Antimicrobial resistance profile of extended-spectrum beta-lactamases, adenosine-monophosphate-cyclic, and carbapenemase-producing Gram-negative bacteria isolated from domestic animals

Background and Aim: The production of beta-lactamase enzymes, such as extended-spectrum beta-lactamase (ESBL), adenosine-monophosphate-cyclic (AmpC), and Klebsiella pneumoniae carbapenemase (KPC), is one of the most important mechanisms of bacterial resistance to antimicrobials. Gram-negative bacteria show significant resistance due to various intrinsic and acquired factors. These intrinsic factors include low permeability of the outer membrane, various efflux systems, and the production of beta-lactamases, while acquired factors include chromosomal mutation and acquisition of resistance genes by horizontal transfer. Mobile elements such as plasmids, integrative conjugative elements, mobilizable islands, or transposable elements are involved in horizontal transfer. At present, the Gram-negative pathogens of most concern are Acinetobacter baumannii, Pseudomonas aeruginosa, and those belonging to the Enterobacteriaceae family (e.g., Escherichia coli, K. pneumoniae, and Proteus mirabilis). This study aimed to evaluate the profile of antimicrobial resistance and the production of the enzymes ESBL, AmpC, and KPC, in 21 gram-negative bacteria isolated from domestic animals treated at the University Veterinary Hospital (HVU) of the Federal University of Western Bahia (UFOB). Materials and Methods: The biological samples (21) were inoculated to brain heart infusion broth, blood agar, and MacConkey agar and incubated for 24-72 h at 37°C. Gram staining and identification through biochemical tests and matrix-associated laser desorption/ionization time-of-flight mass spectrometry were conducted. To evaluate the antimicrobial resistance profile, the disk diffusion method was used, and 25 antibiotics were employed. For the detection of ESBL, the disk approximation method was applied using chromogenic agar. The presence of KPC was observed using chromogenic agar and the Hodge test. For AmpC evaluation, the disk approximation method was used. Results: The most isolated agent was E. coli (66.66%, 14/21), followed by K. pneumoniae and P. mirabilis (both 14.29%, 3/21), and then Pasteurella spp. (4.76%, 1/21). The bacterial isolates showed high levels of resistance against clindamycin, penicillin, imipenem, polymyxin, cefoxitin, gentamycin, cefotaxime, ceftazidime, cephalothin, ceftriaxone, ciprofloxacin, trimethoprim/sulfamethoxazole, chloramphenicol, and tetracycline. The best effectiveness rates were observed for cefepime, streptomycin, amoxicillin-clavulanate, aztreonam, nalidixic acid, tobramycin, levofloxacin, amikacin, and meropenem. All biological isolates showed multiple resistance to at least three of the antibiotics tested (3/25), and some showed resistance to 24 of the antibiotics tested (24/25). Among the 21 pathogens analyzed, 8 were ESBL producers (38.09%); of these, 6 were identified as E. coli (28.57%), and 2 were identified as K. pneumoniae (9.52%). Two strains of K. pneumoniae produced both ESBL and KPC. None of the isolates were producers of AmpC. Conclusion: The results found in the present work raise concern about the level of antimicrobial resistance among pathogens isolated from domestic animals in Brazil. The results highlight the need for the development and implementation of anti-resistance strategies to avoid the dissemination of multiresistant pathogens, including the prudent use of antimicrobials and the implementation of bacterial culture, antimicrobial sensitivity, and phenotypic tests for the detection of beta-lactamase enzymes in bacteria isolated from animals.

790. Evaluation of an Enhanced CPE Screening Program in an Acute Care Hospital in South Korea

Background Carbapenemase-producing Enterobacteriaceae (CPE) poses a great challenge in infection control in healthcare settings. A screening and contact precautions are recommended to prevent the spread of CPE among patients. However, screening strategies differ among countries and healthcare facilities. Methods In September 2018, we launched a CPE screening program at a 660-bed hospital in South Korea, which targeted previously colonized patients, patients with history of admission < 1 month or transferred patients or ICU-admitted patients. Once patients were identified to have CPE, they were isolated in a single room. After a CPE outbreak in July-Aug 2019, the enhanced screening program was implemented, which included patients with additional risk factors (exposure to hospitals in the past 6 months, receipt of hemodialysis or invasive procedures or rehabilitation) combined with weekly screening in ICU-admitted patients. Screening methods changed from two consecutive rectal screening swabs with chromogenic agar to initial screening with Xpert-Carba-R PCR, followed by one or two consecutive tests with chromogenic agar. We compared the CPE incidence in screening and clinical cultures before and after the enhanced screening program introduction (Sep 2018-Nov 2020). Results A total of 14,318 (2,178 vs. 12,140) were screened among 49,980 admitted patients and screening compliance increased from 18.6% to 94.5%. The number of CPE detection increased from 44 to 154 cases and the proportion of CPE-positive screening per 1000 admissions increased 0.6 to 2.2. However, the number of clinical CPE cultures decreased from 11 to 3 (Figure). Among screened patients, time-to-positivity was markedly reduced by 1.9 days (2.96 vs. 1.02 days) during the post-period. Additional 70 patients were detected: 36 due to serial screening in the ICUs and 34 due to enhanced on-admission screening. Factors significantly associated with positive screening were previous exposure to hospital (OR 3.5; 95% CI 1.7-7.1) and receipt of hemodialysis (OR 4.3; 95%CI 1.9-9.2). CPE isolates and carbapenemase genes were diverse (Figure). Trends in CPE detection in screening and clinical samples (upper), and bacterial species with detected carbapenemase genes (lower). Conclusion The study results showed that the enhanced screening program enabled us to identify the previously undetected CPE colonized patients and to decrease clinical CPE cultures. Disclosures All Authors: No reported disclosures

How Accurate Are Veterinary Clinicians Employing Flexicult Vet for Identification and Antimicrobial Susceptibility Testing of Urinary Bacteria?

Antibiotics are frequently used for treating urinary tract infections (UTI) in dogs and cats. UTI often requires time-consuming and expensive antimicrobial susceptibility testing (AST). Alternatively, clinicians can employ Flexicult Vet, an affordable chromogenic agar with added antibiotics for in-clinic AST. We investigated how well veterinary microbiologists and clinicians, without any prior experience, employ Flexicult Vet for the identification and AST of the most common canine and feline urinary pathogenic bacteria. We prepared 47 monoculture plates containing 10 bacterial species. The test’s mean accuracy was 75.1% for bacteria identification (84.6% and 68.7% for microbiologists and clinicians, respectively) and 79.2% for AST (80.7% and 78.2%). All evaluators employed Flexicult Vet with the accuracies over 90% for the distinctively colored bacteria like Escherichia coli (red), Enterococcus faecalis (turquoise), and Proteus spp. (pale brown). However, the evaluators’ experience proved important in recognizing lightly colored bacteria like Staphylococcus pseudintermedius (accuracies of 82.6% and 40.3%). Misidentifications of E. faecium additionally worsened AST performance since bacterial intrinsic resistance could not be considered. Finally, only 33.3% (3/9) of methicillin-resistant S. pseudintermedius (MRSP) were correctly detected. To conclude, Flexicult Vet proved reliable for certain urinary pathogens. In contrast, light-colored bacteria (e.g., Staphylococcus), often misidentified, require a standard AST.

Role of Candida glabrata as nosocomial pathogen and its susceptibility to Fluconazole, Voriconazole, Caspofungin, Micafungin and Amphotericin B

Candida has different types that could cause bloodstream infections. A total number of 150 samples were collected from candidemia patients and examined. The Candida spp. Species isolated from blood samples were analysed. These were identified by culturing the species using different media, namely the chromogenic agar test. Then, the virulence factors of all samples were tested. The Candida glabrata isolates were tested with six commercial antifungal drugs. C. glabrata 67 (44.6%), C. albicans 34 (22.6%), C. krusei 18 (12%), C. tropicalis 17 (11.3%), and C. parasilosis 14 (9.3%). the production of phospholipase ranged between 0.63-0.99 mm. It was found that 96% of the species showed phospholipase activity in aerobic conditions. The protease activities of Candida spp. Isolates were experimentally tested by area of inhibition around the colonies, where 59.3% had the double (++) protease activity, 31.4% with (+) grade, and 9.3% had (–) grade or clear zone around the colony. The hemolytic capacity ranged from 0.69-0.89 in the optimum aerobic environments. Finally, 38.33% of the isolated Candida spp. were positive and 61.67% negative for biofilm formation. Out of the total positive Candida spp. for biofilm formation, 21.73% were strong biofilm producers, and 78.27% were weak. Minimum fungicidal concentration (MFC) of Fluconazole for C. glabrata isolates was not appropriate (NA) due to the occurrence of low inhibition tested for species. Micafungin exhibited the lowest fungicidal activity against C. glabrata ranging from 0.03 - 0.125, while Fluconazole showed the highest.

Neonatal Candidaemia in Tertiary Care Hospitals in Dhaka- A Comparison of Different Carbohydrate Assimilation Methods and Chromagar Technique for Speciation

Background: Nosocomial candidiasis are becoming increasingly important worldwide. Candida is a major causative agent of health care associated bloodstream infections, and lately non-albican Candida species are increasingly isolated from blood samples. Some of the Candida species have intrinsic and acquired resistance to the limited arsenal of antifungals; therefore early speciation is essential for the timely initiation of effective antifungal therapy. Methods: A hospital based cross-sectional study was conducted to evaluate the performance of different carbohydrate assimilation tests and commercially available HiCrome Candida Differential Media (CHROMagar) for the identification of Candida in the four tertiary level hospitals in Dhaka. Results: A total of 58 yeasts samples was included in this study. Non-albicans Candida accounted for 100% of the isolates of which C. tropicalis was the predominant species (81.03%) followed by C. parapsilosis (12.07%), C. auris (5.17%) and C. dubliniensis (1.72%). Swab auxanographic technique and microtitre plate based miniaturized CHO assimilation methods were equally effective in identification of Candida sp. in comparison to CHO impregnated YNB plate method (98.28% and 100% vs 89.66%). Conclusion: By using Chromogenic agar 75.86% yeasts were identified but it could not give the conclusive differentiating color between the species of C. parapsilosis and C. auris.

Comparison of rapid and conventional methods for investigating of mecA presence in Staphylococcus Species

Objectives: Taking the determination of mecA gene by polymerized chain reaction (PCR) method as a reference in determining methicillin resistance in Staphylococcus species, we aimed at comparing the reliability levels of disk diffusion, latex agglutination test and chromogenic agar use methods. Methods: This prospective study was conducted on 228 Staphylococcus strains isolated between January 2020 and December 2020 in Samsun Training and Research Hospital. Disk diffusion, latex agglutination and chromogen agar medium methods were applied along with the polymerized chain reaction (PCR) method. Results: The mecA gene was detected in 47 of the isolates (20.6%) by the PCR method, and these isolates were accepted as methicillin-resistant. When the PCR result was taken as a reference, the sensitivity of the disk diffusion method became 100%, and specificity became 45.9%; sensitivity of latex agglutination was determined as 80.9%, and specificity as 70.2%; sensitivity of chromogenic agar as 85.1% and its specificity was found to be 95%. Only in S. aureus isolates, the highest sensitivity and specificity rate (100% and 88%, respectively) belonged to chromogenic agar. Conclusion: Chromogenic agar provides more reliable data for S. aureus isolates, and the combined use of all three methods does not significantly increase reliability. doi: https://doi.org/10.12669/pjms.37.5.4274 How to cite this:Gorgun S, Isler H, Turgut MC. Comparison of rapid and conventional methods for investigating of mecA presence in Staphylococcus Species. Pak J Med Sci. 2021;37(5):---------. doi: https://doi.org/10.12669/pjms.37.5.4274 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Diagnostic Performance of Various Methodologies for Group B Streptococcus Screening in Pregnant Woman in China

Maternal vaginal/rectal colonization of group B streptococcus (GBS) is a main risk for neonatal invasive infection. Efficient determination of GBS colonization in pregnant women is crucial. This study aimed to investigate the prevalence of GBS carriage and evaluate the diagnostic performance of six methodologies for GBS screening conducted in China, including blood agar plate, liquid chromogenic medium, and loop-mediated isothermal amplification (LAMP) without pre-enrichment, chromogenic agar plate with pre-enrichment, and GBS antigen detection without and with pre-enrichment in comparison with the standard reference method (Lim broth-enriched subculture with plating on 5% sheep blood agar). Vaginal/rectal swabs were collected from 1,281 pregnant women at 35–37 weeks of gestation. Of them, 309 were taken in triplicate, one for Lim broth-enriched subculture, one for blood agar plate, and the third for GBS antigen detection (Reagent W); 177 were acquired in duplicate, one for Lim broth-enriched subculture and the other for GBS antigen detection (Reagent H); 502 were obtained in duplicate, one for Lim broth-enriched subculture and the other for liquid chromogenic medium; 158 were collected in duplicate, one for Lim broth-enriched subculture and the other for LAMP; and 135 were inoculated in Lim broth-enriched for GBS antigen detection (Reagent W) and subculture with chromogenic agar plate and 5% blood agar plate. The overall prevalence of GBS carriage was 10.1% (130/1,281, 95% CI: 8.5–12.1%) according to the standard reference method. Compared with the standard reference method, the LAMP had excellent performance of sensitivity (100%, 95%CI: 83.4–100%), specificity (94%, 95%CI: 88.1–97.1%), and Yoden index (0.940); as well as the blood agar plate with sensitivity (81.5%, 95%CI: 61.3–93.0%), specificity (100%, 95%CI: 98.3–100.0%), and Yoden index (0.815). The other four methods were not sufficient to reach the threshold in terms of sensitivity or specificity compared to the standard reference method. Furthermore, for LAMP, results can be obtained within 0.5–1 h, while for blood agar plate, which needed 24–48 h, and further identification was required. Our data suggested that the performance of LAMP was highly comparable to the standard Lim broth-enriched subculture and LAMP is considered as an alternative for fast and accurate GBS screening.

β-Galactosidase-Producing Isolates in Mucoromycota: Screening, Enzyme Production, and Applications for Functional Oligosaccharide Synthesis

β-Galactosidases of Mucoromycota are rarely studied, although this group of filamentous fungi is an excellent source of many industrial enzymes. In this study, 99 isolates from the genera Lichtheimia, Mortierella, Mucor, Rhizomucor, Rhizopus and Umbelopsis, were screened for their β-galactosidase activity using a chromogenic agar approach. Ten isolates from the best producers were selected, and the activity was further investigated in submerged (SmF) and solid-state (SSF) fermentation systems containing lactose and/or wheat bran substrates as enzyme production inducers. Wheat bran proved to be efficient for the enzyme production under both SmF and SSF conditions, giving maximum specific activity yields from 32 to 12,064 U/mg protein and from 783 to 22,720 U/mg protein, respectively. Oligosaccharide synthesis tests revealed the suitability of crude β-galactosidases from Lichtheimia ramosa Szeged Microbiological Collection (SZMC) 11360 and Rhizomucor pusillus SZMC 11025 to catalyze transgalactosylation reactions. In addition, the crude enzyme extracts had transfructosylation activity, resulting in the formation of fructo-oligosaccharide molecules in a sucrose-containing environment. The maximal oligosaccharide concentration varied between 0.0158 and 2.236 g/L depending on the crude enzyme and the initial material. Some oligosaccharide-enriched mixtures supported the growth of probiotics, indicating the potential of the studied enzyme extracts in future prebiotic synthesis processes.