- Volume 164, Issue 10, 2018

The current paradigm for hospital outbreak detection and investigation is based on methodology first developed over 150 years ago. Daily surveillance to detect patients positive for pathogens of particular importance for nosocomial infection is supported by epidemiological investigation to determine their relationship in time and place, and to identify any other factor that could link them. The antibiotic resistance pattern is commonly used as a surrogate for bacterial relatedness, although this lacks sensitivity and specificity. Typing may be used to define bacterial relatedness, although routine methods lack sufficient discriminatory power to distinguish relatedness beyond the level of bacterial clones. Ultimately, the identification of an outbreak remains a predominately subjective process reliant on the intuition of experienced infection control professionals. Here, we propose a redesign of hospital outbreak detection and investigation in which bacterial species associated with nosocomial transmission and infection undergo routine prospective whole-genome sequencing. Further investigation is based on the probability that isolates are associated with an outbreak, which is based on the degree of genetic relatedness between isolates. Evidence is provided that supports this model based on studies of MRSA (methicillin-resistant Staphylococcus aureus), together with the benefits of a ‘Sequence First’ approach. The feasibility of implementation is discussed, together with residual barriers that need to be overcome prior to implementation.

Sucrose is naturally synthesized by many cyanobacteria under high salt conditions, which can be applied to produce this widely used feedstock. To improve sucrose production with the moderate halo-tolerant cyanobacterium Synechocystis sp. PCC 6803, we identified and biochemically characterized the sucrose-degrading invertase. Inactivating the invertase encoding gene sll0626 (inv) significantly increased cellular sucrose levels; interestingly sucrose over-accumulation was also observed under NaCl-free conditions. The subsequent inactivation of inv in the mutant ΔggpS, which cannot synthesize the major compatible solute glucosylglycerol, resulted in further enhanced sucrose accumulation in the presence of 1.5 % NaCl. Then, inv mutation was introduced into the previously obtained sucrose-producing strain WD25 (Du W, Liang F, Duan Y, Tan X, Lu X. Metab Eng 2013;19:17–25), which resulted in almost 40 % higher sucrose accumulation. These findings show that invertase is an interesting target in obtaining efficient sucrose production in cyanobacterial host cells.

Tolyporphins are structurally diverse tetrapyrrole macrocycles produced by the cyanobacterial culture HT-58-2. Although tolyporphins were discovered over 25 years ago, little was known about the microbiology of the culture. The studies reported herein expand the description of the community of predominantly alphaproteobacteria associated with the filamentous HT-58-2 cyanobacterium and isolate a dominant bacterium, Porphyrobacter sp. HT-58-2, for which the complete genome is established and growth properties are examined. Fluorescence in situ hybridization (FISH) analysis of the cyanobacterium–microbial community with a probe targeting the 16S rRNA of Porphyrobacter sp. HT-58-2 showed fluorescence emanating from the cyanobacterial sheath. Although genes for the biosynthesis of bacteriochlorophyll a (BChl a) are present in the Porphyrobacter sp. HT-58-2 genome, the pigment was not detected under the conditions examined, implying the absence of phototrophic growth. Comparative analysis of four Porphyrobacter spp. genomes from worldwide collection sites showed significant collinear gene blocks, with two inversions and three deletion regions. Taken together, the results enrich our understanding of the HT-58-2 cyanobacterium–microbial culture.

Coral diseases contribute to the decline of coral reefs globally and threaten the health and future of coral reef communities. Acute Montipora white syndrome (aMWS) is a tissue loss disease that has led to the mortality of hundreds of Montipora capitata colonies in Kāne‘ohe Bay, Hawai‘i in recent years. This study describes the analysis of coral-associated bacterial communities using high-throughput sequencing generated by the PacBio RSII platform. Samples from three health states of M. capitata (healthy, healthy-diseased and diseased) were collected during an ongoing aMWS outbreak and a non-outbreak period and the bacterial communities were identified to determine whether a shift in community structure had occurred between the two periods. The bacterial communities associated with outbreak and non-outbreak samples were significantly different, and one major driver was a high abundance of operational taxonomic units (OTUs) identified as Escherichia spp. in the outbreak sequences. In silico bacterial source tracking suggested this OTU was likely from sewage contamination of livestock, rather than human, origin. The most abundant coliform OTU was a culturable E. fergusonii isolate, strain OCN300, however, it did not induce disease signs on healthy M. capitata colonies when used in laboratory infection trials. In addition, screening of the sequencing output found that the most abundant OTUs corresponded to previously described M. capitata pathogens. The synergistic combination of known coral pathogens, sewage contaminants and other stressors, such as fluctuating seawater temperatures and bacterial pathogens, have the potential to escalate the deterioration of coral reef ecosystems.

To understand the effects triggered by Mn2+ on Deinococcus radiodurans, the proteome patterns associated with different growth phases were investigated. In particular, under physiological conditions we tested the growth rate and the biomass yield of D. radiodurans cultured in rich medium supplemented or not with MnCl₂. The addition of 2.5–5.0 µM MnCl₂ to the medium neither altered the growth rate nor the lag phase, but significantly increased the biomass yield. When higher MnCl₂ concentrations were used (10–250 µM), biomass was again found to be positively affected, although we did observe a concentration-dependent lag phase increase. The in vivo concentration of Mn2+ was determined in cells grown in rich medium supplemented or not with 5 µM MnCl₂. By atomic absorption spectroscopy, we estimated 0.2 and 0.75 mM Mn2+ concentrations in cells grown in control and enriched medium, respectively. We qualitatively confirmed this observation using a fluorescent turn-on sensor designed to selectively detect Mn2+ in vivo. Finally, we investigated the proteome composition of cells grown for 15 or 19 h in medium to which 5 µM MnCl₂ was added, and we compared these proteomes with those of cells grown in the control medium. The presence of 5 µM MnCl₂ in the culture medium was found to alter the pI of some proteins, suggesting that manganese affects post-translational modifications. Further, we observed that Mn2+ represses enzymes linked to nucleotide recycling, and triggers overexpression of proteases and enzymes linked to the metabolism of amino acids.

Salmonella enterica serovar Typhimurium (S. typhimurium) can cause food- and water-borne illness with diverse clinical manifestations. One key factor for S. typhimurium pathogenesis is the alternative sigma factor σE, which is encoded by the rpoE gene and controls the transcription of genes required for outer-membrane integrity in response to alterations in the bacterial envelope. The canonical pathway for σE activation involves proteolysis of the antisigma factor RseA, which is triggered by unfolded outer-membrane porins (OMPs) and lipopolysaccharides (LPS) that have accumulated in the periplasm. This study reports new stress factors that are able to activate σE expression. We demonstrate that UVA radiation induces σE activity in a pathway that is dependent on the stringent response regulator ppGpp. Survival assays revealed that rpoE has a role in the defence against lethal UVA doses that is mediated by functions that are dependent on and independent of the alternative sigma factor RpoS. We also report that the envelope stress generated by phage infection requires a functional rpoE gene for optimal bacterial tolerance and that it is able to induce σE activity in an RseA-dependent fashion. σE activity is also induced by hypo-osmotic shock in the absence of osmoregulated periplasmic glucans (OPGs). It is known that the rpoE gene is not essential in S. typhimurium. However, we report here two cases of the conditional lethality of rpoE mutations in this micro-organism. We demonstrate that rpoE mutations are not tolerated in the absence of OPGs (at low to moderate osmolarity) or LPS O-antigen. The latter case resembles that of the prototypic Escherichia coli strain K12, which neither synthesizes a complete LPS nor tolerates null rpoE mutations.