Antibiotic resistance

Vet Microbiol, 1996 Apr, 49(3-4), 209 - 17
Serogroups, toxins and antibiotic resistance of Escherichia coli strains isolated from diarrhoeic lambs in Spain; Blanco J et al.; One hundred and forty-four Escherichia coli strains isolated from 144 diarrhoeic lambs (5 to 21 days old) from 38 flocks in Spain were serotyped and investigated for production of enterotoxins (LT and STa), verotoxins (VT1 and VT2), cytotoxic necrotizing factors (CNF1 and CNF2), alpha-haemolysin (Hly) and enterohaemolysin (EntHly), for necrotic and lethal activities and for antibiotic resistance . The strains belonged to 39 different serogroups; however, 58% were of one of 13 serogroups (O4, O6, O7, O8, O9, O11, O23, O26, O77, O80, O101, O103 and O161) and only four of them (O8, O9, O11 and O77) accounted for 34% of strains . In total 10 (7%) toxigenic strains were detected: two LT+, two VT1+ EntHly+, four VT1+ EntHly-, one CNF1+ Hly+ and one CNF2+ . The highest percentages of antibiotic resistance were reached in the group of antibiotics (tetracycline, streptomycin, sulphadiazine, ampicillin, kanamycin, neomycin, chloramphenicol, trimethoprim and cotrimoxazole) that are most generally used by Spanish veterinary clinics . We conclude that E . coli strains isolated from diarrhoeic lambs are not generally toxigenic and belong to a large number of serogroups.

J Bacteriol, 1996 Apr, 178(8), 2334 - 42
Directed mutagenesis of the Rhodobacter capsulatus puhA gene and orf 214: pleiotropic effects on photosynthetic reaction center and light-harvesting 1 complexes; Wong DK et al.; Rhodobacter capsulatus puhA mutant strains containing either a nonpolar, translationally in-frame deletion or a polar insertion of an antibiotic resistance cartridge were constructed and evaluated for their photosynthetic growth properties, absorption spectroscopy profiles, and chromatophore protein compositions . Both types of mutants were found to be incapable of photosynthetic growth and deficient in the reaction center (RC) and light-harvesting 1 (LH1) complexes . The translationally in-frame puhA deletion strains were restored to the parental strain phenotypes by complementation with a plasmid containing the puhA gene, whereas the polar puhA mutants were not . Analogous nonpolar and polar disruptions of orf 214 (located immediately 3' of the puhA gene) were made, and the resultant mutant strains were evaluated as described above . The strain containing the nonpolar deletion of orf 214 exhibited severely impaired photosynthetic growth properties and had greatly reduced levels of the RC and LH1 complexes . Complementation of this strain with a plasmid that expressed orf 214 from the nifHDK promoter restored photosynthetic growth capability, as well as the RC and LH1 complexes . The polar disruption of orf 214 yielded cells that were incapable of photosynthetic growth and had even lower levels of the RC and LH1 complexes, and complementation in trans with orf 214 only marginally improved these deficiencies . These results indicate that orf 214 and at least one additional gene located 3' of orf 214 are required to obtain the RC and LH1 complexes, and transcription read-through from the puhA superoperon is necessary for optimal expression of these new photosynthesis genes.

J Bacteriol, 1996 Apr, 178(8), 2216 - 23
Autoactivation of the marRAB multiple antibiotic resistance operon by the MarA transcriptional activator in Escherichia coli; Martin RG et al.; Transcriptional activation of the promoters of the mar/soxRS regulons by the sequence-related but independently inducible MarA and SoxS proteins renders Escherichia coli resistant to a broad spectrum of antibiotics and superoxide generators . Here, the effects of MarA and SoxS on transcription of the marRAB promoter itself were assayed in vitro by using a minimal transcription system and in vivo by assaying beta-galactosidase synthesized from marR::lacZ fusions . Purified MarA and MalE-SoxS proteins stimulated mar transcription about 6- and 15-fold, respectively, when the RNA polymerase/DNA ratio was 1 . Purified MarA bound as a monomer to a 16-bp "marbox" located 69 to 54 nucleotides upstream of a putative RNA initiation site . Deletion of the marbox reduced MarA-mar binding 100-fold, abolished the stimulatory effects of MarA and SoxS on transcription in vitro, and reduced marR::lacZ synthesis about 4-fold in vivo . Deletion of upstream DNA adjoining the marbox reduced MarA binding efficiency 30-fold and transcriptional activation 2- to 3-fold, providing evidence for an accessory marbox . Although MarA and the mar operon repressor, MarR, bound to independent sites, they competed for promoter DNA in band shift experiments . Assays of marR::lacZ transcriptional fusions in marRAB deletion or soxRS deletion strains showed that the superoxide generator paraquat stimulates mar transcription via soxRS and that salicylate stimulates mar transcription both by antagonizing MarR and by a MarR-independent mechanism . Thus, transcription of the marRAB operon is autorepressed by MarR and autoactivated by MarA at a site that also can be activated by SoxS.

J Fam Pract, 1996 Apr, 42(4), 357 - 61
Antibiotics and upper respiratory infection: do some folks think there is a cure for the common cold; Mainous AG 3rd et al.; BACKGROUND: Symptomatic treatment is the only recommended therapy for the uncomplicated "common cold." The purpose of this study was to examine the use of antibiotics and other prescription medications for the common cold in a Medicaid population seen in ambulatory care settings . METHODS: A cross-sectional sample of Kentucky Medicaid claims from July 1, 1993, through June 30, 1994, was analyzed . Subjects were patients seen in an ambulatory setting for the common cold, defined as acute nasopharyngitis . A total of 1439 individuals were seen for 2171 separate outpatient and emergency department encounters for the common cold . Outpatient visits accounted for 99% (2144) of the encounters . RESULTS: Patients in 35% (752) of the encounters did not fill a prescription for medication, 6% (129) filled a prescription for an antihistamine or other symptomatic medication, and 60% (1290) filled a prescription for an antibiotic for the common cold . Nineteen different antibiotics, 54% of which were amoxicillin, were prescribed for the common cold . Less than 2% of the encounters had a secondary diagnosis of either acute sinusitis or otitis media . These encounters were not more likely than the total sample to receive antibiotics . Adults were more likely than children to receive an antibiotic (P<.001), and urban physicians were more likely than rural physicians to prescribe antibiotics (P=.02) . A conservative estimate of the annual cost of antibiotic prescribing for the common cold in the United States was $37.5 million . CONCLUSIONS: A majority of persons receiving medical care for the common cold are given prescriptions for an unnecessary antibiotic . Unchecked, this practice may lead to greater antibiotic resistance and unnecessary use of health care resources . Future research should focus on the ability to institute behavioral changes for treatment of the common cold in both closed systems (eg, managed care) and open systems (eg, general community of physicians).

J Clin Microbiol, 1996 Mar, 34(3), 628 - 33
Determination of stability of Brucella abortus RB51 by use of genomic fingerprint, oxidative metabolism, and colonial morphology and differentiation of strain RB51 from B . abortus isolates from bison and elk; Jensen AE et al.; Brucella abortus RB51 and isolates from cattle, bison, and elk were characterized by pulsed-field gel electrophoresis and standard techniques for biotyping Brucella species, which included biochemical, morphological, and antigenic techniques, phage susceptibility, and antibiotic resistance . The objectives were to ascertain the stability of RB51 and to differentiate RB51 from other brucellae . Genomic restriction endonuclease patterns produced by pulsed-field gel electrophoresis demonstrated a unique fingerprint for RB51 relative to other brucellae . Comparisons of the oxidative metabolic profiles of RB51 after time in vivo (14 weeks) and in vitro (75 passages) showed no change in characteristic patterns of oxygen uptake on selected amino acid and carbohydrate substrates . Strain RB51 was biotyped as a typical rough B . abortus biovar 1 (not strain 19) after animal passage or a high number of passages in vitro and remained resistant to rifampin or penicillin and susceptible to tetracycline . No reactions with A or M antiserum or with a monoclonal antibody to the O antigen of Brucella lipopolysaccharides were detected; however, RB51 agglutinated with R antiserum . The results indicate that the genomic fingerprint and rough colonial morphology of RB51 are stable characteristics and can be used to differentiate this vaccine strain from Brucella isolates from cattle, bison, and elk.

RNA, 1996 Mar, 2(3), 254 - 63
A translational fidelity mutation in the universally conserved sarcin/ricin domain of 25S yeast ribosomal RNA; Liu R et al.; Recent evidence suggests that ribosomal RNAs have functional roles in translation . We describe here a new ribosomal RNA mutation that causes translational suppression and antibiotic resistance in eukaryotic cells . Using random mutagenesis of the cloned ribosomal RNA gene and in vivo selection, we isolated a C --> U mutation in the universally conserved sarcin/ricin domain in Saccharomyces cerevisiae 25S ribosomal RNA . This mutation changes the putative CG pair, which closes the GAGA tetraloop in the sarcin/ricin domain, into a weaker UG pair without eliminating ribosomal sensitivity to ricin . We show that suppression of several UGA, UAG, and frameshift mutations is evident when a portion of the cellular ribosomal RNA contains the C --> U mutation . Cells that contain essentially all mutant ribosomal RNA grow only 10% slower than the wild-type, but show increased suppression as well as resistance to paramomycin, G418, and hygromycin, and sensitivity to cycloheximide . Our results provide genetic evidence for the participation of the sarcin/ricin loop in maintaining translational accuracy and are discussed in terms of a hypothesis that this ribosomal RNA region normally undergoes a conformational change during translation.

Behav Brain Res, 1996, 73(1-2), 51 - 8
A molecular analysis of vesicular amine transport; Liu Y et al.; To package classical neurotransmitters into vesicles so that their release can be regulated by activity, neuronal cells express a set of specific vesicular transport proteins . We have used selection in MPP+ to clone the cDNAs encoding two vesicular monoamine transporters, the first members of this novel gene family that now also includes the vesicular transporter for acetylcholine . The sequences show similarity to several bacterial antibiotic resistance proteins, further supporting a role in detoxification and possibly Parkinson's disease . The two vesicular amine transporters show differences in their affinity for substrates, their turnover number and their pharmacology . In particular, the proteins differ in their interactions with the potent inhibitor tetrabenazine and with amphetamines, accounting for several classic pharmacological observations . Since the subcellular localization of the transport proteins determines the site of monoamine storage and the site of monoamine storage appears to differ from other classical transmitters, we have also raised polyclonal antibodies to the transporters and used these to demonstrate localization in dense core vesicles rather than synaptic vesicles . In addition to the implications for monoamine release, these observations also indicate a vesicular amine transporter as the first integral membrane protein restricted to the regulated secretory pathway.

Scand J Gastroenterol Suppl, 1996, 215, 82 - 9
Eradication of Helicobacter pylori: omeprazole in combination with antibiotics; Axon AT et al.; Until recently, the mainstay of treatment for Helicobacter pylori infection was either dual therapy, using omeprazole with amoxycillin or clarithromycin, or traditional triple therapy comprising bismuth and two antibiotics . Success with these treatment strategies has, however, varied widely between centres . Furthermore, the side-effects reported for bismuth triple therapy and the 2-week treatment period recommended have limited its popularity . These drawbacks have thus stimulated research aimed at identifying better drug combinations, with a simpler dosage for a shorter period, fewer side-effects, and greater and more consistent efficacy . A number of studies have now been undertaken using an acid inhibitor in combination with two antibiotics . Omeprazole, a highly effective acid pump inhibitor, has been investigated most extensively in this context, and is markedly effective in eradicating H . pylori when combined with any two of clarithromycin, a nitroimidazole and amoxycillin . These omeprazole triple therapy combinations provide eradication rates that are usually in the range of 85-95%, when assessed on a per protocol basis . Side-effects are minor and rarely interfere with compliance . Increasingly, these combinations are being given in a twice-daily dosage, making them more acceptable for the patient, and the dosage of antibiotics, in some cases, can be reduced . Furthermore, 1 week of treatment has been shown to be effective . In a few patients, however, even these highly effective eradication regimens fails, and anecdotal reports suggest that, once this has happened, other treatments are often similarly ineffective . Failure is not simply a matter of antibiotic resistance because patients with resistant organisms are often cured . In some patients, poor compliance, antibiotic resistance, coccoid bacterial forms, or the presence of sanctuary sites may be the cause of failure, in others, it has been suggested that pretreatment with an acid inhibitor may be the explanation . Research into these particular areas will be required, unless a new and universally effective drug combination can be identified.

S Afr Med J, 1996 Jan, 86(1), 45 - 9
Unexpectedly high strain diversity of Mycobacterium tuberculosis in a high-incidence community; Warren R et al.; OBJECTIVE: To characterise Mycobacterium tuberculosis strains present in a community experiencing an epidemic, in order to establish whether a high rate of transmission results in low strain diversity . DESIGN: Sputum specimens collected for 18 months; IS6110-based DNA fingerprinting . SETTING: The communities of Ravensmead and Uitsig, Cape Town, South Africa . PARTICIPANTS: Three hundred and thirty-four pulmonary tuberculosis patients attending the Local Authority Health Care Clinic . MAIN OUTCOME MEASURE: DNA fingerprinting . RESULTS: A total of 334 M . tuberculosis isolates were characterised by IS6110-based DNA fingerprinting; 209 strains were identified, 199 having 5 or more insertions . Forty of these strains were present in 2 or more patients (clustering--126 patients in total), which indicates a recent transmission rate of 30% . The 163 unique strains suggest reactivation of latent infections . Computer analysis showed a high degree of strain diversity, and a common progenitor could only be linked to 33% of the strains . Clustering was shown in 50% of drug-resistant isolates . CONCLUSIONS: The low rate of transmission (30%) and the high degree of strain diversity (209 strains) was unexpected and unexplained, given the high burden of disease in this community . The clustering of drug-resistant strains suggests that transmission, rather than lack of compliance, drives the spread of antibiotic resistance in this community . Preliminary indications are that BCG vaccination, while having little effect on the incidence of tuberculosis in this community, may have altered the strain dynamics.

Cleve Clin J Med, 1996 Jan-Feb, 63(1), 16 - 30
Community-acquired pneumonia: an update; Meeker DP et al.; Despite the discovery of new pathogens and the evolving problem of antibiotic resistance, the basic trends in community-acquired pneumonia remain remarkably constant . This article reviews the common pathogens, new pathogens, their clinical presentations, the diagnostic workup, the decision to hospitalize, antibiotic resistance, and antibiotic choices.

J Bacteriol, 1996 Jan, 178(1), 306 - 8
AcrAB efflux pump plays a major role in the antibiotic resistance phenotype of Escherichia coli multiple-antibiotic-resistance (Mar) mutants; Okusu H et al.; Multiple-antibiotic-resistance (Mar) mutants of Escherichia coli are resistant to a wide variety of antibiotics, and increased active efflux is known to be responsible for the resistance to some drugs . The identity of the efflux system, however, has remained unknown . By constructing an isogenic set of E . coli K-12 strains, we showed that the marR1 mutation was incapable of increasing the resistance level in the absence of the AcrAB efflux system . This experiment identified the AcrAB system as the major pump responsible for making the Mar mutants resistant to many agents, including tetracycline, chloramphenicol, ampicillin, nalidixic acid, and rifampin.

Gene, 1995 Dec 29, 167(1-2), 333 - 4
Plasmids with a kanamycin-resistance gene for site-directed mutagenesis using the oligodeoxyribonucleotide-directed dual amber method; Hashimoto-Gotoh T et al.; Plasmid vectors carrying lacZ' and kanamycin-resistance (KmR) genes were constructed for site-directed mutagenesis (SDM) using the oligodeoxyribonucleotide (oligo)-directed dual amber (ODA) method {Hashimoto-Gotoh et al., Gene 152 (1995) 271-276} . The plasmids, designated pKF16k, pKF17k, pKF18k and pKF19k, correspond to the previously reported chloramphenicol resistant (CmR) ODA plasmids, pKF16c, pKF17c, pKF18c and pKF19c, respectively, but contain dual amber (am) codons in KmR instead of the CmR gene . The SDM procedure using the KmR ODA plasmids is essentially the same as that with CmR ODA plasmids, which utilizes two oligo primers for in vitro DNA synthesis, one (selection primer) for dual am reversions and the other (mutagenic primer) for the target site . The KmR ODA plasmids yield 5-10-times more DNA per culture volume as compared to the CmR ODA plasmids, and one can prepare selection agar medium simply by spreading Km solution on dried agar plate at a final concentration of 50-100 micrograms/ml; due to the broad range of selecting antibiotic resistance.

Mol Gen Genet, 1995 Dec 20, 249(6), 622 - 8
Directed inactivation of the psbI gene does not affect photosystem II in the cyanobacterium Synechocystis sp . PCC 6803; Ikeuchi M et al.; PsbI is a small, integral membrane protein component of photosystem II (PSII), a pigment-protein complex in cyanobacteria, algae and higher plants . To understand the function of this protein, we have isolated the psbI gene from the unicellular cyanobacterium Synechocystis sp . PCC 6803 and determined its nucleotide sequence . Using an antibiotic-resistance cartridge to disrupt and replace the psbI gene, we have created mutants of Synechocystis 6803 that lack the PsbI protein . Analysis of these mutants revealed that absence of the PsbI protein results in a 25-30% loss of PSII activity . However, other PSII polypeptides are present in near wild-type amounts, indicating that no significant destabilization of the PSII complex has occurred . These results contrast with recently reported data indicating that PsbI-deficient mutants of the eukaryotic alga Chlamydomonas reinhardtii are highly light-sensitive and have a significantly lower (80-90%) titer of the PSII complex . In Synechocystis 6803, PsbI-deficient cells appear to be slightly more photosensitive than wild-type cells, suggesting that this protein, while not essential for PSII biogenesis or function, plays a role in the optimization of PSII activity.

Mol Gen Genet, 1995 Dec 10, 249(4), 375 - 90
Gene silencing in transgenic tobacco hybrids: frequency of the event and visualization of somatic inactivation pattern; Schmulling T et al.; We have investigated the stability of the expression of different T-DNA-borne genes in hybrid tobacco lines . These lines were constructed to rescue rolC-induced male sterility in kanamycin-resistant P35s-rolC transgenic tobacco plants by expression of rolC antisense genes . Using five different tester lines, a total of 158 hybrids was obtained . We observed inactivation of transgene expression in 20% of the F1 progeny and in 35% of the backcrossed F2 progeny, as indicated by the loss of kanamycin resistance . In 3% of all crosses complete loss of antibiotic resistance was noted, while in most affected hybrid progeny only part of the population became kanamycin sensitive . Single genes could be selectively inactivated on T-DNAs harboring several genes . Gene inactivation was not restricted to one of the two T-DNAs examined . Somatic silencing, visualized by a cell-specific 35SGUSINT marker gene, occurred in a random fashion or exhibited an inherited specific pattern . The type of somatic silencing pattern observed indicated developmental control of the process . Two phenotypic classes could be distinguished with respect to frequency and timing of the inactivation process . Rapid gene inactivation, occurring within a few weeks after germination of hybrid seedlings, was characterized by complete methylation of restriction sites in the promoter of the silenced gene, resetting of gene expression during meiosis, heredity of the developmentally controlled program of gene silencing in subsequent generations, and rapid reactivation of gene expression after genetic separation of the different T-DNAs . In contrast, a slow type of gene inactivation was of a more stochastic nature and was recognized only in hybrids of the backcrossed F2 generation . In this case the degree of promoter methylation, which could extend beyond the T-DNA borders, was not correlated with the reduction in steady-state poly(A)+ mRNA levels, the silenced state was transmitted through meiosis and reactivation lasted several generations . The implications of the observations for our understanding of the gene inactivation process are discussed.

Gene, 1995 Dec 1, 166(1), 175 - 6
Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes; Kovach ME et al.; Four new antibiotic-resistant derivatives of the broad-host-range (bhr) cloning vector pBBR1MCS have been constructed . These new plasmids have several advantages over many of the currently available bhr vectors in that: (i) they are relatively small (< 5.3 kb), (ii) they possess an extended multiple cloning site (MCS), (iii) they allow direct selection of recombinant plasmid molecules in Escherichia coli via disruption of the LacZ alpha peptide, (iv) they are mobilizable when the RK2 transfer functions are provided in trans and (v) they are compatible with IncP, IncQ and IncW group plasmids, as well as with ColE1- and P15a-based replicons.

J Bacteriol, 1995 Dec, 177(23), 6798 - 803
Subunit interactions and protein stability in the cyanobacterial light-harvesting proteins; Plank T et al.; Strain 4R is a phycocyanin-minus mutant of the unicellular cyanobacterium Synechocystis sp . strain 6803 . Although it lacks the light-harvesting protein phycocyanin, 4R has normal levels of phycocyanin (cpc) transcripts . Sequence analysis of the cpcB gene encoding the phycocyanin beta subunit shows an insertion mutation in 4R that causes early termination of translation . Other work has shown that the phycocyanin alpha subunit and the linker proteins encoded on the cpc transcripts are all functional in 4R, yet the defective phycocyanin beta subunit results in the complete absence of the alpha subunit and the linkers . Phycocyanin-minus mutants were constructed in a wild-type background by interruption of cpcB and cpcA with an antibiotic resistance gene and were compared with the 4R strain . Immunoblot analysis of the mutants demonstrated that interruption of one subunit was accompanied by a complete absence of the unassembled partner subunit . Phycocyanin assembly begins with the formation of the alpha beta heterodimer (the monomer) and continues through higher-order trimeric and hexameric aggregates that associate with linker proteins to form the phycobilisome rods . The results in this paper indicate that monomer formation is a critical stage in the biliprotein assembly pathway and that unassembled subunits are subject to stringent controls that prevent their appearance in vivo.

Nucleic Acids Res, 1995 Nov 11, 23(21), 4234 - 8
Nonsense suppressor and antisuppressor mutations at the 1409-1491 base pair in the decoding region of Escherichia coli 16S rRNA; Gregory ST et al.; Using a genetic selection for suppressors of a UGA nonsense mutation in trpA, we have isolated a G to A transition mutation at position 1491 in the decoding region of 16S rRNA . This suppressor displayed no codon specificity, suppressing UGA, UAG and UAA nonsense mutations and +1 and -1 frameshift mutations in lacZ . Subsequent examination of a series of mutations at G1491 and its base-pairing partner C1409 revealed various effects on nonsense suppression and frameshifting . Mutations that prevented Watson-Crick base pairing between these residues were observed to increase misreading and frameshifting . However, double mutations that retained pairing potential produced an antisuppressor or hyperaccurate phenotype . Previous studies of antibiotic resistance mutations and antibiotic and tRNA footprints have placed G1491 and C1409 near the site of codon-anticodon pairing . The results of this study demonstrate that the nature of the interaction of these two residues influences the fidelity of tRNA selection.

Gene, 1995 Nov 7, 165(1), 141 - 2
New plasmids carrying antibiotic-resistance cassettes; Reece KS et al.; A series of new plasmid vectors is described that carry gene cassettes imparting resistance to the antibiotics chloramphenicol (CmR), kanamycin (KmR), tetracycline (TcR) and spectinomycin/streptomycin (Sp/SmR) . The gene cassettes are symmetrically flanked by several restriction sites . In addition, several restriction sites that are normally found internal to the gene cassettes have been eliminated, thereby expanding the number of restriction enzymes available to excise an intact antibiotic-resistance gene . The gene cassettes are carried by high-copy-number plasmids that confer ampicillin resistance (ApR).

Biochem Cell Biol, 1995 Nov-Dec, 73(11-12), 1167 - 77
Antibiotic resistance mechanisms of mutant EF-Tu species in Escherichia coli; Kraal B et al.; Analysis of antibiotic-resistant EF-Tu mutants has revealed a connection between resistance and structural elements that participate in the GTPase switching mechanism . Both random and site-directed mutagenesis methods have yielded sets of purified mutant EF-Tu resistant to kirromycin (kirT) or pulvomycin (pulT) . All kirT mutations cluster in the interface of domain 1 and 3 of EF-Tu in its GTP-bound conformation, not in that of EF-Tu.GDP . Other evidence also suggests that kirromycin binds to the interface of wild-type EF-Tu, thereby jamming the GTPase switch . Various functional studies reveal two subsequent resistance mechanisms . The first hinders kirromycin binding to EF-Tu.GTP and the second occurs after GTP hydrolysis by rejection of bound kirromycin . All pulT mutations cluster in the three-domain junction interface of EF-Tu . GTP (which is an open hole in EF-Tu.GDP) and destabilize a salt-bridge network . Pulvomycin may bind nearby and overlap with tRNA binding . Mutations show that a D99-R230 salt bridge is not essential for the transduction of the GTPase switch signal from domain 1 . In vivo and in vitro studies reveal that pulvomycin sensitivity is dominant over resistance . This demands a revision of the current view of the mechanism of pulvomycin inhibition of protein synthesis and may support a translation model with two EF-Tus on the ribosome . Several mutant EF-Tu species display altered behaviour towards aminoacyl-tRNA with interesting effects on translational accuracy . KirT EF-Tu(A375T) is able to reverse the streptomycin-dependent phenotype of a ribosomal protein S12 mutant strain to streptomycin sensitivity.

J Infect, 1995 Nov, 31(3), 225 - 7
Isolation of Arcobacter butzleri from a neonate with bacteraemia; On SL et al.; We describe the case of a neonate with bacteraemia from whom the recently described organism Arcobacter butzleri was isolated . This appears to be the first report of the organism causing neonatal infection . Clinical details suggest that the infection was contracted in utero, although the mother showed no evidence of disease before delivery . Treatment of the preterm infant was ultimately successful in resolving the infection but the organism proved resistant to a wide range of antibiotics . Similar patterns of antibiotic resistance were also observed in 39 reference and field strains of the genus Arcobacter . These findings, combined with available data on the distribution of Arcobacter species, suggest that these organisms may be important human pathogens . Optimized methods for their isolation and identification are therefore required so as to ascertain their role in human disease.

J Bacteriol, 1995 Nov, 177(22), 6440 - 8
The hglK gene is required for localization of heterocyst-specific glycolipids in the cyanobacterium Anabaena sp . strain PCC 7120; Black K et al.; Mutant strain 543 of the cyanobacterium Anabaena sp . strain PCC 7120 was originally isolated as a Fox- mutant following chemical mutagenesis . Ultrastructural analysis shows that in nitrogen-replete media the vegetative cells of the mutant are more cylindrical and have thicker septa than those of the wild type, while in nitrogen-free media the mutant heterocysts lack the normal glycolipid layer external to the cell wall . Although this layer is absent, strain 543 heterocysts nevertheless contain heterocyst-specific glycolipids, as determined by thin-layer chromatography . The mutation in strain 543 is in a gene we have named hglK, encoding a protein of 727 amino acids . The wild-type HglK protein appears to contain four membrane-spanning regions followed by 36 repeats of a degenerate pentapeptide sequence, AXLXX . The mutation in strain 543 introduces a termination codon immediately upstream of the pentapeptide repeat region . A mutant constructed by insertion of an antibiotic resistance cassette near the beginning of the hglK gene has the same phenotype as strain 543 . We propose that hglK encodes a protein necessary for the localization of heterocyst glycolipids and that this function requires the pentapeptide repeats of the HglK protein.

J Biol Chem, 1995 Oct 27, 270(43), 25798 - 804
Identification of residues involved in substrate recognition by a vesicular monoamine transporter; Merickel A et al.; To identify the residues involved in substrate recognition by recently cloned vesicular monoamine transporters (VMAT1 and VMAT2), we have mutagenized the conserved residues in a cytoplasmic loop between transmembrane domains two and three of VMAT2 . Although studies of related bacterial antibiotic resistance proteins indicate an important functional role for this region, we found no effect of these mutations on VMAT2 activity . However, replacement of aspartate 33 in the first predicted transmembrane domain with an asparagine (D33N) eliminates transport . D33N shows normal levels of expression and normal binding at equilibrium to the potent inhibitor reserpine . However, in contrast to wild-type VMAT2, serotonin inhibits reserpine binding to D33N very poorly, indicating a specific defect in substrate recognition . Replacement of three serine residues in transmembrane domain three with alanine (Stmd3A) shows a similarly selective but even more profound defect in substrate recognition . The results suggest that by analogy to receptors and plasma membrane transporters for monoamines, the cationic amino group of the ligand interacts with an asparte in the first transmembrane domain of VMAT2 and hydroxyl groups on the catechol or indole ring interact with a group of serines in the third transmembrane domain . Importantly, D33N and Stmd3A retain coupling to the proton electrochemical gradient as measured by the delta microH(+)-induced acceleration of reserpine binding . This indicates that substrate recognition can be separated from coupling to the driving force.

Nucleic Acids Res, 1995 Oct 25, 23(20), 4073 - 80
The expression of biologically active human p53 in Leishmania cells: a novel eukaryotic system to produce recombinant proteins; Zhang WW et al.; We have investigated the use of Leishmania cells as a novel eukaryotic expression system for the production of recombinant protein . These cells are easy to maintain, requiring no CO2 incubator or shaker, and can be grown in standard tissue culture media . Leishmania cells can be readily transfected with plasmid DNA by electroporation and transformants selected with antibiotic resistance . Recent studies have shown that it is possible to express foreign genes in Leishmania for the purpose of understanding the biology of this protozoan cell . In the present study we report the use of this system as a means of producing a biologically functional human p53 protein . The conformation of the p53 protein is critical for its ability to bind specific DNA sequences . It is demonstrated that Leishmania-synthesized human p53 is phosphorylated and can bind specifically to its enhancer DNA sequence . These data demonstrate that Leishmania may represent a simple eukaryotic expression system for the production of biologically active recombinant proteins.

J Assoc Physicians India, 1995 Oct, 43(10), 679 - 84
Reassessment of frequency of occurrence of typhoid fever and cost efficacy analysis of antibiotic therapy; Sridhar CB et al.; Typhoid fever has assumed importance due to the increased incidence of drug resistance in India . The exact magnitude of the problem is not accurately known . The objective of this study was to collect retrospectively the data on the incidence and frequency of typhoid fever among hospital admissions at St . Johns Medical College Hospital (SJMCH), Bangalore during the year 1987 to 1992 and also to study the sensitivity pattern and the use of antibiotics . The study was also aimed at comparison of immunogenicity and tolerance of conventional vaccine to the newer polysaccharide vaccine . It was found that the incidence of typhoid fever showed change from epidemic to endemic situation with outbreaks of epidemics . Sensitivity pattern also showed change during the same period and antibiotic resistance was increasingly demonstrated from 1989 . Cost per patient and total cost to the hospital due to increased admissions also showed progressive increase . The polysaccharide vaccine (recently made available in India) had very low adverse reaction profile with higher immunogenicity as compared to conventional vaccine . Its single dose effect with long lasting immunity indicates it probable usefulness in the eradication of disease.

Arch Biochem Biophys, 1995 Sep 10, 322(1), 291 - 4
A superoxide dismutase mimic protects sodA sodB Escherichia coli against aerobic heating and stationary-phase death; Benov L et al.; Superoxide appears to be a major cause of stationary-phase death and heat kill . In support of this conclusion are the following observations: (a) Stationary-phase death was apparent in the sodA sodB, but not in the superoxide dismutase (SOD)-competent parental strain; (b) Stationary phase death in the sodA sodB strain was dioxygen-dependent; (c) A manganic porphyrin, which catalyzes the dismutation of superoxide, protected the sodA sodB strain against stationary-phase death; (d) Heating the sodA sodB strain to 42 degrees C caused a loss of viability not seen with the SOD-competent parental strain and preventable by the manganic porphyrin . Exposure to aerobic conditions induced antibiotic resistance in the sodA sodB, but not in the parental strain and the manganic porphyrin prevented that induction . This again indicates its ability to substitute for SOD in Escherichia coli.

Salud Publica Mex, 1995 Sep-Oct, 37(5), 408 - 16
{Risk factors for antitubercular drug resistance in Chiapas, Mexico}; Alvarez-Gordillo GC et al.; OBJECTIVES . To determine risk factors for antibiotic resistance in patients with pulmonary tuberculosis in four Health Jurisdictions of the state of Chiapas . MATERIAL AND METHODS . A case-control study was carried out in patients diagnosed by acid fast smear during 1992 . A questionnaire was applied which included variables related to the diagnosis, treatment and follow-up of the patients . Sputum samples were collected for culture and sensitivity tests . A case of drug-resistant pulmonary tuberculosis was defined as the presence of culture colonies showing resistance to one or more drugs . The control group was patients with negative smears and cultures or positive cultures for M . tuberculosis sensitive to the specific drugs . RESULTS . Of the total of 18 individuals reported to have positive cultures, 13 (72.2%) were resistant to one or more drugs . Resistance to two or more drugs was found in 10 of them of which three were resistant to five antituberculosis drugs . The most frequent resistance was to isoniazid (77%) . Risk factors for resistance encountered in this patient population were monotherapy (OR = 34.2), abandonment of treatment (OR = 6.86), a prolonged period of illness (OR = 6.40), delay in diagnosis and a history of prior therapy (OR = 28.3) . CONCLUSIONS: The high proportion of patients resistant to antituberculosis therapy poses a public health problem and is a clear consequence of the problems arising from inadequate treatment.

Genetics, 1995 Aug, 140(4), 1247 - 58
Alterations in ribosomal protein RPS28 can diversely affect translational accuracy in Saccharomyces cerevisiae; Anthony RA et al.; Three small-subunit ribosomal proteins shown to influence translational accuracy in Saccharomyces cerevisiae are conserved in structure and function with their procaryotic counterparts . One of these, encoded by RPS28A and RPS28B (RPS28), is comparable to bacterial S12 . The others, encoded by sup44 (RPS4) or, sup46 and YS11A (RPS13), are homologues of procaryotic S5 and S4, respectively . In Escherichia coli, certain alterations in S12 cause hyperaccurate translation or antibiotic resistance that can be counteracted by other changes in S5 or S4 that reduce translational accuracy . Using site-directed and random mutagenesis, we show that different changes in RPS28 can have diametrical influences on translational accuracy or antibiotic sensitivity in yeast . Certain substitutions in the amino-terminal portion of the protein, which is diverged from the procaryotic homologues, cause varying levels of nonsense suppression or antibiotic sensitivity . Other alterations, found in the more conserved carboxyl-terminal portion, counteract SUP44- or SUP46-associated antibiotic sensitivity, mimicking E . coli results . Although mutations in these different parts of RPS28 have opposite affects on translational accuracy or antibiotic sensitivity, additive phenotypes can be observed when opposing mutations are combined in the same protein.

Gene, 1995 Jul 4, 160(1), 63 - 7
Improved antibiotic-resistance gene cassettes and omega elements for Escherichia coli vector construction and in vitro deletion/insertion mutagenesis; Alexeyev MF et al.; Several antibiotic-resistance gene cassettes and omega elements for Escherichia coli vector construction include the aacC1, aadA+, bla, cat, nptII and tet gene cassettes, and also the omega-Gm, omega-Sm, omega-Ap, omega-Cm, omega-Km and omega-Tc elements . Both cassettes and elements are flanked by pBluescriptII plasmid multiple cloning sites (MCS) duplicated in inverted (symmetric MCS) or direct (tandem MCS) orientation . Genes that were modified in order to remove sites for the most common restriction endonucleases from their coding regions (except aacC1 and aadA+) were used for cassette and omega-element construction.

Microb Drug Resist, 1995 Summer, 1(2), 137 - 42
Epidemiologic aspects on antibiotic resistance; Hoiby N; The increasing usage of antibiotics has selected for resistant bacteria . Spread of such bacteria may follow mathematical models of infectious diseases, taking into consideration the number of infectious individuals, the number of susceptible individuals, and the effective contact rate between individuals from these two groups . According to calculations of the theoretical epidemic curves, the highest incidence of individuals infected with a resistant bacterial strain is present when the prevalence of patients and carriers is approximately 20-80% . Moreover, the rapidity of the spread of an epidemic increases drastically if the total number of individuals in the exposed group increases and also if the contact rate increases . This means that precautions to stop an epidemic spread of a resistant bacterial strain in a given group of individuals should be undertaken early when the prevalence is below 20% . Efficient precautions consist of cohort isolation, decrease of the number of individuals in exposed groups by subdivision into several smaller groups, and decrease of contact rates by hygienic precautions . Examples are given where such precautions have proven efficient.

J Bacteriol, 1995 Jul, 177(14), 4176 - 8
Regulation of the multiple antibiotic resistance (mar) regulon by marORA sequences in Escherichia coli; Martin RG et al.; The mar operon and adjacent sequences were subcloned on a low-copy-number plasmid to identify essential regulatory elements . A 1.1-kbp fragment containing 133 bp of the operator-promoter region (marO), the full marRA gene sequences, and only 10 of 72 marB codons provided a dela mar strain with normal repressibility and inducibility and the ability to beget mar constitutive mutants.

Yeast, 1995 Jun 15, 11(7), 629 - 40
In vivo cloning by homologous recombination in yeast using a two-plasmid-based system; Degryse E et al.; In order to reduce the number of classical DNA manipulation and ligation steps in the generation of yeast expression plasmids, a series of vectors is described which facilitate the assembly of such plasmids by the more efficient 'recombination in vivo' technique . Two sets of vectors were developed . The first set, called 'expression vectors', contains an expression cassette with a yeast promoter and the PGK terminator separated by a polylinker, and an Escherichia coli replicon . Subcloning in these vectors of a DNA fragment generates a 'transfer vector' which is compatible with the second set of E . coli-yeast shuttle vectors . This set of 'recombination vectors' contains a cassette for a functional copy of a gene complementing a host strain auxotrophy or a bacterial gene conferring an antibiotic resistance to the plasmid-bearing host . Plasmid copy numbers can be modulated through the use of URA3 or URA3-d as the selective marker together with an ARS/CEN and the 2 microns replicon . Integration of the cloned DNAs into the yeast linearized replicative vectors occurs by recombination between homologous flanking sequences during transformation in yeast or E . coli . All the vectors contain the origin of replication of phage f1 and allow the generation of single-stranded DNA in E . coli for sequencing or site-directed mutagenesis.

Proc Natl Acad Sci U S A, 1995 Jun 6, 92(12), 5456 - 60
Binding of purified multiple antibiotic-resistance repressor protein (MarR) to mar operator sequences; Martin RG et al.; Elevated expression of the marORAB multiple antibiotic-resistance operon enhances the resistance of Escherichia coli to various medically significant antibiotics . Transcription of the operon is repressed in vivo by the marR-encoded protein, MarR, and derepressed by salicylate and certain antibiotics . The possibility that repression results from MarR interacting with the marO operator-promoter region was studied in vitro using purified MarR and a DNA fragment containing marO . MarR formed at least two complexes with marO DNA, bound > 30-fold more tightly to it than to salmon sperm DNA, and protected two separate 21-bp sites within marO from digestion by DNase I . Site I abuts the downstream side of the putative -35 transcription-start signal and includes 4 bp of the -10 signal . Site II begins 13 bp downstream of site I, ending immediately before the first base pair of marR . Site II, approximately 80% homologous to site I, is not required for repression since a site II-deleted mutant (marO133) was repressed in trans by wild-type MarR . The absence of site II did not prevent MarR from complexing with the site I of marO133 . Salicylate bound to MarR (Kd approximately 0.5 mM) and weakened the interaction of MarR with sites I and II . Thus, repression of the mar operon, which curbs the antibiotic resistance of E . coli, correlates with the formation of MarR-site I complexes . Salicylate appears to induce the mar operon by binding to MarR and inhibiting complex formation, whereas tetracycline and chloramphenicol, which neither bind MarR nor inhibit complex formation, must induce by an indirect mechanism.

Afr J Med Med Sci, 1995 Jun, 24(2), 125 - 30
The menace of beta-lactamase production on antibiotic prescription in community acquired-infections in Nigeria; Oyelese AO et al.; Antibiotic resistance is a major clinical problem in the management of infectious diseases . The production of beta-lactamases by pathogens of all grades which has spread extensively during the last decade has further narrowed down the choice of antibiotics . Those antibiotics that are efficacious are costly and not readily available . The purposes of this study therefore were: To evaluate the incidence of beta-lactamase producing organisms responsible for common community-acquired infections . To evaluate the incidence of bacterial resistance to commonly prescribed antibiotics in the general practice . Nitrocefin strip was used to test each isolate for beta-lactamase production . All isolates were tested against five commonly prescribed antibiotics and a new oral cephalosporin . A nationwide survey revealed that 78% of community-acquired pathogens produced beta-lactamases while more than 50% of most community isolated showed in-vitro resistance to most commonly prescribed antibiotics . We conclude that treatment of bacterial infections are becoming more difficult and more costly . There is need therefore to continually review the susceptibility profiles of community-acquired pathogens.

J Bacteriol, 1995 Jun, 177(12), 3414 - 9
Characterization of MarR, the repressor of the multiple antibiotic resistance (mar) operon in Escherichia coli; Seoane AS et al.; The marRAB operon is one of two operons in the mar locus of Escherichia coli that are divergently transcribed from a central regulatory region, marO . The marRAB operon, transcribed from marOII, controls intrinsic resistance or susceptibility to multiple antibiotics and is inducible by structurally unrelated compounds such as tetracycline and chloramphenicol (S . P . Cohen, H . Hachler, and S . B . Levy, J . Bacteriol . 175:1484-1492, 1993) . To clarify the role of the operon in response to environmental signals, its transcription was studied under different conditions, using a marOII-lacZ transcriptional fusion introduced into the chromosome of wild-type or mar-deleted cells . In wild-type cells, uncoupling agents (such as carbonyl cyanide m-chlorophenylhydrazone) and different redox-cycling compounds (e.g., menadione and plumbagin) induced expression from the marOII-lacZ fusion two- to sevenfold . In the mar-deleted strain, LacZ expression from the fusion was 10-fold higher than in wild-type cells . This activity was temperature sensitive (3-fold lower at 42 than at 30 degrees C) and decreased 20-fold with the introduction of the gene for MarR . Structurally different compounds which induce the mar operon in wild-type cells reversed the MarR repression of marOII-lacZ expression . To determine the size of MarR, it was fused to MalE as a MarR fusion protein of 144 amino acids {MarR(144)} or of 125 amino acids (deleted of 19 amino acids at the N terminus) {MarR(125)} . Only the MarR(144) fusion showed repressor ability . The purified MarR(144) fusion, but not the MarR(125) fusion, bound specifically to marO in vitro, as revealed by gel retardation, with an apparent dissociation constant of 5 x 10(-9) M . MarR, therefore, controls expression of the marRAB operon presumably by binding to marO . MarR repression in cells can be reversed by different compounds, facilitating the response of bacteria to multiple environmental stress conditions.

Pediatr Clin North Am, 1995 Jun, 42(3), 497 - 507
Mechanisms of bacterial resistance; Burns JL; Antibiotic resistance is a growing problem in clinical pediatrics . Many of the agents traditionally used to treat pediatric pathogens are becoming less effective because of increasing bacterial resistance . In addition, many more children are immunocompromised because of primary or acquired immunodeficiencies and because of advances in cancer chemotherapy and transplantation . These children are being admitted to hospitals where they may be exposed to multiply resistant nosocomial pathogens . An improved understanding of the mechanisms of antibiotic resistance and the development of treatment strategies to prevent the emergence of resistance will be increasingly required in pediatrics.

J Hosp Infect, 1995 Jun, 30 Suppl, 15 - 25
How best to utilize limited resources; Forder AA; South Africa's new health policy embraces the primary health care (PHC) approach for all its peoples and will include good primary, secondary and tertiary care . The policy will hope to provide the highest possible standards of care, yet be of a scale and complexity that the country can sustain into the future . There will almost certainly be rationalization of many of the tertiary teaching hospitals, with inevitable cut-backs in their budgets . This in turn could carry the risk of damage to the fabric of these institutions, which might be impossible to repair . Medicines offer a simple, cost-effective answer to many health problems in Africa, provided they are available, accessible, affordable and properly used . A looming problem in African drug markets is inefficiency and waste . The use of counterfeit medicines has reached unparalleled heights . It is vital that there should be a competent, honest, accountable and independent national drug regulatory authority, secured in law, to provide the necessary infrastructure for the acquisition of sound medicines . Medicines are central to a sound national health policy, but there is great public concern about their costs . Anti-infective drugs are amongst the most widely used class of drugs in the world . Inappropriate use of these agents is widespread and guidelines need to be established for their correct use . The control of all medicines in South Africa is governed by the Medicines & Related Substance Act of 1965 . The Medicines Control Council is mandated to ensure that all medicines (including antibiotics) available to the public are efficacious, safe and of high quality . An informally-constituted Antibiotic Study Group has been established in order to monitor aspects of antibiotic therapy that impinge on more general issues of public health, country-wide . The Antibiotic Study Group has instituted an Antibiotic Surveillance Programme to monitor the development of antibiotic resistance nationally . In addition the majority of the tertiary teaching hospitals have comparable in-house antibiotic control policies to help prevent such resistance and to cut costs . These issues need to be debated and resolved . Once in place and working effectively, they will in the long-term supply the most cost-effective means of providing health care for all.

Gene, 1995 May 26, 158(1), 9 - 14
Gene disruption in Escherichia coli: TcR and KmR cassettes with the option of Flp-catalyzed excision of the antibiotic-resistance determinant; Cherepanov PP et al.; Two cassettes with tetracycline-resistance (TcR) and kanamycin-resistance (KmR) determinants have been developed for the construction of insertion and deletion mutants of cloned genes in Escherichia coli . In both cassettes, the resistance determinants are flanked by the short direct repeats (FRT sites) required for site-specific recombination mediated by the yeast Flp recombinase . In addition, a plasmid with temperature-sensitive replication for temporal production of the Flp enzyme in E . coli has been constructed . After a gene disruption or deletion mutation is constructed in vitro by insertion of one of the cassettes into a given gene, the mutated gene is transferred to the E . coli chromosome by homologous recombination and selection for the antibiotic resistance provided by the cassette . If desired, the resistance determinant can subsequently be removed from the chromosome in vivo by Flp action, leaving behind a short nucleotide sequence with one FRT site and with no polar effect on downstream genes . This system was applied in the construction of an E . coli endA deletion mutation which can be transduced by P1 to the genetic background of interest using TcR as a marker . The transductant can then be freed of the TcR if required.

Mol Med, 1995 May, 1(4), 436 - 46
The MarR repressor of the multiple antibiotic resistance (mar) operon in Escherichia coli: prototypic member of a family of bacterial regulatory proteins involved in sensing phenolic compounds; Sulavik MC et al.; BACKGROUND: The marR gene of Escherichia coli encodes a repressor of the marRAB operon, a regulatory locus controlling multiple antibiotic resistance in this organism . Inactivation of marR results in increased expression of marA, which acts at several target genes in the cell leading to reduced antibiotic accumulation . Exposure of E . coli to sodium salicylate (SAL) induces marRAB operon transcription and antibiotic resistance . The mechanism by which SAL antagonizes MarR repressor activity is unclear . MATERIALS AND METHODS: Recombinant plasmid libraries were introduced into a reporter strain designed to identify cloned genes encoding MarR repressor activity . Computer analysis of sequence databases was also used to search for proteins related to MarR . RESULTS: A second E . coli gene, MprA, that exhibits MarR repressor activity was identified . Subsequent database searching revealed a family of 10 proteins from a variety of bacteria that share significant amino acid sequence similarity to MarR and MprA . At least four of these proteins are transcriptional repressors whose activity is antagonized by SAL or by phenolic agents structurally related to SAL . CONCLUSIONS: The MarR family is identified as a group of regulatory factors whose activity is modulated in response to environmental signals in the form of phenolic compounds . Many of these agents are plant derived . Some of the MarR homologs appear more likely to control systems expressed in animal hosts, suggesting that phenolic sensing by bacteria is important in a variety of environments and in the regulation of numerous processes.

J Bacteriol, 1995 Apr, 177(7), 1655 - 61
Activation of multiple antibiotic resistance and binding of stress-inducible promoters by Escherichia coli Rob protein; Ariza RR et al.; Multiple antibiotic resistance in Escherichia coli can be mediated by induction of the SoxS or MarA protein, triggered by oxygen radicals (in the soxRS regulon) or certain antibiotics (in the marRAB regulon), respectively . These small proteins (SoxS, 107 residues; MarA, 127 residues) are homologous to the C terminus of the XylS-AraC family of proteins and are more closely related to a approximately 100-residue segment in the N terminus of Rob protein, which binds the right arm of the replication origin, oriC . We investigated whether the SoxS-MarA homology in Rob might extend to the regulation of some of the same inducible genes . Overexpression of Rob indeed conferred multiple antibiotic resistance similar to that known for SoxS and MarA (against chloramphenicol, tetracycline, nalidixic acid, and puromycin), as well as resistance to the superoxide-generating compound phenazine methosulfate . The Rob-induced antibiotic resistance depended only partially on the micF antisense RNA that down-regulates the OmpF outer membrane porin to limit antibiotic uptake . Similar antibiotic resistance was conferred by expression of a Rob fragment containing only the N-terminal 123 residues that constitute the SoxS-MarA homology . Both intact Rob and the N-terminal fragment activated expression of stress genes (inaA, fumC, sodA) but with a pattern distinct from that found for SoxS and MarA . Purified Rob protein bound a DNA fragment containing the micF promoter (50% bound at approximately 10(-9) M Rob) as strongly as it did oriC, and it bound more weakly to DNA containing the sodA, nfo, or zwf promoter (50% bound at 10(-8) to 10(-7) M) . Rob formed multiple DNA-protein complexes with these fragments, as seen previously for SoxS . These data point to a DNA-binding gene activator module used in different protein contexts.

Infect Immun, 1995 Apr, 63(4), 1521 - 8
Virulence of a Porphyromonas gingivalis W83 mutant defective in the prtH gene; Fletcher HM et al.; In a previous study we cloned and determined the nucleotide sequence of the prtH gene from Porphyromonas gingivalis W83 . This gene specifies a 97-kDa protease which is normally found in the membrane vesicles produced by P . gingivalis and which cleaves the C3 complement protein under defined conditions . We developed a novel ermF-ermAM antibiotic resistance gene cassette, which was used with the cloned prtH gene to prepare an insertionally inactivated allele of this gene . This genetic construct was introduced by electroporation into P . gingivalis W83 in order to create a protease-deficient mutant by recombinational allelic exchange . The mutant strain, designated V2296, was compared with the parent strain W83 for proteolytic activity and virulence . Extracellular protein preparations from V2296 showed decreased proteolytic activity compared with preparations from W83 . Casein substrate zymography revealed that the 97-kDa proteolytic component as well as a 45-kDa protease was missing in the mutant . In in vivo experiments using a mouse model, V2296 was dramatically reduced in virulence compared with the wild-type W83 strain . A molecular survey of several clinical isolates of P . gingivalis using the prtH gene as a probe suggested that prtH gene sequences were conserved and that they may have been present in multiple copies . Two of 10 isolates did not hybridize with the prtH gene probe . These strains, like the V2296 mutant, also displayed decreased virulence in the mouse model . Taken together, these results suggest an important role for P . gingivalis proteases in soft tissue infections and specifically indicate that the prtH gene product is a virulence factor.

Microbiology, 1995 Apr, 141 ( Pt 4), 831 - 41
Inducible expression of heterologous genes targeted to a chromosomal platform in the cyanobacterium Synechococcus sp . PCC 7942; Geerts D et al.; High-level, inducible expression of heterologous genes in the cyanobacterium Synechococcus sp . strain PCC 7942 was obtained using the Escherichia coli trc promoter and lacI repressor . The petE gene of Anabaena sp . strain PCC 7937 encoding plastocyanin precursor protein and the E . coli uidA gene encoding beta-glucuronidase were initially placed under the control of the trc promoter and lacI repressor by cloning into the E . coli pTrc99C expression vector and were introduced into the chromosomal platform for integration in metF (PIM) of the Synechococcus R2-PIM9 recipient strain . These pTrc99C-derived constructs often gave rise to transformants that did not contain a complete insert gene, probably because of gene conversion events . Selection of the desired Synechococcus R2-PIM9 transformants was vastly improved using the new pTrcIS vector that contains the aadA gene encoding streptomycin resistance as an extra antibiotic resistance marker . The influence of IPTG concentration and induction time on gene expression with the E . coli trc/lacI system in Synechococcus was determined using beta-glucuronidase as a reporter . The Anabaena PCC 7937 petE gene in Synechococcus was expressed to a high level upon induction with IPTG as shown by RNA and immunoblot analysis . The general usability of pTrcIS as a cloning vector for inducible heterologous gene expression in Synechococcus was confirmed by the introduction of several more genes.

J Mol Biol, 1995 Mar 10, 246(5), 595 - 608
Determination of the binding sites of RepA, a replication initiator protein of the basic replicon of the IncN group plasmid pCU1; Papp PP et al.; A 2kb DNA region of the broad-host-range plasmid pCU1 carries all of the information essential for the stable maintenance of the plasmid and to express the same host-range specificity . It was predicted that the protein required to initiate replication from at least one of the three origins of the plasmid is encoded by the longest open-reading frame (ORF239) of the three overlapping in-frame ORFs located within the 2 kb region . The product of ORF239 has been named RepA . The initiator protein was overexpressed, purified and used for in vitro binding studies . Gel mobility shift experiments were performed to localize RepA binding sites . The DNA sequence protected by the bound RepA molecule(s) was determined by DNase I footprinting and 19 of a 20 bp long sequence that is part of the protected sequence were located in two clusters flanking the repA gene . A plasmid created by linking a 310 bp fragment (nucleotides 238 to 547) of the 2 kb region to the antibiotic resistance genes carried by the omega fragment, can be maintained stably if the RepA protein is supplied in trans . We conclude that this 310 bp DNA fragment, which consists of a short G+C and a long A+T rich region and the cluster of five RepA binding sites, carries a functional origin of the plasmid-protein dependent replication . The position of this origin indicates that it is oriB, one of the three origins previously identified by electron microscopy . The second cluster of RepA binding sites is downstream of the repA gene and consists of 14 sites that are in inverted orientation compared with the binding sites located in the oriB region . They are part of the region that was shown formerly to be involved in controlling the copy number of the plasmid . In contrast to oriB, binding of RepA to neither the oriS nor oriV region was detected.

Mol Biochem Parasitol, 1995 Mar, 70(1-2), 45 - 58
Expression of GARP, a major surface glycoprotein of Trypanosoma congolense, on the surface of Trypanosoma brucei: characterization and use as a selectable marker; Hehl A et al.; Procyclic and epimastigote forms of Trypanosoma congolense express an immunodominant glutamic acid/alanine-rich protein (GARP) that covers the parasite surface . Although GARP shows no sequence similarity to procyclins from T . brucei, the general characteristics of the two sets of surface glycoproteins suggest that they have analogous functions, in much the same way that variant surface glycoproteins with unrelated primary sequences fulfil the same function in bloodstream form trypanosomes . Since T . brucei and T . congolense do not follow the same pathway through the tsetse fly, one possible function of procyclins might be to direct parasites to the correct compartments . As a first step towards testing this hypothesis, we have produced stably transformed procyclic forms of T . brucei in which the GARP coding region has been integrated into a procyclin expression site . GARP can be detected on the surface of these transgenic trypanosomes, uniformly distributed within the endogenous procyclin coat, but there are differences in post-translational modification when it is expressed in T . brucei rather than in T . congolense . The fact that GARP is readily accessible to antibodies which were raised against a bacterial fusion protein led us to examine its potential as a selectable surface marker for transfection . We have established a rapid and simple procedure for isolating stable transformants that provides an alternative to conventional methods of selection for antibiotic resistance.

J Bacteriol, 1995 Feb, 177(3), 530 - 5
Identification of new genes regulated by the marRAB operon in Escherichia coli; Seoane AS et al.; Random TnphoA and TnlacZ translational fusions were introduced into an Escherichia coli strain with a deletion of the multiple antibiotic resistance (mar) locus, complemented in trans by a temperature-sensitive plasmid bearing the mar locus with a constitutively expressed mar operon . Five gene fusions (two with lacZ and three with phoA) regulated by the mar operon were identified by increased or decreased marker enzyme activity following loss of the complementary plasmid at the restrictive temperature . Expression of LacZ from both lacZ fusions increased in the presence of the mar operon; expression from the three phoA fusions was represented by the mar operon . The lacZ fusions were mapped at 31.5 and 14 min on the Escherichia coli chromosome . One of the phoA fusions was located at 51.6 min while the two others mapped at 77 min . Cloning and sequencing of a portion of the fused genes showed all of them to be different . The phoA fusions at 77 min were located in a recently identified gene, slp, a lipoprotein of unknown function (D.M . Alexander and A . C . St . John, Mol . Microb . 11:1059-1071, 1994) . The others showed no homology with any known genes of E . coli . The insertions caused small but reproducible changes in the antibiotic susceptibility profile . This approach has enabled the identification of new genes in E . coli which are regulated by the marRAB operon and involved in the Mar phenotype.

Mol Microbiol, 1995 Feb, 15(4), 593 - 600
Mobile gene cassettes and integrons: capture and spread of genes by site-specific recombination; Hall RM et al.; An integron is a genetic unit that includes the determinants of the components of a site-specific recombination system capable of capturing and mobilizing genes that are contained in mobile elements called gene cassettes . An integron also provides a promoter for expression of the cassette genes, and integrons thus act both as natural cloning systems and as expression vectors . The essential components of an integron are an int gene encoding a site-specific recombinase belonging to the integrase family, an adjacent site, attI, that is recognized by the integrase and is the receptor site for the cassettes, and a promoter suitably oriented for expression of the cassette-encoded genes . The cassettes are mobile elements that include a gene (most commonly an antibiotic-resistance gene) and an integrase-specific recombination site that is a member of a family of sites known as 59-base elements . Cassettes can exist either free in a circularized form or integrated at the attI site, and only when integrated is a cassette formally part of an integron . A single site-specific recombination event involving the integron-associated attI site and a cassette-associated 59-base element leads to insertion of a free circular cassette into a recipient integron . Multiple cassette insertions can occur, and integrons containing several cassettes have been found in the wild . The integrase also catalyses excisive recombination events that can lead to loss of cassettes from an itegron and generate free circular cassettes . Due to their ability to acquire new genes, integrons have a clear role in the evolution of the genomes of the plasmids and transposons that contain them.

Biochim Biophys Acta, 1995 Jan 19, 1246(2), 109 - 27
Contribution of mutant analysis to the understanding of enzyme catalysis: the case of class A beta-lactamases; Matagne A et al.; Class A beta-lactamases represent a family of well studied enzymes . They are responsible for many antibiotic resistance phenomena and thus for numerous failures in clinical chemotherapy . Despite the facts that five structures are known at high resolution and that detailed analyses of enzymes modified by site-directed mutagenesis have been performed, their exact catalytic mechanism remains controversial . This review attempts to summarize and to discuss the many available data.

J Biol Chem, 1995 Jan 13, 270(2), 775 - 80
Substitution of Asp for Asn at position 132 in the active site of TEM beta-lactamase . Activity toward different substrates and effects of neighboring residues; Osuna J et al.; Using a random, combinatorial scheme of mutagenesis directed against the conserved SDN region of TEM beta-lactamase, and selective screening in ampicillin-plates, we obtained the N132D mutant enzyme . The kinetic characterization of this mutant indicated relatively small effects compared to the wild-type . Both pK1 and pK2 for catalysis were decreased about 1 unit relative to the pK's for the wild type . This effect was predominantly due to changes in Km . In contrast to the wild-type, the pH-rate profiles of the mutant showed that Km for several side chain-containing penicillin substrates increases when the pH is above 5.5 . 6-Aminopenicillanic acid, which lacks a side chain, did not show this effect . With benzylpenicillin, ampicillin, and carbenicillin, kcat for the mutant showed a similar pH dependence as the wild type . With 6-aminopenicillanic acid, kcat for the mutant was greater than that for the wild type . The nature of the 104 side chain may affect the environment of Asp132; double mutants N132D/E104X (where X can be Q or N) are unable to confer antibiotic resistance to bacterial cells . The computed contact interactions from modeling substrate complexes between benzylpenicillin or 6-aminopenicillanic acid with the N132D mutant confirmed the importance of the protonation state of residue Asp132 for the complex stability with side chain-containing substrates . The data indicate that the contact between the side chain of residue 132 and the substrate is relevant for the ground state recognition, but because of close contact with several important groups in its neighborhood, residue 132 is also indirectly involved in the catalytic step of the wild-type enzyme.

J Bacteriol, 1995 Jan, 177(2), 459 - 61
D-serine deaminase is a stringent selective marker in genetic crosses; Maas WK et al.; The presence of the locus for D-serine deaminase (dsd) renders bacteria resistant to growth inhibition by D-serine and enables them to grow with D-serine as the sole nitrogen source . The two properties permit stringent selection in genetic crosses and make the D-serine deaminase gene an excellent marker, especially in the construction of strains for which the use of antibiotic resistance genes as selective markers is not allowed.

Antimicrob Agents Chemother, 1995 Jan, 39(1), 185 - 91
PCR mapping of integrons reveals several novel combinations of resistance genes; Levesque C et al.; The integron is a new type of mobile element which has evolved by a site-specific recombinational mechanism . Integrons consist of two conserved segments of DNA separated by a variable region containing one or more genes integrated as cassettes . Oligonucleotide probes specific for the conserved segments have revealed that integrons are widespread in recently isolated clinical bacteria . Also, by using oligonucleotide probes for several antibiotic resistance genes, we have found novel combinations of resistance genes in these strains . By using PCR, we have determined the content and order of the resistance genes inserted between the conserved segments in the integrons of these clinical isolates . PCR mapping of integrons can be a useful epidemiological tool to study the evolution of multiresistance plasmids and transposons and dissemination of antibiotic resistance genes.

Antimicrob Agents Chemother, 1995 Jan, 39(1), 155 - 62
Expression of antibiotic resistance genes in the integrated cassettes of integrons; Collis CM et al.; Plasmids containing cloned integron fragments which differ only with respect to either the sequence of the promoter(s) or the number and order of inserted cassettes were used to examine the expression of resistance genes encoded in integron-associated gene cassettes . All transcripts detected commenced at the common promoter P(ant), and alterations in the sequence of P(ant) affected the level of resistance expressed by cassette genes . When both P(ant) and the secondary promoter P2 were present, transcription from both promoters was detected . When more than one cassette was present, the position of the cassette in the array influenced the level of antibiotic resistance expressed by the cassette gene . In all cases, the resistance level was highest when the gene was in the first cassette, i.e., closest to P(ant), and was reduced to different extents by the presence of individual upstream cassettes . In Northern (RNA) blots, multiple discrete transcripts originating at P(ant) were detected, and only the longer transcripts contained the distal genes . Together, these data suggest that premature transcription termination occurs within the cassettes . The most abundant transcripts appeared to contain one or more complete cassettes, and is possible that the 59-base elements found at the end of the cassettes (3' to the coding region) not only function as recombination sites but may also function as transcription terminators.

Immunogenetics, 1995, 42(3), 181 - 7
Efficient nonhomologous and homologous recombination in scid cells; Buhler B et al.; The severe combined immunodeficiency (scid) mutation affects both coding joint formation during immunoglobulin and T-cell receptor V(D)J recombination and double-strand break repair . We analyzed scid cells for their ability to undergo other types of DNA end joining: nonhomologous and homologous recombination . Using plasmid constructs carrying antibiotic resistance genes, we observed that the efficiency of nonhomologous integration in scid cells was equal to that in wildtype cell lines . In addition, there was no obvious difference in the fidelity of the integration and in the expression of the resistance genes . Moreover, scid cells were able to carry out homologous recombination of extrachromosomal substrates just as well as wildtype cells . These results suggest a mechanistic difference between nonhomologous integration and homologous recombination on the one hand and V(D)J recombination and double-strand break repair on the other.

Prog Brain Res, 1995, 105, 273 - 81
The transport of neurotransmitters into synaptic vesicles; Peter D et al.; Using selection in the neurotoxin MPP+, we have isolated a cDNA encoding vesicular amine transport . The transporter protects against MPP+ by sequestering the toxin in vesicles, away from its primary site of action in mitochondria . Unexpectedly, two distinct but highly related genes encode vesicular amine transport in the adrenal gland and the central nervous system . The sequence of both predicts twelve transmembrane domains and weak homology to a class of bacterial antibiotic resistance proteins . The two human genes occur on different chromosomes . In addition, the two transporters show a number of differences in function, including substrate specificity and the interaction with one inhibitor and the amphetamines.

Arch Intern Med, 1994 Dec 12-26, 154(23), 2666 - 77
Efficacy of pneumococcal vaccination in adults . A meta-analysis of randomized controlled trials; Fine MJ et al.; BACKGROUND: Because of the prevalence of pneumococcal pneumonia, the substantial morbidity and mortality associated with many pneumococcal infections, and an increase in the incidence of antibiotic resistance among pneumococcal isolates, considerable efforts for disease prevention have been made using a polyvalent polysaccharide pneumococcal vaccine . Despite numerous clinical trials of the vaccine, its efficacy in the prevention of pneumococcal infections and other clinically relevant medical outcomes in adults remains uncertain . METHODS: To assess quantitatively the efficacy of pneumococcal vaccination, a MEDLINE literature search, manual reviews of article bibliographies, and communications with pneumococcal vaccine investigators were used to identify randomized controlled trials of the pneumococcal vaccine . Independent review of 594 articles revealed nine randomized trials with 12 vaccine and control study groups that evaluated clinically relevant outcomes in adults . To estimate a summary effect size for all outcomes, Mantel-Haenszel odds ratios (ORs) and Dersimonian and Laird rate differences (RDs) and their associated 95% confidence intervals (CIs) were computed . RESULTS: Summary ORs demonstrated a statistically significant protective effect of the vaccine for four pneumococcal infection-related outcomes: definitive pneumococcal pneumonia (OR = 0.34; 95% CI = 0.24 to 0.48), definitive pneumococcal pneumonia for vaccine-containing pneumococcal antigen types only (vaccine types only) (OR = 0.17; 95% CI = 0.09 to 0.33), presumptive pneumococcal pneumonia (OR = 0.47; 95% CI = 0.35 to 0.63), and presumptive pneumococcal pneumonia (vaccine types only) (OR = 0.39; 95% CI = 0.26 to 0.59) . The summary RDs, which account for heterogeneity among studies, confirmed a statistically significant protective effect for two of these same outcomes: definitive pneumococcal pneumonia (RD = 4/1000; 95% CI = 0/1000 to 7/1000) and definitive pneumococcal pneumonia (vaccine types only) (RD = 8/1000; 95% CI = 1/1000 to 16/1000) . Summary ORs and RDs failed to demonstrate a protective effect for pneumonia (all causes), bronchitis, and mortality (all causes) or mortality due to pneumonia or pneumococcal infection . Subgroup analyses showed that for all four pneumococcal infection-related outcomes, vaccine efficacy differed for high- and low-risk subjects, demonstrating efficacy for low-risk subjects and lack of efficacy for high-risk subjects . CONCLUSIONS: Pneumococcal vaccination appears efficacious in reducing bacteremic pneumococcal pneumonia in low-risk adults . However, evidence from randomized controlled trials fails to demonstrate vaccine efficacy for pneumococcal infection-related or other medical outcomes in the heterogeneous group of subjects currently labeled as high risk.

J Bacteriol, 1994 Dec, 176(24), 7754 - 6
Analysis of the genetic requirements for inducible multiple-antibiotic resistance associated with the mar locus in Escherichia coli; Sulavik MC et al.; A series of novel genetic constructs derived from the marRAB operon was used to determine the role of this gene cluster in salicylate-inducible multiple-antibiotic resistance in Escherichia coli . Our findings indicate that regulated antibiotic resistance associated with this locus requires only the products of marR and marA, without any neighboring genes.

J Bacteriol, 1994 Dec, 176(24), 7735 - 9
The incN plasmid replicon: two pathways of DNA polymerase I-independent replication; Kim HY et al.; The 2,053-bp broad-host-range incompatibility group N replicon of plasmid pCU1 has two components: a region of 1,200 bp that is sufficient for its replication in Escherichia coli PolA+ and PolA- hosts and a regulatory region called the group I iteron region that contains 13 39-bp iterons . Within the 1,200-bp region, there are three replication origins, two of which, called oriB and oriS, function in PolA+ and PolA- hosts and a third, called oriV, which functions only in PolA+ hosts . The region also specifies a protein called RepA . We now show that both oriB and oriS can function in a delta polA strain but that in such a strain, only oriB has an absolute requirement for RepA . oriS can function without RepA and polymerase I provided that the iteron region is deleted and that in this circumstance, it is the only origin, the usage of which is detected . The requirements for oriB usage can thus be distinguished from those for oriS usage . The oriB region can be recovered as a plasmid only if RepA is provided in trans . These complex features of this replicon are also shown to be shared by the IncN replicons of other antibiotic resistance plasmids . Functionally distinguishable origins in a small replicon may be a way of endowing such a replicon with a broad host range.

Eur J Clin Microbiol Infect Dis, 1994 Dec, 13(12), 1015 - 22
Non-canonical mechanisms of antibiotic resistance; Martinez JL et al.; Although the current in vitro methods used for detection and analysis of the phenotypes of antibiotic resistance in the laboratory are well established, other resistance mechanisms of resistance exist which may escape detection using the standard approach . The present article reviews some of these mechanisms which are grouped under the term 'non-canonical mechanisms' of antibiotic resistance . Such mechanisms include gene dosage, heterologous induction or selection, populational resistance and synergism between mechanisms of low resistance . The role of these mechanisms in the failure of therapy is discussed.

Southeast Asian J Trop Med Public Health, 1994 Dec, 25(4), 698 - 701
The use of surgical antibiotic prophylaxis in seven Malaysian hospitals; Lim VK et al.; A survey on the use of antibiotics in surgical prophylaxis was carried out in seven Malaysian hospitals . Details of antibiotic prescriptions were obtained through questionnaires completed by the prescriber . A total of 430 such prescriptions was analysed . A large number of different antibiotic regimens were used for a variety of surgical procedures . The majority of prescriptions (70%) were issued for procedures where such prophylaxis was probably not necessary . Antibiotics were also often prescribed for durations that were longer than necessary . There is an urgent need to educate surgeons and standardize surgical prophylactic regimens in order to reduce cost and combat the emergence of antibiotic resistance.

J Bacteriol, 1994 Oct, 176(20), 6262 - 9
Dual regulation of inaA by the multiple antibiotic resistance (mar) and superoxide (soxRS) stress response systems of Escherichia coli; Rosner JL et al.; The roles of the marRAB (multiple antibiotic resistance) operon and soxRS (superoxide response) genes in the regulation of inaA, an unlinked weak-acid-inducible gene, were studied . inaA expression was estimated from the beta-galactosidase activity of a chromosomal inaA1::lacZ transcriptional fusion . marR mutations that elevate marRAB transcription and engender multiple antibiotic resistance elevated inaA expression by 10- to 20-fold over that of the wild-type . Similarly, one class of inaA constitutive mutants that mapped to the mar region were multiply antibiotic resistant . Overexpression of marA alone on a multicopy plasmid caused high constitutive expression of inaA in a strain with an extensive (39-kbp) marRAB deletion . Salicylate, an inducer of marRAB and of an unidentified mar-independent antibiotic resistance system, induced inaA by 6-fold . A portion of this induction was also mar independent . Two soxRS constitutive mutants that were tested showed elevated levels of inaA . Paraquat, an inducer of the soxRS system, elevated inaA expression by 6- to 9-fold . This induction was soxRS dependent and not mar dependent, whereas induction of inaA by salicylate was not dependent on soxRS . Paraquat induced resistance to norfloxacin in the mar-deleted strain but not in a soxRS-deleted strain . Thus, induction of multiple antibiotic resistance and inaA by salicylate occurs via mar and an unidentified pathway, while induction by paraquat occurs via soxRS.

Trends Microbiol, 1994 Oct, 2(10), 416 - 21
Resistance to drugs targeting protein synthesis in mycobacteria; Bottger EC; Many antibiotics exert their effects by interfering with protein synthesis . Studies of the molecular mechanisms of antibiotic resistance in clinical strains of mycobacteria have revealed mutations in ribosomal RNAs . This type of acquired resistance was previously unknown in bacterial pathogens and was made possible because mycobacteria have only a single set of rRNA genes.

Genetika, 1994 Sep, 30(9), 1141 - 5
{Cloning and expression of the B1 hordein gene of barley (Hordeum vulgare L.) in cells of the cyanobacterium Synechocystis sp . PCC6803 and Escherichia coli}; Chemeresiuk NN et al.; The hordein B1 gene of Hordeum vulgare L . (variety Donetskii 4) was cloned in cells of Escherichia coli and cyanobacterium Synechocystis sp . PCC6803 . For cloning in E . coli, a 2.3-kb Hpa I/EcoR I DNA fragment carrying the hordein B1 gene was inserted into plasmid pBSM13(-), controlled by a lac promoter . The constructed recombinant plasmid pBSB1 provides hordein B1 gene expression in transformed E . coli cells . To introduce the hordein B1 gene into the genome of Synechocystis sp . PCC6803, an integrative vector, containing a fragment of cyanobacterium chromosomal DNA with inserted Tn5 antibiotic resistance genes (KmR, BleoR, StpR) was constructed . A Pvu II-fragment of pBSB1, containing hordein B1 gene under control of the lac promoter, was cloned in the Sma I-site of gene BleoR . The resulting recombinant plasmid was used to transform cyanobacterial cells . One KmR transformants contained a DNA fragment yielding a positive signal during Southern blotting with a {32P}-labeled DNA fragment carrying hordein B1 gene . Western blotting with polyclonal antibodies to barley hordein revealed hordein B1 gene expression in Synechocystis sp . PCC6803.

Heart Lung, 1994 Sep-Oct, 23(5), 363 - 7
Antibiotic use and antibiotic resistance in the intensive care unit: are we curing or creating disease?
Kollef MH.
Antibiotics represent one of the most commonly prescribed medical therapies for hospitalized patients . The practice of "spiralling empiricism" has increasingly led to the unnecessary administration of antibiotics, resulting in the emergence of infections with antibiotic-resistant bacteria that are associated with increased rates of patient mortality . Ventilator-associated pneumonia due to antibiotic-resistant bacteria has become recognized as an important problem resulting from prior antibiotic exposure . Health professionals must be aware of this problem and avoid the unnecessary administration of these drugs . Future research efforts should be aimed at improving our ability to diagnose and exclude infections and to develop better strategies for antibiotic administration in the intensive care unit setting.

Todays OR Nurse, 1994 Sep-Oct, 16(5), 7 - 12
Bugs and drugs: antibiotic resistance in the 1990s; Rickman LS; 1 . Bacteria have been versatile in circumventing the mechanisms by which antibiotics work; in the 1990s there has been a world-wide resurgence of antibiotic-resistant bacterial diseases . 2 . Resistance to certain antibiotics develops in bacteria through three basic processes: mutation, transduction, and conjugation . 3 . Infection-control programs, if combined with intensive infection-control measures and control of antibiotic use, may be effective at reducing the transmission of resistant pathogens.

Plasmid, 1994 Sep, 32(2), 222 - 7
DNA sequence of direct repeats of the sulI gene of plasmid pSa; Valentine CR et al.; The restriction enzyme and genetic map of the antibiotic-resistance region of plasmid pSa is related to Tn21 integrons by the insertion of 5.4 kb containing a chloramphenicol resistance gene (catII) and a 1.1-kb direct repeat . We report here the nucleotide sequences of both copies of the repeat with adjoining sequences . They were identical for 1065 bp and contained the entire coding sequence of the sulfanilamide resistance gene, sulI . Since only the first copy of the repeat confers sulfonamide resistance, this leads to the conclusion that no promoter was available for the second copy . The sequence of the pSa sulI gene was identical to several published sulI sequences from other plasmids . The first junction point of the catII-containing insert was identical to the sequence for pDG0100; the second junction occurred farther into the 3'-conserved segment of integrons than does that of pDG0100 . A recent report of these junction sequences for pSa and pDG0100 differs from our sequences by one nucleotide . Two additional differences were an insert of 41 bases and a single base insertion between sulI and ORF341 in our sequence . Our sequenced regions have been assigned GenBank Accession Nos . UO4277 and UO4278 for the first and second sulI genes of pSa, respectively.

Curr Genet, 1994 Aug, 26(2), 179 - 83
Highly-efficient transformation of the homobasidiomycete Schizophyllum commune to phleomycin resistance; Schuren FH et al.; Regulatory sequences of the glyceraldehyde-3-phosphate-dehydrogenase (GPD) gene from the homobasidiomycete Schizophyllum commune were fused to the coding sequence of the ble gene from Streptoalloteichus hindustanus, which codes for a phleomycin-binding protein . The resulting construct transformed S . commune to phleomycin resistance at a high frequency (up to 10(4) transformants/microgram DNA per 10(7) protoplasts) when regeneration was done in 0.5 M MgSO4 . A similar construct with regulatory sequences from Aspergillus nidulans failed to give transformants, showing the importance of homologous regulatory sequences for the expression of genes in S . commune . The homologous GPD promoter could be deleted up to position -130 without any effect on the number of phleomycin-resistant transformants . This is the first effective stable transformation system in a homobasidiomycete employing antibiotic resistance.

Antimicrob Agents Chemother, 1994 Aug, 38(8), 1773 - 9
Genetic relationship between soxRS and mar loci in promoting multiple antibiotic resistance in Escherichia coli; Miller PF et al.; Multiple antibiotic resistance in Escherichia coli has typically been associated with mutations at the mar locus, located at 34 min on the E . coli chromosome . A new mutant, marC, isolated on the basis of a Mar phenotype but which maps to the soxRS (encoding the regulators of the superoxide stress response) locus located at 92 min, is described here . This mutant shares several features with a known constitutive allele of the soxRS gene, prompting the conclusion that it is a highly active allele of this gene . The marC mutation has thus been given the designation soxR201 . This new mutant was used to examine the relationship between the mar and sox loci in promoting antibiotic resistance . The results of these studies indicate that full antibiotic resistance resulting from the soxR201 mutation is partially dependent on an intact mar locus and is associated with an increase in the steady-state level of mar-specific mRNA . In addition, paraquat treatment of wild-type cells is shown to increase the level of antibiotic resistance in a dose-dependent manner that requires an intact soxRS locus . Conversely, overexpression of MarA from a multicopy plasmid results in weak activation of a superoxide stress response target gene . These findings are consistent with a model in which the regulatory factors encoded by the marA and soxS genes control the expression of overlapping sets of target genes, with MarA preferentially acting on targets involved with antibiotic resistance and SoxS directed primarily towards components of the superoxide stress response . Furthermore, compounds frequently used to induce the superoxide stress response, including paraquat, menadione, and phenazine methosulfate, differ with respect to the amount of protection provided against them by the antibiotic resistance response.

Zh Mikrobiol Epidemiol Immunobiol, 1994 Aug-Sep, Suppl 1, 87 - 91
{A comparative evaluation of the persistence characteristics of Escherichia isolated from different econiches}; Gritsenko VA et al.; The multiple evaluation of the persistence characteristics, including antilysozyme, anti-interferon and anticomplement activity, as well as other biological properties, such as adhesiveness, colicinogenicity and resistance to antibiotics, was carried out in 173 E . coli strains isolated from water, healthy and sick children . This evaluation revealed that each group of E . coli, depending on the source of its isolation, had its characteristic set of properties (or bioprofiles) to be analyzed, making it different from other bacterial populations . The comparative intergroup analysis showed differences between E . coli isolated from children with pathological conditions (enteric coli-bacteriosis, pyelonephritis) and E . coli isolated from water and feces of healthy children . These differences were manifested by more pronounced persistence characteristics . Dispersion analysis, having confirmed this feature, revealed that the most labile characteristics of E.coli, subject to the influence of ecological conditions, were their markers of persistence and antibiotic resistance . The results of factor analysis made it possible to unite the above mentioned properties which determined, together with adhesiveness, pathogenic potential of these bacteria.

Mol Gen Genet, 1994 Jul 25, 244(2), 189 - 96
Chloroplast targeting of spectinomycin adenyltransferase provides a cell-autonomous marker for monitoring transposon excision in tomato and tobacco; Scofield SR et al.; Antibiotic resistance genes can act as either cell autonomous or non-cell autonomous genetic markers with which to monitor the excision of plant transposons . To convert spectinomycin resistance from a non-cell autonomous resistance to cell autonomous resistance, a transit peptide for chloroplast localization from a petunia ribulose bisphosphate carboxylase (rbcS) gene was fused in-frame to the aadA gene, which confers spectinomycin and streptomycin resistance . Constructs were generated in which the expression of this chimeric gene was prevented by the presence, in the 5' untranslated leader, of the maize transposons Activator (Ac) or Dissociation (Ds) . When progeny of tobacco or tomato plants transformed with these constructs were germinated on spectinomycin-containing medium, germinally revertant and somatically variegated individuals could be distinguished.

Infect Control Hosp Epidemiol, 1994 Jul, 15(7), 472 - 7
Antibiotic resistance is selected primarily in our patients; Pechere JC; The potential for bacterial resistance probably existed prior to the arrival of humans on earth and bacterial populations isolated before the antibiotic era surely contained antibiotic-resistant organisms . Antibiotic resistance has undergone an explosive development following the introduction of antibiotics in medical practice and in agriculture, and there is no doubt that the higher prevalence of bacterial resistance is closely related to human activities . Strict infection control policies limit the risk of patient-to-patient transmission of resistant as well as susceptible bacteria.

Nucleic Acids Res, 1994 Jun 11, 22(11), 2071 - 8
Characterisation of specific and secondary recombination sites recognised by the integron DNA integrase; Recchia GD et al.; Integrons determine a site-specific recombination system which is responsible for the acquisition of genes, particularly antibiotic resistance genes . The integrase encoded by integrons recognises two distinct classes of recombination sites . The first is the family of imperfect inverted repeats, known as 59-base elements, which are associated with the mobile gene cassettes . The second consists of a single site into which the cassettes are inserted . This site, here designated attI, is located adjacent to the int gene in the recipient integron structure . The attI site has none of the recognisable features of members of the 59-base element family except for a seven-base core site, GTTRRRY, at the recombination crossover point . Using a conduction assay to quantitate site activity, the sequence required for maximal attI site activity was confined to a region of > 39 and < or = 70 bases . Both integrative and excisive site-specific recombination events involving attI and a 59-base element site were demonstrated, but no evidence for events involving two attI sites was obtained . Integrase-mediated recombination between a 59-base element and several secondary sites in pACYC184 with the consensus GNT occurred at low frequency, and such events could potentially lead to insertion of gene cassettes at many non-specific sites.

J Bacteriol, 1994 Jun, 176(11), 3257 - 68
Transposon Tn5090 of plasmid R751, which carries an integron, is related to Tn7, Mu, and the retroelements; Radstrom P et al.; Integrons confer on bacterial plasmids a capability of taking up antibiotic resistance genes by integrase-mediated recombination . We show here that integrons are situated on genetic elements flanked by 25-bp inverted repeats . The element carrying the integron of R751 has three segments conserved with similar elements in Tn21 and Tn5086 . Several characteristics suggest that this element is a transposon, which we call Tn5090 . Tn5090 was shown to contain an operon with three open reading frames, of which two, tniA and tniB, were predicted by amino acid similarity to code for transposition proteins . The product of tniA (559 amino acids) is a probable transposase with 25% amino acid sequence identity to TnsB from Tn7 . Both of these polypeptides contain the D,D(35)E motif characteristic of a protein family made up of the retroviral and retrotransposon IN proteins and some bacterial transposases, such as those of Tn552 and of a range of insertion sequences . Like the transposase genes in Tn552, Mu, and Tn7, the tniA gene was followed by a gene, tniB, for a probable ATP-binding protein . The ends of Tn5090, like those of most other elements producing D,D(35)E proteins, begin by 5'-TG and also contains a complex structure with four 19-bp repeats at the left end and three at the right end . Similarly organized repeats have been observed earlier at the termini of both Tn7 and phage Mu, where they bind their respective transposases and have a role in holoenzyme assembly . Another open reading frame observed in Tn5090, tniC, codes for a recombinase of the invertase/resolvase family, suggesting a replicative transposition mechanism . The data presented here suggest that Tn5090, Tn7, Tn552, and Mu form a subfamily of bacterial transposons which in parallel to many insertion sequences are related to the retroelements.

FASEB J, 1994 Jun, 8(9), 639 - 45
Bcl-2 inhibits glucocorticoid-induced apoptosis but only partially blocks calcium ionophore or cycloheximide-regulated apoptosis in S49 cells; Caron-Leslie LA et al.; Many non-Hodgkins B-cell lymphomas possess a deregulated bcl-2 gene resulting in a phenotype that is apparently resistant to programmed cell death (apoptosis) . We have used a mouse lymphoma cell line (S49.1) that undergoes apoptosis in response to a variety of stimuli to determine the effect of bcl-2 expression on induction of apoptosis . S49 cells were stably transfected with recombinant amphotrophic retroviruses carrying either a G418 antibiotic resistance gene alone (S49-NEO) or this gene in combination with a bcl-2 complementary DNA (S49-Bcl-2) . Three different agents previously shown to activate apoptosis by different pathways in S49 cells (dexamethasone, the calcium ionophore A23187, and cycloheximide) were used to examine the effect of bcl-2 expression on cell growth and apoptosis caused by multiple signal transduction pathways . Dexamethasone (DEX) treatment inhibited cell growth and stimulated cell death in S49-NEO cells . Although S49-Bcl-2 cells exhibited a similar antiproliferative response, they failed to die in response to steroid treatment . Western blot analysis revealed no difference in the levels of glucocorticoid receptor protein in the two cell lines, and both responded to glucocorticoid with a profound inhibition of protein synthesis . Cycloheximide (CX) and A23187 also had antiproliferative and cell killing effects in both cell types, although higher concentrations of each agent were needed to kill S49-Bcl-2 cells . To determine whether the loss of viability in response to these drugs was due to apoptosis, cells were examined morphologically and DNA integrity was examined by gel electrophoresis.(ABSTRACT TRUNCATED AT 250 WORDS)

EMBO J, 1994 Jun 1, 13(11), 2483 - 92
Crystal structure and site-directed mutagenesis of a bleomycin resistance protein and their significance for drug sequestering; Dumas P et al.; The antibiotic bleomycin, a strong DNA cutting agent, is naturally produced by actinomycetes which have developed a resistance mechanism against such a lethal compound . The crystal structure, at 2.3 A resolution, of a bleomycin resistance protein of 14 kDa reveals a structure in two halves with the same alpha/beta fold despite no sequence similarity . The crystal packing shows compact dimers with a hydrophobic interface and involved in mutual chain exchange . Two independent solution studies (analytical centrifugation and light scattering) showed that this dimeric form is not a packing artefact but is indeed the functional one . Furthermore, light scattering also showed that one dimer binds two antibiotic molecules as expected . A crevice located at the dimer interface, as well as the results of a site-directed mutagenesis study, led to a model wherein two bleomycin molecules are completely sequestered by one dimer . This provides a novel insight into antibiotic resistance due to drug sequestering, and probably also into drug transport and excretion.

Gene, 1994 May 16, 142(2), 225 - 30
Three selectable markers for transformation of Ustilago maydis; Gold SE et al.; Although Ustilago maydis is readily amenable to molecular genetic experimentation, few antibiotic-resistance markers are available for DNA-mediated transformation . This poses constraints on experiments involving targeted gene disruption and complementation . To address this problem, we constructed vectors using one of three additional genes as dominant selectable markers for transformation . Two genes, sat-1 (encoding streptothricin acetyltransferase) and Sh-ble (encoding a phleomycin-resistance polypeptide), are of bacterial origin and have been engineered for expression in Ustilago sp . The third gene encodes an allele of U . maydis beta-tubulin that confers resistance to the fungicide benomyl.

Gene, 1994 May 3, 142(1), 49 - 54
Diversity and relative strength of tandem promoters for the antibiotic-resistance genes of several integrons; Levesque C et al.; The integron is a new type of mobile element containing one or more antibiotic-resistance-encoding genes site-specifically integrated as cassettes . The integrated genes are expressed from a common promoter region located in an adjacent conserved segment . Sequence analysis has revealed the existence of four versions of the integron promoters . In this study, we have determined the relative strength of the different integron promoters and compared their activity with that of the tac promoter . Each version of the promoter was cloned upstream from a promoter-less chloramphenicol acetyltransferase-encoding gene (cat) in plasmid pKK232-8 . CAT activity was used to measure transcriptional expression from the promoters of the antibiotic-resistance operon . The strongest promoter is the version (TTGACAN17TAAACT) found in plasmid R388 and in transposon Tn1696 . This promoter is six times more efficient than the derepressed tac promoter.

J Clin Microbiol, 1994 May, 32(5), 1318 - 21
Oligonucleotide (GTG)5 as a marker for Mycobacterium tuberculosis strain identification; Wiid IJ et al.; Culture of Mycobacterium tuberculosis provides no information on the identity of a strain or the distribution of such a strain in the community . Strain identification of M . tuberculosis can help to address important epidemiological questions, e.g., the origin of an infection in a patient's household or community, whether reactivation of infection is endogenous or exogenous in origin, and the spread and early detection of organisms with acquired antibiotic resistance . To research this problem, strain identification must be reliable and accurate . Although genetic identification techniques already exist, it is valuable to have genetic identification techniques based on a number of genetic markers to improve the accurate identification of M . tuberculosis strains . We show that oligonucleotide (GTG)5 can be successfully applied to the identification of M . tuberculosis strains . This technique may be particularly useful in cases in which M . tuberculosis strains have few or no insertion elements (e.g., IS6110) or in identifying other strains of mycobacteria when informative probes are lacking.

J Bacteriol, 1994 May, 176(9), 2525 - 31
Lipid synthesis in mycobacteria: characterization of the biotin carboxyl carrier protein genes from Mycobacterium leprae and M . tuberculosis; Norman E et al.; The causative agents of leprosy and tuberculosis, Mycobacterium leprae and Mycobacterium tuberculosis, have a lipid-rich cell envelope which contributes to virulence and antibiotic resistance . Acyl coenzyme A carboxylase, which catalyzes the first committed step of lipid biosynthesis, consists in mycobacteria of two subunits, one of which is biotinylated . Genes from M . leprae and M . tuberculosis encoding a biotinylated protein have been cloned and sequenced . Analysis of the derived protein sequences demonstrated the presence of biotin-binding sites and putative ATP-bicarbonate interactions sites, consistent with the proteins having a biotin carboxylase function as well as their being biotin carrier proteins.

Med J Aust, 1994 Apr 4, 160(7), 395 - 7
Tuberculosis: medical students at risk; Wilkins D et al.; In 1979 an outbreak of tuberculosis occurred in medical students at the University of Sydney . Eight of 35 Mantoux-negative students who attended the autopsy of an immunosuppressed patient with unsuspected active tuberculosis became infected and one developed clinical disease . A report of the incident was prepared for publication because it supported the then controversial University policy of recommending BCG vaccination to medical and dental students in a country where the reported prevalence of tuberculosis is very low . The report was never published, mainly in order to protect the privacy of the individual students involved, but also because it was felt by the administration of the time that it might undermine confidence in infection control procedures in the autopsy room . The original report, updated and reproduced here, suggested that tuberculosis might be an emerging nosocomial problem . This has been all too clearly realised since its re-emergence as an opportunistic infection in AIDS patients . Worldwide, the problem of antibiotic resistance in Mycobacterium tuberculosis provides an added risk of a return to the situation which prevailed early this century when tuberculosis was a major occupational risk for young health care workers . Infection often restricted career choices, even in those whose disease was relatively benign . Our purpose in bringing this incident to light after so many years is to point out the relevance of the extensive studies of the problem which were conducted in the 1930s and 1940s to the current situation and to suggest that health care students are vulnerable to airborne infections as well as those spread by inoculation injuries . In retrospect, our 1979 conclusions about prospects for preventing nosocomial tuberculosis appear optimistic.

Mol Microbiol, 1994 Apr, 12(2), 217 - 29
Evolution of antibiotic resistance: several different amino acid substitutions in an active site loop alter the substrate profile of beta-lactamase; Palzkill T et al.; In order to understand how TEM-1 beta-lactamase substrate specificity can be altered by mutation, amino acid residues 161 through to 170 were randomly mutagenized to sample all possible amino acid substitutions . The 161-170 region includes a portion of an omega loop structure, which is involved in the formation of the active-site pocket . The percentage of random sequences that provide bacterial resistance to either ampicillin or to the extended-spectrum cephalosporin ceftazidime was determined . It was found that the sequence requirements for wild-type levels of ampicillin resistance are much more stringent than the sequence requirements for ceftazidime resistance . Surprisingly, more than 50% of all amino acid substitutions in the 161-170 region result in levels of ceftazidime resistance at least three times greater than wild type . In addition, by increasing the level of the selection for ceftazidime resistance, substitutions that result in a greater than 100-fold increase in ceftazidime resistance were identified . Characterization of altered beta-lactamase enzymes indicated that while their catalytic efficiency (kcat/Km) for ceftazidime hydrolysis is higher, the enzymes are poorly expressed relative to wild-type TEM-1 beta-lactamase.

Biotechniques, 1994 Apr, 16(4), 626 - 8, 630-3
Phagemid pSIT permits efficient in vitro mutagenesis and tightly controlled expression in E . coli; Andreansky M et al.; A new phagemid vector, pSIT, was constructed that allows both oligonucleotide-directed mutagenesis and tightly controlled, high-level expression of proteins in Escherichia coli . An efficient rate of mutagenesis is achieved by taking advantage of the double oligonucleotide primer technique . In addition to the mutagenic primer, a second oligonucleotide primer conferring antibiotic resistance to the mutant DNA strand is annealed to single-strand DNA . Selection for the antibiotic thus increases the frequency of mutants . High-level and tightly controlled expression of heterologous proteins is enabled by utilizing a very strong hybrid T7lac promoter and lac repressor in conjunction with T7 RNA polymerase, as well as a high copy number of the vector . The pSIT phagemid permits overexpression of proteins and their mutants without subclonings from mutagenic to expression constructs, which saves time, especially when multiple mutations of the same protein are proposed . A retroviral proteinase precursor, toxic for E . coli, was successfully expressed to a high level, and a series of mutants of this protein was readily obtained.

Plasmid, 1994 Mar, 31(2), 166 - 83
Identification of a gene encoding the replication initiator protein of the Streptomyces integrating element, pSAM2; Hagege J et al.; pSAM2 is an 11-kilobase integrating element from Streptomyces ambofaciens which was previously shown to generate single-stranded DNA during replication, indicating that it probably replicates by a rolling-circle replication (RCR) mechanism . Two separate regions are involved in its replication, one of which was shown to contain the plus origin of replication (ds origin) . We report here the study of the second region . Its nucleotide sequence was determined and analysed for open reading frames (ORFs) . Three putative ORFs were identified: orf183 (183 amino acids (aa)), orf50 (50 aa), and repSA (459 aa) . orf183 is not necessary for replication . The function of orf50 is unknown . repSA is essential for pSAM2 replication; it could encode a protein, RepSA, presenting similarities to the replication initiator proteins (Rep) of elements that replicate by an RCR mechanism . A derivative consisting of repSA, the region containing ds origin, a Streptomyces antibiotic resistance marker, and pBR322, could replicate in Streptomyces, further demonstrating that this ORF encodes the major replication protein of pSAM2 . repSA might be co-transcribed with the genes involved in integration and excision of pSAM2.

Genetics, 1994 Mar, 136(3), 867 - 77
A suppressor of a mating-type limited zygotic lethal allele also suppresses uniparental chloroplast gene transmission in Chlamydomonas monoica; VanWinkle-Swift K et al.; Uniparental inheritance of Chlamydomonas chloroplast genes is thought to involve modification of maternal (mt+) chloroplast genomes to protect against a nuclease that is activated after gamete fusion . The mating-type limited mtl-1 mutant strain of Chlamydomonas monoica is unable to protect mt(+)-derived chloroplast DNA . Zygotes homozygous for mtl-1 lose all chloroplast DNA and fail to germinate . We have selected for suppression of this zygote-specific lethality, and have obtained 20 mutant strains that produce viable homozygotes despite the continued presence of the mtl-1 allele . Genetic analysis indicates that the suppressor mutations are all recessive alleles at a single locus (sup-1) which is unlinked to mtl-1 . Crosses between sup-1 strains carrying distinctive chloroplast antibiotic resistance markers also show predominantly biparental chloroplast gene transmission . Chloroplast nucleoids of both parental origins (stained with the DNA-specific fluorochrome, DAPI) are retained in the zygotes homozygous for sup-1 . The data are compatible with the idea that the sup-1 (suppressor of uniparental inheritance) locus may encode a chloroplast DNA nuclease that is expressed from both parental genomes.

SCI Nurs, 1994 Mar, 11(1), 7 - 12
Strategies for the management and control of antibiotic resistant organisms on a spinal cord injury unit; Gardenhire MH et al.; Individuals with a spinal cord injury (SCI) are at an increased risk of infection and colonization . Frequent lengthy hospitalizations, invasive procedures, and skin breakdown contribute to this risk . Intermittent antibiotic use influences the emergence of antibiotic resistance in these organisms . As a result, there is risk of transmission of these antibiotic resistant organisms (ARO) . This article describes the application of a continuous quality improvement model to evaluate ARO management strategies in a SCI unit . A conservative, labor intensive, crisis management approach to the control of ARO was replaced with a more cost effective prospective plan . The new strategies were aimed at control rather than eradication and included collaborative, multidisciplinary planning and improved resource utilization . Efforts have been successful and have resulted in the control of ARO.

Braz J Med Biol Res, 1994 Feb, 27(2), 349 - 56
The role of GPI-PLC in Trypanosoma brucei; Webb H et al.; The glycosylphosphatidylinositol-specific phospholipase C (GPI-PLC) from Trypanosoma brucei exhibits exquisite specificity for the GPI-anchor of the variant specific glycoprotein (VSG) . However the evidence that it is involved in VSG metabolism in the living trypanosome is circumstantial; it shows the same life cycle stage regulated expression as the VSG, no feasible alternative substrate has been identified, and it metabolises the VSG efficiently in vitro and in vivo on hypotonic lysis . Against these considerations are the observations that the GPI-PLC is found on the cytoplasmic face of vesicles so it could not gain access to the VSG through normal vesicle fusion and that the accelerated loss of VSG from bloodstream forms on differentiation to procyclic forms occurs through the action of a protease . To try to determine the role of the GPI-PLC, a homozygous null mutant T . brucei has been constructed . The null mutant was created by replacement of the entire gene at both alleles with selectable antibiotic resistance markers in procyclic form trypanosomes . The GPI-PLC gene is not usually expressed in procyclic forms and so, as would be expected, the null procyclics display no obvious phenotype . The null procyclics have been used to infect tsetse flies and it remains to be seen whether it is possible for them to differentiate to bloodstream forms and, if so, what the antigenic variation phenotype of the null bloodstream forms would be.

J Bacteriol, 1994 Jan, 176(1), 143 - 8
Repressor mutations in the marRAB operon that activate oxidative stress genes and multiple antibiotic resistance in Escherichia coli; Ariza RR et al.; Resistance to multiple antibiotics and certain oxidative stress compounds was conferred by three independently selected mutations (marR1, soxQ1, and cfxB1) that mapped to 34 min on the Escherichia coli chromosome . Mutations at this locus can activate the marRAB operon, in which marR encodes a putative repressor of mar transcription and marA encodes a putative transcriptional activator of defense genes against antibiotics and oxidants . Overexpression of the wild-type MarR protein reversed the phenotypes (antibiotic resistance and increased antioxidant enzyme synthesis) of all three mutants . DNA sequence analysis showed that, like marR1, the other two mutations were alterations of marR: a 285-bp deletion in cfxB1 and a GC-->AT transition at codon 70 (Ala-->Thr) in soxQ1 . All three mutations cause increased amounts of mar-specific RNA, which supports the hypothesis that MarR has a repressor function in the expression of the marRAB operon . The level of mar RNA was further induced by tetracycline in both the marR1 and soxQ1 strains but not in the cfxB1 deletion mutant . In the cfxB1 strain, the level of expression of a truncated RNA, with or without tetracycline exposure, was the same as the fully induced level in the other two mutants . Overproduction of MarR in the cfxB1 strain repressed the transcription of the truncated RNA and restored transcriptional inducibility by tetracycline . Thus, induction of the marRAB operon results from the relief of the repression exerted by MarR . The marRAB operon evidently activates both antibiotic resistance and oxidative stress genes.

J Antimicrob Chemother, 1994 Jan, 33(1), 25 - 32
Genetic structures associated with spread of the type Ia trimethoprim-resistant dihydrofolate reductase gene amongst Escherichia coli strains isolated in the Nottingham area of the United Kingdom; Towner KJ et al.; DNA probes for specific integrase genes were used to study 122 R plasmids encoding the predominant trimethoprim-insusceptible type Ia dihydrofolate reductase (DHFR) found in clinical isolates of Escherichia coli . The predominance of the type Ia DHFR was thought to result from the location of its gene on transposon Tn7, but of trimethoprim R plasmids carrying this gene that were collected between 1978 and 1983, between 1987 and 1988, and during 1992, only 49/60 (81.6%), 30/43 (69.8%) and 9/19 (47.4%) respectively hybridized with a probe for the Tn7 integrase gene . It has been suggested that novel genetic elements termed 'integrons' may play an important role in the dissemination of antibiotic resistance genes . Known integrons encode an integrase similar to that encoded by transposon Tn21, and 28 Tn7-negative plasmids (10/60 from 1978-83, 10/43 from 1987-8 and 8/19 from 1992) showed homology with a probe specific for the Tn21 integrase gene . Six plasmids were negative with both probes . It is concluded that Tn7 has played an important role in the dissemination of the gene encoding the type Ia DHFR amongst clinical isolates of E . coli in the Nottingham region of the UK, but that other genetic structures, some of which seem to have an integrase function similar to that of known integrons, may be playing an increasingly significant role.

Scand J Gastroenterol Suppl, 1994, 201, 11 - 5
The role of Helicobacter pylori in peptic ulcer disease; O'Connor HJ; There is now a persuasive body of evidence linking Helicobacter pylori infection and peptic ulcer disease . Over 90% of duodenal ulcer and 70% of gastric ulcer patients are infected with H . pylori . Only a minority of infected patients develop ulcers, however, and host cofactors, rather than H . pylori strain, are probably critical to the development of peptic ulcer in infected individuals . Conversely, not all ulcers are associated with H . pylori, and in these cases enterogastric reflux and non-steroidal anti-inflammatory drug ingestion may be important . Eradication of H . pylori dramatically reduces ulcer relapse, effectively curing the disease . Eradication may also accelerate duodenal ulcer healing . Triple therapy with bismuth and antibiotics is effective against H . pylori, but there are problems with side effects, compliance and antibiotic resistance . Encouraging results are emerging on the efficacy and safety of omeprazole/antibiotic combination therapy, and this novel approach to H . pylori eradication is an exciting development . H . pylori has established itself as a pivotal factor in peptic ulcer disease and an effective helicobactericidal regimen is now the most rational and cost-effective treatment.

Vet Microbiol, 1994 Jan, 38(3), 193 - 201
Serotypes, toxins and antibiotic resistance of Escherichia coli strains isolated from diarrhoeic and healthy rabbits in Spain; Blanco JE et al.; One hundred and ten Escherichia coli strains isolated from diarrhoeic and healthy rabbits from 50 Spanish commercial farms were serotyped and investigated for production of enterotoxins (LT and STa), verotoxins (VT), cytotoxic necrotizing factors (CNF1 and CNF2), alpha-haemolysin (Hly) and enterohaemolysin (EntHly), for necrotic and lethal activities and for antibiotic resistance . Six serogroups (O2, O26, O49, O92, O103 and O128) accounted for 81% (67 of 83) and 26% (7 of 27) respectively of E . coli strains isolated from diarrhoeic and healthy rabbits (P < 0.001) . The most common serotypes found among E . coli strains associated with diarrhoeic rabbits in order of frequency were: O103:K-:H2, O49:K?:H2, O26:K-:H-, O26:K-:H11, O128:K?:H-, O92:K-:H2 and O2:K5:H6 . E . coli strains belonging to the same serotype but from different farms usually showed a distint antibiotics resistance pattern . Only one strain, of serotype O2:K5:H6 was toxigenic (CNF1+, Hly+, necrotic and lethal).

Rev Mal Respir, 1994, 11(4), 415 - 7
{Diffuse nodular dissemination of thoracic actinomycosis}; Kessler R et al.; Thoracic actinomycosis is a rare infection but sensitive to penicillin G which is the antibiotic of choice . We report a case of thoracic actinomycosis which was characterised by a relapse, probably linked to antibiotic resistance following treatment with Tetracycline . This relapse presented as diffuse nodular dissemination, of miliary type, of which there are only a few examples in the literature.

Mol Gen Genet, 1993 Dec, 241(5-6), 627 - 36
Analysis of the frequency of inheritance of transposed Ds elements in Arabidopsis after activation by a CaMV 35S promoter fusion to the Ac transposase gene; Long D et al.; The Ac/Ds transposon system of maize shows low activity in Arabidopsis . However, fusion of the CaMV 35S promoter to the transposase gene (35S::TPase) increases the abundance of the single Ac mRNA encoded by Ac and increases the frequency of Ds excision . In the experiments reported here it is examined whether this high excision frequency is associated with efficient re-insertion of the transposon . This was measured by using a Ds that carried a hygromycin resistance gene (HPT) and was inserted within a streptomycin resistance gene (SPT) . Excision of Ds therefore gives rise to streptomycin resistance, while hygromycin resistance is associated with the presence of a transposed Ds or with retention of the element at its original location . Self-fertilisation of most individuals heterozygous for Ds and 35S::TPase produced many streptomycin-resistant (strep(r)) progeny, but in many of these families a small proportion of strep(r) seedlings were also resistant to hygromycin (hyg(r)) . Nevertheless, 70% of families tested did give rise to at least one strep(r), hyg(r) seedling, and over 90% of these individuals carried a transposed Ds . In contrast, the Ac promoter fusion to the transposase gene (Ac::TPase) produced fewer strep(r)hyg(r) progeny, and only 53% of these carried a transposed Ds . However, a higher proportion of the strep(r) seedlings were also hyg(r) than after activation by 35S::TPase . We also examined the genotype of strep(r), hyg(r) seedlings and demonstrated that after activation by 35S::TPase many of these were homozygous for the transposed Ds, while this did not occur after activation by Ac::TPase . From these and other data we conclude that excisions driven by 35S::TPase usually occur prior to floral development, and that although a low proportion of strep(r) progeny plants inherit a transposed Ds, those that do can be efficiently selected with an antibiotic resistance gene contained within the element . Our data have important implications for transposon tagging strategies in transgenic plants and these are discussed.

J Bacteriol, 1993 Dec, 175(24), 7856 - 62
Salicylate induction of antibiotic resistance in Escherichia coli: activation of the mar operon and a mar-independent pathway; Cohen SP et al.; Since the growth of wild-type Escherichia coli in salicylate results in a multiple antibiotic resistance phenotype similar to that of constitutive mutants (Mar) of the chromosomal mar locus, the effect of salicylate on the expression of the marRAB operon was investigated . The amount of RNA hybridizing with a mar-specific DNA probe was 5 to 10 times higher in wild-type cells grown with sodium salicylate (5.0 mM) than in untreated controls . Untreated Mar mutants had three to five times more mar-specific RNA than wild-type cells did . When a Mar mutant was treated with salicylate, a 30- to 50-fold increase of mar-specific RNA was seen . In wild-type cells bearing a mar promoter-lacZ fusion on the chromosome, salicylate increased beta-galactosidase activity by sixfold . Thus, salicylate induces transcription of the marRAB operon . Other inducers of phenotypic multiple antibiotic resistance, e.g., benzoate, salicyl alcohol, and acetaminophen, but not acetate, also increased transcription from the mar promoter but to a lesser extent than did salicylate . Both in wild-type and mar-deficient strains, growth in salicylate resulted in increased antibiotic resistance, decreased permeation of the outer membrane to cephaloridine, increased micF transcription, and decreased amounts of OmpF . However, the magnitude of these changes was generally greater in wild-type (mar-containing) cells . Thus, salicylate and other compounds can induce transcription of the mar operon and, presumably, give rise to multiple antibiotic resistance via this pathway . However, salicylate can also activate an unidentified, mar-independent pathway(s) which engenders multiple antibiotic resistance.

Antimicrob Agents Chemother, 1993 Nov, 37(11), 2379 - 84
Antibiotic preparations contain DNA: a source of drug resistance genes?
Webb V, Davies J.
Fluorescence measurements and polymerase chain reaction amplification of streptomycete 16S ribosomal DNA sequences were used to show that a number of antibiotic preparations employed for human and animal use are contaminated with chromosomal DNA of the antibiotic-producing organism . The DNA contains identifiable antibiotic resistance gene sequences; the uptake of this DNA by bacteria and its functional incorporation into bacterial replicons would lead to the generation of antibiotic resistance determinants . We propose that the presence of DNA encoding drug resistance in antibiotic preparations has been a factor in the rapid development of multiple antibiotic resistance in bacteria.

Mol Microbiol, 1993 Nov, 10(4), 823 - 8
Secondary-sites for integration mediated by the Tn21 integrase; Francia MV et al.; The integrase encoded by the integron of the transposon Tn21 can mediate the site-specific fusion of two plasmids if there is a recombination hot spot (59 bp element) in one of them and the sequence GWTMW in the other . The use of this latter, loosely defined site explains how antibiotic-resistance genes could first become associated with integrons.

Cancer Res, 1993 Oct 15, 53(20), 4900 - 6
Expression of a rat glutathione-S-transferase complementary DNA in rat mammary carcinoma cells: impact upon alkylator-induced toxicity; Schecter RL et al.; The role of glutathione-S-transferase (GST) in alkylator drug resistance has been studied in MatB rat mammary carcinoma cells . A series of GST transfectant cell lines was established by using an expression vector containing the complementary DNA for the rat GST Yc gene under regulation of the SV40 early region promoter and the antibiotic resistance plasmid pSV2neo . Transfectant cell lines expressing up to 4-fold higher total GST activity than in the parental wild type cell line were identified . Southern blot analysis confirmed a DNA fragment corresponding in size to the transfected GST Yc complementary DNA . Wild type MatB cells contain very low levels of Yc protein, whereas the Yc+ clones showed greatly increased amounts of the Yc subunit . The effect of increased GST Yc activity on the sensitivity of the transfected clones to various cytotoxic agents was assessed by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cell survival assay . The clones expressing recombinant GST Yc were more resistant to melphalan (6- to 12-fold), mechlorethamine (10- to 16-fold), and chlorambucil (7- to 30-fold) . In late passage populations of the GST Yc+ clones that had been grown over a period of 14 months under continuous selection in G418, GST activity was decreased and it was paralleled by a decrease in Yc protein . These late passage clones with diminished GST Yc content also demonstrate a partial reversion toward the wild type phenotype as determined by cytotoxicity assays using melphalan, mustargen, and chlorambucil . Interstrand DNA cross-links induced by mechlorethamine were significantly lower at 0, 2, and 20 h posttreatment in one of the GST Yc+ clones when compared to wild type MatB cells . These studies indicate that GST Yc overexpression can confer resistance to alkylating agents and that this correlates with inhibition of DNA cross-link formation.

Genitourin Med, 1993 Oct, 69(5), 364 - 9
The incidence of gonorrhoea and the antibiotic sensitivity of gonococci in Australia, 1981-1991 . The Australian Gonococcal Surveillance Programme; Transformation of the acidophilic heterotroph Acidiphilium facilis by electroporation; Division of Bioresources Science, Faculty of Agriculture, Okayama University, JapanWe constructed a cloning vector for use in the acidophilic heterotroph Acidiphilium facilis . The vector pAH101 (8.8 kb) was constructed from a 6.1 kb restriction fragment of the Acidiphilium plasmid pAH1 and a pUC19 carrying a beta-lactamase gene . The antibiotic resistance gene was efficiently expressed in A . facilis . Several factors which influenced the transformation efficiency were optimized, resulting in a transformation efficiency of up to 3 x 10(3) transformants per microgram of plasmid DNA at a field strength of 10 kV/cm with a 7.0 ms pulse.

Plasmid, 1993 Sep, 30(2), 159 - 62
Effects of plasmid pBR322 on respiratory and ATPase activities in Escherichia coli; Eisenbraun MD et al.; The effects of antibiotic resistance plasmids on respiratory and ATPase activities were investigated in Escherichia coli . The rates of oxygen consumption coupled to the oxidation of succinate and NADH, ATP hydrolysis, and ATP-Pi exchange were measured in isolated membrane vesicles prepared from HB101 strains that contained derivatives of plasmid pBR322 . The rates of oxygen consumption coupled to the oxidation of NADH were independent of the presence or absence of plasmids in the strains from which the vesicles were prepared . In contrast, the mean rates of oxygen consumption coupled to the oxidation of succinate, ATP hydrolysis, and ATP-Pi exchange were 140-292% higher in vesicles made from plasmid-containing strains than from plasmid-free HB101 and independent of the expression of the plasmid-encoded tetracycline/H+ antiporter.

J Diarrhoeal Dis Res, 1993 Sep, 11(3), 143 - 7
Antibiotic resistance pattern of heat-labile enterotoxin (LT) producing Escherichia coli isolated from children with diarrhoea in Bangladesh: clonal relationships among isolates with different resistant phenotypes; Faruque SM et al.; Fifty-six heat-labile, enterotoxin-producing (LT+) Escherichia coli isolated from 33 children less than 5 years of age with diarrhoea were analysed for resistance to antibiotics, plasmid contents, and clonal relationships among isolates by ribosomal RNA (rRNA) fingerprinting (ribotyping) . Fifty-five (98.2%) of the LT+ isolates were resistant either to tetracycline alone (48.2%) or to tetracycline and one or more other antibiotic, i.e . ampicillin, streptomycin, chloramphenicol, trimethoprim-sulfamethoxazole, or nalidixic acid . Most of the isolates harboured one or more plasmid but antibiotic resistance patterns did not always correlate with particular plasmid patterns . Ribotyping of the isolates using the restriction endonuclease EcoRI revealed a total of 7 different ribotypes, and ribotypes were shared by E . coli isolates with different antibiotic resistant phenotypes . The results indicate that in Bangladesh at least 7 different clones of LT+ E . coli acquired resistance to one or more different antibiotics in various combinations . However, a similar drug resistance pattern was not mediated by the same set of plasmids in all strains . The mechanism for the emergence and spread of antibiotic resistance among E . coli should be investigated further in Bangladesh, where LT+ E . coli is an important agent of early childhood diarrhoea.

J Bacteriol, 1993 Aug, 175(15), 4641 - 51
Double helicase II (uvrD)-helicase IV (helD) deletion mutants are defective in the recombination pathways of Escherichia coli; Mendonca VM et al.; The Escherichia coli helD (encoding helicase IV) and uvrD (encoding helicase II) genes have been deleted, independently and in combination, from the chromosome and replaced with genes encoding antibiotic resistance . Each deletion was verified by Southern blots, and the location of each deletion was confirmed by P1-mediated transduction . Cell strains containing the single and double deletions were viable, indicating that helicases II and IV are not essential for viability . Cell strains lacking helicase IV (delta helD) exhibited no increase in sensitivity to UV irradiation but were slightly more resistant to methyl methanesulfonate (MMS) than the isogenic wild-type cell strain . As expected, cell strains containing the helicase II deletion (delta uvrD) were sensitive to both UV irradiation and MMS . The introduction of the helicase IV deletion into a delta uvrD background had essentially no effect on the UV and MMS sensitivity of the cell strains analyzed . The double deletions, however, conferred a Rec- mutant phenotype for conjugational and transductional recombination in both recBC sbcB(C) and recBC sbcA backgrounds . The Rec- mutant phenotype was more profound in the recBC sbcB(C) background than in the recBC sbcA background . The recombination-deficient phenotype indicates the direct involvement of helicase II and/or helicase IV in the RecF pathway {recBC sbcB(C) background} and RecE pathway (recBC sbcA background) of recombination . The modest decrease in the recombination frequency observed in single-deletion mutants in the recBC sbcB(C) background suggests that either helicase is sufficient . In addition, helicase IV has been overexpressed in a tightly regulated system . The data suggest that even modest overexpression of helicase IV is lethal to the cell.

Gene, 1993 Jul 15, 129(1), 17 - 25
Construction of new beta-glucuronidase cassettes for making transcriptional fusions and their use with new methods for allele replacement; Metcalf WW et al.; Five cassettes carrying uidA, encoding beta-glucuronidase, were made for the construction of insertion mutants with transcriptional fusions to uidA . Three uidA cassettes contain antibiotic-resistance genes, for chloramphenicol (Cm), for kanamycin (Km) and neomycin (Nm), or for streptomycin (Sm) and spectinomycin (Sp) . Some cause polar insertions while others provide a promoter for downstream gene expression . The expression of these uidA cassettes was compared to the expression of lacZ at the same site in phnD, a phosphate-regulated gene for phosphonate use . Several phn::uidA or phn::lacZ insertions were recombined onto the chromosome to test mutational effects and to measure gene expression in single copy . This was done using one of three methods for allele replacement . A new method involved recombination of mutations in M13 onto the chromosome by infection of an Escherichia coli rep mutant that fails to propagate single-stranded DNA phages . Merodiploid recombinants were selected using a resistance marker carried by the M13 phage; segregants lacking M13 sequences were then selected as deoxycholate-resistant (DocR) ones . An improved method for recombination of mutations in pir-dependent, oriR6K vectors involved the use of plasmids containing genes for tetracycline resistance (TcR) . Merodiploid recombinants were selected by conjugative transfer of such plasmids into a recipient lacking pir (encoding the pi protein of the R6K plasmid); segregants lacking vector sequences were subsequently selected as Tc-sensitive ones . Both procedures are efficient and allow for recombining marked as well as unmarked mutations onto the chromosome . In addition, some insertions with an antibiotic-resistance marker were directly recombined onto the chromosome by transformation of a recD mutant with linear DNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Plasmid, 1993 Jul, 30(1), 39 - 50
The partial 3'-conserved segment duplications in the integrons In6 from pSa and In7 from pDGO100 have a common origin; Stokes HW et al.; Integrons are genetic elements which are capable of acquiring genes by site-specific recombination . The most common integron structure consists of two conserved segments flanking a variable region where many different antibiotic resistance genes have been found . The integrons In6 and In7, present in the plasmids pSa and pDGO100, respectively, are unusual in that they include a duplication of the sulI gene which is located within the integron 3'-conserved segment . To further investigate the structure of these integrons, the DNA sequence of the segment located between the two sulI genes was determined . In In7 this segment is 2822 bases long and includes a trimethoprim resistance gene, dhfrX, at one end . The corresponding region in In6 is 4.5 kb and is nearly identical to the In7 segment over the first 2105 bases . In the region unique to In6, a cat gene, conferring chloramphenicol resistance, has replaced the dhfrX gene of In7 . This location thus represents a second variable region where different antibiotic resistance genes are found, but the way in which genes become associated with this second variable region is not known . The overall similarity of the structures of In6 and In7 suggests that the additional DNA segments found in these integrons have a common origin, and a possible mechanism for the origin of integrons with partial 3'-conserved segment duplications is presented.

Can J Vet Res, 1993 Jul, 57(3), 146 - 51
Characterization of Escherichia coli isolated from cases of avian colibacillosis; Allan BJ et al.; Forty-four western Canadian isolates of Escherichia coli associated with colibacillosis of turkeys and chickens were examined for serotype, antibiotic resistance, and production of aerobactin . The isolates belonged to fourteen O serogroups, with 39% of the strains being non-typeable . A high frequency of resistance to tetracycline, kanamycin, neomycin, cephalothin, streptomycin and erythromycin was observed . Most isolates produced aerobactin . Ten E . coli belonging to serogroups O1, O2 and O78 were also examined for pili production, hemagglutination, serum sensitivity, production of iron-regulated outer membrane proteins (IROMPS), and virulence . All isolates examined produced pili, exhibited mannose-sensitive hemagglutination of avian red blood cells and produced IROMPS under iron-restricted growth conditions . The five isolates of serogroup O1 and O2 were resistant to killing by turkey serum and were highly virulent . Only two of the five isolates of serogroup O78 were serum resistant . No correlation between serum resistance and virulence was observed in serogroup O78.

J Bacteriol, 1993 Jun, 175(12), 3784 - 9
Amplification of the bacA gene confers bacitracin resistance to Escherichia coli; Cain BD et al.; An Escherichia coli genomic library was constructed in order to facilitate selection for genes which confer bacitracin resistance through amplification . One of the plasmids from the library, plasmid pXV62, provided a high level of bacitracin resistance for E . coli . Deletion and nucleotide sequence analyses of bacitracin resistance plasmid pXV62 revealed that a single open reading frame, designated the bacA gene, was sufficient for antibiotic resistance . The bacA gene mapped to approximately 67 min on the E . coli chromosome by proximity to a previously mapped locus . The deduced amino acid sequence of the bacA-encoded protein suggests an extremely hydrophobic protein of 151 amino acids, approximately 65% of which were nonpolar amino acids . E . coli cells containing plasmid pXV62 have increased isoprenol kinase activity . The physical characteristics of the deduced protein and enhanced lipid kinase activity suggest that the bacA gene may confer resistance to bacitracin by phosphorylation of undecaprenol.

Gene, 1993 May 15, 127(1), 53 - 61
The Azotobacter chroococcum hydrogenase gene cluster: sequences and genetic analysis of four accessory genes, hupA, hupB, hupY and hupC; Tibelius KH et al.; The Azotobacter chroococcum chromosome contains a region spanning about 14 kb associated with hydrogen-uptake (Hup) activity . The small and large subunits of the hydrogenase are encoded by the structural genes hupS and hupL . Two other genes, hupD and hupE, are located 8.9 kb downstream from hupL and are required for the formation of a catalytically active hydrogenase . In this study, we determined the nucleotide sequence of a 3.8-kb region immediately upstream from hupD . This revealed four additional closely linked ORFs which we designated hupA, hupB, hupY and hupC; these genes potentially encode polypeptides with predicted masses of 12.6, 33.3, 80.4 and 9.0 kDa, respectively . This cluster of genes was shown to be essential for hydrogenase activity by insertion mutagenesis using antibiotic-resistance gene cassettes and a Tn5 derivative carrying a promoterless lacZ gene . A 10.5-kb fragment of DNA beginning 3.4 kb downstream from hupL, and including the sequenced region, was able to complement hupA and hupY mutants, supporting earlier evidence for a promoter downstream from hupSL . The deduced amino acid sequences of hupA, hupB and hupC are homologous to the Escherichia coli hypA, hypB and hypC gene products, respectively . Of particular interest is the fact that there is no homologue of the hupY gene product in the E . coli hyp operon . Mutations in hupY or hupB had little effect on beta-galactosidase activity in a strain also carrying a hupL::lacZ fusion, showing that hupY and hupB are not major factors in regulating the transcription of the hydrogenase structural genes.

J Bacteriol, 1993 May, 175(10), 2888 - 94
Overexpression of the MarA positive regulator is sufficient to confer multiple antibiotic resistance in Escherichia coli; Gambino L et al.; A genetic approach was undertaken to identify normal bacterial genes whose products function to limit the effective concentration of antibiotics . In this approach, a multicopy plasmid library containing cloned Escherichia coli chromosomal sequences was screened for transformants that showed increased resistance to a number of unrelated antibiotics . Three such plasmids were identified, and all contained sequences originating from the mar locus . DNA sequence analysis of the minimal complementation unit revealed that the resistance phenotype was associated with the presence of the marA gene on the plasmids . The putative marA gene product is predicted to contain a helix-turn-helix DNA binding domain that is very similar to analogous domains found in three other E . coli proteins . One such similarity was to the SoxS gene product, the elevated expression of which has previously been associated with the multiple antibiotic resistance (Mar) phenotype . Constitutive expression of marA conferred antibiotic resistance even in cells carrying a deletion of the chromosomal mar locus . We have also found that transformants bearing marA plasmids show a significant reduction in ompF translation but not transcription, similar to previously described mar mutants . However, this reduction in ompF expression plays only a minor role in the resistance mechanism, suggesting that functions encoded by genes unlinked to mar must be affected by marA . These results suggest that activation of marA is the ultimate event that occurs at the mar locus during the process that results in multiple antibiotic resistance.

Mol Gen Mikrobiol Virusol, 1993 May-Jun, (3), 12 - 5
{Transposon Tn5 and its derivatives used in genetic analysis of bacteria}; Katsy EI; The review deals with the derivatives of transposon Tn5 carrying new genes of antibiotic resistance . The derivatives were constructed for mobilization of genetically labeled replicons, for direct selection of mutants having lost the marked plasmids, for obtaining the genes with the strong constitutive or regulated expression, for isolation of conditional mutations, for faster physical mapping of megaplasmids.

FEMS Microbiol Lett, 1993 May 1, 109(1), 33 - 8
Rapid and efficient selection of recombinant site-directed mutants of Bradyrhizobium japonicum by colony hybridization; Fu C et al.; Due to the high incidence of spontaneous antibiotic resistance and slow growth of Bradyrhizobium japonicum strains, screening for site-directed mutants is cumbersome and time-consuming . A rapid method for selection of recombinant site-directed mutants of B . japonicum was developed . A kanamycin (Km) and a spectinomycin (Sp) cassette were each used to replace DNA fragments in the chromosome by homologous recombination . The primary new features of this method involve a simple plate selection for the antibiotic (Km or Sp) resistant mutants, then colony streaking, and lysis for DNA hybridization on a nitrocellulose filter enabling direct identification of the recombinant site-directed mutants . This method has permitted us to quickly and easily identify a large number of positive recombinant mutants from a large number of individual colonies . The procedure eliminates the need to first isolate genomic DNA from each mutant for Southern hybridization . All of the tested site-directed mutants from this method were confirmed to exhibit the expected mutant phenotype.

Bull Acad Natl Med, 1993 May, 177(5), 729 - 37; discussion 737-8
{Resources for the campaign against nosocomial infection in hospitals}; Regnier B; Since 1973, the Ministry of Health has recommended to set up Infection Control Committees which became statutory in 1988 . They have many responsibilities including the survey of nosocomial infection rate, the implementation of appropriate control strategies, the control of "antibiotic resistance", the training of staff, the production and the evaluation of written procedures for patients care, isolation, sterilization..., as well as they are in charge of the protection of health care workers against occupational risk of infection . Although, such committees have been appointed in more than 85% of hospitals, their true activity and the rate of nosocomial infection remain much less well known . The very low proportion of beds actually involved in a "surveillance" program, the scarcity of specific personnel, the insufficiency of institutional program of training, and the height prevalence of bacterial resistance to antibiotics are all of concern . Recently, the Ministry of Health has intensified its requirements and recommendations . However, resources allocated to nosocomial infection control remain unsatisfactory and it makes mandatory that each hospital generate a program which should be recognized as a priority . It is obvious that willingness is not sufficient and that the appointment of nosocomial infection control teams is warranted with appropriately trained personnel . However it is fair te recognize that the cost-effectiveness evaluation of various strategies remains to be done.

J Antimicrob Chemother, 1993 Apr, 31(4), 463 - 71
Resistance of yeasts to azole-derivative antifungals; Odds FC; There are relatively few antifungal agents available for the treatment of systemic mycoses . The incidence of these infections, particularly among the immunocompromised, has increased significantly in recent years . Amphotericin B, flucytosine and the azole-derivatives--fluconazole, itraconazole and ketoconazole--are the only drugs of value in the treatment of systemic yeast infections currently available . To date resistance among individual yeast species or strains has only been a serious problem with flucytosine . However, resistance among Candida spp . to orally administered azole-derivatives has been observed . The frequency with which resistance has been described in clinical practice among yeasts differs considerably between the three azole antifungal agents . Fluconazole has been implicated in emergent resistance more frequently than ketoconazole, and ketoconazole more often than itraconazole . It must be a matter for concern that, by analogy with the known emergence of antibiotic-resistance among bacteria, that the widespread use of a drug inactive against a particular species may lead to an increased incidence of such infections . An international epidemiological survey is required to establish the extent and degree of resistance to the azole antifungals.

J Infect Dis, 1993 Apr, 167(4), 975 - 8
DNA fragment length polymorphism analysis of Mycobacterium tuberculosis isolates by arbitrarily primed polymerase chain reaction; Palittapongarnpim P et al.; Strain identification of Mycobacterium tuberculosis would prove whether transmission had occurred between individuals . A method to characterize strains of M . tuberculosis has been developed utilizing polymerase chain reaction (PCR) . Purified chromosomal DNA of cultured clinical samples of M . tuberculosis were subjected to PCR using short (10-12 nucleotide) oligonucleotide primers . PCR products visualized after agarose gel electrophoresis and ethidium bromide staining demonstrated that different strains of M . tuberculosis give different banding patterns . This technique was used to confirm the relationship between cases of tuberculosis in several clusters, prove the lack of relationship between 2 isolates with the same antibiotic-resistance pattern, confirm a suspected mislabeling event, and suggest the source of infection in a case of tuberculous meningitis . This method is rapid and simple and does not require radioactive probes.

J Bacteriol, 1993 Mar, 175(5), 1484 - 92
Genetic and functional analysis of the multiple antibiotic resistance (mar) locus in Escherichia coli; Cohen SP et al.; A 7.8-kbp fragment of chromosomal DNA from a region controlling multiple antibiotic resistance (Mar) in Escherichia coli has been sequenced . Within the fragment is a potential divergent promoter region including marO, which contains two pairs of direct repeats, suggesting possible operator-regulatory sites . To the left of marO (region I) are one or two transcriptional units with three putative open reading frames (ORFs) encoding 64, 157, and 70 amino acids . To the right (region II) is a transcriptional unit containing three putative ORFs (ORF125/144, ORF129, and ORF72) . Of six independent Mar mutants, four had mutations within the ORF encoding the first putative protein (ORF125/144) downstream of marO, including three different single-point mutations and an IS2 insertion . One of the other mutations occurred in marO (20-bp duplication), and the other occurred in a site in marO or ORF144 (a 1-bp change) . All six mutations led to increased transcription of the region II transcript . High-copy-number plasmids containing marO and the adjacent ORF125/144 region from a wild-type source but not from a Mar mutant reduced the antibiotic resistance of a Mar mutant to levels comparable to those of wild-type cells . High-copy-number plasmids containing wild-type marO alone caused an increase in resistance to tetracycline, chloramphenicol, and norfloxacin in a wild-type strain . The nature of the Mar mutations and the results of the complementation studies suggest that ORF125/144 encodes a repressor (designated MarR) which acts at marO . The second ORF (ORF129), designated marA, would encode a protein, MarA, whose sequence shows strong similarity to those of a family of positive transcriptional regulators . A Tn5 insertion in marA inactivated the multiresistance phenotype of Mar mutants . The function of ORF72, designated marB, encoding the third putative protein in the operon, and that of other ORFs detected within the 7.8-kb fragment have not yet been determined.

Mol Microbiol, 1993 Feb, 7(3), 407 - 17
Superinfection immunity of mycobacteriophage L5: applications for genetic transformation of mycobacteria; Donnelly-Wu MK et al.; Mycobacteriophage L5 is a temperate phage of the mycobacteria that forms stable lysogens in Mycobacterium smegmatis . We show here that the 183-amino-acid product of L5 gene 71 confers immunity to L5 superinfection, is required for maintenance of the lysogenic state and contains a helix-turn-helix DNA-binding motif--properties associated with repressors of temperate phages . We have utilized these observations to demonstrate the use of L5 gene 71 as a selectable marker for genetic transformation of the mycobacteria . Significantly, the use of L5 gene 71 as a selectable gene avoids the requirement for antibiotic-resistance genes providing an important tool for manipulation of the pathogens Mycobacterium tuberculosis and Mycobacterium avium, and for the construction of recombinant BCG vaccines.

Aust Fam Physician, 1993 Feb, 22(2), 125 - 31
STDs and the overseas traveller; Rowbottom J; Although most HIV and STD patients acquire their infections in Australia there is an increase in the numbers diagnosed with these infections after international travel . Risks to the sexual health of the travellers and their subsequent partners are discussed and suggestions made for minimising risksPIP: Modern international travel contributed greatly to the global AIDS pandemic . About 500,000 Australians have sexual intercourse in the Philippines and Thailand annually . Many do not practice safer sex . A significant potential means of HIV entering the Australian heterosexual population is unprotected intercourse with prostitutes in Southeast Asia . The median HIV prevalence rate in female prostitutes in Thailand is 15% . Other sexually transmitted diseases (STDs) also pose a risk to Australian travelers . In 1991, in Victoria, 44% of gonorrhea cases were heterosexual males who had acquired gonorrhea abroad . Sex workers transmitted gonorrhea to 68% of these cases . A history of gonorrhea or chancroid increases the risk of HIV transmission . Other cofactors of HIV transmission are genital warts and genital herpes, both of which are common in Australia . Various types of men have taken great risks overseas, which places their partners at risk when they return . Since it is not easy to identify the type of persons who places himself at risk when abroad, physicians should discuss sexual risks with any patient who plans to travel overseas or who has returned . Women experience more severe consequences of STDs (e.g., pelvic inflammatory disease) than men because they are more likely to be asymptomatic in the early stages . Women should know that the risk of HIV transmission is high in Africa, Southeast Asia, and some areas in the US . Physicians should know behavioral risk factors (e.g., heavy drinking or drug use) . They should remind homosexual men to practice safer sexual practices abroad, even though they may be better informed than heterosexual men . Physicians need to tell travelers that a prostitute is having a health certificate does not guarantee that she does not have STDs or HIV . Further, a healthy appearance does not equate STD-free status . Travelers should carry condoms with them . Physicians should refrain from prescribing prophylactic antibiotics to minimize antibiotic resistance .

J Bacteriol, 1993 Feb, 175(4), 1126 - 33
Transferable Streptomyces DNA amplification and coamplification of foreign DNA sequences; Hornemann U et al.; The 8.8-kb amplifiable unit of DNA of Streptomyces achromogenes subsp . rubradiris, AUD-Sar 1, which carries 0.8-kb terminal direct repeats and a spectinomycin resistance determinant, can mediate high-level amplification of an AUD-Sar 1-derived 8.0-kb DNA sequence not only in S . achromogenes but also in the heterologous host Streptomyces lividans . This was seen upon introduction of AUD-Sar 1 into chloramphenicol-sensitive strains of S . lividans via the temperature-sensitive (39 degrees C) plasmid pMT660, which contains the thiostrepton resistance gene tsr . Following the cultivation of transformants at 39 degrees C on media containing spectinomycin, a number of strains which were unable to grow on thiostrepton and which carried the amplified 8.0-kb DNA sequence as arrays of 200 to 300 copies of tandem 8.0-kb repeats were found . Chloramphenicol-resistant strains of S . lividans did not yield amplified sequences under similar conditions . Studies with plasmids carrying inserted antibiotic resistance genes at two sites of AUD-Sar 1 yielded coamplified sequences which contain the inserted DNA . Transformation with a plasmid carrying a 1.0-kb deletion in AUD-Sar 1 followed by growth under similar conditions yielded a 7.0-kb repeated DNA sequence . Southern analysis revealed the absence of vector sequences located on the right side of AUD-Sar 1 in the input plasmids in all examined DNA samples of amplified strains . In contrast, a majority of the samples revealed the presence at unit copy level of AUD-Sar 1 left-adjacent sequences which are part of the input plasmids and in several samples the presence of certain vector sequences located near them . The results suggest input plasmid integration into the S . lividans chromosome prior to the generation of the amplified sequences and the deletion of AUD-Sar 1 adjacent sequences.

J Bacteriol, 1993 Feb, 175(3), 826 - 39
Insertion derivatives containing segments of up to 16 amino acids identify surface- and periplasm-exposed regions of the FhuA outer membrane receptor of Escherichia coli K-12; Koebnik R et al.; The FhuA receptor in the outer membrane of Escherichia coli K-12 is involved in the uptake of ferrichrome, colicin M, and the antibiotic albomycin and in infection by phages T1, T5, and phi 80 . Fragments of up to 16 amino acid residues were inserted into FhuA and used to determine FhuA active sites and FhuA topology in the outer membrane . For this purpose antibiotic resistance boxes flanked by symmetric polylinkers were inserted into fhuA and subsequently partially deleted . Additional in-frame insertions were generated by mutagenesis with transposon Tn1725 . The 68 FhuA protein derivatives examined contained segments of 4, 8, 12, 16, and 22 additional amino acid residues at 34 different locations from residues 5 to 646 of the mature protein . Most of the FhuA derivatives were found in normal amounts in the outer membrane fraction . Half of these were fully active toward all ligands, demonstrating proper insertion into the outer membrane . Seven of the 12- and 16-amino-acid-insertion derivatives (at residues 378, 402, 405, 415, 417, 456, and 646) were active toward all of the ligands and could be cleaved by subtilisin in whole cells, suggesting a surface location of the extra loops at sites which did not affect FhuA function . Two mutants were sensitive to subtilisin (insertions at residues 511 and 321) but displayed a strongly reduced sensitivity to colicin M and to phages phi 80 and T1 . Four of the insertion derivatives (at residues 162, 223, 369, and 531) were cleaved only in spheroplasts and probably form loops at the periplasmic side of the outer membrane . The number and size of the proteolytic fragments indicate cleavage at or close to the sites of insertion, which has been proved for five insertions by amino acid sequencing . Most mutants with functional defects were affected in their sensitivity to all ligands, yet frequently to different degrees . Some mutants showed a specifically altered sensitivity to a few ligands; for example, mutant 511-04 was partially resistant only to colicin M, mutant 241-04 was reduced in ferrichrome and albomycin uptake and showed a reduced colicin M sensitivity, and mutant 321-04 was fully resistant to phage T1 and partially resistant to phage phi 80 . The altered residues define preferential binding sites for these ligands . Insertions of 4 to 16 residues at positions 69, 70, 402, 530, 564, and 572 resulted in strongly reduced amounts of FhuA in the outer membrane fraction, varying in function from fully active to inactive . These results provide the basis for a model of FhuA organization in the outer membrane.

Mol Biol Rep, 1993 Feb, 17(2), 101 - 14
Stable expression of antibiotic resistance genes using a promoter fragment of the U1 snRNA gene; Asselbergs FA et al.; As U1 snRNA is produced in all mammalian cell types, antibiotic resistance genes driven by this promoter would be ideally suited as genetic selection markers . However, although the U1 snRNA gene is transcribed by RNA polymerase II, its native product is not a messenger RNA, but a splicing cofactor . To test whether this promoter could nevertheless produce a functional mRNA, sensitive reporter genes expressing resistance to the antibiotics hygromycin-B and bleomycin were constructed with either the U1 snRNA promoter or the SV40 early promoter . Resistant cell lines could only be obtained with constructs equipped with a functional polyadenylation signal . With the U1 snRNA promoter about three times fewer colonies were obtained than with the SV40 early promoter . Another potential advantage of the U1 snRNA promoter is that, in contrast to the promoters commonly used to express genetic selection markers, the enhancer-like element contained in the U1 snRNA promoter had only a minimal stimulative effect, only detectable with the most sensitive methods, on an adjacent mRNA-producing gene . The U1 snRNA promoter was also capable of expressing bleomycin resistance in the context of a self-inactivating retrovirus vector, whereby it was discovered that the mouse 3T3 cells used in this experiment were 10 times more sensitive to bleomycin than human or hamster cell lines.

J Bacteriol, 1993 Feb, 175(4), 1026 - 31
Posttranscriptional repression of Escherichia coli OmpF protein in response to redox stress: positive control of the micF antisense RNA by the soxRS locus; Chou JH et al.; The soxRS regulon is a cornerstone of the adaptive defense systems of Escherichia coli against oxidative stress . Unexpectedly, activation of this regulon also enhances bacterial resistance to multiple antibiotics that seem unrelated to oxygen radicals . We previously correlated this multiple antibiotic resistance with a reduced rate of synthesis of the OmpF outer membrane porin that does not affect the OmpC or OmpA porins . Studies presented here, with operon and gene fusions of ompF to lacZ, show that the soxRS-dependent repression of OmpF is achieved posttranscriptionally . We also show posttranscriptional repression of OmpF mediated by the soxQ1 mutation, which maps to the marA locus . These repressions are dependent on the micF gene, which encodes a small RNA partially complementary to the 5' end of the ompF message . Northern (RNA) blotting experiments show that micF transcription is strongly inducible by the superoxide-generating agent paraquat in a manner that depends completely on the soxRS locus . The soxR-constitutive and soxQ1 mutations elevate the expression of micF in the absence of redox stress . However, the antibiotic resistance mediated by a soxR-constitutive mutation was only partially reversed upon deletion of micF . The soxRS regulon therefore includes other components that contribute to general antibiotic resistance, although the relation of this phenotype to oxidative stress remains to be established.

Nucleic Acids Res, 1993 Jan 25, 21(2), 191 - 5
The ble resistance gene as a new selectable marker for Trypanosoma brucei: fly transmission of stable procyclic transformants to produce antibiotic resistant bloodstream forms; Jefferies D et al.; We describe here the stable transformation of Trypanosoma brucei using a new selectable marker for kinetoplastid protozoa, the Sh ble, or phleomycin, resistance gene . A plasmid containing this gene targeted to the tubulin gene locus by homologous sequences was introduced into procyclic trypanosomes by electroporation and cells selected for antibiotic resistance . Southern analysis of stable transformants showed that the plasmid had been integrated into the tubulin locus by homologous recombination . Analysis of bloodstream stage transformants, produced by transmission through the vector Glossina, showed that the resistance gene was conserved and expressed in these forms in the absence of selective drug pressure . In both procyclic and bloodstream forms, transcription of the ble gene appears to originate from the upstream tubulin promoter, despite the presence of a VSG promoter in the integrated construct . The generation of stable bloodstream transformants for the first time will facilitate the study of gene function and expression during the trypanosome life cycle, and aid in the investigation of genetic exchange in these organisms.

Scand J Gastroenterol Suppl, 1993, 196, 34 - 7
Compliance, adverse events and antibiotic resistance in Helicobacter pylori treatment; Malfertheiner P; The highest H . pylori eradication rates have been reported with triple therapy, using metronidazole with amoxycillin or tetracycline, and colloidal bismuth subcitrate or bismuth subsalicylate . The use of such therapies, however, may be impeded by a number of major disadvantages, including reduced patient compliance, the incidence of adverse events and primary or acquired antibiotic resistance . Patient compliance is a particular problem with triple therapy owing to the quantity of drugs taken, treatment duration and regimen complexity; the eradication rate is reduced from 96% to 69% when only 60% of the medication is taken . The risk of adverse events resulting from the inclusion of antibiotics in the regimen is increased in triple therapy, and this generates reluctance in many practitioners to prescribe such therapy despite its well-documented efficacy . An important cause of antibiotic failure lies in the development of H . pylori resistance; between 6% and 27% of H . pylori strains are primarily resistant to the 5-nitroimidazoles--metronidazole and tinidazole--both of which are used in triple therapy . In contrast, no resistance of H . pylori to amoxycillin has been reported . The combination of an acid pump inhibitor with a single antibiotic represents a promising novel therapy for H . pylori-associated peptic ulcer disease . Treatment with omeprazole and amoxycillin could provide both rapid healing of ulcers and eradication of H . pylori, coupled with few adverse events, good drug compliance and low ulcer relapse rates, and may replace triple therapy as first-line medication.

Antibiot Khimioter, 1993 Jan, 38(1), 46 - 53
{Current strategy and tactics of etiotropic therapy of acute intestinal infections in children}; Miliutina LN et al.; Efficacy of various types of etiotropic therapy in 1646 children with acute intestinal infections (AIIs) treated in the Infectious Department in 1977-1989 was analyzed by comparison with the data on drug resistance of the pathogens . It was shown advisable to apply etiotropic therapy only to patients with AIIs of invasive genesis with a differential approach to the drug choice and an account of the disease severity and phase, patient age, properties of the drugs and pathogens . Follow-up of the pathogen drug resistance by quarterly reports on antibiotic resistance of the circulating pathogens was found expedient . Efficacy of the reserve drugs in treatment of severe and general AIIs and advantages of phage monotherapy and phage combined therapy in patients with AIIs of the moderate and low severity by comparison with the use of antibiotics and chemotherapeutics were also shown . The first experience with the use of original complex immunoglobulin for enteral administration in therapy of AIIs proved promising.

Am J Gastroenterol, 1992 Dec, 87(12), 1716 - 27
Meta-analysis of the efficacy of antibiotic therapy in eradicating Helicobacter pylori; Chiba N et al.; Despite numerous Helicobacter pylori treatment studies, the optimum regimen(s) for its eradication remain unclear . Our objective was to determine systematically which regimen(s) gave the best pooled eradication rates, by using meta-analysis methodology . A total of 27 studies were identified . Pooled eradication rates for single (18.6%), double (48.2%), and triple therapy (82.3%) were statistically highly different (p < 0.0005) . Eradication rates with amoxicillin (23.0%) and bismuth compounds (19.6%) were equivalent . Combined treatment with bismuth+metronidazole was better than bismuth+amoxicillin (55.1% vs . 43.7%, p = 0.049) . Triple therapy with bismuth+metronidazole+tetracycline gave a statistically higher eradication rate (94.1%) than bismuth+metronidazole+amoxicillin (73.1%, p < 0.0005) . Despite increased side effects with multiple antibiotic regimens, patients tolerated these well, without significant drop-out . The combination of bismuth, metronidazole, and tetracycline gives the best eradication rate, but the optimal doses and duration of treatment have yet to be determined . Further studies are necessary to explore factors such as antibiotic resistance and drug compliance as important factors affecting antibiotic efficacy.

Mol Gen Mikrobiol Virusol, 1992 Nov-Dec, (11-12), 3 - 8
{Genetic control of tetracycline resistance in bacteria}; Anisimova LA et al.; Modern data on prevalence, structural and functional organization of the tetracycline resistance determinants in bacteria are reviewed . The three mechanisms of the antibiotic resistance are the tetracycline efflux, the ribosomal protection and the antibiotic modification . The problems of evolution of tetracycline resistance genes are discussed.

Antibiot Khimioter, 1992 Nov, 37(11), 3 - 5
{Effect of protoplasting on antibiotic activity and resistance in the strain Streptomyces hygroscopicus 155}; Ivanova IV et al.; The influence of protoplasting and protoplast regeneration in the presence of polyethylene glycol on antibiotic activity, components of antibiotic complexes and antibiotic resistance in Streptomyces hygroscopicus 155 was studied . It was shown that the protoplasting and protoplast regeneration influenced the antibiotic activity . The protoplast fusion resulted in increased isolation of variants with higher antibiotic activity . The processes also affected the components of the antibiotic complexes but had no effect on the strain resistance to some antibiotics.

Pediatr Cardiol, 1992 Oct, 13(4), 198 - 203
Bacterial endocarditis in children: trends in its diagnosis, course, and prognosis; Hansen D et al.; In a population-based study of 41 children with bacterial endocarditis (BE), diagnosed in the period 1970 through 1989 in eastern Denmark, we analyzed trends in the diagnosis of BE and in mortality, and searched for possible prognostic factors . During this period the delay in diagnosis from first symptom to treatment did not change, but the delay from admission to treatment was significantly prolonged from 0 to 3 days, despite the introduction of echocardiography (ECHO) . There was a significant improvement in the prognosis, the mortality rate having decreased from 40 to 0% {95% confidence limits: 12-74 vs . 0-26 (0.01 less than p less than 0.02)} . The improved prognosis was not explained by changes in the etiology or pattern of antibiotic resistance and may reflect a milder course of BE in children . Children with "mild anomalies"--such as bicuspid aortic valve (n = 5), coarctation of the aorta (n = 2), and prolapse of the mitral valve (n = 2)--had a significantly poorer prognosis than children with other forms of congenital heart disease (CHD) (p = 0.004), a reminder of the importance of suspecting BE in all children with unexplained long-lasting or intermittent fever, because some may have unrecognized "mild" CHD.

Mol Microbiol, 1992 Oct, 6(19), 2825 - 32
Surface exclusion specificity of the TraT lipoprotein is determined by single alterations in a five-amino-acid region of the protein; Harrison JL et al.; The TraT protein is a highly cell-surface-exposed lipoprotein specified by F-like plasmids that confers serum resistance and blocks the conjugative transfer of plasmids to cells bearing identical or closely related plasmids, a process known as surface exclusion . The TraT protein specified by the antibiotic-resistance plasmid R6-5 was purified to apparent homogeneity . When added to mating mixtures, TraT blocked the transfer of plasmids belonging to Surface Exclusion Group IV (Sfx IV) but had no significant effect on the transfer of plasmids belonging to other groups . Additionally, the purified protein has a protective effect on bacterial cells incubated in serum, indicating that it does not have to be located on the cell surface to mediate serum resistance . To localize regions of the protein that were responsible for surface exclusion specificity, the amino acid sequence of the TraT protein specified by CoIB2-K98 (Sfx II) was determined by cloning and sequencing of the corresponding gene . Comparison of the derived sequence with those of the F and R100-1 proteins indicated that surface exclusion specificity of TraT is determined by single alterations in a five-amino-acid region (residues 116-120) . This was confirmed by segment swapping experiments in which the specificity of the R6-5 TraT protein (Sfx IV) was switched to that of the CoIB2-K98 protein (Sfx II) . Our results suggest that the region defined by residues 116-120 is located on the external face of the outer membrane and interacts specifically with the donor cell in surface exclusion.

Genetics, 1992 Oct, 132(2), 413 - 29
Nonrandom distribution of chloroplast recombination events in Chlamydomonas reinhardtii: evidence for a hotspot and an adjacent cold region; Newman SM et al.; Intermolecular recombination of Chlamydomonas chloroplast genes has been analyzed in sexual crosses and following biolistic transformation . The pattern and position of specific exchange events within 15 kb of the 22-kb inverted repeat have been mapped with respect to known restriction fragment length polymorphism markers that distinguish the chloroplast genomes of the interfertile species Chlamydomonas reinhardtii and Chlamydomonas smithii . Recombinant progeny were selected from two- and three-factor crosses involving point mutations conferring herbicide (dr) and antibiotic resistance (er and spr) in the psbA, 23S and 16S ribosomal RNA genes, respectively . Exchange events were not randomly distributed over the 15-kb region, but were found to occur preferentially in a 0.7-kb sequence spanning the 3' end of the psbA gene and were much less common in an adjacent region of ca . 2.0 kb . These findings are corroborated by data showing that the dr mutation is unlinked genetically (3% recombination/kb) to the er and spr rRNA mutations, which are themselves linked and show ca . 1% recombination/kb . This discrepancy is significant since the dr-er and er-spr intervals are about the same length (ca . 7 kb) . During chloroplast transformation, the 0.7-kb recombination hotspot also functions as a preferential site for exchange events leading to the integration of donor psbA gene sequences . The 0.7-kb hotspot region contains four classes of 18-37-bp direct repeats also found in other intergenic regions, but no open reading frame . Using deletion constructs in a chloroplast transformation assay, the hotspot was localized to a 500-bp region that lacks most of these repeats, which suggests that the repeats themselves are not responsible for the increased recombination frequency . Within this region, a 400-bp sequence is highly conserved between the chloroplast genomes of C . reinhardtii and C . smithii and includes several structural motifs characteristic of recombination hotspots in other systems.

FEMS Microbiol Lett, 1992 Oct 1, 76(1-2), 127 - 34
Oligomerization-mediated activation of plasmid-borne genes in Escherichia coli; Weber PC; A plasmid carrying a weakly expressed neomycin phosphotransferase (neo) gene from the transposable element Tn5 was found to confer elevated levels of antibiotic resistance on its host cell when it existed in a non-monomeric state . This activation of the neo gene appeared to be a generalized effect which can be exerted on any plasmid-encoded gene, since specific sequences were not required for enhanced neo expression, and the activity of a plasmid-borne chloramphenicol acetyltransferase gene could be similarly induced by oligomerization . The potential role that multiple origins of replication present in such oligomeric plasmids play in these observed increases in gene expression is discussed.

J Antibiot (Tokyo), 1992 Sep, 45(9), 1481 - 91
The distribution of DNA sequences hybridizing with antibiotic production and resistance gene probes within type strains and wild isolates of Streptomyces species; Phillips L et al.; Total DNA preparations from 74 antibiotic-producing type strains and 102 natural Streptomyces isolates were examined by dot blots for homology to 6 antibiotic production and resistance genes . Pattern diversity of hybridizations decreased as stringency increased from 65% to 85% . There were 146 unique profiles at 65% stringency with 13 repeated patterns, whilst there were only 14 unique and 11 repeated profiles at 85% stringency . Most of the strains which hybridized at 85% reacted with one or two probes although a few strains showed multiple homologies . This data was used to cluster strains and the groups defined were examined for phenotypic antibiotic resistance . Producers of certain classes of antibiotics clustered to specific groups and some gene homologies were more common amongst strains which produced similar antibiotics.

J Appl Bacteriol, 1992 Sep, 73(3), 229 - 36
Survival strategies of plasmid-carrier and plasmidless Escherichia coli strains under illuminated and non-illuminated conditions, in a fresh water ecosystem; Barcina I et al.; A comparative study, in illuminated and non-illuminated systems, was made to determine the survival strategies of plasmid-carrier and plasmidless bacteria in sterile river water . Two strains of Escherichia coli from river water were selected: one plasmidless, EC1, and one antibiotic-resistant strain, EC7, which showed plasmid bands . By matings with EC7 as donor and E . coli K12 strain J62 as recipient, transconjugants were generated, the J62(7) strain, which showed both antibiotic resistance and plasmid bands . Ethidium bromide curing of the EC7 strain generated the EC7(2) strain which showed a partial loss of resistance and a reorganization of plasmid bands . Under non-illuminated conditions the total number of cells detected by direct count and the number of culturable cells (injured and non-injured cells) remained practically constant throughout the period of incubation . In the illuminated systems, however, the number of cfu decreased in four of the five strains studied . The greatest decreases are those of the J62 strain, followed by those of the J62(7), EC1, EC7(2) and EC7 strains . Differences in survival strategies as a consequence of the presence or absence of plasmids are discussed.

J Virol Methods, 1992 Aug, 38(2), 195 - 204
Isolation of a cell line for rapid and sensitive histochemical assay for the detection of herpes simplex virus; Stabell EC et al.; A cell line which can be used in a simple, sensitive, and rapid histochemical assay was isolated for detection of herpes simplex virus (HSV) . The cell line was derived by selection of G418 resistant colonies following co-transfection of baby hamster kidney cells with a plasmid which contains a G418 antibiotic resistance marker and a plasmid which contains the Escherichia coli LacZ gene placed behind an inducible HSV promoter . The promoter is from HSV-1UL39 which encodes ICP6, the large subunit of ribonucleotide reductase (RR1) . This promoter has a number of features which make it ideal for the detection of HSV . First, there is no constitutive expression from this promoter in uninfected cells . Second, activation of the promoter appears to be specific for HSV . Third, expression from this promoter occurs within hours after infection . Fourth, this promoter is strongly transactivated by the virion associated trans-activator protein VP16 . As early as six hours after infection HSV-infected cells can be detected by histochemical staining for beta-galactosidase activity . Infected cells stain intensely blue whereas uninfected cells show no staining, and a single infected cell can easily be recognized in a microscopic field of uninfected cells . Both HSV-1 and HSV-2 are detected with this cell line, but after infection with human cytomegalovirus (HCMV), varicella zoster virus (VZV), adenovirus, and sindbis virus no blue cells were detected . Quantitation of HSV-1 stocks on this cell line by counting blue cell forming units (BFU) reveals that the number of BFU/ml closely approximates the number of plaque forming units (PFU)/ml as determined by plaque assays on the parent cell line . This cell line should provide a useful adjunct in the diagnostic virology laboratory for the rapid detection of HSV in clinical specimens.

EMBO J, 1992 Jul, 11(7), 2707 - 15
An endonuclease with multiple cutting sites, Endo.SceI, initiates genetic recombination at its cutting site in yeast mitochondria; Nakagawa K et al.; Endo.SceI is a mitochondrial sequence-specific endonuclease which has multiple cutting sites . In order to examine the possible role of Endo.SceI in homologous recombination, we analyzed the mode of recombination upon mating using antibiotic resistance markers on the mitochondrial genome . The segregation of a marker located very close to one of the Endo.SceI cutting sites showed a disparity (polarized segregation, i.e . gene conversion) . This gene conversion depended on the presence of the functional Endo.SceI gene . In vivo cutting of mitochondrial DNA upon mating was detected at the cutting site in the antibiotic marker region, which also depended on the Endo.SceI activity . These results suggest that mitochondrial recombination is induced by cleavage of mitochondrial DNA by this sequence-specific endonuclease . This is the first demonstration that a sequence-specific endonuclease with multiple cutting sites induces genetic recombination.

Infect Immun, 1992 Jul, 60(7), 2863 - 9
Construction of Cu-Zn superoxide dismutase deletion mutants of Brucella abortus: analysis of survival in vitro in epithelial and phagocytic cells and in vivo in mice; Tatum FM et al.; Cu-Zn superoxide dismutase (SOD) deletion mutants of Brucella abortus S2308, a virulent strain, and S19, a vaccine strain, were generated by gene replacement . A deletion plasmid, pBA delta sodknr, was constructed by excising the Cu-Zn SOD gene (Cu-Zn sod) from a 2.3-kb B . abortus DNA fragment of plasmid pBA20-1527 and inserting a 1.4-kb DNA fragment encoding kanamycin resistance into the Cu-Zn sod excision site . The deletion plasmid was introduced into B . abortus by electroporation, and Southern blot analysis confirmed that the antibiotic resistance fragment had replaced Cu-Zn sod in kanamycin-resistant colonies . The survival and growth of Cu-Zn SOD mutant strains were compared with that of the parental strains in HeLa cells and in the mouse macrophagelike cell line J774 . The survival and growth of the Cu-Zn SOD mutant strains were similar to those of their respective parental strains in HeLa and J774 cell lines . The kinetics of infection with these strains were examined in BALB/c mice . The splenic levels of the S19 Cu-Zn SOD mutant recovered from intraperitoneally infected BALB/c mice were approximately 10-fold lower than those of the parental strain through 26 days postinfection . Thereafter, infection sharply declined in both groups, and by 105 days postinfection, no organisms were detected . The splenic levels of the S2308 Cu-Zn SOD mutant were lower than those of wild-type S2308-infected mice . The spleen weights of mice infected with the S2308 Cu-Zn SOD mutant were consistently lower than those of wild-type S2308-infected mice . These results suggest that the antioxidant enzyme Cu-Zn SOD plays a role in the survival and pathogenicity of B . abortus in vivo.

Postgrad Med J, 1992 Jul, 68(801), 549 - 57
Eradication of Helicobacter pylori: therapies and clinical implications; O'Connor HJ; This review presents a critical evaluation of the role of Helicobacter pylori eradication in the management of peptic ulcer disease and non-ulcer dyspepsia . On current evidence, H . pylori eradication therapy seems likely to emerge as the most rational and cost-effective treatment for duodenal ulcer . The role of H . pylori eradication in the treatment of gastric ulcer and non-ulcer dyspepsia is unclear and requires further study . The emerging problem of antibiotic resistance in H . pylori is of major clinical importance and a prime cause of treatment failure . There is increasing evidence of a link between H . pylori and gastric cancer but it is premature to recommend large-scale eradication of H . pylori as a valid strategy for the primary prevention of gastric cancer . The search continues for the ideal H . pylori eradication regimen.

Avian Dis, 1992 Jul-Sep, 36(3), 679 - 84
Relationship of complement resistance and selected virulence factors in pathogenic avian Escherichia coli; Wooley RE et al.; Complement resistance, antibiotic resistance profiles, and virulence profiles of 80 Escherichia coli isolates from the intestines of normal chickens (40 isolates) and chickens diagnosed as having colisepticemia (40 isolates) were compared . Differences were observed between the two groups for antibiotic resistance, siderophore production, presence of type 1 pili, complement resistance, motility, and size of plasmids . The systemic isolates were more likely to have siderophores and type 1 pili, and to be complement-resistant and motile than were the intestinal isolates . No differences between the two groups were observed for colicin production . Further comparison of the 10 most complement-resistant isolates from the systemic group and 10 most complement-sensitive isolates from the intestinal group revealed a correlation between an isolate's resistance to complement and its ability to kill embryos, express type 1 pili, and be motile . Virulence of avian E . coli strains appears to be correlated with complement resistance and the interaction of this resistance with the ability to produce type 1 pili and be motile.

Gene, 1992 Jun 15, 115(1-2), 85 - 91
A novel, highly efficient gene-cloning system in Micromonospora applied to the genetic analysis of fortimicin biosynthesis; Hasegawa M; We have developed a gene-cloning system in Micromonospora olivasterospora, a fortimicin A (astromicin) producer . Plasmids of Micromonospora from two strains of M . olivasterospora were used for construction of the vectors . Two antibiotic-resistance genes, nmrA and nmrB, cloned from a neomycin-producing Micromonospora, were introduced into these plasmids for the selection of transformants . In a new protoplasting protocol for lysozyme-resistant bacteria, protoplasts of M . olivasterospora were found in short-time incubation with lysozyme and transformed efficiently, indicating that the method was suitable to shotgun cloning . Using this system, seven biosynthetic genes for fortimicin A were cloned . Their physical maps revealed that at least four of these genes were clustered . Analysis of a cosmid library of M . olivasterospora showed that eleven biosynthetic genes and a self-defense gene existed in a region of approx . 25 kb of DNA.

Gene, 1992 Jun 15, 115(1-2), 61 - 5
Transposition of Tn5096 and related transposons in Streptomyces species; Baltz RH et al.; IS493 is an insertion sequence isolated from Streptomyces lividans by a method designed to 'trap' transposable elements . IS493 was converted to functional transposons by cloning antibiotic-resistance-encoding genes between ORF-A and ORF-B of IS493 or near the left-end inverted repeat of the element . Tn5096 transposed relatively randomly in several Streptomyces species . Tn5096 can be introduced into streptomycetes on temperature-sensitive vectors by protoplast transformation, FP43-mediated transduction, or by conjugation from Escherichia coli . We have shown that additional genes can be inserted in Tn5096 without disrupting transposition, and that Tn5096 insertions in a tylosin (Ty)-producing strain of Streptomyces fradiae frequently cause no deleterious effects on Ty production . A promoter probe transposon, Tn5099, containing a promoterless xylE gene, transposed in Streptomyces griseofuscus and S . fradiae, and transcriptional fusions were readily identified.

Biotechnology (N Y), 1992 May, 10(5), 570 - 3
Stabilization of Streptomyces lividans by homologous recombinational insertion; Kaiser P et al.; We have developed a system for the introduction and maintenance of novel tandem repeats in the chromosome of Streptomyces lividans 66 . This was achieved by introducing, via transformation, Escherichia coli "suicide" vectors carrying manipulated S . lividans DNA fragments . Selection for antibiotic resistance markers carried on such plasmids permitted the isolation and maintenance of mutant strains containing novel tandem repeats formed by the integration into the chromosome of the plasmids, via homologous recombination between plasmid-borne chromosomal sequences and identical sequences on the chromosome . When novel repeats were introduced, and maintained, in regions of the chromosome which become deleted in unstable strains of S . lividans, those deletion events were blocked . Surprisingly, such strains were also 10 to 20-fold more stable than the parent even in the absence of selection . In stable regions of the chromosome, the maintenance of novel repeats had no obvious effect on the deletion events . This strategy could be generally applicable to industrial strains of Streptomyces, where instability is a common problem.

Mol Gen Genet, 1992 May, 233(1-2), 184 - 92
Mutations in bglY, the structural gene for the DNA-binding protein H1 of Escherichia coli, increase the expression of the kanamycin resistance gene carried by plasmid pGR71; Bertin P et al.; bglY mutants of Escherichia coli K12 which show higher levels of kanamycin resistance (Kmr) in the presence of plasmid pGR71 have been previously described . In this work, we show that this increased resistance to an aminoglycoside antibiotic is not due either to low drug uptake or to alteration of its target, the ribosome . The copy number of plasmid pGR71 is not modified . The fact that increased antibiotic resistance is observed with only some of the Kmr determinants used in this study suggests a specific role for the bglY gene product . Moreover, for one such determinant, a higher level of resistance was observed when it was inserted in the chromosome but not when harbored by a plasmid . This discrepancy can be explained by the twin transcriptional-loop model, which proposes that transcription can lead to local variation in topology . A kan-lacZ fusion was constructed from the Kmr gene of plasmid pGR71 and inserted into a low copy number vector . Assay of beta-galactosidase in wild-type and mutant strains showed that expression of the antibiotic resistance gene was directly affected by H1 protein, the bglY gene product.

J Biol Chem, 1992 Apr 25, 267(12), 8377 - 82
Identification of a single base change in ribosomal RNA leading to erythromycin resistance; Vannuffel P et al.; The molecular basis of a mutation conferring an erythromycin-resistance phenotype was explored, as an approach to the role of 23 S rRNA in the peptidyl-transferase activity of 50 S ribosomal subunits . Mutagenization of an Escherichia coli strain, which carried the multicopy plasmid pLC7-21 containing the rrnH operon, led to the production of an erythromycin-resistant strain . Plasmid pBFL1 isolated from this mutant was able to transform the sensitive RecA- strain EM4 and to induce a "dissociated" type of antibiotic resistance . Two ribosome populations occurred in EM4/pBFL1: normal particles coded for by the seven rrn chromosomal genes and mutated particles containing rRNA of plasmid origin . The latter particles displayed in vitro lower affinity and susceptibility to erythromycin than wild type particles . The mutation within plasmid pBFL1 was mapped by a multiple primer extension technique . Three synthetic primers were used to sequence the central loop in domain V of 23 S rRNA, leading to identification of a C to U transition at position 2611 . This base change was proved to be responsible for the erythromycin-resistance phenotype by the plasmid-plasmid marker rescue technique . A molecular explanation for the rrn mutations leading, respectively, to undissociated and to dissociated types of resistance to the MLSb (macrolide-lincosamide-synergimycin B) group of antibiotics is proposed . These results and some literature data support the notion that rRNA bases involved in antibiotic resistance play a conformational role in the ribosomal binding sites for the MLSb antibiotics.

Curr Genet, 1992 Apr, 21(4-5), 331 - 8
Trans-acting factors and properly positioned DNA elements repress mating-type genes in fission yeast; Ekwall K et al.; Repression of the mating-type P genes at the silent mat2-P locus in fission yeast is dependent on four cis-acting DNA elements, two on each side of the coding sequences . The mechanism by which these elements exert their influence on the mating-type promoter is studied here by insertion of a bacterial antibiotic resistance gene at several positions in the silent region . The behavior of the resistance gene itself, and the changes its insertion causes in mating-type expression, reveal that the repressive elements have a limited range of action and that the four elements have unequal effects on gene expression . Repression of the antibiotic resistance gene inside the silent region leads to an antibiotic-sensitive phenotype and facilitates the selection of resistant mutants . These mutants can de-repress the resistance gene at other positions than the one used for their selection . Strong antibiotic resistance correlates with derepression of the plasmid-borne mating-type cassette . These data argue that mat2-P repression is dependent on trans-acting factors and the positioning of the repressive DNA elements, but less dependent on the nature of the affected promoter.

Mol Gen Genet, 1992 Mar, 232(1), 7 - 11
Clustering of genes involved in nitrate assimilation in the cyanobacterium Synechococcus; Luque I et al.; A region of the genome of the cyanobacterium Synechococcus R2, that bears a cluster of genes involved in nitrate assimilation, has been cloned and the relative positions of some of the genes in the region have been determined . Mutations generated by insertion of an antibiotic-resistance gene cassette into the gene encoding nitrite reductase are associated with reduced expression of nitrate reductase; cotranscription of nitrate assimilation genes in the cluster is inferred from this finding.

Gene, 1992 Mar 1, 112(1), 117 - 22
Cloning and sequence of a gene encoding macrotetrolide antibiotic resistance from Streptomyces griseus; Plater R et al.; A gene (nonR) conferring tetranactin resistance on the macrotetrolide-sensitive strain, Streptomyces lividans TK64, was isolated during a shotgun cloning experiment, in which chromosomal fragments from Streptomyces griseus were ligated into the vector pIJ699 and then introduced by transformation into S . lividans TK64 . The sequence (3326 bp) of the cloned DNA revealed three complete open reading frames (ORFs) and one incomplete ORF encoded on one strand of the DNA . The nonR gene (designated here ORFA) encodes a polypeptide of 279 amino acids (Mr 30610) and contains a putative active site motif, GXSXG, characteristic of serine proteases and esterases . A functional role for the nonR gene product may involve the inactivation of the antibiotic through hydrolysis of one or more ester linkages in the macrotetrolide ring . The deduced product of the incomplete ORFX lying adjacent to ORFA showed 27.9% sequence identity with the C-terminal region of rat mitochondrial enoyl-CoA hydratase, and is possibly a macrotetrolide biosynthetic enzyme.

J Bacteriol, 1992 Feb, 174(4), 1333 - 8
Functional interactions within 23S rRNA involving the peptidyltransferase center; Douthwaite S; A molecular genetic approach has been employed to investigate functional interactions within 23S rRNA . Each of the three base substitutions at guanine 2032 has been made . The 2032A mutation confers resistance to the antibiotics chloramphenicol and clindamycin, which interact with the 23S rRNA peptidyltransferase loop . All three base substitutions at position 2032 produce an erythromycin-hypersensitive phenotype . The 2032 substitutions were compared with and combined with a 12-bp deletion mutation in domain II and point mutations at positions 2057 and 2058 in the peptidyltransferase region of domain V that also confer antibiotic resistance . Both the domain II deletion and the 2057A mutation relieve the hypersensitive effect of the 2032A mutation, producing an erythromycin-resistant phenotype; in addition, the combination of the 2032A and 2057A mutations confers a higher level of chloramphenicol resistance than either mutation alone . 23S rRNAs containing mutations at position 2058 that confer clindamycin and erythromycin resistance become deleterious to cell growth when combined with the 2032A mutation and, additionally, confer hypersensitivity to erythromycin and sensitivity to clindamycin and chloramphenicol . Introduction of the domain II deletion into these double-mutation constructs gives rise to erythromycin resistance . The results are interpreted as indicating that position 2032 interacts with the peptidyltransferase loop and that there is a functional connection between domains II and V.

Plasmid, 1992 Jan, 27(1), 52 - 64
Plasmid-mediated resistance to tellurite: expressed and cryptic; Walter EG et al.; The ability of some bacteria to grow in the presence of high concentrations of tellurium compounds has been recognized for almost 100 years . Since then, interest in this phenomenon has generated a slow but steady trickle of literature . In the past few years, the use of modern techniques in molecular biology has led to a dramatic increase in our understanding of the genetics of several bacterial determinants for resistance to tellurium compounds . These determinants are frequently found to be encoded by plasmids which carry multiple antibiotic resistance determinants . Our understanding of the biochemistry of these systems remains limited . In this article, the history of the study of bacterial resistance to tellurium compounds is briefly reviewed . This is followed by an analysis of the recent developments in the study of plasmid-mediated resistance determinants . Finally, preliminary investigations on the possible mechanisms of bacterial resistance to tellurium compounds are presented.

Plant Cell, 1992 Jan, 4(1), 39 - 45
Long regions of homologous DNA are incorporated into the tobacco plastid genome by transformation; Staub JM et al.; We investigated the size of flanking DNA incorporated into the tobacco plastid genome alongside a selectable antibiotic resistance mutation . The results showed that integration of a long uninterrupted region of homologous DNA, rather than of small fragments as previously thought, is the more likely event in plastid transformation of land plants . Transforming plasmid pJS75 contains a 6.2-kb DNA fragment from the inverted repeat region of the tobacco plastid genome . A spectinomycin resistance mutation is encoded in the gene of the 16S rRNA and, 3.2 kb away, a streptomycin resistance mutation is encoded in exon II of the ribosomal protein gene rps12 . Transplastomic lines were obtained after introduction of pJS75 DNA into leaf cells by the biolistic process and selection for the spectinomycin resistance marker . Homologous replacement of resident wild-type sequences resulted in integration of all, or almost all, of the 6.2-kb plastid DNA sequence from pJS75 . Plasmid pJS75, which contains engineered cloning sites between two selectable markers, can be used as a plastid insertion vector.

Rev Elev Med Vet Pays Trop, 1992, 45(3-4), 260 - 2
Susceptibility to antibiotics of Escherichia coli strains isolated from diarrhoeic and non-diarrhoeic livestock in Trinidad; Adesiyun AA et al.; The sensitivity of strains of Escherichia coli isolated from calves, piglets, lambs and kids in Trinidad to seven antibiotics was determined . Two hundred and sixty-four (91.3%) of 289 strains isolated from diarrhoeic animals and 173 (87.4%) of 198 strains from non-diarrhoeic animals exhibited resistance to one or more antibiotics . The difference was not statistically significant (P > or = 0.05; X2) . Regardless of health status, isolates from lambs were least resistant (75.0%) and those from piglets most resistant (96.7%) and the difference was significant (P < or = 0.001; X2) . Strains of E . coli were most resistant to streptomycin (81.3%) and tetracycline (78.9%) and least resistant to chloramphenicol (4.3%) and gentamycin (4.7%) . The predominant antibiotic resistance pattern for isolates from all sources was streptomycin-tetracycline (27.9%) . It was concluded that the widespread prevalence of resistance to antibiotics reflects their misuse in the local environment.

Genetica, 1992, 86(1-3), 99 - 111
Natural genetic engineering in evolution; Shapiro JA; The results of molecular genetics have frequently been difficult to explain by conventional evolutionary theory . New findings about the genetic conservation of protein structure and function across very broad taxonomic boundaries, the mosaic structure of genomes and genetic loci, and the molecular mechanisms of genetic change all point to a view of evolution as involving the rearrangement of basic genetic motifs . A more detailed examination of how living cells restructure their genomes reveals a wide variety of sophisticated biochemical systems responsive to elaborate regulatory networks . In some cases, we know that cells are able to accomplish extensive genome reorganization within one or a few cell generations . The emergence of bacterial antibiotic resistance is a contemporary example of evolutionary change; molecular analysis of this phenomenon has shown that it occurs by the addition rearrangement of resistance determinants and genetic mobility systems rather than by gradual modification of pre-existing cellular genomes . In addition, bacteria and other organisms have intricate repair systems to prevent genetic change by sporadic physicochemical damage or errors of the replication machinery . In their ensemble, these results show that living cells have (and use) the biochemical apparatus to evolve by a genetic engineering process . Future research will reveal how well the regulatory systems integrate genomic change into basic life processes during evolution.

J Bacteriol, 1992 Jan, 174(2), 525 - 9
Identification of the gene (aroK) encoding shikimic acid kinase I of Escherichia coli; Lobner-Olesen A et al.; DNA sequence analysis has revealed that an unidentified open reading frame (ufr1) is present immediately preceding the aroB gene of Escherichia coli . The predicted protein product of urf1 contains a consensus ATP-binding-site sequence and shows 34% amino acid homology to shikimate kinase II in a 97-amino-acid region . Inactivation of urf1 by insertion of an antibiotic resistance gene had a polar effect on aroB, indicating that these two genes constitute a transcriptional unit . The auxotrophic requirements of a strain mutant for both urf1 and aroL (encoding shikimate kinase II) are consistent with shikimate kinase deficiency . We propose that urf1 encodes shikimate kinase I and that it be designated aroK.

APMIS, 1991 Dec, 99(12), 1103 - 10
Prevalence of antibiotic-resistant Escherichia coli in Danish pigs and cattle; Aalbaek B et al.; The present paper reports on the antibiotic resistance of E . coli isolated from Danish piglets and calves in 1987-1988, and compares the results with similar investigations performed during the periods 1971-1972 and 1977-1978 . Rectal swabs from 52 piglets and from 78 calves were examined . All the animals studied harboured resistant E . coli . This is a significant increase compared to the previously conducted investigations . The number of strains having three or more resistance markers did not differ significantly from the previous findings . The spectrum of resistance markers among Danish piglets and calves had increased through all three investigations and resistance to chloramphenicol was still found to be considerable 10 years after the withdrawal of chloramphenicol as a therapeutic drug for farm animals in Denmark in 1978 . Certain resistance patterns (sulfonamide + streptomycin, sulfonamide + streptomycin + tetracycline, sulfonamide + streptomycin + tetracycline + ampicillin) were found to be shared by numerous strains, suggesting a genetic linkage of the resistance markers.

Biochem Biophys Res Commun, 1991 Nov 27, 181(1), 95 - 9
Thymic nuclear matrix associated activity is not V(D)J recombinase; Pandey VN et al.; It was previously reported that nuclear matrix isolated from young rat thymus contained an activity that supported V(D)J recombination at a high efficiency (Dave et al., BIOCHEMISTRY 30: 4763-4767, 1991) . A similar type of activity is also detected in the matrix prepared from fetal calf thymus . However, restriction enzyme mapping analyses of the recombined product clearly suggest that the double antibiotic resistance exhibited by the matrix treated plasmid substrate is not a consequence of V(D)J signal sequence recombination.

J Virol, 1991 Nov, 65(11), 5910 - 20
Extracellular vaccinia virus formation and cell-to-cell virus transmission are prevented by deletion of the gene encoding the 37,000-Dalton outer envelope protein; Blasco R et al.; There are two types of infectious vaccinia virus particles: intracellular naked virions and extracellular enveloped virions (EEV) . To determine the biological role of the enveloped form of vaccinia virus, we produced and characterized a mutant that is defective in EEV formation . The strategy involved replacement by homologous recombination of the gene F13L, encoding a 37,000-Da protein (VP37) that is specific for the outer envelope of EEV, with a selectable antibiotic resistance marker, the Escherichia coli gpt gene . Initial experiments, however, suggested that such a mutation was lethal or prevented plaque formation . By employing a protocol consisting of high-multiplicity passages of intracellular virus from the transfected cells and then limiting dilution cloning, we succeeded in isolating the desired mutant, which was defective in production of plaques and extracellular virus but made normal amounts of intracellular naked virions . Electron microscopic examination indicated that the mutant virus particles, unlike wild type, were neither wrapped with Golgi-derived membranes nor associated with the cell surface . The absence of VP37 did not prevent the transport of the viral hemagglutinin to the plasma membrane but nevertheless abrogated both low-pH- and antibody-mediated cell fusion . These results indicate that VP37 is required for EEV formation and also plays a critical role in the local cell-to-cell transmission of vaccinia virus, perhaps via enveloped virions attached to or released from the cell membrane . By contrast, a mutated virus with a deletion of the K4L open reading frame, which is a homolog of the VP37 gene, was not defective in formation of plaques or EEV.

J Burn Care Rehabil, 1991 Nov-Dec, 12(6), 521 - 4
The role of gentamicin iontophoresis in the treatment of burned ears; Desai MH et al.; Ear cartilage heals slowly, and limited vascularity in cartilage precludes use of systemic antibiotics . Iontophoresis electrically induces drugs in solution to migrate into target tissues . Fifteen patients were randomized to receive gentamicin iontophoresis (n = 7) plus dressing changes every 6 hours and cleaning or routine care alone (n = 8) for treatment of ear burns . There were no differences between the groups in incidence of chondritis (43% vs 50%) or cartilage loss (11% vs 16%) . However, gentamicin-resistant organisms developed in 29% of the patients who received iontophoresis, but in none of the patients in the control group (p less than 0.05) . To identify the etiology of the resistant organisms, 10 New Zealand white rabbits receive 7 cm2 contact burns to each ear . Gentamicin iontophoresis was performed on one ear, and the other ear served as the control . Serum gentamicin levels were always subtherapeutic . Additionally, gentamicin tissue levels in both the treated and control ears were subtherapeutic . Gentamicin iontophoresis appears to offer no additional salutary effects beyond those that are provided by routine care and may encourage the development of antibiotic resistance.

Mol Gen Genet, 1991 Nov, 230(1-2), 65 - 74
Targeted disruption of chloroplast genes in Chlamydomonas reinhardtii; Newman SM et al.; We have developed an efficient procedure for the disruption of Chlamydomonas chloroplast genes . Wild-type C . reinhardtii cells were bombarded with microprojectiles coated with a mixture of two plasmids, one encoding selectable, antibiotic-resistance mutations in the 16S ribosomal RNA gene and the other containing either the atpB or rbcL photosynthetic gene inactivated by an insertion of 0.48 kb of yeast DNA in the coding sequence . Antibiotic-resistant transformants were selected under conditions permissive for growth of non-photosynthetic mutants . Approximately half of these transformants were initially heteroplasmic for copies of the disrupted atpB or rbcL genes integrated into the recipient chloroplast genome but still retained photosynthetic competence . A small fraction of the transformants (1.1% for atpB; 4.3% for rbcL) were nonphotosynthetic and homoplasmic for the disrupted gene at the time they were isolated . Single cell cloning of the initially heteroplasmic transformants also yielded nonphotosynthetic segregants that were homoplasmic for the disrupted gene . Polypeptide products of the disrupted atpB and rbcL genes could not be detected using immunoblotting techniques . We believe that any nonessential Chlamydomonas chloroplast gene, such as those involved in photosynthesis, should be amenable to gene disruption by cotransformation . The method should prove useful for the introduction of site-specific mutations into chloroplast genes and flanking regulatory sequences with a view to elucidating their function.

Acta Virol, 1991 Nov, 35(6), 519 - 25
Transformation and genomic restriction mapping of Rochalimaea spp; Reschke DK et al.; Transformation procedures using electroporation were established for Rochalimaea quintana . Several cosmid/plasmids possessing the RK2 or RSF1010 origin of replication were successfully inserted . Plasmid retention and replication were verified by antibiotic resistance and Southern blot analysis . The highest level of transformation was obtained at a voltage field strength of 12.5 kV/cm with a pulse time of 10 miliseconds . Transformation efficiency was low (0.3%) with approximately 10(5) transformants/microgram of DNA . One construct, designated pAG10, reached sufficient levels in R . quintana to be isolated by density gradient centrifugation . Analysis of this plasmid after several cycles of growth in R . quintana revealed no obvious modifications . Physical maps of Rochalimaea spp . chromosomal DNA using pulse-field electrophoresis are being developed . Digestion of R . vinsonii chromosomal DNA with NotI or SfiI resulted in three and one fragments, respectively . When R . quintana was digested in a similar manner, both NotI and SfiI produced four fragments . Double digestion of R . quintana DNA with NotI and SfiI yield seven fragments ranging in size from 11 to 925 kb . Summing the fragments indicate an approximate genome size of 2.1 x 10(6) bp for R . vinsonii and 1.7 x 10(6) bp for R . quintana chromosomal DNA.

Nucleic Acids Res, 1991 Sep 25, 19(18), 4943 - 8
Rescue of end fragments of yeast artificial chromosomes by homologous recombination in yeast; Hermanson GG et al.; Yeast artificial chromosomes (YACs) provide a powerful tool for the isolation and mapping of large regions of mammalian chromosomes . We developed a rapid and efficient method for the isolation of DNA fragments representing the extreme ends of YAC clones by the insertion of a rescue plasmid into the YAC vector by homologous recombination . Two rescue vectors were constructed containing a yeast LYS2 selectable gene, a bacterial origin of replication, an antibiotic resistance gene, a polylinker containing multiple restriction sites, and a fragment homologous to one arm of the pYAC4 vector . The 'end-cloning' procedure involves transformation of the rescue vector into yeast cells carrying a YAC clone, followed by preparation of yeast DNA and transformation into bacterial cells . The resulting plasmids carry end-specific DNA fragments up to 20 kb in length, which are suitable for use as hybridization probes, as templates for direct DNA sequencing, and as probes for mapping by fluorescence in situ hybridization . These vectors are suitable for the rescue of end-clones from any YAC constructed using a pYAC-derived vector . We demonstrate the utility of these plasmids by rescuing YAC-end fragments from a human YAC library.

Zh Mikrobiol Epidemiol Immunobiol, 1991 Sep, (9), 2 - 4
{The possibility of creating a vaccinal strain of Brucella abortus 19-BA with multiple antibiotic resistance}; Gorelov VN et al.; The possibility of preparing B . abortus vaccine strain 19-BA with multiresistance to antibiotics was shown . The strain was obtained by the spontaneous induction of resistance to rifampicin with the subsequent transformation of nonconjugative hybrid plasmid pOVI which, in addition to rifampicin resistance, governed the resistance of brucellae to tetracycline, doxycycline, ampicillin, and streptomycin . Experiments on guinea pigs demonstrated the immunization with both multiresistant vaccine strain GSA1 and B . abortus initial vaccine strain 19-BA.

J Bacteriol, 1991 Sep, 173(17), 5532 - 8
marA, a regulated locus which controls expression of chromosomal multiple antibiotic resistance in Escherichia coli; Hachler H et al.; Stable chromosomal multiple-antibiotic-resistant (Mar) mutants of Escherichia coli, derived by exposing susceptible cells to low concentrations of tetracycline or chloramphenicol, express cross-resistance to structurally unrelated antibiotics . The entire resistance phenotype is reversed to susceptibility by insertion of transposon Tn5 into a locus, designated marA, near 34 min on the chromosome (A . M . George and S . B . Levy, J . Bacteriol . 155:541-548, 1983) . Strains in which 39 kbp of chromosomal DNA, including marA, had been deleted were unable to produce Mar mutants . The deletion strain could be complemented in trans by introduction of intact marA+ on plasmid F'506 . Junction fragments from a strain containing marA::Tn5 were cloned, exploiting kanamycin resistance on Tn5 for selection . They were used as probes to search a phasmid library of E . coli K-12 for recombinants containing the marA+ region . Two phasmids which contained regions hybridizing to this probe were identified and shown to complement delta marA in a deletion strain . From one phasmid, several marA-containing fragments were cloned: those of greater than or equal to 7.8 kbp restored the ability to form Mar mutants in a deletion strain . These Mar mutants were shown to be dependent on the cloned marA fragment . Chromosomal as well as recombinant Mar mutants showed increased expression of a marA-specific mRNA species of about 1.4 kb, which was barely or not detectable in wild-type strains . Exposure of mutants and, to a lesser extent, parental strains to tetracycline or chloramphenicol resulted in elevated levels of mRNA which hybridized to the marA probe . These results indicate that the marA locus is needed for production of Mar mutants and is regulated, responding to at least two antibiotics to which it controls resistance.

Nucleic Acids Res, 1991 Aug 25, 19(16), 4479 - 84
Molecular characterization of the soxRS genes of Escherichia coli: two genes control a superoxide stress regulon; Amabile-Cuevas CF et al.; The soxR locus of Escherichia coli K12 mediates transcriptional activation of a complex oxidative stress regulon in response to superoxide-generating (redox-cycling) agents . We have cloned the soxR locus, which is positioned near the uvrA gene at 92.2 min on the genetic map, by monitoring complementation of a delta soxR mutation . Subclones from the soxR region in the delta soxR strain simultaneously restored cellular resistance to the redox-cycling agent phenazine methosulfate and inducibility of at least two of the regulon proteins, glucose-6-phosphate dehydrogenase and endonuclease IV, by paraquat, another redox-cycling agent . DNA sequence analysis revealed the presence of two genes involved in activating the soxR regulon . These genes, named soxR and soxS, are arranged divergently with their 5' ends separated by only 85 bp . The predicted 12.9-kDa SoxS protein is related to the AraC family of one-component gene regulators, but corresponds only to the putative DNA-binding regions of these proteins . The 17.1-kDa SoxR protein bears significant homology only to the MerR family of proteins including a predicted DNA-binding helix-turn-helix and a cluster of cysteine residues positioned similarly to those that regulate the activity of MerR in response to Hg2+ . This suggests that SoxR could be a metal-binding gene regulator that acts as the intracellular sensor for superoxide . SoxS is evidently the proximal activator of the regulon genes: antibiotic resistance and high-level expression of at least three of the regulon proteins was effected in vivo by the individual expression of SoxS, but not of SoxR, whether or not the cells were exposed to paraquat . These data, together with the recently reported paraquat-inducibility of the soxS gene (Wu, I., and Weiss, B . (1990) J . Bacteriol . 173, 2864-2871), indicate that SoxR and SoxS may constitute a novel type of two-component regulatory system in which the two proteins act sequentially to activate transcription of the various regulon genes in response to superoxide stress.

Mol Microbiol, 1991 Aug, 5(8), 2053 - 62
Repression and induction of the nag regulon of Escherichia coli K-12: the roles of nagC and nagA in maintenance of the uninduced state; Plumbridge JA; The nag regulon located at 15.5 min on the Escherichia coli chromosome consists of two divergent operons, nagE and nagBACD, encoding genes involved in the uptake and metabolism of N-acetylglucosamine . Null mutations have been created in each of the genes by insertion of antibiotic resistance cartridges . The phenotypes of the strains carrying the insertions in nagE, B and A were consistent with the previous identification of gene products: nagE, EII(Nag), the N-acetylglucosamine specific transporter of the phosphotransferase system and nagB and nagA, the two enzymes necessary for the degradation of N-acetylglucosamine . Insertions in the nagC result in derepression of the nag genes, which is consistent with earlier observations that the nagC gene encodes the repressor of the regulon . Insertions in nagA also provoke a derepression, implying that nagA has a role in the regulation of the expression of the nag regulon as well as in the degradation of the amino-sugars . N-acetylglucosamine-6-phosphate, the intracellular product of N-acetylglucosamine transport and the substrate of the nagA gene product, is shown to be an inducer of the regulon and this suggests how nagA mutations result in derepression: the absence of N-acetylglucosamine-6-phosphate deacetylase allows N-acetylglucosamine-6-phosphate to accumulate and induce the regulon.

Antimicrob Agents Chemother, 1991 Aug, 35(8), 1562 - 9
Analysis of genetic localization of the type I trimethoprim resistance gene from Escherichia coli isolated in Finland; Heikkila E et al.; Among a collection of clinical Escherichia coli isolates, the type I dihydrofolate reductase (DHFR) mediating trimethoprim resistance was generally observed to be chromosomally determined . Only a minority of isolates carried the type I DHFR gene simultaneously on a plasmid . The majority of E . coli isolates studied also hybridized with a probe specific for the transposition gene tnsC of transposon Tn7; and in most of these isolates, Tn7 was found to be inserted into a preferred site in the E . coli chromosome . A minority of isolates that harbored the type I DHFR gene in the chromosome lacked a complete Tn7 . Some of these harbored the type I DHFR gene inserted in a structure similar to that containing the gene for streptomycin resistance in Tn21 . In the other isolates that were negative for a complete Tn7, the sequences upstream of the type I DHFR gene were demonstrated to be homologous to those flanking the type I DHFR gene in Tn7 . This could indicate that the antibiotic resistance region of Tn7 may occur independently of this transposon . In two isolates, no sequences resembling Tn7 or Tn21 were found adjacent to the type I DHFR gene.

Orthop Clin North Am, 1991 Jul, 22(3), 363 - 71
Mechanisms of musculoskeletal sepsis; Gristina AG et al.; Musculoskeletal infection is extraordinarily resistant to treatment . Bacterial colonization is discussed as well as host defense systems and antibiotic resistance.

J Bacteriol, 1991 Jul, 173(14), 4433 - 9
Activation of oxidative stress genes by mutations at the soxQ/cfxB/marA locus of Escherichia coli; Greenberg JT et al.; Exposure of Escherichia coli to superoxide-generating drugs, such as menadione or paraquat, uniquely induces approximately 40 proteins, nine of which are under the positive control of the soxR locus (at min 92) . We report here that certain mutations at a separate locus that we have named soxQ (at min 34) confer some of the phenotypes seen in soxR-constitutive strains, including resistance to menadione . A previously known mutation called cfxB, identified through antibiotic resistance, is likely an allele of soxQ . The soxQ1 and cfxB mutations cause transcriptional activation of the genes that encode Mn-containing superoxide dismutase, glucose 6-phosphate dehydrogenase, and the soi-17/19::lac and soi-28::lac fusions . These genes are also activated by soxR, but the soxQ1 and cfxB mutations increase the synthesis of seven other proteins not influenced by soxR . Moreover, the soxQ1- and cfxB-dependent phenotypes do not depend on the soxR gene, and gene induction by soxR in response to redox stress does not depend on the soxQ locus . As well as increasing cellular resistance to some oxidants, the soxQ1 and cfxB mutations confer elevated resistance to various antibiotics, probably via diminished expression of outer membrane protein OmpF . The marA1 multiple-antibiotic resistance mutation (also at min 34) behaves like a weak allele of soxQ but probably resides in a nearby gene that, with soxQ, is part of a regulatory complex . We propose that soxQ helps control some oxidative stress proteins as part of another regulon that responds to an unknown environmental signal.

J Bacteriol, 1991 Jun, 173(12), 3724 - 31
Direct and general selection for lysogens of Escherichia coli by phage lambda recombinant clones; Henry MF et al.; We report a simple in vivo technique for introducing an antibiotic resistance marker into phage lambda . This technique could be used for direct selection of lysogens harboring recombinant phages from the Kohara lambda bank (a collection of ordered lambda clones carrying Escherichia coli DNA segments) . The two-step method uses homologous recombination and lambda DNA packaging to replace the nonessential lambda DNA lying between the lysis genes and the right cohesive (cos) end with the neomycin phosphotransferase (npt) gene from Tn903 . This occurs during lytic growth of the phage on a plasmid-containing host strain . Neomycin-resistant (npt+) recombinant phages are then selected from the lysates containing the progeny phage by transduction of a polA1 lambda lysogenic host strain to neomycin resistance . We have tested this method with two different Kohara lambda phage clones; in both cases, neomycin resistance cotransduced with the auxotrophic marker carried by the lambda clone, indicating complete genetic linkage . Linkage was verified by restriction mapping of purified DNA from a recombinant phage clone . We also demonstrate that insertion of the npt+ recombinant phages into the lambda prophage can be readily distinguished from insertion into bacterial chromosomal sequences.

Trends Pharmacol Sci, 1991 Jun, 12(6), 227 - 32
Beta-lactamase inhibitors and reversal of antibiotic resistance; Sutherland R; The resistance of bacteria to beta-lactam antibiotics is usually associated with production of the enzyme, beta-lactamase, which inactivates the beta-lactam molecule . In the long search for inhibitors of bacterial beta-lactamase the first clinically useful agent, clavulanic acid, was isolated as a metabolite of Streptomyces clavuligerus . Robert Sutherland describes the background to the demonstration of clinical efficacy of combinations of clavulanic acid and other agents with penicillins which has confirmed beta-lactamase inhibitors as one solution to the problems posed by beta-lactamase-producing bacteria.

Am J Obstet Gynecol, 1991 Jun, 164(6 Pt 2), 1789 - 93
New treatments for Chlamydia trachomatis; Jones RB; Standard regimens of tetracycline, doxycycline, or erythromycin, if compiled with, appear to be effective against Chlamydia trachomatis infections under most circumstances . However, the organism may sometimes persist despite what would seem to be adequate therapy . How often this occurs, to what extent noncompliance is the issue, and the role antibiotic resistance plays remain to be determined . Among newer antibiotics, azithromycin appears to be effective in the treatment of uncomplicated urogenital C . trachomatis infections . Single-dose therapy with azithromycin may be especially useful in overcoming compliance problems associated with the treatment of sexually transmitted disease.

Infect Immun, 1991 Jun, 59(6), 1888 - 92
R plasmid in Escherichia coli O103 coding for colonization of the rabbit intestinal tract; Reynaud A et al.; One rabbit pathogenic Escherichia coli strain, belonging to serogroup O103, harbors a self-transferable 117-kb plasmid (pREC-1) encoding resistance to several antibiotics . The role of this R plasmid in the colonization of the digestive tract in specific-pathogen-free (E . coli O103-free) rabbits was studied . Five-week-old rabbits were inoculated with the wild-type strain, with its variant cured of the plasmid, with an E . coli K-12 strain, or with an untypeable E . coli strain from a healthy rabbit . No symptoms and no mortality were observed in animals inoculated with strains without the plasmid pREC-1, but 87.5% of the rabbits infected by the wild strain died, generally with bloody diarrhea, between days 5 and 15 postinfection . The weight gain of animals was strongly reduced . Transfer of the plasmid to the cured strain or to nonvirulent strains led these strains to induce the same pathology but with a lower mortality . Colonization of the gut by the O103 strain and symptoms of bloody diarrhea are thus related to the presence of the pREC-1 plasmid . The GV strain, which does not produce classical heat-labile enterotoxin or heat-stable enterotoxin and is not invasive, could be considered an enteropathogenic E . coli-like strain . The presence of a conjugative plasmid such as pREC-1 encoding both antibiotic resistance and virulence determinants in O103 E . coli from rabbits could represent a prominent epidemiological hazard under selective pressure by antibiotic therapy.

Mol Microbiol, 1991 Jun, 5(6), 1511 - 8
Mutations in the aphA-2 gene of transposon Tn5 mapping within the regions highly conserved in aminoglycoside-phosphotransferases strongly reduce aminoglycoside resistance; Blazquez J et al.; Aminoglycoside-phosphotransferases contain several conserved amino acid sequence motifs . Using hydroxylamine we have obtained five independent missense mutations within the aphA-2 gene of transposon Tn5 . Four of the mutations dramatically reduced antibiotic resistance . Two were identical and included the replacement of His-188 with Tyr . One other resulted from the replacement of Gly-189 with Asp . These three mutations map within the first of the conserved motifs . The replacement of Asp-261 with Asn maps to the third of these structural motifs . A mutation diminishing but not eliminating aminoglycoside resistance resulted from replacement of the conserved Val-36 with Met . By site-directed mutagenesis three additional mutants were obtained: His-188 was replaced with Leu and Ser, and Arg-211 within the second conserved motif was substituted by Gly . All three showed reduced levels of resistance to kanamycin . Our results show that these conserved motifs are essential for the biological activity of aminoglycoside phosphotransferases.

Rev Invest Clin, 1991 Apr-Jun, 43(2), 113 - 8
{The use of antibiotics at the outpatient clinic of the "Salvador Zubirán" National Institute of Nutrition}; Garcia-Rubi E et al.; We conducted a survey of the prescription of antibiotics among the outpatient clinics of the Instituto Nacional de la Nutricion "Salvador Zubiran", a third level hospital in Mexico City . We made an auditory of the medical record and prescriptions given to every patient treated for an infectious episode, accounting for at least six questions to evaluate the quality of the prescription: 1) if the patient should had received antibiotics; 2) if the antibiotic prescribed was adequate; 3) if the dose was sufficient; 4) if the frequency of administration was correct; 5) if the route of administration was adequate; and 6) if the length of the treatment was sufficient . We validated the concordance among two evaluators and found that it was 89% for the whole questionnaire . In the evaluation we found that the patients should had received antibiotic 94% of the time: the antibiotic selected was a right choice 80% of the time; the dose was adequate 45% of the time; the frequency of administration was adequate 70% of the time; the route of administration was adequate 79% of the time; and the length of treatment was adequate 38% of the time . The worst findings were seen in two of the most important issues of antibiotic prescription which directly affect the appearance of antibiotic resistance.

Am J Gastroenterol, 1991 Mar, 86(3), 354 - 6
Use of plasmid profiles in the investigation of a patient with Helicobacter pylori infection and peptic ulcer disease; Dworkin BM et al.; Plasmids may effect bacterial virulence and antibiotic resistance, and serve as epidemiologic markers . In this study, plasmid DNA profiles of serial isolates of Helicobacter pylori showed persistence of an identical strain of this organism in a patient with duodenal ulcer disease . Three control strains of H . pylori isolated from other patients contained plasmids different from each other and from that of the original patient; two of these strains had two plasmids each . These data have important implications for further study of the epidemiology and pathogenesis of H . pylori-related diseases.

Antibiot Khimioter, 1991 Mar, 36(3), 30 - 1
{Prospects for using DNA probes for rapid diagnosis of antibiotic resistance in clinical strains of enteric bacteria}; Vakulenko SV; Aminoglucoside resistance patterns of clinical strains of enteric bacteria isolated from inpatients of Moscow clinics were determined . APH(3')-I and AAC(3)-II were shown to be the most frequent . The aphA1 and aacC2 genes encoding the enzymes were cloned from the R plasmid of the transconjugant of the E . coli clinical strains . DNA probes based on the determined nucleotide sequences of the cloned genes were constructed and used in DNA-DNA hybridization experiments . The results on the occurrence of APH(3')-I and AAC(3)-II in the strains tested were confirmed by the DNA-DNA hybridization . Prospects for developing a set of DNA probes for rapid diagnosis of antibiotic resistance are discussed.

Biotechniques, 1991 Feb, 10(2), 150 - 2
A method of tagging specific-purpose linkers with an antibiotic-resistance gene for linker mutagenesis using a selectable marker; Bingle WH et al.; A method is described for tagging double-stranded linkers with an antibiotic-resistance gene permitting the direct selection of specific linker insertions during random linker-insertion mutagenesis; after selection, the antibiotic-resistance marker is removed leaving the linker-encoded restriction site in the target gene . The method is simple, relying on commercially available linkers and DNA-modifying enzymes, but retains considerable target site flexibility . The tagged linkers can be inserted at blunt-ended sites created in the target gene with DNase I or at sites created by restriction enzyme digestion leaving blunt ends, 5'-CG or 5'-GATC extensions . The advantages of such an approach over the use of standard antibiotic-resistance gene cartridges is discussed.

Gene, 1991 Jan 2, 97(1), 137 - 42
Cloning of tlrD, a fourth resistance gene, from the tylosin producer, Streptomyces fradiae; Zalacain M et al.; In addition to tlrA, tlrB and tlrC, which were previously cloned by others, a fourth antibiotic-resistance gene (tlrD) has been isolated from Streptomyces fradiae, a producer of tylosin (Ty), and cloned in Streptomyces lividans . Like tlrA, tlrD encodes an enzyme that methylates the N6-amino group of the A2058 nucleoside within 23S ribosomal RNA . However, whereas the tlrA protein dimethylates that nucleoside, the tlrD product generates N6-monomethyladenosine . The genes also differ in their mode of expression: tlrA is inducible, whereas tlrD is apparently expressed constitutively, and it has been confirmed that the tlrA-encoded enzyme can add a second methyl group to 23S rRNA that has already been monomethylated by the tlrD-encoded enzyme . Presumably, that is what happens in S . fradiae.

Mol Gen Genet, 1991 Jan, 225(1), 168 - 76
Analysis of a high frequency transformation system for Ophiostoma ulmi, the causal agent of Dutch elm disease; Royer JC et al.; A transformation system for Ophiostoma ulmi (Buism.) Nannf . was developed and analyzed . Protoplasts were generated from actively budding yeastlike cells by digestion with NovoZym 234 in MgSO4 after pretreatment with 2-mercaptoethanol . Protoplast regeneration was most efficient when 0.6 M sucrose was used as the osmoticum . Several plasmids containing fusions between fungal promoters and a bacterial gene for hygromycin phosphotransferase successfully transformed O . ulmi to hygromycin resistance . One of these vectors, pPS57, which contains a promoter for isopenicillin N synthetase from Penicillium chrysogenum, consistently conferred the greatest resistance to hygromycin . Linearization of the vector and inclusion of 2-mercaptoethanol in the transformation reaction resulted in enhanced transformation efficiency . Approximately 4 x 10(3) transformants/micrograms DNA per 10(7) protoplasts were obtained using the optimized procedure . Southern hybridization after alternating field and standard electrophoresis suggested random insertion of tandem repeats (some greater than 250 kb) into the fungal chromosomes . Antibiotic resistance was stable through mitosis . However, expression of the transforming DNA after meiosis was highly variable.

Scand J Infect Dis Suppl, 1991, 78, 64 - 70
Treatment failures with broad-spectrum antibiotics; Norrby SR; Three main reasons for clinical failures of antibiotic therapy are reviewed and discussed: antibiotic resistance, pharmacokinetic properties of the drug used and host factors . Of these, antibiotic resistance is the easiest to study but probably not the most common cause of unsuccessful therapy . Inability of an antibiotic to achieve sufficient concentrations at the site of an infection is most likely also an uncommon reason for lack of response to treatment . In most cases, it seems justified to assume that host factors are the most important explanation when clinical failures of antibiotic therapy are encountered . However, firm data to prove these assumptions are lacking and it is imperative that analysis of failures be included in all clinical trials; in doing such analyses it is likely that considerably more valuable information will be generated than just by analysis of frequencies of favourable therapy outcome.

J Basic Microbiol, 1991, 31(2), 127 - 34
Genetic recombination in Streptomyces michiganensis DSM 40,015 revealed three genes responsible for the formation of melanin; Held T et al.; By UV and NTG mutagenesis numerous auxotrophic, antibiotic-resistant and melanin-negative mutants were isolated from Streptomyces michiganensis DSM 40,015 which proved to possess a efficient photoreactivation system . Using a mel- test strain with three auxotrophic markers and a antibiotic resistance for crosses with numerous prototrophic mel- strains three classes of mutants (melA, melB and melC) could be found . This classification was further supported by a series of crosses . The melC locus seemed to correspond to the melC locus of S . glaucescens which contains the tyrosinase structural gene.

Arch Exp Veterinarmed, 1991, 45(1), 149 - 54
{The occurrence and the importance of enterotoxins from strains of Escherichia coli from bovine mastitis}; Alkaff O et al.; Baby mouse and Y1 cell culturing tests were conducted into 117 Escherichia (E.) coli strains from mastitis of cattle to clear up enterotoxin formation . The rate of the latter was found to be relatively low, with enterotoxins recorded from only 7 strains (6%) . No correlations were established between enterotoxin formation, on the one hand, and antibiotic resistance as well as biochemical activities of E . coli isolates, on the other . The question still remains open, if enterotoxin-forming strains derive their udder pathogenicity from endotoxins or with support by enterotoxins.

Semin Dermatol, 1990 Dec, 9(4), 250 - 4
Topical and systemic antibiotics: is there a rationale?
Noble WC.
Topical and systemic antibiotic therapy is common in dermatology, yet it is hard to find a rationale for a particular route in some diseases . Any rational choice needs to take efficacy, cost, and the likelihood of antibiotic resistance into account . Aspects of these factors are discussed in relation to the therapy of infectious and noninfectious bacterial skin diseases.

J Clin Microbiol, 1990 Dec, 28(12), 2726 - 32
Cefotaxime-resistant Nocardia asteroides strains are isolates of the controversial species Nocardia farcinica; Wallace RJ Jr et al.; A recent study of Nocardia asteroides revealed that 95% of clinical strains had one of five antibiotic resistance patterns . We found the pattern of resistance to cefotaxime and cefamandole in 19% of 200 clinical N . asteroides isolates . Isolates with this drug resistance pattern were from numerous geographic sources and were associated with significant clinical disease (56% of patients had disseminated infections) . Phenotypic studies revealed that these isolates were relatively homogeneous and matched previous descriptions and reference strains of the controversial species N . farcinica . Growth at 45 degrees C, acid production from rhamnose, ability to utilize acetamide as a nitrogen and carbon source, and resistance to tobramycin and cefamandole were features of N . farcinica that could be tested in the clinical laboratory and allowed their distinction from N . asteroides . The serious nature of disease due to N . farcinica and its resistance to the newer cephalosporins suggest a clinical need for laboratory identification of this species . (Current tests used in clinical laboratories do not distinguish N . farcinica from N . asteroides.) This is the first recognition that N . farcinica has a specific drug resistance pattern and confirms the previously described concept that drug resistance patterns of N . asteroides may be associated with specific taxonomic groups.

Biotechniques, 1990 Dec, 9(6), 694, 696 - 7
Easy selection of recombinant clones; Saris PE et al.; We have developed a polymerase chain reaction (PCR)-based procedure to facilitate the selection of recombinant clones . The insert to be cloned is ligated to an antibiotic resistance marker . The ligation product is amplified by PCR, followed by standard cloning procedure into a bacterial vector . The selection for the antibiotic resistance coded by the PCR product ensures 100% insertion frequency, eliminating the screening of the transformants.

Acta Paediatr Jpn, 1990 Dec, 32(6), 610 - 4
K1 antigen, serotype and antibiotic susceptibility of Escherichia coli isolated from cerebrospinal fluid, blood and other specimens from Japanese infants; Fujita K et al.; K1 antigens, serotypes and antibiotic susceptibilities of Escherichia coli isolates from neonates and infants were investigated . The presence of K1 antigen was tested by the K1-specific phage method . The number of K1 positive strains was 27 (84%) of 32 isolates from cerebrospinal fluid, 11 (25%) of 44 from blood and 4 (22%) of 18 from other specimens . Fourteen (33%) of the K1 positive strains were serotyped as O16:H6, and 8, 7 and 5 were serotyped as O18ac:H7, O1:H7 and O7:H-, respectively . One of 5 of the K1 negative strains were distributed into 30 different combinations of O and H antigens . The ampicillin resistance rates were 19% in K1 positive strains and 45% in K1 negative ones . The incidence of chloramphenicol resistance was the same in K1 positive and negative strains (21%) . Ampicillin resistance was not noted in O16:H6 strains, but the incidence of antibiotic resistance was high (65% to ampicillin and 53% to chloramphenicol) in the rough-type strains.

Antibiot Khimioter, 1990 Dec, 35(12), 11 - 3
{Isolation and characteristics of rifampicin-resistant mutants of Streptomyces erythraeus strain}; Nastasiak IN et al.; Erythromycin-producing strains of S . erythraeus were characterized with respect to formation of spontaneous and induced rifampicin-resistant mutants . It was shown that the frequency of spontaneous rifampicin-resistant mutants formed by various strains amounted to 0.9.10(-8) = 9.1.10(-7) . In some events the exposure to nitrosoguanidine increased the frequency of such mutants by 2 orders of magnitude . The rifampicin-resistant mutants differed in antibiotic resistance . It was found that a significant part of the rifampicin-resistant mutants became sensitive to heating (19.1 per cent) and lost the ability to form aeromycelium (21.8 per cent).

Genetics, 1990 Dec, 126(4), 875 - 88
Transformation of chloroplast ribosomal RNA genes in Chlamydomonas: molecular and genetic characterization of integration events; Newman SM et al.; Transformation of chloroplast ribosomal RNA (rRNA) genes in Chlamydomonas has been achieved by the biolistic process using cloned chloroplast DNA fragments carrying mutations that confer antibiotic resistance . The sites of exchange employed during the integration of the donor DNA into the recipient genome have been localized using a combination of antibiotic resistance mutations in the 16S and 23S rRNA genes and restriction fragment length polymorphisms that flank these genes . Complete or nearly complete replacement of a region of the chloroplast genome in the recipient cell by the corresponding sequence from the donor plasmid was the most common integration event . Exchange events between the homologous donor and recipient sequences occurred preferentially near the vector:insert junctions . Insertion of the donor rRNA genes and flanking sequences into one inverted repeat of the recipient genome was followed by intramolecular copy correction so that both copies of the inverted repeat acquired identical sequences . Increased frequencies of rRNA gene transformants were achieved by reducing the copy number of the chloroplast genome in the recipient cells and by decreasing the heterology between donor and recipient DNA sequences flanking the selectable markers . In addition to producing bona fide chloroplast rRNA transformants, the biolistic process induced mutants resistant to low levels of streptomycin, typical of nuclear mutations in Chlamydomonas.

Biotechniques, 1990 Nov, 9(5), 570 - 2, 574, 576-7
Optimization of routine transformation of Escherichia coli with plasmid DNA; Huff JP et al.; Methods to optimize resources and transformation efficiency of routine daily transformations of DH1 Escherichia coli prepared by three calcium chloride methods were investigated and compared with polyethylene glycol and Hanahan methods . The benefit of a heat-shock step, a preplating incubation step to allow expression of antibiotic resistance, use of log phase bacteria and prolonged storage of bacteria were investigated using pBR322 and pUC18 plasmid DNAs . Bacteria prepared by CaCl2 methods consistently gave efficiencies of 4 x 10(6) transformants/microgram of plasmid DNA or better and were overall the most labor- and resource-efficient methods . Use of log phase bacteria, a heat shock and an incubation step were found to be beneficial for freshly prepared bacteria for all methods . Prolonged storage of up to 30 days of bacteria prepared by the CaCl2 methods was beneficial, resulting in a sustained increase in transformation efficiency when selection was by ampicillin but not when by tetracycline resistance . Also found when using bacteria stored three days or longer was an increased transformation efficiency of stationary vs . log phase bacteria and an unchanged or even increased efficiency when the preplating incubation step was omitted . The Hanahan methods were the most labor and resource intensive and routinely gave efficiencies of 2 x 10(7) . Higher efficiencies of 10(8) were obtained only with repeated trial and error and were not consistently reproducible . The polyethylene glycol method consistently gave efficiencies of 2 x 10(7), and bacteria could easily be prepared daily or frozen with a minimal decrease in efficiency.

Mol Cell Biol, 1990 Nov, 10(11), 5849 - 56
Repair and recombination of X-irradiated plasmids in Xenopus laevis oocytes; Sweigert SE et al.; Plasmid DNA substrates were X-irradiated and injected into the nuclei of Xenopus laevis oocytes . After incubation for 20 h, DNA was recovered from the oocytes and analyzed simultaneously for repair and for intermolecular homologous recombination by electrophoresis and bacterial transformation . Oocyte-mediated repair of DNA strand breaks was observed with both methods . Using a repair-deficient mutant Escherichia coli strain and its repair-proficient parent as hosts for the transformation assay, we also demonstrated that oocytes repaired oxidative-type DNA base damage induced by X-rays . X-irradiation of a circular DNA stimulated its potential to recombine with a homologous linear partner . Recombination products were detected directly by Southern blot hybridization and as bacterial transformant clones expressing two antibiotic resistance markers originally carried separately on the two substrates . The increase in recombination was dependent on X-ray dose . There is some suggestion that lesions other than double-strand breaks contribute to the stimulation of oocyte-mediated homologous recombination . In summary, oocytes have considerable capacity to repair X-ray-induced damage, and some X-ray lesions stimulate homologous recombination in these cells.

Exp Cell Res, 1990 Nov, 191(1), 71 - 5
Expression of human chromosomal proteins HMG-14 and HMG-17 in Saccharomyces cerevisiae; Srikantha T et al.; The cDNAs coding for human chromosomal proteins HMG-14 and HMG-17 were cloned into yeast expression vector pBM150, under the control of the Gal10 promoter . Northern analysis of transformed yeast cells revealed that both cDNAs were efficiently transcribed . Western analysis indicated that the mRNAs were translated into authentic proteins . Expression of human HMG proteins in yeast cell did not produce detectable phenotypic changes, as measured by the growth rate of the yeast cells under a variety of conditions . The antibiotic resistance of the transfected cells was similar to that of control cells, suggesting that the presence of HMG did not affect the expression of actively transcribed genes . However, examination of the protein profile on two-dimensional polyacrylamide gel electrophoresis revealed differences between control and HMG-transfected cells.

J Bacteriol, 1990 Nov, 172(11), 6348 - 54
Intramolecular transposition by a synthetic IS50 (Tn5) derivative; Tomcsanyi T et al.; We report the formation of deletions and inversions by intramolecular transposition of Tn5-derived mobile elements . The synthetic transposons used contained the IS50 O and I end segments and the transposase gene, a contraselectable gene encoding sucrose sensitivity (sacB), antibiotic resistance genes, and a plasmid replication origin . Both deletions and inversions were associated with loss of a 300-bp segment that is designated the vector because it is outside of the transposon . Deletions were severalfold more frequent than inversions, perhaps reflecting constraints on DNA twisting or abortive transposition . Restriction and DNA sequence analyses showed that both types of rearrangements extended from one transposon end to many different sites in target DNA . In the case of inversions, transposition generated 9-bp direct repeats of target sequences.

Indian J Med Res, 1990 Nov, 91, 437 - 9
Incidence of R-plasmids in toxigenic & non-toxigenic strains of Escherichia coli from children with diarrhoea; Chakravarti A et al.; Toxigenic (30) and non-toxigenic (30) multiresistant strains of Esch . coli were studied for the incidence of R-plasmids . ST+, LT+ and ST+ LT+ plasmids were produced by 66.7, 30 and 3.3 per cent of toxigenic strains, respectively . ACST was the predominant resistance pattern in both the types . Incidence of R-plasmid in the toxigenic strains was 73.3 per cent as compared to 60 per cent in the non-toxigenic strains . Non-conjugative R-plasmid was present in 10 per cent of the non-toxigenic strain . In 13.3 per cent of toxigenic Esch . coli strains along with the transfer of antibiotic resistance, the ability to produce enterotoxin was also conferred on the recipient . This suggests that the genes coding for antibiotic resistance and enterotoxin are frequently associated and can be transferred together in vitro.

Sci China B, 1990 Nov, 33(11), 1341 - 9
Recombinant bivalent live vaccines against neonatal colibacillosis in piglets; Chen TM et al.; The genes of colonization factor K88 and avirulent heat-labile enterotoxin LT A-B+ of enterotoxigenic E . coli (ETEC) have been ligated, and a vaccine strain containing recombinant plasmid that efficiently expresses two antigens has been obtained . Using the activity of beta-galactosidase as a selection marker instead of drug resistance, another bivalent vaccine strain with the same expression level has also been constructed . The vaccine strains have no toxicity and do not cause any adverse reactions . In challenge study and field trials, a high degree of protection from colibacillosis was afforded to piglets when their dams were immunized orally or parenterally . Practice of bivalent live vaccines including colonization factor and enterotoxin antigens and without antibiotic resistance gene shows effective.

Med J Aust, 1990 Oct 15, 153(8), 493 - 5
The turtle's revenge: a case of soft tissue Mycobacterium chelonae infection; Miller AC et al.; A case of cutaneous Mycobacterium chelonae infection with sporotrichoid spread and extensive antibiotic resistance is presented . Control of this problem was only achieved after extensive debridement and grafting of the involved limb . The importance of surgery in management is discussed and the literature is reviewed.

Tierarztl Prax, 1990 Oct, 18(5), 501 - 2
{Antibiotic resistance of important bacteria from clinical and dissection material of the Institute for Poultry Diseases}; Gerlach H; 631 bacterial strains isolated during 1989 from many avian species were tested for their antibiotic sensitivity . The sensitivity ranged from 34.9 to 92.9%.

J Bacteriol, 1990 Sep, 172(9), 5130 - 4
Transformation of Rochalimaea quintana, a member of the family Rickettsiaceae; Reschke DK et al.; Rochalimaea quintana is the only member of the family Rickettsiaceae that can be grown in vitro . Because of its relationship to the other members of this family, techniques developed to transform R . quintana might be applicable to the obligate intracellular bacteria of the Rickettsiaceae . These procedures are critical to understanding mechanisms of pathogenesis and the nature of obligate intracellular growth . A transformation procedure for R . quintana has been established by using electroporation techniques . Several cosmids or plasmids with replicons RK2 and RSF1010 have been successfully used to transform this organism . Transformants were obtained by selection for antibiotic resistance to chloramphenicol or kanamycin . Plasmid retention and replication has been verified by Southern blot analysis and chloramphenicol acetyltransferase assay . Experimentation with different voltage field strengths and pulse times indicate that 12.5 kV/cm at 10 ms (25 microF and 400 omega) was optimal, giving a transformation frequency of approximately 0.3% and 3 x 10(5) transformants per microgram of DNA.

FEMS Microbiol Lett, 1990 Sep 1, 59(1-2), 133 - 7
Detection of small genotypic changes in Escherichia coli by pyrolysis mass spectrometry; Goodacre R et al.; Pyrolysis mass spectrometry (Py-MS) has been used to discriminate between four very closely related strains of Escherichia coli; a parent strain UB5021 and three derivatives each containing one of the antibiotic resistance plasmids, pBR322, pACYC184 or R388.

Gene, 1990 Sep 1, 93(1), 61 - 6
Development of a cloning system in Mycoplasma pulmonis; Mahairas GG et al.; A system suitable for recombinant DNA manipulation in mycoplasmas was developed using the cloned antibiotic-resistance genes of Tn4001 and Tn916 . An integrative plasmid containing one of the resistance markers was inserted into the genome of Mycoplasma pulmonis to form a recipient strain . This was accomplished by transformation and homologous recombination between chromosomal DNA sequences cloned onto the integrative plasmid . A second vector, the cloning vector, containing the same plasmid replicon and alternate resistance marker, carried cloned foreign DNA . When transformed into mycoplasmal recipients, homologous recombination between plasmid sequences resulted in integration of the cloning vector and foreign DNA . A Brucella abortus gene coding for a 31-kDa protein and the P1 structural gene and operon from Mycoplasma pneumoniae were introduced to examine the feasibility of developing mycoplasma as cloning hosts . Recombinant plasmids as large as 20 kb were inserted into M . pulmonis, and the integrated foreign DNA was stably maintained . The maximum size of clonable DNA was not determined, but plasmids larger than 22 kb have not been transformed into mycoplasmas using polyethylene glycol . Also the size of genome (800-1200 kb) may affect the stability of larger inserts of foreign DNA . This system is applicable to any mycoplasma capable of transformation, homologous recombination and expression of these resistance markers . Because of their lack of a cell wall, mycoplasmas may be useful cloning hosts for membrane or excreted protein genes from other sources.

J Chemother, 1990 Aug, 2(4), 238 - 40
Heavy metals, chlorine and antibiotic resistance in Escherichia coli isolates from ambulatory patients; Aguiar JM et al.; The susceptibility of Escherichia coli strains isolated from ambulatory patients to heavy metals, chlorine and antibiotics was evaluated . It seemed that heavy metal resistance was associated with antibiotic resistance . On the other hand, chlorine resistant strains seemed to be more sensitive to antibiotics . Clinical and ecological implications of these results are discussed.

J Med Microbiol, 1990 Aug, 32(4), 223 - 6
Resistance to metal ions and antibiotics in Escherichia coli isolated from foodstuffs; Grewal JS et al.; Of 39 strains of Escherichia coli isolated from foodstuffs, all were resistant to at least one of a panel of four metallic ions tested . The most common resistance (94.9%) was against cadmium, followed by arsenate (76.9%), silver (71.8%) and mercury (61.5%) . Multiple resistance to three (35.9%) or four (38.5%) metals was seen more often than resistance to two (18%) or one (7.7%) metal only . The opposite trend was seen in antibiotic resistance; resistance to one (30%) or two (49%) antibiotics was more common than to three or more antibiotics (13%) . Resistance to kanamycin correlated with resistance to silver and cadmium ions and resistance to ampicillin or cephalothin was, with one exception, associated with resistance to cadmium ions.

Plant Cell, 1990 Aug, 2(8), 701 - 7
Preferential transposition of the maize element Activator to linked chromosomal locations in tobacco; Jones JD et al.; The autonomous maize transposon Activator (Ac) has been used in maize for gene isolation by tagging and may prove similarly useful in other species . To test the feasibility of gene tagging with heterospecific transposons, we have examined three key genetic properties of a slightly modified Ac in tobacco . First, we show that frequencies of germinal excision of this Ac element from the antibiotic resistance gene streptomycin phosphotransferase can be comparable with or slightly lower than in maize . Second, we show that about half of the progeny carrying a germinal excision product also carry a transposed Ac . Last, we have mapped transposed Ac locations relative to the streptomycin transferase gene excision product and have shown that as in maize Ac in tobacco preferentially transposes to genetically linked sites.

Proc Natl Acad Sci U S A, 1990 Jul, 87(14), 5415 - 9
A biological price of antibiotic resistance: major changes in the peptidoglycan structure of penicillin-resistant pneumococci; Garcia-Bustos J et al.; Pneumococcal strains with greatly elevated levels of resistance to penicillin have by now been described with increasing frequency worldwide . The mechanism of antibiotic resistance in these strains involves the molecular remodeling of cell wall synthetic enzymes (penicillin binding proteins) . We have now analyzed the peptidoglycan structures of 10 penicillin-susceptible and 10 penicillin-resistant clinical isolates (4 of intermediate and 6 of high level resistance) with a high-resolution HPLC technique . Cell wall peptidoglycan of the susceptible strains contained monomeric and oligomeric forms of primarily (70% or more) linear stem peptides with the sequence of L-Ala-D-iGln-L-Lys-D-Ala (where iGln is isoglutamine) . In contrast, the major peptide species (70% or more) of resistant cell walls were abnormal branched-stem peptides carrying Ala-Ser or Ala-Ala dipeptides on the epsilon-amino groups of the stem peptide lysine residues . The structural alteration in the peptidoglycan was not related to serotype, date, or site of isolation but showed strong correlation with penicillin resistance and was cotransformed with high-level penicillin resistance during genetic transformation . We suggest that the remodeling of the active site of penicillin binding proteins in the resistant bacteria, which results in the reduced affinity for penicillin, also changes the substrate preference of these enzymes for the more hydrophobic branched peptides (instead of linear peptides) for cell wall synthesis.

J Bacteriol, 1990 Jul, 172(7), 3745 - 57
Structural and functional characterization of tnpI, a recombinase locus in Tn21 and related beta-lactamase transposons; Mercier J et al.; A novel discrete mobile DNA element from Tn21 from the plasmid R100.1 is described, and its mobilization function was confirmed experimentally . In addition, the element behaves as a recombinase-active locus (tnpI) which facilitates insertions of antibiotic resistance genes as modules or cassettes at defined hot spots or integration sites . A similar tnpI sequence was detected by DNA hybridization in a series of beta-lactamase transposons and plasmids and localized on their physical maps . The genetic function of the locus cloned from Tn21 into pACYC184 was tested for conduction and integration into the plasmids R388 and pOX38Km, and the results suggested recombinase-integrase activity and recA independence . DNA sequence analysis of the tnpI locus revealed no inverted or direct terminal repeats or transposition features of class I and class II transposons . The coding capacity revealed three putative open reading frames encoding 131, 134, and 337 amino acids . Orf3 encoded a putative polypeptide product of 337 amino acids that shared highly significant identity with the carboxyl region of integrase proteins . A comparison and an alignment of the tnpI locus from Tn21 and its flanking sequences identified similar sequences in plasmids and in transposons . The alignment revealed discrete nucleotide changes in these tnpI-like loci and a conserved 3' and 5' GTTA/G hot spot as a duplicated target site . Our data confirm the remarkable ubiquity of tnpI associated with antibiotic resistance genes . We present a model of transposon modular evolution into more complex multiresistant units via tnpI and site-specific insertions, deletions, and DNA rearrangements at this locus.

Nature, 1990 Jun 21, 345(6277), 739 - 43
Transposition of an antibiotic resistance element in mycobacteria; Martin C et al.; Bacterial resistance to antibiotics is often plasmid-mediated and the associated resistance genes encoded by transposable elements . Mycobacteria, including the human pathogens Mycobacterium tuberculosis and M . leprae, are resistant to many antibiotics, and their cell-surface structure is believed to be largely responsible for the wide range of resistance phenotypes . Antibiotic-resistance plasmids have so far not been implicated in resistance of mycobacteria to antibiotics . Nevertheless, antibiotic-modifying activities such as aminoglycoside acetyltransferases and phosphotransferases have been detected in fast-growing species . beta-lactamases have also been found in most fast- and slow-growing mycobacteria . To date no mycobacterial antibiotic-resistance genes have been isolated and characterized . We now report the isolation, cloning and sequencing of a genetic region responsible for resistance to sulphonamides in M . fortuitum . This region also contains an open reading frame homologous to one present in Tn1696 (member of the Tn21 family) which encodes a site-specific integrase . The mycobacterial resistance element is flanked by repeated sequences of 880 base pairs similar to the insertion elements of the IS6 family found in Gram+ and Gram- bacteria . The insertion element is shown to transpose to different sites in the chromosome of a related fast-growing species, M . smegmatis . The characterization of this element should permit transposon mutagenesis in the analysis of mycobacterial virulence and related problems.

Int J Cancer, 1990 Jun 15, 45(6), 1105 - 12
Selective immortalization of a phenotypically distinct epithelial cell type by microinjection of SV40 DNA into cultured human milk cells; Bartek J et al.; An immortal cell line, MMSV-1, has been developed which exhibits many features of the common luminal epithelial cell of the human mammary gland . The cell line was developed by microinjection of SV40 DNA into individual cells in selected colonies in cultures of human milk epithelial cells . Immunohistochemical staining shows that the MMSV-1 cells express keratins 7, 8, 18 and 19 homogeneously in organized filaments which lead into well-developed desmosomes . They do not express vimentin or keratins found in stratified epithelia or keratin 14 found in basal cells in the mammary gland . The PEM mucin, recognized by the antibody HMFG-I, is also expressed and appears to be processed normally . Fibronectin is detected but shows the punctate pattern typical of cultured normal milk epithelial cells . MMSV-1 cells show a reduced requirement for added growth factors, including cyclic AMP-elevating agents, but do not grow in agar or form tumours in nude mice . Since the transfected cells could be selected on the basis of an extended in vitro life span, antibiotic resistance markers were not introduced and the cells remain sensitive to hygromycin and neomycin.

Mol Microbiol, 1990 Jun, 4(6), 1019 - 28
Integration host factor affects expression of two genes at the conjugal transfer origin of plasmid R100; Dempsey WB et al.; Integration host factor (IHF) binds to two sites near the origin of transfer of the conjugative antibiotic resistance plasmid, R100 . DNase I footprinting shows that one site is immediately adjacent to oriT and the gene X promoter, and another is adjacent to the traM promoter . A third site, known only from retardation gels, is near the traJ promoter . The relative promoter activities of genes X, traJ and traM are reduced in himA mutants (IHF-), as measured by chloramphenicol-resistance assays . Transcript analyses by Northern blots showed a reduction in size of the principal gene X and traJ transcripts in the absence of IHF.

J Vasc Surg, 1990 Jun, 11(6), 793 - 8
The vascular endothelial cell as a vehicle for gene therapy; Callow AD; Increasing knowledge that has accumulated during the past decade reveals that the endothelial cell plays a far larger role than that traditionally assigned to it, namely maintenance of the fluid state of the blood . Unraveling the complex interreactions of the endothelial cell with the other cellular and molecular components of the arterial wall, as well as with the blood and its cellular and particulate components, is leading to better understanding of anastomotic hyperplasia and recurrent stenosis after endarterectomy and balloon angioplasty . More recently with the newly acquired techniques of inserting genetic material into the vascular endothelial cell many new therapeutic possibilities may be developed . Important to the technique of seeding vascular grafts, and the possible use of the genetically modified endothelial cell for gene therapy systems, are the needs to identify the origin of the cell originally seeded and ways to increase their number . Retroviral vectors and genetically conferred antibiotic resistance provide these means.

Orv Hetil, 1990 May 6, 131(18), 971 - 4
{Immunohistochemical studies in contaminated small bowel syndrome}; Kiss Z et al.; This study was undertaken to evaluate the number of the immunoglobulin producing cells in the lamina propria of the small intestine by immunocytochemical techniques, using peroxidase-antiperoxidase (PAP) complex in normacid patients with CSBS . 25 patients were studied including 13 patients with bacterial overgrowth, where the bacterial concentration was higher than 10(4) colony forming units/ml, and 12 subjects with normal bacterial concentration, served as control . The patients with CSBS were treated with antibiotics according to the antibiotic resistance . After treatment the luminal bacterial concentrations was lower than 10(4) cfu/ml in 7 of 13 patients (CSBS I . group) . In 6 patients the bacterial concentration remained high (CSBS II . group) . The immunoglobulin producing cells were determined in biopsy specimens taken from the lower part of duodenum . The number of the IgA and IgM producing immunocytes was significantly decreased only in the CSBS II . group . Our results show that temporary immunological alterations may play an important role in the mechanism of the recurrent CSBS.

Indian J Med Res, 1990 May, 91, 185 - 8
Antibiotic resistance of coliforms in drinking water in rural areas; Ramteke PW et al.; The antibiotic sensitivity of 197 coliform sp . isolated from drinking water in five rural areas was studied . Twelve strains (6.1%) showed multiple antibiotic resistance, three (1.5%) of which were able to transfer the resistances to an Escherichia coli K-12 recipient . It seems unlikely that the occurrence of transmissible multiple antibiotic resistance among coliforms in drinking water in the areas studied poses a significant public health risk.

Mikrobiol Zh, 1990 May-Jun, 52(3), 66 - 70
{The virulence and antibiotic resistance of Escherichia isolated from man}; Bondarenko VI et al.; The virulence and antibiotic resistance of the same 75 strains of pathogenic Escherichia (PE) has been studied . Simultaneous determination of virulence in three tests (keratoconjuctival, intranasal and with the infection of cell culture) has permitted increasing detection of virulence strains to 97.4% . Differences of PE in O-antigen as well as differences of strains depending on the source of their isolation (a sick or a healthy carrier) have not an essential effect on the PE virulence . Drug resistance of PE was induced by neomycin (the highest degree) and by other antibiotics (arranged in the descending order): mycin, streptomycin, laevomycetin, polymyxin, tetracyclin and gentamycin.

J Bacteriol, 1990 May, 172(5), 2267 - 72
Genetic exchange of transposon and integrative plasmid markers in Mycoplasma pulmonis; Mahairas GG et al.; Matings of genetically marked derivatives of Mycoplasma pulmonis resulted in the exchange of chromosomal DNA and the appearance of doubly marked transconjugants . Transposons Tn916 and Tn4001, and a series of integrative plasmids derived from their cloned antibiotic resistance genes, were used to construct antibiotic-resistant mycoplasmal derivatives to examine this phenomenon at the molecular level . Genetic exchange occurred on agar surfaces at frequencies ranging from 3.3 X 10(-4) to 6.4 X 10(-8) transconjugants per CFU . Examination of chromosomal DNA from transconjugants by hybridization revealed that the transposons or integrated plasmids were in the same chromosomal locations as in the parental strains, indicating that exchange involved the transfer of chromosomal DNA and homologous recombination . Transfer was not affected by DNase, polyethylene glycol, EDTA, or calcium chloride but was affected by treatment of either parent with trypsin . Mixing of mating strains before plating had no effect on mating frequencies, but mating did occur in liquid media . The ability to exchange chromosomal markers was limited to selected strains of M . pulmonis; mating did not occur with Acholeplasma laidlawii or M . gallisepticum . Heat and UV inactivation studies revealed that nonviable cells could act as donors in matings . The evidence presented supports a conjugationlike mechanism involving specific trypsin-sensitive membrane components.

Aust Fam Physician, 1990 May, 19(5), 723 - 4, 727, 730-1
Antibiotic resistance in general practice; Hammond L; With the addition of several new oral antibiotics to those already available, there is the implication that bacterial resistance has become a significant problem in general practice . To test this hypothesis it is worth examining the susceptibilities of the major bacterial pathogens isolated from community based infections.

Int J STD AIDS, 1990 May, 1(3), 191 - 4
Evaluation of the significance of Mycoplasma hominis and Ureaplasma urealyticum in female genital tract infection--a retrospective case note study; Crawshaw SC et al.; Routine screening for sexually transmitted diseases in new patients attending the Genitourinary Clinic in Stoke-on-Trent includes a culture for Mycoplasma hominis (MH) and Ureaplasma urealyticum (UU) . A retrospective study was carried out on 400 female patients to ascertain whether there were any significant differences between the group positive for MH and UU and the negative control group . The positive group were found to be younger on average, but to have similar sexual histories to the negative control group . An association was found between the presence of genital mycoplasmas and Gardnerella vaginalis . An odourous vaginal discharge was more common in the positive group . Erythromycin was ineffective in eradicating the organisms in 62.5% of patients with MH, and 70% of those with UU . Continuing work is required to identify those women in whom the presence of MH or UU could have pathogenic effects . Treatment regimens for this group of women need to be carefully reassessed, in the light of increasing antibiotic resistance.

Eur J Biochem, 1990 Apr 20, 189(1), 67 - 72
Methylation of 23S ribosomal RNA due to carB, an antibiotic-resistance determinant from the carbomycin producer, Streptomyces thermotolerans; Zalacain M et al.; A resistance gene, carB, originally isolated from the carbomycin-producing organism, Streptomyces thermotolerans, confers on Streptomyces lividans high-level resistance to the drug . However, ribosomes from S . lividans expressing carB show only moderate resistance to this macrolide in vitro, although they are highly resistant to the action of lincosamide antibiotics . The carB product monomethylates the amino group of the adenosine residue located at position 2058 in 23S ribosomal RNA . In contrast, ribosomes from S . lividans expressing ermE, in which 23S RNA is dimethylated at this same position, are much more highly resistant to macrolides and insensitive to lincosamides.

J Econ Entomol, 1990 Apr, 83(2), 519 - 25
Host plant resistance and linear furanocoumarin content of Apium accessions; Trumble JT et al.; Linear furanocoumarin contents and antibiotic resistance to Liriomyza trifolii (Burgess) were documented for Apium species being investigated in a celery breeding program . In no-choice tests, L . trifolii fed more, produced more offspring, and had the highest pupal and adult productivity on the widely planted cultivar 'Tall Utah' 52-70R (Apium graveolens L.) . Antibiotic effects of the commercial cultivar 'Tall Utah' 52-70 HK and University of California families 87A-147 and 87A-338, derived from A . chilense Hook and Arn., were intermediate . Only A . nodiflorum (L.) Lag (accession 87A-236) did not allow survival beyond the larval stage . Concentrations of the carcinogenic and mutagenic linear furanocoumarins varied by location within plants (leaves usually greater than petioles), by specific compound (trend: psoralen less than xanthotoxin less than bergapten or isopimpinellin), and between accessions . A . nodiflorum had the lowest foliar levels of phototoxic furanocoumarins (11.8 micrograms/g fresh weight) and the best potential for use in the breeding program . Foliar levels of phototoxic furanocoumarins (psoralen, bergapten, and xanthotoxin) in plants 87A-147-3 (406 micrograms/g), 87A-147-2 (292.9 micrograms/g), and the family 87A-338 (265.9 micrograms/g) were 22.6, 16.3, and 14.8 times higher, respectively, than the concentration known to produce contact dermatitis (18 micrograms/g) . Even with such variability in concentration, the foliar content of linear furanocoumarins (individually or total) and L . trifolii adult production were not correlated.

Antimicrob Agents Chemother, 1990 Apr, 34(4), 642 - 50
The dhfrI trimethoprim resistance gene of Tn7 can be found at specific sites in other genetic surroundings; Sundstrom L et al.; The dhfrI gene, mediating high-level trimethoprim resistance, was earlier found only on Tn7 . Evidence is given here for an alternative location of this gene at a site identical to sites observed earlier for dhfrII on plasmid R388, dhfrV on pLMO20, and aadA on Tn21 . All these genes and dhfrI are precisely inserted as discrete GTTA-flanked elements at distinct loci in very conserved surrounding sequences . One of these dhfrI insertions was observed to occur in association with a similarly inserted aadA nucleotidyltransferase gene, which mediates streptomycin and spectinomycin resistance . Close to the insertion site, there is an open reading frame translating into a 337-amino-acid peptide which shows striking similarities to recombinases of the integrase family, sulI, the sulfonamide resistance gene, is very often found close to the insertion point forming a genetic surrounding, originally observed as a part of Tn21-like transposons . The alleged integration mechanism thus provides a recombination pathway for the genetic linkage of sulfonamide and other antibiotic resistance genes, including the most frequently encountered gene for trimethoprim resistance, dhfrI . Furthermore, the newly observed location of dhfrI could shed light on the evolution of the antibiotic resistance region of Tn7, which could be able to take up genes by the same mechanism as that of Tn21-like transposons.

EMBO J, 1990 Apr, 9(4), 1275 - 81
Genetic elements involved in Tn21 site-specific integration, a novel mechanism for the dissemination of antibiotic resistance genes; Martinez E et al.; Transposon Tn21 codes for a site-specific integration system, which is probably a novel recombination mechanism, responsible for the acquisition of resistance genes in this widespread family of transposons . Using insertion and deletion mutagenesis we have identified the genetic loci of the various recombination hot-spots (RHS) and of the gene product (the integrase) that catalyses the reaction . The site of recombination has been localized in two of the RHSs to the DNA sequence GTTAG, which is present at the 3' termini of a loosely conserved palindromic sequence of approximately 59 bp . This 59 bp sequence, which flanks the inserted genes in a number of naturally occurring transposons, is the only element required in cis for the recombination reaction.

Mol Gen Mikrobiol Virusol . 1990 Mar;(3):32.
{A new site-specific endodeoxyribonuclease EcoHI}; Glatman LI et al.; A new sitespecific endonuclease of the II class EcoHI has been isolated from Escherichia coli strain and characterized . Restriction endonuclease EcoHI recognises the nucleotide sequence C C (C/G) G G with the cleavage site between the fourth and fifth nucleotide . It is an isoshizomer of the restriction endonuclease CauII . The yield of enzyme is 2500 units of activity per 1 g of biomass . The producing strain Escherichia coli HI is nonpathogenic, easily grown with the antibiotic resistance markers permitting to cultivate the strain under selective conditions.

Hautarzt, 1990 Feb, 41(2), 94 - 7
{Experiences with oxytetracycline treatment of non-gonorrhea urethritis caused by Ureaplasma urealyticum}; Elsner P et al.; In a retrospective study, the efficacy of tetracycline therapy was assessed in 48 men with non-gonococcal urethritis who only harbored Ureaplasma urealyticum in their urethras . After 2 weeks of therapy with 2.0 g oxytetracycline per day, U . urealyticum was still found in the urethra of 8 patients (17%) . Urethritis was still present in 8 patients (17%) according to clinical criteria and in 11 patients (23%) according to microscopic findings . The persistence of U . urealyticum in the urethra did not correlate with the persistence of urethritis to a statistically significant degree . Therapy compliance, antibiotic resistance in a few cases, reinfections in 2 cases and a special host-parasite relationship are discussed with respect to the treatment failures.

Arch Geschwulstforsch, 1990, 60(2), 129 - 32
Verapamil effect on the accumulation of doxorubicin in the leukemia P388 cells with induced antibiotic resistance; Moroz LV et al.; Using mice BDF1 it has been shown that the period of retention of Doxorubicin (Dx) is shorter in the leukemia P388 cells with induced antibiotic resistance (P388/Dx) as compared to P388 cells sensitive to Dx . Administration of Verapamil (Vp) to animals leads to an increase of Dx concentration in the leukemia P388/Dx cells during a 240 min observation period . Vp promotes the therapeutic effect of Dx on P388/Dx bearing mice . It can be suggested that the mechanism of Vp action consists in the damaged Dx elimination from cells with induced resistance, since Vp doesn't change the period of circulation of the antibiotic in the blood plasma of mice.

Intensive Care Med, 1990, 16 Suppl 3, S206 - 11
The emergence of antibiotic resistance: myths and facts in clinical practice; Hamilton-Miller JM; Selection pressure, caused by the use of antibiotics--especially in hospitals--is the main factor responsible for the emergence of antibiotic-resistant bacteria . Resistance can arise endogenously by mutation (one-step, as found for Mycobacterium tuberculosis to rifampicin, or multi-step, as in gonococci to benzylpenicillin), or exogenously by transfer of R-factors . Mechanisms of resistance may involve a decrease in permeability, chemical modification of the antibiotic, or a change in the affinity of the target site . There are many misconceptions concerning the incidence, nature and spread of antibiotic resistance, and some of the most important of these are discussed . The emergence and spread of resistance can be controlled by adhering to antibiotic policies and by preventing or controlling outbreaks of infection . The importance of resistant organisms can be diminished by the development of new antibiotic agents, preferably containing new chemical entities.

Yi Chuan Xue Bao, 1990, 17(1), 38 - 45
{The selective isolation of cosmid clones by homologous recombination in Escherichia coli--a cosmid clone containing t complex linkage DNA sequence of mouse was isolated}; Chai JH; A procedure for the selective isolation of specific cosmid clones by homologous recombination between cosmid clones of genomic library and a probe DNA sequence cloned in a plasmid in vivo has been developed . The cosmid library was constructed in a rec- host cell strain and packaged into phage particles in vivo . The rec+ host cells containing a DNA sequence used as selection probe cloned in the pUC plasmid were infected by packaged cosmid phage particles . There is no homology between cosmid and the plasmid vectors . After a period of 1-3 hr . for the recombination to take place, the probe plasmids were integrated into cosmid, in which the DNA sequence are homologous with the probe, by homologous recombination . The cosmids are then packaged in vivo and transferred into a rec- cell strain . The specific cosmid clones were selected by double antibiotic resistance carried by both vectors . The probe plasmid can be excised by lambda excision enzyme by using superinfection with red+ phage . After packaging in vivo, these cosmid revertants can be identified on Xgal plate . A cosmid clone containing the t complex linkage DNA sequence of mouse was selected by using the procedure above with a probe derived from microdissected metaphase chromosome.

Rev Ig Bacteriol Virusol Parazitol Epidemiol Pneumoftiziol Bacteriol Virusol Parazitol Epidemiol, 1990 Jan-Mar, 35(1), 65 - 70
{The biochemical characteristics and antibiotic resistance phenotypes of strains of H . influenza isolated from the CSF of patients with meningitis}; Mihancea N et al.; The paper reports on the biochemical characteristics and the antibiotic resistant phenotypes in 18 strains of H . influenza (17 b serotype and a strain of c serotype) isolated from the CSF of the patients with purulent meningitis . The following were found: --all the 16 viable strains have homogeneous biochemical characteristics, typical for H . influenza; --the viable strains were: 7 biotype, 4 biotype II, 1 biotype III, 3 biotype IV and 1 biotype VII; --68.8% of the strains had the following antibiotic resistant phenotypes: Ap, Km, Rf, ApKm, ApRf, ApKmTc, ApKmRfT and ApCmFrKmRfTc; --the strains of the biotypes II and IV have the highest resistance to antibiotics, as concerns frequency and phenotype; --the frequency of the resistant strains is of 56.2% for ampicilline, 18.7% for tetracycline, 31.2% for rifampicine and 6.2% for cholarmphenicol.

J Egypt Public Health Assoc, 1990, 65(3-4), 291 - 303
A suggested vaccine formulation for the control of pneumococcal meningitis in Egypt; Guirguis NI et al.; Mortality rates of pneumococcal meningitis ranges from 13-60% in different parts of the world . Reports of pneumococci with multiple antibiotic resistance add urgency to the need for developing means of primary prevention . A 14-valent pneumococcal vaccine was licensed in 1977, and a 23-valent pneumococcal vaccine in 1983 . In the present work 131 strains of pneumococci isolated from meningitis cases in Egypt were identified and serotyped by Quellung reaction . The most frequently isolated serotype was serotype 1 (32%) . Serotypes 6A, 9L, 12A, 19A and 29 were next in prevalence . The age groups 0-18 years were the most frequently affected groups (79%) and over 18 years of age were only 21% of total cases . A vaccine formulation is suggested to have a coverage rate against pneumococcal meningitis cases of 79.3% and 89.3% of a proposed 14-valent vaccine and 23-valent vaccine versus to coverage rate of 48% and 54% of 14-valent and 23-valent International vaccines respectively . According to the age distribution of cases and the isolated serotypes a vaccination could be recommended during the first two years of age in order to protect the most frequently infected groups.

Chin J Biotechnol, 1990, 6(1), 19 - 26
The construction of a non-antibiotic resistant ETEC vaccine strain containing K88ac and LT-B antigens; Huang PT et al.; By inserting the LacZ gene into the plasmid containing ETEC LT-B antigen gene we obtained a recombinant plasmid which could express the beta-galactosidase and LT-B antigen, but has the Tc resistant gene . The K88ac antigen gene was then inserted into the BamHI site of the Tc resistant region in the above plasmid . The final recombinant plasmid has no antibiotic resistance gene but can stably express both LT-B and K88ac antigens, as well as the beta-galactosidase as a tracking marker.

Scand J Infect Dis Suppl, 1990, 73, 23 - 9
Antibiotic resistance in Europe and the current use of antibiotics in severe pediatric infections; Levy J; The prevalence of antibiotic resistance among bacteria responsible for severe infections in childhood is increasing . In H . influenzae, clinically relevant resistance to ampicillin and chloramphenicol is caused by plasmid mediated enzymatic drug inactivation whereas penicillin resistant pneumococci emerge as the result of alterations in their penicillin binding proteins . In N . meningitidis both mechanisms have recently been implicated in penicillin resistance . Although antibiotic resistant H . influenzae, S . pneumoniae and, to much less extent, N . meningitidis have a widespread distribution, marked geographical variations exist in their prevalence . In areas where these drug resistant bacteria are reported, cefotaxime or ceftriaxone should be used for the empiric treatment of suspected severe bacterial infections in childhood.

Biull Eksp Biol Med, 1989 Dec, 108(12), 714 - 6
{The effect of finoptin on the accumulation of doxorubicin in leukemia P388 cells with induced resistance to the antibiotic}; Moroz LV et al.; Using male mice BDF1, it has been shown that the retention period of doxorubicin (DOX) is shorter in the leukemia P 388 cells with induced antibiotic resistance (P 388/DOX) as compared to the P 388 cells, sensitive to DOX . Administration of finoptin (FP) to animals leads to the increase of DOX concentration in the leukemia P 388/DOX cells during 240 min observation . FP promotes the therapeutic effect of DOX on mice bearing leukemia P 388/DOX . It can be suggested that the mechanism of FP action is the damaged DOX elimination from cells with induced resistance, since FP doesn't change the period of antibiotic circulation in the murine blood plasma.

Mol Microbiol, 1989 Dec, 3(12), 1669 - 83
A novel family of potentially mobile DNA elements encoding site-specific gene-integration functions: integrons; Stokes HW et al.; A family of novel mobile DNA elements is described, examples of which are found at several independent locations and encode a variety of antibiotic resistance genes . The complete elements consist of two conserved segments separated by a segment of variable length and sequence which includes inserted antibiotic resistance genes . The conserved segment located 3' to the inserted resistance genes was sequenced from Tn21 and R46, and the sequences are identical over a region of 2026 bases, which includes the sulphonamide resistance gene sull, and two further open reading frames of unknown function . The complete sequences of both the 3' and 5' conserved regions of the DNA element have been determined . A 59-base sequence element, found at the junctions of inserted DNA sequences and the conserved 3' segment, is also present at this location in the R46 sequence . A copy of one half of this 59-base element is found at the end of the sull gene, suggesting that sull, though part of the conserved region, was also originally inserted into an ancestral element by site-specific integration . Inverted or direct terminal repeats or short target site duplications, both of which are characteristics of class I and class II transposons, are not found at the outer boundaries of the elements described here . Furthermore, the conserved regions do not encode any proteins related to known transposition proteins, except the DNA integrase encoded by the 5' conserved region which is implicated in the gene insertion process . Mobilization of this element has not been observed experimentally; mobility is implied from the identification of the element in at least four independent locations, in Tn21, R46 (IncN), R388 (IncW) and Tn1696 . The definitive features of these novel elements are (i) that they include site-specific integration functions (the integrase and the insertion site); (ii) that they are able to acquire various gene units and act as an expression cassette by supplying the promoter for the inserted genes . As a consequence of acquiring different inserted genes, the element exists in a variety of forms which differ in the number and nature of the inserted genes . This family of elements appears formally distinct from other known mobile DNA elements and we propose the name DNA integration elements, or integrons.

J Mol Biol, 1989 Oct 20, 209(4), 645 - 53
Isolation of temperature-sensitive mutants of 16 S rRNA in Escherichia coli; Triman K et al.; Temperature-sensitive mutants have been isolated following hydroxylamine mutagenesis of a plasmid containing Escherichia coli rRNA genes carrying selectable markers for spectinomycin resistance (U1192 in 16 S rRNA) and erythromycin resistance (G2058 in 23 S rRNA) . These antibiotic resistance alleles, originally identified by Morgan and co-workers, enable us to follow expression of cloned rRNA genes in vivo . Recessive mutations causing the loss of expression of the cloned 16 S rRNA gene were identified by the loss of the ability of cells to survive on media containing spectinomycin . The mutations were localized by in vitro restriction fragment replacement followed by in vivo marker rescue and were identified by DNA sequence analysis . We report here seven single-base alterations in 16 S rRNA (A146, U153, A350, A359, A538, A1292 and U1293), five of which produce temperature-sensitive spectinomycin resistance and two that produce unconditional loss of resistance . In each case, loss of ribosomal function can be accounted for by disruption of base-pairing in the secondary structure of 16 S rRNA . For the temperature-sensitive mutants, there is a lag period of about two generations between a shift to the restrictive temperature and cessation of growth, implying that the structural defects cause impairment of ribosome assembly.

Genetics, 1989 Oct, 123(2), 281 - 92
Antibiotic resistance mutations in the chloroplast 16S and 23S rRNA genes of Chlamydomonas reinhardtii: correlation of genetic and physical maps of the chloroplast genome; Harris EH et al.; Mutants resistant to streptomycin, spectinomycin, neamine/kanamycin and erythromycin define eight genetic loci in a linear linkage group corresponding to about 21 kb of the circular chloroplast genome of Chlamydomonas reinhardtii . With one exception, all of these mutants represent single base-pair changes in conserved regions of the genes encoding the 16S and 23S chloroplast ribosomal RNAs . Streptomycin resistance can result from changes at the bases equivalent to Escherichia coli 13, 523, and 912-915 in the 16S gene, or from mutations in the rps12 gene encoding chloroplast ribosomal protein S12 . In the 912-915 region of the 16S gene, three mutations were identified that resulted in different levels of streptomycin resistance in vitro . Although the three regions of the 16S rRNA mutable to streptomycin resistance are widely separated in the primary sequence, studies by other laboratories of RNA secondary structure and protein cross-linking suggest that all three regions are involved in a common ribosomal neighborhood that interacts with ribosomal proteins S4, S5 and S12 . Three different changes within a conserved region of the 16S gene, equivalent to E . coli bases 1191-1193, confer varying levels of spectinomycin resistance, while resistance to neamine and kanamycin results from mutations in the 16S gene at bases equivalent to E . coli 1408 and 1409 . Five mutations in two genetically distinct erythromycin resistance loci map in the 23S rDNA of C . reinhardtii, at positions equivalent to E . coli 2057-2058 and 2611, corresponding to the rib3 and rib2 loci of yeast mitochondria respectively . Although all five mutants are highly resistant to erythromycin, they differ in levels of cross-resistance to lincomycin and clindamycin . The order and spacing of all these mutations in the physical map are entirely consistent with our genetic map of the same loci and thereby validate the zygote clone method of analysis used to generate this map . These results are discussed in comparison with other published maps of chloroplast genes based on analysis by different methods using many of the same mutants.

Antonie Van Leeuwenhoek, 1989 Oct, 56(3), 273 - 82
Stability of Escherichia coli strains harboring recombinant plasmids for L-threonine production; Nudel BC et al.; Escherichia coli recombinant strains bearing the thr operon have been previously selected for threonine production and phenotypically classified according to antibiotic resistance properties (Nudel et al . 1987) . Further analysis of those strains permitted the isolation and restriction mapping of two different plasmids of 13 kb and 18.6 kb . The smaller one, which expressed tetracycline resistance gave better results on threonine accumulation but it was rather unstable when grown without antibiotic pressure . Therefore, other hosts were transformed with those plasmids to improve stability . A threonine-auxotrophic strain was a better host for plasmid maintenance and expression of thr operon . Host influence in plasmid-mediated threonine production was studied in terms of specific yields (the ratios of threonine accumulated to biomass values) and of plasmid maintenance (percent of AprTcr clones after cultivation in non selective media) . We also determined that semisynthetic media of defined composition were better than rich media for threonine expression, due to feed-back controls exerted by undesired catabolites accumulated in complex media.

Tubercle, 1989 Sep, 70(3), 159 - 70
Genetic transformation of BCG; Lugosi L et al.; Two substrains of BCG, the Pasteur and Japanese, were successfully transformed with E . coli-mycobacteria shuttle plasmids, constructed from the E . coli plasmid, pIJ666 and the M . fortuitum plasmid, pAL5000 . Individual plasmids (pYUB13, pYUB14) were obtained that contain selectable antibiotic resistance markers for kanamycin and chloramphenicol resistance that can replicate in both E . coli and BCG . Transformation of two substrains of BCG was successfully accomplished in 8/14 experiments by means of electroporation, and assessed by the growth of kanamycin-resistant colonies . The E . coli plasmid pIJ666 alone was unable to effect transformation . The results suggest that the M . fortuitum sequences required for transformation function as an origin of replication in BCG . The introduction, persistence and the identity of the plasmids were monitored by re-isolation from consecutive subcultures and restriction analysis . The variables associated with transformation, including the age, viability, and glycine pretreatment of BCG cultures, as well as the electroporation parameters on transformation frequencies are analysed . Consecutive transformations of BCG with plasmid DNA isolated from a BCG transformant increased the efficiency from the level of 10(1)-10(2) obtained with the initial library to 10(3)-10(4) colonies/micrograms DNA with functional pYUB plasmids . The hybrid plasmids were genetically stable and maintained expression of kanamycin resistance in continuous subcultures containing kanamycin for 250 generations . The introduction and stable expression of foreign DNA in BCG on a plasmid vector establishes a basis for the construction of polyvalent recombinant BCG vaccine vehicles expressing not only putative protective mycobacterial antigens, but also antigens for other infectious and malignant diseases.

Biochemistry, 1989 Aug 22, 28(17), 6960 - 9
Directed alteration of the D1 polypeptide of photosystem II: evidence that tyrosine-161 is the redox component, Z, connecting the oxygen-evolving complex to the primary electron donor, P680; Metz JG et al.; In photosystem II, electrons are sequentially extracted from water at a site containing Mn atoms and transferred through an intermediate carrier (Z) to the photooxidized reaction-center chlorophyll (P680+) . Two polypeptides, D1 and D2, coordinate the primary photoreactants of the reaction center . Recently Debus et al . {Debus, R.J., Barry, B.A., Babcock, G.T., & McIntosh, L . (1988) Proc . Natl . Acad . Sci . U.S.A . 85, 427-430}, have suggested that Z is a tyrosine residue located at position 161 of the D1 protein . To test this proposal, we have engineered a strain of the cyanobacterium Synechocystis PCC 6803 to produce a D1 polypeptide in which Tyr-161 has been replaced by phenylalanine . Wild-type Synechocystis PCC 6803 contains three nonidentical copies of the psbA gene which encode the D1 polypeptide . In the mutant strain, two copies were deleted by replacement with antibiotic-resistance genes, and site-directed mutations were constructed in a cloned portion of the remaining gene (psbA-3), carrying a third antibiotic-resistance gene downstream . Transformants were selected for antibiotic resistance and then screened for a photoautotrophy-minus phenotype . The mutant genotype was verified by complementation tests and by amplification and sequencing of genomic DNA . Cells of the mutant cannot evolve oxygen and, unlike the wild type, are unable to stabilize, with high efficiency, the charge-separated state in the presence of hydroxylamine and DCMU {3-(3,4-dichlorophenyl)-1,1-dimethylurea} . Analyses by optical and EPR spectroscopy of reaction centers purified from this mutant indicate that Z can no longer be photooxidized and, instead, a chlorophyll cation radical, Chl+, is produced in the light . In the wild type, charge recombination between Z+ and the reduced primary quinone electron acceptor QA- occurs with a t1/2 of 80 ms . In the mutant, charge recombination between Chl+ and QA- occurs with a t1/2 of 1 ms . From these observations, we conclude that Z is indeed Tyr-161 of the D1 polypeptide.

Mol Gen Genet, 1989 Aug, 218(2), 364 - 6
A new procedure for the targeted inactivation of essential bacterial genes; Lukacsovich T et al.; A new method is described for the exchange of a plasmid encoded mutant gene with its chromosomal counterpart . The method is based on positive selection and is applicable for the exchange of essential genes . The main features of the method are: (1) cloning of an antibiotic resistance marker (without its promoter) downstream of the cloned target gene, thus forming an artificial operon; (2) inactivating the target gene (and consequently also the antibiotic resistance gene) by inserting a strong transcriptional termination signal into it; and (3) selection for double recombinants by means of the antibiotic resistance gene.

Mol Gen Genet, 1989 Jul, 218(1), 161 - 8
Transmission of the yeast mitochondrial genome to progeny: the impact of intergenic sequences; Piskur J; In a previous publication it was shown that the output of yeast mitochondrial loci lacking nearby intergenic sequences (encompassing ori/rep elements) was reduced in crosses to strains with wild-type mtDNAs . In the present work, mitochondrial genomes carrying the intergenic deletions were marked at unlinked loci by introducing specific antibiotic resistance mutations against erythromycin, oligomycin and paromomycin . These marked genomes were used to follow the output of unlinked regions of the genome from crosses between the intergenic deletion mutants and wild-type strains . Transmission of genetically unlinked markers in coding regions was substantially reduced when an intergenic deletion was present on the same genome . In general the transmission of the antibiotic markers was the same as or slightly higher than the corresponding intergenic marker . These results indicate that the presence of an intergenic deletion in the regions studied impairs the transmission to progeny of a mitochondrial genome as a whole . More specifically, the results suggest that ori/rep sequences, present in the regions that have been deleted, confer a competitive advantage over genomes lacking a full complement of such sequences . These results support the hypothesis that intergenic sequences, and specifically ori/rep elements, have a biological role in the mitochondrial genome . However, because of the exclusive presence of ori/rep sequences in the genus Saccharomyces, it may be that these sequences evolved in (or invaded) the mitochondrial genome relatively late in the evolution of the yeasts.(ABSTRACT TRUNCATED AT 250 WORDS)

G E N, 1989 Jul-Sep, 43(3), 194 - 201
Heterogeneous plasmid population from enterotoxigenic Escherichia coli strains isolated in Venezuelan children with acute diarrhea; Urbina G et al.; Thirty eight enterotoxigenic Escherichia coli (ETEC) isolated from children with acute diarrhea were analyzed in order to assess the possible associations among enterotoxigenicity, antibiotic resistance and other plasmid-mediated virulence properties such as CoIV, Hly and CFA/I . Eighty four percent of ETEC strains were multiresistant . Twenty strains (52.63%) were able to transfer one or more properties studied and 92.68% of the transconjugants were multiresistant . The simultaneous transfer of genes encoding ST enterotoxin and CoIV, Hly or CFA/I was very low (1.82%) . The plasmid analysis revealed the presence of a heterogeneous enterotoxigenic (Ent) plasmid population . Additionally, the existence of a conjugative plasmid of approximately 31 megadaltons (Md) of molecular weight encoding for ST and resistance to ampicillin, kanamycin and streptomycin was found . However, this plasmid was not present in all isolates . These results show a diversity of Ent plasmid population which is probably a consequence of the indiscriminate use of antibiotics and the molecular mechanism of transposition of ST and drug-resistance in the evolution of bacterial strains.

Nucleic Acids Res, 1989 Jun 26, 17(12), 4441 - 54
Efficient oligonucleotide-directed construction of mutations in expression vectors by the gapped duplex DNA method using alternating selectable markers; Stanssens P et al.; An efficient method for the construction of multiple mutations in a sequential manner is described . It is based on the gapped duplex DNA approach to oligonucleotide-directed mutagenesis (Kramer et al . 1984, Nucl . Acids Res . 12, 9441-9456) and a set of newly constructed phasmid vectors . These are characterized by the following features . Presence of the phage fl replication origin permits ready conversion to the single stranded DNA form . An amber mutation within, alternatively, the bla or cat gene provides a means for ready selection of the strand into which the mutagenic oligonucleotide has been incorporated . By means of the alternating antibiotic resistance markers any number of mutations can be constructed in consecutive rounds of mutagenesis . The optional presence of gene expression signals allows the direct overproduction of structurally altered proteins without re-cloning . Both the mutagenesis and expression aspects were tested using the lacZ gene as a model.

Curr Genet, 1989 Jun, 15(6), 421 - 8
Molecular transformation of Fusarium solani with an antibiotic resistance marker having no fungal DNA homology; Marek ET et al.; A vector was constructed for transformation of the plant pathogenic fungus Fusarium solani . The promoter 35Sp, from cauliflower mosaic virus, was fused to the bacterial gene APH(3')II, which confers resistance to the aminoglycoside antibiotic G418 . Two transformation procedures were developed: one using isolated fungal protoplasts, the other using germinated fungal spores . A transformation frequency of 3.3 G418-resistant colonies were obtained per microgram DNA . Of 14 colonies analyzed, 12 had vector sequences integrated into their high molecular weight DNA, and 2 were untransformed . Integration was sometimes accompanied by rearrangements of both the vector and flanking fungal DNAs . Primer-extension analysis of the mRNA from one transformant revealed two putative transcription initiation sites in the chimeric APH(3')II gene . Both sites differed from the normal initiation site in plants . This vector will be useful in transformation systems in which integration by non-homologous recombination is desired.

J Bacteriol, 1989 Jun, 171(6), 3449 - 57
Insertional mutagenesis by random cloning of antibiotic resistance genes into the genome of the cyanobacterium Synechocystis strain PCC 6803; Labarre J et al.; The facultative heterotrophic cyanobacterium Synechocystis sp . strain PCC 6803 was transformed by HaeII Cmr fragments ligated at random to HaeII DNA fragments of the host genome . A similar transformation was done with an AvaII Kmr marker ligated to AvaII host DNA fragments . Integration of the resistance markers into the host genome led to a high frequency of stable Kmr and Cmr transformants . Physical analysis of individual transformants indicated that this result was due to homologous recombination by conversionlike events leading to insertion of the Cmr (or Kmr) gene between two HaeII (or AvaII) sites of the host genome, with precise deletion of the host DNA between these sites . In contrast, integrative crossover of circular DNA molecules with homology to the host DNA is very rare in this cyanobacterium . Strain PCC 6803 was shown to have about 12 genomic copies per cell in standard growth conditions, which complicates the detection of recessive mutations induced by chemical or UV mutagenesis . Random disruption of the host DNA by insertional transformation provides a convenient alternative to transposon mutagenesis in cyanobacteria and may help to overcome the difficulties encountered in generating recessive mutants by classical mutagenesis.

Mol Gen Genet, 1989 May, 217(1), 111 - 7
Isolation and characterization of a conditional replication mutant of the antibiotic resistance factor R1 affected in the gene of the replication protein repA; Ortega S et al.; In vitro mutagenesis with hydroxylamine of a ParD- miniderivative of R1, pAB174, yielded mutants that were less stable in the cell than pAB174 . Some of these mutants had a thermosensitive phenotype . The replication of pAB2623, one of the thermosensitive mutants, was inhibited in the cell at the restrictive temperature of 42 degrees C . The efficiency of the RepA protein of pAB2623 to promote replication of R1 in an in vitro assay was greatly reduced . Sequence analysis indicated that the repA gene of pAB2623 contains, close to its 3' end, two GC-AT transitions, separated by a single base, that change two consecutive codons of the gene . These results indicate that the phenotype of the mutant is the consequence of a mutated RepA protein and is consistent with the requirement of RepA for the in vivo replication of this plasmid.

FEMS Microbiol Lett, 1989 May, 50(1-2), 93 - 6
Sewage dilution and loss of antibiotic resistance and virulence determinants in E . coli; Gonzalo MP et al.; The possession of potential virulence factors (serum resistance, aerobactin production, colicin production) and antibiotic resistance was evaluated in 418 E . coli strains isolated from river water . The strains were isolated from 11 points showing different levels of contamination . From the data obtained, it can be concluded that bacteria from less contaminated water present less antibiotic resistance and virulence factors than those isolated from highly contaminated water . This situation suggests that E . coli strains producing plasmid encoded antibiotic resistance and/or virulence factors, survive less well in aquatic environments without selective pressure) than the sensitive non-virulent ones.

J Bacteriol, 1989 May, 171(5), 2886 - 8
Derepression of conjugal transfer of the antibiotic resistance plasmid R100 by antisense RNA; Dempsey WB; Conjugal transfer of the normally repressed antibiotic resistance plasmid R100 was derepressed by fragments of R100 that carried the traJ promoter and the traJ leader but lacked the finP promoter.

J Infect Dis, 1989 Apr, 159(4), 708 - 16
Diversity and sources of rapidly growing mycobacteria associated with infections following cardiac surgery; Wallace RJ Jr et al.; Eighty-nine isolates of rapidly growing mycobacteria associated with cardiac bypass-related infections were characterized . Isolates from sporadic infections belonged to eight taxonomic groups and displayed numerous multilocus enzyme genotypes, plasmid profiles, and heavy metal and antibiotic resistance patterns . Compared with 449 noncardiac wound isolates, 45 sporadic cardiac isolates were more likely to be Mycobacterium fortuitum and M . smegmatis and less likely to be M . chelonae . About 80% of cardiac and noncardiac isolates were from southern coastal states . Eight outbreaks of cardiac bypass-related infections were identified . Strains from each outbreak were genotypically distinctive, and five outbreaks involved more than one strain . In two outbreaks, isolates from environmental sources and noncardiac infections were similar or identical to isolates from sternal wound infections . The heterogeneity of these isolates suggests that most isolates are unrelated and are derived from local environmental sources rather than from contaminated commercial surgical materials or devices.

J Bacteriol, 1989 Apr, 171(4), 1942 - 51
Structure of the cutinase gene and detection of promoter activity in the 5'-flanking region by fungal transformation; Soliday CL et al.; The cutinase gene from Fusarium solani f . sp . pisi (Nectria hematococa) was cloned and sequenced . Sau3A fragments of genomic DNA from the fungus were cloned in a lambda Charon 35 vector . When restriction fragments generated from the inserts were screened with 5' and 3' probes from cutinase cDNA, a 5.5-kilobase SstI fragment hybridized with both probes, suggesting the presence of the entire cutinase gene . A 2,818-base pair segment was sequenced, revealing a 690-nucleotide open reading frame that was identical to that found in the cutinase cDNA with a single 51-base pair intron . Transformation vectors were constructed containing a promoterless gene for hygromycin resistance, which was translationally fused to flanking sequences of the cutinase gene . When protoplasts and mycelia were transformed with these vectors, hygromycin-resistant transformants were obtained . Successful transformation was assessed by Southern blot analysis by using radiolabeled probes for the hygromycin resistance gene and the putative promoter . The results of Southern blot analysis indicated that the plasmid had integrated into the Fusarium genome and that the antibiotic resistance was a manifestation of the promoter activity of the cutinase flanking sequences . Transformation of Colletotrichum capsici with the same construct confirmed the promoter activity of the flanking region and the integration of the foreign DNA . Transformation and deletion analysis showed that promoter activity resided within the 360 nucleotides immediately 5' to the cutinase initiation codon.

Genetika, 1989 Apr, 25(4), 626 - 34
{Characterization of multiple changes of antibiotic resistance characters in Streptomyces coelicolor A3(2)}; Fedorenko VA et al.; Among mutants of Streptomyces coelicolor A3(2) studied which were sensitive to chloramphenicol (Cmls), strains sensitive to a number of antibiotics (ristomycin, tetracycline, polymyxin, lincomycin) amount of 46% . Antibiotic-sensitive mutants are capable to form different classes of resistant revertants with frequency varying from 10(-2) to 10(-6) in independent strains . Ristomycin-sensitive clones (Rims) have been found to occur with high frequency in Cmls strains and Cmlr revertants . Mutations mediating the Rims phenotype are mapped in a locus linked to the gene for resistance to chloramphenicol . The results obtained are discussed, in accordance with the notion about possible role of cml mutation in induction of secondary mutational changes in the genome of S . coelicolor A3(2).

J Bacteriol, 1989 Apr, 171(4), 1775 - 80
Transformation of Mycoplasma pulmonis: demonstration of homologous recombination, introduction of cloned genes, and preliminary description of an integrating shuttle system; Mahairas GG et al.; The transposons Tn916 and Tn4001 and a series of integrating plasmids derived from their antibiotic resistance genes were used to examine polyethylene glycol-mediated transformation of Mycoplasma pulmonis . Under optimal conditions, Tn916 and Tn4001 could be introduced into M . pulmonis at frequencies of 1 x 10(-6) and 5 x 10(-5) per CFU, respectively . Integrating plasmids were constructed with the cloned antibiotic resistance determinants of Tn916 and Tn4001, a pMB1-derived plasmid replicon, and mycoplasmal chromosomal DNA and were used to examine recombinational events after transformation into M . pulmonis . Under optimal conditions, chromosomal integrations could be recovered at a frequency of 1 x 10(-4) to 1 x 10(-6) per CFU, depending on the size and nature of the chromosomal insert and the parental plasmid . Integrated plasmids were stable in the absence of selection and could be rescued in Escherichia coli along with adjacent mycoplasma DNA . These studies provide the first direct evidence of a recombination system in the Mollicutes and describe the first E . coli-M . pulmonis shuttle vectors.

Mol Microbiol, 1989 Apr, 3(4), 561 - 70
Sense and antisense transcripts of traM, a conjugal transfer gene of the antibiotic resistance plasmid R100; Dempsey WB; The region of the antibiotic resistance plasmid R100 that encodes the plasmid-specific transfer gene traM has two tandemly aligned promoters separated by 145 nucleotides . The principal transcripts are 705 and 562 nucleotides long . Minor transcripts are 1550 and 1700 nucleotides long . The 705-base transcript appears to encode an 11 kD traM protein . The 562-base transcript does not encode a detectable protein . When subcloned on short fragments, the promoter for the 562-base transcript initiates efficiently but that for the 705 site does not . The 3' ends of the 705 and 562 base transcripts end inside the traJ ORF . Thus they provide additional sense RNA to compete with traJ for finP, the antisense translational regulator of traJ . A model is proposed for the participation of these sense and antisense transcripts in the control of expression of the traJ gene.

Science, 1989 Mar 10, 243(4896), 1357 - 60
Selection for precise chromosomal targeting of a dominant marker by homologous recombination; Dorin JR et al.; The antibiotic resistance gene neomycin phosphotransferase (neo) has been precisely targeted to a chromosomal region close to the cystic fibrosis (CF) locus on chromosome 7 . The chromosomal target was the expressed SV40 array integrated at chromosome 7, band q31-q35 in a human-mouse hybrid cell line that contains chromosome 7 as the only human component . Stringent selection for neo expression by homologous recombination (3 of 11 correctly targeted) was achieved by fusing the SV40 large T antigen gene, in frame, to neo in a promoterless construct, such that G418 resistance depended on endogenous promoter function and read-through transcription . Chromosome-mediated gene transfer (CMGT) with G418 selection was then used generate mouse hybrids that carried the targeted locus intact, but retained only a fragment of human chromosome 7 . This gene targeting strategy will access new regions of the human (or other mammalian) genome, create precise mutations efficiently by gene disruption, and potentially restore normal gene function by mutation correction.

Microbiol Rev, 1989 Mar, 53(1), 1 - 24
A collection of strains containing genetically linked alternating antibiotic resistance elements for genetic mapping of Escherichia coli; Singer M et al.; We present a collection of 182 isogenic strains containing genetically linked antibiotic resistance elements located at approximately 1-min intervals around the Escherichia coli chromosome . At most positions both Tn10 (Tetr) and TN10kan (Kanr) elements are available, so that the collection contains a linked set of alternating antibiotic resistance markers . The map position of each insertion has been aligned to the E . coli genetic map as well as to the Kohara ordered clone bank . These strains are designed to be used in a rapid two-step mapping system in E . coli . In the first step, the mutation is localized to a 5- to 15-min region of the chromosome by Hfr mapping with a set of Hfr strains containing either Tn10 or Tn10kan elements located 20 min from their respective origins of transfer . In the second step, the mutation is localized to a 1-min region by P1 transduction, with a collection of isogenic insertion strains as donors . We discuss the uses of this collection of strains to map and eventually to clone a variety of mutations in E . coli.

J Bacteriol, 1989 Mar, 171(3), 1435 - 44
Defective gamma subunit of ATP synthase (F1F0) from Escherichia coli leads to resistance to aminoglycoside antibiotics; Humbert R et al.; A strain of Escherichia coli which was derived from a gentamicin-resistant clinical isolate was found to be cross-resistant to neomycin and streptomycin . The molecular nature of the genetic defect was found to be an insertion of two GC base pairs in the uncG gene of the mutant . The insertion led to the production of a truncated gamma subunit of 247 amino acids in length instead of the 286 amino acids that are present in the normal gamma subunit . A plasmid which carried the ATP synthase genes from the mutant produced resistance to aminoglycoside antibiotics when it was introduced into a strain with a chromosomal deletion of the ATP synthase genes . Removal of the genes coding for the beta and epsilon subunits abolished antibiotic resistance coded by the mutant plasmid . The relationship between antibiotic resistance and the gamma subunit was investigated by testing the antibiotic resistance of plasmids carrying various combinations of unc genes . The presence of genes for the F0 portion of the ATP synthase in the presence or absence of genes for the gamma subunit was not sufficient to cause antibiotic resistance . alpha, beta, and truncated gamma subunits were detected on washed membranes of the mutant by immunoblotting . The first 247 amino acid residues of the gamma subunit may be sufficient to allow its association with other F1 subunits in such a way that the proton gate of F0 is held open by the mutant F1.

Gene, 1989 Feb 20, 75(2), 271 - 88
Complete nucleotide sequence and gene organization of the broad-host-range plasmid RSF1010; Scholz P et al.; We present the complete nucleotide sequence of RSF1010, a naturally occurring broad-host-range plasmid belonging to the Escherichia coli incompatibility group Q and encoding resistance to streptomycin and sulfonamides . A molecule of RSF1010 DNA consists of 8684 bp and has a G + C content of 61% . Analysis of the distribution of translation start and stop codons in the sequence has revealed the existence of more than 40 open reading frames potentially capable of encoding polypeptides of 60 or more amino acids . To date, products of eleven such potential RSF1010 genes have been identified through the application of controlled expression vector systems, and for eight of them, the reading frame has been confirmed by N- and/or C-terminal amino acid sequence determinations on the purified proteins . The sequencing results are discussed in relation to the systems of replication, host range, conjugal mobilization and antibiotic resistance determinants associated with the RSF1010 plasmid.

Mol Endocrinol, 1989 Feb, 3(2), 305 - 14
Transcriptional regulation by estrogen of episomal prolactin gene regulatory elements; Seyfred MA et al.; As a first step in defining the role of chromatin structure in steroid-regulated gene transcription, we have established a steroid-responsive minichromosome system that contains the 5' upstream regulatory region of the rat PRL gene (PRL) from -10 to -1960-basepairs fused to the antibiotic resistance gene, Tn5 . The hybrid gene was inserted into a bovine papilloma virus (BPV) vector and then transfected into GH3 cells . Southern analysis of total genomic DNA revealed that the PRL-Tn5-BPV DNA existed in the cells as unrearranged episomes or minichromosomes at a level of 25-100 copies/cell . We monitored the estrogen responsiveness of the minichromosome-based PRL regulatory regions by measuring Tn5 mRNA levels . Treatment of GH3 cells for 48 h with 10 nM 17 beta-estradiol (E2) increased Tn5 mRNA levels 3- to 6-fold over those in untreated cells . Concurrently, endogenous PRL mRNA levels were induced 8- to 15-fold . Using nuclear run-on assays, it was found that E2 increased PRL-Tn5 transcription rates approximately 3-fold over those in untreated cells . The induced transcription was mediated through the PRL elements and not through any other cis-acting elements within the minichromosome . The PRL elements that contain a functional enhancer are located 3' downstream of the BPV early gene promoters in the minichromosome . However, there was no detectable effect of the PRL enhancer on BPV early gene transcription . Thus, we have established a minichromosome system containing the transcriptional regulatory elements of the rat PRL gene that responds to E2 in a manner very similar to the endogenous rat PRL gene.

Zentralbl Mikrobiol, 1989, 144(2), 97 - 101
Occurrence of multiple antibiotic resistance in Azotobacter chroococcum; Sindhu SS et al.; Of 117 strains of Azotobacter chroococcum, isolated from local soils the antibiotic resistance pattern to ten widely used antibiotics was determined by antibiotic disk and agar plate dilution method . Over 95% of the strains were resistant to 10 micrograms ml-1 concentration of ampicillin, chloramphenicol, erythromycin and tetracycline and 70% or more were resistant to kanamycin, nalidixic acid, rifampicin, streptomycin and trimethoprim . 1 to 8% of the strains showed resistance upto 400 micrograms ml-1 concentration of 5 antibiotics (ampicillin, chloramphenicol, kanamycin, streptomycin and tetracycline) . The intrinsic resistance to the 10 antibiotics was generally high in Azotobacter chroococcum strains.

Trans R Soc Trop Med Hyg, 1989 Jan-Feb, 83(1), 45 - 8
Antibiotic resistance in the tropics . 3 . Medical responsibilities of the pharmaceutical industry with respect to use of antibiotics in the tropics; Salter AJ; The pharmaceutical industry has three major medical responsibilities: the efficacy and safety of its products, the accuracy of the statements it makes about them, and the provision to governments and health workers of full and proper information concerning these products . The development of new antibiotics is very costly, and their provision to Third World countries alone can never be financially rewarding; furthermore, only about 20% of world-wide pharmaceutical sales are to Third World countries . The industry's interest in developing drugs for exclusive or major use in such countries is declining . However, support from industry for the World Health Organization's action programme on essential drugs is growing, and this should help to provide drugs more cheaply to the poorer countries of the world.

Microbiol Immunol, 1989, 33(6), 441 - 7
Spreading of streptomycin antibiotic resistance gene in Escherichia coli plasmids demonstrated by Southern blot analysis; Hajnal A et al.; Plasmids from E . coli strains of 38 donors were transconjugated to common recipient SY663 Escherichia coli K12 . The restriction patterns of the isolated plasmids were highly heterogenous . However, the streptomycin (Sm) resistance genes of the plasmids were identical or closely homologous in 29 of the 33 plasmids conferring Sm resistance . These data were based on Southern blot analysis, using the Sm resistance gene (encoding aminoglycoside phosphoryl transferase) as probe cut out from pBP1 plasmid . Our data suggest an extensive spreading of streptomycin resistance gene of this type.

Vision Res, 1989, 29(8), 907 - 14
Production of bovine rhodopsin by mammalian cell lines expressing cloned cDNA: spectrophotometry and subcellular localization; Nathans J et al.; Cloned cDNA encoding bovine rhodopsin has been recombined into an expression vector and cotransfected with an antibiotic resistance plasmid into cultured human embryonic kidney cells . The resulting cell lines produce 100-200 micrograms of bovine opsin per liter of saturated tissue culture medium (10(9) cells) . Incubation in vitro with 11-cis retinal produces a photolabile pigment the absorbance spectrum of which is indistinguishable from that of bona fide bovine rhodopsin . Expressed rhodopsin accumulates in the plasma membrane as determined by immunoelectron microscopy.

Arch Exp Veterinarmed, 1989, 43(5), 779 - 82
{Antibiotic resistance of mycoplasmas from cell cultures}; Polster U; Investigations were conducted on 36 mycoplasma isolates to elucidate their resistance behaviours to kanamycin, gentamycin, neomycin, tylosin, oxytetracycline, chloramphenicol, and tiamulin . Only the latter proved to be highly effective, whereas all 36 strains were absolutely resistant to kanamycin, gentamycin, and neomycin, while 16 strains were resistant to all of the above antibiotics but tiamulin . Comparison of resistograms was found to provide some limited way of comparing strains and possibilities for detection of sources of contamination . Tiamulin may be used for improvement of cell cultures on account of its good cellular compatibility and high effectiveness on mycoplasmas.

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