Matrix-assisted Laser Desorption/ionization Time-of-flight Research Articles

Can infectious omphalitis in piglets be clinically diagnosed during the first three days of life?

BackgroundGlobally, the increase in antimicrobial resistance is of great concern. In Denmark, the pig sector is accountable for the majority of antimicrobial usage in animals. As new-born piglets are at risk of developing infectious omphalitis, and many pigs are treated by antimicrobials within the first days of life, an early and accurate diagnosis of the disease is imperative to maintain animal welfare and reduce the antimicrobial usage. The aim of the present study was to compare histopathological and microbiological findings in piglets clinically diagnosed with and without omphalitis during the first three days after birth.ResultsA total of 98 case piglets with omphalitis and 98 control piglets without omphalitis, based on clinical examinations during the first three days of life, were included. Of the 196 piglets, 79 (38 cases and 41 controls) presented histopathological omphalitis. Post mortem, the volume of the umbilicus was significantly enlarged in cases compared to controls (p = 0.01). Except for a group of bacteria unidentified by matrix-assisted laser desorption ionisation time-of-flight (MALDI-TOF) (p = 0.02), no apparent association was found between histopathologically diagnosed omphalitis and the prevalence of specific pathogens (p > 0.05). The frequency of histopathologically diagnosed omphalitis tended to increase with increasing age in both cases and controls. The frequency of piglets with both histopathological omphalitis and arthritis/synovitis was significantly different among cases and controls (p = 0.05). This was due to all controls, and none of the cases, with arthritis/synovitis presented histopathological omphalitis.ConclusionThe clinical differentiation between omphalitis cases and controls did not correspond to the histological diagnosis of omphalitis in zero-to-three days old piglets. An inaccurate clinical diagnosis complicates prudent use of antimicrobials in pig herds. In addition, animal welfare may be hampered in infected piglets due to lack of treatment and in healthy piglets due to the antimicrobial effect on the gut microbiome.

Kumada-Tamao Catalyst-Transfer Condensation Polymerization of AB2 Monomer: Synthesis of Well-Defined Hyperbranched Poly(thienylene-phenylene).

The synthesis of well-defined hyperbranched aromatic polymer by Kumada-Tamao catalyst-transfer condensation polymerization of AB2 monomer is investigated. Grignard monomer 2 is generated by treatment of 2-(3,5-dibromophenyl)-3-hexyl-5-iodothiophene (1) with 1.0 equivalent of isopropylmagnesium chloride in THF at 0°C for 1h and subsequently polymerized with Ni(dppe)Cl2 at room temperature for 1h. The molecular weight of the obtained polymer increases linearly up to ≈30000 in proportion to the ratio of [consumed 2] /[Ni(dppe)Cl2]0 and in proportion to the conversion of 2, while a narrow molecular weight distribution is maintained (Mw/Mn ≤ 1.12). The matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrum shows almost a single series of peaks due to polymer with hydrogen at one end and bromine at the other, as in the case of Kumada-Tamao catalyst transfer condensation polymerization of AB monomers. The degree of branching (DB) of the obtained polymers is 0.70-0.75, irrespective of the degree of polymerization. These results indicate that the polymerization of 2 proceeds in a chain-growth polymerization manner through the intramolecular catalyst transfer mechanism, affording hyperbranched polymer with higher DB than the theoretical DB value of 0.5 in conventional polycondensation of AB2 monomers.

Identifying falsified COVID-19 vaccines by analysing vaccine vial label and excipient profiles using MALDI-ToF mass spectrometry

The rapid development and worldwide distribution of COVID-19 vaccines is a remarkable achievement of biomedical research and logistical implementation. However, these developments are associated with the risk of a surge of substandard and falsified (SF) vaccines, as illustrated by the 184 incidents with SF and diverted COVID-19 vaccines which have been reported during the pandemic in 48 countries, with a paucity of methods for their detection in supply chains. In this context, matrix-assisted laser desorption ionisation-time of flight (MALDI-ToF) mass spectrometry (MS) is globally available for fast and accurate analysis of bacteria in patient samples, offering a potentially accessible solution to identify SF vaccines. We analysed the COVISHIELD™ COVID-19 vaccine; falsified versions of which were found in India, Myanmar and Uganda. We demonstrate for the first time that analysis of spectra from the vaccine vial label and its adhesive could be used as a novel approach to detect falsified vaccines. Vials tested by this approach could be retained in the supply chain since it is non-invasive. We also assessed whether MALDI-ToF MS could be used to distinguish the COVISHIELD™ vaccine from surrogates of falsified vaccines and the effect of temperature on vaccine stability. Both polysorbate 80 and L-histidine excipients of the genuine vaccine could be detected by the presence of a unique combination of MALDI-ToF MS peaks which allowed us to distinguish between the genuine vaccines and falsified vaccine surrogates. Furthermore, even if a falsified product contained polysorbate 80 at the same concentration as used in the genuine vaccine, the characteristic spectral profile of polysorbate 80 used in genuine products is a reliable internal marker for vaccine authenticity. Our findings demonstrate that MALDI-ToF MS analysis of extracts from vial labels and the vaccine excipients themselves can be used independently to detect falsified vaccines. This approach has the potential to be integrated into the national regulatory standards and WHO’s Prevent, Detect, and Respond strategy as a novel effective tool for detecting falsified vaccines.

Quantitative MALDI-TOF Mass Spectrometry of Star-Shaped Polylactides Based on Chromatographic Hyphenation.

The end groups of three- and four-arm star-shaped polylactides (PLA) with trimethylolpropane and pentaerythritol core structures were functionalized with acetic acid. Reaction products with different degrees of functionalization were analyzed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Additional gradient elution liquid adsorption chromatography (GELAC) measurements were performed to determine the degree of functionalization. This technique enabled clear separation and sufficient quantification of the formed species. These chromatographic data could be used inversely to quantify mass spectrometric results, which are usually biased by the unknown ionization probabilities of different polymer end group structures. Our results showed that, in this particular case, the peak intensity in the MALDI-TOF mass spectra can be used to semiquantitatively determine the degree of functionalization in incompletely functionalized multiarm PLA.

Decoding MexB efflux pump genes: structural, molecular, and phylogenetic analysis of multidrug-resistant and extensively drug-resistant Pseudomonas aeruginosa.

Emerging drug resistance in Pseudomonas aeruginosa is of great concern in clinical settings. P. aeruginosa activates its efflux-pump system in order to evade the effect of antibiotics. The current investigation aims to detect MexB genes in P. aeruginosa, their structural and molecular analysis and their impact on antimicrobial susceptibility profiling. A total of 42 clinical specimens were aseptically collected from hospitalized patients who had underlying infections related to medical implants. Matrix-assisted laser desorption ionization-time of flight (MALDI-ToF) were used for the identification of isolates. The methods used in this study were antibiotic susceptibility profiling, minimum inhibitory concentration (MIC), polymerase chain reaction (PCR), sanger sequencing, phylogenetic analysis, MolProbity score, Ramachandran plot analysis and multiple sequence alignment. The highest resistance was shown by P. aeruginosa against cefoperazone (67%), gentamycin and amikacin (66%) each, followed by cefotaxime (64%). The prevalence of multi-drug resistant (MDR) and extensively drug resistant (XDR) was 57% and 12%, respectively. The presence of an active efflux-pump system was indicated by the MexB genes found in most of the resistant isolates (p<0.05). Following addition of efflux pump inhibitor carbonyl cyanide m-chlorophenyl hydrazone (CCCP), a significant decrease (p<0.05) in MIC was observed in resistance, that revealed the presence of active efflux pump system. Phylogenetic analysis revealed evolutionary relationships with the P. aeruginosa strains isolated in Switzerland, Denmark and Germany. Protein domain architecture revealed that MexB gene proteins were involved in particular efflux pump function. Protein sequences aligned by multiple sequence alignment revealed conserved regions and sequence variants, which suggested antibiotic translocation and evolutionary divergence. These highly conserved regions could be used for diagnostic purposes of efflux pump MexB genes. To avoid their spread in hospital settings, responsible authorities ought to begin rigorous initiatives in order to reduce the prevalence of multi-drug resistant, extensively drug resistant, and efflux pump carrying isolates in clinical settings.

Screening of Candida auris upon admission to an intensive care unit in the United Arab Emirates.

Candida auris screening is one of the crucial components of infection prevention and control measures to curb the spread of C. auris. However, previous research has yielded various results on the effectiveness of C. auris screening according to region, type of healthcare facility, and patient group in the various levels of endemicity. We aimed to assess the clinical effectiveness of routine C. auris screening upon admission to the intensive care unit (ICU). This retrospective study involved 3356 ICU patients during pre-screening (N = 1658, June 2020 to November 2021) and post-screening period (N = 1698, June 2022 to November 2023). Swabs for C. auris screening were taken from axilla and groin, then cultured, and identified using either matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry or VITEK 2 YST identification card. We compared the positivity rates of C. auris during the pre- and post-screening period. Of the 1698 patients who underwent C. auris screening, only 4 (0.2 %) tested positive. The positivity rates before and after the introduction of the screening program were 2.92 cases per 1000 patient days (36/12333) and 2.36 (21/8909), respectively, without significant difference (P = 0.435). Interrupted times series analysis showed no immediate (P = 0.775) or sustained (P = 0.484) effects from the screening program. Subgroup analysis of patients in the medical ICU showed similar results. Universal C. auris screening upon ICU admission identified a small number of cases. Instead, targeted screening and regular point prevalence surveys, along with other infection prevention and control measures may be employed. Further studies in various settings with diverse testing strategies are needed to establish the most effective approach for C. auris screening.

The impact of ligand chain length on the HER performance of atomically precise Pt6(SR)12 nanoclusters.

Atomically precise metal cluster-based electrocatalysts have been paid significant attention for an efficient hydrogen evolution reaction (HER). Herein, we have synthesized atomically precise Pt6(SR)12 nanoclusters using 3-mercaptopropionic acid (MPA), 6-mercaptohexanoic acid (MHA), 8-mercaptooctanoic acid (MOA), and 11-mercaptoundecanoic acid (MUA) thiol ligands in aqueous media at room temperature to understand the impact of ligand chain length on the HER performance. The composition of Pt6(SR)12 metal clusters was confirmed by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. Electrochemical studies confirmed that the HER performance of Pt6(SR)12 NCs is notably affected by the ligand chain length, and Pt6(MPA)12 exhibits an overpotential of 19 mV at a current density of 10 mA cm-2, which is several-fold higher than the Pt NCs developed in the recent past. The interfacial charge transfer kinetics and the HER performance decrease with the increase in the chain length of the thiol ligands. Density functional theory calculations showed that the Gibbs free energy for hydrogen adsorption decreases with decreasing chain length of the thiol ligand. The ligands used to synthesize Precise metal clusters for electrocatalysis play an essential role in their efficiency.

Synthesis and Properties of Symmetrical and Asymmetrical Polyferrocenylmethylenes.

The synthesis of differently substituted polyferrocenylmethylenes (PFM) via ring-opening transmetalation polymerization (ROTP) is reported. A number of novel, symmetrically and asymmetrically substituted carba[1]magnesocenophanes have been prepared, which were used as precursors and allowed investigations of the influence of different substitution patterns on the PFM polymer properties. The novel carba[1]magnesocenophanes have been fully characterized by 1H and 13C{1H} NMR spectroscopy, and structurally authenticated by single-crystal X-ray diffraction (SC-XRD). The corresponding PFMs were obtained with molecular weights in a range of Mw=1900 g mol-1 up to 15100 g mol-1 and corresponding dispersities of Đ=1.48 to 1.81. All PFMs have been fully characterized by 1H and 13C{1H} NMR spectroscopy, as well as by gel permeation chromatography (GPC), thermal gravimetric analysis (TGA) and matrix assisted laser desorption ionization-time-of-flight (MALDI-ToF) mass spectrometry. Pyrolysis of spyrocyclic substituted PFM resulted in a ceramic yield above 50 %, while the obtained residue was characterized by powder X-ray diffraction (PXRD).

Performance of the VITEK® MS system for the identification of filamentous fungi in a microbiological laboratory in Chile

Filamentous fungi are an emergent cause of severe infections in immunocompromised patients. Timely and accurate identification is crucial to initiate appropriate therapy. Traditional identification methods are time-consuming, labor-intensive, and operator-dependent. Matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry is a rapid and easy-to-perform identification method. The effectiveness of a commercial MALDI-TOF MS platform to identify filamentous fungi in a clinical laboratory was evaluated. The study included 67 fungal isolates from 35 species/species complexes, which were identified and confirmed in mycology reference laboratories; 32 derived from clinical samples, 34 from strain collections and one was an ATCC strain. The study used the VITEK® MS system (v3.2.0 database), after sample extraction by VITEK® MS Mould Kit. Results were classified as “correct species”, “correct species complex”, “correct genus”and “no identification”. VITEK® MS correctly identified 91.0% of isolates (58.2% to species, 29.9% to species complex, and 1.5% to genus level only). In 82%, the result matched the species/species complex identified by reference methods. No misidentifications were observed. The kit was rapid and easy to use. In conclusion, the VITEK® MS system showed a high capability to accurately identify filamentous fungi in a clinical laboratory.

Distinguishing Daratumumab from Endogenous Monoclonal Proteins in Serum from Multiple Myeloma Patients Using an Automated Mass Spectrometry System.

Therapeutic monoclonal antibodies (t-mAbs) may interfere with electrophoresis-based methods used to monitor multiple myeloma (MM), which can create inaccurate results. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry is an alternative to gels distinguishing between endogenous M-proteins and t-mAbs based on molecular mass. Serum samples (n = 109) from 34 MM patients receiving Dara-KRd were collected 14 or 28 days postdaratumumab administration. Samples were analyzed using the EXENT® Analyzer that combines automated immunopurification and MALDI-TOF MS for the isotyping and quantification of monoclonal immunoglobulins. Daratumumab was identified in 103 out of 109 samples (94.5%). In all IgGλ (n = 8), IgAκ (n = 8), and IgAλ (n = 2) patients, the M-protein and daratumumab were detected. Of the IgGκ patients (n = 18), 5 patients had a total of 6 samples where the M-protein was detected but daratumumab was not. There was no difference in the detection rate of daratumumab between samples taken 14 and 28 days postadministration with the median daratumumab concentration being 0.95 and 0.54 g/L, respectively. A precision study was also performed on 25 replicates containing 1 g/L daratumumab in serum where a coefficient of variation of 4.2% was observed as determined by the EXENT Analyzer. The Immunoglobulin Isotypes (GAM: IgG, IgA, and IgM) for the EXENT Analyzer detected and distinguished a daratumumab kappa light chain peak from an M-protein light chain peak in MM patient serum when resolved by the mass spectrometer.

Penetration of Vegetable Oils into Textured Hair Fibers: Integrating Molecular Matrix Assisted Laser Desorption Ioni-Zation Time-of-Flight Mass Spectroscopy (MALDI TOF/TOF MS) Analysis with Mechanical Measurements

The promotion of natural beauty has empowered women with textured hair to embrace their natural hair texture and opt for bleaching as a means of style variation. However, bleaching exacerbates the inherent fragility of this hair type, necessitating treatments to partially restore its mechanical properties. Vegetable oils, renowned for their strengthening properties, were evaluated regarding (I) their ability to penetrate both virgin and bleached textured hair using Matrix-Assisted Laser Desorption Ionization (MALDI) time-of-flight (TOF) analysis, and (II) their effects by way of tensile and fatigue tests. The MALDI–TOF results revealed groups of oil molecules in the cortical region of the bleached textured hair. The tensile test results, in turn, showed that the oil treatments were unable to alter the mechanical properties of the hair. Conversely, the fatigue test showed an increase in resistance in the virgin hair, most likely attributed to a lubrication effect in the outermost portions of the cortex and cuticles. In the bleached hair, a reduction in resistance was noted following the treatment with the oils. Comparative analysis with a previous study on straight hair by our group suggests that external molecules diffuse more homogeneously in straight hair than in textured hair. The unique cortical structure of textured hair creates two areas with distinct diffusion zones, resulting in the irregular distribution of external materials and different effects compared to straight hair.

Evaluation of a New Tandem Mass Spectrometry Method for Sickle Cell Disease Newborn Screening.

In France, sickle cell disease newborn screening (SCD NBS) has been targeted to at-risk regions since 1984, but generalization to the whole population will be implemented from November 2024. Although tandem mass spectrometry (MS/MS) is already used for the NBS of several inherited metabolic diseases, its application for SCD NBS has not been widely adopted worldwide. The aim of this study was to evaluate a dedicated MS/MS kit (Targeted MS/MS Hemo, ZenTech, LaCAR Company, Liege, Belgium) for SCD NBS and to compare the results obtained with those from an NBS reference center using matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) and cation-exchange high-performance liquid chromatography (CE-HPLC, Variant NBS, Biorad Laboratories, Inc., Hercules, CA, USA) as confirmatory method. The MS/MS Hemo kit was used according to the manufacturer's instructions and performed on a Waters Xevo TQ-D (Waters Corporation, USA). The software provided by the manufacturer was used for the calculation and analysis of peptide signal ratios. Among the 1333 samples, the results of 1324 samples were consistent with the HPLC and/or MALDI-TOF results (1263 FA, 50 FAS, 7 FAC, 1 FAO-Arab, and 3 FS). All the discordant results (one FAS on MS/MS vs. FA in CE-HPLC, one FA on MS/MS vs. FAS in CE-HPLC, seven FS on MS/MS vs. FAS in CE-HPLC) were corrected after modifying the peptide signal ratios thresholds, allowing the MS/MS Hemo kit to achieve near-100% sensitivity and specificity for SCD NBS. In conclusion, the MS/MS Hemo kit appears to be an effective method for SCD NBS, particularly for laboratories already equipped with MS/MS technology. However, these results should be confirmed in a larger cohort including a greater number of positive samples for SCD.

Serovars, virulence factors, and antimicrobial resistance profile of non-typhoidal Salmonella in the human-dairy interface in Northwest Ethiopia: A one health approach.

Non-typhoidal Salmonella (NTS) is a zoonotic pathogen that exerts huge public health and economic impacts in the world. The severity of illness is mainly related to the serovars involved, the presence of virulence genes, and antimicrobial resistance (AMR) patterns. However, data are scarce on serovars, virulence genes, and AMR among NTS identified from the human-dairy interface in Northwest Ethiopia. Thus, this study investigated the serovars, common virulence genes, and AMR patterns of NTS isolates in the area. The study was conducted from June 2022 to August 2023 among randomly selected 58 dairy farms. A total of 362 samples were processed to detect NTS using standard bacteriological methods. The presumptive positive colonies were confirmed by Matrix-Assisted Laser Desorption Ionization-Time-of-Flight (MALDi-ToF). Polymerase chain reaction (PCR) was used to detect virulence genes, including invA and spvC. A slide agglutination test according to the White-Kauffmann-Le Minor scheme was employed to identify the serovars of the NTS isolates. The Kirby-Bauer disk diffusion method was used to assess the antimicrobial susceptibility patterns. Of the processed samples (362), 28 (7.7%) NTS isolates were detected. When distributed among samples, the proportions were 11.9%, 10.5%, 10.3%, 5.2%, 4.3%, and 1.7% among cows' feces, dairy farm sewage, pooled raw milk, milk container swabs, milkers' stool, and milkers' hand swab samples, respectively. Six serovars were detected with the dominancy of S. Uganda (39.3%), followed by S. enterica subsp. diarizonae (25.0%) and S. Typhimurium (21.4%). Among the 28 NTS isolates, 100% and 21.4% had the virulence genes invA and spvC, respectively. The susceptibility profile showed that 89.3% of the NTS isolates were resistant to at least one antimicrobial agent and 46.4% were resistant to three or more classes of antimicrobials (multidrug-resistant). Among antimicrobials, isolates were highly resistant to ampicillin (57.1%), followed by tetracycline (42.9%) and chloramphenicol (35.7%). On the other hand, the NTS isolates were 100%, 96.4%, and 96.4% susceptible to ceftriaxone, azithromycin, and norfloxacin, respectively. In conclusion, we detected NTS from humans, dairy cows, raw milk, dairy utensils, and the environment (sewage), showing the potential of the human-dairy farm-environment nexus in the NTS circulation. These further highlight that the interface is a good point of intervention in the control and prevention of NTS infection. The susceptibility profiles of the isolate necessitate interventions including the prudent use of the antimicrobials.

“Clicking” Amphiphilic Block Copolymers onto POSS Core: A General Approach for “Star-like” Polymers with Different Symmetry

“Star-like” macromolecules have been widely studied because of their unique physical and chemical properties, which play a great role in many important application fields. In this study, a library of star-like amphiphilic block copolymers consisting of a polyhedral oligomeric silsesquioxane (POSS) core and polyethylene glycol-bolck-polystyrene (PEGm-b-PSn) diblock copolymer arms with an exact arm number were prepared through a robust copper-catalysed azide-alkyne cycloadditions (CuAAC) and strain-promoted azide-alkyne cycloaddition (SPAAC) click reactions. Moreover, homo- and mikto-arms star polymers with different compositions were obtained by precisely regulating the central symmetry of the POSS core. The target polymers and intermediate products were characterized by nuclear magnetic resonance (NMR), size exclusion chromatography (SEC), Fourier transform infrared (FT-IR) spectroscopy, and matrix-assisted laser desorption ionization time-of-flight (MALDI-ToF) mass spectrometry. This study demonstrates the chemical robustness of the novel POSS-based building block and establishes a general and efficient approach to prepare star-like macromolecules, especially for asymmetric heteroarm star-like macromolecules, which serve as important model compounds in the study of their bulk properties as well as self-assembling behaviors.

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.

Integration of MALDI glycotyping and NMR analysis to uncover an O-antigen substructure from pathogenic Escherichia coli O111

Escherichia coli O111 is a critical pathogenic E. coli serotype that causes severe, potentially fatal complications. Despite its reported variation, only one structure of the O-antigen polysaccharide from E. coli O111 has been reported. Here, a substructure of the O-antigen from E. coli O111 was characterized using matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry and NMR analysis. MALDI glycotyping revealed differing O-antigen repeating unit masses of Δm/z 787 and 828 in the E. coli strains and lipopolysaccharides from the O111 serogroup. This variation was caused by the replacement of the hexose residue with hexosamine in the repeating units, which was further confirmed by LIFT-TOF/TOF analysis. Structural elucidation of the O111 substructure by NMR analysis further demonstrated replacement of the hydroxyl group with an N-acetyl group on the terminal glucose residue of the O-antigen pentasaccharide repeating unit. To our knowledge, this study is the first to provide a detailed structural analysis of a new O-antigen substructure from the E. coli O111 serogroup.

Bacterial isolates and antibiotic sensitivity in canine bacterial keratitis in Korea.

To analyze bacterial isolates associated with canine bacterial keratitis and their antibiotic susceptibility patterns in Korea, focusing on multidrug resistance (MDR) and identifying effective antibiotic combinations for clinical treatment. A total of 146 dogs diagnosed with suspected bacterial keratitis between October 2022 and October 2023 in Korea, with 157 eye samples collected for analysis. Eye samples were cultured to isolate bacteria, and antibiotic susceptibility testing was performed using the minimum inhibitory concentration (MIC) method. Bacterial identification was conducted using Matrix-Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF). The study assessed the efficacy of individual antibiotics and combination therapies. Bacteria were isolated in 55.4% of the samples. The most common genera were Staphylococcus species (48.5%, 48/99), Streptococcus species (13.1%, 13/99), Pseudomonas species (9.1%, 9/99), and Escherichia coli (9.1%, 9/99). Amikacin (84.8%) showed the highest antibiotic susceptibility, while doxycycline exhibited the lowest (17.2%). The most effective antibiotic combinations were amikacin-moxifloxacin (93%). MDR isolates accounted for 52.5% (52/99) of the total bacterial samples. Staphylococcus species were the most common isolates, with 52.5% showing MDR, underscoring the need to curb antibiotic misuse. While antibiotics like amikacin demonstrated high susceptibility rates, their use should be reserved for resistant infections to prevent further resistance development. Rather than focusing solely on finding effective combinations of antibiotics, it is crucial to consider alternative treatment strategies that offer more sustainable solutions. Rather than relying on antibiotic combinations, attention should shift to sustainable alternatives to treat bacterial keratitis and reduce antibiotic dependence in clinical practice.

Evaluation of virulence genes in Proteus strains isolated from diabetic foot infections and urinary tract infections.

Proteus species are frequently isolated from urinary tract infections (UTIs) and diabetic foot infections (DFIs). We aimed to evaluate the presence of virulence genes in P. mirabilis and P. vulgaris strains isolated from DFI and UTI. A total of 78 Proteus isolates (57 P. mirabilis and 21 P. vulgaris) collected from patients were studied. The isolates were identified using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometer. The presence of virulence-associated genes (hlyA, mrpA, atfA, pmfA, hmpA, ptaA, ureA, ureC, zapA, ireA, rsbA, flaA, and ucaA) was evaluated by polymerase chain reaction (PCR). 46 isolates were obtained from wound cultures, and 32 were obtained from the midstream urine cultures. All virulence genes, except hlyA, were detected in the study. ureA was the most detected gene in both UTI (100%) and DFI isolates (84.8%). The distributions of ureC, flaA, hpmA, ireA, rsbA, pmfA, zapA, ucaA, ptaA, atfA, and mrpA genes in DFI and UTI isolates were as follows: 82.6% and 96.9%, 71.7% and 93.8%, 69.6% and 93.8%, 69.6% and 96.9%, 69.6% and 96.9, 67.4% and 81.3%, 65.2% and 43.8%, 54.3 and 71.9%, 34.8% and 96.9%, 26.1 and 93.8%, and 23.9% and 46.9%, respectively. We demonstrated that P. mirabilis and P. vulgaris express a variety of virulence genes related to pathogenicity. All virulence genes were found to be more frequent in UTI isolates except zapA. There is limited data on the virulence factors of Proteus species in DFIs. Further studies are needed to investigate virulence genes in wound isolates.

The Clinical Impression of NDM-producing Acinetobacter baumannii in Intensive Care Units of the University Referral Hospital in North India.

Carbapenem-resistant Acinetobacter baumannii (CRAb), a major public health threat, causes severe infections in Intensive Care Unit (ICU) patients. It resists β-lactam antibiotics through mechanisms like New Delhi metallo-beta-lactamase (NDM). In ICU patients, 69 Acinetobacter species were isolated from 86 non-fermenting Gram-negative bacilli. Isolates were identified using biochemical methods and Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS), and carbapenem resistance detection was done by both phenotypic (mCIM and eCIM) and molecular methods. Out of 66 A. baumannii, 61 were carbapenem-resistant, with 20 confirmed as NDM producers. NDM-positive isolates exhibited higher resistance and were associated with significant mortality (75%). NDM-positive Acinetobacter isolates are significant ICU pathogens with poor outcomes. Key risk factors include prolonged ICU stays, prior antimicrobial use, and inadequate therapy. Early detection and infection control are crucial. NDM-positive Acinetobacter infections in ICU patients are linked to poor outcomes, highlighting the need for early detection and control measures. Singh S, Verma A, Venkatesh V, Verma S, Reddy DH, Agrawal A. The Clinical Impression of NDM-producing Acinetobacter baumannii in Intensive Care Units of the University Referral Hospital in North India. Indian J Crit Care Med 2024;28(11):1044-1049.

Genomic characterization of Pantoea dispersa A003 isolated from a clinical patient

IntroductionPantoea dispersa is a Gram-negative bacterium generally considered as a plant pathogen and rarely causes human infections. To date, there have been 13 studies that have documented clinical infections linked to P. dispersa, with a primary emphasis on the initial identification of this pathogen. The genomic features and the mechanisms underlying the pathogenesis of P. dispersa remain largely uninvestigated. In the present study, we describe a clinical infection caused by P. dispersa and provide the first genomic analysis of this bacterium.MethodsThe bacterial strain designated A003 was isolated from blood samples. Preliminary identification of strain A003 was conducted using matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) technology (VITEK® MS, bioMérieux, France). Biochemical and antimicrobial susceptibility assessments were carried out utilizing the VITEK-2 compact automated microbial analysis system (bioMérieux, France). Additionally, whole genome sequencing and subsequent analysis were performed to further elucidate the potential pathogenicity.ResultsThe bacterial isolate was cultured overnight at 35°C in a CO2-enriched environment on Columbia blood agar, resulting in the appearance of a white, smooth, Gram-negative rod-shaped bacterium with diameters ranging from 1 to 2 mm. Identification of strain A003 was achieved with a high confidence level of 99.9% as P. dispersa using MALDI-TOF mass spectrometry. The average nucleotide identity (ANI) value between strain A003 and the reference strain P. dispersa DSM 30073T was 98.08%, while the DNA–DNA hybridization (DDH) value was 84.10% (Formula 2) based on genome sequencing data, which further confirmed that A003 belonged to the P. dispersa species. Comprehensive analysis revealed the presence of 372 virulence factors, 15 antibiotic resistance genes, 222 carbohydrate-active enzymes, and 666 genes related to Type III secretion system (T3SS) effector proteins, as identified through the core databases of VFDB (Virulence Factors of Pathogenic Bacteria), CARD (Comprehensive Antibiotic Resistance Database), CAZY (Carbohydrate-Active enZYmes Database), and T3SS. The virulence factors included type IV pili (TFP), type VI secretion system, flagella, iron uptake system, and ompA, which are implicated in bacterial pathogenicity. The antibiotic resistance profile indicated resistance to fluoroquinolones, cephalosporins, penams, macrolides, and aminoglycosides, as annotated by the CARD database.ConclusionThe draft genome sequenced represents the inaugural genomic sequence of P. dispersa derived from clinical sources. This advancement may enhance clinical practitioners’ comprehension of the organism’s clinical attributes and serve as a foundational resource for future research into its virulence, antibiotic resistance, and host–pathogen interactions.