Detection of Cronobacter spp . ( formerly Enterobacter sakazakii ) from medicinal plants and spices in Syria

Introduction: Cronobacter spp. (formerly Enterobacter sakazakii) is an emerging food-borne pathogen that causes severe meningitis, sepsis, and necrotizing enterocolitis in neonates and infants. These infections have been reported from different parts of the world. The epidemiology and reservoir of Cronobacter spp. are still unknown, and most strains have been isolated from clinical specimens and from a variety of foods, including cheese, meat, milk, vegetables, grains, spices, and herbs. Methodology: Our study aimed to detect and isolate Cronobacter spp. from different Syrian samples of spices, medicinal herbs and liquorices, depending on the pigment production and biochemical profile of isolates and PCR technique. This PCR method, which provides a powerful tool for rapid, specific, and sensitive detection of Cronobacter spp., is considered a reliable alternative to traditional bacteriological methods. Results and conclusions: This study revealed that the percentage of Cronobacter spp. was 94%, 52%, and 32% in liquorice, spices and medicinal herbs, respectively. In addition, it assured that the optimal enhancing growth temperature was 44°C, and optimal enhancing growth pH was 5.

(formerly Enterobacter sakazakii) is a non-spore forming, Gram-negative rodshaped bacterium, approximately 3 x 1 μm in size.It is motile with peritrichousflagellae anaerobes facultative.It belongs to the family Enterobacteriaceae and genus Enterobacter, which contains a number of species, among which the differentiation is based on biochemical reactions and serological and molecular techniques [1].Cronobacter spp., E. agglomerans, and E. cloacae are considered the main species of this genus that are frequently isolated from clinical samples and food products [2].Cronobacter spp. is catalase positive, oxidase negative, and generally positive for α-D-glucosidase [3].It reduces nitrates, utilizes citrates, hydrolyzes esculin and arginine, and produces acid from D-glucose, D-sucrose, D-raffinose, D-melibiose, D-cellobiose, D-mannitol, D-mannose, L-rhamnose, L-arabinose, D-xylose, D-trehalose, galacturonate and D-maltose, and it is also generally positive for acetoin production (Voges-Proskauer test) and negative for the methyl red test [4].Traditional culture methods for identifying Cronobacter spp.were laborious and time-consuming and required steps of enrichment and biological tests that take 6 to 7 days to complete.PCR assay is considered a rapid, sensitive, specific and more reliable method for early detection of pathogen bacteria, including Cronobacter spp.[5].
The first case attributed to this organism occurred in 1958 in England and it took its name from Riichi Sakazakii, a Japanese microbiologist [6].Cronobacter spp. was first described as a "yellow-pigmented Enterobacter cloacae" by Urmenyi and Franklin in 1961 [6].In 1980, Farmer et al. designated it as a unique species [2].The differentiation between Cronobacter spp.and E. cloacae was based on differences in biochemical reactions, the ability of Cronobacter spp.colonies to produce yellow pigments, and by DNA-DNA hybridization.Cronobacter spp.formed a microbiological hazard in the infant food chain with historic high morbidity and mortality in neonates [7].Therefore, the name Cronobacter gen.nov.was proposed after the Greek mythological god Cronos, who was described as swallowing his children at birth [8].This genus contains the species C. sakazakii sp.nov.; C. malonaticus sp.nov.; C. muytjensii sp.nov.; C. dublinensis sp.nov.; and C. turicensis sp.nov.[9].This group of bacteria are considered opportunistic pathogens that have been associated with severe forms of necrotizing enterocolitis [10] and meningitis [11], especially in neonates, and catheter-associated infections in elderly and immunocompromised people, with a mortality rate varying from 40% to 80% [12]; however, this figure has declined to about 20% in recent years [13].
Among all cases, about half of the patients die within one week of the onset of the infections and about 94% of the meningitis survivors exhibit severe neurological complications [14].The International Commission for Microbiological Specification for Foods has ranked Cronobacter spp. as "Severe hazard for restricted populations, life-threatening or substantial chronic sequelae for long duration" [15].Also, the US Food and Drug Administration has issued an alert to health-care professionals about the risk associated with Cronobacter spp.infections among neonates fed with milk-based infant formula.The alert stated that the most effective way to avoid Cronobacter spp.infections in premature babies and neonates is to prevent contamination of infant milk formula during production and bottle preparation [16].The Food and Agriculture Organization of the United Nations (FAO) and the World Health Organization (WHO) promote research that investigates ways to reduce the levels of Cronobacter spp. in reconstituted powdered infant formula, e.g., ensuring strict timetemperature control on rehydration; decreasing the time of feeding; adding inhibitors; using biopreservatives and acidification; combining treatments; and promoting research to gain a better understanding of the ecology, taxonomy, characteristics and virulence of Cronobacter spp.The information gathered from this research will be important for the interpretation of epidemiology data and undertaking further risk assessments.More complex risk assessments were initiated in two FAO/WHO expert meetings and will be completed and expanded by the Joint FAO/ WHO Expert Meetings on Microbiological Risk Assessment (JEMRA) group [17].However, knowledge of the etiological and ecological characteristics of Cronobacter spp. is sparse and its occurrence in factories that produce infant formulas and in hospital kitchens has not been studied in depth.Cronobacter spp.repeatedly have been reported as remarkably resistant to osmotic stress and dryness and moderately thermotolerant as some encapsulated Cronobacter spp.were still recoverable from desiccated infant formula after storage for up to 2.5 years [18].The composition of dry foods and infant formula combined with their low water activity significantly affected the survival of Cronobacter spp. in these foods [19].Previously in Syria, traditional medicine used some herbs, such as cuminum cyminum and pimpinella anisum, as additives to infant formula to treat some enterogastric confusions.The presence of Cronobacter spp. in these herbs was not yet been completely studied.The aim of this study is to analyze the contribution of vegetative foods, including medicinal plants, (e.g., spices and medicinal plants purchased from a Syrian traditional market) in the morbidity of adults with Cronobacter spp.

Food samples
A total of 144 different samples were collected from different locations across Damascus, Syria, and its countryside.The samples composed of 68 medicinal plants, 16 liquorice, and 60 (Tables 1 and  2).

Detection, isolation and identification of Cronobacter spp.
The procedure of ISO/TS 22964:2006 for detection of Cronobacter spp. was followed [20].Typical colonies that appeared yellow on tryptic soy agar (TSA) were picked and subjected to further characterization by using microscope, biochemicals, and PCR analysis.and studying its ability to grow at different temperatures and pH degrees.

Morphology
Yellow colonies from TSA were examined by microscope after staining with Gram stain to study their morphology, and by dark field microscope to study their motility.

Growth at different temperatures and pH degrees
Six typical yellow colonies confirmed as Cronobacter spp.were chosen for further analysis as follows: two from medicinal plants (C.acutifolia and M. chamomilla) two from spices (curry and black pepper) and two from liquorice.Each of these colonies was then subcultured in 3 ml BPW at 44° for 24 hours.Then, to determine the optimal temperature and the optimal pH level for enhancing the growth of bacteria, 0.6 x 109 cfu/ml of each sample was re-cultured at a fixed pH (7.0) and at different temperatures as follows: 25-30-37-44-48°C for 24 hours.Optical densities (O.D) were read at 600 nm.Next, using the same conditions mentioned above, the optimal enhancing growth temperature was fixed, and different  pH levels (pH = 3-5-7-9) were examined to assess the optimal pH level at the optimal temperature, in.Each sample was processed five times in this manner.

Results and discussion
This research isolated Cronobacter spp.from different spices and medicinal plants.There are many differences in phenotype among the isolates of Cronobacter spp; thus it is difficult to confirm the identity of isolates by using only one method or one set of specific PCR primers [24].Therefore, this study depended on the use of chromogenic, biochemical, and molecular techniques for detection, isolation, and identification of Cronobacter spp.from food samples.Our results concur with those described by Farmer and colleagues [2], who showed that freshly isolated Cronobacter spp.produce two distinct colony morphologies, the first type dry and the second type mucoid.
All isolated strains of Cronobacter spp.appeared by microscopical examination as Gram-negative, rod shaped, and motile bacteria.They were also lactose fermenting, catalase positive, oxidase negative, αglucosidase positive, methyl red negative, and Voges-Proskauer positive.However, several previous studies have produced conflicting results [25].
PCR was performed by using three pairs of specific primers as described above and all strains of Cronobacter spp.showed a correct-sized amplification product of 1680, 282, and 251 bp according to the primers EsAg, SG, and SI respectively (Figure 1).However, no amplification product was obtained for all E. cloacae strains by using the same primers (100% specificity).
It was observed that the highest percentage of Cronobacter spp.(≈ 94%) was found in liquorice (herbal drink), while the percentage of Cronobacter spp.found in spices and medicinal plants was about 52% and 32%, respectively [Table 3].The large amount of these bacteria in liquorice (roots) corresponds s with the similar large amount of Cronobacter spp.present in the roots of some agriculture plants such as corn, cucumber, rough lemon, and tomato [26,27,28,29].
The medicinal plants and spices that were positive for the presence of Cronobacter spp.are listed in table [4].In addition, biochemical test results for these isolates are given in the table.
When the typical colonies were confirmed by biochemical tests and PCR, characterization of optimal growth temperatures and pH levels were determined as described in the methodology section.Figures 2 and 3 show the effects of different temperatures and different pH levels, respectively, on enhancing the growth of bacteria.It was observed that the optimal temperature was 44˚C and the optimal pH level was 5.
These results conform with the fact that the pH level of an infant's stomach is 5 [30], which would allow the growth of Cronobacter spp., whereas this bacteria likely would not survive at the pH level found in an adult's stomach (pH3).Most studies recommend not preparing food for infants at room temperature for longer than 20 minutes as doing so allows bacteria to grow very quickly [31].These findings reflect the possibility of coexisting Cronobacter spp. in foods other than infant formula, infant food, and milk powder.Despite the death of the bacteria at high temperatures, secreted endotoxins which tolerate high temperatures used in food preparation still remain and can cause illness.
Our results are in agreement with those reported in a review of endophytic bacteria in agriculture crops by Hallmann et al., 1997, who noted that the genus Enterobacter is associated with the phytic flora [32].Also Enterobacter species have been isolated from corn roots and stems [26], cucumber roots [27], rough lemon roots [28], and grapevine stems [33].Mossel and Struijk hypothesized that just as the primary reservoir for the coliform E. coli is feces, the reservoir for E. sakazakii (Cronobacter spp.), in addition to other coliforms, such as Klebsiella oxytoca, K. pneumoniae, E. cloacae and Citrobacter species, may primarily be environmental and from plant materials [34].Iversen and Forsythe hypothesize that the principal environmental sources of E. sakazakii (Cronobacter spp.) are water, soil and vegetables, and  [36].However, our results differ from those of several reports that have implicated rehydrated powdered infant formula as a source of Cronobacter spp. in neonatal infections [10,37].It might be that infant formula and infant foods become contaminated at certain stages during the processing, particularly after sterilization during vitamin or supplement fortification steps.Furthermore it is worthwhile to mention that Cronobacter spp.may be associated with the foods other than infant formula, infant food, and milk powder.These results are in agreement with those reported by Forsythe and Friedemann, who emphasize that the majority of E. sakazakii (Cronobacter spp.) isolates are from plant sources, irrespective of the claims of most studies which confirmed that powdered infant formula is a source of this pathogen [38,39].
Our results also indicate that plants possibly incarnate the major reservoir of Cronobacter spp., and its ability to survive in dry foods, herbs, spices and the general manufacturing environment may be due to its thermotolerant and osmotolerant nature [40].
The high association of this pathogen with herbs and spices suggests that extra precautions should be taken when home medications containing herbs or herbal beverages are given to infants to allay gastrointestinal disturbance.Extra care should also be taken in hospital kitchens when preparing meals for immuno-compromized persons who may be at risk for Cronobacter spp.infection from contaminated.

Table 1 .
Medicinal plants that were used in the study

Table 2 .
Spices that were used in the study

Table 3 .
Percentage of the presence of Cronobacter spp. in the specimens

Table 4 .
Positive specimens for Cronobacter spp.and their biochemical tests

Table 5 .
[35]imens used in the experiments of temperature and pH Brackets refer to the specimen lab ID number a secondary means of contamination may be vectors such as flies and rodents[13].Cottyn et al. analyzed rice harvested from various sites in the Philippines for bacterial flora; of the 428 bacterial isolates examined, 184 were Gram-positive and 244 were Gram-negative.The most prevalent (25%) of the Gram-negative isolates were from the family Enterobacteriaceae, with the genus Pantoea and Enterobacter predominating.Four seed lots yielded 20 E. sakazakii (Cronobacter spp.) isolates and five lots yielded 9 isolates of E. cloacae[35].In another study, the highest percentage of Cronobacter spp.isolates (39%) was found in herbs and spices; for instance, the four samples tested of a traditional herbal drink (liquorice) contained Cronobacter spp.(100%) while 11 out of 15 samples (73.3%) of mixed spices contained Cronobacter spp. *