Diagnostic Microbiology Research Articles

Intervention with impact: Reduced isolation of methicillin-resistant Staphylococcus pseudintermedius from dogs following the introduction of antimicrobial prescribing legislation in Germany.

Legislation was introduced in Germany in 2018, requiring bacterial culture and antimicrobial susceptibility testing before the prescription of fluoroquinolones and third-generation cephalosporins to dogs. We hypothesised that, following this intervention, the number of clinical samples testing positive for methicillin-resistant Staphylococcus pseudintermedius (MRSP) would reduce. Reports of S. pseudintermedius isolated from canine clinical samples by three German veterinary diagnostic microbiology laboratories during the 38 months before the introduction of the legislation and the 46 months after were compared. Bacterial identification was performed by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry, and antimicrobial susceptibility testing followed recognised recommendationsbut with changes during the study period. Among a total of 120,571 S. pseudintermedius isolates, MRSP accounted for 7.1% overall. Following the legislative intervention, monthly submissions yielding S. pseudintermedius increased at all three laboratories. The MRSP percentage was lower in the period after the intervention in two of the three laboratories (p < 0.001); in the third laboratory, there was no change between periods, but a year-on-year reduction in MRSP percentages occurred after the intervention (p = 0.0004). Changing susceptibility testing methods limited the direct comparison of resistance patterns among laboratories. The reduction in MRSP in canine clinical samples following the introduction of this legislation suggests a positive impact of compulsory laboratory testing on reducing antimicrobial resistance.

Comparative Genomic Analysis of Cutibacterium spp. Isolates in Implant-Associated Infections.

Bacteria of the genus Cutibacterium are Gram-positive commensals and opportunistic pathogens that represent a major challenge in the diagnosis and treatment of implant-associated infections (IAIs). This study provides insight into the distribution of different sequence types (STs) of C. acnes, and the presence of virulence factors (VFs) in 64 Cutibacterium spp. isolates from suspected or confirmed IAIs obtained during routine microbiological diagnostics. Fifty-three C. acnes, six C. avidum, four C. granulosum, and one C. namnetense isolate, collected from different anatomical sites, were included in our study. Using whole-genome sequencing and a single-locus sequencing typing scheme, we successfully characterized all C. acnes strains and revealed the substantial diversity of STs, with the discovery of six previously unidentified STs. Phylotype IA1, previously associated with both healthy skin microbiome and infections, was the most prevalent, with ST A1 being the most common. Some minor differences in STs' distribution were observed in correlation with anatomical location and association with infection. A genomic analysis of 40 investigated VFs among 64 selected strains showed no significant differences between different STs, anatomical sites, or infection-related and infection undetermined/unlikely groups of strains. Most differences in VF distribution were found between strains of different Cutibacterium spp., subspecies, and phylotypes, with CAMP factors, biofilm-related VFs, lipases, and heat shock proteins identified in all analyzed Cutibacterium spp.

Občutljivost za antibiotike pri povzročiteljih nezapletenega cistitisa v Sloveniji

Background: Acute uncomplicated cystitis in women is a common reason for antibiotic treatment in the outpatient clinic of family doctors. Microbiological diagnostics is not indicated; treatment is generally empirical and based on guidelines and recommendations. Therefore, we do not have representative data on the incidence and sensitivity of pathogens from regular laboratory work. Special targeted research is needed.&#x0D; Methods: We present the results of the prospective research conducted in four regional laboratories of the Centre for Medical Microbiology NLZOH between 15 September 2017 and 31 December 2019. We included 110 female patients with suspected acute uncomplicated cystitis who completed a questionnaire and submitted urine samples for microbiological examination.&#x0D; Results: The result of urine examination was positive in 79 patients (71.8%). Among all isolates, the most common bacterium was Escherichia coli (74%), followed by Staphylococcus saprophyticus (10%), and others. The susceptibility of E. coli to trimethoprim with sulfamethoxazole was 85.5%, to nitrofurantoin 98.4%, and to fosfomycin 100%. S. saprophyticus isolates were susceptible to the tested antibiotics except for fosfomycin, against which the bacterium is intrinsically resistant. The total susceptibility of all isolates was 85.8% - 88.2% for trimethoprim with sulfamethoxazole, 88.1% - 95.2% for nitrofurantoin, 73.8% - 80.8% for fosfomycin, 72.6% for ampicillin and amoxicillin, 97.7% for cefadroxil, and 98.9% for amoxicillin with clavulanate acid. A comparison with the results of monitoring bacterial resistance as part of the regular work of microbiological laboratories shows significant differences for most of the tested antibiotics.&#x0D; Conclusion: The research results show a relatively good susceptibility of the causative agents of acute uncomplicated cystitis to oral antibiotics, the only exception being amoxicillin.

831. Majocchi’s Granuloma – A Single Institution Retrospective Review

Abstract Background Majocchi’s granuloma (MG) is an uncommon deep fungal folliculitis predominantly caused by dermatophytes. Available data regarding predisposing comorbidities/risk factors, clinical characteristics, offending microbiologic pathogens, diagnostics, pathologic findings, and treatment approaches has been inferred from historical cases. Given these limitations in the existing literature of MG, we sought to conduct a detailed review of our institutional experience with biopsy-confirmed cases of MG. Methods Following IRB approval, we retrospectively identified and analyzed a multicenter cohort of adult patients diagnosed with MG between September 1992 and September 2022 at Mayo Clinic campuses (Figure 1). Data extracted included demographics, comorbid conditions, presenting clinical characteristics, diagnostic, microbiologic, and biopsy characteristics, treatment details, and outcomes. Figure 1 Flow Diagram show exclusion of patients from the final cohort. Results A total of 147 patients with MG with the accompanying baseline characteristics met study criteria (Table 1). One-hundred and five patients were male with a median age of 55.6 years. Immunosuppressant and topical corticosteroid use were common prior to development of MG (Table 1). Dermatologic lesions and their sites of involvement did not differ based on the immune status of patients (Table 2). Trichophyton rubrum was the most common causative pathogen of MG, in addition to other dermatophytes and fungal organisms (Table 3a). Treatment duration for all prescribed agents was median 31.5 days. Oral terbinafine was the most frequently utilized agent and clinical resolution was achieved in 96.6% of cases (Table 3b). Baseline Characteristics of 147 Patients with Majocchi’s Granuloma Diagnostic, Microbiologic, and Biopsy Characteristics, Treatment Characteristics and Outcomes Conclusion While rare and clinically variable in presentation, the diagnosis of MG requires careful clinical examination and history-taking, histopathologic confirmation, and frequently fungal culture. Most cases of MG in our study responded well to a course of oral terbinafine at a median of 31.5 days of treatment. Disclosures All Authors: No reported disclosures

Rapid identification of pathogens in blood serum via Raman tweezers in combination with advanced processing methods.

Pathogenic microbes contribute to several major global diseases that kill millions of people every year. Bloodstream infections caused by these microbes are associated with high morbidity and mortality rates, which are among the most common causes of hospitalizations. The search for the "Holy Grail" in clinical diagnostic microbiology, a reliable, accurate, low cost, real-time, and easy-to-use diagnostic method, is one of the essential issues in clinical practice. These very critical conditions can be met by Raman tweezers in combination with advanced analysis methods. Here, we present a proof-of-concept study based on Raman tweezers combined with spectral mixture analysis that allows for the identification of microbial strains directly from human blood serum without user intervention, thus eliminating the influence of a data analyst.

Fracture related Infection - Challenges in definition and diagnosis

In the field of orthopedics and trauma surgery, the rise of periprosthetic joint infections following joint replacement and fracture-related infections (FRI) has become a growing concern. The recent establishment of a definitive definition for FRI aimed to standardize diagnosis and treatment approaches while considering unique aspects of implant-associated infections in the presence of concomitant bone fractures.Diagnosing FRI can be challenging due to the varied clinical symptoms, and confirmatory criteria may not always be evident, necessitating additional diagnostic measures. Blood markers like leukocyte count, C-reactive protein (CRP), and erythrocyte sedimentation rate (ESR) have limited specificity. Although novel biomarkers such as D-dimer and interleukin-6 (IL-6) show potential, they require further investigation. The use of microbiological diagnostics with tissue samples and sonication has improved pathogen detection. Cross-sectional imaging techniques like CT scans and MRI scans help evaluate bone status, soft tissue infiltration, and abscesses. Nuclear medicine techniques are accurate but may not always be practical in routine clinical practice.Histopathological interpretation for FRI remains less standardized compared to periprosthetic joint infections (PJI). FRI diagnosis requires the identification of visible microorganisms in deep tissue specimens and the quantification of polymorphonuclear neutrophils (PMNs).The defined concept of FRI has opened doors for better diagnostic and treatment approaches. However, challenges persist, especially in preoperative diagnosis, particularly for cases with unclear clinical presentations. Future endeavors aimed at optimizing diagnostic procedures and establishing a histopathological classification for FRI could lead to improved treatment recommendations and outcomes.

Assessment of four in vitro phenotypic biofilm detection methods in relation to antimicrobial resistance in aerobic clinical bacterial isolates.

The lack of standardized methods for detecting biofilms continues to pose a challenge to microbiological diagnostics since biofilm-mediated infections induce persistent and recurrent infections in humans that often defy treatment with common antibiotics. This study aimed to evaluate diagnostic parameters of four in vitro phenotypic biofilm detection assays in relation to antimicrobial resistance in aerobic clinical bacterial isolates. In this cross-sectional study, bacterial strains from clinical samples were isolated and identified following the standard microbiological guidelines. The antibiotic resistance profile was assessed through the Kirby-Bauer disc diffusion method. Biofilm formation was detected by gold standard tissue culture plate method (TCPM), tube method (TM), Congo red agar (CRA), and modified Congo red agar (MCRA). Statistical analyses were performed using SPSS version 17.0, with a significant association considered at p<0.05. Among the total isolates (n = 226), TCPM detected 140 (61.95%) biofilm producers, with CoNS (9/9) (p<0.001) as the predominant biofilm former. When compared to TCPM, TM (n = 119) (p<0.001) showed 90.8% sensitivity and 70.1% specificity, CRA (n = 88) (p = 0.123) showed 68.2% sensitivity and 42% specificity, and MCRA (n = 86) (p = 0.442) showed 65.1% sensitivity and 40% specificity. Juxtaposed to CRA, colonies formed on MCRA developed more intense black pigmentation from 24 to 96 hours. There were 77 multi-drug-resistant (MDR)-biofilm formers and 39 extensively drug-resistant (XDR)-biofilm formers, with 100% resistance to ampicillin and ceftazidime, respectively. It is suggested that TM be used for biofilm detection, after TCPM. Unlike MCRA, black pigmentation in colonies formed on CRA declined with time. MDR- and XDR-biofilm formers were frequent among the clinical isolates.

Detection and characterization of putative hypervirulent Klebsiella pneumoniae isolates in microbiological diagnostics

Hypervirulent Klebsiella pneumoniae strains (hvKp) can cause invasive community-acquired infections in healthy patients of all ages. In this study, the prevalence of putative hvKp in a German tertiary center was investigated and hvKp were characterized by phenotypic and molecular assays. All K. pneumoniae isolates in routine microbiological diagnostics from a single center were screened by string-testing over a period of 6 months. String-test positive (≥ 0.5 mm) isolates were re-evaluated on different media and under various conditions (aerobe, anaerobe). For string-test positive isolates, genes (magA, iutA, rmpA and rmpA2) associated with hypermucoviscosity and hypervirulence were amplified by multiplex PCR. PCR-positive isolates were subjected to whole-genome sequencing and sedimentation and biofilm formation assays. From 1310 screened K. pneumoniae isolates in clinical routine 100 isolates (7.6%) were string test positive. From these, 9% (n = 9) were defined as putative hvKp (string-test+/PCR+). Highest rate of string-test-positive isolates was observed on MacConkey agar under aerobic conditions. Amongst these nine putative hvKp isolates, the international lineage ST23 carrying hvKp-plasmid pKpVP-1 was the most common, but also a rare ST86 with pKpVP-2 was identified. All nine isolates showed hypermucoviscosity and weak biofilm formation. In conclusion, 9% of string-positive, respectively 0.69% of all K. pneumoniae isolates from routine were defined as putative hypervirulent. MacConkey agar was the best medium for hvKp screening.

Multicenter evaluation of BioFire JI panel related to improved microbiological diagnostics on acute osteoarticular infections

Microbiological diagnosis of osteoarticular infections (OI) is crucial for a successful treatment. A prospective multicenter study including 262 synovial fluids with suspicion of acute OI was performed between July 2021 and October of 2022. BioFire Joint Infection Panel multiplex-PCR test was performed and results were compared with conventional cultures of synovial fluid specimens. In total, 136 microorganisms were detected, and fourteen samples were positive for more than one microorganism. In monomicrobial infections (n = 87) agreement with culture was 69%. In 26 samples, the multiplex PCR yield an additional positive result when culture result was negative. It helped in the detection of fastidious microorganisms as K. kingae and N. gonorrhoeae. This multiplex PCR has proven to be a useful technique that can be used for patients with high suspicion of acute OI in a rapid and automated manner.

Structured approach for infected prosthesis

Endoprosthesis infections represent amajor challenge for doctors and patients. Due to the increase in endoprosthesis implantation because of the increasing life expectancy, an increase in endoprosthesis infections is to be expected. In addition to infection prophylaxis, methods of infection control become highly relevant, especially in the group of geriatric and multimorbid patients. The aim is to reduce the high 1‑year mortality from prosthesis infections through astructured algorithm. Prosthesis infections can basically be divided into early and late infections. According to the criteria of the International Consensus Meeting, alate infection is defined as the occurrence more than 30days after implantation. With respect to the planned approach, the (p)TNM classification offers an orientation. In the early postoperative interval the clinical appearance is crucial as in this phase neither laboratory parameters nor an analysis of synovial fluid show ahigh sensitivity. It is fundamental that, apart from patients with sepsis, environment diagnostics should be initiated. If alate infection is suspected, in addition to radiological diagnostics (X-ray, skeletal scintigraphy and if necessary, computed tomography, CT), laboratory (C-reactive protein, CRP, leukocytes, blood sedimentation, and if necessary, interleukin‑6, procalcitonin) and microbiological diagnostics (arthrocentesis with synovial analysis and microbiology) are indicated; however, in addition to the arthrocentesis result, the clinical appearance is crucial in cases where an exclusion cannot be confirmed by laboratory parameters. If an infection is confirmed, the treatment depends on the spectrum of pathogens, the soft tissue situation and the comorbidities, including amultistage procedure with temporary explantation and, if necessary, implantation of an antibiotic-containing spacer is necessary. Aprosthesis preservation using the debridement, antibiotics and implant retention (DAIR) regimen is only appropriate in an acute infection situation. Basically, radical surgical debridement should be carried out to reduce the pathogen load and treatment of apossible biofilm formation for both early and late infections. The subsequent antibiotic treatment (short or long interval) should be coordinated with the infectious disease specialists. Astructured approach for prosthesis infections oriented to an evidence-based algorithm provides asufficient possibility of healing. An interdisciplinary approach involving cooperation between orthopedic and infectious disease specialists has proven to be beneficial. Surgical treatment with the aim of reducing the bacterial load by removing the biofilm with subsequent antibiotic treatment is of intrinsic importance.

Microbiological diagnostics: From traditional to molecular genetic methods: A literature review

The development of new and optimisation of known cultural and molecular genetic methods for accurate species identification of microorganisms is an urgent and practically necessary task that is receiving great attention from researchers. The purpose of this study was to analyse and systematise theoretical scientific data on methods of microbial identification and assess their main advantages and disadvantages. For this purpose, a systematic review of 53 randomised research papers published between 2018 and 2023 was conducted. The search for publications using the keywords “microbiome”, “microbiological diagnostics”, “identification of microorganisms”, “sequencing”, and “omics technologies” in the title or text of the research paper was carried out in Web of Science, Scopus, PubMed, and Google Scholar. The study provides generalised information on traditional and modern methods of microbial identification. It is established that the advantages of traditional diagnostic methods include the possibility of preserving the obtained microorganisms for further research, in particular, for determining their antibiotic sensitivity. Modern molecular genetic methods open up new possibilities for the accurate identification of microorganisms, including those that are difficult to incubate using traditional cultural methods. The use of these methods allows obtaining detailed data on the genetic structure and diversity of microorganisms, which is important in many fields, including microbiology, medicine, and ecology. However, molecular methods are more sensitive to contamination or errors in the sample collection and processing. Moreover, omics technologies (genomics, transcriptomics, proteomics, and metabolomics) open up new opportunities for studying cells at more complex levels of life organisation. In everyday clinical practice, the microbiological method of isolating microbial cultures remains the main one, as it is the only one that allows determining not only the cause of the infection but also the antibiotic sensitivity. However, cultural methods for studying fastidious microorganisms are quite limited. Thus, the results obtained contribute to the development of fast and accurate methods for identifying, classifying, and systematising microorganisms, which facilitates the processes of diagnosis and development of strategies for the control and treatment of infectious diseases in laboratories and clinical institutions

Evolution of Diagnostic and Forensic Microbiology in the Era of Artificial Intelligence.

Diagnostic microbiology plays a vital role in managing infectious diseases, combating antimicrobial resistance, and containment of outbreaks. During the fourth industrial revolution, when artificial intelligence (AI) became an essential part of our day-to-day lives, its integration into healthcare would further revolutionize our knowledge and potential. Although in the budding stage, AI with machine learning is being increasingly utilized in various aspects of diagnostic microbiology. It can handle large datasets that are difficult to analyze manually. Researchers have developed and demonstrated several machine-learning algorithms for interpreting bacterial cultures, conducting image analysis for microbial detection, and predicting antimicrobial susceptibility patterns. Thus, AI may most likely be the ultimate solution to the ever-increasing demand for improved results with shorter turnaround times. AI can also assist forensic microbiologists in crime scene investigations, as it can guide individual identification, cause and time since death, and manner of death. This review summarizes the application of AI in diagnostic microbiology for performing diverse sets of microbial investigations and is an essential aid in forensic microbiology.

A head-to-head comparison of three MALDI-TOF mass spectrometry systems with 16S rRNA gene sequencing.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has revolutionized diagnostics in culture-based microbiology. Commonly used MALDI-TOF MS systems in clinical microbiology laboratories are MALDI Biotyper (Bruker Daltonics) and Vitek MS (bioMérieux), but recently the new EXS2600 (Zybio) has been launched. This study aimed to evaluate the performance of the three devices by comparing the results to 16S rRNA gene sequencing. A set of 356 previously collected difficult-to-identify bacteria was tested in parallel with the three systems. Only the direct smear method and simple formic acid extraction were applied. Valid results were achieved for 98.6%, 94.4%, and 93.3% of all isolates by MALDI Biotyper, EXS2600, and Vitek MS, respectively. Of all valid results, agreement with sequencing data was achieved in 98.9%, 98.5%, and 99.7% by MALDI Biotyper, EXS2600, and Vitek MS, respectively. Considering only the isolates with valid measurements at the single-species level, misidentification rates were 0%, 2.6%, and 1.1% for MALDI Biotyper, EXS2600, and Vitek MS, respectively. Apart from minor performance differences, our data demonstrate that the three systems provide comparable results and are suitable for use in medical diagnostic laboratories.

Agroterrorism involving biological agents and related threats in Poland and Europe in the context of the COVID-19 pandemic and the war in Ukraine

With the growing threat of agroterrorism and the highest level of risk in Poland and the European region since the Biological and Toxin Weapons Convention (1972) and the Additional Protocols to the Geneva Conventions (1977) came into force, it is important to analyse the challenges in the area of biosecurity and food security and make recommendations. The analysis carried out by the author of this article indicates that the COVID-19 pandemic has contributed to the dissemination of knowledge of the basics of microbiology and epidemiology and to the increased availability of low-cost, portable microbiological diagnostics, which may also have negative effects. The analysis took into account the possibility of foreign intelligence influencing food production in Poland, e.g. through disinformation via social media. Conclusions of the analysis include: expanding monitoring of the expert community and social media, strengthening the vigilance of food producers and agricultural experts, simulating introduction scenarios, studying radicalisation processes and using epidemiological assessment tools in case of alarming events.

Detection, Identification and Diagnostic Characterization of the Staphylococcal Small Colony-Variant (SCV) Phenotype.

While modern molecular methods have decisively accelerated and improved microbiological diagnostics, phenotypic variants still pose a challenge for their detection, identification and characterization. This particularly applies if they are unstable and hard to detect, which is the case for the small-colony-variant (SCV) phenotype formed by staphylococci. On solid agar media, staphylococcal SCVs are characterized by tiny colonies with deviant colony morphology. Their reduced growth rate and fundamental metabolic changes are the result of their adaptation to an intracellular lifestyle, regularly leading to specific auxotrophies, such as for menadione, hemin or thymidine. These alterations make SCVs difficult to recognize and render physiological, biochemical and other growth-based methods such as antimicrobial susceptibility testing unreliable or unusable. Therefore, diagnostic procedures require prolonged incubation times and, if possible, confirmation by molecular methods. A special approach is needed for auxotrophy testing. However, standardized protocols for SCV diagnostics are missing. If available, SCVs and their putative parental isolates should be genotyped to determine clonality. Since their detection has significant implications for the treatment of the infection, which is usually chronic and relapsing, SCV findings should be specifically reported, commented on, and managed in close collaboration with the microbiological laboratory and the involved clinicians.

IVDR: Analysis of the Social, Economic, and Practical Consequences of the Application of an Ordinance of the In Vitro Diagnostic Ordinance in Switzerland.

IVDR regulation represents a major challenge for diagnostic microbiology laboratories. IVDR complicates a broad range of aspects and poses a risk given the high diversity of pathogens (including rare but highly virulent microbes) and the large variety of samples submitted for analysis. The regular emergence of new pathogens (including Echovirus E-11, Adenovirus 41, Monkeypox virus, Alongshan virus, and Enterovirus D68, as recent examples in Europe in the post SARS-CoV-2 era) is another factor that makes IVDR regulation risky, because its detrimental effect on production of in-house tests will negatively impact knowledge and expertise in the development of new diagnostic tests. Moreover, such regulations negatively impact the availability of diagnostic tests, especially for neglected pathogens, and has a detrimental effect on the overall costs of the tests. The increased regulatory burden of IVDR may thereby pose an important risk for public health. Taken together, it will have a negative impact on the financial balance of diagnostic microbiology laboratories (especially small ones). The already-high standards of quality management of all ISO-accredited and Swissmedic-authorized laboratories render IVDR law of little value, at least in Switzerland, while tremendously increasing the regulatory burden and associated costs. Eventually, patients will need to pay for diagnostic assays outside of the framework of their insurance in order to obtain a proper diagnostic assessment, which may result in social inequity. Thus, based on the risk assessment outlined above, the coordinated commission for clinical microbiology proposes adjusting the IvDO ordinance by (i) introducing an obligation to be ISO 15189 accredited and (ii) not implementing the IvDO 2028 milestone.

Evaluation of six commercial and in-house phenotypic tests for detection of AmpC β-lactamases: is routine detection possible?

Phenotypic characterization of the prevalent AmpC β-lactamases in clinical isolates is essential for making informed empirical decisions and critical for strengthening antimicrobial stewardship programmes. This study focused on assessing six assays, two in-house and four commercial phenotypic tests for detection of AmpC, to study the feasibility of making its detection a routine diagnostic microbiology laboratory activity. A total of 116 non-duplicate Gram-negative bacteria that were resistant to third-generation cephalosporins and amoxicillin/clavulanic acid, and resistant or susceptible to piperacillin/tazobactam and carbapenems, were screened by cefoxitin discs for AmpC. These isolates were subjected to two in-house (AmpC Tris-EDTA and disc approximation) methods and four commercial tests: D69C AmpC Detection Set; D72C ESBL, AmpC & Carbapenemase Detection Set; combination disc test: ESBL + AmpC Screen Disc Kit; and AmpC MIC Test Strip for confirmation of AmpC production. Ten whole-genome-sequenced AmpC-confirmed Gram-negative isolates were used as positive controls and one as a negative control. The prevalence of AmpC β-lactamases was 16%. Escherichia coli was a major carrier of plasmid-mediated AmpC (26.5%), followed by Klebsiella pneumoniae (23.4%). Phenotypically, 61% of AmpCs were detected by Tris-EDTA (accuracy: 73.8%), 76% by disc approximation (accuracy: 89.2%), 75% by the D69C AmpC Detection Set (accuracy: 95.4%), 74% by the D72C AmpC, ESBL & Carbapenemase Detection Set (accuracy: 95.4%), 76% by the combination disc test (accuracy: 95.4%) and 63% by AmpC MIC Test Strip (accuracy: 87.7%). The sensitivity and specificity of D69C were 97.9% and 88.2%, respectively, and 95.9% and 93.8% for the combination disc test, while for the disc approximation test and D72C they were 93.9% and 75%, and 93.9% and 100%, respectively. Screening by cefoxitin screening was less sensitive (75%) and specific (25%). Disc approximation and the combination disc test detect AmpC in Enterobacterales and also Pseudomonas aeruginosa and Acinetobacter species. We recommend the in-house disc approximation test and the commercial D69C, as well as the combination disc test, as excellent tools for detection of AmpC. The cefoxitin test overcalls AmpC and cannot be considered a good stand-alone test for AmpC detection.

Molecular Identification, Effector genes and Virulence of Isolates of &lt;i&gt;Parastagonospora nodorum&lt;/i&gt; from Altai Krai (Russia)

Septoria blotches represent some of the most harmful wheat diseases that encompass all grain-producing countries worldwide. They are caused mainly by fungi of the genera Parastagonospora and Zymoseptoria. The aims of the research were to analyze the nucleotide sequences of two phylogenetically informative DNA loci, ITS and tub2, of Parastagonospora nodorum isolates from the Altai Krai, to detect the presence of Tox1, Tox3, ToxA effector genes and to study the virulence of the isolates. Microscopic analysis of fragments of affected plant tissue was used for primary identification of the Septoria blotch. The causative agent from the studied plant samples was identified as P. nodorum. Fungal colonies cultivated on potato-glucose agar displayed a well-developed light brown velvety mycelium with dark periphery (mixed type) and numerous pycnidia exhibiting high sporulation capacity. Microbiological diagnostics were complemented by molecular genetic studies. Sequencing of ITS and tub2 loci isolates revealed complete genetic identity of all eight studied monoconidial isolates obtained from different samples. PCR-based detection of tree Tox-genes demonstrated that the only Tox3 was present in the geno-types of 80 tested P. nodorum monoconidial isolates. The ToxA and Tox1 genes were not found in the studied isolates. The virulence of P. nodorum isolates was evaluated in laboratory conditions using an isolated wheat leaves assay. Mixture of four isolates from spring wheat and one isolate from winter wheat, were characterized as virulent. When spring and winter wheat cultivars were infected with two isolates obtained from oats, pathogenic but avirulent properties were detected. The isolate from triticale was avirulent to winter wheat cultivars and virulent to spring wheat cultivars.

Comparison of automatic methods MALDI-TOF, VITEK2 and manual methods for the identification of intestinal microbial communities on the example of samples from alpacas (Vicugna pacos).

Universally, in microbiological diagnostics the detection of live bacteria is essential. Rapid identification of pathogens enables appropriate remedial measures to be taken. The identification of many bacteria simultaneously facilitates the determination of the characteristics of the accompanying microbiota and/or the microbiological complexity of a given environment. The effectiveness of the VITEK2 Compact automated microbial identification system and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS), analytical profile index (API) and Remel RapID tests were compared in identification of bacteria isolated from the alpaca gastrointestinal tract. Most isolates were Gram-positive, such as Bacillus cereus, Bacillus flexus, Bacillus licheniformis, Bacillus pumilus and Bacillus subtilis; Enterococcus faecium, Enterococcus gallinarum, Enterococcus hirae and Enterococcus casseliflavus; Staphylococcus aureus, Staphylococcus equorum, Staphylococcus lentus, Staphylococcus pseudintermedius and Staphylococcus sciuri; Paenibacillus amylolyticus; Cellulosimicrobium cellulans; Leuconostoc mesenteroides; Clostridium perfringens; Corynebacterium stationis, Corynebacterium xerosis, and Corynebacterium diphtheriae (the last only isolated manually by API Coryne and the VITEK2 system and Corynebacteria (CBC) card). Corynebacterium diphtheriae was misidentified by MALDI-TOF MS as Candida lipolytica (currently Yarrowia lipolytica). Gram-positive and Gram-variable Micrococcus luteus were also isolated. Gram-negative Enterobacter cloacae, Enterobacter gergoviae, Enterobacter hormaechei and Enterobacter ludwigii; E. coli; Klebsiella pneumoniae subsp. pneumoniae; Citrobacter braakii and Citrobacter freundii; Serratia liquefaciens, Serratia odorifera and Serratia marcescens; Morganella morganii subsp. morganii; Providencia alcalifaciens; Pseudomonas aeruginosa; Stenotrophomonas maltophilia; Moraxella osloensis; and Ochrobactrum intermedium were also found. The yeasts Candida albicans, Candida haemulonii and Candida ciferrii were also present. MALDI-TOF MS enabled the identification of pathogens and opportunistic pathogens from the alpaca gut which may represent a high risk to human and animal health.

Microbiologic Analysis of Hand Infections: A Prospective Study.

Background: Hand infections are a common problem in emergency departments. Staphylococcus aureus is the main pathogen of both hand and blood stream infections. Therefore, the aim of the present study was to evaluate the frequency and impact of bacteremia in patients with hand infections to improve the microbiologic diagnostics. Patients and Methods: A prospective study of 90 patients with acute hand infections without antimicrobial pre-treatment was performed. Blood cultures were taken pre-operatively. If positive, transesophageal echocardiography was performed to rule out infectious endocarditis. Tissue samples were microbiologically processed using standardized culture media. If negative, a broad-spectrum bacterial 16S ribosomal RNA (rRNA) gene polymerase chain reaction (PCR) was applied. The etiology and location of the infection, the length of hospital stay, the number of surgical interventions, and the inflammatory parameters were obtained. Results: Six patients with bacteremia (6.6%) were diagnosed, after animal bites (n = 3) and intra-articular empyema (n = 3). Pathogens included Staphylococcus pettenkoferi, Pasteurella multocida, Staphylococcus epidermidis, Staphylococcus aureus, and Bacteroides pyogenes. No case of infective endocarditis was detected. Patients with bacteremia required more surgical interventions (p = 0.002), had a longer hospital stay (p < 0.001), higher plasma C-reactive protein (CRP; p = 0.016), and a higher age (p = 0.002) compared with those without bacteremia. In 14 cases (15.6%) no pathogen was detected by culture, whereas the subsequent broad-spectrum PCR diagnosed three cases (21.4%). Conclusions: Pre-operative blood cultures in patients with hand infections are important to detect bacteremia as an essential marker of clinical severity. Those blood cultures are indicated after deep animal bites and joint empyema. A precise identification of pathogens is fundamental for an effective treatment of hand infections, for which a 16S rRNA gene PCR can contribute in culture-negative tissue samples.