Acid stress management by Cronobacter sakazakii

Abstract

Cronobacter sakazakii is a foodborne pathogenic microorganism associated with sporadic cases of neonatal meningitis, necrotising enterocolitis, septicaemia, bloody diarrhoea and brain abscesses acquired through the consumption of contaminated powdered infant formula (PIF). This study aimed to investigate the growth of C. sakazakii DPC6529, a particularly stress tolerant clinical isolate, in acidified laboratory media and PIF. The possibility of a stationary-phase acid tolerance response (ATR) was also investigated. C. sakazakii DPC6529 grew in LB broth acidified to pH4.2 with hydrochloric acid (HCl) and was capable of relatively fast growth in PIF acidified to pH5.0 with HCl, representing the stomach pH reported for newborns and infants. Moreover, bacterial growth in LB broth supplemented with 1% (w/v) glucose gave rise to a stationary-phase ATR which resulted in enhanced survival against a subsequent acid challenge at pH3.0. A transposon mutagenesis approach was used to shed light on some of the molecular mechanisms involved in the response C. sakazakii DPC6529 to normally lethal acid exposures. The data suggests that repairing damage in proteins and nucleic acids, posttranscriptional modification of tRNA molecules and maintenance of the integrity of the cellular envelope are key processes in the defence against acid stress. Clones carrying transposon insertions in genes encoding the envelope stress response regulators CpxR and OmpR were identified as acid-sensitive mutants. Further analyses of the ompR defective mutant and its complemented counterpart evidenced that OmpR is a key player in the response of C. sakazakii to acid stress, although it was not essential to mount an active stationary-phase ATR, at least under the tested conditions. The ability of C. sakazakii DPC6529 to grow in acid environments and to develop an adaptive stationary-phase ATR may allow for its survival or even proliferation within the infant gastrointestinal tract after consumption of contaminated milk formulae.

Keywords: Acid stress; Acid tolerance response; C. sakazakii; Powdered infant formula; Transposon mutagenesis; ompR.