Thomas Köller

Mode of Expression and Functional Characterization of FCT-3 Pilus Region-Encoded Proteins in Streptococcus pyogenes Serotype M49

ABSTRACT The human pathogen Streptococcus pyogenes (group A streptococcus [GAS]) pilus components, suggested to play a role in pathogenesis, are encoded in the variable FCT (fibronectin- and collagen-binding T-antigen) region. We investigated the functions of sortase A (SrtA), sortase C2 (SrtC2), and the FctA protein of the most prevalent type 3 FCT region from a serotype M49 strain. Although it is considered a housekeeping sortase, SrtAs activity is involved in pilus formation in addition to its essentiality for GAS extracellular matrix protein binding, host cell adherence/internalization, survival in human blood, and biofilm formation. SrtC2 activity is crucial for pilus formation but dispensable for the other phenotypes tested in vitro. FctA is the major pilus backbone protein, simultaneously acting as the M49 T antigen, and requires SrtC2 and LepA, a signal peptidase I homologue, for monomeric surface expression and polymerization, respectively. Collagen-binding protein Cpa expression supports pilus formation at the pilus base. Immunofluorescence microscopy and fluorescence-activated cell sorting analysis revealed several unexpected expression patterns, as follows: (i) the monomeric pilus protein FctA was found exclusively at the old poles of GAS cells, (ii) FctA protein expression increased with lower temperatures, and (iii) FctA protein expression was restricted to 20 to 50% of a given GAS M49 population, suggesting regulation by a bistability mode. Notably, disruption of pilus assembly by sortase deletion rendered GAS serotype M49 significantly more aggressive in a dermonecrotic mouse infection model, indicating that sortase activity and, consequently, pilus expression allow a subpopulation of this GAS serotype to be less aggressive. Thus, pilus expression may not be a virulence attribute of GAS per se.

Rapid degradation of Streptococcus pyogenes biofilms by PlyC, a bacteriophage-encoded endolysin

OBJECTIVES Streptococcus pyogenes, or Group A streptococcus (GAS), has a propensity to colonize human tissues and form biofilms. Significantly, these biofilms are a contributing mechanism of antibiotic treatment failure in streptococcal disease. In this study, we evaluate a streptococcal-specific bacteriophage-encoded endolysin (PlyC), which is known to lyse planktonic streptococci, on both static and dynamic streptococcal biofilms. METHODS PlyC was benchmarked against antibiotics for MIC, MBC and minimum biofilm eradication concentration (MBEC). A biomass eradication assay based on crystal violet staining of the biofilm matrix was also used to quantify the anti-biofilm properties of PlyC. Finally, conventional fluorescence microscopy and laser scanning confocal microscopy were used to study the effects of PlyC on static and dynamic biofilms of GAS. RESULTS PlyC and antibiotics had similar MIC (range 0.02-0.08 mg/L) and MBC (range 0.02-1.25 mg/L) values on planktonic GAS. However, when GAS grew in biofilms, the MBEC values for antibiotics rose to clinically resistant values (≥400 mg/L) whereas PlyC had MBEC values two orders of magnitude lower by mass and four orders of magnitude lower by molarity than the conventional antibiotics. Laser scanning confocal microscopy revealed that PlyC destroys the biofilm as it diffuses through the matrix in a time-dependent fashion. CONCLUSIONS Our findings indicate that while streptococcal cells within a biofilm rapidly become refractory to traditional antibiotics, the biofilm matrix is readily destroyed by the lytic actions of PlyC.

The Streptococcus pyogenes Serotype M49 Nra-Ralp3 Transcriptional Regulatory Network and Its Control of Virulence Factor Expression from the Novel eno ralp3 epf sagA Pathogenicity Region

ABSTRACT Many Streptococcus pyogenes (group A streptococcus [GAS]) virulence factor- and transcriptional regulator-encoding genes cluster together in discrete genomic regions. Nra is a central regulator of the FCT region. Previous studies exclusively described Nra as a transcriptional repressor of adhesin and toxin genes. Here transcriptome and proteome analysis of a serotype M49 GAS strain and an isogenic Nra mutant of this strain revealed the complete Nra regulon profile. Nra is active in all growth phases tested, with the largest regulon in the transition phase. Almost exclusively, virulence factor-encoding genes are repressed by Nra; these genes include the GAS pilus operon, the capsule synthesis operon, the cytolysin-mediated translocation system genes, all Mga region core virulence genes, and genes encoding other regulators, like the Ihk/Irr system, Rgg, and two additional RofA-like protein family regulators. Surprisingly, our experiments revealed that Nra additionally acts as a positive regulator, mostly for genes encoding proteins and enzymes with metabolic functions. Epidemiological investigations revealed strong genetic linkage of one particular Nra-repressed regulator, Ralp3 (SPy0735), with a gene encoding Epf (extracellular protein factor from Streptococcus suis). In a serotype-specific fashion, this ralp3 epf gene block is integrated, most likely via transposition, into the eno sagA virulence gene block, which is present in all GAS serotypes. In GAS serotypes M1, M4, M12, M28, and M49 this novel discrete genetic region is therefore designated the eno ralp3 epf sagA (ERES) pathogenicity region. Functional experiments showed that Epf is a novel GAS plasminogen-binding protein and revealed that Ralp3 activity counteracts Nra and MsmR regulatory activity. In addition to the Mga and FCT regions, the ERES region is the third discrete chromosomal pathogenicity region. All of these regions are transcriptionally linked, adding another level of complexity to the known GAS growth phase-dependent regulatory network.

Environmental Acidification Drives S. pyogenes Pilus Expression and Microcolony Formation on Epithelial Cells in a FCT-Dependent Manner

Group A Streptococcus (GAS, Streptococcus pyogenes) is a Gram-positive human pathogen responsible for a diverse variety of diseases, including pharyngitis, skin infections, invasive necrotizing fasciitis and autoimmune sequelae. We have recently shown that GAS cell adhesion and biofilm formation is associated with the presence of pili on the surface of these bacteria. GAS pilus proteins are encoded in the FCT (Fibronectin- Collagen-T antigen) genomic region, of which nine different variants have been identified so far. In the present study we undertook a global analysis of GAS isolates representing the majority of FCT-variants to investigate the effect of environmental growth conditions on their capacity to form multicellular communities. For FCT-types 2, 3, 5 and 6 and a subset of FCT-4 strains, we observed that acidification resulting from fermentative sugar metabolism leads to an increased ability of the bacteria to form biofilm on abiotic surfaces and microcolonies on epithelial cells. The higher biofilm forming capacity at low environmental pH was directly associated with an enhanced expression of the genes encoding the pilus components and of their transcription regulators. The data indicate that environmental pH affects the expression of most pilus types and thereby the formation of multicellular cell-adhering communities that assist the initial steps of GAS infection.

Streptococcus pyogenes biofilms—formation, biology, and clinical relevance

Streptococcus pyogenes (group A streptococci, GAS) is an exclusive human bacterial pathogen. The virulence potential of this species is tremendous. Interactions with humans range from asymptomatic carriage over mild and superficial infections of skin and mucosal membranes up to systemic purulent toxic-invasive disease manifestations. Particularly the latter are a severe threat for predisposed patients and lead to significant death tolls worldwide. This places GAS among the most important Gram-positive bacterial pathogens. Many recent reviews have highlighted the GAS repertoire of virulence factors, regulators and regulatory circuits/networks that enable GAS to colonize the host and to deal with all levels of the host immune defense. This covers in vitro and in vivo studies, including animal infection studies based on mice and more relevant, macaque monkeys. It is now appreciated that GAS, like many other bacterial species, do not necessarily exclusively live in a planktonic lifestyle. GAS is capable of microcolony and biofilm formation on host cells and tissues. We are now beginning to understand that this feature significantly contributes to GAS pathogenesis. In this review we will discuss the current knowledge on GAS biofilm formation, the biofilm-phenotype associated virulence factors, regulatory aspects of biofilm formation, the clinical relevance, and finally contemporary treatment regimens and future treatment options.

Surveillance of Food- and Smear-Transmitted Pathogens in European Soldiers with Diarrhea on Deployment in the Tropics: Experience from the European Union Training Mission (EUTM) Mali

Introduction. Since 2013, European soldiers have been deployed on the European Union Training Mission (EUTM) in Mali. From the beginning, diarrhea has been among the most “urgent” concerns. Diarrhea surveillance based on deployable real-time PCR equipment was conducted between December 2013 and August 2014. Material and Methods. In total, 53 stool samples were obtained from 51 soldiers with acute diarrhea. Multiplex PCR panels comprised enteroinvasive bacteria, diarrhea-associated Escherichia coli (EPEC, ETEC, EAEC, and EIEC), enteropathogenic viruses, and protozoa. Noroviruses were characterized by sequencing. Cultural screening for Enterobacteriaceae with extended-spectrum beta-lactamases (ESBL) with subsequent repetitive sequence-based PCR (rep-PCR) typing was performed. Clinical information was assessed. Results. Positive PCR results for diarrhea-associated pathogens were detected in 43/53 samples, comprising EPEC (n = 21), ETEC (n = 19), EAEC (n = 15), Norovirus (n = 10), Shigella spp./EIEC (n = 6), Cryptosporidium parvum (n = 3), Giardia duodenalis (n = 2), Salmonella spp. (n = 1), Astrovirus (n = 1), Rotavirus (n = 1), and Sapovirus (n = 1). ESBL-positive Enterobacteriaceae were grown from 13 out of 48 samples. Simultaneous infections with several enteropathogenic agents were observed in 23 instances. Symptoms were mild to moderate. There were hints of autochthonous transmission. Conclusions. Multiplex real-time PCR proved to be suitable for diarrhea surveillance on deployment. Etiological attribution is challenging in cases of detection of multiple pathogens.

Mucosa‐attached bacterial community in Crohn's disease coheres with the clinical disease activity index

In inflammatory bowel diseases (IBD), microbial communities often become imbalanced suggesting abnormal microbial-gut interactions. In this study, we analysed the mucosa-attached gut microbiota from 26 Crohns disease (CD) patients using 16S rRNA gene amplicon sequencing. The samples were stratified according to their disease activity (Crohns disease activity index, CDAI). The different disease activity categories had a comparable bacterial richness. Bacterial communities of patients in remission and intermediate CDAI (0-220) were relatively similar and dominated by the genus Bacteroides (>40%). The bacterial composition of patients assigned to a high CDAI category was dominated by Pelomonas (25%) and Flavobacterium (13%) but had a low relative abundance of Bacteroidetes (4%). This indicates the presence of specific abundant bacterial taxa at different CDAI levels. In addition, bacterial communities were also significantly influenced when a tumour necrosis factor (TNF)-α inhibitor was applied or by the local mucosal inflammation level. As a consequence, a shift of the microbial composition may also indicate a change of the disease activity in CD patients.

Low Enteric Colonization with Multidrug-Resistant Pathogens in Soldiers Returning from Deployments- Experience from the Years 2007-2015.

This assessment describes the enteric colonization of German soldiers 8–12 weeks after returning from mostly but not exclusively subtropical or tropical deployment sites with third-generation cephalosporin-resistant Enterobacteriaceae, vancomycin-resistant enterococci (VRE), and methicillin-resistant Staphylococcus aureus (MRSA). Between 2007 and 2015, 828 stool samples from returning soldiers were enriched in nonselective broth and incubated on selective agars for Enterobacteriaceae expressing extended-spectrum beta-lactamases (ESBL), VRE and MRSA. Identification and resistance testing of suspicious colonies was performed using MALDI-TOF-MS, VITEK-II and agar diffusion gradient testing (bioMérieux, Marcy-l’Étoile, France). Isolates with suspicion of ESBL were characterized by ESBL/ampC disc-(ABCD)-testing and molecular approaches (PCR, Sanger sequencing). Among the returnees, E. coli with resistance against third-generation cephalosporins (37 ESBL, 1 ESBL + ampC, 1 uncertain mechanism) were found in 39 instances (4.7%). Associated quinolone resistance was found in 46.2% of these isolates. Beta-lactamases of the blaCTX-M group 1 predominated among the ESBL mechanisms, followed by the blaCTX-M group 9, and blaSHV. VRE of vanA-type was isolated from one returnee (0.12%). MRSA was not isolated at all. There was no clear trend regarding the distribution of resistant isolates during the assessment period. Compared with colonization with resistant bacteria described in civilians returning from the tropics, the colonization in returned soldiers is surprisingly low and stable. This finding, together with high colonization rates found in previous screenings on deployment, suggests a loss of colonization during the 8- to 12-week period between returning from the deployments and assessment.

Screening agars for MRSA: evaluation of a stepwise diagnostic approach with two different selective agars for the screening for methicillin-resistant Staphylococcus aureus (MRSA)

BackgroundColonization with methicillin-resistant Staphylococcus aureus (MRSA) poses a hygiene risk that does not spare field hospitals or military medical field camps during military deployments. Diagnostic options for unambiguously identifying MRSA isolates are usually scarce in military environments. In this study, we assessed the stepwise application of two different selective agars for the specific identification of MRSA in screening analyses.MethodsNasal swabs from 1541 volunteers were subjected to thioglycollate broth enrichment and subsequently screened on CHROMagar MRSA selective agar for the identification of MRSA. The MRSA identity of suspicious-looking colonies was confirmed afterwards or excluded by another selective agar, chromID MRSA. All isolates from the selective agars with MRSA-specific colony morphology were identified by biochemical methods and mass spectrometry.ResultsThe initial CHROMagar MRSA screening identified suspicious colonies in 36 out of 1541 samples. A total of 25 of these 36 isolates showed MRSA-like growth on chromID agar. Out of these 25 isolates, 24 were confirmed as MRSA, while one isolate was identified as Staphylococcus kloosii. From the 11 strains that did not show suspicious growth on chromID agar, 3 were methicillin-sensitive Staphylococcus aureus (MSSA, with one instance of co-colonization with Corynebacterium spp.), 2 were confirmed as MRSA (with 1 instance of co-colonization with MSSA), 2 were lost during passaging and could not be re-cultured, one could not be identified by the applied approaches, and the remaining 3 strains were identified as Staphylococcus saprophyticus, Staphylococcus hominis (co-colonized with Macrococcus caseolyticus) and Staphylococcus cohnii, respectively.ConclusionsThe application of the selective agar CHROMagar MRSA alone proved to be too non-specific to allow for a reliable diagnosis of the presence of MRSA. The combined use of two selective agars in a stepwise approach reduced this non-specificity with an acceptably low loss of sensitivity. Accordingly, such a stepwise screening approach might be an option for resource-restricted military medical field camps.

Nosocomial infection due to Enterococcus cecorum identified by MALDI-TOF MS and Vitek 2 from a blood culture of a septic patient.

We report the case of a nosocomial infection due to Enterococcus cecorum isolated from a blood culture of a 75-year-old septic male patient. Matrix-assisted laser desorption–ionization time-of-flight mass spectrometry (MALDI-TOF MS) and Vitek 2 succeeded in identification of the isolate.