Mechanisms of Bacterial Resistance to Antibiotics in Infections of COPD Patients

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1 Current Drug Targets, 2011, 12, Mechanisms of Bacterial Resistance to Antibiotics in Infections of COPD Patients Jennelle M. Kyd *, John McGrath and Ajay Krishnamurthy CQ University Australia, Rockhampton Qld. 4702, Australia Abstract: A key characteristic of airway inflammation in chronic obstructive pulmonary disease (COPD) is the persistent presence of bacteria in the lower airways. The most commonly isolated bacteria in the lower respiratory tract of COPD patients are nontypeable Haemophilus influenzae, Moraxella catarrhalis and Streptococcus pneumoniae, with growing evidence of the significance of Pseudomonas aeruginosa infections in severe COPD disease. This review focuses on the antibiotic resistant mechanisms associated with the gram-negative bacteria H. influenzae and M. catarrhalis and comparison with P. aeruginosa infection because of the recent evidence of its significance in patients with severe COPD disease. These mechanisms of resistance to β-lactams in H. influenzae and M. catarrhalis are mostly associated with serine β-lactamases of class A type, whereas P. aeruginosa strains exhibit a much broader repertoire with class A-D type mechanisms. Other mechanisms of antibiotic resistance include membrane permeability, efflux pump systems and mutations in antimicrobial targets. Antimicrobial resistance within biofilm matrices appears to be different to the mechanisms observed when the bacteria are in the planktonic state. P. aeruginosa exhibits a more numerous and diverse range of antimicrobial resistance mechanisms in comparison to M. catarrhalis and H. influenzae. The recognition that P. aeruginosa is associated with exacerbations in patients with more severe COPD and that turnover in infecting strains is detected (unlike in cystic fibrosis patients), then further investigation is required to better understand the contribution of antimicrobial resistance and other virulence mechanisms to poor clinical outcomes to improve therapeutic approaches. Keywords: Gram-negative bacteria, Pseudomonas aeruginosa, β-lactamases, antimicrobial resistance, COPD, biofilms, efflux pumps. INTRODUCTION Bacterial Infections in COPD A key characteristic of airway inflammation in chronic obstructive pulmonary disease (COPD) is the persistent presence of bacteria in the lower airways, even during quiescent stages between acute exacerbations. Various studies report that the most commonly isolated bacteria in the lower respiratory tract of COPD patients are nontypeable Haemophilus influenzae, Moraxella catarrhalis and Streptococcus pneumoniae, with approximately 40 50% of acute exacerbations of COPD being directly linked to a bacterial infection [1]. Improvements in study methods such as bronchoscopic sampling of the lower respiratory tract to compliment the many sputum culture studies and techniques to investigate genetic diversity, alterations in expression and structure of bacterial components have assisted in improving the understanding of infection dynamics associated with exacerbations [1-3]. There is clear evidence that the acquisition of a new bacterial strain provides a two-fold increase in the risk of an exacerbation [1], with this increase in risk associated with all three predominant bacterial pathogens. This new infection induces immune and inflammatory responses that are clinically associated with an acute exacerbation. Pseudomonas aeruginosa is also isolated in sputum cultures from adults with COPD but the significance of this *Address correspondence to this author at the CQ University Australia, Rockhampton Qld. 4702, Australia; Tel: ; Fax: ; j.kyd@cqu.edu.au organism in these clinical settings is not as well defined and its significance is only now being realised. Infection with multi-antibiotic resistant bacteria, and particularly Pseudomonas aeruginosa in the more severe stages of COPD, is associated with accelerating the progression of the lung disease and bronchial obstruction [4]. Recent studies reported that essentially two distinct patterns of carriage of P. aeruginosa are observed in adults with COPD: a short-term colonization that was cleared and a long-term persistent infection [2, 5]. While there is clear evidence of the coincidence of the acquisition of P. aeruginosa in association with an exacerbation, the exacerbations caused by P. aeruginosa were more likely to occur in patients with more advanced COPD. Recent antibiotic therapy and the use of mechanical ventilation for an exacerbation has also been identified as being a risk factor for P. aeruginosa infection [6-8]. It would appear that only a subset of patients become chronically colonized with P. aeruginosa. There has been some conjecture that perhaps colonisation and exacerbation patterns in COPD would be similar to that observed in cystic fibrosis (CF). An initial study in a COPD patient subset suggested that patterns of infection and evolution were the same [9]. However, a recent larger molecular epidemiology and population biology 10-year prospective study of P. aeruginosa in COPD revealed that of the 134 P. aeruginosa COPD isolates collected, 60 were unrelated bacterial clones [2]. The study concluded that intraclonal microevolution and frequent turnover or loss of clones was typical for infections with P. aeruginosa in COPD, differing from the epidemiological pattern of chronic carriage by the same P. aeruginosa clone in patients with cystic fibrosis. This sporadic and intermittent infection pattern is similar to the patterns of turnover for the other bacteria associated with exacerbations in COPD /11 $ Bentham Science Publishers Ltd.

2 522 Current Drug Targets, 2011, Vol. 12, No. 4 Kyd et al. Although there are frequently other bacteria isolated from COPD patient lower airways, these four constitute the predominant etiological agents associated with the exacerbations for which antibiotic therapies might be initiated. This paper will outline the mechanisms of bacterial resistance to antibiotics, focusing on those associated with the gram-negative bacteria, with an emphasis on P. aeruginosa infection in COPD due to the more recent evidence of its significance in patients with severe disease. ANTIBIOTIC THERAPIES Clinical practice decisions about whether or not to treat with antibiotics when bacterial infection is suspected varies. The results from controlled trials vary as to the benefit of the prophylactic use of antibiotics [10], but this use is not recommended as it has been shown to have no effect on the overall frequency of exacerbations in COPD [11]. Antibiotics are only recommended for use in treating exacerbations that show increased purulent sputum or require mechanical ventilation, with many studies recommending antibiotic use only when bacterial infection is confirmed [12]. A number of antibiotics are preferred for use in treating exacerbations. They include trimethoprim-sulfamethoxazole, doxycycline, amoxicillin-clavulanate, and ampicillin with azithromycin, clarithromycin, and levofloxacin recommended for more severe lung infections or where resistance to the other drugs has been identified [13]. In general, a stratified risk management approach is recommended [14]. Sethi and Murphy recommend that for patients with uncomplicated COPD, an advanced macrolide (azithromycin, clarithromycin), ketolide (telithromycin), cephalosporin (cefuroxime, cefpodoxime, or cefdinir) or doxycycline should be considered, but not amoxicillin because of the high incidence of β-lactamase production among H. influenzae and M. catarrhalis. For complicated COPD, antibiotic choices would be a fluoroquinolone (moxifloxacin, gemifloxacin, gatfloxacin, or levofloxacin) or amoxicillin-clavulanate. Clinical trials investigating the efficacy and importance of utilizing antibiotics for the treatment of acute exacerbations in COPD has yielded varying results, with a number of studies appearing to demonstrate minimal apparent benefit. Sethi [1] has proposed that this lack of benefit might be attributable to study design and end point parameters, citing other mucosal infections, such as otitis media and sinusitis, as also not always demonstrating significant differences in outcomes in treatment groups over the placebo controls. Other contributing factors that appear to diminish the potential effectiveness of antibiotic therapies in clinical trial studies are the possible introduced type 2 errors. About 50% of exacerbations are believed to be non-bacterial in cause and in situations where the rate of antibiotic resistance of the pathogen causing bacterial exacerbations is low, along with poor penetration by some drugs into the bronchial tissues and fluids, all are factors that influence the power of the study and the outcome. Combining therapies, such as inhaled steroids and longacting inhaled beta agonists, mucolytics and prophylactic antibiotics have been shown to reduce the frequency of exacerbations; though there is uncertainty about how and when mucolytic and prophylactic antibiotic treatments should be used [15]. In addition, most clinical trials investigating antibiotic treatment of exacerbations of COPD, do not determine relationships that inform the choice of antibiotic in treating an exacerbation, that is, the relationship between the in vitro efficacy to different antibiotics and the clinical outcome of the antibiotic treatment. With recognition of the significance of P. aeruginosa infections in patients with more severe COPD, the choice of antibiotic therapy should be reviewed. Tobramycin is a commonly used antibiotic for the treatment of P. aeruginosa infections. Recently, Dal Negro et al. demonstrated a therapeutic role for aerosol administration of tobramycin in COPD patients colonized with multiresistant P. aeruginosa [16]. This effect was believed to be mediated through reducing eosinophilic inflammation and related to aerosol tobramycin-induced effects on persistent inflammatory damage rather than on direct antimicrobial activity. Macrolide antibiotics can improve airway inflammation in diffuse panbronchiolitis, a complex pulmonary disease most commonly encountered among Asian populations. The condition is characterized by progressive chronic inflammatory sinobronchial disease with mixed obstructive and restrictive pulmonary function with many patients with severe disease infected with P. aeruginosa [17]. While the macrolide clarithromycin improves pulmonary function and controls a diverse range of sputum bacteria in patients with this condition [18], in COPD patients oral clarithromycin had no significant effect on sputum neutrophil numbers or cytokine levels in patients with moderate-to-severe stable COPD [19] suggesting that the inflammatory responses associated with the disease differ between the two conditions. ANTIMICROBIAL RESISTANCE Within the context of COPD, the antimicrobial resistance of the predominant gram-negative bacteria, H. influenzae and M. catarrhalis, have focused predominantly on β-lactamase producing strains. Surveillance studies that include a study of 77 H. influenzae and 30 M. catarrhalis isolates collected between in patients with community-acquired pneumonia (including COPD patients) and another of 143 H. influenzae and 62 M. catarrhalis pediatric isolates collected between both showed similar high numbers of strains producing β-lactamase circulating within the community, affecting susceptibility to this class of antimicrobial, but with sufficient multiple oral treatment options available to which these microbes were susceptible [20, 21]. Most M. catarrhalis strains are β-lactamase producing, but the number of β-lactamase producing H. influenzae strains varies by geographical location ranging from as low as 3% in Germany to as high as 65% in South Korea [22] and is reported to be approximately 17% overall from a worldwide study of 8,523 isolates [23]. The level of colonization or infection by multi-drug resistant bacteria in COPD patients is quite significant. A four-year study in France of COPD patients requiring intubation and mechanical ventilation yielded that although only 30% (260/857) of the patients had bacteria isolated during their exacerbation, 26% (69/260) of these patients were infected with multiple-drug resistant bacteria [8]. These bacteria were predominantly methicillin-resistant Staphylo-

3 Bacterial Resistance to Antibiotics Current Drug Targets, 2011, Vol. 12, No Changes to surface target expression. e.g. penicillin binding proteins Changes to cell permeability. e.g. porin expression Production of enzymes that destroy the antibiotic. e.g. -lactamases Active removal of toxic components including antibiotics. e.g efflux pumps Fig. (1). Schematic of mechanisms of resistance. Bacteria may: change the expression of or mutate the structure of a surface target component, such as the penicillin binding protein; alter cell permeability, such as changes to porin expression; produce enzymes that destroy the antibiotics, such as b-lactamases; or utilize efflux pumps that actively remove toxic components. coccus aureus, ceftazidime- or imipenem-resistant P. aeruginosa, Acinetobacter baumannii, Stenotrophomonas maltophilia, and extended-spectrum beta-lactamase-producing Gram-negative bacilli. MECHANISMS OF RESISTANCE Bacterial resistance to antimicrobials is generally a function of outer membrane permeability and a reduction in ability of the antibiotic to access its target. This is mediated through such mechanisms as the acquisition of genes that: (1) express enzymes, such as β-lactamases, that seek to destroy the antibiotics, neutralizing their action; (2) that initiate metabolic pathways that alter bacterial cell walls, changing either the binding target of the antimicrobial or cell permeability through down-regulation or mutation of genes for membrane or metabolic components; or (3) establish efflux pumps to expel the antibiotic from the cell before it exerts its effect (Fig. 1). The presence, range of activity, level of production of β-lactamases, the presence and efficiency of efflux mechanisms, the expression of porins and the affinity of the antibiotic to penicillin-binding protein (PBP) target sites all contribute to variations in the spectrum of antibiotic resistance. In general, horizontal gene transfer mechanisms are responsible for the presence of genes associated with antibiotic resistance or the regulation of expression of bacterial components. These genes can be located on plasmids, in transposons or integrons or are part of the bacteria s chromosomal DNA that may respond to changed regulatory signals (Table 1). There is also strong evidence that the formation of biofilms associated with chronic infections provides a physical barrier to antibiotic penetration. Mechanisms of antimicrobial resistance have been the subject of other recent reviews, including reviews specific for some of these pathogens [23, 28-31]. Of significant concern are the increasing levels of multidrug-resistant P. aeruginosa with many clinical isolates in the United States exhibiting resistance profiles to the more routinely used drugs such as tobramycin, gentamycin, ciprofloxacin, cefotaxime, ceftazidime and piperacillin [28]. β-lactamases Acquisition or expression of β-lactamases is a common form of resistance that mediates its mode of action through rupturing the amide bond of the β-lactam ring (Reviewed in [30, 31]). There are four classes of β-lactamases (A D) associated with the functional classification of the enzyme Table 1. Summary of Genetic Basis for Antibiotic Resistance Gene Presence Type Plasmids Transposons Integrons Chromosomal Description Circular DNA capable of replication that allows rapid synthesis of multiple copies of genes, including genes for antibiotic resistance determinants [24]. DNA that can move from site to site including between chromosomal and plasmid locations. The antibiotic resistance gene is often flanked by insertion sequence elements [25]. Integrons are assembly platforms that incorporate exogenous DNA open reading frames using site-specific recombination that ensures correct expression. These insertions are converted to functional genes. Essential elements include an integrase; a primary recombination site; and a promoter that directs transcription [26]. Bacterial chromosomal DNA may encode proteins that provide a resistance mechanism. An example is the genes that mediate small molecule efflux. These remove toxic compounds from the bacterium, including a capacity to remove antibiotics [27].

4 524 Current Drug Targets, 2011, Vol. 12, No. 4 Kyd et al. substrate and inhibitor profiles with classes A, C and D operating through a serine-based mechanism and class B or (metallo-β-lactamases) requiring zinc for their action. M. catarrhalis strains exhibit a greater than 90% expression of β-lactamases that have a penicillinase-type profile, but are very susceptible to β-lactamase inhibitors [32]. M. catarrhalis produces novel β-lactamases, called BRO, that have two type classifications, BRO-1 (predominant) and BRO-2. The β-lactamase-produced in these strains is encoded by the bla gene and exhibit differences in their susceptibility patterns although their sequences differ by only minor amino acid changes [33-35]. The penicillin G MICs for BRO-1 isolates were found to be significantly higher than those for BRO-2 isolates [34], with resistance to clarithromycin, tetracycline and trimethoprim sulphamethoxazole relatively low at was 1.1%, 2.2% and 1.1%, respectively, of 90 clinical isolates. They exhibit relatively weak activity in comparison with other β-lactamases and production is constitutive, that is, not regulated by the presence of antibiotics [36]. BRO enzymes are encoded for by chromosomal genes, but appear to be readily transferred by conjugation, although the exact process for this is not known. The uptake of the genes associated with M. catarrhalis β-lactamases exhibited one of the fastest spreads of any known β-lactamase [37]. This β-lactamase does not appear to share nucleotide sequence homology with other known serine β-lactamases of class A type, but does have similarities in the amino acid sequence [35]. The origin remains unknown, but the gene G+C content differs from that of the M. catarrhalis genome and the adjacent open reading frames. It is believed that the spread has been via horizontal gene transfer as there is no evidence of a transposon-encoded β-lactamase [35]. In general, H. influenzae exhibits resistance to β-lactams via either production of a β-lactamase or the presence of altered penicillin-binding proteins. Most resistant strains possess only one of these mechanisms, but a few strains will possess both and are usually known as β-lactamase-positive amoxicillin-clavulanate-resistant [22, 38, 39]. There are two types of β-lactamases produced by H. influenzae, TEM and ROB [23], with both being plasmid-mediated class A serine β-lactamases with similar substrate profiles and, like the M. catarrhalis β-lactamases, are susceptible to β-lactamase inhibitors [40-42]. The bla TEM gene in H. influenzae is believed to have been transposed from Enterobacteriaceae onto cryptic plasmids in Haemophilus [43] and there are two different TEM-specifying plasmid groups: the small nonconjugative plasmids with one bla TEM resistance determinant and larger conjugative plasmids with multiple resistant genes [23]. Similarly, the ROB-1 gene is found on small plasmids and there is an association between its presence and higher cefaclor MICs (Minimum Inhibitory Concentrations) [40]. It appears that concerns over enrichment of the H. influenzae population for resistance to extended spectrum cephalosporins as a result of selective pressure from cefaclor use may not be a problem, with ROB-1 mutants that produce additional spectrum activity exhibiting decreased hydrolyzing ability for cefaclor [44]. P. aeruginosa exhibits a far more complex and extensive repertoire of β-lactamase genes and gene products than either H. influenzae or M. catarrhalis (Table 2). With recognition of the significance of P. aeruginosa infection in exacerbations of COPD patients with more severe disease and evidence that there is turnover of infecting strains, this complex repertoire has the potential to challenge physicians to determine the most appropriate therapy. The class A extended spectrum β-lactamases (ESBLs) include TEM, SHV, PER, VEB, GES/IBC, BEL and CTX-M types [45-52], all with low common genetic identity but with similar hydrolysis profiles. The dissemination of the genes encoding Table 2. Classes of β-lactamases in P. aeruginosa β-lactamase Class Description Types Common Antibiotic Resistances TEM SHV Class A Extended Spectrum (ESBL) PER VEB GES/IBC BEL CTX-M Varies by type but includes: penicillin, amoxicillin, ampicillin, cefzolin, cefuroxim, methicillin, oxacillin, cefepime. PSE-1 (CARB-2) Class A Carbenicillin hydrolyzing PSE-4 (CARB-1) CARB-3 CARB-4 Additional to above, varying susceptibilities to cefpirome and axtreonam. Class B Metallo (MBL) Zn 2+ requiring Carbapenem-hydrolyzing enzymes As for class A and includes imipenem, meropenem and ertapenem and cephamycins. Are not susceptible to inhibitors such as clavulanic acid. Class C Cephalosporinase AmpC but involves several genes (AmpR, AmpG and AmpDs) Cephamycins as well as Class A groups. Increased resistance occurs in presence of β-lactams such as imipenem and cefoxitin. Class D Oxacillinase Ceflazidine-hydrolyzing OXA genes Defotaxime, defepime, defpirome, aztreonam, oxacillin, methicillin and cloxacillin.

5 Bacterial Resistance to Antibiotics Current Drug Targets, 2011, Vol. 12, No these enzymes plays an important role in antibiotic resistance dissemination, with plasmids and integrons important contributing factors in this dissemination. Plasmid localization has been shown for most of the genes encoding the TEM and SHV enzymes, whereas genes encoding VEB- and GES-type enzymes are usually located in class 1 integrons, and an additional route of dissemination is provided by some genes being on transposons [53-56]. The diversity of these locations provides multiple mechanisms for gene mobility and can result in ESBL-encoding genes being present on both plasmids and the chromosome. There are also Class A carbenicillin hydrolysing β- lactamases with Pseudomonas-specific enzyme (PSE) types that include PSE-1 (CARB-2), PSE-4 (CARB-1), CARB-3 and CARB-4 with PSE-1, PSE-4 and CARB-3 closely related but only 86 % homologous with CARB-4 [57, 58]. It is believed that the P. aeruginosa bla CARB-4 gene was acquired from other bacterial species. Strains producing these enzymes have variable susceptibility to cefepime, cefpirome and aztreonam, but 100 % susceptibility towards ceftazidime and carbapenems. The class B metallo-β-lactamases Class B (MBLs) are the carbapenem-hydrolysing enzymes, or carbapenemases, requiring the presence of Zn 2+ in their active centre [59]. These confer resistance to all β-lactams, including the carbapenems imipenem and meropenem, but not to aztreonam. β-lactamase inhibitors, such as clavulanic acid, do not inhibit and instead chelators, such as EDTA, are required to suppress their activity [60]. MBL-producing strains are often multi-drug resistant, causing infections that are difficult to treat and associated with high mortality rates [59]. This has led to the use of drugs such as colistin and polymyxin B, which are older and more toxic [61, 62]. P. aeruginosa can produce a molecular class C inducible chromosome-encoded AmpC β-lactamase (cephalosporinase) that is usually expressed in low levels but can increase significantly in the presence of inducing β-lactams (especially imipenem and cefoxitin) by up-regulation of multiple pathways [63]. Weak AmpC inducers, such as penicillins and cephalosporins, can cause selection of high level AmpC producers during treatment and AmpC β- lactamase activity is not inhibited by β-lactamase inhibitors such as clavulanic acid [64, 65]. Several genes are involved in AmpC regulation and the process is linked with peptidoglycan recycling [66]. AmpR, a member of the LysR family, is related to peptidoglycan processing and is contiguous to AmpC yet divergently transcribed, and is necessary for this β-lactamase induction [67, 68]. AmpG, is considered to encode signal molecules associated with AmpC induction and is a transmembrane protein that acts as a permease for 1,6-anhydromurapeptide [69, 70]. Repression of AmpC expression is associated with a cytosolic N-acetylanhydromuramyl-L-alanine amidase endcoded by AmpD, but it also has a major role in peptidoglycan catabolism products [66, 71]. Mutations in AmpD is one of the most common mechanisms for causing AmpC hyperproduction and β-lactam resistance, but in P. aeruginosa there are three AmpD homologues, AmpD, AmpDh2 and AmpDh3, enabling a stepwise upregulation mechanism of AmpC [72-74]. It provides for an unusual sequentially-amplified chromosomally-encoded resistance mechanism that ultimately leads to constitutive hyperexpression with a triple mutant able to exhibit more than 1000-fold (compared to the wild-type) depressed expression. This provides P. aeruginosa with a biologically efficient and effective resistance mechanism. Oxacillinase (OXA) type enzymes are molecular class D and provide resistance to carboxypenicillins and ureidopenicillins, excluding ceftazidime [42, 57]. The ceftazidime hydrolysing extended-spectrum OXA are very clinically signifycant, with a hydrolysis spectrum that includes cefotaxime, cefepime, cefpirome, aztreonam and moxalactam and their activity is not suppressed by β-lactamase inhibitors (except for OXA-18) [31, 75]. There is an extensive repertoire of OXA genes that are generally considered to fall into one of five groups [76] with most of the extended-spectrum OXAs encoded by plasmid- or integron-located genes contributing to their easy dissemination and high prevalence [77]. Alternate β-lactam Resistance Mechanisms Both H. influenzae and P. aeruginosa also have the capacity to modify the β-lactam target site through alteration of the penicillin-binding proteins (PBP). Mutations in the ftsi gene that are associated with PBP-3 substitutions vary across clinical isolates and can cluster within geographical regions but which specific mutations may be associated with the level of β-lactamase negative ampicillin-resistance (BLNAR) in H. influenzae are not conclusive (reviewed in [23]). This is because of the diversity of substitutions. In contrast, alterations to PBPs in P. aeruginosa are the rarest of the β-lactam resistance mechanisms but alterations in PBP-3 (overproduction) and PBP-4 (mutation) have been reported [78]. Membrane Permeability The Role of Porins Porins of gram-negative bacteria enable either specific or general conductance of components across the bacterial outer membrane. These proteins can frequently be involved in antimicobial resistance (reviewed in [79]). H. influenzae expresses a major outer membrane porin called P2 [80]. Porin alterations that are associated with a decrease in outer membrane permeability for H. influenzae have been shown to only partially contribute to resistance to ampicillin, penicillin, cephalothin and chloramphenicol [81, 82] and this has not been considered a major mechanism of antimicrobial resistance in H. influenzae, despite P2 proteins exhibiting significant sequence variations between strains [80]. The only well characterized porin of M. catarrhalis is M35 [83, 84] and recently Jetter et al. reported that ciprofloxacin, levofloxacin and moxifloxacin, cefuroxime, imipenem, amoxicillin-clavulanate, ampicillin and amoxicillin demonstrated differences in the MIC between the wildtype and the M35 mutant strains but no differences for penicillin G, ceftriaxone, meropenem, erythromycin, doxycycline, gentamicin, and vancomycin [85]. In particular, ampicillin and amoxicillin had 2.5 to 2.9-fold MIC increases in the M35 mutants, providing strong evidence for the role of this porin in membrane permeability and the potential of strains lacking M35 expression is a previously unknown mechanism of aminopenicillin resistance in M. catarrhalis. In contrast, deficiency in P. aeruginosa porin OprD and its association with imipenem-resistance has been exten-

6 526 Current Drug Targets, 2011, Vol. 12, No. 4 Kyd et al. sively studied [31, 78, 86]. This porin forms a specific channel that enables the passage of basic amino acids and carbapenems and while mutational loss of OprD determines resistance to carbapenems, it was only functional when chromosomal AmpC β-lactamase was expressed [87], demonstrating cooperation between the two mechanisms. In contrast, PBPs were not found to have a role in imipenem resistance in conjunction with alterations in OprD [86]. Efflux Pump Systems Efflux systems are widely distributed among gramnegative bacteria, and are linked to the upregulation of certain outer membrane proteins. They are an added mechanism of β-lactam resistance and to the development of multiple resistances to all strategic antipseudomonal antibiotics [30, 31]. The AcrAB efflux pump for H. influenzae was first described in 1997 and is constitutively produced by Haemophilus [88]. Similar to other resistance-nodulationcell division transporters, AcrB associates with AcrA, a periplasmic membrane fusion protein, and the outer membrane channel TolC [89]. AcrAB efflux has not been shown to have an important role in the efflux of ß-lactam antibiotics, but is associated with the efflux of macrolide antibiotics. However, some BLNAR strains with high ampicillin resistance have combined resistance mechanisms in PBP3 and in the AcrAB efflux pump [90]. AcrAB provides an important inhibition mechanism against the macrolides and ketolides, therefore is it speculated that the use of these antibiotics may select for over expressing AcrAB strains and hence increase or augment resistance to ß-lactam antibiotics. An understanding of these mechanisms and the potential to contribute to the resistance to antimicrobial therapies against P. aeruginosa infections in COPD patients requires further investigation. In P. aeruginosa the efflux system is linked to several outer membrane proteins that constitute different efflux pump systems. These are MexAB OprM, MexCD OprJ, MexEF OprN, MexXY OprM and MexVW- OprM, with fluoroquinolones the only substrate common to each [91, 92]. Efflux pump associated fluoroquinolone resistance is also linked with mutations in the DNA gyrase and topoisomerase IV genes. A single mutation in gyra with a wild-type level of the MexAB-OprM efflux pump had 128 times higher fluoroquinolone resistance than cells lacking the MexAB-OprM [93]. Type III secretion system virulence is also associated with poor patient outcomes. In the study by Wong-Beringer et al., in clinical isolates the exos cytotoxin gene occurred more frequently than those encoding only exou, with exou + isolates more likely to be fluoroquinoloneresistant, exhibit both a gyra mutation and an efflux pump over-expressed phenotype. This suggests that exou + and fluoroquinolone resistance may be co-selected traits that result in highly virulent and resistant strains [94]. Peroxide stress has also been shown to contribute to antibiotic resistance through sensing of this stress by MexR, a protein that is a negative regulator of MexAB-oprM through gene suppression [95]. Cys-residues of MexR are redox-active, leading to dissociation from promoter DNA and depression of the mexab-oprm operon and hence, increased antibiotic resistance. Many antibiotics generate oxidative stress through hydroxyl radical formation [96]. In general, over expression of efflux pumps contribute to a wide range of resistance mechanisms in P. aeruginosa and in addition to the fluoroquinolones, include β-lactams, aminoglycosides and polymyxin B [87]. The interplay between various systems of antimicrobial resistance and the frequent association between multiple mechanisms and poor clinical parameters highlights the potential importance of monitoring the infectious isolates in patients with COPD and P. aeruginosa infection. Biofilms There is indirect evidence that H. influenzae may form biofilms in the lower airway of COPD patients. Peroxiredoxin-glutaredoxin is present in greater abundance in H. influenzae biofilm compared to planktonic bacterial cultures and has been shown to be involved in biofilm formation [97]. This study by Murphy et al. showed that some COPD patients developed antibodies to peroxiredoxin-glutaredoxin post-infection indicating that it is expressed by H. influenzae during infection of the human respiratory tract and therefore may be indicative of bacterial biofilm formation within these patients. A mouse model of COPD has also yielded evidence of the ability of H. influenzae to form biofilms in the lungs [98]. The ability of antimicrobials to exert their effect on biofilms is generally investigated in in vitro studies. In a study by Kaji et al., levelfloxacin and gatifloxacin (fluoroquinolones), ampicillin (penicillin group), cefotaxine (cephalosporin) and erythromycin and clarithromycin (macrolides) were assessed for effectiveness in vitro against both β- lactamase-negative ampicillin-susceptible and ampicillinresistant H. influenzae strains in mature biofilms. Only the fluoroquinolones were able to inhibit biofilm formation and gatifloxacin was able to completely kill bacteria within the biofilm [99]. The study by Roveta et al., demonstrated that the fourth generation fluoroquinolone, moxifloxacin, inhibited biofilm synthesis and induced slime disruption, but was less effective on mature biofilms [100]. In another study, the improved clinical outcomes in chronic lung disease observed with long-term, low-dose azithromycin may be associated with inhibiting bacterial biofilms in these patients [101]. Understanding biofilm formation is important to developing effective methods of treatment. Both proteinaceous adhesins and extracellular DNA contribute to H. influenzae biofilm cohesion, with this matrix contributing to antibiotic resistance [102, 103]. Biofilms containing M. catarrhalis have been found in the middle ear of OM patients [104] and the capacity of M. catarrhalis to form biofilms has been demonstrated in in vitro assays [ ]. To date there have been no reports of evidence of biofilm formation in patients with COPD, so this as a mechanism of resistance to or avoidance of the actions of antibiotics remains unknown. However, the production of β-lactamase by M. catarrhalis has been shown to confer levels of antibiotic protection to pathogens such as S. pneumoniae. Budhani et al. showed that penicillin-sensitive pneumococcus remained susceptible to a range of β-lactam antibiotics in biofilms [108], but when grown with β- lactamase-positive M. catarrhalis the pneumococcus was protected [109]. The levels of β-lactamase activity differed between cell-free supernatants of broth culture and in biofilm effluent, with levels in the biofilm significantly lower,

7 Bacterial Resistance to Antibiotics Current Drug Targets, 2011, Vol. 12, No indicating a difference in production and ability to confer resistance between the planktonic and biofilm states. The ability of P. aeruginosa to form biofilms during infection has been well documented, particularly in association with chronic lung infection in CF patients. Various mechanisms for the antimicrobial resistance associated with biofilms have been proposed and tested, including the role of exclusion by the exopolysaccharide matrix, limited oxygen and reduced metabolic activity within the biofilms [110]. Although this prevents some antibiotics accessing the bacteria or exerting their effects, antibiotics such as tobramycin and ciprofloxacin are able to penetrate the biofilms but fail to effectively kill the bacteria [111, 112]. Therefore limited antibiotic diffusion is not the only protective mechanism of biofilms. Cells near the air interface in antibiotic-treated biofilms show antibiotic-mediated lysis and further investigation confirmed that oxygen limitation and low metabolic activity in the interior of the biofilm, rather than poor antibiotic penetration, correlated with antibiotic tolerance within some P. aeruginosa biofilms [112]. NdvB has been identified as a gene important in antimicrobial resistance in biofilms, acting via the interaction of ndvb-derived glucans with tobramycin [113]. Within the repertoire of antimicrobial resistance mechanisms that exist for P. aeruginosa, Zhang and Mah [114] have recently identified a novel efflux pump that is more highly expressed in biofilm cells. When the PA genes were deleted, the biofilms were more sensitive to tobramycin, gentamicin and ciprofloxacin. In addition, they were able to demonstrate that the combination of ndvb and PA increased resistance compared to their single resistance levels, indicating that these contribute through two different mechanisms. In contrast, expression of mexab-oprm and mexcd-oprj efflux systems appear to decrease over time in the developing biofilm and none of the four efflux pumps, MexAB- OprM, MexCD-OprJ, MexEF-OprN, and MexXY, increased expression in P. aeruginosa biofilms indicating that they do not appear to play a role in the antibiotic-resistant phenotype of P. aeruginosa biofilms [115]. The other structural element of biofilms recently shown to have a role in antimicrobial resistance is the extracellular DNA matrix that is believed to function as a structural support to maintain biofilm architecture. The extracellular DNA was shown to have antimicrobial activity at physiologically relevant concentrations, by causing cell lysis through chelating cations that stabilize the lipopolysaccharide and outer membrane [116]. In a cationic-limited environment, DNA-induced expression of PA3552-PA3559, the PhoPQ- and PmrAB-regulated cationic antimicrobial peptide resistance operon, resulting in up to a >2500-fold increase in resistance to cationic antimicrobial peptides and 640-fold increased resistance to aminoglycosides. This confirmed an important role for the extracellular DNA in the biofilm matrix in forming cation gradients and inducible antibiotic resistance. HYPERMUTATION P. aeruginosa strains have also been categorized by their ability to hypermutate. Some strains have been shown to have up to a 1,000-fold spontaneous mutation rate that can be attributed to defects in genes involved in DNA repair or error avoidance [117]. Hypermutation has been found to be a key factor for the development of multiple-antimicrobial resistance associated with P. aeruginosa in chronic lung infections, mostly associated with CF [118, 119]. This may have important consequences for the treatment of chronic infections, but further studies are required to determine the significance of these strains as contributing to antibiotic resistance in strains associated with infections in COPD. CONCLUSION Essentially, the challenge associated with selecting appropriate antimicrobial therapy for exacerbations caused by bacterial infection in patients with COPD can be complicated by the presence of antimicrobial resistance mechanisms. Although M. catarrhalis has been a bacterium that exhibited one of the most rapid transmission of the genes responsible for β-lactamase, it does not appear to have adopted an extensive array of β-lactamase resistance mechanisms. In contrast, to H. influenzae utilizes mechanisms associated with ß-lactamases, penicillin-binding proteins (PBPs) and efflux pumps. Both these bacteria may also be able to utilise antimicrobial mechanisms associated with biofilm formation, however, the formation of biofilms in COPD patients still requires further investigation. The mechanisms of resistance to β-lactams in H. influenzae and M. catarrhalis are mostly associated with serine β-lactamases of class A type, whereas P. aeruginosa strains exhibit a much broader repertoire with class A-D type mechanisms. P. aeruginosa appears capable of exhibiting a range of effective mechanisms of antibiotic resistance that include alterations membrane permeability, efflux pump systems and mutations in antimicrobial targets. The mechanisms by which bacteria within a P. aeruginosa biofilm matrix confers antibiotic resistance differs from when the bacteria are in the planktonic state. 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