Phenotypic characterisation of Listeria monocytogenes in cow milk from three catchment areas of Goliati Area in Thyolo District, Malawi

Background: School closures were mandated in response to COVID-19 as a nonpharmaceutical intervention. Influenza A was widespread in Wisconsin at the time of mandate, March 18, 2020, allowing its evaluation for influenza interruption.

Methods: Daily percent positivity of influenza A among primary care patients who received a rapid influenza diagnostic test was evaluated throughout two influenza seasons while schools were open and closed.

Results: Median deviation from baseline of percent positivity was significantly lower when schools were closed than when open (-2.25% vs 1.08%; H=7.84; P=0.005). Median deviation in percent positivity for the eight days following statewide school closure was -20.0.

Discussion: Unprecedented closures of all Wisconsin schools was associated with a rapid decline in influenza A. Additional evaluations within other jurisdictions are warranted.

Introduction

Milk is the fluid that is secreted by mammals for the nourishment of their offspring’s. Since humans began to domesticate lactating animals, milk and milk products have been part of the human diet. It is considered one of the most complete sources of nutrients for human beings because of its diverse components such as proteins, vitamins and minerals that are important in human nutrition [1]. However due to its high nutritive value, neutral pH and high water activity, raw milk serve as an excellent growth medium for different microorganisms whose multiplication depends mainly on temperature, other competing microorganisms and their metabolic products [2]. Raw milk also creates good conditions for a variety of spoilage and thus growth of potentially pathogenic microorganisms.

Listeria monocytogenes is one of the most important pathogens of public health concerns that can contaminate milk. It is a Gram positive, facultative, intracellular, non-spore forming, motile, rod shaped bacterium that causes Listeriosis in humans, which is manifested by gastroenteritis, meningitis and meningo-encephalitis septicemia especially in people with compromised immunity, including the elderly, pregnant women and newborns [3-6]. The bacterium is widespread in nature and can survive and grow under low temperatures and pH, high salt and bile concentration, oxidative stress, carbon starvation and other adverse conditions making it a potential hazard in foods [7]. Currently 13 serotypes of Listeria monocytogenes have been identified with serotypes 1/2a, 1/2b, 1/2c and 4b are responsible for human Listeriosis [8].

Listeria monocytogenes has been isolated from different raw and ready to eat foods and in raw milk and dairy products in different countries [9]. Several cases of Listeriosis in humans have been reported, sometimes with high case fatality rates of up to 30% [10-12]. In Malawi, it is estimated that 23% of respondents of a survey titled ‘consumers attitudes and willingness to pay for safer milk in Malawi consumed raw milk, however, there is limited data that can be used for the qualitative and quantitative assessment of the risk of Listeria monocytogenes infections related to the consumption of raw milk and milk products in Malawi. The objective of this study was to determine the presence of Listeria monocytogenes in raw milk that is produced from Goliati in Thyolo district in Malawi by isolating Listeria monocytogenes according to phenotypic characterization.

Materials and methods

Study location and design

Goliati is located in the southern part of Malawi with the population of approximately 28,000 people [13]; it is one of the milk producing areas. This milk is used for commercial and domestic purposes and it is distributed across Malawi by the Lilongwe dairy limited. This is a cross-sectional study where the milk samples were collected between 19th to 23rd November, 2020.

Sampling method and sample size

The study employed convenient sampling in the selection of the three milk bulking centres for sample collection for the following reasons; A sampling frame of all the milk bulking centres in Thyolo District was not readily available from the district council. The other reason was that these milk bulking centres were convenient in that they are close to the study laboratories, minimizing transportation cost and a time-reduction for the samples get to the laboratory.

The following bulking centres were recruited in this study; Goliati dairy bulking centre which had 450 dairy farmers within its catchment area, Namahoya dairy bulking centre which had 500 dairy farmers within its catchment area and Super dairy bulking centre which had 250 dairy farmers within its catchment area.

Each bulking centre represented a cluster of milk from dairy farmers who sold their milk at these centres. It was estimated that on average, it takes about three days for all the farmers within the catchment area of each bulking centre to supply their milk. Thus, we postulated that in an ideal situation, milk samples collected on three consecutive days from each bulking centre will represent all the milk that had been collected from the catchment area of that bulking centre. This explains why three samples were collected per each milk bulking centre. In total we had nine raw milk samples, three samples per each bulking Centre and two pasteurized milk samples processed by Lilongwe dairy company as our controls.

Sample collection

At each bulking centre, 500mLs of raw milk samples were collected per day for three consecutive days. This was done in order to capture all raw milk supplied by the farmers for each bulking centre. The samples were stored at a 40C for transportation to the laboratory [14]. Table 1 shows a summary of the codes used to collect the samples from the three bulking areas.

Sample preparation

Sample serial dilutions: 1ml of each of all the nine (9) raw milk samples was serially diluted until a dilution factor of 10¯6 was reached. This means that for each sample, we had the following concentrations of the sample: 10¯0, 10¯1, 10¯2, 10¯3 , 10¯4, 10¯5 and 10¯6 but due to the shortage of culturing plates, we randomly selected the following dilutions for primary culturing: 10¯0, 10¯1, 10¯3 and 10¯6 [15].

Preparation of the primary culture

0.05µl sample from each test tube of 10¯0, 10¯1,10¯3 and 10¯6 dilutions were transferred to the labelled Nutrient agar (Techno PharmChem, India) and Brain heart infusion agar (Oxoid L.T.D Basingstroke, Hampshire, England) petri dishes. Sterile stick cotton swabs were used to plate the sample using quantitative streaking method. Another two plates labelled Nutrient agar and Brain heart infusion agar were used to culture the control sample which was Long life processed milk produced by the Lilongwe dairy industry, Malawi. After culturing all petri dishes, they were incubated at 36°C for 48 hours for the growth of microorganisms [16].

Preparation of the secondary (Sub) culture

Isolated colonies from the selected primary culture plates were sub cultured on Brain heart infusion agar using sterile loops. Isolation streaking technique was employed for sub culturing. Brain heart infusion agar (Oxoid L.T.D Basingstroke, Hampshire, England) was used for sub culturing to due lack of selective medium, PALCAM agar. The sub cultured petri dishes were incubated at 36°C for 48 hours for the growth of a pure culture [17].

Gram staining

Colonies were isolated from pure culture using sterile plastic inoculating loops and they were placed on clean, labelled microscope slides with a drop of normal saline (Aculife Healthcare Pvt. L.T.D – India). Colonies were mixed with normal saline to make thick smear. The prepared smear was allowed to dry in air and then the dry smear was fixed by placing the slides on a slide warmer with the smear side facing up.

Heat fixed smear slides were placed on staining rack over the sink and the smear was covered with the primary stain Crystal violet (Cypress Diagnostics-Belgium) which was allowed to remain on the slides for a minute. After a minute primary stain was washed away by rinsing the slides with running tap water and slides were shaken off to remove excess water. Then the smear was flooded with a Mordant gram’s iodine solution (Cypress Diagnostics-Belgium) and was left for a minute. The slides were rinsed with running tap water to remove iodine solution on the slides. The decolorizing agent Acetone alcohol (Merck Chemicals (pvt) L.T.D - USA) was applied to the smear and waited for approximately 20 seconds. The slides were washed with tap water to remove acetone alcohol. Then the slides were flooded with counterstain, Safranin (Cypress Diagnostics-Belgium) and it stayed there for 20 seconds. Lastly the slides were washed and then heat dried. After this they were observed under microscope and recorded. During microscopy, a 100x objective lens was used [18].

Catalase test

The test was executed by transferring the suspected colonies from our sub culture using sterile wire loop to labelled microscopic slides. Then one drop of 3% hydrogen peroxide (Glassworld and Chemical Suppliers, Cape Town – South Africa) was placed on the slides and observations were made and documented. This test procedure was carried out on all the samples and normal saline was used as control [19].

Haemolysis test

The test was done by isolating suspected colonies from the subculture using sterile wire loop and these were cultured on Blood agar medium (Oxiod Ltd, Basingstroke, Hampshire-England) plates. Then the plates were incubated at 36 0 C for 48 hours and then observations were made and documented [20].

Motility test

The test was carried out by isolating suspected colonies using sterile wire loop and stabbing into prepared Motility Indole Ornithine (Becton, Dickinson and Company – India) test tubes. Then the tubes were incubated at 37oC for 48 hours after which observations were made and recorded [21].

Ethics review of the protocol

The study was ethically reviewed and approved by The Malawi University of Science and Technology Research and Ethics Committee (MUSTREC). It was certified under the following reference number: U10/2020/008.

Statistical analysis

The data were analysed in R. Descriptive measures of Listeria monocytogenes counts were made and compared across the centers and concentration levels. A test of normality of the counts was also made to check normality distribution of the data for further analysis of variance.

Results

During primary culturing, all the 3 samples per each milk bulking center were cultured at 4 different concentration levels hence there were 72 replicates with 24 replicates from each center. The mean Listeria monocytogenes colony count was 178 (35,600 CFU/ml of raw milk) with standard deviation of 74 counts (14,800 CFU/ml of raw milk). The mean Listeria monocytogenes colony count was slightly higher at center 1 than centers 2 and 3. Center 2 and 3 that had almost equal mean colony counts of Listeria monocytogenes in the milk samples as shown in Figure 1.

Figure 1: Mean counts of Listeria monocytogenes by Centre of the sample.

The results also indicate higher mean Listeria monocytogenes counts in nutrient agar of 182 counts (36,400 CFU/ml of raw milk) than in Brain heart infusion agar which had a mean Listeria monocytogenes colony count of 174 counts (34,800 CFU/ml of raw milk). The distribution of the counts by concentration level and the media type are shown in Figure 2.

Figure 2: Mean Listeria monocytogenes counts by concentration level of medium

The results indicate that an increase in the concentration in which Listeria monocytogenes were cultured was associated with increased growth of colonies of Listeria monocytogenes.

The results in Table 2, indicate that at concentration levels 10^0 and 10^-1 (higher concentrations), the mean Listeria monocytogenes counts were slightly higher in nutrient agar medium than in Brain heart infusion agar. This is because Brain Heart Infusion agar is highly selective compared to nutrient agar [16,17]. The trend was different at lower concentration levels 10^-3 and 10^-6 where the mean counts were slightly lower in nutrient agar medium than in Brain heart infusion agar, thus this might suggest possible contamination or a technical flaw in the serial dilution method.

Further analysis was done to check if mean Listeria monocytogenes colony counts were significantly different at independent centers from where the milk samples were drawn. In Figure 3, are boxplots for counts at the three centers from where milk samples were obtained. The figure shows slightly higher mean Listeria monocytogenes colony counts for centre 1 than median Listeria monocytogenes colony counts for center2 and centre3 which are almost the same.

Figure 3: Boxplots for Listeria monocytogenes at the three centres.

Normality assumptions and analysis of variance

Data were checked for normality distribution assumptions. The data were independently obtained from the three centres. Here, the only assumptions checked were Normality distribution assumptions of data and homogeneity of variance assumptions using Leven’s test.

In Figure 4, the normality assumption was validated through a QQ-plot as most data points are within the reference line. The Leven’s test validates the equality of variance assumptions (p-value=0.8734).

Figure 4: Testing normality distribution of Listeria monocytogenes counts.

The analysis of variance results indicate that the mean Listeria monocytogenes counts were not significantly different at the three centres (p-value=0.987). However, at different concentration levels, the mean Listeria monocytogenes colony counts are significantly different (p-value<0.0001). Turkeys multiple pairwise comparison showed that the only pair whose mean counts were not significantly different were concentration levels 10^-6 and 10^-3 with adjusted (p-value=0.1331). The mean Listeria monocytogenes colony counts were significantly different at the rest of the pairs of concentration levels (p-value<0.0001).

Comparison of primary culture results with sub-culture results

Samples that were randomly selected for sub-culturing in Brain heart infusion agar indicate a mean Listeria monocytogenes count of 101.2 (20,440 CFU/ml) at concentration level of 10^-6 and 99.8 counts (19,960 CFU/ml) cat concentration level 10^-3. When these results were compared to primary culturing counts obtained from nutrient agar and brain heart infusion agar media at concentration levels 10^-3 and 10^-6, it shows that the mean Listeria monocytogenes colony counts in sub-culturing are lower than mean counts in primary culturing. This is due to the fact that primary cultures are prone to microbial contamination compared to secondary cultures hence the noted higher mean counts in primary cultures [22]. Different from these results, standard milk samples that were obtained from market places showed lower mean Listeria monocytogenes colony counts of 55 and 64 in nutrient agar and Brain heart infusion agar respectively. This is inevitable as milk processing which include pasteurization has been shown to result in log10 or more reduction of Listeria monocytogenes [23] Table 3.

Results of Gram staining test, Haemolysis test, Motility test and Catalase test for isolates from secondary (sub) culture per each sample

Thus, Tables 4-7 indicates that the isolates that were obtained and microbiologically analysed were gram positive, catalase-positive, orthinine negative, indo negative but motile. These isolates also exhibited beta haemolysis on blood agar suggestive of Listeria monocytogenes.

There was also growth of colonies on control samples whose isolates were exhibiting phenotypic features that were similar to the isolates from the raw milk.

Discussion

The colony forming units found in this study which were enumerated from all the raw milk samples and the control samples used in this study were above 10,000 CFU/ml and according to the European legislation containing microbiological food safety criteria for Listeria monocytogenes which stipulates that the presence of more than 100 CFU/ml of Listeria monocytogenes is injurious to health, hence this means that this finding requires special attention from policy makers and milk producers to ensure the production of safe and quality milk[24].

There was no statistical significant difference in the mean colony forming units per milliliter (CFU/ml) across the three milk bulking centers suggesting that the level of contamination was almost equal across the centers and probably they employ similar milk handling procedures when collecting milk from farmers.

It has been shown that there is a 100% growth rate of colony forming units suggestive of Listeria monocytogenes in all the three milk bulking centers This is not a true reflection of the actual individual level prevalence as bulk tank raw milk represented a cluster of milk from individual farmers within the catchment area of each bulk tank. Thus, the contamination of raw milk with Listeria monocytogenes is attributed to the entire catchment area supplying the bulk centre. This explains why this finding is different from the prevalence’s that have been reported in several studies; the prevalence was reported at 41.6% in Syria [25], the prevalence was reported at 6.5% in the USA [26], the prevalence was reported at an average of 6.8% in Iran [27-29], the prevalence was reported at 5.9% in morocco [30] and the prevalence was reported at 1.4% in Latvia [31]. It is essential to note that these prevalence’s were based on samples of individual farmers contrary to the design that was used in this study.

The phenotypic findings that have been reported in this study are similar to those that were reported from samples of Kerman, Iran [2]. In order to ascertain specific species or strains of Listeria monocytogenes, they employed genotypic techniques which were not used in this study due to lack of reagents. This explains why we only managed to phenotypically isolate listeria monocytogenes.

The isolation of Listeria monocytogenes in this study shows that there is contamination of raw milk with Listeria monocytogenes. The following risk factors have implicated in necessitating raw milk contamination with Listeria monocytogenes; defective disinfection of teats before milking, lack of correct management of barn and silage, insufficient hygiene practice in the environment and a low level of cleanliness among cows [32]. Milk handling processes during storage and transportation have also been shown to contribute to milk contamination with various microorganisms [33]. Despite showing the presence of Listeria monocytogenes, this study did not focus on the local factors that leads to milk contamination with this microorganism.

In conclusion, it is essential to appreciate that there is Listeria monocytogenes contamination of raw milk in all the three catchments areas that supplied milk to the three milk bulking centres. The isolates are a cluster or group level finding suggesting that individual level studies are needed to ascertain the exact prevalence of Listeria monocytogenes contamination. There is also a need to do studies that will focus on genotypic and virulent profiling of the strains of Listeria monocytogenes that are in circulation in these catchment areas and also ascertain the factors that facilitate local contamination of milk with listeria monocytogenes. This will have an impact on public health planning and interventions.

Authors contributions

CM and AM were involved in the study conceptualisation, sample collection, laboratory analysis and interpretation of results.

YK and MK were involved in providing technical guidance in the laboratory.

DM was involved in statistical analysis and interpretation

GB, MM, PAMN, AGM, WM and PC were involved in the reviewing of the entire study from the stage of the proposal to this final article.

TEM was the overall supervisor of the study.

Financial support

The study was supported by the Directorate of Research and Post Graduate Studies at the Malawi University of Science and Technology.

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