The inoculum effect of antibiotics against CTX-M-extended-spectrum ß-lactamase-producing Escherichia coli.

BACKGROUND: Questions remain regarding the use of the cephalosporins to treat infections caused by extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli. For example, should ceftazidime or cefepime be used to treat infections with CTX-M ESBL-producing organisms with low MICs (minimum inhibitory concentrations), according to the new Clinical and Laboratory Standards Institute's (CLSI) recommendations for susceptibility testing? Some studies have reported that in vitro MICs of cephalosporins increase as the inoculum increases, which is the inoculum effect; however, most of the enzymes studied were SHV and TEM. In this study, we aimed to investigate the inoculum effect on ceftazidime, cefepime and four other ß-lactam agents against CTX-M-ESBLs-producing Escherichia coli. METHODS: Antibiotic susceptibilities were determined using broth microdilution MIC methodology according to the CLSI recommended with standard and 100-fold-higher inocula. RESULTS: An inoculum effect on meropenem and cefminox was not detected. The size of the inoculum affected piperacillin/tazobactam activity against only 4 strains, all CTX-M-14 genotypes. The inoculum size affected the activity of ceftazidime, cefepime and cefotaxime against 35%, 85%, 100% of strains, respectively. Among the strains with an inoculum effect, CTX-M-14 was the most common ESBL genotype. CONCLUSIONS: These findings suggest that meropenem is the most active compound against serious infections caused by Escherichia coli producing ESBLs. Cefminox and piperacillin-tazobactam exhibit strong activity against many strains. Until further studies are performed, clinicians should be aware that third- and fourth-generation cephalosporins (such as ceftazidime and cefepime) are not reliable for serious infections even though in vitro tests indicate susceptibility.

Genotype diversity of methicillin-resistant Staphylococcus aureus in Shenyang, China.

BACKGROUND: The aim of this study was to better understand methicillin-resistant Staphylococcus aureus (MRSA) at the molecular level by investigating the genotypic characteristics and evolutionary patterns of MRSA clones in Shenyang, China. METHODS: We analyzed the molecular epidemiology of 60 MRSA isolates in Shenyang, China, between 2002 and 2008, using pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), staphylococcal cassette chromosome mec (SCCmec) typing, and S. aureus protein A (spa) typing. They were examined for their antimicrobial susceptibilities. RESULTS: Among the 60 isolates, ST239 was identified most frequently (34 isolates; 58%), followed by ST5 (20 isolates; 34%). Nine spa types were obtained and 4 PFGE strain families (A, B, C, and D) were resolved. Spa type t030, which corresponded to PFGE genotypes A1, A3, and A4, constituted 45% (27/60) of all isolates; spa type t037, which corresponded to PFGE type A2, accounted for 13% (8/60) of all isolates. These 2 spa genotypes belonged to ST239 and carried SCCmec type III. Isolates genotyped as spa type t002 comprised 27% (16/60) of the study set and included isolates typed as PFGE B1 and B2, ST5, and SCCmec II. Most of MRSA isolates belonging to ST239 were susceptible to trimethoprim-sulfamethoxazole. The minimum inhibitory concentration (MIC50) of vancomycin among MRSA isolates belonging to ST5 (2 mg/l) was higher than that for other isolates (1 mg/l). CONCLUSIONS: These data document 2 major epidemic MRSA clones in Shenyang, China: ST239-MRSA-SCCmec type III-t037/t030 and ST5-MRSA-SCCmec type II-t002.

Antimicrobial resistance, genotypic characterization and pulsed-field gel electrophoresis typing of extended spectrum ß-lactamases-producing clinical Escherichia coli strains in Macao, China.

BACKGROUND: The rise of the production of CTX-M class extended spectrum ß-lactamases (ESBLs) has been well documented in traveling countries but no data are found for Macao, an international travel city. The objectives of this study were to identify the antimicrobial resistance pattern, and determine the prevalence, genotype and clonal relationship of ESBLs in 209 clinical Escherichia coli strains from Macao, China. METHODS: Antimicrobial susceptibility test was performed to determine the resistance patterns of the isolates using the disk diffusion method with 17 antimicrobial agents. Phenotypic detection was screened and confirmed according to the Clinical and Laboratory Standards Institute. Genotypic characterization was detected by isoelectric focusing analysis, polymerase chain reaction and sequencing. The clonal relationship between the different ESBL isolates was studied by pulsed-field gel electrophoresis (PFGE). RESULTS: Imipenem and meropenem exhibited 100% susceptible among 209 strains. Overall, 82.3%, 67.3%, 52.9%, 51.2% and 51.0% of the isolates displayed resistance to ampicillin, tetracycline, ciprofloxacin, sulfamethoxazole trimethoprim and gentamycin. The prevalence rate of ESBLs was 30.1%. Antibiotic resistances were found to be significantly higher among the ESBL producing group compared to non-ESBL producing group. We detected CTX-M-14 to be the major genotypic characterization of ESBLs (76.2%). Two strains showed indistinguishable patterns by PFGE. CONCLUSIONS: The prevalence of antimicrobial resistance is alarming high in Macao. Antimicrobial resistance is significantly higher among the ESBL producing group. This study documented CTX-M-14 as the predominant ESBL type. Although indistinguishable pattern was found between two strains, it was too small to decide whether any of the investigated strains was epidemic. Our findings may be also pertinent for other geographic areas undergoing similar travel characteristics to understand the corresponding effects on bacterial populations.

ISEcp1 element in association with bla(CTX-M) genes of E. coli that produce extended-spectrum ß-lactamase among the elderly in community settings.

INTRODUCTION: This study analyzed the relationship between the ISEcp1 element and bla(CTX-M) genes of Escherichia coli isolates that produce extended-spectrum ß-lactamase (ESBL) in community settings. METHODS: Nineteen E. coli isolates that produced CTX-M-type ß-lactamase were collected from four communities of elderly people in Shenyang, China. Polymerase chain reaction (PCR) amplification and direct sequencing were used to detect the insertion of the ISEcp1 element into the genetic environment of the bla(CTX-M) genes. RESULTS: The ISEcp1 element was associated with several bla(CTX-M) gene types, including CTX-M-14, CTX-M-24, CTX-M-22, and CTX-M-79. Sequence analysis revealed that all of the ISEcp1-like DNA sequences contained the putative promoter region that is involved in CTX-M genes transcription. ISEcp1 insertion sequences were observed 42-127bp upstream of the open reading frames (ORFs) that encode the CTX-M enzymes in all 15 strains. The CTX-M-79 ß-lactamase-encoding gene was observed with a different ISEcp1 insertion site and variable sequences between the ISEcp1 and bla(CTX-M-79) gene. For one strain (T298), the ISEcp1 element was disrupted by IS10. CONCLUSION: This work confirmed that the ISEcp1 elements were closely linked to bla(CTX-M) genes in community isolates from Shenyang, China.

Molecular characteristics and resistant mechanisms of imipenem-resistant Acinetobacter baumannii isolates in Shenyang, China.

The investigation was carried out to elucidate the molecular characteristics and resistant mechanisms of imipenem-resistant Acinetobacter baumannii. Thirty-seven isolates were collected from January 2007 to December 2007. The homology of the isolates was analyzed by both pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). The genes of ß-lactamases, adeB, and class 1 integron were polymerase chain reaction amplified. Genotype analysis of the 37 A. baumannii isolates by PFGE revealed the circulation of four PFGE types (A-D); the A- and B-type accounted for 48.6% and 40.5%, respectively. MLST showed the existence of three allelic profiles. The agar dilution method was carried out to determine the MIC of imipenem, in the absence or presence of carbonyl cyanide m-chlorophenylhydrazone (CCCP, 10 µg/ml). The MICs of the strains to imipenem were between 16 µg/ml and 128 µg/ml. When CCCP was added, a MIC decrease of at least four-fold was observed in 20 isolates, which belonged to the A- or C-type. AdeB and bla (PER-1) genes were each detected in 35 isolates, bla (OXA-23) gene in 34 isolates and bla (OXA-58)-like gene in 24 isolates. All isolates harbored bla (OXA-51)-like genes. No isolates carried the bla (IMP-1) gene. Integron was detected in 25 isolates, which mediated the resistance to aminoglycosides and rifampin. The epidemiologic data suggested that the increasing infection of A. baumannii in our hospital was mainly caused by the inter-hospital spread of two epidemic clones. The AdeABC efflux system may be the important factor that leads to the high level of imipenem-resistance in PFGE A-type.

[Molecular epidemiological study of multi-drug resistant Acinetobacter baumannii].

OBJECTIVE: To determine the possible genetic background and the source of our hospital's 43 clinical isolates of multidrug-resistant Acinetobacter baumannii, and the category of gene cassettes in type 1 integrons of all strains. METHODS: Restriction enzyme Apa I was chosen for all strains in pulsed-field gel electrophoresis (PFGE) methods. Multilocus sequence typing (MLST) was used to compare the allelic profiles of all the strains. PCR method was used for amplify the integrons of all strains. RESULTS: PFGE results showed that 43 strains were divided into four types. A-type and B-type were divided into 4 and 2 subtypes, respectively. The MLST results showed the existing of three allelic profiles: 1-3-3-2-2-7-3, 1-3-3-2-2-11-3, and 1-3-3-2-2-14-3. B-type and D-type of PFGE have the same allelic profile (1-3-3-2-2-11-3). A-type strains were detected mainly in ICU, and in burn unit only found B- and D-type. The same integron was detected in 62.8% of the strains. The constituent ratio of A1, A2, A3, A4, B1, B2, C and D-type was 40.7%, 18.5%, 7.4%, 3.7%, 14.8%, 3.7%, 3.7% and 7.4%, respectively. CONCLUSIONS: The coexistence of multiple cloning system in this region was proved by the PFGE and MLST, and the same clone can evolve to different subtypes when stimulated by different environmental conditions; and the different carrying-situation of the same integron in strains prove the possibility of the change during the evolution of resistance mechanisms.

Pharmacokinetic-pharmacodynamic profiling of four antimicrobials against gram-negative bacteria collected from Shenyang, China.

BACKGROUND: To examine common antimicrobial regimens used in eradicating certain nosocomial gram-negative pathogens and determine which ones are likely to be the most suitable as empirical choices in Shenyang, China. METHODS: A 5000-subject Monte Carlo simulation was conducted to determine the cumulative fraction of response (CFR) for meropenem, imipenem, cefepime, piperacillin/tazobactam and levofloxacin against Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae, Acinetobacter baumannii and Pseudomonas aeruginosa collected in 2006 and 2007 from Shenyang. RESULTS: Meropenem and imipenem had the highest CFRs against the Enterobacteriaceae (97%-100%), followed by cefepime. No antibiotic simulated regimen achieved optimal CFR against P. aeruginosa and A. baumannii. Piperacillin/tazobactam dosed at 4.5 g q8h achieved the lowest CFR against all bacteria. CONCLUSIONS: This study suggests that the carbapenems provide the greatest likelihood of clinical success for the Enterobacteriaceae, and combination therapy might be needed when choosing empirical therapy, especially when A. baumannii or P. aeruginosa are suspected.

[Novel gene sequence variants of pbp2b in penicillin-nonsusceptible Streptococcus pneumonia isolates].

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 Thr(445)--> Ala following the conservative motif SSN, Glu(475)-->Gly and Thr(488)-->Ala/Ser. The exchange of Glu(332)-->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 > or = 3 mg/L) shared the amino acid substitution Ala(618)-->Gly adjacent to third conserved (KTG) motif and the PBP2b sequences of seven PRSP isolates were classified within Baek'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.

Prevalence of rectal carriage of extended-spectrum beta-lactamase-producing Escherichia coli among elderly people in community settings in China.

The importance of community-acquired infections due to extended-spectrum beta-lactamase-producing (ESBL) Escherichia coli has been increasingly recognized in recent years. No comprehensive data are available on the prevalence, risk factors, and genotypes of ESBL production in community residents in China. Rectal samples from 270 elderly people were collected in four communities in Shenyang (China). Colonies were screened by double-disk synergy test for ESBL production and then, ESBLs were characterized by PCR and sequencing. The clonal relatedness of all ESBL-producing isolates was determined by pulsed-field gel electrophoresis. Potential risk factors for rectal carriage of ESBL producers were examined by multivariate analysis. The prevalence of rectal carriage of ESBL-producing E. coli was 7.0%. All 19 ESBL-producing isolates produced CTX-M-type ESBLs, including CTX-M-14 (11 strains), CTX-M-22 (3 strains), CTX-M-79 (3 strains), CTX-M-24 (1 strain), and CTX-M-24 and CTX-M-79 together (1 strain). CTX-M-79 ESBL was first detected worldwide. ESBL-producing strains were clonally unrelated. Appearance of ESBL producers is strongly associated with the use of antibiotics in the past 3 months (odds ratio 3.2, 95% CI 1.1-9.0, P = 0.03). Our results show the importance of the intestinal tract as a reservoir for ESBL-producing isolates in community settings in China and that the use of antibiotics in the past 3 months is clearly linked to rectal carriage of ESBL producers.

Molecular characteristics of penicillin-binding protein 2b, 2x, and 1a sequences in penicillin-nonsusceptible Streptococcus pneumoniae isolates in Shenyang, China.

The aim of this study was to investigate the nature of the amino acid motifs found in penicillin-binding proteins (PBP) 2b, 2x, and 1a of penicillin-nonsusceptible Streptococcus pneumoniae isolates from Shenyang, China, and to obtain information regarding the prevalence of alterations within the motifs or in positions flanking the motifs. For 18 clinical isolates comprising 4 penicillin-susceptible S. pneumoniae, 5 penicillin-intermediate S. pneumoniae, and 9 penicillin-resistant S. pneumoniae. the DNA sequences of PBP2b, PBP2x, and PBP1a transpeptidase domains were determined and then genotyped by multilocus sequence typing. Sequence analysis revealed that most penicillin-nonsusceptible S. pneumoniae isolates (penicillin MIC > or = 1.5 microg/mL and cefotaxime MIC > or = 2 microg/mL) shared identical PBP2b, PBP2x, and PBP1a amino acid profiles. Most penicillin-resistant S. pneumoniae isolates were ST320 (4-16-19-15-6-20-1), the double-locus variant of the Taiwan19F-14 clone. This study will serve as a basis for future monitoring of genetic changes associated with the emergence and spread of beta-lactam resistance in Shenyang, China.