Are Enterobacteriaceae and Enterococcus Isolated from Powdered Infant Formula a Hazard for Infants? A Genomic Analysis.

Powdered infant formulas (PIF) are the most used dietary substitutes that are used in order to supplement breastfeeding. However, PIF are not sterile and can be contaminated with different microorganisms. The objective of this study was to genomically characterize Enterobacteriaceae (ENT) and Enterococcus strains that were isolated from PIF. Strains were identified by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) and whole-genome sequencing (WGS). Genomic typing, detection of virulence, and resistance profiles and genes were performed with the Ridom SeqSphere+ software; the comprehensive antibiotic resistance database (CARD) platform; ResFinder and PlasmidFinder tools; and by the disk diffusion method. Nineteen isolates from PIF were analyzed, including ENT such as Kosakonia cowanii, Enterobacter hormaechei, Franconibacter helveticus, Mixta calida, and lactic acid bacteria such as Enterococcus faecium. The strains exhibited resistance to beta-lactams, cephalosporins, and macrolides. Resistance genes such as AcrAB-TolC, marA, msbA, knpEF, oqxAB, fosA, blaACT-7, blaACT-14,qacJ, oqxAB,aac(6')-Ii, and msr(C); and virulence genes such as astA, cheB, cheR, ompA ompX, terC, ironA, acm, and efaAfm, adem were also detected. All the analyzed strains possessed genes that produced heat-shock proteins, such as IbpA and ClpL. In PIF, the presence of ENT and Enterococcus that are multiresistant to antibiotics-together with resistance and virulence genes-pose a health risk for infants consuming these food products.

Genomic Characterization of Cronobacter spp. and Salmonella spp. Strains Isolated From Powdered Infant Formula in Chile.

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.

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.

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.

Virulence and Antibiotic Resistance Profiles of Cronobacter sakazakii and Enterobacter spp. Involved in the Diarrheic Hemorrhagic Outbreak in Mexico.

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.

Cronobacter sakazakii and Microbiological Parameters in Dairy Formulas Associated With a Food Alert in Chile.

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.

Verificación intralaboratorio de la norma iso 6579: Método horizontal para la detección de salmonella spp. en fórmula en polvo para lactantes. / Intralaboratory verification of iso 6579 standard: horizontal method for the detection of salmonella spp. in powdered infant formula

Las fórmulas en polvo para lactantes no son productos estériles, pudiendo contener microorganismos patógenos capaces de producir enfermedad severa en lactantes. La introducción de estos contaminantes puede ocurrir no sólo durante la fabricación del producto, sino también en su manipulación o almacenamiento[1]. El Código Alimentario Argentino, en su artículo 1340[2], incisos E. A1 y E. A2, establece los criterios microbiológicos aplicables a productos para lactantes que han de consumirse después de añadir un líquido para la población de 0 a 6 meses y para la población de 6 a 12 meses, en los cuales el método de referencia para la determinación de Salmonella spp. es la norma ISO 6579[3]. Al tratarse de una norma validada es importante disponer de protocolos que verifiquen su adecuada aplicación, garantizando así resultados confiables para el control de inocuidad de estos productos. Dichos protocolos pueden ser útiles para orientar a otros laboratorios en la verificación de la implementación del método en cuestión o para desarrollar un protocolo de verificación de otras normas ya validadas. En consideración de lo anterior, se desarrolló un estudio analítico de tipo experimental que permitió constatar, mediante el cálculo del límite de detección, la sensibilidad y la especificidad, que la metodología aplicada en el Laboratorio de Microbiología del Instituto Nacional de Alimentos cumple con los parámetros de rendimiento de la norma ISO 6579[3] (Método horizontal para la detección de Salmonella spp.) para la matriz fórmula en polvo para lactantes, brindando por lo tanto resultados confiables y comparables.

Powdered infant formula is not a sterile product; it may contain pathogenic microorganisms capable of producing diseases in infants. The introduction of these contaminants can occur not only during the product manufacture, but also in its handling or storage. The Argentine Food Code, in its article 1340 (subsections E.1 and E.2), establishes the microbiological criteria applicable to products for infants that are consumed after adding a liquid, for the population from 0 to 6 months and for the population of 6 to 12 months, in which the reference method for the determination of Salmonella spp. is the Standard ISO 6579. In the case of a validated standard it is important to have protocols to verify its proper application, ensuring reliable results for the control of safety of these products. These protocols may be useful to provide guidance to other laboratories in the verification of the implementation of the method in question or to develop a verification protocol to other standards already validated. In consideration of the above, an analytical study of experimental type was developed to verify, through the calculation of the detection limit, the sensitivity and the specificity, that the methodology applied in the Microbiology Laboratory of the National Institute of Food complies with the performance parameters of the ISO 6579 (horizontal method for the detection of Salmonella spp.) for powdered infant formula, providing reliable and comparable results.

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.

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.