Isolates ofP Research Articles

Aminoglycoside resistance in Pseudomonas aeruginosa: the contribution of the MexXY-OprM efflux pump varies between isolates

Introduction. Aminoglycoside antibiotics are widely used to treat infections of Pseudomonas aeruginosa. The MexXY-OprM efflux pump is an important contributor to aminoglycoside tolerance in P. aeruginosa reference strains and expression of the mexXY genes is repressed by the MexZ repressor protein. Direct investigation of the role of this efflux pump in clinical isolates is relatively limited.Hypothesis. The contribution of MexXY-OprM to P. aeruginosa aminoglycoside resistance is isolate-specific.Aim. To quantify the role of MexXY-OprM and its repressor, MexZ, in clinical isolates of P. aeruginosa. Methodology. The mexXY genes were deleted from ten clinical isolates of P. aeruginosa, and the mexZ gene from nine isolates. Antimicrobial susceptibility testing was carried out for commonly used antipseudomonal drugs on the engineered mutants and the isogenic wild-type isolates. RT-qPCR was used to measure expression of the mexX gene.Results. All but one of the mexXY mutants were more susceptible to the clinically used aminoglycosides tobramycin, gentamicin and amikacin but the degree to which susceptibility increased varied greatly between isolates. The mexXY mutants were also more susceptible to a fluoroquinolone, ciprofloxacin. In three isolates with functional MexZ, deletion of mexZ increased expression of mexXY and aminoglycoside tolerance. Conversely, deleting mexZ from six clinical isolates with mexZ sequence variants had little or no effect on expression of mexXY or on aminoglycoside susceptibility, consistent with the variants abolishing MexZ function. Genome analysis showed that over 50 % of 619 clinical isolates had sequence variants predicted to reduce the affinity of MexZ for DNA, likely increasing mexXY expression and hence efflux of aminoglycosides.Conclusion. Our findings show that the interplay between MexXY, MexZ and the level of mexXY expression plays an important role in aminoglycoside resistance in clinical isolates of P. aeruginosa but the magnitude of the contribution of this efflux pump to resistance is isolate-specific.

Activity of ceftolozane/tazobactam against Gram-negative isolates among different infections in Hong Kong: SMART 2017-2019.

Introduction. Ceftolozane/tazobactam was approved by the Drug Office, Department of Health, Government of the Hong Kong Special Administrative Region in 2017.Hypothesis/Gap Statement. Currently the in vitro activity of ceftolozane/tazobactam against Gram-negative pathogens isolated from patients in Hong Kong is undocumented. It would be prudent to document the activity of ceftolozane/tazobactam against Pseudomonas aeruginosa and Enterobacterales isolated from hospitalized patients in Hong Kong.Aim. To describe the in vitro susceptibility of recent clinical isolates of P. aeruginosa and the two most common Enterobacterales species (Klebsiella pneumoniae, Escherichia coli) cultured from respiratory tract, intra-abdominal, urinary tract and bloodstream infection samples to ceftolozane/tazobactam and other commonly used antimicrobial agents.Methodology. CLSI-defined broth microdilution MICs were determined and interpreted for Gram-negative isolates collected in Hong Kong from 2017 to 2019 by the SMART surveillance programme.Results. For P. aeruginosa, 96.7 % of isolates (n=210) were susceptible to ceftolozane/tazobactam, while susceptibility rates were ≥14 % lower to meropenem (82.9 % susceptible), cefepime (82.4 %), ceftazidime (81.4 %), piperacillin/tazobactam (76.7 %) and levofloxacin (79.5 %). Ceftolozane/tazobactam inhibited 85.7 % of piperacillin/tazobactam-nonsusceptible isolates, 80.6-82.1 % of cefepime-, ceftazidime- or meropenem-nonsusceptible isolates, and 75.9 % of multidrug-resistant (MDR) isolates of P. aeruginosa. For K. pneumoniae, 96.1 % of isolates (n=308) were susceptible to ceftolozane/tazobactam compared with meropenem (99.0 % susceptible), piperacillin/tazobactam (93.8 %), cefepime (85.7 %) and ceftazidime (85.4 %). The majority (88.3 %) of ESBL (extended-spectrum β-lactamase) non-CRE (carbapenem-resistant Enterobacterales) phenotype isolates of K. pneumoniae were susceptible to ceftolozane/tazobactam, comparable to piperacillin/tazobactam (85.0 %) but lower than meropenem (100 %). For E. coli, 98.5 % of isolates (n=609) were susceptible to ceftolozane/tazobactam compared to meropenem (99.3 % susceptible), piperacillin/tazobactam (96.7 %), ceftazidime (82.3 %) and cefepime (76.5 %). The majority (96.7 %) of ESBL non-CRE phenotype isolates of E. coli were susceptible to ceftolozane/tazobactam, similar to both meropenem (100 %) and piperacillin/tazobactam (94.5 %).Conclusions. Overall, >96 % of clinical isolates of P. aeruginosa, K. pneumoniae and E. coli collected in Hong Kong in 2017-2019 were susceptible to ceftolozane/tazobactam, while the activity of several commonly prescribed β-lactams was reduced, especially for P. aeruginosa. Continued surveillance of ceftolozane/tazobactam and other agents is warranted.

Phage Infection Restores PQS Signaling and Enhances Growth of a Pseudomonas aeruginosa lasI Quorum-Sensing Mutant.

ABSTRACTChemical communication between bacteria and between bacteria and the bacteriophage (phage) viruses that prey on them can shape the outcomes of phage-bacterial encounters. Quorum sensing (QS) is a bacterial cell-to-cell communication process that promotes collective undertaking of group behaviors. QS relies on the production, release, accumulation, and detection of signal molecules called autoinducers. Phages can exploit QS-mediated communication to manipulate their hosts and maximize their own survival. In the opportunistic pathogen Pseudomonas aeruginosa, the LasI/R QS system induces the RhlI/R QS system, and in opposing manners, these two systems control the QS system that relies on the autoinducer called PQS. A P. aeruginosa ΔlasI mutant is impaired in PQS synthesis, leading to accumulation of the precursor molecule HHQ, and HHQ suppresses growth of the P. aeruginosa ΔlasI strain. We show that, in response to a phage infection, the P. aeruginosa ΔlasI mutant reactivates QS, which, in turn, restores pqsH expression, enabling conversion of HHQ into PQS. Moreover, downstream QS target genes encoding virulence factors are induced. Additionally, phage-infected P. aeruginosa ΔlasI cells transiently exhibit superior growth compared to uninfected cells.IMPORTANCE Clinical isolates of P. aeruginosa frequently harbor mutations in particular QS genes. Here, we show that infection by select temperate phages restores QS, a cell-to-cell communication mechanism in a P. aeruginosa QS mutant. Restoration of QS increases expression of genes encoding virulence factors. Thus, phage infection of select P. aeruginosa strains may increase bacterial pathogenicity, underscoring the importance of characterizing phage-host interactions in the context of bacterial mutants that are relevant in clinical settings.

Genome evolution drives transcriptomic and phenotypic adaptation in Pseudomonas aeruginosa during 20 years of infection.

The opportunistic pathogen Pseudomonas aeruginosa chronically infects the lungs of patients with cystic fibrosis (CF). During infection the bacteria evolve and adapt to the lung environment. Here we use genomic, transcriptomic and phenotypic approaches to compare multiple isolates of P. aeruginosa collected more than 20 years apart during a chronic infection in a CF patient. Complete genome sequencing of the isolates, using short- and long-read technologies, showed that a genetic bottleneck occurred during infection and was followed by diversification of the bacteria. A 125 kb deletion, an 0.9 Mb inversion and hundreds of smaller mutations occurred during evolution of the bacteria in the lung, with an average rate of 17 mutations per year. Many of the mutated genes are associated with infection or antibiotic resistance. RNA sequencing was used to compare the transcriptomes of an earlier and a later isolate. Substantial reprogramming of the transcriptional network had occurred, affecting multiple genes that contribute to continuing infection. Changes included greatly reduced expression of flagellar machinery and increased expression of genes for nutrient acquisition and biofilm formation, as well as altered expression of a large number of genes of unknown function. Phenotypic studies showed that most later isolates had increased cell adherence and antibiotic resistance, reduced motility, and reduced production of pyoverdine (an iron-scavenging siderophore), consistent with genomic and transcriptomic data. The approach of integrating genomic, transcriptomic and phenotypic analyses reveals, and helps to explain, the plethora of changes that P. aeruginosa undergoes to enable it to adapt to the environment of the CF lung during a chronic infection.

Detection of Antiseptic Resistant Genes in Colistin- Resistant Pseudomonas aeruginosa and MDR Klebsiella penumoniae

Objectives: The aim of this study was to detect the existence of developments in resistance to biocide ofPseudomonas aeruginosa resist of colistin and in multi-drug resistance Klebsiella pneumonia in the hospitalenvironment. Materials and Methods: The study included 25 isolates of K. pneumoniae and 30 isolates ofP. aeruginosa. Isolated from different clinical and environmental samples in Baghdad hospitals. Antibioticsensitivity tests and their susceptibility to multiple antibiotic resistance and sensitivity tests were studied forthe most commonly used antiseptics at the preventive level (benzalkonium chloride). The test was carriedout using the micro dilution broth method, following Institution of Clinical and Laboratory Standardsguidelines, PCR was performed for detection of blaTEM, blaSHV , blaCTX-M , qacC/D, qac?E1and qacE betalactamase and antiseptic genes. Results: A high rate of multiple resistance to the most used antibioticswas observed, so the rate of resistance to all antibiotics that was used was 16.0% of P. aeruginosa and 4%of K. pneumoniae possesses comprehensive resistance to all antibiotics that were used and the resiste ofcolistin in P. aeruginosa was 36% .The prevalence of ESBLs was 36.0% and 48.0 % of P.aeruginosa andK.penomoniea respectively ,in addition to their strong ability to form biofilms 80 % in P.aeruginosa and94% in k.pneumoniea and their ability to resist Antiseptics . The percentage of resistance to antiseptic ofbenzilkonium chloride showed the highest concentration of P.aeruginosa was 33.3% and K. pneumoniae37.5% . The result of ESBL and antiseptic genes detection clarify, the percent of production genes were(10%), (40%) blaTEM; (6.66%),(56%)blaSHV ;(33%),(64%) blaCTX-M ;(70%),(44%) qacC/D; ( 80% ),(56%)qac?E1 in P.aeruginosa and K.pneumoniea respectively and no any isolate carried qacE gene. production ofextended spectrum ?-lactamase genes in addition to their strong ability to form biofilms 80 % in p.aeroginosaand 94% in k.pneumoniea and their ability to resist a antiseptics . The percentage of resistance to antisepticof benzilkonium chloride showed the highest concentration of P. aeruginosa was 33.3% and K. pneumoniae37.5%, the result of beta lactamase and antiseptic genes detection clarify PCR was performed for detectionof blaTEM, blaSHV , blaCTX-M , qacC/D, qac?E1and qacE beta lactamase and antiseptic genes . Conclusion:Our observations indicate that there is a significant correlation between the ability of bacteria to resistmultiple antibiotics in addition to their ability to resist the most commonly used antiseptics, due to theirphysiological nature and increased virulence factors.

Emergence of clinical isolates of Pseudomonas asiatica and Pseudomonas monteilii from Japan harbouring an acquired gene encoding a carbapenemase VIM-2.

Pseudomonas asiatica and Pseudomonas monteilii, belonging to the Pseudomonas putida phylogenetic group, are occasionally isolated from clinical samples, partly because they are often misidentified as P. putida in clinical laboratories. There are five reports describing carbapenem-resistant clinical isolates of these species. Carbapenem-resistant strains of P. asiatica and P. monteilii were isolated from stool samples. These isolates were sequenced using Illumina MiSeq and reidentified using average nucleotide identity (ANI) based on comparisons of their whole-genome sequences using the OrthoANI algorithm. The clonal relatedness of the isolates was assessed by pulse-field gel electrophoresis (PFGE). The size of plasmids conveying bla VIM-2 was examined by Southern blotting. A total of six carbapenem-resistant clinical isolates of P. asiatica (two isolates) and P. monteilii (four isolates) were obtained from stool samples from five patients in a Japanese hospital. All isolates harboured blaVIM-2. The two isolates of P. asiatica had a different pattern in the PFGE analysis, with both having a 23 kb plasmid. Of the four isolates of P. monteilii with similar patterns in the PFGE analysis, three had 320 kb plasmids and one had a 240 kb plasmid. The genetic environments of the 320/240 kb and 23 kb plasmids differed. The results strongly indicated that carbapenem-resistant P. asiatica and P. monteilii producing metallo-β-lactamase are emerging in Japan. This is the first report of carbapenem-resistant P. asiatica and P. monteilii in Japan.

Differences in in vitro interactions between macrophages with pathogenic and environmental strains of Prototheca.

Aim: We investigated the interactions between macrophage and different strains of Prototheca. Materials & method: J774A.1 macrophages were infected with clinical isolates of Prototheca ciferrii 18125 and P. ciferrii 50779 and environmental isolate ofP. ciferrii N71. Phagocytosis activities were compared by colony-forming unit assays at 3, 6 and 9 h after infection. Cytokine levels were detected by RT-PCR and ELISA. iNOS protein expression was examined by western blotting. Results: All P. ciferrii strains were phagocytized by macrophages but induced different levels of cytokines in macrophages. Moreover, infected byP. ciferrii N71upregulated much higher iNOS protein expression in J774A.1 than that infected by the clinical strains. Conclusion: Clinical and environmental P. ciferrii strains show differences in their interactions with macrophages, which may be attributed to their virulence.

Curcumin as Efflux Pump Inhibitor Agent for Enhancement Treatment Against Multidrug Resistant Pseudomonas aeruginosa Isolates

Pseudomonas aeruginosa is considered as a developing opportunistic nosocomial pathogen and is well-known for its multidrug resistance that can be efficiently treated by a combination of antibiotics andefflux pump inhibitors (EPI). Therefore, the purpose of this study was to investigate the effect of curcumin as an EPI for the enhancement of the effectiveness of antibiotics against multidrug resistant (MDR) isolates ofP. aeruginosa. Susceptibility patterns of suspected bacteria was determined using the disc diffusion method andresistant bacteria were identified using chromogenic agar and 16S rDNA. The effectsof curcuminon the enhancement of antibiotics’s activity was evaluated usingthe broth microdilution method.The susceptibility patterns for 50 (67.6%) suspectedP. aeruginosaisolates showed that 36 (72%) of these isolateswere resistant to one of the used antibiotics,whereasonly 21 (42%) were MDR. The highest percentage of resistance was observedtoceftazidime (66%) followed by ciprofloxacin and levofloxacin (40%). Only 35 isolates were specified by chromogenic agar and 16S rDNAas P. aeruginosa.The minimal inhibitory concentration (MIC) of 35 isolates for ciprofloxacin resistant was between 4 and128 µg/ml while for ceftazidime was between 64and 512 µg/ml. After the addition of 50 μg/ml curcumin with ciprofloxacin, there wasa significant increase in the sensitivity (p≤ 0.01) of 13 MDR P.aeroginosa isolates whereas no differences in the sensitivity to ceftazidime were recorded before and after addition ofcurcumin. In conclusion, the results of this study show that curcumin can decrease the MIC value of ciprofloxacin in MDR isolates of P. aeruginosaand can be used as a native compound to enhance the treatment of resistant isolates with ciprofloxacin.

Antimicrobial susceptibility of Pasteurella multocida isolated from sheep and pigs in Spain - Short communication.

Pasteurella multocida is responsible for economically important diseases in sheep and pigs. Antimicrobial susceptibility studies are essential for initiating rational and effective empirical therapy of P. multocida infections. In this study we investigated the antimicrobial susceptibility to 18 antimicrobial agents of 156 clinical isolates of P. multocida from sheep (n = 87) and pigs (n = 69) using the microdilution method. Both sheep and pig isolates exhibited low levels of resistance (≤ 15%) to ceftiofur, gentamicin, neomycin, spectinomycin, chlortetracycline, tulathromycin, florfenicol, danofloxacin, and enrofloxacin and trimethoprim/sulphamethoxazole, high resistance rates (> 15% up to 50%) to oxytetracycline, tilmicosin, and tiamulin, and very high resistance rates (> 50%) to tylosin tartrate, clindamycin, and sulphadimethoxine. However, sheep isolates exhibited significantly lower percentages of resistance and lower MIC90 values (P < 0.05) than pig isolates for most of the antimicrobials tested. In addition, sheep isolates exhibited also significantly lower phenotypic antimicrobial resistance diversity (8 resistotypes vs. 30 resistotypes). LAC-LIN-SUL-MAC was the resistotype most frequently detected in sheep (39.1%) and LIN-SUL-MAC in pig isolates (26.1%). The differences in susceptibility patterns could be influenced by the lower use of antimicrobials in the small ruminant industry compared with the pig farming industry.

Antimicrobial susceptibility and virulence genes of clinical and environmental isolates of Pseudomonas aeruginosa.

Background Pseudomonas aeruginosa is ubiquitous, has intrinsic antibiotic resistance mechanisms, and is associated with serious hospital-associated infections. It has evolved from being a burn wound infection into a major nosocomial threat. In this study, we compared and correlated the antimicrobial resistance, virulence traits and clonal relatedness between clinical and fresh water environmental isolates of P. aeruginosa.Methods219 P. aeruginosa isolates were studied: (a) 105 clinical isolates from 1977 to 1985 (n = 52) and 2015 (n = 53), and (b) 114 environmental isolates from different fresh water sources. All isolates were subjected to ERIC-PCR typing, antimicrobial susceptibility testing and virulence factor genes screening.ResultsClinical and environmental isolates of P. aeruginosa were genetically heterogenous, with only four clinical isolates showing 100% identical ERIC-PCR patterns to seven environmental isolates. Most of the clinical and environmental isolates were sensitive to almost all of the antipseudomonal drugs, except for ticarcillin/clavulanic acid. Increased resistant isolates was seen in 2015 compared to that of the archived isolates; four MDR strains were detected and all were retrieved in 2015. All clinical isolates retrieved from 1977 to 1985 were susceptible to ceftazidime and ciprofloxacin; but in comparison, the clinical isolates recovered in 2015 exhibited 9.4% resistance to ceftazidime and 5.7% to ciprofloxacin; a rise in resistance to imipenem (3.8% to 7.5%), piperacillin (9.6% to 11.3%) and amikacin (1.9% to 5.7%) and a slight drop in resistance rates to piperacillin/tazobactam (7.7% to 7.5%), ticarcillin/clavulanic acid (19.2% to 18.9%), meropenem (15.4% to 7.5%), doripenem (11.5% to 7.5%), gentamicin (7.7% to 7.5%) and netilmicin (7.7% to 7.5%). Environmental isolates were resistant to piperacillin/tazobactam (1.8%), ciprofloxacin (1.8%), piperacillin (4.4%) and carbapenems (doripenem 11.4%, meropenem 8.8% and imipenem 2.6%). Both clinical and environmental isolates showed high prevalence of virulence factor genes, but none were detected in 10 (9.5%) clinical and 18 (15.8%) environmental isolates. The exoT gene was not detected in any of the clinical isolates. Resistance to carbapenems (meropenem, doripenem and imipenem), β-lactamase inhibitors (ticarcillin/clavulanic acid and piperacillin/tazobactam), piperacillin, ceftazidime and ciprofloxacin was observed in some of the isolates without virulence factor genes. Five virulence-negative isolates were susceptible to all of the antimicrobials. Only one MDR strain harbored none of the virulence factor genes.ConclusionOver a period of 30 years, a rise in antipseudomonal drug resistance particularly to ceftazidime and ciprofloxacin was observed in two hospitals in Malaysia. The occurrence of resistant environmental isolates from densely populated areas is relevant and gives rise to collective anxiety to the community at large.

Prevalence of Extended Spectrum Betalactamase (ESBL) and Metallobetalactamase (MBL) Producing Pseudomonas aeruginosa and Acinetobacter baumannii Isolated from Various Clinical Samples.

This study was conducted with an objective to find the prevalence of extended spectrum betalactamase (ESBL) and metallobetalactamase (MBL) in P. aeruginosa and A. baumannii isolates obtained from various clinical samples. It was conducted in the Department of Microbiology, Adesh Institute of Medical Sciences and Research, Bathinda, over a period of two years from July 2014 to June 2016. Clinical specimens including urine, pus, blood, high vaginal swabs, respiratory samples, and various body fluids were processed and P. aeruginosa and A. baumannii isolates were identified by standard protocols. Antibiotic sensitivity testing for all isolates was done using Kirby-Bauer disc diffusion method. Disc potentiation test was performed to check ESBL and MBL production in these bacteria. Maximum ESBL positive isolates of P. aeruginosa were observed among pus samples and maximum MBL positive isolates were detected in tracheal aspirates. A. baumannii showed maximum positivity for ESBL and MBL production in endotracheal secretions. This study gives an alarming sign towards high prevalence of cephalosporin and carbapenem resistance due to production of extended spectrum betalactamases and metallobetalactamases, respectively. Early detection, stringent antibiotic policies, and compliance towards infection control practices are the best defenses against these organisms.

Enumerating Antibiotic Susceptibility Patterns of Pseudomonas aeruginosa Isolated from Different Sources in Dhaka City.

Background: Pseudomonas aeruginosa is a ubiquitous free-living bacterium and is responsible for severe nosocomial infections, life-threatening infections in immune compromised persons. The bacterium, along with its natural resistance, can acquire resistance to many antibiotics by a variety of methods.Method:Therefore, to compare the antibiotic sensitivity pattern of Pseudomonas aeruginosa, a total of seventeen isolates of P. aeruginosa were isolated from different sources; for example environmental sources, frozen food sources, clinical sources and medical waste materials. Isolates were confirmed to be P. aeruginosa by cultural and biochemical properties.Result:The isolates were tested against seventeen commercially available antibiotics to observe the antibiotic susceptibility patterns. Imipenem and meropenem were the most potent antibiotics (100% sensitivity) followed by amikacin and piperacillin with maximum sensitivity. Among others, gentamicin, ciprofloxacin, levofloxacin and aztreonam were found to be fairly active. A good number of isolates were intermediately resistant to ceftriaxone. The rates of resistance to aztreonam, cefotaxime and ceftazidime were 11.76%, 82.35% and 5.88% respectively. Complete resistance was observed against penicillin, ampicillin, cefixime and cefpodoxime.Conclusion:It can be concluded that the clinical isolates including isolate from medical waste, were multi-drug resistant than environmental and food isolates indicating the risk of transmission of resistance to the environmental isolates of P. aeruginosa.

Phylogenetics of a Fungal Invasion: Origins and Widespread Dispersal of White-Nose Syndrome.

ABSTRACTGlobalization has facilitated the worldwide movement and introduction of pathogens, but epizoological reconstructions of these invasions are often hindered by limited sampling and insufficient genetic resolution among isolates. Pseudogymnoascus destructans, a fungal pathogen causing the epizootic of white-nose syndrome in North American bats, has exhibited few genetic polymorphisms in previous studies, presenting challenges for both epizoological tracking of the spread of this fungus and for determining its evolutionary history. We used single nucleotide polymorphisms (SNPs) from whole-genome sequencing and microsatellites to construct high-resolution phylogenies of P. destructans. Shallow genetic diversity and the lack of geographic structuring among North American isolates support a recent introduction followed by expansion via clonal reproduction across the epizootic zone. Moreover, the genetic relationships of isolates within North America suggest widespread mixing and long-distance movement of the fungus. Genetic diversity among isolates of P. destructans from Europe was substantially higher than in those from North America. However, genetic distance between the North American isolates and any given European isolate was similar to the distance between the individual European isolates. In contrast, the isolates we examined from Asia were highly divergent from both European and North American isolates. Although the definitive source for introduction of the North American population has not been conclusively identified, our data support the origin of the North American invasion by P. destructans from Europe rather than Asia.

Targeting the Homoserine Dehydrogenase of Paracoccidioides Species for Treatment of Systemic Fungal Infections.

This work evaluated new potential inhibitors of the enzyme homoserine dehydrogenase (HSD) of Paracoccidioides brasiliensis, one of the etiological agents of paracoccidioidomycosis. The tertiary structure of the protein bonded to the analogue NAD, and l-homoserine was modeled by homology. The model with the best output was subjected to gradient minimization, redocking, and molecular dynamics simulation. Virtual screening simulations with 187,841 molecules purchasable from the Zinc database were performed. After the screenings, 14 molecules were selected and analyzed by the use of absorption, distribution, metabolism, excretion, and toxicity criteria, resulting in four compounds for in vitro assays. The molecules HS1 and HS2 were promising, exhibiting MICs of 64 and 32 μg · ml-1, respectively, for the Pb18 isolate of P. brasilensis, 64 μg · ml-1 for two isolates of P. lutzii, and also synergy with itraconazole. The application of these molecules to human-pathogenic fungi confirmed that the HSD enzyme may be used as a target for the development of drugs with specific action against paracoccidioidomycosis; moreover, these compounds may serve as leads in the design of new antifungals.

Identification of 1-((2,4-Dichlorophenethyl)Amino)-3-Phenoxypropan-2-ol, a Novel Antibacterial Compound Active against Persisters of Pseudomonas aeruginosa.

Antibiotics typically fail to completely eradicate a bacterial population, leaving a small fraction of transiently antibiotic-tolerant persister cells intact. Persisters are therefore seen to be a major cause of treatment failure and greatly contribute to the recalcitrant nature of chronic infections. The current study focused on Pseudomonas aeruginosa, a Gram-negative pathogen belonging to the notorious ESKAPE group of pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) and, due to increasing resistance against most conventional antibiotics, posing a serious threat to human health. Greatly contributing to the difficult treatment of P. aeruginosa infections is the presence of persister cells, and elimination of these cells would therefore significantly improve patient outcomes. In this study, a small-molecule library was screened for compounds that, in combination with the fluoroquinolone antibiotic ofloxacin, reduced the number of P. aeruginosa persisters compared to the number achieved with treatment with the antibiotic alone. Based on the early structure-activity relationship, 1-((2,4-dichlorophenethyl)amino)-3-phenoxypropan-2-ol (SPI009) was selected for further characterization. Combination of SPI009 with mechanistically distinct classes of antibiotics reduced the number of persisters up to 106-fold in both lab strains and clinical isolates of P. aeruginosa Further characterization of the compound revealed a direct and efficient killing of persister cells. SPI009 caused no erythrocyte damage and demonstrated minor cytotoxicity. In conclusion, we identified a novel antipersister compound active against P. aeruginosa with promising applications for the design of novel, case-specific combination therapies in the fight against chronic infections.

Urinary Concentrations of Colistimethate and Formed Colistin after Intravenous Administration in Patients with Multidrug-Resistant Gram-Negative Bacterial Infections.

Limited information is available on the urinary excretion of colistin in infected patients. This study aimed to investigate the pharmacokinetics of colistimethate sodium (CMS) and formed colistin in urine in patients with multidrug-resistant (MDR) Gram-negative bacterial infections. A pharmacokinetic study was conducted on 12 patients diagnosed with an infection caused by an extremely drug-resistant (XDR) P. aeruginosa strain and treated with intravenous CMS. Fresh urine samples were collected at 2-h intervals, and blood samples were collected predose (Cmin ss) and at the end of the CMS infusion (Cmax ss) for measurement of concentrations of CMS and formed colistin using high-performance liquid chromatography (HPLC). CMS urinary recovery was determined as the summed amount of CMS and formed colistin recovered in urine for each 2-h interval divided by the CMS dose. There were 12 enrolled patients, 9 of whom were male (75%). Data [median (range)] were as follows: age, 65.5 (37 to 86) years; colistimethate urinary recovery 0 to 6 h, 42.6% (2.9% to 72.8%); range of concentrations of colistin in urine, <0.1 to 95.4 mg/liter; Cmin ss and Cmax ss of colistin in plasma, 0.9 (<0.2 to 1.4) and 0.9 (<0.2 to 1.4) mg/liter, respectively. In 6/12 (50%) patients, more than 40% of the CMS dose was recovered in the urine within the first 6 h after CMS administration. This study demonstrated rapid urinary excretion of CMS in patients within the first 6 h after intravenous administration. In all but one patient, the concentrations of formed colistin in urine were above the MIC for the most predominant isolate of P. aeruginosa in our hospital. Future studies are warranted for optimizing CMS dosage regimens in urinary tract infection (UTI) patients.

Exopolysaccharide-Repressing Small Molecules with Antibiofilm and Antivirulence Activity against Pseudomonas aeruginosa.

Biofilm formation is a universal virulence strategy in which bacteria grow in dense microbial communities enmeshed within a polymeric extracellular matrix that protects them from antibiotic exposure and the immune system. Pseudomonas aeruginosa is an archetypal biofilm-forming organism that utilizes a biofilm growth strategy to cause chronic lung infections in cystic fibrosis (CF) patients. The extracellular matrix of P. aeruginosa biofilms is comprised mainly of exopolysaccharides (EPS) and DNA. Both mucoid and nonmucoid isolates of P. aeruginosa produce the Pel and Psl EPS, each of which have important roles in antibiotic resistance, biofilm formation, and immune evasion. Given the central importance of the EPS for biofilms, they are attractive targets for novel anti-infective compounds. In this study, we used a high-throughput gene expression screen to identify compounds that repress expression of the pel genes. The pel repressors demonstrated antibiofilm activity against microplate and flow chamber biofilms formed by wild-type and hyperbiofilm-forming strains. To determine the potential role of EPS in virulence, pel/psl mutants were shown to have reduced virulence in feeding behavior and slow killing virulence assays in Caenorhabditis elegans The antibiofilm molecules also reduced P. aeruginosa PAO1 virulence in the nematode slow killing model. Importantly, the combination of antibiotics and antibiofilm compounds increased killing of P. aeruginosa biofilms. These small molecules represent a novel anti-infective strategy for the possible treatment of chronic P. aeruginosa infections.

The Mechanism of Killing by the Proline-Rich Peptide Bac7(1–35) against Clinical Strains of Pseudomonas aeruginosa Differs from That against Other Gram-Negative Bacteria

Pseudomonas aeruginosa infections represent a serious threat to worldwide health. Proline-rich antimicrobial peptides (PR-AMPs), a particular group of peptide antibiotics, have demonstrated in vitro activity against P. aeruginosa strains. Here we show that the mammalian PR-AMP Bac7(1-35) is active against some multidrug-resistant cystic fibrosis isolates of P. aeruginosa By confocal microscopy and cytometric analyses, we investigated the mechanism of killing against P. aeruginosa strain PAO1 and three selected isolates, and we observed that the peptide inactivated the target cells by disrupting their cellular membranes. This effect is deeply different from that previously described for PR-AMPs in Escherichia coli and Salmonella enterica serovar Typhimurium, where these peptides act intracellularly after having been internalized by means of the transporter SbmA without membranolytic effects. The heterologous expression of SbmA in PAO1 cells enhanced the internalization of Bac7(1-35) into the cytoplasm, making the bacteria more susceptible to the peptide but at the same time more resistant to the membrane lysis, similarly to what occurs in E. coli The results evidenced a new mechanism of action for PR-AMPs and indicate that Bac7 has multiple and variable modes of action that depend on the characteristics of the different target species and the possibility to be internalized by bacterial transporters. This feature broadens the spectrum of activity of the peptide and makes the development of peptide-resistant bacteria a more difficult process.

Royal Jelly InhibitsPseudomonas aeruginosaAdherence and Reduces Excessive Inflammatory Responses in Human Epithelial Cells

Pseudomonas aeruginosa is a Gram-negative bacterium and causes respiratory infection especially in elderly patients. Royal jelly has been used worldwide as a traditional remedy and as a nutrient; however, the effect against P. aeruginosa is unclear. The aim of this study was to analyze antibacterial, antiadherent, and anti-inflammatory effects of royal jelly against P. aeruginosa. Wild-type strain PAO1 and clinical isolates of P. aeruginosa were used for antibacterial assay and antiadherent assay to abiotic surface and epithelial cells, which are pharynx (Detroit 562) and lung (NCI-H292) epithelial cells. In anti-inflammatory assay, epithelial cells were pretreated with royal jelly before bacterial exposure to investigate its inhibitory effect on interleukin (IL-8) and macrophage inflammatory protein-3α/CCL20 overproduction. Although royal jelly did not have antibacterial activity at concentration of 50% w/v, antiadherent activity was confirmed on the abiotic surface and epithelial cells under concentration of 25%. Pretreatment with royal jelly significantly inhibited overproduction of IL-8 and CCL20 from both cells. These results demonstrated that royal jelly inhibits P. aeruginosa adherence and protects epithelial cells from excessive inflammatory responses against P. aeruginosa infection. Our findings suggested that royal jelly may be a useful supplement as complementary and alternative medicine for preventing respiratory infection caused by P. aeruginosa.

Evaluate the Relationship Between Class 1 Integrons and Drug Resistance Genes in Clinical Isolates of Pseudomonas aeruginosa

Background:The prevalence of resistant Pseudomonas aeruginosa isolates is increasing and it is considered as one of the major public health concerns in the world. The association between integrons and drug resistance has been proven and evidences suggest that integrons are coding and responsible for dissemination of antibiotic resistance among P. aeruginosa isolates.Objective:This study is aimed to evaluate the relationship between class 1 integrons and drug resistance genes in clinical isolates of P. aeruginosa from burn patients.Methods:100 isolates of P. aeruginosa were collected from burn patients hospitalized in the skin ward of Shahid Motahari hospital and susceptibility testing was performed by disk diffusion method (Kirby-Bauer). Then DNA was extracted and PCR technique was performed for the detection of class 1 integrons and drug resistance genes. Then data was analyzed using SPSS software.Results:The most effective antibiotic was polymyxin B with sensitivity 100%, and the most resistance was observed to the ciprofloxacin (93%) and amikacin (67%), respectively. The maximum and lowest frequencies of drug resistance genes belonged to the aac (6 ') - 1, VEB-1 with prevalence rate 93% and 10%, respectively. The statistical Chi-square test did not find any significant correlation between class 1 integrons and drug resistance genes (p˃ 0.05).Conclusion:Although no significant correlation between class 1 integrons and drug resistance was observed, but the resistance rate to antibiotics tested among P. aeruginosa isolates was high. So, surveillance, optimization and strict consideration of antimicrobial use and control of infection are necessary.