Chloramphenicol-resistant Mutants Research Articles

TCA and SSRI Antidepressants Exert Selection Pressure for Efflux-Dependent Antibiotic Resistance Mechanisms in Escherichia coli.

Microbial diversity is reduced in the gut microbiota of animals and humans treated with selective serotonin reuptake inhibitors (SSRIs) and tricyclic antidepressants (TCAs). The mechanisms driving the changes in microbial composition, while largely unknown, is critical to understand considering that the gut microbiota plays important roles in drug metabolism and brain function. Using Escherichia coli, we show that the SSRI fluoxetine and the TCA amitriptyline exert strong selection pressure for enhanced efflux activity of the AcrAB-TolC pump, a member of the resistance-nodulation-cell division (RND) superfamily of transporters. Sequencing spontaneous fluoxetine- and amitriptyline-resistant mutants revealed mutations in marR and lon, negative regulators of AcrAB-TolC expression. In line with the broad specificity of AcrAB-TolC pumps these mutants conferred resistance to several classes of antibiotics. We show that the converse also occurs, as spontaneous chloramphenicol-resistant mutants displayed cross-resistance to SSRIs and TCAs. Chemical-genomic screens identified deletions in marR and lon, confirming the results observed for the spontaneous resistant mutants. In addition, deletions in 35 genes with no known role in drug resistance were identified that conferred cross-resistance to antibiotics and several displayed enhanced efflux activities. These results indicate that combinations of specific antidepressants and antibiotics may have important effects when both are used simultaneously or successively as they can impose selection for common mechanisms of resistance. Our work suggests that selection for enhanced efflux activities is an important factor to consider in understanding the microbial diversity changes associated with antidepressant treatments. IMPORTANCE Antidepressants are prescribed broadly for psychiatric conditions to alter neuronal levels of synaptic neurotransmitters such as serotonin and norepinephrine. Two categories of antidepressants are selective serotonin reuptake inhibitors (SSRIs) and tricyclic antidepressants (TCAs); both are among the most prescribed drugs in the United States. While it is well-established that antidepressants inhibit reuptake of neurotransmitters there is evidence that they also impact microbial diversity in the gastrointestinal tract. However, the mechanisms and therefore biological and clinical effects remain obscure. We demonstrate antidepressants may influence microbial diversity through strong selection for mutant bacteria with increased AcrAB-TolC activity, an efflux pump that removes antibiotics from cells. Furthermore, we identify a new group of genes that contribute to cross-resistance between antidepressants and antibiotics, several act by regulating efflux activity, underscoring overlapping mechanisms. Overall, this work provides new insights into bacterial responses to antidepressants important for understanding antidepressant treatment effects.

Development and characterisation of highly antibiotic resistant Bartonella bacilliformis mutants.

The objective was to develop and characterise in vitro Bartonella bacilliformis antibiotic resistant mutants. Three B. bacilliformis strains were plated 35 or 40 times with azithromycin, chloramphenicol, ciprofloxacin or rifampicin discs. Resistance-stability was assessed performing 5 serial passages without antibiotic pressure. MICs were determined with/without Phe-Arg-β-Napthylamide and artesunate. Target alterations were screened in the 23S rRNA, rplD, rplV, gyrA, gyrB, parC, parE and rpoB genes. Chloramphenicol and ciprofloxacin resistance were the most difficult and easiest (>37.3 and 10.6 passages) to be selected, respectively. All mutants but one selected with chloramphenicol achieved high resistance levels. All rifampicin, one azithromycin and one ciprofloxacin mutants did not totally revert when cultured without antibiotic pressure. Azithromycin resistance was related to L4 substitutions Gln-66 → Lys or Gly-70 → Arg; L4 deletion Δ62–65 (Lys-Met-Tyr-Lys) or L22 insertion 83::Val-Ser-Glu-Ala-His-Val-Gly-Lys-Ser; in two chloramphenicol-resistant mutants the 23S rRNA mutation G2372A was detected. GyrA Ala-91 → Val and Asp-95 → Gly and GyrB Glu474 → Lys were detected in ciprofloxacin-resistant mutants. RpoB substitutions Gln-527 → Arg, His-540 → Tyr and Ser-545 → Phe plus Ser-588 → Tyr were detected in rifampicin-resistant mutants. In 5 mutants the effect of efflux pumps on resistance was observed. Antibiotic resistance was mainly related to target mutations and overexpression of efflux pumps, which might underlie microbiological failures during treatments.

Untargeted metabolomic profiling of amphenicol-resistant Campylobacter jejuni by ultra-high-performance liquid chromatography-mass spectrometry.

Campylobacter jejuni, an important foodborne microorganism, poses severe and emergent threats to human health as antibiotic resistance becomes increasingly prevalent. The mechanisms of drug resistance are hard to decipher, and little is known at the metabolic level. Here we apply metabolomic profiling to discover metabolic changes associated with amphenicol (chloramphenicol and florfenicol) resistance mutations of Campylobacter jejuni. An optimized sample preparation method was combined with ultra-high-performance liquid chromatography-time-of-flight mass spectrometry (UHPLC-TOF/MS) and pattern recognition for the analysis of small-molecule biomarkers of drug resistance. UHPLC-triple quadrupole MS operated in multiple reaction monitoring mode was used for quantitative analysis of metabolic features from UHPLC-TOF/MS profiling. Up to 41 differential metabolites involved in glycerophospholipid metabolism, sphingolipid metabolism, and fatty acid metabolism were observed in a chloramphenicol-resistant mutant strain of Campylobacter jejuni. A panel of 40 features was identified in florfenicol-resistant mutants, demonstrating changes in glycerophospholipid metabolism, sphingolipid metabolism, and tryptophan metabolism. This study shows that the UHPLC-MS-based metabolomics platform is a promising and valuable tool to generate new insights into the drug-resistant mechanism of Campylobacter jejuni.

Durable and broad-spectrum disease protection measure against airborne phytopathogenic fungi by using the detachment action of gelatinolytic bacteria

We had previously obtained collagenolytic/gelatinolytic bacteria, which degrade the fungal extracellular matrix, to establish a novel biological control measure that inhibits germling adhesion of airborne phytopathogenic fungi on the host plant surface. By using barley-Magnaporthe oryzae pathosystem, Chryseobacterium sp. was most effective biocontrol agents as tested. The selected bacteria were evaluated for durable disease protection against M. oryzae on barley leaves by using chloramphenicol-resistant mutants. Chryseobacterium sp. from the soil was less likely to settle on leaf surfaces. Therefore, we tried to manipulate Chryseobacterium sp. to inhabit the leaf’s surface. The gelatin supplementation dramatically improved the settlement of gelatinolytic bacteria Chryseobacterium sp. from the soil, and the disease protection effect lasted for more than 2weeks on barley. Moreover, exploitation of Chryseobacterium sp. for disease protection was extended against other airborne pathogens, Alternaria alternata Japanese pear pathotype on Japanese pear and Colletotrichum orbiculare on cucumber.

High incidence of multiple antibiotic resistant cells in cultures of in enterohemorrhagic Escherichia coli O157:H7

The spontaneous incidence of chloramphenicol (Cam) resistant mutant bacteria is at least ten-fold higher in cultures of enterohemorrhagic Escherichia coli O157:H7 strain EDL933 than in E. coli K-12. It is at least 100-fold higher in the dam (DNA adenine methyltransferase) derivative of EDL933, compared to the dam strain of E. coli K-12, thereby preventing the use of Cam resistance as a marker in gene replacement technology. Genome sequencing of Cam-resistant isolates of EDL933 and its dam derivatives showed that the marR (multiple antibiotic resistance) gene was mutated in every case but not in the Cam-sensitive parental strains. As expected from mutation in the marR gene, the Cam-resistant bacteria were also found to be resistant to tetracycline and nalidixic acid. The marR gene in strain EDL933 is annotated as a shorter open reading frame than that in E. coli K-12 but the longer marR+ open reading frame was more efficient at complementing the marR antibiotic-resistance phenotype of strain EDL933. Beta-lactamase-tolerant derivatives were present at frequencies 10–100 times greater in cultures of marR derivatives of strain EDL933 than the parent strain. Spontaneous mutation frequency to rifampicin, spectinomycin and streptomycin resistance was the same in E. coli O157:H7 and E. coli K-12 strains.

Production of a chloramphenicol-resistant mutant of Lysinibacillus sphaericus by solid state fermentation

A highly active mosquitocidal mutant of Lysinibacillus sphaericus Ahmed 2362, namely, UCR-146, was efficiently produced on cottonseed meal (CSM) medium, using sand as a carrier under solid state fermentation (SSF). The optimum CSM concentration for the highest sporulation and toxin formation was 12%. The maximum toxicity of the tested organism against second instar larvae of Culex pipiens was obtained at 25% moisture content, initial pH 6–7, 1% sodium acetate, 18.9×106 CFU/g inoculum and 6 days incubation period at 30°C. Pilot scale production of UCR-146 under the optimum SSF conditions was assessed in aluminium trays. Spore count, mortality of larvae and LC50 of the final product were 5.5×1010 CFU/g, 72% at 1 part per million (PPM) and 0.54 PPM, respectively. These results were comparable with those obtained from bench-scale production (in flasks). The cost of 1 kg of this bio-larvicide was estimated at US $0.34.

Rapid construction of Campylobacter jejuni deletion mutants

To develop a novel method for rapid construction of Campylobacter jejuni deletion mutants. We used overlapping extension PCR protocol to amplify a target sequence region of Camp. jejuni genomic DNA in which an internal fragment, Cj0618 coding sequence, was replaced by a chloramphenicol resistance cassette. After the resulting PCR product was introduced into electrocompetent Camp. jejuni 81-176, chloramphenicol-resistant mutants in which the wild type allele has been replaced by the deletion cassette were selected. DNA sequencing confirmed precise deletion in the Cj0618 gene. As expected from the previously reported role of Cj0618 in chick colonization, the resulting deletion mutant showed a caecal colonization defect in chick infection. This method can be used for rapid construction of Camp. jejuni deletion mutants. The use of this method should facilitate functional characterization of various Camp. jejuni genes.

Production of the cryptic EefABC efflux pump in Enterobacter aerogenes chloramphenicol-resistant mutants

AcrAB-TolC is the major tripartite multidrug efflux pump in Enterobacter aerogenes while EefABC is a cryptic efflux system. This study was conducted to identify and characterize E. aerogenes mutants producing the EefABC efflux pump. Four spontaneous chloramphenicol-resistant (CMR) mutants were isolated. The expression level of the eefABC promoter and the production of the EefA and B proteins were analysed in the mutants. Antibiotic susceptibilities were compared for wild-type and mutant strains. Efflux activity was investigated using an efflux pump inhibitor. The activation of the eefABC promoter was detected in four CMR mutants. These mutants showed increased resistance to erythromycin and ticarcillin, but not to fluoroquinolones, ketolides and detergents. Two additional efflux proteins were detected in the mutants. The CMR mutants bear no mutation in hns, which encodes a repressor of eefABC. No alteration of porin expression, a phenotype observed in marA or ramA multidrug-resistant mutants, was detected in the mutants. These observations suggest that eefABC activation can occur in vitro independently of the H-NS, MarA or RamA global regulators.

Intrinsic Mechanism Decreases Susceptibility ofEscherichia coli O157:H7 to Multiple Antibiotics

Chloramphenicol-resistant mutants ofEscherichia coli O157:H7 (n = 52) demonstrated multiple resistance to tetracycline, nalidixic acid, and ciprofloxacin. Approximately half of the mutants approached the clinical level of resistance to the antibiotic tetracycline. Mutants demonstrated a wide range of sensitivity to each antibiotic; for tetracycline, some isolates demonstrated a twofold increase, whereas others demonstrated a 15-fold increase in resistance. Continuous exposure to chloramphenicol did not affect the growth of mutants, suggesting that mutation does not have a negative effect on cell survival. Complementation experiments with a functional marR restored antibiotic susceptibility of selected mutants to levels similar to wild-type strains, suggesting that mar mutation was responsible for the decrease in sensitivity. The multiple antibiotic resistance (mar) operon is a global regulator controlling intrinsic resistance toward structurally and functionally unrelated antibiotics and other noxious agents. Antimicrobial use in both human and agricultural practice should be administered in a manner to prevent selection of resistant mutants.

Deletion of the hmc operon of Desulfovibrio vulgaris subsp. vulgaris Hildenborough hampers hydrogen metabolism and low-redox-potential niche establishment.

The hmc operon of Desulfovibrio vulgaris subsp. vulgaris Hildenborough encodes a transmembrane redox protein complex (the Hmc complex) that has been proposed to catalyze electron transport linking periplasmic hydrogen oxidation to cytoplasmic sulfate reduction. We have replaced a 5-kb DNA fragment containing most of the hmc operon by the cat gene. The resulting chloramphenicol-resistant mutant D. vulgaris H801 grows normally when lactate or pyruvate serve as electron donors for sulfate reduction. Growth with hydrogen as electron donor for sulfate reduction (acetate and CO2 as the carbon source) is impaired. These results confirm the importance of the Hmc complex in electron transport from hydrogen to sulfate. Mutant H801 is also deficient in low-redox-potential niche establishment. On plates, colony development takes 14 days longer than colony development of the wild-type strain, when the cells use hydrogen as the electron donor. This result suggests that, in addition to transmembrane electron transport from hydrogen to sulfate, the redox reactions catalyzed by the Hmc complex are crucial in establishment of the required low-redox-potential niche that allows single cells to grow into colonies.

Dynamics of serine/threonine protein kinase activity during the growth of the wild-typeStreptomyces avermitilis strain and its chloramphenicol-resistant mutant

The dynamics of serine/threonine protein kinase activity during the growth of the wild-type Streptomyces avermitilis strain and its chloramphenicol-resistant (Cmlr) pleiotropic mutant with an enhanced production of avermectins was studied by measuring the transfer of radiolabeled phosphate from [gamma-32P]ATP to the serine and threonine residues of proteins in cell-free extracts. In both of the strains studied, radiolabeled phosphate was found to incorporate into polypeptides with molecular masses of 32, 35, 41, 68, 75, 79, 83, and 137 kDa; however, the degree and the dynamics of phosphorylation of particular peptides were different in these strains. The differences revealed could not be accounted for by the interference of ATPases or phosphoprotein phosphatases. The data obtained may be interpreted as evidence that Cmlr mutation activates the protein kinase signalling system of S. avermitilis cells in the early stationary growth phase and thus enhances the production of avermectins and leads to some other physiological changes in the mutant strain.

Novel point mutations in mitochondrial 16S rRNA gene of Chinese hamster cells.

To know the nature and mechanisms of spontaneous mutations in mitochondrial DNA (mtDNA), we determined, by direct cycle sequencing, the nucleotide sequence of the 3' terminal region of the mitochondrial 16S rRNA gene from chloramphenicol-resistant (CAP-R) mutants isolated in Chinese hamster V79 cells. Four different base substitutions were identified in common for the six CAP-R mutants. All mutations were heteroplasmic. One A to G transition was mapped at a site within the putative peptidyl transferase domain, the target region for chloramphenicol, and one G to A transition and two T to G transversions were located within the two different segments which form the stems of the hairpin loop structures attached to this key domain in the predicted secondary structure of 16S rRNA. The mutations detected in this study do not map to the same sites where CAP-R mutations were found previously in mammalian cells. Allele specific-PCR analyses revealed that all four mutations occurred on a single mutant-DNA molecule, but not on several ones independently. Together with the other previous reports, our data suggest that spontaneous mtDNA mutations may not be caused exclusively by oxidative DNA damage at least in 16S rRNA gene.

Large-scale identification of virulence genes from Streptococcus pneumoniae.

Streptococcus pneumoniae is the major cause of bacterial pneumonia, and it is also responsible for otitis media and meningitis in children. Apart from the capsule, the virulence factors of this pathogen are not completely understood. Recent technical advances in the field of bacterial pathogenesis (in vivo expression technology and signature-tagged mutagenesis [STM]) have allowed a large-scale identification of virulence genes. We have adapted to S. pneumoniae the STM technique, originally used for the discovery of Salmonella genes involved in pathogenicity. A library of pneumococcal chromosomal fragments (400 to 600 bp) was constructed in a suicide plasmid vector carrying unique DNA sequence tags and a chloramphenicol resistance marker. The recent clinical isolate G54 was transformed with this library. Chloramphenicol-resistant mutants were obtained by homologous recombination, resulting in genes inactivated by insertion of the suicide vector carrying a unique tag. In a mouse pneumonia model, 1.250 candidate clones were screened; 200 of these were not recovered from the lungs were therefore considered virulence-attenuated mutants. The regions flanking the chloramphenicol gene of the attenuated mutants were amplified by inverse PCR and sequenced. The sequence analysis showed that the 200 mutants had insertions in 126 different genes that could be grouped in six classes: (i) known pneumococcal virulence genes; (ii) genes involved in metabolic pathways; (iii) genes encoding proteases; (iv) genes coding for ATP binding cassette transporters; (v) genes encoding proteins involved in DNA recombination/repair; and (vi) DNA sequences that showed similarity to hypothetical genes with unknown function. To evaluate the virulence attenuation for each mutant, all 126 clones were individually analyzed in a mouse septicemia model. Not all mutants selected in the pneumonia model were confirmed in septicemia, thus indicating the existence of virulence factors specific for pneumonia.

Isolation and characterization of the symbiotic phenotype of antibiotic-resistant mutantsof Frankia from Rhamnaceae

Induced rifampicin-resistant mutants of Frankia were isolated by treatment of spores from strain ChI1 with 2 mg/ml of nitrosoguanidine. The mutagenic treatment was followed by growth for 72 h on non-selective medium to allow the expression of the mutation, before plating on selective medium. Spontaneous rifampicin-resistant mutants were isolated from strain ReI4 and chloramphenicol-resistant mutants were derived from strains ReI6 and TtI42. The mutants grew in medium containing up to 20 μg/ml of antibiotics. They showed similar morphology and growth pattern compared with their parent strains. However, they exhibited differences in symbiotic properties, such as infectivity and nitrogenase activity, from their parent strains.

Isolation and Genetic Analysis of a Chloramphenicol-resistant Mutant ofPleurotus ostreatus

A chloramphenicol-resistant mutant was isolated by mutagenesis with manganese to make a study of the genetics and function of mitochondrial genes in the higher basidiomycete, Pleurotus ostreatus. The resistant mutant obtained was shown to grow well on media containing up to 4mg/ml chloramphenicol. The result of genetic analysis suggested that the origin of the factor for chloramphenicol resistance of the mutant might be mitochondrial.

Survey of Antibiotic Resistance and Dependence inPhytophthora

When culture blocks were incubated on V-8 agar plates containing 300 ppm streptomycin sulfate, mutants appeared as fast-growing sectors within 30 days. Streptomycin-resistant mutants were isolated from the sectors of both A' and A2 mating types of P. parasitica, P. palmivora, P. capsici and P. cinnamomi. Streptomycin also caused mating type change in P. parasitica. Streptomycin-dependent mutants were also isolated from the fast-growing sectors of A2 isolates of P. parasitica and A' and A2 isolates of P. palmivora, P. capsici and P. cinnamomi. Presence of CaCl2 in the medium induced a greater need for streptomycin for the dependent isolates of P. parasitica. Fast-growing sectors also appeared when culture blocks were incubated on medium containing 100 ppm chloramphenicol. Chloramphenicol-resistant mutants were isolated from sectors of A' and A2 mating type of P. parasitica, P. palmivora, P. capsici and P. cinnamomi. By incubating streptomycin-resistant mutants of P. parasitica on medium containing chloramphenicol and vice versa, mutants resistant to both antibiotics were obtained.

Survey of Antibiotic Resistance and Dependence in Phytophthora

When culture blocks were incubated on V-8 agar plates containing 300 ppm streptomycin sulfate, mutants appeared as fast-growing sectors within 30 days. Streptomycin-resistant mutants were isolated from the sectors of both A1 and A2 mating types of P. parasitica, P. palmivora, P. capsici and P. cinnamomi. Streptomycin also caused mating type change in P. parasitica. Streptomycin-dependent mutants were also isolated from the fast-growing sectors of A2 isolates of P. parasitica and A1 and A2 isolates of P. palmivora, P. capsici and P. cinnamomi. Presence of CaCl2 in the medium induced a greater need for streptomycin for the dependent isolates of P. parasitica. Fast-growing sectors also appeared when culture blocks were incubated on medium containing 100 ppm chloramphenicol. Chloramphenicol-resistant mutants were isolated from sectors of A1 and A2 mating type of P. parasitica, P. palmivora, P. capsici and P. cinnamomi. By incubating streptomycin-resistant mutants of P. parasitica on medium containing chloramphenicol and vice versa, mutants resistant to both antibiotics were obtained.

Sequence analysis of mouse mitochondrial chloramphenicol-resistant mutants

The nucleotide sequences of the 3' halves of the mitochondrial 16S rRNA genes from four independent mouse chloramphenicol-resistant (CAP-R) mutants were determined. Each contained a different, single base change that encodes the mutational phenotype. The mitochondrial rRNA gene from the SVA31 CAP-R mutant contains a G-to-A transition at nucleotide 2161 of the noncoding strand; the SVIS CAP-R mutant, a G-to-A transition at position 2375; the LA9 CAP-R mutant, an A-to-T transversion at position 2379; and the SVT2 CAP-R mutant, a T-to-C transition at position 2433. Three of these CAP-R mutants appear to be heteroplasmic as the mtDNA populations contain both wild-type and mutant copies of the rRNA gene. The SVIS CAP-R mutation has not been observed in other mammalian CAP-R mutants, although it occurs at a site homologous to one of the yeast mitochondrial CAP-R mutations. Based upon the locations of the mutated sites within the 16S rRNA, and their proximity to previously analyzed sites of mutations conferring increased inhibitor resistance, all these mutations occur within the ribosomal RNA peptidyltransferase domain. These results provide an explanation for the pleiotropic nature of mitochondrial CAP-R mutations in mammalian cells, particularly the observations that some of the mutant lines are partially respiration deficient.

Hormonal Heterothallism in Phytophthora parasitica: A Novel Mode of Sexual Reproduction?

All the single-spore cultures originating from zoospores and oospores of wild-types of Phytophthora parasitica were sensitive to chloramphenicol and streptomycin. Cultures from zoospores and oospores of chloramphenicol-resistant mutants were resistant to chloramphenicol but not streptomycin, while those from zoospores and oospores of streptomycin-resistant mutants were resistant to streptomycin but not chloramphenicol. When chloramphenicol-resistant A1 (6133Cr) of P. parasitica was paired directly with streptomycin-resistant A2 (6134Sr) of the same species, all single-oospore cultures from three crosses were resistant to either chloramphenicol or streptomycin but not both, suggesting absence of genetic exchange in the progeny. Appearance of A2 resistant to chloramphenicol and A1 resistant to streptomycin was due to a mating-type change during oospore formation because oospores produced by A1 through hormone stimulation also gave rise to A2 cultures, and vice versa. Similar results were obtained when reciprocal crosses involving the same direct and membrane-separated pairings between streptomycin-resistant A1 and chloramphenicol-resistant A2 were studied. When A1 wild-type (6133), sensitive to both chloramphenicol and streptomycin, was paired with A2 double mutant 6134CrSr, resistant to chloramphenicol first and streptomycin later, all single-oospore cultures from three crosses were either sensitive to both chloramphenicol and streptomycin or resistant to both antibiotics, indicating that all single-oospore cultures from direct pairing were of uniparental origin. When A2 isolate 6134Sr, resistant to streptomycin, was paired directly with its A1 variant 6134VCr, resistant to chloramphenicol, all single-oospore cultures from three crosses were resistant to either chloramphenicol or streptomycin but not both, indicating an apparent absence of genetic exchange in the progeny. This ruled out the possibility that absence of genetic exchange in the crosses between two different isolates of P. parasitica might be due to genetic divergence between the strains crossed. We interpret the results of this study to show that all progenies from direct pairings between different mating types of P. parasitica were of uniparental origin, although close linkage or mitochondrial location of the markers cannot as yet be eliminated as possible explanations of the apparent absence of genetic exchange. We propose the term hormonal heterothallism to denote this suggested novel phenomenon.

Isolation of chloramphenicol-resistant mutants of Kluyveromyces lactis and characterization by mitotic segregation analysis of fused hybrids.

Chloramphenicol-resistant mutants of Kluyveromyces lactis were isolated following manganese mutagenesis. Mutants were characterized biochemically in order to determine the effect of CAP on respiration. The genetic basis of the resistance was determined using mitotic segregation analysis of fused hybrids where the transmission of mitochondrial genes from a parent in each parasexual cross was impaired by treatment with ethidium bromide.