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We recently showed that solid-phase peptide synthesis using racemic amino acids yields stereorandomized peptides comprising all possible diastereomers as homogeneous, single-mass products that can be purified by HPLC and that stereorandomization modulates activity, toxicity, and stability of membrane-disruptive cyclic and linear antimicrobial peptides (AMPs) and dendrimers. Here, we tested if stereorandomization might be compatible with target binding peptides with the example of the proline-rich AMP oncocin, which inhibits the bacterial ribosome. Stereorandomization of up to nine C-terminal residues preserved ribosome binding and antibacterial effects including activities against drug-resistant bacteria and protected against serum degradation. Surprisingly, fully stereorandomized oncocin was as active as L-oncocin in dilute growth media stimulating peptide uptake, although it did not bind the ribosome, indicative of an alternative mechanism of action. These experiments show that stereorandomization can be compatible with target binding peptides and can help understand their mechanism of action.

Membrane disruptive α-helical antimicrobial peptides (AMPs) offer an opportunity to address multidrug resistance; however, most AMPs are toxic and unstable in serum. These limitations can be partly overcome by introducing D-residues, which often confers protease resistance and reduces toxicity without affecting antibacterial activity, presumably due to lowered α-helicity. Here, we investigated 31 diastereomers of the α-helical AMP KKLLKLLKLLL. Three diastereomers containing two, three, and four D-residues showed increased antibacterial effects, comparable hemolysis, reduced toxicity against HEK293 cells, and excellent serum stability, while another diastereomer with four D-residues additionally displayed lower hemolysis. X-ray crystallography confirmed that high or low α-helicity as measured by circular dichroism indicated α-helical or disordered structures independently of the number of chirality switched residues. In contrast to previous reports, α-helicity across diastereomers correlated with both antibacterial activity and hemolysis and revealed a complex relationship between stereochemistry, activity, and toxicity, highlighting the potential of diastereomers for property optimization.

Bone and joint infection (BJI) epidemiology and outcomes in solid organ transplant recipients (SOTr) remain largely unknown. We aim to describe BJI in a multi-center cohort of SOTr (Swiss Transplant Cohort Study). All consecutive SOTr with BJI (01.05.2008–31.12.2019) were included. A nested case–control study to identify risk factors for BJI was performed. Among 4482 patients, 61 SOTr with 82 BJI were included, at an incidence of 1.4% (95% CI 1.1–1.7), higher in heart and kidney-pancreas SOTr (Gray’s test p< .01). Although BJI were predominately late events (median of 18.5 months post-SOT), most infections occurred during the first year post-transplant in thoracic SOTr. Diabetic foot osteomyelitis was the most frequent infection (38/82, 46.3%), followed by non-vertebral osteomyelitis (26/82, 31.7%). Pathogens included Gram-positive cocci (70/131, 53.4%), Gram-negative bacilli (34/131, 26.0%), and fungi (9/131, 6.9%). BJI predictors included male gender (OR 2.94, 95% CI 1.26–6.89) and diabetes (OR 2.97, 95% CI 1.34–6.56). Treatment failure was observed in 25.9% (21/81) patients and 1-year mortality post-BJI diagnosis was 14.8% (9/61). BJI remain a rare event in SOTr, associated with subtle clinical presentations, high morbidity and relapses, requiring additional studies in the future.

Food‐safety measures are recommended to solid organ transplant (SOT) recipients. However, the burden of foodborne infections in SOT recipients has not been established. We describe the epidemiology and outcomes of bacterial foodborne infections in a nationwide cohort including 4405 SOT recipients in Switzerland between 2008 and 2018. Participants were prospectively followed for a median of 4.2 years with systematic collection of data on infections, and patient and graft‐related outcomes. We identified 151 episodes of microbiologically confirmed bacterial foodborne infections occurring in median 1.6 years (IQR 0.58–3.40) after transplantation (131 [88%] Campylobacterspp. and 15 [10%] non‐typhoidal Salmonella). The cumulative incidence of bacterial foodborne infections was 4% (95% CI 3.4–4.8). Standardized incidence rates were 7.4 (95% CI 6.2–8.7) and 4.6 (95% CI 2.6–7.5) for Campylobacterand Salmonellainfections, respectively. Invasive infection was more common with Salmonella(33.3% [5/15]) compared to Campylobacter(3.2% [4/125]; p= .001). Hospital and ICU admission rates were 47.7% (69/145) and 4.1% (6/145), respectively. A composite endpoint of acute rejection, graft loss, or death occurred within 30 days in 3.3% (5/151) of cases. In conclusion, in our cohort bacterial foodborne infections were late post‐transplant infections and were associated with significant morbidity, supporting the need for implementation of food‐safety recommendations. Implementation of food‐safety recommendations is supported by data from a large, nationwide, prospective cohort of solid organ transplant recipients that shows that bacterial foodborne infections occur late after transplant and are associated with significant morbidity such as high hospitalization rates.

Food-safety measures are recommended to solid organ transplant (SOT) recipients. However, the burden of foodborne infections in SOT recipients has not been established. We describe the epidemiology and outcomes of bacterial foodborne infections in a nationwide cohort including 4405 SOT recipients in Switzerland between 2008 and 2018. Participants were prospectively followed for a median of 4.2 years with systematic collection of data on infections, and patient and graft-related outcomes. We identified 151 episodes of microbiologically confirmed bacterial foodborne infections occurring in median 1.6 years (IQR 0.58–3.40) after transplantation (131 [88%] Campylobacterspp. and 15 [10%] non-typhoidal Salmonella). The cumulative incidence of bacterial foodborne infections was 4% (95% CI 3.4–4.8). Standardized incidence rates were 7.4 (95% CI 6.2–8.7) and 4.6 (95% CI 2.6–7.5) for Campylobacterand Salmonellainfections, respectively. Invasive infection was more common with Salmonella(33.3% [5/15]) compared to Campylobacter(3.2% [4/125]; p= .001). Hospital and ICU admission rates were 47.7% (69/145) and 4.1% (6/145), respectively. A composite endpoint of acute rejection, graft loss, or death occurred within 30 days in 3.3% (5/151) of cases. In conclusion, in our cohort bacterial foodborne infections were late post-transplant infections and were associated with significant morbidity, supporting the need for implementation of food-safety recommendations.

Posttransplant lymphoproliferative disorder (PTLD) is a serious complication of solid organ transplantation (SOT). Most PTLD cases are associated with Epstein–Barr virus (EBV) infection. The role of antiviral prophylaxis or rituximab therapy for prevention of PTLD in SOT recipients is controversial. In a nationwide cohort, we assessed the incidence, presentation, and outcome of histologically proven PTLD. We included 4765 patients with a follow-up duration of 23 807 person-years (py). Fifty-seven PTLD cases were identified; 39 (68%) were EBV positive (EBV+ PTLD). Incidence rates for EBV+ PTLD at 1, 2, and 3 years posttransplant were 3.51, 2.24, and 1.75/1000 py and 0.44, 0.25, and 0.29/1000 py for EBV– PTLD. We did not find an effect of antiviral prophylaxis on early and late EBV+ PTLD occurrence (early EBV+ PTLD: SHR 0.535 [95% CI 0.199–1.436], p= .264; late EBV+ PTLD: SHR 2.213, [95% CI 0.751–6.521], p= .150). However, none of the patients (0/191) who received a rituximab-containing induction treatment experienced PTLD, but 57 of 4574 patients without rituximab induction developed PTLD. In an adjusted restricted mean survival time model, PTLD-free survival was significantly longer (0.104 years [95% CI 0.077–0.131]) in patients receiving rituximab as induction treatment. This study provides novel data on the association of rituximab induction and reduced risk for PTLD.

Solid organ transplant (SOT) recipients are exposed to respiratory viral infection (RVI) during seasonal epidemics; however, the associated burden of disease has not been fully characterized. We describe the epidemiology and outcomes of RVI in a cohort enrolling 3294 consecutive patients undergoing SOT from May 2008 to December 2015 in Switzerland. Patient and allograft outcomes, and RVI diagnosed during routine clinical practice were prospectively collected. Median follow-up was 3.4 years (interquartile range 1.61–5.56). Six hundred ninety-six RVIs were diagnosed in 151/334 (45%) lung and 265/2960 (9%) non-lung transplant recipients. Cumulative incidence was 60% (95% confidence interval [CI] 53%-69%) in lung and 12% (95% CI 11%-14%) in non-lung transplant recipients. RVI led to 17.9 (95% CI 15.7–20.5) hospital admissions per 1000 patient-years. Intensive care unit admission was required in 4% (27/691) of cases. Thirty-day all-cause case fatality rate was 0.9% (6/696). Using proportional hazard models we found that RVI (adjusted hazard ratio [aHR] 2.45; 95% CI 1.62–3.73), lower respiratory tract RVI (aHR 3.45; 95% CI 2.15–5.52), and influenza (aHR 3.57; 95% CI 1.75–7.26) were associated with graft failure or death. In this cohort of SOT recipients, RVI caused important morbidity and may affect long-term outcomes, underlying the need for improved preventive strategies.

Solid-phase peptide synthesis (SPPS) is usually performed with optically pure building blocks to prepare peptides as single enantiomers. Herein we report that SPPS using racemic amino acids provides stereorandomized (sr) peptides, containing up to billions of different stereoisomers, as well-defined single HPLC peaks, single mass products with high yield, which can be used to investigate peptide bioactivity. To exemplify our method, we show that stereorandomization abolishes the membrane-disruptive effect of α-helical amphiphilic antimicrobial peptides but preserves their antibiofilm effect, implying different mechanisms involving folded versus disordered conformations. For antimicrobial peptide dendrimers by contrast, stereorandomization preserves antibacterial, membrane-disruptive, and antibiofilm effects but reduces hemolysis and cytotoxicity, thereby increasing their therapeutic index. Finally, we identify partially stereorandomized analogues of the last resort cyclic peptide antibiotic polymyxin B with preserved antibacterial activity but lacking membrane-disruptive and lipopolysaccharide-neutralizing activity, pointing to the existence of additional targets.

We sought in this case-control retrospective study to compare posaconazole and isavuconazole (PCZ and IVC, respectively) plasma trough concentration (Ctrough) levels in high-risk allogeneic hematopoietic cell transplant (HCT) recipients who received letermovir (LET) or not. PCZ/IVC Ctroughlevels were not found to be significantly different between cases and controls, as they were 1.31 mg/liter (median) (interquartile range [IQR], 0.90) versus 1.36 mg/liter (IQR, 1.16) (P = 0.31) and 3.20 mg/liter (IQR, 2.40) versus 2.35 mg/liter (IQR, 1.50) (P = 0.17), respectively.

Immunocompromised patients may be at increased risk for complications of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) infection. However, comprehensive data of SARS‐CoV‐2 infection in solid organ transplant (SOT) recipients are still lacking. We performed a multicenter nationwide observational study within the Swiss Transplant Cohort Study (STCS) to describe the epidemiology, clinical presentation, treatment and outcomes of the first microbiologically documented SARS‐CoV‐2 infection among SOT recipients. Overall, 21 patients were included with a median age of 56 years (10 kidney, 5 liver, 1 pancreas, 1 lung, 1 heart and 3 combined transplantations). The most common presenting symptoms were fever (76%), dry cough (57%), nausea (33%), and diarrhea (33%). Ninety‐five percent and 24% of patients required hospital and ICU admission, respectively, and 19% were intubated. After a median of 33 days of follow‐up, 16 patients were discharged, 3 were still hospitalized and 2 patients died. These data suggest that clinical manifestations of SARS‐CoV‐2 infection in middle‐aged SOT recipients appear to be similar to the general population without an apparent higher rate of complications. These results need to be confirmed in larger cohorts. This study describes the epidemiology, clinical presentation, treatment, and outcome of the first 21 solid organ transplant recipients infected with SARS‐CoV‐2 in the Swiss Transplant Cohort Study.

Allergy transfer upon solid organ transplantation has been reported in the literature, although only few data are available as to the frequency, significance, and management of these cases. Based on a review of 577 consecutive deceased donors from the Swisstransplant Donor‐Registry, 3 cases (0.5%) of fatal anaphylaxis were identified, 2 because of peanut and 1 of wasp allergy. The sera of all 3 donors and their 10 paired recipients, prospectively collected before and after transplantation for the Swiss Transplant Cohort Study, were retrospectively processed using a commercial protein microarray fluorescent test. As early as 5 days posttransplantation, newly acquired peanut‐specific IgE were transiently detected from 1 donor to 3 recipients, of whom 1 liver and lung recipients developed grade III anaphylaxis. Yet, to define how allergy testing should be performed in transplant recipients and to better understand the impact of immunosuppressive therapy on IgE sensitization, we prospectively studied 5 atopic living‐donor kidney recipients. All pollen‐specific IgE and >90% of skin prick tests remained positive 7 days and 3 months after transplantation, indicating that early diagnosis of donor‐derived IgE sensitization is possible. Importantly, we propose recommendations with respect to safety for recipients undergoing solid‐organ transplantation from donors with a history of fatal anaphylaxis. Based on the Swisstransplant donor registry, the Swiss‐Transplant‐Cohort‐Study, and a prospective analysis on allergy maintenance in atopic recipients, this study makes new recommendations for the management of allergy transfer upon solid organ transplantation, emphasizing the poor effect of immunosuppression on IgE sensitization and the need of early allergological investigation in the donor and respective recipients.

Solid organ transplant (SOT) candidates may not be immune against potentially vaccine-preventable diseases because of insufficient immunizations and/or limited vaccine responses. We evaluated the impact on vaccine immunity at transplant of a systematic vaccinology workup at listing that included (1) pneumococcal with and without influenza immunization, (2) serology-based vaccine recommendations against measles, varicella, hepatitis B virus, hepatitis A virus, and tetanus, and (3) the documentation of vaccines and serology tests in a national electronic immunization registry (www.myvaccines.ch). Among 219 SOT candidates assessed between January 2014 and November 2015, 54 patients were transplanted during the study. Between listing and transplant, catch-up immunizations increased the patients’ immunity from 70% to 87% (hepatitis A virus, P= .008), from 22% to 41% (hepatitis B virus, P= .008), from 77% to 91% (tetanus, P= .03), and from 78% to 98% (Streptococcus pneumoniae, P= .002). Their immunity at transplant was significantly higher against S. pneumoniae(P= .006) and slightly higher against hepatitis A virus (P= .07), but not against hepatitis B virus, than that of 65 SOT recipients transplanted in 2013. This demonstrates the value of a systematic multimodal serology-based approach of immunizations of SOT candidates at listing and the need for optimized strategies to increase their hepatitis B virus vaccine responses.

New-onset diabetes mellitus after transplantation (NODAT) is a complication following solid organ transplantation (SOT) and may be related to immune or inflammatory responses. We investigated whether single nucleotide polymorphisms (SNPs) within 158 immune- or inflammation-related genes contribute to NODAT in SOT recipients. The association between 263 SNPs and NODAT was investigated in a discovery sample of SOT recipients from the Swiss Transplant Cohort Study (STCS, n1= 696). Positive results were tested in a first STCS replication sample (n2= 489) and SNPs remaining significant after multiple test corrections were tested in a second SOT replication sample (n3= 156). Associations with diabetic traits were further tested in several large general population-based samples (n > 480 000). Only SP110 rs2114592C>Tremained associated with NODAT in the STCS replication sample. Carriers of rs2114592-TThad 9.9 times (95% confidence interval [CI]: 3.22-30.5, P= .00006) higher risk for NODAT in the combined STCS samples (n = 1184). rs2114592C>Twas further associated with NODAT in the second SOT sample (odds ratio: 4.8, 95% CI: 1.55-14.6, P= .006). On the other hand, SP110 rs2114592C>Twas not associated with diabetic traits in population-based samples, suggesting a specific gene-environment interaction, possibly due to the use of specific medications (ie, immunosuppressants) in transplant patients and/or to the illness that may unmask the gene effect.

Surgical site infections (SSI) are common healthcare-associated infections. SSIs after kidney transplantation (K-Tx) can endanger patient and allograft survival. Multicenter studies on this early post-transplant complication are scarce. We analyzed consecutive adult K-Tx recipients enrolled in the Swiss Transplant Cohort Study (STCS) that received a K-tx between May 2008 and September 2020. All data were prospectively collected with the exception of the categorization of SSI that was performed retrospectively according to the Centers for Disease Control and Prevention criteria. A total of 58 out of 3059 (1.9%) K-Tx recipients were affected by SSIs. Deep incisional (15, 25.9%) and organ/space infections (34, 58.6%) predominated. In the majority of SSIs (52, 89.6%) bacteria were detected, most frequently Escherichia coli(15, 28.9%), Enterococcusspp. (14, 26.9%), and coagulase-negative staphylococci (13, 25.0%). A BMI ≥25kg/m2(multivariable OR 2.16, 95% CI 1.07-4.34, P=0.023) and delayed graft function (multivariable OR 2.88, 95% CI 1.56-5.34, P=0.001) were independent risk factors for SSI. In Cox proportional hazard models, SSI was independently associated with graft loss (multivariable HR 3.75, 95% CI 1.35-10.38, P=0.011). In conclusion, SSI was a rare complication after K-Tx. BMI ≥25kg/m2and delayed graft function were independent risk factors. SSI were independently associated with graft loss.

Detrimental and beneficial interactions between co-colonizing bacteria may influence the course of infections. In cystic fibrosis (CF) airways, Staphylococcus aureus prevails in childhood, whereas Pseudomonas aeruginosa progressively predominates thereafter. While a range of interactions has been identified, it is unclear if these represent specific adaptations or correlated responses to other aspects of the environment. Here, we investigate how P. aeruginosa adapts to S. aureus by evolving P. aeruginosa in the presence and absence of S. aureus. P. aeruginosa populations that evolved for 150 generations were sequenced and compared to the ancestor strain. Mutations in the Wsp signaling system were identified in both treatments and likely occurred because of low oxygen availability. Despite showing increased killing activity, wsp mutants were less fit in the presence of S. aureus. In contrast, mutations in lipopolysaccharide (LPS) biosynthesis occurred exclusively in co-cultures with S. aureus and conferred a fitness gain in its presence. Moreover, they increased resistance towards beta-lactam antibiotics. Strikingly, both mutations in wsp and LPS genes are observed in clinical isolates from CF-patients. Our results suggest that P. aeruginosa LPS mutations are a direct consequence of S. aureus imposed selection in vitro.

Detrimental and beneficial interactions between co-colonizing bacteria may influence the course of infections. In cystic fibrosis (CF) airways, Staphylococcus aureusprevails in childhood, whereas Pseudomonas aeruginosaprogressively predominates thereafter. While a range of interactions has been identified, it is unclear if these represent specific adaptations or correlated responses to other aspects of the environment. Here, we investigate how P. aeruginosaadapts to S. aureusby evolving P. aeruginosain the presence and absence of S. aureus. P. aeruginosapopulations that evolved for 150 generations were sequenced and compared to the ancestor strain. Mutations in the Wsp signaling system were identified in both treatments and likely occurred because of low oxygen availability. Despite showing increased killing activity, wspmutants were less fit in the presence of S. aureus. In contrast, mutations in lipopolysaccharide (LPS) biosynthesis occurred exclusively in co-cultures with S. aureusand conferred a fitness gain in its presence. Moreover, they increased resistance towards beta-lactam antibiotics. Strikingly, both mutations in wspand LPS genes are observed in clinical isolates from CF-patients. Our results suggest that P. aeruginosaLPS mutations are a direct consequence of S. aureusimposed selection in vitro.

We aimed to compare the efficacy of ceftazidime-avibactam (CAZ-AVI) versusthe best available therapy (BAT) in solid organ transplant (SOT) recipients with bloodstream infection caused by carbapenemase-producing Klebsiella pneumoniae(CPKP-BSI). A retrospective (2016-2021) observational cohort study was performed in 14 INCREMENT-SOT centers (ClinicalTrials.govidentifier: NCT02852902). Outcomes were 14-day and 30-day clinical success (complete resolution of attributable manifestations, adequate source control and negative follow-up blood cultures) and 30-day all-cause mortality. Multivariable logistic and Cox regression analyses adjusted for the propensity score to receive CAZ-AVI were constructed. Among 210 SOT recipients with CPKP-BSI, 149 received active primary therapy with CAZ-AVI (66/149) or BAT (83/149). Patients treated with CAZ-AVI had higher 14-day (80.7% versus 60.6%, P=0.011) and 30-day (83.1% versus 60.6%, P=0.004) clinical success and lower 30-day mortality (13.25% versus 27.3%, P=0.053) than those receiving BAT. In the adjusted analysis, CAZ-AVI increased the probability of 14-day (adjusted odds ratio [aOR]: 2.65; 95% confidence interval [95%CI]: 1.03-6.84, P=0.044) and 30-day clinical success (aOR: 3.14; 95%CI: 1.17-8.40; P=0.023). In contrast, CAZ-AVI therapy was not independently associated with 30-day mortality. In the CAZ-AVI group, combination therapy was not associated with better outcomes. In conclusion, CAZ-AVI may be considered a first-line treatment in SOT recipients with CPKP-BSI.

There are conflicting data on the role of the lectin pathway of complement activation and its recognition molecules in acute rejection and outcome after transplantation. To help resolve this we analyzed polymorphisms and serum levels of lectin pathway components in 710 consecutive kidney transplant recipients enrolled in the nationwide Swiss Transplant Cohort Study, together with all biopsy-proven rejection episodes and 1-year graft and patient survival. Functional mannose-binding lectin (MBL) levels were determined in serum samples, and previously described MBL2, ficolin 2, and MBL-associated serine protease 2polymorphisms were genotyped. Low MBL serum levels and deficient MBL2diplotypes were associated with a higher incidence of acute cellular rejection during the first year, in particular in recipients of deceased-donor kidneys. This association remained significant (hazard ratio 1.75, 95% confidence interval 1.18–2.60) in a Cox regression model after adjustment for relevant covariates. In contrast, there was no significant association with rates of antibody-mediated rejection, patient death, early graft dysfunction or loss. Thus, results in a prospective multicenter contemporary cohort suggest that MBL2polymorphisms result in low MBL serum levels and are associated with acute cellular rejection after kidney transplantation. Since MBL deficiency is a relatively frequent trait in the normal population, our findings may lead to individual risk stratification and customized immunosuppression.

Solid organ transplant recipients with KIR B haplotypes are associated with protection from cytomegalovirus. This study shows that they area also associated with protection from varicella zoster infection, but not Epstein-Barr virus (another herpes virus) or BK, a polyomavirus.

OBJECTIVEImmunocompromised patients now benefit from a longer life expectancy due to advanced medical techniques, but they are also weakened by aggressive treatment approaches and are at high risk for invasive fungal disease. We determined risk factors associated with an outbreak of invasive filamentous fungal infection (IFFI) among hospitalized hemato-oncological patients.METHODSA retrospective, matched, case-control study was conducted between January 1, 2009, and April 31, 2011, including 29 cases (6 proven, 8 probable, and 15 possible) of IFFI and 102 matched control patients hospitalized during the same time period. Control patients were identified from the hospital electronic database. Conditional logistic regression was performed to identify independent risk factors for IFFI.RESULTSOverall mortality associated with IFFI was 20.7% (8.0%–39.7%). Myelodysplastic syndrome was associated with a higher risk for IFFI compared to chronic hematological malignancies. After adjustment for major risk factors and confounders, >5 patient transfers outside the protected environment of the hematology ward increased the IFFI risk by 6.1-fold. The risk increased by 6.7-fold when transfers were performed during neutropenia.CONCLUSIONThis IFFI outbreak was characterized by a strong association with exposure to the unprotected environment outside the hematology ward during patient transfer. The independent associations of a high number of transfers with the presence of neutropenia suggest that affected patients were probably not sufficiently protected during transport in the corridors. Our study highlights that a heightened awareness of the need for preventive measures during the entire care process of at-risk patients should be promoted among healthcare workers.Infect Control Hosp Epidemiol 2014;00(0): 1–9

The purpose of this study is to provide an overview of bacterial biliary tract infections in liver transplant recipients with a focus on pathogenesis and conservative treatment strategies.

ABSTRACTBAL30072 is a monosulfactam conjugated with an iron-chelating dihydroxypyridone moiety. It is active against Gram-negative bacteria, including multidrug-resistant Pseudomonas aeruginosa. We selected mutants with decreased susceptibilities to BAL30072 in P. aeruginosaPAO1 under a variety of conditions. Under iron-deficient conditions, mutants with overexpression of AmpC β-lactamase predominated. These mutants were cross-resistant to aztreonam and ceftazidime. Similar mutants were obtained after selection at >16× the MIC in iron-sufficient conditions. At 4× to 8× the MIC, mutants with elevated MIC for BAL30072 but unchanged MICs for aztreonam or ciprofloxacin were selected. The expression of ampCand the major efflux pump genes were also unchanged. These BAL30072-specific mutants were characterized by transcriptome analysis, which revealed upregulation of the Fe-dicitrate operon, FecIRA. Whole-genome sequencing showed that this resulted from a single nucleotide change in the Fur-box of the fecIpromoter. Overexpression of either the FecI ECF sigma factor or the FecA receptor increased BAL30072 MICs 8- to 16-fold. A fecImutant and a fecAmutant of PAO1 were hypersusceptible to BAL30072 (MICs < 0.06 μg/ml). The most downregulated gene belonged to the pyochelin synthesis operon, although mutants in pyochelin receptor or synthesis genes had unchanged MICs. The piuCgene, coding for a Fe(II)-dependent dioxygenase located next to the piuAiron receptor gene, was also downregulated. The MICs of BAL30072 for piuCand piuAtransposon mutants were increased 8- and 16-fold, respectively. We conclude that the upregulation of the Fe-dicitrate system impacts the expression of other TonB-dependent iron transporters and that PiuA and PiuC contribute to the susceptibility of P. aeruginosaPAO1 to BAL30072.

The debate on the merits of observational studies as compared with randomized trials is ongoing. We will briefly touch on this subject, and demonstrate the role of cohort studies for the description of infectious disease patterns after transplantation. The potential benefits of cohort studies for the clinical management of patients outside of the expected gain in epidemiological knowledge are reviewed. The newly established Swiss Transplantation Cohort Study and in particular the part focusing on infectious diseases will serve as an illustration.

Background Solid organ transplant (SOT) recipients are a priority group for influenza vaccination and strategies enhancing immunogenicity are needed.Methods We determined adverse reactions, changes in biomarkers of graft function and immune responses to two doses of the AS03-adjuvanted influenza A/09/H1N1 vaccine in 216 SOT recipients and in 138 controls after one dose. Antibody responses were measured by haemag-glutination inhibition and confirmed by microneutralization. We calculated geometric mean titres (GMT) and seroprotection rates (GMT=40).Results Adverse reactions were fewer than in controls and graft function remained unaffected. Seroprotection was achieved by only 70.3% of SOT recipients, with significant differences between groups (lung 43.6%, heart 72.0%, kidney 83.3%, liver 83.3% and pancreas 85%), compared to 87% of controls (P<0.001). The weakest responses (seroprotection 43.5%) were elicited in lung transplant recipients. GMT remained threefold lower (115 versus 340) in SOT recipients than controls. Multivariate analyses identified increasing age, type of transplant and increasing blood levels of mycophenolate as independently associated to weaker responses. In contrast, even high blood levels of calcineurin inhibitors remained without significant influence on vaccine responses.Conclusions The squalene-based adjuvanted A/09/H1N1 vaccine was safe in SOT recipients. However, even two doses of this adjuvanted influenza vaccine did not provide adequate protection for lung transplant recipients and those with high mycophenolate blood levels. Additional prophylactic measures should, therefore, be considered for these high-risk groups.

Herpes simplex virus (HSV) hepatitis is an uncommon cause of acute liver failure (ALF), primarily affecting immunocompromised patients. So far, 148 cases have been published, of which 9 underwent liver transplantation (LT). The reported post-transplant survival is poor, with over 60% dying in the first year. Dosing and duration of antiviral therapy after LT are not established. Concerns include both the risk of hepatic recurrence after LT and emergence of viral resistance during prolonged therapy. HSV DNA plasma levels might be helpful to monitor therapeutic response and guide duration of therapy. We present a case of ALF complicating a primary HSV-1 infection in an immunocompetent host, who required emergency LT. We further discuss the value of measuring serial HSV DNA plasma loads to monitor antiviral therapy.

ABSTRACTIn this study, we investigated the resistance mechanisms to fluoroquinolones of 85 non-cystic fibrosis strains of Pseudomonas aeruginosaexhibiting a reduced susceptibility to ciprofloxacin (MICs from 0.25 to 2 μg/ml). In addition to MexAB-OprM (31 of 85 isolates) and MexXY/OprM (39 of 85), the MexEF-OprN efflux pump (10 of 85) was found to be commonly upregulated in this population that is considered susceptible or of intermediate susceptibility to ciprofloxacin, according to current breakpoints. Analysis of the 10 MexEF-OprN overproducers (nfxCmutants) revealed the presence of various mutations in the mexT(2 isolates), mexS(5 isolates), and/or mvaT(2 isolates) genes, the inactivation of which is known to increase the expression of the mexEF-oprNoperon in reference strain PAO1-UW. However, these genes were intact in 3 of 10 of the clinical strains. Interestingly, ciprofloxacin at 2 μg/ml or 4 μg/ml preferentially selected nfxCmutants from wild-type clinical strains (n= 10 isolates) and from first-step mutants (n= 10) overexpressing Mex pumps, thus indicating that MexEF-OprN represents a major mechanism by which P. aeruginosamay acquire higher resistance levels to fluoroquinolones. These data support the notion that the nfxCmutants may be more prevalent in the clinical setting than anticipated and strongly suggest the involvement of still unknown genes in the regulation of this efflux system.

ABSTRACTSince their initial description 2 decades ago, MexCD-OprJ-overproducing efflux mutants of Pseudomonas aeruginosa(also called nfxBmutants) have rarely been described in the clinical setting. Screening of 110 nonreplicate clinical isolates showing moderate resistance to ciprofloxacin (MIC from 0.5 μg/ml to 4 μg/ml) yielded only four mutants (3.6%) of that type harboring various alterations in the repressor gene nfxB. MexCD-OprJ upregulation correlated with an increased resistance to ciprofloxacin, cefepime, and chloramphenicol in most of the clinical strains, concomitant with a higher susceptibility to ticarcillin, aztreonam, imipenem, and aminoglycosides. Evidence was obtained that this increased susceptibility to aminoglycosides results from the impaired activity of efflux pump MexXY-OprM. Furthermore, MexCD-OprJ upregulation was found to impair bacterial growth and to have a strain-specific, variable impact on rhamnolipid, elastase, phospholipase C, and pyocyanin production. Review of patient files indicated that the four nfxBmutants were responsible for confirmed cases of infection and emerged during long-term therapy with ciprofloxacin. Taken together, these data show that, while rather infrequent among P. aeruginosastrains with low-level resistance to ciprofloxacin, MexCD-OprJ-overproducing mutants may be isolated after single therapy with fluoroquinolones and may be pathogenic.

ABSTRACTIn Pseudomonas aeruginosa, azithromycin has been shown to reduce virulence factor production, to retard biofilm formation, and to exhibit bactericidal effects on stationary-phase cells. In this study we analyzed whether these azithromycin-mediated effects require interaction with the ribosome. We blocked the access of azithromycin to the ribosome in P. aeruginosaPAO1 by expressing the 23S rRNA methylase ErmBP from Clostridium perfringens. Ribosome protection prevented the azithromycin-mediated reduction of elastase and rhamnolipid production, as well as the inhibition of swarming motility. Ribosome protection also prevented the killing of stationary-phase cells, suggesting that the cell-killing effect of azithromycin does not result solely from membrane destabilization. We further show that rhamnolipids are involved in cell killing, probably by increasing the uptake of the hydrophobic azithromycin molecule. These results have important implications for the treatment with azithromycin of patients chronically colonized by P. aeruginosaand might explain the variability in the efficacy of azithromycin treatments.

ABSTRACTIntubated patients frequently become colonized by Pseudomonas aeruginosa, which is subsequently responsible for ventilator-associated pneumonia. This pathogen readily acquires resistance against available antimicrobials. Depending on the resistance mechanism selected for, resistance might either be lost or persist after removal of the selective pressure. We investigated the rapidity of selection, as well as the persistence, of antimicrobial resistance and determined the underlying mechanisms. We selected 109 prospectively collected P. aeruginosatracheal isolates from two patients based on their prolonged intubation and colonization periods, during which they had received carbapenem, fluoroquinolone (FQ), or combined β-lactam-aminoglycoside therapies. We determined antimicrobial resistance phenotypes by susceptibility testing and used quantitative real-time PCR to measure the expression of resistance determinants. Within 10 days after the initiation of therapy, all treatment regimens selected resistant isolates. Resistance to β-lactam and FQ was correlated with ampCand mexCgene expression levels, respectively, whereas imipenem resistance was attributable to decreased oprDexpression. Combined β-lactam-aminoglycoside resistance was associated with the appearance of small-colony variants. Imipenem and FQ resistance persisted for prolonged times once the selecting antimicrobial treatment had been discontinued. In contrast, resistance to β-lactams disappeared rapidly after removal of the selective pressure, to reappear promptly upon renewed exposure. Our results suggest that resistant P. aeruginosais selected in less than 10 days independently of the antimicrobial class. Different resistance mechanisms lead to the loss or persistence of resistance after the removal of the selecting agent. Even if resistant isolates are not evident upon culture, they may persist in the lung and can be rapidly reselected.

ABSTRACTCell-to-cell signaling involving N-acyl-homoserine lactone compounds termed autoinducers (AIs) is instrumental to virulence factor production and biofilm development by Pseudomonas aeruginosa. In order to determine the importance of cell-to-cell signaling during the colonization of mechanically ventilated patients, we collected 442 P. aeruginosapulmonary isolates from 13 patients. Phenotypic characterization showed that 81% of these isolates produced the AI-dependent virulence factors elastase, protease, and rhamnolipids. We identified nine genotypically distinct P. aeruginosastrains. Six of these strains produced AIs [N-butanoyl-homoserine lactone or N-(3-oxo-dodecanoyl)-homoserine lactone] and extracellular virulence factors (elastase, total exoprotease, rhamnolipid, hydrogen cyanide, or pyocyanin) in vitro. Three of the nine strains were defective in the production of both AIs and extracellular virulence factors. Two of these strains had mutational defects in both the lasRand rhlRgenes, which encode the N-acyl-homoserine lactone-dependent transcriptional regulators LasR and RhlR, respectively. The third of these AI-deficient strains was only mutated in the lasRgene. Our observations suggest that most, but not all, strains colonizing intubated patients are able to produce virulence factors and that mutations affecting the cell-to-cell signaling circuit are preferentially located in the transcriptional regulator genes.

ABSTRACTIt remains controversial whether combination therapy, given empirically or as definitive treatment, for Pseudomonas aeruginosabacteremia is associated with a better outcome than monotherapy. The aim of the present study was to compare the rates of survival among patients who received either combination therapy or monotherapy for P. aeruginosabacteremia. We assembled a historical cohort of 115 episodes of P. aeruginosabacteremia treated with empirical antipseudomonal therapy between 1988 and 1998. On the basis of susceptibility testing of the bacteremic P. aeruginosaisolate, we defined categories of empirical treatment, including adequate combination therapy, adequate monotherapy, and inadequate therapy, as well as corresponding categories of definitive therapy. Neither the adequacy of the empirical treatment nor the use of combination therapy predicted survival until receipt of the antibiogram. However, the risk of death from the date of receipt of the antibiogram to day 30 was higher for both adequate empirical monotherapy (adjusted hazard ratio [aHR], 3.7; 95% confidence interval [CI], 1.0 to 14.1) and inadequate empirical therapy (aHR, 5.0; 95% CI, 1.2 to 20.4) than for adequate empirical combination therapy. Compared to adequate definitive combination therapy, the risk of death at 30 days was also higher with inadequate definitive therapy (aHR, 2.6; 95% CI, 1.1 to 6.7) but not with adequate definitive monotherapy (aHR, 0.70; 95% CI, 0.30 to 1.7). In this retrospective analysis the use of adequate combination antimicrobial therapy as empirical treatment until receipt of the antibiogram was associated with a better rate of survival at 30 days than the use of monotherapy. However, adequate combination antimicrobial therapy given as definitive treatment for P. aeruginosabacteremia did not improve the rate of survival compared to that from the provision of adequate definitive monotherapy.

ABSTRACTAt least four broad-spectrum efflux pumps (Mex) are involved in elevated intrinsic antibiotic resistance as well as in acquired multidrug resistance in Pseudomonas aeruginosa. Substrate specificity of the Mex pumps has been shown to be determined by the cytoplasmic membrane component (MexB, MexD, MexF, and MexY) of the tripartite efflux pump system. Alignment of their amino acid sequences with those of the homologous AcrB and AcrD pump proteins of Escherichia colishowed conservation of five charged amino acid residues located in or next to transmembrane segments (TMS). These residues were mutated in the MexF gene by site-directed mutagenesis and replaced by residues of opposite or neutral charge. MexF proteins containing combined D410A and A411G substitutions located in TMS4 were completely inactive. Similarly, the substitutions E417K (next to TMS4) and K951E (TMS10) also caused loss of activity towards all tested antibiotics. The substitution E349K in TMS2 resulted in a MexF mutant protein which was unable to transport trimethoprim and quinolones but retained partial activity for the transport of chloramphenicol. All mutated MexF proteins were expressed at comparable levels when tested by Western blot analysis. It is concluded that charged residues located in or close to TMS are essential for proper function of MexF.

ABSTRACTWe report that 2 μg of azithromycin/ml inhibits the quorum-sensing circuitry of Pseudomonas aeruginosastrain PAO1. Addition of synthetic autoinducers partially restored the expression of the trancriptional activator-encoding geneslasRand rhlRbut not that of the autoinducer synthase-encoding gene lasI. We propose that azithromycin interferes with the synthesis of autoinducers, by an unknown mechanism, leading to a reduction of virulence factor production.

To evaluate the efficacy of low dose fluconazole treatment for the prevention of yeast colonization and infection in severely neutropenic patients.

ABSTRACTThe lasquorum-sensing system of Pseudomonas aeruginosacontrols the expression of elastase and rhamnolipid. We report that starvation can select a mutant producing these virulence factors in spite of a lasRdeletion. Expression of the autoinducer synthase gene rhlIwas increased in this suppressor mutant, suggesting compensation by the rhlsystem. These data show that P. aeruginosacan restore elastase and rhamnolipid production in the absence of a functionallasquorum-sensing system.

Colistin (polymyxin E) is a last-resort antibiotic against multidrug-resistant isolates of Pseudomonas aeruginosa. However, the nephro-toxicity of colistin limits its use, spurring the interest in novel antimicrobial peptides (AMP). Here, we show that the synthetic AMP-dendrimer G3KL (MW 4,531.38 Da, 15 positive charges, MIC = 8 mg/liter) showed faster killing than polymyxin B (Pmx-B) with no detectable resistance selection in P. aeruginosastrain PA14.

&lt;it>Objectives&lt;/it>: In &lt;it>Pseudomonas aeruginosa&lt;/it>, biofilm formation is controlled by a cell-to-cell signalling circuit relying on the secretion of 3-oxo-C&lt;inf>12&lt;/inf>-HSL and C&lt;inf>4&lt;/inf>-HSL. Previous studies suggested that C&lt;inf>4&lt;/inf>-HSL plays no significant role in biofilm formation. However the wild-type PAO1 strain PAO-BI, used as a control in these studies is itself impaired in the production of C&lt;inf>4&lt;/inf>-HSL. We wondered therefore whether the role of C&lt;inf>4&lt;/inf>-HSL in biofilm formation might have been underestimated, and whether azithromycin inhibits biofilm formation by interfering with cell-to-cell signalling. &lt;it>Methods&lt;/it>: We used isogenic mutants of wild-type PAO1 strains PAO-BI and PT5 in a static biofilm model. Biofilm formation was quantified using Crystal Violet staining and exopolysaccharide measurements. &lt;it>Results&lt;/it>: Wild-type strain PAO-BI, as a result of its reduced C&lt;inf>4&lt;/inf>-HSL secretion, produced 40&percnt; less biofilm compared with the wild-type PAO1 strain PT5. Using isogenic mutants of strain PT5 we have shown that whereas a &lt;it>lasI&lt;/it> mutant (deficient in 3-oxo-C&lt;inf>12&lt;/inf>-HSL) produced similar amounts of biofilm to the wild-type, a &lt;it>rhlI&lt;/it> mutant (deficient in C&lt;inf>4&lt;/inf>-HSL) produced 70&percnt; less biofilm. In the latter strain, biofilm formation could be restored by addition of exogenous C&lt;inf>4&lt;/inf>-HSL. Azithromycin, known to reduce the production of both 3-oxo-C&lt;inf>12&lt;/inf>-HSL and C&lt;inf>4&lt;/inf>-HSL, inhibited biofilm formation of wild-type PT5 by 45&percnt;. This inhibition could be reversed by the addition of both cell-to-cell signals. &lt;it>Conclusions&lt;/it>: Our results indicate that C&lt;inf>4&lt;/inf>-HSL also plays a significant role in biofilm formation. Furthermore, we demonstrate the potential of using cell-to-cell signalling blocking agents such as azithromycin to interfere with biofilm formation.