MALDI-TOF MS Research Articles

Genomic characteristics of antimicrobial resistance and virulence factors of carbapenem-resistant Stutzerimonas nitrititolerans isolated from the clinical specimen

BackgroundStutzerimonas nitrititolerans (S. nitrititolerans) is a rare human pathogenic bacterium and has been inadequately explored at the genomic level. Here, we report the first case of carbapenem-resistant S. nitrititolerans isolated from the peritoneal dialysis fluid of a patient with chronic renal failure. This study analyzed the genomic features, antimicrobial resistance, and virulence factors of the isolated strain through whole genome sequencing (WGS).MethodsThe bacterial isolate from the peritoneal dialysis fluid was named PDI170223, and preliminary identification was conducted through Matrix-assisted laser desorption ionization/time of flight mass spectrometry (MALDI-TOF MS). WGS of the strain PDI170223 was performed using the Illumina platform, and a phylogenetic tree was constructed based on the 16S rRNA gene sequences. Antimicrobial susceptibility test (AST) was conducted using the TDR-200B2 automatic bacteria identification/drug sensitivity tester.ResultsS. nitrititolerans may emerge as a human pathogen due to its numerous virulence genes, including those encoding toxins, and those involved in flagellum and biofilm formation. The AST results revealed that the strain is multidrug- and carbapenem-resistant. The antimicrobial resistance genes of S. nitrititolerans are complex and diverse, including efflux pump genes and β⁃lactam resistance genes.ConclusionThe analysis of virulence factors and antimicrobial resistance of S. nitrititolerans provides clinical insight into the pathogenicity and potential risks of this bacterium. It is crucial to explore the mechanisms through which S. nitrititolerans causes diseases and maintains its antimicrobial resistance, thereby contributing to development of effective treatment and prevention strategies.

A-254 Increase of Stenotrophomonas maltophilia Isolates Non-Susceptible to Trimethoprim-Sulfamethoxazole and/or Levofloxacin at Brazilian Hospitals

Abstract Background Stenotrophomonas maltophilia is an emerging multidrug-resistant pathogen that exhibits intrinsic and acquired resistance to a wide variety of antimicrobial agents such as β-lactams, aminoglycosides, quinolone, tetracyclines and disinfectants. In addition to that, the increased resistance to sulfamethoxazole/trimethoprim (SMX/TMP) and Levofloxacin has been reported in the past few years, leading to few therapeutic options. This study aimed to describe the increase of Stenotrophomonas maltophilia clinical isolates recovered from Brazilian hospitals over a 6-year analysis (January 2018 to December 2023). Methods This study design was a retrospective, observational analysis based on microbiological data of emergent BGN-NF isolates detected in several Brazilian tertiary hospitals. The bacterial identification was performed by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF/MS) and the antimicrobial susceptibility profile to trimethoprim-sulfamethoxazole and levofloxacin was determined using disk-diffusion method and epsilometer method, respectively. Only one sample per patient was considered in this analysis. Results During the 6 year period, a total of 1,706 (2018; 221), (2019; 203), (2020; 294), (2021; 276), (2022; 294) and (2023; 418) Stenotrophomonas maltophilia strains were isolated from blood (n=626), respiratory tract (n=365), fluid and secretion (n=235), skin and soft tissue (n=162), urine (n=132), catheter tip (n=112), swab (n=36), bone fragment (n=29) and cerebrospinal fluid (n=9). The major frequency of S. maltophilia isolation occurred among man (52.6%) and the main age range was over 60 years (42.7%), while 0-10 comprised 16.6% of cases. Compared the 6-years period, the resistance rate to trimethoprim-sulfamethoxazole was 2.7%; 5.4%; 5.8%; 2.2%; 5.8%; 7.9% and levofloxacin was 3.6%; 3.4%; 3.1%; 3.3%; 1.7%; 2.9%, respectively. The resistance rate to trimethoprim-sulfamethoxazole and levofloxacin, simultaneously, was 0.23%. Conclusions The incidence rate of S. maltophilia infections has increased significantly, especially in the last year, followed by an increase in the pattern of resistance to trimethoprim-sulfamethoxazole. Regarding trimethoprim-sulfamethoxazole and levofloxacin co-resistance, very low rates were detected in our analysis. Our data point to a scenario wherein the presence of S. maltophilia may have been intensified by the covid-19 pandemic. Regional surveillance reports are extremely important and should be out to understand some aspects of the dynamics of bacterial resistance in both hospital and community settings.

A-288 Active Surveillance Cultures: Frequency of Microorganisms and Phenotypic Resistance Profile From Brazilian Public Hospitals After COVID-19 Pandemic Peak

Abstract Background Surveillance cultures are routinely used by public health authorities to screen for multidrug-resistant bacteria. Recently, the overuse of antibiotics intensified by the COVID-19 pandemic worldwide brought concerns about potential impacts on the acquisition and/or selective pressure of resistance genes, that in general carried by plasmids, which contribute to the rapid resistance spread within specific bacterial populations. The purpose of this study was to determine the prevalence of multidrug-resistant (MDR) organisms recovered from surveillance cultures (rectal, anal, nasal, and axillary) isolated from several Brazilian public hospitals. Methods Surveillance samples were analyzed from 1st January to 31st December 2023. All the swab samples were collected from patients admitted from high-risk settings or transferred from areas with high rates of MDR organisms. All clinical specimens were inoculated onto a selective media (ChromID media, bioMerieux) and incubated at 35±2°C for 18-24 h. The screening test was performed to Gram-negative bacilli (GNB) using the antibiotics ertapenem, imipenem and meropenem and for Gram-positive Cocci (GPC) screening was tested oxacillin (methicillin) and vancomycin using disc diffusion method. Bacteria identification was performed by MALDI-TOF mass spectrometry (Vitek-MS and bioMerieux) and as required, the minimal inhibitory concentrations (MIC) of antibiotics were determined using the Vitek 2 System (bioMerieux). Results A total of 71,064 rectal swab samples were processed. 4,652/71,064 (6.5%) samples showed positive results for the presence of 5.255 MDR microorganisms. Out of which, 4,477/5,255 (85.2%) were Gram-negative Bacilli (GNB) and 778/5,255 (14.8%) were Gram-positive Cocci (GPC). The majority of the isolated carbapenems resistance was Klebsiella spp. [2,791/5,255 (53.1%)], which the Klebsiella pneumoniae was the predominant species, followed by Acinetobacter baumannii [1,268/5,255 (24.1%)], Pseudomonas aeruginosa [263/5,255 (5%)], Enterobacter spp. [96/5255 (1.8%)], Serratia spp. [31/5,255 (0.6%)], Citrobacter spp. [22/5,255 (0.4%)], and others BGN [6/5,255 (0.2%)]. The resistance to vancomycin and methicillin were observed in 10% (525/5,255) of Enterococcus spp. and 4.8% (253/5,255) of Staphylococcus aureus, respectively. Conclusions The study found that K. pneumoniae carbapenem-resistant and Enterococcus spp. vancomycin-resistant were the main Gram-negative and Gram-positive organisms recovered from surveillance culture samples. However, our data draws attention to the increased cases of carbapenem-resistant Acinetobacter baumannii as the second most frequent pathogen isolated from surveillance samples after the last wave of COVID-19 pandemic. Active surveillance culture for target microorganisms followed by contact precautions to minimize the chance of transmitting MDR organisms from colonized to non-colonized patients consist of an important activity of infection control policy.

A-255 Microbiological Profile and Frequency Distribution of Uropathogens in Brazilian Public Hospitals. Data From a Clinical Laboratory (2018-2023)

Abstract Background Urinary tract infections (UTIs) are among the most common infectious diseases in humans and represent a critical health problem, especially due to a significant rise in global antibiotic use in recent years. The purpose of this study was to describe the main uropathogens and determine the antimicrobial resistance profiles among inpatients at public hospitals in the largest city in Brazil, comparing three different epidemiological periods: pre-Covid-19 (2018-2019), Covid-19 (2020-2021) and post-Covid-19 (2022-2023). Methods A 6-year retrospective review of laboratory data from 12 public hospitals of São Paulo city from January 2018 to December 2023. The urine samples were cultivated onto ChromID® CPS agar plates and then incubated at 35°C for 18-24 h. The bacteria identification was performed by MALDI-TOF mass spectrometry (Vitek-MS), and the minimal inhibitory concentrations of antibiotics were determined using the Vitek®2 system. Only positive results with one bacterial species and a colony count ≥ 105 CFU/mL was considered for descriptive analysis. Results In the 6-year period, a total of 58,738 microorganisms from hospitalized patients were included. The frequency of isolates detected during the period were: 33.9% (19,338/58,738) in pre-Covid-19, 30.1% (17,688/58,738) in Covid-19 and 36.9% (21,712/58,738) in post-Covid-19 period. The top ten microorganisms’ prevalence detected in pre-Covid-19, Covid-19 and post-Covid-19 periods were as follow: 23,951 Escherichia coli (47.5%; 37.1%; 37.7%), 9,247 Klebsiella pneumoniae (12.5%; 14.3%; 19.8%), 7,640 Candida spp. (8.8%; 19.2%; 11.7%), 5,476 Enterococcus spp. (8%; 10%; 9.9%), 3,101 Proteus mirabilis (5.56%; 4.9%; 5.4%), 2,166 Pseudomonas aeruginosa (3.3%; 3.5%; 4.1%), 1,252 Coagulase-Negative Staphylococcus (CoNS) (2.5%; 1.6%; 2.2%), 1,166 Enterobacter spp. (2.3%; 2.1%; 1.6%), 1,088 Streptococcus spp. (3.6%; 1.2%; 0.8%) and 758 Acinetobacter spp. (0.8%; 1.5%; 1.5%). Extended spectrum beta-lactamase rates for E. coli were 16.8% and 20.2% and for K. pneumoniae were 65.7% and 65.2% in Covid-19 and Post-Covid-19 periods, respectively. The resistance to carbapenems was observed mainly in Acinetobacter spp. (85.7%, 93.8% and 80.7%), K. pneumoniae (38.3%, 42.6% and 47.3%), Pseudomonas aeruginosa (22.3%, 23.1% and 25%), and Proteus spp. (0.2%, 1.9% and 5%). Between the Gram-positive cocci, the resistance to vancomycin was observed mainly in E. faecium (43.5%, 37.5%, 30.2%) and the resistance to methicillin was detected in S. aureus (30.1%, 35.8%, 34.1%) and CoNS (31.9%, 47.8%, 53.5%). Conclusions In this study, we observed an increase in the incidence of Candida spp. coinciding with the start of the pandemic period. Overall, the frequency of uropathogens isolated from hospital centers increased and exhibited high resistance to various classes of antimicrobial agents. ESBL-producing K. pneumoniae and Carbapenem-resistant Enterobacteriacea, and nonfermenters such as Acinetobacter spp. and P. aeruginosa constitute serious problems in Brazilian hospitals.

A-290 Prevalence and Antimicrobial Resistance Pattern of Microorganisms Recovered From Bloodstream Infections From Public Hospitals of São Paulo City, Brazil

Abstract Background Nosocomial Bloodstream Infections (nBSI) are an important cause of morbidity and mortality. Brazilian studies concerning bloodstream infections are important tools that can bring specific issues related to antimicrobial resistance. The objective of this study was to describe the frequency and antimicrobial susceptibility profile of bacterial isolates from BSI in patients admitted to public hospitals in 2022 and 2023. Methods This study was a retrospective analysis from 12 hospitals (January 2022 to December 2023) based on BSI isolates laboratory data detected by the clinical microbiology department of a medical diagnostic laboratory. The identification and susceptibility tests were performed using MALDI-TOF mass spectrometry (Vitek MS) and Vitek®2 systems, according to the manufacture’s recommendations. Coagulase Negative Staphylococci (CoNS) BSI was only considered in the presence of at least two sets of positive blood cultures with the same organism. Results During the period (January 2022 to December 2023) a total of 19,588 microorganisms isolated from BSI were obtained from 12 hospitals. The frequency of organism group was Gram-positive (10,707; 54.7%), Gram-negative (7,874; 40.2%), yeasts (961; 4.9%) and filamentous (46; 0.2%). The order of prevalence (top ten) organisms was CoNS (5,149; 26.3%); Staphylococcus aureus (2,769; 14.1%); Klebsiella spp. (2,355; 12.0%); Escherichia coli (1,301; 6.6%); Acinetobacter spp. (1,293; 6.6%); Streptococcus spp. (1,125; 5.7%); Enterococcus spp. (1,112; 5.7%); Candida spp. (937; 4.8%); Pseudomonas spp. (749; 3.8%) and Proteus spp. (553; 2.8%). Extended spectrum beta-lactamase rates for E. coli and Klebsiella pneumoniae were 31.6% (268/848) and 74.9% (1027/1371), respectively. The resistance to carbapenems was detected mainly in 74.1% (661/892) of Acinetobacter spp. isolates, 60.7% (832/1371) of Klebsiella pneumoniae; 24.5% (114/465) of P. aeruginosa; 9.7% (34/349) of Proteus spp. and 1.4% (12/828) of Escherichia coli. Antimicrobial resistance was observed mainly in 87.2% of CoNS (2831/3247) and 44.5% of S. aureus (743/1669) to methicillin; 22.9% (146/637) of Enterococccus spp. to vancomycin. Conclusions The frequency of Klebsiella spp. isolates from nBSI has progressively increased and effective infection control measures should be taken to prevent them from spreading. The rates of antimicrobials resistance reported for Klebsiella pneumoniae, Acinetobacter spp., CoNS, S. aureus and Enterococcus spp. reinforce the high resistance rates to antimicrobials in São Paulo city.

A-253 Antimicrobial Susceptibility Pattern in Escherichia coli Isolated From Community-Acquired Urinary Tract Infections in the era Post-Covid-19

Abstract Background Urinary tract infections (UTI) are one of the most common infectious diseases diagnosed in the community as well as in hospitals. Community-acquired urinary tract infections caused by Escherichia coli represent the largest proportion of infectious diseases affecting women and a significant amount of oral antibiotics are prescribed daily to treat UTI in community outpatient clinics. Having knowledge of regional susceptibility data regarding E. coli is very important for selecting an appropriate empirical antibiotic. This study aimed to report the frequency and antimicrobial susceptibility of E. coli isolated from community-acquired urinary tract infections post-Covid-19 pandemic. Methods This study was a retrospective analysis from January to December 2023 based on the analysis of 24,019 uropathogens isolated from 182 outpatient health centers located in several regions of São Paulo city, Brazil. The urine samples were cultivated onto ChromID™CPS® agar plates by semi-quantitative technique using 1µL disposable loop, incubated at 35-37°C and the colony counting were read after 24h and 48h of incubation. The bacteria identification was performed by MALDI-TOF mass spectrometry (Vitek-MS), and the minimal inhibitory concentrations of antibiotics were determined using the Vitek®2 system, according to the manufacture’s recommendations. Only positive results with one bacterial species and a colony count ≥105 CFU/mL was considered for descriptive analysis. Results Of the 24,019 urine samples tested, 71.44% (17,159/24,019) were identified as Escherichia coli. The major frequency of E. coli infection occurred among women (90.48%) and the main age range was over 60 years (38.57%), while 21-30 comprised 16.5% of cases. The antimicrobial resistance profile for E. coli isolates showed 39.9% (6852/17158) of resistance to ciprofloxacin (MIC, >0.5 mg/L), 34.7% (5,921/17,075) to trimethoprim/sulfamethoxazole (MIC, >0.5 mg/L), 37.5% (6,371/17,002) to Amoxicillin/clavulanate (MIC, >8 mg/L), 8.75% (1,501/17,592) to ceftriaxone (MIC, >1 mg/L), 4.88% (837/17,138) to cefepime (MIC, >4 mg/L), 3.64% (616/16,901) to piperacillin/tazobactam (MIC, >8 mg/L), 11.07% (1,900/17,156) of gentamicin (MIC, >2 mg/L), 0.43% (74/17,158) of amikacin (MIC, >8 mg/L) and 2.23% (377/16,909) of fosfomycin (MIC, >16 mg/L). High-level of susceptibility was detected to carbapenems with 99.94% (10/17159) to ertapenem (MIC, ≤0.5 mg/L) and 99.8% (4/17,159) to meropenem (MIC, ≤2 mg/L). The rate of Extended-spectrum beta-lactamase production was 8.1% (1391/17159). Conclusions This data could be used locally with other related samples, to properly interpret significant resistance patterns and choose the most appropriate antimicrobial for empirical treatment of patients with community-acquired urinary tract infections.

B-165 The EXENT System reliably distinguishes daratumumab from endogenous monoclonal immunoglobulins in multiple myeloma patients

Abstract Background The use of therapeutic monoclonal antibodies (t-mAbs) in the treatment of multiple myeloma (MM) has created challenges in the clinical laboratory. Co-migration of t-mAbs (predominantly IgGK) and the endogenous M-protein during serum protein electrophoresis can result in inability to distinguish between a patient’s M protein and the t-mAb. This can hinder accurate assessment of therapeutic responses or may lead to extensive workup with immunofixation electrophoresis and gel-shift assays for accurate confirmation of a complete response, or to exclude the emergence of a new clone. There is a need for an analytically specific routine method to identify t-mAb interference. MALDI-TOF mass spectrometry is an especially promising tool, as due to its ability to identify M proteins’ molecular mass, it has the potential to recognize any t-mAbs with known amino acid composition. We have used the EXENT® Immunoglobulin Isotypes (GAM) Assay for the EXENT Analyzer (The Binding Site, part of Thermo Fisher Scientific) to assess the reproducibility of daratumumab (anti-CD38, IgGK) detection in an analytical study, and assessed the EXENT System’s ability to detect daratumumab in a cohort of MM patients. Methods The expected mass to charge ratio (m/z) of the +2-charge state of daratumumab’s kappa light chain was calculated based on published information. Daratumumab was spiked into normal human serum at 1.0 g/L, and this material was tested in 5 replicates a day over 5 days. In addition, 109 samples from 34 MM patients receiving daratumumab, carfilzomib, lenalidomide and dexamethasone (Dara-KRd) treatment were also tested. Samples were obtained after 14 or 28 days following daratumumab administration in various treatment cycles, ranging from cycle 4 to end-of-treatment. Results The expected m/z for the +2-charge state of daratumumab’s kappa light chain was calculated as 11,691.01 to 11,693.1. In the reproducibility experiment, the median m/z was 11691.3, within the expected range. The difference between the lowest and highest m/z was 1.3 across the 25 replicates. Daratumumab was detected in 103 out of 109 samples (97.3%): 51/57 (89.5%) in IgGK samples, and in all IgGL (23) and IgA (29) samples. In two IgGK patients, where daratumumab was not detected in 2 out of 4 samples and in 1 out of 3 samples, respectively, the patients’ own M proteins were in the proximity (24.8 and 27.1 m/z) of daratumumab’s m/z. In the other three cases where daratumumab was not modeled by the algorithm, the daratumumab peak overlapped with the endogenous M-protein's adduct peak (estimated m/z 11,688) in one sample; it was masked by the shoulder of a large (8.42 g/L) IgGK M-protein in another sample; and no obvious reason was identified in the third sample. There was no difference in the detection rate between 14 days and 28 days post administration. Conclusions The EXENT Immunoglobulin Isotypes (GAM) Assay has successfully identified the presence of daratumumab in approximately 90% of IgGK patient samples in addition to all other (non-IgGK) MM samples. These results demonstrate the potential of the EXENT Immunoglobulin Isotypes (GAM) Assay for recognizing known t-mAb interference.

A-285 Adoption of 2023 CLSI Revised Aminoglycoside Clinical Breakpoints on Carbapenem Resistant Enterobacterales Isolates Identified at a Commercial Reference Laboratory in Korea, Republic of

Abstract Background The incidence of carbapenem resistant Enterobacterales (CRE) has been rapidly increasing as a nationally notifiable communicable disease since 2017 in Korea. The therapeutic options available for these pathogens are extremely limited. Several new drugs developed to treat CRE infections was recently introduced in Korea, but was not widely used. Instead, aminoglycoside is used as one of current available antimicrobials in CRE infections. The Clinical and Laboratory Standards Institute (CLSI) publishes revised aminoglycoside breakpoints in Enterobacterales in 2023. The purpose of this study was to evaluate the adoption of changing the aminoglycoside breakpoint in a commercial reference laboratory. Methods We retrospectively evaluated aminoglycosides (amikacin, gentamicin, tobramycin) for 1,393 CRE isolates not including surveillance culture identified by MALDI-TOF MS (Bruker Daltonik, Bremen, Germany) from January to December in 2023 at a commercial reference laboratory in Korea. Antimicrobial susceptibility testing was performed using a N415 card on the VITEK2 XL (bioMerieux, France). Susceptibilities of aminoglycoside were assessed based on previous breakpoints in CLSI M100-S32 in 2022 as well as the current breakpoints from the most recent CLSI MIC breakpoints in 2023. Results We evaluated 1,393 CRE isolates. Most isolates were Klebsiella pneumoniae [n=876 (62.9%)] and Escherichia coli [n=322 (23.1%)] followed by Citrobacter koseri [n=76 (5.5%)] The new aminoglycoside breakpoints reduced the percentage of strains that were previously judged to be susceptible by CLSI M100-S32 from 87.5% (1,219/1,393) to 71.2% (992/1,393) in amikacin, from 45.5 (634/1,393) to 44.8% (624/1,393) in gentamicin and from 23.3% (323/1,385) to 21.4% (297/1,385) in tobramycin, respectively. In meropenem resistant 56 isolates with MIC≤8 mg/L available of meropenem in combination with a second agent, frequently polymyxins or aminoglycosides, the new aminoglycoside breakpoints reduced the percentage of strains that were previously judged to be susceptible from 92.9% (52/56) to 64.3% (36/56) in amikacin and from 64.3% (36/56) to 60.7% (34/56) in gentamicin, respectively. There was no difference in tobramycin. Conclusions Our results show decreased susceptibility of aminoglycoside especially for amikacin in CRE isolates tested. Amikacin is commonly used to treat CRE infections, either alone or as part of combination therapy. Although our study has no correlation with clinical outcomes, we found microbiology laboratories should adopt the updated breakpoints.

B-089 Pseudo-clonal Increased IgG4 with Polyclonal Distribution Demonstrated by MALDI-TOF-MS in a Pediatric Patient with Tricho-Hepato-Enteric Syndrome (THES)

Abstract Background THES is a rare autosomal recessive syndromic enteropathy caused by pathogenic variants in two genes, the Tetratricopeptide Repeat Domain 37 (TTC37) and Ski2 Like RNA Helicase (SKIV2L), that encode components of the human SKI complex involved in RNA degradation. This disease is characterized by several clinical features with variable penetrance including intractable congenital diarrhea, wooly hair abnormalities, intrauterine growth restriction, facial dysmorphism, immune dysfunction, liver abnormalities, and skin hyperpigmentation. Hypogammaglobulinemia with low IgG and normal to low IgM levels have been described in these cases which may require intravenous immunoglobulin (IVIG) replacement therapy. Some cases have a reduced population of switched memory B cells, T cells with reduced IFN-γ secretion, and hyporesponsive NK cells. Our patient is a 4-year-old female reported to have a homozygous TTC37 mutation with clinical manifestations of TPN dependence, liver fibrosis with portal hypertension, splenomegaly, developmental delay, thrombocytopenia and anemia requiring transfusion, and a history of hypogammaglobinemia requiring IVIG replacement. Patient would go on to develop elevated immunoglobin levels leading to additional testing described below. Methods Serum total protein analysis performed using the using Siemens Atellica CH Total Protein II test (Weichselbaum method using biuret reagent). Serum immunoglobin (IgA, IgG and IgG), IgG subclass, and kappa/lambda free light chain level analysis performed by turbidimetric photometry assay using a Binding Site Optilite analyzer. Serum protein electrophoresis and immunotyping performed using CAPILLARYS 2 capillary zone electrophoresis and immunosubtraction with antisera. Serum protein immunofixation performed using Sebia Hydrasys 2 with Hydragel 9 IF Maxi kit. Confirmation and final determination of serum immunoproteins performed at our reference laboratory using MASS-FIX (MALDI-TOF MS) matrix-assisted laser desorption/ionization-time of flight mass spectrometry after patient serum applied to 5 separate immunoaffinity resins specific to immunoglobulin G, A, M, K, and L for purification. Results Initial testing showed a broadly migrating beta-gamma region IgG kappa monotypic restriction measuring 1.7 g/dL, with predominantly kappa subtraction and trivial lambda subtraction by immunosubtraction; serum free kappa/lambda light chain ratio was increased at 22.94. Serum immunoglobins and free light chains measured (mg/dL): IgA 738 (15 - 160), IgM 48 (50 - 240), IgG 2,805 (405 - 1,160), free kappa 20.42 (0.33 - 1.94), and free lambda 0.89 (0.57 - 2.63). IgG subclasses measured (mg/dL): IgG1 533 (320 - 950), IgG2 174 (50 - 340), IgG3 16.9 (10.0 - 120.0), IgG4 1,221.1 (2.0 - 110.0). Additional serum protein values included (g/dL): total protein 8.10 (6.00 - 8.30), albumin 2.97 (3.43 - 4.84), alpha-1 0.32 (0.21 - 0.44), alpha-2 0.52 (0.54 - 0.97), beta globulin 3.42 (0.65 - 1.03), and gamma globulin 0.87 (0.70 - 2.30). The polyclonal nature of the IgG4 spike was later demonstrated by the MASS-FIX mass spectrograph. Conclusions MASS-FIX is a useful addition to the toolset used in the diagnosis of possible monoclonal gammopathies and protein disorders, especially where broad monotypic restrictions are observed or in patients with abnormal presentations for monoclonal gammopathies. Using current standard techniques, the above case could have been incorrectly classified as a monoclonal process resulting in unnecessary cost and incorrect treatment.

B-169 Combining the EXENT System and LC-MS for the Detection of Monoclonal Immunoglobulins at Low Levels

Abstract Background The EXENT® system is an automated platform designed to quantify monoclonal immunoglobulins in serum. EXENT® combines immunoprecipitation and MALDI-TOF mass spectrometry for the detection of monoclonal immunoglobulins at levels lower than traditional gel based methods. This study was designed to demonstrate if EXENT® prepared samples that were negative could be reflexed to a more sensitive method based on capillary flow Liquid Chromatography- Mass Spectrometry (LC-MS) without any modifications to the EXENT® prepared sample. Methods Patient samples containing a monoclonal immunoglobulin (ranging from 20 to 40 g/L) were diluted into pooled polyclonal serum and the samples were prepared and analyzed by EXENT®. Samples that were negative by EXENT® were reflexed by directly loading EXENT® eluates onto the LC-MS instrument. The abundance of the monoclonal immunoglobulin was defined by the signal-to-noise (S/N) of the light chain peak observed after deconvolution. Results Baseline samples were diluted down to two levels, 0.100 g/L and 0.001 g/L. LC-MS detected the same light chain from the monoclonal immunoglobulin as the EXENT® system in all reflexed samples that were negative by EXENT®. Conclusions LC-MS can detect monoclonal immunoglobulins that can no longer be detected using the EXENT® system by tracking the unique molecular mass of the monoclonal light chain. Reflexing samples to LC-MS did not require additional sample handling resulting in a faster time-to-result than current NGS, NGF, and clonotypic peptide approaches. Furthermore, monitoring the intact monoclonal light chain circumvents enzymatic cleavage to create a unique clonotypic peptide, alleviating a situation where no unique peptide can be generated as has been shown previously by Bergen et al. As a consequence, this reflex-methodology results in a consistent way of measuring the presence of malignant B-cells with high sensitivity.

Identification methods as a factor affecting the performance of clinical microbiology laboratories participating in an external quality assessment program: a cross-sectional, retrospective analysis.

Introduction. Laboratory participation in external quality assessment (EQA) programmes including proficiency testing (PT) is a requirement of clinical laboratory conformance to ISO 15189:2022 Medical laboratories - Requirements for quality and competence. PT is one EQA method whereby laboratories are sent blinded samples for characterization by routine laboratory diagnostic methods. Importantly, PT enables a laboratory's performance to be evaluated in comparison to the standard reference methods and to the performance of other peer laboratories using similar diagnostic methods.Gap statement. The desired outcome of participating in PT is to help laboratories identify possible sources of error in each step of the total testing process and particularly in their test methods during the analytical phase.Aim. This cross-sectional study investigated the impact of using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) compared to conventional phenotypic biochemical testing on laboratory performance in a clinical bacteriology PT scheme.Methodology. During a 6-year period from 2017-2022, the Canadian Microbiology Proficiency Testing implemented 112 PT challenges comprising 22 different sample types and included 61 different bacterial species. This was translated into 5883 graded test events for analysis. Multiple logistic regression techniques were employed to explore the association between the test method employed and laboratory performance. The sample type and aerobic classification of challenge organisms were included as confounding variables.Results. Laboratories using MALDI-TOF MS performed significantly better in characterizing microorganisms than laboratories using phenotypic biochemical testing alone [odds ratio OR = 5.68, confidence interval (CI): 3.92, 8.22] regardless of the sample type and aerobic classification. Notably, our analysis identified a significant association between anaerobic organisms and laboratory performance (OR: 0.24, CI: 0.17-0.35), suggesting that culturing and identifying fastidious organisms remains a significant obstacle for many clinical microbiology laboratories.Conclusions. Although no method is infallible and its performance will depend on the validation and quality assurance procedures, this finding may help the management in the decision for implementing MALDI-TOF MS in the microbiology laboratory. This study highlights the important role PT providers play in the objective assessment of laboratory performance and how it can provide evidence for quality improvement.

Provolone del Monaco PDO cheese: Lactic microflora, biogenic amines and volatilome characterization

One commercial production run of Provolone del Monaco − a long-ripened pasta filata cheese − was followed up to the end of ripening for a total of 20 samples. 371 LAB isolates were subject to genetic characterization followed by 16S rRNA gene sequencing. The dominant species were Lacticaseibacillus casei/paracasei (19.4 %), Streptococcus macedonicus (19.1 %) and Enterococcus faecalis (13.2 %). Strains were screened for features of technological interest or safety relevance. Tyramine-producing cultures were quite common, above all within enterococci. By MALDI TOF Mass Spectrometry, one Lactococcus lactis and one Enterococcus faecium strain proved to be bacteriocin producers.Four further cheese wheels from the same production run at 623 days of ripening were evaluated for volatile organic compounds, biogenic amines, and bacterial community by metagenomic sequencing. Three individual wheel samples shared a rather similar microbiome with Lactobacillus delbrueckii and Streptococcus thermophilus as the most represented species, while the fourth wheel appeared wholly different being dominated by Lentilactobacillus buchneri and St. infantarius. Additionally, this sample had the greatest content of biogenic amines and a different VOCs composition.Given the variance seen among cheese wheels processed and ripened under the same conditions, the search for adjunct cultures in the production of this cheese seems to be of utmost importance.

Emergence and spread of resistant and biofilm-forming Acinetobacter baumannii in critically ill COVID-19 patients

The COVID-19 pandemic has raised concerns beyond the viral infection itself. Bacterial co-infections, particularly those involving Acinetobacter baumannii, have become a significant worry in critically ill COVID-19 patients. A. baumannii is an opportunistic pathogen that can cause nosocomial infections, especially in patients with compromised immune systems. This study investigated 36 A. baumannii isolates obtained from COVID-19 patients during a concurrent outbreak. The isolates were collected over two years through routine medical requests sent to the Clinical Microbiology laboratory. Identification of the strains was confirmed through biochemical tests, the Phoenix BD® Automated Microbiology System, and MALDI-TOF Mass Spectrometry. The study assessed the antimicrobial sensitivity of the isolates, with a specific focus on resistance to the beta-lactam group as well as aminoglycosides. The presence of specific antibiotic resistance genes (blaOXA-23, -24, -51 and -58, blaKPC, blaSHV, blaIMP, blaVIM, aac(6')-Ib, ant(3'')-Ia, and aph(3')-Ia) was investigated using PCR and Sanger DNA sequencing. Biofilm-forming capabilities of the isolates were also evaluated. The findings revealed diverse resistance profiles, with a high prevalence of resistant strains, including resistance to carbapenems. Genetic analysis suggested potential clonal spread of certain strains within the hospital setting. Moreover, a significant proportion of the isolates demonstrated strong biofilm-forming abilities, which can enhance persistence and antibiotic resistance. In conclusion, this study highlights the need for vigilant monitoring and targeted interventions to address bacterial co-infections in COVID-19 patients. The diversity in resistance patterns, potential clonal spread, and robust biofilm-forming abilities among A. baumannii isolates underscore the importance of addressing this issue to better manage and treat critically ill COVID-19 patients.

Methylation of Selected CpG-Sites of the Gene CSF1 as a Factor in Regulation of Its Expression and a Marker of Human Biological Aging

—Age-associated transformation of methylation patterns is considered to be an important predictor of human biological age. Changes in the level of CpG-dinucleotide methylation contribute to a shift in the function of a number of genes, including those associated with the functioning of the immune system. One such gene is CSF1. The protein product of this gene is associated with inflammatory aging, making it an important biomarker of age-related diseases. We studied the methylation profile of the promoter-associated CpG island of the CSF1 gene by MALDI-TOF mass spectrometry. Dependences between the character of CpG-site methylation within the investigated regions and the relative level of the gene mRNA and its protein product in people of different age groups were sought. For two CpG sites, a high level of correlation with the studied parameters is shown. A search for the landing sites of transcription factor binding sites associated with gene transcription showed that these CpG dinucleotides are part of motifs for the NFI family transcription factors and the EGR1 factor. We hypothesize that these CpG sites play an important role in the regulation of CSF1 gene expression.

Evaluation of a nucleic acid amplification system, GENECUBE, for rapid detection of staphylococcal nuc and mecA in blood culture samples

ObjectiveThis study determined whether the GENECUBE rapid nucleic acid amplification test could directly detect nuc and mecA genes in clinical blood culture samples of Staphylococcus and various other pathogens. MethodsBetween September 2020 and December 2021, 537 blood culture samples from 192 patients with suspected bacteremia were tested using conventional assays (MicroScan WalkAway96 or VITEK 2 systems) and GENECUBE nuc and mecA assays. Isolates from samples with discrepant results between the conventional and GENECUBE assays were further evaluated using MALDI-TOF mass spectrometry, disk diffusion testing using cefoxitin, broth microdilution testing using oxacillin, and sequencing for mecA. Bacterial solutions containing a mixture of methicillin-susceptible Staphylococcus aureus (MSSA) and methicillin-resistant Staphylococcus epidermidis (MRSE) were prepared to evaluate the limit of detection (LOD) of mecA. ResultsUsing conventional assays as the reference, the sensitivity, specificity, and positive and negative predictive values (95 % confidence interval) of GENECUBE were 100 % (96.8–100 %), 100 % (99.1–100 %), 100 % (96.8–100 %), and 100 % (99.1–100 %), respectively, for nuc detection and 100 % (96.1–100 %), 98.9 % (97.4–99.6 %), 94.9 % (88.5–98.3 %), and 100 % (99.2–100 %), respectively, for mecA detection. Sequencing analysis of five samples identified as methicillin-sensitive staphylococci using conventional assays and methicillin-resistant staphylococci using GENECUBE revealed the presence of methicillin-resistant isolates in all samples. The estimated LOD of mecA was 104 colony-forming units (CFU)/mL of MRSE with GENECUBE, compared with 105 CFU/mL with conventional assays. ConclusionThe GENECUBE assay accurately detected mecA in positive blood culture samples and had higher sensitivity than conventional assays.

Epidemiology of antimicrobial resistance in commensal E. coli from healthy dairy cattle on a Mediterranean pasture-based system of Australia: A cross-sectional study

This study aimed to determine the prevalence of antimicrobial resistance (AMR) in commensal E. coli from healthy lactating cows and calves in the Mediterranean pasture-based feeding dairy system of Western Australia (WA). Fecal samples were collected from healthy adult lactating cows and healthy calves from dairy farms in WA. Presumptive commensal E. coli was isolated from these samples and confirmed using matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Broth microdilution was used to assess the prevalence and the phenotypic AMR profiles of the E. coli isolates to 8 antimicrobial agents of dairy industry and human importance. The minimum inhibitory concentration (MIC) for each isolate was interpreted using the Epidemiologic Cutoff (ECOFF) and Clinical and Laboratory Standards Institute (CLSI) breakpoints. Genomic characterization provided multi-locus sequence types and AMR genes for a selection of isolates categorised as non-wild type (NWT) by ECOFF values for the combination of ampicillin, trimethoprim-sulfamethoxazole, and tetracycline. From a total of 1,117 fecal samples (633 adult, 484 calf) collected across 26 randomly selected farms, 891 commensal E. coli isolates were recovered (541 adult, 350 calf). Commensal E. coli classified as NWT was highest for ampicillin for both adult (68.8%; 95% CI = 64.7 - 72.7) and calf feces (67.1%; 95% CI = 62.0 - 72.0). A large proportion of tetracycline NWT and trimethoprim-sulfamethoxazole NWT organisms were also identified from calf feces, being 44.0% (95% CI = 38.7 - 49.4) and 24.6% (95% CI = 20.2 - 29.4) respectively. Clinical resistance prevalence was low, being higher for calves than for adult feces (ampicillin (adult: 7.8% (95% CI = 5.7 - 10.3); calf: 30.0% (95% CI = 25.2 - 35.1), tetracycline (adult: 6.3% (95% CI = 4.4 - 8.7); calf: 40.3% (95% CI = 35.1 - 45.6), and trimethoprim-sulfamethoxazole (adult: 2.6% (95% CI = 1.4 - 4.3); calf: 22.0% (95% CI = 17.7 - 26.7)). Commensal E. coli originating from calf feces was significantly higher in NWT prevalence compared with adult feces for ciprofloxacin (P = 0.023), gentamicin (P = 0.02), tetracycline (P < 0.001), and trimethoprim-sulfamethoxazole (P < 0.001). The overall number of antimicrobials an isolate was classified as NWT toward varied among farms and was significantly higher for isolates originating from calf than adult feces (P < 0.001). The strain type and sampling source of the commensal E. coli investigated were both associated with the commonality of the resultant resistance genome. Clinical resistance and NWT classification were highest for ampicillin, tetracycline, and trimethoprim-sulfamethoxazole, all antimicrobials commonly used in the treatment of dairy cattle in Australia. Though highly variable across farms, commensal E. coli isolated from healthy dairy calf feces had significantly higher NWT and multidrug resistance (MDR) prevalence compared with feces from healthy adult lactating dairy cows. The resistant genome identified in MDR isolates, though not always consistent with the phenotype, included QnrS1 and genes encoding AmpC β-lactamase and aminoglycoside phosphotransferase.

Antifungal susceptibility and biofilm production of Candida species- causative agents of female genital tract infections.

Recurrent vulvovaginal candidosis (RVVC) is a chronic infection affecting 8-10% of women worldwide. Biofilm production of the infecting species and reduced sensitivity to antimycotics could contribute to the recurrence of this infection. This study aimed to examine the biofilm production ability and antifungal susceptibility of genital yeast isolates to determine their virulence potential. Matrix-assisted laser desorption in ionization-time of flight mass spectrometry (MALDI-TOF MS) was used to identify 300 Candida species. Using crystal violet method, strains were categorized into non-producers, weak, moderate, and strong biofilm producers (BFP). Antifungal susceptibility testing was performed using commercial Integral System YEASTS Plus test (ISYPT) and broth microdilution method (BMM). MALDI-TOF MS identified 150 Candida albicans, 124 non-albicans Candida (NAC), and 26 Saccharomyces cerevisiae strains. Within 138 (46.0%) BFP, 23 (16.7%) were strong, 44 (31.9%) moderate, and 71 (51.4%) weak. BMM was done for 43 BFP selected isolates with nystatin MIC ˃1.25 μl, fluconazole MIC ˃64 μl, and clotrimazole MIC ˃1.0 μl determined by ISYPT. Compared to all examined isolates, BMM confirmed that: i) C. albicans and NAC BFP showed low sensitivity to fluconazole (12% and 4%, respectively); ii) all BFP showed low sensitivity to nystatin (12.7% C. albicans, 14.5% NAC, and 23.1% S. cerevisiae); iii) clotrimazole in vitro was the most efficient regarding C. albicans and S. cerevisiae strains, but in 4.0% NAC BFP for this antimycotic higher MIC was established. Novel antimycotics or possible combinations of antifungal agents and natural products could be a new treatment option for RVVC.

Discriminating fingerprints of chronic neuropathic pain following spinal cord injury using artificial neural networks and mass spectrometry analysis of female mice serum

Spinal cord injury (SCI) often leads to central neuropathic pain, a condition associated with significant morbidity and is challenging in terms of the clinical management. Despite extensive efforts, identifying effective biomarkers for neuropathic pain remains elusive. Here we propose a novel approach combining matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) with artificial neural networks (ANNs) to discriminate between mass spectral profiles associated with chronic neuropathic pain induced by SCI in female mice. Functional evaluations revealed persistent chronic neuropathic pain following mild SCI as well as minor locomotor disruptions, confirming the value of collecting serum samples. Mass spectra analysis revealed distinct profiles between chronic SCI and sham controls. On applying ANNs, 100% success was achieved in distinguishing between the two groups through the intensities of m/z peaks. Additionally, the ANNs also successfully discriminated between chronic and acute SCI phases. When reflexive pain response data was integrated with mass spectra, there was no improvement in the classification. These findings offer insights into neuropathic pain pathophysiology and underscore the potential of MALDI-TOF MS coupled with ANNs as a diagnostic tool for chronic neuropathic pain, potentially guiding attempts to discover biomarkers and develop treatments.

Bacterial profile of wound site infections and evaluation of risk factors for sepsis among road traffic accident patients from Apex Trauma Centre, Northern India.

Background. Among the most significant yet often ignored health issues worldwide are trauma and accidental injuries. India accounts for 11% of global deaths in road accidents, the highest in the world, according to the World Bank report. There are limited data about the bacterial contamination of road traffic accident (RTA) wounds and their antibiotic susceptibility patterns. Materials and Methods. This prospective study was conducted in a tertiary care centre in northern India from January 2023 to January 2024. Wound deep swabs or aspirates were collected from RTA patients with traumatic injuries at different time intervals. Gram stain and culture were performed, and positive aerobic culture was subjected to antibiotic susceptibility testing. Organism identification was done using MALDI-TOF MS and routine biochemical tests. Blood samples were also collected to rule out bloodstream infections during follow-up if the patient became febrile or showed symptoms of systemic infection. Sepsis was defined in those patients who had two or more scores in the systemic inflammatory response syndrome criteria with a positive microbiological culture. Risk factors were evaluated for sepsis on the basis of the patient's vitals, injury characteristics, procalcitonin, Glasgow Coma Scale (GCS) score, need for mechanical ventilation and complete blood count, which were obtained from the patient's admission file. Results. A total of 189 wound samples were collected, of which 99 (52.38%) samples showed the growth of microorganisms. The aerobic isolates included 69 (69.69%) Gram-negative bacilli, of which the majority were Klebsiella pneumoniae, 28 (28.28%) Gram-positive cocci, of which the majority were Staphylococcus aureus and 2 (2.02%) anaerobic isolates. Among the Gram-negative isolates, none of the isolates were resistant to colistin. All S. aureus isolates were susceptible to vancomycin, teicoplanin and levonadifloxacin. Sepsis developed in 50 (26.45 %) patients. Significant risk factors evaluated for sepsis were a raised procalcitonin level, a low GCS score, a higher injury severity score, the need for mechanical ventilation and a raised quick sequential organ failure assessment score. Conclusion. It is essential to ascertain the profile of microorganisms isolated from RTA wounds in order to reduce antibiotic resistance and deliver efficient treatment.

Purification, characterization, and antimicrobial mechanism of a novel broad-spectrum bacteriocin produced by Lactiplantibacillus plantarum SCA12 from Traditional Chinese Fermented Mustard greens

In this study, Lactiplantibacillus plantarum SCA12, a producer of bacteriocins, derived from traditional Chinese fermented mustard greens was isolated. A novel L. plantarum SCA12-derived bacteriocin, LPsca12, was purified through a multi-step protocol involving macroporous adsorption resin, cation exchange, Sephadex G-25 column chromatography, and reverse-phase high-performance liquid chromatography (RP-HPLC). The molecular mass of LPsca12 was determined to be 1197.0 Da through matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis. The amino acid sequence of LPsca12 was characterized as GLFPSLVRGPR via liquid chromatography-tandem mass spectrometry (LC-MS/MS). Antimicrobial assessment was then carried out, identifying LPsca12 as a broad-spectrum bacteriocin active against both Gram-negative and Gram-positive bacteria, particularly Salmonella paratyphi A. It demonstrates excellent thermal stability (121 °C, 15 min) and maintains its antibacterial activity under acidic conditions (pH 2.0–6.5). Additionally, LPsca12 is sensitive to digestion by pepsin and trypsin. The antimicrobial action of LPsca12 involves compromising the cell membrane integrity, forming pores, and promoting leakage of intracellular contents, which dissipates both the membrane potential (ΔΨ) and cytoplasmic transmembrane pH gradient (ΔpH) of the target cells. These findings suggest that LPsca12 holds promise as a natural biopreservative in food processing.