exoU Genes Research Articles

Molecular detection of ExoU and ExoS virulence genes among clinical isolates of P. aeruginosa recovered from patients attending a private tertiary hospital in southwestern Nigeria

Molecular detection of ExoU and ExoS virulence genes among clinical isolates of P. aeruginosa recovered from patients attending a private tertiary hospital in southwestern Nigeria

Genetic profiling of virulence genes among Pseudomonas Aeruginosa isolates recovered from diseased chickens

Pseudomonas aeruginosa (P. aeruginosa) is regarded as an opportunistic organism for bird species. Also is one can be mentioned as an example of an environment-associated infection that results in severe disease in poultry farms, Indeed, we detected the incidence of P. aeruginosa in chickens and also discovered the implicated virulence genes and antibiotic-resistant mechanism. Two hundred cases were collected from broiler and layer chicken organs (heart blood, heart, and lungs) that suffered from some respiratory manifestation and septicemia in addition to yolk sacs from one-day-old chicks, which were examined bacteriologically and Serologically. The studied isolates were tested against eight antimicrobial discs. Bacterial DNA was analyzed using polymerase chain reaction (PCR) against six virulence genes (lasL, lasB toxA, exoU, exoT, and oprl genes). Eighteen isolates out of 200 (9%) of P. aeruginosa were defined. Serologically the isolates were related to more serogroup (O1,O4,O6, O11 and O12) P. aeruginosa. PCR defined the existence of P. aeruginosa by detection of the 16SRNA gene. All isolates showed 100% multidrug resistance (MDR) to more than one antimicrobial disc (sulfamethoxazole, erythromycin, tetracycline, and ampicillin). Conversely, those isolates exhibited 100% sensitivity to ciprofloxacin, and four of them showed sensitivity to norfloxacin with a percentage of (22.2%). In addition to 15 isolates showed resistance of 83.3% percent to streptomycin and nalidixic acid. By PCR test, all examined P. aeruginosa strains carried four virulence genes (lasI, toxA, exoU, and oprl). However, only three strains carried lasB, and four carried the exoT gene. In conclusion, we confirmed the presence of different factors of virulence for P. aeruginosa and MDR to more than one antimicrobial drug. So, restricting Pseudomonas infection in poultry requires strict biosecurity measures to control and eradicate the infection by P. aeruginosa

High prevalence of multidrug-resistant Pseudomonas aeruginosa carrying integron and exoA, exoS, and exoU genes isolated from burn patients in Ahvaz, southwest Iran: A retrospective study.

Pseudomonas aeruginosa as an opportunistic pathogen produces several virulence factors. This study evaluated the relative frequency of exoenzymes (exo) A, U and S genes and integron classes (I, II, and III) among multi-drug-resistant clinical P. aeruginosa isolates from burn patients in Ahvaz, southwest of Iran. In this cross-sectional study P. aeruginosa isolates were recovered from 355 wound samples. The antimicrobial susceptibility test was done by disk agar diffusion method on Muller-Hinton agar according to the Clinical and Laboratory Standards Institute. MDR isolates were defined if they showed simultaneous resistance to 3 antibiotics. Extensively drug-resistant was defined as nonsusceptibility to at least one agent in all but two or fewer antimicrobial categories. The presence of class I, II, and III integrons and virulence genes was determined using a PCR assay on extracted DNA. Overall, 145 clinical P. aeruginosa isolates were confirmed with biochemical and PCR tests. Overall, 35% (52/145) of the isolates were taken from males and 64% (93/145) from female hospitalized burn patients. The highest resistance rates of P. aeruginosa isolates to antibiotics were related to piperacillin 59% (n = 86/145) and piperacillin-tazobactam 57% (n = 83/145). A total of 100% of isolates were resistant to at least one antibiotic. MDR and XDR P. aeruginosa had a frequency of 60% and 29%, respectively. The prevalence of integron classes I, II, and III in P. aeruginosa was 60%, 7.58%, and 3.44%, respectively. IntI was more common in MDR and XDR P. aeruginosa isolates. In addition, 70(48%) of P. aeruginosa isolates did not harbor integron genes. Besides, exoA, exoS, and exoU in P. aeruginosa had a frequency of 55%, 55%, and 56%, respectively. It was found that P. aeruginosa as a potent pathogen with strong virulence factors and high antibiotic resistance in the health community can cause refractory diseases in burn patients.

Biocontrol of multidrug resistant pathogens isolated from fish farms using silver nanoparticles combined with hydrogen peroxide insight to its modulatory effect

This study was divided into two parts. The first part involved the isolation, and detection of the prevalence and antimicrobial resistance profile of Aeromonas hydrophila, Pseudomonas aeruginosa, and Vibrio species from Nile tilapia fish and marine aquatic water. One hundred freshly dead Nile tilapia fish were collected from freshwater aquaculture fish farms located in Al-Abbassah district, Sharkia Governorate, and 100 samples of marine aquatic water were collected from fish farms in Port Said. The second part of the study focused on determining the in vitro inhibitory effect of dual-combination of AgNPs-H2O2 on bacterial growth and its down regulatory effect on crucial virulence factors using RT-PCR. The highest levels of A. hydrophila and P. aeruginosa were detected in 43%, and 34% of Nile tilapia fish samples, respectively. Meanwhile, the highest level of Vibrio species was found in 37% of marine water samples. Additionally, most of the isolated A. hydrophila, P. aeruginosa and Vibrio species exhibited a multi-drug resistance profile. The MIC and MBC results indicated a bactericidal effect of AgNPs-H2O2. Furthermore, a transcriptional modulation effect of AgNPs-H2O2 on the virulence-associated genes resulted in a significant down-regulation of aerA, exoU, and trh genes in A. hydrophila, P. aeruginosa, and Vibrio spp., respectively. The findings of this study suggest the effectiveness of AgNPs-H2O2 against drug resistant pathogens related to aquaculture.

Molecular investigation of exoU and exoY virulence genes in Pseudomonas aeruginosa collected from hospitalized patients in North of Iran: A descriptive-analytical study

Objective: To investigate the frequency of exoU and exoY genes in patients with Pseudomonas aeruginosa infection. Methods: In this study, 100 clinical isolates of Pseudomonas aeruginosa were collected from patients hospitalized in educational- therapeutic hospitals and were identified using standard microbiological tests. Then, the antibiotic resistance pattern of the isolates was determined by the disk agar diffusion method. The bacterial DNAs were extracted by the alkaline lysis method. Finally, the presence of exoU and exoY genes was evaluated by the PCR test. Results: In this study, 47%, 72%, 29%, 39%, 40%, and 44% of the isolates were non-susceptible to piperacillin, aztreonam, ceftazidime, imipenem, tobramycin, and ciprofloxacin, respectively. In addition, 95% and 93% of the clinical isolates carried the exoU and exoY genes. Blood and fecal isolates had both virulence genes, while only one wound isolate had neither genes. Meanwhile, all urinary isolates contained the exoY gene and only one isolate lacked the exoU gene. Also, 88 isolates simultaneously had both exoU and exoY genes. Conclusions: High prevalence of exoU and exoY genes in this region indicates a significant role of type III secretion system in pathogenesis of Pseudomonas aeruginosa. The type III secretion system may be a suitable target to reduce the pathogenicity of this bacterium.

Pseudomonas aeruginosa from river water: antimicrobial resistance, virulence and molecular typing.

Pseudomonas aeruginosa isolates were recovered from surface river water samples in La Rioja region (Spain) to characterise their antibiotic resistance, molecular typing and virulence mechanisms. Fifty-two P. aeruginosa isolates were isolated from 15 different water samples (45.4%) and belonged to 23 different pulsed-field electrophoresis (PFGE) patterns. All isolates were susceptible to all antibiotics tested, except one carbapenem-resistant P. aeruginosa that showed a premature stop codon in OprD porin. Twenty-two sequence types (STs) (six new ones) were detected among 29 selected P. aeruginosa (one strain with a different PFGE pattern per sample), with ST274 (14%) being the most frequent one. O:6 and O:3 were the predominant serotypes (31%). Seven virulotypes were detected, being 59% exoS-exoY-exoT-exoA-lasA-lasB-lasI-lasR-rhlAB-rhlI-rhlR-aprA-positive P. aeruginosa. It is noteworthy that the exlA gene was identified in three strains (10.3%), and the exoU gene in seven (24.1%), exoS in 18 (62.1%), and both exoS and exoU genes in one strain. High motility ranges were found in these strains. Twenty-seven per cent of strains produced more biofilm biomass, 90% more pyorubin, 83% more pyocyanin and 65.5% more than twice the elastase activity compared with the PAO1 strain. These results highlight the importance of rivers as temporary reservoirs and sources of P. aeruginosa transmission, and show the importance of their epidemiological surveillance in the environment.

Prevalence and antibiogram of Pseudomonas aeruginosa and Staphylococcus aureus clinical isolates from burns and wounds in Duhok City, Iraq.

this study aimed to isolate P. aeruginosa and S. aureus, investigate the antimicrobial resistance of collected isolates, and investigate the distribution of exoU and mecA genes in P. aeruginosa and S. aureus isolates. Out of 150 samples, 32 isolates were identified as P. aeruginosa, 48 isolates were identified as S. aureus. All isolates were checked for AST. Then, a PCR was applied to detect exoU and mecA genes in P. aeruginosa and S. aureus. 12.0% and 29.3% of the samples showed co-isolates and single isolates of studied pathogens, respectively. Regarding burn samples, S. aureus was the most prevalent pathogen (38.0%, 38/100) among males (41.8%, 23/55), followed by P. aeruginosa (27.0%, 27/100) among females (28.9%, 13/45). The highest burn infection rates of S. aureus (50.0%) and P. aeruginosa (32.7%) were recorded among age groups (≥ 50) and (18-49), respectively. Comparatively, wound samples were less infected with these pathogens. P. aeruginosa isolates usually exhibited high resistance to gentamicin, tobramycin, and netilmicin, whereas, imipenem showed low resistance at 46.87%. S. aureus isolates were susceptible to trimethoprim-sulphamethoxazole and rifampin. 56.25% of P. aeruginosa isolates were exoU positive and 37.5% of S. aureus isolates were mecA positive. Results of the cefoxitin inhibition zone with mecA gene amplification, 33.3% isolates were MRSA, 4.2% isolates were nmrMRSA, and 62.5% isolates were MSSA. Most of the resistant isolates of P. aeruginosa carried the exoU gene, 80% resistant isolates to imipenem were exoU positive. S. aureus was more predominant than P. aeruginosa in burns and wounds infections.

Whole-genome sequencing reveals high-risk clones of Pseudomonas aeruginosa in Guangdong, China.

The ever-increasing prevalence of infections produced by multidrug-resistant or extensively drug-resistant Pseudomonas aeruginosa is commonly linked to a limited number of aptly-named epidemical 'high-risk clones' that are widespread among and within hospitals worldwide. The emergence of new potential high-risk clone strains in hospitals highlights the need to better and further understand the underlying genetic mechanisms for their emergence and success. P. aeruginosa related high-risk clones have been sporadically found in China, their genome sequences have rarely been described. Therefore, the large-scale sequencing of multidrug-resistance high-risk clone strains will help us to understand the emergence and transmission of antibiotic resistances in P. aeruginosa high-risk clones. In this study, 212 P. aeruginosa strains were isolated from 2 tertiary hospitals within 3 years (2018-2020) in Guangdong Province, China. Whole-genome sequencing, multi-locus sequence typing (MLST) and antimicrobial susceptibility testing were applied to analyze the genomic epidemiology of P. aeruginosa in this region. We found that up to 130 (61.32%) of the isolates were shown to be multidrug resistant, and 196 (92.45%) isolates were Carbapenem-Resistant Pseudomonas aeruginosa. MLST analysis demonstrated high diversity of sequence types, and 18 reported international high-risk clones were identified. Furthermore, we discovered the co-presence of exoU and exoS genes in 5 collected strains. This study enhances insight into the regional research of molecular epidemiology and antimicrobial resistance of P. aeruginosa in China. The high diversity of clone types and regional genome characteristics can serve as a theoretical reference for public health policies and help guide measures for the prevention and control of P. aeruginosa resistance.

Emergence of hypervirulent Pseudomonas aeruginosa pathotypically armed with co-expressed T3SS effectors ExoS and ExoU

Pseudomonas aeruginosa is a significant pathogen mainly causing healthcare-associated infections (HAIs). Newly emerging high-risk clones of P. aeruginosa with elevated virulence profiles furtherly cause severe community-acquired infections (CAIs). Usually, it is not common for P. aeruginosa to co-carry exoU and exoS genes, encoding two type III secretion system (T3SS) effectors. The pathogenicity mechanism of exoS+/exoU+ strains of P. aeruginosa remains unclear. Here, we provide detailed evidence for a subset of hypervirulent P. aeruginosa strains, which abundantly co-express and secrete the T3SS effectors ExoS and ExoU. The exoS+/exoU+P. aeruginosa strains were available to cause both HAIs and CAIs. The CAI-associated strains could elicit severe inflammation and hemorrhage, leading to higher death rates in a murine acute pneumonia model, and had great virulence potential in establishing chronic infections, demonstrating hypervirulence when compared to PAO1 (exoS+/exoU-) and PA14 (exoS-/exoU+). Both ExoS and ExoU were co-expressed and co-secreted in abundance in exoS+/exoU+ strains. Their abundant protein secretion could boost exoS+/exoU+ strains’ potentials for cytotoxicity in vitro and pathogenicity in vivo. Genomic evidence indicates that exoU acquisition is likely mediated by horizontal gene transfer (HGT) of the pathogenicity island PAPI-2, while deletion of exoU was sufficient to mitigate virulence in the exoS+/exoU+ strains. Furthermore, bioinformatics analysis showed that such exoS+/exoU+P. aeruginosa strains turned out to be widely distributed across the globe. Overall, the research provide detailed evidence for the high virulence and epidemicity of exoS+/exoU+ strains of P. aeruginosa, highlighting an urgent need for surveillance against these high-risk hypervirulent strains.

Decoding Genetic Features and Antimicrobial Susceptibility of Pseudomonas aeruginosa Strains Isolated from Bloodstream Infections.

Pseudomonas aeruginosa is a Gram-negative rod and an etiological factor of opportunistic infections. The infections of this etiology appear mostly among hospitalized patients and are relatively hard to treat due to widespread antimicrobial resistance. Many virulence factors are involved in the pathogenesis of P. aeruginosa infection, the coexistence of which have a significant impact on the course of an infection with a particular localization. The aim of this study was to assess the antimicrobial susceptibility profiles and the frequency of genes encoding selected virulence factors in clinical P. aeruginosa strains isolated from bloodstream infections (BSIs). The following genes encoding virulence factors of enzymatic activity were assessed: lasB, plC H, plC N, nan1, nan2, aprA and phzM. The frequency of the genes encoding the type III secretion system effector proteins (exoU and exoS) and the genes encoding pilin structural subunits (pilA and pilB) were also investigated. The occurrence of virulence-factor genes was assessed using polymerase chain reactions, each in a separate reaction. Seventy-one P. aeruginosa strains, isolated from blood samples of patients with confirmed bacteremia hospitalized at the University Hospital No. 1 of Dr. Antoni Jurasz in Bydgoszcz, Poland, were included in the study. All the investigated strains were susceptible to colistin, while the majority of the strains presented resistance to ticarcillin/clavulanate (71.8%), piperacillin (60.6 %), imipenem (57.7%) and piperacillin/tazobactam (52.1%). The presence of the lasB and plC H genes was noted in all the tested strains, while the plC N, nan2, aprA, phzM and nan1 genes were identified in 68 (95.8%), 66 (93.0%), 63 (88.7%), 55 (77.5%) and 34 (47.9%) isolates, respectively. In 44 (62.0%) and 41 (57.7%) strains, the presence of the exoU and exoS genes was confirmed, while the pilA and pilB genes were noted only in 14 (19.7%) and 3 (4.2%) isolates, respectively. This may be due to the diverse roles of these proteins in the development and maintenance of BSIs. Statistically significant correlations were observed between particular gene pairs’ coexistence (e.g., alkaline protease and neuraminidase 2). Altogether, twenty-seven distinctive genotypes were observed among the studied strains, indicating the vast variety of genetic compositions of P. aeruginosa strains causing BSIs.

Characterization of Uropathogenic Pseudomonas aeruginosa: Serotypes, Resistance Phenotypes, and Virulence Genotypes

Pseudomonas aeruginosa is a major cause of urinary tract infections. This organism has extended resistance to antimicrobials along with multiple virulence factors, making it difficult to treat. In this study, 49 isolates from urine samples were identified as P. aeruginosa and serotyped by the slide agglutination method. The sensitivity of isolates against 10 antipseudomonal drugs was determined. Phenotypically, lipase, protease, hemolysin, and biofilm production were detected. Genes for the type III secretion system, elastase B, and exotoxin A were detected by PCR. Serotype O11 was the most predominant serotype among test isolates. High levels of resistance were observed against ceftazidime, cefepime, piperacillin, and piperacillin/tazobactam while 10.2% of isolates were resistant to amikacin. MDR was detected in 20.4% of the isolates and was significantly associated with strong biofilm producers. About 95.9% and 63.3% of P. aeruginosa isolates had proteolytic and lipolytic activity, respectively. Among the genes detected, the exoY gene was the most prevalent gene (79.6%), while the exoU gene was the least frequent one (10.2%). toxA and lasB genes were amplified in 63.27% and 75.5% of the isolates, respectively. In addition, the exoU gene was significantly associated with MDR isolates. The high incidence of exoS, exoT, exoY, lasB, and toxA genes in uropathogenic P. aeruginosa implies that these genes can be considered markers for virulent isolates. Furthermore, the coexistence of exoU and exoS genes, even in 6% of isolates, poses a significant treatment challenge because those isolates possess both the invasive and cytotoxic properties of both effector proteins.

The antagonistic effect and protective efficacy of gram-positive probiotics Bacillus coagulans to newly identified pathogens Pseudomonas aeruginosa in crucian carp Carassius auratus gibelio

Pseudomonas aeruginosa is a common gram-negative pathogen belonging to the Aeromonas genus. In the present study, dominant bacteria were isolated from diseased crucian carp (Carassius auratus gibelio). The isolate was identified as P. aeruginosa based on biochemical identification and 16S rDNA sequencing. In the histological observation of diseased C. auratus, a large number of vacuoles and inflammatory infiltration appeared in the liver, the structure of intestinal villi was loose, and a large number of inflammatory cells and vacuoles were found in gill. Five virulence genes exoU, exoS, pcrV, exoT and exoY were identified in this P. aeruginosa isolate. Drug sensitivity testing showed that the isolate was susceptible to norfloxacin, ofloxacin and ciprofloxacin. The supernatant of Bacillus coagulans NJ3 exhibited a significant bactericidal effect on isolated P. aeruginosa based on the inhibition zone experiment. The accumulative mortality of C. auratus caused by P. aeruginosa infection could be significantly reduced by adding B. coagulans NJ3 into the aquaculture water. These results indicated that B. coagulans NJ3 could be used as probiotics to prevent the infections of P. aeruginosa in C. auratus. The present study provided a scientific basis for the identification of P. aeruginosa, and treatment for this pathogen infections in fish by using B. coagulans NJ3.

Bloodstream Infections Caused by Klebsiella pneumoniae Carbapenemase-Producing P. aeruginosa Sequence Type 463, Associated With High Mortality Rates in China: A Retrospective Cohort Study.

ObjectivesRecently, KPC-producing P. aeruginosa has rapidly emerged and expanded in East China. Here we described the clinical impact and characteristics of bloodstream infections (BSIs) from the dominant KPC-producing CRPA belonging to Sequence Type (ST) 463.MethodsRetrospective cohort study was performed with CRPA BSI cases from 2019 to 2020 in a hospital in East China. Clinical characteristics, risk factors, and all-course mortality were evaluated. All CRPA isolates had whole-genome sequencing, antimicrobial susceptibility testing, and serum resistance assay. Representative isolates were tested for virulence in a Galleria mellonella infection model.ResultsAmong the 50 CRPA BSI cases, ST463 predominated (48.0%). In multivariate analysis, we found three independent risk factors for fatal outcome: KPC carriage (OR 4.8; CI95% 1.0-23.7; P = 0.05), Pitt bacteremia score (OR 1.3; CI95% 1.0-1.6; P = 0.02), and underlying hematological disease (OR 8.5; CI95% 1.6-46.4; P = 0.01). The baseline clinical variables were not statistically different across STs, however the 28-day mortality was significantly higher in ST463 cases than that in non-ST463 cases (66.7% vs 33.3%, P = 0.03). ExoU and exoS virulence genes coexisted in all ST463 isolates, and the carbapenem resistant gene bla KPC were produced in almost all ST463 isolates, significantly higher than in the non-ST463 group(95.8% vs 7.7%, P<0.001). ST463 CRPA isolates also showed higher resistance rates to antipseudomonal cephalosporins, monobactam, and fluoroquinolones. And ST463 CRPA was confirmed hypervirulence in the larvae model. The genome of one ST463 CRPA strain showed that the bla KPC-2 gene was the sole resistance gene located on a 41,104bp plasmid pZYPA01, carried on a 7-kb composite transposon-like element flanked by two IS26 elements (IS26–Tn3-tnpA–ISKpn27–bla KPC-2–ISKpn6–IS26). Plasmid from various species presented core bla KPC-2 was franked by mobile genetic element ISKpn27 and ISKpn6. ConclusionsIn the ST463 CRPA BSI cohort, the mortality rates were higher than those in the non-ST463 CRPA BSI. The ST463 CRPA clone coharboring the bla KPC and exoU/exoS genes emerged and spread in East China, which might develop to a new threat in the clinic. Our results suggest that the surveillance of the new high-risk clone, ST463 CRPA, should be strengthened in China, even worldwide in the future.

Virulence genotyping of drug resistant Pseudomonas aeruginosa clinical isolates in Egypt using multiplex PCR

Pseudomonas aeruginosa is a serious Gram- negative pathogen capable of causing several nosocomial and community acquired infections. It produces many toxins such as: exotoxin A, pyocyanin, elastase and type III secretion system proteins. P. aeruginosa is genotyped according to the produced type III secretion system protein into invasive and cytotoxic genotypes. This study aimed to determine the prevalent virulence genotypes as well as the prevalence of five virulence genes using multiplex PCR. The antimicrobial resistance of the collected 134 P. aeruginosa isolates was detected using the disc diffusion method. The five exotoxin genes were detected by conventional multiplex PCR. Seventy percent of isolates were multidrug resistant. The phz, lasB, toxA, exoS and exoU genes were detected in 93%, 90%, 88%, 57% and 43% of isolates, respectively. Fifty two percent of isolates represented the invasive genotype and 38.8% represented the cytotoxic genotype. Clinical P. aeruginosa strains exhibit high antimicrobial resistance. The invasive and cytotoxic genotypes are associated with different types of clinical specimens and both genotypes can co-exist in the same isolates. Therefore, following up the antibiotic resistance of P. aeruginosa and its virulence factors and produced toxins is recommended to judge the bacterial virulence in outbreaks and epidemiologic studies.

Prevalence, Virulence, Antimicrobial Resistance, and Molecular Characterization of Pseudomonas aeruginosa Isolates From Drinking Water in China.

Pseudomonas aeruginosa is an important opportunistic pathogen and remains a major threat to the microbial safety of drinking water. There is a lack of comprehensive data on P. aeruginosa contamination in drinking water in China. Therefore, this study aimed to determine the prevalence, genetic diversity, virulence genes, and antimicrobial resistance of P. aeruginosa isolated from mineral water and spring water in China. From January 2013 to January 2014, 314 drinking water samples were collected from 23 cities in China. Of the collected samples, 77 (24.5%) were contaminated with P. aeruginosa, and these comprised 34 raw water (30.4%), 39 activated carbon-filtered water (30.6%), and four final water product (3.9%). A total of 132 P. aeruginosa isolates were obtained, and all of them showed the presence of virulence genes, with the detection rates of ExoU, ExoS, phzM, toxA, and lasB genes being 7.6, 86.3, 95.5, 89.4, and 100%, respectively. All isolates were sensitive to the 14 antibiotics (ciprofloxacin, levofloxacin, ofloxacin, norfloxacin, gentamicin, tobramycin, amikacin, polymyxin B, imipenem, meropenem, aztreonam, ceftazidime, cefepime, and piperacillin/tazobactam) tested. The 132 isolates were categorized into 42 sequence types according to multilocus sequence typing, and ST235 accounted for 8.3% (11) of the total isolates. Thus, this study provides comprehensive data on the prevalence and characteristics of P. aeruginosa in drinking water in China and can aid in developing preventive measures against contamination during the drinking water treatment process.

High mortality by nosocomial infections caused by carbapenem-resistant P. aeruginosa in a referral hospital in Brazil: facing the perfect storm.

Introduction. Carbapenem-resistant Pseudomonas aeruginosa is responsible for increased patient mortality.Gap Statement. Five and 30 day in-hospital all-cause mortality in patients with P. aeruginosa infections were assessed, followed by evaluations concerning potential correlations between the type III secretion system (TTSS) genotype and the production of metallo-β-lactamase (MBL).Methodology. This assessment comprised a retrospective cohort study including consecutive patients with carbapenem-resistant infections hospitalized in Brazil from January 2009 to June 2019. PCR analyses were performed to determine the presence of TTSS-encoding genes and MBL genes.Results. The 30-day and 5-day mortality rates for 262 patients were 36.6 and 17.9 %, respectively. The unadjusted survival probabilities for up to 5 days were 70.55 % for patients presenting exoU-positive isolates and 86 % for those presenting exo-negative isolates. The use of urinary catheters, as well as the presence of comorbidity conditions, secondary bacteremia related to the respiratory tract, were independently associated with death at 5 and 30 days. The exoS gene was detected in 64.8 % of the isolates, the presence of the exoT and exoY genes varied and exoU genes occurred in 19.3 % of the isolates. The exoU genotype was significantly more frequent among multiresistant strains. MBL genes were not detected in 92 % of the isolates.Conclusions. Inappropriate therapy is a crucial factor regarding the worse prognosis among patients with infections caused by multiresistant P. aeruginosa, especially those who died within 5 days of diagnosis, regardless of the genotype associated with TTSS virulence.

High prevalence of atypical virulotype and genetically diverse background among Pseudomonas aeruginosa isolates from a referral hospital in the Brazilian Amazon.

Pseudomonas aeruginosa is an opportunistic pathogen causing different types of infections, particularly in intensive care unit patients. Characteristics that favor its persistence artificial environments are related to its high adaptability, wide arsenal of virulence factors and resistance to several antimicrobial classes. Among the several virulence determinants, T3SS stands as the most important due to the clinical impact of exoS and exoU genes in patient's outcome. The molecular characterization of P. aeruginosa isolates helps in the comprehension of transmission dynamics and enhance knowledge of virulence and resistance roles in infection process. In the present study, we investigated virulence and resistance properties and the genetic background of P. aeruginosa isolated from ICUs patients at a referral hospital in Brazilian Amazon. A total of 54 P. aeruginosa isolates were characterized by detecting 19 virulence-related genes, antimicrobial susceptibility testing, molecular detection of β-lactamase-encoding genes and genotyping by MLST and rep-PCR. Our findings showed high prevalence of virulence-related markers, where 53.7% of the isolates presented at least 17 genes among the 19 investigated (P = 0.01). The rare exoS+/exoU+ cytotoxic virulotype was detected in 55.6% of isolates. Antimicrobial susceptibility testing revealed percentages of antibiotic resistance above 50% to carbapenems, cephalosporins and fluoroquinolones associated to MDR/XDR isolates. Isolates harboring both blaSPM-1 and blaOXA genes were also detected. Genotyping methods demonstrated a wide genetic diversity of strains spread among the different intensive care units, circulation of international MDR/XDR high-risk clones (ST111, ST235, ST244 and ST277) and emergence of seven novel MLST lineages. Finally, our findings highlight the circulation of strains with high virulence potential and resistance to antimicrobials and may be useful on comprehension of pathogenicity process, treatment guidance and establishment of strategies to control the spread of epidemic P. aeruginosa strains.

Molecular detection of type III secretory toxins in Pseudomonas aeruginosa isolates

Pseudomonas aeruginosa has been known as a common unscrupulous pathogen that reasons cause nosocomial infections in patients with immunocompromise. Infection with multi-drug resistant Pseudomonas aeruginosa infection in many patients is a public health problem. The bacterium causes urinary tract infections, respiratory tract infections, skin inflammation and inflammation, soft tissue infections, bacteremia, bone and joint infections, gastrointestinal infections and various systemic infections, especially in patients with severe burns, cancer and AIDS, whose immune systems are suppressed. Among diverse virulence factors, the type III secretion system is known as a significant agent in virulence and development of antimicrobial resistance in P. aeruginosa. A total of 50 isolates of P. aeruginosa were gathered from burn wound and milk specimens. Documentation and antimicrobial susceptibility evidence were performed using the VITEK 2 system. Multiplex PCR was done to detect the secretion toxins-encoding genes. Out of 50 samples: 45/225 (20%) burn wound and 6/120 (5%) raw milk samples were found positive for P. aeruginosa. The multiplex PCR analysis of ExoT and ExoY genes showed that all P. aeruginosa 50 (100%) were positive. The occurrence of the ExoS and ExoU genes was 97.7% and 86.6% among clinical isolates while none of the raw milk isolates harbored the ExoU gene and 60% of them carried the ExoS gene. The results found 20 (40%) of isolates were multidrug resistance and the most effective antibiotics against clinical isolates were Ciprofloxacin and Meropenem. The aim of this study was to prevalence the exotoxin genes encoded type III secretion system and pattern of antimicrobial susceptibility of P. aeruginosa isolated from clinical and raw milk specimens.

Detection of T3SS, oprI, aprA, and pvdA Genes in Clinical Isolates of Pseudomonas aeruginosa obtained from Wound Samples

Background: Pseudomonas aeruginosa employs a varied number o f virulence determinants, predominantly porins, type III secretion system (T3SS), alkaline protease and pigments to manipulate the host to establish infections. These factors contribute significantly to virulence in P. aeruginosa and are worrisome. This study is aimed at identifying the virulence genes in P. aeruginosa isolates from wound swabs of patients at two tertiary hospitals in Osun State, Nigeria. Methods: Altogether , 237 participants consisting of 133 fr o m State Hospital, Osogbo and 104 from General Hospital, Iwo with different types of wounds were enrolled. Swabs from the various wound types were collected, grown on cetrimide agar, and recovered isolates identified using conventional biochemical tests. Chromosomal DNA was extracted by thermal lysis and subjected to polymerase chain reaction using specific primers to affirm biochemical identification and detect the presence of ExoT, ExoS, ExoU, ExoY, oprI, aprA, and pvdA genes. Results: Sixty-one (25.7%) P. aeruginosa isolates were recovered in the study. Based on the different wound types, the highest recovery was from surgical sites of caesarian sections (CS) (37.7%; 23/61) followed by trauma sustained from motorcycle and automobile accidents (36.1%; 22/61) and others wound types (26.2%; 16/71). Fifty-nine of the 61 recovered isolates were successfully amplified by PCR primers that targets P. aeruginosa parugin gene. Of these 59 PCR confirmed P. aeruginosa, the oprI gene was detected in 74.6% (n = 44/59) of isolates ; 18 from Osogbo and 26 from Iwo. No bands were detected for the other genes in all 59 isolates analysed. Conclusion: The prevalence of P. aeruginosa w as highest from surgical sites of caesarian sections, with the rates from Iwo higher than that from Osogbo. Detection of oprL gene in 74.6% of strains is significant as it’s interaction with the peptidoglycan plays a part in the maintenance of the structural integrity of the cell, and may cause infections that impair wound healing.

The Population Structure of Pseudomonas aeruginosa Is Characterized by Genetic Isolation of exoU+ and exoS+ Lineages.

The diversification of microbial populations may be driven by many factors including adaptation to distinct ecological niches and barriers to recombination. We examined the population structure of the bacterial pathogen Pseudomonas aeruginosa by analyzing whole-genome sequences of 739 isolates from diverse sources. We confirmed that the population structure of P. aeruginosa consists of two major groups (referred to as Groups A and B) and at least two minor groups (Groups C1 and C2). Evidence for frequent intragroup but limited intergroup recombination in the core genome was observed, consistent with sexual isolation of the groups. Likewise, accessory genome analysis demonstrated more gene flow within Groups A and B than between these groups, and a few accessory genomic elements were nearly specific to one or the other group. In particular, the exoS gene was highly overrepresented in Group A compared with Group B isolates (99.4% vs. 1.1%) and the exoU gene was highly overrepresented in Group B compared with Group A isolates (95.2% vs. 1.8%). The exoS and exoU genes encode effector proteins secreted by the P. aeruginosa type III secretion system. Together these results suggest that the major P. aeruginosa groups defined in part by the exoS and exoU genes are divergent from each other, and that these groups are genetically isolated and may be ecologically distinct. Although both groups were globally distributed and caused human infections, certain groups predominated in some clinical contexts.