Qiongqiong Yan

Cronobacter species (formerly known as Enterobacter sakazakii) in powdered infant formula: a review of our current understanding of the biology of this bacterium

Cronobacter species (formerly known as Enterobacter sakazakii) are opportunistic pathogens that can cause necrotizing enterocolitis, bacteraemia and meningitis, predominantly in neonates. Infection in these vulnerable infants has been linked to the consumption of contaminated powdered infant formula (PIF). Considerable research has been undertaken on this organism in the past number of years which has enhanced our understanding of this neonatal pathogen leading to improvements in its control within the PIF production environment. The taxonomy of the organism resulted in the recognition of a new genus, Cronobacter, which consists of seven species. This paper presents an up‐to‐date review of our current knowledge of Cronobacter species. Taxonomy, genome sequencing, current detection protocols and epidemiology are all discussed. In addition, consideration is given to the control of this organism in the manufacturing environment, as a first step towards reducing the occurrence of this pathogen in PIF.

Molecular characterization of blaESBL–harboring conjugative plasmids identified in multi-drug resistant Escherichia coli isolated from food-producing animals and healthy humans

Background: Extended-spectrum β-lactamase (ESBL)-encoding genes are frequently mapped to plasmids, yet few of these structures have been characterized at the molecular level, to date. Methods: Eighty-seven ESBL-producing Escherichia coli were isolated from fecal samples of food-producing animals and healthy humans in Switzerland from 2009 to 2011. Plasmid DNA of all isolates was purified. Broth mating assays were carried out individually for 32 isolates to determine if the ESBL marker could be transferred by conjugation. The plasmid sizes were determined by S1-nuclease pulsed-field gel electrophoresis (PFGE) and the plasmids were typed by PCR-based replicon typing. Susceptibility tests by disk diffusion followed with a re-analysis S1-nuclease PFGE and PCRs were performed to confirm plasmid transfer. Microarray was performed to detect additional antibiotic resistance markers and multi-locus sequence typing was also performed in selected donor strains. The phylotypes were identified by triplex PCR. Results: About half (n = 46) of the 87 isolates carried small (<20-kb) plasmids. All selected 32 isolates contained large plasmids (ranging in sizes from 20- to 600-kb). Eleven plasmid replicon types were detected. Of these, IncFIA (n = 5), IncFIB (n = 9), and IncK/B (n = 4) were common. Nine isolates demonstrated the ability to transfer their cefotaxime resistance marker at high transfer rates. Plasmid profile re-analysis of these transconjugants identified 16 plasmids. IncFIB and IncI1 were the most prevalent replicon types. Phylogenetic grouping showed that five of the nine donor strains belonged to phylogroup B1. Nine different sequence types were identified in nine tested donor strains. Conclusion: Characterization of these ESBL-encoding conjugative plasmids extends our understanding on these resistance markers in multi-drug resistant E. coli cultured from healthy human and animal sources.

Nucleotide sequences of 16 transmissible plasmids identified in nine multidrug-resistant Escherichia coli isolates expressing an ESBL phenotype isolated from food-producing animals and healthy humans

OBJECTIVES Nine extended-spectrum β-lactamase (ESBL)-producing Escherichia coli isolated from healthy humans and food-producing animals were found to transfer their cefotaxime resistance marker at high frequency in laboratory conjugation experiments. The objective of this study was to completely characterize 16 transmissible plasmids that were detected in these bacterial isolates. METHODS The nucleotide sequences of all 16 plasmids were determined from transconjugants using next-generation sequencing technology. Open reading frames were assigned using Rapid Annotation using Subsystem Technology and analysed by BLASTn and BLASTp. The standard method was used for plasmid multilocus sequence typing (pMLST) analysis. Plasmid structures were subsequently confirmed by PCR amplification of selected regions. RESULTS The complete circularized nucleotide sequence of 14 plasmids was determined, along with that of a further two plasmids that could not be confirmed as closed. These ranged in size from 1.8 to 166.6 kb. Incompatibility groups and pMLSTs identified included IncI1/ST3, IncI1/ST36, IncN/ST1, IncF and IncB/O, and those of the same Inc types presented a similar backbone structure despite being isolated from different sources. Eight plasmids contained bla(CTX-M-1) genes that were associated with either ISEcp1 or IS26 insertion sequence elements. Six plasmids isolated from humans and chickens were identical or closely related to the IncI1 reference plasmid, R64. CONCLUSIONS These data, based on comparative sequence analysis, highlight the successful spread of blaESBL-harbouring plasmids of different Inc types among isolates of human and food-producing animal origin and provide further evidence for potential dissemination routes.

Genome Sequence of Cronobacter sakazakii SP291, a Persistent Thermotolerant Isolate Derived from a Factory Producing Powdered Infant Formula

ABSTRACT Cronobacter is an opportunistic pathogen associated with meningitis in neonates. Based on long-term surveillance of a powdered infant formula production facility, a persistent and thermotolerant isolate, denoted Cronobacter sakazakii SP291, was detected. Here we report the complete genome along with the sequences of three plasmids identified in this organism.

Comparative Genotypic and Phenotypic Analysis of Cronobacter Species Cultured from Four Powdered Infant Formula Production Facilities: Indication of Pathoadaptation along the Food Chain

ABSTRACT Cronobacter species are opportunistic pathogens commonly found in the environment. Among the seven Cronobacter species, Cronobacter sakazakii sequence type 4 (ST-4) is predominantly associated with recorded cases of infantile meningitis. This study reports on a 26-month powdered infant formula (PIF) surveillance program in four production facilities located in distinct geographic regions. The objective was to identify the ST(s) in PIF production environments and to investigate the phenotypic features that support their survival. Of all 168 Cronobacter isolates, 133 were recovered from a PIF production environment, 31 were of clinical origin, and 4 were laboratory type strains. Sequence type 1 (n = 84 isolates; 63.9%) was the dominant type in PIF production environments. The majority of these isolates clustered with an indistinguishable pulsotype and persisted for at least an 18-month period. Moreover, DNA microarray results identified two phylogenetic lineages among ST-4 strains tested. Thereafter, the ST-1 and -4 isolates were phenotypically compared. Differences were noted based on the phenotypes expressed by these isolates. The ST-1 PIF isolates produced stronger biofilms at both 28°C and 37°C, while the ST-4 clinical isolates exhibited greater swimming activity and increased binding to Congo red dye. Given the fact that PIF is a low-moisture environment and that the clinical environment provides for an interaction between the pathogen and its host, these differences may be consistent with a form of pathoadaptation. These findings help to extend our current understanding of the epidemiology and ecology of Cronobacter species in PIF production environments.

Development of a Custom-Designed, Pan Genomic DNA Microarray to Characterize Strain-Level Diversity among Cronobacter spp.

Cronobacter species cause infections in all age groups; however neonates are at highest risk and remain the most susceptible age group for life-threatening invasive disease. The genus contains seven species:Cronobacter sakazakii, Cronobacter malonaticus, Cronobacter turicensis, Cronobacter muytjensii, Cronobacter dublinensis, Cronobacter universalis, and Cronobacter condimenti. Despite an abundance of published genomes of these species, genomics-based epidemiology of the genus is not well established. The gene content of a diverse group of 126 unique Cronobacter and taxonomically related isolates was determined using a pan genomic-based DNA microarray as a genotyping tool and as a means to identify outbreak isolates for food safety, environmental, and clinical surveillance purposes. The microarray constitutes 19,287 independent genes representing 15 Cronobacter genomes and 18 plasmids and 2,371 virulence factor genes of phylogenetically related Gram-negative bacteria. The Cronobacter microarray was able to distinguish the seven Cronobacter species from one another and from non-Cronobacter species; and within each species, strains grouped into distinct clusters based on their genomic diversity. These results also support the phylogenic divergence of the genus and clearly highlight the genomic diversity among each member of the genus. The current study establishes a powerful platform for further genomics research of this diverse genus, an important prerequisite toward the development of future countermeasures against this foodborne pathogen in the food safety and clinical arenas.

Cronobacter: an emergent pathogen causing meningitis to neonates through their feeds.

The recognition of Cronobacter as a public health concern was raised when powdered infant formula (PIF) was linked to several neonatal meningitis outbreaks. It is an opportunistic pathogen that causes necrotising enterocolitis, infantile septicaemia, and meningitis which carries a high mortality rate among neonates. It has been also linked with cases of infection in adults and elderly. Over the past decade, much focus has been made on developing sensitive and specific characterisation, detection, and isolation methods to ascertain the quality of foods, notably contamination of PIF with Cronobacter and to understand its ability to cause disease. Whole genome sequencing has unveiled several putative virulence factors, yet the full capacity of the pathogenesis of Cronobacter has not yet been elucidated.

Strategies for the identification and tracking of Cronobacter species: an opportunistic pathogen of concern to neonatal health

Cronobacter species are emerging opportunistic food-borne pathogens, which consists of seven species, including C. sakazakii, C. malonaticus, C. muytjensii, C. turicensis, C. dublinensis, C. universalis, and C. condimenti. The organism can cause severe clinical infections, including necrotizing enterocolitis, septicemia, and meningitis, predominately among neonates <4 weeks of age. Cronobacter species can be isolated from various foods and their surrounding environments; however, powdered infant formula (PIF) is the most frequently implicated food source linked with Cronobacter infection. This review aims to provide a summary of laboratory-based strategies that can be used to identify and trace Cronobacter species. The identification of Cronobacter species using conventional culture method and immuno-based detection protocols were first presented. The molecular detection and identification at genus-, and species-level along with molecular-based serogroup approaches are also described, followed by the molecular sub-typing methods, in particular pulsed-field gel electrophoresis and multi-locus sequence typing. Next generation sequence approaches, including whole genome sequencing, DNA microarray, and high-throughput whole-transcriptome sequencing, are also highlighted. Appropriate application of these strategies would contribute to reduce the risk of Cronobacter contamination in PIF and production environments, thereby improving food safety and protecting public health.

A proposed harmonized LPS molecular-subtyping scheme for Cronobacter species.

Cronobacter are opportunistic pathogens, which cause infections in all age groups. To aid the characterization of Cronobacter in foods and environments a harmonized LPS identification scheme for molecular serotyping is needed. To this end, we studied 409 Cronobacter isolates representing the seven Cronobacter species using two previously reported molecular serotyping schemes, described here as Mullane-Jarvis (M-J) and Sun schemes. PCR analysis revealed many overlapping results that were obtained when independently applying the two serotyping schemes. There were complete agreements between the two PCR schemes for Cronobacter sakazakii (Csak) O:1, Csak O:3, and Csak O:7 serotypes. However, only thirty-five of 41 Csak O:4 strains, identified using the M-J scheme, were PCR-positive with the Sun scheme primers. Also the Sun scheme Csak O:5 primers failed to identify this serotype in any of the C. sakazakii strains tested, but did recognize seven Cronobacter turicensis strains, which were identified as Ctur O:3 using the M-J scheme. Similarly, the Sun scheme Csak O:6 primers recognized 30 Cronobacter malonaticus O:2 strains identified with the M-J scheme, but failed to identify this serotype in any C. sakazakii strain investigated. In this report, these findings are summarized and a harmonized molecular-serotyping scheme is proposed which is predicated on the correct identification of Cronobacter species, prior to serotype determination. In summary, fourteen serotypes were identified using the combined protocol, which consists of Csak O:1-O:4, and Csak O:7; Cmal O:1-O:2; Cdub O:1-O:2, Cmuy O:1-O:2, Cuni O:1, as well as Ctur O:1 and Ctur O:3.