Genotypic and Phenotypic Analysis of Enterobacter sakazakii Strains from an Outbreak Resulting in Fatalities in a Neonatal Intensive Care Unit in France

Abstract

In 1994, an outbreak of Enterobacter sakazakii infections occurred in a neonatal intensive care unit in France from 5 May to 11 July. During the outbreak, 13 neonates were infected with E. sakazakii, resulting in 3 deaths. In addition, four symptomless neonates were colonized by E. sakazakii. The strains were subjected to 16S rRNA gene sequence analysis, genotyped using pulsed-field gel electrophoresis, and phenotyped for a range of enzyme activities. E. sakazakii was isolated from various anatomical sites, reconstituted formula, and an unopened can of powdered infant formula. A fourth neonate died from septic shock, attributed to E. sakazakii infection, during this period. However, 16S rRNA gene sequence analysis revealed that the organism was Enterobacter cloacae. There were three pulsotypes of E. sakazakii associated with infected neonates, and three neonates were infected by more than one genotype. One genotype matched isolates from unused prepared formula and unfinished formula. However, no pulsotypes matched the E. sakazakii strain recovered from an unopened can of powdered infant formula. One pulsotype was associated with the three fatal cases, and two of these isolates had extended-spectrum ␤-lactamase activity. It is possible that E. sakazakii strains differ in their pathogenicities, as shown by the range of symptoms associated with each pulsotype.Enterobacter sakazakii is an opportunistic pathogen associated with the ingestion of reconstituted infant formula and is a rare cause of neonatal meningitis, necrotizing enterocolitis (NEC), and sepsis (9, 10, 11, 23). Such cases often occur among low-birth-weight preterm neonates, who are generally more susceptible to gram-negative bacterial sepsis and endotoxemia associated with NEC (1, 26). The International Commission on Microbiological Specifications for Foods (14) has ranked E. sakazakii as a "severe hazard for restricted populations, life-threatening or substantial chronic sequelae or long duration." A number of reported E. sakazakii outbreaks have been attributed to contaminated reconstituted infant formula (4,7,13,18,31). Bowen and Braden (4) reviewed 46 cases of invasive E. sakazakii infections and showed a link between symptoms and birth weight but did not consider cases of NEC.The virulence of E. sakazakii has been studied by Pagotto et al. (23) and Mange et al. (21), who showed the presence of enterotoxins and adhesion to brain cells, respectively. Townsend et al. demonstrated the translocation of E. sakazakii and other intestinal bacteria across the rat intestinal wall in response to the presence of lipopolysaccharide (28). They also demonstrated that E. sakazakii causes chronic-patterned inflammation in the neonatal rat brain, invades capillary endothelial brain cells, is taken up by macrophages, and induces anti-inflammatory cytokine (interleukin-10) expression in vitro and in vivo at various levels according to strain (29). However, these publications did not report the individual case details associated with the isolates under study. Therefor...

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

“…The translocated strains might need a longer infection period to accomplish the strain that was linked to a fatal NEC infection [14]. This suggests that this strain was 507 able to establish a successful infection and has the virulence traits to avoid 508 phagocytic killing.…”

“…HBMEC cell line was associated with a fatal meningitis case [14] and also produces 457 cytopathogenic OMVs [26]. Overall, the HBMEC TEER was stable during the first 4 458 hours of incubation, indicating the stability of the tight junctions and the integrity of 459 the cell line.…”

Study of Cronobacter sakazakii Strains Isolated from Powdered Milk Infant Formula by Phenotypic and Molecular Methods in Iran

“…About 10% -80% mortality rates have been reported, and surviving patients often suffer from neurological problems and sequel. Cronobacter infection outbreaks in NICUs related to contaminate PIF have been described (7,8). Cronobacter sakazakii is considered an opportunistic pathogen that has been implicated in severe forms of meningitis (leading to brain abscess, ventriculitis, infarction, and cyst formation) and necrotizing enterocolitis, especially in neonates, with a mortality rate varying from 40% -80% (9, 10).…”

Optimazation of Protease Produced From Cronobacter Sakazakii R4

“…Each of carbon sources was added to the production medium in a concentration of 1% w/v. Results mentioned in figure (2) showed that the maximum production of protease by C.sakazakii R4 was achieved when glucose was used as a sole source for carbon and energy with specific activity of 40.8 U/mg in the culture filtrate…”

“…Results showed that the isolates all were protease producers. Specific activity of protease in culture filtrates was ranged between 28.2 and 35.8 U/mg protein and it was found that C.sakazakii R4 was the most efficient, as showen in table (2), and it was selected to determine the optimum conditions for protease production. Optimum conditions for protease production…”