Adaptive Resistance Mutations at Suprainhibitory Concentrations Independent of SOS Mutagenesis

Emergence of resistant bacteria during antimicrobial treatment is one of the most critical and universal health threats. It is known that several stress-induced mutagenesis and heteroresistance mechanisms can enhance microbial adaptation to antibiotics. Here, we demonstrate that the pathogen Bartonella can undergo stress-induced mutagenesis despite the fact it lacks error-prone polymerases, the rpoS gene and functional UV-induced mutagenesis. We demonstrate that Bartonella acquire de novo single mutations during rifampicin exposure at suprainhibitory concentrations at a much higher rate than expected from spontaneous fluctuations. This is while exhibiting a minimal heteroresistance capacity. The emerged resistant mutants acquired a single rpoB mutation, whereas no other mutations were found in their whole genome. Interestingly, the emergence of resistance in Bartonella occurred only during gradual exposure to the antibiotic, indicating that Bartonella sense and react to the changing environment. Using a mathematical model, we demonstrated that, to reproduce the experimental results, mutation rates should be transiently increased over 1,000-folds, and a larger population size or greater heteroresistance capacity is required. RNA expression analysis suggests that the increased mutation rate is due to downregulation of key DNA repair genes (mutS, mutY, and recA), associated with DNA breaks caused by massive prophage inductions. These results provide new evidence of the hazard of antibiotic overuse in medicine and agriculture.     

A novel type of colistin resistance genes selected from random sequence space

Antibiotic resistance is a rapidly increasing medical problem that severely limits the success of antibiotic treatments, and the identification of resistance determinants is key for surveillance and control of resistance dissemination. Horizontal transfer is the dominant mechanism for spread of resistance genes between bacteria but little is known about the original emergence of resistance genes. Here, we examined experimentally if random sequences can generate novel antibiotic resistance determinants de novo. By utilizing highly diverse expression libraries encoding random sequences to select for open reading frames that confer resistance to the last-resort antibiotic colistin in Escherichia coli, six de novo colistin resistance conferring peptides (Dcr) were identified. The peptides act via direct interactions with the sensor kinase PmrB (also termed BasS in E. coli), causing an activation of the PmrAB two-component system (TCS), modification of the lipid A domain of lipopolysaccharide and subsequent colistin resistance. This kinase-activation was extended to other TCS by generation of chimeric sensor kinases. Our results demonstrate that peptides with novel activities mediated via specific peptide-protein interactions in the transmembrane domain of a sensory transducer can be selected de novo, suggesting that the origination of such peptides from non-coding regions is conceivable. In addition, we identified a novel class of resistance determinants for a key antibiotic that is used as a last resort treatment for several significant pathogens. The high-level resistance provided at low expression levels, absence of significant growth defects and the functionality of Dcr peptides across different genera suggest that this class of peptides could potentially evolve as bona fide resistance determinants in natura.     

Convergent phenotypic evolution towards fosfomycin collateral sensitivity of Pseudomonas aeruginosa antibiotic-resistant mutants

The rise of antibiotic resistance and the reduced amount of novel antibiotics support the need of developing novel strategies to fight infections, based on improving the use of the antibiotics we already have. Collateral sensitivity is an evolutionary trade-off associated with the acquisition of antibiotic resistance that can be exploited to tackle this relevant health problem. However, different works have shown that patterns of collateral sensitivity are not always conserved, thus precluding the exploitation of this evolutionary trade-off to fight infections. In this work, we identify a robust pattern of collateral sensitivity to fosfomycin in Pseudomonas aeruginosa antibiotic-resistant mutants, selected by antibiotics belonging to different structural families. We characterize the underlying mechanism of the collateral sensitivity observed, which is a reduced expression of the genes encoding the peptidoglycan-recycling pathway, which preserves the peptidoglycan synthesis in situations where its de novo synthesis is blocked, and a reduced expression of fosA, encoding a fosfomycin-inactivating enzyme. We propose that the identification of robust collateral sensitivity patterns, as well as the understanding of the molecular mechanisms behind these phenotypes, would provide valuable information to design evolution-based strategies to treat bacterial infections.     

Mucoidy,Quorum Sensing,Mismatch Repair and Antibiotic Resistance in Pseudomonas aeruginosa from Cystic Fibrosis Chronic Airways Infections

Survival of Pseudomonas aeruginosa in cystic fibrosis (CF) chronic infections is based on a genetic adaptation process consisting of mutations in specific genes, which can produce advantageous phenotypic switches and ensure its persistence in the lung. Among these, mutations inactivating the regulators MucA (alginate biosynthesis), LasR (quorum sensing) and MexZ (multidrug-efflux pump MexXY) are the most frequently observed, with those inactivating the DNA mismatch repair system (MRS) being also highly prevalent in P. aeruginosa CF isolates, leading to hypermutator phenotypes that could contribute to this adaptive mutagenesis by virtue of an increased mutation rate. Here, we characterized the mutations found in the mucA, lasR, mexZ and MRS genes in P. aeruginosa isolates obtained from Argentinean CF patients, and analyzed the potential association of mucA, lasR and mexZ mutagenesis with MRS-deficiency and antibiotic resistance. Thus, 38 isolates from 26 chronically infected CF patients were characterized for their phenotypic traits, PFGE genotypic patterns, mutations in the mucA, lasR, mexZ, mutS and mutL gene coding sequences and antibiotic resistance profiles. The most frequently mutated gene was mexZ (79%), followed by mucA (63%) and lasR (39%) as well as a high prevalence (42%) of hypermutators being observed due to loss-of-function mutations in mutL (60%) followed by mutS (40%). Interestingly, mutational spectra were particular to each gene, suggesting that several mechanisms are responsible for mutations during chronic infection. However, no link could be established between hypermutability and mutagenesis in mucA, lasR and mexZ, indicating that MRS-deficiency was not involved in the acquisition of these mutations. Finally, although inactivation of mucA, lasR and mexZ has been previously shown to confer resistance/tolerance to antibiotics, only mutations in MRS genes could be related to an antibiotic resistance increase. These results help to unravel the mutational dynamics that lead to the adaptation of P. aeruginosa to the CF lung.     

Genomics of Adaptation during Experimental Evolution of the Opportunistic Pathogen Pseudomonas aeruginosa

Adaptation is likely to be an important determinant of the success of many pathogens, for example when colonizing a new host species, when challenged by antibiotic treatment, or in governing the establishment and progress of long-term chronic infection. Yet, the genomic basis of adaptation is poorly understood in general, and for pathogens in particular. We investigated the genetics of adaptation to cystic fibrosis-like culture conditions in the presence and absence of fluoroquinolone antibiotics using the opportunistic pathogen Pseudomonas aeruginosa. Whole-genome sequencing of experimentally evolved isolates revealed parallel evolution at a handful of known antibiotic resistance genes. While the level of antibiotic resistance was largely determined by these known resistance genes, the costs of resistance were instead attributable to a number of mutations that were specific to individual experimental isolates. Notably, stereotypical quinolone resistance mutations in DNA gyrase often co-occurred with other mutations that, together, conferred high levels of resistance but no consistent cost of resistance. This result may explain why these mutations are so prevalent in clinical quinolone-resistant isolates. In addition, genes involved in cyclic-di-GMP signalling were repeatedly mutated in populations evolved in viscous culture media, suggesting a shared mechanism of adaptation to this CF–like growth environment. Experimental evolutionary approaches to understanding pathogen adaptation should provide an important complement to studies of the evolution of clinical isolates.     

Antibiotic resistance in Pseudomonas aeruginosa: mechanisms and alternative therapeutic strategies

Pseudomonas aeruginosa is an opportunistic pathogen that is a leading cause of morbidity and mortality in cystic fibrosis patients and immunocompromised individuals. Eradication of P. aeruginosa has become increasingly difficult due to its remarkable capacity to resist antibiotics. Strains of Pseudomonas aeruginosa are known to utilize their high levels of intrinsic and acquired resistance mechanisms to counter most antibiotics. In addition, adaptive antibiotic resistance of P. aeruginosa is a recently characterized mechanism, which includes biofilm-mediated resistance and formation of multidrug-tolerant persister cells, and is responsible for recalcitrance and relapse of infections. The discovery and development of alternative therapeutic strategies that present novel avenues against P. aeruginosa infections are increasingly demanded and gaining more and more attention. Although mostly at the preclinical stages, many recent studies have reported several innovative therapeutic technologies that have demonstrated pronounced effectiveness in fighting against drug-resistant P. aeruginosa strains. This review highlights the mechanisms of antibiotic resistance in P. aeruginosa and discusses the current state of some novel therapeutic approaches for treatment of P. aeruginosa infections that can be further explored in clinical practice.     

Reverting Antibiotic Tolerance of Pseudomonas aeruginosa PAO1 Persister Cells by (Z)-4-bromo-5-(bromomethylene)-3-methylfuran-2(5H)-one

Background

Bacteria are well known to form dormant persister cells that are tolerant to most antibiotics. Such intrinsic tolerance also facilitates the development of multidrug resistance through acquired mechanisms. Thus persister cells are a promising target for developing more effective methods to control chronic infections and help prevent the development of multidrug-resistant bacteria. However, control of persister cells is still an unmet challenge.

Methodology/Principal Findings

We show in this report that (Z)-4-bromo-5-(bromomethylene)-3-methylfuran-2(5H)-one (BF8) can restore the antibiotic susceptibility of Pseudomonas aeruginosa PAO1 persister cells at growth non-inhibitory concentrations. Persister control by BF8 was found to be effective against both planktonic and biofilm cells of P. aeruginosa PAO1. Interestingly, although BF8 is an inhibitor of quorum sensing (QS) in Gram-negative bacteria, the data in this study suggest that the activities of BF8 to revert antibiotic tolerance of P. aeruginosa PAO1 persister cells is not through QS inhibition and may involve other targets.

Conclusion

BF8 can sensitize P. aeruginosa persister cells to antibiotics.     

Integrated Model of De Novo and Inherited Genetic Variants Yields Greater Power to Identify Risk Genes

De novo mutations affect risk for many diseases and disorders, especially those with early-onset. An example is autism spectrum disorders (ASD). Four recent whole-exome sequencing (WES) studies of ASD families revealed a handful of novel risk genes, based on independent de novo loss-of-function (LoF) mutations falling in the same gene, and found that de novo LoF mutations occurred at a twofold higher rate than expected by chance. However successful these studies were, they used only a small fraction of the data, excluding other types of de novo mutations and inherited rare variants. Moreover, such analyses cannot readily incorporate data from case-control studies. An important research challenge in gene discovery, therefore, is to develop statistical methods that accommodate a broader class of rare variation. We develop methods that can incorporate WES data regarding de novo mutations, inherited variants present, and variants identified within cases and controls. TADA, for Transmission And De novo Association, integrates these data by a gene-based likelihood model involving parameters for allele frequencies and gene-specific penetrances. Inference is based on a Hierarchical Bayes strategy that borrows information across all genes to infer parameters that would be difficult to estimate for individual genes. In addition to theoretical development we validated TADA using realistic simulations mimicking rare, large-effect mutations affecting risk for ASD and show it has dramatically better power than other common methods of analysis. Thus TADA''s integration of various kinds of WES data can be a highly effective means of identifying novel risk genes. Indeed, application of TADA to WES data from subjects with ASD and their families, as well as from a study of ASD subjects and controls, revealed several novel and promising ASD candidate genes with strong statistical support.     

Clonal Expansion during Staphylococcus aureus Infection Dynamics Reveals the Effect of Antibiotic Intervention

To slow the inexorable rise of antibiotic resistance we must understand how drugs impact on pathogenesis and influence the selection of resistant clones. Staphylococcus aureus is an important human pathogen with populations of antibiotic-resistant bacteria in hospitals and the community. Host phagocytes play a crucial role in controlling S. aureus infection, which can lead to a population “bottleneck” whereby clonal expansion of a small fraction of the initial inoculum founds a systemic infection. Such population dynamics may have important consequences on the effect of antibiotic intervention. Low doses of antibiotics have been shown to affect in vitro growth and the generation of resistant mutants over the long term, however whether this has any in vivo relevance is unknown. In this work, the population dynamics of S. aureus pathogenesis were studied in vivo using antibiotic-resistant strains constructed in an isogenic background, coupled with systemic models of infection in both the mouse and zebrafish embryo. Murine experiments revealed unexpected and complex bacterial population kinetics arising from clonal expansion during infection in particular organs. We subsequently elucidated the effect of antibiotic intervention within the host using mixed inocula of resistant and sensitive bacteria. Sub-curative tetracycline doses support the preferential expansion of resistant microorganisms, importantly unrelated to effects on growth rate or de novo resistance acquisition. This novel phenomenon is generic, occurring with methicillin-resistant S. aureus (MRSA) in the presence of β-lactams and with the unrelated human pathogen Pseudomonas aeruginosa. The selection of resistant clones at low antibiotic levels can result in a rapid increase in their prevalence under conditions that would previously not be thought to favor them. Our results have key implications for the design of effective treatment regimes to limit the spread of antimicrobial resistance, where inappropriate usage leading to resistance may reduce the efficacy of life-saving drugs.     

Characterization of Two Macrolide Resistance-Related Genes in Multidrug-Resistant Pseudomonas aeruginosa Isolates

In analyzing the drug resistance phenotype and mechanism of resistance to macrolide antibiotics of clinical Pseudomonas aeruginosa isolates, the agar dilution method was used to determine the minimum inhibitory concentrations (MICs), and PCR (polymerase chain reaction) was applied to screen for macrolide antibiotics resistance genes. The macrolide antibiotics resistance genes were cloned, and their functions were identified. Of the 13 antibiotics tested, P. aeruginosa strains showed high resistance rates (ranging from 69.5–82.1%), and MIC levels (MIC90 > 256 μg/ml) to macrolide antibiotics. Of the 131 known macrolide resistance genes, only two genes, mphE and msrE, were identified in 262 clinical P. aeruginosa isolates. Four strains (1.53%, 4/262) carried both the msrE and mphE genes, and an additional three strains (1.15%, 3/262) harbored the mphE gene alone. The cloned msrE and mphE genes conferred higher resistance levels to three second-generation macrolides compared to two first-generation ones. Analysis of MsrE and MphE protein polymorphisms revealed that they are highly conserved, with only 1–3 amino acids differences between the proteins of the same type. It can be concluded that even though the strains showed high resistance levels to macrolides, known macrolide resistance genes are seldom present in clinical P. aeruginosa strains, demonstrating that a mechanism other than this warranted by the mphE and msrE genes may play a more critical role in the bacteria’s resistance to macrolides.Key words: Pseudomonas aeruginosa, macrolide, resistance gene, mphE, msrE     

Bacterial cis-2-unsaturated fatty acids found in the cystic fibrosis airway modulate virulence and persistence of Pseudomonas aeruginosa

There is an increasing appreciation of the polymicrobial nature of many bacterial infections such as those associated with cystic fibrosis (CF) and of the potentially important role for interspecies interactions in influencing both bacterial virulence and response to therapy. Patients with CF are often co-infected with Pseudomonas aeruginosa and other pathogens including Burkholderia cenocepacia and Stenotrophomonas maltophilia. These latter bacteria produce signal molecules of the diffusible signal factor (DSF) family, which are cis-2-unsaturated fatty acids. We have previously shown by in vitro studies that DSF from S. maltophilia leads to altered biofilm formation and increased resistance to antibiotics by P. aeruginosa; these responses of P. aeruginosa require the sensor kinase PA1396. Here we show that DSF signals are present in sputum taken from patients with CF. Presence of these DSF signals was correlated with patient colonization by S. maltophilia and/or B. cenocepacia. Analysis of 50 clinical isolates of P. aeruginosa showed that each responded to the presence of synthetic DSF by increased antibiotic resistance and these strains demonstrated little sequence variation in the PA1396 gene. In animal experiments using CF transmembrane conductance regulator knockout mice, the presence of DSF promoted P. aeruginosa persistence. Furthermore, antibiotic resistance of P. aeruginosa biofilms grown on human airway epithelial cells was enhanced in the presence of DSF. Taken together, these data provide substantial evidence that interspecies DSF-mediated bacterial interactions occur in the CF lung and may influence the efficacy of antibiotic treatment, particularly for chronic infections involving persistence of bacteria.     

Role of the flagellar hook in the structural development and antibiotic tolerance of Pseudomonas aeruginosa biofilms

Pseudomonas aeruginosa biofilms exhibit an intrinsic resistance to antibiotics and constitute a considerable clinical threat. In cystic fibrosis, a common feature of biofilms formed by P. aeruginosa in the airway is the occurrence of mutants deficient in flagellar motility. This study investigates the impact of flagellum deletion on the structure and antibiotic tolerance of P. aeruginosa biofilms, and highlights a role for the flagellum in adaptation and cell survival during biofilm development. Mutations in the flagellar hook protein FlgE influence greatly P. aeruginosa biofilm structuring and antibiotic tolerance. Phenotypic analysis of the flgE knockout mutant compared to the wild type (WT) reveal increased fitness under planktonic conditions, reduced initial adhesion but enhanced formation of microcolony aggregates in a microfluidic environment, and decreased expression of genes involved in exopolysaccharide formation. Biofilm cells of the flgE knock-out mutant display enhanced tolerance towards multiple antibiotics, whereas its planktonic cells show similar resistance to the WT. Confocal microscopy of biofilms demonstrates that gentamicin does not affect the viability of cells located in the inner part of the flgE knock-out mutant biofilms due to reduced penetration. These findings suggest that deficiency in flagellar proteins like FlgE in biofilms and in cystic fibrosis infections represent phenotypic and evolutionary adaptations that alter the structure of P. aeruginosa biofilms conferring increased antibiotic tolerance.Subject terms: Microbiology, Diseases     

Co-Existence of Multidrug-Resistant and -Susceptible Strains of Pseudomonas aeruginosa from a Single Clinical Isolate

Pseudomonas aeruginosa is a leading cause of hospital-acquired infections and difficult to treat due to acquired-resistance to multiple antibiotics. A pair of strains, M38100A and M38100B, previously identified from a single clinical isolate of P. aeruginosa was investigated to understand phenotypic and genotypic characteristics. Results revealed that the pair of strains was very similar for serum susceptibility, growth rate in a complex medium (Luria–Bertani), RAPD-genotype profiles, status of genes encoding type III secretion toxins, and no extra-chromosomal DNA. However, antibiotic susceptibility of the strain M38100B showed resistant to all tested-antibiotics while the strain M38100A showed susceptible to the same tested-antibiotics as similar levels of P. aeruginosa PAO1. The strain M38100B exhibited no growth in a minimal medium as a sole carbon and nitrogen source of glutamate while the strain M38100A grew well in the same minimal medium. These results suggest that multidrug resistance of the strain M38100B may be caused by multiple mutations on its genomic DNA and a precursor stage for a homogeneous multidrug resistant population.     

Influence of Pseudomonas aeruginosa pvdQ Gene on Altering Antibiotic Susceptibility Under Swarming Conditions

In Pseudomonas aeruginosa PAO1, the pvdQ gene has been shown to have at least two functions. It encodes the acylase enzyme and hydrolyzes 3-oxo-C12-HSL, the key signaling molecule of quorum sensing system. In addition, pvdQ is involved in swarming motility. It is required and up-regulated during swarming motility, which is triggered by high cell densities. As high density bacterial populations also display elevated antibiotics resistance, studies have demonstrated swarm-cell differentiation in P. aeruginosa promotes increased resistance to various antibiotics. PvdQ acts as a signal during swarm-cell differentiation, and thus may play a role in P. aeruginosa antibiotic resistance. The aim of this study was to examine whether pvdQ was involved in modifying antibiotic susceptibility during swarming conditions and to investigate the mechanism by which this occurred. We constructed the PAO1pMEpvdQ strain, which overproduces PvdQ. PAO1pMEpvdQ promotes swarming motility, while PAO1ΔpvdQ abolishes swarming motility. In addition, both PAO1 and PAO1pMEpvdQ acquired resistance to ceftazidime, ciprofloxacin, meropenem, polymyxin B, and gentamicin, though PAO1pMEpvdQ exhibited a twofold to eightfold increase in antibiotic resistance compared to PAO1. These results indicate that pvdQ plays an important role in elevating antibiotic resistance via swarm-cell differentiation and possibly other mechanisms as well. We analyzed outer membrane permeability. Our data also suggest that pvdQ decreases P. aeruginosa outer membrane permeability, thereby elevating antibiotic resistance under swarming conditions. Our results suggest new approaches for reducing P. aeruginosa resistance.     

Identification of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> genes associated with antibiotic susceptibility

Pseudomonas aeruginosa causes acute and chronic infections in humans and these infections are difficult to treat due to the bacteria’s high-level of intrinsic and acquired resistance to antibiotics. To address this problem, it is crucial to investigate the molecular mechanisms of antibiotic resistance in this organism. In this study, a P. aeruginosa transposon insertion library of 17000 clones was constructed and screened for altered susceptibility to seven antibiotics. Colonies grown on agar plates containing antibiotics at minimum inhibitory concentrations (MICs) and those unable to grow at 1/2 MIC were collected. The transposon-disrupted genes in 43 confirmed mutants that showed at least a three-fold increase or a two-fold decrease in susceptibility to at least one antibiotic were determined by semi-random PCR and subsequent sequencing analysis. In addition to nine genes known to be associated with antibiotic resistance, including mexI, mexB and mexR, 24 new antibiotic resistance-associated genes were identified, including a fimbrial biogenesis gene pilY1 whose disruption resulted in a 128-fold increase in the MIC of carbenicillin. Twelve of the 43 genes identified were of unknown function. These genes could serve as targets to control or reverse antibiotic resistance in this important human pathogen.     

Prediction of Antibiotic Resistance Evolution by Growth Measurement of All Proximal Mutants of Beta-Lactamase

The antibiotic resistance crisis continues to threaten human health. Better predictions of the evolution of antibiotic resistance genes could contribute to the design of more sustainable treatment strategies. However, comprehensive prediction of antibiotic resistance gene evolution via laboratory approaches remains challenging. By combining site-specific integration and high-throughput sequencing, we quantified relative growth under the respective selection of cefotaxime or ceftazidime selection in ∼23,000 Escherichia coli MG1655 strains that each carried a unique, single-copy variant of the extended-spectrum β-lactamase gene bla_CTX-M-14 at the chromosomal att HK022 site. Significant synergistic pleiotropy was observed within four subgenic regions, suggesting key regions for the evolution of resistance to both antibiotics. Moreover, we propose PEAR^P and PEAR^R, two deep-learning models with strong clinical correlations, for the prospective and retrospective prediction of bla_CTX-M-14 evolution, respectively. Single to quintuple mutations of bla_CTX-M-14 predicted to confer resistance by PEAR^P were significantly enriched among the clinical isolates harboring bla_CTX-M-14 variants, and the PEAR^R scores matched the minimal inhibitory concentrations obtained for the 31 intermediates in all hypothetical trajectories. Altogether, we conclude that the measurement of local fitness landscape enables prediction of the evolutionary trajectories of antibiotic resistance genes, which could be useful for a broad range of clinical applications, from resistance prediction to designing novel treatment strategies.     

A Window of Opportunity to Control the Bacterial Pathogen Pseudomonas aeruginosa Combining Antibiotics and Phages

The evolution of antibiotic resistance in bacteria is a global concern and the use of bacteriophages alone or in combined therapies is attracting increasing attention as an alternative. Evolutionary theory predicts that the probability of bacterial resistance to both phages and antibiotics will be lower than to either separately, due for example to fitness costs or to trade-offs between phage resistance mechanisms and bacterial growth. In this study, we assess the population impacts of either individual or combined treatments of a bacteriophage and streptomycin on the nosocomial pathogen Pseudomonas aeruginosa. We show that combining phage and antibiotics substantially increases bacterial control compared to either separately, and that there is a specific time delay in antibiotic introduction independent of antibiotic dose, that minimizes both bacterial density and resistance to either antibiotics or phage. These results have implications for optimal combined therapeutic approaches.     

Identification of the Biosynthetic Gene Cluster and an Additional Gene for Resistance to the Antituberculosis Drug Capreomycin

Capreomycin (CMN) belongs to the tuberactinomycin family of nonribosomal peptide antibiotics that are essential components of the drug arsenal for the treatment of multidrug-resistant tuberculosis. Members of this antibiotic family target the ribosomes of sensitive bacteria and disrupt the function of both subunits of the ribosome. Resistance to these antibiotics in Mycobacterium species arises due to mutations in the genes coding for the 16S or 23S rRNA but can also arise due to mutations in a gene coding for an rRNA-modifying enzyme, TlyA. While Mycobacterium species develop resistance due to alterations in the drug target, it has been proposed that the CMN-producing bacterium, Saccharothrix mutabilis subsp. capreolus, uses CMN modification as a mechanism for resistance rather than ribosome modification. To better understand CMN biosynthesis and resistance in S. mutabilis subsp. capreolus, we focused on the identification of the CMN biosynthetic gene cluster in this bacterium. Here, we describe the cloning and sequence analysis of the CMN biosynthetic gene cluster from S. mutabilis subsp. capreolus ATCC 23892. We provide evidence for the heterologous production of CMN in the genetically tractable bacterium Streptomyces lividans 1326. Finally, we present data supporting the existence of an additional CMN resistance gene. Initial work suggests that this resistance gene codes for an rRNA-modifying enzyme that results in the formation of CMN-resistant ribosomes that are also resistant to the aminoglycoside antibiotic kanamycin. Thus, S. mutabilis subsp. capreolus may also use ribosome modification as a mechanism for CMN resistance.     

Chloraminated drinking water does not generate bacterial resistance to antibiotics in Pseudomonas aeruginosa biofilms

Aim: To determine if exposure of Pseudomonas aeruginosa biofilms to chloraminated drinking water can lead to individual bacteria with resistance to antibiotics. Methods and Results: Biofilms of P. aeruginosa PA14 were grown in drinking water in a Kadouri drip‐fed reactor; the biofilms were treated with either 0·5 mg l^‐1 or 1·0 mg l^‐1 of chloramine for 15 or 21 days; control biofilms were grown in water without chloramine. Fewer isolates with antibiotic resistance were obtained from the chloramine‐treated biofilms as compared to the control. Minimum inhibitory concentrations (MIC) for selected antibiotic‐resistant isolates were determined using ciprofloxacin, tobramycin, gentamicin, rifampicin and chloramphenicol. All of the isolates tested had increased resistance over the wildtype to ciprofloxacin, rifampicin and chloramphenicol, but were not resistant to tobramycin or gentamicin. Conclusions: Under these test conditions, there was no detectable increase in antibiotic resistance in P. aeruginosa exposed as biofilms to disinfectant residues in chloraminated drinking water. Significance and Impact of the study: Chloramine in drinking water, while unable to kill biofilm bacteria, does not increase the potential of P. aeruginosa to become resistant to antibiotics.