Assisted Laser Desorption Ionization Time Research Articles

Multidrug-Resistant Staphylococcus sp. and Enterococcus sp. in Municipal and Hospital Wastewater: A Longitudinal Study.

The objective of the study was to detect multidrug-resistant Staphylococcus sp. and Enterococcus sp. isolates in municipal and hospital wastewater and to determine their elimination or persistence after wastewater treatment. Between August 2021 and September 2022, raw and treated wastewater samples were collected at two hospital and two community wastewater treatment plants (WWTPs). In each season of the year, two treated and two raw wastewater samples were collected in duplicate at each of the WWTPs studied. Screening and presumptive identification of staphylococci and enterococci was performed using chromoagars, and identification was performed with the Matrix Assisted Laser Desorption Ionization Time of Flight mass spectrometry (MALDI-TOF MS®). Antimicrobial susceptibility was performed using VITEK 2® automated system. There were 56 wastewater samples obtained during the study period. A total of 182 Staphylococcus sp. and 248 Enterococcus sp. were identified. The highest frequency of Staphylococcus sp. isolation was in spring and summer (n = 129, 70.8%), and for Enterococcus sp. it was in autumn and winter (n = 143, 57.7%). Sixteen isolates of Staphylococcus sp. and sixty-three of Enterococcus sp. persisted during WWTP treatments. Thirteen species of staphylococci and seven species of enterococci were identified. Thirty-one isolates of Staphylococcus sp. and ninety-four of Enterococcus sp. were multidrug-resistant. Resistance to vancomycin (1.1%), linezolid (2.7%), and daptomycin (8.2%/10.9%%), and a lower susceptibility to tigecycline (2.7%), was observed. This study evidences the presence of Staphylococcus sp. and Enterococcus sp. resistant to antibiotics of last choice of clinical treatment, in community and hospital wastewater and their ability to survive WWTP treatment systems.

In vitro assessment of crude oil degradation by Acinetobacter junii and Alcanivorax xenomutans isolated from the coast of Ghana

This study was aimed at using in vitro microcosm experiments to assess crude oil degradation efficiency of Acinetobacter junii and Alcanivorax xenomutans isolated along Ghana's coast. Uncontaminated seawater from selected locations along the coast was used to isolate bacterial species by employing enrichment culture procedures with crude oil as the only carbon source. The isolates were identified by means of the extended direct colony transfer method of the Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectroscopy (MALDI-TOF MS), as Acinetobacter junii, and Alcanivorax xenomutans. Remediation tests showed that Acinetobacter junii yielded degradation efficiencies of 27.59 %, 41.38 % and 57.47 %. Whereas efficiencies of 21.14 %, 32.18 % and 43.68 % were recorded by Alcanivorax xenomutans representing 15, 30 and 45 days respectively. Consortia of Acinetobacter junii, and Alcanivorax xenomutans also yielded 32.18 %, 48.28 % and 62.07 % for the selected days respectively. Phylogenetic characterization using ClustalW and BLAST of sequences generated from the Oxford Nanopore Sequencing technique, showed that the Ghanaian isolates clustered with Alcanivorax xenomutans and Acinetobacter junii species respectively. An analysis of the sequenced data for the 1394-bp portion of the 16S rRNA gene of the isolates revealed >99 % sequence identity with the isolates present on the GenBank database. The isolates of closest identity were Alcanivorax xenomutans and Acinetobacter junii with accession numbers, NR_133958.1 and KJ147060.1 respectively. Acinetobacter junii and Alcanivorax xenomutans isolated from Ghana's coast under pristine seawater conditions have therefore demonstrated their capacity to be used for the remediation of crude oil spills.

Analysis of gene characteristics and core genome characteristics of carbapenem-resistant Enterobacter cloacae in rural residents of Weifang City, Shandong Province

Objective: To investigate the drug-resistant gene characteristics and core genome characteristics of carbapenem-resistant Enterobacter cloacae (CR-ECL) in rural residents of Weifang City, Shandong Province. Methods: Fecal samples were collected from rural community residents in Weifang City, Shandong Province in 2017. Drug-resistant strains were screened using a carbapenem-resistant enterobacter chromogenic medium. CR-ECL positive strains were acquired via Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry(MALDI-TOFMS) analysis. The antibiotic resistance phenotype of CR-ECL was determined using a microbroth dilution assay. Whole genome sequencing (WGS) and analysis were conducted, along with an examination of the immediate vicinity of the blaNDM gene and phylogenetic analysis of the strains. Results: A total of 628 fecal samples were collected and tested, of which 6 were CR-ECL positive (detection rate 0.96%), all exhibiting multiple drug resistance (MDR) phenotypes. Six CR-ECL strains had four MLST genotypes (ST), all of which carried multiple drug resistance genes (blaNDM-1, blaNDM-5, etc.) and virulence genes (acrA, acrB, entB, fepC, etc.). There were mobile genetic elements ISAba125, TN3-IS3000, TN3 and IS5 in the genetic environment surrounding the blaNDM gene. The phylogenetic tree showed that the multi-locus sequence typing of the core genome (cgMLST) was consistent with the single nucleotide polymorphism (SNPs) results. The cgMLST results showed that the allele differences between strains 2BC0101B and 2BC0251B, 2BG0561B and 2BI0221B were 2 and 1, respectively. The SNPs results showed that the above two pairs of bacteria also clustered together. It was found that the strains of chicken fecal samples in the National Center for Biotechnology Information (NCBI) database were located in the center of the evolutionary tree, and the local sequences could be traced back to American human sequences. Conclusion: Multidrug-resistant CR-ECL is detected in rural community residents in Weifang City, Shandong Province.

Enrichment of phosphopeptides by arginine-functionalized magnetic chitosan nanoparticles.

One of the most crucial and prevalent post-translational modifications is the phosphorylation of proteins. The study and examination of protein phosphorylation hold immense importance in comprehending disease mechanisms and discovering novel biomarkers. However, the inherent low abundance, low ionization efficiency, and coexistence with non phosphopeptides seriously affect the direct analysis of phosphopeptides by mass spectrometry. In order to tackle these problems, it is necessary to carry out selective enrichment of phosphopeptides prior to conducting mass spectrometry analysis. Herein, magnetic chitosan nanoparticles were developed by incorporating arginine, and were then utilized for phosphopeptide enrichment. A tryptic digest of β-casein was chosen as the standard substance. After enrichment, combined with matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS), the detection limit of the method was 0.4 fmol. The synthesized magnetic material demonstrated great potential in the detection of phosphopeptides in complex samples, as proven by its successful application in detecting phosphopeptides in skim milk and human saliva samples.

868. Early Organism Identification by Matrix Assisted Laser Desorption Ionization Time Of Flight Mass Spectrometry (MALDI TOF MS) Decreases the Time to Optimal Antibiotic Therapy for Common Bacterial Infections

Abstract Background MALDI-TOF helps in precise identification of microorganisms from positive cultures. It is based on the principle of proteomics and obviates the need for the use of biochemicals. It reduces the turnaround time for reporting microbiologic cultures. Early identification and reporting of the organism, before the availability of antimicrobial susceptibilities, may reduce the time to first antibiotic modification and impact the clinical outcomes of patients. Methods This is a prospective observational two-arm comparative study conducted at a tertiary care centre in North India, with a sample size of 50 patients in the conventional arm and 100 patients in the interventional arm. The workflow of both arms are depicted in figure 1&2. The study population included patients in the medical ward and intensive care units (ICU) with positive cultures. The primary objective of this study was to compare the average time to first antibiotic modification post-culture positivity in the conventional and interventional arms and to compare clinical outcomes of patients in both arms. Escalation, de-escalation and any modification in antibiotics were also recorded. Results The median time to first antibiotic modification was significantly shorter in the intervention arm compared to the conventional arm (2010 mins vs 2905 mins, p=0.002). Overall 44/100 antibiotic modifications were made in the interventional arm out of which 43.2% (19/44) were made based on the MALDI report alone without awaiting susceptibilities. The major type of modification was the de-escalation of antibiotics (47.4%). Mortality rates were 32% in the conventional arm and 27% in the intervention arm, but the difference was not statistically significant (p=0.52) (table 1). Conclusion MALDI-TOF helps in the early modification of antibiotics. The major utility is in the de-escalation of unnecessary antibiotics. Coupled with a robust antimicrobial stewardship programme, this modality can help in reducing antibiotic use. Disclosures All Authors: No reported disclosures

B-179 Development of a Reflex Mass Spectrometric Workflow for Confirmation of IgE and IgD Specificity Paraproteins Utilising the EXENT® Platform

Abstract Background The EXENT® (GAM) assay is a MALDI-TOF (Matrix Assisted Laser Desorption Ionisation Time of Flight) mass spectrometry-based solution which utilises IgG, IgA, IgM, total kappa and total lambda specific paramagnetic beads, which could be used in the investigation of monoclonal proteins in multiple myeloma and other plasma cell disorders (in development by The Binding Site (TBS), part of Thermo Fisher Scientific). Rare cases of IgD and IgE myelomas will be partially typed with the current iteration of the EXENT (GAM) assay. We present here a workflow to confirm the heavy chain specificity of such paraproteins as IgD or IgE using reflex LC-MS analysis of EXENT eluates. Methods Samples known to contain IgD (2) or IgE (3) paraproteins were provided from either routine workload at the immunology laboratory at Birmingham Heartlands Hospital, UK or an internal library of historical myeloma samples. Capillary zone electrophoresis (CZE) and immunofixation (IFE) were performed on Capillarys2™ and Hydrasys2 Scan™ platforms, respectively (Sebia, France). Serum free light chain (sFLC) measurement was performed using Freelite® reagents (The Binding Site, UK) on a Cobas c501 turbidimeter (Roche, Switzerland). Samples were prepared via EXENT (GAM) assay, by separately incubating diluted serum with antisera specific for IgG, IgA, IgM, total kappa and total lambda conjugated to paramagnetic beads. After washes, purified immunoglobulins were treated to reduce and elute the light chain component. Samples were mixed with matrix, spotted onto a MALDI target plate and acquired by MALDI-TOF MS. Once light-chain specificity of the suspected IgD or IgE paraprotein was determined, repeat immuno-precipitation with the same light-chain bead was carried out. Eluted samples were neutralised, transferred to fresh tubes, reduced, alkylated and digested with trypsin for 3 h. After acidification digested samples were analysed by C18 reverse phase UPLC on a BioAccord LC-MS system (Waters, UK). Data was then screened against a database of known immunoglobulin heavy chain peptides to assess the presence of heavy chain peptides specific for each immunoglobulin isotype. Results In each sample tested, the EXENT solution identified at least one monoclonal light chain that was only seen in the total kappa or total lambda immunoprecipitations with no corresponding monoclonal peak(s) at the same mass(es) in the IgG, IgA or IgM specificities. This finding would be consistent with the presence of clonal plasma cells producing either monoclonal free light chains only, or IgD and IgE paraproteins. Subsequent LC-MS and LC-MSe (tandem MS) analysis of reduced, alkylated and tryptically digested eluates corresponding to these suspected IgD and IgE samples produced high quality peptide sequencing data. The data identified multiple IgD or IgE specific peptides in each sample and showed between 40% and 66% sequence coverage for IgD or IgE heavy-chain constant regions. Thus, confirming the presence of IgD or IgE in these samples which had been partially typed by the EXENT solution. Conclusion This data demonstrates that the EXENT (GAM) assay could potentially aid in detection of IgD and IgE paraproteins when combined with LC-MS proteomic techniques, as it allows efficient and facile confirmation of these rare heavy chain isotypes.

Application of MALDI-TOF Mass Spectrometry for the Assessment of Prevalence of Listeria monocytogenes in Raw Milk, Dairy Products and Freshwater Fishes

Background: Foodborne infections like Listeriosis cover several disorders and are a worldwide public health emergency. Listeria monocytogenes has been isolated from various foodstuffs, including milk and fishes. MALDI-TOF mass spectrometry technique is known for its rapid and accurate identification of bacterial organisms. Methods: In the current research, a total of 360 samples comprising raw milk (130), milk products (125) and freshwater fishes (105) were screened for the detection of pathogenic Listeria species by using the USDA method. The recovered Listeria isolates were characterized using conventional set of biochemical analysis along with sugar fermentation tests and further confirmed by MALDI-TOF MS. The virulent nature of pathogenic Listeria isolates was also assessed by in vitro tests like hemolysis on blood agar, CAMP and PI-PLC assay. Result: In current study, on screening 360 animal origin food samples, three isolates were recovered from raw milk samples and identified as Listeria monocytogenes indicating an overall prevalence of Listeria monocytogenes to the tune of 0.83%. Excellent correlation was observed with identification of Listeria species using conventional phenotypic tests and advanced molecular tool Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS) technique. The results depict dependability of advanced technique for rapid and reliable identification of Listeria species.

Fc N-glycosylation of autoreactive Aβ antibodies as a blood-based biomarker for Alzheimer's disease.

Naturally occurring autoantibodies (nAbs) against the pathologic isoform of amyloid beta (Aβ42 ) were found in body fluids and indicate a systemic B cell response that may prevent Alzheimer's disease (AD) onset. N-glycans attached to immunoglobulin G-Fab/Fc fragments are features that influence their mechanism of action. The aim was to study the role of N-glycans in nAbs-Aβ42 . nAbs-Aβ42 were isolated from AD patients and age-/sex-matched controls (n = 40) and immunoglobulin preparations. Glycosylated/deglycosylated nAbs-Aβ42 were analyzed for their effect on Aβ42 's aggregation, toxicity, and phagocytosis. Glycan structure was analyzed using matrix assisted laser desorption ionization time of flight mass spectrometry. Deglycosylation of nAbs-Aβ42 had a major impact on Aβ42 's aggregation/toxicity/phagocytosis. The glycan structure showed considerable differences between AD and controls. We were able to predict disease status with a sensitivity/specificity of 95% (confidence interval [CI]: 76.4-99.7%)/100% (CI: 83.9-100%). N-glycosylation has been identified as a critical attribute maintaining the beneficial effects of autoreactive Aβ antibodies. These data have consequences for the development of monocloncal Aβ antibodies and may open new avenues for diagnostics.

Utilizing Mass Spectrometry to Detect and Isotype Monoclonal Proteins in Urine: Comparison to Electrophoretic Methods.

Matrix assisted laser desorption ionization time of flight mass spectrometry coupled to immune enrichment (MASS-FIX) as an alternative to serum immunofixation electrophoresis has demonstrated increased sensitivity in monoclonal protein (MP) detection with improved laboratory workflow. This study explored similar replacement of urine immunofixation electrophoresis (u-IFE) with urine MASS-FIX (u-MASS-FIX) by method comparison. Residual urine (n = 1008) from Mayo Clinic patients with a known plasma cell disease were assayed neat by u-MASS-FIX analysis. Each sample was paired with the following: u-IFE, urine total protein, urine protein electrophoresis, serum κ/λ free light chain (LC) ratio (rFLC), and serum MASS-FIX (s-MASS-FIX). Analytical sensitivities were measured in pooled urine spiked with daratumumab. u-IFE and u-MASS-FIX had 91% agreement in determining the presence/absence of MPs (Cohen kappa = 0.8200). In discrepant cases, serum rFLC statistically aligned more closely with positive u-MASS-FIX cases than u-IFE. Patients positive by both s-MASS-FIX and u-MASS-FIX had matching MP masses (±20 daltons) in 94% of cases. The u-MASS-FIX spectra further identified κ/λ LC fragments and glycosylated LCs not appreciated on u-IFE. The unconcentrated u-MASS-FIX limit of detection of 0.156 mg/mL was determined equivalent to 100× concentrated u-IFE. u-MASS-FIX is a reliable alternative to u-IFE with the added benefits of LC glycosylation detection and MP mass tracking between serum and urine. Furthermore, u-MASS-FIX is performed using neat urine. Eliminating the need to concentrate urine for u-IFE has potential to increase productivity by decreasing labor minutes per test.

Whole genome sequencing to study antimicrobial resistance and RTX virulence genes in equine Actinobacillus isolates

Actinobacillus equuli is mostly associated with disease in horses and is most widely known as the causative agent of sleepy foal disease. Even though existing phenotypic tools such as biochemical tests, 16S rRNA gene sequencing, and Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS) can be used to identify members of the Actinobacillus genus, these methods struggle to differentiate between certain species and do not allow strain, virulence, and antimicrobial susceptibility typing. Hence, we performed in-depth analysis of 24 equine Actinobacillus isolates using phenotypic identification and susceptibility testing on the one hand, and long-read nanopore whole genome sequencing on the other hand. This allowed to address strain divergence down to the whole genome single nucleotide polymorphism (SNP) level. While lowest resolution was observed for 16S rRNA gene classification, a new multi-locus sequence typing (MLST) scheme allowed proper classification up to the species level. Nevertheless, a SNP-level analysis was required to distinguish A. equuli subspecies equuli and haemolyticus. Our data provided first WGS data on Actinobacillus genomospecies 1, Actinobacillus genomospecies 2, and A. arthritidis, which allowed the identification of a new Actinobacillus genomospecies 1 field isolate. Also, in-depth characterization of RTX virulence genes provided information on the distribution, completeness, and potential complementary nature of the RTX gene operons within the Actinobacillus genus. Even though overall low prevalence of acquired resistance was observed, two plasmids were identified conferring resistance to penicillin-ampicillin-amoxicillin and chloramphenicol in one A. equuli strain. In conclusion our data delivered new insights in the use of long-read WGS in high resolution identification, virulence gene typing, and antimicrobial resistance (AMR) of equine Actinobacillus species.

Pulmonary Nocardiosis Caused by <i>Nocardia otitidiscaviarum</i> and <i></i>Successfully Treated with Ciprofloxacin Based on Bacterial Identification and Antimicrobial Susceptibility Testing

We report a case of pulmonary nocardiosis caused by Nocardia otitidiscaviarum in a man diagnosed with idiopathic thrombocytopenia who was on oral prednisolone therapy. A chest PET/CT examination 13 days after the start of prednisolone therapy revealed a nodular shadow, and it progressively worsened and was accompanied by cavity formation over time. N. otitidiscaviarum was identified in bronchoalveolar lavage fluid by matrix assisted laser desorption ionization time of flight mass spectrometry. The isolate was eventually confirmed to be N. otitidiscaviarum by a 16S rRNA analysis. There was no evidence of disseminated disease. Combination therapy consisting of oral sulfamethoxazole-trimethoprim (SMX-TMP) and intravenous amikacin and imipenem was initiated, but the SMX-TMP was discontinued because of adverse effects. Antimicrobial susceptibility testing revealed that the isolate was non-susceptible to ceftriaxone, imipenem, minocycline, and clarithromycin. The patient declined long-term intravenous therapy. Oral ciprofloxacin was administered instead, and the pulmonary nocardiosis improved after 6 months of therapy. Bacterial identification and antimicrobial susceptibility testing influence the choice of treatment regimens for nocardia infections by N. otitidiscaviarum, which may be resistant to multiple antimicrobial agents.

Emergence of Clonally-Related South Asian Clade I Clinical Isolates of Candida auris in a Greek COVID-19 Intensive Care Unit.

Candida auris has recently emerged as a multidrug-resistant yeast implicated in various healthcare-associated invasive infections and hospital outbreaks. In the current study, we report the first five intensive care unit (ICU) cases affected by C. auris isolates in Greece, during October 2020-January 2022. The ICU of the hospital was converted to a COVID-19 unit on 25 February 2021, during the third wave of COVID-19 in Greece. Identification of the isolates was confirmed by Matrix Assisted Laser Desorption Ionization Time of Flight mass spectroscopy (MALDI-TOF]. Antifungal susceptibility testing was performed by the EUCAST broth microdilution method. Based on the tentative CDC MIC breakpoints, all five C. auris isolates were resistant to fluconazole (≥32 μg/mL), while three of them exhibited resistance to amphotericin B (≥2 μg/mL). The environmental screening also revealed the dissemination of C. auris in the ICU. Molecular characterization of C. auris clinical and environmental isolates was performed by MultiLocus Sequence Typing (MLST) of a set of four genetic loci, namely ITS, D1/D2, RPB1 and RPB2, encoding for the internal transcribed spacer region (ITS) of the ribosomal subunit, the large ribosomal subunit region and the RNA polymerase II largest subunit, respectively. MLST analysis showed that all isolates possessed identical sequences in the four genetic loci and clustered with the South Asian clade I strains. Additionally, PCR amplification and sequencing of the CJJ09_001802 genetic locus, encoding for the "nucleolar protein 58" that contains clade-specific repeats was performed. Sanger sequence analysis of the TCCTTCTTC repeats within CJJ09_001802 locus also assigned the C. auris isolates to the South Asian clade I. Our study confirms that C. auris is an emerging yeast pathogen in our region, especially in the setting of the ongoing COVID-19 worldwide pandemic. Adherence to strict infection control is needed to restrain further spread of the pathogen.

MALDI-TOF-MS for Rapid Detection of Fungi causing Mucormycosis during COVID-19 Pandemic from a Tertiary Care Hospital in Southern India: A Retrospective Cohort Study

Introduction: Mucormycosis is an opportunistic fungal infection that became a public health emergency during the second wave of Coronavirus Disease-2019 (COVID-19). It is an acute, angioinvasive, opportunistic, and emerging mycosis caused by mucormycetes, resulting in a significantly fatal fungal infection among immunocompromised and/or immunosuppressed individuals. Mucormycosis has a specific predilection for blood vessels (angioinvasive) and tissues, leading to extensive necrosis and thromboembolic events. Globally, the incidence rate of mucormycosis varies from 0.005 to 1.7 per million people. Early diagnosis and appropriate therapy are crucial for clinical outcomes. Aim: To evaluate the mycological profile of mucormycosis during the COVID-19 pandemic using automated Matrix Assisted Laser Desorption Ionisation Time of Flight Mass Spectroscopy (MALDI-ToF-MS) in comparison with conventional fungal identification methods. Materials and Methods: A retrospective cohort study was conducted, collecting data from July 2020 to June 2022, involving 1,176 patients from various clinical departments who attended PSG Hospitals affiliated with PSG Institute of Medical Sciences and Research, Coimbatore, Tamil Nadu, India. Data analysis was performed from July 2022 to August 2022. Clinical specimens, including Bronchoalveolar Lavage (BAL), Sputum, Cerebrospinal Fluid (CSF), nasal swabs, tracheal aspirates, pus, blood, corneal scraping, eye discharge, and necrotised tissues, were subjected to Potassium Hydroxide (KOH) mount, conventional culture coupled with micrometry-aided microscopy, and MALDI-ToF-MS among suspected mucormycosis cases. The results were collected in Microsoft Excel, and data analysis was conducted using the International Business Machines Statistical Package for the Social Sciences statistical software (IBM SPSS) version 24.0. The study variables were expressed as descriptive statistics: frequency (N) and percentage (%). The incidence of mucormycosis, mycological profile, and diagnostic utility of conventional micrometry-aided microscopy-assisted culture were compared with the automated MALDI-ToF-MS protein signature method of testing. Results: Fungal elements were positive in 130 (11.05%) out of a total of 1,176 clinical samples, such as necrotised tissue, biopsy, and sputum, in the KOH mount. Fungal culture was positive in 258 (21.94%) out of 1176 samples, with mucormycosis detected in 73 (28.29%) of the culture-positive samples. The incidence of mucormycosis in this study was 73 (6.21%) out of 1176. A total of 101 (8.59%) samples showed positive results for mucormycetes through KOH, culture, and MALDI-ToF-MS. MALDI-ToF-MS could rapidly and specifically identify and confirm the causative agents of mucormycosis at their genus and species levels compared to KOH or culture. Conclusion: Simple, low-cost, and less laborious conventional culture coupled with micrometry-aided microscopy can detect Mucorales at the species level, where KOH microscopy could not differentiate between species. MALDI-ToF-MS can be employed for the rapid detection and confirmation of mucormycosis agents at the genus or species level

Scubcutaneous Mycobacterium marinum infection misdiagnosed as sporotrichosis: A case report

Nontuberculous mycobacteria (NTM), are natural inhabitants of natural waters, engineered water systems, and soils. As a consequence of their ubiquitous distribution, humans are surrounded by these opportunistic pathogens. In this report, we describe a case of scubcutaneous Mycobacterium marinum infection associated with home tropical ornamental fish aquaria. A 43-years-old man reported progressively increasing nodules over his left forearm and hand for more than 7 months. Based on NTM culture, pathological examination, identification by gene sequencing and matrix assisted laser desorption ionization time of flight mass spectrometry, the diagnosis of scubcutaneous NTM infection was confirmed. The patient was treated with itraconazole for suspected sporotrichosis over 1 month. The patient was treated with oral doxycycline hydrochloride capsules (200 mg/day) for 5 months, the nodules had resolved. Home aquarium environments may serve as a possible source of mycobacteria infection in urban family.

Methicillin-resistant and methicillin-sensitive Staphylococcus aureus isolates from skin and nares of Brazilian children with atopic dermatitis demonstrate high level of clonal diversity

Atopic dermatitis (AD) primarily affects the pediatric population, which is highly colonized by S. aureus. However, little is known about the genetic features of this microorganism and other staphylococcal species that colonize AD patients. This study aimed to characterize Staphylococcus spp. isolated from the nares and skin (with and without lesion) of 30 AD and 12 non-AD Brazilian children. Skin and nasal swabs were cultured onto mannitol salt agar, and bacterial colonies were counted and identified by matrix assisted laser desorption ionization time of flight mass spectrometry and polymerase chain reaction (PCR). Antimicrobial susceptibility was evaluated by phenotypic and genotypic tests. In S. aureus isolates, Panton-Valentine leukocidin genes were detected by PCR, and their clonality was assessed by pulsed-field gel electrophoresis and multilocus sequence typing. S. aureus was more prevalent in the nares (P = 0.005) and lesional skin (P = 0.0002) of children with AD, while S. hominis was more frequent in the skin of non-AD children (P < 0.0001). All children in the study, except one from each group, were colonized by methicillin-resistant coagulase-negative Staphylococcus and 24% by methicillin-resistant S. aureus. Despite the great clonal diversity of S. aureus (18 sequence types identified), most AD children (74.1%) were colonized by the same genotype in both niches. High colonization by polyclonal S. aureus isolates was found among children with AD, while S. hominis was more frequent among non-AD children. The high prevalence of methicillin-resistant staphylococcal isolates highlights the importance of continued surveillance, especially when considering empiric antibiotic therapy for the treatment of skin infections in these patients.

A new Fe3+-selective, sensitive, and dual-channel turn-on probe based on rhodamine carrying thiophenecarboxaldehyde: Smartphone application and imaging in living cells

A new dual-channel probe based on rhodamine B derivative (MSB) was successfully designed, synthesized, characterized by Nuclear Magnetic Resonance (NMR) Spectroscopy, Fourier Transform Infrared Spectrophotometer (FTIR), Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS), X-ray Photoelectron Spectroscopy (XPS), and Single Crystal X-rayDiffraction, and the sensing abilities toward Fe3+ cation have been demonstrated and the probe was successfully utilized for fluorescence imaging of Fe3+ in living cells. The probe demonstrated quite fast, sensitive, and selective response to Fe3+ by causing an extreme enhancement in UV–vis and fluorescence spectroscopy techniques in the buffered aqueous media which makes MSB a dual-channel probe. While the color of MSB solution was initially light yellow, it turned pink in the presence of Fe3+, which provided highly selective naked-eye determination among several ions as alkaline, alkaline-earth, and transition metal ions. After that, the probe was easily applied to paper strips and real samples such as drinking waters and supplementary iron tablets for sensing Fe3+ in an aqueous solution. The detection limit (LOD) and the response time of the probe were determined as 4.85x10-9 M and 4 min, respectively, which are quite lower compared with other rhodamine based Fe3+ sensors in the literature. According to Job’s plot, 1H NMR titration, MALDI-TOF MS, XPS, and DFT study techniques, the complexation ratio between MSB and Fe3+ was found as 1:1. Moreover, the spectral response was reversible with alternately addition of Fe3+ or Na2EDTA to the MSB solution. In addition, fluorescence imaging in NIH/3T3 mouse fibroblast cells and studies in real samples with a quite high recovery rate exhibited that the probe is qualified for detection of Fe3+ ion with multiple practical usages.

MALDI-TOF MS for Rapid Analysis of Bacterial Pathogens Causing Urinary Tract Infections in the Riyadh Region.

The successful treatment of bacterial disease is relied on selecting a suitable drug based on the type of bacteria and antimicrobial susceptibility testing. The study’s objective was to identify bacterial isolates from urine samples of patients from the community, followed by antimicrobial susceptibility testing of the isolated bacteria. A total of seventy urine samples were received in the clinical microbiology laboratory; out of which 18 culture-positive cultures and by direct identification using MALDI-TOF MS (Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry) were identified. Of 18 identified bacteria, 17 (94%) were pathogenic. The culture demonstrated that the major species detected in urine samples were Escherichia coli, Klebsiella pneumoniae, Enterococcus faecalis, and Aeromonas caviae. E. coli (72.2%) was the most common bacterium retrieved from urine samples followed by K. pneumoniae (16.6%). Interestingly, all the isolates, except Enterococcus faecalis, were resistant to erythromycin. The isolates 8 of 13 (61.5%) were resistant to both of the cotrimoxazole and tetracycline. We performed MLST (Multi-locus Sequence Typing) typing of 13 E. coli isolates to study their genetic relatedness and diversity. MLST typing of E. coli showed a total of nine different STs (Sequence Types), which showed the diversity among them. ST 129 was the most common ST found in three E. coli isolates. In our study, two isolates with ST 1126 and ST 1432 represented the global clonal complex 155. MALDI-TOF MS provided dependable results for identifying the bacteria up to species level from urine samples by indirect culture methods. Such local surveillances are highly recommended for empirical therapy awareness and determining isolates’ level of resistance.

S6.2d Dermatophytosis due to Trichophyton erinacei caused by contact with African hedgehogs as family pets

S6.2 Resurgence of dermatophytic infections, September 22, 2022, 4:45 PM - 6:15 PMObjectiveTo present a series of cases of various types of dermatophytosis caused by Trichophyton erinacei, caused by contact with African hedgehogs.MethodsPatients with clinical suspicion of ringworm caused by T. erinacei were included in the study. Each patient underwent a mycological study, with direct examinations (KOH), cultures in Sabouraud dextrose agar medium with and without antibiotics. With the cultures obtained, molecular identification was made using the PCR technique and Matrix Assisted Laser Desorption Ionization Time of Flight (MALDI-TOF) proteomics. Once confirmed, the patients were given treatment and follow-up. When the pets (hedgehogs) were accessible, they were cultured with a cytobrush and the quills removed.ResultsIn a period of 3.5 years, 16 verified cases were incorporated, 13 were females (72%) and 3 males (18%), the average age was 18.2 years, with the smallest case being 8 years old and the oldest 45 years old. The tineas manifested as follows: tinea manus 11 cases (68.7%); 2 respective cases of tinea corporis and tinea capitis (18.1 respectively) and one case of Majocchi's granuloma. The average evolution was 1.5 months with the case older than 3 months and the younger 15 days. All cases were given terbinafine at variable times and doses (according to weight and age) and clinical and mycological cure was obtained.DiscussionTineas can have epidemiological changes that are favored by a variety of factors, in general, pet ringworms are due to common domestic animals such as dogs and cats. Due to changing fashions, children and adolescents in recent years have changed pets such as hamsters, and recently hedgehogs, especially African variety (Atelerix albiventris) the latter may have T. erinacei as part of their skin microbiota, a similar dermatophyte or that was part of the T. mentagrophyes complex, in general of low virulence, however, its antigenic variants are little recognized by humans, which makes them present in a very inflammatory form, particularly in the hands, due to the handling of the animal, as well as the deeper inoculation (in the dermis) by the spikes left by the inoculum. So, they can generate deep tineas such as Majocchi's granuloma. In general, they respond well to systemic antifungals such as terbinafine and itraconazole, and sometimes the use of topical or systemic steroids is necessary.ConclusionsThis series of cases is a good example of how a change of habits when having pets can introduce new etiological agents that are poorly recognized antigenically, which give rise to more inflammatory and sometimes profound tineas.

P362 Seasonal trend of fungal flora in water of tertiary care hospital in North India

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PMObjectivesThe study was conducted to assess the seasonal variation of fungal flora in hospital water of a tertiary care hospital in North India.MethodsA total of 200 water samples from the main reservoir, overhead and underground tanks, and taps of critical care units of the hospital were collected. The water samples were filtered by membrane filtration technique (0.22 micron) and cultured on dichloran rose-Bengal chloramphenicol agar with and without benomyl. The plates were incubated for upto 15 days and fungal colonies recovered were sub-cultured on Sabouraud Dextrose Agar and identified by phenotypic methods. Yeasts were identified by Matrix Assisted Laser Desorption Ionization Time of Flight (MALDI-TOF-MS).ResultsMycelial fungi were isolated from 100% of the water samples which included Alternaria, Curvularia, Nigrospora, Penicillium, Aspergillus, Paecilomyces, Scytillidium, and Mycelia sterilia as depicted in Figure 1. Different fungi were prevailing in different water storage units like: Advance eye center— A. fumigatus, A. flavus, Fusarium solani, Alternaria spp. Rhizopus arrhizus, Ustilago spp., mycelia sterilia, Trichosporon spp.; Advance trauma Center—Cladophaialophora spp., Alternaria alternata, Penicillium, spp. A. flavus, A. nidulans, A. fumigatus, F. solani, Rhodotorula spp.: Bone marrow transplant unit— Alterneria alternata, A. niger, A. flavus, A. versicolor, Cladosporium spp., Fonsecaea pedrosoi, Fusarium spp., Nigrospora spherica, Penicillium spp., Rhodotorula spp., Trichosporon ashaii. The seasonal variation of fungal isolation is depicted in Figure 2. Isolation rate of Aspergillus species was 35% in winters, 31% in post-monsoon, 25% in summers. Isolation rate of Penicillium species was 19% in post-monsoon, 16% in winter and 15% in summers. Maximum number of dematiaceous fungi were isolated in summer season with isolation rate of 30% in summers as compared with 21.5% in post-monsoon and 19% in winters. Few yeasts isolated were Rhodotorula, Trichosporon, and Ustilago. Mucorales isolated rarely included Rhizopus, Absidia, Syncephalastrum, and Mucor species. Fungal colony forming units in the water samples ranged from 50 to 450 colony forming units/liter of water.ConclusionThe distribution of fungi in hospital water showed diversity and seasonal variability. Aspergillus species were isolated in maximum number in the winter season, Penicillium species in post-monsoon season and dematiaceous fungi in the winter season. Water as a source of fungal infection in critical care units, remains a relatively neglected area. Water supply could be a source of nosocomial fungal infections. Improving the quality of water by regular testing for fungal contamination and appropriate action to reduce its burden may reduce the hospital-acquired fungal infections.

S1.2c Diagnosis of fungal infections in animals: Combining the old and the new to maximize results

S1.2c Diagnosis of fungal infections in animals: Combining the old and the new to maximize results