Letters in Applied Microbiology ISSN 0266-8254
ORIGINAL ARTICLE
Periodontal disease as reservoir for multi-resistant and hydrolytic enterobacterial species M.O. Gonc¸alves, W.P. Coutinho-Filho, F.P. Pimenta, G.A. Pereira, J.A.A. Pereira, A.L. Mattos-Guaraldi and R. Hirata, Jr Faculdade de Cieˆncias Me´dicas, Disciplina de Microbiologia e Imunologia, Programa de Po´s-graduac¸a˜o em Cieˆncias Me´dicas, Universidade do Estado do Rio de Janeiro, Rio de Janeiro, Brazil
Keywords enteric rods, hydrolytic enzymes, periodontal diseases, resistance to antimicrobial agents, virulence factors. Correspondence Raphael Hirata, Jr, Disciplina de Microbiologia e Imunologia, Faculdade de Cieˆncias Me´dicas, UERJ. Av. 28 de Setembro, 87, Fundos. Ed. Ame´rico Piquet Carneiro, 3 andar. Vila Isabel, Rio de Janeiro, RJ, CEP: 20.551-030, Brazil. E-mail:
[email protected]
2006/0923: received 28 June 2006, revised 24 November 2006 and accepted 28 November 2006 doi:10.1111/j.1472-765X.2007.02111.x
Abstract Aims: This investigation aimed to isolate enteric rods from subgingival sites of patients presenting chronic periodontitis lesions, and to assess antimicrobial resistance and expression of hydrolytic enzymes. Methods and Results: Enterobacteriaceae were isolated from 20% patients, and assayed for antimicrobial susceptibility and hydrolytic enzymes with specificity to different substrates. Isolates comprised seven Enterobacter cloacae (43Æ75%), five Serratia marcescens (31Æ25%), one Klebsiella pneumoniae (6Æ25%), one Enterobacter aerogenes (6Æ25%), one Pantoea agglomerans (6Æ25%), and one Citrobacter freundii (6Æ25%). Gelatinase activity was observed for 75% strains; caseinase and elastase was produced by six and two strains, respectively. DNase, lecithinase and lipase were expressed by S. marcescens. Most of strains were resistant to ampicillin (93Æ75%) and amoxicillin/clavulanic acid (81Æ25%). The majority of strains were susceptible to cephalosporins and aztreonam. Enterobacteria remained susceptible to imipenem, streptomycin and fluoroquinolones. Resistance to gentamicin, amikacin, sulfamethoxazole/thrimethoprim, tetracycline, and chloramphenicol were also observed. Eight strains presented multiple drug resistance. Conclusions: Subgingival sites from periodontal diseases contain multi-resistant and hydrolytic enzyme-producing enterobacteria that may contribute to overall tissue destruction and spreading. Significance and Impact of the Study: Enterobacteria isolated from patients generally considered as healthy individuals poses periodontal diseases as reservoir for systemic infections particularly in immunocompromised and hospitalized hosts.
Introduction The complex oral microbiota, normally in equilibrium in health, contains more than 500 different bacterial species including enteric Gram-negative rods (Gendron et al. 2000). Enterobacterial species have been detected in oral cavity of immunocompromised individuals including bone marrow-transplanted (Galili et al. 1995), and cancer patients submitted to chemotherapeutic procedures (Meurman et al. 1997). Differences in prevalence of enterobacteria in the oral cavity in certain populations 488
may be partially due to ingestion of contaminated drinking water or food, or also by inadequate personal hygiene (Slots et al. 1988; Barbosa et al. 2001). A 4-year longitudinal investigation detected a mean prevalence of 27Æ9% of enteric Gram-negative rods, comprising 57% of enterobacteria, in the oral cavity of healthy pre-school children in Hong Kong (Sedgley et al. 1997). Microbial succession may occur with predominance of some bacterial species in the development of both dental caries and periodontal disease (Gendron et al. 2000). Periodontal diseases are probably the most common chronic
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infectious/inflammatory disorder in adults leading to tooth loss in absence of appropriate treatment. Some procedures, such as administration of antimicrobial agents or mouth rinses containing chlorhexidine might lead to the partial suppression of periodontitis-associated anaerobic microbiota along with overgrowth of enterobacterial species (Slots et al. 1991). In Brazil, 79% of 853 dentate subjects, aged 30– 103 years old, presented periodontal pockets (Susin et al. 2004), which might exert an ideal niche for colonization by facultative Gram-negative bacilli. Studies of subgingival microbiota of Brazilian subjects demonstrated the presence of enteric Gram-negative rods such as Serratia marcescens, Klebsiella oxytoca, Enterobacter sp. and Kluyvera cryocrescens. Most of the isolates exhibited resistance to antibiotics used in the management of periodontal disease (Barbosa et al. 2001). In addition, checkerboard DNA– DNA hybridization of clinical specimens of patients with untreated chronic periodontitis detected the occurrence of unusual species including Escherichia coli (Colombo et al. 2002). Periodontitis lesions is characterized by the predominance of specific Gram-negative bacterial species in gingival crevice, accomplished with formation of a periodontal pocket which greatly favours further accumulation and a shift in the proportional composition of microbiota (Socransky et al. 1998). The participation of enteric rods in the progression of chronic periodontitis and in the failure of periodontal therapy remains unclear. Whether periodontal sites act as a reservoir for potentially virulent and/ or multi-resistant enterobacteria implicated in systemic disease, is also a matter of concern. This study aimed to determine the presence of different enterobacterial species in lesions of chronic periodontitis patients from Rio de Janeiro, Brazil, in addition to the assessment of expression of some extracellular hydrolytic enzymes and antibiotic susceptibility profiles of the bacterial isolates. Materials and methods Bacterial strains and sampling procedures Eighty subjects from dental division of Hospital Marcilio Dias, Rio de Janeiro, Brazil, aged 35–60 years old and presenting at least two periodontal active sites [i.e. probing depth ‡5 mm, with bleeding on probing and other signs of inflammation (purulent discharge, oedema), accompanied by radiographic evidence of alveolar bone loss] were selected for sampling. Teeth were isolated, dried and supragingival biofilm removed with sterile gauze prior to sampling procedure. Sterile paper points were inserted into the periodontal pockets until firm resistance and maintained for 60 s. The paper points were
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pooled, immediately dispensed into test tubes containing MacConkey broth (Merck, Darmstad, Germany), and incubated for 48 h/37C. Aliquots were taken and streaked onto MacConkey agar plates and CLED agar (Merck) and incubated for more 48 h/37C. Colonies of suspected enteric rods were identified as previously described (MacFaddin 2000; Farmer 2003). Briefly, Gramnegative bacilli with oxidase-negative and glucose fermentation profiles (OF) were submitted to biochemical tests including: Methyl red/Voges-Proskauer tests; motility; H2S, indol, and DNase (25C) production; utilization of citrate; decarboxilation of lysine and ornithine; dehydrolization of arginine; fermentation of diverse carbohydrates. Bacterial strains were maintained frozen at )80C in 10% skimmed milk (Difco Labs, Detroit, MI, USA) and on Trypticase soy agar (TSA, Difco) plates during the experiments. Pseudomonas aeruginosa ATCC 27853 was used as control for proteolytic activity. Aeromonas hydrophyla ATCC 7966 was used as positive control for DNase, lipase and lecithinase tests. Escherichia coli ATCC 25922 and ATCC 35218 (beta-lactamase producer strain) were used in the validation of antibiotic susceptibility tests (NCCLS 2005). Distribution of species isolated from periodontal lesions by gender was analysed by chi-square and Fisher’s exact test. Informed consent was obtained from each patient. Exclusion criteria included the utilization of corticosteroids, immunosuppressive agents, and anti-infective chemotherapeutic schemes. In addition, patients submitted to hospitalization within the preceding 6 months were also excluded from the study. The study protocol was approved by The Institutional Review Committee, Hospital Universita´rio Pedro Ernesto/Universidade do Estado do Rio de Janeiro. Antibiotic susceptibility tests The antibiotic susceptibility of enterobacterial isolates by disk diffusion method was performed according to the recommendations of the National Committee for Clinical Laboratory Standards (NCCLS 2005) using disks impregnated with antimicrobials agents (Cefar, Rio de Janeiro, Brazil) included in Tables 1 and 2. Proteolytic activity Proteolytic activities against casein and elastin were observed in nutrient agar (Merck) containing 3% skimmed milk (Difco) or 1% elastin powder (Sigma Chemical Co., St Louis, MO, USA), respectively (Burke et al. 1991). Gelatinase was performed by inoculation of bacterial isolates in nutrient broth containing 12% gelatin (Difco). Gelatin liquefaction was monitored after placing test tubes under room temperature (MacFaddin 2000). Plates
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Antimicrobial resistance (%) Bacterial species
AMP
AMC
CAZ
CRO
ATM
Enterobacter cloacae (n ¼ 7) Enterobacter aerogenes (n ¼ 1) Pantoea agglomerans (n ¼ 1) Serratia marcescens (n ¼ 5) Klebsiella pneumoniae (n ¼ 1) Citrobacter freundii (n ¼ 1) Total (n ¼ 16)
85Æ7 100 100 100 100 100 93Æ75%
85Æ7 100 100 100 0 0 81Æ25%
14Æ3 0 0 0 0 0 6Æ25
14Æ3 0 0 0 0 0 6Æ25
14Æ3 0 0 0 0 0 6Æ25
Table 1 Percentage of resistance among enterobacterial isolates from patients with periodontitis against beta-lactam antibiotics
AMP, ampicillin (10 lg); AMC, amoxicillin–clavulanic acid (20/10 lg); CAZ, ceftazidime (30 lg); CRO, ceftriaxone (30 lg); ATM, aztreonam (30 lg). All strains were susceptible to imipenem (10 lg).
Table 2 Percentage of resistance among enterobacterial isolates from patients with periodontitis against aminoglycosides and other antimicrobial agents Antimicrobial resistance (%) Bacterial species
GM
AM
TET
STX
CM
Enterobacter cloacae (n ¼ 7) Enterobacter aerogenes (n ¼ 1) Pantoea agglomerans (n ¼ 1) Serratia marcescens (n ¼ 5) Klebsiella pneumoniae (n ¼ 1) Citrobacter freundii (n ¼ 1) Total (n ¼ 16)
0 0 0 40 0 0 12Æ5
14Æ3 0 0 20 0 0 12Æ5
28Æ6 0 0 40 0 0 25
0 0 0 20 100 0 12Æ5
14Æ3 0 100 20 0 0 18Æ8
Aminoglycosides: GM, gentamicin (10 lg); AM, amicacin (30 lg); TET, tetracycline (30 lg); STX, sulfamethoxazole/trimethoprim (25 lg); CM, chloramphenicol (30 lg). All strains were susceptible to streptomycin (10 lg), ciprofloxacin (5 lg) and norfloxacin (10 lg).
and test tubes were incubated for 48 h and transferred to room temperature for additional 14 days. DNase activity Bacterial isolates were grown on DNase test agar (Difco) supplemented with 0Æ01% toluidine blue O. Production of DNase was positive with formation of a pink zone around the colonies (Janda and Bottone 1981). Production of lecithinase and lipase Production of lecithinase was revealed in 10% egg yolkenriched TSA plates. A white precipitate around the isolates indicated lecithinase production. Lipase activity was assayed with TSA plates supplemented with 1% Tween 80. Formation of haloes around the colonies revealed lipase production (Edberg et al. 1996). 490
Results Bacterial species and distribution by gender Enteric rods were isolated from periodontal pockets of 16 (20%) from 80 subjects presenting chronic periodontitis. Only one enterobacterial strain was isolated from each of the individuals; 10 strains from 36 female patients and six from 44 males, with relative percentages of 27Æ78% for females and 13Æ63% for males. Percentages of the distribution of isolates by gender was not significant (P > 0Æ05). Enteric rods were identified as Enterobacter cloacae (7 strains), Enterobacter aerogenes (1 strain), Pantoea (Enterobacter) agglomerans (1 strain), S. marcescens (5 strains), Klebsiella pneumoniae (1 strain) and Citrobacter freundii (1 strain). Resistance to antimicrobial agents Antimicrobial resistance of enteric rods isolated from 80 systemically healthy subjects is expressed in Tables 1 and 2. The majority of the bacterial isolates were resistant to ampicillin (93Æ75%) and to amoxicillin/clavulanic acid (81Æ25%). One E. cloacae strain showed resistance to cephalosporins (ceftazidime and ceftriaxone), aztreonam and sulfamethoxazole/trimethoprim. This strain also presented resistance to tetracycline. Klebsiella pneumoniae and C. freundii strains showed resistance to ampicillin and susceptibility to amoxicillin/clavulanic acid, suggesting the production of a nonextended spectrum beta-lactamase, due to its susceptibility to other beta-lactam antibiotics. Most of strains (93Æ75%) were susceptible to cephalosporins and to aztreonam. Resistance to imipenem was not detected among enterobacterial isolates. One E. cloacae isolate was susceptible to all antimicrobial agents tested in this study. Resistance to aminoglycosides was observed for gentamicin and amikacin in a rate of 12Æ5% for each chemotherapeutic. Enterobacteria remained
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susceptible to streptomycin. One S. marcescens strain was concomitantly resistant to both gentamicin and amikacin. Resistance of enterobacteria to other classes of antimicrobial agents was detected for tetracycline (25%), sulfamethoxazol/trimethoprim (12Æ5%) and chloramphenicol (18Æ8%). Resistance to quinolones, ciprofloxacin and norfloxacin, were not detected among enteric rods isolated from periodontal pockets. Extracellular enzymes related to pathogenicity The Table 3 presents the profiles of enzymatic activities to diverse substrates displayed by each enterobacterial Table 3 Enzymatic activities and antimicrobial resistance profiles of enterobacterial strains isolated from periodontal pockets of chronic periodontitis patients
isolate. Most of the strains isolated from sites of periodontal disease were able to produce protein-degrading enzymes. The majority (75%) of strains presented gelatinase activity. Some strains liquefied gelatin only after incubation for 14 days, particularly for Enterobacter species. Caseinase activity was observed for K. pneumoniae and S. marcescens after 48 h incubation. Prolonged periods of incubation (14 days/25C) did not alter the results of caseinolytic activity for other species. Two strains (one K. pneumoniae and one E. cloacae) were capable of degrading elastin. Degradation of this substrate was clearly observed after incubation of the plates for at least 5 days.
Species/isolates/resistance to antimicrobial agents Enterobacter cloacae PcOM 46* AMP, AMC, ATM, CAZ, CRO, TET PcOM 25* AMP, AMC, AM PcOM 26* TET, CM PcOM 5 AMP, AMC PcOM 15 AMP, AMC PcOM 17 AMP, AMC PcOM 37 AMP, AMC Serratia marcescens PcOM 63* AMP, AMC, TET, GM, STX, CM PcOM 68* AMP, AMC, GM, AM PcOM 77* AMP, AMC, TET PcOM 69 AMP, AMC PcOM 75 AMP, AMC Pantoea agglomerans PcOM 23* AMP, AMC, CM Klebsiella pneumoniae PcOM 19* AMP, STX Enterobacter aerogenes PcOM 1 AMP, AMC Citrobacter freundii PcOM 7Æ3 AMP
Enzymatic activity Caseinase Gelatinase Elastase Lipase Lecithinase DNase
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*Strains presenting multiple antimicrobial resistance phenotypes. ª 2007 The Authors Journal compilation ª 2007 The Society for Applied Microbiology, Letters in Applied Microbiology 44 (2007) 488–494
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Serratia marcescens strains showed enzymatic hydrolytic activities to most of substrates. Production of DNase, frequently used in the identification of enterobacteria, was observed only for S. marcescens strains. In addition, S. marcescens presented lipase and lecithinase activities after incubation (72 h/37C). Relationship of multiple resistance phenotypes with expression of enzymatic activities Resistance patterns of the isolates and the expression of hydrolytic enzymes are expressed in Table 3. Multi-resistant phenotypes (resistance to two or more classes of antibiotics), were detected in 50% of the isolates. Resistance to varied antimicrobial agents was not correlated to the production of multiple hydrolytic enzyme activities. One E. cloacae strain presented resistance to most of beta-lactam antibiotics and tetracycline, and did not show hydrolytic activity to any substrate tested. Nevertheless, the S. marcescens isolate presenting the highest multi-resistance level (resistance to five distinct classes of antimicrobial agents) also displayed activity to most of the substrates. Discussion The isolation of enteric rods from subgingival sites of periodontitis patients varies among populations (Slots et al. 1988, 1991; Barbosa et al. 2001). Although present in sites of periodontal destruction little information is available about the putative participation of enteric rods in the pathogenesis of periodontal disease. Herein, six species were isolated in 20% of individuals, corresponding to E. cloacae, S. marcescens, K. pneumoniae, E. aerogenes, P. agglomerans and C. freundii. Micro-organisms K. oxytoca, K. cryocrescens and E. coli (Barbosa et al. 2001; Colombo et al. 2002) were not isolated in this investigation. Microbial proteases are capable of both resisting plasma protease inhibitors and degrading such molecules. Collectively, these enzymes may facilitate the activity of host proteinase cascades, accelerating the inflammation process (Maeda 1996). In this study, enteric rods were shown to produce hydrolytic enzymes that might corroborate with proteolytic activities expressed by recognized periodontal pathogens, during destruction of tooth-supporting tissues. Production of proteolytic enzymes to different substrates was observed among six enterobacterial species isolated from periodontal pockets of systemically healthy subjects. Most of the strains were capable of liquefying gelatin in vitro. Degradation of bovine gelatin suggests production of collagenases by enterobacteria present in periodontal sites. Collagen, an abundant extracellular matrix component, is relatively resistant to proteolytic cleavage by 492
endogenous and exogenous proteinases, except for matrix metalloproteinases (MMPs). Such enzymes are formed as inactive precursors (pro-MMPs) which can be processed to active forms by different microbial proteases, including bacterial elastases (Okamoto et al. 1997). Some enteric rods isolated from active sites of chronic periodontitis were capable of degrading elastin. Elastase activity has been recognized as a molecular marker of periodontal destruction detected in gingival crevicular fluid from adult periodontits, and released after activation of neutrophil phagocytes (Gustafsson et al. 1992). Elastase production has also been considered as a marker of P. aeruginosa invasive strains in the aetiology of lower respiratory tract infections (Janda and Bottone 1981; Beatty et al. 2005). Thus, microbial enzymes might also contribute to the overall elastase activity displayed in gingival crevicular fluid obtained from active periodontal sites. The isolation from periodontal sites of elastin degrading K. pneumoniae strain, a usual pathogen that may cause pneumonia, emphasize the oral cavity as a reservoir of pathogens that may spread and cause severe infections in lower respiratory tract. In this study, lecithinase and/or lipase production was observed for S. marcescens strains. Lipase and lecithinase are potent extracellular enzymes involved in the pathogenesis of some bacterial species due to disturbing effects on host cell membranes. Lecithinase production by S. marcescens strains from periodontal lesions may dysregulate transduction pathways in endothelial cells, platelets and neutrophils leading to uncontrolled production of intracellular mediators and adhesion molecules, thus altering the traffic of granulocytes to the infected tissue, similar to previous observations with Clostridium perfringens (Flores-Diaz and Alape-Giron 2003; Flieger et al. 2004). Serratia marcescens was the sole species capable of degrading DNA among enteric rods isolated from periodontal pockets. DNase production may favour bacterial evasion from innate immune response through the destruction of neutrophil extracellular traps, a complex composed of chromatin and bactericidal granule proteins that kills extracellular bacteria (Brinkmann et al. 2004; Sumby et al. 2005). The multiple enzymatic activities displayed by enteric rods, particularly S. marcescens may contribute to the damage observed at sites of infection where this species make part of a complex microbiota. It is established that enteric Gram-negative rods constitute less than 1% of total cultivable microbiota of subgingival samples (Dahle´n and Wilkinstro¨m 1995). However, administration of some antibiotics may exert selective advantage to many resistant species colonizing periodontal pockets, including in periodontal therapy. In this study, a great proportion of bac-
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terial strains presented resistance to ampicillin and amoxicillin/clavulanic acid, drugs commonly used in treatment of oral infections or in prophylaxis of systemic infection during dental therapy. Production of beta-lactamase among periodontal bacteria was described previously, especially in genus Prevotella (van Winkelhoff et al. 1997; Herrera et al. 2000). One E. cloacae strain presented a broad spectrum resistance to beta-lactam antibiotics, suggesting that periodontal pockets may harbour bacterial strains producing broadened beta-lactam-degrading enzymes. The expression of extended spectrum beta lactamases by oral enterobacterial strains is presently under investigation. Aminoglycoside antibiotics are not usually recommended to infections in oral cavity, and are not used as chemotherapeutic in periodontal therapy. Both aminoglycoside and fluoroquinolones are employed for the empirical treatment of febrile neutropenic patients and in serious infections caused by aerobic and facultative Gram-negative bacilli, including Enterobacteriaceae (Vakulenko and Mobashery 2003; Okimoto et al. 2005). Fluoroquinolones are also recommended in the empirical treatment of community-acquired urinary tract infections in regions were resistance to sulfamethoxazole/trimethoprim raises to 10% or higher (Nickel 2005). Enterobacterial strains isolated from sites with chronic periodontitis remained susceptible to ciprofloxacin and norfloxacin, as previously observed (Barbosa et al. 2001). However, the occurrence of microbial resistance to aminoglycosides in commensal flora of periodontitis lesions alerts for the risk of systemic dissemination of resistant enterobacterial clones in hospitalized patients with periodontal diseases. Expression of multi-drug resistance among the oral enterobacterial isolates demonstrates that systemically adult healthy subjects may harbour multi-resistant clones in sites of chronic periodontal diseases. Antibiotic resistance raised among commensal bacteria is supposed to represent a major feature in the development of resistance within bacterial pathogens. The detection of resistant bacteria in commensal microbiota points periodontal diseases as possible sources for transfer of resistant genes to pathogenic bacteria, particularly in patients submitted to varied chemotherapeutic schemes, as observed for the microbiota of other body sites (Andremont 2003). Approximately 48% of US adults have chronic periodontitis, and similar or higher rates have been reported in other populations. Moderate and advanced periodontitis is more prevalent among older age groups and rates of 70% or more have been reported in varied populations (Albandar 2005). The presence of species of enterobacteria, the virulence factors displayed by these micro-organisms and the resistance to antimicrobial agents commonly used in treatment of severe systemic infections, add a new
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dimension on the importance of dissemination of enteric rods from periodontal pockets to other body sites, especially in compromised nosocomial patients. Acknowledgements This work was supported by grants from CAPES, CNPq, FAPERJ, SR-2/UERJ. Authors are also grateful to Jorge Ary F. Cruz and Ma´rcia M.C.F. Jones for technical assistance. References Albandar, J.M. (2005) Epidemiology and risk factors of periodontal diseases. Dent Clin North Am 49, 517–532. Andremont, A. (2003) Commensal flora may play key role in spreading antibiotic resistance. ASM News 69, 601–607. Barbosa, F.C.B., Mayer, M.P.A. and Saba-Chujfi, E. (2001) Subgingival occurrence and antimicrobial susceptibility of enteric rods and pseudomonads from Brazilian periodontitis patients. Oral Microbiol Immunol 16, 306–310. Beatty, A.L., Malloy, J.L. and Wright, J.R. (2005) Pseudomonas aeruginosa degrades pulmonary surfactant and increases conversion in vitro. Am J Respir Cell Mol Biol 32, 128–134. Brinkmann, V., Reichard, U., Goosmann, C., Fauler, B., Uhlemann, Y., Weiss, D.S., Weinrauch, Y. and Zychlinsky, A. (2004) Neutrophil extracellular traps kill bacteria. Science 303, 1532–1535. Burke, V., Robinson, J.O., Richardson, C.J.L. and Bundell, C.S. (1991) Longitudinal studies of virulence factors of Pseudomonas aeruginosa in cystic fibrosis. Pathology 23, 145–148. Colombo, A.P., Teles, R.P., Torres, M.C., Souto, R., Rosalem, W.J., Mendes, M.C. and Uzeda, M. (2002) Subgingival microbiota of Brazilian subjects with untreated chronic periodontitis. J Periodontol 73, 360–369. Dahle´n, G. and Wilkinstro¨m, M. (1995) Occurrence of enteric rods, staphylococci and Candida in subgingival samples. Oral Microbiol Immunol 10, 42–46. Edberg, S.C., Gallo, P. and Kontnick, C. (1996) Analysis of the virulence characteristics of bacteria isolated from bottled, water cooler, and tap water. Microbial Ecol Health Dis 9, 67–77. Farmer, J.J., Jr (2003) Enterobacteriaceae: introduction and identification. In Manual of Clinical Microbiology ed. Murray, P.R., Baron, E.J., Jorgensen, J.H., Pfaller, M.A. and Yolken, R.H. pp. 636–653. Washington, DC: ASM Press. Flieger, A., Rydzewski, K., Banerji, S., Broich, M. and Heuner, K. (2004) Cloning and characterization of the gene encoding the major cell-associated phospholipase A of Legionella pneumophila, plaB, exhibiting hemolytic activity. Infect Immun 72, 2648–2658.
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