Strategies to enhance rational use of antibiotics in hospital: a guideline by the German Society for Infectious Diseases.

INTRODUCTION: In the time of increasing resistance and paucity of new drug development there is a growing need for strategies to enhance rational use of antibiotics in German and Austrian hospitals. An evidence-based guideline on recommendations for implementation of antibiotic stewardship (ABS) programmes was developed by the German Society for Infectious Diseases in association with the following societies, associations and institutions: German Society of Hospital Pharmacists, German Society for Hygiene and Microbiology, Paul Ehrlich Society for Chemotherapy, The Austrian Association of Hospital Pharmacists, Austrian Society for Infectious Diseases and Tropical Medicine, Austrian Society for Antimicrobial Chemotherapy, Robert Koch Institute. MATERIALS AND METHODS: A structured literature research was performed in the databases EMBASE, BIOSIS, MEDLINE and The Cochrane Library from January 2006 to November 2010 with an update to April 2012 (MEDLINE and The Cochrane Library). The grading of recommendations in relation to their evidence is according to the AWMF Guidance Manual and Rules for Guideline Development. CONCLUSION: The guideline provides the grounds for rational use of antibiotics in hospital to counteract antimicrobial resistance and to improve the quality of care of patients with infections by maximising clinical outcomes while minimising toxicity. Requirements for a successful implementation of ABS programmes as well as core and supplemental ABS strategies are outlined. The German version of the guideline was published by the German Association of the Scientific Medical Societies (AWMF) in December 2013.

First report of mecC MRSA in human samples from Austria: molecular characteristics and clinical data.

Reports of mecC methicillin-resistant Staphylococcus aureus (MRSA) strains have been published from several European countries. We describe the first six mecC MRSA isolates of human origin from Austria and report the application of a rapid PCR test. Candidate isolates (n = 295) received between 2009 and 2013 were investigated phenotypically by cefoxitin screening and streaking on ChromID MRSA plates. The presence of mecC was confirmed in six isolates from blood cultures, wound swabs and screening samples of four female and two male patients (age range 7-89 years) by an in-house PCR method and the new Genspeed MRSA test (Greiner Bio-One, Kremsmünster, Austria). The mecC MRSA were further characterized by whole genome sequencing, multilocus sequence and spa typing. Antimicrobial susceptibility testing was performed by Eucast disk-diffusion method and Vitek 2. The six mecC MRSA isolates were from two clonal lineages (CC130, including a new single-locus variant, and CC599) and four different spa types (t843, t1535, t3256, t5930). Analysis for virulence factor genes yielded lukED, eta, etd2 and edin-B (CC130 isolates) and tst, lukED, eta and sel (ST599 isolates). The Genspeed MRSA test identified mecC in all isolates whereas Vitek 2 failed to detect methicillin resistance in one isolate. The strains were susceptible to a wide range of non-ß-lactam antibiotics. All patients were successfully treated or decolonized. mecC MRSA are present in Austria as colonizers but may also cause infections. Thus, laboratories must choose appropriate test methods such as cefoxitin screening and confirmation using molecular assays specifically targeting mecC.

Temocillin and meropenem to discriminate resistance mechanisms leading to decreased carbapenem susceptibility with focus on OXA-48 in Enterobacteriaceae.

A temocillin minimal inhibitory concentration ≥ 128 mg/L combined with the results of meropenem double disc synergy testing was used to (i) discriminate carbapenemase production from other resistance mechanisms leading to decreased carbapenem susceptibility; and (ii) differentiate Ambler classes in carbapenemase-producing enterobacteriaceae (CPE). The suggested test algorithm discriminated all extended spectrum ß-lactamase/AmpC from CPE isolates, which could further be divided correctly into Ambler classes A and B enzymes as well as OXA-48 in all cases. The algorithm is simple to implement as part of the daily routine in a standard microbiology laboratory with limited access to or resources for molecular biological tools.

The role of pharmacokinetics/pharmacodynamics in setting clinical MIC breakpoints: the EUCAST approach.

Clinical breakpoints are used in clinical microbiology laboratories to categorize microorganisms as clinically susceptible (S), intermediate (I) or resistant (R) dependent on the quantitative antimicrobial susceptibility as indicated by the MIC value determined in a well-defined standard test system. The laboratory report, with the designations of S, I or R for each antimicrobial agent, provides guidance to clinicians with respect to the potential use of agents in the treatment of patients, and clinical breakpoints should therefore distinguish between patients that are likely or unlikely to respond to antimicrobial treatment. In Europe, clinical breakpoints are set by the European Committee on Antimicrobial Susceptibility Testing (EUCAST), following a defined procedure. This includes evaluation of efficacy in experimental settings and clinical studies to derive pharmacodynamic targets such as the fAUC/MIC ratio or %fT > MIC required for efficacy, the pharmacokinetic properties of the agent, Monte Carlo simulations to estimate exposures of the antimicrobial agent in the target patient population and commonly used dosing regimens. The probability of target attainment is subsequently determined for a range of pharmacodynamic targets and the results from the Monte Carlo simulations. The breakpoints derived are subsequently evaluated with respect to the wild-type population of the target microorganisms, specific resistance mechanisms and other relevant data. In this paper, we provide an overview of the EUCAST process and considerations for setting pharmacokinetic/pharmacodynamic breakpoints. These are the breakpoints that in the EUCAST breakpoint tables are referred to as 'non-species-related breakpoints'.

Prevalence of Panton-Valentine leukocidin genes in methicillin-resistant Staphylococcus aureus isolates phenotypically consistent with community-acquired MRSA, 1999-2007, Vienna General Hospital.

The purpose of this study was to investigate methicillin-resistant Staphylococcus aureus (MRSA) isolates with antibiotic resistance restricted to beta-lactam antibiotics and variable resistance to fusidic acid for the presence of Panton-Valentine leukocidin (PVL) genes. Our data show that the selected resistance pattern is found rarely among MRSA isolates in our hospital, but it appears that this phenotype consistent with typical community-acquired (ca) MRSA is indicative of PVL-positive MRSA.

Evaluation of a protocol for molecular broad-range diagnosis of culture-negative bacterial infections in clinical routine diagnosis.

AIM: Introduction of a protocol for broad-range diagnosis of bacterial infections, which remain negative in culture. METHODS AND RESULTS: The new TaqMan real-time PCR assay amplifies part of the 16S rRNA gene. Species are identified by subsequent sequencing and phylogenetic blast analysis. The analytical sensitivity showed to be 50 fg DNA per PCR. The lowest detectable bacterial cell concentration in blood was 1000 CFU per 200 mul EDTA-blood. The utility in clinical routine diagnosis was evaluated by testing 136 clinical specimens. Bacterial pathogens were detected in 33 samples (24.3%) either by culture or molecular diagnosis. In 10 culture negative cases, pathogens such as Mycoplasma timone/orale, Ureaplasma parvum/urealyticum, Treponema pallidum, different streptococci and staphylococci were identified by molecular diagnosis only. CONCLUSIONS: The introduced broad-range real-time PCR protocol showed to be useful in the clinical routine in cases where bacterial infection was highly anticipated but culture remained negative. However, the obtained data have to be always interpreted with caution and in conjunction with the clinical data, crossing-point values and with the Blast result of both the sample and the controls. SIGNIFICANCE AND IMPACT OF THE STUDY: This work introduces a new and well-evaluated broad-range real-time PCR protocol for diagnosis of bacterial infections.

Isolated endocarditis of the pulmonary valve caused by Pasteurella multocida.

We describe the rare case of a patient with an isolated endocarditis of the pulmonary valve caused by Pasteurella multocida. The bacterium was cultured from blood as well as from the excised valve after pulmonary valve replacement. Risk factors were contact with animals and concurrent intravenous drug use.

In vitro activity of fosfomycin alone and in combination with amoxicillin, clarithromycin and metronidazole against Helicobacter pylori compared with combined clarithromycin and metronidazole.

In order to evaluate the suitability of fosfomycin in combination with other agents for the treatment of Helicobacter pylori infections, the susceptibility profiles of 65 H. pylori strains were determined against multiple antimicrobial agents and combinations thereof using the agar dilution method. For fosfomycin alone, the range of minimum inhibitory concentration (MIC) results and the MICs at which 50% and 90% of strains were inhibited were 0.5-32 microg/ml and 2 and 4 microg/ml, respectively. For the combination of fosfomycin with amoxicillin, clarithromycin or metronidazole, the means calculated for the minimum and maximum fractional inhibitory concentration index were 0.70-1.17 and 1.15-2.03, respectively, suggesting partial synergy or indifference in the majority of strains. The combination of clarithromycin and metronidazole showed synergistic activity against 14 of 28 H. pylori strains tested. The in vitro activity results suggest the combination of fosfomycin with either amoxicillin or clarithromycin may be a promising alternative for the treatment of H. pylori infection. However, the clinical efficacy of these regimens remains to be investigated.

Isolation of Bordetella trematum from a diabetic leg ulcer.

BACKGROUND: The species Bordetella trematum (a Gram-negative, opportunistic bacterium) was established in 1996. To date, 10 cases of human infection/colonization with the pathogen have been described. CASE REPORT: The first case of isolation of B. trematum from a diabetic ulcer is reported. Since there are no commercially available kits for identification of the organism, species diagnosis was based on 16S rDNA sequencing. B. trematum disappeared from the ulcer without antimicrobial therapy. CONCLUSION: In the present case, there was no evidence for a causative role of the organism in the diabetic foot infection, which is in agreement with previously published data. However, B. trematum has to be considered when otherwise unclassified Gram-negative rods are isolated from infected diabetic ulcers.

Safety evaluation of piperacillin/tazobactam in very low birth weight infants.

An open, non-comparative study was designed to evaluate the safety and tolerance of parenteral piperacillin/tazobactam in very low birth weight infants. Twenty-seven patients were included for nosocomial sepsis with gram-negative bacteria (n = 4), nosocomial sepsis not responding to the empirical antibiotic regimen (n = 3), suspected necrotizing enterocolitis (n = 17), and infection after abdominal surgery for reasons other than necrotizing enterocolitis (n = 3). No clinical adverse events considered related to the study drug were noted, in particular, no cases of phlebitis, rash or stool changes. Several possibly related, mild and transitory abnormalities of laboratory parameters were observed. No long-lasting effect on the intestinal flora was detected. Seventeen patients (63%) were considered to have a favorable clinical response. This study demonstrates that piperacillin/tazobactam is a safe and well tolerated drug for preterm infants with bacterial infections, particularly those involving the gastrointestinal tract. Comparative clinical trials are warranted to further clarify the microbiological efficacy of piperacillin/tazobactam in this particular patient population.

Antimicrobial susceptibility profiles of clinically relevant blood culture isolates from nine surgical intensive care units, 1996-2000.

In order to elucidate trends in the incidence and susceptibility profiles of causative agents of bacteremia/fungemia in nine surgical intensive care units, a total of 744 isolates obtained during a 5-year period (1996-2000) were studied. The isolates included 698 bacteria and 46 fungi obtained from 523 positive blood cultures, representing 317 episodes of bacteremia/fungemia. Methicillin-resistant Staphylococcus aureus accounted for 2.3 episodes per 1000 surgical ICU admissions in 1996, 1.6 in 1997, 0.3 in 1998, 0.6 in 1999, and 1.7 in 2000. One Enterococcus faecalis (VanA) isolate resistant to both vancomycin and teicoplanin was recovered in 1996. Ciprofloxacin resistance in Pseudomonas aeruginosa decreased from 36% in 1996 to 20% in 2000, and resistance to third-generation cephalosporins decreased from 40% in 1996 to 9% in 2000. In light of differences between these results and those found elsewhere, these findings might prove useful for making infection control policy decisions in intensive care units.

Application of blood-based polymerase chain reaction for detection of Chlamydia pneumoniae in acute respiratory tract infections.

The aim of this study was to investigate whether blood-based polymerase chain reaction could serve as a diagnostic tool to identify individuals with acute respiratory Chlamydia pneumoniae infection. Respiratory specimens and peripheral blood mononuclear cells of 58 patients were analyzed using nested polymerase chain reaction and cell culture. Fifteen patients were polymerase chain reaction-positive for Chlamydia pneumoniae. Nine patients were positive in only the respiratory specimen; two in both the respiratory and blood sample (time intervals between onset of symptoms and sample collection, 3-10 days and 3-4 weeks, respectively); and four in only the blood sample. Detection of Chlamydia pneumoniae DNA in peripheral blood mononuclear cells does not seem to be a suitable marker for acute respiratory Chlamydia pneumoniae infection.

Urease prevents adherence of Helicobacter pylori to Kato III gastric epithelial cells.

The role of urease in Helicobacter pylori adherence to and internalization by Kato III cells was investigated. Kato III cells were incubated with wild-type strains (N6 or P1), with isogenic mutants lacking urease (N6ureB::TnKm or P1ureA::TnMax5) or producing the inactive apoprotein (N6ureG::TnKm), and with urease-positive clones recovered after complementation of N6ureB::TnKm with ureAB. Bacteria were stained with the green fluorescent dye PKH2, and the bacteria load of cells was analyzed by flow cytometry. With mutants lacking urease, the bacteria load was considerably increased, in comparison with the corresponding parental strains (P<.001). With clone K2(3), producing larger amounts of urease than N6, a significant reduction of bacteria load was observed, in comparison with the wild type (P<.001). N6ureG::TnKm showed adherence characteristics similar to those of N6. The role of urease in internalization was not clear. Thus, urease significantly inhibits H. pylori adherence to Kato III cells by a mechanism largely independent of enzymatic activity.

Multicenter comparison trial of DNA extraction methods and PCR assays for detection of Chlamydia pneumoniae in endarterectomy specimens.

The reported rate of detection of Chlamydia pneumoniae DNA within atherosclerotic lesions by PCR varies between 0 and 100%. In this study, identical sets of coded experimental atheroma samples (n = 15) and spiked controls (n = 5) were analyzed by 16 test methods in nine centers by means of PCR. The positive controls were correctly identified to levels of 1, 0.1, and 0.01 inclusion bodies of C. pneumoniae/ml of tissue homogenate by 16 (100%), 11 (69%), and 3 (19%) of the test methods, respectively. Three out of 16 negative controls (19%) were rated positive. Positivity rates for atheroma samples varied between 0 and 60% for the different test methods, with the maximum concordant result for positivity being only 25% for one carotid artery sample. There was no consistent pattern of positive results among the various laboratories, and there was no correlation between the detection rates and the sensitivity of the assay used.

A case of continuous ambulatory peritoneal dialysis peritonitis with an uncommon organism and an atypical clinical course.

This report describes a 46-year-old patient who experienced an atypical course of peritonitis while undergoing continuous ambulatory peritoneal dialysis (CAPD). The first sign of peritonitis was progressive impairment of ultrafiltration with increasing fluid absorption. The patient came to the center after 5 days with leg edemas and 645 leukocytes/microL in the first dialysate outflow. On the same day, the dialysate cell count decreased to 208/microL. During the following days, ultrafiltration failure persisted despite spontaneous normalization of PD-fluid leukocytes. No other clinical symptoms were observed, and the serum C-reactive protein (CRP) level remained normal. Magnetic resonance peritoneography and abdominal radiograph did not show dislocation of the catheter, a dialysate leak, or other causes of ultrafiltration failure. At day 14, fever, diarrhea, and an elevated serum CRP level occurred. Dialysate cultures taken on days 8, 11, and 14 showed growth of NEISSERIA: sicca. After initiation of antibiotic therapy with levofloxacine on day 14 ultrafiltration, clinical symptoms and serum CRP normalized within 3 days. In conclusion, Neisseria sicca should be considered as a rare cause of PD peritonitis. Our case report further illustrates the importance of ultrafiltration failure as an early and main symptom of peritoneal inflammation. The frequently used peritonitis criteria may not apply to cases of mild PD peritonitis.

Susceptibility testing of Candida species: comparison of NCCLS microdilution method with Fungitest.

Fungitest is a new commercially available and easy-to-perform breakpoint test system using six antifungal agents. We compared this test with a modified standard method described by the National Committee for Clinical Laboratory Standards (NCCLS). One hundred isolates of Candida species were tested with both methods. Based on the same breakpoints, the correlation of qualitative results between the reference method and Fungitest was high. Best results were obtained after incubation of Fungitest for 48 h. Overall agreement was high, an excellent correlation was given with amphotericin B and flucytosine (100% and 99%, respectively), whereas itraconazole showed only 86% concordance. When Fungitest was read after 24 h the agreement was lower ranging from 100% to 75%. Some of the breakpoints used with Fungitest differ from the breakpoints recommended by NCCLS, whereas others have not been elaborated by the NCCLS. The adaptation of Fungitest breakpoints to NCCLS and determination of further breakpoints have to be discussed before Fungitest can be recommended for routine use.

Two enzyme immunoassays and PCR for detection of Helicobacter pylori in stool specimens from pediatric patients before and after eradication therapy.

This study of pediatric patients was intended to determine the suitability of stool PCR and two antigen enzyme immunoassays (EIAs; Premier Platinum HpSA and the novel FemtoLab H. pylori), which detect Helicobacter pylori antigens in feces, as pretreatment diagnostic tools and especially as posttreatment control. Forty-nine H. pylori-infected children with dyspepsia received eradication therapy. Successful treatment was determined by a negative [(13)C]urea breath test 4 and 12 weeks after discontinuation of therapy. Fecal specimens were collected prior to eradication therapy as well as 4 weeks after the end of treatment. Successfully treated children delivered stool samples at 6, 8, and 12 weeks posttreatment also. Specimens were examined by seminested PCR and Premier Platinum HpSA and were reexamined by both EIAs as soon as FemtoLab H. pylori was available. In the first test series, the overall sensitivities of PCR and Premier Platinum HpSA were 93.0 and 91.1%, respectively. With specimens collected at 4 weeks after treatment, the respective specificities were 68.8 and 79.3%. After longer follow-up periods, however, they gradually increased to 100 and 96.9%, respectively. In the new test series, Premier Platinum HpSA delivered a considerably lower number of false-positive results (4 versus 18), indicating intertest variations. The overall test sensitivity was 94.6%, and the overall specificity was 97.5%. FemtoLab H. pylori showed an excellent performance with an overall sensitivity and specificity of 98.2 and 98.1%, respectively. Thus, in contrast to PCR, both EIAs were shown to be suitable for early posttreatment control.

Neisseria meningitidis serogroup W-135 primary monarthritis of the hip in an immunocompetent child.

Reported here is the first known case of primary monoarthritis of the hip due to Neisseria meningitidis W-135. The isolate was obtained from an immunocompetent child suffering from acute hip pain as the only symptom upon presentation at the hospital. Meningococcal infection must be considered in the diagnosis of any child presenting with arthritis, even if afebrile and without rash.

Isolation and continuous growth of Chlamydia pneumoniae from arterectomy specimens.

For the purpose of collecting Chlamydia pneumoniae strains of vascular origin that could be grown continuously in vitro, a cell culture system has been established. Using different types of vascular specimens obtained from 38 patients, Chlamydia pneumoniae could be isolated in three (7.9%) cases. The strains were obtained from specimens of the carotid artery, the femoral artery and an infrarenal aneurysm of the abdominal aorta of three male atherosclerosis patients. Thus, viable Chlamydia pneumoniae strains are also present in vascular regions other than those hitherto described.