Surface conditioning of stainless steel coupons with skim milk,buttermilk, and butter serum solutions and its effect on bacterial adherence

In the dairy industry, dairy by-products such as skim milk, buttermilk and butter serum which possess different specific compositions, could contact with processing surfaces to form conditioning layers and subsequently alter bacterial attachment behavior of the surfaces. In order to simulate and elucidate this process, stainless steel coupons were conditioned with skim milk, buttermilk and butter serum solutions. Formed conditioning layers were examined under a confocal laser scanning microscope (CLSM) and the influence of surface conditioning on bacterial adherence was investigated. The results showed that different conditioning layers were formed by different dairy by-products. The layer formed by skim milk, buttermilk and butter serum was the thinnest, medium and the thickest, respectively. The treatment of stainless steel surfaces with skim milk, buttermilk and butter serum could reduce the adherence of dairy-related bacteria (Lactococcus lactis subsp. lactis NBRC 100933, Leuconostoc mesenteroides subsp. cremoris NBRC 107766 and Lactobacillus casei FIRI 108) at different levels. In the majority of cases, the adherence-reducing ability of buttermilk and butter serum was found better than skim milk. While skim milk could reduce bacterial adherence during shorter exposure time (almost of 30 min), buttermilk and butter serum could act during the longer period (up to 720 min). The result suggested that, bacterial adherence-reducing effect of buttermilk and butter serum may correlate to their substances associated with milk fat globule membrane. In order to decrease bacterial adherence, surface conditioning with skim milk, buttermilk and butter serum is recommended. Surface conditioning with skim milk is suitable for short bacterial exposure time (30 min), for a longer period of time (more than 180 min), only surface conditioning with buttermilk and with butter serum is advisable.     

Adherence kinetics, resistance to benzalkonium chloride and microscopic analysis of mixed biofilms formed by Listeria monocytogenes and Pseudomonas putida

Comparison between the resistance to BAC and the microscopic structure between mixed-species biofilms formed by different strains of Listeria monocytogenes and Pseudomonas putida CECT 845 under different scenarios and that obtained by the corresponding monospecies L. monocytogenes biofilm was carried out. The association of P. putida with L. monocytogenes quickens biofilm formation and increases significantly (p < 0.05) the BAC-resistance of the biofilm after 4 days of incubation at 25 °C respecting to that formed by monospecies biofilms. According with the adherence profiles of P. putida, two different patterns of association between both species (A and B) were identified, being type A pattern found in the mixed biofilms much more resistant to BAC. After 11 days of incubation, a destructuration of mixed biofilms occurred in all experimental assays, being in 2 out of 5 experimental cases (4032 and BAC-adapted 5873 on polypropylene) accompanied by a sharp decrease in the number of adhered cells. Microscopic analyses demonstrated that complex three-dimensional microscopic structure showed the highest resistance to BAC (4032-SS). Obtained results clearly highlight that to improve disinfection protocols for assuring food safety, it is necessary to mimick those bacterial association that occur in nature.     

The sanitizing action of essential oil-based solutions against Salmonella enterica serotype Enteritidis S64 biofilm formation on AISI 304 stainless steel

Bacterial adhesion and biofilm formation by Salmonella enterica serovar Enteritidis is a serious concern in the food processing industry; organism persistence in biofilms represents a continual source of contamination. Due to unsuccessful disinfection processes and emerging resistance, conventional control methods are rapidly becoming ineffective, necessitating the development of new control strategies. The following study evaluated the anti-biofilm effect of disinfectant solutions formulated with essential oils (EOs) of peppermint (Mentha piperita) and lemongrass (Cymbopogon citratus) against biofilm formation by S. enterica serotype Enteritidis S64 on stainless steel surface AISI 304 (#4) after 10, 20 and 40 min of contact. A minimum inhibitory concentration (MIC) of 7.8 μL/mL was found for both EOs and disinfectant solutions were formulated based on these MIC values. Ten minutes of sanitizing solution contact significantly reduced (p ≤ 0.05) adhered bacterial populations for both EOs tested. After 20 and 40 min of treatment, cell counts were not detected. Thus, M. piperita and C. citratus EOs can be considered convenient, quality alternatives to the application of conventional sanitizing agents in the food industry; further, use of these EOs addresses the increasing consumer demand for natural products.     

Removal of Listeria monocytogenes, Staphylococcus aureus and Escherichia coli O157:H7 biofilms on stainless steel using scallop shell powder

Biofilms on steel surfaces containing Listeria monocytogenes, Staphylococcus aureus and Escherichia coli O157:H7 continue to threaten dairy and meat processors. In this study, the ability of scallop shell powder (SSP) to remove biofilms formed by these three pathogens on stainless steel plates was examined. Whey powder solution (WPS) and bench wash water (BWW) provided by dairy and meat factories, respectively, were inoculated with L. monocytogenes, S. aureus or E. coli O157:H7 (9 log₁₀ CFU/ml). Stainless steel plates (10 cm²) were placed in the inoculated fluids and incubated at 20 °C at 48 h to form biofilms. After drying and washing in sterile water, the plates were treated with 0.0, 0.25, or 0.50% (w/v) SSP slurries for 1, 5, or 10 min and then quantitatively examined for the three pathogens. Both 0.25 and 0.50% SSP reduced L. monocytogenes on the plates by 4 log CFU/cm² with a 1 min exposure to 0.50% SSP decreasing S. aureus by 5 logs CFU/cm². After 1 min in 0.25 and 0.50% SSP, E. coli O157:H7 populations in WPS and BWW biofilms decreased 4 and 6 log CFU/cm² and 3 and 5 log CFU/cm², respectively. Increasing the concentration of SSP led to significantly increased efficacy against the tested pathogens (P < 0.05). In conclusion, this study showed that SSP slurries could significantly reduce the numbers of L. monocytogenes, S. aureus and E. coli O157:H7 in biofilms on stainless steel surfaces.     

A comparison of saturated steam and superheated steam for inactivation of Escherichia coli O157:H7, Salmonella Typhimurium,and Listeria monocytogenes biofilms on polyvinyl chloride and stainless steel

This study was performed to compare the effectiveness of saturated steam (SS) and superheated steam (SHS) in the inactivation of Escherichia coli O157:H7, Salmonella Typhimurium, and Listeria monocytogenes biofilms on polyvinyl chloride (PVC) and stainless steel. Biofilms were formed on PVC and stainless steel coupons by using a mixture of three strains each of three foodborne pathogens at 25 °C. After biofilm development, PVC and stainless steel coupons were treated with SS at 100 °C and SHS at 125, 150, 175, and 200 °C for 5, 10, 20, and 30 s on both sides. The viable cell numbers of biofilms were significantly (p < 0.05) reduced as SHS temperature and exposure time increased. For all biofilm cells, SHS treatment resulted in an additional log reduction compared to SS treatments. After exposure to 200 °C steam for 30 s or 10 s on PVC or stainless steel, respectively, the numbers of biofilm cells were reduced to below the detection limit (1.48 log). This study demonstrated that SHS treatment effectively reduced populations of biofilm cells and reduced disinfection time compared to SS treatments and further evaluated its potential as an excellent intervention for controlling microbial biofilms and enhancing safety in the food processing industry.     

Use of epifluorescence microscopy to assess the effectiveness of phage P100 in controlling Listeria monocytogenes biofilms on stainless steel surfaces

One source of foodborne listeriosis is related to a virulent strain established in the food-processing environment. We used direct epifluorescence microscopy (DEM) to evaluate the effectiveness of phage P100 in controlling Listeria monocytogenes biofilms on stainless steel surfaces, in wet conditions at room temperature. Biofilms that were allowed to develop for 72 h were subsequently treated with different concentrations of phage (5, 6, 7, and 8 log PFU/ml). L. monocytogenes was monitored up to 48 h, using both DEM and cultivation method. When L. monocytogenes was monitored using a cultivation method, the first significant reduction was observed after phage treatment with 7 and 8 log PFU/ml at 8 h. Subsequently, these treatments achieved undetectable levels of pathogen at 48 h, with a mean reduction of 5.29 log CFU/cm². Conversely, when samples were evaluated using DEM in treatments with 6, 7, and 8 log PFU/ml, although disaggregation of biofilms could be observed after 8 h, viable cells were still present up to 48 h (maximal reduction 1.5 log units). The phage titer remained stable or increased up to 2.59 log units during the study period. In conclusion, phage P100 may provide an adjuvant measure to control L. monocytogenes biofilm on stainless steel surfaces. However, phage treatment must be used in combination with other hygienization measures to increase efficacy. In this study, DEM was a good tool to quickly and accurately assess the real effect of phage P100 on L. monocytogenes biofilms.     

Influence of temperature and surface kind on biofilm formation by Staphylococcus aureus from food-contact surfaces and sensitivity to sanitizers

This study aimed to assess the adhesion, detachment kinetic and biofilm formation of Staphylococcus aureus isolates from food services surfaces on stainless steel and polypropylene surfaces when cultivated in a vegetable-based broth at 7 and 28 °C, and the efficacy of peracetic acid (30 mg/L) and sodium hypochlorite (250 mg/L) in removing the bacterial cells from the matrix of the preformed biofilm. The isolates adhered over 4 Log cfu/cm² regardless the surface kind and incubation temperature. Cell detachment was around 3 Log cfu/cm² over the first six contacts with agar characterizing a high persistence of cells on the tested surfaces. Number of cells (5-7 Log cfu/cm²) needed for biofilm formation was noted at all experimental systems already after 3 days of incubation. A range of 2.0-3.3 and 1.5 to 2.1 Log cfu/cm² was observed in the reduction of cells in biofilm matrix caused by peracetic acid and sodium hypochlorite, respectively. The isolates of S. aureus revealed high capability to adhere and form biofilm on the tested surfaces in both assayed incubation temperature.     

Changes in resistance of Salmonella Typhimurium biofilms formed under various conditions to industrial sanitizers

The aim of this study was to understand how different factors including biofilm age, attachment surface (stainless steel and acrylic) and various growth conditions such as different pH (pH 6 and 7) and temperature (28, 37 and 42 °C) affected the resistance of Salmonella Typhimurium biofilm against industrial sanitizers. The sanitizers tested were quaternary ammonium compounds (QAC, 200 ppm), mixed peroxyacetic acid/organic acids (PAO, 0.1%) and sodium hypochlorite (chlorine, 50 ppm). It was observed that, for biofilms formed at pH 7–37 °C, chlorine was the most effective sanitizer, followed by QAC and PAO. For all conditions tested, attachment surfaces didn't cause any significant difference in biofilm resistance against sanitizers. Increasing biofilm age led to an increase in resistance to sanitizers, although such effect was growth condition- and sanitizer-dependent. The resistance of biofilm formed on stainless steel at pH 6–37 °C increased with increasing biofilm ages. The effect of temperature and pH on biofilm resistance was dependent on biofilm ages. For 168-h biofilm formed at pH 6, the resistance to all three sanitizers was highest for 37 °C, followed by 28 and 42 °C; while biofilm formed at 37 °C for 168 h, pH 6 condition increased biofilm resistance to QAC and PAO, but not chlorine, compared with pH 7. These results indicate that the resistance of biofilms against sanitizers was dependent on biofilm age, temperature, and pH.     

Rhamnolipid and surfactin: Anti-adhesion/antibiofilm and antimicrobial effects

Biofilm growth in food industrial processing environment increases the probability of product's microbial contamination by spoilage and pathogenic microorganisms. Biosurfactants represent a great strategy to reduce biofilms due to their environmental-friendly nature when compared to synthetic surfactants. In order to evaluate biosurfactants potential on controlling biofilms, surfactin and rhamnolipid were tested against Listeria monocytogenes and Pseudomonas fluorescens. Adhesion/biofilm formation on polystyrene and stainless steel 304 surfaces, and physicochemical parameters were used to analyze and discuss findings. Adhesion was reduced up to 79% and 54% on polystyrene, while biofilm formation was reduced up to 83% and 73% on stainless steel surfaces (rhamnolipids and surfactin, respectively). Surfactants altered surfaces physicochemical characteristics. When added to the culture medium, microbial adhesion was inhibited up to 100%, depending on the product. Biosurfactants showed antimicrobial activity. Results demonstrate good potential of tested biomolecules to decrease adhesion and biofilm formation on food-contact surfaces.     

Adhesion of Staphylococcus aureus on stainless steel treated with three types of milk

Staphylococcus aureus has the ability to adhere and to form biofilm on inert surface such as stainless steel commonly used in food industry. The biofilm formed on the surface of milk processing equipments could be a source of dairy products contamination. This contamination causes a food poisoning. In this paper the S. aureus adhesion on stainless steel treated by three types of milk (ultrahigh-temperature (UHT)-treated milk; UHT skimmed milk, UHT semi-skimmed milk) was investigated.Stainless steel was exposed to three types of milk with a different amount of fat component. Contact angles measurements were used to determine the surface physicochemical properties of substratum treated with the three milk products. The hydrophobicity and electron acceptor properties of stainless steel seem to be decreasing with the amount of fat component present in milk but its electron donor property increase with this component. The ability of S. aureus to adhere to stainless steel treated and untreated with milk was also examined. Treatment with the three types of milk reduces bacterial attachment. On treated substratum, the adhesion extent was affected by the type of milk and consequently by the amount of fat component. The lower and the higher adhesion were obtained when the steel was treated by the UHT semi-skimmed milk and UHT skimmed milk respectively. The correlation between physicochemical properties and S. aureus adhesion show that this latter was controlled by hydrophobicity and electron donor properties.The findings of this work can contribute to develop strategies for prevent S. aureus adhesion on stainless steel and biofilm formation. Also they could be taken into account in cleaning and disinfection procedures.     

Antilisterial activity and stability of nanovesicle-encapsulated antimicrobial peptide P34 in milk

Bacillus sp. P34, a strain isolated from aquatic environments of Brazilian Amazon basin, produces a bacteriocin-like substance (BLS) which was encapsulated in nanovesicles prepared from partially purified soy lecithin. The efficiency of free and encapsulated BLS P34 to control the development of L. monocytogenes and maintenance of antimicrobial activity was assessed over time in milk. The antimicrobial activity of free and encapsulated BLS P34 decreased approximately 50% after 4 days of storage (<4 °C) in skim and whole milk. After this period there was not significant loss of activity up to 21 days. The viable counts of Listeria monocytogenes in skim and whole milk containing 3200 AU/ml of free or encapsulated BLS P34 were always lower than those observed in controls without bacteriocin at both 30 °C and 7 °C. At 1600 AU/ml concentration, free and encapsulated BLS P34 were inhibitory to L. monocytogenes in skim milk, when compared with the control at 7 days. Nanovesicle-encapsulated and free BLS P34 shows potential use as biopreservative for application in milk-derived products.     

Antibiotic resistance in Campylobacter jejuni and Campylobacter coli isolated from food in Poland

This study presents the results of investigations on the susceptibility of Campylobacter spp. strains isolated from chicken meat and giblets to fluorochinolones (ciprofloxacin), macrolides (erythromycin), tetracyclines (tetracycline) and aminoglycosides (gentamicin) andV an analysis of the molecular mechanisms of resistance to the selected antibiotics.Between January 2008 and December 2009 a total of 218 samples of chicken meat and giblets from retail trade in Poland were examined. Campylobacter bacteria were found in 143 samples, that is in 65.6% of the total number embraced by the study. Campylobacter coli was the most ubiquitous - its presence was determined in 108 samples out of 143 (75.5%), whereas Campylobacter jejuni was found in 35 of the contaminated samples (24.5%).The results obtained point to the high percentage (97.9%) of Campylobacter isolates resistant to ciprofloxacin. 92 strains (64.3%) were resistant to tetracycline, 14 (9.1%) to erythromycin and only 9 (6.3%) to gentamicin. Moreover, ten out of the 143 resistant Campylobacter strains (7.0%) were found to be resistant to at least three unrelated antibiotics.     

Substratum attachment location and biofilm formation by Bacillus cereus strains isolated from different sources: Effect on total biomass production and sporulation in different growth conditions

Bacillus cereus, an endospore forming human pathogen associated with foodborne diseases, can form biofilms and attach to surfaces of processing equipment in the food industry. It is a consistent source of contamination and/or cross contamination of processed food products. The objective of this study was to understand substratum attachment location and biofilm formation behavior of B. cereus strains under different growth conditions. A total of 60 strains isolated from food, human, or farm and a number of reference strains were used in this study. Substratum attachment locations of these strains in 96-well microtiter plates were highly diversified among these strains. Strains isolated from food showed higher preference to attach at the air-liquid interface during early stage of biofilm formation. To the best of our knowledge, this is the first report showing the level of substratum attachment location and biofilm formation of B. cereus strains isolated from different sources. Substratum properties did not affect biofilm formation location when a number of selected strains were grown on stainless steel coupon, indicating that biofilm formation location might be independent of the type of substratum. Substratum attachment location and biofilm formation related phenotypes such as total biomass production, number of sessile cells, and sporulation were closely correlated. Substratum attachment location and sporulation behavior were strongly affected during biofilm formation under nutrient stress condition. The number of spores was significantly increased in biofilms grown under nutrient stress condition even though total biomass formation was lower. Our results on substratum attachment location and related biofilm formation behavior are substantially important for food industries where different surfaces are prone to B. cereus attachment, particularly for setting up and implementing clean in place (CIP) protocols.     

Biofilm formation by Salmonella Typhimurium and Staphylococcus aureus on stainless steel under either mono- or dual-species multi-strain conditions and resistance of sessile communities to sub-lethal chemical disinfection

Intercellular interactions encountered within and between different bacterial species are believed to play key roles in both biofilm formation and antimicrobial resistance. In this study, Salmonella Typhimurium and Staphylococcus aureus (3 strains per species) were left to form biofilms on stainless steel coupons incubated at 20 °C for 144 h (i.e. 6 days), in periodically renewable growth medium, under either mono- or dual-species conditions. Subsequently, the developed sessile communities were exposed for 6 min to sub-lethal concentrations of: (i) benzalkonium chloride (BC, 50 ppm), (ii) sodium hypochlorite (NaClO, 10 ppm), or (iii) peroxyacetic acid (PAA, 10 ppm). The dominance of each strain in the mono- and dual-species biofilm communities, both before and after disinfection, was monitored by pulsed field gel electrophoresis (PFGE). Results revealed that dual-species conditions led to a significant (ca. 10-fold) reduction in the number of sessile cells for both species, compared to mono-species ones, with interspecies interactions however found to not exert any significant effect on the disinfection resistance of each species as a whole. However, PFGE analysis revealed that the different strains here employed behaved differently with regard to biofilm formation and disinfection resistance, with this effect to be also strongly dependent on the culture conditions (mono-/dual-species) and the disinfectant applied. Such results expand our knowledge on multi-species biofilms formed by foodborne pathogenic bacteria and could hopefully be helpful in our efforts to develop effective elimination strategies and thus improve food safety.     

Production of biofilm by Listeria monocytogenes in different materials and temperatures

Listeria monocytogenes, considered as one of the most important foodborne pathogens, is easily found on surfaces, particularly in the form of a biofilm. Biofilms are aggregates of cells that facilitate the persistence of these pathogens in food processing environments conferring resistance to the processes of cleaning and may cause contamination of food during processing, thus, representing a danger to public health. Little is known about the dynamics of the formation and regulation of biofilm production in L. monocytogenes, but several authors reported that the luxS gene may be a precursor in this process. In addition, the product of the inlA gene is responsible for facilitating the entry of the microorganism into epithelial cells that express the receptor E-cadherin, also participates in surface attachment. Thus, 32 strains of L. monocytogenes isolated from different foods (milk and vegetables) and from food processing environments were analyzed for the presence of these genes and their ability to form biofilms on three different surfaces often used in the food industry and retail (polystyrene, glass and stainless steel) at different temperatures (4, 20 and 30 °C). All strains had the ilnA gene and 25 out of 32 strains (78.1%) were positive for the presence of the luxS gene, but all strains produced biofilm in at least one of the temperatures and materials tested. This suggests that genes in addition to luxS may participate in this process, but were not the decisive factors for biofilm formation. The bacteria adhered better to hydrophilic surfaces (stainless steel and glass) than to hydrophobic ones (polystyrene), since at 20 °C for 24 h, 30 (93.8%) and 26 (81.3%) produced biofilm in stainless steel and glass, respectively, and just 2 (6.2%) in polystyrene. The incubation time seemed to be an important factor in the process of biofilm formation, mainly at 35 °C for 48 h, because the results showed a decrease from 30 (93.8%) to 20 (62.5%) and from 27 (84.4%) to 12 (37.5%), on stainless steel and glass, respectively, although this was not significant (p = 0.3847). We conclude that L. monocytogenes is capable of forming biofilm on different surfaces independent of temperature, but the surface composition may be important factor for a faster development of biofilm.     

Effect of neutral electrolyzed water on lux-tagged Listeria monocytogenes EGDe biofilms adhered to stainless steel and visualization with destructive and non-destructive microscopy techniques

The presence of Listeria monocytogenes in food processing environment is a risk of food contamination by persistent cells due to their ability to attach to stainless steel and other surfaces. We aimed to study biofilms formation of lux-tagged L. monocytogenes EGDe on stainless steel surfaces and their control using neutral electrolyzed water (NEW), where biofilms development was monitored using destructive and non-destructive microscopy techniques. The development of biofilms was monitored for 5 days on stainless steel chips. We used two sources of NEW, commercial (NEW-1) and from a prototype (NEW-2) for treatments of free and biofilm L. monocytogenes EGDe cells. Complete inhibition of L. monocytogenes EGDe free cells was observed after 1 min contact time for both NEW sources, but NEW-1 concentration used (9 mg/L total available chlorine, TAC) was 1.8 times higher. Cells within biofilms were more resistant to NEW compared to planktonic cells. Same concentration of both NEW sources (70 mg/L TAC) exhibited complete inhibition of biofilm cells after 3 min contact time. However, using a sub-lethal dose of 40 mg/L TAC, NEW-2 reduced about 2 log CFU/cm² biofilm cells while NEW-1 inhibited 0.3 log CFU/cm² only. Biofilms formation and antagonistic effect of NEW could be visualized by epifluorescence and scanning electron microscopy, revealing significant biofilms structure. The disinfectant effect of NEW may be attributed to the combined antimicrobial effect of available chlorine and high ORP exhibited by its oxidizing compounds. NEW does not promote metal equipment corrosion due to its neutral pH, and is also environmentally friendly.     

Non starter lactic acid bacteria biofilms: A means to control the growth of Listeria monocytogenes in soft cheese

The possibility to consider non starter lactic acid bacteria (NSLAB) biofilms as a means to control the growth of Listeria monocytogenes in soft cheeses was evaluated. In particular, the aptitude to form biofilm of four NSLAB strains (Lactobacillus plantarum DSM1055, Lactobacillus casei DSM20011, Lactobacillus curvatus DSM20019 and Lactobacillus paracasei DSM20207) was investigated. All tested strains were able to form biofilm on stainless steel and the highest quantities were produced when NSLAB were present simultaneously (about 6 Log CFU cm^?2). Then, experimental cheeses were made in presence of chips containing 7-days NSLAB biofilms and inoculated with L. monocytogenes (about 2 Log CFU g^?1). Results demonstrated that NSLAB biofilms can be considered a useful means to delay the growth of L. monocytogenes in soft cheeses: the maximum cell load attained at the stationary phase was about 6 Log CFU g^?1 in experimental cheeses against about 7 Log CFU g^?1 observed in control samples.     

Mechanism of the combined anti-bacterial effect of green tea extract and NaCl against Staphylococcus aureus and Escherichia coli O157:H7

The mechanism of the combined anti-bacterial effect of green tea extract (GTE) and NaCl against Staphylococcus aureus NBRC 13276 and Escherichia coli O157:H7 was investigated. After treatment for 1 h, GTE was more effective against S. aureus than E. coli O157:H7, and combined GTE/NaCl treatment caused greater cellular damage in S. aureus NBRC 13276, where it was bactericidal, than E. coli O157:H7. Compared to treatment with 1.0 mg/mL GTE, which had no effect on the survival rate of E. coli O157:H7 after 48 h, treatment with 4% NaCl alone caused greater cellular damage. Moreover, bacteria pretreated with NaCl showed delayed growth in the presence of GTE. It is therefore likely that susceptibility of E. coli O157:H7 to GTE was increased by exposure to NaCl. E. coli O157:H7 pretreated with GTE and NaCl did not multiply in the presence of GTE. Visualization of the catechin components of GTE-treated bacteria using an electron microscope and SDS-PAGE analysis of cell proteins showed that GTE attached to proteins on the surface of the bacteria to form high-molecular weight complexes, suggesting the possibility that GTE inhibits the uptake and secretion of substrates and inhibits enzyme activity. Notably, after the GTE treatment for 1 h, both bacterial strains suffered injury but recovered by cultivation in rich medium. The damage and aggregation of proteins caused by GTE treatment were repaired upon treatment with LP diluent.     

Seasonal prevalence of Vibrio species in retail shrimps with an emphasis on Vibrio parahaemolyticus

Seasonal prevalence of Vibrio species in shrimp samples from retail outlets in the South-western part of Iran was studied. A total of 300 samples were analyzed (75 samples in each season). Special attention was paid to the prevalence of total and pathogenic Vibrio parahaemolyticus. All the TCBS isolates were first identified to the genus level with PCR and then identified to the species level using a battery of biochemical reactions and tests. To investigate the pathogenicity of the isolated V. parahaemolyticus, multiplex PCR (tl, tdh and trh genes) was performed. Vibrios were detected during the whole investigation period, depending on the sampling season. They were detected in 18.6% of the winter samples, 64% of the spring samples, 70.6% of the summer samples and 41.3% of the autumn samples. Vibrio calviensis and Vibrio alginolyticus were dominant in samples of different seasons, with the average prevalence of 18.6% and 17.6%, respectively. V. parahaemolyticus was found in 4.0% of the winter samples, 13.3% of the spring samples, 18.6% of the summer samples and 8% of the autumn samples. During the period of this study, two tdh-positive strains were isolated, while no trh-positive V. parahaemolyticus strain was detected in samples of different seasons.     

Direct detection of Salmonella without pre-enrichment in milk, ice-cream and fruit juice by PCR against hilA gene

A novel PCR based assay was devised to specifically detect contamination of any Salmonella serovar in milk, fruit juice and ice-cream without pre-enrichment. This method utilizes primers against hilA gene which is conserved in all Salmonella serovars and absent from the close relatives of Salmonella. An optimized protocol, in terms time and money, is provided for the reduction of PCR contaminants from milk, ice-cream and juice through the use of routine laboratory chemicals. The simplicity, efficiency (time taken 3-4 h) and sensitivity (to about 5-10 CFU/ml) of this technique confers a unique advantage over other previously used time consuming detection techniques. This technique does not involve pre-enrichment of the samples or extensive sample processing, which was a pre-requisite in most of the other reported studies. Hence, this assay can be ideal for adoption, after further fine tuning, by food quality control for timely detection of Salmonella contamination as well as other food-borne pathogens (with species specific primers) in food especially milk, ice-cream and fruit juice.