CTX-M-123, a Novel Hybrid of the CTX-M-1 and CTX-M-9 Group β-Lactamases Recovered from Escherichia coli Isolates in China

The chimeric bla_CTX-M-123 gene was identified in two ceftazidime-resistant Escherichia coli isolates from animals in different Chinese provinces. Like other CTX-M-1/9 group hybrids (CTX-M-64 and CTX-M-132), the ends (amino acids 1 to 135 and 234 to 291) of CTX-M-123 match CTX-M-15 while the central part (122 to 241) matches CTX-M-14. bla_CTX-M-123 is carried on related, but not identical, ∼90-kb IncI1 plasmids in the two isolates, and one isolate simultaneously carries the group 1 bla_CTX-M-55 gene on an additional IncI2 plasmid.     

New Variant of CTX-M-Type Extended-Spectrum β-Lactamases,CTX-M-71, with a Gly238Cys Substitution in a Klebsiella pneumoniae Isolate from Bulgaria

A single Klebsiella pneumoniae strain isolated in a Bulgarian hospital was found to produce CTX-M-71, a new CTX-M variant characterized by one amino acid substitution from glycine to cysteine at position 238 in comparison to CTX-M-15. This exchange decreased the hydrolytic activity of the β-lactamase for cefotaxime, ceftazidime, and cefepime.Since the first reports on CTX-M-type extended-spectrum β-lactamases (ESBLs) in the late 1980s and early 1990s (2, 4, 13), their number has increased to 89 (http://www.lahey.org/studies [last accessed in June 2009]) and they have become the most prevalent ESBL-type worldwide (6).Several amino acid residues of CTX-M β-lactamases have been investigated with respect to their influence on hydrolytic activity. Asn104, Asn132, and Ser237 are thought to fix the cefotaxime substrate within the binding site (5). Arg276 appears to be important for the hydrolysis of cefotaxime, and the substitutions Asp240Gly and Pro167Ser improve the hydrolytic activity of CTX-M enzymes for ceftazidime (5, 17).We analyzed a Klebsiella pneumoniae isolate producing a new CTX-M variant, CTX-M-71, with one amino acid substitution (Gly238Cys) in comparison to CTX-M-15.K. pneumoniae AH24-270 was isolated in September 2003 from the urine of a 29-year-old male patient at the Alexandrovska University Hospital in Sofia, Bulgaria. The patient had been hospitalized since June 2003 due to cranial brain trauma, aspiration pneumonia, and respiratory dysfunction. Antibiotic treatment prior to isolation of the strain included penicillin, metronidazole, amikacin, vancomycin, meropenem, ceftazidime, and piperacillin-tazobactam.The sequencing of the bla_CTX-M gene of K. pneumoniae AH24-270, by using the oligonucleotides CTX-M-1/P1c (5′-TCGTCTCTTCCAGAATAAGG-3′) and CTX-M-1/P2c (5′-AAGGAGAACCAGGAACCACG-3′), revealed one nucleotide exchange from G to T at open reading frame position 721, causing an amino acid substitution from glycine to cysteine at position 238 in comparison to CTX-M-15 (Ambler numbering [1]). This new CTX-M variant was named CTX-M-71. So far, only one other natural CTX-M β-lactamase, namely, CTX-M-34 (accession no. {"type":"entrez-nucleotide","attrs":{"text":"AY515297","term_id":"41079306","term_text":"AY515297"}}AY515297), which also harbored a cysteine at position 238 was described (15). All other CTX-M variants carry a glycine at that position. However, Shimizu-Ibuka et al. introduced in vitro the Gly238Cys exchange in Toho-1 (CTX-M-44) (19).Conjugative plasmid transfer, performed as described previously (11), located bla_CTX-M-71 on a transferable plasmid together with determinants for resistance to tobramycin, gentamicin, and tetracycline (data not shown).The MICs, determined by an agar dilution procedure following Clinical and Laboratory Standards Institute (formerly NCCLS) guidelines (16), are shown in Table ​Table1.1. Interestingly, K. pneumoniae AH24-270 was resistant to ertapenem and had an elevated MIC for meropenem but was susceptible to imipenem. The MICs for meropenem and ertapenem, but not for imipenem and faropenem, were lowered in combination with clavulanic acid. The transconjugant showed no elevated MICs for carbapenems.

TABLE 1.

Antimicrobial susceptibilities of wild-type, transconjugant, and transformant strains

产CTX-M-14和CTX-M-15大肠埃希菌毒力基因分布差异

目的分析尿培养产CTX-M-14和产CTX-M-15大肠埃希菌毒力基因分布的差异。方法收集南京鼓楼医院2012年临床尿培养分离大肠埃希菌162株,双环协同试验检测超广谱β内酰胺酶(ESBLs);采用PCR和DNA测序对blaCTX-M编码基因及毒力基因iut A、omp T、fyu A、fde C、fim H、tra T、cva C、pap、kps MT、PAIs、usp、aer、hly A、cnf和chu A进行分析;用脉冲场凝胶电泳(PFGE)分析产CTX-M-14和产CTX-M-15大肠埃希菌遗传相关性;将细菌分为产CTX-M-14和产CTX-M-15大肠埃希菌2组,统计学分析毒力基因在这2组之间的分布差异。结果 162株大肠埃希菌中,126株产ESBLs,91株产CTX-M酶,其中bla_(CTX-M-14)和bla_(CTX-M-15)编码基因分别检出49株和36株。PFGE分析显示,产CTX-M酶大肠埃希菌具有遗传多样性。毒力基因iut A、fyu A、fim H、tra T及chu A在2组中的检出率均较高(65%);pap、kps MT、omp T在2组中的检出率约为20%~60%;cva C和PAIs在2组中的检出率较低(20%);毒力基因fed C在产CTX-M-14大肠埃希菌组中分布高于产CTX-M-15大肠埃希菌组(P=0.017);未检出aer、hly A和cnf毒力基因。结论在尿培养大肠埃希菌中,产CTX-M-14菌株fed C毒力基因分布较产CTX-M-15菌株更高。     

The Novel CTX-M-116 β-Lactamase Gene Discovered in Proteus mirabilis Is Composed of Parts of the CTX-M-22 and CTX-M-23 Genes

The novel β-lactamase gene bla_CTX-M-116 was identified in a Proteus mirabilis nosocomial isolate recovered from the urine of a patient in Moscow in 2005. DNA sequence analysis showed bla_CTX-M-116 to be a hybrid gene consisting of 5′ bla_CTX-M-23 (nucleotides 1 to 278) and 3′ bla_CTX-M-22 (nucleotides 286 to 876) moieties separated by an intervening putative site of recombination (GTTAAAT). A retrospective analysis of available bla_CTX-M genes in the GenBank database revealed 19 bla_CTX-M genes that display the same hybrid structure.     

High Prevalence of ST131 Isolates Producing CTX-M-15 and CTX-M-14 among Extended-Spectrum-β-Lactamase-Producing Escherichia coli Isolates from Canada

Phenotypic and genotypic methods were used to characterize extended-spectrum-β-lactamase (ESBL)-producing Escherichia coli isolated in 2007 from 11 different Canadian medical centers. Of the 209 ESBL-producing E. coli isolates tested, 148 (71%) produced CTX-M-15, 17 (8%) produced CTX-M-14, 5 (2%) produced CTX-M-3, and 1 produced CTX-M-27. Overall, 96 (46%) of the ESBL producers belonged to clonal complex ST131, with the highest prevalence in Brampton, Calgary, and Winnipeg. ST131 is an important cause of community onset urinary tract infections due to ESBL-producing E. coli across Canada.Since 2000, Escherichia coli producing CTX-M enzymes has emerged worldwide as an important cause of community onset urinary tract infections (UTIs), and this has been called “the CTX-M pandemic” (3). This phenomenon accelerated rapidly, especially during the past 5 years, and today organisms producing these enzymes are the most common type of extended-spectrum β-lactamase (ESBL) producers found in most areas of the world (24). Although several members of the family Enterobacteriaceae that produce CTX-M β-lactamases have been involved in hospital-acquired infections, E. coli producing these enzymes is more likely to be responsible for community onset infections (21).Currently, the most widely distributed CTX-M enzyme is CTX-M-15, which was first detected in E. coli from India in 2001 (10). Multidrug-resistant, CTX-M-15-producing E. coli is emerging worldwide, especially since 2003, as an important pathogen causing both community onset and hospital-acquired infections (6, 14, 20).Two recent studies using multilocus sequencing typing (MLST) identified a single clone of CTX-M-15-producing E. coli, named ST131, in isolates from several countries, including Spain, France, Canada, Portugal, Switzerland, Lebanon, India, Kuwait, and Korea (6, 14). This clone is associated with serogroup O25, belongs to highly virulent phylogenetic group B2, and harbors multidrug-resistant IncFII plasmids. Since those initial studies, isolates of clonal complex ST131 that produce CTX-M-15 have also been reported in several countries, including the United Kingdom (11), Italy (2), Turkey (27), Croatia (12), Japan (25), the United States (8), and Norway (13). Isolates of clonal complex ST131 have also been associated with other types of β-lactamases, as well as ciprofloxacin-resistant E. coli isolates that do not have ESBLs (4, 9, 12, 15).Due to the worldwide emergence of clone ST131 isolates that produce CTX-M β-lactamases, we designed a study to investigate the prevalence and characteristics of this clone in ESBL-producing E. coli isolated from community and hospital settings during 2007 from 11 different Canadian medical centers.(This study was presented at the 26th International Congress of Chemotherapy and Infection in Toronto, Ontario, Canada, 2009 [abstract P179].)Nonrepeat ESBL-producing E. coli was collected over a 1-month period in 2007 from different Canadian medical centers representing 11 cities in six provinces (Table ​(Table1).1). ESBL production was confirmed phenotypically by using the Clinical and Laboratory Standards Institute [CLSI] criteria for ESBL screening and disk confirmation tests (5).

TABLE 1.

ESBL-producing E. coli isolated at various medical centers in Canada

Extended-spectrum-β-lactamase-producing Escherichia coli as a cause of pediatric infections: report of a neonatal intensive care unit outbreak due to a CTX-M-14-producing strain

Little information is available about pediatric infections caused by extended-spectrum-β-lactamase (ESBL)-producing Escherichia coli. We characterized an outbreak caused by a CTX-M-14-producing E. coli isolate in a neonatal intensive care unit (NICU) and studied other infections caused by ESBL-producing E. coli in non-NICU pediatric units. All children ≤4 years old who were infected or colonized by ESBL-producing E. coli isolates between January 2009 and September 2010 were included. Molecular epidemiology was studied by phylogroup analysis, pulsed-field gel electrophoresis (PFGE), and multilocus sequence typing. Antibiotic resistance genes were analyzed by PCR and sequencing. Plasmids were studied by PFGE with S1 nuclease digestion and by incompatibility group analysis using a PCR-based replicon-typing scheme. Of the ESBL-producing E. coli isolates colonizing or infecting the 30 newborns, identical PFGE results were observed for 21 (70%) isolates, which were classified as CTX-M-14-producing E. coli of ST23 phylogroup A. bla(CTX-M-14a) was linked to ISEcp1 and was carried on an ～80-bp IncK plasmid. A smaller ongoing outbreak due to SHV-12-producing ST131 E. coli was also identified in the same NICU. Fifteen additional infections with ESBL-producing E. coli were identified in non-NICU pediatric units, but none was caused by the CTX-M-14-producing E. coli epidemic clone. Overall, CTX-M-14 (71.1%), CTX-M-15 (13.3%), and SHV-12 (13.3%) were the most important ESBLs causing pediatric infections in this study. Infections of newborns with CTX-M-14-producing E. coli were caused by both clonal and nonclonal isolates.     

Existential pain--an entity, a provocation, or a challenge?

"Existential pain" is a widely used but ill-defined concept. Therefore the aim of this study was to let hospital chaplains (n=173), physicians in palliative care (n=115), and pain specialists (n=113) respond to the question: "How would you define the concept existential pain?" A combined qualitative and quantitative content analysis of the answers was conducted. In many cases, existential pain was described as suffering with no clear connection to physical pain. Chaplains stressed significantly more often the guilt issues, as well as various religious questions (P<0.001). Palliative physicians (actually seeing dying persons) stressed more often existential pain as being related to annihilation and impending separation (P<0.01), while pain specialists (seeing chronic patients) more often emphasized that "living is painful" (P<0.01). Thirty-two percent (32%) of the physicians stated that existential suffering can be expressed as physical pain and provided many case histories. Thus, "existential pain" is mostly used as a metaphor for suffering, but also is seen as a clinically important factor that may reinforce existing physical pain or even be the primary cause of pain, in good agreement with the current definition of pain disorder or somatization disorder.     

广州地区肺炎克雷伯菌携带CTX-M-14质粒同源性分析

目的研究广州肺炎克雷伯菌(Klebsiella pneumonia,Kp)CTX-M-14型超广谱β-内酰胺酶(ESBLs)的质粒同源性特征。方法收集2007～2008年广州地区9家医院临床分离的产ESBLs肺炎克雷伯菌,PCR检测ESBLs分子表型;用肠杆菌科基因间重复序列PCR(Enterobacteriaceae repetive intergenic consensus-PCR,ERIC-PCR)分析产CTX-M-14型ESBLs菌株的分子同源性;取产CTX-M-14型ESBLs的Kp的所有非克隆株,通过结合试验、质粒图谱、PCR分析blaCTX-M-14基因环境。结果产ESBLsKp共181株,69.1%(125/181)的产ESBLs株为CTX-M表型;其中产CTX-M-14型ESBLs株的检出率为28.2%(51/181),经ERIC-PCR分析,共分为33个基因型;基因环境显示,75.7%(25/33)blaCTX-M-14位于90 kb的可接合质粒上,其他耐药基因blaSHV、blaDHA-1、blaOXA-1、qnr、aac(6’)-Ib-cr等均未检到;有28株菌的blaCTX-M-14位于ISEcp1-like插入序列下游,ISEcp1-like末端与blaCTX-M-14间距均为42 bp,有2株菌ISEcp1末端与blaCTX-M-14起始区间距也为42 bp;但在ISECP1中间有一个S10插入序列。结论广州地区ESBLs主要分子表型为CTX-M型,主要流行为CTX-M-14型,携blaCTX-M-14质粒的传播,可能是本地区CTX-M型ESBLs高发生率的主要原因。     

志贺菌属中鉴别出产CTX-M-55型菌株

在革兰阴性肠杆菌中,国内有关大肠埃希菌、肺炎克雷伯菌和阴沟肠杆菌B内酰胺酶耐药基因已有较多报道[1-5],但志贺菌属产β内酰胺酶耐药基因的报道鲜见[6].我们对20株志贺菌属进行9种β内酰胺酶基因(TEM、SHV、CARB、LAP、GES、PER、VEB、CTX-M-1群、OXA-1群)检测,经测序和BLASTn比对发现CTX-M-55型,报告如下.     

Residues Distal to the Active Site Contribute to Enhanced Catalytic Activity of Variant and Hybrid β-Lactamases Derived from CTX-M-14 and CTX-M-15

A variety of CTX-M-type extended-spectrum β-lactamases (ESBLs), including hybrid ones, have been reported in China that are uncommon elsewhere. To better characterize the substrate profiles and enzymatic mechanisms of these enzymes, we performed comparative kinetic analyses of both parental and hybrid CTX-M enzymes, including CTX-M-15, -132, -123, -64, -14 and -55, that are known to confer variable levels of β-lactam resistance in the host strains. All tested enzymes were susceptible to serine β-lactamase inhibitors, with sulbactam exhibiting the weakest inhibitory effects. CTX-M-55, which differs from CTX-M-15 by one substitution, A⁷⁷V, displayed enhanced catalytic activity (k_cat/K_m) against expanded-spectrum cephalosporins (ESCs). CTX-M-55 exhibits higher structure stability, most likely by forming hydrophobic interactions between A⁷⁷V and various key residues in different helices, thereby stabilizing the core architecture of the helix cluster, and indirectly contributes to a more stable active site conformation, which in turn shows higher catalytic efficiency and is more tolerant to temperature change. Analyses of the hybrids and their parental prototypes showed that evolution from CTX-M-15 to CTX-M-132, CTX-M-123, and CTX-M-64, characterized by gradual enhancement of catalytic activity to ESCs, was attributed to introduction of different substitutions to amino acids distal to the active site of CTX-M-15. Similarly, the increased hydrolytic activities against cephalosporins and sensitivity to β-lactamase inhibitors, clavulanic acid and sulbactam, of CTX-M-64 were partly due to the amino acids that were different from CTX-M-14 and located at both the C and N termini of CTX-M-64. These data indicate that residues distal to the active site of CTX-Ms contributed to their enhanced catalytic activities to ESCs.     

在中国首次检出CTX-M-15型超广谱β-内酰胺酶基因

目的探讨湖南地区中南大学三所附属医院肠杆菌科细菌产CTX-M型超广谱β-内酰胺酶(extended-spectrumβ-lactamases,ESBLs)的检出率及其基因型。方法采用多重PCR法对产ES- BLs的142株临床分离的肠杆菌进行CTX-M酶基因检测,然后用组特异性引物对CTX-M酶整个开放阅读框进行扩增,PCR产物测序并确定其基因型。结果湖南地区中南大学三所附属医院肠杆菌科细菌产CTX-M型ESBLs检出率为76.8%(109/142):对109株产CTX-M型ESBLs肠杆菌科细菌进行特异性PCR扩增,进行分组,发现CTX-M-1组48株,CTX-M-9组48株,另外13株可能属于CTX-M-2组和CTX-M-8组。根据不同病室不同菌株来源,共测序25株,组特异性PCR产物测序后发现有三种基因型,分别为CTX-M-15型、CTX-M-3型和CTX-M-14型。其中CTX-M-15型6株,CTX-M-3型7株,CTX-M-14型11株。结论本地区CTX-M型ESBLs检出率较高,共检出三种CTX-M酶基因型,并且CTX-M-15型ESBLs是在国内首次被发现。     

Preintegration HIV-1 Inhibition by a Combination Lentiviral Vector Containing a Chimeric TRIM5�� Protein, a CCR5 shRNA, and a TAR Decoy

Human immunodeficiency virus (HIV) gene therapy offers a promising alternative approach to current antiretroviral treatments to inhibit HIV-1 infection. Various stages of the HIV life cycle including pre-entry, preintegration, and postintegration can be targeted by gene therapy to block viral infection and replication. By combining multiple highly potent anti-HIV transgenes in a single gene therapy vector, HIV-1 resistance can be achieved in transduced cells while prohibiting the generation of escape mutants. Here, we describe a combination lentiviral vector that encodes three highly effective anti-HIV genes functioning at separate stages of the viral life cycle including a CCR5 short hairpin RNA (shRNA) (pre-entry), a human/rhesus macaque chimeric TRIM5α (postentry/preintegration), and a transactivation response element (TAR) decoy (postintegration). The major focus on designing this anti-HIV vector was to block productive infection of HIV-1 and to inhibit any formation of provirus that would maintain the viral reservoir. Upon viral challenge, potent preintegration inhibition of HIV-1 infection was achieved in combination vector–transduced cells in both cultured and primary CD34⁺ hematopoietic progenitor cell (HPC)–derived macrophages. The generation of escape mutants was also blocked as evaluated by long-term culture of challenged cells. The ability of this combination anti-HIV lentiviral vector to prevent HIV-1 infection, in vitro, warrants further evaluation of its in vivo efficacy.