Cronobacter sakazakii is an emerging food borne pathogen which has been associated with outbreaks of a rare form of infant meningitis. Although the origin of the microorganism has not been established, several
infection cases have been associated with the consumption of contaminated powdered infant formula (PIF). In the present study, growth characteristics of three C. sakazakii strains isolated from PIF samples and C.
muytjensii strain ATCC 51329, which was formerly the ATCC Preceptrol™ strain for the quality control of
‘Enterobacter sakazakii’ prior to the taxonomic revision, were investigated in Tryptone Soya broth (TSB) and
reconstituted PIF at 4, 10, 25, 37, 45 and 50ºC. The viability of heat treated cells of Cronobacter strains was
evaluated by plating on Violet Red Bile Glucose agar (VRBGA) and the Druggan-Forsythe-Iversen (DFI)
chromogenic agar followed by incubation at 37ºC. These strains were also subjected to higher temperatures
between 52 to 60ºC to measure their thermal tolerance. The mean generation time of all Cronobacter strains
were slightly lower in PIF than in TSB. C. muytjensii ATCC 51329 showed lower generation time in all culture
media and all temperatures compared to the Cronobacter food isolates, but the results were not significantly
different (P>0.05). The results also indicated that combination of PIF: DFI culture media had higher recovery at
all temperatures compared to other combinations. Survival study also indicated that C. muytjensii ATCC 51329
had higher D-value compared to food isolates at all incubation temperatures.
Enterobacter sakazakii previously known as 'yellow-pigmented E. cloacae' has been classified as a new genus 'Cronobacter' based on taxonomic analysis and geno-and phenotypic evaluation. This pathogenic organism has been associated with rare form of infant meningitis and necrotizing enterocolitis (NEC) with high mortality rate (40-80%). Some cases have been linked to the consumption of contaminated powdered infant formula milk (PIF). The objective of this study was to determine the presence of Cronobacter spp. in PIF sold in Malaysia. A selective chromogenic agar, Brilliance Enterobacter sakazakii (DFI, Oxoid), was used for detection of Cronobacter strains. Presumptive Cronobacter isolates were identified using biochemical tests (API 20E and MicrogenTM) and molecular assays (SYBR Green Real-time PCR and 16S ribosomal DNA sequencing). All presumptive Cronobacter strains produced typical blue-green colonies and non-Cronobacter strains produced yellow colonies on Brilliance Enterobacter sakazakii agar (DFI formulation). A total of 12 presumptive isolates were selected from DFI agar and identified with biochemical and molecular tests. The results indicated prevalence of 12.5% C. sakazakii contamination from 72 PIF samples. Molecular detection methods such as Real-time PCR and 16S rDNA proved to have higher identification percentage compared to the biochemical tests. In this study, it was observed that molecular assays were suitable means for sensitive identification of Cronobacter strains in PIF samples.
Pacifier nipples are in permanent contact with saliva and with the oral microflora therefore, act as a favoured site for the growth of biofilms. This research was conducted to identify the bacterial biofilms that has been found on the pacifiers that collected from local child nursery and to analyse the formation of biofilms by Cronobacter sp. during growth in infant formula milk. Pacifiers collected were analysed to obtain colony forming unit (CFU) and isolated bacteria were identified using several biochemical tests according to Bergey's Manual. Biofilm assay of three Cronobacter sp. were conducted using 24 wells microtiter plate and stained with 1% of crystal violet solution at different time interval: 6, 12, 18 and 24 h. The hydrophobicity of the bacterial cell suspension was evaluated using bacterial adhesion to hydrocarbons (BATH) method. Extracellular polymeric substances (EPS) analysis was done to identify percentage of carbohydrate and protein content by using phenol sulphuric acid method and Bradford method, respectively. The results obtained showed that the normal microflora bacteria were the most abundant microorganisms that were found on the pacifier with the main genus isolated was Staphylococcus sp., Enterobacteriaceae sp. and Clostridium sp. Based on biofilm and EPS analysis, Cronobacter sakazakii formed a strong biofilms after 18 h, with carbohydrate was identified as main component of EPS.