In contrast to planktonic cells, bacteria imbedded biofilms are notoriously refractory to treatment by antibiotics or bacteriophage (phage) used alone. Given that the mechanisms of killing differ profoundly between drugs and phages, an obvious question is whether killing is improved by combining antibiotic and phage therapy. However, this question has only recently begun to be explored. Here, in vitro biofilm populations of Pseudomonas aeruginosa PA14 were treated singly and with combinations of two phages and bactericidal antibiotics of five classes. By themselves, phages and drugs commonly had only modest effects in killing the bacteria. However some phage-drug combinations reduced bacterial densities to well below that of the best single treatment; in some cases, bacterial densities were reduced even below the level expected if both agents killed independently of each other (synergy). Furthermore, there was a profound order effect in some cases: treatment with phages before drugs achieved maximum killing. Combined treatment was particularly effective in killing in Pseudomonas biofilms grown on layers of cultured epithelial cells. Phages were also capable of limiting the extent to which minority populations of bacteria resistant to the treating antibiotic ascend. The potential of combined antibiotic and phage treatment of biofilm infections is discussed as a realistic way to evaluate and establish the use of bacteriophage for the treatment of humans.

PulseNet International is a global network dedicated to laboratory-based surveillance for food-borne diseases. The network comprises the national and regional laboratory networks of Africa, Asia Pacific, Canada, Europe, Latin America and the Caribbean, the Middle East, and the United States. The PulseNet International vision is the standardised use of whole genome sequencing (WGS) to identify and subtype food-borne bacterial pathogens worldwide, replacing traditional methods to strengthen preparedness and response, reduce global social and economic disease burden, and save lives. To meet the needs of real-time surveillance, the PulseNet International network will standardise subtyping via WGS using whole genome multilocus sequence typing (wgMLST), which delivers sufficiently high resolution and epidemiological concordance, plus unambiguous nomenclature for the purposes of surveillance. Standardised protocols, validation studies, quality control programmes, database and nomenclature development, and training should support the implementation and decentralisation of WGS. Ideally, WGS data collected for surveillance purposes should be publicly available, in real time where possible, respecting data protection policies. WGS data are suitable for surveillance and outbreak purposes and for answering scientific questions pertaining to source attribution, antimicrobial resistance, transmission patterns, and virulence, which will further enable the protection and improvement of public health with respect to food-borne disease.

Summary Binding of the Origin Recognition Complex (ORC) to replication origins is essential for initiation of DNA replication, but ORC has non-essential functions outside of DNA replication, including in heterochromatic gene silencing and telomere maintenance. Trypanosoma brucei, a protozoan parasite that causes human African trypanosomiasis, uses antigenic variation as a major virulence mechanism to evade the host’s immune attack by expressing its major surface antigen, the Variant Surface Glycoprotein (VSG), in a monoallelic manner. An Orc1/Cdc6 homolog has been identified in T. brucei, but its role in DNA replication has not been directly confirmed and its potential involvement in VSG repression or switching has not been thoroughly investigated. In this study, we show that TbOrc1 is essential for nuclear DNA replication in mammalian-infectious bloodstream and tsetse procyclic forms (BF and PF). Depletion of TbOrc1 resulted in derepression of telomere-linked silent VSGs in both BF and PF, and increased VSG switching particularly through the in-situ transcriptional switching mechanism. TbOrc1 associates with telomere repeats but appears to do so independently of two known T. brucei telomere proteins, TbRAP1 and TbTRF. We conclude that TbOrc1 has conserved functions in DNA replication and is also required to control telomere-linked VSG expression and VSG switching.

We postulate that phenotypic resistance to antibiotics, persistence, is not an evolved (selected-for) character but rather like mutation, an inadvertent product of different kinds of errors and glitches. The rate of generation of these errors is augmented by exposure to these drugs. The genes that have been identified as contributing to the production of persisters are analogous to the so-called mutator genes; they modulate the rate at which these errors occur and/or are corrected. In theory, these phenotypically resistant bacteria can retard the rate of microbiological cure by antibiotic treatment.

Zoonotic transmission of Salmonella infections causes an estimated 11% of salmonellosis annually in the United States. This report describes the epidemiologic, traceback and laboratory investigations conducted in the United States as part of four multistate outbreaks of Salmonella infections linked to small turtles. Salmonella isolates indistinguishable from the outbreak strains were isolated from a total of 143 ill people in the United States, pet turtles, and pond water samples collected from turtle farm A, as well as ill people from Chile and Luxembourg. Almost half (45%) of infections occurred in children aged <5 years, underscoring the importance of the Centers for Disease Control and Prevention recommendation to keep pet turtles and other reptiles out of homes and childcare settings with young children. Although only 43% of the ill people who reported turtle exposure provided purchase information, most small turtles were purchased from flea markets or street vendors, which made it difficult to locate the vendor, trace the turtles to a farm of origin, provide education and enforce the United States federal ban on the sale and distribution of small turtles. These outbreaks highlight the importance of improving public awareness and education about the risk of Salmonella from small turtles not only in the United States but also worldwide.

The maximum exponential growth rate, the Malthusian parameter (MP), is commonly used as a measure of fitness in experimental studies of adaptive evolution and of the effects of antibiotic resistance and other genes on the fitness of planktonic microbes. Thanks to automated, multi-well optical density plate readers and computers, with little hands-on effort investigators can readily obtain hundreds of estimates of MPs in less than a day. Here we compare estimates of the relative fitness of antibiotic susceptible and resistant strains of E. coli, Pseudomonas aeruginosa and Staphylococcus aureus based on MP data obtained with automated multi-well plate readers with the results from pairwise competition experiments. This leads us to question the reliability of estimates of MP obtained with these high throughput devices and the utility of these estimates of the maximum growth rates to detect fitness differences.

Infections caused by pathogens commonly acquired from consumption of food are not always transmitted by that route. They may also be transmitted through contact to animals, other humans or the environment. Additionally, many outbreaks are associated with food contaminated from these non-food sources. For this reason, such presumed foodborne outbreaks are best investigated through a One Health approach working across human, animal and environmental sectors and disciplines. Outbreak strains or clones that have propagated and continue to evolve in non-human sources and environments often show more sequence variation than observed in typical monoclonal point-source outbreaks. This represents a challenge when using whole genome sequencing (WGS), the new gold standard for molecular surveillance of foodborne pathogens, for outbreak detection and investigation. In this review, using recent examples from outbreaks investigated in the United States (US) some aspects of One Health approaches that have been used successfully to solve such outbreaks are presented. These include using different combinations of flexible WGS based case definition, efficient epidemiological follow-up, traceback, surveillance, and testing of potential food and environmental sources and animal hosts.

Trypanosomes contain a unique form of mitochondrial DNA called kinetoplast DNA (kDNA) that is a catenated network composed of minicircles and maxicircles. Several proteins are essential for network replication, and most of these localize to the antipodal sites or the kinetoflagellar zone. Essential components for kDNA synthesis include three mitochondrial DNA polymerases TbPOLIB, TbPOLIC, and TbPOLID). In contrast to other kDNA replication proteins, TbPOLID was previously reported to localize throughout the mitochondrial matrix. This spatial distribution suggests that TbPOLID requires redistribution to engage in kDNA replication. Here, we characterize the subcellular distribution of TbPOLID with respect to the Trypanosoma brucei cell cycle using immunofluorescence microscopy. Our analyses demonstrate that in addition to the previously reported matrix localization, TbPOLID was detected as discrete foci near the kDNA. TbPOLID foci colocalized with replicating minicircles at antipodal sites in a specific subset of the cells during stages II and III of kDNA replication. Additionally, the TbPOLID foci were stable following the inhibition of protein synthesis, detergent extraction, and DNase treatment. Taken together, these data demonstrate that TbPOLID has a dynamic localization that allows it to be spatially and temporally available to perform its role in kDNA replication.M itochondrial DNA (mtDNA) is packaged into protein-DNA complexes called nucleoids. These structures are dynamic macrocomplexes located in the mitochondrial matrix, and they act as units of mtDNA replication and inheritance with composition that can undergo remodeling in response to metabolic stresses (7,23,47). Using bromodeoxyuridine (BrdU) incorporation, nucleoids have been shown to be the sites of mtDNA replication in yeast and mammalian cells (35,39). However, not all nucleoids replicate concurrently; only a subset undergo replication at any given time. With no strict control related to cell cycle progression, the segregation and inheritance of the nucleoid depends upon a membrane-associated apparatus that interacts with the fusion and fission machinery of the mitochondrial organelle network (2,19,31). Lastly, while the protein composition of nucleoids varies among cell types and in response to metabolic conditions, the core proteins of the nucleoid appear to remain constant and include transcription and replication factors, such as mitochondrial transcription factor A, single-stranded binding protein, Twinkle helicase, and the sole mitochondrial DNA polymerase, Pol ␥ (1, 21).One of the most unusual and structurally complex mtDNA genomes is found in trypanosomatid parasites such as Trypanosoma brucei, the causative agent of African sleeping sickness. This extranuclear genome, called kinetoplast DNA (kDNA), is a network composed of thousands of topologically interlocked minicircles and maxicircles that are condensed into a single diskshaped nucleoid structure. Approximately 25 identical maxicircle copies (23 kb) encode a subset of respiratory c...