Antibiotic Resistance, Virulence Factors, Phenotyping, and Genotyping of Non-Escherichia coli Enterobacterales from the Gut Microbiota of Healthy Subjects.

Non-Escherichia coli Enterobacterales (NECE) can colonize the human gut and may present virulence determinants and phenotypes that represent severe heath concerns. Most information is available for virulent NECE strains, isolated from patients with an ongoing infection, while the commensal NECE population of healthy subjects is understudied. In this study, 32 NECE strains were isolated from the feces of 20 healthy adults. 16S rRNA gene sequencing and mass spectrometry attributed the isolates to Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter cloacae, Enterobacter aerogenes, Enterobacter kobei, Citrobacter freundii, Citrobacter amalonaticus, Cronobacter sp., and Hafnia alvei, Morganella morganii, and Serratia liquefaciens. Multiplex PCR revealed that K. pneumoniae harbored virulence genes for adhesins (mrkD, ycfM, and kpn) and enterobactin (entB) and, in one case, also for yersiniabactin (ybtS, irp1, irp2, and fyuA). Virulence genes were less numerous in the other NECE species. Biofilm formation was spread across all the species, while curli and cellulose were mainly produced by Citrobacter and Enterobacter. Among the most common antibiotics, amoxicillin-clavulanic acid was the sole against which resistance was observed, only Klebsiella strains being susceptible. The NECE inhabiting the intestine of healthy subjects have traits that may pose a health threat, taking into account the possibility of horizontal gene transfer.

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.

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.

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.

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.

Antibiotic Susceptibility of Cronobacter spp. Isolated from Clinical Samples.

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

Efficient inhibition of Cronobacter biofilms by chitooligosaccharides of specific molecular weight.

Biofilms enable Cronobacter spp. to contaminate food, infect infants and resist different environmental stresses, especially desiccation, which is the main reason why Cronobacter can survive in powdered infant formula (PIF) for a long time. Considering the high lethality of Cronobacter infection in infants, it is important to find efficient and safe inhibitors of Cronobacter biofilms. In this study, we found that chitooligosaccharides (COS) with a molecular weight of 2000 Da efficiently inhibited Cronobacter biofilms, especially in skim milk broth. The minimum biofilm inhibitory concentration (MBIC77) of COS was as low as 20 µg/mL, which is lower than that reported in most previous studies. Besides, the elimination rate of COS for Cronobacter mature biofilms was 50% when the concentration was 10 mg/mL. COS could significantly inhibit soluble polysaccharide secretion and biofilm cell growth, as well as change the cell membrane permeability of Cronobacter. These might be the possible reasons for COS's efficient inhibition of Cronobacter biofilms. However, during the inhibition, five important genes-related to biofilm formation-flhD, flgJ, luxR, ompA, and wcaJ-were all up-regulated after COS treatment, except the gene bcsA. In summary, our findings showed that COS could be used as an efficient and safe inhibitor against Cronobacter biofilms for better control of Cronobacter contamination and infection.

Molecular and phenotypical characterization of Cronobacter species isolated with high occurrence from oats and linseeds.

The aim of this study was to determine the prevalence Cronobacter from 30 samples of oats and 30 of linseeds commercially available in Brazil. The detection of Cronobacter was as according to the ISO 22964:2017. The isolates were characterized according to their phenotypically using Vitek 2.0 and antibiotic susceptibility profile. Molecular characterization was accomplished by real-time PCR targeting dnaG gene, PCR targeting rpoB gene, multiplex-PCR targeting cgcA gene and fusA allele sequencing. A total of 34 samples (56.7%) contained Cronobacter; 19 (63.3%) of linseeds and 15 (50.0%) of oats. The isolates were identified as C. sakazakii (n = 18, 52.9%), C. dublinensis (n = 7, 20.6%), C. turicensis (n = 6, 17.7%) and C. malonaticus (n = 3, 8.8%). Thirty-four Cronobacter isolates were assigned to 11 different fusA alleles of which 3 were new (169, 170 and 171). The PCR targeting rpoB gene and cgcA gene failed to identify 19 isolates. Seven (20.6%) strains showed resistance or intermediate/resistance to tetracycline, and one (2.9%) strain had intermediate resistance to piperacilin-tazobactam. The presence of Cronobacter in oats and linseeds indicate that these foods can be a potential threat to human health, particularly when preparing food for elderly or immunosuppressed persons. The incorrect use of this foods for feeding of neonates (<6 months) by careers should also be avoided.

Inactivation of Cronobacter malonaticus cells and inhibition of its biofilm formation exposed to hydrogen peroxide stress.

Presence of Cronobacter malonaticus in powdered infant formula (PIF) poses a high risk to infant and public health. Cronobacter malonaticus has been widely distributed in food and food processing environments, and the true origin of C. malonaticus in PIF is poorly understood. Control and prevention of C. malonaticus is necessary for achieving microbial safety of PIF. However, little information about decontamination of C. malonaticus is available. In this study, effects of hydrogen peroxide on inactivation and morphological changes of C. malonaticus cells were determined. Furthermore, inhibitory effects of H2O2 on biofilm formation in C. malonaticus were also performed. Results indicated that H2O2 could completely inactivate C. malonaticus in sterile water with 0.06% H2O2 for 25 min, 0.08% H2O2 for 15 min, and 0.10% for 10 min, respectively, whereas the survival rates of C. malonaticus in tryptic soy broth medium significantly increased with the same treatment time and concentration of H2O2. In addition, morphological changes of C. malonaticus cells, including cell shrinkage, disruption of cells, cell intercession, and leakage of intercellular material in sterile water after H2O2 treatment, were more predominant than those in tryptic soy broth. Finally, significant reduction in biofilm formation by H2O2 was found using crystal violet staining, scanning electron microscopy, and confocal laser scanning microscopy detection compared with control samples. This is the first report to determine the effects of H2O2 on C. malonaticus cells and biofilm formation. The findings provided valuable information for practical application of H2O2 for decontamination of C. malonaticus in dairy processing.

Two Cases of Multi-antibiotic Resistant Cronobacter spp. Infections of Infants in China.

Infections by Cronobacter spp. are hazardous to infants since they can lead to neonatal meningitis, bacteremia, and necrotizing enterocolitis. Cronobacter spp. are frequently resistant to ß-lactam derivatives, macrolides, and aminoglycosides. In addition, multi-resistant strains have also been detected. In China, the isolation rate of Cronobacter spp. from commercial powdered infant formula (PIF) or follow-up formula (FUF) is relatively high. Nevertheless, clinical cases of Cronobacter infection have been ignored to date. Here we describe two cases of Cronobacter infection detected at the Wuhan Women and Children Medical Care Center Hospital (Wuhan City, China). We provide the genomic analysis of the isolates and the antibiotic-resistance profiles of the two strains. The Cronobacter strains identified in this study were not susceptible to third-generation cephalosporins, aminoglycoside, and/or trimethoprim-sulfamethoxazole. Whole genome sequencing revealed various genes known to encode antibiotic resistance. Future studies are needed to determine whether the genes predicted in this study are functional. As with Enterobacter spp., the antibiotic resistance of Cronobacter is a serious issue that requires more attention.

The Effects of Temperature on the Growth and Heat Resistance of Cronobacter spp.

　The growth and survival of Cronobacter isolates were examined under incubation at different temperatures, and their thermal death behavior was investigated at high temperature conditions of above 50℃. Seventy three strains isolated from fresh vegetables, dried foods and soil were tested: 28 of Cronobacter sakazakii, 5 of C. dublienensis, 27 of C. malonaticus and 13 of C. turicensis. All Cronobacter strains grew and multiplied predominantly at 35 and 44℃ until 16 hours of incubation, but showed poor growth at 15℃, and no growth at 5℃. At 48℃, the bacteria grew slightly during 6 to 8 h-incubation but decreased or were inactivated after 16 h-incubation. The heat resistance of Cronobacter spp. was measured under the conditions of 50, 55, 60, 65 and 70℃. Cronobacter strains survived almost without decrement for 30 min at 50℃, but decreased suddenly and perished completely within 10 to 20 min at 55℃ and within 2 - 5 min at above 60℃. Some food materials should be stored below 5℃ until the preparation, and dried food including powdered infant milk formula should be utilized immediately after reconstitution and preparation.

Microbiological quality of selected ready-to-eat leaf vegetables, sprouts and non-pasteurized fresh fruit-vegetable juices including the presence of Cronobacter spp.

Bacteria of the genus Cronobacter are emerging food-borne pathogens. Foods contaminated with Cronobacter spp. may pose a risk to infants or adults with suppressed immunity. This study was aimed at determining the microbiological quality of ready-to-eat (RTE) plant-origin food products available on the Polish market with special emphasis on the prevalence of Cronobacter genus bacteria. Analyses were carried out on 60 samples of commercial RTE type plant-origin food products, including: leaf vegetables (20 samples), sprouts (20 samples) and non-pasteurized vegetable, fruit and fruit-vegetable juices (20 samples). All samples were determined for the total count of aerobic mesophilic bacteria (TAMB) and for the presence of Cronobacter spp. The isolates of Cronobacter spp. were subjected to genetic identification and differentiation by 16S rDNA sequencing, PCR-RFLP analysis and RAPD-PCR and evaluation of antibiotic susceptibility by the disk diffusion assay. The TAMB count in samples of lettuces, sprouts and non-pasteurized fruit, vegetable and fruit-vegetable juices was in the range of 5.6-7.6, 6.7-8.4 and 2.9-7.7 log CFU g-1, respectively. The presence of Cronobacter spp. was detected in 21 (35%) samples of the products, including in 6 (30%) samples of leaf vegetables (rucola, lamb's lettuce, endive escarola and leaf vegetables mix) and in 15 (75%) samples of sprouts (alfalfa, broccoli, small radish, lentil, sunflower, leek and sprout mix). No presence of Cronobacter spp. was detected in the analyzed samples of non-pasteurized fruit, vegetable and fruit-vegetable juices. The 21 strains of Cronobacter spp. isolated from leaf vegetable and sprouts included: 13 strains of C. sakazakii, 4 strains of C. muytjensii, 2 strains of C. turicensis, one strain of C. malonaticus and one strain of C. condimenti. All isolated C. sakazakii, C. muytjensii, C. turicensis and C. malonaticus strains were sensitive to ampicillin, cefepime, chloramphenicol, gentamycin, streptomycin, tetracycline, ciprofloxacin and cotrimoxazol, whereas the C. condimenti isolate showed intermediate resistance to streptomycin and cotrimoxazole.

Isolation, molecular and phenotypic characterization, and antibiotic susceptibility of Cronobacter spp. from Brazilian retail foods.

Several Cronobacter species are opportunistic pathogens that cause infections in humans. The aim of this study was to detect Cronobacter spp. from 90 samples of retail foods in Brazil, and characterize the strains by phenotypic tests, molecular assays and antibiotic susceptibility. Three isolation methodologies were evaluated using different selective enrichments and the isolates were identified using Vitek 2.0, PCRs protocols, fusA allele sequencing and multilocus sequence typing (MLST). Thirty-eight samples (42.2%) contained Cronobacter spp., and the highest percentage was found in flours (66.7%, 20/30), followed by spices and herbs (36.7%, 11/30), and cereal mixes for children (23.3%, 7/30). The 45 isolates included four species: C. sakazakii (n = 37), C. malonaticus (n = 3), C. dublinensis (n = 3), and C. muytjensii (n = 2); that presented 20 different fusA alleles. MLST analysis revealed 32 sequence types (STs), 13 of which were newly identified. All strains were sensitive to all antibiotics (n = 10) tested. The combination of CSB/v enrichment with DFI plating was considered the most efficient for Cronobacter spp. isolation. This study revealed the presence of Cronobacter spp. in foods commercialized in Brazil and the isolates showed a high diversity after MLST analysis and included two strains of the C. sakazakii ST4 neonatal meningitic pathovar.

Surveillance and molecular typing of Cronobacter spp. in commercial powdered infant formula and follow-up formula from 2011 to 2013 in Shandong Province, China.

BACKGROUND: Infection with Cronobacter spp. leads to neonatal meningitis, necrotizing enterocolitis and bacteremia. Cronobacter spp. are reported to comprise an important pathogen contaminating powdered infant formula (PIF) and follow-up formula (FUF), although little is known about the contamination level of Cronobacter spp. in PIFs and FUFs in China. RESULTS: In total, 1032 samples were collected between 2011 and 2013. Forty-two samples were positive, including 1.6% in PIFs and 6.5% in FUFs. The strains were susceptible to most antibiotics except for cefoxitin. Pulsed-field gel electrophoresis after XbaI digestion produced a total of 36 banding patterns. The 38 strains were found in 27 sequence types (STs), of which nine types (ST454 to ST462) had not been reported in other countries. The clinically relevant strains obtained from the 38 isolates in the present study comprised three ST3, two ST4, two ST8 and one ST1. CONCLUSION: The contamination rate in the PIF and FUF has stayed at a relatively high level. The contamination rate of PIF was significantly lower than FUF. The isolates had high susceptibility to the antibiotics tested, except cefoxitin. There were polymorphisms between the Cronobacter spp. as indicated by pulsed-field gel electrophoresis and multilocus sequence typing. Therefore, contamination with Cronobacter spp. remains a current issue for commercial infant formulas in China. © 2016 Society of Chemical Industry.

Cronobacter spp.

The Cronobacter group of pathogens, associated with severe and potentially life-threatening diseases, until recently were classified as a single species, Enterobacter sakazakii. The group was reclassified in 2007 into the genus Cronobacter as a member of the Enterobacteriaceae. This chapter outlines the history behind the epidemiology, analyzes how our understanding of these bacteria has evolved, and highlights the clinical significance the Cronobacter spp. have for neonatal and elderly patient populations and treatment of the associated infections.

Occurrence and Characterization of Cronobacter spp. in Dehydrated Rice Powder from Chinese Supermarket.

Cronobacter spp. are emerging food-borne pathogens and have been identified as causative agents of meningitis and necrotizing enterocolitis in infants. Dehydrated rice is popular with a wide range of people and it is frequently used as a substitute for infant milk powder to baby older than four months. The occurrence of Cronobacter spp. was investigated in 1,012 samples of dehydrated rice powder collected from 14 manufacturers in China during 2010 to 2012. The isolates were identified using fusA allele sequencing and subtyped using pulsed-field gel electrophoresis. Seventy-six samples (7.5%) contained Cronobacter spp. The prevalence among manufacturers ranged from 0-28.8%. The 76 isolates included 4 species [Cronobacter sakazakii (52 isolates) Cronobacter malonaticus (14 isolates), Cronobacter dublinensis (7 isolates), and Cronobacter muytjensii (3 isolates)]. Twenty-three unique fusA alleles and sixty-six PFGE-patterns were detected. All isolated strains were observed to be sensitive or to show intermediate susceptibility to eight tested antimicrobial agents. The study revealed serious contamination of dehydrated rice powder by Cronobacter spp., with prevalence varying among manufacturers in China. Identified Cronobacter species, fusA alleles, and subtypes were diverse.

Evaluation of zebrafish as a model to study the pathogenesis of the opportunistic pathogen Cronobacter turicensis.

Bacteria belonging to the genus Cronobacter spp. have been recognized as causative agents of life-threatening systemic infections, primarily in premature, low-birth weight and/or immune-compromised neonates. Knowledge remains scarce regarding the underlying molecular mechanisms of disease development. In this study, we evaluated the use of a zebrafish model to study the pathogenesis of Cronobacter turicensis LMG 23827(T), a clinical isolate responsible for two fatal sepsis cases in neonates. Here, the microinjection of approximately 50 colony forming units (CFUs) into the yolk sac resulted in the rapid multiplication of bacteria and dissemination into the blood stream at 24 h post infection (hpi), followed by the development of a severe bacteremia and larval death within 3 days. In contrast, the innate immune response of the embryos was sufficiently developed to control infection after the intravenous injection of up to 10(4) CFUs of bacteria. Infection studies using an isogenic mutant devoid of surviving and replicating in human macrophages (ΔfkpA) showed that this strain was highly attenuated in its ability to kill the larvae. In addition, the suitability of the zebrafish model system to study the effectiveness of antibiotics to treat Cronobacter infections in zebrafish embryos was examined. Our data indicate that the zebrafish model represents an excellent vertebrate model to study virulence-related aspects of this opportunistic pathogen in vivo.

Prevalence and Characterization of Cronobacter spp. from Various Foods, Medicinal Plants, and Environmental Samples.

Dairy or non-dairy based products were explored to determine the prevalence, molecular characterization, and antibiotic susceptibility of Cronobacter spp. The isolation was done as per ISO 22964:2006 on chromogenic media followed by further confirmation by biochemical- and 16S rRNA-based identification. From 219 samples, the chromogenic agar assay and biochemical tests yielded presumptive 45 isolates. Among them, only 36 isolates showed 282 bp band amplified from ITS-G gene confirming as Cronobacter sakazakii. The Cronobacter spp. prevalence was highest in herbs and spices (34 %) while environmental samples had contamination rates of 23 % indicating plants as a possible reservoir of this pathogen. All the isolates were resistant to ß-lactam derivatives (68 %), macrolides (88.6 %), and aminoglycosides (79.9 %) but susceptible to phenicoles (31.6 %) and tetracyclines (15 %) derivatives. The results emphasize the screening of plant materials before their incorporation in food matrices.

A Cronobacter turicensis O1 antigen-specific monoclonal antibody inhibits bacterial motility and entry into epithelial cells.

Cronobacter turicensis is an opportunistic foodborne pathogen that can cause a rare but sometimes lethal infection in neonates. Little is known about the virulence mechanisms and intracellular lifestyle of this pathogen. In this study, we developed an IgG monoclonal antibody (MAb; MAb 2G4) that specifically recognizes the O1 antigen of C. turicensis cells. The antilipopolysaccharide antibody bound predominantly monovalently to the O antigen and reduced bacterial growth without causing cell agglutination. Furthermore, binding of the antibody to the O1 antigen of C. turicensis cells caused a significant reduction of the membrane potential which is required to energize flagellar rotation, accompanied by a decreased flagellum-based motility. These results indicate that binding of IgG to the O antigen of C. turicensis causes a direct antimicrobial effect. In addition, this feature of the antibody enabled new insight into the pathogenicity of C. turicensis. In a tissue culture infection model, pretreatment of C. turicensis with MAb 2G4 showed no difference in adhesion to human epithelial cells, whereas invasion of bacteria into Caco-2 cells was significantly inhibited.

Antibacterial activities of plant-derived compounds and essential oils toward Cronobacter sakazakii and Cronobacter malonaticus.

Cronobacter sakazakii and C. malonaticus are opportunistic pathogens that cause infections in children and immunocompromised adults. In the present study, the antibacterial activity of 19 plant-derived compounds, 5 essential oils, and an extract of propolis were assessed against C. sakazakii and C. malonaticus. The effects of most of these antimicrobials have not been reported previously. Both strains were susceptible to thymol, carvacrol, thymoquinone, p-cymene, linalool, camphor, citral, eugenol, and trans-cinnamaldehyde as well as cinnamon, lemongrass, oregano, clove, and laurel essential oils; their minimum inhibitory concentrations varied between 0.1 and 2.0 mg/mL. As an alternative treatment method, vapors of the volatiles were tested as an indirect treatment. Vapors of trans-cinnamaldehyde, eugenol, oregano, and cinnamon essential oils inhibited both tested strains, while vapors of linalool were only active against C. sakazakii. To our knowledge, this study is the first time that the inhibitory activity of the vapors of these compounds and essential oils has been reported against Cronobacter spp.