Phenotypic and genetic analysis of virulence factors in multidrug- sensitive and multidrug-resistant clinical isolates of Pseudomonas aeruginosa

This study aimed to correlate the pattern of antimicrobial susceptibility, phenotypic production of virulence factors, the occurrence of virulence factors genes and the clonal profile of clinical isolates of Pseudomonas aeruginosa of a tertiary hospital in Recife-PE. The 30 clinical isolates (15 multidrug-sensitive (MDS) and 15 multidrug-resistant (MDR)) were analyzed using phenotypic methods to detect virulence factors (alkaline protease, hemolysin, phospholipase C, lipase, and pigments). The detection of the aprA, lasA, lasB, plcH, and toxA genes was performed through specific PCRs, and the clonal profile was assessed using ERIC-PCR. The results revealed cephalosporins being the class eliciting the highest percentage of resistance; the MDR isolates were all resistant. Among the MDS isolates, all were sensitive to carbapenems and quinolones. The MDR isolates produced less virulence factors such as pyocyanin and lipase, and exhibited lower expression of toxA and lasA genes, whereas the MDS isolates produced less hemolysin and phospholipase C. There was no difference between the groups for alkaline protease production and aprA gene expression. All the isolates produced pyocyanin and expressed lasB and plcH genes. A great genetic diversity was found, and it was possible to observe 28 genetic profiles. Clones were present among the MDR isolates. The occurrence of virulence factors in almost all the isolates studied suggests their high level of pathogenicity, demonstrating that this pathogen is capable of accumulating numerous virulence factors, and in some cases, is associated with multidrug resistance, which makes it difficult to treat these infections.

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ANÁLISE COMPARATIVA DOS FATORES DE VIRULÊNCIA DOS ISOLADOS CLÍNICOS E AMBIENTAIS DE PSEUDOMONAS AERUGINOSA

Colloquium Vitae ◽

10.5747/cv.2019.v11.n3.v269 ◽

2019 ◽

Vol 11 (3) ◽

pp. 41-50

Author(s):

Isabela Alves de Souza ◽

Doroti de Oliveira Garcia ◽

Laís Anversa ◽

Renata Katsuko Takayama Kobayashi ◽

Gerson Nakazato ◽

...

Keyword(s):

Public Health ◽

Pseudomonas Aeruginosa ◽

Virulence Factors ◽

Pathogenic Bacteria ◽

Clinical Isolates ◽

Environmental Isolates ◽

High Level

Pseudomonas aeruginosais a very important bacteria for public health because it is present in the environment and clinical infections. The aim of this study was toevaluate the virulence factors such as motility, protease and rhamnolipids in clinical and environmental P. aeruginosaisolates.Twenty-five clinical isolates and ten environmental isolates were analyzed by phenotypic assays and categorized into non-mobile, weakly, moderately and highly mobile strains; and producers of protease and rhamnolipids. The isolates were tested in triplicate on three different days. Environmental isolates produced virulence factors such as motility (Swimming and Twitching), and Ramnolipids significantly higher than clinical isolates.This study alerts us to the high level of pathogenicity of P. aeruginosastrains, mainly environmental strains. For a better understanding of motility and rhamnolipids, virulence factors that are directly associated with the biofilms formation, may favor studies that complement the research aimed at the control of pathogenic bacteria.

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Relevance of resistance levels to carbapenems and integron-borne bla IMP-1, bla IMP-7, bla IMP-10 and bla VIM-2 in clinical isolates of Pseudomonas aeruginosa

Journal of Medical Microbiology ◽

10.1099/jmm.0.010017-0 ◽

2009 ◽

Vol 58 (8) ◽

pp. 1080-1085 ◽

Cited By ~ 31

Author(s):

Wei-Hua Zhao ◽

Gelin Chen ◽

Ribu Ito ◽

Zhi-Qing Hu

Keyword(s):

Pseudomonas Aeruginosa ◽

Resistance Genes ◽

Gene Cassette ◽

Multidrug Resistant ◽

Resistance Mechanisms ◽

Clinical Isolates ◽

Gene Cassettes ◽

Class 1 ◽

High Level ◽

Encoding Genes

Molecular detection and surveillance of the resistance genes harboured by Pseudomonas aeruginosa are becoming increasingly important in assessing and controlling spread and colonization in hospitals, and in guiding the treatment of infections. This study analysed the resistance mechanisms of carbapenem-resistant clinical isolates of P. aeruginosa and identified the associated integron-borne metallo-β-lactamase (MBL)-encoding genes. Twenty-seven imipenem (IPM)-resistant clinical isolates of P. aeruginosa were divided into three groups according to their resistance levels to carbapenems. Strains bearing bla IMP-10 showed extremely high-level resistance to IPM, with MICs of 512–2048 μg ml−1. By comparison, strains bearing bla IMP-1, bla IMP-7 and bla VIM-2 showed an intermediate level of resistance, with MICs of 32–256 μg ml−1. The non-MBL-producing strains showed a low level of resistance, with MICs of 8–32 μg ml−1. The same trend in resistance levels was also observed when resistance to other carbapenems, such as meropenem and panipenem, was determined. DNA sequencing showed that the MBL-encoding gene cassettes were carried by class 1 integrons. The bla IMP-1, bla IMP-7 and bla IMP-10 gene cassettes were preceded by a hybrid P ant promoter, TGGACA-N17-TAAACT, and the bla VIM-2 gene cassette was preceded by a weak promoter, TGGACA-N17-TAAGCT. Most of the MBL-encoding genes were linked to one or two resistance genes encoding aminoglycoside-modifying enzymes, such as aac(6′)Iae, aac(6′)II, aacA7, aacC4, aadA1, aadA2 and aadA6, highlighting the multidrug-resistant properties of these clinical isolates.

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Interplay of Efflux System, ampC, and oprD Expression in Carbapenem Resistance of Pseudomonas aeruginosa Clinical Isolates

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.50.5.1633-1641.2006 ◽

2006 ◽

Vol 50 (5) ◽

pp. 1633-1641 ◽

Cited By ~ 230

Author(s):

John Quale ◽

Simona Bratu ◽

Jyoti Gupta ◽

David Landman

Keyword(s):

Pseudomonas Aeruginosa ◽

Target Genes ◽

Stop Codon ◽

Premature Stop Codon ◽

Carbapenem Resistance ◽

Multidrug Resistant ◽

Clinical Isolates ◽

Carbapenem Resistant ◽

High Level ◽

Increased Activity

ABSTRACT Carbapenems are important agents for the therapy of infections due to multidrug-resistant Pseudomonas aeruginosa; the development of carbapenem resistance hampers effective therapeutic options. To assess the mechanisms leading to resistance, 33 clinical isolates with differing degrees of carbapenem susceptibility were analyzed for the expression of the chromosomal β-lactamase (ampC), the porin that is important for the entry of carbapenems (oprD), and the proteins involved in four efflux systems (mexA, mexC, mexE, and mexX). Real-time reverse transcriptase PCR was performed using primers and fluorescent probes for each of the target genes. The sequencing of regulatory genes (ampR, mexR, nalC, nalD, mexT, and mexZ) was also performed. Diminished expression of oprD was present in all imipenem- and meropenem-resistant isolates but was not required for ertapenem resistance. Increased expression of ampC was not observed in several isolates that were overtly resistant to carbapenems. Increased expression of several efflux systems was observed in many of the carbapenem-resistant isolates. Increased efflux activity correlated with high-level ertapenem resistance and reduced susceptibility to meropenem and aztreonam. Most isolates with increased expression of mexA had mutations affecting nalC and/or nalD. Two isolates with mutations leading to a premature stop codon in mexZ had markedly elevated mexX expressions, although mutations in mexZ were not a prerequisite for overexpression. β-Lactam resistance in clinical isolates of P. aeruginosa is a result of the interplay between diminished production of oprD, increased activity of ampC, and several efflux systems.

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Antimicrobial susceptibility, serotype distribution, virulence profile and molecular typing of piliated clinical isolates of pneumococci from east coast, Peninsular Malaysia

Scientific Reports ◽

10.1038/s41598-021-87428-z ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Nurul Diana Dzaraly ◽

Mohd Nasir Mohd Desa ◽

AbdulRahman Muthanna ◽

Siti Norbaya Masri ◽

Niazlin Mohd Taib ◽

...

Keyword(s):

Phylogenetic Analysis ◽

Antimicrobial Susceptibility ◽

East Coast ◽

Tertiary Hospital ◽

Multidrug Resistant ◽

Peninsular Malaysia ◽

Clinical Isolates ◽

Serotype Distribution ◽

Antimicrobial Susceptibility Test ◽

Conjugate Vaccines

AbstractPilus has been recently associated with pneumococcal pathogenesis in humans. The information regarding piliated isolates in Malaysia is scarce, especially in the less developed states on the east coast of Peninsular Malaysia. Therefore, we studied the characteristics of pneumococci, including the piliated isolates, in relation to antimicrobial susceptibility, serotypes, and genotypes at a major tertiary hospital on the east coast of Peninsular Malaysia. A total of 100 clinical isolates collected between September 2017 and December 2019 were subjected to serotyping, antimicrobial susceptibility test, and detection of pneumococcal virulence and pilus genes. Multilocus sequence typing (MLST) and phylogenetic analysis were performed only for piliated strains. The most frequent serotypes were 14 (17%), 6A/B (16%), 23F (12%), 19A (11%), and 19F (11%). The majority of isolates were resistant to erythromycin (42%), tetracycline (37%), and trimethoprim-sulfamethoxazole (24%). Piliated isolates occurred in a proportion of 19%; 47.3% of them were multidrug-resistant (MDR) and a majority had serotype 19F. This study showed ST236 was the most predominant sequence type (ST) among piliated isolates, which was related to PMEN clone Taiwan19F-14 (CC271). In the phylogenetic analysis, the piliated isolates were grouped into three major clades supported with 100% bootstrap values. Most piliated isolates belonged to internationally disseminated clones of S. pneumoniae, but pneumococcal conjugate vaccines (PCVs) have the potential to control them.

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Susceptibility of the Candida haemulonii Complex to Echinocandins: Focus on Both Planktonic and Biofilm Life Styles and a Literature Review

Journal of Fungi ◽

10.3390/jof6040201 ◽

2020 ◽

Vol 6 (4) ◽

pp. 201

Author(s):

Lívia S. Ramos ◽

Laura N. Silva ◽

Marta H. Branquinha ◽

André L. S. Santos

Keyword(s):

Literature Review ◽

Therapeutic Potential ◽

Multidrug Resistant ◽

Antifungal Drugs ◽

Clinical Isolates ◽

Published Data ◽

Life Styles ◽

Fungal Isolates ◽

Candida Haemulonii ◽

Almost All

Candida haemulonii complex (C. haemulonii, C. duobushaemulonii and C. haemulonii var. vulnera) is well-known for its resistance profile to different available antifungal drugs. Although echinocandins are the most effective class of antifungal compounds against the C. haemulonii species complex, clinical isolates resistant to caspofungin, micafungin and anidulafungin have already been reported. In this work, we present a literature review regarding the effects of echinocandins on this emergent fungal complex. Published data has revealed that micafungin and anidulafungin were more effective than caspofungin against the species forming the C. haemulonii complex. Subsequently, we investigated the susceptibilities of both planktonic and biofilm forms of 12 Brazilian clinical isolates of the C. haemulonii complex towards caspofungin and micafungin (anidulafungin was unavailable). The planktonic cells of all the fungal isolates were susceptible to both of the test echinocandins. Interestingly, echinocandins caused a significant reduction in the biofilm metabolic activity (viability) of almost all fungal isolates (11/12, 91.7%). Generally, the biofilm biomasses were also affected (reduction range 20–60%) upon exposure to caspofungin and micafungin. This is the first report of the anti-biofilm action of echinocandins against the multidrug-resistant opportunistic pathogens comprising the C. haemulonii complex, and unveils the therapeutic potential of these compounds.

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Detection of Extended-Spectrum β-Lactamases in Clinical Isolates of Pseudomonas aeruginosa

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01511-05 ◽

2006 ◽

Vol 50 (9) ◽

pp. 2990-2995 ◽

Cited By ~ 70

Author(s):

Xiaofei Jiang ◽

Zhe Zhang ◽

Min Li ◽

Danqiu Zhou ◽

Feiyi Ruan ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Multidrug Resistant ◽

Clinical Isolates ◽

Screening Methods ◽

Extended Spectrum ◽

Pcr Product ◽

Amoxicillin Clavulanate ◽

Synergy Test ◽

Esbl Producers ◽

Disk Test

ABSTRACT With the occurrence of extended-spectrum β-lactamases (ESBLs) in Pseudomonas aeruginosa being increasingly reported worldwide, there is a need for a reliable test to detect ESBLs in clinical isolates of P. aeruginosa. In our study, a total of 75 clinical isolates of P. aeruginosa were studied. Nitrocefin tests were performed to detect the β-lactamase enzyme; isoelectric focusing electrophoresis, PCR, and PCR product sequencing were designed to further characterize the contained ESBLs. Various ESBL-screening methods were designed to compare the reliabilities of detecting ESBLs in clinical isolates of P. aeruginosa whose β-lactamases were well characterized. Thirty-four of 36 multidrug-resistant P. aeruginosa clinical isolates were positive for ESBLs. bla VEB-3 was the most prevalent ESBL gene in P. aeruginosa in our study. Among the total of 34 isolates that were considered ESBL producers, 20 strains were positive using conventional combined disk tests and 10 strains were positive using a conventional double-disk synergy test (DDST) with amoxicillin-clavulanate, expanded-spectrum cephalosporins, aztreonam, and cefepime. Modifications of the combined disk test and DDST, which consisted of shorter distances between disks (20 mm instead of 30 mm) and the use of three different plates that contained cloxacillin (200 μg/ml) alone, Phe-Arg β-naphthylamide dihydrochloride (MC-207,110; 20 μg/ml) alone, and both cloxacillin (200 μg/ml) and MC-207,110 (20 μg/ml) increased the sensitivity of the tests to 78.8%, 91.18%, 85.29%, and 97.06%.

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ampG Gene of Pseudomonas aeruginosa and Its Role in β-Lactamase Expression

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00009-10 ◽

2010 ◽

Vol 54 (11) ◽

pp. 4772-4779 ◽

Cited By ~ 35

Author(s):

Ying Zhang ◽

Qiyu Bao ◽

Luc A. Gagnon ◽

Ann Huletsky ◽

Antonio Oliver ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Amino Acid ◽

Transmembrane Protein ◽

Amino Acid Identity ◽

Clinical Isolates ◽

Signal Molecules ◽

Carbonyl Cyanide ◽

Increased Sensitivity ◽

Potential Strategy ◽

High Level

ABSTRACT In enterobacteria, the ampG gene encodes a transmembrane protein (permease) that transports 1,6-GlcNAc-anhydro-MurNAc and the 1,6-GlcNAc-anhydro-MurNAc peptide from the periplasm to the cytoplasm, which serve as signal molecules for the induction of ampC β-lactamase. The role of AmpG as a transporter is also essential for cell wall recycling. Pseudomonas aeruginosa carries two AmpG homologues, AmpG (PA4393) and AmpGh1 (PA4218), with 45 and 41% amino acid sequence identity, respectively, to Escherichia coli AmpG, while the two homologues share only 19% amino acid identity. In P. aeruginosa strains PAO1 and PAK, inactivation of ampG drastically repressed the intrinsic β-lactam resistance while ampGh1 deletion had little effect on the resistance. Further, deletion of ampG in an ampD-null mutant abolished the high-level β-lactam resistance that is associated with the loss of AmpD activity. The cloned ampG gene is able to complement both the P. aeruginosa and the E. coli ampG mutants, while that of ampGh1 failed to do so, suggesting that PA4393 encodes the only functional AmpG protein in P. aeruginosa. We also demonstrate that the function of AmpG in laboratory strains of P. aeruginosa can effectively be inhibited by carbonyl cyanide m-chlorophenylhydrazone (CCCP), causing an increased sensitivity to β-lactams among laboratory as well as clinical isolates of P. aeruginosa. Our results suggest that inhibition of the AmpG activity is a potential strategy for enhancing the efficacy of β-lactams against P. aeruginosa, which carries inducible chromosomal ampC, especially in AmpC-hyperproducing clinical isolates.

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Incidence and Molecular Characterization of Carbapenemase Genes in Association with Multidrug-Resistant Clinical Isolates of Pseudomonas aeruginosa from Tertiary Healthcare Facilities in Southwest Nigeria

Current Microbiology ◽

10.1007/s00284-021-02706-3 ◽

2021 ◽

Vol 79 (1) ◽

Author(s):

Oluwatoyin B. Olaniran ◽

Olufemi E. Adeleke ◽

Ahmed Donia ◽

Ramla Shahid ◽

Habib Bokhari

Keyword(s):

Pseudomonas Aeruginosa ◽

Molecular Characterization ◽

Multidrug Resistant ◽

Clinical Isolates ◽

Healthcare Facilities ◽

Southwest Nigeria ◽

Tertiary Healthcare

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Enhancement of pyocyanin production by subinhibitory concentration of royal jelly in Pseudomonas aeruginosa

F1000Research ◽

10.12688/f1000research.27915.3 ◽

2021 ◽

Vol 10 ◽

pp. 14

Author(s):

Dina Auliya Amly ◽

Puspita Hajardhini ◽

Alma Linggar Jonarta ◽

Heribertus Dedy Kusuma Yulianto ◽

Heni Susilowati

Keyword(s):

Pseudomonas Aeruginosa ◽

Bacterial Growth ◽

Biofilm Formation ◽

Royal Jelly ◽

Microtiter Plate ◽

Multidrug Resistant ◽

Clinical Isolates ◽

Antibiotic Tolerance ◽

Subinhibitory Concentration ◽

Pyocyanin Production

Background: Pseudomonas aeruginosa, a multidrug-resistant Gram-negative bacterium, produces pyocyanin, a virulence factor associated with antibiotic tolerance. High concentrations of royal jelly have an antibacterial effect, which may potentially overcome antibacterial resistance. However, in some cases, antibiotic tolerance can occur due to prolonged stress of low-dose antibacterial agents. This study aimed to investigate the effect of subinhibitory concentrations of royal jelly on bacterial growth, pyocyanin production, and biofilm formation of P. aeruginosa. Methods: Pseudomonas aeruginosa ATCC 10145 and clinical isolates were cultured in a royal jelly-containing medium to test the antibacterial activity. Pyocyanin production was observed by measuring the absorbance at 690 nm after 36 h culture and determined using extinction coefficient 4310 M-1 cm-1. Static microtiter plate biofilm assay performed to detect the biofilm formation, followed by scanning electron microscopy. Results: Royal jelly effectively inhibited the viability of both strains from a concentration of 25%. The highest production of pyocyanin was observed in the subinhibitory concentration group 6.25%, which gradually decreased along with the decrease of royal jelly concentration. Results of one-way ANOVA tests differed significantly in pyocyanin production of the two strains between the royal jelly groups. Tukey HSD test showed concentrations of 12.5%, 6.25%, and 3.125% significantly increased pyocyanin production of ATCC 10145, and the concentrations of 12.5% and 6.25% significantly increased production of the clinical isolates. Concentrations of 12.5% and 6.125% significantly induced biofilm formation of P. aeruginosa ATCC 10145, in line with the results of the SEM analysis. Conclusions: The royal jelly concentration of 25% or higher inhibits bacterial growth; however, the subinhibitory concentration increases pyocyanin production and biofilm formation in P. aeruginosa. It is advisable to determine the appropriate concentration of royal jelly to obtain beneficial virulence inhibiting activity.

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