RESUMO
Cronobacter spp. are bacterial pathogens isolated from a wide variety of foods. This study aims at evaluating the occurrence of Cronobacter spp. in low water activity functional food samples, detect the presence of virulence genes, and determine the antibiotic susceptibility of strains. From 105 samples, 38 (36.2%) were contaminated with Cronobacter spp. The species identified by polymerase chain reaction (PCR) and sequencing analyses (rpoB and fusA genes, respectively) were C. sakazakii (60.3%), C. dublinensis (25.4%), C. turincensis (9.5%), and C. malonaticus (4.8%). Nineteen fusA alleles were identified, including four new alleles. The virulence genes were identified by PCR and all isolates were positive for ompX and sodA genes, 60.3% to cpa gene, and 58.7% to hly gene. Using the disk diffusion method, antibiotic susceptibility to twelve antibiotics was assessed twice, separated by a 19-month period. In the first test, the isolates showed diverse antibiotic susceptibility profiles, with nineteen isolates (30.2%) being multi-drug resistant (resistant to three or more antibiotic classes), in the second, the isolates were susceptible to all antibiotics. Cronobacter spp. in functional foods demonstrates the need for continued investigation of this pathogen in foods, and further research is needed to clarify the loss of resistance of Cronobacter strains.
Assuntos
Antibacterianos , Cronobacter , Alimento Funcional , Testes de Sensibilidade Microbiana , Cronobacter/genética , Cronobacter/efeitos dos fármacos , Cronobacter/isolamento & purificação , Cronobacter/classificação , Brasil , Antibacterianos/farmacologia , Microbiologia de Alimentos , Fatores de Virulência/genética , Proteínas de Bactérias/genética , Contaminação de Alimentos/análise , Água , Farmacorresistência Bacteriana/genéticaRESUMO
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
AIMS: The objective of this study was to evaluate the cytotoxic activity of Cronobacter strains isolated from foods (n = 50) and clinical samples (n = 6) in Brazil and genotype selected strains (n = 18) using multi-locus sequence typing (MLST) METHODS AND RESULTS: The cytotoxic activity of C. sakazakii (n = 29), C. dublinensis (n = 13), C. malonaticus (n = 6), C. turicensis (n = 6) and C. muytjensii (n = 2) was screened using Vero, RK13, Hep2c, NCTC clone 929 and BHK-21 cell lines. Selected Cronobacter strains were assigned to C. sakazakii ST 21, C. turicensis ST 252, C. sakazakii ST 647, and three newly assigned STs: C. turicensis STs 738-740. The maximum death caused by non-heat-treated filtrates was 20·4, 86·2, 47·0 and 84·0%, in Vero, RK13, Hep2c and NCTC clone 929 cells, respectively. These were caused by C. sakazakii strains C291 and C292 (ST 494) which had been isolated during neonatal Cronobacter meningitis infection, and C110 (ST 395) isolated from flaxseed flour. Thermal treatment (100°C/20 min) significantly reduced the cytotoxicity activity in NCTC clone 929 and Vero cells (P ≤ 2 × 10-6 ), but not in RK13 (P = 0·12) and Hep2c (P = 0·85), indicating the cytotoxin(s) were probably proteinaceous. Electron microscopy revealed that cytotoxic compounds from C. sakazakii induced several cell death characteristics, including loss of cell-cell contact, microvilli reduction and cellular lysis. Autophagic vacuoles and mitochondrial damage were the most common ultrastructural features observed. CONCLUSIONS: It was concluded that Cronobacter strains, especially C. sakazakii, could produce heat-labile cytotoxic compounds in cell filtrates. SIGNIFICANCE AND IMPACT OF THE STUDY: This study providing insights into the pathogenesis of the Cronobacter genus. Cytotoxins were identified in excreted filtrates of C. sakazakii strains isolated from food and clinical specimens. The presence of Cronobacter strains that can produce cytotoxins in foods can be a potential threat to human health and highlight the need for high levels of hygiene.
Assuntos
Cronobacter/classificação , Cronobacter/patogenicidade , Microbiologia de Alimentos , Meningites Bacterianas/microbiologia , Virulência , Animais , Brasil , Linhagem Celular , Sobrevivência Celular , Chlorocebus aethiops , Cronobacter/genética , DNA Bacteriano , Infecções por Enterobacteriaceae/microbiologia , Genótipo , Interações Hospedeiro-Patógeno , Humanos , Tipagem de Sequências Multilocus , Células VeroRESUMO
The aim of this study was to determine the prevalence Cronobacter from 30 samples of oats and 30 of linseeds commercially available in Brazil. The detection of Cronobacter was as according to the ISO 22964:2017. The isolates were characterized according to their phenotypically using Vitek 2.0 and antibiotic susceptibility profile. Molecular characterization was accomplished by real-time PCR targeting dnaG gene, PCR targeting rpoB gene, multiplex-PCR targeting cgcA gene and fusA allele sequencing. A total of 34 samples (56.7%) contained Cronobacter; 19 (63.3%) of linseeds and 15 (50.0%) of oats. The isolates were identified as C. sakazakii (n = 18, 52.9%), C. dublinensis (n = 7, 20.6%), C. turicensis (n = 6, 17.7%) and C. malonaticus (n = 3, 8.8%). Thirty-four Cronobacter isolates were assigned to 11 different fusA alleles of which 3 were new (169, 170 and 171). The PCR targeting rpoB gene and cgcA gene failed to identify 19 isolates. Seven (20.6%) strains showed resistance or intermediate/resistance to tetracycline, and one (2.9%) strain had intermediate resistance to piperacilin-tazobactam. The presence of Cronobacter in oats and linseeds indicate that these foods can be a potential threat to human health, particularly when preparing food for elderly or immunosuppressed persons. The incorrect use of this foods for feeding of neonates (<6 months) by careers should also be avoided.
Assuntos
Avena/microbiologia , Cronobacter/efeitos dos fármacos , Cronobacter/genética , Linho/microbiologia , Microbiologia de Alimentos , Antibacterianos/farmacologia , Técnicas de Tipagem Bacteriana , Brasil , Cronobacter/classificação , Genes Bacterianos/genética , Humanos , Testes de Sensibilidade Microbiana , Reação em Cadeia da Polimerase em Tempo RealRESUMO
In Bogotá, Colombia, a large number of babies are fed with breast milk substitutes made from corn and plantain starch. We found 34.3% of tested samples to be contaminated with Cronobacter spp.; C. sakazakii was the most recovered species. Our findings underscore the risk for contamination of breast milk substitutes.
Assuntos
Cronobacter , Contaminação de Alimentos , Microbiologia de Alimentos , Doenças Transmitidas por Alimentos/epidemiologia , Doenças Transmitidas por Alimentos/microbiologia , Substitutos do Leite , Colômbia/epidemiologia , Cronobacter/classificação , Cronobacter/genética , Cronobacter/isolamento & purificação , Humanos , Vigilância em Saúde PúblicaRESUMO
Several Cronobacter species are opportunistic pathogens that cause infections in humans. The aim of this study was to detect Cronobacter spp. from 90 samples of retail foods in Brazil, and characterize the strains by phenotypic tests, molecular assays and antibiotic susceptibility. Three isolation methodologies were evaluated using different selective enrichments and the isolates were identified using Vitek 2.0, PCRs protocols, fusA allele sequencing and multilocus sequence typing (MLST). Thirty-eight samples (42.2%) contained Cronobacter spp., and the highest percentage was found in flours (66.7%, 20/30), followed by spices and herbs (36.7%, 11/30), and cereal mixes for children (23.3%, 7/30). The 45 isolates included four species: C. sakazakii (n = 37), C. malonaticus (n = 3), C. dublinensis (n = 3), and C. muytjensii (n = 2); that presented 20 different fusA alleles. MLST analysis revealed 32 sequence types (STs), 13 of which were newly identified. All strains were sensitive to all antibiotics (n = 10) tested. The combination of CSB/v enrichment with DFI plating was considered the most efficient for Cronobacter spp. isolation. This study revealed the presence of Cronobacter spp. in foods commercialized in Brazil and the isolates showed a high diversity after MLST analysis and included two strains of the C. sakazakii ST4 neonatal meningitic pathovar.
Assuntos
Antibacterianos/farmacologia , Cronobacter/genética , Cronobacter/isolamento & purificação , Microbiologia de Alimentos , Técnicas de Tipagem Bacteriana , Técnicas Bacteriológicas/métodos , Brasil , Cronobacter/classificação , Cronobacter/efeitos dos fármacos , Cronobacter sakazakii/genética , Cronobacter sakazakii/isolamento & purificação , Farmacorresistência Bacteriana , Farinha/microbiologia , Tipagem de Sequências Multilocus , Fator G para Elongação de Peptídeos/genética , Fenótipo , Reação em Cadeia da Polimerase , Especiarias/microbiologiaRESUMO
BACKGROUND/AIMS: Cronobacter spp. have been identified as being of considerable risk to neonates. The occurrence of organisms in infant formulas is therefore of considerable interest. METHODS: The occurrence of Cronobacter spp. in infant feeds (formulas and fortified cow's milk) was determined using most probable number (MPN) analysis, and from formula preparation utensils. Ninety-nine samples were analyzed, of which 42 were unopened cans of powdered infant formula (PIF), 25 reconstituted infant formulas in feeding bottles, 27 utensils used in the preparation of infant formula and 5 samples of fortified cow's milk. Presumptive Cronobacter spp. isolates were identified using the 7 allele multilocus sequence typing (MLST) scheme. RESULTS: C. sakazakii, C. malonaticus and C. muytjensii were recovered from PIF. Although the incidence of Cronobacter in PIF was 29% (12/42), the level was low with an average of 0.54 MPN/100 g. According to MLST profiling, C. sakazakii was the most frequently isolated Cronobacter species, and C. sakazakii ST4 (associated with neonatal meningitis) was recovered from 2/42 PIF samples at 0.51 and 0.92 MPN/100 g. CONCLUSIONS: Cronobacter spp. can be isolated from PIF and therefore strict hygienic practices during PIF preparation are important to minimize neonate exposure and reduce the risk of severe infections.
Assuntos
Cronobacter/isolamento & purificação , Contaminação de Alimentos/análise , Microbiologia de Alimentos , Fórmulas Infantis , Alelos , Técnicas de Tipagem Bacteriana , Cronobacter/classificação , Cronobacter/genética , Infecções por Enterobacteriaceae/prevenção & controle , Genótipo , Maternidades , Humanos , Lactente , Fórmulas Infantis/instrumentação , Tipagem de Sequências Multilocus , FenótipoRESUMO
Cronobacter (formerly known as Enterobacter sakazakii) is a genus comprising seven species regarded as opportunistic pathogens that can be found in a wide variety of environments and foods, including powdered infant formula (PIF). Cronobacter sakazakii, the major species of this genus, has been epidemiologically linked to cases of bacteremia, meningitis in neonates, and necrotizing enterocolitis, and contaminated PIF has been identified as an important source of infection. Robust and reproducible subtyping methods are required to aid in the detection and investigation, of foodborne outbreaks. In this study, a pulsed-field gel electrophoresis (PFGE) protocol was developed and validated for subtyping Cronobacter species. It was derived from an existing modified PulseNet protocol, wherein XbaI and SpeI were the primary and secondary restriction enzymes used, generating an average of 14.7 and 20.3 bands, respectively. The PFGE method developed was both reproducible and discriminatory for subtyping Cronobacter species.