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

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

OBJECTIVEThe catheter-related infections caused by mechanical ventilation have become a intractable clinical problem, and it is related to the formation of bacterial biofilm (BF) on the surface of the implanted material. The majority of natural biofilms are formed by multiple bacterial species. However, there always only one or limited species were detected on tracheal tubes removed from intubated neonates by using traditional methods including bacterium culture and antigen detection. The aims of this study were to observe the bacterial communities diversity of BF on endotracheal tube (ETT), and discuss the difference between traditional bacterium culture methods and the use of molecular biology techniques on the basis of denatured gradient gel electrophoresis (DGGE), to provide new ideas for clinical prevention, diagnosis and treatment of bacterial infections.

METHODThirty-five ETTs were obtained from 26 neonates on mechanical ventilator (from October 2012 to March 2013) in Department of Neonatology of Children's Hospital. Among the patients, 18 were boys and 8 girls, and 19 patients were < 37 weeks gestational age and 7 patients ≥ 37 weeks. DGGE profiling of 16S rDNA gene amplicons was used to assess the diversity of the bacterial population by using the software of quantity one. TA Cloning Kit and sequencing were used to investigate the distribution of bacteria and common dominant bacteria in ETT-BF.

RESULTThe mean bands of 35 ETTs cases were 13.8 ± 5.4 from 16S rDNA PCR-DGGE, and the mean Shanon-Wiener indexes was 2.42 ± 0.38. The 16 ETTs were collected in different stages of diseases from the 7 patients. The indwelling days of 6/7 patients' ETTs increased, the Shanon-siener indexes were decreased. Among the 6 cases from different basic illnesses, and there were different Shanon-siener indexes. The result of molecular cloning and sequencing for 24 dominant bands showed that 35 cases (100%) contained Klebsiella SP·, 28 cases (80%) had Pseudomonas SP·, 27 cases (77%) had Streptococcus SP·, and 32 cases (91%) had Uncultured bacterium, while more than 2 bacterial species were found in 34 cases (97%). 28/35 (80%) Klebsiella SP· and 22/27(82%) Streptococcus SP· were accompanied by Pseudomonas SP·. There were 22 positive results of sputum culture from 26 newborns, including 10 strains (45%) of Klebsiella pneumoniae, 2 strains (9%) of Acinetobacter baumannii, Enterobacter cloacae and non-cultured bacterium in each patient (5%), but only one bacterium isolated from every sputum. Eight sputum samples had normal flora only, corresponding to the ETTs on which Klebsiella and other bacterial genuses were found.

CONCLUSIONThe diversity of microbiota in BF on ETT was confirmed. 16S rDNA PCR-DGGE could produce a more complete picture of bacterial community than traditional bacterium culture method. Klebsiella, Pseudomonas and Streptococcus were common dominant bacteria in ETT-BF, and there might be interactions among them in the formation of BF.

Bacteria ; classification ; genetics ; isolation & purification ; Biodiversity ; Biofilms ; Catheter-Related Infections ; microbiology ; DNA, Bacterial ; analysis ; genetics ; Denaturing Gradient Gel Electrophoresis ; Female ; Humans ; Infant, Newborn ; Intubation, Intratracheal ; adverse effects ; Klebsiella ; genetics ; isolation & purification ; Male ; Polymerase Chain Reaction ; Pseudomonas ; genetics ; isolation & purification ; RNA, Ribosomal, 16S ; genetics ; Respiration, Artificial ; adverse effects ; Sputum ; microbiology ; Streptococcus ; genetics ; isolation & purification ; Ventilators, Mechanical ; microbiology

The p38 mitogen-activated protein kinase (MAPK) plays an evolutionarily conserved role in the cellular response to microbial infection and environmental stress. Activation of p38 is mediated through phosphorylation by upstream MAPKK, which in turn is activated by MAPKKK. In the Caenorhabditis elegans, the p38 MAPK (also called PMK-1) signaling pathway has been shown to be required in its resistance to bacterial infection. However, how different upstream MAP2Ks and MAP3Ks specifically contribute to the activation of PMK-1 in response to bacterial infection still is not clearly understood. By using double-stranded RNA-mediated interference (RNAi) and genetic mutants of C. elegans, we demonstrate that C. elegans MOM-4, a mammalian TAK1 homolog, is required for the resistance of C. elegans to a P. aeruginosa infection. We have also found that the MKK-4 of C. elegans is required for P. aeruginosa resistance, but not through the regulation of DLK-1. In summary, our results indicate that different upstream MAPKKKs or MAPKKs regulate the activation of PMK-1 in response to P. Aeruginosa.
Animals ; Caenorhabditis elegans ; enzymology ; genetics ; immunology ; microbiology ; Caenorhabditis elegans Proteins ; genetics ; metabolism ; Disease Resistance ; Enzyme Activation ; MAP Kinase Kinase 1 ; metabolism ; MAP Kinase Signaling System ; Membrane Proteins ; deficiency ; genetics ; metabolism ; Mutation ; Pseudomonas Infections ; enzymology ; Pseudomonas aeruginosa ; physiology ; RNA Interference ; p38 Mitogen-Activated Protein Kinases ; metabolism

OBJECTIVETo investigate drug-resistance and carriage of virulence factors of Pseudomonas aeruginosa (Pa) isolated from children.

METHODThirty-eight strains of Pa were collected and isolated in pediatric clinic during 2006-2009, and tests were undertaken to identify bacteria and susceptibility test was performed using VITEK-2 COMPACT GNI and AST-GN13 cards. The virulence factors were confirmed by using polymerase chain reaction (PCR) and sequencing.

RESULTAll the 38 strains of Pa were resistant to ampicillin, ampicillin/sulbactam, cefazolin, nitrofurantoin, trimethoprim/sulfamethoxazole, resistance rates were 100%. Except for ceftriaxone (60.53%), the resistance rates to other antibiotics were all below 16%. PCR test showed that all the 38 strains of Pa carried exotoxin A(toxA) and nitric oxide reductase A (norA), however, detective ratio of the other virulence factors, exoenzyme Y (exoY) was 84.21% (32/38), exoenzyme S (exoS) 57.89% (22/38), pyocyanin (pyp) 42.11% (16/38), exoenzyme U (exoU) 34.21% (13/38), and 38 strains of Pa did not carry exoenzyme T (exoT) and elastase B (lasB) without exception. By analyzing tests, we discovered that 3 pan-drug resistant strains of Pa were all combination of exo U+/pyp+, there were 4 strains of Pa which were moderately-resistant to imipenem, including exoU+/pyp+/exoY+ (2 isolates), exo U+/pyp+ (1 isolate), and exoY+/exoS+ (1 isolates). It indicated that the drug-resistance rate of exoU+/pyp+ is much higher, compared with exoS+ and exoY+. Molecular epidemiological detection revealed that 2 of 3 extensive-resistance strains of Pa were the same clone, but another one had 96.3% of homology with them.

CONCLUSIONThe above mentioned 34.21% of Pa isolated from children carried virulence factors toxA, norA, exoS, exoY, pyp and exoU. The strains with exoU/pyp had rather high resistance. The strains with pyp had strong toxicity, they easily cause generalized infection, the patients with them had very high mortality.

ADP Ribose Transferases ; genetics ; Bacterial Proteins ; genetics ; Bacterial Toxins ; genetics ; Carrier State ; epidemiology ; microbiology ; Child ; Drug Resistance, Multiple, Bacterial ; genetics ; Exotoxins ; Genes, Bacterial ; Humans ; Microbial Sensitivity Tests ; Polymerase Chain Reaction ; Pseudomonas Infections ; epidemiology ; genetics ; microbiology ; Pseudomonas aeruginosa ; genetics ; isolation & purification ; pathogenicity ; Virulence Factors ; genetics

Bacteria survive in nature by forming biofilms on surfaces and probably most, if not all, bacteria (and fungi) are capable of forming biofilms. A biofilm is a structured consortium of bacteria embedded in a self-produced polymer matrix consisting of polysaccharide, protein and extracellular DNA. Bacterial biofilms are resistant to antibiotics, disinfectant chemicals and to phagocytosis and other components of the innate and adaptive inflammatory defense system of the body. It is known, for example, that persistence of staphylococcal infections related to foreign bodies is due to biofilm formation. Likewise, chronic Pseudomonas aeruginosa lung infections in cystic fibrosis patients are caused by biofilm growing mucoid strains. Gradients of nutrients and oxygen exist from the top to the bottom of biofilms and the bacterial cells located in nutrient poor areas have decreased metabolic activity and increased doubling times. These more or less dormant cells are therefore responsible for some of the tolerance to antibiotics. Biofilm growth is associated with an increased level of mutations. Bacteria in biofilms communicate by means of molecules, which activates certain genes responsible for production of virulence factors and, to some extent, biofilm structure. This phenomenon is called quorum sensing and depends upon the concentration of the quorum sensing molecules in a certain niche, which depends on the number of the bacteria. Biofilms can be prevented by antibiotic prophylaxis or early aggressive antibiotic therapy and they can be treated by chronic suppressive antibiotic therapy. Promising strategies may include the use of compounds which can dissolve the biofilm matrix and quorum sensing inhibitors, which increases biofilm susceptibility to antibiotics and phagocytosis.
Animals ; Antibiotic Prophylaxis ; Biofilms ; drug effects ; growth & development ; Chronic Disease ; Cystic Fibrosis ; microbiology ; Drug Resistance, Microbial ; physiology ; Foreign Bodies ; microbiology ; Humans ; Microbial Consortia ; drug effects ; genetics ; immunology ; Phagocytosis ; Pseudomonas Infections ; microbiology ; Pseudomonas aeruginosa ; drug effects ; genetics ; physiology ; Quorum Sensing ; drug effects ; genetics

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