The detection of viable vegetative cells of Bacillus sporothermodurans using propidium monoazide with semi-nested PCR

Bacillus sporothermodurans produces highly heat-resistant spores that can survive ultra-high temperature (UHT) treatment in milk. Therefore, we developed a rapid, specific and sensitive semi-nested touchdown PCR assay combined with propidium monoazide (PMA) treatment for the detection of viable B. sporothermodurans vegetative cells. The semi-nested touchdown PCR alone proved to be specific for B. sporothermodurans, and the achieved detection limit was 4 CFU/mL from bacterial culture and artificially contaminated UHT milk. This method combined with PMA treatment was shown to amplify DNA specifically from viable cells and presented a detection limit of 10² CFU/mL in UHT milk. The developed PMA-PCR assay shows applicability for the specific detection of viable cells of B. sporothermodurans from UHT milk. This method is of special significance for applications in the food industry by reducing the time required for the analysis of milk and dairy products for the presence of this microorganism.     

Accurate quantification of thermophilic spores in dairy powders

Internationally, there are no official guidelines for the quantification of thermophilic spores in dairy products, which leads to variations in applied methodology. In this study, we assess the heat sensitivity of thermophilic spores, vegetative cells grown under laboratory conditions and spores in German dairy powders to determine appropriate heating conditions for accurate quantification of total thermophilic spores. The heat inactivation effect (80–95 °C) is limited for spores of Anoxybacillus flavithermus and Geobacillus stearothermophilus grown under laboratory conditions. However, for spores originating from whey, whey powder and skimmed milk powder (mostly identified as A. flavithermus), a different trend was observed; spore counts continuously reduced when heating time and temperature increased (80–98 °C, 10–30 min). The results indicate that data obtained using laboratory cultures cannot be extrapolated to commercial powders, and in this case, applying temperatures above 80 °C leads to an underestimation of spore counts in dairy powders.     

Simple membrane filtration method for estimating numbers of Paenibacillus spp. spores in raw milk,using β-galactosidase activity as a selection criterion

Paenibacillus spp. are spore-forming bacteria that adversely affect the quality of dairy products. There is currently no appropriate method for enumerating Paenibacillus spp. spores. We developed a simple membrane filtration method to enumerate Paenibacillus spp. spores in raw milk, using β-galactosidase activity as a selection criterion. Although Paenibacillus spp. spores are relatively small, use of a membrane filter with 0.65-μm pore size allowed us to easily filter raw milk with sufficient recovery. The membrane was put on plates containing X-gal, and detection of β-galactosidase-positive colonies enabled selective enumeration of Paenibacillus spp. spores. We investigated Paenibacillus spp. spore levels in raw milk from six different areas in the Tokachi region, Hokkaido, Japan over 1 year. There were ≤10 spores 100 mL⁻¹ throughout the year, with no significant differences between areas or seasons. Paenibacillus amylolyticus and Paenibacillus odorifer were the predominant species, accounting for 50.6% and 27.4% of the total spores, respectively.     

Concurrence of spores of Clostridium tyrobutyricum,Clostridium beijerinckii and Paenibacillus polymyxa in silage,dairy cow faeces and raw milk

Spores of lactate-fermenting clostridia, known as butyric acid bacteria (BAB), can cause severe quality defects in semi-hard cheeses, called late-blowing. The routine detection method of BAB spores involves most probable number quantification of spores of anaerobic, gas-forming bacteria in a medium containing lactic acid. In this study, BAB spores were detected in 296 samples of soil, maize and grass silage, dairy cow faeces and farm tank milk collected from dairy farms in The Netherlands and the most abundant populations identified. Three major populations were detected, namely Clostridium tyrobutyricum, Clostridium beijerinckii and Paenibacillus spp. Paenibacillus polymyxa was the most abundant species of the latter group. The results indicate that the three spore populations share the same sources and the same contamination route of milk. In contrast to the Clostridium species, P. polymyxa isolates were unable to ferment lactate into butyrate. P. polymyxa spores are presumably unable to cause cheese defects.     

Preconditioning of the stainless steel surface affects the adhesion of Bacillus cereus spores

The adhesion of Bacillus cereus on stainless steel, with and without prior conditioning of the surface (water, skimmed milk, and whole milk) was evaluated. Inocula consisting of a pool of spores of four different B. cereus strains isolated from the dairy industry, and spores of B. cereus ATCC 14579 were used. The pool of B. cereus spores adhered in all conditions evaluated. Higher adhesion of B. cereus spores (4.93 log cfu cm⁻²) was observed when using whole milk as conditioning matrix. However, without prior conditioning, lower adhesion was observed (3.01 log cfu cm⁻²) when the pool of B. cereus spores was inoculated on whole milk, suggesting the interaction between milk fat and microorganism on the stainless steel. The pool of B. cereus spores showed higher adhesion to the surface, possibly due to its greater hydrophobicity (66%) when compared with the B. cereus ATCC 14579 spores (47%).     

Bacillus thermoamylovorans – A new threat to the dairy industry – A review

Bacillus thermoamylovorans is a newly described spore forming bacterium emerging in the dairy industry. There is an increasing threat of B. thermoamylovorans contamination, not only in food ingredients and raw milk, but also in sterilised milk and dairy products. Animal feed, processing equipment, and farm environments are potential entry points for contamination of milk with B. thermoamylovorans. The ability of B. thermoamylovorans to produce lipolytic and β-galactosidase enzymes is linked to food spoilage. B. thermoamylovorans spores have a heat resistance comparable with that of the spores of Bacillus sporothermodurans, indicating the potential to survive ultra-high temperature treatments. However, despite the rise in reports of the isolation of B. thermoamylovorans from dairy samples, there have been few reports on the characterisation of these bacteria. The origin, characteristics and potential issues resulting from B. thermoamylovorans in the dairy industry and potential methods of control are discussed in this review.     

Fast detection and quantification of four dairy propionic acid bacteria in milk samples using real-time quantitative polymerase chain reaction

Propionibacterium freudenreichii is added to vat milk to create the characteristic eyes and typical nutty flavour of Emmentaler Protected Designation of Origin (PDO) cheese, but leads to serious quality defects in other raw milk cheeses from Switzerland. To trace propionic acid bacteria (PAB) in raw milk, we developed and validated a fast quantitative polymerase chain reaction (qPCR)-based method for P. freudenreichii, Propionibacterium thoenii, Propionibacterium jensenii, and Propionibacterium acidipropionici. qPCR-standard curves were linear over five log units down to 10¹ copies per reaction (R ≥ 0.997); efficiencies ranged from 0.83 to 0.97. In spiking experiments, the lower limits of quantification were 10¹–10² cfu mL⁻¹ raw milk. Fifty one vat milk samples were analysed with plate count method and qPCR in parallel. Compared with the classic plate count method, the newly developed qPCR method gave faster and species specific determination of four dairy PAB in milk and yielded comparable quantitative results.     

Quantitative Detection of <Emphasis Type="Italic">Clostridium perfringens</Emphasis> by Real-Time PCR in Raw Milk

Clostridium perfringens causes a broad spectrum of diseases in both humans and animals and is an important cause of foodborne illness. We developed and tested a real-time (Q-)PCR assay for the species-specific detection of C. perfringens that targets the phospholipase C (plc) gene and includes an internal amplification control (IAC), making it possible to identify false-negative results, which are common due to the high level of PCR inhibition by food compounds. The CPplc-IAC real-time PCR (RTi-PCR) assay was 100% selective, as shows with 36 Clostridium strains and 85 non-Clostridium strains, with an analytical sensitivity of 1 genome equivalent (GE) in 23% of the reactions and 10 GE in 100% of the reactions. The quantification was linear (R ² = 0.9990) over a 7-log dynamic range, down to 10 GE, with a PCR efficiency E = 0.841. The applicability of this RTi-PCR assay was assessed in milk samples. The assay detected as few as 300 spores in 25 mL of artificially contaminated raw sheep milk with 78% probability and 30 spores in 25 mL with 50% probability. It also has accuracy of 83.03 to 151.18%, as shown by an evaluation of the correspondence between RTi-PCR assay results and the number of spores per milliliter determinated by standard plating. This RTi-PCR method was effective for the detection and quantification of C. perfringens in milk having an important applicability in the control of this pathogen in the dairy food industry.     

Cycling versus Continuous High Pressure treatments at moderate temperatures: Effect on bacterial spores?

The advantage of using high pressure (HP) cycling treatment compared with continuous HP treatment was investigated for the inactivation of bacterial spores. The effects of parameters such as pulse number, pressure level, treatment temperature, compression and decompression rates, and time between pulses were evaluated. For this purpose, Bacillus subtilis and B. cereus spores (10⁸ and 10⁶ CFU/mL respectively) were suspended in 2-(N-morpholino) ethanesulfonic acid (MES) buffer solution, tryptone salt (TS) buffer solution, or infant milk and treated by HP cycling at 300–400 MPa, at 38–60 °C, for 1–5 pulses. Pressure cycling reduced the number of viable spores by 1.8 and 5.9 log respectively for B. subtilis and B. cereus species. Continuous HP treatments were performed at the same pressure and temperature for similar treatment durations. Our results showed that the spore inactivation ratio was correlated with the cumulative exposure time to pressure rather than to effects of the cycling process. Greater spore inactivation caused by HP cycling was observed only when faster compression and decompression rates were applied, probably due to adiabatic heating. A three-step kinetic model was developed, which seemed to support our hypothesis regarding the mechanisms of inactivation by pressure cycling and continuous HP treatments.Industrial relevanceThe resistance of bacterial spores to HP limits the industrial applications to refrigerated food products. In this study, we investigated the use of pressure cycling as a means to improve spore baroinactivation at moderate temperatures (T < 60 °C). We showed that cycling pressure does not significantly increase bacterial spore inactivation in comparable treatment duration, but certainly increases material fatigue in HP vessels. Thus, under moderate temperature, cycling pressure treatment is not industrially relevant.     

Prevalence and comparison of Streptococcus infantarius subsp. infantarius and Streptococcus gallolyticus subsp. macedonicus in raw and fermented dairy products from East and West Africa

Streptococcus infantarius subsp. infantarius (Sii) and Streptococcus gallolyticus subsp. macedonicus are members of the Streptococcus bovis/Streptococcus equinus complex (SBSEC) associated with human infections. SBSEC-related endocarditis was furthermore associated with rural residency in Southern Europe. SBSEC members are increasingly isolated as predominant species from fermented dairy products in Europe, Asia and Africa. African variants of Sii displayed dairy adaptations to lactose metabolism paralleling those of Streptococcus thermophilus including genome decay. In this study, the aim was to assess the prevalence of Sii and possibly other SBSEC members in dairy products of East and West Africa in order to identify their habitat, estimate their importance in dairy fermentation processes and determine geographic areas affected by this potential health risk. Presumptive SBSEC members were isolated on semi-selective M17 and SM agar media. Subsequent genotypic identification of isolates was based on rep-PCR fingerprinting and SBSEC-specific16S rRNA gene PCR assay. Detailed identification was achieved through application of novel primers enhancing the binding stringency in partial groES/groEL gene amplification and subsequent DNA sequencing. The presence of S. thermophilus-like lacS and lacZ genes in the SBSEC isolates was determined to elucidate the prevalence of this dairy adaptation. Isolates (n = 754) were obtained from 72 raw and 95 fermented milk samples from Côte d'Ivoire and Kenya on semi-selective agar media. Colonies of Sii were not detected from raw milk despite high microbial titers of approximately 10⁶ CFU/mL on M17 agar medium. However, after spontaneous milk fermentation Sii was genotypically identified in 94.1% of Kenyan samples and 60.8% of Kenyan isolates. Sii prevalence in Côte d'Ivoire displayed seasonal variations in samples from 32.3% (June) to 40.0% (Dec/Jan) and isolates from 20.5% (June) to 27.7% (Dec/Jan) present at titers of 10⁶–10⁸ CFU/mL. lacS and lacZ genes were detected in all Kenyan and 25.8% (June) to 65.4% (Dec/Jan) of Ivorian Sii isolates. Regional differences in prevalence of Sii and dairy adaptations were observed, but no clear effect of dairy animal, fermentation procedure and climate was revealed. Conclusively, the high prevalence of Sii in Kenya, Côte d'Ivoire in addition to Somalia, Sudan and Mali strongly indicates a pivotal role of Sii in traditional African dairy fermentations potentially paralleling that of typical western dairy species S. thermophilus. Putative health risks associated with the consumption of high amounts of live Sii and potential different degrees of evolutionary adaptation or ecological colonization require further epidemiologic and genomic investigations, particularly in Africa.     

Persistence strategies of Bacillus cereus spores isolated from dairy silo tanks

Survival of Bacillus cereus spores of dairy silo tank origin was investigated under conditions simulating those in operational dairy silos. Twenty-three strains were selected to represent all B. cereus isolates (n = 457) with genotypes (RAPD-PCR) that frequently colonised the silo tanks of at least two of the sampled eight dairies. The spores were studied for survival when immersed in liquids used for cleaning-in-place (1.0% sodium hydroxide at pH 13.1, 75 °C; 0.9% nitric acid at pH 0.8, 65 °C), for adhesion onto nonliving surfaces at 4 °C and for germination and biofilm formation in milk. Four groups with different strategies for survival were identified. First, high survival (log 15 min kill ≤1.5) in the hot-alkaline wash liquid. Second, efficient adherence of the spores to stainless steel from cold water. Third, a cereulide producing group with spores characterised by slow germination in rich medium and well preserved viability when exposed to heating at 90 °C. Fourth, spores capable of germinating at 8 °C and possessing the cspA gene. There were indications that spores highly resistant to hot 1% sodium hydroxide may be effectively inactivated by hot 0.9% nitric acid. Eight out of the 14 dairy silo tank isolates possessing hot-alkali resistant spores were capable of germinating and forming biofilm in whole milk, not previously reported for B. cereus.     

High-throughput detection of spore contamination in food packages and food powders using tiered approach of ATP bioluminescence and real-time PCR

A rapid and quantitative method for detection of Bacillus spores in food/non-alcoholic beverage packages and food powders has been developed using filtration-based ATP bioluminescence and real-time PCR, targeting the sporulation gene (spo0A). In combination with heat activation, the presence and amount of viable bacterial spores (i.e., Bacillus amyloliquefaciens, Bacillus licheniformis, and Bacillus thuringiensis) was determined within 20 min through ATP signal amplifications. The detection limits of heat activation-ATP bioluminescence assay for B. amyloliquefaciens and B. licheniformis spores on food packages were 1.4 × 10² and 1.0 × 10³ CFU/cm², respectively. In contaminated food powders, B. thuringiensis spores could be detected by the ATP assay within the range of 7.9 × 10⁰ to 3.2 × 10⁴ CFU/mg powder while the PCR detection limit was 614 CFU/mg. Linear relationships between luminescent signal (RLU/mg) and plate count (CFU/mg) were found. The same sample after heat activation-ATP assay could be directly used for real-time PCR as a streamlined detection to confirm the identity of Bacillus spores in food packages and food powders even though some bacterial DNA loss was observed. This tiered approach, filtration-based one-tube ATP luminescence method as a rapid, viable screening and using real-time PCR as confirmation, could serve as a high-throughput tool for the detection of Bacillus spores in the food and beverage industry.     

Detection and enumeration of Lactobacillus helveticus in dairy products

Lactobacillus helveticus, a lactic acid bacterium, is an important species in food fermentation, e.g., cheesemaking, and is considered beneficial to human health. We developed a quantitative real-time polymerase chain reaction (qPCR) method for the detection and quantification of L. helveticus in dairy products. The method uses a set of target-specific PCR primers and a fluorogenic probe and amplifies a part of the pheS gene that encodes the alpha subunit of the phenyalanine-tRNA synthetase. All 24 L. helveticus strains tested were qPCR positive; no signal was observed for 23 strains belonging to closely related species. The limit of detection was ten copies per reaction and the assay covered a linear dynamic range of eight logs. The method was used to detect and enumerate L. helveticus in milk and cheese during ripening; therefore it can be used to study the temporal and spatial distribution of L. helveticus during cheese manufacturing and ripening.     

Effect of thermosonication in a batch system on the survival of spore‐forming bacteria

Thermosonication may help reduce bacteria counts responsible for spoilage in dairy products. Vegetative cells and spores of Geobacillus stearothermophilus, Anoxybacillus flavithermus and Bacillus subtilis (spores only) were treated with either heat alone or thermosonication in a batch system from 0 to 120 s in tryptic soy broth and 2% fat milk at 72 and 73 °C. D‐values for vegetative cells were calculated and were reduced after thermosonication. Maximum reduction in vegetative cells after thermosonication was 1 log after 30–45 s and for spores was ≤0.2 log after 120 s, which may not influence dairy product quality in scale‐up systems.     

Effects of pulsed electric field on fat globule structure,lipase activity,and fatty acid composition in raw milk and milk with different fat globule sizes

To modify the structure of milk fat globules (MFGs), raw milk (RM) with native MFGs, and milk with small (MS) and large (ML) MFGs, were subjected to moderate pulsed electric field (PEF) treatment at 9 and 16 kV·cm⁻¹ for 30 μs. The changes caused by PEF on MFG structure leading to MFG-MFG and MFG-protein aggregation were found to depend on the size, composition and/or previous damage caused by the separation and mixing processes during production of MS and ML, in addition to the intensity of the PEF treatment. Although, PEF treatment had a more pronounced effect on MFGM proteins of MS, these MFG were less affected by PEF due to significant adsorption of milk proteins. The structural changes caused by PEF were accompanied by changes in lipase activity and fatty acid (FA) profiles, i.e. increase in the proportion of C6:0, C8:0, C10 and some long-chain FAs and a decrease in medium and other long chain FAs.Industrial relevancePEF is gaining attention in the dairy industry. However, information regarding the effect of PEF processing on milk components and functionality is still limited. The present investigation contributes to understand the potential of PEF at moderate field intensities to modify the MFGs and consequences for milk stability, lipolysis and composition of FAs. The new insight is relevant in development of new products and ingredients containing milk fat.     

Inactivation of Bacillus cereus spores using a combined treatment of UV-TiO2 photocatalysis and high hydrostatic pressure

Bacillus cereus spores are resistant to high hydrostatic pressure (HHP) processing treatment. A combination of UV-TiO₂ photocatalysis (UVTP for 10 min) and two cycles of 600 MPa HHP treatment for 10 min for the first cycle and 1 min for the second cycle (UVTP-2HHP) at ambient temperature was applied to inactivate B. cereus spores inoculated on a solidified agar matrix (SAM) used as a model matrix. Two cycles of HHP treatment were used as a strategy for induction of spore germination, followed by inactivation. UVTP and 2 cycles of HHP resulted in a 5.0-log CFU/cm² spore reduction (initial spore count was 6.6 log CFU/cm²), including an approximate 0.8-log CFU/cm² reduction due to a synergistic effect. The inactivation mechanism of UVTP pretreatment was related to lipid peroxidation of the spore membrane based on the level of malondialdehyde (MDA) making spores susceptible to the HHP treatment. Flow cytometry and transmission electron microscopic (TEM) analyses showed severe physiological alteration and structural damage to spores after the combined treatment. UVTP and 2 cycles of HHP showed potential for effective inactivation of B. cereus to ensure food safety from B. cereus spores on food products.Practical applicationsInactivation of bacterial spores remains a technical challenge for HHP and other interventions because spores are highly resistant to high pressure. Pretreatment with UVTP followed by two cycles of HHP resulted in reduction in B. cereus spores due to a synergistic effect. This hurdle technology of UVTP and HHP can help food industry in ensuring food safety against the Bacillus spores.     

Optimization of nutrient-induced germination of Bacillus sporothermodurans spores using response surface methodology

Spores of Bacillus sporothermodurans are known to be contaminant of dairy products and to be extremely heat-resistant. The induction of endospore germination before a heat treatment could be an efficient method to inactivate these bacteria and ensure milk stability. In this study, the nutrient-induced germination of B. sporothermodurans LTIS27 spores was studied. Testing the effect of 23 nutrient elements to trigger an important germination rate of B. sporothermodurans spores, only d-glucose, l-alanine, and inosine were considered as strong independent germinants. Both inosine and l-alanine play major roles as co-germinants with several other amino acids. A central composite experimental design with three factors (l-alanine, d-glucose, and temperature) using response surface methodology was used to optimize the nutrient-induced germination. The optimal rate of nutrient-induced germination (100%) of B. sporothermodurans spores was obtained after incubation of spore for 60 min at 35 °C in presence of 9 and 60 mM of d-glucose and l-alanine, respectively. The results in this study can help to predict the effect of environmental factors and nutrients on spore germination, which will be beneficial for screening of B. sporothermodurans in milk after induction their germination. Moreover, the chosen method of optimization of the nutrient-induced germination was efficient in finding the optimum values of three factors.     

Occurrence of β-casomorphins 5 and 7 in commercial dairy products and in their digests following in vitro simulated gastro-intestinal digestion

The occurrence of β-casomorphin-5 (BCM5) and β-casomorphin-7 (BCM7) was investigated in commercial dairy products and in their digests, following in vitro simulated gastro-intestinal digestion (SGID), by means of HPLC–MS. The analysed dairy products were as follows: 10 cheeses (Gorgonzola, Caprino, Brie, Taleggio, Gouda, Fontina, Cheddar and Grana Padano 10-, 15- or 25-m ripened); 4 samples of drinking milk (unprocessed, pasteurised, UHT and in bottle-sterilised); 2 yoghurts and 4 fermented milks containing probiotics; 7 infant formulas; and 4 dried milk-derivatives (skim milk powder, calcium caseinate and milk protein concentrates). β-Casomorphin-5 was not detected in dairy products, either prior to or after SGID. β-Casomorphin-7 was detected only in cheeses with the exception of Taleggio, Caprino and Grana Padano samples. Peptide amount was in the range 0.01–0.15 mg kg⁻¹ the highest level being recovered in Brie sample. Following SGID, BCM7 formed in all dairy samples or increased up to 21.77 mg kg⁻¹ in digests of cheeses. The peptide level ranged from 0.29 to 1.23 mg kg⁻¹in fermented milks and from 3.46 to 22.18 mg kg⁻¹ in dried milk-derivatives. Digests of commercial infant formulas contained BCM7 at concentrations of 0.04–0.21 mg l⁻¹. For the first time, this work reports quantitative values for BCM5 and BCM7 in a range of dairy products providing evidence that, during processing, only proteolytic systems involved in manufacturing and ripening of cheese can potentially hydrolyse β-CN to BCM7. Nevertheless, formation or further release of BCM7 is mainly promoted by the action of gastrointestinal proteinases during in vitro digestion irrespective of the type of dairy product.     

Comparison between novel and standard methods for analysis of free fatty acids in milk – Including relation to rancid flavour

A range of analytic methods for quantifying free fatty acids (FFAs) in milk were compared and further correlated to rancid flavour detected by sensory analysis. Proton transfer reaction–mass spectrometry (PTR–MS) and ¹H nuclear magnetic resonance (NMR) spectrometry were introduced as novel methods for quantifying individual short chain FFAs. Gas chromatography–mass spectrometry (GC–MS) combined with in-solution derivatisation was used for quantifying all individual FFAs, and Bureau of dairy industries (BDI), acyl-CoA-synthetase–acyl-CoA oxidase (ACS–ACOD) assay, Fourier transform infrared spectroscopy were used to estimate the total FFA concentration in 28 milk samples. High correlations (r between 0.76 and 0.86) to rancid off-flavour were found for all analytical methods, and all the individual FFAs were quantitatively inter-correlated. Furthermore, a high correlation was found between PTR–MS and GC–MS quantification of hexanoic acid. For the BDI method, the sensory thresholds rancid off-flavour in farm bulk milk were 1.95 mmol 100 g⁻¹ fat.     

A simple and rapid realtime PCR assay for the detection of Shigella and Escherichia coli species in raw milk

Escherichia coli and Shigella spp. are two major bacterial contaminants in raw milk. Effective screening of the two microbes before starting the production process is a critical step for the quality and safety guarantee of the resulting dairy products. This study reported a rapid and simple realtime PCR assay using a ubiquitous primer and probe set targeting the tuf gene for the detection of E. coli and Shigella spp. An internal amplification control (IAC) was also comprised to indicate false-negative results. The duplex realtime PCR assay showed a high efficiency above 96 % and a detection limit <10 cfu per PCR. When artificially contaminated raw milk samples were further evaluated, the assay performed equally as well as the traditionally cultural-based method, and facilitated quantitative detection of the two microbes in the range from ~10² to ~10⁶ cfu mL^?1 raw milk. The detection limit was ~10² cfu mL^?1 raw milk for either or a mixture of the two strains without pre-enrichment step, and could be <10 cfu per 10 mL of raw milk if a pre-enrichment step was added. Considering the detection effectiveness, time-consumption saving, and economical efficiency, the present duplex realtime PCR assay has a great potential in the application in raw milk for assessing their microbiological quality and safety in relation to E. coli and Shigella spp.