Antibiotic resistance profiles and virulence markers of Pseudomonas aeruginosa strains isolated from composts

Bioresource Technology 102 (2011) 1543–1548

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Antibiotic resistance profiles and virulence markers of Pseudomonas aeruginosa strains isolated from composts Edit Kaszab a, Sándor Szoboszlay a,⇑, Csaba Dobolyi a, Judit Háhn b, Nikoletta Pék a, Balázs Kriszt a a b

}, Hungary Szent István University, Páter Károly 1, H-2103 Gödöllo }, Hungary Regional University Center of Excellence, Páter Károly 1, H-2103 Gödöllo

a r t i c l e

i n f o

Article history: Received 29 December 2009 Received in revised form 2 August 2010 Accepted 7 August 2010 Available online 10 August 2010 Keywords: Compost Pseudomonas aeruginosa Virulence genes Antibiotic resistance

a b s t r a c t The aim of our work was to determine the presence of Pseudomonas aeruginosa in compost raw materials, immature and mature compost, and compost-treated soil. Twenty-five strains of P. aeruginosa were isolated from a raw material (plant straw), immature and mature compost and compost-treated soil samples. The strains were identified using the PCR method for the detection of species specific variable regions of 16S rDNA. Strains were examined for the presence of five different virulence-related gene sequences (exoA, exoU, exoT, exoS and exoY) and their antibiotic resistance profiles were determined. Based on our results, species P. aeruginosa can reach significant numbers (up to 106 MPN/g sample) during composting and 92.0% of the isolated strains carrying at least two gene sequences encoding toxic proteins. Various types of drug resistance were detected among compost originating strains, mainly against third generation Cephalosporins and Carbapenems. Six isolates were able to resist two different classes of antibiotics (third generation Cephalosporins and Carbapenems, wide spectrum Penicillins or Aminoglycosides, respectively). Based on our results, composts can be a source of P. aeruginosa and might be a concern to individuals susceptible to this opportunistic pathogen. Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction Compost may harbor pathogens including bacteria, viruses, fungi, protozoa and helminths that are able to cause infections in humans, animals or plants. Pathogenic microorganisms can originate from raw materials such as municipal solid waste (MSW), sewage sludge, manure (Déportes et al., 1998) or yard trimmings (Pietronave et al., 2004). Although composting is increasingly being used for disposal of such materials as by-products of biogas plants, information about the pathogen content of these materials is limited. Most opportunistic and obligate pathogens are mesophilic bacteria and can be destroyed during the thermophilic phase of composting if specified temperature and time criteria are met (Gerardi and Zimmerman, 2005). Based on EPA (Environmental Protection Agency, USA) recommendations, composting materials should be exposed to a temperature of 55 °C for three days to ensure the destruction of the pathogens (EPA, 1994). With properly managed composting technology, the reduction of human, animal and plant pathogens was demonstrated in several scientific investigations (Russ and Yanko, 1981; Hassen et al., 2001; Ryckeboer et al., 2003). However, inhomogeneity of solid materials in a compost ⇑ Corresponding author. Tel./fax: +36 28 522000. E-mail address: [email protected] (S. Szoboszlay). 0960-8524/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2010.08.027

pile can lead to differences in temperature between the inner and outer zones; therefore, the minimum lethal temperature cannot be ensured throughout the entire compost pile (Nemerow et al., 2008). Furthermore, a laboratory scale investigation has documented survival of mesophilic pathogens under uniformly thermophilic (50–70 °C) conditions (Droffner et al., 1995). Potentially these pathogens can increase in numbers during the final mesophilic (45 °C or cooler) stage of composting. Compost applications to soil can introduce these pathogens into the environment from where they can be transmitted to plants, animals and humans (de Bertoldi et al., 1988) and a few cases have identified compost as the source of infectious bacteria (Bollen and Volker, 1996). Pseudomonas aeruginosa is a ubiquitous bacterium and opportunistic pathogen (Wunderink and Mendoza, 2007). Little is known about the fate of this bacterium in compost and the characteristics of strains inhabiting compost and compost-amended soils. Depending on the number of these bacteria in compost and their virulence and antibiotic resistance properties, the presence of P. aeruginosa might be a concern to susceptible individuals. Many environmental strains of P. aeruginosa are able to resist ampicillin, ceftiofur, florfenicol, sulphachloropyrodazine, sulphadimethoxine and trimethoprim/sulfamethoxazole, chlortetracycline and spectinomycin (Edrington et al., 2009). Although composting was found to be able to degrade several antimicrobials with over 90% efficiency, in some cases, composting had no effect on antibiotic concentrations

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(Dolliver et al., 2008). Therefore, it is possible that antibiotics in compost materials exert a selective pressure on microorganisms leading to antibiotic resistant populations. The level of health concerns regarding P. aeruginosa in compost also depends on the presence of virulence factors in these bacteria. These virulence factors include the production of proteins with toxic effects (Winstanley et al., 2005). Type II (T2SS) and Type III (TTSS) secretion systems are important in the secretion of these effector proteins. In particular, the Type III secretion system of P. aeruginosa has been adapted for the secretion of toxic proteins directly into the cytoplasm of the host cells (Ajayi et al., 2003). These effector proteins can be ADP-ribosylating enzymes, cytotoxins or adenyl-cyclases among others (Sato et al., 2003). Given the limited information on compost-originating P. aeruginosa, the aim of the current study was to detect and enumerate P. aeruginosa in raw materials, during all stages of composting, including immature and mature compost, and in compost-treated soils. In addition, antibiotic resistance features and several virulence markers of P. aeruginosa isolated from these sources were examined. 2. Methods 2.1. Sampling protocol Sixteen raw material samples from eight different industrial plants and factories in Hungary were collected. The samples consisted of plaster, thick slurry, slag, coal fired slime, and flue ash (one sample each), cinder from heat-producing power stations (three samples), vinasse from a distillery (two samples), separate phases of biogas by-products from two biogas plants (four samples) manure and plant straw from two composting plants (one sample each). Based on preliminary laboratory scale investigations, three different mixtures of these raw materials were used for pilot-

scale experiments in open piles (designated A1, A2, A3, B1, B2, B3 and C1, C2, C3) and aerated static piles (designated D, E, F, G, H, I and J). Samples for the isolation of P. aeruginosa were collected from these piles in 2008 and 2009. The main properties of the sampled compost materials are summarized in Table 1. Compost piles were sampled in all stages of composting including the thermophilic phase, with temperatures between 50.2 and 68.0 °C and finished compost. Fifty-four samples were collected from open piles and 24 from aerated static piles. Compost-treated soil samples were collected from six experimental plots treated with compost of animal origin. The solid raw materials, immature and mature compost and compost-treated soil were sampled in accordance with Hungarian Standard (MSZ 21470-1, 1980) under aseptic conditions. 2.2. Isolation and identification of P. aeruginosa Isolation of P. aeruginosa was performed in accordance with Hungarian Standard (MSZ 21470-77, 1988). In the first step, 1 g of the solid samples was inoculated into asparagine broth containing (g/L): L-asparagine, 3; K2HPO4, 1; MgSO47H2O 0.5; glycerol, 10; pH 7.0 and serially diluted in ten-fold increments. Inoculation was done in three replicate tubes to facilitate enumeration by the Most Probable Number (MPN) method (Highsmith and Abshire, 1975). After 48 h at 42 °C, the bacterial suspension was spread onto cetrimide agar (MERCK 105284). Plates were incubated for 24 h at 37 °C, and colonies showing pyocyanin production and trimethylamine odor were inoculated into acetamide broth consisting (g/ L): acetamide, 1; NaCl, 5; KH2PO4, 2; MgSO47H2O, 1; pH 6.8. Acetamide decomposition was verified with Nessler’s reagent. Yellow or brown discoloration and/or precipitation were considered indicative of P. aeruginosa. Confirmation of this identification was done by PCR using primers specific to the V2 and V8 regions of 16S rDNA (Spilker et al.,

Table 1 Main properties of the sampled compost materials. Designation of compost pile

Method of composting

Composition

Microbial inoculation

Sampling Strategy

Number of collected samples

A1, A2, A3

Open piles

55 vol.% plant straw 15 vol.% biogas by-product 30 vol.% manure

Separately (A1, A2, A3)

18

B1, B2, B3

Open piles

40 vol.% plant straw 45 vol.% biogas by-products 15 vol.% flue ash

Separately (B1, B2, B3)

18

C1, C2, C3

Open piles Aerated static piles Aerated static piles

Separately (C1, C2, C3) Average (D)

18

D1, D2, D3

55 vol.% plant straw 15 vol.% manure 15 vol.% vinasse 15 vol.% flue ash Uninoculated control of E, F and G

Thermobifida fusca Bacillus circulans Scytalidium thermophilum Paecilomyces variotii Thermobifida fusca Bacillus circulans Scytalidium thermophilum Paecilomyces variotii Mikrokomp-GeocellÒ inoculum

Average (E)

3

E1, E2, E3

55 vol.% plant straw 15 vol.% biogas by-product 30 vol.% manure

F1, F2, F3

Aerated static piles

40 vol.% plant straw 45 vol.% biogas by-products 15 vol.% flue ash

G1, G2, G3

Aerated static piles Aerated static piles Aerated static piles

55 vol.% plant straw 15 vol.% manure 15 vol.% vinasse 15 vol.% flue ash uninoculated control of I, J and H

J1, J2, J3

Aerated static piles

40 vol.% plant straw 45 vol.% municipal sewage sludge 15 vol.% flue ash

K1, K2, K3

Aerated static piles

55 vol.% plant straw 15 vol.% manure 15 vol.% municipal sewage sludge 15 vol.% flue ash

H1, H2, H3 I1, I2, I3

55 vol.% plant straw 15 vol.% municipal sewage sludge 30 vol.% manure

-

3

Thermobifida fusca Bacillus circulans Scytalidium thermophilum Paecilomyces variotii Thermobifida fusca Bacillus circulans Scytalidium thermophilum Paecilomyces variotii Mikrokomp-GeocellÒ inoculum

Average (F)

3

Average (G)

3

–

Average (H)

3

Thermobifida fusca Bacillus circulans Scytalidium thermophilum Paecilomyces variotii Thermobifida fusca Bacillus circulans Scytalidium thermophilum Paecilomyces variotii Mikrokomp-GeocellÒ inoculum

Average (I)

3

Average (J)

3

Average (K)

3

C/N ratio of compost piles was adjusted between 26 and 33, with 50–70% moisture content.

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Table 2 Primer sequences and reaction parameters of PCR-based examination of compost-originating P. aeruginosa strains. Genes

Primer sequences

16S rDNAa

F: R: F: R: F: R: F: R: F: R: F: R:

exoAb exoSc exoTc exoYc exoUc a b c

50 50 50 50 50 50 50 50 50 50 50 50

GGGGGATCTTCGGACCTCA TCCTTAGAGTGCCCACCCG AAC CAG CTC AGC CAC ATG TC CGC TGG CCC ATT CGC TCC AGC GCT GCG AGG TCA GCA GAG TAT CG TTC GGC GTC ACT GTG GAT GC AAT CGC CGT CCA ACT GCA TGC G TGT TCG CCG AGG TAC TGC TC CGG ATT CTA TGG CAG GGA GG GCC CTT GAT GCA CTC GAC CA CCG TTG TGG TGC CGT TGA AG CCA GAT GTT CAC CGA CTC GC

30 30 30 30 30 30 30 30 30 30 30 30

Amplicon size (bp)

Specification

Reaction parameters

956

Species level identification

95 °C 20 25  (94 °C 25’’ 58 °C 40’’ 72 °C 40’’) 72 °C 10

396

Detection of exotoxin A encoding gene sequence Detection of exotoxin S encoding gene sequence Detection of exotoxin T encoding gene sequence Detection of exotoxin Y encoding gene sequence Detection of exotoxin U encoding gene sequence

95 °C 20 30  (94 °C 10 68 °C 10 72 °C 10 ) 72 °C 70

118 152 289 134

94 °C 20 36  (94 °C 30’’ 58 °C 30’’ 68 °C 10 ) 68 °C 70 94 °C 20 36  (94 °C 30’’ 58 °C 30’’ 68 °C 10 ) 68 °C 70 94 °C 20 36  (94 °C 30’’ 58 °C 30’’ 68 °C 10 ) 68 °C 70 94 °C 20 36  (94 °C 30’’ 58 °C 30’’ 68 °C 10 ) 68 °C 70

Spilker et al., 2004; Atzél et al., 2007; Ajayi et al., 2003.

2004). DNA was prepared with the FastDNATM kit (Qbiogene 6540400) and following the manufacturer’s instructions. Primers and reaction parameters are detailed in Table 2. PCR products were visualized after agarose gel (2%) electrophoresis by staining with ethidium bromide. P. aeruginosa ATCC 27853 served as positive control. 2.3. Antibiotic resistance assays Minimal inhibitory concentrations (MICs) were detected by Etest strips (AB Biodisk, Sweden) containing cefoperazone/sulbactam, cefotaxime, ceftazidime, ceftriaxone, cefepime, piperacillin, imipenem, ciprofloxacin, ofloxacin, and gentamicin representing five different classes of antibiotics. Strains of P. aeruginosa were cultivated on Mueller–Hinton agar (MERCK 105435) in accordance with the recommendations of the Clinical Laboratory Standards Institute (CLSI, 2006) and the instructions of the manufacturer. MIC breakpoints were interpreted according to the categories of Performance Standards (CLSI, 2007) as susceptible, intermediate, or resistant. The quality control strain for the Etest was P. aeruginosa ATCC 27853. Isolates were considered multidrug resistant (MDR) if they displayed simultaneous resistance to two or more antimicrobial drug classes.

2.4. Virulence markers assays Virulence-related gene sequences were detected by PCR with primer pairs and reaction parameters as specified in Table 2. Results were evaluated with agarose gel electrophoresis. Positive control strains were P. aeruginosa ATCC 27853 for exoA, exoS, exoT and exoY and an environmental originated strain, P. aeruginosa P43 (Kaszab et al., 2010) for exoU. 3. Results and discussion 3.1. Frequency of detection of P. aeruginosa P. aeruginosa was not detected in any of the raw materials analyzed, except in plant straw where it reached 103 MPN/g. Of 78 samples from compost piles, 56.4% contained a detectable number of P. aeruginosa. Open pile samples had a 53.7% occurrence frequency, while that of aerated static piles was 62.5%. This finding is in contrast to that of van Heerden et al. (2002), who determined that P. aeruginosa appeared in final compost, but not during maturation. The MPN values ranged from 100 to 106 per g of compost. In compost-treated soil samples the detection frequency of P. aeruginosa

Fig. 1. Changes in temperature and MPN/g numbers of P. aeruginosa during the composting of piles designated A1-C3.

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Table 3 Antibiotic resistance profiles and the frequency of exotoxin encoding gene sequences in compost-originating P. aeruginosa strains.

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a b c

Reference strains of antibiotic resistance and virulence investigations (Kaszab et al., 2010). Compost treated soil samples. Designation of compost piles; grey colored – resistance (CLSI), positive PCR reaction.

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Fig. 2. Detection rate of exotoxin encoding genes exoS, Y, T, U and A of compost originating strains in comparison to clinical originating isolates (Bjorn et al., 1977; Winstanley et al., 2005).

was 83.3%, and the counts were between 100 and 102 MPN per g of soil. These numbers are likely not only due to compost application, but also the soil itself (Green et al., 1974). Time dependent changes of MPN/g numbers and temperature during composting in the cases of the A1, A2, A3, B1, B2, B3 and C1, C2, C3 compost piles are demonstrated in Fig. 1 (data of piles D, E, F, G, H, I and J are not shown). The detectable numbers of P. aeruginosa continuously changed during composting and are greatly – but not completely – influenced by the temperatures of the compost piles. From the 33rd to the 85th day of composting P. aeruginosa was detectable in all compost piles and reached remarkable numbers in compost samples from piles A1, A2 and A3. Therefore, P. aeruginosa was able not only to survive, but also to replicate under thermophilic conditions. A single exposure to P. aeruginosa in water provides a risk of less than 1/10,000 for colonization (Rusin et al., 1997) and the risk of an infection might be lower. With solid materials such as compost, the possible pathways of exposure expand to include inhalation and breaching of the integrity of physical barriers such as skin. The MPN/g numbers (100–106) of P. aeruginosa in compost therefore reached the infectious dose (102–108) determined by Fok (2005); and Lizewski et al. (2002). Based on further investigations of nosocomial infections, asymptomatic phase just like colonization at one or several parts of body cannot be ignored as it can be the first stage of a complex epidemiology (Boldin et al., 2007). 3.2. Antibiotic resistance phenotypes of P. aeruginosa isolates Twenty-five P. aeruginosa strains were isolated from piles A1, A2, A3, H, I and K and their antibiotic resistance profiles and minimal inhibitory concentrations were established (Table 3). Based on our results, the examined fourth generation Cephalosporin cefepime and the synthetic drug class of Fluoroquinolones were completely effective (100.0%) as it was expected from a previous investigation of non-clinical isolates of P. aeruginosa (da Silva et al., 2008). Third generation Cephalosporins were only moderately effective against compost originating strains except the combination of cefoperazone–sulbactam that could completely eradicate the examined strains. Ceftriaxone resistant strains were widespread (48.0%). Cefotaxime and ceftazidime resistant strains were detectable in four and two cases, respectively. The Carbapenems were ineffective against 24.0% of the examined strains. Gentamicin, the applied Aminoglycoside agent that is usually effective against strains of P. aeruginosa isolated from non-clinical environments (Trypathy et al., 2007), showed no effectiveness in two compost originating strains.

Six strains (K2, K4, K5, K13, K24 and K39) were simultaneously resistant to different classes of antibiotics and can thus be considered as multidrug resistant. In these situations two classes of antibiotics (third generation Cephalosporins and Carbapenems, wide spectrum Penicillins or Aminoglycosides) had no effectiveness. This represents the first verification of multiple drug resistant features in compost originating strains of P. aeruginosa. The minimal inhibitory concentrations of the examined antibiotic agents are summarized in Table 3. 3.3. Virulence markers assay P. aeruginosa is able to secrete a large number of virulence associated factors that have great influence on pathogenesis (van Delden, 2004). Since it has been shown that mortality was six times greater in cases of P. aeruginosa expressing the secreted proteins ExoS, ExoT or ExoU (Roy-Burman et al., 2001), the presence of the corresponding genes and also that of the adenylate cyclaseencoding gene exoY (Prasain et al., 2008) of exoA (Atzél et al., 2007) was evaluated in the compost isolates. The exotoxin A encoding gene (exoA), was detected in all cases. The incidences of exoS, exoT, exoY genes were various (Fig. 2). The exoU gene, which is a major contributor to the potential pathogenesis of P. aeruginosa (Lin et al., 2006), was not detected. Only two strains did not carry exoS, exoT, exoY or exoU (Table 3). ExoS and exoT are usually found in invasive strains, while cytotoxic isolates seem to have lost exoS but retained exoT (Fleiszig et al., 1997). Cytotoxicity of non-invasive strains is mainly the result of the action of exoU (Hauser et al., 2002). Therefore we can conclude that the majority of the strains originating from compost can be classified into the invasive group. Considering that 92.0% of the isolated strains of P. aeruginosa originating from compost-related samples have genes encoding two or more different toxic proteins, it is possible that these strains could cause disease in humans. Compared to clinical isolates (Bjorn et al., 1977; Winstanley et al., 2005), the compost isolates had a higher frequency of occurrence of exoS and a lower frequency for exoU (Fig. 2). In the future, the increased number of the examined compost originated strains can help us to highlight these differences as significant. It remains to be determined if the detected virulence genes are expressed by the compost isolates. 4. Conclusions P. aeruginosa was detected in samples taken from plant straw, maturing and final compost and compost-treated soil, and isolated strains exhibited antibiotic resistance and in six cases multidrug

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resistance, and carried gene sequences encoding toxic proteins. The presence of the virulence gene exoS and the absence of exoU in the compost isolates suggest genetic differences between these and clinical isolates. Acknowledgements This work was supported by the Ányos Jedlik Programme (OM 00120/2007) and KMOP-2007-1.1.1. References Ajayi, T., Allmond, L.R., Sawa, T., Wiener-Kronish, J.P., 2003. Single-nucleotidepolymorphism mapping of the Pseudomonas aeruginosa Type III secretion toxins for development of a diagnostic multiplex PCR system. J. Clin. Microbiol. 41, 3526–3531. Atzél, B., Szoboszlay, S., Mikuska, Zs., Kriszt, B., 2007. Comparison of phenotypic and genotypic methods for the detection of environmental isolates of Pseudomonas aeruginosa. Int. J. Hyg. Environ. Health 211, 143–155. Bjorn, M.J., Vasil, M.L., Sadoff, J.C., Iglewski, B.H., 1977. Incidence of exotoxin production by Pseudomonas aeruginosa species. Infect. Immunol. 16, 362–366. Boldin, B., Bonten, M.J.M., Diekmann, O., 2007. Relative effects of barrier precautions and topical antibiotics on nosocomial bacterial transmission: results of multicompartment models. B. Math. Biol. 69, 2227–2248. Bollen, G.J., Volker, D., 1996. Phytohygienic aspects of composting. In: de Bertoldi, M., Sequi, P., Papi, T. (Eds.), The Science of Composting. Blackie Academic & Professional, Glasgow, pp. 233–246. CLSI, 2006. Clinical and Laboratory Standards Institute, Methods for Dilution Antmicrobial Susceptibility Tests for Bacteria That Grow Aerobically. Approved Standard, M7–A7. CLSI, 2007. Clinical and Laboratory Standards Institute, Performance Standards for Antimicrobial Susceptibility Testing; Seventeenth Informational Supplement. M100-S17 27, 1, 1–182. da Silva, M.E.Z., Filho, I.C., Endo, E.H., Nakamura, A.C., Ueda-Nakamura, T., Filho, B.D.P., 2008. Characterisation of potential virulence markers in Pseudomonas aeruginosa isolated from drinking water. A. van Leeuw. J. Microb. 93, 323–334. de Bertoldi, M., Zucconi, F., Civilini, M., 1988. An analysis of the effectiveness of various systems to reduce the pathogen content of the compost product. Biocycle 29, 43–47. Déportes, I., Benoit-Guyod, J.L., Zmirou, D., Bouvier, M.C., 1998. Microbial disinfection capacity of municipal solid waste (MSW) composting. J. Appl. Microbiol. 85, 238–246. Dolliver, H., Gupta, S., Noll, S., 2008. Antibiotic degradation during manure composting. J. Environ. Qual. 37, 1245–1253. Droffner, M.L., Brinton, W.F., Evans, E., 1995. Evidence for the prominence of well characterized mesophilic bacteria in thermophilic (50–70 °C) composting environments. Biomass Bioenerg. 8, 191–195. Edrington, T.S., Fox, W.E., Callaway, T.R., Anderson, R.C., Hoffman, D.W., Nisbet, D.J., 2009. Pathogen prevalence and influence of composted dairy manure application on antimicrobial resistance profiles of commensal soil bacteria. Foodborne Pathog. Dis. 6, 217–224. EPA, 1994. Composting yard trimmings and municipal solid waste. Environmental Protection Agency, Office of Solid Waste and Emergency Response, EPA530-R94-003. Fleiszig, S.M.J., Wiener-Kronish, J.P., Miyazaki, H., Vallas, V., Mostov, K.E., Kanada, D., Sawa, T., Yen, T.S.B., Frank, D.W., 1997. Pseudomonas aeruginosa-mediated cytotoxicity and invasion correlate with distinct genotypes at the loci encoding exoenzyme S. Infect. Immunol. 65, 579–586. Fok, N., 2005. Pseudomonas aeruginosa as a waterborne gastroenteritis pathogen. Environ. Health Rev. (Winter), 121–130. Gerardi, M.H., Zimmerman, M.C., 2005. Wastewater Pathogens. John Wiley & Sons, Inc., New Jersey. Green, S.K., Schroth, M.N., Cho, J.J., Kominos, S.D., Vitanza-Jack, V.B., 1974. Agricultural plants and soil as a reservoir for Pseudomonas aeruginosa. Appl. Microbiol. 28, 987–991.

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