The mycobacterial DNA-binding protein 1 (MDP1) from Mycobacterium bovis BCG influences various growth characteristics

<p>Abstract</p> <p>Background</p> <p>Pathogenic mycobacteria such as <it>M. tuberculosis</it>, <it>M. bovis </it>or <it>M. leprae </it>are characterised by their extremely slow growth rate which plays an important role in mycobacterial virulence and eradication of the bacteria. Various limiting factors influence the generation time of mycobacteria, and the mycobacterial DNA-binding protein 1 (MDP1) has also been implicated in growth regulation. Our strategy to investigate the role of MDP1 in mycobacterial growth consisted in the generation and characterisation of a <it>M. bovis </it>BCG derivative expressing a MDP1-antisense gene.</p> <p>Results</p> <p>The expression rate of the MDP1 protein in the recombinant <it>M. bovis </it>BCG containing the MDP1-antisense plasmid was reduced by about 50% compared to the reference strain <it>M. bovis </it>BCG containing the empty vector. In comparison to this reference strain, the recombinant <it>M. bovis </it>BCG grew faster in broth culture and reached higher cell masses in stationary phase. Likewise its intracellular growth in mouse and human macrophages was ameliorated. Bacterial clumping in broth culture was reduced by the antisense plasmid. The antisense plasmid increased the susceptibility of the bacteria towards Ampicillin. 2-D protein gels of bacteria maintained under oxygen-poor conditions demonstrated a reduction in the number and the intensity of many protein spots in the antisense strain compared to the reference strain.</p> <p>Conclusion</p> <p>The MDP1 protein has a major impact on various growth characteristics of <it>M. bovis </it>BCG. It plays an important role in virulence-related traits such as aggregate formation and intracellular multiplication. Its impact on the protein expression in a low-oxygen atmosphere indicates a role in the adaptation to the hypoxic conditions present in the granuloma.</p

Novel Immunomodulatory Flagellin-Like Protein FlaC in Campylobacter jejuni and Other Campylobacterales

The human diarrheal pathogens Campylobacter jejuni and Campylobacter coli interfere with host innate immune signaling by different means, and their flagellins, FlaA and FlaB, have a low intrinsic property to activate the innate immune receptor Toll-like receptor 5 (TLR5). We have investigated here the hypothesis that the unusual secreted, flagellin-like molecule FlaC present in C. jejuni, C. coli, and other Campylobacterales might activate cells via TLR5 and interact with TLR5. FlaC shows striking sequence identity in its D1 domains to TLR5-activating flagellins of other bacteria, such as Salmonella, but not to nonstimulating Campylobacter flagellins. We overexpressed and purified FlaC and tested its immunostimulatory properties on cells of human and chicken origin. Treatment of cells with highly purified FlaC resulted in p38 activation. FlaC directly interacted with TLR5. Preincubation with FlaC decreased the responsiveness of chicken and human macrophage-like cells toward the bacterial TLR4 agonist lipopolysaccharide (LPS), suggesting that FlaC mediates cross-tolerance. C. jejuni flaC mutants induced an increase of cell responses in comparison to those of the wild type, which was suppressed by genetic complementation. Supplementing excess purified FlaC likewise reduced the cellular response to C. jejuni. In vivo, the administration of ultrapure FlaC led to a decrease in cecal interleukin 1β (IL-1β) expression and a significant change of the cecal microbiota in chickens. We propose that Campylobacter spp. have evolved a novel type of secreted immunostimulatory flagellin-like effector in order to specifically modulate host responses, for example toward other pattern recognition receptor (PRR) ligands, such as LPS. IMPORTANCE Flagellins not only are important for bacterial motility but are major bacterial proteins that can modulate host responses via Toll-like receptor 5 (TLR5) or other pattern recognition receptors. Campylobacterales colonizing the intestinal tracts of different host species harbor a gene coding for an unusual flagellin, FlaC, that is not involved in motility but is secreted and possesses a chimeric amino acid sequence composed of TLR5-activating and non-TLR5-activating flagellin sequences. Campylobacter jejuni FlaC activates cells to increase in cytokine expression in chicken and human cells, promotes cross-tolerance to TLR4 ligands, and alters chicken cecal microbiota. We propose that FlaC is a secreted effector flagellin that has specifically evolved to modulate the immune response in the intestinal tract in the presence of the resident microbiota and may contribute to bacterial persistence. The results also strengthen the role of the flagellar type III apparatus as a functional secretion system for bacterial effector proteins

Closely related Campylobacter jejuni strains from different sources reveal a generalist rather than a specialist lifestyle

Background: Campylobacter jejuni and Campylobacter coli are human intestinal pathogens of global importance. Zoonotic transmission from livestock animals or animal-derived food is the likely cause for most of these infections. However, little is known about their general and host-specific mechanisms of colonization, or virulence and pathogenicity factors. In certain hosts, Campylobacter species colonize persistently and do not cause disease, while they cause acute intestinal disease in humans. Results: Here, we investigate putative host-specificity using phenotypic characterization and genome-wide analysis of genetically closely related C. jejuni strains from different sources. A collection of 473 fresh Campylobacter isolates from Germany was assembled between 2006 and 2010 and characterized using MLST. A subset of closely related C. jejuni strains of the highly prevalent sequence type ST-21 was selected from different hosts and isolation sources. PCR typing of strain- variable genes provided evidence that some genes differed between these strains. Furthermore, phenotypic variation of these strains was tested using the following criteria: metabolic variation, protein expression patterns, and eukaryotic cell interaction. The results demonstrated remarkable phenotypic diversity within the ST-21 group, which however did not correlate with isolation source. Whole genome sequencing was performed for five ST-21 strains from chicken, human, bovine, and food sources, in order to gain insight into ST-21 genome diversity. The comparisons showed extensive genomic diversity, primarily due to recombination and gain of phage-related genes. By contrast, no genomic features associated with isolation source or host were identified. Conclusions: The genome information and phenotypic data obtained in vitro and in a chicken infection model provided little evidence of fixed adaptation to a specific host. Instead, the dominant C. jejuni ST-21 appeared to be characterized by phenotypic flexibility and high genetic microdiversity, revealing properties of a generalist. High genetic flexibility might allow generalist variants of C. jejuni to reversibly express diverse fitness factors in changing environments

A virulence factor as a therapeutic: the probiotic Enterococcus faecium SF68 arginine deiminase inhibits innate immune signaling pathways

The probiotic bacterial strain Enterococcus faecium SF68 has been shown to alleviate symptoms of intestinal inflammation in human clinical trials and animal feed supplementation studies. To identify factors involved in immunomodulatory effects on host cells, E. faecium SF68 and other commensal and clinical Enterococcus isolates were screened using intestinal epithelial cell lines harboring reporter fusions for NF-κB and JNK(AP-1) activation to determine the responses of host cell innate immune signaling pathways when challenged with bacterial protein and cell components. Cell-free, whole-cell lysates of E. faecium SF68 showed a reversible, inhibitory effect on both NF-κB and JNK(AP-1) signaling pathway activation in intestinal epithelial cells and abrogated the response to bacterial and other Toll-like receptor (TLR) ligands. The inhibitory effect was species-specific, and was not observed for E. avium, E. gallinarum, or E. casseliflavus. Screening of protein fractions of E. faecium SF68 lysates yielded an active fraction containing a prominent protein identified as arginine deiminase (ADI). The E. faecium SF68 arcA gene encoding arginine deiminase was cloned and introduced into E. avium where it conferred the same NF-κB inhibitory effects on intestinal epithelial cells as seen for E. faecium SF68. Our results indicate that the arginine deiminase of E. faecium SF68 is responsible for inhibition of host cell NF-κB and JNK(AP-1) pathway activation, and is likely to be responsible for the anti-inflammatory and immunomodulatory effects observed in prior clinical human and animal trials. The implications for the use of this probiotic strain for preventive and therapeutic purposes are discussed

Metabolic Characteristics of Porcine LA-MRSA CC398 and CC9 Isolates from Germany and China via Biolog Phenotype MicroArrayTM

Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) is an important zoonotic pathogen, often multi-resistant to antimicrobial agents. Among swine, LA-MRSA of clonal complex (CC) 398 dominates in Europe, Australia and the Americas, while LA-MRSA-CC9 is the main epidemic lineage in Asia. Here, we comparatively investigated the metabolic properties of rare and widespread porcine LA-MRSA isolates from Germany and China using Biolog Phenotype MicroArray technology to evaluate if metabolic variations could have played a role in the development of two different epidemic LA-MRSA clones in swine. Overall, we were able to characterize the isolates’ metabolic profiles and show their tolerance to varying environmental conditions. Sparse partial least squares discriminant analysis (sPLS-DA) supported the detection of the most informative substrates and/or conditions that revealed metabolic differences between the LA-MRSA lineages. The Chinese LA-MRSA-CC9 isolates displayed unique characteristics, such as a consistently delayed onset of cellular respiration, and increased, reduced or absent usage of several nutrients. These possibly unfavorable metabolic properties might promote the ongoing gradual replacement of the current epidemic LA-MRSA-CC9 clone in China with the emerging LA-MRSA-CC398 lineage through livestock trade and occupational exposure. Due to the enhanced pathogenicity of the LA-MRSA-CC398 clone, the public health risk posed by LA-MRSA from swine might increase further

Antimicrobial Resistance and Virulence of Methicillin-Resistant Staphylococcus aureus from Human, Chicken and Environmental Samples within Live Bird Markets in Three Nigerian Cities

Background: Methicillin-resistant Staphylococcus aureus (MRSA) has emerged as a major threat to public health. This study investigated the occurrence of MRSA in humans, chickens, chicken meat and environmental samples within poultry farms and live bird markets in southwestern Nigeria. Methods: MRSA were isolated using selective culture and tested for antimicrobial susceptibility by broth microdilution. Selected isolates were characterized by whole genome sequencing (WGS). From WGS data, spa, dru, multilocus sequence typing (MLST) and SCCmec types, but also virulence and antimicrobial resistance genes, were identified. Results: Fifty-six MRSA isolates were detected in 734 samples. They showed resistance to β-lactams (100%), tetracycline (60.7%), ciprofloxacin (33.9%), erythromycin (28.6%), gentamicin (32.1%), and trimethoprim/sulfamethoxazole (10.7%). All 30 isolates investigated by WGS carried mecA, dfrG, and tet(38) genes. Other resistance genes detected were blaZ (83.3%), fosB (73.3%), tet(K) (60.0%), aacA-aphD (36.6%), aphA3 (33.3%), msr(A) (30.0%), mph(C) (30.0%), dfrS1 (3.3%), and sat4 (3.3%). Seven spa types (t091, t314, t657, t1476, t2331, t4690 and t12236), four known (dt9aw, dt10ao, dt10cj, and dt11a) and two novel (dt10dr and dt11dw) dru types, as well as five sequence types (ST8, ST121, ST152, ST772 and ST789) were found among the MRSA isolates. All ST121 isolates carried an SCCmec type IV cassette and were not dru-typeable. ST152 and ST121 were found only in specific sample categories within defined locations, while ST8 and ST772 were distributed across most sample categories and locations. Three SCCmec types, IVa, V and Vc, were identified. All MRSA isolates possessed virulence genes including aur, clpP, coa, fnbA, esaA, hly, hla, ica, isdA, srtB, sspA, and vWbp, among others. The toxic shock syndrome toxin gene (tst) was not detected in any isolate, whereas the Pantone–Valentine leukocidin genes lukF-PV/lukS-PV were present in all ST121, all ST772, and all but one ST152 isolates. Conclusion: The results of this study (i) showed that chicken meat is contaminated by MRSA and (ii) suggested that live bird markets may serve as focal points for the dissemination of MRSA within the community

Closely related Campylobacter jejuni strains from different sources reveal a generalist rather than a specialist lifestyle

Background: Campylobacter jejuni and Campylobacter coli are human intestinal pathogens of global importance. Zoonotic transmission from livestock animals or animal-derived food is the likely cause for most of these infections. However, little is known about their general and host-specific mechanisms of colonization, or virulence and pathogenicity factors. In certain hosts, Campylobacter species colonize persistently and do not cause disease, while they cause acute intestinal disease in humans. Results: Here, we investigate putative host-specificity using phenotypic characterization and genome-wide analysis of genetically closely related C. jejuni strains from different sources. A collection of 473 fresh Campylobacter isolates from Germany was assembled between 2006 and 2010 and characterized using MLST. A subset of closely related C. jejuni strains of the highly prevalent sequence type ST-21 was selected from different hosts and isolation sources. PCR typing of strain-variable genes provided evidence that some genes differed between these strains. Furthermore, phenotypic variation of these strains was tested using the following criteria: metabolic variation, protein expression patterns, and eukaryotic cell interaction. The results demonstrated remarkable phenotypic diversity within the ST-21 group, which however did not correlate with isolation source. Whole genome sequencing was performed for five ST-21 strains from chicken, human, bovine, and food sources, in order to gain insight into ST-21 genome diversity. The comparisons showed extensive genomic diversity, primarily due to recombination and gain of phage-related genes. By contrast, no genomic features associated with isolation source or host were identified. Conclusions: The genome information and phenotypic data obtained in vitro and in a chicken infection model provided little evidence of fixed adaptation to a specific host. Instead, the dominant C. jejuni ST-21 appeared to be characterized by phenotypic flexibility and high genetic microdiversity, revealing properties of a generalist. High genetic flexibility might allow generalist variants of C. jejuni to reversibly express diverse fitness factors in changing environments

Host-dependent factors in the pathogenicity and persistence of Salmonella serovars in animals and man

Salmonella enterica führt deutschlandweit zu den zweithäufigsten lebensmittelbedingten Infektionskrankheiten. Auffällig sind diesbezüglich die Heterogenität des Wirtsspektrums und die Variabilität der Krankheitsverläufe. Zur Aufklärung möglicher Wirts- und Serovar-abhängiger Faktoren, die zur unterschiedlichen Adaptation und Pathogenese von Salmonella beitragen, wurden systematisch vergleichende Infektionsstudien mit Salmonella Serovaren unterschiedlichen Wirtsspektrums in intestinalen Epithel- und Makrophagenzellen porcinen, aviären und humanen Ursprungs als repräsentative Zelltypen der frühen Infektionsphase durchgeführt. Die Wirts-Pathogen- Interaktionen wurden hinsichtlich Invasivität und Persistenz der Serovare, daraus resultierender zytotoxischer und inflammatorischer Effekte sowie der genomweiten Genexpressionsänderung des Wirtes untersucht. Unter Berücksichtigung aller Faktoren ergaben sich Phänotypen, die wichtige Hinweise auf Mechanismen zur Wirtsadaptation und Pathogenese liefern. Die Spezies- und Zelltyp-übergreifend hohen Invasionsraten der Breitspektrum Serovare S. Typhimurium und S. Enteritidis verursachten eine starke proinflammatorische Antwort sowie im verhältnismäßig hohen Maße zytotoxische Effekte, was in vivo zu einer starken Inflammation und örtlichen Begrenzung der Infektion führen würde, wie sie etwa bei der humanen Gastroenteritis durch Breitspektrum- Serovare zu beobachten ist. Dagegen stellte sich S. Choleraesuis insbesondere in den porcinen Zelllinien als schwach invasiv dar, persistierte jedoch in den meisten Zelllinien ähnlich gut wie die Breitspektrum-Serovare und führte dementsprechend zu einer mittleren NF-κB Aktivierung und Zytotoxizität. Diesbezüglich stellten sich die verwendeten porcinen Zelllinien auch bei hohen MOIs als sehr robust heraus, die zudem auf verschiedene synthetische pathogen- associated molecular patterns (PAMPs) wie Flagellin, PGN-Fragmenten und LPS nur schwach bis mäßig reagierten. In vivo würde die beobachtete schwache Invasivität und NF-κB Aktivierung von S. Choleraesuis vor allem in porcinen Zellen und die relativ gute Persistenz, die durch das robuste Verhalten der porcinen Zelllinien jedoch weitestgehend toleriert wird, auf eine „sanfte“ Infektion ohne starke Inflammation hindeuten, die damit eine systemische Ausbreitung ermöglicht, wie sie für S. Choleraesuis im Schwein beschrieben wurde. Die Geflügel-beschränkten Serovare S. Gallinarum und S. Pullorum invadierten und persistierten dagegen insgesamt verhältnismäßig schlecht. Dementsprechend verursachten sie kaum zytotoxische Effekte. Auffällig war diesbezüglich auch die hohe Sensibilität der Hühnermakrophagen, die bereits bei einer moderaten MOI um 5 bei höher invasiven Serovaren abstarben. Demnach begünstigen die Charakterisitka von S. Gallinarum und S. Pullorum trotz eines relativ hohen inflammatorischen Potentials das Überleben in sehr sensibel reagierenden Hühnermakrophagen, die für eine systemische Infektion, wie sie für S. Gallinarum und S. Pullorum im Huhn beschrieben sind, notwendig wären. Untersuchungen mit synthetischen PAMPs sowie entsprechenden Salmonella Deletions-mutanten mit beeinträchtigter Flagellensynthese und Peptidoglycanrecycling deuteten zudem vor allem auf die initiale Interaktion mit Epithelzellen eher als die mit Makrophagen als diskriminierenden Faktor für eine Spezies-spezifische Anpassung und Pathogenität hin. Zusammenfassend verdeutlichen die Ergebnisse dieser Arbeit, dass die Wirtsadaptation von Salmonella neben einer möglichen bakteriellen Adaptation insbesondere aus immunologischen oder anatomischen Eigenschaften resultiert, die einem Serovar, das im Vergleich zu Breitspektrum-Serovaren Einschränkungen der Virulenz oder des Metabolismus aufweist, in bestimmten Spezies eine erfolgreiche Infektion und Persistenz ermöglichen.Salmonella enterica is the second most prevalent causative agent of foodbourne diseases in Germany. With regard to their host range and pathogenicity, Salmonella is a group of strong heterogenicity. To identify possible host- and serovar-specific determinants involved in adaptation and different pathogenicities of Salmonella, we systematically compared infections with Salmonella serovars of different host-ranges in intestinal epithelial and macrophage-like cells of porcine, avian and human origin as representative of the early affected cell types during infection. Host-pathogen interactions were analyzed with regard to invasiveness and persistence of the serovars, cytotoxic effects and host immune responses as well as whole genome expression patterns of the host. The outcomes of these assays revealed certain phenotypes suggesting possible mechanisms for host adaptation and pathogenicity. The high invasion rates observed for broad host-range serovars S. Typhimurium and S. Enteritidis correlated with a strong proinflammation und resulted in relatively strong cytotoxic effects, which suggest a strong inflammation and a local limitation of the infection in vivo, consistent with observations of human gastroenteritis due to broad host-range serovars. In contrast, S. Choleraesuis strains were weakly invasive, especially in porcine cells, but showed similar levels of persistence in most cell lines as the broad host- range serovars. The resulting innate immune responses were correspondingly intermediate, with average NF-κB activation levels and associated cytotoxicity. The porcine cell lines were found to be even at high infective doses extremely robust and responded to synthetic pathogen-associated molecular patterns (PAMPs) with only average or weak NF-kB activation. The lower invasiveness and NF-κB activation of the swine-adapted serovar S. Choleraesuis in porcine cells but the fairly high-level persistence which is tolerated by these host cells would result in a “soft” infection without a strong inflammation in vivo that allows this serovar to spread systemically as has been described for S. Choleraesuis infections in pigs. The poultry- restricted Salmonella serovars Gallinarum and Pullorum showed the lowest invasion rates and persisted poorly, independent of the cell type or species. This correlated with no or only very weak cytotoxic effects. In this context we observed a strong sensitivity of chicken macrophages with early onset of cell cytoxicity at even moderate MOI of 5 with the more highly invasive serovars. These observations suggest the characteristics for Salmonella serovars Gallinarum and Pullorum enabling them to survive despite a relatively high inflammatory potential. This conclusion would also be consistent with the patterns of systemic infections reported for S. Gallinarum / S. Pullorum infections in chickens. In addition, screening of the cell lines of different species with synthetic PAMPs like flagellin, PGN-fragments or LPS as well as Salmonella mutants with disrupted synthesis of flagella and recycling of peptidogylcan indicate the epithelial cell layer rather than interactions with macrophages as discriminating factor enabling a species-specific host adaptation and pathogenicity. Rather than an adaptation to a particular host, this study suggests host adaptation is the result of a particular host providing the necessary prerequisites, anatomical or immunological, which permit successful infection and persistence of serovars which may have virulence or metabolic disadvantages compared to broad host-range serovars

Plasmid-Coded Linezolid Resistance in Methicillin-Resistant Staphylococcus&nbsp;aureus from Food and Livestock in Germany

Resistance of methicillin-resistant Staphylococcus&nbsp;aureus (MRSA) from food and livestock to last resort antibiotics such as linezolid is highly concerning, since treatment options for infections in humans might be diminished. Known mechanisms of linezolid resistance include point mutations in the 23S rRNA gene and in the ribosomal proteins L3, L4 and L22 as well as an acquisition of the cfr, optrA or poxtA gene. The objective of our study was to characterize antimicrobial resistance (AMR) determinants and phylogenetic relationships among linezolid-resistant (LR-) MRSA from food and livestock. In total, from more than 4000 incoming isolates in the years 2012 to 2021, only two strains from 2015 originating from pig samples exhibited linezolid resistance in the antimicrobial susceptibility testing with MICs of &ge;8 mg/L. These LR-MRSA were characterized in detail by whole-genome sequencing and phylogenetic analyses using cgMLST. The LR-MRSA strains showed resistances to ten and eight different antibiotics, respectively. Both strains harbored plasmid-coded cfr genes mediating the linezolid resistance. The cfr genes showed identical sequences in both strains. In addition to the cfr gene, genes for phenicol and clindamycin resistance were detected on the respective plasmids, opening the possibility for a co-selection. The LR-MRSA differed distantly in the phylogenetic analyses and also to other MRSA from pig samples in the year 2015. In conclusion, the occurrence of LR-MRSA in food and livestock seems to be very rare in Germany. However, carriage of plasmids with linezolid resistance determinants could lead to further linezolid-resistant strains by horizontal gene transfer

Phylogenetic Tracking of LA-MRSA ST398 Intra-Farm Transmission among Animals, Humans and the Environment on German Dairy Farms

Methicillin-resistant Staphylococcus&nbsp;aureus (MRSA) are a major threat to human and animal health, causing difficult-to-treat infections. The aim of our study was to evaluate the intra-farm transmission of livestock-associated (LA) MRSA sequence type (ST) 398 isolates on German dairy farms. A total of 115 LA-MRSA ST398 isolates originating from animals, humans and the environment of six dairy farms were analyzed by whole-genome sequencing and core genome multilocus sequence typing. Phylogenetic clusters of high allelic similarity were detected on all dairy farms, suggesting a MRSA transmission across the different niches. On one farm, closely related isolates from quarter milk samples (QMS), suckers of calf feeders and nasal cavities of calves indicate that MRSA may be transferred by feeding contaminated milk to calves. Detection of related MRSA isolates in QMS and teat cups (4/6 farms) or QMS and human samples (3/4 farms) pointed out a transmission of MRSA between cows during the milking process and a potential zoonotic risk. In conclusion, LA-MRSA ST398 isolates may spread between animals, humans and the environment on dairy farms. Milking time hygiene and other internal biosecurity measures on farms and pre-treatment of milk before feeding it to calves may reduce the risk of MRSA transmission