Partial sequence analysis of a tet(O) plasmid from a multiple-drug-resistant clinical isolate of Campylobacter jejuni revealed 10 genes or pseudogenes encoding different aminoglycoside inactivating enzymes, transposaselike genes, and multiple unknown genes from a variety of pathogenic and commensal bacteria. The plasmid could be mobilized by a P incompatibility group plasmid into Escherichia coli, where it apparently integrated into the chromosome and expressed high-level resistance to multiple aminoglycoside antibiotics. This work provides new information about both the nature of drug resistance in C. jejuni and the ability of C. jejuni to exchange genes with other bacterial species.Campylobacter jejuni is among the most frequent causes of diarrheal disease worldwide (2, 23), and the incidence is particularly high in Thailand, where both antimicrobial resistance and multidrug resistance are increasing problems (6). Although the levels of resistance to the macrolides erythromycin and azithromycin have remained low, the incidence of resistance to the quinolones has risen to 96% in a 1998 study of U.S. military personnel (22). Plasmid-mediated resistance to tetracycline, chloramphenicol, and kanamycin has been well documented in C. jejuni (25,31,29), but no other plasmidencoded resistance genes have been reported, other than a very recent report of streptomycin and streptothricin resistance (12). Recently, comparison of the sequences of transferable tet(O) plasmids from a C. jejuni strain and a C. coli strain isolated on different continents more than 20 years apart revealed high conservation (4, 5). Surveys of clinical isolates of C. jejuni in Thailand in our laboratories have revealed a high incidence of both multiple drug resistance and plasmids (unpublished data). Given these observations, we hypothesized that at least some of these antibiotic resistances in recent Thai isolates were plasmid encoded. Here we describe the partial genetic structure of a multiple-drug-resistant plasmid from a Thai clinical isolate of C. jejuni. The plasmid is related to other tet(O) plasmids but is novel in that it also contains multiple aminoglycoside-inactivating enzymes and transposon-like sequences from a variety of bacteria that have not previously been reported in C. jejuni.(This work was done in partial fulfillment of the requirements for a Ph.D. degree from Mahidol University by W.N.) MATERIALS AND METHODSBacterial strains and plasmids. C. jejuni strain CG8245 was isolated from a diarrhea case of a U.S. soldier deployed to Thailand during a military exercise in 1999. Escherichia coli DH5␣ was used as the host for cloning experiments, and pBluescript (Stratagene, La Jolla, CA) was used as the cloning vector. For conjugation experiments with another C. jejuni strain, a mutant of C. jejuni NCTC 11168 that had been insertionally inactivated with a chloramphenicol resistance (cat) cassette (32) in the Cj1316c gene was used as the recipient (P. Guerry, unpublished data). For conjugation experiments with E. coli, E. coli C600(RK...

Serology to detect antibodies to Helicobacter pylori is not frequently used as a diagnostic tool in developing countries. When compared to a commercial ELISA, an ELISA constructed and validated in Thailand had a higher sensitivity (98% vs. 85%), specificity (76% vs. 66%), and negative predictive value (97% vs. 76%) for the detection of H. pylori infection among 104 patients with dyspepsia evaluated by endoscopy. The positive predictive value was 88% for both tests. Serum antibody levels fell significantly 5-8 months after eradication of infection in 8 Thai patients (P = .009). By 8 years of age, > 50% of Thai persons living in urban and rural locations were seropositive. The low negative predictive value of the commercial ELISA limits the usefulness of this assay as a diagnostic tool in Thailand and suggests a need to reevaluate H. pylori serologic tests when used in populations living in developing countries.

N-Methylhydantoin amidohydrolase, an ATP-dependent amidohydrolase involved in microbial degradation of creatinine, was purified 70-fold to homogeneity, with a 62% overall recovery, and was crystallized from Pseudomonas putida 77. The enzyme has a relative molecular mass of 300000. It is a tetramer of two identical small subunits (Mr 70000) and two identical large subunits (M, 80000). The enzyme requires ATP for the amidohydrolysis of N-methylhydantoin and vice versa. Mg2+, Mn2+ or Co2+, and K', NK' , Rb+ or Cs+, were absolutely required concomitantly for the enzyme activity as divalent and monovalent cations, respectively. The K , and V,,, values for N-methylhydantoin were 32 pM and 9.0 pmol . min-' . mg protein-'. The hydrolysis of amide compounds and coupled hydrolysis of ATP were observed with hydantoin, ~~-5-methylhydantoin, glutarimide and succimide in addition to N-methylhydantoin. 2-Pyrrolidone, 2-oxazolidone, 8-valerolactam, 2,4-thiazolidinedione, 2-imidazolidone, 0-5-oxoproline methyl ester, DL-5-oxoproline methyl ester, and naturally occurring pyrimidine compounds, i.e. dihydrouracil, dihydrothymine, uracil, and thymine, effectively stimulated ATP hydrolysis by the enzyme without undergoing detectable self-hydrolysis.Keywords. N-Methylhydantoin amidohydrolase ; ATP-dependent amidohydrolase ; creatinine metabolism ; Pseudomonas putida.An ATP-dependent amidohydrolase, N-methylhydantoin amidohydrolase, which catalyzes the reaction presented in Scheme 1, was first found in Pseudomonasputidu 77 [I, 21. The enzyme catalyzes the second step in the degradation route from creatinine to glycine, via N-methylhydantoin, N-carbamoylsarcosine, and sarcosine as successive intermediates [l-91. NMethylhydantoin amidohydrolase resembles dihydropyrimidinase, which is widely distributed from microorganisms to mammals [lo-161, in that both enzymes hydrolyze hydantoin compounds. In contrast to N-methylhydantoin amidohydrolase, however, dihydropyrimidinase does not require ATP for the hydrolysis of its substrates.Enzymes that catalyze this type of ATP-dependent amidohydrolysis reaction include 5-oxoprolinase [17-211, urea amidolyase [22, 231, L-isomer-specific hydantoinase [24] and non-stereospecific hydantoinase [25], in addition to N-methylhydantoin amidohydrolase. Through extensive studies by Meister and coworkers, it has been shown that rat kidney 5-oxoprolinase is involved in the metabolism of glutathione [26], and that phosphorylation of the substrate is involved in the reaction mechanism 127-291. The rat kidney 5-oxoprolinase catalyzes the uncoupled hydrolysis of ATP in the presence of several structural analogs of 5-oxo-~-proline, such as L-2-imidazolidone-4-carboxylate, dihydroorotate, etc. [18, 21, 301, and is considered to be composed of two subunits identical in molecular mass [21]. The bacterial 5-oxoproIinase, however, easily separates into two components on purification and the overall reaction only proceeds after remixing of the components [31]. Urea amidolyase from Saccharomyces cerevisiae catalyzes success...

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