Phenotypic Tests Research Articles

Comparative evaluation of phenotypic methods to detect methicillin resistance with mecA gene detection by PCR in Staphylococcus aureus

It is crucial to employ precise, reliable, and rapid detection methods to avoid the indiscriminate use of antimicrobial drugs and make informed decisions regarding appropriate treatment and the execution of efficient measures to prevent infections.: A group of 112 isolates from different clinical specimens, initially identified as using the cefoxitin disc diffusion test, underwent additional phenotypic tests such as the Oxacillin E strip and MRSA CHROM agar. These methods were then evaluated and compared with gene detection via PCR, which is regarded as the gold standard. Of the 112 isolates identified by the cefoxitin disc diffusion test, 101 (90.2%) tested positive for the gene. The Oxacillin E strip had a sensitivity of 98% and a specificity of 91%, while MRSA CHROM agar showed a sensitivity of 96.03% and a specificity of 82%. Among the 101 mec A-positive isolates, 44.5% met the CDC definition criteria for . : Our study concludes that phenotypic methods, including the cefoxitin disc diffusion test, are not completely reliable in detecting methicillin resistance in According to our results, combining the cefoxitin disc diffusion test with the oxacillin E strip is an effective approach for detecting in resource-constrained settings. Given the advantages of PCR, it is recommended to perform PCR for gene detection on a regular basis to identify strains in important clinical specimens.

Rapid detection of carbapenemase production in Aeromonas using phenotypic tests based on colorimetric microtube assay.

Antibiotic resistance, particularly carbapenem resistance, poses a significant global health threat due to the limited availability of effective antibiotics. Carbapenem-resistant Aeromonas are increasingly recognized for their role in various infections, necessitating rapid and accurate detection methods. This study aimed to evaluate several phenotypic tests, including the Carba NP test (CNPt), Carba NP-direct test (CNPd), and Blue-Carba test (BCT), for their effectiveness in rapidly detecting carbapenemase production in Aeromonas. These tests target both the chromosomally encoded CphA metallo-β-lactamase (MBL) and acquired carbapenemases. Additionally, a modified phenotypic test called the Colony-Carba NP test (c-CNPt) was introduced to enhance sensitivity and specificity. A retrospective analysis was conducted on 131 clinically conserved Aeromonas strains harboring identified carbapenem resistance genes, using CNPt, CNPd, BCT, and the newly developed c-CNPt and EDTA-Colony-Carba NP test (ec-CNPt). The stability of c-CNPt reagents stored at -80°C was also assessed. Additionally, a prospective study conducted from July 2021 to November 2023 evaluated 152 Aeromonas isolates to determine the clinical applicability of these tests. Our results demonstrated that CNPd and BCT achieved 100% sensitivity and specificity, surpassing the traditional CNPt, which showed only 63.6% sensitivity for Aeromonas strains. The c-CNPt also showed 100% sensitivity and specificity, with the ec-CNPt effectively differentiating between MBL and serine carbapenemase types. Stability tests confirmed that c-CNPt reagents could be stored at -80℃ for up to 1 year without performance degradation. These findings highlight the practicality and reliability of these phenotypic tests for routine laboratory use, providing a rapid and cost-effective method for detecting carbapenemase production.The rapid detection of carbapenemase production in Aeromonas is of paramount importance due to the significant clinical and public health implications associated with antibiotic resistance. The development and validation of rapid phenotypic tests such as the Colony-Carba NP test (c-CNPt) and the EDTA-Colony-Carba NP test (ec-CNPt) are crucial advancements in the field. These tests offer a highly sensitive and specific method for detecting carbapenemase production in Aeromonas, including the differentiation between metallo-β-lactamase and serine carbapenemases. The c-CNPt and ec-CNPt are cost-effective, easy to perform, and provide rapid results, making them suitable for routine clinical use. Additionally, the stability of the reagents ensures their practicality for long-term application in various healthcare settings. Implementing these phenotypic tests in clinical laboratories can significantly enhance the early detection and appropriate treatment of carbapenem-resistant Aeromonas infections.

Determination of antimicrobial resistance genes in patients with infected miscarriage. A rapid identification and optimized empirical therapy

BACKGROUND: The infectious factor during pregnancy remains the leading cause not only of maternal mortality, but also of premature pregnancy loss and subsequent reproductive function impairment. Patients with infected miscarriage represent a special cohort in terms of complexity of clinical decision-making, while the choice of antimicrobial chemotherapy is particularly difficult in conditions of accumulation of scientific knowledge about the main microbial agents. For adequate treatment, it is essential to use all modern methods of microbiota diagnosis and conduct regular monitoring of microorganism resistance in order to update empirical schemes of antibacterial therapy. AIM: The aim of this study was to evaluate the correlation relationship between antimicrobial resistance genes and identified microbial agents in patients with infected miscarriage in order to optimize empirical antimicrobial chemotherapy. MATERIALS AND METHODS: We prospectively studied 42 samples of genital tract discharge in patients with infected miscarriage. The classical culture method was used to determine microorganism sensitivity to standard antibacterial drugs by the disc diffusion test. A molecular genetic study of the species composition of clinically significant microbial agents and genes of resistance to β-lactam and glycopeptide antibiotics was performed using Femoflor Screen and BakRezista GLA test systems (“DNA-Technology” LLC., Russia), respectively. RESULTS: In the cultural study, one microbial agent was identified in 59.5% of cases, two in 40.0% of cases, and three in 7.1% of cases. The following microorganisms were most frequently identified: Enterococcus spp. in 25.0% of cases, Esherichia coli in 16.7% of cases, and Candida spp. in 11.7% of cases. During the molecular genetic study, Lactobacillus spp. were identified in 64.3% of the samples, the microorganisms associated with bacterial vaginosis in 45.3% of the samples, Candida spp. in 23.8% of the samples, and Herpesviridae family viruses in 14.3% of the samples. In 45.2% of the samples, one to 14 genes of resistance to β-lactam and glycopeptide antibiotics were identified, while Tem (19.0%), Oxa-40-like (19.0%), Stx-M1 (14.3%), Ges (14.3%), Oxa-51-like (11.9%), and Van A/B (9.5%) were most frequently determined in the samples positive for resistance genes. Comparison between phenotypic susceptibility testing and genetic prediction for antibiotics resistance demonstrated that the specificity varied from 96.4 to 100%, and the sensitivity from 40.0 to 85.7%. CONCLUSIONS: The use of molecular genetic diagnostic methods allows for the identification of clinically significant urogenital tract infections and the detection of markers of their resistance to β-lactam antibiotics and glycopeptides in the shortest possible time. The data obtained make it possible to optimize the initial antimicrobial chemotherapy.

Comprehensive pathogen identification and antimicrobial resistance prediction from positive blood cultures using nanopore sequencing technology

BackgroundBlood cultures are essential for diagnosing bloodstream infections, but current phenotypic tests for antimicrobial resistance (AMR) provide limited information. Oxford Nanopore Technologies introduces nanopore sequencing with adaptive sampling, capable of real-time host genome depletion, yet its application directly from blood cultures remains unexplored. This study aimed to identify pathogens and predict AMR using nanopore sequencing.MethodsIn this cross-sectional genomic study, 458 positive blood cultures from bloodstream infection patients in central Taiwan were analyzed. Parallel experiments involved routine microbiologic tests and nanopore sequencing with a 15-h run. A bioinformatic pipeline was proposed to analyze the real-time sequencing reads. Subsequently, a comparative analysis was performed to evaluate the performance of species identification and AMR prediction.ResultsThe pipeline identified 76 species, with 88 Escherichia coli, 74 Klebsiella pneumoniae, 43 Staphylococcus aureus, and 9 Candida samples. Novel species were also discovered. Notably, precise species identification was achieved not only for monomicrobial infections but also for polymicrobial infections, which was detected in 23 samples and further confirmed by full-length 16S rRNA amplicon sequencing. Using a modified ResFinder database, AMR predictions showed a categorical agreement rate exceeding 90% (3799/4195) for monomicrobial infections, with minimal very major errors observed for K. pneumoniae (2/186, 1.1%) and S. aureus (1/90, 1.1%).ConclusionsNanopore sequencing with adaptive sampling can directly analyze positive blood cultures, facilitating pathogen detection, AMR prediction, and outbreak investigation. Integrating nanopore sequencing into clinical practices signifies a revolutionary advancement in managing bloodstream infections, offering an effective antimicrobial stewardship strategy, and improving patient outcomes.

Artesunate remarkably alleviates interstitial cystitis/bladder pain syndrome in a murine model: Novel molecular signature deciphered by bulk tissue and single-cell transcriptomic analysis.

Interstitial cystitis/bladder pain syndrome is a chronic pain syndrome of elusive etiology, accompanied by lower urinary tract symptoms. Over the past decades, many studies have been carried out for exploration of more effective therapies against IC/BPS. However, the results have been inconsistent, probably due to the multifactorial nature of IC/BPS. We establish a model of IC/BPS in mice by combining protamine sulfate /lipopolysaccharide and phenylephrine. Typical histological changes and symptoms were observed. We then explored the effectiveness of artesunate (ART), which has been reported to alleviate autoimmune diseases. Phenotypic tests demonstrated a significant reduction in symptoms. Histological staining showed pathological improvement. WGCNA identified three gene modules specifically related to IC/BPS, and six genes were identified as hub genes. CIBERSORT analysis showed that the activated NK cells seem to be decreased in IC modeling group and partially restored in IC + ART group, whereas the resting NK cells showed the opposite trend. Single-cell transcriptomic analysis elaborated on the changing trends of subgroups of infiltrated immune cells, including T cells, NK cells, and dendritic cells. Our study represents our effort in establishing a reliable and reproducible IC/BPS murine model, and the first study using scRNA-seq in exploring the immune microenvironment of the IC/BPS murine model, and the possible molecular mechanisms of ART treatment in IC/BPS. Further studies are needed to confirm the effect of ART in IC/BPS patients.

Phenotypic and genotypic characteristics of Trueperella abortisuis recovered from the feline reproductive tract

Large numbers of infections and effective treatment or control depend on the ability to identify the causative agents of animal infections. Here we describe a study of an isolate DG22/1209/12 obtained from vaginal pus exudate collected from a Siamese cat. The isolate was believed to be a member of Trueperella abortisuis, which is related to problems with reproduction. The most important was to identify this isolate by applying different techniques of diagnosis. It included phenotypic testing, Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS), and molecular techniques such as amplification and sequencing of the 16S rRNA gene, intergenic spacer region (ISR), and the gap and tuf genes. Phenotypic tests confirmed the identification of DG22/1209/12 as T. abortisuis. Finally, the identification was confirmed with MALDI-TOF MS analysis which demonstrated log (score) value of 2.09 indicating a secure genus identification, probable species identification on T. abortisuis. Molecular analysis revealed a high sequence identity of 99.9 %, 99.5 %, 99.4 %, and 99.5 % for the 16S rRNA gene, ISR, gap, and tuf genes, respectively, for both the isolate DG22/1209/12 and the reference isolate T. abortisuis DSM 19515T. The study highlights the effectiveness of combining phenotypic and genomic methods to accurately identify bacterial pathogens in veterinary medicine, leading towards focused intervention strategies.

Consensus of the Italian Primary Immunodeficiency Network on the use and interpretation of genetic testing for the diagnosis of inborn errors of immunity (IEI)

BackgroundInborn errors of immunity (IEIs) are more than 500 different rare congenital disorders of the immune system characterized by susceptibility to infections and immune dysregulation. The significant overlap of the clinical features among the different forms may lead to diagnostic delay. High throughput sequencing techniques may allow a timely genetic definition. Guidelines for the use and the interpretation of genetic testing produced by the American College of Medical Genetics and Genomics (ACMG) and the European Society of Human Genetics (ESHG) do not cover specificities for the application to IEIs. ObjectiveThe aim of this consensus study is to define the best approach to genetic testing for IEIs. MethodsA panel of experts in the context of the Italian Primary Immunodeficiency Network (IPINet) composed a list of statements that were evaluated using Delphi method. ResultsThe experts recommend that genetic testing for IEIs should be offered to selected patients with warning signs for IEIs and highlight the crucial role of thorough phenotyping and functional tests for the conclusive diagnosis of IEI. Comprehensive educational programs targeted to health care professionals and the public should be developed to increase IEIs awareness and reduce the diagnostic delay. Ethical issues should be pondered over the diagnostic advantages of genetic tests requested for diagnostic purposes. ConclusionAdherence to the guidelines on the use and interpretation of genetic testing for the diagnosis of IEIs should help limiting the inappropriate use of these techniques and reduce the risk of misdiagnosis and apprehension for inconclusive genetic results.

Prevalence of Atypical Bacteria in Patients from Different Paediatric Age Groups Diagnosed with a Respiratory Disease.

Atypical bacterial pathogens present the ability to induce pulmonary damage. At present, there are no available phenotypic diagnosis tests that achieve up to 100% reliability. Therefore, clinicians must utilise molecular techniques for the detection and identification of these pathogens. The main objective of this research was to evaluate the prevalence of atypical bacteria in paediatric patients from different age groups. A total of 609 clinical samples were collected from paediatric patients who presented with an adverse respiratory condition during the period from March 2021 to February 2024. DNA was extracted from the samples, and end-point PCR was performed to detect atypical bacteria. Statistical analyses were performed to evaluate the bacterial prevalence and assess clinical data from newborns and mothers that could be related to RDS. A total of 139 patients exhibited at least one atypical organism (22.82%). Ureaplasma parvum was more prevalent in neonates, while M. pneumoniae and C. pneumoniae were more prevalent in older infants. Atypical bacteria can be present in all seasons of the year, but their prevalence increases during hot weather. Mixed infections due to atypical bacteria may occur. The risk factors related to the development of RDS are prematurity, low weight, and orotracheal intubation.

Interrelation Between Pathoadaptability Factors and Crispr-Element Patterns in the Genomes of Escherichia coli Isolates Collected from Healthy Puerperant Women in Ural Region, Russia.

Escherichia coli is a commensal and opportunistic bacterium widely distributed around the world in different niches including intestinal of humans and animals, and its extraordinary genome plasticity led to the emergence of pathogenic strains causing a wide range of diseases. E. coli is one of the monitored species in maternity hospitals, being the main etiological agent of urogenital infections, endometriosis, puerperal sepsis, and neonatal diseases. This study presents a comprehensive analysis of E. coli isolates obtained from the maternal birth canal of healthy puerperant women 3-4 days after labor. According to whole genome sequencing data, 31 sequence types and six phylogenetic groups characterized the collection containing 53 isolates. The majority of the isolates belonged to the B2 phylogroup. The data also includes phenotypic and genotypic antibiotic resistance profiles, virulence factors, and plasmid replicons. Phenotypic and genotypic antibiotic resistance testing did not demonstrate extensive drug resistance traits except for two multidrug-resistant E. coli isolates. The pathogenic factors revealed in silico were assessed with respect to CRISPR-element patterns. Multiparametric and correlation analyses were conducted to study the interrelation of different pathoadaptability factors, including antimicrobial resistance and virulence genomic determinants carried by the isolates under investigation. The data presented will serve as a valuable addition to further scientific investigations in the field of bacterial pathoadaptability, especially in studying the role of CRISPR/Cas systems in the E. coli genome plasticity and evolution.

Multilocus sequence typing of clinical Burkholderia pseudomallei isolates from Cambodia.

Melioidosis is a neglected tropical disease caused by Burkholderia pseudomallei, endemic to Southeast Asia and Northern Australia. Despite its increasing global public health and clinical significance, the molecular epidemiology of melioidosis and genetic diversity of B. pseudomallei in Cambodia remains poorly understood. This study aims to elucidate the genetic diversity and antibiotic susceptibility profiles of B. pseudomallei isolates responsible for melioidosis in humans. For this purpose, 14 clinical isolates cryopreserved at the Medical Biology Laboratory at Institut Pasteur du Cambodge from 2016 to 2020 were subjected to antimicrobial susceptibility testing and Multilocus Sequence Typing (MLST). Phenotypic testing revealed that 92.86% (13/14) of the isolates were sensitive to all tested antibiotics, while one isolate exhibited resistance to trimethoprim-sulfamethoxazole. MLST analysis resolved our isolates into 14 unique Sequence Types (STs), including 10 previously documented in Southeast Asia. Notably, ST1858, ST2064, ST2065, and ST2066 were identified as novel STs, while ST54, ST99, ST211, and ST1359 were reported in Cambodia for the first time in this study. Comparing our MLST data with available sequences on PubMLST (n = 165), our study unveiled a high genetic diversity of B. pseudomallei in Cambodia. The identified STs were closely associated with isolates from other Southeast Asian countries, particularly Thailand, Vietnam, and Malaysia. In conclusion, this study provided insight into the genetic diversity among B. pseudomallei clinical isolates in Cambodia and their close genetic association with Southeast Asian isolates. To further our understanding, a One Health approach, incorporating human, environmental (mainly soil), and animal compartments, is essential to decipher the epidemiology of B. pseudomallei in Cambodia.

Phenotypic Resistance to Rifampicin of Mycobacterium tuberculosis with the rpoB Leu430Pro Mutation

The objective: to determine the role of the rpoB Leu430Pro mutation in the degree of phenotypic resistance of Mycobacterium tuberculosis to rifampicin.Subjects and Methods. The WHO classifies the rpoB Leu430Pro mutation of Mycobacterium tuberculosis as a borderline resistance mutation but of clinical significance. From an array of cultures, we selected and analyzed 14 isolates of M. tuberculosis with discrepancies in the results of molecular genetic and phenotypic testing, carrying only this mutation in rpoB. For all samples, the phenotypic resistance level (MIC) of these isolates to rifampicin was determined using BACTEC MGIT 960 and Middlebrook 7H11 medium.Results. It was found out that 12 of 14 isolates had the rifampicin MIC below the current critical concentration when tested by both methods, thus they were phenotypically sensitive. One isolate was resistant when tested with Middlebrook 7H11 but susceptible when tested with BACTEC MGIT 960. Only one isolate which had the additional rpoB F425L mutation demonstrated high-level phenotypic resistance when tested by the same tests. Our data indicate that the clinical significance of this mutation requires clarification since even a decrease in the critical concentration of rifampicin does not lead to unambiguous results of molecular genetic and phenotypic tests. It is necessary to standardize the use of various molecular genetic tests and principles of their clinical interpretation to optimize strategies for managing patients with tuberculosis caused by M. tuberculosis with the rpoB Leu430Pro mutation.

Investigation of Antibiotic Resistance of E. coli Associated with Farm Animal Feces with Participation of Citizen Scientists.

This paper presents the findings of a large-scale study on antibiotic resistance in bacteria found in farm animal feces across Russia. The study included 6578 samples of farm animal manure from 13 regions in Russia, with the help of citizen scientists. Molecular and microbiological methods were used to analyze 1111 samples of E. coli. The microbiological analysis focused on culturing the microorganisms present in the fecal samples on selective media for E. coli and evaluating the sensitivity of the bacteria to different antibiotics, including ampicillin, tetracycline, chloramphenicol, cefotaxime, and ciprofloxacin. The molecular analysis involved isolating the genomic DNA of the bacteria and conducting PCR assays to detect the vanA, vanB, and mcr-1 antibiotic resistance genes. The results demonstrated significant differences in antibiotic sensitivity of the samples that are morphologically identical to E. coli from different regions. For example, 98.0% and 82.5% of E. coli and other fecal bacterial isolates from the Omsk and Vologda regions lacked antibiotic resistance genes, while 97.7% of samples from the Voronezh region possessed three resistance genes simultaneously. The phenotypic antibiotic sensitivity test also revealed regional differences. For instance, 98.1% of fecal bacterial samples from cattle in the Udmurt Republic were sensitive to all five antibiotics tested, whereas 92.8% of samples from the Voronezh region showed resistance to all five antibiotics. The high level of antibiotic resistance observed may be attributed to their use in farming practices. The distinctive feature of our research is that comprehensive geographical coverage was achieved by using a citizen science platform. Citizen scientists, specifically students from colleges and universities, were responsible for the collection and initial analysis of samples. The project attracted 3096 student participants, enabling the collection and analysis of a significant number of samples from various locations in Russia.

Campylobacter sputorum subsp. bovis subsp. nov., isolated from cattle, and an emended description of Campylobacter sputorum.

Six urease-negative Campylobacter strains were isolated from cattle faeces over a 19-month period from 2009 to 2010. These strains were initially identified as Campylobacter sputorum by 16S rRNA gene and atpA typing. Initial studies characterizing these strains by multilocus sequence typing and genome sequencing further supported their classification as C. sputorum but indicated that these strains form a divergent clade within the species. A polyphasic study was undertaken here to clarify their taxonomic position. Phylogenetic analyses were performed based on 16S rRNA gene sequences and the concatenated sequences of 330 core genes, with the latter analysis also placing the six strains into a clade distinct from the three C. sputorum biovars. Pairwise digital DNA-DNA hybridization values identified these strains as C. sputorum, and the pairwise average nucleotide identity values were consistent with those observed between current Campylobacter subspecies pairs. Standard phenotypic testing was also performed. All strains are microaerobic, anaerobic, motile, Gram-negative and oxidase- and catalase-positive; cells are curved rods or spirals. Strains can be distinguished from the C. sputorum biovars by the presence of alkaline phosphatase activity and triphenyltetrazolium chloride reduction and absence of nitrate reduction. The data presented here show that these strains represent a novel subspecies within C. sputorum, for which the name C. sputorum subsp. bovis subsp. nov. (type strain RM8705T=LMG 32300T=CCUG 75470T) is proposed.

A novel LAMP-based assay for the identification of Streptococcus pneumoniae and Streptococcus pseudopneumoniae in clinical isolates.

The Gram-positive bacteria Streptococcus pneumoniae is part of the Streptococcus mitis group (SMG) and causes life-threatening infections, such as pneumonia, sepsis, and meningitis. The closely related Streptococcus pseudopneumoniae has recently been shown to cause respiratory tract infections, as well as invasive infections, especially in patients with comorbidities. Due to the genetic and phenotypic similarities of species belonging to the SMG, the identification of S. pneumoniae and S. pseudopneumoniae is difficult and unreliable using phenotypic tests, as well as matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and 16S rRNA sequencing. In this study, a loop-mediated isothermal amplification (LAMP)-based assay was developed using molecular markers specific for S. pneumoniae (SPN0001) and S. pseudopneumoniae (SPPN_RS10375). The LAMP assay was evaluated using a collection of SMG clinical isolates, concluding that the method provides a correct, reliable, and fast identification of both S. pneumoniae and S. pseudopneumoniae clinical isolates.

A case of CDKL5 deficiency disorder with a novel intragenic multi-exonic duplication

We present a case of suspected CDKL5 deficiency disorder (CDD) in which a novel intragenic multi-exonic duplication in the CDKL5 gene was identified using next-generation sequencing and multiple ligation-dependent probe amplification. This duplication was assumed to result in a shift of the reading frame and the introduction of a premature stop codon. This case highlights the importance of careful phenotyping and comprehensive genetic testing to detect rare structural variants in CDD patients.

Leveraging large-scale Mycobacterium tuberculosis whole genome sequence data to characterise drug-resistant mutations using machine learning and statistical approaches

Tuberculosis disease (TB), caused by Mycobacterium tuberculosis (Mtb), is a major global public health problem, resulting in > 1 million deaths each year. Drug resistance (DR), including the multi-drug form (MDR-TB), is challenging control of the disease. Whilst many DR mutations in the Mtb genome are known, analysis of large datasets generated using whole genome sequencing (WGS) platforms can reveal new variants through the assessment of genotype-phenotype associations. Here, we apply tree-based ensemble methods to a dataset comprised of 35,777 Mtb WGS and phenotypic drug-susceptibility test data across first- and second-line drugs. We compare model performance across models trained using mutations in drug-specific regions and genome-wide variants, and find high predictive ability for both first-line (area under ROC curve (AUC); range 88.3–96.5) and second-line (AUC range 84.1–95.4) drugs. To aggregate information from low-frequency variants, we pool mutations by functional impact and observe large improvements in predictive accuracy (e.g., sensitivity: pyrazinamide + 25%; ethionamide + 10%). We further characterise loss-of-function mutations observed in resistant phenotypes, uncovering putative markers of resistance (e.g., ndh 293dupG, Rv3861 78delC). Finally, we profile the distribution of known DR-associated single nucleotide polymorphisms across discretised minimum inhibitory concentration (MIC) data generated from phenotypic testing (n = 12,066), and identify mutations associated with highly resistant phenotypes (e.g., inhA − 779G > T and 62T > C). Overall, our work demonstrates that applying machine learning to large-scale WGS data is useful for providing insights into predicting Mtb binary drug resistance and MIC phenotypes, thereby potentially assisting diagnosis and treatment decision-making for infection control.

A comparison of two MALDI-TOF MS based assays for the detection of carbapenemases in Enterobacterales

Carbapenem resistant (CRE) and carbapenemase producing Enterobacterales (CPE) in particular, represent a major threat for healthcare systems worldwide. Rapid, reliable, and easy to perform assays are required to enable targeted and effective therapy. MALDI-TOF MS based carbapenemase diagnostics has potential advantages over molecular and phenotypic sensitivity tests, especially in terms of time to result. So far, only one mass spectrometry (MS)-based carbapenemase test system is commercially available for routine use. The aim of this study was to compare the performance of the established system to a novel MS-based test to identify CPE isolates. Forty consecutive CRE isolates (70% CPEs) were pre-screened for carbapenemase activity by routine laboratory methods. Isolates then were tested using the to date only IVD CE certified MALDI-TOF MS carbapenemase detection assay (MBT STAR-Carba IVD Kit; Bruker Daltonics) and a novel test designed for the recently launched EXS2600 MALDI-TOF MS system (Carbapenemase Activity Kit; Zybio). Valid results were obtained for 93% and 85% isolates by the Bruker and the Zybio assay, respectively. Sensitivities, specificities, positive and negative predictive values were 92%, 91%, 96%, and 83% for the Bruker assay and 96%, 64%, 85%, and 88% for the Zybio assay. There are notable differences concerning the handling of the assays. In summary, both systems featured high sensitivities for the detection of carbapenemases, but the Bruker assay yielded less false-positive results. There are advantages and disadvantages concerning the handling for each system, but both proved to be suitable for the use in a routine laboratory.

Characterizing Escherichia coli carrying plasmid-mediated AmpC β-lactamases to optimize detection in a diagnostic laboratory setting.

Plasmid-mediated AmpC β-lactamases are a cause of acquired cephalosporin resistance in Gram-negative bacteria. However, consensus regarding the optimal detection method is yet to be achieved and varies depending on local epidemiology and laboratory capacity. We determined the acquired genotypic resistance mechanisms of 250 Escherichia coli isolates with a positive AmpC screen, defined as cefoxitin MIC ≥ 8 mg/L and a positive AmpC double- disc diffusion test, using in-house designed high-resolution melt PCR, detecting plasmid-acquired genes from the CIT and DHA families. A proportion of these isolates (n = 170, 68%) underwent further genotypic characterization using whole- genome sequencing (WGS). Of 250 isolates with a positive screening test, 72 (28.8%) were determined to carry an acquired AmpC gene. There was 100% concordance between PCR and WGS in the identification of acquired AmpC genes. The phenotypic criteria were then assessed to determine their utility in predicting acquired AmpC gene carriage. Criteria 1 (cefoxitin MIC > 8 mg/L plus ceftriaxone MIC > 1 mg/L and/or ceftazidime MIC > 1 mg/L) yielded a sensitivity of 93.1% and a specificity of 47.8%. Criteria 2 (cefoxitin MIC > 16 mg/L plus ceftriaxone MIC > 4 mg/L) had a sensitivity of 33.3% and a specificity of 98.9%. DHA genes, whose expression may be induced following antibiotic exposure, were present in 19% of isolates testing susceptible to ceftriaxone (MIC ≤ 1 mg/L) and were significantly more likely than CIT genes to be detected in susceptible isolates (P < 0.0001). These findings highlight the importance of using genotypic methods to detect acquired AmpC resistance in E. coli isolates that meet phenotypic screening criteria.IMPORTANCEDetection of transmissible AmpC resistance remains a challenging problem for diagnostic laboratories, especially in Escherichia coli where the expression of its intrinsic AmpC gene can result in phenotypic resistance patterns indistinguishable from plasmid-mediated resistance. In conjunction with whole- genome sequencing (WGS), we describe the development and performance of a novel melt-curve PCR to identify the two most prevalent plasmid-mediated AmpC gene families: CIT and DHA. We then describe phenotypic testing algorithms that incorporate this PCR and can differentiate these from non-acquired resistance in E. coli. It is important to distinguish these, not only to spare patients from unnecessarily being treated with infection control precautions, but also to identify plasmid-mediated genes, especially of the DHA family, that have been associated with inducible drug resistance to third- generation cephalosporins.

High frequency of acquired virulence factors in carbapenemase-producing Klebsiella pneumoniae isolates from a large German university hospital, 2013-2021.

Carbapenemase-producing Klebsiella pneumoniae (CP-Kp) isolates are a public health concern as they can cause severe hospital-acquired infections that are difficult to treat. It has recently been shown that CP-Kp can take up virulence factors from hypervirulent K. pneumoniae lineages. In this study, 109 clinical CP-Kp isolates from the University Hospital Cologne were examined for the presence of acquired virulence factors using whole-genome sequencing and phenotypic tests, and results were linked to clinical data. The virulence factor iuc was present in 18/109 of the CP-Kp isolates. Other acquired virulence factors, such as ybt, cbt, iro, rmpA/rmpA2, peg-344, and hypervirulence-associated capsule types were detected in various combinations among these isolates. The iuc-positive isolates produced OXA-232 (n = 7), OXA-48 (n = 6), OXA-48+NDM (n = 3), NDM, and KPC (each n = 1), and 7/18 isolates were resistant to ceftazidime-avibactam, colistin, and/or cefiderocol. Four isolates carried hybrid plasmids that harbored acquired virulence factors alongside the carbapenemase genes blaNDM-1/5 or blaOXA-48. In 15/18 patients, iuc-positive CP-Kp were isolated from a clinically manifest infection site. Among these, four patients had osteomyelitis, and four patients died from pneumonia with OXA-232-producing ST231 isolates, three of them as part of an outbreak. In conclusion, acquired virulence factors are frequently detected in various combinations in carbapenemase-producing K. pneumoniae isolates in Germany, warranting continuous monitoring of infections caused by these strains.

Psychrobacter sanguinis infection in a pediatric patient with craniopharyngioma identified in a blood culture by 16S rRNA sequencing

AbstractPsychrobacter species are gram‐negative bacteria in the Moraxellaceae family. These bacteria are considered rare opportunistic human pathogens, and the infection sites include blood, cerebral spinal fluid, wounds, urine, the ears, and the eyes. Few cases of human infection by these species have been described previously. We report a case of a 10‐year‐old boy with postneurosurgical bacteremia due to Psychrobacter sanguinis infection. This infection was difficult to identify using routine biochemical phenotypical tests. Sequencing of 16S rRNA was performed to identify this pathogen. The patient was successfully treated with antibiotics. In conclusion, P. sanguinis infections are rare but should be considered when cultures remain negative for common pathogens.