Targeted AntiBiotics for Chronic pulmonary diseases (TARGET ABC): can targeted antibiotic therapy improve the prognosis of Pseudomonas aeruginosa-infected patients with chronic pulmonary obstructive disease, non-cystic fibrosis bronchiectasis, and asthma? A multicenter, randomized, controlled, open-label trial.

BACKGROUND: Pseudomonas aeruginosa infection is seen in chronic pulmonary disease and is associated with exacerbations and poor long-term prognosis. However, evidence-based guidelines for the management and treatment of P. aeruginosa infection in chronic, non-cystic fibrosis (CF) pulmonary disease are lacking. The aim of this study is to investigate whether targeted antibiotic treatment against P. aeruginosa can reduce exacerbations and mortality in patients with chronic obstructive pulmonary disease (COPD), non-CF bronchiectasis, and asthma. METHODS: This study is an ongoing multicenter, randomized, controlled, open-label trial. A total of 150 patients with COPD, non-CF bronchiectasis or asthma, and P. aeruginosa-positive lower respiratory tract samples will be randomly assigned with a 1:1 ratio to either no antibiotic treatment or anti-pseudomonal antibiotic treatment with intravenous beta-lactam and oral ciprofloxacin for 14 days. The primary outcome, analyzed with two co-primary endpoints, is (i) time to prednisolone and/or antibiotic requiring exacerbation or death, in the primary or secondary health sector, within days 20-365 from study allocation and (ii) days alive and without exacerbation within days 20-365 from the study allocation. DISCUSSION: This trial will determine whether targeted antibiotics can benefit future patients with chronic, non-CF pulmonary disease and P. aeruginosa infection in terms of reduced morbidity and mortality, thus optimizing therapeutic approaches in this large group of chronic patients. TRIAL REGISTRATION: ClinicalTrials.gov NCT03262142 . Registered on August 25, 2017.

Clinical impact of a commercially available multiplex PCR system for rapid detection of pathogens in patients with presumed sepsis.

BACKGROUND: Timely identification of pathogens is crucial to minimize mortality in patients with severe infections. Detection of bacterial and fungal pathogens in blood by nucleic acid amplification promises to yield results faster than blood cultures (BC). We analyzed the clinical impact of a commercially available multiplex PCR system in patients with suspected sepsis. METHODS: Blood samples from patients with presumed sepsis were cultured with the Bactec 9240 system (Becton Dickinson, Heidelberg, Germany) and aliquots subjected to analysis with the LightCycler SeptiFast (SF) Test (Roche Diagnostics, Mannheim, Germany) at a tertiary care centre. For samples with PCR-detected pathogens, the actual impact on clinical management was determined by chart review. Furthermore a comparison between the time to a positive blood culture result and the SF result, based on a fictive assumption that it was done either on a once or twice daily basis, was made. RESULTS: Of 101 blood samples from 77 patients, 63 (62%) yielded concordant negative results, 14 (13%) concordant positive and 9 (9%) were BC positive only. In 14 (13%) samples pathogens were detected by SF only, resulting in adjustment of antibiotic therapy in 5 patients (7,7% of patients). In 3 samples a treatment adjustment would have been made earlier resulting in a total of 8 adjustments in all 101 samples (8%). CONCLUSION: The addition of multiplex PCR to conventional blood cultures had a relevant impact on clinical management for a subset of patients with presumed sepsis.

Risk factors for negative blood cultures in adult medical inpatients--a retrospective analysis.

BACKGROUND: The identification of clinical factors associated with negative blood cultures could help to avoid unnecessary blood cultures. C-reactive protein (CRP) is a well-established inflammation marker commonly used in the management of medical inpatients. METHODS: We studied the association of clinical factors, CRP levels and changes of CRP documented prior to blood culture draws with the absence of bacteremia for hospitalized medical patients. RESULTS: In the retrospective analysis of 710 blood cultures obtained from 310 medical inpatients of non-intensive-care wards during one year (admission blood cultures obtained in the emergency room were excluded), the following retrospectively available factors were the only independent predictors of blood cultures negative for obligate pathogens: a good clinical condition represented by the lowest of three general nursing categories (OR 4.2, 95% CI 1.8 - 9.5), a CRP rise > 50 mg/L documented before the blood culture draw (OR 2.0 95% CI 1.8-9.5) and any antibiotic treatment in the previous seven days (OR 2.0, 95% CI 1.1-3.5). CONCLUSION: Including the general clinical condition, antibiotic pre-treatment and a substantial rise of CRP into the decision, whether or not to obtain blood cultures from medical inpatients with a suspected infection, could improve the diagnostic yield.

Leg ulcers and abscesses caused by Serratia marcescens.

Cutaneous infections caused by S. marcescens, a gram-negative bacillus belonging to the family of Enterobacteriaceae, are uncommon but may be predisposed by immunocompromised conditions or pre-damaged skin. A 73-year-old man presented with multiple ulcers and painful nodules on the lower right leg as well as abscesses on the right malleolus lateralis. He had been treated with oral penicillin without success. Due to chronic obstructive pulmonary disease, he was receiving a systemic therapy with corticosteroids. In addition, he had a post-thrombotic syndrome of the lower right leg. Serratia marcescens was the only microorganism isolated from all cultures performed. After a microbial sensitivity test, ertapenem 1 g/day was given intravenously for 10 days, followed by oral administration of ciprofloxacin 500 mg 1-0-1 for a further 7 days. This therapy resulted in the resolution of all lesions. This rare skin infection with S. marcescens needs specific microbiological diagnosis and adapted antibiosis.

Fatal pneumonia caused by Panton-Valentine Leucocidine-positive methicillin-resistant Staphylococcus aureus (PVL-MRSA) transmitted from a healthy donor in living-donor liver transplantation.

Severe infections are the most dangerous complications in liver transplantation and their prevention is one of the major goals. A 60-year-old Saudi-Arabian female with decompensated hepatitis C liver cirrhosis received a right-lobe liver graft from her healthy daughter. After 9 days, the patient developed a rapidly progressive necrotizing pneumonia that was fatal in spite of extracorporal lung assist. The pneumonia was due to a Panton-Valentine Leucocidine-positive (PVL) methicillin-resistant Staphylococcus aureus (MRSA), or "community-acquired" MRSA, that had not been detectable in the patient preoperatively. The same strain of PVL-MRSA could be demonstrated in the nares of the asymptomatic donor, but not of other relatives, patients, or medical staff. These findings strongly suggest transmission of PVL-MRSA from the donor to the recipient. This case demonstrates a previously unknown, and potentially fatal, risk in living-donor liver transplantation: transmission of a severe infection from a healthy donor to the recipient.

Prevalence of and risk factors for carriage of Panton-Valentine leukocidin-positive methicillin-resistant Staphylococcus aureus among residents and staff of a German nursing home.

The aims of this cross-sectional study were to determine the prevalence of and risk factors for carriage of Panton-Valentine leukocidin-producing methicillin-resistant Staphylococcus aureus (PVL-MRSA) in residents and personnel of a nursing home in Germany. In this study, PVL-MRSA carriage status among nursing home residents was associated with risk factors reflecting their dependence on nursing care. No specific risk factors were detected among staff.

Treatment of implant-associated infections with moxifloxacin: an animal study.

The efficacy of moxifloxacin in the treatment of an implant-associated infection by Staphylococcus aureus was compared with vancomycin in an animal study. The femoral medullary cavity of 36 Wistar rats was contaminated with S. aureus (ATCC 29213) and a metal device was implanted. After treatment for 14 days with moxifloxacin (2 x 10 mg/kg/day) or vancomycin (2 x 15 mg/kg/day), the bacterial counts (colony-forming units) in the periprosthetic bone, the soft tissue and the implant-associated biofilm were measured. Compared with the control group, moxifloxacin achieved a highly significant decrease in the microbial counts in the bone and soft tissue and in the biofilm (P<0.001). Moreover, the efficacy of moxifloxacin was significantly greater than that of vancomycin (P<0.01). Vancomycin did not reduce the microbial count significantly compared with the control group (P>0.05). The results justify further investigations of the treatment of implant-associated infections due to S. aureus with moxifloxacin.

Green fluorescent protein for detection of the probiotic microorganism Escherichia coli strain Nissle 1917 (EcN) in vivo.

Probiotic microorganisms are defined as viable nutritional agents conferring benefit to the health of the human host. Especially, Escherichia coli strain Nissle 1917 (EcN) was shown to be equally effective as mesalazine in the maintenance of remission in ulcerative colitis (UC). Presumably, the therapeutic effect of EcN is linked to the presence of the strain in the region of interest; however, it remains difficult to follow the orally administered strain on its passage through the complex microbial environment of the intestine in vivo, inhabited dominantly by various E. coli strains, using traditional culturing methods. In this study we transformed EcN and a wild-type E. coli from a laboratory rat (EcR) with a plasmid carrying a gfp gene (pUC-gfp) to obtain EcN- and EcR-GFP to allow in vivo detection without alteration of strain-specific characteristics. Analysis of different strain-specific characteristics included the measurement of stimulation of IL-8 secretion and adhesion in vitro using the epithelial cell line HT-29. The kinetics of intestinal distribution in mice and colonization properties in rats following oral administration was studied in vivo. Detectability of the strain in histologic specimens was analysed using fluorescence microscopy and immunohistochemistry. The identity of fluorescent E. coli strains isolated from stool samples, Peyer's patches (PP) and mesenteric lymph nodes (MLN) was determined by REP-PCR. We were able to demonstrate that EcN and EcN-GFP do not differ in stimulation of IL-8 secretion or adhesion to HT-29 cells. In vivo, EcN-GFP colonies were readily detectable by fluorescence microscopy in luminal samples and also by immunohistochemistry in histological sections allowing analysis of the kinetics of the intestinal passage following oral administration. Translocation of fluorescent and non-fluorescent bacteria into PP and MLN was noted at 6 h post oral administration. EcN-GFP was detectable initially for 14 days in faecal samples of rats, while EcR-GFP was detectable throughout the whole experiment (45 days). Challenge with ampicillin at day 45 demonstrated continuing presence of EcN-GFP in small numbers by reappearing fluorescent colonies. The plasmid was not stable in vivo since non-fluorescent EcN colonies were detected also in faecal samples by REP-PCR. In summary, transformation of EcN to obtain EcN-GFP in our study had no detectable influence on the probiotic microorganism regarding adhesion on and induction of IL-8 secretion of HT-29 cells and allows the detection in mixed microbial environments in vivo but the stability of EcN-GFP in vivo is limited.

Clinical determinants for the recovery of fungal and mezlocillin-resistant pathogens from bile specimens.

We conducted a retrospective analysis of all bile specimens obtained for routine cultures from January 1995 through December 1999 at our tertiary care hospital. Results of microbiologic testing were linked to clinical parameters gathered by means of chart review. A total of 722 isolates were cultured from 345 of 454 bile specimens obtained from 288 individual patients. Prior receipt of a >7-day course of antibiotics (odds ratio [OR], 5.7), extensive leukocytosis (leukocyte count, >20,000 cells/microL) on admission (OR, 7.8), endoscopic or percutaneous biliary manipulation during the previous 14 days (OR, 2.9), and treatment in an internal medicine ward (OR, 2.5) were independent factors significantly associated (Pless-than-or-eq, slant.05) with recovery of Candida species from bile specimens. Culture of mezlocillin-resistant bacteria from bile specimens was independently associated with the specimen having been obtained >1 week after admission (OR, 3.8), lack of history of endoscopic biliary drainage (OR, 3.2), and high serum aspartate aminotransferase levels (>72 U/L) on admission (OR, 2.6). Prospective studies are warranted to evaluate accordingly adjusted empiric therapies for biliary infections.

Disseminated Cladophialophora bantiana infection in a heart transplant recipient.

Cerebral phaeohyphomycosis caused by Cladophialophora bantiana, a dematiaceous fungus, is a rare disease. The majority of cases have been reported among immunocompetent patients; only 4 cases have been published that describe transplantation patients. The overall prognosis is poor. Surgical therapy in combination with chemotherapy with itraconazole is recommended. We report the case of a heart transplant recipient with cutaneous, cerebral, and lung manifestation of Cladophialophora bantiana who died despite surgical and systemic, high-dosage itraconazole treatment.

Combined skin disinfection with chlorhexidine/propanol and aqueous povidone-iodine reduces bacterial colonisation of central venous catheters.

OBJECTIVE: Central venous catheter (CVC)-related infections may be caused by micro-organisms introduced from the skin surface into deeper tissue at the time of CVC insertion. The optimal disinfection regimen to avoid catheter-related infections has not yet been defined. This study compares three different approaches. DESIGN: Prospective randomised trial. SETTING: A tertiary care hospital. PATIENTS AND PARTICIPANTS: One hundred nineteen patients scheduled electively to receive 140 CVCs. INTERVENTIONS: Skin disinfection was performed with either povidone-iodine 10% (PVP-iodine), chlorhexidine 0.5%/propanol 70%, or chlorhexidine 0.5%/propanol 70% followed by PVP-iodine 10%. Prior to disinfection, a swab from the site of insertion was taken for culture. CVCs were removed if no longer needed or infection was suspected. All catheters were cultured quantitatively after removal. MEASUREMENT AND RESULTS: Bacteria could be isolated from 20.7% of the catheter tips. Bacterial growth was found in 30.8% of the catheters placed after skin disinfection with povidone-iodine, in 24.4% after disinfection with propanol/chlorhexidine and in 4.7% after disinfection with propanol/chlorhexidine followed by povidone-iodine ( p=0.006). In 15 cases, the same organism was isolated from the skin swab and the catheter tip. Ten of these paired isolates showed the same pattern in a pulsed-field gel electrophoresis analysis. CONCLUSIONS: Skin disinfection with propanol/chlorhexidine followed by PVP-iodine was superior in the prevention of microbial CVC colonisation compared to either of the regimens alone. These results support the concept that catheter infections can originate from bacterial translocation at the time of catheter insertion.

Ablation of the sympathetic nervous system decreases gram-negative and increases gram-positive bacterial dissemination: key roles for tumor necrosis factor/phagocytes and interleukin-4/lymphocytes.

The sympathetic nervous system is intensely activated during bacteremia, but its immediate influence on the bacterial tissue burden remains unclear. We demonstrate that prior ablation of the sympathetic nervous system decreases this dissemination of Pseudomonas aeruginosa or Escherichia coli through a mechanism of increased secretion of peritoneal tumor necrosis factor, improved phagocytic response of peritoneal cells, and increased influx of monocytes into the peritoneal cavity. When gram-positive Staphylococcus aureus strains were used, sympathectomy increased the bacterial tissue burden, which was caused by a reduction in corticosterone tonus, and decreased both interleukin-4 secretion from peritoneal cells and the influx of lymphocytes into the peritoneal cavity. In both models, the peritoneal wall was the critical border for systemic infection. These results show the dual role of the sympathetic nervous system in sepsis. It can be favorable or unfavorable, depending on the innate immune effector mechanisms necessary to overcome infection.

Mutant prevention concentration of nalidixic acid, ciprofloxacin, clinafloxacin, levofloxacin, norfloxacin, ofloxacin, sparfloxacin or trovafloxacin for Escherichia coli under different growth conditions.

OBJECTIVES: We used two different strains of Escherichia coli, E.coli ATCC 25922 and a recent urinary isolate from a clinical sample, to investigate in vitro how the MIC and mutant prevention concentration (MPC) are affected by different temperatures (37 or 20 degrees C) or oxygen tension (aerobic or anaerobic atmosphere; MIC, MIC(an); MPC, MPC(an)). MATERIALS AND METHODS: MIC and MPC for E.coli ATCC 25922 and the clinical isolate were determined on agar containing ciprofloxacin or levofloxacin, and for the ATCC strain on agar supplemented with nalidixic acid, norfloxacin, ofloxacin, sparfloxacin, trovafloxacin or clinafloxacin. RESULTS: Results for the ATCC strain and the clinical strain for ciprofloxacin or levofloxacin were similar. The MPC values for E.coli ATCC 25922 were 2 x MIC (trovafloxacin), 4 x MIC (ciprofloxacin, norfloxacin, ofloxacin), 8 x MIC (clinafloxacin, levofloxacin), 16 x MIC (sparfloxacin) and 32 x MIC (nalidixic acid) at 37 degrees C and under aerobic conditions. Generally, a 37 degrees C aerobic atmosphere was associated with the highest MPC values. As an exception, both the MIC and the MPC of ciprofloxacin were higher under anaerobic versus aerobic conditions (MIC(an) approximately 8 x MIC; MPC(an) = 4 x MPC) for both E.coli isolates. Irrespective of the quinolone or growth conditions, the MIC for mutants was 1-256 x wild-type MIC. Calculated from published serum half-lives and the MPC values from this study, a putative selection period, in which resistant mutants might be selected, was calculated to be 14 h for nalidixic acid, 16 h for norfloxacin and ciprofloxacin, 28 h for ofloxacin, 30 h for trovafloxacin, 35 h for levofloxacin, 40 h for clinafloxacin, and 120 h for sparfloxacin. CONCLUSIONS: As calculated from our model in respect to the length of the selection period, long serum half-lives of recently developed compounds could not be compensated for by a more favourable activity in terms of MPC. Higher concentrations of ciprofloxacin may be required under an anaerobic atmosphere to prevent the emergence of resistant mutants among 10(10) cfu.

Comparison of BacT/Alert and BACTEC NR 860 blood culture systems in a laboratory not continuously staffed.

OBJECTIVE: To evaluate the performance of continuously working blood culture systems in a discontinuous laboratory system. METHODS: The systems used were BacT/Alert (Organon Teknika Corp., Durham, NC) and BACTEC NR 860 (Becton Dickinson Diagnostic Instruments, Sparks, Md) in a comparison in a laboratory staffed 8 1/2 h on Mondays to Fridays and 4 1/2 h on Saturdays. Blood culture bottles (BacT/Alert aerobic and anaerobic, BACTEC NR 26 A and NR 27 A) were received thrice daily. RESULTS: From 1824 pairs of blood culture vials, 110 clinically significant microorganisms were recovered by both BACTEC and BacT/Alert, 43 by BACTEC alone, and 33 by BacT/Alert alone. The differences between the systems in total recovery and in recovery of individual species were not statistically significant. The average detection times were 13.36 h for BACTEC and 13.93 h for BacT/Alert (P>0.1). These times represent only 35.6% (BACTEC) and 32.6% (BacT/Alert) of the total timespans from collection of blood to informing the ward of a positive result (tcrd, clinically relevant detection time). If 24 h per day blood culture processing conditions and continuous transport of vials to the laboratory had been available, these percentages would have risen to 87% (BACTEC) and 87.5% (BacT/Alert). Under such 'ideal' conditions, ttrd could have been reduced by 22.16 h using BACTEC and by 26.81 h using BacT/Alert. The BacT/Alert system showed more false-positive results than the BACTEC system (80 (4.39%) versus 23 (1.26%), P<0.001). CONCLUSIONS: No time benefit for detection of positive blood cultures is gained with continuously measuring systems, if loading and processing of vials is organized discontinuously, as in our laboratory.

Bacteria ingestion by blowfly larvae: an in vitro study.

BACKGROUND: Maggot debridement therapy is the medical use of live fly larvae for cleaning chronic and infected wounds, removing devitalized tissue and decreasing the risk of infection. Maggot-derived proteins are able to kill bacteria, and proteolytic enzymes are responsible for the liquefying of necrotic tissue. OBJECTIVE: The aim of this study is to investigate bacterial ingestion by larvae roaming free on bacterial agar, compared to those larvae contained within vinyl bags. METHODS: Free-roaming sterile larvae of Lucilla sericata and larvae contained in vinyl bags were fed on Escherichia coli producing green fluorescent protein (GFP). The time interval to the onset of fluorescent maggots was determined. At different time intervals, maggots were sacrificed, washed in sterile saline, sagittally cut in frozen sections and examined under a microscope with UV light. RESULTS: After feeding on GFP-labelled E. coli, maggots roaming free on bacterial lawn agar demonstrated fluorescence after 3 min, maggots entrapped in vinyl bags after 25 min. In the sagittal frozen sections, the highest fluorescent intensity was detected in the larvae's rostral part of the alimentary tract, the crop and the anterior midgut. CONCLUSION: In an in vitro setting, digestion and ingestion of whole or disintegrated bacteria is accomplished within minutes. The vinyl bag's permeable membrane clearly causes a delay of this process.

Direct detection and differentiation of Legionella spp. and Legionella pneumophila in clinical specimens by dual-color real-time PCR and melting curve analysis.

A dual-color LightCycler PCR assay targeting the 16S rDNA gene of Legionella spp. was established. By using two pairs of hybridization probes, Legionella spp. and Legionella pneumophila could be detected and differentiated simultaneously. With 26 culture-positive and 42 culture-negative respiratory specimens from patients with atypical pneumonia, 100% sensitivity and specificity was observed for L. pneumophila.

Septicemia due to Acinetobacter junii.

Acinetobacter spp. are considered to be emerging nosocomial pathogens. Acinetobacter junii is a rare cause of disease in humans and was associated mainly with bacteremia in preterm infants and pediatric oncologic patients. In this report we describe a case of catheter-related infection by A. junii in an adult oncologic patient. Application of molecular methods for precise species identification of Acinetobacter spp. will help to further clarify their role as human pathogens.

A C-terminal 18 amino acid deletion in MarR in a clinical isolate of Escherichia coli reduces MarR binding properties and increases the MIC of ciprofloxacin.

As described recently, the different degree of fluoroquinolone resistance in a pair of sequential clinical isolates of Escherichia coli was due to the increased expression of the regulatory gene marA as a consequence of an 18 amino acid C-terminal deletion in the repressor MarR (MarR Delta). To further investigate the molecular mechanism of the loss of repressor function, we purified recombinant wild-type and mutated MarR, and tested their respective ability to form dimers and their specific DNA binding properties to the operator region marO. The dimerization capacity was analysed by non-reducing SDS-PAGE and by disuccinimidyl suberate-mediated cross-linking of the recombinant proteins. The binding of MarR was studied using the recombinant proteins and DNA probes containing the two identified binding sites in marO in the presence or absence of specific and non-specific DNA fragments. Dimerization of MarR Delta was reduced compared with MarR: the dimer portion was 33.8% (MarR) and 12.4% (MarR Delta) at a protein concentration of 10 IM. In mobility-shift assays MarR Delta showed a highly reduced complex formation. Footprinting analysis confirmed reduced binding of MarR Delta to its target sites, compared with MarR. The biochemical data are in full agreement with the crystal structure of MarR, which shows that the N- and C-terminal regions of MarR contribute to dimer formation. The data also indicate a major role of the MarR dimer as opposed to the monomer in DNA binding.

Increase in MICs of ciprofloxacin in vivo in two closely related clinical isolates of Enterobacter cloacae.

The mechanisms of fluoroquinolone resistance in two isolates of Enterobacter cloacae, Ecl#1 and Ecl#2, from the same patient and with identical pulsed-field gel electrophoresis patterns, have been analysed. MICs of ciprofloxacin were 0.25 and 1 mg/L for Ecl#1 and Ecl#2, respectively. Ecl#2 was also more resistant to chloramphenicol and organic solvents. The quinolone resistance determining regions of gyrA/B and parC/E, and the marORA and acrB genes, were sequenced. Expression of marR, acrB, soxS, robA, ramA and fis was analysed by northern blotting. The activity of a 90 bp E. cloacae mar promoter fragment was examined with the reporter plasmid pIGJ-1mar. Sequencing the gyrAB and parCE genes revealed a single amino acid substitution in GyrA (corresponding to position 83 in GyrA of Escherichia coli) in Ecl#1 and Ecl#2 (Phe83) compared with reference strain E. cloacae DSMZ 3264 (Thr83). Ecl#2 accumulated significantly less norfloxacin and displayed higher levels of expression of marR and acrB than Exl#1, indicative of greater fluoroquinolone efflux activity. Sequencing gyrB, parC/E and marORA, and northern blotting of robA, ramA and fis, did not reveal any further differences between the two strains. No homologue of soxRS was detected in E. cloacae. Expression of GFP from pIGJ1-mar in Ecl#2 was higher than in Ecl#1. In these two closely related clinical isolates of E. cloacae, a target mutation in GyrA (Ecl#1 and Ecl#2) and increased fluoroquinolone efflux by AcrAB (Ecl#2) contribute to the resistance phenotypes, corroborating findings in vitro and in vivo about the sequential development of fluoroquinolone resistance.