Biofilm formation on enteral feeding tubes by Cronobacter sakazakii, Salmonella serovars and other Enterobacteriaceae

WHO (2007) recommended that to reduce microbial risks, powdered infant formula should be reconstituted with water at temperatures >70 °C, and that such feeds should be used within 2 h of preparation. However, this recommendation does not consider the use of enteral feeding tubes which can be in place for more than 48 h and can be loci for bacterial attachment. This study determined the extent to which 29 strains of Cronobacter sakazakii, Salmonella serovars, other Enterobacteriaceae and Acinetobacter spp. can adhere and grow on enteral feeding tubes composed of polyvinyl chloride and polyurethane. The study also included silver-impregnated tubing which was expected to have antibacterial activity. Bacterial biofilm formation by members of the Enterobacteriaceae was ca. 105-106 cfu/cm after 24 h. Negligible biofilm was detected for Acinetobacter gensp. 13; ca. 10 cfu/cm, whereas Cr. sakazakii strain ATCC 12868 had the highest biofilm cell density of 107 cfu/cm. Biofilm formation did not correlate with capsule production, and was not inhibited on silver-impregnated tubing. Bacteria grew in the tube lumen to cell densities of 107 cfu/ml within 8 h, and 109 cfu/ml within 24 h. It is plausible that in vivo the biofilm will both inoculate subsequent routine feeds and as the biofilm ages, clumps of cells will be shed which may survive passage through the neonate's stomach. Therefore biofilm formation on enteral feeding tubes constitutes a risk factor for susceptible neonates

Multilocus sequence typing of Cronobacter sakazakii and Cronobacter malonaticus reveals stable clonal structures with clinical significance which do not correlate with biotypes

Background: The Cronobacter genus (Enterobacter sakazakii) has come to prominence due to its association with infant infections, and the ingestion of contaminated reconstituted infant formula. C. sakazakii and C. malonaticus are closely related, and are defined according their biotype. Due to the ubiquitous nature of the organism, and the high severity of infection for the immunocompromised, a multilocus sequence typing (MLST) scheme has been developed for the fast and reliable identification and discrimination of C. sakazakii and C. malonaticus strains. It was applied to 60 strains of C. sakazakii and 16 strains of C. malonaticus, including the index strains used to define the biotypes. The strains were from clinical and non-clinical sources between 1951 and 2008 in USA, Canada, Europe, New Zealand and the Far East. Results: This scheme uses 7 loci; atpD, fusA, glnS, gltB, gyrB, infB, and pps. There were 12 sequence types (ST) identified in C. sakazakii, and 3 in C. malonaticus. A third (22/60) of C. sakazakii strains were in ST4, which had almost equal numbers of clinical and infant formula isolates from 1951 to 2008. ST8 may represent a particularly virulent grouping of C. sakazakii as 7/8 strains were clinical in origin which had been isolated between 1977 - 2006, from four countries. C. malonaticus divided into three STs. The previous Cronobacter biotyping scheme did not clearly correspond with STs nor with species. Conclusion: In conclusion, MLST is a more robust means of identifying and discriminating between C. sakazakii and C. malonaticus than biotyping. The MLST database for these organisms is available online at http://pubmlst.org/cronobacter

Detergent and sanitizer stresses decrease the thermal resistance of Enterobacter sakazakii in infant milk formula

Infant milk formula has been identified as a potential source of Enterobacter sakazakii. This bacterium can cause a severe form of neonatal meningitis and necrotizing entercolitis. This study determined the effect of acid, alkaline, chlorine and ethanol stresses on the thermal inactivation of E. sakazakii in infant milk formula. Stressed cells were mixed with reconstituted powdered infant milk formula (PIMF) at temperatures between 52 and 58°C for various time periods or mixed with PFMF prior to reconstitution with water at temperatures between 50 and 100°C. The D- and z-values of the cells were determined using linear regression analysis. Detergent and sanitizer stresses decreased the thermal resistance of E. sakazakii in powdered and reconstituted infant milk formula. The values for Z)- acid, alkaline, chlorine and ethanol stressed E. sakazakii at 52-58°C were 14.57-0.54, 12.07-0.37, 10.08-0.40 and 11.61-0.50 min, respectively. The values of alkaline, chlorine and ethanol stressed cells were significantly lower than those of unstressed cells. Only the z-value (4.4°C) of ethanol stressed E. sakazakii was significantly different than that of unstressed cells (4.12°C). Reconstitution at 60°C did not significantly reduce the number of pre-stressed E. sakazakii cells compared with unstressed control cells, whereas significant decreases were obtained at 70°C. Using water at 70°C during the preparation of reconstituted PIMF before feeding infants, may be a suitable and applicable means of reducing the risk of E. sakazakii in the formula. The results of this study may be of use to regulatory agencies, infant milk producers and infant caregivers to design heating processes to eliminate E. sakazakii that may be present in infant milk formula

Comparison of methods for the microbiological identification and profiling of cronobacter species from ingredients used in the preparation of infant formula

Cronobacter spp. (formerly Enterobacter sakazakii) can be isolated from a wide range of foods and environments, and its association with neonatal infections has drawn considerable attention from regulatory authorities. The principle route of neonatal infection has been identified as the ingestion of contaminated infant formula. A number of methods have been developed to identify Cronobacter spp., however these were before the most recent (2012 ) taxonomic revision of the genus into seven species. In this study, phenotyping, protein profiling and molecular methods were used to identify Cronobacter strains which had been recently isolated from ingredients used in the preparation of infant formula. Pulsed field gel electrophoresis revealed that different Cronobacter strains had been recovered from the same food products. All isolates were identified as C sakazakii according to four genus specific PCR-probes and protein profiling using MALDI-TOF analysis. However, 16S rDNA sequence analyses and fusA allele sequencing gave more accurate identification: four strains were C sakazakii, one strain was C malonaticus and the remaining strain was C universalis. Multilocus sequence typing showed the strains were different sequence types. These results demonstrate the presence of different Cronobacter species in food ingredients used in the preparation of infant formula, and also the need for molecular identification and profiling methods to be revised according to taxonomic revisions

Capsular profiling of the Cronobacter genus and the association of specific Cronobacter sakazakii and C. malonaticus capsule types with neonatal meningitis and necrotizing enterocolitis

Background: Cronobacter sakazakii and C. malonaticus can cause serious diseases especially in infants where they are associated with rare but fatal neonatal infections such as meningitis and necrotising enterocolitis. Methods: This study used 104 whole genome sequenced strains, covering all seven species in the genus, to analyse capsule associated clusters of genes involved in the biosynthesis of the O-antigen, colanic acid, bacterial cellulose, enterobacterial common antigen (ECA), and a previously uncharacterised K-antigen. Results: Phylogeny of the gnd and galF genes flanking the O-antigen region enabled the defining of 38 subgroups which are potential serotypes. Two variants of the colanic acid synthesis gene cluster (CA1 and CA2) were found which differed with the absence of galE in CA2. Cellulose (bcs genes) were present in all species, but were absent in C. sakazakii sequence type (ST) 13 and clonal complex (CC) 100 strains. The ECA locus was found in all strains. The K-antigen capsular polysaccharide Region 1 (kpsEDCS) and Region 3 (kpsMT) genes were found in all Cronobacter strains. The highly variable Region 2 genes were assigned to 2 homology groups (K1 and K2). C. sakazakii and C. malonaticus isolates with capsular type [K2:CA2:Cell+] were associated with neonatal meningitis and necrotizing enterocolitis. Other capsular types were less associated with clinical infections. Conclusion: This study proposes a new capsular typing scheme which identifies a possible important virulence trait associated with severe neonatal infections. The various capsular polysaccharide structures warrant further investigation as they could be relevant to macrophage survival, desiccation resistance, environmental survival, and biofilm formation in the hospital environment, including neonatal enteral feeding tubes

The speciation and genotyping of Cronobacter isolates from hospitalised patients

The World Health Organization (WHO) has recognised all Cronobacter species as human pathogens. Among premature neonates and immunocompromised infants, these infections can be life-threatening, with clinical presentations of septicaemia, meningitis and necrotising enterocolitis. The neurological sequelae can be permanent and the mortality rate as high as 40 – 80 %. Despite the highlighted issues of neonatal infections, the majority of Cronobacter infections are in the elderly population suffering from serious underlying disease or malignancy and include wound and urinary tract infections, osteomyelitis, bacteraemia and septicaemia. However, no age profiling studies have speciated or genotyped the Cronobacter isolates. A clinical collection of 51 Cronobacter strains from two hospitals were speciated and genotyped using 7-loci multilocus sequence typing (MLST), rpoB gene sequence analysis, O-antigen typing and pulsed- field gel electrophoresis (PFGE). The isolates were predominated by C. sakazakii sequence type 4 (63 %, 32/51) and C. malonaticus sequence type 7 (33 %, 17/51). These had been isolated from throat and sputum samples of all age groups, as well as recal and faecal swabs. There was no apparent relatedness between the age of the patient and the Cronobacter species isolated. Despite the high clonality of Cronobacter , PFGE profiles differentiated strains across the sequence types into 15 pulsotypes. There was almost complete agreement between O-antigen typing and rpoB gene sequence analysis and MLST profiling. This study shows the value of applying MLST to bacterial population studies with strains from two patient cohorts, combined with PFGE for further discrimination of strains

Cronobacter sakazakii clinical isolates overcome host barriers and evade the immune response

Cronobacter sakazakii is the most frequently clinically isolated species of the Cronobacter genus. However the virulence factors of C. sakazakii including their ability to overcome host barriers remains poorly studied. In this study, ten clinical isolates of C. sakazakii were assessed for their ability to invade and translocate through human microvascular endothelial cells (HBMEC). Their ability to avoid phagocytosis in human macrophages U937 and human brain microglial cells was investigated. Additionally, they were tested for serum sensitivity and the presence of the Cronobacter plasminogen activation gene (cpa) gene, which is reported to confer serum resistance. Our data showed that the clinical C. sakazakii strains invaded and translocated through Caco-2 and HBMEC cell lines and some strains showed significantly higher levels of invasion and translocation. Moreover, C. sakazakii was able to persist and even multiply in phagocytic macrophage and microglial cells. All strains, except one, were able to withstand human serum exposure, the single serum sensitive strain was also the only one which did not encode for the cpa gene. These results demonstrate that C. sakazakii clinical host immune response indicating their capacity to cause diseases such as necrotizing enterocolitis (NEC) and meningitis. Our data showed for the first time the ability of C. sakazakii clinical isolates to survive and multiply within human microglial cells. Additionally, it was shown that C. sakazakii clinical strains have the capacity to translocate through the Caco-2 and HBMEC cell lines paracellularly