Disease-Associated Mutations in Tau Encode for Changes in Aggregate Structure Conformation.

The accumulation of tau fibrils is associated with neurodegenerative diseases, which are collectively termed tauopathies. Cryo-EM studies have shown that the packed fibril core of tau adopts distinct structures in different tauopathies, such as Alzheimer's disease, corticobasal degeneration, and progressive supranuclear palsy. A subset of tauopathies are linked to missense mutations in the tau protein, but it is not clear whether these mutations impact the structure of tau fibrils. To answer this question, we developed a high-throughput protein purification platform and purified a panel of 37 tau variants using the full-length 0N4R splice isoform. Each of these variants was used to create fibrils in vitro, and their relative structures were studied using a high-throughput protease sensitivity platform. We find that a subset of the disease-associated mutations form fibrils that resemble wild-type tau, while others are strikingly different. The impact of mutations on tau structure was not clearly associated with either the location of the mutation or the relative kinetics of fibril assembly, suggesting that tau mutations alter the packed core structures through a complex molecular mechanism. Together, these studies show that single-point mutations can impact the assembly of tau into fibrils, providing insight into its association with pathology and disease.

In Vitro and In Vivo Evidence towards Fibronectin's Protective Effects against Prion Infection.

A distinctive signature of the prion diseases is the accumulation of the pathogenic isoform of the prion protein, PrPSc, in the central nervous system of prion-affected humans and animals. PrPSc is also found in peripheral tissues, raising concerns about the potential transmission of pathogenic prions through human food supplies and posing a significant risk to public health. Although muscle tissues are considered to contain levels of low prion infectivity, it has been shown that myotubes in culture efficiently propagate PrPSc. Given the high consumption of muscle tissue, it is important to understand what factors could influence the establishment of a prion infection in muscle tissue. Here we used in vitro myotube cultures, differentiated from the C2C12 myoblast cell line (dC2C12), to identify factors affecting prion replication. A range of experimental conditions revealed that PrPSc is tightly associated with proteins found in the systemic extracellular matrix, mostly fibronectin (FN). The interaction of PrPSc with FN decreased prion infectivity, as determined by standard scrapie cell assay. Interestingly, the prion-resistant reserve cells in dC2C12 cultures displayed a FN-rich extracellular matrix while the prion-susceptible myotubes expressed FN at a low level. In agreement with the in vitro results, immunohistopathological analyses of tissues from sheep infected with natural scrapie demonstrated a prion susceptibility phenotype linked to an extracellular matrix with undetectable levels of FN. Conversely, PrPSc deposits were not observed in tissues expressing FN. These data indicate that extracellular FN may act as a natural barrier against prion replication and that the extracellular matrix composition may be a crucial feature determining prion tropism in different tissues.

Innate Immune Status of Glia Modulates Prion Propagation in Early Stage of Infection.

Prion diseases are progressive neurodegenerative disorders affecting humans and various mammals. The prominent neuropathological change in prion-affected brains is neuroinflammation, histopathologically characterized by reactive gliosis surrounding prion deposition. The cause and effect of these cellular responses are still unclear. Here we investigate the impact of innate immune responses on prion replication using in vitro cell culture models. Hamster-adapted transmissible mink encephalopathy prions, hyper (HY) and drowsy (DY) strains, were assayed for accumulation of pathogenic prion protein (PrPSc) in primary glial cultures derived from 8-day-old hamster pups. The kinetics of PrPSc accumulation largely depended on prion strain and brain regions from where glial cells originated. Glial cells derived from the cerebellum were susceptible to HY, but resistant to DY strain as determined by western blot analysis, immunocytochemistry, and animal bioassay. Glial cells from the cerebral cortex were, however, refractory to both strains. PrPSc accumulation was affected by innate immune modulators. Priming glial cells with lipopolysaccharide decreased prion replication, whereas pre-treatment with dexamethasone, inhibiting innate immunity, increased susceptibility to DY infection. Our results suggest that neuroinflammation resulting from prion infection is a response to resolve and/or prevent prion propagation in the brain. It implies a therapeutic potential of innate immune modulation in the early stages of prion disease.

Beta-endoproteolysis of the cellular prion protein by dipeptidyl peptidase-4 and fibroblast activation protein.

The cellular prion protein (PrPC) converts to alternatively folded pathogenic conformations (PrPSc) in prion infections and binds neurotoxic oligomers formed by amyloid-ß α-synuclein, and tau. ß-Endoproteolysis, which splits PrPC into N- and C-terminal fragments (N2 and C2, respectively), is of interest because a protease-resistant, C2-sized fragment (C2Sc) accumulates in the brain during prion infections, seemingly comprising the majority of PrPSc at disease endpoint in mice. However, candidates for the underlying proteolytic mechanism(s) remain unconfirmed in vivo. Here, a cell-based screen of protease inhibitors unexpectedly linked type II membrane proteins of the S9B serine peptidase subfamily to PrPC ß-cleavage. Overexpression experiments in cells and assays with recombinant proteins confirmed that fibroblast activation protein (FAP) and its paralog, dipeptidyl peptidase-4 (DPP4), cleave directly at multiple sites within PrPC's N-terminal domain. For wild-type mouse and human PrPC substrates expressed in cells, the rank orders of activity were human FAP ~ mouse FAP > mouse DPP4 > human DPP4 and human FAP > mouse FAP > mouse DPP4 >> human DPP4, respectively. C2 levels relative to total PrPC were reduced in several tissues from FAP-null mice, and, while knockout of DPP4 lacked an analogous effect, the combined DPP4/FAP inhibitor linagliptin, but not the FAP-specific inhibitor SP-13786, reduced C2Sc and total PrPSc levels in two murine cell-based models of prion infections. Thus, the net activity of the S9B peptidases FAP and DPP4 and their cognate inhibitors/modulators affect the physiology and pathogenic potential of PrPC.

Prion-like strain effects in tauopathies.

Tau is a microtubule-associated protein that plays crucial roles in physiology and pathophysiology. In the realm of dementia, tau protein misfolding is associated with a wide spectrum of clinicopathologically diverse neurodegenerative diseases, collectively known as tauopathies. As proposed by the tau strain hypothesis, the intrinsic heterogeneity of tauopathies may be explained by the existence of structurally distinct tau conformers, "strains". Tau strains can differ in their associated clinical features, neuropathological profiles, and biochemical signatures. Although prior research into infectious prion proteins offers valuable lessons for studying how a protein-only pathogen can encompass strain diversity, the underlying mechanism by which tau subtypes are generated remains poorly understood. Here we summarize recent advances in understanding different tau conformers through in vivo and in vitro experimental paradigms, and the implications of heterogeneity of pathological tau species for drug development.

Prion protein with a mutant N-terminal octarepeat region undergoes cobalamin-dependent assembly into high-molecular weight complexes.

The cellular prion protein (PrPC) has a C-terminal globular domain and a disordered N-terminal region encompassing five octarepeats (ORs). Encounters between Cu(II) ions and four OR sites produce interchangeable binding geometries; however, the significance of Cu(II) binding to ORs in different combinations is unclear. To understand the impact of specific binding geometries, OR variants were designed that interact with multiple or single Cu(II) ions in specific locked coordinations. Unexpectedly, we found that one mutant produced detergent-insoluble, protease-resistant species in cells in the absence of exposure to the infectious prion protein isoform, scrapie-associated prion protein (PrPSc). Formation of these assemblies, visible as puncta, was reversible and dependent upon medium formulation. Cobalamin (Cbl), a dietary cofactor containing a corrin ring that coordinates a Co3+ ion, was identified as a key medium component, and its effect was validated by reconstitution experiments. Although we failed to find evidence that Cbl interacts with Cu-binding OR regions, we instead noted interactions of Cbl with the PrPC C-terminal domain. We found that some interactions occurred at a binding site of planar tetrapyrrole compounds on the isolated globular domain, but others did not, and N-terminal sequences additionally had a marked effect on their presence and position. Our studies define a conditional effect of Cbl wherein a mutant OR region can act in cis to destabilize a globular domain with a wild type sequence. The unexpected intersection between the properties of PrPSc's disordered region, Cbl, and conformational remodeling events may have implications for understanding sporadic prion disease that does not involve exposure to PrPSc.

Distinct populations of highly potent TAU seed conformers in rapidly progressing Alzheimer's disease.

Although genetic factors play a main role in determining the risk of developing Alzheimer's disease (AD), they do not explain extensive spectrum of clinicopathological phenotypes. Deposits of aggregated TAU proteins are one of the main predictors of cognitive decline in AD. We investigated the hypothesis that variabilities in AD progression could be due to diverse structural assemblies (strains) of TAU protein. Using sensitive biophysical methods in 40 patients with AD and markedly different disease durations, we identified populations of distinct TAU particles that differed in size, structural organization, and replication rate in vitro and in cell assay. The rapidly replicating, distinctly misfolded TAU conformers found in rapidly progressive AD were composed of ~80% misfolded four-repeat (4R) TAU and ~20% of misfolded 3R TAU isoform with the same conformational signatures. These biophysical observations suggest that distinctly misfolded population of 4R TAU conformers drive the rapid decline in AD and imply that effective therapeutic strategies might need to consider not a singular species but a cloud of differently misfolded TAU conformers.

Pathologic tau conformer ensembles induce dynamic, liquid-liquid phase separation events at the nuclear envelope.

BACKGROUND: The microtubule-associated protein tau forms aggregates in different neurodegenerative diseases called tauopathies. Prior work has shown that a single P301L mutation in tau gene, MAPT, can promote alternative tau folding pathways that correlate with divergent clinical diagnoses. Using progressive chemical denaturation, some tau preparations from the brain featured complex transitions starting at low concentrations of guanidine hydrochloride (GdnHCl) denaturant, indicating an ensemble of differently folded tau species called conformers. On the other hand, brain samples with abundant, tangle-like pathology had simple GdnHCl unfolding profile resembling the profile of fibrillized recombinant tau and suggesting a unitary conformer composition. In studies here we sought to understand tau conformer progression and potential relationships with condensed liquid states, as well as associated perturbations in cell biological processes. RESULTS: As starting material, we used brain samples from P301L transgenic mice containing tau conformer ensembles that unfolded at low GdnHCl concentrations and with signatures resembling brain material from P301L subjects presenting with language or memory problems. We seeded reporter cells expressing a soluble form of 4 microtubule-binding repeat tau fused to GFP or YFP reporter moieties, resulting in redistribution of dispersed fluorescence signals into focal assemblies that could fuse together and move within processes between adjacent cells. Nuclear envelope fluorescent tau signals and small fluorescent inclusions behaved as a demixed liquid phase, indicative of liquid-liquid phase separation (LLPS); these droplets exhibited spherical morphology, fusion events and could recover from photobleaching. Moreover, juxtanuclear tau assemblies were associated with disrupted nuclear transport and reduced cell viability in a stable cell line. Staining for thioflavin S (ThS) became more prevalent as tau-derived inclusions attained cross-sectional area greater than 3 µm2, indicating (i) a bipartite composition, (ii) in vivo progression of tau conformers, and (iii) that a mass threshold applying to demixed condensates may drive liquid-solid transitions. CONCLUSIONS: Tau conformer ensembles characterized by denaturation at low GdnHCl concentration templated the production of condensed droplets in living cells. These species exhibit dynamic changes and develop in vivo, and the larger ThS-positive assemblies may represent a waystation to arrive at intracellular fibrillar tau inclusions seen in end-stage genetic tauopathies.

Cellular Biology of Tau Diversity and Pathogenic Conformers.

Tau accumulation is a prominent feature in a variety of neurodegenerative disorders and remarkable effort has been expended working out the biochemistry and cell biology of this cytoplasmic protein. Tau's wayward properties may derive from germline mutations in the case of frontotemporal lobar degeneration (FTLD-MAPT) but may also be prompted by less understood cues-perhaps environmental or from molecular damage as a consequence of chronological aging-in the case of idiopathic tauopathies. Tau properties are undoubtedly affected by its covalent structure and in this respect tau protein is not only subject to changes in length produced by alternative splicing and endoproteolysis, but different types of posttranslational modifications that affect different amino acid residues. Another layer of complexity concerns alternate conformations-"conformers"-of the same covalent structures; in vivo conformers can encompass soluble oligomeric species, ramified fibrillar structures evident by light and electron microscopy and other forms of the protein that have undergone liquid-liquid phase separation to make demixed liquid droplets. Biological concepts based upon conformers have been charted previously for templated replication mechanisms for prion proteins built of the PrP polypeptide; these are now providing useful explanations to feature tau pathobiology, including how this protein accumulates within cells and how it can exhibit predictable patterns of spread across different neuroanatomical regions of an affected brain. In sum, the documented, intrinsic heterogeneity of tau forms and conformers now begins to speak to a fundamental basis for diversity in clinical presentation of tauopathy sub-types. In terms of interventions, emphasis upon subclinical events may be worthwhile, noting that irrevocable cell loss and ramified protein assemblies feature at end-stage tauopathy, whereas earlier events may offer better opportunities for diverting pathogenic processes. Nonetheless, the complexity of tau sub-types, which may be present even within intermediate disease stages, likely mitigates against one-size-fits-all therapeutic strategies and may require a suite of interventions. We consider the extent to which animal models of tauopathy can be reasonably enrolled in the campaign to produce such interventions and to slow the otherwise inexorable march of disease progression.

Diverse, evolving conformer populations drive distinct phenotypes in frontotemporal lobar degeneration caused by the same MAPT-P301L mutation.

Tau protein accumulation is a common denominator of major dementias, but this process is inhomogeneous, even when triggered by the same germline mutation. We considered stochastic misfolding of human tau conformers followed by templated conversion of native monomers as an underlying mechanism and derived sensitive conformational assays to test this concept. Assessments of brains from aged TgTauP301L transgenic mice revealed a prodromal state and three distinct signatures for misfolded tau. Frontotemporal lobar degeneration (FTLD)-MAPT-P301L patients with different clinical phenotypes also displayed three signatures, two resembling those found in TgTauP301L mice. As physicochemical and cell bioassays confirmed diverse tau strains in the mouse and human brain series, we conclude that evolution of diverse tau conformers is intrinsic to the pathogenesis of this uni-allelic form of tauopathy. In turn, effective therapeutic interventions in FTLD will need to address evolving repertoires of misfolded tau species rather than singular, static molecular targets.

Proteasomal Inhibition Redirects the PrP-Like Shadoo Protein to the Nucleus.

The Shadoo protein (Sho) exhibits homology to the hydrophobic region of the cellular isoform of prion protein (PrPC). As prion-infected brains gradually accumulate infectivity-associated isoforms of prion protein (PrPSc), levels of mature endogenous Sho become reduced. To study the regulatory effect of the proteostatic network on Sho expression, we investigated the action of lactacystin, MG132, NH4Cl, and 3-methyladenine (3-MA) in two cell culture models. In primary mixed neuronal and glial cell cultures (MNGCs) from transgenic mice expressing wild-type Sho from the PrP gene promoter (Tg.Sprn mice), lactacystin- and MG132-mediated inhibition of proteasomal activity shifted the repertoire of Sho species towards unglycosylated forms appearing in the nuclei; conversely, the autophagic modulators NH4Cl and 3-MA did not affect Sho or PrPC glycosylation patterns. Mouse N2a neuroblastoma cells expressing Sho under control of a housekeeping gene promoter treated with MG132 or lactacystin also showed increased nuclear localization of unglycosylated Sho. As two proteasomal inhibitors tested in two cell paradigms caused redirection of Sho to nuclei at the expense of processing through the secretory pathway, our findings define a balanced shift in subcellular localization that thereby differs from the decreases in net Sho species seen in prion-infected brains. Our data are indicative of a physiological pathway to access Sho functions in the nucleus under conditions of impaired proteasomal activity. We also infer that these conditions would comprise a context wherein Sho's N-terminal nucleic acid-binding RGG repeat region is brought into play.

Dual MicroRNA to Cellular Prion Protein Inhibits Propagation of Pathogenic Prion Protein in Cultured Cells.

Prion diseases are fatal transmissible neurodegenerative disorders affecting humans and various mammals. In spite of intensive efforts, there is no effective cure or treatment for prion diseases. Cellular forms of prion protein (PrPC) is essential for propagation of abnormal isoforms of prion protein (PrPSc) and pathogenesis. The effect of an artificial dual microRNA (DmiR) on PrPC suppression and resultant inhibition of prion replication was determined using prion-infectible cell cultures: differentiated C2C12 culture and primary mixed neuronal and glial cells culture (MNGC). Processing of DmiR by prion-susceptible myotubes, but not by reserve cells, in differentiated C2C12 culture slowed prion replication, implying an importance of cell type-specific PrPC targeting. In MNGC, reduction of PrPC with DmiR was effective for suppressing prion replication. MNGC lentivirally transduced with non-targeting control miRNAs (scrambled) reduced prion replication at a level similar to that with a synthetic analogue of viral RNA, poly I:C. The results suggest that a synergistic combination of the immunostimulatory RNA duplexes (miRNA) and PrPC silencing with DmiR might augment a therapeutic potential of RNA interference.

Toll-like receptor-mediated immune response inhibits prion propagation.

Prion diseases are progressive neurodegenerative disorders affecting humans and various mammals. The prominent neuropathological change in prion diseases is neuroinflammation characterized by activation of neuroglia surrounding prion deposition. The cause and effect of this cellular response, however, is unclear. We investigated innate immune defenses against prion infection using primary mixed neuronal and glial cultures. Conditional prion propagation occurred in glial cultures depending on their immune status. Preconditioning of the cells with the toll-like receptor (TLR) ligand, lipopolysaccharide, resulted in a reduction in prion propagation, whereas suppression of the immune responses with the synthetic glucocorticoid, dexamethasone, increased prion propagation. In response to recombinant prion fibrils, glial cells up-regulated TLRs (TLR1 and TLR2) expression and secreted cytokines (tumor necrosis factor-α, interleukin-1ß, interleukin-6, granulocyte-macrophage colony-stimulating factor, and interferon-ß). Preconditioning of neuronal and glial cultures with recombinant prion fibrils inhibited prion replication and altered microglial and astrocytic populations. Our results provide evidence that, in early stages of prion infection, glial cells respond to prion infection through TLR-mediated innate immunity.

Establishment and characterization of Prnp knockdown neuroblastoma cells using dual microRNA-mediated RNA interference.

Prion diseases are fatal transmissible neurodegenerative disorders. In the pathogenesis of the disease, the cellular prion protein (PrPC) is required for replication of abnormal prion (PrPSc), which results in accumulation of PrPSc. Although there have been extensive studies using Prnp knockout systems, the normal function of PrPC remains ambiguous. Compared with conventional germline knockout technologies and transient naked siRNA-dependent knockdown systems, newly constructed durable chained-miRNA could provide a cell culture model that is closer to the disease status and easier to achieve with no detrimental sequelae. The selective silencing of a target gene by RNA interference (RNAi) is a powerful approach to investigate the unknown function of genes in vitro and in vivo. To reduce PrPC expression, a novel dual targeting-microRNA (miRdual) was constructed. The miRdual, which targets N- and C- termini of Prnp simultaneously, more effectively suppressed PrPC expression compared with conventional single site targeting. Furthermore, to investigate the cellular change following PrPC depletion, gene expression analysis of PrPC interacting and/or associating genes and several assays including proliferation, viability and apoptosis were performed. The transcripts 670460F02Rik and Plk3, Ppp2r2b and Csnk2a1 increase in abundance and are reported to be involved in cell proliferation and mitochondrial-mediated apoptosis. Dual-targeting RNAi with miRdual against Prnp will be useful for analyzing the physiological function of PrPC in neuronal cell lines and may provide a potential therapeutic intervention for prion diseases in the future.

Mouse neuronal cells expressing exogenous bovine PRNP and simultaneous downregulation of endogenous mouse PRNP using siRNAs.

Prion diseases, which are called transmissible spongiform encephalopathies (TSEs), comprise a group of fatal infectious neurodegenerative disorders. Investigation of prion strains and generation of species dependent TSE model are necessary to understand pathogenesis of the disease. To establish a BSE-specific in vitro cell culture model, N2a and GT1 mouse neuronal cell lines were generated to express the bovine prion protein by transfection of the bovine prion gene (Prnp). In addition, the endogenous mouse prion protein was suppressed in N2a, NbP, GT1 and GbP cell lines using the siRNA duplexes, siRNA1 and siRNA2 that target the N- and C-termini of murine Prnp, respectively. Both siRNA1 and siRNA2 effectively decreased murine prion protein levels by more than 80% and the downregulation efficacy was increased in siRNA dose-dependent manner. The greatest downregulation was observed 48 h after siRNA delivery. The moPrnp knockdown NbP and GbP cell lines and the Prnp-targeting siRNA technique established in the present study would be useful tools for dissecting the basic mechanisms of prion infection, especially for BSE.

Assessment of antibiotic resistance phenotype and integrons in Salmonella enterica serovar Typhimurium isolated from swine.

Salmonella enterica serovar Typhimurium (S. Typhimurium) isolated and identified from swine were subjected for the analysis of antibiotic resistance pattern and clinically important class 1 and 2 integrons. In addition, S. Typhimurium isolates exhibiting ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, tetracycline and florfenicol (ACSSuTF) resistance pattern as described in most Salmonella enterica serotype Typhimurium definitive type 104 (DT104) were characterized by polymerase chain reaction. All the isolates were resistant to more than four antibiotics and showed the highest resistance to streptomycin (94.1%), followed by tetracycline (90.1%), ampicillin (64.7%), chloramphenicol (56.8%) and gentamicin (54.9%). MIC value for the ten isolates ranged between 0.125-2 mug/ml for ciprofloxacin. Among the beta-lactams used, only one of the isolate exhibited resistance to ceftiofur (MIC 8 microg/ml). Sixty eight percent of these multi drug resistance (MDR) S. Typhimurium isolates carried clinically important class 1 integron with 1kb (aadA) and/or 2kb (dhfrXII-orfF-aadA2) resistance gene cassettes. This study reports the increasing trend of multi drug resistance (MDR) S. Typhimurium with clinically important class 1 integron in pigs. In addition, emergence of the ACSSuTF-type resistance in S. Typhimurium PT other than DT104 may limit the use of resistance gene markers in its detection methods by PCR.

Chitosan microspheres containing Bordetella bronchiseptica antigens as novel vaccine against atrophic rhinitis in pigs.

The immune-stimulating activities of Bordetella bronchiseptica antigens containing dermonecrotoxin (BBD) loaded in chitosan microspheres (CMs) have already been reported in vitro and in vivo with a mouse alveolar macrophage cell line (RAW264.7) and mice. Therefore, this study attempted to demonstrate the successful induction of mucosal immune responses after the intranasal administration of BBD loaded in CMs (BBD-CMs) in colostrum-deprived pigs. The BBD was introduced to the CMs using an ionic gelation process involving tripolyphosphate (TPP). Colostrum-deprived pigs were then directly immunized through intranasal administration of the BBD-CMs. A challenge with a field isolate of B. bronchiseptica was performed ten days following the final immunization. The BBD-specific IgG and IgA titers, evident in the nasal wash and serum from the vaccinated pigs, increased with time (p<0.05). Following the challenge, the clinical signs of infection were about 6-fold lower in the vaccinated pigs compared with the nonvaccinated pigs. The grades for gross morphological changes in the turbinate bones from the vaccinated pigs were also significantly lower than the grades recorded for the nonvaccinated pigs (p<0.001). Therefore, the mucosal and systemic immune responses induced in the current study would seem to indicate that the intranasal administration of BBD-CMs may be an effective vaccine against atrophic rhinitis in pigs.

Development of multiplex polymerase chain reaction assays for detecting enterotoxigenic Escherichia coli and their application to field isolates from piglets with diarrhea.

Fimbriae and enterotoxins are major virulence factors associated with enterotoxigenic Escherichia coli (ETEC). In this study, 3 sets of multiplex polymerase chain reaction (mPCR) assays targeting fimbriae, enterotoxins, and other adherence factors were developed for detecting ETEC. A total number of 188 E. coli field isolates were examined, and percentages of E. coli strains carrying each virulence factors were as follows: F4 (7.45%), F5 (29.79%), F6 (6.38%), F18 (15.43%), F41 (3.72%), STa (10.11%), STb (20.74%), LT (9.57%), Stx2e (2.13%), EAST1 (42.02%), F1 (67.55%), AIDA-I (2.66%), and pAA (7.45%). Of the 188 E. coli field isolates examined, 25.53% were found to be pathogenic ETEC, having both fimbriae and enterotoxins. However, the ratio increased to 44.68% when the presence of other adhesins was considered as criteria for virulence. Among the adherence factors, F1 was found to be the most prevalent. AIDA-I and pAA were also found with similar ratio as compared with other virulence factors. In addition, virulence patterns carrying these alternate adhesive genes with enterotoxins were detected with significant ratio. Therefore, it is desirable that alternate adhesins be considered as markers for diagnosis of ETEC.

Characterization of TEM-, SHV- and AmpC-type beta-lactamases from cephalosporin-resistant Enterobacteriaceae isolated from swine.

Extended-spectrum beta-lactamase (ESBL) and AmpC-producing Enterobacteriaceae are an increasing problem in human medicine and an emerging problem in the veterinary field. Our study, therefore, focused on assessing the prevalence of beta-lactamases isolated from swine. Sixty-six Salmonella enterica serovar Typhimurium (S. Typhimurium), 33 Salmonella enterica serovar Enteritidis (S. Enteritidis), 26 Klebsiella pneumonia (K. pneumoniae) and 130 Escherichia coli (E. coli) pig isolates collected from 1999-2006 were screened for beta-lactam resistance by the disk diffusion test (DDT) and micro-broth dilution. Among the isolates, five E. coli and five K. pneumoniae exhibited reduced susceptibility to the cephalosporins tested. PCR, plasmid profiling and Southern blot hybridization showed the presence of multiple beta-lactamases in these isolates of animal origin. Hybridization patterns of the DHA-1 specific probe indicated that dissemination of DHA-1 related beta-lactamases could be attributed to plasmids of one common size among the enteric microbes of animal origin. To the best of our knowledge, this study reports the first identification of SHV-28 and DHA-1 from microbes of animal origin.