Rosa roxburghii Tratt Pomace Crude Extract Inactivates Cronobacter sakazakii Isolated from Powdered Infant Formula.

Cronobacter sakazakii is an important foodborne pathogen in powder infant formula (PIF). The objective of this study was to evaluate the inactivation effect of Rosa roxburghii Tratt pomace crude extract (RRPCE) on C. sakazakii isolated from PIF and to reveal the mechanism of action. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were used to evaluate the inhibitory activity of RRPCE against C. sakazakii. The inhibitory mechanism was revealed from the perspective of effects of RRPCE on intracellular adenosine 5'-triphosphate (ATP), reactive oxygen species (ROS), membrane potential, protein and nucleic acid leakage, and cell morphology of C. sakazakii. The inactivation effects of RRPCE on C. sakazakii in biofilms on stainless steel, tinplate, glass, silica gel, polyethylene terephthalate, and polystyrene to evaluate its potential as a natural disinfectant. The results showed that the MIC and MBC of RRPCE against C. sakazakii were 7.5 and 15 mg/mL, respectively. After treatments with RRPCE, intracellular ATP content decreased significantly while intracellular ROS level increased significantly (p < 0.05). The cell membrane depolarization, large leakage of proteins and nucleic acids, and severely damaged cell morphology also occurred in C. sakazakii treated with RRPCE. In addition, a 20-minute treatment with 2 MIC (15 mg/mL) of RRPCE could inactivate all C. sakazakii (from 6.10 to 6.40 CFU/mL) in biofilms on all six contact surfaces. Our findings suggest that RRPCE is ideal for the inactivation of C. sakazakii and has the potential to be used as a natural disinfectant for the inactivation of PIF packaging materials and containers.

Depletion of reactive oxygen species induced by beetroot (Beta vulgaris) extract leads to apoptosis-like death in Cronobacter sakazakii.

This research aimed to disclose the antibacterial activity of beetroot extract (Beta vulgaris) against Cronobacter sakazakii and its possible mechanisms. We evaluated its antibacterial activity by measuring the minimum inhibitory concentration (MIC) and time-kill kinetics. We also evaluated the intracellular ATP levels, bacterial apoptosis-like death (ALD), and reactive oxygen species (ROS) levels to reveal the possible antibacterial mechanisms. Our results showed that the MIC of beetroot extract against C. sakazakii was 25 mg/mL and C. sakazakii (approximately 8 log cfu/mL) was completely inhibited after treatment with 2 MIC of beetroot extract for 3 h. Beetroot extract reduced intracellular ATP levels and facilitated characteristics of ALD in C. sakazakii, such as membrane depolarization, increased intracellular Ca2+ levels, phosphatidylserine externalization, caspase-like protein activation, and DNA fragmentation. Additionally, and different from most bacterial ALD caused by the accumulation of ROS, beetroot extract reduced the intracellular ROS levels in C. sakazakii. Our experimental data provide a rationale for further research of bacterial ALD and demonstrate that beetroot extract can inhibit C. sakazakii in food processing environments.

Bacteroides fragilis ameliorates Cronobacter malonaticus lipopolysaccharide-induced pathological injury through modulation of the intestinal microbiota.

Cronobacter has attracted considerable attention due to its association with meningitis and necrotizing enterocolitis (NEC) in newborns. Generally, lipopolysaccharide (LPS) facilitates bacterial translocation along with inflammatory responses as an endotoxin; however, the pathogenicity of Cronobacter LPS and the strategies to alleviate the toxicity were largely unknown. In this study, inflammatory responses were stimulated by intraperitoneal injection of Cronobacter malonaticus LPS into Sprague-Dawley young rats. Simultaneously, Bacteroides fragilis NCTC9343 were continuously fed through gavage for 5 days before or after injection of C. malonaticus LPS to evaluate the intervention effect of B. fragilis. We first checked the morphological changes of the ileum and colon and the intestinal microbiota and then detected the generation of inflammatory factors, including tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1ß), interleukin-6 (IL-6), and interleukin-10 (IL-10) and the expression of Toll-like receptor 4 (TLR4), occludin, claudin-4, and iNOs. The results indicated that C. malonaticus LPS exacerbated intestinal infection by altering gut microbe profile, tight junction protein expression, and releasing inflammatory factors in a time- and dose-dependent manner. Intriguingly, treatment with B. fragilis obviously diminished the pathological injuries and expression of TLR4 caused by C. malonaticus LPS while increasing gut microbes like Prevotella-9. We note that Shigella, Peptoclostridium, and Sutterella might be positively related to C. malonaticus LPS infection, but Prevotella-9 was negatively correlated. The results suggested that the intestinal microbiota is an important target for the prevention and treatment of pathogenic injuries induced by C. malonaticus LPS.

Case report of Curtobacterium isolated from a catheter tip sample misidentified as Cronobacter.

The Curtobacterium genus is a member of the family Microbacteriaceae, and Curtobacterium species are recognized as plant pathogens. The aim of this study was to investigate a dubious result of species identification for an infection located on a catheter tip of a patient with Covid-19. A strain isolated from a catheter tip sample, identified by VITEK® 2 as Cronobacter spp., was submitted to polyphasic analysis: Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) using VITEK® MS, real-time polymerase chain reaction targeting dnaG gene, and 16S rRNA full gene Sanger sequencing analysis for confirmation. The strain presented negative result using qPCR and could not identified by MALDI-TOF MS. 16S rRNA full gene Sanger sequencing analysis identified the strain as Curtobacterium spp. The Gram-variable characteristic (Gram-negative instead of Gram-positive) of the isolated strain was the responsible for the misidentification by VITEK® 2 and VITEK® MS did not identify the strain. 16S rRNA full gene sequencing analysis identified the strain as Curtobacterium genus, but other complementary techniques are necessary to identify at species level.

Contaminación microbiólogica en fórmulas infantiles en polvo en dos hospitales de Honduras / Microbiological contamination in children's powdered formulas in two Honduran hospitals

OBJETIVOS: Determinar la existencia de contaminación por patógenos en fórmulas infantiles en polvo (FIP) procesadas en los dos hospitales públicos más grandes de Honduras y evaluar las condiciones de procesamiento de sus servicios de fórmulas infantiles (SFI). MÉTODOS: Estudio exploratorio realizado en dos etapas: ') Evaluación presencial de las condiciones de procesamiento de las FIP de los dos SFI; 2) Recolección y análisis de las muestras FIP para el aislamiento de Cronobacter spp. y enterobacterias. RESULTADOS: La evaluación de los SFI mostró debilidades en diferentes aspectos como infraestructura, almacenamiento, capacitación y registros. Cincuenta muestras fueron recolectadas en total de cinco marcas originarias de seis países. El 38% se encontraban en uso durante el muestreo y 62% fueron recolectadas de latas selladas. Se comprobó la presencia de Cronobacter spp. en 4% (2/50) del total de muestras, una proveniente de cada hospital. Se elaboró y entregó un manual de procesamiento de FIP a cada hospital participante. CONCLUSIONES: Existe contaminación de Cronobacter spp., Klebsiella y Acinetobacter en los dos hospitales hondureños; resultado similar a los estimados en Chile (5%) y Cuba (',6%). Es necesaria la implementación del manual de procesamiento FIP y el monitoreo de estos y otros microorganismos patógenos.

OBJECTIVES: Determine the existence of pathogen contamination in powdered infant formulas (PIF) processed in the two largest public hospitals in Honduras and evaluate the processing conditions of their infant formula services (IFS). METHODS: Exploratory study executed in two stages: ') faceto-face evaluation of the processing conditions of the PIF of the two IFS; 2) Collection and analysis of the PIF samples for Cronobacter spp. and Enterobacteriaceae isolation. RESULTS: The evaluation of the IFS showed weaknesses in different aspects such as infrastructure, storage, training and keeping records. In total, fifty samples were collected, representing five brands from six countries. Thirty eight percent of samples were collected from cans in use during sampling and 62% were collected from sealed cans. The presence of Cronobacter spp. was detected in 4% (2/50) of the total samples, one from each hospital. A PIF processing manual was prepared and delivered to each participating hospital. CONCLUSIONS: Contamination of Cronobacter spp., Klebsiella and Acinetobacter existed in two evaluated Honduran hospitals; results similar to others in Chile (5%) and Cuba ('.6%). It is necessary to implement the PIF processing manual and monitor these and other pathogenic microorganisms

Antibiotic Susceptibility of Cronobacter spp. Isolated from Clinical Samples.

Cronobacter spp. have been recognized as causative agents of various severe infections in pre-term or full-term infants as well as elderly adults suffering from serious underlying disease or malignancy. A surveillance study was designed to identify antibiotic resistance among clinical Cronobacter spp. strains, which were isolated from patients of two hospitals between May 2007 and August 2013. Altogether, 52 Cronobacter spp. isolates were analyzed. Although MALDI-TOF mass spectrometry recognized all Cronobacter sakazakii and Cronobacter malonaticus strains, it could not identify Cronobacter muytjensii strain. Nevertheless, all strains were identified as Cronobacter spp. using multilocus sequence typing (MLST). Strains were tested against 17 types of antibiotics, using the standard microdilution method according to the 2018 European Committee on Antimicrobial Susceptibility Testing criteria. Three Cronobacter species were identified as C. sakazakii (n = 33), C. malonaticus (n = 18), and C. muytjensii (n = 1); all isolates were susceptible to all tested antibiotics. All strains were PCR-negative for bla TEM, bla SHV, and bla CTX-M ß-lactamase genes, as well. Even though the results of this study showed that Cronobacter spp. isolates were pan-susceptible, continued antibiotic resistance surveillance is warranted.Cronobacter spp. have been recognized as causative agents of various severe infections in pre-term or full-term infants as well as elderly adults suffering from serious underlying disease or malignancy. A surveillance study was designed to identify antibiotic resistance among clinical Cronobacter spp. strains, which were isolated from patients of two hospitals between May 2007 and August 2013. Altogether, 52 Cronobacter spp. isolates were analyzed. Although MALDI-TOF mass spectrometry recognized all Cronobacter sakazakii and Cronobacter malonaticus strains, it could not identify Cronobacter muytjensii strain. Nevertheless, all strains were identified as Cronobacter spp. using multilocus sequence typing (MLST). Strains were tested against 17 types of antibiotics, using the standard microdilution method according to the 2018 European Committee on Antimicrobial Susceptibility Testing criteria. Three Cronobacter species were identified as C. sakazakii (n = 33), C. malonaticus (n = 18), and C. muytjensii (n = 1); all isolates were susceptible to all tested antibiotics. All strains were PCR-negative for bla TEM, bla SHV, and bla CTX-M ß-lactamase genes, as well. Even though the results of this study showed that Cronobacter spp. isolates were pan-susceptible, continued antibiotic resistance surveillance is warranted.

Characterization of Biofilm Formation by Cronobacter spp. Isolates of Different Food Origin under Model Conditions.

Cronobacter spp. are opportunistic human pathogens that cause serious diseases in neonates and immunocompromised people. Owing to their biofilm formation on various surfaces, both their detection and their removal from production plants constitute a major challenge. In this study, food samples were randomly collected in Austria and examined for the presence of Cronobacter spp. Presumptive isolates were identified by a polyphasic approach. Five percent of the samples were positive for C. sakazakii and 2.4% for C. dublinensis. Individual growth of the isolates was characterized based on lag time, growth rate, and generation time. During an incubation period of 6 to 72 h, biofilm formation of 11 selected isolates was quantified under model conditions by a crystal violet staining assay with 96-well plates with different carbon sources (lactose, glucose, maltose, sucrose, and sodium acetate) and NaCl levels and under variable temperature and pH conditions. Biofilm formation was more pronounced at lactose concentrations between 0.25 and 3% compared with 5% lactose, which lead to thinner layers. C. sakazakii isolate C7, isolated from infant milk powder, was the strongest biofilm producer at 10 mM Mg2+ and 5 mM Mn2+, 0.5% sodium acetate, at pH levels between 7 and 9 at 37°C for 24 h. C. sakazakii strain C6 isolated from a plant air filter was identified as a moderate biofilm former and C. sakazakii strain DSM 4485, a clinical isolate, as a weak biofilm former. Based on PCR detection, genes bcsA, bcsB, and bcsG encoding for cellulose could be identified as markers for biofilm formation. Isolates carrying bcsA and bcsB showed significantly stronger biofilm formation than isolates without these genes ( P < 0.05), in strong correlation with the results obtained in the crystal violet assay. Further investigations using confocal laser scanning microscopy revealed that extracellular polymeric substances and glycocalyx secretions were the dominating components of the biofilms and that the viable fraction of bacteria in the biofilm decreased over time.

Characterization of the pathogenicity of clinical Cronobacter malonaticus strains based on the tissue culture investigations.

Cronobacter malonaticus is a member of the genus Cronobacter which is considered an opportunistic pathogen. The significance of C. malonaticus has recently increased since it was documented to be involved in several serious neonatal infections. However, the virulence factors of C. malonaticus including their ability to adhere, invade and overcome host barriers have not been studied before. Unlike previous Cronobacter research, this study is mainly focused on C. malonaticus and is aimed to investigate its virulence characteristics that enable this species to cause adult and neonatal infections. Altogether, 20 strains were included in this study (19 clinical and one environmental strain). Our data showed that the clinical C. malonaticus has an ability to adhere and invade Caco-2, HBMEC, A549 and T24 cell lines. Moreover, the result showed that certain strains of C. malonaticus (including 1827 and 2018) were able to persist well in macrophages. However, ST7 strains 1827 and 2018 proved to be the most invasive strains among all used strains. The CDC strain 1569 (ST307) which was isolated from the blood of a fatal neonatal case showed also significant results in this study as it was able to invade all used human cells and survive and replicate within microphages. Finally, the findings of this study confirm the potential ability of C. malonaticus to cause serious infections in neonates or adults such as necrotising enterocolitis, meningitis, bacteraemia, pneumonia and urinary tract infection.

Occurrence of virulence factors in Cronobacter sakazakii and Cronobacter malonaticus originated from clinical samples.

BACKGROUND: Cronobacter spp. are Gram-negative, facultative-anaerobic, non-spore forming, enteric coliform bacteria, which belongs to the Enterobacteriaceae family. Cronobacter spp. are opportunistic pathogens that have brought rare but life-threatening infections such as meningitis, necrotizing enterocolitis and bloodstream infections in neonates and infants. Information on the diversity, pathogenicity and virulence of Cronobacter species obtained from various sources is still relatively scarce and fragmentary. The aim of this study was to examine and analyse different pathogenicity and virulence factors among C. sakazakii and C. malonaticus strains isolated from clinical samples. METHODS: The thirty-six clinical Cronobacter strains have been used in this study. This bacterial collection consists of 25 strains of C. sakazakii and 11 strains of C. malonaticus, isolated from different clinical materials. Seven genes (ompA, inv, sip, aut, hly, fliC, cpa) were amplified by PCR. Moreover, the motility and the ability of these strains to adhere and invade human colorectal adenocarcinoma (HT-29) and mouse neuroblastoma (N1E-115) cell lines were investigated. RESULTS: Our results showed that all tested strains were able to adhere to both used cell lines, HT-29 and N1E-115 cells. The invasion assay showed that 66.7% (24/36) of isolates were able to invade N1-E115 cells while 83% (30/36) of isolates were able to invade HT-29 cells. On the average, 68% of the C. sakazakii strains exhibited seven virulence factors and only 18% in C. malonaticus. All strains amplified ompA and fliC genes. The other genes were detected as follow: sip 97% (35/36), hlyA 92% (33/36), aut 94% (34/36), cpa 67% (24/36), and inv 69% (25/36). CONCLUSIONS: C. sakazakii and C malonaticus strains demonstrate the diversity of the virulence factors present among these pathogens. It is necessary to permanently monitor the hospital environment to appropriately treat and resolve cases associated with disease. Furthermore, in-depth knowledge is needed about the source and transmission vehicles of pathogens in hospitals to adopt pertinent prevention measures.

[Study on identification of Cronobacter spp. species in 11 areas in China].

OBJECTIVE: To compare different methods on the identification of Cronobacter (C.) spp. species and to choose an optimum one. METHODS: Biochemical test, 16S rRNA and fusA sequencing methods were carried out. RESULTS: When using the biochemical test, 105 strains showed six different conditions but C. turicensis and C. universalis could not be effectively identified. Under the 16S rRNA sequencing analysis, all the strains were divided into 5 groups but C. sakazakii and C. malonaticus were tangled. Finally, all the strains were identified into 58 C. sakazakii, 30 C. malonaticus, 11 C. dublinensis, 5 C. turicensis, 1 C. muytjensii, under the fusA sequencing analysis. CONCLUSION: Currently, fusA sequencing analysis seemed an effective method for identifying the species of Cronobacter. Since fusA sequencing analysis method was less intuitive, another method for rapid testing should be developed.

Identificação de Cronobacter spp. em queijos e perfil de suscetibilidade antimicrobiana / Identification of Cronobacter spp. in cheeses and the antimicrobial susceptibility profile

Cronobacter spp. é um patógeno oportunista que pode causar infecções em indivíduos de qualquer idade, cuja incidência é maior em neonatos, pacientes imunocomprometidos e idosos. Neste estudo Cronobacter spp. foi pesquisado em 90 amostras de queijos (30 do tipo Minas Frescal, 30 do tipo Prato e 30 do tipo Prato fatiado). As espécies isoladas foram identificadas, e foi avaliado o perfil de suscetibilidade a antimicrobianos. A pesquisa foi realizada utilizando-se pré-enriquecimento em água peptonada tamponada, enriquecimento seletivo no caldo lauril sulfato triptose contendo vancomicina, isolamento no meio Enterobacter sakazakii Isolation Agar e identificação no Vitek 2.0. As espécies foram identificadas por meio de múltipla PCR com alvo no gene cgcA. O antibiograma foi realizado pela técnica de difusão em ágar (Kirby-Bauer). Cronobacter spp. foi isolada em uma (1,1 %) amostra de queijo tipo Minas Frescal, identificada como C. sakazakii que apresentou sensibilidade a todos os antimicrobianos testados. Cronobacter spp. pode nمo representar risco à saْde dos indivíduos pelo consumo de queijos produzidos com leite pasteurizado. Entretanto, a presença de Cronobacter spp. em uma amostra de queijo demonstra falhas na produção, o que reforça a necessidade de maior adesão às Boas Práticas de Fabricação.

Cronobacter spp. is an opportunistic pathogen that may cause infections in individuals ofany age, and the highest incidence occurs in neonates, immunocompromised patients andelderly persons. This study investigated Cronobacter spp. occurrence in 90 cheese samples(30 “Minas Frescal” type cheeses, 30 “Prato” type and 30 sliced “Prato” type). The isolatedspecies were identified and the antimicrobial susceptibility profile of the isolated strains wasevaluated. The microbiological assay was performed with pre-enrichment in buffered peptonewater, and selective-enrichment in modified lauryl sulphate tryptose broth containingvancomycin. Isolation and identification were done in Enterobacter sakazakii Isolation Agar andin Vitek 2.0, respectively. The species identification was performed by multiple PCR targetingcgaA gene. Antibiogram was done using agar diffusion method (Kirby-Bauer). Cronobacter spp.was isolated from one (1.1 %) sample of “Minas Frescal” type cheese, identified as C. sakazakii which was sensitive to all of tested antimicrobials. Cronobacter spp. does not represent arisk to the individuals health by consuming cheeses made from pasteurized milk. However,the presence of Cronobacter spp. in one sample of analyzed cheese indicates failures in their production, reinforcing the need for following the Good Manufacturing Practices.

Pesquisa, identificação e perfil de suscetibilidade antimicrobiana de Cronobacter spp. em produtos destinados à alimentação infantil / Detection, identification and antimicrobial susceptibility profile of Cronobacter spp. in products for infant feeding

Cronobacter spp. emergiu como perigo microbiológico em fórmulas infantis desidratadas (FID), responsável por infecções graves em neonatos. Contudo, muitos pacientes não ingeriram FID, o que indica que outros alimentos podem atuar como veículo do patógeno. Os objetivos deste estudo foram pesquisar Cronobacter spp. em alimentos infantis, identificar as espécies e avaliar o perfil de suscetibilidade aos antimicrobianos das cepas isoladas. Foram analisadas 47 amostras pré-cozidas de cereais à base de grãos, amidos de milho e farinhas lácteas. A pesquisa foi realizada com pré-enriquecimento em água peptonada tamponada, enriquecimento seletivo no Cronobacter Screening Broth, isolamento por meio de Enterobacter sakazakii Isolation Agar e identificação no Vitek 2.0. A identificação das espécies foi realizada por reação em cadeia pela polimerase com alvo nos genes rpoB e cgcA. O antibiograma foi realizado pelo método de difusão em ágar (Kirby-Bauer). Cronobacter spp. foi identificada em 11 amostras (23,4 %). Oito cepas foram identificadas como C. sakazakii (72,7 %), duas como C. malonaticus (18,2 %) e uma como C. dublinensis (9,1 %). Apenas uma cepa de C. malonaticus apresentou resistência intermediária a ciprofloxacina. Os produtos destinados à alimentação infantil avaliados podem apresentar risco, no caso destes alimentos serem ingeridos por pacientes pertencentes ao grupo de risco, como neonatos e idosos.

Cronobacter spp. emerged as a microbiological hazard in powdered infant formulas (PIF)causing severe infections in newborns. However, among these patients many of them had not ingested PIF indicating that other foods categories might be as the pathogen vehicle. This study aimed at investigating Cronobacter spp. in infant foods, identifying the species and evaluatingthe antimicrobial susceptibility profile of the isolated strains. Forty-seven samples of precooked grain-based cereals, corn starch and milk flours were analyzed. The microbiological analysis was performed with pre-enrichment in buffered peptone water, followed by selective-enrichment in Cronobacter Screening Broth, isolation in Enterobacter sakazakii Isolation Agar and identificationin Vitek 2.0. The identification of species was performed by polymerase chain reaction targetingrpoB and cgaA genes. The antibiogram was carried out using the agar diffusion method(Kirby-Bauer). Cronobacter spp. was identified in 11 samples (23.4 %). Eight strains wereidentified as C. sakazakii (72.7 %), two as C. malonaticus (18.2 %) and one as C. dublinensis (9.1 %). Only one C. malonaticus strain showed an intermediate resistance to ciprofloxacin. The evaluated samples produced for infant feeding might cause hazard when ingested by patients belonging tothe risk group as newborns and elderly.

Pesquisa, identificação e perfil de suscetibilidade antimicrobiana de Cronobacter spp. em produtos destinados à alimentação infantil / Detection, identification and antimicrobial susceptibility profile of Cronobacter spp. in products for infant feeding

Cronobacter spp. emergiu como perigo microbiológico em fórmulas infantis desidratadas (FID), responsável por infecções graves em neonatos. Contudo, muitos pacientes não ingeriram FID, o que indica que outros alimentos podem atuar como veículo do patógeno. Os objetivos deste estudo foram pesquisar Cronobacter spp. em alimentos infantis, identificar as espécies e avaliar o perfil de suscetibilidade aos antimicrobianos das cepas isoladas. Foram analisadas 47 amostras pré-cozidas de cereais à base de grãos, amidos de milho e farinhas lácteas. A pesquisa foi realizada com pré-enriquecimento em água peptonada tamponada, enriquecimento seletivo no Cronobacter Screening Broth, isolamento por meio de Enterobacter sakazakii Isolation Agar e identificação no Vitek 2.0. A identificação das espécies foi realizada por reação em cadeia pela polimerase com alvo nos genes rpoB e cgcA. O antibiograma foi realizado pelo método de difusão em ágar (Kirby-Bauer). Cronobacter spp. foi identificada em 11 amostras (23,4 %). Oito cepas foram identificadas como C. sakazakii (72,7 %), duas como C. malonaticus (18,2 %) e uma como C. dublinensis (9,1 %). Apenas uma cepa de C. malonaticus apresentou resistência intermediária a ciprofloxacina. Os produtos destinados à alimentação infantil avaliados podem apresentar risco, no caso destes alimentos serem ingeridos por pacientes pertencentes ao grupo de risco, como neonatos e idosos.

Cronobacter spp. emerged as a microbiological hazard in powdered infant formulas (PIF) causing severe infections in newborns. However, among these patients many of them had not ingested PIF indicating that other foods categories might be as the pathogen vehicle. This study aimed at investigating Cronobacter spp. in infant foods, identifying the species and evaluating the antimicrobial susceptibility profile of the isolated strains. Forty-seven samples of precooked grain-based cereals, corn starch and milk flours were analyzed. The microbiological analysis was performed with pre-enrichment in buffered peptone water, followed by selective-enrichment in Cronobacter Screening Broth, isolation in Enterobacter sakazakii Isolation Agar and identification in Vitek 2.0. The identification of species was performed by polymerase chain reaction targeting rpoB and cgaA genes. The antibiogram was carried out using the agar diffusion method (Kirby-Bauer). Cronobacter spp. was identified in 11 samples (23.4 %). Eight strains were identified as C. sakazakii (72.7 %), two as C. malonaticus (18.2 %) and one as C. dublinensis (9.1 %). Only one C. malonaticus strain showed an intermediate resistance to ciprofloxacin. The evaluated samples produced for infant feeding might cause hazard when ingested by patients belonging to the risk group as newborns and elderly.

Identificação de Cronobacter spp. em queijos e perfil de suscetibilidade antimicrobiana / Identification of Cronobacter spp. in cheeses and the antimicrobial susceptibility profile

Cronobacter spp. é um patógeno oportunista que pode causar infecções em indivíduos de qualquer idade, cuja incidência é maior em neonatos, pacientes imunocomprometidos e idosos. Neste estudo Cronobacter spp. foi pesquisado em 90 amostras de queijos (30 do tipo Minas Frescal, 30 do tipo Prato e 30 do tipo Prato fatiado). As espécies isoladas foram identificadas, e foi avaliado o perfil de suscetibilidade a antimicrobianos. A pesquisa foi realizada utilizando-se pré-enriquecimento em água peptonada tamponada, enriquecimento seletivo no caldo lauril sulfato triptose contendo vancomicina, isolamento no meio Enterobacter sakazakii Isolation Agar e identificação no Vitek 2.0. As espécies foram identificadas por meio de múltipla PCR com alvo no gene cgcA. O antibiograma foi realizado pela técnica de difusão em ágar (KirbyBauer). Cronobacter spp. foi isolada em uma (1,1 %) amostra de queijo tipo Minas Frescal, identificada como C. sakazakii que apresentou sensibilidade a todos os antimicrobianos testados. Cronobacter spp. pode não representar risco à saúde dos indivíduos pelo consumo de queijos produzidos com leite pasteurizado. Entretanto, a presença de Cronobacter spp. em uma amostra de queijo demonstra falhas na produção, o que reforça a necessidade de maior adesão às Boas Práticas de Fabricação.

Cronobacter spp. is an opportunistic pathogen that may cause infections in individuals of any age, and the highest incidence occurs in neonates, immunocompromised patients and elderly persons. This study investigated Cronobacter spp. occurrence in 90 cheese samples (30 “Minas Frescal” type cheeses, 30 “Prato” type and 30 sliced “Prato” type). The isolated species were identified and the antimicrobial susceptibility profile of the isolated strains was evaluated. The microbiological assay was performed with pre-enrichment in buffered peptone water, and selective-enrichment in modified lauryl sulphate tryptose broth containing vancomycin. Isolation and identification were done in Enterobacter sakazakii Isolation Agar and in Vitek 2.0, respectively. The species identification was performed by multiple PCR targeting cgaA gene. Antibiogram was done using agar diffusion method (Kirby-Bauer). Cronobacter spp. was isolated from one (1.1 %) sample of “Minas Frescal” type cheese, identified as C. sakazakii which was sensitive to all of tested antimicrobials. Cronobacter spp. does not represent a risk to the individuals health by consuming cheeses made from pasteurized milk. However, the presence of Cronobacter spp. in one sample of analyzed cheese indicates failures in their production, reinforcing the need for following the Good Manufacturing Practices.

The speciation and genotyping of Cronobacter isolates from hospitalised patients.

The World Health Organization (WHO) has recognised all Cronobacter species as human pathogens. Among premature neonates and immunocompromised infants, these infections can be life-threatening, with clinical presentations of septicaemia, meningitis and necrotising enterocolitis. The neurological sequelae can be permanent and the mortality rate as high as 40-80%. Despite the highlighted issues of neonatal infections, the majority of Cronobacter infections are in the elderly population suffering from serious underlying disease or malignancy and include wound and urinary tract infections, osteomyelitis, bacteraemia and septicaemia. However, no age profiling studies have speciated or genotyped the Cronobacter isolates. A clinical collection of 51 Cronobacter strains from two hospitals were speciated and genotyped using 7-loci multilocus sequence typing (MLST), rpoB gene sequence analysis, O-antigen typing and pulsed-field gel electrophoresis (PFGE). The isolates were predominated by C. sakazakii sequence type 4 (63%, 32/51) and C. malonaticus sequence type 7 (33%, 17/51). These had been isolated from throat and sputum samples of all age groups, as well as recal and faecal swabs. There was no apparent relatedness between the age of the patient and the Cronobacter species isolated. Despite the high clonality of Cronobacter, PFGE profiles differentiated strains across the sequence types into 15 pulsotypes. There was almost complete agreement between O-antigen typing and rpoB gene sequence analysis and MLST profiling. This study shows the value of applying MLST to bacterial population studies with strains from two patient cohorts, combined with PFGE for further discrimination of strains.

Structural mechanisms of DNA binding and unwinding in bacterial RecQ helicases.

RecQ helicases unwind remarkably diverse DNA structures as key components of many cellular processes. How RecQ enzymes accommodate different substrates in a unified mechanism that couples ATP hydrolysis to DNA unwinding is unknown. Here, the X-ray crystal structure of the Cronobacter sakazakii RecQ catalytic core domain bound to duplex DNA with a 3' single-stranded extension identifies two DNA-dependent conformational rearrangements: a winged-helix domain pivots ∼90° to close onto duplex DNA, and a conserved aromatic-rich loop is remodeled to bind ssDNA. These changes coincide with a restructuring of the RecQ ATPase active site that positions catalytic residues for ATP hydrolysis. Complex formation also induces a tight bend in the DNA and melts a portion of the duplex. This bending, coupled with translocation, could provide RecQ with a mechanism for unwinding duplex and other DNA structures.

Influence of FkpA variants on survival and replication of Cronobacter spp. in human macrophages.

Members of the genus Cronobacter are responsible for cases of meningitis and bacteremia with high fatality rates in neonates. Macrophage uptake of invading microbes is an innate process, and it has been proposed that macrophage infectivity potentiator (Mip) like proteins enhance the ability of pathogens to survive within macrophages. Cronobacter harbor the mip-like gene fkpA, but its role in intracellular survival of these bacteria in human macrophages has not yet been studied. Application of gentamicin exclusion assays and human THP-1 macrophage cells revealed significant differences in the capablility of Cronobacter species to survive and replicate within macrophages. Analysis to the amino acid level revealed both length and sequence variations in FkpA proteins among species. In this study, we addressed the possible influence of FkpA variants in intracellular survival of Cronobacter spp. in human macrophages, by knocking out the fkpA genes in two different Cronobacter strains and subsequent complementation with variants of the fkpA genes. Our results provide strong evidence that, in Cronobacter spp., FkpA must be considered a virulence factor, but its influence on macrophage survival and replication varies among strains and/or species due to the presence of amino acid variations.

Incidence of Cronobacter spp. infections, United States, 2003-2009.

During 2003-2009, we identified 544 cases of Cronobacter spp. infection from 6 US states. The highest percentage of invasive infections occurred among children <5 years of age; urine isolates predominated among adults. Rates of invasive infections among infants approximate earlier estimates. Overall incidence of 0.66 cases/100,000 population was higher than anticipated.

Re-examination of the taxonomic status of Enterobacter helveticus, Enterobacter pulveris and Enterobacter turicensis as members of the genus Cronobacter and their reclassification in the genera Franconibacter gen. nov. and Siccibacter gen. nov. as Franconibacter helveticus comb. nov., Franconibacter pulveris comb. nov. and Siccibacter turicensis comb. nov., respectively.

Recently, a taxonomical re-evaluation of the genus Enterobacter, based on multi-locus sequence typing (MLST) analysis, has led to the proposal that the species Enterobacter pulveris, Enterobacter helveticus and Enterobacter turicensis should be reclassified as novel species of the genus Cronobacter. In the present work, new genome-scale analyses, including average nucleotide identity, genome-scale phylogeny and k-mer analysis, coupled with previously reported DNA-DNA hybridization values and biochemical characterization strongly indicate that these three species of the genus Enterobacter are not members of the genus Cronobacter, nor do they belong to the re-evaluated genus Enterobacter. Furthermore, data from this polyphasic study indicated that all three species constitute two new genera. We propose reclassifying Enterobacter pulveris and Enterobacter helveticus in the genus Franconibacter gen. nov. as Franconibacter pulveris comb. nov. (type strain 601/05(T) = LMG 24057(T) = DSM 19144(T)) and Franconibacter helveticus comb. nov. (type strain 513/05(T) = LMG 23732(T) = DSM 18396(T)), respectively, and Enterobacter turicensis in the genus Siccibacter gen. nov. as Siccibacter turicensis comb. nov. (type strain 508/05(T) = LMG 23730(T) = DSM 18397(T)).