In vitro evaluation of ceftaroline alone and in combination with tobramycin against hospital-acquired meticillin-resistant Staphylococcus aureus (HA-MRSA) isolates

The aim of this study was to evaluate the in vitro activity of ceftaroline and its potential for synergy with tobramycin in comparison with vancomycin against a collection of hospital-acquired meticillin-resistant Staphylococcus aureus (HA-MRSA), including isolates with reduced susceptibility to glycopeptides. Ceftaroline, vancomycin, daptomycin and linezolid susceptibilities were determined for 200 HA-MRSA isolates. Four randomly selected strains [including one vancomycin-intermediate S. aureus (VISA) and one heteroresistant VISA (hVISA)] were evaluated in time–kill experiments with ceftaroline and vancomycin alone or combined with tobramycin at 0.25 and 0.5 times the minimum inhibitory concentration (MIC). MICs for 50% and 90% of the organisms (MIC₅₀ and MIC₉₀, respectively) were both 1 mg/L for ceftaroline and were 1 mg/L and 2 mg/L, respectively, for vancomycin. The same ceftaroline MIC ranges (0.25–2 mg/L) were observed for isolates recovered from respiratory tract samples, blood or skin. In time–kill experiments, no synergy was observed at 0.25× MIC against any tested isolates with either ceftaroline or vancomycin. In contrast, the combination of ceftaroline plus tobramycin at 0.5× MIC was synergistic against the two MRSA strains and the hVISA but was indifferent against the VISA isolate. In conclusion, ceftaroline demonstrated antimicrobial activity independently of the specimen source and exhibited lower MICs than vancomycin. Finally, at sub-MIC levels, ceftaroline plus tobramycin displayed significantly greater activity than vancomycin plus tobramycin against MRSA (P < 0.01).     

Application of pharmacokinetic/pharmacodynamic modelling and simulation for the prediction of target attainment of ceftobiprole against meticillin-resistant Staphylococcus aureus using minimum inhibitory concentration and time–kill curve based approaches

The purpose of this report was to compare two different methods for dose optimisation of antimicrobials. The probability of target attainment (PTA) was calculated using Monte Carlo simulation to predict the PK/PD target of fT_>MIC or modelling and simulation of time–kill curve data. Ceftobiprole, the paradigm compound, activity against two MRSA strains was determined, ATCC 33591 (MIC = 2 mg/L) and a clinical isolate (MIC = 1 mg/L). A two-subpopulation model accounting for drug degradation during the experiment adequately fit the time–kill curve data (concentration range 0.25–16× MIC). The PTA was calculated for plasma, skeletal muscle and subcutaneous adipose tissue based on data from a microdialysis study in healthy volunteers. A two-compartment model with distribution factors to account for differences between free serum and tissue interstitial space fluid concentration appropriately fit the pharmacokinetic data. Pharmacodynamic endpoints of fT_>MIC of 30% or 40% and 1- or 2-log kill were used. The PTA was >90% in all tissues based on the PK/PD endpoint of fT_>MIC >40%. The PTAs based on a 1- or 2-log kill from the time–kill experiments were lower than those calculated based on fT_>MIC. The PTA of a 1-log kill was >90% for both MRSA isolates for plasma and skeletal muscle but was slightly below 90% for subcutaneous adipose tissue (both isolates ca. 88%). The results support a dosing regimen of 500 mg three times daily as a 2-h intravenous infusion. This dose should be confirmed as additional pharmacokinetic data from various patient populations become available.     

Prevalence of clinical meticillin-resistant Staphylococcus aureus (MRSA) with high-level mupirocin resistance in Shanghai and Wenzhou,China

A total of 803 clinical meticillin-resistant Staphylococcus aureus (MRSA) isolates obtained from Shanghai and Wenzhou in China were subjected to a screening test by disk diffusion for detection of mupirocin resistance. Among the 803 strains, 53 (6.6%) were mupirocin-resistant. Of these 53 strains, all were discovered by the agar dilution method and polymerase chain reaction (PCR) to be high-level mupirocin-resistant and to harbour the mupA gene. Plasmid DNA hybridisation and curing experiments disclosed that mupA was located on a large plasmid varying in size between 23.0 kb and 52.4 kb in all strains. Susceptibility testing of 10 antibiotics revealed that resistance rates between the Shanghai isolates and the Wenzhou isolates to trimethoprim/sulfamethoxazole and rifampicin differed significantly. Molecular typing by pulsed-field gel electrophoresis (PFGE), staphylococcal chromosomal cassette mec (SCCmec) and staphylococcal protein A (spa) revealed that PFGE A–SCCmec IIIA–spa t030 and PFGE B–SCCmec IIIA–spa t030 represented all of the Wenzhou strains, whereas PFGE N–SCCmec I–spa t318, PFGE P–SCCmec III–spa t037, PFGE I–SCCmec III–spa t037 and PFGE M–SCCmec IIIA–spa t002 were the predominant profiles among Shanghai isolates. These findings indicated that high-level mupirocin resistance mediated by plasmids prevailed in the clinical mupirocin-resistant MRSA from Shanghai and Wenzhou and was mainly related to the transmission of clones.     

Ceftolozane/tazobactam activity tested against Gram-negative bacterial isolates from hospitalised patients with pneumonia in US and European medical centres (2012)

During 2012, a total of 2968 isolates were consecutively collected from 59 medical centres in the USA and 15 European countries from hospitalised patients with pneumonia. Ceftolozane/tazobactam (tazobactam at a fixed concentration of 4 mg/L) and comparator agents were tested by reference methods, and MIC endpoints were interpreted by CLSI (2013) and EUCAST (2013) breakpoint criteria. Pseudomonas aeruginosa was the most common isolated pathogen (1019 strains; 34.3%), and ceftolozane/tazobactam was the most active β-lactam tested against P. aeruginosa (MIC_50/90, 0.5/4 mg/L; 94.1% inhibited at ≤8 mg/L). P. aeruginosa exhibited moderate susceptibility to meropenem (MIC_50/90, 0.5/>8 mg/L; 73.7% susceptible), ceftazidime (MIC_50/90, 2/>32 mg/L; 73.6% susceptible), cefepime (MIC_50/90, 4/>16 mg/L; 76.5% susceptible), piperacillin/tazobactam (MIC_50/90, 8/>64 mg/L; 69.5% susceptible), levofloxacin [MIC_50/90, 0.5/>4 mg/L; 69.9/61.0% susceptible (CLSI/EUCAST criteria)] and gentamicin (MIC_50/90, 2/>8 mg/L; 80.7% susceptible). Ceftolozane/tazobactam exhibited activity against many ceftazidime-non-susceptible, meropenem-non-susceptible and piperacillin/tazobactam-non-susceptible, multidrug-resistant (MDR) and extensively drug-resistant (XDR) P. aeruginosa isolates. Ceftolozane/tazobactam was active (MIC_50/90, 0.25/4 mg/L; 94.6% inhibited at ≤8 mg/L) against 1530 Enterobacteriaceae, including activity against many MDR and XDR strains. MDR and XDR prevalence varied widely between countries both for P. aeruginosa (24.1% MDR and 17.1% XDR overall) and Enterobacteriaceae (15.4% MDR and 2.7% XDR overall). All β-lactams had limited activity against Acinetobacter spp. and Stenotrophomonas maltophilia. Ceftolozane/tazobactam demonstrated greater in vitro activity than currently available cephalosporins, carbapenems and piperacillin/tazobactam when tested against P. aeruginosa. In addition, ceftolozane/tazobactam demonstrated greater activity than contemporary cephalosporins and piperacillin/tazobactam when tested against most Enterobacteriaceae.     

Daptomycin activity tested against 164 457 bacterial isolates from hospitalised patients: Summary of 8 years of a Worldwide Surveillance Programme (2005–2012)

We report the results of 8 years (2005–2012) of the Daptomycin Surveillance Programme Worldwide. Consecutive non-duplicate bacterial isolates (prevalence design) were collected from patients with documented infections in 410 medical centres and were susceptibility tested by reference broth microdilution methods. A total of 164 457 Gram-positive isolates were evaluated, including 97 542 Staphylococcus aureus, 21 413 coagulase-negative staphylococci (CoNS), 29 619 enterococci and 15 883 β-haemolytic streptococci. The prevalence of daptomycin-non-susceptible isolates was extremely low for all species in all geographic regions. Overall, the highest occurrence of non-susceptible isolates was observed among CoNS (0.19%), followed by Enterococcus faecium (0.18%), S. aureus (0.05%), Enterococcus faecalis (0.02%) and β-haemolytic streptococci (0.00%). Moreover, no trend towards increased daptomycin resistance (non-susceptibility) was observed for any species in any geographic region during the study interval. Against S. aureus, the daptomycin MIC_50/90 was 0.25/0.5 mg/L in all geographic regions (99.95% susceptible overall). Only 53 daptomycin-non-susceptible S. aureus isolates were observed and the vast majority (49; 92.5%) had a daptomycin MIC value only 1 log₂ dilution above the published susceptible breakpoint. Daptomycin was also active against CoNS (MIC_50/90, 0.25/0.5 mg/L; 99.81% susceptible), E. faecalis (MIC_50/90, 1/2 mg/L; 99.98% susceptible), E. faecium (MIC_50/90, 2/4 mg/L; 99.82% susceptible) including vancomycin-non-susceptible isolates (4521 isolates; MIC_50/90, 2/2 mg/L; 99.76% susceptible), and β-haemolytic streptococci (MIC_50/90, ≤0.06/0.25 mg/L; 100.0% susceptible). In conclusion, daptomycin has remained very active against indicated species worldwide, and no significant year-to-year or regional variation in daptomycin activity has been detected.     

Ceftaroline activity tested against uncommonly isolated Gram-positive pathogens: report from the SENTRY Antimicrobial Surveillance Program (2008–2011)

Ceftaroline was tested against 1859 clinically significant Gram-positive organisms from uncommonly isolated species. The organisms (31 species/groups) were collected from 133 medical centres worldwide over a 4-year period (2008–2011). Coagulase-negative staphylococci were generally susceptible to ceftaroline, with MIC₅₀ values (minimum inhibitory concentration required to inhibit 50% of the isolates) of 0.06–0.5 mg/L. Ceftaroline was active against Micrococcus spp. [minimum inhibitory concentration required to inhibit 90% of the isolates (MIC₉₀) = 0.06 mg/L], but showed more limited potency versus some Corynebacterium spp. and Listeria monocytogenes isolates. Ceftaroline was active against all β-haemolytic streptococci and viridans group streptococcal species/groups listed, with MIC₅₀ and MIC₉₀ values ranging from ≤0.015 mg/L to 0.03 mg/L and from ≤0.015 mg/L to 0.5 mg/L, respectively. Based on these in vitro findings, ceftaroline may have a potential role in the treatment of infections caused by these rarer species as guided by reference MIC test results.     

High efficacy of clofazimine and its synergistic effect with amikacin against rapidly growing mycobacteria

The aim of this study was to determine whether clofazimine, dapsone and cycloserine may be suitable antimicrobial agents for the treatment of infections due to rapidly growing mycobacteria (RGM). The antimicrobial activity of the three drugs against 117 Mycobacterium abscessus isolates, 48 Mycobacterium fortuitum isolates and 20 Mycobacterium chelonae isolates was evaluated based on their broth microdilution minimal inhibitory concentrations (MICs) against the isolates. Clofazimine was highly efficacious against these RGM. The vast majority of M. abscessus, M. fortuitum and M. chelonae isolates (99.1%, 91.7% and 100%, respectively) had clofazimine MICs of ≤1 mg/L. MIC₅₀ values (MIC for 50% of the organisms) of clofazimine against the isolates ranged from 0.25 mg/L to 0.5 mg/L and MIC₉₀ values (MIC for 90% of the organisms) ranged from 0.5 mg/L to 1.0 mg/L. Cycloserine and dapsone had little or no activity against the isolates. The effects of combined application of clofazimine and amikacin on 40 M. abscessus isolates, 48 M. fortuitum isolates and 20 M. chelonae isolates were evaluated. Addition of 0.25× MIC of amikacin for the isolates to clofazimine reduced clofazimine MICs in all of the M. abscessus and M. chelonae isolates and in 48% of the M. fortuitum isolates tested. Clofazimine, either alone or combined with amikacin, may serve as a promising drug for the treatment of RGM infections.     

In vitro antibacterial activity of ceftobiprole against clinical isolates from French teaching hospitals: proposition of zone diameter breakpoints

The aims of this study were to determine the in vitro activity profile of ceftobiprole, a pyrrolidinone cephalosporin, against a large number of bacterial pathogens and to propose zone diameter breakpoints for clinical categorisation according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) minimum inhibitory concentration (MIC) breakpoints. MICs of ceftobiprole were determined by broth microdilution against 1548 clinical isolates collected in eight French hospitals. Disk diffusion testing was performed using 30 μg disks according to the method of the Comité de l’Antibiogramme de la Société Française de Microbiologie (CA-SFM). The in vitro activity of ceftobiprole, expressed by MIC_50/90 (MICs for 50% and 90% of the organisms, respectively) (mg/L), was as follows: meticillin-susceptible Staphylococcus aureus, 0.25/0.5; meticillin-resistant S. aureus (MRSA), 1/2; meticillin-susceptible coagulase-negative staphylococci (CoNS), 0.12/0.5; meticillin-resistant CoNS, 1/2; penicillin-susceptible Streptococcus pneumoniae, ≤0.008/0.03; penicillin-resistant S. pneumoniae, 0.12/0.5; viridans group streptococci, 0.03/0.12; β-haemolytic streptococci, ≤0.008/0.016; Enterococcus faecalis, 0.25/1; Enterococcus faecium, 64/128; Enterobacteriaceae, 0.06/32; Pseudomonas aeruginosa, 4/16; Acinetobacter baumannii, 0.5/64; Haemophilus influenzae, 0.03/0.12; and Moraxella catarrhalis, 0.25/0.5. According to the regression curve, zone diameter breakpoints could be 28, 26, 24 and 22 mm for MICs of 0.5, 1, 2 and 4 mg/L respectively. In conclusion, this study confirms the potent in vitro activity of ceftobiprole against many Gram-positive bacteria, including MRSA but not E. faecium, whilst maintaining a Gram-negative spectrum similar to the advanced-generation cephalosporins such as cefepime. Thus ceftobiprole appears to be well suited for the empirical treatment of a variety of healthcare-associated infections.     

Bactericidal activity of daptomycin versus vancomycin in the presence of human albumin against vancomycin-susceptible but tolerant methicillin-resistant Staphylococcus aureus (MRSA) with daptomycin minimum inhibitory concentrations of 1–2 μg/mL

This study explored the influence of vancomycin tolerance and protein binding on the bactericidal activity of vancomycin versus daptomycin (protein binding 36.9% vs. 91.7%, respectively) against four vancomycin-tolerant methicillin-resistant Staphylococcus aureus (MRSA) [minimum inhibitory concentration/minimum bactericidal concentration (MIC/MBC) = 0.5/16, 1/32, 2/32 and 1/32 μg/mL for vancomycin and 1/1, 1/2, 2/2 and 2/4 μg/mL for daptomycin]. Killing curves were performed with vancomycin/daptomycin concentrations equal to serum peak concentrations (C_max) (65.70/98.60 μg/mL) and trough concentrations (C_min) (7.90/9.13 μg/mL) in the presence and absence of a physiological human albumin concentration (4 g/dL), controlled with curves with the theoretical free drug fraction of vancomycin/daptomycin C_max (41.45/8.18 μg/mL) and C_min (4.98/0.76 μg/mL). Vancomycin C_max and C_min concentrations, regardless of the media, showed a bacteriostatic profile not reaching a reduction of 99% or 99.9% of the initial inocula during the 24-h experimental time period. Daptomycin antibacterial profiles significantly differed when testing C_max and C_min. C_max was rapidly bactericidal (≤4 h) with >5 log₁₀ reduction in the initial inocula for all strains, regardless of the presence or not of albumin or the use of concentrations similar to free C_max. C_min exhibited similar final colony counts at 0 h and 24 h in curves with albumin, but with >3 log colony-forming units (CFU)/mL reduction at ≤4 h for strains with an MIC of 1 μg/mL and ca. 2 log CFU/mL reduction at ≤6 h for strains with an MIC of 2 μg/mL. This activity was significantly higher than the activity of the free C_min fraction. The results of this study reinforce the idea that pharmacodynamics using concentrations calculated using reported protein binding are unreliable. Daptomycin exhibited rapid antibacterial activity against vancomycin-tolerant MRSA isolates even against those with high daptomycin MICs in the presence of physiological albumin concentrations.     

In vitro and intracellular activities of fosfomycin against clinical strains of Listeria monocytogenes

This study was designed to evaluate the potential role of fosfomycin as a therapeutic agent in human listeriosis. The in vitro activity of fosfomycin against 154 Listeria monocytogenes clinical isolates under conditions that mimic the induction of prfA expression was determined and was correlated with fosfomycin intracellular antimicrobial activity. In vitro, partial induction of prfA expression is achieved through bacterial growth in brain–heart infusion agar supplemented with activated charcoal (BHIC). A fosfomycin pharmacokinetic/pharmacodynamic breakpoint of ≤64 mg/L was estimated using a Monte Carlo simulation to assess the success of an intravenous fosfomycin dose of 300 mg/kg/day over 5000 individuals. Eighty strains (51.9%) were susceptible to fosfomycin in BHIC, with minimum inhibitory concentrations (MICs) of ≤64 mg/L; 13 strains (8.4%) had the epidemic clone (EC) marker. In addition, 27 strains (17.5%) had a three doubling dilutions reduction in the MIC from ≥1024 mg/L to 128 mg/L (96–128 mg/L by Etest). The fosfomycin modal MIC is lower under prfA expression. However, this effect is smaller in terms of clinical categorisation of isolates and can be influenced by the serotype and clonal type. In A549 cells, the reductions in bacterial inocula of the two susceptible isolates studied after 1 h and 24 h of incubation with fosfomycin at 0.5× the human maximum serum concentration (C_max) were 45.8% and 46.6%, and 93.8% and 99.1%, respectively. Slightly higher reductions were found with fosfomycin at 1× C_max. The resistant strain tested showed significantly lower reductions in all assays.     

In vitro activity of the siderophore monosulfactam BAL30072 against contemporary Gram-negative pathogens from New York City,including multidrug-resistant isolates

The in vitro activity of BAL30072 was assessed against clinical isolates from NYC hospitals, including isolates from a citywide surveillance study and a collection of isolates with well-characterised resistance mechanisms. BAL30072 was the most active β-lactam against Pseudomonas aeruginosa (MIC_50/90, 0.25/1 μg/mL), Acinetobacter baumannii (MIC_50/90, 4/>64 μg/mL) and KPC-possessing Klebsiella pneumoniae (MIC_50/90, 4/>64 μg/mL). Combining BAL30072 with meropenem resulted in a ≥4-fold decrease in the BAL30072 MIC₉₀ both for A. baumannii and K. pneumoniae. For isolates with a BAL30072 MIC > 4 μg/mL, addition of a sub-MIC concentration of colistin resulted in a four-fold decrease in the BAL30072 MIC in 44% of P. aeruginosa, 82% of A. baumannii and 23% of K. pneumoniae. Using sub-MIC concentrations, BAL30072 plus colistin was bactericidal against 4 of 11 isolates in time–kill studies. BAL30072 MICs were frequently lower for P. aeruginosa and K. pneumoniae when tested using Mueller–Hinton agar versus Iso-Sensitest agar or Mueller–Hinton broth. Against the well-characterised isolates, reduced susceptibility to BAL30072 correlated with mexA and mexX expression (P. aeruginosa), adeB expression (A. baumannii) and presence of SHV-type ESBLs (A. baumannii and K. pneumoniae). BAL30072 shows promising activity against contemporary Gram-negatives, including MDR P. aeruginosa, A. baumannii and K. pneumoniae. Enhanced activity was often present when BAL30072 was combined with meropenem or colistin. BAL30072 MICs were influenced by the testing method, particularly for P. aeruginosa and K. pneumoniae. Further in vivo studies are warranted to determine the potential clinical utility of BAL30072 alone and combined with other agents.     

Selection and characterisation of Staphylococcus aureus mutants with reduced susceptibility to the investigational oxazolidinone MRX-I

MRX-I is a new oxazolidinone antimicrobial under development. In this study, the potential for development of resistance to MRX-I in Staphylococcus aureus was investigated and key mutations were characterised. Determination of spontaneous resistance frequency and the mutant selection window (MSW) were performed with meticillin-susceptible S. aureus (MSSA) ATCC 29213, meticillin-resistant S. aureus (MRSA) ATCC 33591 and two clinical MRSA isolates SA 0016 and SA 0017. Selected resistant mutants were sequenced for 23S rRNA as well as genes encoding the ribosomal proteins L3, L4 and L22. Resistance frequencies for the aforementioned strains were <8.25 × 10⁻¹², <6.33 × 10⁻¹², <2.96 × 10⁻¹³ and <4.52 × 10⁻¹³, respectively, and the MSW of MRX-I was 2–4, 1–4, 1–2 and 1–4 mg/L, respectively. After 30 serial passages, MRX-I minimum inhibitory concentrations (MICs) increased up to 8- to 16-fold both against MSSA and MRSA, whilst linezolid MICs increased 128-fold against MSSA and 16- to 32-fold against MRSA. MRX-I resistance mutations were clustered mainly in 23S rRNA and L3 protein regions. The U2504A transversion in 23S rRNA dominated in MRX-I-resistant mutants. No mutations in L4 and L22 proteins were observed. MRX-I exhibits a low potential to develop resistance in S. aureus, with a reduced resistance propensity compared with linezolid.     

Evaluation of the in vitro activities of ceftobiprole and comparators in staphylococcal colony or microtitre plate biofilm assays

The aim of this study was to evaluate the in vitro efficacy of ceftobiprole and comparator antibiotics, either alone or in combination, in staphylococcal MBEC™ (minimum biofilm eradication concentration) and colony biofilm assays at dilutions of the maximum free-drug plasma concentration attained during clinical use (fC_max). Staphylococci tested included meticillin-susceptible and meticillin-resistant Staphylococcus aureus (n = 6) and Staphylococcus epidermidis (n = 2). Relative to no-drug controls, after 7 days of exposure ceftobiprole concentrations from 1/4 fC_max to fC_max generally decreased CFUs in MBEC or colony biofilms of S. aureus isolates by ca. 1.5 log₁₀ to ≥2.5 log₁₀. Gentamicin reduced colony biofilm CFUs by ≥1.4 log₁₀ at these concentrations with gentamicin-susceptible isolates. Following 7 days of exposure, vancomycin and rifampicin were ineffective as single agents or in combination in the colony model, but yielded CFU decreases from 0 to 5 log₁₀ in the MBEC model. Treatment of biofilms with rifampicin for 7 days yielded rifampicin-resistant mutants, and the selection of rifampicin resistance was inhibited by co-treatment with ceftobiprole. Thus, ceftobiprole alone or in combination demonstrated promising activity against biofilms of meticillin-susceptible and -resistant staphylococci at clinically relevant concentrations. In contrast, vancomycin and rifampicin, two agents used clinically for the treatment of biofilm infections, tested separately or together gave inconsistent results and generally had little impact on cell viability.     

Colistin/daptomycin: an unconventional antimicrobial combination synergistic in vitro against multidrug-resistant Acinetobacter baumannii

The in vitro activity of the combination colistin/daptomycin was evaluated against multidrug-resistant Acinetobacter baumannii clinical isolates. Clonal relationships were assessed by pulsed-field gel electrophoresis. The following synergy studies were undertaken: (i) daptomycin MICs were determined by E-test on Mueller–Hinton agar plates supplemented with a subinhibitory concentration of colistin; and (ii) time–kill methodology using tubes containing an inoculum of 5 × 10⁵ CFU/mL and subinhibitory concentrations of each antibiotic alone or in combination subcultured at 0, 5 and 24 h for colony counting. Synergy was defined as ≥2 log₁₀ CFU/mL decrease of viable colonies compared with colistin alone. Ten colistin-susceptible and four colistin-resistant A. baumannii isolates were tested. Isolates were assigned to nine different clonal types. Enhanced in vitro activity of the combination was detected only against colistin-susceptible isolates; using plates supplemented with colistin, the daptomycin MIC was reduced by 4- to 128-fold. From a total of 30 isolate–concentration combinations in time–kill studies, a synergistic interaction was detected in 16 (53.3%). The combination exhibited synergy against 8 and 12 of these combinations at 5 h and 24 h, respectively. No antagonism was detected. Colistin alone was bactericidal against two colistin-susceptible isolates at 24 h, whereas the combination was bactericidal against 9 colistin-susceptible isolates at 24 h. Against all colistin-resistant isolates, the combination exhibited a static effect and indifference in time–kill studies. Potent in vitro synergistic interactions between colistin and daptomycin provide evidence that this unorthodox combination may be beneficial in the treatment of colistin-susceptible multidrug-resistant A. baumannii.     

Chemical composition and anticancer,antiinflammatory, antioxidant and antimalarial activities of leaves essential oil of Cedrelopsis grevei

The essential oil from Cedrelopsis grevei leaves, an aromatic and medicinal plant from Madagascar, is widely used in folk medicine. Essential oil was characterized by GC–MS and quantified by GC–FID. Sixty-four components were identified. The major constituents were: (E)-β-farnesene (27.61%), δ-cadinene (14.48%), α-copaene (7.65%) and β-elemene (6.96%). The essential oil contained a complex mixture consisting mainly sesquiterpene hydrocarbons (83.42%) and generally sesquiterpenes (98.91%). The essential oil was tested cytotoxic (on human breast cancer cells MCF-7), antimalarial (Plasmodium falciparum), antiinflammatory and antioxidant (ABTS and DPPH assays) activities. C. grevei essential oil was active against MCF-7 cell lines (IC₅₀ = 21.5 mg/L), against P. falciparum, (IC₅₀ = 17.5 mg/L) and antiinflammatory (IC₅₀ = 21.33 mg/L). The essential oil exhibited poor antioxidant activity against DPPH (IC₅₀ > 1000 mg/L) and ABTS (IC₅₀ = 110 mg/L) assays. A bibliographical review was carried out of all essential oils identified and tested with respect to antiplasmodial, anticancer and antiinflammatory activities. The aim was to establish correlations between the identified compounds and their biological activities (antiplasmodial, anticancer and antiinflammatory). According to the obtained correlations, 1,4-cadinadiene (R² = 0.61) presented a higher relationship with antimalarial activity. However, only (Z)-β-farnesene (R² = 0.73) showed a significant correlation for anticancer activity.     

Characterising the post-antifungal effects of micafungin against Candida albicans,Candida glabrata,Candida parapsilosis and Candida krusei isolates

In this study, we measured time–kills and post-antifungal effects (PAFEs) for micafungin against Candida albicans (n = 4), Candida glabrata (n = 3), Candida parapsilosis (n = 3) and Candida krusei (n = 2) isolates and further characterised the PAFEs. Minimum inhibitory concentrations (MICs) were 0.5–1.0 mg/L against C. parapsilosis and 0.008–0.125 mg/L against the other species. Micafungin caused kills >1 log at 1 × MIC, 4 × MIC (range 1.19–3.10 log) and 16 × MIC (2.27–3.68 log), achieving fungicidal levels (≥3 log) against nine isolates. One-hour drug exposure during PAFE experiments resulted in kills of 0.73–2.88 log and 1.72–3.55 log at 4× and 16× MIC, respectively, achieving fungicidal levels against five isolates. Isolates of each species collected 8 h after a 1-h exposure to micafungin (4× and 16× MIC) were hypersusceptible to sodium dodecyl sulphate (SDS) and Calcofluor White. Cells tested during the PAFE period demonstrated cell wall disturbances as evident on electron micrographs as well as significant reductions in adherence to epithelial cells. Phagocytosis by J774 macrophages was significantly enhanced for three PAFE isolates tested. Micafungin is fungicidal and exerts PAFEs that kill diverse Candida spp., disturb cell walls of viable organisms, reduce adherence and enhance susceptibility to phagocytosis.     

Drug resistance patterns in Salmonella enterica subspecies enterica serotype Typhi strains isolated over a period of two decades,with special reference to ciprofloxacin and ceftriaxone

Fluoroquinolone-resistant Salmonella enterica subspecies enterica serotype Typhi are being increasingly reported from the Asian subcontinent. This has been hypothesised to be due to a double mutation in the gyrA gene. A total of 113 S. Typhi strains isolated during 1987–2006 in a tertiary-level hospital of North India were monitored for their antibiotic susceptibility by the disk diffusion and minimum inhibitory concentration (MIC) methods. The study period was arbitrarily divided into four equal parts, each comprising 5 years. The antibiotics tested showed an extremely wide range of MICs during all four periods except for ceftriaxone, which showed no resistance during the study period. However, a gradual increase in the MIC of this drug was observed, i.e. 0.047 mg/L, 0.098 mg/L, 0.211 mg/L and 0.3652 mg/L during the four study periods. Ninety-one percent of the strains isolated in the final study period were observed to have MIC levels ≥0.125 mg/L to ciprofloxacin. Furthermore, gyrA restriction analysis showed no mutation at the two reported sites of the gene, suggesting that the double mutation theory in the development of ciprofloxacin resistance may not be the only mechanism responsible for fluoroquinolone resistance.     

Methodological agreement on the in vitro activity of ceftaroline against cefotaxime-susceptible and -resistant pneumococci

Ceftaroline reportedly has lower minimum inhibitory concentrations (MICs) than established cephalosporins for Streptococcus pneumoniae. We further evaluated this activity using 155 pneumococci chosen by serotype and cefotaxime MIC. MICs were determined by agar dilution on Mueller–Hinton agar and Iso-Sensitest agar and by Etest. Inhibition zones were measured for 5 μg and 30 μg ceftaroline discs using both CLSI/EUCAST and BSAC methodology. Ceftaroline was more active than cefotaxime, with MICs 2–8-fold lower for isolates with cefotaxime MICs of ≤1 mg/L and mostly in the range 0.125–0.5 mg/L for those with cefotaxime MICs of 2 mg/L to ≥16 mg/L. Twelve isolates belonging to serotypes 14 (n = 2), 19A (n = 6) and 19F (n = 4) were ceftaroline-resistant, with MICs of 0.5–1 mg/L. Essential agreement between MIC methods was excellent, with values on Iso-Sensitest agar and Mueller–Hinton agar identical ±1 doubling dilution in all cases, and with 154/155 values identical ±1 doubling dilution between agar dilution and Etest. Nevertheless, 5/11 isolates with agar dilution MICs of 0.5 mg/L (i.e. just resistant) ‘had’ MICs of 0.25 mg/L (just susceptible) by Etest. Inhibition zones also correlated with MICs, but discrimination around the breakpoint MICs was poor irrespective of method and disc type. In summary, the results confirm the good activity of ceftaroline against pneumococci, but susceptibility testing will present challenges in routine laboratories, with discs poorly discriminatory and with Etest prone to give susceptible results for isolates with MICs one doubling dilution above the breakpoint.     

In vitro activity of rifaximin against clinical isolates of Escherichia coli and other enteropathogenic bacteria isolated from travellers returning to the UK

Rifaximin is licensed in the EU and USA for treating travellers’ diarrhoea caused by non-invasive bacteria. Selection for resistance mechanisms of public health significance might occur if these are linked to rifamycin resistance. Rifaximin MICs were determined by agar dilution for 90 isolates each of Escherichia coli, Shigella spp., nontyphoidal Salmonella enterica, typhoidal S. enterica and Campylobacter spp., an additional 60 E. coli with CTX-M ESBLs isolated from patients with travellers’ diarrhoea, and 30 non-diarrhoeal carbapenemase-producing E. coli. Comparators were rifampicin, ciprofloxacin, azithromycin, trimethoprim/sulfamethoxazole and doxycycline. Isolates with rifaximin MICs>32 mg/L were screened for arr genes, and critical rpoB regions were sequenced. Rifaximin was active at ≤32 mg/L against 436/450 (96.9%) diverse Enterobacteriaceae, whereas 81/90 (90%) Campylobacter spp. were resistant to rifaximin at ≥128 mg/L. Rifaximin MICs were ≥128 mg/L for two Shigella and five MDR E. coli producing NDM (n = 3), OXA-48 (n = 1) or CTX-M-15 (n = 1). Two of the five MDR E. coli had plasmids harbouring arr-2 together with bla_NDM, and two (one each with bla_NDM and bla_CTX-M-15) had His526Asn substitutions in RpoB. The rifamycin resistance mechanism remained undefined in one MDR E. coli isolate (with bla_OXA-48) and the two Shigella isolates. Rifaximin showed good in vitro activity against diverse Enterobacteriaceae but was largely inactive against Campylobacter spp. Rifaximin has potential to co-select MDR E. coli in the gut flora, but much stronger associations were seen between ESBL and/or carbapenemase production and resistance to alternative treatments for travellers’ diarrhoea, notably ciprofloxacin and azithromycin.     

Activity of aminocandin (IP960; HMR3270) compared with amphotericin B,itraconazole, caspofungin and micafungin in neutropenic murine models of disseminated infection caused by itraconazole-susceptible and -resistant strains of Aspergillus fumigatus

Aminocandin (IP960; HMR3270; NXL201) is a new echinocandin with broad-spectrum in vitro activity against Aspergillus and Candida spp. We compared the activity of aminocandin with that of amphotericin B (AmB), itraconazole (ITC) and caspofungin (CAS) in murine models of disseminated aspergillosis against three strains of A. fumigatus, two of which were fully susceptible (AF293 and A1163) and one was resistant to ITC (AF91). Mice were rendered temporarily neutropenic or persistently neutropenic with cyclophosphamide and were infected intravenously 3 days later. Temporarily neutropenic mice were treated with either intraperitoneal (i.p.) AmB (5 mg/kg/dose), oral (p.o.) ITC (25 mg/kg/dose), intravenous (i.v.) aminocandin (0.25–10 mg/kg/dose), i.p. aminocandin (1 mg/kg/dose) or solvent control for 9 days. Mice were euthanised 11 days post infection and the kidneys and liver were removed for quantitative culture. Following infection with AF293, only aminocandin 5 mg/kg i.v. yielded 100% survival. Aminocandin 1 mg/kg i.v., AmB 5 mg/kg i.p. or ITC 25 mg/kg p.o. were equivalent (P > 0.05). Aminocandin 5 mg/kg was superior to aminocandin 0.25 mg/kg (P < 0.0001) as well as all controls (P < 0.0001) in reducing mortality. Following infection with AF91, only aminocandin at 5 mg/kg and 1 mg/kg i.v. yielded 100% survival, which was superior to ITC, aminocandin 0.25 mg/kg and controls (all P < 0.0001). In the persistently neutropenic model with A1163, aminocandin, CAS and micafungin (2–10 mg/kg) were all effective at prolonging survival, with some impact on reducing culture burdens, even with alternate-day dosing (4 mg/kg). The only fungicidal regimen was aminocandin 5 mg/kg, which sterilised 40% and 50% of mice following infection with AF293 and AF91, respectively. Aminocandin at doses of ≥1 mg/kg is highly effective in reducing mortality and organ burden in disseminated infection caused by ITC-susceptible and -resistant A. fumigatus.