Screening of specific nucleic acid targets for Cronobacter sakazakii and visual detection by loop-mediated isothermal amplification and lateral flow dipstick method in powdered infant formula

Due to the lack of specific genes for rapid detection methods of Cronobacter sakazakii in food samples, whole genome sequence analysis was performed in this investigation using the basic local alignment search tool. Forty-two DNA fragments unique to C. sakazakii were mined, then primers were designed and screened by PCR and loop-mediated isothermal amplification (LAMP). Two primer sets, CS1 and CS31, were found as specific and stable primers, with their corresponding nucleic acid targets the CSK29544_00235 gene and CSK29544_03484 gene, respectively. Furthermore, compared with 3 genes reported previously, these 2 genes were verified as more specific to C. sakazakii among Cronobacter species, by sequence similarity alignment using Cronobacter MLST databases (http://pubmlst.org/cronobacter). The specificity of the LAMP reaction approached 100% by using 48 bacterial strains, which included 22 C. sakazakii strains. Subsequently, LAMP was combined with visual lateral flow dipstick (LFD) based on the above 2 nucleic acid targets, and was demonstrated as a rapid, efficient method with high specificity. Finally, the detection sensitivity of this assay system for pure cultures and artificially contaminated milk was measured as 4.5 × 10⁰ cfu/mL and 5.7 × 10¹ cfu/g, respectively. Total time to detection for this assay was within 2 h. Thus, the establishment of this LAMP-LFD method shows great significance and potential for rapid detection of C. sakazakii in powdered infant formula.     

Isolation and molecular identification of Cronobacter spp. from powdered infant formula (PIF) in Bangladesh

Cronobacter spp. formerly known as Enterobacter sakazakii is an occasional contaminant of powdered infant formula (PIF). This pathogen has been associated with out-breaks of a rare form of infant meningitis, necrotizing enterocolitis (NEC), bacteremia and neonate deaths. The organism is ranked by the International Commission for Microbiological Specifications for Foods (ICMSF) as a ‘Severe hazard for restricted populations, life threatening or substantial chronic sequelae or long duration’. Present study aimed to isolate Cronobacter spp. from PIF and clinical samples, such as blood, stool and CSF collected from 93 neonates and child patients, age ranged from 0 to 24 months. We did not detect Cronobacter spp. in any of these samples. Later 32 PIF samples collected from retail markets in Bangladesh were tested for the presence of Cronobacter spp. Of these only one was found to be contaminated with Cronobacter sp. This is the first case of Cronobacter contaminated PIF found in Bangladesh to be reported. The organism was successfully identified based on its typical culture characteristics, producing blue-green colonies on chromogenic DFI agar and also by a standardized conventional PCR assay targeting the alpha glucosidase and 16 S rRNA gene sequence of Cronobacter sp. The 16 S rRNA gene was partially sequenced to provide for the phylogenetic analysis of this isolate (DA01) and found to cluster with some other Cronobacter isolates in the phylogram.     

RpoS loss in Cronobacter sakazakii by propagation in the presence of non-preferred carbon sources

This study assessed whether propagation of four Cronobacter sakazakii strains in the presence of various non-preferred substrates (formate, fumarate, propionate, pyruvate and succinate) as sole carbon source can cause loss of RpoS functionality. Growth of C. sakazakii DPC 6525 in the presence of fumarate, propionate and succinate selected for loss of RpoS. rpoS mutants could not be obtained for C. sakazakii DPC 6522, DPC 6526 and DPC 6530. DPC 6525 showed lower growth ability in all carbon sources than the other strains tested and had higher mutagenesis rates, determined by plating on agar media supplemented with rifampicin, than DPC 6522 and DPC 6526 but much lower than DPC 6530. Results demonstrate that growth of C. sakazakii in non-preferred carbon sources can select for RpoS loss and suggest that certain strains are more predisposed to the loss of RpoS due to their reduced growth ability in environments of limited nutrients availability.     

The gyrase B gene as a molecular marker to resolve interspecific relationships within the Acetobacter pasteurianus group and a novel target for species-specific PCR

Members of the Acetobacter pasteurianus are popular acetic acid bacteria (AAB) for the production of vinegar. Neither phenotypic nor the most frequently applied genotypic marker (16S ribosomal DNA) provides sufficient resolution for accurate identification of the AAB strains. In this study, the gyrB gene was used for species discrimination by direct DNA sequencing and as marker in a species-specific PCR assay. All examined A. pasteurianus strains were clearly distinguished from the closely related species by comparative sequence analysis of the gyrB gene. The average sequence similarity for the gyrB gene (82.2 %) among type strains was significantly lower than that of the 16S rRNA sequence (98.2 %). Therefore, the gyrB gene can be proposed as an additional molecular marker for A. pasteurianus and related taxa that provides higher resolution than 16S rRNA. In addition, the species-specific primers were also developed based on the gyrB and 16S rRNA gene sequences, which were then employed for PCR using the template DNA of Acetobacter strains. The PCR primer pairs were shown to be specific for A. pasteurianus, A. peroxydans and papayae. Our data indicate that the phylogenetic relationships in the A. pasteurianus group are easily resolved by direct sequencing of the gyrB gene and combined with species-specific PCR assays.     

Transposon mutagenesis reveals genes involved in osmotic stress and drying in Cronobacter sakazakii

This study characterizes the growth in hyperosmotic media and the resistance to desiccation of a collection of fifteen Cronobacter sakazakii strains. C. sakazakii strains showed similar abilities to grow/persist under osmotic stress conditions to strains from other related Enterobacteriaceae, i.e. Cronobacter muytjensii, Cronobacter malonaticus, Enterobacter gergoviae, Enterobacter cloacae, Enterobacter aerogenes, and S. Typhimurium. Nevertheless, some degree of heterogeneity among C. sakazakii strains could be observed, and in general strains isolated from clinical sources showed the greatest robustness. A transposon mutagenesis approach was used to identify genetic systems involved in the response of C. sakazakii DPC 6529 to hyperosmotic conditions. We obtained evidence that de novo protein synthesis, repair of damage in macromolecules and maintenance of the structure and integrity of the cellular envelope are essential processes for the cell under osmotic stress. Moreover, some metabolic activities are also important, including the synthesis of glutamine as a compatible solute and the regulation of nucleotide and nucleoside pools. The Cpx system, known as an envelope stress response regulator, and the sigma factors RpoN and RpoS seem to be the main signals regulating the bacterial response to hyperosmotic conditions. Among the identified salt-sensitive mutants, only those disrupted in dnaK and dnaJ, encoding two molecular chaperones, were important for C. sakazakii survival under desiccation. This suggests that the systems and proteins involved in the desiccation response differ from those responsible for growth under hyperosmotic conditions, at least under the conditions tested in the current study.     

Heat resistance of Cronobacter sakazakii DPC 6529 and its behavior in reconstituted powdered infant formula

Cronobacter sakazakii is an opportunistic pathogen in neonates which can cause meningitis, septicaemia and enterocolitis related to the consumption of contaminated Powdered Infant Formula (PIF). C. sakazakii has an unusual ability to survive under dry conditions and it could be among the most thermotolerant members of the Enterobacteriaceae. Little is known about how Cronobacter species respond to heat stress and the mechanisms involved in the process. In the current study we determined the heat resistance of a particularly stress tolerant C. sakazakii strain, C. sakazakii DPC 6529, and monitored the behavior of a lux-tagged derivative under different reconstitution and handling scenarios in a commercial brand of PIF. Some of the molecular mechanisms involved in the heat stress response were investigated using a transposon mutagenesis approach. Survival curves of C. sakazakii DPC 6529 in Luria-Bertani (LB) broth and PIF at various temperatures (58, 60, 62 and 64 °C) displayed an upward concavity and were fitted to the non-linear Weibull model. While at the highest treatment temperatures heat resistance was lower in PIF than in LB broth, at lower temperatures no significant differences in heat resistance were observed. Experiments in real time with artificially inoculated PIF reconstituted at different water temperatures (50, 55, 60, 65, 70 °C) and cooled at different rates confirmed that C. sakazakii can survive for long time periods in powdered formula, and is capable of proliferating after reconstitution. The use of water at temperatures between 50 and 65 °C for reconstitution did not provide a significant inactivation of C. sakazakii cells. Reconstitution at 70 °C reduced the bacterium to levels below the detection limit, although survivors were able to proliferate and reached dangerous levels when the reconstituted product was stored for a long time at room temperature. The cooling rate had an important impact on survival and subsequent growth of C. sakazakii, which makes it advisable to avoid rapid cooling of baby formula. Transposon mutagenesis allowed the identification of some of the molecular mechanisms involved in the response of C. sakazakii DPC6529 to heat stress. Genes identified included the Ribosome Maturation Protein RimP and Outer Membrane Porin L (OmpL). Results suggest that de novo protein synthesis, and the uptake of cysteine for the formation of disulfide bonds for protein stabilization, are key processes.     

Use of highly variable gene (yycH) as DNA marker to resolve interspecific relationships within the Lactobacillus casei group and a target for developing novel species-specific PCR primers

Identifying Lactobacillus species group using only phenotypic and genotypic (16S rDNA sequence homology analysis) techniques yields inaccurate results. In this study, the partial yycH gene sequence was used for species discrimination by direct DNA sequencing and as target in a species-specific PCR assay. All examined Lactobacillus strains were clearly distinguished from the closely related species by comparative sequence analysis of the yycH gene. The average sequence similarity for the yycH gene (78.5 %) among type strains was significantly lower than that of the 16S rRNA sequence (99.0 %). Therefore, the yycH gene can be proposed as an additional molecular marker for Lactobacillus casei group that provides higher resolution than 16S rRNA. In addition, the species-specific primers were also developed based on the yycH gene sequences, which were then employed for PCR using the template DNA of Lactobacter strains. The PCR primer pairs were shown to be specific for Lactobacillus paracasei and Lactobacillus rhamnosus. Our data indicate that the phylogenetic relationships in the L. casei group are easily resolved by direct sequencing of the yycH gene and combined with species-specific PCR assays.     

Identification of Lactobacillus spp. in probiotic products by real-time PCR and melting curve analysis

A rapid and reliable technique for identifying the strains of lactic acid bacteria (LAB) was developed in this study, and the 16S ribosomal RNA gene (16S rRNA gene) was used as the target. Several species-specific primer pairs were designed based on the variability of 16S rRNA sequence(s) for differentiating 5 strains of lactobacilli which were added into probiotic products in Taiwan. It was simple to identify Lactobacillus acidophilus and L. delbrueckii by species-specific primers, but it could not be used to distinguish L. casei, L. paracasei and L. rhamnosus. Another PCR approach was developed with hybridization probes which were designed according to the difference among the 16S rRNA genes of L. casei, L. paracasei and L. rhamnosus, and melting curve analysis of the hybridization probe was used to distinguish them. It was found that this approach could identify L. paracasei and L. rhamnosus correctly but not separate L. paracasei from L. casei, the result was due to both of them had the same 16S rRNA sequence. These results suggest that melting curve analysis of PCR approach in this study is a rapid, simple and accurate method in distinguishing the closely related strains of lactobacilli.     

Evaluation of three commercially available real-time PCR based systems for detection of Cronobacter species

In the last few years, various PCR based methods have been developed that enable detection of Cronobacter spp. to the genus and species level. Moreover, several real-time PCR based systems for detection of Cronobacter spp. are available, however, comparative evaluation studies are not available.The current study represents a comparative evaluation of three commercial diagnostic systems, namely the BAX® System PCR Assay Enterobacter sakazakii (DuPont, Qualicon, Wilmington, USA), the Assurance GDS™ Enterobacter sakazakii (BioControl, Bellvue, USA) and the foodproof® Enterobacter sakazakii Detection Kit (Biotecon Diagnostics, Potsdam, Germany) for the rapid identification of Cronobacter spp.Twenty-one target and non-target strains were included in the study and results were compared for specificity and convenience in performance. A specificity of 100% was observed for two of the three real time PCR systems tested, namely the Assurance GDS™ Enterobacter sakazakii and the foodproof® Enterobacter sakazakii Detection Kit for pure cultures as well as artificially contaminated powdered infant formula (PIF) samples.     

Phylogenetic study of Lactobacillus acidophilus group, L. casei group and L. plantarum group based on partial hsp60, pheS and tuf gene sequences

The partial nucleotide sequences encoding elongation factor Tu (tuf gene), 60-kDa heat shock protein (hsp60 gene) and phenylalanyl-tRNA synthase (pheS gene) were determined to assess the suitability as phylogenetic markers for discriminating the closely related species in Lactobacillus acidophilus group, L. casei group and L. plantarum group. A total of 234 lactobacilli were chosen from traditional fermented dairy products that were not exactly assigned to species based on biochemical tests and 16S rRNA gene sequences. The sequencing of partial tuf, hsp60 and pheS gene of all strains was performed, and then, the phylogenetic trees were constructed by neighbor-joining method. Phylogenetic tree revealed three genes provided better resolution of each Lactobacillus species than 16S rDNA, and all of strains were clearly identified as L. casei (63 strains), L. plantarum (58 strains) and L. helveticus (113 strains) by comparison of sequences with the type strains. From our results, the partial sequences of three genes had a higher discriminatory power than 16S rRNA gene sequences and were an alternative molecular tool for the taxonomical analysis of L. casei group, L. plantarum group and L. acidophilus group.     

Real-time PCR targeting OmpA gene for detection of Cronobacter spp. in powdered infant formula

Cronobacter spp. (formerly Enterobacter sakazakii), a pathogen commonly found in powdered infant formula (PIF), is a rare cause of invasive infection with a high mortality rate in neonates. In the present study, a realtime PCR assay was conducted to identify the pathogens in PIF using a TaqMan probe targeting the outer membrane protein A gene (ompA) of Cronobacter spp. The specificity of the PCR assay was tested against 25 strains of Cronobacter spp. and 38 non-Cronobacter bacterial species. The detection limits of this method are 1.0×10² copy/μL in standard plasmid, 1.1 CFU/100 g in PIF through 38 h of enrichment, and 2.8×10² CFU/mL in phosphate-buffered saline (pH 7.0). Based on the detection limits, real-time PCR is more sensitive than simplex and duplex PCR. These methods were successfully applied to actual samples, indicating that this real-time PCR assay can be used for the detection of Cronobacter spp. in PIF.     

Microbiological Quality of Raw Dried Pasta from the German Market,with Special Emphasis on Cronobacter Species

The microbiological quality of 132 dried pasta products available on the German market, originating from 11 different countries, was studied. Sample materials included soft or durum wheat products, some of which produced with other ingredients such as eggs, spices, or vegetables. Parameters included hygiene indicators (aerobic plate count, mold count, the presence of Enterobacteriaceae) and pathogenic/toxinogenic bacterial species (Salmonella spp., Staphylococcus aureus, presumptive Bacillus cereus, and Cronobacter spp.). The overall results of hygiene parameters indicated a satisfactory quality. Salmonella was not found in any sample. Three samples were positive for S. aureus (10² to 10⁴ colony forming unit (CFU)/g). Presumptive B. cereus at levels of 10³ to 10⁴ CFU/g were detected in 3 samples. Cronobacter spp. were isolated from 14 (10.6%) products. Of these, 9 isolates were identified as C. sakazakii, 2 each as C. turicensis and C. malonaticus, and 1 as C. muytjensii. The isolates were assigned to 9 multilocus sequence typing (MLST) sequence types and to 14 different PFGE profiles. Although pasta products are typically cooked before consumption, some consumers, and children in particular, may also eat raw pasta as nibbles. Raw pasta seems to be a relevant source of exposure to dietary Cronobacter spp., although health risks are probably restricted to vulnerable consumers. High numbers of presumptive B. cereus as found in some samples may be a risk after improper storage of cooked pasta products because toxinogenic strains are frequently found within this species.     

Development and application of a sensitive,rapid, and reliable immunomagnetic separation-PCR detection method for Cronobacter spp.

Cronobacter spp. have been linked to clinical cases of infection in both adults and infants. Enrichment of Cronobacter spp. before detection has been necessary but is quite time consuming. Hence, we sought to develop an immunomagnetic separation (IMS) PCR method that could shorten the time of enrichment before the detection of Cronobacter spp. The polyclonal antibody used in this immunomagnetic separation was prepared based on the outer membrane protein A of Cronobacter sakazakii China Center of Industrial Culture Collection 21560 and had high specificity to the target. The primers used in the IMS-PCR method also showed high specificity. The detection limit of IMS-PCR for pure C. sakazakii culture was 5.2 × 10² cfu/mL. Cronobacter sakazakii in artificially contaminated powdered infant formula (PIF) was also detected at a detection limit of 5.2 × 10² cfu/mL. After 8 h of enrichment, the detection limit in PIF was lower than 5.2 × 10¹ cfu/mL. An interference test using Escherichia coli in artificially contaminated PIF showed that the IMS-PCR method developed in this study had a good ability to resist interference. Finally, the IMS-PCR method was applied to the detection of Cronobacter in food samples and was shown to be reliable. Thus, this newly developed IMS-PCR detection method was quite sensitive, rapid, and reliable and could be applied to the detection of Cronobacter in foods.     

Rapid detection and identification of Streptococcus macedonicus by species-specific PCR and DNA hybridisation

The aim of this study was to develop a simple and specific method for the rapid detection and identification of Streptococcus macedonicus. The method was based on polymerase chain reaction (PCR) using species-specific primers derived from the 16S rRNA gene. Specific identification was proven on seven S. macedonicus strains, while 16 strains belonging to different lactic acid bacteria species were tested negative. The PCR assay was capable of detecting 100 pg of S. macedonicus DNA, and it was also efficient on single colonies of the bacterium. Furthermore, the same bacterial strains were used for the specificity evaluation of a S. macedonicus species-specific probe. Neither species-specific PCR nor DNA hybridisation experiments could differentiate Streptococcus waius from S. macedonicus, due to the identity of the 16S rRNA gene of the two species, indicating high phylogenetical relatedness. This was further confirmed by the comparative sequence analysis of the 16S-23S rRNA intergenic regions. It was thus clearly demonstrated that S. waius, recently described as a novel Streptococcus species, is phylogenetically identical to S. macedonicus.     

Prevalence,genetic diversity,and antibiotic susceptibility of <Emphasis Type="Italic">Cronobacter</Emphasis> spp. (<Emphasis Type="Italic">Enterobacter sakazakii</Emphasis>) isolated from <Emphasis Type="Italic">Sunshik</Emphasis>, its ingredients and soils

The prevalence, genetic diversity, and antibiotic susceptibility of Cronobacter species (Enterobacter sakazakii) isolated from sunshik products, its ingredients, and root vegetable farm’s soils were investigated to analyze main reservoirs and contaminated sources of Cronobacter spp. Cronobacter spp. was isolated from 9 of 15 sunshik products, 26 of 72 its ingredients, and 2 of 39 soils. The root vegetables such as sweet potato and carrot showed higher contamination rate (70%) than the other sunshik ingredients. All isolates showed 929 bp band amplified from 16S rRNA and resistant to ampicillin, amoxicillin/clavulanic acid, and cefazolin. All isolates showed diverse random-amplified polymorphic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE) band patterns. However, 3 cases of RAPD banding patterns between clinical strains and isolates from sunshik products and root vegetables (yam, carrot) were related with similarities level of 80%. These studies indicated that root vegetables can be an important contamination source of Cronobacter spp. in sunshik products. Thus, the preparation of root vegetables for manufacturing sunshik products used as a weaning diet was handled with more care than the other sunshik ingredients.     

Two novel analytical methods based on polyclonal and monoclonal antibodies for the rapid detection of Cronobacter spp.: Development and application in powdered infant formula

Cronobacter spp. are opportunistic pathogens, and infections are associated with a high mortality rate. In the current study, monoclonal antibodies (mAbs) and polyclonal antibodies (pAbs) were generated using heat-inactivated C. sakazakii strain ATCC29544 as the immunogen. Following assay optimization, an indirect enzyme-linked immunosorbent assay (ELISA) based on pAbs and a sandwich ELISA based on mAbs and pAbs were established for the detection of Cronobacter spp. The indirect ELISA detected all species of Cronobacter assayed, and the limit of detection (LOD) was established as 10⁵ cfu/mL. In contrast, the sandwich ELISA was specific for C. sakazakii and had greater sensitivity than the indirect ELISA (LOD of 2 × 10⁴ cfu/mL). Following 10 h of enrichment, Cronobacter spp. were detected using either of the two analytical methods in samples inoculated with 1 cfu/100 g powdered infant formula (PIF). The results from this study demonstrated that both of these novel ELISAs were specific, sensitive, and rapid assays for the screening of pathogenic Cronobacter spp. in PIF.     

Species of Cronobacter – A review of recent advances in the genus and their significance in infant formula milk

Enterobacter sakazakii has recently been reclassified as the novel genus Cronobacter. These bacteria have been associated with disease outbreaks and sporadic infections, particularly in premature and immunocompromised infants. Cronobacter spp. can cause foodborne infections such as neonatal meningitis, septicaemia and necrotising enterocolitis. Infant formula milk is the only food source that has been epidemiologically linked to disease outbreaks caused by Cronobacter spp. This holds particular risk for premature and immunocompromised infants. This review focuses on the recent reclassification of Cronobacter and the importance that these pathogens pose to the food industry. The inactivation, inhibition, thermal, osmotic and desiccation tolerances of these bacteria are described, as well as improvements made in the isolation and detection methods of members of Cronobacter from food products.     

Stability and growth characteristics of GFPuv-labeled Cronobacter sakazakii isolated from foods

Stability of ultraviolet green fluorescent protein (GFPuv)-labeled Cronobacter sakazakii and C. muytjensii isolated from foods and the effects of the plasmid GFPuv (pGFPuv) on growth were analyzed. PCR analysis and microscopic observation showed that C. sakazakii and C. muytjensii isolates took up the plasmid into cells and expressed the GFPuv gene. All Cronobacter transformants maintained this plasmid after frozen storage and 2 consecutive subcultures. The C. sakazakii FWHd16 transformant was the most stable, while the C. muytjensii FWHd11 transformant was the least stable. All transformants showed nearly identical growth curves during lag, log and stationary phases, compared to wild type parental isolates. The maximum bacterial growth rates (μ_max) of the transformants and parents were similar, indicating that the presence of pGFPuv in transformants did not affect cell growth. Stable GFPuv-labeled Cronobacter has potential for use in tracking bacterial behavior during food handling and drying.     

Short communication: Roles of outer membrane protein W on survival,cellular morphology,and biofilm formation of Cronobacter sakazakii in response to oxidative stress

Cronobacter species are a group of opportunistic food-borne pathogens that cause rare but severe infections in neonates. Tolerance to environmental stress in Cronobacter is known; however, factors involved in oxidative stress are undefined. In this study, Cronobacter sakazakii survival, cellular morphology, and biofilm formation in response to oxidative stress were evaluated between the wild type (WT) and an outer membrane protein W (OmpW) mutant. The survival rates of ΔOmpW strain after treatment with 1.0 and 1.5 mM hydrogen peroxide were significantly reduced compared with those of WT. Morphological changes, including cell membrane damage and cell fragmentation, in ΔOmpW were more predominant than those in WT. By crystal violet staining, we also observed increased biomass in ΔOmpW biofilms as compared with WT following treatment with 0.5 and 1.0 mM H₂O₂. Biofilms using scanning electron microscopy and confocal laser scanning microscopy further confirmed the structural changes of biofilms between WT and ΔOmpW in response to oxidative stress. The current findings show that OmpW contributed to survival of planktonic cells under oxidative stress and the deletion of OmpW facilitated the biofilm formation in C. sakazakii to adapt to oxidative stress.