MALDI-TOF MS Research Articles

Pioneering Klebsiella Pneumoniae Antibiotic Resistance Prediction With Artificial Intelligence-Clinical Decision Support System–Enhanced Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry: Retrospective Study

Background The rising prevalence and swift spread of multidrug-resistant gram-negative bacteria (MDR-GNB), especially Klebsiella pneumoniae (KP), present a critical global health threat highlighted by the World Health Organization, with mortality rates soaring approximately 50% with inappropriate antimicrobial treatment. Objective This study aims to advance a novel strategy to develop an artificial intelligence-clinical decision support system (AI-CDSS) that combines machine learning (ML) with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), aiming to significantly improve the accuracy and speed of diagnosing antibiotic resistance, directly addressing the grave health risks posed by the widespread dissemination of pan drug-resistant gram-negative bacteria across numerous countries. Methods A comprehensive dataset comprising 165,299 bacterial specimens and 11,996 KP isolates was meticulously analyzed using MALDI-TOF MS technology. Advanced ML algorithms were harnessed to sculpt predictive models that ascertain resistance to quintessential antibiotics, particularly levofloxacin and ciprofloxacin, by using the amassed spectral data. Results Our ML models revealed remarkable proficiency in forecasting antibiotic resistance, with the random forest classifier emerging as particularly effective in predicting resistance to both levofloxacin and ciprofloxacin, achieving the highest area under the curve of 0.95. Performance metrics across different models, including accuracy, sensitivity, specificity, positive predictive value, negative predictive value, and F1-score, were detailed, underlining the potential of these algorithms in aiding the development of precision treatment strategies. Conclusions This investigation highlights the synergy between MALDI-TOF MS and ML as a beacon of hope against the escalating threat of antibiotic resistance. The advent of AI-CDSS heralds a new era in clinical diagnostics, promising a future in which rapid and accurate resistance prediction becomes a cornerstone in combating infectious diseases. Through this innovative approach, we answered the challenge posed by KP and other multidrug-resistant pathogens, marking a significant milestone in our journey toward global health security.

Active Surveillance of Patients Colonized with CRE: A Single-Center Study Based on a Combined Molecular/Culture Protocol

Background/Objectives: Carbapenem-resistant Enterobacteriaceae (CRE) are types of bacteria that need urgent attention globally. Active surveillance programs at hospitals are essential for the early identification of CRE carriers and the timely adoption of infection control measures. We aimed to analyze the epidemiology of CRE identified by multiplex RT-PCR in rectal swabs of patients upon admission to high-risk wards and to compare data obtained from both molecular and culture CRE screening. Methods: A total of 2861 rectal swabs, prospectively collected within 12–24 h of admission, underwent molecular screening for identification of K. pneumoniae carbapenemase (KPC), New Delhi metallo-β-lactamase (NDM), Verona integron-mediated metallo-β-lactamase (VIM), imipenemase (IMP), and OXA-48 (AllplexTM Entero-DR Assay). Only samples that tested positive or invalid underwent culture testing (Agar MacConkey and CHROMID® CARBA plates, bioMérieux, Craponne, France). Results: A total of 118 out of 2861 (about 4%) were positive for at least one carbapenem-resistant gene by a molecular approach (MA), with KPC, NDM, and VIM having the highest prevalence. Culture testing confirmed the presence of carbapenemase in 89 samples (75.4%), showing a disagreement rate of about 25% between the two methods, which, unfortunately, rises up to 60% for VIM. The dominant bacterial species were K. pneumoniae and E. coli (MALDI-TOF mass spectrometry). Conclusions: Our data underlined the need for the molecular screening of CRE carriers in order to implement active surveillance protocol in critical care settings and to improve infection control measures.

Pasteurella multocida from deep nasal swabs and tracheobronchial lavage in bovine calves from Sweden

BackgroundBovine respiratory disease (BRD) is common in intensively raised cattle and is often treated with antibiotics. For practitioners, knowledge of the bacteria involved in an outbreak and their antibiotic susceptibility is warranted. To this end, samples from the upper or lower respiratory tract of calves can be submitted for bacteriological culture and susceptibility testing of relevant isolates. However, it is debated whether isolates from the upper respiratory tract are representative of bacteria causing infections in the lower respiratory tract. In this study, we used MALDI-TOF MS, multilocus sequence typing (MLST) and core-genome multilocus sequence typing (cgMLST) to compare culture results of 219 paired samples (sample pairs) of deep nasal swabs (DNS) and tracheobronchial lavage (TBL). The sample pairs came from 171 calves in 30 calf groups across 25 farms with 48 calves sampled twice.ResultsThe predominant bacterial pathogen was Pasteurella multocida, which was isolated from 37.4% of DNS and 22.4% of TBL. There was no statistically significant difference in isolation frequency of P. multocida between calves considered healthy and those suspected for BRD for DNS (P = 0.778) or TBL (P = 0.410). Among the 49 sample pairs where P. multocida was isolated from TBL, the same species was isolated from DNS in 29 sample pairs (59.2%). Isolates from 28 of these sample pairs were evaluated by MLST, and in 24 pairs (86.0%) P. multocida from DNS and TBL were of the same sequence type (ST). Moreover, cgMLST showed that the genetic distance between isolates within 21 of the 28 sample pairs (75.0%), was less than two alleles, and DNS and TBL isolates were considered identical. In seven sample pairs (25%), the genetic distance was greater, and DNS and TBL isolates were considered nonidentical.ConclusionsPasteurella multocida was readily isolated from DNS and in calves where this species was isolated also from TBL, DNS and TBL isolates were identical in 75% of the sample pairs. This suggests that during an outbreak of BRD, submission of DNS samples from 4 to 6 calves could be a convenient approach for practitioners seeking guidance on P. multocida present in the lower respiratory tract and their antibiotic susceptibility.

First documentation of a clinical multidrug-resistant Enterobacter chuandaensis ST2493 isolate co-harboring blaNDM-1 and two blaKPC-2 bearing plasmids.

The increasing prevalence of carbapenem-resistant Enterobacter cloacae complex (CREC) poses great challenges to infection treatment in the clinical setting. In this study, we reported the emergence of carbapenemase in a rare species, Enterobacter chuandaensis, belonging to the Enterobacter cloacae complex (ECC). We elucidated the genetic characteristics of carbapenem-resistant isolate FAHZZU5885, co-harboring blaNDM-1 and blaKPC-2. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and average nucleotide identity (ANI) analysis were used to identify E. chuandaensis. S1 nuclease pulsed-field gel electrophoresis (S1-PFGE) and Southern blotting were used to clarify the number and size of the plasmids in FAHZZU5885. Antimicrobial phenotypes were identified by antimicrobial susceptibility testing (AST), and the characteristics of the strain were examined with whole-genome sequencing (WGS). The conjugation experiment and stability assay were conducted to verify the transferability and stability of the plasmid carrying carbapenemase-encoding genes. E. chuandaensis FAHZZU5885 was isolated from a perianal swab of a patient admitted to the ICU. This strain simultaneously carried blaNDM-1 and two blaKPC-2 genes. FAHZZU5885 was resistant to most of the tested antibiotics except for amikacin, tigecycline, and colistin. Two blaKPC-2 were located separately on two different plasmids, the ~ 120kb IncFIA-IncFII plasmid and the ~ 80kb IncR plasmid. Both plasmids shared the conserved sequence klcA-korC-ISkpn6-blaKPC-2-ISkpn27-tnpR-tnpA. The blaNDM-1-bearing plasmid had the potential to transfer and can remain stable after successive passages. In addition, the blaNDM-1 was carried on a ~ 80kb IncFII plasmid with the conserved sequence ISAba125-blaNDM-1-ble-trpF-dsbD-cutA-groS-groL. In summary, this study marks the first report of the multidrug-resistant E. chuandaensis strain FAHZZU5885 harboring two blaKPC-2-bearing plasmids, indicating the potential for the further dissemination of carbapenemase-encoding genes in novel species. The findings contribute to enhancing our understanding of CREC strains, emphasizing the need for continued and comprehensive surveillance of this species.

A magnetic epitope-imprinted microsphere used for selective separation and rapid detection of SHV-type β-lactamases in bacteria: a novel strategy of antimicrobial resistance detection

BackgroundThe production of β-lactamases is the most prevalent resistance mechanism for β-lactam antibiotics in Gram-negative bacteria. Presently, over 4900 β-lactamases have been discovered, and they are categorized into hundreds of families. In each enzyme family, amino acid substitutions result in subtle changes to enzyme hydrolysis profiles; in contrast, certain conserved sequences retained by all of the family members can serve as important markers for enzyme family identification.ResultsThe SHV family was chosen as the study object. First, a unique 10-mer peptide was identified as SHV family's epitope by an approach of protein fingerprint analysis. Then, an SHV-specific magnetic epitope-imprinted gel polymer (MEI-GP) was prepared by an epitope surface imprinting technique, and its sorption behavior and recognition mechanism for template epitope and SHV were both elaborated. Finally, the MEI-GP was successfully applied to selectively extract SHV from bacteria, and the extracted SHV was submitted to MALDI-TOF MS for specific determination. By following this strategy, other β-lactamase families can also be specifically detected. According to the molecular weight displayed in mass spectra, the kind of β-lactamase and its associated hydrolysis profile on β-lactams can be easily identified. Based on this, an initial drug option scheme can be quickly formulated for antimicrobial therapy. From protein extraction to medication guidance reporting, the mean time to detection (MTTD) was less than 2 h, which is much faster than conventional phenotype-based methods (at least 16–20 h) and gene-based techniques (usually about 8 h).ConclusionsThis enzyme-specific detection strategy combined the specificity of epitope imprinting with the sensitivity of mass spectrometry, enabling β-lactamase to be selectively extracted from bacteria and clearly presented in mass spectra. Compared with other drug resistance detection methods, this technique has good specificity, high sensitivity (≤ 15 mg of bacteria), a short MTTD (less than 2 h), and simple operation, and therefore has a broad application prospect in clinical medicine.Graphical

Phlebotomine sand fly survey, blood meal source identification, and description of Sergentomyia imihra n. sp. in the central Sahara of Algeria

BackgroundPhlebotomine sand flies (Diptera: Psychodidae) are important vectors of various pathogens, mainly Leishmania parasites. In the Old World, the most important genus in term of pathogens transmission is the genus Phlebotomus, which includes many proven or suspected vectors of several Leishmania species, while the genus Sergentomyia remains so far unproven as a vector of human pathogens. Algeria is one of the most affected countries by human leishmaniasis.MethodsIn the present study, an entomological survey was carried out in two provinces, Ghardaïa and Illizi, located in the north and central Sahara, respectively, where cases of human leishmaniasis are recorded. Our goal was to understand the role of the local sand fly species in the transmission of Leishmania parasites and to analyze their blood meal preferences. Collected sand flies were identified by a combination of morphological and molecular approaches that included DNA-barcoding and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) protein profiling. In addition, female blood meals were analyzed by peptide mass mapping using MALDI-TOF MS.ResultsIn total, 640 sand fly specimens belonging to Phlebotomus and Sergentomyia genera were collected in the two provinces. Sergentomyia antennata and Se. fallax were most abundant species in Ghardaïa, and Ph. papatasi and Ph. alexandri in Illizi. In addition, a new sand fly species was described in Illizi named Sergentomyia (Sergentomyia) imihra n. sp. Blood meal analysis of the engorged females revealed various mammalian hosts, especially goats, but also humans for Phlebotomus papatasi and Ph. alexandri, suggesting that these vector species are opportunistic feeders.ConclusionsIntegrative approach that combined morphological analysis, sequencing of DNA markers, and protein profiling enabled the recognition and description of a new Sergentomyia species, raising the number of the Algerian sand fly fauna to 27 species. Further sand fly surveillance in the central Sahara is recommended to identify the thus-far unknown males of Se. imihra n. sp.Graphical

Variable sensitivity of clinical Candida auris strains to Biocides: implications for infection control in Healthcare Settings

PurposeCandida auris, a multidrug-resistant yeast, poses significant challenges in healthcare settings due to its ability to form biofilms and resistance to common disinfectants. Understanding its susceptibility to biocides used in hospital disinfection practices is crucial for infection control. We investigated the biocide sensitivity of eight clinical C. auris strains from different patients and one reference strain (CDC B11903) using the biocide activity tests.MethodsSpecies identification was confirmed through MALDI-TOF MS, while clade differentiation and phylogenetic classification were determined via whole-genome sequencing. Biofilm formation was assessed using the MTT assay. Antifungal susceptibilities were tested according to CLSI standards. The effectiveness of biocides, including chlorine, chlorhexidine, and benzalkonium chloride, was evaluated through broth microdilution following CLSI standards and quantitative suspension and carrier tests, following EN standards.ResultsAll clinical strains were identified as clade 1, and the reference strain as clade 4, with all exhibiting biofilm formation. Clade 1 strains showed resistance to fluconazole, with MIC values ranging from 8 to 32 µg/ml, while being susceptible to other antifungals. Broth microdilution MIC assays for biocides demonstrated that all strains exhibited resistance to benzalkonium chloride. Chlorine and chlorhexidine showed variable efficacy, dependent on concentration and environmental cleanliness. Alcohol-based hand sanitizers demonstrated effectiveness against C. auris from the first minute of application.ConclusionThe study highlights the variable susceptibility of C.auris to different biocides, underscoring the challenge in eradicating this pathogen from healthcare environments. Our findings advocate for the careful selection of disinfectants in hospital settings, emphasizing the need for high-concentration chlorine and chlorhexidine solutions to combat C. auris, even in especially clean environments.

First Case of Chryseobacterium gleum Post-COVID-19 in a Child with Recurrent Fever

Background and Clinical Significance:Chryseobacterium gleum is a Gram-negative opportunistic and emerging pathogen able to induce systemic manifestations (e.g., peritonitis, pneumonia, urinary tract infections, meningitis) in immunocompromised patients. No data on children have been published. Case Presentation: A 2-year-old child presented in the pediatric ambulatory room with recurrent fever, submandibular lymphadenopathy, and skin rash. Laboratory findings revealed the presence of microcytic anemia with an increase in c-reactive protein. Chest X-ray reported mild accentuation of the bronchial structure, especially on the right side and middle–lower zone. In the peripheral blood smear, anisopoikilocytosis and elliptical red cells were evident. Clinical evaluation revealed the presence of conjunctivitis and polymorphic erythema, hyperemic pharynx and tonsils, SPO2 99%, auscultation of the chest, harsh vesicular murmur all over the area, and some wheezing. Microbiological analysis of sputum and throat swabs revealed the presence of numerous colonies of Chryseobacterium gleum confirmed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS score > 2.2). Conclusions: This is the first case of Chryseobacterium gleum post-COVID in a child. We suggest that a quick identification and an appropriate treatment represent the critical factors able to prevent the adverse outcomes related to C. gleum infection.

Development of a MALDI-TOF MS model for differentiating haemorrhagic septicaemia-causing strains of Pasteurella multocida from other capsular groups.

Pasteurella multocida capsular types A, D, and F cause disease in many animal hosts, including bovine respiratory disease in cattle, which is one of the most globally significant animal diseases. Additionally, P. multocida capsular types B and E cause haemorrhagic septicaemia, a devastating disease primarily of cattle, water buffalo, and bison that develops rapidly with high mortality. Haemorrhagic septicaemia mostly occurs in developing countries and has potential to emerge elsewhere in the world. The diagnosis of haemorrhagic septicaemia currently requires recognition of compatible gross or histologic lesions and serotyping or molecular characterization of strains. In this study, we performed genomic characterization of 84 P. multocida strains, which were then used to develop and validate a matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) biomarker-based method for differentiating non-haemorrhagic septicaemia strains of P. multocida from haemorrhagic septicaemia-causing strains. Haemorrhagic septicaemia strain types B:2,5, E:2,5, and B:3,4 were used to maximize diversity. Three automated classification models were generated and then used to develop an assisted model, which utilized two peaks (6419 and 7729 m/z) to accurately differentiate non-haemorrhagic septicaemia-causing strains from haemorrhagic septicaemia-causing strains of P. multocida. The assisted model performed with 98.2 % accuracy for non-haemorrhagic septicaemia strains, 100 % accuracy for classic B:2,5 and E:2,5 strains, and 84.4 % accuracy for combined haemorrhagic septicaemia-causing strains (B:2,5, E:2,5, and B:3,4) with an overall accuracy of 96.9 %. Our results suggest that MALDI-TOF MS may be used to routinely screen P. multocida isolated from diagnostic cases for initial identification of haemorrhagic septicaemia-causing strains, and to determine whether additional characterizations are warranted.

Enhanced detection of Pythium insidiosum via lipid profiling with matrix-assisted laser desorption ionization time of flight mass spectrometry

Pythiosis is a severe disease in humans and animals globally, caused by the pathogenic oomycete Pythium insidiosum. Early and accurate detection is crucial for effective treatment, but traditional diagnostic methods have limitations. This study presents an alternative approach using Matrix-Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS) for lipid profiling to efficiently identify P. insidiosum. The study involved extracting microbial lipid components using optimized chloroform: methanol biphasic method and creating a lipid profile database with samples from 30 P. insidiosum isolates and 50 various fungi. The methodology was validated on 25 blinded samples for assay detection performance. Unique lipid profiles allowed species-specific identification with high efficiency: scores ≥ 2.682 indicated P. insidiosum, scores ≤ 2.512 suggested fungi, and scores in between pointed to other oomycetes. The assay demonstrated sensitivity, specificity, and accuracy of 100%, 80%, and 88%, respectively, for detecting P. insidiosum. The limited detection specificity was due to false positive samples from closely related Pythium species, which are not a significant clinical concern. The findings show that MALDI-TOF MS lipid profiling can efficiently identify P. insidiosum, offering significant advantages in sample preparation, stability, and reproducibility over protein profile-based methods. This study marks the first instance of lipid profiles being reported for P. insidiosum, paving the way for clinical use in improving accurate detection and facilitating timely treatment interventions.

Polyphasic identification of Vibrio species from aquatic sources using mass spectrometry, housekeeping gene sequencing and whole genome analysis

Accurate bacterial identification is essential for determining the causative agent of an infection, thus facilitating appropriate treatment and management strategies in both human and animal health contexts. Some species in the Vibrio genus are recognized pathogens, associated with a variety of infections. However, identification of these bacteria is oftentimes controversial. Therefore, we aimed to evaluate different identification approaches in terms of their reliability in distinguishing Vibrio species. To achieve this, we selected a set of 40 Vibrio isolates previously recovered from water and floating plastic samples in a large bay environment and identified them employing MALDI-TOF mass spectrometry, and rrs and pyrH gene sequencing. A subset of isolates was also submitted to whole genome sequencing. Overall, MALDI-TOF was found to be a fast-screening methodology for identification, notably at genus-level. However, for better species discrimination, pyrH gene sequencing stood out as a more reliable tool in contrast to rrs gene sequencing and MALDI-TOF, as corroborated by whole genome sequencing analysis.

Prevalence and antibiotics susceptibility of thermotolerant <i>Campylobacter</i> spp. isolated from humans and chickens in the Republic of Guinea

Background.The issue of diarrheal diseases remains relevant for modern health care in all countries. Campylobacteriosis is the most common infectious disease with foodborne transmission and poultry meat is a transmission factor.Materials and methods.724 items of faeces sampled from patients with diarrheal syndrome and 283 samples of faeces of chickens raised on private farms and five poultry farms in the province were studied. For bacteriological method were used selective media. Traditional routine tests (cell morphology, cytochrome oxidase, catalase, hydrolysis of sodium hippurate and indoxyl acetate) and MALDI-ToF mass spectrometry was performed for identification. The susceptibility of strains to antibiotics was analysed using the disc-diffusion method. Results were interpreted according to the EUCAST criteria, versions 2019–2022.Results.Campylobacterspp. was cultured in 65 out of 724 faecal samples from patients with acute diarrhoea, of them 83.08% were identified asC. jejuni, and 16.92% asC. coli. Of the 237Campylobacterstrains from chicken were identified asC. jejuni(54.0%), asC. coli(46.0%).Campylobacterspp. strains from humans were resistant to tetracycline (40.0%), to erythromycin (6.15%), to ciprofloxacin (12.31%). The strains from chickens kept on farms, were resistant to tetracycline in 42.55%, to ciprofloxacin — in 22.70% and to erythromycin — in 11.35%. The strains from chickens kept on private farms were resistant to tetracycline in 4.17%, to ciprofloxacin — in 1.04%, all strains were sensitive to erythromycin.Conclusion.Thus, due to the widespread prevalence ofCampylobacterspp., infectious diseases they cause remain a topical issue. Studying the resistance to antibiotics inCampylobacterspp. among poultry could allow to develop new approaches to confirming the significance of their foodborne nature and to improve the national disease prevention system.

Use of BD BACTEC™ MGIT™ for the detection of non-tuberculous mycobacteria in sanitary water samples

IntroductionThe most commonly used method for the detection of non-tuberculous mycobacteria (NTM) is culture in BD BACTEC™ MGIT™ Mycobacteria Growth Indicator Tubes incubated in an automated growth detection reader BD BACTEC™ MGIT™ 960 Instrument. The system is currently validated for the detection of mycobacteria from clinical specimens but not environmental matrices.MethodsFrom November 2018 to December 2023, 1,369 sanitary water samples from 92 heater–cooler units (HCUs) and 747 sanitary water samples from 489 haemodialysis instruments (dialysis) were concentrated, decontaminated, and cultured on MGIT and solid Lowenstein–Jensen media to evaluate the presence of NTM. NTM-positive cultures (n = 261 HCUs and n = 20 dialysis) were purified by Middlebrook 7H11 agar plate subcultures and identified by MALDI-TOF mass spectrometry technology.ResultsThe purpose of this study was to evaluate the accuracy and reproducibility of the MGIT system on sanitary water from HCU and dialysis, using the two strains most frequently isolated on these devices as sources of NTM during the Emilia- Romagna surveillance programme: M. chimaera (79%) and M. saskatchewanense (100%), respectively. To evaluate the accuracy, sanitary water was spiked with M. chimaera and M. saskatchewanense at the theoretical concentrations of 100 and 10 CFU/mL, and all resulted positive in MGIT tubes. No significant changes in time to positivity were observed when MGIT tubes were inoculated with NTM at the theoretical concentrations of 10 and 100 CFU/mL on 3 consecutive days, indicating that the detection method is reproducible.DiscussionThe MGIT system is suitable for detecting the presence of NTM in sanitary water samples as it was capable of detecting up to 4 CFU/mL for both M. chimaera and M. saskatchewanense. Our results indicate that the MGIT system can be used for NTM detection not only for clinical samples but also for environmental matrices.

Characterization of bla NDM in two Escherichia coli ST1193 clinical isolates in the Gulf region.

Escherichia coli ST1193 is an emerging high-risk clone associated with the production of plasmid-mediated CTX-type extended-spectrum β-lactamases. However, this clone has seldom been found to contain plasmids carrying carbapenemase genes. We report two epidemiologically unlinked multidrug-resistant (MDR) clinical isolates of E. coli ST1193 with plasmids harbouring NDM-type carbapenemase genes from the Gulf region. The isolates were identified by MALDI-TOF MS and antibiotic susceptibility testing was performed using the VITEK 2/Phoenix system. A conjugation experiment was performed to assess the transferability of the resistance plasmids. Genomic DNA of both isolates was subject to Illumina sequencing; one isolate was also sequenced using Oxford Nanopore technology. Bioinformatics analyses were performed to detect antimicrobial resistance genes, and to annotate the genetic context of the NDM genes. Both isolates were resistant to carbapenems using phenotypic tests. Conjugation experiment confirmed that NDM-5-encoding plasmids of both strains could be transferred to the recipient cells. The completed NDM-5-encoding plasmid of E. coli isolate FQ71 was highly similar to several plasmids from ST410 isolates in the NCBI database. Genomic analysis revealed the presence of transposase genes and transposons in the flanking regions of the NDM genes in the plasmids. Since carbapenems constitute first-line agents for the treatment of serious infections caused by ESBL producers, E. coli ST1193 isolates co-producing ESBL and NDM-type carbapenemases represent a serious challenge for antimicrobial stewardship and infection control programmes.

Use of stable isotope combined with intact cell lipidomic by routine MALDI mass spectrometry analysis for rapid drug susceptibility assay in mycobacteria.

Rapid, accurate, and easy-to-perform diagnostic assays are required to address the current need for the diagnosis of resistant pathogens. That is particularly the case for mycobacteria, such as the human pathogen Mycobacterium tuberculosis, which requires up to 2weeks for the determination of the drug susceptibility profile using the conventional broth microdilution method. To address this challenge, we investigated the incorporation of deuterium, the stable isotope of hydrogen, into lipids as a read out of the drug susceptibility profile. Deuterium is incorporated into newly synthesized proteins or lipids in place of hydrogen as bacterial cells grow, increasing the mass of the macromolecules, which can then be observed via matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS). As proof-of-concept, we used the non-pathogenic Mycobacterium smegmatis mc2155 strain, which is susceptible to the aminoglycoside antibiotic kanamycin, and M.smegmatis mc2155 containing the empty vector pVV16, which is kanamycin-resistant. Bacteria were incubated in a culture medium containing 50% of deuterium oxide (D2O) and either 1 or 2 times the minimal inhibitory concentration (MIC50) of kanamycin. Lipids were then analyzed using the MBT lipid Xtract matrix combined with routine MALDI mass spectrometry in the positive ion mode to evaluate the changes in the lipid profile. Using this approach, we were able to distinguish susceptible from resistant bacteria in less than 5h, a process that would take 72 h using the conventional broth microdilution method. We therefore propose a solution for the rapid determination of drug susceptibility profiles using a phenotypic assay combining D2O stable isotope labelling and lipid analysis by routine MALDI mass spectrometry.

Production of bacteriocin-like inhibitory substance (BLIS) by Staphylococcus spp. isolates from dogs.

In the present study, 39 canine isolates of Staphylococcus spp. were tested for antimicrobial substance (AMS) production. Seven AMS producers were identified, whose products exhibited a non-acidic character and a proteinaceous nature, therefore being considered bacteriocin-like inhibitory substances (BLIS). The producer strains of BLIS P1, P16 and I3 showed a broad spectrum of antimicrobial activity. Human, veterinary and plant pathogens, such as Listeria monocytogenes, Klebsiella pneumoniae, Staphylococcus spp. and Clavibacter michiganensis, were among the inhibited micro-organisms, suggesting the potential biotechnological application of these peptides. MALDI-TOF mass spectrometry and 16S rDNA sequencing identified the producer strains of BLIS P1, P16 and I3 as Staphylococcus pseudintermedius P1, Staphylococcus schleiferi P16 and Staphylococcus pseudintermedius I3. The plasmid profile of these strains suggests that the BLIS production is linked to biosynthetic genes located on plasmids. PCR analyses revealed that BLIS P1, P16 and I3 are different from 11 staphylococcins already described in the literature and that their genomic DNAs do not carry the most prevalent staphylococcal enterotoxin genes. The highest levels of BLIS production were achieved after 18-24h of growth of the producer strains in TSB medium. Moreover, BLIS P1 and I3 exhibited high resistance to temperature and pH variations, and BLIS P16 maintained 100% of its activity in almost all conditions tested. The characteristics associated with BLIS P1, P16 and I3 described in this work encourage further investigation of these substances, in addition to this study being the first report of BLIS production by a strain of S. schleiferi.

New Pd(II)-pincer type complexes as potential antitumor drugs: synthesis, nucleophilic substitution reactions, DNA/HSA interaction, molecular docking study and cytotoxic activity.

Two new complexes of Pd(II), [Pd(L1)Cl]Cl (Pd1) and [Pd(L2)Cl]Cl (Pd2), (where L1 = N2,N6-bis(5-methylthiazol-2-yl)pyridine-2,6-dicarboxamide and L2 = N2,N6-di(benzo[d]thiazol-2-yl)pyridine-2.6-dicarboxamide) were synthesized. Characterization of the complexes was performed using elemental analysis, IR, 1H NMR spectroscopy and MALDI-TOF mass spectrometry. The nucleophilic substitution reactions of complexes with L-Methionine (L-Met), L-Cysteine (L-Cys) and guanosine-5'-monophosphate (5'-GMP) were studied by stopped-flow method at physiological conditions (pH = 7.2 and 37 °C). Complex Pd1 was more reactive than Pd2 in all studied reactions, while the order of reactivity of the selected ligands was: L-Met > L-Cys > 5'-GMP. The interaction of complexes with calf thymus-DNA (CT-DNA) was studied by Uv-Vis absorption and fluorescence emission spectroscopy. Competitive binding studies with intercalative agent ethidium bromide (EB) and minor groove binder Hoechst 33258 were performed as well. Both complexes interacted with DNA through intercalation and minor groove binding, where the latter was preferred. Additionally, the interaction of Pd1 and Pd2 complexes with human serum albumin (HSA) was studied employing fluorescence quenching spectroscopy. The results indicate a moderate binding affinity of complexes, with slightly stronger binding of the Pd1. Fluorescence competition experiments with site-markers (eosin Y and ibuprofen) for HSA were used to locate the binding site of Pd1 to the HSA. Additionally, the interaction with DNA and HSA was studied by molecular docking and the revealed results were in good agreement with the experimentally obtained ones. Pd1 complex exhibited cytotoxicity toward human (HCT116) and mouse cell lines (CT26) of colorectal cancer, mouse (4T1) and human (MDA-MB468) breast cancer lines and non-cancerous mouse mesenchymal stem cells (mMSC). In addition, Pd1 complex demonstrated significant selectivity towards cancer cells over non-cancerous mMSC, indicating a high potential to eliminate malignant cells without affecting normal cells. It induced apoptosis in CT26 cells, effectively arrested the cell cycle in the S phase, and selectively down-regulated cyclin D and cyclin E. Moreover, it can alter the expression of cell cycle regulators by increasing p21 and decreasing p-AKT. These findings confirm its ability to disrupt key tumor cell survival signals and suggest that the Pd1 complex is a potent candidate for effective cancer treatment.

Structure and bioactivity of tannin extracts from Taxodium ‘Zhongshanshan’

Abstract Taxodium ‘Zhongshanshan’ is a distinctive tree known for its rapid growth and robust stress resilience. To enhance the utilization of forestry resources, tannin extracts were procured from the barks (BE) and leaves (LE) of Taxodium ‘Zhongshanshan’. The tannin structures were characterized by FT-IR and MALDI-TOF MS, and found to be condensed tannin with a degree of polymerization not exceeding 6. The tannin extracts exhibited a potent ability to scavenge free radicals at capacities of 60–80 % compared to Vitamin C (VC). They inhibited the bacterial strains Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus), with a pronounced effect on the latter. Furthermore, tannin from Taxodium ‘Zhongshanshan’ appears benign and have no significant effect on cell activity.

Clinical characteristics of patients with granulomatous lobular mastitis associated with Corynebacterium parakroppenstedtii infection and drug sensitivity analysis of the isolated strains

BackgroundIt is presently considered that Corynebacterium especially Corynebacterium kroppenstedtii (CK) infection, is one of the important causes of granulomatous lobular mastitis (GLM). However, the pathogen of mastitis in the past two years has been identified as a newly discovered Corynebacterium. But it is unclear whether the pathogen associated with the occurrence of GLM is also this bacterium.MethodsGLM female patients with positive bacterial culture in pus specimens from February 2023 to February 2024 who were identified as CK infection by mass spectrometer were selected as the research objects in this study, and the clinical isolates were identified by 16S rDNA sequencing technology to identify the specific pathogen of GLM-related bacterial infection. Subsequently, the clinical characteristics of the patients were compared with those of GLM patients without bacterial infection during the same period, to explore the effect of this particular type of Corynebacterium infection on disease development in GLM patients. Finally, we tested the minimum inhibitory concentration (MIC) values of antibiotics when inhibiting these separation strains in vitro through the E-Test experiment, to evaluate their medicine sensitivity.ResultsA total of 31 GLM patients initially diagnosed with Corynebacterium kroppenstedtii (CK) infection via MALDI-TOF MS were enrolled in the study. However, subsequent 16S rDNA sequencing revealed that 28 isolates (90.32%) were actually identified as the newly recognized Corynebacterium parakroppenstedtii (CPK). This discovery challenges the conventional belief that CK is the primary pathogen of GLM, suggesting instead that CPK is the predominant pathogen associated with GLM bacterial infections. Comparative analysis of the clinical characteristics between the two groups revealed a significantly higher recurrence rate among CPK-infected GLM patients compared to those without CPK infection, along with elevated prolactin levels (P < 0.05). The sensitivity test results indicated high sensitivity of the isolates to vancomycin, linezolid, and rifampicin.ConclusionIn conclusion, this study highlights that Corynebacterium kroppenstedtii strains isolated from GLM specimens were Corynebacterium parakroppenstedtii, serving as the primary pathogen closely linked to GLM’s occurrence. CPK infection significantly increases the risk of recurrence in GLM patients, with elevated prolactin levels potentially playing a pivotal role in this process. In clinical antimicrobial treatment, antimicrobials other than penicillin and ciprofloxacin may be empirically administered when sensitivity test results are inconclusive.

Association of inflammation with left ventricular mass index in patients with rheumatoid arthritis

Abstract Background Rheumatoid arthritis (RA) is an inflammatory autoimmune disease associated with cardiovascular organ damage. Purpose We explored the association of inflammatory markers with left ventricular (LV) mass index during 22 months of follow-up in patients with RA. Methods Eighty-three outpatients with RA (age 55±12 years, 71 % women) with indication for biological (b) or targeted synthetic (ts) disease modifying antirheumatic drug (DMARD) therapy were examined with echocardiography at baseline and after 22 months. LV mass was indexed for height in the allometric power of 2.7. Serum concentrations of the inflammatory markers, C-reactive protein (CRP), serum amyloid A (SAA) and calprotectin were measured by MALDI-TOF mass spectrometry. Results At baseline, 37% had hypertension, 6% diabetes, 21% obesity and 100% were using b/ts DMARDs. The inflammatory markers decreased during follow-up (all p&amp;lt;0.001), while the LV mass index did not change (33.1±8.1 g/m^2.7 vs. 33.5±7.3 g/m^2.7, p=0.57). The prevalence of hypertension, diabetes and obesity were unchanged at follow-up (p&amp;gt;0.05), but the percentage of patients using b/ts DMARDs had decreased to 83% (p&amp;lt;0.001). In multivariable analyses, higher CRP and SAA at baseline were associated with higher LV mass index at both baseline and follow-up independent of presence of obesity, hypertension, age, sex or treatment with b/ts DMARDs at follow-up (Table 1). Inflammatory markers at follow-up were not associated with LV mass index (Table 1), but use of b/ts DMARDs at follow-up were associated with lower LV mass index after adjusting for inflammatory markers, age, sex, presence of obesity and hypertension (Table 1). Conclusion Higher levels of inflammatory markers at baseline were associated with greater LV mass index in RA patients. The use of immunosuppressive treatment throughout the study period was associated with lower LV mass index at follow-up, pointing to a role of chronic inflammation in LV hypertrophy.