INTRODUCTION
Microbial food safety and food-borne infections are an important public health
concern worldwide. Contaminated food consumption often results in an illness
which is called food borne illness or food poisoning (Saikia
and Joshi, 2010; Arzinz et al., 2011).
The hygiene process criteria for many foodstuff include tests for Salmonella
spp., Listeria spp., Enterobacteriaceae and Staphylococcus aureus
(S. aureus). Members of the genus Salmonella are Gram-negative
and facultative anaerobic, rod-shaped bacteria (Malkawi
and Gharaibeh, 2004). Salmonella is a major food and water borne
pathogenic bacterium which causes an intestinal infection, accompanied by fever,
abdominal cramps and diarrhea which is commonly known as salmonellosis (Rathnayaka,
2011). Salmonella is more often associated with any raw food of animal
origin which may be subject to fecal contamination, such as raw meat, poultry,
fish/seafood, eggs and dairy product (Fadel and Ismail,
2009; McGuinness et al., 2009). Listeria
spp. are ubiquitous bacteria widely distributed in the environment. Among
the seven species of Listeria, only Listeria monocytogenes is
commonly pathogenic for humans. L. monocytogenes is a Gram-positive rod
that is catalase positive and shows a characteristic tumbling motility (Enan,
2006). It has been recognized as a veterinary pathogen and in humans it
causes a disease known as listeriosis that could be very dangerous in older
adults, persons with weakened immune systems, pregnant women and newborns. Food
implicated in outbreaks of listeriosis have included various types of products
such as dairy, meat, vegetable and sea food (Jalali et
al., 2007; Adetunji and Arigbede, 2011).
Bacteria belonging to the Enterobacteriaceae family are the most common bacterial
pathogens associated with gastrointestinal infections, particularly diarrhea.
This bacterial family consists of a large heterogeneous group of facultative
anaerobic, Gram-negative rods and includes species in the groups Escherichia
coli, Salmonella and Shigella, the most common causative
agents of intestinal infections (Rustam et al., 2006).
Therefore, detection of Enterobacteriaceae, rather than for the traditional
Coliform group, is advantageous because Enterobacteriaceae includes some potentially
pathogenic species. Unlike the Coliform group, some Enterobacteriaceae species
are often present in the processing environment. For this reason, enumeration
of Enterobacteriaceae in foods now shows evidence of increasing interest (Rustam
et al., 2006; Feinberg et al., 2009).
S. aureus is a Gram-positive, catalase positive, coagulase positive non
motile coccus bacterium (Ugbogu et al., 2007).
S. aureus is a common cause of a bacterial food borne disease worldwide.
However, quantitative evaluation of S. aureus contamination of food
is not a simple task. Several studies have found S. aureus strains unable
to produce black colonies with a clear halo on the Baird Parker Agar selective
medium in dairy products and many S. aureus display peculiar biochemical
properties (Da Silva et al., 2000). Therefore,
the analysis of S. aureus eventually contaminated food stuff is made
difficult by the risk of overlooking potentially pathogenic strains of S.
aureus just because they have atypical morphological and/or biochemical
characters. To avoid this danger, Harvey and Gilmour prescribe the coagulase
test for all colonies with a diameter greater or less than 1 mm with or without
halo or areas of clarification (Harvey and Gilmour, 1985).
Food borne pathogens are a growing concern for human illness and death. Therefore,
there is an increasing demand to ensure a safe food supply and an urgent need
to implement programmes such as Hazard Analysis Critical Control Points (HACCP)
to monitor the quality of the products concerning produced for the presence
of the pathogens (Nicolas et al., 2007). New method
for the rapid and reliable detection of food borne pathogens are continuously
proposed and improvements in the fields of immunology, molecular biology, automation
and computer technology continue to have a positive effect on the development
of faster, more sensitive and more convenient method in food microbiology (Biswas
et al., 2011; Mandal et al., 2011).
Standardized method (e.g., ISO described method) are acknowledged as the reference
analytical method for official control. They rely on traditional microbiological
culture standard method that are widely used in food analysis laboratories.
These traditional method involve the following basic steps: pre-enrichment,
selective enrichment, selective plating, biochemical screening and serological
confirmation (Mandal et al., 2011). These conventional
method present several difficulties, such as subjectivity in the interpretation
of some biochemical or morphological tests, the possible interference of matrices,
especially when they present high levels of contamination, intense labor, high
cost of supplies and above all, the prolonged time (from 3 to 7 days) needed
to give definitive results, depending on the ability of the organisms to multiply
into visible colonies (Thomas et al., 2009).
In this context, MBS srl (a spin-off of Roma Tre University, Rome, Italy) has
developed an alternative rapid method, called Micro Biological Survey (MBS)
method. It is a fast colorimetric fast system for the detection and the selective
counting of bacteria present in agro-food, in water and in environmental samples.
This method consists of an analytical kit containing disposable, ready-to-use
reaction vials for fast microbiological analyses. The analysis is based on the
change of color of the vial content which is induced by the presence of bacteria.
The analyses can be carried out by untrained personnel and anywhere they are
necessary, without the need for any instrumentation other than a thermostat
which is provided on request. The MBS method measures the catalytic activity
of the redox enzymes in the main metabolic pathways of bacteria (Shultz
and Chan, 2001; Slater, 2003; Antonini
et al., 2007) and allows an unequivocal correlation between the observed
enzymatic activity and the number of viable cells present in the samples. The
time required for a color change is inversely related to the log of bacterial
concentration; like an enzymatic reaction, the greater the number of bacteria,
the faster the color changes (Berlutti et al., 2003).
In a previous study, we carried out the primary validation of the MBS method
for Total Viable Count and for Escherichia coli with almost perfect
agreement between reference method (Bottini et al.,
2011). The objective of the present study was the primary validation of
the qualitative MBS method for Salmonella spp. and Listeria spp.
and the quantitative MBS method for Enterobacteriaceae and S. aureus
in accord with ISO 16140 (2003). Qualitative method are
method of analysis whose responses are either the presence or absence of the
analyte detected either directly or indirectly in a certain amount of sample
as indicated by ISO 16140 (2003). Quantitative method
are method of analysis whose responses are the amount of the analyte measured
either directly or indirectly in a certain amount of sample as indicated by
ISO 16140 (2003). The validation here reported provide
evidence that the new MBS method gave similar results and are in agreement with
the reference method, confirming the reproducibility and specificity of MBS
method.
MATERIALS AND METHODS
This study was conducted at the department of Biology, University Roma Tre during the period from 2009 to 2011.
Bacterial strains: All the strains used in these validations were available at ATCC (American Type Culture Collection): E. coli (ATCC 25992), E. coli O157:H7 (ATCC 35150), C. freundii (ATCC 43864), K. pneumoniae (ATCC 13883), E. cloacae ( ATCC 13047), E. sakazakii (ATCC 51329), S. enteritidis (ATCC 13076), and S. enterica ser. Typhimurium (ATCC 14028), Y. enterocolitica (ATCC 19543), B. cereus (ATCC 11778), B. stearothermophilus (ATCC 24567), B. subtilis (ATCC 6633), L. innocua (ATCC 33090), L. ivanovii (ATCC 19119), L. monocytogenes (ATCC 7644), S. aureus (ATCC 12600), S. epidermidis (ATCC 12228), S. lentus (ATCC 29070), P. aeruginosa (ATCC 27853), R. equi (ATCC 31543), E. faecalis (ATCC 29212), L. delbrueckii subsp. lactis (ATCC 12315), C. perfringens (ATCC 13124), A. niger (ATCC 9642) and S. cerevisiae (ATCC 9763).
Preparation of naturally contaminated food samples with different levels
of contamination: Naturally contaminated food samples were randomly selected
among those found positive by reference method. Four different food matrices
were selected for validation of qualitative method: Raw meat products, vegetables,
pastry and dairy products. Three different food matrices were selected for validation
of quantitative method: Raw meat products, pastry and dairy products. Sterilized
“Baby foods” of the same foodstuff typology were used as a negative
control. Different levels of contamination of naturally contaminated samples
were obtained as follows: 10±0.5 g of naturally contaminated samples
were homogenized in 90 mL of peptone water by a stomacher according to ISO
16140 (2003). Then homogenates were incubated for different times at different
temperatures obtaining different levels of contamination. To verify the equivalence
between the MBS method and the reference method, these samples were simultaneously
tested.
Artificially contaminated food samples: To achieve the number of samples required for statistical evaluation of the chosen parameters, artificially contaminated samples were also used. Food samples, found negative by reference method, were contaminated with a mixture of the above indicated microorganisms from overnight cultures with serial dilutions in sterile saline solution up to 10-8. Ten different dilutions of ten different samples were analyzed for Listeria spp., Salmonella spp., Enterobacteriaceae and S. aureus. Each dilution was tested in duplicate with both the MBS method and the plate counting reference method.
Colorimetric MBS method procedure: The analytical procedure for qualitative and quantitative MBS method is based on colorimetric survey, using a redox indicator of the change of the oxidoreductive state in the reaction medium. For the analysis by the MBS method, ready-to-use MBS vials, sterilized and containing the reagent for the analysis were used. Four different kinds of vials were used: The vials for Listeria spp., Salmonella spp., Enterobacteriaceae and S. aureus analysis. To carry out the analysis, 10 mL of sterile distilled water and 1 mL of the samples were added to a vial of one or another type, depending on the type of analysis to be carried out. The vial was shaken until the entire reagent was dissolved. Later on, the vial was incubated at 37°C for all bacteria. The starting color is blue for vials for Listeria spp. and red for vials for Salmonella spp., Enterobacteriaceae and S. aureus. In the presence of the microorganisms of interest, the colors of vials changed to yellow indicating a positive result. The time taken to turn yellow is inversely related to the bacterial content of the analyzed sample. The persistence of the starting color after 36 h for Listeria spp. and S. aureus, 32 h for Salmonella spp. and 24 h for Enterobacteriaceae indicates a negative result, that is an absence of microorganisms.
Reference method: For Listeria spp. the reference method was
plate count on Agar Listeria Ottaviani and Agosti (ALOA; Sigma, St. Louis,
MO, USA) and Listeria Palcam Agar (PALCAM; Liofilchem, Roseto degli Abbruzzi,
Italy) after 24 h of incubation at 37°C according to ISO
11290-1:1996/Adm 1 (2004). For Salmonella spp. it was plate count
on Xylose Lysine Desoxycholate Agar (XLD, Sigma, St. Louis, MO, USA ) and Brilliant
Green Agar (BGA; Sigma, St. Louis, MO, USA) after 24-48 h of incubation at 37°C
according to ISO 6579:2002/COR 1 (2004). For Enterobacteriaceae
the reference method was plate count on Violet Red Bile Glucose Agar (VRBGA;
Liofilchem, Roseto degli Abbruzzi, Italy) after 24 h of incubation at 37°C.
For S. aureus it was plate Baird-Parker Agar (BPA; Sigma, St. Louis,
MO, USA) after 46-48 h according to ISO 6888-1:1999/Adm 1
(2003). However, it should be kept in mind that, when a food sample is analysed
with a reference method, a pretreatment is always required, according to the
above reported ISO rules. Such pretreatment may vary from sample homogenization
and dilution up to an additional enrichment, according to the different analysis
to be carried out. The whole procedure for analysing a food sample may therefore
last from a minimum of 36 h up to a maximum of 72 h.
Data analysis for qualitative validation: The primary validation of
the qualitative MBS method for Listeria spp. and Salmonella spp.
was made according to ISO 16140 (2003). The relative
performance parameters indicated by the ISO 16140 (2003):
accuracy, specificity and sensitivity were determined. They were calculated
as follows: accuracy AC = ((PA+NA)/N) x 100%; specificity SP = (NA/N-)
x 100%; sensitivity: SE = (PA/N+) x 100%. Where, PA is the agreement
for positive results; NA is the agreement for negative results; N is the total
number of samples; N_ is the total number of negative results with the reference
method (N_=NA+PD); N+ is the total number of positive results with
the reference method (N+ = PA+ND); PD is positive deviation (i.e., false positive
result); ND is negative deviation (i.e., false negative result) (ISO
16140, 2003).
Data analysis for quantitative validation: Data analysis was carried
out according to ISO 16140 (2003). Two parameters were
analyzed: linearity and accuracy. The linearity of the method was assessed by
analyzing the correlation using a plot of bacteria concentrations (expressed
as CFU mL-1) against the time taken to change color. The accuracy
was assessed by analyzing the correlation using a plot of bacteria concentrations
(expressed as log CFU mL-1) obtained with the reference method and
with the alternative MBS method (ISO 16140, 2003).
S. aureus confirmative coagulase test: Preparation of samples
for coagulase test was carried out as follows: selected colonies grown on BPA
agar were transferred each with a sterile inoculation loop to different culture
tubes containing Brain Heart Broth (Sigma, St. Louis, MO, USA) and were incubated
at 37°C for 20-24 h; 10 mL of the supernatants of MBS method for S. aureus,
that have changed color from red to yellow, were inoculated each within a vial
containing 0.5 g of Amberlite MB-150 Mixed Bed Exchanger (Sigma, St. Louis,
MO, USA), moderately shacked and left rest for 5-10 min. The confirmative coagulase
test was carried out as follows: a vial with lyophilized rabbit plasma with
EDTA (Sigma, St. Louis, MO, USA) was rehydrated with 3 mL of distilled water
and 0.3 mL of the rehydrated rabbit plasma were pipetted into a sterile culture
tube using a sterile pipette; 0.1 mL of the sample (either coming from culture
tubes containing Brain Heart Broth or from MBS supernatants) was carefully mixed
with the plasma in the sterile culture tube and then incubated at 37°C;
the tubes were checked every hour for coagulation by gently tipping to the side;
the coagulase test was positive if more than 75% of the tube contents had formed
a coherent clot. If the test was negative after 4-6 h, the tube was left in
the incubator and a final assessment was made after 24 h.
RESULTS
Selectivity of MBS method: Preliminary experiments were carried out to determine whether the food matrices may interfere with the MBS method. For this purpose tests on food samples artificially contaminated with target ATCC strains (L. monocytogenes ATCC 7644, S. enterica ser. Typhimurium ATCC 14028, E. coli ATCC 25922 and S. aureus ATCC 12600) were carried out (data not shown). A perfect agreement between reference method and MBS method was observed for all the strains, indicating that no interference came from any of the food matrices utilized (raw meat products, vegetables, pastry and dairy products).
Further preliminary tests were carried out to determine the selectivity of
the different MBS method. The selectivity is defined as the ability of an alternative
method to detect the target analyte from a wide range of strains and the lack
of interference from a relevant range of non-target strains of the alternative
method (ISO 16140, 2003).
| Table 1: |
Selectivity tests |
 |
|
Table 1 reports the selectivity tests indicating the minimum
detection limit (expressed as CFU mL-1) of the MBS method for Listeria
spp., Salmonella spp., Enterobacteriaceae and for S. aureus
towards different ATCC bacterial strains suspended in protonated water.
PRIMARY VALIDATION OF MBS METHOD FOR Listeria spp. AND Salmonella spp. IN QUALITATIVE ASSAYS
The primary validation of the qualitative MBS method for Salmonella
spp. and Listeria spp. was performed according to ISO
16140 (2003).
Table 2 shows the results of analysis on both naturally and artificially contaminated food matrices (raw meat products, vegetable, pastry and dairy products) obtained with MBS method and reference method for Listeria spp. and Salmonella spp. These results indicate the concordance between results obtained with MBS method and reference method to detect Listeria spp. and Salmonella spp. Out of 71 positive samples obtained with the reference method for Listeria spp. 67 were found to be positive (N+) and 4 were found to be negatives (ND) by the MBS method. Out of 17 negative samples with the reference method, 17 were found to be negative (N–) by the MBS method and no positives were found (PD). For Salmonella spp. out of 81 positive samples obtained with the reference method, 79 were found to be positive (N+) and 2 were found to be negatives (ND) by the MBS method. Out of 15 negative samples obtained with the reference method, 12 were found to be negative (N_) and 2 were found to be positives (ND) by the MBS method.
The main performance parameters which the alternative method must demonstrate are the relative accuracy, specificity, sensitivity and selectivity.
| Table 2: |
Results of analysis of food samples (raw meat products, vegetable,
pastry and dairy products) either naturally or artificially contaminated
obtained with MBS method and reference method for (a) Listeria spp.
and (b) Salmonella spp. |
 |
| PA: Positive agreement, NA: Negative agreement, ND: Negative
deviation (false negatives), PD: Positive deviation (false positives), N:
Total number of samples (NA+PA+PD+ND), N+: Total number of positive
results obtained with reference method, N–: Total number
of negative results obtained with reference method |
| Table 3: |
Paired values of relative accuracy (AC), relative sensitivity
(SE) and relative specificity (SP) for MBS method and reference method |
 |
|
Relative accuracy is the degree of correspondence between the results obtained
by the reference method and the results obtained by the alternative method on
identical samples (ISO 16140, 2003). The term “relative
accuracy” used here is complementary to the “accuracy” and “trueness”
as defined in ISO 5725-1:1994/COR 1 (1998). This states
that accuracy is “the closeness of agreement between a test result and
the accepted reference value”, and that trueness is "the closeness of agreement
between the average value obtained from a large series of test results and an
accepted reference value".
Relative sensitivity is the ability of the alternative method to detect the analyte when it is detected by the reference method.
Relative specificity is the ability of the alternative method to not detect the analyte when it is not detected by the reference method.
Table 3 shows the values of the performance parameters for
MBS method calculated using the positive and negative results shown in Table
2 and the same performance parameters for the reference method as reported
in the literature (ISO 11290-1:1996/Adm 1, 2004 for Listeria
and ISO 6579:2002/COR 1, 2004 for Salmonella spp.).
PRIMARY VALIDATION OF QUANTITATIVE MBS METHOD FOR ENTEROBACTERIACEAE AND S. aureus
The primary validations of the quantitative MBS method for S. aureus
and Enterobacteriaceae were performed according to ISO 16140
(2003). The main performance parameters which the alternative method must
demonstrate are: linearity and accuracy.
|
| Fig. 1 (a-b): |
Linearity: Correlation line between analyte (a) Enterobacteriaceae
and (b) S. aureus concentrations with the time taken to change color
with in the MBS method |
Linearity is the ability of the method when used with a given matrix to give
results that are in proportion to the amount of analyte present in the sample,
that is, an increase in analyte corresponds to a linear or proportional increase
in results as indicated by ISO 16140 (2003). This was
achieved graphically as illustrated in Fig. 1 by plotting
bacteria concentrations (expressed as the log of CFU mL-1) obtained
with the reference method with the time occurred for taken to change color with
the identical samples analyzed with MBS method. A linear inverse relationship
between the MBS method and the bacteria concentration, with a correlation factor
(R2) close to 1.00, confirming the linearity of the data can be observed.
Using naturally and artificially contaminated food samples, bacteria concentrations
(expressed as the log of CFU mL-1) obtained with the reference method
are plotted against the time taken to change color with the identical samples
analyzed with MBS method. A linear inverse relationship between the time, taken
to change color with the MBS method and the bacteria concentration could be
observed with Enterobacteriaceae vials and S. aureus vials on three
different food matrices: raw meat products, pastry and dairy products. The correlation
factors (R2) are 0.98 and 0.95 for Enterobacteriaceae and for S.
aureus, respectively.
Accuracy is the degree of correspondence between the response obtained by the
reference method and the response obtained by the alternative method on identical
samples (ISO 16140, 2003). Figure 2
show a perfect correlation between the bacteria number (expressed as log CFU
mL-1) obtained with the traditional counting method and the alternative
MBS method. The straight lines obtained were close to the theoretical y = x
(slope = 1,00), with values of correlation factor (R2), which confirm
the high equivalence between the reference method and the alternative. Using
naturally and artificially contaminated food samples, bacteria numbers (expressed
as the log CFU mL-1) obtained with the reference method are plotted
against the time taken to change color with the identical samples analyzed with
MBS method. A good correlation between the bacteria numbers (expressed as log
CFU mL-1) obtained with the traditional counting method and the alternative
MBS method could be observed. In fact the slopes are close to the theoretical
value of 1.00 (i.e., 0,98 for Enterobacteriaceae and 0.93 for S. aureus).
The correlation factors (R2) are 0.97 and 0.94 for Enterobacteriaceae
and for S. aureus, respectively.
Confirmation tests for S. aureus were performed on all the positive
and negative results. Results obtained using coagulase test were in full agreement
with results obtained by MBS method since all the MBS positive results were
positive in the coagulase tests.
|
| Fig. 2 (a-b): |
Accuracy: Correlation line between alternative MBS method
and reference method (a) Enterobacteriaceae and (b) S. aureus |
DISCUSSION
Rapid and reliable detection of microorganisms in food samples is essential
for prevention of disease, but it is also important to save on the cost of storage
and transportation of infected products, (Rathnayaka, 2011),
therefore, the development of new method for detection and identification of
microorganisms in food, water and environmental samples which give accurate
results and are economically competitive, are always needed. With traditional
count plate method bacteria replication can be observed with the naked eye,
but greater expertise between the operators and operational complexity are required.
On the other hand, alternative method often turn out to be very expensive and
also require highly equipped laboratories (Settanni and Corsetti,
2007; Thacker et al., 1996).
In this context, rapid colorimetric MBS method may play an important role.
This is a fast colorimetric system for the detection and selective counting
of bacteria in agro-food, in water and in environmental samples. Colorimetric
method currently available are mainly based upon microorganisms secondary metabolism
measuring. On the contrary, the MBS method measures the catalytic activity of
redox enzymes of the main metabolic pathways of bacteria (Antonini
et al., 2007; Bottini et al., 2011),
allowing an unequivocal correlation between the observed enzymatic activity
and the number of viable cells present in the samples. The time required for
color change is inversely related to the log of bacterial concentration; like
an enzymatic reaction, the greater the number of bacteria, the faster the color
change (Bottini et al., 2011). Alternative rapid
analytical method, like the MBS method, are allowed by regulatory authorities
once they have been validated against the reference method according to ISO
16140 (2003) and McGuinness et al. (2009).
A previous study provided the evidence that the MBS method for TVC and E.
coli gave similar results and is in agreement with reference method (Bottini
et al., 2011). The purpose of the present study was the primary validation
of qualitative MBS method for Listeria spp. and Salmonella spp.
and of quantitative MBS method for Enterobacteriaceae and S. aureus.
Qualitative method are method of analysis whose responses are either the presence
or absence of the analyte detected either directly or indirectly in a certain
amount of sample as indicated by ISO 16140 (2003). Quantitative
method are method of analysis whose responses are the amount of the analyte
measured either directly or indirectly in a certain amount of sample as indicated
by ISO 16140 (2003). Both the MBS qualitative and quantitative
method were demonstrated to be very selective and all showed a high reliability
and correlation with traditional count plate method. No interference due to
food matrices was observed.
In particular, quantitative MBS method for Enterobacteriaceae and S. aureus demonstrated high linearity (results are in proportion to the amount of analyte present in the sample) and accuracy (correspondence between the results obtained by the reference method and the results obtained by the alternative method on identical samples). It should also be noted that results obtained using coagulase test were in full agreement with results obtained by the MBS method on S. aureus, demonstrating that the well known variability of the morphological and biochemical properties of naturally occurring S. aureus strains did not influence the exact quantification by MBS method of S. aureus cells present in the food samples.
For qualitative analysis, a perfect correspondence between the MBS method and
the reference method for Listeria spp. and Salmonella spp. was
observed when ATCC reference strains were inoculated into different food matrices.
On the contrary, a limited number of discrepancies between MBS method and reference
method were observed when the same food matrices were contaminated by naturally
occurring strains of Listeria and Salmonella. This phenomenon
may lead to either false positive or false negative results; however these discrepancies
may be attributed either to the MBS method or to the reference method. It should
be kept in mind that the apparent false negatives or false positives become
real false negatives or false positives only when an independent reference method
has been proven to be true. The paired values for relative accuracy, sensitivity
and specificity for MBS method here reported are lower than the same parameters
reported for the reference method (ISO 11290-1:1996/Adm 1,
2004; ISO 6579:2002/COR 1, 2004). As a matter of
fact, a small percentage of false negatives and false positives were observed
using the reference method in a inter-laboratory experiments (ISO
11290-1:1996/Adm 1, 2004; ISO 6579:2002/COR 1, 2004).
For these reasons, the very same ISO documents stated that when the reference
method indicates a positive result, supplementary analysis to prove whether
there is a real presence or not of pathogenic bacteria should be carried out
(ISO 11290-1:1996/Adm 1, 2004; ISO
6579:2002/COR 1, 2004).
Although, we have here demonstrated that relative accuracy, sensitivity and specificity for MBS method for Listeria spp. and Salmonella spp. are more reliable than the respective reference method, an independent analysis should be carried out when a positive result is found.
CONCLUSIONS
The validations of the MBS method for Listeria spp., Salmonella spp., Enterobacteriaceae and S. aureus give similar results and are in agreement with the reference method according to ISO rules. MBS method could therefore become a valid support for the control procedures for all the food farming companies willing to do a microbiological screening of their products to ensure complete hygienic production.
ACKNOWLEDGMENTS
This study was supported by a grant from Italian Ministry of University and Research (Decreto No. 2933/2006 ex art.11 DM 593/2000) and by an EUROTRANS-BIO grant B01/0558/X13. Mrs. Marta Macchia, Ilaria D’Elia, Federica Pattofatto and Monia Sanza are gratefully acknowledged for helpful assistance in carrying out experiments.
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