Putrescine Reduces Antibiotic-Induced Oxidative Stress as a Mechanism of Modulation of Antibiotic Resistance in Burkholderia cenocepacia

Communication of antibiotic resistance among bacteria via small molecules is implicated in transient reduction of bacterial susceptibility to antibiotics, which could lead to therapeutic failures aggravating the problem of antibiotic resistance. Released putrescine from the extremely antibiotic-resistant bacterium Burkholderia cenocepacia protects less-resistant cells from different species against the antimicrobial peptide polymyxin B (PmB). Exposure of B. cenocepacia to sublethal concentrations of PmB and other bactericidal antibiotics induces reactive oxygen species (ROS) production and expression of the oxidative stress response regulator OxyR. We evaluated whether putrescine alleviates antibiotic-induced oxidative stress. The accumulation of intracellular ROS, such as superoxide ion and hydrogen peroxide, was assessed fluorometrically with dichlorofluorescein diacetate, while the expression of OxyR and putrescine synthesis enzymes was determined in luciferase assays using chromosomal promoter-lux reporter system fusions. We evaluated wild-type and isogenic deletion mutant strains with defects in putrescine biosynthesis after exposure to sublethal concentrations of PmB and other bactericidal antibiotics. Exogenous putrescine protected against oxidative stress induced by PmB and other antibiotics, whereas reduced putrescine synthesis resulted in increased ROS generation and a parallel increased sensitivity to PmB. Of the 3 B. cenocepacia putrescine-synthesizing enzymes, PmB induced only BCAL2641, an ornithine decarboxylase. This study reveals BCAL2641 as a critical component of the putrescine-mediated communication of antibiotic resistance and as a plausible target for designing inhibitors that would block the communication of such resistance among different bacteria, ultimately reducing the window of therapeutic failure in treating bacterial infections.     

Computer-aided design of agents that inhibit the cep quorum-sensing system of Burkholderia cenocepacia

Recent research has provided evidence that interference with bacterial cell-to-cell signaling is a promising strategy for the development of novel antimicrobial agents. Here we report on the computer-aided design of novel compounds that specifically inhibit an N-acyl-homoserine lactone-dependent communication system that is widespread among members of the genus Burkholderia. This genus comprises more than 30 species, many of which are important pathogens of animals and humans. Over the past few years, several Burkholderia species, most notably Burkholderia cenocepacia, have emerged as important opportunistic pathogens causing severe pulmonary deterioration in persons with cystic fibrosis. As efficient treatment of Burkholderia infections is hampered by the inherent resistance of the organisms to a large range of antibiotics, novel strategies for battling these pathogens need to be developed. Here we show that compounds targeting the B. cenocepacia signaling system efficiently inhibit the expression of virulence factors and attenuate the pathogenicity of the organism.     

Clinafloxacin for Treatment of Burkholderia cenocepacia Infection in a Cystic Fibrosis Patient

Respiratory infection with Burkholderia cenocepacia is associated with accelerated decline in lung function and increased mortality in cystic fibrosis (CF) patients (A. M. Jones, M. E. Dodd, J. R. W. Govan, V. Barcus, C. J. Doherty, J. Morris, and A. K. Webb, Thorax 59:948–951, 2004, http://dx.doi.org/10.1136/thx.2003.017210). B. cenocepacia often possesses innate resistance to multiple antimicrobial classes, making eradication uncommon in established infection (P. B. Davis, Am J Respir Crit Care Med 173:475–482, 2006, http://dx.doi.org/10.1164/rccm.200505-840OE). We report the use of clinafloxacin in a CF patient with advanced B. cenocepacia infection, present pharmacokinetic (PK) data, and discuss the potential therapeutic role of clinafloxacin in patients with this condition.     

Aerosol Phage Therapy Efficacy in Burkholderia cepacia Complex Respiratory Infections

Phage therapy has been suggested as a potential treatment for highly antibiotic-resistant bacteria, such as the species of the Burkholderia cepacia complex (BCC). To address this hypothesis, experimental B. cenocepacia respiratory infections were established in mice using a nebulizer and a nose-only inhalation device. Following infection, the mice were treated with one of five B. cenocepacia-specific phages delivered as either an aerosol or intraperitoneal injection. The bacterial and phage titers within the lungs were assayed 2 days after treatment, and mice that received the aerosolized phage therapy demonstrated significant decreases in bacterial loads. Differences in phage activity were observed in vivo. Mice that received phage treatment by intraperitoneal injection did not demonstrate significantly reduced bacterial loads, although phage particles were isolated from their lung tissue. Based on these data, aerosol phage therapy appears to be an effective method for treating highly antibiotic-resistant bacterial respiratory infections, including those caused by BCC bacteria.     

Cell Wall Recycling-Linked Coregulation of AmpC and PenB β-Lactamases through ampD Mutations in Burkholderia cenocepacia

In many Gram-negative pathogens, mutations in the key cell wall-recycling enzyme AmpD (N-acetyl-anhydromuramyl-l-alanine amidase) affect the activity of the regulator AmpR, which leads to the expression of AmpC β-lactamase, conferring resistance to expanded-spectrum cephalosporin antibiotics. Burkholderia cepacia complex (Bcc) species also have these Amp homologs; however, the regulatory circuitry and the nature of causal ampD mutations remain to be explored. A total of 92 ampD mutants were obtained, representing four types of mutations: single nucleotide substitution (causing an amino acid substitution or antitermination of the enzyme), duplication, deletion, and IS element insertion. Duplication, which can go through reversion, was the most frequent type. Intriguingly, mutations in ampD led to the induction of two β-lactamases, AmpC and PenB. Coregulation of AmpC and PenB in B. cenocepacia, and likely also in many Bcc species with the same gene organization, poses a serious threat to human health. This resistance mechanism is of evolutionary optimization in that ampD is highly prone to mutations allowing rapid response to antibiotic challenge, and many of the mutations are reversible in order to resume cell wall recycling when the antibiotic challenge is relieved.     

Antibacterial Activity of Eravacycline (TP-434), a Novel Fluorocycline,against Hospital and Community Pathogens

Eravacycline (TP-434 or 7-fluoro-9-pyrrolidinoacetamido-6-demethyl-6-deoxytetracycline) is a novel fluorocycline that was evaluated for antimicrobial activity against panels of recently isolated aerobic and anaerobic Gram-negative and Gram-positive bacteria. Eravacycline showed potent broad-spectrum activity against 90% of the isolates (MIC₉₀) in each panel at concentrations ranging from ≤0.008 to 2 μg/ml for all species panels except those of Pseudomonas aeruginosa and Burkholderia cenocepacia (MIC₉₀ values of 32 μg/ml for both organisms). The antibacterial activity of eravacycline was minimally affected by expression of tetracycline-specific efflux and ribosomal protection mechanisms in clinical isolates. Furthermore, eravacycline was active against multidrug-resistant bacteria, including those expressing extended-spectrum β-lactamases and mechanisms conferring resistance to other classes of antibiotics, including carbapenem resistance. Eravacycline has the potential to be a promising new intravenous (i.v.)/oral antibiotic for the empirical treatment of complicated hospital/health care infections and moderate-to-severe community-acquired infections.     

DNA microarray-based detection and identification of Burkholderia mallei,Burkholderia pseudomallei and Burkholderia spp.

We developed a rapid oligonucleotide microarray assay based on genetic markers for the accurate identification and differentiation of Burkholderia (B.) mallei and Burkholderia pseudomallei, the agents of glanders and melioidosis, respectively. These two agents were clearly identified using at least 4 independent genetic markers including 16S rRNA gene, fliC, motB and also by novel species-specific target genes, identified by in silico sequence analysis. Specific hybridization signal profiles allowed the detection and differentiation of up to 10 further Burkholderia spp., including the closely related species Burkholderia thailandensis and Burkholderia-like agents, such as Burkholderia cepacia, Burkholderia cenocepacia, Burkholderia vietnamiensis, Burkholderia ambifaria, and Burkholderia gladioli, which are often associated with cystic fibrosis (CF) lung disease. The assay was developed using the easy-to-handle and economical ArrayTube? (AT) platform. A representative strain panel comprising 44 B. mallei, 32 B. pseudomallei isolates, and various Burkholderia type strains were examined to validate the test. Assay specificity was determined by examination of 40 non-Burkholderia strains.     

Strains of Burkholderia cenocepacia genomovar IIIA possessing the cblA gene that are distinct from ET12

Three strains of Burkholderia cenocepacia genomovar IIIA that were polymerase chain reaction positive for cblA, bcrA, and the epidemic strain marker, but were distinct from representatives of ET12 by pulsed-field gel electrophoresis, are described. One of these strains was shown to express cable pili by electron microscopy.     

Activity of Tobramycin against Cystic Fibrosis Isolates of Burkholderia cepacia Complex Grown as Biofilms

Pulmonary infection with Burkholderia cepacia complex in cystic fibrosis (CF) patients is associated with more-rapid lung function decline and earlier death than in CF patients without this infection. In this study, we used confocal microscopy to visualize the effects of various concentrations of tobramycin, achievable with systemic and aerosolized drug administration, on mature B. cepacia complex biofilms, both in the presence and absence of CF sputum. After 24 h of growth, biofilm thickness was significantly reduced by exposure to 2,000 μg/ml of tobramycin for Burkholderia cepacia, Burkholderia multivorans, and Burkholderia vietnamiensis; 200 μg/ml of tobramycin was sufficient to reduce the thickness of Burkholderia dolosa biofilm. With a more mature 48-h biofilm, significant reductions in thickness were seen with tobramycin at concentrations of ≥100 μg/ml for all Burkholderia species. In addition, an increased ratio of dead to live cells was observed in comparison to control with tobramycin concentrations of ≥200 μg/ml for B. cepacia and B. dolosa (24 h) and ≥100 μg/ml for Burkholderia cenocepacia and B. dolosa (48 h). Although sputum significantly increased biofilm thickness, tobramycin concentrations of 1,000 μg/ml were still able to significantly reduce biofilm thickness of all B. cepacia complex species with the exception of B. vietnamiensis. In the presence of sputum, 1,000 μg/ml of tobramycin significantly increased the dead-to-live ratio only for B. multivorans compared to control. In summary, although killing is attenuated, high-dose tobramycin can effectively decrease the thickness of B. cepacia complex biofilms, even in the presence of sputum, suggesting a possible role as a suppressive therapy in CF.     

Inducible resistance of fish bacterial pathogens to the antimicrobial peptide cecropin B

Cecropin B is a cationic antimicrobial peptide originally isolated from the diapausing pupae of the giant silk moth, Hylphora cecropia. Cecropin B elicits its antimicrobial effects through disruption of the anionic cell membranes of gram-negative bacteria. Previous work by our laboratory demonstrated that a constitutively expressed cecropin B transgene conferred enhanced resistance to bacterial infection in medaka. The development of antibiotic resistance by pathogenic bacteria is a growing problem. The potential for fish bacterial pathogens to develop resistance to cecropin B was addressed in this study. Four fish bacterial pathogens were selected for the study based on their importance in aquaculture. Vibrio anguillarum, Vibrio vulnificus, and Yersinia ruckeri all exhibited inducible resistance to cecropin B. The inducible resistance of these three pathogens was correlated with reversible changes in their ultrastructures, as observed by scanning electron microscopy. V. anguillarum was demonstrated to become more adhesive to a CHSE-214 cell monolayer and to cause increased cumulative mortality in medaka following exposure to cecropin B. This work demonstrates that the resistance of fish bacterial pathogens to cecropin B is inducible and suggests that resistance to other cationic antimicrobial peptides may occur through similar means. The observed changes in ultrastructure and infectivity suggest that resistance to antimicrobial peptides is an integral part of the pathogenesis of fish gram-negative bacterial pathogens.     

The New Macrolide-Lincosamide-Streptogramin B Resistance Gene erm(45) Is Located within a Genomic Island in Staphylococcus fleurettii

Genome alignment of a macrolide, lincosamide, and streptogramin B (MLS_B)-resistant Staphylococcus fleurettii strain with an MLS_B-susceptible S. fleurettii strain revealed a novel 11,513-bp genomic island carrying the new erythromycin resistance methylase gene erm(45). This gene was shown to confer inducible MLS_B resistance when cloned into Staphylococcus aureus. The erm(45)-containing island was integrated into the housekeeping gene guaA in S. fleurettii and was able to form a circular intermediate but was not transmissible to S. aureus.     

Experimental Bacteriophage Therapy Increases Survival of Galleria mellonella Larvae Infected with Clinically Relevant Strains of the Burkholderia cepacia Complex

The Burkholderia cepacia complex (BCC) is a group of bacterial pathogens that are highly antibiotic resistant and associated with debilitating respiratory infections. Although bacteriophages of the BCC have been isolated and characterized, no studies have yet examined phage therapy against the BCC in vivo. In a caterpillar infection model, we show that BCC phage therapy is an alternative treatment possibility and is highly effective under specific conditions.Since the development and mass production of antibiotics in the 1940s, people have looked to these “miracle drugs” to treat bacterial infections. Unfortunately, the escalation of antibiotic resistance is threatening our ability to adequately combat these bacterial infections. In order to develop more-effective alternative antimicrobial therapies, the utilization of bacteriophages as bioagents against pathogenic bacteria is being examined. Recent experimental findings have shown that phage therapy has the potential to efficaciously address one of our most critical emerging health care problems: antibiotic-resistant bacterial infections.Chronic bacterial colonization of the major airways resulting in debilitating pulmonary infections and immune response exacerbations is the major cause of morbidity and mortality in cystic fibrosis (CF) patients (9). In the late 1970s and early 1980s, members of the Burkholderia cepacia complex (BCC) emerged as a group of opportunistic bacterial pathogens primarily affecting CF patients (for a review, see the work of Mahenthiralingam et al. [15]). In these patients, life-threatening infection with BCC bacteria is confounded by the fact that there are few therapeutic options available. Effective treatment of BCC infections is difficult due to the microorganisms'' very high level of intrinsic resistance to many antibiotics and their ability to develop further resistance during therapy (26). Most BCC species are resistant to all classes of commonly used chemical antibiotics (1, 20). Triple-combination antibiotic therapy is believed to provide the most effective therapy against BCC infections (1); however, it is important to note that even when susceptibility to antibiotics is demonstrated in vitro, aggressive therapy often does not result in clinical improvement or even a reduction in bacterial numbers (9, 20). Although there are notable regional differences in the prevalences of species-specific BCC infections in CF patients (21), B. cenocepacia isolates are associated with higher prevalences and higher rates of morbidity and mortality (16, 27).In this study, we examine B. cenocepacia strains K56-2 and C6433, both of which are BCC strains that have spread epidemically among patients with CF (14).Multiple studies have demonstrated the effectiveness of phage therapy in animal models for the treatment of various bacterial pathogens, including Escherichia coli (24, 25), Pseudomonas aeruginosa (10, 18, 32), Klebsiella pneumoniae (31), Campylobacter jejuni (13), Enterococcus spp. (4, 30), and Staphylococcus aureus (5, 17). Although several BCC phages have recently been isolated and characterized (8, 22, 28, 29), to date there have been no published reports demonstrating the efficacy of phage therapy for BCC infections. Here, we demonstrate the efficacy of phages for treatment of fatal infection caused by clinical isolates of the BCC, using the Galleria mellonella infection model (23). Although G. mellonella has been used extensively for the study of human pathogens, including P. aeruginosa (12, 19), Bacillus cereus (7), and Francisella tularensis (2), there are few examples of antimicrobial agents being tested in this system.BCC phage KS4-M is an uncharacterized phenotypic mutant of double-stranded DNA tailed Myoviridae phage KS4 (22), which, unlike its parent, is able to lyse liquid cultures of Burkholderia cenocepacia K56-2. High titers of phage KS4-M were prepared by adding KS4-M phage to K56-2 (pregrown for ∼3.5 h at 30°C in one-half-strength Luria-Bertani broth), and the mixture was shaken at 30°C until complete bacterial lysis was observed. Preparations were purified by centrifugation (1 × 10⁴ × g for 10 min) and supernatant filtration (pore size, 0.45 μm). Phage KS4 was prepared as previously described (22). Following incubation of liquid B. cenocepacia K56-2 with KS4-M, the optical density at 600 nm (OD₆₀₀) was 0.09, while the sample inoculated with KS4 had an OD₆₀₀ of 0.44 and the uninfected control had an OD₆₀₀ of 0.48. The concentrations of bacteria remaining in the samples were determined to be 6.9 × 10⁶ CFU/ml for the KS4-M-infected culture, 1.6 × 10⁸ CFU/ml for the KS4-infected culture, and 2.1 × 10⁸ CFU/ml for the uninfected control. Concomitantly, the KS4-M-infected sample produced more phage, having a titer of 2 × 10⁷ PFU/ml, than the KS4-infected sample (2 × 10⁵ PFU/ml).Three novel BCC phages were isolated from soil obtained from the Muttart Conservatory (Edmonton, Alberta, Canada), using a procedure similar to that described previously (22), involving KS12 from soil planted to Dietes grandiflora (wild iris) and KS14 and DC1 from soil planted to a Dracaena sp. (dragon tree). We determined the host ranges of newly isolated KS12, KS14, and DC1 phages against a previously utilized BCC panel comprising 24 strains, representing nine different species of the BCC (6, 14, 46). In addition to this strain panel, three additional BCC strains were tested for host range in this study: B. cenocepacia PC184 and Cep511 (LMG 18830) and Burkholderia vietnamiensis DBO1 (ATCC 29424). KS12 was isolated with B. cenocepacia strain K56-2 and, of the 27 strains in the BCC panel tested, is able to form plaques only on K56-2 and Burkholderia multivorans C5274. KS14 was isolated using B. multivorans strain C5393 and has a considerably broader host range, capable of producing plaques on 8 of the 27 strains tested. These eight strains are B. multivorans strains C5393 and C5274; B. cenocepacia strains 715J, C6433, C5424, and PC184; Burkholderia dolosa strain 21443; and Burkholderia ambifaria strain 17828. DC1 was isolated using B. cepacia strain LMG18821, and this phage is able to form plaques on B. cenocepacia strains K56-2, C6433, PC184, and Cep511 and Burkholderia stabilis strain LMG18870. KS14 and DC1 are double-stranded-DNA-tailed myoviridae (data not shown).Phages were tested for their ability to persist in the hemolymph of uninfected G. mellonella larvae over time (Fig. ​(Fig.1).1). All phage preparations were applied to a Detoxi-Gel endotoxin-removing column (Pierce Biotechnology, Rockford, IL) and serially diluted in 10 mM MgSO₄ plus 1.2 mg/ml ampicillin prior to injection. Ampicillin was present in the inoculum to prevent infection with bacteria naturally present on the surfaces of the larvae. A 250-μl Hamilton syringe fitted with a reproducibility adapter was used to inject 5-μl aliquots into G. mellonella. Following injection, larvae were placed in a static incubator in the dark at 30°C. A 20-gauge needle was used to withdraw hemolymph samples from larvae for isolation of phage and bacteria on BCSA medium (11). At various times, 10 μl of hemolymph was collected from five larvae and combined; this sample was then serially diluted in suspension medium and plated with the appropriate BCC strain for detection and quantification of plaques. Larvae injected with phage KS4-M alone showed no decrease in number of phage collected from the hemolymph over a 48-hour period (Fig. ​(Fig.1).1). These results are also representative for phages DC1 and KS4, whose phage titers did not decline in the hemolymph over time. However, larvae injected with KS12 or KS14 alone showed a decline in number of phage isolated over a 48-hour period.Open in a separate windowFIG. 1.Bacteriophage persistence in G. mellonella larvae. Uninfected larvae were injected with individual phages (KS4 [▪], KS4-M [♦], DC1 [line without symbol], KS12 [□], or KS14 [×]). Equal volumes of hemolymph were collected from five worms at each time point, combined, serially diluted, and plated with B. cenocepacia K56-2 or B. cenocepacia C6433 for quantification. Each point represents the average for three trials, and the standard deviations are indicated.Phages were tested for their ability to rescue BCC-infected G. mellonella larvae from death. Bacteria were prepared for injection as previously described (23). Larvae were scored as dead or alive at 48 h postinfection (p.i.) at 30°C. Untreated larvae received a control injection of 10 mM MgSO₄ plus 1.2 mg/ml ampicillin in place of phage, and uninfected larvae received a control injection of 10 mM MgSO₄ plus 1.2 mg/ml ampicillin and a subsequent injection of phage to measure any potentially lethal effects of the physical injection process. Larvae infected with ∼2.5 × 10³ CFU of B. cenocepacia strain K56-2 had a mortality rate of nearly 100% at 48 h p.i. As shown in Table ​Table1,1, larvae treated with KS4-M immediately following infection showed increased survival rates, and this rescue was dependent upon the number of phage used in the therapeutic dose. The optimal phage dose in this experiment was approximately 2.5 × 10³ PFU, resulting in a multiplicity of infection (MOI) of 1. At this dose, 60% of the larvae were alive at 48 h p.i., whereas none of the untreated larvae were viable at 48 h p.i. Despite in vitro growth differences in liquid culture, phage KS4 showed results similar to those for KS4-M in its ability to treat K56-2-infected larvae (data not shown).

TABLE 1.

Mortality rates of phage-treated BCC-infected G. mellonella

Preclinical Characterization and In Vivo Efficacy of GSK8853, a Small-Molecule Inhibitor of the Hepatitis C Virus NS4B Protein

The hepatitis C virus (HCV) NS4B protein is an antiviral therapeutic target for which small-molecule inhibitors have not been shown to exhibit in vivo efficacy. We describe here the in vitro and in vivo antiviral activity of GSK8853, an imidazo[1,2-a]pyrimidine inhibitor that binds NS4B protein. GSK8853 was active against multiple HCV genotypes and developed in vitro resistance mutations in both genotype 1a and genotype 1b replicons localized to the region of NS4B encoding amino acids 94 to 105. A 20-day in vitro treatment of replicons with GSK8853 resulted in a 2-log drop in replicon RNA levels, with no resistance mutation breakthrough. Chimeric replicons containing NS4B sequences matching known virus isolates showed similar responses to a compound with genotype 1a sequences but altered efficacy with genotype 1b sequences, likely corresponding to the presence of known resistance polymorphs in those isolates. In vivo efficacy was tested in a humanized-mouse model of HCV infection, and the results showed a 3-log drop in viral RNA loads over a 7-day period. Analysis of the virus remaining at the end of in vivo treatment revealed resistance mutations encoding amino acid changes that had not been identified by in vitro studies, including NS4B N56I and N99H. Our findings provide an in vivo proof of concept for HCV inhibitors targeting NS4B and demonstrate both the promise and potential pitfalls of developing NS4B inhibitors.     

Multiple Genetic Mutations Associated with Polymyxin Resistance in Acinetobacter baumannii

We studied polymyxin B resistance in 10 pairs of clinical Acinetobacter baumannii isolates, two of which had developed polymyxin B resistance in vivo. All polymyxin B-resistant isolates had lower growth rates than and substitution mutations in the lpx or pmrB gene compared to their parent isolates. There were significant differences in terms of antibiotic susceptibility and genetic determinants of resistance in A. baumannii isolates that had developed polymyxin B resistance in vivo compared to isolates that had developed polymyxin B resistance in vitro.     

Characterization of In40 of Enterobacter aerogenes BM2688, a Class 1 Integron with Two New Gene Cassettes,cmlA2 and qacF

Enterobacter aerogenes BM2688, which is resistant to multiple antibiotics, and its aminoglycoside-susceptible derivative BM2688-1 were isolated from the same clinical sample. Strain BM2688 harbored plasmid pIP833, which carries a class 1 integron, In40, containing (in addition to qacEΔ1 and sul1, which are characteristic of class 1 integrons) four gene cassettes: aac(6′)-Ib, qacF, cmlA2, and oxa-9. The cmlA2 gene had 83.7% identity with the previously described nonenzymatic chloramphenicol resistance cmlA1 gene. The qacF gene conferred resistance to quaternary ammonium compounds and displayed a high degree of similarity with qacE (67.8% identity) which, however, has been found as part of a cassette with a very different 59-base element. The oxa-9 gene was not expressed due to a lack of promoter sequences. Study of the antibiotic-susceptible derivative BM2688-1 indicated that a 3,148-bp deletion between the 3′ end of the aac(6′)-Ib gene and the 3′ conserved segment of In40 was responsible for the loss of resistance. The occurrence of this DNA rearrangement, which did not involve homologous sequences, suggests that the In40 integrase could promote recombination at secondary sites.Integrons are genetic elements that can integrate, by site-specific recombination, gene cassettes, usually antibiotic resistance genes, between two conserved segments (14, 28). The 5′ conserved segment contains the int gene encoding the integrase which catalyzes site-specific recombination. In class 1 integrons, the 3′ conserved segment carries qacEΔ1, a functional deletion derivative of the qacE gene, which specifies resistance to antiseptics and disinfectants; the sul-1 gene, which confers sulfonamide resistance; and an open reading frame (ORF; ORF5) of unknown function (13, 22, 39). Integrons are often part of transposons or plasmids of various incompatibility groups. Cassettes are individual mobile units composed of a gene and of a short inverted repeat called the 59-base element, located at the 3′ end of the gene, which is recognized by the integrase IntI (4). Over the last 50 years the spread of antibiotic resistance genes occurred by integration and excision of cassettes into integrons (4, 5). Multiple cassette insertions can occur, and more than 40 distinct cassettes have been identified (28). Genes other than those conferring antibiotic resistance have been described, such as qacE, which encodes an exporter protein mediating resistance to antiseptics and disinfectants (22, 27). Cassettes are always integrated in the same orientation and are transcribed from a promoter located in the 5′ conserved segment; the cmlA1 gene, however, carries its own promoter (1, 37).Enterobacter aerogenes BM2688, which is resistant to multiple antibiotics, and its aminoglycoside-susceptible derivative BM2688-1 were isolated from the same clinical sample. Since an aac(6′)-Ib gene was detected in both strains by PCR, we attempted to determine the genetic event responsible for the loss of resistance in BM2688-1. We report on the characterization of a new integron, In40, located on a large plasmid in E. aerogenes BM2688. This element contained four cassettes including two new genes, qacF and cmlA2. Analysis of E. aerogenes BM2688-1 indicated that the loss of aminoglycoside resistance resulted from an unusual recombination event in In40.     

Distribution of capsular serotypes and macrolide resistance mechanisms among macrolide-resistant Streptococcus pneumoniae isolates in Korea

The mechanism of macrolide resistance was investigated in 251 Streptococcus pneumoniae isolates with reduced susceptibility to erythromycin (erythromycin-resistant S. pneumoniae [ERSP]) collected during the period from 2000 to 2004 in Korea. Among these strains, erm(B) was the most prevalent pneumococcal macrolide resistance genotype. In particular, dual mechanisms of both the erm(B) and mef(A) genes were detected in 77 (30.7%) of 251 ERSP isolates. All of the 77 ERSP isolates, with dual erm(B) and mef(A), showed resistance to 2 or more antimicrobial agents, including penicillin, cefotaxime, clindamycin, tetracycline, and levofloxacin. Serotypes 19F, 23F, 19A, 14, 11A, 6B, 6A, and 9V accounted for 73.3% of ERSP isolates. Most of the strains with serotypes 19F (77.2%) or 19A (87.5%) had the dual erm(B) and mef(A) genes. The prevalence and spread of serotype 19F or 19A isolates may have contributed to the high rate of macrolide-resistant pneumococci in Korea. In addition, we identified the emergence of a macrolide-nonsusceptible nonvaccine serotype 35B, which carries mef(A)-mediated resistance to macrolides. These findings emphasize the need for a continuous monitoring of macrolide-resistant S. pneumoniae in Korea.     

New Trimethoprim-Resistant Dihydrofolate Reductase Cassette,dfrXV, Inserted in a Class 1 Integron

The nucleotide sequence of a plasmid-borne trimethoprim resistance gene from a commensal fecal Escherichia coli isolate revealed a new dihydrofolate reductase gene, dfrXV, which occurred as a gene cassette integrated in a site-specific manner in a class 1 integron. The new gene shows 84% nucleotide identity and the predicted protein shows 90% amino acid identity with dfrI and DHFR type I, respectively. Genes for spectinomycin resistance, aadA1 [ant (3′′)-Ia], and sulfonamide resistance, sulI, were located downstream of dfrXV in a manner identical to that in pLMO229.Trimethoprim is an antimicrobial agent used on its own or in combination with sulfamethoxazole in the treatment of infections caused by gram-negative organisms. Trimethoprim selectively inhibits the bacterial dihydrofolate reductase (DHFR), thus preventing the reduction of dihydrofolate to tetrahydrofolate (8). The most common mechanism of resistance to trimethoprim in enterobacteria is the production of an additional plasmid-mediated DHFR which, unlike the chromosomal enzyme, is less sensitive to inhibition by trimethoprim (5). Sixteen trimethoprim resistance enzymes have been identified in enterobacteria and have been characterized and grouped on the basis of their nucleotide sequences and kinetic properties. The largest of these groups and by far the most prevalent are the type I-like enzymes, which include dfrI, dfrIb, dfrV, dfrVI, and dfrVII (14). This enzyme group is characterized by an open reading frame (ORF) of 157 amino acid residues, and the members of this group share between 64 and 88% amino acid sequence identity in this ORF (14). The majority of these enzymes have been found as gene cassettes inserted into the recombinationally active sites of integrons (22). In a survey of trimethoprim resistance in South Africa, 357 isolates of gram-negative, aerobic, commensal fecal flora were probed with oligonucleotide probes to determine the prevalence of DHFR resistance genes within the population (2, 3). Hybridization experiments revealed that contrary to all previous data, the most prevalent DHFR was type Ib (21.8%), followed by types VII (18.8%), I (14.6%), VIII (12.9%), XIII (12.3%), V (7.8%), and XII (0.3%) (1, 3). Forty-six of 357 isolates did not hybridize to any of the DHFR probes. One of these isolates, Escherichia coli UI14, which is highly resistant to trimethoprim (MIC, >2,048 μg/ml), was shown to transfer a 101-kb plasmid (pUK2317) which confers resistance to trimethoprim, spectinomycin, tetracycline, and sulfonamides to a recipient strain, E. coli J62-2, by conjugation (4).     

Phospholipase A1 Modulates the Cell Envelope Phospholipid Content of Brucella melitensis,Contributing to Polymyxin Resistance and Pathogenicity

A subset of bacterial pathogens, including the zoonotic Brucella species, are highly resistant against polymyxin antibiotics. Bacterial polymyxin resistance has been attributed primarily to the modification of lipopolysaccharide; however, it is unknown what additional mechanisms mediate high-level resistance against this class of drugs. This work identified a role for the Brucella melitensis gene bveA (BMEII0681), encoding a predicted esterase, in the resistance of B. melitensis to polymyxin B. Characterization of the enzymatic activity of BveA demonstrated that it is a phospholipase A1 with specificity for phosphatidylethanolamine (PE). Further, lipidomic analysis of B. melitensis revealed an excess of PE lipids in the bacterial membranes isolated from the bveA mutant. These results suggest that by lowering the PE content of the cell envelope, BveA increases the resistance of B. melitensis to polymyxin B. BveA was required for survival and replication of B. melitensis in macrophages and for persistent infection in mice. BveA family esterases are encoded in the genomes of the alphaproteobacterial species that coexist with the polymyxin-producing bacteria in the rhizosphere, suggesting that maintenance of a low PE content in the bacterial cell envelope may be a shared persistence strategy for association with plant and mammalian hosts.