No abstract available.
Amino Acid Substitution ; Korea ; Penicillin-Binding Proteins ; Penicillins ; Streptococcus

BACKGROUND: The development of new drugs or alternative therapies effective against methicillin-resistant Staphylococcus aureus (MRSA) is of great importance, and various natural anti-MRSA products are good candidates for combination therapies. We evaluated the antibacterial activities of a Phellinus baumii ethyl acetate extract (PBEAE) and its synergistic effects with beta-lactams against MRSA. METHODS: The broth microdilution method was used to determine the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of the PBEAE. The PBEAE synergistic effects were determined by evaluating the MICs of anti-staphylococcal antibiotic mixtures, with or without PBEAE. Anti-MRSA synergistic bactericidal effects of the PBEAE and beta-lactams were assessed by time-killing assay. An ELISA was used to determine the effect of the PBEAE on penicillin binding protein (PBP)2a production. RESULTS: The MICs and MBCs of PBEAE against MRSA were 256-512 and 1,024-2,048 microg/mL, respectively. The PBEAE significantly reduced MICs of all beta-lactams tested, including oxacillin, cefazolin, cefepime, and penicillin. However, the PBEAE had little or no effect on the activity of non-beta-lactams. Time-killing assays showed that the synergistic effects of two beta-lactams (oxacillin and cefazolin) with the PBEAE were bactericidal in nature (Deltalog10 colony forming unit/mL at 24 hr: 2.34-2.87 and 2.10-3.04, respectively). The PBEAE induced a dose-dependent decrease in PBP2a production by MRSA, suggesting that the inhibition of PBP2a production was a major synergistic mechanism between the beta-lactams and the PBEAE. CONCLUSIONS: PBEAE can enhance the efficacy of beta-lactams for combined therapy in patients infected with MRSA.
Acetates/chemistry ; Agaricales/*chemistry/metabolism ; Anti-Infective Agents/chemistry/*pharmacology ; Drug Synergism ; Enzyme-Linked Immunosorbent Assay ; Methicillin-Resistant Staphylococcus aureus/*drug effects/metabolism ; Microbial Sensitivity Tests ; Penicillin-Binding Proteins/analysis/metabolism ; Plant Extracts/chemistry/*pharmacology ; beta-Lactams/*pharmacology

Penicillin expandase, also known as deacetoxycephalosporin C synthase (DAOCS), is an essential enzyme involved in cephalosporin C biosynthesis. To evaluate the catalytic behaviors of penicillin expandase under high penicillin G concentration and to identify mutants suitable for industrial applications, the specific activities of wild-type DAOCS and several mutants with increased activities toward penicillin G were determined by HPLC under high penicillin G concentrations. Their specific activity profiles were compared with theoretical predictions by different catalytic dynamics models. We evaluated the specific activities of wild-type DAOCS and previous reported high-activity mutants H4, H5, H6 and H7 at concentrations ranging from 5.6 to 500 mmol/L penicillin G. The specific activities of wild-type DAOCS and mutant H4 increased as penicillin G concentration increased, but decreased when concentrations of substrate go above 200 mmol/L. Other mutants H5, H6 and H7 showed more complex behaviors under high concentration of penicillin G. Among all tested enzymes, mutant H6 showed the highest activity when concentration of penicillin G is above 100 mmol/L. Our results revealed that the substrate inhibition to wild-type DAOCS' by penicillin G is noncompetitive. Other DAOCS mutants showed more complex trends in their specific activities at high concentration of penicillin G (>100 mmol/L), indicating more complex substrate inhibition mechanism might exist. The substrate inhibition and activity of DAOCS mutants at high penicillin G concentration provide important insight to help select proper mutants for industrial application.
Catalysis ; Intramolecular Transferases ; genetics ; Mutation ; Penicillin G ; pharmacology ; Penicillin-Binding Proteins ; genetics ; Streptomyces ; enzymology ; genetics

OBJECTIVETo study the relationship between nasal carriage and Staphylococcus aureus (S. aureus) infection in hospitalized children.

METHODSFifty-six hospitalized children infected with S. aureus were recruited in this study. Nasal swabs were collected and cultured, and the nasal carriage rate of S. aureus was examined. PVL virulence gene and mecA resistance gene were both detected in clinical strains and nasal carriage strains by PCR.

RESULTSTwenty-two (39%) of the 56 children had nasal carriage of S. aureus, and most of them (18 cases) were younger than one year. Among these 22 children, 11 (50%) had previous hospitalization over the past year. In the infected strains, the rate of methicillin-resistant S. aureus (MRSA) was 29% (16/56), while it was 32% (7/22) in carriage strains. The mecA positive results in clinical strains were consistent with the results in nasal carriage strains. Among 5 PVL-positive nasal carriage strains, 4 (90%) could be matched with their clinical strains, all of which were MRSA.

CONCLUSIONSNasal carriage is a potential risk factor for S. aureus infection. Nosocomial transmission may lead to nasal carriage, which can cause S. aureus infection. The isolation rate of MRSA is high in hospitalized children infected with S. aureus, which implies that more attention is needed for this situation. The isolates from noses may be clonally identical to the isolates from clinical secretions, and the homology between them needs to be confirmed by multi-locus sequence typing.

Bacterial Proteins ; genetics ; Carrier State ; microbiology ; Child ; Child, Hospitalized ; Child, Preschool ; Female ; Humans ; Infant ; Infant, Newborn ; Male ; Methicillin-Resistant Staphylococcus aureus ; isolation & purification ; Nose ; microbiology ; Penicillin-Binding Proteins ; Staphylococcal Infections ; microbiology ; Staphylococcus aureus ; isolation & purification

BACKGROUND/AIMS: This study aims to identify the gene mutation pattern associated with antibiotic resistance for mainly used antibiotics in Helicobacter pylori strains isolated from Koreans. MATERIALS AND METHODS: Seventy-one H. pylori strains were isolated from gastric mucosal biopsy specimens. The specimens were cultivated and the resistance to 5 antibiotics were assessed by using agar gel dilution method. DNA sequencing was carried out to detect the resistance-related gene mutations. RESULTS: A point mutation at A2143G of 23S rRNA was observed in all of the clarithromycin resistant strains, but tetracycline resistant strains were not found. Substitution N562Y in penicillin binding protein 1 were observed in an amoxicillin resistant strain (minimum inhibitory concentration [MIC] 2.0microg/mL). Eleven (57.8%) out of 19 levofloxacin resistant strains showed amino acid substitution at N87K (8 strains), N87I, A88V and D91N in GyrA. The truncation in rdxA was detected in 8 (25.0%) out of 32 metronidazole resistant strains. Two out of the 7 patients who failed in first-line treatment of clarithromycin and amoxicillin showed A2143G mutation. CONCLUSIONS: 23S rRNA mutation is closely related to the failure of eradication, however, the fact that five people who have no gene mutation failed eradication implies that other factors are related. As MIC levels in clarithromycin and levofloxacin resistance strains are getting higher, their appropriate gene mutation is more correlated.
Agar ; Amino Acid Substitution ; Amoxicillin ; Anti-Bacterial Agents ; Biopsy ; Clarithromycin ; Drug Resistance, Microbial* ; Helicobacter pylori* ; Humans ; Levofloxacin ; Metronidazole ; Penicillin-Binding Proteins ; Point Mutation ; Sequence Analysis, DNA ; Tetracycline

BACKGROUND/AIMS: This study aims to identify the gene mutation pattern associated with antibiotic resistance for mainly used antibiotics in Helicobacter pylori strains isolated from Koreans. MATERIALS AND METHODS: Seventy-one H. pylori strains were isolated from gastric mucosal biopsy specimens. The specimens were cultivated and the resistance to 5 antibiotics were assessed by using agar gel dilution method. DNA sequencing was carried out to detect the resistance-related gene mutations. RESULTS: A point mutation at A2143G of 23S rRNA was observed in all of the clarithromycin resistant strains, but tetracycline resistant strains were not found. Substitution N562Y in penicillin binding protein 1 were observed in an amoxicillin resistant strain (minimum inhibitory concentration [MIC] 2.0microg/mL). Eleven (57.8%) out of 19 levofloxacin resistant strains showed amino acid substitution at N87K (8 strains), N87I, A88V and D91N in GyrA. The truncation in rdxA was detected in 8 (25.0%) out of 32 metronidazole resistant strains. Two out of the 7 patients who failed in first-line treatment of clarithromycin and amoxicillin showed A2143G mutation. CONCLUSIONS: 23S rRNA mutation is closely related to the failure of eradication, however, the fact that five people who have no gene mutation failed eradication implies that other factors are related. As MIC levels in clarithromycin and levofloxacin resistance strains are getting higher, their appropriate gene mutation is more correlated.
Agar ; Amino Acid Substitution ; Amoxicillin ; Anti-Bacterial Agents ; Biopsy ; Clarithromycin ; Drug Resistance, Microbial* ; Helicobacter pylori* ; Humans ; Levofloxacin ; Metronidazole ; Penicillin-Binding Proteins ; Point Mutation ; Sequence Analysis, DNA ; Tetracycline

BACKGROUND/AIMS: A worldwide increase in amoxicillin resistance in Helicobacter pylori is having an adverse effect on eradication therapy. In this study, we investigated the mechanism of the amoxicillin resistance of H. pylori in terms of amino acid substitutions in penicillin-binding protein 1 (PBP1). METHODS: In total, 150 H. pylori strains were isolated from 144 patients with chronic gastritis, peptic ulcers, or stomach cancer. The minimum inhibitory concentrations (MICs) of the strains were determined with a serial 2-fold agar dilution method. The resistance breakpoint for amoxicillin was defined as >0.5 microg/mL. RESULTS: Nine of 150 H. pylori strains showed amoxicillin resistance (6%). The MIC values of the resistant strains ranged from 1 to 4 microg/mL. A PBP1 sequence analysis of the resistant strains revealed multiple amino acid substitutions: Val16-->Ile, Val45-->Ile, Ser414-->Arg, Asn562-->Tyr, Thr593-->Ala, Gly595-->Ser, and Ala599-->Thr. The natural transformation of these mutated genes into amoxicillin-sensitive strains was performed in two separate pbp1 gene segments. A moderate increase in the amoxicillin MIC was observed in the segment that contained the penicillin-binding motif of the C-terminal portion, the transpeptidase domain. CONCLUSIONS: pbp1 mutation affects the amoxicillin resistance of H. pylori through the transfer of the penicillin-binding motif.
Adult ; Amino Acid Sequence ; *Amino Acid Substitution ; Amoxicillin/*pharmacology ; Anti-Bacterial Agents/*pharmacology ; Female ; Helicobacter Infections/drug therapy ; Helicobacter pylori/*chemistry/*drug effects/genetics ; Humans ; Male ; Microbial Sensitivity Tests ; Middle Aged ; *Penicillin Resistance/genetics ; Penicillin-Binding Proteins/*chemistry/genetics ; Republic of Korea ; Sequence Analysis, Protein ; Transformation, Genetic

OBJECTIVETo develop a rapid multi-residue assay for detecting 16 demanded by the European Union (EU).

METHODSA recombinant penicillin-binding protein (PBP) 2x* from Streptococcus pneumoniae R6 was expressed in vitro and six β-lactams were conjugated to HRP by four methods. A rapid multi-residue assay for β-lactams was established with PBP2x* and HRP-conjugate.

RESULTSPBP2x* was expressed and purified successfully and the ideal HRP-conjugate was identified. The multi-residue assay was developed. After optimization, penicillin G, ampicillin, amoxicillin, cloxacillin, dicloxacillin, oxacillin, nafcillin, cephalexin, ceftiofur, cefalonium, cefquinome, cefazolin, cefoperazone, cephacetrile, and cephapirin can be detected at levels below MRL in milk with simple pretreatment.

CONCLUSIONThis assay developed can detect all 16 β-lactams demanded by the European Union (EU). The whole procedure takes only 45 min and can detect 42 samples and the standards with duplicate analysis.

Animals ; Milk ; chemistry ; Penicillin-Binding Proteins ; metabolism ; beta-Lactams ; analysis ; metabolism

OBJECTIVETo study the clinical and molecular characteristics of methicillin-resistant Staphylococcus aureus (MRSA) infection in children.

METHODA total of 37 MRSA strains were isolated from hospitalized patients in Children's Hospital of Fudan University from March 2009 to November 2011. The clinical characteristics were investigated by a cohort study. Furthermore, the mecA, Panton-Valentine leucocidin (PVL) genes were detected by polymerase chain reaction (PCR), and the genotypes of SCCmec were determined by multiplex PCR.

RESULT(1) Among the 37 MRSA isolates, infections with 21 were acquired from hospital (HA-MRSA), and 16 isolates were acquired from community (CA-MRSA). (2) In the study, MRSA frequently caused respiratory tract infection, and most of the strains were isolated from intensive care unit (ICU). (3) CA-MRSA was most frequently associated with skin and soft tissue infections (SSTI), suppurative tonsillitis, even pneumonia and septicemia. HA-MRSA infection was more aggressive, most frequently associated with pneumonia, septicemia, and central nervous system (CNS) infections, such as meningitis. In children with fever caused by HA-MRSA or CA-MRSA infection, HA-MRSA showed a longer duration of fever, for 10.5 days. C-reactive protein (CRP) level caused by HA-MRSA (63.00 mg/L) was higher than CA-MRSA (9.50 mg/L) , and there were statistically significant differences between the groups (t = 2.5670, P < 0.05). However, there were no statistically significant differences between the groups in white blood cell count (WBC) or procalcitonin (PCT) level. (4) Among 37 MRSA isolates, the whole isolates were mecA gene positive (100%). SCCmec genotyping results showed that the most frequent SCCmec types were type III, 17 isolates, the others including type IV 8 isolates, type II1 isolates, nontypable 11 isolates, type I and type V were not found in this group. Therein, among 21 HA-MRSA isolates, SCCmec III was the most common, 15 isolates, type IV 1 isolates, nontypable 5 isolates; among 16 CA-MRSA isolates, SCCmec type IV was the most common, 7 isolates, type III 2 isolates, type II 1 isolate, nontypable 6 isolates. (5) Among the 37 MRSA isolates, 28 were PVL gene positive; and among 21 HA-MRSA isolates, 17 were PVL gene positive; Among 16 CA-MRSA isolates, 11 were PVL gene positive; There were no statistically significant differences between the groups (χ(2) = 0.735, P > 0.05) .

CONCLUSIONCompared with CA-MRSA, HA-MRSA infection was more aggressive, and induced higher C reactive protein; the dominant epidemic strains of CA-MRSA was SCCmec type IV, and HA-MRSA was SCCmec type III; the positive rate of PVL gene was high.

Adolescent ; Anti-Bacterial Agents ; pharmacology ; Bacterial Proteins ; genetics ; Bacterial Toxins ; genetics ; Bacterial Typing Techniques ; Child ; Child, Preschool ; China ; epidemiology ; Cohort Studies ; Community-Acquired Infections ; epidemiology ; microbiology ; Cross Infection ; epidemiology ; microbiology ; DNA, Bacterial ; genetics ; Female ; Genotype ; Humans ; Infant ; Infant, Newborn ; Male ; Methicillin ; pharmacology ; Methicillin Resistance ; genetics ; Methicillin-Resistant Staphylococcus aureus ; classification ; genetics ; isolation & purification ; Penicillin-Binding Proteins ; Staphylococcal Infections ; epidemiology ; microbiology

BACKGROUND: This study compared three non-molecular methods for the detection of methicillin-resistance directly from blood cultures containing Staphylococcus aureus: penicillin-binding protein (PBP) 2a latex agglutination (LA), PBP2a immunochromatographic assay (ICA) and MRSA chromogenic medium (CM). METHODS: Fifty methicillin-resistant S. aureus (MRSA) and 50 methicillin-susceptible S. aureus (MSSA) were seeded into blood-culture bottles. When isolates returned a positive signal, 5 mL of culture was added to serum separator tubes and centrifuged at 1,300 g for 10 min. The pellets were then used as the inoculum for the PBP2a LA, MRSA-CM and PBP2a ICA. The pure colony was used for PBP2a LA test, additionally. RESULTS: The respective sensitivities and specificities were 98 and 100% for PBP2a ICA, and 100 and 100% for MRSA-CM in direct detection of MRSA from positive blood culture. The results of PBP2a LA test using pure colony were entirely compatible with those by mecA gene PCR but the PBP2a LA test using the pellets directly isolated from positive blood culture showed sometimes ambiguous agglutination; its sensitivity and specificity were 78 and 100%, if ambiguous results were scored as negative, and were 90 and 92%, if ambiguous results were scored as positive, respectively. CONCLUSION: For direct detection of MRSA in positive blood culture, MRSA-CM and PBP2a ICA provided excellent results. The PBP2a LA test using pure colony also gave excellent results but the PBP2a LA test by the direct method using pellet of positive blood culture was slightly less sensitive than the other two methods.
Adenosine ; Agglutination ; Immunochromatography ; Latex ; Methicillin Resistance ; Methicillin-Resistant Staphylococcus aureus ; Penicillin-Binding Proteins ; Polymerase Chain Reaction ; Seeds ; Sensitivity and Specificity ; Staphylococcus