Typing and susceptibility of bacterial isolates from the fidaxomicin (OPT-80) phase II study for C. difficile infection

BackgroundClostridium difficile infection (CDI) has been increasing in incidence and severity in recent years, coincident with the spread of a “hypervirulent” strain, REA type BI (ribotype 027, PFGE NAP 1). Exacerbating the problem has been the observation that metronidazole may be showing decreased effectiveness, particularly in the more severe cases. Fidaxomicin is an 18-membered macrocycle currently in phase 3 trials for the treatment of C. difficile infection (CDI). An open-label, phase II study in CDI patients has been completed and the clinical results published. C. difficile organisms were isolated from patient stool specimens and typed by restriction endonuclease analysis (REA) in order to determine the frequency and susceptibility of the C. difficile isolates and their response to treatment.MethodsFecal samples were plated on CCFA agar for isolation of C. difficile. These isolates were tested for susceptibility to fidaxomicin, vancomycin, and metronidazole using CLSI agar dilution methods and were typed by REA.ResultsC. difficile was isolated from 38 of 49 subjects and 16 (42%) were the epidemic C. difficile BI group. The BI strain was distributed approximately equally in the three dosing groups. Overall antibiotic susceptibilities were consistent with the previously reported MIC₉₀ values for the three antibiotics tested, but the MIC₉₀ of BI strains was two dilutions higher than non-BI strains for metronidazole and vancomycin (for both antibiotics, MIC₉₀ was 2 μg/mL vs. 0.5 μg/mL, P < 0.01 for metronidazole, P = NS for vancomycin). Clinical cure for BI isolates (11/14, 79%) was not significantly different from non-BI isolates (21/22, 95%).ConclusionThese results underscore the high prevalence of the BI epidemic strain and demonstrate that mild to moderate CDI infection as well as severe disease can be caused by these strains. Fidaxomicin cure rates for subjects with BI and with non-BI strains are similar, although the small numbers of subjects preclude a robust statistical comparison.     

Efficacy and safety of telavancin in clinical trials: a systematic review and meta-analysis

Introduction

The epidemiology and antibiotic resistance of Staphylococcus aureus have evolved, underscoring the need for novel antibiotics, particularly against methicillin-resistant S. aureus (MRSA). Telavancin is a bactericidal lipoglycopeptide with potent activity against Gram-positive pathogens.

Objective

To systematically review and synthesize the available evidence from randomized controlled trials (RCTs) evaluating telavancin in the treatment of patients with infections due to Gram-positive organisms with the methodology of meta-analysis.

Results

Six RCTs comparing telavancin with vancomycin were included; 4 (2229 patients) referred to complicated skin and soft tissue infections (cSSTIs) and 2 (1503 patients) to hospital-acquired pneumonia (HAP). Regarding cSSTIs, telavancin and vancomycin showed comparable efficacy in clinically evaluable patients (odds ratio [OR] = 1.10 [95% confidence intervals: 0.82–1.48]). Among patients with MRSA infection, telavancin showed higher eradication rates (OR = 1.71 [1.08–2.70]) and a trend towards better clinical response (OR = 1.55 [0.93–2.58]). Regarding HAP, telavancin was non-inferior to vancomycin in terms of clinical response in two Phase III RCTs; mortality rates for the pooled trials were comparable with telavancin (20%) and vancomycin (18.6%). Pooled data from cSSTIs and HAP studies on telavancin 10 mg/kg indicated higher rates of serum creatinine increases (OR = 2.22 [1.38–3.57]), serious adverse events (OR = 1.53 [1.05–2.24]), and adverse event-related withdrawals (OR = 1.49 [1.14–1.95]) among telavancin recipients.

Conclusion

Telavancin might be an alternative to vancomycin in cases of difficult-to-treat MRSA infections. The potent antistaphylococcal activity of telavancin should be weighted against the potential for nephrotoxicity.     

Distribution of Clostridium difficile strains from a North American,European and Australian trial of treatment for C. difficile infections: 2005–2007

Clostridium difficile is a widely distributed pathogen with multiple strain types as determined by restriction endonuclease analysis (REA) and by PCR ribotyping, two well-characterized typing systems. In this study, REA typing was performed on 894 C. difficile isolates from patients enrolled from 16 countries on three continents in two large, recently conducted clinical treatment trials of C. difficile infection. REA group BI (Ribotype 027) isolates were the most common strains identified and were widely distributed throughout North America, but restricted to three of thirteen countries in Europe. REA group J (Ribotype 001) isolates were the most common strains identified in Europe and non-specific REA groups (historically less frequent) were the most common strains identified in Australia. REA groups BI, J, G and CF correlated with specific PCR ribotypes whereas more than one ribotype was found within REA groups Y, BK, and K. International surveillance of C. difficile strains is important to document the changing epidemiology of this enteric pathogen that continues to cause healthcare facility outbreaks and sporadic infections in other settings.     

Diarylacylhydrazones: Clostridium-selective antibacterials with activity against stationary-phase cells

Current antibiotics for treating Clostridium difficile infections (CDI), that is, metronidazole, vancomycin and more recently fidaxomicin, are mostly effective but treatment failure and disease relapse remain as significant clinical problems. The shortcomings of these agents are attributed to their low selectivity for C. difficile over normal gut microflora and their ineffectiveness against C. difficile spores. This Letter reports that certain diarylacylhydrazones identified during a high-throughput screening/counter-screening campaign show selective activity against two Clostridium species (C. difficile and Clostridium perfringens) over common gut commensals. Representative examples are shown to possess activity similar to vancomycin against clinical C. difficile strains and to kill stationary-phase C. difficile cells, which are responsible for spore production. Structure–activity relationships with additional synthesised analogues suggested a protonophoric mechanism may play a role in the observed activity/selectivity and this was supported by the well-known protonophore carbonyl cyanide m-chlorophenyl hydrazone (CCCP) showing selective anti-Clostridium effects and activity similar to diarylacylhydrazones against stationary-phase C. difficile cells. Two diarylacylhydrazones were shown to be non-toxic towards human FaDu and Hep G2 cells indicating that further studies with the class are warranted towards new drugs for CDI.     

Synergistic interactions in two-drug and three-drug combinations (thymol,EDTA and vancomycin) against multi drug resistant bacteria including E. coli

Combinations of two or more drugs, which affect different targets, have frequently been used as a new approach against resistant bacteria. In our work we studied the antimicrobial activity (MIC, MBC) of individual drugs (the phenolic monoterpene thymol, EDTA and vancomycin), of two-drug interactions between thymol and EDTA in comparison with three-drug interactions with vancomycin against sensitive and resistant bacteria. Thymol demonstrated moderate bactericidal activity (MBC between 60 and 4000 μg/ml) while EDTA only exhibited bacteriostatic activity over a range of 60–4000 μg/ml. MICs of vancomycin were between 0.125 and 16 μg/ml against Gram-positive and between 32 and 128 μg/ml against Gram-negative bacteria. Checkerboard dilution and time-kill curve assays were performed to evaluate the mode of interaction of several combinations against Methicillin-resistant Staphylococcus aureus (MRSA NCTC 10442) and Escherichia coli (ATCC 25922). Checkerboard data indicate indifferent interaction against Gram-positive (FICI = 1–1.3) and synergy against Gram-negative bacteria (FICI ≈ 0.4), while time kill analyses suggest synergistic effect in different combinations against both types of bacteria. It is remarkable that the combinations could enhance the sensitivity of E. coli to vancomycin 16-fold to which it is normally insensitive. We have provided proof for the concept, that combinations of known antibiotics with modern phytotherapeutics can expand the spectrum of useful therapeutics.     

Synergy of aminoglycoside antibiotics by 3-Benzylchroman derivatives from the Chinese drug Caesalpinia sappan against clinical methicillin-resistant Staphylococcus aureus (MRSA)

The in vitro antimicrobial activities of three 3-Benzylchroman derivatives, i.e. Brazilin (1), Brazilein (2) and Sappanone B (3) from Caesalpinia sappan L. (Leguminosae) were assayed, which mainly dealt with synergistic evaluation of aminoglycoside and other type of antibiotics against methicillin-resistant Staphylococcus aureus (MRSA) by the three compounds through the Chequerboard and Time-kill curve methods. The results showed that Compounds 1–3 alone exhibited moderate to weak activity against methicillin-susceptible S. aureus (MSSA) and other standard strains by MICs/MBCs ranged from 32/64 to >1024/>1024 μg/ml, with the order of activity as 1 > 2 > 3. Chequerboard method showed significant anti-MRSA synergy of 1/Aminoglycosides (Gentamicin, Amikacin, Etimicin and Streptomycin) combinations with (FICIs)₅₀ at 0.375–0.5. The combined (MICs)₅₀ values (μg/ml) reduced from 32–128/16–64 to 4–8/4–16, respectively. The percent of reduction by MICs ranged from 50% to 87.5%, with a maximum of 93.8% (1/16 of the alone MIC). Combinations of 2 and 3 with Aminoglycosides and the other antibiotics showed less potency of synergy. The dynamic Time-killing experiment further demonstrated that the combinations of 1/aminoglycoside were synergistically bactericidal against MRSA. The anti-MRSA synergy results of the bacteriostatic (Chequerboard method) and bactericidal (time-kill method) efficiencies of 1/Aminoglycoside combinations was in good consistency, which made the resistance reversed by CLSI guidelines. We concluded that the 3-Benzylchroman derivative Brazilin (1) showed in vitro synergy of bactericidal activities against MRSA when combined with Aminoglycosides, which might be beneficial for combinatory therapy of MRSA infection.     

Sensitivity to antibiotics of Clostridium difficile toxigenic nosocomial strains

Clostridium difficile is the etiological agent of diarrhoea and colitis, especially in elderly patients. The incidence of these diseases has increased during the last 10 years. Emergence of so-called hypervirulent strains is considered as one of the main factors responsible for the more severe disease and changed profile of sensitivity to antimicrobial agents. The aim of this work was to determine the sensitivity profile of toxigenic strains of C. difficile in the Czech Republic in 2011–2012 to selected antibiotics. The antibiotics clindamycin, metronidazole, vancomycin and amoxicillin with clavulanic acid were used for this purpose. Isolates cultured on Brazier's C. difficile selective agar were analysed for the presence of toxin genes using Xpert detection system. Xpert analysis revealed that 33 strains carried the genes for toxins tcdB, cdt and tcdCΔ117, thus showing characteristics typical for the hypervirulent ribotype 027/PFGE type NAP1/REA type B1. The remaining 29 strains carried only the gene for toxin B (tcdB) and not cdt and tcdCΔ117. Our results indicate the higher susceptibility of C. difficile hypertoxigenic strains to three out of four tested antibiotics (except vancomycin) than it is for the other toxigenic strains. We found that only 10.34 % of other toxigenic strains were resistant to clindamycin, and no resistance was found in all other cases. All the isolates were sensitive to amoxicillin/clavulanic acid in vitro. However, its use is not recommended for therapy of infections caused by C. difficile.     

Salicylanilide diethyl phosphates: Synthesis,antimicrobial activity and cytotoxicity

A series of 27 salicylanilide diethyl phosphates was prepared as a part of our on-going search for new antimicrobial active drugs. All compounds exhibited in vitro activity against Mycobacterium tuberculosis, Mycobacterium kansasii and Mycobacterium avium strains, with minimum inhibitory concentration (MIC) values of 0.5–62.5 μmol/L. Selected salicylanilide diethyl phosphates also inhibit multidrug-resistant tuberculous strains at the concentration of 1 μmol/L. Salicylanilide diethyl phosphates also exhibited mostly the activity against Gram-positive bacteria (MICs ⩾1.95 μmol/L), whereas their antifungal activity is significantly lower. The IC₅₀ values for Hep G2 cells were within the range of 1.56–33.82 μmol/L, but there is no direct correlation with MICs for mycobacteria.     

Molecular epidemiology of Clostridium difficile strains from nosocomial-acquired infections

The purpose of this study is to analyze isolates of Clostridium difficile from patients with nosocomial acquired infection in respect to their molecular type and antimicrobial susceptibility. Fifty-nine randomly selected clinical isolates were characterized. Molecular typing was performed by rep-PCR (DiversiLab). Isolates were tested by disk diffusion towards 11 different antibiotics. All isolates were susceptible to metronidazole and vancomycin. Fifty five (93 %) isolates were resistant to erythromycin and fifty six (95 %) exhibited resistance to both clindamycin and moxifloxacin. Twenty rep-PCR types were identified, but most clinical isolates formed four major rep-PCR clusters (A₁ 24/59, 40 %; A₂ 20/59, 33 %; A₃ 5/59, 8 %; A₄ 3/59, 5 %). These results show high genetic variability, which demonstrate clearly the complexity of the strains of C. difficile and also show an increasing rate of resistance to fluoroquinolones in our region emphasizing the importance of implementing surveillance programs in order to prevent further spread of resistance in C. difficile.     

Identification and characterization of a phospholipase A2 from the venom of the Saw-scaled viper: Novel bactericidal and membrane damaging activities

Phospholipase A₂ (PLA₂), a common toxic component of snake venom, has been implicated in various pharmacological effects. In this study, a basic myotoxic PLA₂, named EcTx-I was isolated from Echis carinatus snake venom by using gel filtration on Superdex G-75, and reverse phase HPLC on C18 and C8 Sepharose columns. PLA₂, EcTx-I was 13,861.72 molecular weight as estimated by MALDI-TOF (15 kD by SDS-PAGE), and consisted of 121 amino acid residues cross-linked by seven disulfide bonds. The N-terminal sequences revealed significant homology with basic myotoxic PLA₂s from other snake venoms. The purified PLA₂ EcTx-I was evaluated (250 μg/ml) for bactericidal activity of a wide variety of human pathogens against Burkholderia pseudomallei (KHW&TES), Enterobacter aerogenes, Escherichia coli, Proteus vulgaris, Proteus mirabilis, Pseudomonas aeruginosa and Staphylococcus aureus. EcTx-I showed strong antibacterial activity against B. pseudomallei (KHW) and E. aerogenes among the tested bacteria. Other Gram-negative and Gram-positive bacteria showed only a moderate effect. However, the Gram-positive bacterium E. aerogenes failed to show any effect on EcTx-I protein at tested doses. The most significant bacteriostatic and bactericidal effect of EcTx-I was observed at MICs of >15 μg/ml against (B. pseudomallei, KHW) and MICs >30 μg/ml against E. aerogenes. Mechanisms of bactericidal and membrane damaging effects were proved by ultra-structural analysis. EcTx-I was able to induce cytotoxicity on THP-1 cells in vitro as well as lethality in BALB/c mice. EcTx-I also induced mild myotoxic effects on mouse skin, but was devoid of hemolytic effects on human erythrocytes up to 500 μg/ml. It is shown that the toxic effect induced by E. carinatus venom is due to the presence of myotoxic PLA₂ (EcTx-I). The result also corroborates the hypothesis of an association between toxic and enzymatic domains. In conclusion, EcTx-I displays a heparin binding C-terminal region, which is probably responsible for the cytotoxic and bactericidal effects.     

Molecular typing and antimicrobial susceptibility testing to six antimicrobials of <Emphasis Type="Italic">Clostridium difficile</Emphasis> isolates from three Czech hospitals in Eastern Bohemia in 2011–2012

In 2011–2012, a survey was performed in three regional hospitals in the Czech Republic to determine the incidence of Clostridium difficile infections (CDIs) and to characterize bacterial isolates. C. difficile isolates were characterized by PCR ribotyping, toxin genes detection, multiple-locus variable-number tandem-repeat analysis (MLVA), and antimicrobial susceptibility testing to fidaxomicin, vancomycin, metronidazole, clindamycin, LFF571, and moxifloxacin using agar dilution method. The incidence of CDI in three studied hospitals was 145, 146, and 24 cases per 100,000 inhabitants in 2011 and 177, 258, and 67 cases per 100,000 inhabitants in 2012. A total of 64 isolates of C. difficile was available for molecular typing and antimicrobial susceptibility testing. 60.9% of the isolates were classified as ribotype 176. All 41 isolates of ribotypes 176 and 078 were positive for the presence of binary toxin genes. Ribotype 176 also carried 18-bp deletion in the regulatory gene tcdC. Tested isolates of C. difficile were fully susceptible to vancomycin and metronidazole, whereas 65.1% of the isolates were resistant to moxifloxacin. MLVA results indicated that isolates from three different hospitals were genetically related, suggesting transmission between healthcare facilities.     

Defining the Vulnerable Period for Re-Establishment of Clostridium difficile Colonization after Treatment of C. difficile Infection with Oral Vancomycin or Metronidazole

Background

Clostridium difficile is an anaerobic, spore-forming bacterium that is the most common cause of healthcare-associated diarrhea in developed countries. A significant proportion of patients receiving oral vancomycin or metronidazole for treatment of Clostridium difficile infection (CDI) develop recurrences. However, the period of vulnerability to re-establishment of colonization by C. difficile after therapy is not well defined.

Principal Findings

In a prospective study of CDI patients, we demonstrated that most vancomycin-treated patients maintained inhibitory concentrations of vancomycin in stool for 4 to 5 days after therapy, whereas metronidazole was only detectable during therapy. From the time of elimination of the antibiotics to 14 to 21 days after therapy, a majority of stool suspensions supported growth of C. difficile and deep 16S rRNA sequencing demonstrated persistent marked alteration of the indigenous microbiota. By 21 to 28 days after completion of CDI treatment, a majority of stool suspensions inhibited growth of C. difficile and there was evidence of some recovery of the microbiota.

Conclusions

These data demonstrate that there is a vulnerable period for re-establishment of C. difficile colonization after CDI treatment that begins within a few days after discontinuation of treatment and extends for about 3 weeks in most patients.     

Generation of novel cationic antimicrobial peptides from natural non-antimicrobial sequences by acid-amide substitution

Background

The accessory gene regulator (agr) is a quorum sensing cluster of genes which control colonization and virulence in Staphylococcus aureus. We evaluated agr function in community- (CA) and healthcare-associated (HA) MRSA, to compare the pharmacodynamics and bactericidal activity of vancomycin against agr functional and dysfunctional HA-MRSA and CA-MRSA.

Methods

40 clinical isolates of MRSA from the Canadian Nosocomial Infection Surveillance Program were evaluated for delta-haemolysin production, as a surrogate marker of agr function. Time kill experiments were performed for vancomycin at 0 to 64 times the MIC against an initial inoculum of 10⁶ and 10⁸ cfu/ml of agr functional and dysfunctional CA-MRSA and HA-MRSA and these data were fit to a hill-type pharmacodynamic model.

Results

15% isolates were agr dysfunctional, which was higher among HA-MRSA (26.3%) versus CA-MRSA (4.76%). Against a low initial inoculum of 10⁶ cfu/ml of CA-MRSA, vancomycin pharmacodynamics were similar among agr functional and dysfunctional strains. However, against a high initial inoculum of 10⁸ cfu/ml, killing activity was notably attenuated against agr dysfunctional CA-MRSA (USA400) and HA-MRSA (USA100). CA-MRSA displayed a 20.0 fold decrease in the maximal reduction in bacterial counts (Emax) which was 3.71 log₁₀ CFU/ml for agr functional vs. 2.41 log₁₀ CFU/ml for agr dysfunctional MRSA (p = 0.0007).

Conclusions

Dysfunction in agr was less common among CA-MRSA vs. HA-MRSA. agr dysfunction demonstrated an impact on vancomycin bactericidal activity and pharmacodynamics against a high initial inoculum of CA-MRSA and HA-MRSA, which may have implications for optimal antimicrobial therapy against persistent, difficult to treat MRSA infections.     

Characteristics of the Clostridium difficile cell envelope and its importance in therapeutics

Clostridium difficile infection (CDI) is a challenging threat to human health. Infections occur after disruption of the normal microbiota, most commonly through the use of antibiotics. Current treatment for CDI largely relies on the broad‐spectrum antibiotics vancomycin and metronidazole that further disrupt the microbiota resulting in frequent recurrence, highlighting the need for C. difficile‐specific antimicrobials. The cell surface of C. difficile represents a promising target for the development of new drugs. C. difficile possesses a highly deacetylated peptidoglycan cell wall containing unique secondary cell wall polymers. Bound to the cell wall is an essential S‐layer, formed of SlpA and decorated with an additional 28 related proteins. In addition to the S‐layer, many other cell surface proteins have been identified, including several with roles in host colonization. This review aims to summarize our current understanding of these different C. difficile cell surface components and their viability as therapeutic targets.     

Effect of the Inoculum Size on Carbapenem Susceptibilities of β-Lactamase-Negative,Ampicillin-Resistant <Emphasis Type="Italic">Haemophilus influenzae</Emphasis>

A higher inoculum size of β-lactamase-positive Haemophilus influenzae is reported to increase minimum inhibitory concentrations (MICs) for β-lactams. However, the effect of inoculum size of β-lactamase-negative, ampicillin-resistant H. influenzae (BLNAR) on MICs for carbapenems has not been investigated. This study evaluated the effect of inoculum size on MICs for carbapenems and other β-lactams in nine clinical isolates of BLNAR. The MICs were determined by both the standard method described by the Clinical and Laboratory Standards Institute (final inoculum size of 5 × 10⁵ colony-forming units [CFU]/ml) and a modified method (final inoculum size of 5 × 10⁶ CFU/ml) using viable cell counts. The findings showed that the higher inoculum size increased MICs for imipenem, meropenem, panipenem, biapenem, ampicillin, ceftazidime, and ceftriaxone. The inoculum effect (4 log₂ dilution or a greater increase in the MIC) with imipenem, meropenem, panipenem, and biapenem was found in three, five, two, and two isolates, respectively. The magnitude of the inoculum effect for panipenem significantly increased with the levels of MICs, but correlation between them for the others was not statistically significant. The mutations of penicillin-binding protein genes had little relevance to the reduced susceptibility to carbapenems or to the magnitude of the inoculum effect. These results suggest that MIC determination using turbidity can produce interpretive errors in the antimicrobial susceptibility testing of BLNAR for carbapenems because of their inoculum effect. Thus, accurate adjustment of inoculum size, such as viable cell count, is helpful for confirming the true MICs when the isolates are interpreted as “resistant” by turbidity-based MIC determination.     

Characterization of vaginal lactobacilli in women after kidney transplantation

Limited number of publications described vaginal microflora after kidney transplantation. Our PubMed search revealed only 18 publications including words “vaginal bacteria &; kidney transplant” in the period of 1978–2011. The aim of this study was to characterize lactobacilli isolated from vaginal swabs of women after kidney transplantation, compared with healthy women. Eighteen renal transplant recipients (mean age 36.1) and 20 healthy women (mean age 36.0) were evaluated. Lactobacilli were cultured on MRS and Columbia blood agars. Biochemical identification with API 50 CHL (bioMerieux, Marcy L’Etoile, France) and multiplex PCR according to Song et al. was performed. Lactobacilli were tested for production of H₂O₂. Minimal inhibitory concentrations (MICs) of selected antimicrobial agents were determined with E-tests (bioMerieux, Marcy L’Etoile, France) and interpreted with CLSI and EUCAST criteria. No bacterial vaginosis was found among studied women. Two strains of group I were identified as Lactobacillus delbrueckii; 18 strains as Lactobacillus gasseri and 15 strains as Lactobacillus crispatus. Only 3 strains from group II were not identified by species-specific mPCR. Group IV was represented with 2 unidentified strains. Vaginal lactobacilli isolated from healthy women represented more homogenous group compared with heterogenous renal transplant recipients. Biochemical identification of lactobacilli by API 50 CHL kits was concordant with mPCR results only in 7 cases (17.5%), all 7 strains were identified as L. crispatus. Majority (93%) of lactobacilli were H₂O₂ producers. All isolated lactobacilli (100%) demonstrated high resistance to metronidazole (MIC > 256 μg/ml). Only 2 strains resistant to vancomycin (MICs: 32 and 256 μg/ml respectively), in the study and control group, and one to moxifloxacin (MIC = 32 μg/ml), were found. Resistance to metronidazole and vancomycin was concordant in CLSI and EUCAST (2010) criteria. Although significant differences between lactobacilli isolated from vaginas of kidney transplant and healthy women were not demonstrated, we demonstrated strains resistant to metronidazole, vancomycin and moxifloxacin in groups of examined women. Our study was performed on a small group of kidney transplant recipients and further more detailed molecular studies on a larger group of patients are required to confirm our results.     

Surveillance and trends of antimicrobial resistance among clinical isolates of anaerobes in Kuwait hospitals from 2002 to 2007

The susceptibility trends for all anaerobes processed by the Anaerobe Reference Laboratory against various antibiotics were determined by using data for 2557 isolates referred by all government hospitals in Kuwait from 2002 to 2007. MIC were determined for the following anti-anaerobic antibiotics: amoxicillin–clavulanic acid, clindamycin, imipenem, meropenem, metronidazole, penicillin, piperacillin, piperacillin–tazobactam and vancomycin (for Gram-positive anaerobes only), using E-test method. The commonest isolates were Bacteroides fragilis (36.8%), followed by Peptostreptococcus spp. (21.9%), Bacteroides ovatus (15.5%) and Prevotella bivia (12.1%). In addition, Prevotella oralis and other Bacteroides spp. represented 8.5% and 8.1% of total number of isolates, respectively. Resistance rate varied among the antimicrobial agents and the species tested. The β-lactams, with the exception of penicillin, were the most active drugs. Piperacillin–tazobactam was the only antimicrobial agent to which all the isolates were uniformly susceptible. Imipenem and metronidazole were highly active with resistance rate of only <5% recorded against most isolates. However, 42.8, 55.8 and 9.3% of Clostridium difficile isolates were resistant to imipenem, clindamycin and meropenem, respectively. It is noteworthy that from 2002 to 2007, there was a gradual increase in resistance rates to clindamycin, amoxicillin–clavulanic acid and piperacillin among B. fragilis. Periodic surveillance of antibiotic resistance among the anaerobic bacteria is recommended as a guide to empiric antibiotic use and formulation of guideline for appropriate choice of antimicrobial therapy in anaerobic infections.     

In vitro activity of salicylamide derivatives against vancomycin-resistant enterococci

A series of 13 salicylamide derivatives was assessed for antibacterial activity against three isolates of vancomycin-resistant Enterococcus faecalis (VRE) and Enterococcus faecalis ATCC 29212 as a quality standard. The minimum inhibitory concentration was determined by the broth microdilution method with subsequent subcultivation of aliquots to assess minimum bactericidal concentration. The growth kinetics was established by the time-kill assay. Ampicillin, ciprofloxacin, tetracycline and vancomycin were used as the reference antibacterial drugs. Three of the investigated compounds showed strong bacteriostatic activity against VRE (0.199–25?µM) comparable to or more potent than ampicillin and ciprofloxacin. In addition, these compounds were tested for synergistic effect with vancomycin, ciprofloxacin and tetracycline, while 5-chloro-2-hydroxy-N-[4-(trifluoromethyl)phenyl]benzamide showed the highest potency as well as synergistic activity with vancomycin against VRE 368. Screening of the cytotoxicity of the most effective compounds was performed using human monocytic leukemia THP-1 cells, and based on LD₅₀ values, it can be stated that the compounds have insignificant toxicity against human cells.     

Analysis of diverse β-lactamases presenting high-level resistance in association with OmpK35 and OmpK36 porins in ESBL-producing Klebsiella pneumoniae

Emerging extensively drug-resistant (XDR) Klebsiella pneumoniae due to the production of β-lactamases and porin loss is a substantial worldwide concern. This study aimed to elucidate the role of outer membrane porin (OMP) loss, AmpC, and carbapenemases among extended-spectrum β-lactamase (ESBL)-producing K. pneumoniae strains with XDR phenotype. This study analyzed 79 K. pneumoniae from several clinical sources and detected ESBLs in 29 strains co-harbored with other β-lactamases using standard microbiological practices and phenotypic procedures. Minimum inhibitory concentrations (MICs) were determined against several antibiotics using Microscan WalkAway plus. OMP analysis was carried out using sodium dodecyl sulfate–polyacrylamide gel electrophoresis. ESBL, AmpC, and carbapenemase genes were detected using molecular methods. The microbiological analysis discovered 29 (36.7%) ESBL strains of K. pneumoniae, which showed the co-existence of 7 (24.1%) AmpC β-lactamases and 22 (75.9%) carbapenemases. Porin loss of OmpK35 was observed in 13 (44.8%) and OmpK36 in 8 (27.5%) K. pneumoniae strains. The strains were significantly associated with the intensive care unit (ICU) (p = 0.006) and urinary sources (p = 0.004). The most commonly detected gene variants in each β-lactamase class included 16 (55.2%) bla_CTX-M?1, 7 (100%) bla_CYM-2, 11 (50%) bla_NDM-1, and integron-1 was detected in 21/29 (72.4%) strains. MICs of cephalosporin, fluoroquinolone, carbapenem, aminoglycoside, and β-lactam combinations demonstrated a high number of XDR strains. Tigecycline (2 µg/mL MIC₅₀ and >32 µg/mL MIC₉₀) and colistin (1 µg/mL MIC₅₀ and 8 µg/mL MIC₉₀) presented lower resistance. ESBL K. pneumoniae strains with OmpK35 and OmpK36 porin loss demonstrate conglomerate resistance mechanisms with AmpC and carbapenemases, leading to emerging XDR and pan drug resistance.     

Metronidazole Susceptibility in Clostridium difficile Isolates Recovered from Cases of C. difficile -associated Disease Treatment Failures and Successes

Despite high clinical efficacy, a small number of patients with Clostridium difficile -associated disease (CDAD) do not respond to treatment with metronidazole. We looked for evidence of metronidazole resistance in human C. difficile isolates from 632 patients with CDAD treated with metronidazole, 14(2%) of whom failed treatment. C. difficile isolates were available from 10 of the metronidazole-failure cases and were matched with isolates from 20 contemporary control CDAD patients who responded to treatment with metronidazole. The mean (±SD) MIC of metronidazole-failure-associated C. difficile isolates was similar to the mean (±SD) MIC of isolates from metronidazole-success cases (E-test; 0.23±0.21 vs 0.29±0.19μg/mL, P=0.4). Restriction endonuclease analysis typing revealed that no particular C. difficile strain was associated with metronidazole treatment failure. Clinical CDAD treatment failures with metronidazole could not be attributed to decreased susceptibility of the causativeC. difficle isolate to metronidazole.