Emergent methods for inactivation of Cronobacter sakazakii in foods: A systematic review and meta-analysis.

Cronobacter sakazakii is a potentially pathogenic bacterium that is resistant to osmotic stress and low aw, and capable of persisting in a desiccated state in powdered infant milks. It is widespread in the environment and present in various products. Despite the low incidence of cases, its high mortality rates of 40 to 80 % amongst neonates make it a microorganism of public health interest. This current study performed a comparative assessment between current reduction methods applied for C. sakazakii in various food matrices, indicating tendencies and relevant parameters for process optimization. A systematic review and meta-analysis were conducted, qualitatively identifying the main methods of inactivation and control, and quantitatively evaluating the effect of treatment factors on the reduction response. Hierarchical clustering dendrograms led to conclusions on the efficiency of each treatment. Review of recent research trend identified a focus on the potential use of alternative treatments, with most studies related to non-thermal methods and dairy products. Using random-effects meta-analysis, a summary effect-size of 4-log was estimated; however, thermal methods and treatments on dairy matrices displayed wider dispersions - of τ2 = 8.1, compared with τ2 = 4.5 for vegetal matrices and τ2 = 4.0 for biofilms. Meta-analytical models indicated that factors such as chemical concentration, energy applied, and treatment time had a more significant impact on reduction than the increase in temperature. Non-thermal treatments, synergically associated with heat, and treatments on dairy matrices were found to be the most efficient.

Diversity of Cronobacter genus isolated between 1970 and 2019 on the American continent and genotyped using multi-locus sequence typing.

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.

Proteomics profiles of Cronobacter sakazakii and a fliF mutant: Adherence and invasion in mouse neuroblastoma cells.

Cronobacter sakazakii is an opportunistic foodborne pathogen associated with necrotizing enterocolitis, bacteremia, and meningitis in infants. A comparative proteomic study of C. sakazakii ATCC BAA-894 (CS WT) and a fliF::Tn5 mutant was performed, including the ability of both strains to adhere to and invade N1E-115 cells. To achieve this goal, a nonmotile C. sakazakii‬ ATCC BAA-894 fliF::Tn5 (CS fliF::Tn5) strain was generated using an EZ-Tn5 Tnp Transposome kit. Analysis of differential protein expression showed that 81.49% (361/443) of the proteins were expressed in both strains, 8.35% (37/443) were exclusively expressed in the CS WT strain, and 10.16% (45/443) were exclusively expressed in the CS fliF::Tn5 strain. The main exclusively expressed proteins in the CS WT strain were classified into the "cell motility" and "signal transduction mechanisms" subcategories. The proteins exclusively expressed in the CS fliF::Tn5 strain were classified into the following subcategories: "intracellular trafficking, secretion, and vesicular transport", "replication, recombination, and repair", "nucleotide transport and metabolism", "carbohydrate transport and metabolism", "coenzyme transport and metabolism", and "lipid transport and metabolism". Expression of the Cpa protein was detected in both strains, but Cpa was more abundant in the CS WT strain than in the CS fliF::Tn5 strain. A significant increase (p = 0.0001) in adherence to N1E-115 cells was observed in the nonmotile CS fliF::Tn5 strain (31.3 × 106 CFU/mL) compared to the CS WT strain (14.5 × 106 CFU/mL). Additionally, the CS WT strain showed a 0.17% invasion frequency in N1E-115 cells, which was significantly higher (p = 0.01) than that of the nonmotile CS fliF::Tn5 strain. In conclusion, the proteins involved in the motility were mainly identified by proteomic analysis in the CS WT strain compared to the CS fliF::Tn5 strain. Our data indicate that flagella are required to promote the invasion of N1E-115 cells and that the absence of flagella significantly increases the adherence to N1E-115 cells.

Multi-locus sequence typing and antimicrobial susceptibility profile of Cronobacter sakazakii and Cronobacter malonaticus isolated from corn-based farinaceous foods commercialized in Brazil.

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.

Microbiological Quality of Powdered Infant Formula in Latin America.

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.

Adaptação homóloga e heteróloga de células sésseis de Cronobacter sakazakii a óleos essenciais / Homologous and heterologous adaptation of Cronobacter sakazakii sessile cells to essential oils

Cronobacter sakazakii é patogênico, importante contaminante de fórmulas infantis, que apresenta tolerância aos sanificantes comerciais. Assim, os óleos essenciais se destacam. O objetivo desse trabalho foi avaliar a adaptação homologa e heteróloga de células sésseis de C. sakazakii aos óleos de canela, ho wood e menta. C. sakazakii foi adaptado a concentrações subletais dos óleos e desafiado a diferentes concentrações dos mesmos, determinando-se a adaptação homóloga. Já para avaliação da adaptação heteróloga os óleos foram diferentes daqueles usados na adaptação. De modo geral a adaptação tornou as células sésseis mais sensíveis aos óleos, entretanto, houve adaptação homóloga para o óleo de canela. Também foi observada adaptação heteróloga. C. sakazakii na forma séssil foi capaz de desenvolver adaptação homóloga e heteróloga.(AU)

Adaptação homóloga e heteróloga de células sésseis de Cronobacter sakazakii a óleos essenciais / Homologous and heterologous adaptation of Cronobacter sakazakii sessile cells to essential oils

Cronobacter sakazakii é patogênico, importante contaminante de fórmulas infantis, que apresenta tolerância aos sanificantes comerciais. Assim, os óleos essenciais se destacam. O objetivo desse trabalho foi avaliar a adaptação homologa e heteróloga de células sésseis de C. sakazakii aos óleos de canela, ho wood e menta. C. sakazakii foi adaptado a concentrações subletais dos óleos e desafiado a diferentes concentrações dos mesmos, determinando-se a adaptação homóloga. Já para avaliação da adaptação heteróloga os óleos foram diferentes daqueles usados na adaptação. De modo geral a adaptação tornou as células sésseis mais sensíveis aos óleos, entretanto, houve adaptação homóloga para o óleo de canela. Também foi observada adaptação heteróloga. C. sakazakii na forma séssil foi capaz de desenvolver adaptação homóloga e heteróloga.

Fatal Cronobacter sakazakii Sequence Type 494 Meningitis in a Newborn, Brazil.

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.

Occurrence and molecular characterization of different virulence-associated genes of Cronobacter sakazakii isolates from some foods and dust samples / Ocorrência e caracterização molecular de diferentes genes associados à virulência de Cronobacter sakazakiidetectados em alguns alimentos e amostras de poeira

Among the Cronobacter genus, Cronobacter sakazakii is the most common species posing a severe health risk for newborns, infants and children. Some infant formulas, cereal-based foods, and food production environments may be the potential reservoirs of C. sakazakii. This pathogen possesses different virulence factors encoded by different virulence genes. Therefore, characterizing these genes is important for distinguishing pathogenic strains from nonpathogenic ones. The objective of this study was to characterize some virulence genes [OmpA, OmpX, zpx, and Cpa] by real-time polymerase chain reaction (PCR) in C. sakazakii isolates from a total of 120 samples (20 each of milk powder, starch, rice flour, semolina, infant formula and dust samples from food production environments). Overall, 13 isolates (7 from milk powder, 2 rice flour, 1 semolina, and 3 dust) were cultured, identified by bioMérieux API® 20E test kit, and then subjected to real-time PCR application for screening the target virulence-associated genes. Our results showed that all of 13 isolates were positive for the virulence genes OmpA, OmpX, zpx, and Cpa. In summary, our study revealed that some of the analyzed foods and environmental samples were contaminated with pathogenic C. sakazakii with its virulence-associated markers, far above the allowable limit; and therefore, this level of contamination may pose a severe health threat for newborns, infants, and children.(AU)

Dentre o gênero Cronobacter, Cronobacter sakazakii é a espécie mais comum que representa um grave risco para a saúde dos recém-nascidos, bebês e crianças. Algumas formulas infantis para crianças, alimentos a base de cereais e locais de produção de alimentos, foram considerados como potenciais locais de contaminação de C. sakazakii. Este patógeno possui diferentes agentes de virulência codificados por diferentes genes de virulência. Portanto, a caracterização dos genes é importante para distinguir as cepas patogênicas das não patogênicas. O objetivo deste estudo foi caracterizar os diferentes genes de virulência [OmpA, OmpX, zpx, and Cpa] em C. sakazakii isolados em um total de 120 amostras (20 de cada uma delas - leite em pó, amido, farinha de arroz, sêmola, comida para bebês e amostras de poeira provenientes dos ambientes de produção de alimentos) por reação em cadeia da polimerase em tempo real (RT-PCR). No total, foram cultivadas 13 estirpes (7 de leite em pó, 2 de farinha de arroz, 1 de sêmola e 3 poeiras) e depois identificadas pelo kit de teste bio Mérieux API® 20E. As estirpes identificadas foram submetidas ao PCR em tempo real para caracterizar os genes alvo associados à virulência. Os resultados mostraram que todos os genes C. sakazakii isolados eram patogênicos e positivos às OmpA, OmpX, zpx e Cpa com um padrão de coexistência. Em resumo, o nosso estudo revelou que os alimentos e os ambientes de produção de alimentos analisados constituíam uma ameaça à saúde dos recém-nascidos, bebês e crianças devido à contaminação por C. sakazakii patogênico como marcadores associados à virulência.(AU)

Isolation, molecular and phenotypic characterization, and antibiotic susceptibility of Cronobacter spp. from Brazilian retail foods.

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.

Reevaluation of a Suspected Cronobacter sakazakii Outbreak in Mexico.

In 2010, two infants became ill at a hospital in Mexico. Subsequently, a range of clinical, environmental, and powdered and rehydrated infant formula isolates were identified by using a combination of phenotyping and PCR probes. The strains were clustered according to pulsed-field gel electrophoresis. The causative agent was reported as Cronobacter sakazakii, with powdered infant formula (PIF) identified as the likely source of the infections. This new study further characterized the isolates from this outbreak by using multilocus sequence typing and whole genome sequencing of selected strains. Though four PIF isolates and one hospital environmental isolate were identified as C. sakazakii sequence type 297 by multilocus sequence typing, they were isolated 6 months prior to the outbreak. Genotypic analyses of patient isolates identified them as Enterobacter hormaechei and Enterobacter spp. The pulsed-field gel electrophoresis profile of the Enterobacter spp. isolates matched those of isolates from previously unopened tins of PIF. E. hormaechei was only isolated from the two infants and not PIF. The reevaluation of this outbreak highlights the need for accurate detection and identification assays, particularly during outbreak investigations in which incorrect identifications may mislead the investigation and attribution of the source. Though the species responsible for the symptoms could not be determined, this outbreak demonstrated the possible transmission of Enterobacter spp. from PIF to infants. These are possibly the first reported cases of Enterobacter spp. infection of infants from bacterial-contaminated PIF.

Riesgo de contaminación por Cronobacter Sakazakii en leches en polvo para la nutrición de lactantes / Risk of Cronobacter Sakazakii contamination in powdered milk for infant nutrition

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.

Identificação e perfil de sensibilidade de Enterobacter spp. isolados de leite bovino cru / Identification and sensitivity profile of Enterobacter spp. isolated from raw cow milk / Identificación y perfil de la sensibilidad de Enterobacter spp. aislados de leche cruda de vaca

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.

Stress tolerant virulent strains of Cronobacter sakazakii from food.

BACKGROUND: Cronobacter sakazakii is considered as an emerging foodborne pathogen. The aim of this study was to isolate and characterize virulent strains of Cronobacter sakazakii from food samples of Bangladesh. RESULT: Six (6) Cronobacter sakazakii was isolated and identified from 54 food samples on the basis of biochemical characteristics, sugar fermentation, SDS-PAGE of whole cell protein, plasmid profile and PCR of Cronobacter spp. specific genes (esak, gluA, zpx, ompA, ERIC, BOX-AIR) and sequencing. These strains were found to have moderately high antibiotic resistance against common antibiotics and some are ESBL producer. Most of the C. sakazakii isolates were capable of producing biofilm (strong biofilm producer), extracellular protease and siderophores, curli expression, haemolysin, haemagglutinin, mannose resistant haemagglutinin, had high cell surface hydrophobicity, significant resistance to human serum, can tolerate high concentration of salt, bile and DNase production. Most of them produced enterotoxins of different molecular weight. The isolates pose significant serological cross-reactivity with other gram negative pathogens such as serotypes of Salmonella spp., Shigella boydii, Shigella sonnei, Shigella flexneri and Vibrio cholerae. They had significant tolerance to high temperature, low pH, dryness and osmotic stress. CONCLUSION: Special attention should be given in ensuring hygiene in production and post-processing to prevent contamination of food with such stress-tolerant virulent Cronobacter sakazakii.

Stress tolerant virulent strains of Cronobacter sakazakii from food

BACKGROUND: Cronobacter sakazakii is considered as an emerging foodborne pathogen. The aim of this study was to isolate and characterize virulent strains of Cronobacter sakazakii from food samples of Bangladesh. RESULT: Six (6) Cronobacter sakazakii was isolated and identified from 54 food samples on the basis of biochemical characteristics, sugar fermentation, SDS-PAGE of whole cell protein, plasmid profile and PCR of Cronobacter spp. specific genes (esak, gluA, zpx, ompA, ERIC, BOX-AIR) and sequencing. These strains were found to have moderately high antibiotic resistance against common antibiotics and some are ESBL producer. Most of the C. sakazakii isolates were capable of producing biofilm (strong biofilm producer), extracellular protease and siderophores, curli expression, haemolysin, haemagglutinin, mannose resistant haemagglutinin, had high cell surface hydrophobicity, significant resistance to human serum, can tolerate high concentration of salt, bile and DNase production. Most of them produced enterotoxins of different molecular weight. The isolates pose significant serological cross-reactivity with other gram negative pathogens such as serotypes of Salmonella spp., Shigella boydii, Shigella sonnei, Shigella flexneri and Vibrio cholerae. They had significant tolerance to high temperature, low pH, dryness and osmotic stress. CONCLUSION: Special attention should be given in ensuring hygiene in production and post-processing to prevent contamination of food with such stress-tolerant virulent Cronobacter sakazakii.

Efecto de la temperatura de refrigeración y calentamiento de fórmulas lácteas Infantiles en el crecimiento de cronobacter sakazakii / Effect of temperature cooling and heating of powered infant formula on the growth of cronobacter sakazakii

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)

First clinical isolates of Cronobacter spp. (Enterobacter sakazakii) in Argentina: characterization and subtyping by pulsed-field gel electrophoresis.

Cronobacter species are opportunistic pathogens associated with severe infections in neonates and immunocompromised infants. From January 2009 through September 2010, two cases of neonatal infections associated with Cronobacter malonaticus and one case associated with Cronobacter sakazakii, two of them fatal, were reported in the same hospital. These are the first clinical isolates of Cronobacter spp. in Argentina. The objective of this work was to characterize and subtype clinical isolates of Cronobacter spp. in neonate patients, as well as to establish the genetic relationship between these isolates and the foodborne isolates previously identified in the country. Pulsed-field gel electrophoresis analysis showed a genetic relationship between the C. malonaticus isolates from two patients. Different results were found when the pulsed-field gel electrophoresis patterns of clinical isolates were compared with those deposited in the National Database of Cronobacter spp.

First clinical isolates of Cronobacter spp. (Enterobacter sakazakii) in Argentina: characterization and subtyping by pulsed-field gel electrophoresis / First clinical isolates of Cronobacter spp. (Enterobacter sakazakii) in Argentina: characterization and subtyping by pulsed-field gel electrophoresis.

Cronobacter species are opportunistic pathogens associated with severe infections in neonates and immunocompromised infants. From January 2009 through September 2010, two cases of neonatal infections associated with Cronobacter malonaticus and one case associated with Cronobacter sakazakii, two of them fatal, were reported in the same hospital. These are the first clinical isolates of Cronobacter spp. in Argentina. The objective of this work was to characterize and subtype clinical isolates of Cronobacter spp. in neonate patients, as well as to establish the genetic relationship between these isolates and the foodborne isolates previously identified in the country. Pulsed-field gel electrophoresis analysis showed a genetic relationship between the C. malonaticus isolates from two patients. Different results were found when the pulsed-field gel electrophoresis patterns of clinical isolates were compared with those deposited in the National Database of Cronobacter spp.