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Disk diffusion testing, quality control guidelines, and antimicrobial spectrum of HR810, a fourth-generation cephalosporin, in clinical microbiology laboratories.

HR810 is a new, very broad-spectrum cephalosporin with significant activity against members of the family Enterobacteriaceae, pseudomonads, gram-positive cocci, and anaerobes that is generally greater than the third-generation cephalosporins (99.6% of 4,128 clinical facultative enteric isolates were inhibited by less than or equal to 8.0 micrograms of HR810 per ml). Tests and statistical methods to establish in vitro antimicrobial susceptibility test criteria favor tentative breakpoints of greater than or equal to 18 mm (less than or equal to 8.0 micrograms/ml) as susceptible and less than or equal to 14 mm (greater than or equal to 32 micrograms/ml) as resistant. This provides a 93.7 to 98.3% absolute interpretive accuracy. Several preliminary ranges for zone sizes obtained with quality control organisms are proposed for the 30-micrograms HR810 disk diffusion test used during the clinical trials.

Bacteria↗

Synthesis, characterization and antimicrobial activity of Fe(II), Zn(II), Cd(II) and Hg(II) complexes with 2,6-bis(benzimidazol-2-yl) pyridine ligand.

2,6-Bis(benzimidazol-2-yl)pyridine (L) ligand and complexes [M(L)Cl(2)] and [Fe(L)(2)](ClO(4))(2) (M=Zn, Cd, Hg) have been synthesized. The geometries of the [M(L)Cl(2)] complexes were derived from theoretical calculation in DGauss/DFT level (DZVP basis set) on CACHE. The central M(II) ion is penta-coordinated and surrounded by N(3)Cl(2) environment, adopting a distorted trigonal bipyramidal geometry. The ligand is tridentate, via three nitrogen atoms to metal centre and two chloride ions lie on each side of the distorted benzimidazole ring. In the [Fe(L)(2)](ClO(4))(2) complex, the central Fe(II) ion is surrounded by two (3N) units, adopting a octahedral geometry. The elemental analysis, molecular conductivity, FT-Raman, FT-IR (mid-, far-IR), (1)H, and (13)C NMR were reported. The antimicrobial activities of the free ligand, its hydrochloride salt, and the complexes were evaluated using the disk diffusion method in dimethyl sulfoxide (DMSO) as well as the minimal inhibitory concentration (MIC) dilution method, against 10 bacteria and the results compared with that for gentamycin. Antifungal activities were reported for Candida albicans, Kluyveromyces fragilis, Rhodotorula rubra, Debaryomyces hansenii, Hanseniaspora guilliermondii, and the results were referenced against nystatin, ketaconazole, and clotrimazole antifungal agents. In most cases, the compounds tested showed broad-spectrum (Gram positive and Gram negative bacteria) activities that were either more effective than or as potent as the references. The binding of two most biologically effective compounds of zinc and mercury to calf thymus DNA has also been investigated by absorption spectra.

Anti-Bacterial Agents↗

"All-in-one-plate" E-test and disk diffusion susceptibility co-testing for multiresistant Acinetobacter baumannii.

Multiresistant Acinetobacter baumannii requires ancillary manual susceptibility testing with the E-test and disk diffusion when tested with the VITEK-2 system (bioMérieux, Marcy l'Etoile, France). In the study presented here, the E-test and disk diffusion were combined in a single plate, and the method was verified by comparing categorical agreement of combined and control plates. There were no very major, major or minor errors, and similar results were obtained for all ten representative bacterial strains used as controls. Co-testing is thus feasible, accurate and reproducible, and it merits evaluation with other bacterial species.

Acinetobacter baumannii↗

Detection of heterogeneous, intermediate-vancomycin-resistant Staphylococcus aureus (hVISA) using low-concentration vancomycin disks.

Heterogeneous, intermediate-vancomycin-resistant Staphylococcus aureus (hVISA) represents a threat of an incurable infection since the first report in 1997. The method used to detect hVISA isolates is a population analysis profile (PAP); however, it is impractical for routine laboratory analysis. We therefore tested a simple, reliable and inexpensive method for the detection of hVISA. Eighteen isolates of hVISA and 22 of vancomycin-sensitive S. aureus (VSSA) were included. The organisms were tested by the disk diffusion method, using 15-microg vancomycin disks on four different media: Mueller-Hinton agar (MHA), MHA plus 2% NaCI (MHAS), Brain Heart Infusion agar (BHA), and BHA plus 2% NaCl (BHAS). In addition, two different inoculum sizes, bacterial suspensions adjusted to 0.5 and 2.0 McFarland, were tested. The inhibition zone was read independently by three medical technologists after incubation at 37 degrees C for 24 and 48 hours. The use of MHAS with an inoculum size of 2.0 McFarland and 48-hour incubation period yielded the highest sensitivity (94.4%), specificity (81.8%), positive predictive value (80.9%), and negative predictive value (94.7%). The disk diffusion test with 15-microg vancomycin disk is simple and may be used as a screening method for the detection of hVISA.

Culture Media↗

In vitro diffusion bed, 3-day repeat challenge 'capacity' test for antimicrobial wound dressings.

The aim of this study was to develop an in vitro wound infection model that allows the comparison of the bacterial kill rate of antimicrobial wound dressings over the course of 3 days, with renewed microbial challenges each day, under realistic wound-like conditions. A test bed model of a moderately exuding wound was constructed from a hydrogel containing releasable foetal calf serum (FCS), and cellulose discs dosed with test microbes (Methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa) suspended in 50% FCS applied at the interface between the test dressing and the hydrogel test bed. Freshly prepared discs were used to challenge the same dressing over a 23-hour period for a course of 3 days. Different test dressings produced differing kill rates, allowing quantitative comparison of both their immediate activity and their capacity to continue working over 3 days, within a fluid-donating system similar to the situation in vivo. The reported method has significant advantages over established test procedures since it enables the researcher to assess the antimicrobial capacity of wound dressings to continue working under conditions that match those encountered in wounds. These key conditions are those that would be expected to impede the action of the dressing and protect the infecting organisms.

Anti-Infective Agents↗

[Disk diffusion method for fluconazole susceptibility testing of Candida spp. isolates].

In order to standardize and evaluate a disk diffusion method with visual reading to detect in vitro fluconazole susceptibility of yeast, 1193 clinical isolates were tested. These included 584 Candida albicans, 196 Candida parapsilosis, 200 Candida tropicalis, 113 Candida glabrata, 50 Candida krusei and 50 Candida spp. and other opportunistic yeasts. The disks were manufactured in the INEI-ANLIS "Dr. Carlos G. Malbrán. The disk diffusion method results were compared to MIC results obtained by the reference CLSI M27-A2 broth microdilution method modified by EUCAST. The interpretative breakpoints for in vitro susceptibility testing of fluconazole were established at: zone diameter > or =16 mm for MIC < or =8 microg/ml (susceptible isolates), between 9 and 15 mm for MIC = 16-32 microg/ml (susceptible dose-dependent isolates), and < or =8 mm for MIC > or =64 microg/ml (resistant isolates). Overall agreement between the two methods was 94.7%, with 0.2% very major errors, and 0.3% major errors. Inter - and intralaboratory agreement was good. The disk diffusion method for drug susceptibility testing of Candida spp. isolates is inexpensive, reliable and reproducible. However, when the inhibition zone diameter is < or =15 mm, it is advisable to test the isolate by the reference microdilution method.

Antifungal Agents↗

Urinary bactericidal activity of oral antibiotics against common urinary tract pathogens in an ex vivo model.

BACKGROUND: In this investigation, the urine samples obtained in a single oral-dose pharmacokinetic study were examined for their bactericidal activity against a range of relevant urinary tract pathogens. METHODS: Six healthy volunteers received a single oral dose of ten oral antibiotics available in Croatia. Urine samples were taken every 2 h during the whole dosing interval of the particular antibiotic. The urinary bactericidal activity was tested by determination of urinary bactericidal titers. RESULTS: All antibiotics showed a significant urinary bactericidal activity against non-extended spectrum beta-lactamase Escherichia coli and Proteus mirabilis. Fluoroquinolones displayed high and persisting levels of urinary bactericidal activity against all gram-negative bacteria and Staphylococcus saprophyticus. CONCLUSIONS: Average urinary bactericidal activity can be predicted from in vitro susceptibility testing, but we expect that there will be patients with a low level of urinary bactericidal activity.

Acetamides↗

[Drug resistance of phage types of Salmonella typhimurium (author's transl)].

Antimicrobial susceptibility testing (disk diffusion test) and phage typing (method according to Anderson, performed by Prof. Brandis, Bonn) of 703 strains of Salmonella typhimurium from humans, isolated in Essen and surrounding during 1972 to 1975, was performed to dertermine whether characteristic patterns of drug resistance were associated with a single phage type or not. The most frequently isolated phage types are phage type 17 (12.2%), 12 (10.1%), 49 (8.1%), 15a (5.7%), 2 (3,3%) and the untypable phage type (46.7%). Resistance to tetracycline (45%) was most common, followed by resistance to sulfanilamide (19%), ampicillin (9%), and chloramphenicol (7%). Resistance to other antimicrobial agents (trimethoprim/sulfamethoxazol, gentamicin, colistin) was quite rare. Antibiograms of different phage types were found to be different, not only as far as the ratio of sensitive to resistant strains is concerned, but also for the ratio single-resistant to multiple-resistant strains. These differences were found to be statistically significant (chi-square-test, table 2). Comparison of antimicrobial resistance to tetracycline showed, that the portion of resistant strains was about 70% for the untypable phage types, 40% for phage type 17 and 7% for phage type 12. Among isolates of the phage type 49 multiple resistance was most common. The combination of resistance determinants is specified in figure 3. A possible interrelation between resistance pattern and the presence of R-plasmids in isolated strains is discussed.

Anti-Bacterial Agents↗

Molecular characterization of pESI-like megaplasmids in Salmonella Infantis from poultry in Lebanon.

UNLABELLED: Salmonella enterica serovar Infantis has emerged as a globally disseminated multidrug-resistant (MDR) pathogen, largely driven by the spread of the plasmid of emerging Salmonella Infantis (pESI)-like megaplasmid. In our study, we investigated the prevalence, antimicrobial resistance (AMR) phenotypes, and genomic features of S. Infantis isolates collected from poultry farms in Lebanon. A total of 72 isolates were recovered during a nationwide surveillance effort, among which 67 (93%) were MDR based on antimicrobial susceptibility testing (disk diffusion and broth microdilution) results, including resistance to critically important agents such as quinolones, and highly important classes such as tetracyclines and sulfonamides. Whole-genome sequencing was performed on 19 isolates selected through a stratified approach to encompass all identified AMR phenotypes; this analysis revealed a conserved pESI-like backbone together with MDR-associated determinants, including sul1, tet(A), and aadA. Plasmid marker analysis confirmed the presence of pESI in the majority of isolates, with plasmid-associated genes (ardA and trbA) and replicon markers (IncP and IncFIB(pN55391)) among the most prevalent. Comparative plasmid alignments with representative pESI sequences from Italy, Turkey, and the United States revealed strong conservation of the backbone alongside regional variation in AMR gene content. These findings highlight the role of poultry production systems in Lebanon as reservoirs for pESI-like megaplasmids and MDR S. Infantis, underscoring the zoonotic and public health risks posed at the human-animal-environment interface. Strengthened surveillance, antimicrobial stewardship, and biosecurity interventions are urgently needed to mitigate the spread of MDR S. Infantis within agriculture and beyond. IMPORTANCE: The emergence of plasmid of emerging Salmonella Infantis (pESI)-like megaplasmids has transformed Salmonella Infantis into a globally distributed multidrug-resistant (MDR) clone with the capacity to persist in livestock and disseminate resistance genes across ecological boundaries. Our study provides the first genomic characterization of pESI-positive S. Infantis from poultry farms in Lebanon, a region with high antimicrobial usage and limited stewardship frameworks. By integrating phenotypic susceptibility testing and whole-genome sequencing, we demonstrate that Lebanese isolates harbor conserved pESI-like backbone markers together with antimicrobial resistance determinants, aligning them with internationally circulating lineages. Comparative analysis with isolates from Italy, Turkey, and the United States highlights both the evolutionary stability and geographic diversity of pESI. These findings emphasize the urgent need for integrated surveillance and stewardship strategies to curb the spread of MDR S. Infantis and reduce the zoonotic risk at the human-animal-environment interface.

Animals↗

Use of boronic acid disk methods to detect the combined expression of plasmid-mediated AmpC beta-lactamases and extended-spectrum beta-lactamases in clinical isolates of Klebsiella spp., Salmonella spp., and Proteus mirabilis.

A study using boronic acid (BA), an AmpC enzyme inhibitor, was designed to detect the combined expression of plasmid-mediated AmpC beta-lactamases (pAmpCs) and extended-spectrum beta-lactamases (ESBLs) in bacterial isolates naturally lacking chromosomal ampC genes. A total of 122 Klebsiella spp., Salmonella spp., and Proteus mirabilis isolates producing or nonproducing pAmpCs and/or ESBLs were analyzed. Detection of genes encoding ESBLs and AmpCs was confirmed by polymerase chain reaction (PCR) followed by sequencing of PCR products. A > or = 5-mm increase in zone diameter for i) cefoxitin (FOX) and/or cefotetan (CTT) containing BA versus FOX and/or CTT alone was considered positive for AmpC; ii) ceftazidime (CAZ)-clavulanate (CA) and/or cefotaxime (CTX)-CA tested in combination with BA versus CAZ and/or CTX containing BA was considered positive for ESBL. The disk tests of FOX and/or CTT alone and with BA detected 98.4% of organisms producing pAmpCs. All of the 21 pAmpC and ESBL coproducers were accurately detected ESBL by the disk tests of CTX-CA and/or CAZ-CA containing BA and CTX and/or CAZ containing BA. In conclusion, The BA disk test using Clinical and Laboratory Standards Institute methodology is simple and very efficient method to detect pAmpC and ESBL in organisms naturally lacking chromosomal AmpC enzymes. In particular, the method accurately detects the isolates that harbor both AmpCs and ESBLs.

Bacterial Proteins↗

In vitro screening of five local medicinal plants for antibacterial activity using disc diffusion method.

Medicinal plants have many traditional claims including the treatment of ailments of infectious origin. In the evaluation of traditional claims, scientific research is important. The objective of the study was to determine the presence of antibacterial activity in the crude extracts of some of the commonly used medicinal plants in Malaysia, Andrographis paniculata, Vitex negundo, Morinda citrifolia, Piper sarmentosum, and Centella asiatica. In this preliminary investigation, the leaves were used and the crude extracts were subjected to screening against five strains of bacteria species, Methicillin Resistant Staphylococcus aureus (MRSA), Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa and Escherichia coli, using standard protocol of Disc Diffusion Method (DDM). The antibacterial activities were assessed by the presence or absence of inhibition zones and MIC values. M. citrifolia, P. sarmentosum and C. asiatica methanol extract and A. paniculata (water extract) have potential antibacterial activities to both gram positive S. aureus and Methicillin Resistant S. aureus (MRSA). None of the five plant extracts tested showed antibacterial activities to gram negative E. coli and K. pneumoniae, except for A. paniculata and P. sarmentosum which showed activity towards P. aeruginosa. A. paniculata being the most potent at MIC of 2 g/disc. This finding forms a basis for further studies on screening of local medicinal plant extracts for antibacteria properties.

Anti-Bacterial Agents↗

Comparison of antimicrobial susceptibility testing of Campylobacter spp. by the agar dilution and the agar disk diffusion methods.

The correlation and the level of agreement between the standardized agar dilution and the agar disk diffusion methods for antimicrobial susceptibility testing of Campylobacter were investigated. A high-level agreement between the two methods was evident for aminoglycosides and fluoroquinolones, while a low-level agreement was observed for other antibiotics.

Agar↗

Accelerated detection of extended-spectrum beta-lactamases in clinical isolates of Enterobacteriaceae.

A prospective study is carried out to evaluate the performance of a protocol for the accelerated detection of extended-spectrum beta-lactamases (ESBLs) in clinical isolates of Escherichia coli, Klebsiella pneumoniae and other Gram-negative bacteria. A modified double-disc test (MDDT) is incorporated in a Gram-negative template for routine susceptibility testing. The MDDT identified accurately ESBLs in all isolates subsequently confirmed as ESBL-producers by the standard Clinical Laboratory Standards Institute (CLSI) combined disc method. Of 1213 isolates tested, 98 (8%) were positive for ESBLs by MDDT and 95 (7.8%) were positive by the CLSI method. ESBLs were detected in 48 (7.8%) E. coli, 21 (8%) K. pneumoniae, 12 (5.8%) Proteus mirabilis, 13 (18.8%) Providencia stuartii and four (6.8%) Enterobacter cloacae isolates. Time required for ESBL detection by the MDDT method was one day. The protocol described provides a simple, rapid and low-cost method for early detection of ESBLs in Gram-negative bacteria.

Anti-Bacterial Agents↗

Epidemiologic cutoff values for antimicrobial agents against Aeromonas salmonicida isolates determined by frequency distributions of minimal inhibitory concentration and diameter of zone of inhibition data.

OBJECTIVE: To develop epidemiologic cutoff values by use of frequency distributions for susceptibility to 4 antimicrobial agents when tested against a representative population of a major aquaculture pathogen, Aeromonas salmonicida. SAMPLE POPULATION: 217 typical and atypical A salmonicida isolates obtained from 20 states and 12 countries. PROCEDURES: Species identification of A salmonicida isolates was confirmed by detection of specific nucleotide sequences by use of a PCR assay. Minimal inhibitory concentration (MIC) and diameter of the zone of inhibition for oxytetracycline, ormetoprim-sulfadimethoxine, oxolinic acid, and florfenicol were determined for each isolate in accordance with standardized antimicrobial susceptibility testing methods that have been approved by the Clinical and Laboratory Standards Institute for bacterial isolates from aquatic animals. Susceptibility data were tabulated in a scattergram and analyzed by use of error rate bounding. RESULTS: Susceptibility tests for oxytetracycline, ormetoprim-sulfadimethoxine, and oxolinic acid revealed 2 distinct populations of bacteria. Isolates tested against florfenicol clustered into a single population. Oxolinic acid susceptibility data revealed higher MICs in the non-United States A salmonicida isolates. Slow-growing (atypical) A salmonicida isolates were generally more susceptible than typical isolates for all antimicrobials, except oxolinic acid. CONCLUSIONS AND CLINICAL RELEVANCE: Use of frequency distributions of susceptibility results to develop epidemiologic cutoff values appears to be applicable to aquatic isolates. Frequency distributions of susceptibility results for A salmonicida revealed clear divisions between isolate susceptibilities. This type of data, considered in conjunction with pharmacokinetic and efficacy data, may be useful for developing clinical breakpoints for use in aquaculture.

Aeromonas salmonicida↗

[Evaluation and utility of the E-test and Neo-Sensitabs methods in studying fluconazole yeast susceptibility].

Standardized broth dilution methods are cumbersome for routine use in a clinical laboratory to study antifungal yeast susceptibility. Recently, the CLSI has standardized a disk diffusion method faster and more suitable to study fluconazole and voriconazole susceptibility. The objectives of the present study were to determine: a) the suitability of the Neo-Sensitabs tablets to study fluconazole susceptibility; b) whether Mueller-Hinton agar with methylene blue (MHAG-AM) could be used in the E-test method; and c) the interaction of the methylene blue with RPMI medium. A total of 84 blood stream yeast isolates were used (25 C. albicans, 7 C. parapsilosis, 10 C. tropicalis, 12 C. glabrata, 7 C. krusei, 4 C. lusitaniae and 19 C. neoformans). The methylene blue makes sharper inhibition zones both in MHAG-AM and RPMI media. With fluconazole Neo-Sensitabs tablets, the lowest percentage of very major errors was found in MHAG-AM and the greatest in RPMIG. In both diffusion methods and culture media, the very major errors were found in C. albicans, C. tropicalis (only with Neo-Sensitabs) and C. glabrata. The percentage of fluconazole-resistant strains was lower in the media that contained glucose (2%). Neo-Sensitabs tablets are a reliable alternative to the dilution methods to detect fluconazole susceptibility. In the case of resistance, more studies are required; nevertheless, inhibition zone > or =17 mm should be applied to define fluconazole resistance.

Antifungal Agents↗

Silver as biocides in burn and wound dressings and bacterial resistance to silver compounds.

Silver products have been used for thousands of years for their beneficial effects, often for hygiene and in more recent years as antimicrobials on wounds from burns, trauma, and diabetic ulcers. Silver sulfadiazine creams (Silvazine and Flamazine) are topical ointments that are marketed globally. In recent years, a range of wound dressings with slow-release Ag compounds have been introduced, including Acticoat, Actisorb Silver, Silverlon, and others. While these are generally accepted as useful for control of bacterial infections (and also against fungi and viruses), key issues remain, including importantly the relative efficacy of different silver products for wound and burn uses and the existence of microbes that are resistant to Ag+. These are beneficial products needing further study, although each has drawbacks. The genes (and proteins) involved in bacterial resistance to Ag have been defined and studied in recent years.

Bacteria↗

Prevalence, molecular characterization and antimicrobial resistance of thermophilic campylobacter isolates from cattle, hens, broilers and broiler meat in south-eastern Italy.

Eleven cattle farms, 8 layer farms, 7 broiler farms and 30 broiler meat samples were investigated in south-eastern Italy throughout 2003 to evaluate the prevalence, the molecular type and antimicrobial resistance of thermophilic Campylobacters. A total of 398 samples were analysed. One Campylobacter isolate for each positive faecal swab and three isolates per positive broiler meat sample were selected for further analysis. Multiplex PCR was performed for species-level identification and PCR-RFLP of the flagellin A gene for genotyping. Resistance to 14 antimicrobials was studied in 188 Campylobacter isolates. Prevalence of campylobacters was high both on farms (100%) and in food samples (73%). On 4/11 cattle farms and on 10/15 poultry farms more than one species was isolated. The presence of more than one genotype was found on 8/11 cattle farms, on 10/15 poultry farms and in 8/22 Campylobacter-positive food samples. High rates of resistance to quinolone were observed: 9/31 (29%) C. jejuni bovine isolates, 4/22 (18%) C. jejuni poultry isolates, and 14/26 (54%) C. coli poultry isolates. Resistance to sulphamethoxazole-trimethoprim was also observed frequently: 18/26 (69%) of the avian C. coli strains, 25/31 (80%) of the C. jejuni strains isolated from poultry and 15/22 (68%) of those isolated from cattle were resistant. There was a significant difference between the rate of resistance to macrolides of C. coli and C. jejuni isolated in poultry, which amounted to 23% and 3%, respectively. This study provided data on the prevalence and antimicrobial resistance of thermophilic campylobacters in south-eastern Italy and confirmed that flaA-typing is an efficient tool to study the epidemiology of Campylobacter strains in short-term investigations.

Animals↗

Antimicrobial susceptibility of clinical isolates of Haemophilus influenzae to ampicillin-sulbactam.

A total of 1092 clinical isolates of Haemophilus influenzae (306 type b; 786 non-type-b), from five medical centers were obtained during 1987 and 1988. Disk diffusion antimicrobial susceptibilities were obtained for all isolates, and broth microdilution susceptibilities were obtained for 502 isolates. Beta-lactamase was produced by 34.3% of type-b and 22.1% of non-type-b isolates, with some geographic variations. Using disk diffusion antimicrobial susceptibility testing, all isolates were susceptible to ampicillin-sulbactam, ceftriaxone, cefuroxime, and rifampin; two isolates were resistant to chloramphenicol. Whether tested using a fixed ratio of ampicillin to sulbactam of 2:1 or a fixed concentration of sulbactam, the ampicillin-sulbactam combination demonstrated good activity against clinical isolates of H. influenzae. Only 8 of the 1092 isolates did not produce beta-lactamase but demonstrated MICs of greater than or equal to 2 micrograms/ml for ampicillin.

Ampicillin↗