Multiplex PCR Assay Targeting a Diguanylate Cyclase-Encoding Gene,
            <i>cgcA</i>
            , To Differentiate Species within the Genus Cronobacter

Carter, L.; Lindsey, Laura Anne; Grim, Christopher J.; Sathyamoorthy, Venugopal; Jarvis, Karen G.; Gopinath, Gopal; Lee, C.; Sadowski, J. A.; Trach, Larisa H.; Pava‐Ripoll, Monica; McCardell, Barbara A.; Tall, Ben D.; Hu, Lan

doi:10.1128/aem.02898-12

Cited by 56 publications

(63 citation statements)

References 19 publications

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“…Although C. sakazakii and C. malonaticus were found to be closely related and difficult to distinguish by 16S rDNA gene sequence analysis, a MLST scheme reported by Baldwin et al (2009) was shown to support the separation of these two species (Yan et al, 2012). Other supporting evidence which suggested that C. malonaticus and C. sakazakii are separate species were the single nucleotide polymorphisms identified for two genes, rpoB and cgcA, as reported by Stoop et al (2009) and Carter et al (2013). Their work led to the development of two species-specific end-point PCR assay schemes that are now widely used for speciation of isolates.…”

Section: Molecular Characterization Of Strainssupporting

confidence: 48%

Cronobacter spp. – opportunistic food-borne pathogens. A review of their virulence and environmental-adaptive traits

Jaradat

Mousa

Elbetieha

et al. 2014

Journal of Medical Microbiology

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The genus Cronobacter consists of a diverse group of Gram-negative bacilli and comprises seven species: Cronobacter sakazakii, Cronobacter malonaticus, Cronobacter muytjensii, Cronobacter turicensis, Cronobacter dublinensis, Cronobacter universalis and Cronobacter condimenti. Cronobacter are regarded as opportunistic pathogens, and have been implicated in newborn and infant infections, causing meningitis, necrotizing enterocolitis and bacteraemia or sepsis. Cronobacter virulence is believed to be due to multiple factors. Some strains were found to produce diarrhoea or cause significant fluid accumulation in suckling mice. Two iron acquisition systems (eitCBAD and iucABCD/iutA), Cronobacter plasminogen activator gene (cpa), a 17 kb type VI secretion system (T6SS), and a 27 kb filamentous haemagglutinin gene (fhaBC) and associated putative adhesins locus are harboured on a family of RepFIB-related plasmids (pESA3 and pCTU1), suggesting that these are common virulence plasmids; 98 % of 229 tested Cronobacter strains possessed these plasmids. Even though pESA3 and pCTU1 share a common backbone composed of the repA gene and eitCBAD and iucABCD/iutA gene clusters, the presence of cpa, T6SS and FHA loci depended on species, demonstrating a strong correlation with the presence of virulence traits, plasmid type and species. Other factors were observed, in that Cronobacter form biofilms, and show unusual resistance to heat, dry and acid stress growth conditions. The outer-membrane protein A is probably one of the bestcharacterized virulence markers of Cronobacter. Furthermore, it was reported that Cronobacter employ phosphatidylinositide 3-kinase/Akt signalling, which activates protein kinase C-a and impairs the host cell's mitogen-activated protein kinase pathway, in order to invade cells. Cronobacter can also use immature dendritic cells and macrophages to escape the immune response. This review addresses the various virulence and environmental-adaptive characteristics possessed by members of the genus Cronobacter.

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Section: Molecular Characterization Of Strainssupporting

confidence: 48%

Cronobacter spp. – opportunistic food-borne pathogens. A review of their virulence and environmental-adaptive traits

Jaradat

Mousa

Elbetieha

et al. 2014

Journal of Medical Microbiology

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“…as previously described by Carter et al (2013) (Table 1). Amplification procedure involved an initial denaturation for 3 min at 94 ºC followed by 25 cycles of 30 sec at 94 °C, 30 sec at 62 °C and 60 sec at 72 °C and finally 5 min at 72 °C for elongation (Carter et al, 2013).…”

Section: Typing Of Cronobacter Sppmentioning

confidence: 99%

Prevalence and identification by multiplex polymerase chain reaction patterns of Cronobacter spp. isolated from plant-based foods

Aksu

Altunatmaz

Issa³

et al. 2016

Food Sci. Technol

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Cronobacter spp. involves a group of opportunistic pathogens that cause meningitis in newborns, immunosuppressed individuals with a mortality rate of 50-80%. Seven species like C. sakazakii, C. malonaticus, C. muytjensii, C. turicensis, C. dublinensis, C. universalis, C. condimenti are included in this genus which has been a subject of research especially in the bacteriologic analysis of baby foods. However, since these species were detected also in prepared foodstuffs. The objective of this study was to assert the presence of Cronobacter spp. in foodstuff offered for sale in Turkey. A total of 151 prepared foodstuffs including a variety of spice, flour, instant soup were purchased from different sales points. The presence of Cronobacter spp. were investigated in these samples. Cronobacter suspected isolates which were obtained by microbiological analyses were confirmed by PCR targeted to gyrB gene and were then identified by multiplex PCR. Prevalence of Cronobacter spp was estimated to be 17.88%. Out of 27 Cronobacter spp. isolates obtained, 13(48.1%), 6(22.2%), 5(18.5%), 3(11.1%) belonged to C. sakazakii, C. muytjensii, C. turicensis, C. malonaticus species, respectively. Consequently, the presence of the bacteria in widely consumed foodstuff revealed that Cronobacter spp. is subject to monitoring due to its opportunistic nature in terms of public health concern.Keywords: Cronobacter spp.; flour; instant soup; PCR; spice.Practical Application: Cronobacter spp in plant-based foods may compose health risks.

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“…To identify Cronobacter strains at the genus, species, and serotype levels, the proposed schema for Cronobacter spp. (29) was applied using molecular detection techniques: an alpha-glucosidase-based PCR assay was performed to confirm the bacterial genus (37), and the species of the Cronobacter strains were identified using rpoB and cgcA as the gene targets for the PCR amplification (7,38). Only strains that were confirmed to be C. sakazakii were then subjected to multiplex PCR to identify the serotype.…”

Section: Methodsmentioning

confidence: 99%

“…Therefore, the genus and species of a collection of 76 strains that were previously identified as C. sakazakii at our institute by classical culture methods, according to the ISO standard in reference 25, were verified using a PCR method (7,37,38). Thereafter, strains were serotyped using a multiplex PCR method (18,20).…”

Section: Identification Of C Sakazakii Serotypesmentioning

confidence: 99%

Simultaneous Rapid Detection and Serotyping of Cronobacter sakazakii Serotypes O1, O2, and O3 by Using Specific Monoclonal Antibodies

Scharinger

Dietrich

Kleinsteuber

et al. 2016

Appl Environ Microbiol

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Cronobacter sakazakii is a foodborne pathogen associated with rare but often lethal infections in neonates. Powdered infant formula (PIF) represents the most frequent source of infection. Out of the identified serotypes (O1 to O7), O1, O2, and O3 are often isolated from clinical and PIF samples. Serotype-specific monoclonal antibodies (MAbs) suitable for application in enzyme immunoassays (EIAs) for the rapid detection of C. sakazakii have not yet been developed. In this study, we created specific MAbs with the ability to bind to C. sakazakii of serotypes O1, O2, and O3. Characterization by indirect EIAs, immunofluorescence, motility assays, and immunoblotting identified lipopolysaccharide (LPS) and exopolysaccharide (EPS) as the antigenic determinants of the MAbs. The established sandwich EIAs were highly sensitive and were able to detect between 2 ؋ 10 3 and 9 ؋ 10 6 CFU/ml. Inclusivity tests confirmed that 93% of serotype O1 strains, 100% of O2 strains, and 87% of O3 strains were detected at low cell counts. No cross-reactivity with >100 strains of Cronobacter spp. and other Enterobacteriaceae was observed, except for that with C. sakazakii serotype O3 and Cronobacter muytjensii serotype O1. Moreover, the sandwich EIAs detected C. sakazakii in PIF samples artificially contaminated with 1 to 10 bacterial cells per 10 g of sample after 15 h of preenrichment. The use of these serotype-specific MAbs not only allows the reliable detection of C. sakazakii strains but also enables simultaneous serotyping in a simple sandwich EIA method. Cronobacter spp. are Gram-negative opportunistic foodborne pathogens of the family Enterobacteriaceae that cause rare but severe infections in patients of all age groups. In adults, Cronobacter spp. are often associated with nosocomial infections, including pneumonia, septicemia, wound infections, and osteomyelitis, while causing invasive disease in young infants and neonates (1-4). Among the seven identified Cronobacter species, C. sakazakii, C. malonaticus, C. muytjensii, C. turicensis, C. dublinensis, C. condimenti, and C. universalis (5-8), C. sakazakii plays a prominent role due to it causing life-threatening infections in neonates (9-11). Clinically manifested infections present as necrotizing enterocolitis, sepsis, and meningitis, with a mortality rate as high as 80% (1, 12, 13). Although C. sakazakii has been isolated from a variety of different plant-and animal-based food products (14, 15), the presence in powdered infant formula (PIF) seems crucial in the infection of neonates (9, 12, 16). According to an established O-antigen serotyping scheme based on rabbit antisera and a PCRbased serotyping method (17-21), seven serotypes (O1 to O7) have been identified for C. sakazakii. Serotypes O1 and O2 seem to be most prevalent in PIF samples and in clinical cases, whereas serotype O3 has been isolated quite frequently from PIF but not as often from clinical cases (19,(22)(23)(24).Today, the contamination of PIF by C. sakazakii is being detected using conventional microbiological m...

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Multiplex PCR Assay Targeting a Diguanylate Cyclase-Encoding Gene, cgcA , To Differentiate Species within the Genus Cronobacter

Cited by 56 publications

References 19 publications

Cronobacter spp. – opportunistic food-borne pathogens. A review of their virulence and environmental-adaptive traits

Cronobacter spp. – opportunistic food-borne pathogens. A review of their virulence and environmental-adaptive traits

Prevalence and identification by multiplex polymerase chain reaction patterns of Cronobacter spp. isolated from plant-based foods

Simultaneous Rapid Detection and Serotyping of Cronobacter sakazakii Serotypes O1, O2, and O3 by Using Specific Monoclonal Antibodies

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