Ciprofloxacin-induced persister-cells in Campylobacter jejuni.

Campylobacter jejuni is a major bacterial foodborne-pathogen. Ciprofloxacin is an important antibiotic for the treatment of C. jejuni, albeit high rates of fluoroquinolone resistance have limited its usefulness. Persister-cells are transiently antibiotic-tolerant fractions of bacterial populations and their occurrence has been associated with recalcitrant and persistent bacterial infections. Here, time-kill assays with ciprofloxacin (200×MIC, 25 µg ml-1) were performed in C. jejuni strains 81-176 and RM1221 and persister-cells were found. The frequency of survivors after 8 h of ciprofloxacin exposure was approx. 10-3 for both strains, while after 22 h the frequency was between 10-5-10-7, depending on the strain and growth-phase. Interestingly, the stationary-phase cultures did not display more persister-cells compared to exponential-phase cultures, in contrast to what has been observed in other bacterial species. Persister-cells after ampicillin exposure (100×MIC, 200 µg ml-1) were not detected, implying that persister-cell formation in C. jejuni is antibiotic-specific. In attempts to identify the mechanism of ciprofloxacin persister-cell formation, stringent or SOS responses were not found to play major roles. Overall, this study reports ciprofloxacin persister-cells in C. jejuni and challenges the notion of persister-cells as plainly dormant non-growing cells.

Phenotypic resistance and the dynamics of bacterial escape from phage control.

The canonical view of phage - bacterial interactions in dense, liquid cultures is that the phage will eliminate most of the sensitive cells; genetic resistance will then ascend to restore high bacterial densities. Yet there are various mechanisms by which bacteria may remain sensitive to phages but still attain high densities in their presence - because bacteria enter a transient state of reduced adsorption. Importantly, these mechanisms may be cryptic and inapparent prior to the addition of phage yet result in a rapid rebound of bacterial density after phage are introduced. We describe mathematical models of these processes and suggest how different types of this 'phenotypic' resistance may be elucidated. We offer preliminary in vitro studies of a previously characterized E. coli model system and Campylobacter jejuni illustrating apparent phenotypic resistance. As phenotypic resistance may be specific to the receptors used by phages, awareness of its mechanisms may identify ways of improving the choice of phages for therapy. Phenotypic resistance can also explain several enigmas in the ecology of phage-bacterial dynamics. Phenotypic resistance does not preclude the evolution of genetic resistance and may often be an intermediate step to genetic resistance.

Visualizing a complete Siphoviridae member by single-particle electron microscopy: the structure of lactococcal phage TP901-1.

Tailed phages are genome delivery machines exhibiting unequaled efficiency acquired over more than 3 billion years of evolution. Siphophages from the P335 and 936 families infect the Gram-positive bacterium Lactococcus lactis using receptor-binding proteins anchored to the host adsorption apparatus (baseplate). Crystallographic and electron microscopy (EM) studies have shed light on the distinct adsorption strategies used by phages of these two families, suggesting that they might also rely on different infection mechanisms. Here, we report electron microscopy reconstructions of the whole phage TP901-1 (P335 species) and propose a composite EM model of this gigantic molecular machine. Our results suggest conservation of structural proteins among tailed phages and add to the growing body of evidence pointing to a common evolutionary origin for these virions. Finally, we propose that host adsorption apparatus architectures have evolved in correlation with the nature of the receptors used during infection.

Structure and molecular assignment of lactococcal phage TP901-1 baseplate.

P335 lactococcal phages infect the gram(+) bacterium Lactococcus lactis using a large multiprotein complex located at the distal part of the tail and termed baseplate (BP). The BP harbors the receptor-binding proteins (RBPs), which allow the specific recognition of saccharidic receptors localized on the host cell surface. We report here the electron microscopic structure of the phage TP901-1 wild-type BP as well as those of two mutants bppL (-) and bppU(-), lacking BppL (the RBPs) or both peripheral BP components (BppL and BppU), respectively. We also achieved an electron microscopic reconstruction of a partial BP complex, formed by BppU and BppL. This complex exhibits a tripod shape and is composed of nine BppLs and three BppUs. These structures, combined with light-scattering measurements, led us to propose that the TP901-1 BP harbors six tripods at its periphery, located around the central tube formed by ORF46 (Dit) hexamers, at its proximal end, and a ORF47 (Tal) trimer at its distal extremity. A total of 54 BppLs (18 RBPs) are thus available to mediate host anchoring with a large apparent avidity. TP901-1 BP exhibits an infection-ready conformation and differs strikingly from the lactococcal phage p2 BP, bearing only 6 RBPs, and which needs a conformational change to reach its activated state. The comparison of several Siphoviridae structures uncovers a close organization of their central BP core whereas striking differences occur at the periphery, leading to diverse mechanisms of host recognition.