RESUMO
Cronobacter species are opportunistic pathogens that are capable of causing morbidity and mortality, particularly in infants. Although the transmission dynamics involved in Cronobacter infections remain largely unknown, contaminated powdered infant formula (PIF) has been linked to 30% of Cronobacter sakazakii cases involving invasive illness in infants. As several lines of evidence have implicated the domestic environment in PIF contamination, we undertook a microbiological survey of homes (N = 263) across the US. Cronobacter spp. and C. sakazakii were isolated from 36.1% and 24.7% of US homes, respectively, with higher recovery rates observed for floor and kitchen surfaces. Multi-locus sequence typing indicated that the dominant strain was C. sakazakii ST4, the sequence type most commonly associated with neonatal meningitis. For comparison purposes, retail foods (N = 4,009) were also surveyed, with the highest contamination frequencies (10.1%-26.3%) seen for nut products, seeds, and grains/baked goods/flours. The sequence type profile of isolates recovered from homes mirrored that of isolates recovered from retail foods, with increased representation of ST1, ST4, ST13, ST17, and ST40. Analysis of 386 whole genomic sequences revealed significant diversity. Redundancies were only observed for isolates recovered from within the same domicile, and there were no identical matches with sequences archived at the NCBI pathogen database. Genes coding for putative virulence and antibiotic resistance factors did not segregate with clinically significant sequence types. Collectively, these findings support the possibility that contamination events occurring within the home should not be overlooked as a contributor to community-onset Cronobacter infections. IMPORTANCE: Cronobacter sakazakii is an opportunistic pathogen that can cause significant morbidity and mortality in neonates. Its transmission dynamics are poorly understood, though powered infant formula (PIF) is thought to be the major transmission vehicle. How the PIF becomes contaminated remains unknown. Our survey shows that roughly 1/4 of US homes are contaminated with Cronobacter sakazakii, particularly in the kitchen setting. Our analyses suggest that the domestic environment may contribute to contamination of PIF and provides insights into mitigating the risk of transmission.
Assuntos
Cronobacter sakazakii , Microbiologia de Alimentos , Fórmulas Infantis , Cronobacter sakazakii/genética , Cronobacter sakazakii/isolamento & purificação , Cronobacter sakazakii/classificação , Estados Unidos , Humanos , Fórmulas Infantis/microbiologia , Tipagem de Sequências Multilocus , Genoma Bacteriano , Lactente , Infecções por Enterobacteriaceae/microbiologia , Infecções por Enterobacteriaceae/transmissão , Características da Família , GenômicaRESUMO
We examined the effects of elevated temperatures and biocides on survivability of food isolates of Cronobacter spp. (C. sakazakii) and concomitant enterobacteriaceae obtained in microbiological control of infant nutrition products. Increased resistance of certain strains of Cronobacter, Enterobacter cloacae, and Pantoea spp. to thermal processing was revealed. Salmonella, Pantoea, and Cronobacter bacteria were least sensitive to antimicrobial action of chlorine-containing agents. The above properties varied in the strains of the same species. Specifically, only two of three examined isolates of Cronobacter spp. demonstrated lower sensitivity to heat in comparison with the enterobacterial test-cultures of other species.
Assuntos
Cloro , Cronobacter , Desinfetantes , Microbiologia de Alimentos , Desinfetantes/farmacologia , Cronobacter/efeitos dos fármacos , Cronobacter/isolamento & purificação , Cloro/farmacologia , Enterobacteriaceae/efeitos dos fármacos , Enterobacteriaceae/isolamento & purificação , Temperatura Alta , Humanos , Cronobacter sakazakii/efeitos dos fármacos , Cronobacter sakazakii/isolamento & purificação , Testes de Sensibilidade Microbiana , Salmonella/efeitos dos fármacos , Salmonella/isolamento & purificação , Enterobacter cloacae/efeitos dos fármacos , Enterobacter cloacae/isolamento & purificaçãoRESUMO
How timely identification and determination of pathogen species in pathogen-contaminated foods are responsible for rapid and accurate treatments for food safety accidents. Herein, we synthesize four aggregation-induced emissive nanosilicons with different surface potentials and hydrophobicities by encapsulating four tetraphenylethylene derivatives differing in functional groups. The prepared nanosilicons are utilized as receptors to develop a nanosensor array according to their distinctive interactions with pathogens for the rapid and simultaneous discrimination of pathogens. By coupling with machine-learning algorithms, the proposed nanosensor array achieves high performance in identifying eight pathogens within 1 h with high overall accuracy (93.75-100%). Meanwhile, Cronobacter sakazakii and Listeria monocytogenes are taken as model bacteria for the quantitative evaluation of the developed nanosensor array, which can successfully distinguish the concentration of C. sakazakii and L. monocytogenes at more than 103 and 102 CFU mL-1, respectively, and their mixed samples at 105 CFU mL-1 through the artificial neural network. Moreover, eight pathogens at 1 × 104 CFU mL-1 in milk can be successfully identified by the developed nanosensor array, indicating its feasibility in monitoring food hazards.
Assuntos
Microbiologia de Alimentos , Listeria monocytogenes , Aprendizado de Máquina , Listeria monocytogenes/isolamento & purificação , Cronobacter sakazakii/isolamento & purificação , Dióxido de Silício/química , Sistemas Automatizados de Assistência Junto ao Leito , Animais , Leite/microbiologia , Leite/química , Técnicas Biossensoriais , Redes Neurais de ComputaçãoRESUMO
As outbreaks of foodborne illness caused by the opportunistic pathogen Cronobacter sakazakii (Cs) continue to occur, particularly in infants consuming powdered infant formula (PIF), the need for sensitive, rapid, and easy-to-use detection of Cs from food and food processing environments is increasing. Here, we developed bioluminescent reporter bacteriophages for viable Cs-specific, substrate-free, rapid detection by introducing luciferase and its corresponding substrate-providing enzyme complex into the virulent phage ΦC01. Although the reporter phage ΦC01_lux, constructed by replacing non-essential genes for phage infectivity with a luxCDABE reporter operon, produced bioluminescence upon Cs infection, the emitted signal was quickly decayed due to the superior bacteriolytic activity of ΦC01. By truncating the membrane pore-forming protein holin and thus limiting its function, the bacterial lysis was delayed and the resultant engineered reporter phage ΦC01_lux_Δhol could produce a more stable and reliable bioluminescent signal. Accordingly, ΦC01_lux_Δhol was able to detect at least an average of 2 CFU/ml of Cs artificially contaminated PIF and Sunsik and food contact surface models within a total of 7 h of assays, including 5 h of pre-enrichment for Cs amplification. The sensitive, easy-to-use, and specific detection of live Cs with the developed reporter phage could be applied as a novel complementary tool for monitoring Cs in food and food-related environments for food safety and public health.
Assuntos
Técnicas Bacteriológicas , Bacteriófagos , Cronobacter sakazakii , Microbiologia de Alimentos , Medições Luminescentes , Proteínas Virais , Cronobacter sakazakii/genética , Cronobacter sakazakii/isolamento & purificação , Técnicas Bacteriológicas/métodos , Bacteriófagos/genética , Bacteriófagos/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo , Microbiologia de Alimentos/métodos , Genoma Viral/genética , Deleção de Sequência , Medições Luminescentes/métodos , Sensibilidade e EspecificidadeRESUMO
One immunomagnetic separation (IMS) assay based on immunomagnetic beads (IMBs) has been evaluated as a potential pretreatment tool for the separation and enrichment of target bacteria. In this study, we successfully immobilized antibodies onto magnetic bead surfaces to form IMBs through biotin and a streptavidin (SA) system to capture viable but nonculturable (VBNC) Cronobacter sakazakii (C. sakazakii) from dairy products. Various parameters that affected the capture efficiency (CE) of IMS, including the number of antibodies, IMBs dose, incubation time, magnetic separation time, and immunoreaction temperature, were systematically investigated. We further determined the optimal enrichment conditions for different dairy substrates to ensure maximum enrichment of target pathogens in the system. An IMS technique combining improved propidium monoazide (PMAxx) and droplet digital PCR (ddPCR) was established to detect the pathogenic VBNC C. sakazakii. The IMS-PMAxx-ddPCR method after IMBs enrichment showed higher accuracy when the VBNC C. sakazakii was under 1 Log10 copies/g. The detection limit for this method in a background of powdered infant formula (PIF) was 5.6 copies/g. In summary, the developed IMS-PMAxx-ddPCR method has great potential for the analysis and detection of VBNC bacteria in food.
Assuntos
Cronobacter sakazakii/crescimento & desenvolvimento , Cronobacter sakazakii/isolamento & purificação , Laticínios/microbiologia , Separação Imunomagnética/métodos , Azidas/química , Cronobacter sakazakii/química , Cronobacter sakazakii/genética , Contaminação de Alimentos/análise , Microbiologia de Alimentos , Fórmulas Infantis/microbiologia , Viabilidade Microbiana , Reação em Cadeia da Polimerase , Propídio/análogos & derivados , Propídio/químicaRESUMO
INTRODUCTION: Cronobacter sakazakii is an opportunistic Gram-negative, rod-shaped bacterium which may be a causative agent of meningitis in premature infants and enterocolitis and bacteremia in neonates and adults. While there have been multiple cases of C. sakazakii infections, there have been no acute cholangitis cases reported in humans. CASE PRESENTATION: An 81-year-old male with a past medical history of basal cell carcinoma, alcoholic liver cirrhosis, transjugular intrahepatic portosystemic shunt procedure, complicated by staphylococcus bacteremia, pituitary tumor, glaucoma, and hypothyroidism presented to the emergency room with the complaint of diffuse and generalized 10/10 abdominal pain of 1 day's duration. There was a concern for pancreatitis, acute cholangitis, and possible cholecystitis, and the patient underwent a percutaneous cholecystostomy tube placement. Blood cultures from admission and biliary fluid cultures both grew C. sakazakii. The patient was treated with a carbapenem and clinically improved. CONCLUSIONS: The case study described a patient with multiple medical comorbidities that presented with C. sakazakii bacteremia and cholangitis. While this bacterium has been implicated in other infections, we believe this is the first time the bacteria is being documented to have caused acute cholangitis.
Assuntos
Bacteriemia/diagnóstico , Colangite/diagnóstico , Cronobacter sakazakii/isolamento & purificação , Infecções por Enterobacteriaceae/diagnóstico , Idoso de 80 Anos ou mais , Antibacterianos/uso terapêutico , Bacteriemia/complicações , Bacteriemia/terapia , Carbapenêmicos/uso terapêutico , Colangite/microbiologia , Colangite/terapia , Colecistostomia/métodos , Cronobacter sakazakii/patogenicidade , Drenagem/métodos , Infecções por Enterobacteriaceae/complicações , Infecções por Enterobacteriaceae/terapia , Humanos , Masculino , Infecções Oportunistas/diagnóstico , Infecções Oportunistas/microbiologia , Infecções Oportunistas/terapia , Reação em Cadeia da Polimerase/métodos , Tomografia Computadorizada por Raios X/métodos , Resultado do TratamentoRESUMO
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.
Assuntos
Cronobacter/classificação , Cronobacter/isolamento & purificação , Infecções por Enterobacteriaceae/epidemiologia , Doenças Transmitidas por Alimentos/microbiologia , Fórmulas Infantis/microbiologia , Cronobacter/genética , Cronobacter sakazakii/genética , Cronobacter sakazakii/isolamento & purificação , Bases de Dados Factuais , Infecções por Enterobacteriaceae/microbiologia , Variação Genética/genética , Humanos , Lactente , Recém-Nascido , Tipagem de Sequências Multilocus , Fator G para Elongação de Peptídeos/genética , Estados Unidos/epidemiologiaRESUMO
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.
Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Cronobacter/classificação , Cronobacter/isolamento & purificação , Farmacorresistência Bacteriana , Alimentos Marinhos/microbiologia , Antibacterianos/farmacologia , Cefalotina/farmacologia , China , Cloranfenicol/farmacologia , Cronobacter sakazakii/classificação , Cronobacter sakazakii/isolamento & purificação , Contaminação de Alimentos , Microbiologia de Alimentos , Variação Genética , Tipagem de Sequências Multilocus , Prevalência , Sorotipagem , Tetraciclina/farmacologia , Trimetoprima/farmacologiaRESUMO
The purpose of this study was to elucidate the antibacterial activity and possible mechanism of action of Amaranthus tricolor crude extract (ATCE) against Cronobacter sakazakii isolated from powdered infant formula (PIF). The antibacterial activity of ATCE was assessed by measuring the diameter of inhibition zone (DIZ), minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC). The possible mechanism of action of ATCE was revealed by analyzing the effects of ATCE on growth curves and changes in cell membrane potential, intracellular pH, content of bacterial protein and genomic DNA, and cell morphology. Finally, ATCE was applied to the disinfection of C. sakazakii in biofilm on stainless steel tube. The results showed that the DIZ, MIC, and MBC of ATCE against C. sakazakii strains were from 14.35 ± 0.67 to 14.84 ± 0.67 mm, 20 mg/mL, and 40 mg/mL, respectively. Treatment with ATCE ended the logarithmic growth phase of C. sakazakii, and led to depolarization of the cell membranes, reducing intracellular pH and bacterial protein and genomic DNA contents, and resulting in cytoplasmic leakage and deformation. In addition, ATCE effectively inactivated C. sakazakii in biofilm, reducing viable bacteria by approximately 6.5 log cfu/mL bacterial count after treatment with 1 MIC (1 MIC = 20 mg/mL) of ATCE for 20 min at 25°C. Our findings showed that ATCE inactivated C. sakazakii strains isolated from PIF and has potential as a natural disinfectant to reduce the contamination of PIF by C. sakazakii.
Assuntos
Amaranthus/química , Biofilmes/efeitos dos fármacos , Misturas Complexas/farmacologia , Cronobacter sakazakii/efeitos dos fármacos , Microbiologia de Alimentos , Fórmulas Infantis/microbiologia , Biofilmes/crescimento & desenvolvimento , Membrana Celular/efeitos dos fármacos , Misturas Complexas/isolamento & purificação , Cronobacter sakazakii/crescimento & desenvolvimento , Cronobacter sakazakii/isolamento & purificação , Cronobacter sakazakii/ultraestrutura , Humanos , Potenciais da Membrana/efeitos dos fármacos , Testes de Sensibilidade MicrobianaRESUMO
Cronobacter sakazakii is an important opportunistic food-borne pathogen, and it can cause severe diseases with main symptoms including neonatal meningitis, necrotizing enterocolitis, and sepsis. For the achievement of practical and convenient detection of viable C. sakazakii, a simple and robust strategy based on the cascade signal amplification of RT-PCR triggered G-quadruplex DNAzyme catalyzed reaction was firstly used to develop an effective and sensitive DNAzyme electrochemical assay. Without viable C. sakazakii in the samples there are no RT-PCR and DNAzyme products, which can cause a weak electrochemical response. Once viable C. sakazakii exists in the samples, an obvious enhancement of the electrochemical response can be achieved after the target signal is amplified by RT-PCR and the resulting DNAzyme, which catalyze the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) by H2O2 with the assistance of the cofactor hemin. Our novel assay can be performed in a range of 2.4 × 107 CFU mL-1 to 3.84 × 104 CFU mL-1 (R2 = 0.9863), with a detection limit of 5.01 × 102 CFU mL-1. Through the assay of 15 real samples, electrochemical detection assay provided the same results as conventional detection methods. Therefore, detection of viable C. sakazakii based on G-quadruplex DNAzyme electrochemical assay with RT-PCR demonstrates the significant advantages of high sensitivity, low cost and simple manipulation over existing approaches and offers an opportunity for potential application in pathogen detection.
Assuntos
Cronobacter sakazakii/isolamento & purificação , DNA Bacteriano/análise , DNA Catalítico/química , Técnicas Eletroquímicas/métodos , Quadruplex G , Benzidinas/química , Cronobacter sakazakii/química , DNA Bacteriano/química , Contaminação de Alimentos/análise , Hemina/química , Peróxido de Hidrogênio/química , Fórmulas Infantis/análise , Fórmulas Infantis/microbiologia , Limite de Detecção , Oxirredução , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The aim of this study was to evaluate the microbiological quality of 45 samples of corn-based farinaceous foods commercialized in Brazil. The bacteriological analysis performed were: detection of Salmonella and Cronobacter, and enumeration of faecal coliforms and Bacillus cereus. The Cronobacter isolates were phenotypically characterized by Vitek 2.0 and the antibiotic susceptibility profile. Molecular characterization was accomplished by real-time PCR targeting dnaG gene and MLST. No sample presented contamination by Salmonella or B. cereus (<102 UFC/g). Faecal coliforms were detected in two (4.4%) samples but in low concentration (≤23.0 MPN/g), and 20 samples (44.4%) contained Cronobacter. Twenty-nine unique Cronobacter isolates were identified as C. sakazakii (n = 18), C. malonaticus (n = 2); that presented 11 different fusA alleles, including new fusA 183. MLST analysis revealed 17 sequence types (STs), six of which were newly identified (ST687-690, 693, and 694). Resistance or intermediary resistance were found to ceftazidime (15.0%), aztreonam (15.0%), nalidixic acid (15.0%), nitrofurantoin (15.0%), cefepime (10.0%), gentamicin (5.0%), and tetracycline (5.0%). The presence of Cronobacter in corn-based farinaceous foods could be a significant risk to infants as these products are used as alternatives to commercially available infant formula. Strategies to manage the risk of Cronobacter infections due to the consumption of these alternative feeds need to be developed by the regulatory agencies.
Assuntos
Cronobacter sakazakii/isolamento & purificação , Cronobacter/isolamento & purificação , Farmacorresistência Bacteriana Múltipla , Tipagem de Sequências Multilocus , Zea mays/microbiologia , Antibacterianos/farmacologia , Aztreonam/farmacologia , Brasil , Cefepima/farmacologia , Ceftazidima/farmacologia , Cronobacter/crescimento & desenvolvimento , Cronobacter sakazakii/crescimento & desenvolvimento , Contaminação de Alimentos/análise , Manipulação de Alimentos , Microbiologia de Alimentos , Gentamicinas , Fórmulas Infantis/análise , Fórmulas Infantis/microbiologia , Testes de Sensibilidade Microbiana , Ácido Nalidíxico/farmacologia , Nitrofurantoína/farmacologia , Tetraciclina/farmacologiaRESUMO
Cronobacter sakazakii is a food-borne pathogen carried in milk powder that can cause severe bacteremia, enterocolitis, and meningitis in newborns, which can lead to death of newborns. Preventing infection by this pathogen is significant to the health of newborns. Since infants and young children are the main target group of C. sakazakii, it is considered that maternal immunity can enhance the protection of newborns. Previous studies showed that two proteins of C. sakazakii (GroEL and OmpX) exhibited high expression levels and elicited strong immune reactions, suggesting their potential as vaccine candidates. In this study, GroEL and OmpX were recombinantly expressed in Escherichia coli and purified as immunogens to immunize pregnant rats. Three days after birth, the progeny were challenged with C. sakazakii to determine the protective effect of maternal immunity on the offspring. The results showed that immunization during pregnancy decreased bacterial load in the brain and blood, reduced brain and intestine damage, and significantly increased specific antibody titers in the offspring. Immunization with the recombinant proteins significantly increased cytokine levels in the serum of the progeny. The group whose mothers were immunized with OmpX produced more IL-4, while the group whose mothers were immunized with GroEL produced more IFN-γ, indicating that the immunogens enhanced the Th2 and Th1 responses, respectively. However, although the immune response was induced by both proteins, only the offspring of the pregnant rats immunized with OmpX or OmpX/GroEL mixture showed delayed death, possibly because immunization with OmpX led to a stronger humoral immune response in the offspring, suggesting that OmpX was a better vaccine candidate than GroEL. This study first reported that exposure to C. sakazakii proteins during pregnancy could improve the offspring's ability to resist infection caused by this pathogen.
Assuntos
Proteínas da Membrana Bacteriana Externa/imunologia , Vacinas Bacterianas/imunologia , Chaperonina 60/imunologia , Cronobacter sakazakii/imunologia , Infecções por Enterobacteriaceae/prevenção & controle , Imunidade Materno-Adquirida , Animais , Anticorpos Antibacterianos/sangue , Carga Bacteriana , Proteínas da Membrana Bacteriana Externa/genética , Encéfalo/patologia , Chaperonina 60/genética , Cronobacter sakazakii/genética , Cronobacter sakazakii/isolamento & purificação , Cronobacter sakazakii/fisiologia , Citocinas/sangue , Infecções por Enterobacteriaceae/imunologia , Infecções por Enterobacteriaceae/microbiologia , Feminino , Imunogenicidade da Vacina , Intestinos/patologia , Gravidez , Ratos , Proteínas Recombinantes/imunologia , Vacinas Sintéticas/imunologiaRESUMO
Bovine mastitis is a common inflammatory disease caused by various factors. The main factor of mastitis is pathogenic microorganism infection, such as Staphylococcus aureus, Escherichia coli, and Streptococcus. Cronobacter sakazakii (C. sakazakii) is a newly discovered pathogenic bacteria in milk products, which seriously threat human health in recent years. At present, it has not been reported that the pathogenesis of mastitis is caused by C. sakazakii. This study investigated the inflammation of mammary gland epithelium, which was induced by C. sakazakii for the first time. We focused on bacterial isolation, histological observation, AIM2 inflammasome pathways, endoplasmic reticulum stress, and apoptosis. The results showed that C. sakazakii-induced inflammation caused damage of tissue, significantly increased the production of pro-inflammatory cytokines (including TNF-α, IL-1ß, and IL-6), activated the AIM2 inflammasome pathway (increased the expression of AIM2 and cleaved IL-1ß), and induced endoplasmic reticulum stress (increased the expression of ERdj4, Chop, Grp78) and apoptosis (increased the ratio of Bax/Bcl-2, a marker of apoptosis). In conclusion, it is suggested that it maybe inhibite AIM2 inflammasome pathways and alleviate endoplasmic reticulum stress (ER stress) against the C. sakazakii-induced inflammation.
Assuntos
Cronobacter sakazakii , Proteínas de Ligação a DNA/metabolismo , Estresse do Retículo Endoplasmático , Infecções por Enterobacteriaceae/metabolismo , Inflamação/metabolismo , Mastite Bovina , Leite/microbiologia , Animais , Bovinos , Linhagem Celular , Cronobacter sakazakii/imunologia , Cronobacter sakazakii/isolamento & purificação , Citocinas/metabolismo , Chaperona BiP do Retículo Endoplasmático , Células Epiteliais , Feminino , Mastite Bovina/imunologia , Mastite Bovina/microbiologia , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Cronobacter turicensis is a food-borne pathogen found in dairy products. It has been reported to cause bacteremia and enteritis in immunocompromised people, especially infants. Cronobacter turicensis has been isolated from various food sources, and contaminated powdered infant formula was found to be the most common source of infection among infants. Although some gene targets are used for the identification of C. turicensis, they are not specific at the species level. In this study, we analyzed the genome sequence of C. turicensis by bioinformatics and identified 13 specific gene targets. Primer sets targeting these sequences were designed and selected based on their specificity. Finally, primer set CT11, targeting gene CTU_19580, which codes for a hypothetical protein, was selected for development of the PCR assay because it alone produced positive PCR results for C. turicensis. To our knowledge, this is the first time that this gene target has been used to develop PCR detection assays for C. turicensis. The specific PCR assay had detection limits as low as 760 fg/µL for genomic DNA (approximately 158 copies/µL of DNA) and could detect C. turicensis in powdered infant formula with initial cell concentrations as low as 8.5 cfu per 10 g of powdered infant formula after 10 h of enrichment. Thus, this PCR assay is highly sensitive and can be used for rapid detection of C. turicensis.
Assuntos
Cronobacter sakazakii/isolamento & purificação , DNA Bacteriano/análise , Marcação de Genes , Genoma Bacteriano , Reação em Cadeia da Polimerase/métodos , Sequência de Bases , Biologia Computacional , Cronobacter sakazakii/classificação , Cronobacter sakazakii/genéticaRESUMO
Cronobacter sakazakii is a xerotolerant neonatal pathogen epidemiologically linked to powdered infant food formula, often resulting in high mortality rates. Here, we used transcriptome sequencing (RNA-seq) to provide transcriptional insights into the survival of C. sakazakii in desiccated conditions. Our RNA-seq data show that about 22% of the total C. sakazakii genes were significantly upregulated and 9% were downregulated during desiccation survival. When reverse transcription-quantitative PCR (qRT-PCR) was used to validate the RNA-seq data, we found that the primary desiccation response was gradually downregulated during the tested 4 hours of desiccation, while the secondary response remained constitutively upregulated. The 4-hour desiccation tolerance of C. sakazakii was dependent on the immediate microenvironment surrounding the bacterial cell. The removal of Trypticase soy broth (TSB) salts and the introduction of sterile infant formula residues in the microenvironment enhanced the desiccation survival of C. sakazakii SP291. The trehalose biosynthetic pathway encoded by otsA and otsB, a prominent secondary bacterial desiccation response, was highly upregulated in desiccated C. sakazakiiC. sakazakii SP291 ΔotsAB was significantly inhibited compared with the isogenic wild type in an 8-hour desiccation survival assay, confirming the physiological importance of trehalose in desiccation survival. Overall, we provide a comprehensive RNA-seq-based transcriptional overview along with confirmation of the phenotypic importance of trehalose metabolism in Cronobacter sakazakii during desiccation.IMPORTANCECronobacter sakazakii is a pathogen of importance to neonatal health and is known to persist in dry food matrices, such as powdered infant formula (PIF) and its associated production environment. When infections are reported in neonates, mortality rates can be high. The success of this bacterium in surviving these low-moisture environments suggests that Cronobacter species can respond to a variety of environmental signals. Therefore, understanding those signals that aid the persistence of this pathogen in these ecological niches is an important step toward the development of strategies to reduce the risk of contamination of PIF. This research led to the identification of candidate genes that play a role in the persistence of this pathogen in desiccated conditions and, thereby, serve as a model target to design future strategies to mitigate PIF-associated survival of C. sakazakii.
Assuntos
Cronobacter sakazakii/genética , Infecções por Enterobacteriaceae/microbiologia , RNA Bacteriano/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cronobacter sakazakii/classificação , Cronobacter sakazakii/isolamento & purificação , Cronobacter sakazakii/metabolismo , Humanos , Fórmulas Infantis/microbiologia , RNA Bacteriano/metabolismo , Análise de Sequência de RNA , Transcrição Gênica , Trealose/metabolismoRESUMO
BACKGROUND: Cronobacter species are associated with severe foodborne infections in neonates and infants, with particular pathovars associated with specific clinical presentations. However, before 2008 the genus was regarded as a single species named Enterobacter sakazakii which was subdivided into 8 phenotypes. This study re-analyzed, using multi-locus sequence typing (MLST) and whole genome sequence with single nucleotide polymorphism analysis (WGS-SNP), 52 strains which had been identified as Enterobacter sakazakii as according to the convention at the time of isolation. These strains had been isolated from dairy product imports into China from 9 countries between 2005 and 6. Bioinformatic analysis was then used to analyze the relatedness and global dissemination of these strains. RESULT: FusA allele sequencing revealed that 49/52 strains were Cronobacter sakazakii, while the remaining 3 strains were Escherichia coli, Enterobacter cloacae, and Franconibacter helveticus. The C. sakazakii strains comprised of 8 sequence types (STs) which included the neonatal pathovars ST1, ST4 and ST12. The predominant sequence type was ST13 (65.3%, 32/49) which had been isolated from dairy products imported from 6 countries. WGS-SNP analysis of the 32 C. sakazakii ST13 strains revealed 5 clusters and 5 unique strains which did not correlate with the country of product origin. CONCLUSION: The mis-identification of E. coli, E. cloacae and F. helveticus as Cronobacter spp. reinforces the need to apply reliable methods to reduce the incidence of false positive and false negative results which may be of clinical significance. The WGS-SNP analysis demonstrated that indistinguishable Cronobacter strains within a sequence type can be unrelated, and may originate from multiple sources. The use of WGS-SNP analysis to distinguishing of strains within a sequence type has important relevance for tracing the source of outbreaks due to Cronobacter spp.
Assuntos
Cronobacter sakazakii/genética , DNA Bacteriano/isolamento & purificação , Laticínios/microbiologia , Proteínas de Bactérias/genética , China , Cronobacter sakazakii/classificação , Cronobacter sakazakii/isolamento & purificação , DNA Bacteriano/química , DNA Bacteriano/metabolismo , Microbiologia de Alimentos , Humanos , Tipagem de Sequências Multilocus , Fator G para Elongação de Peptídeos/genética , Filogenia , Polimorfismo de Nucleotídeo Único , Sorogrupo , Sequenciamento Completo do GenomaRESUMO
Cronobacter sakazakii is a foodborne pathogen that can cause a rare, septicemia, life-threatening meningitis, and necrotizing enterocolitis in infants. In general, standard methods for pathogen detection rely on culture, plating, colony counting and polymerase chain reaction DNA-sequencing for identification, which are time, equipment and skill demanding. Recently, nanoparticle- and surface-based immunoassays have increasingly been explored for pathogen detection. We investigate the functionalization of gold nanoparticles optimized for irreversible and specific binding to C. sakazakii and their use for spectroscopic detection of the pathogen. We demonstrate how 40-nm gold nanoparticles grafted with a poly(ethylene glycol) brush and functionalized with polyclonal antibodies raised against C. sakazakii can be used to specifically target C. sakazakii. The strong extinction peak of the Au nanoparticle plasmon polariton resonance in the optical range is used as a label for detection of the pathogens. Individual binding of the nanoparticles to the C. sakazakii surface is also verified by transmission electron microscopy. We show that a high degree of surface functionalization with anti-C. sakazakii optimizes the detection and leads to a detection limit as low as 10 CFU/mL within 2 h using a simple cuvette-based UV-Vis spectrometric readout that has great potential for further optimization.
Assuntos
Cronobacter sakazakii/imunologia , Cronobacter sakazakii/isolamento & purificação , Infecções por Enterobacteriaceae/microbiologia , Ouro/imunologia , Nanopartículas Metálicas , Animais , Cronobacter sakazakii/patogenicidade , Infecções por Enterobacteriaceae/diagnóstico , Humanos , Lactente , Limite de Detecção , Reação em Cadeia da Polimerase , CoelhosRESUMO
Cronobacter sakazakii is an opportunistic foodborne pathogen that can infect newborns through powdered infant formula (PIF). In this study, we developed a novel enhanced lateral flow immunoassay (LFA) with enhanced sensitivity for detection of C. sakazakii in PIF by the naked eye. The proposed strategy for signal enhancement of the traditional LFA used concentrated gold nanoparticles (AuNP) as the enhancer to conjugate with capture antibodies, which could increase the immobilized capture antibodies concentration at the detection zone to improve capture efficiency. Besides, the detection signal was further amplified by accumulated AuNP as the C. sakazakii labeled with AuNP probes was captured by antibodies conjugated with enhancer at the test line. We also studied the effect of different concentrations of capture antibodies and concentrated AuNP on detection performance, and found that 2.2 mg/mL of capture antibodies and 0.06 nM concentrated AuNP were the optimal combination that could avoid a false-positive signal and maximally amplify the detection signal of the enhanced LFA. Using this strategy, the detection sensitivity of the enhanced LFA was 103 cfu/mL and improved 100-fold compared with traditional LFA. The strip was highly specific to C. sakazakii, and the time for detection of C. sakazakii in PIF was shortened by 3 h. In summary, the enhanced LFA developed by the addition of concentrated AuNP as the enhancer can be used as a sensitive, rapid, visual qualitative and point-of-care test method for detecting target analytes.
Assuntos
Cronobacter sakazakii/isolamento & purificação , Contaminação de Alimentos/análise , Imunoensaio/métodos , Fórmulas Infantis/microbiologia , Cronobacter sakazakii/crescimento & desenvolvimento , Ouro/química , Imunoensaio/instrumentação , Fórmulas Infantis/análise , Nanopartículas Metálicas/química , Pós/análise , Especificidade da EspécieRESUMO
A sensitive electrochemical immunosensing platform for the detection of Cronobacter sakazakii was developed using a graphene oxide/gold (GO/Au) composite. Transmission electron microscopy showed that the Au nanoparticles, with an average size of <â¯30â¯nm, were well dispersed on the GO surface. For the detection of C. sakazakii, a polyclonal anti-C. sakazakii antibody (IgG) was covalently immobilized to the Au nanoparticles on the surface of the GO/Au composite coated glassy carbon electrode (GCE). The electrochemical sensing performance of immunofunctionalized GCE was characterized by cyclic voltammetry and differential pulse voltammetry. Under optimized conditions, in pure culture there was a linear relationship between electrical signal and C. sakazakii levels over the range 2.0â¯×â¯102-2.0â¯×â¯107 cfu/mL (R2 =â¯0.999), with a detection limit of 2.0â¯×â¯101 cfu/mL. The total analytical time was 15â¯min per sample. The C. sakazakii electrochemical immunosensing assay was able to successfully detect 2.0â¯×â¯101 cfu/mL of C. sakazakii in artificially contaminated powdered infant formula without any enrichment or pre-enrichment steps. Furthermore, the recovery rates of the C. sakazakii electrochemical immunosensing assay following spiking of powdered infant formula with different concentrations of C. sakazakii (cfu/mL) were 82.58% at 2.0â¯×â¯101 cfu/mL, 84.86% at 2.0â¯×â¯102 cfu/mL, and 95.40% at 2.0â¯×â¯103 cfu/mL. The C. sakazakii electrochemical immunosensing assay had good selectivity, reproducibility, and reactivity compared with other Cronobacter spp. and/or pathogens belonging to other genera, indicating its significant potential in the clinical diagnosis of C. sakazakii.