Aims
Listeria species may colonize and persist in food processing facilities for prolonged periods of time, despite hygiene interventions in place. To understand the genetic factors contributing to ...persistence of Listeria strains, this study undertook a comparative analysis of seven persistent and six presumed non‐persistent strains, isolated from a single food processing environment, to identify genetic markers correlating to promoting persistence of Listeria strains, through whole genome sequence analysis.
Methods and Results
A diverse pool of genetic markers relevant to hygiene tolerance was identified, including disinfectant resistance markers qacH, emrC and the efflux cassette bcrABC. Both persistent and presumed non‐persistent cohorts encoded a range of stress resistance markers, including heavy metal resistance, oxidative and pH stress, although trends were associated with each cohort (e.g., qacH and cadA1C resistance was more frequently found in persistent isolates). Persistent isolates were more likely to contain mutations associated with attenuated virulence, including a truncated InlA. Plasmids and transposons were widespread between cohorts.
Conclusions
Results suggest that no single genetic marker identified was universally responsible for a strain's ability to persist. Persistent strains were more likely to harbour mutation associated with hypovirulence.
Significance and Impact of the Study
This study provides additional insights into the distribution of genetic elements relevant to persistence across Listeria species, as well as strain virulence potential.
The emergence and spread of antimicrobial‐resistant (AMR) bacteria in natural environments is a major concern with serious implications for human and animal health. The aim of this study was to ...determine the prevalence of AMR Escherichia coli (E. coli) in wild birds and mammalian species. Thirty faecal samples were collected from each of the following wildlife species: herring gulls (Larus argentatus), black‐headed gulls (Larus ridibundus), lesser black‐back gulls (Larus fuscus), hybrid deer species (Cervus elaphus x Cervus nippon) and twenty‐six from starlings (Sturnus vulgaris). A total of 115 E. coli isolates were isolated from 81 of 146 samples. Confirmed E. coli isolates were tested for their susceptibility to seven antimicrobial agents by disc diffusion. In total, 5.4% (8/146) of samples exhibited multidrug‐resistant phenotypes. The phylogenetic group and AMR‐encoding genes of all multidrug resistance isolates were determined by PCR. Tetracycline‐, ampicillin‐ and streptomycin‐resistant isolates were the most common resistant phenotypes. The following genes were identified in E. coli: blaTEM , strA, tet(A) and tet(B). Plasmids were identified in all samples that exhibited multidrug‐resistant phenotypes. This study indicates that wild birds and mammals may function as important host reservoirs and potential vectors for the spread of resistant bacteria and genetic determinants of AMR.
Spontaneous activity in the sensory periphery drives infant brain activity and is thought to contribute to the formation of retinotopic and somatotopic maps 1–3. In infant rats during active (or REM) ...sleep, brainstem-generated spontaneous activity triggers hundreds of thousands of skeletal muscle twitches each day 4; sensory feedback from the resulting limb movements is a primary activator of forebrain activity 1. The rodent whisker system, with its precise isomorphic mapping of individual whiskers to discrete brain areas, has been a key contributor to our understanding of somatotopic maps and developmental plasticity 5–7. But although whisker movements are controlled by dedicated skeletal muscles 8, 9, spontaneous whisker activity has not been entertained as a contributing factor to the development of this system 10. Here we report in 3- to 6-day-old rats that whiskers twitch rapidly and asynchronously during active sleep; furthermore, neurons in whisker thalamus exhibit bursts of activity that are tightly associated with twitches but occur infrequently during waking. Finally, we observed barrel-specific cortical activity during periods of twitching. This is the first report of self-generated, sleep-related twitches in the developing whisker system, a sensorimotor system that is unique for the precision with which it can be experimentally manipulated. The discovery of whisker twitching will allow us to attain a better understanding of the contributions of peripheral sensory activity to somatosensory integration and plasticity in the developing nervous system 11–13.
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► Whiskers twitch rapidly and asynchronously in sleeping infant rats ► Whisker twitches result from activation of dedicated whisker skeletal muscles ► Whisker twitches trigger bursts of neural activity in whisker thalamus ► Barrel cortex exhibits discrete activation during periods of twitching
The structure and function of both adherens (AJ) and tight (TJ) junctions are dependent on the cortical actin cytoskeleton. The zonula occludens (ZO)-1 and -2 proteins have context-dependent ...interactions with both junction types and bind directly to F-actin and other cytoskeletal proteins, suggesting ZO-1 and -2 might regulate cytoskeletal activity at cell junctions. To address this hypothesis, we generated stable Madin-Darby canine kidney cell lines depleted of both ZO-1 and -2. Both paracellular permeability and the localization of TJ proteins are disrupted in ZO-1/-2-depleted cells. In addition, immunocytochemistry and electron microscopy revealed a significant expansion of the perijunctional actomyosin ring associated with the AJ. These structural changes are accompanied by a recruitment of 1-phosphomyosin light chain and Rho kinase 1, contraction of the actomyosin ring, and expansion of the apical domain. Despite these changes in the apical cytoskeleton, there are no detectable changes in cell polarity, localization of AJ proteins, or the organization of the basal and lateral actin cytoskeleton. We conclude that ZO proteins are required not only for TJ assembly but also for regulating the organization and functional activity of the apical cytoskeleton, particularly the perijunctional actomyosin ring, and we speculate that these activities are relevant both to cellular organization and epithelial morphogenesis.
ZO-1 binds numerous transmembrane and cytoplasmic proteins and is required for assembly of both adherens and tight junctions, but its role in defining barrier properties of an established tight ...junction is unknown. We depleted ZO-1 in MDCK cells using siRNA methods and observed specific defects in the barrier for large solutes, even though flux through the small claudin pores was unaffected. This permeability increase was accompanied by morphological alterations and reorganization of apical actin and myosin. The permeability defect, and to a lesser extent morphological changes, could be rescued by reexpression of either full-length ZO-1 or an N-terminal construct containing the PDZ, SH3, and GUK domains. ZO-2 knockdown did not replicate either the permeability or morphological phenotypes seen in the ZO-1 knockdown, suggesting that ZO-1 and -2 are not functionally redundant for these functions. Wild-type and knockdown MDCK cells had differing physiological and morphological responses to pharmacologic interventions targeting myosin activity. Use of the ROCK inhibitor Y27632 or myosin inhibitor blebbistatin increased TER in wild-type cells, whereas ZO-1 knockdown monolayers were either unaffected or changed in the opposite direction; paracellular flux and myosin localization were also differentially affected. These studies are the first direct evidence that ZO-1 limits solute permeability in established tight junctions, perhaps by forming a stabilizing link between the barrier and perijunctional actomyosin.
Polarized epithelia assemble into sheets that compartmentalize organs and generate tissue barriers by integrating apical surfaces into a single, unified structure. This tissue organization is shared ...across organs, species, and developmental stages. The processes that regulate development and maintenance of apical epithelial surfaces are, however, undefined. Here, using an intestinal epithelial-specific knockout (KO) mouse and cultured epithelial cells, we show that the tight junction scaffolding protein zonula occludens-1 (ZO-1) is essential for development of unified apical surfaces in vivo and in vitro. We found that U5 and GuK domains of ZO-1 are necessary for proper apical surface assembly, including organization of microvilli and cortical F-actin; however, direct interactions with F-actin through the ZO-1 actin-binding region (ABR) are not required. ZO-1 lacking the PDZ1 domain, which binds claudins, rescued apical structure in ZO-1–deficient epithelia, but not in cells lacking both ZO-1 and ZO-2, suggesting that heterodimerization with ZO-2 restores PDZ1-dependent ZO-1 interactions that are vital to apical surface organization. Pharmacologic F-actin disruption, myosin II motor inhibition, or dynamin inactivation restored apical epithelial structure in vitro and in vivo, indicating that ZO-1 directs epithelial organization by regulating actomyosin contraction and membrane traffic. We conclude that multiple ZO-1–mediated interactions contribute to coordination of epithelial actomyosin function and genesis of unified apical surfaces.
Tight junctions (TJs) regulate the paracellular movement of ions, macromolecules and immune cells across epithelia. Zonula occludens (ZO)-1 is a multi-domain polypeptide required for the assembly of ...TJs. MDCK II cells lacking ZO-1, and its homolog ZO-2, have three distinct phenotypes: reduced localization of occludin and some claudins to the TJs, increased epithelial permeability, and expansion of the apical actomyosin contractile array found at the apical junction complex (AJC). However, it is unclear exactly which ZO-1 binding domains are required to coordinate these activities. We addressed this question by examining the ability of ZO-1 domain-deletion transgenes to reverse the effects of ZO depletion. We found that the SH3 domain and the U5 motif are required to recruit ZO-1 to the AJC and that localization is a prerequisite for normal TJ and cytoskeletal organization. The PDZ2 domain is not required for localization of ZO-1 to the AJC, but is necessary to establish the characteristic continuous circumferential band of ZO-1, occludin and claudin-2. PDZ2 is also required to establish normal permeability, but is not required for normal cytoskeletal organization. Finally, our results demonstrate that PDZ1 is crucial for the normal organization of both the TJ and the AJC cytoskeleton. Our results establish that ZO-1 acts as a true scaffolding protein and that the coordinated activity of multiple domains is required for normal TJ structure and function.
The function of occludin remains elusive. Proposed roles include maintenance of tight junction barriers, signaling and junction remodeling. To investigate a potential role in mediating ...cytokine-induced changes in barrier properties, we measured barrier responses to interferon-γ plus TNFα in control, occludin-overexpressing and occludin knockdown MDCK II monolayers. MDCK cells show a complex response to cytokines characterized by a simultaneous increase in the transepithelial electrical resistance and a decrease in the barrier for large solutes. We observed that overexpression of occludin increased and occludin knockdown decreased sensitivity to cytokines as assessed by both these parameters. It is known that caveolin-1 interacts with occludin and is implicated in several models of cytokine-dependent barrier disruption; we found that occludin knockdown altered the subcellular distribution of caveolin-1 and that partitioning of caveolin into detergent-insoluble lipid rafts was influenced by changing occludin levels. Knockdown of caveolin decreased the cytokine-induced flux increase, whereas the increase in the electrical barrier was unaltered; the effect of double knockdown of occludin and caveolin was similar to that of occludin single knockdown, consistent with the possibility that they function in the same pathway. These results demonstrate that occludin is required for cells to transduce cytokine-mediated signals that either increase the electrical barrier or decrease the large solute barrier, possibly by coordinating the functions of caveolin-1.
1 Institute for Food Safety and Hygiene, Vetsuisse Faculty, University of Zurich, CH-8057, Zurich, Switzerland
2 Centre for Food Safety and Food-borne Zoonomics, UCD Veterinary Sciences Centre, ...University College Dublin, Belfield, Dublin 4, Ireland
3 US Food and Drug Administration, Laurel, MD 20708, USA
4 Quality and Safety Department, Nestlé Research Centre, Vers-chez-les-Blanc, CH-1000 Lausanne, Switzerland
Correspondence Roger Stephan stephanr{at}fsafety.unizh.ch
Enterobacter sakazakii is an opportunistic pathogen that can cause infections in neonates. This study further clarifies the taxonomy of isolates described as E. sakazakii and completes the formal description of the proposed reclassification of these organisms as novel species and subspecies within a proposed novel genus, Cronobacter gen. nov. E. sakazakii was first defined in 1980, however recent polyphasic taxonomic analysis has determined that this group of organisms consists of several genomospecies. In this study, the phenotypic descriptions of the proposed novel species are expanded using Biotype 100 and Biolog Phenotype MicroArray data. Further DNA–DNA hybridization experiments showed that malonate-positive strains within the E. sakazakii genomospecies represent a distinct species, not a subspecies. DNA–DNA hybridizations also determined that phenotypically different strains within the proposed species, Cronobacter dublinensis sp. nov., belong to the same species and can be considered as novel subspecies. Based on these analyses, the following alternative classifications are proposed: Cronobacter sakazakii gen. nov., comb. nov. type strain ATCC 29544 T (=NCTC 11467 T ); Cronobacter malonaticus sp. nov. type strain CDC 1058-77 T (=LMG 23826 T =DSM 18702 T ); Cronobacter turicensis sp. nov. type strain z3032 T (=LMG 23827 T =DSM 18703 T ); Cronobacter muytjensii sp. nov. type strain ATCC 51329 T (=CIP 103581 T ); Cronobacter dublinensis sp. nov. type strain DES187 T (=LMG 23823 T =DSM 18705 T ); Cronobacter dublinensis subsp. dublinensis subsp. nov. type strain DES187 T (=LMG 23823 T =DSM 18705 T ); Cronobacter dublinensis subsp. lausannensis subsp. nov. type strain E515 T (=LMG 23824=DSM 18706 T ), and Cronobacter dublinensis subsp. lactaridi subsp. nov. type strain E464 T (=LMG 23825 T =DSM 18707 T ).
Abbreviations: f-AFLP, fluorescent-amplified fragment length polymorphism
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of the members of the genus Cromobacter gen. nov. described in this paper are the following: Cronobacter sakazakii gen. nov., comb. nov. ATCC 29544 T , EF059843 ; Cronobacter malonaticus sp. nov. CDC 1058-77 T , EF059881 ; Cronobacter turicensis sp. nov. z3032 T , EF059891 ; Cronobacter muytjensii sp. nov. ATCC 51329 T , EF059845 ; Cronobacter dublinensis sp. nov. DES187 T , EF059892 ; Cronobacter dublinensis subsp. lausannensis subsp. nov. E515 T , EF059841 ; Cronobacter dublinensis subsp. lactaridi subsp. nov. E464 T , EF059838 , and Cronobacter genomospecies 1 strain NCTC 9529, EF059877 .
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