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Axenfeld-Rieger anomaly (ARA) is a specific ocular disorder that is frequently associated with other systemic abnormalities. PITX2 and FOXC1 variants explain the majority of individuals with Axenfeld-Rieger syndrome (ARS) but leave ~30% unsolved. Here, we present pathogenic/likely pathogenic variants in nine families with ARA/ARS or similar phenotypes affecting five different genes/regions. USP9X and JAG1 explained three families each. USP9X was recently linked with syndromic cognitive impairment that includes hearing loss, dental defects, ventriculomegaly, Dandy-Walker malformation, skeletal anomalies (hip dysplasia), and other features showing a significant overlap with FOXC1 -ARS. Anterior segment anomalies are not currently associated with USP9X , yet our cases demonstrate ARA, congenital glaucoma, corneal neovascularization, and cataracts. The identification of JAG1 variants, linked with Alagille syndrome, in three separate families with a clinical diagnosis of ARA/ARS highlights the overlapping features and high variability of these two phenotypes. Finally, intragenic variants in CDK13 , BCOR , and an X chromosome deletion encompassing HCCS and AMELX (linked with ocular and dental anomalies, correspondingly) were identified in three additional cases with ARS. Accurate diagnosis has important implications for clinical management. We suggest that broad testing such as exome sequencing be applied as a second-tier test for individuals with ARS with normal results for PITX2/FOXC1 sequencing and copy number analysis, with attention to the described genes/regions.

Background: The malaria parasite Plasmodium falciparum has an unusually euchromatic genome with poorly conserved positioning of nucleosomes in intergenic sequences and poorly understood mechanisms of gene regulation. Variant histones and histone modifications determine nucleosome stability and recruit trans factors, but their combinatorial contribution to gene regulation is unclear., Results: Here, we show that the histone H3 acetylations H3K18ac and H3K27ac and the variant histone Pf H2A.Z are enriched together at regulatory sites upstream of genes. H3K18ac and H3K27ac together dynamically mark regulatory regions of genes expressed during the asexual life cycle. In contrast, H3K4me1 is depleted in intergenic sequence and dynamically depleted upstream of expressed genes. The temporal pattern of H3K27ac and H3K18ac enrichment indicates that they accumulate during S phase and mitosis and are retained at regulatory sequences until at least G1 phase and after cessation of expression of the cognate genes. We integrated our ChIPseq data with existing datasets to show that in schizont stages H3K18ac, H3K27ac and Pf H2A.Z colocalise with the transcription factor PfAP2-I and the bromodomain protein PfBDP1 and are enriched at stably positioned nucleosomes within regions of exposed DNA at active transcriptional start sites. Using transient transfections we showed that sequences enriched with colocalised H3K18ac, H3K27ac and Pf H2A.Z possess promoter activity in schizont stages, but no enhancer-like activity., Conclusions: The dynamic H3 acetylations define P. falciparum regulatory sequences and contribute to gene activation. These findings expand the knowledge of the chromatin landscape that regulates gene expression in P. falciparum.

Introduction : Bromodomains (BRDs) bind to acetylated lysine residues, often on histones. The BRD proteins can contribute to gene regulation either directly through enzymatic activity or indirectly through recruitment of chromatin-modifying complexes or transcription factors. There is no evidence of direct orthologues of the Plasmodium falciparum BRD proteins (PfBDPs) outside the apicomplexans. PfBDPs are expressed during the parasite's life cycle in both the human host's blood and in the mosquito. PfBDPs could also prove to be promising targets for novel antimalarials, which are urgently required to address increasing drug resistance. Areas covered : This review discusses recent studies of the biology of PfBDPs, current target-based strategies for PfBDP inhibitor discovery, and different approaches to the important step of validating the specificity of hit compounds for PfBDPs. Expert opinion : The novelty of Plasmodium BRDs suggests that they could be targeted by selective compounds. Chemical series that showed promise in screens against human BRDs could be leveraged to create targeted compound libraries, as could hits from P. falciparum phenotypic screens. These targeted libraries and hits could be screened in target-based strategies aimed at discovery and optimization of novel inhibitors of PfBDPs. A key task for the field is to generate parasite assays to validate the hit compounds' specificity for PfBDPs.

Dermatofibroma sarcoma protuberans (DFSP) is a rare, locally aggressive soft tissue sarcoma with a tendency for recurrence after excision. Although reports of unilateral orbital and bilateral eyelid disease exist, there have been no prior reports of DFSP with bilateral orbital involvement and no previously described cases of DFSP associated with transient optic neuropathy. The authors present a case report of a 34-year-old woman with a giant scalp DFSP involving the bilateral orbits. Despite radical resection with 5 cm margins where possible, multiple positive margins remained including deep positive margins at the bilateral superomedial retroseptal soft tissue. The patient completed adjuvant radiation for surgically unresectable disease. This case highlights the challenge of achieving local control given the disease extent and infiltration of the bilateral eyelids and orbits. This is the first reported case of DFSP with bilateral orbital involvement and associated transient optic neuropathy.

The pathogenic nature of malaria infections is due in part to the export of hundreds of effector proteins that actively remodel the host erythrocyte. The Plasmodium translocon of exported proteins (PTEX) has been shown to facilitate the trafficking of proteins into the host cell, a process that is essential for the survival of the parasite. The role of the auxiliary PTEX component PTEX88 remains unclear, as previous attempts to elucidate its function through reverse genetic approaches showed that in contrast to the core components PTEX150 and HSP101, knockdown of PTEX88 did not give rise to an export phenotype. Here, we have used biochemical approaches to understand how PTEX88 assembles within the translocation machinery. Proteomic analysis of the PTEX88 interactome showed that PTEX88 interacts closely with HSP101 but has a weaker affinity with the other core constituents of PTEX. PTEX88 was also found to associate with other PV-resident proteins, including chaperones and members of the exported protein-interacting complex that interacts with the major virulence factor PfEMP1, the latter contributing to cytoadherence and parasite virulence. Despite being expressed for the duration of the blood-stage life cycle, PTEX88 was only discretely observed at the parasitophorous vacuole membrane during ring stages and could not always be detected in the major high molecular weight complex that contains the other core components of PTEX, suggesting that its interaction with the PTEX complex may be dynamic. Together, these data have enabled the generation of an updated model of PTEX that now includes how PTEX88 assembles within the complex., (© 2018 Federation of European Biochemical Societies.)

The ability of Plasmodium parasites to egress from their host red blood cell is critical for the amplification of these parasites in the blood. Previous forward chemical genetic approaches have implicated the subtilisin-like protease (SUB1) and the cysteine protease dipeptidyl aminopeptidase 3 (DPAP3) as key players in egress, with the final step of SUB1 maturation thought to be due to the activity of DPAP3. In this study, we have utilized a reverse genetics approach to engineer transgenic Plasmodium falciparum parasites in which dpap3 expression can be conditionally regulated using the glmS ribozyme based RNA-degrading system. We show that DPAP3, which is expressed in schizont stages and merozoites and localizes to organelles distinct from the micronemes, rhoptries and dense granules, is not required for the trafficking of apical proteins or processing of SUB1 substrates, nor for parasite maturation and egress from red blood cells. Thus, our findings argue against a role for DPAP3 in parasite egress and indicate that the phenotypes observed with DPAP3 inhibitors are due to off-target effects.

The malaria parasite, Plasmodium falciparum, displays the P. falciparum erythrocyte membrane protein 1 (PfEMP1) on the surface of infected red blood cells (RBCs). We here examine the physical organization of PfEMP1 trafficking intermediates in infected RBCs and determine interacting partners using an epitope-tagged minimal construct (PfEMP1B). We show that parasitophorous vacuole (PV)-located PfEMP1B interacts with components of the PTEX (Plasmodium Translocon of EXported proteins) as well as a novel protein complex, EPIC (Exported Protein-Interacting Complex). Within the RBC cytoplasm PfEMP1B interacts with components of the Maurer's clefts and the RBC chaperonin complex. We define the EPIC interactome and, using an inducible knockdown approach, show that depletion of one of its components, the parasitophorous vacuolar protein-1 (PV1), results in altered knob morphology, reduced cell rigidity and decreased binding to CD36. Accordingly, we show that deletion of the Plasmodium berghei homologue of PV1 is associated with attenuation of parasite virulence in vivo.

Plasmodium falciparum parasites, the causative agents of malaria, modify their host erythrocyte to render them permeable to supplementary nutrient uptake from the plasma and for removal of toxic waste. Here we investigate the contribution of the rhoptry protein RhopH2, in the formation of new permeability pathways (NPPs) in Plasmodium -infected erythrocytes. We show RhopH2 interacts with RhopH1, RhopH3, the erythrocyte cytoskeleton and exported proteins involved in host cell remodeling. Knockdown of RhopH2 expression in cycle one leads to a depletion of essential vitamins and cofactors and decreased de novo synthesis of pyrimidines in cycle two. There is also a significant impact on parasite growth, replication and transition into cycle three. The uptake of solutes that use NPPs to enter erythrocytes is also reduced upon RhopH2 knockdown. These findings provide direct genetic support for the contribution of the RhopH complex in NPP activity and highlight the importance of NPPs to parasite survival.

When in their human hosts, malaria parasites spend most of their time housed within vacuoles inside erythrocytes and hepatocytes. The parasites extensively modify their host cells to obtain nutrients, prevent host cell breakdown and avoid the immune system. To perform these modifications, malaria parasites export hundreds of effector proteins into their host cells and this process is best understood in the most lethal species to infect humans, Plasmodium falciparum. The effector proteins are synthesized within the parasite and following a proteolytic cleavage event in the endoplasmic reticulum and sorting of mature proteins into the correct vesicular trafficking pathway, they are transported to the parasite surface and released into the vacuole. The effector proteins are then unfolded before extrusion across the vacuole membrane by a unique translocon complex called Plasmodium translocon of exported proteins. After gaining access to the erythrocyte cytoplasm many effector proteins continue their journey to the erythrocyte surface by utilising various membranous structures established by the parasite. This complex trafficking pathway and a large number of the effector proteins are unique to Plasmodium parasites. This pathway could, therefore, be developed as new drug targets given that protein export and the functional role of these proteins are essential for parasite survival. This review explores known and potential drug targetable steps in the protein export pathway and strategies for discovering novel drug targets., (Copyright © 2016 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.)

Objectives: To investigate a potential outbreak of high-level azithromycin resistant (HL-AziR) gonococcal infections diagnosed in eight patients attending a sexual health clinic in Leeds, North England, between November 2014 and March 2015., Methods: Eight cases of infection with gonococci exhibiting azithromycin minimum inhibitory concentrations (MICs) ≥256 mg/L were identified from patients in Leeds as part of the routine service provided by the Sexually Transmitted Bacteria Reference Unit. All patient records were reviewed to collate epidemiological and clinical information including evaluation of patient management. Whole-genome sequencing (WGS) was performed on seven gonococcal isolates to determine Neisseria gonorrhoeae multiantigen sequence type (NG-MAST), WGS comparison and mutations in the 23S rRNA genes., Results: All patients were heterosexual (five male, three female) from a range of ethnic backgrounds and from the Leeds area. Three patients were linked by partner notification. All patients were infected at genital sites and two women had pharyngeal infection also. Six patients received the recommended first-line therapy for uncomplicated gonorrhoea, one was treated for pelvic inflammatory disease and one received spectinomycin followed later by ciprofloxacin. Test of cure was achieved in seven patients and confirmed successful eradication. All seven isolates sequenced were identical by NG-MAST and WGS comparison, and contained an A2143G mutation in all four 23S rRNA alleles., Conclusions: Epidemiological and microbiological investigations confirm that an outbreak of a gonococcal strain showing HL-AziR is ongoing in the North of England. Every effort should be made to identify and curtail dissemination of this strain as it presents a significant threat to the current recommended front-line dual therapy., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/)

Pathogenesis of malaria infections is linked to remodeling of erythrocytes, a process dependent on the trafficking of hundreds of parasite-derived proteins into the host erythrocyte. Recent studies have demonstrated that the Plasmodium translocon of exported proteins (PTEX) serves as the central gateway for trafficking of these proteins, as inducible knockdown of the core PTEX constituents blocked the trafficking of all classes of cargo into the erythrocyte. However, the role of the auxiliary component PTEX88 in protein export remains less clear. Here we have used inducible knockdown technologies in P. falciparum and P. berghei to assess the role of PTEX88 in parasite development and protein export, which reveal that the in vivo growth of PTEX88-deficient parasites is hindered. Interestingly, we were unable to link this observation to a general defect in export of a variety of known parasite proteins, suggesting that PTEX88 functions in a different fashion to the core PTEX components. Strikingly, PTEX88-deficient P. berghei were incapable of causing cerebral malaria despite a robust pro-inflammatory response from the host. These parasites also exhibited a reduced ability to sequester in peripheral tissues and were removed more readily from the circulation by the spleen. In keeping with these findings, PTEX88-deficient P. falciparum-infected erythrocytes displayed reduced binding to the endothelial cell receptor, CD36. This suggests that PTEX88 likely plays a specific direct or indirect role in mediating parasite sequestration rather than making a universal contribution to the trafficking of all exported proteins.

Background: The European Gonococcal Antimicrobial Surveillance Programme performs antimicrobial resistance surveillance and is coordinated by the European Centre for Disease Prevention and Control. This study used epidemiological and behavioral data combined with the gonococcal susceptibility profiles to determine risk factors associated with harboring resistant gonococci in Europe., Methods: From 2009 to 2011, gonococcal isolates from 21 countries were submitted to the European Gonococcal Antimicrobial Surveillance Programme for antimicrobial susceptibility testing. Patient variables associated with resistance to azithromycin, cefixime, and ciprofloxacin were identified using univariate and multivariable logistic regression analyses of odds ratios. Geometric means for ceftriaxone and cefixime minimum inhibitory concentrations (MICs) were compared for patients of different sexual orientation and sex., Results: A total of 5034 gonococcal isolates were tested from 2009 to 2011. Isolates exhibiting resistance to cefixime (MIC > 0.125 mg/L) and ciprofloxacin (MIC > 0.5 mg/L) were significantly associated with infection in heterosexuals (males only for ciprofloxacin), older patients (>25 years of age), or those without a concurrent chlamydial infection in the multivariable analysis. The geometric mean of cefixime and ceftriaxone MICs decreased from 2009 to 2011, most significantly for men who have sex with men, and isolates from male heterosexuals exhibited the highest MICs in 2011., Conclusions: The linking of epidemiological and behavioral data to the susceptibility profiles of the gonococcal isolates has allowed those at higher risk for acquiring antimicrobial resistant Neisseria gonorrhoeae to be identified. Improved data numbers and representativeness are required before evidence-based risk groups can be identified, and subsequent focused treatments or public health intervention strategies can be initiated with confidence.

Neisseria gonorrhoeae has consistently developed resistance to antimicrobials used therapeutically for gonorrhoea and few antimicrobials remain for effective empiric first-line therapy. Since 2009 the European gonococcal antimicrobial surveillance programme (Euro-GASP) has been running as a sentinel surveillance system across Member States of the European Union (EU) and European Economic Area (EEA) to monitor antimicrobial susceptibility in N. gonorrhoeae. During 2011, N. gonorrhoeae isolates were collected from 21 participating countries, and 7.6% and 0.5% of the examined gonococcal isolates had in vitro resistance to cefixime and ceftriaxone, respectively. The rate of ciprofloxacin and azithromycin resistance was 48.7% and 5.3%, respectively. Two (0.1%) isolates displayed high-level resistance to azithromycin, i.e. a minimum inhibitory concentration (MIC) ≥256 mg/L. The current report further highlights the public health need to implement the European response plan, including further strengthening of Euro-GASP, to control and manage the threat of multidrug resistant N. gonorrhoeae.

Purpose: To describe an ophthalmic phenotype in children at relatively early stages of Wolfram syndrome., Methods: Quantitative ophthalmic testing of visual acuity, color vision, automated visual field sensitivity, optic nerve pallor and cupping, and retinal nerve fiber layer (RNFL) thickness assessed by optical coherence tomography (OCT) was performed in 18 subjects 5-25 years of age. Subjects were also examined for presence or absence of afferent pupillary defects, cataracts, nystagmus, and strabismus., Results: Subnormal visual acuity was detected in 89% of subjects, color vision deficits in 94%, visual field defects in 100%, optic disk pallor in 94%, abnormally large optic nerve cup:disk ratio in 33%, thinned RNFL in 100%, afferent pupillary defects in 61%, cataracts in 22%, nystagmus in 39%, and strabismus in 39% of subjects. RNFL thinning (P < 0.001), afferent pupillary defects (P = 0.01), strabismus (P = 0.04), and nystagmus (P = 0.04) were associated with more severe disease using the Wolfram United Rating Scale., Conclusions: Children and adolescents with Wolfram syndrome have multiple ophthalmic markers that correlate with overall disease severity. RNFL thickness measured by OCT may be the most reliable early marker., (Copyright © 2014 American Association for Pediatric Ophthalmology and Strabismus. Published by Elsevier Inc. All rights reserved.)

During the blood stages of malaria, several hundred parasite-encoded proteins are exported beyond the double-membrane barrier that separates the parasite from the host cell cytosol. These proteins have a variety of roles that are essential to virulence or parasite growth. There is keen interest in understanding how proteins are exported and whether common machineries are involved in trafficking the different classes of exported proteins. One potential trafficking machine is a protein complex known as the Plasmodium translocon of exported proteins (PTEX). Although PTEX has been linked to the export of one class of exported proteins, there has been no direct evidence for its role and scope in protein translocation. Here we show, through the generation of two parasite lines defective for essential PTEX components (HSP101 or PTEX150), and analysis of a line lacking the non-essential component TRX2 (ref. 12), greatly reduced trafficking of all classes of exported proteins beyond the double membrane barrier enveloping the parasite. This includes proteins containing the PEXEL motif (RxLxE/Q/D) and PEXEL-negative exported proteins (PNEPs). Moreover, the export of proteins destined for expression on the infected erythrocyte surface, including the major virulence factor PfEMP1 in Plasmodium falciparum, was significantly reduced in PTEX knockdown parasites. PTEX function was also essential for blood-stage growth, because even a modest knockdown of PTEX components had a strong effect on the parasite's capacity to complete the erythrocytic cycle both in vitro and in vivo. Hence, as the only known nexus for protein export in Plasmodium parasites, and an essential enzymic machine, PTEX is a prime drug target.

Plasmodium parasites remodel their vertebrate host cells by translocating hundreds of proteins across an encasing membrane into the host cell cytosol via a putative export machinery termed PTEX. Previously PTEX150, HSP101 and EXP2 have been shown to be bona fide members of PTEX. Here we validate that PTEX88 and TRX2 are also genuine members of PTEX and provide evidence that expression of PTEX components are also expressed in early gametocytes, mosquito and liver stages, consistent with observations that protein export is not restricted to asexual stages. Although amenable to genetic tagging, HSP101, PTEX150, EXP2 and PTEX88 could not be genetically deleted in Plasmodium berghei, in keeping with the obligatory role this complex is postulated to have in maintaining normal blood-stage growth. In contrast, the putative thioredoxin-like protein TRX2 could be deleted, with knockout parasites displaying reduced grow-rates, both in vivo and in vitro, and reduced capacity to cause severe disease in a cerebral malaria model. Thus, while not essential for parasite survival, TRX2 may help to optimize PTEX activity. Importantly, the generation of TRX2 knockout parasites that display altered phenotypes provides a much-needed tool to dissect PTEX function., (© 2013 John Wiley & Sons Ltd.)

Objectives: After a trend of increasing incidence of gonorrhoea in the 1990s, by 2004 the incidence was declining in England, but continuing to increase in Wales. This prompted an investigation of the epidemiology of gonorrhoea in Wales to inform future prevention and control measures., Methods: As an extension to Gonococcal Resistance to Antimicrobials Surveillance Programme, between May 2005 and September 2006, 540 consecutive gonococcal isolates were collected from three microbiology laboratories in South Wales. Isolates were typed using Neisseria gonorrhoeae Multi Antigen Sequence Typing tested for susceptibility to therapeutic agents and demographic and behavioural data were collected retrospectively from patient notes., Results: 163 sequence types (STs) were identified in 475 N gonorrhoeae isolates from 502 patient episodes. The most frequently observed STs (>20 isolates) were: 2, 752, 471, 249 and 8, all of which were susceptible to the antimicrobial agents tested. A significant association between ST and sexual orientation was identified, the most frequently observed STs occurring in young (median age <25 years) heterosexuals. STs 147, 4, 1634 and 64 predominated in men who have sex with men., Conclusions: We confirm the existence of common STs across the UK, as well as identify a number of types that were novel to Wales. Discrete sexual networks were identified, the most localised being in young heterosexuals. Molecular typing provides a method for identifying local clusters of gonorrhoea, and could assist in the implementation and evaluation of targeted interventions.

Treatment of gonorrhoea is threatened by antimicrobial resistance, and decreased susceptibility and resistance to recommended therapies is emerging in Europe. Current associations between resistance and molecular type remain poorly understood. Gonococcal isolates (n=1,066) collected for the 2009 and 2010 European Gonococcal Antimicrobial Surveillance Programme were typed by Neisseria gonorrhoeae multi-antigen sequence typing (NG-MAST). A total of 406 sequence types (STs) were identified, 125 of which occurred in ≥two isolates. Seven major genogroups of closely related STs (varying by ≤1% at just one of the two target loci) were defined. Genogroup 1407 (G1407), observed in 20/21 countries and predominant in 13/21 countries, accounted for 23% of all isolates and was associated with decreased susceptibility to cefixime and resistance to ciprofloxacin and raised minimum inhibitory concentrations for ceftriaxone and azithromycin. Genogroup 225 (G225), associated with ciprofloxacin resistance, was observed in 10% of isolates from 19/21 countries. None of the other genogroups were associated with antimicrobial resistance. The predominance of a multidrug-resistant clone (G1407) in Europe is worrying given the recent reports of recommended third generation cephalosporins failing to treat infections with this clone. Identifying associations between ST and antimicrobial resistance aids the understanding of the dissemination of resistant clones within a population and could facilitate development of targeted intervention strategies.

Neisseria gonorrhoeae antimicrobial susceptibility is monitored in the European Union (EU) and the European Economic Area (EEA) by the European gonococcal antimicrobial surveillance programme (Euro-GASP). Results from 17 EU/EEA Member States in 2009 showed that 5% of isolates had decreased susceptibility to cefixime, an upward trend in the minimum inhibitory concentrations of ceftriaxone and a high prevalence of resistance to ciprofloxacin (63%)and azithromycin (13%). These results are of public health value and highlight the need for healthcare professionals to be aware of possible cefixime treatment failures. Euro-GASP is being implemented in additional EU/EEA Member States to achieve greater representativeness. In addition, Euro-GASP aims to set up a system which will allow biannual reporting of antimicrobial resistance in the EU/EEA, with a transition from centralised towards decentralised testing,and will link epidemiological data to laboratory data to enhance surveillance. The benefits of this approach include more timely detection of emerging trends in gonococcal resistance across the EU/EEA and the provision of a robust evidence base for informing national and European guidelines for therapy.

Objectives: The emergence of decreased susceptibility to third-generation, extended-spectrum cephalosporins in Neisseria gonorrhoeae and associated treatment failures highlights the need to consider alternatives for future therapeutic use, such as gentamicin., Methods: The three laboratories surveying gonococcal antimicrobial susceptibility as part of the European Network for Sexually Transmitted Infections Surveillance compared agar dilution and Etest to determine gentamicin MICs and performed the first survey of gentamicin susceptibility on 1366 gonococcal isolates from 17 European Union/European Economic Area (EU/EAA) countries in 2009., Results: Sentinel surveillance of gentamicin susceptibility showed that 95% of European isolates were within a narrow MIC range (4-8 mg/L), with 79% showing an MIC of 8 mg/L. Most countries showed little variation, but wider MIC ranges were observed in Greece (1-16 mg/L) and France, Norway and Sweden (2-16 mg/L). While MICs for both methods generally differed by just one doubling dilution, they were lower by Etest., Conclusions: This is the first reported evidence that the European gonococcal population susceptibility to gentamicin is similar to that reported in other world regions. Clinical trials to evaluate the therapeutic efficacy of gentamicin may be warranted.

Objective: To perform a European sentinel surveillance study for antimicrobial resistance (AMR) in Neisseria gonorrhoeae as part of the European Surveillance of Sexually Transmitted Infections Programme., Methods: From 2006 to 2008 17 countries participated in the AMR surveillance programme. The susceptibility of a total of 3528 consecutive isolates was tested using the agar dilution breakpoint technique or Etests for ciprofloxacin, penicillin, tetracycline, azithromycin, spectinomycin and ceftriaxone. Nitrocefin was used to detect β-lactamase activity., Results: Rates of resistance to ciprofloxacin, the previously recommended treatment, were high across Europe (42-52%), indicating that usage is no longer appropriate. Although resistance to the currently recommended treatment, ceftriaxone, was not demonstrated, a concerning upward drift in the minimal inhibitory concentration (MIC) distribution was identified since an earlier European study in 2004. No resistance to spectinomycin was seen, whereas azithromycin resistance varied from 2% to 7% and isolates from Scotland (n=4) and Ireland (n=1) showed high-level resistance (MIC >256 mg/l). High-level resistance to tetracycline and penicillin remained relatively constant at 16% and 12%, respectively., Conclusions: AMR is an ongoing problem in Europe, with high rates of resistance to many previously recommended therapeutic agents observed in many European countries. Continual European and global surveillance of AMR in N gonorrhoeae is essential to monitor for increasing, emerging and high-level resistance to therapeutically relevant agents and to inform treatment guidelines so optimum treatments are administered.

Background: Gonorrhoea has been among the easiest infections to cure with antibiotics. Nevertheless, emerging resistance has driven repeated treatment shifts. Decreased cephalosporin susceptibility is now being reported. We examined cephalosporin MIC trends for Neisseria gonorrhoeae in the UK and undertook pharmacodynamic analyses to predict efficacy against strains with raised MICs., Methods: Neisseria gonorrhoeae isolates were collected annually in a structured surveillance from 26 genitourinary medicine clinics in England and Wales. MICs were determined by agar dilution and confirmed by Etests. Pharmacodynamic modelling was performed for cefixime and ceftriaxone with Monte Carlo simulations., Results: There was a progressive emergence of small numbers of gonococci with cephalosporin MICs of 0.125-0.25 mg/L; these were not seen before 2005 but, for ceftriaxone and cefixime, respectively, accounted for 0.4% (95% confidence interval 0.2%-1.1%) and 2.8% (1.6%-4.8%) of the 1253 isolates collected in 2008; such MICs are 16-64 times the modal values for the species. Pharmacodynamic analysis was complicated by evidence that cephalosporins need a longer period with the free drug level above MIC than the 7-10 h required for penicillin G; nevertheless, pharmacodynamic analyses predict that failures with the standard 400 mg cefixime po and 250 mg ceftriaxone im regimens become likely around the present MIC maxima., Conclusions: Gonococci with ceftriaxone and cefixime MICs of 0.125-0.25 mg/L are accumulating in the UK. These MICs lie on the edge of likely responsiveness to current regimens, which need review. Possible responses include: (i) higher cephalosporin doses; (ii) multidose cephalosporin regimens; (iii) multidrug regimens; (iv) microbiologically directed treatment; or, in the future, (v) drug cycling. The practicalities of these approaches are discussed.

High-level azithromycin resistance (AZM-HR), defined as a MIC of > or = 256 mg/liter, emerged in Neisseria gonorrhoeae in the United Kingdom in 2004. To determine the mechanism of this novel phenotype, isolates from the United Kingdom that were AZM-HR (n, 19), moderately AZM resistant (MICs, 2 to 8 mg/liter) (n, 26), or sensitive (MICs, 0.12 to 0.25 mg/liter) (n, 4) were screened for methylase (erm) genes and for mutations in the mtrR promoter region, associated with efflux pump upregulation. All AZM-resistant isolates and 12 sensitive isolates were screened for mutations in domain V of each 23S rRNA allele. All AZM-HR isolates contained the A2059G mutation (Escherichia coli numbering) in three (3 isolates) or four (16 isolates) 23S rRNA alleles. Most (22/26) moderately AZM resistant isolates contained the C2611T mutation in at least 3/4 alleles. The remainder contained four wild-type alleles, as did 8/12 sensitive isolates, while one allele was mutated in the remaining four sensitive isolates. Serial passage of AZM-sensitive colonies on an erythromycin-containing medium selected AZM-HR if the parent strain already contained mutation A2059G in one 23S rRNA allele. The resultant AZM-HR strains contained four mutated alleles. Eight isolates (five moderately AZM resistant and three AZM-HR) contained mutations in the mtrR promoter. No methylase genes were detected. This is the first evidence that AZM-HR in gonococci may result from a single point mutation (A2059G) in the peptidyltransferase loop in domain V of the 23S rRNA gene. Mutation of a single allele is insufficient to confer AZM-HR, but AZM-HR can develop under selection pressure. The description of a novel resistance mechanism will aid in screening for the AZM-HR phenotype.

Objective: To examine the molecular epidemiology of syphilis in Scotland., Methods: Ulcer specimens were collected from 85 patients with infectious syphilis. Typing of Treponema pallidum was performed using a method that examines variation in two loci; the number of 60-basepair repeats within the arp gene and sequence variation in the tpr genes., Results: Patients were predominately white men who have sex with men (MSM). Treponemal DNA was detected in 75 specimens and a total of six subtypes were identified from 58 typeable specimens (77%). The most common subtypes were 14d (44/58, 76%), followed by 14e (7/58, 12%), 14j (3/58, 5%), 14b (2/58, 3%), 14p and 14k (1/58, 2%)., Conclusions: This study shows that subtype 14d is the predominant subtype circulating in Scotland and there is a surprising level of genetic diversity within the Scottish MSM community.

Background: The aim of this study was to investigate the effect of amoxicillin therapy of poultry flocks upon the persistence of commensal Campylobacter spp. and the incidence of antibiotic resistance., Methods: Four poultry flocks naturally colonized with Campylobacter were treated with amoxicillin and monitored before, during and up to 4 weeks post-treatment. The numbers of Campylobacter were determined and the isolates speciated and typed by flaA short variable region (SVR) sequence analysis and PFGE. The susceptibility of the isolates to antibiotics, presence of the Cj0299 gene encoding a beta-lactamase and beta-lactamase production (nitrocefin hydrolysis) were also determined., Results: Amoxicillin-resistant Campylobacter were isolated from Flock 1 before and during treatment, but Campylobacter were not detected afterwards. Flock 2 was colonized by amoxicillin-susceptible strains throughout sampling. No amoxicillin-resistant isolates arose during or after treatment. Flock 3 contained amoxicillin-susceptible and -resistant types pre-treatment. Resistant isolates were detected during treatment, while antibiotic-susceptible isolates re-emerged at 3 weeks post-treatment. All Campylobacter isolates from Flock 4 were amoxicillin resistant, irrespective of sampling time. All but one of the 82 amoxicillin-resistant (MICs 16 to >128 mg/L) Campylobacter jejuni and Campylobacter coli tested for the presence of Cj0299 carried the gene and all of these produced beta-lactamase. Co-amoxiclav remained active against amoxicillin-resistant isolates., Conclusions: Amoxicillin therapy had little effect on the numbers of amoxicillin-resistant commensal Campylobacter except for one flock where amoxicillin-resistant Campylobacter temporarily dominated. Amoxicillin therapy did not select amoxicillin-resistant isolates from a previous susceptible strain. Co-amoxiclav remained active against amoxicillin-resistant isolates.

Treatment failure with standard Helicobacter pylori eradication regimes may require the use of 'rescue' therapies containing fluoroquinolones or rifamycins. The susceptibilities of H. pylori in the UK to such antimicrobials are unknown; therefore, this study aimed to determine the frequencies and molecular markers of resistance. Ciprofloxacin and rifampicin susceptibilities were determined by Etest and/or disc diffusion for 255 isolates of H. pylori, including 171 isolates from adult dyspeptic patients with refractive infections. Mutations in known resistance-determining regions of gyrA and rpoB were determined. The ciprofloxacin resistance rate was 7.5 %, and gyrA mutations, predominantly at codon position 91, were identified in most resistant isolates. One isolate (<1 %) had an unequivocal rifampicin-resistant phenotype by Etest yet had no associated mutations in the rpoB gene. As resistance rates were low in H. pylori isolates, including those from patients with refractive infections, it was concluded that fluoroquinolones or rifamycins might be considered in the UK for inclusion in 'rescue' therapies.

Objectives: This study aimed to investigate the origin of high-level azithromycin resistance that emerged in isolates of Neisseria gonorrhoeae in England and Wales in 2007, and to establish methods for identifying high-level azithromycin resistance., Methods: The Gonococcal Resistance to Antimicrobials Surveillance Programme (GRASP) data from 2001-07 were examined for emerging trends in azithromycin susceptibility. Further to the identification of six high-level azithromycin-resistant isolates in GRASP 2007, an additional 102 isolates were selected on the basis of azithromycin susceptibility and geographic origin from the GRASP 2006 and 2007 collections. Susceptibility testing by Etest and disc diffusion was performed on all 108 isolates and 75 of these were typed by N. gonorrhoeae multiantigen sequence typing., Results: A slight drift towards higher MICs of azithromycin was observed in the gonococcal population since 2001. Of greater concern was the first example of a shift to high-level resistance observed in six isolates in 2007. All six isolates were sequence type 649, which was not observed in any of the lower-level azithromycin-resistant isolates from 2007 or in any isolates tested from the same geographical locations. Contact tracing data for one patient suggested a link with Scotland. Disc diffusion testing of all 108 isolates showed that azithromycin, but not erythromycin, discs can differentiate between low-level and high-level resistance., Conclusions: High-level azithromycin resistance has emerged in England and Wales. Contact tracing and typing data suggest this may have originated from Scotland. Surveillance of azithromycin resistance will be key in controlling its further dissemination.

Objectives: The aim of this study was to investigate the persistence of Campylobacter species, strain types, antibiotic resistance and mechanisms of tetracycline resistance in poultry flocks treated with chlortetracycline., Methods: Three commercially reared broiler flocks, naturally colonized with Campylobacter, were treated with chlortetracycline under experimental conditions. The numbers of Campylobacter isolated, and the species, flaA short variable region allele, and antimicrobial resistance of isolates were determined., Results: For two of three flocks, tetracycline-resistant strains predominated prior to chlortetracycline exposure. Presence of the antibiotic had no discernible effect on the numbers or types of Campylobacter and the tetracycline-resistant strains persisted in numbers similar to those observed before treatment. With all flocks, some faecal samples were obtained that contained no Campylobacter, irrespective of exposure to chlortetracycline; this was more common as the birds grew older. For the third flock, tetracycline-resistant Campylobacter were in the minority of samples before and during exposure to chlortetracycline, but at sampling times after this, no resistant strains were found in the treated (or untreated) birds, irrespective of exposure to the antibiotic. All tetracycline-resistant isolates (MICs 16 to >128 mg/L) contained tet(O) and, for some isolates, this was transferable to Campylobacter jejuni 81116. The efflux pump inhibitor PAbetaN reduced the MICs of tetracycline for these isolates by 4-fold, suggesting that an intact efflux pump, presumably CmeABC, is required for high-level tetracycline resistance., Conclusions: Our data indicate that chlortetracycline treatment does not eradicate tetracycline-resistant Campylobacter spp. from poultry. However, if a low number of resistant isolates are present, then the antibiotic pressure appears insufficient to select such strains as the dominant population.

The benefits of using a multiplex detection polymerase chain reaction (PCR) assay for Helicobacter pylori speciation and 2 real-time probe hybridization assays determining clarithromycin and tetracycline susceptibilities in gastric biopsies from 171 dyspeptic patients were investigated. Overall, 70 of 71 H. pylori culture-positive biopsies were PCR positive. For the 100 culture-negative biopsies, PCR identified a further 29 H. pylori positives (17% overall) and presence of resistance markers for clarithromycin (20/28) and tetracycline (2/28). The results demonstrated that PCR testing was valuable in providing improved detection rates and antibiotic susceptibility information when H. pylori culture was unsuccessful.

Antibiotic resistance is a key factor in the failure of Helicobacter pylori eradication therapy, yet few sentinel schemes exist to monitor trends in resistance at local, national or international levels. This study aimed, over a six-year period, to monitor resistance levels of H. pylori in England and Wales to the four antibiotics used in its treatment. A total of 1,310 isolates from Gwynedd in north Wales and from mid-Essex in south-east England were collected from 2000 to 2005 and tested for susceptibilities to metronidazole, clarithromycin, amoxicillin and tetracycline. Overall, metronidazole and clarithromycin resistance rates were 28.6% and 8.3% in Gwynedd and significantly higher (36.3%, p=0.0031, and 12.7%, p=0.0112) in mid-Essex. Rates of resistance to metronidazole and clarithromycin increased in both areas over this six-year period. Resistance rates were higher in female compared with male patients (38.1% vs 26.6% for metronidazole, p<0.0001, and 12.9% vs 7.5% for clarithromycin, p=0.0024), and were higher in patients <45 years compared with those ?45 years (44.0% vs 29.0% for metronidazole, p=0.0002, and 15.0% vs 9.4% for clarithromycin, p=0.0233). This study highlights the importance of antibiotic resistance surveillance in H. pylori for providing information on local resistance rates for test and treat strategies.

Helicobacter pylori is an important global human pathogen and there is growing evidence from PCR assays that contaminated drinking water might be a possible source of infection in some circumstances. There are no validated protocols for direct isolation but various culture media have been developed for possible environmental sampling. Our aim here was to investigate how inter-strain variation might affect the interpretation of results with such media. Two laboratory adapted reference strains and four recent clinical isolates were tested on four solid media and in ten liquid media. Considerable variation was found between strains in their ability to recover on the different media after stress exposure (suspension in sterile tap water). Generally, clinical isolates were less robust than the laboratory-adapted strains and, overall, the former required longer recovery times. Our findings highlighted the importance of using a range of isolates for evaluations, as examination of laboratory-adapted strains alone did not provide an accurate representation of the utility of media that may be used to recover H. pylori from water.

Stool antigen-testing allows non-invasive detection of Helicobacter pylori that is indicative of active infection. Three commercial kits are currently marketed in the UK for stool antigen-testing. The aim of this study was to conduct a comparative evaluation of the performances of each of these tests, compared with culture and histological examination of gastric biopsies, for pre-treatment diagnosis of infection in an adult dyspeptic population in south-east England. Examination of 112 stool samples by the Premier Platinum HpSA ELISA (Meridian Diagnostics) and by the Amplified IDEIA HpStAR ELISA (DakoCytomation) kits demonstrated that the latter was more sensitive (81.3 versus 93.8%, respectively) and specific (91.7 versus 100.0%, respectively). Additionally, the IDEIA HpStAR was easier to interpret, with OD readings of positive and negative results being far from the recommended cut-off, whereas equivocal results that were generated by the HpSA kit were difficult to interpret. Additional testing of 87 of the 112 stools by the ImmunoCard STAT! HpSA kit (Meridian Diagnostics) demonstrated that this test was easier to perform than ELISA and was more sensitive than the HpSA kit but, compared with the IDEIA HpStAR kit, the ImmunoCard test was less sensitive (87.8 versus 95.9%, respectively) and specific (89.4 versus 100.0%, respectively). Furthermore, the ImmunoCard test generated weakly positive results, correlating with lower OD readings for both ELISA kits, that were difficult to interpret. The Amplified IDEIA HpStAR kit is therefore the most sensitive and specific of the three tests that are available for pre-treatment, non-invasive detection of H. pylori in stool samples in an English adult dyspeptic population.

Mutations in the NAD(P)H flavin oxidoreductase gene (frxA) are thought to contribute to the development of metronidazole resistance in Helicobacter pylori. To test this further, 44 frxA sequences in 18 patient isolate sets of H. pylori were examined including a unique collection comprising separated Mtz-sensitive (MtzS) and Mtz-resistant (MtzR) subpopulations pre-treatment and matched MtzR strains post-treatment. Sequences of frxA contained frameshift mutations that led to premature protein truncation in at least one strain from most (17/18) patient sets. These mutations were present in all strains, irrespective of Mtz resistotype in 13/18 patients. Frameshift due to a single adenine deletion at nucleotide 53 was the most common mutation and was present in isolates from 11/18 patients. A novel real-time (LightCycler) PCR-based probe hybridization melting-point assay applied to a further 119 isolates confirmed that the frameshift-53 mutation occurred frequently, in 20% of isolates, and could be present in MtzS as well as MtzR strains (42% vs 58%). This study demonstrates that frameshift mutations occur in MtzS strains as well as in MtzR strains, and are thus unlikely to cause Mtz resistance.

Reports on Helicobacter pylori and extragastric diseases have almost doubled this year compared with last year, bearing witness to the persistent scientific interest in this branch of Helicobacter-related pathology. Data belong increasingly to the area of vascular medicine, as well as hematology, dermatology, pediatrics and other fields. Unfortunately, these studies show overall controversial results, due to the impact of several confounding factors, and to the difficulty of recruiting homogeneous patient populations. Furthermore, many studies continue to be conducted on Helicobacter species other than H. pylori, focusing on animal models of gastroenterological illnesses which may retain strong similarities with human diseases. In this paper, taxonomy, detection and characterisation of Helicobacter spp. will be reviewed, together with the most important data issued this year on other Helicobacters and animal models.

Background: Bacteria have been implicated in the pathogenesis of inflammatory bowel disease. Helicobacter species have been shown to cause colitis in animal models and have been identified in human diarrhoeal illness and Crohn's disease., Aim: To determine whether Helicobacter species are present in human inflammatory bowel disease tissue., Methods: Thirty patients undergoing colonoscopy for clinical reasons were studied. Nine had Crohn's disease, 11 had ulcerative colitis and 10 had histologically normal colons. Tissue was snap-frozen at -70 degrees C. DNA was extracted and examined by five different polymerase chain reaction (PCR) assays that were either genus or species specific for Helicobacter., Results: Analyses of colonic biopsies by two Helicobacter genus-specific PCR assays, two H. pylori-specific assays and a PCR assay designed to amplify fragments of 'H. heilmannii'-like organisms demonstrated that product was not generated by any test. Internal control PCR demonstrated that PCR results for the five assays were not negative due to the presence of residual substances inhibitory to PCR., Conclusions: Helicobacter species were not identified in this study, using multiple PCRs to eliminate the problems of non-specific cross-reaction. This suggests that Helicobacter species do not play a role in the pathogenesis of inflammatory bowel disease.

Cag pathogenicity island-containing Helicobacter pylori (type I) induces signal transduction pathways resulting in tyrosine phosphorylation of proteins adjacent to the site of bacterial adhesion on host gastric epithelial cells. Conventional block PCR-restriction fragment length polymorphism (RFLP) and real-time LightCycler (LC) PCR hybridization assays, validated by direct sequencing, were designed to test for the presence of three nucleotide sequences corresponding to tyrosine phosphorylation motifs (TPMs) A, B, and C in 84 isolates of H. pylori type I from patients in England. Overall, the PCR assays demonstrated that one or more TPMs were present in 62 strains (75%). Motif A was common (71% of strains), whereas motifs B and C were rarer (8% of strains). Strains lacking a TPM were typically vacuolating cytotoxin genotype vacA m2. Motif A was widely distributed in relation to disease severity and was more commonly (but not significantly [P = 0.071]) associated with gastric ulcer than with duodenal ulcer (86 versus 56%). The LC hybridization assay provided a rapid means of detecting all three motifs, but RFLP analysis was more specific for TPM-A. TPMs provide novel additional strain markers for defining cagA variation, including identification of RFLP types within TPM-A. The presence of a particular TPM was not of direct diagnostic value, either singly or in combination, but the higher proportion of TPM-A strains in gastric ulcer patients merits further investigation.

A novel PCR assay (HHLO-16) to screen for presence of 'Helicobacter heilmannii'-like organisms (HHLO) direct from gastric biopsies is described. As 'H. heilmannii' is generally uncultivable, diagnosis of infection is reliant on histology; thus prevalence may be underestimated. Analysis of an HHLO histology-positive human gastric biopsy and 15 gastric biopsies from domestic cats demonstrated that the HHLO-16 assay was more sensitive than an alternative available species-specific PCR assay. Further testing of 131 gastric biopsies from dyspeptic patients demonstrated an HHLO prevalence rate of 2.3% in Southeast England. Subsequent combination of the HHLO-16 assay with a H. pylori-specific PCR assay in a multiplex format (HpHh assay), and repeat analysis of the 131 biopsies showed the HpHh assay was as sensitive as each individual test. This novel PCR assay provides simple concomitant testing of dyspeptic patients for both HHLOs and H. pylori, thereby rapidly identifying individuals requiring eradication therapy.

Metronidazole resistance in Helicobacter pylori reportedly occurs by mutational inactivation of the oxygen-insensitive nitroreductase gene rdxA. Nucleotide sequences of rdxA were determined in a set of 46 isolates from 19 dyspeptic patients from the UK. The study set comprised matched isolates that were either metronidazole susceptible (four) or mixed metronidazole susceptible and metronidazole resistant (15) before therapy and metronidazole resistant post-therapy (10) in the 11 patients that were followed up. Various mutation types were identified in rdxA of metronidazole-resistant strains (post-treatment) that were absent in matched metronidazole-susceptible strains (pre-treatment). However, rdxA sequences from pre-treatment metronidazole-resistant and metronidazole-susceptible subpopulations were identical in 11 of 15 patients. Thus, mutations in rdxA may not always be essential for metronidazole resistance. Future examination of rdxA expression at the transcription and translational level may provide further insight into the role of this locus in metronidazole action and resistance in H. pylori.

Aims: To develop and evaluate a novel multiplex PCR assay that enables definition of Helicobacter pylori vacA allelic type in a single reaction., Methods and Results: Application of the one-step system to DNA extracts from 22 cultures of known vacA genotype demonstrated that it was highly accurate. Analysis of 15 matched gastric biopsy/culture pairs generated exactly correlating genotype profiles. vacA genotypes were determined from an additional 62/70 gastric biopsies from dyspeptic patients of known H. pylori positive status by the one-step assay, compared with 63/70 by the original two-reaction test. Types s1/m1, s1/m2 and s2/m2 were identified in 51.9%, 31.2% and 16.9% of biopsies, respectively., Conclusions: The multiplex PCR system developed enables rapid one-step vacA genotyping that is accurate, easy to interpret and more economical than the alternative multiple-reaction tests. Application of this system to gastric biopsies from patients in South-east England demonstrated that s1/m1 was the most common genotype, while s1/m2 and s2/m2 were less prevalent., Significance and Impact of the Study: This simple one-step system can be applied direct to antral gastric biopsies without the need for culture, thereby facilitating rapid surveillance of vacA genotype in relation to geographical location and disease status.

A novel PCR detection assay that amplifies the Helicobacter pylori-specific vacuolating cytotoxin gene (vacA) and thus enables rapid diagnosis of infection is described. Additionally, a real-time probe hybridization melting point analysis assay to detect all three mutations in the 23S rRNA gene associated with clarithromycin resistance was applied directly to antral gastric biopsy samples. Comparison with culture and an alternative PCR assay targeting the 16S rrn gene showed that the vacA assay was sensitive and specific when tested on biopsy samples from 121 patients. Clarithromycin susceptibilities could be determined in the majority (92.3%) of culture-positive gastric biopsy samples analyzed, four of which generated melting peaks indicative of clarithromycin resistance by either an A-->G or A-->C mutation. The presence of the mutations correlated with the clarithromycin disk diffusion sensitivities of matched cultures. This PCR-based system was simple to perform and could be completed in 3 to 4 h, thereby overcoming the delays associated with conventional culture methods for H. pylori identification and susceptibility testing.

One-hundred and twenty-one isolates of Salmonella enterica serotype Montevideo, representing different biotypes and incidents of infection detected in the UK between 1977 and 1995, were analysed by EcoRI ribotyping, PvuII ribotyping and IS200 fingerprinting. Among the isolates examined, 7 EcoRI ribotypes, 5 PvuII ribotypes and 55 IS200 profile types were recognized and 4 arbitrary groups defined. All 33 isolates of biotype 2d belonged to EcoRI/PvuII ribotype 1/1 and IS200 lineage A and comprised Group I. The other 88 isolates of biotype 10di and its variants were assigned to Groups II-IV. All 27 isolates in Group II were of EcoRI/PvuII ribotype 2/2 and IS200 lineage B. Among the 43 isolates in Group III, 42 of which were of EcoRI/PvuII ribotype 3/3, IS200 analysis identified 38 profiles in lineages C-I. Six EcoRI/PvuII ribotypes and 8 IS200 profiles, mostly in lineages C-E, were recognized among the 18 isolates in Group IV. The combined use of biotyping and ribotyping, and to some extent IS200 profiling, has enhanced our understanding of the clonal structure of serotype Montevideo and provides a basis for further study.

Thirteen isolates of Salmonella serotype Glostrup (antigenic formula, 6.8:z10:e,n,z15) from various sources and countries were analysed by ribotyping and IS200 fingerprinting. Both methods provided a high index of strain discrimination by allowing detection of three ribotypes and eight IS200 fingerprints which, though generally related, were readily distinguishable. The findings of this analysis confirm the usefulness of ribotyping and IS200 fingerprinting for studying the epidemiology of rarely isolated salmonellae of serogroup C.

One-hundred-and-thirteen isolates of Salmonella serotype Thompson from diverse sources in seven countries were characterized by PvuII ribotyping and IS200 fingerprinting. Ten PvuII ribotypes were observed. The predominant PvuII ribotype 1 represented a major clone of world-wide distribution but was not found in Australia; PvuII ribotypes 2 and 3 represented minor clones. HincII ribotyping discriminated subtypes within PvuII ribotype 1: HincII ribotype 1 was distributed widely but HincII ribotype 2 was found mainly in Scottish isolates. None of 101 isolates of PvuII ribotypes 1-3 contained copies of IS200. All 12 isolates of PvuII ribotypes 4-10 were from Australia and 7 of them contained copies of IS200 of 5 different profiles. These results suggest the existence of at least two lineages of Salmonella Thompson with a different geographical distribution. The finding that most isolates from man and poultry in Scotland belonged to the same ribotype (PvuII 1/HincII 2) and were IS200-negative suggests that poultry is an important source of human infection in Scotland.