These results significantly advance our understanding of the within-host population biology of P. aeruginosa during infection of patients with cystic fibrosis, and provide in vivo evidence for a link between pyocyanin production and patient morbidity.

There is growing concern about the relevance of in vitro antimicrobial susceptibility tests when applied to isolates of P. aeruginosa from cystic fibrosis (CF) patients. Existing methods rely on single or a few isolates grown aerobically and planktonically. Predetermined cut-offs are used to define whether the bacteria are sensitive or resistant to any given antibiotic 1 . However, during chronic lung infections in CF, P. aeruginosa populations exist in biofilms and there is evidence that the environment is largely microaerophilic 2 . The stark difference in conditions between bacteria in the lung and those during diagnostic testing has called into question the reliability and even relevance of these tests 3 .Artificial sputum medium (ASM) is a culture medium containing the components of CF patient sputum, including amino acids, mucin and free DNA. P. aeruginosa growth in ASM mimics growth during CF infections, with the formation of self-aggregating biofilm structures and population divergence 4,5,6 . The aim of this study was to develop a microtitre-plate assay to study antimicrobial susceptibility of P. aeruginosa based on growth in ASM, which is applicable to both microaerophilic and aerobic conditions. An ASM assay was developed in a microtitre plate format. P. aeruginosa biofilms were allowed to develop for 3 days prior to incubation with antimicrobial agents at different concentrations for 24 hours. After biofilm disruption, cell viability was measured by staining with resazurin. This assay was used to ascertain the sessile cell minimum inhibitory concentration (SMIC) of tobramycin for 15 different P. aeruginosa isolates under aerobic and microaerophilic conditions and SMIC values were compared to those obtained with standard broth growth. Whilst there was some evidence for increased MIC values for isolates grown in ASM when compared to their planktonic counterparts, the biggest differences were found with bacteria tested in microaerophilic conditions, which showed a much increased resistance up to a >128 fold, towards tobramycin in the ASM system when compared to assays carried out in aerobic conditions. The lack of association between current susceptibility testing methods and clinical outcome has questioned the validity of current methods 3 . Several in vitro models have been used previously to study P. aeruginosa biofilms 7,8 . However, these methods rely on surface attached biofilms, whereas the ASM biofilms resemble those observed in the CF lung 9 . In addition, reduced oxygen concentration in the mucus has been shown to alter the behavior of P. aeruginosa 2 and affect antibiotic susceptibility 10. Therefore using ASM under microaerophilic conditions may provide a more realistic environment in which to study antimicrobial susceptibility. Video LinkThe video component of this article can be found at https://www.jove.com/video/3857/ Protocol Preparation of Artificial Sputum Medium (ASM)1. Add 4 g DNA from fish sperm to 250 ml sterile water very slowly over a period of several hours. The DNA tak...

Campylobacter is the most common cause of foodborne bacterial illness worldwide. Faecal contamination of meat, especially chicken, during processing represents a key route of transmission to humans. There is a lack of insight into the mechanisms driving C. jejuni growth and survival within hosts and the environment. Here, we report a detailed analysis of C. jejuni fitness across models reflecting stages in its life cycle. Transposon (Tn) gene-inactivation libraries were generated in three C. jejuni strains and the impact on fitness during chicken colonisation, survival in houseflies and under nutrient-rich and –poor conditions at 4 °C and infection of human gut epithelial cells was assessed by Tn-insertion site sequencing (Tn-seq). A total of 331 homologous gene clusters were essential for fitness during in vitro growth in three C. jejuni strains, revealing that a large part of its genome is dedicated to growth. We report novel C. jejuni factors essential throughout its life cycle. Importantly, we identified genes that fulfil important roles across multiple conditions. Our comprehensive screens showed which flagella elements are essential for growth and which are vital to the interaction with host organisms. Future efforts should focus on how to exploit this knowledge to effectively control infections caused by C. jejuni.

BackgroundPseudomonas aeruginosa populations within the cystic fibrosis lung exhibit extensive phenotypic and genetic diversification. The resultant population diversity is thought to be crucial to the persistence of infection and may underpin the progression of disease. However, because cystic fibrosis lungs represent ecologically complex and hostile environments, the selective forces driving this diversification in vivo remain unclear. We took an experimental evolution approach to test the hypothesis that sub-inhibitory antibiotics can drive diversification of P. aeruginosa populations. Replicate populations of P. aeruginosa LESB58 were cultured for seven days in artificial sputum medium with and without sub-inhibitory concentrations of various clinically relevant antibiotics. We then characterised diversification with respect to 13 phenotypic and genotypic characteristics.ResultsWe observed that higher population diversity evolved in the presence of azithromycin, ceftazidime or colistin relative to antibiotic-free controls. Divergence occurred due to alterations in antimicrobial susceptibility profiles following exposure to azithromycin, ceftazidime and colistin. Alterations in colony morphology and pyocyanin production were observed following exposure to ceftazidime and colistin only. Diversification was not observed in the presence of meropenem.ConclusionsOur study indicates that certain antibiotics can promote population diversification when present in sub-inhibitory concentrations. Hence, the choice of antibiotic may have previously unforeseen implications for the development of P. aeruginosa infections in the lungs of cystic fibrosis patients.

Campylobacter jejuni is the leading cause of foodborne bacterial gastroenteritis with contaminated poultry meat its main source. Control of C. jejuni is a priority for the poultry industry but no vaccines are available and their development hampered by poor understanding of the immunobiology of C. jejuni infection. Here we show the functional role of B lymphocytes in response to C. jejuni in the chicken through depletion of the B lymphocyte population (bursectomy) followed by challenge. B lymphocyte depletion has little effect on bacterial numbers in the ceca, the main site of colonisation, where C. jejuni persist to beyond commercial slaughter age, but reduces clearance from the small intestine. In longer-term experiments we show antibody leads to reduction in C. jeuni numbers in the ceca by nine weeks post infection. Whilst we did not examine any protective role to re-challenge, it illustrates the difficulty in producing a vaccine in a young, immunologically naïve host. We believe this is first study of functional immunity to C. jejuni in chicken and shows antibody is ineffective in clearing C. jejuni from the ceca within the production lifetime of chickens, although is involved in clearance from the small intestine and longer-term clearance from the ceca.