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PubMed2026

Molecular surveillance of foodborne bacterial pathogens and resistome in food products from Hong Kong.

Foodborne infections pose an increasing public health challenge worldwide. The problem has been aggravated by the dissemination of antimicrobial resistance genes among zoonotic pathogens, which results in a sharp increase in antibiotic resistance rate recorded among the major foodborne pathogens. To obtain an overview of the extent to which food products purchased in the markets in Hong Kong were contaminated by foodborne pathogens, we collected 95 raw meat samples from wet markets and isolated 236 bacterial strains of various species, with Escherichia coli being the most dominant species (131 strains). Contamination of food products by multiple foodborne pathogens was commonly observed. These include both Gram-positive and Gram-negative bacteria that exhibit various levels of resistance, with some possessing multiple clinically important antibiotic resistance genes. Seventeen bacterial strains of various species isolated from three food samples were comprehensively analysed by the Oxford Nanopore R10.4 technology. Novel conjugative plasmids carrying antimicrobial resistance gene-bearing mobile genetic elements were commonly detectable in the test strains. Some of the plasmids were shown to have originated from other environmental sources or other bacterial species, indicating that raw foods in the local market may serve as a reservoir of resistance-encoding genetic elements from which such elements are disseminated to various microbial pathogens. These findings suggest a need to perform periodic but comprehensive surveillance of multidrug-resistant bacterial pathogens and the major antimicrobial resistance genes in common food products, so as to disrupt the transmission routes of such organisms and the resistance-encoding genetic elements that they harbour.

Hong Kong↗
PubMed1987

Characterization and mobilization of nonconjugative plasmids encoding resistance to streptomycin and sulfanilamide.

Most of nonconjugative streptomycin (Sm)- and sulfanilamide (Su)- resistance of clinical isolates belonging to various species of Enterobacteriaceae and Pseudomonas were encoded by an Inc Q plasmid, molecular size of which was 5.5 Md. The SmSu plasmids were efficiently mobilized by Inc P plasmids between E. coli strains. Inc I group and Inc F group plasmids could mobilize the Inc Q plasmids at lower efficiencies. The Inc Q plasmid was also mobilized to various species of Enterobacteriaceae at high frequencies without accompanying the conjugative Inc P plasmid; as a result, most of the SmSu-resistant transconjugants were nontransferable. The above results may explain the wide distribution of nonconjugative SmSu strains among clinical isolates.

Conjugation, Genetic↗
PubMed1989

Characterization of Rhizobium phaseoli Sym plasmid regions involved in nodule morphogenesis and host-range specificity.

Two nodulation regions from the symbiotic plasmid (pSym) of Rhizobium phaseoli CE-3 were identified. The two regions were contained in overlapping cosmids pSM927 and pSM991. These cosmids, in a R. phaseoli pSym-cured strain background, induced ineffective nodules on Phaseolus vulgaris roots. Transconjugants of Rhizobium meliloti harbouring pSM991 induced nodule-like structures on bean roots, suggesting that this cosmid contains host-range determinants. Analysis of deletions and insertional mutations in the sequences of pSM991 indicated that the genes responsible for the induction and development of nodules in P. vulgaris are organized in two regions 20 kb apart. One region, located in a 6.8 kb EcoRI fragment, includes the common nodABC genes. The other region, located in a 3.5 kb EcoRI fragment, contains information required for host-range determination.

Blotting, Southern↗
PubMed1992

DNA sequence determination and functional characterization of the OCT-plasmid-encoded alkJKL genes of Pseudomonas oleovorans.

The alkBFGHJKL and alkST operons encode enzymes that allow Pseudomonas putida (oleovorans) to metabolize alkanes. In this paper we report the nucleotide sequence of a 4592 bp region of the alkBFGHJKL operon encoding the AlkJ, AlkK and AlkL polypeptides. The alkJ gene encodes a protein of 59 kilodaltons. The predicted amino acid sequence shows significant homology with four flavin proteins: choline dehydrogenase, a glucose dehydrogenase and two oxidases. AlkJ is membrane-bound and converts aliphatic medium-chain-length alcohols into aldehydes. The properties of AlkJ suggest that it is linked to the electron transfer chain. AlkJ is necessary for growth on alkanes only in P. putida alcohol dehydrogenase (AlcA) mutants. AlkK is homologous to a range of proteins which act by an ATP-dependent covalent binding of AMP to their substrate. This list includes the acetate, coumarate and long-chain fatty acid CoA ligases. The alkK gene complements a fadD mutation in Escherichia coli, which shows that it indeed encodes an acyl-CoA synthetase. AlkK is a 60 kilodalton protein located in the cytoplasm. AlkL is homologous to OmpW, a Vibrio cholerae outer membrane protein of unknown function, and a hypothetical polypeptide encoded by ytt4 in E. coli. AlkL, OmpW and Ytt4 all have a signal peptide and end with a sequence characteristic of outer membrane proteins. The alkL gene product was found in the outer membrane of E. coli W3110 containing the alk-genes. The alkL gene can be deleted without a clear effect on growth rate. Its function remains unknown. The G+C content of the alkJKL genes is 45%, identical to that of the alkBFGH genes, and significantly lower than the G+C content of the OCT-plasmid and the P. putida chromosome.

Alcohol Dehydrogenase↗
PubMed1990

Characterization and physical mapping of plasmids of black-pigmented Bacteroides.

Eight species of black-pigmented Bacteroides (BPB) were examined for plasmid content. Three classes of plasmids with molecular sizes of 16.0 kilobase (kb), 5.4 kb and 5.0 kb were found in 7 of 125 Bacteroides intermedius strains. One strain each of Porphyromonas (former Bacteroides) asaccharolyticus and of Bacteroides levii were also shown to have plasmids. However, no plasmids were detected in 90 strains of Porphyromonas (Bacteroides) gingivalis. The plasmids were purified and digested with restriction endonucleases for restriction mapping. The maps obtained in this study have given us information on the scheme of constructing recombinants which may be usable as shuttle vectors, by the recombination of BPB and Escherichia coli plasmids.

Bacteroides↗
PubMed1996

Characterization of a 4.2-kb plasmid isolated from periodontopathic spirochetes.

Oral anaerobic treponemes are assoicated with active periodontal disease and may comprise up to 57% of the microbiota in periodontal pockets. Four treponeme strains (designated U2a, U2b, U9b, and U9c) isolated from clincial cases were found to harbor a new 4.2-kb plasmid when plasmid DNA was extracted and purified employing the Qiagen Plasmid Kit. This plasmid differs from the smaller plasmids (2.0-, 2.6-, and 2.7-Kb) reported previously by others in Treponema denticola. The newly discovered 4.2-kb plasmid was found to be the same in all four treponeme strains by restriction endonuclease analysis. It is a circular plasmid since restriction with PstI, Pvu II, Sma I, Xma I, Ava 1 or Bam HI produced a single band of the same size. Bacterial strain U2b was shown to be Treponema socranskii and U9c to be T. denticola. The plasmid is designated "pTS1". The presence of the same plasmid in different species of the treponemes isolated from the same patient suggests the possibility of a naturally occurring genetic transfer system within the oral spirochetes or their ability to take up and maintain mobilizable plasmids.

DNA, Bacterial↗
PubMed1976

Characterization and replication control of plasmids from Enterobacter cloacae DF 13.

It has been shown previously that Enterobacter clocacae DF13 harbours at least five different size classes of plasmids. A 45 x 10(6)-Mr self-transmissible R factor determining resistance against tetracyclin, sulfanilamide, streptomycin and chloramphenicol, a 6.0 x 10(6)-Mr bacteriocinogenic factor without sex factors activity and cryptic plasmids in the size classes of Mr 1.3 x10(6), 2.8 x 10(6) and 8.0 x 10(6) respectively. The present work deals with the determination of the homogeneity and molecular relationship of 1.3 x 10(6)-Mr (mini) and 2.8 x 10(6)-Mr (midi) cryptic plasmids and the 6.0 x 10(6)-Mr (maxi) bacteriocinogenic factor, their kinetics of replication and their replication control in response to inhibition of protein synthesis.

Base Sequence↗
PubMed1994

Assembly of functional skeletal muscle troponin complex in Escherichia coli.

The production of multi-subunit proteins of eukaryotic origin in Escherichia coli usually relies on the different subunits being expressed individually and the protein being reassembled in vitro. Here we describe the construction and characterization of plasmids capable of coexpressing the three subunits of chicken skeletal muscle troponin complex in E. coli. We demonstrate that the troponin subunits assembled in the cytoplasm of E. coli cell are fully functional. The troponin complex was purified to homogeneity in high yields. When reconstituted into actin filaments, the complex assembled in vivo was capable of regulating the myosin ATPase with a calcium dependence that was identical to the complex reconstituted in vitro. These results demonstrate that the coexpression of the subunits of a protein complex can prevent the accumulation of denatured proteins in inclusion granules.

Animals↗
PubMed1999

Molecular methods for the analysis of Clostridium perfringens relevant to food hygiene.

Clostridium perfringens continues to be a common cause of food-borne disease. It produces an enterotoxin (CPE) which is released upon lysis of the vegetative cell during sporulation in the intestinal tract. Catering premises with insufficient cooling and reheating devices often seem to be the cause of outbreaks of C. perfringens food poisoning. Typing of C. perfringens is of great importance for investigating sources of food poisoning cases and for studying the epidemiology of this microorganism. This report describes the examination of 155 C. perfringens isolates by molecular methods. Isolates were taken from 10 food poisoning outbreaks and cases (n = 34, food and fecal isolates) and from meat and fish pastes (n = 121). Isolates were characterized by plasmid profiling, ribotyping, and/or macrorestriction analysis by pulsed-field gel electrophoresis (PFGE). Results show that all three methods are suitable for classifying C. perfringens isolates below the species level. Ribopatterns and PFGE patterns can be interpreted more easily than plasmid profiling results and can be recommended for contamination studies and epidemiologic investigation of food poisonings associated with C. perfringens.

Animals↗
PubMed1999

Genetic manipulation of Kluyveromyces lactis linear DNA plasmids: gene targeting and plasmid shuffles.

Genetic manipulation of yeast linear DNA plasmids, particularly of k1 and k2 from the non-conventional dairy yeast Kluyveromyces lactis, has been advanced by the recent establishment of DNA transformation-mediated one-step gene disruption and allele replacement techniques. These methods provide the basis for a strategy for the functional analysis of plasmid genes and DNA elements. By use of double selection regimens, these single-gene procedures have been extended to effect disruption of individual genes on plasmid k2 and transplacement of a functional copy onto plasmid k1, resulting in the production of yeast strains with an altered plasmid composition. This cytoplasmic gene shuffle system facilitates the introduction of specifically modified alleles into k1 or k2 in order to study the function, expression (from UCS promoters) and regulation of cytoplasmic linear plasmid genes. Additionally, identification, characterization and localization of plasmid gene products of interest are made possible by shuffling GFP-, epitope- or affinity purification-tagged alleles between k2 and k1. The gene shuffle approach can also be used for vector development and heterologous protein expression in order to exploit the biotechnical potential of the K. lactis k1/k2 system in yeast cell factory research.

Biological Evolution↗
PubMed2000

Mutations at arg486 and glu571 in human topoisomerase IIalpha confer resistance to amsacrine: relevance for antitumor drug resistance in human cells.

Human topoisomerase II, a nuclear protein involved in chromosome segregation, is the target of amsacrine and other clinically important anticancer drugs. The enzyme is expressed as alpha and beta isoforms whose mutation/down-regulation has been implicated in drug resistance. To understand the role of target mutations in cellular drug resistance, we have used yeast to select and characterize plasmid-borne human topoisomerase IIalpha mutants resistant to amsacrine. Single point changes of Glu571 to Lys (E571K) or Arg486 to Lys (R486K) in the conserved PLRGK motif, both of which reside in the GyrB homology domain of human topoisomerase IIalpha, were frequently selected and could be shown in vivo to confer >25-fold and >100-fold resistance, respectively, to amsacrine and approximately 3-fold cross-resistance to etoposide. Highly purified E571K and R486K human topoisomerase IIalpha proteins required 100-fold higher levels of amsacrine to induce DNA cleavage similar to that of wild-type protein, consistent with a resistance mechanism involving reduced cleavable complex formation. Our functional studies of the R486K mutation, previously identified in two amsacrine-resistant human cell lines and in human biopsy material, establish unequivocally that it confers resistance, and suggest mechanisms for its phenotypic expression in vivo. These results differ significantly from previous work using yeast topoisomerase II as a model system: introduction of the equivalent mutation to R486K (R476K) into the yeast enzyme did not give amsacrine resistance. We conclude that species-specific differences in topoisomerase II enzymes can affect the drug resistance phenotype of particular mutations and highlight the need to study the relevant human homolog.

Amsacrine↗
PubMed1977

Characterization of an R-plasmid associated with ampicillin resistance in Shigella dysenteriae type 1 isolated from epidemics.

Ampicillin-resistant strains of Shigella dysenteriae type 1 isolated in epidemics in Mexico, Central America, and Bangla Desh were examined for the presence of plasmid deoxyribonucleic acid (DNA) by gel electrophoresis. All strains contained a heterogeneous population of plasmids. Transfer experiments to Escherichia coli K-12 indicated that the ampicillin resistance determinant (Ap(r)) was located on a 5.5-megadalton (Mdal) plasmid identical in all Shiga strains examined, as judged by DNA hybridization and by its molecular properties. This 5.5-Mdal plasmid contained the ampicillin transposon (TnA) sequences. There was not a high degree of homology between the Shiga Ap(r) plasmid DNA and DNA obtained from Ap(r)Salmonella typhi strains isolated from typhoid epidemics in Mexico, previous to the dysentery outbreaks. Although low, the degree of reassociation observed indicated that probably part of the TnA sequence was present in S. typhi DNA. The DNA hybridization experiments showed, in addition, that there was a high degree of homology among Ap(r) plasmids isolated from different enterobacteria, and this identity was confirmed by restriction endonuclease activity. These results together with their similarities in molecular and replicative properties indicate that the Ap(r) plasmids, as was suggested for the Sm(r) Su(r) plasmids, possibly evolved once and then epidemiologically spread in the Enterobacteriaceae.

Ampicillin↗
PubMed1978

Genetic and molecular characterization of an Escherichia coli plasmid coding for hydrogen sulfide production and drug resistance.

An H(2)S-producing variant of Escherichia coli (strain 142) isolated from a urinary tract infection was found to be resistant to high levels of tetracycline, ampicillin, streptomycin, and sulfonamide. The H(2)S trait segregated spontaneously at a frequency of 2.5 x 10(-3). No segregation was observed for the drug resistance determinants. Neither ethidium bromide nor acridine orange affected the rate of segregation of the drug resistance determinants or the trait for H(2)S production. Antibiotic resistance and hydrogen sulfide production were conjugally transferred to E. coli K-12 recipients at a frequency of approximately 10(-5) per donor cell. Antibiotic resistance and hydrogen sulfide production were also transduced as a single unit with phage P1L4. Genetic data, based on the segregation of resistance determinants and the H(2)S trait among transconjugant and transductant classes, suggested the presence of two R plasmids. Plasmid DNA was isolated by cesium chloride-ethidium bromide centrifugation. Two plasmid species were detected by agarose gel electrophoresis of purified plasmid DNA, a large molecule of about 80 x 10(6) daltons (designated pSR12) and a small molecular species of approximately 5.5 x 10(6) daltons (designated pSR13). Transformation studies using purified plasmid DNA showed that the large pSR12 plasmid confers resistance to ampicillin, tetracycline, and streptomycin and also carries the gene(s) for H(2)S production. The small pSR13 plasmid confers resistance to streptomycin and sulfonamide.

Conjugation, Genetic↗
PubMed1989

Comparative in vitro and in vivo activities of piperacillin combined with the beta-lactamase inhibitors tazobactam, clavulanic acid, and sulbactam.

Tazobactam (YTR-830H), a novel beta-lactamase inhibitor, was compared with clavulanic acid and sulbactam for enhancement of the activity of piperacillin against beta-lactamase-producing, piperacillin-resistant clinical isolates. Piperacillin MICs were determined in media containing a fixed concentration of 2 or 4 micrograms of the inhibitors per ml. The higher concentration was generally more effective. Tazobactam was superior to sulbactam in enhancing the spectrum and potency of piperacillin. Although the calvulanic acid combination was more potent, tazobactam was effective for a similar spectrum of resistant gram-negative clinical isolates containing beta-lactamase. MICs were reduced to the susceptible range for Escherichia coli, Klebsiella pneumoniae, Proteus spp., Salmonella spp., and Shigella spp. Combinations with tazobactam and sulbactam, but not clavulanic acid, were effective against Morganella spp. Some antagonism of the activity of piperacillin was observed with clavulanic acid but not with tazobactam or sulbactam. The inhibitors were similarly effective with piperacillin against beta-lactamase-positive Staphylococcus spp. and the Bacteroides fragilis group. Piperacillin-tazobactam was more effective against a broader spectrum of gram-negative enteric bacteria than ticarcillin plus clavulanic acid was. Combinations with tazobactam or clavulanic acid had a broader spectrum of activity than combinations with sulbactam against bacteria that produce characterized plasmid-mediated enzymes of clinical significance. In particular, piperacillin with tazobactam or clavulanic acid, but not with sulbactam, inhibited TEM-1, TEM-2, and SHV-1 enzymes. In vitro activity was reflected in vivo. Tazobactam and clavulanic acid were superior to sulbactam in enhancing the therapeutic efficacy of piperacillin in mice infected with beta-lactamase-positive E. coli, K. pneumoniae, Proteus mirabilis, and Staphylococcus aureus. Only combinations with tazobactam and sulbactam were effective against the Morganella infection. Tazobactam has a good potential for enhancing the clinical efficacy of piperacillin.

Animals↗
PubMed2001

Molecular characterization of a novel plasmid-encoded cefotaximase (CTX-M-12) found in clinical Klebsiella pneumoniae isolates from Kenya.

Nine Klebsiella pneumoniae isolates, six from blood and three from cerebrospinal fluid of newborn babies at Kenyatta National Hospital, Nairobi, Kenya, were analyzed for the mechanism of cephalosporin resistance. By using pulsed-field gel electrophoresis of XbaI-digested chromosomal DNA, all the nine isolates were found to be clonal. PCR and direct sequencing revealed a novel extended-spectrum beta-lactamase, which we designated CTX-M-12. It has a more potent hydrolytic activity against cefotaxime than against ceftazidime and a pI of 9.0 and is encoded on a large self-transferable ca. 160-kbp plasmid.

Amino Acid Substitution↗
PubMed2002

Characterization of a novel plasmid-mediated cephalosporinase (CMY-9) and its genetic environment in an Escherichia coli clinical isolate.

An Escherichia coli strain, HKYM68, which showed resistance to broad-spectrum cephalosporins was isolated from a sputum specimen in Japan. The high-level resistance of the strain to ceftazidime, cefpirome, and moxalactam was carried by a self-transferable plasmid. The beta-lactamase gene responsible for the resistance was cloned and sequenced. The deduced amino acid sequence of this gene product, CMY-9, had a single amino acid substitution (E85D), the residue reported to be part of the recognition site for the R1 side chain of beta-lactams, compared with the amino acid sequence of CMY-8 and also had 78% identity with the amino acid sequence of CepH, a chromosomal cephalosporinase of Aeromonas hydrophila. A sul1-type class 1 integron containing an aacA1-orfG gene cassette was identified upstream of bla(CMY-9) and ended with a truncated 3' conserved segment. The following 2.1 kb was almost identical to the common region of integrons In6 and In7 and the integron of pSAL-1, except that orf513 encoding a putative transposase was identified instead of orf341 due to addition of a single nucleotide. bla(CMY-9) was closely located downstream of the end of the common region. These observations are indicative of the exogenous derivation of bla(CMY-9) from some environmental microorganisms such as aeromonads.

Anti-Bacterial Agents↗