Outlier Strain Research Articles

Determination of the two-component systems regulatory network reveals core and accessory regulations across Pseudomonas aeruginosa lineages.

Pseudomonas aeruginosa possesses one of the most complex bacterial regulatory networks, which largely contributes to its success as a pathogen. However, most of its transcription factors (TFs) are still uncharacterized and the potential intra-species variability in regulatory networks has been mostly ignored so far. Here, we used DAP-seq to map the genome-wide binding sites of all 55 DNA-binding two-component systems (TCSs) response regulators (RRs) across the three major P. aeruginosa lineages. The resulting networks encompass about 40% of all genes in each strain and contain numerous new regulatory interactions across most major physiological processes. Strikingly, about half of the detected targets are specific to only one or two strains, revealing a previously unknown large functional diversity of TFs within a single species. Three main mechanisms were found to drive this diversity, including differences in accessory genome content, as exemplified by the strain-specific plasmid in IHMA87 outlier strain which harbors numerous binding sites of conserved chromosomally-encoded RRs. Additionally, most RRs display potential auto-regulation or RR-RR cross-regulation, bringing to light the vast complexity of this network. Overall, we provide the first complete delineation of the TCSs regulatory network in P. aeruginosa that will represent an important resource for future studies on this pathogen.

ExlA Pore-Forming Toxin: Localization at the Bacterial Membrane, Regulation of Secretion by Cyclic-Di-GMP, and Detection In Vivo.

ExlA is a highly virulent pore-forming toxin that has been recently discovered in outlier strains from Pseudomonas aeruginosa. ExlA is part of a two-partner secretion system, in which ExlA is the secreted passenger protein and ExlB the transporter embedded in the bacterial outer membrane. In previous work, we observed that ExlA toxicity in a host cell was contact-dependent. Here, we show that ExlA accumulates at specific points of the outer membrane, is likely entrapped within ExlB pore, and is pointing outside. We further demonstrate that ExlA is maintained at the membrane in conditions where the intracellular content of second messenger cyclic-di-GMP is high; lowering c-di-GMP levels enhances ExlB-dependent ExlA secretion. In addition, we set up an ELISA to detect ExlA, and we show that ExlA is poorly secreted in liquid culture, while it is highly detectable in broncho-alveolar lavage fluids of mice infected with an exlA+ strain. We conclude that ExlA translocation is halted at mid-length in the outer membrane and its secretion is regulated by c-di-GMP. In addition, we developed an immunological test able to quantify ExlA in biological samples.

Clinical and molecular characteristics of carbapenem non-susceptible Escherichia coli: A nationwide survey from Oman.

The prevalence of carbapenem-resistant Enterobacterales (CRE) in the Arabian Peninsula is predicted to be high, as suggested from published case reports. Of particular concern, is carbapenem-resistant E. coli (CR-EC), due to the importance of this species as a community pathogen. Herein, we conducted a comprehensive molecular characterization of putative CR-EC strains from Oman. We aim to establish a baseline for future molecular monitoring. We performed whole-genome sequencing (WGS) for 35 putative CR-EC. Isolates were obtained from patients at multiple centers in 2015. Genetic relatedness was investigated using several typing approaches such as MLST, SNP calling, phylogroup and CRISPR typing. Maxiuium likelihood SNP-tree was performed by RAxML after variant calling and removal of recombination regions with Snippy and Gubbins, respectively. Resistance genes, plasmid replicon types, virulence genes, and prophage were also characterised. The online databases CGE, CRISPRcasFinder, Phaster and EnteroBase were used for the in silico analyses. Screening for mutations in genes regulating the expression of porins and efflux pump as well as mutations lead to fluoroquinolones resistance were performed with CLC Genomics Workbench. The genetic diversity suggests a polyclonal population structure with 21 sequence types (ST), of which ST38 being the most prevalent (11%). SNPs analysis revealed possible transmission episodes. Whereas, CRISPR typing helped to spot outlier strains belonged to phylogroups other than B2 which was CRISPR-free. The virulent phylogroups B2 and D were detected in 4 and 9 isolates, respectively. In some strains bacteriophages acted as vectors for virulence genes. Regarding resistance to β-lactam, 22 were carbapenemase producers, 3 carbapenem non-susceptible but carbapenemase-negative, 9 resistant to expanded-spectrum cephalosporins, and one isolate with susceptibility to cephalosporins and carbapenems. Thirteen out of the 22 (59%) carbapenemase-producing isolates were NDM and 7 (23%) were OXA-48-like which mirrors the situation in Indian subcontinent. Two isolates co-produced NDM and OXA-48-like enzymes. In total, 80% (28/35) were CTX-M-15 producers and 23% (8/35) featured AmpC. The high-risk subclones ST131-H30Rx/C2, ST410-H24RxC and ST1193-H64RxC were detected, the latter associated with NDM. To our knowledge, this is the first report of ST1193-H64Rx subclone with NDM. In conclusion, strains showed polyclonal population structure with OXA-48 and NDM as the only carbapenemases in CR-EC from Oman. We detected the high-risk subclone ST131-H30Rx/C2, ST410-H24RxC and ST1193-H64RxC. The latter was reported with carbapenemase gene for the first time here.

Comprehensive annotations of the mutational spectra of SARS-CoV-2 spike protein: a fast and accurate pipeline.

Infecting millions of people, the SARS‐CoV‐2 is evolving at an unprecedented rate, demanding advanced and specified analytic pipeline to capture the mutational spectra. In order to explore mutations and deletions in the spike (S) protein — the most‐discussed protein of SARS‐CoV‐2 — we comprehensively analyzed 35,750 complete S protein‐coding sequences through a custom Python‐based pipeline. This GISAID‐collected dataset of until 24 June 2020 covered six continents and five major climate zones. We identified 27,801 (77.77% sequences) mutated strains compared to reference Wuhan‐Hu‐1 wherein 84.40% of these strains mutated by only a single amino acid (aa). An outlier strain (EPI_ISL_463893) from Bosnia and Herzegovina possessed six aa substitutions. We also identified 11 residues with high aa mutation frequency, and each contains four types of aa variations. The infamous D614G variant has spread worldwide with ever‐rising dominance and across regions with different climatic conditions alongside L5F and D936Y mutants, which have been documented throughout all regions and climate zones, respectively. We also found 988 unique aa substitutions spanned across 660 residues, which differed significantly among different continents (p = .003) and climatic zones (p = .021) as inferred with the Kruskal–Wallis test. Besides, 17 in‐frame deletions at four sites adjacent to receptor‐binding‐domain were determined that may have a possible impact on attenuation. This study provides a fast and accurate pipeline for identifying mutations and deletions from the large dataset for coding and also non‐coding sequences as evidenced by the representative analysis on existing S protein data. By using separate multi‐sequence alignment, removing ambiguous sequences and in‐frame stop codons, and utilizing pairwise alignment, this method can derive both synonymous and non‐synonymous mutations (strain_ID reference aa:mutation position:strain aa). We suggest that the pipeline will aid in the evolutionary surveillance of any SARS‐CoV‐2 encoded proteins and will prove to be crucial in tracking the ever‐increasing variation of many other divergent RNA viruses in the future. The code is available at https://github.com/SShaminur/Mutation-Analysis.

Genomic insights into the phylogeny of Bacillus strains and elucidation of their secondary metabolic potential

The genus Bacillus constitutes a plethora of species that have medical, environmental, and industrial applications. While genus Bacillus has been the focus of several studies where genomic data have been used to resolve many taxonomic issues, there still exist several ambiguities. Through the use of in-silico genome-based methods, we tried to resolve the taxonomic anomalies of a large set of Bacillus genomes (n = 178). We also proposed species names for uncharacterized strains and reported genome sequence of a novel isolate Bacillus sp. RL. In the hierarchical clustering on genome-to-genome distances, we observed 11 distinct monophyletic clusters and investigated the functional pathways annotated as the property of these clusters and core-gene content of the entire dataset. Thus, we were able to assert the possible outlier strains (n = 17) for this genus. Analyses of secondary metabolite potential of each strain helped us unravel still unexplored diversity for various biosynthetic genes.

Rapid and solitary production of mono-rhamnolipid biosurfactant and biofilm inhibiting pyocyanin by a taxonomic outlier Pseudomonas aeruginosa strain CR1

Biosurfactant - Rhamnolipids (RLs) and antibacterial toxin - pyocyanin (PYO) produced by Pseudomonas aeruginosa strains have great potential for biotechnological applications. Generally, RLs are produced as a mixture of di-rhamnolipids (di-RLs) and mono-rhamnolipids (mono-RLs). Mono-RLs possess superior emulsification and antimicrobial properties and are costlier than di-RLs. In this study, a taxonomic outlier P. aeruginosa strain CR1 isolated from rhizosphere soil was explored for mono-RLs and PYO production. Phylogenetically strain CR1 resembles avirulent outlier P. aeruginosa strain ATCC9027, lacks archetypical virulence genes and harbors unique pathways for the synthesis of solely mono-RLs and PYO. Strain CR1 produced RL biosurfactant which efficiently emulsified hydrocarbons, showed hemolysis and inhibited Bacillus subtilis. At 37 °C, strain CR1 exclusively produced 21.77 g L−1 and 19.22 g L−1 rhamnolipid in glycerol amended Luria Bertani (LB) medium and basal medium amended with rice bran oil, respectively after 54 h growth. Besides RL production was unaffected under varying nitrogen sources. Structural characterization using FTIR, TLC, and LC–MS confirmed that strain CR1 exclusively produced mono-RLs, majorly dominated by Rha-C10-C10, Rha-C10-C8, and CH3-Rha-C12:2-C10:1. The compound was stable over a wide pH range (4–12), salinity (25%) and 100 °C indicating its applicability under harsh environmental conditions. In addition, strain CR1 produced 4.5 μg mL−1 PYO, which could efficiently inhibit biofilm formation by Bacillus species. The environmental outlier strain CR1 can be used for the industrial production of biotechnologically important mono-RLs and PYO.

Comparative Genomic Analyses Reveal Core-Genome-Wide Genes Under Positive Selection and Major Regulatory Hubs in Outlier Strains of Pseudomonas aeruginosa.

Genomic information for outlier strains of Pseudomonas aeruginosa is exiguous when compared with classical strains. We sequenced and constructed the complete genome of an environmental strain CR1 of P. aeruginosa and performed the comparative genomic analysis. It clustered with the outlier group, hence we scaled up the analyses to understand the differences in environmental and clinical outlier strains. We identified eight new regions of genomic plasticity and a plasmid pCR1 with a VirB/D4 complex followed by trimeric auto-transporter that can induce virulence phenotype in the genome of strain CR1. Virulence genotype analysis revealed that strain CR1 lacked hemolytic phospholipase C and D, three genes for LPS biosynthesis and had reduced antibiotic resistance genes when compared with clinical strains. Genes belonging to proteases, bacterial exporters and DNA stabilization were found to be under strong positive selection, thus facilitating pathogenicity and survival of the outliers. The outliers had the complete operon for the production of vibrioferrin, a siderophore present in plant growth promoting bacteria. The competence to acquire multidrug resistance and new virulence factors makes these strains a potential threat. However, we identified major regulatory hubs that can be used as drug targets against both the classical and outlier groups.

Antibiotic susceptibility testing of Mycoplasma hyopneumoniae field isolates from Central Europe for fifteen antibiotics by microbroth dilution method.

Mycoplasma hyopneumoniae infections are responsible for significant economic losses in the swine industry. Commercially available vaccines are not able to inhibit the colonisation of the respiratory tract by M. hyopneumoniae absolutely, therefore vaccination can be completed with antibiotic treatment to moderate clinical signs and improve performances of the animals. Antibiotic susceptibility testing of M. hyopneumoniae is time-consuming and complicated; therefore, it is not accomplished routinely. The aim of this study was to determine the in vitro susceptibility to 15 different antibiotics of M. hyopneumoniae isolates originating from Hungarian slaughterhouses and to examine single-nucleotide polymorphisms (SNPs) in genes affecting susceptibility to antimicrobials. Minimum inhibitory concentration (MIC) values of the examined antibiotics against 44 M. hyopneumoniae strains were determined by microbroth dilution method. While all of the tested antibiotics were effective against the majority of the studied strains, high MIC values of fluoroquinolones (enrofloxacin 2.5 μg/ml; marbofloxacin 5 μg/ml) were observed against one strain (MycSu17) and extremely high MIC values of macrolides and lincomycin (tilmicosin, tulathromycin and lincomycin >64 μg/ml; gamithromycin 64 μg/ml; tylosin 32 μg/ml and tylvalosin 2 μg/ml) were determined against another, outlier strain (MycSu18). Amino acid changes in the genes gyrA (Gly81Ala; Ala83Val; Glu87Gly, according to Escherichia coli numbering) and parC (Ser80Phe/Tyr; Asp84Asn) correlated with decreased antibiotic susceptibility to fluoroquinolones and a SNP in the nucleotide sequence of the 23S rRNA (A2059G) was found to be associated with increased MIC values of macrolides. The correlation was more remarkable when final MIC values were evaluated. This study presented the antibiotic susceptibility profiles of M. hyopneumoniae strains circulating in the Central European region, demonstrating the high in vitro efficacy of the tested agents. The observed high MIC values correlated with the SNPs in the examined regions and support the relevance of susceptibility testing and directed antibiotic therapy.

CAMP and Vfr Control Exolysin Expression and Cytotoxicity of Pseudomonas aeruginosa Taxonomic Outliers.

The two-partner secretion system ExlBA, expressed by strains of Pseudomonas aeruginosa belonging to the PA7 group, induces hemorrhage in lungs due to disruption of host cellular membranes. Here we demonstrate that the exlBA genes are controlled by a pathway consisting of cAMP and the virulence factor regulator (Vfr). Upon interaction with cAMP, Vfr binds directly to the exlBA promoter with high affinity (equilibrium binding constant [Keq] of ≈2.5 nM). The exlB and exlA expression was diminished in the Vfr-negative mutant and upregulated with increased intracellular cAMP levels. The Vfr binding sequence in the exlBA promoter was mutated in situ, resulting in reduced cytotoxicity of the mutant, showing that Vfr is required for the exlBA expression during intoxication of epithelial cells. Vfr also regulates function of type 4 pili previously shown to facilitate ExlA activity on epithelial cells, which indicates that the cAMP/Vfr pathway coordinates these two factors needed for full cytotoxicity. As in most P. aeruginosa strains, the adenylate cyclase CyaB is the main provider of cAMP for Vfr regulation during both in vitro growth and eukaryotic cell infection. We discovered that the absence of functional Vfr in the reference strain PA7 is caused by a frameshift in the gene and accounts for its reduced cytotoxicity, revealing the conservation of ExlBA control by the CyaB-cAMP/Vfr pathway in P. aeruginosa taxonomic outliers.IMPORTANCE The human opportunistic pathogen Pseudomonas aeruginosa provokes severe acute and chronic human infections associated with defined sets of virulence factors. The main virulence determinant of P. aeruginosa taxonomic outliers is exolysin, a membrane-disrupting pore-forming toxin belonging to the two-partner secretion system ExlBA. In this work, we demonstrate that the conserved CyaB-cAMP/Vfr pathway controls cytotoxicity of outlier clinical strains through direct transcriptional activation of the exlBA operon. Therefore, despite the fact that the type III secretion system and exolysin are mutually exclusive in classical and outlier strains, respectively, these two major virulence determinants share similarities in their mechanisms of regulation.

Phylogenetic analysis of emergent Streptococcus pneumoniae serotype 22F causing invasive pneumococcal disease using whole genome sequencing.

Since implementation of the 13-valent polyvalent conjugate vaccine (PCV13) in Canada during 2010, the proportion of PCV13 serotypes causing invasive pneumococcal disease (IPD) has declined from 55% (n = 1492) in 2010 to 31% (n = 764) in 2014. A concurrent increase of non-PCV13 serotypes has occurred and 22F has become the most prevalent serotype in Canada increasing from 7% (n = 183) to 11% (n = 283). Core single nucleotide variant phylogenetic analysis was performed on 137 Streptococcus pneumoniae serotype 22F isolates collected across Canada from 2005–2015. Six phylogenetic lineages (n = 117) were identified among a serotype 22F/ST433 clonal complex (CC), including a recently expanding erythromycin-resistant clone. Erythromycin-resistance was observed in 25 isolates possessing ermB, mef or a 23S rRNA A2061G point mutation; 2 penicillin-resistant isolates had recombinant pbp1a, pbp2a and/or pbp2x; 3 tetracycline-resistant isolates contained tetM; and 1 isolate was multidrug-resistant. Virulence factor analysis indicated a high level of homogeneity among the 22F/ST433 clonal complex strains. A group of 6 phylogenetic outlier strains had differing MLST, antimicrobial resistance and molecular profiles suggestive of capsule switching events. While capsule switch events among S. pneumoniae serotype 22F has been observed, increasing prevalence of S. pneumoniae serotype 22F can be attributed to an evolving homogenous clone expanding nationally through local transmission events.

Population genomic analysis uncovers environmental stress-driven selection and adaptation of Lentinula edodes population in China.

The elucidation of genome-wide variations could help reveal aspects of divergence, domestication, and adaptation of edible mushrooms. Here, we resequenced the whole genomes of 39 wild and 21 cultivated strains of Chinese Lentinula edodes, the shiitake mushroom. We identified three distinct genetic groups in the Chinese L. edodes population with robust differentiation. Results of phylogenetic and population structure analyses suggest that the cultivated strains and most of the wild trains of L. edodes in China possess different gene pools and two outlier strains show signatures of hybridization between groups. Eighty-four candidate genes contributing to population divergence were detected in outlier analysis, 18 of which are involved in response to environmental stresses. Gene enrichment analysis of group-specific single nucleotide polymorphisms showed that the cultivated strains were genetically diversified in biological processes related to stress response. As the formation of fruiting bodies is a stress-response process, we postulate that environment factors, such as temperature, drove the population divergence of L. edodes in China by natural or artificial selection. We also found phenotypic variations between groups and identified some wild strains that have potential to diversify the genetic pool for improving agricultural traits of L. edodes cultivars in China.

Cryptic Diversity of Malassezia pachydermatis from Healthy and Diseased Domestic Animals.

Malassezia pachydermatis is part of the normal cutaneous microbiota of wild and domestic carnivores. However, under certain conditions this yeast can overproliferate and cause several diseases in its host, mainly otitis and dermatitis in dogs. The aim of this study was to conduct a molecular characterization of M. pachydermatis isolates from healthy and diseased domestic animals, in order to assess the molecular diversity and phylogenetic relationship within this species. The large subunit (LSU) and the internal transcribed spacer (ITS) of ribosomal RNA, chitin synthase 2 (CHS2) and β-tubulin genes from sixteen strains isolated from dogs, cats, a goat, a pig and a horse were sequenced. A different number of types of sequences were identified for each target gene, including some types described for the first time. Five sequence types were characterized for the LSU, eleven for the ITS region, nine for CHS2 and eight for β-tubulin. A multilocus analysis was performed including the four genes, and the resulting phylogenetic tree revealed fifteen genotypes. Genotypes were distributed in two well-supported clades. One clade comprised strains isolated from different domestic animals and a strongly supported cluster constituted by strains isolated from cats. The second clade included strains isolated mainly from dogs and an outlier strain isolated from a horse. No apparent association could be observed between the health status of the animal hosts and concrete strains. The multilocus phylogenetic analysis is a useful tool to assess the intraspecific variation within this species and could help understand the ecology, epidemiology and speciation process of M. pachydermatis.

Insights into epidemiology of human parvovirus B19 and detection of an unusual genotype 2 variant, Bulgaria, 2004 to 2013.

The present study aimed to determine the role of human parvovirus В19 (B19V) as an aetiological agent in measles and rubella negative fever/rash patients from Bulgaria between 2004 and 2013. A total of 1,266 sera from all over the country were tested for B19V IgM antibodies and all positives were further investigated by polymerase chain reaction (PCR). Overall, 280 sera (22%) were B19V IgM positive and 227 of these (81%) were also PCR positive. The highest number of IgM positives was found among five to nine year-old children (27%). Eight infected women gave birth to healthy children; one fetus was aborted with hydrops fetalis. Of the 55 genetic sequences obtained, 54 belonged to genotype 1a and one grouped as a genotype 2 outlier. Phylogenetic analysis of all available genotype 2 sequences covering the 994 nucleotide non-structural protein 1(NS1)/capsid viral protein 1 (VP1) unique region junction, showed that only one other sequence grouped with the outlier strain, forming a clearly distinct and well-supported cluster of genotype 2 (between-group genetic distance: 3.32%). In accordance with B19V nomenclature, this cluster may represent a new subgenotype 2b. The study showed that B19V infections may be falsely identified as rubella or measles in ca 22% of cases, emphasising the need for laboratory confirmation.

Re-emergence of a genetic outlier strain of equine arteritis virus: Impact on phylogeny

Equine arteritis virus (EAV) is the causative agent of equine viral arteritis (EVA), a respiratory and reproductive disease of equids, which is notifiable in some countries including the Great Britain (GB) and to the OIE. Herein, we present the case of a persistently infected stallion and the phylogenetic tracing of the virus strain isolated. Discussing EAV occurrence and phylogenetic analysis we review features, which may aid to harmonise and enhance the classification of EAV.

The 2014 Ebola Virus Outbreak in West Africa: Current Perspectives for Prevention and Treatment

In 2014, West Africa has been the scene of the worst Ebola virus outbreak in history, with no end in sight [1-4]. This health crisis is reportedly associated with an outlier strain of Zaire Ebola virus [4]. While the first infections were reported in December of last year, the number of cases has literally exploded during the summer months of 2014, affecting mainly Guinea, Liberia and Sierra Leone, with a few cases in Mali, Nigeria and Senegal. According to the World Health Organization (WHO), as many as 13703 people have been infected by the Ebola virus as of October 27, resulting in 4922 fatalities [5]. The real figures are most likely higher as the healthcare systems in these countries have been overwhelmed by the large number of cases, the limited resources and the lack of basic facilities. While the local Ebola outbreaks in Senegal and Nigeria were declared over in October, the transmission remained persistent and widespread in Guinea, Liberia, and Sierra Leone, especially in the capital cities of these countries. Notably, on a smaller scale, there has also been in 2014 a separate and independent outbreak of Ebola virus in the Democratic Republic of the Congo, which involved 66 cases and resulted in 49 deaths so far, although the situation there seems to remain under relative control [5].

The 2014 Ebola virus disease outbreak in West Africa.

On 23 March 2014, the World Health Organization issued its first communiqué on a new outbreak of Ebola virus disease (EVD), which began in December 2013 in Guinée Forestière (Forested Guinea), the eastern sector of the Republic of Guinea. Located on the Atlantic coast of West Africa, Guinea is the first country in this geographical region in which an outbreak of EVD has occurred, leaving aside the single case reported in Ivory Coast in 1994. Cases have now also been confirmed across Guinea as well as in the neighbouring Republic of Liberia. The appearance of cases in the Guinean capital, Conakry, and the transit of another case through the Liberian capital, Monrovia, presents the first large urban setting for EVD transmission. By 20 April 2014, 242 suspected cases had resulted in a total of 147 deaths in Guinea and Liberia. The causative agent has now been identified as an outlier strain of Zaire Ebola virus. The full geographical extent and degree of severity of the outbreak, its zoonotic origins and its possible spread to other continents are sure to be subjects of intensive discussion over the next months.

Approximated Repair Methods for Outlier Strain Data from Hole-Drilling Residual Measurements

The current ASTM E 837 standard gives the standardized procedure for the evaluation of uniform and non uniform residual stresses, that is, stresses that do not vary and vary significantly with depth from the specimen surface, respectively. For non uniform stresses, the standard states that many small increments should be done in order to have a stable calculation of the residual stress profile. In addition, it states that irregularities as well as outlier strain points should be investigated and if necessary, the hole-drilling test should be repeated. In some applications outside the laboratory, the availability to repeat a test with outlier points is not possible. In these cases, the standard does not show the more appropriated way which should be followed to use only valid measured data (without outliers). For this reason, a stress profile corresponding to a shot peening test was simulated and one hole step was included in different hole depths as outlier point in order to evaluate the feasibility of some proposed ways of computation. These ways were: (a) following the ASTM procedure but replacing outlier strain points by linear interpolation of neighboring good ones, (b) following the ASTM procedure but replacing outlier strain points by a parabola interpolation of neighboring good ones and (c) using cumulative relaxation strain functions and only good measured points. Statistical criteria were also introduced and developed in order to identify outlier points. Results show a practical procedure to detect outlier points in experimental strain data.

Hepatitis B Virus (HBV) Infection and Recombination between HBV Genotypes D and E in Asymptomatic Blood Donors from Khartoum, Sudan

Sudan is a highly endemic area for hepatitis B virus (HBV), and >5% of blood donors are chronically infected. To examine potential strategies to improve HBV blood safety, 404 replacement donor samples previously screened for HBV surface antigen (HBsAg) were tested for antibody to HBV core (anti-HBc), anti-surface antigen (anti-HBs), and HBV DNA. Of 145 anti-HBc-containing samples (36%) identified, 16 retested were HBsAg positive (11%). Anti-HBs was detected in 43/77 (56%) anti-HBc-reactive samples. Six samples were HBsAg(-)/anti-HBc(+)/anti-HBs(+) and contained HBV DNA, meeting the definition of occult HBV infection (OBI). OBIs had low HBV DNA loads (<10 IU/ml) and were genotype B (n = 1) or genotype D (n = 5). Pre-S/S and/or whole genome sequences were obtained from 47 randomly selected HBsAg-positive donors added to the previous 16. Genotype E was identified in 27 strains (57.5%), genotype D in 19 strains (40.5%), and genotype A2 in 1 strain (2%). Two outlier strains within genotype D ultimately were identified as recombinants of genotypes D and E with identical recombination points, suggesting circulating, infectious, recombinant strains. Anti-HBc screening does not appear to be a sustainable blood safety strategy because of the cost and the negative impact on the Sudanese blood supply, even when reduced by anti-HBs testing. Being at the junction between two main African HBV genotypes, genetic recombination occurred and became part of the molecular epidemiology of HBV in Sudan.

Evolutionary Changes of the flhDC Flagellar Master Operon in Shigella Strains

Shigella strains are nonmotile. The master operon of flagellar synthesis, flhDC, was analyzed for genetic damage in 46 Shigella strains representing all known serotypes. In 11 strains (B1, B3, B6, B8, B10, B18, D5, F1B, D10, F3A, and F3C) the flhDC operon was completely deleted. PCR and sequence analysis of the flhDC region of the remaining 35 strains revealed many insertions or deletions associated with insertion sequences, and the majority of the strains were found to be defective in their flhDC genes. As these genes also play a role in regulation of non-flagellar genes, the loss may have other consequences or be driven by selection pressures other than those against flagellar motility. It has been suggested that Shigella strains fall mostly into three clusters within Escherichia coli, with five outlier strains, four of which are also within E. coli (G. M. Pupo, R. Lan, and P. R. Reeves, Proc. Natl. Acad. Sci. USA 97:10567-10572, 2000). The distribution of genetic changes in the flhDC region correlated very well with the three clusters and outlier strains found using housekeeping gene DNA sequences, enabling us to follow the sequence of mutational change in the flhDC locus. Two cluster 2 strains were found to have unique flhDC sequences, which are most probably due to recombination during the exchange of the adjacent O-antigen gene clusters.

Molecular evolutionary relationships of enteroinvasive Escherichia coli and Shigella spp.

Enteroinvasive Escherichia coli (EIEC), a distinctive pathogenic form of E. coli causing dysentery, is similar in many properties to bacteria placed in the four species of Shigella. Shigella has been separated as a genus but in fact comprises several clones of E. coli. The evolutionary relationships of 32 EIEC strains of 12 serotypes have been determined by sequencing of four housekeeping genes and two plasmid genes which were used previously to determine the relationships of Shigella strains. The EIEC strains were grouped in four clusters with one outlier strain, indicating independent derivation of EIEC several times. Three of the four clusters contain more than one O antigen type. One EIEC strain (an O112ac:H- strain) was found in Shigella cluster 3 but is not identical to the Shigella cluster 3 D2 and B15 strains with the same O antigen. Two forms of the virulence plasmid pINV have been identified in Shigella strains by using the sequences of ipgD and mxiA genes, and all but two of our EIEC strains have pINV A. The EIEC strains were grouped in two subclusters with a very low level of variation, generally not intermingled with Shigella pINV A strains. The EIEC clusters based on housekeeping genes were reflected in the plasmid gene sequences, with some exceptions. Two strains were found in the pINV B form by using the ipgD sequence, with one strain having an mxiA sequence similar to the divergent sequence of D1. Clearly, EIEC and Shigella spp. form a pathovar of E. coli.