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.     

Building PulseNet Latin America and Caribbean Salmonella regional database: First conclusions of genetic subtypes of S. Typhi,S. Typhimurium and S. Enteritidis circulating in six countries of the region

PulseNet Latin America and Caribbean Network (PulseNet LA and C) works together with PulseNet International sharing molecular epidemiologic information for the recognition and investigation of foodborne disease outbreaks.The participants of PulseNet LA and C perform standardized pulsed field gel electrophoresis (PFGE) protocols and analysis generating data that is incorporated into Regional Databases. In this study we present the relationship and distribution of genetic subtypes of Salmonella enterica ser. Typhimurium (STM), Salmonella enterica ser. Enteritidis (SE), and Salmonella enterica ser. Typhi (ST) human isolates circulating in six countries of the Region between 2005 and 2009, from the analysis of the Salmonella Database.The 70 ST isolates analyzed were diverse and none of the countries shared the same PFGE profiles with XbaI enzyme. These results show a high genetic diversity among the strains studied and provide background to trace future outbreaks and travel related cases. In the analysis of 550 STM isolates, we found 10 patterns shared at least between two countries, suggesting the need of further studies of attribution to the source of origin. Only one of these PFGE patterns was associated with a known outbreak. Among 225 SE isolates, a predominant subtype was identified, that grouped 83.5% of the isolates and was associated with foodborne outbreaks in five of the six countries; showing the need to use other subtyping techniques for this serovar.The continuous update of PulseNet LA and C Salmonella Regional Database provides an important tool for the laboratory based surveillance of the serovars analyzed, for the prevention and control of foodborne outbreaks, and for the detection of emerging strains in the Region.     

Comparison of methods for the microbiological identification and typing of Cronobacter species in infant formula

Cronobacter species represent an emerging opportunistic foodborne pathogen associated with meningitis and necrotizing enterocolitis in infants. Current evidence indicates that powdered infant formula (PIF) is the main source of Cronobacter contamination. A total of 75 strains of Cronobacter spp. from different geographic regions, as well as from PIF processing environments, were identified and typed with different methods, including biochemical profiling by the API 20E system (bioMérieux, Marcy l’Etoile, France), protein profiling by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), and genotypic profiling by ribotype. Analysis by MALDI-TOF MS and biochemical identification was more accurate compared with ribotype analysis. However, MALDI-TOF MS typing and ribotype analysis showed more discriminatory ability compared with biochemical phenotyping. In conclusion, MALDI-TOF MS is a rapid and reliable tool to identify Cronobacter spp. in PIF and has the potential to trace dissemination of Chronobacter along the production chain.     

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.     

Detection of Cronobacter species in powdered infant formula by probe-magnetic separation PCR

Cronobacter species are opportunistic foodborne pathogens associated with serious infections in preterm neonates and infants. Based on the epidemiological research, infant formula products are considered to be the main source of infections from this organism. Therefore, accurate methods are required for detection of Cronobacter species. In this study, the specific probe and primers for detection of this organism were designed and verified. The probe-magnetic beads were prepared for sequence capture, followed by PCR assay to detect the target gene. This probe-magnetic separation PCR assay could detect as few as 10³ cfu/mL of Cronobacter in artificially contaminated infant formulas in less than 4 h. The combination of magnetic beads and PCR showed the potential for the detection of Cronobacter in infant formulas and may have applications in the dairy industry.     

Whole genome sequencing as a typing tool for foodborne pathogens like Listeria monocytogenes – The way towards global harmonisation and data exchange

BackgroundVarious molecular typing methods are used for the surveillance of foodborne pathogens and outbreak investigations, differing widely in information content and discriminatory power. Presently, not least because of the rapid technological development, the focus is shifting to whole genome sequencing (WGS) as an analytical tool. As a result of globalisation of food trade, a comprehensive understanding of the association between the occurrence of human infections and causative pathogens has to be established to monitor and to prevent their spread. In this respect, the accuracy of WGS clearly supersedes that of previous tools.Scope and approachOur review describes the status quo of WGS in surveillance and outbreak investigations of foodborne pathogens through the example of Listeria monocytogenes. It highlights the value of WGS in trace-back of infections to food sources and provides an overview of methods used for data generation (wet lab) and analysis (dry lab). Altogether, progress but also challenges in the worldwide practical application of WGS for bacterial typing are described.Key findings and conclusionsThe current status of WGS differs widely between countries and even laboratory sites. A consensus has to be found concerning methods, quality measures, thresholds for data generation and analysis as well as rules for data sharing. International harmonisation is going to be indispensable on the way to data exchangeability which will finally support global control of foodborne pathogens.     

Prevalence of Staphylococcus aureus and methicillin-resistant S. aureus (MRSA) in food samples associated with foodborne illness in Alberta, Canada from 2007 to 2010

Consumption of foods containing Staphylococcus aureus can cause severe gastro-intestinal illness. Given the fact that over the past decade, Canada has seen increasing rates of methicillin-resistant S. aureus (MRSA) carriage and infection, the objective of this study was to investigate the impact of methicillin-susceptible S. aureus (MSSA) and MRSA on foodborne illness in Alberta, Canada. Between January 2007 and December 2010, there were 693 food samples associated with foodborne investigations submitted to the Alberta Provincial Laboratory for Public Health (ProvLab). These foods were screened for: Bacillus cereus, Clostridium perfringens, S. aureus, Aeromonas spp., Campylobacter spp., Escherichia coli O157:H7, Salmonella, Shigella spp., and Yersinia spp. S. aureus was identified in 10.5% (73/693) of samples, and of these, 59% (43/73) were co-contaminated with at least one other organism on the screening panel. The S. aureus positive samples included 29 meat, 20 prepared foods containing meat, 11 prepared foods not containing meat, 10 dairy, and three produce. Methicillin-resistance was not detected in any isolates tested. These findings indicate that the presence of S. aureus in food associated with foodborne investigations is a cause for concern, and although MRSA was not found, the potential for outbreaks exists, and ongoing surveillance should be sustained.     

The Cronobacter sp. in milk and dairy products: Detection and typing

Cronobacter is a group of important foodborne pathogens that have been implicated in meningitis, sepsis and necrotising enterocolitis, especially in neonates through consumption of contaminated powdered infant formula (PIF). The detection of PIF contamination is of great concern to the infant formula industry. In this article, phenotypic and genotypic methods for detecting and typing Cronobacter are discussed in relation to minimising and controlling the risk of Cronobacter sp. contamination. To reliably detect and type Cronobacter strains, the use of conventional microbiological methods in combination with molecular assays is recommended.     

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.     

Comprehensive molecular,probiotic, and quorum-sensing characterization of anti-listerial lactic acid bacteria,and application as bioprotective in a food (milk) model

Listeria monocytogenes is a major foodborne pathogen that adversely affects the food industry. In this study, 6 anti-listerial lactic acid bacteria (LAB) isolates were screened. These anti-listerial LAB isolates were identified via 16S rRNA gene sequencing and analyzed via repetitive extragenic palindromic-PCR. Probiotic assessment of these isolates, comprising an evaluation of the antibiotic susceptibility, tolerance to lysozyme, simulated gastric and intestinal juices, and gut conditions (low pH, bile salts, and 0.4% phenol), was carried out. Most of the isolates were resistant to streptomycin, vancomycin, gentamycin, kanamycin, and ciprofloxacin. All of the isolates were negative for virulence genes, including agg, ccf, cylA, cylB, cylLL, cylLS, cylM, esp, and gelE, and hemolytic activity. Furthermore, autoinducer-2 (a quorum-sensing molecule) was detected and quantified via HPLC with fluorescence detection after derivatization with 2,3-diaminonaphthalene. Metabolites profiles of the Lactobacillus sakei D.7 and Lactobacillus plantarum I.60 were observed and presented various organic acids linked with antibacterial activity. Moreover, freeze-dried cell-free supernatants from Lb. sakei (55 mg/mL) and Lb. plantarum (40 mg/mL) showed different minimum effective concentration (MEC) against L. monocytogenes in the food model (whole milk). In summary, these anti-listerial LAB isolates do not pose a risk to consumer health, are eco-friendly, and may be promising candidates for future use as bioprotective cultures and new probiotics to control contamination by L. monocytogenes in the food and dairy industries.     

RE-PCR variability and toxigenic profile of food poisoning, foodborne and soil-associated Bacillus cereus isolates from Brazil

Twenty-three Bacillus cereus isolates from food poisoning outbreaks associated with a diarrheal-type syndrome, fourteen foodborne isolates not associated with food poisoning and fifteen isolates from Brazilian soil samples were analyzed for the presence and genetic diversity (by RE-PCR) of the virulence genes ces (emetic toxin, cereulide), plcR-papR (pleiotropic regulator PlcR and peptide PapR), nheA (a component of the NHE complex), bceT (diarrheal enterotoxin bc-D-ENT), gyrB (B subunit of DNA gyrase), cytK-2 (necrotic enterotoxin cytotoxin K-2), and plcA (phosphatidylcholine-specific phospholipase C). Additionally, these isolates were phenotypically characterized for motility, hemolytic and lecithinase activities, as well as HBL enterotoxin production. The group of isolates associated with food poisoning had the highest occurrence of the phenotypically analyzed factors and the most frequent occurrence and highest genetic diversity of the plcR-papR, nheA, bceT, cytK-2, plcA, and gyrB genes. An analysis of molecular variance (AMOVA), in which all loci were analyzed, demonstrated that the genetic variation intragroup of isolates (92%) was significantly higher than that intergroup (8%) (P < 0.05). These results were corroborated by an analysis of the genetic differentiation between the groups, which was low/moderate, the result of a high degree of allele sharing. Our results suggest that B. cereus isolates with the potential to cause food poisoning outbreaks do not have a specific genetic profile characterized by the presence of a particular gene or allele among the genes assessed. On the contrary, different combinations of genes encoding virulence factors may be present in different isolates of B. cereus that potentially cause food poisoning outbreaks.     

Prevalence and characterization of foodborne pathogens from Australian dairy farm environments

The ability of foodborne pathogens to gain entry into food supply systems remains an ongoing concern. In dairy products, raw milk acts as a major vehicle for this transfer; however, the sources of pathogenic bacteria that contaminate raw milk are often not clear, and environmental sources of contamination or the animals themselves may contribute to the transfer. This survey examined the occurrence of 9 foodborne pathogens in raw milk and environments of 7 dairy farms (3 bovine, 3 caprine, and 1 ovine farm) in summer and autumn, in Victoria, Australia. A total of 120 samples were taken from sampling points common to dairy farms, including pasture, soil, feed, water sources, animal feces, raw milk, and milk filters. The prevalence of the Bacillus cereus group, Campylobacter, Clostridium perfringens, Cronobacter, Shiga-toxigenic Escherichia coli, Listeria, Salmonella, coagulase-positive staphylococci (CPS), and Yersinia enterocolitica across the farms was investigated. The 2 most prevalent bacteria, which were detected on all farms, were the B. cereus group, isolated from 41% of samples, followed by Cl. perfringens, which was isolated from 38% of samples. The highest occurrence of any pathogen was the B. cereus group in soil, present in 93% of samples tested. Fecal samples showed the highest diversity of pathogens, containing 7 of the 9 pathogens tested. Salmonella was isolated from 1 bovine farm, although it was found in multiple samples on both visits. Out of the 14 occurrences where any pathogen was detected in milk filters, only 5 (36%) of the corresponding raw milk samples collected at the same time were positive for the same pathogen. All of the CPS were Staphylococcus aureus, and were found in raw milk or milk filter samples from 6 of the 7 farms, but not in other sample types. Pathogenic Listeria species were detected on 3 of the 7 farms, and included 4 L. ivanovii-positive samples, and 1 L. monocytogenes-positive water sample. Shiga-toxigenic Escherichia coli were identified in fecal samples from 3 of the 7 farms and in a single raw milk sample. Cronobacter species were identified on 4 of the 7 farms, predominantly in feed samples. No Y. enterocolitica was detected. Results of this study demonstrate high standards of pathogen safety across the 7 farms, with a low incidence of pathogens detected in raw milk samples. Monitoring feed contamination levels may help control the spread of bacterial species such as Cl. perfringens and B. cereus through the farm environment, which is a natural reservoir for these organisms.     

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.     

Extended and global phylogenetic view of the Bacillus cereus group population by combination of MLST, AFLP, and MLEE genotyping data

The Bacillus cereus group of bacteria includes species that can cause food-poisoning or spoilage, such as B. cereus, as well as Bacillus anthracis, the cause of anthrax. In the present report we have conducted a multi-datatype analysis using tools from the HyperCAT database (http://mlstoslo.uio.no/) that we recently developed, combining data from multilocus sequence typing (Tourasse et al., 2010), amplified fragment length polymorphism, and multilocus enzyme electrophoresis typing techniques. We provide a comprehensive snapshot of the B. cereus group population, incorporating 2213 isolates including 450 from food and dairy products, in the form of both phylogenetic supertrees and superclusters of genetically closely related isolates. Our main findings include the detection of phylogenetically separated groups of isolates possibly representing novel evolutionary lineages within the B. cereus group, a putative new branch of B. anthracis, as well as new groups of related strains containing both environmental and clinical isolates. In addition, the multi-datatype analysis revealed to a larger extent than previously recognized that food-borne isolates can share identical genotyping profiles with strains from various other origins. Altogether, the global analysis confirms and extends the results underlining the opportunistic nature of B. cereus group organisms, and the fact that isolates responsible for disease outbreaks and contamination of foodstuffs can originate from various genetic backgrounds.     

Invited review: Stress resistance of Cronobacter spp. affecting control of its growth during food production

Members of the Cronobacter genus include food-borne pathogens that can cause infections in infants, with a mortality rate as high as 40 to 80%. The high fatality rate of Cronobacter and its isolation from numerous types of food, especially from powdered infant formula, demonstrate the serious nature of this organism. The source tracking of Cronobacter spp. and the analysis of high-frequency species from different sources are helpful for a more targeted control. Furthermore, the persistence during food processing and storage may be attributed to strong resistance of Cronobacter spp. to environment stresses such as heat, pH, and desiccation. There are many factors that support the survival of Cronobacter spp. in harsh environments, such as some genes, regulatory systems, and biofilms. Advanced detection technology is helpful for the strict monitoring of Cronobacter spp. In addition to the traditional heat treatment, many new control techniques have been developed, and the ability to control Cronobacter spp. has been demonstrated. The control of this bacteria is required not only during manufacture, but also through the selection of packaging methods to reduce postprocessing contamination. At the same time, the effect of inactivation methods on product quality and safety must be considered. This review considers the advances in our understanding of environmental stress response in Cronobacter spp. with special emphasis on its implications in food processing.     

Prevalence of staphylococcal enterotoxins, toxin genes and genetic-relatedness of foodborne Staphylococcus aureus strains isolated in the Marmara Region of Turkey

Staphylococcus aureus is a major foodborne pathogen and it has the ability to produce a number of extracellular toxins. We analyzed 1070 food samples obtained from retail markets and dairy farms in the Marmara Region of Turkey for the presence of S. aureus. Out of 147 isolates, 92 (62.6%) were enterotoxigenic. PCR was used to investigate the presence of staphylococcal enterotoxin genes (sea, seb, sec, sed, see, seg, seh, sei, sej, sek, sel, sem, sen, seo, sep, seq and seu), exfoliative toxin genes (eta and etb) and the toxic − shock syndrome toxin gene (tst). The PCR results showed that 53.3% of the isolates contained staphylococcal enterotoxin-like (SEl) toxin genes (seg, seh, sei, sej, sek, sel, sem, sen, seo, sep, seq and seu) which were more frequent than classical enterotoxin genes (sea to see). Furthermore, seo, sei, sem, seg, seu and sec were found in 37.0, 32.7, 30.4, 29.3, 29.3 and 27.2% of the isolates, respectively. The tst gene was detected and confirmed by DNA sequencing in 9 isolates. The presence of eta and etb were not found in the isolates. Enterotoxigenic capabilities of isolates with SEA-SEE were investigated by ELISA. Enterotoxigenic S. aureus isolates produced one to three enterotoxins, with the most frequently produced types being enterotoxin A and C. There was a correlation of 72.1% between production of a specific toxin and the presence of the respective genes. PFGE analysis was used to identify genetic-relatedness of enterotoxigenic S. aureus isolates and the results revealed that 13 groups of isolates from different or the same origin that contained the same genes showed 100% homology with indistinguishable band patterns. The other enterotoxigenic isolates showed related band patterns with 72-86% homology in sea-, 61-90% homology in sec-, 80-96% homology in seh-, and 69-96% homology in sep-positive isolates. To our knowledge, this is the first study to examine enterotoxins and related gene contents of S. aureus food isolates in the Marmara Region of Turkey.     

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.     

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.     

Simultaneous quantitative detection of viable Escherichia coli O157:H7, Cronobacter spp., and Salmonella spp. using sodium deoxycholate-propidium monoazide with multiplex real-time PCR

Escherichia coli O157:H7, Cronobacter spp., and Salmonella spp. are common food-borne pathogens in milk that may cause serious diseases. In the present study, we established a simple, rapid, and specific method to simultaneously detect viable E. coli O157:H7, Cronobacter spp., and Salmonella spp. in milk. Three specific genes, fliC from E. coli O157:H7, cgcA from Cronobacter spp., and invA from Salmonella spp., were selected and used to design primers and probes. False-positive results were eliminated with the use of a combined sodium deoxycholate (SD) and propidium monoazide (PMA) treatment. Using the optimized parameters, this SD-PMA treatment combined with multiplex real-time PCR (SD-PMA-mRT-PCR) detected E. coli O157:H7, Cronobacter spp. and Salmonella spp. respectively, at 10² cfu/mL in pure culture or artificially spiked skim milk samples. A reasonable recovery rate (from 100 to 107%) for detection of viable bacteria using the SD-PMA-mRT-PCR assay was obtained in the presence of dead bacteria at 10⁷ cfu/mL. The SD-PMA-mRT-PCR method developed in this study can accurately detect and monitor combined contamination with E. coli O157:H7, Cronobacter spp., and Salmonella spp. in milk and milk products.