Escherichia coli K-12 mutants hyperproducing chromosomal beta-lactamase by gene repetitions.

Escherichia coli K-12 ampicillin-resistant mutants hyperproducing chromosomal beta-lactamase arose spontaneously from strains carrying ampA1 ampC(+). Such mutants were found even in a recA background. Two Amp(r)-100 strains were analyzed genetically. The Amp(r)-100 resistance level of both strains could be transduced by direct selection for ampicillin resistance. Several classes of ampicillin-resistant transductants were found that differed from one another in the beta-lactamase activity and the ampicillin resistance mediated by an ampA1 ampC(+)-carrying strain. The data suggested that beta-lactamase hyperproduction was due to repetitions of the chromosomal amp genes. The size of the repeated region was calculated from cotransduction estimates, using the formula of Wu (Genetics 54:405-410, 1966), and was found to be about 1 min in one strain and 1.5 min in the other. Second-step Amp(r)-400 mutants were isolated from an Amp(r)-100 strain. The resistance of these mutants was apparently also due to repetitions, each mediating a resistance to about 10 mug/ml. Mutants of wild-type strains that were moderately resistant to ampicillin also gave rise to intermediate-resistance classes, suggesting repetitions of the wild-type amp alleles. F' factors hyperproducing chromosomal beta-lactamase by gene repetitions were constructed. They mediated levels of ampicillin resistance comparable to that of naturally occurring resistance plasmids. The expression of beta-lactamase hyperproduction was not affected by the presence of ampA and ampC alleles in trans and did not act in trans on the other alleles.     

Resistance of Escherichia coli to penicillins: fine-structure mapping and dominance of chromosomal beta-lactamase mutations.

Seven Escherichia coli K-12 mutants with a lowered chromosomal beta-lactamase activity were analyzed genetically. The beta-lactamase-negative mutants isolated from ampA1-carrying strains (resistant to 10 microgram of ampicillin per ml) all carried genetic lesions very close to the ampA1 mutation, which was still present. In an earlier report, two of the mutations mediating a beta-lactamase-negative phenotype (L. G. Burman, T. Park, E. B. Linstr?m, and H. G. Boman, J. Bacteriol. 116:123-130, 1973) were shown to have occurred in the structural gene for beta-lactamase, designated ampC. It is suggested that all beta-lactamase-negative mutants studied here were altered in ampC. The relative order of ampC mutations was (ampC1, ampC8)-ampC9-(ampC12, ampC14)-ampC11, and the gene order was found to be ampC-1mpA-purA. The ampA1 allele was dominant over its wild-type allele but acted only cis and not trans, suggesting that ampA is the promoter or operator region for ampC. A gene dosage effect was found for strains homozygous for ampA+ ampC+ or ampA1 ampC+. Heterozygotes carrying the ampC8 allele on the chromosome showed an apparent derepression of the episomal ampC allele, suggesting a role for beta-lactamase in its own regulation.     

Physical size of the donor locus and transmission of Haemophilus influenzae ampicillin resistance genes by deoxyribonucleic acid-mediated transformation.

The properties of donor deoxyribonucleic acid (DNA) from three clinical isolates and its ability to mediate the transformation of competent Rd strains to ampicillin resistance were examined. A quantitative technique for determining the resistance of individual Haemophilus influenzae cells to ampicillin was developed. When this technique was used, sensitive cells failed to tolerate levels of ampicillin greater than 0.1 to 0.2 mug/ml, whereas three resistant type b beta-lactamase-producing strains could form from the colonies in 1- to 3-mug/ml levels of the antibiotic. DNA extracted from the resistant strains elicited transformation of the auxotrophic genes in a multiply auxotrophic Rd strain. For two of the donors, transformation to ampicillin resistance occurred after the uptake of a single DNA molecule approximately 104-fold less frequently than transformation of auxotrophic loci and was not observed to occur at all with the third. The frequency of transformation to ampicillin resistance was two- to fivefold higher in strain BC200 (Okinaka and Barnhart, 1974), which was cured of a defective prophage. All three clinical ampicillin-resistant strains were poor recipients, but the presence of the ampicillin resistant genes in strain BC200 did not reduce its competence. Sucrose gradients of DNA from ampicillin-resistant transformants of BC200 and from the original ampicillin-resistant strains showed that: (i) all the DNA preparations had high molecular weights; (ii) donor activity for ampicillin resistance sedimented heterogeneously and in parallel with genome DNA up to the highest molecular weights observed (100 x 106 to 200 x 106); and (iii) genetic transformation of ampicillin resistance from strain BC200-amp90383 required the physical integrity of a linearly integrated segment of DNA having a molecular weight of about 25 x 106 to 30 x 106.     

Mutation in Escherichia coli K-12 Mediating Spherelike Envelopes and Changed Tolerance to Ultraviolet Irradiation and Some Antibiotics

In a mutation experiment with a rough, ampicillin-resistant strain of Escherichia coli K-12, two smooth ampicillin-sensitive mutants were isolated. One of the mutants (with the envA gene) was recently described. The second mutant (strain D23) with the envB gene which has been mapped to a position close to streptomycin resistance (strA) at 64 min is described. The envB gene gives rise to spherelike cells. Electron microscopy revealed an abnormal septum formation and a circular distribution of the nuclear material. Strain D23 (with envB) showed a changed resistance to several antibiotics as well as an increased tolerance to ultraviolet irradiation. The envB gene decreased the ampicillin resistance mediated by the ampA gene (at 82 min), but no effect was found on episomally mediated penicillin resistance.     

Molecular epidemiology of antimicrobial-resistant commensal Escherichia coli strains in a cohort of newborn calves

Pulsed-field gel electrophoresis (PFGE) was used to investigate the dissemination and diversity of ampicillin-resistant (Amp(r)) and nalidixic acid-resistant (Nal(r)) commensal Escherichia coli strains in a cohort of 48 newborn calves. Calves were sampled weekly from birth for up to 21 weeks and a single resistant isolate selected from positive samples for genotyping and further phenotypic characterization. The Amp(r) population showed the greatest diversity, with a total of 56 different genotype patterns identified, of which 5 predominated, while the Nal(r) population appeared to be largely clonal, with over 97% of isolates belonging to just two different PFGE patterns. Distinct temporal trends were identified in the distribution of several Amp(r) genotypes across the cohort, with certain patterns predominating at different points in the study. Cumulative recognition of new Amp(r) genotypes within the cohort was biphasic, with a turning point coinciding with the housing of the cohort midway through the study, suggesting that colonizing strains were from an environmental source on the farm. Multiply resistant isolates dominated the collection, with >95% of isolates showing resistance to at least two additional antimicrobials. Carriage of resistance to streptomycin, sulfamethoxazole, and tetracycline was the most common combination, found across several different genotypes, suggesting the possible spread of a common resistance element across multiple strains. The proportion of Amp(r) isolates carrying sulfamethoxazole resistance increased significantly over the study period (P < 0.05), coinciding with a decline in the most common genotype pattern. These data indicate that calves were colonized by a succession of multiply resistant strains, with a probable environmental source, that disseminated through the cohort over time.     

Isolation of nonsense suppressor mutants in Pseudomonas.

A strain of Escherichia coli harboring the drug resistance plasmid RP1 was treated with the mutagen N-methyl-N-nitro-N-nitro-N-nitrosoguanidine, and mutants were isolated in which ampicillin resistance had been lost due to an amber mutation in the plasmid. One of these mutants was again treated, and a strain was isolated in which tetracycline resistance was also lost due to an amber mutation in the plasmid. The plasmid containing amber mutations in the genes amp and tet was named pLM2. This plasmid could be transferred to strains of Pseudomonas aeruginosa, P. phaseolicola, and P. pseudoalcaligenes. Mutants resistant to ampicillin and tetracycline could not be obtained from P. phaseolicola carrying pLM2. However, strains of E. coli, P. aeruginosa, and P. pseudoalcaligenes carrying the plasmid did produce mutants simultaneously resistant to both antibiotics. All of the mutants of E. coli had developed nonsense suppressors since they became phenotypically lac+, although harboring a lac amber mutation, and formed plaques with amber mutants of phages PRR1 and PRD1 that attack organisms carrying RP1. Approximately 20% of the resistant mutants of P. aeruginosa and P. pseudoalcaligenes were sensitive to the amber mutant of PRD1. These mutants were of variable stability and grew somewhat more slowly than their parent strains. One of the suppressor mutants of P. pseudoalcaligenes, designated ERA(pLM2)S4, was used for the isolation of nonsense mutants of bacteriophage PHA6, a virus having a segmented genome of double-stranded ribonucleic acid and an envelope of lipids and proteins.     

Mapping of a new genetic locus responsible for ampicillin resistance in Escherichia coli.

The ampicillin resistance locus of three different ampicillin-resistant, temperature-sensitive Escherichia coli mutants was mapped between proC and purE and does not correspond to any of the known genes in this region. The mutant gene responsible for the temperature sensitivity and consequent morphological changes in each mutant strain was not located in the same 5-min region, even though the two mutants characteristics co-reverted at a very high frequency.     

Ferric citrate transport in Escherichia coli requires outer membrane receptor protein fecA.

Mutants of Escherichia coli K-12 AB2847 and of E. coli K-12 AN92 were isolated which were unable to grow on ferric citrate as the sole iron source. Of 22 mutants, 6 lacked an outer membrane protein, designated FecA protein, which was expressed by growing cells in the presence of 1 mM citrate. Outer membranes showed an enhanced binding of radioactive iron, supplied as a citrate complex, depending on the amount of FecA protein. The FecA protein was the most resistant of the proteins involved in ferric irion iron translocation across the outer membrane (FhuA = TonA, FepA, Cir, or 83K proteins) to the action of pronase P. It is also shown that previously isolated fec mutants (G. C. Woodrow et al., J. Bacteriol. 133:1524-1526, 1978) which are cotransducible with argF all lack the FecA protein. They were termed fecA to distinguish them from the other ferric citrate transport mutants, now designated fecB, which mapped in the same gene region at 7 min but were not cotransducible with ArgF. E. coli W83-24 and Salmonella typhimurium, which are devoid of a citrate-dependent iron transport system, lacked the FecA protein. It is proposed that the FecA protein participates in the transport of ferric citrate.     

Characterization and genetic mapping of fructose phosphotransferase mutations in Pseudomonas aeruginosa

Pseudomonas aeruginosa transports and phosphorylates fructose via a phosphoenolpyruvate-dependent fructose phosphotransferase system (PTS). Mutant strains deficient in both PTS activity and glucose-6-phosphate dehydrogenase activity were isolated and were used to select mannitol-utilizing revertant strains singly deficient in PTS activity. These mutants were unable to utilize fructose as a carbon source and failed to accumulate exogenously provided [14C]fructose, and crude cell extracts lacked phosphoenolpyruvate-dependent fructose PTS activity. Thus, the PTS was essential for the uptake and utilization of exogenously provided fructose by P. aeruginosa. Mutations at a locus designated pts, which resulted in a loss of PTS activity, exhibited 57% linkage to argF at 55 min on the chromosome in plasmid R68.45-mediated conjugational crosses. The pts mutations in four independently isolated mutant strains exhibited from 11 to 20% linkage to argF, and one of these mutations exhibited 3% linkage to lys-9015 in phage F116L-mediated transductional crosses.     

Mutant of Escherichia coli with Anomalous Cell Division and Ability to Decrease Episomally and Chromosomally Mediated Resistance to Ampicillin and Several Other Antibiotics

In a mutation experiment with a rough, ampicillin-resistant strain, we isolated two smooth mutants which were both sensitive to ampicillin and carried defects in the cell envelope. One of the strains (with the envA gene) is hindered in its completion of septa and forms chains of cells. The envA gene has been mapped to a position between leu and proB, at 2 to 4 min. The envA gene decreased the resistance mediated by both episomal and chromosomal genes for resistance to several antibiotics. During growth the envA mutant was characterized by abnormal ratios between viable count or cell count and optical density. The ratio between viable count and optical density was affected during shift-up and shift-down experiments. When compared to the parent strain, the envA mutant was found to be more resistant to ultraviolet irradiation on plates. Prestarvation for tryptophan had a protective effect against irradiation both on the parent strain and the envA mutant.     

Physical mapping and expression of hybrid plasmids carrying chromosomal beta-lactamase genes of Escherichia coli K-12.

Hybrid plasmids carrying the ampC gene of Escherichia coli K-12 that codes for the chromosomal beta-lactamase were physically studied. The ampC gene was mapped to a deoxyribonucleic acid segment encompassing 1,370 base pairs. The mapping was facilitated by the isolation of a plasmid carrying an insertion of the transposable element gamma delta (gamma delta) close to ampC. The ampA1 mutation, which increases the expression of ampC by a factor of about 20, was localized to a 370-base pair segment of the 1,370-base pair deoxyribonucleic acid segment that contains the ampC gene. Using a minicell protein labeling system, it was seen that plasmids carrying either ampA+, ampC, or ampA1 and ampC coded for a 36,000-dalton protein which comigrated with purified chromosomal beta-lactamase. In cells carrying plasmids that bore the ampA1 allele, the production of this protein was greater. In addition, a protein with a slightly higher molecular weight (38,000) was expressed by both ampA+ ampC and ampA1 ampC plasmids in this protein labeling system. This protein might represent a precursor form of chromosomal beta-lactamasee. From E. coli K-12 strains carrying the ampA1 allele, second-step mutants were isolated that hyperproduced chromosomal beta-lactamase. By reciprocal recombination, plasmid derivatives were isolated that carried these mutations. Two second-step regulatory mutations mapped within the same 370-base pair region as ampA1. This piece of deoxyribonucleic acid therefore contains ampA, a control sequence region for ampC.     

Resistance of Escherichia coli to penicillins. VI. Purification and characterization of the chromosomally mediated penicillinase present in ampA-containing strains

The chromosomally mediated penicillinase present in three strains of Escherichia coli K-12 has been purified and characterized. Two of the strains carried the ampA gene and the third the wild-type allele. The purification involves release of the enzyme by spheroplast formation, dialysis, chromatography on sulfoethyl cellulose, and chromatography on hydroxylapatite. Enzyme from the two mutants appeared homogeneous in polyacrylamide gel electrophoresis. Enzyme from the wild-type strain gave two bands. Immunologically, the enzymes from all three strains were identical. Ultracentrifugation gave a homogeneous peak with a sedimentation coefficient of 3.4S. Gel filtration gave an estimated molecular weight of 29,000. The N-terminal amino acid residue was found to be alanine. Complete amino acid analysis showed a lack of cysteine. Ultraviolet spectra were recorded at three different pH values. The extinction coefficient at 280 nm is 21.0 for a 1% solution at pH 6.8. The optimal pH is 7.3. With enzyme from one of the resistant mutants, the following K(m) and turnover number values were obtained: for penicillin G, 12 mum and 2,080; for d-ampicillin, 6 mum and 83; for cephalosporin C, 217 mum and 18,400. The effect of different salts on the enzyme activity was tested. Under many conditions the enzyme was found to be unstable.     

Plasmid characterization and pulsed-field electrophoretic analysis demonstrate that ampicillin-resistant strains of Salmonella enteritidis phage type 6a are derived from Salm. enteritidis phage type 4

Plasmid incompatibility studies have demonstrated that strains of Salmonella enteritidis phage type (PT) 6a resistant to ampicillin possess a 36 megadalton incompatibility group (Inc) X plasmid coding for resistance to ampicillin which is capable of converting strains of Salm. enteritidis belonging to PTs 1 and 4 to PT 6a, and PT 8 to PT 13. However, pulsed-field gel electrophoresis (PFGE) has demonstrated that all clinical isolates of PT 6a have a characteristic Xba I pulsed-field profile which is distinct from that of PT 1 and which can only be differentiated from that of PT 4 by the presence of plasmid-associated fragments of less than 45 kb. It is concluded that ampicillin-resistant strains of Salm. enteritidis PT 6a are derived from strains of Salm. enteritidis PT 4 by acquisition of an Inc X ampicillin resistance plasmid.     

Effects of low concentrations of ampicillin in feed on the intestinal Escherichia coli of chicks

Ampicillin in low concentrations (1.7 and 5 g t^-1) was incorporated in the feed given to 1-d-old chicks for 2 weeks. This was sufficient to select, in the intestinal contents, resistant Escherichia coli strains for which the minimum inhibitory concentration of ampicillin was > 100 μg ml^-1. Different clones of E. coli were identified by their biotype, pattern of resistance to antibacterial agents and plasmid profile (designated P-P types). The experiment was repeated twice and the average proportion of ampicillin-resistant P-P types among 72 isolates of E. coli from chicks given feed containing 0, 1.7 and 5 g ampicillin t^-1 were 10%, 31% and 46% respectively.     

marA, a regulated locus which controls expression of chromosomal multiple antibiotic resistance in Escherichia coli.

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.     

Effect of bovine lactoferrin on the minimum inhibitory concentrations of ampicillin and trimethoprim-sulfamethoxazole for clinical Shigella spp. strains

Here, we determined the effect of bovine lactoferrin (bLF) on the minimum inhibitory concentration (MIC) of ampicillin and trimethoprim-sulfamethoxazole in Shigella . Using a microdilution method, the MIC was determined in the presence or absence of bovine lactoferrin (10 mg/mL) on 88 Shigella strains (56 Shigella flexneri , 15 Shigella boydii , 13 Shigella sonnei , and 4 Shigella dysenteriae ) previously isolated from peruvian children <2 years old. A fold change of 2 or more in MIC values was considered significant. For ampicillin, 67 (76%) strains were highly resistant; one-third of the strains (32%) showed a decrease in ampicillin MIC in the presence of LF. This was more typical of MIC values in less resistant strains. For 7 (8%) ampicillin-resistant strains, the decrease in the MIC resulted in the strains reaching the cutoff for susceptible in the presence of bLF. For trimethoprim-sulfamethoxazole, 93% of the isolates (n = 82) were highly resistant and only 4 isolates (5%) decreased their MIC in the presence of bLF. None of the trimethoprim-sulfamethoxazole resistant strains became susceptible in the presence of LF. The decrease in the MIC in the presence of bLF seems to depend on the mechanisms of action of each antibiotic. In vivo studies are needed to further evaluate bLF as a coadjuvant to antibiotic treatment of resistant Shigella.     

革兰氏阴性杆菌氟喹诺酮耐药株的实验研究

对８２株临床分离的氟喹诺酮药物（Ｆｌｕｏｒｏｑｕｉｎｏｌｏｎｅｓ,ＦＱＮＳ）耐药菌进行了多种抗生素敏感试验。结果显示８２株耐药菌对５种药物耐药率分别为：诺氟沙星９７．５６％、依诺沙星９６．３４％、罗美沙星９５．１２％、氧氟沙星９２．６８％、环丙沙星８７．８％。３２株菌对ＦＱＮＳ存在较普遍的交叉耐药现象,对氨苄青霉素、庆大霉素、丁胺卡那霉素及头孢菌素类的多重耐药率也较高。８２株菌中有４３株菌携带质粒（５２．４４％）。质粒消除及转化实验结果表明：质粒与ＦＱＮＳ耐药性无关。     

Mapping nonselectable genes of Escherichia coli by using transposon Tn10: location of a gene affecting pyruvate oxidase.

Mutants of Escherichia coli K-12 deficient in pyruvate oxidase were isolated by screening for the production of 14CO2 from [1-14C]pyruvate by the method of Tabor et al. (J. Bacteriol. 128:485-486, 1976). One of these lesions (designated poxA) decreased the pyruvate oxidase activity to 10 to 15% of the normal level but grew well. To map this nonselectable mutation, we isolated strains having transposon Tn10 inserted into the chromosome close to the poxA locus and mapped the transposon. These insertions were isolated by the following procedure: (i) pools of Tn10 insertions into the chromosomes of two different Hfr strains were prepared by transposition from a lambda::Tn10 vector; (ii) these Tn10-carrying strains were then mated with a poxA recipient strain, and tetracycline-resistant (Tetr) recombinants were selected; (iii) the Tetr recombinants were then screened for 14CO2 production from [1-14C]pyruvate. This method was shown to give a greater than 40-fold enrichment of insertions of Tn10 near the poxA gene as compared with transduction. Calculations indicate that a similar enrichment should be expected for other genes. The enrichment is due to the much greater map interval over which strong linkage between selected and unselected markers is found in conjugational crosses as compared with transductional crosses. The use of Hfr conjugative transfer allows isolation of transposon insertions closely linked to a nonselectable gene by scoring hundreds rather than thousands of colonies. Using a Tn10 insertion greater than 98% cotransduced with the poxA locus, we mapped the poxA gene on the E. coli genetic map. The poxA locus is located at 94 min, close to the psd locus. The clockwise gene order is ampA, poxA, psd, purA. The poxA mutation is recessive and appears to be a regulatory gene.     

Cloning of Pseudomonas aeruginosa algG, which controls alginate structure.

The biochemical mechanism by which alpha-L-guluronate (G) residues are incorporated into alginate by Pseudomonas aeruginosa is not understood. P. aeruginosa first synthesizes GDP-mannuronate, which is used to incorporate beta-D-mannuronate residues into the polymer. It is likely that the conversion of some beta-D-mannuronate residues to G occurs by the action of a C-5 epimerase at either the monomer (e.g., sugar-nucleotide) or the polymer level. This study describes the results of a molecular genetic approach to identify a gene involved in the formation or incorporation of G residues into alginate by P. aeruginosa. Mucoid P. aeruginosa FRD1 was chemically mutagenized, and mutants FRD462 and FRD465, which were incapable of incorporating G residues into alginate, were independently isolated. Assays using a G-specific alginate lyase from Klebsiella aerogenes and 1H-nuclear magnetic resonance analyses showed that G residues were absent in the alginates secreted by these mutants. 1H-nuclear magnetic resonance analyses also showed that alginate from wild-type P. aeruginosa contained no detectable blocks of G. The mutations responsible for defective incorporation of G residues into alginate in the mutants FRD462 and FRD465 were designated algG4 and algG7, respectively. Genetic mapping experiments revealed that algG was closely linked (greater than 90%) to argF, which lies at 34 min on the P. aeruginosa chromosome and is adjacent to a cluster of genes required for alginate biosynthesis. The clone pALG2, which contained 35 kilobases of P. aeruginosa DNA that included the algG and argF wild-type alleles, was identified from a P. aeruginosa gene bank by a screening method that involved gene replacement. A DNA fragment carrying algG was shown to complement algG4 and algG7 in trans. The algG gene was physically mapped on the alginate gene cluster by subcloning and Tn501 mutagenesis.