Speed, energy and area optimized early output quasi-delay-insensitive array multipliers.

Multiplication is a widely used arithmetic operation that is frequently encountered in micro-processing and digital signal processing. Multiplication is implemented using a multiplier, and recently, QDI asynchronous array multipliers were presented in the literature utilizing delay-insensitive double-rail data encoding and four-phase return-to-zero (RTZ) handshaking and four-phase return-to-one (RTO) handshaking. In this context, this article makes two contributions: (i) the design of a new asynchronous partial product generator, and (ii) the design of a new asynchronous half adder. We analyze the usefulness of the proposed partial product generator and the proposed half adder to efficiently realize QDI array multipliers. When the new partial product generator and half adder are used along with our indicating full adder, significant reductions are achieved in the design metrics compared to the optimum QDI array multiplier reported in the literature. The cycle time is reduced by 17%, the area is reduced by 16.1%, the power is reduced by 15.3%, and the product of power and cycle time is reduced by 29.6% with respect to RTZ handshaking. On the other hand, the cycle time is reduced by 13%, the area is reduced by 16.1%, the power is reduced by 15.2%, and the product of power and cycle time is reduced by 26.1% with respect to RTO handshaking. Further, the RTO handshaking is found to be preferable to RTZ handshaking to achieve slightly improved optimizations in the design metrics. The QDI array multipliers were realized using a 32/28nm complementary metal oxide semiconductor (CMOS) process technology.

Speed and energy optimized quasi-delay-insensitive block carry lookahead adder.

We present a new asynchronous quasi-delay-insensitive (QDI) block carry lookahead adder with redundant carry (BCLARC) realized using delay-insensitive dual-rail data encoding and 4-phase return-to-zero (RTZ) and 4-phase return-to-one (RTO) handshaking. The proposed QDI BCLARC is found to be faster and energy-efficient than the existing asynchronous adders which are QDI and non-QDI (i.e., relative-timed). Compared to existing asynchronous adders corresponding to various architectures such as the ripple carry adder (RCA), the conventional carry lookahead adder (CCLA), the carry select adder (CSLA), the BCLARC, and the hybrid BCLARC-RCA, the proposed BCLARC is found to be faster and more energy-optimized. The cycle time (CT), which is expressed as the sum of the worst-case times taken for processing the data and the spacer, governs the speed. The product of average power dissipation and CT viz. the power-cycle time product (PCTP) defines the low power/energy efficiency. For a 32-bit addition, the proposed QDI BCLARC achieves the following reductions in design metrics on average over its counterparts when considering RTZ and RTO handshaking: i) 20.5% and 19.6% reductions in CT and PCTP respectively compared to an optimum QDI early output RCA, ii) 16.5% and 15.8% reductions in CT and PCTP respectively compared to an optimum relative-timed RCA, iii) 32.9% and 35.9% reductions in CT and PCTP respectively compared to an optimum uniform input-partitioned QDI early output CSLA, iv) 47.5% and 47.2% reductions in CT and PCTP respectively compared to an optimum QDI early output CCLA, v) 14.2% and 27.3% reductions in CT and PCTP respectively compared to an optimum QDI early output BCLARC, and vi) 12.2% and 11.6% reductions in CT and PCTP respectively compared to an optimum QDI early output hybrid BCLARC-RCA. The adders were implemented using a 32/28nm CMOS technology.

Within-host spatiotemporal dynamics of systemic Salmonella infection during and after antimicrobial treatment.

OBJECTIVES: We determined the interactions between efficacy of antibiotic treatment, pathogen growth rates and between-organ spread during systemic Salmonella infections. METHODS: We infected mice with isogenic molecularly tagged subpopulations of either a fast-growing WT or a slow-growing ΔaroC Salmonella strain. We monitored viable bacterial numbers and fluctuations in the proportions of each bacterial subpopulation in spleen, liver, blood and mesenteric lymph nodes (MLNs) before, during and after the cessation of treatment with ampicillin and ciprofloxacin. RESULTS: Both antimicrobials induced a reduction in viable bacterial numbers in the spleen, liver and blood. This reduction was biphasic in infections with fast-growing bacteria, with a rapid initial reduction followed by a phase of lower effect. Conversely, a slow and gradual reduction of the bacterial load was seen in infections with the slow-growing strain, indicating a positive correlation between bacterial net growth rates and the efficacy of ampicillin and ciprofloxacin. The viable numbers of either bacterial strain remained constant in MLNs throughout the treatment with a relapse of the infection with WT bacteria occurring after cessation of the treatment. The frequency of each tagged bacterial subpopulation was similar in the spleen and liver, but different from that of the MLNs before, during and after treatment. CONCLUSIONS: In Salmonella infections, bacterial growth rates correlate with treatment efficacy. MLNs are a site with a bacterial population structure different to those of the spleen and liver and where the total viable bacterial load remains largely unaffected by antimicrobials, but can resume growth after cessation of treatment.

Distinguishable epidemics of multidrug-resistant Salmonella Typhimurium DT104 in different hosts.

The global epidemic of multidrug-resistant Salmonella Typhimurium DT104 provides an important example, both in terms of the agent and its resistance, of a widely disseminated zoonotic pathogen. Here, with an unprecedented national collection of isolates collected contemporaneously from humans and animals and including a sample of internationally derived isolates, we have used whole-genome sequencing to dissect the phylogenetic associations of the bacterium and its antimicrobial resistance genes through the course of an epidemic. Contrary to current tenets supporting a single homogeneous epidemic, we demonstrate that the bacterium and its resistance genes were largely maintained within animal and human populations separately and that there was limited transmission, in either direction. We also show considerable variation in the resistance profiles, in contrast to the largely stable bacterial core genome, which emphasizes the critical importance of integrated genotypic data sets in understanding the ecology of bacterial zoonoses and antimicrobial resistance.

Prevalence of Salmonella enterica in poultry and eggs in Uruguay during an epidemic due to Salmonella enterica serovar Enteritidis.

Salmonella enterica serovar Enteritidis (S. Enteritidis) is frequently associated with food-borne disease worldwide. Poultry-derived products are a major source. An epidemic of human infection with S. Enteritidis occurred in Uruguay, and to evaluate the extent of poultry contamination, we conducted a nationwide survey over 2 years that included the analysis of sera from 5,751 birds and 12,400 eggs. Serological evidence of infection with Salmonella group O:9 was found in 24.4% of the birds. All positive sera were retested with a gm flagellum-based enzyme-linked immunosorbent assay, and based on these results, the national prevalence of S. Enteritidis infection was estimated to be 6.3%. Salmonellae were recovered from 58 of 620 pools made up of 20 eggs each, demonstrating a prevalence of at least 1 in every 214 eggs. Surprisingly, the majority of the isolates were not S. Enteritidis. Thirty-nine isolates were typed as S. Derby, 9 as S. Gallinarum, 8 as S. Enteritidis, and 2 as S. Panama. Despite the highest prevalence in eggs, S. Derby was not isolated from humans in the period of analysis, suggesting a low capacity to infect humans. Microarray-based comparative genomic hybridization analysis of S. Derby and S. Enteritidis revealed more than 350 genetic differences. S. Derby lacked pathogenicity islands 13 and 14, the fimbrial lpf operon, and other regions encoding metabolic functions. Several of these regions are present not only in serovar Enteritidis but also in all sequenced strains of S. Typhimurium, suggesting that these regions might be related to the capacity of Salmonella to cause food-borne disease.

Salmonella enterica serovar typhimurium trxA mutants are protective against virulent challenge and induce less inflammation than the live-attenuated vaccine strain SL3261.

In Salmonella enterica serovar Typhimurium, trxA encodes thioredoxin 1, a small, soluble protein with disulfide reductase activity, which catalyzes thiol disulfide redox reactions in a variety of substrate proteins. Thioredoxins are involved as antioxidants in defense against oxidative stresses, such as exposure to hydrogen peroxide and hydroxyl radicals. We have made a defined, complete deletion of trxA in the mouse-virulent S. Typhimurium strain SL1344 (SL1344 trxA), replacing the gene with a kanamycin resistance gene cassette. SL1344 trxA was attenuated for virulence in BALB/c mice by the oral and intravenous routes and when used in immunization experiments provided protection against challenge with the virulent parent strain. SL1344 trxA induced less inflammation in murine spleens and livers than SL3261, the aroA mutant, live attenuated vaccine strain. The reduced splenomegaly observed following infection with SL1344 trxA was partially attributed to a reduction in the number of both CD4(+) and CD8(+) T cells and B lymphocytes in the spleen and reduced infiltration by CD11b(+) cells into the spleen compared with spleens from mice infected with SL3261. This less severe pathological response indicates that a trxA mutation might be used to reduce reactogenicity of live attenuated vaccine strains. We tested this by deleting trxA in SL3261. SL3261 trxA was also less inflammatory than SL3261 but was slightly less effective as a vaccine strain than either the SL3261 parent strain or SL1344 trxA.

Salmonella enterica serovar Typhimurium mutants completely lacking the F(0)F(1) ATPase are novel live attenuated vaccine strains.

The F(0)F(1) ATPase plays a central role in both the generation of ATP and the utilisation of ATP for cellular processes such as rotation of bacterial flagella. We have deleted the entire operon encoding the F(0)F(1) ATPase, as well as genes encoding individual F(0) or F(1) subunits, in Salmonella enteric serovar Typhimurium. These mutants were attenuated for virulence, as assessed by bacterial counts in the livers and spleens of intravenously infected mice. The attenuated in vivo growth of the entire atp operon mutant was complemented by the insertion of the atp operon into the malXY pseudogene region. Following clearance of the attenuated mutants from the organs, mice were protected against challenge with the virulent wild type parent strain. We have shown that the F(0)F(1) ATPase is important for bacterial growth in vivo and that atp mutants are effective live attenuated vaccines against Salmonella infection.

The enzyme phosphoglucomutase (Pgm) is required by Salmonella enterica serovar Typhimurium for O-antigen production, resistance to antimicrobial peptides and in vivo fitness.

The enzyme phosphoglucomutase (Pgm) catalyses the interconversion of glucose 1-phosphate and glucose 6-phosphate and contributes to glycolysis and the generation of sugar nucleotides for biosynthesis. To assess the role of this enzyme in the biology of the pathogen Salmonella enterica serovar Typhimurium we have characterized a pgm deletion mutant in strain SL1344. Compared to SL1344, SL1344 pgm had impaired growth in vitro, was deficient in the ability to utilize galactose as a carbon source and displayed reduced O-antigen polymer length. The mutant was also more susceptible to antimicrobial peptides and showed decreased fitness in the mouse typhoid model. The in vivo phenotype of SL1344 pgm indicated a role for pgm in the early stages of infection, most likely through deficient O-antigen production. Although pgm mutants in other pathogens have potential as live attenuated vaccine strains, SL1344 pgm was not sufficiently attenuated for such use.

Redundancy in the requirement for the glycolytic enzymes phosphofructokinase (Pfk) 1 and 2 in the in vivo fitness of Salmonella enterica serovar Typhimurium.

To assess the role of the glycolytic enzyme phosphofructokinase (Pfk) in the in vivo fitness of the pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium) we have generated single and double gene deletion mutants of the two known isoforms of this enzyme, pfkA and pfkB. In a mouse model of typhoid fever, bacterial counts in the spleen and liver were similar between wild type and single pfkA and pfkB mutant-infected mice. However, a double pfkAB mutant was significantly attenuated for growth in vivo. This defect was complemented by provision of either pfkA or pfkB on pBR322. Together these data show that Pfk activity is required for the full in vivo fitness of S. Typhimurium with functional redundancy between pfkA and pfkB. The level of attenuation of the pfkAB double mutant was not sufficient for its consideration as a live attenuated vaccine strain.

Critical assessment of an in vitro bovine respiratory organ culture system: a model of bovine herpesvirus-1 infection.

A bovine in vitro organ culture (BIVOC) system was evaluated as a model to study host and pathogen events during the course of bovine herpesvirus-1 infection. Upper respiratory tract epithelium, from slaughtered animals, was cultured in an air-liquid interface system and integrity, viability, and TNF-alpha gene expression of tissue explants were monitored over 72h in the presence or absence of infection by bovine herpesvirus type 1 (BHV-1). Uninfected explants maintained viability and integrity over the 72h time course although histological signs of degeneration were first visible from 24h of culture. Explants were productively infected with BHV-1 and typical, dose dependent, cytopathic changes were observed in response to infection. Regulation of TNF-alpha gene expression in uninfected explants varied over time and was region-specific but there was significant down-regulation of TNF-alpha gene expression at 2h post-infection when compared to uninfected controls at the same time point. Taking caveats into consideration the BIVOC system shows promise as a tool for analysis of immediate or early events in host-pathogen interaction.

Deletion of tolA in Salmonella Typhimurium generates an attenuated strain with vaccine potential.

The Gram-negative Tol-Pal system of envelope proteins plays a key role in maintaining outer membrane integrity and contributes to the virulence of several pathogens. We have investigated the role of one of these proteins, TolA, in the biology of Salmonella enterica serovar Typhimurium. Deletion of tolA rendered strain SL1344 more susceptible to killing by bile and human serum. In addition the mutant had impaired membrane integrity and displayed alterations in LPS production. The tolA mutant was highly attenuated in mouse infections via the oral and intravenous routes. Importantly, each phenotype displayed by the mutant was complemented by provision of tolA in trans. The tolA gene therefore contributes to virulence, membrane integrity, LPS production and bile and serum resistance in S. enterica serovar Typhimurium SL1344. Finally, immunization with the tolA mutant provided significant protection against subsequent challenge with wild-type SL1344. The Tol-Pal system is therefore a potential target in the development of novel attenuated live vaccines against Salmonella and other Gram-negative pathogens.

The cellular Toll-like receptor 4 antagonist E5531 can act as an agonist in horse whole blood.

Sepsis and endotoxaemia are important causes of morbidity and mortality in humans. Research on sepsis focuses on rodent models most of which are poorly responsive to lipopolysaccharide (LPS), and thus do not mimic very well the high sensitivity of humans. Therefore, there is a need to develop more clinically relevant models. Horses suffer from a similar endotoxaemic syndrome to humans with high morbidity and mortality. LPS analogues that act as antagonists at Toll-like receptor 4 (TLR4) are being developed as novel treatments for endotoxaemia. Due to differences in recognition of ligands by TLR4 from different mammalian species, individual LPS molecules may act as agonists in some species and antagonists in others. The synthetic lipid A analogue E5531 is an antagonist at TLR4 in humans and mice, but its effects at TLR4 from other species are unknown. In the studies reported here, Escherichia coli LPS is a full agonist on equine bone marrow macrophage-like cells and its effects are antagonised by E5531. Similarly, E. coli LPS is an agonist and E5531 an antagonist on monocytes isolated from peripheral blood of healthy horses and human embryonic kidney (HEK) cells, transiently transfected to express horse TLR4 and its associated cell surface proteins MD2 and CD14. In contrast, both E. coli LPS and E5531 behave as agonists in horse whole blood by inducing production of equivalent amounts of the inflammatory mediator prostaglandin. This finding suggests that modification of E5531 may occur in whole blood, for example, deacylation, which alters its activity. This comparative study has revealed a novel pharmacological action of E5531 and emphasises the importance of extending studies of this nature beyond the normal rodent models.

Campylobacter jejuni gene expression in the chick cecum: evidence for adaptation to a low-oxygen environment.

Transcriptional profiling of Campylobacter jejuni during colonization of the chick cecum identified 59 genes that were differentially expressed in vivo compared with the genes in vitro. The data suggest that C. jejuni regulates electron transport and central metabolic pathways to alter its physiological state during establishment in the chick cecum.

Oxidative and nitrosative responses of the chicken macrophage cell line MQ-NCSU to experimental Salmonella infection.

Phagocytes limit replication or kill ingested organisms by producing toxic reactive oxygen and nitrogen species via NADPH oxidase and inducible nitric oxide synthase (iNOS). The present experiments were to investigate the production and the possible roles of superoxide, hydrogen peroxide (H2O2) and nitric oxide (NO) in the MQ-NCSU chicken macrophage cell line infected with Salmonella in vitro. After infection, intracellular Salmonella viable counts remained constant until 24 h post infection (PI) and started to decline from 48 h PI. Infection of cells with S. Typhimurium, S. Enteritidis and S. Gallinarum, as well as exposure to S. Enteritidis LPS induced low, but significant concentrations of superoxide 1 to 2 h PI, as determined by reduction of ferricytochrome c. There was no difference in superoxide production in infected cells and control cells after 4 h. Increased H2O2 was observed from cells infected with all the different Salmonella species between 2 and 3 h of infection. Nitrite was always greater in infected cells compared to uninfected cells at all times. However, Salmonella was not completely eliminated from the cells though these cells are capable of eliciting a noticeable oxidative burst response and great nitrosative responses, indicating that a strong oxidative burst (and other mechanism/s) is essential for the elimination of intracellular Salmonella.

Mutagenesis of Streptococcus equi and Streptococcus suis by transposon Tn917.

Genetic tools for studying streptococci are much less sophisticated than those that are available for many other bacterial genera. In this paper, we describe the development of a transposon mutagenesis system that we have used to mutate two important veterinary streptococci, Streptococcus equi and Streptococcus suis. The system uses a temperature-sensitive suicide vector to deliver Tn917 via electroporation, transposing Tn917 into the chromosomal DNA of the two streptococci. The transposon insertions can be rescued from the streptococcal chromosomes by plasmid rescue and selection in E. coli, with subsequent insertion site analysis by DNA sequencing. Transposition appeared to have occurred in an essentially random fashion when chromosomal DNA of S. suis and S. equi mutants was analysed by Southern blotting. However, when analysis of 60 S. equi mutants was carried out using the S. equi genome sequence database, 60% of transposon insertions had occurred within a 15 kb region of the genome whereas the other insertions appeared to have occurred essentially randomly. This finding suggests that Southern blot analysis for assessing the randomness of transposon libraries may need to be interpreted with caution. However, this observation notwithstanding, the Tn917 based system described in this paper will facilitate the study of S. suis and S. equi.

New aspects of the role of MR/P fimbriae in Proteus mirabilis urinary tract infection.

Proteus mirabilis, a common cause of urinary tract infection (UTI), produces a number of different fimbriae including mannose-resistant Proteus-like fimbriae (MR/P). The precise role of different P. mirabilis fimbriae in ascending UTI has not yet been elucidated. In this study, a clinical isolate of P. mirabilis and an isogenic mutant unable to express MR/P were tested using different experimental approaches. They were tested for their ability to cause infection in an ascending co-infection model of UTI and in a haematogenous model in the mouse. In both models, the mutant was less able than the wild-type strain to colonise the lower and upper urinary tracts although infectivity was not abolished. In vitro adherence to uroepithelial cells was also assessed. Significant differences in adherence between both strains were observed at 1 h but not at 15 min post infection. We have also shown that a wild-type strain carries two copies of the mrpA gene. These data reinforce the importance of MR/P fimbriae in P. mirabilis UTI although other virulence factors may be necessary for efficient colonisation and development of infection.

Overexpression, purification, crystallization and data collection on the Bordetella pertussis wlbD gene product, a putative UDP-GlcNAc 2'-epimerase.

The Boredetella pertussis wlbD gene product is a putative uridine-5-diphosphate N-acetylglucosamine (UDP-GlcNAc) 2'-epimerase involved in Band A lipopolysaccharide biosynthesis. The wlbD gene is homologous to Escherichia coli rffE (32% identical), an established UDP-GlcNAc 2'-epimerase that is involved in enterobacterial common antigen (ECA) formation. The structure of the rffE protein reveals an unexpected role for a bound sodium ion in orientating a substrate-binding alpha-helix in the enzyme active site. Whilst key active-site residues in rffE are present in the wlbD sequence, the sodium-binding residues outside the active site are absent. This raises questions about the modulation of enzyme activity in these two enzymes. The wlbD gene from B. pertussis has been cloned and overexpressed in E. coli and the resulting protein has been purified to homogeneity. In the current study, crystals of the mutant Gln339Arg wlbD enzyme have been obtained by sitting-drop vapour diffusion. Uncomplexed Gln339Arg and UDP-GlcNAc complex data sets have been collected in-house on a rotating-anode generator to 2.1 A. Combined, the data sets identify the space group as P2(1)2(1)2(1), with unit-cell parameters a = 78, b = 91, c = 125 A, alpha = beta = gamma = 90 degrees. The asymmetric unit contains two monomers and 53% solvent.