Cronobacter Dublinensis Research Articles

Diverse Genotypes of Cronobacter spp. Associated with Dairy Farm Systems in Jiangsu and Shandong Provinces in China.

Cronobacter spp. are the most concerning foodborne pathogen in infant formula milk powder. Currently, there are many reports on the prevalence of Cronobacter spp. in infant formula milk and its processing environment, but there are few studies on the prevalence of Cronobacter spp. on dairy farms. We have, therefore, undertaken this study to investigate and track genomic epidemiology of Cronobacter spp. isolates from Chinese dairy farms in the provinces of Jiangsu and Shandong. In this study, forty Cronobacter spp. strains, consisting of thirty Cronobacter sakazakii, eight Cronobacter malonaticus, and two Cronobacter dublinensis, were obtained from 1115 dairy farm samples (raw milk, silage, bedding, and feces), with a prevalence rate of 3.57%. These isolates were classified into 10 Cronobacter serotypes and 31 sequence types (STs), including three novel STs which were isolated for the first time. Notably, pathogenic Cronobacter STs 7, 8, 17, 60, and 64, which are associated with clinical infections, were observed. Antimicrobial susceptibility testing showed that all the Cronobacter spp. were highly resistant to cephalothin and fosfomycin, which was consistent with the antimicrobial genotype. All isolates carried core virulence genes related to adherence, invasion, endotoxin, immune evasion, secretion system, and regulation. Approximately half the isolates were also able to produce a strong biofilm. Twenty-one prophages and eight plasmids were detected, with the most common prophage being Cronobacter_ENT47670 and the most common plasmid being IncFIB (pCTU1). In addition, two isolates harbored the transmissible locus of stress tolerance (tLST) which confers high environmental persistence. Phylogenetic analysis showed strong clustering by species level and sequence types. Isolates from different sources or regions with a similar genomic background suggests the cross-contamination of Cronobacter spp. The presence of diverse genotypes of Cronobacter spp. in dairy farms in Jiangsu and Shandong provinces indicates that surveillance of Cronobacter spp. on dairy farms should be strengthened, to prevent and control transmission and ensure the quality and safety of raw dairy products.

Complete Genome Sequence of Cronobacter dublinensis Bacteriophage vB_Cdu_VP8.

Cronobacter dublinensis is a Gram-negative pathogen that is capable of causing infection in humans. In this announcement, we describe the characterization of bacteriophage vB_Cdu_VP8, which is able to lyse a Cronobacter dublinensis strain. Related to phages belonging to the genus Muldoonvirus, such as Muldoon and SP1, vB_Cdu_VP8 contains 264 predicted protein-coding genes and 3 tRNAs.

Microbial inoculants with inorganic fertilizers slacken the chlorosis impact on Kalmegh [Andrographis paniculata (Burm. F.) Wall ex., Nees] improve yields and quality traits

A common problem witnessed in Kalmegh [Andrographis paniculata (Burm. F.) Wall ex., Nees] plants are yellowing of leaves, when cultivated during rainy season which drastically affects the yield and quality of andrographolide. In the present study, four strains of bioinoculants (Pseudomonas monteilii, Cedecea devisae, Cronobacter dublinensis, Advenella species) were scrutinized along with combinations of inorganic fertilizers to identify the best bioinoculant-fertilizer combination which can curb the effect of yellowing in Kalmegh plants most efficiently as well as enhance its biomass and secondary metabolite content. Minimum percentage of yellowing of leaves was recorded in the plantlets inoculated with bioinoculants (Cedecea devisae) and N:P:K applied at the ratio of 80:60:40 kg ha−1. Among all the treatments, Cedecea devisae (T5) gave maximum fresh and dry herb yields. The results showed a significant variation in photosynthetic pigments along with the treatments. Maximum andrographolide yield was recorded in the plantlets inoculated with Cedecea devisae which was 63.07% more compared to control. Moreover, the concentration of andrographolide in different parts of Kalmegh plants followed the trend-leaves>stem>inflorescence>flowers>seeds>seed husk>root. Except soil pH, other analysed soil parameters of the post harvest soil were significantly improved. Significant enhancement of the soil dehydrogenase, alkaline and acid phosphatase enzymes activity was 14.8% to 54.0%, 57.0% to 234%, and 51.6% to 276% greater, respectively over control. Principal component analysis revealed a strong positive interrelationship between vegetative attributes, soil enzyme activity, biomass yield and andrographolide content. We can suggest from our study that, bioinoculants especially free living nitrogen fixers (Cedecea davisae) along with recommended doses of NPK can effectively control the problem of yellowing (chlorosis) in Kalmegh plants during rainy season.

Evaluation of the Effect of PGPR Strains on Tomato Growth and Suppression of Ralstonia Wilt Disease

The tomato (Lycopersicum esculentum Mill.) has substantial scope for development due to its high economic value and large export potential. Ralstonia syzygii subsp. Indonesiensis (RSI) is the cause of bacterial wilt disease which attacks the vascular system in Solanaceae. It can cause large losses in yield and has caused global concern because of its widespread distribution and attack on many important crops. The aim of this research was to identifiy and characterize the ability of indigenous rhizobacterial isolates to control RSI and promote tomato growth. The PGPR traits studied were production of hydrogen cyanide, siderophores, biosurfactant, and ammonia, and protease activity. Bacterial identification was performed using 16S rRNA. Our findings revealed that the strains identified shared some similarities with Bacillus thuringiensis strain ATCC 10792 (IR.2.3.5), B. mycoides strain ATCC 6462 (IR.1.3.4), Bacillus thuringiensis strain IAM 12077 (IR.2.2. 1), Serratia ficaria strain DSM 4569 (IR.3.1.4), Enterobacter oryzendophyticus strain REICA_082 (IR.2.2.7), Cronobacter dublinensis subsp. lausannensis strain E515 (IR.2.2.5) and S. rubidaea strain DSM 4480 (IR.2.2.6). All of the isolates were tested for a variety of abilities related to growth promotion and biocontrol.
 Keywords: Ralstonia wilt; 16S rRNA identification; PGPR traits

Cronobacter spp. in Commercial Powdered Infant Formula Collected From Nine Provinces in China: Prevalence, Genotype, Biofilm Formation, and Antibiotic Susceptibility.

The purpose of this study was to investigate the prevalence of Cronobacter spp. in commercial powdered infant formula (PIF) from nine provinces in China from March 2018 to September 2020, and to reveal the genotype, biofilm-forming ability, and antibiotic susceptibility of these isolates. A total of 27 Cronobacter strains, consisting of 22 Cronobacter sakazakii strains, 3 Cronobacter malonaticus strains, 1 Cronobacter turicensis strain, and 1 Cronobacter dublinensis strain, were isolated from 3,600 commercial PIF samples with a prevalence rate of 0.75%. Compared with the other 8 provinces, PIF from Shaanxi province had a higher prevalence rate (1.25%) of Cronobacter spp. These isolates were divided into 14 sequence types (STs), and 6 Cronobacter serotypes. The main Cronobacter STs were ST4, ST1, and ST64, and the dominant Cronobacter serotype was C. sakazakii serotype O2. Approximately 88.89% of Cronobacter isolates had a strong ability (OD595 > 1) to form biofilms on tinplate, among which the strains with ST4 were more dominant. All isolates were susceptible to ampicillin-sulbactam, ceftriaxone, cefotaxime, sulfadiazine, sulfadoxine, trimethoprim-sulfamethoxazole, gentamicin, tetracycline, ciprofloxacin, and colistin, while 55.56 and 96.30% isolates were resistant to cephalothin and vancomycin, respectively. Taken together, our findings highlighted the contamination status and characterization of Cronobacter spp. in commercial PIF from nine provinces of China, and provided guidance for the effective prevention and control of this pathogen in the production of PIF.

Plant growth-promoting rhizobacteria Shewanella putrefaciens and Cronobacter dublinensis enhance drought tolerance of pearl millet by modulating hormones and stress-responsive genes.

Drought is a major abiotic stress that affects crop productivity. Endophytic bacteria have been found to alleviate the adverse effects of drought on plants. In the present study, we evaluated the effects of two endophytic bacteria Shewanella putrefaciens strain MCL-1 and Cronobacter dublinensis strain MKS-1 on pearl millet (Pennisetum glaucum (L.) R. Br.) under drought stress conditions. Pearl millet plants were grown under three water levels: field capacity (FC), mild drought stress (MD), and severe drought stress (SD). The effects of inoculation on plant growth, physiological attributes, phytohormone content, and drought stress-responsive genes were assessed. The inoculation of pearl millet seeds with endophytes significantly improved shoot and root dry weight and root architecture of plants grown under FC and drought stress conditions. There was a significant increase in relative water content and proline accumulation in the inoculated plants. Among the phytohormones analyzed, the content of ABA and IAA was significantly higher in endophyte-treated plants under all moisture regimes than in uninoculated plants. C. dublinensis-inoculated plants had higher GA content than uninoculated plants under all moisture regimes. The expression level of genes involved in phytohormone biosynthesis (SbNCED, SbGA20oX, and SbYUC) and coding drought-responsive transcription factors (SbAP2, SbSNAC1 and PgDREB2A) was significantly higher under SD in endophyte-inoculated plants than in uninoculated plants. Thus, these endophytic bacteria presumably enhanced the tolerance of pearl millet to drought stress by modulating root growth, plant hormones, physiology and the expression of genes involved in drought tolerance.

Molecular Characterization of Gut Bacterial Flora of Honeybee (Apis Mellifera Adansonii) From Some Selected Apiaries in Ogun State, Nigeria

Research related to physiology and pathology of honey bees in particular Apis mellifera adansonii has attracted a lot of attention. The present study is aimed to determine honeybee (Apis mellifera adansonii) gut microbiome from Apiary in Olabisi Onabanjo University and Osoba Avenue Odo-Epo, Odogbolu Local Government. Twenty (20) honeybees workers (A. mellifera) were collected into a small vile containing sugar powder from the apiary located in OOU and Osoba Avenue at Odo-Epo during rainy season in July and transported to Zoology and Environmental Biology laboratory in OOU and kept in ice-cubes (-50C) till daybreak. Standard microbiological analysis for isolation of bacteria was used, adopting Clinical Laboratory Standard Institute procedures. The phylogenetic analyses based on the 16S rDNA gene were further used to characterize the organism in order to establish relationships among them. The results showed microbiota of the studied samples includes; Cedeca davisae, Cronobacter dublinensis, Enterobacter aerogenes, Kluyvera cryocrescens, Klebsiella oxytoca, Providencia vermicola, Salmonella enteric, Providencia alcalifaciens, Serratia nematodiphila, Pseudomonas plecoqlossicida, Klebsiella michiganensis, Serratia marcenscens, Pseudomonas aeruginosa, Escherichia coli, Aeromonas hydrophila and Enterobacter asburiae. Klebsiella spp. was more abundant and prominent in the digestive guts of honeybee workers both in OOU and Osoba Avenue, Odo Epo. The result of the percentage identity and closest accession of the isolates revealed that, Enterobacter aerogenes had the closest accession number and with highest percentage identity of (99%). The findings from this study showed that microbiota component communities of A. mellifera adansonii in OOU were composed of more Gram-negative bacteria than Gram-positive bacteria in Odo Epo.

Diversity of Cronobacter genus isolated between 1970 and 2019 on the American continent and genotyped using multi-locus sequence typing.

This study aimed to evaluate the Cronobacter spp. strains isolated on the American continent and characterized using multi-locus sequence typing (MLST) available in the PubMLST database and current literature. From 465 Cronobacter spp. strains, the majority (n=267, 57.4%) was from North America, mainly from USA (n=234) and 198 (42.6%) were from South America, mainly from Brazil (n=196). A total of 232 (49.9%) were isolated from foods, 102 (21.9%) from environmental, 87 (18.7%) from clinical, 27 (5.8%) from PIF, one from water (0.2%) and 16 (3.5%) from unknown sources. A total of five species were represented: Cronobacter sakazakii (374, 80.4%), Cronobacter malonaticus (41, 8.8%), Cronobacter dublinensis (29, 6.2%), Cronobacter turicensis (16, 3.5%) and Cronobacter muytjensii (5, 1.1%). The strains with complete MLST profile (n=345) were assigned to 98 STs, a ratio of 3.5 strain by ST found and the calculated Simpson`s index was 0.93. The strains showed a high diversity and after eBURST analysis, 30 STs (n=189) formed 12 single and/or double-locus variant clonal complexes (CC). A total of 38 STs (38.7%) were associated with clinical cases of infection, including well established C. sakazakii CC 1, 4, 8 and 83; C. malonaticus ST60, 307, 394 and 440; and C. sakazakii ST 12 and 494.

Analysis of the Molecular Diversity Among Cronobacter Species Isolated From Filth Flies Using Targeted PCR, Pan Genomic DNA Microarray, and Whole Genome Sequencing Analyses.

Cronobacter species are opportunistic pathogens capable of causing life-threatening infections in humans, with serious complications arising in neonates, infants, immuno-compromised individuals, and elderly adults. The genus is comprised of seven species: Cronobacter sakazakii, Cronobacter malonaticus, Cronobacter turicensis, Cronobacter muytjensii, Cronobacter dublinensis, Cronobacter universalis, and Cronobacter condimenti. Despite a multiplicity of genomic data for the genus, little is known about likely transmission vectors. Using DNA microarray analysis, in parallel with whole genome sequencing, and targeted PCR analyses, the total gene content of two C. malonaticus, three C. turicensis, and 14 C. sakazaki isolated from various filth flies was assessed. Phylogenetic relatedness among these and other strains obtained during surveillance and outbreak investigations were comparatively assessed. Specifically, microarray analysis (MA) demonstrated its utility to cluster strains according to species-specific and sequence type (ST) phylogenetic relatedness, and that the fly strains clustered among strains obtained from clinical, food and environmental sources from United States, Europe, and Southeast Asia. This combinatorial approach was useful in data mining for virulence factor genes, and phage genes and gene clusters. In addition, results of plasmidotyping were in agreement with the species identity for each strain as determined by species-specific PCR assays, MA, and whole genome sequencing. Microarray and BLAST analyses of Cronobacter fly sequence datasets were corroborative and showed that the presence and absence of virulence factors followed species and ST evolutionary lines even though such genes were orthologous. Additionally, zebrafish infectivity studies showed that these pathotypes were as virulent to zebrafish embryos as other clinical strains. In summary, these findings support a striking phylogeny amongst fly, clinical, and surveillance strains isolated during 2010–2015, suggesting that flies are capable vectors for transmission of virulent Cronobacter spp.; they continue to circulate among United States and European populations, environments, and that this “pattern of circulation” has continued over decades.

Evaluation of inhibitory activities of two medicinal plant extracts Parkia biglobosa and Lonicera japonica against spoilage microorganisms isolated from mulberry fruit

Two medicinal plants, African Locust Bean (Parkia biglobosa) and Lonicera japonica, traditionally used in West Africa and China were investigated against four spoilage microorganisms: Bacillus subtilis, Cronobacter dublinensis, Pantoea agglomerans, and Bacillus sp., isolated from mulberry fruit. The aqueous extract of the P. biglobosa (ALB) was potent with average inhibitory zones against four spoilage microorganisms recorded as (Means ± SD) 6.5 to 19.5 mm as well as 6.5 to 15.5 mm for L. japonica (HSF) at concentrations of 400 mg/ml. The aqueous extracts of P. biglobosa (54%) inhibited decay better than L. japonica (66%). Decay situations of the medicinal plants with 1-MCP emerged higher than the control group 78%, 72%, and 68% in ALB + 1-MCP, HSF + 1-MCP, and deionized water, respectively. Therefore, two plant extracts against spoilage microorganisms isolated from mulberry fruit had good inhibitory activities, but their mixture with 1-MCP could not preserve the mulberry fruits in limited storage time. Practical applications Advance in the bacteria control using a natural control like medicinal plants against plant infections was investigated. The present study demonstrated the potentials of African locust bean and L. japonica as a novel biocontrol agent to the extent of preserving the postharvest decay of mulberry fruit in limited storage time at room temperature. The results and findings obtained in this study support the potential application of these two plants in mulberry fruit preservation as well as in traditional therapeutics. In addition, the combination with 1-MCP also serves as the potential to extend the shelf life of mulberry fruit in order to limit the use of pesticides in postharvest treatments. This study reports for the first time isolated microorganisms from mulberry fruit and the developments of edible coatings utilizing plant extracts from different continents against their activities.

Close structural relationship of the O-antigens of various Cronobacter species containing 3-deoxy-d-manno-oct-2-ulosonic acid

Some O-polysaccharides (O-antigens) of the lipopolysaccharides of bacteria of the genus Cronobacter contain 3-deoxy-d-manno-oct-2-ulosonic acid. O-Polysaccharides of Cronobacter turicensis G3874 and Cronobacter muytjensii G3886 containing this unit were found to be structurally related to each other and to those of Cronobacter dublinensis HPB 3169 and Cronobacter sakazakii G2706 (serotype O5) studied earlier. The O-polysaccharides differ in the N-acyl group on a 3-amino-3,6-dideoxy-d-glucose residue [(R)-3-hydroxybutanoyl vs. acetyl] or in the presence or absence of the O-acetyl group on an l-rhamnose residue only. Structures of the new O-antigens were established by chemical methods and by 1D and 2D 1H and 13C NMR spectroscopy applied to polysaccharides and pentasaccharides obtained by mild alkaline O-deacylation and mild acid hydrolysis of the isolated lipopolysaccharides, respectively.

Contamination of Cronobacter spp. in Chinese Retail Spices.

Crononbacter spp. is an opportunistic foodborne pathogen that causes infections in neonates, infants, and immunocompromised adults. Although the contamination of spices with Cronobacter has been previously reported in some countries, there have been no studies on Cronobacter contamination in China. Therefore, this study aimed to investigate the prevalence of Cronobacter spp. in Chinese retail spices. Fifty-six packaged Chinese spices were collected from different markets, and 32 of these were found to be contaminated with Cronobacter. Five species were identified from the 54 isolates of the 32 positive samples: Cronobacter sakazakii (n = 35), Cronobacter muytjensii (n = 8), Cronobacter malonaticus (n = 6), Cronobacter turicensis (n = 3), and Cronobacter dublinensis (n = 2). Pulsed-field gel electrophoresis demonstrated high genetic diversity, as 53 PFGE profiles were revealed for the 54 isolates. Multilocus sequence typing analysis revealed 46 sequence types, and of these, 26 were newly identified. Most of the isolates were sensitive to antibiotics (n = 15), with the exception of cefazolin. This study revealed that the contamination of Chinese retail spices by Cronobacter spp. poses a potential risk to the consumer.

Thermal resistance ofCronobacter sakazakiiisolated from baby food ingredients of dairy origin in liquid medium

Milk and whey powders are commonly used ingredients in powdered infant formula (PIF) and follow-up formula (FUF). In this study, Cronobacter sakazakii and Cronobacter dublinensis both of dairy origin and a reference strain, Cronobacter muytjensii, ATCC 51329 were investigated for thermal inactivation (D and z values). Heat resistance of the pathogen was studied between 52 and 60 °C in trypic soy broth. Among the strains, C. muytjensii ATCC 51329 was the most heat-resistant strain at lower temperatures (52 and 54 °C). The D-values at 52 and 54 °C of C. muytjensii ATCC 51329 were 33.30 (±1.17) and 6.79 (±1.05) min, respectively. At higher temperatures, e.g., 56, 58, and 60 °C, C. sakazakii strains 131 and 807 exhibited the highest D values. The D-values at 56 and 58 °C were 4.73 (±0.40) min for C. sakazakii 131 and 2.30 (±0.26) min for C. sakazakii 807. The D-values at 60 °C for C. sakazakii 131 and 807 were 1.17 (±0.03) and 1.14 (±0.02) min, respectively. The z-value of the reference strain (C. muytjensii ATCC 51329) was lower than the other strains. The findings of this study will be very helpful in understanding the heat resistance of C. sakazakii and guarding against this pathogen in PIF and FUF. Practical applications The results presented will play a significant role in eliminating Cronobacter from powdered infant formula and follow-up formula and ensuring safety against this pathogen.

Use of a Pan-Genomic DNA Microarray in Determination of the Phylogenetic Relatedness among Cronobacter spp. and Its Use as a Data Mining Tool to Understand Cronobacter Biology.

Cronobacter (previously known as Enterobacter sakazakii) is a genus of Gram-negative, facultatively anaerobic, oxidase-negative, catalase-positive, rod-shaped bacteria of the family Enterobacteriaceae. These organisms cause a variety of illnesses such as meningitis, necrotizing enterocolitis, and septicemia in neonates and infants, and urinary tract, wound, abscesses or surgical site infections, septicemia, and pneumonia in adults. The total gene content of 379 strains of Cronobacter spp. and taxonomically-related isolates was determined using a recently reported DNA microarray. The Cronobacter microarray as a genotyping tool gives the global food safety community a rapid method to identify and capture the total genomic content of outbreak isolates for food safety, environmental, and clinical surveillance purposes. It was able to differentiate the seven Cronobacter species from one another and from non-Cronobacter species. The microarray was also able to cluster strains within each species into well-defined subgroups. These results also support previous studies on the phylogenic separation of species members of the genus and clearly highlight the evolutionary sequence divergence among each species of the genus compared to phylogenetically-related species. This review extends these studies and illustrates how the microarray can also be used as an investigational tool to mine genomic data sets from strains. Three case studies describing the use of the microarray are shown and include: (1) the determination of allelic differences among Cronobacter sakazakii strains possessing the virulence plasmid pESA3; (2) mining of malonate and myo-inositol alleles among subspecies of Cronobacter dublinensis strains to determine subspecies identity; and (3) lastly using the microarray to demonstrate sequence divergence and phylogenetic relatedness trends for 13 outer-membrane protein alleles among 240 Cronobacter and phylogenetically-related strains. The goal of this review is to describe microarrays as a robust tool for genomics research of this assorted and important genus, a criterion toward the development of future preventative measures to eliminate this foodborne pathogen from the global food supply.

Duplex Real-Time PCR Method for the Differentiation of Cronobacter sakazakii and Cronobacter malonaticus

Cronobacter sakazakii and Cronobacter malonaticus are the most common species of Cronobacter , so it is necessary to detect the two species as soon as possible in surveillance programs. We developed a real-time PCR method for identifying C. sakazakii and C. malonaticus from the genus Cronobacter . In this study, the two pairs of primers and probes were designed, targeting 16S rRNA and fusA, respectively. The specificity of the real-time PCR assay was validated with 112 strains of Cronobacter , including 56 C. sakazakii , 32 C. malonaticus , 16 Cronobacter dublinensis , 6 Cronobacter turicensis , and 2 Cronobacter muytjensii . The results showed that C. sakazakii and C. malonaticus were all correctly identified, consistent with the results of another method by analyzing the clustering of the fusA sequence. The detection limit for pure culture was 102 CFU/ml and 103 CFU/g for artificially contaminated rehydrated powdered infant formula. Therefore, the developed real-time PCR was a rapid, sensitive, and reliable method for the identification of C. sakazakii and C. malonaticus .

Characterization of Cronobacter spp. isolated from food of plant origin and environmental samples collected from farms and from supermarkets in the Czech Republic

The Cronobacter genus (previously known as Enterobacter sakazakii) comprises seven species (Cronobacter sakazakii, Cronobacter malonaticus, Cronobacter muytjensii, Cronobacter turicensis, Cronobacter dublinensis, Cronobacter universalis and Cronobacter condimenti) which cause serious infections in neonates and immunocompromised people. Most of the documented outbreaks of these bacteria have been associated with consumption of contaminated powdered infant formula. The plant environment is considered to be the natural habitat of these bacteria. Therefore, a total number of 563 samples of vegetables, fruit, water and environmental swabs were collected from local farms and supermarkets in the Czech Republic and investigated for the presence of Cronobacter spp. The obtained 45 isolates (8.0%) were further characterized by phenotyping (antimicrobial resistance, capsule and pigment production) and genotyping (fusA sequencing, MLST, PCR-serotyping) methods. Most of the Cronobacter isolates (42.2%) were identified as C. sakazakii, followed by C. turicensis (31.1%), C. dublinensis (22.2%), C. malonaticus (2.2%) and C. universalis (2.2%). The 25 identified sequence types, out of which 17 were unique for only one strain, indicated a high diversity of strains. C. sakazakii sequence type 4 (ST 4), which has been associated with many cases of meningitis, was isolated only in one case. A strong association of C. turicensis and C. dublinensis with the plant environment can be deduced from our results.

Prevalence and subtyping of Cronobacter species in goat milk powder factories in Shaanxi province, China

Cronobacter spp. are opportunistic pathogens that can cause serious diseases in neonates and infants via consumption of contaminated milk powder. To determine Cronobacter spp. contamination status, 632 samples, including 15 evaporated milk, 45 intermediate powder, 150 finished products, and 422 manufacturing environment samples, were collected from 3 goat milk powder factories in Shaanxi province, China, from July 2013 to April 2014. The recovered Cronobacter isolates were subtyped using pulsed-field gel electrophoresis to trace the potential dissemination routes during the whole production processing. Sixty-seven Cronobacter spp. isolates were recovered. The prevalence rates in manufacturing environment, intermediate powder, and finished products were 92.5, 6.0, and 1.5%, respectively. The predominant species were Cronobacter sakazakii (88.1%); no Cronobacter turicensis, Cronobacter condimenti, or Cronobacter dublinensis were detected. Sixty-seven Cronobacter isolates were grouped in 26 clusters by pulsed-field gel electrophoresis, and substantial genetic similarity was observed among isolates from different sampling sites in the same factory. Isolates in the main clusters were commonly recovered from intermediate powder, floor powder, and shoes. These data indicated that air, powder, and personnel movement were potential routes for Cronobacter dissemination, and manufacturing environment is the key control point for Cronobacter contamination.

Occurrence and Characterization of Cronobacter spp. in Dehydrated Rice Powder from Chinese Supermarket.

Cronobacter spp. are emerging food-borne pathogens and have been identified as causative agents of meningitis and necrotizing enterocolitis in infants. Dehydrated rice is popular with a wide range of people and it is frequently used as a substitute for infant milk powder to baby older than four months. The occurrence of Cronobacter spp. was investigated in 1,012 samples of dehydrated rice powder collected from 14 manufacturers in China during 2010 to 2012. The isolates were identified using fusA allele sequencing and subtyped using pulsed-field gel electrophoresis. Seventy-six samples (7.5%) contained Cronobacter spp. The prevalence among manufacturers ranged from 0-28.8%. The 76 isolates included 4 species [Cronobacter sakazakii (52 isolates) Cronobacter malonaticus (14 isolates), Cronobacter dublinensis (7 isolates), and Cronobacter muytjensii (3 isolates)]. Twenty-three unique fusA alleles and sixty-six PFGE-patterns were detected. All isolated strains were observed to be sensitive or to show intermediate susceptibility to eight tested antimicrobial agents. The study revealed serious contamination of dehydrated rice powder by Cronobacter spp., with prevalence varying among manufacturers in China. Identified Cronobacter species, fusA alleles, and subtypes were diverse.

Multilocus Sequence Typing of Cronobacter Strains Isolated from Retail Foods and Environmental Samples.

Cronobacter spp. are bacterial pathogens that affect children and immunocompromised adults. In this study, we used multilocus sequence typing (MLST) to determine sequence types (STs) in 11 Cronobacter spp. strains isolated from retail foods, 29 strains from dust samples obtained from vacuum cleaners, and 4 clinical isolates. Using biochemical tests, species-specific polymerase chain reaction, and MLST analysis, 36 strains were identified as Cronobacter sakazakii, and 6 were identified as Cronobacter malonaticus. In addition, one strain that originated from retail food and one from a dust sample from a vacuum cleaner were identified on the basis of MLST analysis as Cronobacter dublinensis and Cronobacter turicensis, respectively. Cronobacter spp. strains isolated from the retail foods were assigned to eight different MLST sequence types, seven of which were newly identified. The strains isolated from the dust samples were assigned to 7 known STs and 14 unknown STs. Three clinical isolates and one household dust isolate were assigned to ST4, which is the predominant ST associated with neonatal meningitis. One clinical isolate was classified based on MLST analysis as Cronobacter malonaticus and belonged to an as-yet-unknown ST. Three strains isolated from the household dust samples were assigned to ST1, which is another clinically significant ST. It can be concluded that Cronobacter spp. strains of different origin are genetically quite variable. The recovery of C. sakazakii strains belonging to ST1 and ST4 from the dust samples suggests the possibility that contamination could occur during food preparation. All of the novel STs and alleles for C. sakazakii, C. malonaticus, C. dublinensis, and C. turicensis determined in this study were deposited in the Cronobacter MLST database available online ( http://pubmlst.org/cronobacter/).

Structure of fructans from three strains of Cronobacter dublinensis

Partially O-acetylated fructan was isolated by phenol—water extraction of cells of Cronobacter dublinensis G2732 followed by anion-exchange chromatography on DEAE-Toyopearl 650M. Structure of the isolated fructan was established by 2D 1H and 13C NMR spectroscopy. The same fructan but lacking O-acetyl groups was isolated from two other C. dublinensis strains, G3947 and G4061. Genes for the fructan biosynthesis were not found at the typical Cronobacter O-antigen gene cluster located between the conserved genes galF and gnd and are evidently located elsewhere on the chromosome.