Pseudomonas aeruginosa is an important pathogenic bacterium of nosocomial infections. The microbe easily produce biofilm which brings us much difficulties in clinical treatment. The formation processes of biofilm, including the stages of early bacteria planting, mushroom-like structure forming and extracellular matrix producing, are regulated by a series of molecules and genes. And quorum sensing system of the microbe is responsible for regulation of the whole process of biofilm formation. According to the process of biofilm formation and the mimitat associated regulation mechanism, several anti-biofilm therapeutic strategies have been applied in clinical medicine, and some novel drugs and methods are developed.
Biofilms ; growth & development ; Gene Expression Regulation, Bacterial ; Polysaccharides, Bacterial ; metabolism ; Pseudomonas Infections ; drug therapy ; microbiology ; Pseudomonas aeruginosa ; genetics ; physiology ; Quorum Sensing ; genetics ; physiology

PURPOSE: Two Korean nationwide studies showed that metallo-beta-lactamases (MBLs)-producing-Pseudomonas spp. are not rare. The aim of this study was to assess the trends of MBL-producing isolates among imipenem-resistant isolates of Pseudomonas spp. MATERIALS AND METHODS: Imipenem-resistant clinical isolates were collected from 23 hospitals and one commercial laboratory participating in the KONSAR program in 2005. Polymerase chain reaction (PCR) was used to detect MBL genes. RESULTS: Alleles of MBL genes were detected in 10.8% of 415 Pseudomonas aeruginosa and 66.7% of 12 P. putida isolates from 18 of 24 hospitals/laboratory. Among the 14 IMP-1-like and 39 VIM-2-like MBLs, emergence of IMP-6 was detected for the first time. CONCLUSION: Prevalence of MBL-producing P. aeruginosa has not significantly increased, but IMP-6 emerged in P. aeruginosa.
Anti-Bacterial Agents/pharmacology ; Electrophoresis, Gel, Pulsed-Field ; Humans ; Imipenem/pharmacology ; Korea ; Polymerase Chain Reaction ; Pseudomonas Infections/*microbiology ; Pseudomonas aeruginosa/drug effects/genetics/*metabolism ; beta-Lactamases/genetics/*metabolism

To investigate the distribution of the genes of two major metallo-beta-lactamases (MBL; i.e., IMP and VIM) and class 1 integrons (intI) in the clinical imipenem-resistant Pseudomonas aeruginosa, a total of 65 isolates, from a university hospital in Sichuan between December 2004 and April 2005 were screened for MBL genes by PCR using primers specific for bla ( IMP-1 ), bla ( VIM ) and bla ( VIM-2 ) genes. The MBL-positive isolates were further assessed for class 1 integrons by PCR using specific primers. The nucleotide sequences of several PCR products were also determined. The results revealed that the bla ( VIM ) gene was found in 81.5% (53/65) of all isolates, bla ( VIM-2 ) gene was found in only 1 isolate and the intI gene was observed in 45.3% (24/53) of bla ( VIM )-positive isolates. One isolate carried simultaneously both bla ( IMP-1 ) and intI genes, and to the best of our knowledge this is the first report of such isolate in southwest China. These observations highlight that the genes for VIM beta-lactamase and class 1 integrons were predominantly present among the imipenem-resistant P. aeruginosa tested, confirming the current widespread threat of imipenem-resistant, integron-borne P. aeruginosa.
Anti-Bacterial Agents/pharmacology ; China ; DNA Primers/chemistry ; Drug Resistance, Bacterial ; Gene Expression Regulation, Bacterial ; Imipenem/*pharmacology ; Integrons ; Microbial Sensitivity Tests ; Models, Genetic ; Pseudomonas Infections/genetics ; Pseudomonas Infections/*microbiology ; Pseudomonas aeruginosa/*metabolism ; Sequence Analysis, DNA ; beta-Lactamases/*metabolism

OBJECTIVETo investigate class I integrons and integrated gene cassettes in metalloenzyme-producing Pseudomonas aeruginosa.

METHODSA total of 68 isolated clinical strains of Pseudomonas aeruginosa were subjected to PCR analysis with primers specific for bla(IMP-1) and bla(VIM). The positive strains then underwent examination for class I integrons and integrated gene cassettes with PCR with primers specific to class I integrase ((IntI)1) and integrated gene cassettes, followed by sequence analysis for some of the positive strains.

RESULTSOnly 1 isolated strain showed positive results for both bla(IMP-1) and bla IntI1 detection. Fifty-five strains were positive for bla(VIM), including 26 positive for bla (IntI)1. Of the 26 bla (IntI)1-positive strains, only 18 contained integrated gene cassettes, which were classified into 5 types according to agarose gel electrophoresis.

CONCLUSIONIt is the first time to identify IMP-1-producing Pseudomonas aeruginosa carring bla(Int)1 in West China. The class I integrons were widespread in these Pseudomonas aeruginosa and 69.2% of them carry the gene cassettes. These findings provide useful insights into the clinical spread of these drug-resistant genes.

Bacterial Proteins ; genetics ; metabolism ; DNA, Bacterial ; analysis ; genetics ; Drug Resistance, Bacterial ; genetics ; Electrophoresis, Agar Gel ; Humans ; Integrons ; genetics ; Polymerase Chain Reaction ; Pseudomonas Infections ; microbiology ; Pseudomonas aeruginosa ; enzymology ; genetics ; isolation & purification ; Species Specificity ; beta-Lactamases ; genetics ; metabolism

OBJECTIVETo study the clinical significance of virulence genes exo U and exo S of type III secretion system (TTSS) of Pseudomonas aeruginosa (PA).

METHODSOne hundred and eighty-nine clinical isolates of PA were collected from five hospitals. The incidence of virulence genes exo U and exo S in PA were determined with PCR. Minimum inhibitory concentration of anti-bacterial drug for PA was determined with microdilution method. The clinical features and outcomes of 60 hospitalized patients colonized or infected with exo U+/exo S- positive or exo U-/exo S+ positive PA isolated from sputum were analyzed retrospectively. Data were processed with chi-square test.

RESULTSAmong the 189 PA isolates, 85.2% (161/189) harbored TTSS genes, including exo U-/exo S+ type (120 isolates), exo U+/exo S- type (31 isolates), exo U-/exo S- type (7 isolates), and exo U+/exo S+ type (3 isolates). 72.0% (72/100) isolates from sputum and 81.5% (44/54) isolates from blood belonged to exo U-/exo S+ genotype. Compared with those of TTSS-negative isolates, the antimicrobial resistance of TTSS-positive isolates to cefoperazone/sulbactam, ceftazidime, amikacin, and cefepime were lower (with χ² value respectively 10.1, 16.1, 9.3, 33.8, P values all below 0.01). The antimicrobial resistance to all examined drug between exo U-/exo S+ type and exo U+/exo S- type isolates was close (with χ² values from 0.08 to 2.04, P values all above 0.05). Patients detected with exo U+/exo S- positive PA isolated from sputum were significantly associated with PA infection, and they usually had history of tracheal intubation, ICU hospitalization, and combined use of drugs for anti-infection treatment. Patients detected with exo U-/exo S+ positive PA isolated from sputum were significantly associated with PA colonization, which had basic lung disease and better outcome than the former infection type.

CONCLUSIONSThe TTSS exists in most clinical isolates of PA. Detection of exo U or exo S of PA isolated from sputum is helpful for the analysis of clinical features and outcome of patients.

ADP Ribose Transferases ; genetics ; metabolism ; Bacterial Proteins ; genetics ; metabolism ; Bacterial Secretion Systems ; genetics ; Bacterial Toxins ; genetics ; metabolism ; Drug Resistance, Bacterial ; Genes, Bacterial ; Humans ; Microbial Sensitivity Tests ; Pseudomonas Infections ; microbiology ; Pseudomonas aeruginosa ; genetics ; isolation & purification ; pathogenicity ; Retrospective Studies ; Virulence

Bronchiectasis is a chronic lung disorder and a number of bacterial pathogens are involved. However, 30%-40% of sputum and purulent samples in good quality failed to grow any pathogenic bacteria, making it difficult to confirm the pathogen. In this study, we collected bronchoalveolar lavage fluid from a bronchiectasis patient undergoing acute exacerbation, and sent for 16S rDNA pyrosequencing by a 454 GS Junior machine. Metagenomic analysis showed the composition of bacterial community in sample was complex. More than a half of reads (51.3%) were from Pseudomonas aeruginosa. This result was corresponding with the culture result but came out 2 d earlier, which is meaningful for early diagnosis and treatment. The detection with 16S rDNA pyrosequencing technology is more sensitive and rapid than routine culture, and can detect the co-infection or symbiosis in airway, giving us a novel and convenient approach to perform rapid diagnosis.
Bronchiectasis ; microbiology ; Bronchoalveolar Lavage Fluid ; chemistry ; microbiology ; Early Diagnosis ; Female ; Humans ; Metagenome ; genetics ; Metagenomics ; methods ; Middle Aged ; Pseudomonas Infections ; microbiology ; Pseudomonas aeruginosa ; genetics ; isolation & purification ; RNA, Ribosomal, 16S ; genetics ; Time Factors

BACKGROUNDCarbapenems are an important class of drugs for the treatment of Pseudomonas aeruginosa (P. aeruginosa) infections. However, carbapenem resistance has been commonly observed in nonfermenter species of bacteria. The purpose of this study was to investigate the molecular epidemiology and carbapenem resistant mechanisms of P. aeruginosa isolated from a surgical intensive care unit (SICU) in China.

METHODSThe molecular typing was analyzed by REP-PCR. Enzyme activity was measured with a 260 nm wavelength spectrophotometer. The levels of outer membrane proteins OprD and OprN were measured by Western blotting. The levels of mexA gene transcriptional expression were measured by quantitative real-time PCR. The metallo-beta-lactamase genes IMP, VIM, SPM, GES, and GIM were amplified by PCR. DNA fragments were sequenced by an automated ABI PRISM 3700.

RESULTSForty-two strains resistant to carbapenems isolated from a SICU were analyzed. REP-PCR revealed 34 belonging to type A, a predominant strain in this SICU. But we did not find metallo-beta-lactamases IMP, VIM, SPM, GES, or GIM genes by PCR. With a three-dimensional extract test, we found 34 strains producing high levels of AmpC enzymes. We also observed the activity of beta-lactamases enzymes in the imipenem resistant group, which was statistically different from the sensitive group. Western blotting revealed that 23 strains showed loss of OprD, 18 strains had decreased OprD expression, and 14 strains expressed OprN. We discovered 27 strains that overexpressed mexA by quantitative real-time PCR, and the resistance rate to meropenem was statistically different between the overexpressing group and the low-expressing group. Nucleotide sequences and deduced amino acid sequence analysis revealed that eight strains carried mutations in the mexR gene operon down regulating MexAB-OprM. The nucleotide sequences of mexR genes from PA36, PA41 and PA48 were submitted to the Genebank with accession numbers of AY899299, AY899300, and AY899301.

CONCLUSIONSThere was a predominant strain in the SICU of our hospital. Imipenem resistance is mainly mediated by OprD deficiency or loss, and high activity AmpC enzymes. Overexpression of MexAB-OprM is one of the mechanisms of meropenem resistance, which are partly upregulated by mutations in the mexR gene. The expression of MexEF-OprN also plays an important role in the carbapenem resistance.

Bacterial Proteins ; genetics ; metabolism ; Blotting, Western ; Carbapenems ; pharmacology ; China ; Humans ; Intensive Care Units ; Microbial Sensitivity Tests ; Pseudomonas Infections ; microbiology ; Pseudomonas aeruginosa ; drug effects ; genetics ; Real-Time Polymerase Chain Reaction ; beta-Lactamases ; genetics ; metabolism

Objective: To identify the antibiotic resistance, virulence genes, and sequence types of Pseudomonas aeruginosa (P. aeruginosa) strains isolated from blood. Methods: From November 2014 to December 2021, a total of 94 nonrepetitive P. aeruginosa isolates were obtained from blood samples of patients at the First Affiliated Hospital of Shandong First Medical University in Shandong Province, China. The bacteria were identified using matrix-assisted laser desorption ionization time of flight mass spectrometry. Antibiotic resistance of the P. aeruginosa isolates was detected using Vitek 2 Compact system. Polymerase chain reaction (PCR) was conducted for the 18 virulence genes, and multi locus sequence typing (MLST) was performed to identify the sequence types of the P. aeruginosa strains. The resistance rates and distributions of virulence genes between carbapenem resistant pseudomonas aeruginosa (CRPA) and carbapenem susceptible pseudomonas aeruginosa (CSPA) isolates were compared using the Chi-square test. Results: Among 94 P. aeruginosa isolates, 19 (20.2%) isolates were found to be multidrug resistant (MDR) bacteria, of which 17 were CRPA isolates and 2 were CSPA isolates. All strains contained more than 10 virulence genes. Except for exoU gene, the detection rate of other genes was above 83%. MLST analysis revealed a total of 66 different STs, including 59 existing STs and 7 novel STs. Among them, ST244 (n=11, 11.7%) and ST270 (n=7, 7.4%) were the dominant STs. Although these two types of isolates harbored the same virulence genes, the resistance rates to carbapenem were different. 54.5% (6/11) ST244 isolates were CRPA but all 7 ST270 isolates were CSPA. Conclusion: Although the resistance rates of P. aeruginosa strains isolated from blood were at a low level, some MDR and CRPA isolates were detected. As the high virulence gene detection rates and genetic diversity were found for P. aeruginosa strains isolated from blood, close attention should be paid to avoid transmission and outbreaks.
Humans ; Pseudomonas aeruginosa/genetics* ; Multilocus Sequence Typing ; Molecular Epidemiology ; Pseudomonas Infections/microbiology* ; Microbial Sensitivity Tests ; Hospitals ; Carbapenems/pharmacology* ; Drug Resistance, Multiple, Bacterial/genetics* ; Anti-Bacterial Agents/pharmacology* ; beta-Lactamases

Objective: To identify the antibiotic resistance, virulence genes, and sequence types of Pseudomonas aeruginosa (P. aeruginosa) strains isolated from blood. Methods: From November 2014 to December 2021, a total of 94 nonrepetitive P. aeruginosa isolates were obtained from blood samples of patients at the First Affiliated Hospital of Shandong First Medical University in Shandong Province, China. The bacteria were identified using matrix-assisted laser desorption ionization time of flight mass spectrometry. Antibiotic resistance of the P. aeruginosa isolates was detected using Vitek 2 Compact system. Polymerase chain reaction (PCR) was conducted for the 18 virulence genes, and multi locus sequence typing (MLST) was performed to identify the sequence types of the P. aeruginosa strains. The resistance rates and distributions of virulence genes between carbapenem resistant pseudomonas aeruginosa (CRPA) and carbapenem susceptible pseudomonas aeruginosa (CSPA) isolates were compared using the Chi-square test. Results: Among 94 P. aeruginosa isolates, 19 (20.2%) isolates were found to be multidrug resistant (MDR) bacteria, of which 17 were CRPA isolates and 2 were CSPA isolates. All strains contained more than 10 virulence genes. Except for exoU gene, the detection rate of other genes was above 83%. MLST analysis revealed a total of 66 different STs, including 59 existing STs and 7 novel STs. Among them, ST244 (n=11, 11.7%) and ST270 (n=7, 7.4%) were the dominant STs. Although these two types of isolates harbored the same virulence genes, the resistance rates to carbapenem were different. 54.5% (6/11) ST244 isolates were CRPA but all 7 ST270 isolates were CSPA. Conclusion: Although the resistance rates of P. aeruginosa strains isolated from blood were at a low level, some MDR and CRPA isolates were detected. As the high virulence gene detection rates and genetic diversity were found for P. aeruginosa strains isolated from blood, close attention should be paid to avoid transmission and outbreaks.
Humans ; Pseudomonas aeruginosa/genetics* ; Multilocus Sequence Typing ; Molecular Epidemiology ; Pseudomonas Infections/microbiology* ; Microbial Sensitivity Tests ; Hospitals ; Carbapenems/pharmacology* ; Drug Resistance, Multiple, Bacterial/genetics* ; Anti-Bacterial Agents/pharmacology* ; beta-Lactamases

OBJECTIVE: To evaluate the in vitro activity of ceftazidime-avibactam (CAZ-AVI) alone or in combination with colistin (COL) against clinically isolated extensively drug-resistant Pseudomonas aeruginosa (XDR-PA). METHODS: Minimum inhibitory concentration (MIC) of 16 clinical XDR-PA isolates was determined by broth dilution method and chessboard design when CAZ-AVI and COL were used alone or in combination, then the combined inhibitory concentration index (FICI) was calculated. Class A [Klebsiella pneumoniae carbapenemase β-lactamase (bla_KPC), Guiana extended-spectrum β-lactamase (bla_GES)], Class B [imipenemase β-lactamase (bla_IMP), Verona-Integronmetallo β-lactamase (bla_VIM), New Delhi metallo β-lactamase (bla_NDM), German imipenemase β-lactamase (bla_GIM), Sao Paulo metallo-β-lactamase (bla_SPM)], Class C [AmpC β-lactamase (bla_AmpC)], Class D [oxacillinase β-lactamase (bla_OXA)] β-lactamase-related resistance genes were detected by polymerase chain reaction. Drug-resistant mutation frequencies of each strain were determined on a drug-containing plate. The time kill curves of three XDR-PA were plotted by colony counting method. A biofilm model was established in vitro, and the synergistic effect of CAZ-AVI and COL on biofilm inhibition was detected by methythiazolyl tetrazolium assay (MTT). RESULTS: The MICs of 16 XDR-PA for CAZ-AVI ranged from 1 mg/L to 128 mg/L, and three of the isolates showed resistance (MIC > 8 mg/L). The FICI range of CAZ-AVI combined with COL was 0.312-1.000. Four isolates were synergistic, while the other 12 isolates were additive. Three isolates resistant to CAZ-AVI contained Class B resistance genes such as bla_IMP and bla_VIM, while 13 susceptible isolates carried resistance genes belonging to Class A, C or D. The logarithm values of mutation frequencies of drug resistance in CAZ-AVI group, COL group and combination group were -4.81±0.88, -7.06±0.69 and -9.70 (-9.78, -9.53), respectively. There were significant differences among the three groups (H = 33.601, P < 0.001), and between every two groups (adjusted P < 0.05). In time kill curves, the phytoplankton load of three XDR-PA decreased more than 6 log CFU/L when these two drugs were used together, and number of PA1819 planktonic bacteria decreased more than 5.1 log CFU/L compared with monotherapy group. Viable quantity in biofilm (A₄₉₀) of normal saline group, CAZ-AVI group, COL group and CAZ-AVI-COL group were 0.665±0.068, 0.540±0.072, 0.494±0.642 and 0.317±0.080, respectively. There was significant difference between the other two groups (all P < 0.001), except for that between CAZ-AVI group and COL group (P = 0.109). CONCLUSIONS CAZ-AVI combined with COL can effectively improve the bactericidal effect of each drug alone on XDR-PA. The regimen can also reduce the production of drug-resistant bacteria and inhibit the formation of biofilm. Therefore, it is a potential treatment for XDR-PA infection.
Anti-Bacterial Agents/therapeutic use* ; Azabicyclo Compounds/therapeutic use* ; Ceftazidime/therapeutic use* ; Colistin/therapeutic use* ; Drug Combinations ; Drug Resistance, Bacterial/genetics* ; Microbial Sensitivity Tests ; Pseudomonas Infections/drug therapy* ; Pseudomonas aeruginosa ; beta-Lactamases