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PubMed1990

Single base alterations upstream of the E. coli 16S rRNA coding region result in temperature-sensitive 16S rRNA expression.

We have isolated three new temperature-sensitive mutants of 16S rRNA, using the U1192 spectinomycin resistance as a selectable marker. These differ from our previously characterized ts mutants in that they map in the upstream leader region of the rRNA precursor (at positions -13, -30 and -59). The relative distribution of plasmid and chromosome-derived 16S rRNA is similar between 30S subunits, 70S ribosomes and polysomes at the permissive and restrictive temperatures. Processing of the 5' end of the RNA does not appear to be affected by the mutations. Second-site suppressors were found, and all of these except one (which is within 16S rRNA) were also due to point mutations in the upstream leader.

Base Sequence↗
PubMed1987

Antibiotic dependence in a strain of Neisseria pharyngis.

A strain of Neisseria pharyngis was isolated in pure culture from a specimen of sputum. Growth was markedly enhanced by sub-lethal concentrations of rifampicin, spectinomycin, erythromycin and clindamycin. The effect was temporary with tetracycline. The possible mechanisms of this phenomenon are discussed.

Adult↗
PubMed1984

In vitro insertional mutagenesis with a selectable DNA fragment.

A new method for in vitro insertional mutagenesis of genes cloned in Escherichia coli is presented. This simple procedure combines the advantages of in vitro DNA linker mutagenesis with those of in vivo transposition mutagenesis. It makes use of the omega fragment, a 2.0-kb DNA segment consisting of an antibiotic resistance gene (the Smr/Spcr gene of the R100.1 plasmid) flanked by short inverted repeats carrying transcription and translation termination signals and synthetic polylinkers. The omega fragment is inserted into a linearized plasmid by in vitro ligation, and the recombinant DNA molecules are selected by their resistance to streptomycin and spectinomycin. The omega fragment terminates RNA and protein synthesis prematurely, thus allowing the definition and mapping of both transcription and translation units. Because of the symmetrical structure of omega, the same effect is obtained with insertions in either orientation. The antibiotic resistance gene can be subsequently excised from the mutated molecules, leaving behind its flanking restriction site(s).

Base Sequence↗
PubMed1994

Fusidic acid-resistant mutants define three regions in elongation factor G of Salmonella typhimurium.

We have sequenced fusA, the gene coding for elongation factor G (EF-G), in 18 different mutants of Salmonella typhimurium selected as fusidic acid resistant (FuR). In addition, we have sequenced two previously described FuR mutants from Escherichia coli. In all cases, the resistance is due to a mutation in one of three separate regions in fusA. The three clusters of mutant sites superimpose on regions that are well conserved, suggesting that they are of a more general functional importance. To further classify the mutants, we have measured the minimal inhibitory concentration (MIC) for Fu and for two other antibiotics which interfere with translocation on the ribosome, kanamycin (Km) and spectinomycin (Sp). The levels of resistance to Fu for each of the mutants are significantly higher than in the wild type (wt), and vary by about one order of magnitude between the highest and the lowest. Most of the mutants are also more resistant to Km than the wt, although the level of resistance is low and the variation small. In contrast, about half of the mutants are more sensitive to Sp than the wt, with only one being more resistant. Only three of the twenty mutants behave like the wt with respect to the non-selected phenotypes, KmR and SpR.

Bacterial Proteins↗
PubMed1995

Construction and evaluation of new drug-resistance cassettes for gene disruption mutagenesis in Streptococcus pneumoniae, using an ami test platform.

Although drug-resistance markers have been used frequently for gene-disruption mutagenesis in Streptococcus pneumoniae, none has yet been shown to be free of dependence on local transcription for its expression. Indeed, the erythromycin-resistance marker (erm), originating in pAM beta 1, has been used as an indicator of local transcription on several occasions. A procedure is demonstrated for evaluation of the autonomous expression of such a marker by placing it in a consistent background, at the pneumococcal ami (aminopterin resistance) locus, in combination with active or inactive alleles of the ami promotor (pA). Using this test platform, a chloramphenicol-resistance marker (cat) and a spectinomycin-resistance marker used in streptococcal gene disruption studies and derived from pJS3 and pDL269, respectively, were shown to depend on local transcriptional signals for expression when placed in the pneumococcal chromosome as single-copy genes. To overcome this limitation, new drug-resistance cassettes were designed and constructed, using pA as a model for synthetic promoters for the erm and cat genes. Both new cassettes were shown, by the same procedure, to be expressed after insertion in the pneumococcal chromosome, independent of local transcription. A new insertion-duplication vector, pEVP3, incorporating the new cat cassette and a lacZ reporter derived from pTV32, was also constructed. The ami test platform was used to demonstrate both the autonomous expression of cat and the reporter function of lacZ in chromosomal copies of pEVP3.

Amino Acid Sequence↗
PubMed1993

Adaptation of the Sensititre broth microdilution technique to antimicrobial susceptibility testing of Mycoplasma hyopneumoniae.

A broth microdilution technique is described for determining the antimicrobial susceptibility of Mycoplasma hyopneumoniae, using commercially prepared Sensititre plates. Twenty-five field isolates and two reference strains (J & 232), were tested against seven antimicrobials. Field isolates were tested in duplicate and reference strains, four times to estimate reproducibility. Ninety-seven percent of the duplicate MIC results for the field isolates were in agreement, or within one log2 dilution. Similar results were obtained with the reference strains. The isolates were susceptible to lincomycin-spectinomycin, tylosin and oxytetracycline or resistant to amoxycillin, apramycin and erythromycin. Susceptibility to furaltadone varied. This method retains the accuracy and reproducibility of broth MIC determinations, while avoiding the lengthy preparation of antimicrobial dilutions normally associated with more traditional methods.

Amoxicillin↗
PubMed1988

Antimicrobial agent resistance in Neisseria gonorrhoeae in St. Paul Minnesota.

In a prospective survey of 283 Neisseria gonorrhoeae isolated in St. Paul, Minnesota, 5% were found to produce beta-lactamase, and 11% were resistant to penicillin by chromosomal mediation, thus indicating a hyperendemic level of resistance according to Centers for Disease Control guidelines. A significant level of chromosomal resistance (30%) was noted for tetracycline, and MICs for spectinomycin approached the upper limits of susceptibility for a large number of isolates. The need for uniformity in agar dilution test methodology is discussed, and the variability in antimicrobial agent interpretive breakpoint criteria is illustrated by comparing penicillin breakpoints published by the Centers for Disease Control with those of the National Committee for Clinical Laboratory Standards as applied to the study isolates.

Ceftriaxone↗
PubMedDate not supplied

Cefmetazole and trospectomycin in vitro susceptibility testing interpretive criteria and quality control guidelines for Neisseria gonorrheae.

Cefmetazole and trospectomycin were tested in a multilaboratory trial to establish Neisseria gonorrhoeae susceptibility testing criteria and quality control (QC) guidelines. Cefmetazole was active against the penicillinase-producing isolates and has an MIC90 of 16 micrograms/ml, the breakpoint MIC previously used for nonfastidious species. However, a single-dose gonorrhea regimen (1 g i.m.) would require a lower less than or equal to 2 micrograms/ml breakpoint with a correlate zone (greater than or equal to 33 mm) consistent with similarly used cephamycins (cefoxitin and cefotetan). An intermediate category was proposed for MICs greater than 2-4 micrograms/m (28-32 mm) pending more clinical experience with higher and/or prolonged cefmetazole dosing regimens. Trospectomycin was active (MIC90, 8 micrograms/ml) against all spectinomycin-susceptible gonococci. A susceptible breakpoint MIC of less than or equal to 16 micrograms trospectomycin per milliliter was proposed with a correlate zone diameter of greater than or equal to 17 mm. An intermediate category was also suggested for trospectomycin at 32 micrograms/ml (14-16 mm). QC guidelines were established for 30-micrograms cefmetazole and 30-micrograms trospectomycin disk diffusion tests and the GC agar base MICs using a multilaboratory study design consistent with National Committee for Clinical Laboratory Standards (NCCLS) M23-T guidelines. Both drugs were stable in GC agar plates for 21 days stored at 2 degrees-5 degrees C.

Cefmetazole↗
PubMedDate not supplied

In vitro antibacterial activity of trospectomycin (U-63,366F) against anaerobic bacteria and aerobic gram-positive cocci in Chile.

The in vitro activity of trospectomycin sulfate was compared with those of several antimicrobials, against 301 anaerobic bacteria and 613 aerobic Gram-positive cocci. Trospectomycin was about 4- to 32-fold more active than was spectinomycin. Trospectomycin exhibited consistently good activity against all Bacteroides fragilis group isolates, except Bacteroides vulgatus, and against all other anaerobes comparable or higher to that of clindamycin. The trospectomycin's activity was most similar to that of vancomycin, even against methicillin-resistant Staphylococcus aureus and methicillin-resistant Staphylococcus epidermidis.

Anti-Bacterial Agents↗
PubMed2004

A plasmid-encoded class 1 integron carrying sat, a putative phosphoserine phosphatase gene and aadA2 from enterotoxigenic Escherichia coli O159 isolated in Japan.

A class 1 integron was detected in a single multidrug-resistant strain of enterotoxigenice Escherichia coli (ETEC) O159 after examination of 23 clinical E. coli isolates. This isolate was resistant to streptomycin, kanamycin, gentamicin, chloramphenicol and ampicillin. Sequencing of the class 1 integron identified three-gene cassettes. The first is the streptothricin acetyltransferase gene, sat, which confers resistance to streptothricin. The second is an ORF whose product is a putative phosphoserine phosphatase (PSP), and the last is an aminoglycoside adenyltransferase gene, aadA2, which confers resistance to streptomycin and spectinomycin. The putative PSP gene product was found to be 39%, 38%, 28%, and 27% identical to PSP gene products of Vibrio vulnificus CMCP6, V. vulnificus YJ016, Pseudomonas syringae, and P. aeruginosa, respectively. Southern-blot hybridization showed that this integron is located on a 90 kb plasmid. This is the first report identifying a putative PSP gene in an integron.

Acetyltransferases↗
PubMed2005

Cryptic plasmid pRK2 from Escherichia coli W: sequence analysis and segregational stability.

Cryptic plasmid pRK2 of the strain Escherichia coli W (ATCC 9637), an ancestor of production strains for penicillin G acylase, was sequenced and characterized. Based on the data on replication region and origin (ori sequence AAC, 924-926nt), the plasmid was classified as ColE1-like plasmid. DNA sequence analysis revealed five orfs hypothetical products of which shared a significant sequence similarity with putative proteins encoded by DNA of plasmid pColE1. orf1 codes for protein Rom involved in the control of plasmid replication, orfs 2-5 code for putative mobilization proteins (Mob A-D) that show a high level of similarity with the ones encoded by DNA of plasmids pColE1 and pLG13 (E. coli), pECL18 and pEC01 (Enterobacter cloacae), pSFD10 (Salmonella choleraesuis), and pScol7 (Shigella sonnei). Recombinant plasmids pRS11 (4.91kbp), pRS12 (4.91kbp), pRS2 (2.996kbp), and pRS3 (2.623kbp) that bear the Spectinomycin resistance determinant (Spc(R)) were prepared on the basis of nucleotide sequence of pRK2. These constructs are stably maintained in the population of E. coli cells grown in the absence of the selection pressure for 63 generations. The copy number of Spc(R) constructs in E. coli host grown in antibiotic-free LB medium ranges from 25 to 40 molecules per chromosomal equivalent.

Bacterial Proteins↗
PubMed2005

Development of a shuttle vector for Moraxella catarrhalis.

Efforts to perform genetic analysis in Moraxella catarrhalis have been hampered by the lack of a cloning vector. M. catarrhalis strain E22 was previously shown to contain plasmid pLQ510 which lacked a selectable antibiotic resistance marker. Several methods were used to eliminate unnecessary DNA from pLQ510. Then, a 1.2 kb spectinomycin resistance cartridge, a multiple cloning site, and the origin of replication from pACYC184 were cloned into this plasmid backbone to obtain the 7.2 kb plasmid pWW102B. This new plasmid could replicate in M. catarrhalis as well as in both Escherichia coli and Haemophilus influenzae. This shuttle vector was used to clone and express two different M. catarrhalis genes, respectively, encoding an adhesin and a protein involved in serum resistance. When these two plasmids were introduced into appropriate M. catarrhalis mutants, they complemented the phenotypic deficiency of each mutant. This is the first report of functional complementation in trans in this pathogen.

Adhesins, Bacterial↗
PubMed1976

Differential response of chlamydial and ureaplasma-associated urethritis to sulphafurazole (sulfisoxazole) and aminocyclitols.

91 men with non-gonococcal urethritis (N.G.U.) were randomly treated with either sulphafurazole (sulfisoxazole), 500 mg orally q.i.d. for 10 days, or an aminocyclitol (streptomycin or spectinomycin), 2 g intramuscularity for 1 to 3 doses at 12 h intervals. Initial urethral cultures were positive for Chlamydia trachomatis (C) in 36 (40%). Ureaplasma urealyticum (U) was isolated from the urethra or urine from20 (95%) of 21 White men in a first episode of N.G.U. who had negative chlamydia cultures. Sulphafurazole, active against C. trachomatis but not U. urealyticum in vitro, produced a clinical response in 7 of 7 men with C+U- N.G.U. and 5 of 19 with C-U+ N.G.U. (P less than 0-01). Aminocyclitols, active against U. urealyticum but relatively inactive against C. trachomatis in vitro produced a clinical response in 0 of 6 men with C+U-N.G.U., 9 of 11 men with C-U+N.G.U. from whom ureaplasma was eradicated (P less than 0-01), and 0 of 8 with C-U+ N.G.U. from whom ureaplasma was not eradicated. C+U+ N.G.U. responded poorly to both antimicrobials alone. These results support the aetiological importance of both C. trachomatis and U. urealyticum in N.G.U.

Administration, Oral↗
PubMed2002

The 27.8-kb R-plasmid pTET3 from Corynebacterium glutamicum encodes the aminoglycoside adenyltransferase gene cassette aadA9 and the regulated tetracycline efflux system Tet 33 flanked by active copies of the widespread insertion sequence IS6100.

We determined the complete nucleotide sequence of the 27.8-kb R-plasmid pTET3 from Corynebacterium glutamicum LP-6 which encodes streptomycin, spectinomycin, and tetracycline resistance. The antibiotic resistance determinant of pTET3 comprises an intI1-like gene, which was truncated by the insertion sequence IS6100, and the novel aminoglycoside adenyltransferase gene cassette aadA9. The deduced AADA9 protein showed 61% identity and 71% similarity to AADA6 of integron In51 from Pseudomonas aeruginosa. In addition, pTET3 carries the novel repressor-regulated tetracycline resistance determinant Tet 33 which revealed amino acid sequence homology to group 1 tetracycline efflux systems. The highest level of similarity was observed to the tetracycline efflux protein TetA(Z) from the C. glutamicum plasmid pAG1 with 65% identical and 77% similar amino acids. Each antibiotic resistance region of pTET3 is flanked by identical copies of the widespread insertion sequence IS6100 initially identified in Mycobacterium fortuitum. Transposition assays with a cloned copy of IS6100 revealed that this element is transpositionally active in C. glutamicum. These data suggest a central role of IS6100 in the evolutionary history of pTET3 by mediating the cointegrative assembly of resistance gene-carrying DNA segments.

Amino Acid Sequence↗
PubMed1986

Eliminating organisms against Treponema hyodysenteriae in the gut of mice.

Ta:CF#1 mice lost their high susceptibility to Treponema hyodysenteriae infection after being mingled with Slc:ICR mice which had no susceptibility to the infection. Ta:CF#1 mice also lost susceptibility to the infection after being treated orally with fecal suspensions from Slc:ICR mice; treatment with heated (70 degrees C, 15 min) or filtered (pore size 0.2 micron) fecal suspensions did not cause a loss of susceptibility. Furthermore, even after the infection had established, Ta:CF#1 mice became free from T. hyodysenteriae and cecal lesions by oral treatment with the fecal suspensions. When Slc:ICR mice were treated with spectinomycin throughout the experiment, they became susceptible to the infection. These studies indicate that there exist eliminating organisms against T. hyodysenteriae in the gut of mice.

Animals↗
PubMed1976

Diagnosis and treatment of pelvic inflammatory disease and gonorrhea in the emergency department.

One hundred emergency department charts on cases of suspected pelvic inflammatory disease (PID) were reviewed to determine method of diagnosis and treatment. Seventy-three gonococcus smears, 60 gonococcus cultures, and 53 VDRL tests for syphilis were done. Of these, 20 smears, 19 cultures, and 1 VDRL were positive. All 100 patients were treated for gonorrhea with either spectinomycin, tetracycline, ampicillin or penicillin. The author concludes there is no need to procure VDRL tests on all patients with suspected gonorrhea, nor do gonococcus cultures on all patients with suspected PID. Also, since only 39% of patients had a positive gonococcus smear or culture, penicillin may not always be the appropriate treatment.

Ampicillin↗
PubMed1989

Trospectomycin enhances surface phagocytosis of Bacteroides and Staphylococcus by altering the bacterial glycocalyx.

The influence of trospectomycin on phagocytosis of Bacteriodes thetaiotaomicron, Bacteroides fragilis and Staphylococcus epidermidis was studied in the presence or absence of glycocalyx isolated from these microorganisms. Bacteria were grown with or without 0.25 or 0.5 of the minimal inhibitory concentration (MIC) of trospectomycin, a new chemically synthesized analog of spectinomycin. Surface phagocytosis by human polymorphonuclear leukocytes (PMNL) was determined using a modified fluorochrome assay. Subinhibitory concentrations of trospectomycin significantly enhanced surface phagocytosis of Bacteroides and Staphylococcus. When homologous or heterologous isolated glycocalyx was added to trospectomycin treated bacteria prior to incubation with PMNL, phagocytosis was reduced to levels observed in the untreated bacteria. Addition of glycocalyx to untreated strains produced no significant reduction of phagocytosis. The glycocalyx preparations were free of lipopolysaccharide and did not affect PMNL viability.

Anti-Bacterial Agents↗
PubMed1973

The incidence of antibiotic resistance and other characteristics amongst Escherichia coli strains causing fatal infection in chickens: the utilization of these characteristics to study the epidemiology of the infection.

Of 173 epidemiologically unrelated strains of Escherichia coli isolated from the pericardial sac of chickens that had died from infection with these organisms in England in 1972, approximately 1 year after the introduction of legislation forbidding the routine use of feeds containing ;therapeutic' antibiotics, 83.8% were resistant to sulphonamides, 31.2% to tetracyclines, 20.8% to furazolidone, 18.5% to streptomycin, 2.9% to spectinomycin and 1.2% to ampicillin; none of the strains were resistant to chloramphenicol, neomycin, polymixin, trimethoprim or nalidixic acid. The sulphonamide resistance and possibly some of the resistance to other agents might have been the consequence of sulphonamides being exempted from the legislation. Much of the resistance, with the exception of that to furazolidone, was of the transferable type. Many strains possessed transfer factors in the absence of any known transferable characteristic. Colicine production was twice as common in the pathogenic strains as in a collection of strains isolated from the faeces of healthy chickens; about half of it was transferable.By means of serology, antibiotic resistance and other markers, it was found that several different kinds of E. coli were usually incriminated in any one outbreak of E. coli infection in broiler chickens. Sometimes the same kinds of E. coli were found in outbreaks in consecutive crops of chickens on the same farm. New kinds, too, appeared to be brought in by replacement chickens.

Ampicillin↗