Identification of a novel gene product that promotes survival of Mycobacterium smegmatis in macrophages

Background

Bacteria of the suborder Corynebacterineae include significant human pathogens such as Mycobacterium tuberculosis and M. leprae. Drug resistance in mycobacteria is increasingly common making identification of new antimicrobials a priority. Mycobacteria replicate intracellularly, most commonly within the phagosomes of macrophages, and bacterial proteins essential for intracellular survival and persistence are particularly attractive targets for intervention with new generations of anti-mycobacterial drugs.

Methodology/Principal Findings

We have identified a novel gene that, when inactivated, leads to accelerated death of M. smegmatis within a macrophage cell line in the first eight hours following infection. Complementation of the mutant with an intact copy of the gene restored survival to near wild type levels. Gene disruption did not affect growth compared to wild type M. smegmatis in axenic culture or in the presence of low pH or reactive oxygen intermediates, suggesting the growth defect is not related to increased susceptibility to these stresses. The disrupted gene, MSMEG_5817, is conserved in all mycobacteria for which genome sequence information is available, and designated Rv0807 in M. tuberculosis. Although homology searches suggest that MSMEG_5817 is similar to the serine:pyruvate aminotransferase of Brevibacterium linens suggesting a possible role in glyoxylate metabolism, enzymatic assays comparing activity in wild type and mutant strains demonstrated no differences in the capacity to metabolize glyoxylate.

Conclusions/Significance

MSMEG_5817 is a previously uncharacterized gene that facilitates intracellular survival of mycobacteria. Interference with the function of MSMEG_5817 may provide a novel therapeutic approach for control of mycobacterial pathogens by assisting the host immune system in clearance of persistent intracellular bacteria.     

Microscopy,Culture, and Quantitative Real-Time PCR Examination Confirm Internalization of Mycobacteria in Plants

The environment is a reservoir of nontuberculous mycobacteria and is considered a source of infection for animals and humans. Mycobacteria can persist in different types of environments for a relatively long time. We have studied their possible internalization into plant tissue through intact, as well as damaged, root systems of different types of plants grown in vitro and under field conditions. The substrate into which plants were seeded was previously contaminated with different strains of Mycobacterium avium (10⁸ to 10¹⁰ cells/g of soil) and feces from animals with paratuberculosis. We detected M. avium subsp. avium, hominissuis, and paratuberculosis in the stems and leaves of the plants by both culture and real-time quantitative PCR. The presence of mycobacteria in the plant tissues was confirmed by microscopy. The concentration of mycobacteria found inside plant tissue was several orders of magnitude lower (up to 10⁴ cells/g of tissue) than the initial concentration of mycobacteria present in the culture medium or substrate. These findings led us to the hypothesis that plants may play a role in the spread and transmission of mycobacteria to other organisms in the environment.     

Application of Fluorescent Protein Expressing Strains to Evaluation of Anti-Tuberculosis Therapeutic Efficacy In Vitro and In Vivo

The slow growth of Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), hinders development of new diagnostics, therapeutics and vaccines. Using non-invasive real-time imaging technologies to monitor the disease process in live animals would facilitate TB research in all areas. We developed fluorescent protein (FP) expressing Mycobacterium bovis BCG strains for in vivo imaging, which can be used to track bacterial location, and to quantify bacterial load in live animals. We selected an optimal FP for in vivo imaging, by first cloning six FPs: tdTomato, mCherry, mPlum, mKate, Katushka and mKeima, into mycobacteria under either a mycobacterial Hsp60 or L5 promoter, and compared their fluorescent signals in vitro and in vivo. Fluorescence from each FP-expressing strain was measured with a multimode reader using the optimal excitation and emission wavelengths for the FP. After normalizing bacterial numbers with optical density, the strain expressing L5-tdTomato displayed the highest fluorescence. We used the tdTomato-labeled M. bovis BCG to obtain real-time images of pulmonary infections in living mice and rapidly determined the number of bacteria present. Further comparison between L5-tdTomato and Hsp60-tdTomato revealed that L5-tdTomato carried four-fold more tdTomato gene copies than Hsp60-tdTomato, which eventually led to higher protein expression of tdTomato. Evaluating anti-TB efficacy of rifampicin and isoniazid therapy in vitro and in vivo using the L5-tdTomato strain demonstrated that this strain can be used to identify anti-TB therapeutic efficacy as quickly as 24 h post-treatment. These M. bovis BCG reporter strains represent a valuable new tool for evaluation of therapeutics, vaccines and virulence.     

Comparative Proteomics Identifies Host Immune System Proteins Affected by Infection with Mycobacterium bovis

Mycobacteria of the Mycobacterium tuberculosis complex (MTBC) greatly impact human and animal health worldwide. The mycobacterial life cycle is complex, and the mechanisms resulting in pathogen infection and survival in host cells are not fully understood. Eurasian wild boar (Sus scrofa) are natural reservoir hosts for MTBC and a model for mycobacterial infection and tuberculosis (TB). In the wild boar TB model, mycobacterial infection affects the expression of innate and adaptive immune response genes in mandibular lymph nodes and oropharyngeal tonsils, and biomarkers have been proposed as correlates with resistance to natural infection. However, the mechanisms used by mycobacteria to manipulate host immune response are not fully characterized. Our hypothesis is that the immune system proteins under-represented in infected animals, when compared to uninfected controls, are used by mycobacteria to guarantee pathogen infection and transmission. To address this hypothesis, a comparative proteomics approach was used to compare host response between uninfected (TB-) and M. bovis-infected young (TB+) and adult animals with different infection status [TB lesions localized in the head (TB+) or affecting multiple organs (TB++)]. The results identified host immune system proteins that play an important role in host response to mycobacteria. Calcium binding protein A9, Heme peroxidase, Lactotransferrin, Cathelicidin and Peptidoglycan-recognition protein were under-represented in TB+ animals when compared to uninfected TB- controls, but protein levels were higher as infection progressed in TB++ animals when compared to TB- and/or TB+ adult wild boar. MHCI was the only protein over-represented in TB+ adult wild boar when compared to uninfected TB- controls. The results reported here suggest that M. bovis manipulates host immune response by reducing the production of immune system proteins. However, as infection progresses, wild boar immune response recovers to limit pathogen multiplication and promote survival, facilitating pathogen transmission.     

Animal board invited review: OneARK: Strengthening the links between animal production science and animal ecology

Wild and farmed animals are key elements of natural and managed ecosystems that deliver functions such as pollination, pest control and nutrient cycling within the broader roles they play in contributing to biodiversity and to every category of ecosystem services. They are subjected to global changes with a profound impact on the natural range and viability of animal species, the emergence and spatial distribution of pathogens, land use, ecosystem services and farming sustainability. We urgently need to improve our understanding of how animal populations can respond adaptively and therefore sustainably to these new selective pressures. In this context, we explored the common points between animal production science and animal ecology to identify promising avenues of synergy between communities through the transfer of concepts and/or methodologies, focusing on seven concepts that link both disciplines. Animal adaptability, animal diversity (both within and between species), selection, animal management, animal monitoring, agroecology and viability risks were identified as key concepts that should serve the cross-fertilization of both fields to improve ecosystem resilience and farming sustainability. The need for breaking down interdisciplinary barriers is illustrated by two representative examples: i) the circulation and reassortment of pathogens between wild and domestic animals and ii) the role of animals in nutrient cycles, i.e. recycling nitrogen, phosphorus and carbon through, for example, contribution to soil fertility and carbon sequestration. Our synthesis identifies the need for knowledge integration techniques supported by programmes and policy tools that reverse the fragmentation of animal research toward a unification into a single Animal Research Kinship, OneARK, which sets new objectives for future science policy. At the interface of animal ecology and animal production science, our article promotes an effective application of the agroecology concept to animals and the use of functional diversity to increase resilience in both wild and farmed systems. It also promotes the use of novel monitoring technologies to quantify animal welfare and factors affecting fitness. These measures are needed to evaluate viability risk, predict and potentially increase animal adaptability and improve the management of wild and farmed systems, thereby responding to an increasing demand of society for the development of a sustainable management of systems.     

Stably Luminescent Staphylococcus aureus Clinical Strains for Use in Bioluminescent Imaging

In vivo bioluminescent imaging permits the visualization of bacteria in live animals, allowing researchers to monitor, both temporally and spatially, the progression of infection in each animal. We sought to engineer stably luminescent clinical strains of Staphylococcus aureus, with the goal of using such strains in mouse models. The gram-positive shuttle vector pMAD was used as the backbone for an integration plasmid. A chloramphenicol resistance gene, a modified lux operon from Photorhabdus luminescens, and approximately 650 bp of homology to the chromosome of the USA300 S. aureus strain NRS384 were added, generating plasmid pRP1195. Electroporation into strain RN4220 followed by temperature shift led to integration of pRP1195 into the chromosome. The integrated plasmid was transferred to clinical strains by phage transduction. Luminescent strains displayed no in vitro growth defects. Moreover, luminescence was stable in vitro after three rounds of subculture over 48 hours of growth in the absence of antibiotics. Mice were infected with a luminescent strain of NRS384 in skin and intravenous models. In a mouse skin model, luminescent bacteria were present in lesions that formed and cleared over the course of several days, and in an intravenous model, bacteria inoculated in the mouse tail vein were observed spreading to multiple tissues. No statistically significant difference in virulence was observed between NRS384 and the luminescent strain in either infection model. These preliminary data suggest that this luminescent USA300 strain is suitable for use in mouse models. Similar strains were engineered using other sequenced clinical strains. Because these strains are stably luminescent, they should prove useful in animal models of infection.     

Relative Sensitivity of Conventional and Real-Time PCR Assays for Detection of SFG Rickettsia in Blood and Tissue Samples from Laboratory Animals

Studies on the natural transmission cycles of zoonotic pathogens and the reservoir competence of vertebrate hosts require methods for reliable diagnosis of infection in wild and laboratory animals. Several PCR-based applications have been developed for detection of infections caused by Spotted Fever group Rickettsia spp. in a variety of animal tissues. These assays are being widely used by researchers, but they differ in their sensitivity and reliability. We compared the sensitivity of five previously published conventional PCR assays and one SYBR green-based real-time PCR assay for the detection of rickettsial DNA in blood and tissue samples from Rickettsia- infected laboratory animals (n = 87). The real-time PCR, which detected rickettsial DNA in 37.9% of samples, was the most sensitive. The next best were the semi-nested ompA assay and rpoB conventional PCR, which detected as positive 18.4% and 14.9% samples respectively. Conventional assays targeting ompB, gltA and hrtA genes have been the least sensitive. Therefore, we recommend the SYBR green-based real-time PCR as a tool for the detection of rickettsial DNA in animal samples due to its higher sensitivity when compared to more traditional assays.     

Isolation and Excision of Murine Aorta; A Versatile Technique in the Study of Cardiovascular Disease

Cardiovascular disease is a broad term describing disease of the heart and/or blood vessels. The main blood vessel supplying the body with oxygenated blood is the aorta. The aorta may become affected in diseases such as atherosclerosis and aneurysm. Researchers investigating these diseases would benefit from direct observation of the aorta to characterize disease progression as well as to evaluate efficacy of potential therapeutics. The goal of this protocol is to describe proper isolation and excision of the aorta to aid investigators researching cardiovascular disease. Isolation and excision of the aorta allows investigators to look at gross morphometric changes as wells as allowing them to preserve and stain the tissue to look at histologic changes if desired. The aorta may be used for molecular studies to evaluate protein and gene expression to discover targets of interest and mechanisms of action. This technique is superior to imaging modalities as they have inherent limitations in technology and cost. Additionally, primary isolated cells from a freshly isolated and excised aorta can allowing researchers to perform further in situ and in vitro assays. The isolation and excision of the aorta has the limitation of having to sacrifice the animal however, in this case the benefits outweigh the harm as it is the most versatile technique in the study of aortic disease.     

Highly Sensitive Real-Time In Vivo Imaging of an Influenza Reporter Virus Reveals Dynamics of Replication and Spread

The continual public health threat posed by the emergence of novel influenza viruses necessitates the ability to rapidly monitor infection and spread in experimental systems. To analyze real-time infection dynamics, we have created a replication-competent influenza reporter virus suitable for in vivo imaging. The reporter virus encodes the small and bright NanoLuc luciferase whose activity serves as an extremely sensitive readout of viral infection. This virus stably maintains the reporter construct and replicates in culture and in mice with near-native properties. Bioluminescent imaging of the reporter virus permits serial observations of viral load and dissemination in infected animals, even following clearance of a sublethal challenge. We further show that the reporter virus recapitulates known restrictions due to host range and antiviral treatment, suggesting that this technology can be applied to studying emerging influenza viruses and the impact of antiviral interventions on infections in vivo. These results describe a generalizable method to quickly determine the replication and pathogenicity potential of diverse influenza strains in animals.     

Enhanced Detection of Tuberculous Mycobacteria in Animal Tissues Using a Semi-Nested Probe-Based Real-Time PCR

Bovine tuberculosis has been tackled for decades by costly eradication programs in most developed countries, involving the laboratory testing of tissue samples from allegedly infected animals for detection of Mycobacterium tuberculosis complex (MTC) members, namely Mycobacterium bovis. Definitive diagnosis is usually achieved by bacteriological culture, which may take up to 6–12 weeks, during which the suspect animal carcass and herd are under sanitary arrest. In this work, a user-friendly DNA extraction protocol adapted for tissues was coupled with an IS6110-targeted semi-nested duplex real-time PCR assay to enhance the direct detection of MTC bacteria in animal specimens, reducing the time to achieve a diagnosis and, thus, potentially limiting the herd restriction period. The duplex use of a novel β-actin gene targeted probe, with complementary targets in most mammals, allowed the assessment of amplification inhibitors in the tissue samples. The assay was evaluated with a group of 128 fresh tissue specimens collected from bovines, wild boars, deer and foxes. Mycobacterium bovis was cultured from 57 of these samples. Overall, the full test performance corresponds to a diagnostic sensitivity and specificity of 98.2% (CI_P95% 89.4–99.9%) and 88.7% (CI_P95% 78.5–94.7%), respectively. An observed kappa coefficient was estimated in 0.859 (CI_P95% 0.771–0.948) for the overall agreement between the semi-nested PCR assay and the bacteriological culture. Considering only bovine samples (n = 69), the diagnostic sensitivity and specificity were estimated in 100% (CI_P95% 84.0–100%) and 97.7% (CI_P95% 86.2–99.9%), respectively. Eight negative culture samples exhibiting TB-like lesions were detected by the semi-nested real-time PCR, thus emphasizing the increased potential of this molecular approach to detect MTC-infected animal tissues. This novel IS6110-targeted assay allows the fast detection of tuberculous mycobacteria in animal specimens with very high sensitivity and specificity, being amenable and cost effective for use in the routine veterinary diagnostic laboratory with further automation possibilities.     

Instability of the morpholine-degradative phenotype in mycobacteria isolated from activated sludge

The stability of the morpholine-degradative phenotype was studied in a group of nine distinct strains of mycobacteria, all isolated from an activated sludge plant treating morpholine.
Variants incapable of morpholine degradation arose from all strains at high frequency following growth under non-selective conditions. However, strains differed with respect to the frequency at which Mor⁻ variants arose. No spontaneous reversion to wild type could be demonstrated. Six of the nine mycobacterial strains contained plasmids, each had a different plasmid profile. The plasmid profiles of wild type and Mor⁻ variants were compared. In no case could loss of the ability to grow on morpholine be correlated with loss of a complete plasmid. However, evidence is presented which suggests that in two strains loss of the morpholine-degradative phenotype may be correlated with a decrease in the size of a very large plasmid implying deletion of the DNA encoding morpholine degradation. The techniques employed to screen for plasmids in mycobacteria are discussed.     

An Investigation of Electron Spin Resonance in Wild Type Chlamydomonas reinhardi and Mutant Strains Having Impaired Photosynthesis

The electron spin resonance signals of wild type Chlamydomonas reinhardi and three mutant strains having impaired photosynthesis have been investigated. The wild type strain generates two different electron spin resonance signals. Signal I is obtained without illumination (i.e., dark signal) whereas signal II is generated preferentially only by red light. Signal I is missing from wild type cells that have been cultured in the dark, but it returns after these dark-grown cells have been illuminated. Chloroplast fragments obtained from the three mutant strains cannot photoreduce TPN. Two of the strains lack the dark signal I while the third strain has both signal I and signal II. Other studies have revealed that the two mutant strains which lack signal I give no Hill reaction but that they can photoreduce TPN if supplied with an artificial reductant. The mutant strain which has both electron spin resonance signals can carry out the Hill reaction, yet it too will not photoreduce TPN unless reductant is supplied. The electron spin resonance signals generated by the wild type and mutant strains are discussed in terms of the pathway of TPN photoreduction, and it is suggested that signal I is associated with one of the two light-dependent phases of this pathway.     

Relationship between Phylogenetic Groups, Genotypic Clusters, and Virulence Gene Profiles of Escherichia coli Strains from Diverse Human and Animal Sources

Escherichia coli strains in water may originate from various sources, including humans, farm and wild animals, waterfowl, and pets. However, potential human health hazards associated with E. coli strains present in various animal hosts are not well known. In this study, E. coli strains from diverse human and animal sources in Minnesota and western Wisconsin were analyzed for the presence of genes coding for virulence factors by using multiplex PCR and biochemical reactions. Of the 1,531 isolates examined, 31 (2%) were found to be Shiga toxin-producing E. coli (STEC) strains. The majority of these strains, which were initially isolated from the ruminants sheep, goats, and deer, carried the stx_1c and/or stx_2d, ehxA, and saa genes and belonged to E. coli phylogenetic group B1, indicating that they most likely do not cause severe human diseases. All the STEC strains, however, lacked eae. In contrast, 26 (1.7%) of the E. coli isolates examined were found to be potential enteropathogenic E. coli (EPEC) strains and consisted of several intimin subtypes that were distributed among various human and animal hosts. The EPEC strains belonged to all four phylogenetic groups examined, suggesting that EPEC strains were relatively widespread in terms of host animals and genetic background. Atypical EPEC strains, which carried an EPEC adherence factor plasmid, were identified among E. coli strains from humans and deer. DNA fingerprint analyses, done using the horizontal, fluorophore-enhanced repetitive-element, palindromic PCR technique, indicated that the STEC, potential EPEC, and non-STEC ehxA-positive E. coli strains were genotypically distinct and clustered independently. However, some of the potential EPEC isolates were genotypically indistinguishable from nonpathogenic E. coli strains. Our results revealed that potential human health hazards associated with pathogenic E. coli strains varied among the animal hosts that we examined and that some animal species may harbor a greater number of potential pathogenic strains than other animal species.     

Antimicrobial Susceptibility of Standard Strains of Nontuberculous Mycobacteria by Microplate Alamar Blue Assay

In this study, 24 standard nontuberculous mycobacteria (NTM) species strains including 12 slowly growing mycobacteria strains and 12 rapidly growing mycobacteria strains were subjected to drug susceptibility testing using microplate Alamar Blue assay-based 7H9 broth. The most active antimicrobial agents against the 24 NTM strains were streptomycin, amikacin, the fluoroquinolones, and the tetracyclines. Mycobacterium chelonae, Mycobacterium abscessus, Mycobacterium bolletii, and Mycobacterium simiae are resistant to most antimicrobial agents. The susceptibility results of this study from 24 NTM standard strains can be referenced by clinicians before susceptibility testing for clinical isolates is performed or when conditions do not allow for susceptibility testing. The application of broth-based methods is recommended by the Clinical and Laboratory Standards Institute, and the documentation of the susceptibility patterns of standard strains of mycobacteria can improve the international standardization of susceptibility testing methods.     

Live en face imaging of aortic valve leaflets under mechanical stress

Soft tissues, such as tendons, skin, arteries, or lung, are constantly subject to mechanical stresses in vivo. None more so than the aortic heart valve that experiences an array of forces including shear stress, cyclic pressure, strain, and flexion. Anisotropic biaxial cyclic stretch maintains valve homeostasis; however, abnormal forces are implicated in disease progression. The response of the valve endothelium to deviations from physiological levels has not been fully characterized. Here, we show the design and validation of a novel stretch apparatus capable of applying biaxial stretch to viable heart valve tissue, while simultaneously allowing for live en face endothelial cell imaging via confocal laser scanning microscopy (CLSM). Real-time imaging of tissue is possible while undergoing highly characterized mechanical conditions and maintaining the native extracellular matrix. Thus, it provides significant advantages over traditional cell culture or in vivo animal models. Planar biaxial tissue stretching with simultaneous live cell imaging could prove useful in studying the mechanobiology of any soft tissue.     

Rapid and Accurate Identification of Mycobacterium tuberculosis Complex and Common Non-Tuberculous Mycobacteria by Multiplex Real-Time PCR Targeting Different Housekeeping Genes

Rapid and accurate identification of mycobacteria isolates from primary culture is important due to timely and appropriate antibiotic therapy. Conventional methods for identification of Mycobacterium species based on biochemical tests needs several weeks and may remain inconclusive. In this study, a novel multiplex real-time PCR was developed for rapid identification of Mycobacterium genus, Mycobacterium tuberculosis complex (MTC) and the most common non-tuberculosis mycobacteria species including M. abscessus, M. fortuitum, M. avium complex, M. kansasii, and the M. gordonae in three reaction tubes but under same PCR condition. Genetic targets for primer designing included the 16S rDNA gene, the dnaJ gene, the gyrB gene and internal transcribed spacer (ITS). Multiplex real-time PCR was setup with reference Mycobacterium strains and was subsequently tested with 66 clinical isolates. Results of multiplex real-time PCR were analyzed with melting curves and melting temperature (T _m) of Mycobacterium genus, MTC, and each of non-tuberculosis Mycobacterium species were determined. Multiplex real-time PCR results were compared with amplification and sequencing of 16S-23S rDNA ITS for identification of Mycobacterium species. Sensitivity and specificity of designed primers were each 100?% for MTC, M. abscessus, M. fortuitum, M. avium complex, M. kansasii, and M. gordonae. Sensitivity and specificity of designed primer for genus Mycobacterium was 96 and 100?%, respectively. According to the obtained results, we conclude that this multiplex real-time PCR with melting curve analysis and these novel primers can be used for rapid and accurate identification of genus Mycobacterium, MTC, and the most common non-tuberculosis Mycobacterium species.     

Wild Animal Mycobacterial Isolates

Thirteen strains of mycobacteria isolated from deer and various species of wild birds were analysed by gas chromatography (GG) for cellular fatty acids and by thin-layer chromatography (TLG) for polar lipids. These strains were compared to reference strains of Mycobacterium avium, M. para tuberculosis and M. mal-moense. All the examined strains exhibited a generally similar fatty acid pattern characterized by relatively large amounts of hexadenca-noate (16:0), octadecenoate (18:1), octadecanoate (18:0) and 10-me-thyl-octadecanoate (tuberculostearic acid, 10-Me-18:0). Several additional acids were also generally present but in smaller amounts. By means of small but distinct differences in fatty acid composition, the wild animal isolates could be distinguished from both M. paratuber-culosis and M. malmoense but not from M. avium. The TLG polar lipid patterns on the other hand separated the wild animal isolates into 2 distinct groups of complex and simple polar lipid composition which corresponded to the morphologically smooth and rough types, respectively. The complex patterns of the smooth strains were comparable to those of the M. avium serovars whereas both the rough wild animal isolates and all the M. paratuber-culosis strains showed a simple pattern of polar lipids. Both fatty acid profiles and TLG polar lipid patterns support allocation of the wild animal isolates to the MAIS complex. Moreover, the 2 chemical techniques, particularly the GC procedure, are very useful for a more rapid and precise identification of the slow-growing wild animal mycobacterial isolates which have hitherto been characterized on basis of vague criteria.     

Wild birds and urban pigeons as reservoirs for diarrheagenic <Emphasis Type="Italic">Escherichia coli</Emphasis> with zoonotic potential

In order to describe the role of wild birds and pigeons in the transmission of shiga toxigenic Escherichia coli (STEC) and enteropathogenic Escherichia coli (EPEC) to humans and other animals, samples were collected from cloacae and oropharynx of free-living wild birds and free-living pigeons. Two STEC (0.8%) and five EPEC strains (2.0%) were isolated from wild birds and four EPEC strains (2.0%) were recovered from pigeons. Serogroups, sequence types (STs) and virulence genes, such as saa, iha, lpfA _O113, ehxA, espA, nleB and nleE, detected in this study had already been implicated in human and animal diseases. Multidrug resistance (MDR) was found in 25.0% of the pigeon strains and in 57.0% of the wild bird strains; the wild birds also yielded one isolate carrying extended-spectrum β-lactamases (ESBLs) gene bla _CTX-M-8. The high variability shown by PFGE demonstrates that there are no prevalent E. coli clones from these avian hosts. Wild birds and pigeons could act as carriers of multidrug-resistant STEC and EPEC and therefore may constitute a considerable hazard to human and animal health by transmission of these strains to the environment.     

Phenotypic differentiation of bifidobacteria of human and animal origins.

The phenotypes of 153 strains belonging or related to the genus Bifidobacterium were studied. These organisms included 38 collection strains and 115 wild strains (41 strains of human origin, 56 strains of animal origin, and 18 strains obtained from rivers or sewage). Our phenotypic analysis revealed seven main groups that were subdivided into 20 subgroups. Seven subgroups contained no type or collection strain. Among the human strains, the type strains of Bifidobacterium pseudocatenulatum and B. catenulatum fell into group I, which contained the type strains of B. adolescentis (subgroup Ib), B. dentium (subgroup Ic), and B. angulatum (ungrouped). The type strain of B. breve belonged to subgroup IIIa1, and the type strains of B. infantis and B. longum fell into subgroup IIIb1. Group VII comprised only wild strains that were isolated from human infant feces. Among the animal strains, group II consisted mainly of bifidobacteria that were isolated from pig feces and contained the type strains of B. suis (subgroup IIb), B. thermophilum (subgroup IIf), B. choerinum, and B. boum (ungrouped). Wild strains belonging to group V were isolated from pig, calf, cow, and chicken feces; this included the type strains of B. animalis (subgroup Va), B. magnum (subgroup Vb), B. pseudolongum, and B. globosum (subgroup Vc). The strains of human origin (groups I, III, and VII) were well separated from the animal strains (groups II, IV, and V). It was not surprising that the wild strains isolated from surface water or sewage were distributed in the animal groups as well as the human groups. Thus, bifidobacteria can be considered to be successful indicators of human or animal fecal pollution when they are correctly classified. The acidification patterns were not adequate to differentiate Bifidobacterium species, as determined previously by Mitsuoka (Bifidobacteria Microflora 3:11-28, 1984) and Scardovi (p. 1418-1434, in P. H. A. Sneath, N. S. Mair, M. E. Sharpe, and J. G. Holt, ed., Bergey's Manual of Systematic Bacteriology, vol. 2, 1986). However, enzymatic tests furnished new taxonomic criteria for the genus.     

Catecholates and mixed catecholate hydroxamates as artificial siderophores for mycobacteria

Different mono-, bis- or triscatecholates and mixed mono- or biscatecholate hydroxamates were synthesized as potential siderophores for mycobacteria. Siderophore activity was tested by growth promotion assays using wild type strains and iron transport mutants of mycobacteria as well as Gram-negative bacteria. Some triscatecholates and biscatecholate hydroxamates were active in mutants of Mycobacterium smegmatis deficient in mycobactin and exochelin biosynthesis or exochelin permease, respectively, indicating an uptake route independent of the exochelin/mycobactin pathway. Structure activity relationships were studied. Ampicillin conjugates of some of these compounds were inactive against mycobacteria but active against Gram-negative bacteria.