Development of In Vitro and Ex Vivo Biofilm Models for the Assessment of Antibacterial Fibrous Electrospun Wound Dressings.

Increasing evidence suggests that the chronicity of wounds is associated with the presence of bacterial biofilms. Therefore, novel wound care products are being developed, which can inhibit biofilm formation and/or treat already formed biofilms. A lack of standardized assays for the analysis of such novel antibacterial drug delivery systems enhances the need for appropriate tools and models for their characterization. Herein, we demonstrate that optimized and biorelevant in vitro and ex vivo wound infection and biofilm models offer a convenient approach for the testing of novel antibacterial wound dressings for their antibacterial and antibiofilm properties, allowing one to obtain qualitative and quantitative results. The in vitro model was developed using an electrospun (ES) thermally crosslinked gelatin-glucose (GEL-Glu) matrix and an ex vivo wound infection model using pig ear skin. Wound pathogens were used for colonization and biofilm development on the GEL-Glu matrix or pig skin with superficial burn wounds. The in vitro model allowed us to obtain more reproducible results compared with the ex vivo model, whereas the ex vivo model had the advantage that several pathogens preferred to form a biofilm on pig skin compared with the GEL-Glu matrix. The in vitro model functioned poorly for Staphylococcus epidermidis biofilm formation, but it worked well for Escherichia coli and Staphylococcus aureus, which were able to use the GEL-Glu matrix as a nutrient source and not only as a surface for biofilm growth. On the other hand, all tested pathogens were equally able to produce a biofilm on the surface of pig skin. The developed biofilm models enabled us to compare different ES dressings [pristine and chloramphenicol-loaded polycaprolactone (PCL) and PCL-poly(ethylene oxide) (PEO) (PCL/PEO) dressings] and understand their biofilm inhibition and treatment properties on various pathogens. Furthermore, we show that biofilms were formed on the wound surface as well as on a wound dressing, indicating that the demonstrated methods mimic well the in vivo situation. Colony forming unit (CFU) counting and live biofilm matrix as well as bacterial DNA staining together with microscopic imaging were performed for biofilm quantification and visualization, respectively. The results showed that both wound biofilm models (in vitro and ex vivo) enabled the evaluation of the desired antibiofilm properties, thus facilitating the design and development of more effective wound care products and screening of various formulations and active substances.

Identification and Antibiotic Resistance Assessment of Ensifer adhaerens YX1, a Vitamin B12 -Producing Strain Used as a Food and Feed Additive.

This study provides phenotypic and molecular analyses of the antibiotic resistance of Ensifer adhaerens strain YX1 (CICC 11008s), a strain that was identified using a polyphasic taxonomy approach. The antibiotic resistance profile of E. adhaerens YX1 was assessed using the Clinical & Laboratory Standards Inst. (CLSI) method. The strain was susceptible to ciprofloxacin, levofloxacin, norfloxacin, ofloxacin, gentamicin, tobramycin, chloramphenicol, tetracycline, imipenem, and ceftazidime, and resistant to kanamycin, streptomycin, fosfomycin, and nitrofurantoin. The antibiotic resistance genes nsfA, nsfB, fosA, aph, and aadA1 were not detected in E. adhaerens YX1 via PCR using gene-specific primers. Subsequently, the genome sequence of E. adhaerens was screened for antibiotic genes. Although no antibiotic resistance genes were identified using the ResFinder database, five genes copies of one resistance gene, adeF, were detected using the Comprehensive Antibiotic Resistance Database (CARD). The results of this study will be useful for understanding the phenotypic and genotypic aspects of E. adhaerens antibiotic resistance. No safety issues were identified for E. adhaerens YX1 in terms of antibiotic resistance. Performing similar studies will be conducive to the safety assessment and control of the use of E. adhaerens in the food and feed industry. PRACTICAL APPLICATION: Few relevant reports are currently available regarding antibiotic resistance assessments or other safety evaluations for Ensifer adhaerens. Because of a lack of relevant information on the safety of this bacterium, including the genetic basis of antibiotic resistance in the production strain, it has not been recommended for use in the "qualified presumption of safety" (QPS) list and subsequent updated lists. The current study shows no safety issue of E. adhaerens YX1 in terms of its antibiotic resistance. These results are important as they provide an initial basis for an understanding of the antibiotic resistance/susceptibility of E. adhaerens YX1 (CICC 11008s), which produces vitamin B12 and is widely used in the food and feed industry.

Assessment of antibiotic resistance in bacteriophage-insensitive Klebsiella pneumoniae.

This study was design to evaluate the physiological properties of bacteriophage-insensitive Klebsiella pneumoniae (BIKP) mutants in association with the antibiotic cross-resistance, ß-lactamase activity, and gene expression. Klebsiella pneumoniae ATCC 23357(KPWT), ciprofloxacin-induced antibiotic-resistant K. pneumoniae ATCC 23357 (KPCIP), and clinically isolated antibiotic-resistant K. pneumoniae 10263 (KPCLI) were used to isolate BIKP mutants against KPB1, PBKP02, PBKP21, PBKP29, PBKP33, and PBKP35. PBKP35-induced mutants, including bacteriophage-insensitive K. pneumoniae ATCC 23357 (BIKPWT), ciprofloxacin-induced K. pneumoniae ATCC 23357 (BIKPCIP), and clinically isolated antibiotic-resistant K. pneumoniae CCARM 10263 (BIKPCLI). BIKPWT, BIKPCIP, and BIKPCLI were resistant to Klebsiella bacteriophages, KPB1, PBKP02, PBKP21, PBKP29, and PBKP33. The antibiotic cross-resistance to cefotaxime, cephalothin, chloramphenicol, ciprofloxacin, erythromycin, kanamycin, levofloxacin, and nalidixic acid was observed in BIKPWT. The relative expression levels of vagC was increased by more than 8-folds in BIKPWT, corresponding to the increased ß-lactamase activity. The aac(6')-Ib-cr was overexpressed in BIKP mutants, responsible for aminoglycoside and quinolone resistance. The phage-resistant mutants decreased the antibiotic susceptibilities in association with ß-lactamase activity and antibiotic resistance-related gene expression. The results pointed out the cross-resistance of BIKP mutants to antibiotics, which might be considered when applying for the therapeutic use of bacteriophage.

Effects and time-kill assessment of amoxicillin used in combination with chloramphenicol against bacteria of clinical importance.

With the emergence of multidrug-resistant organisms in an era when drug development faces challenges causing pharmaceutical companies to curtail or abandon research on anti-infective agents, the use of combined existing antimicrobial agents may be an alternative. This study evaluated the effects of combining amoxicillin and chloramphenicol, to which many bacteria have become resistant, in vitro against Gram positive and Gram negative bacteria by agar diffusion, checkerboard and time-kill assays. The test isolates were susceptible to amoxicillin with minimum inhibitory concentrations (MICs) ranging between 0.448 and 500 µg/ml and between 1.953 and 31.25 µg/ml for chloramphenicol. Upon combining these agents, there was a drastic reduction in their MICs indicating an increased antibacterial activity that showed synergistic interaction against all the bacteria. At the highest concentrations, the inhibition zones ranges were 20.33-38.33±0.58 µg/ml for amoxicillin, 27.67-37.67±0.58 µg/ml for chloramphenicol and 31.67-39.33±0.58 µg/ml for the combined agents. The fractional inhibitory concentration indices (FICIs) showed synergy ranging from 0.129 to 0.312 while FICIs for additive interaction were between 0.688 and 1.0. There was no antagonistic interaction. At the 1/2MICs of the combined antibiotics, all the tested bacteria, except for Klebsiella pneumoniae ATCC 4352, Proteus vulgaris CSIR 0030 and Enterococcus cloacae ATCC 13047 were eliminated before 24 h. At the MICs, all the tested bacteria were eliminated except Enterococcus cloacae ATCC 13047 which was almost totally eliminated. Post-antibiotic assessment after 48 h showed that all the cultures were sterile except for that of Enterococcus cloacae ATCC 13047. The lack of antagonism between these antibacterial agents in checkerboard and time-kill assays suggested that combining amoxicillin with chloramphenicol can provide an improved therapy in comparison to the use of each antibiotic individually. The study indicates the potential beneficial value of combining amoxicillin and chloramphenicol in the treatment of microbial infections in clinical settings.

Short communication: In vivo screening platform for bacteriocins using Caenorhabditis elegans to control mastitis-causing pathogens.

This study aimed to develop an in vivo screening platform using Caenorhabditis elegans to identify a novel bacteriocin for controlling the mastitis-causing pathogen Staphylococcus aureus strain RF122 in dairy cows. Using Bacillus spp. isolated from traditional Korean foods, we developed a direct in vivo screening platform that uses 96-well plates and fluorescence image analysis. We identified a novel bacteriocin produced by Bacillus licheniformis strain 146 (lichenicin 146) with a high in vivo antimicrobial activity using our liquid C. elegans-Staph. aureus assay. We also determined the characteristics of lichenicin 146 using liquid chromatography-mass spectrometry and confirmed that it shared homologous sequences with bacteriocin family proteins. In addition, RNA-sequencing analysis revealed genes encoding cell surface or membrane proteins (SAB0993c, SAB0150, SAB0994c, and SAB2375c) that are involved in the bactericidal activity of lichenicin 146 against Staph. aureus strain RF122 infection as well as those encoding transcriptional regulators (SAB0844c and SAB0133). Thus, our direct in vivo screening platform facilitates simple, convenient, cost-effective, and reliable screening of potential antimicrobial compounds with applications in the dairy field.

Assessment of altered binding specificity of bacteriophage for ciprofloxacin-induced antibiotic-resistant Salmonella Typhimurium.

This study describes a new effort toward understanding the interaction mechanisms between antibiotic-resistant Salmonella Typhimurium and phages. The antibiotic susceptibility, ß-lactamase activity, bacterial motility, gene expression, and lytic activity were evaluated in ciprofloxacin-induced antibiotic-sensitive Salmonella Typhimurium (ASST(CIP)) and ciprofloxacin-induced antibiotic-resistant S. Typhimurium (ARST(CIP)), which were compared to the wild-type strains (ASST(WT) and ARST(WT)). The MIC values of ampicillin, norfloxacin, chloramphenicol, and tetracycline were significantly increased to > 512, 16, 16, and 256 µg/ml, respectively, in the ARST(CIP). The lowest and highest extracellular lactamase activities were observed in ASST(WT) (6.85 µmol/min/ml) and ARST(CIP) (48.83 µmol/min/ml), respectively. The acrA, lpfE, and hilA genes were significantly upregulated by more than tenfold in both ASST(CIP) and ARST(CIP). The induction of multiple antibiotic resistance resulted from the increased efflux pump activity (AcrAB-TolC). The highest phage adsorption rates were more than 95 % for ASST(WT), ASST(CIP), and ARST(WT), while the lowest adsorption rate was 52 % for ARST(CIP) at 15 min of infection. The least lytic activity of phage was 20 % against the ARST(CIP), followed by ASST(CIP) (30 %). The adsorption rate of phage against ARST(CIP) was 52 % at 15 min of infection, which resulted in the decrease in lytic activity (12 %). Understanding the interaction of phage and bacteria is essential for the practical application of phage to control and detect antibiotic-resistant bacteria. The results provide useful information for understanding the binding specificity of phages for multiple antibiotic-resistant pathogens.

Characterization of cost with respect to nutritional upshift in the media composition along with sublethal doses of transcriptional and translational inhibitor.

Expression of unnecessary proteins is known to reduce the growth rate of Escherichia coli. This reduction in growth rate, due to the diversion of cellular resources from making essential proteins, is called cost. Cellular resources depend upon the macromolecular content of the cell. Cost phenomenon lies in the process of transcription and translation. The effect of an upshift in the nutritional content of the medium on the cost process of natural lac proteins is not looked upon. Which process of gene expression, transcription, or translation is more important for the cost process is not clear. The current study indicated that cost process is not associated with the upshift mechanism. Cost process was not observed in presence of rifampicin but was detected in existence of chloramphenicol. The current study will help in better understanding of the cost process.

Safety assessment and probiotic evaluation of Enterococcus faecium YF5 isolated from sourdough.

Enterococcus faecium YF5, a strain previously isolated from sourdough, was assessed for safety and probiotic potential. Its virulence and antibiotic resistant phenotypes (cytolysin and gelatinase production, antibiotic susceptibility) and genes (cylA, gelE, ace, agg, esp, and vanA) were surveyed. Results indicated that the tested virulence determinants were nontoxic. In addition, E. faecium YF5 was sensitive to 3 antibiotics such as amoxicillin, vancomycin, and chloramphenicol. Furthermore, results of in vivo animal acute oral toxicity of E. faecium YF5 studies were similar to the control group that indicated no abnormalities. In addition, E. faecium YF5 stably survived in low pH, bile salts, gastric, and intestinal fluids in vitro. Moreover, E. faecium YF5 was found to adhere to human colon cancer cell line HT-29 at 3.39 (±0.67) × 10(5) CFU/mL. When cocultured with pathogenic organisms (Enterobacter sakazakii CMCC45402, Escherichia coli CMCC44102, enterohemorrhage Escherichia coli O157: H7 CMCC44828, Salmonella Typhimurium CMCC50071, Shigella flexneri 301, and Shigella sonnei ATCC 29930) and 2 gram-positive strains (Listeria monocytogenes CMCC54001 and Staphylococcus aureus CMCC 26003), it inhibited these foodborne pathogens with exception of S. aureus. Therefore, E. faecium YF5 can be regarded as a safe strain and it may be used as a probiotic preparation or for microecologics.

Real-time assessment of the microbial quality of retail shrimp using CO2 evolution rate.

A real-time CO(2) evolution rate (CER) method together with conventional cultural and sensory techniques were utilized to determine the microbial quality and shelf life of several types of shrimp products: chloramphenicol (CAP) treated, imported farm raised, and domestic wild caught. Treatment with CAP was used to create different bacterial loads in shrimp samples to demonstrate the ability and sensitivity of the CER method for differentiating the bacterial activity in samples. Samples were divided into control (nontreated) and 0, 10, and 30 ppm of CAP treatment groups and stored at 4°C. The CER was recorded with a microrespirometer, and aerobic plate counts (APCs), olfactory sensory analyses, and pH measurements were recorded daily until spoilage occurred. The real-time CER results were highly correlated with the APCs (R(2) = 0.93) and readily distinguished the onset of spoilage in each of the treatment groups. CAP treatment at 10 and 30 ppm increased the sample shelf life by 2 and 3 days, respectively, compared with the nontreated samples. Untreated domestic wild-caught shrimp had a shelf life 1 day longer than that of the untreated imported farm-raised shrimp. No pattern of change in pH was noted throughout the storage period. When the olfactory sensory scores reached the marginally acceptable level, the mean CER was 27.23 µl/h/g and the mean APC was 5.78 log CFU/g. A cutoff CER of 25.0 µl/h/g was therefore selected to define acceptable raw shrimp. The CER method was a highly effective and sensitive real-time method for determining the microbial quality of raw shrimp.

Spatial and temporal patterns in antimicrobial resistance of Salmonella Typhimurium in cattle in England and Wales.

Salmonella is the second most commonly reported human foodborne pathogen in England and Wales, and antimicrobial-resistant strains of Salmonella are an increasing problem in both human and veterinary medicine. In this work we used a generalized linear spatial model to estimate the spatial and temporal patterns of antimicrobial resistance in Salmonella typhimurium in England and Wales. Of the antimicrobials considered we found a common peak in the probability that an S. typhimurium incident will show resistance to a given antimicrobial in late spring and in mid to late autumn; however, for one of the antimicrobials (streptomycin) there was a sharp drop, over the last 18 months of the period of investigation, in the probability of resistance. We also found a higher probability of resistance in North Wales which is consistent across the antimicrobials considered. This information contributes to our understanding of the epidemiology of antimicrobial resistance in Salmonella.

Flora conjuntival y sensibilidad antibiótica en pacientes a ser sometidos a cirugía de catarata y LASIK / Antibiotic susceptibility of bacterial conjunctival flora isolated from patients undergoing LASIK and cataract surgery

Objetivo: Conocer y proveer de evidencia acerca de la sensibilidad de la flora bacteriana normal de la superficie ocular aislada en dos diferentes grupos sociales y etáreos de pacientes prontos a someterse a cirugía de Catarata y LASIK en nuestro país. Materiales y Métodos: Se cultivaron muestran conjuntivales de 221 pacientes previo a LASIK y de 180 pacientes de un grupo de Cataratas. De haber un cultivo positivo se realizó aislamiento e identificación bacteriana utilizando la técnica de difusión en disco de Kirby-Bauer para doce antibióticos. El análisis estadístico se hizo con chi-cuadrado y el test exacto de Fisher. Resultados: Hubo 66,8 por ciento de cultivos positivos, más frecuentemente gran positivos. SCN fue el aislado en mayor porcentaje (92,2 por ciento) y mostró una alta sensibilidad a Cloramfenicol, Tobramicina, Moxifloxacino y Gatifloxacino, intermedia para Levofloxacino, Gentamicina y Ciprofloxacino y menor para Eritromicina, Oxacilina, Cefalotina y Ceftriaxona (p<0,01). Todos los cultivos fueron sensibles a Vancomicina. No hubo diferencia estadísticamente significativa entre ambos grupos. Conclusiones: 1. Por primera vez nuestros resultados muestran que la flora y sensibilidad antibiótica son similares en pacientes a ser sometidos a LASIK y Catarata, siendo los SCN los más frecuentemente encontrados en ambos grupos de pacientes. 2. Las bacterias más comúnmente aisladas permanecen altamente sensibles a Cloramfenicol, Tobramicina, Moxifloxacino y Gatifloxacino.

Purpose: To know and provide a background on antibiotic susceptibility of normal ocular surface bacterial flora isolated from two different social and age groups of patients undergoing LASIK and cataract surgery in our country. Material and Methods: Conjunctival samples of 221 patients in a LASIK group and 180 patients in a cataract surgery group were cultivated. When there were a positive cultures, isolation and identification of the bacteria were made and antibiotic susceptibility tests were carried out, using the Kirby-Bauer disc diffusion technique for twelve antibiotics. Statistical analysis was performed using chi-square and exact Fisher test. Results: There were 66.8 percent of positive cultures, most of them gram positives. The most frequently isolated bacteria were the CNS (92,2 percent) that showed high sensitivity for Chloramphenicol, Tobramycin, Moxifloxacin and Gatifloxacin, intermedia for Levofloxacin, Gentamicin and Ciprofloxacin and lowest for Erytomycin, Oxacillin, Cefalotin and Ceftriaxone (p<0,01). All the cultures were susceptible to Vancomycin. There was not statistically difference between LASIK and cataract group. Conclusions: 1. For the first time, our results have shown that the conjunctival flora and its sensitivity to antibiotics are similar in the conjunctival flora of the patients undergoing LASIK surgery and Cataract, being CNS the bacterium most frequently found in both different groups of patients. 2. The most frequently isolated conjunctival bacteria remained highly sensitive to Chloramphenicol, Tobramycin, Moxifloxacin and Gatifloxacin.

Sensitivity of Haloquadratum and Salinibacter to antibiotics and other inhibitors: implications for the assessment of the contribution of Archaea and Bacteria to heterotrophic activities in hypersaline environments.

Antibiotics and bile salts have been used to differentiate between heterotrophic activity of halophilic Archaea and Bacteria in saltern ponds. In NaCl-saturated brines of crystallizer ponds, most activity was attributed to Archaea. Following the recent isolation of Haloquadratum, the dominant archaeon in the salterns (reported to be sensitive to chloramphenicol and erythromycin), and the discovery of Salinibacter, a representative of the Bacteria, in the same ecosystem, reevaluation of the earlier data is required. The authors measured amino acid incorporation by Haloquadratum and Salinibacter suspended in crystallizer brine to investigate the suitability of antibiotics and bile salts to distinguish between archaeal and bacterial activities. The amino acid uptake rate per cell in Salinibacter was two orders of magnitude lower than that of Haloquadratum under the same conditions. Salinibacter was inhibited by chloramphenicol, erythromycin, and deoxycholate, but not by taurocholate. Erythromycin did not inhibit incorporation by Haloquadratum, but moderate inhibition was found by chloramphenicol at 10-50 microg mL(-1). Deoxycholate was highly inhibitory, but only partial inhibition was obtained in the presence of 25 microg mL(-1) taurocholate. Inhibition by chloramphenicol and taurocholate increased with increasing salt concentration. Erythromycin and taurocholate proved most valuable to differentiate between archaeal and bacterial activities in saltern brines.

[Electrooptic analysis of Escherichia coli cell suspension for assessment of antibacterial activity of levomycetin].

Influence of chloramphenicol on electrophysiologic charateristics of Escherichia coli strains susceptible (K-12 strain) and resistant (pBR-325 strain) to it has been studied. It has been shown that incubation of susceptible bacteria with chloramphenicol leads to significant change of magnitute of electrooptic (EO) signal. Significant changes in orientantional spectra of suspensions of susceptible to chloramphenicol cells incubated with different concentrations of antibiotic were observed only on first five frequencies of orienting electric field (10 - 1000 kHz). Maximal change of EO signal occurred at chloramphenicol concentration 35 mg/ml and it didn't depend on the time of antibiotic exposure. Incubation of resistant strain pBR-325 with chloramphenicol did not lead to change of EO parameters of cell suspension. Potential for use of electrophysical analytic methods for assessment of antibacterial activity of chloramphenicol to control effect of antibiotics on microorganisms has been proposed.

Resistance pattern and assessment of phenicol agents' minimum inhibitory concentration in multiple drug resistant Chryseobacterium isolates from fish and aquatic habitats.

AIMS: To assess the susceptibility of Chryseobacterium isolates of fish and aquatic habitats to antimicrobial compounds. Special attention was paid to the resistance to chloramphenicol and florfenicol, a phenicol derivative recently licensed for use in veterinary medicine and fish farming. METHODS AND RESULTS: Sixty-seven Chryseobacterium spp. isolates and reference strains, originating mainly from different aquatic habitats, were tested using the disk-diffusion method. In addition, agar dilution was used for assessing minimum inhibitory concentration of chloramphenicol and florfenicol. In spite of (i) conditions that hampered properly standardized experiments and (ii) the heterogeneity of the isolates resulting in some aberrant values in diffusion, correlation between the two methods was confirmed. Most of the isolates exhibited considerable multiresistance to most antimicrobial drug families, and many were clearly resistant to phenicols. Molecular investigations conducted on 10 strains selected for high resistance to florfenicol did not establish the existence of floR or cmlA genes currently reported in the literature as responsible for florfenicol resistance. Nevertheless, when an efflux pump inhibitor, phenyl-arginin-beta-naphthylamide, was combined with diffusion tests, drug susceptibility to florfenicol was restored, suggesting that Chryseobacterium's resistance to this molecule is under the control of efflux mechanisms. CONCLUSIONS: Constitutive multiresistance to antibiotics is common in chryseobacteria isolated from the aquatic environment. Although no gene related to the floR family could be detected, efflux mechanisms could partly support the resistance to phenicols. SIGNIFICANCE AND IMPACT OF THE STUDY: These results explain the difficulty of treatment and clearly reflect the properties previously reported in Chryseobacterium isolates of human origin. Because several species have been involved in opportunistic infections in humans, the possible role of aquatic organisms as a source of infection should be considered.

Susceptibility testing of Haemophilus influenzae--an international collaborative study in quality assessment.

In order to compare the prevalence of antibiotic resistance in different geographical areas, it is necessary to ensure that agreement is achieved between laboratories on the assignment of strains to 'susceptible' and 'resistant' categories. An international quality assessment study, involving 15 laboratories in eight countries, was performed to investigate the standard of performance of the susceptibility testing of Haemophilus influenzae. One hundred and fifty strains of H. influenzae were distributed from the London Hospital Medical College (LHMC) to all laboratories who were asked to test the susceptibility of the strains to ampicillin, chloramphenicol, tetracycline, trimethoprim, cephalosporins and ciprofloxacin. Laboratories were also asked to provide the details of methodology to test the susceptibility. Significant discrepancy between the LHMC and the participating laboratories appeared in the detection of resistance to ampicillin (especially beta-lactamase-negative strains resistant to ampicillin) as well as the assignment of susceptibility and resistance to chloramphenicol, tetracycline and trimethoprim. Often these reflected the use of inappropriate breakpoints which led to erroneous assignment of susceptibility. Other variations including disc content, medium and supplement, inoculum as well as failure to measure zone sizes properly also led to some repeating anomalies.

A method for production of 13C/15N double labelled RNA in E. coli, and subsequent in vitro synthesis of ribonucleotide 5' triphosphates.

In this paper we describe an enhanced method for the large scale production of high quality 13C/15N labelled NTPs. High amounts of labelled RNA was obtained from E. coli cells grown in 13C/15N enriched medium and treated with chloramphenicol. Total RNA was extracted from spheroplasted cells in the presence of SDS and proteinase K and subsequently degraded to NMPs by nuclease P1 and high concentrations of nuclease S1 in a low salt buffer. To avoid non-specific degradation of the RNA, nuclease digestion was performed in a short term reaction on native, not heat-denatured RNA. CMP, AMP, GMP and UMP were chromatographically separated and converted to the corresponding NTPs by a mixture of kinases in the presence of a coupled redox system based on thioredoxin and dithiothreitol. The quality of the 13C/15N labelled NTPs was tested by in vitro transcription.

[Assessment of the sensitivity of Neisseria meningitidis strains to benzylpenicillin using the disc diffusion method]. / Otsenka chuvstvitel'nosti shtammov Neisseria meningitidis k benzilpenitsillinu disko-diffuzionnym metodom.

The sensitivity of 235 N. meningitidis strains to 5 antibiotics was estimated by the diameter of growth inhibition zones according to the criteria recommended by a Laboratory (Marseilles, France) collaborating with the WHO. All the strains proved to be sensitive to benzylpenicillin when disks containing 10 and 2 IU of the antibiotic were used. The strains were also shown to be sensitive to chloramphenicol and tetracycline. 95.7 and 7.7 per cent of the strains were sensitive to rifampicin and oleandomycin, respectively. When the strain sensitivity was assayed with the disks containing 10 and 2 IU of benzylpenicillin by the more severe criteria recommended by J. Saez-Nieto et al., significant changes were detected: meningococci with relative resistance to benzylpenicillin were detected in various regions of this country and the number of such strains was found to have a tendency to slightly increase.

Antimicrobial susceptibility testing of Streptococcus pneumoniae: quality assessment results.

Six strains of Streptococcus pneumoniae were distributed to 405 United Kingdom laboratories who were asked to test the susceptibility of the strains to penicillin, tetracycline, chloramphenicol and erythromycin and to provide details of methodology to test the standards of susceptibility testing. High error rates were seen only in failure to detect moderate resistance to penicillin (12%) and resistance to chloramphenicol (16%). Increased error rates were associated with several methods or practices. These included the use of certain culture media; failure to standardise the inoculum; inoculation by loop rather than by swab; failure to use control organisms; failure to measure zone sizes; the use of discs containing a high content of penicillin to test susceptibility to penicillin, and the use of high content discs for testing erythromycin, tetracycline, and chloramphenicol.

Antimicrobial susceptibility testing of Haemophilus influenzae: trial organised as part of United Kingdom national external quality assessment scheme for microbiology.

Six strains of Haemophilus influenzae were distributed to 417 United Kingdom laboratories who were asked to test susceptibility of the strains to ampicillin, augmentin, tetracycline, chloramphenicol, and trimethoprim and to test for beta lactamase production. Laboratories were also asked to provide details of their methods by completing a questionnaire. The incidence of reports recording sensitive strains as resistant was 8% (ampicillin), 7% (augmentin), 3% (tetracycline), 1% (chloramphenicol), and 12% (trimethoprim). The incidence of reports recording resistant strains as sensitive was 9% (ampicillin), (2% with beta lactamase producing strains, 24% with non-beta lactamase producing strains), 51% (augmentin), 10% (tetracycline), 20% (chloramphenicol), and 3% (trimethoprim). High error rates were associated with several methods or practices. These included use of general purpose growth media rather than susceptibility testing media and failure to add lysed blood to the media when testing trimethoprim susceptibility; standardise the inoculum; use suitable control strains; and the use of high content discs for testing chloramphenicol, tetracycline, and ampicillin.

The assessment of interaction between drugs by the Autobac 1 system.

A study has been undertaken on the applicability of the Autobac 1 system to the checker board method to determine the interaction of combined drugs on bacterial populations. Preliminary results are reported which show that the Autobac makes easier, accurate, flexible and rapid such a method. In order to improve the significance and facilitate technical operations and data processing of this test additional equipment and device are suggested.