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1.  In vitro analysis of flufenamic acid activity against Candida albicans biofilms 
In a recent high-throughput screen against specific Candida albicans drug targets, several compounds that exhibited non-specific antifungal activity were identified, including the non-steroidal anti-inflammatory drug flufenamic acid (FFA). This study sought to determine the effect of different doses of FFA, alone or in combination with fixed concentrations of the standard antifungal agents amphotericin B (AmB), caspofungin (CAS) or fluconazole (FLU), for the prevention and treatment of C. albicans biofilms. Biofilms were formed in a 96-well microplate followed by evaluation of antifungal activity using the XTT assay. FFA concentrations of ≥512 mg/L demonstrated >80% prevention of biofilm formation. FFA concentrations of 1024 mg/L demonstrated >85% reduction of mature biofilms. When FFA (≥8 mg/L) was used in combination with FLU (32 mg/L), antifungal activity increased to 99% for the prevention of biofilm formation. Similarly, when a FFA concentration of ≥8 mg/L was used in combination with either AmB (0.25 mg/L) or CAS (0.125 mg/L), antifungal activity also increased up to 99% for the prevention of biofilm formation. The inhibitory effect of FFA on C. albicans biofilms has not been reported previously, therefore these findings suggest that FFA in combination with traditional antifungals might be useful for the treatment and prevention of C. albicans biofilms.
PMCID: PMC3902125  PMID: 24156913
Candida albicans; Biofilm; Flufenamic acid; Antifungal
2.  Pharmacokinetics and efficacy of liposomal polymyxin B in a murine pneumonia model☆ 
International journal of antimicrobial agents  2013;42(6):10.1016/j.ijantimicag.2013.07.009.
Polymyxin B (PB) is increasingly used as the last treatment for multidrug-resistant (MDR) Gram-negative bacterial infections. In this study, serum and epithelial lining fluid (ELF) pharmacokinetics and the efficacy of a PB liposomal formulation were investigated. Two groups of 24 Swiss Webster mice were intravenously administered PB liposomes or PB aqueous solution at ca. 3 mg/kg. Serum and ELF samples were collected for up to 6 h to quantify major PB components. Three groups of neutropenic mice (n = 6/group) were infected with a clinical MDR Pseudomonas aeruginosa strain followed by intravenous administration of PB liposomes or PB aqueous solution at 3 mg/kg every 6 h or sham (drug-free) liposomes every 6 h. Bacterial burden in animal lung tissues was quantified after 24 h of therapy and was compared using one-way ANOVA. Survival of infected animals over time (n = 10/group) was evaluated by Kaplan–Meier analysis and log-rank test. In the pharmacokinetic study, the AUC ratio in ELF between liposome and aqueous solution groups ranged from 4.6 to 11.1 for various major PB components. In the efficacy study, for strain PA 9019 a significantly lower bacterial burden was seen in the liposomal group (3.8 ± 0.7 vs. 7.9 ± 0.8 log10 CFU/g in the aqueous solution group), which subsequently prolonged survival of infected animals. In this study, treatment with a PB liposomal formulation yielded higher drug penetration into pulmonary ELF, which resulted in superior efficacy. However, further investigations on the clinical utility of the PB liposomal formulation are warranted.
PMCID: PMC3849129  PMID: 24016799
Multidrug resistance; Pulmonary; Pseudomonas aeruginosa; Epithelial lining fluid
3.  Fluoroquinolone susceptibility in Mycobacterium tuberculosis after pre-diagnosis exposure to older- versus newer-generation fluoroquinolones☆ 
Fluoroquinolone exposure before tuberculosis (TB) diagnosis is common. We anticipated that exposure to older-generation fluoroquinolones is associated with greater fluoroquinolone MICs in Mycobacterium tuberculosis than exposure to newer agents. A nested case–control study was performed among newly diagnosed TB patients reported to the Tennessee Department of Health (January 2002–December 2009). Each fluoroquinolone-resistant case (n = 25) was matched to two fluoroquinolone-susceptible controls (n = 50). Ciprofloxacin and ofloxacin were classified as older-generation fluoroquinolones; levofloxacin, moxifloxacin and gatifloxacin were considered newer agents. There was no difference between median ofloxacin MIC for isolates from 9 patients exposed only to older fluoroquinolones, 25 exposed only to newer fluoroquinolones, 6 exposed to both and 35 fluoroquinolone-unexposed patients (Kruskal–Wallis, P = 0.35). Using multivariate proportional odds logistic regression adjusting for age and sex, duration of exposure to newer fluoroquinolones was independently associated with higher MIC (OR = 1.79, 95% CI 1.22–2.64), but duration of exposure to older fluoroquinolones was not (OR = 0.94, 95% CI 0.50–1.78). Isolates from patients exposed only to newer fluoroquinolones tended to have mutations at gyrA codons 90, 91 or 94 more frequently than those exposed only to older fluoroquinolones (44% vs. 11%). We were surprised to find that duration of exposure to newer fluoroquinolones, but not older ones, was independently associated with higher ofloxacin MIC. This suggests that the mutant selection window lower boundary is likely to have clinical relevance; caution is warranted when newer fluoroquinolones are prescribed to patients with TB risk factors.
PMCID: PMC3780576  PMID: 23806638
Drug-resistant tuberculosis; Genotypic resistance; Moxifloxacin; Levofloxacin; Ciprofloxacin
4.  Characteristics of antimicrobial studies registered in the USA through ClinicalTrials.Gov 
Increasing rates of antimicrobial-resistant infections and the dwindling pipeline of new agents necessitate judicious, evidence-based antimicrobial prescribing. Clinical trials represent a vital resource for establishing evidence of safety and efficacy, which are crucial to guiding antimicrobial treatment decisions. The objective of this study was to comprehensively evaluate the characteristics of antimicrobial research studies registered in Primary outcome measures, funding sources, inclusion criteria and the reporting of study results were evaluated for 16 055 antimicrobial studies registered in as of mid 2012. Interventional studies accounted for 93% of registered antimicrobial studies. Clinical trials of drugs (82%) and biologics (9%) were most common. Antibacterial, antiviral and antifungal studies accounted for 43%, 41% and 16% of drug trials, respectively. Among interventional drug trials, 73% featured randomised allocation to study arms and 71% included measures of safety and/or efficacy as primary endpoints. Children were eligible for enrolment in 26% of studies. Among the studies, 60% were sponsored primarily by non-profit organisations, 30% by industry and 10% by the federal government. Only 7% of studies reported results; however, 71% of these were sponsored primarily by industry. Antimicrobial studies commonly incorporated elements of high-quality trial design, including randomisation and safety/efficacy endpoints. Publication of study results and updating of should be encouraged for all studies, with particular attention paid to research sponsored by non-profit organisations and governmental agencies. Leveraging the application of these data to guide the careful selection of antimicrobial agents will be essential to preserve their utility for years to come.
PMCID: PMC3714326  PMID: 23726436
Antibiotic; Antibacterial; Antiviral; Antifungal; Anti-infective agents
5.  Characterisation and clinical features of Enterobacter cloacae bloodstream infections occurring at a tertiary care university hospital in Switzerland: is cefepime adequate therapy? 
Despite many years of clinical experience with cefepime, data regarding the outcome of patients suffering from bloodstream infections (BSIs) due to Enterobacter cloacae (Ecl) are scarce. To address the gap in our knowledge, 57 Ecl responsible for 51 BSIs were analysed implementing phenotypic and molecular methods (microarrays, PCRs for bla and other genes, rep-PCR to analyse clonality). Only two E. cloacae (3.5%) were ESBL-producers, whereas 34 (59.6%) and 18 (31.6%) possessed inducible (Ind-Ecl) or derepressed (Der-Ecl) AmpC enzymes, respectively. All isolates were susceptible to imipenem, meropenem, gentamicin and ciprofloxacin. Der-Ecl were highly resistant to ceftazidime and piperacillin/tazobactam (both MIC90 ≥ 256 µg/mL), whereas cefepime retained its activity (MIC90 of 3 µg/mL). rep-PCR indicated that the isolates were sporadic, but Ecl collected from the same patients were indistinguishable. In particular, three BSIs initially due to Ind-Ecl evolved (under ceftriaxone or piperacillin/tazobactam treatment) into Der-Ecl because of mutations or a deletion in ampD or insertion of IS4321 in the promoter. These last two mechanisms have never been described in Ecl. Mortality was higher for BSIs due to Der-Ecl than Ind-Ecl (3.8% vs. 29.4%; P = 0.028) and was associated with the Charlson co-morbidity index (P = 0.046). Using the following directed treatments, patients with BSI showed a favourable treatment outcome: cefepime (16/18; 88.9%); carbapenems (12/13; 92.3%); ceftriaxone (4/7; 57.1%); piperacillin/tazobactam (5/7; 71.4%); and ciprofloxacin (6/6; 100%). Cefepime represents a safe therapeutic option and an alternative to carbapenems to treat BSIs due to Ecl when the prevalence of ESBL-producers is low.
PMCID: PMC4018813  PMID: 23313399
Cefepime; Bacteraemia; Outcome; Enterobacter; AmpC; AmpD
6.  Emergence of extended-spectrum β-lactam resistance among Escherichia coli at a US academic children’s hospital is clonal at the sequence type level for CTX-M-15, but not for CMY-2 
Resistance to extended-spectrum β-lactams is increasing worldwide among Escherichia coli and has been linked to a small number of emergent clones (e.g. ST38, ST131 and ST405) recovered from extraintestinal infections in community and hospital settings. There are, however, limited data about the relative contributions of bacterial strains, plasmids and β-lactamase genes to extended-spectrum β-lactam resistance in paediatric infections. We performed an extensive molecular analysis of phylogenetic, virulence and antibiotic resistance-related properties of 49 previously reported serial E. coli isolates recovered during 1999–2007 at Seattle Children’s Hospital (Seattle, WA). Class C enzyme CMY-2 and class A enzyme CTX-M-15 were the most prominent extended-spectrum β-lactam resistance enzymes in the collection, first appearing in this patient population in 2001 and 2003, respectively, and then steadily increasing in frequency over the remainder of the study period. Among 19 CMY-2-positive isolates, 16 distinct STs were detected (D = 98.25%, 95% CI 96–100.25%), indicating that CMY spread is non-clonal at the host strain level. In contrast, among ten CTX-M-15-positive isolates, three STs were detected (D = 37.78%, 95% CI 2.36–73.20%), of which eight represented the worldwide-disseminated ST131 lineage, consistent with clonal spread of CTX-M-15-associated resistance. fimHTR subtyping of ten ST131 isolates (including two CTX-M-negative isolates) revealed that, within ST131, carriage of allele fimHTR30 correlated with CTX-M-15 positivity, whilst carriage of non-fimHTR30 alleles correlated with carriage of non-CTX-M enzymes. Thus, spread of CMY-2 is non-clonal at the ST level, but clonal spread of CTX-M-15 may be associated with a specific fimHTR-defined sublineage of ST131.
PMCID: PMC3627824  PMID: 23434250
Antibiotic resistance; Escherichia coli; ST131; Plasmid; CTX-M; CMY
7.  Antimalarial drug resistance: a review of the biology and strategies to delay emergence and spread 
The emergence of resistance to former first-line antimalarial drugs has been an unmitigated disaster. In recent years, artemisinin class drugs have become standard and they are considered an essential tool for helping to eradicate the disease. However, their ability to reduce morbidity and mortality and to slow transmission requires the maintenance of effectiveness. Recently, an artemisinin delayed-clearance phenotype was described. This is believed to be the precursor to resistance and threatens local elimination and global eradication plans. Understanding how resistance emerges and spreads is important for developing strategies to contain its spread. Resistance is the result of two processes: (i) drug selection of resistant parasites; and (ii) the spread of resistance. In this review, we examine the factors that lead to both drug selection and the spread of resistance. We then examine strategies for controlling the spread of resistance, pointing out the complexities and deficiencies in predicting how resistance will spread.
PMCID: PMC3610176  PMID: 23394809
Malaria; Artemisinin; Drug resistance; Plasmodium falciparum
8.  Peptide nucleic acids inhibit growth of Brucella suis in pure culture and in infected murine macrophages 
International journal of antimicrobial agents  2013;41(4):10.1016/j.ijantimicag.2012.11.017.
Peptide nucleic acids (PNAs) are single-stranded, synthetic nucleic acid analogues containing a pseudopeptide backbone in place of the phosphodiester sugar–phosphate. When PNAs are covalently linked to cell-penetrating peptides (CPPs) they readily penetrate the bacterial cell envelope, inhibit expression of targeted genes and cause growth inhibition both of Gram-positive and Gram-negative bacteria. However, the effectiveness of PNAs against Brucella, a facultative intracellular bacterial pathogen, was unknown. The susceptibility of a virulent Brucella suis strain to a variety of PNAs was assessed in pure culture as well as in murine macrophages. The studies showed that some of the PNAs targeted to Brucella genes involved in DNA (polA, dnaG, gyrA), RNA (rpoB), cell envelope (asd), fatty acid (kdtA, acpP) and protein (tsf) synthesis inhibit the growth of B. suis in culture and in macrophages after 24 h of treatment. PNA treatment inhibited Brucella growth by interfering with gene expression in a sequence-specific and dose-dependent manner at micromolar concentrations. The most effective PNA in broth culture was that targeting polA at ca. 12 μM. In contrast, in B. suis-infected macrophages, the most effective PNAs were those targeting asd and dnaG at 30 μM; both of these PNAs had little inhibitory effect on Brucella in broth culture. The polA PNA that inhibits wild-type B. suis also inhibits the growth of wild-type Brucella melitensis 16M and Brucella abortus 2308 in culture. This study reveals the potential usefulness of antisense PNA constructs as novel therapeutic agents against intracellular Brucella.
PMCID: PMC3834731  PMID: 23305655
Peptide nucleic acid (PNA); Brucella; Antibiotic resistance; Antisense; Murine macrophage
9.  Current management of prosthetic joint infections in adults: results of an Emerging Infections Network survey 
There is a dearth of guidance on the management of prosthetic joint infections (PJIs), in particular because of the lack of high-quality evidence for optimal antibiotics. Thus, we designed a nine-question survey of current practices and preferences among members of the Emerging Infections Network, a CDC-sponsored network of infectious diseases physicians, which was distributed in May 2012. In total, 556 (47.2%) of 1178 network members responded. As first-line antibiotic choice for MSSA PJI, 59% of responders indicated oxacillin/nafcillin, 33% cefazolin and 7% ceftriaxone; the commonest alternative was cefazolin (46%). For MRSA PJI, 90% preferred vancomycin, 7% daptomycin and 0.8% ceftaroline; the commonest alternative was daptomycin (65%). Antibiotic selection for coagulase-negative staphylococci varied depending on meticillin susceptibility. For staphylococcal PJIs with retained hardware, most providers would add rifampicin. Propionibacterium is usually treated with vancomycin (40%), penicillin (23%) or ceftriaxone (17%). Most responders thought 10–19% of all PJIs were culture-negative. Culture-negative PJIs of the lower extremities are usually treated with a vancomycin/fluoroquinolone combination, and culture-negative shoulder PJIs with vancomycin/ceftriaxone. The most cited criteria for selecting antibiotics were ease of administration and the safety profile. A treatment duration of 6–8 weeks is preferred (by 77% of responders) and is mostly guided by clinical response and inflammatory markers. Ninety-nine percent of responders recommend oral antibiotic suppression (for varying durations) in patients with retained hardware. In conclusion, there is considerable variation in treatment of PJIs both with identified pathogens and those with negative cultures. Future studies should aim to identify optimum treatment strategies.
PMCID: PMC3572796  PMID: 23312602
Prosthetic joint infection; Osteomyelitis; Staphylococcus aureus; Antibiotic; Treatment
10.  Carbapenem-associated multidrug-resistant Acinetobacter baumannii are sensitised by aztreonam in combination with polyamines 
Carbapenem-associated multidrug-resistant Acinetobacter baumannii (MDR-Ab) are common among clinical isolates worldwide and are a major therapeutic challenge. Previously it was shown that exogenous polyamines (spermine and spermidine) enhanced susceptibility to β-lactams but induced resistance to polymyxins in Pseudomonas aeruginosa. This study aimed to explore the possible availability of exogenous polyamines in treating carbapenem-associated MDR-Ab. The effects of polyamines on the growth rate of A. baumannii, minimal inhibitory concentrations (MICs) of antibiotics, and time–kill and checkerboard assays were determined. Roles of lipopolysaccharide (LPS) and β-lactamase activity of A. baumannii were also assessed for the polyamine effects. Growth of A. baumannii was unaffected at 4 mM spermine and 16 mM spermidine alone, but was significantly inhibited by a subinhibitory concentration of aztreonam (AZT) (8 μg/mL) and those concentrations of the polyamines. MICs to AZT alone (≥128 μg/mL) were reduced to the range 0.25–8 μg/mL in combination with polyamines in all carbapenem-associated MDR-Ab. MICs to penicillins, but not to ceftazidime and meropenem, were also significantly reduced, whilst MICs to other antibiotics, including polymyxin B, were unaffected in combination with polyamines for all tested A. baumannii. Polyamine effects on AZT were strongly synergistic with bactericidal activity and were retained at concentrations of 5 mM MgCl2 (or CaCl2) or 200 mM NaCl. Roles of LPS and β-lactamase in the polyamine effects were excluded. Overall results suggest that AZT in combination with polyamines may be useful for the treatment of carbapenem-associated MDR-Ab.
PMCID: PMC3530022  PMID: 23148986
Polyamines; β-Lactam susceptibility; Acinetobacter baumannii
11.  Invasive fungal infections in patients with cancer in the intensive care unit 
International journal of antimicrobial agents  2012;39(6):10.1016/j.ijantimicag.2011.11.017.
Invasive fungal infections (IFIs) have emerged as a major cause of morbidity and mortality among critically ill patients. Cancer patients admitted in the intensive care unit (ICU) have multiple risk factors for IFIs. The vast majority of IFIs in the ICU are due to Candida spp. The incidence of invasive candidiasis (IC) has increased over the last decades, especially in the ICU. A shift in the distribution of Candida spp. from Candida albicans to non-albicans Candida spp. has been observed both in ICUs and oncology units in the last two decades. Timely diagnosis of IC remains a challenge despite the introduction of new microbiology techniques. Delayed initiation of antifungal therapy is associated with increased mortality. Therefore, prediction rules have been developed and validated prospectively in order to identify those ICU patients at high risk for IC and likely to benefit from early treatment. These rules, however, have not been validated in cancer patients. Similarly, major clinical studies on the efficacy of newer antifungals typically do not include cancer patients. Despite the introduction of more potent and less toxic antifungals, mortality of IFIs among cancer patients remains high. In recent years, aspergillosis and mucormycosis have also emerged as significant causes of morbidity and mortality among ICU patients with haematological cancer.
PMCID: PMC3855365  PMID: 22337064
Fungal infections; Cancer patients; Intensive care unit
12.  Molecular mechanisms of antibiotic resistance in diarrhoeagenic Escherichia coli isolated from children 
Diarrhoeagenic Escherichia coli (DEC) are an important cause of diarrhoea in children and are associated with high antibiotic resistance. However, there are few studies on the molecular mechanisms of resistance in this group of bacteria. The aim of this study was to determine the mechanisms associated with antibiotic resistance in the most common phenotypes of DEC. A total of 369 E. coli strains [commensal strains and DEC from children with (‘DEC-diarrhoea’) or without (‘DEC-control’) diarrhoea] isolated from children aged <1 year in periurban districts of Lima, Peru, were analysed. In total, 154 ampicillin-resistant strains (36 commensals, 33 DEC-control and 85 DEC-diarrhoea) were studied by PCR for the most prevalent resistance mechanisms to ampicillin, trimethoprim/sulfamethoxazole (SXT), tetracycline and chloramphenicol as well as for integrase types 1 and 2. In additional, restriction fragment length polymorphism was performed for SXT-resistant strains. Commensal strains were more frequently resistant to nalidixic acid and ciprofloxacin (68% and 28%, respectively) than DEC strains (23% and 2%, respectively) (P < 0.05). DEC-diarrhoea strains were more frequently SXT-resistant (78%) compared with DEC-control strains (65%) and commensal strains (60%) (P < 0.05). The most frequent mechanisms of antibiotic resistance in DEC strains were: for β-lactams, blaTEM (31%; 37/118); for SXT, sul2 (48%; 49/103); for tetracycline, tetA (27%; 23/84); and for chloramphenicol, cat (80%; 28/35). The genes sul1 and dfrA1, related to SXT resistance, were more frequent in the DEC-diarrhoea group (41% and28%, respectively) than in the other two groups (P < 0.05). There was a high diversity of resistance genes in DEC, including symptomatic strains.
PMCID: PMC3505230  PMID: 23078917
Antibiotics; Antibiotic resistance mechanism; Children; Diarrhoeagenic E. coli; Commensal E. coli
13.  sarA inactivation reduces vancomycin-intermediate and ciprofloxacin resistance expression by Staphylococcus aureus 
International journal of antimicrobial agents  2009;34(2):10.1016/j.ijantimicag.2009.01.018.
It is known that multiple genome-wide transcriptional changes often accompany the development of antimicrobial resistance and occur in response to challenge with antimicrobial agents. We now show that inactivation of the staphylococcal accessory gene regulator sarA, which controls at least tens of genes in Staphylococcus aureus, leads to dramatic reductions in vancomycin and ciprofloxacin resistance in vancomycin-intermediate and ciprofloxacin-resistant strains of S. aureus. This is particularly evident when judged by antimicrobial-gradient plate analysis or population analysis profiles. Whilst the intact sarA cistron is required for full vancomycin resistance expression by vancomycin-intermediate S. aureus (VISA), sarA expression as determined by real-time polymerase chain reaction was found to be VISA strain-dependent. Reductions in vancomycin resistance expression levels following sarA inactivation do not necessarily include an alteration in autolysis. Expression of sarR, the negative regulator of sarA, was downregulated in two VISA mutants, and transcription of the alternative sigma factor sigB was downregulated in one VISA strain. This study contributes to a growing body of evidence demonstrating the importance of loci previously identified to control virulence in the regulation of clinically relevant antibiotic resistance mechanisms.
PMCID: PMC3831611  PMID: 19324528
Staphylococcus aureus; VISA; Ciprofloxacin; Resistance; sarA
14.  Skin and soft tissue concentrations of tedizolid (formerly torezolid), a novel oxazolidinone, following a single oral dose in healthy volunteers 
Plasma concentrations of antimicrobial drugs have long been used to correlate exposure with effect, yet one cannot always assume that unbound plasma and tissue concentrations are similar. Knowledge about unbound tissue concentrations is important in the development of antimicrobial drugs, since most infections are localised in tissues. Therefore, a clinical microdialysis study was conducted to evaluate the distribution of tedizolid (TR-700), the active moiety of the antimicrobial prodrug tedizolid phosphate (TR-701), into interstitial fluid (ISF) of subcutaneous adipose and skeletal muscle tissues following a single oral 600 mg dose of tedizolid phosphate in fasting conditions. Twelve healthy adult subjects were enrolled. Two microdialysis probes were implanted into the thigh of each subject, one into the vastus medialis muscle and one into subcutaneous adipose tissue. Probes were calibrated using retrodialysis. Dialysate samples were collected every 20 min for 12 h following a single oral dose of 600 mg tedizolid phosphate, and blood samples were drawn over 24 h. Unbound tedizolid levels in plasma were similar to those in muscle and adipose tissue. The ratios of unbound (free) AUC in tissues over unbound AUC in plasma (fAUCtissue/fAUCplasma) were 1.1 ± 0.2 and 1.2 ± 0.2 for adipose and muscle tissue, respectively. The median half-life was 8.1, 9.2 and 9.6 h for plasma, adipose tissue and muscle tissue, respectively. Mean protein binding was 87.2 ± 1.8%. The study drug was very well tolerated. The results of this study show that tedizolid distributes well into ISF of adipose and muscle tissues. Unbound levels of tedizolid in plasma, adipose tissue and muscle tissue were well correlated. Free plasma levels are indicative of unbound levels in the ISF of muscle and adipose tissues.
PMCID: PMC3789129  PMID: 22584101
Microdialysis; Tissue distribution; Tedizolid; Pharmacokinetics
15.  Human β-defensin-3 alters, but does not inhibit, the binding of Porphyromonas gingivalis haemagglutinin B to the surface of human dendritic cells 
Human β-defensin-3 (HBD3) is a small, cationic, host defence peptide with broad antimicrobial activities and diverse innate immune functions. HBD3 binds to many microbial antigens and, in this study, we hypothesised that the known binding of HBD3 to Porphyromonas gingivalis recombinant haemagglutinin B (rHagB) alters, but does not inhibit, the binding of rHagB to human dendritic cells. To test this, human myeloid dendritic cells were incubated for 5 min with rHagB, HBD3 + rHagB (10:1 molar ratio), HBD3 or 0.1 M phosphate-buffered saline (PBS) (pH 7.2) and were then rapidly fixed and processed for confocal microscopy and ultramicrotomy. rHagB and HBD3 could be detected with primary monoclonal mouse antibody to rHagB (MoAb 1858) or polyclonal rabbit antibody to HBD3 (P241) and secondary fluorescent-labelled anti-mouse or anti-rabbit antibodies (confocal microscopy) or protein A–colloidal gold (immunoelectron microscopy). In cells incubated with rHagB only, fluorescence and protein A–colloidal gold were seen at the cell surface and throughout the cytoplasm. In cells incubated with HBD3 + rHagB, fluorescence was observed only at the cell surface in a ‘string of pearls’ configuration. Overall, these results suggest that HBD3 binding to rHagB alters, but does not inhibit, the binding of rHagB to human myeloid dendritic cells.
PMCID: PMC3371132  PMID: 22578747
Defensins; Human β-defensin-3; HBD3; Porphyromonas gingivalis; Haemagglutinin B; Dendritic cells; Confocal microscopy
16.  Database screening and in vivo efficacy of antimicrobial peptides against meticillin-resistant Staphylococcus aureus USA300 
Natural antimicrobial peptides (AMPs) are promising candidates for developing a generation of new antimicrobials to meet the challenge of antibiotic-resistant pathogens such as meticillin-resistant Staphylococcus aureus (MRSA). To facilitate the search for new candidates, we have utilised the Antimicrobial Peptide Database (APD), which contains natural AMPs from bacteria, fungi, plants and animals. This study demonstrates the identification of novel templates against MRSA by screening 30 peptides selected from the APD. These peptides are short (<25 residues), cysteine-free, cationic and represent candidates from different biological sources such as bacteria, insects, arachnids, tunicates, amphibians, fish and mammals. Six peptides, including ascaphin-8, database-screened antimicrobial peptide 1 (DASamP1), DASamP2, lycotoxin I, maculatin 1.3 and piscidin 1, were found to exert potent antimicrobial activity against an MRSA USA300 isolate. Although five of the six peptides showed broad-spectrum antibacterial activity, DASamP1 displayed killing of MRSA in vitro but not of Escherichia coli, Bacillus subtilis or Pseudomonas aeruginosa. In addition, DASamP1 suppressed early biofilm formation in a mouse model of catheter-associated MRSA infection. DASamP1 is a novel, short and potent peptide that will be a useful starting template for further developing novel anti-MRSA peptides.
PMCID: PMC3320733  PMID: 22445495
Antimicrobial peptides; Biofilms; Meticillin-resistant Staphylococcus aureus
17.  Effect of vinyl sulfone inhibitors of cysteine proteinases on Tritrichomonas foetus infection 
Tritrichomonas foetus is a sexually transmitted protozoon that causes genital inflammation and adverse pregnancy outcomes in cattle. Cysteine proteinases (CPs) released by T. foetus degrade immunoglobulin G (IgG) antibodies, complement component 3 and matrix proteins as well as inducing apoptosis of bovine genital epithelial cells. In this study, the efficacies of the vinyl sulfone CP inhibitors K11777 and WRR-483 were tested against CPs of T. foetus. The activity of secreted T. foetus CPs in culture supernatants was decreased in the presence of vinyl sulfone inhibitors. Inhibitor K11777 reduced the in vitro cytopathogenic effects of T. foetus in bovine foetal trophoblast cells, which are relevant target cells since this pathogen interferes with pregnancy. Pre-treatment of T. foetus prior to intravaginal inoculation diminished genital infection in a murine model. Therefore, vinyl sulfone CP inhibitors reduce several effects of T. foetus-secreted CPs, including cytotoxicity on relevant target host cells and genital infection in a murine model. These inhibitors have potential as chemotherapeutic agents against bovine trichomoniasis. Generalisation to human trichomoniasis requires further study.
PMCID: PMC3279618  PMID: 22104282
Tritrichomonas foetus; Trichomonad cysteine proteinase; Cysteine proteinase inhibitors
18.  Contributions of sigB and sarA to distinct multiple antimicrobial resistance mechanisms of Staphylococcus aureus 
Multiple antimicrobial resistance in Staphylococcus aureus can result from mutations leading to reduced susceptibility to Pine oil-based cleaners (PSRS) as well as following growth with the non-steroidal anti-inflammatory salicylate. We now define the contributions of alternative sigma factor (sigB) and staphylococcal accessory regulator (sarA) to these mechanisms. We conclude that sarA plays a more prominent role than sigB in overall intrinsic multiple antimicrobial resistance. Both genes have similar effects on intrinsic vancomycin resistance, and the salicylate-inducible mechanism is not sigB- or sarA-dependent. Furthermore, analyses determined that altered expression of sigB and sarA is not responsible for the salicylate-inducible mechanism, and sarA upregulation is associated with the PSRS phenotype.
PMCID: PMC3551609  PMID: 16777384
Staphylococcus aureus; Multiple antimicrobial resistance; Alternative sigma factor; Staphylococcal accessory regulator
19.  Molecular diversity in mechanisms of carbapenem resistance in paediatric Enterobacteriaceae 
Development of carbapenem resistance in Enterobacteriaceae has impacted Clinical and Laboratory Standards Institute (CLSI) guidelines, infection control approaches and treatment strategies. The clinical, phenotypic and genotypic characteristics of carbapenem-resistant Enterobacteriaceae (CRE) infections at paediatric referral centres are not well described. CRE were identified through the clinical microbiology laboratory at Seattle Children’s Hospital (Seattle, WA). Clinical data were retrieved from medical records. Resistance testing, polymerase chain reaction (PCR) for resistance determinants, and Escherichia coli transformation were carried out for each isolate. Multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) were used to characterise strain relatedness. PCR amplification and sequencing as well as sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) were used to investigate porin alterations. Six CRE isolates were identified between 2002 and 2010. Significant molecular diversity was documented in their mechanisms of resistance, including plasmid-mediated serine carbapenemase (KPC) and metallo-β-lactamase (IMP), chromosomally-encoded β-lactamase (SME) and porin alterations with extended-spectrum β-lactamases. Patients had underlying health conditions and were from geographically diverse regions. In one case, PFGE of serial isolates documented the development of resistance in a previously susceptible strain. Molecular investigation of this strain identified insertion of the genetic mobile element insertion sequence ISEcp1 in the ompK36 gene, conferring a functional porin alteration as demonstrated by SDS-PAGE. This is the first description of porin disruption by ISEcp1 in a CTX-M-15-positive isolate. This is the largest report of paediatric CRE to date. This diverse description of demographic, phenotypic and molecular characteristics highlights the challenge of CRE infections in high-risk paediatric patients and that attention to emerging resistance mechanisms (including membrane alteration) at paediatric referral centres is essential.
PMCID: PMC3237943  PMID: 22055532
Carbapenem resistance; Enterobacteriaceae; Porin; ISEcp1
20.  Bioactivity of WLBU2 peptide antibiotic in combination with bioerodible polymer 
WLBU2 is a peptide antibiotic designed for broad antimicrobial activity, including bacteria associated with periodontal disease. Although periodontitis is associated with various systemic conditions, ranging from cardiovascular disease to preterm birth, local therapy is needed to treat the source of infection. Biodegradable polymers are often used to control locally the amount and rate of delivery of drugs. In the present study, a bioerodible association polymer comprising cellulose acetate phthalate (CAP) and Pluronic® F-127 (PF-127) was explored for its interaction with WLBU2. The intrinsic antimicrobial activity of CAP/PF-127 and the combined effects of the polymer and WLBU2 were examined using Streptococcus gordonii, a species involved in early colonisation of tooth surfaces. The polymer blend alone had dose-dependent bacteriostatic properties, resulting in a ≥2 log decrease in colonies at the highest concentrations tested, possibly due to the hydrophobicity of CAP disrupting the surface of bacteria. When WLBU2 was combined with CAP/PF-127, an apparent binding of peptide to polymer significantly decreased the activity compared with free WLBU2, which functions like other cationic peptides by destabilising the bacterial membrane. Formulation with sucrose as an excipient, which reduced the interaction between WLBU2 and polymer, restored the bactericidal activity of the peptide antibiotic as reflected by a >3 log decrease in S. gordonii. WLBU2 can be locally delivered using CAP/PF-127 as a release vehicle, with the peptide’s bactericidal activity dominating the polymer’s bacteriostatic effect.
PMCID: PMC3206138  PMID: 21920706
Anti-infectives; Antimicrobial peptide; Periodontitis; Polymeric drug carrier; Streptococcus gordonii; WLBU2
21.  In vitro anti-HIV-1 activity of salicylidene acylhydrazide compounds 
Salicylidene acylhydrazide compounds have been shown to inhibit bacterial pathogens, including Chlamydia and Neisseria gonorrhoeae. If such compounds could also target HIV-1, their potential use as topical microbicides to prevent sexually transmitted infections would be considerable. We determined the in vitro anti-HIV-1 activity, cytotoxicity and mechanism of action of several salicylidene acylhydrazides.
Inhibitory activity was assessed using TZMbl cells and primary peripheral blood mononuclear cells (PBMCs) as targets for HIV-1 infection. Anti-viral activity was measured against cell-free and cell-associated virus and in vaginal fluid and semen simulants. Since the anti-bacterial activity of salicylidene acylhydrazides is reversible by Fe2+, we determined whether Fe2+ and other cations could reverse the anti-HIV-1 activity of the compounds. We also employed real-time PCR to determine the stage affected in the HIV-1 replication cycle.
We identified four compounds with 50% HIV-1 inhibitory concentrations of 1 to 7 μM. In vitro toxicity varied but was generally limited. Activity was similar against three R5 clade B primary isolates and whether targets for virus replication were TZMbl cells or PBMCs. Compounds inhibited cell-free and cell-associated virus and were active in vaginal fluid and semen simulants. Fe2+, but not other cations, reversed the anti-HIV-1 effect. Finally, inhibitory effect of the compounds occurred at a post-integration step.
We identified salicylidene acylhydrazides with in vitro anti-HIV-1 activity in the μM range. The activity of these compounds against other sexually transmitted pathogens makes them potential candidates to formulate for use as a broad-spectrum topical genital microbicide.
PMCID: PMC3438335  PMID: 22819150
Salicylidene acylhydrazides; HIV; microbicide; iron chelation
22.  Effect of colistin exposure and growth phase on the surface properties of live Acinetobacter baumannii cells examined by atomic force microscopy 
The diminishing antimicrobial development pipeline has forced the revival of colistin as a last line of defence against infections caused by multidrug-resistant Gram-negative ‘superbugs’ such as Acinetobacter baumannii. The complete loss of lipopolysaccharide (LPS) mediates colistin resistance in some A. baumannii strains. Atomic force microscopy was used to examine the surface properties of colistin-susceptible and -resistant A. baumannii strains at mid-logarithmic and stationary growth phases in liquid and in response to colistin treatment. The contribution of LPS to surface properties was investigated using A. baumannii strains constructed with and without the lpxA gene. Bacterial spring constant measurements revealed that colistin-susceptible cells were significantly stiffer than colistin-resistant cells at both growth phases (P < 0.01), whilst colistin treatment at high concentrations (32 mg/L) resulted in more rigid surfaces for both phenotypes. Multiple, large adhesive peaks frequently noted in force curves captured on colistin-susceptible cells were not evident for colistin-resistant cells. Adhesion events were markedly reduced following colistin exposure. The cell membranes of strains of both phenotypes remained intact following colistin treatment, although fine topographical details were illustrated. These studies, conducted for the first time on live A. baumannii cells in liquid, have contributed to our understanding of the action of colistin in this problematic pathogen.
PMCID: PMC3433558  PMID: 21925844
Atomic force microscopy; Colistin; Acinetobacter baumannii; Morphology; Surface properties
23.  Will new generations of modified antimicrobial peptides improve their potential as pharmaceuticals? 
The concept of antimicrobial peptides (AMPs) as potent pharmaceuticals is firmly established in the literature, and most research articles on this topic conclude by stating that AMPs represent promising therapeutic agents against bacterial and fungal agents. Indeed, early research in this field showed that AMPs were diverse in nature, had high activities with low minimal inhibitory concentrations, had broad spectrums of activity against bacterial, fungal and viral pathogens, and could easily be manipulated to alter their specificities, reduce their cytotoxicities and increase their antimicrobial activities. Unfortunately, commercial development of these peptides, for even the simplest of applications, has been very limited. With some peptides there are obstacles with their manufacture, in vivo efficacy and in vivo retention. More recently, the focus has shifted. Contemporary research now uses a more sophisticated approach to develop AMPs that surmount many of these prior obstacles. AMP mimetics, hybrid AMPs, AMP congeners, cyclotides and stabilised AMPs, AMP conjugates and immobilised AMPs have all emerged with selective or ‘targeted’ antimicrobial activities, improved retention, or unique abilities that allow them to bind to medical or industrial surfaces. These groups of new peptides have creative medical and industrial application potentials to treat antibiotic-resistant bacterial infections and septic shock, to preserve food or to sanitise surfaces both in vitro and in vivo.
PMCID: PMC3159164  PMID: 21733662
Antimicrobial peptide mimotopes; Hybrid antimicrobial peptides; Antimicrobial peptide congeners; Stabilised antimicrobial peptides; Antimicrobial peptide conjugates; Immobilised antimicrobial peptides; Cyclotides
24.  Differences in biofilm formation and antimicrobial resistance of Pseudomonas aeruginosa isolated from airways of mechanically ventilated patients and cystic fibrosis patients 
Pseudomonas aeruginosa biofilms exhibit increased antimicrobial resistance compared with planktonic isolates and are implicated in the pathogenesis of both acute and chronic lung infections. Whilst antibiotic choices for both infections are based on planktonic antibiotic susceptibility results, differences in biofilm-forming ability between the two diseases have not previously been explored. The aim of this study was to compare differences in biofilm formation and antibiotic resistance of P. aeruginosa isolated from intubated patients and from patients with chronic pulmonary disease associated with cystic fibrosis (CF). The temporal evolution of antibiotic resistance in clonal P. aeruginosa strains isolated from CF patients during periods of chronic infection and acute pulmonary exacerbation was also evaluated. Biofilm formation and biofilm antibiotic susceptibilities were determined using a modified microtitre plate assay and were compared with antibiotic susceptibility results obtained using traditional planktonic culture. Clonality was confirmed using random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) analysis. Pseudomonas aeruginosa isolates collected from intubated patients produced substantially more biofilms compared with CF isolates. There was considerable heterogeneity in biofilm-forming ability among the CF isolates and this was unrelated to pulmonary status. Biofilm antibiotic resistance developed rapidly among clonal CF isolates over time, whilst traditional antibiotic resistance determined using planktonic cultures remained stable. There was a significant positive correlation between imipenem/cilastatin and ceftazidime resistance and biofilm-forming ability. The variability in biofilm-forming ability in P. aeruginosa and the rapid evolution of biofilm resistance may require consideration when choosing antibiotic therapy for newly intubated patients and CF patients.
PMCID: PMC3176759  PMID: 21382698
Pseudomonas aeruginosa; Bacterial biofilm; Antimicrobial resistance; Mechanical ventilation; Cystic fibrosis
25.  Mouse salivary glands and human β-defensin-2 as a study model for antimicrobial gene therapy: technical considerations☆ 
Transduction of salivary glands with antimicrobial peptide genes has great potential for oral infection control. Our ultimate goal is to introduce antimicrobial peptide genes into salivary glands that secrete these peptides into saliva to control bacterial/fungal infection in the oral cavity. However, an animal study model to test this potential has not been established. Therefore, we determined to test (i) whether the potent antimicrobial peptide human β-defensin-2 (hBD-2) can be overexpressed in saliva after transduction of salivary glands and (ii) whether oral fungal infection can be developed in a NOD/SCID murine model. Lentiviral vector SIN18cPPTRhMLV bearing hBD-2 cDNA was introduced into SCID mouse submandibular glands via cannulation. Reverse transcription polymerase chain reaction (RT-PCR), immunohistochemistry or enzyme-linked immunosorbent assay (ELISA) were performed to detect hBD-2 expression in glands or in saliva. Candida albicans 613p was inoculated orally into SCID mice to establish oral candidiasis. Whilst expression of hBD-2 was detected in mouse salivary glands by RT-PCR and immunohistochemistry 1 day or 1 week following delivery of lentivirus, hBD-2 was not detected in saliva. There was recoverable C. albicans from the oral cavity and gastrointestinal tract 4 days to 4 weeks after infection, but there was no establishment of observable oral candidiasis in SCID mice under a stereomicroscope. Our data indicate that lentiviral vectors transduce mouse salivary glands, but not at a sufficient level to allow hBD-2 detection in saliva. Other vectors for gene transduction and additional treatment of SCID mice to establish oral candidiasis are needed in order to utilise mouse salivary glands to test antimicrobial gene therapy.
PMCID: PMC3285981  PMID: 16963233
hBD-2; Lentiviral vectors; Mouse salivary glands; Candida albicans; SCID mice

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