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Yersiniabactin, a siderophore with a high affinity to iron, has been described as a potential virulence factor in Enterobacteriaceae. Klebsiella aerogenes is a Gram-negative rod known to cause invasive infection in very low birth weight infants but is an unusual pathogen to cause outbreaks in neonatal intensive care units (NICU).We performed a retrospective analysis of all patients colonized with K. aerogenes in our NICU from September to December 2018. Each infant with an occurrence of K. aerogenes in any microbiological culture was defined as a case. Clinical data were taken from medical charts. K. aerogenes isolates were genotyped using whole-genome sequencing combined with core genome multilocus sequencing type analysis. Yersiniabactin production was evaluated by luciferase assay.In total 16 patients were colonized with K. aerogenes over the 3-month period and 13 patients remained asymptomatic or developed late-onset neonatal sepsis from another pathogen. Three patients developed necrotizing enterocolitis, 2 complicated by sepsis and 1 of them died. All symptomatic patients were premature infants with low birth weight. Genetic sequencing confirmed an outbreak with the same strain, all samples expressed the high-pathogenicity island, necessary for the production of yersiniabactin. Six exemplary cases were proven to produce yersiniabactin in vitro.This is the first report of an outbreak of a yersiniabactin-producing K. aerogenes strain causing invasive infection in preterm infants. We hypothesize that, due to improved iron uptake, this strain was associated with higher virulence than non-yersiniabactin-producing strains. Extended search for virulence factors and genetic sequencing could be pivotal in the management of NICU outbreaks in the future.

The transperineal approach is preferred to reduce prostate biopsy (PB)-related infections. Fluoroquinolones are suspended for prophylaxis of PB in the European Union; therefore, alternative antibiotics based on local resistance, or targeted prophylaxis, in conjunction with povidone-iodine rectal preparation are recommended for transrectal PB.

This case-control study investigated the long-term evolution of multidrug-resistant bacteria (MDRB) over a 5-year period associated with the use of selective oropharyngeal decontamination (SOD) in the intensive care unit (ICU). In addition, effects on health care-associated infections and ICU mortality were analysed.We investigated patients undergoing mechanical ventilation 48 h in 11 adult ICUs located at 3 campuses of a university hospital. Administrative, clinical, and microbiological data which were routinely recorded electronically served as the basis. We analysed differences in the rates and incidence densities (ID, cases per 1000 patient-days) of MDRB associated with SOD use in all patients and stratified by patient origin (outpatient or inpatient). After propensity score matching, health-care infections and ICU mortality were compared.5034 patients were eligible for the study. 1694 patients were not given SOD. There were no differences in the incidence density of MDRB when SOD was used, except for more vancomycin-resistant Enterococcus faecium (0.72/1000 days vs. 0.31/1000 days, p 0.01), and fewer ESBL-producing Klebsiella pneumoniae (0.22/1000 days vs. 0.56/1000 days, p 0.01). After propensity score matching, SOD was associated with lower incidence rates of ventilator-associated pneumonia and death in the ICU but not with ICU-acquired bacteremia or urinary tract infection.Comparisons of the ICU-acquired MDRB over a 5-year period revealed no differences in incidence density, except for lower rate of ESBL-producing Klebsiella pneumoniae and higher rate of vancomycin-resistant Enterococcus faecium with SOD. Incidence rates of ventilator-associated pneumonia and death in the ICU were lower in patients receiving SOD.

The Gram-negative bacterium Klebsiella pneumoniae is a frequent pathogen causing outbreaks in neonatal intensive care units. Some Enterobacteriaceae can acquire the ability to sequester iron from infected tissue by secretion of iron-chelating compounds such as yersiniabactin. Here we describe an outbreak and clinical management of infections because of a highly virulent yersiniabactin-producing, nonmultiresistant K. pneumoniae strain in a neonatal intensive care unit. Outbreak investigation and effectiveness assessment of multidisciplinary infection control measurements to prevent patient-to-patient transmission of highly pathogenic K. pneumoniae were undertaken.Outbreak cases were identified by isolation of K. pneumoniae from blood or stool of infants. Clinical data were abstracted from medical charts. K. pneumoniae isolates were genotyped using whole genome sequencing, and yersiniabactin production was evaluated by luciferase assay.Fourteen cases were confirmed with 8 symptomatic and 6 colonized patients. Symptomatic patients were infants of extremely low gestational and chronologic age with fulminant clinical courses including necrotizing enterocolitis and sepsis. Whole genome sequencing for bacterial isolates confirmed the presence of an outbreak. All outbreak isolates produced yersiniabactin.Yersiniabactin-producing K. pneumoniae can display a high pathogenicity in extremely premature infants with low chronologic age. This outbreak also underlines the considerable potential of today's infection control systems for recognizing and controlling nosocomial infections in highly vulnerable populations.

To analyse the therapeutic efficacy of various phytotherapeutics and their antimicrobial compounds with regard to strain specificity and dose dependence. A representative strain collection of 40 uropathogenic bacteria isolated from complicated and uncomplicated urinary tract infection was subjected to various virulence assays (bacterial growth, mannose-sensitive agglutination, and motility) to determine the therapeutic impact of various compounds with antimicrobial activity. We tested proanthocyanidins (PAC), d-mannose, rosemary extract (Canephron®), and isothiocyanates (Angocin®). d-mannose efficiently blocked the adhesive properties of all type 1 fimbriae-positive isolates in low concentration (0.2%), but showed no bacteriostatic effect. PAC also actively blocked agglutination, but the concentration varied considerably among isolates. Escherichia coli required the highest concentration (10%), while Enterobacter cloacae responded to low concentrations (0.1%). Allyl isothiocyanates not only impaired agglutination in all tested isolates, but also had a dramatic impact on flagella-mediated motility in Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis (p

The genus Escherichia is composed of several species and cryptic clades, including E. coli, which behaves as a vertebrate gut commensal, but also as an opportunistic pathogen involved in both diarrheic and extra-intestinal diseases. To characterize the genetic determinants of extra-intestinal virulence within the genus, we carried out an unbiased genome-wide association study (GWAS) on 370 commensal, pathogenic and environmental strains representative of the Escherichia genus phylogenetic diversity and including E. albertii (n = 7), E. fergusonii (n = 5), Escherichia clades (n = 32) and E. coli (n = 326), tested in a mouse model of sepsis. We found that the presence of the high-pathogenicity island (HPI), a ~35 kbp gene island encoding the yersiniabactin siderophore, is highly associated with death in mice, surpassing other associated genetic factors also related to iron uptake, such as the aerobactin and the sitABCD operons. We confirmed the association in vivo by deleting key genes of the HPI in E. coli strains in two phylogenetic backgrounds. We then searched for correlations between virulence, iron capture systems and in vitro growth in a subset of E. coli strains (N = 186) previously phenotyped across growth conditions, including antibiotics and other chemical and physical stressors. We found that virulence and iron capture systems are positively correlated with growth in the presence of numerous antibiotics, probably due to co-selection of virulence and resistance. We also found negative correlations between virulence, iron uptake systems and growth in the presence of specific antibiotics (i.e. cefsulodin and tobramycin), which hints at potential “collateral sensitivities” associated with intrinsic virulence. This study points to the major role of iron capture systems in the extra-intestinal virulence of the genus Escherichia., PLoS Genetics, 16 (10), ISSN:1553-7390, ISSN:1553-7404

Antimicrobial peptides (AMPs) represent important facets of the immune system controlling infectious diseases. However, pathogens show varying susceptibilities to AMPs. This study investigates the susceptibilities of strains of Streptococcus mutans (SM), Actinomyces naeslundii (AN), and Lactobacillus spp. (LB) towards AMPs and if there are correlations between the appearance of such high-risk strains and clinical caries status. Plaque samples were collected from patients along with clinical examinations. Bacterial strains were identified via selective media, matrix-assisted laser desorption/ionization analysis-time of flight (MALDI-TOF), and arbitrary-primed-PCR (AP-PCR). Each strain was tested for susceptibility to LL-37, HBD-2, HNP-1, and HNP-3 or phosphate-buffered saline as negative control in a biofilm model on hydroxylapatite discs. Survival rates and resulting risk classification for each strain were determined. Correlations were calculated between the number of high-risk strains (all/S. mutans) appearing in patients and their clinical caries status. Forty-seven patients were included with mean DMFT values of 11.4 ± 8.7. A total of 8 different SM, 30 LB, and 47 AN strains were detected. One-way ANOVA indicated that type/concentration of AMPs had major influence on reductions of Lactobacilli and Actinomyces. Seventeen strains of AN, 2 of SM, and 6 of LB had low susceptibilities to AMPs. The number of such strains in patients showed significant positive correlations to the DMFT values (all p = 0.001; r = 0.452; S. mutans p

We hypothesized that local infection plays a critical role in the pathogenesis of medication-related osteonecrosis of the jaw (MRONJ). Recent developments in molecular methods have revolutionized new approaches for the rapid detection of microorganisms including those difficult to culture. The aim of our study is to identify the bacterial profiles in MRONJ by microbiological culture and polymerase chain reactions (PCR). A retrospective analysis was performed on MRONJ patients from 2008 to 2014. The bacterial profile from MRONJ bone samples was determined using microbiological culture and PCR. Ninety five patients fulfilled the inclusion criteria with mean age of 69.85 ± 8.71 years. A female predilection was detected. The mandible was more commonly affected than maxilla. Tooth extraction was the frequent triggering factor. Breast cancer was the primary cause for administration and intravenous bisphosphonates were the most commonly administrated antiresorptive drugs. The majority of patients were classified as stage 2. Posterior teeth were most commonly affected. Based on bone culture results, the most common microorganism were both actinomyces and mixed flora. PCR confirmed the presence of actinomyces in 55 patients. Our data suggest that PCR might be an innovative method for detection of microorganisms difficult to culture using traditional microbiological techniques.

In September 2018, a child who had returned from Somalia to Germany presented with cutaneous diphtheria by toxigenic Corynebacterium diphtheriae biovar mitis. The child’s sibling had superinfected insect bites harbouring also toxigenic C. diphtheriae. Next generation sequencing (NGS) revealed the same strain in both patients suggesting very recent human-to-human transmission. Epidemiological and NGS data suggest that the two cutaneous diphtheria cases constitute the first outbreak by toxigenic C. diphtheriae in Germany since the 1980s.

Successful bacterial pathogens produce an array of virulence factors that allow subversion of the immune system and persistence within the host. For example, uropathogenic Escherichia coli strains, such as CFT073, express Toll/IL-1 receptor-containing (TIR-containing) protein C (TcpC), which impairs TLR signaling, thereby suppressing innate immunity in the urinary tract and enhancing persistence in the kidneys. Here, we have reported that TcpC also reduces secretion of IL-1β by directly interacting with the NACHT leucin-rich repeat PYD protein 3 (NLRP3) inflammasome, which is crucial for recognition of pathogens within the cytosol. At a low MOI, IL-1β secretion was minimal in CFT073-infected macrophages; however, IL-1β release was markedly increased in macrophages infected with CFT073 lacking tcpC. Induction of IL-1β secretion by CFT073 and tcpC-deficient CFT073 required the NLRP3 inflammasome. TcpC attenuated activation of the NLRP3 inflammasome by binding both NLRP3 and caspase-1 and thereby preventing processing and activation of caspase-1. Moreover, in a murine urinary tract infection model, CFT073 infection rapidly induced expression of the NLRP3 inflammasome in the bladder mucosa; however, the presence of TcpC in WT CFT073 reduced IL-1β levels in the urine of infected mice. Together, these findings illustrate how uropathogenic E. coli use the multifunctional virulence factor TcpC to attenuate innate immune responses in the urinary tract.

In this single-blinded, prospective, randomized, controlled clinical trial, the effectiveness of an ozone gas or NaOCl/CHX disinfection protocol was compared within the root canal treatment of apical periodontitis. Sixty permanent teeth were randomly allocated to one technique. The clinical/radiographic assessment criteria included clinical symptoms, periapical index (PAI), and size of the apical lesion. In both groups, the root canal was mechanically cleaned and irrigated with NaCl and EDTA. Ozone gas (32 g m−3) or NaOCl (3 %) was applied followed by a 1-week inter-appointment dressing (Ca(OH)2). As final disinfection, ozone gas (ozone group) or CHX 2 % (NaOCl group) was applied. Microbial samples were taken after preparing the access cavity, after chemo-mechanical treatment and after inter-appointment dressing by sterile paper points. Microbial identification was performed by mass spectroscopy (MALDI-TOF-MS) and 16S-rRNA gene sequencing. The treated teeth were blindly re-evaluated after 6/12 months. Success rates, the decrease in PAI, the size of apical lesions and bacterial reduction were compared between groups (Fischer’s exact test, Mann-Whitney U test). There were no significant differences between the success rates (ozone group: 96.2/95.5 % after 6/12 months; NaOCl group: 95.5/95.2 % after 6/12 months). The differences in the decreases in PAI values and apical lesion sizes were also insignificant after 6 and 12 months. The bacterial reduction showed no significant differences between groups after chemo-mechanical treatment and after inter-appointment dressing. The most commonly found bacterial genera were Streptococcus spp., Parvimonas spp. and Prevotella spp. The here used ozone gas and NaOCl/CHX protocols showed no difference in bacterial reduction in the sampled areas of the root canals. Within the limitations of the study, ozone gas seems to be a possible alternative disinfection agent within the root canal treatment of apical periodontitis.

The incidence of candidemia caused by Candida albicans and Candida glabrata is constantly increasing and is accompanied by the rising use of the few available antifungals. The widespread use of echinocandins and azoles for the treatment of invasive candidemia has enhanced the development of antifungal resistance, resulting in an increasing health care problem. Hence, the rapid detection of resistant strains is required. This study aimed to evaluate the detection of C. albicans and C. glabrata strains resistant to caspofungin by the matrix-assisted laser desorption ionization Biotyper antibiotic susceptibility test rapid assay (MBT ASTRA). This novel semiquantitative technique facilitates the detection of caspofungin-resistant strains within 6 h. MBT ASTRA results were compared to the data obtained by the use of Clinical and Laboratory Standards Institute (CLSI) guidelines. Clinical isolates of C. albicans (n = 58) and C. glabrata (n = 57) were analyzed by MBT ASTRA and the CLSI microdilution method. Antifungal susceptibility testing against caspofungin by the CLSI microdilution method classified the C. albicans isolates into 36 susceptible and 22 resistant strains and the C. glabrata isolates into 5 susceptible, 33 resistant, and 19 intermediate strains. For C. albicans, the comparison of MBT ASTRA and the CLSI method revealed an excellent categorical agreement of 100%. A sensitivity and a specificity between MBT ASTRA and the CLSI microdilution method of 94% and 80%, respectively, were detected for C. glabrata strains, based on categorical agreement. In conclusion, the results obtained by MBT ASTRA indicate that this is a very promising approach for the rapid detection of Candida isolates resistant to caspofungin.

The key to success of extraintestinal pathogenic Escherichia coli (ExPEC) to colonize niches outside the intestinal tract and to establish infection is the coordinated action of numerous virulence and fitness factors. Intense research revealed not only an arsenal of unique virulence determinants with specific action, but also the multi-functionality of single elements. Especially iron uptake systems of ExPEC proved to be of prime importance. Apart from iron acquisition they optimize certain virulence properties. Here we analyzed the contribution of the salmochelin siderophore system to the ability of ExPEC to form biofilms. In the same iron limited environment, ExPEC displayed a distinct transcriptional profile of siderophore systems. During biofilm formation the iroN gene coding for the specific receptors of the siderophore salmochelin was highly upregulated. Almost no induction was observed during planctonic growth. Disruption of iroN resulted in a reduction of almost 50% in biofilm production. Efficient biofilm formation was not affected in a salmochelin synthesis mutant. Thus, the contribution of IroN is independent from the ability to produce salmochelin. Enhanced expression of IroN did not increase significantly the capacity to form biofilms in ExPEC. Interestingly, the additional expression of IroN or even the acquisition of the entire salmochelin system was not able to improve biofilm formation in a poor biofilm producer like a laboratory E. coli K12 strain. However, complementation with only IroN in an ExPEC iroA deletion mutant was able to restore biofilm formation. The contribution of IroN to biofilm formation appears to require a certain background found in ExPEC, but not in E. coli K12. This study identified the contribution of IroN to biofilm formation and highlights the multi-functional role of iron uptake systems in ExPEC.

We report on two new cases Bordetella trematum in chronic ulcers. Literature shows that this species is infrequently isolated from clinical samples. Only a few cases have been reported so far. B. trematum may be underdiagnosed because of its problematic identification. Today, MALDI-TOF MS allows for an accurate discrimination of these bacteria. Overall, B. trematum was typically associated with severe skin and soft tissue infections, particularly in diabetic patients. The intrinsic resistance to some antibiotics commonly used to treat these infections makes the isolation of B. trematum significant.

Received 20 March 2014; returned 4 June 2014; revised 9 July 2014; accepted 15 July 2014Background: Bloodstream infections (BSIs) caused by enterobacteria remain a leading cause of mortality inpatients with chemotherapy-induced neutropenia. The rate and type of colonization and infection with ESBL-producing Enterobacteriaceae (ESBL-E) and their mode of transmission in German cancer centres is largelyunknown.Methods: We performed a prospective, observational study at five German university-based haematologydepartments. Participating sites screened for intestinal ESBL-E colonization within 72 h of admission, every10+2 days thereafter and before discharge. Three of the five centres performed contact isolation for patientscolonized or infected with ESBL-E. Molecular characterization of resistance mechanisms and epidemiologicaltyping of isolates by repetitive extragenic palindromic PCR (rep-PCR) and PFGE was performed to assess straintransmission between patients.Results: Between October 2011 and December 2012, 719 hospitalizations of 497 haematological high-riskpatients comprising 20143 patient-days were analysed. Mean duration of in-hospital stay was 36.6 days(range: 2–159 days). ESBL-E were identified from screening samples (82.8%Escherichia coliand 14.6%Klebsiella pneumoniae) in 55/497 patients (11.1%; range by centre: 5.8%–23.1%). PFGE and rep-PCR revealedonlyasinglecaseofpotentialcross-transmissionamongtwopatientscolonizedwithK.pneumoniae.Sixepisodesof BSI with ESBL-E were observed. Multivariate analysis revealed previous colonization with ESBL-E as the mostimportant risk factor for BSI with ESBL-E (OR 52.00; 95% CI 5.71–473.89).Conclusions: Even though BSI with ESBL-E is still rare in this high-risk population, colonization rates are substan-tial and vary considerably between centres. In-hospital transmission of ESBL-E as assessed by molecular typingwas the exception.Keywords: febrile neutropenia, bacteraemia, bloodstream infections, intestinal colonization, infection control, contact isolation

With the increasing prevalence of multidrug-resistant Gram-negative bacteria, rapid identification of the pathogen and its individual antibiotic resistance is crucial to ensure adequate antiinfective treatment at the earliest time point. Matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry for the identification of bacteria directly from the blood culture bottle has been widely established; however, there is still an urgent need for new methods that permit rapid resistance testing. Recently, a semiquantitative MALDI-TOF mass spectrometry-based method for the prediction of antibiotic resistance was described. We evaluated this method for detecting nonsusceptibility against two β-lactam and two non-β-lactam antibiotics. A collection of 30 spiked blood cultures was tested for nonsusceptibility against gentamicin and ciprofloxacin. Furthermore, 99 patient-derived blood cultures were tested for nonsusceptibility against cefotaxime, piperacillin-tazobactam, and ciprofloxacin in parallel with MALDI-TOF mass spectrometry identification from the blood culture fluid. The assay correctly classified all isolates tested for nonsusceptibility against gentamicin and cefotaxime. One misclassification for ciprofloxacin nonsusceptibility and five misclassifications for piperacillin-tazobactam nonsusceptibility occurred. Identification of the bacterium and prediction of nonsusceptibility was possible within approximately 4 h.

Pathogenic microbes have evolved sophisticated molecular strategies to subvert host defenses. Here we show that virulent bacteria interfere directly with Toll-like receptor (TLR) function by secreting inhibitory homologs of the Toll/interleukin-1 receptor (TIR) domain. Genes encoding TIR domain containing-proteins (Tcps) were identified in Escherichia coli CFT073 (TcpC) and Brucella melitensis (TcpB). We found that TcpC is common in the most virulent uropathogenic E. coli strains and promotes bacterial survival and kidney pathology in vivo. In silico analysis predicted significant tertiary structure homology to the TIR domain of human TLR1, and we show that the Tcps impede TLR signaling through the myeloid differentiation factor 88 (MyD88) adaptor protein, owing to direct binding of Tcps to MyD88. Tcps represent a new class of virulence factors that act by inhibiting TLR- and MyD88-specific signaling, thus suppressing innate immunity and increasing virulence.

Extraintestinal pathogenic Escherichia coli (ExPEC) are a major cause of urinary tract infections, sepsis, and neonatal meningitis. A variety of virulence factors in these strains is encoded by mobile genetic elements, such as transposons or pathogenicity islands (PAIs). Using subtractive cloning of ExPEC genomes, we recently detected short DNA fragments, which were significantly associated with the extraintestinal virulent phenotype. In this study, we identified four novel genomic DNA regions of the highly virulent uropathogenic E. coli strain JS299 carrying these previously identified DNA fragments. Characterization of the partial sequences of the genomic DNA regions revealed complex DNA arrangements with variable genetic compositions regarding the G+C contents and codon usage patterns. The prevalence of 15 previously uncharacterized genes was determined in a collection of clinical ExPECs and commensal E. coli strains by means of DNA microarray analyses. From this, 13 novel DNA sequences were demonstrated to be significantly associated with extraintestinal virulent strains, and thus may represent new virulence traits. Beside genes predicted to play a role in metabolic functions, such as sucrose utilization (scr), we identified DNA sequences shared by both ExPEC and enteropathogenic E. coli (EPEC). These sequences were significantly more prevalent among ExPECs when compared to commensal E. coli isolates. Our results support the idea of a considerable genetic variability among ExPEC strains and suggest that the novel genomic determinants described in this study may contribute to the ExPEC virulence.

The high-pathogenicity island (HPI) is a genomic island essential for the mouse-virulence phenotype in Yersinia and indispensable for pathogenicity of Yersinia and certain pathotypes of Escherichia coli. In contrast to most genomic islands, the HPI is a functional island widely disseminated among members of the family of Enterobacteriaceae. The HPI-encoded phage P4-like integrase together with excisionase and recombination sites make up the genetic mobility module of the island, while the siderophore yersiniabactin biosynthesis and uptake system comprises its functional part with respect to fitness and pathogenicity. The HPI-integrase promotes integration of the island into attB sites represented by three to four asn tDNAs in Yersinia pestis and E. coli. An additional enzyme, excisionase, is essential for efficient excision of the HPI from the initial site of integration. Furthermore a unique type of HPI has been characterized in the E. coli strain ECOR31 carrying a functional conjugative mating pair formation (Mpf) and a DNA-processing system, both of which are characteristic of integrative and conjugative elements (ICE). A model of conjugative transfer for the dissemination of HPIs is proposed in which the excised HPI is mobilized to a new recipient either trapped by a transmissive asn tDNA-carrying plasmid or autonomously as an ICE named ICEEcl.

In this study we tested 74 Salmonella strains of all eight Salmonella groups and were able to demonstrate the presence of two high-pathogenicity island types in strains of Salmonella groups IIIa, IIIb, and VI. Most high-pathogenicity island-positive isolates produced yersiniabactin under iron-limited conditions and were positive for the high-molecular-weight proteins HMWP1 and HMWP2.

Antibiotic resistance in Gram-negative microorganisms is an increasing health care problem. The rapid detection of such resistance is crucial for starting an early specific therapy and to enable initiation of the required hygiene measures. With continued emphasis on reducing the cost of laboratory testing, only economical/low-cost approaches have a chance of being implemented. During recent years, matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) has been developed to be a standard method in microbiology laboratories for the rapid and cost-efficient identification of microorganisms. Extending the usage of MALDI-TOF MS in the clinical microbiology laboratory to the area of resistance testing is an attractive option. Quantitative MALDI-TOF MS using an internal standard facilitates the measurement of the quantity of peptides and small proteins within a spectrum. These quantities correlate to the number of microorganisms and therefore to the growth of a microorganism. The comparison of growth in the presence or absence of an antibiotic allows for analysis of the susceptibility behavior of a strain. Here, we describe a novel method and its application in the analysis of 108 Klebsiella sp. isolates. After 1 h of incubation at a meropenem concentration of 8 μg/ml, a sensitivity of 97.3% and a specificity of 93.5% were achieved (compared to Etest results).

Fluorescence in situ hybridisation (FISH) targeted to ribosomal RNA is well established for studies in environmental microbiology. Initial applications of this technique in the field of medical microbiology showed that FISH is also a suitable means for the rapid, reliable and cultivation-independent identification of bacterial pathogens. In particular, for infectious diseases that follow a fulminant live-threatening course, such as sepsis or necrotising fasciitis (NF), a fast and reliable detection technique is of great importance. This study describes the development of an rRNA-targeted oligonucleotide set covering more than 95% of the pathogens associated with NF. These probes were tested with a broad collection of target and non-target organisms and found to be highly specific. Subsequently, the FISH approach was applied for the direct detection of bacterial pathogens in clinical samples. Two cases of NF and one case of streptococcal toxic shock syndrome (STSS) were analysed. FISH correctly identified almost all pathogens present in the samples examined within 2–3 h. However, Proteus mirabilis, which was identified in one sample by conventional methods was detected as a rod-shaped bacteria but could not be identified by FISH, since no specific probe was available for this particular organism. In contrast, identification of pathogens in these samples by conventional laboratory methods took 48–72 h. Furthermore, in one patient with pre-sampling antimicrobial therapy bacteria could not be grown from any of the samples. FISH unequivocally revealed the presence of Streptococcus pyogenes in affected tissue samples from this patient. In an experimental setting we demonstrated that FISH readily identifies S. pyogenes cells rendered non-cultivable by antibiotic treatment. Thus, FISH holds great promise for rapid identification of pathogens in fulminant infections such as NF, particularly in cases when pre-sampling antimicrobial therapy hampers culture of the causative agent.

Against the background of increasing numbers of resistant microorganisms, the fast and cost-efficient detection of microbial resistance is an important clinical requirement for optimal therapeutic intervention. Current routine assays take at least 5 h, but in most cases an overnight incubation is necessary to identify resistant isolates. The usage of matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) profiling in combination with growth media containing isotopically labeled amino acids facilitates the detection of resistant microorganisms after 3 h or less directly from the profile spectrum. Growing microorganisms incorporate isotopically labeled amino acids, increasing protein masses and thereby leading to mass shifts of their corresponding peaks in the profile spectra. In the presence of antibiotics, only resistant microorganisms are able to grow and to incorporate the labeled amino acids. This leads to a difference in the mass spectra of susceptible and resistant isolates, allowing their differentiation. In the presented study, we demonstrated the applicability of this novel approach for the detection of methicillin-resistant Staphylococcus aureus and tested different bioinformatics approaches for automated data interpretation.

A 32-year-old female African patient presented to the emergency department for abdominal pain. She had been diagnosed with HIV infection 3 years earlier. She was ART-naive with a CD4-T cell count of 456/ll and a viral load of 626 cp/ml. Her C-reactive protein level was elevated at 84.3 mg/l, and no other laboratory abnormality was noted. Surgery for suspected appendicitis was performed. Histopathological work-up revealed phlegmonous appendicitis with localized eosinophilic infiltration and necrotic epitheloid granulomas (Fig. 1a, b). Differential diagnoses included eosinophilic gastroenteritis, hematologic malignancy, and mycobacterial infection. Finally, three species of intestinal worms, Schistosoma mansoni (Fig. 1c), Strongyloides stercoralis (Fig. 1d), and Dicrocoelium dendriticum (Fig. 1e) were detected in a stool sample. Antihelminthic treatment with praziquantel (1,800 mg on days 1 and 8) and albendazole (800 mg qd on days 1–3 and 8–10) was administered. Further stool samples were negative, and the patient remained asymptomatic. The CD4-Tcell counts measured 1 and 4 months later were somewhat higher at 675 and 633/ll, respectively. More than two billion individuals are estimated to be infected with one or more helminth species [1, 2] and over 22 million are HIV-coinfected [3, 4], especially in sub-Saharan Africa. Typical clinical signs and symptoms or laboratory abnormalities are often absent, despite the frequency of typical histopathologic alterations such as eosinophilic granulomas in chronic schistosomiasis [5]. Helminthic infections are not considered to be opportunistic infections. However, helminth-induced immunomodulation may affect the CD4 count [3–6]. The benefit of deworming in helminth/HIV-coinfection is suggested by several studies, but its effect may vary by individual helminth species [4]. Routine screening for tropical parasites according to travel or origin is recommended by current HIV treatment guidelines [7], reflecting the traveland migration-related increasing overlap of tropical medicine and HIV care also in developed countries [8]. S. Sammet M. Huber U. Seybold (&) Sektion Klinische Infektiologie, Medizinische Klinik und Poliklinik IV, Klinikum der Universitat, Ludwig-Maximilians-Universitat Munchen, Pettenkoferstr. 8a, 80336 Munchen, Germany e-mail: useybold@med.uni-muenchen.de

Reported here is the first case of the North American strain Yersinia enterocolitica serogroup O8, biotype 1B acting as a pathogen in Germany. The strain was isolated from the stool specimen of a 4-year-old boy who presented with persistent abdominal pain, fever and diarrhea accompanied by leukocytosis, anemia and an elevated sedimentation rate. The typical virulence characteristics identified in the isolated strain included the virulence plasmid pYV, the high pathogenicity island and mouse virulence.

Background Enteric pathogens need to grow efficiently in the gut lumen in order to cause disease and ensure transmission. The interior of the gut forms a complex environment comprising the mucosal surface area and the inner gut lumen with epithelial cell debris and food particles. Recruitment of neutrophils to the intestinal lumen is a hallmark of non-typhoidal Salmonella enterica infections in humans. Here, we analyzed the interaction of gut luminal neutrophils with S. enterica serovar Typhimurium (S. Tm) in a mouse colitis model. Results Upon S. Tmwt infection, neutrophils transmigrate across the mucosa into the intestinal lumen. We detected a majority of pathogens associated with luminal neutrophils 20 hours after infection. Neutrophils are viable and actively engulf S. Tm, as demonstrated by live microscopy. Using S. Tm mutant strains defective in tissue invasion we show that pathogens are mostly taken up in the gut lumen at the epithelial barrier by luminal neutrophils. In these luminal neutrophils, S. Tm induces expression of genes typically required for its intracellular lifestyle such as siderophore production iroBCDE and the Salmonella pathogenicity island 2 encoded type three secretion system (TTSS-2). This shows that S. Tm at least transiently survives and responds to engulfment by gut luminal neutrophils. Gentamicin protection experiments suggest that the life-span of luminal neutrophils is limited and that S. Tm is subsequently released into the gut lumen. This “fast cycling” through the intracellular compartment of gut luminal neutrophils would explain the high fraction of TTSS-2 and iroBCDE expressing intra- and extracellular bacteria in the lumen of the infected gut. Conclusion In conclusion, live neutrophils recruited during acute S. Tm colitis engulf pathogens in the gut lumen and may thus actively engage in shaping the environment of pathogens and commensals in the inflamed gut., PLoS ONE, 7 (4), ISSN:1932-6203

Few developments in microbiological diagnostics have had such a rapid impact on species level identification of microorganisms as matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS). Conventional differentiation methods rely on biochemical criteria and require additional pre-testing and lengthy incubation procedures. In comparison, MALDI-TOF MS can identify bacteria and yeast within minutes directly from colonies grown on culture plates. This radically new, methodically simple approach profoundly reduces the cost of consumables and time spent on diagnostics. The reliability and accuracy of the method have been demonstrated in numerous studies and different systems are already commercially available. Novel applications of the system besides microbial species level identification are also being explored. This includes identification of pathogens from positive blood cultures or directly from patient samples, such as urine. Currently, intriguing MALDI-TOF MS developments are being made regarding the phenotypic detection of certain antibiotic resistance mechanisms, e.g., β-lactamases and carbapenemases. This mini review provides an overview of the literature in the field and also includes our own data and experiences gathered from over 4 years of routine MALDI-TOF MS use in a university hospital’s microbiological diagnostics facility.

Infections due to extraintestinal pathogenic E. coli (ExPEC) are very common in humans as well as in animals. In humans ExPEC infections include urinary tract infections (UTI), septicemia, and wound infections, which result in significant morbidity, mortality, and substantial healthcare costs. In view of the increasing number of ExPEC infections caused by more and more resistant strains, effective prevention would be desirable. Given the rising treatment costs, a vaccine may be cost-effective in selected patient groups, such as women with recurrent UTI, patients with neurologic disorders impairing bladder function and men with prostate hyperplasia. Previous vaccine studies used single target proteins or whole inactivated ExPEC cells. Here, we describe a vaccine system for oral application based on artificial multiple subunit vaccine proteins. Those multi-epitope proteins are composed of predicted epitopes derived from ExPEC virulence-associated proteins. As ExPEC are known to form intracellular biofilms in the urothelium and can also resist killing by non-activated macrophages, T-cell responses are supposed to be an important measure to counteract these stages of ExPEC during infection. Therefore, a live bacterial antigen delivery system based upon the Salmonella type-III secretion system (T3SS) was used in this study to directly deliver the vaccine proteins into the cytoplasm of the host cells. Epitope-rich domains of the proteins FyuA, IroN, ChuA, IreA, Iha, and Usp were expressed in an attenuated Salmonella enterica serovar Typhimurium strain and translocated into target cells for extended periods of time inducing a strong T-cell response. No significant antibody titre increase against the secreted vaccine proteins could be detected in vaginal wash or serum. Despite that, one of the vaccine proteins was able to significantly reduce bacterial load in the challenge model of intraperitoneal sepsis. This study shows that a vaccine encompassing distinct epitopes of virulence-associated ExPEC proteins (i) can be applied for a T3SS-dependent vaccination strategy, (ii) elicits T-cell responses and (iii) confers protection after a single application.

Summary Staphylococcus aureus carriage increases the risk of infection. Demographic and microbiological data from adult patients with nasal S . aureus carriage were analysed in order to define effect modifiers of this association. Predictors for growth of S . aureus from clinical cultures were identified in a case–control study using bivariate and multi-variate logistic regression analysis. Between 1 January 2005 and 1 April 2009, 645 patients with nasal S . aureus colonization and documented follow-up of ≥90 days were identified; 159 (25%) patients were found to carry meticillin-resistant S . aureus (MRSA). The median age of patients was 58 years, and 421 (65%) were male. During the subsequent 90 days, one or more clinical cultures were positive for S . aureus in 131 patients (20%). Multi-variate analysis identified a prior history of any S . aureus positive culture [adjusted odds ratio (aOR) 2.4, 95% confidence interval (CI) 1.5–3.8; P =0.0005) as an independent predictor of subsequent S . aureus infection. MRSA colonization was a predictor of infection in patients aged >40 years (aOR 2.5, 95% CI 1.4–4.1; P =0.0004), and even more so in patients aged ≤40 years (aOR 12.4, 95% CI 3.0–51; P =0.0005). Age >40 years was an additional independent risk factor for meticillin-susceptible S . aureus carriers (aOR 3.0, 95% CI 1.2–7.8; P =0.02) but not for MRSA carriers. Preferential screening of patients at high risk for MRSA carriage and subsequent infection, as well as the absence of a universal policy for the use of decolonization regimens, may partly explain the relatively high risk of S . aureus infection in the patient population. MRSA carriers and older patients with recurrent S . aureus positive cultures may gain the greatest benefit from routine decolonization measures.

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is widely used for rapid and reliable identification of bacteria and yeast grown on agar plates. Moreover, MALDI-TOF MS also holds promise for bacterial identification from blood culture (BC) broths in hospital laboratories. The most important technical step for the identification of bacteria from positive BCs by MALDI-TOF MS is sample preparation to remove blood cells and host proteins. We present a method for novel, rapid sample preparation using differential lysis of blood cells. We demonstrate the efficacy and ease of use of this sample preparation and subsequent MALDI-TOF MS identification, applying it to a total of 500 aerobic and anaerobic BCs reported to be positive by a Bactec 9240 system. In 86.5% of all BCs, the microorganism species were correctly identified. Moreover, in 18/27 mixed cultures at least one isolate was correctly identified. A novel method that adjusts the score value for MALDI-TOF MS results is proposed, further improving the proportion of correctly identified samples. The results of the present study show that the MALDI-TOF MS-based method allows rapid (

The rapid identification of the potentially toxigenic Corynebacterium species, C. diphtheriae, C. ulcerans and C. pseudotuberculosis is essential for diagnosis and treatment of diphtheria and diphtheria-like diseases. We used matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) in comparison with classical microbiological and molecular methods on 116 Corynebacterium strains. All 90 potentially toxigenic Corynebacterium strains collected by the German National Consiliary Laboratory on Diphtheria in a period of more than ten years were correctly identified by MALDI-TOF MS. We propose an algorithm for fast and reliable diagnosis of diphtheria incorporating MALDI-TOF MS, real-time tox PCR and Elek testing.

Extraintestinal pathogenic Escherichia coli (ExPECs) possess an armament of virulence factors to colonize and infect the host, such as adhesins, toxins, capsules and iron-uptake systems. Recently, we could identify a novel virulence factor of ExPECs that interferes with the innate immune response of the host by interrupting the NF-κB signaling pathway. This protein named TcpC shows considerable homology to motifs of the Tir domain of Toll-like receptors. Here we demonstrate that the tcpC gene is widely distributed among clinical ExPEC isolates with almost half of the E. coli strains from patients suffering pyelonephritis shown to be tcpC positive as compared to only 8% in commensal isolates. However, this gene is only present in phylogenetic group B2 strains. Interestingly, the tcpC gene is strongly associated with presence of the high-pathogenicity island (HPI). The phylogenetic history of the tcpC gene, in the E. coli reference collection (ECOR) and other well-defined E. coli strains, compared to the phylogenetic histories of the HPI and the strains, showed that the tcpC gene (i) is scattered among various B2 subgroups with specific O-types, (ii) has a phylogeny incongruent with the strain phylogeny, but (iii) congruent with the HPI phylogenetic history. This, together with the strong conservation of the tcpC gene, indicates a very recent introduction of this virulence factor into E. coli by horizontal gene transfer which occurred "en bloc" with the HPI at one major hot spot of recombination in the E. coli genome. The present data provide evidence for a strong impact of homologous recombination events in the spread of the TcpC virulence trait among E. coli.

In this study, we investigated the variability of MutS among Pseudomonas aeruginosa recovered from cystic fibrosis (CF) patients. Sequencing of the mutS gene of 15 hypermutable P. aeruginosa isolates obtained from different patients revealed high rates of nucleotide substitutions as compared to that of strain PAO1. Significantly more synonymous than non-synonymous nucleotide substitutions have been found, indicating that generally MutS is highly conserved. The functional analysis of MutS variants by complementation of a PAO1 mutS mutant revealed 5 isolates with a defective MutS due to frameshift mutations or amino acid substitutions. This work supports the hypothesis that the respiratory tract of CF patients represents an environment that favors the selection of highly adaptive mutator phenotypes.

Among 1,102 recent Escherichia coli clinical isolates, clonal group A was identified in 17 of 20 (U.S. and non-U.S.) geographic locales, mainly among U.S. isolates (9% vs. 3%; p < 0.001) and those resistant to trimethoprim-sulfamethoxazole (10% vs. 1.7%; p < 0.001). The extensive antimicrobial resistance and virulence profiles of clonal group A may underlie its recent widespread emergence.

This report presents the case of a patient with a massive pyelonephritis and a urosepsis caused by Streptococcus pneumoniae . This case is unusual as the focus was distant from the respiratory tract, the usual primary site of infection caused by this organism. No other primary site of infection was documented.

We present the case of a 68-year-old diabetic woman who has been suffering from chronic urinary tract infections, recurring over a period of at least 5 years, caused by a slowly growing metabolically deficient dwarf mutant (MDD) of Escherichia coli. This MDD strain was auxotrophic for histidine, was resistant to multiple antibiotics, and showed atypical growth behavior. Colonies were tiny on routine media but were able to revert to normal growth after extended incubation. This strain was identified as E. coli by 16S ribosomal DNA sequencing, and virulence factor profiles were determined by PCR. Seven MDD isolates collected over the 5-year period were grown from midstream urine to significant colony counts and shown to belong to the same clonal group by pulsed-field gel electrophoresis and enterobacterial repetitive intergenic consensus PCR. These MDDs were repeatedly misidentified by biochemical methods due to their slow growth and atypical colony morphology. This case highlights the importance of recognizing MDDs of Enterobacteriaceae in patients with chronic infections. To our knowledge this is the first report of an MDD of E. coli causing a chronic urinary tract infection.

The chromosomal iroBCDEN gene cluster first described for Salmonella enterica is involved in the uptake of catecholate-type siderophore compounds. An orthologous gene cluster has recently been detected in Escherichia coli strains which cause extraintestinal disease. This E. coli iroBCDEN gene cluster has an impact on virulence and has been reported to be located in a pathogenicity island on the chromosome. In this study we characterized an iro gene cluster of a uropathogenic E. coli isolate which is located on a transmissible plasmid related to the R64 plasmid of S. enterica . This cluster is highly homologous to the chromosomal iro cluster of E. coli . When introduced into an E. coli fepA cir fiu aroB mutant, IroN, but not IroBCDE, mediated the utilization of structurally related catecholate siderophores, including 2,3-dihydroxybenzoyl- l -serine, 2,3-dihydroxybenzoyl- d -ornithine, 2,3-dihydroxybenzoic acid, and enterochelin. This study supports the idea of an ongoing horizontal transfer of putative virulence factors and the mobilization of single virulence gene clusters, which lead to a modular assembly of virulence determinants such as pathogenicity islands.

The Yersinia high-pathogenicity island (HPI) encodes an iron uptake system mediated by the siderophore yersiniabactin (Ybt) and confers the virulence of highly pathogenic Yersinia species. This HPI is also widely distributed in human pathogenic members of the family of Enterobacteriaceae , above all in extraintestinal pathogenic Escherichia coli (ExPEC). In the present study we demonstrate a highly significant correlation of a functional HPI and extraintestinal virulence in E. coli . Moreover, using a mouse infection model, we show for the first time that the HPI contributes to the virulence of ExPEC.

Importance Although chronic meningococcemia is an uncommon disorder, it is of great importance to clinicians across multiple disciplines because it presents similarly to reactive, neoplastic, or rheumatic disorders. Ruling out chronic meningococcemia, however, represents a diagnostic challenge because routine microbiological investigations frequently fail to identify Neisseria meningitidis . Although treatment with corticosteroids might be helpful in various conditions, corticosteroid treatment may lead to severe complications in underlying chronic meningococcemia. Observations We describe a patient with a history of recurrent fever, arthralgia, and disseminated skin lesions. The patient was assumed to have Sweet syndrome and was treated with corticosteroids. Subsequently the patient developed meningococcal meningitis and was admitted to the neurointensive care unit. Chronic meningococcemia was confirmed retrospectively by nonroutine polymerase chain reaction and silver staining of skin biopsy specimens. Immunologic workup revealed decreased IgG subclass 3. Conclusions and Relevance Consideration of chronic meningococcemia is important when a patient presents with a history of fever and disseminated skin lesions. Polymerase chain reaction testing of skin biopsy specimens should be performed more systematically if the results of routine microbiological investigations remain unrevealing. In addition, silver staining of skin lesions can help establish the diagnosis. Eventually, testing for immune deficiencies should more routinely follow a confirmed diagnosis of chronic meningococcemia.

Clinical samples obtained over a period of 8 months ( n = 2,624) were processed in parallel with the BACTEC 460TB system, with the MGIT 960 system, and in Löwenstein-Jensen (LJ) medium, resulting in the recovery of 127 mycobacteria. Recovery rates in combinations of the BACTEC 460TB or MGIT 960 system with LJ were, respectively, 94.7 and 94.7% for Mycobacterium tuberculosis complex ( n = 57) and 91.4 and 70.0% for nontuberculous mycobacteria ( n = 70). Contamination rates, elevated in the MGIT 960 system, were associated with patients (cystic fibrosis) and type of material but not with transport time. Detection time was reduced in the MGIT 960 system.

Production of the siderophore yersiniabactin is associated with virulence in Yersinia species. The genes for biosynthesis and uptake of yersiniabactin are located on a high-pathogenicity island (HPI). The distribution and functioning of the Yersinia HPI were assessed in different Enterobacteriaceae strains isolated from blood cultures and urine samples. In total, 550 clinical isolates from 10 Enterobacteriaceae species were investigated by polymerase chain reaction and DNA hybridization. The Yersinia HPI was most prevalent in Escherichia coli (overall prevalence, 72.3%) and, to a lesser extent, in Klebsiella oxytoca (58.3%), Citrobacter species (25%), Klebsiella pneumonia (17.7%), and Enterobacter species (12.2%). The production of the siderophore yersiniabactin was also demonstrated in these HPI-positive strains by use of a reporter gene bioassay. These findings indicate that the HPI of Yersinia is distributed and functions in clinical specimens of different Enterobacteriaceae species.

Shiga toxin-producing Escherichia coli (STEC) strains cause a wide spectrum of diseases in humans. In this study, we tested 206 STEC strains isolated from patients for potential virulence genes including stx , eae , and enterohemorrhagic E. coli hly . In addition, all strains were examined for the presence of another genetic element, the high-pathogenicity island (HPI). The HPI was first described in pathogenic Yersinia species and encodes the pesticin receptor FyuA and the siderophore yersiniabactin. The HPI was found in the genome of distinct clonal lineages of STEC, including all 31 eae -positive O26:H11/H − strains and 7 of 12 eae -negative O128:H2/H − strains. In total, the HPI was found in 56 (27.2%) of 206 STEC strains. However, it was absent from the genome of all 37 O157:H7/H − , 14 O111:H − , 13 O103:H2, and 13 O145:H − STEC isolates, all of which were positive for eae . Polypeptides encoded by the fyuA gene located on the HPI could be detected by using immunoblot analysis in most of the HPI-positive STEC strains, suggesting the presence of a functional yersiniabactin system. The HPI in STEC was located next to the tRNA gene asnT . In contrast to the HPI of other pathogenic enterobacteria, the HPI of O26 STEC strains shows a deletion at its left junction, leading to a truncated integrase gene int . We conclude from this study that the Yersinia HPI is disseminated among certain clonal subgroups of STEC strains. The hypothesis that the HPI in STEC contributes to the fitness of the strains in certain ecological niches rather than to their pathogenic potential is discussed.