Exploring the root canal microbiome in previously treated teeth: A comparative study of diversity and metabolic pathways across two geographical locations.

AIM: To analyse and compare the root canal microbiome present in root-filled teeth of two different geographical populations, and to study their functional potential using a next-generation sequencing approach. METHODOLOGY: Sequencing data obtained from surgical specimens from previously treated teeth with periapical bone loss from Spain and USA were included in the study. Taxa were classified using SILVA v.138 database. Differences in genera abundances among the 10 most abundant genera were evaluated using a Kruskal-Wallis test. Alpha diversity indices were calculated in mothur. The Shannon and Chao1 indices were used. Analyses of similarity (ANOSIM) to determine differences in community composition were done in mothur, with Bonferroni correction for multiple comparisons. p-Values < .05 were considered statistically significant. Identification of enriched bacteria function prediction in the study groups (KEGG pathways) was carried out by linear discriminant analysis effect size (LEfSe) via Python 3.7.6. RESULTS: A greater alpha-diversity (Shannon and Chao1 indices) was observed from samples obtained in Spain (p = .002). Geography showed no significant effects on community composition via an ANOSIM using Bray-Curtis dissimilarities (R = 0.03, p = .21). Bacterial functional analysis prediction obtained by PICRUSt showed that 5.7% KEGG pathways differed between the Spain and US samples. CONCLUSIONS: The taxonomic assessment alone does not fully capture the microbiome's differences from two different geographical locations. Carbohydrate and amino acid metabolism were enriched in samples from Spain, while samples from USA had a higher representation of pathways related to nitrogen, propanoate metabolism, and secretion systems.

Genomic heterogeneity of Dichelobacter nodosus within and between UK sheep flocks and between age groups within a flock.

BACKGROUND: Footrot and interdigital dermatitis are endemic infectious diseases in all sheep farming regions, impairing welfare and production. The development of efficacious vaccines against the primary causative pathogen has been hampered by the extensive antigenic diversity of Dichelobacter nodosus. Understanding the heterogeneity of the pathogen within and between flocks is essential if the feasibility of bespoke vaccine production is to be assessed for use in the U.K. RESULTS: In this study 56 ewe and lamb isolates from 9 flocks were compared by D. nodosus serogroup and Multi Locus Sequence Type which provides significantly enhanced discriminatory power for molecular epidemiology. Serogroup heterogeneity between flocks ranged from two to five unique serogroups per flock. Three flocks contained isolates of two serogroups, two flocks contained isolates of three serogroups and one flock included isolates of five serogroups. Analysis of 25 isolates from one flock with high prevalence of lameness, identified that serogroup and sequence type was significantly correlated with age. Significantly higher proportion of lambs were infected with serogroup B (principally ST85) as opposed to serogroup H (principally ST86), which predominated amongst adult sheep. CONCLUSIONS: Genomic heterogeneity of the pathogen was significantly lower within flock compared to heterogenicity observed between flocks. Furthermore, this study indicates that within a flock, the host-pathogen dynamics and susceptibility to particular D. nodosus strains may be age dependent.

The Effect of Season and Weather on Orthopaedic Trauma: Consult Volume Is Significantly Correlated with Daily Weather.

INTRODUCTION: On-call orthopedic clinicians have long speculated that daily consult volume is closely correlated with weather. While prior studies have demonstrated a relationship between weather and certain fracture types, the effect of weather on total orthopaedic consult volume has not yet been examined. The aim of this study was to investigate this relationship. METHODS: We retrospectively reviewed orthopaedic consult data from 405 consecutive days at an urban, level one trauma center. The number, mechanism of injury, and type of consult were collected, along with daily weather data (temperature, wind, and precipitation). Statistical analysis was then performed to determine the relationship between weather and orthopaedic trauma consults. RESULTS: A total of 4543 consults were received during the study period. There was a significant difference in total number of consults between months of the year (p<0.001). A post hoc analysis revealed that this was due to increased volume in the summer months relative to the winter months (i.e., August 13.7 consults/day; January 9.3 consults/day). Average daily temperature and consult volume were also positively correlated (p<0.001, r= 0.30). While there was no significant association between precipitation and total consult volume, when there was over 0.25 inches of rain, there were less penetrating trauma (p=0.034) and motorcycle collision consults (p=0.013). CONCLUSION: Weather parameters, specifically average temperature and precipitation, were found to be associated with daily orthopedic consult type and volume. Additionally, consult volume varies significantly between months of the year. Because trauma centers are often resource scarce, this is an important relationship to understand for proper resource allocation.

Seasonal metabolic analysis of marine sediments collected from Moreton Bay in South East Queensland, Australia, using a multi-omics-based approach.

Anthropogenic effects of urban density have altered natural ecosystems. Such changes include eutrophication of freshwater and adjoining coastal habitats, and increased levels of inorganic nutrients and pollutants into waterways. In Australia, these changes are intensified by large-scale ocean-atmospheric events, leading to considerable abiotic stress on the natural flora and fauna. Bacterial communities in marine sediments from Moreton Bay (South East Queensland, Australia) were examined in order to assess the impact of rainfall changes, chemical pollution, and subsequent abiotic stress on living organisms within a marine ecosystem. Sediments were collected during the wet and dry seasons and analyzed using bacterial metagenomics and community metabolomics techniques. Physicochemical data were also analyzed to account for biological variance that may be due to non-rainfall-based abiotic stresses. Wet-dry seasonality was the dominant control on bacterial community structure and metabolic function. Changes in the availability of nutrients, organic matter and light appeared to be the major seasonal stressors. In contrast, urban and industrial pollutants appeared to be minor stressors at the sites sampled. During the wet season, the bacterial community composition reflected organisms that utilize biogeochemical pathways with fast kinetics, such as aerobic metabolism, direct assimilation of inorganic compounds, and primary production. The transition to the dry season saw the bacterial community composition shift towards organisms that utilize more complex organic energy sources, such as carbohydrates and fatty acids, and anaerobic redox processes.

Comparisons of bacterial and archaeal communities in the rumen and a dual-flow continuous culture fermentation system using amplicon sequencing.

Dual-flow continuous culture (CC) fermenters are commonly used to study rumen fermentation in vitro. Research using culture-based and oligonucleotide techniques has shown that certain microbial populations within fermenters may be maintained at abundances similar to those observed in vivo. In this study, bacterial and archaeal communities in the rumen of dairy cattle and in a dual-flow CC fermentation system were compared using high-throughput amplicon sequencing targeting the V4 hypervariable region of 16S rRNA. We hypothesized that the in vitro system harbored a comparable bacterial and archaeal community to that observed in the rumen. Members of the Bacteroidetes and Firmicutes made up the 2 most abundant phyla in the rumen, inoculum, and fermenters and did not differ among sample types (P > 0.10). Similarly, Prevotellaceae, the most abundant family in all 3 sample types, did not differ based on source (P = 0.80). However, beta diversity analyses revealed that bacterial and archaeal communities differed between fermenters and rumen samples (P ≤ 0.001), but fermenter bacterial and archaeal communities stabilized by day 4 of each period. While the overall bacterial and archaeal community differs between natural rumens and those detected in in vitro fermenter systems, several prominent taxa were maintained at similar relative abundances suggesting that fermenters may provide a suitable environment in which to study shifts among the predominant members of the microbial community.

A multi-omics based ecological analysis of coastal marine sediments from Gladstone, in Australia's Central Queensland, and Heron Island, a nearby fringing platform reef.

The impact of anthropogenic factors arising from point and non-point pollution sources at a multi commodity marine port and its surrounding ecosystems were studied using sediment samples collected from a number of onshore (Gladstone Harbour and Facing Island) and offshore (Heron Island and Fitzroy Reefs) sites in Australia's Central Queensland. Sediment samples were analyzed for trace metals, organic carbon, polycyclic aromatic hydrocarbons (PAH), emerging chemicals of concern (ECC) and sterols. Similarly, the biological and biochemical interaction between the reef and its environment was analyzed by the multi-omic tools of next-generation sequencing characterization of the bacterial community and microbial community metabolic profiling. Overall, the trace elements were observed at the lower end of the Australian environmental guideline values at the offshore sites, while higher values were observed for the onshore locations Nickel and copper were observed above the high trigger value threshold at the onshore sites. The levels of PAH were below limits of detection across all sites. However, some of the ECC and sterols were observed at higher concentrations at both onshore and offshore locations, notably, the cholesterol family sterols and 17α-ethynylestradiol. Multi-omic analyses also indicated possible thermal and photo irradiation stressors on the bacterial communities at all the tested sites. The observed populations of γ-proteobacteria were found in combination with an increased pool of fatty acids that indicate fatty acid synthesis and utilisation of the intermediates of the shikimate pathways. This study demonstrates the value of applying a multi-omics approach for ecological assessments, in which a more detailed assessment of physical and chemical contaminants and their impact on the community bacterial biome is obtained.

Amplicon-based taxonomic characterization of bacteria in urban and peri-urban roof-harvested rainwater stored in tanks.

Overall, 26% of Australian households use rainwater tanks as a source of potable and nonpotable water. Limited information is available on the total bacterial communities in tank water. Therefore, identification of dominant bacterial communities, diversity, and their distribution is important in understanding the microbial quality of tank water. In this study, the abundance and diversity of bacterial communities in 88 tank water samples collected from the urban areas of Brisbane (n=44) and the peri-urban center of Currumbin (n=44) in Southeast Queensland, Australia were determined using amplicon-based Illumina next-generation sequencing. In addition, the SourceTracker program was used to identify the sources of fecal contamination in tank water samples. Sequence reads were also analyzed to detect potential bacterial pathogenic genera in the tank water samples collected. Differences in sample coverage, alpha diversity, and richness did not differ significantly between the Brisbane and Currumbin tank water samples. Comamonadaceae and Planctomycetaceae were the most abundant families in all tank water samples. Curvibacter was the most abundant genus in all tank water samples. SourceTracker revealed that around 34% (Brisbane) and 43% (Currumbin) of tank water samples had a signature for bird fecal contamination. The potential opportunistic pathogenic genera including Burkholderia, Chromobacterium, Clostridium, Legionella, Mycobacterium, Nocardia, and Pseudomonas were most prevalent in tank water samples. Next-generation sequencing can be used as an initial screening tool to identify a wide array of potential pathogenic genera in tank water samples followed by quantifying specific pathogen(s) of interest using more sensitive molecular assays such as quantitative PCR (qPCR).

Toolbox Approaches Using Molecular Markers and 16S rRNA Gene Amplicon Data Sets for Identification of Fecal Pollution in Surface Water.

In this study, host-associated molecular markers and bacterial 16S rRNA gene community analysis using high-throughput sequencing were used to identify the sources of fecal pollution in environmental waters in Brisbane, Australia. A total of 92 fecal and composite wastewater samples were collected from different host groups (cat, cattle, dog, horse, human, and kangaroo), and 18 water samples were collected from six sites (BR1 to BR6) along the Brisbane River in Queensland, Australia. Bacterial communities in the fecal, wastewater, and river water samples were sequenced. Water samples were also tested for the presence of bird-associated (GFD), cattle-associated (CowM3), horse-associated, and human-associated (HF183) molecular markers, to provide multiple lines of evidence regarding the possible presence of fecal pollution associated with specific hosts. Among the 18 water samples tested, 83%, 33%, 17%, and 17% were real-time PCR positive for the GFD, HF183, CowM3, and horse markers, respectively. Among the potential sources of fecal pollution in water samples from the river, DNA sequencing tended to show relatively small contributions from wastewater treatment plants (up to 13% of sequence reads). Contributions from other animal sources were rarely detected and were very small (<3% of sequence reads). Source contributions determined via sequence analysis versus detection of molecular markers showed variable agreement. A lack of relationships among fecal indicator bacteria, host-associated molecular markers, and 16S rRNA gene community analysis data was also observed. Nonetheless, we show that bacterial community and host-associated molecular marker analyses can be combined to identify potential sources of fecal pollution in an urban river. This study is a proof of concept, and based on the results, we recommend using bacterial community analysis (where possible) along with PCR detection or quantification of host-associated molecular markers to provide information on the sources of fecal pollution in waterways.

Predominant populations of indigenous soybean-nodulating Bradyrhizobium japonicum strains obtained from organic farming systems in Minnesota.

AIMS: Bradyrhizobium from organic fields in Minnesota were isolated and genotyped to assess diversity of soybean-bradyrhizobia in organic farming systems that can be used to improve soybean productivity. METHODS AND RESULTS: Soil samples were collected from 25 organic fields in Minnesota during May to July 2012. Soybean (cv. Lambert) was used as a host to trap indigenous bradyrhizobia in each sample. Genetic diversity of Bradyrhizobium strains (n=733) was determined using the horizontal, fluorophore-enhanced, repetitive extragenic palindromic-PCR (HFERP) DNA fingerprinting technique and the soybean-bradyrhizobia were classified into 79 different genotypes. Of these, 15 dominant genotypes were found and were highly similar (>92% fingerprint similarity) to serotypes USDA 127 (40.4%), USDA 4 (31.8%) and USDA 123 (15.5%), which were the three main populations of soybean-bradyrhizobia in organic fields. CONCLUSIONS: Bradyrhizobium japonicum serogroup USDA 4 strains were found to make up a previously unrecognized, predominant rhizobial population in the organic farming soils examined. The relative abundance of strain USDA 4 was negatively correlated with that of USDA 127 and this relationship may be influenced by the levels of NO3 -N and other soil edaphic factors. SIGNIFICANCE AND IMPACT OF THE STUDY: The local community of bradyrhizobia can be affected by applying inoculant bacteria to organic fields. Based on these results, soybean production in organic farms may be improved by displacing strains similar to USDA 4 with those better at nitrogen fixation and competitive ability than indigenous strains.

Low CHD5 expression activates the DNA damage response and predicts poor outcome in patients undergoing adjuvant therapy for resected pancreatic cancer.

The DNA damage response (DDR) promotes genome integrity and serves as a cancer barrier in precancerous lesions but paradoxically may promote cancer survival. Genes that activate the DDR when dysregulated could function as useful biomarkers for outcome in cancer patients. Using a siRNA screen in human pancreatic cancer cells, we identified the CHD5 tumor suppressor as a gene, which, when silenced, activates the DDR. We evaluated the relationship of CHD5 expression with DDR activation in human pancreatic cancer cells and the association of CHD5 expression in 80 patients with resected pancreatic adenocarcinoma (PAC) by immunohistochemical analysis with clinical outcome. CHD5 depletion and low CHD5 expression in human pancreatic cancer cells lead to increased H2AX-Ser139 and CHK2-Thr68 phosphorylation and accumulation into nuclear foci. On Kaplan-Meier log-rank survival analysis, patients with low CHD5 expression had a median recurrence-free survival (RFS) of 5.3 vs 15.4 months for patients with high CHD5 expression (P=0.03). In 59 patients receiving adjuvant chemotherapy, low CHD5 expression was associated with decreased RFS (4.5 vs 16.3 months; P=0.001) and overall survival (OS) (7.2 vs 21.6 months; P=0.003). On multivariate Cox regression analysis, low CHD5 expression remained associated with worse OS (HR: 3.187 (95% CI: 1.49-6.81); P=0.003) in patients undergoing adjuvant chemotherapy. Thus, low CHD5 expression activates the DDR and predicts for worse OS in patients with resected PAC receiving adjuvant chemotherapy. Our findings support a model in which dysregulated expression of tumor suppressor genes that induce DDR activation can be utilized as biomarkers for poor outcome.

Application of Illumina next-generation sequencing to characterize the bacterial community of the Upper Mississippi River.

AIMS: A next-generation, Illumina-based sequencing approach was used to characterize the bacterial community at ten sites along the Upper Mississippi River to evaluate shifts in the community potentially resulting from upstream inputs and land use changes. Furthermore, methodological parameters including filter size, sample volume and sample reproducibility were evaluated to determine the best sampling practices for community characterization. METHODS AND RESULTS: Community structure and diversity in the river was determined using Illumina next-generation sequencing technology and the V6 hypervariable region of 16S rDNA. A total of 16,400 operational taxonomic units (OTUs) were observed (4594 ± 824 OTUs per sample). Proteobacteria, Actinobacteria, Bacteroidetes, Cyanobacteria and Verrucomicrobia accounted for 93.6 ± 1.3% of all sequence reads, and 90.5 ± 2.5% belonged to OTUs shared among all sites (n = 552). Among nonshared sequence reads at each site, 33-49% were associated with potentially anthropogenic impacts upstream of the second sampling site. Alpha diversity decreased with distance from the pristine headwaters, while rainfall and pH were positively correlated with diversity. Replication and smaller filter pore sizes minimally influenced the characterization of community structure. CONCLUSIONS: Shifts in community structure are related to changes in the relative abundance, rather than presence/absence of OTUs, suggesting a 'core bacterial community' is present throughout the Upper Mississippi River. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is among the first to characterize a large riverine bacterial community using a next-generation-sequencing approach and demonstrates that upstream influences and potentially anthropogenic impacts can influence the presence and relative abundance of OTUs downstream resulting in significant variation in community structure.

Microbial source tracking to identify human and ruminant sources of faecal pollution in an ephemeral Florida river.

AIMS: Levels and sources of faecal indicator bacteria (FIB) in an ephemeral Florida river were assessed under different rainfall/flow patterns to explore the effects of rainfall on water quality. METHODS AND RESULTS: Quantitative PCR for sewage markers [human-associated Bacteroides HF183 and human polyomaviruses (HPyVs)] and PCR for ruminant faecal markers were used to explore contamination sources. Escherichia coli, faecal coliform and enterococci levels consistently exceeded recreational water quality criteria, and sediment FIB levels were about 100-fold higher compared with water. HPyVs detections cooccurred with HF183, which was frequently detected near septic systems. Ruminant markers were detected only in livestock-grazing areas. Significantly greater faecal coliform and E. coli concentrations were observed under no-flow conditions and the levels of faecal coliforms in water column and sediments were negatively correlated with duration since last rain event. CONCLUSIONS: Septic systems and cattle grazing in this watershed contributed to the formation of FIB reservoirs in sediments, which were persistent following prolonged rainfall. SIGNIFICANCE AND IMPACT OF THE STUDY: Ephemeral water bodies that flow only under the direct influence of recent rainfall are rarely studied. FIB levels in the New River in Florida were greater during dry weather than wet weather, which contrasts with most observations and may be attributed to bacterial reservoirs formed in still pool, sediments and water-saturated soils in this subtropical environment.

Colonic motor dysfunction in human diabetes is associated with enteric neuronal loss and increased oxidative stress.

BACKGROUND: Gastrointestinal dysfunction is very common in diabetic patients. We assessed the changes in the colonic enteric nervous system using colectomy specimens and intestinal biopsies from diabetic subjects and age-matched controls. METHODS: In control and diabetic colons, we determined the total ganglion area (hematoxylin-eosin staining), changes in neuronal markers-protein gene product 9.5, peripherin, neuronal nitric oxide synthase (nNOS), neuropeptide Y (NPY), choline acetyl transferase (ChAT) and vasoactive intestinal peptide (by immunostaining), apoptosis (cleaved caspase-3 staining) and reduced glutathione levels. Superoxide dismutase mRNA was determined in enteric ganglia isolated by laser capture micro dissection. Isometric muscle recording was used to assess contraction and relaxation responses of colonic circular muscle strips. Apoptosis in enteric neurons under hyperglycemia in vitro was determined by cleaved caspase-3 Western blotting and protective effects of lipoic acid were evaluated. KEY RESULTS: Diabetic subjects had higher incidence of lower gastrointestinal symptoms like constipation and diarrhea at baseline prior to surgery. Diabetic ganglia displayed significant decrease in ganglion size due to enhanced apoptosis and loss of peripherin, nNOS, NPY, and ChAT neurons. Reduced glutathione levels in the diabetic colon (HbA1C > 7%) were significantly less than the control, indicating increased oxidative stress. Colonic circular muscle strips from diabetic subjects showed impaired contraction and relaxation responses compared with the healthy controls. Hyperglycemia-induced cleaved caspase-3 in enteric neurons was reversed by lipoic acid. CONCLUSIONS & INFERENCES: Our data demonstrate loss of enteric neurons in the colon due to increased oxidative stress and apoptosis which may cause the motility disturbances seen in human diabetes. Antioxidants may be of therapeutic value for preventing motility disorders in diabetes.

Neurocognitive deficits in decision-making and planning of patients with DSM-III-R borderline personality disorder.

BACKGROUND: Repeated, self-damaging behaviour occurring in the context of borderline personality disorder (BPD) may reflect impairments in decision-making and planning cognition. However, there has been no systematic neuropsychological examination of these particular cognitive functions in patients diagnosed with BPD. Such investigations may improve our understanding of the possible role of brain dysfunction in BPD and improve the characterization of the psychological difficulties associated with this disorder. METHOD: Forty-two psychiatric patients with a diagnosis of DSM-III-R BPD (41 of whom gave a history of self-harm), without a history of specified 'psychoses' or current major affective disorder, were clinically assessed before completing computerized tasks of decision-making and planning previously shown to be sensitive to frontal lobe dysfunction, and tests of spatial and pattern visual recognition memory previously shown to be sensitive to frontal lobe damage and temporal lobe damage respectively. The performance of the BPD patient group was compared with that of a non-clinical control group consisting of 42 subjects. RESULTS: The performance of the BPD patients on the decision-making task was characterized by a pattern of delayed and maladaptive choices when choosing between competing actions, and by impulsive, disinhibited responding when gambling on the outcome of their decisions. BPD patients also showed impairments on the planning task. There was no evidence of impaired visual recognition memory. Additional analyses suggested only limited effects of current medication and history of previous substance use disorder. CONCLUSIONS: These findings suggest that BPD is associated with complex impairments in dissociable cognitive processes mediated by circuitry encompassing the frontal lobes. These impairments may mediate some of the behavioural changes evident in BPD. Further work is needed to examine the specificity of these findings.

Genetic diversity and response to IFN of the NS3 protease gene from clinical strains of the hepatitis C virus.

The N-terminal one-third of the hepatitis C virus nonstructural gene 3 (NS3) codes for a serine protease. To investigate natural genetic diversity of this enzyme a nested PCR reaction was developed to obtain NS3 protease sequence data directly from patient strains. This data was used to determine genetic diversity, phylogenetic and evolutionary rates, and selection of variants by interferon therapy. The potential effect of genetic diversity on enzyme structure using molecular modeling was also attempted. Results show significant variability in clinical HCV strains at both the nucleotide (30.2% for 1a and 25.8% for 1b) and amino acid sequences (11.0% for 1a and 9.9% for 1b). Phylogenic analysis shows two distinct clades with two HCV isolates grouping as a sister clade to 1b. Structural analysis reveals that most mutations lie in the N-terminus of the enzyme. When strains were sorted as to whether or not the patient had received antiviral therapy, no difference was found in the number or locations of mutations in 1a strains. However, 1b strains demonstrated an overall drop in the number of positions that were mutated. This study demonstrates significant differences among natural strains that may pose a problem for structure based drug development.

Genetic approaches to studying protein synthesis: effects of mutations at Psi516 and A535 in Escherichia coli 16S rRNA.

A genetic system for the study of ribosomal RNA function and structure was developed. First, the ribosome binding sequence of the chloramphenicol acetyltransferase gene and the message binding sequence of 16S ribosomal RNA were randomly mutated and alternative highly functional sequences were selected and characterized. From this set of mutants, a single clone was chosen and subjected to a second round of mutagenesis to optimize the specificity of the system. In the resulting system, plasmid-encoded ribosomes efficiently and exclusively translate specific mRNA containing the appropriate ribosome binding sequences. This system allows facile isolation and analysis of mutations that would normally be lethal and allows direct selection of rRNA mutants with predetermined levels of ribosome function. The system was used to examine the effects of mutations at the sole pseudouridine (Psi) in Escherichia coli 16S rRNA which is located at position 516 of the conserved 530 loop. The nucleotide opposite Psi516 in the hairpin, A535, was also mutated. The data show that a pyrimidine (Psi or C) is required at position 516, while substitutions at position 535 reduce ribosome function by < 50%. A requirement for base pair formation between Psi516 and A535 was not indicated.

Comparison of results generated by serotyping, pulsed-field restriction analysis, ribotyping, and repetitive-sequence PCR used to characterize penicillin-resistant pneumococci from the United States.

One hundred forty-seven isolates of Streptococcus pneumoniae with high-level penicillin resistance collected during a national surveillance program in the United States were characterized by serotyping, pulsed-field restriction analysis, ribotyping, and repetitive-sequence (BOX element) PCR. The results generated by each method were compared by frequency of association to examine whether relationships existed between the various typing methods and statistically to determine association with the geographic source of the isolate or the age of the patient from whom the isolate was obtained. When the data were examined by pairwise analysis of individual strain classifications produced by each typing method, no statistically significant relationships between strain type, geographic location, or patient age were identified, suggesting that distinct clones of penicillin-resistant S. pneumoniae have been widely distributed throughout the United States. However, we did observed shared expression of two or three typing markers at a high frequency (>50%) among clusters of strains, indicating a certain level of concordance between the various typing methods used to classify penicillin-resistant S. pneumoniae.

Augmentation with sulpiride for a schizophrenic patient partially responsive to clozapine.

OBJECTIVE: Schizophrenic patients who are only partially responsive to clozapine pose a therapeutic challenge. In these circumstances some clinicians would consider adding in a second antipsychotic. We present a case report and review evidence for the efficacy of such augmentation strategies. METHOD: Single case report and literature review. RESULTS: The total number of patients in studies and case reports of combining clozapine with other antipsychotics is small. There has been only one randomized controlled trial. This found the addition of sulpiride to clozapine resulted in clinical improvement in some patients. CONCLUSION: Further randomized controlled studies of augmentation of clozapine therapy are needed to provide scientific justification for this clinical practice.

Aerobic activity of Escherichia coli alcohol dehydrogenase is determined by a single amino acid.

Expression of the alcohol dehydrogenase gene, adhE, in Escherichia coli is anaerobically regulated at both the transcriptional and the translational levels. To study the AdhE protein, the adhE(+) structural gene was cloned into expression vectors under the control of the lacZ and trp(c) promoters. Wild-type AdhE protein produced under aerobic conditions from these constructs was inactive. Constitutive mutants (adhC) that produced high levels of AdhE under both aerobic and anaerobic conditions were previously isolated. When only the adhE structural gene from one of the adhC mutants was cloned into expression vectors, highly functional AdhE protein was isolated under both aerobic and anaerobic conditions. Sequence analysis revealed that the adhE gene from the adhC mutant contained two mutations resulting in two amino acid substitutions, Ala267Thr and Glu568Lys. Thus, adhC strains contain a promoter mutation and two mutations in the structural gene. The mutant structural gene from adhC strains was designated adhE*. Fragment exchange experiments revealed that the substitution responsible for aerobic expression in the adhE* clones is Glu568Lys. Genetic selection and site-directed mutagenesis experiments showed that virtually any amino acid substitution for Glu568 produced AdhE that was active under both aerobic and anaerobic conditions. These findings suggest that adhE expression is also regulated posttranslationally and that strict regulation of alcohol dehydrogenase activity in E. coli is physiologically significant.