Pseudomonas aeruginosa associated pulmonary infections and in vitro amplification virulent rhamnolipid (rhlR) gene / Infecções pulmonares associadas a Pseudomonas aeruginosa e gene ramnolipídeo virulento (rhlR) da amplificação in vitro

Background: Pseudomonas aeruginosa is a common opportunistic pathogenic bacterium with the ability to develop a strong communication pathway by quorum sensing system and different virulent factors. Among the various important secretions of P. aeruginosa rhamnolipid is important biological detergent, believed to be involved in the development of the biofilm and intercellular communication. It readily dissolves the lung surfactants that are then easily catalyzed by the phospholipases and in this way is involved in the acute pulmonary infection. Objective: research work was designed to investigate virulence and gene associated with virulence in P. aeruginosa responsible for pulmonary infections. Methods: In current study polymerase chain reaction (PCR) was used for the detection of the rhlR (rhamnolipid encoding) gene of isolated strains. A number of assays were performed that ensured its virulent behavior. Disc diffusion method was used to check its antibiotic resistance. Isolated strains were resistant to a number of antibiotics applied. Result: It was found that males are more prone to respiratory infections as compared to females. Male members with age of 44-58 and 59-73 are at a higher risk, while females with age of 44-58 are also at a risk of pulmonary infections. Antibiotic resistance was observed by measuringzone of inhibition in strains GCU-SG-M4, GCU-SG-M3, GCU-SG-M5, GCU-SG-M2, GCU-SG-M1 and GCU-SG-M6. GCU-SG-M2 was resistant to fluconazole (FLU), clarithromycin (CLR), cefixime (CFM) and Penicillin (P10). No zone of inhibition was observed. But it showed unusual diffused zone around the Ak and MEM antibiotic discs. rhl R gene and 16s rRNA gene were characterized and analyzed. Conclusion: Findings from current study would help[...].

Antecedentes: Pseudomonas aeruginosa é uma bactéria patogênica oportunista comum, com a capacidade de desenvolver uma forte via de comunicação pelo sistema de detecção de quorum e diferentes fatores virulentos. Entre as várias secreções importantes de P. aeruginosa rhamnolipid, há um importante detergente biológico, que se acredita estar envolvido no desenvolvimento do biofilme e na comunicação intercelular. Dissolve rapidamente os surfactantes pulmonares que são facilmente catalisados pelas fosfolipases e, dessa maneira, estão envolvidos na infecção pulmonar aguda. Objetivo: O trabalho de pesquisa foi desenhado para investigar a virulência e o gene associado à virulência em P. aeruginosa responsável por infecções pulmonares. Métodos: No presente estudo, a reação em cadeia da polimerase (PCR) foi utilizada para a detecção do gene rhlR (codificação ramnolipídeo) de cepas isoladas. Foram realizados vários ensaios que garantiram seu comportamento virulento. O método de difusão em disco foi utilizado para verificar sua resistência a antibióticos. As estirpes isoladas foram resistentes a vários antibióticos aplicados. Resultado: Verificou-se que os homens são mais propensos a infecções respiratórias em comparação às mulheres. Membros do sexo masculino com idade entre 44 e 58 e 59 e 73 anos correm maior risco, enquanto mulheres com idade entre 44 e 58 anos também correm risco de infecções pulmonares. A resistência aos antibióticos foi observada medindo a zona de inibição nas cepas GCU-SG-M4, GCU-SG-M3, GCU-SG-M5, GCU-SG-M2, GCU-SG-M1 e GCU-SG-M6. O GCU-SG-M2 foi resistente ao fluconazol (FLU), claritromicina (CLR), cefixima (CFM) e penicilina (P10). Nenhuma zona de inibição foi observada. Mas se notou uma zona difusa incomum ao redor dos discos antibióticos Ak e MEM. Os genes rhl R e 16s rRNA foram caracterizados e analisados. Conclusão: As conclusões do presente estudo ajudariam a aumentar a conscientização sobre a resistência a antibióticos de, [...].

Modulation of oxidative and nitrosative stress attenuates microvascular hyperpermeability in ovine model of Pseudomonas aeruginosa sepsis.

In sepsis, microvascular hyperpermeability caused by oxidative/nitrosative stress (O&NS) plays an important role in tissue edema leading to multi-organ dysfunctions and increased mortality. We hypothesized that a novel compound R-107, a modulator of O&NS, effectively ameliorates the severity of microvascular hyperpermeability and preserves multi-organ function in ovine sepsis model. Sepsis was induced in twenty-two adult female Merino sheep by intravenous infusion of Pseudomonas aeruginosa (PA) (1 × 1010 CFUs). The animals were allocated into: 1) Control (n = 13): intramuscular injection (IM) of saline; and 2) Treatment (n = 9): IM of 50 mg/kg R-107. The treatment was given after the PA injection, and monitored for 24-h. R-107 treatment significantly reduced fluid requirement (15-24 h, P < 0.05), net fluid balance (9-24 h, P < 0.05), and water content in lung/heart/kidney (P = 0.02/0.04/0.01) compared to control. R-107 treatment significantly decreased lung injury score/modified sheep SOFA score at 24-h (P = 0.01/0.04), significantly lowered arterial lactate (21-24 h, P < 0.05), shed syndecan-1 (3-6 h, P < 0.05), interleukin-6 (6-12 h, P < 0.05) levels in plasma, and significantly attenuated lung tissue 3-nitrotyrosine and vascular endothelial growth factor-A expressions (P = 0.03/0.002) compared to control. There was no adverse effect in R-107 treatment. In conclusion, modulation of O&NS by R-107 reduced hyperpermeability markers and improved multi-organ function.

BSA Hydrogel Beads Functionalized with a Specific Aptamer Library for Capturing Pseudomonas aeruginosa in Serum and Blood.

Systemic blood stream infections are a major threat to human health and are dramatically increasing worldwide. Pseudomonas aeruginosa is a WHO-alerted multi-resistant pathogen of extreme importance as a cause of sepsis. Septicemia patients have significantly increased survival chances if sepsis is diagnosed in the early stages. Affinity materials can not only represent attractive tools for specific diagnostics of pathogens in the blood but can prospectively also serve as the technical foundation of therapeutic filtration devices. Based on the recently developed aptamers directed against P. aeruginosa, we here present aptamer-functionalized beads for specific binding of this pathogen in blood samples. These aptamer capture beads (ACBs) are manufactured by crosslinking bovine serum albumin (BSA) in an emulsion and subsequent functionalization with the amino-modified aptamers on the bead surface using the thiol- and amino-reactive bispecific crosslinker PEG4-SPDP. Specific and quantitative binding of P. aeruginosa as the dedicated target of the ACBs was demonstrated in serum and blood. These initial but promising results may open new routes for the development of ACBs as a platform technology for fast and reliable diagnosis of bloodstream infections and, in the long term, blood filtration techniques in the fight against sepsis.

Extrathoracic multiple trauma dysregulates neutrophil function and exacerbates pneumonia-induced lung injury.

BACKGROUND: Forty percent of critically ill trauma patients will develop an infectious complication. Pneumonia is the most common cause of death of trauma patients surviving their initial insult. We previously demonstrated that polytrauma (PT), defined as two or more severe injuries in at least two areas of the body, induces emergency hematopoiesis characterized by accelerated myelopoiesis in the bone marrow and increased myeloid cell frequency in the peripheral tissues. We hypothesized that PT alone induces priming of neutrophils, resulting in hyperactivation upon secondary exposure to bacteria and causing acute lung injury and increased susceptibility to secondary exposure to Pseudomonas aeruginosa pneumonia. METHODS: C57BL/6 mice were subjected to PT consisting of a lower extremity pseudofracture, liver crush injury, and 15% blood-volume hemorrhage. Pneumonia was induced by intratracheal injection of 5 × 106 CFU live P. aeruginosa or 1 × 107 of heat-killed P. aeruginosa (HKPA). For reactive oxygen species (ROS), studies polymorphonuclear neutrophils (PMNs) were isolated by immunomagnetic bead negative selection and stimulated ex-vivo with HKPA. Reactive oxygen species production was measured by immunofluorescence. For histology, lung sections were stained by hematoxylin-eosin and analyzed by a blinded grader. RESULTS: Polytrauma induced persistent changes in immune function at baseline and to secondary infection. Pneumonia after injury resulted in increased mortality (60% vs. 5% p < 0.01). Blood neutrophils from PT mice had higher resting (unstimulated) ROS production than in naive animals (p < 0.02) demonstrating priming of the neutrophils following PT. After intratracheal HKPA injection, bronchoalveolar lavage PMNs from injured mice had higher ROS production compared with naive mice (p < 0.01), demonstrating an overexuberant immunopathologic response of neutrophils following PT. CONCLUSION: Polytrauma primes neutrophils and causes immunopathologic PMN ROS production, increased lung injury and susceptibility to secondary bacterial pneumonia. These results suggest that trauma-induced immune dysfunction can cause immunopathologic response to secondary infection and suggests neutrophil-mediated pulmonary damage as a therapeutic target for posttrauma pneumonia.

The lytic polysaccharide monooxygenase CbpD promotes Pseudomonas aeruginosa virulence in systemic infection.

The recently discovered lytic polysaccharide monooxygenases (LPMOs), which cleave polysaccharides by oxidation, have been associated with bacterial virulence, but supporting functional data is scarce. Here we show that CbpD, the LPMO of Pseudomonas aeruginosa, is a chitin-oxidizing virulence factor that promotes survival of the bacterium in human blood. The catalytic activity of CbpD was promoted by azurin and pyocyanin, two redox-active virulence factors also secreted by P. aeruginosa. Homology modeling, molecular dynamics simulations, and small angle X-ray scattering indicated that CbpD is a monomeric tri-modular enzyme with flexible linkers. Deletion of cbpD rendered P. aeruginosa unable to establish a lethal systemic infection, associated with enhanced bacterial clearance in vivo. CbpD-dependent survival of the wild-type bacterium was not attributable to dampening of pro-inflammatory responses by CbpD ex vivo or in vivo. Rather, we found that CbpD attenuates the terminal complement cascade in human serum. Studies with an active site mutant of CbpD indicated that catalytic activity is crucial for virulence function. Finally, profiling of the bacterial and splenic proteomes showed that the lack of this single enzyme resulted in substantial re-organization of the bacterial and host proteomes. LPMOs similar to CbpD occur in other pathogens and may have similar immune evasive functions.

Bacterial behavior in human blood reveals complement evaders with some persister-like features.

Bacterial bloodstream infections (BSI) are a major health concern and can cause up to 40% mortality. Pseudomonas aeruginosa BSI is often of nosocomial origin and is associated with a particularly poor prognosis. The mechanism of bacterial persistence in blood is still largely unknown. Here, we analyzed the behavior of a cohort of clinical and laboratory Pseudomonas aeruginosa strains in human blood. In this specific environment, complement was the main defensive mechanism, acting either by direct bacterial lysis or by opsonophagocytosis, which required recognition by immune cells. We found highly variable survival rates for different strains in blood, whatever their origin, serotype, or the nature of their secreted toxins (ExoS, ExoU or ExlA) and despite their detection by immune cells. We identified and characterized a complement-tolerant subpopulation of bacterial cells that we named "evaders". Evaders shared some features with bacterial persisters, which tolerate antibiotic treatment. Notably, in bi-phasic killing curves, the evaders represented 0.1-0.001% of the initial bacterial load and displayed transient tolerance. However, the evaders are not dormant and require active metabolism to persist in blood. We detected the evaders for five other major human pathogens: Acinetobacter baumannii, Burkholderia multivorans, enteroaggregative Escherichia coli, Klebsiella pneumoniae, and Yersinia enterocolitica. Thus, the evaders could allow the pathogen to persist within the bloodstream, and may be the cause of fatal bacteremia or dissemination, in particular in the absence of effective antibiotic treatments.

Area under the curve achievement of once daily tobramycin in children with cystic fibrosis during clinical care.

BACKGROUND: The area under the concentration-time curve over 24 hours (AUC24 ) is frequently utilized to monitor tobramycin exposure in children with cystic fibrosis (CF). An understanding of exposure target achievement during clinical implementation of an AUC24 based approach in children is limited. METHODS: A retrospective chart review was performed in children with CF treated with once daily tobramycin and drug concentration monitoring at a pediatric CF center. During clinical care AUC24 was estimated using a traditional log-linear regression approach (LLR). AUC24 was also estimated retrospectively using a pharmacokinetic model-based Bayesian forecasting approach (BF). AUC24 achievement after both approaches were compared. RESULTS: In 77 treatment courses (mean age, 12.7 ± 5.0 years), a target AUC24 100 to 125 mg h/L was achieved after starting dose in 21 (27%) and after initial dose adjustment in 35 (45%). In the first 7 days of treatment, 24 (32%) required ≥3 dose adjustments, and the mean number of drug concentrations measured was 7.1 ± 3.2. Examination of a BF approach demonstrated adequate prediction of measured tobramycin concentrations (median bias -2.1% [95% CI -3.1 to -1.4]; median precision 7.6% [95% CI, 7.1%-8.2%]). AUC24 estimates utilizing the BF approach were higher than the LLR approach with a mean difference of 6.4 mg h/L (95% CI, 4.8 to 8.0 mg h/L). CONCLUSIONS: Achievement of a narrow AUC24 target is challenging during clinical care, and dose individualization is needed in most children with CF. Implementing a BF approach for estimating AUC24 in children with CF is supported.

Assessment of Silver Levels in a Closed-Incision Negative Pressure Therapy Dressing: In Vitro and In Vivo Study.

Objective: In recent years, reticulated open-cell foam-based closed-incision negative pressure therapy (ROCF-ciNPT) has shown effectiveness in management of various postoperative incisions. These dressings consist of a skin interface layer that absorbs fluid from the skin surface and reduces the potential for microbial colonization within the dressing by means of ionic silver. This study examines the ability of silver to reduce the bioburden within the dressing as well as the localized effect due to potential silver mobility. Approach: Ability of silver to reduce bioburden within the ROCF-ciNPT dressing was assessed using Staphylococcus aureus, Pseudomonas aeruginosa, and Candida spp. Furthermore, silver mobility was assessed using an in vitro skin model to study the zone of inhibition along with released silver quantification. Using a porcine model, diffusion of silver into blood and tissue was studied using emission spectrometry and histology. Results: Microbial growth in the ROCF-ciNPT dressing was significantly reduced (∼2.7-4.9 log reduction) compared to a silver-free negative control. No zone of inhibition was observed for microbial colonies for up to 7 days with minimal localized silver release (<5.5 ppm release). In vivo studies demonstrated no measurable concentration (<0.2 µg/g) of silver in the blood, urine, feces, kidney, and liver tissue biopsy. Innovation: This study provides an important insight into silver concentration and mobility within the ROCF-ciNPT dressing, given emerging concerns associated with potential silver cytotoxicity. Conclusion: These results indicate the concentration of silver (0.019% silver by weight) in the ROCF-ciNPT dressings has been adequate to reduce bioburden within the skin interface layer, while severely limiting the amount of silver leaching out.

Construction of Genomic Library and High-Throughput Screening of Pseudomonas aeruginosa Novel Antigens for Potential Vaccines.

Hospital-acquired infections with Pseudomonas aeruginosa have become a great challenge in caring for critically ill and immunocompromised patients. The cause of high mortality is the presence of multi-drug resistant (MDR) strains, which confers a pressing need for vaccines. Although vaccines against P. aeruginosa have been in development for more than several decades, there is no vaccine for patients at present. In this study, we purified genomic DNA of P. aeruginosa from sera of patients affected, constructed genome-wide library with random recombinants, and screened candidate protein antigens by evaluating their protective effects in vivo. After 13-round of screening, 115 reactive recombinants were obtained, among which 13 antigens showed strong immunoreactivity (more than 10% reaction to PcrV, a well-characterized V-antigen of P. aeruginosa). These 13 antigens were: PpiA, PtsP, OprP, CAZ10_34235, HmuU_2, PcaK, CarAd, RecG, YjiR_5, LigD, KinB, RtcA, and PscF. In vivo studies showed that vaccination with PscF protected against lethal P. aeruginosa challenge, and decreased lung inflammation and injury. A genomic library of P. aeruginosa could be constructed in this way for the first time, which could not only screen candidate antigens but also in a high-throughput way. PscF was considered as an ideal promising vaccine candidate for combating P. aeruginosa infection and was supported for further evaluation of its safety and efficacy.

Neonatal infection and passive acquisition of serum total IgG and reactive with "Streptococcus" B, anti-LPS of "Klebsiella spp" and "Pseudomonas spp" antibodies in twins.

OBJECTIVE To describe the concentration of total and specific IgG antibodies anti-Streptococcus B, anti-lipopolysaccharide of Klebsiella spp, and anti-lipopolysaccharide of Pseudomonas spp in the umbilical cord of newborn(NB) twins and to analyze the association between neonatal infection and antibody concentration in the umbilical cord blood. METHODS A prospective cross-sectional study of a cohort of NB twins admitted during the period of 20 months. Patients with malformations and mothers with infection were excluded. Variables analyzed: gestational age(GA); birth weight(BW); antibody concentrations in umbilical cord blood; infection episodes. We used the paired Student t-test, Spearman correlation, and generalized estimation equation. RESULTS 57 pairs of twins were included, 4 excluded, making the sample of 110 newborns. GA=36±1.65weeks and BW=2304.8±460g(mean±SD). Antibody concentrations in twins(mean±SD): total IgG=835.71±190.73mg/dL, anti-StreptococcusB IgG=250.66±295.1 AU/mL, anti-lipopolysaccharide of Pseudomonas spp IgG=280.04±498.66 AU/mL and anti-lipopolysaccharide of Klebsiella spp IgG=504.75±933.93 AU/mL. There was a positive correlation between maternal antibody levels and those observed in newborns(p <0.005). The transplacental transfer of maternal total IgG and anti-LPS Pseudomonas IgG antibodies was significantly lower at NB GA <34 weeks(p <0.05). Five newborns were diagnosed with an infection. Infants with infection had significantly lower total IgG concentration(p <0.05). CONCLUSION This study showed a positive correlation between maternal and newborn antibodies levels. In infants younger than 34 weeks there is less transfer of total IgG and anti-LPS Pseudomonas IgG. The highest incidence of infection in the newborn group who had significantly lower total IgG serum antibodies reinforces the importance of anti-infectious protection afforded by passive immunity transferred from the mother.

Clinical characteristics and predictors of community-acquired pseudomonas aeruginosa sepsis and nontyphoidal salmonella sepsis in infants: A matched Case-Control study.

BACKGROUND: Pseudomonas aeruginosa and nontyphoidal Salmonella (NTS) species may cause enteric illness with sepsis in infancy. The clinical predictors distinguishing the two pathogens have not been comprehensively evaluated in this population in Taiwan. METHODS: A retrospective matched case-control study was conducted in a teaching hospital in southern Taiwan from January 1, 2003 to January 30, 2019. The patients with community-acquired P. aeruginosa sepsis were matched at a ratio of 1:2 by age and gender with controls (who developed NTS sepsis). RESULTS: A total of 21 infants with community-acquired P. aeruginosa sepsis were identified; of these, 12 (57.1%) were male, and the mean ± standard deviation of age was 6.95 ± 2.47 months. Two independent predictors indicative of P. aeruginosa sepsis, as identified by multivariate analysis using conditional logistic regression, were hemoglobin level (Hb) (matched odds ratio [mOR], 0.155; 95% confidence interval [CI], 0.027-0.900; p = 0.038) and platelet count (mOR, 0.988, 95% CI, 0.976-1.000; p = 0.049). The areas under the receiver operating characteristic (ROC) curves of Hb and platelet count for P. aeruginosa sepsis prediction were 0.855 and 0.803, respectively. With cut-off values for Hb of 10.7 g/dL and platelet count of 173,000/µL, the predictors had maximal diagnostic accuracy. CONCLUSION: Most patients with P. aeruginosa sepsis are less than one year old. A lower hemoglobin level and a lower platelet count are significant predictors of P. aeruginosa sepsis. These findings should help to reshape the policy of empirical antibiotics in infants with sepsis.

Neonatal infection and passive acquisition of serum total IgG and reactive with Streptococcus B, anti-LPS of Klebsiella spp and Pseudomonas spp antibodies in twins

SUMMARY OBJECTIVE To describe the concentration of total and specific IgG antibodies anti-Streptococcus B, anti-lipopolysaccharide of Klebsiella spp, and anti-lipopolysaccharide of Pseudomonas spp in the umbilical cord of newborn(NB) twins and to analyze the association between neonatal infection and antibody concentration in the umbilical cord blood. METHODS A prospective cross-sectional study of a cohort of NB twins admitted during the period of 20 months. Patients with malformations and mothers with infection were excluded. Variables analyzed: gestational age(GA); birth weight(BW); antibody concentrations in umbilical cord blood; infection episodes. We used the paired Student t-test, Spearman correlation, and generalized estimation equation. RESULTS 57 pairs of twins were included, 4 excluded, making the sample of 110 newborns. GA=36±1.65weeks and BW=2304.8±460g(mean±SD). Antibody concentrations in twins(mean±SD): total IgG=835.71±190.73mg/dL, anti-StreptococcusB IgG=250.66±295.1 AU/mL, anti-lipopolysaccharide of Pseudomonas spp IgG=280.04±498.66 AU/mL and anti-lipopolysaccharide of Klebsiella spp IgG=504.75±933.93 AU/mL. There was a positive correlation between maternal antibody levels and those observed in newborns(p <0.005). The transplacental transfer of maternal total IgG and anti-LPS Pseudomonas IgG antibodies was significantly lower at NB GA <34 weeks(p <0.05). Five newborns were diagnosed with an infection. Infants with infection had significantly lower total IgG concentration(p <0.05). CONCLUSION This study showed a positive correlation between maternal and newborn antibodies levels. In infants younger than 34 weeks there is less transfer of total IgG and anti-LPS Pseudomonas IgG. The highest incidence of infection in the newborn group who had significantly lower total IgG serum antibodies reinforces the importance of anti-infectious protection afforded by passive immunity transferred from the mother.

RESUMO OBJETIVOS Descrever o título de anticorpos IgG total e específico anti-Streptococcus B, anti-lipopolissacarídeos(LPS) de Klebsiella e Pseudomonas no cordão umbilical em gêmeos e analisar a possível associação entre os títulos desses anticorpos e a ocorrência de infecção. MÉTODOS Estudo prospectivo transversal de uma coorte de recém-nascidos (RN) gemelares em 20 meses. Excluídos: malformação, infecção congênita ou materna. Variáveis estudadas: idade gestacional(IG); peso de nascimento(PN); título de anticorpos e episódios de infecção. Foram utilizados testes t-Student pareado, correlação de Spearman e equações de estimação generalizadas. RESULTADOS Elegíveis 59 pares de gêmeos, excluídos 4 e incluídos 55 pares (n=110RN). A IG foi 36±1,65semanas e o PN foi 2304,8±460g (média±DP). Concentrações de anticorpos dos RN(média±DP): IgG total=835,71±190,73 mg/dL, IgG anti-Streptococcus B=295,1±250,66 UA/mL, IgG anti-LPS Pseudomonas=280,04±498,66 UA/mL e IgG anti-LPS Klebsiella=504,75± 933,93UA/mL. Houve correlação positiva entre níveis de anticorpos maternos e aqueles observados nos RN (p<0,005). A transferência transplacentária de anticorpos maternos IgG total e IgG anti-LPS Pseudomonas foi significativamente menor em RN IG < 34semanas (p<0,05). Foram diagnosticados 5 RN com infecção. Os RN que apresentaram infecção tinham concentração de IgG total significativamente menor (p<0,05). CONCLUSÕES Na população estudada existe correlação entre os anticorpos maternos e os níveis de anticorpos no RN. Nos gêmeos menores que 34 semanas há menor transferência de IgG total e IgG anti-LPS Pseudomonas. Nos RN com infecção a concentração de IgG total é significativamente menor, o que demonstra a maior vulnerabilidade e risco de infecção dessa população e a importância da imunidade passiva transferida pela placenta.

Pseudomonas aeruginosa antibody response in cystic fibrosis decreases rapidly following lung transplantation.

BACKGROUND: Specific Pseudomonas aeruginosa (PA) precipitating immunoglobulin G antibodies in serum are correlated with PA biofilm infection and are used as diagnostic and prognostic markers in cystic fibrosis (CF). The aim of this study was to examine the change of PA antibody response in CF patients after bilateral sequential lung transplantation (LTx). METHODS: PA antibodies and airway bacteriology were retrospectively evaluated in 20 chronically infected CF patients, who underwent LTx between 2001 and 2016 at Rigshospitalet, Copenhagen. Yearly precipitin counts from one year before LTx and up to five years after LTx were compared. Monthly airway cultures were examined in the five-year period after LTx. In addition, crossed immunoelectrophoresis (CIE) were analysed for each patient for antigenic similarities from time of infection, pre-LTx and post-LTx. RESULTS: All patients experienced a significant drop in PA antibodies from one year pre-LTx to one year post-LTx (p < 0.0001). The PA antibody level did not differ between those, who became reinfected immediately after LTx, and those, who did not. No patients regained the high pre-LTx precipitin levels in the following five years. The antigenic specificities of the sera post-LTx were in each patient similar to the antigenic specificities at the beginning of infection indicating a decades long memory of their immune response like an "immunological fingerprint". CONCLUSIONS: After LTx a significant and continuous reduction in PA antibodies was observed. The reduction was independent of immediate reinfection after LTx. A novel three-factor explanatory model is presented.

Association of Serum TGF-ß1 Levels with Different Clinical Phenotypes of Cystic Fibrosis Exacerbation.

PURPOSE: Cystic Fibrosis (CF) is a multi-organ genetic disorder and Transforming Growth Factor (TGF-ß1) is a modifier gene which modulates lung pathology in CF. There is great phenotypic variability among CF patients who even have similar genotype. The aim of the present study was to associate the serum levels of TGF-ß1 with several clinical phenotypes of CF. METHODS: The diagnosed cases of CF were recruited and the blood sample was withdrawn at different time points: during exacerbation (n = 26), non-exacerbation (n = 9) and after antibiotic therapy (n = 11). The concentration of the total TGF-ß1 in serum was measured with commercial ELISA kit. The ΔF508 mutation was assessed by the Amplification Refractory Mutation System (ARMS-PCR). RESULTS: The levels of TGF-ß1 were increased in exacerbation phase (119.89 ± 29.64 ng/mL), infection with P. aeruginosa (121.8 ± 28.83 ng/mL) and in subjects with ΔF508 mutation (139.2 ± 19.59 ng/mL). The levels of TGF-ß1 in CF patients with Allergic Bronchopulmonary Aspergillosis (ABPA) (109.97 ± 27.71 ng/mL) were decreased as compared to CF patients without ABPA (123.55 ± 30.20 ng/mL). It was observed that the serum levels of TGF-ß1 were decreased significantly after antibiotic therapy (p < 0.05). CONCLUSIONS: The present study has determined that the serum levels of TGF-ß1 vary with the type of infections, ΔF508 CFTR mutation, presence of ABPA and response to therapy.

An unusual community-acquired invasive and multi systemic infection due to ExoU-harboring Pseudomonas aeruginosa strain: Clinical disease and microbiological characteristics.

Community-acquired Pseudomonas aeruginosa infections are rare. Most cases involve patients either with underlying immunosuppression or structural chronic lung diseases. We report here an atypical case of a severe community-acquired invasive infection due to a hypervirulent ExoU-producing strain, in an immunocompetent patient.

Dosage Regimens for Meropenem in Children with Pseudomonas Infections Do Not Meet Serum Concentration Targets.

There have been literature reports that some recommended meropenem dosage regimens may fail to meet therapeutic targets in some high-risk children and adults. We evaluated this observation in children using literature studies conducted in infants and children. Observed and, as necessary, simulated data from the literature were combined, yielding a data set of 288 subjects (1 day to ~ 17 years). A population pharmacokinetic model was fit to the data and then used to simulate the recommended dosing regimens and estimate the proportion of subjects achieving recommended target exposures. A two-compartment model best fit the data with weight, postnatal age, gestational age, and serum creatinine as covariates. The US Food and Drug Administration (FDA)-approved dosing regimens achieved targets in ~ 90% or more of subjects less than 3 months of age for organisms with minimum inhibitory concentration (MIC)'s of 2 and 4 mg/L; however, only 68.4% and 41.7% of subjects older than 3 months and weighing < 50 kg achieved target exposures for organisms with MIC's of 2 and 4 mg/L, respectively [Correction added on January 23, 2020, after first online publication: "> 3 months" corrected to "less than 3 months".]. Moreover, for subjects weighing more than 50 kg, only 41.3% and 17% achieved these respective targets. Simulation studies were used to explore the impact of changing dose, dosing interval, and infusion duration on the likelihood of achieving therapeutic targets in these groups. Our findings illustrate that current dosing recommendations for children over 3 months of age fail to meet therapeutic targets in an unacceptable fraction of patients. Further investigation is needed to develop new dosing strategies in these patients.

Dissociation of systemic and mucosal autoimmunity in cystic fibrosis.

BACKGROUND: Pseudomonas aeruginosa accounts for ~80% of cystic fibrosis (CF) airway infection. It shows a remarkable correlation with presence of autoantibody to bactericidal/permeability-increasing protein (BPI), which is not understood. In this study, we sought to better understand the characteristics of systemic and mucosal autoimmunity and their relation to humoral immunity to P. aeruginosa. METHODS: Antibody titers and isotypes to BPI and P. aeruginosa were characterized in sera and bronchoalveolar lavage (BAL) of adult and pediatric CF patients (n = 131), by ELISA and/or immunoblot. RESULTS: Serum BPI autoantibodies were common (~43%) in adult while rare (≪5%) in pediatric (≤18 yrs) CF patients. Serum BPI IgG autoantibodies were of high avidity and strongly correlated with anti-P. aeruginosa IgG responses. A parallel relationship was observed with IgA, but not IgG, responses in adult and pediatric CF patient in the BAL. Thus, BAL IgA anti-BPI antibodies were independent of age and correlated with the presence of BPI cleavage in BAL. CONCLUSIONS: IgG and IgA autoreactivity to BPI in CF patients was demonstrated in serum and BAL, respectively, and correlated with the isotype of the antibody response to P. aeruginosa. The co-occurrence of anti-BPI and anti-P. aeruginosa IgA in the BAL, but not serum, of pediatric CF patients suggests that BPI tolerance is broken in the P. aeruginosa-infected airway and that serologic IgG autoantibodies are later induced, potentially through a separate pathway. The relationship between P. aeruginosa, BPI cleavage, and IgA autoantibodies in the BAL suggests a role for cryptic epitope generation in the breaking of tolerance.

Sepsis 2019: What Surgeons Need to Know.

The definition of sepsis continues to be as dynamic as the management strategies used to treat this. Sepsis-3 has replaced the earlier systemic inflammatory response syndrome (SIRS)-based diagnoses with the rapid Sequential Organ Failure Assessment (SOFA) score assisting in predicting overall prognosis with regards to mortality. Surgeons have an important role in ensuring adequate source control while recognizing the threat of carbapenem-resistance in gram-negative organisms. Rapid diagnostic tests are being used increasingly for the early identification of multi-drug-resistant organisms (MDROs), with a key emphasis on the multidisciplinary alert of results. Novel, higher generation antibiotic agents have been developed for resistance in ESKCAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) organisms while surgeons have an important role in the prevention of spread. The Study to Optimize Peritoneal Infection Therapy (STOP-IT) trial has challenged the previous paradigm of length of antibiotic treatment whereas biomarkers such as procalcitonin are playing a prominent role in individualizing therapy. Several novel therapies for refractory septic shock, while still investigational, are gaining prominence rapidly (such as vitamin C) whereas others await further clinical trials. Management strategies presented as care bundles continue to be updated by the Surviving Sepsis Campaign, yet still remain controversial in its global adoption. We have broadened our temporal and epidemiologic perspective of sepsis by understanding it both as an acute, time-sensitive, life-threatening illness to a chronic condition that increases the risk of mortality up to five years post-discharge. Artificial intelligence, machine learning, and bedside scoring systems can assist the clinician in predicting post-operative sepsis. The public health role of the surgeon is key. This includes collaboration and multi-disciplinary antibiotic stewardship at a hospital level. It also requires controlling pharmaceutical sales and the unregulated dispensing of antibiotic agents globally through policy initiatives to control emerging resistance through prevention.

Elevated serum procalcitonin predicts Gram-negative bloodstream infections in patients with burns.

BACKGROUND: Many studies have suggested that procalcitonin can predict bloodstream infection and also distinguish between Gram-negative, Gram-positive and fungal infections after burn. However, up to now, there is no literature on serum procalcitonin level of multidrug-resistant pathogens and non-multidrug-resistant pathogens among Gram-negative bloodstream infections after burn. The purpose of this study is to explore the value of serum procalcitonin in identifying Gram-negative bloodstream infection in patients with febrile critical burn and then to investigate the difference of serum procalcitonin level between multidrug-resistant pathogens and non-multidrug-resistant pathogens among Gram-negative bloodstream infections after burn. METHODS: Patients with febrile critical burn admitted to the burn department of our hospital from 1 January 2014 to 1 August 2018 were retrospectively analysed. Patients with positive blood culture whose blood samples were collected for simultaneous blood culture and procalcitonin testing were enrolled. All strains were identified by an automatic microorganism analyser, and procalcitonin was analysed by an automatic electrochemiluminescence immunoassay. RESULTS: Overall, a total of 119 patients with positive blood culture met the inclusion criteria. There were 64 Gram-negative bacilli, 38 Gram-positive bacteria, 8 C. albicans and 9 polymicrobial bloodstream infections. The median procalcitonin value in Gram-negative bloodstream infections (2.67 ng/mL, interquartile range (IQR) 1.58-6.08) was significantly higher than that in Gram-positive bloodstream infections (1.04 ng/mL, IQR 0.35-1.60, P < 0.01), or C. albicans bloodstream infections (1.09 ng/mL, IQR 0.82-2.30, P < 0.05). Receiver operating characteristic curve (ROC) analysis showed that in addition to polymicrobial bloodstream infections, the area of procalcitonin under the curve distinguishing Gram-negative bloodstream infections from all other blood culture-positive bloodstream infections was 0.761, the best critical value was 1.73 ng/mL, the sensitivity was 73%, the specificity was 74%, the positive predictive value was 80%, the negative predictive value was 67%, The level of procalcitonin was significantly higher in multidrug-resistant Gram-negative bacilli (A. baumannii, Klebsiella pneumoniae and Pseudomonas aeruginosa) (2.76 ng/mL, IQR 2.01-7.76) than in non-multidrug-resistant bacilli (1.01 ng/mL, IQR 0.58-1.56, P < 0.01). CONCLUSION: Elevated serum procalcitonin can identify Gram-negative bloodstream infections in patients with febrile critical burn. In Gram-negative bloodstream infections, high procalcitonin levels may be associated with multidrug-resistant Gram-negative bacilli (A. baumannii, K. pneumoniae and P. aeruginosa).

Dynamic Emergence of Mismatch Repair Deficiency Facilitates Rapid Evolution of Ceftazidime-Avibactam Resistance in Pseudomonas aeruginosa Acute Infection.

Strains of Pseudomonas aeruginosa with deficiencies in DNA mismatch repair have been studied in the context of chronic infection, where elevated mutational rates ("hypermutation") may facilitate the acquisition of antimicrobial resistance. Whether P. aeruginosa hypermutation can also play an adaptive role in the more dynamic context of acute infection remains unclear. In this work, we demonstrate that evolved mismatch repair deficiencies may be exploited by P. aeruginosa to facilitate rapid acquisition of antimicrobial resistance in acute infection, and we directly document rapid clonal succession by such a hypermutating lineage in a patient. Whole-genome sequencing (WGS) was performed on nine serially cultured blood and respiratory isolates from a patient in whom ceftazidime-avibactam (CZA) resistance emerged in vivo over the course of days. The CZA-resistant clone was differentiated by 14 mutations, including a gain-of-function G183D substitution in the PDC-5 chromosomal AmpC cephalosporinase conferring CZA resistance. This lineage also contained a substitution (R656H) at a conserved position in the ATPase domain of the MutS mismatch repair (MMR) protein, and elevated mutational rates were confirmed by mutational accumulation experiments with WGS of evolved lineages in conjunction with rifampin resistance assays. To test whether MMR-deficient hypermutation could facilitate rapid acquisition of CZA resistance, in vitro adaptive evolution experiments were performed with a mutS-deficient strain. These experiments demonstrated rapid hypermutation-facilitated acquisition of CZA resistance compared with the isogenic wild-type strain. Our results suggest a possibly underappreciated role for evolved MMR deficiency in facilitating rapid adaptive evolution of P. aeruginosa in the context of acute infection.IMPORTANCE Antimicrobial resistance in bacteria represents one of the most consequential problems in modern medicine, and its emergence and spread threaten to compromise central advances in the treatment of infectious diseases. Ceftazidime-avibactam (CZA) belongs to a new class of broad-spectrum beta-lactam/beta-lactamase inhibitor combinations designed to treat infections caused by multidrug-resistant bacteria. Understanding the emergence of resistance to this important new drug class is of critical importance. In this work, we demonstrate that evolved mismatch repair deficiency in P. aeruginosa, an important pathogen responsible for significant morbidity and mortality among hospitalized patients, may facilitate rapid acquisition of resistance to CZA in the context of acute infection. These findings are relevant for both diagnosis and treatment of antimicrobial resistance emerging in acute infection in the hypermutator background and additionally have implications for the emergence of more virulent phenotypes.