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Comparison of screening methods for TEM- and SHV-derived extended-spectrum beta-lactamase detection.

OBJECTIVE: To compare common extended-spectrum beta-lactamase (ESBL) screening methods and beta-lactams for their ability to detect TEM- and SHV-related ESBL enzymes. METHODS: This study compared disk diffusion testing by NCCLS methodology, the Jarlier double disk test, a disk-on-disk test, a modified three-dimensional test and the E test method for their sensitivity and specificity in detecting TEM- and SHV-related ESBL producers. Three negative and 22 positive controls were studied. These were two Klebsiella pneumoniae and 23 Escherichia coli transconjugants. Seventeen beta-lactam antibiotics were tested: cefamandole, cefotetan, cefoxitin, cefuroxime, cefixime, cefoperazone, cefotaxime, cefpodoxime, cefsulodin, ceftazidime, ceftibuten, ceftizoxime, ceftriaxone, moxalactam, cefepime, cefpirome and aztreonam. RESULTS: NCCLS disk diffusion was 14% sensitive with ceftriaxone, 36% with cefotaxime, 64% with aztreonam, 68% with cefpodoxime, and 73% with ceftazidime. Cefoperazone, cefamandole, cefpodoxime and cefpirome showed 91% sensitivity using the Jarlier test. Using the disk-on-disk test, cefsulodin showed 95% sensitivity, and cefoperazone, cefepime and cefamandole showed 91% sensitivity. With the modified three-dimensional test, cefoperazone, cefpodoxime and cefpirome showed 91% sensitivity. CONCLUSIONS: For practical reasons, we would recommend use of either the Jarlier test or the commercial cephalosporin disks containing clavulanic acid to screen for ESBL producers. Cefoperazone, cefamandole, cefpodoxime and cefpirome showed good sensitivity across the methods tested.

Anti-Bacterial Agents↗

Antibiotic sensitivity pattern of pneumococci relatively insensitive to penicillin and cephalosporin antibiotics.

Penicillin-insensitive pneumococci, of 10 serotypes, which had been isolated in Australia and New Guinea from healthy carriers and patients with pneumococcal infections, were shown to be relatively resistant to penicillin G, penicillin V, methicillin, cloxacillin, cephaloridine and cephalothin, compared with pneumococci fully sensitive to penicillin G. Most penicillin-insensitive pneumococci either were fully sensitive to ampicillin or showed a slight decrease in sensitivity. The size of inoculum, measured in viable units, had little effect on the minimal inhibitory concentration of penicillin G; this applied to both penicillin-insensitive and penicillin-sensitive pneumococci. In bacterial tests, the minimal bactericidal concentration of penicillin G either equalled or exceeded the minimal inhibitory concentration.

Ampicillin↗

A rapid staphylococcal coagglutination technic to differentiate group A from other streptococcal groups.

A rapid staphylococcal coagglutination technic for differentiating group A from other streptococcal groups properly classified 500 isolates of beta-hemolytic streptococci when compared with the results of a reference capillary precipitin test. Presumptive classification of the same 500 isolates by bacitracin susceptibility produced a significant number of incorrect identifications. The findings further document the inherent inaccuracy of the bacitracin susceptibility test and suggest that the staphylococcal coagglutination technic may be a simple, accurate, inexpensive alternative for identification of group A streptococci.

Agglutination Tests↗

A chromosomal ars operon homologue of Pseudomonas aeruginosa confers increased resistance to arsenic and antimony in Escherichia coli.

Operons encoding homologous arsenic-resistance determinants (ars) have been discovered in bacterial plasmids from Gram-positive and Gram-negative organisms, as well as in the Escherichia coli chromosome. However, evidence for this arsenic-resistance determinant in the medically and environmentally important bacterial species Pseudomonas aeruginosa is conflicting. Here the identification of a P. aeruginosa chromosomal ars operon homologue via cloning and complementation of an E. coli ars mutant is reported. The P. aeruginosa chromosomal ars operon contains three potential ORFs encoding proteins with significant sequence similarity to those encoded by the arsR, arsB and arsC genes of the plasmid-based and E. coli chromosomal ars operons. The cloned P. aeruginosa chromosomal ars operon confers augmented resistance to arsenic and antimony oxyanions in an E. coli arsB mutant and in wild-type P. aeruginosa. Expression of the operon was induced by arsenite at the mRNA level. DNA sequences homologous with this operon were detected in some, but not all, species of the genus Pseudomonas, suggesting that its conservation follows their taxonomic-based evolution.

Adenosine Triphosphatases↗

BpeAB-OprB, a multidrug efflux pump in Burkholderia pseudomallei.

Burkholderia pseudomallei, the causative agent of melioidosis, is intrinsically resistant to a wide range of antimicrobial agents, including beta-lactams, aminoglycosides, macrolides, and polymyxins. An operon, bpeR-bpeA-bpeB-oprB, which encodes a putative repressor, a membrane fusion protein, an inner membrane protein, and an outer membrane protein, respectively, of a multidrug efflux pump of the resistance-nodulation-division family was identified in B. pseudomallei. The divergently transcribed bpeR gene encodes a putative repressor protein of the TetR family which probably regulates the expression of the bpeAB-oprB gene cluster. Comparison of the MICs and minimal bactericidal concentrations of antimicrobials for bpeAB deletion mutant KHW Delta bpeAB and its isogenic wild-type parent, KHW, showed that the B. pseudomallei BpeAB-OprB pump is responsible for the efflux of the aminoglycosides gentamicin and streptomycin, the macrolide erythromycin, and the dye acriflavine. Antibiotic efflux by the BpeAB-OprB pump was dependent on a proton gradient and differs from that by the AmrAB-OprA pump in that it did not efflux the aminoglycoside spectinomycin or the macrolide clarithromycin. The broad-spectrum efflux pump inhibitor MC-207,110 did not potentiate the effectiveness of the antimicrobials erythromycin and streptomycin in B. pseudomallei.

Aminoglycosides↗

Production of bacteriocin inhibitory to Listeria species by Enterococcus hirae.

A bovine intestinal bacterial isolate, identified as Enterococcus hirae, was found to produce a bacteriocin (designated hiraecin S) inhibitory to Listeria monocytogenes and other Listeria spp. Identification to species level was determined by comprehensive biochemical and morphological tests which were verified by DNA-DNA homology assays. The antimicrobial agent was inactivated by pronase and papain and was insensitive to catalase. The antimicrobial activity was not due to hydrogen peroxide or acid formation, nor was lysozyme or muramidase activity observed in cell-free bacteriocin preparations. Inhibition of selected gram-negative bacteria was not observed. Other enterococci were sensitive to the bacteriocin, and except for Listeria spp., no other gram-positive bacteria tested were inhibited.

Animals↗

Antifungal activity of ring poly-chlorinated pyrimidines: structure activity relationships.

A total of 48 ring chlorinated pyrimidines was screened against strains of five fungi by the disc-plate method, in liquid culture, and for activity of the vapors of the compounds. Correlations of the results obtained by the three methods were made, and structure to activity relationships were discussed. The outstanding members of this series were found to be 2,4,5-trichloropyrimidine, 4,5,6-trichloropyrimidine, and 2-chloromethyl-4,5,6-trichloropyrimidine, in this screening system.

Antifungal Agents↗

Diverse phenotypes resulting from polyphosphate kinase gene (ppk1) inactivation in different strains of Helicobacter pylori.

Connections among biochemical pathways should help buffer organisms against environmental stress and affect the pace and trajectory of genome evolution. To explore these ideas, we studied consequences of inactivating the gene for polyphosphate kinase 1 (ppk1) in strains of Helicobacter pylori, a genetically diverse gastric pathogen. The PPK1 enzyme catalyzes synthesis of inorganic polyphosphate (poly P), a reservoir of high-energy phosphate bonds with multiple roles. Prior analyses in less-fastidious microbes had implicated poly P in stress resistance, motility, and virulence. In our studies, ppk1 inactivation caused the expected near-complete absence of poly P (>250-fold decrease) but had phenotypic effects that differed markedly among unrelated strains: (i) poor initial growth on standard brain heart infusion agar (five of six strains tested); (ii) weakened colonization of mice (4 of 5 strains); (iii) reduced growth on Ham's F-12 agar, a nutritionally limiting medium (8 of 11 strains); (iv) heightened susceptibility to metronidazole (6 of 17 strains); and (v) decreased motility in soft agar (1 of 13 strains). Complementation tests confirmed that the lack of growth of one Deltappk1 strain on F-12 agar and the inability to colonize mice of another were each due to ppk1 inactivation. Thus, the importance of ppk1 to H. pylori differed among strains and the phenotypes monitored. We suggest that quantitative interactions, as seen here, are common among genes that affect metabolic pathways and that H. pylori's high genetic diversity makes it well suited for studies of such interactions, their underlying mechanisms, and their evolutionary consequences.

Animals↗

Characterization of methicillin-resistant Staphylococcus aureus isolated from dogs in Korea.

Twelve strains of the methicillin-resistant Staphylococcus aureus (MRSA) recovered from hospitalized dogs were analyzed for in vitro antimicrobial susceptibility and virulence, and were genetically characterized by pulsed-field gel electrophoresis (PFGE). Antibiotic susceptibility test showed that nearly all isolates were resistant to beta-lactam antibiotics tested and all the strains were fully susceptible to glycopeptides. There were no inhibitory activities among the aminoglycosides. The 50% lethal dose (LD50) was determined by intraperitoneal injection of cell suspensions and estimated by the Spearman-Kärber method. The mouse lethality of MRSA and methicillin-susceptible S. aureus (MSSA) was not significantly different in both normal and cyclophosphamide-treated mice (p>0.05), indicating that they were equally virulent. There was a great difference in the incidence of toxin production between the MRSA and MSSA group; 83.3% (10 of 12) of the MRSA and 14.3% (1 of 7) of the MSSA were toxin producers. The predominant types produced by MRSA was B. All the MRSA strains were capsular type 5 producers, while of 7 MSSA strains, four were type 5, one for type 8, and two were nontypeable. Based on the PFGE analysis, the 12 MRSA isolates generated 9 to 11 fragments in the size range of <48.5 to 630.5 kb, and yielded 6 different patterns. The results indicated that production of toxin and capsule type do not play a role in the pathogenicity to mouse and PFGE is a valuable tool for the characterization of MRSA. This report is the first such cases in the veterinary literature in Korea and may indicate the frequent emergence of MRSA in veterinary clinic hereafter.

4-Quinolones↗