algD Gene Research Articles

Regulate the Risk Factors of Pseudomonas aeruginosa by some of Sigma Factor Genes

In this research, a study was conducted isolating and identification of Pseudomonas aeruginosa bacteria from clinical samples from various pathological conditions such as burns , otitis media , urinary tract infection , wounds, eye infection, blood infection, catheter infection, throat , vaginal infection and surgical. The present study includes 313 samples collected from patients who attend Alsader Medical City and Al- Hakim General Hospital during the period extended From September 2023 to December 2023 ( men and women ) with age groups between (1 -77) years. The identification of bacterial isolates were detected by classical and molecular technique (PCR), were the frequency among males 163 (52.07% ) was more than that in female 150 (47.92). The samples distribution is made according age group , it appears high for the following high (15.97) and (12.77) with group (16-30) years of male and female respectively . The results revealed that 57/313 specimens of the total number of samples are Pseudomonas aeruginosa by identified according to their morphological, physiological and biochemical properties. Also with molecular identification by using four primers wre the results showed 16S rRNA gene was detected in 57/57 (100%) P. aeruginosa isolates , the Las biofilm gene was detected in 43/57 (75.43%) , the algD gene was detected in 41/57 (71.92%) and 41/57 samples show positive results with the oprL gene. Six types of antibiotics were also tested to determine the resistance of the samples to them.

Study on molecular epidemiology of carbapenem resistant Pseudomonas aeruginosa and related genes of quorum sensing signal system

This study aims to investigate the drug resistance, regulation mechanism of quorum sensing system, expression of related virulence genes, and epidemiological characteristics of carbapenem-resistant Pseudomonas aeruginosa (CRPA).In this study, Polymerase chain reaction amplification was performed to evaluate carbapenemase genes, OprD2 gene, quorum sensing system, and related virulence genes. Bacterial genotypes were analyzed using multilocus sequence typing and evolutionary analysis was conducted based on the goeBURST algorithm. The results demonstrated that a total of 47 CRPA strains were collected in this study, primarily from respiratory specimens in the ICU. Drug sensitivity results showed that the resistance rates of the 47 CRPA strains were highest for imipenem (97.87 %). The loss of OprD2 may be the main factor contributing to carbapenem resistance in our hospital's CRPA strains.All isolates tested positive for the quorum sensing system genes lasI and rhlI/R, and the virulence gene lasB was detected in all isolates, while the algD gene was detected in 19.15 % of the isolates. Among the 47 strains, 6 were untypeable, and the 41 strains with 28 different sequence types were clustered into three clonal complexes (BG1, BG2, and BG3).In conclusion, the CRPA isolates from our hospital exhibit high genetic diversity, with the deletion of the OprD2 gene possibly being the primary determinant of carbapenem resistance in Pseudomonas aeruginosa.Moreover, Las and RhI systems play a key role in quorum sensing signal system. Further research and development of drugs targeting quorum sensing signaling system may provide valuable guidance for the treatment of CRPA.

Identification of algD and oprL genes in Pseudomonas aeruginosa isolated from multiple environments and different local sources in Iraq

The aim of this study was to investigate the spread of the genes algD and oprL in P. aeruginosa within the genetic structure of environmental sources of isolates and various clinical studies, and to evaluate a successful alternative for diagnosing Pseudomonas aeruginosa (P. aeruginosa) among various sources. In this study, 100 samples were collected from various clinical and environmental sources. Twenty-eight isolates of P. aeruginosa bacteria were isolated. Nine pathological samples were taken from wounds, burns, blood, and ear infections in three governorates: Salah al-Din, Baghdad, and Kirkuk. Five isolates of P. aeruginosa bacteria were collected from contaminated water from the Salah Al-Din and Baghdad governorates. The isolates were diagnosed phenotypically through the colony's shape, colour, odour and interaction with Gram stain. Additionally, the VITEK 2 system was used to confirm the diagnosis of the isolates. The results show that all the isolates belonged to the bacteria P. aeruginosa. The results of the molecular diagnosis using a polymerase chain reaction (PCR) technique for three genes with specialised primers (16SrRNA, algD, and oprL) show that most of the bacterial isolates from different sources belong to the type P. aeruginosa because they contain the specific gene 16SrRNA except for five isolates (M5, M7, M8, M9 and W1). Four of these belong to isolates with a pathogenic source and one was isolated from water. The results of the PCR for the algD gene show that all isolates contain the gene except for four diseased samples. Samples M5, M7, M8, and M9 were without the gene, while all isolates had the oprL gene. A sequencing analysis of the DNA was then performed for the isolates that were not associated with the specialised primers of the above genes to ensure their genera (M5, M7, M8, and M9), in addition to a sample of 15 isolated from soil (S6) that was associated with all special primers for the same genes for the purpose of comparison. The results show that isolates M5, M7, M8 and M9 were diseased isolates belonging to Escherichia coli, while the 15 isolated from soil (S6) were diagnosed as P. aeruginosa.

Determine Biofilm Genes in Pseudomonas Aeruginosa Isolated from Clinical and Environmental Samples

Objective: Isolates of Pseudomonas aeruginosa are an extremely adaptable bacterium that causes opportunistic diseases because of its varied metabolic pathways, genes, virulence factors, and considerable antibiotic resistance. Methods: A total of 293 samples were collected from different places: 193 samples (% 65.87) of human samples and 100 samples (% 34.13) of wastewater samples in the period between 3rd September to 15th November 2023). Bacterial isolates were identified according to microscopic, cultural, and genetic characteristics. Antibiotic susceptibility of bacterial isolates was determined against twelve of the selected antibiotics. The biofilm production was done by using phenotypic ways (Congo red agar and Microtiter plate methods) as well as genotypic ways by detection of biofilm genes (algD, pelf, and pslD genes). Results: Hundred-forty eight bacterial isolates were obtained, and sixty of these isolates were identified as Pseudomonas spp. (40.9%), twenty-six isolates of E. coli (17.9%), seventeen isolates of K. pneumoniae (11.3%), and forty-five isolates were beyond to other types of bacteria (30.1%), and out of sixty isolates of Pseudomonas spp., forty-two were identified as P. aeruginosa isolates. P. aeruginosa isolates revealed various resistance levels to antimicrobial agents gradually, ranging from 83.87% to Trimethoprim-sulfamethoxazole (SXT) to 3.22% to Aztreonam (AZT). Biofilm production by using the Congo red method showed that 27 isolates (64.28%) were positive results, while in the microtiter method, all forty-two isolates were positive (100%), the genetic detection showed that the AlgD gene was recognized in thirty-one isolates (73.8%), followed by Pelf and PslD genes in four isolates each (9.5%). Conclusion: The isolation percentage showed a high occurrence of multi-drug resistance biofilm forming Pseudomonas spp. isolates which could be a critical indicator. Methods of biofilm detection showed that the microtiter plate method has accuracy more than the Congo red method; as well AlgD gene was prevalent compared with both other genes Pelf, and PslD.

Molecular Detection of Virulence Genes Associated With Multi- Drug Resistant Pseudomonas Aeruginosa Isolated from Clinical and Environmental Samples within Maiduguri

Pseudomonas aeruginosa is an ubiquitous opportunistic pathogen having numerous virulence factors and the ability to acquire multidrug resistant traits. This study aimed to determine the presence of the virulence genes algD and lasB in multidrug resistant Pseudomonas aeruginosa isolated from clinical and environmental samples within Maiduguri. A total of 200 samples were collected from four sources namely; Farm soil at root region of groundnut, abattoir waste water, commercial boreholes and urine of urinary tract infected patients. All positive growths on cetrimide agar were further identified biochemically. Antibiotic profile of confirmed Pseudomonas aeruginosa was determined using Kirby-Baeur disc diffusion method. The genes algD and lasB were detected in the multidrug resistant isolates. The result also showed that 90.90%, 81.81% and 81.81% of the urine isolates were resistant to nalidixic acid, chloramphenicol and tetracycline respectively while highest sensitivity was observed against amikacin at 81.81%. Highest resistance among environmental samples was observed in abattoir wastewater with 95.24% resistance to chloramphenicol while least resistance was observed in borehole isolates with 0.00% resistance to Gentamicin, Amikaicin, Meropenem and Aztreonam. According to the findings of this study also, there is no significant difference in habouring virulence genes among the isolates of the different sources. All the multidrug resistant isolates were found to habour both algD and LasB genes. In conclusion, P. aeruginosa was found in all of the studied sources and widespread of algD and LasB genes in the multidrug resistant isolates from all the sources.

Comparing antibiotic resistance and virulence profiles of Enterococcus faecium, Klebsiella pneumoniae, and Pseudomonas aeruginosa from environmental and clinical settings

Antibiotic resistance and virulence profiles of Enterococcus faecium, Klebsiella pneumoniae, and Pseudomonas aeruginosa, isolated from water sources collected in informal settlements, were compared to clinical counterparts. Cluster analysis using repetitive extragenic palindromic sequence-based polymerase chain reaction (REP-PCR) indicated that, for each respective species, low genetic relatedness was observed between most of the clinical and environmental isolates, with only one clinical P. aeruginosa (PAO1) and one clinical K. pneumoniae (P2) exhibiting high genetic similarity to the environmental strains. Based on the antibiograms, the clinical E. faecium Ef CD1 was extensively drug resistant (XDR); all K. pneumoniae isolates (n = 12) (except K. pneumoniae ATCC 13883) were multidrug resistant (MDR), while the P. aeruginosa (n = 16) isolates exhibited higher susceptibility profiles. The tetM gene (tetracycline resistance) was identified in 47.4 % (n = 6 environmental; n = 3 clinical) of the E. faecium isolates, while the blaKPC gene (carbapenem resistance) was detected in 52.6 % (n = 7 environmental; n = 3 clinical) and 15.4 % (n = 2 environmental) of the E. faecium and K. pneumoniae isolates, respectively. The E. faecium isolates were predominantly poor biofilm formers, the K. pneumoniae isolates were moderate biofilm formers, while the P. aeruginosa isolates were strong biofilm formers. All E. faecium and K. pneumoniae isolates were gamma (γ)-haemolytic, non-gelatinase producing (E. faecium only), and non-hypermucoviscous (K. pneumoniae only), while the P. aeruginosa isolates exhibited beta (β)-haemolysis and produced gelatinase. The fimH (type 1 fimbriae adhesion) and ugE (uridine diphosphate galacturonate 4-epimerase synthesis) virulence genes were detected in the K. pneumoniae isolates, while the P. aeruginosa isolates possessed the phzM (phenazine production) and algD (alginate biosynthesis) genes. Similarities in antibiotic resistance and virulence profiles of environmental and clinical E. faecium, K. pneumoniae, and P. aeruginosa, thus highlights the potential health risks posed by using environmental water sources for daily water needs in low-and-middle-income countries.

Characterization of β-lactamase and virulence genes in Pseudomonas aeruginosa isolated from clinical, environmental and poultry sources in Bangladesh.

The emergence and spread of multidrug-resistant pathogens like Pseudomonas aeruginosa are major concerns for public health worldwide. This study aimed to assess the prevalence of P. aeruginosa in clinical, environmental, and poultry sources in Bangladesh, along with their antibiotic susceptibility and the profiling of β-lactamase and virulence genes using standard molecular and microbiology techniques. We collected 110 samples from five different locations, viz., BAU residential area (BAURA; n = 15), BAU Healthcare Center (BAUHCC; n = 20), BAU Veterinary Teaching Hospital (BAUVTH; n = 22), Poultry Market (PM; n = 30) and Mymensingh Medical College Hospital (MCCH; n = 23). After overnight enrichment in nutrient broth, 89 probable Pseudomonas isolates (80.90%) were screened through selective culture, gram-staining and biochemical tests. Using genus- and species-specific PCR, we confirmed 22 isolates (20.0%) as P. aeruginosa from these samples. Antibiogram profiling revealed that 100.0% P. aeruginosa isolates (n = 22) were multidrug-resistant isolates, showing resistance against Doripenem, Penicillin, Ceftazidime, Cefepime, and Imipenem. Furthermore, resistance to aztreonam was observed in 95.45% isolates. However, P. aeruginosa isolates showed a varying degree of sensitivity against Amikacin, Gentamicin, and Ciprofloxacin. The blaTEM gene was detected in 86.0% isolates, while blaCMY, blaSHV and blaOXA, were detected in 27.0%, 18.0% and 5.0% of the P. aeruginosa isolates, respectively. The algD gene was detected in 32.0% isolates, whereas lasB and exoA genes were identified in 9.0% and 5.0% P. aeruginosa isolates. However, none of the P. aeruginosa isolates harbored exoS gene. Hence, this study provides valuable and novel insights on the resistance and virulence of circulating P. aeruginosa within the clinical, environmental, and poultry environments of Bangladesh. These findings are crucial for understanding the emergence of β-lactamase resistance in P. aeruginosa, highlighting its usefulness in the treatment and control of P. aeruginosa infections in both human and animal populations.

In vitro evaluation of biofilm phenotypic and genotypic characteristics among clinical isolates of Pseudomonas aeruginosa in Hamadan, West of Iran.

Due to high antimicrobial resistance and biofilm-forming ability, Pseudomonas aeruginosa is one of the seriously life-threatening agents causing chronic and nosocomial infections. This study was performed to determine the antibiotic resistance pattern, biofilm formation, and frequency of biofilm-related genes in P. aeruginosa strains. In total, 123 P. aeruginosa isolates were collected from different clinical sources. Antimicrobial susceptibility testing (AST) was performed to detect multidrug-resistant P. aeruginosa (MDRPA) isolates. To evaluate the biofilm-forming isolates, the microtiter plate (MTP) method was carried out. Also, the prevalence of biofilm genotype patterns, including pslA, pslD, pelA, pelF, and algD genes, was detected by polymerases chain reaction (PCR). According to our findings, the highest resistance and susceptibility rates were found in ceftazidime with 74.7% (n = 92) and ciprofloxacin with 42.2% (n = 52), respectively. In our study, the highest level of antibiotic resistance belonged to wound isolates which meropenem had the most antibacterial activity against them. In total, 86.1% (n = 106) P. aeruginosa isolates were determined as MDRPA, of which 61.3% (n = 65) were able to form strong biofilm. The highest and lowest frequency of biofilm-related genes among biofilm producer isolates belonged to pelF with 82.1% (n = 101) and algD with 55.2% (n = 68), respectively. The findings of the conducted study indicate a significant relationship between MDRPA and biofilm genotypic/phenotypic patterns, suggesting the necessity of a careful surveillance program in hospital settings.

Diversity of biofilm-specific antimicrobial resistance genes in Pseudomonas aeruginosa recovered from various clinical isolates.

The resistance of Pseudomonas aeruginosa to antibiotics offers a significant challenge in the treatment of patients. This study aimed to investigate the antimicrobial resistance profile, biofilm-specific antimicrobial resistance genes, and genetic diversity of P. aeruginosa recovered from clinical samples. Totally 47 non-duplicate isolates of P. aeruginosa were recovered from various clinical samples. toxA, algD, ndvB, and tssC1 genes were detected in biofilm-producing isolates. The DNA sequences of the toxA and tssC1 genes were analyzed, by creating phylogenetic trees. The findings revealed that 30 (63.8%) of the isolates tested positive for Extended spectrum β-lactamase (ESBL), whereas 31 (65.9%) tested positive for Metallo-β-lactamase (MBL) and all of the isolates presented the toxA genes, and 19.1%,17%, 6.3% presented by algD, ndvB and tssC1 genes. Besides, the phylogenetic trees of the toxA and tssC1 gene isolates suggested a genotype that was closely aligned with others. Gene sequencing similarity revealed 99% identity with other isolates deposited in GenBank. The occurrence of toxA was most prevalent. One isolate was recorded as a novel isolate in the global gene bank as a locally isolated strain from the city of Erbil that has never been identified in global isolates due to genetic variation.

Murepavadin induces envelope stress response and enhances the killing efficacies of β-lactam antibiotics by impairing the outer membrane integrity of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a ubiquitous opportunistic pathogen that can cause a variety of acute and chronic infections. The bacterium is highly resistant to numerous antibiotics. Murepavadin is a peptidomimetic antibiotic that blocks the function of P. aeruginosa lipopolysaccharide (LPS) transport protein D (LptD), thus inhibiting the insertion of LPS into the outer membrane. In this study, we demonstrated that sublethal concentrations of murepavadin enhance the bacterial outer membrane permeability. Proteomic analyses revealed the alteration of protein composition in bacterial inner and outer membranes following murepavadin treatment. The antisigma factor MucA was upregulated by murepavadin. In addition, the expression of the sigma E factor gene algU and the alginate synthesis gene algD was induced by murepavadin. Deletion of the algU gene reduces bacterial survival following murepavadin treatment, indicating a role of the envelope stress response in bacterial tolerance. We further demonstrated that murepavadin enhances the bactericidal activities of β-lactam antibiotics by promoting drug influx across the outer membrane. In a mouse model of acute pneumonia, the murepavadin-ceftazidime/avibactam combination showed synergistic therapeutic effect against P. aeruginosa infection. In addition, the combination of murepavadin with ceftazidime/avibactam slowed down the resistance development. In conclusion, our results reveal the response mechanism of P. aeruginosa to murepavadin and provide a promising antibiotic combination for the treatment of P. aeruginosa infections.IMPORTANCEThe ever increasing resistance of bacteria to antibiotics poses a serious threat to global public health. Novel antibiotics and treatment strategies are urgently needed. Murepavadin is a novel antibiotic that blocks the assembly of lipopolysaccharide (LPS) into the Pseudomonas aeruginosa outer membrane by inhibiting LPS transport protein D (LptD). Here, we demonstrated that murepavadin impairs bacterial outer membrane integrity, which induces the envelope stress response. We further found that the impaired outer membrane integrity increases the influx of β-lactam antibiotics, resulting in enhanced bactericidal effects. In addition, the combination of murepavadin and a β-lactam/β-lactamase inhibitor mixture (ceftazidime/avibactam) slowed down the resistance development of P. aeruginosa. Overall, this study demonstrates the bacterial response to murepavadin and provides a new combination strategy for effective treatment.

DETECTION OF BIOFILM PRODUCTION AMONG MBLS-PRODUCING PSEUDOMONAS AERUGINOSA CARRYING ALGD, PELF, PSLD GENES ISOLATED FROM BURN CENTER IN AL-NAJAF GOVERNORATE, IRAQ

Background: Pseudomonas aeruginosa is a worldwide cause of chronic and acute infections of burn wounds. Aim of study: Detection of algD, pelF, and pslD genes encoding biofilm formation among isolates of Pseudomonas aeruginosa that producing metallo β-lactamases. Materials and Methods: 93 isolates of Pseudomonas aeruginosa were isolated from 362 inpatients with infected burn wounds at the burn center in Al-Najaf Governorate, Iraq, from September 2022 to February 2023. Results: Pseudomonas aeruginosa isolates were identified microscopically, phenotypically, culturally, and biochemically then by using the double-disc synergy method to test their capacity to produce metallo β-lactamases. Out of the 25 MBLs-producers, 22 (88%) were biofilm-producing isolates when tested by tissue culture plate method, 4 (16%), 10 (40%), and 8 (32%) were, respectively, weak, medium, and strongly biofilm producers. 25 metallo β-lactamases-producers under study, which had previously been evaluated using biofilm phenotypic method, were subjected to the investigation of biofilm encoding genes by using polymerase chain reaction technique. This molecular technique detected 21 (84%) isolates carried single and multiple biofilm encoding genes, 10(40%) isolates had algD gene, 6 (24%) isolates harbored algD and pslD genes and 5(20%) isolates carried algD, pelF and pslD genes. The single pslD and pelF genes were not detected among test isolates. Conclusions: There was a high prevalence of biofilmproducers among metallo β-lactamases-producing P. aeruginosa isolates. In addition, a clear positive correlation was observed between the multiple genes encoding biofilm and the degree of biofilm production among metallo β-lactamases-producers and close association between metallo β-lactamases and biofilms production, which explains the prevalence of carbapenems resistance and biofilm-production in this study because biofilm provides the appropriate environment for antibiotic resistance in general and carbapenems in particular

Detection of exopolysaccharides (algD, pelF, and pslD) genes in burn wound Pseudomonas aeruginosa isolates

Abstract Background: Pseudomonas aeruginosa biofilm is an essential component of virulence that plays a significant role in antimicrobial resistance and chronic burn wound infections. Objective: This study aimed to investigate the biofilm formation capacity of P. aeruginosa isolated from chronic burn wound from January to May 2022 by biochemical and molecular techniques. Materials and Methods: Quantification of biofilm was performed based on tube method for local isolates of P. aeruginosa after growing on brain heart Broth. The genes encoding exopolysaccharides (algD, pelF, and pslD) were targeted by using conventional polymerase chain reaction (PCR). Results: The results showed that 92.6% of isolates were biofilm former, interestingly 68% of isolates were considered as strong former comparing with other biofilm categories. Gel electrophoresis result of PCR products presented clear bands for algD and psID genes with percentages (96%) and (3.7%) respectively. However, there was no PCR product for pelf gene among all isolates. Conclusion: The prevalence of algD, the large operon necessary for alginate production, was high among P. aeruginosa biofilm producer in this study and it can be an essential agent in the pathogenicity of P. aeruginosa burn wound infections comparing with other biofilm genes (pelF and pslD) of exopolysaccharide structure.

Effect of curcumin nanoparticles and alcoholic extract of Falcaria vulgaris on the growth rate, biofilm, and gene expression in Pseudomonas aeruginosa isolated from burn wound infection.

This study aimed to investigate the effect of Curcumin nanoparticles and alcoholic extract of Falcaria vulgaris on the growth rate, biofilm, and gene expression in Pseudomonas aeruginosa isolated from burn wound infection. The alcoholic extract of Falcaria vulgaris was purchased from Pasargad Company. Curcumin nanoparticles were synthesized. Antibacterial activity of Curcumin nanoparticles and alcoholic extract of Falcaria vulgaris was investigated by microdilution method alone and in combination. Biofilm inhibitory was investigated by microtitrplate method. Effect of Curcumin nanoparticles and alcoholic extract of Falcaria vulgaris were evaluated on algD gene expression via Real-Time PCR. Cytotoxicity was evaluated by MTT assay on HDF cell line. Then, the data were analyzed using SPSS software. Synthesized Curcumin nanoparticles were approved by Fourier Transform Infrared (FTIR), and Scanning Electron Microscope. The alcoholic extract of Falcaria Vulgaris showed significant antibacterial activity against multidrug resistance (MDR) P. aeruginosa isolates at a concentration of 156.25µg/mL. Moreover, MIC of the curcumin nanoparticle for isolates was 625µg/mL. Based on fraction inhibition concentration, synergy, and the additive effect were shown against %7.7, and %93.3 of MDRs, respectively. The sub-MIC concentration of the binary compound reduced biofilms and algD gene expression in P. aeruginosa isolates. The Biological function of HDF cell lines was desirable after the effect of the binary compound. Regarding our results, this combination can be suggested as a promising agent in terms of biofilm inhibitory and antimicrobial properties.

Molecular detection of different virulence factors genes harbor pslA, pelA, exoS, toxA and algD among biofilm-forming clinical isolates of Pseudomonas aeruginosa.

Pseudomonas aeruginosa (P. aeruginosa) is considered as the foremost cause of hospital-acquired infections due to its innate and plasmid-mediated resistance to multiple antibiotics making it a multi-drug resistant (MDR) pathogen. This study aimed to determine the biofilm formation ability and the presence of different virulence factors genes (pslA, pelA, exoS, toxA and algD) among biofilm-forming strains of P. aeruginosa clinical isolates from burn units in Ismailia Hospitals, Egypt. In our cross-sectional study, one hundred and twenty-six (126) non-duplicate clinical P. aeruginosa isolates were recovered from 450 clinical specimens from burn units in Ismailia Hospitals. The antibiotic sensitivity of strong and moderate biofilm producer isolates was investigated using the disc diffusion method. The isolated bacteria were tested for their ability to form biofilm using a microtiter plate assay. The expression of (pslA, pelA, exoS, toxA and algD) genes in biofilm producers isolates was detected using PCR. The MPA detected 80% (95 /126) isolates as biofilm producers, 18% (22/126) were strong biofilm producers, 34% (43/126) were moderate biofilm producers, 28% (35/126) were weak biofilm producers and 20% (31/126) non-biofilm producers. Susceptibility pattern analysis of biofilm-forming P. aeruginosa isolates (95) detected that 60% (68/ 95) were multi-drug resistant isolates (MDR). Resistance to all used antibiotics and multidrug resistance was higher among biofilm-producing than non-biofilm-producing strains, but the difference was statistically non-significant. Investigation of virulence factors associated genes revealed that 96%, 94%, 86.4%, 80.0% and 74% of the biofilm producers isolates were harboring algD, pslA, pel A, toxA and exoS gene, respectively. The present study confirmed that antimicrobial resistance and virulence genes were more prominent in biofilm-producing P. aeruginosa than in non-biofilm-producers.

Snapshot of Phenotypic and Molecular Virulence and Resistance Profiles in Multidrug-Resistant Strains Isolated in a Tertiary Hospital in Romania.

A current major healthcare problem is represented by antibiotic resistance, mainly due to multidrug resistant (MDR) Gram negative bacilli (GNB), because of their extended spread both in hospital facilities and in the community's environment. The aim of this study was to investigate the virulence traits of Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa MDR, XDR, and PDR strains isolated from various hospitalized patients. These GNB strains were investigated for the presence of soluble virulence factors (VF), such as hemolysins, lecithinase, amylase, lipase, caseinase, gelatinase, and esculin hydrolysis, as well as for the presence of virulence genes encoding for VF involved in adherence (TC, fimH, and fimA), biofilm formation (algD, ecpRAB, mrkA, mrkD, ompA, and epsA), tissue destruction (plcH and plcN), and in toxin production (cnfI, hlyA, hlyD, and exo complex). All P. aeruginosa strains produced hemolysins; 90% produced lecithinase; and 80% harbored algD, plcH, and plcN genes. The esculin hydrolysis was detected in 96.1% of the K. pneumoniae strains, whereas 86% of them were positive for the mrkA gene. All of the A. baumannii strains produced lecithinase and 80% presented the ompA gene. A significant association was found between the number of VF and the XDR strains, regardless of the isolation sources. This study opens new research perspectives related to bacterial fitness and pathogenicity, and it provides new insights regarding the connection between biofilm formation, other virulence factors, and antibiotic resistance.

The Inhibitory Effects of Lactobacillus Agilis Against Pseudomonas Aeruginosa Biofilm Formation and Evaluation of PpyR (PA2663) and algD Gene Expression

Background and Aims: Hospital infections and their antibiotic resistance have become a global concern recently. One of the most prominent factors in hospital infections is Pseudomonas aeruginosa (P. aeruginosa), which can become resistant to many antibiotics due to its ability to form biofilms. Recently, scientists have tried to replace antibiotic therapy with alternative therapies such as probiotics which can reduce or eliminate the pathogenic bacteria's ability to form biofilms. Therefore, the present study revealed that some genes, such as algD and PpyR, were involved in biofilm formation in P. aeruginosa. Furthermore, the inhibitory effect of the supernatant of lactobacillus agilis on the biofilm formation of P. aeruginosa was evaluated in the current study.
 Materials and Methods: In this study, the effect of the supernatant of probiotic Lactobacillus agilis on the biofilm formation of P. aeruginosa and also the expression of two genes effective in biofilm formation (algD and PpyR) were investigated. Antibiograms were performed to detect the most resistant bacteria since there is a link between biofilm formation and antibiotic resistance. Further, the effects of probiotics on the expression of PpyR and algD genes were discussed.
 Results: Results showed that the biofilm formation of P. aeruginosa was significantly reduced in the presence of lactobacillus agilis.
 Conclusions: According to the current study, it could be concluded that because of antibiotics resistance and their associated mechanisms, probiotics could be used as a replacement for antibiotics in many treatments.

Prevalence of Quorum Sensing and Virulence Factor Genes Among Pseudomonas aeruginosa Isolated from Patients Suffering from Different Infections and Their Association with Antimicrobial Resistance.

Antimicrobial resistance and virulence genes play important roles in increasing the severity of Pseudomonas aeruginosa infections, especially in hospitalized patients with high antibiotic pressure. Most genes that encode Pseudomonas aeruginosa virulence factors are controlled and regulated by the quorum sensing (QS) system. The aim of this study was to investigate the frequency of some virulence genes (rhlR, rhlI, lasR, lasI, lasB, toxA, aprA, algD, ExoS, and plcH genes) and their association with antibiotic resistance. Antimicrobial susceptibility was determined by Kirby-Bauer agar disk diffusion method. A total of 125 clinical isolates of P. aeruginosa were tested for some virulence genes using polymerase chain reaction (PCR). The highest resistance was observed against cefepime (92.8%). Multi-drug resistant (MDR) P. aeruginosa represented 63.2% of total isolates with high distribution among wound isolates (21/79, 26.3% of MDR isolates). LasB was the most prevalent virulence gene among the tested isolates (89.6%) followed by aprA (85.6%), exoS (84%), algD (80%), toxA (76.8%), and plcH (75.2). Furthermore, a significant association (P < 0.05) among most of the tested virulence genes and MDR isolates was found. The presence of more than 5 virulence genes was highly observed among wound infections, otitis media, and respiratory tract infection isolates. The complex association of virulence genes including QS system regulating genes with antibiotic resistance indicates the importance of the tested factors in the progression of infections, which is considered a great challenge for the health-care team with the need for specific studies for each area having different antibiotic resistance profiles and the development of effective treatment strategies such as anti-virulent and quorum sensing inhibiting drugs against P. aeruginosa infections.

Influence pH on virulence genes of Pseudomonas aeruginosa analyzed by RT-PCR method

PurposeThe purpose of this study was to determine the influence of environmental pH on production of biofilms and virulence genes expression in Pseudomonas aeruginosa.Design/methodology/approachAmong 303 clinical and environmental samples 109 (61 + 48) isolates were identified as clinical and environmental P. aeruginosa isolates, respectively. Clinical samples were obtained from patients in the Al-Yarmouk hospital in Baghdad city, Iraq. Waste water from Al-Yarmouk hospital was used from site before treatment unit to collect environmental samples. The ability of producing biofilm at various pH levels was examined by microtiter plate and the prevalence of Alg D, Psl A and Pel A was determined by quantitative real time-polymerase chain reaction (qRT-PCR).FindingsThis study showed that the ability of clinical and environmental isolates to biofilm development was observed in 86.9% and 85.42% of clinical and environmental isolates, respectively. As well as, the environmental P. aeruginosa isolates showed the highest biofilm production at pH 7. Clinical isolates showed the highest genes expression of Alg D, Psl A and Pel A as compared to environmental isolates with pH change. In general, both clinical and environmental isolates formed biofilm and carried AlgD, PslA and PelA genes. Also, alkaline pH was favored for biofilm production.Originality/valueThere are very few studies done to find out the influence of environmental pH on production of biofilms and virulence genes expression in Pseudomonas aeruginosa. This study is unique as it has highlighted the influence of environmental pH on the ability of clinical and environmental isolates to biofilm development and genes expression.

Expression of algD Gene in Single- and Dual-Species Biofilms of &lt;i&gt;Pseudomonas aeruginosa&lt;/i&gt; and &lt;i&gt;Staphylococcus aureus Under Starvation Stress

Dual-species biofilms of Pseudomonas aeruginosa and Staphylococcus aureus generate difficultto-treat illnesses. Nutrition stress in biofilms affects physiology, microbial metabolism, and species interactions, impacting bacteria growth and survival. Furthermore, the function of alginate, which is encoded by the algD gene, in the production of biofilms has been established. The present study aimed at investigating the impact of starvation on algD gene expression in single-species biofilm of P. aeruginosa and dual-species biofilms of P. aeruginosa and S. aureus from hospital sewage. A total of six P. aeruginosa and six S. aureus isolates were obtained from the microbiology laboratory at the Department of Biology, College of Science, University of Baghdad, Iraq. These isolates are multidrug-resistant and were obtained from various hospital sewage stations in Baghdad city. P. aeruginosa and S. aureus isolates were co-cultured as singleand dual-species biofilms in full-strength brain heart infusion broth (BHIB) and 1000-fold diluted BHIB. In order to evaluate the level of expression of the algD gene in P. aeruginosa that had been treated to starvation, the quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was utilized. The results demonstrated that starvation stress significantly (P&lt; 0.05) up regulated the expression of algD in single-species biofilm (3.117 to 4.532-fold). However, starvation stress down regulated the algD expression in dual-species biofilm (0.001 and 0.901-fold). In conclusion, malnutrition up regulated algD expression in single-species P. aeruginosa biofilms but down regulated it in dual-biofilms. This work helps create biofilm-related disease treatments.

Evaluation of the inhibitory effects of TiO2 nanoparticle and Ganoderma lucidum extract against biofilm-producing bacteria isolated from clinical samples

The emergence of multidrug-resistant pathogens leads to treatment failure. So, the need for new antibacterial drugs is urgent. We evaluated the antibacterial and antibiofilm effects of titanium dioxide (TiO2) nanoparticles (NPs) and Ganoderma extract against biofilm-producing Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus (MRSA) by microbroth dilution and crystal violet assays. The combined effect of these compounds was studied using the checkerboard method. The OD260 was measured to assess the destruction of the membrane permeability. The expression of biofilm-related genes (iacA and algD) was investigated by real-time PCR. MRSA isolate was more susceptible to test compounds. The OD260 increased and algD gene was down-regulated after treatment with TiO2 NPs and a combination of TiO2 NPs and Ganoderma extract. iacA gene did not affect by test compounds. Overall, these findings revealed that nanoparticles and natural substances might represent the potential candidates to develop promising antibacterial agents, especially against Gram-positive bacteria.