Oudessa Kerro Dego

Rapid antimicrobial susceptibility testing (AST) is crucial for combating antimicrobial resistance and guiding effective therapy. Conventional phenotypic AST methods are often time-consuming, requiring 18–72 h for results, while genotypic approaches often depend on pre-existing knowledge of resistance markers. Although nucleic acid-based phenotypic AST methods have reduced turnaround time, attempts to shorten antibiotic exposure often produce only small differences between susceptible and resistant bacteria. Consequently, most loop-mediated isothermal amplification (LAMP)-based phenotypic AST assays still require prolonged antibiotic exposure (up to 4 h) or rely on sophisticated high-resolution nucleic acid detection, such as digital LAMP, to accurately detect these subtle changes, limiting their simplicity. Therefore, there is a critical need to develop a rapid assay with both short antibiotic exposure times and a simple readout. To address this unmet need, we developed PMAxx dye-assisted colorimetric Dual DNAzyme-LAMP (PD-cDDLAMP), a rapid nucleic acid-based phenotypic AST method that enhances suppression of nucleic acid amplification from antibiotic-susceptible bacteria through the selective binding of photoreactive DNA-crosslinker dye (PMAxx) to the DNA of membrane-compromised cells. This strategy enables reliable discrimination between antibiotic-resistant bacteria (“signal-on”) and antibiotic-susceptible bacteria (“signal-off”) after only 30 min of antibiotic exposure using a user-friendly colorimetric DNAzyme-LAMP readout system. PD-cDDLAMP achieved 91.7% sensitivity, 83.3% specificity, and 87.5% accuracy in detecting Escherichia coli exposed to ampicillin and tetracycline, outperforming current methods by reducing antibiotic exposure time to just 30 min. The method was further validated in spiked milk samples, demonstrating its potential for on-site AST in dairy farming. PD-cDDLAMP’s simplicity, speed, and reduced workflow complexity make it a promising tool for rapid AST. IMPORTANCE: Rapid, reliable antimicrobial susceptibility testing (AST) is essential for guiding targeted therapy and combating the escalating threat of antimicrobial resistance (AMR). However, current phenotypic ASTs are too slow for urgent decision-making, and genotypic assays cannot fully capture emerging or unknown resistance mechanisms. The PMAxx dye-assisted colorimetric dual DNAzyme-loop-mediated isothermal amplification (PD-cDDLAMP) assay introduced in this work provides a rapid nucleic acid-based phenotypic AST capable of distinguishing antibiotic-resistant from susceptible bacteria within 30 min of antibiotic exposure. By combining PMAxx-mediated suppression of nucleic acid amplification in susceptible cells with equipment-free colorimetric DNAzyme-LAMP readout, PD-cDDLAMP bridges the gap between speed and phenotypic accuracy. Its high diagnostic performance, compatibility with complex matrices such as milk, and minimal instrumentation requirements position this method as a promising point-of-care tool for clinical microbiology laboratories and on-site testing environments. PD-cDDLAMP has the potential to significantly shorten time-to-result while maintaining reliability, thereby supporting antimicrobial stewardship.

Background: Mycoplasma bovis mastitis is an important disease of dairy cows that causes substantial economic losses. However, its prevalence in different states in the United States (US), including Tennessee, is not well known. Furthermore, recent studies showed a high prevalence of bovine hemotropic mycoplasmas in US dairy farms. Objectives: Determine the prevalence of M. bovis in bulk tank milk (BTM) of dairy farms in Tennessee and evaluate the genetic diversity, virulence factors, and antimicrobial resistance genes of the identified isolates. In addition, the prevalence of Mycoplasma wenyonii and Candidatus Mycoplasma haemobos in the bulk tank milk was determined. Methods: Seventy-five BTM samples were collected from 59 dairy farms. Of the 59 farms, 56 are in Tennessee and the remaining 3 farms are in the neighboring states, Georgia (n = 2) and Alabama (n = 1). Milk samples were tested using bacterial culture, PCR, and qPCR. M. bovis isolates were genetically characterized by pangenome analysis. Results: Of the 56 farms, 3 (5.3%) were positive for M. bovis by bacterial culture and 43 (76.7%) were positive by PCR. Pangenome analysis showed clustering of current isolates with mastitis isolates from the US, Israel, and Europe. Of 75 BTM samples tested by qPCR, 42 (56%) and 51 (68%) were positive for M. wenyonii and C. M. haemobos, respectively. Conclusions: M. bovis intramammary infection is prevalent in Tennessee dairy farms.

Nucleases and 5′ nucleotidase (5′-NT) play essential roles in cell biology and are often associated with bacterial virulence. In Mycoplasma spp., which have limited metabolic capacities and rely on nutrient availability, these enzymes are of significant importance for nucleotide salvage. This study explores the potential role of 2 membrane-associated lipoproteins, the major nuclease MnuA and 5′-NT, in Mycoplasma bovis mastitis. Mutants deficient in MnuA (mnuA::Tn) and in 5’-NT (0690::Tn) were identified through genome-wide transposon mutagenesis of M. bovis PG45 type strain and their fitness and virulence were assessed both in vitro, in axenic medium, and in vivo, using murine and cow mastitis models. The mnuA::Tn mutant demonstrated reduced nuclease activity, while 0690::Tn exhibited slow log-phase growth and impaired hydrolase activity towards nucleotides as well as deoxynucleotides (dAMP and dGMP). In comparison to the parent strain, the 0690::Tn mutant displayed markedly reduced fitness, as evidenced by a significant decrease or even absence in post-challenge mycoplasma counts in murine and cow mammary tissues, respectively. Moreover, the 0690::Tn mutant failed to induce mastitis in both experimental models. Conversely, the mnuA::Tn mutant induced inflammation in murine mammary glands, characterized by neutrophil infiltration and increased expression of major inflammatory genes. In cows, the mnuA::Tn was able to cause an increase in somatic cell counts in a manner comparable to the wild type, recruit neutrophils, and induce mastitis. Collectively, these findings provide complementary insights, revealing that disruption of 5′-NT significantly attenuated M. bovis pathogenicity, whereas a MnuA-deficient mutant retained the ability to cause mastitis.

Klebsiella species commonly reside in dairy cattle guts and are consistently exposed to beta-lactam antibiotics, including ceftiofur, which are frequently used on the U.S. dairy farms. This may impose selection pressure and result in the emergence of extended-spectrum beta-lactamase (ESBL)-producing strains. However, information on the status and antimicrobial resistance (AMR) profile of ESBL- Klebsiella spp. in the U.S. dairy farms is largely unknown. This study aimed to determine the prevalence and AMR profile of ESBL- Klebsiella spp. and the factors affecting their occurrence in dairy cattle farms. Rectal fecal samples ( n = 508) and manure, feed, and water samples ( n = 64) were collected from 14 dairy farms in Tennessee. Samples were directly plated on CHROMagar ESBL, and presumptive Klebsiella spp. were confirmed using matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Antimicrobial susceptibility testing was performed on the isolates against panels of 14 antimicrobial agents from 10 classes using minimum inhibitory concentration. Of 572 samples, 57 (10%) were positive for ESBL- Klebsiella spp. The fecal prevalence of ESBL- Klebsiella spp. was 7.2% (95% CI: 6.5–8.0). The herd-level fecal prevalence of ESBL- Klebsiella spp. was 35.7% (95% CI: 12.7–64.8). The fecal prevalence of ESBL- Klebsiella spp. was significantly higher in calves than in cows and higher in cows with higher parity (≥3) as compared to cows with low parity ( P < 0.001). Most (96.5%, n = 57) ESBL- Klebsiella spp. were resistant to ceftriaxone. The highest level of acquired co-resistance to ceftriaxone in ESBL- Klebsiella spp. was to sulfisoxazole (66.7%; 38/57). About 19% of ESBL-K lebsiella spp. were multidrug resistant. The presence of ESBL-producing Klebsiella spp. in dairy cattle, feed, and water obtained from troughs could play a crucial epidemiological role in maintaining and spreading the bacteria on farms and serving as a point source of transmission. IMPORTANCE: We collected 572 samples from dairy farms, including rectal feces, manure, feed, and water. We isolated and identified extended-spectrum beta-lactamase (ESBL)- Klebsiella spp. and conducted an antimicrobial susceptibility test and analyzed different variables that may be associated with ESBL- Klebsiella spp. in dairy farms. The results of our study shed light on how ESBL- Klebsiella spp. are maintained through fecal-oral routes in dairy farms and possibly exit from the farm into the environment. We determine the prevalence of ESBL- Klebsiella spp. and their antimicrobial susceptibility profiles, underscoring their potential as a vehicle for multiple resistance gene dissemination within dairy farm settings. We also collected data on variables affecting their occurrence and spread in dairy farms. These findings have significant implications in determining sources of community-acquired ESBL- Enterobacteriaceae infections and designing appropriate control measures to prevent their spread from food animal production systems to humans, animals, and environments.

Introduction: The rising prevalence of Extended-Spectrum Beta-Lactamase (ESBL)-producing Klebsiella species (spp.) poses a significant threat to human and animal health and environmental safety. To address this pressing issue, a comprehensive study was undertaken to elucidate the burden and dissemination mechanisms of ESBL-Klebsiella spp. in dairy cattle farms.Methods: Fifty-seven Klebsiella species were isolated on CHROMagar™ ESBL plates and confirmed with MADLI-TOF MS and whole genome sequenced from 14 dairy farms.Results and discussion: Six families of beta-lactamase (bla) (bla_CTX−M, bla_SHV, bla_TEM, bla_OXY, bla_OXA, and bla_SED) were detected in ESBL-Klebsiella spp. genomes. Most (73%) of isolates had the first three types of beta-lactamase genes, with bla_SHV being the most frequent, followed by bla_CTX−M. Most (93%) isolates harbored two or more bla genes. The isolates were genotypically MDR, with 26 distinct types of antibiotic resistance genes (ARGs) and point mutations in gyrA, gyrB, and parC genes. The genomes also harbored 22 different plasmid replicon types, including three novel IncFII. The IncFII and Col440I plasmids were the most frequent and were associated with bla_CTXM−27 and qnrB19 genes, respectively. Eighteen distinct sequence types (STs), including eight isolates with novel STs of K. pneumoniae, were detected. The most frequently occurring STs were ST353 (n = 8), ST469 (n = 6), and the novel ST7501 (n = 6). Clusters of ESBL-Klebsiella strains with identical STs, plasmids, and ARGs were detected in multiple farms, suggesting possible clonal expansion. The same ESBL variant was linked to identical plasmids in different Klebsiella STs in some farms, suggesting horizontal spread of the resistance gene. The high burden and dual spread mechanism of ESBL genes in Klebsiella species, combined with the emergence of novel sequence types, could swiftly increase the prevalence of ESBL-Klebsiella spp., posing significant risks to human, animal, and environmental health. Immediate action is needed to implement rigorous surveillance and control measures to mitigate this risk.

Background: The lineage 4 (L4) of Mycobacterium tuberculosis (MTB) is not only globally prevalent but also locally dominant, surpassing other lineages, with lineage 2 (L2) following in prevalence. Despite its widespread occurrence, factors influencing the expansion of L4 and its sub-lineages remain poorly understood both at local and global levels. Therefore, this study aimed to conduct a pan-genome and identify genomic signatures linked to the elevated prevalence of L4 sublineages among extrapulmonary TB (EPTB) patients in western Ethiopia. Methods: A cross-sectional study was conducted at an institutional level involving confirmed cases of extrapulmonary tuberculosis (EPTB) patients from August 5, 2018, to December 30, 2019. A total of 75 MTB genomes, classified under lineage 4 (L4), were used for conducting pan-genome and genome-wide association study (GWAS) analyses. After a quality check, variants were identified using MTBseq, and genomes were de novo assembled using SPAdes. Gene prediction and annotation were performed using Prokka. The pan-genome was constructed using GET_HOMOLOGUES, and its functional analysis was carried out with the Bacterial Pan-Genome Analysis tool (BPGA). For GWAS analysis, Scoary was employed with Benjamini-Hochberg correction, with a significance threshold set at p-value ≤ 0.05. Results: The analysis revealed a total of 3,270 core genes, predominantly associated with orthologous groups (COG) functions, notably in the categories of ‘[R] General function prediction only’ and ‘[I] Lipid transport and metabolism’. Conversely, functions related to ‘[N] Cell motility’ and ‘[Q] Secondary metabolites biosynthesis, transport, and catabolism’ were primarily linked to unique and accessory genes. The pan-genome of MTB L4 was found to be open. Furthermore, the GWAS study identified genomic signatures linked to the prevalence of sublineages L4.6.3 and L4.2.2.2. Conclusions: Apart from host and environmental factors, the sublineage of L4 employs distinct virulence factors for successful dissemination in western Ethiopia. Given that the functions of these newly identified genes are not well understood, it is advisable to experimentally validate their roles, particularly in the successful transmission of specific L4 sublineages over others.

Mycoplasma bovis has recently been identified increasingly in dairy cows causing huge economic losses to the dairy industry. M. bovis is a causative agent for mastitis, pneumonia, endometritis, endocarditis, arthritis, otitis media, and many other clinical symptoms in cattle. However, some infected cows are asymptomatic or may not shed the pathogen for weeks to years. This characteristic of M. bovis, along with the lack of adequate testing and identification methods in many parts of the world until recently, has allowed the M. bovis to be largely undetected despite its increased prevalence in dairy farms. Due to growing levels of antimicrobial resistance among wild-type M. bovis isolates and lack of cell walls in mycoplasmas that enable them to be intrinsically resistant to beta-lactam antibiotics that are widely used in dairy farms, there is no effective treatment for M. bovis mastitis. Similarly, there is no commercially available effective vaccine for M. bovis mastitis. The major constraint to developing effective intervention tools is limited knowledge of the virulence factors and mechanisms of the pathogenesis of M. bovis mastitis. There is lack of quick and reliable diagnostic methods with high specificity and sensitivity for M. bovis. This review is a summary of the current state of knowledge of the virulence factors, pathogenesis, clinical manifestations, diagnosis, and control of M. bovis mastitis in dairy cows.

Escherichia coli O157:H7, Salmonella and Staphylococcus aureus are common foodborne pathogens. We determined the prevalence of E. coli O157:H7 and Salmonella in feces and milk and the prevalence of S. aureus in milk from dairy cattle and camels in the Borana pastoral community in the Southern Oromia Region of Ethiopia. Paired individual cow composite (pooled from all quarters in equal proportions) milk and fecal samples were collected from cows (n = 154) and camels (n = 158). Samples were cultured on bacterial isolation and identification media. E. coli O157:H7 and Salmonella isolates were further tested for susceptibility against nine antimicrobial drugs. Different risk factors associated with hygienic milking practices were recorded and analyzed for their influence on the prevalence of these bacteria in milk and feces. The prevalence of E. coli O157:H7 and Salmonella in feces were 3.9% and 8.4%, respectively, in cows, and 0.6% and 2.5%, respectively, in camels. E. coli O157:H7 and Salmonella were detected in the composite milk samples of 2.6% and 3.9% of the cows, respectively, and 0% and 1.3% of the camels, respectively. S. aureus was detected in composite milk samples of 33.4% of the cows and 41.7% of the camels. All E. coli O157:H7 (n = 11) and Salmonella (n = 25) isolates from both animal species and sample types were resistant to at least one antimicrobial drug. Multidrug resistance was observed in 70% (7/10) of the E. coli O157:H7 fecal and milk isolates from cows and 33.3% (2/6) of the Salmonella fecal and milk isolates from camels. The prevalence of these bacteria in feces and milk was not affected by risk factors associated with milking practices. Given the very close contact between herders and their animals and the limited availability of water for hand washing and udder cleaning, these bacteria are most likely present in all niches in the community. Improving community awareness of the need to boil milk before consumption is a realistic public health approach to reducing the risk of these bacteria.

Introduction: The extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae, such as Escherichia coli, are emerging as a serious threat to global health due to their rapid spread and their multidrug-resistant (MDR) phenotypes. However, limited information is available regarding the prevalence and antimicrobial resistance (AMR) profile of ESBL-E. coli in the United States dairy farms. This study aimed to determine the prevalence and AMR pattern of ESBL-E. coli in East Tennessee dairy cattle farms.Methods: Rectal fecal samples from dairy cattle (n = 508) and manure (n = 30), water (n = 19), and feed samples (n = 15) were collected from 14 farms. The presumptive E. coli was isolated on CHROMagar™ ESBL and confirmed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). Antimicrobial susceptibility testing was performed on the ESBL-E. coli isolates.Results and discussion: From 572 fecal and farm environmental samples, a total of 233 (41%, n = 572) ESBL-E. coli were identified. The prevalence of fecal ESBL-E. coli was 47.5% (95% CI: 46.2–49.2). The within-farm prevalence of ESBL-E. coli ranged from 8 to 100%. Recent treatment history with third-generation cephalosporins (3GC), cow parity ≥3, and calves were the independent risk factors associated (P < 0.05) with fecal carriage of ESBL-E. coli. Overall, 99.6% (n = 231) ESBL-E. coli tested were phenotypically resistant to at least one of the 14 antimicrobial agents tested. The most common AMR phenotypes were against beta-lactam antibiotics, ampicillin (99.1%; n = 231 isolates), and ceftriaxone (98.7%, n = 231). Most ESBL-E. coli isolates (94.4%) were MDR (resistance to ≥3 antimicrobial classes), of which 42.6% showed co-resistance to at least six classes of antimicrobials. ESBL-E. coli isolates with concurrent resistance to ceftriaxone, ampicillin, streptomycin, tetracycline, sulfisoxazole, and chloramphenicol are widespread and detected in all the farms. The detection of MDR ESBL-E. coli suggests that dairy cattle can be a reservoir for these bacteria, highlighting the associated public health risk.