AsaGEI2d: a new variant of a genomic island identified in a group of Aeromonas salmonicida subsp. salmonicida isolated from France, which bears the pAsa7 plasmid.

Genomic islands (Aeromonas salmonicida genomic islands, AsaGEIs) are found worldwide in many isolates of Aeromonas salmonicida subsp. salmonicida, a fish pathogen. To date, five variants of AsaGEI (1a, 1b, 2a, 2b and 2c) have been described. Here, we investigate a sixth AsaGEI, which was identified in France between 2016 and 2019 in 20 A. salmonicida subsp. salmonicida isolates recovered from sick salmon all at the same location. This new AsaGEI shares the same insertion site in the chromosome as the other AsaGEI2s as they all have a homologous integrase gene. This new AsaGEI was thus named AsaGEI2d, and has five unique genes compared to the other AsaGEIs. The isolates carrying AsaGEI2d also bear the plasmid pAsa7, which was initially found in an isolate from Switzerland. This plasmid provides resistance to chloramphenicol thanks to a cat gene. This study reveals more about the diversity of the AsaGEIs.

Identification of mcr-1 and a novel chloramphenicol resistance gene catT on an integrative and conjugative element in an Actinobacillus strain of swine origin.

The aim of this study was to characterize a mcr-1-carrying integrative and conjugative element (ICE) in a novel Pasteurellaceae-like bacteria of swine origin. The mcr-1-positive GY-402 strain, recovered from a pig fecal sample, was subjected to whole genome sequencing with the combination of Illumina Hiseq and MinION platforms. Genome-based taxonomy revealed that strain GY-402 exhibited highest ANI value (84.89 %) to Actinobacillus succinogenes, which suggested that it represented a novel Actinobacillus species. Sequence analysis revealed that mcr-1 was clustered with eight other resistance genes in the MDR region of a novel ICE element, named ICEAsp1. Inverse PCR and mating assays showed that ICEAsp1 is active and transferrable. In addition, six circular forms mediated by four ISApl1 elements were detected with different inverse PCR sets, indicating that flexible composite transposons could be formed by pairwise combinations of multiple IS copies. Cloning experiment and phylogenetic analysis revealed that the novel Cat protein, designated CatT, belongs to type-A family and confers resistance to chloramphenicol. In conclusion, this is, to the best of our knowledge, the first report of mcr-1 gene on ICE structure and also in Pasteurellaceae bacteria. The diverse composite transposons mediated by multicopy IS elements may facilitate the dissemination of different resistance genes.

Horizontal genetic exchange of chromosomally encoded markers between Campylobacter jejuni cells.

Many epidemiological studies provide us with the evidence of horizontal gene transfer (HGT) contributing to the bacterial genomic diversity that benefits the bacterial populations with increased ability to adapt to the dynamic environments. Campylobacter jejuni, a major cause of acute enteritis in the U.S., often linked with severe post-infection neuropathies, has been reported to exhibit a non-clonal population structure and comparatively higher strain-level genetic variation. In this study, we provide evidence of the HGT of chromosomally encoded genetic markers between C. jejuni cells in the biphasic MH medium. We used two C. jejuni NCTC-11168 mutants harbouring distinct antibiotic-resistance genes [chloramphenicol (Cm) and kanamycin (Km)] present at two different neutral genomic loci. Cultures of both marker strains were mixed together and incubated for 5 hrs, then plated on MH agar plates supplemented with both antibiotics. The recombinant cells with double antibiotic markers were generated at the frequency of 0.02811 ± 0.0035% of the parental strains. PCR assays using locus-specific primers confirmed that transfer of the antibiotic-resistance genes was through homologous recombination. Also, the addition of chicken cecal content increased the recombination efficiency approximately up to 10-fold as compared to the biphasic MH medium (control) at P < 0.05. Furthermore, treating the co-culture with DNase I decreased the available DNA, which in turn significantly reduced recombination efficiency by 99.92% (P < 0.05). We used the cell-free supernatant of 16 hrs-culture of Wild-type C. jejuni as a template for PCR and found DNA sequences from six different genomic regions were easily amplified, indicating the presence of released chromosomal DNA in the culture supernatant. Our findings suggest that HGT in C. jejuni is facilitated in the chicken gut environment contributing to in vivo genomic diversity. Additionally, C. jejuni might have an active mechanism to release its chromosomal DNA into the extracellular environment, further expediting HGT in C. jejuni populations.

The spread of chloramphenicol-resistant Neisseria meningitidis in Southeast Asia.

OBJECTIVES: Invasive disease caused by Neisseria meningitidis is a significant health concern globally, but our knowledge of the prevailing serogroups, antimicrobial susceptibility patterns, and genetics of N. meningitidis in Southeast Asia is limited. Chloramphenicol resistance in N. meningitidis has rarely been reported, but was first described in isolates from Vietnam in 1998. We aimed to characterise eight chloramphenicol resistant meningococcal isolates collected between 2007 and 2018 from diagnostic microbiology laboratories in Cambodia, Thailand and the Lao People's Democratic Republic (Laos). METHODS: Whole-genome sequencing was used to generate genome sequences from 18 meningococcal isolates including the eight chloramphenicol resistant isolates. We identified antimicrobial resistance genes present in these strains, and examined the phylogenetic relationships between strains. RESULTS: The eight resistant strains all contain the same chloramphenicol resistance gene first described in 1998, and are closely related to each other. Strains resistant to penicillin, tetracycline, and ciprofloxacin were also observed, including a chloramphenicol-resistant strain which has acquired penicillin and ciprofloxacin resistance. CONCLUSIONS: This study suggests that chloramphenicol-resistant N. meningitidis is more widespread than previously thought, and that the previously-identified resistant lineage is now found in multiple countries in Southeast Asia.

Molecular detection of chloramphenicol-florfenicol resistance (cfr) genes among linezolid resistant MRSA isolates in Sokoto State, Nigeria / Detecção molecular de genes de resistência ao cloranfenicol-florfenicol (cfr) entre isolados de MRSA resistentes a linezolida no estado de Sokoto, Nigéria

Objective: we investigated previous literatures for documentation of the trend in Sokoto, Nigeria and found none. We deemed it fit to determine the frequency of linezolid resistance mediated by cfr gene among MRSA isolates from Sokoto State-owned hospitals. Methods: Bacterial species identification was carried out with Microgen™ Staph-ID System kit (Microgen, Surrey, UK). Disc agar diffusion method (Modified Kirby-Bauer's) following Clinical and Laboratory Standards Institute (CLSI 2018) guidelines was used in antimicrobial susceptibility testing. The results were interpreted and managed using WHONET 5.6 software (WHO, Switzerland). Oxacillin resistant screening agar base (ORSAB) culture was used to determine phenotypic methicillin resistance. Polymerase chain reaction (PCR) was carried out to determine the presence of cfr-gene. Results: A total of 81 S. aureus isolates were phenotypically identified. Of this number, 46.91% (38/81) were MRSA; Healthcare workers (39.5%), Outpatient (28.9%), In patient (21%), Security men and Cleaners (5.3% each). Importantly linezolid resistance rate among the MRSA isolates was 44.7%. Analysis of antimicrobial susceptibility profile also showed a multiple antibiotics resistance burden of MDR (5.9%), possible XDR (47.1%), XDR (41.1%) and PDR (5.9%) amongst LR-MRSA. About 52.9% (9/17) of LR-MRSA harbored the cfr gene. Conclusions: This is the first report to document cfr gene in LR-MRSA strains in Sokoto. The cfr gene was found among the studied LR-MRSA strains and if cfr-mediated linezolid resistance is not properly checked, its phenotypic expression may result in an outbreak of multiple antibiotic resistant strains.

Objetivo: avaliar a incidência de resistência linezolida cfr-mediada entre os isolados de MRSA dos hospitais do Estado de Sokoto. Métodos: A identificação das espécies bacterianas foi realizada com Microgen™ Staph-ID System kit (Microgen, Surrey, UK). Método de difusão em ágar de disco (Kirby-Bauer modificado) seguindo as diretrizes do Clinical and Laboratory Standards Institute (CLSI 2018). O resultado foi interpretado e gerido com WHONET 5.6 (OMS, Suíça) software. A cultura ORSAB (Oxacillin resistant screening agar) foi utilizada para determinar a resistência fenotípica à meticilina. A PCR foi realizada para determinar a presença de cfr-gene. Resultados: um total de 81 isolados de S. aureus foi identificada fenotipicamente. Desse número, 46,91% (38/81) eram de MRSA; Profissionais de saúde (39,5%), Ambulatoriais (28,9%), Em paciente (21%), Homens de segurança e Limpadores (5,3% cada). A taxa de resistência linezolida entre os isolados de MRSA foi de 44,7%. A análise do perfil de sensibilidade antimicrobiana também mostrou uma carga de resistência a antibióticos múltiplos de MDR (5,9%), possível XDR (47,1%), XDR (41,1%) e PDR (5,9%) entre LR-MRSA. Um total de, 52,9% (9/17) da LR-MRSA abrigava o gene cfr. Conclusões: Este é o primeiro relatório a documentar o cfr-gen nas estirpes LR-MRSA em Sokoto. O gene cfr está presente entre as cepas estudadas de LR-MRSA, e se a resistência cfr-mediated linezolida não for adequadamente verificada, sua expressão fenotípica pode resultar em um surto de múltiplas cepas resistentes a antibióticos.

Split intein-mediated selection of cells containing two plasmids using a single antibiotic.

To build or dissect complex pathways in bacteria and mammalian cells, it is often necessary to recur to at least two plasmids, for instance harboring orthogonal inducible promoters. Here we present SiMPl, a method based on rationally designed split enzymes and intein-mediated protein trans-splicing, allowing the selection of cells carrying two plasmids with a single antibiotic. We show that, compared to the traditional method based on two antibiotics, SiMPl increases the production of the antimicrobial non-ribosomal peptide indigoidine and the non-proteinogenic aromatic amino acid para-amino-L-phenylalanine from bacteria. Using a human T cell line, we employ SiMPl to obtain a highly pure population of cells double positive for the two chains of the T cell receptor, TCRα and TCRß, using a single antibiotic. SiMPl has profound implications for metabolic engineering and for constructing complex synthetic circuits in bacteria and mammalian cells.

Whole transcriptome analysis and gene deletion to understand the chloramphenicol resistance mechanism and develop a screening method for homologous recombination in Myxococcus xanthus.

BACKGROUND: Myxococcus xanthus DK1622 is a model system for studying multicellular development, predation, cellular differentiation, and evolution. Furthermore, it is a rich source of novel secondary metabolites and is widely used as heterologous expression host of exogenous biosynthetic gene clusters. For decades, genetic modification of M. xanthus DK1622 has mainly relied on kanamycin and tetracycline selection systems. RESULTS: Here, we introduce an alternative selection system based on chloramphenicol (Cm) to broaden the spectrum of available molecular tools. A chloramphenicol-resistant growth phase and a chloramphenicol-susceptible growth phase before and after chloramphenicol-induction were prepared, and later sequenced to identify specific genes related to chloramphenicol-repercussion and drug-resistance. A total of 481 differentially expressed genes were revealed in chloramphenicol-resistant Cm5_36h and 1920 differentially expressed genes in chloramphenicol-dormant Cm_8h. Moreover, the gene expression profile in the chloramphenicol-dormant strain Cm_8h was quite different from that of Cm5_36 which had completely adapted to Cm, and 1513 differentially expression genes were identified between these two phenotypes. Besides upregulated acetyltransferases, several transporter encoding genes, including ABC transporters, major facilitator superfamily transporters (MFS), resistance-nodulation-cell division (RND) super family transporters and multidrug and toxic compound extrusion family transporters (MATE) were found to be involved in Cm resistance. After the knockout of the most highly upregulated MXAN_2566 MFS family gene, mutant strain DK-2566 was proved to be sensitive to Cm by measuring the growth curve in the Cm-added condition. A plasmid with a Cm resistance marker was constructed and integrated into chromosomes via homologous recombination and Cm screening. The integration efficiency was about 20% at different concentrations of Cm. CONCLUSIONS: This study provides a new antibiotic-based selection system, and will help to understand antibiotic resistance mechanisms in M. xanthus DK1622.

Outbreaks of a Methicillin-Resistant Staphylococcus aureus Clone ST398-t011 in a Hungarian Equine Clinic: Emergence of Rifampicin and Chloramphenicol Resistance After Treatment with These Antibiotics.

Between July 2011 and May 2016, a total of 40 Staphylococcus aureus strains originating from 36 horses were confirmed as methicillin resistant (methicillin-resistant Staphylococcus aureus [MRSA]) in a university equine clinic. An additional 10 MRSA strains from 36 samples of clinic workers were obtained in October 2017. The first equine isolate represented the sequence type ST398, spa-type t011, and SCCmec IV. This isolate was resistant to a wide spectrum of antimicrobial agents. MRSA strains with the same genotype and with very similar resistance profiles were isolated on 21 more occasions from September 2013 to September 2014. A second outbreak occurred from May 2015 until May 2016. The first isolate in this second outbreak shared the same genotype, but was additionally resistant to chloramphenicol. The second isolate from August 2015 also showed resistance to rifampicin. The clone was isolated 18 times. Most of the human isolates shared the same genotype as the isolates from horses and their resistance patterns showed only slight differences. We can conclude that the MRSA-related cases at the Department and Clinic of Equine Medicine were all nosocomial infections caused by the same clonal lineage belonging to the clonal complex 398. The clonal complex 398 of equine origin is reported for the first time in Hungary. In addition, our observation of the emergence of new resistance to antimicrobial agents within the clonal lineage after treatment with antibiotics is of concern. Strict hygiene regulations have been introduced to lower the incidence of MRSA isolation and the related clinical disease.

Tracking of single tRNAs for translation kinetics measurements in chloramphenicol treated bacteria.

Chloramphenicol is a broad-spectrum antibiotic targeting the protein synthesis machinery by binding to the bacterial ribosome. Chloramphenicol has been considered a classic general inhibitor of translation, blocking the accommodation of aa-tRNA into the A site of the large ribosomal subunit. However, recent studies suggest that this proposed mechanism is a simplification and that the effect of chloramphenicol on mRNA translation is much more dynamic. By tracking single dye-labelled elongator and initiator tRNAs in Escherichia coli cells treated with chloramphenicol, we observe the direct effect of chloramphenicol on translation kinetics. We find clear indications of slow but significant mRNA translation on drug bound ribosomes.

Putative antibiotic resistance genes present in extant Bacillus licheniformis and Bacillus paralicheniformis strains are probably intrinsic and part of the ancient resistome.

Whole-genome sequencing and phenotypic testing of 104 strains of Bacillus licheniformis and Bacillus paralicheniformis from a variety of sources and time periods was used to characterize the genetic background and evolution of (putative) antimicrobial resistance mechanisms. Core proteins were identified in draft genomes and a phylogenetic analysis based on single amino acid polymorphisms allowed the species to be separated into two phylogenetically distinct clades with one outlier. Putative antimicrobial resistance genes were identified and mapped. A chromosomal ermD gene was found at the same location in all B. paralichenformis and in 27% of B. licheniformis genomes. Erythromycin resistance correlated very well with the presence of ermD. The putative streptomycin resistance genes, aph and aadK, were found in the chromosome of all strains as adjacent loci. Variations in amino acid sequence did not correlate with streptomycin susceptibility although the species were less susceptible than other Bacillus species. A putative chloramphenicol resistance gene (cat), encoding a novel chloramphenicol acetyltransferase protein was also found in the chromosome of all strains. Strains encoding a truncated CAT protein were sensitive to chloramphenicol. For all four resistance genes, the diversity and genetic context followed the overall phylogenetic relationship. No potentially mobile genetic elements were detected in their vicinity. Moreover, the genes were only distantly related to previously-described cat, aph, aad and erm genes present on mobile genetic elements or in other species. Thus, these genes are suggested to be intrinsic to B. licheniformis and B. paralicheniformis and part of their ancient resistomes. Since there is no evidence supporting horizontal transmission, these genes are not expected to add to the pool of antibiotic resistance elements considered to pose a risk to human or animal health. Whole-genome based phylogenetic and sequence analysis, combined with phenotypic testing, is proposed to be suitable for determining intrinsic resistance and evolutionary relationships.

Insertion sequences in the CRISPR-Cas system regulate horizontal antimicrobial resistance gene transfer in Shigella strains.

Multidrug-resistant (MDR) Shigella strains are an enormous threat to public health. Antimicrobial resistance genes are frequently located on plasmids, phages and integrons, which enter bacterial cells by horizontal gene transfer (HGT). CRISPR-Cas systems are adaptive prokaryotic immune systems in bacteria that confer resistance to foreign genetic material such as phages and other mobile genetic elements. However, this may come at a cost of inhibiting the acquisition of other beneficial genes through HGT. This study investigated how Shigella strains regulate the activity of the CRISPR-Cas system spontaneously when they require an exogenous gene necessary for survival. Insertion sequence (IS) elements were identified in cas genes, such as IS600 in cse2, ISSfl2 in cas6e and IS629 in cse1-cas3. The number of spacers in CRISPR-Cas arrays in strains containing an IS was less than that for strains with no IS. Interestingly, fewer spacers were also found in MDR Shigella isolates. Furthermore, an antimicrobial-resistant strain was constructed by electrotransformation of a resistance plasmid in order to detect changes in the CRISPR-Cas system. It was found that the cse2 gene had a new IS (IS600) in the antimicrobial-resistant strain. Bioinformatics analyses showed that the IS600 insertion hotspot was TGC-GGC in the cse2 gene, and the tertiary structure of the Cse2 protein was different with IS600. IS600 caused a five-order of magnitude decrease in relative expression of the cse2 gene. This study sheds mechanistic light on CRISPR-Cas-mediated HGT of antimicrobial resistance genes in Shigella spp. isolates.

Multidrug-Resistant Acinetobacter baumannii Chloramphenicol Resistance Requires an Inner Membrane Permease.

Acinetobacter baumannii is a Gram-negative organism that is a cause of hospital-acquired multidrug-resistant (MDR) infections. A. baumannii has a unique cell surface compared to those of many other Gram-negative pathogens in that it can live without lipopolysaccharide (LPS) and it has a high content of cardiolipin in the outer membrane. Therefore, to better understand the cell envelope and mechanisms of MDR A. baumannii, we screened a transposon library for mutants with defective permeability barrier function, defined as a deficiency in the ability to exclude the phosphatase chromogenic substrate 5-bromo-4-chloro-3-indolylphosphate (XP). We identified multiple mutants with mutations in the ABUW_0982 gene, predicted to encode a permease broadly present in A. baumannii isolates with increased susceptibility to the ribosome-targeting antibiotic chloramphenicol (CHL). Moreover, compared to other known CHL resistance genes, such as chloramphenicol acyltransferase genes, we found that ABUW_0982 is the primary determinant of intrinsic CHL resistance in A. baumannii strain 5075 (Ab5075), an important isolate responsible for severe MDR infections in humans. Finally, studies measuring the efflux of chloramphenicol and expression of ABUW_0982 in CHL-susceptible Escherichia coli support the conclusion that ABUW_0982 encodes a single-component efflux protein with specificity for small, hydrophobic molecules, including CHL.

Construction of a New Phage Integration Vector pFIV-Val for Use in Different Francisella Species.

We recently identified and described a putative prophage on the genomic island FhaGI-1 located within the genome of Francisella hispaniensis AS02-814 (F. tularensis subsp. novicida-like 3523). In this study, we constructed two variants of a Francisella phage integration vector, called pFIV1-Val and pFIV2-Val (Francisella Integration Vector-tRNAVal-specific), using the attL/R-sites and the site-specific integrase (FN3523_1033) of FhaGI-1, a chloramphenicol resistance cassette and a sacB gene for counter selection of transformants against the vector backbone. We inserted the respective sites and genes into vector pUC57-Kana to allow for propagation in Escherichia coli. The constructs generated a circular episomal form in E. coli which could be used to transform Francisella spp. where FIV-Val stably integrated site specifically into the tRNAVal gene of the genome, whereas pUC57-Kana is lost due to counter selection. Functionality of the new vector was demonstrated by the successfully complementation of a Francisella mutant strain. The vectors were stable in vitro and during host-cell infection without selective pressure. Thus, the vectors can be applied as a further genetic tool in Francisella research, expanding the present genetic tools by an integrative element. This new element is suitable to perform long-term experiments with different Francisella species.

Genetic characterization of phenicol-resistant Escherichia coli and role of wild-type repressor/regulator gene (acrR) on phenicol resistance.

The genetic basis for phenicol resistance was examined in 38 phenicol-resistant clinical Escherichia coli isolates from poultry. Out of 62 isolates, 38 showed resistance for chloramphenicol and nine for florfenicol, respectively. Each strain also demonstrated resistance to a variety of other antibiotics. Molecular detection revealed that the incidence rates of the cat1, cat2, flo, flo-R, cmlA, and cmlB were 32, 29, 18, 13, 0, and 0%, respectively. Nineteen strains were tolerant to organic solvents. PCR amplification of the complete acrR (regulator/repressor) gene of five isolates revealed the amino acid changes in four isolates. DNA sequencing showed the non-synonymous mutations which change the amino acid, silent mutation, and nucleotide deletion in four isolates. MY09C10 showed neither deletion nor mutation in nucleotide. The AcrA protein of the AcrAB multidrug efflux pump was overexpressed in these strains. Complementation with a plasmid-borne wild-type acrR gene reduced the expression level of AcrA protein in the mutants and partially restored antibiotic susceptibility one- to fourfold. This study shows that mutations in acrR are an additional genetic basis for phenicol resistance.

Integrated Genomic and Proteomic Analyses of High-level Chloramphenicol Resistance in Campylobacter jejuni.

Campylobacter jejuni is a major zoonotic pathogen, and its resistance to antibiotics is of great concern for public health. However, few studies have investigated the global changes of the entire organism with respect to antibiotic resistance. Here, we provide mechanistic insights into high-level resistance to chloramphenicol in C. jejuni, using integrated genomic and proteomic analyses. We identified 27 single nucleotide polymorphisms (SNPs) as well as an efflux pump cmeB mutation that conferred modest resistance. We determined two radical S-adenosylmethionine (SAM) enzymes, one each from an SNP gene and a differentially expressed protein. Validation of major metabolic pathways demonstrated alterations in oxidative phosphorylation and ABC transporters, suggesting energy accumulation and increase in methionine import. Collectively, our data revealed a novel rRNA methylation mechanism by a radical SAM superfamily enzyme, indicating that two resistance mechanisms existed in Campylobacter. This work provided a systems biology perspective on understanding the antibiotic resistance mechanisms in bacteria.

Effect of bio-electrochemical system on the fate and proliferation of chloramphenicol resistance genes during the treatment of chloramphenicol wastewater.

Bioelectrochemical systems can effectively degrade antibiotics, but there is the need to better understand the fate of antibiotic resistance bacteria and antibiotic resistance genes during the bioelectrochemical degradation of antibiotics. In this study, a BES was developed as a platform to investigate the fate of chloramphenicol resistance bacteria (CRB) and the expression of chloramphenicol resistance genes (CRGs) under different operating conditions during chloramphenicol biodegradation. The results indicated that chloramphenicol was effectively removed and chloramphenicol removal efficiency could be improved under less chloramphenicol concentration and more negative cathode potential. Higher chloramphenicol concentration enhanced the enrichment of CRB and expression of CRGs. Furthermore, the abundances of CRB were enhanced under more negative cathode potential, the expression of CRGs under less negative cathode potential were induced. However, both the enrichment of CRB and expression of CRGs could be moderated under a medium cathode potential. This result could provide the scientific reference for research about the fate of antibiotic resistance genes in bioelectrochemical systems.

Chloramphenicol acetyltransferase-a new selectable marker in stable nuclear transformation of the red alga Cyanidioschyzon merolae.

In this study, we have shown the applicability of chloramphenicol acetyltransferase as a new and convenient selectable marker for stable nuclear transformation as well as potential chloroplast transformation of Cyanidioschyzon merolae-a new model organism, which offers unique opportunities for studding the mitochondrial and plastid physiology as well as various evolutionary, structural, and functional features of the photosynthetic apparatus.

Long-range transcriptional interference in E. coli used to construct a dual positive selection system for genetic switches.

We have investigated transcriptional interference between convergent genes in E. coli and demonstrate substantial interference for inter-promoter distances of as far as 3 kb. Interference can be elicited by both strong σ(70) dependent and T7 promoters. In the presented design, a strong promoter driving gene expression of a 'forward' gene interferes with the expression of a 'reverse' gene by a weak promoter. This arrangement allows inversely correlated gene expression without requiring further regulatory components. Thus, modulation of the activity of the strong promoter alters expression of both the forward and the reverse gene. We used this design to develop a dual selection system for conditional operator site binding, allowing positive selection both for binding and for non-binding to DNA. This study demonstrates the utility of this novel system using the Lac repressor as a model protein for conditional DNA binding, and spectinomycin and chloramphenicol resistance genes as positive selection markers in liquid culture. Randomized LacI libraries were created and subjected to subsequent dual selection, but mispairing IPTG and selection cues in respect to the wild-type LacI response, allowing the isolation of a LacI variant with a reversed IPTG response within three rounds of library generation and dual selection.