Inhibitory effects of Listeria selective enrichment media on growth characteristics of L. ivanovii

Transcription

1 Available online at Procedia Engineering 32 (2012) I-SEEC2011 Inhibitory effects of Listeria selective enrichment media on growth characteristics of L. ivanovii P. Supanivatin, S. Kosonpisit, R. Liamkaew, N. Saeteaw, A. Thipayarat Department of Food Engineering, King Mongkut s University of Technology Thonburi, Bangkok, 10140, Thailand Elsevier use only: Received 30 September 2011; Revised 10 November 2011; Accepted 25 November Abstract Various standard methods by different food regulatory bodies have been proposed different selective enrichment to grow and isolate Listeria monocytogenes in food samples. Different studies have demonstrated a variety of enrichment strategies to effectively improve the isolation rate of Listeria. However, very limited research was dedicated to understand the kinetics of Listeria spp. growth under imposed sublethal stresses by common selective agents used in those conventional enrichment media. A comprehensive list of primary and secondary enrichment media (i.e., UVM, LEB, SEB, HFB, PB, DFB, BLEB, MOPS-BLEB and FB) were used to study L. ivanovii multiplication at 37 o C incubation for 24 hr. The medium concentrations were varied at four levels; 25%, 50%, 100% and 200% to accentuate the inhibitory effect of indigenous selective agents. The net normalized growth rate and the total cell count were utilized to evaluate the lethality of each selective media and survivability of L. ivanovii. The toxicity of the media to L. ivanovii was escalated significantly and produced negative impact on growth as the strength of the enrichment medium concentration increased (25% to 200%). The negative growth effect was more pronounced for all secondary enrichment media used. Only did LEB and SEB return positive net growth rate and produce the least negative impact on the cell multiplicity Published by Elsevier Ltd. Selection and/or peer-review under responsibility of I-SEEC2011 Open access under CC BY-NC-ND license. Keywords: Listeria ivanovii; Primary enrichment broth; Secondary enrichment broth; Selective agents; Broth concentrations * Corresponding author. Tel.: ; fax: address: athipaya@yahoo.com Published by Elsevier Ltd. doi: /j.proeng Open access under CC BY-NC-ND license.

2 P. Supanivatin et al. / Procedia Engineering 32 (2012) Introduction It has been reported that several standard Listeria media do not always execute successful recovery of sublethally injured Listeria in many types of food samples [1]. The false negative situation incurs a serious health threat to general public in the sense that the injured bacteria might later recover and contribute to the development of listeriosis outbreaks [2]. In standard enrichment media for culture Listeria, selective agents (e.g., acriflavine, nalidixic acid and lithium chloride) are included to inhibit growths of non-listeria organisms [3]. These inhibitors to some degree negatively affect the growth of Listeria spp. as well [3, 4]. The inappropriate strength of selective agents in the enrichment broth can produce many cases of false negativity. The application of ISO enrichment procedure has been shown to fail the detection of any positive L. monocytogenes contamination in fresh salmon and salmon trout samples [5]. Also many authors demonstrated the common Listeria enrichment medium (i.e., Fraser broth) returned dismal recoveries of injured Listeria spp. [5, 6]. Nevertheless, several studies have also shown that using appropriate enrichment procedures can increase the isolation rate of Listeria in many food samples [7, 8, 9, 10, 11, 12, 13, 14] The objective of this study is to evaluate the influence of various standard enrichment broths and investigate the effect of selective enrichment media concentrations on the growth of L. ivanovii. 2. Materials and methods 2.1 Bacterial Strains Listeria ivanovii used in this research was isolated from local food industry. L. ivanovii culture was prepared in shake tubes using Tryptic Soy Broth (TSB) and incubated to reach the final cell density at 10 9 CFU/mL. Serial dilutions were done to achieve the desired initial cell concentration at 10 4 CFU/mL. The stain of Listeria spp. was confirmed and enumerated in Tryptic Soy Agar (TSA) using the microinoculation technique (MIT) as described elsewhere [15, 16, 17]. 2.2 Selective enrichment broths preparation Selective enrichment media concentration was varied at different strengths (i.e., 0.25X, 0.50X, 1.00X and 2.00X of typical concentration). Selective enrichment media was separated into primary enrichment and secondary enrichment steps. Selective enrichment media for primary enrichment included Half Fraser broth (HFB), modified UVM enrichment broth (UVM), Buffered Listeria enrichment broth plus selective agents (BLEB), Selective enrichment broth (SEB), Listeria enrichment broth (LEB), Demi Fraser broth (DFB) and Palcam broth (PB). Selective enrichment media for secondary enrichment were Fraser enrichment broth (FB) and Morpholinepropanesulfonic acid-buffered Listeria enrichment broth (MOPS). Five hundred microlitres of Listeria ivanovii were pipetted into small reactors containing 5 ml of aforementioned selective enrichment broth. The liquid cultures were mixed well and incubated at 35±2 C. Total cell count was performed at various stages of incubation (i.e., 0, 1, 2, 4, 6, 8, 10, 12, 14, 18 and 24 hours). 2.3 Total cell count Since the experiments were carried out using pure culture, total plate count (spread plate technique) was utilized to monitor cell multiplication in the suspended cell cultivation. The TSA was prepared by gently dissolving 20 g of premixed TSA powder in a 500 ml of distilled water. The TSA was autoclaved

3 114 P. Supanivatin et al. / Procedia Engineering 32 (2012) at 121 C for 15 min. The sterilized TSA was solidified in 96-well microtiter plates. Each well contained 150 μl of TSA and miniaturized spread plate technique was validated against the regular Petri dish method (described elsewhere) [16, 17]. 2.4 Micro Inoculation Culture The use of 96-well microtiter plate allows the reduction of sampling volumes to facilitate micro-scale reaction [15, 16, 17]. Hence, the sampling volumes inoculated on the TSA in microwell were fixed at 5 μl per sample. Triplicate repetition was applied to each sample for statistical purpose. The micro inoculation culture was kept in a hot air incubator at 35±2ºC. 2.5 Comparison of cultivation ability of selective media To evaluate the suitable enrichment media of the primary and secondary steps, growth profiles of L. ivanovii were analyzed to extract two indicating parameters (the net difference between growth and death rates and total cell change). The net rate difference of L. ivanovii growth and cell declining stages were calculated. The higher value of net rate difference indicates the better growth on that substrate. The positive values represent net cell growth whereas the negative values were not desirable for cell multiplication. The other indicator was the total cell change derived from the difference between the initial relative cell density and the maximum relative cell density. This parameter was to specify the maximum cell density achieved by culturing cell in that particular substrate. 2.6 Statistical analysis All data were analyzed at p<0.05 for significant values by ANOVA and Duncan s multiple range tests [18]. 3. Results and Discussion 3.1 Batch growth profiles of L. ivanovii in various selective media Several authors suggested using primary enrichment to recover low numbers of Listeria spp. from foods and food plant environmental samples [3, 19]. The rationale was to avoid the possibility of poor cell resuscitation or false negativity due to strong inhibitory effect from powerful selective enrichment substrates. Fig 3.1 and 3.2 helped demonstrate the inhibitory effect from 7 common primary selective enrichment and 2 other secondary enrichment substrates. The profiles of the batch growth kinetics elucidated the different degrees of toxicity from different substrates. Most of selective enrichment media in the primary step seemed to sustain L. ivanovii growth at the initial stage of incubation (the first 4-10 hr) except for PB and DFB. After this initial phase, the selective agents induced negative growth effect on L. ivanovii where the total colony count started to decline see Fig 3.1. As the incubation progressed to 24 hr, the viable cell counts were reset to the initial cell concentration or less in some instances. For the effect of concentration variation, the growth profiles of L. ivanovii in the primary selective enrichment demonstrated mixed results. For certain treatments (such as UVM, HFB, BLEB and DFB), the incremental changes of substrate concentration from 0.25X to 2X only marginally affect the growth profiles where the rest of the primary enrichment substrate showed significant influences, especially at higher medium concentrations. Therefore, the concentration of the primary selective enrichment substrate was able to be adjusted to favour the growth of target Listeria strains (in this case L. ivanovii). For most

4 P. Supanivatin et al. / Procedia Engineering 32 (2012) primary enrichment media, the regular strength (1X concentration treatments) allowed fast initial cell amplification especially SEB and LEB. Perhaps, the interaction between proper nutrient requirement (addition of enzymatic digested soybean) and minimal usage of selective agents contributed to good growth response to these selective enrichment media. However, these two recipes were highly sensitive to concentration. Lower concentrations of SEB and LEB were proven to be more favourable to L. ivanovii growth. (a) UVM (b) SEB (c) LEB (d) HFB (e) BLEB (f) PB (g) DFB Fig. 3.1 The growth kinetic of Listeria ivanovii in primary enrichment between the different concentrations of selective enrichment media at 0.25X ( ), 0.50X ( ), 1.00X ( ) and 2.00X ( )

5 116 P. Supanivatin et al. / Procedia Engineering 32 (2012) High toxicity of the primary enrichment media was observed on the PB and DFB treatments in Fig 3.1(f) and 3.1(g). The concentration of DFB only slightly affected the total viable colony count whereas the lethality from PB was highly sensitive to concentration. The highest lethality of PB was possibly derived from high concentration of lithium chloride (10 g/l) [20], which was more than 3-fold increase of what normally used in HFB and DFB, with the addition of polymixin B and ceftazidime. Weaker dilution of PB was able to mitigate the lethality effect of regular strength PB (1X concentration), see Fig 3.1(f). It was possible to reduce the concentration if isolation of Listeria from other competing microbes was still effective. (a) FB (b) MOPS Fig. 3.2 The growth kinetic of Listeria ivanovii in secondary enrichment between the different concentrations of selective enrichment media at 0.25X ( ), 0.50X ( ), 1.00X ( ) and 2.00X ( ) The growth characteristic of L. ivanovii on secondary enrichment media (FB and MOPS) did not show significant intensity of the lethality rate and produced rather similar growth kinetics as observed in the primary enrichment step, see Fig 3.2. The FB did return flatter growth profiles and showed slightly negative growth regardless of concentrations used; see Fig 3.2 (a). For MOPS treatment, there was an indication of some growth initially; however, the cell density returned to initial cell concentration as the cultivation progressed, see Fig 3.2 (b). Considering the L. ivanovii growth profile of MOPS and other primary selective enrichment substrates (including BLEB, SEB, and LEB) that contain similar ingredients of carbon and nitrogen sources, there were always some increases of cell density at the initial phase. Presumably at the beginning of the selective enrichment, the L. ivanovii inoculums were active and the composition of the carbon and nitrogen sources were appropriate and easily absorbable. This fast nutrient assimilation contributes to early growth. It took some delay time for the selective agents to diffuse into the metabolism and produce negative effect in reduction cell density. 3.2 Comparison of growth ability on selective enrichment substrates To further analyze the complex growth patterns in selective enrichment step, two derivatives representing growth characteristics of L. ivanovii in different selective enrichment media were formulated. The net difference of the growth rate and death rate was proposed to indicate the appropriateness of the selective media for L. ivanovii enrichment. Fig 3.3 screened out the confusion by analysing the growth profiles and dividing the growth patterns into positive slopes and negative slopes to calculate the net difference of multiplication rate. The higher values of this parameter indicate favourable growth pattern in the enrichment step. Among different substrates investigated, SEB and LEB showed the highest values of this indicator and the concentration did play some role in improving the growth ability of L. ivanovii using

6 P. Supanivatin et al. / Procedia Engineering 32 (2012) these media, see Fig 3.3. The least preferable medium for L. ivanovii growth was the PB followed by DFB. This finding agreed well with the prior conclusion from the previous topic. The total cell change provided insightful information to indicate optimal medium for selective enrichment. Fig 3.4 showed different degrees of negative total cell changes. Basically only SEB and LEB return net positive cell density after 24 h of incubation. For SEB and LEB, only treatments with diluted concentration of the two media (less than regular strength as described in the manufacturers preparation methods) produced positive net cell density. This plot helps accentuate the practicality of both primary and secondary enrichment substrates. More selectivity aspect in applying these selective media merits consideration. The selectivity study should provide meaningful information to select the appropriate dilution and find the most effective selective substrate to multiply the number of target microbes while separating competing bacteria at the same time during cell cultivation. Net difference between growth and death rates (1/h) Total cell change (log CFU/ml) Type of selective enrichment broth Fig. 3.3 Intrinsic colony growth kinetic Type of selective enrichment broth Fig. 3.4 lethally of substrate 4. Conclusions The inhibitory effect of seven most common primary enrichment substrates and two other secondary enrichment media was investigated using L. ivanovii as a Listeria model. The cultivation using both enrichment media produced rather similar growth characteristics and did not affect significantly on survivability of L. ivanovii. However, two primary media (i.e., SEB and LEB) in this list did support L. ivanovii multiplication fairly well. PB, on the other hand, was the most lethal enrichment substrate creating a significant reduction of cell density after 24 h incubation. The higher lithium salt content with the addition of two extra selective components (polymixin B and ceftazidime) was hypothesized to cause such potent inhibitory effect. This paper introduced two indicating parameters (i.e., net difference value between growth and death rates and total cell change) to compare the effectiveness of different media. The evaluation of growth characteristics using these indicators provided an insight into medium selection and optimization processes. Acknowledgements We are gratefully acknowledgement the Royal Golden Jubilee Ph.D. Program (RGJ) for their graduate scholarship support or The Thailand Research Fund- Master research Grants (TRF- MAG).

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