Extended Spectrum Beta-Lactamase (ESBL) Produced by Gram-Negative Bacteria in Trinidad and Tobago.

Gram-negative bacterial infections are a global health problem. The production of beta-lactamase is still the most vital factor leading to beta-lactam resistance. In Trinidad and Tobago, extended spectrum beta-lactamase (ESBL) production has been detected and reported mainly in the isolates of Klebsiella pneumoniae and Escherichia coli and constitutes a public health emergency that causes high morbidity and mortality in some patients. In this literature review, the authors cover vast information on ESBL frequency and laboratory detection using both conventional and molecular methods from clinical data. The aim is to make the reader reflect on how the actual knowledge can be used for rapid detection and understanding of the spread of antimicrobial resistance problems stemming from ESBL production among common Gram-negative organisms in the health care system.

The role of the super-relaxed state of myosin in human metabolism.

BACKGROUND: The super-relaxed state of myosin (SRX) plays a fundamental role in maintaining the low resting metabolic rate of skeletal muscle. Our previous work on this state has been in animal models. Piperine is a small molecule that has been shown to destabilize the SRX in rabbit fast twitch fibers. METHODS: Here we extend this work to human muscle obtained from biopsies of the vastus lateralis of both lean and obese subjects. The slow release of nucleotides by myosin in the SRX was measured by incubating permeable fibers in a fluorescent analog of ATP and chasing with ATP. RESULTS: The fraction of myosin heads in the SRX was 0.48 ± 0.04 with a lifetime of 148 ± 5 s in lean subjects and a fraction of 0.41 ± 0.05 and a lifetime of 176 ± 7 s in obese subjects. Addition of 100 µM piperine decreased the SRX population by 43 ± 7% in lean subjects and 36 ± 7% in obese subjects, with little change in lifetimes. Addition of piperine to human cardiac cells had no effect on the SRX, a requirement for a drug to treat metabolic diseases. CONCLUSIONS: In human muscle the SRX and its responses to piperine are similar to those seen previously, with no significant differences between muscles from lean and obese subjects. Thus analogs of piperine that have greater specificity could provide effective treatment for metabolic diseases. The SRX provides a potential mechanism contributing to the large dynamic range of metabolic rate.

Genetic characteristics and molecular epidemiology of vancomycin-resistant Enterococci isolates from Caribbean countries.

Emergence of vancomycin-resistant Enterococci (VRE) that first appeared on the stage about three decades ago is now a major concern worldwide as it has globally reached every continent. Our aim was to simply undertake a multinational study to delineate the resistance and virulence genes of clinical isolates of VRE isolates from the Caribbean. We employed both conventional (standard microbiological methods including use of E-test strips, chromogenic agar) and molecular methods (polymerase chain reactions-PCR, pulsed-field gel electrophoresis-PFGE and multilocus sequence typing-MLST) to analyze and characterize 245 Enterococci species and 77 VRE isolates from twelve hospitals from eight countries in the Caribbean. The PCR confirmed and demonstrated the resistance and virulence genes (vanA and esp) among all confirmed VRE isolates. The PFGE delineated clonally related isolates from patients from the same country and other countries in the region. The main sequence types of the VRE isolates from the region included STs 412, 750, 203, 736 and 18, all from the common ancestor for clonal complex 17 (CC17). Despite this common ancestor and association of outbreaks of this lineage clones, there has been no reports of outbreaks of infection by VRE in several hospitals in the Caribbean.

The link between Proteus mirabilis, environmental factors and autoantibodies in rheumatoid arthritis.

Rheumatoid arthritis (RA) is a relatively common and potentially disabling immune-mediated inflammatory systemic disease, predominantly affecting women and characterised by multiple small joint arthritis. Extensive data supports the roles of genetic, environmental and microbial factors in the triggering and development of this disease. Proteus mirabilis is considered as the main microbial culprit in the causation of RA. The evidence for the role of these microbes in RA and their links with commonly associated autoantibodies such as rheumatoid factors and anti-citrullinated peptide antibodies have been elucidated together with their relations with some of the non-microbial environmental factors which have been implicated in the aetiopathogenesis of RA. The most likely mechanism in the development of RA is "molecular mimicry" where Proteus antigens were found to share homologous sequences, which cross-react with certain self-antigens present in synovial tissues. This could raise possibilities for implementing a new therapeutic strategy in the treatment of RA.

Molecular characterization of vancomycin-resistant Enterococcus faecium isolates from Bermuda.

Molecular characteristics of vancomycin resistant enterococci isolates from Bermuda Island is currently unknown. This study was conducted to investigate phenotypic and genotypic characteristics of VRE isolates from Bermuda Island using the chromogenic agar, E-tests, polymerase chain reaction (PCR), pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Eighteen E. faecium isolates were completely analyzed and were all resistant to vancomycin, susceptible to linezolid and quinupristin/dalfopristin, positive for vanA and esp genes. The MLST analysis confirmed most isolates were of the sequence types linked to clonal complex 17 (CC17) that is widely associated with outbreaks in hospitals. Infection control measures, antibiotic stewardship, and surveillance activities will continue to be a priority in hospital on the Island.

The Link Between Klebsiella and Ankylosing Spondylitis in Worldwide Geographical Locations.

Ankylosing spondylitis (AS) is a world-wide chronic inflammatory disease of the axial skeleton most likely caused by a microbial factor in genetically susceptible individuals. Over the last 40 years extensive data has been produced which shows that the majority of patients with AS possess the HLA-B27 genetic marker. Significantly elevated levels of Klebsiella antibodies have been demonstrated in 1556 AS patients in 16 different countries with various geographical locations. Other evidence for the link between Klebsiella and AS include increased fecal isolation rates of Klebsiella microbes in AS patients together with shared molecular and immunological cross-reactivity features existing between Klebsiella antigens and HLA-B27 and collagens I, III and IV. Anti-Klebsiella measures could possibly be included with the currently used medical treatment in the management of patients with AS.

Fundamental immunological problems associated with "transmissible spongiform encephalopathies".

"Bovine spongiform encephalopathy", "scrapie", as well as Creutzfeldt-Jakob disease and kuru belong to a group of related neurological conditions termed "transmissible spongiform encephalopathies". These diseases are based on the LD50 measurement whereby saline brain homogenates are injected into experimental animals and when 50% of them develop symptoms, this is considered as transmission of the disease, but the gold standard for diagnosis is autopsy examination. However, an untenable assumption is being made in that saline brain homogenates do not cause tissue damage but it is known since the time of Pasteur, that they give rise to "post-rabies vaccination allergic encephalomyelitis". This is the fundamental flaw in the diagnosis of these diseases. A way forward, however, is to examine infectious agents, such as Acinetobacter which show molecular mimicry with myelin and elevated levels of antibodies to this microbe are found in multiple sclerosis patients and animals affected by "bovine spongiform encephalopathy".

Raised incidence of ankylosing spondylitis among Inuit populations could be due to high HLA-B27 association and starch consumption.

Ankylosing spondylitis (AS) is a chronic inflammatory arthritis mainly affecting the spinal joints. It would appear that the most likely causative agent in the development of AS is an environmental factor in the genetically susceptible, HLA-B27 positive, individuals. Extensive data from several countries support the notion that Klebsiella pneumonia bacteria are the most likely culprit in the causation of AS. These microbes possess antigens which resemble HLA-B27 and spinal collagens. Increased intake of high-starch diet is directly proportional to the gut-associated bacterial load, especially in the large intestine, and among these microbial agents, Klebsiella is considered as one of the main constituting components. Therefore, a low-starch diet intake alongside the currently used medical therapeutic modalities could be beneficial in the management of patients with early AS. It is suggested that a change in the dietary habits from high protein, low-starch marine components to the Westernized high-starch diet among the Inuit peoples of Alaska and Canada could be considered as one of the main contributing factors in the increased prevalence of AS during the last few decades within this genetically unmixed native population.

The myosin inhibitor blebbistatin stabilizes the super-relaxed state in skeletal muscle.

The super-relaxed state of myosin (SRX), in which the myosin ATPase activity is strongly inhibited, has been observed in a variety of muscle types. It has been proposed that myosin heads in this state are inhibited by binding to the core of the thick filament in a structure known as the interacting-heads motif. The myosin inhibitor blebbistatin has been shown in structural studies to stabilize the binding of myosin heads to the thick filament, and here we have utilized measurements of single ATP turnovers to show that blebbistatin also stabilizes the SRX in both fast and slow skeletal muscle, providing further support for the proposal that myosin heads in the SRX are also in the interacting-heads motif. We find that the SRX is stabilized using blebbistatin even in conditions that normally destabilize it, e.g., rigor ADP. Using blebbistatin we show that spin-labeled nucleotides bound to myosin have an oriented spectrum in the SRX in both slow and fast skeletal muscle. This is to our knowledge the first observation of oriented spin probes on the myosin motor domain in relaxed skeletal muscle fibers. The spectra for skeletal muscle with blebbistatin are similar to those observed in relaxed tarantula fibers in the absence of blebbistatin, demonstrating that the structure of the SRX is similar in different muscle types and in the presence and absence of blebbistatin. The mobility of spin probes attached to nucleotides bound to myosin shows that the conformation of the nucleotide site is closed in the SRX.

The role of Klebsiella in Crohn's disease with a potential for the use of antimicrobial measures.

There is a general consensus that Crohn's disease (CD) develops as the result of immune-mediated tissue damage triggered by infections with intestinal microbial agents. Based on the results of existing microbiological, molecular, and immunological studies, Klebsiella microbe seems to have a key role in the initiation and perpetuation of the pathological damage involving the gut and joint tissues in patients with CD. Six different gastroenterology centres in the UK have reported elevated levels of antibodies to Klebsiella in CD patients. There is a relationship between high intake of starch-containing diet, enhanced growth of gut microbes, and the production of pullulanases by Klebsiella. It is proposed that eradication of these microbes by the use of antibiotics and low starch diet, in addition to the currently used treatment, could help in alleviating or halting the disease process in CD.

The link between ankylosing spondylitis, Crohn's disease, Klebsiella, and starch consumption.

Both ankylosing spondylitis (AS) and Crohn's disease (CD) are chronic and potentially disabling interrelated conditions, which have been included under the group of spondyloarthropathies. The results of a large number of studies support the idea that an enteropathic pathogen, Klebsiella pneumoniae, is the most likely triggering factor involved in the initiation and development of these diseases. Increased starch consumptions by genetically susceptible individuals such as those possessing HLA-B27 allelotypes could trigger the disease in both AS and CD by enhancing the growth and perpetuation of the Klebsiella microbes in the bowel. Exposure to increased levels of these microbes will lead to the production of elevated levels of anti-Klebsiella antibodies as well as autoantibodies against cross-reactive self-antigens with resultant pathological lesions in the bowel and joints. Hence, a decrease of starch-containing products in the daily dietary intake could have a beneficial therapeutic effect on the disease especially when used in conjunction with the currently available medical therapies in the treatment of patients with AS and CD.

The role of Acinetobacter in the pathogenesis of multiple sclerosis examined by using Popper sequences.

Multiple sclerosis (MS) is an autoimmune neurological disorder. The role of 'Acinetobacter' has been examined using the method of Karl Popper and involves nine "Popper sequences". (1) The frequency of MS increases with latitudes in the Northern Hemisphere, and the reverse is found in the Southern Hemisphere. (2) Sinusitis is found frequently at colder latitudes. (3) Sinusitis occurs frequently in patients with MS. (4) Specific sequences of bovine myelin when injected into experimental animals will produce a neurological disorder resembling MS which is called "experimental allergic encephalomyelitis". (5) Computer analysis of myelin shows molecular mimicry with sequences found in Acinetobacter. (6) Antibodies to Acinetobacter bacteria are found in MS patients. (7) Acinetobacter bacteria are located on human skin and in the nasal sinuses. (8) IgA antibodies are preferentially elevated in the sera of MS patients, thereby suggesting the trigger microbe is acting across a mucosal surface probably located in the nasal sinuses. (9) Only Acinetobacter bacteria and no other microbes evoke statistically significant titres of antibodies in MS patients. These nine Popper sequences suggest that MS is most probably caused by infections with Acinetobacter bacteria in the nasal sinuses, and this could have therapeutic implications.

Root-specific transcript profiling of contrasting rice genotypes in response to salinity stress.

Elevated salinity imposes osmotic and ion toxicity stresses on living cells and requires a multitude of responses in order to enable plant survival. Building on earlier work profiling transcript levels in rice (Oryza sativa) shoots of FL478, a salt-tolerant indica recombinant inbred line, and IR29, a salt-sensitive cultivar, transcript levels were compared in roots of these two accessions as well as in the roots of two additional salt-tolerant indica genotypes, the landrace Pokkali and the recombinant inbred line IR63731. The aim of this study was to compare transcripts in the sensitive and the tolerant lines in order to identify genes likely to be involved in plant salinity tolerance, rather than in responses to salinity per se. Transcript profiles of several gene families with known links to salinity tolerance are described (e.g. HKTs, NHXs). The putative function of a set of genes identified through their salt responsiveness, transcript levels, and/or chromosomal location (i.e. underneath QTLs for salinity tolerance) is also discussed. Finally, the parental origin of the Saltol region in FL478 is further investigated. Overall, the dataset presented appears to be robust and it seems likely that this system could provide a reliable strategy for the discovery of novel genes involved in salinity tolerance.

Rheumatoid arthritis, Proteus, anti-CCP antibodies and Karl Popper.

Rheumatoid arthritis (RA) is a crippling joint disease affecting over 20 million people worldwide. The cause of RA is most probably linked to the triad of microbial trigger, genetic association and autoimmunity and can be explained using the philosophical method of Karl Popper or Popperian sequences. Ten "Popper sequences" have been identified which point to the urinary microbe Proteus mirabilis as the cause of RA: Popper sequence 1 establishes that HLA-DR4 lymphocytes injected into a rabbit evoke specific antibodies against Proteus bacteria. Popper sequence 2 establishes that antibodies to Proteus bacteria are present in RA patients from 14 different countries. Popper sequence 3 establishes that antibodies to Proteus bacteria in RA patients are disease specific since no such antibodies are found in other conditions. Popper sequence 4 establishes that when RA patients have high titres of antibodies to Proteus such bacteria are found in urinary cultures. Popper sequence 5 establishes that only Proteus bacteria and no other microbes evoke significantly elevated antibodies in RA patients. Popper sequence 6 establishes that the "shared epitope" EQR(K)RAA shows "molecular mimicry" with the sequence ESRRAL found in Proteus haemolysin. Popper sequence 7 establishes that Proteus urease contains a sequence IRRET which has "molecular mimicry" with LRREI found in collagen XI of hyaline cartilage. Popper sequence 8 establishes that sera obtained from RA patients have cytopathic properties against sheep red cells coated with the cross-reacting EQR(K)RAA and LRREI self-antigen peptides. Popper sequence 9 establishes that Proteus sequences in haemolysin and urease as well as the self antigens, HLA-DR1/4 and collagen XI, each contain an arginine doublet, thereby providing a substrate for peptidyl arginine deiminase (PAD) to give rise to citrulline, which is the main antigenic component of CCP, antibodies to which are found in early cases of RA. Popper sequence 10 establishes that antibodies to Proteus come not only from sequences crossreacting to self antigens but also from non-crossreacting sequences, thereby indicating that active RA patients have been exposed to infection by Proteus. The ten Popper sequences establish that RA is most probably caused by Proteus upper urinary tract infections, which can possibly be treated with anti-Proteus therapy.

Comparing genomic expression patterns across plant species reveals highly diverged transcriptional dynamics in response to salt stress.

BACKGROUND: Rice and barley are both members of Poaceae (grass family) but have a marked difference in salt tolerance. The molecular mechanism underlying this difference was previously unexplored. This study employs a comparative genomics approach to identify analogous and contrasting gene expression patterns between rice and barley. RESULTS: A hierarchical clustering approach identified several interesting expression trajectories among rice and barley genotypes. There were no major conserved expression patterns between the two species in response to salt stress. A wheat salt-stress dataset was queried for comparison with rice and barley. Roughly one-third of the salt-stress responses of barley were conserved with wheat while overlap between wheat and rice was minimal. These results demonstrate that, at transcriptome level, rice is strikingly different compared to the more closely related barley and wheat. This apparent lack of analogous transcriptional programs in response to salt stress is further highlighted through close examination of genes associated with root growth and development. CONCLUSION: The analysis provides support for the hypothesis that conservation of transcriptional signatures in response to environmental cues depends on the genetic similarity among the genotypes within a species, and on the phylogenetic distance between the species.

Detection and validation of single feature polymorphisms using RNA expression data from a rice genome array.

BACKGROUND: A large number of genetic variations have been identified in rice. Such variations must in many cases control phenotypic differences in abiotic stress tolerance and other traits. A single feature polymorphism (SFP) is an oligonucleotide array-based polymorphism which can be used for identification of SNPs or insertion/deletions (INDELs) for high throughput genotyping and high density mapping. Here we applied SFP markers to a lingering question about the source of salt tolerance in a particular rice recombinant inbred line (RIL) derived from a salt tolerant and salt sensitive parent. RESULTS: Expression data obtained by hybridizing RNA to an oligonucleotide array were analyzed using a statistical method called robustified projection pursuit (RPP). By applying the RPP method, a total of 1208 SFP probes were detected between two presumed parental genotypes (Pokkali and IR29) of a RIL population segregating for salt tolerance. We focused on the Saltol region, a major salt tolerance QTL. Analysis of FL478, a salt tolerant RIL, revealed a small (< 1 Mb) region carrying alleles from the presumed salt tolerant parent, flanked by alleles matching the salt sensitive parent IR29. Sequencing of putative SFP-containing amplicons from this region and other positions in the genome yielded a validation rate more than 95%. CONCLUSION: Recombinant inbred line FL478 contains a small (< 1 Mb) segment from the salt tolerant parent in the Saltol region. The Affymetrix rice genome array provides a satisfactory platform for high resolution mapping in rice using RNA hybridization and the RPP method of SFP analysis.

Structural dynamics of the actomyosin complex probed by a bifunctional spin label that cross-links SH1 and SH2.

We have used a bifunctional spin label (BSL) to cross-link Cys(707) (SH1) and Cys(697) (SH2) in the catalytic domain of myosin subfragment 1 (S1). BSL induces the same weakened ATPase activity and actin-binding affinity that is observed when SH1 and SH2 are cross-linked with pPDM, which traps an analog of the post-hydrolysis state A.M.ADP.P. Electron paramagnetic resonance showed that BSL reports the global orientation and dynamics of S1. When bound to actin in oriented muscle fibers in the absence of ATP, BSL-S1 showed almost complete orientational disorder, as reported previously for the weakly bound A.M.ADP. In contrast, helical order is observed for the strongly bound state A.M. Saturation transfer electron paramagnetic resonance showed that the disorder of cross-linked S1 on actin is nearly static on the microsecond timescale, at least 30 times slower than that of A.M.ADP. We conclude that cross-linked S1 exhibits rotational disorder comparable to that of A.M.ADP, slow rotational mobility comparable to that of A.M, and intermediate actin affinity. These results support the hypothesis that the catalytic domain of myosin is orientationally disordered on actin in a post-hydrolysis state in the early stages of force generation.

Array-based genotyping and expression analysis of barley cv. Maythorpe and Golden Promise.

BACKGROUND: Golden Promise is a salt-tolerant spring barley closely related to Maythorpe. Salt tolerance in Golden Promise has been attributed to a single mutation at the Ari-e locus (on 5H) resulting from irradiation of Maythorpe. Golden Promise accumulates lower shoot Na+ compared to Maythorpe when growing under saline conditions. This study focused on elucidating the genetic basis and mechanisms involved in this difference. RESULTS: The level of polymorphism between the two genotypes was explored using the Barley1 GeneChip for single feature polymorphisms (SFPs) and an oligonucleotide pool assay for single nucleotide polymorphisms (SNPs). Polymorphism analyses revealed three haplotype blocks spanning 6.4 cM on chromosome 1H, 23.7 cM on chromosome 4H and 3.0 cM on 5H. The Barley1 GeneChip was used to examine transcript abundance in different tissues and stages during development. Several genes within the polymorphic haplotype blocks were differentially regulated. Additionally, a more global difference in the jasmonic acid pathway regulation was detected between the two genotypes. CONCLUSION: The results confirm that Golden Promise and Maythorpe are genetically very closely related but establish that they are not isogenic, as previously reported, due to three polymorphic haplotype blocks. Transcriptome analysis indicates that the response of the two genotypes to salinity stress is quite different. Additionally, the response to salinity stress in the roots and shoot tissue is strikingly different.

Large-scale expression profiling and physiological characterization of jasmonic acid-mediated adaptation of barley to salinity stress.

Barley (Hordeum vulgare L.) is a salt-tolerant member of the Triticeae. Recent transcriptome studies on salinity stress response in barley revealed regulation of jasmonic acid (JA) biosynthesis and JA-responsive genes by salt stress. From that observation and several other physiological reports, it was hypothesized that JA is involved in the adaptation of barley to salt stress. Here we tested that hypothesis by applying JA to barley plants and observing the physiological responses and transcriptome changes. Photosynthetic and sodium ion accumulation responses were compared after (1) salinity stress, (2) JA treatment and (3) JA pre-treatment followed by salinity stress. The JA-pre-treated salt-stressed plants accumulated strikingly low levels of Na(+) in the shoot tissue compared with untreated salt-stressed plants after several days of exposure to stress. In addition, pre-treatment with JA partially alleviated photosynthetic inhibition caused by salinity stress. Expression profiling after a short-term exposure to salinity stress indicated a considerable overlap between genes regulated by salinity stress and JA application. Three JA-regulated genes, arginine decarboxylase, ribulose 1.5-bisphosphate carboxylase/oxygenase (Rubisco) activase and apoplastic invertase are possibly involved in salinity tolerance mediated by JA. This work provides a reference data set for further study of the role of JA in salinity tolerance in barley and other plants species.

From rabies to transmissible spongiform encephalopathies: an immune-mediated microbial trigger involving molecular mimicry could be the answer.

The concept of experimental allergic encephalomyelitis (EAE) being linked to both rabies post-vaccination encephalomyelitis and multiple sclerosis (MS) has raised the intriguing question whether animal studies carried out for the induction and transmission of transmissible spongiform encephalopathies (TSEs) using brain antigens including prions do have a similar immunopathogenetic mechanism. Although an essential link between autoimmunity and MS has been well established, its role in the pathogenesis of TSEs is generally lacking. However, auto-antibodies to myelin proteins and/or other neuronal antigens such as neurofilaments and prion proteins have been reported in animals with bovine spongiform encephalopathy (BSE) and scrapie as well as in patients with Creutzfeld-Jakob disease (CJD) and kuru. Acinetobacter has been suggested as a possible triggering microbial factor in the initiation of the autoimmune responses in these diseases because bacterial molecular sequences resemble brain antigens, especially in animals affected with BSE and patients with MS and CJD. These possibilities need to be evaluated further with longitudinal prospective studies carried out on larger numbers of animals or humans with such diseases. The transplantation of saline suspensions of brain homogenates will evoke immunological responses and therefore, the results in the study of MS and other neurological diseases have to be interpreted with caution.