Mycobacteria

Among the costs attributed to Mycobacterium avium ssp. paratuberculosis (MAP) infection in dairy cattle, the effects on reproduction and culling are the least documented. To estimate the cost of MAP infections and Johne's disease in a dairy herd, the rates of calving and culling were calculated for cows in each stage of MAP infection relative to uninfected cows. Data from 6 commercial dairy herds, consisting of 2,818 cows with 2,754 calvings and 1,483 cullings, were used for analysis. Every cow in each study herd was tested regularly for MAP, and herds were followed for between 4 and 7 yr. An ordinal categorical variable for Johne's disease status [test-negative, low-positive (low-shedding or ELISA-positive only), or high-shedding] was defined as a time-dependent variable for all cows with at least 1 positive test result or 2 negative test results. A Cox regression model, stratified on herd and controlling for the time-dependent infection variable, was used to analyze time to culling. Nonshedding animals were significantly less likely to be culled in comparison with animals in the low-shedding or ELISA-positive category, and highshedding animals had nonsignificantly higher culling rates than low-shedding or ELISA-positive animals. Time to calving was analyzed using a proportional rates model, an analog to the Andersen-Gill regression model suitable for recurrent event data, stratifying on herd and weighted to adjust for the dependent censoring caused by the culling effects described above. Highshedding animals had lower calving rates in comparison with low-shedding or ELISA-positive animals, which tended to have higher calving rates than test-negative animals.

Microbial survival in dynamic environments requires the ability to successfully respond to abrupt changes in osmolarity. The mechanosensitive channel of large conductance (MscL) is a ubiquitous channel that facilitates the survival of bacteria and archaea under severe osmotic downshock conditions by relieving excess turgor pressure in response to increased membrane tension. A prominent structural feature of MscL, the cytoplasmic C-terminal domain, has been suggested to influence channel assembly and function. In this report, we describe the X-ray crystal structure and electrophysiological properties of a C-terminal domain truncation of the Mycobacterium tuberculosis MscL (MtMscLΔC). A crystal structure of MtMscLΔC solubilized in the detergent n-dodecyl-β-D-maltopyranoside reveals the pentameric, closed state-like architecture for the membrane spanning region observed in the previously solved full-length MtMscL. Electrophysiological characterization demonstrates that MtMscLΔC retains mechanosensitivity, but with conductance and tension sensitivity more closely resembling full length EcMscL than MtMscL. This study establishes that the C-terminal domain of MtMscL is not required for oligomerization of the full-length channel, but rather influences the tension sensitivity and conductance properties of the channel. The collective picture that emerges from these data is that each MscL channel structure has characteristic features, highlighting the importance of studying multiple homologs. Mechanosensitive (MS) channels transduce mechanical stimuli into a variety of cellular responses. MS channels are widely distributed through all kingdoms of life, including bacteria and archaea, where they play a prominent role in maintaining proper osmoregulation 1-5. Sudden changes in osmolarity, such as osmotic downshock during exposure to hypotonic conditions, increases turgor pressure across the cell membrane. This in turn increases the tension within the lipid bilayer and activates a network of MS channels which respond by opening typically nonselective pores in the membrane to release cell contents and alleviate the pressure 1,5-7. Bacterial MS channels are characterized by their relatively high and non-selective conductances, ranging from ~0.1 nS to ~3 nS under defined experimental conditions 6-8 , which is up to several orders of magnitude greater than typical for ion-selective channels 3,6,9. Of these channels, the mechanosensitive channel of large conductance (MscL) exhibits the largest conductance, with an estimated open state pore diameter of ~25-35 Å-large enough for the passage of molecules up to 9 kDa 4,7,10,11. In view of the high conductance yet small subunit size of MscL (Escherichia coli MscL (EcMscL) has 136 residues; Fig. 1), the oligomeric nature of this channel was appreciated from its initial discovery 11. Considerable efforts have been made to establish the molecular mechanism of MscL gating 3,12 , employing a variety of techniques including electrophysiology, biochemical and genetic studies, molecular dynamics

Bacteria can proliferate perpetually without ageing, but they also face conditions where they must persist. Mycobacteria can survive for a long period. This state appears during mycobacterial diseases such as tuberculosis and leprosy, which are chronic and develop after long-term persistent infections. However, the fundamental mechanisms of the long-term living of mycobacteria are unknown. Every Mycobacterium species expresses Mycobacterial DNA-binding protein 1 (MDP1), a histone-like nucleoid associated protein. Mycobacterium smegmatis is a saprophytic fast grower and used as a model of mycobacterial persistence, since it shares the characteristics of the long-term survival observed in pathogenic mycobacteria. Here we show that MDP1-deficient M. smegmatis dies more rapidly than the parental strain after entering stationary phase. Proteomic analyses revealed 21 upregulated proteins with more than 3-fold in MDP1-deficient strain, including DnaA, a replication initiator, NDH, a NADH dehydrogenase that catalyzes downhill electron transfer, Fas1, a critical fatty acid synthase, and antioxidants such as AhpC and KatG. Biochemical analyses showed elevated levels of DNA and ATP syntheses, a decreased NADH/NAD + ratio, and a loss of resistance to oxidative stress in the MDP1-knockout strain. This study suggests the importance of MDP1-dependent simultaneous control of the cellular functions in the long-term survival of mycobacteria.

Interleukin-10 is an immunomodulatory cytokine that has been implicated, along with IFN-c, in the disease sequelae of mycobacterial infection. In order to investigate the role of IL-10 in marsupial disease models we sequenced and characterised the IL10 gene in the tammar wallaby (Macropus eugenii) and rufous hare-wallaby (Lagorchestes hirsutus). An isoform IL-10D3, in which an in-frame deletion of exon 3 occurs, was discovered in both macropod species. Analysis of wallaby and other reported marsupial IL-10 homologs suggests that while marsupial IL-10 is comparable to that of human IL-10, the predicted IL-10D3 protein may play a more complicated role in the modulation of IL-10-directed responses. Expression of the canonical gene and splicing variant was confirmed in both wallabies, and the rufous hare-wallaby showed differential expression across lymph node, spleen and liver, with isoform expression detected in the lymph node. This characterisation and expression of IL-10 in de novo tissues provides a basis for further study into the role of IL-10 in disease models in marsupials.

Rapidly growing mycobacteria (RGM) inhabit soil and water but certain strains represent a health risk for human
and animals. Both clinical and soil RGM may be under selection pressure for resistance to tetracycline (TET) antibiotics,
since tetracyclines are administrated to humans and farm animals, and TET residues enter soil through manuring;
however, resistance to TET and the presence of TET-resistance genes have been assessed only in clinical isolates. We
were therefore interested in comparing soil and clinical RGM in terms of TET resistance and the presence of TETresistance
genes. We used 44 RGM from grasslands with different exposure to animal manure, and 38 clinical RGM
from Czech hospitals. There was no difference between the clinical and soil isolates in TET resistance, with >50%
resistant isolates in both groups. otr(A), otr(B), tet(K), tet(L) or tet(M) were not detected in any soil or clinical isolate.
In contrast, most isolates harbored tet(V) and tap, both encoding mycobacterial efflux pumps, including species where
these genes have never been evidenced before. The phylogeny of tet(V) correlated with isolates’ BOX-PCR profiles,
suggesting that this gene evolved along with mycobacterial genomes as a part of the intrinsic resistome. In certain
cases, tet(V) and/or tap were found in TET-sensitive isolates, or inversely, were not found in resistant strains. Concluding,
intrinsic efflux pumps may be more important for TET resistance than horizontally transferred genes in both soil and
clinical RGM. Their simple presence, however, does not attest to resistance, and therefore their diversity, function and
expression merit further research.

Tuberculosis (TB) is a serious infectious disease caused by a bacterial pathogen. Mortality from tuberculosis was estimated at 1.5 million deaths worldwide in 2013. Development of new TB drugs is needed to not only to shorten the medication period but also to treat multidrug resistant and extensively drug-resistant TB. Mycobacterium tuberculosis (Mtb) grows slowly and only multiplies once or twice per day. Therefore, conventional drug screening takes more than 3 weeks. Additionally, a biosafety level-3 (BSL-3) facility is required. Thus, we developed a new screening method to identify TB drug candidates by utilizing luciferase-expressing recombinant Mycobacterium bovis bacillus Calmette-Guéren (rBCG). Using this method, we identified several candidates in 4 days in a non-BSL-3 facility. We screened 10,080 individual crude extracts derived from Actinomyces and Streptomyces and identified 137 extracts which possessed suppressive activity to the luciferase of rBCG. Among them, 41 compounds inhibited the growth of both Mtb H37Rv and the extensively drug-resistant Mtb (XDR-Mtb) strains. We purified the active substance of the 1904-1 extract, which possessed strong activity toward rBCG, Mtb H37Rv, and XDR-Mtb but was harmless to the host eukaryotic cells. The MIC of this substance was 0.13 μg/ml, 0.5 μg/ml, and 2.0-7.5 μg/ml against rBCG, H37Rv, and 2 XDR-strains, respectively. Its efficacy was specific to acid-fast bacterium except for the Mycobacterium avium intracellular complex. , 24390106 (S Matsumoto), 24501015 (Y Ozeki).

Collagen deposition within granulomas formed after Mycobacterium avium infection was analysed on histological sections stained with Masson’s trichrome using acquired computerized image analysis and a program that was specifically designed for that purpose. Comparison was made between immunocompetent C57BL⁄ 6 mice and mice genetically deficient in the inducible nitric oxide (NO) synthase gene (iNOS– ⁄ – mice) infected with either a highly virulent strain or a moderately virulent strain of M. avium. iNOS-deficient mice were more resistant to the highly virulent strain than control C57B1⁄ 6 mice, but both strains were equally susceptible to the less virulent M. avium strain. Collagen distribution in the granuloma was found in the cuff surrounding the granuloma in an area rich in lymphoid cells as well as inside the granuloma itself, conferring a mesh-like structure within that lesion. It was seen that iNOS– ⁄ – mice induced a higher collagen deposition than C57BL ⁄ 6 mice and that such collagen deposition varied with the mycobacterial strain used to
infect the animals.

Environmental contamination with Mycobacterium avium subsp. paratuberculosis (MAP) is thought to be one of the primary sources of infection for dairy cattle. The exact link between fecal shedding of MAP by individual cows and environmental contamination levels at the herd level was explored with a cross-sectional analysis of longitudinally collected samples on 3 dairy farms. Composite samples from multiple environmental sites in 3 commercial dairy herds in the Northeast US were cultured quarterly for MAP, providing 1131 samples (133 (11.8%) were culture-positive), and all adult animals in the herds were tested biannually by fecal culture (FC), for 6 years. Of the environmental sites sampled, manure storage areas and shared alleyways were most likely to be culture-positive. Environmental sample results were compared to FC results from either the concurrent or previous sampling date at both the herd and the pen level. At the herd level, a 1 log unit increase in average fecal shedding increased the odds of a positive non-pen environmental sample by a factor of 6 and increased the average amount of MAP in non-pen samples by 2.9 cfu/g. At the pen level, a 1 log unit increase in average fecal shedding in the pen increased the odds of a positive environment by a factor of 2.4 and the average amount of MAP was increased by 3.5 cfu/g. We were not able to model the relationship between non-pen environmental sample status and the distance between shedding animals and the sample's location, and neighboring pens did not significantly affect the results of the pen-level analysis. The amount of MAP in pen-level samples and the probability of a pen testing positive for MAP were both positively but non-significantly correlated with the number of animals in the pen shedding >30 cfu/g of MAP. At least 6 environmental samples met the criteria for the U.S. Voluntary Bovine Johne's Disease Control Program on 47 of the 72 sampling dates; of these, 19 of the 47 FC-positive sampling dates were positive by the 6-sample environmental testing method, resulting in a herd sensitivity of 0.40 (95% CI: 0.26-0.54). None of the 3 FC-negative sampling dates produced positive environmental samples. Although environmental sampling can be * Corresponding author at: S2-064 Schurman Hall,

Johne's disease is a worldwide concern, as it causes huge economic losses. The etiological agent, Mycobac-terium avium ssp. paratuberculosis (MAP), has limited genetic diversity, impeding efforts to understand transmission and distribution of strain types. Whole-genome sequencing was previously performed on a representative set of MAP isolates from Canadian dairy herds and 9 divergent clades were identified. Four clades were of particular interest, as they were either MAP types rarely reported in North America, or they represented a substantial proportion of isolates recovered from dairy farms in Canada. One clade included type I/III isolates, whereas the remaining clades included type II isolates. Variant sites in the MAP genome are often separated by thousands of base pairs, limiting use of single nucleotide polymorphism (SNP)-based genotyp-ing on a single genomic region. Therefore, a SNP-PCR assay was developed to facilitate interrogation of 5 SNP in 2 distant regions of the genome, linking them together in a single PCR reaction for subsequent Sanger sequencing. This high-throughput assay enabled discrimination of 602 MAP isolates from 264 herds (from all 10 provinces). More than 1 isolate was cultured from 133 herds, 14 of which included multiple subtypes. A previously identified dominant type included 87% of isolates, whereas the Bison type was more widespread than previously reported. The latter type and isolates from a second clade of interest were overrepresented in Québec and Saskatchewan, respectively. In conclusion , the distribution and relative frequency of MAP subtypes within Canadian dairy herds were assessed using a novel SNP-based typing assay. These findings will contribute to understanding the clinical relevance and transmission dynamics of MAP in this population and elsewhere.

The growth in C57Bl/6 mice of seven distinct Mycobacterium avium strains was not exacerbated by the disruption of the inducible (type 2) nitric oxide synthase regardless of the virulence of the strain. The susceptibility of this panel of M. avium strains to reactive nitrogen intermediates in a cell-free system, namely the exposure to acidified nitrite or to the NO donor NOC-18, showed that there is no correlation between strain virulence and the corresponding minimal bactericidal and minimal inhibitory concentrations for those compounds determined in vitro.

AmpliBASE MT™ is an online databank of highresolution DNA fingerprints representing fluorescent amplified fragment length polymorphism (FAFLP) profiles or amplitypes developed for the Mycobacterium tuberculosis complex strains from 48 different countries. AmpliBASE MT™ is based on a relational database management system that is hyperlinked to visualize genotyping results in the form of DNA fingerprint images for individual strains. A flexible search system based on systematic comparisons of fragment sizes in base pairs allows inter-laboratory comparison of FAFLP profiles. Besides this, the database also displays previously published data on IS6110 profiles, spoligotypes, MIRU-VNTRs and large sequence polymorphisms along with the FAFLP records that will give the overall comparisons. Being the first of its kind, AmpliBASE MT™ is expected to be a very helpful tool in strengthening the concept of 'geographic genomics' and will be very helpful to molecular epidemiologists and those interested in diagnostic development for tuberculosis.

Currently, the Chilean authority has implemented a
National Eradication Program for bovine tuberculosis (bTB),
aimed at controlling and eradicating the disease in Chile. The
area under study has a low within-herd prevalence, has a relatively
low number of infected herds, and is one of the major
milk and beef producing areas in the country. However, so far,
no attempts at eradicating the disease have been successful. It
has been suggested that the diagnostic tests used were either
not sensitive or specific enough. In addition, previous studies
have shown that a great number of herds are infected with
Mycobacterium avium subsp. paratuberculosis (MAP). The
present study estimates the effect of MAP infection under field
conditions, on the diagnostic sensitivity and specificity of routine
tests in live animals for Mycobacterium bovis infection
diagnosis in cattle. In general, the estimations of test accuracy
observed an increase in the sensitivity and specificity on
MAP-infected animals for tuberculin test but observed a
decrease in the sensitivity of gamma interferon tests for
MAP-infected cattle. These results are different from those
of previous studies considering the role of MAP infection as
an interfering infection. More research is needed in order to
understand the complex interactions of the different
mycobacteria that can be found infecting production

Rapidly growing mycobacteria (RGM) are common soil saprophytes, but certain strains cause infections in human and animals. The
infections due to RGM have been increasing in past decades and are often difficult to treat. The susceptibility to antibiotics is regularly
evaluated in clinical isolates of RGM, but the data on soil RGM are missing. The objectives of this study was to isolate RGM from four
grassland soils with different impact of manuring, and assess their resistance to antibiotics and the ability to grow at 37°C and 42°C.
Since isolation of RGM from soil is a challenge, a conventional decontamination method (NaOH/malachite green/cycloheximide) and a
recent method based on olive oil/SDS demulsification were compared. The olive oil/SDS method was less efficient, mainly because of
the emulsion instability and plate overgrowing with other bacteria. Altogether, 44 isolates were obtained and 23 representatives of
different RGM genotypes were screened. The number of isolates per soil decreased with increasing soil pH, consistently with previous
findings that mycobacteria were more abundant in low pH soils. Most of the isolates belonged to the Mycobacterium fortuitum group.
The majority of isolates was resistant to 2-4 antibiotics. Multiresistant strains occurred also in a control soil that has a long history
without the exposure to antibiotic-containing manure. Seven isolates grew at 37°C, including the species M. septicum and M. fortuitum
known for infections in humans. This study shows that multiresistant RGM close to known human pathogens occur in grassland soils
regardless the soil history of manuring.

The authors describe a study of the tuberculosis (TB) incidence in cattle exposed to low doses of radiation resulting from the Chernobyl (pronounced ‘Chornobyl’ in Ukrainian) nuclear plant catastrophe in 1986. The purpose of the study was to determine if ionising radiation influences the number of outbreaks of bovine TB and their severity on farms in the Kyiv, Cherkasy and Chernigiv regions of Ukraine. These farms are all located within a 200 km radius of Chernobyl and have had low-dose radiation pollution. Pathological and blood samples were taken from cattle in those regions that had positive TB skin tests. Mycobacterium spp. were isolated, differentiated by PCR, analysed and tested in guinea-pigs and rabbits. Species differentiation showed a significant percentage of atypical mycobacteria, which resulted in the allergic reactions to tuberculin antigen in the skin test. Mixed infection of M. bovis and M. avium subsp. hominissuis was found in three cases. The results concluded that low-dose radiation plays a major role in the occurrence of bovine TB in regions affected by the Chernobyl nuclear disaster.

Buruli ulcer is a bacterial disease, alternatively called Bairnsdale ulcer, Searls ulcer, or Daintree ulcer. The tropical disease causes blood clots on the skin and, when untreated, leads to severe ulceration. The infectious disease caused by a bacterium called Mycobacterium ulcerans. It is the third most common mycobacterial disease after tuberculosis and leprosy.

The current treatment option primarily involves courses of antibiotics. More serious or untreatable cases require surgical intervention. To improve current treatment regimes, scientists are investigating alternative approaches; this article assesses some of these approaches alongside current practices.

The TSE'S or transmissible spongiform enchephalopathies, include bovine spongiform encephalopathy (also called BSE or " mad cow disease "), Creutzfeldt– Jakob disease (CJD) in humans, and " scrapie " in sheep or goats (caprine spongiform encephalopathy). They remain a mystery, their cause still hotly debated. Current mad cow diagnosis lies solely in the detection of late appearing " prions " , an acronym for hypothesized, geneless, misfolded proteins, somehow claimed to cause the disease. Yet laboratory preparations of prions contain other things, which could include unidentified bacteria or viruses. And the only real evidence that prion originator Stanley Prusiner had in his original paper that the disease agent behind " Scrapie " in sheep and goats was devoid of DNA or RNA– was based upon the fact that he couldn't find any. Furthermore, the rigors of prion purification alone, might, in and of themselves, have killed any causative microorganism and Heino Dringer, who did pioneer work on their nature, candidly predicts " it will turn out that the prion concept is wrong. " Roels and Walravens as well as Hartly traced Mad Cow to Mycobacterium bovis. Moreover, epidemiologic maps of the origins and peak incidence of Mad Cow in the UK, suggestively match those of England's areas of highest bovine tuberculosis, the Southwest. The neurotaxic potential of bovine tuberculosis has for some time been well known. By 1911 Alois Alzheimer called attention to " a characteristic condition of the cortical tissue which Fischer referred to as 'spongy cortical wasting " in Alzheimer's disease (AD). But behind AD, Fischer suspected a microbe called Streptothrix which was constantly being mistaken and confused for tuberculosis. Our present investigation of the TSEs clearly shows cell-wall-deficient (CWD) tubercular mycobacteria present, verified by molecular analysis, ELISA, PCR and microscopy to cause spongiform encephalopathy.

Se comunican dos casos clínicos poco frecuentes de micobacteriosis cutáneas; uno adquirido en la comunidad y uno en medio hospitalario, ambos pacientes del género masculino, con dermatosis crónicas constituidas por úlceras, fístulas y placas de aspecto papilomatoso. El diagnóstico fue tardío y la evolución no fue satisfactoria, a pesar de prescribir tratamiento adecuado contra Mycobacterium chelonae. El propósito de este artículo es mostrar la manifestación clínica de las lesiones para sospecha diagnóstica temprana y oportuna de las diferentes disciplinas médicas que participan en la atención de los pacientes con estas infecciones.

This paper reports the two rare clinical cases of cutaneous mycobacterial infections. One of them acquired the infection within the community while the other was a noso-comial case, both cases were male with chronic ulcers, fistulae and papilloma-like lesions. In both cases diagnosis was late and evolution, despite correct Mycobacterium chelonae treatment, was unsatisfactory. The purpose of this paper is to show the clinical presentation and accurate suspicion of infection of the different medical areas involved in its management.

Introduction: Secondary bacterial infections of chest tubes placed to drain tuberculous pleural effusions are common but has not been studied before. The aim of this study is to estimate the frequency of these secondary bacterial infections in patients with suspected drain infections based on clinical signs and symptoms and to assess the prevalence, microbiological and biochemical profile of their pleural fluid samples and the effect of the presence of the tuberculous effusion and the ongoing anti-TB chemotherapy on this prevalence. Methods: This is a multicenter retrospective observational study. We enrolled 690 patients with confirmed TB effusion being treated with first class anti-TB drugs and chest tube drain. Strict exclusion criteria were applied. Patients were divided into two groups, group A (220 patients), who had suspected secondary bacterial infection of their chest tube drains and who developed signs or symptoms of drain infections. Group B (470 patients), who had no suspected secondary bacterial infection in their chest tube drains. Pleural fluid samples and cultures and insertion site samples were taken from both groups then cytochemical and microbiological profiles were assessed. Results: In biochemical analysis: there were statistically significant differences between groups A and B regarding the levels of LDH (1466.9 ± 6169.4 U/l versus 255.2 ± 445.4 U/l; P < 0.001) which was significantly higher in group A. In the other hand, glucose was significantly higher in group B (109.4 ± 82.2 mg/dl versus 131.6 ± 76.4 mg/dl; P < 0.001). In Cytological analysis, the percentages of total nucleated cells (mainly neutrophils) were significantly higher in group A, while the percentages of lymphocytes were significantly higher in group B. In microbiological analysis, pleural fluid cultures were positive in 34% of group A samples and were positive in only 6% of group B samples. Insertion site cultures were positive in 14% of group A samples and 4.2% of group B samples. The PPV and NPV of chest tubes drain culture for insertion site infection for group A were 36.5% and 97.5% respectively, and for group B were 37.5% and 97.8% respectively. Gram positive bacteria were the most prevalent bacterial isolates from both groups A and B, 74% and 48% respectively. Staphylococcus aureus species were the predominant bacteria in both groups A and B, 63% and 42.8% respectively. Gram negative bacteria were more prevalent in group B (42%) with Pseudomonas species were the most common gram-negative bacteria (21%). Conclusion: Clinical features is a good evidence for the presence of secondary bacterial infection of chest tube drains in patients being treated for tuberculous effusion and this was confirmed by bacterial cultures. In addition, the presence of ongoing pleural mycobacterial infection and anti-TB drug treatment has not altered the causative bacteria profile from what has been reported for other cases. Staphylococcus aureus and Pseudomonas species being the more prevalent organisms.