Multicentre investigation of carbapenemase-producing Escherichia coli and Klebsiella pneumoniae in German hospitals.

Aim of this study was to determine the incidence and molecular epidemiology of carbapenemase-producing Escherichia coli and Klebsiella pneumoniae in Germany. E. coli and K. pneumoniae isolates from clinical samples which were non-susceptible to carbapenems were collected in laboratories serving 20 hospitals throughout Germany from November 2013 to April 2014. The isolates were tested for the presence of carbapenemases by PCR and phenotypic methods and typed by multilocus sequence typing. Risk factors including a previous hospitalization abroad were analysed. Carbapenemases were detected in 24 isolates from 22 patients out of 464,514 admissions. Carbapenemases included OXA-48 (n=14), KPC-2 (n=8) and NDM-1 (n=2). Except for two K. pneumoniae isolates with ST101, all OXA-48 producing strains belonged to different clones. In contrast, half of KPC-2 producing K. pneumoniae were of ST258 and both NDM-1 producing strains were of ST11. Compared to carbapenem-susceptible controls, patients with carbapenemase-producing strains differed by a significantly higher proportion of males, a higher proportion of isolates from wound samples and a more frequent previous stay abroad in univariate analysis. This multicentre study demonstrated an incidence of carbapenemase-producing E. coli and K. pneumoniae from clinical samples in Germany of 0.047 cases per 1000 admissions. OXA-48 was more frequent than KPC-2 and NDM-1 and showed a multiclonal background.

Detection of significant bacteriuria by use of the iQ200 automated urine microscope.

In the microbiology laboratory, there is an augmented need for rapid screening methods for the detection of bacteria in urine samples, since about two-thirds of these samples will not yield any bacteria or will yield insignificant growth when cultured. Thus, a reliable screening method can free up laboratory resources and can speed up the reporting of a negative urine result. In this study, we have evaluated the detection of leukocytes, bacteria, and a new sediment indicator, the "all small particles" (ASP), by an automated instrument, the iQ200 urine analyzer, to detect negative urine samples that can be excluded from culture. A coupled automated strip reader (iChem Velocity), enabling the detection of nitrite and leukocyte esterase, was tested in parallel. In total, 963 urine samples were processed through both conventional urine culture and the iQ200/iChem Velocity workstation. Using the data, a multivariate regression model was established, and the predicted specificity and the possible reduction in urine cultures were calculated for the indicators and their respective combinations (leukocytes plus bacteria plus ASP and leukocyte esterase plus nitrite). Among all options, diagnostic performance was best using the whole microscopic content of the sample (leukocytes plus bacteria plus ASP). By using a cutoff value of ≥ 10(4) CFU/ml for defining a positive culture, a given sensitivity of 95% resulted in a specificity of 61% and a reduction in urine cultures of 35%. By considering the indicators alone, specificity and the culture savings were both much less satisfactory. The regression model was also used to determine possible cutoff values for running the instrument as part of daily routine. By using a graphical representation of all combinations possible, we derived cutoff values for leukocyte, bacterial, and ASP count, which should enable the iQ200 microscope to screen out approximately one-third of the urine samples, significantly reducing the workload in the microbiology laboratory.

Increased T helper type 17 response to pathogen stimulation in patients with primary sclerosing cholangitis.

UNLABELLED: T helper (Th)17 cells are important for host defense against bacteria and fungi, but are also involved in the pathogenesis of autoimmune diseases. In primary sclerosing cholangitis (PSC), bile fluid is frequently colonized with pathogens and its strong association with inflammatory bowel disease suggests the contribution of pathogen responses to disease pathogenesis. Interleukin (IL)-17A, the signature cytokine of Th17 cells, was recently described to promote inflammation and fibrosis within the liver. Therefore, we investigated Th17 immune response to pathogens in patients with PSC. Bile fluid was obtained by endoscopic retrograde cholangiography, and bacterial and fungal species grew in the majority of samples. In addition, bacterial RNA was stained in liver sections using 16sRNA fluorescence in situ hybridization and was detected in the portal tracts in 12 of 13 tested PSC patients. Bacteria grown from patients' bile fluid were then used to stimulate peripheral blood mononuclear cells (PBMCs) and to assess their Th17 response. Compared to healthy controls or primary biliary cirrhosis patients, PBMCs from PSC patients manifested significantly higher frequencies of Th17 and Th1/Th17 cells after pathogen stimulation. The highest frequencies of Th17 cells were detected after stimulation with Candida albicans, a pathogen that has been linked to disease progression. Immunohistochemically, IL-17A-expressing lymphocytes were detected within the periductal areas of PSC patients. Th17 induction was also noted after stimulation of Toll-like receptor 5 or 7, but not of other pattern recognition receptors tested, pointing to signaling pathways potentially involved in Th17 induction in PSC. CONCLUSION: We demonstrate an increased Th17 response to microbial stimulation in patients with PSC. These data should prompt further studies investigating the link between pathogen responses, inflammation, and fibrosis in patients with PSC.

Radiography-based score indicative for the pathogenicity of bacteria in odontogenic infections.

OBJECTIVE: To develop a new radiography-based score to assess the potential of bacteria to cause odontogenic infections derived from the occurrence of bacteria at small or large radiographical lesions. MATERIALS AND METHODS: The patients analyzed were a sub-population from a large randomized clinical trial comparing moxifloxacin and clindamycin in the treatment of inflammatory infiltrates and odontogenic abscesses. Routine radiographs were used to analyze the area of the periapical radiolucent lesions. Lesions were stratified by their radiographically measured area as large (>9 mm(2)) or small (≤9 mm(2)). A risk ratio was calculated for each species from the frequency of their occurrence in large vs in small lesions. RESULTS: Fifty-one patients, 19 with abscesses and 32 with infiltrates, were evaluated. Overall, the radiographical lesion areas ranged from 0.4-46.2 mm(2) (median = 9 mm(2)). An increased risk (risk ratio >1) to occur at large abscess lesions was observed for Prevotella (P.) oralis, P. buccae, P. oris, P. intermedia, Fusobacterium nucleatum and Streptococcus (Strep.) anginosus group. An increased risk to occur at large infiltrate lesions was found for Strep. salivarius, Strep. parasanguis, Strep. anginosus group, Capnocytophaga spp., Neisseria (N.) sicca, Neisseria spp., Staphylococcus (Staph.) aureus, P. intermedia, P. buccae, Prevotella spp. and P. melaninogenica. CONCLUSIONS: The radiography-based score suggests that certain Prevotella spp., F. nucleatum and Strep. anginosus groups play a crucial role in the pathogenesis of odontogenic abscesses, and that various streptococci, Neisseria spp., Capnocytophaga spp., Staph. aureus and Prevotella spp. are involved in the pathogenesis of odontogenic infiltrates.

Aspergillus galactomannan antigen for diagnosis and treatment monitoring in cerebral aspergillosis.

Invasive aspergillosis, especially neuroaspergillosis, predominantly affects immunocompromised patients such as transplant recipients. Early diagnosis and subsequent early initiation of therapy may improve final outcome. In cerebral aspergillosis, samples for culture or histopathology, the diagnostic reference standard, are often impossible to obtain without risk. Because of these limitations, serological, nucleic acid amplification tests such as polymerase chain reaction or enzyme immunoassay from cerebrospinal fluid appear advantageous for early diagnosis and probably also for therapy monitoring. We report on the successful detection and treatment monitoring by serial testing of galactomannan in cerebrospinal fluid in a patient with neuroaspergillosis after heart transplant.

Microbiological analysis of a prospective, randomized, double-blind trial comparing moxifloxacin and clindamycin in the treatment of odontogenic infiltrates and abscesses.

The objective of this study was to identify the oral pathogens found in odontogenic infections, to determine their susceptibilities to amoxicillin-clavulanic acid (AMC), clindamycin (CLI), doxycycline (DOX), levofloxacin (LVX), moxifloxacin (MXF), and penicillin (PEN), and to search for associations between specific pathogens and types of infection. Swabs from patients enrolled in a randomized, double-blind phase II trial comparing MXF with CLI for the treatment of odontogenic abscesses or inflammatory infiltrates were cultured on media for aerobes and anaerobes. All bacterial isolates were identified at the species level. Overall, 205 isolates were cultured from 71 patients: 77 viridans group streptococci, 56 Prevotella spp., 19 Neisseria spp., 17 Streptococcus anginosus group isolates and hemolytic streptococci, 15 other anaerobes, and 21 other bacteria. Ninety-eight percent of pathogens were susceptible to MXF, 96% to AMC, 85% to LVX, 67% to PEN, 60% to CLI, and 50% to DOX. S. anginosus group and hemolytic streptococci were found significantly more frequently (P = 0.04) in patients with abscesses (12/95) than in patients with infiltrates (5/110). In four patients with infiltrates who failed to respond to CLI therapy, three isolates of the Streptococcus mitis group and four Neisseria spp. resistant to CLI were found. In this study, S. anginosus group and hemolytic streptococci were clearly associated with odontogenic abscesses. Our analysis suggests that viridans group streptococci and Neisseria spp. play a decisive role in the etiology of odontogenic infiltrates. The high in vitro activity of MXF against odontogenic bacteria corresponds well to its clinical results in the treatment of odontogenic abscesses and infiltrates.

Comparative efficacy and safety of moxifloxacin and clindamycin in the treatment of odontogenic abscesses and inflammatory infiltrates: a phase II, double-blind, randomized trial.

Moxifloxacin penetrates well into oromaxillary tissue and covers the causative pathogens that show an increasing resistance to standard antibiotics. Clinical reports suggest that moxifloxacin may be effective for the treatment of odontogenic infections that can lead to serious complications. The objective of this prospective, randomized, double-blind, multicenter study was to compare the efficacies and safeties of moxifloxacin and clindamycin for the medical treatment of patients with gingival inflammatory infiltrates and as an adjuvant therapy for patients with odontogenic abscesses requiring surgical treatment. Patients received either 400 mg moxifloxacin per os once daily or 300 mg clindamycin per os four times daily for 5 days consecutively. The primary efficacy endpoint was the percent reduction in patients' perceived pain on a visual analogue scale at days 2 to 3 from baseline. Primary analysis included 21 moxifloxacin- and 19 clindamycin-treated patients with infiltrates and 15 moxifloxacin- and 16 clindamycin-treated patients with abscesses. The mean pain reductions were 61.0% (standard deviation [SD], 46.9%) with moxifloxacin versus 23.4% (SD, 32.1%) with clindamycin (P = 0.006) for patients with infiltrates and 55.8% (SD, 24.8%) with moxifloxacin versus 42.7% (SD, 48.5%) with clindamycin (P = 0.358) for patients with abscesses. A global efficacy assessment at days 2 to 3 and 5 to 7 showed faster clinical responses with moxifloxacin in both abscess and infiltrate patients. Rates of adverse events were lower in moxifloxacin- than in clindamycin-treated patients. In patients with inflammatory infiltrates, moxifloxacin was significantly more effective in reducing pain at days 2 to 3 of therapy than clindamycin. No significant differences between groups were found for patients with odontogenic abscesses.

Characterization of Enterobacter cloacae pneumonia: a single-center retrospective analysis.

OBJECTIVES: Enterobacter cloacae (E. cloacae) is a Gram-negative rod commonly found on intensive care units (ICU) causing severe infections with high mortality. Specific characteristics of E. cloacae pneumonia, however, have not been identified. DESIGN: Evaluation of clinical and microbiological records of patients with positive respiratory samples for E. cloacae was performed by a 1-year retrospective study in a large university hospital. RESULTS: Ninety-seven of 115 eligible patients with E. cloacae-positive respiratory samples developed pneumonia. Patients were predominantly male (68%), older (median age = 62 years), and immunodeficient (54%). Seventy-eight percent required ICU admission, of which 97% required mechanical ventilation. Ventilator-associated pneumonia (VAP) occurred in 58%. Those who developed E. cloacae VAP had undergone twice as many surgical procedures under translaryngeal intubation prior to VAP onset (89 vs. 48%, P < 0.0001). Overall, E. cloacae VAP mortality was 24%. In E. cloacae VAP patients, presence of translaryngeal tubes (P = 0.02) and female gender (P = 0.0003) were associated with poor survival. Multivariate analysis confirmed male sex as a protective factor (relative risk: 0.39; P = 0.007). CONCLUSION: Enterobacter cloacae causes VAP with high mortality, predominantly in women. Risk factors for E. cloacae pneumonia seem to match those for VAP. The presence of translaryngeal endotracheal tubes seems to be the specific factor for E. cloacae VAP.

Invasive aspergillosis caused by Aspergillus terreus in a living donor liver transplant recipient successfully treated by caspofungin.

Invasive aspergillosis is one of the most severe complications after liver transplantation characterised by early dissemination of disease and high mortality. Recent data show that the prognosis is diminishing even further when Aspergillus terreus, a strain resistant to standard treatment with amphotericin, is isolated. We report a high risk liver transplant recipient with multiple co-morbidities including renal failure and allograft dysfunction in whom pulmonary aspergillosis due to A. terreus was successfully treated by the echinocandin antifungal agent caspofungin.

A 56-Year-Old Woman with Chronic Hepatitis C Liver Disease and Meningitis due to Streptococcus equi subsp. Zooepidemicus.

Streptococcus equi subsp. zooepidemicus (S. equi subsp. zooepidemicus), which carries the Lancefield group C antigen, is an uncommon human pathogen. It is considered an opportunistic commensal of the equine upper respiratory tract and causes invasive infections in immunocompromised hosts, following close contact to infected horses. Meningitis caused by S. equi subsp. zooepidemicus is a rare infectious disease with high rates of complications. We present the case of a 56-year-old female with acutely altered mental status following three days of fever and vomiting. For several months, she was taking care of horses. The most relevant preexisting illnesses were chronic hepatitis C infection and traumatic paraplegia due to spinal cord injury 30 years ago. Laboratory evaluation on admission revealed leukocytosis, hyponatremia, and elevated C-reactive protein. Cerebral CT scan showed diffuse cerebral edema. Whereas cerebrospinal fluid real-time PCR assay for common pathogens was negative, cultures showed S. equi subsp. zooepidemicus. She recovered fully after intravenous administration of ceftriaxone for four weeks. This is one of only few reported cases of S. equi subsp. zooepidemicus meningitis and the first case in chronic hepatitis C infection. Our case supports the necessity for extended microbiological examination especially in immunocompromised patients if PCR examination for common pathogens is negative.

Suitability of current typing procedures to identify epidemiologically linked human Giardia duodenalis isolates.

BACKGROUND: Giardia duodenalis is a leading cause of gastroenteritis worldwide. Humans are mainly infected by two different subtypes, i.e., assemblage A and B. Genotyping is hampered by allelic sequence heterozygosity (ASH) mainly in assemblage B, and by occurrence of mixed infections. Here we assessed the suitability of current genotyping protocols of G. duodenalis for epidemiological applications such as molecular tracing of transmission chains. METHODOLOGY/PRINCIPAL FINDINGS: Two G. duodenalis isolate collections, from an outpatient tropical medicine clinic and from several primary care laboratories, were characterized by assemblage-specific qPCR (TIF, CATH gene loci) and a common multi locus sequence typing (MLST; TPI, BG, GDH gene loci). Assemblage A isolates were further typed at additional loci (HCMP22547, CID1, RHP26, HCMP6372, DIS3, NEK15411). Of 175/202 (86.6%) patients the G. duodenalis assemblage could be identified: Assemblages A 25/175 (14.3%), B 115/175 (65.7%) and A+B mixed 35/175 (20.0%). By incorporating allelic sequence heterozygosity in the analysis, the three marker MLST correctly identified 6/9 (66,7%) and 4/5 (80.0%) consecutive samples from chronic assemblage B infections in the two collections, respectively, and identified a cluster of five independent patients carrying assemblage B parasites of identical MLST type. Extended MLST for assemblage A altogether identified 5/6 (83,3%) consecutive samples from chronic assemblage A infections and 15 novel genotypes. Based on the observed A+B mixed infections it is estimated that only 75% and 50% of assemblage A or B only cases represent single strain infections, respectively. We demonstrate that typing results are consistent with this prediction. CONCLUSIONS/SIGNIFICANCE: Typing of assemblage A and B isolates with resolution for epidemiological applications is possible but requires separate genotyping protocols. The high frequency of multiple infections and their impact on typing results are findings with immediate consequences for result interpretation in this field.

Rapid identification of bacteria from positive blood culture bottles by use of matrix-assisted laser desorption-ionization time of flight mass spectrometry fingerprinting.

Early and adequate antimicrobial therapy has been shown to improve the clinical outcome in bloodstream infections (BSI). To provide rapid pathogen identification for targeted treatment, we applied matrix-assisted laser desorption-ionization time of flight (MALDI-TOF) mass spectrometry fingerprinting to bacteria directly recovered from blood culture bottles. A total of 304 aerobic and anaerobic blood cultures, reported positive by a Bactec 9240 system, were subjected in parallel to differential centrifugation with subsequent mass spectrometry fingerprinting and reference identification using established microbiological methods. A representative spectrum of bloodstream pathogens was recovered from 277 samples that grew a single bacterial isolate. Species identification by direct mass spectrometry fingerprinting matched reference identification in 95% of these samples and worked equally well for aerobic and anaerobic culture bottles. Application of commonly used score cutoffs to classify the fingerprinting results led to an identification rate of 87%. Mismatching mostly resulted from insufficient bacterial numbers and preferentially occurred with Gram-positive samples. The respective spectra showed low concordance to database references and were effectively rejected by score thresholds. Spiking experiments and examination of the respective study samples even suggested applicability of the method to mixed cultures. With turnaround times around 100 min, the approach allowed for reliable pathogen identification at the day of blood culture positivity, providing treatment-relevant information within the critical phase of septic illness.

Penetration of moxifloxacin into rat mandibular bone and soft tissue.

OBJECTIVE: Based on its in vitro activity and spectrum of activity, the new 8-methoxyquinolone antibiotic moxifloxacin (MXF) seems suited for the antibiotic therapy of odontogenic infections. Penetration into the relevant tissue is another prerequisite for clinical efficacy. For this reason, the levels of MXF in plasma, soft tissue, and mandibular bone were determined in an animal model with Wistar rats. MATERIAL AND METHODS: Samples of 49 rats were analyzed. Tissue samples were homogenized and proteins were precipitated. The pharmacokinetic evaluation was conducted based on non-compartmental analysis. RESULTS: The concentration-time courses of tissues show a more plateau-shaped curve compared to plasma. Calculated AUC (area under the curve) ratios tissue:plasma were M. masseter:plasma = 2.64 and mandibles:plasma = 1.13. CONCLUSIONS: Administration of antibiotics is considered an important part of therapy during and/or after surgical procedures in the maxillofacial area. Because of the good penetration into bone and muscle tissues demonstrated in Wistar rats, MXF might be an option for clinical application in this indication.

Evaluation of a new screen agar plate for detection and presumptive identification of Enterobacteriaceae producing extended-spectrum beta-lactamases.

A new agar screen plate for extended-spectrum beta-lactamase (ESBL) detection was evaluated with 50 clinical isolates of ESBL-producing Enterobacteriaceae species: Enterobacter cloacae (n = 10), Escherichia coli (n = 10), Klebsiella oxytoca (n = 3), Klebsiella pneumoniae (n = 25), and Proteus mirabilis (n = 2). Fecal samples were artificially inoculated with 2 concentrations (25 and 250 colony forming units [CFU]/plate) of the test strains and then applied to the new agar screen plates. By this approach, the new agar formula detected growth that was suggestive of ESBL activity in 44 of 50 (88%) and 50 of 50 (100%) of ESBL strains with 25 and 250 CFU/plate, respectively. A limitation of the agar screen plates was a lack of some specificity. Among 15 strains with resistant phenotypes other than ESBL (K1 producers of K. oxytoca, 6 strains; 9 strains with AmpC phenotype), growth was recorded in 7 (25 CFU/plate) and 11 (250 CFU/plate) of 15 strains. In conclusion, the new agar screen plate is a sensitive and convenient method to directly screen for ESBL organisms in rectal swabs or stool samples, with the potential for incorporation into routine clinical laboratory service.

Disseminated Mycobacterium haemophilum infection as initial manifestation of AIDS.

Mycobacterium haemophilum has rarely been implicated in human disease. The organisms have been isolated mainly in patients with human immunodeficiency virus (HIV) disease or transplant recipients. We describe the first case of a disseminated M. haemophilum infection as initial manifestation of AIDS in Europe.

Comparison of BDPhoenix and VITEK2 automated antimicrobial susceptibility test systems for extended-spectrum beta-lactamase detection in Escherichia coli and Klebsiella species clinical isolates.

The present study compares the ability to detect extended-spectrum beta-lactamases (ESBL) among a collection of 34 ESBL producing clinical isolates belonging to Escherichia coli and Klebsiella species with two new rapid susceptibility and identification instruments-VITEK2 (bioMérieux, Marcy l'Etoile, France) vs. BDPhoenix (BD Biosciences, Sparks, MD). ESBL content in these isolates was previously characterized on the basis of PCR amplification and sequencing results which were used as the reference method in our evaluation. BDPhoenix correctly determined the ESBL outcome for all strains tested (100% detection rate), whereas VITEK2 was not able to detect the ESBL status in 5 isolates (85% detection rate). Detailed analysis revealed that the discrepancies were mainly observed with 'difficult-to-detect' strains. Misidentification was either due to low oximino cephalosporin MIC in these strains or was associated with pronounced 'cefotaximase' or 'ceftazidimase' phenotypes. Klebsiella oxytoca chromosomal beta-lactamase (K1) is phenotypically quite similar to ESBL enzymes. In order to evaluate whether the K1 and ESBL enzymes could be discriminated, we expanded our analysis by 8 clinical K. oxytoca strains with K1 phenotypes. VITEK2 gave excellent identification of these strains whereas 7 out of 8 were falsely labeled ESBL-positive by the BDPhoenix system.

Pathology of fatal traumatic and nontraumatic clostridial gas gangrene: a histopathological, immunohistochemical, and ultrastructural study of six autopsy cases.

We prospectively investigated six fatal cases of clostridial gas gangrene using autopsy, histology, immunohistochemistry, microbiology, and scanning electron microscopy. The causative pathogen was Clostridium perfringens in four cases, C. sordellii in one case, and a mixed infection with both C. perfringens and C. sordellii in one case. According to the previous medical history and autopsy findings, clostridial infection was related to trauma in three cases. Characterized by extensive tissue necrosis and total absence of an accompanying leukocyte infiltration and tissue inflammatory response, the histopathological picture of clostridial gas gangrene is distinctly different from other bacterial infections. In medicolegal casework, the proof of the source of infection and the portal of entry of the responsible pathogen is not always an easy task, especially in the absence of trauma.