Search

Epidemic methicillin-resistant Staphylococcus aureus types 15 and 16 (EMRSA-15 and EMRSA-16) are the dominant types of MRSA found in UK hospitals, but accurate designation of strains has been difficult. Restriction fragment length polymorphism (RFLP) profiles of seven core virulence genes were used to classify unambiguously isolates of MRSA from St George's Hospital into two groups corresponding to EMRSA-15 and EMRSA-16. Variants of both EMRSA-15 and EMRSA-16 isolates occurred that had lost virulence genes encoded on mobile genetic elements. EMRSA-16 isolates had core gene profiles identical to a cluster of previously characterised MSSA (methicillin-sensitive S. aureus) isolates from St George's Hospital, suggesting that they have arisen from this source, or that loss of the accessory genetic element encoding methicillin resistance is frequent. EMRSA-15 and EMRSA-16 strains were distinct from other MRSA strains previously identified in UK hospitals, and always carried a mobile genetic element encoding multiple superantigens. These results contribute to the understanding of the types of MRSA found in UK hospitals, how they vary and how they arose.

Anthrax toxin is responsible for the severe symptoms and death associated with inhalational anthrax (Fig. 1Fig. 1). Inhaled Bacillus anthracis spores are phagocytosed by macrophages, transported to the lymph nodes where germination occurs up to 60days later, and the new vegetative bacteria produce the toxin. While antibiotics kill the vegetative bacteria, they are of limited benefit if a threshold level of anthrax toxin has already been produced. By the time a patient shows obvious symptoms of disease, this level has been reached. Treatment options for these patients are limited and anti-toxin therapies are urgently needed, especially in the wake of the recent bioterrorist attacks involving anthrax.Fig. 1Chest radiograph showing widened mediastinum caused by inhalation anthrax, taken 22 hours before death. Photo courtesy of CDC/ P.S. Brachman.View Large Image | Download PowerPoint SlideAnthrax toxin has three interacting components: protective antigen (PA), which is the major component of the anthrax vaccine; lethal factor (LF); and edema factor (EF). PA is necessary for transporting the other factors into the host cell; it binds to human cells and is cleaved and activated by host proteases, allowing it to form a heptamer. The PA heptamer then binds either LF or EF and the complex is taken up by receptor-mediated endocytosis. The acidified environment within the endosome triggers PA to act as a pore and transport LF and EF into the cytosol, where they induce macrophage cell death and inhibit phagocytosis, respectively.Three new studies have boosted the search for anti-toxin therapies by clarifying important interactions between these components and their targets. Bradley et al. [1xIdentification of the cellular receptor for anthrax toxin. Bradley, K.A. et al. Nature. 2001; 414: 225–229Crossref | PubMed | Scopus (652)See all References][1] have identified the human cellular receptor that binds PA, anthrax toxin receptor (ATR). The PA-binding domain was predicted by its resemblance to those found on integrins. Importantly, a large synthetic peptide containing this domain protected culture cells from the effects of anthrax toxin. This discovery shows that inhibiting PA–ATR binding could be of therapeutic benefit and will speed up the design of further inhibitors.Mourez et al. [2xDesigning a polyvalent inhibitor of anthrax toxin. Mourez, M. et al. Nat. Biotechnol. 2001; 19: 958–961Crossref | PubMed | Scopus (221)See all References][2] screened a phage display library and identified a peptide that binds to PA and inhibits its interaction with LF and OF. As the peptide bound weakly to PA, a molecule consisting of multiple peptide copies on a flexible backbone was synthesized. At high concentration, this molecule prevented PA–LF interaction and protected rats from toxin-induced death.Finally, LF is a protease that targets mitogen-activated protein kinase kinases (MAPKKs), which control cell-signalling pathways, affecting release of inflammatory mediators. Although it is not clear how MAPKK+ LF induces macrophage lysis, Pannifer et al. [3xCrystal structure of the anthrax lethal factor. Pannifer, A.D. et al. Nature. 2001; 414: 229–233Crossref | PubMed | Scopus (315)See all References][3] have now crystallized the LF protein and show its very high specificity for MAPKK binding. This suggests that inhibition of this interaction would block the activity of LF and could be exploited therapeutically.These studies demonstrate there are at least three steps necessary for anthrax toxin activity that could be exploited successfully as targets for inhibitors. A combination of inhibitors might be particularly effective therapy for patients with symptoms of inhalational anthrax. Along with antibiotic treatment of individuals exposed to spores, successful treatment of patients with symptoms would reduce much of the public's terror in response to anthrax bioterrorism, reducing its effectiveness as a biological weapon.

Methicillin-resistant Staphylococcus aureus (MRSA) bacteria are opportunistic, zoonotic pathogens presenting major challenges for health care providers. The pig-adapted lineage, MRSA ST398, is now recognized worldwide ([8][1]), and pigs are increasingly considered an important source of community

A putative operon encoding a probable zinc-responsive regulatory element (zur) and components of an ABC-type transporter (mreA mreB) have been characterized in Staphylococcus aureus. The zur gene was inactivated but apparently this did not alter Zn(2+) uptake. Expression of mreAB zur is at a low level under a range of ion conditions. To allow inducible expression of the operon, a construct was made placing it under the control of the IPTG-inducible P(spac) promoter. Using this approach, it was shown that zur is able to repress expression of the entire operon in a Zn(2+)-dependent manner, and that mreA and mreB are likely to be involved in high-affinity ion uptake. zur has no apparent role in pathogenicity in a lesion model of S. aureus infection.

Iron is an essential nutrient for all cells, but in the human body, the distribution of iron is tightly controlled. Iron is rapidly sequestered away by transport proteins, such as transferrin and lactoferrin, and stored in intracellular protein complexes (ferritin) and haemoglobin. The resulting very low levels of freely available iron prevent the colonization or infection of the host by many types of bacteria. In response, pathogenic bacteria have evolved complex high-affinity mechanisms for the uptake of nutritional iron in order to survive.Peptic ulcers are thought to be caused by the slow-growing, spiral bacterium Helicobacter pylori. Although 50% of the population are colonized by this organism, clearance of H. pylori from the gut of patients with peptic ulcers is therapeutically beneficial. However, antimicrobial regimes have proved to be complex owing to poor secretion of agents into the gut, inactivation in the acid environment of the stomach and bacterial resistance. Now, Velayudhan et al.1xIron acquisition and virulence in Helicobacter pylori: a major role for FeoB, a high-affinity ferrous iron transporter. Velayudhan, J. et al. Mol. Microbiol. 2000; 37: 274–286Crossref | PubMed | Scopus (153)See all References1, using the completed bacterial genome sequence as a guide, have constructed knockout mutants in four genes predicted to be involved in H. pylori iron-uptake pathways. Using this approach, they identified a single gene, feoB, which is necessary for high-affinity uptake of iron. Most importantly, this gene is essential for bacterial colonization of the mouse gut model.The FeoB protein is probably located in the cytoplasmic membrane and relies on an unidentified mechanism to transfer iron from the extracellular environment to the periplasmic space. The FeoB pathway is expressed constitutively, and functions both in iron- restricted and iron-replete conditions. These results not only suggest that uptake of iron by H. pylori is necessary for colonization of the gut, but that surprisingly, this process is dependent on a single protein. This nutritional ‘Achilles heel’ for the bacterium could be a new target for therapies to aid clearance of these organisms, alleviating peptic ulcer discomfort as well as reducing the incidence of gastric carcinoma.

The accessory gene regulator (agr) and staphylococcal accessory regulator (sar) loci are important regulators of toxin production in Staphylococcus aureus. In this study we examined how environmental conditions degree of aeration and salt concentration - affect the transcription and translation of mRNAs for alpha-haemolysin (Hla) and serine protease (Ssp) via these pathways and influence the stability of these proteins. Using Northern analysis, we have confirmed earlier observations that sarA is involved in the upregulation of RNAIII, the effector molecule encoded by the agr locus. However, this effect was abolished in highly aerated cultures. While sarA does appear to have an up-regulatory effect on hla transcription that is independent of agr, we propose that the PC1839 (sarA) mutant produces less alpha-haemolysin activity mainly as a result of post-translational inactivation by proteases. The most obvious phenotypic feature of PC1839 (sarA) is the upregulation of proteases. In this study we show that ssp is repressed by SarA at the transcriptional level. Western analysis using an anti-alpha-haemolysin antibody identified a major breakdown product that is only present in the supernatant of strains that are overexpressing serine protease. We have also confirmed that agr exerts a significant regulatory influence on hla at the level of translation, as well as transcription. Finally, the addition of salt upregulates ssp transcription and dramatically downregulates transcription of hla, and is an example of an environmental parameter that affects toxin production independently of agr and sarA. How environmental signals are transduced to control alpha-haemolysin and serine protease production, activity and stability at multiple levels are discussed.

Tst, the gene for toxic shock syndrome toxin-1 (TSST-1), is part of a 15.2 kb genetic element in Staphylococcus aureus that is absent in TSST-1-negative strains. The prototype, in RN4282, is flanked by a 17 nucleotide direct repeat and contains genes for a second possible superantigen toxin, a Dichelobacter nodosus VapE homologue and a putative integrase. It is readily transferred to a recA recipient, and it always inserts into a unique chromosomal copy of the 17 nucleotide sequence in the same orientation. It is excised and circularized by staphylococcal phages phi13 and 80alpha and replicates during the growth of the latter, which transduces it at very high frequency. Because of its site and orientation specificity and because it lacks other identifiable phage-like genes, we consider it to be a pathogenicity island (PI) rather than a transposon or a defective phage. The tst element in RN4282, near tyrB, is designated SaPI1. That in RN3984 in the trp region is only partially homologous to SaPI1 and is excised by phage 80 but not by 80alpha. It is designated SaPI2. These PIs are the first in any gram-positive species and the first for which mobility has been demonstrated. Their mobility may be responsible for the spread of TSST-1 production among S. aureus strains.

Background Meticillin-resistant Staphylococcus aureus (MRSA) infections remain important medical and veterinary challenges. The MRSA isolated from dogs and cats typically belong to dominant hospital-associated clones, in the UK mostly EMRSA-15 (CC22 SCCmecIV), suggesting original human-to-animal transmission. Nevertheless, little is known about host-specific genetic variation within the same S. aureus lineage. Hypothesis/objectives To identify host-specific variation amongst MRSA CC22 SCCmecIV by comparing isolates from pets with those from in-contact humans using whole-genome microarray. Methods Six pairs of MRSA CC22 SCCmecIV from human carriers (owners and veterinary staff) and their respective infected in-contact pets were compared using a 62-strain whole-genome S. aureus microarray (SAM-62). The presence of putative host-specific genes was subsequently determined in a larger number of human (n = 47) and pet isolates (n = 93) by PCR screening. Results Variation in mobile genetic elements (MGEs) occurred frequently and appeared largely independent of host and in-contact pair. A plasmid (SAP078A) encoding heavy-metal resistance genes (arsR, arsA, cadA, cadC, mco and copB) was found in three of six human and none of six animal isolates. However, only two of four resistance genes were associated with human hosts (P = 0.015 for arsA and cadA). Conclusions and clinical importance The variation found amongst MGEs highlights that genetic adaptation in MRSA continues. However, host-specific MGEs were not detected, which supports the hypothesis that pets may not be natural hosts of MRSA CC22 and emphasizes that rigorous hygiene measures are critical to prevent contamination and infection of dogs and cats. The host specificity of individual heavy-metal resistance genes warrants further investigation into different selection pressures in humans and animals.

Invasive aspergillosis is a devastating infection with 30–40% mortality. Immune mechanisms involved in recognition and elimination of aspergillus are incompletely understood. Mannose-binding lectin (MBL) deficiency occurs in 10–15% of the population and has been identified as a susceptibility factor in animal models of aspergillosis. We hypothesise there is an association between human MBL deficiency and invasive aspergillosis. In this multi-centre case-control study, serum MBL concentrations were measured in 65 patients with probable or proven invasive aspergillosis (as defined by the 2002 EORTC/NIH-MSG criteria) and in 79 immunocompromised controls with fever not due to aspergillosis. MBL concentrations were measured using a commercially available, technically straightforward solid phase ELISA (Hycult Biotechnology, The Netherlands). Median MBL concentrations and the frequency of MBL deficiency were compared using the Mann-Whitney and Fisher’s exact tests. Cases and controls were well matched in terms of age, gender, ethnicity and cause of immunocompromise. The assay performed well with a mean coefficient of variation of 7%. Patients with invasive aspergillosis had significantly lower MBL concentrations and a greater frequency of MBL deficiency than controls (65% vs. 33%, p = 0.0002, MBL deficiency cut-off 500ng/ml). MBL deficiency was significantly more frequent in cases than in controls at all MBL concentrations ≤500ng/ml. This is the first study to identify a strong association between MBL deficiency and invasive aspergillosis in immunocompromised patients. Because MBL concentrations do not consistently change during acute infection we conclude that MBL deficiency predisposes to invasive aspergillosis. Routine measurement of MBL levels is within the capability of most diagnostic laboratories. These data suggest that replacement therapy with recombinant human MBL may be an opportunity to prevent or mitigate the poor outcome of invasive aspergillosis.

Protein translocation into host epithelial cells by infecting enteropathogenic Escherichia coli Wolff, C. et al. (1998) Mol. Microbiol. 28, 143–155 The complete sequence of the locus of enterocyte effacement (LEE) from enteropathogenic Escherichia coli E2348/69 Elliott, S.J. et al. (1998) Mol. Microbiol . 28, 1–4

Tuberculosis is one of the three infectious diseases with the highest fatality rate. However, only about 10% of individuals infected with the causative organism, Mycobacterium tuberculosis, show clinical symptoms. Those that succumb to disease are often immunocompromised, or are thought to have a predisposed genetic susceptibility. Those with an adequate immune response are able to control the infection. An adequate response requires the migration of macrophages to the site of infection, phagocytosis of the bacteria, priming of CD4+ and CD8+ T cells to release INF-γ, and killing of the internalized organisms. At present, the signals necessary for the important step of macrophage recruitment to the site of infection are unknown.Recently, a mouse with a knockout in the gene encoding chemokine receptor 2 (CCR2) was shown to be defective in macrophage trafficking to inflammatory sites. CCR2 is expressed by many leukocytes and responds to the monocyte chemoattractant proteins (MCPs) 1–5. CCR2 is therefore a candidate for playing a role in the early stages of M. tuberculosis control, and Peters et al.1xChemokine receptor 2 serves an early and essential role in resistance to Mycobacterium tuberculosis. Peters, W. et al. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7958–7963Crossref | PubMed | Scopus (193)See all References1 have now developed the CCR2−/− mouse model to test this hypothesis.CCR2−/− mice were significantly more susceptible to M. tuberculosis disease. Bacterial numbers in the lung were elevated and the CCR2−/− mice lost more weight and mobility. To investigate the reasons for enhanced disease, the distribution of cells in the lung and lymph nodes was determined. The lungs of CCR2−/− mice had decreased numbers of macrophages, dendritic cells, and primed CD4+ and CD8+ T cells. Less macrophages and dendritic cells in the lymph nodes were also seen. On the other hand, the lungs of infected CCR2−/− mice showed larger infiltrations of neutrophils.These results show that CCR2 has a role in the control of M. tuberculosis infection, probably via recruitment of the appropriate cells to the site of infection. Further studies will now be directed at this early stage of the immune response and the importance of cell recruitment. Furthermore, CCR2 is polymorphic in humans, suggesting it could account for the increased susceptibility of some patients to disease. In the future, development of these findings should result in improved approaches to identify and control infection in patients susceptible to active tuberculosis.

Burkholderia pseudomallei causes melioidosis, an emerging disease in tropical areas such as South-east Asia and northern Australia, with a high fatality rate. It usually presents as a severe pneumonia or septicaemia, although subclinical infection is also seen, and relapses are common. Infection often responds poorly to antibiotic treatment, and alternative therapies are urgently required.The reservoir of B. pseudomallei is soil and stagnant water. The closely related species, B. thailandensis, is also found in these environments but is not pathogenic in humans. In a Syrian hamster model of septicaemia, the lethal dose that kills 50% of animals tested (LD50) of B. pseudomallei is ten organisms, whereas for B. thailandensis it is 106. Reckseidler et al. 1xDetection of bacterial virulence genes by subtractive hybridization: identification of capsular polysaccharide of Burkholderia pseudomallei as a major virulence determinant. Reckseidler, S.L et al. Infect. Immun. 2001; 69: 34–44Crossref | PubMed | Scopus (184)See all References1 have now exploited this difference, using subtractive hybridization to identify genes that are present in B. pseudomallei and not B. thailandensis, with the aim of identifying those necessary for virulence. One of the unique genes identified showed weak sequence homology to a Pseudomonas aeruginosa glycosyltransferase that is involved in capsule synthesis. Following this clue, the team then constructed a strain of B. pseudomallei with a mutation in this gene (wbpX). In the hamster model, the LD50 of the mutant was 3.5×105 organisms, thus demonstrating the role of wbpX in pathogenesis.As it was hypothesized that wbpX was also involved in capsule synthesis, the authors visualized the bacterial capsule using immunogold staining, and compared wild-type to mutant strains. The mutant strain was found to be deficient in type I O-polysaccharide (O-PS) moieties found on the cell surface. Further sequencing of the B. pseudomallei chromosome around wbpX identified a large cluster of genes, all of which are either predicted or proven to be necessary for type I O-PS synthesis or transport. These genes showed sequence homology to group 3 capsule genes found in other pathogens such as Neisseria meningitidis, Haemophilus influenzae, and Escherichia coli. Furthermore, this region has a G+C content that is unusually low for B. pseudomallei, suggesting it is encoded on a pathogenicity island that has been horizontally acquired from another bacteria. It is therefore possible that the acquisition of these genes has enabled B. pseudomallei to become a pathogen.Capsules often make good targets for bacterial vaccines. Before a vaccine to melioidosis can be developed, it will be necessary to prove that antibody to group 3 capsule is protective, and that protective antibodies are induced after vaccination. If successful, such a vaccine might be particularly useful for visitors to endemic countries, as well as preventing disease in high-risk populations and relapse in seropositive patients.

Pathogenicity islands (PIs) are discrete segments of bacterial chromosomal DNA that encode multiple virulence genes. PIs are present in some strains but entirely absent in others, and are presumed to have been acquired by horizontal transfer from another species at some point in their evolution. Many PIs in many types of pathogenic bacteria have now been identified. For example, the LEE (locus of enterocyte effacement) PI is a 35.6 kb fragment of DNA, carried by only some strains of E. coli, that encodes genes causing bacterial attachment to intestinal epithelium and effacement – the formation of lesions characterized by loss of microvilli and induction of a cellular pedestals. LEE is found in enteropathogenic E. coli (EPEC), which cause diarrhoea in infants, and enterohaemorrhagic E. coli (EHEC), including serotype O157 strains, which cause severe foodpoisoning outbreaks.For these infections to occur, LEE genes are not only necessary, but they must be expressed appropriately. Not surprisingly then, Elliott et al. have confirmed that a major regulatory locus of LEE gene expression is found on the LEE PI which they have called ler (LEE encoded regulator) 1xThe locus of enterocyte effacement (LEE)-encoded regulator controls the expression of both LEE- and non-LEE-encoded virulence factors in enteropathogenic and enterohemorrhagic Escherichia coli. Elliott, S.J et al. Infect. Immun. 2000; 68: 6115–6126Crossref | PubMed | Scopus (212)See all References1. However, it was not predicted that ler would be an important regulator of non-LEE genes, including the control of the induction of another enterotoxin and repression of fimbria. Therefore, these results show that the LEE PI has a dual nature. Not only does LEE encode the genes necessary for attachment and effacement, but also has the ability to dictate a more global bacterial virulence response, switching on and off other virulence genes as required.

The accessory gene regulator (agr) locus is the major regulator of virulence-factor gene expression in staphylococci, and controls the upregulation of multiple extracellular proteins including toxins and proteases. The agr locus is autoinduced by a quorum sensing mechanism: it encodes a peptide pheromone that is secreted by the bacterial cell, and, once extracellular levels accumulate to a threshold level (quorum), they are detected by the agr signal transduction system, activating the gene regulation response. Because peptides produced by different staphylococcal species and subspecies inhibit the signal transduction step, such inhibitory peptides are proposed adjuncts to antibacterial therapy, and can be administered in vivo to reduce disease.Now, Qin and colleagues1xEffects of Enterococcus faecalis fsr genes on production of gelatinase and a serine protease and virulence. Qin, X. et al. Infect. Immun. 2000; 68: 2579–2586Crossref | PubMed | Scopus (201)See all References1 have reported that a similar mechanism for controlling virulence factor expression is present in Enterococcus faecalis, an important cause of hospital acquired infection. The E. faecalis regulator (fsr) locus is highly homologous to agr, it is autoregulated, and controls the upregulation of the exoproteins gelatinase and serine protease. Importantly, fsr mutants are attenuated in virulence. Although there is still much to do, this discovery suggests that similar signal transduction systems to agr might be widespread in pathogenic Gram-positive bacteria. These organisms are particularly difficult to treat in hospitals, because of their resistance to multiple antibiotics, so adjunct therapies that impact on virulence might be particularly useful. Whether a broad spectrum approach to encompass all Gram-positive bacteria can be developed awaits further characterization of this locus and investigation of other pathogens.

Group A streptococci (GAS) cause ‘strep throat’ and, occasionally, invasive diseases such as rheumatic fever and necrotizing fasciitis (flesh-eating disease). In several continents, epidemic outbreaks of GAS disease lasting 1–3 years have been observed, and it has been assumed that one (or a few) particularly ‘fit’ strains are responsible. However, a possible cause of increased fitness or host resistance in epidemic strains does not appear to be associated with obvious GAS virulence genes, and the reason for these outbreaks had remained a mystery. Musser and colleagues1xRapid selection of complement-inhibiting protein variants in group A Streptococcus epidemic waves. Hoe, N.P. et al. Nat. Med. 1999; 5: 924–929Crossref | PubMed | Scopus (57)See all References, 2xNonpolar inactivation of the hypervariable streptococcal inhibitor of complement gene (sic) in serotype M1 Streptococcus pyogenes significantly decreases mouse mucosal colonization. Lukomski, S. et al. Infect. Immun. 2000; 68: 535–542Crossref | PubMed | Scopus (89)See all References have now suggested a new hypothesis for the development of epidemic waves of GAS infection, which includes a role for the sic gene (streptococcal inhibitor of complement).The sequence of the sic gene, in strains that had been isolated during several epidemics, showed it to be highly polymorphic. Mutations predominantly led to amino acid changes or radical replacements, whereas synonymous changes were rarer. Furthermore, many of the mutations represented single-event changes from other isolates of the same epidemic. Armed with these observations, they have now shown that: (1) a GAS-strain constructed with a sic deletion colonized the mouse throat less efficiently than the wild-type strain2xNonpolar inactivation of the hypervariable streptococcal inhibitor of complement gene (sic) in serotype M1 Streptococcus pyogenes significantly decreases mouse mucosal colonization. Lukomski, S. et al. Infect. Immun. 2000; 68: 535–542Crossref | PubMed | Scopus (89)See all References2; (2) mice and humans produce antibodies directed towards the Sic protein2xNonpolar inactivation of the hypervariable streptococcal inhibitor of complement gene (sic) in serotype M1 Streptococcus pyogenes significantly decreases mouse mucosal colonization. Lukomski, S. et al. Infect. Immun. 2000; 68: 535–542Crossref | PubMed | Scopus (89)See all References2; (3) mutant forms of Sic protein inhibit complement1xRapid selection of complement-inhibiting protein variants in group A Streptococcus epidemic waves. Hoe, N.P. et al. Nat. Med. 1999; 5: 924–929Crossref | PubMed | Scopus (57)See all References1; and (4) in a mouse throat-infection model, only 1% of GAS show sic mutations at 45 days, whereas, at 54 days, 74% of GAS have sic mutations. sic mutations are rare in vitro, suggesting that variation is selected for in the throat1xRapid selection of complement-inhibiting protein variants in group A Streptococcus epidemic waves. Hoe, N.P. et al. Nat. Med. 1999; 5: 924–929Crossref | PubMed | Scopus (57)See all References1 – after sufficient time for an anti-Sic response to be generated.These results suggest that the Sic protein is necessary for efficient colonization, that infected hosts produce antibodies to this protein, and that GAS strains that produce variant, but functional, Sic proteins have a selective advantage in the throat. It is envisaged that these mutants then infect new hosts during the epidemic, and further mutants are selected. Therefore, these epidemic waves of GAS disease appear to result from a highly heterogeneous population of strains that are evolving by rapid natural selection to evade the human humoral response.

Multilocus sequence typing (MLST) involves comparing the sequences of five to seven housekeeping genes in order to epidemiologically type bacterial and parasitic strains. Identification of point mutations and small, horizontal recombinations allows a series of allele numbers to be assigned to each strain. The real advantage of this typing method is that sequence information is highly reproducible, and easily deposited into growing banks of MLST sequence on the internet (for example, see http://mlst.zoo.ox.ac.uk). Furthermore, each laboratory will not need to obtain reference strains or conduct extensive validation before typing their own strains.MLST typing has highlighted the different mutation rates in various bacteria, and this makes the technique suitable for different purposes in each species. This is emphasized by two recent publications.Neisseria meningitidis has a rapid mutation rate that is clearly observed in housekeeping genes. The mutations are stable enough to be useful for tracking epidemiological outbreaks. Feavers and colleagues1xMultilocus sequence typing and antigen gene sequencing in the investigation of a meningococcal disease outbreak. Feavers, I.M. et al. J. Clin. Microbiol. 1999; 37: 3883–3887PubMedSee all References1 applied MLST to a recent outbreak of meningococcal meningitis in a British university, and were able to demonstrate that more than one clone was responsible, and that several other clones were circulating in the student population without causing disease. They have also suggested that it might be possible to MLST-type an isolate that could not actually be cultured, as PCR amplification might be possible.In contrast, the mutation rate in housekeeping genes of Yersinia is extremely low. Achtman and colleagues2xYersinia pestis, the cause of plague, is a recently emerged clone of Yersinia pseudotuberculosis. Achtman, M. et al. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14043–14048Crossref | PubMed | Scopus (598)See all References2 showed that all strains of Y. pestis tested were identical, and very similar to Y. pseudotuberculosis strains. Y. enterocolitica strains are also highly conserved but are quite distinct from the former two species. This supports other evidence that Y. pestis and Y. pseudotuberculosis are closely related and should be re-classified as two subspecies. Clinical and historical considerations have prevented this so far. As the mutation rate is so low, Achtman and colleagues2xYersinia pestis, the cause of plague, is a recently emerged clone of Yersinia pseudotuberculosis. Achtman, M. et al. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14043–14048Crossref | PubMed | Scopus (598)See all References2 were able to estimate the point at which Y. pestis is likely to have diverged from Y. pseudotuberculosis. Their estimate suggests a date of between 1500 and 20 000 years ago, some time prior to the first plague pandemic in the sixth century.Potentially, MLST could be applied to any two genomes in order to determine their relatedness, provided that there is enough background information available to make meaningful interpretations. This process is under way for a number of microbes, aided by the rapid deposition and dissemination of information on the internet.

Lipopolysaccharide (LPS), a component of the Gram-negative cell wall, triggers monocytes and macrophages in the systemic circulation to release proinflammatory cytokines. However, in the mucosa, commensal bacteria release large amounts of LPS without provoking inflammation and disease.Uropathogenic E. coli express P-type fimbriae that are essential for the induction of cytokine release from uroepithelial cells. It was thought that the fimbriae brought LPS into close contact with the epithelial cells to allow triggering. However, studies of the relative roles of LPS and P-type fimbriae have been hampered because LPS is essential for E. coli viability. Hedlund et al. have sought to overcome this difficulty by constructing strains that produce defective LPS1xP fimbriae-dependent, lipopolysaccharide-independent activation of epithelial cytokine responses. Hedlund, M. et al. Mol. Microbiol. 1999; 33: 693–703Crossref | PubMed | Scopus (75)See all References1. They have shown that these defective strains can still activate epithelial cells, but only if they express P-type fimbriae. This suggests that P-type fimbriae activate epithelial cells independently of LPS.Furthermore, LPS activates monocytes and macrophages via membrane-bound CD14. The authors could not detect CD14 protein or mRNA in a uroepithelial cell line or in epithelial cells in sections of healthy human urinary-tract tissue. They have concluded that uroepithelial cells are poor LPS responders, and that uropathogenic E. coli overcome this by using a unique activation mechanism triggered by P-type fimbriae.These fimbriae could represent a unique target for the therapy of E. coli urinary tract infections (UTIs), provided that the infection is not invasive.

Bacteriophages (bacterial viruses) encode many important bacterial toxins, including diphtheria toxin in Corynebacterium diphtheriae, enterotoxin A and Panton-Valentine leukocidin in Staphylococcus aureus, and cholera toxin in Vibrio cholerae. Only the bacterial strains that have the bacteriophage genome stably inserted into their genome (lysogenic bacteria) encode the toxin genes. A second cluster of genes essential for V. cholerae virulence is now reported to be encoded on a bacteriophage genome1xA bacteriophage encoding a pathogenicity island, a type-IV pilus and a phage receptor in cholera bacteria. Karaolis, D.K.R. et al. Nature. 1999; 399: 375–379Crossref | PubMed | Scopus (263)See all References1. These genes include the type IV pilus, which is necessary for disease and intestinal colonization. Intriguingly, type IV pilus is also the receptor for the bacteriophage, CTXφ, which carries the cholera toxin gene. Infection of V. cholerae with the CTXφ bacteriophage cannot occur unless the strain is already lysogenic for the pilus-carrying phage, VPIφ . Many bacteria are multiply lysogenic, and these viral genomes probably account for a significant amount of bacterial strain-to-strain variation. Lysogenic bacteria are a source of potential new bacteriophage viruses in the environment, and in the laboratory VPIφ could infect V. cholerae serotypes not normally associated with pathogenicity1xA bacteriophage encoding a pathogenicity island, a type-IV pilus and a phage receptor in cholera bacteria. Karaolis, D.K.R. et al. Nature. 1999; 399: 375–379Crossref | PubMed | Scopus (263)See all References1. As these pathophages can turn non-pathogenic strains of bacteria into pathogens, we need to be aware of their host ranges, their ability to spread to other strains, and the risks associated with the use of live attenuated bacterial vaccine strains that are susceptible to toxin-carrying bacteri phages.

Staphylococcus aureus is one of the most common causes of both nosocomial- and community-acquired infection, causing bacteraemia, surgical site and wound infections, pneumonia, endocarditis, osteomyelitis, toxic shock syndrome, food poisoning and many other diseases. Strains that are resistant to multiple antibiotics are prevalent in many hospitals, and treatment options are often limited. Over 30 toxins and virulence factors produced by S. aureus have been described and are thought to contribute to its ability to adapt to so many environments in vivo. Streptococcus pneumoniae is the major cause of bacterial pneumonia but also causes meningitis and otitis media in children. It too produces a number of extracellular toxins and adhesins. Both are low G+C Gram-positive bacteria, a group that is phylogenetically different to the Gram-negative bacteria whose pathogenic mechanisms have been elucidated in far more detail.Two papers describe the application of signature tagged mutagenesis (STM) to S. aureus (by Pathogenesis Corporation, CA, USA)1xStaphylococcus aureus genetic loci impacting growth and survival in multiple infection environments. Coulter, S.N. et al. Mol. Microbiol. 1998; 30: 393–404Crossref | PubMed | Scopus (196)See all References1 and S. pneumoniae (by Glaxo Wellcome, Verona, Italy)2xLarge-scale identification of virulence genes from Streptococcus pneumoniae. Polissi, A. et al. Infect. Immun. 1998; 66: 5620–5629PubMedSee all References2, pathogens whose genomic sequences are not available in the public domain. STM allows the large-scale identification of bacterial genes that are required for virulence in vivo. A pathogenic strain is randomly mutagenized, using transposons or homologous recombination, and is labelled with specific tags. A collection of mutants with different tags are simultaneously inoculated into an animal model and the resulting isolates are screened for weak or non-survivors. Although this method is biased against the isolation of mutants in extracellular toxins and essential genes, both studies have been strengthened by the use of more than one animal model.In S. aureus 237, and in S. pneumoniae 126, attenuated strains were identified. Although some of the genes encoded proteins that had been previously identified as virulence determinants, many of the remaining genes were involved in transportation or biosynthesis of amino acids, peptides and energy metabolism, emphasizing the need for bacteria to survive in vivo to cause infection. Mutations in regions that did not have significant homology to the genomic databases might represent novel virulence determinants.The most unexpected and fascinating result generated from both studies is that only a fraction of genes that were required for virulence in one animal model were also required in other models (10% of S. aureus in all three models, 70% of S. pneumoniae in both models). This shows that in different infection types, bacteria are exposed to different environmental conditions and rely on different factors for survival and disease generation.Therefore, we must conclude that virulence mechanisms of S. aureus and S. pneumoniae are highly dependent on the location and type of infection. In order to further our understanding of the pathogenic processes involved, we must regard each infection type as unique rather than making generalizations about disease caused by a particular bacterial species.

Influence of the TonB energy-coupling protein on efflux-mediated multidrug resistance in Pseudomonas aeruginosa Zhao, Q. et al . (1998) Antimicrob. Agents Chemother . 42, 2225–2231

Recombinational exchanges at the capsular polysaccharide biosynthetic locus lead to frequent serotype changes among natural isolates of Streptococcus pneumoniae Coffey, T.J. et al. (1998) Mol. Microbiol . 27, 73–83

Hyperglycaemia as a result of diabetes mellitus or acute illness is associated with increased susceptibility to respiratory infection with Staphylococcus aureus. Hyperglycaemia increases the concentration of glucose in airway surface liquid (ASL) and promotes the growth of S. aureus in vitro and in vivo. Whether elevation of other sugars in the blood, such as fructose, also results in increased concentrations in ASL is unknown and whether sugars in ASL are directly utilised by S. aureus for growth has not been investigated. We obtained mutant S. aureus JE2 strains with transposon disrupted sugar transport genes. NE768(fruA) exhibited restricted growth in 10 mM fructose. In H441 airway epithelial-bacterial co-culture, elevation of basolateral sugar concentration (5–20 mM) increased the apical growth of JE2. However, sugar-induced growth of NE768(fruA) was significantly less when basolateral fructose rather than glucose was elevated. This is the first experimental evidence to show that S. aureus directly utilises sugars present in the ASL for growth. Interestingly, JE2 growth was promoted less by glucose than fructose. Net transepithelial flux of d-glucose was lower than d-fructose. However, uptake of d-glucose was higher than d-fructose across both apical and basolateral membranes consistent with the presence of GLUT1/10 in the airway epithelium. Therefore, we propose that the preferential uptake of glucose (compared to fructose) limits its accumulation in ASL. Pre-treatment with metformin increased transepithelial resistance and reduced the sugar-dependent growth of S. aureus. Thus, epithelial paracellular permeability and glucose transport mechanisms are vital to maintain low glucose concentration in ASL and limit bacterial nutrient sources as a defence against infection. Electronic supplementary material The online version of this article (doi:10.1007/s00018-014-1635-y) contains supplementary material, which is available to authorized users.

Background Microarray-based Comparative Genomic Hybridisation (CGH) has been used to assess genetic variability between bacterial strains. Crucial for interpretation of microarray data is the availability of a reference to compare signal intensities to reliably determine presence or divergence each DNA fragment. However, the production of a good reference becomes unfeasible when microarrays are based on pan-genomes. When only a single strain is used as a reference for a multistrain array, the accessory gene pool will be partially represented by reference DNA, although these genes represent the genomic repertoire that can explain differences in virulence, pathogenicity or transmissibility between strains. The lack of a reference makes interpretation of the data for these genes difficult and, if the test signal is low, they are often deleted from the analysis. We aimed to develop novel methods to determine the presence or divergence of genes in a Staphylococcus aureus multistrain PCR product microarray-based CGH approach for which reference DNA was not available for some probes. Results In this study we have developed 6 new methods to predict divergence and presence of all genes spotted on a multistrain Staphylococcus aureus DNA microarray, published previously, including those gene spots that lack reference signals. When considering specificity and PPV (i.e. the false-positive rate) as the most important criteria for evaluating these methods, the method that defined gene presence based on a signal at least twice as high as the background and higher than the reference signal (method 4) had the best test characteristics. For this method specificity was 100% and 82% for MRSA252 (compared to the GACK method) and all spots (compared to sequence data), respectively, and PPV were 100% and 76% for MRSA252 (compared to the GACK method) and all spots (compared to sequence data), respectively. Conclusion A definition of gene presence based on signal at least twice as high as the background and higher than the reference signal (method 4) had the best test characteristics, allowing the analysis of 6-17% more of the genes not present in the reference strain. This method is recommended to analyse microarray data that partially lack a reference signal.