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.

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

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

Analysis of the cellulose synthase operon genes, bcsA, bcsB, and bcsC in Cronobacter species: Prevalence among species and their roles in biofilm formation and cell-cell aggregation.

Cronobacter species are emerging food-borne pathogens that cause severe sepsis, meningitis, and necrotizing entercolitis in neonates and infants. Bacterial pathogens such as Escherichia coli and Salmonella species produce extracellular cellulose which has been shown to be involved in rugosity, biofilm formation, and host colonization. In this study the distribution and prevalence of cellulose synthase operon genes (bcsABZC) were determined by polymerase chain reaction (PCR) analysis in 231 Cronobacter strains isolated from clinical, food, environmental, and unknown sources. Furthermore, bcsA and bcsB isogenic mutants were constructed in Cronobacter sakazakii BAA894 to determine their roles. In calcofluor binding assays bcsA and bcsB mutants did not produce cellulose, and their colonial morphotypes were different to that of the parent strain. Biofilm formation and bacterial cell-cell aggregation were significantly reduced in bcsA and bcsB mutants compared to the parental strain. bcsA or bcsAB PCR-negative strains of C. sakazakii did not bind calcofluor, and produced less biofilm and cell-cell aggregation compared to strains possessing bcsAB genes. These data indicated that Cronobacter bcsABZC were present in all clinical isolates and most of food and environmental isolates. bcsA and bcsB genes of Cronobacter were necessary to produce cellulose, and were involved in biofilm formation and cell-cell aggregation.

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

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

Presence of AmpC beta-lactamases, CSA-1, CSA-2, CMA-1, and CMA-2 conferring an unusual resistance phenotype in Cronobacter sakazakii and Cronobacter malonaticus.

Here we describe the presence of two very similar but unusual variants of AmpC cephalosporinase in each Cronobacter sakazakii and C. malonaticus isolates conferring resistance exclusively to first generation cephalosporins. During a survey on the antibiotic resistance patterns of C. sakazakii and C. malonaticus strains isolated from a milk powder production facility, originally two different phenotypes regarding the susceptibility/resistance for the two beta-lactam antibiotics ampicillin (amp) and cephalothin (ceph) were observed: (i) isolates being susceptible for both antibiotics (amp(S)/ceph(S)), and (ii) strains exhibiting susceptibility to ampicillin but resistance to cephalothin (amp(S)/ceph(R)). The latter phenotype (amp(S)/ceph(R)) was observed in the majority of the environmental strains from the facility. Analysis of whole genome sequences of C. sakazakii revealed a gene putatively coding for an AmpC beta-lactamase. Consequently, the ampC genes from both species and both phenotypes were subjected to a cloning approach. Surprisingly, when expressed in Escherichia coli, all transformants exhibited the amp(S)/ceph(R) phenotype regardless of (i) the phenotypic backgrounds or (ii) the AmpC amino acid sequences of the original strains from which the clones were derived. The novel AmpC beta-lactamases were designated CSA-1 and CSA-2 (from C. sakazakii) and CMA-1 and CMA-2 (from C. malonaticus). The observed variations in the minimum inhibitory concentration (MIC) levels for cephalothin (wt compared to transformants) suggest that this feature is a target of a yet unknown regulatory mechanism present in the natural Cronobacter background but absent in the neutral E. coli host.

Multiplex PCR assay targeting a diguanylate cyclase-encoding gene, cgcA, to differentiate species within the genus Cronobacter.

In a comparison to the widely used Cronobacter rpoB PCR assay, a highly specific multiplexed PCR assay based on cgcA, a diguanylate cyclase gene, that identified all of the targeted six species among 305 Cronobacter isolates was designed. This assay will be a valuable tool for identifying suspected Cronobacter isolates from food-borne investigations.

Use of rpoB gene sequence analysis for phylogenetic identification of Cronobacter species.

Here, we report the RNA polymerase beta-subunit gene (rpoB) as a new molecular marker for the identification of the Cronobacter species. The results indicated that members of the Cronobacter genus are more easily discriminated by rpoB sequencing than 16S rRNA sequencing, and reliable identification could be achieved by rpoB gene sequence comparison.