Dihydrofolate Reductase Type Research Articles

Phytochemical constituents of Inula britannica as potential inhibitors of dihydrofolate reductase: A strategic approach against shigellosis

Shigella dysenteriae type 1 is considered as an epidemic in different developing countries, which is responsible for the most severe form of bacterial dysentery. It habitually can develop to the most severe form of dysentery with deadly complications. Development of drugs against this disease is still ongoing. Therefore, we used in silico studies to screen the Inula britannica phytocompounds that are used in traditional Chinese and Kampo Medicines and have activities against different diseases. Spinacetin, eupatin, chrysoeriol and diosmetin were successfully passed through the docking-based screening and absorption, distribution, metabolism, excretion and toxicity (ADMET) filtration. The estimated docking affinities of eupatin, diosmetin, chrysoeriol and spinacetin with Dihydrofolate reductase type 1 (DHFR-1), were −6.5, −6.5, −6.3 and −6.1 kcal/mol, respectively. Which were selected for further investigations based on their favorable ADME/Tox characteristics. Then, the 100 ns molecular dynamics (MD) simulations of apo DHFR, spinacetin-DHFR, eupatin-DHFR, chrysoeriol-DHFR and diosmetin-DHFR complexes were carried out. The RMSD fluctuations of the spinacetin, eupatin, chrysoeriol and diosmetin inside the binding site were explored. Subsequently, the effect of binding Spinacetin, eupatin, chrysoeriol and diosmetin upon the dynamic stability of protein was assessed. Additionally, Principal Component Analysis (PCA) and Hydrogen bond analysis was performed for the apo protein and the protein ligand complexes. The results revealed that chrysoeriol and eupatin has good inhibitory effects against DHFR-1 as treatment for Shigella dysenteriae type when compared to other compounds under study. Hence this study implies that eupatin and chrysoeriol are a significantly potential drug like molecule for the treatment of Shigellosis and must undergo validation through in vivo and in vitro experiments. Communicated by Ramaswamy H. Sarma

Molecular Characterization Of Multidrug Resistant Salmonella Enterica Isolated From Poultry

In this study, Atotal of 180 samples from poultry meat: 60 fresh slaughtered poultry , 60 frozen poultry meat, 60 poultry organs (liver and gizzard) were obtained randomly from slaughter houses, restaurants and supermarkets at Sharqia governorate. The collected samples were examined for the presence of S.enterica. Biochemical and serological identification were applied, Biochemical and serological examination revealed that incidence of S.enterica was 8.3 % (15 isolates) from all samples. Molecular characterization using multiplex PCR technique was applied for confirming the serotyping of S.enterica. From these 15 isolates , S.typhiurium was the most isolated serovar represented in 9 of 15 isolates (60%), S.paratyphi A was 3 of 15 isolates (20%), S.enteritidis was 2 of 15 isolates (13.3%) and S.kentucky was 1 of 15 isolates (6.7%), Invitro sensitivity of S.enterica isolates to various antimicrobial agents revealed that the incidence of resistance to enrofloxacin was (1/15) (6.6%),streptomycin was (4/15) (26.6%), ceftriaxone (11/15) (73.3%), cefpodoxime was (11/15) ( 73.3%) , ceftazidime was (10/15) (66.7%), ampicillin was (12/15) (80%) , chloramphenicol was (0/15) (0%), cefotaxime was (12/15) ( 80%), tetracycline was (6/15) (40%), ciprofloxacin was (0/15)( 0%), nalidixic acid was (5/15) (33.3%) and amoxicillin-clavulanic acid was (9/15) ( 60%) ,The molecular characterization of β-lactamase resistance genes in MDR S.enterica isolates showed presence of blaCMY gene in 2 of 15 isolates (13.3%), blaCTX-M in 12 isolates (80%), blaTEM in 6 isolates (40%), where blaSHV and blaOXA genes could not be identified, Molecular characterization of plasmid mediated quinolone resistance genes in multidrug-resistant S.enterica isolates revealed the presence of qnrA in 7 of 15 isolates (46.7%),qnrB in 3 of 15 isolates (20%) and qnrS in 1 of 15 isolates (6.7%). Class 1 integron was detected in 2 of 15 MDR S.enterica (13.3%), DNA-sequencing results for the inserted gene cassettes identified 3 types of class 1 integrons. The identified antibiotic resistance genes were dihydrofolate reductase types : dfrA15 and dfrA17 which confer resistance to trimethoprim and aminoglycoside adenyltransferase type aadA2 which confer resistance to streptomycin and spectinomycin, All strains were negative for class 2 integrons.

Analysis of Integron-associated Gene Cassettes in Clinical Acinetobacter Calcoaceticus-Baumannii Complex Isolates from Central Taiwan

Purpose: Acinetobacter calcoaceticus-baumannii complex (ABC) has become a major infectious pathogen in Taiwan, and mobile DNA such as integrons and transposons often contribute to its multiple antibiotic resistance characteristics. Methods: A clinical epidemiological study of the cassette contents of the class 1 integron of ABC isolates in two central Taiwan hospitals was performed. Results: It was found that 78% (39/50) of strains possessed the IntI1 integrase gene, while four major different sizes of class 1 integron cassette arrays were found: 3.0 kb (aacC1-ORF1-ORF2- ORF3-aadA1) (aacC1: aminoglycoside 3'-N-acetyltransferase; aadA1: aminoglycoside 3'-adenyltransferase), 2.5 kb (aacA4- catB8-aadA1) (aacA4: aminoglycoside 6'-N-acetyl-transferase), 2.0 kb (dhfrXII-ORF-aadA2) (dhfrXII: dihydrofolate reductase; aadA2: aminoacyl-adenyltransferase A) and 1.2 kb (dhfrAI-ORF) (dhfrAI: dihydrofolate reductase type AI). Six different combinations of cassette arrays were also noted. The co-relationships between antibiotic-resistant phenotypes and genotypic combinations of cassettes were analyzed. As the 2.5-kb cassette array was found to be the most common in this study, the flanking regions of the specific integron of ABC were also analyzed by inverse PCR. In addition to antibiotic resistance genes, such as aphA1 (kanamycin resistance gene) (AphA1: aminoglycosidephosphotransferase), the transposase gene (tnpIS15/tnpAIS26) was found in the 2.5-kb cassette array flanking regions. Furthermore, PCR-RFLP confirmed that these flanking regions were conserved in all of the examined isolates. Conclusion: The SXT and GM resistance phenotypes of ABC isolates could be used in clinical screening for the IntI1- associated genotype. ABC isolates that carry a 2.5-kb cassette array have become dominant in Central Taiwan, and dissemination of this specific type of integron may derive from the involvement of mobile DNA.

Molecular characterization of enterohemorrhagic Escherichia coli isolates from cattle.

ABSTRACTA total of 21 (4.3%) enterohemorrhagic E. coli strains were isolated by biochemical tests and identification of the eae+stx1+stx2+ genotype from 490 stool samples obtained from calves with diarrhea during 1-year period from a major farm in Tehran, Iran. All of the strains showed resistance to ampicillin, ciprofloxacin, trimethoprim, streptomycin, chloramphenicol and tetracycline, while 19% showed resistance to gentamicin. Out of 21 EHEC strains, 11 (53%) harbored class 1 integron. Two different amplification products, which were approximately 750 and 1,700 bp in size, were obtained from amplified variable regions (in-F/in-R primers) in 3 (14.3%) and 4 (19%) of the EHEC isolates, which corresponded to dfrA7(dihydrofolate reductase type I) and dfrA1/aadA1(dihydrofolate reductase/aminoglycoside adenyltransferase) resistance gene cassettes, respectively, and this was confirmed by sequencing. Genotyping analysis revealed a total of 16 pulsotypes that corresponded to 16 isolates with the similarity indices of 62% and 30% for the most and least similar isolates, respectively, 9 of which harbored class 1 integron. Analysis of pulsotypes showed an extensive diversity among the isolates harboring integron, which is indicative of a lack of any significant genetic relatedness among the isolates. No obvious relation could be deduced between integron content and special pulsotypes. The little data available on the genotyping patterns of EHEC isolates from cattle and their resistance gene contents emphasize the need to establish genotyping databases in order to monitor and source track the source of emergence and spread of new resistant and integron-carrying genotypes.

Genetic analysis of multiple antimicrobial resistance in Salmonella isolated from diseased broilers in Egypt

To date, no information has been available on the molecular bases of antimicrobial resistance in Salmonella spp. from poultry in Egypt or even in Africa. Therefore, the objective of this study was to analyze, at the molecular level, the mechanisms of multidrug-resistance in isolates of Salmonella recovered from diseased broilers in Egypt. Twenty-one Salmonella isolates were identified; 13 of these isolates were Salmonella enterica serovar Enteritidis and eight Salmonella enterica serovar Typhimurium. 17 (81%). Salmonella isolates displayed multidrug resistance phenotypes, particularly against ampicillin, streptomycin, spectinomycin, kanamycin, tetracycline, chloramphenicol, and trimethoprim/sulfamethoxazole. PCR and DNA sequencing identified class 1 integrons in nine (42.9%) isolates and class 2 integrons in three (14.3%) isolates. The identified resistance genes within class 1 integrons were aminoglycoside adenyltransferase type A, aadA1, aadA2 and aadA5 and dihydrofolate reductase type A, dfrA1, dfrA5, dfrA12, dfrA15 and dfrA17. The β-lactamase encoding genes bla(TEM-1) and bla(CMY-2) and florfenicol resistance gene floR were also identified. Furthermore, the tetracycline resistance gene tet(A) was identified in 14 (66.7%) Salmonella isolates. To the best of our knowledge, this is the first report of the molecular basis of antimicrobial resistance in Salmonella spp. isolated from poultry in Africa.

Genetic analysis of antimicrobial resistance in Escherichia coli isolated from diarrheic neonatal calves

This study was carried out to screen and analyze the genetic basis of antimicrobial resistance in Escherichia coli strains isolated from neonatal calf diarrhea in Egypt. A total of 182 isolates of E. coli recovered from 91 diarrheic neonatal calves were analyzed for antimicrobial susceptibilities, the presence of class 1 and class 2 integrons and antimicrobial resistance genes. Nineteen isolates (10.4%) showed multidrug resistance phenotypes and harbored at least three antimicrobial resistance genes. PCR screening detected class 1 integrons in 19 isolates (10.4%) and class 2 integrons in 2 isolates (1.1%). The identified antimicrobial resistance genes within class 1 integrons were dihydrofolate reductase types: dfrA1, dfrA12, dfrA15 and dfrA17, which confer resistance to trimethoprim; aminoglycoside adenyltransferase types: aadA1, aadA2, aadA5, aadA7 and aadA23, which confer resistance to streptomycin and spectinomycin; and aminoglycoside acetyltransferase gene, aac(3)-Id, which confers resistance to gentamicin and sisomicin. Furthermore, many β-lactamases encoding genes, plasmid-mediated quinolone resistance genes and florfenicol resistance gene were identified in this study. To the best of our knowledge, this is the first report for molecular characterization of antimicrobial resistance in E. coli isolated from diarrheic neonatal calves in Africa.

Particular interaction between pyrimethamine derivatives and quadruple mutant type dihydrofolate reductase of Plasmodium falciparum: CoMFA and quantum chemical calculations studies

Comparative molecular field analysis (CoMFA) was performed on twenty-three pyrimethamine (pyr) derivatives active against quadruple mutant type (Asn51Ile, Cys59Arg, Ser108Asn, Ile164Leu) dihydrofolate reductase of Plasmodium falcipaarum (PfDHFR). The represented CoMFA models were evaluated based on the various three different probe atoms, Csp3 (+1), Osp3 ( − 1) and H (+1), resulting in the best model with combined three types of probe atoms. The statistical results were = 0.702, Spress = 0.608, = 0.980, s = 0.156, and = 0.698 which can explain steric contribution of about 50%. In addition, an understanding of particular interaction energy between inhibitor and surrounding residues in the binding pocket was performed by using MP2/6-31G(d,p) quantum chemical calculations. The obtained results clearly demonstrate that Asn108 is the cause of pyr resistance with the highest repulsive interaction energy. Therefore, CoMFA and particular interaction energy analyses can be useful for identifying the structural features of potent pyr derivatives active against quadruple mutant type PfDHFR.

Genomic analysis of a multidrug-resistant strain of enterohaemorrhagic Escherichia coli O157 : H7 causing a family outbreak in Japan

A family outbreak of enterohaemorrhagic Escherichia coli (EHEC) O157:H7 infection occurred in October 2003 in the Hiroshima prefecture, Japan. Four isolates of EHEC O157:H7, 03064, 03065, 03066 and 03067, were recovered from a 1-year-old daughter, mother, father and 3-year-old daughter, respectively. All EHEC O157:H7 isolates were positive for Stx1 and Stx2 Shiga toxins. Surprisingly, DNA fingerprinting profiles obtained by PFGE showed that the first isolate, 03064, had unique XbaI and BlnI profiles that differed from the other three isolates. Also, plasmid analysis results revealed that isolate 03064 contained an extra plasmid larger than the classic large plasmid of EHEC O157, pO157 (93.6 kb). This new plasmid was named pMDR157. Furthermore, isolate 03064 showed a multidrug-resistance (MDR) phenotype against streptomycin, spectinomycin, co-trimoxazole (trimethoprim/sulfamethoxazole), ampicillin and tetracycline; the other isolates were completely sensitive to these antibiotics. Molecular analysis of the MDR phenotype in this unique strain revealed the presence of a class 1 integron containing two gene cassettes: a dihydrofolate reductase type 1 gene (dfrI), which confers resistance to trimethoprim, and an aminoglycoside adenyltransferase gene (aadA1), which confers resistance to streptomycin and spectinomycin. Southern blot hybridization showed that the class 1 integron was located in the extra plasmid, pMDR157. The ampicillin resistance was found to be due to the presence of the TEM-1-type beta-lactamase gene. The MDR phenotype was transferred successfully to E. coli HB101 by conjugation, indicating that both the class 1 integron and the TEM-1 beta-lactamase were located on the conjugative transferable plasmid, pMDR157. To the authors' knowledge, this is the first report of the identification of a beta-lactamase gene in EHEC O157.

Molecular characterization of a multidrug-resistant strain of enteroinvasive Escherichia coli O164 isolated in Japan

Enteroinvasive Escherichia coli (EIEC) O164 strain RIMD05091045 was isolated from a travelling patient suffering from diarrhoea at the Osaka airport quarantine facility in Japan. The strain showed multidrug resistance against streptomycin, spectinomycin, co-trimoxazole (trimethoprim/sulfamethoxazole) and ampicillin, and reduced susceptibility to ciprofloxacin. Molecular characterization of the multidrug-resistance phenotype revealed the presence of a class 1 integron containing three genes, a dihydrofolate reductase type XII gene, dfrXII, which confers resistance to trimethoprim, an aminoglycoside adenyltransferase gene, aadA2, which confers resistance to streptomycin and spectinomycin, and an ORF of unknown function. Southern blot hybridization and conjugation experiments showed that the class 1 integron was located on a transferable plasmid that was less than 90 kb in size. The resistance of EIEC O164 to ampicillin was found to be due to the presence of TEM-1 beta-lactamase. On the other hand, a single mutation that has not previously been described, P158-to-S, was detected downstream of the quinolone-resistance-determining region of parC of topoisomerase IV and may be responsible for the reduced susceptibility to ciprofloxacin in this strain.

Characterization of multi-drug resistant Salmonella typhi isolated from Pakistan.

Thirty-nine strains of Salmonella typhi, isolated in 1995 from four Districts in Pakistan, Rawalpindi, Islamabad, Kharian and Jehlem, were catalogued and examined. Chromosomal DNA from each isolate was digested with XbaI restriction endonuclease and subjected to pulsed-field gel electrophoresis. Three clonal variants comprising of 17-19 DNA fragments were identified. Antibiotic susceptibility testing identified that 37 of the S. typhi were resistant to chloramphenicol, trimethoprim and ampicillin. These antibiotic resistance genes were found to be located on one of four plasmids belonging to incompatibility group IncHI1 and ranging in size from 150-175 Kb. The genes responsible for this resistance in each case were the chloramphenicol acetyltransferase (CAT) type I, the dihydrofolate reductase (DHFR) type VII and the beta-lactamase TEM-1 respectively.

New gene cassettes for trimethoprim resistance, dfr13, and Streptomycin-spectinomycin resistance, aadA4, inserted on a class 1 integron.

In a previous survey of 357 trimethoprim-resistant isolates of aerobic gram-negative bacteria from commensal fecal flora, hybridization experiments showed that 25% (90 of 357) of the isolates failed to hybridize to specific oligonucleotide probes for dihydrofolate reductase types 1, 2b, 3, 5, 6, 7, 8, 9, 10, and 12. Subsequent cloning and sequencing of a plasmid-borne trimethoprim resistance gene from one of these isolates revealed a new dihydrofolate reductase gene, dfr13, which occurred as a cassette integrated in a site-specific manner in a class 1 integron. The gene product shared 84% amino acid identity with dfr12 and exhibited a trimethoprim inhibition profile similar to that of dfr12. Gene probing experiments with an oligonucleotide probe specific for this gene showed that 12.3% (44 of 357) of the isolates which did not hybridize to probes for other dihydrofolate reductases hybridized to this probe. Immediately downstream of dfr13, a new cassette, an aminoglycoside resistance gene of the class AADA ¿ANT(3")(9)-I, which encodes streptomycin-spectinomycin resistance, was identified. This gene shares 57% identity with the consensus aadA1 (ant(3")-Ia) and has been called aadA4 (ant(3")-Id). The 3' end of the aadA4 cassette was truncated by IS26, which was contiguous with a truncated form of Tn3. On the same plasmid, pUK2381, a second copy of IS26 was associated with sul2, which suggests that both integrase and transposase activities have played major roles in the arrangement and dissemination of antibiotic resistance genes dfr13, aadA4, bla(TEM-1), and sul2.

Conjugative mobilization of the rolling-circle plasmid pIP823 from Listeria monocytogenes BM4293 among gram-positive and gram-negative bacteria.

We determined the sequence and genetic organization of plasmid pIP823, which contains the dfrD gene; dfrD confers high-level trimethoprim resistance to Listeria monocytogenes BM4293 by synthesis of dihydrofolate reductase type S2. pIP823 possessed all the features of the pUB110/pC194 plasmid family, whose members replicate by the rolling-circle mechanism. The rep gene encoded a protein identical to RepU, the protein required for initiation of the replication of plasmids pTB913 from a thermophilic Bacillus sp. and pUB110 from Staphylococcus aureus. The mob gene encoded a protein with a high degree of amino acid identity with the Mob proteins involved in conjugative mobilization and interplasmidic recombination of pTB913 and pUB110. The host range of pIP823 was broad and included L. monocytogenes, Enterococcus faecalis, S. aureus, Bacillus subtilis, and Escherichia coli. In all these species, pIP823 replicated by generating single-stranded DNA and was stable. Conjugative mobilization of pIP823 was obtained by self-transferable plasmids between L. monocytogenes and E. faecalis, between L. monocytogenes and E. coli, and between strains of E. coli, and by the streptococcal conjugative transposon Tn1545 from L. monocytogenes to E. faecalis, and from L. monocytogenes and E. faecalis to E. coli. These data indicate that the gene flux observed in nature from gram-positive to gram-negative bacteria can occur by conjugative mobilization. Our results suggest that dissemination of trimethoprim resistance in Listeria spp. and acquisition of other antibiotic resistance determinants in this species can be anticipated.

Increase in incidence of resistance to ampicillin, chloramphenicol and trimethoprim in clinical isolates of Salmonella serotype Typhimurium with investigation of molecular epidemiology and mechanisms of resistance.

Antimicrobial resistance patterns of Salmonella serotype Typhimurium isolates obtained during the period 1987-1994 were examined and the molecular epidemiology and the mechanisms of resistance to ampicillin, chloramphenicol and trimethoprim were investigated in 24 strains isolated during 1994. Resistance to ampicillin increased from 18% to 78%, to chloramphenicol from 15% to 78%, to tetracycline from 53% to 89% and to co-trimoxazole from 3% to 37%, whereas resistance to norfloxacin remained at 0%. Of Salmonella serotype Typhimurium strains isolated during 1994, all ampicillin-resistant strains had an MIC > 256 mg/L, except one strain in which the MIC was 64 mg/L. Twelve strains (52%) had a TEM-type beta-lactamase, nine (39%) a CARB-type beta-lactamase and two strains (8%) had an OXA-type beta-lactamase. Chloramphenicol acetyl-transferase activity was detected in only nine (47%) of 19 chloramphenicol resistant strains, whereas all eight trimethoprim-resistant strains produced a dihydrofolate reductase type Ia enzyme. Three different epidemiological groups were defined by either low-frequency restriction analysis of chromosomal DNA and pulsed-field gel electrophoresis or repetitive extragenic palindromic-PCR. The latter technique provided an alternative, rapid and powerful genotyping method for S. Typhimurium. Although quinolones provide a good therapeutic alternative, the multiresistance of S. Typhimurium is of public health concern and it is important to continue surveillance of resistance levels and their mechanisms.

Molecular analysis of and identification of antibiotic resistance genes in clinical isolates of Salmonella typhi from India.

A representative sample of 21 Salmonella typhi strains isolated from cultures of blood from patients at the Christian Medical College and Hospital, Vellore, India, were tested for their susceptibilities to various antimicrobial agents. Eleven of the S. typhi strains possessed resistance to chloramphenicol (256 mg/liter), trimethoprim (64 mg/liter), and amoxicillin (>128 mg/liter), while four of the isolates were resistant to each of these agents except for amoxicillin. Six of the isolates were completely sensitive to all of the antimicrobial agents tested. All the S. typhi isolates were susceptible to cephalosporin agents, gentamicin, amoxicillin plus clavulanic acid, and imipenem. The antibiotic resistance determinants in each S. typhi isolate were encoded by one of four plasmid types. Plasmid-mediated antibiotic resistance genes were identified with specific probes in hybridization experiments; the genes responsible for chloramphenicol, trimethoprim, and ampicillin resistance were chloramphenicol acetyltransferase type I, dihydrofolate reductase type VII, and TEM-1 beta-lactamase, respectively. Pulsed-field gel electrophoresis analysis of XbaI-generated genomic restriction fragments identified a single distinct profile (18 DNA fragments) for all of the resistant isolates. In comparison, six profiles, different from each other and from the resistance profile, were recognized among the sensitive isolates. It appears that a single strain containing a plasmid conferring multidrug-resistance has emerged within the S. typhi bacterial population in Vellore and has been able to adapt to and survive the challenge of antibiotics as they are introduced into clinical medicine.

Molecular characterization of trimethoprim resistance in salmonellas isolated in Sicily, 1985–1988

The occurrence of trimethoprim (Tp) resistance in salmonellas isolated from humans and water samples in Sicily between 1985 and 1988 has been investigated and the Tp resistance mechanisms have been further characterized on the basis of hybridization with probes for the dihydrofolate reductase (DHFR) genes types I, II, IV and V. Of 765 strains examined, high level (> 1000 mg/l) resistance to Tp was identified in 23 strains (3%). In 22 of these strains, such resistance was associated with resistance to sulphonamides. Six serovars with Tp-resistant strains were identified, Salmonella typhimurium (14 strains), S. enteridis (2), S. agona (2), S. mbandaka (2), S. virchow (2), S. indiana (1). In all strains with high level Tp resistance, resistance to this antimicrobial was plasmid-encoded, in most strains by plasmids with MWs ranging from 70-100 MDa. On the basis of restriction endonuclease analysis, four different categories of Tp resistance plasmids were identified in Tp-resistant strains of S. typhimurium. Hybridization with the DHFR I probe was observed in three strains of Tp-resistant S. typhimurium and two strains of Tp-resistant S. enteritidis; in contrast, in none of the strains tested was there any detectable hybridization with the probes for DHFR types II, IV and V. It is concluded that the DHFR type I resistance mechanism, common in Tp-resistant enterobacteria in many European countries, is relatively uncommon in Tp-resistant salmonellas isolated in Sicily. Furthermore, the DHFR V resistance mechanism, previously identified in strains of Shigella sonnei isolated in Sicily and associated with travellers from Sri Lanka, has not yet appeared in salmonellas in Sicily.

In vivoR–plasmid transfer in a patient with a mixed infection of shigella dysentery

Transfer of shigella R-plasmids in vivo has seldom been demonstrated. Strains of Shigella dysenteriae type 1 and Shigella flexneri type 5b were isolated from a Bulgarian traveller who visited Vietnam and developed dysentery, which was treated with trimethoprim/sulfamethoxazole (TMP/SMZ) for a short time. Both species of shigellae are unusual in Bulgaria where strains of S. sonnei predominate. Both shigella strains were multiresistant to the same antimicrobial agents. Each strain contained a 48-kilobase plasmid that conferred the entire resistance phenotype to a susceptible Escherichia coli. Restriction endonuclease patterns of plasmid DNA from the respective strains were identical. Transmissible plasmids of the same resistance phenotypes and restriction patterns were isolated from the patient's colonic E. coli. Transconjugants hybridized to a dihydrofolate reductase type I-DNA probe. These studies support the hypothesis that R-plasmid transfer may occur between non-pathogenic, faecal strains and pathogenic shigellae, a process that may have been facilitated by inadequate treatment with TMP/SMZ at the onset of the illness.

Nucleotide sequence of dihydrofolate reductase type VI

The complete sequence of the type VI dihydrofolate reductase (DHFR) gene from plasmid pUK672 was determined. The structural gene coded for a polypeptide of 157 amino acids which had a deduced mol. wt of 17,424. Comparison with amino-acid sequences of the type I, type V and Escherichia coli K12 chromosomal DHFRs showed that there was 63%, 61% and 31% homology respectively. Putative RNA polymerase and ribosomal binding sites were identified proximal to the initiation codon and a feature consistent with transcription termination was present distal to the coding region. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis showed that the enzyme had a subunit mol. wt of 17,500.

Resistance to trimethoprim and 2,4-diamino-6,7-diisopropyl-pteridine ( [formula omitted]) in Pasteurella haemolytica

Thirteen strains of Pasteurella haemolytica resistant to moderate levels of trimethoprim (MICs from 8 to 64 μg/ml and 0 129 (MICs from 16 to 64 μg/ml) were isolated from bovine specimens. Two strains, CNP330 and CNP334, were studied and found to harbour various plasmids but all attempts to cure trimethoprim resistance were unsuccessful. Resistance characters were not transferable to Escherichia coli or the Pasteurella multocida by conjugation and to E. coli by transformation. The resistance gene(s) was therefore tentatively assigned to a chromosomal location and cloned in E. coli where it conferred trimethoprim resistance. Trans-complementation analysis of a dihydrofolate reductase-deficient mutant of E. coli showed that trimethoprim resistance was secondary to synthesis of a dihydrofolate reductase. DNA/DNA hybridization of the hybrid plasmid and of strains CNP330 and CNP334 with probes specific for dihydrofolate reductase types I to V were negative, indicating that cross-resistance to trimetrhoprim and 0 129 in P. haemolytica was due to the acquisition by P. haemolytica of a new resistance determinant.

Cloning of the type Ib trimethoprim-resistant dihydrofolate reductase gene and preparation of a specific biotinylated DNA probe

A 3.0-kb PstI/HindIII fragment encoding the type Ib trimethoprim-resistant dihydrofolate reductase (DHFR) was cloned into pUC18. Following successive subcloning, the type Ib DHFR gene was identified within a 500 bp HincII fragment. The 500 bp fragment was purified, biotinylated and tested for its suitability as a specific DNA probe in non-isotopic hybridisation experiments with standard plasmids carrying genes for the available known Gram-negative DHFR types. Under conditions of high stringency the probe was specific for the type Ib gene and hence will be useful for monitoring the spread of this particular trimethoprim resistance gene.

Molecular characterization of trimethoprim resistance inShigella sonneiin Sicily

During the 3-year period 1985-7, all strains of Shigella sonnei isolated in Catania, Sicily, showed a high level of resistance to trimethoprim (Tp) which was invariably associated with resistance to other antibiotics. Plasmid analysis showed 18 different electropherotypes: 35 of 37 strains harboured a plasmid of 70 Megadaltons (MDa), and 29 of 37 strains a plasmid of 130 MDa. Restriction endonuclease fingerprinting of purified 70 MDa plasmid DNA from different strains demonstrated that these plasmids were similar but not identical. In some strains with transferable Tp resistance, DNA hybridization analysis demonstrated the presence of gene coding for the production of dihydrofolate reductase (DHFR) type V. In contrast, there was no detectable hybridization with DNA probes specific for genes coding for DHFR types I, II and IV. This is the first report of the DHFR type V gene outside Sri Lanka.