Malka Halpern

Culturable Psychrotrophic Bacterial Communities in Raw Milk and Their Proteolytic and Lipolytic Traits

ABSTRACT During cold storage after milk collection, psychrotrophic bacterial populations dominate the microflora, and their extracellular enzymes, mainly proteases and lipases, contribute to the spoilage of dairy products. The diversity, dynamics, and enzymatic traits of culturable psychrotrophs in raw milk from four farms were investigated over a 10-month period. About 20% of the isolates were found to be novel species, indicating that there is still much to be learned about culturable psychrotrophs in raw milk. The psychrotrophic isolates were identified and classified in seven classes. Three classes were predominant, with high species richness (18 to 21 species per class) in different seasons of the year: Gammaproteobacteria in spring and winter, Bacilli in summer, and Actinobacteria in autumn. The four minor classes were Alphaproteobacteria, Betaproteobacteria, Flavobacteria, and Sphingobacteria. The dominant classes were found in all four dairies, although every dairy had its own unique “bacterial profile.” Most but not all bacterial isolates had either lipolytic or both lipolytic and proteolytic activities. Only a few isolates showed proteolytic activity alone. The dominant genera, Pseudomonas and Acinetobacter (Gammaproteobacteria), showed mainly lipolytic activity, Microbacterium (Actinobacteria) was highly lipolytic and proteolytic, and the lactic acid bacteria (Lactococcus and Leuconostoc) displayed very minor enzymatic ability. Hence, the composition of psychrotrophic bacterial flora in raw milk has an important role in the determination of milk quality. Monitoring the dominant psychrotrophic species responsible for the production of heat-stable proteolytic and lipolytic enzymes offers a sensitive and efficient tool for maintaining better milk quality in the milk industry.

Pathogen reservoirs: Chironomid egg masses and Vibrio cholerae

Cholera is a severe diarrhoeal disease triggered by a toxin produced by specific biotypes of the bacterium Vibrio cholerae that are pathogenic only to humans. There seems to be no chronic state of the disease and the natural reservoir of the pathogen is environmental. Here we show that egg masses of the non-biting midge Chironomus sp. (Diptera) harbour V. cholerae and act as its sole carbon source, thereby providing a possible natural reservoir for the cholera bacterium.

Chironomid Egg Masses as a Natural Reservoir of Vibrio cholerae Non-O1 and Non-O139 in Freshwater Habitats

Cholera is a diarrheal disease caused by the gram-negative bacterium Vibrio cholerae, and an estimated 120,000 deaths from cholera occur globally every year. The natural reservoir of the bacterium is environmental. A recent report indicated an association between V. cholerae and chironomid egg masses. Chironomids, the “non-biting midges” (Diptera; Chironomidae), are the most widely distributed and frequently the most abundant insects in freshwater. Females attach egg masses, each containing hundreds of eggs encased in a layer of gelatin, to the water’s edge where bacteria are abundant and may encounter the nutrient-rich substrate. Here we report the isolation of non-O1 and non-O139 V. cholerae from chironomid egg masses from different freshwater bodies in Israel, India, and Africa. In a yearly survey in Israel, chironomid populations were found to peak biannually, and it seemed that those peaks were followed by subsequent bacterial growth and disappearance during the winter in the Mediterranean region. The bacterial population rose as water temperature surpassed 25°C. Thirty-five different serogroups of V. cholerae were identified among the bacteria isolated from chironomids, demonstrating population heterogeneity. Two strains of V. cholerae O37 and O201 that were isolated from chironomid egg masses in Zanzibar Island were NAG-ST positive. Our findings support the hypothesis that the association found between chironomids and the cholera bacteria is not a rare coincidence, indicating that chironomid egg masses may serve as yet another potential reservoir for V. cholerae.

Bacterial communities in floral nectar.

Floral nectar is regarded as the most important reward available to animal-pollinated plants to attract pollinators. Despite the vast amount of publications on nectar properties, the role of nectar as a natural bacterial habitat is yet unexplored. To gain a better understanding of bacterial communities inhabiting floral nectar, culture-dependent and -independent (454-pyrosequencing) methods were used. Our findings demonstrate that bacterial communities in nectar are abundant and diverse. Using culture-dependent method we showed that bacterial communities of nectar displayed significant variation among three plant species: Amygdalus communis, Citrus paradisi and Nicotiana glauca. The dominant class in the nectar bacterial communities was Gammaproteobacteria. About half of the isolates were novel species (< 97% similarities of the 16S rRNA gene with known species). Using 454-pyrosequencing we demonstrated that nectar microbial community are distinct for each of the plant species while there are no significant differences between nectar microbial communities within nectars taken from different plants of the same species. Primary selection of the nectar bacteria is unclear; it may be affected by variations in the chemical composition of the nectar in each plant. The role of the rich and diverse nectar microflora in the attraction-repulsion relationships between the plant and its nectar consumers has yet to be explored.

Fish as reservoirs and vectors of Vibrio cholerae.

Vibrio cholerae, the etiologic agent of cholera, is autochthonous to various aquatic environments, but despite intensive efforts its ecology remains an enigma. Recently, it was suggested that copepods and chironomids, both considered as natural reservoirs of V. cholerae, are dispersed by migratory waterbirds, thus possibly distributing the bacteria between water bodies within and between continents. Although fish have been implicated in the scientific literature with cholera cases, as far as we know, no study actually surveyed the presence of the bacteria in the fish. Here we show for the first time that fish of various species and habitats contain V. cholerae in their digestive tract. Fish (n = 110) were randomly sampled from freshwater and marine habitats in Israel. Ten different fish species sampled from freshwater habitats (lake, rivers and fish ponds), and one marine species, were found to carry V. cholerae. The fish intestine of Sarotherodon galilaeus harboured ca. 5×103 V. cholerae cfu per 1 gr intestine content—high rates compared with known V. cholerae cfu numbers in the bacterias natural reservoirs. Our results, combined with evidence from the literature, suggest that fish are reservoirs of V. cholerae. As fish carrying the bacteria swim from one location to another (some fish species move from rivers to lakes or sea and vice versa), they serve as vectors on a small scale. Nevertheless, fish are consumed by waterbirds, which disseminate the bacteria on a global scale. Moreover, V. cholerae isolates had the ability to degrade chitin, indicating a commensal relationship between V. cholerae and fish. Better understanding of V. cholerae ecology can help reduce the times that human beings come into contact with this pathogen and thus minimize the health risk this poses.

Molecular analysis of bacterial communities in raw cow milk and the impact of refrigeration on its structure and dynamics

The impact of refrigeration on raw cow milk bacterial communities in three farm bulk tanks and three dairy plant silo tanks was studied using two methods: DGGE and cloning. Both methods demonstrated that bacterial taxonomic diversity decreased during refrigeration. Gammaproteobacteria, especially Pseudomonadales, dominated the milk after refrigeration. Farm samples and dairy plant samples differed in their microbial community composition, the former showing prevalence of Gram-positive bacteria affiliated with the classes Bacilli, Clostridia and Actinobacteria, the latter showing prevalence of Gram-negative species belonging to the Gammaproteobacteria class. Actinobacteria prevalence in the farm milk samples immediately after collection stood at about 25% of the clones. A previous study had found that psychrotolerant Actinobacteria identified in raw cow milk demonstrated both lipolytic and proteolytic enzymatic activity. Thus, we conclude that although Pseudomonadales play an important role in milk spoilage after long periods of cold incubation, Actinobacteria occurrence may play an important role when assessing the quality of milk arriving at the dairy plant from different farms. As new cooling technologies reduce the initial bacterial counts of milk to very low levels, more sensitive and efficient methods to evaluate the bacterial quality of raw milk are required. The present findings are an important step towards achieving this goal.

Vibrio cholerae Hemagglutinin/Protease Degrades Chironomid Egg Masses

ABSTRACT Cholera is a severe diarrheal disease caused by specific serogroups of Vibrio cholerae that are pathogenic to humans. The disease does not persist in a chronic state in humans or animals. The pathogen is naturally present as a free-living organism in the environment. Recently, it was suggested that egg masses of the nonbiting midge Chironomus sp. (Diptera) harbor and serve as a nutritive source for V. cholerae, thereby providing a natural reservoir for the organism. Here we report that V. cholerae O9, O1, and O139 supernatants lysed the gelatinous matrix of the chironomid egg mass and inhibited eggs from hatching. The extracellular factor responsible for the degradation of chironomid egg masses (egg mass degrading factor) was purified from V. cholerae O9 and O139 and was identified as the major secreted hemagglutinin/protease (HA/P) of V. cholerae. The substrate in the egg mass was characterized as a glycoprotein. These findings show that HA/P plays an important role in the interaction of V. cholerae and chironomid egg masses.

Culturable and VBNC Vibrio cholerae: interactions with chironomid egg masses and their bacterial population.

Vibrio cholerae, the etiologic agent of cholera, is autochthonous to various aquatic environments. Recently, it was found that chironomid (nonbiting midges) egg masses serve as a reservoir for the cholera bacterium and that flying chironomid adults are possible windborne carriers of V. cholerae non-O1 non-O139. Chironomids are the most widely distributed insect in freshwater. Females deposit egg masses at the waters edge, and each egg mass contains eggs embedded in a gelatinous matrix. Hemagglutinin/protease, an extracellular enzyme of V. cholerae, was found to degrade chironomid egg masses and to prevent them from hatching. In a yearly survey, chironomid populations and the V. cholerae in their egg masses followed phenological succession and interaction of host–pathogen population dynamics. In this report, it is shown via FISH technique that most of the V. cholerae inhabiting the egg mass are in the viable but nonculturable (VBNC) state. The diversity of culturable bacteria from chironomid egg masses collected from two freshwater habitats was determined. In addition to V. cholerae, representatives of the following genera were isolated: Acinetobacter, Aeromonas, Klebsiella, Shewanella, Pseudomonas, Paracoccus, Exiguobacterium, and unidentified bacteria. Three important human pathogens, Aeromonasveronii, A. caviae, and A. hydrophila, were isolated from chironomid egg masses, indicating that chironomid egg masses may be a natural reservoir for pathogenic Aeromonas species in addition to V. cholerae. All isolates of V. cholerae were capable of degrading chironomid egg masses. This may help explain their host–pathogen relationship with chironomids. In contrast, almost none of the other bacteria that were isolated from the egg masses possessed this ability. Studying the interaction between chironomid egg masses, the bacteria inhabiting them, and V. cholerae could contribute to our understanding of the nature of the V. cholerae–egg mass interactions.

Leucobacter chironomi sp. nov., a chromate-resistant bacterium isolated from a chironomid egg mass

A Gram-positive, rod-shaped, aerobic, non-motile, yellow-pigmented bacterial strain, designated MM2LB(T), was isolated from a chironomid egg mass that was sampled in a waste stabilization pond in northern Israel. 16S rRNA gene sequence analysis indicated that the isolate should be placed in the genus Leucobacter (family Microbacteriaceae). Levels of 16S rRNA gene sequence similarity between strain MM2LB(T) and the type strains of recognized Leucobacter species were 97.1 % or lower. The dominant cellular fatty acids of strain MM2LB(T) were anteiso-C(15 : 0), iso-C(16 : 0) and anteiso-C(17 : 0), which are typical of members of the genus Leucobacter. Strain MM2LB(T) grew at 17-37 degrees C, in the presence of 0-7.0 % NaCl and at pH 4.0-9.5. Cell-wall amino acids were alanine, glycine, threonine, 2,4-diaminobutyric acid, glutamic acid and gamma-aminobutyric acid in a B-type cross-linked peptidoglycan. The quinone system consisted of menaquinones MK-11, MK-10 and MK-12. The novel isolate was able to grow in medium containing 18 mM Cr(VI). On the basis of phenotypic properties and phylogenetic distinctiveness, strain MM2LB(T) is considered to represent a novel species of the genus Leucobacter, for which the name Leucobacter chironomi sp. nov. is proposed. The type strain is MM2LB(T) (=LMG 24399(T)=DSM 19883(T)).

Do honeybees shape the bacterial community composition in floral nectar

Floral nectar is considered the most important reward animal-pollinated plants offer to attract pollinators. Here we explore whether honeybees, which act as pollinators, affect the composition of bacterial communities in the nectar. Nectar and honeybees were sampled from two plant species: Amygdalus communis and Citrus paradisi. To prevent the contact of nectar with pollinators, C. paradisi flowers were covered with net bags before blooming (covered flowers). Comparative analysis of bacterial communities in the nectar and on the honeybees was performed by the 454-pyrosequencing technique. No significant differences were found among bacterial communities in honeybees captured on the two different plant species. This resemblance may be due to the presence of dominant bacterial OTUs, closely related to the Arsenophonus genus. The bacterial communities of the nectar from the covered and uncovered C. paradisi flowers differed significantly; the bacterial communities on the honeybees differed significantly from those in the covered flowers’ nectar, but not from those in the uncovered flowers’ nectar. We conclude that the honeybees may introduce bacteria into the nectar and/or may be contaminated by bacteria introduced into the nectar by other sources such as other pollinators and nectar thieves.