Dynamics of the human gut phageome during antibiotic treatment.

Bacterial viruses contribute to the dynamics of the microbiome communities, as they are involved in the horizontal gene transfer. Previously we studied changes in the gut microbiome of the two healthy individuals over the course of a 6-days antibiotics treatment and subsequent 28 days recovery time (Willmann et al., 2015). Now, from the same samples, the virus-like particles were isolated and sequenced. As the phage sequences are currently poorly represented in reference databases, the reads had to be assembled, annotated and their abundance had to be evaluated via reads mapping. We analyzed and compared patterns of changes in abundance of the phage scaffolds and scaffolds with antibiotics resistant genes, in both phage and whole-genome metagenomic sets. We observed an increase in abundance of scaffolds carrying antibiotic-resistant genes in response to the treatment.

Whole-genome sequencing enabling the detection of a colistin-resistant hypermutating Citrobacter werkmanii strain harbouring a novel metallo-ß-lactamase VIM-48.

Citrobacter spp. harbouring metallo-ß-lactamases (MBLs) have been reported from various countries and different sources, but their isolation from clinical specimens remains a rare event in Europe. MBL-harbouring Enterobacteriaceae are considered a major threat in infection control as therapeutic options are often limited to colistin. In this study, whole-genome sequencing was applied to characterise five clinical isolates of multidrug-resistant Citrobacter werkmanii obtained from rectal swabs. Four strains possessed a class 1 integron with a novel blaVIM-48 MBL resistance gene and the aminoglycoside acetyltransferase gene aacA4, whilst one isolate harboured a blaIMP-8 MBL. Resistance to colistin evolved in one strain isolated from a patient who had received colistin orally for 8 days. Genomic comparison of this strain with a colistin-susceptible pre-treatment isolate from the same patient revealed 66 single nucleotide polymorphisms (SNPs) and 26 indels, indicating the presence of a mutator phenotype. This was confirmed by the finding of a SNP in the mutL gene that led to a significantly truncated protein. Additionally, an amino acid change from glycine to serine at position 53 was observed in PmrA. Mutations in the pmrA gene have been previously described as mediating colistin resistance in different bacterial species and are the most likely reason for the susceptibility change observed. To the best of our knowledge, this is the first description of a colistin-resistant Citrobacter spp. isolated from a human sample. This study demonstrates the power of applying next-generation sequencing in a hospital setting to trace and understand evolving resistance at the level of individual patients.

Emergence of Citrobacter freundii carrying IMP-8 metallo-ß-lactamase in Germany.

Metallo-ß-lactamases (MBLs) in Enterobacteriaceae are an increasing problem worldwide. This report describes the isolation of Citrobacter freundii carrying IMP-8 MBL from three patients during the period from March 2012 until March 2013 in Germany. The bla IMP-8 enzyme is predominantly found in Asia, where IMP-8 has spread to various enterobacterial species causing serious infections. To our best knowledge, this is the first report of bla IMP-8 habouring Enterobacteriaceae in Europe.

Evaluation of phenotypic detection methods for metallo-ß-lactamases (MBLs) in clinical isolates of Pseudomonas aeruginosa.

Metallo-beta-lactamase (MBL) production in Pseudomonas aeruginosa is a growing issue across the globe. Fast and reliable diagnostic tools are needed for appropriate implementation of infection control measures. In this study we evaluated the performance of three commercial combined disk tests, two EDTA based in-house combined disk tests and the Carba NP test in comparison to molecular detection of MBL genes on 133 meropenem non-susceptible non-duplicate P. aeruginosa clinical isolates. The meropenem/DPA based commercial KPC + MBL-confirm ID kit (Rosco Diagnostica, Denmark) and the MASTDISCS™ ID carbapenemase (Enterobacteriaceae) detection disc set (MAST Diagnostics, UK) showed sensitivities of 31.1 % and 28.8 % and specificities of 69.3 % and 79.6 %, respectively. The total MBL confirm kit (Rosco Diagnostica, Denmark) contains imipenem/DPA and imipenem/EDTA combination disks. Evaluation of the single disk combinations revealed 84.4 % sensitivity and 81.8 % specificity for the imipenem/DPA assay and 86.7 % sensitivity and 51.1 % specificity for the imipenem/EDTA test. Applying both tests simultaneously resulted in a slightly higher sensitivity of 88.9 % but a lower specificity of 48.9 % when compared to the single tests alone. The Carba NP test showed 93.3 % sensitivity and 96.6 % specificity. All phenotypic combined disk tests lacked either sensitivity or specificity for the detection of MBL in P. aeruginosa. The Carba NP test showed excellent test properties, but suffers from drawbacks in handling and high costs. The optimal diagnostic approach needs to be chosen depending on the epidemiological situation, laboratory resources and availability of molecular confirmation tests.

Postinfectious irritable bowel syndrome: follow-up of a patient cohort of confirmed cases of bacterial infection with Salmonella or Campylobacter.

BACKGROUND: Gastrointestinal infections have been proposed to predict subsequent irritable bowel syndrome (IBS) but large-scale infectious events are rare and long-term data are missing. METHODS: We identified 576 individuals with a Salmonella or Campylobacter infection between 2000 and 2009 that were followed by a short postal questionnaire asking for the presence of current symptoms in 2010. In case of agreement (n = 90), an extended postinfectious (PI)-IBS questionnaire was mailed including the Hospital Anxiety Depression Scale and the Patient Health Questionnaire. KEY RESULTS: A total of 189 patients reported back (36%); 98 had a Salmonella and 91 had a Campylobacter infection, of which 56 reported persistent symptoms (9.7% of the initial sample). Fifty-one patients returned the PI-IBS questionnaire. Of 48 patients with complete data, 15 reported no or mild symptoms of abdominal pain or discomfort while 17 had moderate and 16 severe symptoms. Twenty-two met Rome IBS criteria, 14 (29%) reported GI symptoms before the infection. Patients with moderate and/or severe PI-IBS symptoms were significantly more often females, were more often infected by Salmonella than by Campylobacter, had more severe symptoms during the initial infection, and had more often GI symptoms prior to the infection. They reported higher anxiety, depression, and somatisation scores, but were not different with respect to acute stool habits. CONCLUSIONS & INFERENCES: Nearly 10% of patients with an intestinal bacterial infection report postinfectious symptoms up to 10 years after the infectious event. They represent a clinically important population with high psychiatric comorbidity and somatic symptom burden.

C-terminal amino acid residues of the trimeric autotransporter adhesin YadA of Yersinia enterocolitica are decisive for its recognition and assembly by BamA.

The Bam complex is a highly conserved multiprotein machine essential for the assembly of ß-barrel outer membrane proteins. It is composed of the essential outer membrane protein BamA and four outer membrane associated lipoproteins BamB-E. The Yersinia enterocolitica Adhesin A (YadA) is the prototype of trimeric auotransporter adhesins (TAAs), consisting of a head, stalk and a ß-barrel membrane anchor. To investigate the role of BamA in biogenesis of TAAs, we expressed YadA in a BamA-depleted strain of Escherichia coli, which resulted in degradation of YadA. Yeast-two-hybrid experiments and immunofluorescence studies revealed that BamA and YadA interact directly and colocalize. As BamA recognizes the C-terminus of OMPs, we exchanged the nine most C-terminal amino acids of YadA. Substitution of the amino acids in position 1, 3 or 5 from the C-terminus with glycine resulted in DegP-dependent degradation of YadA. Despite degradation all YadA proteins assembled in the outer membrane. In summary we demonstrate that (i) BamA is essential for biogenesis of the TAA YadA, (ii) BamA interacts directly with YadA, (iii) the C-terminal amino acid motif of YadA is important for the BamA-dependent assembly and differs slightly compared with other OMPs, and (iv) BamA and YadA colocalize.

Life-threatening pneumonia caused by human cytomegalovirus and Mycoplasma pneumoniae coinfection in a young, immunocompetent patient.

A young, previously healthy and immunocompetent patient was transferred to our hospital to recover a suspected Ascaris worm from his gall bladder. Although the diagnosis of Ascaris infection could not be confirmed, the patient suffered from cholecystitis. To our surprise, the respiratory situation of the patient deteriorated within 24 h under antibiotic therapy and he had to be transferred to the intensive care unit for mechanical respiration. Human cytomegalovirus (HCMV) was isolated directly from a bronchoalveolar lavage (BAL) sample, and Mycoplasma pneumoniae DNA was detected by PCR in an enrichment culture of the same BAL sample. Serology for HCMV and M. pneumoniae clearly supported a primary/post-primary infection for both agents (IgM detection, increase of IgG titres and, in the case of HCMV, a low avidity index of only 22 %). Therefore, we assumed that a rare HCMV and M. pneumoniae coinfection was the aetiology of the fulminant pneumonia. Under broad antibiotic and antiviral treatment, the situation of the patient improved only very slowly.

Trimer stability of YadA is critical for virulence of Yersinia enterocolitica.

Yersinia adhesin A (YadA) is a trimeric autotransporter adhesin with multiple functions in host-pathogen interactions. The aim of this study was to dissect the virulence functions promoted by YadA in vitro and in vivo. To accomplish this, we generated Yersinia enterocolitica O:8 mutants expressing point mutations in YadA G389, a highly conserved residue in the membrane anchor of YadA, and analyzed their impact on YadA expression and virulence functions. We found that point mutations of YadA G389 led to impaired transport, stability, and surface display of YadA. YadA G389A and G389S mutants showed comparable YadA surface expression, autoagglutination, and adhesion to those of wild-type YadA but displayed reduced trimer stability and complement resistance in vitro and were 10- to 1,000-fold attenuated in experimental Y. enterocolitica infection in mice. The G389T, G389N, and G389H mutants lost trimer stability, exhibited strongly reduced surface display, autoagglutination, adhesion properties, and complement resistance, and were avirulent (>10,000-fold attenuation) in mice. Our data demonstrate that G389 is a critical residue of YadA, required for optimal trimer stability, transport, surface display, and serum resistance. We also show that stable trimeric YadA protein is essential for virulence of Y. enterocolitica.

Emergence of increasing linezolid-resistance in enterococci in a post-outbreak situation with vancomycin-resistant Enterococcus faecium.

During 2004 and at the start of 2005 a university hospital in Southwest Germany was affected by an extensive outbreak of vancomycin-resistant Enterococcus faecium (VRE). Although the outbreak was contained, linezolid-resistant enterococci emerged during and after the outbreak as the usage of linezolid became more common. Linezolid resistance was no longer limited to VRE. Nosocomial spread of linezolid-resistant but vancomycin-susceptible E. faecium was detected and these strains also emerged in patients without prior drug exposure. Linezolid should therefore be used with caution and the susceptibility of isolates monitored over time. Isolation precautions and screening of contacts should be considered to avoid spread of resistant isolates.

Discrimination between epidemic and non-epidemic glycopeptide-resistant E. faecium in a post-outbreak situation.

Vancomycin-resistant enterococci (VRE) have been isolated in increasing numbers. Hospital-adapted VRE exhibit relatively high pathogenicity by expressing factors like enterococcal surface protein (Esp), which facilitates epidemic spread. By contrast, 'community-acquired' VRE show low pathogenicity and non-epidemic features. In 2004 and 2005 an extended outbreak of VRE occurred at a university hospital in Southwestern Germany and an infection control programme was implemented to confine the outbreak. Pulsed-field gel electrophoresis (PFGE), esp PCR, multiple-locus variable number of tandem repeat analysis (MLVA), purK1 typing and multiple-locus sequence typing (MLST) were performed on representative VRE isolates. Twenty-six non-epidemic and two epidemic VRE types (MLST203, MLST280) were identified by PFGE. Seven of the non-outbreak VRE types were esp gene negative, whereas 19 non-outbreak and both epidemic VRE types were esp positive. Eight MLVA types were identified. MLVA type 1 included five PFGE types and MLVA type 159 included 16 PFGE types. Currently there is no efficient method available to identify non-epidemic VRE and avoid unnecessary isolation of patients. More than 50% non-epidemic clones were esp positive; nevertheless, esp PCR appears to be the most promising approach to identify non-epidemic VRE.

The Yersinia enterocolitica invasin protein promotes major histocompatibility complex class I- and class II-restricted T-cell responses.

Yersinia enterocolitica invasin (Inv) protein confers internalization into and expression of proinflammatory cytokines by host cells. Both events require binding of Inv to beta1 integrins, which initiates signaling cascades including activation of focal adhesion complexes, Rac1, mitogen-activated protein kinase, and NF-kappaB. Here we tested whether Inv might be suitable as a delivery molecule and adjuvant if used as a component of a vaccine. For this purpose, hybrid proteins composed of Inv and ovalbumin (OVA) were prepared, applied as a coating to microparticles, and used for vaccination. Fusion of OVA to Inv did not significantly disturb the ability of Inv to promote host cell binding, internalization, and interleukin-8 (IL-8) secretion when applied as a coating to microparticles. The microparticles were used for vaccination of mice adoptively transferred with OVA-specific T cells from OT-1 or DO11.10 mice. Administration of OVA-Inv-coated microparticles induced OVA-specific T-cell responses. OVA-specific CD4 T cells produced both gamma interferon (IFN-gamma) and IL-4 as determined by enzyme-linked immunosorbent assay. Likewise, pronounced OVA-specific CD8 T-cell responses associated with IFN-gamma production were observed. Together, these results suggest that Inv might be an attractive tool in vaccination as it confers both host cell uptake and adjuvant activity by engagement of beta1 integrins of host cells, which leads to CD4 as well as CD8 T-cell responses.

Innate defenses of the intestinal epithelial barrier.

The innate immune system plays a crucial role in maintaining the integrity of the intestine and protecting the host against a vast number of potential microbial pathogens from resident and transient gut microflora. Mucosal epithelial cells and Paneth cells produce a variety of antimicrobial peptides (defensins, cathelicidins, crytdinrelated sequence peptides, bactericidal/permeabilityincreasing protein, chemokine CCL20) and bacteriolytic enzymes (lysozyme, group IIA phospholipase A2) that protect mucosal surfaces and crypts containing intestinal stem cells against invading microbes. Many of the intestinal antimicrobial molecules have additional roles of attracting leukocytes, alarming the adaptive immune system or neutralizing proinflammatory bacterial molecules. Dysfunction of components of the innate immune system has recently been implicated in chronic inflammatory bowel diseases such as Crohn's disease and ulcerative colitis, illustrating the pivotal role of innate immunity in maintaining the delicate balance between immune tolerance and immune response in the gut.

Metallo-beta-lactamase expressing multi-resistant Acinetobacter baumannii transmitted in the operation area.

Outbreaks of Acinetobacter baumannii demonstrating multiple antibiotic resistance, including meropenem resistance, have been described as severe therapeutic problems. Here we describe a monoclonal outbreak of infection and colonization with multidrug-resistant A. baumannii over a two-month period. Resistance to meropenem was mediated by expression of a metallo-beta-lactamase enzyme. Four of 14 patients showed clinical signs of infection and two died. Contamination of the environment, water, or instruments were excluded as causes of the outbreak. All patients, except one, underwent surgery in a specific operation theatre where surgery of contamination class IV (infected, dirty) was performed. Although individual surgeon error was eliminated, analyses of the patients' histories suggested that bacterial transmission had occurred during surgery. Five patients showed signs of A. baumannii infection and two of these patients suffered from large abdominal wounds infected with a high density of A. baumannii requiring repeated revisions. Presumably, these revisions favoured the transmission of A. baumannii, which is remarkably resistant to various environmental stresses including soaps, disinfectants and dry conditions. No case of meropenem-resistant A. baumannii had been observed in the hospital before the outbreak. Interestingly, the resistant bacteria appear to have been imported by a patient returning from West Africa. This indicates that, similar to MRSA, multiresistant A. baumannii may be introduced by patients from foreign hospitals. The outbreak was stopped in the following months by reinforcing standard procedures and by taking all necessary precautions such as patient isolation, and finally only one new case was detected.

An abundance of Escherichia coli is harbored by the mucosa-associated bacterial flora of interleukin-2-deficient mice.

Mice deficient in interleukin-2 are well suited for use as an animal model for inflammatory bowel disease. Raised under specific-pathogen-free conditions, interleukin-2-deficient mice develop an inflammatory bowel disease resembling ulcerative colitis in humans. The finding that colitis was attenuated when the mice were kept under germfree conditions implies that the resident intestinal flora is involved in the pathogenesis of colitis. The present study addresses the composition of the mucosa-associated bacterial flora in colon samples from interleukin-2-deficient mice that developed colitis. This was investigated by comparative 16S ribosomal DNA (rDNA) sequence analysis and fluorescence in situ hybridization using rRNA-targeted fluorescent probes to quantify the bacterial populations of the mucosa-associated flora. The investigations revealed distinct differences in the bacterial composition of the mucosa-associated flora between interleukin-2-deficient mice and healthy controls. Fluorescence in situ hybridization identified up to 10% of the mucosa-associated flora in interleukin-2-deficient mice as Escherichia coli, whereas no E. coli was detected in the mucosa from healthy wild-type mice. This finding was consistent with the results from comparative 16S rDNA analysis. About one-third of the clones analyzed from 16S rDNA libraries of interleukin-2-deficient mice represented Enterobacteriaceae, whereas none of the clones analyzed from the healthy controls harbored 16S rDNA from Enterobacteriaceae. The abundance of E. coli in the colonic mucosa of interleukin-2-deficient mice strongly suggests a participation in the pathogenesis of colitis in the interleukin-2-deficient mouse model for inflammatory bowel disease.

Gene expression patterns of epithelial cells modulated by pathogenicity factors of Yersinia enterocolitica.

Epithelial cells express genes whose products signal the presence of pathogenic microorganisms to the immune system. Pathogenicity factors of enteric bacteria modulate host cell gene expression. Using microarray technology we have profiled epithelial cell gene expression upon interaction with Yersinia enterocolitica. Yersinia enterocolitica wild-type and isogenic mutant strains were used to identify host genes modulated by invasin protein (Inv), which is involved in enteroinvasion, and Yersinia outer protein P (YopP) which inhibits innate immune responses. Among 22 283 probesets (14,239 unique genes), we found 193 probesets (165 genes) to be regulated by Yersinia infection. The majority of these genes were induced by Inv, whose recognition leads to expression of NF-kappa B-regulated factors such as cytokines and adhesion molecules. Yersinia virulence plasmid (pYV)-encoded factors counter regulated Inv-induced gene expression. Thus, YopP repressed Inv-induced NF-kappa B regulated genes at 2 h post infection whereas other pYV-encoded factors repressed host cell genes at 4 and 8 h post infection. Chromosomally encoded factors of Yersinia, other than Inv, induced expression of genes known to be induced by TGF-beta receptor signalling. These genes were also repressed by pYV-encoded factors. Only a few host genes were exclusively induced by pYV-encoded factors. We hypothesize that some of these genes may contribute to pYV-mediated silencing of host cells. In conclusion, the data demonstrates that epithelial cells express a limited number of genes upon interaction with enteric Yersinia. Both Inv and YopP appear to modulate gene expression in order to subvert epithelial cell functions involved in innate immunity.

No evidence of Wolbachia endosymbiosis with Loa loa and Mansonella perstans.

Endosymbiotic Wolbachia bacteria from different filarial species, including major pathogens of humans such as Wuchereria bancrofti, Brugia malayi and Onchocerca volvulus, seem to play an important role in the development, viability and fertility of these worms. Wolbachia trigger inflammatory host responses as well as adverse reactions against standard treatment regimens and are therefore under investigation as novel treatment targets. We investigated whether Wolbachia are also endosymbiotic in Loa loa and Mansonella perstans. In both male and female adult L. loa, we found no evidence of bacteria by light or transmission electron microscopy. Furthermore, Wolbachia-specific PCR was negative in both L. loa and M. perstans microfilariae. The absence of Wolbachia in both filarial species therefore discourages the use of antibiotics as an adjunct or alternative approach to current treatment concepts for both loiasis and mansonelliasis perstans.

Microflora reactive IL-10 producing regulatory T cells are present in the colon of IL-2 deficient mice but lack efficacious inhibition of IFN-gamma and TNF-alpha production.

BACKGROUND: Inflammatory bowel disease in interleukin 2 (IL-2) deficient (IL-2(-/-)) mice is triggered by the intestinal microflora and mediated by CD4(+) T cells. AIMS: To determine the characteristics of microflora specific intestinal T cells, including migration and cytokine production. METHODS: Intestinal T cell populations and cytokine mRNA expression of specific pathogen free (SPF) and germ free (GF) IL-2(-/-) and IL-2(+/+) mice were compared by flow cytometry and reverse transcription-polymerase chain reaction. Cytokine production of intestinal mononuclear cells on stimulation with microflora antigens was assessed by ELISA. In vivo migration of T cells was assessed by adoptive transfer of (51)Cr labelled CD4(+)CD25(-)alpha beta(+) T cells. The ability of intestinal T cell lines to promote colitis was determined by adoptive transfer experiments. RESULTS: SPF IL-2(-/-) mice produced higher interferon gamma (IFN-gamma) and tumour necrosis factor alpha mRNA levels than GF IL-2(-/-) mice, which was accompanied by an increased number of CD4(+)alpha beta T cells in the colon. Tracking of (51)Cr labelled and adoptively transferred T cells revealed an increased MAdCAM-1 dependent but VCAM-1 independent recruitment of these cells into the colon of SPF IL-2(-/-) mice. Colon lamina propria lymphocytes (LPL) from SPF IL-2(-/-) mice showed increased spontaneous IFN-gamma production in vitro. On stimulation with bacterial microflora antigens, intraepithelial lymphocytes and LPL did not produce IFN-gamma, but high quantities of IL-10, which did not suppress IFN-gamma production. Bacterial antigen specific cell lines established from colon LPL of SPF IL-2(-/-) mice with colitis showed a regulatory T cell-like cytokine profile and only marginally modulated the course of colitis and survival of IL-2(-/-) mice. CONCLUSIONS: Our results suggest that microflora reactive regulatory T cells are present in the colon of SPF IL-2(-/-) mice. However, IL-10 produced by these cells did not significantly modulate a possible secondary proinflammatory CD4 Th1 cell population to produce IFN-gamma.