The AmpC inhibitor, Syn2190, can be used to reveal extended-spectrum beta-lactamases in Escherichia coli

AmpC beta-lactamases are not inhibited by clavulanic acid and could potentially mask detection of extended-spectrum beta-lactamases (ESBLs) using the Clinical and Laboratory Standards Institute confirmatory test. Syn2190 (1,5-dihydroxy-4-pyridone monobactam) selectively inhibits AmpC, but not ESBLs. Fifty-four MicroScan ESBL screen-positive strains of Escherichia coli and an unrelated group of 20 cefoxitin-nonsusceptible E. coli strains were tested with the confirmatory ceftazidime-cefotaxime-clavulanate disk method with or without 4 microg/mL of Syn2190 in the agar. Without Syn2190, 8 (14.8%) of 54 E. coli isolates and 0 of 20 cefoxitin-nonsusceptible E. coli isolates were confirmed. With Syn2190, an additional 9 (16.6%) of 54 of the MicroScan screen-positive E. coli isolates and 6 (30%) of 20 of the cefoxitin-nonsusceptible E. coli isolates were found. Multiplex polymerase chain reaction and sequence analysis confirmed the presence of the plasmid-associated beta-lactamase gene bla(CMY-2) in the 2 available MicroScan-screened E. coli isolates and in 5 of 6 of the cefoxitin-resistant group. These data suggest that in the presence of AmpC, ESBLs in E. coli may not be detected by the currently recommended confirmatory test.     

Effect of conalbumin on the activity of Syn 2190, a 1,5 dihydroxy-4-pyridon monobactam inhibitor of AmpC beta-lactamases

Syn 2190, a 1,5 dihydroxy-4-pyridon monobactam inhibitor of AmpC enzymes, was tested against beta-lactamase-producing bacteria with piperacillin, piperacillin-tazobactam and ceftazidime as partner drugs. In the presence of conalbumin as an iron chelator, Syn 2190 potentiated these drugs against most AmpC producers, although Klebsiella spp. with plasmidic AmpC enzymes were an exception. Potentiation was much weaker without conalbumin, suggesting that Syn 2190 exploits a ferric uptake pathway, as do catecholic cephalosporins. Syn 2190 had little ability to potentiate partner drugs against strains with other beta-lactamase types but, with conalbumin, increased the activity of piperacillin-tazobactam against Escherichia coli transconjugants producing various class A or D enzymes.     

In Vitro and In Vivo Activities of Syn2190, a Novel β-Lactamase Inhibitor

Syn2190, a monobactam derivative containing 1,5-dihydroxy-4-pyridone as the C-3 side chain, is a potent inhibitor of group 1 beta-lactamase. The concentrations of inhibitor needed to reduce the initial rate of hydrolysis of substrate by 50% for Syn2190 against these enzymes were in the range of 0.002 to 0.01 microM. These values were 220- to 850-fold lower than those of tazobactam. Syn2190 showed in vitro synergy with ceftazidime and cefpirome. This synergy was dependent on the concentration of the inhibitor against group 1 beta-lactamase-producing strains, such as Pseudomonas aeruginosa, Enterobacter cloacae, Citrobacter freundii, and Morganella morganii. However, against beta-lactamase-derepressed mutants of P. aeruginosa, the MICs of ceftazidime plus Syn2190 were not affected by the amount of beta-lactamase, and the values were the same for the parent strains. The MICs at which 50% of isolates are inhibited (MIC(50)s) of ceftazidime plus Syn2190 were 2- to 16-fold lower than those of ceftazidime alone for ceftazidime-resistant, clinically isolated gram-negative bacteria. Similarly, the MIC(50)s of cefpirome plus Syn2190 were two- to eightfold lower for cefpirome-resistant clinical isolates. The synergies of Syn2190 plus ceftazidime or cefpirome observed in vitro were also reflected in vivo. Syn2190 improved the efficacies of both cephalosporins in both a murine systemic infection model with cephalosporin-resistant rods and urinary tract infection models with cephalosporin-resistant P. aeruginosa.     

临床常见产AmpC β-内酰胺酶菌株中染色质ampC共同序列的研究

目的分析临床常见AmpC β-内酰胺酶（简称AmpC酶）产酶菌株中染色质ampC的基因序列,从而为AmpC酶的分子生物学检测以及其调控机制研究提供理论依据。方法 57株临床常见AmpC酶产酶菌株分离自医院感染患者样本,抽提细菌染色质DNA,聚合酶链反应（PCR）扩增ampC基因并连接入pMD19-T载体,双链测序后比对同种细菌之间和不同种细菌之间染色质ampC基因的同源性和共同序列。根据共同序列设计引物,进一步利用该引物检测染色质ampC。结果 57株细菌的基因组中,使用PCR扩增出染色质ampC基因41株,并成功测定了其ampC基因的序列。比对后发现大肠埃希菌、阴沟肠杆菌、鲍曼不动杆菌和产气肠杆菌的染色质ampC菌种内有很高的同源性,但细菌之间的同源性较低。根据共同序列设计出菌种特异性PCR引物,能够有效的鉴定出染色质ampC基因。结论大肠埃希菌、阴沟肠杆菌、鲍曼不动杆菌和产气肠杆菌各自染色质ampC具有高度同源性,其菌种特异性的ampC引物可用来检测其染色质ampC的存在。     

Multicity outbreak of carbapenem-resistant Acinetobacter baumannii isolates producing the carbapenemase OXA-40

During 2005 we detected a multicity outbreak of infections or colonization due to high-level imipenem-resistant Acinetobacter baumannii (MIC, 64 microg/ml). One hundred isolates from diverse sources were obtained from seven acute-care hospitals and two extended-care facilities; 97% of the isolates belonged to one clone. Susceptibility testing of the first 42 isolates (January to April 2005) revealed broad resistance profiles. Half of the isolates were susceptible to ceftazidime, with many isolates susceptible only to colistin. The level of AmpC beta-lactamase expression was stronger in isolates resistant to ceftazidime. PCR and subsequent nucleotide sequencing analysis identified bla(OXA-40). The presence of an OXA-40 beta-lactamase in these isolates correlated with the carbapenem resistance. By Southern blot analysis, a bla(OXA-40)-specific probe revealed that the gene was both plasmid and chromosomally located. This is the first time in the United States that such carbapenem resistance in A. baumannii has been attributable to a carbapenemase.     

Epidemiology of conjugative plasmid-mediated AmpC beta-lactamases in the United States

A sample of 752 resistant Klebsiella pneumoniae, Klebsiella oxytoca, and Escherichia coli strains from 70 sites in 25 U.S. states and the District of Columbia was examined for transmissibility of resistance to ceftazidime and the nature of the plasmid-mediated beta-lactamase involved. Fifty-nine percent of the K. pneumoniae, 24% of the K. oxytoca, and 44% of the E. coli isolates transferred resistance to ceftazidime. Plasmids encoding AmpC-type beta-lactamase were found in 8.5% of the K. pneumoniae samples, 6.9% of the K. oxytoca samples, and 4% of the E. coli samples, at 20 of the 70 sites and in 10 of the 25 states. ACT-1 beta-lactamase was found at eight sites, four of which were near New York City, where the ACT-1 enzyme was first discovered; ACT-1 beta-lactamase was also found in Massachusetts, Pennsylvania, and Virginia. FOX-5 beta-lactamase was also found at eight sites, mainly in southeastern states but also in New York. Two E. coli strains produced CMY-2, and one K. pneumoniae strain produced DHA-1 beta-lactamase. Pulsed-field gel electrophoresis and plasmid analysis suggested that AmpC-mediated resistance spread both by strain and plasmid dissemination. All AmpC beta-lactamase-containing isolates were resistant to cefoxitin, but so were 11% of strains containing transmissible SHV- and TEM-type extended-spectrum beta-lactamases. A beta-lactamase inhibitor test was helpful in distinguishing the two types of resistance but was not definitive since 24% of clinical isolates producing AmpC beta-lactamase had a positive response to clavulanic acid. Coexistence of AmpC and extended-spectrum beta-lactamases was the main reason for these discrepancies. Plasmid-mediated AmpC-type enzymes are thus responsible for an appreciable fraction of resistance in clinical isolates of Klebsiella spp. and E. coli, are disseminated around the United States, and are not so easily distinguished from other enzymes that mediate resistance to oxyimino-beta-lactams.     

112株鲍曼不动杆菌的临床分布及耐药性分析

目的了解临床分离的112株鲍曼不动杆菌的分布特点及耐药情况。方法用K-B纸片扩散法检测鲍曼不动杆菌的耐药率,以超广谱β-内酰胺酶(ESBLs)确证试验和三维试验检测鲍曼不动杆菌ESBLs、AmpC酶产酶情况与变化。结果临床感染鲍曼不动杆菌对13种抗生素耐药严重,ESBLs与AmpC酶检出率分别为1.8%和71.4%,对亚胺培南耐药率最低,为9.8%。结论邢台市人民医院临床分离的鲍曼不动杆菌流行株主要是产AmpC酶,且呈多重耐药性,应重视合理使用抗生素,减少多重耐药菌株的产生。     

Cloning, nucleotide sequencing, and analysis of the gene encoding an AmpC beta-lactamase in Acinetobacter baumannii

A clinical strain of Acinetobacter baumannii (strain Ab RYC 52763/97) that was isolated during an outbreak in our hospital and that was resistant to all beta-lactam antibiotics tested produced three beta-lactamases: a TEM-1-type (pI, 5.4) plasmid-mediated beta-lactamase, a chromosomally mediated OXA-derived (pI, 9.0) beta-lactamase, and a presumptive chromosomal cephalosporinase (pI, 9.4). The nucleotide sequence of the chromosomal cephalosporinase gene shows for the first time the gene encoding an AmpC beta-lactamase in A. baumannii. In addition, we report here the biochemical properties of this A. baumannii AmpC beta-lactamase.     

鲍曼不动杆菌的高产AmpC酶、ESBLs及其耐药性分析

目的了解鲍曼不动杆菌高产AmpC酶、超广谱β-内酰胺酶（ESBLs）产生情况及其耐药谱。方法用K—B法进行药物敏感性检测,按表型确证试验检测ESBLs,按表型筛选法检测高产AmpC酶。结果93株鲍曼不动杆菌主要分离自呼吸内科、神经外科,以痰或咽拭子、创面或伤口分泌物、尿3类标本多见,检测到15株（16．1％）产高产AmpC酶菌株,10株（10．8％）产ESBLs菌株,产酶株均呈多重耐药,对第3代头孢菌素、青霉素类及氨曲南耐药率明显高于非产酶菌株,未发现亚胺培南耐药株。结论产高产AmpC酶、产ESBLs酶是鲍曼不动杆菌多重耐药的原因之一,应加强对产酶株的检测,以指导临床合理应用抗生素,防止其暴发流行。     

Diversity of extended-spectrum beta-lactamases and class C beta-lactamases among cloacal Escherichia coli Isolates in Belgian broiler farms

A total of 295 ceftiofur-resistant Escherichia coli isolates were obtained from 489 cloacal samples collected at five different Belgian broiler farms with the aim to evaluate the diversity of this resistance at the farm level. Strains were examined for resistance against beta-lactam antibiotics and other antimicrobial agents by using disk diffusion tests. Three different beta-lactam resistance phenotypes suggested the presence of an extended-spectrum beta-lactamase (ESBL), a class C beta-lactamase, or the combination of an ESBL with a class C beta-lactamase. Seventy-six percent of these isolates also showed acquired resistance to other antimicrobial agents. After genotyping by repetitive extragenic palindromic-PCR, 51 unrelated E. coli strains were selected for further analyses. Isoelectric focusing and sequencing of the amplicons obtained in PCRs for the detection of genes encoding broad-spectrum beta-lactamase enzymes revealed the following ESBLs: TEM-52 (13.2%), TEM-106 (2%), CTX-M-1 (27.4%), CTX-M-2 (7.8%), CTX-M-14 (5.9%), and CTX-M-15 (2%). The only plasmidic AmpC beta-lactamase found in this study was the CMY-2 enzyme (49%). Mutations in the promoter and attenuator regions of the chromosomal ampC gene were found only in association with bla CMY-2 genes and ESBL genes. The combination of an ESBL (CTX-M-1) with a plasmidic AmpC beta-lactamase (CMY-2) was found in 7.8% of the isolates. These data show that ceftiofur-resistant E. coli strains are often present in cloacal samples of broilers at the farm level in Belgium. The diversity of broad-spectrum beta-lactamases among these isolates is high, and they may act as a reservoir of ESBL and ampC genes.     

A nosocomial outbreak of Acinetobacter baumannii isolates expressing the carbapenem-hydrolysing oxacillinase OXA-58

OBJECTIVE: The aim of this study was to analyse the spread of the bla(OXA-58) gene as a source of carbapenem resistance in Acinetobacter baumannii in the burns unit of a university hospital in Toulouse in 2003-2004. METHODS: Six carbapenem-resistant A. baumannii isolates from six patients, and a carbapenem-resistant environmental A. baumannii isolate were collected in the burns unit of the Rangueil hospital (Toulouse). Susceptibility tests were carried out by disc diffusion and agar dilution methods. The detection of the bla(OXA-58) gene was conducted by PCR followed by sequence analysis. Plasmids were extracted and hybridized with a probe specific for bla(OXA-58). DNA fingerprints were obtained by pulsed-field gel electrophoresis of ApaI- or SmaI-digested chromosomal DNA of the tested strains. RESULTS: The multidrug-resistant clinical isolates had a similar 30 kb plasmid that encoded the carbapenem-hydrolysing beta-lactamase OXA-58. These isolates were clonally related. The unrelated environmental carbapenem-resistant A. baumannii isolate had a similar bla(OXA-58)-carrying plasmid, suggesting spread of this gene. CONCLUSIONS: A novel oxacillinase was the source of carbapenem resistance in the A. baumannii isolates. Its gene was plasmid-, but not integron-borne.     

Mechanisms of beta-lactam resistance in Pseudomonas aeruginosa: prevalence of OprM-overproducing strains in a French multicentre study (1997)

One hundred and forty-three non-repetitive strains of Pseudomonas aeruginosa were collected in 13 French hospitals in 1997. A decreased susceptibility or resistance to ticarcillin (MIC > 16 mg/L) was found in 61 isolates (43%) and this was attributed to three major mechanisms: (i) overexpression of OprM and hence related efflux components such as MexAB or MexXY (42.6%), (ii) production of acquired beta-lactamase (29.5%) and (iii) overexpression of chromosomally encoded AmpC cephalosporinase (21.3%). Four of seven 'intrinsically' resistant strains (11.5%) with normal amounts of OprM were shown to produce low levels of AmpC, whereas in three isolates no resistance mechanism to beta-lactams could be identified. Overproduction of OprM thus appears as an important mechanism of ticarcillin resistance in French isolates of P. aeruginosa.     

大肠埃希菌超广谱β-内酰胺酶与AmpC酶耐药性分析

目的研究本地区大肠埃希菌产超广谱β-内酰胺酶与AmpC酶耐药性。方法收集临床分离无重复大肠埃希菌共116株,利用双纸片协同法检测超广谱β-内酰胺酶(ESBLs);利用3-氨基苯酚硼酸对AmpCβ-内酰胺酶的抑制作用检测表型AmpC酶。K-B法测定细菌对抗生素的耐药性。结果116株大肠埃希菌ESBLs阳性43株(37.1%),ESBLs与AmpC酶同时阳性10株(8.6%),表型未检出AmpC酶单独阳性株。大肠埃希菌的产酶株及非产酶株对碳青霉烯类100%敏感,但产酶株(尤其是同时产ESBLs与AmpC酶)比非产酶株耐药率高,多重耐药性更常见。结论从本院患者送检标本分离的大肠埃希菌中发现AmpC酶,应引起临床重视。碳青霉烯类药物可作为治疗产酶细菌感染的首选药物。     

重症监护病房中多重耐药鲍曼不动杆菌耐药基因型及同源性分析

目的分析22株重症监护病房(ICU)中多重耐药鲍曼不动杆菌耐药表型,耐药基因型及基因同源性。方法菌株鉴定采用Vitek-32细菌鉴定仪,药物敏感试验采用K-B法。三维试验检测ESBLs和AmpC酶,协同试验检测金属酶(MBL)。PCR检测各类β-内酰胺酶类、氨基糖苷类抗菌药耐药基因及喹诺酮类相关gyrA基因。脉冲场凝胶电泳(PFGE)进行基因同源性分析。结果 (1)对亚胺培南耐药率最低为4.5%,头孢哌酮/舒巴坦耐药率较低为22.7%,对其他所测抗菌药耐药率均大于90%。(2)产AmpC酶21株占95.5%;产ESBLs和MBL各5株,分别占22.7%。(3)100%携带AmpC酶基因、TEM-1型广谱β-内酰胺酶基因、aacA4氨基糖苷乙酰转移酶基因;aacC1、aadA1型氨基糖苷乙酰转移酶基因各占95.5%;有gyrA型DNA旋转酶基因突变;各1株检测到OXA-23型碳青霉烯酶基因、PER型ESBLs基因和aphA1型氨基糖苷乙酰转移酶基因,分别占4.5%;未检测到SHV型β-内酰胺酶基因、VEB型ESBL基因I、MP型和VIM型金属酶基因、OXA-24型碳青霉烯酶基因。(4)PFGE结果显示20株出现完全相同条带,具有高度同源性,另2株各自为独立株。结论 22株多重耐药鲍曼不动杆菌中存在一个克隆流行株,携带AmpC酶基因、TEM-1型β-内酰胺酶基因及aacA4、aacC1、aadA1型氨基糖苷类修饰酶基因,高产AmpC酶,治疗应首选亚胺培南。     

Confirmation of extended-spectrum beta-lactamase-producing Serratia marcescens: preliminary report from Taiwan

Although Serratia marcescens is a common cause of nosocomial infection in Taiwan, strains producing extended-spectrum beta-lactamases (ESBLs) are rare. We detected four clinical isolates of S. marcescens from Taiwan that exhibited resistance to cefotaxime (MICs, > 256 microg/ml) and cefepime (MICs, > or = 32 microg/ml), but were susceptible to imipenem and meropenem. Transconjugants revealed similar MIC profiles when compared to the parental strains. Isoelectric focusing revealed one major transferable beta-lactamase (pI 8.4), which was further identified as CTX-M-3 by polymerase chain reaction and gene sequencing. An AmpC-like enzyme (pI 8.8) was not transferable. All four isolates had significant MIC reductions of > or =3 log(2) dilutions for cefepime in the presence of clavulanic acid, compatible with the presence of an ESBL (CTX-M-3). Clavulanate did not significantly reduce the cefotaxime MIC for one isolate that may co-produce high-level AmpC beta-lactamase (pI 8.8). Since high-level AmpC expression has minimal effect on the activity of cefepime, isolates co-producing AmpC beta-lactamase may be recognized as additional ESBL producers by using cefepime as an ESBL screening agent.     

Characterization of CMY-type beta-lactamases in clinical strains of Proteus mirabilis and Klebsiella pneumoniae isolated in four hospitals in the Paris area

We isolated five clinical strains (three Proteus mirabilis and two Klebsiella pneumoniae) with beta-lactam resistance phenotypes consistent with production of an AmpC-type beta-lactamase. The predicted amino acid sequences of the enzymes were typical of class C beta-lactamases. The enzymes were identified as CMY-2, CMY-4 and a new CMY-variant beta-lactamase, CMY-12. The AmpC beta-lactamases from the two K. pneumoniae isolates were found to be encoded on self-transferable plasmids. The genes encoding the AmpC-type beta-lactamase produced by the three P. mirabilis isolates were chromosomal. Four of the five clinical isolates were from patients transferred from Greece, Algeria and Egypt; one of the K. pneumoniae strains was recovered from a French patient. PFGE analysis and rep-PCR fingerprinting showed that the two P. mirabilis isolates from Greek patients were closely related.     

Constitutive high expression of chromosomal beta-lactamase in Pseudomonas aeruginosa caused by a new insertion sequence (IS1669) located in ampD

The expression of chromosomal AmpC beta-lactamase in Pseudomonas aeruginosa is negatively regulated by the activity of an amidase, AmpD. In the present study we examined resistant clinical P. aeruginosa strains and several resistant variants isolated from in vivo and in vitro biofilms for mutations in ampD to find evidence for the genetic changes leading to high-level expression of chromosomal beta-lactamase. A new insertion sequence, IS1669, was found located in the ampD genes of two clinical P. aeruginosa isolates and several biofilm-isolated variants. The presence of IS1669 in ampD resulted in the expression of high levels of AmpC beta-lactamase. Complementation of these isolates with ampD from the reference P. aeruginosa strain PAO1 caused a dramatic decrease in the expression of AmpC beta-lactamase and a parallel decrease of the MIC of ceftazidime to a level comparable to that of PAO1. One highly resistant, constitutive beta-lactamase-producing variant contained no mutations in ampD, but a point mutation was observed in ampR, resulting in an Asp-135-->Asn change. An identical mutation of AmpR in Enterobacter cloacae has been reported to cause a 450-fold higher AmpC expression. However, in many of the isolates expressing high levels of chromosomal beta-lactamase, no changes were found in either ampD, ampR, or in the promoter region of ampD, ampR, or ampC. Our results suggest that multiple pathways may exist leading to increased antimicrobial resistance due to chromosomal beta-lactamase.     

Evaluation of the chromogenic Cica-beta-Test for detecting extended-spectrum, AmpC and metallo-beta-lactamases

BACKGROUND: Extended-spectrum, metallo- and AmpC beta-lactamases usually are sought subsequently to susceptibility testing, meaning that producers are not identified until 72 h after a clinical specimen is taken. Chromogenic tests might usefully shorten this delay, and we investigated the Cica-beta-Test for this purpose. METHODS: Reference and clinical strains with known beta-lactamases, or controls, were grown with a cefpodoxime disc to promote conservation of resistance. The cultures were then tested with nitrocefin and with the Cica-beta-Test, which examines for hydrolysis of the chromogenic oxyimino-cephalosporin HMRZ-86 with and without specific inhibitors of extended-spectrum, metallo- and AmpC beta-lactamases. RESULTS: were scored, as colour changes from yellow to red, with the tester blinded to the strain identity and the mechanism(s) present. Results Proportions of extended-spectrum, metallo- and AmpC beta-lactamase producers correctly identified by the Cica-beta-Test were 85%, 77% and 72%, respectively. Such performance should be achievable if testing colonies from a primary culture plate, 24 h after a specimen was taken. Greater precision, albeit at more delay, would be achieved if results were read in conjunction with antibiogram data available 48 h after the specimen was taken. Limitations were frequent confusion of Klebsiella oxytoca hyperproducing K1 enzyme with AmpC hyperproducers, and that isolates with NMC-A or KPC carbapenemases were wrongly inferred to have AmpC enzymes. CONCLUSIONS: The Cica-beta-Test has the potential to provide useful therapeutic guidance, identifying isolates with potent beta-lactamases and informing early therapy; it will also help to monitor beta-lactamase epidemiology among multiresistant strains.