Effect of colony morphology variation of Burkholderia pseudomallei on intracellular survival and resistance to antimicrobial environments in human macrophages in vitro

Sarunporn Tandhavanant,Sharon J Peacock,Aunchalee Thanwisai,Sunee Korbsrisate,Narisara Chantratita,Nicholas Pj Day,Direk Limmathurotsakul

doi:10.1186/1471-2180-10-303

Abstract

BackgroundPrimary diagnostic cultures from patients with melioidosis demonstrate variation in colony morphology of the causative organism, Burkholderia pseudomallei. Variable morphology is associated with changes in the expression of a range of putative virulence factors. This study investigated the effect of B. pseudomallei colony variation on survival in the human macrophage cell line U937 and under laboratory conditions simulating conditions within the macrophage milieu. Isogenic colony morphology types II and III were generated from 5 parental type I B. pseudomallei isolates using nutritional limitation. Survival of types II and III were compared with type I for all assays.ResultsMorphotype was associated with survival in the presence of H2O2 and antimicrobial peptide LL-37, but not with susceptibility to acid, acidified sodium nitrite, or resistance to lysozyme, lactoferrin, human neutrophil peptide-1 or human beta defensin-2. Incubation under anaerobic conditions was a strong driver for switching of type III to an alternative morphotype. Differences were noted in the survival and replication of the three types following uptake by human macrophages, but marked strain-to strain-variability was observed. Uptake of type III alone was associated with colony morphology switching.ConclusionsMorphotype is associated with phenotypes that alter the ability of B. pseudomallei to survive in adverse environmental conditions.

Highlights

Primary diagnostic cultures from patients with melioidosis demonstrate variation in colony morphology of the causative organism, Burkholderia pseudomallei
Prior to observation of intracellular replication in macrophages, extracellular growth of B. pseudomallei was compared between 3 isogenic morphotypes cultured in trypticase soy broth (TSB)
Replication of isogenic B. pseudomallei morphotypes in macrophages Evaluation of the initial B. pseudomallei-macrophage cell interaction using a multiplicity of infection (MOI) of 25:1 demonstrated that 3.0% of the bacterial inoculum was associated with macrophages at 2 h

Summary

Introduction

Primary diagnostic cultures from patients with melioidosis demonstrate variation in colony morphology of the causative organism, Burkholderia pseudomallei. This study investigated the effect of B. pseudomallei colony variation on survival in the human macrophage cell line U937 and under laboratory conditions simulating conditions within the macrophage milieu. Burkholderia pseudomallei is an environmental Gramnegative bacterium that causes a severe and often fatal disease called melioidosis. This is an important cause of sepsis in south-east Asia and northern Australia, a geographic distribution that mirrors the presence of B. pseudomallei in the environment [1]. Macrophages have several strategies to control bacterial infection, including bacterial killing following uptake through the action of reactive oxygen and reactive nitrogen compounds, antimicrobial peptides and lysozomal enzymes. B. pseudomallei can invade and replicate in primary human macrophages [8,9,10]

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