or clarithromycin (500 mg twice daily for 10 days) [ 43 ]. Other clinical studies have also confirmed the safety and tolerability of telithromycin 800 mg administered for 5 – 10 days [ 44 ]. Once a day dosing schedules and shorter courses may promote patient adherence to therapy, and this in turn could delay the development of resistance.Although this study provides valuable information on the overall antimicrobial profile of bacteria causing AECB, care should be taken when interpreting data related to specific demographics. The prevalence of species could not be calculated in this study as a major limitation, inherent to most surveillance studies, is the requirement for collecting centres to fulfil a specified quota of isolates over a defined time period (1 year). If, for instance, a centre managed to fulfil the quota for  S. pneumoniae  isolates from patients with community-acquired pneumonia, it could then only send  H. influenzae  from patients with AECB to fulfil the quota for this organism. In addition, atypical pathogens were not sampled and they can represent up to 10% of the causative pathogens [ 28 ].S. pneumoniaeH. influenzaeIn summary, the data presented here demonstrate that telithromycin has good  in vitro  activity against  H. influenzae ,  S. pneumoniae , and  M. catarrhalis , respiratory pathogens commonly isolated in AECB. It is as active as or more active than agents that are currently used in this clinical setting. Additionally, although not shown here, telithromycin has better  in vitro  activity against atypical pathogens than other agents; an important advantage in this clinical setting as these pathogens may represent 10% of AECB associated infections.in vitroH. influenzaeS. pneumoniaeM. catarrhalisin vitroThe development of resistance will always be a threat to the usefulness of antibacterial compounds, however surveillance studies such as PROTEKT allow the rapid detection and characterization of resistance mechanisms and highlight the need for and examine the  in vitro  efficacy of newer antibacterial agents. Providing careful surveillance for the development of resistance is maintained, telithromycin currently offers a useful agent in the treatment of AECB.in vitro


Industrial development may result in the increase of environmental risks. The enzymatic transformation of polluting compounds to less toxic or even innocuous products is an alternative to their complete removal. In this regard, a number of different redox enzymes are able to transform a wide variety of toxic pollutants, such as polynuclear aromatic hydrocarbons, phenols, azo dyes, heavy metals, etc. Here, novel information on chromate reductases, enzymes that carry out the reduction of highly toxic Cr(VI) to the less toxic insoluble Cr(III), is discussed. In addition, the properties and application of bacterial and eukaryotic proteins (lignin-modifying enzymes, peroxidases and cytochromes) useful in environmental enzymology is also discussed.

Introduction Chromate reductases are a group of enzymes that catalyze the reduction of toxic and carcinogenic Cr(VI) to the less soluble and less toxic Cr(III). These proteins have recently raised enormous interest because of their central role in mediating chromium toxicity and their potential use in bioremediation and biocatalysis. Chromate (Cr(VI)) is generated as by-product of various industrial processes such as leather tanning, chrome-plating, pigment production and thermonuclear weapon manufacture [ 1 ]. Its high water solubility facilitates a rapid leaching, provoking a wide dispersion capable to contaminate drinking water supplies. Therefore, the characterization of enzymes that reduce chromate, as well as the study of their induction patterns and gene expression are relevant to complete our understanding of chromium metabolism in order to minimize the toxicity of this compound in the environment. The chromate-reducing activities have been located in the cell membrane or in the cytoplasm of many bacteria [ 2 ]. Their ubiquities in many different organisms suggest that they might share a common role in, for example, physiological redox sensing or detoxification. Recently, two novel dimeric flavoproteins with chromate reductase activity, ChrR (from  Pseudomonas putida ) and YieF (from  E. coli ) have been purified and characterized [ 1 ]. These enzymes were able to transform chromate to the less toxic Cr(III). However, while ChrR was not a pure two-electron reducer of chromate, YieF was able to catalyze a three-electron reduction. The role of ChrR and YieF in protection against chromate toxicity was also investigated and the results suggested that both enzymes may have an important role in protection against chromate toxicity [ 1 ]. The ability of some microorganism and their enzymes to remove toxic pollutants has been recently reviewed [ 3 - 6 ]. The identification and characterization of the degradative pathways functioning in microorganism have been the starting point for biotechnological and environmental applications [ 3 ].
Introduction
Chromate reductases are a group of enzymes that catalyze the reduction of toxic and carcinogenic Cr(VI) to the less soluble and less toxic Cr(III). These proteins have recently raised enormous interest because of their central role in mediating chromium toxicity and their potential use in bioremediation and biocatalysis. Chromate (Cr(VI)) is generated as by-product of various industrial processes such as leather tanning, chrome-plating, pigment production and thermonuclear weapon manufacture [ 1 ]. Its high water solubility facilitates a rapid leaching, provoking a wide dispersion capable to contaminate drinking water supplies. Therefore, the characterization of enzymes that reduce chromate, as well as the study of their induction patterns and gene expression are relevant to complete our understanding of chromium metabolism in order to minimize the toxicity of this compound in the environment.The chromate-reducing activities have been located in the cell membrane or in the cytoplasm of many bacteria [ 2 ]. Their ubiquities in many different organisms suggest that they might share a common role in, for example, physiological redox sensing or detoxification. Recently, two novel dimeric flavoproteins with chromate reductase activity, ChrR (from  Pseudomonas putida ) and YieF (from  E. coli ) have been purified and characterized [ 1 ]. These enzymes were able to transform chromate to the less toxic Cr(III). However, while ChrR was not a pure two-electron reducer of chromate, YieF was able to catalyze a three-electron reduction. The role of ChrR and YieF in protection against chromate toxicity was also investigated and the results suggested that both enzymes may have an important role in protection against chromate toxicity [ 1 ].Pseudomonas putidaE. coliThe ability of some microorganism and their enzymes to remove toxic pollutants has been recently reviewed [ 3 - 6 ]. The identification and characterization of the degradative pathways functioning in microorganism have been the starting point for biotechnological and environmental applications [ 3 ].Discussion The intensive industrial and agricultural development has been considered as responsible for a widespread contamination of soil, air and groundwater with toxic pollutants, which are harmful for human health and the environment [ 6 ]. These contaminants enter the environment through different paths, which may include direct application, combustion processes and natural emissions. Major contaminants are polycyclic aromatic hydrocarbons (PAHs), petroleum hydrocarbons, phenols, polychlorinated biphenyls, azo dyes, organophosphorus pesticides and heavy metals [ 3 ]. In particular, Cr(VI) is a common pollutant due to the use of chromium compounds in tanning and other industries. Chromate shares structural similarities with sulphate ion (SO 4  -2 ) and may be introduced in eukaryotic and bacterial cells by the sulphate transport system [ 1 ]. In bacteria, flavoenzymes such as glutathione reductase reduce Cr(VI) by a one electron transfer leading to the formation of the highly unstable radical Cr(V) and the flavin semiquinone form of the enzyme. Both species undergo a further redox cycle in which Cr(VI) is re-generated by one-electron transfer to oxygen, producing and accumulating reactive oxygen species (ROS). The appearance of relatively large quantities of ROS, and the consequent oxidative stress are responsible for the toxic effects and cellular damage attributable to the presence of Cr(VI). On the other hand, trivalent chromium Cr(III) is water insoluble, less bio-available and less toxic [ 1 ]. Thus, the strategies employed to eliminate chromate toxicity would involve its reduction to Cr(III) by chemical or biological means. While chemical methods are expensive at the large scale required to decontaminate waste sites, microorganisms are commonly used for environmental purposes through the exploitation of their natural catalytic activities. Enzymatic treatments have a minimal impact on ecosystems, as they present no risk of biological contamination. Furthermore, enzymes can act over a wide range of pH, temperature and ionic strength and also may be active in the presence of high concentrations of organic solvents in which major pollutant molecules are soluble [ 6 ]. Several bacterial enzymes that can be used in bioremediation have been described; they include mainly oxidative enzymes such as mono-and dioxygenases but their use is restricted by the need of cofactors, which can only be efficiently regenerated inside the microorganism [ 6 ]. In the last two decades bioremediation has explored the use of the catalytic machinery of white rot fungi to remove toxic pollutants. White rot fungi comprise all those fungi capable to degrade lignin, a polyphenolic polymer highly resistant to bacterial biodegradation. Many strains from the genera  Pleurotus, Bjerkandera, Phanerochaete , and  Trametes  produce extracellular enzymes with ligninolytic activity. These enzymes are often referred to as lignin-modifying enzymes and include mainly Lignin peroxidase, Manganese dependent peroxidase and laccase [ 4 ], though some authors have reported other related enzymes such as a Mn-independent MnP activity [ 7 ]. Besides lignin-modifying enzymes, several other enzymes such as the heme-containing peroxidases, chloroperoxidase and horseradish peroxidase, and the non-enzymatic hemeproteins, hemoglobin and cytochrome  c , are able to oxidize organic compounds in the presence of hydrogen peroxide. An interesting feature of these enzymes is their remarkable low specificity towards substrates that arises from their own catalytic mechanism.  In vivo , peroxidases use endogenous low-molecular weight compounds, called mediators, to generate free radicals capable to carry out a wide variety of reactions such as oxidations, bond cleavage, hydroxylations, polymerization and demethylation [ 4 ]. Several research efforts have been focused on the ability of peroxidases to degrade pollutants such as PAH's, azo dyes and organophosphorus pesticides [ 8 - 10 ]. Strong regulations have been established to push the industrial sector to develop new programs destined to a greater environmental care. Nowadays, industry is strongly dependent on petroleum and its derivatives as a source for raw materials and energy. There are still large reserves of crude oil, which are heavy oils with a high content of sulphur and heavy metals. The use of these fuels generate a great pollution, being one of their most important environmental impacts the formation of the acid rain which takes place by the sulphur oxide production during combustion. Redox enzymes may encounter fields of application not only in the bioremediation of polluted environments, but also in the development of novel clean technologies to avoid or diminish the environmental contamination. Biocatalytic methods for sulphur removal from straight-run diesel fuel have been developed [ 11 ]. The removal of heavy metals from the petrophorphyrin-rich fraction of asphaltenes has also been reported [ 12 , 13 ]. Thus, enzymes can play an important role in the development of alternative or complementary biotechnological processes with potential application in polluting industries. Despite their potential application in bioremediation and clean processes, the activity of oxidative enzymes may be limited, among other factors, by the low bioavailability of the pollutants and by the relatively low operational stability of the enzyme under the environmental conditions required to carry out the bioremediation. Several strategies to increase the catalytic activity of peroxidases have been proposed, including chemical modification of the enzyme [ 14 , 15 ] and genetic tools [ 16 ]. Recently, with the cloning and expression in suitable hosts, larger amounts of the desired enzymes may be produced, facilitating their characterization and their direct use in environmental applications. Further, through the use of novel techniques such as directed molecular evolution [ 17 ], proteins designed specifically for bioremediation could be made available in a not distant future. In the last few years there has been an extensive research in the application of laboratory evolution for tailoring redox enzymatic systems (laccases, peroxidases, cytochrome P450 monooxygenases) to improve their activities and stabilities against temperature or organic solvents [ 18 - 20 ]. The application of this powerful approach for bioremediation issues is coming up, but first a big effort in the high-throughput (HTP) screening methodology must be done. So far, little has been reported on the optimisation of suitable HTP for the detection of xenobiotics [ 21 ]. Therefore, the success in the enzyme evolution for environmental issues will be highly dependent on the automation of HTP. Microbial genomics is a new emerging field that enables us to look at parts of the environment that were, until recently, masked to us. Present estimations suggest that more than 99% of the microorganisms in most environments (also those subjected to chronic contamination) are not amenable to grow in pure culture, and thus very little is known about their enzymatic activities. We can now access the genomes of non-culturable microorganisms through creating the "so-called" metagenomic-libraries and identify protein-coding genes and biochemical pathways that will shed some light on their properties and function [ 22 ]. New enzymatic systems found in contaminated areas can be used as parental types for some rounds of directed evolution with the main aim of improving the catalytic performance for their use towards solving a broad range of environmental problems.
Discussion
The intensive industrial and agricultural development has been considered as responsible for a widespread contamination of soil, air and groundwater with toxic pollutants, which are harmful for human health and the environment [ 6 ]. These contaminants enter the environment through different paths, which may include direct application, combustion processes and natural emissions. Major contaminants are polycyclic aromatic hydrocarbons (PAHs), petroleum hydrocarbons, phenols, polychlorinated biphenyls, azo dyes, organophosphorus pesticides and heavy metals [ 3 ]. In particular, Cr(VI) is a common pollutant due to the use of chromium compounds in tanning and other industries. Chromate shares structural similarities with sulphate ion (SO 4  -2 ) and may be introduced in eukaryotic and bacterial cells by the sulphate transport system [ 1 ]. In bacteria, flavoenzymes such as glutathione reductase reduce Cr(VI) by a one electron transfer leading to the formation of the highly unstable radical Cr(V) and the flavin semiquinone form of the enzyme. Both species undergo a further redox cycle in which Cr(VI) is re-generated by one-electron transfer to oxygen, producing and accumulating reactive oxygen species (ROS). The appearance of relatively large quantities of ROS, and the consequent oxidative stress are responsible for the toxic effects and cellular damage attributable to the presence of Cr(VI). On the other hand, trivalent chromium Cr(III) is water insoluble, less bio-available and less toxic [ 1 ]. Thus, the strategies employed to eliminate chromate toxicity would involve its reduction to Cr(III) by chemical or biological means.While chemical methods are expensive at the large scale required to decontaminate waste sites, microorganisms are commonly used for environmental purposes through the exploitation of their natural catalytic activities. Enzymatic treatments have a minimal impact on ecosystems, as they present no risk of biological contamination. Furthermore, enzymes can act over a wide range of pH, temperature and ionic strength and also may be active in the presence of high concentrations of organic solvents in which major pollutant molecules are soluble [ 6 ].Several bacterial enzymes that can be used in bioremediation have been described; they include mainly oxidative enzymes such as mono-and dioxygenases but their use is restricted by the need of cofactors, which can only be efficiently regenerated inside the microorganism [ 6 ]. In the last two decades bioremediation has explored the use of the catalytic machinery of white rot fungi to remove toxic pollutants. White rot fungi comprise all those fungi capable to degrade lignin, a polyphenolic polymer highly resistant to bacterial biodegradation. Many strains from the genera  Pleurotus, Bjerkandera, Phanerochaete , and  Trametes  produce extracellular enzymes with ligninolytic activity. These enzymes are often referred to as lignin-modifying enzymes and include mainly Lignin peroxidase, Manganese dependent peroxidase and laccase [ 4 ], though some authors have reported other related enzymes such as a Mn-independent MnP activity [ 7 ]. Besides lignin-modifying enzymes, several other enzymes such as the heme-containing peroxidases, chloroperoxidase and horseradish peroxidase, and the non-enzymatic hemeproteins, hemoglobin and cytochrome  c , are able to oxidize organic compounds in the presence of hydrogen peroxide. An interesting feature of these enzymes is their remarkable low specificity towards substrates that arises from their own catalytic mechanism.  In vivo , peroxidases use endogenous low-molecular weight compounds, called mediators, to generate free radicals capable to carry out a wide variety of reactions such as oxidations, bond cleavage, hydroxylations, polymerization and demethylation [ 4 ]. Several research efforts have been focused on the ability of peroxidases to degrade pollutants such as PAH's, azo dyes and organophosphorus pesticides [ 8 - 10 ].Pleurotus, Bjerkandera, PhanerochaeteTrametescIn vivoStrong regulations have been established to push the industrial sector to develop new programs destined to a greater environmental care. Nowadays, industry is strongly dependent on petroleum and its derivatives as a source for raw materials and energy. There are still large reserves of crude oil, which are heavy oils with a high content of sulphur and heavy metals. The use of these fuels generate a great pollution, being one of their most important environmental impacts the formation of the acid rain which takes place by the sulphur oxide production during combustion. Redox enzymes may encounter fields of application not only in the bioremediation of polluted environments, but also in the development of novel clean technologies to avoid or diminish the environmental contamination. Biocatalytic methods for sulphur removal from straight-run diesel fuel have been developed [ 11 ]. The removal of heavy metals from the petrophorphyrin-rich fraction of asphaltenes has also been reported [ 12 , 13 ]. Thus, enzymes can play an important role in the development of alternative or complementary biotechnological processes with potential application in polluting industries.Despite their potential application in bioremediation and clean processes, the activity of oxidative enzymes may be limited, among other factors, by the low bioavailability of the pollutants and by the relatively low operational stability of the enzyme under the environmental conditions required to carry out the bioremediation. Several strategies to increase the catalytic activity of peroxidases have been proposed, including chemical modification of the enzyme [ 14 , 15 ] and genetic tools [ 16 ]. Recently, with the cloning and expression in suitable hosts, larger amounts of the desired enzymes may be produced, facilitating their characterization and their direct use in environmental applications. Further, through the use of novel techniques such as directed molecular evolution [ 17 ], proteins designed specifically for bioremediation could be made available in a not distant future. In the last few years there has been an extensive research in the application of laboratory evolution for tailoring redox enzymatic systems (laccases, peroxidases, cytochrome P450 monooxygenases) to improve their activities and stabilities against temperature or organic solvents [ 18 - 20 ]. The application of this powerful approach for bioremediation issues is coming up, but first a big effort in the high-throughput (HTP) screening methodology must be done. So far, little has been reported on the optimisation of suitable HTP for the detection of xenobiotics [ 21 ]. Therefore, the success in the enzyme evolution for environmental issues will be highly dependent on the automation of HTP.Microbial genomics is a new emerging field that enables us to look at parts of the environment that were, until recently, masked to us. Present estimations suggest that more than 99% of the microorganisms in most environments (also those subjected to chronic contamination) are not amenable to grow in pure culture, and thus very little is known about their enzymatic activities. We can now access the genomes of non-culturable microorganisms through creating the "so-called" metagenomic-libraries and identify protein-coding genes and biochemical pathways that will shed some light on their properties and function [ 22 ]. New enzymatic systems found in contaminated areas can be used as parental types for some rounds of directed evolution with the main aim of improving the catalytic performance for their use towards solving a broad range of environmental problems.Conclusion The even more strict regulations on hazard wastes has forced to the development of new environmentally compatible strategies to substitute or complement the conventional ones. Chemical technologies are expensive when applied to large scale and in many cases are technically not feasible. On the other side, by exploitation of the huge diversity of natural activities and metabolic pathways presented by microorganisms, new strategies can be envisaged. The use of oxidative enzymes as biocatalysts for environmental purposes presents a promising potential due to their low specificity and low energetic requirements. However, further characterization of new biocatalysts is needed. The use of novel technologies such as molecular directed evolution may have a large impact in the tailoring and further application of enzymes not only for bioremediation but also for the development of friendly environmental technologies.
Conclusion
The even more strict regulations on hazard wastes has forced to the development of new environmentally compatible strategies to substitute or complement the conventional ones. Chemical technologies are expensive when applied to large scale and in many cases are technically not feasible. On the other side, by exploitation of the huge diversity of natural activities and metabolic pathways presented by microorganisms, new strategies can be envisaged. The use of oxidative enzymes as biocatalysts for environmental purposes presents a promising potential due to their low specificity and low energetic requirements. However, further characterization of new biocatalysts is needed. The use of novel technologies such as molecular directed evolution may have a large impact in the tailoring and further application of enzymes not only for bioremediation but also for the development of friendly environmental technologies.