Genome evolution drives transcriptomic and phenotypic adaptation in Pseudomonas aeruginosa during 20 years of infection

The opportunistic pathogen Pseudomonas aeruginosa chronically infects the lungs of patients with cystic fibrosis (CF). During infection the bacteria evolve and adapt to the lung environment. Here we use genomic, transcriptomic and phenotypic approaches to compare multiple isolates of P. aeruginosa collected more than 20 years apart during a chronic infection in a CF patient. Complete genome sequencing of the isolates, using short- and long-read technologies, showed that a genetic bottleneck occurred during infection and was followed by diversification of the bacteria. A 125 kb deletion, an 0.9 Mb inversion and hundreds of smaller mutations occurred during evolution of the bacteria in the lung, with an average rate of 17 mutations per year. Many of the mutated genes are associated with infection or antibiotic resistance. RNA sequencing was used to compare the transcriptomes of an earlier and a later isolate. Substantial reprogramming of the transcriptional network had occurred, affecting multiple genes that contribute to continuing infection. Changes included greatly reduced expression of flagellar machinery and increased expression of genes for nutrient acquisition and biofilm formation, as well as altered expression of a large number of genes of unknown function. Phenotypic studies showed that most later isolates had increased cell adherence and antibiotic resistance, reduced motility, and reduced production of pyoverdine (an iron-scavenging siderophore), consistent with genomic and transcriptomic data. The approach of integrating genomic, transcriptomic and phenotypic analyses reveals, and helps to explain, the plethora of changes that P. aeruginosa undergoes to enable it to adapt to the environment of the CF lung during a chronic infection.

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Combinatorial quorum sensing in Pseudomonas aeruginosa allows for novel cheating strategies

Microbiology ◽

10.1099/mic.0.000941 ◽

2020 ◽

Vol 166 (8) ◽

pp. 777-784 ◽

Cited By ~ 1

Author(s):

James Gurney ◽

Sheyda Azimi ◽

Sam P. Brown ◽

Stephen P. Diggle

Keyword(s):

Pseudomonas Aeruginosa ◽

Quorum Sensing ◽

Type Species ◽

Natural Populations ◽

Opportunistic Pathogen ◽

Growth Media ◽

Public And Private ◽

Content Type ◽

Private Goods ◽

Link Type

In the opportunistic pathogen Pseudomonas aeruginosa, quorum sensing (QS) is a social trait that is exploitable by non-cooperating cheats. Previously it has been shown that by linking QS to the production of both public and private goods, cheats can be prevented from invading populations of cooperators and this was described by Dandekar et al. (Science 2012;338:264–266) as ‘a metabolic incentive to cooperate’. We hypothesized that P. aeruginosa could evolve novel cheating strategies to circumvent private goods metabolism by rewiring its combinatorial response to two QS signals (3O-C12-HSL and C4-HSL). We performed a selection experiment that cycled P. aeruginosa between public and private goods growth media and evolved an isolate that rewired its control of cooperative protease expression from a synergistic (AND-gate) response to dual-signal input to a 3O-C12-HSL-only response. We show that this isolate circumvents metabolic incentives to cooperate and acts as a combinatorial signalling cheat, with higher fitness in competition with its ancestor. Our results show three important principles: first, combinatorial QS allows for diverse social strategies to emerge; second, restrictions levied by private goods are not sufficient to explain the maintenance of cooperation in natural populations; and third, modifying combinatorial QS responses could result in important physiological outcomes in bacterial populations.

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From genotype to phenotype: adaptations of Pseudomonas aeruginosa to the cystic fibrosis environment

Microbial Genomics ◽

10.1099/mgen.0.000513 ◽

2021 ◽

Vol 7 (3) ◽

Author(s):

Laura Camus ◽

François Vandenesch ◽

Karen Moreau

Keyword(s):

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Persistent Infection ◽

Chronic Infection ◽

Respiratory Function ◽

Type Species ◽

Genetic Mutations ◽

Chronic Infections ◽

Content Type ◽

Link Type

Pseudomonas aeruginosa is one of the main microbial species colonizing the lungs of cystic fibrosis patients and is responsible for the decline in respiratory function. Despite the hostile pulmonary environment, P. aeruginosa is able to establish chronic infections thanks to its strong adaptive capacity. Various longitudinal studies have attempted to compare the strains of early infection with the adapted strains of chronic infection. Thanks to new ‘-omics’ techniques, convergent genetic mutations, as well as transcriptomic and proteomic dysregulations have been identified. As a consequence of this evolution, the adapted strains of P. aeruginosa have particular phenotypes that promote persistent infection.

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MexXY RND pump of Pseudomonas aeruginosa PA7 effluxes bi-anionic β-lactams carbenicillin and sulbenicillin when it partners with the outer membrane factor OprA but not with OprM

Microbiology ◽

10.1099/mic.0.000971 ◽

2020 ◽

Vol 166 (11) ◽

pp. 1095-1106 ◽

Cited By ~ 1

Author(s):

Manu Singh ◽

Ellen M. E. Sykes ◽

Yanqi Li ◽

Ayush Kumar

Keyword(s):

Pseudomonas Aeruginosa ◽

Antibiotic Resistance ◽

Outer Membrane ◽

Type Species ◽

State Of The Art ◽

Efflux System ◽

Content Type ◽

Link Type ◽

Resistance Nodulation Division ◽

First Time

Antibiotic resistance in Pseudomonas aeruginosa is a serious concern in healthcare systems. Among the determinants of antibiotic resistance in P. aeruginosa , efflux pumps belonging to the resistance–nodulation–division (RND) family confer resistance to a broad range of antibacterial compounds. The MexXY efflux system is widely overexpressed in P. aeruginosa isolates from cystic fibrosis (CF) patients. MexXY can form functional complexes with two different outer membrane factors (OMFs), OprA and OprM. In this study, using state-of-the-art genetic tools, the substrate specificities of MexXY–OprA and MexXY–OprM complexes were determined. Our results show, for the first time, that the substrate profile of the MexXY system from P. aeruginosa PA7 can vary depending on which OM factor (OprM or OprA) it complexes with. While both MexXY–OprA and MexXY–OprM complexes are capable of effluxing aminoglycosides, the bi-anionic β-lactam molecules carbenicillin and sulbenicillin were found to only be the substrate of MexXY–OprA. Our study therefore shows that by partnering with different OMF proteins MexY can expand its substrate profile.

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Anthranilate Acts as a Signal to Modulate Biofilm Formation, Virulence, and Antibiotic Tolerance of Pseudomonas aeruginosa and Surrounding Bacteria

Microbiology Spectrum ◽

10.1128/spectrum.01463-21 ◽

2022 ◽

Author(s):

Hyeon-Ji Hwang ◽

Xi-Hui Li ◽

Soo-Kyoung Kim ◽

Joon-Hee Lee

Keyword(s):

Pseudomonas Aeruginosa ◽

Antibiotic Resistance ◽

Biofilm Formation ◽

Chronic Infection ◽

Antibiotic Tolerance ◽

Content Type ◽

Before And After

Pseudomonas aeruginosa is a notorious pathogen with high antibiotic resistance, strong virulence, and ability to cause biofilm-mediated chronic infection. We found that these characteristics change profoundly before and after the time when anthranilate is produced as an “anthranilate peak”.

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Characterization of the φCTX-like Pseudomonas aeruginosa phage Dobby isolated from the kidney stone microbiota

Access Microbiology ◽

10.1099/acmi.0.000002 ◽

2019 ◽

Vol 1 (1) ◽

Cited By ~ 2

Author(s):

Genevieve Johnson ◽

Alan J. Wolfe ◽

Catherine Putonti

Keyword(s):

Pseudomonas Aeruginosa ◽

Urinary Tract ◽

Human Body ◽

Kidney Stone ◽

Type Species ◽

Phage Group ◽

Content Type ◽

Link Type ◽

Abundance And Diversity ◽

Cultured Bacteria

Bacteriophages (phages) are vital members of the human microbiota. They are abundant even within low biomass niches of the human body, including the lower urinary tract. While several prior studies have cultured bacteria from kidney stones, this is the first study to explore phages within the kidney stone microbiota. Here we report Dobby, a temperate phage isolated from a strain of Pseudomonas aeruginosa cultured from a kidney stone. Dobby is capable of lysing clinical P. aeruginosa strains within our collection from the urinary tract. Sequencing was performed producing a 37 152 bp genome that closely resembles the temperate P. aeruginosa phage φCTX, a member of the P2 phage group. Dobby does not, however, encode for the cytotoxin CTX. Dobby’s genome was queried against publicly available bacterial sequences identifying 44 other φCTX-like prophages. These prophages are integrated within the genomes of P. aeruginosa strains from a variety of environments, including strains isolated from urine samples and other niches of the human body. Phylogenetic analysis suggests that the temperate φCTX phage species is widespread. With the isolation of Dobby, we now have evidence that phages are members of the kidney stone microbiota. Further investigation, however, is needed to determine their abundance and diversity within these communities.

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Prevalence of Mycoplasma genitalium fluoroquinolone-resistance markers, and dual-class-resistance markers, in asymptomatic men who have sex with men

Journal of Medical Microbiology ◽

10.1099/jmm.0.001429 ◽

2021 ◽

Vol 70 (9) ◽

Author(s):

Teck-Phui Chua ◽

Kaveesha Bodiyabadu ◽

Dorothy A. Machalek ◽

Suzanne M. Garland ◽

Catriona S. Bradshaw ◽

...

Keyword(s):

Antibiotic Resistance ◽

Type Species ◽

Health Centre ◽

Mycoplasma Genitalium ◽

Fluoroquinolone Resistance ◽

Content Type ◽

Link Type ◽

Resistance Markers ◽

Sex With Men ◽

Dual Class

Introduction. Failure of fluoroquinolones, the principal treatment option for macrolide-resistant Mycoplasma genitalium infections, has recently emerged. This is of particular concern for men who have sex with men (MSM), who have high proportions of macrolide-resistant M. genitalium infections. Treatment failure with moxifloxacin is likely the result of single nucleotide polymorphisms (SNPs) in parC, whilst concurrent gyrA mutations may play a role. Gap Statement. The levels of fluoroquinolone resistance and dual-class (i.e. macrolide and fluoroquinolone) resistance in M. genitalium among asymptomatic MSM is unknown. Aim. To (i) determine the proportion of fluoroquinolone resistance and dual-class resistance in M. genitalium infections among asymptomatic MSM, (ii) explore any clinical and behavioural associations with fluoroquinolone resistance, and (iii) determine the distribution of antibiotic resistance among M. genitalium mgpB sequence types (STs). Methodology. M. genitalium positive samples (N=94) were obtained from 1001 asymptomatic MSM enrolled in a study at Melbourne Sexual Health Centre (Carlton, Australia) between August 2016 and September 2017. Sanger sequencing was performed to determine the proportion of M. genitalium infections with SNPs in parC that have previously been associated with failure of moxifloxacin (corresponding to amino changes S83I, D83R, D87Y and D87N) and in gyrA (corresponding to amino acid changes M95I, D99N, D99Y and D99G). Associations between clinical/behavioural factors and parC SNPs were examined. Strain typing was performed by sequencing a portion of the mgpB gene. Results. The proportion of MSM with infections harbouring parC and gyrA SNPs was 13.0 % [95 % confidence interval (CI): 6.8–23.2 %] and 4.7 % (95 % CI: 1.1–13.4 %), respectively; dual-class resistance was 13.0 %. No significant clinical/behavioural associations were found. Antibiotic resistance was not restricted to specific mgpB STs. Conclusion. One in eight (13 %) of asymptomatic MSM with M. genitalium had an infection with dual-class-resistance mutations. Typing by mgpB sequence suggested fluoroquinolone resistance is arising from independent mutation events. This study illustrates that asymptomatic MSM may act as a reservoir for antibiotic-resistant M. genitalium .

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bla OXA-48-like genome architecture among carbapenemase-producing Escherichia coli and Klebsiella pneumoniae in the Netherlands

Microbial Genomics ◽

10.1099/mgen.0.000512 ◽

2021 ◽

Vol 7 (5) ◽

Author(s):

Antoni P. A. Hendrickx ◽

Fabian Landman ◽

Angela de Haan ◽

Sandra Witteveen ◽

Marga G. van Santen-Verheuvel ◽

...

Keyword(s):

Escherichia Coli ◽

Antibiotic Resistance ◽

The Netherlands ◽

Resistance Genes ◽

Type Species ◽

Antibiotic Resistance Genes ◽

Gene Content ◽

Content Type ◽

E Coli ◽

Link Type

Carbapenem-hydrolysing enzymes belonging to the OXA-48-like group are encoded by bla OXA-48-like alleles and are abundant among Enterobacterales in the Netherlands. Therefore, the objective here was to investigate the characteristics, gene content and diversity of the bla OXA-48-like carrying plasmids and chromosomes of Escherichia coli and Klebsiella pneumoniae collected in the Dutch national surveillance from 2014 to 2019 in comparison with genome sequences from 29 countries. A combination of short-read genome sequencing with long-read sequencing enabled the reconstruction of 47 and 132 complete bla OXA-48-like plasmids for E. coli and K. pneumoniae , respectively. Seven distinct plasmid groups designated as pOXA-48-1 to pOXA-48-5, pOXA-181 and pOXA-232 were identified in the Netherlands which were similar to internationally reported plasmids obtained from countries from North and South America, Europe, Asia and Oceania. The seven plasmid groups varied in size, G+C content, presence of antibiotic resistance genes, replicon family and gene content. The pOXA-48-1 to pOXA-48-5 plasmids were variable, and the pOXA-181 and pOXA-232 plasmids were conserved. The pOXA-48-1, pOXA-48-2, pOXA-48-3 and pOXA-48-5 groups contained a putative conjugation system, but this was absent in the pOXA-48-4, pOXA-181 and pOXA-232 plasmid groups. pOXA-48 plasmids contained the PemI antitoxin, while the pOXA-181 and pOXA-232 plasmids did not. Furthermore, the pOXA-181 plasmids carried a virB2-virB3-virB9-virB10-virB11 type IV secretion system, while the pOXA-48 plasmids and pOXA-232 lacked this system. A group of non-related pOXA-48 plasmids from the Netherlands contained different resistance genes, non-IncL-type replicons or no replicons. Whole genome multilocus sequence typing revealed that the bla OXA-48-like plasmids were found in a wide variety of genetic backgrounds in contrast to chromosomally encoded bla OXA-48-like alleles. Chromosomally localized bla OXA-48 and bla OXA-244 alleles were located on genetic elements of variable sizes and comprised regions of pOXA-48 plasmids. The bla OXA-48-like genetic element was flanked by a direct repeat upstream of IS1R, and was found at multiple locations in the chromosomes of E. coli . Lastly, K. pneumoniae isolates carrying bla OXA-48 or bla OXA-232 were mostly resistant for meropenem, whereas E. coli bla OXA-48, bla OXA-181 and chromosomal bla OXA-48 or bla OXA-244 isolates were mostly sensitive. In conclusion, the overall bla OXA-48-like plasmid population in the Netherlands is conserved and similar to that reported for other countries, confirming global dissemination of bla OXA-48-like plasmids. Variations in size, presence of antibiotic resistance genes and gene content impacted pOXA-48, pOXA-181 and pOXA-232 plasmid architecture.

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An antipathogenic compound that targets the OxyR peroxide sensor in Pseudomonas aeruginosa

Journal of Medical Microbiology ◽

10.1099/jmm.0.001341 ◽

2021 ◽

Vol 70 (4) ◽

Author(s):

Hyo-Young Oh ◽

Shivakumar S. Jalde ◽

In-Young Chung ◽

Yeon-Ji Yoo ◽

Hye-Jeong Jang ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Drug Resistance ◽

Stress Responses ◽

Type Species ◽

Reduced Form ◽

Infection Model ◽

Redox Cycle ◽

Content Type ◽

Link Type ◽

Computer Based

Introduction. Antipathogenic or antivirulence strategy is to target a virulence pathway that is dispensable for growth, in the hope to mitigate the selection for drug resistance. Hypothesis/Gap Statment. Peroxide stress responses are one of the conserved virulence pathways in bacterial pathogens and thus good targets for antipathogenic strategy. Aim. This study aims to identify a new chemical compound that targets OxyR, the peroxide sensor required for the full virulence of the opportunistic human pathogen, Pseudomonas aeruginosa . Methodology. Computer-based virtual screening under consideration of the ‘eNTRy’ rules and molecular docking were conducted on the reduced form of the OxyR regulatory domain (RD). Selected hits were validated by their ability to phenocopy the oxyR null mutant and modulate the redox cycle of OxyR. Results. We first isolated three robust chemical hits that inhibit OxyR without affecting prototrophic growth or viability. One (compound 1) of those affected the redox cycle of OxyR in response to H2O2 treatment, in a way to impair its function. Compound 1 displayed selective antibacterial efficacy against P. aeruginosa in Drosophila infection model, without antibacterial activity against Staphylococcus aureus . Conclusion. These results suggest that compound 1 could be an antipathogenic hit inhibiting the P. aeruginosa OxyR. More importantly, our study provides an insight into the computer-based discovery of new-paradigm selective antibacterials to treat Gram-negative bacterial infections presumably with few concerns of drug resistance.

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Study of quorum-sensing LasR and RhlR genes and their dependent virulence factors in Pseudomonas aeruginosa isolates from infected burn wounds

Access Microbiology ◽

10.1099/acmi.0.000211 ◽

2021 ◽

Vol 3 (3) ◽

Author(s):

Aya Ahmad Elnegery ◽

Wafaa Kamel Mowafy ◽

Tarek Ahmed Zahra ◽

Noha Tharwat Abou El-Khier

Keyword(s):

Pseudomonas Aeruginosa ◽

Quorum Sensing ◽

Proteolytic Activity ◽

Virulence Factors ◽

Biofilm Formation ◽

Type Species ◽

Assay Method ◽

Content Type ◽

Burn Wounds ◽

Link Type

Background. Pseudomonas aeruginosa (P. aeruginosa) is an opportunistic pathogen responsible for burn-wound infection. High incidence, infection severity and increasing resistance characterize P. aeruginosa -induced burn infection. Purpose. To estimate quorum-sensing (QS)-dependent virulence factors of P. aeruginosa isolates from burn wounds and correlate it to the presence of QS genes. Methods. A cross-sectional descriptive study included 50 P . aeruginosa isolates from burn patients in Mansoura University Plastic and Burn Hospital, Egypt. Antibiotic sensitivity tests were done. All isolates were tested for their ability to produce biofilm using a micro-titration assay method. Protease, pyocyanin and rhamnolipid virulence factors were determined using skimmed milk agar, King’s A medium and CTAB agar test, respectively. The identity of QS lasR and rhlR genes was confirmed using PCR. Results. In total, 86 % of isolates had proteolytic activity. Production of pyocyanin pigment was manifested in 66 % of isolates. Altogether, 76 % of isolates were rhamnolipid producers. Biofilm formation was detected in 96 % of isolates. QS lasR and rhlR genes were harboured by nearly all isolates except three isolates were negative for both lasR and rhlR genes and two isolates were positive for lasR gene and negative for rhlR gene. Forty-nine isolates were considered as extremely QS-proficient strains as they produced QS-dependent virulence factors. In contrast, one isolate was a QS deficient strain. Conclusions. QS affects P. aeruginosa virulence-factor production and biofilm in burn wounds. Isolates containing lasR and rhlR seem to be a crucial regulator of virulence factors and biofilm formation in P. aeruginosa whereas the lasR gene positively regulates biofilm formation, proteolytic activity, pyocyanin production and rhamnolipid biosurfactant synthesis. The QS regulatory RhlR gene affects protease and rhamnolipid production positively.

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Phylum barrier and Escherichia coli intra-species phylogeny drive the acquisition of antibiotic-resistance genes

Microbial Genomics ◽

10.1099/mgen.0.000489 ◽

2021 ◽

Vol 7 (8) ◽

Author(s):

Marie Petitjean ◽

Bénédicte Condamine ◽

Charles Burdet ◽

Erick Denamur ◽

Etienne Ruppé

Keyword(s):

Escherichia Coli ◽

Antibiotic Resistance ◽

Resistance Genes ◽

Type Species ◽

Antibiotic Resistance Genes ◽

Resistance Pattern ◽

Content Type ◽

E Coli ◽

Link Type ◽

Specific Distribution

Escherichia coli is a ubiquitous bacterium that has been widely exposed to antibiotics over the last 70 years. It has adapted by acquiring different antibiotic-resistance genes (ARGs), the census of which we aim to characterize here. To do so, we analysed 70 301 E. coli genomes obtained from the EnteroBase database and detected 1 027 651 ARGs using the AMRFinder, Mustard and ResfinderFG ARG databases. We observed a strong phylogroup and clonal lineage specific distribution of some ARGs, supporting the argument for epistasis between ARGs and the strain genetic background. However, each phylogroup had ARGs conferring a similar antibiotic class resistance pattern, indicating phenotypic adaptive convergence. The G+C content or the type of ARG was not associated with the frequency of the ARG in the database. In addition, we identified ARGs from anaerobic, non- Proteobacteria bacteria in four genomes of E. coli , supporting the hypothesis that the transfer between anaerobic bacteria and E. coli can spontaneously occur but remains exceptional. In conclusion, we showed that phylum barrier and intra-species phylogenetic history are major drivers of the acquisition of a resistome in E. coli .

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