Matrix-assisted Laser Desorption Ionization-time Research Articles

Pseudomonas otitidis bacteremia in an immunocompromised patient with cellulitis: case report and literature review

BackgroundPseudomonas otitidis belongs to the genus Pseudomonas and causes various infections, including ear, skin, and soft tissue infections. P. otitidis has a unique susceptibility profile, being susceptible to penicillins and cephalosporins but resistant to carbapenems, due to the production of the metallo-β-lactamase called POM-1. This revealed genetic similarities with Pseudomonas aeruginosa, which can sometimes lead to misidentification.Case presentationWe report the case of a 70-year-old Japanese male who developed cellulitis and bacteremia during chemotherapy for multiple myeloma. He was initially treated with meropenem, but blood culture later revealed gram-negative bacilli identified as P. otitidis using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Carbapenem resistance was predicted from previous reports; therefore, we switched to dual therapy with levofloxacin and cefepime, and favorable treatment results were obtained.ConclusionThis is the first reported case of P. otitidis cellulitis and bacteremia in an immunocompromised patient. Carbapenems are typically used in immunocompromised patients and P. otitidis is often resistant to it. However, its biochemical properties are similar to those of Pseudomonas aeruginosa; therefore, its accurate identification is critical. In the present study, we rapidly identified P. otitidis using MALDI-TOF MS and switched from carbapenems to an appropriate antimicrobial therapy, resulting in a successful outcome.

Seed protein biotyping in Amaranthus species: a tool for rapid identification of weedy amaranths of concern

BackgroundThe Amaranthus genus contains at least 20 weedy and invasive species, including Amaranthus palmeri (palmer’s amaranth) and Amaranthus tuberculatus (tall waterhemp), two species of regulatory concern in North America, impacting production and yield in crops like corn, soybean and cotton. Amaranthus tuberculatus is regulated in Canada with limited establishment, while current climate models predict a range expansion of A. palmeri impacting crop growing areas in Ontario, Quebec and Manitoba. Since many Amaranthus species are similar in their morphology, especially at the seed stage, this demands the development of additional methods that can efficiently aid in the detection and identification of these species. Protein biotyping using Matrix-Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS) has been traditionally used to identify microorganism species, races and pathotypes. Major protein fractions extracted from an organism, ionized and run through a biotyper using mass spectrometry, result in protein spectra that represent a fingerprint at the species or lower taxonomic rank, providing an efficient molecular diagnostics method. Here we use a modified protein biotyping protocol to extract major protein fractions from seeds of the family Brassicaceae to test our protocol, and then implemented the standardized approach in seeds from Amaranthus species. We then created a database of Amaranthus protein spectra that can be used to test blind samples for a quick identification of species of concern.ResultsWe generated a protein spectra database with 16 Amaranthus species and several accessions per species, spanning target species of regulatory concern and species which are phylogenetically related or easily confused at the seed stage due to phenotypic plasticity. Testing of two Amaranthus blind sample seed sets against this database showed accuracies of 100% and 87%, respectively.ConclusionsOur method is highly efficient in identifying Amaranthus species of regulatory concern. The mismatches between our protein biotyping approach and phenotypic identification of seeds are due to absence of the species in the database or close phylogenetic relationship between the species. While A. palmeri cannot be distinguished from A. watsonii, there is evidence these two species have the same native range and are closely related.

Nocardia pseudobrasiliensis infection in a patient with arthritis

Nocardia pseudobrasiliensis is a new taxon constituting an emerging species of human pathogenic Nocardia, which shares morphological features with N. brasiliensis. However, N. pseudobrasiliensis is more invasive and more easily disseminated, and it exhibits distinctive antibiotic susceptibility. Few clinical cases related to N. pseudobrasiliensis infection have been reported, and N. pseudobrasiliensis hydrarthrosis has not been described. Here, we analyzed the case information, diagnostic process, treatment, and prognosis of a patient with N. pseudobrasiliensis hydrarthrosis who received treatment in Zhejiang Provincial People's Hospital. Magnetic resonance imaging showed joint cavity effusion and soft tissue swelling with high signal on proton density-fat saturated images and low signal on T1-weighted images. Oil microscopy revealed abundant acid-fast–positive filaments in hydrarthrosis puncture fluid. The pathogen was identified as N. pseudobrasiliensis by matrix-assisted laser desorption ionization–time of flight mass spectrometry. In contrast to the 100% ciprofloxacin resistance displayed by N. brasiliensis, this clinical isolate of N. pseudobrasiliensis was completely susceptible. In summary, this is the first report of N. pseudobrasiliensis in joint effusion from a patient with arthritis.

Performance assessment of the Bruker Biotyper MALDI-TOF MS for the identification of difficult-to-identify viridans group streptococci.

Differentiating Streptococcus pneumoniae among nonpneumococcal viridans group streptococci (VGS) is challenging in conventional laboratories. Therefore, we aimed to evaluate the performance of the latest Bruker Biotyper matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) system in identifying VGS by comparing the results to those of the specific gene sequencing approach. Clinical isolates were initially identified using the BD Phoenix system to identify Streptococcus species. The optochin test was used to distinguish nonpneumococcal VGS from S. pneumoniae. The species of individual reference strains and clinical isolates were determined by comparing the sequences of the 16S rDNA, gyrB, sodA, groESL, or coaE genes with those in the GenBank sequence databases. We evaluated the performance of the Bruker Biotyper MALDI-TOF MS in VGS identification using two different machines with three databases. We collected a total of 103 nonpneumococcal VGS and 29 S. pneumoniae blood isolates at a medical center in northern Taiwan. Among these isolates, only seven could not be identified at the species level by the specific gene sequencing approach. We found that none of the nonpneumococcal VGS isolates were misidentified as pneumococci by the latest Biotyper system, and vice versa. However, certain strains, especially those in the mitis and bovis groups, could still not be correctly identified. The latest Bruker Biotyper 4.1 (DB_10833) showed significant improvement in identifying VGS strains. However, a specific gene sequencing test is still needed to precisely differentiate the species of strains in the mitis and bovis groups.

Prevalence of carbapenemases among Gram-negative bacteria in Tunisia: first report of KPC-2 producing Acinetobacter baumannii.

The rapid evolution of the antibacterial resistance problem worldwide, including the Mediterranean countries, constitutes a real threat to public health. This study aims to characterize carbapenemase encoding genes among Gram-negative bacteria collected from some Tunisian hospitals. Twenty-two clinical carbapenem-resistant Gram-negative bacteria were recovered, and identified by the matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) method. Antibiotic resistance was tested by disk diffusion method on Muller-Hinton Agar. The minimum inhibitory concentration (MIC) for imipenem was revealed by the E-test method. Carbapenemase encoding genes were screened by polymerase chain reaction (PCR). Genetic relatedness was performed by the pulsed field gel electrophoresis (PFGE) method. Our isolates, identified as K. pneumoniae (n = 7), P. mirabilis (n = 1), A. baumannii (n = 13), and P. aeruginosa (n = 1), presented high MIC values for imipenem. Enterobacerales were resistant to carbapenems due to OXA-48 production. Only, four K. pneumoniae harbored the blaNDM-1 gene. VIM-2 production was detected in P. aeruginosa. However, OXA-23 production was observed in A. baumannii isolates, one of which co-produced the KPC-2 enzyme that was identified for the first time in Tunisia in this species. A high genetic diversity was demonstrated by pulsed-field gel electrophoresis in K. pneumoniae and A. baumannii after XbaI and ApaI digestion respectively. Our findings highlight the spread of various unrelated clones of carbapenemase-producers in some Tunisian hospitals as well as the spread of several carbapenemase types.

199. Fast Detection of Ceftazidime-avibactam-resistant Enterobacterales with VITEK-MSTM Incorporating a Direct-on-target Microdroplet Growth Assay

Abstract Background The direct-on-target microdroplet growth assay (DOT-MGA) was evaluated for its ability to rapidly detect ceftazidime-avibactam resistant Enterobacterales. Methods In this study, 47 non-duplicate CRE isolates were used to determine unequivocally the performance of ceftazidime-avibatam resistance detection of 7 organisms found in clinical isolates. The isolates were incubated for 3 to 6 h in the presence and absence of 8 µg/mL ceftazidime-avibatam in the broth applied as microdroplets directly on to the matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) target spots. Following the incubation period, broth was separated from the microbes by pipette. MALDI-TOF MS was employed to evaluate the growth of isolates in each spot and to identify the microbes simultaneously. The micro-broth dilution method, recommended by EUCAST was used as a reference method. A PCR-based assay was carried out to evaluate the carbapenem resistance mechanisms by screening for the presence of various β-lactamase-encoding genes. Results It was found that a 3 h incubation time was insufficient for MS to identify isolates. At this time-point, the identification of valid results of the growth control was only 25.0%. Therefore, the study of 3 h incubation times was not continued. For ceftazidime-avibactam-resistant Enterobacter strains, 83.7% of growth controls were successfully detected after the 4 h-time-point of incubation when the sensitivity and specificity of drug resistance detection were both 100%. Following incubation for 5 h, the rate of growth controls, sensitivity and specificity were 98.0%, 100% and 100%. When the incubation was extended to 6 h, the rate of growth controls, sensitivity and specificity reached an ideal 100%. Conclusion The VITEK MS-based DOT-MGA is a fast, convenient and accurate in detecting Enterobacterales that is resistant to ceftazidime-avibatam within 5 to 6 h. The assay can also determine the resistance status of an isolate and the potential novel resistant mechanisms involved. Disclosures All Authors: No reported disclosures

A Retrospective Observational Study on the Comparison of Different Diagnostic Modalities of Post-COVID Mucormycosis.

Background Mucormycosis, attributed to a group of molds known as mucormycosis, is a rare yet life-threatening fungal infection often colloquially referred to as black fungus. While its incidence notably surged during the second wave of COVID-19 infections in India, it's essential to recognize that mucormycosis was a significant concern even before the advent of the pandemic. Understanding the prevalence and characteristics of this infection in the pre-COVID era provides a crucial context for evaluating its impact and dynamics during the pandemic. Multiple diagnostic methods, such as potassium hydroxide (KOH) mount, culture, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), and histopathological examination (HPE), are available for identifying this lethal infection. The primary objective of this study is to ascertain the sensitivity of various diagnostic methods for mucormycosis and to analyze the comparative effectiveness of microbiological versus histopathological diagnoses. Methods We conducted a retrospective observational study spanning six months, from May 2021 to October 2021, encompassing all mucormycosis cases meeting the inclusion criteria and diagnosed via histopathological examination (HPE) in the departments of pathology and microbiology. Microbiological tests were performed prior to the histopathological examinations. Sensitivity was assessed through statistical analysis, and the relationship between microbiological and histopathological diagnoses was evaluated using the chi-square test. Results Biopsy samples of 77 patients were collected, comprising 56 male and 21 female patients. Regarding age distribution, most patients fell within the 41-60 age bracket, while the smallest proportion was over 60 years old. The sensitivity and specificity of histopathological diagnosis, confirmed with periodic acid-Schiff (PAS) and Grocott-Gomori's methenamine silver (GMS) staining, both recorded a flawless 100%. KOH mount sensitivity stood at 88.3%, while fungal culture and MALDI-TOF exhibited sensitivities of 75.3%. Histopathological analysis revealed that 17% of cases displayed minimal fungal hyphae alongside necrotic tissue, whereas 58% exhibited abundant fungal hyphae accompanied by inflammatory cells. Additionally, absolute neutrophilia was observed in 55% of cases. Conclusions In our study, histopathology and KOH mount emerged as not only compassionate but also cost-effective diagnostic tools for identifying mucormycosis. The economic aspect of these diagnostic methods is highlighted in the results section to provide a comprehensive understanding of their cost-effectiveness. Additionally, we utilized MALDI-TOF MS as a straightforward, economically viable, and expeditious method specifically for confirming the fungal subtype in mucormycosis cases. The rationale behind choosing either MALDI-TOF MS or KOH for the diagnosis is elucidated, contributing to a clearer interpretation of our diagnostic approach. Furthermore, our findings indicate that absolute neutrophilia consistently manifests in 55% of mucormycosis cases.

Bioremediation potential of cadmium-resistant bacteria isolated from water samples of rivulet Holy Kali Bein, Punjab, India

The present study was conducted with the goal of isolation, characterization, optimization and exploitation of the cadmium (Cd) bioremediation potential of Cd-resistant bacterial strains from the Holy Kali Bein, Punjab, India. From biochemical analysis, protein-based identification by using Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectroscopy (MALDI-TOF) and 16S rRNA sequencing the isolate KCd7 identified as Aeromonas caviae, KCd8 as Exiguobacterium indicum and isolate KCd9 as Acinetobacter haemolyticus. The isolates KCd7, KCd8 and KCd9 exhibited high degree of Cd resistance, up to 400, 400 and 500 mg/L respectively, with multiple antibiotic resistances (MAR) index above 0.0, 0.08 and 0.016 respectively. The optimal conditions were recorded as: initial metal concentration 150 mg/L, temperature 37 °C for all three isolates, pH 8 for KCd7 and pH7.5 for KCd8 and KCd9. Results showed that the isolates KCd7, KCd8 and KCd9 can remove 100% within 168 h and 70% within 168 h and 100% within 144 h of Cd respectively. The study’s results demonstrated that the bacterial isolate KCd9 (Acinetobacter haemolyticus) is more effective than other bacterial isolates since it can completely eliminate cadmium from the aqueous medium in just 144 h and it came to the conclusion that these bacteria have the potential for bioremediation of cadmium.

Get to Know Your Neighbors: Characterization of Close Bacillus anthracis Isolates and Toxin Profile Diversity in the Bacillus cereus Group.

Unexpected atypical isolates of Bacillus cereus s.l. occasionally challenge conventional microbiology and even the most advanced techniques for anthrax detection. For anticipating and gaining trust, 65 isolates of Bacillus cereus s.l. of diverse origin were sequenced and characterized. The BTyper3 tool was used for assignation to genomospecies B. mosaicus (34), B. cereus s.s (29) and B. toyonensis (2), as well as virulence factors and toxin profiling. None of them carried any capsule or anthrax-toxin genes. All harbored the non-hemolytic toxin nheABC and sphygomyelinase spH genes, whereas 41 (63%), 30 (46%), 11 (17%) and 6 (9%) isolates harbored cytK-2, hblABCD, cesABCD and at least one insecticidal toxin gene, respectively. Matrix-assisted laser desorption ionization-time of flight mass spectrometry confirmed the production of cereulide (ces genes). Phylogeny inferred from single-nucleotide polymorphisms positioned isolates relative to the B. anthracis lineage. One isolate (BC38B) was of particular interest as it appeared to be the closest B. anthracis neighbor described so far. It harbored a large plasmid similar to other previously described B. cereus s.l. megaplasmids and at a lower extent to pXO1. Whereas bacterial collection is enriched, these high-quality public genetic data offer additional knowledge for better risk assessment using future NGS-based technologies of detection.

High prevalence of Panton-Valentine Leucocidin among Staphylococcus coagulans isolated from dogs in Rio de Janeiro.

The purpose of this study was to characterize the capacity for biofilm formation, antimicrobial resistance rates, and search for genetic determinants of resistance and virulence in the species. Strains were collected from asymptomatic and infected dogs. Identification was conducted using matrix-assisted laser desorption ionization-time of flight (MALDI-TOF), antimicrobial susceptibility using disk diffusion and PCR targeting mecA. Biofilm formation was evaluated on a microtiter plate assay. A total of 27 strains were selected for whole-genome sequencing. We identified 111 Staphylococcus coagulans. The highest number was obtained from infected dogs. The highest resistance rates were observed for penicillin, gentamicin, and ciprofloxacin/erythromycin. Twelve strains were characterized as resistant to methicillin. All isolates had the ability to form biofilm and were strong producers. Among Methicillin Resistant Staphylococcus coagulans (MRSC), SCCmec types IIIA, and Vc were identified. Acquired resistance genes, such as aac(6')-aph(2''), tet(K), blaZ, qacG, qacJ, and erm(C) were found. Different virulence genes were identified. Of note, Panton-Valentine Leucocidin was highly prevalent among the isolates. Staphylococcus coagulans had a high isolation rate among infected dogs and demonstrated significant resistance to commonly used antibiotics such as penicillin and gentamicin.

Occurrence and Antimicrobial Resistance among Staphylococci Isolated from the Skin Microbiota of Healthy Goats and Sheep.

Staphylococci colonize the skin and mucous membranes of different animals. The purpose of this study was to determine the staphylococcal composition of the skin microbiota of healthy, non-vet visiting, and antimicrobially non-treated sheep and goats. In total, 83 strains (44 from goats and 39 from sheep) were isolated and identified using matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS). The diversity of the isolated Staphylococcus species was relatively high, and only coagulase-negative staphylococci (CoNS) were isolated. In sheep, S. vitulinus (9/39, 23.1%) was the most common species, followed by S. equorum (8/39, 20.5%), S. lentus (7/39, 17.9%), S. sciuri (6/39, 15.4%), S. xylosus (6/39, 15.4%), S. warneri (1/39, 2.6%), S. simulans (1/39, 2.6%), and S. nepalensis (1/39, 2.6%). In the goats, the most common species was S. sciuri, which was detected in 13 (29.5%) animals. The goat skin was also inhabited by S. equorum (7/44, 15.9%), S. vitulinus (6/44, 13.6%), S. cohnii (5/44, 11.4%), S. lentus (4/44, 9.1%), S. suscinus (3/44, 6.8%), S. caprae, (2/44, 4.5%), S. auricularis (2/44, 4.5%), S. warneri (1/44, 2.3%), and S. xylosus (1/44, 2.3%). Only one S. xylosus strain of goat origin carried the enterotoxin gene (sea). Antimicrobial resistance was not common among the isolated staphylococci. Only 31 (37.3%) strains were resistant to at least one antimicrobial agent, with the highest frequency of resistance to penicillin (16.8%), followed by clindamycin (9.6%), erythromycin (8.4%), moxifloxacin (8.4%), and tetracycline (7.2%). All isolates were susceptible to eight antibiotics (amikacin, gentamycin, ciprofloxacin, levofloxacin, rifampicin, chloramphenicol, trimethoprim-sulfamethoxazole, and tigecycline), representing six different classes. Three isolates displayed a multi-resistance phenotype (MDR): the goat isolates S. cohnii and S. sciuri, as well as the ewe isolate S. xylosus. The MDR S. cohnii isolate was found to be methicillin-resistant and carried the mecA gene. Moreover, the staphylococci isolated from the healthy animals carried genes conferring resistance to β-lactams (mecA, blaZ), tetracyclines (tetL, tetK), macrolides (ermB, ermC), lincosamides (lnu), and fluoroquinolones (grlA). However, the prevalence of these genes was low.

Bioactive compound and chemical characterization of lactic acid bacteria from fermented food as bio-preservative agents to control food-borne pathogens

Context: Lactic acid bacteria (LAB), which are found in many fermented foods, are known to produce antimicrobial compounds that play a vital role in food bio-preservation. Aims: To screen, identify, characterize, and determine the secondary metabolites of LAB isolated from Thai fermented foods that are beneficial against Escherichia coli and Staphylococcus aureus. Methods: The antimicrobial activity of cell free supernatant (CFS) was evaluated by agar well diffusion assay, and determining the minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC). Bacterial strains were identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), and chemical compound was conducted by gas chromatography and mass spectrometry (GC-MS). Additionally, microbial dynamic and bile salt tolerance were assessed. Results: Seven of the 90 lactic acid isolates from Thai fermented foods, showed antibacterial activity against E. coli and S. aureus in the 12.00–16.00 mm inhibitory zone. It was identified that the bacteria were Lactobacillus pentosus, Lactobacillus farciminis, Lactobacillus brevis, and Lactobacillus plantarum. The best antibacterial activity was represented by LBST1861 strain, which also provided bile salt resistance at 0.3% for 24 hours and had MIC and MBC values of 12.5 mg/mL and 50.0 mg/mL against S. aureus and E. coli, respectively. Furthermore, the GC-MS discovered a total of 16 chemical compounds that may be used to limit microbial growth and has a potential to be employed as a bio-preservative. Conclusions: The most potent strain of LBST1861 strain against S. aureus and E. coli as L. plantarum, isolated from fermented foods in Thailand, generated significant bioactive chemicals that can be applied to promote food products.

Study of mercury resistance and Fourier transform infrared spectroscopy-based metabolic profiling of a potent Bacillus tropicus strain from forest soil.

Mercury (Hg) is a highly toxic heavy metal and Hg-resistant indigenous bacterial isolates may offer a green and cost-effective bioremediation strategy to counter Hg contamination. In this study, a potent Hg-resistant bacterium was isolated from the forest soil of a bird sanctuary. Identification using matrix-assisted laser desorption ionization-time of flight mass spectrometry depicted the isolate as a strain of Bacillus tropicus, validated by morphological, biochemical, and molecular studies. The isolate demonstrated biological Hg removal efficiency and capacity of 50.67% and 19.76 mg g-1 , respectively. The plasmid borne resistance determinant, merA, encoding mercuric reductase, was detected in the bacterium endowing it with effective Hg volatilization and resistance capability. A Fourier-transform infrared spectroscopic comparative metabolic profiling revealed the involvement of various functional groups like -COOH, -CH2 , -OH, PO4 - and so on, resulting in differential spectral patterns of the bacterium both in control and Hg-exposed situations. A temporal variance in metabolic signature was also observed during the early and mid-log phase of growth in the presence of Hg. The bacterium described in this study is the first indigenous Hg-resistant strain isolated from the Uttar Dinajpur region, which could be further explored and exploited as a potent bioresource for Hg remediation.

Current Fungal Taxonomy and Developments in the Identification System.

A functional identification system is the core and basis of fungal taxonomy, which provides sufficient diagnostic characteristics for species delimitation. Phenotype-based identification systems have exhibited significant drawbacks, such as being laborious and time-consuming. Thus, a molecular-based identification system (rDNA, DNA fingerprint, etc.) is proposed for application to fungi that lack reliable morphological characteristics. High Throughput Sequencing also makes great contributions to fungal taxonomy. However, the formal naming of nonculturable fungi from environmental sequencing is a significant challenge. Biochemical profile-based identification systems have outstanding value in fungal taxonomy and can occasionally be indispensable. This method utilizes biomarker metabolites and proteins that are expected to be unequivocal and stable. Of these, Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry has become the method of choice for chemotaxonomy. In sum, these described identification systems cannot solve all problems of species delimitation, and considerable attention to the updating of fungal nomenclature, standardization of techniques, knowledge sharing, and dissemination will be necessary.

Escherichia coli Isolation, Prevalence, and Multi Drug Resistance from a Poultry Farm in Sendafa Town, Central Ethiopia

Background: Escherichia coli is bacteria that exist as commensal in the intestine of animals and humans, but pathogenic strains cause disease in chickens. The growth of antimicrobial resistance in E. coli is one of major concern worldwide. Objective: This study aimed to assess the prevalence, and Multi drug resistance profile and determine the potential risk factor of E. coli isolates from chickens and chicken’s environment in Sendafa town, central Ethiopia. Methods: A cross-sectional study was carried out from January to August 2023 in Sendafa town. Purposive sampling techniques were used based on the presence of clinical symptoms suggestive of E. coli in chicken farms. Different types of samples were collected including 207 cloacae swabs, 8 feed, and 8 water from different farms. E. coli isolation and identification were done using bacterial culture, and biochemical, and confirmed using the Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI TOF MS). Antimicrobial susceptibility testing (AST) was done using the Kirby Bauer disc diffusion method. Data were entered in Microsoft Excel and analyzed with descriptive statistics using SPSS version 20. Result: Out of a total of 223 samples of cloacae swab, feed, and water 71 (31.83 %) were found to be E. coli suspected by biochemical and finally confirmed with Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI TOF MS). All confirmed isolates were subjected to 12 antimicrobial agents to check their susceptibility. From 71 tested Escherichia coli isolates we found resistant patterns of E. coli to 62(87.3%) Amoxicillin, 44(62%) Tetracycline, and 39(54.9%) Streptomycin respectively. Multi-drug resistance was observed in this study for two or more than resistant detected. Some isolate was sensitive to 60(84.5%) Trimethoprim Sulphamethoxazole, 57(80.3%) Norfloxacin, 56(78.9 %) Ceftriaxone, and 53(74.6%) Meropenem respectively. Intermediate antibiotics were observed on 32(45.1 %) Sulfonamides, and 23(32.4%) Gentamicin for Escherichia coli isolate. Risk factors were analyzed with descriptive statistics. In the study, the difference in E. coli prevalence by age and among sample types was not statistically significant (P>0.05). Between kebele and farm, there was a statistically significant difference in E. coli prevalence (P <0.05). Conclusion: In this study, a high incidence of E. coli and antibiotic resistance to commonly used antibiotics including Amoxicillin, Tetracycline, and Streptomycin in poultry were found. This implies that there is the existence of practices that accelerate antimicrobial resistance in the sampled chickens therefore, appropriate use of antimicrobial agents, good biosecurity measures, and hygiene practices in chicken farms are important.

Closing the Gap in Proteomic Identification of Histoplasma capsulatum: A Case Report and Review of Literature.

Histoplasmosis is a globally distributed systemic infection caused by the dimorphic fungus Histoplasma capsulatum (H. capsulatum). This fungus can cause a wide spectrum of clinical manifestations, and the diagnosis of progressive disseminated histoplasmosis is often a challenge for clinicians. Although microscopy and culture remain the gold standard diagnostic tests for Histoplasma identification, matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) has emerged as a method of microbial identification suitable for the confirmation of dimorphic fungi. However, to our knowledge, there are no entries for H. capsulatum spectra in most commercial databases. In this review, we describe the case of disseminated histoplasmosis in a patient living with HIV admitted to our university hospital that we failed to identify by the MALDI-TOF method due to the limited reference spectrum of the instrument database. Furthermore, we highlight the utility of molecular approaches, such as conventional polymerase chain reaction (PCR) and DNA sequencing, as alternative confirmatory tests to MALDI-TOF technology for identifying H. capsulatum from positive cultures. An overview of current evidence and limitations of MALDI-TOF-based characterization of H. capsulatum is also presented.

Contamination rate of rare bacterial species detected by MALDI-TOFMS: a retrospective cohort study

When rare bacterial species are identified in blood cultures, determining the clinical significance is sometimes difficult. This study aimed to analyze the clinical significance of rare bacterial species detected in blood cultures using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOFMS) by comparing their contamination rates with those of common species. We retrospectively analyzed medical records of adult patients with positive blood cultures at Kyoto City Hospital from 2014 to 2022. Rare species were defined by low detection rates and few PubMed reports. Of 4880 microorganisms identified from 3441 individuals, 1150 (23.6%) were classified as contamination. Meanwhile, 24 rare microorganisms were identified, of which 14 (58.3%) were classified as contamination, which was significantly higher than common species (odds ratio 4.56, 95% confidence Interval 1.88–11.50, P < 0.001). These findings may help in determining the clinical significance of rare bacterial species in blood cultures with few reported cases.

Molecular epidemiology and antibiotic resistance of Pseudomonas aeruginosa isolated from blood in a hospital in Shandong Province from 2014 to 2021

Objective: To identify the antibiotic resistance, virulence genes, and sequence types of Pseudomonas aeruginosa (P. aeruginosa) strains isolated from blood. Methods: From November 2014 to December 2021, a total of 94 nonrepetitive P. aeruginosa isolates were obtained from blood samples of patients at the First Affiliated Hospital of Shandong First Medical University in Shandong Province, China. The bacteria were identified using matrix-assisted laser desorption ionization time of flight mass spectrometry. Antibiotic resistance of the P. aeruginosa isolates was detected using Vitek 2 Compact system. Polymerase chain reaction (PCR) was conducted for the 18 virulence genes, and multi locus sequence typing (MLST) was performed to identify the sequence types of the P. aeruginosa strains. The resistance rates and distributions of virulence genes between carbapenem resistant pseudomonas aeruginosa (CRPA) and carbapenem susceptible pseudomonas aeruginosa (CSPA) isolates were compared using the Chi-square test. Results: Among 94 P. aeruginosa isolates, 19 (20.2%) isolates were found to be multidrug resistant (MDR) bacteria, of which 17 were CRPA isolates and 2 were CSPA isolates. All strains contained more than 10 virulence genes. Except for exoU gene, the detection rate of other genes was above 83%. MLST analysis revealed a total of 66 different STs, including 59 existing STs and 7 novel STs. Among them, ST244 (n=11, 11.7%) and ST270 (n=7, 7.4%) were the dominant STs. Although these two types of isolates harbored the same virulence genes, the resistance rates to carbapenem were different. 54.5% (6/11) ST244 isolates were CRPA but all 7 ST270 isolates were CSPA. Conclusion: Although the resistance rates of P. aeruginosa strains isolated from blood were at a low level, some MDR and CRPA isolates were detected. As the high virulence gene detection rates and genetic diversity were found for P. aeruginosa strains isolated from blood, close attention should be paid to avoid transmission and outbreaks.

Milk as diagnostic fluid for udder health management.

Mastitis is the major disease affecting milk production of dairy cattle, and milk is an obvious substrate for the detection of both the inflammation and its causative infectious agents at quarter, cow, or herd levels. In this review, we examine the use of milk to detect inflammation based on somatic cell count (SCC) and other biomarkers, and for the detection of mastitis pathogens through culture-based and culture-free methods. The use of SCC at a cow or bulk milk level to guide udder health management in lactation is well-established, and SCC is increasingly used to guide selective dry cow treatment. Other markers of inflammation include electrical conductivity, which is used commercially, and markers of disease severity such as acute phase proteins but are not pathogen-specific. Some pathogen-specific markers based on humoral immune responses are available, but their value in udder health management is largely untested. Commercial pathogen detection is based on culture or polymerase chain reaction, with other tests, for example, loop-mediated isothermal amplification or 16S microbiome analysis still at the research or development stage. Matrix-assisted laser desorption ionisation time of flight (MALDI-ToF) is increasingly used for the identification of cultured organisms whilst application directly to milk needs further development. Details of test sensitivity, specificity, and use of the various technologies may differ between quarter, cow, and bulk milk applications. There is a growing array of diagnostic assays that can be used to detect markers of inflammation or infection in milk. The value of some of these methods in on-farm udder health improvement programs is yet to be demonstrated whilst methods with proven value may be underutilised.

A side-by-side comparison of the new VITEK MS PRIME and the MALDI Biotyper sirius in the clinical microbiology laboratory

PurposeThis study aims to evaluate the performance of two latest generation matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) systems in routine laboratory settings, focusing on turnaround time (TAT), time to results (TTR), hands-on time, and identification rate.MethodsWe conducted a time and motion study on three workflow scenarios to simulate different laboratory settings. Overall, 618 bacterial isolates from a tertiary hospital’s laboratory were processed using the VITEK MS PRIME (bioMérieux) and the MALDI Biotyper sirius (Bruker Daltonics) and their corresponding databases VITEK IVD Database 3.2 and MBT reference library 12.ResultsThe target preparation process showed no significant difference in TAT, but the Biotyper workflow had a shorter hands-on time by 3 to 6 min. In the measurement process, TTR was three to five times shorter for the Biotyper sirius while hands-on time was significantly shorter for VITEK MS PRIME (approximately 1.5 min per target). The identification rate without retesting was 97.9% for VITEK MS PRIME and 98.9% for Biotyper sirius. Both systems achieved 100% agreement at genus and 96.2% at species level.ConclusionBoth systems exhibited excellent identification rates for routine bacterial isolates. Due to its high speed, the Biotyper sirius is suited for laboratories with high sample throughput and a workflow designed for processing larger batches. The VITEK MS PRIME, with its “load and go” system accommodating up to 16 targets, reduces hands-on time, making it a reasonable choice for laboratories with fewer identifications overall but a higher number of targets and a workflow designed for parallel processing on different workstations.