Phage lytic enzymes as therapy for antibiotic-resistant Streptococcus pneumoniae infection in a murine sepsis model

Isabel Jado1, Rubens López2,*, Ernesto García2, Asunción Fenoll1, Julio Casal1 and Pedro García2 on behalf of the Spanish Pneumococcal Infection Study Network§

1 Centro Nacional de Microbiología, Instituto de Salud Carlos III, 28220 Majadahonda, Madrid; 2 Departamento de Microbiología Molecular, Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, 28040 Madrid, Spain

Received 16 July 2003; returned 15 September 2003; revised 18 September 2003; accepted 25 September 2003


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objectives: Phage-coded lysins, i.e. murein hydrolases, are enzymes that destroy the cell wall of bacteria. A rapid killing of Streptococcus pneumoniae in the nasopharynx of mice has been described recently using a phage-coded murein hydrolase (enzybiotic). The in vivo effects of a dose-ranging treatment, using either of the phage-coded lytic enzymes Cpl-1 lysozyme or the Pal amidase, have been investigated here in a murine sepsis model.

Methods: Purified Pal amidase and/or Cpl-1 lysozyme were used alone or in combination. These enzymes were injected intraperitoneally at different times after challenge with 5 x 107 cfu of a type 6B, antibiotic-resistant S. pneumoniae clinical isolate.

Results: Animals challenged with 5 x 107 cfu of this strain alone died within 72 h, whereas a single intraperitoneal injection of Cpl-1 or Pal (200 µg; 1100 U) administered 1 h after the bacterial challenge was sufficient to effectively protect the mice, according to unpaired t-test (P < 0.0001). Bacteraemia in unprotected mice reached colony counts >107 cfu/mL, whereas the mean colony count in lysin-protected animals was <106 cfu/mL over time and ultimately became undetectable. Interestingly, a synergic effect in vivo was observed with the combined use of 2.5 µg each of Cpl-1 and Pal.

Conclusions: Our findings suggest strongly that phage lysins protect animals from bacteraemia and death. Moreover, the simultaneous attack of the pneumococcal peptidoglycan by a lysozyme and an amidase leads to a remarkable effect through enhanced destruction of the bacterial cell wall. The benefits of therapy with enzybiotics against pneumococcus reported here might warrant the examination of alternative strategies for the treatment of diseases caused by clinically relevant pathogens.

Keywords: enzybiotics, pneumococcus, phage, lytic enzymes


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Streptococcus pneumoniae is an important human pathogen. It is currently a leading cause of pneumonia, meningitis and bloodstream infections in the elderly and is one of the main pathogens responsible for middle ear infections in children. In addition, pneumococcal ß-lactam antibiotic resistance has increased to 44% in the USA1 and 41% in Spain.2 Most of this resistance has resulted from the spread of multiply resistant clones that originated through interspecific gene transfer events involving pneumococci and closely related species colonizing the same habitat (e.g. the nasopharynx), leading to the acquisition of, for example, low-affinity penicillin-binding proteins.3 It has been reported previously that among the mechanisms of DNA transfer, lysogenic conversion by bacteriophages appears to be advantageous in several bacterial systems. Moreover, the potential use of bacteriophages for therapy and prophylaxis of antibiotic-resistant bacteria has been also suggested.4

The last step of phage infection is the release of mature phage particles by hydrolysis of the bacterial cell wall. The biological properties of several lytic and temperate phages infecting S. pneumoniae have been reviewed.5 Most often, phages encode murein-degrading enzymes that hydrolyse either the glycosidic linkages between the amino sugars of the peptidoglycan (glucosaminidases, lysozymes), the N-acetylmuramoyl–L-alanine amide bond between the glycan strand and the cross-linking peptide (amidases), or the inter-peptide bridge linkages (endopeptidases).6 Peptidoglycan (murein) hydrolases represent one of the most widely distributed enzyme families in nature, and their participation in a series of important biological events (cell division, daughter cell separation, the irreversible effects caused by ß-lactam antibiotics, etc.) is well recognized.

In S. pneumoniae, we have analysed in detail the lytic enzymes encoded by four different bacteriophages: Cp-1,7 HB-3,8 EJ-19 and Dp-1.10 Genes encoding these lytic enzymes have been cloned by taking advantage of their homology with the lytA gene encoding the host LytA amidase. Sequence comparisons revealed that the host- and phage-coded enzymes have a highly similar C-terminal domain that is responsible for the binding to the choline residues present in the teichoic acids of the cell wall substrate.1113 We proposed that this aminoalcohol served as an element of selective pressure to preserve the substrate-recognition domain of most of the pneumococcal lytic enzymes characterized so far. The enzyme coded by Cp-1 has been identified as a lysozyme (Cpl-1), whereas those coded by Dp-1, HB-3 and EJ-1 (Pal, Hbl and Ejl, respectively) are amidases (Figure 1).



View larger version (46K):
[in this window]
[in a new window]
 
Figure 1. Characteristics of the Cpl-1 lysozyme and the Pal amidase. (a) Electron micrographs of the podovirus Cp-1 and the siphovirus Dp-1, from which the lytic genes were isolated. (b) Schematic representation of the pneumococcal cell wall, indicating the chemical bonds cleaved by the lytic enzymes used in this work. (c) A scheme of the structural organization of the Cpl-1 and Pal proteins. White squares represent the seven C-terminal repeats involved in recognition of the choline residues present in the cell wall substrate. Black and striped rectangles correspond to the N-terminal domains where the active centre of the enzymes resides.10,20 (d) SDS–PAGE analyses of the purified Pal (by 10% SDS–PAGE, left) and Cpl-1 (by 15% SDS–PAGE, right) lytic enzymes. Lanes 1 and 3, total cell extracts from E. coli DH5{alpha} (pMSP11) and DH5{alpha} (pCIP100), respectively; lanes 2 and 4, purified Pal and Cpl-1 obtained by affinity chromatography on DEAE-cellulose. G, N-acetylglucosamine; M, N-acetylmuramic acid.

 
Phage lytic enzymes have recently been used successfully as tools to destroy the cell wall of pathogenic bacteria such as Streptococcus pyogenes (GAS),14 S. pneumoniae15 and Bacillus anthracis,16 which has led to the designation of these muralytic proteins as enzybiotics.14 The idea of exploiting a phage lytic enzyme that specifically recognizes choline, which presumably will account for target specificity, has been successfully used to eliminate the nasopharyngeal carriage of pneumococci.15 We decided to expand upon previous work in the pneumococcal system by studying the potential of phage amidases and lysozymes to rescue (cure) bacteraemia produced in a murine model by an antibiotic-resistant 6B strain of S. pneumoniae, the most common serotype isolated from children with bacteraemia.17


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The study was performed in accordance with the prevailing regulations regarding the care and use of laboratory animals in the European Union.

Bacteria, bacteriophages and growth conditions

The strain used in this study was S. pneumoniae 541, a serotype 6B clinical isolate resistant to ß-lactam antibiotics (penicillin MIC and MBC, 2 and 4 mg/L, respectively), was selected for this study on the basis of its resistance to ß-lactams and its virulence in mice.18 S. pneumoniae was grown in C medium19 supplemented with yeast extract (0.8 mg/mL; Difco Laboratories) (C+Y medium) at 37°C without shaking and the growth was monitored with a Hach 2100N nephelometer. Escherichia coli DH5{alpha} (pCIP100)20 and DH5{alpha} (pMSP11),10 grown in Luria–Bertani (LB) medium at 37°C with shaking, were the sources for production of Cpl-1 and Pal, respectively.

Purification of Cpl-1 and Pal

Cpl-1 and Pal enzymes were purified by affinity chromatography in DEAE-cellulose as previously described for other choline-binding proteins.21 Typically, E. coli cultures harbouring either pCIP100 or pMSP11 were grown in LB medium supplemented with ampicillin (100 mg/L) and 0.4 mM isopropyl-ß-D-thiogalactopyranoside at 37°C for 24 h with shaking, harvested by centrifugation and broken in a French press cell. The crude extracts were ultracentrifuged (100 000g, for 1 h at 4°C) and the total soluble protein in the supernatant was loaded onto a DEAE-cellulose column (8 x 2.5 cm) and washed with three volumes of 20 mM sodium phosphate buffer pH 6.9 (or pH 6.0 for Cpl-1) containing 1.5 M NaCl. The same buffer containing 2% choline was then added and fractions were recovered. The purity of the lytic enzymes was checked by SDS–PAGE (Figure 1d) and by western blot analysis (not shown), as already reported.10,20 The purified enzymes were dialysed against 20 mM sodium phosphate buffer pH 6.9 (or pH 6.0 for Cpl-1). Finally, the enzyme solutions were adjusted to 1 mg/mL, and filter-sterilized using 0.20 µm Iwaki membrane filters. Purified Cpl-1 and Pal enzymes had approximately the same specific activity (110 000 U/mg) (see below).

Preparation of radioactively labelled cell walls and assay for enzymatic activity

Pneumococcal cell walls labelled with [methyl-3H]choline were prepared by biosynthetic labelling of the bacteria. The standard assay conditions for the hydrolysis of the labelled pneumococcal cell walls, as well as the determination of the enzymatic activities, have been described elsewhere.22 Briefly, pneumococcal cell walls were incubated with enzymatic extracts or purified proteins at 37°C for 10 min and the reaction stopped upon the addition of formaldehyde and BSA. Non-digested murein was separated by centrifugation at 12 000g for 15 min, and the digested (solubilized) substrate was quantified by measuring the radioactivity in the supernatant. One unit of enzymatic activity is defined as the amount of enzyme that catalyses the hydrolysis of 1 µg (~715 cpm) of pneumococcal cell walls in 10 min.

Animals

Eight- to 12-week-old female BALB/c mice weighing 19 to 22 g were used.

Determination of MLD and challenge dose

Groups of five mice per experiment were injected intraperitoneally with different inocula ranging from 105 to 108 cfu/mL to determine the minimal dose that produces 100% mortality rate over a 7 day follow-up period. Experimental details of this procedure have been documented recently.18

Immune response to bacteria and phage lytic enzymes

Immune response to specific 6B capsular polysaccharide was measured by a standardized ELISA protocol (Workshop at the CDC, 1996).18 Serum samples were obtained by cardiac puncture 10 days after bacterial challenge with S. pneumoniae 541 strain and enzybiotics. Specific IgG antibodies to capsular 6B polysaccharide were determined for preimmune and immune sera.

Statistical analysis

Unpaired t-test values (P) were calculated for survival of mice or not with the lytic enzymes using a program available from http://www.physics. csbsju.edu/stats/t-test.html. P values of <0.001 were considered highly significant.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Rescue of mice from antibiotic-resistant S. pneumoniae bacteraemia using either a phage-coded lysozyme or an amidase

The use of pneumococcal lysins to produce bacterial killing in in vitro assays, the so-called ‘lysis from without’, was previously shown to be successful in liberating the phage progeny from bacterial strains deficient in lytic enzymes.23 The effect of Cpl-1 lysozyme or Pal amidase on the pneumococcal strain 541 was tested here using the mouse sepsis model of infection. Cpl-1 (39.1 kDa) and Pal (34.6 kDa) enzymes have homologous C-terminal choline-binding domains, whereas their different N-terminal moieties determine the biochemical specificity (Figure 1). We first determined that inoculation intraperitoneally with 5 x 107 cfu (2x MLD) of the clinical isolate S. pneumoniae 541 was sufficient to produce a near 100% mortality rate over a 3 day follow-up period (Figure 2a) (mean survival rate per mouse and standard deviation of the mean were 7.14 x 10–2 and 0.26, respectively). Next, we performed a set of experiments with single doses of Cpl-1 or Pal administered intraperitoneally shortly (10 min) after the challenge with 2x MLD of bacteria. After 24 h a dose-dependent effect on the state of the animals was visible. When 40 µg of either Cpl-1 or Pal was used a pronounced therapeutic effect was observed: all mice were fully recovered and the establishment of bacteraemia was greatly reduced (unpaired t-test, P < 0.001) (unpublished data), indicating that both enzymes were capable of destroying the infecting bacteria in the peritoneal cavity. This test shows that, in the short-term, the intraperitoneal cavity mimics the intranasal model of infection previously developed for S. pneumoniae.15



View larger version (12K):
[in this window]
[in a new window]
 
Figure 2. Rescue of mice from lethal S. pneumoniae 541 infection by Pal amidase and Cpl-1 lysozyme. Mice were inoculated intraperitoneally with 5 x 107 cfu at time 0. (a) One hour later, 200 µg (open circles), 10 µg (filled circles) or 5 µg of Pal (open diamonds), or 200 µg (open triangles), 10 µg (filled triangles) or 5 µg (crosses) of Cpl-1 were injected into the mouse peritoneal cavity. Control mice (filled squares) were treated only with pneumococcus. Each symbol represents the average of three experiments. (b and c) At the indicated times, bacterial counts (cfu/mL) in four mice either untreated (b) or treated with 200 µg of Cpl-1 (c) were determined from peripheral blood samples (5 µL) taken from the caudal vein. The arrow indicates the moment at which the lysin was injected (1 h after challenge). In (b) and (c) each curve indicates the bacteraemic counts of a single mouse. Black crosses indicate a dead animal.

 
Experimental controls (not shown) demonstrated that high bacteraemic rates (>5 x 105 cfu/mL) were normally attained ~45 min after the challenge, reaching a plateau 15 min later. Consequently, the injection of the phage lytic enzymes was carried out at least 1 h after the inoculation of pneumococci. The minimal effective dose of Cpl-1 and Pal (10 µg per mouse) (according to unpaired t-test, P < 0.0001) was enough to protect two to three out of four mice tested (Figure 2a). Lytic enzyme doses <10 µg were not protective; all the animals died. Heat-inactivated Cpl-1 or Pal did not show any protective effect under the conditions used. Moreover, toxic effects were not observed upon intraperitoneal administration of 200 µg of either Cpl-1 or Pal (not shown).

Rescue is associated with a significant decrease in bacterial titre

The mean bacterial titre in the blood of mice inoculated with 5 x 107 cfu S. pneumoniae 541 was ~107–108 cfu/mL until death (Figure 2b). Since the rise in the colony counts in blood is directly related to an increase in the probability of death,17 the ability of Pal or Cpl-1 to reduce colony counts in blood was investigated. When 200 µg of either lysin were injected intraperitoneally 1 h after the challenge, a drop of ~4 log units was already observed 2 h later, followed by small fluctuations of bacterial titre, whereas after 4–5 days, bacterial clearance was complete, or nearly so (Figure 2c).

Synergic effect of a combination of phage lytic enzymes

A recent study using S. pneumoniae cells revealed the existence of a synergic killing effect between Cpl-1 and Pal in vitro.24 We decided to analyse the combined effect of these enzymes in the pneumococcal sepsis model. The combined use of 2.5 µg of each Cpl-1 and Pal injected intraperitoneally 1 h after the challenge with the S. pneumoniae 541 strain completely rescued the mice from infection (Figure 3a), whereas a single injection of 5 µg of either Cpl-1 or Pal did not protect the animals, as shown above (Figure 2a). These findings were statistically significant according to an unpaired t-test (P < 0.0001). This synergic effect of the lysozyme and the amidase has been tested using various amounts and application times, with similar results. Again, mice rescue was associated with a drop in bacterial counts in blood a few hours after injection of both lysins (Figure 3b). When the mice were treated with 2.5 µg of both Cpl-1 and Pal, a near complete bacterial clearance was achieved by the sixth day, although a transitory increase in bacterial counts took place by day 2–3.



View larger version (15K):
[in this window]
[in a new window]
 
Figure 3. Synergic therapeutic effect of the combined use of Pal and Cpl-1. (a) One hour after injection of mice with 5 x 107 cfu (at time 0), 2.5 µg each of Pal and Cpl-1 (filled circles), or 5 µg of either Pal (open circles) or Cpl-1 (filled squares) were injected into the mouse peritoneal cavity and survival of mice was followed. (b) The effect of the combined therapy (2.5 µg each of Pal and Cpl-1 administered intraperitoneally 1 h after challenge) in bacteraemia of four mice was estimated as cfu/mL at different times after injection of the lytic enzymes. In (b) each curve indicates the bacteraemia counts of a single mouse.

 
Effect of delay of lysin injection

We carried out additional experiments to determine whether Cpl-1 and/or Pal were capable of protecting mice when injected several hours after the bacterial lethal challenge. Thus, at various time points, ranging from 1 to 6 h, mice received a single intraperitoneal injection of 200 µg of Cpl-1 or Pal. The results of these treatments demonstrated that Cpl-1 and Pal cured mice even when inoculated up to 2 h after S. pneumoniae 541 injection. When the enzymes were injected with a more prolonged delay the protective effect of both enzybiotics was not statistically significant according to unpaired t-test (P > 0.05). When injection was performed 4 h after the challenge, the bacteraemia was already too high and the protective effect was limited to prolonging the life of the severely ill mice for several days, after which time all the animals died (not shown). When treated with 200 µg of either lysin 2 h after challenge, three out of four animals treated with Pal had no bacteraemia (or very low levels) by the sixth day (Figure 4b), corroborating the healthy state of these mice. In the case of Cpl-1, the protective effect extended to the four animals assayed although the bacteraemia level was maintained for a longer time (up to 8 days in some cases) (Figure 4a).



View larger version (15K):
[in this window]
[in a new window]
 
Figure 4. Protective effects of mice from lethal S. pneumoniae 541 infection by delayed administration of Pal or Cpl-1. Mice were injected intraperitoneally with 5 x 107 cfu at time 0 and 2 h later they were treated with 200 µg of either Cpl-1 (a) or Pal (b) and bacteraemia was analysed. Black crosses indicate a dead animal. Each curve indicates the bacteraemic counts of a single mouse.

 
Immune response to Pal and Cpl-1

To address the question of an eventual immune response of mice as a result of protein injection, we inoculated mice with a second dose of Cpl-1 or Pal at different times after the bacterial challenge. The immediate consequence of this second intraperitoneal injection was a decreasing bacteraemia profile of ~2–3 log units, although complete recovery or death appears to depend on the clinical outcome of the animals at the time of second injection (data not shown). Furthermore, we checked the titre of antibodies 10 days after injection with the standard challenge dose of 541 strain and, immediately, 200 µg of Cpl-1 or Pal. Titres of specific IgG antibodies to serotype 6B polysaccharide were measured in parallel for the hyperimmune serum, raised against the 6B S. pneumoniae strain, and preimmune serum. ELISA titres were eight times higher in hyperimmune than preimmune serum (not shown). Immune serum was reactive to the corresponding lysin and several pneumococcal proteins as indicated by western blot analysis (Figure 5). Interestingly, preliminary experiments suggested that when these animals received a second dose of bacterial challenge and enzybiotics 10 days after the first injections, all mice recovered fully. Moreover, no signs of anaphylaxis or adverse side effects were observed (data not shown).



View larger version (15K):
[in this window]
[in a new window]
 
Figure 5. Western blot analysis of immune sera from mice challenged with pneumococci, and injected 10 min later with 200 µg of either Cpl-1 or Pal. Mice were bled 10 days after the challenge to obtain the corresponding sera. Lanes 1 and 4, Cpl-1 (3 µg); lanes 2 and 5, crude sonicated extract prepared from S. pneumoniae 541; lanes 3 and 6, Pal (3 µg). Sera were used at a dilution of 1/250. The molecular size of the standards is indicated on the right.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Pneumococcal bacteraemia is most often fatal for several groups of patients, mainly the immunocompromised, the elderly and children <2 years old. Bacteria usually gain entry into the bloodstream from the nasopharynx, where a carrier state is likely to play a major role in the transmission of S. pneumoniae. It has been postulated recently that the Pal amidase can control and eliminate nasopharyngeal colonization and, consequently, might reduce or prevent infection by this pathogen.15 Similar experimental approaches have been used to detect and kill B. anthracis16 and group A streptococcus infections.14 The results reported here provide new data on the protection offered by phage lytic enzymes by using a mouse sepsis model. The strain 541 used in our experiments, belonging to serotype 6B, was isolated in Spain and is resistant to penicillin, tetracycline, chloramphenicol and erythromycin.18

The ability of Cpl-1 lysozyme and Pal amidase to cure bacteraemic animals is a protein-specific function, as indicated by the observation that rescue did not occur with heat-inactivated enzymes. Irrespective of the enzymatic activity of the enzyme used, the experiments presented here revealed that a single intraperitoneal injection of the corresponding lysin rescued mice from death due to S. pneumoniae even when bacteraemia was already well established (Figure 2). Under the experimental conditions used, we found that effective protection takes place with both enzymes through a wide range of enzyme concentrations or time of intraperitoneal injection after challenge. We observed that effective protection was achieved in mice when a single dose of at least 10 µg of Cpl-1 or Pal was administered 1 h after the MLD of S. pneumoniae 541 strain was inoculated (Figure 2a). The protective effect of both enzybiotics was notable even when the enzymes were injected 2 h after pneumococcal challenge, although the extent of this effect was related to the pre-treatment outcome. Moreover, a complete pneumococcal clearance was observed in most surviving mice after a 6–8 day follow-up period (Figure 4). A single intraperitoneal injection of either lytic enzyme, delayed up to 4 h after challenge (Figure 4), has a partial protective effect by prolonging the life of the severely ill mice for several days. Provided that mice are most susceptible to pneumococcal infections,25 the results obtained in these experiments are encouraging, since our findings and those of others16,26 support the development in a near future of modified procedures to improve the use of lysins in a more efficient way. For example, intravenous administration of the enzymes may confer a superior protection to animals when used later after the challenge.

Our results demonstrate that the simultaneous use of both enzymes, rather than being competitive, enhanced the destruction of the cell wall, and hence shows a synergic lytic action on S. pneumoniae in the murine sepsis model used here. The positive interaction of the two muralytic enzymes could be due to the increased access of these enzymes, with different catalytic domains, to their cleavage sites (Figure 1), as recently suggested.24 Survival rates in the animals that received both enzymes were significantly higher than those of animals receiving only Cpl-1 or Pal (Figure 3). Both enzymes, as most of the rest of cell wall lytic enzymes of the pneumococcal system, specifically recognize the choline component of the envelope of this bacterium through their C-terminal domain.5 The experiments reported here extend these observations to the sepsis model, and also reveal the lack of toxicity of the pneumococcal enzybiotics in mice. Furthermore, the combined use of two lytic enzymes would make more unlikely the intrinsic resistance of the bacteria to these proteins that target essential cell wall molecules, as previously suggested.16 These findings, together with the observations12 that these enzymes exhibit a modular design that facilitates the construction of chimeric enzymes in which bacterial specificities and activities can be combined by enzymatic swapping,27 suggest that these lysins are adaptable for use with different pathogens.

Antibiotic therapeutic effect in infected mice requires an appropriate dosing interval of administration to maintain pharmacological concentrations in serum. Most interestingly, a single intraperitoneal injection of a lytic enzyme(s) was sufficient for a complete clearance of invading pneumococci from surviving mice (Figures 24). This property, referred to as therapeutic efficacy, provides a rapid and specific lytic activity, making these proteins very promising candidates in current antimicrobial therapies.

It has recently been emphasized that during the long history of using phages administered by different routes in several countries there have been virtually no reports of serious complications related to their use.28 As phages are common entities in the environment and regularly consumed in foods, in theory the development of neutralizing antibodies should not be a significant obstacle during the initial treatment of acute infections because the kinetics of phage action or lytic enzymes, as illustrated in this work (Figure 5), are much faster than is the host’s production of neutralizing antibodies.28 In addition, as reported here, improved survival upon lysin injection may enable the host to synthesize neutralizing anti-capsular antibodies that should be protective against a new challenge with a pneumococcal strain of the same or of a cross-reactive serotype. However, it would be prudent to further ensure the safety of lytic enzymes as therapeutic tools before using them as therapeutic agents.

The emergence of antibiotic-resistant bacteria, such as many pneumococci that have spread over several continents, has highlighted the need to explore the potential therapeutic applications of alternative therapies, such as the utilization of bacteriophages,26 or the use of phage-coded lytic enzymes.14 A better understanding of the biology of phages, combined with the availability of highly purified phage lytic enzymes and the experience attained in several Eastern European countries where rigorously controlled trials are currently being carried out,4,28 should facilitate the development of these new therapies.


    Acknowledgements
 
We thank J. L. García for helpful comments and for the critical reading of the manuscript, and W. Sanders for correcting the English version. This work was supported by grants from the Dirección General de Investigación Científica y Técnica (BCM2000-1002) and from Redes Temáticas de Investigación Cooperativa (G03/103 and C03/14) (Ministerio de Sanidad y Consumo).

Spanish Pneumococcal Infection Study Network (G03/103). General coordination: Román Pallarés (rpallares{at}bell.ub.es). Participants and centres: Ernesto García (Centro de Investigaciones Biológicas, Madrid); Julio Casal, Asunción Fenoll, Adela G. de la Campa (Centro Nacional de Microbiología, Instituto de Salud Carlos III, Madrid); Emilio Bouza (Hospital Gregorio Marañón, Madrid); Fernando Baquero (Hospital Ramón y Cajal, Madrid); Francisco Soriano, José Prieto (Fundación Jiménez Díaz y Facultad de Medicina de la Universidad Complutense, Madrid); Román Pallarés, Josefina Liñares (Hospital Universitari de Bellvitge, Barcelona); Javier Garau, Javier Martínez Lacasa (Hospital Mutua de Terrassa, Barcelona); Cristina Latorre (Hospital Sant Joan de Deu, Barcelona); Emilio Pérez-Trallero (Hospital Donostia, San Sebastián); Juan García de Lomas (Hospital Clínico, Valencia); and Ana Fleites (Hospital Central de Asturias).


    Footnotes
 
* Corresponding author. Tel: +34-91-8373112; Fax: +34-91-5360432; E-mail: ruben{at}cib.csic.es Back

§ Members of the Spanish Pneumococcal Infection Study Network are listed in the Acknowledgements. Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Musser, J. M. & Kaplan, S. L. (2001). Pneumococcal research transformed. New England Journal of Medicine 345, 1206–7.[Free Full Text]

2 . Oteo, J., Alós, J. I. & Gómez-Garcés, J. L. (2001). Antimicrobial resistance of Streptococcus pneumoniae in 1999–2000 in Madrid (Spain): multicenter surveillance study. Journal of Antimicrobial Chemotherapy 47, 215–8.[Abstract/Free Full Text]

3 . Hakenbeck, R., Grebe, T., Zänher, D. et al. (1999). ß-lactam resistance in Streptococcus pneumoniae: penicillin-binding proteins and non-penicillin-binding proteins. Molecular Microbiology 33, 673–8.[CrossRef][ISI][Medline]

4 . Carlton, R. M. (1999). Phage therapy: past history and future prospects. Archivum Immunologiae & Therapiae Experimentalis 47, 267–74.

5 . García, P., Martín, A. C. & López, R. (1997). Bacteriophages of Streptococcus pneumoniae: a molecular approach. Microbial Drug Resistance 3, 165–76.[ISI][Medline]

6 . López, R., García, E., García, P. et al. (1997). The pneumococcal cell wall degrading enzymes: a modular design to create new lysins? Microbial Drug Resistance 3, 199–211.[ISI][Medline]

7 . García, J. L., García, E., Arrarás, A. et al. (1987). Cloning, purification, and biochemical characterization of the pneumococcal bacteriophage Cp-1 lysin. Journal of Virology 61, 2573–80.[ISI][Medline]

8 . Romero, A., López, R. & García, P. (1990). Characterization of the pneumococcal bacteriophage HB-3 amidase: cloning and expression in Escherichia coli. Journal of Virology 64, 137–42.[ISI][Medline]

9 . Díaz, E., López, R. & García, J. L. (1992). EJ-1, a temperate bacteriophage of Streptococcus pneumoniae with a Myoviridae morphotype. Journal of Bacteriology 174, 5516–25.[Abstract]

10 . Sheehan, M. M., García, J. L., López, R. et al. (1997). The lytic enzyme of the pneumococcal phage Dp-1: a chimeric lysin of intergeneric origin. Molecular Microbiology 25, 717–25.[ISI][Medline]

11 . Fernández-Tornero, C., López, R., García, E. et al. (2001). A novel solenoid fold in the cell wall anchoring domain of the pneumococcal virulence factor LytA. Nature Structural Biology 8, 1020–4.[CrossRef][ISI][Medline]

12 . García, E., García, J. L., García, P. et al. (1988). Molecular evolution of lytic enzymes of Streptococcus pneumoniae and its bacteriophages. Proceedings of the National Academy of Sciences, USA 85, 914–8.[Abstract]

13 . Hermoso, J. A., Monterroso, B., Albert, A. et al. (2003). Structural basis for selective recognition of pneumococcal cell wall by modular endolysin from phage Cp-1. Structure 11, 1239–49.[CrossRef][ISI][Medline]

14 . Nelson, D., Loomis, L. & Fischetti, V. A. (2001). Prevention and elimination of upper respiratory colonization of mice by group A streptococci by using a bacteriophage lytic enzyme. Proceedings of the National Academy of Sciences, USA 98, 4107–12.[Abstract/Free Full Text]

15 . Loeffler, J. M., Nelson, D. & Fischetti, V. A. (2001). Rapid killing of Streptococcus pneumoniae with a bacteriophage cell wall hydrolase. Science 294, 2170–2.[Abstract/Free Full Text]

16 . Schuch, R., Nelson, D. & Fischetti, V. A. (2002). A bacteriolytic agent that detects and kills Bacillus anthracis. Nature 418, 884–9.[CrossRef][ISI][Medline]

17 . Yuste, J., Giménez, M. J., Jado, I. et al. (2001). Enhanced decrease of blood colony counts by specific anti-pneumococcal antibodies in the presence of subinhibitory concentrations of amoxicillin. Journal of Antimicrobial Chemotherapy 48, 594–5.[Free Full Text]

18 . Casal, J., Aguilar, L., Jado, I. et al. (2002). Effects of specific antibodies against Streptococcus pneumoniae on pharmacodynamic parameters of ß-lactams in a mouse sepsis model. Antimicrobial Agents and Chemotherapy 46, 1340–4.[Abstract/Free Full Text]

19 . Lacks, S. (1966). Integration efficiency and genetic recombination in pneumococcal transformation. Genetics 53, 207–35.[Free Full Text]

20 . Sanz, J. M., Díaz, E. & García, J. L. (1992). Studies on the structure and function of the N-terminal domain of the pneumococcal murein hydrolases. Molecular Microbiology 6, 921–31.[ISI][Medline]

21 . Sánchez-Puelles, J. M., Sanz, J. M., García, J. L. et al. (1992). Immobilization and single-step purification of fusion proteins using DEAE-cellulose. European Journal of Biochemistry 203, 153–9.[Abstract]

22 . Mosser, J. L. & Tomasz, A. (1970). Choline-containing teichoic acid as a structural component of pneumococcal cell wall and its role in sensitivity to lysis by an autolytic enzyme. Journal of Biological Chemistry 245, 287–98.[Abstract/Free Full Text]

23 . Ronda, C., López, R., Tapia, A. et al. (1977). Role of the pneumococcal autolysin (murein hydrolase) in the release of phage progeny bacteriophage and in the bacteriophage-induced lysis of the host cells. Journal of Virology 21, 366–74.[ISI][Medline]

24 . Loeffler, J. M. & Fischetti, V. A. (2003). Synergistic lethal effect of a combination of phage lytic enzymes with different activities on penicillin-sensitive and -resistant Streptococcus pneumoniae strains. Antimicrobial Agents and Chemotherapy 47, 375–7.[Abstract/Free Full Text]

25 . Briles, D. E., Crain, M. J., Gray, B. M. et al. (1992). Strong association between capsular type and virulence for mice among human isolates of Streptococcus pneumoniae. Infection and Immunity 60, 111–6.[Abstract]

26 . Biswas, B., Adhya, S., Washart, P. et al. (2002). Bacteriophage therapy rescues mice bacteremic from a clinical isolate of vancomycin-resistant Enterococcus faecium. Infection and Immunity 70, 204–10.[Abstract/Free Full Text]

27 . Díaz, E., López, R. & García, J. L. (1990). Chimeric phage-bacterial enzymes: a clue to the modular evolution of genes. Proceedings of the National Academy of Sciences, USA 87, 8125–9.[Abstract]

28 . Sulakvelidze, A., Alavidze, Z. & Morris, J. G. (2001). Bacteriophage therapy. Antimicrobial Agents and Chemotherapy 45, 649–59.[Free Full Text]