Laser Desorption Ionization-time Of Flight Research Articles

Carbapenemase Genes, Virulence Genes, and Molecular Epidemiology of Carbapenem-Resistant Klebsiella pneumoniae Derived From Bloodstream Infections

To investigate the clinical characteristics and molecular epidemiology of carbapenem-resistant Klebsiella pneumoniae (CRKP) isolated from patients with bloodstream infections in a large tertiary-care general hospital in Southwest China. A total of 131 strains of non-repeating CRKP were collected from the blood cultures of patients who had bloodstream infections in 2015-2019. The strains were identified by VITEK-2, a fully automated microbial analyzer, and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. The minimum inhibitory concentration (MIC) was determined by microbroth dilution method. The common carbapenemase resistant genes and virulence factors were identified by PCR. Homology analysis was performed by multilocus sequencing typing. Whole genome sequencing was performed to analyze the genomic characteristics of CRKP without carbapenemase. The 131 strains of CRKP showed resistance to common antibiotics, except for polymyxin B (1.6% resistance rate) and tigacycline (8.0% resistance rate). A total of 105 (80.2%) CRKP strains carried the Klebsiella pneumoniae carbapenemase (KPC) resistance gene, 15 (11.4%) strains carried the New Delhi Metallo-β-lactamase (NDM) gene, and 4 (3.1%) isolates carried both KPC and NDM genes. Sequence typing (ST) 11 (74.0%) was the dominant sequence type. High detection rates for mrkD (96.2%), fimH (98.5%), entB (100%), and other virulence genes were reported. One hypervirulent CRKP strain was detected. The seven strains of CRKP that did not produce carbapenemase were shown to carry ESBL or AmpC genes and had anomalies in membrane porins OMPK35 and OMPK36, according to whole genome sequencing. In a large-scale tertiary-care general hospital, CRKP mainly carries the KPC gene, has a high drug resistance rate to a variety of antibiotics, and possesses multiple virulence genes. Attention should be paid to CRKP strains with high virulence.

Clinical and microbiological profile of Viridans group streptococcal bacteraemia; experience from South India.

Viridans Group Streptococci (VGS) are a group of distinct species that can cause bacteraemia and other invasive infections. They are also among the common organisms causing infective endocarditis. Data on the epidemiology and clinical profile of VGS is limited, especially from India. We conducted an electronic medical record-based retrospective analysis of patients with VGS bacteraemia admitted to our hospital between January 2012 to December 2021. Blood cultures were incubated by BacT/ALERT system and bacterial identification and susceptibility testing were done by using the VITEK 2 microbial identification system. Susceptibility test reporting was as per Clinical and Laboratory Standards Institute (CLSI) guidelines. The incidence, clinical profile, source of bacteraemia, co-morbidities and antimicrobial resistance among VGS bacteraemia were analyzed. VGS were isolated in 219 patients, accounting for 3.2% of positive blood cultures during the period studied. The median age of the patients was 58 years and 69% were males. Diabetes mellitus was the most common co-morbidity (55%) followed by chronic kidney disease and chronic liver disease. Patients with haematological malignancy and neutropenia were few. Intra-abdominal infections were the most common source of infection and was noted in 26%. Infective endocarditis was diagnosed in only 10% of the cases. Streptococcus mitis was the most common species isolated followed by S. gallolyticus and S. sanguinis. 9.58% of the isolates could not be identified up to the species level. Overall penicillin susceptibility was 71% and ceftriaxone susceptibility was 92%, with individual species variation. In-hospital mortality was 19%. VGS are an important cause of bacteraemia and was associated with 19% mortality in our study. High rates of penicillin and ceftriaxone resistance are a reason of concern. Molecular diagnostics like matrix assisted laser desorption ionization-time of flight (MALDI-TOF) identification must be increasingly applied for species identification considering that a substantial number of isolates were not identified to species level.

The Role of Matrix-Assisted Laser Desorption Ionization-Time of Flight- Mass Spectrometry (MALDI-TOF-MS) in the Identification of Non-fermenting Gram-Negative Bacilli from Clinical Isolates in a Tertiary Care Hospital

Non-fermenting Gram-negative bacilli (NFGNB) are a heterogeneous group of aerobic non-spore forming bacilli. Matrix-Assisted Laser Desorption Ionization-Time of Flight- Mass Spectrometry (MALDI-TOF-MS) is a quick, precise diagnostic method that could identify the isolates at the species level. The aim of the study is to identify the non-fermenters isolated from various clinical infections by MALDI-TOF analysis and to detect the antibiogram of these isolates. The non-fermenters were isolated from various clinical specimens, plated on Blood agar and MacConkey agar and incubated at 37⁰C for 24 hours. Identification was done on the basis of colony morphology, Gram stain, and MALDI-TOF Analysis. Antimicrobial Susceptibility Testing was done by Kirby- Bauer Disc Diffusion Technique using commercially available antibiotics on Muller-Hinton Agar. A total of 360 samples were studied. The majority of the non-fermenters were from Blood/ Body fluids (39.72%). Acinetobacter spp (31.67%) was the most common isolate, followed by Pseudomonas spp (22.50%). Other non-fermenters like Stenotrophomonas maltophilia, Burkholderia spp, Elizabethkingia spp, Achromobacter spp, Chryseobacterium spp, Ralstonia spp, Pandoraea spp, Rhizobium radiobacter, Sphingomonas spp, Ochrobactrum spp, Myroides odoratimimus and Brevundimonas spp were rarely isolated non-fermenters. Most of the strains were multi drug resistant to commonly used antibiotics like Cephalosporins, Aminoglycosides and Carbapenems. Our results showed that identifying non-fermenters using MALDI-TOF MS is a reliable and rapid technique that shortens the time to initiate appropriate therapy and even the length of the hospital stay.

Multidimensional mass profiles increase confidence in bacterial identification when using low-resolution mass spectrometers.

Field-forward analytical technologies, such as portable mass spectrometry (MS), enable essential capabilities for real-time monitoring and point-of-care diagnostic applications. Significant and recent investments improving the features of miniaturized mass spectrometers enable various new applications outside of small molecule detection. Most notably, the addition of tandem mass spectrometry scans (MS/MS) allows the instrument to isolate and fragment ions and increase the analytical specificity by measuring unique chemical signatures for ions of interest. Notwithstanding these technological advancements, low-cost, portable systems still struggle to confidently identify clinically significant organisms of interest, such as bacteria, viruses, and proteinaceous toxins, due to the limitations in resolving power. To overcome these limitations, we developed a novel multidimensional mass fingerprinting technique that uses tandem mass spectrometry to increase the chemical specificity for low-resolution mass spectral profiles. We demonstrated the method's capabilities for differentiating four different bacteria, including attentuated strains of Yersinia pestis. This approach allowed for the accurate (>92%) identification of each organism at the strain level using de-resolved matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) data to mimic the performance characteristics of miniaturized mass spectrometers. This work demonstrates that low-resolution mass spectrometers, equipped with tandem MS acquisition modes, can accurately identify clinically relevant bacteria. These findings support the future application of these technologies for field-forward and point-of-care applications where high-performance mass spectrometers would be cost-prohibitive or otherwise impractical.

Enhanced exopolysaccharide production in gamma irradiated Bacillus subtilis: A biofilm-mediated strategy for ZnO nanoparticles removal

Biofilm-mediated strategy was studied for ZnO nanoparticle removal from aqueous media. Bacillus subtilis isolated from the soil rhizosphere was selected based on its high viscosity (133 Pa/s) of the cultivated culture and biofilm formation. The bacterium was exposed to gamma-irradiation to enhance EPS production along with its cultivation in EPS-producing media. The results show an increase in viscosity that reached 160 Pa/s at 2 kGy. EPS production increased from 4.45 to 7.95 mg/mL and the protein/carbohydrate ratio increased from 3 to 4.4 which reflects the stickiness of EPS. Thermal Gravimetric Analysis (TGA) showed 2 phase weight loss for gamma irradiated EPS and defined protein peaks when characterized using Matrix Assisted Laser Desorption Ionization-Time of Flight (MALDI-TOF). Native and gamma-irradiated Bacillus subtilis cells with their enhanced EPS were grown as a biofilm on sterile waste gauze fabric, Scanning Electron Microscopy (SEM) showed an increased biofilm attachment in gamma-irradiated samples. The latter was used for the removal of ZnO NP from aqueous media. Energy dispersive X-ray (EDX) mapping confirms that ZnO NPs were entrapped within the carbon and oxygen elements forming the biofilm with net intensities of 14.04, 1713, and 1190, respectively. The results confirm that biofilm-mediated strategy is effective in nanoparticles removal.

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.

Ceria-based ceramics surface investigation

Ceria-based nanopowders and ceramics have been prepared and their surfaces were investigated by a set of methods. Depending on the method used: HRTEM-(HAADF-STEM)-EDS, XPS, ICP-MS, SEM-EDS, LDI-TOF, it is possible to estimate the amount of dopants included in the composition of the ceria-containing sample. The method of laser desorption/ionization time of flight (LDI-TOF) mass spectrometry was used to establish the composition of powders and ceramics. Based on structural cationic fragments in the mass spectra, the surface composition was estimated. It is shown that the surface composition according to XPS and LDI data for powders are in good agreement.

A New Hemoglobin Variant: Hb Tangshan [HBA1: c.239C > T, CD79(GCG > GTG)(Ala > Val)] Detected by MALDI-TOF MS

In this report we decribed a new α-chain variant found during the measurement of hemoglobin A1c (Hb A1c) using matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS). MALDI-TOF MS analysis detected an α-chain variant with a mass of 15,155 Da. However, this Hb variant was not detected during Hb A1c measurement by cation-exchange high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE) methods. Sanger sequencing validated the presence of a heterozygous missense mutation [HBA1: c.239C > T, CD79(GCG > GTG)(Ala > Val)]. The observed 28 Da mass difference exactly matches the theoretical mass difference (28 Da) resulting from the substitution of alanine (89.079) with valine (117.133). As this represents the initial documentation of the mutation, we named it Hb Tangshan after the proband’s residence.

Non-thermal plasma radiation-induced changes in antibiotic susceptibility and protein profile of Staphylococcus aureus.

Plasma radiation is a widely used technique for sterilization or decontamination in various industries, as well as in some healthcare settings such as dentistry. The primary aim of this study was to assess the potential of plasma radiation to create a new population of Staphylococcus aureus cells with distinct characteristics that could lead to novel healthcare challenges. A homemade non-thermal plasma apparatus was applied and the effects of plasma treatment on S. aureus ATCC25923 was assessed. Plasma radiation was applied under controlled conditions to ensure that some bacterial cells remained viable. The treatment was repeated 10 times, with each round followed by a recovery phase to collect any surviving bacterial cells. To assess the potential changes in the bacterial population, we examined the antibiotic susceptibility pattern, micro-structural characteristics using scanning electron microscopy (SEM), and total protein profile using the matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) technique. The experimental results revealed slight variations in the antibiotic susceptibility patterns of certain cell wall agents (imipenem, cephalothin, and cefepime), as well as in the MALDI-TOF spectra. However, no changes were observed in the SEM images. The insufficient application of non-thermal plasma in bacterial decontamination may lead to physiological changes that could enrich or select certain subpopulations of S. aureus.

Detection of Carbapenemase Encoding Gene and Resistance to Cefiderocol in Hospital and Community eXtensive Drug Resistance and Carbapenem-Resistant Pseudomonas aeruginosa Strains in Morocco.

Pseudomonas aeruginosa (Pa) remains among clinically-significant Gram-negative species. The carbapenems are often the last resort for treating infections due to multidrug resistant isolates such as Pa. The carbapenems' efficacy is increasingly compromised by the emergence and the rapid spread of Pa carrying carbapenemases which represent a serious threat to public health. This study aimed to establish the resistance profile and to identify carbapenemase genes in isolates with imipenem resistant phenotypes. Among 134 Pa isolates collected both in the community (46) and hospital (88) from January 2021 to December 2021 in Morocco, 18 (8 were from the community and 10 from the hospital settings) were carbapenem resistant. The identification of these strains has been confirmed using matrix assisted laser desorption ionization-time of flight (MALDI-TOF). The antibiotic susceptibility testing against 16 antibiotics was carried out and interpreted according to the recommendations of the European Committee on Antimicrobial Susceptibility Testing (2021). The worrying antibiotics resistance profiles, which spread to cefiderocol for two isolates, were obtained for all isolates, which were eXtensive Drug Resistance showing highly resistant to all antibiotic categories tested, even to ceftolozane-tazobactam. Colistin (100% susceptible) and cefiderocol (88.88%) were the most active agents against carbapenem-resistant Pa (CRPa). Phenotypic detection by NP-CARBA and NG-CARBA tests of metallo‑β‑lactamase (MβL) production was confirmed by PCR amplification and sequencing. Three CRPa isolates coharboring blaVIM-2-blaNDM-1 (two isolates) and blaVIM-2-blaIMP-8 (one isolate) genes were detected. In this study, we describe the coexistence of these MβL genes and the cefiderocol resistance in CRPa strains in Morocco. The alarming antibiotic resistance patterns of all these CRPa isolates and their resistance genes emphasize the importance of antimicrobial susceptibility testing in the choice of antibiotics for treating Pa infections.

Efficient Mass Spectrometry Peak Detection by Combining Resolution Enhancement and Image Segmentation

Mass spectrometry data may be affected by random noise and baseline drift due to experimental instruments and conditions, posing significant challenges for detecting spectral peaks, particularly when identifying weak and separating overlapping peaks. To increase the sensitivity and enhance the resolution, we propose a mass spectral peak detection algorithm that integrates resolution enhancement and image segmentation. Initially, the extended Mexican hat wavelet is proposed by integrating the peak sharpening method with its wavelet. This approach accurately transforms mass spectra into wavelet space using the continuous wavelet transform. Subsequently, the triangular single-peak thresholding method, a more suitable threshold segmentation approach for spectral analysis, is introduced to identify ridges in the two-dimensional wavelet space. Compared to traditional Otsu and its improved variants, long-tailed single-peaked histograms are more effectively processed by this method with lower computational complexity, enabling faster identification of segmentation thresholds and image segmentation. Ultimately, peak positions are determined by utilizing ridge and valley lines in wavelet space along with the original spectrum. To evaluate the performance of the peak recognition algorithm, two metrics are introduced: the receiver operating characteristic (ROC) curve and the balanced F score (F1 score). When compared to multi-scale peak detection (MSPD), continuous wavelet transform and image segmentation (CWT-IS), the developed approach is more suitable for weak and highly overlapping peaks. The robustness and practicality of the method are verified through peak detection using matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectra.

The Effect of Corticosteroids on Sinus Microbiota in Chronic Rhinosinusitis Patients with Nasal Polyposis.

Chronic rhinosinusitis with nasal polyposis (CRSwNP) is a multifactorial disease with no known single cause, but it is thought that bacteria play a role in the disease process. This pilot study aims to assess the longitudinal effect of corticosteroid therapy on sinus microbiota in chronic rhinosinusitis patients with nasal polyposis (CRSwNP). A longitudinal prospective case-control study was done on patients with CRSwNP and healthy controls. Patients with CRSwNP were randomly allocated to a corticosteroids and antibiotics treatment group (CRSwNP-SA) or a corticosteroid-only treatment group (CRSwNP-S). Data were collected at three-time points (before treatment, 1, and 3 months after treatment). Specimens were cultured and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) was used as a bacterial detection method. Data from 29 patients with CRSwNP (16 CRSwNP-SA and 13 CRSwNP-S) was compared to 15 healthy subjects. Patients reported significant symptom improvement initially (1 month), but not in the long-term (3 months). This result was found in both treatment groups, whether or not antibiotics were used. After 3 months from treatment, the prevalence of Corynebacterium genera tended to increase in the CRSwNP-SA, while Staphylococcus and Gram-negative genera (Pseudomonas) tended to increase in the CRSwNP-S. Smoking, aspirin sensitivity, and previous endoscopic sinus surgery were found to be co-factors significantly associated with the response to systemic corticosteroid therapy. In this pilot study, both treatment options were effective to improve symptoms in the short-term but not in the long-term, and were not linked to any clear sinus microbiota response. As a result, this study supports the avoidance of systemic antibiotics without evidence of active infection.

Isolation of Malassezia yeasts from dogs with gastrointestinal disease undergoing duodenal endoscopy.

To culture Malassezia and other fungi from the duodenum of dogs with gastrointestinal signs undergoing routine endoscopic examination. Quantitative microbial culture was performed on duodenal juice aspirated from dogs with suspected enteropathy during routine upper gastrointestinal endoscopy; samples were cultured on Sabouraud's dextrose agar (30, 32 and 37°C) and modified Dixon agar (32°C) for 14 days. Isolates were identified phenotypically and by matrix-assisted laser desorption ionisation-time of flight, and internal transcribed spacer sequencing. Yeast presence was also evaluated by cytological and histopathological examination of smears and biopsy specimens. Forty-five dogs were recruited with chronic inflammatory enteropathy (n=38), granulomatous colitis (n=2), gastric adenocarcinoma (n=2), duodenal small cell lymphoma (n=1) and idiopathic severe gastrointestinal haemorrhage (n=2). Fungi were cultured from 14 dogs: Malassezia pachydermatis was isolated from eight [chronic inflammatory enteropathy (n=7) (along with Candida albicans n=1); granulomatous colitis (n=1)] and Malassezia sympodialis from another (gastric adenocarcinoma). Five dogs with chronic inflammatory enteropathy yielded other yeasts (C. albicans, Candida glabrata, Kazachstania slooffiae, Kazachstania telluris, Pichia kudriavzevii [syn. C. krusei]). Yeasts were never observed in histopathological specimens. Fluorescent microscopical examination of cytological specimens showed yeast in only one case, from which K. slooffiae was subsequently isolated. Based on a literature search, this is the first report of isolation of M. pachydermatis, M. sympodialis, K. slooffiae and K. telluris from the canine duodenum. Further studies are needed to determine whether these are resident or transient fungi in the canine duodenum and whether their presence has a pathogenic effect on the host.

Genetic characterization of Escherichia coli and Klebsiella spp. from humans and poultry in Nigeria.

The emergence of antibiotic resistance in livestock, especially food-producing animals, is of major public health importance as a result of the possibility of these bacteria entering the food chain. In this study, the genetic characteristics of antibiotic-resistant Escherichia coli and Klebsiella spp. isolates from humans and poultry in Edo state, Nigeria, were investigated. In April 2017, 45 Klebsiella spp. and 46 E. coli isolates were obtained from urine, clinical wounds, nasal and chicken faecal samples. Isolates were recovered and identified as previously described. Species identification was achieved by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry and ribosomal multilocus sequence typing. Antimicrobial susceptibility testing was carried out using the Kirby-Bauer method for 12 antibiotics. A double disc synergy test was used to screen for extended-spectrum beta-lactamse (ESBL) production. Whole genome sequencing was performed for strain characterization of the isolates. Thirteen Klebsiella spp. isolates yielded positive results by the ESBL phenotypic test and harboured ESBL genes. Of the 46 E. coli isolates, 21 human and 13 poultry isolates were resistant to at least one of the tested antibiotics. Four human E. coli isolates harboured ESBL genes and revealed positive results when applying ESBL double disc synergy tests. ESBL genes in the Klebsiella spp. and E. coli isolates include bla CTX-M-15 and bla SHV-28. Whole genome-based core gene multilocus sequence typing of the Klebsiella spp. and E. coli isolates revealed a close relatedness among the isolates. An integrated 'One Health' surveillance system is required to monitor transmission of antimicrobial resistance in Nigeria.

Kawal: A fermented food as a source of Bacillus strain producing antimicrobial peptides

Kawal is a fermented food condiment obtained by spontaneous fermentation of Senna obtusifolia leaves. Fermentative microbiota's consists of microorganisms producing a diversity of bioactive molecules. Thus, research of novel microbial products capable of inhibiting or eliminating plant and food pathogens is an absolute necessity. In this study, 126 bacterial strains were isolated from Kawal. Phenotypical, biochemical and genotypic methods were used to determine their genus or species. Forty-three (43) of them belonging to the genus Bacillus were selected for their antibacterial and antifungal properties. Their ability to produce lipopeptides (fengycins, surfactins, iturins, kurstakins) was investigated using HPLC and Matrix-assisted Laser Desorption Ionization-Time of Flight (MALDI-ToF-MS). Molecular characterization of 16S rRNA identified 10 species belonging to the genus Bacillus: B. subtilis, B. licheniformis, B. amyloliquefaciens, B. velezensis, B. spizizenii, B. wiedmannii, B. tropicus, B. cereus, B. tequilensis and B. safensis. MALDI-ToF-MS analysis revealed a diversity of antimicrobial molecules. Among all Bacillus strains tested, 84.44% produced surfactin, 22.22% produced fengycin, 11.11% produced iturin and 2.22% produced kurstakin. The results of this study demonstrated the high diversity of lipopeptides among kawal Bacillus strains and their antimicrobial capacity. In view of the potentialities that have been highlighted in these strains, some of them can be used in the bioconservation or in the bioprotection of plants.

Endocarditis due to Aggregatibacter Segnis: a rare case report

BackgroundAs a member of the HACEK group, Aggregatibacter segnis (A. segnis) is a fastidious Gram-negative coccobacillus that resides in the human oropharyngeal flora. Infective endocarditis caused by A. segnis is rarely reported.Case presentationA 31-year-old male was admitted to our hospital for a 3-month history of intermittent high fever, chills, and chest distress. On presentation, he was febrile and tachycardic but otherwise with stable vital signs. Physical examination revealed systolic murmurs in the aortic and mitral valve areas. Pitting edema was evident in the lower extremities. Transthoracic echocardiography demonstrated multiple vegetations in the mitral and aortic valves. Severe regurgitation of the aortic valve and left heart dysfunction were also detected. With the suspicion of infective endocarditis and heart failure, we immediately performed microbiological tests and arranged the cardiac replacement surgery. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry and metagenomic next-generation sequencing (mNGS) identified A. segnis from the bloodstream. While the surgical specimen culture was negative, the mNGS was positive for A. segnis. The patient was treated with ceftriaxone for four weeks and discharged. He remained clinically well, with laboratory results restored.ConclusionThis is the first report of A. segnis infective endocarditis that combined MALDI-TOF and metagenomic next-generation sequencing in the diagnosis. The hypothesis-independent molecular techniques can outperform conventional tools to prevent diagnostic delay.

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.

Association between Mastitis Occurrence in Dairy Cows and Bedding Characteristics of Compost-Bedded Pack Barns.

Compost-bedded pack barns (CB) are receiving increasing attention as a housing system that can potentially improve the welfare of dairy cows. This study characterized the frequency and profile of pathogens isolated from clinical (CM) and subclinical (SCM) mastitis in dairy cows housed in CB. It evaluated the association between mastitis occurrence and bedding characteristics in CB systems. Over six months, seven dairy herds were visited monthly for milk and bedding sample collections. Milk samples from mastitis cases were submitted to microbiological identification by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF MS). Bedding samples were submitted to physical-chemical (pH, organic matter, moisture, and carbon to nitrogen ratio) and microbiological counting (total bacterial counts, coliforms, streptococci, and staphylococci) analyses. Regression analysis was used to determine the association between mastitis occurrence and CB characteristics. Our results showed that Escherichia coli and environmental streptococci were the most frequently isolated pathogens from CM cases, while Staphylococcus chromogenes and contagious pathogens (Staphylococcus aureus and Streptococcus agalactiae) were the most commonly isolated from SCM cases. Bedding moisture content was positively associated with the incidence of CM. The bedding carbon to nitrogen ratio was negatively associated with the incidence of SCM, and the bedding total bacteria counts tended to be associated with the incidence of SCM. Bedding counts of coliforms positively associated with the prevalence of SCM. Our results can support decision-makers in the dairy industry seeking strategies for bedding management and mastitis control.

Peptide Spectra Analysis of DNA Starvation/Stationary Phase Protection-Like Protein Found in Hospital-Acquired ESBL Producing Escherichia coli by MALDI-TOF Technique

The present study aimed to investigate the hospital-acquired Extended Spectrum β-Lactamase production (ESBL) producing Escherichia coli, to identify the significant ESBL peptide spectra after exposure to ceftazidime and amoxicillin/clavulanic acid, and to identify the resistant peptide spectra after exposure to ciprofloxacin. ESBL production and the minimal inhibitory concentration of ciprofloxacin were investigated using the Epsilometer strip test. Their colonies were collected to analyze the peptide spectra by VITEK® Mass Spectrometry. The significant difference in spectrum intensity for suspected ESBL peptide spectra, and DNA starvation/stationary phase protection (Dps) -like peptide spectra were also determined. Among 228 isolates, the high ESBL-producing E. coli which exhibited high ciprofloxacin resistance was demonstrated as 22.63%. The significant reduction of spectrum intensity after exposure to ceftazidime and amoxicillin/clavulanic acid was only detected at spectrum 9714 Da in the high ESBL-producing group (P ≤0.001). Among four selected high ESBL producers, only one significant spectrum located at 9063 Da was detected in the high ciprofloxacin-resistant group (P ≤0.001) after being exposed to ceftazidime and ciprofloxacin. The highest percentage increase of spectrum 9063 Da was 69.42%. Multidrug resistance was demonstrated in the tested E. coli isolates, especially β-lactam resistance which may be caused by their ESBL production. It may be said that the peptide spectrum located at 9714, and 9063 Da were suspected as the ESBL indicative peptide spectrum in the high ESBL producers, and as the Dps indicative peptide spectrum in the high ciprofloxacin-resistant isolates. Keywords: Peptide spectra analysis, DNA starvation/stationary phase protection like protein, Hospital-acquired infection, Extended spectrum β-lactamase, Escherichia coli, Matrix-Assisted Laser Desorption Ionization-Time Of Flight

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.