Comparison of E-test with other conventional susceptibility testing methods for ciprofloxacin and gentamicin against gram negative enteric bacilli.

BACKGROUND: Increasing antibiotic resistance in Gram negative bacteria has led to the need for a faster and reliable method for determining antimicrobial susceptibility testing. In a resource poor setting like ours, it's also important to look for methods that will be clinically and economically beneficial to the patient. AIM: This study was aimed at evaluating the Epsilometer test (E-test) and conventional methods for determining antimicrobial susceptibility of isolates of Gram-negative enteric bacteria to ciprofloxacin and gentamicin. METHODS: Disc diffusion, E-test, broth dilution and agar dilution methods were performed on 54 bacterial isolates. RESULTS: Using the E-test, 88.9% of bacterial isolates were resistant to ciprofloxacin, 92.6% were resistant using broth microdilution, 96.3% were resistant using agar dilution and 72.2% were resistant using disc diffusion. Minimum inhibitory concentration (MIC50) of isolates for gentamicin showed significant difference for all the techniques (p < 0.05) while MIC90 for gentamicin and MIC50 and MIC90 for ciprofloxacin for all the techniques had no significant difference (p > 0.05). Both E-test and broth dilution methods showed high levels of agreement (p > 0.05), there were low levels of agreement between E-test and agar dilution method (p < 0.05), especially at MIC50. CONCLUSION: The E-test can therefore be considered a reliable method to determine antimicrobial susceptibility testing and it gives results which are at least as accurate as those obtained by the broth dilution method.

High levels of multidrug resistance in clinical isolates of Gram-negative pathogens from Nigeria.

In Nigeria, quinolones and ß-lactam antibiotics are widely used to treat bacterial infections. This study aimed to identify the prevalence of resistance to these drugs and to determine the mechanisms of resistance to these agents. In total, 134 non-duplicate, Gram-negative enteric isolates of 13 species from different hospitals were investigated for susceptibility to a panel of antibiotics, carriage of plasmid-mediated quinolone and ß-lactam resistance genes, production of extended-spectrum ß-lactamases (ESBLs), and mutations within topoisomerase genes. The level of resistance to all antibiotics tested was extremely high, with minimum inhibitory concentrations for 90% of the organisms (MIC(90) values) of ≥ 256 µg/mL for all drugs. Of the 134 isolates, 92 had mutations within the quinolone resistance-determining region (QRDR) of gyrA or within gyrA and parC. In addition, the plasmid-mediated quinolone resistance genes qnrA, qnrB, aac(6')-Ib-cr and qepA were identified. The qnrD allele, which has previously only been found in Salmonella isolates from China, was identified in two Proteus isolates and one Pseudomonas isolate. Of the 134 isolates, 23 (17.2%) carried aac(6')-Ib-cr, 11 (8.2%) carried a qnr variant and 5 (3.7%) were positive for qepA. Twenty-eight isolates (20.9%) produced ESBL variants, with a CTX-M variant being carried by 25 isolates (18.7%). In addition, six isolates (4.5%) carried ampC variants [ACT-1 (1 isolate), DHA-1 (4 isolates) and CMY-2 (1 isolate)]. This study demonstrates a very high level of multidrug resistance amongst Gram-negative enteric bacilli isolated from different sites from patients in Nigerian hospitals as well as the presence of a variety of plasmid-associated resistance genes, including some identified from Africa for the first time.

Antibiotic susceptibility patterns of Pseudomonas aeruginosa to available antipseudomonal drugs in Ibadan, Nigeria.

Pseudomonas aeruginosa has been reported to be a leading cause ofnosocomial infections. Resistance of this notorious bacterium to commonly used antimicrobial agents is becoming an increasing clinical problem and a recognized public health threat because there are limited number of antimicrobial agents including the antipseudomonal penicillins, cephalosporins, carbapenems, aminoglycosides and fluoroquinolones with reliable activity against it. This study was therefore carried out, using Bauer-Kirby method, to determine the antibiotic susceptibility patterns of Pseudomonas aeruginosa isolates from in-patients and out-patients attending the University College Hospital, Ibadan in Nigeria between June 2004 and May 2006. The isolation rate of Pseudomonas aeruginosa in clinical specimens was found to be 16.8% with the highest occurrence of 41.9% in ear swab followed by 39.3% occurrence in wound swab. The susceptibility pattern showed that 78.3% were sensitive to amikacin and 72.0% to ciprofloxacin. The isolates from the in-patients showed higher resistance to all the antibiotics tested than the isolates from the out-patients, most especially amikacin and ciprofloxacin. However, no consistent antibiotic susceptibility pattern could be established for this pathogenic bacterium based on sources. In conclusion, the Pseudomonas aeruginosa species harboured by in-patients showed higher rates of antibiotic resistance than those of the out-patients. Also amikacin and ciprofloxacin were the two antibiotics found to be most potent against this pathogen.

In vitro antimicrobial properties of coconut oil on Candida species in Ibadan, Nigeria.

The emergence of antimicrobial resistance, coupled with the availability of fewer antifungal agents with fungicidal actions, prompted this present study to characterize Candida species in our environment and determine the effectiveness of virgin coconut oil as an antifungal agent on these species. In 2004, 52 recent isolates of Candida species were obtained from clinical specimens sent to the Medical Microbiology Laboratory, University College Hospital, Ibadan, Nigeria. Their susceptibilities to virgin coconut oil and fluconazole were studied by using the agar-well diffusion technique. Candida albicans was the most common isolate from clinical specimens (17); others were Candida glabrata (nine), Candida tropicalis (seven), Candida parapsilosis (seven), Candida stellatoidea (six), and Candida krusei (six). C. albicans had the highest susceptibility to coconut oil (100%), with a minimum inhibitory concentration (MIC) of 25% (1:4 dilution), while fluconazole had 100% susceptibility at an MIC of 64 microg/mL (1:2 dilution). C. krusei showed the highest resistance to coconut oil with an MIC of 100% (undiluted), while fluconazole had an MIC of > 128 microg/mL. It is noteworthy that coconut oil was active against species of Candida at 100% concentration compared to fluconazole. Coconut oil should be used in the treatment of fungal infections in view of emerging drug-resistant Candida species.

Genetic analysis of tetracycline-resistant plasmids in enteropathogenic Escherichia coli isolated from patients in Nigeria.

Genetic analysis of antibiotic-resistant plasmids from 102 serologically defined strains of enteropathogenic Escherichia coli from Nigeria was carried out. All the isolates were screened for susceptibility to antibiotics, and 47 were found resistant to tetracycline. A total of 138 plasmids was isolated by agarose gel electrophoresis. Transformation and conjugation experiments showed that 57.4% of the resistant strains carried R-plasmids ranging in sizes from 2 to 46 x 10(6) daltons. Plasmid-determined resistance to tetracycline, ampicillin and streptomycin was found. Restriction endonuclease analysis of three of the commonest plasmids: p1679, p529 and p1479 revealed relatedness with respect to function and structure. The DNA segment on which TcR gene is located on each of them was identified by cloning into the vector plasmid pGL101. The recombinant plasmids pOADI and pOAD2 gave full expression of TcR gene when transformed into E. coli DHI. Furthermore, the tetracycline-resistant strains were examined for their phenotypic behaviour with respect to tetracycline and its lipophilic analogs.

Cloning and characterization of the tetracycline resistance determinant from enteropathogenic Escherichia coli p1479.

As part of an epidemiological study, R plasmids coding for tetracycline resistance were isolated from enteropathogenic Escherichia coli. A 1.4kb Pst 1 fragment of one of them p1479 (size 5.7kb) has been cloned into plasmid pGL101. This recombinant plasmid containing the tetracycline gene was then transformed into E. coli DHI where the tetracycline resistant gene was fully expressed. Attempts to develop this clone to a diagnostic probe is now in progress.

Preliminary epidemiological studies on tetracycline resistant plasmids isolated from enteric bacteria in Nigeria.

In an epidemiological investigation on the genetic determinants responsible for tetracycline resistance in Nigeria, 518 isolates of enteric bacteria from hospitals and clinics were screened for susceptibility to antibiotics. 305 (58.8%) were resistant to tetracycline. The commonest resistance pattern that involved tetracycline resistance was tetr ampr sxtr Smr. Of the 305 isolates, 207 (67.8%) transferred resistant plasmids to Escherichia coli K-12. Altogether, 12 types of plasmids were isolated depending on the phenotypes of antibiotics resistant character borne on the plasmids; they ranged in sizes between 3 to 180 kilobases. The plasmids were evenly distributed in the country. Thus R plasmids are a major reason for resistance to tetracycline encountered in Nigeria.

Isolation and properties of creatine kinase from the breast muscle of tropical fruit bat, Eidolon helvum (Kerr).

Creatine kinase, from fruit bat breast muscle, has been purified to homogeneity. The mol. wt of the enzyme was estimated to be about 78,000-80,000 with two subunits of 42,500. There are nine thiol residues/mol of the enzyme and two of these react readily with DTNB leading to total inactivation of the enzyme. The metal ion specificity was in order Mg2+ greater than Zn2+ greater than Co2+. Initial velocity and product inhibition studies of the reverse reaction are consistent with sequential reaction but of either rapid equilibrium random or ordered type.