Salmonella is a significant foodborne pathogen that causes human illness and comprises numerous serotypes. This study investigated the prevalence of Salmonella co-contamination with multiple serotypes within single food samples in China. Of 3,570 samples analyzed, 550 (15.4%) tested positive for Salmonella. Using a combined approach of serological typing via slide agglutination tests and molecular characterization by multilocus sequence typing (MLST), we found that 18.5% (102/550) of Salmonella-positive samples harbored more than one serotype. Specifically, 86 samples contained two distinct serotypes, 11 samples contained three, and 5 samples contained four. The highest frequencies of multi-serotype contamination were identified in animal-derived foods: poultry meat (20.5%), livestock meat (19.5%), and aquatic products (16.7%). Notably, certain serogroup and serotype co-occurrence patterns appeared preferentially associated with specific food categories. This widespread phenomenon of multi-serotype contamination presents significant challenges: it not only complicates outbreak traceback investigations but may also potentiate synergistic pathogenic effects. Therefore, we recommend that systematic detection and characterization of multi-serotype contamination be adopted, as it is critical for improving the accuracy and comprehensiveness of public health risk assessments.

Contamination of food by Cronobacter spp. and their biofilms on stainless steel surfaces in food processing facilities pose serious public health risks. Phage cocktails can be used to inactivate Cronobacter spp. and eliminate biofilms while reducing the risk of monophage-induced bacterial resistance. In this study, six lytic phages were isolated from environmental water samples, all exhibiting a broad host range against Cronobacter strains. A phage cocktail, combining individual phages at equal concentrations (1 x 108 PFU/mL), completely inhibited the growth of four representative Cronobacter strains-C. sakazakii ST256 (clinical isolate), C. sakazakii ST4, C. malonaticus ST60, and C. malonaticus ST211 (foodborne strains)-after 24 h at 37 degrees C. The phage cocktail also effectively inhibited biofilm formation in all four Cronobacter strains and their mixtures after 48 h and 72 h of incubation at 37 degrees C, respectively. Furthermore, the phage cocktail demonstrated efficacy in eradicating preexisting Cronobacter biofilm on stainless steel surfaces. These findings highlight the potential of this phage cocktail as an effective biocontrol agent against Cronobacter in food processing.

Burkholderia gladioli pathovar cocovenenans (BGC), which is a high morbidity and mortality foodborne pathogens because of produce a lethal toxin (Bongkrekic acid, BA), is one of the best known members of Burkholderia gladioli (BG). Methods for rapid detection and accurate differentiation of BGC and BG are urgently needed because of the difficulty and complexity, especifically no noncultural techniques for discriminating BGC due to lack of specific targets. PCR-based methods with suitable specific targets can achievable rapid detection due to the accuracy, sensitivity, and specificity. In this study, new species and pathovar-specific target genes for rapid detection of BGC and BG in food, was identified using pan-genome analysis. A total of 2098 Burkholderia genome sequences, containing those of 223 Burkholderia gladioli and 31 species of other Burkholderia, were utilized to identify BGC and BG specific targets. One BGC-specific genes (EDD84_14,360) and two BG-specific genes (EDD84_30,365 and EDD84_11,630) were obtained. Based on these specific genes, real-time PCR methods for the rapid detection of BGC and BG were developed respectively. High specificty (100%) were observed when appraised various produce or non-produce bongkrekic acid of BG and 56 non-BG species. Moreover, these real-time PCR methods could accurately detect BGC and BG even when mixed with different concentrations of non-target interfering bacteria (Burkholderia cepacia). The methods could successfully detect 108-103 CFU/g of BGC and BG in artificially contaminated rice noodles without enrichment, and effectively screen of BGC and BG within 24 h from natural food samples with enrichment step in GVC broth which the corresponding sensitivity, specificity and efficiency values were 100%. These results revealed that the real-time PCR methods based on three new pathovar and species-specific molecular targets provide a promising approach with high accuracy and sensitivity to the rapid detection and differentiation of BGC and BG strains in foods.

Cronobacter spp. are emerging foodborne pathogens that cause severe diseases. However, information on Cronobacter contamination in quick-frozen foods in China is limited. Therefore, we studied the prevalence, molecular characterization, and antimicrobial susceptibility of Cronobacter in 576 quick-frozen food samples collected from 39 cities in China. Cronobacter spp. were found in 18.75% (108/576) of the samples, and the contamination degree of the total positive samples was 5.82 MPN/g. The contamination level of frozen flour product samples was high (44.34%). Among 154 isolates, 109 were C. sakazakii, and the main serotype was C. sakazakii O1 (44/154). Additionally, 11 serotypes existed among four species. Eighty-five sequence types (STs), including 22 novel ones, were assigned, indicating a relatively high genetic diversity of the Cronobacter in this food type. Pathogenic ST148, ST7, and ST1 were the main STs in this study. ST4, epidemiologically related to neonatal meningitis, was also identified. All strains were sensitive to cefepime, tobramycin, ciprofloxacin, and imipenem, in which the resistance to cephalothin was the highest (64.94%).Two isolates exhibited multidrug resistance to five and seven antimicrobial agents, respectively. In conclusion, these findings suggest that the comparatively high contamination level of Cronobacter spp. in quick-frozen foods is a potential risk warranting public attention.

This study explored the prevalence of Cronobacter spp. in wet rice and flour products from Guangdong province, China, the molecular characteristics and antimicrobial susceptibility profiles of the isolates were identified. Among 249 samples, 100 (40.16%) were positive for Cronobacter spp., including 77 wet rice and 23 wet flour products. Eleven serotypes were characterized among 136 isolates with C. sakazakii O2 (n = 32) predominating. Forty-nine MLST patterns were assigned, 15 of which were new. C. sakazakii ST4 (n = 17) was the dominant ST, which is previously reported to have caused three deaths; followed by C. malonaticus ST7 (n = 15), which is connected to adult infections. All strains presented susceptibility to ampicillin/sulbactam, imipenem, aztreonam and trimethoprim/sulfamethoxazole. The isolates showed maximum resistance to cephalothin, and the resistance and intermediate rates were 91.91% and 3.68%, each. Two strains, croM234A1 and croM283-1, displayed resistance to three antibiotics. High contamination level and predominant number of pathogenic STs of Crono-bacter in wet rice and flour products implied a potential risk to public healthiness. This survey could provide comprehensive information for establishing more targeted control methods for Cronobacter spp.

We report fatal neonatal necrotizing enterocolitis in China caused by Cronobacter sakazakii capsular profile K1:CA1, sequence type 64, and CRISPR type 197. Phylodynamic analyses indicated that the strain originated from the ancient, widespread, and antimicrobial drug-sensitive CRISPR sublineage b. Enhanced surveillance and pathogenesis research on this organism are required.

阪崎克罗诺杆菌(Cronobacter sakazakii)是一种重要的条件致病菌,容易感染新生儿和早产儿并引起坏死性小肠结肠炎(NEC)、败血症和脑膜炎,其致死率高达40％～80％.因其特有的致病性、生物膜形成能力,同时对高渗透压、低pH、高温、氧化、干燥等有较强的抵抗力,可以在食品加工生产过程中长期存活,具有潜在的食品安全风险.该研究综述并对比了近5年关于阪崎克罗诺杆菌物理防控(如脉冲电场、微波、高压、射频、紫外光、真空干燥等)和化学防控方法(如植物提取物及衍生成分、生物活性肽/乳及乳制品衍生成分、碳水化合物等)的相关研究,综合讨论了各种防治方法及其抑菌效果.然而,每种控制方法在其剂量和应用方法方面都有其局限性,已有的物理和化学防控之间的协同效应,这可能有助于降低食物中不合阪崎克罗诺杆菌的风险.

Cronobacter spp. are opportunistic pathogenic bacteria that cause severe diseases in neonates and infants. Bacteriophages are novel antibacterial agents with a potential to control this pathogen. In the current study, a novel lytic Cronobacter phage, vB_CtuP_A24, was isolated from a river in Guangzhou, China. The phage was characterized by a short, non-contractile tail and a long head and identified as a new member of the family Podoviridae. Complete genome sequence analysis of this phage indicated that its genome contained 75,106 bp of DNA, an average GC content of 44.05%, and 108 predicted open reading frames (ORFs). The annotated ORFs were associated with phage structure, packaging, host lysis, DNA manipulation, transcription, and additional functions. Genes responsible for antibiotic resistance, virulence, and toxic effects were not present. Cronobacter phage vB_CtuP_A24 is a novel lytic phage that can lyse five Cronobacter spp. It was stable over a wide range of temperatures (25-60C) and pH values (pH 4-11) and displayed a short latent period (approximately 10 min) and a large burst size (500 plaque-forming units (PFUs)/cell). In Luria-Bertani (LB) broth, phage A24 effectively inhibited the growth of C. dublinensis cro280B, C. sakazakii 465G, and C. malonaticus cro695W up to 9, 10, and 12 h, respectively, while in infant milk formulas, it inhibited two strains, C. sakazakii 465G and C. malonaticus cro695W, up to 24 h at 37 degrees C, with maximum reduction levels of approximately 5.12 +/- 3.95 and 7.38 +/- 3.03 log10 colony-forming unit (CFU)/mL, respectively. In lettuce, the reduction of all three strains was highly significant at 25 degrees C. However, the growth of C. dublinensis cro280B and C. sakazakii 465G were not significantly inhibited at 4 degrees C. In conclusion, Cronobacter spp. phage vB_CtuP_A24, which has lytic ability against five Cronobacter species, stability under different environments, and shows potential as a promising biocontrol agent against Cronobacter spp. in food production.

Cronobacter spp. are important foodborne pathogens that are a threat to people of all ages, but especially neonates and infants. Bacteriophages are biological agents that are potentially useful for the control of foodborne pathogens. However, there has been little research on the control of C. malonaticus and C. turicensis using bacteriophages. In the present study, a novel lytic phage vB_CtuP_B1 (hereafter referred to as B1)-which can simultaneously lyse C. malonaticus and C. turicensis- was isolated from river water in Guangzhou, China, and was used in the control of Cronobacter contaminated food. The phage has a short tail, and has been identified as a new species of Kayfunavirus based on genomic and phylogenetic analyses. One-step growth and stability assays revealed that phage B1 has a very short latent period (<5 min) and a large burst size (4006 pfu/cell), and is highly stable between 25 and 60 degrees C and between pH 5 and 11. Its genome encodes two lytic proteins, but does not contain any genes responsible for antibiotic resistance and virulence factors. In broth, the phage B1 completely inhibited the growth of C. malonaticus cro2475W and C. turicensis cro1541A1-1 for up to 6 h. On lettuce, phage B1 reduced the viable count of C. turicensis cro1541A1-1 to below the detection limit for bacteria on lettuce (<10 cfu/mL) after 6 h at 4 degrees C and 2 h at 25 degrees C, and also significantly reduced the viable count of C. malonaticus cro2475W at those temperatures. In powdered infant formula, the viable counts of both the phagetreated bacterial hosts were significantly reduced after 2 h of storage at 4 degrees C or 37 degrees C. Furthermore, phage B1 reduced the viable count of C. turicensis cro1541A1-1 to below the detection limit (<10 cfu/mL) from 4 h to 24 h at 37 degrees C. It significantly inhibited the growth of C. turicensis cro1541A1-1 than that of C. malonaticus cro2475W (P < 0.05). In conclusion, phage B1 with high stability and strong lytic ability is potentially useful for controlling C. malonaticus and C. turicensis.

Cronobacter species (Cronobacter spp.) are important foodborne pathogens that can infect and cause serious life-threatening diseases in infants and immunocompromised elderly. This study aimed to acquire data on Cronobacter spp. contamination of aquatic products in China from 2011 to 2016. In total, 800 aquatic products were tested, and the overall contamination rate for Cronobacter spp. was 3.9% (31/800). The average contamination level of the positive samples was 2.05 MPN/g. Four species and nine serotypes were identified among 33 isolates, of which the C. sakazakii serogroup O1 (n = 9) was the primary serotype. The majority of Cronobacter spp. strains harbored highest resistance against cephalothin (84.8%), followed by tetracycline (6.1%), trimethoprim/sulfameth-oxazole (3.0%) and chloramphenicol (3.0%). Two isolates were resistant to three antibiotics. In total, 26 sequence types and 33 CRISPR types (including 6 new STs and 26 new CTs) were identified, which indicates the extremely high diversity of Cronobacter spp. in aquatic products. Pathogenic C. sakazakii ST4, ST1, and C. malonaticus ST7 were also observed. Overall, this large-scale study revealed the relatively low prevalence and high genetic diversity of Cronobacter spp. in aquatic products in China, and the findings provide valuable information that can guide the establishment of effective measures for the control and precaution of Cronobacter spp. in aquatic products during production processes.

Cronobacter spp. are important foodborne pathogens that infections occur in all age groups, especially cause serious life-threatening diseases in infants. This study aimed to acquire data on Cronobacter spp. contamination of meat and meat products (n = 588) in China during 2011 to 2016, and investigated the use of CRISPR typing technology as an approach for characterizing the genetics of Cronobacter spp. The overall contamination rate for Cronobacter spp. was determined to be 9.18% (54/588). Of the positive samples, 90.74% (49/54) had <10 MPN/g, with duck samples had a relatively high contamination rate (15.69%, 8/51) and highest contamination level (28.90 MPN/g). Four species and nine serotypes were identified among 69 isolates, of which C. sakazakii was the major species (n = 50) and C. sakazakii serogroup O1 and O2 (n = 17) were the primary serotypes. The majority of Cronobacter spp. strains were found to be susceptible to most antibiotics except exhibited high resistance to cephalothin (76.81%, 53/69), and total two multi-drug resistant C. sakazakii strains were isolated from duck. The genetic diversity of Cronobacter spp. was remarkably high, as evidenced by the identification of 40 sequence types (STs) and 60 CRISPR types (CTs). C. sakazakii ST64 (n = 7) was the predominant genotype and was further divided into two sub-lineages based on CRISPR diversity, showing different antibiotic resistance profile. These results demonstrate that CRISPR typing results have a good correspondence with bacterial phenotypes, and it will be a tremendously useful approach for elucidating inter-subtyping during molecular epidemiological investigations while interpreting the divergent evolution of Cronobacter. The presence of Cronobacter spp. in meat and meat product is a potential threat to human public health.

Cronobacter spp. are foodborne pathogens that can infect and cause life-threatening diseases in all age groups, particularly in infants and immunocompromised elderly. This study aimed to investigate the prevalence, antibiotic susceptibility, and molecular characteristics of Cronobacter spp. isolates in edible mushrooms collected from 44 cities in China. In total, 668 edible mushrooms were collected from traditional retail markets and supermarkets and were analyzed by quantitative methods, PCR-based serotyping, multilocus sequence typing (MLST), and antibiotic susceptibility testing. Among the 668 samples tested, 89 (13.32%) were positive for Cronobacter spp., and the contamination levels exceeded the 110 most probable number (MPN)/g in 13.48% (12/89) of the samples. Flammulina velutipes samples had the highest contamination rate of 17.54% (37/211), whereas Hypsizygus marmoreus samples had the lowest contamination rate of 3.28% (2/61). Ten serotypes were identified among 115 isolates, of which the C. sakazakii serogroup O1 (n = 32) was the primary serotype. MLST indicated that there was quite high genetic diversity in Cronobacter spp. and 72 sequence types were identified, 17 of which were new. Notably, C. sakazakii ST148 (n = 10) was the most prevalent, followed by C. malonaticus ST7 (n = 5). Antibiotic susceptibility testing revealed that the majority of Cronobacter spp. strains were susceptible to the 16 antibiotics tested. However, a portion of isolates exhibited relatively high resistance to cephalothin, with resistance and intermediate rates of 93.91 and 6.09%, respectively. One isolate (cro300A) was multidrug-resistant, with resistance to five antibiotics. Overall, this large-scale study revealed the relatively high prevalence and high genetic diversity of Cronobacter spp. on edible mushrooms in China, indicating a potential public health concern. To our knowledge, this is the first large-scale and systematic study on the prevalence of Cronobacter spp. on edible mushrooms in China, and the findings can provide valuable information that can guide the establishment of effective measures for the control and precaution of Cronobacter spp on edible mushrooms during production processes.

Cronobacter strains harboring CRISPR-Cas systems are important foodborne pathogens that cause serious neonatal infections. CRISPR typing is a new molecular subtyping method to track the sources of pathogenic bacterial outbreaks and shows a promise in typing Cronobacter, however, this molecular typing procedure using routine PCR method has not been established. Therefore, the purpose of this study was to establish such methodology, 257 isolates of Cronobacter sakazakii, C. malonaticus, and C. dublinensis were used to verify the feasibility of the method. Results showed that 161 C. sakazakii strains could be divided into 129 CRISPR types (CTs), among which CT15 (n = 7) was the most prevalent CT followed by CT6 (n = 4). Further, 65 C. malonaticus strains were divided into 42 CTs and CT23 (n = 8) was the most prevalent followed by CT2, CT3, and CT13 (n = 4). Finally, 31 C. dublinensis strains belonged to 31 CTs. There was also a relationship among CT, sequence type (ST), food types, and serotype. Compared to multi-locus sequence typing (MLST), this new molecular method has greater power to distinguish similar strains and had better accordance with whole genome sequence typing (WGST). More importantly, some lineages were found to harbor conserved ancestral spacers ahead of their divergent specific spacer sequences; this can be exploited to infer the divergent evolution of Cronobacter and provide phylogenetic information reflecting common origins. Compared to WGST, CRISPR typing method is simpler and more affordable, it could be used to identify sources of Cronobacter food-borne outbreaks, from clinical cases to food sources and the production sites.

We report a case of meningitis in a neonate in China, which was caused by a novel multidrug-resistant Cronobacter sakazakii strain, sequence type 256, capsular profile K1:CA1. We identified genetic factors associated with bacterial pathogenicity and antimicrobial drug resistance in the genome and plasmids. Enhanced surveillance of this organism is warranted.

萤火虫荧光素酶(Firefly Luciferase,FL)是ATP快速检测技术的核心组件,通过荧光强度与ATP浓度的对应关系,在食品行业检测微生物数量发挥着重大作用.本文为实现荧光素酶在重组毕赤酵母GS115中的异源表达,通过从载体pGL2-control中扩增荧光素酶基因luc,克隆到真核表达载体pPIC9K中,线性化后电击转化毕赤酵母GS1l5菌株,筛选阳性重组菌株.在甲醇的诱导下进行酶的表达,对粗酶液进行生物活性发光分析,然后对粗酶进行超滤、阴离子层析和分子筛凝胶层析三步纯化.甲醇诱导表达96 h发现胞外和胞内粗酶液均有相对较高的酶发光活性,酶活分别为1.45×106 RLU/mL和1.58× 109 RLU/mL.SDS-PAGE与Western blot分析重组荧光蛋白大小约为70 ku,最终纯化得到的荧光素酶,其比活为7.0× 108 RLU/mg,纯化倍数达到19.3倍,产量为48 mg/L.以上结果表明,荧光素酶能够在毕赤酵母表达系统获得较好的表达和纯化效果.

A newly identified lytic Cronobacter phage, GW1, was isolated from the Pearl River of Guangzhou, China. GW1 had a double-stranded DNA genome of 39,695 nucleotides with an average GC content of 53.18 %. Among the 49 open reading frames (ORFs) identified, genes for rRNA, tRNA, antibiotic resistance, and virulence factors were not found in the phage genome. The morphology, genomic features, and phylogenetic position of GW1 revealed that it represents a new species in the genus T7virus. This novel lytic Cronobacter phage may provide an alternative for phage therapy and biocontrol against Cronobacter.

Cronobacter spp. is a foodborne pathogen that causes life-threatening and invasive diseases, such as necrotizing enterocolitis, meningitis, and sepsis. In this study, we aimed to investigate the prevalence, molecular characteristics and antimicrobial resistance of Cronobacter spp. in raw vegetables marketed in China. Based on dietary habits in China, 403 raw vegetables that could be eaten without additional cooking were collected. Of the 403 samples tested, 122 (30.27%) were positive for Cronobacter spp., and the contamination levels exceeded 110 most probable number (MPN)/g for 16.39% (20/122) of the samples. Coriander samples had the highest contamination rate of 52.81%, and the MPN values of 19.15% of positive coriander samples exceeded 100 MPN/g. Eleven serotypes were identified among 171 isolates, with Cronobacter sakazakii serogroup O1 (41 isolates) being the dominant serotype. Molecular characterization indicated that there was quite high genetic diversity in Cronobacter spp., and multilocus sequence typing analyses yielded 106 sequence types (STs), 55 of which were newly identified. Notably, the most prevalent ST (eight isolates) was C. malonaticus ST60, which appeared in a recent clinical infectious disease study in China. Five C. sakazakii ST4, seven C. malonaticus ST7, and three C. sakazakii ST8 confirmed as pathogenic STs in other countries were also detected in this study. Furthermore, all isolates were susceptible to amikacin, amoxicillin-clavulanic, cefepime, ciprofloxacin, and imipenem, but some isolates exhibited a high ratio of resistance to cephalothin (59.65%). In this study, the high contamination rate and the detection of pathogenic and new STs in raw vegetables indicated potential hazards to customers. To the best of our knowledge, this is the first report to provide valuable information on the contamination status of Cronobacter spp. in vegetables that can be eaten raw in China.

Cronobacter species are foodborne pathogens that can cause neonatal meningitis, necrotizing enterocolitis, and sepsis; they have unusual abilities to survive in environmental stresses such as acid stress. However, the factors involved in acid stress responses and biofilm formation in Cronobacter species are poorly understood. In this study, we investigated the role of grxB on cellular morphology, acid tolerance, surface hydrophobicity, auto-aggregation (AAg), motility, and biofilm formation in Cronobacter sakazakii. The deletion of grxB decreased resistance to acid stresses, and notably led to weaker surface hydrophobicity, AAg, and biofilm formation under normal and acid stress conditions, compared with those of the wild type strain; however, motility was unaffected. Therefore, grxB appears to contribute to the survival of C. sakazakii in acid stresses and biofilm formation. This is the first report to provide valuable evidence for the role of grxB in acid stress responses and biofilm formation in C. sakazakii.

ABSTRACT Cronobacter strains harboring the CRISPR-Cas system are important foodborne pathogens causing serious neonatal infections. However, the specific role of the CRISPR-Cas system in bacterial evolution remains relatively unexplored. In this study, we investigated the impact of the CRISPR-Cas system on Cronobacter evolution and obtained 137 new whole-genome Cronobacter sequences by next-generation sequencing technology. Among the strains examined ( n = 240), 90.6% (193/213) of prevalent species Cronobacter sakazakii , Cronobacter malonaticus , and Cronobacter dublinensis strains had intact CRISPR-Cas systems. Two rare species, Cronobacter condimenti ( n = 2) and Cronobacter universalis ( n = 6), lacked and preserved the CRISPR-Cas system at a low frequency (1/6), respectively. These results suggest that the presence of one CRISPR-Cas system is important for a Cronobacter species to maintain genome homeostasis for survival. The Cronobacter ancestral strain is likely to have harbored both subtype I-E and I-F CRISPR-Cas systems; during the long evolutionary process, subtype I-E was retained while subtype I-F selectively degenerated in Cronobacter species and was even lost by the major Cronobacter pathovars. Moreover, significantly higher CRISPR activity was observed in the plant-associated species C . dublinensis than in the virulence-related species C. sakazakii and C . malonaticus . Similar spacers of CRISPR arrays were rarely found among species, suggesting intensive change through adaptive acquisition and loss. Differentiated CRISPR activity appears to be the product of environmental selective pressure and might contribute to the bidirectional divergence and speciation of Cronobacter . IMPORTANCE This study reports the evolutionary history of Cronobacter under the selective pressure of the CRISPR-Cas system. One CRISPR-Cas system in Cronobacter is important for maintaining genome homeostasis, whereas two types of systems may be redundant and not conducive to acquiring beneficial DNA for environmental adaptation and pathogenicity. Differentiated CRISPR activity has contributed to the bidirectional divergence and genetic diversity of Cronobacter . This perspective makes a significant contribution to the literature by providing new insights into CRISPR-Cas systems in general, while further expanding the roles of CRISPR beyond conferring adaptive immunity and demonstrating a link to adaptation and species divergence in a genus. Moreover, our study provides new insights into the balance between genome homeostasis and the uptake of beneficial DNA related to CRISPR-based activity in the evolution of Cronobacter .

We report a case of meningitis in a neonate in China, which was caused by a novel multidrug-resistant Cronobacter sakazakii strain, sequence type 256, capsular profile K1:CA1. We identified genetic factors associated with bacterial pathogenicity and antimicrobial drug resistance in the genome and plasmids. Enhanced surveillance of this organism is warranted.