Antimicrobial drug resistance in Salmonella: problems and perspectives in food- and water-borne infections

Strains of Salmonella spp. with resistance to antimicrobial drugs are now widespread in both developed and developing countries. In developed countries it is now increasingly accepted that for the most part such strains are zoonotic in origin and acquire their resistance in the food-animal host before onward transmission to humans through the food chain. Of particular importance since the early 1990s has been a multiresistant strain of Salmonella typhimurium definitive phage type (DT) 104, displaying resistance to up to six commonly used antimicrobials, with about 15% of isolates also exhibiting decreased susceptibility to ciprofloxacin. Mutations in the gyrA gene in such isolates have been characterised by a PCR LightCycler-based gyrA mutation assay, and at least four different mutations have been identified. Multiple resistance (to four or more antimicrobials) is also common in the poultry-associated pathogens Salmonella virchow and Salmonella hadar, with an increasing number of strains of these serotypes exhibiting decreased susceptibility to ciprofloxacin. Multiple resistance is also being found in other serotypes in several other European countries, and has been associated with treatment failures. For Salmonella typhi, multiple drug resistance is now the norm in strains originating in the Indian subcontinent and south-east Asia. Such multiresistant strains have been responsible for several epidemics and some of these have been associated with contaminated water supplies. Furthermore, an increasing number of multiresistant strains of S. typhi are now exhibiting decreased susceptibility to ciprofloxacin, with concomitant treatment failures. In developed countries antimicrobial resistance in zoonotic salmonellas has been attributed to the injudicious use of antimicrobials in food-producing animals. It is hoped that the application of Codes of Practice for the use of such agents, which have been prepared by the pharmaceutical industry in response to widespread international concern about the development of drug resistance in bacterial pathogens, will now result in a widespread reduction in the incidence of drug-resistant salmonellas in food production animals and humans on an international scale.     

Inhibition of influenza virus RNA (PB2 mRNA) expression by a modified DNA enzyme.

DNA enzymes are RNA-cleaving single stranded DNA molecules. The structure and the catalytic domain of a DNA enzyme were determined by Santro et al. in 1997. In this study, we have designed several types of DNA enzymes (PB2Dz) targeted to the PB2 mRNA translation initiation region of influenza A virus, and examined their cleavage kinetics, nuclease resistance, and a luciferase gene reporter assay. Using a synthetic substrate, these DNA enzymes were shown to have cleavage activity that is dependent on the length of the substrate recognition domain. To confer serum nuclease resistance to the DNA enzymes, we designed a new type of DNA enzyme that has the N3'-P5' phosphoramidate modification (PB2Dz-N) at each terminal. We examined the activity of this DNA enzyme in vivo. The DNA enzymes used in this study inhibited the expression of the PB2-luciferase gene in COS cells. These results suggest that DNA enzymes are potentially useful as gene inactivating agents of influenza A virus.     

Typhoid fever: facing the challenge of resistant strains

The introduction of chloramphenicol in 1948 revolutionised the outcome of typhoid fever but chloramphenicol-resistant strains of Salmonella enterica serotype Typhi were reported just two years later. Resistance followed also the introduction of ampicillin and co-trimoxazole. During the second half of the 1980s, strains resistant to the three first-line antimicrobial agents, chloramphenicol, ampicillin and co-trimoxazole emerged and spread rapidly throughout the Indian subcontinent and South East Asia. During the 1990s when fluoroquinolones had become a first-line treatment for typhoid fever, these multi drug resistant (MDR) strains acquired an additional resistance to nalidixic acid with decreased susceptibilities to ciprofloxacin (CIPDS) (MIC range, 0.125-1 mg/l). Considerable data have now accumulated to suggest that infections due to CIPDS strains may not respond satisfactorily to therapy with ciprofloxacin or ofloxacin. Furthermore, identification of such CIPDS strains in clinical laboratories is not easy without determination of MIC of ciprofloxacin. Recently, several isolates highly resistant to ciprofloxacin or to extended-spectrum cephalosporins of Asian origin have been reported.     

Do we need an intravenous fluoroquinolone?

Intravenous ciprofloxacin, the first parenteral fluoroquinolone available in this country, represents another class of antimicrobial agents from which physicians must choose when treating nosocomial infections. Fluoroquinolones are bactericidal antimicrobial agents that act by inhibiting DNA gyrase. They are active in vitro against most gram-negative bacteria and methicillin-susceptible staphylococci. Activity against anaerobic bacteria and streptococci is poor. The rapid development of bacterial resistance in centers with high quinolone usage is of great concern. Resistance develops most commonly in Pseudomonas aeruginosa and staphylococci. Resistance emerges most often when quinolones are used to treat chronic infections or in patients with poorly drained abscesses, necrotic tissue, or indwelling catheters. Clinical trials have shown ciprofloxacin to be as effective as ceftazidime in the treatment of infections caused by gram-negative bacteria. Although the overall frequency of side effects to fluoroquinolones is low, seizures and allergic reactions have been attributed to their use. Ciprofloxacin inhibits the metabolism of theophylline, and morbidity and death have been reported in patients taking the two drugs concomitantly. Parenteral fluoroquinolones should be reserved for the treatment of gram-negative bacterial infections in patients in whom standard agents cannot be used.     

Characterization of the DNA polymerases induced by a group of herpes simplex virus type I variants selected for growth in the presence of phosphonoformic acid

Five independently derived variants of a herpes simplex virus type I (HSV-1) strain were plaque purified from a virus population passaged in 1 mM phosphonoformic acid (PFA). The DNA polymerase induced by the parent and PFA-resistant viruses were purified and characterized. No differences were observed among the enzymes with respect to their chromatographic properties, specific activities, or polypeptides resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The variant enzymes exhibited levels of PFA resistance which ranged from 15- to 25-fold. Resistance to PFA was always associated with a similar degree of resistance to its congener phosphonoacetic acid, but cross-resistance to beta-phenylphosphonoacetic acid was only seen with two of the five variant enzymes. PFA and pyrophosphate were mutually competitive in PPi exchange reactions, but in DNA synthetic reactions the levels of resistance to PFA and PPi were not equal. The apparent affinities of the enzymes for Mg2+ did parallel their affinities for PFA. Km values of dNTPs were about 2-fold higher than the parent virus enzyme for all of the variant enzymes except one which was 4-fold higher. The processivity of polymerization was apparently unaffected by the enzyme changes related to PFA resistance although one variant enzyme had a lower value. Resistance among the variant enzymes to the triphosphates of 9-(2-hydroxyethoxymethyl)guanine and 2',3'-dideoxyguanosine was directly related to the level of resistance to PFA. The data presented here indicated that (i) PFA resistance may result from several types of active site alterations, since the PFA-resistant enzymes were of three kinetically distinct types. Also, additional enzyme alterations, probably unrelated to PFA resistance, were detected in one enzyme. (ii) PFA and PPi possess some different binding determinants within the active center of herpes simplex virus type I DNA polymerase. (iii) PFA and the triphosphates of 9-(2-hydroxyethoxymethyl)guanine and 2',3'-dideoxyguanosine may have a common ultimate inhibitory mechanism.     

Sulfa and trimethoprim-like drugs – antimetabolites acting as carbonic anhydrase,dihydropteroate synthase and dihydrofolate reductase inhibitors

Abstract

Recent advances in microbial genomics, synthetic organic chemistry and X-ray crystallography provided opportunities to identify novel antibacterial targets for the development of new classes of antibiotics and to design more potent antimicrobial compounds derived from existing antibiotics in clinical use for decades. The antimetabolites, sulfa drugs and trimethoprim (TMP)-like agents, are inhibitors of three families of enzymes. One family belongs to the carbonic anhydrases, which catalyze a simple but physiologically relevant reaction in all life kingdoms, carbon dioxide hydration to bicarbonate and protons. The other two enzyme families are involved in the synthesis of tetrahydrofolate (THF), i.e. dihydropteroate synthase (DHPS) and dihydrofolate reductase. The antibacterial agents belonging to the THF and DHPS inhibitors were developed decades ago and present significant bacterial resistance problems. However, the molecular mechanisms of drug resistance both to sulfa drugs and TMP-like inhibitors were understood in detail only recently, when several X-ray crystal structures of such enzymes in complex with their inhibitors were reported. Here, we revue the state of the art in the field of antibacterials based on inhibitors of these three enzyme families.     

Antibiotic Exposure in a Low-Income Country: Screening Urine Samples for Presence of Antibiotics and Antibiotic Resistance in Coagulase Negative Staphylococcal Contaminants

Development of antimicrobial resistance has been assigned to excess and misuse of antimicrobial agents. Staphylococci are part of the normal flora but are also potential pathogens that have become essentially resistant to many known antibiotics. Resistances in coagulase negative staphylococci (CoNS) are suggested to evolve due to positive selective pressure following antibiotic treatment. This study investigated the presence of the nine most commonly used antimicrobial agents in human urine from outpatients in two hospitals in Ghana in relation to CoNS resistance. Urine and CoNS were sampled (n = 246 and n = 96 respectively) from patients in two hospitals in Ghana. CoNS were identified using Gram staining, coagulase test, and MALDI-TOF/MS, and the antimicrobial susceptibility to 12 commonly used antimicrobials was determined by disk diffusion. Moreover an analytical method was developed for the determination of the nine most commonly used antimicrobial agents in Ghana by using solid-phase extraction in combination with HPLC-MS/MS using electron spray ionization. The highest frequency of resistance to CoNS was observed for penicillin V (98%), trimethoprim (67%), and tetracycline (63%). S. haemolyticus was the most common isolate (75%), followed by S. epidermidis (13%) and S. hominis (6%). S. haemolyticus was also the species displaying the highest resistance prevalence (82%). 69% of the isolated CoNS were multiple drug resistant (≧4 antibiotics) and 45% of the CoNS were methicillin resistant. Antimicrobial agents were detected in 64% of the analysed urine samples (n = 121) where the most frequently detected antimicrobials were ciprofloxacin (30%), trimethoprim (27%), and metronidazole (17%). The major findings of this study was that the prevalence of detected antimicrobials in urine was more frequent than the use reported by the patients and the prevalence of resistant S. haemolyticus was more frequent than other resistant CoNS species when antimicrobial agents were detected in the urine.     

Antimicrobial enzymes: An emerging strategy to fight microbes and microbial biofilms

With the increasing prevalence of antibiotic resistance, antimicrobial enzymes aimed at the disruption of bacterial cellular machinery and biofilm formation are under intense investigation. Several enzyme-based products have already been commercialized for application in the healthcare, food and biomedical industries. Successful removal of complex biofilms requires the use of multi-enzyme formulations that contain enzymes capable of degrading microbial DNA, polysaccharides, proteins and quorum-sensing molecules. The inclusion of anti-quorum sensing enzymes prevents biofilm reformation. The development of effective complex enzyme formulations is urgently needed to deal with the problems associated with biofilm formation in manufacturing, environmental protection and healthcare settings. Nevertheless, advances in synthetic biology, enzyme engineering and whole DNA-Sequencing technologies show great potential to facilitate the development of more effective antimicrobial and anti-biofilm enzymes.     

The liver/erythrocyte pyruvate kinase gene complex [Pk-1] in the mouse: regulatory gene mutations.

Nine enzyme activity variants and one charge variant of liver/erythrocyte pyruvate kinase have been found amongst laboratory and wild mice. Four of the enzyme activity variants were previously reported to be caused by allelic differences in the structural gene, Pk-1s. Analysis of two putative regulatory gene mutations is now reported, both of which map at, or close to, the structural gene on chromosome 3. One of these mutations, in the inbred strain SWR, is tissue specific, affecting enzyme concentration in the liver but not the erythrocyte the other, which arose in a mutation experiment, doubles the enzyme concentration in both tissues. The organization and the nomenclature in the [Pk-1] gene complex are discussed and are compared with the organization of other comprehensively analysed gene complexes in the mouse.     

G6PD lozere and trinacria-like

Summary Two new G6PD variants have been found in red blood cells of the members of a French family originating from Lozere. The father is hemizygous for an electrophoretically fast variant with mild enzyme deficiency (50–60% of normal). The abnormal paternal G6PD gene is segregating in his daughter who is double heterozygous for maternal and paternal variants. This mutant enzyme, different from previously described variants is designated as Gd Lozère. The mother is heterozygous for another G6PD variant. Two sons are hemizygous for this latter mutant enzyme characterized by a moderate deficiency (25–30% of normal) and slower electrophoretic mobility with some slightly altered kinetic properties. This G6PD has been identified as Gd Trinacria like.These two abnormal enzymes are not associated with any hemolytic problem. Case reported is the first showing the segregation of two new mutant enzymes, distinct from common G6PD variants, among the members of the same family.     

Prevalence of Primary Fluoroquinolone Resistance Among Clinical Isolates of Helicobacter pylori at a University Hospital in Southern Taiwan

Background:  Fluoroquinolone-containing therapy is effective in eradicating Helicobacter pylori . However, the resistance rate of H. pylori to fluoroquinolones in Taiwan has not yet been reported. In this study, we aimed to investigate the susceptibility to antibiotics commonly used in eradication schedules and fluoroquinolones in H. pylori .
Methods:  A total of 210 clinical isolates of H. pylori were collected from April 1998 to September 2007 from patients in southern Taiwan. The in vitro activities of six antimicrobial agents were determined by the agar dilution method and Etest. The mutations in quinolone resistance-determining regions of gyrA and gyrB were investigated by direct sequencing.
Results:  Overall, 5.7% of the isolates were resistant to ciprofloxacin and levofloxacin. The resistance rate to amoxicillin, clarithromycin, metronidazole, and tetracycline was 1.0% (two of 210), 9.5% (20 of 210), 27.6% (58 of 210), and 0.5% (one of 210), respectively. The resistance rate to either ciprofloxacin or to levofloxacin increased from 2.8% (1998–2003) to 11.8% (2004–2007). The mutations in gyrA at N87 or D91 had an impact on primary fluoroquinolone resistance in H. pylori . Garenoxacin, but not moxifloxacin, had a good in vitro inhibitory effect against ciprofloxacin/levofloxacin-resistant strains compared with objective minimal inhibitory concentration values.
Conclusions:  Drug resistance to ciprofloxacin and levofloxacin in H. pylori collected from 2004 to 2007 increased significantly compared with resistance level observed during 1998–2003. The continuous surveillance of quinolone resistance among H. pylori is important in this area.     

一株洛菲不动杆菌对碳青霉烯类抗生素耐药机制的研究

目的研究洛菲不动杆菌对亚胺培南、美洛培南耐药的分子机制。方法K-B纸片琼脂扩散法检测洛菲不动杆菌B69对头孢他啶、头孢曲松、环丙沙星、阿米卡星的耐药性,琼脂对倍稀释法检测B69对亚胺培南、美洛培南的最低抑菌浓度;PCR扩增OXA、IMP、VIM型碳青霉烯酶基因,测序确定耐药基因型别;粗提酶水解亚胺培南纸片试验检测酶活性。结果洛菲不动杆菌B69具有多重耐药性;PCR扩增IMP基因阳性,经测序为IMP-8;粗提酶水解亚胺培南。结论产IMP-8型金属β-内酰胺酶是洛菲不动杆菌B69对碳青霉烯耐药的重要机制。     

A novel NDP-6-deoxyhexosyl-4-ulose reductase in the pathway for the synthesis of thymidine diphosphate-D-fucose.

The serotype-specific polysaccharide antigen of Actinobacillus actinomycetemcomitans Y4 (serotype b) consists of D-fucose and L-rhamnose. Thymidine diphosphate (dTDP)-D-fucose is the activated nucleotide sugar form of D-fucose, which has been identified as a constituent of structural polysaccharides in only a few bacteria. In this paper, we show that three dTDP-D-fucose synthetic enzymes are encoded by genes in the gene cluster responsible for the synthesis of serotype b-specific polysaccharide in A. actinomycetemcomitans. The first and second steps of the dTDP-D-fucose synthetic pathway are catalyzed by D-glucose-1-phosphate thymidylyltransferase and dTDP-D-glucose 4,6-dehydratase, which are encoded by rmlA and rmlB in the gene cluster, respectively. These two reactions are common to the well studied dTDP-L-rhamnose synthetic pathway. However, the enzyme catalyzing the last step of the dTDP-D-fucose synthetic pathway has never been reported. We identified the fcd gene encoding a dTDP-4-keto-6-deoxy-D-glucose reductase. After purifying the three enzymes, their enzymatic activities were analyzed by reversed-phase high performance liquid chromatography. In addition, nuclear magnetic resonance analysis and gas-liquid chromatography analysis proved that the fcd gene product converts dTDP-4-keto-6-deoxy-D-glucose to dTDP-D-fucose. Moreover, kinetic analysis of the enzyme indicated that the Km values for dTDP-4-keto-6-deoxy-D-glucose and NADPH are 97.3 and 28.7 microM, respectively, and that the enzyme follows the sequential mechanism. This paper is the first report on the dTDP-D-fucose synthetic pathway and dTDP-4-keto-6-deoxy-D-glucose reductase.     

Alu-Alu recombination results in a duplication of seven exons in the lysyl hydroxylase gene in a patient with the type VI variant of Ehlers-Danlos syndrome.

The type VI variant of the Ehlers-Danlos syndrome (EDS) is a recessively inherited connective-tissue disorder. The characteristic features of the variant are muscular hypotonia, kyphoscoliosis, ocular manifestations, joint hypermobility, skin fragility and hyperextensibility, and other signs of connective-tissue involvement. The biochemical defect in most but not all patients is a deficiency in lysyl hydroxylase activity. Lysyl hydroxylase is an enzyme that catalyzes the formation of hydroxylysine in collagens and other proteins with collagen-like amino acid sequences. We have recently reported an apparently homozygous large-duplication rearrangement in the gene for lysyl hydroxylase, leading to the type VI variant of EDS in two siblings. We now report an identical, apparently homozygous large duplication in an unrelated 49-year-old female originally analyzed by Sussman et al. Our simple-sequence-repeat-polymorphism analysis does not support uniparental isodisomy inheritance for either of the two duplications. Furthermore, we indicate in this study that the duplication in the lysyl hydroxylase gene is caused by an Alu-Alu recombination in both families. Cloning of the junction fragment of the duplication has allowed synthesis of appropriate primers for rapid screening for this rearrangement in other families with the type VI variant of EDS.     

Emerging cystic fibrosis pathogens: incidence and antimicrobial resistance

We examined the frequency of isolation and the antimicrobial resistance of Burkholderia cepacia complex, Stenotrophomonas maltophilia and Achromobacter xylosoxidans in cystic fibrosis patients from 2000 to 2004. Strains susceptibility to tobramycin, piperacillin/tazobactam, imipenem, gentamicin, ciprofloxacin and ceftazidime was determined by disc diffusion assay. B. cepacia complex showed a very high resistance also to ciprofloxacin reaching 100% in 2004. S. maltophilia and A. xvylosoxidans showed high rates of antimicrobial resistance both aminoglycoside and ciprofloxacin. It is very important to monitor the percentage of isolation of these species over time to verify strains resistance to antibiotics and also to test new combinations of antimicrobial agents.     

Synthesis of the antibiotic cortalcerone from D-glucose using pyranose 2-oxidase and a novel fungal enzyme, aldos-2-ulose dehydratase.

Using two enzymes purified from the white-rot fungus, Polyporus obtusus, 5% solutions of D-glucose have been quantitatively converted in vitro into D-arabino-hexos-2-ulose (D-glucosone) and subsequently into a compound having antimicrobial activity. The antibiotic has been shown by nuclear magnetic resonance and mass spectroscopy to be chemically identical to a previously described fungal metabolite known as cortalcerone. Based on kinetic analysis of the synthetic process, a pathway for the biosynthesis of cortalcerone is proposed, involving both chemical rearrangement and enzymically catalyzed steps. Two enzymes, pyranose 2-oxidase and a previously uncharacterized D-arabino-hexos-2-ulose-utilizing enzyme, may be sufficient for the biosynthesis of cortalcerone from glucose in vivo. The D-arabino-hexos-2-ulose-utilizing enzyme dehydrates certain aldosuloses and has been named aldos-2-ulose dehydratase. The enzyme, which appears to be a dimer of 95-kDa subunits, has been purified 450-fold. Additional properties of aldos-2-ulose dehydratase are described, including its apparent ability to catalyze two different steps in the proposed biosynthetic pathway for cortalcerone.     

A novel locus conferring fluoroquinolone resistance in Staphylococcus aureus.

Fluoroquinolones such as ciprofloxacin and ofloxacin are potent antimicrobial agents that antagonize the A subunit of DNA gyrase. We selected and mapped a novel fluoroquinolone resistance gene on the Staphylococcus aureus chromosome. Resistant mutants were selected with ciprofloxacin or ofloxacin and were uniformly localized to the A fragment of chromosomal DNA digested with SmaI and arrayed by pulsed-field gel electrophoresis. Several mutants (cfxB, ofxC) were genetically mapped between the thr and trp loci in the A fragment. A majority of A fragment fluoroquinolone resistance mutations were associated with reduced susceptibility to novobiocin, an antagonist of the B subunit of DNA gyrase. Two genes previously associated with fluoroquinolone resistance, the gyrA gene of DNA gyrase and the norA gene (associated with decreased drug accumulation), were localized to the G and D fragments, respectively. Thus, the fluoroquinolone resistance mutations in the A fragment are distinct from previously identified fluoroquinolone resistance mutations in gyrA and norA. Whether mutations in the A fragment after a second topoisomerase or another gene controlling supercoiling or affect drug permeation is unknown.     

Increased sensitivity to quinolone antibacterials can be engineered in human topoisomerase IIalpha by selective mutagenesis

A potential region of drug-DNA interaction in the A subunit of DNA gyrase has previously been identified from crystallographic studies. The local amino acid sequence has been compared with similar regions in yeast topoisomerase II and human topoisomerase IIalpha. Three non- conserved, potentially solvent-accessible residues at positions 762, 763 and 766 in human topoisomerase IIalpha lie between well-conserved regions. The corresponding residues in GyrA (83, 84 and 87) have a high frequency of mutation in quinolone-resistant bacteria. Mutations in human topoisomerase IIalpha have been generated in an attempt to engineer ciprofloxacin sensitivity into this enzyme: M762S, S763A and M766D (each mutated to the identical amino acid present in gyrase), along with an M762S/S763A double mutant and a triple mutant. These enzymes were introduced into a temperature-sensitive yeast strain, deficient in topoisomerase II, for in vivo studies, and were overproduced for in vitro studies. The M766D mutation renders the enzyme incapable of supporting the temperature-sensitive strain at a non-permissive temperature. However, both M766D and the triple mutant enzymes can be overproduced and are fully active in vitro. The double mutant was impaired in its ability to cleave DNA and had reduced catalytic activity. The triple mutation confers a three-fold increase in sensitivity to ciprofloxacin in vitro and similar sensitivities to a range of other quinolones. The activity of the quinolone CP-115,953, a bacterial and eukaryotic topoisomerase II poison, was unaffected by any of these mutations. Mutations in this region were found to increase the sensitivity of the enzyme to the DNA intercalating anti-tumour agents m-AMSA and ellipticine, but confer resistance to the non-intercalating agents etoposide, teniposide and merbarone, an effect that was maximal in the triple mutant. We have therefore shown the importance of this region in determining the sensitivity of topoisomerase II to drugs and have engineered increased sensitivity to quinolones.     

<Superscript>1</Superscript>H, <Superscript>13</Superscript>C and <Superscript>15</Superscript>N resonance assignments of the new lysostaphin family endopeptidase catalytic domain from <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

Lysostaphin family endopeptidases, produced by Staphylococcus genus, are zinc-dependent enzymes that cleave pentaglycine bridges of cell wall peptidoglycan. They act as autolysins to maintain cell wall metabolism or as toxins and weapons against competing strains. Consequently, these enzymes are compelling targets for new drugs as well as are potential antimicrobial agents themselves against Staphylococcus pathogens, which depend on cell wall to retain their immunity against antibiotics. The rapid spread of methicillin and vancomycin-resistant Staphylococcus aureus strains draws demand for new therapeutic approaches. S. aureus gene sa0205 was found to be implicated in resistance to vancomycin and synthesis of the bacteria cell wall. The gene encodes for a catalytic domain of a lysostaphin-type endopeptidase. We aim to obtain the structure of the Sa0205 catalytic domain, the first solution structure of the catalytic domain of the lysostaphin family enzymes. In addition, we are to investigate the apparent binding of the second zinc ion, which has not been previously reported for the enzyme group. Herein, we present the backbone and side chain resonance assignments of Sa0205 endopeptidase catalytic domain in its one and two zinc-bound forms.     

Demystification of Chester porphyria: a nonsense mutation in the Porphobilinogen Deaminase gene

The porphyrias arise from predominantly inherited catalytic deficiencies of specific enzymes in heme biosynthesis. All genes encoding these enzymes have been cloned and several mutations underlying the different types of porphyrias have been reported. Traditionally, the diagnosis of porphyria is made on the basis of clinical symptoms, characteristic biochemical findings, and specific enzyme assays. In some cases however, these diagnostic tools reveal overlapping findings, indicating the existence of dual porphyrias with two enzymes of heme biosynthesis being deficient simultaneously. Recently, it was reported that the so-called Chester porphyria shows features of both variegate porphyria and acute intermittent porphyria. Linkage analysis revealed a novel chromosomal locus on chromosome 11 for the underlying genetic defect in this disease, suggesting that a gene that does not encode one of the enzymes of heme biosynthesis might be involved in the pathogenesis of the porphyrias. After excluding candidate genes within the linkage interval, we identified a nonsense mutation in the porphobilinogen deaminase gene on chromosome 11q23.3, which harbors the mutations causing acute intermittent porphyria, as the underlying genetic defect in Chester porphyria. However, we could not detect a mutation in the coding or the promotor region of the protoporphyrinogen oxidase gene that is mutated in variegate porphyria. Our results indicate that Chester porphyria is neither a dual porphyria, nor a separate type of porphyria, but rather a variant of acute intermittent porphyria. Further, our findings largely exclude the possibility that a hitherto unknown gene is involved in the pathogenesis of the porphyrias.