The biochemical differentiation of Enterobacter sakazakii genotypes

Background  

Enterobacter sakazakii is an emergent pathogen that has been associated with neonatal infections through contaminated powdered infant milk formula. The species was defined by Farmer et al. (1980) who described 15 biogroups according to the biochemical characterization of 57 strains. This present study compares genotypes (DNA cluster groups based on partial 16S rDNA sequence analysis) with the biochemical traits for 189 E. sakazakii strains.     

Genome Sequence of Cronobacter sakazakii BAA-894 and Comparative Genomic Hybridization Analysis with Other Cronobacter Species

Background

The genus Cronobacter (formerly called Enterobacter sakazakii) is composed of five species; C. sakazakii, C. malonaticus, C. turicensis, C. muytjensii, and C. dublinensis. The genus includes opportunistic human pathogens, and the first three species have been associated with neonatal infections. The most severe diseases are caused in neonates and include fatal necrotizing enterocolitis and meningitis. The genetic basis of the diversity within the genus is unknown, and few virulence traits have been identified.

Methodology/Principal Findings

We report here the first sequence of a member of this genus, C. sakazakii strain BAA-894. The genome of Cronobacter sakazakii strain BAA-894 comprises a 4.4 Mb chromosome (57% GC content) and two plasmids; 31 kb (51% GC) and 131 kb (56% GC). The genome was used to construct a 387,000 probe oligonucleotide tiling DNA microarray covering the whole genome. Comparative genomic hybridization (CGH) was undertaken on five other C. sakazakii strains, and representatives of the four other Cronobacter species. Among 4,382 annotated genes inspected in this study, about 55% of genes were common to all C. sakazakii strains and 43% were common to all Cronobacter strains, with 10–17% absence of genes.

Conclusions/Significance

CGH highlighted 15 clusters of genes in C. sakazakii BAA-894 that were divergent or absent in more than half of the tested strains; six of these are of probable prophage origin. Putative virulence factors were identified in these prophage and in other variable regions. A number of genes unique to Cronobacter species associated with neonatal infections (C. sakazakii, C. malonaticus and C. turicensis) were identified. These included a copper and silver resistance system known to be linked to invasion of the blood-brain barrier by neonatal meningitic strains of Escherichia coli. In addition, genes encoding for multidrug efflux pumps and adhesins were identified that were unique to C. sakazakii strains from outbreaks in neonatal intensive care units.     

阪崎克罗诺杆菌噬菌体JC01的筛选和特性

【背景】阪崎克罗诺杆菌是一种食源性病原体,摄入受污染的婴儿配方奶粉(powdered infant formula, PIF)常引起新生儿坏死性小肠结肠炎和脑膜炎,对早产儿和免疫功能低下的婴儿健康甚至生命产生严重威胁。噬菌体具有特异性杀菌性,可成为一种新型生物防控制剂预防阪崎克罗诺杆菌和其他食源性致病菌的污染。【目的】从污水中分离出感染阪崎克罗诺杆菌噬菌体,评价其生物学特性、基因组生物信息及在婴儿配方奶粉中杀菌作用。【方法】采用双层琼脂法分离鉴定噬菌体,并测定其酸碱稳定性、温度稳定性、宿主范围和一步生长曲线,透射电镜观察形态,对其基因组进行二代测序和裂解功效测定。【结果】从污水中分离到一株新的能裂解阪崎克罗诺杆菌的噬菌体JC01,具有二十面立体对称头部和非收缩的长尾,噬菌体JC01基因组为双链DNA,由61 736 bp组成,预测有76个开放阅读框(open reading frame, ORF),不含有tRNA,基因组中不含有耐药基因和毒力基因。系统发育分析及基因比对分析显示噬菌体JC01是全新的噬菌体,归类于有尾噬菌体纲(Caudoviricetes)卡金斯病毒科(Casjensviridae)雅昆病毒属(Jacunavirus),被命名为Jacunavirus 01。噬菌体JC01在不同温度(−20-40 °C)和pH 5.0-9.0范围内稳定,且对婴儿配方奶粉污染的阪崎克罗诺杆菌具有良好的杀菌效果。【结论】JC01噬菌体是裂解阪崎克罗诺杆菌的新噬菌体,作为生物安全防控剂在食品生产和加工方面具有很大潜力。     

Genotyping and Source Tracking of Cronobacter sakazakii and C. malonaticus Isolates from Powdered Infant Formula and an Infant Formula Production Factory in China

Cronobacter spp. (formerly defined as Enterobacter sakazakii) are opportunistic bacterial pathogens of both infants and adults. In this study, we analyzed 70 Cronobacter isolates from powdered infant formula (PIF) and an infant formula production facility in China to determine possible contamination routes. The strains were profiled by multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), PCR-based O-antigen serotyping, and ompA and rpoB sequence analyses. The isolates were primarily Cronobacter sakazakii (66/70) or Cronobacter malonaticus (4/70). The strains were divided into 38 pulsotypes (PTs) using PFGE and 19 sequence types (STs) by MLST. In contrast, rpoB and ompA sequence analyses divided the strains into 10 overlapping clusters each. PCR serotyping of the 66 C. sakazakii and 4 C. malonaticus strains resulted in the identification of four C. sakazakii serotypes (O1, O2, O4, and O7) and a single C. malonaticus serotype, O2. The dominant C. sakazakii sequence types from PIF and an infant formula production factory in China were C. sakazakii clonal complex 4 (CC4) (n = 19), ST1 (n = 14), and ST64 (n = 11). C. sakazakii CC4 is a clonal lineage strongly associated with neonatal meningitis. In the process of manufacturing PIF, the spray-drying, fluidized-bed-drying, and packing areas were the main areas with Cronobacter contamination. C. sakazakii strains with the same pulsotypes (PT3 and PT2) and sequence types (ST1 and ST64) were isolated both from processing equipment and from the PIF finished product.     

Isolation of Cronobacter spp. (formerly Enterobacter sakazakii) from infant food, herbs and environmental samples and the subsequent identification and confirmation of the isolates using biochemical, chromogenic assays, PCR and 16S rRNA sequencing

Background  

Cronobacter spp. (formerly Enterobacter sakazakii), are a group of Gram-negative pathogens that have been implicated as causative agents of meningitis and necrotizing enterocolitis in infants. The pathogens are linked to infant formula; however, they have also been isolated from a wide range of foods and environmental samples.     

Occurrence of Cronobacter spp. in retail foods

Aims: To study the occurrence of Cronobacter spp. in foods and to investigate the phenotypic properties of the strains isolated. Methods and Results: A total of 53 strains of Cronobacter spp. isolated from 399 food samples were identified using conventional biochemical methods and MALDI‐TOF mass spectrometry. Foods of plant origin were the most frequently contaminated samples. No Cronobacter spp. were found in infant milk formula, wheat‐based infant food, pasteurized and raw cow milk, mincemeat, chicken, chickpea and potato dumpling powder. The individual species were identified as Cronobacter sakazakii (54·7%), Cronobacter malonaticus (28·4%), Cronobacter dublinensis (7·5%), Cronobacter muytjensii (7·5%) and Cronobacter turicensis (1·9%). Cronobacter sakazakii and C. malonaticus belong to biotype 1, 2, 2a, 3, 4 and 5, 5a, respectively. Cronobacter dublinensis strains were subdivided into biotypes 6 and 12. All strains were resistant to erythromycin and two of them were resistant to both erythromycin and tetracycline. Conclusions: Cronobacter spp. were isolated from various food samples pre‐eminently of plant origin and dried food ingredients. Significance and Impact of the Study: These findings will increase and detail our knowledge of the presence and diversity of Cronobacter spp. in foods.     

Adhesive properties of Enterobacter sakazakii to human epithelial and brain microvascular endothelial cells

Background  

Enterobacter sakazakii is an opportunistic pathogen that has been associated with sporadic cases and outbreaks causing meningitis, necrotizing enterocolitis and sepsis especially in neonates. However, up to now little is known about the mechanisms of pathogenicity in E. sakazakii. A necessary state in the successful colonization, establishment and ultimately production of disease by microbial pathogens is the ability to adhere to host surfaces such as mucous membranes, gastric and intestinal epithelial or endothelial tissue.     

A robotic DNA purification protocol and real-time PCR for the detection of Enterobacter sakazakii in powdered infant formulae

Background  

Enterobacter sakazakii is the causative agent of rare but severe food-borne infections associated with meningitis, necrotizing enterocolitis and sepsis in infants. Rehydrated powdered infant formulae have been implicated as the source of infection in several outbreaks and sporadic cases. In this work, a real time fluorescence resonance energy transfer PCR assay incorporating an internal amplification control (IAC) was developed for the specific detection of E. sakazakii in foods. Performance of the assay, coupled to an automated DNA extraction system and the E. sakazakii ISO-IDF (TS 22964/RM 210) enrichment procedure, was evaluated on infant formulae and samples from production environment.     

Comparative Analysis of Genome Sequences Covering the Seven Cronobacter Species

Background

Species of Cronobacter are widespread in the environment and are occasional food-borne pathogens associated with serious neonatal diseases, including bacteraemia, meningitis, and necrotising enterocolitis. The genus is composed of seven species: C. sakazakii, C. malonaticus, C. turicensis, C. dublinensis, C. muytjensii, C. universalis, and C. condimenti. Clinical cases are associated with three species, C. malonaticus, C. turicensis and, in particular, with C. sakazakii multilocus sequence type 4. Thus, it is plausible that virulence determinants have evolved in certain lineages.

Methodology/Principal Findings

We generated high quality sequence drafts for eleven Cronobacter genomes representing the seven Cronobacter species, including an ST4 strain of C. sakazakii. Comparative analysis of these genomes together with the two publicly available genomes revealed Cronobacter has over 6,000 genes in one or more strains and over 2,000 genes shared by all Cronobacter. Considerable variation in the presence of traits such as type six secretion systems, metal resistance (tellurite, copper and silver), and adhesins were found. C. sakazakii is unique in the Cronobacter genus in encoding genes enabling the utilization of exogenous sialic acid which may have clinical significance. The C. sakazakii ST4 strain 701 contained additional genes as compared to other C. sakazakii but none of them were known specific virulence-related genes.

Conclusions/Significance

Genome comparison revealed that pair-wise DNA sequence identity varies between 89 and 97% in the seven Cronobacter species, and also suggested various degrees of divergence. Sets of universal core genes and accessory genes unique to each strain were identified. These gene sequences can be used for designing genus/species specific detection assays. Genes encoding adhesins, T6SS, and metal resistance genes as well as prophages are found in only subsets of genomes and have contributed considerably to the variation of genomic content. Differences in gene content likely contribute to differences in the clinical and environmental distribution of species and sequence types.     

16S rRNA gene based analysis of <Emphasis Type="Italic">Enterobacter sakazakii</Emphasis> strains from different sources and development of a PCR assay for identification

Background  

E. sakazakii is considered to be an opportunistic pathogen, implicated in food borne diseases causing meningitis or enteritis especially in neonates and infants. Cultural standard identification procedures for E. sakazakii include the observation of yellow pigmentation of colonies and a positive glucosidase activity. Up to now, only one PCR system based on a single available 16S rRNA gene sequence has been published for E. sakazakii identification. However, in our hands a preliminary evaluation of this system to a number of target and non-target strains showed significant specificity problems of this system. In this study full-length 16S rRNA genes of thirteen E. sakazakii strains from food, environment and human origin as well as the type strain ATCC 51329 were sequenced. Based on this sequence data a new specific PCR system for E. sakazakii was developed and evaluated.     

Comparison of two chromogenic media and evaluation of two molecular based identification systems for Enterobacter sakazakii detection

Background  

Enterobacter sakazakii is a foodborne pathogen that has been associated with sporadic cases and outbreaks causing meningitis, necrotizing enterocolitis and sepsis especially in neonates. The current FDA detection method includes two enrichment steps, the subculturing of the second enrichment broth on a selective agar (VRBG), a further subculturing of selected grown colonies on TSA and the subsequent biochemical identification of yellow-pigmented colonies by API20E. However, there is a strong need for simplified methods for isolation and identification of E. sakazakii. In this study, two chromogenic media, which allow to indicate presumptive E. sakazakii colonies by the alpha glucosidase activity, as well as a newly developed 1,6-alpha-glucosidase based conventional PCR assay and a rRNA oligonucleotide probe based commercial test system for identification of presumptive E. sakazakii were evaluated on 98 target and non-target strains. The methods were compared with respect to specifiCity aspects.     

Identification of Enterobacter sakazakii from closely related species: The use of Artificial Neural Networks in the analysis of biochemical and 16S rDNA data

Background  

Enterobacter sakazakii is an emergent pathogen associated with ingestion of infant formula and accurate identification is important in both industrial and clinical settings. Bacterial species can be difficult to accurately characterise from complex biochemical datasets and computer algorithms can potentially simplify the process.     

Structures and genetics of Kdo-containing O-antigens of Cronobacter sakazakii G2706 and G2704, the reference strains of serotypes O5 and O6

Cronobacter sakazakii G2706 and G2704 are the reference strains of serotypes O5 and O6 in the serological classification of this species proposed recently. Mild acid degradation of the lipopolysaccharides of both strains resulted in cleavage of the O-polysaccharide chains at the acid-labile linkage of 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) to yield oligosaccharides representing repeating units of the O-polysaccharides. The oligosaccharides and alkali-degraded lipopolysaccharides were studied by sugar analysis along with 1D and 2D ¹H and ¹³C NMR spectroscopy, and the following O-polysaccharide structures were established:The structure of strain G2706 is unique among the known bacterial polysaccharide structures, whereas that of strain G2704 is identical to the structure of Cronobacter malonaticus 3267 [MacLean, L. L.; Vinogradov, E.; Pagotto, F.; Farber, J. M.; Perry, M. B. Biochem. Cell Biol.2009, 87, 927–932], except for that the latter lacks O-acetylation. Putative functions of the genes in the O-antigen gene clusters of C. sakazakii strains studied are in agreement with the O-polysaccharide structures.     

Identification of genomic differences between Campylobacter jejuni subsp. jejuni and C. jejuni subsp. doylei at the nap locus leads to the development of a C. jejuni subspeciation multiplex PCR method

Background  

The human bacterial pathogen Campylobacter jejuni contains two subspecies: C. jejuni subsp. jejuni (Cjj) and C. jejuni subsp. doylei (Cjd). Although Cjd strains are isolated infrequently in many parts of the world, they are obtained primarily from human clinical samples and result in an unusual clinical symptomatology in that, in addition to gastroenteritis, they are associated often with bacteremia. In this study, we describe a novel multiplex PCR method, based on the nitrate reductase (nap) locus, that can be used to unambiguously subspeciate C. jejuni isolates.     

Lactoferrin and free secretory component of human milk inhibit the adhesion of enteropathogenic Escherichia coli to HeLa cells

Background  

Diarrhoea caused by Escherichia coli is an important cause of infant morbidity and mortality in developing countries. Enteropathogenic Escherichia coli (EPEC) is considered one of the major causes of diarrhoea in children living in developing countries. The ability of diarrhoeagenic strains of E. coli to adhere to and colonize the intestine is the first step towards developing the disease. EPEC strains adhere to enterocytes and HeLa cells in a characteristic pattern known as localized adherence.     

Untreated and enzyme‐modified bovine whey products reduce association of Salmonella Typhimurium,Escherichia coli O157:H7 and Cronobacter malonaticus (formerly Enterobacter sakazakii) to CaCo‐2 cells

Aims: Adhesion of a micro‐organism to a cell surface is often considered to be the first step in pathogenesis. Inhibiting this process may have therapeutic effects in vivo. This study investigates the inhibitory effects of various bovine whey products on the association of Salm. Typhimurium, E. coli O157:H7 and C. malonaticus (formerly Enterobacter sakazakii) to the human CaCo‐2 cell line. Invasion of CaCo‐2 cells by Salm. Typhimurium and C. malonaticus was also examined. Methods and Results: Infection assays were performed by incubating pathogenic acteria with CaCo‐2 cells in the presence of untreated (UT) or enzyme‐modified (EM) whey products. Associated micro‐organisms were directly quantified by plate counts. Invasion of CaCo‐2 cells by Salm. Typhimurium and C. malonaticus in the presence/absence of test materials was also quantified using gentamicin protection assays. At a concentration of 40 mg ml^?1, some UT whey products reduced association and invasion, but this effect was enhanced following hydrolysis with porcine pancreatic lipase. Conclusions: Both UT and EM sweet whey protein concentrates (WPCs) were found to be particularly effective inhibitors of association and invasion. All EM whey products significantly (P < 0·05) inhibited invasion of C. malonaticus into epithelial cells, causing a 2‐log reduction in the quantity of these micro‐organisms internalized. Significance and Impact of the Study: The present study suggests that whey products can inhibit association to and invasion of CaCo‐2 cells by selected micro‐organisms and may be useful in the treatment and/or prevention of foodborne infections.     

Evolution and diversity of Rickettsia bacteria

Background  

Rickettsia are intracellular symbionts of eukaryotes that are best known for infecting and causing serious diseases in humans and other mammals. All known vertebrate-associated Rickettsia are vectored by arthropods as part of their life-cycle, and many other Rickettsia are found exclusively in arthropods with no known secondary host. However, little is known about the biology of these latter strains. Here, we have identified 20 new strains of Rickettsia from arthropods, and constructed a multi-gene phylogeny of the entire genus which includes these new strains.     

Genetical and functional investigation of fliC genes encoding flagellar serotype H4 in wildtype strains of Escherichia coli and in a laboratory E. coli K-12 strain expressing flagellar antigen type H48

Background  

Serotyping of O-(lipopolysaccharide) and H-(flagellar) antigens is a wideley used method for identification of pathogenic strains and clones of Escherichia coli. At present, 176 O- and 53 H-antigens are described for E. coli which occur in different combinations in the strains. The flagellar antigen H4 is widely present in E. coli strains of different O-serotypes and pathotypes and we have investigated the genetic relationship between H4 encoding fliC genes by PCR, nucleotide sequencing and expression studies.     

Characterization of N -acylhomoserine lactone-degrading bacteria associated with the Zingiber officinale (ginger) rhizosphere: Co-existence of quorum quenching and quorum sensing in Acinetobacter and Burkholderia

Background  

Cell-to-cell communication (quorum sensing (QS)) co-ordinates bacterial behaviour at a population level. Consequently the behaviour of a natural multi-species community is likely to depend at least in part on co-existing QS and quorum quenching (QQ) activities. Here we sought to discover novel N -acylhomoserine lactone (AHL)-dependent QS and QQ strains by investigating a bacterial community associated with the rhizosphere of ginger (Zingiber officinale) growing in the Malaysian rainforest.