Disk Susceptibility Testing Research Articles

Extended-Spectrum Beta-Lactamase (ESBL)–Producing Gram-negative Isolates from Urine and Wound Specimens in a Tertiary Health Facility in Southern Nigeria

Purpose : To determine the prevalence of extended spectrum beta lactamase (ESBL) producing Gram negative bacteria causing wound and urinary tract infections among in- and out-patients in a tertiary health facility. Methods : The presence of ESBL was determined among 230 Gram negative bacilli isolated from wound (105) and urine (125) specimens from in- and out-patients who attended University of Benin Teaching Hospital (UBTH) for treatment using the double disc synergy method. Disc susceptibility test was performed on all isolates using standard techniques. Results : There was no significant difference in the prevalence of ESBL production between isolates from wound (47.6 %) and urine (41.6 %) as well as between in-patients (48.8 %) and out–patients (39.38 %). Enterobacter species were the most prevalent producers of ESBL from both wound and urine specimens as well as from both in- and out–patients. Bacterial isolates that produced ESBL were more resistant to amoxicillin-clavulanate, ceftazidime, ceftriaxone, cefotaxime, gentamicin, ciprofloxacin and ofloxacin compared to non-ESBL producers. Conclusion : A high prevalence (44.3%) of ESBL producing Gram–negative bacteria was observed among the patients, with Enterobacter species being the most prevalent. Prudent use of antibacterial agents is advocated to stem the tide. Keywords : Extended-spectrum beta-lactamase, Enterobacter species, Wound, Urine, Gram negative bacteria

Manual for reporting on antimicrobial resistance within the framework of Directive 2003/99/EC and Decision 2013/652/EU for information deriving from the year 2014

EFSA Supporting PublicationsVolume 12, Issue 2 771E Technical reportOpen Access Manual for reporting on antimicrobial resistance within the framework of Directive 2003/99/EC and Decision 2013/652/EU for information deriving from the year 2014 European Food Safety Authority (EFSA), European Food Safety Authority (EFSA)Search for more papers by this author European Food Safety Authority (EFSA), European Food Safety Authority (EFSA)Search for more papers by this author First published: 27 February 2015 https://doi.org/10.2903/sp.efsa.2015.EN-771Citations: 1 Published date: 27 February 2015 Question number: EFSA-Q-2015-00011 AboutPDF ToolsExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat References CLSI (Clinical and Laboratory Standards Institute), 2006. Methods for antimicrobial dilution and disk susceptibility testing of infrequently isolated or fastidious bacteria; approved guideline. CLSI document M45-A2. Clinical and Laboratory Standards Institute, Wayne, PA, USA. CLSI (Clinical and Laboratory Standards Institute), 2007. Performance standards for antimicrobial susceptibility testing; 17th informational supplement. CLSI document M100-S17 Clinical and Laboratory Standards Institute, Wayne, PA, USA. CLSI (Clinical and Laboratory Standards Institute), 2008. Performance standards for antimicrobial disk and dilution susceptibility tests for bacteria isolated from animals; approved standard— Third Edition. CLSI document M31-A3. Clinical and Laboratory Standards Institute, Wayne PA, USA. ECDC (European Centre for Disease Prevention and Control), EFSA (European Food Safety Authority) and EMA (European Medicines Agency), 2015. ECDC/EFSA/EMA first joint report on the integrated analysis of the consumption of antimicrobial agents and occurrence of antimicrobial resistance in bacteria from humans and food-producing animals. ECDC/EFSA/EMA, Stockholm, Sweden/Parma, Italy/London, UK. EFSA Journal 2015; 13(1):4006, 114 pp. doi:10.2903/j.efsa.2015.4006 EFSA (European Food Safety Authority), 2012. Technical specifications for the harmonised monitoring and reporting of antimicrobial resistance in MRSA in food-producing animals and food. EFSA Journal 2012; 10(10):2897, 56 pp. doi:10.2903/j.efsa.2012.2897 EFSA (European Food Safety Authority), 2014. Technical specifications on randomised sampling for harmonised monitoring of antimicrobial resistance in zoonotic and commensal bacteria. EFSA Journal 2014; 12(5):3686, 33 pp. doi:10.2903/j.efsa.2014.3686 EFSA (European Food Safety Authority), 2015. Data dictionaries – guidelines for reporting data on zoonoses, antimicrobial resistance and food-borne outbreaks using the EFSA data models for the Data Collection Framework (DCF) to be used in 2015, for 2014 data. EFSA supporting publication, in press. ISO (International Organization for Standardization), 2006. ISO 20776-1:2006 Clinical laboratory testing and in vitro diagnostic test systems – Susceptibility testing of infectious agents and evaluation of performance of antimicrobial susceptibility test devices – Part 1: Reference method for testing the in vitro activity of antimicrobial agents against rapidly growing aerobic bacteria involved in infectious diseases. International Organization for Standardization, 19 pp. Available online: https://www.iso.org/obp/ui/#iso:std:41630:en Noordhuizen JPTM, Frankena K, Thrusfield MV and Graat EAM, 2001. Application of quantitative methods in veterinary epidemiology. Wageningen Press, Wageningen, The Netherlands. Rothman KJ, 1986. Modern epidemiology. Little, Brown & Co, Boston, MA, USA, 299– 304. Citing Literature Volume12, Issue2February 2015771E ReferencesRelatedInformation

Application of Artificial Intelligence to the Prediction of the Antimicrobial Activity of Essential Oils.

Essential oils (EOs) are vastly used as natural antibiotics in Complementary and Alternative Medicine (CAM). Their intrinsic chemical variability and synergisms/antagonisms between its components make difficult to ensure consistent effects through different batches. Our aim is to evaluate the use of artificial neural networks (ANNs) for the prediction of their antimicrobial activity. Methods. The chemical composition and antimicrobial activity of 49 EOs, extracts, and/or fractions was extracted from NCCLS compliant works. The fast artificial neural networks (FANN) software was used and the output data reflected the antimicrobial activity of these EOs against four common pathogens: Staphylococcus aureus, Escherichia coli, Candida albicans, and Clostridium perfringens as measured by standardised disk diffusion assays. Results. ANNs were able to predict >70% of the antimicrobial activities within a 10 mm maximum error range. Similarly, ANNs were able to predict 2 or 3 different bioactivities at the same time. The accuracy of the prediction was only limited by the inherent errors of the popular antimicrobial disk susceptibility test and the nature of the pathogens. Conclusions. ANNs can be reliable, fast, and cheap tools for the prediction of the antimicrobial activity of EOs thus improving their use in CAM.

Cephalosporinases associated with outer membrane vesicles released by Bacteroides spp. protect gut pathogens and commensals against β-lactam antibiotics.

ObjectivesTo identify β-lactamase genes in gut commensal Bacteroides species and to assess the impact of these enzymes, when carried by outer membrane vesicles (OMVs), in protecting enteric pathogens and commensals.MethodsA deletion mutant of the putative class A β-lactamase gene (locus tag BT_4507) found in the genome of the human commensal Bacteroides thetaiotaomicron was constructed and a phenotypic analysis performed. A phylogenetic tree was built from an alignment of nine Bacteroides cephalosporinase protein sequences, using the maximum likelihood method. The rate of cefotaxime degradation after incubation with OMVs produced by different Bacteroides species was quantified using a disc susceptibility test. The resistance of Salmonella Typhimurium and Bifidobacterium breve to cefotaxime in liquid culture in the presence of B. thetaiotaomicron OMVs was evaluated by measuring bacterial growth.ResultsThe B. thetaiotaomicron BT_4507 gene encodes a β-lactamase related to the CepA cephalosporinase of Bacteroides fragilis. OMVs produced by B. thetaiotaomicron and several other Bacteroides species, except Bacteroides ovatus, carried surface-associated β-lactamases that could degrade cefotaxime. β-Lactamase-harbouring OMVs from B. thetaiotaomicron protected Salmonella Typhimurium and B. breve from an otherwise lethal dose of cefotaxime.ConclusionsThe production of membrane vesicles carrying surface-associated β-lactamases by Bacteroides species, which constitute a major part of the human colonic microbiota, may protect commensal bacteria and enteric pathogens, such as Salmonella Typhimurium, against β-lactam antibiotics.

<i>In Vitro</i> Antibacterial Activity of <i>Eclipta alba</i> (L.) Hassk

The antibacterial activity of Eclipta alba against three gram positive and five gram negative bacterial strains was investigated. The fresh whole plants were collected from Chidambaram, Tamil Nadu. Petroleum ether, chloroform, ethyl acetate, acetone, methanol and aqueous extracts at different concentrations (1, 2, 5, 10 mg/ml) were used to investigate the antibacterial activity. NCCL standards were strictly followed to perform antibacterial disc susceptibility test using disc diffusion method. The extracts showed varying degree of inhibitory potential against all the tested bacteria. Methanol extract of the plant had higher inhibitory action against Staphylococcus aureus, Bacillus subtilis, Proteus mirabilis and Pseudomonas fluroescens.

Synthesis, spectroscopic, anticancer, antibacterial and antifungal studies of Ni(II) and Cu(II) complexes with hydrazine carboxamide, 2-[3-methyl-2-thienyl methylene

Schiff’s base ligand(L) hydrazine carboxamide, 2-[3-methyl-2-thienyl methylene] and its metal complexes have been synthesized and characterized by elemental analysis, molar conductance, various spectroscopic techniques such as electronic, IR, 1H NMR, mass, EPR. Molar conductance of complexes in DMF solution corresponds to non-electrolyte. Complexes have general composition [M(L)2X2], where M=Ni(II) and Cu(II), X=Cl−, NO3−, CH3COO− and ½SO42−. On the basis of above spectral studies, an octahedral geometry has been assigned for Ni(II) complexes and tetragonal geometry for Cu(II) complexes except [Cu(L)2SO4] which possesses five coordinated trigonal bipyramidal geometry. These metal complexes were also tested for their anticancer, antibacterial and antifungal activities to assess their inhibition potential. Anticancer activity of ligand and its metal complexes were evaluated using SRB fluorometric assay and Adriamycin (ADR) was applied as positive control. Schiff’s base ligand and its metal complexes were screened for their antibacterial and antifungal activity against Escherichia coli, Bacillus cereus and Aspergillus niger, Aspergillus flavus, respectively. Kirby-Bauer single disk susceptibility test was used for antibacterial activity and well diffusion method for antifungal activity of the compounds on the used fungi.

<i>In Vitro</i> Antibacterial Activity of <i>Eclipta alba</i> (L.) Hassk

The antibacterial activity of Eclipta alba against three gram positive and five gram negative bacterial strains was investigated. The fresh whole plants were collected from Chidambaram, Tamil Nadu. Petroleum ether, chloroform, ethyl acetate, acetone, methanol and aqueous extracts at different concentrations (1, 2, 5, 10 mg/ml) were used to investigate the antibacterial activity. NCCL standards were strictly followed to perform antibacterial disc susceptibility test using disc diffusion method. The extracts showed varying degree of inhibitory potential against all the tested bacteria. Methanol extract of the plant had higher inhibitory action against Staphylococcus aureus, Bacillus subtilis, Proteus mirabilis and Pseudomonas fluroescens.

Distribution of Methicillin-resistant Staphylococcus aureus (MRSA) in RAW meat and fish samples in Korea

Methicillin-resistant Staphylococcus aureus (MRSA) in raw meat and fish samples in Korea was investigated. A total of 209 samples was analyzed. Antimicrobial disk susceptibility testing with oxacillin (OX) was used to detect MRSA in 74 S. aureus isolates (35.4%), 7 of which showed resistance towards OX. The mecA gene was identified in the 7 isolates with OX resistance and was expressed in all 7 isolates. Pulsed-field gel electrophoresis was used to analyze genetic homologies among the 7 MRSA isolates. No genetic correlations were detected among the isolates.

EFFECT OF NORFLOXACIN ON ANTIBIOTIC RESISTANCE OF ESCHERICIA COLI: COMPARISON BETWEEN SEQUENCING BATCH REACTOR AND SEQUENCING BATCH MEMBRANE BIOREACTOR

Seven antibiotics including norfloxacin (NOR) were tested via disk susceptibility test on E. coli culture isolated from the MLSS of the two types of lab-scale sequencing batch reactors (SBR): a common SBR and an SBR with microfiltration membrane (SB-MBR) for treatment of synthetic municipal wastewater. The same experiment treating the NOR-added wastewater to examine the possible induction of resistance to itself and the other antibiotics. The MLSS from Bangkok’s municipal wastewater treatment plant was employed as an initial seed and the susceptible E. coli culture (TISTR780) were spiked daily into both reactors. The reactors were continuously operated under 2hr/2hr of aeration/non-aeration cycle and resistances to antibiotics of E. coli in MLSS were monitored. When NOR was not added, the SB-MBR showed lower percentages of resistant E. coli than the SBR did to amoxicillin/clavulanic acid, amikacin, nalidixic acid, tetracycline and chloramphenicol. Oppositely, the SB-MBR treating the NORadded wastewater appeared to promote resistances of E. coli to nalidixic acid, sulfamethoxasole and tetracycline probably due to a long SRT and low DO compared to that of SBR. Although its mechanism should be analyzed with molecular techniques in further studies, this NOR-induced expression of resistance resulted in a higher occurrence of multidrug resistant E. coli in the SBMBR than that in the SBR.

Synthesis, spectroscopic, anticancer and antibacterial studies of Ni(II) and Cu(II) complexes with 2-carboxybenzaldehyde thiosemicarbazone

Ni(II) and Cu(II) complexes of 2-carboxybenzaldehyde thiosemicarbazone (L) were synthesized and investigated by their spectral and analytical data. These newly synthesized complexes have a composition of M(L)X(H2O)2 (where M=Ni(II), Cu(II) and X=Cl−, NO3-, CH3COO−) and (L) is the tridentate Schiff base ligand. The ligand and its complexes have been characterized on the basis of analytical, molar conductivity, magnetic susceptibility measurements, FT-IR, ESR, 1H NMR and electronic spectral analysis. All the compounds were non-electrolytic in nature. On the basis of spectral studies an octahedral geometry has been assigned for Ni(II) and a tetragonal geometry for Cu(II) complexes. The ligand and its metal complexes were screened for their anticancer studies against human breast cancer cell lines MCF-7 and calculated minimum inhibitory concentration and also for antibacterial activity using Kirby–Bauer single disk susceptibility test.

Comparison of phenotypic methods for the detection of IMP producing enterobacteriaceae in queensland

Background The prevalence of carbapenemase producing enterobacteriaceae (CPE), particularly IMP producers (IMP), in Australia is increasing. Optimal methods for detection of IMP CPE are unknown. We compare six phenotypic methods to determine the most sensitive method for the detection of IMP CPE. Methods We tested 25 molecularly confirmed IMP CPE producers, including 16 Enterobacter cloacae , collected from Queensland Health hospitals. The following methods were performed: disc susceptibility testing (DST), minimum inhibitory concentrations (MICs) by E-test, broth microdilution (BMD) and Vitek 2, modified hodge test (MHT) and Carba NP test. Results Meropenem MIC range (MIC 50/90 ) was 0.25->32 (4/16), 0.25-32(2/16), 1->16(>16/>16) for BMD, E-test and Vitek 2, respectively. Seventeen percent of isolates had a Vitek 2 meropenem MIC between 1-2 |xg/mL. Using EUCAST breakpoints, all isolates tested resistant by Ertapenem E-test and all except for one isolate was resistant by ertapenem DST. CLSI breakpoints increased detection of CPE with meropenem and imipenem. Carba NP test and MHT provided positive results for all isolates. Conclusion: Our results indicate that isolates with a Vitek 2 meropenem MIC of ≥1 μg/mL should undergo further phenotypic testing. Ertapenem DST, ertapenem E-test, Carba NP test and MHT are the most sensitive methods for detection of IMP CPE.

Comparative phytochemical and anti-bacterial studies of two indigenous medicinal plants Curcuma caesia Roxb. and Curcuma aeruginosa Roxb

Background: Traditional medicinal plants could serve as a good supply of new dependable, biodegradable, renewable drugs and can be utilised for its anti‑bacterial activity directly or indirectly. Aims: To evaluate the phytochemical and anti‑bacterial properties of two morphologically similar indigenous medicinal plants Curcuma caesia and Curcuma aeruginosa belonging to the family Zingiberaceae. Materials and Methods: Evaluation of rhizome extracts using methanol was performed for the presence of active principles. Qualitative analysis was carried out for diverse phytoconstituents. Different concentrations (1.25, 2.5, 5.0 mg/ml) of hexane, chloroform, ethyl acetate, acetone, methanol and water serial extracts from the rhizome of C. caesia and C. aeruginosa were tested against Gram positive (Staphylococcus aureus, Streptococcus haemolyticus and Bacillus cereus) and Gram negative (Salmonella typhi, Enterobacter aerogens, Vibrio cholerae, Pseudomonas aeruginosa and Serratia marcescens) bacteria. National Committee for Clinical Laboratory Standard (NCCL) standards were strictly followed to perform anti‑bacterial disc susceptibility test using disc diffusion method. Statistical Analysis: All the values of the results were expressed as means of two independent experiments ± standard deviation. Results: Phytochemical screening of these two plants confirmed the presence of various bioactive substances and thus validating its use in herbal remedies. Anti‑bacterial studies showed varying degree of inhibitory action against all the tested bacteria. Among the Gram positive bacteria, acetone extract of C. caesia showed maximum activity against S. aureus and hexane extract of C. aeruginosa exhibited maximum activity against B. cereus. In Gram negative bacteria, chloroform extract of C. caesia showed maximum inhibitory action against S. marcescens, whereas the methanol extract of C. aeruginosa showed higher inhibitory action against S. typhi. Conclusions: The findings about present study suggest that the rhizome extract possess excellent anti‑bacterial potential that can be used for therapeutic purposes for many bacterial infectious diseases with proper evaluation procedures. The present study validated the use of these plants in traditional medicine and recommends for making new pharmaceuticals for therapeutic needs. Key words: Anti‑bacterial activity, Curcuma aeruginosa, Curcuma caesia, medicinal plants, plant extracts

Value of phenotypic and genotypic identification of Acinetobacter baumannii isolates from two hospitals in Jordan

Acinetobacter baumannii isolates have been recovered from hospitalized patients over the past few years from two hospitals in Jordan. Phenotypic and biochemical characterization in addition to disc susceptibility testing of all clinical isolates indicated that all of them were belonging to A. baumannii. A high degree of conservation of both the ITS length and the ITS sequence was observed, and their identities were further confirmed by amplified ribosomal DNA gene restriction analysis (ARDRA). The application of ARDRA for the identification of Acinetobacter species has several advantages over phenotypic identification. ARDRA considered rapid and reliable and universally applicable method for identification of most of the Acinetobacter genomic species, thus contribute to better understanding of the clinical importance and epidemiology of isolates.

Microbiology of neonatal septicemia in a tertiary hospital in Benin City, Nigeria

Septicemia is a major cause of death in neonates. Prompt diagnosis and effective treatment is necessary to treat patients with septicemia. However, the prevalence, etiology, and antibiotic susceptibility vary with location and time. This study aimed at determining the prevalence of neonatal septicemia and the effect of age and gender on this prevalence. In addition, the antibacterial susceptibility of etiologic agents was also determined. Blood samples were collected from 534 neonates (322 males and 212 females) between 1 day and 28 days of age with signs and symptoms of septicemia. The blood samples were processed to diagnose septicemia. Identification of bacterial isolates and disc susceptibility testing were performed using standard techniques. Age and gender did not significantly affect the prevalence of neonatal septicemia (p = 0.554 and 0.127, respectively). Klebsiella species were the predominant microorganism causing neonatal septicemia, in males and within the first 14 days of life. Fluoroquinolones, gentamicin, and β-lactams (with the exception of cloxacillin) were the most active antibacterial agents. An overall neonatal septicemia prevalence rate of 38.95% was observed. Klebsiella species was the most predominant isolate causing neonatal septicaemia. The β-lactam antibiotics recommended in susceptibility testing and the data collected in this study will be helpful in empiric therapy of neonatal septicemia.

Chlorhexidine activity against bacterial biofilms

A biofilm is a complex microbiological ecosystem deposited on surfaces. Microorganisms in form of biofilms are of particular clinical concern because of the poor response to antimicrobial treatments. This study aimed to determine whether bacterial and fungal biofilms are able to resist the antimicrobial activity of chlorhexidine, a powerful antiseptic widely used in the hospital environment. Disk diffusion and susceptibility tests were conducted in accordance with Clinical and Laboratory Standards Institute standards for the determination of biofilm inhibitory concentration. Chlorhexidine was tested first at a minimum inhibitory concentration and then at higher concentrations when it was not able to destroy the biofilm. The plates were developed with a solution of 0.1% crystal violet, and readings were made at an optical density of 570 nm. Chlorhexidine demonstrated excellent antimicrobial activity for most microorganisms tested in their free form, but was less effective against biofilms of Acinetobacter baumannii, Escherichia coli, methicillin-resistant Staphylococcus aureus, and Pseudomonas aeruginosa. This study confirms that microorganisms in biofilms have greater resistance to chlorhexidine, likely owing to the mechanisms of resistance conferred to the structure of biofilms.

Microbiology of otitis media among children attending a tertiary hospital in Benin City, Nigeria

Otitis media has a worldwide prevalence. Though it is more common in children, the aetiologies and antibiogram varies with age, time and geographical location. A total of 220 children less than 18 years with signs and symptoms of otitis media were recruited for this study. Middle ear discharge were collected, processed and microbial isolates identified using standard microbiological techniques. Disc susceptibility tests were performed on bacterial isolates. Gender and age did not significantly affect the prevalence of otitis media pathogens (P = 0.8310 and P = 0.8272, respectively). Pseudomonas aeruginosa (33.33%) were the most prevalent microbial agent of otitis media followed by Staphylococcus aureus(23.19%) while Citrobacter species and Aspergillus niger were the least prevalent with a prevalence of 0.48% each. In relation to gender, P. aeruginosa were the most prevalent followed by S. aureus in males. In females, S. aureus (32.18%) predominated. Only S. aureus was significantly associated with female gender (odd ratio (OR) = 2.422 95% confidene interval (CI) = 1.261, 4.65, P = 0.0113). Tetracycline, erythromycin, cloxacillin and amoxicillin were poorly active against the bacterial isolates. Other antibacterial agents exhibited good activity against the bacterial isolates. In conclusion, an overall prevalence of 84.55% of culture-positive otitis media was observed in this study with P. aeruginosa as the predominant aetiologic agent. Though, the antibacterial agents exhibited good activities, prudent use of antibacterial agents are advocated. Key words: Otitis media, children, tertiary hospital, Benin City.

P129 Duration of colonisation with extended-spectrum beta-lactamase-producing enterobacteria

The emergence of multidrug-resistant (MDR) Acinetobacter baumannii has become a serious medical problem worldwide. To clarify the genetic and epidemiological properties of MDR A. baumannii strains isolated from a medical setting in Nepal, 246 Acinetobacter spp. isolates obtained from different patients were screened for MDR A. baumannii by antimicrobial disk susceptibility testing. Whole genomes of the MDR A. baumannii isolates were sequenced by MiSeq™ (Illumina), and the complete genome of one isolate (IOMTU433) was sequenced by PacBio RS II. Phylogenetic trees were constructed from single nucleotide polymorphism concatemers. Multilocus sequence types were deduced and drug resistance genes were identified. Of the 246 Acinetobacter spp. isolates, 122 (49.6%) were MDR A. baumannii, with the majority being resistant to aminoglycosides, carbapenems and fluoroquinolones but not to colistin and tigecycline. These isolates harboured the 16S rRNA methylase gene armA as well as blaNDM-1, blaOXA-23 or blaOXA-58. MDR A. baumannii isolates belonging to clonal complex 1 (CC1) and CC2 as well as a novel clonal complex (CC149) have spread throughout a medical setting in Nepal. The MDR isolates harboured genes encoding carbapenemases (OXA and NDM-1) and a 16S rRNA methylase (ArmA).

Clinical evaluation of cefoxitin in surgery

Cefoxitin (CFX) was administered to 13 patients who were admitted to the surgical ward of the hospital. The results of the study as follows; Disc susceptibility tests of the 11 strains isolated from the patients to antibiotic were performed and 10 strains showed good susceptibility to CFX. The clinical response was good in all 4 patients with surgical infections. It is noted that 1 patient with mixed infection caused by E. coli, Clostridium sp. and Bacteroides sp. showed good response to CFX. Out of 9 patients treated with CFX for prophylaxis of postoperative infections, 8 patients responded effectively to CFX, and clinical efficacy rate was 88.9%. No side effects were noted due to CFX. It is considered from these results of the study that CFX is a highly effective and safe drug for treatment of infections and for the prevention of postoperative infections in surgery.

Vibrio ordalii antimicrobial susceptibility testing—Modified culture conditions required and laboratory-specific epidemiological cut-off values

The present study aimed to determine oxytetracycline (OTC), florfenicol (FLO) and oxolinic acid (OXO) MICs and zone diameters for 24 Chilean Vibrio ordalii isolates using the methods for broth dilution susceptibility testing of bacteria isolated from aquatic animals and the methods for antimicrobial disk susceptibility testing of bacteria isolated from aquatic animals guidelines published by the Clinical Laboratory Standards Institute (CLSI). The results were then used in a normalized resistance interpretation (NRI) analysis to establish tentative laboratory-specific epidemiological cut-off (ECOFF) values. MIC results were similar at the two tested temperatures (22 °C and 18 °C). At 18 °C, the NRI analysis of OTC, FLO and OXO MIC data calculated laboratory-specific ECOFF values and non-wild-type (NWT) rates to be ≤4 mg/l (24%), ≤16 mg/l (4%) and ≤8 mg/l (25%), respectively. Tests performed with all V. ordalii isolates following the officially recommended incubation temperature (22 °C) revealed difficulties in measuring inhibition zone diameters. When disk diffusion tests were performed using Mueller-Hinton agar with 1% NaCl (MHA-1) at 18 °C the inhibition zone diameter distributions showed the formation of WT populations which could be defined using NRI analysis. For OTC the laboratory-specific ECOFF value was ≥38 mm with NWT rate of 16.7%. For FLO and OXO, the laboratory-specific ECOFF values were ≥38 and ≥40, respectively, generating NWT rates of 25 and 46%, respectively. Although the CLSI suggests testing Vibrio spp. on MHA-1 at 22 °C, we found measurements of the 24 isolates were better defined and normally distributed at 18 °C. This is the first study determining the MIC and disk diffusion test of V. ordalii isolated from diseased salmonids, where laboratory-specific ECOFF values could be established. Also resistance to OTC, FLO and OXO among some Chilean isolates was demonstrated.

SCREENING OF ANTIBACTERIAL AND CYTOTOXIC ACTIVITY OF EXTRACTS FROM EPIDERMIS AND EPIDERMAL MUCUS OF BARBONYMUS SCHWANENFELDII (TINFOIL BARB FISH)

Aim: The main objective of this work is to increase the utilization of by-products like mucus and skin from Tinfoil barb fish in order to isolate antibacterial protein against gram positive and gram negative microorganism. Materials and Methods: The fish epidermis and epidermal mucus samples were extracted with crude, ethanol and dichloromethane to identify potential antimicrobial agents including the ethanol and dichloromethane soluble compounds. Preliminary screening for antimicrobial activity of the extracts were tested against two Gram-positive bacteria Bacillus cereus (MTCC 1306)and Staphylococcus aureus(MTCC 3160) and two Gram-negative bacteria Shigellaboydii and E.coli(MTCC 1303) using standardized disc susceptibility test method recommended by the British Society of Antimicrobial Chemotherapy(BSAC). The activity was measured in terms of zone of inhibition in mm. Also Mouse fibroblast 3T3 L1 cell lines were used to measure cytotoxic activity. Minimum cell viability was shown by sample of 10 mg/ml concentration of the epidermal mucus. Result: The ethanol extract exhibited a bactericidal activity and inhibited the growth of Bacillus cereus, Staphylococcus aureus, Shigellaboydii and E.coli. The dichloromethane exhibited activity against Bacillus cereus, Staphylococcus aureus, E.coli and no activity against Shigellaboydii. The aqueous showed no bactericidal activity for any of the human pathogen tested. These results show the presence of antimicrobial agent and the role of fish byproducts like mucus and epidermis in antimicrobial protection. Conclusion: the present result suggested that the mucus and epidermal extract of Barbonymus schwanenfeldii (Tinfoil barb fish) may be a potent source of antimicrobial agent for human pathogen.