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. 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. 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. 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.

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

The effectiveness of 13 disinfectants used in hospitals, day-care centers, and food service kitchens in killing Enterobacter sakazakii in suspension, dried on the surface of stainless steel, and in biofilm was determined. E. sakazakii exhibited various levels of resistance to the disinfectants, depending on the composition of the disinfectants, amount and type of organic matrix surrounding cells, and exposure time. Populations of planktonic cells suspended in water (7.22 to 7.40 log CFU/ml) decreased to undetectable levels ( cells inoculated and dried on stainless steel > cells in biofilms on stainless steel. Findings show that disinfectants routinely used in hospital, day-care, and food service kitchen settings are ineffective in killing some cells of E. sakazakii embedded in organic matrices.

Effectiveness of disinfectants in killing Enterobacter sakazakii in suspension, dried on the surface of stainless steel, and in a biofilm.

The genus Cronobacter consists of a diverse group of Gram-negative bacilli and comprises seven species: Cronobacter sakazakii, Cronobacter malonaticus, Cronobacter muytjensii, Cronobacter turicensis, Cronobacter dublinensis, Cronobacter universalis and Cronobacter condimenti. Cronobacter are regarded as opportunistic pathogens, and have been implicated in newborn and infant infections, causing meningitis, necrotizing enterocolitis and bacteraemia or sepsis. Cronobacter virulence is believed to be due to multiple factors. Some strains were found to produce diarrhoea or cause significant fluid accumulation in suckling mice. Two iron acquisition systems (eitCBAD and iucABCD/iutA), Cronobacter plasminogen activator gene (cpa), a 17 kb type VI secretion system (T6SS), and a 27 kb filamentous haemagglutinin gene (fhaBC) and associated putative adhesins locus are harboured on a family of RepFIB-related plasmids (pESA3 and pCTU1), suggesting that these are common virulence plasmids; 98% of 229 tested Cronobacter strains possessed these plasmids. Even though pESA3 and pCTU1 share a common backbone composed of the repA gene and eitCBAD and iucABCD/iutA gene clusters, the presence of cpa, T6SS and FHA loci depended on species, demonstrating a strong correlation with the presence of virulence traits, plasmid type and species. Other factors were observed, in that Cronobacter form biofilms, and show unusual resistance to heat, dry and acid stress growth conditions. The outer-membrane protein A is probably one of the best-characterized virulence markers of Cronobacter. Furthermore, it was reported that Cronobacter employ phosphatidylinositide 3-kinase/Akt signalling, which activates protein kinase C-α and impairs the host cell's mitogen-activated protein kinase pathway, in order to invade cells. Cronobacter can also use immature dendritic cells and macrophages to escape the immune response. This review addresses the various virulence and environmental-adaptive characteristics possessed by members of the genus Cronobacter.

Cronobacter spp.--opportunistic food-borne pathogens. A review of their virulence and environmental-adaptive traits.

Enterobacter sakazakii is an opportunistic pathogen and an occasional contaminant in powdered infant formula. Interaction between specific probiotics and E. sakazakii may reduce the risk of infection. The aim of this study was to characterize in vitro the ability of probiotics (alone and in combinations) to inhibit, compete with and displace the adhesion of E. sakazakii to immobilized human mucus and to assess their capacity to aggregate with pathogen. Specific probiotic strains have proved to aggregate E. sakazakii cells and, through competitive exclusion, inhibition and displacement of the adhered pathogen, were able to inhibit E. sakazakii action on intestinal mucus. The ability to inhibit and to displace adhered pathogen depended on both the probiotic and the pathogen, suggesting that several complementary mechanisms are involved in the processes. We suggest that the selection of specific probiotic strains and their combinations may be a useful means of counteracting E. sakazakii contamination in infant formula and thus to reduce the risk of emerging infection. This approach may also allow the development of new probiotic combinations to counteract the risks associated with other pathogens by improving the intestinal barrier against pathogens.

Specific probiotic strains and their combinations counteract adhesion of Enterobacter sakazakii to intestinal mucus

Enterobacter sakazakii and Salmonella in powdered infant formula

The association of powdered infant formula with cases of severe Enterobacter sakazakii infections in immunocompromised and premature neonates has led to a need to learn about the basic behavior of this emerging pathogen in food systems and the environment. The current study examines the microorganism's stationary-phase acid resistance using 12 strains that had been previously used to characterize its thermal resistance. Acid resistance was determined by initially culturing the isolates for 18 h in brain heart infusion broth (BHI) at 36 °C, transferring the cells to tryptic soy broth (TSB) adjusted to pH 3.0 and 3.5, and determining E. sakazakii survival over the course of 5 h incubation at 36 °C. At pH 3.5, 10 of the 12 strains showed less than a 1 log cycle decline over the 5-h incubation period, with the most acid sensitive strain showing an approximate 3.5 log cycle decline. At pH 3.0, the decline over the 5-h incubation period ranged from 4.9 to >6.3 log cycles; however, substantial diversity was evident when the 1-h/pH 3.0 results were compared. The effect of habituating the cells to a moderately acidic environment was determined by growing the strains in TSB with 0% (nonacidogenic) and 1% glucose (acidogenic), transferring the cells to acidified (pH 3.0) BHI, and determining E. sakazakii survival over the course of 5 h of incubation at 36 °C. While there was diversity observed among the strains, in general the stationary-phase acid resistances of several of the strains were enhanced, at least transitorily, by growth in the acidogenic medium. No apparent correlation between the stationary-phase relative acid resistances of the strains based on the 1-h/pH 3.0 acid inactivation values and the previously reported thermal D-values was observed.

M.K. Porteous

Papers

Acid Resistance of Twelve Strains of Enterobacter sakazakii, and the Impact of Habituating the Cells to an Acidic Environment