Serotype Diversity and Antimicrobial Resistance among Salmonella enterica Isolates from Patients at an Equine Referral Hospital.

Although Salmonella enterica can produce life-threatening colitis in horses, certain serotypes are more commonly associated with clinical disease. Our aim was to evaluate the proportional morbidity attributed to different serotypes, as well as the phenotypic and genotypic antimicrobial resistance (AMR) of Salmonella isolates from patients at an equine referral hospital in the southern United States. A total of 255 Salmonella isolates was obtained from clinical samples of patients admitted to the hospital between 2007 and 2015. Phenotypic resistance to 14 antibiotics surveilled by the U.S. National Antimicrobial Resistance Monitoring System was determined using a commercially available panel. Whole-genome sequencing was used to identify serotypes and genotypic AMR. The most common serotypes were Salmonella enterica serotype Newport (18%), Salmonella enterica serotype Anatum (15.2%), and Salmonella enterica serotype Braenderup (11.8%). Most (n = 219) of the isolates were pansusceptible, while 25 were multidrug resistant (≥3 antimicrobial classes). Genes encoding beta-lactam resistance, such as blaCMY-2, blaSHV-12, blaCTX-M-27, and blaTEM-1B, were detected. The qnrB2 and aac(6')-Ib-cr genes were present in isolates with reduced susceptibility to ciprofloxacin. Genes encoding resistance to gentamicin (aph(3')-Ia, aac(6')-IIc), streptomycin (strA and strB), sulfonamides (sul1), trimethoprim (dfrA), phenicols (catA), tetracyclines [tet(A) and tet(E)], and macrolides [ere(A)] were also identified. The main predicted incompatibility plasmid type was I1 (10%). Core genome-based analyses revealed phylogenetic associations between isolates of common serotypes. The presence of AMR Salmonella in equine patients increases the risk of unsuccessful treatment and causes concern for potential zoonotic transmission to attending veterinary personnel, animal caretakers, and horse owners. Understanding the epidemiology of Salmonella in horses admitted to referral hospitals is important for the prevention, control, and treatment of salmonellosis.IMPORTANCE In horses, salmonellosis is a leading cause of life-threatening colitis. At veterinary teaching hospitals, nosocomial outbreaks can increase the risk of zoonotic transmission, lead to restrictions on admissions, impact hospital reputation, and interrupt educational activities. The antimicrobials most often used in horses are included in the 5th revision of the World Health Organization's list of critically important antimicrobials for human medicine. Recent studies have demonstrated a trend of increasing bacterial resistance to drugs commonly used to treat Salmonella infections. In this study, we identify temporal trends in the distribution of Salmonella serotypes and their mechanisms of antimicrobial resistance; furthermore, we are able to determine the likely origin of several temporal clusters of infection by using whole-genome sequencing. These data can be used to focus strategies to better contain the dissemination and enhance the mitigation of Salmonella infections and to provide evidence-based policies and guidelines to steward antimicrobial use in veterinary medicine.

Contamination, distribution and pathogenicity of Toxocara canis and T. cati eggs from sandpits in Tokyo, Japan.

The contamination, distribution and pathogenicity of Toxocara canis and T. cati eggs in sandpits in the Tokyo metropolitan area, Japan, are described. A total of 34 sandpits were examined, 14 of which were contaminated with T. cati eggs, as assessed by the floatation method and polymerase chain reaction (PCR) analysis. Two naturally contaminated sandpits were investigated to determine the vertical and horizontal distribution of eggs, and an inverse relationship between the sand depth and number of eggs was observed. To examine the pathogenicity of the eggs, three ICR mice were inoculated with 300 eggs, which were recovered from sandpits. The mice exhibited eosinophilia in the peripheral blood and IgG antibody production in the sera after 3 weeks of infection. Most migrating larvae were recovered from carcasses, although three were found in the brains of two infected mice. These three larvae were determined to be T. canis by PCR, revealing that not only T. cati, but also T. canis eggs could be found in sandpits and, further, that eggs recovered from sandpits have the ability to invade a paratenic host.

Cell cycle regulation in bacteria.

Significant progress has been made in the study of ftsZ expression and the topology of FtsZ protein localization in Escherichia coli cells. Exciting results on the identification of new genes required for chromosome resolution and partitioning after the completion of DNA synthesis have also been reported. A recent area of study is asymmetric cell division and its role in differentiation in Bacillus subtilis and Caulobacter crescentus. Biochemical activities of bacterial cell division gene products are also beginning to be addressed.

Association between an HLA haplotype and low responsiveness to schistosomal worm antigen in man.

Immune responsiveness to schistosomal worm antigen was investigated in the individuals infected with Schistosoma japonicum by measuring the antigen-specific proliferative response of the peripheral T lymphocytes in vitro. Out of 57 infected individuals, 10 (17.5%) were low responders, whereas 47 (82.5%) were high responders to this antigen. The strong association between the HLA-Aw24-Bw52-Dw12 haplotype and the low responder group was demonstrated. Because the association of the low responder group was strongest with the HLA-D specificity, Dw12, and because the HLA-D region was assumed to be comparable to the I region of the murine H-2 complex, it was suggested that the low responsiveness to schistosomal worm antigen was controlled by a single dominant immune suppression gene that was in strong linkage disequilibrium with HLA-Dw12.

Formation mechanism studies of phenylene-bridged periodic mesoporous organosilicas (PMOs).

The formation of phenylene-bridged periodic mesoporous organosilicas (PMOs) in the presence of three diblock copolymers differing in hydrophobic hydrocarbon chain lengths was investigated. Hexaethylene glycol octadecylether (C(18)(EO)(6)), hexaethylene glycol hexadecylether (C(16)(EO)(6)), and hexaethylene glycol dodecylether (C(12)(EO)(6)) were chosen in order to obtain insight on the influence of the hydrocarbon chain length on the mesostructure. 1,4-Bis(triethoxysilyl)benzene (BTEB) was used as organosilica precursor under mild acidic conditions. The reactions were followed by in situ small-angle X-ray diffraction (SAXD) on-time in a capillary flow setup. It was found that during the reaction the formation of different structures was observed, which is ascribed to the hydrolysis and condensation of the organosilica precursor. In all cases, different structures evolve with time and phase transitions are observed during the measurements independent of the hydrocarbon chain length.

Evaluation of enamel crystallites in subsurface lesion by microbeam X-ray diffraction.

Early caries lesion is a demineralization process that takes place in the top 0.1 mm layer of tooth enamel. In this study, X-ray microbeam diffraction was used to evaluate the hydroxyapatite crystallites in the subsurface lesion of a bovine enamel section and the results are compared with those obtained by transversal microradiography, a method commonly used for evaluation of tooth mineral. Synchrotron radiation from SPring-8 was used to obtain a microbeam with a diameter of 6 microm. Wide-angle X-ray diffraction reports the amount of hydroxyapatite crystals, and small-angle X-ray scattering reports that of voids in crystallites. All three methods showed a marked decrease in the enamel density in the subsurface region after demineralization. As these diffraction methods provide structural information in the nanometre range, they are useful for investigating the mechanism of the mineral loss in early caries lesion at a nanometre level.

Nuclear encoding of a chloroplast RNA polymerase sigma subunit in a red alga.

A chloroplast RNA polymerase sigma factor is encoded by a nuclear gene, sigA, in the red alga Cyanidium caldarium RK-1. The encoded protein functions as an RNA polymerase sigma factor in vitro and it is localized to the chloroplast in vivo. SigA shows high sequence similarity to the sigma factors of cyanobacteria, which is indicative of the ancestral endosymbiotic event and subsequent transfer of the sigA gene to the nuclear genome.

An improved method for recovery of muscle-stage larvae from mice infected with Toxocara canis.

We report a modified digestion method that improves the recovery of Toxocara canis larvae from skeletal muscle. Minced muscle tissue from infected mice was incubated in artificial gastric juice for 48 hr at 37 C, and ethanol was added for the second 24 hr. This procedure allowed the larvae to be identified and counted more quickly than with the standard digestion method. This method allows measurement of the total number of larvae present in muscle tissue following oral intubation of embryonated eggs, although it does not permit counting of live larvae.

Schistosoma japonicum egg antigen-specific T cell lines in man. Induction of helper and suppressor T cell lines and clones in vitro in a patient with chronic schistosomiasis japonica.

T cell lines (TCLs) specific for Schistosoma japonicum egg antigen were established from a patient with chronic schistosomiasis japonica to investigate the regulatory mechanism of S.japonicum egg antigen-driven T cell responses in man. All five TCLs tested were CD2+, CD4+, CD8-, and were strongly proliferative only to S. japonicum egg antigen in the absence of exogenous IL-2. All but one TCL produced IL-2-like lymphokines in vitro, indicating their helper T cell functions. One TCL, SjE-3, failed to produce IL-2-like lymphokines. Moreover, this TCL suppressed the specific proliferation of autologous peripheral blood lymphocytes to S. japonicum egg antigen. This TCL produced a soluble suppressor factor(s). These functional diversities among established TCLs were also confirmed by cloned T cells. Our observations might suggest that the regulatory system through helper and suppressor T-T interactions somehow involved in T cell responses to the egg antigen in human chronic schistosomiasis japonica.

Clonal expansion of CD8+ cytotoxic T lymphocytes against human T cell lymphotropic virus type I (HTLV-I) genome products in HTLV-I-associated myelopathy/tropical spastic paraparesis patients.

Short-term culture of peripheral blood mononuclear cells (PBMC) derived from patients with human T cell lymphotropic virus type I-associated myelopathy (HAM)/tropical spastic paraparesis resulted in dominance by DR+ activated CD8+ T cells. Variations in the T cell receptor (TCR) V alpha and V beta chains in these cells were analyzed, and in all 10 patients examined, 2-3 V gene families were dominant in both TCR V alpha and V beta. In five patients we examined, cultured lymphocytes contained cytotoxic lymphocytes for p40tax (patients HAM2, 3, 7, and 8) or env protein (patient HAM4) of human T lymphotropic virus type I. In patients HAM2 and HAM8, cultured lymphocytes contained a large proportion of V beta 8+ CD8+ and/or V beta 12+ CD8+ cells. The sequence of V beta 8+ and V beta 12+ cDNA revealed that they were oligoclonal with identical or similar sequences in each patient. Elimination experiments with monoclonal antibodies for TCR V beta 8 and V beta 12 showed that they were CD8+ cytotoxic T lymphocytes (CTL) for p40tax. In addition, flow cytometry and sequencing analysis of uncultured PBMC revealed that in HAM2, V beta 8+ CTL and their precursors account for 7% and V beta 12+ CTL and their precursors account for 18% of total CD8+ cells. This indicates the presence of two markedly expanded clones in vivo. No common dominant TCR V alpha or V beta were observed among 10 HAM patients analyzed.

A microbeam X-ray diffraction study of insulin spherulites.

The peptide hormone insulin forms a spherical aggregate, called a spherulite, at low pH and high temperature. A spherulite is composed of a core and many fibrils extending from it. These fibrils are thought to be amyloid fibers with a beta-sheet structure. In the present study, spherulites with a diameter of 50-100 microm were examined by X-ray fiber diffraction using a 6 microm beam. When a spherulite was scanned with the microbeam and the observed diffraction patterns were arranged in a two-dimensional array, the direction of the scatter was centrosymmetric, demonstrating a symmetric growth of fibrils. There were diffraction peaks at Bragg spacings of 23 nm, 3.3 nm and 1.2 nm in the direction perpendicular to the fibrils and 0.48 nm along the fibrils. The 0.48 nm reflection shows that the hydrogen bonds between beta-strands are along the fibril. The 23 nm reflection corresponds to the separation between fibrils, the 3.3 nm reflection is due to the arrangement of protofilaments, and the 1.2 nm reflection arises from the arrangement of peptide chains. On the basis of these results, a model of a fibril with an extended insulin molecule is proposed.

Identification of a Plasmid-Mediated Quinolone Resistance Gene in Salmonella Isolates from Texas Dairy Farm Environmental Samples.

A recent increase in plasmid-mediated quinolone resistance (PMQR) has been detected among Salmonella isolated from humans in the United States, and it is necessary to determine the sources of human infection. We had previously isolated Salmonella from dairy farm environmental samples collected in Texas, and isolates were tested for anti-microbial susceptibility. Two isolates, serotyped as Salmonella Muenster, showed the discordant pattern of nalidixic acid susceptibility and intermediate susceptibility to ciprofloxacin. For this project, whole-genome sequencing of both isolates was performed to detect genes associated with quinolone resistance. The plasmid-mediated qnrB19 gene and IncR plasmid type were identified in both isolates. To our knowledge, this is the first report of PMQR in Salmonella isolated from food animals or agricultural environments in the United States.

Expression of glucocorticoid receptors in non-neoplastic lymphoid follicles and B cell type malignant lymphomas.

OBJECTIVE: To evaluate the expression of human glucocorticoid receptors (hGRs), such as hGR (4H2), hGR-alpha, and hGR-beta, in non-neoplastic lymphoid follicles and B cell type malignant lymphomas. METHODS: The expression of hGRs in non-neoplastic lymphoid follicles and malignant lymphomas, including diffuse large cell lymphoma, mantle cell lymphoma, and follicular lymphoma, was examined immunohistochemically. HGR (4H2) expression was confirmed by double immunostaining of tissues and in isolated cells from tonsillar germinal centres, and by immunoelectronmicroscopy. RESULTS: In secondary lymphoid follicles of any non-neoplastic diseases--such as chronic tonsillitis, reactive lymphadenitis, and Kimura's disease--the germinal centre cells often expressed hGR (4H2) and hGR-alpha. Double immunocytochemical staining of isolated germinal centre cells showed that the majority of hGR (4H2) positive cells were CD20 positive B cells, and that follicular dendritic cells also expressed hGR. Immunoelectronmicroscopy revealed the presence of nuclear hGR (4H2) in the binucleated follicular dendritic cells and germinal centre cells. The frequency of hGR (4H2) expression in diffuse large B cell lymphoma was higher, that in mantle cell lymphoma was lower, and that in follicular lymphoma was intermediate among the types of malignant lymphoma. The hGR (4H2) expression was less frequent in cases of grade I follicular lymphoma. CONCLUSIONS: There are differences in hGR expression between the germinal centre and the mantle zone in non-neoplastic lymphoid follicles, and differences of hGR (4H2) expression among the types of malignant lymphoma and grades of follicular lymphoma, which probably contribute to the different steroid sensitivities.

DNA-binding specificity and dimerization of the DNA-binding domain of the PEND protein in the chloroplast envelope membrane.

The PEND protein is a DNA-binding protein in the inner envelope membrane of a developing chloroplast, which may anchor chloroplast nucleoids. Here we report the DNA-binding characteristics of the N-terminal basic region plus leucine zipper (bZIP)-like domain of the PEND protein that we call cbZIP domain. The basic region of the cbZIP domain diverges significantly from the basic region of known bZIP proteins that contain a bipartite nuclear localization signal. However, the cbZIP domain has the ability to dimerize in vitro. Selection of binding sites from a random sequence pool indicated that the cbZIP domain preferentially binds to a canonical sequence, TAAGAAGT. The binding site was also confirmed by gel mobility shift analysis using a representative binding site within the chloroplast DNA. These results suggest that the cbZIP domain is a unique DNA-binding domain of the chloroplast protein.

Prevention of interstitial fibrosis of renal allograft by angiotensin II blockade.

We previously confirmed that losartan (LOS), an angiotensin-II (A-II) receptor blocker, diminished plasminogen activator inhibitor-1 (PAI-1) in cyclosporine (CsA)-treated renal graft recipients. Because PAI-1 is known to correlate with tissue fibrosis, we speculated that LOS would have the potential to prevent renal graft interstitial fibrosis. In this study, we focused our attention on the LOS-induced histopathologic changes in renal grafts. Out of 24 CsA-treated normotensive kidney transplanted patients, 8 began to take 25 to 50 mg/day of LOS soon after kidney transplantation (group 1). Eight did so 2 years after kidney transplantation (group 2). Eight received no ARBs as a control group (group 3). PAI-1 levels were monitored every 3 months for 2 years. Renal graft biopsy was performed on all participants, with informed consent, before and 2 years after the onset of this study. The biopsy specimens were stained with periodic acid-methenamine-silver (PAM)-Masson stain for light-microscopic examination. Fibrotic areas in each biopsy specimen were measured using the LUZEX-III image analyzing system. Statistical analysis was performed using Student's t-test. When we considered the pre-value of PAI-1 in each patient as 100%, the mean percent value of PAI-1 at 2 years after the onset of this study of groups 1, 2, and 3 were 81.5 +/- 10.3%, 90.1 +/- 12.5%, and 116.8 +/- 11.9%, respectively (P < .01 groups 1 and 2 vs group 3). Light-microscopic examination revealed less remarkable renal interstitial fibrosis among LOS administered groups. A-II blockade may be a key to prevent renal graft interstitial fibrosis.

Signal transduction in the cell cycle regulation of Caulobacter differentiation.

Caulobacter crescentus differentiates to form a new cell type during asymmetric cell division. Recent results indicate that signal transduction pathways mediated by protein kinases and essential response regulators play a central role in the regulation of development and cell division in response to cell cycle checkpoints.

Characterization of unstable poly (A)-RNA in Caulobacter crescentus.

A significant amount of poly(A)-RNA in Caulobacter crescentus is located on polysomes and the size distribution of this polysomal poly(A)-RNA is small compared to the total pulse-labeled RNA in these cells. These observations suggest that the poly (A)-RNA represents a subset of small messenger RNA molecules. Poly (A)-RNA, and presumably the poly (A) portion of these molecules is extremely unstable: as assayed by binding to oligo (dT)-cellulose the poly (A)-RNA turns over with a chemical half-life of 15--20 s compared to a half-life of approx. 2 min for total cellular messenger RNA. The presence of adenosine in hydrolysates of poly(A) tracts showed that these sequences are located at the 3'-OH end of the RNA. The ratios of AMP/adenosine in the samples confirmed that the length of the A-tracts is approx. 13-17 nucleotides (Ohta, N., Sanders, M. and Newton, A. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 2343--2346)

Novel structural difference between nopaline- and octopine-type trbJ genes: construction of genetic and physical map and sequencing of trb/traI and rep gene clusters of a new Ti plasmid pTi-SAKURA.

We constructed a gene library of a nopaline-type Ti plasmid (called pTi-SAKURA) which was newly isolated from Agrobacterium tumefaciens MAFF301001 of a Japanese cherry tree. The partial sequencing data, which were distributed over the entire plasmid genome, made it possible to assign typical Ti-encoded genes including trb and rep gene clusters. The trb/traI and rep gene clusters were sequenced completely. All the genes in the regions except trbJ were homologous with the corresponding genes on octopine-type Ti plasmids, based on both ORF size and sequence similarity. The trbJ on pTi-SAKURA is similar to that of an octopine-type Ti, but has an extra 282-base segment in its central domain. The above gene organization and sequences suggest a divergence of Ti plasmid during evolution in relation to Rhizobium plasmids, and is discussed in this paper.

Transcriptional regulation of a periodically controlled flagellar gene operon in Caulobacter crescentus.

Temporal regulation of flagellar gene expression in Caulobacter crescentus has been examined by a detailed analysis of the flbG-flaJ-flbH-flaK hook operon. The approximate location of the promoter for this 4.4 X 10(3) base-pair transcriptional unit was determined by deletion mapping, and the flaK gene was shown by nucleotide sequencing to code for the hook protein. flaK messenger RNA was quantified by S1 nuclease mapping with an internal restriction fragment of the gene as the 5'-labeled DNA probe. The results of these assays provide the first direct evidence that periodic expression of a flagellar gene in the C. crescentus cell cycle is regulated at the transcriptional level. The effect of altering the time of gene duplication in the cell cycle was examined by subcloning the complete hook operon on a plasmid that replicates throughout the S phase. The normal periodicity of flaK transcription and translation was maintained in this merodiploid strain, which suggests that replication alone is not sufficient to initiate flagellar gene expression. We also show that the three adjacent transcriptional units III, IV and V are required in trans for transcription of the book operon, and we discuss the possible role of these genes in the hierarchical regulation of the flagellar gene expression.

Inhibition of plasminogen activator inhibitor-1 by angiotensin II receptor blockers on cyclosporine-treated renal allograft recipients.

INTRODUCTION: We previously showed that proteinuria from a renal graft was significantly decreased by administration of losartan potassium, an angiotensin II receptor blockers (ARB). To further evaluate the mechanism, we performed another clinical study focusing on the change in plasma plasminogen activator inhibitor-1 (PAI-1) levels among cyclosporine (CyA)-treated renal allograft recipients. METHODS: Among 12 hypertensive CyA-treated kidney transplant patients, four received 25 to 50 mg/day of losartan; four, 4 to 8 mg/day of candesartan cilexetil; and another four, 20 to 40 mg/day of nifedipine. Four CyA-treated kidney-transplanted patients without hypertension were selected as a control group. Informed consent was obtained from all participants. PAI-1 and serum creatinine (S-Cr) levels were monitored every 3 months for 1 year. RESULTS: Considering the pretreatment of PAI-1 as 100%, the mean percent of PAI-1 at 1 year after the onset of study for losartan, candesartan, nifedipine, and control groups were 78.6 +/- 6.7%, 81.4 +/- 8.0%, 96.7 +/- 7.6%, and 110.4 +/- 9.2%, respectively. The ARB groups demonstrated significant differences from the control group (P < .01), while the nifedipine group did not. S-Cr levels among ARB-administered groups were increased slightly but temporarily. As for S-Cr levels, no significant differences were seen among the four groups. CONCLUSIONS: Control of hypertension itself is important for all renal graft recipients; however, PAI-1 reduction by ARBs was thought to be a key for renal preservation. We expect that ARBs will contribute to prolonged renal allograft survival.