Emergence of tetracycline resistance due to a multiple drug resistance plasmid in Vibrio cholerae O139

Abstract Of the 173 clinical strains of Vibrio cholerae O139 isolated from India, Bangladesh, and Thailand tested, six strains from India were resistant to tetracycline, ampicillin, chloramphenicol, kanamycin, and gentamicin. These six strains harbored a self-transmissible plasmid that mediated resistance to tetracycline, ampicillin, chloramphenicol, kanamycin, gentamicin, sulfamethoxazole, trimethoprim, and O/129. The multiple drug resistance plasmids were 200 kb in size and belonged to the incompatibility group C. Although a majority of the O139 strains (94.8%) were highly resistant to streptomycin, sulfamethoxazole, trimethoprim, and O/129, the tetracycline-susceptible strains so far tested were plasmid-negative. The data suggest the existence of two distinct multiple antimicrobial agent resistance (MAR) patterns in V. cholerae O139.     

Detection of the aminoglycosidestreptothricin resistance gene cluster ant(6)-sat4-aph(3 ')-III in commensal viridans group streptococci.

High-level aminoglycoside resistance was assessed in 190 commensal erythromycin-resistant alpha-hemolytic streptococcal strains. Of these, seven were also aminoglycoside-resistant: one Streptococcus mitis strain was resistant to high levels of kanamycin and carried the aph(3 ')-III gene, four S. mitis strains were resistant to high levels of streptomycin and lacked aminoglycoside-modifying enzymes, and two S. oralis strains that were resistant to high levels of kanamycin and streptomycin harbored both the aph(3 ')-III and the ant(6) genes. The two S. oralis strains also carried the ant(6)-sat4- aph(3 ' ')-III aminoglycoside-streptothricin resistance gene cluster, but it was not contained in a Tn5405-like structure. The presence of this resistance gene cluster in commensal streptococci suggests an exchange of resistance genes between these bacteria and enterococci or staphylococci.     

Cloning of the Escherichia coli K-12 dihydrofolate reductase gene following Mu-mediated transposition

The dihydrofolate reductase structural gene, folA, has been cloned into the multicopy vector pBR322 following the gene's enrichment by bacteriophage Mu-mediated transposition. Strains carrying the resultant plasmid, pJFMS, produce 25 to 30 times more dihydrofolate reductase than control strains. Consequently they are resistant to trimethoprim, an inhibitor of this enzyme. This elevation in enzyme production is due to an increase in the number of folA gene copies per cell. The higher yield of dihydrofolate reductase obtained will be extremely useful for purifying and characterising this trimethoprim-sensitive chromosomally derived enzyme. The plasmid will also be invaluable for studying the structure, function and regulation of dihydrofolate reductase.     

A multiresistant clone of Shiga toxin-producing Escherichia coli O118:[H16] is spread in cattle and humans over different European countries

Multiresistant Shiga toxin-producing Escherichia coli (STEC) O118:H16 and O118 nonmotile strains (designated O118:[H16]) were detected by examination of 171 STEC isolates for their antimicrobial sensitivity. Of 48 STEC O118:[H16] strains, 98% were resistant to sulfonamide, 96% were resistant to streptomycin, 79% were resistant to kanamycin, 75% were resistant to tetracycline, 67% were resistant to ampicillin, 60% were resistant to chloramphenicol, 48% were resistant to trimethoprim, and 10% each were resistant to gentamicin and nalidixic acid. Nalidixic acid resistance and reduced susceptibility to ciprofloxacin were associated with the mutation gyrA(LEU-83). The STEC O118:[H16] strains were found to belong to a single genetic clone as investigated by multilocus enzyme electrophoresis and by multilocus sequence analysis of E. coli housekeeping genes. The STEC O118:[H16] strains originated from humans and cattle and were isolated in seven different countries of Europe between 1986 and 1999. Strains showing multiresistance to up to eight different antimicrobials predominated among the more recent STEC O118:[H16] strains. The genes in parentheses were associated with resistance to kanamycin (aphA1-Ia), chloramphenicol (catA1), tetracycline [tet(A)], and ampicillin (bla(TEM-1)). Class 1 integrons containing sulI (sulfonamide resistance), aadA1a (streptomycin resistance), or dfrA1 (trimethoprim resistance)-aadA1a gene cassettes were detected in 28 strains. The bla(TEM-1b) gene was present in 18 of 21 strains that were examined by nucleotide sequencing. Class 1 integrons and bla(TEM) genes were localized on plasmids and/or on the chromosome in different STEC O118:[H16] strains. Hybridization of XbaI-digested chromosomal DNA separated by pulsed-field gel electrophoresis revealed that bla(TEM) genes were integrated at different positions in the chromosome of STEC O118:[H16] strains that could have occurred by Tn2 insertion. Our data suggest that strains belonging to the STEC O118:[H16] clonal group have a characteristic propensity for acquisition and maintenance of resistance determinants, thus contrasting to STEC belonging to other serotypes.     

Phenotypic and genetic characterization of multidrug-resistant <Emphasis Type="Italic">Salmonella</Emphasis> Infantis strains isolated from broiler chicken meats in Turkey

Twenty Salmonella Infantis strains resistant against kanamycin, tetracycline, neomycin, spectinomycin, sulphonamide, nalidixic acid and trimethoprim were selected for this study out of 103 Salmonella strains isolated from broiler samples collected from several markets in the Bolu and Ankara regions of Turkey. The resistance genes aadA1, aphA1, sul1, tet(A), dfrA5/dfrA14 and gyrA were determined for these multidrug-resistant S. Infantis strains. S. Infantis strains contained a mega plasmid with the molecular size of 206 kb. The strains were divided into three groups according to the pulsed field gel electrophoresis patterns of XbaI digested chromosomal DNA. A Ser83→Tyr83 point mutation was found in the gyrA gene of all quinolone-resistant isolates. Filter mating experiments showed that 206 kb plasmid transferred nalidixic acid resistance associated with class I integrons.     

Conjugative trimethoprim resistance in Staphylococcus aureus

A multiply resistant Staphylococcus aureus isolate, WBG7410, harbours plasmids of 38, 26, 2.8, 2.4 and 1.9 kb and transfers trimethoprim and kanamycin resistance at high frequencies by conjugation. The transconjugants contained the 38-kb plasmid, pWBG707, and the 2.8-kb plasmid. Plasmid pWBG707 was shown to encode trimethoprim resistance, was conjugative and mobilised at high frequencies the 2.8-kb plasmid which presumably encodes kanamycin resistance. Plasmid pWBG707 was isolated mostly in the open circular form and analysis with EcoRI restriction endonuclease suggests that pWBG707 is a new conjugative plasmid distinct from the other conjugative plasmids reported in S. aureus.     

Yeast mutants sensitive to trimethoprim

Wild-type strains of Saccharomyces cerevisiae are resistant to growth inhibition by the folate antagonist trimethoprim. A mutant strain sensitive to trimethoprim was isolated. It was found to be sensitive to both ultraviolet light and X-irradiation. Genetic tests revealed that it was allelic with a known radiation-sensitive strain of Saccharomyces cerevisiae, rad 6-1. Strains harbouring a variety of mutant alleles conferring radiation-sensitivity were tested for sensitivity to trimetroprim. It was found that rad 6-1 and each of the four known alleles of rad 18 conferred sensitivity to the drug, but all other rad mutants tested were trimethoprim-resistant. All trimethoprim-sensitive strains, including double mutants of rad 6 rad 18, gave rise to trimethoprim-resistant outgrowths at a rather high frequency (∼ 10⁻⁵). Several resistant outgrowths were analysed. A wide variation in phenotype with respect to UV-sensitivity was found. Genetical analysis revealed that resistance to trimethoprim resulted from torward mutations at separate loci rather than back mutations of rad 6 or rad 18 alleles.     

Occurrence of heterogeneous R-plasmids during two concurrent epidemics due to multidrug-resistantSalmonella oranienburg

During two different epidemics that started in August–September, 1980, 140 and 6 multidrug-resistant strains ofSalmonella oranienburg were isolated from a Children's Hospital in New Delhi (epidemic 1) and H.N. Das Hospital in Bombay (epidemic 2) respectively. Out of 140, 123 strains showed high levels of resistance to ampicillin, kanamycin, streptomycin, sulfamethoxazole, tetracycline, and trimethoprim. All six strains of epidemic 2 and two strains of epidemic 1 were also found resistant to chloramphenicol in addition to the above antibiotics. The genetic characterization of conjugative R-plasmids harbored by these strains revealed that, while strains of epidemic 1 carry a fi^–, 96 megadalton, an unclassified plasmid, the strains isolated during epidemic 2 contain a fi^–, 62 megadalton plasmid of incompatibility group I₁. All the six strains of epidemic 2 were found to produce bacteriocin of Col Ib group. Plasmid transfer studies revealed that the genes for antibiotic resistance and bacteriocin production were coded on a single plasmid of 62 megadalton. The data show the significance of detailed genetic analysis while dealing with the clonal outbreaks due to same resistant serotypes.     

Characterization of an R-plasmid dihydrofolate reductase with a monomeric structure

A plasmid-encoded dihydrofolate reductase that originated in a clinical isolate of Salmonella typhimurium (phage type 179) moderately resistant to trimethoprim has been isolated and characterized. The dihydrofolate reductase (called type III) was purified to homogeneity using a combination of gel filtration, hydrophobic chromatography, and methotrexate affinity chromatography. Polyacrylamide gel electrophoresis under denaturing and nondenaturing conditions indicated that the enzyme is a 16,900 molecular weight monomeric protein. Kinetic analyses showed that trimethoprim is a relatively tight binding inhibitor (Ki = 19 nM) competitive with dihydrofolate. The enzyme is also extremely sensitive to methotrexate inhibition (Ki = 9 pM) and has a high affinity for dihydrofolate (Km = 0.4 microM). The sequence of the first 20 NH2-terminal residues of the protein shows 50% homology with the trimethoprim-sensitive chromosomal Escherichia coli dihydrofolate reductase and suggests that the two enzymes may be closely related. This is the first example of a plasmid encoding for a monomeric dihydrofolate reductase only moderately resistant to trimethoprim, and a resistance mechanism, dependent in part on the high dihydrofolate affinity of the type III enzyme, is proposed.     

Purification and characterization of dihydrofolate reductase from wild-type and trimethoprim-resistant Mycobacterium smegmatis

Dihydrofolate reductase (DHFR) from extracts of Mycobacterium smegmatis strain mc2(6) and trimethoprim-resistant mutant mc2(26) was purified to homogeneity. In crude extracts, the specific activity of the enzyme from the trimethoprim resistant strain was comparable to that from the sensitive strain. The DHFR from both sources was purified using affinity chromatography on MTX-Sepharose followed by Mono Q FPLC. The enzyme has an apparent molecular mass of 23 kDa from gel filtration on Sephadex G-100 and from SDS-PAGE. Amino terminal sequence analysis showed homology with DHFRs from a subset of other gram-positive organisms. The purified enzyme from the trimethoprim-sensitive organism exhibited Km values for H2folate and NADPH of 0.68 +/- 0.2 microM and 21 +/- 4 microM, respectively. The Km values for H2folate and NADPH for the enzyme from the drug-resistant organism were 1.8 +/- 0.4 microM and 5.3 +/- 1.5 microM, respectively. A kcat of 4.5 sec-1 was determined for the DHFR from both sources. The enzyme from both sources was competitively inhibited by pyrimethamine and trimethoprim. The Ki value of trimethoprim, for the enzyme from the drug-resistant organism was about six-fold higher than for the enzyme from drug-sensitive strain. Our data suggest that mutation of DHFR contributes to trimethoprim resistance in the mc2(26) strain of M. smegmatis.     

A new mechanism of plasmid trimethoprim resistance. Characterization of an inducible dihydrofolate reductase

The dihydrofolate reductase encoded by plasmid pUK1123, which confers only a moderate level of trimethoprim resistance on its host, has been isolated and characterized. This enzyme, designated type IV, differs markedly from all previously described plasmid dihydrofolate reductases. It has a relatively high molecular weight of 46,700 as measured by gel filtration and, unlike previous plasmid dihydrofolate reductases, its synthesis is induced in the presence of increasing concentrations of trimethoprim. It is only slightly resistant to trimethoprim but is competitively inhibited by this drug with an inhibitor binding constant of 63 nM. In addition, the enzyme has a relatively low affinity for the substrate, dihydrofolate (Km = 37 microM). This is the first report of a plasmid trimethoprim resistance mechanism resulting from the induced synthesis of a large molecular weight dihydrofolate reductase which is only slightly resistant to trimethoprim. The possible origins of the type IV enzyme are discussed.     

Incidence of resistance to ampicillin,chloramphenicol, kanamycin,tetracycline and trimethoprim of Salmonella strains isolated in The Netherlands during 1975–1980

From 1975–1980, about 130 000 Salmonella strains isolated from various sources were tested for resistance to ampicillin, chloramphenicol, kanamycin, tetracycline and trimethoprim. Following the ban on incorporation of tetracycline in animal feeds for nutritive purposes, tetracycline resistance in S. typhimurium and S. panama strains of porcine origin dropped from about 90% in 1974 for both species, to about 34% and 1%, respectively, in 1980. The incidence of resistance in human strains concurrently decreased from about 80% in 1974 to 25% and 1%, respectively, in 1980.The build-up of multiple resistance in bovine S. dublin and S. typhimurium strains, already started in 1973–1974, has continued. Recently, phage type 193 S. typhimurium strains have become predominant and they are invariably resistant to ampicillin, chloramphenicol, tetracycline, kanamycin, neomycin, streptomycin, sulphonamide and trimethoprim. Up to now, type 193 strains were hardly encountered in human patients, but the number of human isolates is slowly increasing.A fairly large number of multiply resistant strains belonging to S. oranienburg, S. schwarzengrund, S. typhimurium and, recently, S. krefeld have been isolated from adoptive children from the Far East.     

Antimicrobial susceptibilities of Aeromonas spp. isolated from environmental sources

Aeromonas spp. are ubiquitous aquatic bacteria that cause serious infections in both poikilothermic and endothermic animals, including humans. Clinical isolates have shown an increasing incidence of antibiotic and antimicrobial drug resistance since the widespread use of antibiotics began. A total of 282 Aeromonas pure cultures were isolated from both urban and rural playa lakes in the vicinity of Lubbock, Texas, and several rivers in West Texas and New Mexico. Of these, at least 104 were subsequently confirmed to be independent isolates. The 104 isolates were identified by Biolog and belonged to 11 different species. The MICs of six metals, one metalloid, five antibiotics, and two antimicrobial drugs were determined. All aeromonads were sensitive to chromate, cobalt, copper, nickel, zinc, cefuroxime, kanamycin, nalidixic acid, ofloxacin, tetracycline, and sulfamethoxazole. Low incidences of trimethoprim resistance, mercury resistance, and arsenite resistance were found. Dual resistances were found in 5 of the 104 Aeromonas isolates. Greater numbers of resistant isolates were obtained from samples taken in March versus July 2002 and from sediment versus water. Plasmids were isolated from selected strains of the arsenite- and mercury-resistant organisms and were transformed into Escherichia coli XL1-Blue MRF'. Acquisition of the resistance phenotypes by the new host showed that these resistance genes were carried on the plasmids. Mercury resistance was found to be encoded on a conjugative plasmid. Despite the low incidence of resistant isolates, the six playa lakes and three rivers that were sampled in this study can be considered a reservoir for antimicrobial resistance genes.     

Transposition of penicillinase determinants in methicillin-resistant Staphylococcus aureus

Abstract A methicillin-resistant Staphylococcus aureus (MRSA) typical of those being isolated in Australian hospitals has been studied. It contains two plasmids, one of 1.4 megadalton (MDa) and one of 18 MDa. When selection is made for resistance to nucleic acid-binding (NAB) compounds in mixed-culture transfer, two types of transcipients are obtained; those containing an 18-MDa plasmid and resistant to NAB compounds, trimethoprim and aminoglycosides such as gentamicin and kanamycin and those having a 22 MDa plasmid and the additional phenotype of penicillinase production. The penicillinase determinants on the 22-MDa plasmid have been found to transpose to the chromosome and from the chromosome to an 18-MDa plasmid similar to that found in the original isolate. Restriction enzyme analysis has shown that a 7.3-kilobase pair (kb) element is involved. This has been designated Tn 3852 .     

Transfer of plasmid-borne resistance from a multiply-resistant Staphylococcus aureus isolate,WBG1022

Staphylococcus aureus isolate, WBG1022, was resistant to penicillin, kanamycin, neomycin, streptomycin, chloramphenicol, trimethoprim, cadmium, and ethidium bromide and harbored plasmids of 34.5, 24.5, 4.4, 3.2, and 2.6 kilobases. The plasmids were transferred in mixed-culture transfer and conjugation experiments. No resistance phenotype was associated with the 2.6-kb plasmid. The 3.2-kb and 4.4-kb plasmids encoded chloramphenicol and streptomycin resistance respectively. The 24.5-kb plasmid, pWBG626, encoded joint resistance to penicillin, kanamycin, neomycin, and ethidium bromide. Resistance to trimethoprim and cadmium were chromosomal. The 34.5-kb plasmid, pWBG661, had no resistance phenotype but was found to be conjugative. It also mobilized the 4.4-kb and 24.5-kb plasmids in WBG1022. Restriction endonuclease analysis of pWBG661 with EcoRI, ClaI, PvuII, and BglII restriction enzymes demonstrated that pWBG661 was identical to two previously isolated S. aureus conjugative plasmids, p WBG620 and pWBG637, that also lack resistance phenotypes.     

The isolation and characterization of Campylobacter jejuni subsp. jejuni from domestic geese (Anser anser)

AIMS: The objectives of this study were to determine the presence of thermophilic Campylobacter spp. in free range domestic geese, and to characterize isolated strains using phenotyping criteria and SDS-PAGE of whole-cell proteins. METHODS AND RESULTS: Forty cloacal swabs from two different flocks of domestic geese were examined. All Camp. jejuni strains isolated from geese were biotyped using the Lior biotyping scheme. Twelve Camp. jejuni isolates were also tested for their susceptibility to 17 different antibacterial agents by a disc diffusion METHOD: Fourteen of the isolates were also subjected to SDS-PAGE. All of the geese examined were found to harbour Camp. jejuni. Six geese carried more than one species of Campylobacter. All strains examined were susceptible to various antibiotics but resistant to penicillin G and cephalothin. Eleven strains (92%) were resistant to sodium cefuroxime, and eight (67%) were resistant to cloxacillin, ampicillin and colistin sulphate. Three strains (25%) were resistant to tetracycline, and one strain was resistant to sulfamethoxazole/trimethoprim and kanamycin. Nine strains were subtyped as Camp. jejuni subsp. jejuni biotype II and the remaining ones as biotype I. There were 96% and 100% similarities between all the strains examined by SDS-PAGE. CONCLUSION: This study showed that Camp. jejuni were common in the intestinal tract of domestic geese. SIGNIFICANCE AND IMPACT OF THE STUDY: Geese should be considered as potential reservoirs for human and animal campylobacteriosis. The antibiotic resistance data from this study also showed that fluoroquinolone resistance, which appears to be a problem in poultry isolates in some countries, is not yet a problem in these geese.     

Liposome-mediated transformation of tobacco mesophyll protoplasts by an Escherichia coli plasmid.

An Escherichia coli plasmid, pLGV23neo, carrying a kanamycin resistance gene expressed in plant cells, was encapsulated into negatively charged liposomes prepared by the reverse phase evaporation technique. These liposomes were induced to fuse with tobacco mesophyll protoplasts by polyethyleneglycol treatment. Kanamycin-resistant clones were reproducibly isolated from transfected cultures at an average frequency of 4 X 10(-5). Plants regenerated from these resistant colonies were confirmed to be transformed according to three criteria. Protoplasts isolated from their leaves were resistant to 100 micrograms/ml kanamycin. The enzyme aminoglycoside 3'-phosphotransferase II encoded by the plasmid pLGV23neo was detected in leaf extracts. Approximately 3-5 copies of the gene encoding for kanamycin resistance were inserted in the genome of at least one of the studied transformants. The restriction pattern of inserted DNA was best explained by assuming a tandem integration of the pPLGV23neo sequences, implying an homologous recombination event between these sequences during transformation. Kanamycin resistance was transmitted as a single dominant nuclear marker to the progeny of resistant plants after selfing or cross-pollination with the wild-type.     

Antibiotic resistance of bacteria in raw and biologically treated sewage and in groundwater below leaking sewers

More than 750 isolates of faecal coliforms (>200 strains), enterococci (>200 strains) and pseudomonads (>340 strains) from three wastewater treatment plants (WTPs) and from four groundwater wells in the vicinity of leaking sewers were tested for resistance against 14 antibiotics. Most, or at least some, strains of the three bacterial groups, isolated from raw or treated sewage of the three WTPs, were resistant against penicillin G, ampicillin, vancomycin, erythromycin, triple sulfa and trimethoprim/sulfamethoxazole (SXT). Only a few strains of pseudomonads or faecal coliforms were resistant against some of the other tested antibiotics. The antibiotic resistances of pseudomonads, faecal coliforms and enterococci from groundwater varied to a higher extent. In contrast to the faecal coliforms and enterococci, most pseudomonads from all groundwater samples, including those from non-polluted groundwater, were additionally resistant against chloramphenicol and SXT. Pseudomonads from sewage and groundwater had more multiple antibiotic resistances than the faecal coliforms or the enterococci, and many pseudomonads from groundwater were resistant to more antibiotics than those from sewage. The pseudomonads from non-polluted groundwater were the most resistant isolates of all. The few surviving faecal coliforms in groundwater seemed to gain multiple antibiotic resistances, whereas the enterococci lost antibiotic resistances. Pseudomonads, and presumably, other autochthonous soil or groundwater bacteria, such as antibiotic-producing Actinomyces sp., seem to contribute significantly to the gene pool for acquisition of resistances against antibiotics in these environments.     

Molecular epidemiology of plasmid patterns in Shigella dysenteriae type I obtained from an outbreak in West Bengal (India)

Abstract Multiple antibiotic-resistant Shigella dysenteriae type 1 isolates from a recent epidemic in West Bengal (India) showed identical plasmid patterns. All isolates were resistant to ampicillin (Am), chloramphenicol (Cm), tetracycline (Tc), streptomycin (Sm) and trimethoprim (Tp) and contained 6 plasmids, ranging from 2.5–120 kb. The Am resistance determinant was located on the 120 kb plasmid. This plasmid was unstable when the S. dysenteriae strains were grown above 37°C. The Bangladesh strains of S. dysenteriae type 1 showed identical plasmid patterns, except that many isolates were Am-sensitive and lacked the 120 kb plasmid. In strains from both Bangladesh and West Bengal, predominantly group-B plasmids conferred resistance to Cm and Tc. Comparisons of Eco R1 fragments generated from the total plasmid DNA content of each strain support the view that the plasmids present in the S. dysenteriae type 1 strains isolated from all recent epidemics in India and Bangladesh were identical.