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
This review considers how research in China has progressed our understanding and subsequent improved control of Cronobacter. This emergent bacterial pathogen is associated with neonatal infections through the ingestion of contaminated prepared feed. The review includes large-scale surveys of various sources of the organism, including infant formula production facilities. The analysis of over 20,000 samples is presented. Over 10,000 being from powdered infant formula and other infant foods as well as environmental sampling of production facilities, the remaining being from food, food ingredients, and human carriage. A major advance in China was adopting DNA-sequence-based methods (that is, multilocus sequence typing, clustered regularly interspaced short palindromic repeats-cas array profiling, and single-nucleotide polymorphism analysis) for the identification and genotyping of the organism. These methods have considerably advanced our understanding of the taxonomy, ecology, and virulence of this organism. In turn, this has improved source tracking of the organism both in infant formula production facilities and epidemiological investigations. Furthermore, whole-genome sequencing has revealed a range of virulence and persistence mechanisms as well as plasmid-borne multidrug resistance traits. China now has reliable and robust methods for accurate microbial source tracking of Cronobacter for use both in the food production environment and epidemiological analysis.
Assuntos
Cronobacter , Microbiologia de Alimentos/métodos , China , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Cronobacter/genética , Cronobacter/isolamento & purificação , Cronobacter/patogenicidade , Cronobacter sakazakii/classificação , Cronobacter sakazakii/genética , Cronobacter sakazakii/patogenicidade , Infecções por Enterobacteriaceae/epidemiologia , Infecções por Enterobacteriaceae/transmissão , Genótipo , Humanos , Lactente , Alimentos Infantis/microbiologia , Fórmulas Infantis/microbiologia , Recém-Nascido , Tipagem de Sequências Multilocus , VirulênciaRESUMO
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
Cronobacter spp. have been recognized as causative agents of various severe infections in pre-term or full-term infants as well as elderly adults suffering from serious underlying disease or malignancy. A surveillance study was designed to identify antibiotic resistance among clinical Cronobacter spp. strains, which were isolated from patients of two hospitals between May 2007 and August 2013. Altogether, 52 Cronobacter spp. isolates were analyzed. Although MALDI-TOF mass spectrometry recognized all Cronobacter sakazakii and Cronobacter malonaticus strains, it could not identify Cronobacter muytjensii strain. Nevertheless, all strains were identified as Cronobacter spp. using multilocus sequence typing (MLST). Strains were tested against 17 types of antibiotics, using the standard microdilution method according to the 2018 European Committee on Antimicrobial Susceptibility Testing criteria. Three Cronobacter species were identified as C. sakazakii (n = 33), C. malonaticus (n = 18), and C. muytjensii (n = 1); all isolates were susceptible to all tested antibiotics. All strains were PCR-negative for bla TEM, bla SHV, and bla CTX-M ß-lactamase genes, as well. Even though the results of this study showed that Cronobacter spp. isolates were pan-susceptible, continued antibiotic resistance surveillance is warranted.Cronobacter spp. have been recognized as causative agents of various severe infections in pre-term or full-term infants as well as elderly adults suffering from serious underlying disease or malignancy. A surveillance study was designed to identify antibiotic resistance among clinical Cronobacter spp. strains, which were isolated from patients of two hospitals between May 2007 and August 2013. Altogether, 52 Cronobacter spp. isolates were analyzed. Although MALDI-TOF mass spectrometry recognized all Cronobacter sakazakii and Cronobacter malonaticus strains, it could not identify Cronobacter muytjensii strain. Nevertheless, all strains were identified as Cronobacter spp. using multilocus sequence typing (MLST). Strains were tested against 17 types of antibiotics, using the standard microdilution method according to the 2018 European Committee on Antimicrobial Susceptibility Testing criteria. Three Cronobacter species were identified as C. sakazakii (n = 33), C. malonaticus (n = 18), and C. muytjensii (n = 1); all isolates were susceptible to all tested antibiotics. All strains were PCR-negative for bla TEM, bla SHV, and bla CTX-M ß-lactamase genes, as well. Even though the results of this study showed that Cronobacter spp. isolates were pan-susceptible, continued antibiotic resistance surveillance is warranted.
Assuntos
Antibacterianos/farmacologia , Cronobacter/classificação , Cronobacter/efeitos dos fármacos , Técnicas de Tipagem Bacteriana , Cronobacter sakazakii/classificação , Cronobacter sakazakii/efeitos dos fármacos , Cronobacter sakazakii/genética , Farmacorresistência Bacteriana Múltipla , Genótipo , Infecções por Bactérias Gram-Negativas/microbiologia , Humanos , Testes de Sensibilidade Microbiana , Tipagem de Sequências Multilocus , Polônia , Reação em Cadeia da PolimeraseRESUMO
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 has been documented as a cause of life-threating infections, predominantly in neonates. We conducted a multicenter study to assess the occurrence of C. sakazakii across Europe and the extent of clonality for outbreak detection. National coordinators representing 24 countries in Europe were requested to submit all human C. sakazakii isolates collected during 2017 to a study center in Austria. Testing at the center included species identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, subtyping by whole-genome sequencing (WGS), and determination of antimicrobial resistance. Eleven countries sent 77 isolates, including 36 isolates from 2017 and 41 historical isolates. Fifty-nine isolates were confirmed as C. sakazakii by WGS, highlighting the challenge of correctly identifying Cronobacter spp. WGS-based typing revealed high strain diversity, indicating absence of multinational outbreaks in 2017, but identified 4 previously unpublished historical outbreaks. WGS is the recommended method for accurate identification, typing, and detection of this pathogen.
Assuntos
Cronobacter sakazakii , Infecções por Enterobacteriaceae/epidemiologia , Infecções por Enterobacteriaceae/microbiologia , Antibacterianos/farmacologia , Cronobacter sakazakii/classificação , Cronobacter sakazakii/efeitos dos fármacos , Cronobacter sakazakii/genética , Farmacorresistência Bacteriana , Infecções por Enterobacteriaceae/história , Europa (Continente)/epidemiologia , Genoma Bacteriano , Genômica/métodos , História do Século XXI , Humanos , Recém-Nascido , Masculino , Testes de Sensibilidade Microbiana , Tipagem Molecular , Tipagem de Sequências Multilocus , Filogenia , Vigilância em Saúde Pública , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Sequenciamento Completo do GenomaRESUMO
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
Goat milk powder is a nutritious and easy-to-store product that is highly favored by consumers. However, the presence of contaminating bacteria and their metabolites may significantly affect the flavor, solubility, shelf life, and safety of the product. To comprehensively and accurately understand the sanitary conditions in the goat milk powder production process and potential threats from bacterial contamination, a combination of Pacific Biosciences single molecule real-time sequencing and droplet digital PCR was used to evaluate bacterial contamination in seven goat milk powder samples from three dairies. Ten phyla, 119 genera, and 249 bacterial species were identified. Bacillus, Paenibacillus, Lactococcus, and Cronobacter were the primary genera. Bacillus cereus, Lactococcus lactis, Alkaliphilus oremlandii, and Cronobacter sakazakii were the dominant species. With droplet digital PCR, 6.3 × 104 copies per g of Bacillus cereus and 1.0 × 104 copies per g of Cronobacter spp. were quantified, which may increase the risk of food spoilage and the probability of foodborne illness and should be monitored and controlled. This study offers a new approach for evaluating bacterial contamination in goat milk powder and supplies a reference for the assessment of food safety and control of potential risk, which will be of interest to the dairy industry.
Assuntos
Bactérias , Contaminação de Alimentos/análise , Cabras , Leite , Animais , Bactérias/classificação , Bactérias/genética , Bactérias/isolamento & purificação , Cronobacter sakazakii/classificação , Cronobacter sakazakii/genética , Microbiologia de Alimentos , Leite/microbiologia , Reação em Cadeia da PolimeraseRESUMO
We describe a case of infection with Cronobacter sakazakii sequence type 494 causing bacteremia and meningitis in a hospitalized late premature infant in Brazil. We conducted microbiological analyses on samples of powdered infant formula from the same batch as formula ingested by the infant but could not identify the source of contamination.
Assuntos
Cronobacter sakazakii/classificação , Cronobacter sakazakii/genética , Infecções por Enterobacteriaceae/diagnóstico , Infecções por Enterobacteriaceae/microbiologia , Meningites Bacterianas/diagnóstico , Meningites Bacterianas/microbiologia , Bacteriemia , Encéfalo/patologia , Brasil , Infecções por Enterobacteriaceae/transmissão , Contaminação de Alimentos , Microbiologia de Alimentos , Humanos , Lactente , Recém-Nascido , Imageamento por Ressonância Magnética , Meningites Bacterianas/transmissão , Tipagem de Sequências MultilocusRESUMO
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
ãThe four types of chromogenic selective media that are commercially available in Japan were compared for establishing a Japanese standard method for detecting Cronobacter spp. based on ISO/TS 22964:2006. When assessed using 9 standard Cronobacter spp. strains and 29 non-Cronobacter strains, Enterobacter sakazakii isolation agar, ChromocultTM Enterobacter sakazakii agar, CHROMagarTM E. sakazakii, and XM-sakazakii agar demonstrated excellent inclusivity and exclusivity. Using the ISO/TS 22964:2006 method, the recovered numbers of 38 Cronobacter spp. strains, including 29 C. sakazakii isolates obtained from each medium, were equivalent, indicating that there was no significant difference (p > 0.05) among the four types of chromogenic selective media. Thus, we demonstrated that these four chromogenic selective media are suitable alternatives when using the standard method for detecting Cronobacter spp. in Japan, based on the ISO/TS 22964:2006.
Assuntos
Compostos Cromogênicos , Cronobacter sakazakii/classificação , Cronobacter sakazakii/crescimento & desenvolvimento , Meios de Cultura , Antibacterianos/farmacologia , Compostos Cromogênicos/química , Compostos Cromogênicos/metabolismo , Contagem de Colônia Microbiana , Cronobacter sakazakii/efeitos dos fármacos , Cronobacter sakazakii/metabolismo , Meios de Cultura/química , Microbiologia de AlimentosRESUMO
Cronobacter spp. cause infant disease, several cases have been associated with powdered infant formulae (PIF). In the early 2000s, contamination of German PIF with these opportunistic pathogens was quite common. Before 2008, all isolates Cronobacter spp. had been classified as Enterobacter sakazakii, therefore little is known about species diversity within such isolates. Genetic, serologic, and biochemical traits of 80 Cronobacter isolates, originally obtained 2003-2006 within infant food surveys in Germany, were reassessed in this study. By sequencing of the fusA gene, all isolates were unambiguously assigned to two species, C. sakazakii (n = 73) and C. malonaticus (n = 7). PCR serotyping identified five C. sakazakii serotypes and two C. malonaticus serotypes, biochemical profiling yielded five biogroups. PFGE analysis also showed high heterogeneity in both species. Multilocus sequence typing of 26 selected isolates yielded 16 different sequence types (ST), including C. sakazakii ST 1 (n = 6) and the highly virulent ST 4 (n = 2). The results suggest that just two, but highly heterogeneous species were responsible for the Cronobacter contamination problem which challenged the German PIF industry in the beginning of this century. This fact may have influenced the success of efforts to identify and eliminate sources of contamination.
Assuntos
Cronobacter sakazakii/isolamento & purificação , Cronobacter/classificação , Cronobacter/genética , Microbiologia de Alimentos , Fórmulas Infantis/microbiologia , Técnicas de Tipagem Bacteriana , Cronobacter/isolamento & purificação , Cronobacter sakazakii/classificação , Cronobacter sakazakii/genética , Genótipo , Alemanha , Humanos , Lactente , Tipagem de Sequências Multilocus , Fator G para Elongação de Peptídeos/genética , Reação em Cadeia da Polimerase , Estudos Retrospectivos , SorotipagemRESUMO
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 .
Assuntos
Cronobacter sakazakii/classificação , Cronobacter/classificação , Humanos , Fórmulas Infantis , RNA Ribossômico 16S/genética , Reação em Cadeia da Polimerase em Tempo RealRESUMO
AIM: Cronobacter sakazakii sequence types 1, 4, 8 and 12 are associated with outbreaks of neonatal meningitis and necrotizing enterocolitis infections. However clonality results in strains which are indistinguishable using conventional methods. This study investigated the use of clustered regularly interspaced short palindromic repeats (CRISPR)-cas loci profiling for epidemiological investigations. MATERIALS & METHODS: Seventy whole genomes of C. sakazakii strains from four clonal complexes which were widely distributed temporally, geographically and origin of source were profiled. RESULTS & CONCLUSION: All strains encoded the same type I-E subtype CRISPR-cas system with a total of 12 different CRISPR spacer arrays. This study demonstrated the greater discriminatory power of CRISPR spacer array profiling compared with multilocus sequence typing, which will be of use in source attribution during Cronobacter outbreak investigations.
Assuntos
Proteínas de Bactérias/genética , Cronobacter sakazakii/isolamento & purificação , Endonucleases/genética , Infecções por Enterobacteriaceae/microbiologia , Proteínas de Bactérias/metabolismo , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Cronobacter sakazakii/classificação , Cronobacter sakazakii/enzimologia , Cronobacter sakazakii/genética , Endonucleases/metabolismo , Infecções por Enterobacteriaceae/epidemiologia , Feminino , França/epidemiologia , Genoma Bacteriano , Humanos , Lactente , Recém-Nascido , Masculino , FilogeniaRESUMO
Franconibacter (Enterobacter, Cronobacter) pulveris bacteria share several typical characteristics with, and hence pose a challenge for the detection of, Cronobacter sakazakii, an emerging opportunistic pathogen, which can cause severe infections in neonates. A structurally variable O-specific polysaccharide (OPS) called O antigen provides the major basis for the typing of Gram-negative bacteria. We investigated the structure and genetics of the O antigen of F. pulveris G3872 (designated O1). An OPS was isolated by mild alkaline degradation of the LPS, whereas the same polysaccharide and its oligosaccharide fragments were obtained by mild acid degradation. Studies by sugar analysis and NMR spectroscopy showed that the OPS contained d-ribose, l-rhamnose (l-Rha) and a rarely occurring monosaccharide 4-deoxy-d-arabino-hexose, and the OPS structure was established. The O-antigen gene cluster of F. pulveris G3872 between JUMPStart and gnd genes includes putative genes for glycosyltransferases, ATP-binding cassette (ABC)-transporter genes wzm and wzt, and genes for the synthesis of l-Rha, but no genes for the synthesis of 4-deoxy-d-arabino-hexose. A mutation test with the wzm gene confirmed that the OPS is synthesized and exported by the ABC-transporter-dependent pathway. A trifunctional transferase was suggested to catalyse formation of two glycosidic linkages and add a methyl group to the non-reducing end of the OPS to terminate the chain elongation. A carbohydrate-binding module that presumably recognizes the terminal methyl-modified monosaccharide was found at the C-terminus of Wzt. Primers specific for F. pulveris G3872 were designed based on the wzm gene, which has potential to be used for identification and detection of the O1 serogroup.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Enterobacteriaceae/metabolismo , Antígenos O/química , Antígenos O/genética , Oligossacarídeos/química , Sequência de Bases , Cronobacter sakazakii/classificação , Cronobacter sakazakii/imunologia , Enterobacteriaceae/genética , Enterobacteriaceae/imunologia , Antígenos O/imunologia , Análise de Sequência de DNARESUMO
Cronobacter sakazakii is an opportunistic human-pathogenic bacterium known to cause acute meningitis and necrotizing enterocolitis in neonates and immunocompromised individuals. This human-pathogenic microorganism has been isolated from a variety of food and environmental samples, and has been also linked to foodborne outbreaks associated with powdered infant formula (PIF). The U.S. Food and Drug Administration have a policy of zero tolerance of these organisms in PIF. Thus, this agency utilizes the presence of these microorganisms as one of the criteria in implementing regulatory actions and assessing adulteration of food products of public health importance. In this study, we recovered two isolates of Cronobacter from the 91 environmental swab samples during an investigation of sporadic case of foodborne illness following conventional microbiological protocols. The isolated typical colonies were identified using VITEK2 and real-time PCR protocols. The recovered Cronobacter isolates were then characterized for species identification by sequencing the 16S rRNA locus. Further, multilocus sequence typing (MLST) was accomplished characterizing seven known C. sakazakii-specific MLST loci (atpD, fusA, glnS, gltB, gyrB, infB, and pps). Results of this study confirmed all of the recovered Cronobacter isolates from the environmental swab samples to be C. sakazakii. The MLST profile matched with the published profile of the complex 31 of C. sakazakii. Thus, rRNA and 7-loci MLST-based sequencing protocols are robust techniques for rapid detection and differentiation of Cronobacter species, and these molecular diagnostic tools can be used in implementing successful surveillance program and in the control and prevention of foodborne illness.
Assuntos
Cronobacter sakazakii/isolamento & purificação , Infecções por Enterobacteriaceae/microbiologia , Doenças Transmitidas por Alimentos/microbiologia , Cronobacter sakazakii/classificação , Cronobacter sakazakii/genética , DNA Bacteriano/genética , Microbiologia Ambiental , Humanos , Filogenia , RNA Ribossômico 16S/genéticaRESUMO
Cronobacter sakazakii is a foodborne pathogen associated with rare but often lethal infections in neonates. Powdered infant formula (PIF) represents the most frequent source of infection. Out of the identified serotypes (O1 to O7), O1, O2, and O3 are often isolated from clinical and PIF samples. Serotype-specific monoclonal antibodies (MAbs) suitable for application in enzyme immunoassays (EIAs) for the rapid detection of C. sakazakii have not yet been developed. In this study, we created specific MAbs with the ability to bind toC. sakazakii of serotypes O1, O2, and O3. Characterization by indirect EIAs, immunofluorescence, motility assays, and immunoblotting identified lipopolysaccharide (LPS) and exopolysaccharide (EPS) as the antigenic determinants of the MAbs. The established sandwich EIAs were highly sensitive and were able to detect between 2 × 10(3)and 9 × 10(6)CFU/ml. Inclusivity tests confirmed that 93% of serotype O1 strains, 100% of O2 strains, and 87% of O3 strains were detected at low cell counts. No cross-reactivity with >100 strains of Cronobacter spp. and other Enterobacter iaceae was observed, except for that with C. sakazakii serotype O3 and Cronobacter muytjensii serotype O1. Moreover, the sandwich EIAs detected C. sakazakii in PIF samples artificially contaminated with 1 to 10 bacterial cells per 10 g of sample after 15 h of preenrichment. The use of these serotype-specific MAbs not only allows the reliable detection of C. sakazakii strains but also enables simultaneous serotyping in a simple sandwich EIA method.
Assuntos
Anticorpos Antibacterianos/imunologia , Anticorpos Monoclonais/imunologia , Cronobacter sakazakii/classificação , Cronobacter sakazakii/isolamento & purificação , Sorogrupo , Sorotipagem/métodos , Humanos , Técnicas Imunoenzimáticas/métodos , Sensibilidade e Especificidade , Fatores de TempoRESUMO
Cronobacter sakazakii (formerly Enterobacter sakazakii) is an opportunistic pathogen that causes meningitis, sepsis, and necrotizing enterocolitis in neonates and infants through consumption of contaminated milk-based foods. In this study, the prevalence of C. sakazakii in 705 retail milk-based infant and baby food samples was investigated in 12 cities in Shaanxi, China, in 2010 and 2012. One hundred and nineteen samples (16.9%) were C. sakazakii positive. The isolates were further characterized for antimicrobial susceptibility to 14 antibiotics, pulsed-field gel electrophoresis profiles, and presence of the virulence genes. Samples of brand W, Y, A, and G in 2010 and 2012 were C. sakazakii positive. All isolates recovered in 2010 and 2012 were susceptible to levofloxacin and cefoperazone. In 2012, no isolate was resistant to gentamicin, cefoxitin, chloramphenicol, gatifloxacin, ciprofloxacin, and ceftriaxone. Antibiotic resistance of the isolates was most commonly found to rifampicin, amoxicillin-clavulanic acid, streptomycin, tetracycline, and ampicillin in both 2010 and 2012, except to trimethoprim/sulfamethoxazole in 2012. Pulsed-field gel electrophoresis profiles indicated that C. sakazakii isolates were genotypically diverse, although these isolates were prevalent in infant and baby foods with the same brand. A total of 34 virulence gene profiles of the C. sakazakii isolates in 2010 and 2012 were detected. Isolates that co-carried hly-ompX-eitCBAD-iucABCD/iutA genes in 2012 were significantly (p < 0.05) more prevalent than those in 2010. The results added new epidemiological evidence for the widespread occurrence of C. sakazakii in retail milk-based infant and baby foods and this should be an indicator of potential health risk for consumers.
Assuntos
Cronobacter sakazakii/isolamento & purificação , Alimentos em Conserva/microbiologia , Alimentos Infantis/microbiologia , Fórmulas Infantis/microbiologia , Substitutos do Leite , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , China , Cronobacter sakazakii/classificação , Cronobacter sakazakii/efeitos dos fármacos , Cronobacter sakazakii/genética , Farmacorresistência Bacteriana , Farmacorresistência Bacteriana Múltipla , Eletroforese em Gel de Campo Pulsado , Inspeção de Alimentos , Alimentos em Conserva/economia , Genes Bacterianos , Humanos , Lactente , Alimentos Infantis/economia , Fórmulas Infantis/economia , Testes de Sensibilidade Microbiana , Substitutos do Leite/economia , Tipagem Molecular , Filogenia , Análise Espaço-Temporal , Fatores de Virulência/genética , Fatores de Virulência/metabolismoRESUMO
BACKGROUND: Cronobacter sakazakii is a member of the genus Cronobacter that has frequently been isolated from powdered infant formula (PIF) and linked with rare but fatal neonatal infections such as meningitis and necrotising enterocolitis. The Cronobacter MLST scheme has reported over 400 sequence types and 42 clonal complexes; however C. sakazakii clonal complex 4 (CC4) has been linked strongly with neonatal infections, especially meningitis. There have been a number of reported Cronobacter outbreaks over the last three decades. The largest outbreak of C. sakazakii was in a neonatal intensive care unit (NICU) in France (1994) that lasted over 3 months and claimed the lives of three neonates. The present study used whole genome sequencing data of 26 isolates obtained from this outbreak to reveal their relatedness. This study is first of its kind to use whole genome sequencing data to analyse a Cronobacter outbreak. METHODS: Whole genome sequencing data was generated for 26 C. sakazakii isolates on the Illumina MiSeq platform. The whole genome phylogeny was determined using Mugsy and RaxML. SNP calls were determined using SMALT and SAMtools, and filtered using VCFtools. RESULTS: The whole genome phylogeny suggested 3 distant clusters of C. sakazakii isolates were associated with the outbreak. SNP typing and phylogeny indicate the source of the C. sakazakii could have been from extrinsic contamination of reconstituted infant formula from the NICU environment and personnel. This pool of strains would have contributed to the prolonged duration of the outbreak, which was up to 3 months. Furthermore 3 neonates were co-infected with C. sakazakii from two different genotype clusters. CONCLUSION: The genomic investigation revealed the outbreak consisted of an heterogeneous population of C. sakazakii isolates. The source of the outbreak was not identified, but probably was due to environmental and personnel reservoirs resulting in extrinsic contamination of the neonatal feeds. It also indicated that C. sakazakii isolates from different genotype clusters have the ability to co-infect neonates.
Assuntos
Cronobacter sakazakii/genética , Infecção Hospitalar , Surtos de Doenças , Infecções por Enterobacteriaceae/epidemiologia , Infecções por Enterobacteriaceae/microbiologia , Unidades de Terapia Intensiva Neonatal , Cronobacter sakazakii/classificação , Infecções por Enterobacteriaceae/história , França/epidemiologia , Genoma Bacteriano , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala , História do Século XX , Humanos , Lactente , Recém-Nascido , Filogenia , Polimorfismo de Nucleotídeo ÚnicoRESUMO
Cronobacter is a genus of widespread, opportunistic, foodborne pathogens that can result in serious illnesses in at-risk infants because of their immature immunity and high dependence on powdered formula, which is one of the foods most often contaminated by this pathogen. However, limited information is available regarding the pathogenesis and the specific virulence factors of this species. In this study, the virulences of 42 Cronobacter sakazakii isolates were analyzed by infecting neonatal SD rats. A comparison of the typing patterns of the isolates enabled groups with close relationships but that exhibited distinct pathogenesis to be identified. Among these groups, 2 strains belonging to the same group but showing distinct virulences were selected, and 2-DE was applied to identify differentially expressed proteins, focusing on virulence-related proteins. A total of 111 protein spots were identified using matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF/TOF-MS), and 89 were successfully identified. Further analysis suggested that at least 11 of these proteins may be involved in the pathogenesis of this pathogen. Real-time PCR was carried out to further confirm the differential expression pattern of the genes, and the results indicated that the mRNA expression levels were consistent with the protein expression levels. BIOLOGICAL SIGNIFICANCE: The virulence factors and pathogenesis of Cronobacter are largely unknown. In combination with animal toxicological experiments and subtyping results of C. sakazakii, comparative proteomics analysis was performed to comprehensively evaluate the differentially expressed proteins of two isolates that exhibited distinct virulence but were closely related. These procedures made it possible to identify the virulence-related of factors of Cronobacter. Among the 89 total identified proteins, at least 11 show virulence-related potential. This work provides comprehensive candidates for the further investigation of the pathogenesis of Cronobacter.