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NEW MICROBIOLOGICA, 34, 357-370, 2011
Bacteriocin production and resistance to drugs
are advantageous features for Lactobacillus
acidophilus La-14, a potential probiotic strain
Svetoslav Dimitrov Todorov1, Danielle Nader Furtado1, Susana Marta Isay Saad2,
Bernadette Dora Gombossy de Melo Franco1
1Universidade de São Paulo, Faculdade de Ciências Farmacêuticas,
Departamento de Alimentos e Nutrição Experimental, São Paulo, SP, Brasil;
2Universidade de São Paulo, Faculdade de Ciências Farmacêuticas,
Departamento de Tecnologia Bioquímico-Farmacêutica, São Paulo, SP, Brasil
L. acidophilus La-14 produces bacteriocin active against L. monocytogenes ScottA (1600 AU/ml) in MRS broth at 30°C
or 37°C. The bacteriocin proved inhibitory to different serological types of Listeria spp. Antimicrobial activity was
completely lost after treatment of the cell-free supernatant with proteolytic enzymes. Addition of bacteriocin pro-
duced by L. acidophilus La-14 to a 3 h-old culture of L. monocytogenes ScottA repressed cell growth in the following
8h. Treatment of stationary phase cells of L. monocytogenes ScottA (107-108 CFU/ml) by the bacteriocin resulted in
growth inhibition.
Growth of L. acidophilus La-14 was not inhibited by commercial drugs from different generic groups, including non-
steroidal anti-inflammatory drugs (NSAID) containing diclofenac potassium or ibuprofen arginine. Only one non-an-
tibiotic drug tested, Atlansil (an antiarrhythmic agent), had an inhibitory effect on L. acidophilus La-14 with MIC of
2.5 mg/ml. L. acidophilus La-14 was not affected by drugs containing sodium or potassium diclofenac. L. acidophilus
La-14 shows a good resistance to several drugs and may be applied in combination for therapeutic use. KEY WORDS: Lactobacillus acidophilus, Probiotic, Bacteriocin, Medicaments
Received January 01, 2011
Accepted May 19, 2011
etables, fruits, meat, fish, human and animal gas-trointestinal tract (GIT) (Todorov, 2009). Bacteriocins are ribosomally synthesized anti- Probiotics are defined as ‘live microorganisms bacterial peptides and are usually active against that, when administered in adequate amounts, genetically related species. They have been confer a health benefit on the host’ (FAO/WHO, grouped into 4 classes based on their structure 2001). The best known examples of probiotic and mode of action (Heng et al., 2007). In the last
foods are fermented milks and yogurts, which are two decades several reports focused on the pro- generally consumed within days or weeks of man- duction of bacteriocins from lactic acid bacteria ufacture (Nagpal et al., 2007), as well as other
isolated from different fermented products, veg- dairy products, including cheeses (Cruz et al.,
2009b) and ice-creams (Cruz et al., 2009a).
Besides better growth and survival during food
Corresponding author
manufacturing and storage and in the GIT, pro- tection against acid, bile, and gastrointestinal en- zymes, and adhesion to intestinal epithelium, an- timicrobial properties and antibiotic resistance Departamento de Alimentos e Nutrição Experimental could be considered factors that might be im- Av. Prof. Lineu Prestes 580 Bloco 14,05508-000 - São Paulo - SP, Brasil portant in maintaining probiotic efficacy (Ranadheera et al., 2010).
S.D. Todorov, D.N. Furtado, S.M.I. Saad, B.D. Gombossy de Melo Franco
Probiotic Lactobacillus species have been impli-
diseases and allergic disorders (Ezendam and van cated in a variety of beneficial roles for the hu- Loveren, 2008; Ghadimi et al., 2008; He et al.,
man body, including maintenance of the normal intestinal microbiota, pathogen interference, ex- Apart from competition for binding sites, pro- clusion and antagonism, immunostimulation and duction of hydrogen peroxide and bacteriocins immunomodulation, anticarcinogenic and an- play a key role in competitive exclusion and pro- timutagenic activities, deconjugation of bile acids, biotic properties (Boris and Barbes, 2000; and lactase release in vivo (Klaenhammer, 1988;
Guarner and Malagelada, 2003; Shah, 2007; Burton, 2002; Galdeano et al., 2007). Although
Tuohy et al., 2003). Consequently, the potential
the role of bacteriocins and their significance in health-promoting effect of dairy products that in- controlling the proliferation of pathogenic bac- corporate Lactobacillus species and other probi-
teria in the intestinal tract is questionable (Brink otic organisms has stimulated considerable re- et al., 2006), several reports on bacteriocins ac-
search (Buriti et al., 2005). Lactobacillus aci-
tive against Gram-negative bacteria (Ivanova et
dophilus La-14 (Danisco) is a commercially avail-
al., 1998; Messi et al., 2001; Caridi, 2002; Todorov
able potential probiotic strain of human origin and Dicsk, 2005a; Todorov and Dicks, 2005b; and has been deposited in the American Type Todorov and Dicks, 2005c) aroused a renewed in- Culture Collection as SD5212 (Danisco).
terest in these peptides and their interaction with In a double-blind, randomized, controlled trial intestinal pathogens. Only few papers reported with 83 healthy volunteers aged 18 up to 72 years bacteriocin production and potential probiotic who received two capsules per day of the test properties of lactic acid bacteria isolated from product containing 10 log CFU of bacteria in a different ecological niches (Van Reenen et al.,
maltodextrin carrier, L. acidophilus La-14 was ad-
1998; Todorov and Dicks, 2005a; Todorov and ministered to 9 of those volunteers. The serum Dicks, 2005c; Todorov and Dicks, 2006; Todorov IgG was reported to increase significantly in those et al., 2006; Powell et al., 2007; Todorov et al.,
volunteers in an early response compared with 2007; Todorov et al., 2008; Todorov and Dicks,
controls (P=0.01) 7 days after the second vaccine 2008). Probably, bacteriocin production increas- administration. Since IgG are involved in im- es the chances for the probiotic strain to survive mune memory, L. acidophilus La-14 was sug-
in the competing GIT environment. In fact, ac- gested to possibly contribute to disease preven- cording to O’Flaherty and Klaenhammer (2010), tion in the long term (Paineau et al., 2008).
there is strong evidence from in vitro studies that
Probiotic lactic acid bacteria may prevent the probiotic bacteria are able to make use of an- use of certain antibiotics in animal feeds (Park et
timicrobial effects in vivo.
al., 2002) and if carefully selected, control the
The survival of probiotic bacteria in the human or proliferation of pathogenic bacteria that may animal GIT is a complex process and involves the lead to diarrhoea and other clinical disorders, availability of nutrients, type of diet, interactions such as cancer and inflammatory bowel disease with autochthonous bacteria in the GIT, adhe- (Fooks et al., 1999).
sion properties and auto-aggregation and co-ag- They may offer a safe and practical means of mod- gregation characteristics of the probiotic cells.
ulating the function and metabolic activity of the Survival of probiotics in the GIT of patients treat- human intestinal microbiota, excluding pathogens ed for the chronic illnesses that become depend- and helping to keep the gut homeostasis by influ- ent on permanent drug treatment may be less ef- encing the mucosal immune system (Morita et al.,
fective. Recent studies on potential probiotics 2006). Recent clinical and animal studies have have shown that these bacteria may be affected supported the hypothesis that lactobacilli, partic- by non-antibiotic drugs (Boris and Barbes, 2000; ularly certain selected strains with immunomod- Todorov et al., 2007; Botes et al., 2008; Todorov
ulatory properties, can modify the responses of and Dicks, 2008; Carvalho et al., 2009).
the host, thereby inducing beneficial effects This article focuses on the investigation into bac- (Ezendam and van Loveren, 2008; Shida and teriocin production by the potential probiotic Nanno, 2008). Recently, there has been much in- strain of L. acidophilus La-14 and determination
terest in the use of probiotic bacteria for treating of some aspects of bacteriocin mode of action.
Bacteriocin production and resistance to drugs are advantageous features for Lactobacillus acidophilus La-14
The effect of selected drugs from different gener- was used as sensitive strain. In addition, several ic groups on growth of L. acidophilus La-14 was
Gram-positive and Gram-negative bacterial strains were used for determination of spectrum activity. These strains were cultured in MRS orBHI broth, as shown in Table 1, at 30°C or 37°C, MATERIALS AND METHODS
Strains and media
Effect of enzymes, pH, detergents and
L. acidophilus La-14 was provided by Danisco
temperature on bacteriocin activity
(Dangé, France). The strain was grown in MRS Cell-free supernatants of L. acidophilus La-14, ob-
broth (Difco) at 37oC for 24 h. The test microor- tained by centrifugation (8.000 x g, 10 min, 4°C)
ganisms used in this study and their culturing of a 18 h culture in MRS broth at 37°C, were ad- condition are listed in Table 1. All strains were justed to pH 6.0 with 1 N NaOH. Samples of 2 stored at -80°C in MRS broth supplemented with ml were incubated for 2 h in the presence of 1.0 mg/ml (final concentration) Proteinase type XIV(Roche), Proteinase (Roche), α-chymotrypsin Test for bacteriocin production
(Roche), catalase (Roche) and α-amylase (Roche), L. acidophilus La-14 was tested for antimicrobial
and then tested for antimicrobial activity using compounds production against Listeria monocy-
ScottA, using the agar spot-test (Todorov,
2008). Activity was expressed as arbitrary units
TABLE 1 - Spectrum of activity of the antibacterial
(AU)/ml. One AU was defined as the reciprocal of compound produced by Lactobacillus acidophilus
the highest serial twofold dilution showing a clear La-14.
zone of growth inhibition of the indicator strain(Todorov, 2008). The antimicrobial effect of lac- Test microorganisms
tic acid was eliminated by adjusting the pH of the produced by
supernatants to 6.0 with sterile 1 N NaOH. To L. acidophilus
rule out the effect of proteolytic enzymes and La-14 (diameter
H O the cell-free supernatant was heated at 80oC of the inhibition
Listeria monocytogenes
Confirmation of the identity
of L. acidophilus La-14
L. acidophilus La-14 was identified to genus-lev-
el according to its physiological and biochemical characteristics, as described by Stiles and Holzapfel (1997). Carbohydrate fermentation re- (Biomérieux, Marcy-l’Etiole, France). Results were compared to carbohydrate fermentation pattern listed in Bergey’s Manual of Systematic Bacteriology (Sneath et al., 1986).
Dynamics of bacteriocin production
MRS broth was inoculated with an 18h-old cul- ture (2 %, v/v) of L. acidophilus La-14 and incu-
bated at 37°C without agitation. Antimicrobial Listeria innocua ATCC 33090 (BHI, 37°C)
activity (AU/ml) of the bacteriocin, and changes Listeria sakei ATCC 15521 (MRS, 37°C)
in pH and optical density (at 600 nm) of the cul- Staphylococcus aureus ATCC 6538 (BHI, 37°C)
tures, were determined at 3 h and 1 h intervals, Staphylococcus aureus ATCC 29213 (BHI, 37°C)
Bacillus cereus ATCC 11778 (BHI, 37°C)
respectively for 48 h. L. monocytogenes ScottA
S.D. Todorov, D.N. Furtado, S.M.I. Saad, B.D. Gombossy de Melo Franco
the agar-spot test method. Samples of plain MRS Adsorption study of the bacteriocin
added of the listed enzymes in same concentra- to the producer cells
tions were used as controls. In a separate exper- The ability of a bacteriocin to adsorb to produc- iment, the effect of SDS, Tween 20, Tween 80, er cells was studied according to the method de- urea, Na-EDTA and NaCl (1%, m/v, v/v) on bac- scribed by Yang et al. (1992). After 18 h of growth
teriocin stability were determined as described at 37°C, the culture pH was adjusted to pH 6.0, by Todorov and Dicks (2006). The same chemicals the cells harvested (10 000 x g, 15 min, 4°C) and
were applied as controls in plain MRS and incu- washed with sterile 0.1 M phosphate buffer (pH bated in similar conditions. The effect of pH on 6.5). The cells were re-suspended in 10 ml 100 the bacteriocin stability was determined by ad- mM NaCl (pH 2.0), stirred for 1 h at 4°C and then justing the cell-free supernatant to pH 2.0 up to harvested (12 000 x g, 15 min, 4°C). The cell-free
12.0 with sterile 1 N HCl or 1 N NaOH. After 2 h supernatant was neutralized to pH 7.0 with ster- of incubation at 37°C, the samples were read- ile 1 N NaOH and tested for activity as described justed to pH 6.5 with sterile 1 N HCl or 1 N NaOH and the activity was determined as described be-fore (Klaenhammer, 1998). The effect of temper- Susceptibility of L. acidophilus La-14
ature on the bacteriocin stability was tested by to medicaments
heating the cell-free supernatants to 30, 37, 45, L. acidophilus La-14 was tested for susceptibility
60 and 100°C. Residual bacteriocin activity was to commercially available drugs [analgesic, com- tested after 30, 60 and 120 min at each of these bination of analgesics and vasoconstrictor, nar- temperatures, as described before (Todorov and cotic analgesic, antipyretic, anorexiant/sympath- Dicks, 2006). As control, plain MRS broth was omimetic, antiarrhythmic, antibiotic, antiemet- exposed to the same temperatures and pH and ic, antifungal agents, antihistaminic, antihyper- tested against L. monocytogenes ScottA
tensive (Alpha blocker, Angiotensin ConvertingEnzyme (ACE) inhibitor), antitussives (central Growth of the test-microorganisms
and peripheral mode of action), association of in the presence of bacteriocin produced
analgesic/antipyretic, antihistaminic and decon- by L. acidophilus La-14
gestant, contraceptive, diuretic, histamine H2-re- A 20 ml aliquot of bacteriocin-containing filter- ceptor antagonist that inhibits stomach acid pro- sterilized (0.20 µm, Minisart®, Sartorius) super- duction (Proton pump inhibitor), hypolipidemic, natant (pH 6.0) was added to a 100 ml culture of mucolytic agent, non-steroidal anti-inflammato- L. monocytogenes ScottA in early exponential
ry drug (NSAID), proton pump inhibitor, selec- tive serotonin reuptake inhibitor (SSRI) antide- Optical density readings (at 600 nm) were record- pressant, thiazide diuretic] was determined (Table 3). Strains were inoculated separately into 10 mlMRS broth (Difco) and incubated at 37°C for 18 Determination of the reduction of viable
h and imbedded into MRS soft agar (1.0%, w/v, cells of test microorganisms in presence of
Difco) at 106 CFU/ml. Ten µl of each drug was bacteriocin produced by L. acidophilus La-14
spotted onto the surface of the agar. The plates Cells of an early stationary phase (18h-old) cul- were examined for the presence of inhibition ture of L. monocytogenes ScottA were harvested
zones after 24 h of incubation at 37°C. The drugs (5000 x g, 5 min, 4°C), washed twice with sterile presenting the inhibition zones larger than 2 mm saline water and re-suspended in 10 ml of sterile were subjected to the determination of the min- saline water. Equal volumes of the cell suspen- imal inhibition concentration, using serial sions and filter-sterilized (0.20 m, Minisart®, twofold dilutions of the medicaments. For the Sartorius) cell-free supernatant of L. acidophilus
test, 10 µl of each dilution were spotted onto the La-14 containing bacteriocin were mixed. Viable surface of the agar, previously imbedded with L.
cell numbers were determined before and after acidophilus La-14. The plates were incubated for
incubation for 1 h at 37°C by plating onto MRS 24 h at 37°C and examined for inhibition zones.
agar. Cell suspension of L. monocytogenes ScottA
Those presenting inhibition zones above 2 mm without added bacteriocins served as controls. Bacteriocin production and resistance to drugs are advantageous features for Lactobacillus acidophilus La-14
of bacteriocin produced by L. acidophilus La-14
(approx. 400 AU/ml) were recorded after 3 h of Identification of the L. acidophilus
La-14 strain
Based on the biochemical test and API50CHL,
Spectrum of activity
the identity of the strain grown from the com- The bacteriocin produced by L. acidophilus La-
mercial available lyophilized product of Danisco 14 proved inhibitory to different serotypes of L.
(Dangé, France) was confirmed to be L. aci-
innocua and L. monocytogenes listed in Table 1.
However, no activity was recorded against
Staphylococcus aureus, Lactobacillus sakei and
Bacteriocin production
Bacillus cereus.
No significant differences in growth and produc-tion of bacteriocin were observed when the strain Effect of enzymes, pH, detergents and
L. acidophilus La-14 was cultured for 24 h in MRS
temperature on bacteriocin activity
broth at 30°C or at 37°C. At this two incubation Treatment with α-amylase and lipase did not temperatures, activity against L. monocytogenes
change the antimicrobial activity (Table 2).
ScottA was 1600 AU/ml. All further experiments Activity of the bacteriocin produced by L. aci-
were conducted at 37°C, since strain L. aci-
dophilus La-14 was not affected by 1% SDS,
dophilus La-14 is a potential probiotic strain.
Tween 20, Tween 80, Urea, EDTA or NaCl (Table Production of bacteriocin by L. acidophilus La-
2). Bacteriocin produced by L. acidophilus La-14
14 was detected at maximum levels (1600 AU/ml) remained stable after incubation for 2 h at pH after 16 h and remained stable up to 24 h of fer- Stability of bacteriocin produced by L. aci-
After 24 h, the activity against L. monocytogenes
dophilus La-14 was recorded after 120 min at 25,
ScottA decreased and was progressively reduced 30, 45, 60 or 100oC (Table 2). Heating at 121°C to 400 AU/ml at 48 h of incubation (Fig. 1).
for 20 min did not inactivate the bacteriocin, but During this period, the medium pH of L. aci-
caused a reduction of activity, as smaller inhibi- dophilus La-14 culture decreased from 6.40 to
tion zone against L. monocytogenes ScottA were
4.25 and the cell density increased from 0.022 to observed (Table 2). Treatment of bacteriocin at 7.35 (as detected at 39 h) and decreased slightly pH 6.0 at 121°C for 20 min resulted in a decreased to 0.669 in the following 9 h (Fig. 1). Low levels FIGURE 1 - Production of bacteriocin by Lactobacillus acidophilus La-14 in MRS broth (pH 6.5, 37°C). Antimicrobial
activity is presented as AU/ml (bars) against Listeria monocytogenes ScottA. Changes in optical density (-
-) and pH
(--) are indicated. Standard deviation recorded from three repeats was less that 5% and is not indicated.
S.D. Todorov, D.N. Furtado, S.M.I. Saad, B.D. Gombossy de Melo Franco
TABLE 2 - Effect of enzymes, detergents, NaCl,
(OD600nm ≈ 0.044) repressed cell growth in the fol- temperature and pH on the stability of the
lowing 8 h and slightly increased in the next 4 h antibacterial compound produced by Lactobacillus
(Fig. 2), but no viable cells were recorded in 6, 8, acidophilus La-14.
and 10 h. Levels of 102-103 CFU/ml for L. mono-
ScottA were recorded at 12 and 14 h,
Test microorganism
pointing the bacteriostatic mode of action of thisbacteriocin against this test microorganism. L. monocytogenes L. monocytogenes
724 serotype 4b
Reduction in CFU/ml of L. monocytogenes
ScottA after exposure to bacteriocin produced
by L. acidophilus
Treatment of stationary phase cells of L. mono-
cytogenes ScottA (107-108 CFU/ml) with the bac-
teriocin produced by L. acidophilus La-14 result-
ed in growth inhibition. After 1 h of contact, low
levels (101-102 CFU/ml) of viable cells of L. mono-
cytogenes ScottA were detected. No significant
changes in cell numbers of L. monocytogenes
ScottA were recorded in the untreated (control)
Adsorption study of the bacteriocin to the pro-
ducer cells
After treatment of the cell suspension of L. aci-
dophilus La-14 with 100 mM NaCl (pH 2.0) for 1
h, no adsorption of the bacteriocin was record-
ed, showing that this bacteriocin probably does Activity was expressed as: + = presence of inhibition zone ≥2 mm diameter, not adhere to the producer cell surface. Sensitivity of L. acidophilus La-14 to drugs
Only two antibiotics (Amoxil and Urotrobel) and
Growth of the test-microorganisms
the non-antibiotic drug Atlansil (an antiarrhyth- in the presence of bacteriocin produced
mic agent) inhibited growth of L. acidophilus La-
by L. acidophilus La-14
14 in a MIC of <0.5 mg/ml, 5.0 mg/ml and 2.5 Addition of bacteriocin produced by L. aci-
mg/ml, respectively (Table 3). Growth of L. aci-
dophilus La-14 obtained from a 24 h old culture,
dophilus La-14 was not inhibited by other
to a 3-h-old culture of L. monocytogenes ScottA
medicaments belonging to different generic FIGURE 2 - Effect of bacteriocin
produced by Lactobacillus aci-
dophilus La-14 on growth of
Listeria monocytogenes ScottA.
Arrow indicates the time of the
addition of the bacteriocin.
Bacteriocin production and resistance to drugs are advantageous features for Lactobacillus acidophilus La-14
TABLE 3 - Effect of commercial drugs on the growth of Lactobacillus acidophilus La-14.
Active substance
Medication group
L. acidophilus La-14
antagonist that inhibits stomach acid production (Proton pump inhibitor) (Alpha blocker)/Treatment of benign prostatic hyperplasia continue
S.D. Todorov, D.N. Furtado, S.M.I. Saad, B.D. Gombossy de Melo Franco
TABLE 3 - Effect of commercial drugs on the growth of Lactobacillus acidophilus La-14.
Active substance
Medication group
L. acidophilus La-14
(Angiotensin-converting enzyme (ACE) inhibitor) Bacteriocin production and resistance to drugs are advantageous features for Lactobacillus acidophilus La-14
groups, including non-steroidal anti-inflamma- sequence of the antimicrobial molecule.
tory drugs (NSAID) containing diclofenac potas- Production of bacteriocins may be considered an sium or ibuprofen arginine, and drugs containing advantage for the probiotic strains, since this an- sodium or potassium diclofenac (Table 3). timicrobial compound will give them a benefit in
the competition with the GIT pathogens, such as
L. monocytogenes. Previous reports have shown
that several probiotic and potential probioticstrains are bacteriocin producers (Todorov and Similar levels of bacteriocin production were Dicks, 2005a; Todorov and Dicks, 2005c; Todorov recorded for L. acidophilus La-14 when cultured
et al., 2005; Todorov and Dicks, 2006; Todorov et
for 24 h in MRS broth at 30°C or at 37°C. This is al., 2006; Powell et al., 2007; Todorov et al., 2007;
in agreement with the results recorded for other Botes et al., 2008a; Botes et al., 2008b; Todorov
bacteriocins (Todorov and Dicks, 2006). Optimal and Dicks, 2008; Todorov et al., 2008; Todorov
levels of other bacteriocins were recorded in growth media that supported high biomass pro- Activity against pathogens is one of the impor- duction, e.g. MRS and TGE (Biswas et al., 1991;
tant properties a probiotic strain ought to pos- Ray et al., 1992; Yang and Ray, 1994). However,
sess. The antimicrobial ability of the potential during cultivation of L. acidophilus La-14 in MRS
probiotic strain Lactobacillus acidophilus La-14
broth, the reduction in bacteriocin activity levels against some enteropathogens, such as Listeria
was recorded at pH values below 5.1, suggesting monocytogenes, was assayed in this study. The
that production is blocked in these conditions.
overnight culture of L. acidophilus La-14 showed
Only genetic studies on the expression of the strong inhibition action towards Listeria spp.
genes encoding the bacteriocin production can (Table 1). Moreover, treated supernatant (with- out peroxide and lactic acid) also showed anti- Similar results were observed for other bacteri- ocins (Todorov and Dicks, 2005b). The decreased These observations suggest that L. acidophilus
activity by the end of the monitored period might La-14 produced bacteriocins to inhibit the test be explained by degradation of the bacteriocin by pathogens. Some authors have reported that pro- extracellular proteolytic enzymes, as a previous- duction of bacteriocins by lactobacilli is relative- ly similar decrease in activity was shown for bac- ly common, and may contribute to their colo- teriocins produced by Lactobacillus plantarum
nization of habitats and their competitive edge ST414BZ (Todorov and Dicks, 2006), Pediococcus
over other bacteria (Garriga et al., 1993). The an-
acidilactici NRRL B5627 (Anastasiadou et al.,
timicrobial activity of lactic acid bacteria may be 2008a) and Pediococcus pentosaceus (Anastasia -
due to a number of factors including decreased dou et al., 2008b). From a metabolic point of view,
pH levels, competition for substrates, and the pro- this trend is characteristic of a primary metabo- duction of substances with a bactericidal or bac- lite production, as observed for several bacteri- teriostatic action, including bacteriocins (Parente ocins produced by Pediococcus spp. (Bhunia et
al., 1988; Ray et al., 1989; Bhunia et al., 1991;
Our results (Table 2) suggest that bacteriocin Anastasiadou et al., 2008a; Anastasiadou et al.,
produced by L. acidophilus La-14 does not be-
long to group IV bacteriocins (Klaenhammer, The bacteriocin produced by L. acidophilus La-
1988; Heng et al., 2007) and the carbohydrate
14 showed the inhibitory spectrum summarised or lipids are not involved in the structure of the in Table 1. It is important to highlight the activi- active molecule or molecular complex.
ty against L. monocytogenes, an important hu-
According to De Vuyst and Vandamme (1994), man and food pathogen. Based on strong activi- most bacteriocins are polypeptides. Some ex- ty against L. monocytogenes, the bacteriocin pro-
ceptions are those classified in group IV duced by L. acidophilus La-14 is probably a class
(Klaenhammer, 1988; Heng et al., 2007), such as
IIa bacteriocin (Klaenhammer, 1988; Heng et al.,
carnocin 54, produced by Leuconostoc carnosum
2007), but this preliminary conclusion needs to be (Keppler et al., 1994), which is example of amy-
confirmed by determination of the amino-acid S.D. Todorov, D.N. Furtado, S.M.I. Saad, B.D. Gombossy de Melo Franco
The bacteriocin produced by L. acidophilus La-
Based on results of inhibition of L. monocyto-
14 was not affected by the presence of selected genes ScottA by bacteriocin produced by L. aci-
chemicals (Table 2). In a similar experiment with dophilus La-14, most probably this bacteriocin
bacteriocins produced by P. acidilactici HA-6111-
exhibit bacteriostatic mode of action. Possibly, 2 and HA-5692-3 (Albano et al., 2007), exposure
development of bacteriocin resistance in L. mono-
to Triton-100 or Triton X-114 caused a reduction cytogens ScottA is the reason for the detection of
in bacteriocins activity. Similar results were also viable cells at 12 and 14 h. Other reason for the reported for plantaricin 423 (Verellen et al., 1998),
reduction of efficacy of bacteriocin produced by pediocin AcH (Biswas et al., 1991), lactacin B
L. acidophilus La-14 against L. monocytogens
(Barefoot and Klaenhammer, 1984) and lactocin Scott A may be the protein degradation by pro- 705 (Vignolo et al., 1995). However, the effect of
teolytic enzymes, bacteriocin aggregation or sim- SDS or Triton X-100 seems to be bacteriocin de- ply full utilisation of the added bacteriocin in the pendent, as the activity of plantaricin C19 (Atrih et al., 2001), pediocin ST18 (Todorov and Dicks,
When stationary phase cells of L. monocytogenes
2005d), plantaricin ST31 (Todorov et al., 1999),
ScottA (107-108 CFU/ml) were treated with the and bozacin B14 (Ivanova et al., 2000) did not de-
bacteriocin produced by L. acidophilus La-14, low
crease when treated with these compounds.
levels (101-102 CFU/ml) of viable cells of test mi- The bacteriocin produced by L. acidophilus La-
14 was stable at pH rangeing from 2.0 to 12.0 changes in cell numbers of L. monocytogenes
(Table 2). This is a remarkable finding, as sever- ScottA were recorded in the untreated (control) al other studies have shown a reduced activity of sample. Previously, a similar effect regarding bac- bacteriocins exposed to pH 12.0, such as P. acidi-
teriocins HA-6111-2 and HA-5692-3 produced by lactici HA-6111-2 and HA-5692-3 (Albano et al.,
P. acidilactici to E. faecium HKLHS was reported
2007), and pediocin PA-1 (Bhunia et al., 1988;
by Albano et al (2007).
Gonzales and Kunka, 1987). The loss of activity No adsorption of the bacteriocin to the producer may be ascribed to proteolytic degradation or cells was recorded. Similar observation were re- protein aggregation (Aasen et al., 2000; Parente
ported for plantaricin ST31 (Todorov et al., 1999),
and Riccardi, 1994; Parente et al., 1994; De Vuyst
pediocin ST18 (Todorov and Dicks, 2005d), bac- et al., 1996).
teriocins HA-6111-2 and HA-5692-3 (Albano et
The bacteriocin produced by L. acidophilus La-
al., 2007) and bozacin B14 (Ivanova et al., 2000).
14 was thermostable (Table 2). The antimicrobial Patients taking probiotics are often treated for activity of pediocin PA-1 was unaffected by heat- other illnesses. It is thus important to determine ing at 80°C for 60 min, and at 100°C for 10 min, the effect of medicaments on the growth of pro- and the effect of 121°C for 15 min was contro- versial, as values of residual activity of 6% and Only two antibiotics (Amoxil and Urotrobel) and 60% have been reported (Yang and Ray, 1994).
the non-antibiotic drug Atlansil inhibited growth Purified pediocin PA-1 at pH 5 remained stable of L. acidophilus La-14 in a MIC of <0.5 mg/ml,
when stored at 4°C and at 25°C, but not at pH 5.0 mg/ml and 2.5 mg/ml, respectively (Table 3).
7.0. The peptide remained stable at -20°C, inde- Growth of L. acidophilus La-14 was not inhibited
pendent of storage at pH 5.0 or 7.0 (Fimland et al.,
by other medicaments belonging to different 2002). These authors have shown that heat re- generic groups, including non-steroidal anti-in- sistance of pediocin PA-1 produced by P. parvulus
flammatory drugs (NSAID) containing diclofenac was pH dependent. At pH 6.0, 84% activity was potassium or ibuprofen arginine, and drugs con- lost when heated at 121°C for 15 min. No activi- taining sodium or potassium diclofenac (Table ty was recorded when the same experiment was 3). A previous study reported that sodium di- done with pediocin PA-1 adjusted to pH 7.0 and clofenac inhibited the growth of L. plantarum
8.0. However, at pH 4.0, only 11 % of the activity ST8KF and ST341LD, Enterococcus faecium
was lost. Pediocin is more heat sensitive at low- ST311LD and Leuconostoc mesenteroides subsp.
er pH. The same results were recorded for other mesenteroides ST33LD and that dimenhydrinate
pediocins and enterocins (Bhunia et al., 1988;
was inhibitory to Lactobacillus plantarum ST8KF
Moreno et al., 2003).
(Todorov and Dicks, 2008). In another study, Bacteriocin production and resistance to drugs are advantageous features for Lactobacillus acidophilus La-14
potassium diclofenac and ibuprofen inhibited the of the active drugs were not determined, ham- growth of Lactococcus lactis subsp. lactis HV219
pering the correct evaluation of their activity (Todorov et al., 2007). Anti-inflammatory drugs,
against L. casei Shirota in the human body, es-
moderate diuretics and neuroleptics containing pecially when used on a daily basis by patients potassium or sodium diclofenac, ibuprofen, tri- with chronic diseases. The correct evaluation of amterene hydrochlorothiazide and thioridazine possible interactions between medicaments and hydrochloride acted as inhibitors of the growth of probiotic bacteria depends on the determination Lactobacillus plantarum, Lactobacillus rhamno-
sus, Lactobacillus paracasei and Lactobacillus pen-
In another study by Botes et al. (2008a), a similar
tosus strains isolated from boza and evaluated as
experiment was performed (Botes et al., 2008b) to
probiotics (Todorov et al., 2008).
verify the effect of drugs over the same probiotic Dimenhydrinanat inhibited the growth of Lacto -
strains (E. mundtii ST4SA and L. plantarum 423)
bacillus rhamnosus ST462BZ and Lactobacillus
and using the same commercial probiotic strains plantarum ST664BZ (Todorov et al., 2008). It is,
as controls (L. casei Shirota, L. johnsonii La1 and
however, important to mention that the concen- L. rhamnosus GG). As previously reported (Botes
tration of these substances is critical for their in- et al., 2008a), the authors (Botes et al., 2008b)
hibitory mode of action on the probiotic LAB. As evaluated the effect of commercial antibiotics and shown by Carvalho et al. (2009) L. casei Shirota
non-antibiotic drugs on a few probiotic strains, and L. casei LC01 were inhibited by non-steroidal
without establishing the MIC of this agents. The anti-inflammatory drugs (NSAID) containing di- authors studied the effect of selected drugs on the clofenac potassium or ibuprofen arginine. In ad- adhesion to Caco-2 cell line to evaluate E. mundtii
dition, L. casei Shirota was affected by selective
ST4SA and L. plantarum 423 as probiotics.
serotonin reuptake inhibitors (SSRI) antidepres- The mechanism of the inhibitory effect against sant containing paroxetine and antiarrhythmic probiotic LAB and other GIT-related bacteria medication containing amiodarone. L. casei LC01
needs to be related to the chemical composition was inhibited by hypolipidemic medication con- of drugs. A simple recommendation would be not taining simvastatin. The levels of MIC for these to apply a drug presenting an inhibitory effect on drugs on the growth of L. casei Shirota and L. ca-
the probiotic LAB at the same time, since the sei LC01 were reported (Carvalho et al., 2009). It
drug will have a negative effect on the probiotic is important to point out that L. acidophilus La-
cells, resulting in decreased viability. 14 showed good resistance to several drugs, and The application of drugs along with probiotic cul- may be applied in combination with them in the tures needs to be reconsidered, regarding the pos- sibility of a negative interaction. The drug MIC Botes et al. (2008b) reported that L. casei Shirota
on the survival and growth of probiotic bacteria was inhibited by several commercial antibiotics is an important cross point. This type of drug (ciprofloxacin, amoxicillin, cefadroxil, rox- must not be taken by the patient permanently.
ithromycin, doxycycline and norfloxacin). Anti- The daily dose for this drug needs to be linked with the MIC against probiotic LAB. Especially (Coxflam), Ibuprofen (Dolocyl, Adco-Ibuprofen), important are drugs used in the treatment of potassium diclofenac (Cataflam) and pred- chronic diseases. Some of the drugs tested in this nisolone (Preflam) also inhibited the growth, in a study showed an MIC of 2.5 mg/ml (Atlansil, an antiarrythmic drug normally used for long cours- Pinmed, that contains paracetamol, codeine es of treatment). Administration of these drugs phosphate and promethazine HCl, misclassified needs caution, when done together with probiot- as an analgesic instead of an antitussive agent, ic cultures, especially with L. acidophilus La-14,
was also inhibitory to L. casei Shirota. The same
since they are applied on a daily basis and an ac- authors also reported the inhibitory effect of cumulation of the active substances in the GIT is Pynmed (Botes et al., 2008b), which is more like-
highly possible. However, this will also increase ly due to the presence of alcohol in the formula- the inhibitory effect of the drug on L. acidophilus
tion than to the drug itself. An important point La-14 and therefore result in a reduction of the vi- is that in the study of Botes et al. (2008b) the MIC
S.D. Todorov, D.N. Furtado, S.M.I. Saad, B.D. Gombossy de Melo Franco
Influence of growth conditions on the production Dr. Svetoslav D. Todorov was supported by PVE
of a bacteriocin, pediocin AcH, by Pediococcus
grant from CAPES, Ministry of Education, Brazil.
acidilactici H. Appl. Environ. Microbiol. 57, 1265-
Authors are grateful to Danisco, Dangé, France for
BORIS S., BARBES C. (2000). Role played by lactobacil- providing Lactobacillus acidophilus La-14 strain,
li in controlling the population of vaginal to Prof. Maria Teresa Destro and Dr. Eb Chiarini
pathogens. Microbs Infect. 4, 543-546.
(Universidade de São Paulo, Faculdade de Ciências
Farmacêuticas, Departamento de Alimentos e
(2008a). Adhesion of the probiotic strains Nutrição Experimental) for providing the Listeria
Enterococcus mundtii ST4SA and Lactobacillus
monocytogenes strains used in this study.
plantarum 423 to Caco-2 cells under conditions
simulating the intestinal tract, and in the presence
of antibiotics and anti-inflammatory medicaments.
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PattyLoulookedoutthedoor.Shewaswaitingforhergrandson,Robert,to come. She hadn’t seen him since her ninetieth birthday party threemonths earlier, when the whole family had come out to Brookhaven, Mis-sissippi, to celebrate with her. Robert came up from New Orleans to seeher only three or four times a year, and she was looking forward to seeinghim. She looked out at the sky. There were four buzz

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