Mycobacterium Avium Isolates Research Articles

Isolation of Mycobacterium avium ssp. paratuberculosis and other non-tuberculous mycobacteria from head lymph nodes of wild ruminants and badgers in Switzerland.

The family Mycobacteriaceae contains over 188 species, most of which are saprophytic non-tuberculous mycobacteria (NTM). In wildlife, a variety of different NTM can be found, with different reports about their pathogenic potential. A pathogenic member of NTM is Mycobacterium avium ssp. paratuberculosis (MAP), which can infect farmed and wild ruminants. It causes paratuberculosis which is an economically important chronic disease. Infected farm animals are considered to be the source of infection in wild animals. Wildlife, on the other hand, is thought to be a reservoir for certain members of the Mycobacterium tuberculosis complex (MTBC), such as M. caprae, which causes tuberculosis in cattle and red deer. Switzerland implemented a surveillance program for tuberculosis in wild animals in 2014. Here, we describe the results from the mycobacterial culture of lymph node samples collected from red deer, roe deer, chamois, ibex, and badgers collected within this surveillance program from 2020 to 2022. Overall, samples from 548 animals were checked macroscopically for tuberculosis-like lesions. In total, 88 animals (16.1%), which either had lesions in their lymph nodes or were male and aged older than 5 years, were investigated using mycobacterial culture. In total, 25 animals (28.4%) were positive for NTM, while no MTBC was detected. The most often identified NTM was M. vaccae, followed by M. avium. Most animals positive for NTM did not show any macroscopic lesions. Furthermore, MAP was isolated from the head lymph nodes of two male red deer. Neither of the two MAP-positive animals had any macroscopic lesions in their head lymph nodes or any other signs of disease. The shooting sites of the two MAP-positive animals were located in Alpine pastures used for grazing of cattle during summer, which confirms that species transmission can occur when contaminated pastures are used by different species. In agreement with other studies, the occurrence of MAP in red deer was quite low. However, so far, MAP was mostly isolated from feces and intestinal lymph nodes of wild animals. This is the first detection of MAP in the head lymph nodes of red deer in Switzerland.

In Vitro Effect of Three-Antibiotic Combinations plus Potential Antibiofilm Agents against Biofilm-Producing Mycobacterium avium and Mycobacterium intracellulare Clinical Isolates.

Patients with chronic pulmonary diseases infected by Mycobacterium avium complex (MAC) often develop complications and suffer from treatment failure due to biofilm formation. There is a lack of correlation between in vitro susceptibility tests and the treatment of clinical isolates producing biofilm. We performed susceptibility tests of 10 different three-drug combinations, including two recommended in the guidelines, in biofilm forms of eight MAC clinical isolates. Biofilm developed in the eight isolates following incubation of the inoculum for 3 weeks. Then, the biofilm was treated with three-drug combinations with and without the addition of potential antibiofilm agents (PAAs). Biofilm bactericidal concentrations (BBCs) were determined using the Vizion lector system. All selected drug combinations showed synergistic activity, reducing BBC values compared to those treated with single drugs, but BBC values remained high enough to treat patients. However, with the addition of PAAs, the BBCs steadily decreased, achieving similar values to the combinations in planktonic forms and showing synergistic activity in all the combinations and in both species. In conclusion, three-drug combinations with PAAs showed synergistic activity in biofilm forms of MAC isolates. Our results suggest the need for clinical studies introducing PAAs combined with antibiotics for the treatment of patients with pulmonary diseases infected by MAC.

Early-stage findings in an experimental calf model infected with Argentinean isolates of Mycobacterium avium subsp. paratuberculosis

Mycobacterium avium subsp. paratuberculosis (MAP) is an important pathogen that causes granulomatous enteritis known as Johne’s disease or paratuberculosis (PTB). In this study an experimental model of calves infected with Argentinean isolates of MAP for 180 days was used to provide more data of the early PTB stages. Calves were challenged by oral route with MAP strain IS900-RFLPA (MA; n = 3), MAP strain IS900-RFLPC (MC; n = 2) or mock infected (MI; n = 2), and response to infection was evaluated through peripheral cytokine expression, MAP tissue distribution and histopathological early-stage findings. Specific and varied levels of IFN-γ were only detected at 80 days post-infection in infected calves. These data indicate that specific IFN-γ is not a useful indicator for early detection of MAP infection in our calf model. At 110 days post-infection, TNF-α expression was higher than IL-10 in 4 of the 5 infected animals and a significant decrease of TNF-α expression was detected in infected vs. non-infected calves. All calves challenged were identified as infected by mesenteric lymph node tissue culture and real time IS900 PCR. In addition, for lymph nodes samples, the agreement between these techniques was almost perfect (κ = 0.86). Colonization of tissues and levels of tissue infection varied between individuals. Evidence of early MAP dissemination to extraintestinal tissues such as the liver was detected by culture in one animal (MAP strain IS900-RFLPA). In both groups microgranulomatous lesions were observed predominantly in the lymph nodes, with giant cells present only in the MA group. In summary, the findings described herein may indicate that local MAP strains induced specific immune responses with particularities that could suggest differences in their biological behavior. Further studies should be carried out in order to obtain an in-depth understanding of the influence of MAP strains in host-pathogen interactions and the outcome of disease.

Letter to the Editor: In Vitro Activity of Oxazolidinone Against Nontuberculous Mycobacteria in China.

Letter to the Editor: In Vitro Activity of Oxazolidinone Against Nontuberculous Mycobacteria in China.

Differences in Drug-Susceptibility Patterns between Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium chimaera Clinical Isolates: Prospective 8.5-Year Analysis by Three Laboratories.

Background: It has been suggested that Mycobacterium avium, Mycobacterium intracellulare, and M. chimaera have differential drug susceptibility patterns. We prospectively analyzed and compared the drug susceptibility patterns among these species over an 8.5-year period. Methods: A microdilution method (Slomyco®) was performed for drug susceptibility testing of 402 M. avium, 273 M. intracellulare, and 139 M. chimaera clinical isolates. Results: M. avium showed significantly higher resistance to moxifloxacin, ciprofloxacin, rifampicin, ethambutol, streptomycin, linezolid, cotrimoxazole, and clarithromycin. M. avium also showed higher minimum inhibitory concentrations (MIC) than M. intracellulare and M. chimaera against all drugs except ethionamide, to which M. intracellulare and M. chimaera showed greater resistance. Conclusions: Our series demonstrated differential drug resistance patterns among the most frequent M. avium complex species. M. avium was more resistant than M. intracellulare and M. chimaera versus eight antibiotics and showed greater MIC values to most of the antibiotics studied. These data suggest that knowledge of the local distribution and susceptibility profiles of these pathogens is essential for adequate clinical management.

Immunogenicity of chitosan coupled heat killed and fusion protein vaccine of Mycobacterium avium subsp. paratuberculosis

In search for better vaccine candidates for Johne's disease, chitosan nanoparticle coupled heat killed whole cell and fusion protein (Antigen 85A+85B) vaccines were developed using a local isolate of Mycobacterium avium subsp. paratuberculosis. Immunization studies in goats indicated that the heat killed whole cell vaccine and the fusion protein vaccine were found to be safe and resulted in Th1 and Th2 responses. Antibody response was significantly higher in the heat killed whole cell immunized animals than those received the fusion protein. Significant increase in heat killed whole cell MAP and fusion protein specific IFN-γ gene expression was detected in the immunized animals in contrast to the controls. The response was significantly higher with the heat killed whole cell MAP than the fusion protein. The adjuvant effect of chitosan nanoparticles was clearly evident in the second trial wherein the animals were observed for a longer period of 24 weeks. With increased IFN-γ and antibody levels, vaccinated animals responded well as compared to the control ones and the response was better in heat killed whole cell vaccine.

Subunit vaccine protects against a clinical isolate of Mycobacterium avium in wild type and immunocompromised mouse models

The nontuberculous mycobacteria (NTM) Mycobacterium avium is a clinically significant pathogen that can cause a wide range of maladies, including tuberculosis-like pulmonary disease. An immunocompromised host status, either genetically or acutely acquired, presents a large risk for progressive NTM infections. Due to this quietly emerging health threat, we evaluated the ability of a recombinant fusion protein ID91 combined with GLA-SE [glucopyranosyl lipid adjuvant, a toll like receptor 4 agonist formulated in an oil-in-water stable nano-emulsion] to confer protection in both C57BL/6 (wild type) and Beige (immunocompromised) mouse models. We optimized an aerosol challenge model using a clinical NTM isolate: M. avium 2-151 smt, observed bacterial growth kinetics, colony morphology, drug sensitivity and histopathology, characterized the influx of pulmonary immune cells, and confirmed the immunogenicity of ID91 in both mouse models. To determine prophylactic vaccine efficacy against this M. avium isolate, mice were immunized with either ID91 + GLA-SE or bacillus Calmette–Guérin (BCG). Immunocompromised Beige mice displayed a delayed influx of innate and adaptive immune cells resulting in a sustained and increased bacterial burden in the lungs and spleen compared to C57BL/6 mice. Importantly, both ID91 + GLA-SE and BCG vaccines significantly reduced pulmonary bacterial burden in both mouse strains. This work is a proof-of-concept study of subunit vaccine-induced protection against NTM.

Isolation of Mycobacterium avium and other nontuberculous mycobacteria in chickens and captive birds in peninsular Malaysia

BackgroundMycobacterium avium complex (MAC) causes a chronic infectious in the birds known as avian mycobacteriosis. Almost all species of the birds are susceptible to MAC which consists of two closely related species of mycobacteria, that is, M. avium and M. intracellulare. This study aimed to determine the occurrence of Mycobacterium avium subsp. avium (MAA) in chickens and captive birds in selected states of Peninsular Malaysia.ResultsA 300 fecal samples were collected from village chickens (n = 100), layer chickens (n = 100) and captive birds (n = 100). Fecal samples were split into two aliquots for microbiological and molecular detection of MAA. Microbiology detection consisted of microscopy (Ziehl-Neelsen staining) and culture of samples decontaminated with 1% Cetylperidinium chloride and vancomycin, nalidixic acid and amphotericin B (VNA) antibiotic cocktail [vancomycin (VAN) 100 μg/ml, nalidixic acid (NAL) 100 μg/ml and amphotericin B (AMB) 50 μg/ml] onto Löwenstein-Jensen (L-J). Molecular detection (PCR-IS901) was performed to detect MAA DNA from the feces and PCR-16S rRNA and IS901 for identification of genus Mycobacterium and Mycobacterium avium sub species avium isolated onto L-J. All samples (296) were AFB negative smear. M. avium was isolated in 0.3% (1/296) samples by culture and detected in 2.5% (6/242) samples by PCR (IS901). Other mycobacteria were found in 1.7% (5/296) chickens. Of five isolates, two were identified as Mycobacterium terrae and M. engbaekii and remaining isolates were not sequenced. Birds positive for M. avium included White Pelican (n = 1) Black Hornbill (n = 1), Macaw (n = 2), Cockatoo (n = 2) and village chicken (n = 1).ConclusionIt is concluded that chickens and birds were infected with M. avium in selected areas of Peninsular Malaysia. Although, PCR is rapid, reliable and cost effective method for detection of M. avium in a subclinical stage, the culture of the avian feces should still be used as a reference test for the diagnosis of avian tuberculosis.

1654. Evaluation of a rapid detection method of clarithromycin resistance genes in Mycobacterium avium using the Amplification Refractory Mutation System-Loop-Mediated Isothermal Amplification method

BackgroundClarithromycin (CLR) is the key drug in multidrug therapy for Mycobacterium avium complex (MAC) diseases and the only drug for which drug susceptibility is correlated with a clinical response in these diseases. In the case of CLR-resistant MAC, a point mutation is present at either position 2058 or 2059 of the peptidyl transferase active center in the domain V region of 23S rRNA at the macrolide binding site. Using conventional investigation, we clarified the correlation between drug susceptibility testing and mutation of drug resistance genes. In this study, we adapted a rapid detection method using the amplification refractory mutation system (ARMS)-loop-mediated isothermal amplification (LAMP) to identify a mutation in the 23S rRNA gene in M. avium isolates (Figure 1). Furthermore, we evaluated the usefulness as point-of-care testing (POCT) technology using clinical isolates.Figure 1. The designs of CLR resistance A2058G mutant-type mismatch primers used for the ARMS-LAMP assay. a) A strand-displacing DNA polymerase extends the DNA from FIP while separating from the DNA chain. The primer F3 binds to its complementary region on the DNA to displace the newly synthesized DNA. An analogous reaction is performed by BIP and B3. α (α = A, wild type; G, A2058G) and β (β = A, wild type; C, A2058G) are indicated by the point mutation at position 2058 of the 23S rRNA gene. The bold area indicates the mismatched base C (cytosine). b) The synthesized DNA self-anneals because of the complementary region at both ends and forms ‘dumbbell’ structures. c) After repeated rounds, a complementary region on the same chain is amplified. MethodsPrimers for ARMS-LAMP were designed using PrimerExplorerV5 software based on the nucleotide sequence data for 23S rRNA in M. avium strain 104 (Figure 2). Using the minimum inhibitory concentration of CLR, drug susceptibility was determined for 18 clinical M. avium isolates. Of these, eight CLR-susceptible and 10 CLR-resistant strains were analyzed by sequencing the 23S rRNA gene and ARMS-LAMP.Figure 2. Alignment of the nucleotide sequences including the domain V region of 23S rRNA at the macrolide binding site. The constructed LAMP primer sets are shown in solid boxes (forward primers, F1-3) and dashed boxes (backward primers, B1-3). The bold area indicates the point mutation at position 2058 or 2059 of the 23S rRNA gene. ResultsSequence analysis revealed that all eight CLR-sensitive strains tested were wild type, whereas all 10 CLR-resistant strains were mutants. Using ARMS-LAMP, no amplification with the mutant-type mismatch primer sets (MTPS) was observed in the eight wild-type strains, but amplification was observed with MTPS in the 10 mutant strains (Table 1).Table 1. MICs of CLR and results of ARMS–LAMP using Mycobacterium avium isolates. ConclusionThe developed rapid detection method for the CLR resistance gene using ARMS-LAMP can determine drug resistance in a few hours without the need for special equipment. ARMS-LAMP may be a new clinically beneficial POCT technology for examination that is novel and extremely practical.Disclosures All Authors: No reported disclosures

Isolation and molecular typing of Mycobacterium avium subsp. paratuberculosis from faeces of dairy cows.

Mycobacterium avium subsp. paratuberculosis (MAP) is the cause of paratuberculosis mainly in domestic and wild ruminants; paratuberculosis is also known as Johne's disease. This disease is endemic all over the world generating significant economic losses, especially in dairy herds, although, MAP is the cause of infection in many other species including primates. Currently, MAP mycobacteria are recognized as pathogens transmitted by food. They are a potential threat to animal and human health. Infected animals excreting mycobacteria with faeces are the main source of MAP. The development of control strategies and disease control are based on determination of the genetic diversity of the MAP strains causing Johne's disease. This study describes 43 strains isolated from a herd of dairy cows located in northern Poland. The types of MAP were determinted based on the polymorphism analysis of two insertion fragments: IS900 and IS1311. The polymorphism of IS900 was analyzed with the use of a PCR multiplex according to Collins' method and the IS1311 polymorphism with the use of the PCR-REA method. Based on the differences observed, the strains isolated were classified into two MAP types, cattle (C-type) and sheep (S-type), with the predominance of the cattle type.

Molecular isolation and identification of Mycobacterium avium subsp. hominissuis in Didelphis virginiana from Hidalgo, Mexico

Objective: To isolate and identify the exact species of the genus Mycobacterium from Didelphis (D.) virginiana, and the direct implications of this bacterium to public health and veterinary medicine. Methods: Thirty-one D. virginiana were captured and necropsied in Hidalgo, Mexico. Tissue samples were collected to culture mycobacteria present and examine individual specimens’ histopathology. Mycobacterium identification was obtained through the application of amplification and sequencing of 16S rDNA techniques. Results: Three strains were isolated and identified as Mycobacterium (M.) avium subsp. hominissuis by utilizing M. avium complex- specific primers. Granulomatous lesions were observed in the subpleural zone (granuloma grade Π ) and bronchial (granuloma grade I ) of the lungs of D. virginiana with positive isolation. Conclusions: Three strains of M. avium subsp. hominissuis, from lung tissue samples of D. virginiana were identified. This subspecies of M. avium has important implications in public health and veterinary medicine.

Moxifloxacin resistance and genotyping of Mycobacterium avium and Mycobacterium intracellulare isolates in Japan

BackgroundAlthough fluoroquinolones are considered as alternative therapies of pulmonary Mycobacterium avium complex (MAC) disease, the association between fluoroquinolone resistance and MAC genotypes in clinical isolates from individuals not previously treated for MAC infection is not fully clear. MethodsTotals of 154 M. avium isolates and 35 Mycobacterium intracellulare isolates were obtained from treatment-naïve patients with pulmonary MAC disease at the diagnosis of MAC infection at 8 hospitals in Japan. Their susceptibilities of moxifloxacin were determined by broth microdilution methods. Moxifloxacin-resistant isolates were examined for mutations of gyrA and gyrB. Variable numbers of tandem repeats (VNTR) assay was performed using 15 M. avium VNTR loci and 16 M. intracellulare VNTR loci. ResultsMoxifloxacin susceptibility was categorized as resistant and intermediate for 6.5% and 16.9%, respectively, of M. avium isolates and 8.6% and 17.1% of M. intracellulare isolates. Although the isolates of both species had amino acid substitutions of Thr 96 and Thr 522 at the sites corresponding to Ser 95 in the M. tuberculosis GyrA and Gly 520 in the M. tuberculosis GyrB, respectively, these substitutions were observed irrespective of susceptibility and did not confer resistance. The VNTR assays showed revealed three clusters among M. avium isolates and two clusters among M. intracellulare isolates. No significant differences in moxifloxacin resistance were observed among these clusters. ConclusionsAlthough resistance or intermediate resistance to moxifloxacin was observed in approximately one-fourth of M. avium and M. intracellulare isolates, this resistance was not associated with mutations in gyrA and gyrB or with VNTR genotypes.

Novel external quality assurance scheme for drug susceptibility testing of non-tuberculous mycobacteria: a multicentre pilot study.

Our aim was to conduct a multicentre study involving laboratories participating in the European TB Reference Laboratory Network aiming to develop a pilot external quality assessment (EQA) scheme for drug susceptibility testing (DST) of non-tuberculous mycobacteria (NTM). The study comprised a survey using a structured questionnaire followed by a pilot EQA round using identical panels of 10 Mycobacterium avium (MAV) and Mycobacterium abscessus (MAB) isolates. EQA results were received from 16 laboratories utilizing the broth microdilution method. Consensus modal MIC values were determined, and essential and categorical agreement rates were calculated. Twenty-four out of 31 laboratories (77.4%) reported DST for NTM routinely. Essential agreement ranged from 78.8% (amikacin) to 96.2% (linezolid) for MAV and from 76.0% (amikacin) to 100% (doxycycline) for MAB. Categorical agreement ranged from 56.8% (moxifloxacin) to 100% (clarithromycin) for MAV and from 53.6% (linezolid) to 100% (doxycycline) for MAB. Our results show that interlaboratory reproducibility of DST for NTM is insufficient, highlighting the need for expanding EQA schemes. As EQAs for Mycobacterium tuberculosis complex have led to more reliable and reproducible DST, we propose to follow a similar approach for clinically relevant NTM.

Protection efficacy of Argentinian isolates of Mycobacterium avium subsp. paratuberculosis with different genotypes and virulence in a murine model

Paratuberculosis is a chronic disease caused by Mycobacterium avium subsp. paratuberculosis (Map). The disease causes economic losses and, therefore, it is imperative to follow proper control strategies, which should include an effective vaccine. Several strategies have assessed the virulence and immune response of Map strains that could be used as a vaccine. This study evaluates the degree of virulence, immune response, and protection of Argentinian strains of Map with different genotype in a murine model. Four local isolates (Cattle type) with different genotypes (analyzed by MIRU-VNTR and SSRs) were selected and evaluated in a virulence assay in BALB/c mice. This assay allowed us to differentiate virulent and low-virulence Map strains. The less virulent strains (1543/481 and A162) failed to induce a significant production of the proinflammatory cytokine IFNg, whereas the virulent strain 6611 established infection along with a proinflammatory immune response. On the other hand, the virulent strain 1347/498 was efficient in establishing a persistent infection, but failed to promote an important Th1 response compared with 6611 at the evaluated time. We selected the low-virulence strain 1543/498 as a live vaccine and the virulent strain 6611 as a live and inactivated vaccine in a protection assay in mice. Strain 1543/481 failed to protect the animals from challenge, whereas strain 6611, in its live and inactivated form, significantly reduced the CFUs count in the infected mice, although they had different immunological response profiles. The inactivated virulent strain 6611 is a potential vaccine candidate against paratuberculosis to be tested in cattle.

Single-nucleotide polymorphisms related to fluoroquinolone and aminoglycoside resistance in Mycobacterium avium isolates.

ObjectiveThe relationships between fluoroquinolone and aminoglycoside resistance and single-nucleotide polymorphisms (SNPs) in gyrA, gyrB, and rpsL genes were investigated in 95 clinical isolates of Mycobacterium avium from China.MethodsFluoroquinolone and aminoglycoside resistance were determined by the broth microdilution method. GyrA, gyrB, and rpsL were sequenced, SNPs were identified, and the corresponding amino acid mutations were recorded.ResultsThe M. avium isolates displayed high levels of ofloxacin (93.68%), ciprofloxacin (92.63%), and streptomycin (65.26%) resistance. Moxifloxacin (18.95%) and amikacin (2.11%) were highly active against the strains. Fluoroquinolone resistance involving gyrA and gyrB gene mutations was identified. For gyrA, the most frequent SNPs were T→C (71/95, 74.74%), followed by A→G (64/95, 67.37%) and T→C (62/95, 65.26%). The amino acid mutations occurred mainly at Gly2444Asp (GGT→GAT) (20/95, 21.05%), Ala2445Ser (GCC→TCC) (20/95, 21.05%), Ala2447Val (GCC→GTC) (20/95, 21.05%), Val2449Ile (GTC→ATC) (20/95, 21.05%), and Glu2450Gln (GAA→CAA) (20/95, 21.05%). Prominent SNPs in gyrB included A→C (69/95, 72.63%), C→T (51/95, 53.68%), and T→G (29/95, 30.53%), and their amino acid substitutions were Ile2160Val (ATT→GTT) (21/95, 22.11%), Ile2160Met (ATT→ATG) (20/95, 21.05%), and Ile2273Leu (ATC→CTC) (11/95, 11.58%). Among the strains with aminoglycoside resistance, SNPs in rpsL were identified mostly at position G→A (73/95, 76.84%). G→C (21/95, 22.11%) was commonly seen. The amino acid mutations primarily involved Ala1539985Thr (GCC→ACC) (19/95, 20.00%), His1539992Asp (CAC→GAC) (19/95, 20.00%), and Gln-1539983Glu (CAG→GAG) (18/95, 18.95%).ConclusionOur study provides valuable information that could be used for the future diagnosis and treatment of M. avium disease.

Isolation, Identification, and Characterization of a New Highly Pathogenic Field Isolate of Mycobacterium avium spp. avium.

Avian tuberculosis is a chronic, contagious zoonotic disease affecting birds, mammals, and humans. The disease is most often caused by Mycobacterium avium spp. avium (MAA). Strain resources are important for research on avian tuberculosis and vaccine development. However, there has been little reported about the newly identified MAA strain in recent years in China. In this study, a new strain was isolated from a fowl with symptoms of avian tuberculosis by bacterial culture. The isolated strain was identified to be MAA by culture, staining, and biochemical and genetic analysis, except for different colony morphology. The isolated strain was Ziehl-Zeelsen staining positive, resistant to p-nitrobenzoic acid, and negative for niacin production, Tween-80 hydrolysis, heat stable catalase and nitrate production. The strain had the DnaJ gene, IS1245, and IS901, as well. Serum agglutination indicated that the MAA strain was of serotype 1. The MAA strain showed strong virulence via mortality in rabbits and chickens. The prepared tuberculin of the MAA strain had similar potency compared to the MAA reference strain and standard tuberculin via a tuberculin skin test. Our studies suggested that this MAA strain tends to be a novel subtype, which might enrich the strain resource of avian tuberculosis.

Mycobacteriosis in peafowl: Analysis of four cases

Peafowl (Pavo cristatus), similarly to other Galliformes, are particularly susceptible to infection by Mycobacterium avium. Peafowl differ from other Galliformes in the clinical image of the infection, with dominating respiratory signs. Occurrence of severe and sustained dyspnoea in peafowl raises suspicion of mycobacteriosis, which, however, is not always easy to confirm. In the cases described here, mycobacteria were detected in direct swabs from the trachea of two individuals, and cultures were conducted on the Löwenstein- Jensen medium. In one individual, no mycobacteria were found in tracheal swabs stained by the Ziehl-Neelsen method, despite the presence of clear clinical signs. The fourth case was a young bird submitted for necropsy. The cause of death was a mechanical trauma, but scarce caseous nodules typical of mycobacteriosis were found in the liver, spleen and lungs. The Mycobacterium avium isolates obtained from those cases were compared using (CCG)4-based PCR. A high similarity of three isolates of Mycobacterium avium subsp. avium was observed, two of which were derived from peafowl originating from the same farm, while the isolate from the fourth bird differed significantly and was identified by sequencing as Mycobacterium avium subsp. paratuberculosis..

The impact of protein extraction protocols on the performance of currently available MALDI-TOF mass spectrometry for identification of mycobacterial clinical isolates cultured in liquid media

BackgroundProtein extraction step is particularly important for identification of mycobacterial isolates by MALDI-TOF mass spectrometry (MS) because of its thick and solid cell wall. This study compared the performance of MALDI-TOF MS for identification of mycobacterial clinical isolates cultured in liquid media between heating-based protocol and non-heating protocol. MethodsClinical mycobacterial isolates cultured in liquid media were prospectively analyzed. Reference identification was real-time PCR and restriction fragment length polymorphism. The specimens prepared by heating protocol and non-heating protocol were tested using MALDI Biotyper (Bruker Daltonics, Bremen, Germany) and Vitek MS (bioMérieux, Marcy l'Etoile, France), respectively. ResultsAmong the 206 clinical specimens prepared by heating method, identification rates were 90.3% and 60.7% in MALDI Biotyper and Vitek MS, respectively. Identification accuracy of MALDI Biotyper and Vitek MS was 100% for the isolates of Mycobacterium tuberculosis complex (MTBC), Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium abscessus and Mycobacterium fortuitum. Among the 121 clinical specimens prepared by non-heating method, identification rate for MALDI Biotyper and Vitek MS were 61.2% and 69.4%, respectively. Identification accuracy of MALDI Biotyper/Vitek MS were 92.9%/94.1% for MTBC, 92.9%/100% for M. avium, 90%/100% for M. intracellulare, 100%/100% for M. abscessus and 100%/100% for M. fortuitum. ConclusionsThe performance of MALDI-TOF MS for identification of mycobacterial clinical isolates is affected by protein extraction protocol. For best performance, protein extraction protocol should be chosen considering the MALDI-TOF MS system. In the present study, heating protocol with MALDI Biotyper system showed reliable identification results for mycobacterial clinical isolates.

The isolation and molecular characterization of Mycobacterium avium subsp. paratuberculosis in Shandong province, China.

BackgroundMycobacterium avium subspecies paratuberculosis (Map) causes Johne’s disease in domestic and wild ruminants. It has been a debate that whether Map can cause Crohn’s disease in human. To our knowledge there is no report about molecular characterization of Map in China, although several Map strains have been reported in other country. The objectives of this study was to know the recent prevalence of Johne’s disease in dairy farms in Shandong province, and have a better understanding of genotypic distribution of Map in China.MethodsJohne’s disease was detected from 1038 individuals in 19 dairy farms by ELISA. Map in fecal and milk specimens was identified by Ziehl-Neelsen staining and confirmed using PCR-REA. In addition, frozen sections of ileum and mesenteric lymph nodes from two Map shedding cows were performed to observe the histopathological changes. Next-generation sequencing technology was performed to get whole genome sequences.ResultA total of 121 (11.7 %) animals were positive for Map antibody from 1038 sera tested, and 11 (57.9 %) dairy herds were positive for Map antibody. Typically histopathologic changes were observed in mesenteric lymph nodes. We have successfully isolated two Map strains, which both were Map-C. The current genome-wide analysis showed that the genome size of our isolates are respectively 4,750,273 and 4,727,050 bp with a same G + C content of 69.3 %, and the numbers of single nucleotide polymorphisms (SNPs) against Map K-10 are respectively 292 and 296.ConclusionMap is a prevalent pathogen among dairy cattle in China. This study successfully isolated two Map strains from one Chinese dairy herd with signs of diarrhoea, and identified that the two isolates were both Map-C. Furthermore, these isolates were most closely related to Map K-10.

Antimicrobial susceptibility testing and genotyping of Mycobacterium avium isolates of two tertiary tuberculosis designated hospital, China

Background: Mycobacterium avium is frequently isolated from clinical samples, while the bacteriological features of M. avium clinical isolates from China have never been well defined. Methods: A total of 50 M. avium isolates were recruited from two tertiary tuberculosis designated hospitals, one located in Beijing whereas another in Fujian Province, which are northern and southern parts of China, respectively. Subspecies identification was conducted by sequencing the variable 3′ end of the hsp65 gene. The susceptibility against 15 antimicrobial agents, widely administered for the treatment of non-tuberculosis mycobacteria (NTM) infections, was tested by broth microdilution assay. Variable number of tandem repeats (VNTR) assay was also performed using the 16-loci genotyping method. Results: All of the 50 M. avium isolates were identified as M. avium subsp. hominissuis. The drug susceptibility test revealed that clarithromycin (98%, 49/50) and moxifloxacin (86%, 43/50) had the best antimicrobial activities in vitro against the M. avium isolates. The overall Hunter–Gaston Discriminatory Index (HGDI) value for the VNTR typing was 0.95. However, the genotyping method yielded much greater discriminative power for isolates of northern China than that of southern China (1.00 V.S. 0.86, P<0.05). Conclusion: M. avium subsp. hominissuis is the dominate subspecies among M. avium clinical isolates in China. The 16-loci VNTR genotyping method is more discriminative in Beijing than in Fujian Province. The bacteriological features of M. avium isolates from different regions of China demonstrated dramatic variations, and stressed the importance of building up knowledge from the local isolates.