Characterization of Acinetobacter baumannii and meropenem-resistant Pseudomonas aeruginosa in Canada: results of the CANWARD 2007-2009 study

A total of 66 (0.35% of overall isolates) Acinetobacter baumannii and 102 (0.55%) meropenem-resistant Pseudomonas aeruginosa were identified among 18 538 isolates collected from medical centers across Canada during the 2007-2009 period. A. baumannii was most frequently recovered from patients in intensive care units (ICUs; 42.4%) and was isolated mostly from blood cultures (53.0%) and respiratory tract specimens (33.3%). Colistin, meropenem, and amikacin were the most active agents against A. baumannii strains (≥ 92.4% coverage). Gentamicin, levofloxacin, and tigecycline were also active against this bacterial species (MIC(50) 1, 0.12, and 0.5 μg/mL, respectively). Multidrug resistance (MDR; resistance to ≥ 3 antimicrobial classes) was noted in only 4 strains (6.1%), and molecular typing revealed 6 clusters of 2 isolates per cluster that displayed >85% similarity on the dendrogram. Meropenem-resistant P. aeruginosa isolates were primarily obtained from patients in ICUs (40.2%) and the most prevalent specimen types were those collected from the respiratory tract (63.7%), followed by blood cultures (18.6%). Most of the meropenem-resistant P. aeruginosa were resistant to all antimicrobial agents tested, and low susceptibility rates were observed for levofloxacin (8.8%) and gentamicin (28.4%). Amikacin and colistin were active against 67.7% and 88.2% of the isolates, respectively. A total of 68.6% (n = 70) of meropenem-resistant P. aeruginosa were MDR. Pulsed-field gel electrophoresis analysis revealed 94 unique isolates and 2 small clusters (6 and 4 isolates, 1 hospital each). In summary, MDR A. baumannii are rare in Canada and, conversely, meropenem-resistant P. aeruginosa were mostly MDR; however, there was minimal clonal spread among these nonfermentative bacilli.     

Prevalence and characterization of extended-spectrum β-lactamase- and AmpC β-lactamase-producing Escherichia coli: results of the CANWARD 2007-2009 study

The national prevalence of extended-spectrum β-lactamase (ESBL)-producing (2007: 3.4%, 2008: 4.9%, 2009: 4.3%) and AmpC β-lactamase (AmpC)-producing (2007: 0.8%, 2008: 3.2%, 2009: 2.7%) Escherichia coli in Canadian hospitals have fluctuated from 2007 to 2009. Rates of co-resistance to non-lactam agents are elevated, and multidrug-resistant (MDR) phenotype were observed among E. coli strains producing ESBLs (83.3% MDR) and AmpCs (31.0%). The majority (>98%) of isolates remained susceptible to colistin, tigecycline, amikacin, and the carbapenems. CMY-2 encoding gene was detected in 52.9% of AmpC-producing strains, while bla(CTX-M-15) (65.2%) was the predominant ESBL genotype. A total of 50.3% of ESBL-producing E. coli and 21.4% of AmpC producers belonged to the ST131 clone. In conclusion, ESBL- and AmpC-producing E. coli are established in Canadian hospitals; and although the prevalence rates of these isolates remain low, they are often MDR and associated with the ST131 clone.     

The inability of Streptococcus mutans and Lactobacillus acidophilus to form a biofilm in vitro on dentine pretreated with ozone

Background: The use of ozone therapy in the treatment of dental caries is equivocal. The aim of this study was to use an in vitro model to determine the effects of prior ozone application to dentine on biofilm formation and to measure any associated reduction in bacteria viability. Methods: Twenty dentine discs were bonded to the bases of 5 mL polycarbonate screw top vials. Ten dentine discs were infused with ozone for 40 seconds, 10 samples remained untreated as a control. The vials were filled with nutrient medium, sterilized and placed into the outflow from a continuous chemostat culture of Streptococcus mutans and Lactobacillus acidophilus for four weeks. At the conclusion of the experiment bacterial growth was monitored by taking optical density readings of the growth medium in each vial and the outer surface of the dentine specimens were examined by scanning electron microscopy as shown by SEM analysis. Results: Ozone infusion prevented biofilm formation on all the treated samples while there was substantial biofilm present on the control specimens. While the average optical density of the control specimens was almost twice that of the ozone infused dentine (0.710 for the control with a SD of 0.288 and 0.446 for the ozonated samples with a SD of 0.371), the results were not significant (p > 0.05). Conclusions: This preliminary study has shown that the infusion of ozone into non‐carious dentine prevented biofilm formation in vitro from S. mutans and L. acidophilus over a four‐week period. The possibility exists that ozone treatment may alter the surface wettability of dentine through reaction with organic constituents.     

Antibacterial Activity of Ultrashort Cationic Lipo-β-Peptides

Previously reported d,l-lipo-α-peptides and their lipo-β-peptide counterparts (C16-KGGK, C16-KAAK, C16-KKKK, and C12-KLLK) were studied, and the lipo-β-peptides were found to retain antimicrobial activity. Likewise, no significant changes in antimicrobial activity were found upon activity comparisons with d,l-amino acid-based lipopeptides or any l-amino acid lipopeptides. As a defined amphipathic structure is unlikely to form with such short molecules and as similar activities were obtained from all lipopeptides, we suspect that the action of membrane permeation is retained.The rise of antibiotic-resistant microbes has prompted interest in novel therapeutics with new modes of action, including antimicrobial lipopeptides. Naturally occurring lipopeptides produced by bacteria, yeasts, and fungi with largely antifungal activity exist, but some also show antibacterial activity (2, 5, 7). Native lipopeptides are cyclic and anionic and contain short peptide portions of six or seven d- and l-amino acids that are toxic to mammalian cells due to a lack of selectivity (3, 10). However, studies of synthetic lipopeptides formed from acylated antimicrobial peptides report a marked improvement in bioactivity against bacteria (1, 4, 6). Recently, a series of short lipopeptides were synthesized from biologically inactive d,l cationic tetrapeptides and found to possess cell-lysing activity against a variety of gram-positive and gram-negative bacteria, with both aliphatic chain length and peptide sequence determining cell type selectivity (7). This study aims to investigate the biological activity of short cationic lipo-β-peptides on the basis of previously reported lipo-α-peptides (7). As with incorporation of d-enantiomers, peptidomimetics incorporating β-amino acids offer the potential benefit of metabolic and enzymatic stability against proteases, one of the major drawbacks in peptide-based drug development (11).American Type Culture Collection (ATCC) strains as well as clinical isolates from the Canadian Intensive Care Unit (CAN-ICU) study were used, including Staphylococcus aureus ATCC 29213, methicillin-resistant Staphylococcus aureus ATCC 33592, Staphylococcus epidermidis ATCC 14990, methicillin-resistant Staphylococcus epidermidis (MRSE) (cefazolin MIC, >32 μg/ml) CAN-ICU 61589, Enterococcus faecalis ATCC 29212, Enterococcus faecium ATCC 27270, Streptococcus pneumoniae ATCC 49619, Escherichia coli ATCC 25922, E. coli (gentamicin-resistant) CAN-ICU 61714, E. coli (amikacin MIC, 32 μg/ml) CAN-ICU 63074, Pseudomonas aeruginosa ATCC 27853, P. aeruginosa (gentamicin-resistant) CAN-ICU 62308, Stenotrophomonas maltophilia CAN-ICU 62584, Acinetobacter baumannii CAN-ICU 63169, and Klebsiella pneumoniae ATCC 13883 (13).Both the lipo-α-peptides and the lipo-β-peptides (Table ​(Table1)1) investigated in this study were synthesized by solid-phase peptide synthesis using standard 9-fluorenylmethoxy carbonyl chemistry on Rink amide-4-methylbenzhydrylamine hydrochloride salt resin. Palmitic acid and lauric acid were conjugated to the tetrapeptides via modified solid-phase methods. TBTU [2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate] (3 eq), lipophilic acid (3 eq), and diisopropylethylamine (9 eq) reacted in a solution of 45% CH₂Cl₂ in dimethylformamide, and the process was repeated twice. Lipopeptide cleavage in 95% trifluoroacetic acid was achieved, followed by purification on reversed-phase C₁₈ silica. The homogeneity and identity of the synthetic peptides were assessed by electrospray ionization-mass spectrometry, ¹H nuclear magnetic resonance, and ¹³C nuclear magnetic resonance.

TABLE 1.

Antimicrobial activities of ultrashort cationic lipopeptides^a

In Vitro Activity of Colistin (Polymyxin E) against 3,480 Isolates of Gram-Negative Bacilli Obtained from Patients in Canadian Hospitals in the CANWARD Study, 2007-2008

The in vitro activity of colistin was evaluated versus 3,480 isolates of gram-negative bacilli using CLSI broth microdilution methods. The MIC₉₀ of colistin was ≤2 μg/ml against a variety of clinically important gram-negative bacilli, including Escherichia coli, Klebsiella spp., Enterobacter spp., Acinetobacter baumannii, and Pseudomonas aeruginosa. All multidrug-resistant (n = 76) P. aeruginosa isolates were susceptible to colistin (MIC, ≤2 μg/ml). These data support a role for colistin in the treatment of infections caused by multidrug-resistant P. aeruginosa.Colistin (polymyxin E) is a polypeptide antimicrobial originally discovered in 1949 that targets the bacterial cell membrane (4). The use of colistin was largely abandoned by the early 1980s due to toxicity concerns (nephrotoxicity and neurotoxicity) (4, 9). However, there has recently been renewed interest in using polymyxins, including colistin, for the treatment of infections caused by gram-negative bacilli (especially Pseudomonas aeruginosa and Acinetobacter baumannii) due to their activity versus multidrug-resistant (MDR) isolates (4, 9). There have been no current national surveillance studies published evaluating the in vitro activity of colistin versus a variety of nosocomial gram-negative bacterial isolates. The purpose of this study was to evaluate the in vitro activity of colistin against gram-negative bacilli obtained from patients in Canadian hospitals (CANWARD).(These data were presented at the 48th Annual Interscience Conference on Antimicrobial Agents and Chemotherapy- Infectious Diseases Society of America 46th Annual Meeting, Washington, DC, 25 to 28 October 2008 [13].)Twelve sentinel hospital sites (10 in 2008) located in major population centers in 7 of the 10 provinces in Canada participated in the CANWARD study. These sites were geographically distributed in a population-based fashion. From January 2007 through December 2008, inclusive, each study site was asked to submit clinical isolates (consecutive, one per patient, per infection site) from inpatients and outpatients with respiratory (n = 200 per year), urine (n = 100 per year), wound/intravenous (n = 50 per year), and bloodstream (n = 360 per year) infections. Only isolates that were deemed clinically significant were submitted. Isolate identification was performed by the submitting site using local criteria. Where indicated, identification was confirmed at the reference site. Isolates were shipped on Amies semisolid transport medium to the coordinating laboratory (Health Sciences Centre, Winnipeg, Canada), where they were then subcultured on appropriate media and stocked in skim milk at −80°C.Following two subcultures from frozen stock, the in vitro activity of colistin (colistin sulfate) was determined by broth microdilution in accordance with the Clinical and Laboratory Standards Institute (CLSI) guidelines (2, 3). Colistin MIC interpretive standards were defined according to CLSI breakpoints (2). At present, colistin susceptibility breakpoints (CLSI) only exist for P. aeruginosa and A. baumannii.Extended-spectrum β-lactamase (ESBL)-producing Escherichia coli and K. pneumoniae isolates were defined as isolates having a positive ESBL confirmatory test, as described by CLSI (2). MDR P. aeruginosa isolates were defined as isolates demonstrating resistance to antimicrobials from three or more different classes. The number that appears in the following tables after the MDR designation indicates the number of different classes to which isolates were resistant (e.g., MDR3 indicates P. aeruginosa isolates that were resistant to at least one antimicrobial agent from three different classes). For the purpose of this report, the four antimicrobial classes considered were aminoglycosides (amikacin and gentamicin), fluoroquinolones (ciprofloxacin and levofloxacin), cefepime and piperacillin- tazobactam (considered together as one class), and carbapenems (meropenem). Colistin was not used in the classification of MDR isolates.The in vitro activity of colistin was evaluated against 3,480 isolates of gram-negative bacilli from the specimen sources blood (51.6%), urine (25.8%), respiratory (18.1%), and wound (4.5%) and ward types emergency room (31.6%), medical (31.4%), intensive care unit (15.2%), clinic/office (14.4%), and surgical (7.4%). The MIC distribution of colistin, stratified by bacteria species, is presented in Table ​Table1.1. Over 90% of E. coli and K. pneumoniae isolates (including 163 ESBL producers) had a MIC of ≤1 μg/ml. Twenty-nine of 31 A. baumannii isolates (93.5%) were susceptible to colistin (MIC, ≤2 μg/ml). In contrast, colistin demonstrated minimal activity (MIC₅₀, ≥8 μg/ml) versus Proteus mirabilis, Serratia marcescens, and Stenotrophomonas maltophilia (Table ​(Table11).

TABLE 1.

MIC distribution of colistin versus 3,480 isolates of gram-negative bacilli obtained from patients in Canadian hospitals

Cationic Amphiphiles,a New Generation of Antimicrobials Inspired by the Natural Antimicrobial Peptide Scaffold

Naturally occurring cationic antimicrobial peptides (AMPs) and their mimics form a diverse class of antibacterial agents currently validated in preclinical and clinical settings for the treatment of infections caused by antimicrobial-resistant bacteria. Numerous studies with linear, cyclic, and diastereomeric AMPs have strongly supported the hypothesis that their physicochemical properties, rather than any specific amino acid sequence, are responsible for their microbiological activities. It is generally believed that the amphiphilic topology is essential for insertion into and disruption of the cytoplasmic membrane. In particular, the ability to rapidly kill bacteria and the relative difficulty with which bacteria develop resistance make AMPs and their mimics attractive targets for drug development. However, the therapeutic use of naturally occurring AMPs is hampered by the high manufacturing costs, poor pharmacokinetic properties, and low bacteriological efficacy in animal models. In order to overcome these problems, a variety of novel and structurally diverse cationic amphiphiles that mimic the amphiphilic topology of AMPs have recently appeared. Many of these compounds exhibit superior pharmacokinetic properties and reduced in vitro toxicity while retaining potent antibacterial activity against resistant and nonresistant bacteria. In summary, cationic amphiphiles promise to provide a new and rich source of diverse antibacterial lead structures in the years to come.The rise in antibiotic resistance among pathogenic bacteria and the declining rate of novel drug discovery are common concerns in medicine (66), driving research into new antibacterial classes and novel drugs in order to maintain the existing ability to treat infectious diseases, especially those caused by multidrug-resistant (MDR) organisms (49, 51).While the enzymatic inhibitors from which many of our strongest antibiotics are derived are highly effective in the microbial world, higher-order organisms do not appear to rely entirely on such selective inhibitors (27). These organisms instead produce a number of broad-range antimicrobial peptides (AMPs), which do not target any single molecule or process but instead associate with cellular membranes, resulting in depolarization, lysis, and cell death through a disruption of the membrane topology. A subset of these peptides is able to translocate into the cell and disrupt cellular processes, such as protein and DNA synthesis (33). AMPs play a key role in the human immune system, and mutations affecting their production and expression have been linked to diseases such as morbus Kostmann and Crohn''s disease (56, 75).Membrane targeting offers advantages over standard methods of drug design and antibiotic activity due to the wide variety of active structures and a reduced development of resistance mechanisms (78). Nevertheless, potential cytotoxicity to the host cells remains a major unsolved challenge (43). Mutants resistant to AMPs have been developed in the laboratory (54); however, such mutants may be hypersusceptible to conventional antibiotics as well as demonstrate reduced growth compared to wild-type strains (77). The lack of a specific cellular target is another significant advantage of AMPs, as activity toward Gram-positive and Gram-negative bacteria, fungi, and viruses has been reported (22, 26, 81, 82). The development of AMPs as pharmaceutical agents shows great promise, with a variety of natural and synthetic compounds currently in development (26). However, natural AMPs often suffer from a variety of pharmacokinetic shortcomings, including poor bioavailability, low metabolic stability, and formulation difficulties due to their size and the high number of amide bonds, which has driven research toward the creation of partially and wholly synthetic analogues. This review will examine recent research on AMPs and their mimics in an attempt to elucidate the underlying pharmacophore shared between them and highlight the current challenges in AMP-based drug design.     

Influence of human urine on the in vitro activity and postantibiotic effect of ciprofloxacin against Escherichia coli

The purpose of this investigation was to study the effects of human urine on the minimum inhibitory concentration (MIC) and the postantibiotic effect (PAE) of ciprofloxacin against Escherichia coli. MICs and the PAE were performed in Mueller-Hinton broth (MHB; pH 7.3 and 5.5) and in human urine (pH 5.5 and 7.3). In urine, pH 5.5, MICs increased 64-fold (from 0.016 to 1.024 micrograms/ml) and the PAE was abolished (from 101.6 to 3.7 min), when compared to MHB, pH 7.3. An acidic pH demonstrated the greatest effect on reduced susceptibility and PAE. Using ciprofloxacin concentrations adjusted for a higher MIC obtained in pH-adjusted urine and MHB, PAE values were similar for urine and MHB (approximately 280 min at 40 x MIC). This study demonstrated that the MIC and PAE of ciprofloxacin against E. coli are influenced by human urine and in particular its pH.     

Effect of interventions on prescribing of antimicrobials for prophylaxis in obstetric and gynecologic surgery

The effect of interventions on the conformity of physicians with guidelines for the appropriate use of antimicrobial prophylaxis in obstetric and gynecologic surgery is reported. Guidelines on the appropriate use of antimicrobial prophylaxis in common obstetric and gynecologic surgical procedures were developed in late 1986 by the antibiotic subcommittee at a 1100-bed tertiary-care teaching facility. The guidelines were not adopted immediately by the department of obstetrics and gynecology (OB-GYN). An audit of the medical records of women who had received antimicrobial therapy for abdominal and vaginal hysterectomies and emergency cesarean sections during January through March 1987 showed that cefoxitin was used in 68% of the cases instead of the less expensive and equally efficacious cefazolin as recommended in the guidelines. The projected annual cost of this nonconformity was $26,500. After the subcommittee informed the physicians about the guidelines and the audit results, the OB-GYN department adopted the guidelines. A second audit performed one year later showed that cefazolin was used in the recommended manner in 93% of cases; projected annual cost savings were $25,000. Both audits showed that prophylactic treatment was inappropriately prolonged in 6% of cases. Substantial cost savings were realized by minimizing inappropriate antimicrobial drug use through efforts to promote rational and cost-effective therapy.