Neonate Brain Abscess by Cronobacter spp: A Powdered Infant Formula's Opportunistic Pathogen / Abscesso cerebral neonatal por Cronobacter spp: Um patógeno oportunista de fórmula láctea infantil

Cronobacter spp is an opportunistic pathogen that can cause severe neonatal infections, including septicemia, meningitis, and necrotizing enterocolitis. Over 90% of these infections outbreaks are associated with the increased consumption of powdered infant formula (PIF). We report the case of a male neonate born at term with adequate weight for gestational age, fed by breast milk and PIF. He was admitted to the ICU at 22 days old due to fever, irritability, impaired consciousness, and food intolerance. Investigation revealed an extensive abscess in the right frontoparietal region, that was surgically drained. The culture of the purulent material showed the growth of Cronobacter spp. Guided antibiotic was kept for 21 days. The patient presented a good clinical outcome, without neurological deficits. The microbiological powdered formula contamination by Enterobacter sakazakii (Cronobacter spp) can lead to a higher risk of severe infections in infants. Children may present sepsis, sensory alteration, and refractory seizures. An early brain image should be considered for symptomatic infants. The mortality rate ranges from 40 to 80%, and 74% of survivors have an adverse neurological outcome. From 1997­2013, there were reports of 6 outbreaks of E. sakazakii disease in Brazil. According to the World Health Organization (WHO), this disease is undernotified, and active foodborne surveillance systems are less than ideal. To better address this problem, in some countries, the notification is mandatory, and the adoption of stricter sanitary measures by regulatory agencies are proposed.

O Cronobacter spp é um patógeno oportunista que pode causar infecções neonatais graves, incluindo septicemia, meningite e enterocolite necrosante. Mais de 90% dessas infecções estão associadas ao aumento do consumo de fórmula infantil em pó (FIP). Relatamos o caso de um recém-nascido do sexo masculino, nascido a termo, com peso adequado para a idade gestacional, alimentado com leitematerno e FIP. Ele foi internado na UTI com 22 dias de idade devido a febre, irritabilidade, comprometimento da consciência e intolerância alimentar. A investigação revelou abscesso extenso na região frontoparietal direita, que foi drenada cirurgicamente. A cultura do material purulento mostrou o crescimento de Cronobacter spp. Antibiótico guiado foimantido por 21 dias. O paciente apresentou bom resultado clínico, sem déficits neurológicos. A contaminação microbiológica da fórmula em pó pela Enterobacter sakazakii (Cronobacter spp) pode levar a um risco aumentado de infecções graves neonatais. As crianças podem apresentar sepse, alterações sensoriais e crise convulsiva refratária. Uma imagem cerebral precoce deve ser considerada nestes pacientes. A taxa de mortalidade varia de 40 a 80%, e 74% dos sobreviventes têm um quadro neurológico sequelar. Entre 1997 e 2013, houve relatos de 6 surtos de E. sakazakii no Brasil. De acordo com a Organização Mundial de Saúde (OMS), esta doença está subnotificada e os sistemas de vigilância alimentar são insuficientes. Para resolver esse problema, em alguns países, a notificação é obrigatória e a adoção de medidas sanitárias mais rigorosas é exigida pelas agências reguladoras.

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.

Isolation, molecular and phenotypic characterization of Cronobacter spp. in ready-to-eat salads and foods from Japanese cuisine commercialized in Brazil.

The aim of this study was to detect Cronobacter from 30 samples of ready-to-eat (RTE) salads and 30 foods from Japanese cuisine as commercially available in Brazil. The detection of Cronobacter was as according to the ISO standard 22964:2017. The isolates were phenotypically characterized by Vitek 2.0 and the antibiotic susceptibility profile was determined using the standardized agar disc diffusion method. Molecular characterization was accomplished by real-time PCR targeting dnaG gene, multiplex-PCR targeting cgcA gene, and fusA allele sequencing. Twenty-seven samples (45.0%) contained Cronobacter, 14 (23.3%) samples of foods from Japanese cuisine and 13 (21.7%) samples of RTE salads. Twenty-nine unique Cronobacter isolates were selected from the 27 positive samples and were identified as C. sakazakii (n = 18), C. malonaticus (n = 8), and C. dublinensis (n = 3). A high genetic diversity was observed, with 29 Cronobacter strains being assigned to 11 different fusA alleles, a ratio of 2.6 strains by fusA allele was found. The cgcA multiplex-PCR failed to identify many of the Cronobacter isolates at the species level. Four (13.8%) Cronobacter isolates were resistant to one or more antibiotics tested (n = 12). The presence of Cronobacter in RTE foods could be a potential threat to human health and highlights the need for high levels of hygiene, particularly when preparing food for elderly, immunosuppressed persons or adults with prior underlying pathology. Epidemiological surveillance agencies should be aware of the risk that these RTE foods may represent, for these groups.

Cronobacter spp. in Common Breast Milk Substitutes, Bogotá, Colombia / Agente biológico perjudicial para la salud

En Bogotá Colombia, un gran número de bebés son alimentados con sustitutos de la leche materna elaborados con almidón de maíz y plátano. Encontramos que el 34.3% de las muestras analizadas estaban contaminadas con Cronobacter spp, ; C.sakazakii fue la especie más recuperada. Estos hallazgos subrayan el riesgo de contaminación de los sustitutos de la leche materna

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.

Phenotypic characterization of Cronobacter spp. strains isolated from foods and clinical specimens in Brazil.

Several Cronobacter species are opportunistic pathogens that cause infections in humans. This study evaluated the phenotypic characteristics of 57 Cronobacter strains (C. sakazakii n=41, C. malonaticus n=10, C. dublinensis n=4, and C. muytjensii n=2) isolated from food (n=54) and clinical specimens (n=3) in Brazil. These strains included sequence types (ST): ST395-ST398, ST402, ST413 and ST433-ST439, isolated from food samples, and three C. malonaticus clinical strains previous isolated from an outbreak which were ST394 (n=1) and ST440 (n=2). Strains were tested for capsule production, biofilm formation, protease activity, hemolytic activity, cell-cell aggregation, and desiccation resistance. Capsule formation was observed with all Cronobacter strains. Forty-four (77.2%) strains showed proteolytic activity on milk agar. All strains showed ß-hemolysis against erythrocytes from guinea pig, horse and rabbit. Using erythrocytes from sheep, the majority of strains (53/57; 92.9%) showed α-hemolysis and the remaining, ß-hemolysis. All Cronobacter strains produced weak biofilms in microtiters polystyrene plates, which were independent of temperature (4, 25 and 37°C) and/or growth conditions. In glass tubes, formation of either a moderate or strong biofilm was observed in 15/57 (26.3%), 19/57 (33.3%) and 27/57 (47.4%), at 4, 25 and 37°C, respectively. Desiccation treatment decreased Cronobacter viability by 1.55 to >3.87Log10CFU/mL. Cell-cell aggregation was observed in 17 (29.8%) strains. This study showed that the Cronobacter species evaluated showed differing phenotypes, independent of their origin (clinical or not) and ST. Further studies are necessary to elucidate the factors affecting phenotype expression. This may identify novel bacterial targets that could be useful in the development of strategies to control Cronobacter in food chain and to prevent cases of infections.

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.

Investigation on the Factors Affecting Cronobacter sakazakii Contamination Levels in Reconstituted Powdered Infant Formula.

INTRODUCTION: Certain strains of Cronobacter sakazakii can cause serious invasive infections in children, mainly those <2 months old and fed with powdered infant formula (PIF). The infectious dose of C. sakazakii is unknown but evidence suggests that it is approximately 1000 colony forming units (CFU). PIF is currently considered safe if its end-product C. sakazakii level is <1 CFU/g. In this study, we determined the lag time, generation time (GT), and growth rate of five pooled C. sakazakii isolates to evaluate the factors affecting contamination levels in reconstituted PIF. METHODS: 1.71 log CFU/ml of C. sakazakii were inoculated into 100 and 3000 ml of reconstituted PIF and incubated at 22 and 35°C. Growth was evaluated over a 24-h period. ComBase was used for modeling. RESULTS: In 3000 ml, the growth rate was 0.45 ± 0.02 log CFU/h with a lag phase of 3 ± 0.05 h and GT of 0.67 h at 22°C, while the growth rate was 0.73 ± 0.01 log CFU/h with a lag phase of 0.45 ± 0.03 h and GT of 0.41 h at 35° C. CONCLUSION: Cronobacter sakazakii grows rapidly in reconstituted PIF, especially at 35° C.

Pesquisa de Cronobacter spp. em fórmulas infantis desidratadas / Screening the dehydrated powdered infant formulae for Cronobacter spp

Cronobacter spp. é uma bactéria oportunista associada a surtos de infecção em neonatos e criançasem virtude de consumo de fórmulas infantis desidratadas (FID). Neste contexto, o setor reguladortem criado normas específicas para o controle destes agentes patogênicos nas fórmulas infantis. Nesteestudo foi pesquisada a ocorrência de Cronobacter spp. em 60 amostras de FID comercializadas no Riode Janeiro, Brasil. Foram analisadas 30 amostras de fórmulas infantis para lactantes (0-6 meses) e 30 defórmulas infantis de seguimento para lactantes (> 6 meses) seguindo-se a metodologia de cultivo descritano Bacteriological Analytical Manual Online–FDA (2012). A identificação das colônias característicasfoi realizada com uso de kits ID32E, API20E e do sistema Vitek 2.0; e pela reação da polimerase emcadeia (PCR) com alvo no gene gluA. Nenhuma amostra apresentou contaminação por Cronobacterspp. Concluiu-se que a ocorrência de Cronobacter spp. em FID parece ser baixa, o que indica que osprodutores estão cumprindo o disposto nas normas brasileiras vigentes de forma a evitar a contaminaçãodos produtos por este micro-organismo...

Screening the dehydrated powdered infant formulae for Cronobacter spp / Pesquisa de Cronobacter spp. em fórmulas infantis desidratadas

Cronobacter spp. is an opportunistic bacterium that is associated with infection outbreaks in neonates and infants due to the consumption of powdered infant formulas (PIF). In this context, the official regulator sector has created the specific standards for the industries to control these pathogens in the PIF. The present study aimed at evaluating the occurrence of Cronobacter spp. in 60 samples of PIF commercialized in Rio de Janeiro, Brazil. Thirty samples of infant formulas (0-6 months) and 30 samples of follow-up infant formulas (> 6 months) were analyzed according to the conventional methodologies described in the Bacteriological Analytical Manual Online-FDA (2012). The identification of characteristic colonies was performed using kits ID32E, API20E andVitek 2.0 system and polymerase chain reaction (PCR) targeting the gluA gene. No sample showed contamination by Cronobacter spp. The occurrence of Cronobacter spp. in PIF seems to be low, indicating that the producers are complying with the provisions of the Brazilian standards in force to avoid the product contamination by this microorganism.(AU)

Cronobacter spp. é uma bactéria oportunista associada a surtos de infecção em neonatos e crianças em virtude de consumo de fórmulas infantis desidratadas (FID). Neste contexto, o setor regulador tem criado normas específicas para o controle destes agentes patogênicos nas fórmulas infantis. Neste estudo foi pesquisada a ocorrência de Cronobacter spp. em 60 amostras de FID comercializadas no Rio de Janeiro, Brasil. Foram analisadas 30 amostras de fórmulas infantis para lactantes (0-6 meses) e 30 de fórmulas infantis de seguimento para lactantes (> 6 meses) seguindo-se a metodologia de cultivo descrita no Bacteriological Analytical Manual Online–FDA (2012). A identificação das colônias características foi realizada com uso de kits ID32E, API20E e do sistema Vitek 2.0; e pela reação da polimerase em cadeia (PCR) com alvo no gene gluA. Nenhuma amostra apresentou contaminação por Cronobacter spp. Concluiu-se que a ocorrência de Cronobacter spp. em FID parece ser baixa, o que indica que os produtores estão cumprindo o disposto nas normas brasileiras vigentes de forma a evitar a contaminação dos produtos por este micro-organismo.(AU)

Phenotypic profiles and detection of target genes by PCR in isolates from different sources and reference strains, identified as Cronobacter spp. (Enterobacter sakazakii)

Cronobacter, formerly known as Enterobacter sakazakii, is a novel genus of the Enterobacteriaceae family recognized as a cause of high number of fatal cases in neonates, after consuming infant formula. The conventional methods for detecting these organisms are time-consuming and lack sensitivity. The ISO/TS 22964:2006 is the most recently standardized methodology for detecting Cronobacter in powderedinfant formula. This study aimed at confirming the Brazilian isolates previously identified as E. sakazakiias Cronobacter spp. by biochemical assays, and also to compare characteristics of 37 Cronobacter andnon-Cronobacter isolates; and the miniaturized kits and the ISO/TS methodology were evaluated. A conventional PCR protocol targeting dna G was also developed and a previously described gluA targeting protocol was used. The majority of the Brazilian isolates were not confirmed as Cronobacter spp., and the selective enrichment step of ISO/TS methodology was inhibitory to some Cronobacter strains. The ID 32 Ewas the most reliable kit. The PCR protocol targeting gluA showed consistent results with ID 32E and the developed dnaG PCR protocol was 100% sensitive and specific. Thus, the PCR protocols targeting gluA and dnaG might be used to complement the Cronobacter spp. detection or identification after performing the conventional isolation and identification methods. (AU)

Phenotypic profiles and detection of target genes by PCR in isolates from different sources and reference strains, identified as Cronobacter spp. (Enterobacter sakazakii)

Cronobacter, formerly known as Enterobacter sakazakii, is a novel genus of the Enterobacteriaceae family recognized as a cause of high number of fatal cases in neonates, after consuming infant formula. The conventional methods for detecting these organisms are time-consuming and lack sensitivity. The ISO/TS 22964:2006 is the most recently standardized methodology for detecting Cronobacter in powderedinfant formula. This study aimed at confirming the Brazilian isolates previously identified as E. sakazakiias Cronobacter spp. by biochemical assays, and also to compare characteristics of 37 Cronobacter andnon-Cronobacter isolates; and the miniaturized kits and the ISO/TS methodology were evaluated. A conventional PCR protocol targeting dna G was also developed and a previously described gluA targeting protocol was used. The majority of the Brazilian isolates were not confirmed as Cronobacter spp., and the selective enrichment step of ISO/TS methodology was inhibitory to some Cronobacter strains. The ID 32 Ewas the most reliable kit. The PCR protocol targeting gluA showed consistent results with ID 32E and the developed dnaG PCR protocol was 100% sensitive and specific. Thus, the PCR protocols targeting gluA and dnaG might be used to complement the Cronobacter spp. detection or identification after performing the conventional isolation and identification methods.

Primer aislamiento de Cronobacter spp (Enterobacter sakazakii) en fórmula láctea en polvo producida en Chile / First isolation of Cronobacter spp (Enterobacter sakazakii) in powdered baby formula in Chile

La familia Enterobacteriaceae contempla agentes comunes en las enfermedades transmitidas por los alimentos. De esta familia Cronobacter spp, es considerado un patógeno que afecta principalmente a recién nacidos, los que pueden adquirirla a través de fórmulas lácteas infantiles en polvo contaminadas. Objetivo: Pesquisar Cronobacter spp, en una fórmula láctea en polvo producida en Chile. Material y método: En julio de 2008, se obtuvieron 80 muestras desde una planta ubicada en la Región de Los Lagos. Para el aislamiento de Cronobacter spp, se utilizó el método descrito en la norma ISO/TS 22964. Las muestras fueron analizadas en el Laboratorio de Alimentos del Instituto de Medicina Preventiva Veterinaria de la Universidad Austral de Chile. Resultados: Del total de muestras analizadas, el 5 por ciento (4 / 80) fueron confirmadas como Cronobacter spp, constituyendo así el primer reporte de esta bacteria en Chile. Conclusión: Cronobacter spp, está presente en una fórmula láctea en polvo producida en Chile. Su presencia en este producto representa un riesgo que no es considerado en el actual Reglamento Sanitario de los Alimentos de nuestro país.

The family Enterobacteriaceae includes agents that are commonly transmitted through food. Within this family, Cronobacter spp, is considered to be a pathogen that primarily affects newborns, which can acquire it through contaminated powdered formula. Objective: Isolate Cronobacter spp, in a powdered milk formula produced in Chile. Material and method: In July of 2008, 80 samples were obtained from a factory in the Los Lagos Region. To isolate Cronobacter spp, the methods described in the ISO/TS 22964 specifications were used. The samples were analyzed at the Food Laboratory at the Institute of Preventative Veterinary Medicine and the Universidad Austral de Chile. Results: Of all analyzed samples, 5 percent (4/80) were contaminated with Cronobacter spp, which constitutes the first report of this bacterium in Chile. Conclusion: Cronobacter spp is present in powdered baby formula produced in Chile. It’s presence in this product represents a risk that is not considered in the current food safety standards in this country.

Ocorrência de Cronobacter spp. (Enterobacter sakazakii) em alimentos infantis adquiridos em um hospital público / Occurrence of Cronobacter spp. (Enterobacter sakazakii) in infant food purchased in a public hospital

Cronobater spp. (E. sakazakii) é considerada um micro-organismo oportunista que vem ganhando atenção de autoridades de Saúde Pública, pelo crescente número de surtos de infecção em recém–nascidos e lactentes. A bactéria está associada a casos raros, com alta taxa de mortalidade, podendo causar meningites, enterocolite necrosante e septicemia. Cronobacter spp. tem ampla disseminação, porém apenas as fórmulas lácteas infantis em pó foram, epidemiologicamente, associadas às doenças causadas por esse agente. No presente estudo foi avaliada a ocorrência de Cronobacter spp. em alimentos destinados às crianças de 0-36 meses de idade, adquiridos em lactário de um hospital público do município de São Paulo. Vinte e seis amostras de fórmulas reconstituídas e 24 produtos em pó foram analisados segundo a metodologia da ISO. Cronobacter spp. foi detectada em uma amostra (3,8 por cento) reconstituída de alimento infantil à base de farinha de milho e em quatro desse produto em pó (16,7 por cento). A bactéria não foi detectada nas fórmulas infantis destinadas às crianças de 0-6 meses, contudo sua presença em outros alimentos infantis pode contribuir para a contaminação do ambiente e dos utensílios dos lactários por meio da contaminação cruzada.