OBJECTIVETo investigate the resistance genes and antibiotic resistance patterns against beta-lactams in Pseudomonas aeruginosa prevalent in burn ward.

METHODSK-B method was performed to test bacterial resistance patterns against 9 species of beta-lactams in Pseudomonas aeruginosa isolated from wounds and dressings of the patient in burn wards. Seven species of resistance genes against beta-lactams were detected with PCR. Tazobactam-inhibited piperacillin resistance test was performed to study whether the above strains produce extended spectrum beta-lactams.

RESULTSAll 12 strains of bacteria with resistance genes detected were resistant to penicillin and cephalosporins (100%), among them 11 were resistant to all antibiotics. Tazobactam-inhibited piperacillin resistance test demonstrated that all strains with resistance genes were ESBLs.

CONCLUSIONHigh incidence of beta-lactams resistance genes is found in Pseudomonas aeruginosa isolated from burn ward, and they have close relationship with the occurrence of multiple drug-resistance.

Burn Units ; Burns ; microbiology ; Genes, Bacterial ; Humans ; Pseudomonas aeruginosa ; drug effects ; genetics ; isolation & purification ; beta-Lactam Resistance ; genetics

OBJECTIVETo analyze the isolation and the in vitro susceptibility of P. aeruginosa to antibiotics in our burn ward.

METHODSFive hundred and thirty six burn patients admitted to our ward from 1997 to 2003 were enrolled in the study, and the wound excretion, the tips of the venous catheter, the subeschar tissue samples, and the blood samples were collected for bacterial identification and antibiotic susceptibility test with VITEK-AMS system.

RESULTSThe isolation rate of P. aeruginosa from 1997 to 2003 was 24.51%, 23.94%, 21.01%, 40.06%, 36.17%, 46.76% and 55.72%, respectively. The antibiotic effect of the third generation of Cephalosporins against the said bacteria showed a tendency to decline. The susceptibility rate to Cefoperazone, Ceftazidime and Cefotaxime were respectively 71%, 66% and 79% in 1997; 47%, 25%, 39% in 1998; 22%, 16%, 25% in 2002; The third generation cephalosporins had almost lost their antibiotic activity against P. aeruginosa in 2003, with the susceptibility rate to Cefotaxime lowered to 2%. The susceptibility rate to Imipenem from 1997 to 2003 was 76%, 33%, 45%, 11%, 41%, 31%and 4%, respectively.

CONCLUSIONThe isolation rates of P. aeruginosa were steady during the period from 1997 to 1999, and they began to increase in 2000. The bacterial resistance to antibiotics increased gradually in recent years, and the strains of P. aeruginosa had become multi-drug resistant.

Anti-Bacterial Agents ; pharmacology ; Burn Units ; Burns ; microbiology ; Drug Resistance, Multiple, Bacterial ; Humans ; In Vitro Techniques ; Microbial Sensitivity Tests ; Pseudomonas Infections ; microbiology ; Pseudomonas aeruginosa ; drug effects ; isolation & purification

Aged ; Cross Infection ; microbiology ; Female ; Humans ; Intensive Care Units ; Male ; Microbial Sensitivity Tests ; Pneumonia, Bacterial ; microbiology ; Pseudomonas Infections ; microbiology ; Pseudomonas aeruginosa ; classification ; isolation & purification

BACKGROUND: Delayed entry of blood culture bottles is inevitable when microbiological laboratories do not operate for 24 hr. There are few studies reported for prestorage of these bottles. The growth dynamics of Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa were investigated with respect to various preincubation conditions. METHODS: Fifteen or 150 colony-forming units (CFU) of bacteria were inoculated into standard aerobic or anaerobic blood culture bottles. Bottles were preincubated at 25degrees C or 37degrees C for 0, 2, 4, 8, 12, 24, or 48 hr. The time to detection (TTD) then was monitored using the BacT/Alert 3D system (bioMerieux Inc., USA). RESULTS: Significant difference in TTD was observed following preincubation for 8 hr at 25degrees C vs. 4 hr at 37degrees C for S. aureus, 4 hr at 25degrees C vs. 4 hr at 37degrees C for E. coli, 12 hr at 25degrees C vs. 4 hr at 37degrees C for P. aeruginosa, compared to no preincubation (P<0.005). TTD values did not vary significantly with bacterial CFU or with aerobic or anaerobic bottle type. The BacT/Alert 3D system returned false negatives following preincubation of P. aeruginosa for 48 hr at 25degrees C or 24 hr at 37degrees C. CONCLUSIONS: TTD was mainly affected by preincubation temperature and duration rather than by input CFU quantity or bottle type for the 3 experimental bacteria.
Bacteriological Techniques/instrumentation/*methods ; Culture Media ; Escherichia coli/growth & development/*isolation & purification ; Pseudomonas aeruginosa/growth & development/*isolation & purification ; Staphylococcus aureus/growth & development/*isolation & purification ; Temperature ; Time Factors

OBJECTIVETo develop a high-throughput diagnostic method with suspension array technique for detecting pathogenic microbes.

METHODSThe probes and positive controls of 56 kinds of pathogenic microbes were designed, synthesized, and used to detect pathogenic microbes with suspension array technique.

RESULTSFluorescence signals of 56 positive controls were higher than those of the negative controls, and there was no cross-reaction between the probes and positive controls of different microbes.

CONCLUSIONBased on suspension array technique, the high-throughput diagnostic method may be useful in clinical detection of pathogenic microbes.

Animals ; Communicable Diseases ; microbiology ; virology ; Enterovirus ; isolation & purification ; Humans ; Microbiological Techniques ; Mumps virus ; isolation & purification ; Oligonucleotide Array Sequence Analysis ; methods ; Pseudomonas aeruginosa ; isolation & purification

BACKGROUND: Carbapenem-resistant Pseudomonas aeruginosa (CRPA) and Acinetobacter baumannii (CRAB) are the leading causes of nosocomial infections. A rapid and sensitive test to detect CRPA and CRAB is required for appropriate antibiotic treatment. We optimized a loop-mediated isothermal amplification (LAMP) assay to detect the presence of bla(VIM-2), bla(IMP-1), and bla OXA-23, which are critical components for carbapenem resistance. METHODS: Two sets of primers, inner and outer primers, were manually designed as previously described. The LAMP buffer was optimized (at 2mM MgSO4) by testing different concentrations of MgSO4. The optimal reaction temperature and incubation time were determined by using a gradient thermocycler. Then, the optimized bla(VIM-2), bla(IMP-1), and bla(OXA-23) LAMP reactions were evaluated by using 120 P. aeruginosa and 99 A. baumannii clinical isolates. RESULTS: Only one strain of the 100 CRPA isolates harbored bla(IMP-1), whereas none of them harbored bla(VIM-2). These results indicate that the acquisition of bla(VIM-2) or bla(IMP-1) may not play a major role in carbapenem resistance in Korea. Fifty two strains of the 75 CRAB isolates contained bla(OXA-23), but none contained bla(VIM-2) and bla(IMP-1) alleles. CONCLUSIONS: Our results demonstrate the usefulness of LAMP for the diagnosis of CRPA and CRAB.
Acinetobacter baumannii/genetics/*isolation & purification ; Anti-Bacterial Agents/*pharmacology ; Carbapenems/*pharmacology ; Drug Resistance, Bacterial/*genetics ; *Genes, Bacterial ; Nucleic Acid Amplification Techniques ; Pseudomonas aeruginosa/genetics/*isolation & purification ; Sensitivity and Specificity

The 16S rDNA specific primers were designed for rapid detection of Pseudomonas aeruginosa (PA) by the fluorescence quantitative PCR (FQ-PCR) assay, based upon multiple sequence alignment and phylogenetic tree analysis of the 16S rDNAs of over 20 bacteria. After extraction of PA genomic DNA, the target 16S rDNA fragment was amplified by PCR with specific primers, and used to construct recombinant pMDT-Pfr plasmid, the dilution gradients of which were subjected to the standard quantitation curve in FQ-PCR assay. Different concentrations of PA genomic DNA were detected by FQ-PCR in a 20microL of reaction system with SYBR Green I. At the same time, various genomic DNAs of Staphylococcus aureus, Salmonella typhi, Shigella flexneri, Proteus vulgaris, Staphylococcus epidermidis, Escherichia coli, and Mycobacterium tuberculosis were used as negative controls to confirm specificity of the FQ-PCR detection assay. Results demonstrated that the predicted amplified product of designed primers was of high homology only with PA 16S rDNA, and that sensitivity of the FQ-PCR assay was of 3.6pg/microL of bacterial DNA or (2.1 x 10(3) +/- 3.1 x 10(2)) copies/microL of 16S rDNA, accompanied with high specificity, and that the whole detection process including DNA extraction could be completed in about two hours. In contrast to traditional culture method, the FQ-PCR assay targeting 16S rDNA gene can be used to detect PA rapidly, which exhibits perfect application prospect in future.
Base Sequence ; DNA, Ribosomal ; genetics ; Fluorescence ; Molecular Sequence Data ; Polymerase Chain Reaction ; methods ; Pseudomonas aeruginosa ; genetics ; isolation & purification ; RNA, Ribosomal, 16S ; genetics

OBJECTIVETo investigate the relationship between the drug resistance of Pseudomonas aeruginosa (PA) isolated from burn patients wounds and its mobile genetic elements, including plasmid, transposon, and integron.

METHODSThirty-two strains of PA were isolated from wounds exudate of hospitalized burn patients in Ningbo No. 2 Hospital. PA drug sensitivity was determined using GNS-448 drug sensitivity card and K-B tests. The genetic markers of plasmid, transposon and integron including traA, traF, tnpA, tnpU, merA, int I 1 were amplified by PCR and verified by gene sequencing.

RESULTSDrug resistant rate of 32 PA strains to gentamicin, amikacin, cefoperazone/sulbactam, ciprofloxacin was 43.7%, 32.0%, 46.8%, 49.9%, respectively. PA drug resistant rates to piperacillin, cefotaxime, ceftazidime, cefepime, aztreonam, piperacillin/tazobactam, levofloxacin, imipenem and meropenem were all above 56.0%. Seventeen out of 32 PA strains were found to carry transposon and (or) integron genetic markers. One strain was positive for both tnpA and merA, 8 strains were positive for both merA and int I 1, 1 strain was only positive for tnpA, 2 strains were only positive for merA, and 5 strains were positive for int I 1 only.

CONCLUSIONSPA isolated from burn wounds of hospitalized patients in Ningbo No. 2 Hospital is seriously drug resistant, which may relate with its high positive rate of mobile genetic elements of transposon and (or) integron.

Anti-Bacterial Agents ; pharmacology ; Burns ; microbiology ; DNA Transposable Elements ; Drug Resistance, Multiple, Bacterial ; genetics ; Humans ; Integrons ; Microbial Sensitivity Tests ; Plasmids ; Pseudomonas aeruginosa ; drug effects ; genetics ; isolation & purification

OBJECTIVETo investigate drug-resistant genes associated with beta-lactams and aminoglycosides in clinically isolated Pseudomonas aeruginosa.

METHODSTwenty strains of Pseudomonas aeruginosa were isolated from wound excretion of hospitalized burn patients. The strains resistant to 14 antibiotics were selected for detection of 16 kind of drug-resistant genes (TEM, SHV, OXA-10 cluster, PER, VEB, GES, CARB, CTX-M- I, IMP, VIM, SPM, GIM, DHA, MOX, FOX, oprD2) and 6 kind of aminoglycoside modification genes (aac(3)- I, aac(3)-II, aac(6')-I, aac(6')-II, ant (3")- I , ant(2")- I) in them by PCR.

RESULTSAmong the 20 strains resistant to beta-lactam , all of them were TEM and GES positive (100%), oprD2 gene depletion in 5 strains (25%). All other genes were negative. Among aminoglycoside resistant genes, 20 strains were aac (6') - I positive (100%), 7 were ant (2") - I positive (35%), and negative for other stains.

CONCLUSIONThere were very high existence rates of TEM, GES and aac (6')- I genes in Pseudomonas aeruginosa isolated from clinical burn patients. The fact that GES-5 gene has also been detected in Pseudomonas aeruginosa, suggesting this organism is highly drug resistant in our burn unit.

Aminoglycosides ; pharmacology ; Drug Resistance, Multiple, Bacterial ; genetics ; Humans ; Pseudomonas aeruginosa ; drug effects ; genetics ; isolation & purification ; beta-Lactam Resistance ; genetics ; beta-Lactams ; pharmacology

OBJECTIVETo analyze the drug resistance and drug resistance genes of imipenem-resistant Pseudomonas aeruginosa (IRPA) strains isolated from burn wards.

METHODSFrom June 2011 to June 2012, 30 strains of IRPA were isolated from wound excretion, sputum, and venous catheter attachment from burn patients hospitalized in Guangzhou Hospital of Integrated Traditional Chinese and Western Medicine. Drug resistance of the IRPA to 12 antibiotics commonly used in clinic, including ceftazidime, amikacin, ciprofloxacin, etc., was tested with K-B paper agar disk diffusion method. Metallo-β-lactamase (MBL)-producing IRPA was detected by synergism test with imipenem-2-mercaptoethanol. Plasmid of IRPA was extracted, and it was inserted into competent cells, producing transformation strains (TSs). Drug resistance of TSs to imipenem and the MBL-producing TSs were detected. The genes blaIMP, blaVIM, blaOXA-1, blaOXA-2 and blaOXA-10 of IRPA and the TSs were detected by polymerase chain reaction. The drug resistance of IRPA producing MBL or OXA enzyme was summed up.

RESULTSThe sensitive rates of the 30 strains of IRPA to the 12 antibiotics were equal to or above 60.0%. Six strains of MBL-producing IRPA were screened. Twenty-four TSs were resistant to imipenem, and 6 strains among them were MBL-producing positive. Among the 30 strains of IRPA, 6 strains and their corresponding TSs carried blaVIM; 20 strains and their corresponding TSs carried blaOXA-10; no strain was detected to carry blaIMP, blaOXA-1 or blaOXA-2. Two strains and their corresponding TSs were detected carrying both blaVIM and blaOXA-10. No significant difference of drug resistance was observed between strains producing only MBL or OXA enzyme, with the same high resistance to β-lactam antibiotics and some degree of sensitivity to aminoglycoside antibiotics. Strains producing enzymes MBL and OXA were all resistant to the 12 antibiotics.

CONCLUSIONSIRPA strains isolated from burn wards of Guangzhou Hospital of Integrated Traditional Chinese and Western Medicine are multidrug-resistant, and they mainly produce type B and D carbapenemases.

Burns ; microbiology ; Cross Infection ; microbiology ; Drug Resistance, Multiple, Bacterial ; genetics ; Humans ; Imipenem ; Microbial Sensitivity Tests ; Pseudomonas aeruginosa ; drug effects ; genetics ; isolation & purification