Phenotypic profile of priority multiresistant Аcinetobacter baumannii sequence types (ST 1167, ST 944, ST 208)

Introduction. About 1,000,000 cases of infections caused by Acinetobacter spp. per year are registered globally, making up 1.8% of all the cases of hospital-acquired infections. In compliance with long-term studies carried out in in this country and abroad, Acinetobacter baumannii is a clinically important representative of the Acinetobacter genus. Intraspecific typing of microorganisms is an integral part of a clinical microbiologist's contribution to scoring the outbreaks of purulent-septic infections within the sphere of HAI surveillance. Most of the practicing microbiological laboratories cannot use genotypic typing methods because of their high costs.Objective. Developing a test panel for intraspecific identification of A. baumannii sequence types (ST 1167, ST 944, ST 208) based on their phenotypic properties.Materials and methods. Intraspecific membership of 74 A. baumannii strains obtained from four multipurpose health settings of a large industrial centre was studied using a genetic method (multilocus sequence typing) and a suite of phenotypic methods (biochemical tests, biofilmogenous capacity, growth inhibition zones to antibacterial drugs, sensitivity to aniline dyes, disinfectants and Acinetobacter bacteriophage) was studied.Results. Phenotypic features of three predominant A. baumannii sequence types (ST 1167, 944, 208) were determined.Discussion. An efficacious economy set of differentiating tests allowing identification of intraspecific features of A. baumannii multiresistant strains was сreated.Conclusion. The test panel will enable the laboratories that cannot use sequencing methods to conduct intraspecific differentiation of common A. baumannii sequence types as part of microbiological monitoring.

IDENTIFICATION OF METHICILLIN RESISTANT STAPHYLOCOCCUS AUREUS USING TOUCHDOWN PCR AND PHENOTYPIC METHODS FROM PATIENTS AND HOSPITALS ENVIRONMENTS IN DIFFERENT IRAQI CITIES

The study was aimed to evaluate the prevalence of MRSA in some Iraqi hospitals and determine the most powerful methods for identification of MRSA, in order to achieve the, 278 samples were collected from different hospitals in Iraq in various intervals, 204 out of 287 were identified as Staphylococcus aureus by conventional cultural methods and microscopic characteristics and 177 isolates are identified as MRSA by using HiCrome MeReSa Agar Base medium, but 154 of 177 (87%) isolates are methicillin resistance in sensitivity test. MRSA isolates were highly resistant to β-lactam antibiotics and considered multidrug resistant (MDR) in percent of (94.9%). Touchdown PCR used to identify the isolates, 97.05% were identified as Staphylococcus aureus, while 80.88% as MRSA.

Antibiotic sensitivity of Pseudomonas aeruginosa strains isolated from milk of highly productive dairy cows

Pseudomonas aeruginosa is a free-living bacterium that is conditionally pathogenic in natural conditions, but when ingested by an animal can cause severe infectious diseases, depending on the location and stage of infection, increasing the time of convalescence. It is naturally resistant to a number of widely used antibacterial drugs: fluoroquinolones, tetracyclines, chloramphenicol; it is capable of forming a biofilm. The aim of this work was to assess the level of sensitivity of strains isolated from cow's milk to the main groups of antibiotics with clinically significant anti-pseudomonasal activities. The work provides microbiological monitoring data for 2020-2021. During this period, 350 milk samples were taken from Holstinized black-and-white cows of the dairy direction of various lactation ages. All isolates were characterized by generally accepted phenotypic methods, with confirmation of biochemical properties. A number of antibiotics selected in this work comply with the recommendations of EUCAST. Sensitivity testing of the isolated strains was carried out by the disco-diffuse method. Pseudomonas aeruginosa was detected in 69 milk samples, which accounted for 19.7% of the total number of milk samples. Pseudomonas Aeruginosa was isolated as a monoculture in 42.03% of cases, in association with gram (-) bacteria in 20.29% and gram (+) in 37.68%. The percentage of pigmented strains in our work was 98.55% of all isolated strains, and 1.45% of poorly pigmented. During the research work, it was found that more than 90% of the strains were resistant to cefepime, the rest showed partial resistance. Therefore, it can be recommended for use only after a correction for sensitivity to this drug. The aminoglycoside group drugs, amikacin and gentamicin, had the highest activity - over 90% against the isolated Ps.a. strains. The results obtained indicate that the isolated strains of Ps.a. they showed high resistance to representatives of the cephalosporin group, which is also increasing with respect to fluoroquinolones, which is a serious problem in the spread of antibiotic resistance.

Evaluation of phenotypic methods for the detection of carbapenemases applicable to low complexity laboratories

The spread of carbapenemase-producing gram-negative bacilli is a global public health problem. Several authors have proposed phenotypic assays to presumptively detect these enzymes applicable to low and medium complexity laboratories. In the present study, we have developed and compared different phenotypic techniques using strains genetically identified as carbapenemase-producing. All the tested methods detected the presence of carbapenemases. The carbapenem inactivation method (MIC) and the modified carbapenem inactivation method with and without EDTA (mMIC-eMIC) were the simplest and easiest to interpret but their disadvantage was on the time required to obtain results. The direct Carba NP and Carba-Blue colourimetric methods were the fastest but they depend on reagent preparation and accurate pH adjustment of the solutions. Synergy methods with EDTA discs, boronic acid and the Triton Hodge Test (THT) require technical expertise to evaluate true synergism. Whereas, the Disk Carbapenemase Test (DCT) was the method that presented the greatest technical difficulties.

Beta-lactam antibiotics and extended spectrum beta-lactamases

Extended spectrum beta-lactamases (ESBLs) are enzymes produced by bacteria, mostly members of the family Enterobacteriaceae commonly Escherichia coli and Klebsiella pneumoniae. ESBLs hydrolyze the beta-lactam ring of beta-lactam antibiotics making these antibiotics ineffective therefore rendering the bacteria resistance against beta-lactam antibiotics. The global upsurge of ESBLs producing bacteria causing both hospital and community acquired infections mostly urinary tract infections, pneumonia and bloodstream infections, threatens the effectiveness of infectious diseases treatment. ESBL families; TEM, SHV and CTX-M are globally disseminated and frequently detected in clinical isolates as well as colonization and contamination isolates. Various laboratory detection methods of ESBLs producing Gram negative bacteria are available. These methods; phenotypic methods, automated methods and molecular-based methods are varying in sensitivity and specificity, need of technical expertise, and rapidness. Therefore, they should be clearly understood before being employed for routine or research use for detection of ESBLs production among Enterobacteriaceae. In addition, understanding the mode of action and mechanisms of resistance to beta-lactam antibiotics, and the epidemiology of ESBLs producing bacteria is of paramount.

Assessment of the Genetic Diversity of Chrysanthemum Cultivars Using SSR Markers

Chrysanthemums are undoubtedly one of the most popular flowering plants in the world. Their exceptional importance in Asian culture resulted in the global popularization of this species, which resulted in the high interest of breeders. Chrysanthemums can be divided into three groups: small-flowered, mid-flowered, and large-flowered. The exceptional economic importance and a large number of varieties make them problematic to identify, resulting in a less efficient breeding process. In the case of chrysanthemums, genotypes are almost impossible to distinguish by using phenotypic methods due to the high variation in morphological characteristics, even when they belong to the same group. The aim of the study was to evaluate the genetic diversity of 97 chrysanthemum cultivars using 14 selected SSR markers. Large-flowered varieties (Angali and Rosee D’une) were characterized by the smallest mutual distance, and the greatest distance was between large-flowered (Impact Rood) and small-flowered (Conaco Yellow) varieties. All methods of visualizing the results reveal a clear distinctiveness of small-flowered cultivars, except for the cultivars from the Moira series.

Comparative Performance of Genomic Methods for the Detection of Pyrazinamide Resistance and Heteroresistance in Mycobacterium tuberculosis

Background: Pyrazinamide is an important component of both drug-susceptible and drug-resistant tuberculosis treatment regimens. Although approximately 50% of rifampicin resistant isolates are also resistant to pyrazinamide, pyrazinamide susceptibility testing is not routinely performed due to the challenging nature of the assay. We investigated the diagnostic accuracy of genotypic and phenotypic methods, and explored the occurrence of pyrazinamide heteroresistance. Methods: We assessed pyrazinamide susceptibility among 358 individuals enrolled in the South African EXIT-RIF cohort using Sanger and targeted deep sequencing (TDS) of the pncA gene, whole genome sequencing (WGS), and phenotypic drug-susceptibility testing. We calculated the diagnostic accuracy of the different methods, and investigated the prevalence and clinical impact of pncA heteroresistance. True pyrazinamide susceptibility status was assigned to each isolate using the Koser classification and expert rules. Results: We observed 100% agreement across genotypic methods for detection of pncA fixed mutations, only TDS confidently identified three isolates (0.8%) with minor variants. For the 355 (99.2%) isolates that could be assigned true pyrazinamide status with confidence, phenotypic DST had a sensitivity of 96.5% (95% CI: 93.8-99.3%) and specificity of 100% (95% CI: 100-100%); both Sanger sequencing and WGS had a sensitivity of 97.1% (95% CI: 94.6-99.6%) and specificity of 97.8% (95% CI: 95.7-99.9%); and TDS, sensitivity of 98.8% (95% CI: 97.2-100%) and specificity of 97.8% (95% CI: 95.7-99.9%). Conclusions: We demonstrate high sensitivity and specificity for pyrazinamide susceptibility testing among all assessed genotypic methods. The prevalence of pyrazinamide heteroresistance in Mtb isolates was lower than that identified for other first-line drugs.

The Emergence of Linezolid-resistant Staphylococcus Epidermidis in the COVID-19 Hospitalized Intubated Patients in North Khorasan, Iran

BackgroundIn the COVID-19 pandemic from 2019 to date, we confront secondary bacterial and viral infections in SARS-CoV2 infected patients, especially hospitalized patients. Coagulase-negative staphylococci, are commensals of the human body and can lead to infections in immunocompromised patients. The antimicrobial resistance is increasingly reported in coagulase-negative staphylococci, especially in Staphylococcus epidermidis. One of the most critical problems is resistance to linezolid in S. epidermidis, observed in Europe since 2014. The aim of this study was to evaluation of bacterial Co-infections and determination of antimicrobial resistance pattern of co-infection isolated strains in North Khorasan, Iran, in the last six-month period. MethodsAfter microbiological evaluation of pulmonary samples of hospitalized intubated patients with signs of bacterial pneumonia, we found co-infection in 11 of 185 patients with S. epidermidis, S. aureus, and Acinetobacter baumani, respectively. Interestingly seven of nine S. epidermidis isolates were linezolid resistant. For identification of the isolates at the species level, we used phenotypic methods and also the Polymerase Chain Reaction (PCR) for the atlE gene. Selected isolates were characterized by determining their antimicrobial resistance patterns and using molecular methods including SCCmec typing, detection of ica, mecA, vanA, and cfr genes. ResultsAll isolates were resistant to methicillin, and Seven isolates were resistant to linezolid. It should be noted that all nine isolates were positive for the ica gene. Nine of 11 isolated have belonged to the SCCmec I, and two belonged to the SCCmec IV. It should be noted that all patients had the underlying disease and six patients died.ConclusionThe increasing linezolid resistance in bacterial strains becomes a real threat for patients, and monitoring such infections combined with surveillance and infection prevention programs is very important to decrease the number of linezolid-resistant staphylococcal strains.

1361. Actinotignum schaalii an Under-recognized Cause of Infections: A Case Series in Calgary from 2012 - 2020

Background Actinotignum schaalii is a gram-positive rod that is a fastidious commensal of the urogenital tract. Infections with A. schaalii are underdiagnosed previously because phenotypic methods fail to identify it. Both MALDI-TOF mass spectrometry and 16S rRNA sequencing allow definitive identification of this opportunistic emerging pathogen. A. schaalii is an infrequent but important cause of UTIs in the elderly, particularly with urological abnormalities. The spectrum of invasive disease caused by A. schaalii is not well characterized; however, it has been isolated in severe infections including necrotizing skin and soft tissue infections, bacteremia, osteomyelitis, and endocarditis. We used a population-based approach to characterize and describe the clinical and microbiological features of invasive A. schaalii infections in our region. Methods All adult and pediatric cases enrolled had microbiological isolates of Actinotignum schaalii recovered from blood cultures, sterile fluids and tissue cultures from Jan 2012 to Dec 2020 by APL, a regional centralized microbiology laboratory serving the Calgary Zone in Alberta, Canada. Clinical data were retrieved and linked from administrative health databases, chart review and the laboratory information system. Standard descriptive statistics were used. Results We identified 84 unique A. schaalii infections, 35 were from bloodstream, 32 soft tissue, 7 post-operative infections. Median age and Charslon comorbidity score was higher in BSI. 54.3% of patient with BSI had a genitourinary pathology, with 51.4% caused by a complicated urinary infection, while soft and skin tissue infections caused 65.3% of non-BSI. Using EUCAST MIC cut-offs, 48% and 100% of the isolates were resistant to clindamycin and metronidazole, respectively. In contrast, all specimens were susceptible to penicillin. Hospitalization and 90-days mortality were higher in the BSI group. Conclusion A. schaalii is an anaerobic opportunistic pathogen that can cause life-threatening invasive infections, particularly in older adults with underlying genitourinary pathology. BSI were associated with higher rates of hospitalization and mortality. In contrast, patients with A. schaalii isolated from cutaneous sources were younger and had better clinical outcomes. Disclosures All Authors: No reported disclosures