Correction: Macrophages, but not neutrophils, are critical for proliferation of Burkholderia cenocepacia and ensuing host-damaging inflammation.

[This corrects the article DOI: 10.1371/journal.ppat.1006437.].

Macrophages, but not neutrophils, are critical for proliferation of Burkholderia cenocepacia and ensuing host-damaging inflammation.

Bacteria of the Burkholderia cepacia complex (Bcc) can cause devastating pulmonary infections in cystic fibrosis (CF) patients, yet the precise mechanisms underlying inflammation, recurrent exacerbations and transition from chronic stages to acute infection and septicemia are not known. Bcc bacteria are generally believed to have a predominant extracellular biofilm life style in infected CF lungs, similar to Pseudomonas aeruginosa, but this has been challenged by clinical observations which show Bcc bacteria predominantly in macrophages. More recently, Bcc bacteria have emerged in nosocomial infections of patients hospitalized for reasons unrelated to CF. Research has abundantly shown that Bcc bacteria can survive and replicate in mammalian cells in vitro, yet the importance of an intracellular life style during infection in humans is unknown. Here we studied the contribution of innate immune cell types to fatal pro-inflammatory infection caused by B. cenocepacia using zebrafish larvae. In strong contrast to the usual protective role for macrophages against microbes, our results show that these phagocytes significantly worsen disease outcome. We provide new insight that macrophages are critical for multiplication of B. cenocepacia in the host and for development of a fatal, pro-inflammatory response that partially depends on Il1-signalling. In contrast, neutrophils did not significantly contribute to disease outcome. In subcutaneous infections that are dominated by neutrophil-driven phagocytosis, the absence of a functional NADPH oxidase complex resulted in a small but measurably higher increase in bacterial growth suggesting the oxidative burst helps limit bacterial multiplication; however, neutrophils were unable to clear the bacteria. We suggest that paradigm-changing approaches are needed for development of novel antimicrobials to efficiently disarm intracellular bacteria of this group of highly persistent, opportunistic pathogens.

Modulation of Membrane Influx and Efflux in Escherichia coli Sequence Type 131 Has an Impact on Bacterial Motility, Biofilm Formation, and Virulence in a Caenorhabditis elegans Model.

Energy-dependent efflux overexpression and altered outer membrane permeability (influx) can promote multidrug resistance (MDR). The present study clarifies the regulatory pathways that control membrane permeability in the pandemic clone Escherichia coli sequence type 131 (ST131) and evaluates the impact of efflux and influx modulations on biofilm formation, motility, and virulence in the Caenorhabditis elegans model. Mutants of two uropathogenic E. coli (UPEC) strains, MECB5 (ST131; H30-Rx) and CFT073 (ST73), as well as a fecal strain, S250 (ST131; H22), were in vitro selected using continuous subculture in subinhibitory concentrations of ertapenem (ETP), chloramphenicol (CMP), and cefoxitin (FOX). Mutations in genes known to control permeability were shown for the two UPEC strains: MECB5-FOX (deletion of 127 bp in marR; deletion of 1 bp and insertion of an IS1 element in acrR) and CFT073-CMP (a 1-bp deletion causing a premature stop in marR). We also demonstrated that efflux phenotypes in the mutants selected with CMP and FOX were related to the AcrAB-TolC pump, but also to other efflux systems. Alteration of membrane permeability, caused by underexpression of the two major porins, OmpF and OmpC, was shown in MECB5-ETP and mutants selected with FOX. Lastly, our findings suggest that efflux pump-overproducing isolates (CMP mutants) pose a serious threat in terms of virulence (significant reduction in worm median survival) and host colonization. Lack of porins (ETP and FOX mutants) led to a high level of antibiotic resistance in an H30-Rx subclone. Nevertheless, this adaptation created a physiological disadvantage (decreased motility and ability to form biofilm) associated with a low potential for virulence.

A Simple and Safe Protocol for Preparing Brucella Samples for Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry Analysis.

We describe a simple protocol to inactivate the biosafety level 3 (BSL3) pathogens Brucella prior to their analysis by matrix-assisted laser desorption ionization-time of flight mass spectrometry. This method is also effective for several other bacterial pathogens and allows storage, and eventually shipping, of inactivated samples; therefore, it might be routinely applied to unidentified bacteria, for the safety of laboratory workers.

A MALDI-TOF MS database with broad genus coverage for species-level identification of Brucella.

Brucella are highly infectious bacterial pathogens responsible for a severely debilitating zoonosis called brucellosis. Half of the human population worldwide is considered to live at risk of exposure, mostly in the poorest rural areas of the world. Prompt diagnosis of brucellosis is essential to prevent complications and to control epidemiology outbreaks, but identification of Brucella isolates may be hampered by the lack of rapid and cost-effective methods. Nowadays, many clinical microbiology laboratories use Matrix-Assisted Laser Desorption Ionization-Time Of Flight mass spectrometry (MALDI-TOF MS) for routine identification. However, lack of reference spectra in the currently commercialized databases does not allow the identification of Brucella isolates. In this work, we constructed a Brucella MALDI-TOF MS reference database using VITEK MS. We generated 590 spectra from 84 different strains (including rare or atypical isolates) to cover this bacterial genus. We then applied a novel biomathematical approach to discriminate different species. This allowed accurate identification of Brucella isolates at the genus level with no misidentifications, in particular as the closely related and less pathogenic Ochrobactrum genus. The main zoonotic species (B. melitensis, B. abortus and B. suis) could also be identified at the species level with an accuracy of 100%, 92.9% and 100%, respectively. This MALDI-TOF reference database will be the first Brucella database validated for diagnostic and accessible to all VITEK MS users in routine. This will improve the diagnosis and control of brucellosis by allowing a rapid identification of these pathogens.

Surface lipoproteome of Mycoplasma hominis PG21 and differential expression after contact with human dendritic cells.

AIM: To assess the lipoproteins that are involved in the interaction between Mycoplasma hominis and human dendritic cells. MATERIALS & METHODS: The surface lipoproteome of M. hominis PG21 was characterized by using Triton X-114 extraction and LC-MS/MS identification. The transcriptional changes in lipoprotein genes upon contact with human dendritic cells were determined by using reverse transcription quantitative PCR after identification of reference genes suitable for normalization. RESULTS: A large-scale overexpression of lipoprotein genes was observed with 21 upregulated transcripts. Seven genes of unknown function were M. hominis species specific and six genes were putatively associated with increased nutrient capture from the host cell and adhesion. CONCLUSION: M. hominis regulates lipoprotein gene expression and may use species-specific mechanisms during the host colonization process.

Zebrafish embryos as a model to study bacterial virulence.

In recent years the zebrafish has gained enormous attention in infection biology, and many protocols have been developed to study interaction of both human and fish pathogens, including viruses, fungi, and bacteria, with the host. Especially the extraordinary possibilities for live imaging of disease processes in the transparent embryos using fluorescent bacteria and cell-specific reporter fish combined with gene knockdown, transcriptome, and genetic studies have dramatically advanced our understanding of disease mechanisms. The zebrafish embryo is amenable to study virulence of both extracellular and facultative intracellular pathogens introduced through the technique of microinjection. Several protocols have been published that address the different sites of injection, antisense strategies, imaging, and production of transgenic fish in detail. Here we describe a protocol to study the virulence profiles, ranging from acute fatal to persistent, of bacteria belonging to the Burkholderia cepacia complex. This standard operating protocol combines simple survival assays, analysis of bacterial kinetics, analysis of the early innate immune response with qRT-PCR, and the use of transgenic reporter fish to study interactions with host phagocytes, and is also applicable to other pathogens.