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Development and evaluation of a triplex droplet digital PCR method for differentiation of , and BCG.Frontiers in Microbiology 2024Tuberculosis, caused by complex (MTBC), remains a global health concern in both human and animals. However, the absence of rapid, accurate, and highly sensitive...
INTRODUCTION
Tuberculosis, caused by complex (MTBC), remains a global health concern in both human and animals. However, the absence of rapid, accurate, and highly sensitive detection methods to differentiate the major pathogens of MTBC, including , , and BCG, poses a potential challenge.
METHODS
In this study, we have established a triplex droplet digital polymerase chain reaction (ddPCR) method employing three types of probe fluorophores, with targets (targeting CFP-10-ESAT-6 gene of RD1 and Rv0222 genes of RD4), (targeting CFP-10-ESATs-6 gene of RD1), and BCG (targeting Rv3871 and Rv3879c genes of ΔRD1), respectively.
RESULTS
Based on optimization of annealing temperature, sensitivity and repeatability, this method demonstrates a lower limit of detection (LOD) as 3.08 copies/reaction for , 4.47 copies/reaction for and 3.59 copies/reaction for BCG, without cross-reaction to , , , , , , , , and , and showed repeatability with coefficients of variation (CV) lower than 10%. The method exhibits strong milk sample tolerance, the LOD of detecting in spike milk was 5 × 10 CFU/mL, which sensitivity is ten times higher than the triplex qPCR. 60 clinical DNA samples, including 20 milk, 20 tissue and 20 swab samples, were kept in China Animal Health and Epidemiology Center were tested by the triplex ddPCR and triplex qPCR. The triplex ddPCR presented a higher sensitivity (11.67%, 7/60) than that of the triplex qPCR method (8.33%, 5/60). The positive rates of , , and BCG were 1.67, 10, and 0% by triplex ddPCR, and 1.67, 6.67, and 0% by triplex qPCR, with coincidence rates of 100, 96.7, and 100%, respectively.
DISCUSSION
Our data demonstrate that the established triplex ddPCR method is a sensitive, specific and rapid method for differentiation and identification of , , and BCG.
PubMed: 38946908
DOI: 10.3389/fmicb.2024.1397792 -
Microbial Pathogenesis Jun 2024Close contact between cats and humans increases the risk of transmission of zoonotic pathogens, through bites and scratches due to the complexity of microorganisms in...
Close contact between cats and humans increases the risk of transmission of zoonotic pathogens, through bites and scratches due to the complexity of microorganisms in the oral and nail microbiotas of felines. This study investigated the presence of bacteria and fungi in the oral cavity and claws of 100 apparently healthy cats using conventional and selective microbiological culture media, and next-generation sequencing (NGS) and mass spectrometry (MALDI-TOF MS). Furthermore, antimicrobial susceptibility testing of bacteria isolates was performed by disc diffusion method. In total, 671 bacteria and 33 yeasts were identified by MALDI-TOF MS. Neisseria animaloris (10.8%), Staphylococcus felis (8.5%), and Pasteurella multocida (7%) were the most prevalent bacteria in oral cavity samples (n=343), while the most common yeast (n=19) was Candida albicans (68.4%). Staphylococcus pettenkoferi (13.4%), Staphylococcus felis (6.4%), and Staphylococcus simulans (5.8%) were the prevalent bacteria identified in the claw samples (n=328), while Rhodotorula mucilaginosa (57.2%) was the most common yeast (n=14). NGS predominantly identified the genera Moraxella, Neisseria, Pasteurella, and Fusobacterium in oral cavity samples, whereas enterobacteria and staphylococci were prevalent in nail bed samples. In addition, the genera Capnocytophaga and Bartonella were identified, which have been described in serious human infections secondary to feline aggressions. Levofloxacin, marbofloxacin, and amoxicillin/clavulanic acid were the most effective drugs against the main groups of bacteria identified. Multidrug resistance was observed in 17% of the bacterial isolates. Furthermore, three staphylococci harboring the methicillin resistance gene mecA were identified. We highlight the complexity of microorganisms inhabiting the oral/claw microbiotas of cats, the high resistance rate of the isolates to conventional antimicrobial agents, and the zoonotic risk of aggressions caused by bites and scratches from domestic cats.
PubMed: 38944215
DOI: 10.1016/j.micpath.2024.106765 -
Frontiers in Veterinary Science 2024An increase in chronic, non-responsive bovine respiratory disease (BRD) infections in North American feedlot cattle is observed each fall, a time when cattle are... (Review)
Review
An increase in chronic, non-responsive bovine respiratory disease (BRD) infections in North American feedlot cattle is observed each fall, a time when cattle are administered multiple antimicrobial treatments for BRD. A number of factors are responsible for BRD antimicrobial treatment failure, with formation of biofilms possibly being one. It is widely accepted that biofilms play a role in chronic infections in humans and it has been hypothesized that they are the default lifestyle of most bacteria. However, research on bacterial biofilms associated with livestock is scarce and significant knowledge gaps exist in our understanding of their role in AMR of the bacterial BRD complex. The four main bacterial species of the BRD complex, , , , and are able to form biofilms and there is evidence that at least retains this ability . However, there is a need to elucidate whether their biofilm-forming ability contributes to pathogenicity and antimicrobial treatment failure of BRD. Overall, a better understanding of the possible role of BRD bacterial biofilms in clinical disease and AMR could assist in the prevention and management of respiratory infections in feedlot cattle. We review and discuss the current knowledge of BRD bacteria biofilm biology, study methodologies, and their possible relationship to AMR.
PubMed: 38933702
DOI: 10.3389/fvets.2024.1353551 -
Microorganisms May 2024Respiratory diseases arising from co-infections involving () and (Mo) pose a substantial threat to the sheep industry. This study focuses on the isolation and...
Respiratory diseases arising from co-infections involving () and (Mo) pose a substantial threat to the sheep industry. This study focuses on the isolation and identification of the strain extracted from the lung tissue of an argali hybrid sheep infected with Mo. Kunming mice were used as a model to assess the pathogenicity of . Subsequently, whole genome sequencing (WGS) of was conducted using the Illumina NovaSeq PE150 platform. The whole genome sequencing analysis involved the construction of an evolutionary tree to depict conserved genes and the generation of a genome circle diagram. identified as serotype A, was named SHZ01. Our findings reveal that SHZ01 infection induces pathological manifestations, including hemorrhage and edema, in mice. The phylogenetic tree of conserved genes analyzing from different countries and different host sources indicates close relatedness between the SHZ01 strain and the 40540 strain (A:12), originating from turkeys in Denmark. The genome of SHZ01 comprises 2,378,508 base pairs (bp) with a GC content of 40.89%. Notably, this strain, designated , exhibits two distinct gene islands and harbors a total of 80 effector proteins associated with the Type III Secretion System (T3SS). The SHZ01 strain harbors 82 virulence genes and 54 resistance genes. In the SHZ01 strain, the proteins, genes, and related GO and KEGG pathways have been annotated. Exploring the relationship between these annotations and the pathogenicity of the SHZ01 strain would be valuable. This study holds great significance in further understanding the pathogenesis and genetic characteristics of the sheep-derived SHZ01 strain. Additionally, it contributes to our understanding of respiratory diseases in the context of co-infection.
PubMed: 38930454
DOI: 10.3390/microorganisms12061072 -
Journal of Applied Microbiology Jun 2024This study aimed to assess the pharmacokinetic/pharmacodynamic (PK/PD) targets of danofloxacin to minimize the risk of selecting resistant P. multocida mutants and to...
AIMS
This study aimed to assess the pharmacokinetic/pharmacodynamic (PK/PD) targets of danofloxacin to minimize the risk of selecting resistant P. multocida mutants and to identify the mechanisms underlying their resistance in an in vitro dynamic model, attaining the optimum dosing regimen of danofloxacin to improve its clinical efficacy based on the mutant selection window (MSW) hypothesis.
METHODS AND RESULTS
Danofloxacin at seven dosing regimens and five days of treatment were simulated to quantify the bactericidal kinetics and enrichment of resistant mutants upon continuous antibiotic exposure. The magnitudes of PK/PD targets associated with different efficacies were determined in the model. The 24 h danofloxacin area under the concentration-time curve to MIC ratios (AUC24h/MIC) associated with bacteriostatic, bactericidal and eradication effects against P. multocida were 34, 52, and 64 h. This translates to average danofloxacin concentrations (Cav) over 24 h being 1.42, 2.17, and 2.67 times the MIC, respectively. An AUC/MIC-dependent antibacterial efficacy and AUC/MPC (mutant prevention concentration)-dependent enrichment of P. multocida mutants in which maximum losses in danofloxacin susceptibility occurred at a simulated AUC24h/MIC ratio of 72 h (i.e. Cav of 3 times the MIC). The overexpression of efflux pumps (acrAB-tolC) and their regulatory genes (marA, soxS, and ramA) was associated with reduced susceptibility in danofloxacin-exposed P. multocida. The AUC24h/MPC ratio of 19 h (i.e. Cav of 0.8 times the MPC) was determined to be the minimum mutant prevention target value for the selection of resistant P. multocida mutants.
CONCLUSIONS
The emergence of P. multocida resistance to danofloxacin exhibited a concentration-dependent pattern and was consistent with the MSW hypothesis. The current clinical dosing regimen of danofloxacin (2.5 mg kg-1) may have a risk of treatment failure due to inducible fluoroquinolone resistance.
PubMed: 38925653
DOI: 10.1093/jambio/lxae154 -
Pathogens (Basel, Switzerland) Jun 2024This study aimed to enhance our understanding of the agreement between two sampling methods for the detection of bovine respiratory disease (BRD) pathogens in calves...
Comparing Occurrence of Bovine Respiratory Pathogens Detected by High-Throughput Real-Time PCR in Nasal Swabs and Non-Endoscopic Bronchoalveolar Lavage Samples from Dairy and Veal Calves.
This study aimed to enhance our understanding of the agreement between two sampling methods for the detection of bovine respiratory disease (BRD) pathogens in calves using high-throughput real-time qPCR (ht-RT-qPCR). In total, 233 paired nasal swab (NS) and non-endoscopic bronchoalveolar lavage (nBAL) samples were collected from 152 calves from 12 Danish cattle herds. In 202 of the observations, the calves were examined using a standardized clinical protocol. Samples were tested for three viruses (bovine respiratory syncytial virus, bovine corona virus, and influenza D virus) and six bacteria (, , , Mycoplasma species, , and ). The results showed age-related differences in disease and pathogen occurrence, with the highest detection rates in calves aged 35 days or older. Poor to moderate agreement was found between the NS and nBAL results. The presence of in both NS and nBAL in younger calves and in nBAL in older calves was associated with clinical BRD. There was a potential link between BRD and influenza D virus in older calves, although it was only found in one herd in a small sample size. Overall, NS was a relatively poor predictor of pathogens in the lower respiratory tract. The present study confirms the complexity of pathogen detection in BRD, with marked influences of age and the sampling method on pathogen detection and disease associations.
PubMed: 38921777
DOI: 10.3390/pathogens13060479 -
Emerging Infectious Diseases Jul 2024Pasteurella bettyae is a gram-negative bacillus sporadically involved in human infections; its main reservoirs are cats and dogs. A recent publication suggests the...
Pasteurella bettyae is a gram-negative bacillus sporadically involved in human infections; its main reservoirs are cats and dogs. A recent publication suggests the possibility of sexual transmission leading to genital infections in men who have sex with men. We report 9 cases in France of genital infection among this population.
Topics: Humans; Male; France; Adult; Homosexuality, Male; Pasteurella Infections; Pasteurella; Middle Aged; Young Adult
PubMed: 38916800
DOI: 10.3201/eid3007.240352 -
MSystems Jun 2024The development and growth of animals coincide with the establishment and maturation of their microbiotas. To evaluate the respiratory and fecal microbiotas of beef...
The development and growth of animals coincide with the establishment and maturation of their microbiotas. To evaluate the respiratory and fecal microbiotas of beef calves from birth to weaning, a total of 30 pregnant cows, and their calves at birth, were enrolled in this study. Deep nasal swabs and feces were collected from calves longitudinally, starting on the day of birth and ending on the day of weaning. Nasopharyngeal, vaginal, and fecal samples were also collected from cows, and the microbiotas of all samples were analyzed. The fecal microbiota of calves was enriched with during the first 8 weeks of life, before being displaced by genera associated with fiber digestion, and then increasing in diversity across time. In contrast, the diversity of calf respiratory microbiota generally decreased with age. At birth, the calf and cow nasal microbiotas were highly similar, indicating colonization from dam contact. This was supported by microbial source-tracking analysis. The structure of the calf nasal microbiota remained similar to that of the cows, until weaning, when it diverged. The changes were driven by a decrease in and an increase in genera typically associated with bovine respiratory disease, including , , and . These three genera colonized calves early in life, though was initially transferred from the cow reproductive tract. Path analysis was used to model the interrelationships of calf respiratory and fecal microbiotas. It was observed that respiratory and fecal UCG-005 negatively affected the abundance of or .IMPORTANCEIn beef cattle production, bovine respiratory disease (BRD) accounts for most of the feedlot morbidities and mortalities. Metaphylaxis is a common management tool to mitigate BRD, however its use has led to increased antimicrobial resistance. Novel methods to mitigate BRD are needed, including microbiota-based strategies. However, information on the respiratory bacteria of beef calves prior to weaning was limited. In this study, it was shown that the microbiota of cows influenced the initial composition of both respiratory and fecal microbiotas in calves. While colonization of the respiratory tract of calves by BRD-associated genera occurred early in life, their relative abundances increased at weaning, and were negatively correlated with respiratory and gut bacteria. Thus, microbiotas of both the respiratory and gastrointestinal tracts have important roles in antagonism of respiratory pathogens and are potential targets for enhancing calf respiratory health. Modulation may be most beneficial, if done prior to weaning, before opportunistic pathogens establish colonization.
PubMed: 38899874
DOI: 10.1128/msystems.00238-24 -
Animals : An Open Access Journal From... May 2024Pasteurellosis, a disease caused by , is responsible for economic losses in rabbit industrial farms due to rhinitis, conjunctivitis, pneumonia, metritis, mastitis,...
Pasteurellosis, a disease caused by , is responsible for economic losses in rabbit industrial farms due to rhinitis, conjunctivitis, pneumonia, metritis, mastitis, orchitis, subcutaneous abscesses, otitis, encephalitis, and septicaemic forms. Although the occurrence of the disease is conditioned by predisposing factors that affect the rabbit immune response, the strains of involved in the infection may have a different pathogenic ability. Therefore, typing of strains spread among the rabbits is important to assess their pathogenic potential. The aim of this study is to investigate the strains responsible for disease in rabbit industrial farms. A total of 114 strains identified from different lesions were serotyped. Additionally, the presence of virulence-associated genes was investigated using three PCR (polymerase chain reaction) protocols. Capsular type A was prevalently found in strains from respiratory lesions while types D and F in those from metritis, mastitis, and other lesions. Different associations between some virulence-associated genes and both capsular type and lesions found in rabbits were detected. The presence of 8 virulence-associated genes seems to increase the occurrence of metritis. In addition, strains belonging to capsular type A and responsible for respiratory disorders especially, were found equipped with 10 and 11 virulence-associated genes. Nevertheless, the presence of strains responsible only for rhinitis was also detected among the latter, suggesting that the pathogenic ability of the bacteria depends on the expression rather than the presence of a gene.
PubMed: 38891615
DOI: 10.3390/ani14111569 -
Cureus May 2024This report describes a patient with pneumonia. The patient was a man in his 70s with significant comorbid conditions, including chronic obstructive pulmonary disease...
Successful Management of Pasteurella multocida Pneumonia in a Chronic Obstructive Pulmonary Disease Patient: A Case Report Highlighting the Importance of Tailored Antibiotic Therapy.
This report describes a patient with pneumonia. The patient was a man in his 70s with significant comorbid conditions, including chronic obstructive pulmonary disease (COPD), and is an example of the diverse presentations of infections increasingly found in the literature. The novelty of this case lies in the manifestation of pneumonia in a patient with underlying respiratory conditions and its successful management, outlining a unique clinical scenario and a tailored therapeutic approach. A 71-year-old male with a medical history of COPD, asthma, tremors, hypertension, and arthritis presented to the emergency department with progressive shortness of breath, productive cough, and chest tightness. The initial diagnosis was COPD exacerbation and left lower lobe pneumonia, for which a regimen of ceftriaxone and azithromycin was initiated. The patient's condition was further complicated by the persistence of symptoms. Following sputum culture analysis, infection was identified. Consequently, the antibiotic regimen was tailored, transitioning the patient to doxycycline, which led to substantial clinical improvement, enabling discharge with a 10-day course of oral doxycycline. This case elucidates the importance of precise microbiological diagnosis in patients with complex respiratory conditions, as it guides more targeted antibiotic therapy. It highlights the need for clinical vigilance for atypical pathogens like in patients with COPD exacerbations, especially when conventional treatment strategies yield suboptimal responses. The successful resolution of the pneumonia underscores the effectiveness of antibiotic stewardship guided by sputum culture findings.
PubMed: 38868278
DOI: 10.7759/cureus.60210