Effect of detergents on the chloramphenicol inactivation process by resistent bacteria

The effect of detergents, i. e. cationic, anionic, nonionic and polyelectrolytes of the cationic type on the efficacy of chloramphenicol against resistant strains of E. coli and Staph. aureus was studied. It was found that the detergent effect on inactivation of chloramphenicol by the bacterial resistant strains was inconsistent. The cationic detergents and in particular chlorhexidine had the most pronounced inhibitory effect. In subbacteriostatic concentrations they significantly suppressed inactivation of chloramphenicol in the cells of E. coli and Staph. aureus. The anionic detergents and polyelectrolytes of the cationic type in the above concentrations were effective only with respect to Staph. aureus. It is noted that the detergents increased the activity of chloramphenicol against E. coli and Staph. aureus.     

Effects of chronic triclosan exposure upon the antimicrobial susceptibility of 40 ex-situ environmental and human isolates

BACKGROUND: Triclosan (TCS) exposure of Escherichia coli selects for tolerant clones, mutated in their enoyl-acyl carrier protein reductase (FabI). It has been inferred that this phenomenon is widespread amongst bacterial genera and might be associated with resistance to third party agents. METHODS: Ex-situ, low passage isolates of enteric, human axilla, human oral origin and bacteria isolated from a domestic drain, together with selected type cultures were exposed to escalating concentrations of TCS over 10 passages using a gradient plate technique. One fresh faecal isolate of E. coli was included as a positive control. TCS susceptibility was determined for all strains before and after exposure, whilst enteric isolates were additionally assessed for susceptibility towards chlorhexidine, tetracycline, chloramphenicol, nalidixic acid and ciprofloxacin, and the oral isolates towards chlorhexidine, tetracycline and metronidazole. RESULTS: Triclosan exposure of E. coli markedly decreased TCS susceptibility. TCS susceptibility also decreased for Klebsiella oxytoca, Aranicola proteolyticus and Stenotrophomonas maltophilia. Susceptibility of the remaining 35 strains to TCS and the other test agents remained unchanged. CONCLUSIONS: These data suggest that selection for high level resistance by TCS exposure is not widespread and appears to be confined to certain enteric bacteria, especially E. coli. Change in TCS susceptibility did not affect susceptibility towards chemically unrelated antimicrobials. SIGNIFICANCE AND IMPACT: Acquired high-level TCS resistance is not a widespread phenomenon.     

Inactivation of chloramphenicol and florfenicol by a novel chloramphenicol hydrolase

Chloramphenicol and florfenicol are broad-spectrum antibiotics. Although the bacterial resistance mechanisms to these antibiotics have been well documented, hydrolysis of these antibiotics has not been reported in detail. This study reports the hydrolysis of these two antibiotics by a specific hydrolase that is encoded by a gene identified from a soil metagenome. Hydrolysis of chloramphenicol has been recognized in cell extracts of Escherichia coli expressing a chloramphenicol acetate esterase gene, estDL136. A hydrolysate of chloramphenicol was identified as p-nitrophenylserinol by liquid chromatography-mass spectroscopy and proton nuclear magnetic resonance spectroscopy. The hydrolysis of these antibiotics suggested a promiscuous amidase activity of EstDL136. When estDL136 was expressed in E. coli, EstDL136 conferred resistance to both chloramphenicol and florfenicol on E. coli, due to their inactivation. In addition, E. coli carrying estDL136 deactivated florfenicol faster than it deactivated chloramphenicol, suggesting that EstDL136 hydrolyzes florfenicol more efficiently than it hydrolyzes chloramphenicol. The nucleotide sequences flanking estDL136 encode proteins such as amidohydrolase, dehydrogenase/reductase, major facilitator transporter, esterase, and oxidase. The most closely related genes are found in the bacterial family Sphingomonadaceae, which contains many bioremediation-related strains. Whether the gene cluster with estDL136 in E. coli is involved in further chloramphenicol degradation was not clear in this study. While acetyltransferases for chloramphenicol resistance and drug exporters for chloramphenicol or florfenicol resistance are often detected in numerous microbes, this is the first report of enzymatic hydrolysis of florfenicol resulting in inactivation of the antibiotic.     

Genetic transformation of Streptococcus pneumoniae by DNA cloned into the single-stranded bacteriophage f1.

A Staphylococcus aureus plasmid derivative, pFB9, coding for erythromycin and chloramphenicol resistance was cloned into the filamentous Escherichia coli phage f1. Recombinant phage-plasmid hybrids, designated plasmids, were isolated from E. coli and purified by transformation into Streptococcus pneumoniae. Single-stranded DNA was prepared from E. coli cells infected with two different plasmids, fBB101 and fBB103. Introduction of fully or partially single-stranded DNA into Streptococcus pneumoniae was studied, using a recipient strain containing an inducible resident plasmid. Such a strain could rescue the donor DNA marker. Under these marker rescue conditions, single-stranded fBB101 DNA gave a 1% transformation frequency, whereas the double-stranded form gave about a 31% frequency. Transformation of single-stranded fBB101 DNA was inhibited by competing double-stranded DNA and vice versa, indicating that single-stranded DNA interacts with the pneumococcus via the same binding site as used by double-stranded DNA. Heteroduplexed DNA containing the marker within a 70- or 800-base single-stranded region showed only slightly greater transforming activity than pure single-stranded DNA. In the absence of marker rescue, both strands of such imperfectly heteroduplexed DNA demonstrated transforming activity. Pure single-stranded DNA demonstrated low but significant transforming activity into a plasmid-free recipient pneumococcus.     

Overproduction from a cellulase gene with a high guanosine-plus-cytosine content in Escherichia coli.

A recombinant exoglucanase was expressed in Escherichia coli to a level that exceeded 20% of total cellular protein. To obtain this level of overproduction, the exoglucanase gene coding sequence was fused to a synthetic ribosome-binding site, an initiating ATG, and placed under the control of the leftward promoter of bacteriophage lambda contained on the runaway replication plasmid vector pCP3 (E. Remaut, H. Tsao, and W. Fiers, Gene 22:103-113, 1983). With the exception of an inserted asparagine adjacent to the initiating ATG, the highly expressed exoglucanase is identical to the native exoglucanase. The overproduced exoglucanase can be isolated easily in an enriched form as insoluble aggregates, and exoglucanase activity can be recovered by solubilization of the aggregates in 6 M urea or 5 M guanidine hydrochloride. Since the codon usage of the exoglucanase gene is so markedly different from that of E. coli genes, the overproduction of the exoglucanase in E. coli indicates that codon usage may not be a major barrier to heterospecific gene expression in this organism.     

Overproduction from a cellulase gene with a high guanosine-plus-cytosine content in Escherichia coli

A recombinant exoglucanase was expressed in Escherichia coli to a level that exceeded 20% of total cellular protein. To obtain this level of overproduction, the exoglucanase gene coding sequence was fused to a synthetic ribosome-binding site, an initiating ATG, and placed under the control of the leftward promoter of bacteriophage lambda contained on the runaway replication plasmid vector pCP3 (E. Remaut, H. Tsao, and W. Fiers, Gene 22:103-113, 1983). With the exception of an inserted asparagine adjacent to the initiating ATG, the highly expressed exoglucanase is identical to the native exoglucanase. The overproduced exoglucanase can be isolated easily in an enriched form as insoluble aggregates, and exoglucanase activity can be recovered by solubilization of the aggregates in 6 M urea or 5 M guanidine hydrochloride. Since the codon usage of the exoglucanase gene is so markedly different from that of E. coli genes, the overproduction of the exoglucanase in E. coli indicates that codon usage may not be a major barrier to heterospecific gene expression in this organism.     

Overproduction of spinach betaine aldehyde dehydrogenase in Escherichia coli. Structural and functional properties of wild-type, mutants and E. coli enzymes.

Betaine aldehyde dehydrogenase (BADH) catalyzes the last step in the synthesis of the osmoprotectant glycine betaine from choline. Although betaine aldehyde has been thought to be a specific substrate for BADH, recent studies have shown that human and sugar beet BADHs also catalyze the oxidation of omega-aminoaldehydes. To characterize the kinetic and stability properties of spinach BADH, five kinds of expression vectors encoding full length, mature, E103Q, E103K, and chimera BADHs were constructed. These enzymes together with Escherichia coli BADH were expressed in E. coli and purified. The affinities for betaine aldehyde were similar in the spinach and E. coli BADHs, whereas those for omega-aminoaldehydes were higher in spinach BADH than in E. coli BADH. A chimera BADH in which part of the Rossmann type fold in the spinach BADH was replaced with that of E. coli BADH, showed properties which resembled spinach BADH more than E. coli BADH. The spinach E103K mutant was almost inactive, whereas the E103Q mutant showed a similar activity for the oxidation of betaine aldehyde to that of wild type BADH, but a lower affinity for omega-aminoaldehydes. All spinach BADHs were dimers whereas E. coli BADH was a tetramer. E. coli BADH was more stable at high temperature than spinach BADHs. The E103Q mutant was most labile to high temperature. These properties are discussed in relation to the structure of spinach BADH.     

Characterization of Chloramphenicol Acetyltransferase from Chloramphenicol-resistant Staphylococcus aureus

Chloramphenicol-resistant strains of Staphylococcus aureus contain an inducible enzyme which inactivates chloramphenicol by acetylation in the presence of acetyl coenzyme A. The products of acetylation are chromatographically indistinguishable from those obtained with chloramphenicol-resistant Escherichia coli harboring an R factor. The kinetics of induction of chloramphenicol acetyltransferase are complicated by the inducer's effect on protein biosynthesis and its fate as chloramphenicol 3-acetate, which is not an inducer of the enzyme. The E. coli and S. aureus enzymes have been compared, with the conclusion that they are identical with respect to molecular weight (approximately 78,000) and pH optimum (7.8), but differ with respect to heat stability, substrate affinity, electrophoretic mobility, and immunological reactivity. Antiserum prepared against enzyme from E. coli contains precipitating antibody, which inactivates the E. coli enzyme, but neither precipitates nor neutralizes the activity of S. aureus enzyme.     

Differential actions of chlorhexidine on the cell wall of Bacillus subtilis and Escherichia coli

Chlorhexidine is a chlorinated phenolic disinfectant used commonly in mouthwash for its action against bacteria. However, a comparative study of the action of chlorhexidine on the cell morphology of gram-positive and gram-negative bacteria is lacking. In this study, the actions of chlorhexidine on the cell morphology were identified with the aids of electron microscopy. After exposure to chlorhexidine, numerous spots of indentation on the cell wall were found in both Bacillus subtilis and Escherichia coli. The number of indentation spots increased with time of incubation and increasing chlorhexidine concentration. Interestingly, the dented spots found in B. subtilis appeared mainly at the hemispherical caps of the cells, while in E. coli the dented spots were found all over the cells. After being exposed to chlorhexidine for a prolonged period, leakage of cellular contents and subsequent ghost cells were observed, especially from B subtilis. By using 2-D gel/MS-MS analysis, five proteins related to purine nucleoside interconversion and metabolism were preferentially induced in the cell wall of E. coli, while three proteins related to stress response and four others in amino acid biosynthesis were up-regulated in the cell wall materials of B. subtilis. The localized morphological damages together with the biochemical and protein analysis of the chlorhexidine-treated cells suggest that chlorhexidine may act on the differentially distributed lipids in the cell membranes/wall of B. subtilis and E. coli.     

Modification of in vitro metabolism of T-2 toxin by esterase inhibitors.

A shuttle vector that can replicate in both Streptococcus spp. and Escherichia coli has been constructed by joining the E. coli plasmid pACYC184 (chloramphenicol and tetracycline resistance) to the streptococcal plasmid pGB305 (erythromycin resistance). The resulting chimeric plasmid is designated pSA3 (chloramphenicol, erythromycin, and tetracycline resistance) and has seven unique restriction sites: EcoRI, EcoRV, BamHI, SalI, XbaI, NruI, and SphI. Molecular cloning into the EcoRI or EcoRV site results in inactivation of chloramphenicol resistance, and cloning into the BamHI, SalI, or SphI site results in inactivation of tetracycline resistance in E. coli. pSA3 was transformed and was stable in Streptococcus sanguis and Streptococcus mutans in the presence of erythromycin. We have used pSA3 to construct a library of the S. mutans GS5 genome in E. coli, and expression of surface antigens in this heterologous host has been confirmed with S. mutans antiserum. A previously cloned determinant that specifies streptokinase was subcloned into pSA3, and this recombinant plasmid was stable in the presence of a selective pressure and expressed streptokinase activity in E. coli, S. sanguis (Challis), and S. mutans.     

Heterologous gene expression in Bacteroides fragilis.

Bacteroides fragilis and other gastrointestinal tract Bacteroides are unusual gram-negative eubacteria in that genes from other gram-negative eubacteria are not expressed when introduced into these organisms. To analyze gene expression in Bacteroides, expression vector and promoter probe (detection) vector systems were developed. The essential feature of the expression vector was the incorporation of a Bacteroides insertion sequence element, IS4351, which possesses promoter activity directed outward from its ends. Genes inserted into the multiple cloning site downstream from an IS4351 DNA fragment were readily expressed in B. fragilis. The chloramphenicol acetyltransferase (cat) structural gene from Tn9 was tested and conferred chloramphenicol resistance on B. fragilis. Both chloramphenicol resistance and CAT activity were shown to be dependent on the IS4351 promoters. Similar results were obtained with the Escherichia coli beta-glucuronidase gene (uidA) but activity was just 30% of the levels seen with cat. Two tetracycline resistance determinants, tetM from Streptococcus agalactiae and tetC from E. coli, also were examined. tetC did not result in detectable tetracycline resistance but the gram-positive tetM gene conferred high-level resistance to tetracycline and minocycline in Bacteroides hosts. Based on the cat results, promoter probe vectors containing the promoterless cat gene were constructed. These vectors were used to clone random B. fragilis promoters from partial genomic libraries and the recombinants displayed a range of CAT activities and chloramphenicol MICs in B. fragilis hosts. In addition, known E. coli promoters (Ptet, Ptac, Ptrc, Psyn, and P1P2rrnB) were tested for activity in B. fragilis. No chloramphenicol resistance or CAT activity was observed in B. fragilis with these promoters.     

禽源致病性大肠埃希菌的耐药性与中草药有效成分的抑菌活性测定

以28株合肥地区禽源致病性大肠埃希菌为实验材料,采用K-B纸片琼脂扩散法检测禽源致病性大肠埃希菌的耐药情况。同时采用平板打孔法测定盐酸小檗碱、绿原酸、靛玉红和丹参酮ⅡA 4种中草药有效成分的抑菌活性。结果表明,28株禽源致病性大肠埃希菌对17种抗菌药物均呈现不同程度的耐药性,对β-内酰胺类、氨基糖苷类、四环素类和喹诺酮类抗菌药物的耐药率分别介于0%～92.86%、14.29%～50.00%、78.57%～100%和57.14%～71.43%。中草药有效成分盐酸小檗碱和丹参酮ⅡA对大肠埃希菌具有较好的抑制活性,抑菌率分别为92.86%（26/28）和89.29%（25/28）。     

Temperature-sensitive Chloramphenicol Acetyltransferase from Escherichia coli Carrying Mutant R Factors

Bacteria carrying temperature-sensitive mutant R factors for chloramphenicol resistance were isolated. In the presence of chloramphenicol, these bacteria grew at 34 C but not at 43 C. The mutations in the chloramphenicol resistance gene of the R factors affected neither the resistance of the bacteria to dihydrostreptomycin and tetracycline nor the stability of the R factors at 43 C. The chloramphenicol acetyltransferase obtained from Escherichia coli K-12 carrying the mutant R factors was heat-labile as compared with that from a strain carrying the wild-type R factor. We could not find chloramphenicol acetyltransferase activity in 17 chloramphenicol-sensitive and 5 -resistant strains (selected in vitro) of E. coli examined. The results strongly suggest that the chloramphenicol resistance gene of the R factors is the structural gene of the chloramphenicol acetyltransferase rather than the genome controlling the expression of a chromosomal determinant for the enzyme. Furthermore, the studies confirm that the existence of the chloramphenicol acetyltransferase is the primary cause of chloramphenicol resistance of bacteria carrying the R factor. Both the enzyme activity producing the monoacetyl derivative from chloramphenicol and the subsequent formation of the diacetate from the monoacetyl product were heat-labile to the same degree. The results suggest that only one enzyme participates in two steps of chloramphenicol acetylation.     

Some properties of two erythromycin-dependent strains of Escherichia coli

Strains of Escherichia coli can be isolated that require erythromycin for growth. With one strain, AM, a range of antibiotics, including chloramphenicol, tetracycline, spectinomycin, kasugamycin and rifampicin, will substitute for erythromycin on solid and in liquid media; nalidixic acid supports growth in liquid but not on solid media. With a second strain, 103, chloramphenicol, tetracycline and spectinomycin support growth in liquid media but on solid medium only chloramphenicol substitutes for erythromycin. In media of higher than normal ionic strength, strain AM, but not strain 103, can grow in the absence of antibiotics. Possible reasons for these complex phenotypes are discussed.     

Role of protein synthesis in the survival of carbon-starved Escherichia coli K-12.

In a typical Escherichia coli K-12 culture starved for glucose, 50% of the cells lose viability in ca. 6 days (Reeve et al., J. Bacteriol. 157:758-763, 1984). Inhibition of protein synthesis by chloramphenicol resulted in a more rapid loss of viability in glucose-starved E. coli K-12 cultures. The more chloramphenicol added (i.e., the more protein synthesis was inhibited) and the earlier during starvation it was added, the greater was its effect on culture viability. Chloramphenicol was found to have the same effect on a relA strain as on an isogenic relA+ strain of E. coli. Addition of the amino acid analogs S-2-aminoethylcysteine, 7-azatryptophan, and p-fluorophenylalanine to carbon-starved cultures to induce synthesis of abnormal proteins had an effect on viability similar to that observed when 50 micrograms of chloramphenicol per ml was added at zero time for starvation. Both chloramphenicol and the amino acid analogs had delayed effects on viability, compared with their effects on synthesis of normal proteins. The need for protein synthesis did not arise from cryptic growth, since no cryptic growth of the starving cells was observed under the conditions used. From these and previous results obtained from work with peptidase-deficient mutants of E. coli K-12 and Salmonella typhimurium LT2 (Reeve et al., J. Bacteriol. 157:758-763, 1984), we concluded that a number of survival-related proteins are synthesized by E. coli K-12 cells as a response to carbon starvation. These proteins are largely synthesized during the early hours of starvation, but their continued activity is required for long-term survival.     

Low level of cross-resistance between triclosan and antibiotics in Escherichia coli K-12 and E. coli O55 compared to E. coli O157

Misuse of biocides has encouraged the emergence of resistance and cross-resistance in certain strains. This study investigated resistance of triclosan-adapted Escherichia coli K-12 and E. coli O55 to antimicrobial agents and compared these to E. coli O157:H7. Cross-resistance in E. coli K-12 and E. coli O55 was observed however to a lesser extent than in E. coli O157:H7. Triclosan-adapted E. coli K-12 demonstrated cross-resistance to chloramphenicol, whereas triclosan-adapted E. coli O55 exhibited resistance to trimethoprim. In comparison, E. coli O157:H7 was resistant to chloramphenicol, tetracycline, amoxicillin, amoxicillin/clavulanic acid, trimethoprim, benzalkonium chloride and chlorohexidine suggesting strain specific rather than general resistance mechanisms.     

The extended promoters for two outer membrane lipoprotein genes of Borrelia spp. uniquely include a T-rich region

OspA and B proteins of Borrelia burgdorferi and Vmp proteins of Borrelia hermsii are abundant outer membrane lipoproteins, whose expression varies with the environment. The genes for these proteins have the '-35' and '-10' elements of a sigma70-type promoter. Deletions of the promoters for these genes were analysed with a chloramphenicol acetyltransferase (CAT) reporter gene and plasmid constructs that were stably maintained in Escherichia coli or transiently transfected into B. burgdorferi. Reporter expression was measured as susceptibility of transformed E. coli cells to chloramphenicol and the CAT activity of E. coli and B. burgdorferi lysates in vitro. Presence of the '-10' element was essential for full activity in both B. burgdorferi and E. coli. Upstream of the '-35' elements of the ospAB and vmp promoters were tracts with Ts in 16 of 20 positions for B. burgdorferi and 18 of 20 positions for B. hermsii. Deletion of the T-rich region from the ospAB or vmp promoter caused a greater reduction of CAT activity in B. burgdorferi than in E. coli. The findings indicate that ospAB and vmp promoters are extended promoters with two parts: (i) a core region containing typical '-35' and '-10' elements and (ii) a unique T-rich region.     

The effect of alpha-lactalbumin and beta-lactoglobulin hydrolysates on the metabolic activity of Escherichia coli JM103

Bovine milk proteins alpha-lactalbumin (alpha-la) and beta-lactoglobulin (beta-lg) were hydrolysed with seven different proteolytic enzymes, and the effect of various hydrolysates on a genetically modified luminous Escherichia coli JM103 was tested in vitro with a bioluminescence assay for bacterial growth and metabolism. Undigested proteins did not inhibit the activity of tested E. coli JM103 at a concentration as high as 0.1 g ml-1. At the same concentrations, alpha-la hydrolysed with pepsin or trypsin and beta-lg hydrolysed with alcalase, pepsin or trypsin, showed a lower metabolic activity during the first 8 h of growth. The activity of E. coli JM103 in the presence of 25 mg ml-1 alpha-la or beta-lg hydrolysed with pepsin and trypsin was only 21% of the control after incubation for 6 h. The preliminary results indicated that ultrafiltration through 10 kDa and 1 kDa molecular mass cut-off membranes may be used to enrich bacteriostatic properties.