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The objective of this study was to characterize Cronobacter spp. and related organisms isolated from powder dairy products intended for consumption by adults and older adults using whole-genome sequencing (WGS), and to identify genes and traits that encode antibiotic resistance and virulence. Virulence (VGs) and antibiotic resistance genes (ARGs) were detected with the Comprehensive Antibiotic Resistance Database (CARD) platform, ResFinder, and MOB-suite tools. Susceptibility testing was performed using disk diffusion. Five presumptive strains of Cronobacter spp. were identified by MALDI-TOF MS and ribosomal MLST. Three C. sakazakii strains were of the clinical pathovar ST1, one was ST31, and the remaining isolate was C. malonaticus ST60. In addition, Franconibacter helveticus ST345 was identified. The C. sakazakii ST1 strains were further distinguished using core genome MLST based on 2831 loci. Moreover, 100% of the strains were resistant to cefalotin, 75% to ampicillin, and 50% to amikacin. The C. sakazakii ST1 strains were multiresistant (MDR) to four antibiotics. Additionally, all the strains adhered to the N1E-115 cell line, and two invaded it. Eighteen ARGs mainly involved in antibiotic target alteration and antibiotic efflux were detected. Thirty VGs were detected and clustered as flagellar proteins, outer membrane proteins, chemotaxis, hemolysins, and genes involved in metabolism and stress. The pESA3, pSP291-1, and pCMA1 plasmids were detected, and the prevalent mobile genetic elements (MGEs) were ISEsa1, ISEc52, and IS26. The isolates of C. sakazakii and C. malonaticus exhibited multiresistance to antibiotics, harbored genes encoding various antibiotic resistance proteins, and various virulence factors. Consequently, these contaminated powdered dairy products pose a risk to the health of hypersensitive adults.

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The reference material (RM) is a technical requirement for the quality assurance of analytical results and proficiency tests or interlaboratory comparisons. Microbiological RMs are most available in the dehydrated form, mainly by freeze-drying, and maintaining bacterial survival after preparation is a challenge. Thus, obtaining the most resistant cells is essential. Considering that bacteria present cross-response to dehydration after being submitted to an array of stress conditions, this study aimed to evaluate the influence of growth conditions on enterobacteria for the production of mixed microbiological RMs by freeze-drying in skim milk powder. Salmonella enterica serovar Enteritidis, Cronobacter sakazakii, Escherichia coli, and Citrobacter freundii were grown in a minimal medium with 0.5 M NaCl and 0 to 5.0 mM of manganese sulfate (MnSO4) until stationary phase. Salmonella Enteritidis presented an increased resistance to dehydration in the presence of Mn, while C. sakazakii was the most resistant to freeze-drying and further storage for 90 days. Mixed microbiological RMs were produced by freeze-drying and containing Salmonella Enteritidis and coliforms in skim milk powder with 100 mM of trehalose and the Salmonella survival rate was 91.2 to 93.6%. The mixed RM was stable after 30 days at -20 °C, and Salmonella and coliforms were detected by different methods being, the Rambach Agar the best for the bacterial differentiation. The results showed that the culture conditions applied in this study resulted in bacterial cells being more resistant to dehydration, freeze-drying, and stabilization for the production of mixed microbiological RMs more stable and homogeneous.

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This study characterized five Cronobacter spp. and six Salmonella spp. strains that had been isolated from 155 samples of powdered infant formula (PIF) sold in Chile and manufactured in Chile and Mexico in 2018-2020. Two strains of Cronobacter sakazakii sequence type (ST) ST1 and ST31 (serotypes O:1 and O:2) and one strain of Cronobacter malonaticus ST60 (O:1) were identified. All Salmonella strains were identified as Salmonella Typhimurium ST19 (serotype O:4) by average nucleotide identity, ribosomal multilocus sequence typing (rMLST), and core genome MLST (cgMLST). The C. sakazakii and C. malonaticus isolates were resistant to cephalothin, whereas the Salmonella isolates were resistant to oxacillin and ampicillin. Nineteen antibiotic resistance genes were detected in the C. sakazakii and C. malonaticus isolates; the most prevalent were mcr-9.1, blaCSA , and blaCMA . In Salmonella, 30 genes encoding for aminoglycoside and cephalosporin resistance were identified, including aac(6')-Iaa, ß-lactamases ampH, ampC1, and marA. In the Cronobacter isolates, 32 virulence-associated genes were detected by WGS and clustered as flagellar proteins, outer membrane proteins, chemotaxis, hemolysins, invasion, plasminogen activator, colonization, transcriptional regulator, survival in macrophages, use of sialic acid, and toxin-antitoxin genes. In the Salmonella strains, 120 virulence associated genes were detected, adherence, magnesium uptake, resistance to antimicrobial peptides, secretion system, stress protein, toxin, resistance to complement killing, and eight pathogenicity islands. The C. sakazakii and C. malonaticus strains harbored I-E and I-F CRISPR-Cas systems and carried Col(pHHAD28) and IncFIB(pCTU1) plasmids, respectively. The Salmonella strains harbored type I-E CRISPR-Cas systems and carried IncFII(S) plasmids. The presence of C. sakazakii and Salmonella in PIF is a health risk for infants aged less than 6 months. For this reason, sanitary practices should be reinforced for its production and retail surveillance.

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Cronobacter sakazakii is an enteropathogen that causes neonatal meningitis, septicemia, and necrotizing enterocolitis in preterm infants and newborns with a mortality rate of 15 to 80%. Powdered and dairy formulas (P-DF) have been implicated as major transmission vehicles and subsequently the presence of this pathogen in P-DF led to product recalls in Chile in 2017. The objective of this study was to use whole genome sequencing (WGS) and laboratory studies to characterize Cronobacter strains from the contaminated products. Seven strains were identified as C. sakazakii, and the remaining strain was Franconibacter helveticus. All C. sakazakii strains adhered to a neuroblastoma cell line, and 31 virulence genes were predicted by WGS. The antibiograms varied between strains. and included mcr-9.1 and bla CSA genes, conferring resistance to colistin and cephalothin, respectively. The C. sakazakii strains encoded I-E and I-F CRISPR-Cas systems, and carried IncFII(pECLA), Col440I, and Col(pHHAD28) plasmids. In summary, WGS enabled the identification of C. sakazakii strains and revealed multiple antibiotic resistance and virulence genes. These findings support the decision to recall the contaminated powdered and dairy formulas from the Chilean market in 2017.

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ABSTRACT: Cronobacter is a bacterial genus that includes seven species, and the species Cronobacter sakazakii is most related to meningitis and septicemia in infants associated with powdered infant formula (PIF). The objectives of this study were to evaluate the presence of C. sakazakii and to determine the microbiological quality of PIF for infant consumption. To do this, a total of 128 PIF samples were analyzed in four brands and countries (Chile, Mexico, Holland, and Brazil), considering three types of PIF: premature (PIF1), infant (PIF2), and follow-up (PIF3). Aerobic plate counts (APC) and Enterobacteriaceae (ENT) were assessed in accordance with Chilean official standards. The outer membrane protein A (ompA) gene was amplified to detect Cronobacter spp. and the fusA gene was amplified to identify C. sakazakii by using the PubMLST Web site and BLAST (NCBI). The antibiotic resistance profile was performed according to the Clinical and Laboratory Standards Institute standards. The pathogen was quantified by the most probable number (MPN). The results showed that APC median values for PIF1, PIF2, and PIF3 were 3.2, 4.9, and 4.8 log CFU g-1, respectively. The APC were higher in PIF2 (P < 0.01) from Holland (P < 0.01) in the commercial brand 4 (P < 0.01). The ENT median values in PIF1, PIF2, and PIF3 were 1.8, 1.5, and 1.7 log CFU g-1, respectively. Five strains of C. sakazakii and one strain of Cronobacter malonaticus were identified as having values between 0.023 and 2.3 MPN/g. All strains (100%) harbored the ompA, plasminogen activator (cpa), and hemolysin (hly) virulence genes. To conclude, C. sakazakii was found in four PIF samples from four Chilean products and one from Mexico, which is distributed throughout America. C. sakazakii strains exhibit virulence factors and resistance to ampicillin, thus posing a risk when PIFs are consumed by infants.

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Cronobacter spp. are bacterial pathogens that cause neonatal meningitis, septicemia, and necrotizing enterocolitis in infants with a lethality rate of 40-80%. Powdered infant formulas (PIF) have been implicated as the main vehicles of transmission. This pathogen can also cause infection through contaminated expressed breast milk, and it has been recovered from neonatal feeding tubes of neonates not fed reconstituted PIF and milk kitchen areas. This study analyzed antibiotic resistance profiles and the tissue virulence tests of Cronobacter sakazakii and Enterobacter spp. recovered from PIF, infant fecal matter's, and milk kitchen environment involved in a diarrheic hemorrhagic outbreak in 2011 in Mexico. The strains isolated from the outbreak had similar antibiotic resistance profiles and pathogenicity irrespective of isolation site, however, C. sakazakii strains isolated from PIF showed significantly higher invasive profiles than Enterobacter spp. (p = 0.001) and 83% were resistant to more than one antibiotic. The findings of this study can be used to complement existing information to better control Cronobacter and Enterobacter spp. contamination in PIF production, prevent its transmission, and improve infant food safety.

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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.

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The objective of this study was to evaluate the presence of Cronobacter sakazakii and microbiological parameters in dairy products associated with a food alert. Ninety dairy product samples were analyzed, including seven commercial brands and two product types (liquid and powdered) from four countries. Aerobic plate count (APC) and Enterobacteriaceae count were performed according to Chilean standards. Cronobacter spp. and C. sakazakii were identified by polymerase chain reaction real time amplification of rpoB and cgcA genes and the genotype by multilocus sequence typing. Eighty-eight percent of dairy products showed APC higher than the detection limit. Fifty percent of liquid commercial brand samples contained APC: 2.6, 2.3, 1.1, and 2.9 CFU/mL in brands A, C, E, and G, respectively. Results for powdered commercial brands were 3.0, 3.6, and 5.7 CFU/g in brands B, D, and F, respectively. Maximum count (5.7 CFU/g) occurred in brand F dairy product manufactured in Chile. Enterobacteriaceae were found in 55% of the samples, 64% in liquid and 51% in powdered commercial brands. In 50% of brands B, D, and E, samples contained 2.9, 2.8, and 2.7 log CFU/g, respectively. Only liquid commercial brands from the United States had Enterobacteriaceae values between 0.1 and 4.5 CFU/mL. Seventeen suspicious strains were isolated and nine were identified as Enterobacter spp. Only eight suspicious strains from four powdered commercial brands (Chile and Singapore) were confirmed as C. sakazakii by rpoB and cgcA gene amplification and fusA sequencing. C. sakazakii prevalence in the analyzed samples was 8.8%. There were 11% of powdered milk brands that contained APC between 4.0 and 4.7 log CFU/g and 55% of the samples contained Enterobacteriaceae. C. sakazakii was found in dairy products manufactured in Chile and Singapore. On the basis of this information, the Chilean Ministry of Health (RSA) decreed a national and international food alert and recalled all the product batches that resulted positive in the present study from supermarkets and pharmacies.

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Cronobacter spp. have been responsible for severe infections in infants associated with consumption of powdered infant formula and follow-up formulae. Despite several risk assessments described in published studies, few approaches have considered the tremendous variability in cell response that small micropopulations or single cells can have in infant formula during storage, preparation or post process/preparation before the feeding of infants. Stochastic approaches can better describe microbial single cell response than deterministic models as we prove in this study. A large variability of lag phase was observed in single cell and micropopulations of ≤50 cells. This variability increased as the heat shock increased and growth temperature decreased. Obviously, variability of growth of individual Cronobacter sakazakii cell is affected by inoculum size, growth temperature and the probability of cells able to grow at the conditions imposed by the experimental conditions should be taken into account, especially when errors in bottle-preparation practices, such as improper holding temperatures, or manipulation, may lead to growth of the pathogen to a critical cell level. The mean probability of illness from initial inoculum size of 1 cell was below 0.2 in all the cases and for inoculum size of 50 cells the mean probability of illness, in most of the cases, was above 0.7.

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Introduction: Cronobacter spp. is a bacterial genus that includes 7 species; Cronobacter sakazakii is the clinical specie that is the most reported and associated with meningitis and septicemia in infants. Given that it is transmitted by powdered infant formula (PIF), the WHO recommends that this product be free of Cronobacter, whereas the Chilean Food Sanitary Regulation (RSA) does not consider it. Objective: To assess the risk of C. sakazakii in PIF for consumption by infants. Methodology: A total of 72 PIF samples were analyzed using three brands originating from three countries. Aerobic plate count (APC), Enterobacteriaceae (ENT), and most probable number (MPN) were performed using the methodology described by Puch and Ito (2001). Cronobacter differential agar was used to isolate strains (DFI, Oxoid, England), and the ID32E biochemical kit (Biomeriux, France) was used for phenotyping. The pathogen was identified and genotyped by multilocus sequence typing (MLST) based on the criteria found at http://www.pubmlst.org/cronobacter. Results: Median APC for step 1 and preterm PIF was 300 CFU/g (10-36 000) and 650 CFU (70-30 000), respectively and was higher in Chilean PIF (p=0.016). There were no significant differences for type, country or PIF brand in 75 CFU/g (10-36 000) and 200 CFU/g (10-1 000) ETN (p>0.05). Two strains from two different lots with characteristic strains in DFI agar were identified as C. sakazakii with 0.23 and 2.3 MPN/g. In addition, Franconibacter helveticus, specie closely related to Cronobacter spp, was found in two other strains. Conclusions: The prevalence of Cronobacter sakazakii in all the samples was 2.7% isolated only in PIFs manufactured in Chile. The absence of Cronobacter spp in 25 g must be included in the Chilean RSA.

Introducción: Cronobacter spp es un género bacteriano con 7 especies, siendo C. sakazakii la especie clínica más reportada y asociada a meningitis y septicemia en lactantes. Es transmitida por leche en polvo (LP) por lo que la OMS recomienda su ausencia en este producto. En Chile, el reglamento sanitario de los alimentos (RSA) no lo considera. Objetivo: Evaluar el riesgo por Cronobacter sakazakii en LP destinadas al consumo de lactantes. Metodología: Se analizaron 72 muestras de LP de 3 marcas y 3 países. El recuento de bacterias mesófilas (RAM), Enterobacteriaceae (ENT) y número más probable (NMP) se realizó con la metodología de Puch and Ito (2001). Se utilizó agar diferencial Cronobacter para aislamiento (DFI, Oxoid, England) y kit bioquímico ID32E (Biomeriux, Francia) para fenotipo. El patógeno fue identificado y genotipificado por multilocus sequence typing (MLST) utilizando criterios de http://www.pubmlst.org/cronobacter. Resultados: La medianas de RAM para LP etapa 1 y prematuros fueron 300 UFC/g (10-36 000) y 650 UFC/g (70-30 000), siendo mayor en las muestras de Chile (p=0,016). Para ENT de 75 UFC/g (10-1 060) y 200 UFC/g (30-1 000), no existiendo diferencias significativas por tipo, país o marca de LP (p>0,05). Dos cepas de 2 lotes diferentes características en agar DFI se identificaron como C. sakazaki con 0,23 y 2,3 NMP/g. Además de Franconibacter helveticus en otras 2 cepas, especie relacionada estrechamente con Cronobacter spp. Conclusiones: La prevalencia de C. sakazakii en todas las muestras fue de 2,7% y aisló sólo en LP de elaborados en Chile. La ausencia de Cronobacter spp en 25 g debe ser incorporado en el RSA de Chile.

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A produção e o consumo de leite e derivados vêm aumentando anualmente no Brasil. A emergência do consumo de leite cru está relacionada à busca por produtos que mantenham preservadas suas propriedades nutricionais. Esta falsa ideia aumenta o risco do consumo de leite e derivados contaminados por micro-organismos patogênicos. As bactérias do gênero Enterobacter são amplamente distribuídas na natureza e fazem parte da microbiota do trato gastrointestinal humano e animal.  Enterobacter cloacae, Enterobacter aerogenes, Pantoea agglomerans e Cronobacter sakazakii são apontadas como patógenos oportunistas. Nesse sentido, o estudo buscou a identificação e diferenciação bioquímica de espécies de Enterobacter spp. a partir de isolados de leite bovino cru, a caracterização do perfil de sensibilidade a antimicrobianos e a pesquisa de genes de resistência. A partir de 104 cepas de Enterobacter spp. foram identificadas E. cloacae (39), Pantoea agglomerans (23), Cronobacter sakazakii (18) e E. aerogenes (9). Destas, 26% apresentaram resistência a três ou mais antimicrobianos. O gene blaTEM foi encontrado em 27% dos isolados. Dessa forma, este estudo alerta quanto ao risco de infecções por micro-organismos resistentes, potencialmente patogênicos, associados ao consumo de leite bovino cru.

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A produção e o consumo de leite e derivados vêm aumentando anualmente no Brasil. A emergência do consumo de leite cru está relacionada à busca por produtos que mantenham preservadas suas propriedades nutricionais. No entanto, este hábito aumenta os riscos do consumo de leite e derivados contaminados por micro-organismos patogênicos. As bactérias do gênero Enterobacter são amplamente distribuídas na natureza e fazem parte da microbiota do trato gastrintestinal humano e animal. Enterobacter cloacae, Enterobacter aerogenes, Pantoea agglomerans e Cronobacter sakazakii são considerados patógenos oportunistas. Nesse sentido, o presente estudo investigou a ocorrência e a diferenciação bioquímica de espécies de Enterobacter isolados de leite bovino cru, a caracterização do perfil de sensibilidade aos antimicrobianos e a pesquisa de genes de resistência. Do total de 104 cepas de Enterobacter spp. foram identificadas E. cloacae (n=39), Pantoea agglomerans (n=23), Cronobacter sakazakii (n=18) e E. aerogenes (n=9). Destas, 26% apresentaram resistência a três ou mais antimicrobianos. O gene blaTEM foi encontrado em 27% dos isolados. Este achado alerta quanto ao risco de infecções por micro-organismos ambientais resistentes, associados ao consumo de leite bovino cru.

The production and consumption of milk and dairy products is growing fast in Brazil. The increase consumption of raw milk is related to the interest for products that keep conserved their nutritional properties. However, this habit increases the risks of consumption of milk and dairy products contaminated with pathogenic microorganisms. Enterobacter spp. is widely distributed in nature and is part of the commensal microbiota of the intestinal tracts of humans and animal. Enterobacter cloacae, Enterobacter aerogenes, Pantoea agglomerans and Cronobacter sakazakii are recognized as opportunistic pathogens. The current study investigated the occurrence and the differentiation of Enterobacter species isolated from raw cow milk, the characterization of sensitivity profile to antimicrobials and presence of resistant genes. From a total of 104 strains of Enterobacter spp. were identified E. cloacae (n=39), Pantoea agglomerans (n=23), Cronobacter sakazakii (n=18) and E. aerogenes (n=9). Among these, 26% were resistant to three or more antimicrobials. The blaTEM gene was found in 27% of the isolates. Here, we highlighted the risk of infection by potential environmental microorganisms, resistant to some antimicrobials, associated with consumption of raw cow milk.

La producción y el consumo de leche y productos lácteos está aumentando anualmente en Brasil. El surgimiento del consumo de leche cruda se relaciona con la búsqueda de productos que conservan sus propiedades nutricionales. Esta conducta aumenta riesgos del consumo de leche y productos lácteos contaminados con microorganismos patógenos. Las bacterias del género Enterobacter son ampliamente distribuidas en la naturaleza y forma parte de los microorganismos comensales del aparato gastrointestinal humano y animal. Enterobacter cloacae, Enterobacter aerogenes, Pantoca agglomerans y Cronobacter sakazakii son citadas como patógenos oportunistas. De esta manera, este estudio investigó la ocurrencia y diferenciación bioquímica de Enterobacter spp. aislados de leche cruda de ganado bobino, la caracterización del perfil de sensibilidad frente a los antimicrobianos y la detección de los genes de resistencia. Del total de 104 cepas de Enterobacter spp. se identificaron E. cloacae (n=39), Pantoea agglomerans (n=23), Cronobacter sakazakii (n=18), E. aerogenes (n=9). De estos, 26% mostraron resistencia a tres o más antimicrobianos. En 27% de las cepas el gen blaTEM fue detectado. Por lo tanto, este hallazgo advierte sobre el riesgo de infecciones por microorganismos resistentes ambientales, asociados con el consumo de leche cruda de vaca.

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Cronobacter sakazakii (C. sakazakii) es un microorganismo perteneciente a la familia Enterobacteriaceae asociado con patologías como meningitis y septicemia en recién nacidos. Las fórmulas lácteas infantiles (FLI) han sido reconocidas como el vehículo más importante de transmisión de C. sakazakii, por condiciones inadecuadas durante su producción o preparación. El objetivo de la presente investigación fue evaluar el efecto de la temperatura de refrigeración y el calentamiento de las FLI en el crecimiento de C. sakazakii. Las muestras de FLI reconstituidas se contaminaron con 102, 104 y 106 UFC/mL de C. sakazakii y se refrigeraron durante cuatro horas a 4°C, 6°C y 10°C, posterioriormente, se calentaron en baño hidrotérmico hasta 37°C. Las muestras fueron sembradas al tiempo “0” (postrefrigeración) y luego del calentamiento hasta 37°C en placas con agar cromogénico y se incubaron a 35°C por 24h. Para el análisis de los datos se utilizó la prueba de Wilcoxon. Según los datos obtenidos no hubo variación significativa en la población de C. sakazakii en las FLI luego de 4 horas en refrigeración a 4°C, 6°C y 10°C; sin embargo, al calentar las fórmulas hasta 37°C, hubo en todos los casos una reducción de un ciclo logarítmico en la población de C. sakazakii con un valor de p = 0.0056 y Z= 2.752. Se concluye que el calentamiento a 37°C post-refrigeracion de las muestras, disminuye la población de C. sakazakii en FLI (p<0,05)(AU)

Cronobacter sakazakii (C. sakazakii) is a microorganism belonging to the family Enterobacteriaceae associated with diseases such as meningitis and sepsis in newborns. The powdered infant formula (PIF) have been recognized as the most important vehicle of transmission of C. sakazakii, by inadequate conditions during production or during its preparation. The aim of this investigation was to assess the effect of the cooling temperature and the heating of PIF in the growth of C. sakazakii. For this, PIF reconstituted samples were contaminated with 102, 104 and 106 CFU / mL of C. sakazakii and refrigerated for four hours at 4 ° C, 6 ° C or 10 ° C after this hydrothermal bath heated to 37 ° C. Samples were cultured at time “0” (after- cooling) and then heating to 37 ° C in chromogenic agar plates and incubated at 35 ° C for 24h . For data analysis we used the Wilcoxon test. According to the data there was no significant variation in the population of C. sakazakii in PIF after 4 hours in the refrigerator at 4 ° C, 6°C and 10 ° C , however , by heating the formulas to 37 ° C , in all cases there was a reduction of one log cycle in population C. sakazakii with a value of p = 0.0056 and Z = 2.752 . We conclude that heating at 37°C post- cooling of the samples, decreases the population of C. sakazakii in FLI ( p < 0,05 )(AU)

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