Powdered Infant Formula Samples Research Articles

Group selective aptamers: Broad-spectrum recognition of target groups in Cronobacter species and implementation of electrochemical biosensors as receptors

Aptamers are a versatile class of receptors with a high affinity and selectivity for specific targets. Although their ability to recognize individual targets has been extensively studied, some scenarios require the development of receptors capable of identifying all target groups. This study investigated the use of aptamers to achieve the broad-spectrum recognition of groups instead of individual targets. Aptamers were screened for selectively distinct groups of Cronobacter species associated with foodborne diseases. Seven Cronobacter spp. were divided into Group A (C. sakazakii, C. malonaticus, C. turicensis, and C. muytjensii) and Group B (C. dublinensis, C. condimenti, and C. universalis). Aptamers with exclusive selectivity for each group were identified, allowing binding to the species within their designated group while excluding those from the other group. The screened aptamers demonstrated reliable affinity and specificity with dissociation constants ranging from 1.3 to 399.7 nM for Group A and 4.0–24.5 nM for Group B. These aptamers have also been successfully employed as receptors in an electrochemical biosensor platform, enabling the selective detection of each group based on the corresponding aptamer (limit of detection was 7.8 and 3.2 CFU for Group A and Group B, respectively). The electrochemical sensor effectively detected the extent of infection in each group in powdered infant formula samples. This study highlights the successful screening and application of group-selective aptamers as sensing receptors, emphasizing their potential for diverse applications in different fields such as food safety, environmental monitoring, and clinical diagnostics, where the selective biosensing of target groups is crucial.

A Method to Directly Identify Cronobacter sakazakii in Liquid Medium by MALDI-TOF MS.

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry has been widely used as an emerging technology for the rapid identification of microorganisms. Cronobacter sakazakii (C. sakazakii) is a food-borne pathogen of particular importance to the powdered infant formula (PIF) processing environment due to its high lethality in infants. However, the traditional solid spotting detection method of pretreating samples for MALDI-TOF MS leads only to qualitative detection of C. sakazakii. We developed a new, low-cost, robust liquid spotting pretreatment method and used a response surface methodology to optimize its parameters. The applicability, accuracy, and quantitative potential were measured for different types of samples. The optimal parameters of this method were as follows: a volume of 70% formic acid of 25 μL, treatment with ultrasound at 350 W for 3 min, and a volume of acetonitrile added of 75 μL. These conditions led to the highest identification score for C. sakazakii (1926.42 ± 48.497). This method was found to detect bacteria accurately and reproducibly. When 70 strains of C. sakazakii isolates were analyzed with this method, the identification accuracy was 100%. The detection limit of C. sakazakii in environmental and PIF samples was 4.1 × 101 cfu/mL and 2.72 × 103 cfu/mL, respectively.

Visual detection of Cronobacter sakazakii on a microfluidic chip fabricated by a 3D molding method.

Cronobacter sakazakii (C. sakazakii) is a pathogenic bacterium associated with life-threatening neonatal infections that have been linked to contaminated powdered infant formula (PIF). Most C. sakazakii testing is still limited in microbiology laboratories due to the need for sophisticated equipment and professional technicians. Microfluidic chips combined with isothermal amplification analysis are shown to be one of the most attractive microbiological on-site detection platforms. In this study, PDMS microfluidic chips were fabricated by a simple 3D molding method and sealed with "PDMS glue". The chip consisted of an inlet, a microchannel, six reaction wells, and six vent holes. And based on the 16S rRNA and ITS genes of C. sakazakii, we have successfully proposed a multiplex competitive annealing mediated isothermal amplification (mCAMP) assay on the microfluidic chip for the visual detection of C. sakazakii in PIF samples. The primers were fixed in the reaction wells of the chip before detection, which can be preserved for 60 days at 4 °C. The results showed that the established mCAMP assay had high specificity, and the limit of detection was 2.2 × 103 CFU g-1. With enrichment culture, even if the initial inoculation level is 1 CFU g-1, the mCAMP assay can still detect the presence of C. sakazakii in spiked PIF samples. The test results are visible to the naked eye, which is suitable for rapid analysis in resource-limited settings.

Cronobacter spp. in Commercial Powdered Infant Formula Collected From Nine Provinces in China: Prevalence, Genotype, Biofilm Formation, and Antibiotic Susceptibility.

The purpose of this study was to investigate the prevalence of Cronobacter spp. in commercial powdered infant formula (PIF) from nine provinces in China from March 2018 to September 2020, and to reveal the genotype, biofilm-forming ability, and antibiotic susceptibility of these isolates. A total of 27 Cronobacter strains, consisting of 22 Cronobacter sakazakii strains, 3 Cronobacter malonaticus strains, 1 Cronobacter turicensis strain, and 1 Cronobacter dublinensis strain, were isolated from 3,600 commercial PIF samples with a prevalence rate of 0.75%. Compared with the other 8 provinces, PIF from Shaanxi province had a higher prevalence rate (1.25%) of Cronobacter spp. These isolates were divided into 14 sequence types (STs), and 6 Cronobacter serotypes. The main Cronobacter STs were ST4, ST1, and ST64, and the dominant Cronobacter serotype was C. sakazakii serotype O2. Approximately 88.89% of Cronobacter isolates had a strong ability (OD595 > 1) to form biofilms on tinplate, among which the strains with ST4 were more dominant. All isolates were susceptible to ampicillin-sulbactam, ceftriaxone, cefotaxime, sulfadiazine, sulfadoxine, trimethoprim-sulfamethoxazole, gentamicin, tetracycline, ciprofloxacin, and colistin, while 55.56 and 96.30% isolates were resistant to cephalothin and vancomycin, respectively. Taken together, our findings highlighted the contamination status and characterization of Cronobacter spp. in commercial PIF from nine provinces of China, and provided guidance for the effective prevention and control of this pathogen in the production of PIF.

A novel photoreactive DNA-binding dye for detecting viable Klebsiella pneumoniae in powdered infant formula

In addition to Cronobacter spp., Klebsiella pneumoniae is another opportunistic bacterial pathogen present in powdered infant formula (PIF) that can cause pneumonia, septicemia, and other diseases. In this study, a rapid and specific method based on a fluorescence probe was developed for detecting viable K. pneumoniae in PIF samples via the combination of recombinase-aided amplification (RAA) with thiazole orange monoazide (TOMA) dye (the TOMA-RAA assay hereafter). As a novel photosensitive DNA-intercalating dye, TOMA was used to penetrate bacterial cells, including both dead and viable cells, as verified by confocal laser scanning microscopy and fluorescent emission spectrometry. Importantly, the RAA assay exhibited good performance in detecting K. pneumoniae within 40 min at 39°C. Under optimal conditions, the TOMA-RAA assay can detect as low as 2.6 × 103 cfu/mL of K. pneumoniae in pure culture and 2.3 × 104 cfu/g of K. pneumoniae in spiked PIF sample. After 3 h of pre-enrichment, 3 × 100 cfu/g of K. pneumoniae can be detected. Furthermore, the TOMA-RAA assay displayed an excellent anti-interference ability to nontarget bacteria. In short, the proposed method has great potential application for the rapid and accurate detection of viable K. pneumoniae in PIF.

Aerobic spore-forming bacteria in powdered infant formula: Enumeration, identification by MALDI-TOF mass spectrometry (MS), presence of toxin genes and rpoB gene typing

This study aimed to assess the counts and biodiversity characterization of aerobic sporeforming bacteria (ASB) in powdered infant formula (PIF). Fifty-four (n = 54) samples of PIF of three brands were analyzed for mesophilic aerobic bacteria, and ASB counts. ASB isolated from PIF were assessed for their ability to produce spoilage enzymes and hemolytic activity and further identified by MALDI-TOF mass spectrometry. Then, the isolates were subjected to rpoB gene typing and assessment of bceT, entFM, nhe (A, B, C), and hbl (A, B, C) toxin genes. The main species isolated were B. licheniformis (54%), followed by B. cereus (33%) and B. subtilis (5%). The ASB counts ranged from 1 to 4 log CFU/g, and the mean was 2.9 log CFU/g for mesophilic aerobic sporeforming bacteria (MSC) and 2.5 log CFU/g for thermophilic aerobic sporeforming bacteria (TSC). Most PIF samples presented MSC and TSC counts between 2 and 3 log CFU/g. A total of 13%, 50%, and 37% of the samples presented MSC counts from <2 log CFU/g, between 2 and 3 log CFU/g and between 3 and 4 log CFU/g, respectively. Among the ASB isolates, 97% had protease, 84% hydrolyzed starch, 66% had hemolytic activity, and 61% had lecithinase activity. A total of 44 out of 120 isolates harbored at least one toxin gene; 56% for B. cereus, 34% for B licheniformis, and less than 5% for B. subtilis, B pumilus, and L. sphaericus. All B. cereus isolates harbored the nhe gene, 60% entFM, 44% cytK, 32% bceT, and 28% hbl genes. Besides, 17% of B. licheniformis harbored nhe. A small proportion of B. subtilis, B. pumilus, and L. sphaericus carried toxin genes. The rpoB based phylogenetic tree provided high resolution among Bacillus species. The findings of this study provide insights into the phenotypic and genotypic biodiversity of Bacillus present in PIF.

UV-VIS spectrophotometric-assisted chemometric calibration models for simultaneous determination of thiamine and pyridoxine vitamins in powdered infant formula

Thiamine and pyridoxine, which are hydrophilic B vitamins, are compounds that play an important role in many biochemical events for the health of infants and adults. Recently, because of the complexity of food and pharmaceutical composition, chemometric models are preferred to obtain relevant information from multivariate measured data. The aim of this study was to determine the simultaneous quantitative analysis of thiamine and pyridoxine in powdered infant formula and pharmaceutical formulations by the UV-visible spectrophotometric method coupled with chemometric calibration models (partial least squares (PLS) and principal component regression (PCR)). These vitamins were extracted by addition of trichloroacetic acid (precipitation of proteins) after samples of powdered infant formula was dissolved in Milli-Q water. Factorial design was used to prepare the calibration (25 samples) and validation (8 samples) sets containing mixture of thiamine and pyridoxine within their linear range (2.5-25 μg/mL). Spectra of obtained mixtures and samples were recorded between wavelengths of 200-360 nm with the intervals Δλ=1 nm. In regression models created by using PLS and PCR algorithm, the results had acceptable recovery 98.14% for both compounds. These chemometric models predict the quantification of both, thiamine and pyridoxine in powdered infant formula samples via interpreting the UV-Vis spectrum. The average thiamine and pyridoxine contents of powdered infant formula samples were found to be 5.02±0.065 μg/g and 4.32±0.086 μg/g for PLS model and 4.98±0.078 μg/g and 4.03±0.091 μg/g for PCR model, respectively.

A simple and sensitive aptasensor with rolling circle amplification for viable Cronobacter sakazakii detection in powdered infant formula

Cronobacter sakazakii is a foodborne, emerging opportunistic pathogen that causes severe bacteremia, necrotizing enterocolitis, and sepsis with a mortality rate of up to 80%. In this study, we developed a simple and sensitive fluorescent turn-off aptasensor with rolling circle amplification assay for viable C. sakazakii detection in powdered infant formula. The results showed that the proposed aptasensor has good performance and specificity for detecting viable C. sakazakii in pure culture and powdered infant formula samples within 3 h. Under the optimal reaction conditions, there is a linear relationship between fluorescent intensity at 490 nm and logarithmic concentration of C. sakazakii in the range of 2.7 × 105 to 2.7 × 102 cfu/mL, with a limit of detection of 2.7 × 102 cfu/mL in pure culture. The proposed aptasensor achieved a recovery of 104 to 111% in pure culture, and 96 to 107% in spiked powdered infant formula samples. The proposed aptasensor does not require complicated DNA extraction steps or antibodies, and can be performed at 37°C, making it a convenient and sensitive strategy for C. sakazakii detection.

Point-of-care and visual detection of Salmonella spp. and Cronobacter spp. by multiplex loop-mediated isothermal amplification label-based lateral flow dipstick in powdered infant formula

In this study, the invasion protein gene for Salmonella spp. and the 16S–23S rRNA internal transcribed spacer sequence for Cronobacter spp. were used as two species-specific targets simultaneously amplified using labelled primers in the multiplex loop-mediated isothermal amplification (mLAMP) reaction. Subsequently, the modified amplification products were monitored by lateral flow dipstick (LFD) through dual immunoreactions. The accumulation of gold nanoparticles produced characteristic red bands on the LFD, enabling simultaneous visual inspection. Under optimised conditions, the mLAMP-LFD assay showed high specificity. The detection limits of Salmonella spp. and Cronobacter spp. in powdered infant formula (PIF) were as low as 4.3 cfu g−1 and 2.8 cfu g−1, respectively. The whole process can be accomplished in less than 1 h. Moreover, the investigation on 80 PIF samples indicated that the assay performed good feasibility in real samples. Thus, this method could be an effective tool for detecting Salmonella spp. and Cronobacter spp.

Sensitive dual readout assays based on rolling circle amplification for fluorescent and colorimetric detection of Cronobacter spp. in powdered infant formula

Cronobacter spp. are important opportunistic foodborne pathogens in powdered infant formula (PIF). In this study, a sensitive and specific assay was developed for fluorescent and colorimetric detection of Cronobacter spp. in PIF samples by using ligase chain reaction and rolling circle amplification (LCR-RCA) dual-amplification strategy. A pair of gap-padlock probes was designed to induce specific genomic DNA recognition and initiate the LCR. Hyperbranched rolling circle amplification (HRCA) combined with fluorescent dye and linear rolling circle amplification (LRCA) with AuNP probes were performed to achieve fluorescent or colorimetric detection of Cronobacter spp. The limits of detection (LODs) of the proposed assays were 8.6 × 101 CFU/mL (fluorescence) and 7.5 × 102 CFU/mL (colorimetric) in pure culture and 9.2 × 102 CFU/mL (fluorescence) and 8.4 × 103 CFU/mL (colorimetric) in spiked PIF samples, respectively. After 5–7 h enrichment, the proposed methods could detect as low as 100 CFU/mL bacteria in pure culture and PIF sample. The recovery of both methods was in the range of 89.4%–103.8% in pure culture or spiked PIF samples. The proposed assay has the potential to be a rapid and efficient method for detection of Cronobacter spp. in PIF.

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.

Microbiological Quality of Powdered Infant Formula in Latin America

Cronobacter is a bacterial genus that includes seven species, and the species Cronobacter sakazakii is most related to meningitis and septicemia in infants associated with powdered infant formula (PIF). The objectives of this study were to evaluate the presence of C. sakazakii and to determine the microbiological quality of PIF for infant consumption. To do this, a total of 128 PIF samples were analyzed in four brands and countries (Chile, Mexico, Holland, and Brazil), considering three types of PIF: premature (PIF1), infant (PIF2), and follow-up (PIF3). Aerobic plate counts (APC) and Enterobacteriaceae (ENT) were assessed in accordance with Chilean official standards. The outer membrane protein A (ompA) gene was amplified to detect Cronobacter spp. and the fusA gene was amplified to identify C. sakazakii by using the PubMLST Web site and BLAST (NCBI). The antibiotic resistance profile was performed according to the Clinical and Laboratory Standards Institute standards. The pathogen was quantified by the most probable number (MPN). The results showed that APC median values for PIF1, PIF2, and PIF3 were 3.2, 4.9, and 4.8 log CFU g-1, respectively. The APC were higher in PIF2 (P < 0.01) from Holland (P < 0.01) in the commercial brand 4 (P < 0.01). The ENT median values in PIF1, PIF2, and PIF3 were 1.8, 1.5, and 1.7 log CFU g-1, respectively. Five strains of C. sakazakii and one strain of Cronobacter malonaticus were identified as having values between 0.023 and 2.3 MPN/g. All strains (100%) harbored the ompA, plasminogen activator (cpa), and hemolysin (hly) virulence genes. To conclude, C. sakazakii was found in four PIF samples from four Chilean products and one from Mexico, which is distributed throughout America. C. sakazakii strains exhibit virulence factors and resistance to ampicillin, thus posing a risk when PIFs are consumed by infants.

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.

Sensitive and visual detection of Cronobacter spp. in powdered infant formula by saltatory rolling circle amplification method

Cronobacter species are opportunistic foodborne pathogens involved in neonatal infections from powdered infant formula (PIF). In this study, a novel saltatory rolling circle amplification (SRCA) method has been developed for detecting Cronobacter spp. in PIF. 16 of Cronobacter spp. strains and 32 of non-Cronobacter strains were examined by using the DNA-directed RNA polymerase subunit beta (rpoB) gene. All Cronobacter strains were identified in pure cultures, whereas no SRCA products were found in non-Cronobacter strains. The detection limits were 3.4 × 102 CFU/mL through observing the white precipitate by naked eye, while 3.4 × 101 CFU/mL by fluorescence visualization. After enrichment, the detection limits of SRCA method in PIF were 8.7 × 100 CFU/g and 8.7 × 10−1 CFU/g by visualization with white precipitate and fluorescence, respectively. Compared with the conventional PCR method, the SRCA has at least 100-fold higher sensitivity and 100-fold lower detection limit. In addition, 85 of PIF samples were investigated for the Cronobacter contamination, and the results were 100% sensitivity, 97.62% specificity and 97.65% accuracy compared with the ISO method. Therefore, the results show that SRCA is a sensitive and visual detection method for the detection of Cronobacter spp. in PIF with great potential.

Validation of Biomode S.A. Probe4CronobacterTM for the Identification of Cronobacter spp.

Background: Probe4Cronobacter test kit is based on the use of a fluorescence-labeled peptide nucleic acid probe (PNA) allied to fluorescence microscopy. A sample is taken after a 24 h enrichment of rehydrated 30 g portions of powdered infant formula (PIF). The method uses ready to use dropper solutions applied directly in the sample. This simple process takes less than 2 h to provide a result. In the presence of Cronobacter species, bright red rod-shaped cells will be visible under a fluorescence microscope. Objective: Probe4Cronobacter validation as a new method for the detection Cronobacter species in Powdered Infant Formula (PIF) under the AOAC Performance Tested MethodsSM (License No. 081702). Methods: The validation study encompassed matrix comparison study, inclusivity and exclusivity testing and robustness studies (stability, kit variation, and ruggedness). Results: The inclusivity and exclusivity testing (50 and 35 strains, respectively) yielded no false negative or false positive results. Probe4Cronobacter was compared to the ISO/TS 22964:2006 in 30 g of PIF samples within method comparison in an unpaired study. A total of 30 samples with both low and high level of inoculation were analyzed by Probe4Cronobacter and compared to the same number of samples screened by ISO/TS 22964:2006. No statistically significant differences between presumptive and confirmed results or between candidate and reference method results were observed. Robustness studies showed a high level of consistency and integrity of the kit when different parameters were varied. The deviation conditions tested did not affect the performance of the kit. Conclusions: Probe4Cronobacter test kit has shown to be a accurate, highly sensitive and robust methods for the detection of Cronobacter spp. in PIF samples.

Prevalence of Bacillus cereus in powdered infant and powdered follow-up formula in China

Outbreaks and sporadic cases of foodborne disease caused by Bacillus cereus have been reported in China, including cases involving infants and children. However, the prevalence of B. cereus contamination in foods is not clearly known. In China, no legal microbiological criteria for B. cereus in powdered infant formula (PIF) and powdered follow-up formula (PFUF) currently exists. The purpose of the study was to investigate the prevalence of Bacillus cereus in PIF and PFUF sold in China and to provide data that could be used for risk assessment and management to ensure the safety of these kinds of products. As part of the national surveillance program, 6656 samples of PIF and PFUF were collected quarterly from urban and rural markets in 31 provincial-level administrative units in China in 2014. The results show that 7.53% of the samples were contaminated with B. cereus ≥10 CFU/g and 1.11% of the samples were contaminated above 100 CFU/g. There were no statistical differences of B. cereus contamination levels between PIF and PFUF or between samples taken from retail, wholesale and online outlets. The prevalence of B. cereus-positive samples differed between sampling regions and production regions. The occurrence of B. cereus >10 CFU/g of samples produced in summer and autumn, which were 8.80% and 9.18%, respectively, were higher than those in spring and winter, which were 6.67% and 6.32%, respectively. The study indicated a need to conduct further research on B. cereus contamination in powdered formula products in China before microbiological criteria are developed.

Isolation of Antibiotic-Resistant Micrococcus aloeverae from Milk Powder

The emergence of antibiotic resistance in humans via the food chain has become an area of great concern. Antibiotic resistance gene transfer from a food-borne bacteria or probiotic agent to a human gut microbe can lead to a serious food safety issue. Such seemingly harmless bacteria or probiotics can potentially become a threat to human health if they carry mobile genes conferring antibiotic resistance. The recent trend of probiotic application in powdered infant formula and milk products and the presently known contamination of these products by certain opportunistic Enterobacteriaceae bacteria serves as the rationale for evaluating their safety. In this study, 25 milk powder samples and 25 powdered infant formula samples were analyzed for the presence of antibiotic resistant bacteria of family Enterobacteriaceae using an isolation technique. Out of the 50 samples screened, an isolate was detected in one milk powder sample. Its biochemical characteristics, Gram character, antibiotic susceptibility, and motility were analyzed. It was found to be a Gram-positive, non-motile, catalase-positive and oxidase-negative microbe. The isolate was resistant to Rifampicin and Vancomycin, but susceptible towards Streptomycin, Ciprofloxacin, Ceftriaxone and Gentamycin. Molecular characterization by 16S rDNA sequencing identified the isolate as Micrococcus aloeverae. The unique occurrence of Micrococcus aloeverae in milk powder elicits an area of food safety concern for such samples. To the best of our knowledge, this study is the first to report antibiotic resistance by Micrococcus aloeverae. Although it may be a non-pathogenic microbe, its resistance towards antibiotics cannot be ignored. The possibility of Micrococcus aloeverae serving as a potential vehicle for transfer of antibiotic resistance genes to human gut microbiota represents a prospective food safety concern.

Prevalence, Molecular Characterization, and Antibiotic Susceptibility of Cronobacter sakazakii Isolates from Powdered Infant Formula Collected from Chinese Retail Markets.

Cronobacter sakazakii is an opportunistic pathogen that causes severe infections in neonates and infants through contaminated powdered infant formula (PIF). Therefore, the aim of this study was a large-scale study on determine the prevalence, molecular characterization and antibiotic susceptibility of C. sakazakii isolates from PIF purchased from Chinese retail markets. Two thousand and twenty PIF samples were collected from different institutions. Fifty-six C. sakazakii strains were isolated, and identified using fusA sequencing analysis, giving a contamination rate of 2.8%. Multilocus sequence typing (MLST) was more discriminatory than other genotyping methods. The C. sakazakii isolates were divided into 14 sequence types (STs) by MLST, compared with only seven clusters by ompA and rpoB sequence analysis, and four C. sakazakii serotypes by PCR-based O-antigen serotyping. C. sakazakii ST4 (19/56, 33.9%), ST1 (12/56, 21.4%), and ST64 (11/56, 16.1%) were the dominant sequence types isolated. C. sakazakii serotype O2 (34/56, 60.7%) was the primary serotype, along with ompA6 and rpoB1 as the main allele profiles, respectively. Antibiotic susceptibility testing indicated that all C. sakazakii isolates were susceptible to ampicillin-sulbactam, cefotaxime, ciprofloxacin, meropenem, tetracycline, piperacillin-tazobactam, and trimethoprim-sulfamethoxazole. The majority of C. sakazakii strains were susceptible to chloramphenicol and gentamicin (87.5 and 92.9%, respectively). In contrast, 55.4% C. sakazakii strains were resistant to cephalothin. In conclusion, this large-scale study revealed the prevalence and characteristics of C. sakazakii from PIF in Chinese retail markets, demonstrating a potential risk for neonates and infants, and provide a guided to effective control the contamination of C. sakazakii in production process.

Detection of Mycobacterium avium subspecies paratuberculosis in powdered infant formula using IS900 quantitative PCR and liquid culture media

Mycobacterium avium subspecies paratuberculosis (MAP) has been implicated in Crohn's disease in humans resulting in public concern over the presence of MAP in powdered infant formula, which could contribute towards early human exposure to MAP or MAP components. Testing of representative powdered infant formula samples using effective tests is required to provide information on contamination of infant formula with MAP, so that consumers can make informed decisions. This study aimed to test representative powdered infant formula samples for the presence of MAP using a quantitative PCR and liquid culture method. For this purpose, an efficient DNA extraction method was developed and an optimum decontamination protocol for culture method was identified. A total of 122 powdered infant formula samples were tested, comprising 72 brands produced by 12 manufacturers from 9 countries. Powdered infant formula samples were reconstituted and centrifuged to separate the casein pellet, cream layer and whey fraction. A sensitive qPCR test was performed on DNA extracted from the casein pellet. In addition, the cream layer and casein pellet were cultured in liquid media, following decontamination with the optimum protocol. Of the 122 samples tested, 6 were positive for MAP DNA but none were positive for growth in culture at 12 and 20 weeks. The limit of detection of the quantitative PCR was less than 5 MAP organisms per 1.5g milk powder. The methods developed in the study could be used for quality assurance testing for infant formula and calf milk replacers. The low contamination level of MAP and absence of viable forms in our study suggests a relatively low risk of exposure of infants to MAP components.

Salmonella detection in powdered dairy products using a novel molecular tool

In this study, we developed a rapid, specific, and sensitive loop-mediated isothermal amplification technique combined with a lateral flow dipstick (LAMP-LFD) method to detect Salmonella targeting the siiA gene in powdered infant formula (PIF). The specificity of the detection method (LAMP-LFD) approached 100% using 21 Salmonella and 31 non-Salmonella bacterial strains. This detection method exhibited high sensitivity limits for pure cultures at 3.7 cfu/mL and in PIF at 2.2 cfu/g without enrichment. To evaluate the applicability of the LAMP-LFD method, we detected 60 positive PIF samples and 20 negative PIF samples. The results showed that the method of LAMP-LFD had a high diagnostic specificity of 100% for detection of Salmonella in PIF. To reduce incidence of LAMP contamination, we applied propidium monoazide (PMA) to eliminate carryover contamination of LAMP. At the same time, we found that PMA does not affect observation of LFD for measurement of LAMP signal. The results verified that the method of LAMP-LFD targeting the siiA gene is rapid, accurate, and sensitive for Salmonella detection in PIF, and that PMA shows great potential to be widely used to eliminate the amplicon contamination risk generated by the highly sensitive LAMP reaction in the detection process.