Domestication of the Dromedary Revisited and Its Consequences for Legislation as to Keeping Livestock or Pet Animals.

Being in an advanced stage of domestication is a newly proposed requirement to decide which animals can be safely kept by humans. Dutch legislators were the first to apply it and other European countries may be tempted to adopt a similar approach. Unexpectedly, the Dutch assessors considered the dromedary (Camelus dromedarius) as being insufficiently domesticated and this species will therefore no longer be able to be kept as a production animal from 2024 onwards. In a recent publication on this topic, we showed that the domestication of the dromedary is actually very advanced. In this paper, we apply the same criteria that were used by the Dutch assessors to determine the degree of domestication, taking into account the most recent scientific developments in this area, even though it should be noted that these criteria have neither been peer-reviewed, nor published in an international scientific journal. For the sake of comparison, and in order to validate the procedure, we also applied these criteria to the house cat. The results confirm that the dromedary is highly domesticated, but also that the house cat (Felis silvestris catus) is at most semi-domesticated. Obviously, we agree with the decision of the Dutch legislators to place the house cat on the positive list, but our analysis demonstrates that this was decided on false grounds. Our analysis makes it clear that the requirement of being in an advanced stage of domestication is not suitable. Instead of maintaining this requirement, we recommend implementing evidence-based, peer-reviewed methods to decide which animals can be kept by humans, and to include species specific-guidelines in the legislation on how this can be achieved safely.

The Flourishing Camel Milk Market and Concerns about Animal Welfare and Legislation.

The worldwide dromedary milk production has increased sharply since the beginning of this century due to prolonged shelf life, improved food-safety and perceived health benefits. Scientific confirmation of health claims will expand the market of dromedary milk further. As a result, more and more dromedaries will be bred for one purpose only: the highest possible milk production. However, intensive dromedary farming systems have consequences for animal welfare and may lead to genetic changes. Tighter regulations will be implemented to restrict commercialization of raw milk. Protocols controlling welfare of dromedaries and gene databases of milk-dromedaries will prevent negative consequences of intensive farming. In countries where dromedaries have only recently been introduced as production animal, legislators have limited expertise on this species. This is exemplified by an assessment on behalf of the Dutch government, recommending prohibiting keeping this species from 2024 onwards because the dromedary was deemed to be insufficiently domesticated. Implementation of this recommendation in Dutch law would have devastating effects on existing dromedary farms and could also pave the way for adopting similar measures in other European countries. In this paper it is shown that the Dutch assessment lacks scientific rigor. Awareness of breeders and legislators for the increasing knowledge about dromedaries and their products would strengthen the position of dromedaries as one of the most adapted and sustainable animals.

Low-water activity foods: increased concern as vehicles of foodborne pathogens.

Foods and food ingredients with low water activity (a(w)) have been implicated with increased frequency in recent years as vehicles for pathogens that have caused outbreaks of illnesses. Some of these foodborne pathogens can survive for several months, even years, in low-a(w) foods and in dry food processing and preparation environments. Foodborne pathogens in low-a(w) foods often exhibit an increased tolerance to heat and other treatments that are lethal to cells in high-a(w) environments. It is virtually impossible to eliminate these pathogens in many dry foods or dry food ingredients without impairing organoleptic quality. Control measures should therefore focus on preventing contamination, which is often a much greater challenge than designing efficient control measures for high-a(w) foods. The most efficient approaches to prevent contamination are based on hygienic design, zoning, and implementation of efficient cleaning and sanitation procedures in the food processing environment. Methodologies to improve the sensitivity and speed of assays to resuscitate desiccated cells of foodborne pathogens and to detect them when present in dry foods in very low numbers should be developed. The goal should be to advance our knowledge of the behavior of foodborne pathogens in low-a(w) foods and food ingredients, with the ultimate aim of developing and implementing interventions that will reduce foodborne illness associated with this food category. Presented here are some observations on survival and persistence of foodborne pathogens in low-a(w) foods, selected outbreaks of illnesses associated with consumption of these foods, and approaches to minimize safety risks.

Genotypic and phenotypic characterisation of a collection of Cronobacter (Enterobacter sakazakii) isolates.

Enterobacter sakazakii has been identified as the causative agent of serious neonatal infections, associated with high mortality rate. In many cases, powdered infant formula (PIF) has been identified as the source of infection. Recently, E. sakazakii was proposed to be classified in a new genus, Cronobacter. Since knowledge on this pathogen is still incomplete, there is a need for molecular characterization schemes in order to help with epidemiological investigation and evaluate strain variability. The objectives of this study were to combine genotypic (pulsed-field gel electrophoresis [PFGE], 16S rRNA gene sequencing, and automated ribotyping) methods with traditional phenotypic biochemical methods to characterize a collection of Cronobacter isolates from various origins. In addition, the relative growth dynamics were compared by estimating the growth rates for each isolate in non-selective broth (BHI) at 25 degrees C and 37 degrees C. According to biochemical test profiles the majority of isolates were identified as Cronobacter sakazakii, which seemed to be the most common species distributed in the environment of PIF production plants. Furthermore, the PFGE technique displayed very high discriminatory power as 61 distinct pulsotypes were revealed among the 150 Cronobacter isolates. Combining information on sample origin and pulse type, 64 isolates were deemed as unique strains. Although genetic typing data for the strains clearly delineated them into clusters closely corresponding to biochemical speciation results, it was not without discrepancies as some strains did not group as predicted. Important for quantitative risk assessment is the fact that despite the high genetic heterogeneity observed for this collection, most Cronobacter strains displayed similar growth rates irrespective of species designation.

Comparison of two optical-density-based methods and a plate count method for estimation of growth parameters of Bacillus cereus.

Quantitative microbiological models predicting proliferation of microorganisms relevant for food safety and/or food stability are useful tools to limit the need for generation of biological data through challenge testing and shelf-life testing. The use of these models requires quick and reliable methods for the generation of growth data and estimation of growth parameters. Growth parameter estimation can be achieved using methods based on plate counting and methods based on measuring the optical density. This research compares the plate count method with two optical density methods, namely, the 2-fold dilution (2FD) method and the relative rate to detection (RRD) method. For model organism Bacillus cereus F4810/72, the plate count method and both optical density methods gave comparable estimates for key growth parameters. Values for the maximum specific growth rate (mu(max)) derived by the 2FD method and by the RRD method were of the same order of magnitude, but some marked differences between the two approaches were apparent. Whereas the 2FD method allowed the derivation of values for lag time (lambda) from the data, this was not possible with the RRD method. However, the RRD method gave many more data points per experiment and also gave more data points close to the growth boundary. This research shows that all three proposed methods can be used for parameter estimation but that the choice of method depends on the objectives of the research.

Improving the enrichment procedure for Enterobacteriaceae detection.

The current ISO standard method for detection of Enterobacteriaceae (21528-1:2004) includes enrichment in EE broth which has been shown to be inhibitory to some members of this family, notably Cronobacter spp. A shortened procedure omitting the EE broth has been proposed, however competition from Gram-positive flora may be detrimental to the effective recovery of low levels of target organisms in some sample matrices. In this study we investigated novel cost effective modifications, designed to improve ISO 21528-1:2004 for the detection of Enterobacteriaceae. Initial experiments used a worse-case scenario involving stressed Enterobacteriaceae strains known to grow poorly in laboratory media as well as representative background competitors from powdered milk. The interaction between the Enterobacteriaceae and their competitors was characterised and additives to enhance the growth of target strains over non-target strains were investigated. Supplementation of BPW with 40 microM 8-hydroxyquinoline, 0.5 gL(-1) ammonium iron(III) citrate, 0.1 gL(-1) sodium deoxycholate and 0.1 gL(-1) sodium pyruvate (BPW-S) improved the recovery of Enterobacteriaceae from artificially and naturally contaminated samples. This improvement of the pre-enrichment broth may also be of interest for methods designed to detect specific foodborne pathogens belonging to the Enterobacteriaceae (e.g. Salmonella spp., Cronobacter spp.) that require a pre-enrichment step in BPW.

Phylogeny and prediction of genetic similarity of Cronobacter and related taxa by multilocus sequence analysis (MLSA).

Multilocus sequence analysis (MLSA) based on recN, rpoA and thdF genes was done on more than 30 species of the family Enterobacteriaceae with a focus on Cronobacter and the related genus Enterobacter. The sequences provide valuable data for phylogenetic, taxonomic and diagnostic purposes. Phylogenetic analysis showed that the genus Cronobacter forms a homogenous cluster related to recently described species of Enterobacter, but distant to other species of this genus. Combining sequence information on all three genes is highly representative for the species' %GC-content used as taxonomic marker. Sequence similarity of the three genes and even of recN alone can be used to extrapolate genetic similarities between species of Enterobacteriaceae. Finally, the rpoA gene sequence, which is the easiest one to determine, provides a powerful diagnostic tool to identify and differentiate species of this family. The comparative analysis gives important insights into the phylogeny and genetic relatedness of the family Enterobacteriaceae and will serve as a basis for further studies and clarifications on the taxonomy of this large and heterogeneous family.

A rapid and reliable alternative to ISO 21528-1:2004 for detection of Enterobacteriaceae.

Current legislation in Europe uses the Enterobacteriaceae as a parameter in process hygiene criteria for various food products and refers to the corresponding ISO standard (ISO 21528-1:2004) as mandatory analytical method for this purpose. The ISO procedure includes an enrichment step in EE ("Enterobacteriaceae Enrichment") broth, but it has been reported recently that some isolates of Enterobacteriaceae do not grow well or will even die off in this broth, which could lead to false negative results. To determine if this trait is common among the Enterobacteriaceae, a collection of 95 strains was screened for growth in EE broth. Inhibition was observed with 9 strains (7 Cronobacter sakazakii, 1 Cronobacter malonaticus and 1 Enterobacter amnigenus). Factors affecting cell death were found to be related mainly to the inclusion of bile salts and dyes in this medium. In a second step, an alternative method omitting the EE broth was evaluated using 326 samples, comprising 8 different food matrices and environmental samples from the corresponding manufacturing sites. Positive results were obtained for 235 samples using the ISO standard method and 232 samples using the alternative shortened method. No significant difference was found between the results for the two methods. It is proposed that the standard method for detection of Enterobacteriaceae is revised accordingly.

Cronobacter gen. nov., a new genus to accommodate the biogroups of Enterobacter sakazakii, and proposal of Cronobacter sakazakii gen. nov., comb. nov., Cronobacter malonaticus sp. nov., Cronobacter turicensis sp. nov., Cronobacter muytjensii sp. nov., Cronobacter dublinensis sp. nov., Cronobacter genomospecies 1, and of three subspecies, Cronobacter dublinensis subsp. dublinensis subsp. nov., Cronobacter dublinensis subsp. lausannensis subsp. nov. and Cronobacter dublinensis subsp. lactaridi subsp. nov.

[Enterobacter] sakazakii is an opportunistic pathogen that can cause infections in neonates. This study further clarifies the taxonomy of isolates described as [E.] sakazakii and completes the formal description of the proposed reclassification of these organisms as novel species and subspecies within a proposed novel genus, Cronobacter gen. nov. [E.] sakazakii was first defined in 1980, however recent polyphasic taxonomic analysis has determined that this group of organisms consists of several genomospecies. In this study, the phenotypic descriptions of the proposed novel species are expanded using Biotype 100 and Biolog Phenotype MicroArray data. Further DNA-DNA hybridization experiments showed that malonate-positive strains within the [E.] sakazakii genomospecies represent a distinct species, not a subspecies. DNA-DNA hybridizations also determined that phenotypically different strains within the proposed species, Cronobacter dublinensis sp. nov., belong to the same species and can be considered as novel subspecies. Based on these analyses, the following alternative classifications are proposed: Cronobacter sakazakii gen. nov., comb. nov. [type strain ATCC 29544(T) (=NCTC 11467(T))]; Cronobacter malonaticus sp. nov. [type strain CDC 1058-77(T) (=LMG 23826(T)=DSM 18702(T))]; Cronobacter turicensis sp. nov. [type strain z3032(T) (=LMG 23827(T)=DSM 18703(T))]; Cronobacter muytjensii sp. nov. [type strain ATCC 51329(T) (=CIP 103581(T))]; Cronobacter dublinensis sp. nov. [type strain DES187(T) (=LMG 23823(T)=DSM 18705(T))]; Cronobacter dublinensis subsp. dublinensis subsp. nov. [type strain DES187(T) (=LMG 23823(T)=DSM 18705(T))]; Cronobacter dublinensis subsp. lausannensis subsp. nov. [type strain E515(T) (=LMG 23824=DSM 18706(T))], and Cronobacter dublinensis subsp. lactaridi subsp. nov. [type strain E464(T) (=LMG 23825(T)=DSM 18707(T))].

Development of a novel screening method for the isolation of "Cronobacter" spp. (Enterobacter sakazakii).

A differential medium, "Cronobacter" screening broth, has been designed to complement agars based on hydrolysis of chromogenic alpha-glucopyranoside substrates. The broth was evaluated using 329 Enterobacteriaceae strains (229 target isolates), spiked/naturally contaminated samples, and a parallel comparison with current methods for raw materials, line/end products, and factory environment samples.

Enterobacter pulveris sp. nov., isolated from fruit powder, infant formula and an infant formula production environment.

Six Gram-negative, facultatively anaerobic, non-spore-forming, coccoid rod-shaped isolates were obtained from fruit powder (n=3), infant formula (n=2) and an infant formula production environment (n=1) and investigated in a polyphasic taxonomic study. Comparative 16S rRNA gene sequence analysis combined with rpoB gene sequence analysis allocated the isolates to the family Enterobacteriaceae. The highest rpoB gene sequence similarities (91.2-95.8%) were obtained with Enterobacter helveticus, Enterobacter radicincitans, Enterobacter turicensis and Enterobacter sakazakii and the phylogenetic branch formed by these species was supported by a high bootstrap value. Biochemical data revealed that the isolates could be differentiated from their nearest neighbours by their ability to utilize melibiose, sucrose, D-arabitol, mucate and 1-O-methyl-alpha-galactopyranoside and their negative reactions for D-sorbitol utilization and the Voges-Proskauer test. On the basis of the phylogenetic analyses, DNA-DNA hybridization data, and unique physiological and biochemical characteristics, it is proposed that the isolates represent a novel species of the genus Enterobacter, Enterobacter pulveris sp. nov. The type strain is 601/05(T) (=LMG 24057(T)=DSM 19144(T)).

Identification of "Cronobacter" spp. (Enterobacter sakazakii).

A taxonomic reclassification of the neonatal pathogen Enterobacter sakazakii to consist of five species within a new genus, "Cronobacter," has recently been proposed. The correct identification of these organisms is important to clinicians. Therefore, using 312 Enterobacteriaceae, including 210 "Cronobacter" strains, the reliabilities of biochemical and genetic confirmation tests were investigated. All "Cronobacter" isolates were positive using dnaG and gluA PCR protocols, and all expressed alpha-glucosidase activity. ID32E v3.0 identified 99.5% of "Cronobacter" isolates (as the nearest match to E. sakazakii).

Comparison of the phenotyping methods ID 32E and VITEK 2 compact GN with 16S rRNA gene sequencing for the identification of Enterobacter sakazakii.

A total of 34 isolates (28 Enterobacter sakazakii and 6 Enterobacteriaceae) from infant formulae, milk powder, and the production environment of milk powder factories were identified using ID 32E and VITEK 2 compact GN systems (bioMérieux, France). The ID 32E version 3.0 and VITEK 2 compact GN version 01.01b correctly identified 100% (28) of the Enterobacter sakazakii isolates tested, whereas the previous software version 2.0 for ID 32E showed only 71.4% correct results. None of the non-E. sakazakii isolates tested were misidentified as E. sakazakii with either of the identification systems used.

The taxonomy of Enterobacter sakazakii: proposal of a new genus Cronobacter gen. nov. and descriptions of Cronobacter sakazakii comb. nov. Cronobacter sakazakii subsp. sakazakii, comb. nov., Cronobacter sakazakii subsp. malonaticus subsp. nov., Cronobacter turicensis sp. nov., Cronobacter muytjensii sp. nov., Cronobacter dublinensis sp. nov. and Cronobacter genomospecies 1.

BACKGROUND: Enterobacter sakazakii is an opportunistic pathogen that can cause infections such as necrotizing enterocolitis, bacteraemia, meningitis and brain abscess/lesions. When the species was defined in 1980, 15 biogroups were described and it was suggested that these could represent multiple species. In this study the taxonomic relationship of strains described as E. sakazakii was further investigated. RESULTS: Strains identified as E. sakazakii were divided into separate groups on the basis of f-AFLP fingerprints, ribopatterns and full-length 16S rRNA gene sequences. DNA-DNA hybridizations revealed five genomospecies. The phenotypic profiles of the genomospecies were determined and biochemical markers identified. CONCLUSION: This study clarifies the taxonomy of E. sakazakii and proposes a reclassification of these organisms.

Salmonella detection in probiotic products.

The presence of large amounts of probiotic bacteria in a sample may interfere with the detection of undesirable microorganisms. To illustrate this, infant formula with various strains of probiotic bacteria and the corresponding probiotic culture powders and premixes were artificially contaminated with low levels of Salmonella. Recovery of Salmonella was generally very poor when the conventional pre-enrichment procedure using buffered peptone water (BPW) was applied. However, this problem was overcome by adding antimicrobial compounds to selectively suppress the growth and/or metabolic activity of the probiotic bacteria and increasing the buffering capacity of the pre-enrichment broth. It is recommended that these analytical constraints are already addressed during the development phase of new probiotic products.