Summary: Treatment of infectious diseases becomes more challenging with each passing year. This is especially true for infections caused by the opportunistic pathogen Pseudomonas aeruginosa, with its ability to rapidly develop resistance to multiple classes of antibiotics. Although the import of resistance mechanisms on mobile genetic elements is always a concern, the most difficult challenge we face with P. aeruginosa is its ability to rapidly develop resistance during the course of treating an infection. The chromosomally encoded AmpC cephalosporinase, the outer membrane porin OprD, and the multidrug efflux pumps are particularly relevant to this therapeutic challenge. The discussion presented in this review highlights the clinical significance of these chromosomally encoded resistance mechanisms, as well as the complex mechanisms/pathways by which P. aeruginosa regulates their expression. Although a great deal of knowledge has been gained toward understanding the regulation of AmpC, OprD, and efflux pumps in P. aeruginosa, it is clear that we have much to learn about how this resourceful pathogen coregulates different resistance mechanisms to overcome the antibacterial challenges it faces.

Antibacterial-Resistant Pseudomonas aeruginosa: Clinical Impact and Complex Regulation of Chromosomally Encoded Resistance Mechanisms

Drug efflux pumps play a key role in drug resistance and also serve other functions in bacteria. There has been a growing list of multidrug and drug-specific efflux pumps characterized from bacteria of human, animal, plant and environmental origins. These pumps are mostly encoded on the chromosome, although they can also be plasmid-encoded. A previous article in this journal provided a comprehensive review regarding efflux-mediated drug resistance in bacteria. In the past 5 years, significant progress has been achieved in further understanding of drug resistance-related efflux transporters and this review focuses on the latest studies in this field since 2003. This has been demonstrated in multiple aspects that include but are not limited to: further molecular and biochemical characterization of the known drug efflux pumps and identification of novel drug efflux pumps; structural elucidation of the transport mechanisms of drug transporters; regulatory mechanisms of drug efflux pumps; determining the role of the drug efflux pumps in other functions such as stress responses, virulence and cell communication; and development of efflux pump inhibitors. Overall, the multifaceted implications of drug efflux transporters warrant novel strategies to combat multidrug resistance in bacteria.

Efflux-Mediated Drug Resistance in Bacteria

The ability of bacteria to control the biophysical properties of their membrane phospholipids allows them to thrive in a wide range of physical environments. Bacteria precisely adjust their membrane lipid composition by modifying the types of fatty acids that are produced by the biosynthetic pathway and altering the structures of pre-existing phospholipids. The recycling of phospholipids that are used as intermediates in the biosynthesis of other major membrane components is also crucial to bilayer stability in dividing cells. Here, the principal genetic and biochemical processes that are responsible for membrane lipid homeostasis in bacteria are reviewed.

Membrane lipid homeostasis in bacteria.

Several pathogens associated with chronic infections, including Pseudomonas aeruginosa in cystic fibrosis pneumonia, Haemophilus influenzae and Streptococcus pneumoniae in chronic otitis media, Staphylococcus aureus in chronic rhinosinusitis and enteropathogenic Escherichia coli in recurrent urinary tract infections, are linked to biofilm formation. Biofilms are usually defined as surface-associated microbial communities, surrounded by an extracellular polymeric substance (EPS) matrix. Biofilm formation has been demonstrated for numerous pathogens and is clearly an important microbial survival strategy. However, outside of dental plaques, fewer reports have investigated biofilm development in clinical samples. Typically biofilms are found in chronic diseases that resist host immune responses and antibiotic treatment and these characteristics are often cited for the ability of bacteria to persist in vivo. This review examines some recent attempts to examine the biofilm phenotype in vivo and discusses the challenges and implications for defining a biofilm phenotype

https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1462-5822.2009.01323.x

Evolving concepts in biofilm infections

Biofilms are surface-attached groups of microbial cells encased in an extracellular matrix that are significantly less susceptible to antimicrobial agents than non-adherent, planktonic cells. Biofilm-based infections are, as a result, extremely difficult to cure. A wide range of molecular mechanisms contribute to the high degree of recalcitrance that is characteristic of biofilm communities. These mechanisms include, among others, interaction of antimicrobials with biofilm matrix components, reduced growth rates and the various actions of specific genetic determinants of antibiotic resistance and tolerance. Alone, each of these mechanisms only partially accounts for the increased antimicrobial recalcitrance observed in biofilms. Acting in concert, however, these defences help to ensure the survival of biofilm cells in the face of even the most aggressive antimicrobial treatment regimens. This review summarises both historical and recent scientific data in support of the known biofilm resistance and tolerance mechanisms. Additionally, suggestions for future work in the field are provided.

Molecular mechanisms of biofilm-based antibiotic resistance and tolerance in pathogenic bacteria

A 10-min method for preparation of highly electrocompetent Pseudomonas aeruginosa cells: Application for DNA fragment transfer between chromosomes and plasmid transformation

For many bacteria, cloning and expression systems are either scarce or nonexistent. We constructed several mini-Tn7 vectors and evaluated their potential as broad-range cloning and expression systems. In bacteria with a single chromosome, including Pseudomonas aeruginosa, Pseudomonas putida and Yersinia pestis, and in the presence of a helper plasmid encoding the site-specific transposition pathway, site- and orientation-specific Tn7 insertions occurred at a single attTn7 site downstream of the glmS gene. Burkholderia thailandensis contains two chromosomes, each containing a glmS gene and an attTn7 site. The Tn7 system allows engineering of diverse genetic traits into bacteria, as demonstrated by complementing a biofilm-growth defect of P. aeruginosa, establishing expression systems in P. aeruginosa and P. putida, and 'GFP-tagging' Y. pestis. This system will thus have widespread biomedical and environmental applications, especially in environments where plasmids and antibiotic selection are not feasible, namely in plant and animal models or biofilms.

A Tn7-based broad-range bacterial cloning and expression system

Broad host-range mini-Tn7 vectors facilitate integration of single-copy genes into bacterial chromosomes at a neutral, naturally evolved site. Here we present a protocol for employing the mini-Tn7 system in bacteria with single attTn7 sites, using the example Pseudomonas aeruginosa. The procedure involves, first, cloning of the genes of interest into an appropriate mini-Tn7 vector; second, co-transfer of the recombinant mini-Tn7 vector and a helper plasmid encoding the Tn7 site-specific transposition pathway into P. aeruginosa by either transformation or conjugation, followed by selection of insertion-containing strains; third, PCR verification of mini-Tn7 insertions; and last, optional Flp-mediated excision of the antibiotic-resistance selection marker present on the chromosomally integrated mini-Tn7 element. From start to verification of the insertion events, the procedure takes as little as 4 d and is very efficient, yielding several thousand transformants per microgram of input DNA or conjugation mixture. In contrast to existing chromosome integration systems, which are mostly based on species-specific phage or more-or-less randomly integrating transposons, the mini-Tn7 system is characterized by its ready adaptability to various bacterial hosts, its site specificity and its efficiency. Vectors have been developed for gene complementation, construction of gene fusions, regulated gene expression and reporter gene tagging.

mini-Tn7 insertion in bacteria with single attTn7 sites: example Pseudomonas aeruginosa.

Background
Traditional gene replacement procedures are still time-consuming. They usually necessitate cloning of the gene to be mutated, insertional inactivation of the gene with an antibiotic resistance cassette and exchange of the plasmid-borne mutant allele with the bacterial chromosome. PCR and recombinational technologies can be exploited to substantially accelerate virtually all steps involved in the gene replacement process.

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An improved method for rapid generation of unmarked Pseudomonas aeruginosa deletion mutants

Pseudomonas aeruginosa biofilms are extremely recalcitrant to antibiotic treatment. Treatment of cystic fibrosis patients with azithromycin (AZM) has shown promise. We used DNA microarrays to identify differentially expressed transcripts in developing P. aeruginosa biofilms exposed to 2 mug/ml AZM. We report that transcripts for multiple restriction-nodulation-cell division (RND) efflux pumps, known to be involved in planktonic antibiotic resistance, and transcripts involved in type III secretion were upregulated in the resistant biofilms that developed in the presence of AZM. Interestingly, the MexAB-OprM and MexCD-OprJ efflux pumps, but not type III secretion, appear to be integral to biofilm formation in the presence of AZM, as evidenced by the fact that a mutant deleted in both mexAB-oprM and mexCD-oprJ was unable to form a biofilm in the presence of AZM. A mutant deleted in type III secretion was still able to form biofilms in the presence of drug. Furthermore, single mexAB-oprM- and mexCD-oprJ-null mutants were able to form a biofilm in the presence of drug, indicating that either of the pumps can confer resistance to AZM during biofilm development. In contrast to planktonically grown cells, where no mexC expression was detectable regardless of the presence of AZM, biofilms exhibited induction of mexC expression from the outset of their formation, but only in the presence of AZM. mexA, which is constitutively expressed in planktonic cells, was uniformly expressed in biofilms regardless of the presence of AZM. These data indicate that the MexCD-OprJ pump acts as a biofilm-specific mechanism for AZM resistance.

Kyoung-Hee Choi

Papers

A 10-min method for preparation of highly electrocompetent Pseudomonas aeruginosa cells: Application for DNA fragment transfer between chromosomes and plasmid transformation

A Tn7-based broad-range bacterial cloning and expression system

mini-Tn7 insertion in bacteria with single attTn7 sites: example Pseudomonas aeruginosa.

An improved method for rapid generation of unmarked Pseudomonas aeruginosa deletion mutants

Molecular Basis of Azithromycin-Resistant Pseudomonas aeruginosa Biofilms