Chromogenic Medium Research Articles

MALDI-TOF mass spectrometry identification and antifungal susceptibility testing of yeasts causing vulvovaginal candidiasis (VVC) in Tebessa (Northeastern Algeria).

Vulvovaginal candidiasis (VVC) alongside with antifungal resistance are becoming a major clinical problem in recent years. A prospective study aimed to evaluate the diversity of yeast strains associated with VVC in Tebessa city (northeastern Algeria) and investigate their susceptibility patterns. Over two months, yeasts were isolated on chromogenic medium from twenty-nine non-pregnant women with symptomatic VVC. The isolates were characterized with MALDI-TOF MS and antifungal susceptibility testing was performed for nine antifungal drugs using SensititreTM YeastOneTM YO10. Twenty-nine non-duplicate yeasts were recovered and the mass spectrometry profiles showed reliable scores of which four genera and five different species were identified. Candida albicans accounted for 65.5 % (n = 19) of the total number of isolates, followed by C. glabrata with 20.7% (n = 6). For the remaining non-albicans Candida (NCA) species, Kluyveromyces marxianus with 6.9% (n = 2), Pichia kudriavzevii and Saccharomyces cerevisiae with one isolate each. The antifungal susceptibilities showed wild type MICs of C. albicans to amphotericin B, azoles and echinocandins. In addition, four C. albicans isolates were resistant to flucytosine. For C. glabrata isolates, 100% non-WT phenotype was found for both posaconazole and itraconazole. For the very first time, the obtained outcomes bring out new data concerning the epidemiology of yeasts causing VVC in Algeria and their antimicrobial susceptibility profiles.

Fecal carriage of extended-spectrum β-lactamase-producing enterobacterales from hospitals and community settings in Gaza Strip, Palestine

BackgroundThe fecal carriage of extended-spectrum β-lactamase-producing Enterobacterales (ESBL-PE) is a major driver of the global spread of these antibiotic resistance determinants. Here we determined the rate of fecal ESBL-PE carriage in pediatric hospitals and community-serving healthcare centers serving adults and children in the Gaza Strip, Palestine.MethodsA total of 373 fecal and rectal samples were collected from different hospitals and clinics in Gaza. The antibiotic susceptibility was determined using the disk diffusion method and interpreted according to CLSI guidelines. The bacterial isolates were tested for ESBL production using phenotypic methods (double disk synergy test and growth on selective chromogenic media). BlaCTX−M, blaSHV, and blaTEM genes were sought by PCR.ResultsOut of the 373 isolates tested, 138 (37%) were considered ESBL positive as revealed by phenotypic tests. The prevalence of ESBLs among hospitalized patients was 39.1% (hospital setting) whereas, among outpatients attending community healthcare centers, it was 35.1% (community setting). ESBL production among Escherichia coli, Klebsiella pneumoniae, Citrobacter freundii, Proteus mirabilis, and Klebsiella aerogenes isolates was 52.8%, 39.1%, 26.7%, 2.8%, and 2.1% respectively. Meropenem and amikacin were the most effective antibiotics against ESBL producers (68.9% and 73.6% susceptibility, respectively), while only 15.2%, 22.5%, and 24.6% remained susceptible to ceftazidime, cefotaxime, and ceftriaxone, respectively. Out of 138 phenotypically ESBL-positive isolates, 98 randomly chosen were screened for blaCTX−M, blaTEM, and blaSHV genes. The prevalence rate of blaCTX−M was 45.9%, while blaTEM and blaSHV genes were detected in 16.8% and 5.2% of CTX-M-negative isolates (corresponding mostly for K. pneumoniae isolates in the case of SHV-PCR), respectively.ConclusionsThe study revealed an alarmingly high prevalence of fecal carriage of ESBL-producing Enterobacterales among hospitalized children but also in the community of the Gaza Strip. In addition, 30% of ESBL-producers were already resistant to carbapenems, the treatment of choice of infections with ESBL-producers.

Performance of CHROMagar ESBL media for the surveillance of extended-spectrum cephalosporin-resistant Enterobacterales (ESCrE) from rectal swabs in Botswana.

Introduction. Lack of laboratory capacity hampers consistent national antimicrobial resistance (AMR) surveillance. Chromogenic media may provide a practical screening tool for detection of individuals colonized by extended-spectrum beta-lactamase (ESBL)-producing organisms.Hypothesis. CHROMagar ESBL media represent an adequate screening method for the detection of extended-spectrum cephalosporin-resistant Enterobacterales (ESCrE), isolated from rectal swabs.Aim. To evaluate the performance of CHROMagar ESBL media to accurately identify ESCrE isolates from rectal swab samples attained from hospitalized and community participants.Methodology. All participants provided informed consent prior to enrolment. Rectal swabs from 2469 hospital and community participants were inoculated onto CHROMagar ESBL. The performance of CHROMagar ESBL to differentiate Escherichia coli and Klebsiella spp., Enterobacter spp. and Citrobacter spp. (KEC spp.) as well as select for extended-spectrum cephalosporin resistance were compared to matrix-assisted laser desorption/ionization-time-of-flight MS (MALDI-TOF-MS) and VITEK-2 automated susceptibility testing.Results. CHROMagar ESBL had a positive and negative agreement of 91.2 % (95 % CI, 88.4-93.3) and 86.8 % (95 % CI, 82.0-90.7) for E. coli and 88.1 % (95 % CI 83.2-92.1) and 87.6 % (95 % CI 84.7-90.2) for KEC spp. differentiation, respectively, when compared to species ID by MALDI-TOF-MS. When evaluated for phenotypic susceptibilities (VITEK-2), 88.1 % (714/810) of the isolates recovered on the selective agar exhibited resistance to third-generation cephalosporins.Conclusion. The performance characteristics of CHROMagar ESBL media suggest that they may be a viable screening tool for the identification of ESCrE from hospitalized and community participants and could be used to inform infection prevention and control practices in Botswana and potentially other low-and middle-income countries (LMICs). Further studies are required to analyse the costs and the impact on time-to-result of the media in comparison with available laboratory methods for ESCrE surveillance in the country.

Fungal diversity and drug susceptibility of the oral mycobiome of domestic dogs.

The purpose of this study was to characterize the variety and diversity of the oral mycobiome of domestic dogs and to identify the commensal and potentially pathogenic fungi present. Two hundred fifty-one buccal swabs from domestic dogs were obtained and struck onto a chromogenic fungal growth medium that distinguishes between fungal species based on colony color and morphology. After isolating and harvesting single colonies, genomic DNA was extracted from pure cultures. PCR was used to amplify a fungal-specific variable rDNA region of the genome, which was then sent for sequencing. Sequencing results were input into the NCBI BLAST database to identify individual components of the oral mycobiome of tested dogs. Of the 251 dogs swabbed, 73 had cultivable fungi present and 10 dogs had multiple fungal species isolated. Although the dogs did not show signs of oral infections at the time, we did find fungal species that cause pathogenicity in animals and humans. Among fungal isolates, Malassezia pachydermatis and species from the genus Candida were predominant. Following fungal isolate identification, antifungal drug susceptibility tests were performed on each isolate toward the medically important antifungal drugs including fluconazole, ketoconazole, and terbinafine. Drug susceptibility test results indicated that a large number of isolates had high MIC values for all three drugs. Exploring the oral mycobiome of dogs, as well as the corresponding drug susceptibility profiles, can have important implications for canine dental hygiene, health, and medical treatment. Identifying the microorganisms within the canine mouth can illustrate a common pathway for fungal pathogens of One Health concern to spread from our canine companions to humans.

Active surveillance for carbapenem-resistant Acinetobacter baumannii (CRAB) carriage.

Carbapenem-resistant Acinetobacter baumannii (CRAB) poses a significant threat in healthcare settings, necessitating robust infection control measures. To identify the most effective sampling method for CRAB carriage detection, we conducted a comparative analysis between skin sampling using a pre-moistened sponge, rectal swabs, and respiratory specimens. Our study encompassed both acute care and post-acute care hospitals. The results revealed that the skin sponge method, when plated on selective chromogenic media, demonstrated the highest sensitivity, exceeding 90% in both settings. Conversely, the commonly employed methods of rectal swabs and respiratory samples combined to exhibit sensitivity levels below 40%. Based on these findings, we recommend implementing the skin sponge method as the preferred approach for CRAB screening in healthcare facilities. This recommendation aims to optimize infection control strategies and mitigate the spread of CRAB in healthcare settings.IMPORTANCEOur study's results provide promising evidence for the incorporation of a high-sensitivity carbapenem-resistant Acinetobacter baumannii (CRAB) screening method in healthcare settings. Such an approach could prove beneficial in enhancing infection prevention and control measures, leading to improved patient outcomes and potentially alleviating the burden of CRAB in healthcare systems.

Molecular diagnosis, phylogenetic analysis, and antifungal susceptibility profiles of Candida species isolated from neutropenic oncological patients

BackgroundNeutropenia is the most important cause of life-threatening invasive fungal infections (IFIs). Here, we studied the frequency and antifungal susceptibility profiles of Candida species that colonized or caused infections among neutropenic patients with solid or hematological malignancies.MethodsA total of 362 clinical samples were collected from 138 patients. After initial isolation using a mix of mycological methods, isolates were screened using chromogenic culture media. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was applied for molecular identification. Positive or suspected cases were confirmed using the reference method of sequencing. Antifungal susceptibility testing for voriconazole and caspofungin was carried out using the microbroth dilution method. An in-silico assay was applied for phylogenetic analysis.ResultsThirty-four Candida strains were isolated. C. albicans (47.06%) and C. glabrata (29.41%) were the most frequent strains. Antifungal treatment reduced the chance of Candida colonization by almost 76% in neutropenic patients (OR: 1.759; 95% CI: 1.349 to 2.390; p value: 0.000). An unusual and non-resistant strain, C. lambica, was reported from the bloodstream of a 56-year-old man with hematologic malignancy (HM). Eight isolates were non-susceptible, and one isolate was resistant to voriconazole. Also, four isolates were non-susceptible to caspofungin.ConclusionWe can conclude that there is a cause-and-effect relationship between neutropenia, HM background, and Candida species separated from neutropenic patients, which can lead to possible infections. Further and repetitive studies are recommended using different molecular methods for better prediction and management of fungal infections in neutropenic patients.

Concerning the presumptive identification of Candida kefyr on Uriselect™4 agar.

Non-albicans Candida species, such as Candida kefyr, are emerging pathogens. Chromogenic media are highly useful for the diagnosis of urinary tract infections (UTIs). The aim was to describe the behavior of this specie on a non-specific chromogenic medium. A retrospective study of cases of candiduria detected in the Microbiology laboratory of the Virgen de las Nieves Hospital in Granada (Spain) between 2016 and 2021 (N=2,130). Urine samples were quantitatively seeded on non-selective UriSelect™4 chromogenic agar. Between 2016 and 2021, C. kefyr was the seventh most frequent Candida species responsible for candiduria in our setting (n=15). The macroscopic appearance of C. kefyr colonies, punctiform and bluish, allowed the direct identification of these microorganisms. This study provides the first description of the specific behavior of C. kefyr on UriSelect™4 agar, which differentiates it from other Candida species based on its enzymatic characteristics.

<i>Streptococcus agalactiae</i> in asymptomatic bacteriuria in pregnant women

BACKGROUND: Asymptomatic bacteriuria caused by group B gram-positive streptococcus, Streptococcus agalactiae, in pregnant women is a leading cause of sepsis in newborns and young children, transmitted from their mothers. In the present study, the frequency of S. agalactiae inoculation from the urine of pregnant women with asymptomatic bacteriuria was studied with the determination of the sensitivity of isolated strains to antibiotics.
 AIMS: This study aimed to investigate the inoculation of group B streptococcus in asymptomatic bacteriuria of pregnant women and to determine the sensitivity of the isolated cultures of the pathogen to the main antibiotics.
 MATERIALS AND METHODS: The analysis involved bacteriological studies of 2,559 urine samples collected from pregnant women. Urine was inoculated on UriSelect 4 Medium chromogenic medium with subsequent identification of S. agalactiae using a strepto-latex test or a CAMP test with a Staphylococcus aureus culture. The sensitivity of microorganisms to antibacterial drugs was determined using the disk diffusion method. Data analysis was conducted using the Statistica 12.0 software package.
 RESULTS: Out of 2,559 urine samples studied, asymptomatic bacteriuria was detected in 317 samples (12.4%), and 330 strains of microorganisms were isolated. S. agalactiae was isolated by culture from 108 pregnant women (4.2% of the total subjects), with 50 cases (16%) showing asymptomatic bacteriuria caused by S. agalactiae. The high significance of Escherichia coli in asymptomatic bacteriuria in pregnant women was shown, with a lesser extent of involvement observed with Enterococcus spp. Furthermore, associations of S. agalactiae with other microorganisms were identified. The high sensitivity of S. agalactiae strains to clindamycin, to a lesser extent to levofloxacin, chloramphenicol, and, especially, to erythromycin, has been demonstrated.
 CONCLUSIONS: The study revealed a high culturability of group B streptococcus in asymptomatic bacteriuria in pregnant women. Low sensitivity of isolated streptococci to erythromycin, moderate sensitivity to levofloxacin and chloramphenicol, and high sensitivity to clindamycin were also revealed.

Use of a chromogenic medium with and without selective enrichment to screen for carbapenemase-producing Enterobacterales (CPE) from canine and feline fecal specimens during an outbreak of NDM-5-producing Escherichia coli.

Carbapenemase-producing Enterobacterales (CPE) are one of the most urgent threats to human healthcare globally. Descriptions of CPE outbreaks in veterinary hospitals suggest the need for screening strategies for CPE from companion animals. Our aim was to optimize a chromogenic agar method with and without selective enrichment to isolate CPE from companion animal feces in an ongoing outbreak of New Delhi metallo-β-lactamse-5 Escherichia coli. A limit of detection (LOD) assay for spiked canine and feline feces was performed for both methods using a carbapenamase-producing E. coli (24213-18); the LOD (1.5 × 103 cfu/g of feces) was equivalent to that reported for human fecal specimens. We screened 1,247 companion animal fecal specimens for carriage of CPE by 1) direct plating to chromogenic agar and 2) plating to chromogenic agar following selective enrichment. Twenty-one specimens were positive for CPE by both direct culture and enrichment culture. No specimens were positive with selective enrichment and negative by direct culture. A selective enrichment step did not result in any increased recovery of CPE from companion animals, which suggests that enrichment broth may not be necessary for outbreak surveillance testing. It is important to continue to validate methods for the detection of CPE in companion animals as outbreaks become more common in veterinary facilities.

Poultry slaughterhouse wastewater as reservoirs for spreading extended-spectrum beta-lactamase-producing Escherichia coli in Abakaliki, Nigeria

Abstract. Ugbo EN, Jacob JI, Effendi MH, Witaningrum AM, Agumah BN, Ugbo AI, Moses BI. 2023. Poultry slaughterhouse wastewater as reservoirs for spreading extended-spectrum beta-lactamase-producing Escherichia coli in Abakaliki, Nigeria. Biodiversitas 24: 4960-4966. The emergence of antimicrobial and multi-resistance extended-spectrum beta-lactamase-producing organisms (ESBL) from the environment and hospitals is a serious global health concern. This research focused on the role of poultry slaughterhouse wastewater as a reservoir for spreading extended-spectrum beta-lactamase-producing Escherichia coli. Exactly 110 poultry slaughterhouses wastewater was collected using sterile universal containers from abattoirs in Abakaliki. The samples were analyzed microbiological for the presence of E. coli using standard techniques. The E. coli isolates found were subjected to a sensitivity test to antibiotics using the Kirby-Bauer disc diffusion method and further tested for ESBL-producing E. coli using a double disc synergy test and chromogenic media Brilliance ESBL agar. Of 110 wastewater samples analyzed, 55 (50.0%) were positive for E. coli contamination. The E. coli isolates showed antibiotic resistance ranging between 21.8% (amoxicillin clavulanic acid) to 69.1% (tetracycline), and good susceptibility was observed on cefepime (72.7%). Thus, among 55 E. coli isolates, 11(20.0%) were ESBL-producing E. coli with resistance patterns as follows: TET-STX-CAZ-CTX-FEP (four isolates); TET-STX-CAZ-CTX (four isolates); TET-STX-CAZ-CTX-FEP-CRO (three isolates), and average multidrug resistance index of 0.7. This study revealed that poultry slaughterhouse wastewater was a reservoir for multidrug resistance ESBL-producing E. coli. Therefore, properly treating wastewater from poultry production or farms before disposing into the drainage channel or water bodies is highly recommended to avoid spreading ESBL-producing organisms from animal waste products to humans and their environment.

Rapid and automated screening of carbapenemase- and ESBL-producing Gram-negative bacteria from rectal swabs using chromogenic agar media and the ScanStation device.

The ScanSation 100 device (Interscience, France) is an incubator allowing real-time detection of bacterial colony growth by frequently imaging agar plates over time, counting CFU, and detecting colony color. This study evaluated its performance for the early detection of carbapenemase-producing bacteria (CPB) and extended-spectrum β-Lactamase-producing bacteria (ESBL-PB) from rectal swabs inoculated on CHROMagar mSuperCARBA and ESBL media, respectively. Rectal screening ESwabs collected from patients admitted to Grenoble University Hospital between January and June 2021 were analyzed. After inoculation, chromogenic media were incubated for 24 h in the automaton, with image acquisition every 30 min. ScanStation results were compared to visual observations of the plates after 24 h of incubation. In total, 501 rectal swabs were tested. ScanStation showed 100% positive percent agreement (PPA) for the detection of CPB and ESBL-PB, whereas the PPA of color categorization ranged between 45% and 100%. Negative percent agreement (NPA) ranged between 70% and 98%. Negative predictive values (NPVs) were 100% for both bacterial groups, whereas positive predictive values (PPVs) were 70.3% for CPB and 81.0% for ESBL-PB. Importantly, real-time screening allowed detection of the first suspected colony within 10-14 h of growth, on average, whereas visual observation is usually only performed once a day after 18-24 h of incubation. Our study demonstrates the potential use of early images to accelerate the detection of CPB and ESBL-PB and implement effective and timely infection control measures. IMPORTANCE The ScanStation 100 device is an incubator able to follow the real-time growth of bacterial colonies on agar plates through digital imaging, allowing users to sort plates according to the presence or absence of colonies, and to distinguish their color using four numeric color filters. Real-time screening shows that first colony detection is possible much earlier (after 10-14 h of growth, on average), whereas visual observation is usually performed only once a day after 18-24 h of incubation. The ScanStation device, combined with chromogenic agar media, is an efficient automated screening method to accelerate the detection of Gram-negative multidrug-resistant bacteria in laboratories that do not have access to larger laboratory automation systems. Our study shows that setting the image acquisition to one or two early images may allow for the detection of positive samples that were inoculated in the morning, by the end of the working day.

CHARACTERISTICS OF ROOT CANAL MICROBIOTA IN ACUTE PULPITIS OF TEMPORARY TEETH

Aims. To determine the representatives of the microbiota of the root canals seeded in acute forms of pulpitis of temporary teeth among children of the Transcarpathian region, taking into account the endemic features of the Transcarpathian region and its influence on the course of the carious process.
 Materials and methods. Collection of biomaterial was carried out using transport tubes with Amies transport medium. The biomaterial was delivered to the laboratory within 2-4 hours after selection. In order to evaluate the microbiota of the root canals, cultivation of biomaterial was carried out on differential diagnostic nutrient media. The material was sown by the method of sector sowing according to Gold: Saburo for the cultivation of microscopic mushrooms; blood agar (MPA + 5% blood) Streptococcus and Neisseria bacteria; bacteria of the family Enterobacteriaceae Endo and Levin; yolk-salt agar with mannitol bacteria of the genus Staphylococcus; enterococcus agar for isolation of enterococci (Farmaktiv, Ukraine). Additionally, the biomaterial was sown on a chromogenic medium (bioMerieux, France). Microorganisms were identified by cultural, morphological and biochemical characteristics using API-test systems (bioMerieux). The results of the quantitative and qualitative evaluation of the microbiota of the oral cavity were expressed in CFU/ml.
 Results and discussion. Today, in the daily practice of a dentist, among issues of diagnosis, prevention and treatment of dental diseases, the study of the microbiota of the oral cavity, which is a collection of different taxonomic groups of microbes that inhabit the oral cavity and enter into biochemical, immunological and other relationships with macroorganism and each other.
 At the same time, the microbial landscape of the oral cavity is represented by two types of microbiota: obligate, that is, one that is constantly present in the oral cavity and is mainly saprophytic, and provides metabolic processes and the function of protecting the body from virulent infectious agents, and facultative, which is represented opportunistic microorganisms, which, when immune protection is reduced, acquire aggressive properties and contribute to the development of diseases [1].
 Conclusion. Opportunistic bacteria play a significant role in the development of inflammatory diseases. Considering the fact that pulpitis is an inflammatory reaction caused by the activity of opportunistic microflora, the study of the characteristics of the microbiota of root canals is relevant, which will help to improve the selection of antiseptic agents for medicinal treatment of root canals in order to obtain positive delayed treatment results.

Quantitative Stool Culture of Candida in Autism Spectrum Disorder Patients

Abstract Background Autism spectrum disorder (ASD) is a category of neurodevelopmental disorders characterized by social and communication impairment and restricted or repetitive behaviors. Aim of the Work to estimate the quantity of Candida in autism spectrum disorder (ASD) patients compared to normal children and to find the association between Candida colony count and severity of ASD. Patients and Methods The study was carried out on 40 children, diagnosed as autism and 40 normal children as control group recruited from Pediatrics and adolescent Psychiatric Clinic at Ain Shams University Pediatrics Hospitals and Main Microbiology lab in Clinical Pathology Department of Ain Shams University. The study was conducted on a wide age group ranging from 3 to 14 years, (mean age of 6.30±2.40 years), Stool sample was collected from each patient in a sterile container, cultured on Sabouraud Dextrose Agar (SDA) and colony count was determined. Identification of isolated Candida species was done using chromogenic media. Results The study revealed statistically significant difference in Candida isolation rate among patient and control group with p-value (p = 0.006). Candida species were isolated from 23 patients (57.5%) and from 10 child in control group (25%), But detected that stool count was nonsignificant in severe autistic group compared to mild to moderate autistic group with p-value more than 0.05 NS. There were 14 patients (60.9%) with C. Albicans; 2 patients (8.7%) C. Glabrata; 6 patients (26.1%) C.Krusie and one patient (4.3%) C. Utilis, among candida species. Conclusion ASD is more common among male than female; ratio about 4:1. The high rate of intestinal colonization by Candida species in ASD children may represent a part of syndrome related to immune system disorders that may play a role in the pathogenesis of ASD. C. albicans was the predominant isolate than other non-Candida albicans species in ASD. The degree of candida colonization represented as quantitative colony count is not related to the severity of CARS among ASD patients.

Epidemiology of Candida Species Colonizing Mucosae of HIV-Infected Patients in Two Healthcare Centers of Cameroon During 2018-2020

Aims: This study aimed to investigate Candida species colonizing HIV-infected patients in Bafoussam and Yaounde in Cameroon.
 Study Design: Cross-sectional study.
 Place and Duration of Study: Yaoundé Central Hospital (YCH) and Bafoussam Regional Hospital (BRH) between October 2018 and December 2020.
 Methodology: We included 804 HIV-infected (681 women; 123 men; age range 21-81 years). Vaginal discharge, oral swab, stools, and urine were collected, and mycological diagnosis including direct macroscopic and microscopic analyses, culture on Sabouraud chloramphenicol medium, culture on chromogenic medium, germ tube test, evidence of chlamydospores production, biochemical analysis was performed. Yeast isolates were identified using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS).
 Results: Eight hundred and four patients were recruited, and 2754 samples were collected. The colonization frequency was 17.35%, and 513 yeasts were isolated. Overall, Candida albicans 251 (48.92%) was the most frequently isolated. Non-albicans Candida (NAC) isolates have been classified into 16 species, including Candida krusei (14.23%), Candida glabrata (9.94%), Candida parapsilosis (8.18%), and Candida tropicalis (7.99%) as the major ones. There was a relationship (P-value= 0.00) between antiretroviral therapy and Candida species colonization.
 Conclusion: The results provide information on the epidemiology of Candida species in HIV-infected patients in Cameroon.

Prevalence of extended-spectrum β-lactamase and carbapenem-resistant Klebsiella pneumoniae in clinical samples.

To assess the prevalence of these resistant strains in the overall isolates of Klebsiella pneumoniae (K. pneumoniae) in hospital settings. This retrospective study was conducted from November 2020 to November 2021. The identification and antibiotic susceptibility testing were performed using standard laboratory methods according to the EUCAST standards. The detection of ESBL and carbapenemase production was performed using phenotypic methods such as E-test, combined-disk test with various inhibitors (ROSCO Diagnostica A/S), chromogenic medium for the detection of ESBL/carbapenemase-producing Enterobacteriaceae (CPE) isolates, and the VITEK 2 Compact system (BioMerieux). 944 isolates of K. pneumoniae were detected in various clinical specimens. Among these, ESBL-producing strains were detected in 349/944 (37%), whereas carbapenem- resistant strains in 188/944 (20%) of the isolates. The remaining isolates (407/944 [43%]) belonged to the wild type. ESBL isolates were the most common in wound swabs (138 [39.5%]), whereas CRKP isolates in screening samples (110 [58.5%]). The majority of ESBL isolates were detected in surgical departments (105 [30.1%]), whereas CRKP isolates in adult intensive care unit departments (79 [42.%]). Our results show an increasing frequency of CRKP strains. This presents a significant issue in terms of infection prevention and control in hospital settings.

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.

Phenotypic and genomic characteristics of oxacillin-susceptible mecA-positive Staphylococcus aureus, rapid selection of high-level resistance to beta-lactams.

The aim of this study is to describe the phenotypic and genetic properties of oxacillin-susceptible methicillin-resistant Staphylococcus aureus (OS-MRSA) isolates and their beta-lactam resistant derivatives obtained after selection with oxacillin. A collection of hospital- (HA-) and community-acquired (CA-) MRSA was screened for oxacillin susceptibility. Antibiotic susceptibility testing, population analysis profile (PAP), mecA expression analysis, and whole genome sequencing (WGS) were performed for 60 mecA-positive OS-MRSA isolates. Twelve high-level beta-lactam resistant derivatives selected during PAP were also subjected to WGS. OS-MRSA were more prevalent among CA-MRSA (49/205, 24%) than among HA-MRSA (11/575, 2%). OS-MRSA isolates belonged to twelve sequence types (ST), with a predominance of ST22-t223-SCCmecIVc and ST59-t1950-SCCmecV lineages. OS-MRSA were characterized by mecA promoter mutations at - 33 (C→T) or - 7 (G→T/A) along with PBP2a substitutions (S225R or E246G). The basal and oxacillin-induced levels of mecA expression in OS-MRSA isolates were significantly lower than those in control ST8-HA-MRSA isolates. Most of the OS-MRSA isolates were heteroresistant to oxacillin. High-level beta-lactam resistant OS-MRSA derivatives selected with oxacillin carried mutations in mecA auxiliary factors: relA (metabolism of purines), tyrS, cysS (metabolism of tRNAs), aroK, cysE (metabolism of amino acids and glycolysis). Cefoxitin-based tests demonstrated high specificity for OS-MRSA detection. The highest positive predictive values (PPV > 0.95) were observed for broth microdilution, the VITEK® 2 automatic system, and chromogenic media. Susceptibility testing of CA-MRSA requires special attention due to the high prevalence of difficult-to-detect OS-MRSA among them. Mis-prescription of beta-lactams for the treatment of OS-MRSA may lead to selection of high-level resistance and treatment failures.

Mastitis prevention and control: Integration of microbiological and management approaches

Mastitis is a common cause of reduced dairy productivity in cows and poor milk quality. This paper considers a set of approaches based on microbiological studies and risk analysis aimed at reducing the number of cows with mastitis and improving milk quality. The study is aimed at examining microbial agents isolated from the milk of sick cows and means of ensuring udder health, and identifying the main dangers that arise due to errors in the implementation of mastitis prevention measures. The study included microbiological tests of milk, wipes and udder dipping using chromogenic media. For a qualitative risk assessment, a survey of consulting managers and veterinarians was conducted. The paper presents the results of microbiological tests of 115 samples of milk, 45 reusable napkins, and 111 samples of udder dips using conventional and chromogenic media. The most common microorganisms in milk samples were Streptococcus spp. 34.4% (S. agalactiae and S. disgalactiae), Staphylococcus aureus 31.1%, and Escherichia coli 28.9%. Staphylococcus aureus and Streptococcus spp. it is associated with infectious mastitis, and Escherichia coli – with sanitary mastitis. To reduce the incidence of mastitis, it is important to apply targeted measures aimed at various categories of pathogens. Udder wipes were contaminated with pathogens associated with the development of mastitis. The use of laundry detergent containing bactericidal components is crucial to minimise contamination of reusable udder wipes. Among the dips under study, 40.6% of the samples had a complete bactericidal effect, and 9.9% had no bactericidal effect. 3 factors of high risk of environmental mastitis and 6 factors of sanitary mastitis were identified. The results of the studies were tested on two dairy farms. Correction of high-risk risk factors led to an increase in the quality of milk in terms of somatic cells and the number of bacteria. The results obtained can be useful for improving the system of prevention and optimisation of mastitis treatment on dairy farms

Chromogenic culture media complements diagnostic cytology in the visual identification of pathogenic skin bacteria in dogs and cats.

In dogs and cats, bacterial skin infections (pyoderma and otitis externa) are a common cause for visiting the veterinary clinic. The most frequent skin pathogens are Staphylococcus pseudintermedius, Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa, often requiring different therapeutic antibiotic protocols. Unfavorably, existing diagnostics based on cytology cannot reveal bacterial species but only bacterial shapes such as cocci or rods. This microscopic limitation could be overcome by clinical translation of affordable chromogenic media, which enable species identification based on bacterial colonies growing in different colors and sizes. In this study, we determined how well inexperienced general veterinary clinicians identified bacterial pathogens from the skin and ears on two commercial (Chromatic™ MH and Flexicult® Vet) and one custom-made Mueller Hinton agar-based chromogenic medium. For this purpose, four veterinarians evaluated 100 unique samples representing 10 bacterial species. On average, clinicians correctly identified between 72.1 and 86.3% of bacterial species. Colony colors developed quickly on the Chromatic™ MH medium, leading to the highest 81.6% identification accuracy after 24 h incubation. However, Flexicult® Vet exhibited the highest accuracy of 86.3% after prolonged 48 h incubation. Evaluators easily recognized bacteria displaying uniquely colored colonies like green-brown Pseudomonas aeruginosa, blue Enterococcus faecalis, orange-brown Proteus spp., and red Escherichia coli. Oppositely, staphylococci shared uncharacteristically pale pink colonies causing misidentifications among the genus, deteriorating overall accuracy by around 10 percentage points (from 90.9%). Another reason for identification errors was the evaluators' inexperience, reflected in not recognizing colony size differences. For example, although Streptococcus canis exhibited the tiniest colonies, the species was frequently mistaken for other cocci. Finally, around 10% of errors were negligence-related slips due to unconsidered sample history. To conclude, the introduction of chromogenic media into veterinary clinics can significantly complement diagnostics in skin inflammations by identifying pathogen species in around 80% of cases. The extra information may help in therapeutic dilemmas on antibiotics and standard antimicrobial susceptibility testing. Additional personnel training and evaluation help by visuals, flowcharts, checklists, and, if necessary, microbiologists could further improve identification accuracy.

A membrane filtration method for the enumeration of Escherichia coli in bathing water and other waters with high levels of background bacteria.

The presence and level of faecal indicator bacteria are important factors in estimating the microbiological quality of surface water and the risk of human infection upon exposure to this water. Until 2014, ISO 9308-1:2000 was available and used to enumerate faecal indicator Escherichia coli in bathing water. In 2014, this ISO was technically revised and replaced by ISO 9308-1:2014. This ISO introduced a less selective method for enumeration of E. coli that allows non-specific growth from waters containing high levels of bacteria, such as surface waters. This implies that currently there is no suitable reference membrane filtration method for the compliance monitoring of official bathing sites for E. coli according to the European Bathing Water Directive. Here, the performance characteristics of three chromogenic culture media, namely Tryptone Bile X-glucuronide (TBX) agar, Chromogenic Coliform Agar (CCA), and CHROMagar E. coli/Coliform (ECC) were investigated at 44 °C for water with varying levels of bacteria according to ISO 13843:2017. Based on performance characteristics, colony counts, and practical usage, TBX appeared the most suitable culture medium for the enumeration of E. coli in bathing water and other waters with high levels of background bacteria, such as surface water in agricultural areas and wastewater discharge points.