Axel Kola

Epitope mapping of a C5a neutralizing mAb using a combined approach of phage display, synthetic peptides and site-directed mutagenesis.

BACKGROUND The anaphylatoxin C5a is a powerful proinflammatory protein generated on activation of the complement system. Recently, we described an anti-hC5a neoepitope specific mAb, mAb 2925, which was raised against the nonapeptide ISHKDMQLG (C5a-(65-73). This mAb is unique in that it recognizes both hC5a and hC5adesArg, even when it is denatured. It inhibits binding of [125I]C5a to its receptor on Bt2-cAMP differentiated U937 cells. OBJECTIVES To define the epitope of mAb 2925, we used a combined approach of a bacteriophage random octapeptide library, synthetic peptides and site-directed mutagenesis. STUDY DESIGN First a phage peptide library was screened with the anti C5a mAb 2925. Then synthetic peptides were synthesized with respect to the sequence information yielded from the phage approach, and used for binding studies. Site-directed mutagenesis was performed to confirm the results from the mapping experiments. RESULTS AND CONCLUSION Most phages selected by biotinylated Fab 2925 displayed sequences on the minor coat protein which correspond to residues within the C-terminus of human C5a. A first consensus motif comprised amino acids His-Lys or His-Arg, which allowed us to define position 67 and 68 as part of the epitope. A second consensus motif was selected, comprising Arg/Lys-Trp-Trp. This motif did not match any residues within the C5a C-terminus. However, when expressed together with the consensus motif His-Arg, as in HRWWXXXX or in HRXKWWXX, binding of these peptides to Fab 2925 increased as compared to peptides expressing the His-Arg motif only. Thus, the Arg/Lys-Trp-Trp motif serves to stabilize the binding of His-Arg to mAb 2925. Synthetic peptide studies revealed further N-terminal residues Ile65 and Ser66 as part of the epitope. A C5a mutant with an exchange Lys68Glu (C5aGlu68) confirmed the participation of Lys68 as a contact residue within the epitope of mAb 2925. Hence, the epitope recognized by mAb 2925 is linear and comprises residues Ile65, Ser66, His67, and Lys68. Thus, we could demonstrate for the first time that a mAb inhibits C5a receptor binding through specific interaction with receptor binding residues of the ligand.

Impact of Bacterial and Fungal Donor Organ Contamination in Lung, Heart–Lung, Heart and Liver Transplantation

We investigated to which extent bacterial and fungal donor organ contamination (DOC) caused posttransplant nosocomial infections (NI) in solid organ transplant (Tx) recipients. Between January 2002 to December 2003 (lung and heart Tx) and October 2003 to September 2004 (liver Tx), NIs were determined according to modified CDC criteria for NIs for all transplantations performed at Hannover Medical School. Organisms of the same species cultured from donor organs and infected transplantees were genotyped if available. Out of 282 solid organ recipients (140 lung-Tx, 16 heart-lung-Tx, 51 heart-Tx, 75 liver-Tx), 150 recipients (53.2%) received contaminated donor organs. Incidences of NIs were 33.7% in lung, 68.8% in heart-lung, 21.6% in heart, and 28% in liver recipients. In 11 out of 282 transplantees (3.9%, CI 95% 2.0–6.9%) organisms of NIs and of contamination of the donor organ were of the same species. Even if assuming five missing pairs of organisms as genetically identical, incidences of DOC-related posttransplant infections were only between 1.3% (CI95% 0.0; 7.2) in liver-Tx and 18.8% (CI95% 4; 45.6) in heart-lung Tx, and DOC related mortality was 0.4% (CI95% 0.0;1.9). Despite high DOC rates, posttransplant infections due to DOC were rare under the condition of adequate preoperative antibiotic prophylaxis and aseptic organ retrievement.

Is there an association between nosocomial infection rates and bacterial cross transmissions

Objective:Surveillance data of nosocomial infection rates are increasingly used for public reporting and interhospital comparisons. Approximately 15% of nosocomial infections on intensive care units are the result of patient-to-patient transmissions of the causative organisms. These exogenous infections could be prevented by adherence to basic infection control measures. The association between bacterial cross transmissions and nosocomial infection rates was analyzed. Design:Prospective cohort study during 24 months. Setting:Eleven intensive care units from two university hospitals. Patients:All inpatients. Interventions:None. Measurements and Main Results:Primary isolates of six indicator organisms (Acinetobacter baumannii, Enterococcus faecalis and E. faecium, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus) cultured from clinical samples or methicillin-resistant S. aureus surveillance testing of all inpatients were genotyped. Indistinguishable isolates in ≥2 patients defined potential episodes of transmissions. Surveillance of nosocomial infection rates was performed according to the German nosocomial infection surveillance system, Krankenhaus Infektions Surveillance System. Transmission events and nosocomial infection rates were pooled by intensive care unit to calculate Spearmans rank-correlation test. During 100,781 patient days, 100,829 microbiological specimens from 24,362 patients were sampled (average investigation density: 1.0 sample per patient and day) and 3419 primary indicator organisms were cultured. Altogether, 462 transmissions (incidence density of 4.6 transmissions per 1000 patient days; range, 1.4–8.4 days) and 1216 nosocomial infections (incidence density of 12.1 per 1000 patient days; range, 6.2–16.6 days) were discerned. Correlation analysis was unable to reveal any association between the incidence of cross transmissions and nosocomial infections, duration of hospitalization, or device use. Conclusions:Differences in nosocomial infection rates between study intensive care units are not explained solely by cross transmissions. Other factors, like the severity of the patients underlying diseases, the patients endogenous flora, or invasive procedures, likely have a dominant effect on the magnitude of nosocomial infection rates.

A SELECTION SYSTEM TO STUDY C5A-C5A-RECEPTOR INTERACTIONS : PHAGE DISPLAY OF A NOVEL C5A ANAPHYLATOXIN, FOS-C5AALA27

Binding and effector domains of the human anaphylatoxin C5a have been determined by either site directed mutagenesis or synthetic peptide studies. However, the lack of specific selection methods, which allow direct investigation of C5a-C5a-receptor interaction made these studies laborious. To overcome these limitations we have constructed a novel Fos-C5a expressed on the tip of a filamentous phage. To guarantee for a free C-terminus which is required for C5a activity C5a cDNA was cloned into the phagemid vector pJuFo. Helper phage infection of pJuFc-C5a transformed cells resulted in a mutant phage displaying Fos-C5a on its surface. However studies with Bt2cAMP differentiated U937 cells revealed that phage displayed Fos-C5a is functional inactive. Subsequently we replaced a nonconserved cysteine residue at position 27 by alanine and obtained Fos-C5aAla27. Both the purified and the phage displayed Fos-C5aAla27 proteins were functional active and induced enzyme release from differentiated U937 cells. In addition, purified Fos-C5aAla27 exhibited the same binding profile as compared to rhC5a. Fos-C5aAla27 displaying phages were mixed with phage harboring only the pJuFo plasmid at a ratio of 10(6). After four successive rounds of panning on differentiated U937 cells Fos-C5aAla27 phages were enriched to 100% as shown by C5a-specific ELISA. We expect this approach to prove helpful for studying C5a-C5a-receptor interactions. i.e. to screen C5a libraries for high affinity binders with agonistic or antagonistic properties directly on cells.

Clinical impact of infections caused by ESBL-producing E. coli and K. pneumoniae

The outcome of infections occurring at different anatomic sites caused by ESBL-producing E. coli or K. pneumoniae was retrospectively analysed for a 3-y period. 23 cases were compared to 46 controls with infections caused by third-generation cephalosporin-susceptible strains matched by age, severity of illness and duration of hospitalization before onset of infection. Only 27.8% of cases received appropriate empirical antimicrobial therapy compared with 79.6% of controls (ORpaired=0.053, p=0.001). This did not result in higher costs of antibiotic therapy, a longer median post-infection hospital stay or higher mortality in cases of patients with urinary tract or wound infections. In cases of patients with respiratory tract and bloodstream infections (RTI/BSI), median costs of definitive antibiotic treatment were significantly higher than in controls (325 vs 58.9 Euros, p=0.002). Moreover, more case patients with RTI/BSI had a post-infection stay exceeding the 75th percentile of 15 d on ICU and of 18 d in hospital, respectively (50% vs 6.67%, p=0.034). There was no difference in in-hospital mortality between case and control patients with RTI/BSI (25% vs 20%, p=1.0).

An infection with linezolid-resistant S. aureus in a patient with left ventricular assist system

We report an infection with a linezolid-resistant S. aureus in a patient with a left ventricular assist system. Linezolid should be used with caution when invasive devices or foreign materials are in place or therapeutic courses last longer than 14 d. Previous cases of linezolid-resistant S. aureus are summarized.

Mortality and molecular epidemiology associated with extended-spectrum β-lactamase production in Escherichia coli from bloodstream infection

Background The rate of infections due to extended-spectrum β-lactamase (ESBL)-producing Escherichia coli is growing worldwide. These infections are suspected to be related to increased mortality. We aimed to estimate the difference in mortality due to bloodstream infections (BSIs) with ESBL-positive and ESBL-negative E. coli isolates and to determine the molecular epidemiology of our ESBL-positive isolates. Materials and methods We performed a cohort study on consecutive patients with E. coli BSI between 2008 and 2010 at the Charité University Hospital. Collected data were ESBL production, basic demographic parameters, and underlying diseases by the Charlson comorbidity index (CCI). The presence of ESBL genes was analyzed by polymerase chain reaction (PCR) and sequencing. Phylogenetic groups of ESBL-positive E. coli were determined by PCR. Risk factors for mortality were analyzed by multivariable regression analysis. Results We identified 115 patients with BSI due to E. coli with ESBL phenotype and 983 due to ESBL-negative E. coli. Fifty-eight percent (n=67) of the ESBL-positive BSIs were hospital-acquired. Among the 99 isolates that were available for PCR screening and sequencing, we found mainly 87 CTX-M producers, with CTX-M-15 (n=55) and CTX-M-1 (n=21) as the most common types. Parameters significantly associated with mortality were age, CCI, and length of stay before and after onset of BSI. Conclusion The most common ESBL genotypes in clinical isolates from E. coli BSIs were CTX-M-15 (58%) and CTX-M-1 (22%). ESBL production in clinical E. coli BSI isolates was not related to increased mortality. However, the common occurrence of hospital-acquired BSI due to ESBL-positive E. coli indicates future challenges for hospitals.

Analysis of the C5a anaphylatoxin core domain using a C5a phage library selected on differentiated U937 cells.

The human anaphylatoxin C5a is a 74-amino acid comprising polypeptide with a plethora of biological functions. Site directed mutagenesis studies suggest that several residues within the core and the C-terminus mediate the interaction with the C5a receptor. However, the contribution of particular core residues to receptor binding remained to be clarified. By means of the phage display technique, the loop between positions 35-40 was randomly mutated and the resulting C5a[35-40] fusion phage library affinity selected on C5a receptor expressing U937 cells. After five rounds of affinity enrichment, residues Arg37 and Arg40 were preferably selected. Enrichment was as high as 100% for Arg37 and 79% for Arg40. No significant enrichment of consensus residues could be obtained for positions 35, 36, 38 and 39. The core mutant C5a[A35E36R37A38S39R40], in which only Arg37/40 and Ala38 are of the native C5a sequence, was as potent as native C5a in both receptor binding and enzyme release examined on U937 cells. In contrast, replacement of Arg40 as in the mutant C5a[Q35E36R37I38L39N40] resulted in a 10-fold decrease in both binding and functional activities. Thus, selected out of a multiplicity of possibilities by the natural binding partner, Arg37 as well as Arg40 appear to be anchor residues in binding to the C5a receptor.

Sepsis Caused by Extended-Spectrum Beta-Lactamase (ESBL)-Positive K. pneumoniae and E. coli: Comparison of Severity of Sepsis, Delay of Anti-Infective Therapy and ESBL Genotype

Infections with extended-spectrum beta-lactamase-producing Enterobacteriaceae (ESBL-E) are associated with increased mortality. Outcome differences due to various species of ESBL-E or ESBL genotypes are not well investigated. We conducted a cohort study to assess risk factors for mortality in cases of ESBL-E bacteremia (K. pneumoniae or E. coli) and the risk factors for sepsis with organ failure. All consecutive patients of our institution from 2008 to 2011 with bacteremia due to ESBL-E were included. Basic epidemiological data, underlying comorbidities, origin of bacteremia, severity of sepsis and delay of appropriate anti-infective treatment were collected. Isolates were PCR-screened for the presence of ESBL genes and plasmid-mediated AmpC β-lactamases. Cox proportional hazard regression on mortality and multivariable logistic regression on risk factors for sepsis with organ failure was conducted. 219 cases were included in the analysis: 73.1% due to E. coli, 26.9% due to K. pneumoniae. There was no significant difference in hospital mortality (ESBL-E. coli, 23.8% vs. ESBL-K. pneumoniae 27.1%, p = 0.724). However, the risk of sepsis with organ failure was associated in cases of K. pneumoniae bacteremia (OR 4.5, p<0.001) and patients with liver disease (OR 3.4, p = 0.004) or renal disease (OR 6.8, p<0.001). We found significant differences in clinical presentation of ESBL-E bacteremia due to K. pneumoniae compared to E. coli. As K. pneumoniae cases showed a more serious clinical presentation as E. coli cases and were associated with different risk factors, treatment and prevention strategies should be adjusted accordingly.

High admission prevalence of fluoroquinolone resistance in third-generation cephalosporin-resistant Enterobacteriaceae in German university hospitals

Objectives Fluoroquinolone resistance (FQR) in third-generation cephalosporin-resistant Enterobacteriaceae (3GCRE) presents serious limitations to antibiotic therapy. The aim of this study was to investigate whether the FQR proportion among 3GCRE differs between community-acquired (CA) and hospital-acquired (HA) isolates. Methods In a prospective observational study covering 2014 and 2015, we monitored the occurrence of 3GCRE in adult hospitalized patients in six German university hospitals. 3GCRE clinical isolates were subdivided into CA and HA. Multivariable analysis identified factors associated with in vitro non-susceptibility to ciprofloxacin. Results The dataset included 5721 3GCRE isolates of which 52.9% were HA and 52.7% exhibited FQR. Interestingly, the FQR proportion was higher in CA 3GCRE than in HA 3GCRE (overall, 60.1% versus 46.2%, respectively, P < 0.001). Multivariable analysis adjusting for age confirmed community acquisition as a risk factor for FQR [adjusted rate ratio (aRR) 1.33, 95% CI 1.17-1.53]. Escherichia coli and Klebsiella spp. were associated with a much higher FQR proportion than other Enterobacteriaceae species (aRR 8.14, 95% CI 6.86-9.65 and aRR 7.62 with 95% CI 6.74-8.61, respectively). Conclusions The high FQR proportion observed among CA 3GCRE, particularly in E. coli and Klebsiella spp., indicates that selection pressure in the outpatient setting needs to be addressed with antibiotic stewardship and other interventions in order to limit further spread of MDR.