Prevalence Of Cronobacter Research Articles

Relevance of genetic causes and environmental adaptation of Cronobacter spp. isolated from infant and follow-up formula production factories and retailed products in China: A 7-year period of continuous surveillance based on genome-wide analysis

The possible contamination routes, environmental adaptation, and genetic basis of Cronobacter spp. in infant and follow-up formula production factories and retailed products in mainland China have been determined by laboratory studies and whole-genome comparative analysis in a 7-year nationwide continuous surveillance spanning from 2012 to 2018. The 2-year continuous multicenter surveillance of the production process (conducted in 2013 and 2014) revealed that the source of Cronobacter spp. in the dry-blending process was the raw dry ingredients and manufacturing environment (particularly in the vibro sieve and vacuum cleaner), while in the combined process, the main contamination source was identified as the packing room. It is important to note that, according to the contamination control knowledge obtained from the production process surveillance, the contamination rate of retail powdered infant formula (PIF) and follow-up formula (FUF) products in China decreased significantly from 2016 onward, after improving the hygiene management practices in factories. The prevalence of Cronobacter spp. in retailed PIF and FUF in China in 2018 was dramatically reduced from 1.55 % (61/3925, in 2012) to an average as low as 0.17 % (13/7655 in 2018). Phenotype determination and genomic analysis were performed on a total of 90 Cronobacter spp. isolates obtained from the surveillance. Of the 90 isolates, only two showed resistance to either cefazolin or cefoxitin. The multilocus sequence typing results revealed that C. sakazakii sequence type 1 (ST1), ST37, and C. malonaticus ST7 were the dominant sequence types (STs) collected from the production factories, while C. sakazakii ST1, ST4, ST64, and ST8 were the main STs detected in the retailed PIF and FUF nationwide. One C. sakazakii ST4 isolate (1.1 %, 1/90) had strong biofilm-forming ability and 13 isolates (14.4 %, 13/90) had weak biofilm-forming ability. Genomic analysis revealed that Cronobacter spp. have a relatively stable core-genome and an increasing pan-genome size. Plasmid IncFIB (pCTU3) was prevalent in this genus and some contained 14 antibacterial biocide- and metal-resistance genes (BMRGs) including copper, silver, and arsenic resistant genes. Plasmid IncN_1 was predicted to contain 6 ARGs. This is the first time that a multi-drug resistance IncN_1 type plasmid has been reported in Cronobacter spp. Genomic variations with respect to BMRGs, virulence genes, antimicrobial resistance genes (ARGs), and genes involved in biofilm formation were observed among strains of this genus. There were apparent differences in copies of bcsG and flgJ between the biofilm-forming group and non-biofilm-forming group, indicating that these two genes play key roles in biofilm formation. The findings of this study have improved our understanding of the contamination characteristics and genetic basis of Cronobacter spp. in PIF and FUF and their production environment in China and provide important guidance to reduce contamination with this pathogen during the production of PIF and FUF.

Diverse Genotypes of Cronobacter spp. Associated with Dairy Farm Systems in Jiangsu and Shandong Provinces in China.

Cronobacter spp. are the most concerning foodborne pathogen in infant formula milk powder. Currently, there are many reports on the prevalence of Cronobacter spp. in infant formula milk and its processing environment, but there are few studies on the prevalence of Cronobacter spp. on dairy farms. We have, therefore, undertaken this study to investigate and track genomic epidemiology of Cronobacter spp. isolates from Chinese dairy farms in the provinces of Jiangsu and Shandong. In this study, forty Cronobacter spp. strains, consisting of thirty Cronobacter sakazakii, eight Cronobacter malonaticus, and two Cronobacter dublinensis, were obtained from 1115 dairy farm samples (raw milk, silage, bedding, and feces), with a prevalence rate of 3.57%. These isolates were classified into 10 Cronobacter serotypes and 31 sequence types (STs), including three novel STs which were isolated for the first time. Notably, pathogenic Cronobacter STs 7, 8, 17, 60, and 64, which are associated with clinical infections, were observed. Antimicrobial susceptibility testing showed that all the Cronobacter spp. were highly resistant to cephalothin and fosfomycin, which was consistent with the antimicrobial genotype. All isolates carried core virulence genes related to adherence, invasion, endotoxin, immune evasion, secretion system, and regulation. Approximately half the isolates were also able to produce a strong biofilm. Twenty-one prophages and eight plasmids were detected, with the most common prophage being Cronobacter_ENT47670 and the most common plasmid being IncFIB (pCTU1). In addition, two isolates harbored the transmissible locus of stress tolerance (tLST) which confers high environmental persistence. Phylogenetic analysis showed strong clustering by species level and sequence types. Isolates from different sources or regions with a similar genomic background suggests the cross-contamination of Cronobacter spp. The presence of diverse genotypes of Cronobacter spp. in dairy farms in Jiangsu and Shandong provinces indicates that surveillance of Cronobacter spp. on dairy farms should be strengthened, to prevent and control transmission and ensure the quality and safety of raw dairy products.

Prevalence and molecular characterization of Cronobacter species in Egyptian table eggs and egg-based desserts; special insight on Cronobacter sakazakii

The occurrence of Cronobacter spp. was investigated in table eggs and egg-based desserts obtained from retail stores. Cronobacter was isolated from 57 out of 180 (31.7%) (95% Confidence Interval [CI]: 26–43%) examined samples of eggs and egg-based desserts. The prevalence of Cronobacter spp. was significantly higher in farm eggs (67%) (P < 0.05) than in Balady eggs (23%), cream cake (27%) and small-scale ice cream (27%). Cronobacter (C.) sakazakii and C. muytjensii were the predominant isolates obtained in the present study. All C. sakazakii isolates were molecularly confirmed with higher incidence in small-scale ice cream (62.5%).

Identification and antibiotic resistance of Cronobacter spp. isolated from dried edible mushrooms.

Cronobacter spp. is an important foodborne pathogen that can cause life-threatening diseases in infants and immunocompromised adults. The present study was carried out to understand the prevalence and characterization of Cronobacter spp. in dried edible mushrooms in Jiangsu province, China. Cronobacter isolates were identified and genotyped by multilocus sequence typing (MLST); the antimicrobial susceptibility of Cronobacter strains was determined by the disk diffusion method; the biofilm formation ability of Cronobacter spp. was assessed using the microtiter plate method. The overall prevalence of Cronobacter spp. in dried edible mushrooms was 14.8%, with the highest contamination rate of after 37.2% found in Auricularia auricular. The Cronobacter isolates were identified as C. sakazakii (n=26), C. malonaticus (n=2), C. dublinensis (n=2) and C. turicensis (n=1). The MLST scheme produced 20 sequence types (STs), two of which were newly identified. ST148 was the most prevalent ST (n=5), followed by ST4 (n=3), ST17 (n=3), ST64 (n=3), and ST540 (n=2). One (3.2%) and 15 (48.4%) Cronobacter isolates were resistant to tetracycline and meropenem, respectively. In contrast, all of the tested isolates were susceptible to the remaining 14 antibiotics. Moreover, 20 (64.5%) Cronobacter isolates showed weak ability to produce biofilm, but no isolates showed strong or moderate biofilm-forming ability. PRACTICAL APPLICATION: Our findings revealed a high genetic diversity of Cronobacter spp. in dried edible mushrooms and provided new epidemiological evidence for the widespread existence of Cronobacter spp. in such products. The presence of Cronobacter spp. in dried edible mushrooms may pose potential risks to human health and enhancing the hygiene of such products are necessary to ensure food safety.

Prevalence, characterization, and antibiotic susceptibility of Cronobacter spp. in a milk powder processing environment: The first reported case in Serbia.

Cronobacter spp. are opportunistic foodborne pathogens that most often infect neonates and infants through contaminated powdered infant formula. No reports have been published in Serbia on the prevalence of Cronobacter spp. in powdered milk production environments. Consequently, this study aimed to determine the prevalence, molecular characterization, antimicrobial susceptibility, and biofilm‐forming ability of Cronobacter spp. isolated from a milk powder plant. Hundred samples were collected from the production facility. Fifteen Cronobacter sakazakii strains were isolated and identified, giving a contamination rate of 15%. Using multi‐locus sequence typing, the isolates were divided into five sequence types (STs). Cronobacter sakazakii ST4 (50%), ST1 (16.67%), and ST83 (16.67%) were the dominant STs isolated. A novel sequence type (ST759) was identified and registered in the Cronobacter MLST database. The results of the antibiotic susceptibility testing indicated that C. sakazakii strains were susceptible to piperacillin/tazobactam, ampicillin/sulbactam, and amoxicillin/clavulanate, especially to meropenem and cefotaxime. Most of the ST4 showed moderate‐to‐strong biofilm‐forming ability. The presence of clinically relevant isolates (ST4, ST1, ST83, and ST8) revealed that the production plant is likely a potential concern for public health. Finally, finding new sequence types like the one detected in this study (ST759) underlines evolving genetic changes in C. sakazakii.

Prevalence of Cronobacter spp. in various foodstuffs and identification by multiplex PCR

Abstract Cronobacter spp. are opportunistic pathogens isolated from many different type of food and environmental samples and may cause serious health problems and even death in newborns, children and elders. The purpose of the study was to determine occurrence and prevalence of Cronobacter spp. in animal originated food, ready-to-eat food, fruits and vegetables, sold in Istanbul and its vicinity and to perform molecular typing of the isolated agents by multiplex PCR. Analyses were conducted on a total of 219 food samples including 11 confectionary, 50 fruits and vegetables, 52 dairy products, 50 meat and meat products and 56 ready-to-eat food. Suspected isolates were validated by PCR targeted to gyrB gene and multiplex PCR was performed for identification. Overall analyses revealed a prevalence of 5.48% for Cronobacter spp. in all food products assessed. Prevalence of the bacteria was 9%, 1.9% and 17.8% in confectionary, dairy products and ready-to-eat food, respectively whereas no bacteria were detected in fruits and vegetables, meat and meat products. Out of a total of 12 isolates determined, 8 (66%), 3 (25%) and 1 (8%) were identified as C.sakazakii, C.muytjensii and C.malonaticus , respectively and nine isolates belonged to cereal-based balls (meatless cig kofte).

Prevalence, Antibiotic Susceptibility, and Molecular Characterization of Cronobacter spp. Isolated From Edible Mushrooms in China.

Cronobacter spp. are foodborne pathogens that can infect and cause life-threatening diseases in all age groups, particularly in infants and immunocompromised elderly. This study aimed to investigate the prevalence, antibiotic susceptibility, and molecular characteristics of Cronobacter spp. isolates in edible mushrooms collected from 44 cities in China. In total, 668 edible mushrooms were collected from traditional retail markets and supermarkets and were analyzed by quantitative methods, PCR-based serotyping, multilocus sequence typing (MLST), and antibiotic susceptibility testing. Among the 668 samples tested, 89 (13.32%) were positive for Cronobacter spp., and the contamination levels exceeded the 110 most probable number (MPN)/g in 13.48% (12/89) of the samples. Flammulina velutipes samples had the highest contamination rate of 17.54% (37/211), whereas Hypsizygus marmoreus samples had the lowest contamination rate of 3.28% (2/61). Ten serotypes were identified among 115 isolates, of which the C. sakazakii serogroup O1 (n = 32) was the primary serotype. MLST indicated that there was quite high genetic diversity in Cronobacter spp. and 72 sequence types were identified, 17 of which were new. Notably, C. sakazakii ST148 (n = 10) was the most prevalent, followed by C. malonaticus ST7 (n = 5). Antibiotic susceptibility testing revealed that the majority of Cronobacter spp. strains were susceptible to the 16 antibiotics tested. However, a portion of isolates exhibited relatively high resistance to cephalothin, with resistance and intermediate rates of 93.91 and 6.09%, respectively. One isolate (cro300A) was multidrug-resistant, with resistance to five antibiotics. Overall, this large-scale study revealed the relatively high prevalence and high genetic diversity of Cronobacter spp. on edible mushrooms in China, indicating a potential public health concern. To our knowledge, this is the first large-scale and systematic study on the prevalence of Cronobacter spp. on edible mushrooms in China, and the findings can provide valuable information that can guide the establishment of effective measures for the control and precaution of Cronobacter spp on edible mushrooms during production processes.

Contamination and characterization of multiple pathogens in powdered formula at retail collected between 2014 and 2015 in China

A total of 119 powdered formula samples collected from supermarkets, E-shops and online maternal counters in 16 provinces in China between 2014 and 2015 were analyzed for Cronobacter, Bacillus cereus, Clostridium sporogenes, Salmonella and Staphylococcus aureus. Antimicrobial susceptibility of Cronobacter was also tested by a broth microdilution method. Virulence genes related to emetic and diarrheal toxins production harbored by B. cereus were detected by multiplex PCR. The results showed that the prevalence of Cronobacter, B. cereus, C. sporogenes contamination were 3.4%, 36.1% and 9.2%, respectively. The contamination level of Cronobacter ranged from 0.36 MPN/100 g to 15 MPN/100 g. All Cronobacter isolates cultured from powdered formula tested were susceptible to all antimicrobials agents tested except one isolate which showed intermediate resistance to ciprofloxacin. The prevalence of B. cereus contamination in goat-based powdered formula was higher than that of milk-based powdered formula. A large number of B. cereus isolates (93.0%) carried nhe- and entFM-encoding genes (65.1%). Subtyping results demonstrated that the frequency of nheA, nheB, nheC were 86.1%, 88.4%, 39.5%, while 27.9%, 25.6%, 20.9% were positive for hblA, hblC, hblD, respectively. The cytK gene was detected in 25.6% of B. cereus isolates. There were 5 isolates identified as carrying both hblA/C/D and nheA/B/C and being potentially toxic. These data pointed to the need to improve the hygienic and manufacturing practices during powdered formula production in order to reduce the outbreak burden of foodborne disease risk in China.

Occurrence and prevalence of Cronobacter spp. in plant and animal derived food sources: a systematic review and meta-analysis.

Cronobacter species are motile, non-spore forming, Gram negative emerging opportunistic pathogens mostly associated with bacteremia, meningitis, septicemia, brain abscesses and necrotizing enterocolitis in infected neonates, infants and immunocompromised adults. Members of the genus Cronobacter are previously associated with powdered infant formula although the main reservoir and routes of contamination are yet to be ascertained. This study therefore aim to summarize occurrence and prevalence of Cronobacter spp. from different food related sources. A retrospective systematic review and meta-analysis of peer reviewed primary studies reported between 2008 and 2014 for the occurrence and prevalence of Cronobacter spp. in animal and plant related sources was conducted using “Cronobacter isolation”, “Cronobacter detection” and “Cronobacter enumeration” as search terms in the following databases: Web of Science (Science Direct) and ProQuest. Data extracted from the primary studies were then analyzed with meta-analysis techniques for effect rate and fixed effects was used to explore heterogeneity between the sources. Publication bias was evaluated using funnel plot. A total of 916 articles were retrieved from the data bases of which 28 articles met inclusion criteria. Cronobacter spp. could only be isolated from 103 (5.7 %) samples of animal related food while 123 (19 %) samples of plant related food samples harbors the bacteria. The result of this study shows that occurrence of Cronobacter was more prevalent in plant related sources with overall prevalence rate of 20.1 % (95 % CI 0.168–0.238) than animal originated sources with overall prevalence rate of 8 % (95 % CI 0.066–0.096). High heterogeneity (I2 = 84) was observed mostly in plant related sources such as herbs, spices and vegetables compared to animal related sources (I2 = 82). It could be observed from this study that plant related sources serve as reservoir and contamination routes of Cronobacter spp.

Detection of Foodborne Bacterial Pathogens from Individual Filth Flies

There is unanimous consensus that insects are important vectors of foodborne pathogens. However, linking insects as vectors of the pathogen causing a particular foodborne illness outbreak has been challenging. This is because insects are not being aseptically collected as part of an environmental sampling program during foodborne outbreak investigations and because there is not a standardized method to detect foodborne bacteria from individual insects. To take a step towards solving this problem, we adapted a protocol from a commercially available PCR-based system that detects foodborne pathogens from food and environmental samples, to detect foodborne pathogens from individual flies.Using this standardized protocol, we surveyed 100 wild-caught flies for the presence of Cronobacter spp., Salmonella enterica, and Listeria monocytogenes and demonstrated that it was possible to detect and further isolate these pathogens from the body surface and the alimentary canal of a single fly. Twenty-two percent of the alimentary canals and 8% of the body surfaces from collected wild flies were positive for at least one of the three foodborne pathogens. The prevalence of Cronobacter spp. on either body part of the flies was statistically higher (19%) than the prevalence of S. enterica (7%) and L.monocytogenes (4%). No false positives were observed when detecting S. enterica and L. monocytogenes using this PCR-based system because pure bacterial cultures were obtained from all PCR-positive results. However, pure Cronobacter colonies were not obtained from about 50% of PCR-positive samples, suggesting that the PCR-based detection system for this pathogen cross-reacts with other Enterobacteriaceae present among the highly complex microbiota carried by wild flies. The standardized protocol presented here will allow laboratories to detect bacterial foodborne pathogens from aseptically collected insects, thereby giving public health officials another line of evidence to find out how the food was contaminated when performing foodborne outbreak investigations.

Detection of foodborne bacterial pathogens from individual filth flies.

There is unanimous consensus that insects are important vectors of foodborne pathogens. However, linking insects as vectors of the pathogen causing a particular foodborne illness outbreak has been challenging. This is because insects are not being aseptically collected as part of an environmental sampling program during foodborne outbreak investigations and because there is not a standardized method to detect foodborne bacteria from individual insects. To take a step towards solving this problem, we adapted a protocol from a commercially available PCR-based system that detects foodborne pathogens from food and environmental samples, to detect foodborne pathogens from individual flies.Using this standardized protocol, we surveyed 100 wild-caught flies for the presence of Cronobacter spp., Salmonella enterica, and Listeria monocytogenes and demonstrated that it was possible to detect and further isolate these pathogens from the body surface and the alimentary canal of a single fly. Twenty-two percent of the alimentary canals and 8% of the body surfaces from collected wild flies were positive for at least one of the three foodborne pathogens. The prevalence of Cronobacter spp. on either body part of the flies was statistically higher (19%) than the prevalence of S. enterica (7%) and L.monocytogenes (4%). No false positives were observed when detecting S. enterica and L. monocytogenes using this PCR-based system because pure bacterial cultures were obtained from all PCR-positive results. However, pure Cronobacter colonies were not obtained from about 50% of PCR-positive samples, suggesting that the PCR-based detection system for this pathogen cross-reacts with other Enterobacteriaceae present among the highly complex microbiota carried by wild flies. The standardized protocol presented here will allow laboratories to detect bacterial foodborne pathogens from aseptically collected insects, thereby giving public health officials another line of evidence to find out how the food was contaminated when performing foodborne outbreak investigations.

Isolation of Cronobacter spp. isolates from infant formulas and their survival in the production process of infant formula

Over a 24-month surveillance, three Cronobacter strains, NC041, NC830, and NC1006, were isolated from 77 powder infant formulas (3.90%). No Cronobacter was detected in liquid milk. The prevalence of Cronobacter in the prefinal product and packaged final product was 3.70% and 4.35%, respectively. The isolated Cronobacter strains were subjected to several lethal challenges including the pH, drying, disinfectant, and simulated infant formulas manufacturing process (SIFMP). The results indicated that they exhibited unusual resistance to the dry stress and disinfectant. In SIFMP, Cronobacter isolates were inoculated into three possible contamination entry points involving the stages prior to heating, drying, and filling, respectively. No Cronobacter could survive the heating. However, a high level (105 CFU/ml) of Cronobacter was detected in the samples after the inoculation at the drying point. Furthermore, the survival of Cronobacter was observed during the storage at 10°C after 7 days. The results support the hypothesis that Cronobacter contamination occurs at the stages of drying and filling, and highlight the need to improve the disinfecting measures in the manufacturing process.

Prevalence of Cronobacter spp. in a powdered infant formula processing environment

From November 2005 to August 2006, microbiological analyses were performed in a processing plant for powdered infant formula (PIF) to estimate the occurrence and dissemination of Cronobacter spp. and the contamination levels of Enterobacteriaceae in the processing environment. In total 867 samples were collected from the dry processing environment including powders from vacuum cleaners and filtering (sieving) machines, fluids from the drains and swabs from contact surfaces. In total 35 samples in 14 out of 35 tested locations were positive for Cronobacter spp. Positive samples were found in environmental powders (94.3%) and two fluids (5.7%). The Cronobacter spp. prevalence was highest in powders from vacuum cleaners with 28.0% positive samples on a single location. While powders from the sensitive agglomeration section or production (sieving) had contamination rates of 10.7% or below 3.4%, respectively. In addition vacuum powder samples from the filling and can filling line were positive with 5.3 and 8.0%, respectively. High levels (> 500 cfu/g) of Enterobacteriaceae were found in seven locations out of 21 tested powder locations. This fact emphasizes the importance of monitoring of the processing environment to implement effective measures to improve the safety and hygiene in PIF, a very sensitive product.

Temporal and Spatial Distribution ofCronobacterIsolates in a Milk Powder Processing Plant Determined by Pulsed-Field Gel Electrophoresis

A milk powder processing line was sampled for the presence of Enterobacteriaceae and the opportunistic neonatal pathogen Cronobacter at six different sampling sites during an 11-month period. The highest number of Enterobacteriaceae-positive samples was recovered from the raw milk concentrate before pasteurization (78.2%) and from nonproduct samples of the processing line (86.5%), which included swabs from the drying tower and screw conveyers, swabs from the explosion chamber, waste water after the automated cleaning-in-place procedure, air filter cut-outs, and floor samples underneath the outlet of the packaging machine. The prepackaged and packaged final product was contaminated at a rate of 6.6% and 7.1%, respectively. The prevalence of Cronobacter in the prefinal product and the prepackaged and packaged final product was 14.3%, 3.8%, and 2.1%, respectively. Pulsed-field gel electrophoresis (PFGE) analysis of 133 Cronobacter isolates yielded 40 different PFGE profiles. Long-term persistence in the processing line of some of these PFGE profiles was observed. Comparison of the PFGE profiles recovered at different sampling sites revealed the supply air as a potential source for extrinsic Cronobacter contamination. In addition, recovery of the same PFGE profiles before and after CIP events followed by heat treatment indicated the inefficiency of these hygiene measures to completely eliminate Cronobacter from all areas of the processing line. This information provides an essential basis for developing control and prevention strategies concerning this opportunistic pathogen.