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

Neonatal enteral feeding tubes as loci for colonisation by members of the Enterobacteriaceae

Background The objective of this study was to determine whether neonatal nasogastric enteral feeding tubes are colonised by the opportunistic pathogen Cronobacter spp. (Enterobacter sakazakii) and other Enterobacteriaceae, and whether their presence was influenced by the feeding regime. Methods One hundred and twenty-nine tubes were collected from two neonatal intensive care units (NICU). A questionnaire on feeding regime was completed with each sample. Enterobacteriaceae present in the tubes were identified using conventional and molecular methods, and their antibiograms determined. Results The neonates were fed breast milk (16%), fortified breast milk (28%), ready to feed formula (20%), reconstituted powdered infant formula (PIF, 6%), or a mixture of these (21%). Eight percent of tubes were received from neonates who were 'nil by mouth'. Organisms were isolated from 76% of enteral feeding tubes as a biofilm (up to 107 cfu/tube from neonates fed fortified breast milk and reconstituted PIF) and in the residual lumen liquid (up to 107 Enterobacteriaceae cfu/ml, average volume 250 µl). The most common isolates were Enterobacter cancerogenus (41%), Serratia marcescens (36%), E. hormaechei (33%), Escherichia coli (29%), Klebsiella pneumoniae (25%), Raoultella terrigena (10%), and S. liquefaciens (12%). Other organisms isolated included C. sakazakii (2%),Yersinia enterocolitica (1%),Citrobacter freundii (1%), E. vulneris (1%), Pseudomonas fluorescens (1%), and P. luteola (1%). The enteral feeding tubes were in place between 48 h (13%). All the S. marcescens isolates from the enteral feeding tubes were resistant to amoxicillin and co-amoxiclav. Of additional importance was that a quarter of E. hormaechei isolates were resistant to the 3rd generation cephalosporins ceftazidime and cefotaxime. During the period of the study, K. pneumoniae and S. marcescens caused infections in the two NICUs. Conclusion This study shows that neonatal enteral feeding tubes, irrespective of feeding regime, act as loci for the bacterial attachment and multiplication of numerous opportunistic pathogens within the Enterobacteriaceae family. Subsequently, these organisms will enter the stomach as a bolus with each feed. Therefore, enteral feeding tubes are an important risk factor to consider with respect to neonatal infections

Survival and growth of Cronobacter species (Enterobacter) sakazakii in wheat-based infant follow on formulas

Aim: To determine the survival and growth characteristics of Cronobacter species (Enterobacter sakazakii) in infant wheat-based formulas reconstituted with water, milk, grape juice or apple juice during storage. Methods and Results: Infant wheat-based formulas were reconstituted with water, UHT milk, pasteurized grape or apple juices. The reconstituted formulas were inoculated with C. sakazakii and C. muytjensii and stored at 4, 25 or 37°C for up to 24h. At 25 and 37°C, Cronobacter grew more (>5 logio) in formulas reconstituted with water or milk than those prepared with grape or apple juices (ca. 2-3 logio). The organism persisted, but did not grow in any formulas stored at 4°C. Formulas reconstituted with water and milk decreased from pH 6.0 to 4.8-5.0 after 24h, whereas the pH of the formulas reconstituted with fruit juices remained at their initial pH values, ca. pH 4.8-5.0. Conclusion: C sakazakii and C muytjensii can grow in reconstituted wheat-based formulas. If not immediately consumed, these formulas should be stored at refrigeration temperatures to reduce the risk of infant infection. Significance and Impact of the Study: The results of this study will be of use to regulatory agencies and infant formula producers to recommend storage conditions that reduce the growth of Cronobacter in infant wheat-based formulas

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 Cronobacter genus: ubiquity and diversity

Members of the Cronobacter genus (formerly Enterobacter sakazakii) have become associated with neonatal infections and in particular contaminated reconstituted infant formula. However this is only one perspective of the organism since the majority of infections are in the adult population, and the organism has been isolated from the enteral feeding tubes of neonates on non-formula diets. In recent years methods of detection from food and environmental sources have improved, though accurate identification has been problematic. The need for robust identification is essential in order to implement recent Codex Alimentarius Commission (2008) and related microbiological criteria for powdered infant formula (PIF; intended target age 0-6 months). Genomic analysis of emergent pathogens is of considerable advantage in both improving detection methods, and understanding the evolution of virulence. One ecosystem for Cronobacter is on plant material which may enable the organism to resist desiccation, adhere to surfaces, and resist some antimicrobial agents. These traits may also confer survival mechanisms of relevance in food manufacturing and also virulence mechanisms

Cronobacter spp. as emerging causes of healthcare-associated infection

Background: Until recently, members of the Cronobacter genus (formerly known as Enterobacter sakazakii) were a relatively unknown cause of nosocomial infections. However, their association with infant infections, particularly through the consumption of contaminated reconstituted infant formula in neonatal intensive care units, has resulted in international efforts to improve neonatal health care. Aim: To investigate current understanding of this emergent group of bacterial pathogens and the steps taken to reduce neonatal infection. Methods: A literature review was undertaken to determine current knowledge of the Cronobacter genus with respect to recent taxonomic revisions, sources and clinical relevance. Findings: The majority of severe neonatal meningitis infections are associated with one of the 10 Cronobacter spp., the clonal complex known as C. sakazakii sequence type 4. International efforts by the Food and Agriculture Organization–World Health Organization (WHO) to reduce the risk of neonatal infection by this organism have resulted in improved microbiological safety of powdered infant formula (PIF), but revised guidelines for feeding practices have been problematic. In addition, the majority of infections occur in the adult population and the sources are unknown. Conclusion: International improvements in the microbiological safety of PIF and advice on feeding practices have focused on improving neonatal health care following the heightened awareness of Cronobacter infections in this particular age group. These measures are also likely to reduce neonatal exposure to other opportunistic bacterial pathogens, but a number of unresolved issues remain with respect to the practicalities of feeding premature neonates safely while following WHO advice