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The objective of this study was to better understand the in vitro activity of flomoxef against clinical extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae. A total of 401 ESBL-producing isolates, including 196 Escherichia coli , 124 Klebsiella pneumoniae and 81 Proteus mirabilis , were collected consecutively from 21 hospitals in China in 2013. Minimum inhibitory concentrations (MICs) were determined by broth microdilution methods. Phenotypic identification of ESBL production was detected as recommended by the Clinical and Laboratory Standards Institute (CLSI). ESBL genes were detected by PCR and sequencing. Flomoxef, doripenem, meropenem, ertapenem, cefmetazole and piperacillin/tazobactam exhibited good activity against ESBL-producing isolates, with susceptibility rates >90%. Tigecycline showed good activity against E. coli and K. pneumoniae (100% and 97.6%, respectively). Cefotaxime and cefepime showed very low activities against ESBL-producing isolates, with susceptibility rates of 0–0.8% and 1.0–13.6%, respectively. bla CTX-M were the major ESBL genes, with occurrence in 99.5% of E. coli , 91.1% of K. pneumoniae and 97.5% of P. mirabilis . bla CTX-M-14 was the predominant ESBL gene, detected in 46.9% (188/401) of the isolates, followed by bla CTX-M-15 (21.4%), bla CTX-M-55 (17.2%), bla CTX-M-65 (12.7%) and bla CTX-M-3 (6.7%). Flomoxef exhibited excellent activity against the different CTX-M-type ESBL-producing isolates, with MIC 50 and MIC 90 values of 0.064–0.125 μg/mL and 0.25–0.5 μg/mL, respectively. Against the isolates solely producing CTX-M-14, -15, -55, -3 or -65, flomoxef showed susceptibility rates of 98.6%, 98.0%, 98.1%, 100.0% and 97.4%, respectively. In conclusion, flomoxef showed good activity against ESBL-producing Enterobacteriaceae and may be a choice to treat infections caused by these isolates in China. [ABSTRACT FROM AUTHOR]

Abstract: Here we evaluated the frequency of occurrence and antimicrobial susceptibility patterns of Gram-negative bacteria isolated from patients hospitalised with pneumonia in medical centres in the USA (n =28) and Europe and the Mediterranean region (EMR) (n =25) in 2009–2012. Susceptibility testing was performed by reference broth microdilution methods. Overall, 12851 isolates were collected (6873/5978 in USA/EMR). The same top 11 organisms were observed in both geographic regions, but in different rank orders, and Gram-negative organisms represented 61.5/76.1% of strains in USA/EMR. Pseudomonas aeruginosa was the most frequently isolated Gram-negative organism in both regions (20.9/20.9% of cases in USA/EMR) and showed reduced susceptibility to most antimicrobials tested, including ceftazidime (79.6/68.7% susceptibility in USA/EMR), meropenem (76.3/65.8%) and piperacillin/tazobactam (72.9/63.9%). Klebsiella spp. was isolated from 9.7/11.6% of cases and showed extended-spectrum β-lactamase (ESBL) phenotype rates of 19.5/35.1% in USA/EMR. Meropenem and amikacin were active against 62.3/78.7% and 60.8/85.2% of ESBL phenotype Klebsiella spp. from USA/EMR, respectively. Enterobacter spp. ranked fourth in the USA (5.9%) and sixth in EMR (5.5%), whereas Escherichia coli ranked fifth in the USA (5.5%) and third in EMR (11.8%). Acinetobacter spp. and Stenotrophomonas maltophilia combined were isolated from 8.0/10.7% of cases in USA/EMR. A significant increase in P. aeruginosa susceptibility to meropenem and a significant decrease in gentamicin susceptibility among Klebsiella spp. were noted in EMR. These results confirm that very few agents remain broadly active against the most frequently isolated Gram-negative organisms from patients with pneumonia in US and EMR medical centres. [Copyright &y& Elsevier]

Abstract: Prompt detection of infections caused by Enterobacteriaceae that produce therapeutically important β-lactamases [metallo-β-lactamases (MBLs), serine carbapenemases, acquired AmpC and CTX-M extended-spectrum β-lactamases (ESBLs)] is crucial for infection prevention and control and surveillance purposes, and, more contentiously, also for effective patient management. A novel TaqMan PCR assay was developed to detect genes encoding IMP, VIM, NDM, SPM, SIM and GIM MBLs. Published PCR assays for acquired genes encoding CTX-M ESBLs and AmpC β-lactamases were updated and adapted to the TaqMan format, respectively. A published TaqMan assay for serine carbapenemase genes was used. Assay specificity was tested using a panel of 59 isolates with known acquired genes from the four different β-lactamase groupings. The four TaqMan assays correctly identified the most clinically relevant acquired β-lactamase genes in the panel of 59 resistant Enterobacteriaceae, which included 3 VIM-, 7 NDM- and 12 IMP-producers. Consecutive, non-duplicate isolates of Enterobacteriaceae from 965 urinary and 343 blood cultures during 2010 were then screened for β-lactamase genes using these TaqMan assays. Amongst the urinary and blood culture isolates tested, 69 CTX-M-producers and 21 acquired AmpC β-lactamase-producers were identified; the CTX-M rate amongst blood culture isolates (9.3%) broadly reflects the UK national average. During the study period, one Klebsiella pneumoniae isolate producing an NDM carbapenemase was identified from a wound sample. The assays developed and/or used will enable the future surveillance and the rapid detection and appropriate early treatment of infections caused by Gram-negative bacteria producing clinically important β-lactamases, including carbapenemases. [Copyright &y& Elsevier]

Abstract: The impact of extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae of food animal origins on human health has caught considerable attention worldwide. Intestinal Escherichia coli obtained from healthy food animals (pigs, cattle and poultry) in China were tested for the presence of ESBL genes. CTX-M-producing isolates were further characterised by pulsed-field gel electrophoresis (PFGE), phylogenetic grouping, genetic environment analysis, conjugation and plasmid replicon typing. A total of 127 of the 896 E. coli isolates showed reduced susceptibility to cefotaxime (minimal inhibitory concentration≥2μg/mL). bla CTX-M genes were detected in 111 of the 127 isolates. The most common CTX-M types were CTX-M-14 (n =40), CTX-M-55 (n =29) and CTX-M-65 (n =22), followed by CTX-M-27, -15, -98, -24, -3, -102 and -104. CMY-2 was detected in two isolates. High clonal diversity was found amongst CTX-M-producing isolates. Insertion sequence ISEcp1 was observed 42bp upstream of the start codon of all CTX-M-9 group genes, whereas the spacer region between the right inverted repeats and CTX-M-1 group genes varied from 45bp to 127bp. Most bla CTX-M genes were transferable by conjugation. IncFII, IncI1, IncFIB, IncN and IncA/C replicons were detected in 28, 21, 7, 5 and 1 of the 70 transconjugants carrying bla CTX-M, respectively. This study demonstrates that commensal E. coli from healthy food animals can be important reservoirs of bla CTX-M genes and may contribute to the dissemination and transfer of these β-lactamase genes throughout China. [Copyright &y& Elsevier]

Abstract: Worldwide, resistance of Gram-negative micro-organisms to third-generation cephalosporins and carbapenems owing to β-lactamases is an increasing problem. Although the CTX-M, TEM and SHV extended-spectrum β-lactamases (ESBLs) are most widely disseminated, other β-lactamase families have also recently emerged, such as plasmid-mediated AmpC β-lactamases and carbapenemases. Here we describe a new set of multiplex polymerase chain reactions (PCRs) with one amplification protocol enabling detection of 25 prevalent β-lactamase families, including ESBLs, carbapenemases, plasmid-mediated AmpC β-lactamases and OXA β-lactamases. [Copyright &y& Elsevier]

Abstract: The objective of this study was to investigate the distribution and susceptibility of aerobic and facultative Gram-negative bacilli (GNB) isolated from patients with intra-abdominal infections (IAIs) in China. From 2002 to 2009, minimum inhibitory concentrations of 14 antibiotics for 3420 aerobic and facultative GNB from up to eight hospitals in six cities were determined by the broth microdilution method. Enterobacteriaceae comprised 82.9% (2834/3420) of the total isolates, with Escherichia coli (49.2%) being the most commonly isolated species followed by Klebsiella pneumoniae (17.0%), Enterobacter cloacae (5.8%) and Citrobacter freundii (2.3%). Amongst the antimicrobial agents tested, the three carbapenems (ertapenem, imipenem and meropenem) were the most active agents against Enterobacteriaceae, with susceptibility rates of 96.1–99.6% (2002–2009), 98.2–100% (2002–2009) and 99.6–100% (2002–2004), respectively, followed by amikacin (86.8–95.1%) and piperacillin/tazobactam (84.5–94.3%). Susceptibility rates of all tested third- and fourth-generation cephalosporins against Enterobacteriaceae declined by nearly 30%, with susceptibility rates of 40.2%, 39.1%, 56.3% and 51.8% in 2009 for ceftriaxone, cefotaxime, ceftazidime and cefepime, respectively. The occurrence of extended-spectrum β-lactamases increased rapidly, especially for E. coli (from 20.8% in 2002 to 64.9% in 2009). Susceptibility of E. coli to ciprofloxacin decreased from 57.6% in 2002 to 24.2% in 2009. The least active agent against Enterobacteriaceae was ampicillin/sulbactam (SAM) (25.3–44.3%). In conclusion, Enterobacteriaceae were the major pathogens causing IAIs, and carbapenems retained the highest susceptibility rates over the 8-year study period. Third- and fourth-generation cephalosporins, fluoroquinolones and SAM may not be ideal choices for empirical therapy of IAIs in China. [ABSTRACT FROM AUTHOR]

Abstract: This study was designed to determine the in vitro activities of several antimicrobial agents against well-characterised CTX-M-producing Escherichia coli strains isolated from clinical specimens. Minimum inhibitory concentrations (MICs) were determined for 202 extended-spectrum β-lactamase (ESBL)-producing E. coli using microbroth dilution and Vitek methods according to Clinical and Laboratory Standards Institute criteria. Molecular characterisation was performed using isoelectric focusing and polymerase chain reaction (PCR) with sequencing, whilst strain relatedness was determined by pulsed-field gel electrophoresis (PFGE) using XbaI. Of the 202 ESBL-producing E. coli, 2 produced VEB-1, 12 produced TEM-52, 32 produced SHV types (including SHV-2 and -12) and 156 produced CTX-M types (including CTX-M-2, -3, -14, -15, -24, -27 and -30). The MIC50 and MIC90 (MIC for 50% and 90% of the organisms, respectively) for the antimicrobial agents tested were, respectively: piperacillin/tazobactam (TZP), 32mg/L and >256mg/L; ciprofloxacin, 16mg/L and 32mg/L; gentamicin, 8mg/L and 128mg/L; amikacin, 2mg/L and 16mg/L; ertapenem, 0.03mg/L and 0.12mg/L; imipenem, 0.03mg/L and 0.12mg/L; meropenem, 0.03mg/L and 0.03mg/L; and tigecycline 0.12mg/L and 0.5mg/L. Vitek Legacy and Vitek 2 failed to detect TZP resistance in 91 (90%) and 75 (74%) of 101 TZP-resistant ESBL-producing strains, respectively, especially CTX-M-15-producing isolates that co-produced OXA-1. The carbapenems, amikacin and tigecycline had good in vitro activities against multiresistant CTX-M-producing E. coli. We recommend that laboratories using Vitek should employ alternative susceptibility testing methods for TZP before reporting the activity of this agent against ESBL-producing E. coli. [Copyright &y& Elsevier]

Abstract: Bacteria harbouring extended-spectrum β-lactamases (ESBLs), derived by mutation from TEM-1, TEM-2 or SHV-1 β-lactamases, have been described world-wide. The in vitro activities of these enzymes against β-lactam antibiotics, including oral cephalosporins, are well recognised. The aim of this investigation was to assess the bactericidal activity of oral β-lactam antibiotics available in Croatia (amoxicillin/clavulanate, cephalexin, cefuroxime, cefadroxil and ceftibuten), in biological fluids against isogenic Escherichia coli strains producing broad-spectrum (TEM-1, TEM-2 and SHV-1) and extended-spectrum β-lactamases (SHV-2, SHV-3, SHV-4, SHV-5, SHV-12). Bactericidal activity of oral β-lactams in plasma and urine was tested in time-kill experiments and by determining bactericidal titres at different time intervals post-dose. The killing rate of antibiotics in urine was slower than in plasma, but faster than in Mueller–Hinton broth. High bactericidal titres in urine were only maintained throughout the whole dosing interval by ceftibuten against strains producing broad-, SHV-2 and SHV-3 β-lactamases. The older generation cephalosporins can be considered for the therapy of urinary tract infections caused by E. coli harbouring TEM-1, TEM-2 and SHV-1 β-lactamases but a shorter dosing interval is needed. Ceftibuten can be recommended with caution in ESBL producing E. coli except those producing SHV-4, SHV-5 and SHV-12 that confer resistance to it. If these enzymes are produced, fluoroquinolones or carbapenems could be considered. [Copyright &y& Elsevier]

A Klebsiella pneumoniae strain was isolated from a sputum specimen of a patient in the intensive care unit in 1999 in Shanghai Huashan Hospital, China. The isolate was confirmed as an extended-spectrum β-lactamase (ESBL) producing strain by double-disk synergy test. The results of susceptibility test showed that it was resistant to most β-lactams (including third generation cephalosporins) and non-β-lactam antimicrobial agents. Transconjugants were obtained at a frequency of 10−4. A plasmid of about 60 kb was obtained from the transconjugant by plasmid extraction. Three major nitrocefin-hydrolysing bands with pIs of 5.4, 8.2 and 8.4, were shown in extracts of the transconjugant. Partial gene amplification products of blaTEM, blaSHV, and CTX-M-1group gene were obtained from the isolate as well as its transconjugant. The entire blaTEM, blaSHV, and blaCTX-M in the transconjugant were amplified by PCR and the PCR products were cloned into a pHSG398 vector. Afterwards, the susceptibility of transformants and activities of β-lactamases of transformants on antibiotics were tested. The PCR products were directly sequenced, analysed and identified as TEM-1, SHV-12, and CTX-M-3 genes. These results confirm that this strain of Klebsiella pneumoniae produces SHV-12, CTX-M-3 ESBLs and TEM-1 β-lactamase, encoded by one single plasmid, which is responsible for the resistance of this strain to most β-lactams. [Copyright &y& Elsevier]

Of 50 extended-spectrum β-lactamase-producing (ESBL) isolates of Klebsiella pneumoniae collected from six hospitals in Hubei Province, 20 were found to produce CTX-M ESBLs (40%). Sequence analysis of the six isolates carrying blaCTX-M genes revealed that they all harboured blaCTX-M-3. In the majority of isolates, blaCTX-M genes were within large conjugative plasmids. A high degree of diversity of the RAPD types revealed that all the isolates carrying CTX-M were genetically unrelated and horizontal transfer of plasmids was probably the main mechanism of blaCTX-M spread. This is the first report of the occurrence of CTX-M-3 ESBLs in central China; previously this enzyme was identified only in Europe. A more comprehensive survey of ESBL types from China is urgently needed. [Copyright &y& Elsevier]

Klebsiella pneumoniae 2207, from Durban, was resistant to cefoxitin and β-lactamase inhibitor combinations as well as oxyimino-aminothiazolyl cephalosporins. β-Lactamases with isoelectric points of 5.4, 5.6, 7.6, 8.2 and 8.4 were found. DNA hybridisation identified two BamHI and three HindIII fragments carrying blaTEM, and two SalI fragments carrying blaSHV. At least two genes encoded TEM-1 enzyme; one blaSHV copy encoded SHV-5 but the other determined SHV-23, a novel SHV-5 variant with conservative amino-acid substitutions far from the catalytic site. The pI 8.4 activity was an AmpC-type enzyme. Determinants of the pI 5.6 and 7.6 activities were not identified. [Copyright &y& Elsevier]