Use Of Chromogenic Media Research Articles

Zoonotic Staphylococcus spp. among domestic animals in Ukraine: antibiotic resistance and diagnostic approaches

Staphylococcus pseudintermedius and S. aureus are common infectious agents with zoonotic potential, capable of acquiring antibiotic resistance and causing hospital-acquired infections. This study aimed to assess the prevalence of S. pseudintermedius among dogs in Ukraine and explore the possibility of using chromogenic media for accurate identification and differentiation of S. aureus and S. pseudintermedius. The data obtained indicate a higher prevalence of nasal carriage (44.8%) of Staphylococcus spp. in healthy dogs compared to ear carriage (28.5%). Among the isolated strains in nasal swabs, S. pseudintermedius was most often detected (24.1%), S. aureus was detected less often (5.2%). Among the samples taken from sick dogs, 36.5% were found to be Staphylococcus spp., of which 20.5% contained S. pseudintermedius and 4.5% S. aureus. Among the studied cats, 36.8% were found to contain Staphylococcus spp., S. pseudintermedius – 10.5%, and S. aureus – 5.3%. Antibiotic resistance profiles were different: S. pseudintermedius was resistant to the combination of trimethoprim-sulfamethoxazole, as well as to penicillin and erythromycin, while S. aureus was resistant to penicillin. It should be noted that only one coagulase-positive S. aureus isolate obtained from healthy animals was resistant to oxacillin. The use of chromogenic media demonstrated the possibility of successful differentiation of S. aureus and S. pseudintermedius based on differences in culture properties. In addition, various combinations of microbiological tests were evaluated, resulting in the development of two schemes that included specific media and additional reactions to improve identification accuracy. The combination of CHROMagar™ Orientation with the coagulase detection reaction demonstrated higher sensitivity (100%) for the detection of S. pseudintermedius compared to the combination of mannitol salt agar (MSA) with the coagulase detection reaction (75%). In addition, the PCR reaction was valuable for the definitive identification of coagulase-positive staphylococci (CoPS). These comprehensive results shed light on the prevalence, species distribution, antibiotic resistance patterns, and diagnostic approaches for S. aureus in dogs and cats in Ukraine, emphasizing the importance of developing reliable surveillance and control strategies.

Usefulness of Chromogenic Media with Fluconazole Supplementation for Presumptive Identification of Candida auris.

Introduction:Candida auris is a major threat to public health. Rapid detection is essential for early treatment and transmission control. The use of chromogenic media allows the presumptive identification of this new species. The aim of this study is to describe the morphological characteristics of C. auris colonies on three commercial chromogenic media. Methods: Nineteen C. auris isolates from different countries/clades and 18 isolates of other species were cultivated in CHROMagarTM Candida Plus, HiCromeTM Candida, CHROMagar-Candida, and fluconazole-supplemented (32 mg/L) CHROMagar-Candida media. Results: On CHROMagarTM Candida Plus and HiCromeTM Candida, C. auris isolates from Colombia, Venezuela, India, Korea, and Japan displayed blue-shaded colonies, while isolates from Spain and Germany exhibited light pink shades with a bluish halo. All isolates showed white to pink colonies on CHROMagar-Candida. On CHROMagar Candida supplemented with fluconazole, whilst C. auris, C. glabrata, or C. krusei showed a similar pink color at 48 h incubation, phenotypic differentiation was possible by the rough, paraffin-like texture or the intense purple color acquired by C. krusei and C. glabrata, respectively. Moreover, in this medium, the presence of C. auris in combination with other species of similar color was not limiting for its early identification, due to this medium selecting only strains resistant to this antifungal. Conclusions: The use of chromogenic media such as CHROMagarTM Candida Plus facilitates a presumptive identification of C. auris. However, this identification can be difficult in the presence of mixed cultures. In these cases, the use of CHROMagarTM Candida medium with 32 mg/L fluconazole offers better performance for the identification of C. auris by inhibiting fluconazole-susceptible strains and selecting rare or high fluconazole MIC (>32 mg/L) isolates.

Engineered colorimetric detection of Staphylococcus aureus extracellular proteases

This work describes the engineering of a feasible, sensitive, and specific colorimetric assay for the detection of Staphylococcus aureus (S. aureus) extracellular protease production, using a tailored agar-based readout platform. Firstly, S. aureus protease production was evaluated using different culture media. Maximum proteolytic activity was achieved after 24 h of incubation in brain heart infusion (BHI) broth at 37 °C, as confirmed using azo-casein and Sigma’s non-specific protease activity assays. Secondly, to enhance proteolysis detection, novel agar-based platform readouts for S. aureus proteases were developed. Clear proteolytic zones were observed after 48 h of incubation at 37 °C, using an agar-agar platform supplemented with 3% skim milk as a non-specific protease substrate. Thirdly, the colorimetric visualization of S. aureus proteolytic zones was evaluated using three different techniques, namely, the use of chromogenic media, dye flooding over the platform, and protease staining prior to testing. Colorimetric sensing of proteolysis was achieved by applying a Bromocresol Purple-colored protease solution on a skim milk plate count agar–PEG 4000 readout platform. Color change (yellow to burgundy) indicated a positive readout after 15 min of incubation. The lowest limit of S. aureus detection was 102 CFU mL−1. Moreover, direct detection of S. aureus BHI culture was achieved by sensing the pH change because of protease production, using bromothymol blue dye. A light-green color represented a positive readout. A direct relationship between S. aureus culture concentration and color change was observed, with a detection limit as low as 103 CFU mL−1. Examination of blind samples of Escherichia coli and S. aureus showed the potential of this assay to specifically detect S. aureus in situ. Therefore, the developed approach is anticipated to help detect S. aureus for biomedical applications.

An assay for determining minimal concentrations of antibiotics that drive horizontal transfer of resistance

Ability to understand the factors driving horizontal transfer of antibiotic resistance from unknown, harmless bacteria to pathogens is crucial in order to tackle the growing resistance problem. However, current methods to measure effects of stressors on horizontal gene transfer have limitations and often fall short, as the estimated endpoints can be a mix of both the number of transfer events and clonal growth of transconjugants. Our aim was therefore to achieve a proper strategy for assessing the minimal concentration of a stressor (exemplified by tetracycline) that drives horizontal transfer of antibiotic resistance from a complex community to a model pathogen. Conditions were optimized to improve a culture-based approach using the bacterial community of treated sewage effluent as donor, and fluorescent, traceable Escherichia coli as recipient. Reduced level of background resistance, differentiation of isolates as well as decreased risk for measuring effects of selection were achieved through the use of chromogenic medium, optimization of conjugation time as well as applying a different antibiotic for isolation of transconjugants than the one tested for its ability to drive transfer. Using this assay, we showed that a very low concentration of tetracycline, 10μg/L i.e. 150 times below the minimal inhibitory concentration of the recipient, promoted horizontal transfer of multiple antibiotic-resistance determinants. Higher concentrations favoured selection of a tetracycline-resistance phenotype along with a decline in the number of detectable transfer events. The described method can be used to evaluate different environmental conditions and factors that trigger horizontal dissemination of mobile resistance elements, eventually resulting in the formation of drug-resistant pathogens.

Rapid identification of Aeromonas species in stool samples with chromogenic media and matrix-assisted laser desorption ionization-time of flight mass spectrometry: an institutional experience

To evaluate the routine use of chromogenic media together with matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF) in gastrointestinal infections caused by Aeromonas spp. as rapid, practical, cost-effective and reliable methods. Materials and methods: Between August 2007 and March 2012, a total of 13,194 stool specimens of patients were included in the study. The stool samples and the reference strains were inoculated onto Cefsulodin-Irgasan-Novobiocin agar, sheep blood agar, eosin-methylene blue agar, and Hektoen enteric agar media together with CHROMagar Salmonella Plus. All the Salmonella and Aeromonas suspected colonies were identified with an automated system (Phoenix) and MALDI-TOF. Results: Some of the pink colonies resembling Salmonella were identified as Aeromonas spp. without any discordance (100%) between the systems. A total of 86 Aeromonas strains were identified: 30 A. caviae, 27 A. hydrophila, 16 A. veronii, 11 A. sobria, and 2 A. salmonicida. When analyzed macroscopically, those Aeromonas species had prominent colony appearance differences from classically detected Salmonella on CHROMagar Salmonella Plus media. Conclusion: A combination of CHROMagar Salmonella Plus and MALDI-TOF will help to detect Aeromonas species in 24 h in a cost-effective, practical, and reliable manner.

Les fongémies mixtes. À propos de quatre cas tunisiens

Fungemia is classically caused by a single species and the detection of more than one species in blood samples is uncommon. We report four cases of mixed fungemia (MF) diagnosed in the parasitology-mycology laboratory of Farhat-Hached hospital in Sousse, Tunisia. The MF episodes occurred in two neonates and two adults suffering from acute myeloid leukemia. Two fungal species were detected concomitantly within the same blood culture in all cases. Species combination was detected by the subculture of the blood culture on Candida ID(®) chromogenic medium in three cases and on Sabouraud agar in one case. Predisposing factors were: indwelling catheters (4/4), broad-spectrum antibiotics (3/4), neutropenia (2/4), exclusive parenteral nutrition (2/4) and Candida colonization (1/4). Patients presented febrile sepsis with no response to broad-spectrum antibiotic therapy in all cases. Outcome under antifungal treatment was favorable in two cases and the two other patients died. MF appears similar to the more common monomicrobial fungemia. The use of chromogenic media in routine can improve the detection of MF episodes allowing appropriate antifungal therapy.

An Implementation Strategy for the Use of Chromogenic Media in the Rapid, Presumptive Identification of Candida Species

The purpose of this study was to devise an algorithm utilizing chromogenic media for the detection and presumptive identification of Candida species in a clinical microbiology laboratory. In order to select one chromogenic medium, a limited evaluation of 3 commercially available yeast chromogenic media: BBL CHROMagar Candida, Bio- Rad CandiSelect 4, and Oxoid OCCA, was conducted with 12 C. albicans, 8 C. glabrata, 10 C. tropicalis, and 9 C. krusei. The presumptive species identification after 48 hours of incubation on BBL and Bio-Rad media were comparable, with more than 80% of the Candida species correctly identified, and both were superior to Oxoid. An algorithm based on use of chromogenic media for the rapid presumptive identification of C. albicans, C. tropicalis, C. krusei, and C. glabrata was developed for routine use in the microbiology laboratory. A presumptive identification is available within 24 to 48 hours utilizing this algorithm.

Use of chromogenic medium in the isolation of yeasts from clinical specimens

Over a 1 year period 3296 specimens submitted for fungal culture were plated onto routine mycological media (RM) and CHROMagar Candida (CaC) to evaluate the capability of CaC to improve on RM. With RM, cultures producing single yeast isolates were identified from 802 specimens. CaC produced similar results, with 76 % agreement. Of 761 specimens that yielded a single Candida species by RM, 615 (81 %) produced one or more yeast isolates using CaC. Of concern, 132 negative CaC cultures corresponded to specimens that yielded C. albicans alone on RM. When yeasts were recovered, CaC correctly identified 98 % of C. albicans, 93 % of Candida tropicalis, 96 % of Candida glabrata and 100 % of Candida krusei based on typical colours. CaC did potentially improve on RM by detecting yeasts in 91 specimens that yielded none by routine methods. CaC was noted to recover more yeast isolates than RM when mixed cultures were detected. Overall, the role of CaC in improving RM appears limited.

Evaluation of ChromocultR enterococci agar for the isolation and selective enumeration of Enterococcus spp. in broilers

To investigate the productivity and specificity of a new chromogenic enterococci selective medium (Chromocult enterococci agar) recently developed by Merck. The study was carried out comparing Chromocult enterococci agar with MRS agar (Merck), a basal lactic acid bacteria medium in current use. A total of 216 faecal samples from poultry were collected and enterococci populations were counted. Likewise, 100 randomly selected strains were identified for each medium. The differences found between the two media were analysed and discussed. A good sensitivity of 98% was obtained for Chromocult agar and all false-positive isolates obtained were identified as Leuconostoc spp. However significant differences (P<0.01) were obtained between the enterococci species isolation rates identified from these two media, suggesting the poor growth of some species in Chromocult enterococci agar. Viable counts of Enterococcus spp. obtained with MRS agar were significantly higher than those obtained with Chromocult enterococci agar. The use of chromogenic media for microbiological analysis is increasing. Independent studies are important to evaluate newly developed chromogenic media.