- Volume 165, Issue 5, 2019

Bacterial persisters are a subpopulation of cells that exhibit phenotypic resistance during exposure to a lethal dose of antibiotics. They are difficult to target and thought to contribute to the long treatment duration required for tuberculosis. Understanding the molecular and cellular biology of persisters is critical to finding new tuberculosis drugs that shorten treatment. This review focuses on mycobacterial persisters and describes the challenges they pose in tuberculosis therapy, their characteristics and formation, how persistence leads to resistance, and the current approaches being used to target persisters within mycobacterial drug discovery.

The protozoan Cryptosporidium is notorious for its resistance to chlorine disinfection, a mainstay of water treatment. Human infections, mainly of the small intestine, arise from consumption of faecally contaminated food or water, environmental exposure, and person-to-person or animal-to-person spread. Acute gastrointestinal symptoms can be prolonged but are usually self-limiting. Problems arise with immune-deficient, including malnourished, people including chronic diarrhoea, hepato-biliary tree and extra-gastrointestinal site infection, and few options for treatment or prevention exist. Although genomics has enabled refined classification, identification of chemotherapeutic targets and vaccine candidates, and putative factors for host adaption and pathogenesis, their confirmation has been hampered by a lack of biological tools.

The Type VI secretion system (T6SS) is a protein nanomachine that is widespread in Gram-negative bacteria and is used to translocate effector proteins directly into neighbouring cells. It represents a versatile bacterial weapon that can deliver effectors into distinct classes of target cells, playing key roles in inter-bacterial competition and bacterial interactions with eukaryotic cells. This versatility is underpinned by the ability of the T6SS to deliver a vast array of effector proteins, with many distinct activities and modes of interaction with the secretion machinery. Recent work has highlighted the importance and diversity of interactions mediated by T6SSs within polymicrobial communities, and offers new molecular insights into effector delivery and action in target cells.

Members of the Gram-negative bacterial genus Photorhabdus are all highly insect pathogenic and exist in an obligate symbiosis with the entomopathogenic nematode worm Heterorhabditis. All members of the genus produce the small-molecule 3,5-dihydroxy-4-isopropyl-trans-stilbene (IPS) as part of their secondary metabolism. IPS is a multi-potent compound that has antimicrobial, antifungal, immunomodulatory and anti-cancer activities and also plays an important role in symbiosis with the nematode. In this study we have examined the response of Photorhabdus itself to exogenous ectopic addition of IPS at physiologically relevant concentrations. We observed that the bacteria had a measureable phenotypic response, which included a decrease in bioluminescence and pigment production. This was reflected in changes in its transcriptomic response, in which we reveal a reduction in transcript levels of genes relating to many fundamental cellular processes, such as translation and oxidative phosphorylation. Our observations suggest that IPS plays an important role in the biology of Photorhabdus bacteria, fulfilling roles in quorum sensing, antibiotic-competition advantage and maintenance of the symbiotic developmental cycle.

Biofilm model systems are used to study biofilm growth and predict the effects of anti-biofilm interventions within the human oral cavity. Many in vitro biofilm model systems use a confocal laser scanning microscope (CLSM) in conjunction with image analysis tools to study biofilms. The aim of this study was to evaluate an in-house developed image analysis software program that we call BAIT (Biofilm Architecture Inference Tool) to quantify the architecture of oral multi-species biofilms following anti-biofilm interventions using a microfluidic biofilm system. Differences in architecture were compared between untreated biofilms and those treated with water (negative control), sodium gluconate (‘placebo’) or stannous fluoride (SnF2). The microfluidic system was inoculated with pooled human saliva and biofilms were developed over 22 h in filter-sterilized 25 % pooled human saliva. During this period, biofilms were treated with water, sodium gluconate, or SnF2 (1000, 3439 or 10 000 p.p.m. Sn2+) 8 and 18 h post-inoculation. After 22 h of growth, biofilms were stained with LIVE/DEAD stain, and imaged by CLSM. BAIT was used to calculate biofilm biovolume, total number of objects, surface area, fluffiness, connectivity, convex hull porosity and viability. Image analysis showed oral biofilm architecture was significantly altered by 3439 and 10 000 p.p.m. Sn2+ treatment regimens, resulting in decreased biovolume, surface area, number of objects and connectivity, while fluffiness increased (P<0.01). In conclusion, BAIT was shown to be able to measure the changes in biofilm architecture and detects possible antimicrobial and anti-biofilm effects of candidate agents.

Xenorhabdus species are symbionts of entomopathogenic nematodes and pathogens of susceptible insects. Nematodes enter insect hosts and perforate the midgut to invade the haemocoel where Xenorhabdus bacteria are released transitioning to their pathogenic stage. During nematode invasion microbes from the insect gut translocate into the haemocoel. Different species of nematodes carrying specific strains of Xenorhabdus can also invade the same insect. Xenorhabdus species thereby compete for nutrients and space with both related strains and non-related gut microbes. While Xenorhabdus species produce diverse antimicrobial compounds in complex media, their functions in insect hosts are not well understood. We show that Xenorhabdus szentirmaii produced ngrA-dependent antibiotics that were active against both gut-derived microbes and Xenorhabdus nematophila whereas antibiotics of X. nematophila were not active against X. szentirmaii . X. nematophila growth was inhibited in co-cultures with wild-type X. szentirmaii in medium that mimics insect haemolymph. An antibiotic-deficient strain of X. szentirmaii was created by inactivating the ngrA gene that encodes the enzyme that attaches the 4′ phosphopantetheinyl moiety to non-ribosomal peptide synthetases involved in antibiotic biosynthesis. X. nematophila growth was not inhibited in co-cultures with the ngrA strain. The growth of X. nematophila was suppressed in Manduca sexta co-injected with wild-type X. szentirmaii and X. nematophila . In contrast, growth of X. nematophila was not suppressed in M. sexta co-injected with the ngrA strain. Two unique compounds were detected by MALDI-TOF MS analysis in haemolymph infected with the wild-type but not with the ngrA strain. Finally, killing of M. sexta was delayed in insects infected with the ngrA strain. These findings indicate that in the insect host X. szentirmaii produces ngrA-dependent products involved in both interspecies competition and virulence.

The combinatorial action of osmotic (OS) and heat (HS) shocks on the composition of soluble cytosol carbohydrates and membrane lipids was studied. For the first time it was demonstrated that the combinatorial effect of these shocks led to the non-additive response – an increase in the trehalose level, characteristic for HS, but at the same time suppression of glycerol production, uncharacteristic of the OS response. In addition, combinatorial action resulted in a new effect – increase in the mannitol level, which was not typical for the individual HS or OS responses. On the contrary, a general pattern of change was observed in the composition of membrane lipids in response to both individual HS and OS, and their combinations, which was a twofold increase in the proportion of phosphatidic acids. At the same time, the mechanism of alteration in the degree of unsaturation of membrane phospholipids was not involved in adaptation. The response to combinatorial shocks includes the accumulation of trehalose and mannitol, and increase in the proportion of phosphatidic acids in membrane lipids.