Intereactions between doripenem and clavulanate ­ Application of minimal inhibitory concentration analysis and cytometry flow for bactericidal studies

Background: In view of the current low efficacy of bacterial infection treatment the common trend towards searching for antibiotic systems exhibiting synergistic action is well justified. Among carbapenem analogues a particularly interesting option is provided by combinations of clavulanic acid with meropenem, which have proven to be especially effective. Results: Determination of the minimal inhibitory concentration (MIC) along with the method based on flow cytometry constitutes an important tool in the identification of bacterial sensitivity to active substances. Within this study the inhibitory effect of doripenem, clavulanic acid and the doripenem-clavulanate acid system was analyzed in relation to such bacteria as Salmonella enteritidis, Salmonella typhimurium, Staphylococcus aureus, Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, Proteus vulgaris, Clostridium butyricum and Clostridium pasteurianum, Acinetobacter baumannii, Enterobacter aerogenes. The lowest MIC, amounting to 0.03 µg/mL, was observed for the doripenem-clavulanate acid system in the case of E. coli ATCC 25922. In turn, the lowest MIC for doripenem applied alone was recorded for K. pneumoniae ATCC 31488, for which it was 0.1 µg/mL. The strain which proved to be most resistant both to doripenem and the doripenem-clavulanate acid system, was A. baumannii, with MIC of 32 µg/mL (clinical isolate) and 16 µg/mL (reference strain). Cytometric analysis for P. aeruginosa ATCC 27853 and S. aureus ATCC 25923 showed changes in cells following exposure to limiting concentrations of the active substance. Conclusions: Analysis of MIC supplies important information concerning microbial sensitivity to active substances, mainly in terms of limiting concentrations causing mortality or vitality of the tested species, which is essential when selecting appropriate antibiotic therapy.

Resistant mechanism of β-lactam antibiotic of invasive Streptococcus pneumoniae / 中华检验医学杂志

Objective To understand the drug resistance and antibiotic resistance mechanism ofβ-lactam antibiotics of invasive Streptococcus pneumoniae isolated from Shanghai Children′s Hospital, provides the reference for the rational use of antimicrobial agents.Methods This study is based on the research of the mechanism of drug resistance.62 isolates of invasive Streptococcus pneumoniae were collected from Shanghai Children′s Hospital from January 2005 to December 2011.Minimum inhibitory concentrations ( MIC) of strains to 9 antimicrobial agents were determined by E-test method.The penicillin binding protein coding genes pbp2x, pbp2b, and pbp1a of Streptococcus pneumoniae were amplified by PCR.Then, the correlation between the gene mutation andβ-lactam antibiotics resistant level were analyzed.The murM gene of Streptococcus pneumoniae was amplified by PCR and the correlation of mutation and β-lactam antibiotics resistant level was analyzed.Results Out of 62 strains of invasive Streptococcus pneumoniae from children, the detection rate of penicillin resistant Streptococcus pneumoniae was 43.6% (27/62).Between penicillin intermediate Streptococcus pneumoniae ( PISP ) ( 100%, 25/25 ) and penicillin sensitive Streptococcus pneumoniae (PSSP) (3/10), the difference of gene mutation rate near the pbp2b conserved sequence was statistically significant (χ2 =21.875, P<0.01).The same situation occurred between penicillin resistant Streptococcus pneumoniae (PRSP)(100%, 27/27)and PSSP (3/10) (χ2 =23.310, P<0.01).Also the difference of gene mutation rate of PISP (84%, 21/25) vs PSSP (0) and PSSP (0) vs PRSP (85.2%, 23/27) near or in the pbp2x conserved sequence were statistically significant (χ2 =21.000, P <0.01;χ2 =22.513,P<0.01).The difference of gene mutation rate near the pbp1a conserved sequence and Insertion sequence, which were statistically significant, occurred between PISP and PSSP (χ2 =13.22,P<0.01), between PRSP and PSSP (χ2 =37.000,P<0.01), between PISP and PRSP (χ2 =10.211,P=0.001). MurM gene mutation rate was statistically significant different between the 2 group penicillin MIC≥8 mg/L or ceftriaxone MIC≥2 mg/L group (95.8%, 23/24) and penicillin MIC<8 mg/L or ceftriaxone MIC<2 mg/L group (0) (χ2 =56.2,P =0.002 6).Conclusions The resistance phenomenon of invasive Streptococcus pneumoniae in Shanghai Children′s Hospital is serious.The gene mutations of pbps and murM play a role in amide in the beta of antibiotic resistance, and there is a certain correlation with the antibiotic resistance level.

Substitutions in Penicillin-Binding Protein 1 in Amoxicillin-Resistant Helicobacter pylori Strains Isolated from Korean Patients

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.

Analysis of penicillin-binding proteins (PBPs) in carbapenem resistant Acinetobacter baumannii.

Background & objectives : Acinetobacter baumannii is a Gram-negative, cocco-bacillus aerobic pathogen responsible for nosocomial infections in hospitals. In the recent past A. baumannii 0had developed resistance against β-lactams, even against carbapenems. Penicillin-binding proteins (PBPs) are crucial for the cell wall biosynthesis during cell proliferation and these are the target for β-lactams. Therefore, the present study was carried out to identify the PBPs in three (low, intermediate and high MICs) groups of carbapenem resistant isolates strains of A. baumannii. Methods: ATCC 19606 and 20 β-lactam resistant isolates of A. baumannii were obtained. Selective identification of the PBPs was done using Bocillin FL, a non-radioactive fluorescent derivative of penicillin. Results: The fluorescence emission from Bocillin-tag in SDS-PAGE gel of native strain identified eight PBPs, with apparent molecular weight of 94, 65, 49, 40, 30, 24, 22 and 17 kDa, however, these PBPs revealed alteration in carbapenem-resistant isolates. Interpretation & conclusions: A comparative analysis of PBPs in the resistant isolates with those of ATCC revealed a decreased expression of all PBPs except that of 65 and 17 kDa PBPs which were marginally downregulated, and simultaneous appearance of new 28 kDa PBP (in low and intermediate resistant isolates) and 36 kDa in high meropenem resistant group of A. baumannii. The present study indicated an association between alteration in PBPs and β-lactam resistance in A. baumannii.

Novel gene sequence variants of pbp2b in penicillin-nonsusceptible Streptococcus pneumonia isolates / 中华内科杂志

Objective To investigate the alternations in gene/amino acid sequence of penicillin-binding protein (PBP) 2b from clinical isolates of penicillin-nonsusceptible Streptococcus pneumonia(PNSP) in this region.Methods 24 strains of Streptococcus pneumonia were collected from January to December 2006.The antibiotics susceptibility of these strains was detected.PCR amplification and direct sequencing of pbp2b genes were performed.The sequence variations of PBP genes of the PNSP in this region were studied with sequence BLAST analysis.Results Three prominent substitutions were common tO 13 PNSP isolates with minimal inhibitory concentration(MIC) at least 0.1 mg/L.These included the replacement of Thr445→Ala following the conservative motif SSN,Glu475→Gly and Thr488→Ala/Ser.The exchange of Glu332→Gly was identified in 12 PNSP isolates of which the MIC was at least 0.25 mg/L.Seven penicillin resistant Streptococcus pneumonia (PRSP) isolates (MIC≥3 mg/L)shared the amino acid substitution Ala618→Gly adiacent to third conserved (KTG) motif and the PBP2b sequences of seven PRSP isolates were classified within Back's group Ⅱ and were very similar to those of the Korean J77 isolate.Novel gene and amino acid sequence variants in isolate 14,15,8,11 and 24 was identified in this study and these gene sequences have been deposited in the GenBank database and assigned accession no.EU035970,EU056919,EU056920,EU056921 and EU106886.Conclusion Analysis of pbp2b genes revealed highly similar patterns of nucleotide and amino acid sequence variation among most resistant isolates.while penicillin intermediate Streptococcus pneumonia might be associated with novel gene sequence variants.

Unique variations of pbp2x sequences in clinical Streptococcus pneumoniae isolates and penicillin and cefotaxime resistance / 中华微生物学和免疫学杂志

Objective To investigate the alternations in gene/amino acid sequence of penicillin-binding protein (PBP)2x from clinical isolates of Streptococcus pneumoniae, and to find the reasons for the rapid surge of penicillin and cefotaxime nonsusceptibility among pneumococcal isolates from Shenyang. Methods Thirty-four strains of Streptococcus pneumoniae were collected from January 2006 to February 2007. The antibiotics susceptibility of these strains was detected. PCR amplification and direct sequencing of pbp2x genes were performed. The sequence variations of PBP genes of the penicillin nonsusceptible Streptococcus pneumoniae(PNSP) in this region were studied by BLAST analysis. Results Two prominent substitutions were common to 12 PNSP isolates for which the MIC of penicillin resistance and cefotaxime were at least 0.5 mg/L, which included the replacement of Thr338→Ala in the first conservative motif STMK and Leu546→Val adjacent to third conservative motif KSG. The importance of the exchange of His394→Leu was identified in one PNSP isolate 15. The remarkable finding in this study was Met342→Ile following the first conservative motif STMK. pbp2x sequences of eight PRSP isolates shared Lys501-Glu505-Thr507 substitutions which might be served as a unique marker for PRSP in this region. Novel gene and amino acid sequence variants in 17 isolate were identified in this study, and these sequences have been deposited in the GenBank database and assigned accession No. EU044831, EU089706-EU089709, EU106881-EU106884 and EU124672. Conclusion It is likely that the emergence of penicillin and cefotaxime nonsusceptible Streptococcus pneumoniae in Shenyang might be associated with novel gene sequence variants.

Amino acid substitution or insertion patterns in penicillin-binding protein 2 in Neisseria gonorrhoeae isolates with reduced susceptibility to ceftriaxone / 中华皮肤科杂志

Objective To investigate the amino acid patterns in penicillin-binding protein 2(PBP2)in Neisseria gonorrhoeae isolates with reduced susceptibility to ceftriaxonc.and the relationship between the amino acid patterns and reduced ceftriaxone susceptibility.Methods DNA was extracted from 13 clinical isolates of N.gonorrhoeae.including 11 strains with decreased susceptibility to ceftriaxone and 2 sensitive isolates.The full-length penA gene encoding the penicillin-binding protein 2 was amplified and sequenced.BLASTn and BLASTx programs were used to assess the insertion and substitution patterns of nucleotides in penA gene and of amino acids in PBP2,respectively.Results BLASTn analysis revealed insertion or substitution of 18-38 nucleotides in the penA gene of gonococcal isolates with reduced ceftriaxone susceptibility.As shown by BLASTX analysis.there were five patterns of amino acid substitution or insertion in PBP2 of the 11 isolates with reduced ceftriaxone susceptibility.However.mosaic structure of PBP2 was not found in any of these isolates.Conclusion Mosaic PBP2 seems not to be the major factor contributing to the decrease in susceptibility of N.gonorrhoeae to ceftriaxone.

The relationship between penicillin-binding protein genes restriction polymorphism and penicillin susceptibility in Streptococcus pneumoniae / 中华检验医学杂志

Objective To indicate the restriction profiles of pbps in Streptococcus pneumoniae(S.pneumoniae), and relationship between pbps profiles and the penicillin MIC.Methods The E-test MIC method was used to determinate penicillin susceptibility of 132 S.pneumoniae strains consisting of 69 penicillin susceptible S.pneumoniae (PSSP) strains and 63 nonsusceptible S.pneumoniae (PNSP) strains. Furthermore, we compared these strains by detecting restriction fragment length polymorphism (RFLP) of the PBPs genes pbp1a, pbp2b and pbp2x.Results The RFLP results showed that 9 genotypes were founded for pbp1a, in which 2 were detected from PSSP and some PNSP strains. The other 7 ones were founded mainly in the PNSP with penicillin MIC≥0.25 ?g/ml. Ten genotypes were founded for pbp2b, in which 3 were detected from PSSP and some PNSP strains. The other 7 ones, similar with pbp1a, were founded mainly in the PNSP with penicillin MIC≥0.25 ?g/ml. Thirty-one restrictive patterns were founded for pbp2x. Seventeen patternss from them were detected in PSSP, and 13 ones were founded only in PSSP. The other 14 patterns all were covered PNSP strains. A total of 47 patterns were found according to the three pbps types. Twenty-three patterns from them were detected in PSSP, and 17 ones were founded only in PSSP. The other 24 patterns all were detected in PNSP.Conclusions Results of the study are consistent with the concept that mutations in PBP1a, PBP2b and PBP2x play an important role in the development of resistance to ?-lactam antibiotics by S.pneumoniae. In the meantime, the profiles of pbps can predict penicillin susceptibility.