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

Phenotypic and Genotypic Characteristics of Antimicrobial Resistance in Citrobacter freundii Isolated from Domestic Ducks (Anas platyrhynchos domesticus) in Bangladesh.

Antimicrobial resistance (AMR) in Citrobacter freundii poses a serious challenge as this species is one of the sources of nosocomial infection and causes diarrheal infections in humans. Ducks could be the potential source of multidrug-resistant (MDR) C. freundii; however, AMR profiles in C. freundii from non-human sources in Bangladesh have remained elusive. This study aimed to detect C. freundii in domestic ducks (Anas platyrhynchos domesticus) in Bangladesh and to determine their phenotypic and genotypic antibiotic susceptibility patterns. A total of 150 cloacal swabs of diseased domestic ducks were screened using culturing, staining, biochemical, polymerase chain reaction (PCR), and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) to detect C. freundii. Phenotypic and genotypic antibiotic susceptibility patterns were done by the disk diffusion method and PCR, respectively. In total, 16.67% (25/150) of the samples were positive for C. freundii. C. freundii isolates showed a range of 20% to 96% resistance to cefotaxime, gentamicin, levofloxacin, ciprofloxacin, cotrimoxazole, tetracycline, ampicillin, and cephalexin. More than 60% of the isolates were phenotypically MDR, and the index of multiple antibiotic resistance ranged from 0.07 to 0.79. Genes encoding resistance to beta-lactams [blaTEM-1-88% (22/25), blaCMY-2-56% (14/25), blaCMY-9-8% (2/25), and blaCTX-M-14-20% (5/25)], sulfonamides [sul1-52% (13/25), sul2-24% (6/25)], tetracyclines [tetA-32% (8/25) and tetB-4% (1/25)], aminoglycosides [aacC4-16% (4/25)], and fluoroquinolones [qnrA-4% (1/25), qnrB-12% (3/25), and qnrS-4% (1/25)] were detected in the isolated C. freundii. To the best of our knowledge, this is the first study in Bangladesh to detect MDR C. freundii with their associated resistance genes from duck samples. We suggest addressing the burden of diseases in ducks and humans and associated AMR issues using the One Health approach.

The Effect of a Planetary Health Diet on the Human Gut Microbiome: A Descriptive Analysis.

In 2019, researchers from the EAT-Lancet Commission developed the 'Planetary Health (PH) diet'. Specifically, they provided recommendations pertaining to healthy diets derived from sustainable food systems. Thus far, it has not been analysed how such a diet affects the human intestinal microbiome, which is important for health and disease development. Here, we present longitudinal genome-wide metagenomic sequencing and mass spectrometry data on the gut microbiome of healthy volunteers adhering to the PH diet, as opposed to vegetarian or vegan (VV) and omnivorous (OV) diets. We obtained basic epidemiological information from 41 healthy volunteers and collected stool samples at inclusion and after 2, 4, and 12 weeks. Individuals opting to follow the PH diet received detailed instructions and recipes, whereas individuals in the control groups followed their habitual dietary pattern. Whole-genome DNA was extracted from stool specimens and subjected to shotgun metagenomic sequencing (~3 GB per patient). Conventional bacterial stool cultures were performed in parallel and bacterial species were identified with matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. We analysed samples from 16 PH, 16 OV, and 9 VV diet patterns. The α-diversity remained relatively stable for all dietary groups. In the PH group, we observed a constant increase from 3.79% at inclusion to 4.9% after 12 weeks in relative abundance of Bifidobacterium adolescentis. Differential PH abundance analysis highlighted a non-significant increase in possible probiotics such as Paraprevotella xylaniphila and Bacteroides clarus. The highest abundance of these bacteria was observed in the VV group. Dietary modifications are associated with rapid alterations to the human gut microbiome, and the PH diet led to a slight increase in probiotic-associated bacteria at ≥4 weeks. Additional research is required to confirm these findings.

Isolation of Carbapenem and Colistin Resistant Gram-Negative Bacteria Colonizing Immunocompromised SARS-CoV-2 Patients Admitted to Some Libyan Hospitals.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has had a devastating effect, globally. We describe, for the first time, the occurrence of carbapenem-resistant bacteria colonizing SARS-CoV-2 patients who developed hospital-associated infections with carbapenemase-producing, Gram-negative bacteria at some isolation centers of SARS-CoV-2 in the eastern part of Libya. In total, at first, 109 samples were collected from 43 patients, with the samples being recovered from oral (n = 35), nasal (n = 45), and rectal (n = 29) cavities. Strain identification was performed via matrix assisted laser desorption ionization-time of flight (MALDI-TOF). Antibiotic susceptibility testing was carried out on Mueller-Hinton agar, using the standard disk diffusion method. MIC determination was confirmed via E-TEST and microdilution standard methods. A molecular study was carried out to characterize the carbapenem and colistin resistance in Gram-negative bacterial strains. All of the positive results were confirmed via sequencing. Klebsiella pneumoniae (n = 32), Citrobacter freundii (n = 21), Escherichia coli (n = 7), and Acinetobacter baumannii (n = 21) were the predominant isolated bacteria. Gram-negative isolates were multidrug-resistant and carried different carbapenem resistance-associated genes, including NDM-1 (56/119; 47.05%), OXA-48 (15/119; 12.60%), OXA-23 (19/119; 15.96%), VIM (10/119; 8.40%), and the colistin resistance mobile gene mcr-1 (4/119; 3.36%). The overuse of antimicrobials, particularly carbapenem antibiotics, during the SARS-CoV-2 pandemic has led to the emergence of multidrug-resistant bacteria, mainly K. pneumoniae, A. baumannii, and colistin-resistant E. coli strains. Increased surveillance as well as the rational use of carbapenem antibiotics and, recently, colistin are required to reduce the propagation of multidrug-resistant strains and to optimally maintain the efficacy of these antibiotics. IMPORTANCE In this work, we describe, for the first time, the occurrence of carbapenem-resistant bacteria colonizing COVID-19 patients who developed hospital-associated infections with carbapenemase-producing, Gram-negative bacteria at some isolation centers of COVID-19 in the eastern part of Libya. Our results confirmed that the overuse of antimicrobials, such as carbapenem antibiotics, during the COVID-19 pandemic has led to the emergence of multidrug-resistant bacteria, mainly K. pneumoniae and A. baumannii, as well as colistin resistance.

Comparative protein analysis of two maize genotypes with contrasting tolerance to low temperature

BackgroundLow temperature (LT) stress is one of the major environmental stress factors affecting the growth and yield of maize (Zea mays L.). Hence, it is important to unravel the molecular mechanisms behind LT stress tolerance to improve molecular breeding in LT tolerant genotypes. In the present study, two maize genotypes viz. Gurez local from Kashmir Himalaya and tropical grown GM6, were dissected for their LT stress response in terms of accumulation of differentially regulated proteins (DRPs). Leaf proteome analysis at three-leaf stage of maize seedlings subjected to LT stress of 6 °C for a total of 12 h duration was performed using two dimensional gel electrophoresis (2D-PAGE) followed by subsequent identification of the proteins involved.ResultsAfter MALDI-TOF (Matrix-assisted laser desorption/ionization-time of flight) and bioinformatics analysis, 19 proteins were successfully identified in Gurez local, while as 10 proteins were found to get successful identification in GM6. The interesting observations from the present investigation is the identification of three novel proteins viz. threonine dehydratase biosynthetic chloroplastic, thylakoidal processing peptidase 1 chloroplastic, and nodulin-like protein, whose role in abiotic stress tolerance, in general, and LT stress, in particular, has not been reported so far. It is important to highlight here that most of LT responsive proteins including the three novel proteins were identified from Gurez local only, owing to its exceptional LT tolerance. From the protein profiles, obtained in both genotypes immediately after LT stress perception, it was inferred that stress responsive protein accumulation and their expression fashion help the Gurez local in seedling establishment and withstand unfavorable conditions as compared to GM6. This was inferred from the findings of pathway enrichment analysis like regulation of seed growth, timing of floral transition, lipid glycosylation, and aspartate family amino acid catabolic processes, besides other key stress defense mechanisms. However, in GM6, metabolic pathways enriched were found to be involved in more general processes including cell cycle DNA replication and regulation of phenylpropanoid metabolism. Furthermore, majority of the qRT-PCR results of the selected proteins demonstrated positive correlation between protein levels and transcript abundance, thereby strengthening our findings.ConclusionsIn conclusion, our findings reported majority of the identified proteins in Gurez local exhibiting up-regulated pattern under LT stress as compared to GM6. Furthermore, three novel proteins induced by LT stress were found in Gurez local, requiring further functional validation. Therefore, our results offer more insights for elucidating the molecular networks mediating LT stress tolerance in maize.

Genome-wide association study for single nucleotide polymorphism associated with mural and cumulus granulosa cells of PCOS (polycystic ovary syndrome) and non-PCOS patients

BackgroundThe genetic make-up of local granulosa cells and their function in the pathophysiology of polycystic ovary syndrome (PCOS) is crucial to a full comprehension of the disorder. The major purpose of this study was to compare the Single Nucleotide Polymorphism (SNP) of cumulus granulosa cells (CGCs) and mural granulosa cells (MGCs) between healthy individuals and women with PCOS using genome-wide association analysis (GWA). A case–control study was conducted in a total of 24 women diagnosed with PCOS and 24 healthy non-PCOS women of reproductive age aggregated into 4 samples of 6 patients each. GWA studies entail several processes, such as cell separation, cellular DNA extraction, library preparation followed by interpretation using bioinformatics databases. SNP locations were identified by reference gene also involves the use of Matrix-assisted laser desorption/ionisation-time of flight (MALDI-TOF) mass spectrometry (MS) (MALDI-TOF-MS) for the first sorting. Hybridization with the gene chip was followed by reading the SNP genotypes according to the publications in the literature. TASSEL (Trait Analysis by aSSociation, Evolution and Linkage) program and methods were used for GWA studies.ResultsAn aggregate of 21,039 SNP calls were obtained from our samples. Genes of autoimmune illnesses, obesity, inflammatory illnesses, nervous system diseases such as retinitis pigmentosa, autism, neural tube defects, and Alzheimer's disease; and various malignancies such as lung cancer, colorectal cancer, breast cancer were also identified in these cells. Gene ranking score reveals that granulosa cells carry key genes of neurological system and reproductive systems especially in brain and testis, respectively.ConclusionsMural and Cumulus Granulosa cells were shown to have the PCOS directly and indirectly related genes MMP9, PRKAA2, COMT and HP. We found that the expression of ARID4B, MUC5AC, NID2, CREBBP, GNB1, KIF2C, COL18A1, and HNRNPC by these cells may contribute to PCOS.Graphical abstract

Matrix-Assisted Laser Desorption Ionization Time of Flight (MALDI-TOF) as an Indispensable Tool in Diagnostic Bacteriology: A Comparative Analysis With Conventional Technique.

Owing to its accurate diagnosis, rapid turnaround time, cost effectivity, and less rates of error, Matrix-assisted Laser Desorption Ionization Time of Flight (MALDI-TOF) has replaced most of the phenotypic methods of identification. Thus, the objective of this study was to compare and evaluate MALDI-TOF MS to conventional biochemical-to identify bacterial microorganisms. Different bacterial species isolated from 2010 to 2018 (pre-MALDI-TOF era), using routine bio-chemicals were compared to bacterial species isolated from 2019 to August 2021 (post MALDI-TOF), using MALDI-TOF, in the microbiology laboratory of a tertiary care hospital in North India. Chi-Square test (χ2) was used for the evaluation of bacterial identification between biochemical tests and MALDI-TOF MS association with a 95% confidence interval, considering wrong identification in genera or at a species level. Many different and new genera and species of bacteria could be identified using MALDI-TOF, which was not possible using only routine manual bio-chemicals like Kocuria rhizophilus, Rothia mucilaginosa,Enterococcus casseliflavus, Enterococcus gallinarum, Leuconostoc, Leclercia adecarboxylata, Raoultella ornithological, Cryseobacterium indologenes. Conclusion: Each of the newly identified bacteria played an important role in deciding treatment. Wide use of the MALDI-TOF system will not only strengthen diagnostic stewardship but also encourage antimicrobial stewardship programs.

Biofilm formation and antibiotic resistance profiles of water-borne pathogens

Water sources (surface water, drinking water, rivers, and ponds) are significant reservoirs for transmitting antibiotic-resistant bacteria. In addition, these waters are an important public health problem because they are suitable environments for transferring antibiotic resistance genes between bacterial species. Our study aimed to assess the prevalence of Extended-spectrum beta-lactamase (ESBL) producing isolates in water samples, the susceptibility of the isolates to the specified antibiotics, the determination of biofilm ability, antibiotic resistance genes, and the molecular typing of the isolates. For this purpose, Polymerase chain reaction (PCR) and Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) analyses were used. Out of 70 isolates, 15 (21%) were ESBL producing, and sent for the MALDI-TOF analysis, where Escherichia coli, Acinetobacter calcoaceticus, Enterobacter bugandensis, Acinetobacter pittii, Pseudomonas aeruginosa, Acinetobacter junii, Pseudomonas oleovorans, and Enterobacter ludwigigii were identified. Moreover, colistin resistance genes (mcr 1/2/6, mcr 4, mcr 5, mcr 3/7, and mcr 8), ESBL-encoding genes (blaSHV, blaTEM, and blaCTX-M) and carbapenemase genes (blaNDM, blaOXA-48, and blaKPC) using molecular analysis (PCR) were confirmed. The colistin resistance gene was detected at 80% (12/15) in the isolates obtained. The distribution of these isolates according to resistance genes was found as mcr 1/2/6 4 (20%), mcr 3/7 3 (13%), and mcr 5 (40%). Additionally, the isolates harbored blaSHV(6.6%) and blaTEM (6.6%) genes. However, blaNDM, blaOXA-48, blaKPC, and blaCTX-M genes were not detected in any isolates. According to the Congo red agar method, seven (46.6%) isolates showed negative biofilm ability, and eight (53.3%) showed moderate biofilm ability. However, the microplate method detected weak biofilm in 53.3% of the isolates. In conclusion, this study provides evidence for the existence of multidrug-resistant bacteria that co-exist with mcr and ESBL genes in water sources. These bacteria can migrate to other environments and pose increasing threats to public health.

The need for fast and accurate detection of dermatomycosis.

Dermatomycosis of the hair, skin or nails are one of the most common fungal infection worldwide. Beyond permanent damage to the affected area, the risk of severe dermatomycosis in immunocompromised people can be life-threatening. The potential risk of delayed or improper treatment highlights the need for a rapid and accurate diagnosis. However, with traditional methods of fungal diagnostics such as culture, a diagnosis can take several weeks. Alternative diagnostic technologies have been developed which allow for an appropriate and timely selection of an antifungal treatment, preventing nonspecific over-the-counter self-medication. Such techniques include molecular methods such as PCR, real-time PCR, DNA microarray, next-generation sequencing, in addition to matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. Molecular methods can help close the 'diagnostic gap' observed with traditional cultures and microscopy and allow for a rapid detection of dermatomycosis with increased sensitivity and specificity.

Lipase-Catalysed Polymerization of Eutectic Mixtures.

Aiming to reduce the toxicity and operational costs often associated to chemical processes, the enzymatic synthesis is applied herein as a sustainable route for producing polyesters. The use of NADES' (Natural Deep Eutectic Solvents) components as a source of monomers for the synthesis of polymers through lipase-catalyzed esterification in an anhydrous medium is detailed for the first time. Three NADES composed by glycerol and an organic base, or acid, were used to produce polyesters, through polymerization reactions catalyzed by Aspergillus oryzae lipase. High polyester conversion rates (above 70 %), containing at least 20 monomeric units (glycerol:organic acid/base (1 : 1)), were observed by matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) analysis. The NADES monomers' capacity for polymerization, along with their non-toxicity, cheap cost, and simplicity of production, sets up these solvents as a greener and cleaner approach for the synthesis of high value-added products.

Rapid, Easy, and Reliable Identification of Nocardia sp. by MALDI-TOF Mass Spectrometry, VITEK®-MS IVD V3.2 Database, Using Direct Deposit

The reference methods for Nocardia identification are based on gene sequencing. These methods are time-consuming and not accessible for all laboratories. Conversely, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry is easy to use and widely available in clinical laboratories, but for Nocardia identification, the VITEK®-MS manufacturer recommends a tedious step of colony preparation that is difficult to integrate into a laboratory workflow. This study aimed to evaluate Nocardia identification by MALDI-TOF VITEK®-MS using direct deposit with the VITEK®-PICKMETM pen and a formic acid-based protein extraction directly onto the bacterial smear on a 134 isolates collection; this identification was compared to the results from molecular reference methods. For 81.3% of the isolates, VITEK®-MS delivered an interpretable result. The overall agreement with the reference method was 78.4%. Taking only the species included in the VITEK®-MS in vitro diagnostic V3.2 database into account, the overall agreement was significantly higher, 93.7%. VITEK®-MS rarely misidentified isolates (4/134, 3%). Among the 25 isolates that produced no result with the VITEK®-MS, 18 were expected, as Nocardia species were not included in the VITEK®-MS V3.2 database. A rapid and reliable Nocardia identification using direct deposit by VITEK®-MS is possible by combining the use of the VITEK®-PICKMETM pen and a formic acid-based protein extractiondirectly onto the bacterial smear.

Fungal communities on alpine cheese rinds in Southern Switzerland

BackgroundThe biodiversity of the mycobiota of soft cheese rinds such as Brie or Camembert has been extensively studied, but scant information is available on the fungi colonizing the rinds of cheese produced in the Southern Switzerland Alps. This study aimed at exploring the fungal communities present on rinds of cheese matured in five cellars in Southern Switzerland and to evaluate their composition with regards to temperature, relative humidity, type of cheese, as well as microenvironmental and geographic factors. We used macro- and microscopical morphology, matrix-assisted laser desorption/ionization–time of flight (MALDI-TOF) mass spectrometry, and sequencing to characterize the fungal communities of the cheeses, and compared them with metabarcoding targeting the ITS region.ResultsIsolation by serial dilution yielded 201 isolates (39 yeasts and 162 filamentous fungi) belonging to 9 fungal species. Mucor and Penicillium were dominant, with Mucor racemosus, M. lanceolatus, P. biforme, and P. chrysogenum/rubens being the most frequent species. All but two yeast isolates were identified as Debaryomyces hansenii. Metabarcoding detected 80 fungal species. Culture work and metabarcoding produced comparable results in terms of similarity of the fungal cheese rind communities in the five cellars.ConclusionsOur study has shown that the mycobiota on the rinds of the cheeses studied is a comparatively species-poor community influenced by temperature, relative humidity, type of cheese, and manufacturing steps, as well as microenvironmental and possibly geographic factors.

Whole-Cell MALDI-ToF MS Coupled with Untargeted Metabolomics Facilitates Investigations of Microbial Chemical Interactions.

The emergence of drug-resistant pathogens necessitates the development of new countermeasures. In this regard, the introduction of probiotics to directly attack or competitively exclude pathogens presents a useful strategy. Application of this approach requires an understanding of how a probiotic and its target pathogen interact. A key means of probiotic-pathogen interaction involves the production of small molecules called natural products (NPs). Here, we report the use of whole-cell matrix-assisted laser desorption/ionization time-of-flight (MALDI-ToF) mass spectrometry to characterize NP production by candidate probiotics (mouse airway microbiome isolates) when co-cultured with the respiratory pathogen Burkholderia. We found that a Bacillus velezensis strain inhibits growth of and elicits NP production by Burkholderia thailandensis. Dereplication of known NPs detected in the metabolome of this B. velezensis strain suggests that a previously unannotated bioactive compound is involved. Thus, we present the use of whole-cell MALDI as a broadly applicable method for screening the NP composition of microbial co-cultures; this can be combined with other -omics methods to characterize probiotic-pathogen and other microbe-microbe interactions.

Spondylodiscitis veroorzaakt door Trueperella bernardiae: een casusbespreking

Spondylodiscitis due to Trueperella bernardiae: a case report To date, Trueperella bernardiae has rarely been detected in microbiological cultures. However, current literature describes the potential of this pathogen to cause different types of infections. The low prevalence rates may be biased by the use of older detection methods. With new techniques, such as matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) using mass spectrometry, T. bernardiae can be identified more easily. Thereby, the potency of this bacteria is shown. This case report reveals a novel presentation of T. bernardiae in a patient with acute to chronic back pain.

Direct Prosthetic Joint Infection Diagnosis from Sonication Fluid Inoculated in Blood Culture Bottles by Direct MALDI-TOF Mass Spectrometry

An accurate and fast microbiological diagnosis is key for a proper management and results when facing prosthetic joint infection (PJI). The purpose of this study is to assess the role of direct Matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry (MS) for early identification of the pathogens causing PJI from sonication fluid inoculated in blood culture bottles (BCB-SF). This prospective multicentric study included 107 consecutive patients from February 2016 to February 2017. Among them, 71 prosthetic joint revision surgeries were undergone for aseptic and 36 for septic reasons. Prostheses were sonicated and the resulting fluid inoculated into blood culture bottles, regardless the suspicion for infection. We assessed the diagnostic performance of direct MALDI-TOF MS identification of the pathogens in BCB-SF and compared it with periprosthetic tissue and conventional sonication fluid cultures. The sensitivity of direct MALDI-TOF MS of BCB-SF (69%) was higher compared to conventional sonication fluid (69% vs. 64%, p > 0.05) or intraoperative tissue cultures (69% vs. 53%, p = 0.04), especially for patients receiving antimicrobial treatment. This approach also reduced the time for identification but the specificity was compromised (100% vs. 94%) and polymicrobial infections were missed. In conclusion, BCB-SF improves the sensitivity and reduces the time of PJI diagnosis when used in combination with conventional cultures under strict sterility conditions.

Molecular Characterization and Clinical Relevance of Taxonomic Reassignment of Staphylococcus schleiferi Subspecies into Two Separate Species, Staphylococcus schleiferi and Staphylococcus coagulans.

Staphylococcus schleiferi is an opportunistic pathogen in humans and dogs. Recent taxonomic reassignment of its subspecies (S. schleiferi subsp. schleiferi and S. schleiferi subsp. coagulans) into two separate species (S. schleiferi and S. coagulans) lacks supporting data for diagnostic implications and clinical relevance. We aimed to confirm the reclassification of S. schleiferi by using genomic and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) data for a large set of isolates from humans and animals to investigate their molecular epidemiology and clinical relevance. Routine MALDI-TOF analysis and Illumina sequencing were performed on 165 S. schleiferi isolates from the Netherlands. With 33 publicly available genomes, the study included 198 genomes from 149 dogs, 34 humans, and 15 other sources. The Type Strain Genome Server was used to identify species in the genomes, and the MALDI-TOF MS database was extended to improve species differentiation. MALDI-TOF did not discriminate between S. schleiferi and S. coagulans. Genome phylogeny distinguished the two species in two monophyletic clusters. S. schleiferi isolates originated from humans, while S. coagulans isolates were found in animals and three human isolates clustering with the animal isolates. The sialidase B gene (nanB) was a unique marker gene for S. schleiferi, whereas the chrA gene was exclusive for S. coagulans. The mecA gene was exclusively detected in S. coagulans, as were the lnu(A), blaZ, erm(B/C), tet(O/M), and aac(6')-aph(2'') genes. The MALDI-TOF database extension did not improve differentiation between the two species. Even though our whole-genome sequencing-based approach showed clear differentiation between these two species, it remains critical to identify S. schleiferi and S. coagulans correctly in routine diagnostics. IMPORTANCE This study clearly shows that S. schleiferi is a concern in human hospital settings, whereas S. coagulans predominantly causes infections in animals. S. coagulans is more resistant to antibiotics and can sometimes transmit to humans via exposure to infected dogs. Even though genome-based methods can clearly differentiate the two species, current diagnostic methods used routinely in clinical microbiology laboratories cannot distinguish the two bacterial species.

Frequency and diversity of carbapenemase-producing Enterobacterales recovered from untreated wastewater impacted by selective media containing cefotaxime and meropenem in Ohio, USA.

As safe agents of last resort, carbapenems are reserved for the treatment of infections caused by multidrug-resistant organisms. The impact of β-lactam antibiotics, cefotaxime, and meropenem on the frequency and diversity of carbapenemase-producing organisms recovered from environmental samples has not been fully established. Therefore, this methodological study aimed at determining β-lactam drugs used in selective enrichment and their impact on the recovery of carbapenemase-producing Enterobacterales (CPE) from untreated wastewater. We used a longitudinal study design where 1L wastewater samples were collected weekly from wastewater treatment plant (WWTP) influent and quarterly from contributing sanitary sewers in Columbus, Ohio USA with 52 total samples collected. Aliquots of 500 mL were passed through membrane filters of decreasing pore sizes to enable all the water to pass through and capture bacteria. For each sample, the resulting filters were placed into two modified MacConkey (MAC) broths, one supplemented with 0.5 μg/mL of meropenem and 70 μg/mL of ZnSO4 and the other supplemented with 2 μg/mL cefotaxime. The inoculated broth was then incubated at 37° C overnight, after which they were streaked onto two types of correspondingly-modified MAC agar plates supplemented with 0.5 μg/mL and 1.0 μg/mL of meropenem and 70 μg/mL of ZnSO4 and incubated at 37°C overnight. The isolates were identified based on morphological and biochemical characteristics. Then, up to four distinct colonies of each isolate's pure culture per sample were tested for carbapenemase production using the Carba-NP test. Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (MS) MALDI-TOF MS was used to identify carbapenemase-producing organisms. In total 391 Carba-NP positive isolates were recovered from the 52 wastewater samples: 305 (78%) isolates had blaKPC, 73 (19%) carried blaNDM, and 14 (4%) harbored both blaKPC and blaNDM resistance genes. CPE genes of both blaKPC and blaNDM were recovered in both types of modified MAC broths, with 84 (21%) having a blaKPC gene, 22 (6%) carrying blaNDM and 9 (2%) harbored both a blaKPC and blaNDM of isolates recovered from MAC medium incorporated with 0.5ug/mL meropenem and 70ug/mL ZnSO4. The most prevalent isolates were Klebsiella pneumoniae, Escherichia coli, and Citrobacter spp.

Draft Genome Sequences of Spacecraft-Associated Microbes Isolated from Six NASA Missions.

Whole-genome sequencing can be used to better understand and assess the functional abilities of microorganisms isolated from spacecraft hardware and associated surfaces for planetary protection (PP) purposes. We sequenced 191 isolates from 6 spaceflight missions with PP requirements and identified them using Illumina-based sequencing methods and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry.

Unexplained reproductive failure in a Thoroughbred mare with a uterine infection by Arcanobacterium hippocoleae

SummaryEndometritis is recognised as one of the major causes of reproductive failure, specifically infertility and abortions, in mares. Infectious agents are the most common cause of endometritis, mainly bacteria. Here, we describe a case of an unexplained reproductive failure caused by an uncommon bacterium, A. hippocoleae, in a 15‐year‐old Thoroughbred mare with an oscillating reproductive history from Southern Brazil. Strain A. hippocoleae_LBV077/21 was isolated from the mare's uterus swab and identified by MALDI‐TOF (Matrix‐Assisted Laser Desorption Ionisation – Time of Flight) and 16 S rDNA sequencing. The antimicrobial susceptibility test showed that the strain was susceptible to ampicillin, ceftiofur, cephalexin, ciprofloxacin, rifampicin, penicillin, sulfamethoxazole/trimethoprim, tetracycline and vancomycin. Partial 16 S rDNA sequence phylogenetic analysis indicated a well‐supported position of A. hippocoleae_LBV077/21 in an exclusive clade of A. hippocoleae, whilst all other Arcanobacterium species were positioned in a separated clade, highlighting the lower syntenic relationship of the species A. hippocoleae to the other Arcanobacterium species. The present study reaffirms the importance of the isolation and identification of infectious agents in mares in cases of reproductive failure because uncommon agents can impede successful treatment and the improvement of reproductive failures.

Modulation of Capsid Dynamics in Bromoviruses by the Host and Heterologous Viral Replicase.

The three genomic and a single subgenomic RNA of Cowpea chlorotic mottle virus (CCMV), which is pathogenic to plants, is packaged into three morphologically indistinguishable icosahedral virions with T=3 symmetry. The two virion types, C1V and C2V, package genomic RNAs 1 (C1) and 2 (C2), respectively. The third virion type, C3+4V, copackages genomic RNA3 and its subgenomic RNA (RNA4). In this study, we sought to evaluate how the alteration of native capsid dynamics by the host and viral replicase modulate the general biology of the virus. The application of a series of biochemical, molecular, and biological assays revealed the following. (i) Proteolytic analysis of the three virion types of CCMV assembled individually in planta revealed that, while retaining the structural integrity, C1V and C2V virions released peptide regions encompassing the N-terminal arginine-rich RNA binding motif. In contrast, a minor population of the C3+4V virion type was sensitive to trypsin-releasing peptides encompassing the entire capsid protein region. (ii) The wild-type CCMV virions purified from cowpea are highly susceptible to trypsin digestion, while those from Nicotiana benthamiana remained resistant, and (iii) finally, the matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) analysis evaluated the relative dynamics of C3+4V and B3+4V virions assembled under the control of the homologous versus heterologous replicase. The role of viral replicase in modulating the capsid dynamics was evident by the differential sensitivity to protease exhibited by B3+4V and C3+4V virions assembled under the homologous versus heterologous replicase. Our results collectively conclude that constant modulation of capsid dynamics by the host and viral replicase is obligatory for successful infection. IMPORTANCE Infectious virus particles or virions are considered static structures and undergo various conformational transitions to replicate and infect many eukaryotic cells. In viruses, conformational changes are essential for establishing infection and evolution. Although viral capsid fluctuations, referred to as dynamics or breathing, have been well studied in RNA viruses pathogenic to animals, such information is limited among plant viruses. The primary focus of this study is to address how capsid dynamics of plant-pathogenic RNA viruses, namely, Cowpea chlorotic mottle (CCMV) and Brome mosaic virus (BMV), are modulated by the host and viral replicase. The results presented have improved and transformed our understanding of the functional relationship between capsid dynamics and the general biology of the virus. They are likely to provide stimulus to extend similar studies to viruses pathogenic to eukaryotic organisms.

Bacterial culture and susceptibility of samples taken from septic foot lesions of adult beef cattle.

Lameness is an economically important and common disease of cattle, and foot disease is the most common cause of lameness in cattle. Limited data is available regarding lameness in cow-calf operations. Describe the bacteria most commonly isolated from septic lesions of the feet of adult beef cattle and the antimicrobial susceptibility patterns of the isolated bacteria. Fifty-four adult cattle from cow-calf operations and diagnosed with a sole abscess or distal interphalangeal joint sepsis were enrolled. Prospective observational study. Abscess fluid from a convenience sample of clinical cases was cultured. Isolated bacteria were identified using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry or 16s rRNA gene sequencing. Antimicrobial susceptibility profiling was performed on isolates when a bacterial species was identified from ≥5 samples. Fifty of the 54 samples were polymicrobial. Trueperella pyogenes (22/54), Streptococcus uberis (16/54), and Bacteroides pyogenes (14/54) were the most commonly isolated bacteria. Eighty-one of 96 tested isolates were resistant to at least 1 antimicrobial; multidrug resistance was identified in 37/96 isolates. Oxytetracycline (50/96), tylosin (40/96), and florfenicol (37/96) resistance was commonly identified. Resistance to ceftiofur (5/96) was rare. Septic processes of the foot in these adult beef cattle frequently were polymicrobial. Most of the isolated bacteria were resistant to at least 1 antimicrobial with over one-third being multidrug resistant. Although simple sole abscesses do not require antimicrobial treatment, deep septic processes of the foot often are treated with antimicrobials. Culture and susceptibility of deep septic lesions may guide judicious antimicrobial usage.