OBJECTIVETo observe the effect of amino acid substitution in conserved sequence of penicillin-binding protein (PBP) 1A, 2B, 2X on antimicrobial activity of beta-lactams against Streptococcus pneumoniae (SP).

METHODMinimal inhibitory concentration (MIC) of 6 beta-lactams was determined by the E-test in 59 SP strains. The penicillin-binding protein genes pbp1a, 2b, 2x in every SP strain were amplified by nested-polymerase chain reaction (nPCR), then the PCR products were sequenced using automatic genetic analyzer directly. To analyze the amino acid substitutions, the DNA sequences were converted to protein sequences and aligned by Clustalx software. According to amino acid substitution in conserved sequence of PBP2B, 3 phenotypes were observed, including: PBP2B phenotype I (no amino acid substitution); PBP2B phenotype II (Glutamine 432-->Leucine and/or Threonine 445/451-->Alanine/Serine, Glutamic 481-->Glycine, 1 strain had proline insertion between residues 431/432); PBP2B phenotype III (Alanine 624-->Glycine with the addition of phenotype II). According to amino acid substitution in conserved sequence of PBP1A, 3 phenotypes were observed, including: PBP1A phenotype I (no amino acid substitution); PBP1A phenotype II (Threonine 574-->Asparagine, Serine 575-->Threonine, Glutamine 576-->Glycine, Phenylalanine 577-->Tyrosine, 574TSQF-->NTGY); PBP1A III (Threonine 371-->Alanine/Serine, Proline 432-->Threonine with the addition of 574TSQF-->NTGY). According to amino acid substitution in conserved sequence of PBP2X, 4 phenotypes were observed, including: PBP2X phenotype I (no amino acid substitution); PBP2X phenotype II (Histidine 394-->Leucine or Threonine 338-->Alanine); PBP2X phenotype III (Threonine 338-->Alanine, Isoleucine 371-->Threonine, Arginine 384-->Glycine and Leucine 546-->Valine); PBP2X phenotype IV (Methionine 339-->Phenylalanine, Methionine 400-->Threonine with the addition of PBP2X phenotype III).

RESULTAmong 59 SP strains antibacterial activities distribution (sensitive strains, intermediate strains and resistant strains) of 6 beta-lactams were penicillin (12, 29, 18); amoxicillin(49, 9, 1); cefuroxime (16, 16, 27); ceftriaxone (47, 1, 11); cefotaxime (47, 3, 9); imipenem (49, 10, 0). beta-lactam antibiotics insensitive strains (intermediate + resistant strain) in PBP2B phenotype III, PBP1A phenotype III, PBP2X phenotype III and IV were significantly increased, the MIC(50) of these strains were significantly higher than that of the others.

CONCLUSIONThe amino acid substitutions in or vicinal conserved sequence of PBP of SP increase MIC for beta-lactam antibiotics.

Amino Acid Substitution ; Aminoacyltransferases ; genetics ; Anti-Bacterial Agents ; pharmacology ; Bacterial Proteins ; genetics ; Microbial Sensitivity Tests ; Penicillin-Binding Proteins ; genetics ; Peptidyl Transferases ; genetics ; Streptococcus pneumoniae ; drug effects ; beta-Lactam Resistance ; genetics ; beta-Lactams ; pharmacology

Humans ; Microbial Sensitivity Tests ; Pneumoconiosis ; microbiology ; Sputum ; microbiology ; beta-Lactam Resistance ; beta-Lactamases ; analysis

BACKGROUND: Klebsiella oxytoca strain exhibiting an unusual inducible beta-lactam resistance phenotype was isolated from a wound specimen of a patient at a university hospital in August 2002. The isolate was resistant to ampicillin, ampicillin-sulbactam, cephalothin, cefoxitin, and demonstrated reduced inhibition zone diameters for ceftazidime in combination with clavulanate versus those for ceftazidime when tested alone. METHODS: Antimicrobial susceptibilities were tested using the Etest and disk diffusion method. AmpC beta-lactamase production was determined by modified Hodge test. The disk antagonism method was used to detect inducibility of beta-lactamase. Conjugation experiments were performed by the filter mating method using the recipient Escherichia coli J53 Azir strain. PCR and DNA sequencing of DHA-specific PCR products were tested. RESULTS: The double disk synergy test was negative and the modified Hodge test was positive for the K. oxytoca isolate. Antagonism was observed between cefoxitin and oxyimino-cephalosporins. Sequence analysis of the DHA-specific PCR products revealed that they were identical to the amino acid sequence of the DHA-1 beta-lactamase. Transfer of the resistance by conjugation experiments was successful. CONCLUSIONS: We found a plasmid-mediated DHA-1 beta-lactamase-producing K. oxytoca possessing an unusual inducible beta-lactam resistance phenotype was found in a university hospital in Korea. The resistance phenotype was conferred by DHA-1 encoded by a self-transferable plasmid.
Amino Acid Sequence ; Ampicillin ; beta-Lactam Resistance ; beta-Lactamases* ; Cefoxitin ; Ceftazidime ; Cephalothin ; Clavulanic Acid ; Diffusion ; Escherichia coli ; Humans ; Klebsiella oxytoca* ; Klebsiella* ; Korea ; Phenotype ; Plasmids ; Polymerase Chain Reaction ; Sequence Analysis ; Sequence Analysis, DNA ; Wounds and Injuries

OBJECTIVETo study the infectious strains of bacteria in our burn ward in recent 5 years, and analyze their antibiotic resistance.

METHODSBacteria were isolated from the wound excretions of 306 burn patients hospitalized during 2001 to 2006 for analyzing their strains and their antibiotic resistance.

RESULTS378 strains were Grams positive bacteria, among them Staphylococcus aureus was the predominant strain. Further analysis showed that methicillin resistant staphylococcus aureus (MRSA) ranked the first in occurrence, followed by methicillin-resistance Staphylococcus epidermidis (MRSE) and Enterococcus fecalis, 338 strains were Gram negative bacteria, and among them Acinetobacter baumannii was predominant, and Enterobacter cloacae and Pseudomonas aeruginosa ranked the 2nd and 3rd. Twelve strains were fungi.

CONCLUSIONDrug resistance to antibiotics in our burn ward may be related to the beta-lactamases from acinetobacter baumannii and multiple-drug-resistance of MRSA.

Acinetobacter baumannii ; drug effects ; isolation & purification ; Adolescent ; Adult ; Aged ; Aged, 80 and over ; Anti-Bacterial Agents ; pharmacology ; Burn Units ; Burns ; microbiology ; Child ; Child, Preschool ; Drug Resistance, Multiple, Bacterial ; Female ; Humans ; Infant ; Male ; Methicillin-Resistant Staphylococcus aureus ; drug effects ; isolation & purification ; Microbial Sensitivity Tests ; Middle Aged ; Young Adult ; beta-Lactam Resistance

OBJECTIVETo construct prokaryotic expression systems of Streptococcus pneumoniae ciaH and ciaR genes,and to determine their correlation with drug resistance.

METHODSThe total length of ciaH and ciaR genes was amplified by PCR and their prokaryotic expression systems were established by using routine genetic engineering technique. SDS-PAGE was applied to measure the outputs of target recombinant proteins rCiaH and rCiaR. Rabbits antisera and IgGs against rCiaH and rCiaR were prepared. The resistance to penicillin and cefotaxime of S.pneumoniae strains was examined after CiaH and CiaR were extracellularly and intracellularly blocked by the IgGs.

RESULTThe homogeneity of nucleotide and amino acid sequences of the cloned ciaH and ciaR genes with the reported sequences was 99.9-100% and 100%, respectively. The recombinant bacteria E.coli BL21DE3pET42a-ciaH and E.coli BL21DE3pET42a-ciaR were able to express the target recombinant proteins rCiaH and rCiaR with efficiency. The outputs of rCiaH and rCiaR were 33% and 45% of the total bacterial proteins, respectively. The double immunodiffusion titers of rCiaH antiserum,rCiaR antiserum,rCiaH-IgG and rCiaR-IgG were 1:4,1:4,1:1 and 1:1, respectively. After CiaH was extracellularly or intracellularly blocked by CiaH-IgG, and CiaR was intracellularly blocked by CiaR-IgG, the penicillin-sensitive or cefotaxime-sensitive strains developed resistance to the two antibiotics; but the blocks did not change that of penicillin-resisting or cefotaxime-resisting strains.

CONCLUSIONThe prokaryotic expression systems of S. pneumoniae ciaH/ciaR genes have been successfully constructed in this study. Both the CiaH and CiaR may be involved in penicillin and cefotaxime resistance of the bacterium.

Animals ; Bacterial Proteins ; genetics ; metabolism ; Cefotaxime ; pharmacology ; Escherichia coli ; genetics ; metabolism ; Gene Expression Regulation, Bacterial ; Genetic Vectors ; Penicillin Resistance ; genetics ; Protein Kinases ; genetics ; metabolism ; Rabbits ; Recombinant Fusion Proteins ; genetics ; metabolism ; Signal Transduction ; Streptococcus pneumoniae ; drug effects ; enzymology ; genetics ; beta-Lactam Resistance ; genetics

OBJECTIVETo investigate the occurrence and drug resistance of extended-spectrum beta-lactamases (ESBLs)-producing strains of Shigella in pediatric patients, so as to provide information for clinical treatment.

METHODA total of 59 strains of Shigella were isolated from stool specimens of hospitalized children with shigellosis from January 2004 to December 2008. The broth dilution test recommended by Clinical and Laboratory Standards Institute (CLSI) was performed to detect the ESBLs producers. Susceptibility test was carried out by agar dilution method. Escherichia coli ATCC25922 and Klebsiella pneumonia ATCC700603 were used as quality control strains.

RESULTOf the 59 isolates, 21 (35.6%) strains were identified as ESBLs producers. All of the 21 strains were detected by cefotaxime and cefotaxime/clavulanic acid, only 5 (23.8%) were detected by ceftazidime and ceftazidime/clavulanic acid. Both ESBLs and non-ESBLs producers showed high resistance to penicillins. The resistance of ESBLs-producing strains to third and fourth-generation cephalosporins, aztreonam was significantly higher than that of non-ESBLs-producing strains, as well as sulphonamides and quinolones. The drugs sensitive to ESBLs producers were imipenem, meropenem, piperacillin/tazobactam, cefoperazone/sulbactam and cefoxitin, with resistance rate of 0.0%, 0.0%, 14.3%, 9.5%, 14.3%, respectively.

CONCLUSIONThe prevalence of ESBLs-producing Shigella in pediatric patients is at a high level in this area, and the enzyme-producing strains are multidrug resistant. It is recommended that the detection of ESBLs in Shigella should be carried out by microbiological laboratories. Any of the above 5 antibiotics of low resistance should be used according to the patient's condition.

Child ; Feces ; microbiology ; Humans ; Microbial Sensitivity Tests ; Shigella ; Shigella dysenteriae ; drug effects ; isolation & purification ; beta-Lactam Resistance

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 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

To study the resistant mechanism and clinical significance of pseudomonas aeruginosa to beta-lactam antibiotics, the outer membrane permeability rate of 30 P. aeruginosa strains to 5 beta-lactam antibiotics was measured and their production of beta-lactamase and the beta-lactamase genes they carried detected. Furthermore, the relationship between the permeability, beta-lactamase and the clinical effects of beta-lactam antibiotics was observed. By using 14C-penicillin and liquid-scintillant isotope assay, the affinity of penicillin binding proteins (PBPS) was measured and their roles in the resistant mechanism studied. It was revealed that the permeability rate was higher in sensitive strains than in resistant ones (P < 0.05). All strains harbored 1-4 beta-lactamase genes and produced beta-lactamase. Higher permeability rate and higher degree of stability to beta-lactamase indicated better clinical therapeutic effects. The affinity of PBPs changed little without regard to the permeability and beta-lactamase. These results suggested that the permeability of outer membrane and beta-lactamase, but not PBPs, played important roles in the resistant mechanism of P. aeruginosa to beta-lactam antibiotics and affected the clinical therapeutic effectiveness of some patients.
Anti-Bacterial Agents ; pharmacology ; Bacterial Outer Membrane Proteins ; metabolism ; Humans ; Microbial Sensitivity Tests ; Permeability ; Pseudomonas aeruginosa ; drug effects ; beta-Lactam Resistance ; genetics ; beta-Lactamases ; metabolism ; beta-Lactams ; pharmacology

To study the resistant mechanism and clinical significance of pseudomonas aeruginosa to beta-lactam antibiotics, the outer membrane permeability rate of 30 P. aeruginosa strains to 5 beta-lactam antibiotics was measured and their production of beta-lactamase and the beta-lactamase genes they carried detected. Furthermore, the relationship between the permeability, beta-lactamase and the clinical effects of beta-lactam antibiotics was observed. By using 14C-penicillin and liquid-scintillant isotope assay, the affinity of penicillin binding proteins (PBPS) was measured and their roles in the resistant mechanism studied. It was revealed that the permeability rate was higher in sensitive strains than in resistant ones (P < 0.05). All strains harbored 1-4 beta-lactamase genes and produced beta-lactamase. Higher permeability rate and higher degree of stability to beta-lactamase indicated better clinical therapeutic effects. The affinity of PBPs changed little without regard to the permeability and beta-lactamase. These results suggested that the permeability of outer membrane and beta-lactamase, but not PBPs, played important roles in the resistant mechanism of P. aeruginosa to beta-lactam antibiotics and affected the clinical therapeutic effectiveness of some patients.
Anti-Bacterial Agents/*pharmacology ; Bacterial Outer Membrane Proteins/metabolism ; Microbial Sensitivity Tests ; Permeability ; Pseudomonas aeruginosa/*drug effects ; beta-Lactam Resistance/*genetics ; beta-Lactamases/metabolism ; beta-Lactams/*pharmacology