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.

Evaluation of commercial probiotic lactic cultures against biofilm formation by Cronobacter sakazakii

BACKGROUND/AIMS: Cronobacter sakazakii, an emergent pathogen is considered as a major concern to infants and neonates fed on reconstituted powdered infant milk formula. In conjunction with many other factors, biofilm forming capacity adds to its pathogenic potential. In view of the facts that infants are at highest risk to C. sakazakii infections, and emerging antibiotic resistance among pathogens, it is imperative to evaluate probiotic cultures for their efficacy against C. sakazakii. Therefore, pure probiotic strains were isolated from commercial probiotic products and tested for their antimicrobial and anti-biofilm activities against C. sakazakii. METHODS: A total of 6 probiotic strains were tested for their antibiotic susceptibility followed by antimicrobial activity using cell-free supernatant (CFS) against C. sakazakii. The inhibitory activity of CFS against biofilm formation by C. sakazakii was determined using standard crystal violet assay and microscopic observations. RESULTS: All the probiotic strains were sensitive to ampicillin, tetracycline, vancomycin and carbenicillin whereas most of the strains were resistant to erythromycin and novobiocin. Four of the 6 probiotic derived CFS possessed antimicrobial activity against C. sakazakii at a level of 40 μL. A higher biofilm inhibitory activity (>80%) was observed at initial stages of biofilm formation with weaker activity during longer incubation upto 48 hours (50%–60%). CONCLUSIONS: The study indicated the efficacy of isolated commercial probiotics strains as potential inhibitor of biofilm formation by C. sakazakii and could be further explored for novel bioactive molecules to limit the emerging infections of C. sakazakii.

Examine the Correlation between Heat Shock Protein IbpA and Heat Tolerance in Cronobacter sakazakii / 生物医学与环境科学（英文）

We used a proteomic approach to identify IbpA in Cronobacter sakazakii (C. sakazaki), which is related to heat tolerance in this strain. The abundance of IbpA in C. sakazakii strains strongly increased after heat shock. C. sakazakii CMCC 45402 ibpA deletion mutants were successfully constructed. The C. sakazakii CMCC 45402 ΔibpA and wild-type strains could not be distinguished based on colony morphology on LB agar plates or biochemical assays. The growth of the C. sakazakii CMCC 45402 ΔibpA mutant in heat shock conditions was indistinguishable from that of the isogenic wild-type, but showed greater heat resistance than E. coli O157:H7 strain CMCC 44828. This study suggests that the absence of a single ibpA gene has no obvious effect on the phenotype or heat resistance of the strain C. sakazakii CMCC 45402.

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.

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 / 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.

Cronobacter carriage in neonate and adult intestinal tracts / 生物医学与环境科学（英文）

A total of 7 Cronobacter strains were isolated from 703 fecal samples collected in Jinan from June 13 to December 30, 2011, with the positive rate of Cronobacter spp. being 1.0% (95% confidence interval 0.6%-1.4%). Three Cronobacter sakazakii stains were isolated from 157 fecal samples of healthy neonates (95% confidence interval 0.4%-5.5%). This number was slightly higher than that isolated from 273 fecal samples of healthy adults, in which 1 strain of C. sakazakii and 1 strain of Cronobacter malonaticus were isolated, and that from 173 fecal samples of adults with acute diarrhea, in which 1 strain of C. sakazakii and 1 strain of C. malonaticus were isolated, but the differences were not statistically significant (P>0.05). The Cronobacter isolates were all from different genetic sources. It should be noted that Cronobacter carriage may cause infection under certain conditions, especially in neonates.

Work of adhesion of dairy products on stainless steel surface

The adhesion of the solids presents in food can difficult the process of surface cleaning and promotes the bacterial adhesion process and can trigger health problems. In our study, we used UHT whole milk, chocolate based milk and infant formula to evaluate the adhesion of Enterobacter sakazakii on stainless steel coupons, and we determine the work of adhesion by measuring the contact angle as well as measured the interfacial tension of the samples. Inaddition we evaluated the hydrophobicity of stainless steel after pre-conditioning with milk samples mentioned. E. sakazakii was able to adhere to stainless steel in large numbers in the presence of dairy products. The chocolate based milk obtained the lower contact angle with stainless steel surface, higher interfacial tension and consequently higher adhesion work. It was verified a tendency of decreasing the interfacial tension as a function of the increasing of protein content. The pre-conditioning of the stainless steel coupons with milk samples changed the hydrophobic characteristics of the surfaces and became them hydrophilic. Therefore, variations in the composition of the milk products affect parameters important that can influence the procedure of hygiene in surface used in food industry.

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.

Cronobacter sakazakii Infection Induced Fatal Clinical Sequels Including Meningitis in Neonatal ICR Mice / 한국실험동물학회지

Cronobacter sakazakii (C. sakazakii), formerly Enterobacter sakazakii, is an emerging pathogen associated with the ingestion of contaminated reconstituted formula that causes serious illnesses such as bacteremia, septicemia, necrotizing enterocolitis, meningitis and death in low-birth-weight preterm neonatal infants. The objective of this study was to develop an animal model for human neonatal C. sakazakii infections. We acquired timed-pregnant ICR mice and allowed them to give birth naturally. On postnatal day 3.5, each pup was administered orally a total dose of approximately 107 CFU C. sakazakii strain 3439. Mice were observed twice daily for morbidity and mortality. At postnatal day 10.5, the remaining pups were euthanized, and brain, liver, and cecum were excised and analyzed for the presence of C. sakazakii. C. sakazakii was isolated from cecum and other tissues in inoculated mice. In the tissues of C. sakazakii infected mice, meningitis and gliosis were detected in brain. In this study, we confirmed the neonatal ICR mice may be used a very effective animal model for human neonatal C. sakazakii infections.

Occurrence of Enterobacter sakazakii in some meat products sold in Assiut governorate

A total number of 75 samples including 25 samples each of luncheon, minced frozen beef and sausage were collected from Assiut city [Upper Egypt] and examined bacteriologically for the presence of E. sakazakii and other Enterobacteriaceae genera and species by using the FDA enrichment procedure. In addition, a polymerase chain reaction [PCR] method for the rapid detection of E. sakazakii was adapted by using a set of primers designed from a region of the 16S ribosomal RNA gene. E. sakazakii was detected in 7 [28%] of sausage, but none of the luncheon and minced frozen beef samples were positive for E. sakazakii. Moreover, one false positive isolate was observed with FDA method. In contrast, PCR was highly sensitive and specific to E. sakazakii. E. cloacae were isolated from all samples, while E. agglomerans was detected only in luncheon samples. Also, 43 isolates related to family Enterobacteriaceae could be isolated including Citrobacter freundii, Escherichia coli, Hafnia alvei, Klebsiella pneumoniae, K. oxytoca, Kozaenae, K. planticola and Serratia marcescens. The public health importance of E. sakazakii and suggested control measures were discussed

Aislamiento e identificación de Enterobacter sakazakii en lactarios de Bogotá / Isolation and identification of Enterobacter sakazakii in milk feeders of Bogotá, D. C

Introducción. Enterobacter sakazakii es un patógeno oportunista emergente de alto riesgo, responsable de meningitis grave y enterocolitis necrosante. El principal vehículo de transmisión de esta bacteria son los productos infantiles deshidratados, debido a su contaminación después del tratamiento térmico. Objetivo. Identificar cepas de E. sakazakii en muestras de lactarios recolectadas en la ciudad de Bogotá, D.C. Materiales y métodos. Se analizaron 222 muestras de 9 lactarios, de superficies estériles y no estériles, utensilios empleados para la preparación de biberones y operarios. Se realizó recuento de coliformes totales y detección de E. sakazakii utilizando el protocolo propuesto por la Food and Drug Administration y por el Sistema Automático Bax® Dupont Qualicon. Resultados. De las 222 muestras recolectadas en las clínicas de Bogotá, se reportó que 27,4% (61) de las muestras analizadas presentaban coliformes totales; se detectó la presencia de E. sakazakii en 3,6% por el método automatizado de PCR BAX Dupont a partir de muestras de biberones y superficies. Conclusiones. Se demostró la presencia de E. sakazakii en lactarios en Colombia. Debido a que este microorganismo es un patógeno oportunista de alto riesgo para neonatos y que está asociado a las prácticas higiénicas en los lactarios, la información de este estudio puede ser útil para la toma de medidas profilácticas que reduzcan el riesgo de contaminación con este patógeno para la población infantil y, también, aporta información importante para la salud pública.

Introduction: Enterobacter sakazakii is an emergent opportunistic pathogen of high risk responsible of severe meningitis and necrotizing enterocolitis in neonates and infants with underlying medical conditions. One of the principal transmission vehicles for the transmission of these bacteria, is the infant dehydrated formula after exposing them to the heating treatment. Objective: To identify strains of E. sakazakii in milk feeders’ samples from Bogotá. Materials and methods: 222 samples from 9 milk feeders including sterile and non sterile surfaces, utensils used for the formula preparation and food handlers were analyzed. Total coliforms counts and identification of E. sakazakii was done using the FDA protocol and the automatic system Bax ® Dupont Qualicon. Results: From de 222 samples collected from hospitals in Bogotá, it was reported that 27.4% (61) had total coliforms, and the presence of E. sakazakii was detected in 3.6% (8) from one feeding bottle and surfaces. Conclusion: The presence of E. sakazakii strains was reported in Colombian milk feeders. Because this microorganism is a high risk opportunistic pathogen for newborn infants, usually associated with hygiene practices in milk feeders, the information of this research could be useful to develop preventive measurements to reduce the risk of contamination in the infant population and provides important public health information.

Study on the automatic ribotyping for Enterobacter sakazakii / 中华预防医学杂志

<p><b>OBJECTIVE</b>To analyze the ribotyping fingerprint of Enterobacter sakazakii (E. sakazakii) isolated from food and its typing power.</p><p><b>METHODS</b>Two standard strains and twenty-eight isolates of E.sakazakii were analyzed by the DuPont Riboprinter(TM) microbial characterization system. The relevant database was established and the fingerprint patterns were analyzed with BioNumerics software.</p><p><b>RESULTS</b>This system grouped two standard strains and twenty-eight E.sakazakii isolates into 26 ribotypes, and four ribotypes included two strains respectively, the other twenty-two strains showed different ribotypes. The lowest similarity was 31.58%. The number of bands by ribotyping was approximately ten and the molecular weight of these bands ranged from 1 to 50 kb. By the clustering program in BioNumerics, these isolates could be grouped into four clusters.</p><p><b>CONCLUSION</b>The automatic ribotyping method is convenient and fast in E.sakazakii typing.</p>

Primer estudio de Enterobacter sakazakii en alimentos en Cuba / First study on Enterobacter sakazakii in foodstuffs in Cuba

Enterobacter sakazakii es uno de los microorganismos patógenos emergentes que han hecho su aparición en los últimos años, fundamentalmente como contaminante ocasional de las fórmulas infantiles en polvo. En países en vías de desarrollo no existen suficientes investigaciones sobre este microorganismo, en Cuba este constituye el primer estudio realizado. Conocer la presencia de E. sakazakii en muestras de leche en polvo de importación. Se analizaron 60 muestras de leche en polvo, entera y descremada, provenientes de nueve países, se siguió la técnica recomendada por autoridades reguladoras de EE.UU., con la inclusión de pruebas bioquímicas convencionales como alternativa antes de aplicar el sistema de identificación API 20E. Se empleó el agar hierro Kligler y el agar citrato de Simmons; en aquellas cepas con imagen sugestiva de Enterobacter se comprobó la obtención de triptofano a partir de indol, reacción de Voges Proskauer y rojo de metilo, descarboxilación de lisina y ornitina, dihidrólisis de la arginina, producción de ácido de sacarosa, dulcitol, adonitol, rafinosa y sorbitol. Se obtuvo crecimiento de enterobacterias en 26 muestras. Una sola cepa dio resultado presuntivo de E. sakazakii por pruebas bioquímicas, la misma se confirmó por API 20E para 1,6 por ciento de positividad. En general la calidad microbiológica de las muestras de leche estudiadas fue buena. La técnica empleada para determinar E. sakazakii en muestras de leche en polvo, con la inclusión de pruebas bioquímicas convencionales, es factible de realizar en Cuba, para minimizar los costos asociados a la utilización de API 20E. Sería importante continuar estos estudios en áreas hospitalarias, especialmente aquellas donde se preparan fórmulas lácteas para recién nacidos.

Enterobacter sakazakii is an emerging pathogen that has been isolated in milk powder preparations for infant in the last few years. Practically there is no research works about this microorganism in developing countries; in Cuba this is the first study. To detect the presence of E. sakazakii in imported milk powder samples. Sixty samples of whole and skimmed powdered milk imported from 9 countries were analyzed. Before applying API 20E Kit, the recommended technique by US regulatory bodies including conventional biochemical tests was used. Kligler´s iron agar and Simmons´s citrate agar were employed; other tests such as obtaining of triptophane from indole, Voges Proskauer reaction, methyl red, lysine and ornitine decarboxylation, arginine dihydrolysis, production of sucrose acid, dulcit, adonit, raffinose and sorbitol were performed in presumptive Enterobacter strains. Enterobacteria were isolated in 26 samples. Just one strain was classified as E. sakasakii using the traditional biochemical tests and further confirmed by API 20E for 1.6 percent possitivity of the sample. Generally speaking, the microbiological quality of powered milk samples was good. The technique used to determine E. sakazakii in powered milk samples, including conventional biochemical tests, is feasible to be performed in Cuba in order to reduce costs inherent to the use of API 20E. It is important to conduct these studies in hospital settings, particularly in those areas where milk formulae for neonates are prepared.

Surveillance of Stool Samples for the Presence of Enterobacter sakazakii among Korean People

PURPOSE: Enterobacter sakazakii (E. sakazakii) infections are an important cause of life-threatening meningitis, septicemia, and necrotizing enterocolitis in infants. Dried infant formula milk is an important vehicle for E. sakazakii infection. E. sakazakii was isolated in Korea from dried infant formula milk. Although E. sakazakii infection of infants may occur in Korea, its prevalence has not yet been documented. Therefore, we determined the prevalence of E. sakazakii and documented symptoms. MATERIALS AND METHODS: Between March and October 2006, 1,146 stool samples were collected from patients at Uijeongbu St. Mary's Hospital. Each fecal swab was dissolved in 10mL of buffered peptone solution, and enriched culture was streaked onto Druggan-Forsythe-Iversen (DFI) agar. Presumptive E. sakazakii colonies that exhibited a blue-green color during culture on DFI medium were selected. The identity of colonies that developed yellow pigment during culture on TSA was determined using the Vitek system and PCR. RESULTS: We isolated 4 E. sakazakii strains whose 16S rRNA sequence alignments had a similarity of 99% with those of 3 E. sakazakii ATCC strains. CONCLUSION: This is the first report on isolation of E. sakazakii from stool samples and to document the symptoms of Korean patients.

Enterobacter sakazakii in infants: novel phenomenon in India.

E. sakazakii has been implicated in necrotizing enterocolitis, bloodstream and central nervous system infections, with mortality rates of 40-80%. Two cases of E. sakazakii infections; one preterm very low birth weight neonate with meningitis and a two month infant with bacteraemia, are described for the first time in India. The first baby succumbed to the infection while the other responded to appropriate therapy. Powdered infant milk formulae have been implicated in causing neonatal infections and the first baby was on formula feed with classic signs of sepsis and meningitis. The second infant was on breast feed and probably developed nosocomial E. sakazakii bacteraemia.

Mechanism of beta-lactam Resistance in Gram-negative Rods Isolated from Surgical Site Infection / 감염

BACKGROUND: There have been few studies about the kinds of species causing surgical site infections and their resistance pattern in Korea. An increase of extended-spectrum beta-lactamase (ESBL) producing strains is a worldwide problem. However, there is not enough data on the prevalence of ESBL-producing strains in Korea and the true extent of this problem seems to be under-recognized. METHODS: Minimal inhibitory concentrations of gram-negative bacilli isolated from surgical site infections were tested using the standard agar dilution method according to the National Committee for Clinical Laboratory Standards. To identify and characterize beta-lactamases, we performed conjugation test, isoelectric focusing, Southern hybridization, and polymerase chain reaction. RESULTS: A total of 54 strains of gram-negative enteric bacilli were identified:two strains of Acinetobacter spp., one of Citrobacter freundii, nine of Enterobacter cloacae, one of Enterobacter sakazakii, one of Escherichia coli, two of Klebsiella pneumoniae, one of Morganella morganii, one of Proteus vulgaris, 23 of Pseudomonas aeruginosa, four of Xanthomonas maltophila, and nine of Serratia marcescens. Three strains produced ESBL. CONCLUSION: Various species of gram-negative organisms isolated from surgical site infections showed complex antibiograms to various beta-lactams, even to the new generation of antibiotics. A large proportion of these strains showed conjugally transferable, plasmid-mediated, beta-lactam resistance. Some strains were ESBL-producing. This evidence suggests that there has been a molecular evolution of beta-lactamase genes to a great extent in Korea, possibly due to indiscriminate use of antibiotics.