Food Control 18 (2007) 13221327 www.elsevier.

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Detection of acylated homoserine lactones in gram-negative proteolytic psychrotrophic bacteria isolated from cooled raw milk
Uelinton Manoel Pinto 1, Eliseth de Souza Viana, Maurilio Lopes Martins, Maria Cristina Dantas Vanetti *
Department of Microbiology, Federal University of Vicosa, Vicosa, MG, 36570-000, Brazil Received 8 May 2006; received in revised form 6 September 2006; accepted 12 September 2006

Abstract Through a mechanism called quorum sensing, bacteria are able to express specic genes in response to population density. Cell-to-cell communication in bacteria is mediated by signal molecules such as acylated homoserine lactones (AHLs). This work aimed to detect AHL production in Gram-negative psychrotrophic bacteria isolated from raw milk. A total of 84.9% of the bacteria were identied as AHL producers eliciting a diversity of responses in the AHL-monitor systems. These results demonstrate that AHL-production is common among psychrotrophic bacteria isolated from milk, and indicate that quorum sensing may play an important role in the spoilage of this product. 2006 Elsevier Ltd. All rights reserved.
Keywords: Quorum sensing; Acylated homoserine lactones (AHL); Psychrotrophic bacteria; Raw milk

1. Introduction Gram-negative proteolytic psychrotrophic bacteria are the predominant microorganisms responsible for spoilage of milk and milk products due to their ability to produce thermostable proteases that hydrolyze casein and decrease the yield and sensory quality of dairy products (Dogan & Boor, 2003; Srhaug & Stepaniak, 1997). Some bacteria also secrete lecithinases and lipases that can play a signicant role in the spoilage of these products (Dogan & Boor, 2003). It is known that activity of hydrolytic enzymes is detected at the end of logarithmic phase and at the beginning of the stationary growth phase of these bacteria (Matselis & Roussis, 1998; Rajmohan, Dodd, & Waites, 2002), conditions in which a high cell density is achieved. Bacteria are able to regulate expression of phenotypic characteristics as a function of cell density in a mechanism
Corresponding author. Tel.: +55 31 38992954; fax: +55 3138992573. E-mail address: mvanetti@ufv.br (M.C.D. Vanetti). 1 Present address: 360 Wing Hall, Department of Microbiology, Cornell University, Ithaca, NY 14853, USA. 0956-7135/$ - see front matter 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2006.09.005
*

termed quorum sensing (QS) (Fuqua, Winans, & Greenberg, 1994). In Gram-negative bacteria, this regulation is typically mediated by chemical signals such as N-acyl-Lhomoserine lactones (AHL) (Fuqua et al., 1994; Whitehead, Barnard, Slater, Simpson, & Salmond, 2001). The acyl side chain of dierent AHL can vary from 4 to 18 carbons in length, degree of substitution, and saturation providing specicity to QS systems (Zhu, Chai, Zhong, LI, & Winans, 2003). The key regulatory components of these signaling systems are LuxI-type proteins which act as AHL synthases, LuxR-type proteins which serve as AHL receptors, and AHL-dependent transcription factors (Fuqua & Greenberg, 2002). Examples of phenotypes regulated by AHLs include production of antibiotics, biolm development, competence for DNA uptake, cell dierentiation, bioluminescence, growth, pigment production, conjugal plasmid transfer, virulence gene expression, and production of a range of degradative extracellular enzymes (Smith, Fratamico, & Novak, 2004). Several works have been done to elucidate the role of QS in food spoilage. AHLs have been detected in some food products such as cold-smoked salmon (Gram, Christensen,

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Ravn, Molin, & Givskov, 1999), meat (Bruhn et al., 2004), and bean sprouts (Rasch et al., 2005). In this last work, it was shown that pectinase, protease, cellulase and siderophore-mediated iron chelation were regulated by QS, underlying the importance of this system in spoilage of bean sprouts. Christensen et al. (2003) demonstrated that several hydrolytic enzymes produced by Serratia proteamaculans, a typical member of a food spoilage ora, are regulated by QS. The involvement of QS in meat spoilage process, and biolm formation was suggested by Jay, Vilai, and Hughes (2003). Furthermore, Liu and Griths (2003) pointed out that spoilage of milk by Pseudomonas uorescens is correlated with its ability to produce AHLs and the extracellular protease. However, currently, little is known about signal molecule production, such as AHLs, by bacteria isolated from food products. The AHL detection is based on dierent bacterial bioassays. The use of reporter bacteria, in which the luxI homologue gene responsible for AHL production had been inactivated, has shown to be a valuable system for AHL detection (Ravn, Christensen, Molin, & Givskov, 2001). Then, the expression of a reporter gene is possible only in the presence of exogenous AHLs. Plasmid reporter vectors that respond to activation of LuxR homologues have also been used in Escherichia coli strains (Winson et al., 1998). Because of the specicity of each LuxR homologue, reporter strains display specicity towards dierent AHL molecules, allowing detection of a wide range of AHLs and dierentiation between AHL production patterns (Van Houdt, Aerstsen, Jansen, Quintana, & Michiels, 2004). For instance, LuxR of Vibrio sheri, used in several reporter plasmids such as pSB403, is activated by AHLs with carbon chains of C6 or C8 with or without 3-oxo substitutions (Winson et al., 1998). On the other hand, CviR of Chromobacterium violaceum is sensitive to unsubstituted chains varying in size from C4 to C8, and also is able to detect long-chain AHLs by their ability to inhibit violacein production if an activating AHL is incorporated to the medium (McClean et al., 1997). The TraR of Agrobacterium tumefaciens is sensitive to most 3-oxo AHLs (Shaw et al., 1997). In order to improve the understanding of the process of cell-to-cell communication among bacteria found in milk, this work aimed to evaluate the production of AHL in proteolytic psychrotrophic bacteria isolated from cooled raw milk. 2. Material and methods 2.1. Bacterial strains and culture conditions Gram-negative psychrotrophic bacteria isolated from cooled raw milk (Pinto, 2004) and strains from American type culture collection ATCC (Table 1) were investigated for AHL-production. The bacteria were stored in brain heart infusion (BHI) with the addition of glycerol to 20% v/v and maintained at 80 C. The strains were activated

in Luria Berthani broth (LB) and incubated at 25 C for 24 h with agitation (150 r.p.m.). Ultracompetent E. coli DH5a was transformed with pSB403 (Winson et al., 1998) using standard methods described by Sambrook, Fritsch, and Maniats (1989). Chromobacterium violaceum CV026 (McClean et al., 1997) and Agrobacterium tumefaciens a136 (Fuqua & Winans, 1996) were also used to detect AHL-production. These monitor strains were grown in LB supplemented with appropriate antibiotics (Ravn et al., 2001), and incubated at 28 C during 24 h, with agitation (150 r.p.m.), prior the assay. C. violaceum ATCC 6357 and P. aeruginosa 15442 were used as positive controls in the experiments and the monitor strains as negative control themselves. 2.2. Screening for AHL-production The screening for AHL-production was performed according to Ravn et al. (2001). The tested strains were streaked in parallel to the monitor strains on LB agar plates. E. coli (pSB403) is unable to produce luminescence without exogenous AHL. The luminescence was observed after approximately 3 min for adaptation of the eyes to a darkened room, under weak red light, as a positive result in response to AHL produced by the tested strain. C. violaceum CV026 produces the pigment violacein only in the presence of exogenous AHL. Thus, the pigment production indicated a positive result. Testing for AHL production against A. tumefaciens A136 assay, was done in a similar assay supplementing the LB agar with 50 lg/ml 5-bromo4-chloro-3-indolyl-b-D-galactopyranoside (X-gal). This monitor strain has the lacZ gene fused to the promoter of traI gene, which is regulated by autoinduction. The strain produces a blue pigment in response to AHL when the medium is supplemented with X-gal. All plates were incubated for 24 h at 25 C, except the ones streaked with A. tumefaciens A136 that were incubated up to three days. Long chains AHLs were detected observing the inhibition of the pigment production by the induced monitor strain C. violaceum CV026. LB agar plates were supplemented with 75 nM of N-hexanoyl-L-homoserine lactone (HHL) obtained commercially (Fluka, Switzerland). The psychrotrophic isolates were streaked in the medium and incubated for 24 h at 25 C. Then, the monitor strain was streaked in parallel to the previous strains and the plates were re-incubated at the same conditions. Experiments were repeated at least twice. 2.3. Thin-layer chromatography (TLC) Extracts for TLC were prepared from 100 ml cultures after growth in LB medium for 20 h at 25 C with agitation of 150 r.p.m. Bacteria were removed by centrifugation, the supernatants were extracted twice with equal volumes of ethyl acetate acidied with 0.5% of formic acid, and the combined extracts were dried, ltered, and evaporated to

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Table 1 Response of psychrotrophic proteolytic bacteria isolated from cooled raw milk using three-monitor systems: Chromobacterium violaceum CV 026, Escherichia coli DH5a pSB403 and Agrobacterium tumefaciens A136 Species No. of evaluated strains No. responding in monitor system CV 026 Induced Psychrotrophic proteolytic AHL producers Acinetobacter lwo Aeromonas hydrophila Burkholderia pseudomallei Cedecea lapagei Chryseobacterium miningosepticum Chryseomonas luteola Enterobacter cloacae Enterobacter gergoviae Enterobacter sakasaki Hafnia alvei Klebsiella oxytoca Klebsiella ozanae Moraxella catharralis Pantoea agllomerans Pantoea spp Providencia stuartii Pseudomonas uorescens Pseudomonas putida Pseudomonas stutzeri Rhanella aquatilis Serratia liquefaciens Serratia odorifera Total ATCC strains Aeromonas hydrophila ATCC 7966 Chromobacterium violaceum ATCC 6357 Enterobacter aerogenes ATCC 13048 Pseudomonas uorescens ATCC 13525 Psedomonas aeruginosa ATCC 15442 Serratia liquefaciens ATCC 27592 Total 1 4 2 1 1 2 1 1 2 3 2 2 3 3 1 1 7 2 1 2 9 2 53 1 1 1 1 1 1 7 1 1 Inhibited 1 3 2 1 1 1 1 2 3 2 1 2 2 3 1 2 pSB403 A136

2 2

2 3

5 1 1 7 33

1 1 11

2 1 15

2 9

1 1 1 1 1 6

1 1 1 3

1 2

dryness. Cultures extract were dissolved in 400600 ll of HPLC-grade ethyl acetate. Synthetic AHLs or extract samples dissolved in ethyl acetate, in volumes of 1020 ll were spotted onto c18 reversed-phase TLC plates (aluminum sheets 20 20 cm; RP-18 F254 S, 1.05559. Merck 64271, Darmstadt, Germany) and the chromatogram was developed using a solvent system of methanol/water (60:40, v/v) as described by Shaw et al. (1997). After development, the solvent was evaporated, and the dried plates were overlaid with a culture of monitor strain. A 30 ml overnight culture of E coli (pSB403) or C. violaceum CV026 was used to inoculate 150 ml of LB medium, and the culture was spread over the surface of the developed plates. After the agar had solidied, the plates were incubated overnight at 30 C in a sterilized closed plastic container. AHLs were visualized as bright spots in Eagle Eye II (Stratagene, La Jolla, CA, USA) on plates overlaid with E. coli (pSB403), or purple spots for the ones with C. violaceum CV026.

3. Results and discussion The psycrotrophic proteolytic strains elicited diverse responses in the three AHL-monitor systems, which demonstrate production of dierent AHL molecules. A total of 45 (84.9%) of the psychrotrophic bacteria isolated from milk were identied as AHL producers (Table 1). Because of the specicity requirements of the R protein, the LuxR homologues, most of the detection systems are limited in the range of AHLs to which they respond (Shaw et al., 1997). Therefore, it represents a limitation of the bioassay since bacteria often produce more than one AHL molecule. This limitation can be overcome by using multiple monitor systems. C. violaceum CV026 detected 40 (75.5%) of strains as AHL producers (Table 1). Within the positive results, 11 (20.7%) produced short chain AHL as demonstrated by the induction of the violacein production by the reporter strain (Fig. 1A2), and 33 (62.3%) probably produced long chain AHL (Fig. 1B1). The lack of the pigment in this

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Fig. 1. Examples of agar plate assays for screening for AHL production in proteolytic psychrotrophic bacteria isolated from cooled raw milk. Monitor strains were streaked parallel to the tested bacteria or to themselves as described in Section 2. The blue light seen on plate C2 refers to a positive luminescence response. A1, B2, C1, and D1 refer to negative results. (A1) Chromobacterium violaceum CV026 parallel to itself; (A2) C. violaceum CV026 parallel to Pantoea agllomerans. B1 and B2 refer to C. violaceum CV026 inibition assay; (B1) C. violaceum CV026 parallel to Rhanella aquatilis as a positive result for long chain AHL; (B2) C. violaceum CV026 parallel to Serratia odorifera as a negative result for long chain AHL. (C1) Escherichia coli (pSB403) parallel to itself; (C2) E. coli (pSB403) parallel to Pantoea agllomerans. (D1) Agrobacterium tumefaciens A136 parallel to itself; (D2) A. tumefaciens A136 parallel to Pseudomonas uorescens.

experiment is assumed to be due to specic interference with the AHL-induced production of violacein (McClean et al., 1997). The proteolytic psychrotrophic bacteria isolated from cooled raw milk that were positive in the induction assay

of C. violaceum CV026 may produce AHL compounds with unsubstituted side chains from C4 to C8 in length, since this monitor strain responds to these type of AHLs (McClean et al., 1997). On the other hand, the strains, which responded to the inhibition assay, probably produce

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AHL with side chain from C10 to C14 in length (McClean et al., 1997). Bacteria with positive response in E. coli pSB403 (Fig. 1C2) may produce AHL with carbon side chains of C6 or C8 with or without oxo-substitutions (Winson et al., 1998). E. coli (pSB403) detected 15 (28.3%) and A. tumefaciens A136 detected 9 (17.0%) psychrotrophic isolates as AHL producers. Although Shaw et al. (1997) observed that an A. tumefaciens monitor strain was able to detect AHL with 3-oxo, 3-hydroxy, and 3-unsubstituted side chain, the majority of the isolates evaluated in this work did not induce the A. tumefaciens A136 (Table 1), except the P. uorescens isolates which produced molecules that presented a weak response in the assay (Fig. 1D2). This result indicates that the signal molecule(s) produced by P. uorescens strains were in low concentration or they are dierent from the cognate autoinducer that activates TraR protein in A. tumefaciens. Once A. tumefaciens A136 responded to substances present in culture media in some works performed in our lab (unpublished data), we cannot discard a false positive result. Holden et al. (1999) demonstrated that other molecules besides AHLs could activate biosensors, underlining the importance of chemical characterization of the molecules identied in such bioassays. The bacteria belonging to the Enterobacter genus, including the ATCC 13048, showed a positive response only in the inhibition assay using C. violaceum CV026. The same result occurred with the majority of isolates belonging to the species S. liquefaciens, P. uorescens, A. hydrophila, S. liquefaciens ATCC 27592, and P. uorescens ATCC 13525 (Table 1) indicating that they may produce long side chain AHLs. Hafnia alvei produced short chain AHLs once it induced the reporter strains C. violaceum CV026 (induction assay), and E. coli (pSB403). Bruhn et al. (2004) observed that AHL-producing H. alvei may induce food quality-relevant phenotypes in other bacterial species in the same environment. Therefore, H. alvei may inuence spoilage of food products in the presence of Enterobacteriacea. To further conrm the agar assay results, TLC was performed with AHL extracts from representative strains of A. hydrophila, H. alvei, M. catharralis, Pantoea agllomerans, and P. uorescens, followed by revelation with E. coli (pSB403) (Fig. 2) or C. violaceum CV026 (data not shown). The TLC results indicate the presence of at least one kind of AHL in the extracts of A. hydrophila, H. alvei, M. catharralis, and P. agllomerans, conrming the results of the agar assay. Whan, Dunstall, and Rowe (2000) suggested the involvement of QS in the growth of P. uorescens, a known spoilage organism in milk and milk products. In another work, it was observed that N-benzoyloxycarbonyl-L-homoserine lactone and N-3-oxyhexanoyl-DL-homoserine lactone signicantly reduced the lag phase duration and increased the exponential growth rate of three strains of P. uorescens (Dunstall, Rowe, Wisdom, & Kilpatrick, 2005). Christensen et al. (2003) demonstrated that AHLs

Fig. 2. A representative thin-layer chromatogram of the AHLs present in cell free supernatants of proteolytic psychrotrophic bacteria isolated from cooled raw milk. Samples were chromatographed on c18 reversed-phase thin-layer plates, developed with methanol/water (60:40, vol/vol), and the spots were visualized with the Escherichia coli (pSB403) reporter strain in Eagle Eye II (Stratagene, La Jolla, CA, USA). Lane 1, extract from LB medium as a negative control; lane 2, N-hexanoyl-DL-homoserine lactone standard; lane 37, samples from culture extracts of the following: 3, Aeromonas hydrophila; 4, Pseudomonas uorescens; 5, Pantoea agllomerans; 6, Moraxella catharralis; 7, Hafnia alvei.

are involved in the production of spoilage characteristics in milk samples inoculated with Serratia proteamaculans B5a. Our data demonstrated that AHL-production is common among proteolytic psychrotrophic bacteria isolated from raw milk. Once these organisms were isolated from a common source, the possibility of cross-communication between them is relevant and raises the question of what kind of phenotypes might be regulated when they are growing together, and also if these phenotypes have any relation with food deterioration. The understanding of the role of the QS mechanism in the regulation of spoilage phenotypes in bacteria from food origin is relevant and may be used to create new ways to preserve food products. The control of cell-cell communication usually referred to quorum quenching has been studied and proved to be successful in microorganisms isolated from non-food sources. In the plant pathogen Erwinia carotovora, the attenuation of virulence was achieved by inactivating the signal molecule with an enzyme called AiiA from Bacillus sp 240b1 (Dong, Xu, Li, & Zhang, 2000). Some other works have shown that halogenated furanones from alga Dalisea pulchra can inhibit gene expression mediated by AHL by interfering with the autoinducer binding to the LuxR homologue (Maneeld, Welch, Givskov, Salmond, & Kjelleberg, 2001; Rice, Givskov, Steingerg, & Kjelleberg, 1999). Rasmussen et al. (2005) screened several compounds for their ability to inhibit quorum sensing and it was found that garlic extract was one of the most eective being able to reduce P. aeruginosa biolm tolerance to tobramycin treatment as well as virulence in a Caenorhabditis elegans pathogenesis model. As far as this knowledge can be applied to improve

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food quality and safety more work needs to be done to answer this question. Acknowledgements We thank Professor Paul Williams from Nottingham University (UK) for the donation of plasmid pSB403 and strain C. violaceum CV026, and Professor Stephen C. Winans from Cornell University (USA) for the donation of strain A. tumefaciens A136. Uelinton Manoel Pinto and Maurilio Lopes Martins were supported by the Conselho Nacional de Desenvolvimento Cientco e Tecnologico (CNPq) Braslia, Brazil. Eliseth de Souza Viana was sup ported by the Coordenadoria de Pessoal de Nvel Superior-Capes, Brazil. References
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