Systematic risk ranking of microbiological hazards in infant foods.

Ensuring food safety, particularly for vulnerable groups, like infants and young children, requires identifying and prioritizing potential hazards in food chains. We previously developed a web-based decision support system (DSS) to identify specific microbiological hazards (MHs) in infant and toddler foods through a structured five-step process. This study takes the framework further by introducing systematic risk ranking (RR) steps to rank MH risks with seven criteria: process survival, recontamination, growth opportunity, meal preparation, hazard-food association evidence, food consumption habits of infants and toddlers in the EU, and MH severity. Each criterion is given a semi-quantitative or quantitative score or risk value, contributing to the final MH risk calculation via three aggregation methods: semi-quantitative risk scoring, semi-quantitative risk value, and outranking multi-criteria decision analysis (MCDA). To validate the criteria and ranking approaches, we conducted a case study to rank MH risks in infant formula, compared the results of the three risk ranking methods, and additionally evaluated the ranking results against expert opinions to ensure their accuracy. The results showed strong agreement among the three methods, consistently ranking Salmonella non-Typhi and Cronobacter spp. and Shiga-toxin-producing Escherichia coli as the top MH risks in infant formulae, with minor deviations. When MHs were ranked after an initial hazard identification step, all three methods produced nearly identical MH rankings, reinforcing the reliability of the ranking steps and the selected criteria. Notably, the risk value and MCDA methods provided more informative MH rankings compared to the risk scoring method. The risk value and risk scoring methods were implemented into an online tool, called the MIcrobiological hazards risk RAnking decision support system (Mira-DSS), available at https://foodmicrobiologywur.shinyapps.io/MIcrobial_hazards_RAnking/. In conclusion, our framework enables the ranking of MH risks, facilitating intervention comparisons and resource allocations to mitigate MH risks in infant foods, with potential applicability to broader food categories.

Assessment of the Sensitivity of Foodborne Cronobacter spp. Strains and Other Enterobacteria to Temperature Stresses and Chlorine-Containing Biocides.

We examined the effects of elevated temperatures and biocides on survivability of food isolates of Cronobacter spp. (C. sakazakii) and concomitant enterobacteriaceae obtained in microbiological control of infant nutrition products. Increased resistance of certain strains of Cronobacter, Enterobacter cloacae, and Pantoea spp. to thermal processing was revealed. Salmonella, Pantoea, and Cronobacter bacteria were least sensitive to antimicrobial action of chlorine-containing agents. The above properties varied in the strains of the same species. Specifically, only two of three examined isolates of Cronobacter spp. demonstrated lower sensitivity to heat in comparison with the enterobacterial test-cultures of other species.

Occurrence, genetic characterization, and antibiotic susceptibility of Cronobacter spp. isolated from low water activity functional foods in Brazil.

Cronobacter spp. are bacterial pathogens isolated from a wide variety of foods. This study aims at evaluating the occurrence of Cronobacter spp. in low water activity functional food samples, detect the presence of virulence genes, and determine the antibiotic susceptibility of strains. From 105 samples, 38 (36.2%) were contaminated with Cronobacter spp. The species identified by polymerase chain reaction (PCR) and sequencing analyses (rpoB and fusA genes, respectively) were C. sakazakii (60.3%), C. dublinensis (25.4%), C. turincensis (9.5%), and C. malonaticus (4.8%). Nineteen fusA alleles were identified, including four new alleles. The virulence genes were identified by PCR and all isolates were positive for ompX and sodA genes, 60.3% to cpa gene, and 58.7% to hly gene. Using the disk diffusion method, antibiotic susceptibility to twelve antibiotics was assessed twice, separated by a 19-month period. In the first test, the isolates showed diverse antibiotic susceptibility profiles, with nineteen isolates (30.2%) being multi-drug resistant (resistant to three or more antibiotic classes), in the second, the isolates were susceptible to all antibiotics. Cronobacter spp. in functional foods demonstrates the need for continued investigation of this pathogen in foods, and further research is needed to clarify the loss of resistance of Cronobacter strains.

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

This study aimed to evaluate the Cronobacter spp. strains isolated on the American continent and characterized using multi-locus sequence typing (MLST) available in the PubMLST database and current literature. From 465 Cronobacter spp. strains, the majority (n = 267, 57.4%) was from North America, mainly from USA (n = 234) and 198 (42.6%) were from South America, mainly from Brazil (n = 196). A total of 232 (49.9%) were isolated from foods, 102 (21.9%) from environmental, 87 (18.7%) from clinical, 27 (5.8%) from PIF, one from water (0.2%) and 16 (3.5%) from unknown sources. A total of five species were represented: Cronobacter sakazakii (374, 80.4%), Cronobacter malonaticus (41, 8.8%), Cronobacter dublinensis (29, 6.2%), Cronobacter turicensis (16, 3.5%) and Cronobacter muytjensii (5, 1.1%). The strains with complete MLST profile (n = 345) were assigned to 98 STs, a ratio of 3.5 strain by ST found and the calculated Simpson`s index was 0.93. The strains showed a high diversity and after eBURST analysis, 30 STs (n = 189) formed 12 single and/or double-locus variant clonal complexes (CC). A total of 38 STs (38.7%) were associated with clinical cases of infection, including well established C. sakazakii CC 1, 4, 8 and 83; C. malonaticus ST60, 307, 394 and 440; and C. sakazakii ST 12 and 494.

Advances in our understanding and distribution of the Cronobacter genus in China.

This review considers how research in China has progressed our understanding and subsequent improved control of Cronobacter. This emergent bacterial pathogen is associated with neonatal infections through the ingestion of contaminated prepared feed. The review includes large-scale surveys of various sources of the organism, including infant formula production facilities. The analysis of over 20,000 samples is presented. Over 10,000 being from powdered infant formula and other infant foods as well as environmental sampling of production facilities, the remaining being from food, food ingredients, and human carriage. A major advance in China was adopting DNA-sequence-based methods (that is, multilocus sequence typing, clustered regularly interspaced short palindromic repeats-cas array profiling, and single-nucleotide polymorphism analysis) for the identification and genotyping of the organism. These methods have considerably advanced our understanding of the taxonomy, ecology, and virulence of this organism. In turn, this has improved source tracking of the organism both in infant formula production facilities and epidemiological investigations. Furthermore, whole-genome sequencing has revealed a range of virulence and persistence mechanisms as well as plasmid-borne multidrug resistance traits. China now has reliable and robust methods for accurate microbial source tracking of Cronobacter for use both in the food production environment and epidemiological analysis.

Applicability of Enterobacteriaceae and coliforms tests as indicators for Cronobacter in milk powder factory environments.

The emergence of Cronobacter as an important potential pathogen for newborn children and its occurrence in powdered infant formulae has generated a need to develop new management practices for this food group. This includes reduction of the prevalence of Cronobacter in manufacturing environments which can be a source of Cronobacter. This study was performed to assess the suitability of qualitative and quantitative Enterobacteriaceae and coliforms indicator tests for the presence and prevalence of Cronobacter. Environmental swabs (205) from five milk powder factories were examined. The qualitative indicator tests had good sensitivity but they lacked specificity for reliable routine use. Logistic regression analyses revealed a significant relationship between the quantitative indicator tests and Cronobacter prevalence, where the Enterobacteriaceae count was a slightly stronger predictor for Cronobacter than the coliforms count. The optimum test sensitivity (81%) and specificity (66%) was obtained when the indicator count thresholds were set at ≥1 cfu/cm2. However, since 11% of samples were Cronobacter positive when counts of Enterobacteriaceae and coliforms were less than 1 cfu/cm2, specific testing for Cronobacter is advised in addition to Enterobacteriaceae testing to minimise risk of transfer of Cronobacter from the factory environment into powdered infant formulae products.

Cronobacter spp. isolated from aquatic products in China: Incidence, antibiotic resistance, molecular characteristic and CRISPR diversity.

Cronobacter species (Cronobacter spp.) are important foodborne pathogens that can infect and cause serious life-threatening diseases in infants and immunocompromised elderly. This study aimed to acquire data on Cronobacter spp. contamination of aquatic products in China from 2011 to 2016. In total, 800 aquatic products were tested, and the overall contamination rate for Cronobacter spp. was 3.9% (31/800). The average contamination level of the positive samples was 2.05 MPN/g. Four species and nine serotypes were identified among 33 isolates, of which the C. sakazakii serogroup O1 (n = 9) was the primary serotype. The majority of Cronobacter spp. strains harbored highest resistance against cephalothin (84.8%), followed by tetracycline (6.1%), trimethoprim/sulfameth-oxazole (3.0%) and chloramphenicol (3.0%). Two isolates were resistant to three antibiotics. In total, 26 sequence types and 33 CRISPR types (including 6 new STs and 26 new CTs) were identified, which indicates the extremely high diversity of Cronobacter spp. in aquatic products. Pathogenic C. sakazakii ST4, ST1, and C. malonaticus ST7 were also observed. Overall, this large-scale study revealed the relatively low prevalence and high genetic diversity of Cronobacter spp. in aquatic products in China, and the findings provide valuable information that can guide the establishment of effective measures for the control and precaution of Cronobacter spp. in aquatic products during production processes.

Electricity-free amplification and visual detection of Cronobacter species in powdered infant formula.

Due to the lack of electricity and thermostatic instruments in certain settings for convenient detection of Cronobacter species in powdered infant formula (PIF), a novel investigation was conducted to establish an electricity-free visual detection system for rapid detection of Cronobacter species in PIF. This system included a portable electricity-free heater that could use the exothermic reaction of calcium oxide and water and 3 kinds of phase change materials to supply 3 constant temperatures for immunomagnetic separation, DNA extraction, and loop-mediated isothermal amplification assay. Meanwhile, the amplified reaction combined with hydroxynaphthol blue could achieve rapid visual detection. Primers designed based on the 16S-23S ribosomal RNA internal transcribed spacer were used in loop-mediated isothermal amplification to specifically monitor Cronobacter species, and the detection limit can reach 4.2 × 102 cfu/g in PIF by an electricity-free heater in 2 h 30 min. Moreover, 2 h of pre-enrichment was necessary when the level of the PIF samples with Cronobacter spp. was 100 cfu/g. The stability of the system was evaluated in ambient temperature at 4°C, 25°C, and 37°C. The results suggested that the electricity-free heater can maintain 3 constant temperatures to support different processes. Therefore, this amplification and visual system is applicable for use in many fields for rapid and specific detection of Cronobacter species in PIF.

Labchip-based diagnosis system for on-site application: Sensitive and easy-to-implement detection of single recoverable Cronobacter in infant formula without post-enrichment treatment.

Microfluidic labchips have achieved much advancement in the molecular diagnosis of foodborne pathogens. Whereas difficulties in the flow control during the transportation of liquid fluids can occur and should be overcome. Manipulations of reaction temperature and the complex procedures from sample pre-treatment to analysis in a single chip device are major obstacles for the on-site application. Thus, the efficient temperature control of samples without any flow of reaction fluids in microfluidic channels of plastic chip and the simplest protocol omitting post-enrichment processing steps may overcome these limitations represented by the stability and the complexity, respectively. This study aims to develop a novel type of labchip and thermocycler specialized for the gene amplification in microfluidic channels and to evaluate the detectability by sensing the minimum recoverable level of Cronobacter in powdered infant formula (PIF). We developed a thermocycling device accelerating reactions through dual heating-blocks optimized to control temperatures of samples in microfluidic-channels by direct contact with labchip sequentially and repetitively. The structural design of microfluidic channels was to eliminate interference factors associated with the optical detection of fluorescent signals (without distortion due to air bubbles in the reaction chamber). To improve the applicability, a portable device and simplified operation to allow direct loading of samples in the chip without post-enrichment procedures were also adopted. Detection performance was evaluated by a sensitivity/specificity tests using 50 isolates of Cronobacter. Cross-reactivity tests for non-Cronobacter organisms and gDNA [human, raw materials of PIF (cow, soybean)] showed that there was no interference-factor causing false-positive results. In terms of the applied research conducted by using PIF, the enrichment of samples without broth medium (distilled water) displayed outstanding performance and 12 h of incubation facilitated detecting target at concentration as low as 1 CFU/300 g PIF (as initial contamination level) without post-enrichment treatment. Validation of the operation conditions using 30 commercial PIF products was also consistent. The present study presents a novel approach of microfluidic technology with perspective to not only the performance and the practicability [easy-to-implement protocol, portable materials, cost-effectiveness (the use of a miniaturized plastic chip requires a minimum level of materials)] for on-site diagnosis.

Prevalence and genetic characteristics of Cronobacter spp. from food and human clinical stool samples in Wenzhou, China 2008-2018.

Pathogenic Cronobacter species are responsible for life-threatening illness in neonates. A ten-year comprehensive survey was conducted to examine the population structure and antimicrobial resistant patterns of Cronobacter isolates from food (n = 78) and clinical (n = 12) sources in Wenzhou, China. A total of 90 (4.4%) isolates were recovered from 2051 collected samples. The occurrence of Cronobacter spp. was highest in spices with a rate of 22% (26/119), whereas the lowest contamination rate of 1% was found in powered infant and toddler formula (7/494), special medical infant formula (1/95) and human stool samples (12/1024). Cronobacter strains revealed a high degree of genetic diversity among the isolates tested. Pulsed-field gel electrophoresis (PFGE) distinguished 75 clonal groups, and the biggest cluster consisted of four strains. Multilocus sequence typing (MLST) method displayed 43 sequence types (STs), of which ST1, ST4, ST8, ST64, ST148 and ST201 were most frequently identified. Meanwhile, two new sequence types were discovered and added to the PubMLST international database. Resistance to ceftriaxone, cefotaxiv, amoxicillin, ampicillin, cefoxitin, tetracycline, streptomycin, azithromycin, chloramphenicol, as well as multidrug resistance, was noted. Taken together, this large-scale surveillance study highlights the wide dissemination and diverse molecular features of Cronobacter spp. in Wenzhou China.

Multiplex loop-mediated isothermal amplification-based lateral flow dipstick for simultaneous detection of 3 food-borne pathogens in powdered infant formula.

In this study, we established a rapid, simple, and sensitive method for visual and point-of-care detection of Salmonella spp., Cronobacter spp., and Staphylococcus aureus in powdered infant formula (PIF) based on multiplex loop-mediated isothermal amplification (mLAMP) combined with lateral flow dipstick (LFD). Three different species-specific target genes, siiA of Salmonella spp., internal transcribed space (ITS) of Cronobacter spp., and nuc of Staph. aureus, were applied in the mLAMP with biotin-, digoxin-, and Texas Red-modified forward inner primers and fluorescein isothiocyanate (FITC)-modified backward inner primers. After mLAMP, a large number of modified amplicons were detected with LFD; one end of the amplicon was conjugated to the anti-FITC antibody on gold nanoparticles and the other end to streptavidin (anti-digoxin or anti-Texas Red antibody) on test lines. Visual inspection of the device relies on the presence of a red band formed by accumulation of sandwich composites. The detection limits of this mLAMP-LFD assay for Salmonella spp., Cronobacter spp., and Staph. aureus in PIF without enrichment were 4.2, 2.6, and 3.4 cfu/g, respectively. The whole method can be completed in less than 1 h. Thus, mLAMP-LFD is a rapid and efficient method for simultaneously detecting Salmonella spp., Cronobacter spp., and Staph. aureus in PIF.

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.

Dual-signal amplification strategy: Universal asymmetric tailing-PCR triggered rolling circle amplification assay for fluorescent detection of Cronobacter spp. in milk.

Cronobacter spp. are important opportunistic foodborne pathogens in powdered infant formula that cause many serious diseases in neonates and infants. In this study, a novel assay based on dual signal amplification strategy was developed by coupling asymmetric tailing PCR (AT-PCR) with rolling circle amplification (RCA) for the detection of Cronobacter spp. in milk. The tailing single-stranded DNA was generated through AT-PCR and used to initiate RCA, generating tandem repetitive G-quadruplex sequences. In the presence of the fluorescence dye thioflavin T that could intercalate into the G-quadruplex structures, the fluorescence signal was detected with a microplate reader. The AT-PCR coupled with RCA assay was specific for Cronobacter spp. detection because of the highly specific primers chosen for the AT-PCR. The limits of detection were 4.3 × 101 cfu/mL in pure culture and 4.5 × 102 cfu/mL in spiked milk, respectively. The fixed sequences designed in the hairpin DNA allowed this AT-PCR coupled with RCA assay to serve as a universal platform for the detection of other pathogens by modifying the specificity of the PCR primers.

Prevalence, genetic analysis and CRISPR typing of Cronobacter spp. isolated from meat and meat products in China.

Cronobacter spp. are important foodborne pathogens that infections occur in all age groups, especially cause serious life-threatening diseases in infants. This study aimed to acquire data on Cronobacter spp. contamination of meat and meat products (n = 588) in China during 2011 to 2016, and investigated the use of CRISPR typing technology as an approach for characterizing the genetics of Cronobacter spp. The overall contamination rate for Cronobacter spp. was determined to be 9.18% (54/588). Of the positive samples, 90.74% (49/54) had <10 MPN/g, with duck samples had a relatively high contamination rate (15.69%, 8/51) and highest contamination level (28.90 MPN/g). Four species and nine serotypes were identified among 69 isolates, of which C. sakazakii was the major species (n = 50) and C. sakazakii serogroup O1 and O2 (n = 17) were the primary serotypes. The majority of Cronobacter spp. strains were found to be susceptible to most antibiotics except exhibited high resistance to cephalothin (76.81%, 53/69), and total two multi-drug resistant C. sakazakii strains were isolated from duck. The genetic diversity of Cronobacter spp. was remarkably high, as evidenced by the identification of 40 sequence types (STs) and 60 CRISPR types (CTs). C. sakazakii ST64 (n = 7) was the predominant genotype and was further divided into two sub-lineages based on CRISPR diversity, showing different antibiotic resistance profile. These results demonstrate that CRISPR typing results have a good correspondence with bacterial phenotypes, and it will be a tremendously useful approach for elucidating inter-subtyping during molecular epidemiological investigations while interpreting the divergent evolution of Cronobacter. The presence of Cronobacter spp. in meat and meat product is a potential threat to human public health.

Amphiphilic ligand modified gold nanocarriers to amplify lanthanide loading for ultrasensitive DELFIA detection of Cronobacter.

Conventional dissociation-enhanced lanthanide (Ln3+) fluoroimmunoassays (DELFIAs) using Ln3+ chelate-labeled antibodies as molecular probes exhibit limited sensitivity because of their relatively low Ln3+ labeling ratio per biomolecule. Herein, we applied gold nanoflowers (AuNFs) as amplified nanocarriers to increase the Ln3+ labeling ratio in a single molecular binding event for improving the sensitivity of traditional DELFIA. Two thiolated amphiphilic ligands (thiolated ethylenediaminetetraacetic acid (EDTA) and thiolated acylhydrazine-terminated ligands), consisting of a hydrophobic alkane chain, oligo(ethylene glycol) unit, and functional terminal of the EDTA or acylhydrazine group, were designed for the surface modification of AuNFs. The resultant ligand-coated AuNFs exhibited dual functions of Ln3+ chelation via the EDTA group and oriented attachment of antibodies via the acylhydrazine group. By utilizing 80 nm AuNFs as amplified carriers, we demonstrated that the maximum Eu3+ loading amount reached 1.07 × 104 Eu3+ ions per AuNF, which is approximately two to three orders of magnitude higher than that of traditional molecular probes, thereby amplifying the luminescence signal and enhancing the sensitivity of DELFIA. By combining a magnetic-mediated sandwich-type DELFIA method, the designed amplified AuNF nanoprobes achieved an ultrasensitive luminescence detection of Cronobacter muytjensii with a limit of detection (LOD) of 1.2 × 102 cfu mL-1 in a powdered infant formula. This LOD value was ca. 230-fold lower than that of the traditional colorimetric immunoassay. The designed signal amplification strategy using bifunctional ligand-modified AuNFs as enhanced Ln3+ nanocarriers provided a huge potential for building various ultrasensitive luminescence immunoassays for in vitro biodetection.

Occurrence of total coliforms, Escherichia coli and Cronobacter species in commercially available 20 l bottled drinking water sold in Rio de Janeiro State, Brazil.

Cronobacter infections of infants are commonly regarded as due to the ingestion of contaminated feed. The aim of this study was to determine the occurrence of Cronobacter, total coliforms and Escherichia coli in different brands of natural mineral waters as sold in 20 l returnable bottles in Rio de Janeiro, Brazil. The quantification of total coliforms and E. coli was performed by Most Probable Number. The detection of Cronobacter was as according to the ISO 22964:2017 and Bacteriological Analytical Manual/FDA. Molecular characterization of Cronobacter isolates was performed by real-time PCR and by multi-locus sequence typing. The antibiotic susceptibility profile was determined and biofilm production was evaluated in polystyrene microplates. Total coliforms and E. coli were detected in 13 (39·4%) and 2 (6·1%) of the 33 lots analysed respectively, and were considered unsatisfactory for human consumption according to Brazilian law. One (3·0%) lot showed contamination by C. malonaticus ST440 (Cronobacter MLST Databases accession no. ID 2646). The strain was susceptible to all (n = 13) antibiotics tested and only formed a weak biofilm. Since there is a high consumption of natural mineral waters by elderly and immunosuppressed persons, epidemiological surveillance agencies should be aware of the risk that these waters may represent for these groups. SIGNIFICANCE AND IMPACT OF THE STUDY: Cronobacter malonaticus ST440 was isolated from 20 l bottled drinking natural mineral waters sold in markets in Rio de Janeiro State, Brazil, and can be a potential threat to human health, particularly for neonates. Thirteen lots (39·4%) were unsatisfactory for human consumption due to the presence of total coliforms and/or Escherichia coli.

Identification and Characterization of Cronobacter Strains Isolated from Environmental Samples.

As an emerging food-borne pathogen, Cronobacter species are ubiquitous in the food and environment. In order to know the characteristics of Cronobacter spp. from the environment, we isolated Cronobacter spp. from soil and water, and then studied the molecular typing and antibiotic resistance characteristics of these isolates. In 2016, 141 soil and water samples were collected from farms and Riverside Park in Beijing. Isolates were identified by real-time PCR, 16s rRNA sequencing, and whole-genome sequencing. Molecular subtyping of these isolates was characterized by pulsed-field gel electrophoresis, multilocus sequence typing (MLST), and antibiotic susceptibility tests. Cronobacter species were classified based on fusA sequencing. Twenty-two samples (15.60%) contained Cronobacter spp., and four species were detected, i.e., C. dubliniensis (n = 10), C. sakazakii (n = 6), C. turicensis (n = 4), and C. malonaticus (n = 2). For MLST, 12 types (ST519-ST525, ST533-ST537) were newly identified, indicating high diversity. Most isolates (68.18%) showed resistance to cefazolin. Siccibacter turicensis and Cronobacter both with blue-green colonies on selective media should be respectively identified. Apparently, major Cronobacter species in soil and water samples differed from those in food. Molecular subtyping showed that the environment could not be excluded as a source of Cronobacter infection. The resistance to cefazolin of most isolates indicated natural resistance.

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

Plasmonic ELISA based on DNA-directed gold nanoparticle growth for Cronobacter detection in powdered infant formula samples.

The traditional gold nanoparticle (AuNP) growth-based plasmonic ELISA (pELISA) strictly and directly controlled by reducing reagents can achieve high sensitivity, but it remains fragile toward the surrounding environment. This work developed a sandwich pELISA for Cronobacter detection in powdered infant formula samples by mediating AuNP growth through DNA. In this assay, DNA adsorbed on the surface of gold nanoseeds guided the anisotropic crystal growth with hydroxylamine as a reducing reagent, and the catalase-hydrogen peroxide (Cat-H2O2) system was introduced to bridge the DNA-directed AuNP growth and pELISA, as such DNA can be cleaved into fragments by the hydroxyl radical generated from oxidation of H2O2 through Fenton reagents. Under optimized conditions, the proposed pELISA can qualitatively detect Cronobacter species (Cronobacter muytjensii ATCC 51329) by the naked eye with a cut-off limit of 3 × 105 cfu/mL. This method also revealed a good linear range (3 × 102 to 3 × 107 cfu/mL) for quantitative detection of C. muytjensii ATCC 51329 with a limit of detection of 1.6 × 102 cfu/mL, which is approximately 162.5 times lower than that of horseradish peroxidase-based conventional ELISA (2.6 × 104 cfu/mL). By taking advantage of highly stable DNA-directed AuNP growth, the proposed method shows a good performance in powdered infant formula samples spiked with different concentrations of C. muytjensii ATCC 51329 with average recoveries ranging from 90.79 to 119.09% and coefficient of variation ranging from 4.24 to 9.55%. These values corresponded to an acceptable accuracy and precision for the proposed method. In brief, this work shows potential for screening other analytes in food safety, clinical diagnostics, and environmental monitoring.

Antibiotic resistance, putative virulence factors and curli fimbrination among Cronobacter species.

This study aimed to investigate antibiotic resistance and putative virulence factors among Cronobacter sakazakii isolated from powdered infant formula and other sources. The following 9 cultures (CR1-9) were collected from our culture collection: C. sakazakii and 3 Cronobacter species: C. sakazakii ATCC® 29544™, C. muytjensii ATCC® 51329™, C. turicensis E866 were used in this study. Isolates were subjected to antibiotic susceptibility and the following virulence factors (protease, DNase, haemolysin, gelatinase, motility and biofilm formation) using phenotypic methods. All the bacteria were able to form biofilm on agar at 37 °C and were resistant to ampicillin, erythromycin, fosfomycin and sulphamethoxazole. It was observed from this study that tested strains formed weak and strong biofilm with violet dry and rough (rdar), brown dry and rough (bdar), red mucoid and smooth (rmas) colony morphotypes on Congo red agar. Rdar expresses curli and fimbriae, while bdar expresses curli. Both biofilm colony morphotypes are commonly found in Enterobacteriaceae including Salmonella species. This study also reveals a new colony morphotypes in Cronobacter species. Conclusively, there was correlation between putative virulence factors and antibiotic resistance among the tested bacteria. Further study on virulence and antibiotic resistance genes is hereby encouraged.