Efficiency of enzymes and benzalkonium chloride treatments against Listeria monocytogenes dual-species biofilms determined by fluorescence microscopy and image analysis Rodríguez-López, P., Nimo, V., Carrera-Iglesias, A., Blanco. T and Cabo, M.L.* Marine Research Institute (IIM-CSIC), Vigo (Pontevedra), Spain
*Corresponding author:
[email protected]
OBJECTIVE
BACKGROUND Persistence of Listeria monocytogenes in food-related environments has been considered of a great concern over the last decade (1, 2). It forms part of multispecies biofilm communities (3) which confers L. monocytogenes and accompanying microbiota higher resistance to traditional treatments based on quaternary ammonium compounds (4).
To assess the EFFECTIVENESS of sequentially combined application of CELLULASE (CEL) or PRONASE (PRO) with BENZALKONIUM CHLORIDE (BAC) against the elimination of 168h - L. monocytogenes dual species biofilms potentially present in fish and dairy industries.
MATERIALS AND METHODS 1. BACTERIAL CONSORTIA
3. EXPERIMENTAL DESIGN
Previously isolated from surfaces of food related premises. Fish industry consortium L. monocytogenes A1
According to a first order factorial experimental design,enzymatic treatments were applied followed by a solution of benzalkonium chloride on coupons at concentrations detailed in Tables 1 and 2. Samples were stained using LIVE/DEAD BacLight® Bacterial Viability Kit (Life Technologies) and efficiency of treatments was assessed by epifluorescence microscopy followed by image analysis with Metamorph MM AF (Molecular Devices).
Dairy industry consortium
E. coli A 14
L. monocytogenes G1
P. fluorescens B52
2. DUAL-SPECIES BIOFILM SET UP 103 – 104 CFU/ml of each species were mixed 1:1 and cultured in Tryptic Soy Broth supplemented with 0.6 g/l yeast extract and 2.5 g/l D-glucose on 10 x 10 x 1 mm AISI 316 stainless steel coupons put in 24 well-plates at 25ºC and 100 rpm in saturated humidity conditions.
Encoded values
PRN (UI/l)
BAC (ppm)
Encoded values
CEL (UI/l)
BAC (ppm)
(0,0)
3850
1025
(0,0)
0.11
1025
(-1,-1)
700
50
(-1,-1)
0.02
50
(1,1)
7000
2000
(1,1)
0.2
2000
(-1,1)
700
2000
(-1,1)
0.02
2000
(1,-1)
7000
50
(1,-1)
0.2
50
25ºC / 100 rpm 168 h Listeria monocytogenes
Accompanying microbiota
Additionally, in order to avoid ambiguities among the effects and also to identify specific dispersion effects of the treatments tested, detached viable cultivable cells were also quantified in every experimental set performed.
1 cm2 AISI 316 coupon
Inocula adjusted to 103 – 104 CFU/ml
1:1 Mixture in 24 well plates containing SS coupons
Tables 1 and 2: Concentrations of PRN, CEL and BAC used .
Incubation of biofilms
RESULTS AND DISCUSSION Obtained results were significantly described by empirical equations [1-4] and permits to deduce the following GENERAL and SPECIFIC effects • Occupied area (OA) by living cells (Figures 1A to 4A): a synergic effect between PRN and BAC in decreasing occupied area was demonstrated in fish and dairy consortia. However, sequential application of CEL and BAC even has counter-productive effects against the occupied area by the biofilms. So, BAC antimicrobial activity seems to be somehow annulled by cellulose. Moreover, according to equations there is a slight individual effect of cellulose in decreasing the occupied in the fish consortium was detected, though not significantly lower compared with PRN. This is in contrast with previous results that demonstrated cellulase and other polisaccharidases have anti-biofilm properties (5, 6). Effectiveness of PRN as a mixture of proteolytic enzymes could be also reflected in the biofilm’s architecture probably due to biofilms’ matrix composition which in case of Listeria monocytogenes is mainly of a proteic nature (7). This variation is especially noticeable in the fish consortium where the uniformity of the structure is lost, forming cellular aggregates, as the concentration of PRN rises. Thus, it could be hypothesized presence of PRN changes the biofilm structure from uniformity to aggregates and this permits BAC to reach more number of bacterial cells, thus giving rise to the observed synergic effect.Regarding biofilm sensibility to treatments, fish consortium is more resistant to PRN – BAC treatment that dairy consortium. In fact, besides OA values were close to zero in most of the experimental ambit, maximum values of OA reached were significantly lower (250 µm2) when comparing with those obtained in the fish consortium (around 55000 µm2). • Detached living cells (Figures 1B to 4B): BAC clearly decreases spread viable cultivable cells released from the biofilm, probably as a consequence of its effect as disinfectant. In regard to the enzymes, higher dispersing capacity was demonstrated in the case of cellulase comparing with pronase at high BAC concentrations, probably related with the detected synergy in decreasing the occupied area in this last. Figure 1: Effect of PRN-BAC treatments on L. monocytogenes – E.coli biofilms. (A) Occupied area by living cells and (B) detached living cells after treatment. Figure 2: Effect of PRN-BAC treatments on L. monocytogenes – P. fluorescens biofilms. (A) Occupied area by living cells and (B) detached living cells after treatment. L. monocytogenes A1 - E. coli A14
L. monocytogenes G1 - P. fluorescens B52 L. monocytogenes A1 - E. coli A14
L. monocytogenes G1 - P. fluorescens B52
6x104
2,5x102 5
5x104
4
2,0x102 4
2
2
102
)
BAC
(ppm
SE
00 20
)
50 75 0 3, 29 537,5 1,25 25 78 10 8,75 50 , 5 6 12 1512 56,2 000 BAC 2 17
2A
m)
(ppm)
2
(1) 𝑂𝐴 = 34135 − 8262,98𝐵 − 10314,49𝐸𝐵
(U I/l)
(U I/l)
(U I/l)
2B
0
SE
00 20
70 0
25 10
7 32 000 54 12 46 25 ,50 38 37, 30 50 50 22 62,5 14 75 0 70 85,50 0
PR O NA
(ppm
50
5,0x101
PR ON A
BAC
70 0
25 10
38 50
0
ON
50
70 00
AS
38 50
0
1B
2
70 00
PR
(U I/l)
BAC (pp
SE
50 ,75 50 3 5 29 537, 81,2 025 1 ,75 ,50 7 68 ,25 000 2 12 1 56 15 2 17
PR O NA
104
1A
7 3 000 54 212, 4 25 50 38 637,5 30 50 0 22 62,5 14 75 0 70 85,5 0 0
1,5x102
1
1
2x104
OA ( m )
3
E
3x104
log CFU/ml
log CFU/ml
2
OA ( m )
3
4x104
𝑂𝐴 = 52,16 + 53,12𝐸 − 53,94𝐵 − 62,69𝐸𝐵
Figure 3: Effect of CEL-BAC treatments on L. monocytogenes – E.coli biofilms. (A) Occupied area by living cells and (B) detached living cells after treatment. Figure 4: Effect of CEL-BAC treatments on L. monocytogenes – P. fluorescens biofilms. (A) Occupied area by living cells and (B) detached living cells after treatment.
L. monocytogenes A1 - E. coli A14 6x104
L. monocytogenes G1 - P. fluorescens B52
L. monocytogenes G1 - P. fluorescens B52
L. monocytogenes A1 - E. coli A14 5x104
1e+5
8e+4 6
5
5 2
OA ( m )
log CFU/ml
4e+4 0,2 0,178 0,155 0,133 0,11 0,088 0,065 0,043 0,02
BAC
2000
(ppm
)
50
4B
BAC
0.0 2
25 10
(ppm
00 20
(U I/l)
LU LA SE
0.0 2
1025
0.1 1
2e+4
4A
LU
2. No significant positive effect was found when BAC was combined with CEL. Simultaneous action of these elements promotes an increase of OA values, thus making it inappropriate to be used in real industrial settings 3. To avoid inefficient disinfections, detached living cells should be considered when designing in situ cleaning and disinfection procedures since the propagation of living cells could lead to uncontrolled pathogen spread into different premises increasing the risk of transmission to final food products.
5
3,7
29
0
7,5
53
5
1,2
78
25 ,75 2,50 6,25 10 68 1 5 12 15 17
00 20
BAC (ppm)
4
(3) 𝑂𝐴 = 27839,33 − 8561,57𝐵 + 15104𝐸𝐵
1. PRN – BAC treatments decreases the total occupied area by live cells of mature dual-species biofilms formed by Listeria monocytogenes and E. coli or P. fluorescens. Although antibacterial features of the treatment are more likely to be due to the effect of BAC,PRN appears to act synergically dislodging biofilms’ structure and thus allowing BAC to penetrate easier.
50
)
L CE
CONCLUSIONS
0
CE L
50
0.2
0
LU LA
0.1 1
0
3B
3
1
0.2
CE L
50 5 3,7 0 29 37,5 ,25 5 5 81 02 5 BAC 7 1 268,7 ,50 5 (ppm1 1512 56,2 00 ) 20 17
0 0 ,0 0,0 ,043 2 0 6 0,1 ,088 5 0,1 1 l) 0 3 UI/ ( 0,1 ,155 3 SE 0,2 78 LA
4
2
1
0
3A
2
6e+4
)
104
3
(U I/l
2x104
4
SE
log CFU/ml
3x104
(UI/l) CELLULASE
2
OA ( m )
7
6
4x104
𝑂𝐴 = 44280,89 + 38995,72𝐵
REFERENCES 1. Ferreira, V, et al. (2014) J. Food Prot. 77:150-170. 2. EFSA (2013) EFSA Journal. 11:3129. 3. Shi, X and Zhu, X (2009) Trends Food Sci. Technol. 20:407413. 4. Saá Ibusquiza, P, et al. (2012) Food Control. 25:202-210.
5. Thallinger, B, et al. (2013) Biotechnology Journal. 8:97-109. 6. Orgaz, B, et al. (2006) Enzyme Microb. Technol. 40:51-56. 7. Combrouse, T, et al. (2013) J. Appl. Microbiol. 114:11201131.
ACKNOWLEDGMENTS • This research was financially supported by the Spanish Ministerio de Economía y Competitividad (ENZYMONO, AGL201022212-C02-02). • P. Rodríguez-López acknowledges the financial support from the FPI-MICINN programme (Grant number: BES-2011050544).
