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PubMed1990

Further studies on the inhibition of colonization of the chicken alimentary tract with Salmonella typhimurium by pre-colonization with an avirulent mutant.

Oral administration to newly hatched chickens or to chicks up to 5 days of age with an avirulent, rough, spectinomycin-resistant mutant of Salmonella typhimurium strain F98 inhibited the colonization of a nalidixic acid-resistant mutant of the same strain administered by the same route 1 day later. The second strain passed rapidly through the alimentary tract and persisted in the caeca of only a few chickens. Resistance to colonization did not develop until 24 h after inoculation of the first strain but was still evident if the second strain was inoculated up to 7 days later. Resistance occurred in 5 different breeds of chicken and in chickens reared on 5 different diets. Protection was evident against a very high challenge dose and could be produced by the introduction of small numbers of the first strain. Pre-colonization of chicks with the first strain of F98 reduced faecal excretion of the second strain over many weeks, whether chickens were challenged directly or by contact with other infected chickens. The rough strain F98 produced protection against only a few S. typhimurium strains and not against other serotypes. However, strains of S. infantis and S. heidelberg, chosen because they colonized the chicken alimentary tract better than did F98, produced inhibition of a wider range of serotypes.

Animal Feed↗

PubMed1995

Beta-lactam resistance in normal faecal flora from South Africa.

The genetic and biochemical basis of ampicillin resistance amongst the aerobic Gram-negative commensal faecal flora of healthy volunteers in South Africa has been determined. Amongst 608 ampicillin resistant strains isolated from 320 of the participants, 158 were able to transfer their ampicillin resistant determinants into Escherichia coli K-12 J62-2. Iso-electric focusing of the beta-lactamases, extracted from the transconjugants, demonstrated that ampicillin resistance resulted from the presence of the TEM-1, TEM-2 and SHV-1 beta-lactamases in 94.3%, 2.5% and 3.2% of isolates respectively. Endonuclease restriction digests of the plasmids isolated from the transconjugants showed that the beta-lactamase genes were present on a wide variety of plasmid types; 101 distinct plasmid endonuclease restriction patterns were identified. Transferable ampicillin resistance was associated with resistance to other antibiotics at the following frequencies: trimethoprim (48.7%), streptomycin (35.4%), tetracycline (27.2%), spectinomycin (9.5%), chloramphenicol (3.2%) and gentamicin (1.3%). One antibiotic resistance pattern, ampicillin and trimethoprim, predominated (28%). In total, 77.9% of the plasmids conferred resistance to other antibiotics raising the possibility that use of any of these agents, not simply ampicillin, may contribute to the maintenance of resistance genes.

Adolescent↗

PubMed1996

Characteristics of gonococci isolated from men with urethritis in Dubai.

Neisseria gonorrhoeae were collected from men attending out-patient clinics in Dubai. The susceptibility to a range of therapeutic antibiotics and their auxotype and serotype was determined. The plasmid content of all penicillinase-producing strains was also analysed. Thirty-six strains of N. gonorrhoeae were isolated from specimens collected from 79 patients over a 24-day period. Of the 36 isolates, 9(25%) were penicillinase-producing N. gonorrhoeae (PPNG) and 15 (42%) were chromosomally resistant N. gonorrhoeae (CMRNG). CMRNG exhibited higher levels of resistance to cefuroxime, chloramphenicol, tetracycline and erythromycin than PPNG. All isolates were susceptible to ceftriaxone and spectinomycin. Three (8%) isolates showed reduced susceptibility (MIC, > or = 0.25 mg/l) to ciprofloxacin. Six isolates of PPNG carried the 4.4 MD and three the 3.2 MD penicillinase encoding plasmid. The total gonococcal population was phenotypically diverse, with 12 serovars, 6 auxotypes and 21 A/S classes. Gonorrhoea was found to be a major cause of urethritis in Dubai and the strains exhibited high levels of resistance to penicillin.

Adolescent↗

PubMed1993

Antimicrobial susceptibilities and serotyping of Neisseria gonorrhoeae in southern Africa: influence of geographical source of infection.

One hundred and ninety-two strains of Neisseria gonorrhoeae isolated from migrant mine-workers were tested for their susceptibility to antibiotics, auxotyped and serotyped. Of the total, 93 (48%) were acquired locally and 64 (33%) from different geographical locations. Plasmid-mediated resistance to penicillin was found in 28 (14.6%) strains and was associated predominantly with the presence of 5.0 kb penicillinase encoding plasmid (18/28, 64%). Chromosomal resistance to penicillin (MIC > or = 1 mg/l) was detected in 14 (7.3%) strains. Resistance to tetracycline was chromosomally and not plasmid-mediated. Antibiotic resistance was encountered most commonly among strains acquired in Natal. The overall gonococcal population was sensitive to ceftriaxone, ciprofloxacin, spectinomycin and azithromycin. Nine auxotype/serovar (A/S) classes were encountered among penicillinase-producing N. gonorrhoeae (PPNG) compared to 24 A/S classes among non-PPNG strains. The most common A/S class was NR/IA-6 which accounted for 38% of PPNG and 15% of non-PPNG.

Africa, Southern↗

PubMed2003

A national outbreak of multi-resistant Salmonella enterica serovar Typhimurium definitive phage type (DT) 104 associated with consumption of lettuce.

Between 1 August and 15 September 2000, 361 cases of Salmonella enterica serotype Typhimurium definitive phage type (DT) 104, resistant to ampicillin, chloramphenicol, streptomycin, sulphonamides, spectinomycin and tetracycline (R-type ACSSuSpT), were identified in England and Wales residents. Molecular typing of 258 isolates of S. Typhimurium DT104 R-type ACSSuSpT showed that, although isolates were indistinguishable by pulsed-field gel electrophoresis, 67% (174/258) were characterized by a particular plasmid profile. A statistically significant association between illness and consumption of lettuce away from home was demonstrated (OR = 7.28; 95% CI=2.25-23.57; P=0.0006) in an unmatched case-control study. Environmental investigations revealed that a number of food outlets implicated in the outbreak had common suppliers of salad vegetables. No implicated foods were available for microbiological testing. An environmental audit of three farms that might have supplied salad vegetables to the implicated outlets did not reveal any unsafe agricultural practices. The complexity of the food supply chain and the lack of identifying markers on salad stuffs made tracking salad vegetables back to their origin extremely difficult in most instances. This has implications for public health since food hazard warnings and product withdrawal are contingent on accurate identification of the suspect product.

Bacteriophage Typing↗

PubMed2002

A general method for selection and screening of coiled coils on the basis of relative helix orientation.

We have developed a method for selecting coiled coils that associate with a given relative helix orientation from a randomized pool of proteins. To select for antiparallel dimers, we have designed a model basic region-leucine zipper (bZip) heterodimer capable of binding DNA only when the coiled coil associates with an antiparallel relative helix alignment. The dimerization domain for this bZip heterodimer is the model antiparallel coiled coil Acid-a1-Base-a1 (Oakley, M. G.; Kim, P. S. Biochemistry 1998, 37, 12603), and both monomers contain the GCN4 basic region. Although the basic regions in naturally occurring bZip proteins are located N-terminal to the leucine zipper, we have attached the GCN4 basic region to the C-terminus of Acid-a1 to allow both basic regions to contact DNA in an antiparallel heterodimer. The resulting heterodimer, BR-Base-a1-Acid-a1-BR, can bind to a direct repeat of the GCN4 half-site in vivo, leading to spectinomycin resistance in the transcription interference assay of Elledge et al. (Elledge, S. J.; Sugiono, P.; Guarente, L.; Davis, R. W. Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 3689). A buried interhelical polar interaction between two Asn residues in the Acid-a1-Base-a1 heterodimer is known to specify an antiparallel helix orientation. The position of one of these buried Asn residues was randomized, and bZip heterodimers containing antiparallel coiled coils were selected using the transcription interference assay. All of the selected colonies contained Asn at the randomized position, suggesting that the selection is specific for antiparallel coiled coils.

Amino Acid Sequence↗

PubMed2001

Characterization of a two-component signal transduction system involved in the induction of alkaline phosphatase under phosphate-limiting conditions in Synechocystis sp. PCC 6803.

The gene products of sll0337 and slr0081 in Synechocystis sp. PCC 6803 have been identified as the homologues of the Escherichia coli phosphate-sensing histidine kinase PhoR and response regulator PhoB, respectively. Interruption of sll0337, the gene encoding the histidine protein kinase, by a spectinomycin-resistance cassette blocked the induction of alkaline phosphatase activity under phosphate-limiting conditions. A similar result was obtained when slr0081, the gene encoding the response regulator, was interrupted with a cassette conferring resistance to kanamycin. In addition, the phosphate-specific transport system was not up-regulated in our mutants when phosphate was limiting. Unlike other genes for bacterial phosphate-sensing two-component systems, sll0337 and slr0081 are not present in the same operon. Although there are three assignments for putative alkaline phosphatase genes in the Synechocystis sp. PCC 6803 genome, only sll0654 expression was detected by northern analysis under phosphate limitation. This gene codes for a 149 kDa protein that is homologous to the cyanobacterial alkaline phosphatase reported in Synechococcus sp. PCC 7942 [Ray, J.M., Bhaya, D., Block, M.A. and Grossman, A.R. (1991) J. Bact. 173: 4297-4309]. An alignment identified a conserved 177 amino acid domain that was found at the N-terminus of the protein encoded by sll0654 but at the C-terminus of the protein in Synechococcus sp. PCC 7942.

Alkaline Phosphatase↗

PubMed2003

Antibiotic resistance analysis of fecal coliforms to determine fecal pollution sources in a mixed-use watershed.

Antibiotic resistance analysis was performed on fecal coliform (FC) bacteria from a mixed-use watershed to determine the source, human or nonhuman, of fecal coliform contamination. The study consisted of discriminant analysis of antibiotic resistance patterns generated by exposure to four concentrations of six antibiotics (ampicillin, gentamicin sulfate, kanamycin, spectinomycin dihydrochloride, streptomycin sulfate, and tetracycline hydrochloride). A reference database was constructed from 1125 fecal coliform isolates from the following sources: humans, domestic animals (cats and dogs), agricultural animals (chickens, cattle, and horses), and wild animals. Based on similar antibiotic resistance patterns, cat and dog isolates were grouped as domestic animals and horse and cattle isolates were grouped as livestock. The resulting average rate of correct classification (ARCC) for human and nonhuman isolates was 94%. A total of 800 FC isolates taken from the watershed during either a dry event or a wet event were classified according to source. Human sources contribute a majority (> 50%) of the baseflow FC isolates found in the watershed in urbanized areas. Chicken and livestock sources are responsible for the majority of the baseflow FC isolates found in the rural reaches of the watershed. Stormwater introduces FC isolates from domestic (approximately 16%) and wild (approximately 21%) sources throughout the watershed and varying amounts (up to 60%) from chicken and livestock sources. These results suggest that antibiotic resistance patterns of FC may be used to determine sources of fecal contamination and aid in the direction of water quality improvement.

Animals↗

PubMedDate not supplied

Transposition of Tn554 does not generate a target duplication.

Transposable elements from prokaryotic and eukaryotic organisms are discrete DNA segments bounded by inverted or directly repeated sequences that insert into non-homologous DNA in a reaction that is independent of the general recombination functions of the host. The mechanisms proposed generally involve a staggered double-stranded scission of the target DNA, ligation to the nicked ends of the transposable element, and replication of the element, resulting in the generation of a directly repeated oligonucleotide target sequence flanking the new copy of the element. Most transposons have a relatively low degree of target site specificity coupled with a low insertion frequency. Tn554, a Staphylococcus aureus transposon which specifies resistances to erythromycin and spectinomycin, displays an unusually high degree of insertion specificity. Tn554 transposes with high efficiency to a unique ('primary') site in the S. aureus chromosome and only rarely (less than 10(-6) per transductant) to other, secondary sites. We report here the nucleotide sequences surrounding the junctions of Tn554 in three independent 'primary' insertions and two 'secondary' insertions of the transposon. Two unusual features are revealed: first, the termini of Tn554 contain neither inverted nor directly repeated sequences. Second, transposition of Tn554 does not generate the short direct repeats of the target DNA that are characteristic of other transposable elements. These results suggest that the mechanism of Tn554 insertion may be significantly different from that of other transposons.

Base Sequence↗

PubMed1977

Plasmids mediating resistance to gentamicin and other antibiotics in Enterobacteriaceae from four hospitals in Melbourne.

In twenty-nine gentamicin-resistant strains (mostly Klebsiella species) isolated from four Melbourne hospitals over a six month period, the genes for gentamicin resistance were carried on conjugative plasmids belonging to the rare incompatibility group L. These plasmids conferred resistance to gentamicin and sisomycin but not to amikacin or tobramycin, and were inferred to specify the enzyme aminoglycoside 3-N-acetyltransferase (AAC(3)I or GAT I). In addition, they specified resistance to a number of other antibiotics, including ampicillin and carbenicillin, chloramphenicol, kanamycin and neomycin, spectinomycin and streptomycin, sulphonamides and tetracyclines. Although they had a wide host range amongst the Enterobacteriaceae, none of these plasmids appeared able to transfer to Pseudomonas.

Anti-Bacterial Agents↗

PubMed1985

A conjugative plasmid encoding production of a diffusible pigment and resistance to aminoglycosides and macrolides in Staphylococcus aureus.

A methicillin-resistant strain of Staphylococcus aureus, WG512, was isolated in 1968 and has been shown to harbour a plasmid, pWG14, which encodes the production of a diffusible pigment and resistance to kanamycin, neomycin, streptomycin, lincomycin, erythromycin and spectinomycin. Plasmid pWG14 has only been detected as a slowly migrating band of DNA during agarose-gel electrophoresis, and this was thought to be the open-circular form of the plasmid with an estimated molecular weight of 30-34 Mdal. The transfer properties of this plasmid were compared with a non-conjugative plasmid encoding tetracycline resistance and the class 2, conjugative plasmid described by Archer and Johnston (1983). Plasmid pWG14 shared many of the conjugative properties of the latter plasmid, including the ability to transfer at high frequency on the surface of a filter membrane and to transfer between non-lysogenic strains in the absence of Ca++ and in the presence of citrate of DNase I. However, unlike the class 2 plasmid, pWG14 was able to transfer in broth culture at low frequency and did not mobilise the non-conjugative plasmid. Furthermore, the class 2 plasmid and pWG14 were shown to belong to different incompatibility groups. A derivative of WG512, which had lost the ability to produce the diffusible pigment along with plasmid pWG14 but had retained the resistance determinants of plasmid pWG14 in the chromosome, was used to demonstrate that the conjugative mechanism of pWG14 was a property of the plasmid and not the resistance determinants.

Aminoglycosides↗

PubMed1997

A two-gene ABC-type transport system that extrudes Na+ in Bacillus subtilis is induced by ethanol or protonophore.

A transposition mutant of Bacillus subtilis (designated JC901) that was isolated on the basis of growth inhibition by Na+ at elevated pH, was deficient in energy-dependent Na+ extrusion. The capacity of the mutant JC901 for Na(+)-dependent pH homeostasis was unaffected relative to the wild-type strain, as assessed by regulation of cytoplasmic pH after an alkaline shift. The site of transposition was near the 3'-terminal end of a gene, natB, predicted to encode a membrane protein, NatB. NatB possesses six putative membrane-spanning regions at its C-terminus, and exhibits modest sequence similarity to regions of eukaryotic Na+/H+ exchangers. Sequence and Northern blot analyses suggested that natB forms an operon with an upstream gene, natA. The predicted product of natA is a member of the family of ATP-binding proteins that are components of transport systems of the ATP-binding cassette (ABC) or traffic ATPase type. Expression of the lacZ gene that was under control of the promoter for natB indicated that expression of the operon was induced by ethanol and the protonophore carbonylcyanide p-chlorophenylhydrazone (CCCP), and more modestly, by Na+, and K+, but not by choline or a high concentration of sucrose. Restoration of the natAB genes, cloned in a recombinant plasmid (pJY1), complemented the Na(+)-sensitive phenotype of the mutant JC901 at elevated pH and significantly increased the resistance of the mutant to growth inhibition by ethanol and CCCP at pH 7; ethanol was not excluded, however, from the cells expressing natAB, so ethanol-resistance does not result from NatAB-dependent ethanol efflux. Transformation of the mutant with pJY1 did markedly enhance the capacity for Na+ efflux, which was further stimulated by CCCP. In the absence of CCCP, NatAB-mediated Na+ efflux was stimulated by K+. Concomitant NatAB-dependent K+ uptake occurred, as monitored by 86Rb+ uptake; this uptake was inhibited by CCCP and is thus secondary to the primary, electrogenic Na+ efflux. A B. subtilis mutant strain (BsAJ96) in which most of natA and all of natB was replaced by a spectinomycin-resistance-gene cassette exhibited phenotypic properties identical to JC901 Under anaerobic conditions, using a strain of B. subtilis deleted in atp genes encoding the F1F0-ATPase (BD99-A), glucose energized Na+ exclusion in an arsenate-sensitive manner; this exclusion capacity was absent in a strain deleted both in atp and natAB genes (BsAJ96-A). We conclude that NatAB is an inducible, ABC transport system that catalyses ATP-dependent electrogenic Na+ extrusion without mechanistically coupled proton or K+ uptake. This is a novel mode of Na+ extrusion that is hypothesized to play an inducible role in exclusion of cytotoxic Na+ and in the secondary stimulation of K+ uptake, especially when the function of the membrane as an ion-permeability barrier is compromised by agents such as alcohols or uncouplers.

ATP-Binding Cassette Transporters↗

PubMed1998

Stable albinism induced without mutagenesis: a model for ribosome-free plastid inheritance.

Maternally inherited chlorophyll deficiency, or albinism, is a standard marker in plant cytoplasmic genetics. Its stability is consistent with mutations in the plastid genome. Nuclear mutations inducing plastid ribosome deficiency (PRD) also lead to maternally inherited chlorophyll deficiency. Here we report that stable chlorophyll deficiency can be efficiently generated in cruciferous plants without mutagenesis by a short exposure to spectinomycin, an inhibitor of plastid protein synthesis. We show that the chlorophyll-deficient phenotype results from a deficiency in plastid ribosomes and plastid translation products. Loss of plastid ribosomes is irreversible. The data suggest that mutations are not essential for generating inheritable PRD. It allows the formulation of a more general model in which stable PRD can be induced by a variety of factors that prevent the formation of functional plastid ribosomes. A non-mutational mechanisms for generating inheritable chlorophyll deficiency has implications for the origin and inheritance of green-white variegation in nature.

Arabidopsis↗

PubMed2000

The two largest chloroplast genome-encoded open reading frames of higher plants are essential genes.

The chloroplast genomes of most higher plants contain two giant open reading frames designated ycf1 and ycf2. In tobacco, ycf1 potentially specifies a protein of 1901 amino acids. The putative gene product of the ycf2 reading frame is a protein of 2280 amino acids. In an attempt to determine the functions of ycf1 and ycf2, we have constructed several mutant alleles for targeted disruption and/or deletion of these two reading frames. The mutant alleles were introduced into the tobacco plastid genome by biolistic chloroplast transformation to replace the corresponding wild-type alleles by homologous recombination. Chloroplast transformants were obtained for all constructs and tested for their homoplastomic state. We report here that all transformed lines remained heteroplastomic even after repeated cycles of regeneration under high selective pressure. A balanced selection was observed in the presence of the antibiotic spectinomycin, resulting in maintenance of a fairly constant ratio of wild-type versus transformed genome copies. Upon removal of the antibiotic and therewith release of the selective pressure, sorting out towards the wild-type plastid genome occurred in all transplastomic lines. These findings suggest that ycf1 and ycf2 are functional genes and encode products that are essential for cell survival. The two reading frames are thus the first higher plant chloroplast genes identified as being indispensable.

Chloroplasts↗

PubMed2000

In vitro susceptibility of Escherichia coli strains isolated from diarrhoeic dairy calves to 15 antimicrobial agents.

The in vitro activities of 15 antimicrobial agents against 195 strains of Escherichia coli isolated from dairy calves affected by neonatal diarrhoea were determined. Of these strains 137 produced one or more potential virulence factors (F5, F41, F17, cytotoxic necrotizing factor, verotoxin and the eae gene), but the remaining 58 strains did not produce any of these factors. The overall percentage of resistant strains to streptomycin, tylosin and tetracycline was very high (above 65%). A high level of resistance (from 23 to 50%) to ampicillin, neomycin, kanamycin, spectinomycin, chloramphenicol, sulphadimethoxine and trimethoprim was also detected. The E. coli strains were very susceptible (89-95%) to apramycin and gentamicin and highly susceptible (99-100%) to polymyxin B, florfenicol and nitrofurazone. Some significant differences (P < 0.05) in the frequencies of resistance to some of the antimicrobials tested and in the rates of multi-drug resistance among the strains producing potential virulence factors and non-fimbriated, non-toxigenic, eae-negative strains were found. Most of the strains showed multi-resistance: 76.9% of the isolates were resistant to at least two antibiotics, 67.7% were resistant to at least four antibiotics and 50.3% were resistant to at least six antibiotics.

Animals↗

PubMed1998

Improved cloning vectors for Bifidobacterium spp.

The recombinant plasmids pDLI41, pDGA7 and pDCO7 were constructed by cloning in pDG7, a vector based on Bifidobacterium longum replicon pMB1, the following heterologous genes: Pseudomonas fluorescens lipase, Bacillus licheniformis alpha-amylase and Streptomyces sp. cholesterol oxidase. The hybrid plasmids efficiently transformed Bifidobacterium belonging to five different species. A novel Escherichia coli-Bifidobacterium set of shuttle vectors based on the replicon pMB1 (pLF5, pCLJ15, pSPEC1) featuring chloramphenicol, erythromycin and spectinomycin resistance genetic determinants as selection marker for bifidobacteria, was developed. The plasmid pTRE3, a derivative of pLF5, was the smallest (2.8 kb) Bifidobacterium vector, possessed a convenient multicloning site and presented high structural and segregational stability.

Bifidobacterium↗

PubMed1979

Cefoxitin as a single-dose treatment for urethritis caused by penicillinase-producing Neisseria gonorrhoeae.

Gonococci that resist standard penicillin regimens by production of a penicillinase are now well established in certain areas of the world. Because cefoxitin, a semisynthetic cephamycin, resists gonococcal penicillinase in vitro, we compared procaine penicillin G and cefoxitin in treatment of gonorrhea in an area where 40 per cent of isolates produce penicillinase. One hundred and seven men with culture-proved gonococcal urethritis were given a single dose of either procaine penicillin G, 4.8 million U, or cefoxitin, 2 g, intramuscularly. Both groups took 1 g of probenecid orally; cefoxitin was given with lidocaine to reduce pain at the injection site. In men infected with penicillinase-negative gonococci, both cefoxitin and penicillin were highly effective. Penicillin failed in 77 per cent of men with penicillinase-positive strains, whereas cefoxitin was completely successful. Cefoxitin is an effective alternative to spectinomycin for single-session therapy of urethritis caused by penicillinase-producing Neisseria gonorrhoeae.

Cefoxitin↗

PubMed1970

Structure and function of bacterial ribosomes. XI. Dependence of 50S ribosomal assembly on simultaneous assembly of 30S subunits.

Some spectinomycin-resistant mutants of Escherichia coli are cold-sensitive. They are unable to assemble both 30S and 50S ribosomal subunits at low temperatures. They accumulate two kinds of incomplete particles, related to 30S and 50S subunits respectively. A single mutation, causing an alteration in a 30S ribosomal component, is responsible for these phenotypes. These results show that assembly of 50S subunits in vivo is dependent on simultaneous assembly of 30S subunits. On the other hand, the assembly of 30S subunits appears to be independent of 50S assembly.

Bacterial Proteins↗