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CyTA - Journal of Food
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Inhibition of Salmonella spp. isolated from mango using bacteriocin-like produced by lactobacilliInhibición de Salmonella spp. aislada de mango usando sustancias tipo bacteriocinas producidas por lactobacilos
J. A. Ragazzo-Sancheza; L. Sanchez-Pradoa; P. Gutiérrez-Martíneza; G. Luna-Solanob; B. Gomez-Gilc; M. Calderon-Santoyoa a Instituto Tecnologico de Tepic, Tepic, Nayarit, Mexico b Division de Estudios de Posgrado e Investigacion, Instituto Tecnologico de Orizaba, Orizaba, Veracruz, Mexico c CIAD, Sinaloa, Mexico Online publication date: 08 March 2010
To cite this Article Ragazzo-Sanchez, J. A. , Sanchez-Prado, L. , Gutiérrez-Martínez, P. , Luna-Solano, G. , Gomez-Gil, B.
and Calderon-Santoyo, M.(2009) 'Inhibition of Salmonella spp. isolated from mango using bacteriocin-like produced by lactobacilliInhibición de Salmonella spp. aislada de mango usando sustancias tipo bacteriocinas producidas por lactobacilos', CyTA - Journal of Food, 7: 3, 181 — 187 To link to this Article: DOI: 10.1080/19476330903010276 URL: http://dx.doi.org/10.1080/19476330903010276
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CyTA – Journal of Food Vol. 7, No. 3, November 2009, 181–187
Inhibition of Salmonella spp. isolated from mango using bacteriocin-like produced by lactobacilli Inhibicio´n de Salmonella spp. aislada de mango usando sustancias tipo bacteriocinas producidas por lactobacilos J.A. Ragazzo-Sancheza, L. Sanchez-Pradoa, P. Gutie´rrez-Martı´ neza, G. Luna-Solanoc, B. Gomez-Gilb and M. Calderon-Santoyoa* a Instituto Tecnologico de Tepic, Av. Tecnologico, 2595, Ap. Postal 343, Tepic, Nayarit 63175, Mexico; bCIAD, A.C. Mazatlan Unit., AP. 711, Mazatlan, Sinaloa 82000, Mexico; cInstituto Tecnologico de Orizaba, Division de Estudios de Posgrado e Investigacion, Av. Oriente, 9 no. 852, Orizaba, Veracruz 94320, Mexico
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(Received 25 November 2008; final version received 17 April 2009) Five mangos from every step of the packaging process of mango as well as five samples from tap water and five samples from dip tank water were analyzed for Salmonella. Four presumptive Salmonella spp. strains were isolated from mango fruits and from water used for its washing. When identification of the strains was performed by PCR, only three strains turned out to be positive: Salmonella sp. isolated from tap water (tw), Salmonella sp. isolated from mango Ataulfo after washing (ataw) and Salmonella sp. isolated from mango Tommy Atkins after washing (tmaw). A natural but little inhibition of these Salmonella spp. was observed when single bacteriocin-like substances from Lactobacillus plantarum A6 and from L. fermentum OgiE1 were used (average reduction of 0.78 log 10 CFU). An enhanced effectiveness was observed when combining both bacteriocin-like substances (average reduction of 2.84 log 10 CFU). The addition of NaCl (40 mg/mL) to the single bacteriocin-like substances significantly improved the inhibition of Salmonella spp. (reduction of 2.67 to 2.84 log 10 CFU) but when NaCl was added to the mixture of both bacteriocin-like substances, inhibition of Salmonella spp. was not enhanced (reduction of 2.68 log 10 CFU). Keywords: bacteriocins; gram-negative pathogens; inhibition; mango; Salmonella Cinco mangos de cada etapa del proceso de empacado del mango, ası´ como cinco muestras de agua de la llave y cinco muestras de agua del tanque de lavado por inmersio´n fueron analizadas para Salmonella. Cuatro presuntas cepas de Salmonella spp. fueron aisladas a partir de frutos de mango y del agua utilizada para su lavado. Cuando la identificacio´n de las cepas se realizo´ por PCR, so´lo tres cepas resultaron positivas: Salmonella sp. aislada del agua de la llave (tw), Salmonella sp. aislada del mango Ataulfo despue´s del lavado (ataw) y Salmonella sp. aislada del mango Tommy Atkins despue´s del lavado (tmaw). Cuando so´lo las sustancias tipo bacteriocinas de Lactobacillus plantarum A6 y de L. fermentum OgiE1 fueron utilizadas, se observo´ una ligera inhibicio´n de Salmonella spp. (reduccio´n promedio de 0,78 log 10 UFC). Cuando ambas sustancias tipo bacteriocinas fueron utilizadas en combinacio´n, se observo´ un aumento de la efectividad en la inhibicio´n de Salmonella spp. (reduccio´n promedio de 2,84 log10 UFC). La adicio´n de NaCl (40 mg/mL) a las sustancias tipo bacteriocinas de manera separada, aumento´ la eficacia en la inhibicio´n de Salmonella spp. (reduccio´n de 2,67 a 2,84 log 10 UFC), pero cuando el NaCl se agrego´ a la mezcla de ambas sustancias tipo bacteriocinas, no se observo´ una mejora en la inhibicio´n de Salmonella spp. (reduccio´n de 2,68 log 10 UFC). Palabras clave: bacteriocinas; pato´genos Gram-negativos; inhibicio´n; mango; Salmonella
Introduction Outbreaks of human infections associated with consumption of raw fruits and vegetables have occurred with increased frequency during the past decade. Foods of animal origin, such as poultry, eggs, meat, and dairy products, have been traditionally recognized as vehicles of Salmonella. However, salmonellosis has also been associated with consumption of raw vegetables and fruits such as tomatoes (Guo, Chen, Brackett, & Beuchat, 2001), mangos (Penteado, Eblen, & Miller, 2004; Sivapalasingam, Barrett, *Corresponding author. Email:
[email protected] ISSN 1947-6337 print/ISSN 1947-6345 online Ó 2009 Taylor & Francis DOI: 10.1080/19476330903010276 http://www.informaworld.com
Kimura, Van Duyne, & De Witt, 2003), watermelons (Blostein, 1991), and alfalfa sprouts (Barak, Whitehand, & Charkowski, 2002). The first salmonellosis documented outbreak triggered by consumption of in natura mangos occurred in 1999, in 13 states of the USA, involving 78 people and two deaths. Laboratory findings showed that water samples collected at the farm and one cloacal sample were positive for Salmonella Newport (Penteado et al., 2004; Sivapalasingam et al., 2003). In 2001, another multistate outbreak occurred in USA, triggered by the
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consumption of mangos, this time caused by Salmonella Saint Paul. In mangos imported from Peru and in those imported from Brazil, the epidemiological investigation pointed that the water utilized in the hydrothermal treatment is responsible for the contamination of the fruits (Branquinho Bordini, Asturiano Ristori, Jakabi, & Scala Gelli, 2007). Recent outbreaks showed the importance of incorporating safety hurdles in production of these types of products. Disinfectant agents of chemical origin have been used for controlling pathogen contamination. Furthermore, chemical control has been threatened by consumer preferences and regulatory issues concerning chemical residues in fruits, detrimental effect on the environment, and consequences on the consumer’s health (McAuliffe, Ross, & Hill, 2001). In rapidly developed fields, biological control has shown greater promise in recent years. Bacteriocins produced by lactic acid bacteria (LAB) are peptides or proteins with an antibacterial mode of action towards closely related species and some Gram positive pathogens (McAuliffe et al., 2001; Van Reenen, Chikindas, Zyl, & Dicks, 2003). However, these peptides have rarely been associated to inhibit Gram negative bacteria (Messens & De Vuyst, 2002). The antibiotic activity of bacteriocins against Gram-positive bacteria is based on interaction with the bacterial membrane, pore formation, and disruption. For the nisin and epidermin family of lantibiotics, the membrane-bounded cell wall precursor lipid II has recently been identified as a target in pore formation. However, combinations of bacteriocins with other substances as organic acids, NaCl, quelant agents (EDTA) (Bouttefroy & Milliere, 2000; Branen & Davidson, 2004), or combinations of different bacteriocins (Bouttefroy & Milliere, 2000) induce a greater inhibitory effect than the use of a single bacteriocin. Even the inhibition of Gram negative pathogens has been observed using these methods. Some bacteriocins produced by Lactobacillus spp. have been reported to show an antimicrobial action before Gram negative bacteria (Sanni, Onilude, Ogunbanwo, & Smith, 1999; Suma, Misra, & Varadaraj, 1998), however; little is known concerning the specific inhibitory mechanism. Bacteriocins are attractive as antimicrobial agents because of their GRAS (Generally Recognized as Safe) status established by the FDA (Food and Drugs Administration) (Rodrı´ guez, Martı´ nez, Horn, & Dodd, 2002). The aim of this work was to evaluate the behavior of single or combined (with NaCl salt) bacteriocins-like substances produced by lactobacilli of vegetal origin: Lb. plantarum A6 (Giraud, Brauman, & Keleke, 1991) and Lb. fermentum Ogi E1 (Agati, Guyot, MorlonGuyot, Talamond, & Hounhouigan, 1998; Caldero´n, Loiseau, & Guyot, 2001) against Salmonella spp., isolated from mango (Mangifera indica L var. Tommy Atkins and Ataulfo) produced in Nayarit and destined to exportation.
Materials and methods Bacterial strains The bacteriocin-like producers Lactobacillus plantarum A6 (deposited at the BCCMTM/LMG culture collection under accession number LMG 18053) and Lactobacillus fermentum OgiE1 (I-2028, CNCM, Institut Pasteur) were obtained from the Institut de Recherche pour le Developement (IRD, Montpellier, France). Listeria innocua AST 062 was obtained from Instituto Tecnologico de Veracruz, Veracruz, Mexico. The strains were conserved in 40% glycerol at 7808C. Culture conditions Bacteriocin-like producer strains were grown in 10 6 120 mm culture tubes containing 10 mL of MRS broth (de Man, Rogosa and Sharpe broth) (de Man, Rogosa, & Sharpe, 1960) (Difco, Detroit, MI, USA) at 308C for 24 h. A 10 mL aliquot was inoculated in 150 mL Erlenmeyer flasks containing 100 mL of MRS broth and incubated at 308C for 12 h. Fermented broth was centrifuged at 5000g for 15 min to obtain cell-free supernatant that was neutralized with 0.1 N NaOH to pH 6.5, and microfiltered using 0.2 mm pore size sterile membranes. The bacteriocin-like crude extract was conserved at 7208C until use for inhibition tests. Salmonella spp. isolation and identification Physiological ripened mangos (var. Tommy Atkins and Ataulfo) were obtained from a fruit export agency in El Crucero, Nayarit. According to the International Commission on Microbiological Specifications for Foods (ICMSF, El Crucero, Nayarit, Mexico), a second class sampling plan recommended for Salmonella analysis in fresh vegetables and fruits (n ¼ 5, c ¼ 0, and m ¼ 0) was used (http://www.nap.edu/ openbook.php?record_id¼10690). Then, five mangos from every step of the packaging process (reception, after washing, after hydrothermal and water-cooling treatment, during caliber selection and packaging) were analyzed for Salmonella spp. Additionally, five samples from tap water and dip tank were sampled. All samples were transferred to the laboratory under refrigerated conditions. Salmonella spp. strains were isolated according to the U.S. Food and Drug Administration’s Bacteriological Analytical Manual (www.cfsan.fda. gov/*ebam/bam-5.html#Isol). Whole mangos were placed into sterile plastic bags containing 300 mL of buffered peptone water (BPW) pH 7.0, and its surface was rubbed for 2 min. Then 25 mL of the broth from the rinsing process were inoculated in 225 mL of lactose broth (Difco, Detroit, MI, USA) and incubated at 358C for 24 h. After this period, 10 mL of the preenrichment liquid were inoculated into Rappaport– Vassiliadis broth (Merck, Darmstadt, Germany) and Selenite–Cystine broth (Merck, Darmstadt, Germany),
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CyTA – Journal of Food respectively, and were incubated at 428C for 24 h. Selective plating was carried in Xylose lysine desoxycholate (XLD) agar (Oxoid, Basingstoke, UK), Hektoen enteric (HE) agar (Biokar Diagnostic, Beauvais, France), and Bismuth sulfite (BS) agar (Difco). Colonies resembling Salmonella on isolation media were isolated for presumptive identification by some biochemical tests (TSI, LIA, indole test, and urease). Presumptive Salmonella colonies were identified by PCR. DNA was extracted from typical colonies with Promega Wizard Genomic DNA Purification kit (Promega, Madison, WI, USA). Identification of Salmonella spp. was done based on the methodology of Wang, Cao, and Cerniglia (1997); briefly, specific primers targeting the invA gene (Sal-3 TATCGC CACGTTCGGGCAA, Sal-4 TCGCACCGTCAAA GGAACC) were used in a PCR reaction, consisting in 16.96 mL of water, 1.32 mL of a dNTP mix (2.5 mM each), 2.5 mL of 106 PCR buffer, 1.50 mL of a 25 mM MgCl2 solution, 0.26 mL of each primer (0.25 mg/mL), 0.2 mL of Taq (5 U/mL, Promega, Madison, WI, USA), and 1.0 mL of DNA (50 ng/mL) for a final volume of 25 mL. The amplification protocol was 948C for 2 min followed by 35 cycles of 948C for 1 min, 618C for 1 min and 728C for 1 min with a final extension of 728C for 5 min. PCR products were resolved in a 1.5% agarose electrophoresis gel. Salmonella choleraesuis subsp. choleraesuis CAIM 698 (¼ ATCC 14028) was used as a positive control. A posterior identification of species for strains resulting positive for PCR analysis was performed by additional biochemical tests (Koneman, Allen, Dowell, & Sommers, 1991; MacFadden, 1980). Bacteriocin activity estimation Quantitative activity of bacteriocin-like extracts from culture samples of Lactobacillus plantarum A6 and Lactobacillus fermentum OgiE1 were estimated using a photometric assay on culture tubes (Guerra, Luisa Rua, & Pastrana, 2001). Listeria innocua AST 062 was used as target microorganism. The method consists on the determination of growth inhibition (at 600 nm) of the target organism caused by serial dilutions (1:10, 1:30, 1:50, 1:100, 1:200) of bacteriocin-like crude extracts. Bacteriocin-like extracts were diluted in distilled water. 2.5 mL of diluted extracts were added to sterile tubes. Each tube was inoculated with 2.5 mL of a culture of Listeria innocua AST 062 (diluted to an absorbance of 0.2 at 600 nm) with double concentration of sterile MRS broth. Controls consisted of three tubes in which the diluted bacteriocin was substituted by distilled sterile water. Tubes were incubated for 12 h at 308C. Growth inhibition was measured spectrophotometrically at 600 nm. Dose– response curves were obtained from these data. Bacteriocin activity was calculated as Bacteriocin Units (BU mL71, 1 BU mL71 is equivalent to the amount of bacteriocin needed to obtain 50% growth inhibition (lethal dose 50 (LD50) compared to control tubes)).
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Inhibition tests Bacteriocins-like inhibition against Salmonella spp. was evaluated in 10 6 120 mm culture tubes containing 3 mL nutritive broth. A 50 mL aliquot of a 14 h culture of Salmonella spp. adjusted to 0.6 at 600 nm, was inoculated to the culture broth. The inhibitory effect of bacteriocins-like and NaCl was evaluated according to a factorial design (Table 1). A control containing Salmonella spp. but not bacteriocins-like was performed (Trial 1). Culture tubes were incubated at 378C for 12 h and absorbance (A600 nm) was recorded. A calibration curve (CFU/mL ¼ 798,551 6 A600 nm þ 898.55, R2 ¼ 0.963) was generated using values of absorbance (A600 nm) from 0.05 to 0.8 and mean counts on Violet Red Bile Glucose Agar (VRBGA), for every absorbance value, measurements were performed by triplicate. The calibration curve was then used for routine analysis of samples using the absorbance to generate an estimate of the number of colony forming units (CFU) present. Mean colony forming units (log 10 CFU) were compared by a bifactorial analysis LSD test (p 5 0.05) in SAS for Windows. Results and discussion Detection of Salmonella spp. in mango Even if 42 characteristic colonies for Salmonella strains were found in samples inoculated on XLD, HE and BS agar, only four isolates resulted presumptive for Salmonella after applying some biochemical tests (Table 2). After the PCR was performed for the four presumptive isolates, both mango varieties, Tommy Atkins and Ataulfo, were positive for Salmonella in samples after washing (Figure 1, Table 2). Tap water turned out to be positive for Salmonella spp., although
Table 1. 23 Factorial design used to evaluate the inhibitory effect of bacteriocins-like and NaCl on Salmonella spp. strains isolated from the packaging process of mango Ataulfo and Tommy Atkins. Tabla 1. Disen˜o factorial 23 utilizado para evaluar el efecto inhibitorio de las sustancias tipo bacteriocinas y de NaCl en cepas de Salmonella spp. aisladas del proceso de empacado del mango variedades Ataulfo y Tommy Atkins.
Trial 1 2 3 4 5 6 7 8
Bacteriocin-like by Lb. plantarum A6 (mg/L crude extract)
Bacteriocin-like by Lb. fermentum OgiE1 (mg/L crude extract)
NaCl (g/L)
0 100 0 100 0 100 0 100
0 0 100 100 0 0 100 100
0 0 0 0 40 40 40 40
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J.A. Ragazzo-Sanchez et al. Table 2. Number of strains presumptive or positive for Salmonella isolated from different steps in the packaging process of mango Ataulfo y Tommy Atkins. Tabla 2. Nu´mero de cepas presuntivas positivas a Salmonella aisladas de diferentes etapas en el proceso de empacado del mango variedades Ataulfo y Tommy Atkins.
Packaging process step
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Figure 1. Agarose gel electrophoresis from polymerase chain reaction products amplified from Salmonella spp. strains from the packaging process of mango Ataulfo and Tommy Atkins, using specific primers targeting the invA gene. Line 1: Isolate from mango Tommy Atkins after washing; Line 2: Isolate from dip tank; Line 3: Isolate from mango Ataulfo after washing; Line 4: Isolate from tap water; Line 5: Salmonella choleraesuis subsp. choleraesuis, positive control; Line 6: reagents blank; Line M: molecular weight marker (fx 174/Hae III, Invitrogen life Technologies, bp). Figura 1. Electroforesis en gel de agarosa de productos amplificados de reaccio´n en cadena de la polimerasa de cepas de Salmonella spp. provenientes del proceso de empacado del mango variedades Ataulfo y Tommy Atkins, usando primers especı´ ficos de la region invA. (Lı´ nea 1): cepa aislada del mangoTommy Atkins despue´s del lavado; (Lı´ nea 2): cepa aislada del tanque de inmersio´n para el lavado; (Lı´ nea 3): cepa aislada del mango Ataulfo despue´s del lavado; (Lı´ nea 4): cepa aislada del agua de la llave; (Lı´ nea 5): Salmonella choleraesuis subsp. choleraesuis, control positivo; (Lı´ nea 6): blanco de reactivos; (Lı´ nea M): marcador de peso molecular (fx 174/Hae III, Invitrogen Life Technologies, bp).
Mango Ataulfo from reception Mango Tommy Atkins from reception Mango Ataulfo after washing Mango Tommy Atkins after washing Mango Ataulfo after hydrothermal and water cooling Mango Tommy Atkins after hydrothermal and water cooling Mango Ataulfo during caliber selection Mango Tommy Atkins during caliber selection Mango Ataulfo packaged Mango Tommy Atkins packaged Tap water Dip tank water
Number of isolates presumptive for Salmonella after biochemical tests
Number of isolates positive for Salmonella after PCR analysis
ND
–
ND
–
1
1
1
1
ND
–
ND
–
ND
–
ND
–
ND ND
– –
1 1
1 ND
ND, not detected.
in water samples from dip tank Salmonella spp. was not detected by PCR methods (Figure 1, Table 2). Additional biochemical tests were applied to strains identified as Salmonella spp. by PCR and species were determined as follows: Salmonella enteritidis for Salmonella sp. tw and Salmonella sp. ataw and Salmonella choleraesuis for Salmonella tmaw. Estimation of crude extracts bacteriocin activity Quantitative activity of bacteriocin-like crude extracts from culture samples of Lb. plantarum A6 and Lb. fermentum OgiE1 using Listeria innocua AST 062 as sensible microorganism were estimated as being 143 BU mL71 and 187 BU mL71, respectively. This result shows a good potential for these bacteriocin-like substances for their use in foods systems since similar activities have been reported for nisin, a bacteriocin largely studied and used in some foods, against Listeria strains (Rodrı´ guez et al., 2002). Effect of bacteriocins-like on Salmonella spp The effectiveness of bacteriocins-like substances from Lb. fermentum OgiE1 and Lb. plantarum A6 on Salmonella spp., expressed as the reduction in log 10
ND, no detectado.
CFU comparing with a control without bacteriocins, was shown to be moderate. In fact, there was a statistic difference (p 5 0.05) only between control and treatments with bacteriocins-like substances from Lb. fermentum OgiE1 and Lb. plantarum A6 for Salmonella sp. tmaw. (isolated from mango var. Tommy Atkins after washing). Thus, a reduction of 1.4 and 1.3 log 10 CFU, respectively, was achieved (Table 3). The lowest inhibition (reduction of 0.48 log 10 CFU) was noted for Salmonella sp. ataw (isolated from mango var. Ataulfo after washing). Similar results were obtained when using the bacteriocin from Lb. fermentum OgiE1 (growth reduction between 0.44 and 1.30 log 10 CFU). These results agree with those found by Sanni et al. (1999) and Suma et al. (1998), where some inhibition of Gram negative bacteria was obtained with plantaricin LP84 and with a bacteriocin obtained from the Ogi fermentation, the natural source of Lb. fermentum OgiE1. Nevertheless, this inhibition was performed with purified bacteriocins (Sanni et al., 1999). However, in this study an enhanced inhibition was observed when both bacteriocins were combined,
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Table 3. Effect of bacteriocin-like substances from L. plantarum A6 (P) and L. fermentum OgiE1 (F) and NaCl (N) on microbial growth (log 10 CFU) of Salmonella strains isolated from tap water (tw), mango Ataulfo after washing (ataw) and mango Tommy Atkins after washing (tmaw). Tabla 3. Efecto de las sustancias tipo bacteriocinas de L. plantarum A6 (P) y L. fermentum OgiE1 (F) and NaCl (N) en el crecimiento microbiano (log 10 UFC) de cepas de Salmonella aisladas del agua de la llave (tw) y del mango Ataulfo despue´s del lavado (ataw) y Tommy Atkins despue´s del lavado (tmaw). Key Control P F PþF N PþN FþN PþFþN
Trial
Salmonella sp. tw
Salmonella sp. ataw
Salmonella sp. tmaw
1 2 3 4 5 6 7 8
7.11+0.4a,x 6.59+0.3a,x 6.61+0.4a,x,z 4.11+0.5b,x 4.89+0.6b,x 4.23+0.1b,x 3.92+0.4b,x 4.25+0.6b,x,z
7.18+0.5a,x 6.69+0.6a,x 6.73+0.1a,x 4.65+0.5b,c,x 5.02+0.5b,x 5.02+0.6c,y 5.05+0.5c,y 4.88+0.2b,c,x
7.30+0.1a,x 5.90+0.2b,y 6.00+0.3b,y,z 4.30+0.3c,x 5.08+0.4d,x 4.34+0.1c,x 4.11+0.2c,x 4.43+0.2c,y,z
Different upper case letters (a, b, c, d) on the same column indicate significant differences (p 5 0.05) among treatments according to LSD test. Different upper case letters (x, y, z) on the same row indicate significant differences (p 5 0.05) among strains Salmonella sp. tw, Salmonella sp. ataw, and Salmonella sp. tmaw according to LSD test. Values (log 10 CFU) are the mean of four measurements+S.D.
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Letras diferentes en superı´ ndice (a, b, c, d) en el mismo renglo´n indican diferencias (p 5 0.05) entre tratamientos de acuerdo a la prueba LSD. Letras diferentes en superı´ ndice (x, y, z) en la misma columna indican diferencias (p 5 0,05) entre las cepas Salmonella sp. tw, Salmonella sp. ataw y Salmonella sp. tmaw de acuerdo a la prueba LSD. Los valores (log 10 UFC) son la media de cuatro ana´lisis+D.E.
growth reductions from 3.0 to 2.52 log 10 CFU for Salmonella sp. tw and tmaw (isolated from tap water and mango var. Tommy Atkins) and Salmonella sp. (isolated from mango var. Ataulfo after washing), respectively (Table 3). A synergism between bacteriocins has been reported before, Bouttefroy and Millie`re (2000) found that nisin-curvaticin 13 combinations turned out to have a better bactericidal effect against Listeria monocytogenes ATCC 15313 than the use of a single bacteriocin. Bacteriocins are traditionally characterized by their bacteriostatic or bactericidal activity against related bacterial genera (Lactobacillus, Listeria, Streptococcus, Staphylococcus, Clostridium, and Bacillus) and some are also active against Gram positive food-borne pathogens and spoilage bacteria, but they are not considered against Gram-negative bacteria (Gomez, Cosson, & Deschamps, 1997). Surprisingly, some bacteriocins produced by Lactobacillus (Sanni et al., 1999; Suma et al., 1998) and one produced by Streptococcus spp. CNCM I-841 (Gomez et al., 1997) could inhibit Gram-negative microorganisms such as Escherichia coli and Salmonella. Nevertheless, little is known about their passage through the Salmonella membranes and periplasm, since the Gramnegative’s characteristic cellular wall works as a natural barrier blocking the bacteriocin access to the cytoplasm (McAuliffe et al., 2001). Gao, Van Belkum, and Stiles (1999) determined the outer membrane of Salmonella enterica ser. typhimurium act as a natural barrier against brochocin-C, a peptide bacteriocin produced by Brochothrix campestris ATCC 43754. In this particular case, inhibition was only achieved on EDTA-treated cells, Salmonella enterica lipopolysaccharide mutants and spheroplasts of typhimurium
strains LT2 and SL3600. Concerning Escherichia coli, a reputed Gram-negative food borne pathogen, Duche´ (2007) suggested that colicins reach their targets in susceptible Escherichia coli strains through two envelope protein systems: the Tol system is used by group A colicins and the TonB system by group B colicins. Additionally, the Escherichia coli outer membrane cobalamin transporter, BtuB, forming a 1:1 complex, initiates cellular import of colicin E3. A dogma of colicin biology is that it must unfold or unfurl to span the periplasm, with recent reports claiming this reaction is initiated by receptor binding. Small conformational changes occur in the BtuB cork and barrel domains but are insufficient to form a translocation channel. It was found out the porin OmpF is recruited directly to the BtuB colicin complex to form the translocon. Thus, recruitment is through the natively unfolded region of the colicin translocation domain (Housden, Loftus, Moore, James, & Kleanthous, 2005). Thus, similar proteins systems could be involved in the entry of bacteriocins-like from Lb. fermentum OgiE1 and Lb. plantarum A6 into the Salmonella spp. inner system. Effect of bacteriocins-like in combination with NaCl on Salmonella spp An enhanced inhibition of Salmonella spp. was observed, when adding NaCl to the bacteriocins-like used separately (Trials 2 and 3 and Trials 6 and 7, Table 3). It is important to remark that no statistical differences were obtained between treatments using bacteriocins-like substances combined (Trial 4, Table 3) and treatments using bacteriocins-like substances
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combined with NaCl (Runs 6 and 7, Table 3). The combined effect of both bacteriocins-like substances together with NaCl (Trial 8, Table 3) showed similar growth as the bacteriocins-like substances combined (Trial 4, Table 3). NaCl treatment (Trial 5, Table 3) turned out to have similar Salmonella spp. growth as the treatment where bacteriocin-like substances were combined (Trial 4, Table 3), except for Salmonella sp. tmaw, whose growth was inferior when treated with bacteriocin-like substances (Trial 4, Table 3). Even if a similar inhibition was achieved with NaCl treatment, some later studies applying 4 g/L NaCl solutions on the mango exocarp have shown high values of total weight loss (data not shown), then an application of a mixture of bacteriocin-like substances could be more useful. Some reports explain that Salmonella and E. coli exposure to a sublethal stress that disrupts the outer membrane allows bacteriocins access to the cytoplasmic membrane and leads to an increased sensitivity. These sublethal stresses include hydrostatic pressure (Hugas, Garriga, & Monfort, 2002), heat, freezing, as well as NaCl, Tris and EDTA addition (Branen & Davidson, 2004), organic acids addition: Potassium lactate and sodium diacetate (Stekelenburg, 2003) or eukariotic antimicrobial peptides addition (Lu¨ders, Birkemo, Fimland, Nissen-Meyer, & Nes, 2003). Some authors have suggested that a chelator such as EDTA and sodium triphosphate, can destabilize the cell membranes of bacteria by complexing the divalent cations which act as salt bridges between membrane macromolecules such as lipopolysaccharides. In this manner, bacteriocins access to the Gramnegative bacteria cytoplasm is allowed (Branen & Davidson, 2004). The inhibitory effect was different among the different Salmonella strains isolated from the two varieties of mangos (Tommy Atkins and Ataulfo) and from water samples; these differences confirm sensitivity changes among strains. This fact could be explained by differences in composition and conformation of the cellular structures. This fact confirms it is necessary to perform studies concerning inhibitory treatments in strains of natural occurrence in the food matrix analyzed. Conclusions There are only a few bacteriocins reported to inhibit Gram-negative bacteria, however using these bacteriocins for pathogen control could be interesting. Although the commercial exploitation of bacteriocins to date is mainly restricted to the food applications of nisin and pediocin, potential novel applications for bacteriocins having an inhibitory activity against Gram-negative pathogens continue to be developed. The fact that the bacteriocins-like substances produced by Lactobacillus plantarum A6 and Lactobacillus fermentum Ogi E1 shown a natural inhibiting effect
on Salmonella strains, let us to considerate them for their use as natural additives for foods. In these respects, bacteriocins inhibiting Gram-negative pathogens, deserve further, in-depth scientific attention. Acknowledgements The authors thank J.P. Guyot from IRD for Lb. fermentum Ogi E1 and Lb. plantarum A6 strains, C. Bola´n for technical assistance, P.G. Mendoza Garcı´ a for Listeria innocua AST 062, M.C. Wacher for manuscript revision, and L. Sa´nchezPrado is indebted to COSNET (Mexico) for a research grant.
References Agati, V., Guyot, J.P., Morlon-Guyot, J., Talamond, P., & Hounhouigan, D.J. (1998). Isolation and characterization of new amylolytic strains of Lactobacillus fermentum from fermented maize doughs (mawe` and ogi) from Benin. Journal of Applied Microbiology, 85, 512–520. Barak, J.D., Whitehand, L.C., & Charkowski, A.O. (2002). Differences in attachment of Salmonella enterica serovars and Escherichia coli O157:H7 to alfalfa sprouts. Applied and Environmental Microbiology, 68, 4758–4763. Blostein, J. (1991). An outbreak of Salmonella javiana associated with consumption of watermelon. Journal of Environmental Health, 56, 29–31. Bouttefroy, A., & Milliere, J.B. (2000). Nisin-curvaticin 13 combinations for avoiding the regrowth of bacteriocin resistant cells of Listeria monocytogenes ATCC 15313. International Journal of Food Microbiology, 62, 65–75. Branen, J.K., & Davidson, P.M. (2004). Enhancement of nisin, lysozyme, and monolaurin antimicrobial activities by ethylenediaminetetraacetic acid and lactoferrin. International Journal of Food Microbiology, 90, 63–74. Branquinho Bordini, M.E., Asturiano Ristori, C., Jakabi, M., & Scala Gelli, D. (2007). Incidence, internalization and behavior of Salmonella in mangoes, var. Tommy Atkins. Food Control, 18, 1002–1007. Caldero´n, M., Loiseau, G., & Guyot, J.P. (2001). Nutritional requirements and simplified cultivation medium to study growth and energetics of a sourdough lactic acid bacterium Lactobacillus fermentum Ogi E1 during heterolactic fermentation of starch. Journal of Applied Microbiology, 90, 508–516. de Man, J.C., Rogosa, M., & Sharpe, M.E. (1960). A medium for the cultivation of Lactobacilli. Journal of Applied Bacteriology, 23, 130–135. Duche´, D. (2007). Colicin E2 is still in contact with its receptor and import machinery when nuclease domain enters the cytoplasm. Journal of Bacteriology, 189, 4217– 4222. Gao, Y., Van Belkum, M.J., & Stiles, M.E. (1999). The outer membrane of Gram-negative bacteria inhibits antibacterial activity of brochocin-C. Applied and Environmental Microbiology, 65(10), 4329–4333. Giraud, E., Brauman, A., & Keleke, S. (1991). Isolation and physiological study of an amylolytic strain of Lactobacillus plantarum. Applied Microbiology and Biotechnology, 36, 379–383. Gomez, S., Cosson, C., & Deschamps, A.M. (1997). Evidence for a bacteriocin-like substance produced by a new strain of Streptococcus sp., inhibitory to Gram positive foodborne pathogens. Research in Microbiology, 148, 757– 766. Guerra, N.P., Luisa Rua, M., & Pastrana, L. (2001). Nutritional factors affecting the production of two bacteriocins from lactic acid bacteria on whey. International Journal of Food Microbiology, 70, 267–281.
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CyTA – Journal of Food Guo, X., Chen, J., Brackett, R.E., & Beuchat, L. (2001). Survival of Salmonellae on and in tomato plants from the time of inoculation at flowering and early stages of fruit development through fruit ripening. Applied and Environmental Microbiology, 67(10), 4760–4764. Housden, N.G., Loftus, S.R., Moore, G.R., James, R., & Kleanthous, C. (2005). Cell entry mechanism of enzymatic bacterial colicins: Porin recruitment and the thermodynamics of receptor binding. Proceedings of the National Academy of Sciences of the United States of America, 102(39), 13849–13854. Hugas, M., Garriga, M., & Monfort, J.M. (2002). New mild technologies in meat processing: High pressure as a model technology. Meat Science, 62, 359–371. Koneman, E., Allen, S.D., Dowell, V.R., & Sommers, H.M. (1991). Diagnostico microbiologico (pp. 152–199). Mexico DF: Editorial Medica Panamericana. Lu¨ders, T., Birkemo, G.A., Fimland, G., Nissen-Meyer, J., & Nes, I.F. (2003). Strong synergy between a eukariotic antimicrobial peptide and bacteriocins from lactic acid bacteria. Applied and Environmental Microbiology, 69(3), 1797–1799. MacFadden, J.F. (1980). Biochemical tests for identification of medical bacteria (2nd ed., pp. 36–50). Baltimore, MD: The Williams and Wilkins Co. McAuliffe, O., Ross, R.P., & Hill, C. (2001). Lantibiotics: Structure, biosynthesis and mode of action. FEMS Microbiology Reviews, 25, 285–308. Messens, W., & De Vuyst, L. (2002). Inhibitory substances produced by Lactobacilli isolated from sourdoughs-a review. International Journal of Food Microbiology, 72, 31–43. Penteado, A.L., Eblen, B.S., & Miller, A.J. (2004). Evidence of Salmonella internalization into fresh mangos during simulated postharvest insect disinfestations procedures. Food Protection, 67(1), 181–184.
187
Rodrı´ guez, J.M., Martı´ nez, M.I., Horn, N., & Dodd, H.M. (2002). Heterologous production of bacteriocins by lactic acid bacteria. International Journal of Food Microbiology, 80, 101–116. Sanni, A.I., Onilude, A.A., Ogunbanwo, S.T., & Smith, S.I. (1999). Antagonistic activity of bacteriocin produced by Lactobacillus species from Ogi, an indigenous fermented food. Journal of Basic Microbiology, 39(3), 189–195. Sivapalasingam, S.E., Barrett, A., Kimura, S., Van Duyne, W., & De Witt, M. (2003). A multistate outbreak of Salmonella enterica serotype Newport infections linked to mango consumption: Impact of water dip disinfestation technology. Clinical Infectious Diseases, 37, 1585– 1590. Stekelenburg, F.K. (2003). Enhanced inhibition of Listeria monocytogenes in Frankfurter sausage by the addition of potassium lactate and sodium diacetate mixtures. Food Microbiology, 20, 133–137. Suma, K., Misra, M.C., & Varadaraj, M.C. (1998). Plantaricin LP84, a broad spectrum heat-stable bacteriocin of Lactobacillus plantarum NCIM 2084 produced in a simple glucose broth medium. International Journal of Food Microbiology, 40, 17–25. Van Reenen, C.A., Chikindas, M.L., Zyl, W.H.V., & Dicks, L.M.T. (2003). Characterization and heterologous expression of a class IIa bacteriocin, plantaricin 423 from Lactobacillus plantarum 423, in Saccharomyces cerevisiae. International Journal of Food Microbiology, 81, 29–40. Wang, R.F., Cao, W.W., & Cerniglia, C.E. (1997). A universal protocol for PCR detection of 13 species of foodborne pathogens in foods. Journal of Applied Microbiology, 83, 727–736.
