Prevalent diagnosed HIV in England, Wales and Northern Ireland: adjusted totals 1996 to 2001 and extrapolations to 2004.

OBJECTIVE: To predict trends in diagnosed HIV prevalence by extrapolation to 2004 using data from the annual surveys of individuals receiving HIV-related care in England, Wales and Northern Ireland from 1996 to 2001. METHODS: Data from the annual surveys of prevalent HIV infections diagnosed (SOPHID) were adjusted for under-reporting and non-attendance and separately extrapolated for infections acquired homosexually, heterosexually and by other routes. The data were extrapolated using negative binomial and linear regression models based on the 1996 to 2001 annual surveys. RESULTS: The negative binomial model predicted an increase of 56% in diagnosed HIV prevalence in England, Wales and Northern Ireland between 2001 and 2004. The linear model predicted an increase of 25% for the same time period. The predicted increases are mostly driven by the large rise in the number of new diagnoses, in particular in individuals infected heterosexually. CONCLUSION: Increases in HIV prevalence in England, Wales and Northern Ireland have diverged from a linear trend. Negative binomial modelling of the data predicts that large rises in prevalence will continue during the early 2000s.

Past it? HIV and older people in England, Wales and Northern Ireland.

The majority of those infected and affected by HIV are younger adults. The ability of highly active antiretroviral therapies (HAART) to extend survival means that those infected when younger may reach older age, and future increases in numbers of older individuals living with HIV in England, Wales and Northern Ireland (E,W&NI) are expected. Evidence that older individuals engage in risky sexual behaviours suggests potential for HIV transmission. Data from national HIV/AIDS surveillance systems were reviewed (1997-2001). An older individual is defined as aged 45 years or over. Between 1997 and 2001, 2290 older individuals were diagnosed with HIV; 361 in 1997, rising to 648 in 2001. Heterosexual acquisition accounted for 1073 (47%) infections; 662 were male. Where reported, 666 (65%) older heterosexuals were probably infected in Africa, 144 (14%) in the United Kingdom and 113 (11%) in Asia. There were 1020 (45%) new diagnoses acquired homosexually; white (92%), infected in the United Kingdom (78%). Numbers of older individuals accessing HIV-related services more than doubled between 1997 (2488) and 2001 (5175). In 2001, 2270 (53%) were London residents. Between 1997 and 2001, among HIV-infected older individuals attending genitourinary medicine (GUM) clinics, the proportions previously undiagnosed were 60% and 82% in heterosexual males and females respectively, and for men who have sex with men (MSM), 42%. Numbers of older individuals newly diagnosed with HIV have increased in recent years. The increase in numbers of older individuals accessing HIV-related services were in excess of younger adults. A significant proportion of older HIV-infected female heterosexuals and MSM were undiagnosed. Awareness must be raised among clinicians, and an 'aged response' to HIV is required.

The type IV leader peptidase/N-methyltransferase of Vibrio vulnificus controls factors required for adherence to HEp-2 cells and virulence in iron-overloaded mice.

Vibrio vulnificus expresses a number of potential virulence determinants that may contribute to its ability to cause a severe and rapidly disseminating septicemia in susceptible hosts. We have cloned and characterized two genes encoding products related to components of the type IV pilus biogenesis and general secretory (type II) pathways by complementation of a type IV peptidase/N-methyltransferase (PilD) mutant of Pseudomonas aeruginosa with a V. vulnificus genomic library. One of the genes (vvpD) encodes a protein homologous to PilD and other members of the type IV peptidase family that completely restores this activity in a P. aeruginosa mutant deficient in the expression of PilD. The other gene (vvpC) encodes a homolog of PilC from P. aeruginosa, where it is essential for assembly of type IV pili. Phenotypic characterization of a V. vulnificus vvpD mutant, constructed by allelic exchange, showed that VvpD is required for the expression of surface pili, suggesting that the pili observed on V. vulnificus are of the type IV class. This mutant was also unable to secrete at least three extracellular degradative enzymes, and the localization of one of these (the cytolysin/hemolysin) to the periplasmic space indicates that these proteins are normally exported via the type II secretion pathway. Loss of VvpD resulted in significant decreases in CHO cell cytotoxicity, adherence to HEp-2 cells, and virulence in a mouse model. Capsule formation and serum resistance were not affected in the vvpD mutant, indicating that in addition to capsule, virulence of V. vulnificus requires type IV pili and/or extracellular secretion of several exoenzymes.

Pilus assembly by Agrobacterium T-DNA transfer genes.

Agrobacterium tumefaciens can genetically transform eukaryotic cells. In many bacteria, pili are required for interbacterial DNA transfer. The formation of pili by Agrobacterium required induction of tumor-inducing (Ti) plasmid-encoded virulence genes and growth at low temperature. A genetic analysis demonstrated that virA, virG, virB1 through virB11, and virD4 are the only Ti plasmid genes necessary for pilus assembly. The loss and gain of pili in various mutants correlated with the loss and gain of transferred DNA (T-DNA) transfer functions, which is consistent with the view that Agrobacterium pili are required for transfer of DNA to plant cells in a process similar to that of conjugation.

Cycloclasticus pugetii gen. nov., sp. nov., an aromatic hydrocarbon-degrading bacterium from marine sediments.

Three heterotrophic bacterial strains were isolated from different locations in Puget Sound, Washington, by using biphenyl as the principal carbon source. These strains grow by using a limited number of organic compounds, including the aromatic hydrocarbons naphthalene, phenanthrene, anthracene, and toluene, as sole carbon sources. These aerobic, gram-negative rods are motile by means of single polar flagella. Their 16S rRNA sequences indicate that they are all members of the gamma subdivision of the Proteobacteria. Their closet known relatives are the genera Methylobacter and Methylomonas (genera of methane-oxidizing bacteria), uncultured sulfur-oxidizing symbionts found in marine invertebrates, and clone FL5 containing 16S ribosomal DNA amplified from an environmental source. However, the Puget Sound bacteria do not use methane or methanol as a carbon source and do not oxidize reduced sulfur compounds. Furthermore, a 16S rRNA base similarity comparison revealed that these bacteria are sufficiently different from other bacteria to justify establishment of a new genus. On the basis of the information summarized above, we describe a new genus and species, Cycloclasticus pugetti, for these bacteria; strain PS-1 is the type strain of C. pugetti.

Haemophilus ducreyi attaches to and invades human epithelial cells in vitro.

Haemophilus ducreyi is a sexually transmitted pathogen that causes genital ulcers and inguinal adenopathy. Because chancroidal ulcers are most commonly located on the foreskins of uncircumcised males, we utilized human foreskin epithelial cells (HFECs) to investigate the initial interaction of H. ducreyi with its host. The eight different strains of H. ducreyi that were studied varied in their abilities to attach to these epithelial cells, with six strains consistently attaching to > or = 90% of HFECs and two strains attaching to < 25% of HFECs. The strains with low levels of adherence also failed to exhibit chaining in broth culture and were avirulent in the rabbit model, suggesting that virulence in this model and attachment may be linked. The most adherent strain, LA228R, was further evaluated for its ability to invade HFECs and HEp-2 cells. Scanning electron microscopy and transmission electron microscopy of HFECs after interaction with LA228R produced images consistent with attachment, ingestion into vesicles, and escape from the vesicles into the cytoplasm. In addition, the gentamicin protection assay and inhibition of invasion by cytochalasin B and D indicated that LA228R was able to invade both HFECs and HEp-2 cells. Further examination of the mechanisms involved in the adherence and invasion of H. ducreyi into epithelial cells and their correlation with virulence will provide a better understanding of the pathogenesis of the disease caused by this important pathogen.

The regulation of exopolysaccharide production is important at two levels of nodule development in Rhizobium meliloti.

We show that two exopolysaccharide overproducing Tn5 mutants of Rhizobium meliloti, exoR and exoS, have distinct symbiotic defects. While the exoR mutant is unable to colonize nodules, the exoS mutant retains that ability but varies in its ability to produce nitrogen-fixing nodules. We correlate these defects with different degrees of exopolysaccharide overproduction and growth impairment. We further show that the exoR mutant is able to enter developing infection threads but is unable to invade nodule cells. The exoR mutant gives rise to spontaneous pseudorevertants containing second-site suppressor mutations that decrease exopolysaccharide synthesis. These pseudorevertants form nitrogen-fixing nodules. Although the suppressor mutations have the opposite effect on exopolysaccharide production compared to the exoS::Tn5 mutation, they consistently map to the exoS::Tn5 region and belong to the same genetic complementation group as defined by transposon insertion mutations. The effect of the suppressor mutations on exopolysaccharide production is correlated with effects on the expression of exo genes involved in exopolysaccharide synthesis. Finally, we provide evidence that the exoR gene is not required for the regulation of exopolysaccharide synthesis by ammonia.

Epithelial cell invasion by bovine septicemic Escherichia coli.

Little is known regarding the pathogenesis of Escherichia coli-induced septicemic colibacillosis of calves. To understand the mechanism by which these strains penetrate the intestinal epithelium and gain access to the bloodstream, we examined the potential of bovine septicemic E. coli to invade cultured epithelial cells. By using a gentamicin survival assay, we demonstrated bacterial invasion of Madin-Darby canine kidney (MDCK) cells. Transcytosis of polarized MDCK cell monolayers was also observed, but only when bacteria were added to the basolateral surface. Electron microscopy confirmed the presence of intracellular organisms which appeared to be within membrane-bound vacuoles. The bovine septicemic isolate used in this study expressed the fimbrial adhesion CS31A. To examine the role of CS31A-mediated adherence in invasion and transcytosis of MDCK cell monolayers, a CS31A-deficient mutant was constructed by suicide vector-mediated insertional mutagenesis. Although nonadherent, the mutant showed a level of invasion similar to that of the wild-type parent. E. coli DH5 alpha carrying the cloned CS31A determinant was noninvasive. These findings suggest that expression of CS31A is neither required nor sufficient to mediate invasion.

Mutations in the consensus ATP-binding sites of XcpR and PilB eliminate extracellular protein secretion and pilus biogenesis in Pseudomonas aeruginosa.

The process of extracellular secretion in Pseudomonas aeruginosa requires specialized machinery which is widely distributed among bacteria that actively secrete proteins to the extracellular medium. One of the components of this machinery is the product of the xcpR gene, which is homologous to pilB, a gene encoding a protein essential for the biogenesis of type IV pili. Both XcpR and PilB are characterized by the presence of a conserved ATP-binding motif (Walker sequence). The codons of highly conserved glycine residues within the Walker sequences of xcpR and pilB were altered to encode a serine, and the effects of these substitutions were examined. Bacteria expressing mutant XcpR or PilB were unable to secrete exotoxin A or assemble pili, respectively. In addition, high-level expression of mutant XcpR in wild-type P. aeruginosa led to a pleiotropic extracellular secretion defect, resulting in the periplasmic accumulation of enzymes that are normally secreted from the cell. These studies show that the putative ATP-binding sites of XcpR and PilB are essential for their functions in protein secretion and assembly of pili, respectively. Moreover, the observed dominant negative phenotype of mutant XcpR suggests that this protein functions as a multimer or, alternatively, interacts with another essential component of the extracellular protein secretion machinery.

Rhizobium meliloti mutants with decreased DAHP synthase activity are sensitive to exogenous tryptophan and phenylalanine and form ineffective nodules.

We isolated two Tn5-generated mutants of Rhizobium meliloti whose growth was inhibited by rich medium or by exogenous tryptophan or phenylalanine. These mutants, Rm7479 and Rm7480, belonged to the same genetic complementation group. The mutant locus could not be found on either indigenous megaplasmid but was localized on the chromosome. The mutants formed ineffective nodules on alfalfa plants. They invaded nodules within infection threads and were released into plant cells enclosed within peribacteroid membranes, but once released into the plant cells they failed to differentiate into mature bacteroids. The mutants demonstrated a decrease in total 2-keto-3-deoxy-D-arabino-heptonic acid 7-phosphate synthase (DAHP synthase) activity, which is the first committed step in aromatic biosynthesis. Wild-type genes were isolated that complemented in one case or suppressed in another case, all three mutant phenotypes: growth on rich medium, symbiotic effectiveness, and DAHP synthase activity. Each mutant strain gave rise to linked second-site suppressor mutations that restored growth on rich medium. The suppressor mutants showed restoration of near wild-type DAHP synthase levels. One of the suppressor strains restored effective symbiosis while the other did not. Genetic complementation experiments showed that growth on rich medium, DAHP synthase activity, and effective symbiosis were all affected by the same genetic lesion. These results suggest that normal flux of metabolites through the aromatic biosynthesis pathway is essential for bacteroid development.

Specific oligosaccharide form of the Rhizobium meliloti exopolysaccharide promotes nodule invasion in alfalfa.

Rhizobium meliloti strain SU47 produces both high molecular weight (HMW) and low molecular weight (LMW) forms of an acidic exopolysaccharide, succinoglycan. Genetic studies have shown that succinoglycan is required for alfalfa root nodule invasion. We found that LMW succinoglycan, when applied exogenously to alfalfa roots, restored nodule invasion to exoA, exoB, exoF, and exoH mutants. Nodule initiation signals were not involved, since LMW succinoglycan from R. meliloti nodD1D2D3 and nodA mutants and from luteolin-induced wild-type cultures elicited effects similar to LMW succinoglycan from the uninduced wild-type strain. In contrast, LMW fractions from an exoA mutant, nonsuccinylated LMW succinoglycan, and HMW succinoglycan did not promote invasion, nor did LMW exopolysaccharides from R. leguminosarum bv. trifolii and Rhizobium sp. strain NGR234. LMW succinoglycan could be separated by anion-exchange chromatography into several distinct subfractions differing in repeating subunit multiplicities (monomer, trimer, and tetramer) and charge. When tested singly, only the most charged, tetrameric form was active. These results show that a specific oligosaccharide form of succinoglycan promotes nodule invasion in alfalfa. The implications for the mode of action of succinoglycan are discussed.

Adhesion of Pseudomonas aeruginosa pilin-deficient mutants to mucin.

Attachment of Pseudomonas aeruginosa to epithelial cells or tracheobronchial mucin is mediated by surface adhesins. Pili, composed of monomeric pilin subunits, make up one such class of adhesins. The formation of pili and flagella in P. aeruginosa is under the control of the alternative sigma factor rpoN. Isogenic mutant strains with insertionally inactivated rpoN genes were constructed with strains PAK, 1244, and CF613 and were tested for their ability to adhere to respiratory mucin. All rpoN mutants showed significant reduction of adherence to mucin relative to that of their wild-type parents. In contrast, the adherence of pilin structural gene mutants was similar to the adherence of wild types. These results provide suggestive evidence that P. aeruginosa also binds to mucin via adhesins that are distinct from pilin and are still under the genetic control of rpoN. Unlike the laboratory strain PAK, the clinical strains 1244 and CF613 are capable of agglutinating erythrocytes. The rpoN mutation had a minimal effect on the interaction of bacteria with erythrocytes, indicating that the transcription of a gene(s) specifying the agglutination phenomenon does not utilize rpoN. These findings collectively indicate the existence of several classes of adhesins on the surface of P. aeruginosa that may play an important role in colonization of the human respiratory tract.

The rpoN gene product of Pseudomonas aeruginosa is required for expression of diverse genes, including the flagellin gene.

The product of the rpoN gene is an alternative sigma factor of RNA polymerase which is required for transcription of a number of genes in members of the family Enterobacteriaceae, including those that specify enzymes of nitrogen assimilation, amino acid uptake, and degradation of a variety of organic molecules. We have previously shown that transcription of the pilin gene of Pseudomonas aeruginosa also requires RpoN (K. S. Ishimoto and S. Lory, Proc. Natl. Acad. Sci. USA 86:1954-1957, 1989) and have undertaken a more extensive survey of genes under RpoN control. Strains of P. aeruginosa that carry an insertionally inactivated rpoN gene were constructed and shown to be nonmotile because of the inability of these mutants to synthesize flagellin. The mutation in rpoN had no effect on expression of extracellular polypeptides, outer membrane proteins, and the alginate capsule. However, the rpoN mutants were glutamine auxotrophs and were defective in glutamine synthetase, indicating defects in nitrogen assimilation. In addition, the P. aeruginosa rpoN mutants were defective in urease activity. These findings indicate that the sigma factor encoded by the rpoN gene is used by P. aeruginosa for transcription of a diverse set of genes that specify biosynthetic enzymes, degradative enzymes, and surface components. These rpoN-controlled genes include pili and flagella which are required for full virulence of the organism.

Isolation and properties of pili from spores of Bacillus cereus.

Structures whose morphology is identical to that of bacterial pili have been isolated from spores of Bacillus cereus. The structures are absent from log-phase and sporulating cells. The pili are 6.8 nm in diameter, are of variable length, and appear to emanate randomly from the exosporium. Examination of spores from 12 Bacillus species showed that only those from B. cereus and B. thuringiensis have pili. Although isolated spore pili were shown to be composed of protein, their subunit nature was not discernible due to the extreme insolubility of the structure. An antiserum to spore pili was labeled with ferritin and used to examine the distribution of pilus antigen on the outer spore surface.

Tratamento das feridas anfractuosas e ou infectadas. / Treatment of anfractous and or infectious wounds

Foram estudados 31 pacientes com feridas anfractuosas e/ou contaminadas, bem como com ulceras por pressao, aos quais se aplicou um composto de enzimas proteoliticas, associado a um antimicrobiano em forma topica, na frequencia de uma a duas vezes ao dia. Foram adotadas medidas gerais de apoio como repouso relativo, asseio local e geral e, em alguns casos, procedimentos cirurgicos complementares. Os resultados foram tabulados quanto a idade, sexo, diagnostico e parametros clinicos evolutivos. Foram obtidos resultados favoraveis em todos os casos e na grande maioria dentro das duas primeiras semanas de tratamento

Selective antibacterial action of 2-mercaptoethanol on propionibacteria in skin cultures.

2-Mercaptoethanol applied to the surface of agar medium had a selective antibacterial effect on Propionibacterium acnes and Propionibacterium granulosum without interfering with the growth of Peptococcus saccharolyticus or staphylococci in anaerobic cultures of skin or in pure cultures. In aerobic broth culture, 2-mercaptoethanol inhibited aerobes and stimulated anaerobes, consistent with its action as a reducing agent.

Isolation and characterization of gas vesicles from Microcyclus aquaticus.

Intact gas vesicles of Microcyclus aquaticus S1 were isolated by using centrifugally accelerated flotation of vesicles and molecular sieve chromatography. Isolated gas vesicles were cylindrical organelles with biconical ends and measured 250x100nm. The gas vesicle membrane was composed almost entirely of protein; neither lipid nor carbohydrate was detected, although one mole of phosphate per mole of protein was found. Amino acid analysis indicated that the protein contained 54.6% hydrophobic amino acid residues, lacked sulfur-containing amino acids, and had a low aromatic amino acid content. The protein subunit composition of the vesicles was determined by gel electrophoresis in (i) 0.1% sodium dodecyl sulfate at pH 9.0 and (ii) 5 M urea at pH 2.0. The membrane appeared to consist of one protein subunit of MW 50000 daltons. Charge isomers of this subunit were not detected on urea gels. Antiserum prepared against purified gas vesicles of M. aquaticus S1 crossreacted with the gas vesicles of all other gas vacuolate strains of M. aquaticus, as well as those of Prosthecomicrobium pneumaticum, Nostoc muscorum, and Anabaena flos-aquae, indicating that the gas vesicles of these widely divergent organisms have some antigenic determinants in common.

Gas vesicle assembly in Microcyclus aquaticus.

When observed in the electron microscope intact gas vesicles appeared as transparent areas in whole cells of Microcylus aquaticus, whereas vesicles collapsed by centrifugation were not discernible. Within 5 min of suspending cells containing collapsed vesicles in growth medium, small transparent vesicles were detected. By 15 min the average number of vesicles per cell was 15. This number remained relatively constant while the size of the vesicles increased until they attained their maximum diamtere of 100 nm. At this time the vesicles, interpreted as biconical structures, began to elongate presumably due to the synthesis of the cylindrical midsection. Closely correlated with the time at which vesicles began to elongate was the initiation of smaller vesicles which resulted in a doubling of the number of vesicles per cell by 90 min. This evidence coupled with the isolation of a mutant which assembles only the conical portions of the vesicle suggests that assembly occurs in two distinct stages subject to genetic mutation. Protein and ribonucleic acid synthesis, and presumably adenosine triphosphate formation, were required for gas vesicle assembly. In addition, inhibition of protein or ribonucleic acid synthesis resulted in a loss of extant gas vesicles. Over the time course of our study, deoxyribonucleic acid synthesis was not required for gas vesicle assembly or stability.

Release and recovery of forespores from Bacillus cereus.

A method is described which makes possible the release of immature forespores from sporulating cells at specific stages of development, from the completion of stage III through to mature spore formation. With the aid of zonal density gradient centrifugation, the method makes possible the recovery of quantities of forespores ample for biochemical and physical studies. With the capability to examine forespores and some mother cell components independently, we have established that several enzymes associated with the sporulation process are localized in the newly developed forespores. Studies showed that aspartate aminotransferase and alanine aminotransferase are associated with the forespores, whereas l-alanine dehydrogenase is found only in the mother cell cytoplasm.