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Will the close of the 20th century usher in the post-antibiotic era, a time of 'superbugs' resistant to all known antimicrobial agents? Some authorities believe so--and that bacterial infection could [...]

Focuses on antibiotic resistance of bacteria. Implications of the resistance of bacteria to antibiotics; Benefits of bacteria to the body; Factors that influence the insensitiveness of the bacteria to antibiotics; How resistance genes enable bacteria to survive an attack of antibiotic; How antibiotics promote resistance; How to reverse the resistance of bacteria. INSETS: The antibacterial fad: A new threat;Some actions physicians and consumers can take to limit....

Resistance stems from misguided efforts to try to sterilise out environment Bacterial resistance to multiple antibiotics characterises the present decade. Finding organisms insensitive to over 10 different antibiotics is not [...]

ABSTRACTPersister cells are highly tolerant to different antibiotics and are associated with relapsing infections. In order to understand this phenomenon further, we exposed a transposon library to a lethal concentration of ampicillin, and mutants that survived were identified by transposon sequencing (Tn-Seq). We determined that mutations related to carbon metabolism, cell envelope (cell wall generation and membrane proteins), and stress response have a role in persister cell generation.

ABSTRACTOmadacycline is a novel first-in-class aminomethylcycline with potent activity against important skin and pneumonia pathogens, including community-acquired methicillin-resistant Staphylococcus aureus(MRSA), β-hemolytic streptococci, penicillin-resistant Streptococcus pneumoniae, Haemophilus influenzae, and Legionella. In this work, the mechanism of action for omadacycline was further elucidated using a variety of models. Functional assays demonstrated that omadacycline is active against strains expressing the two main forms of tetracycline resistance (efflux and ribosomal protection). Macromolecular synthesis experiments confirmed that the primary effect of omadacycline is on bacterial protein synthesis, inhibiting protein synthesis with a potency greater than that of tetracycline. Biophysical studies with isolated ribosomes confirmed that the binding site for omadacycline is similar to that for tetracycline. In addition, unlike tetracycline, omadacycline is active in vitroin the presence of the ribosomal protection protein Tet(O).

Previous data from our laboratory suggest a relationship between increased pmrABexpression and virulence in an Escherichia colimouse infection model of pyelonephritis. Competitive infections with wild type and pmrABmutants showed that disruption of pmrABcaused decreased persistence of E. coliwithin the mouse kidney. These results were confirmed with plasmid-mediated complementation of the pmrABmutant. Additionally, increased expression of pmrABfrom this complementing plasmid in a previously attenuated marA-rob-soxStriple mutant displayed increased bacterial persistence in the infection when compared with the triple mutant alone. These findings suggest a role for this two-component regulatory system in the virulence of E. coliin a murine pyelonephritis model.

The efflux pump AcrAB is important in the antibiotic resistance and virulence of several pathogenic bacteria. We report that deletion of the Yersinia pestis AcrAB-TolC homolog leads to increased susceptibility to diverse substrates, including, though unlike in Escherichia coli, the aminoglycosides. Neither is the Y. pestis pump affected by the efflux pump inhibitor phenylalanine-arginine beta-naphthylamide. In mouse plague models, pump deletion does not have a significant effect on tissue colonization.

ABSTRACTThe efflux pump AcrAB is important in the antibiotic resistance and virulence of several pathogenic bacteria. We report that deletion of the Yersinia pestisAcrAB-TolC homolog leads to increased susceptibility to diverse substrates, including, though unlike in Escherichia coli, the aminoglycosides. Neither is the Y. pestispump affected by the efflux pump inhibitor phenylalanine-arginine beta-naphthylamide. In mouse plague models, pump deletion does not have a significant effect on tissue colonization.

SUMMARYAntimicrobials are valuable therapeutics whose efficacy is seriously compromised by the emergence and spread of antimicrobial resistance. The provision of antibiotics to food animals encompasses a wide variety of nontherapeutic purposes that include growth promotion. The concern over resistance emergence and spread to people by nontherapeutic use of antimicrobials has led to conflicted practices and opinions. Considerable evidence supported the removal of nontherapeutic antimicrobials (NTAs) in Europe, based on the “precautionary principle.” Still, concrete scientific evidence of the favorable versus unfavorable consequences of NTAs is not clear to all stakeholders. Substantial data show elevated antibiotic resistance in bacteria associated with animals fed NTAs and their food products. This resistance spreads to other animals and humans—directly by contact and indirectly via the food chain, water, air, and manured and sludge-fertilized soils. Modern genetic techniques are making advances in deciphering the ecological impact of NTAs, but modeling efforts are thwarted by deficits in key knowledge of microbial and antibiotic loads at each stage of the transmission chain. Still, the substantial and expanding volume of evidence reporting animal-to-human spread of resistant bacteria, including that arising from use of NTAs, supports eliminating NTA use in order to reduce the growing environmental load of resistance genes.

ABSTRACTLcrF (VirF), a transcription factor in the multiple adaptational response (MAR) family, regulates expression of the Yersiniatype III secretion system (T3SS). Yersinia pseudotuberculosis lcrF-null mutants showed attenuated virulence in tissue culture and animal models of infection. Targeting of LcrF offers a novel, antivirulence strategy for preventing Yersiniainfection. A small molecule library was screened for inhibition of LcrF-DNA binding in an in vitroassay. All of the compounds lacked intrinsic antibacterial activity and did not demonstrate toxicity against mammalian cells. A subset of these compounds inhibited T3SS-dependent cytotoxicity of Y. pseudotuberculosistoward macrophages in vitro. In a murine model of Y. pseudotuberculosispneumonia, two compounds significantly reduced the bacterial burden in the lungs and afforded a dramatic survival advantage. The MAR family of transcription factors is well conserved, with members playing central roles in pathogenesis across bacterial genera; thus, the inhibitors could have broad applicability.

LcrF (VirF), a transcription factor in the multiple adaptational response (MAR) family, regulates expression of the Yersinia type III secretion system (T3SS). Yersinia pseudotuberculosis lcrF-null mutants showed attenuated virulence in tissue culture and animal models of infection. Targeting of LcrF offers a novel, antivirulence strategy for preventing Yersinia infection. A small molecule library was screened for inhibition of LcrF-DNA binding in an in vitro assay. All of the compounds lacked intrinsic antibacterial activity and did not demonstrate toxicity against mammalian cells. A subset of these compounds inhibited T3SS-dependent cytotoxicity of Y. pseudotuberculosis toward macrophages in vitro. In a murine model of Y. pseudotuberculosis pneumonia, two compounds significantly reduced the bacterial burden in the lungs and afforded a dramatic survival advantage. The MAR family of transcription factors is well conserved, with members playing central roles in pathogenesis across bacterial genera; thus, the inhibitors could have broad applicability.

ABSTRACTMultidrug resistance (MDR) in clinical isolates of Escherichia colican be associated with overexpression of marA, a transcription factor that upregulates multidrug efflux and downregulates membrane permeability. Using random transposome mutagenesis, we found that many chromosomal genes and environmental stimuli affected MarA-mediated antibiotic resistance. Seven genes affected resistance mediated by MarA in an antibiotic-specific way; these were mostly genes encoding unrelated enzymes, transporters, and unknown proteins. Other genes affected MarA-mediated resistance to all antibiotics tested. These genes were acrA, acrB, and tolC(which encode the major MarA-regulated multidrug efflux pump AcrAB-TolC), crp, cyaA, hns, and pcnB(four genes involved in global regulation of gene expression), and the unknown gene damX. The last five genes affected MarA-mediated MDR by altering marAexpression or MarA function specifically on acrA. These findings demonstrate that MarA-mediated MDR is regulated at multiple levels by different genes and stimuli, which makes it both complex and fine-tuned and interconnects it with global cell regulation and metabolism. Such a regulation could contribute to the adaptation and spread of MDR strains and may be targeted to treat antibiotic-resistant E. coliand related pathogens.

ABSTRACTMarA, an AraC/XylS transcriptional regulator in Escherichia coli, affects drug susceptibility and virulence. Two MarA-like proteins have been found in Yersinia pestis: MarA47 and MarA48. Deletion or overexpression of these proteins in the attenuated KIM 1001 Δpgmstrain led to a change in multidrug susceptibility (including susceptibility to clinically relevant drugs). Additionally, lung colonization by the marA47or marA48deletion mutant was decreased about 10-fold in a pneumonic plague mouse model. Complementation of the deletions by replacing the deleted genes on the chromosome restored wild-type characteristics. These findings show that two MarA homologs in Y. pestisaffect antibiotic susceptibility and virulence.

Transposon inactivation of ycgE, a gene encoding a putative transcriptional regulator, led to decreased multidrug susceptibility in an Escherichia coli lon mutant. The multidrug susceptibility phenotype (e.g., to tetracycline and β-lactam antibiotics) required the inactivation of both lon and ycgE. In this mutant, a decreased amount of OmpF porin contributes to the lowered drug susceptibility, with a greater effect at 26°C than at 37°C.

ABSTRACTThirteen spontaneous multiple-antibiotic-resistant (Mar) mutants of Escherichia coliAG100 were isolated on Luria-Bertani (LB) agar in the presence of tetracycline (4 μg/ml). The phenotype was linked to insertion sequence (IS) insertions in marRor acrRor unstable large tandem genomic amplifications which included acrABand which were bordered by IS3or IS5sequences. Five different lonmutations, not related to the Mar phenotype, were also found in 12 of the 13 mutants. Under specific selective conditions, most drug-resistant mutants appearing late on the selective plates evolved from a subpopulation of AG100 with lonmutations. That the lonlocus was involved in the evolution to low levels of multidrug resistance was supported by the following findings: (i) AG100 grown in LB broth had an important spontaneous subpopulation (about 3.7 × 10−4) of lon::IS186mutants, (ii) new lonmutants appeared during the selection on antibiotic-containing agar plates, (iii) lonmutants could slowly grow in the presence of low amounts (about 2× MIC of the wild type) of chloramphenicol or tetracycline, and (iv) a lonmutation conferred a mutator phenotype which increased IS transposition and genome rearrangements. The association between lonmutations and mutations causing the Mar phenotype was dependent on the medium (LB versus MacConkey medium) and the antibiotic used for the selection. A previously reported unstable amplifiable high-level resistance observed after the prolonged growth of Mar mutants in a low concentration of tetracycline or chloramphenicol can be explained by genomic amplification.

ABSTRACTMarA47Ypfrom Yersinia pestis, showing 47% identity to Escherichia coliMarA in its N terminus, caused resistance to antibiotics and to organic solvents when expressed in both E. coliand Y. pestis. Resistance was linked to increased expression of the AcrAB multidrug efflux pump. In four of five spontaneous multidrug-resistant mutants of Y. pestisindependently selected by growth on tetracycline, the marA47Ypgene was overexpressed. The findings suggest that marA47Ypis a marAortholog in Y. pestis.

ABSTRACTThe possible association between triclosan and bacterial susceptibility to antibiotic was examined among staphylococci and several species of gram-negative bacteria (GNB) isolated from the hands of individuals in a community setting. Hand cultures from individuals randomized to using either antibacterial cleaning and hygiene products (including a hand soap containing 0.2% triclosan) or nonantibacterial cleaning and hygiene products for a 1-year period were taken at baseline and at the end of the year. Although there was no statistically significant association between triclosan MICs and susceptibility to antibiotic, there was an increasing trend in the association the odds ratios (ORs) for all species were compared at baseline (OR = 0.65, 95% confidence interval [95%CI] = 0.33 to 1.27) versus at the end of the year (OR = 1.08, 95%CI = 0.62 to 1.97) and for GNB alone at baseline(OR = 0.66, 95%CI = 0.29 to 1.51) versus the end of year (OR = 2.69, 95%CI = 0.78 to 9.23) regardless of the hand-washing product used. Moreover, triclosan MICs were higher in some of the species compared to earlier reports on household, clinical, and industrial isolates, and some of these isolates had triclosan MICs in the range of concentrations used in consumer products. The absence of a statistically significant association between elevated triclosan MICs and reduced antibiotic susceptibility may indicate that such a correlation does not exist or that it is relatively small among the isolates that were studied. Still, a relationship may emerge after longer-term or higher-dose exposure of bacteria to triclosan in the community setting.

Antibiotic resistance has become a major clinical and public health problem within the lifetime of most people living today. Confronted by increasing amounts of antibiotics over the past 60 years, bacteria have responded to the deluge with the propagation of progeny no longer susceptible to them. While it is clear that antibiotics are pivotal in the selection of bacterial resistance, the spread of resistance genes and of resistant bacteria also contributes to the problem. Selection of resistant forms can occur during or after antimicrobial treatment; antibiotic residues can be found in the environment for long periods of time after treatment. Besides antibiotics, there is the mounting use of other agents aimed at destroying bacteria, namely the surface antibacterials now available in many household products. These too enter the environment. The stage is thus set for an altered microbial ecology, not only in terms of resistant versus susceptible bacteria, but also in terms of the kinds of microorganisms surviving in the treated environment. We currently face multiresistant infectious disease organisms that are difficult and, sometimes, impossible to treat successfully. In order to curb the resistance problem, we must encourage the return of the susceptible commensal flora. They are our best allies in reversing antibiotic resistance.

Antibiotic resistance has become a major clinical and public health problem within the lifetime of most people living today. Confronted by increasing amounts of antibiotics over the past 60 years, bacteria have responded to the deluge with the propagation of progeny no longer susceptible to them. While it is clear that antibiotics are pivotal in the selection of bacterial resistance, the spread of resistance genes and of resistant bacteria also contributes to the problem. Selection of resistant forms can occur during or after antimicrobial treatment; antibiotic residues can be found in the environment for long periods of time after treatment. Besides antibiotics, there is the mounting use of other agents aimed at destroying bacteria, namely the surface antibacterials now available in many household products. These too enter the environment. The stage is thus set for an altered microbial ecology, not only in terms of resistant versus susceptible bacteria, but also in terms of the kinds of microorganisms surviving in the treated environment. We currently face multiresistant infectious disease organisms that are difficult and, sometimes, impossible to treat successfully. In order to curb the resistance problem, we must encourage the return of the susceptible commensal flora. They are our best allies in reversing antibiotic resistance.

MarR is a regulator of multiple antibiotic resistance in Escherichia coli. It is the prototypical member of the MarR family of regulatory proteins found in bacteria and archaea that play important roles in the development of antibiotic resistance, a global health problem. Here we describe the crystal structure of the MarR protein, determined at a resolution of 2.3 Å. This is the first reported crystal structure of a member of this newly-described protein family. The structure shows MarR as a dimer with each subunit containing a winged-helix DNA binding motif.

ABSTRACTThe genetic basis for fluoroquinolone resistance was examined in 30 high-level fluoroquinolone-resistant Escherichia coliclinical isolates from Beijing, China. Each strain also demonstrated resistance to a variety of other antibiotics. PCR sequence analysis of the quinolone resistance-determining region of the topoisomerase genes (gyrA/B, parC) revealed three to five mutations known to be associated with fluoroquinolone resistance. Western blot analysis failed to demonstrate overexpression of MarA, and Northern blot analysis did not detect overexpression of soxSRNA in any of the clinical strains. The AcrA protein of the AcrAB multidrug efflux pump was overexpressed in 19 of 30 strains of E. colitested, and all 19 strains were tolerant to organic solvents. PCR amplification of the complete acrR(regulator/repressor) gene of eight isolates revealed amino acid changes in four isolates, a 9-bp deletion in another, and a 22-bp duplication in a sixth strain. Complementation with a plasmid-borne wild-type acrRgene reduced the level of AcrA in the mutants and partially restored antibiotic susceptibility 1.5- to 6-fold. This study shows that mutations in acrRare an additional genetic basis for fluoroquinolone resistance.

ABSTRACTThe mdt(A) gene, previously designatedmef214, from Lactococcus lactissubsp.lactisplasmid pK214 encodes a protein [Mdt(A) (multiple drug transporter)] with 12 putative transmembrane segments (TMS) that contain typical motifs conserved among the efflux proteins of the major facilitator superfamily. However, it also has two C-motifs (conserved in the fifth TMS of the antiporters) and a putative ATP-binding site. Expression of the cloned mdt(A) gene decreased susceptibility to macrolides, lincosamides, streptogramins, and tetracyclines in L. lactisand Escherichia coli, but not in Enterococcus faecalisor inStaphylococcus aureus. Glucose-dependent efflux of erythromycin and tetracycline was demonstrated in L. lactisand in E. coli.

The intense use and misuse of antibiotics are undoubtedly the major forces associated with the high numbers of resistant pathogenic and commensal bacteria worldwide. Both the volume and the way antibiotics are applied contributes to the selection of resistant strains. Still, other social, ecological and genetic factors affect a direct relationship between use and frequency of resistance. Resistant bacteria, following their emergence and evolution in the presence of antibiotics, appear to acquire a ‘life of their own’. They proliferate and maintain the resistance traits even in the absence of antibiotics, thus jeopardizing the reversal of bacterial resistance by simple reduction in antibiotic use. Reversing resistance requires restoration of the former susceptible flora in people and in the environment.

An interdomain hybrid Tet protein consisting of a class C α domain and a class B β domain (Tet(C/B)) lacks detectable efflux ability and provides only minimal levels of resistance to tetracycline (Tc) (3 μg/ml) compared with intact class B (256 μg/ml) and class C (64 μg/ml). Twenty-one independently isolated mutants of the Tet(C/B) protein with increased Tc resistance were generated by random chemical mutagenesis. Nine mutants with a Glu substitution for Gly-152 in helix 5 of the class C α domain produced a resistance of 48 μg/ml, whereas another 9 with an Asp replacement of Gly-247 in helix 8 of the class B β domain mediated resistance at 32 μg/ml. The third type of mutation, found in 3 mutants expressing 24 μg/ml resistance, was a S202F replacement in the putative interdomain cytoplasmic loop of Tet(C/B). The latter underscores a previously unappreciated function of the interdomain cytoplasmic loop. All three types of Tet(C/B) mutant proteins were expressed in amounts comparable with that of the original protein and demonstrated restored energy-dependent efflux of tetracycline. Site-directed mutational analysis demonstrated that a Gly-247 to Asn mutation could also facilitate Tc resistance by the Tet(C/B) hybrid, and a negatively charged side chain at position 152 was required for Tet(C/B) activity. These mutations appear to promote the necessary functional interactions between the interclass domains that do not occur in the Tet(C/B) hybrid protein and suggest a direct association between helix 5 and helix 8 in the function of Tet efflux proteins.

ABSTRACTFluoroquinolone-resistant mutants, selected from a wild-typeEscherichia coliK-12 strain and its Mar mutant by exposure to increasing levels of ofloxacin on solid medium, were analyzed by Northern (RNA) blot analysis, sequencing, and radiolabelled ciprofloxacin accumulation studies. Mutations in the target genegyrA(DNA gyrase), the regulatory gene marR, and additional, as yet unidentified genes (genes that probably affect efflux mediated by the multidrug efflux pump AcrAB) all contributed to fluoroquinolone resistance. Inactivation of the acrABlocus made all strains, including those with target gene mutations, hypersusceptible to fluoroquinolones and certain other unrelated drugs. These studies indicate that, in the absence of the AcrAB pump, gyrase mutations fail to produce clinically relevant levels of fluoroquinolone resistance.

ABSTRACTActive efflux is a useful strategy by which bacteria evade growth inhibition by antibiotics. Certain semisynthetic tetracycline (TC) analogs, substituted at the 13th carbon at C-6 on ring C of the TC molecule, blocked TC efflux as revealed in everted membrane vesicles from class B TC-resistant (Tcr) Escherichia coli(M. L. Nelson, B. H. Park, J. S. Andrews, V. A. Georgian, R. C. Thomas, and S. B. Levy, J. Med. Chem. 36:370–377, 1993). A representative C-13-substituted analog, 13-cyclopentylthio-5-OH-TC (13-CPTC), was shown to competitively inhibit TC translocation by the Tet(B) protein, blocking the uptake of TC into vesicles and therefore the efflux of TC from whole cells. Against TcrE. coli, 13-CPTC, when used in combination with doxycycline, produced synergistic inhibition of growth. 13-CPTC was shown to increase the uptake of [3H]TC into the resistant cells. 13-CPTC alone was a potent growth inhibitor against TC-susceptible (Tcs) and TcrStaphylococcus aureusand enterococci specifying class K or class L efflux-dependent TC resistance mechanisms or, unexpectedly, the class M ribosomal protection mechanism. These findings indicate that derivatives of TC, identified by their ability to block the Tet(B) efflux protein, can restore TC activity against Tcrbacteria bearing either of the two known resistance mechanisms. Blocking drug efflux and increasing intracellular drug concentrations constitute an effective approach to reversing TC resistance and may be generally applicable to other antibiotics rendered ineffective by efflux proteins.

We examined the expression of cytochemical markers of myeloid and monocyte-macrophage differentiation in conjunction with ultrastructural studies of different malignant erythroleukemic cells isolated from mice infected with the Friend polycythemic virus complex (FLV-P). The amounts of fluoride-sensitive and resistant nonspecific esterase activity increased with the progression of malignancy. Isoelectric focusing resolved this enzyme activity into 13 isozymes in the most malignant Friend cell type tested. These same isozymes were found in the adherent cell population of normal spleens. Two of these isozymes were shown to have chloroacetate esterase activity characteristic of granulocytes. Despite these myeloid and monocyte characteristics, light and electron microscopy showed no morphological evidence of differentiation in either of these lineages. This study demonstrates that the Friend erythroleukemic cell contains markers of three different hemopoietic cell types. The expression of myeloid, monocytic, and erythroid traits in these erythroleukemic cells can be used to monitor their malignant progression.

Triclosan (Irgasan) is a broad spectrum antimicrobial agent used in handsoaps, toothpastes, fabrics, and plastics. It inhibits lipid biosynthesis in Escherichia coli, probably by action upon enoyl reductase (FabI) (McMurry L.M., Oethinger M. and Levy S.B. (1988) Nature 394, 531–532). We report here that overexpression of the multidrug efflux pump locus acrAB, or of marAor soxS, both encoding positive regulators of acrAB, decreased susceptibility to triclosan 2‐fold. Deletion of the acrABlocus increased the susceptibility to triclosan approximately 10‐fold. Four of five clinical E. colistrains which overexpressed marAor soxSalso showed enhanced triclosan resistance. The acrABlocus was involved in the effects of triclosan upon both cell growth rate and cell lysis.

When introduced as pairs into irradiated, sterile soils, aPseudomonas fluorescens strain prevented optimal colonization by aP. putida strain. The addition ofP. putida to sterile soil already populated byP. fluorescens impeded growth ofP. putida in that soil. However, addingP. fluorescens to soil populated withR. putida did not prevent growth ofP. fluorescens and caused a decrease in the titer ofP. putida. This “preemptive colonization/exclusion” phenomenon was also observed between two isogenic strains. The degree of exclusion depended on the amount of time between the inoculation of the two strains and which organism was introduced first. These results suggest competition for similar “niches” in soils and a hierarchy of fitness among pseudomonads sharing a soil environment. Autoclaving of the soil removed the competitive colonization betweenPseudomonas spp. observed in irradiated soils. The competitive colonization of irradiated soils by these twoPseudomonas sp. was reflected in their relative survival when introduced into nonsterile soil.

Escherichia coliminicells containing the plasmid pSC101 (-10 kb) or pBR322 (-4 kb) were opsonized and incubated with human neutrophils. The neutrophils responded to the minicells as they would to native E coli:they ingested the minicells, discharged their granule contents into the mini-cell-containing phagosomes, and expressed a respiratory burst. After one hour of incubation, the fate of the ingested plasmid DNA was examined. No DNA degradation was detected by trichloroacetic acid precipitation or agarose gel electrophoresis. Moreover, when pBR322 recovered from ingested minicells was transformed into E coli, no mutations in either of the antibiotic resistance genes carried by the plasmid were detected out of many thousand transformants screened. These findings confirm the surprisingly limited effect of neutrophils on ingested DNA.

Analysis of the protein synthesized by Escherichia coliminicells containing R factors demonstrated a variety of low- and high-molecular-weight polypeptides in sodium dodecyl sulfate (SDS)-polyacrylamide gels. Only half of this protein was released into a soluble fraction on lysis of these minicells. The other half remained associated with the minicell envelope. The efficiency of precursor incorporation into protein and the kinds of proteins synthesized changed with the age of the minicells at the time of harvest. About 1 to 2% of the soluble R factor-coded protein bound to calf thymus, E. coli, or R factor DNA-cellulose. Although most of these proteins were excluded from Sephadex G-100 columns, they migrated chiefly as low-molecular-weight-polypeptides (13,000 to 15,000) in SDS-polyacrylamide gels. Additional DNA-binding proteins that appeared to be higher-molecular-weight peptides were noted in extracts from younger minicells. At least one protein, identified as an SDS band, appeared to bind selectively to R factor DNA-cellulose. Minicells with R factors also contained DNA-binding proteins of cell origin, including the core RNA polymerase. No such binding proteins were found in R−minicells. These studies suggest that: (i) R factors code for proteins that may be involved in their own DNA metabolism; (ii) R factor DNA-binding proteins may be associated with larger host cell DNA-binding proteins or subunits of larger R factor proteins; and (iii) the age of the minicell influences the extent of protein synthesis and the kinds of proteins synthesized by R factors in minicells.

R factor proteins are synthesized in R factor-containing Escherichia coliminicells. Half of this protein remained associated with the minicell membrane upon lysis of the minicells. Over 90% of the membrane-associated protein was extracted by sodium lauryl sarcosinate, suggesting a location of these proteins in the inner membrane. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of these membrane preparations demonstrated the presence of multiple peptides, including a prominent band with a molecular weight of 28,000 to 30,000. A polypeptide of similar size was seen in membrane preparations from minicells harboring R factors from five different compatibility types. This major R factor membrane peptide was seen with R factors repressed or derepressed for pilus synthesis, with and without antibiotic resistances. It was associated with R factor deoxyribonucleic acid in membrane-deoxyribonucleic acid complexes. Its possible role in R factor replication and/or transfer is being investigated.

Escherichia colisensitivity to tetracycline involves transport and accumulation of the antibiotic within the cell by two different uptake systems: an initial rapid uptake, which occurs over the initial 6 min of contact of the cell with tetracycline, and a slower uptake system, which continues indefinitely and whose rate of uptake is 1/10 that of the rapid system. Only the slow uptake system is blocked by inhibitors of energy-driven systems; it appears to be particularly dependent upon energy from oxidative phosphorylation. Although both uptake systems lead to accumulation of intracellular tetracycline and contribute to the cell's sensitivity, the rapid uptake system appears to be the more important. While these studies confirm active transport of tetracycline into the cell, they demonstrate that a critical uptake system which appears insensitive to metabolic inhibitors occurs initially.

In the presence of suboptimal inducing amounts of dimethylsulfoxide or hexamethylenebisacetamide, a major proportion of uncommitted murine erythroleukemia (MEL) cells was found to be precommitted or primed for commitment, which was demonstrated by their rapid commitment to terminal differentiation when recultured for short periods of time (three to six hours) with fresh inducer. These same cells did not commit if left in the original inducer-containing media or if replated in fresh media without inducer. The two inducers could be interchanged in the priming and postpriming period without affecting the commitment event. However, hemin, an agent that induces hemoglobin synthesis without commitment, showed no ability to enhance commitment of a primed cell population. The rapid commitment of primed cells was inhibited by 12-0-tetradecanoylphorbol-13-acetate and cordycepin but not by cycloheximide. The latter finding indicated that this rapid inducer-dependent commitment event required new RNA synthesis but not new protein synthesis. The precommitment state was lost within six hours of the growth of cells in the absence of inducer but could be sustained longer if cells were incubated in cycloheximide. These studies characterize a precommitment state not previously described and one that appears during chemically induced differentiation in the absence of metabolic inhibitors. The stabilization of these precommitted cells by cycloheximide suggests that the reversibility of precommitment involves new protein synthesis. These findings show that MEL cells proceed to terminal differentiation by accumulating unstable products that must be maintained by the inducer until the final commitment event. © 1987 by Grune & Stratton, Inc.

The plasmid pUC18 contains a lacZ a-complementation gene that codes for a small peptide that can complement the AM15 mutation of the Escherichia colilacZ (β-galacto-sidase) gene, converting bacteria carrying that mutated gene from the lac Z-to the lacZ+phenotype. This plasmid was used in experiments designed to study mutagenesis by human neutrophils. E colicarrying pUC18 were incubated with neutrophils under conditions in which little ingestion of the bacteria took place; the plasmid was then isolated and transformed into an E colistrain (BOZO) that carries the lacZAM15 mutation. Of these transformants, 11 of 205,000 were lac-, suggesting that in these 11, α-comple-mentation had been lost through a mutation. No lac-colonies were detected among several hundred thousand BOZO transformed with plasmid isolated from incubations in which phagocytosis could take place, nor from incubations from which neutrophils were omitted. Despite the lac-phenotype of these 11 transformants, plasmids reiso-lated from nine of them showed normal a-complementing ability when transformed into fresh BOZO. These findings indicated that in these nine, the mutations were located in the chromosomes of the transformed BOZO. It thus appears that on exposure to activated neutrophils, a plasmid may acquire a lesion (?mutation) that can somehow be transferred to the genome of a recipient microorganism, resulting in repair of the damaged plasmid accompanied by mutation of the recipient's chromosome. Restriction mapping of the DNA from four of these nine chromosomal mutants suggested that the mutations did not represent major insertions or deletions in the portion of the bacterial chromosome corresponding to the pUC18 lac operon insert, nor in the remainder of the lacZΔM1 5 gene. These results confirm previous work showing that exposure to activated neutrophils can induce mutations in biological systems, and provides an experimental model in which the mechanism of neutrophil-mediated mutagenesis may be examined.1988 by Grune & Stratton, Inc. 0006-4971/88/7102-0028$3.00/0

The resistance of Gram−bacteria to the broad-spectrum antibiotic tetracycline (Tc) results from energy-dependent drug efflux mediated by the tetgene product, the cytoplasmic membrane Tet protein. Amino acid (aa) sequences deduced from total tetnucleotide sequences of three different resistance determinants (classes A, B and C) indicate that the protein products [Tet(A), Tet(B), and Tet(C)] share a common ancestor. Hydropathic analysis of Tet sequences predicts twelve transmembrane segments in each protein, with six occuring in each half of the molecule. More importantly, the linear distributions of these segments in the N- and C-terminal halves are nearly identical, suggesting that the two halves of each Tet protein are related by a process of tandem gene duplication and divergence. Indeed, a variable but significant conservation of sequence was detected among the N- and C-terminal halves for all possible comparisons of the three proteins. Such conservation was not observed within other prokaryotic integral membrane proteins or when other prokaryotic proteins were compared to Tet halves. Similarity, both in sequence and in predicted transmembrane structural organization, strongly suggests that a common aancestor of Tet(A), Tet(B), and Tet(C) arose by duplication of a gene reading frame specifying a transmembrane protein of approximately 200 aa residues. The two halves of Tet proteins correspond to the two domains, α and β, which have distinct, complementary roles in Tc efflux. Nevertheless, selective constraints to function in the cytoplasmic membrane have apparently led to maintenance of similar patterns of secondary structural organization in these complementary domains.

ABSTRACTThe contribution of regulatory genes to fluoroquinolone resistance was studied with clinical Escherichia colistrains bearing mutations in gyrAand parCand with different levels of fluoroquinolone resistance. Expression of marAand soxSwas evaluated by Northern blot analysis of isolates that demonstrated increased organic solvent tolerance, a phenotype that has been linked to overexpression of marA,soxS, and rob. Among 25 cyclohexane-tolerant strains detected by a screen for increased organic solvent tolerance (M. Oethinger, W. V. Kern, J. D. Goldman, and S. B. Levy, J. Antimicrob. Chemother. 41:111–114, 1998), we found 5 Mar mutants and 4 Sox mutants. A further Mar mutant was detected among 11 fluoroquinolone-resistant, cyclohexane-susceptible E. colistrains used as controls. Comparison of the marORsequences of clinical Mar mutants with that of E. coliK-12 (GenBank accession no. M96235) revealed point mutations in marRin all mutants which correlated with loss of repressor function as detected in a marO::lacZtranscriptional assay. We found four other amino acid changes in MarR that did not lead to loss of function. Two of these changes, present in 20 of the 35 sequenced marORfragments, identified a variant genotype of marOR. Isolates with the samegyrAand parCmutations showed increased fluoroquinolone resistance when the mutations were accompanied by overexpression of marAor soxS. These data support the hypothesis that high-level fluoroquinolone resistance involves mutations at several chromosomal loci, comprising structural and regulatory genes.

Penicillin has been used to purify large quantities of minicells.

Salmonella enteritidis14C-endotoxin was recovered predominantly from the nuclear and mitochondrial subcellular fractions of livers and spleens of mice and rats, 3.5 hr and 3 days after intravenous administration. Of the recovered radioactivity, 10 to 20% was present in the liver mitochondrial fraction as high-molecular-weight, biologically active material, suggesting the presence of intact endotoxin. Autoradiographic studies demonstrated nuclear and cytoplasmic labeling in the liver and at least nuclear label in spleen cells. The resistance of rats, as compared to mice, to the induction of amyloidosis does not appear to be based on a difference in subcellular localization of endotoxin within the reticuloendothelial system.

Levy, StuartB. (Institut du Radium, Paris, France). Isologous interference with ultraviolet and X-ray irradiated bacteriophage T2. J. Bacteriol. 87:1330–1338. 1964.—Qualitative and quantitative analysis of the interference capacity of an irradiated T2bacteriophage was made with ultraviolet and X-ray irradiation. Two different effects were found to explain the total interference picture in the ultraviolet-irradiated system: exclusion and depression. Exclusion is the absolute inhibition of infectious phage growth in the bacterial host. Depression is the diminution of burst size in instances where the infectious phage has not been excluded. Both effects were seen when the infectious phage was added after the addition of ultraviolet-irradiated phage. Doses between 1,600 and 2,200 ergs/mm2(survivals, ca. 10−7) showed the greatest exclusion effect (70%). Exclusion was lost between 6,500 and 7,500 ergs/mm2. The depression effect was highest (90%) at lower doses (survivals, ca. 10−6), falling off as the dose range went above 1,600 ergs/mm2or survivals of 10−7. Depression was lost at 3,000 ergs/mm2. X-ray irradiation (both direct and indirect) to survivals less than 10−2showed no interference capacity in the phage irradiated. Indirect X-ray irradiation to survivals between 5 and 10% showed 50% exclusion, but no depression.

Both fi+and fi−type R factors function as intact replicative units when segregated into Escherichia coliminicells. Hybridization studies demonstrate that at least 95% of the deoxyribonucleic acid (DNA) in R+minicells is episomal in origin. About half of the DNA can be extracted in a closed circular form and about 75% of the DNA is membrane associated. DNA, ribonucleic acid, and protein synthesis proceeds in R+minicells in contrast to R−minicell controls. The system offers a unique opportunity to study a relatively small replicative unit in a native cell environment and a simple means of isolating large quantities of episomal DNA.

ABSTRACTA carbapenem-resistant clinical isolate of Escherichia coli, which lacked OmpF and OmpC porins, carried a marRmutation and expressed a functional yedS, a normally nontranslated gene. MarR and YedS are described here as having effects on the ability of this strain to resist carbapenems. Additionally, expression of YedS was regulated by the small RNA MicF in a MarA-dependent way. These findings illustrate how broadly bacteria can mutate within a selective clinical setting, in this case, resistance to carbapenems, by altering three porin genes and one regulatory gene.

ABSTRACTThree Mycobacterium smegmatismutants selected for resistance to triclosan each had a different mutation in InhA, an enoyl reductase involved in fatty acid synthesis. Two expressed some isoniazid resistance. A mutation originally selected on isoniazid also mediated triclosan resistance, as did the wild-type inhAgene on a multicopy plasmid. Replacement of the mutant chromosomalinhAgenes with wild-type inhAeliminated resistance. These results suggest that M. smegmatisInhA, like its Escherichia colihomolog FabI, is a target for triclosan.

THE natural ecology of Escherichia coli and its infectious plasmids is not understood, although there is suggestive evidence that animals may serve as reservoirs for E. coli found in humans1,2. Investigation in this area becomes additionally important in view of the practice of introducing plasmids with pieces of foreign DNA into E. coli3,4. During an examination of the effects of antibiotic-supplemented animal feed on flora of farm animals and human personnel (ref. 5 and S.B.L., G.B.F. and A.B.M., unpublished), a study was initiated to determine if R plasmids and their E. coli hosts were naturally transferred among chickens and from chickens to human handlers. Our results illustrate the spread of antibiotic-resistant organisms from chicken to chicken and from chicken to man.