Molecular characterization of imipenem-resistant Pseudomonas aeruginosa in Hiroshima, Japan

Pseudomonas aeruginosa showing resistance to imipenem were found in 100 of 1,058 strains (9.5%) from six hospitals (a-f) in Hiroshima City, Japan. Of the 100 strains, 14 (14%) were double disk synergy test positive using sodium mercaptoacetic acid disks, and 18 (18%) were bla(IMP-1) or bla(VIM-2) allele positive by polymerase chain reaction (PCR). Among 100 imipenem-resistant strains, 32 were categorized into multi-drug resistant strains, in which 13 were positive for the metallo-beta-lactamase gene. Fifty-one strains (51%) among the 100 imipenem-resistant strains had elevated RND efflux pump activity against levofloxacin. But only 6 of 51 strains were classified as multi-drug resistant strains. The pulsed field gel electrophoresis analysis of the Spe I-digested DNA from the 100 isolates suggested not only clonal spread but spread of heterogeneous clones started to contribute to the prevalence of metallo-beta-lactamase producing P. aeruginosa strains in Japanese hospitals.     

Study of mycoloyl transferase transport across the cell envelope of Corynebacterium glutamicum

A bla(VIM-2) metallo-beta-lactamase determinant, identical to that previously identified in Pseudomonas aeruginosa COL-1 isolate from a French hospital, was detected on a 28-kb plasmid carried by a nosocomial isolate of P. aeruginosa from Verona, Italy. In this plasmid the bla(VIM-2) determinant was inserted into a class 1 integron of original structure, named In72, that contains a partially deleted intI1 integrase gene and two gene cassettes. The first cassette carries an aacA4 aminoglycoside acetyl transferase determinant. The second cassette carries a bla(VIM-2) determinant followed by a partially deleted attC site. The structure of In72 was notably different from that of In56, the bla(VIM-2)-containing integron found in the COL-1 isolate, revealing the existence of molecular heterogeneity among bla(VIM-2)-containing integrons in clinical isolates of P. aeruginosa from Europe.     

福州地区碳青霉烯类耐药鲍曼不动杆菌碳青霉烯酶基因型研究

目的探讨福州地区碳青霉烯类耐药鲍曼不动杆菌(CRAB)碳青霉烯酶基因型的流行情况。方法收集多家医院临床标本中分离得到的107株CRAB。应用K-B纸片扩散法进行药物敏感试验。采用PCR法检测7种碳青霉烯酶基因,包括OXA-23、OXA-24、OXA-51、OXA-58、IMP-1、IMP-4和VIM-2。结果 107株CRAB对除多粘菌素B、米诺环素外的其他所有常见的抗生素均为耐药。碳青霉烯酶基因OXA-51、OXA-23的检出率分别为100.0%(107/107)和87.9%(94/107)。其他OXA-24、OXA-58、IMP-1、IMP-4和VIM-2基因均未检出。结论福州地区临床分离的CRAB耐药现象严重;表达OXA-23基因是CRAB对碳青霉烯类药物耐药的重要机制之一。     

皖北地区耐碳青霉烯鲍曼不动杆菌产金属β-内酰胺酶检测

目的了解皖北地区ICU病房内耐碳青霉烯鲍曼不动杆菌产金属β-内酰胺酶（metallo-β-lactamase,MBL）情况。方法收集皖北地区3家教学医院ICU病房中不动杆菌,K-B纸片扩散法检测其对碳青霉烯类抗生素耐药情况;PCR法检测blaIMP、blaVIM等MBL基因。结果共收集35株耐亚胺培南、美罗培南非重复分离鲍曼不动杆菌菌株,其中9株（25.7%）金属酶初筛阳性;PCR检测5株（5/35,14.3%）产IMP型MBL,1株（1/35,2.9%）VIM型MBL。结论在本地区耐碳青霉烯鲍曼不动杆菌中首次发现IMP和VIM型金属酶,提示产金属酶机制参与本地区鲍曼不动杆菌对碳青霉烯耐药,值得进一步关注。     

Occurrence of a new metallo-beta-lactamase IMP-4 carried on a conjugative plasmid in Citrobacter youngae from the People's Republic of China

During the course of an antimicrobial resistance surveillance programme in Guangzhou, the People's Republic of China, single strains of Citrobacter youngae and Pseudomonas aeruginosa were identified which were resistant to imipenem and found to carry the carbapenemase gene bla(IMP). PCR screening of the citrobacter strain with specific primers for the bla(IMP) type genes gave a 587-bp product which when sequenced gave 100% homology with the bla(IMP-4) sequence reported recently from Acinetobacter spp. The determinant in the C. youngae strain was found to be located on a 156-kb plasmid capable of transfer to Escherichia coli UB1637 by conjugation. Sequencing of the bla(IMP-4) open reading frame in the C. youngae strain and adjacent sequences not only confirmed the presence of bla(IMP-4) but also identified that a conserved core site found within the 59-bp element of integrons was present and the same as the one described in the only other occurrence of bla(IMP-4) in Acinetobacter spp. isolated from an intensive care unit in Hong Kong. This is the second report of transferable carbapenemase genes in Enterobacteriaciae outside of Japan and the first in the People's Republic of China. Under the selective pressure of carbapenems and extended spectrum cephalosporins use we might expect this gene to spread and widespread surveillance should be instituted.     

Identification and antimicrobial susceptibility testing of clinical isolates of nonfermenting gram-negative bacteria by the Phoenix Automated Microbiology System

The Phoenix Automated Microbiology System (Becton Dickinson, Sparks, MD) was evaluated for its ability to identify nonfermenting gram-negative pathogens and measure their drug susceptibility. Isolates producing rare extended-spectrum beta-lactamases (PER-1, IMP-2, VIM-1, and VIM-2) were included in the study. Species identification was compared to that given by the ATB System (bio-Mérieux, Marcy l'Etoile, France), whereas susceptibility results were compared to those produced by a reference broth microdilution test (panels manufactured by Pasco Laboratories, Becton Dickinson). The Phoenix system consistently identified all isolates of Pseudomonas aeruginosa (n = 55) and Stenotrophomonas maltophilia (n = 28), while in other cases species agreement was obtained for 47/53 isolates (Acinetobacter baumannii, 29/31; Pseudomonas putida, 10/11; Burkholderia cepacia, 6/7; and Pseudomonas fluorescens, 2/4). Overall, the Phoenix and ATB systems gave equal results in 130/136 cases (95.6%). For two isolates, consistent identification was obtained at the genus level, thus bringing the cumulative agreement to 97.1%. MIC values (interpreted according to NCCLS guidelines) gave essential and categorical agreement in 94.2% and 93.1% of cases, respectively. Minor and major errors were 5.1% and 5.2%, respectively. No very major errors were produced. The mean time to results (TTR) for the Phoenix system was 14.8 +/- 1.6 h (mean +/- SD), with the shortest TTR being observedfor A. baumannii (13.0 +/- 1.8 h) and the longest one for P. aeruginosa (15.6 +/- 1.2 h). In conclusion, the Phoenix system performed rapidly and correctly in the identification of clinical isolates of important opportunistic pathogens and in measuring their susceptibility to antipseudomonal drugs.     

耐亚胺培南铜绿假单胞菌金属酶的筛选及基因型研究

目的研究重庆医科大学附属第一医院分离的29株耐亚胺培南铜绿假单胞菌中金属酶（Metallo-β-Lactamase,MBL）的基因型分布情况。方法用亚胺培南-EDTA纸片法筛选29株铜绿假单胞菌中产MBL的铜绿假单胞菌,用PCR扩增法检测29株菌中金属酶VIM和IMP基因。结果29株耐亚胺培南的铜绿假单胞菌中,亚胺培南-EDTA纸片法筛选出MBL阳性菌株5株,阳性率为17％。PCR扩增出IMP基因型有4株,阳性率为14％,均为金属酶IMP-9型,未扩增出VIM基因。以PCR法结果为判定标准,亚胺培南-EDTA纸片法敏感性100％,特异性96％。结论IMP-9型为该院分离的这29株耐亚胺培南的铜绿假单胞菌中主要MBL基因型。本实验中所用的亚胺培南一EDTA纸片法能简单有效的筛选出产MBL的铜绿假单胞菌。     

Multiniche screening reveals the clinically relevant metallo-beta-lactamase VIM-2 in Pseudomonas aeruginosa far from the hospital setting: an ongoing dispersion process?

A screening study of the presence of metallo-beta-lactamases (IMP and VIM types and SPM-1) in isolates from different nonhospital sources was conducted, and it revealed the presence of bla(VIM-2), associated with the In58 class 1 integron, in two unrelated Pseudomonas aeruginosa strains from aquatic habitats. The results suggest that the hospital setting was the possible origin of these bla(VIM-2)-carrying strains.     

Identification and genetic characterization of metallo-beta-lactamase-producing strains of Pseudomonas aeruginosa in Tehran, Iran

Metallo-beta-lactamases (MBLs) are being reported with increasing frequency worldwide. The aim of this study was to investigate the prevalence of blalMP-1, blaVIM-1,2 and blaSPM-1 genes encoding metallo-beta-lactamases (MBLs) among a collection of Pseudomonas aeruginosa strains isolated from patients at different hospitals in Tehran and to trace the disseminated clones at these hospitals by pulsed field gel electrophoresis (PFGE). Susceptibility of 610 P aeruginosa to 14 different antibiotics was determined using disc diffusion method. Isolates showing resistance to imipenem and ceftazidime were subjected to micro broth dilution assay to determine their MIC values. The blaIMP-1, blaVIM-1, blaVIM-2, and blaSPM-1, genes were amplified by PCR. Isolates containing blaVIM-1 were analyzed by PFGE. Sixty-eight isolates were resistant to imipenem (MIC > or = 4 microg/ml) of which 16 isolates carried blaVIM-1 gene using PCR assay. No other MBL genes were detected in this study. Three different unrelated patterns were found for isolates containing blaVIM-1 gene by PFGE of which pattern A was predominant. All isolates were susceptible to colistin and polymixin B. blaVIM-1 was the main gene encoding MBL among the isolates of P aeruginosa in our study. Clonal spread of isolates containing blaVIM-1 had occurred at Tehran hospitals. However, heterogeneous clones also were involved in the outbreaks.     

Molecular epidemiology of PER-1 extended spectrum β-lactamase among gram-negative bacteria isolated at a tertiary care hospital

The bla(PER-1) presence was sought by PCR in 289 ceftazidime resistant Gram-negative bacteria isolated at Dokuz Eylul University Hospital (Turkey) between 1998 and 2003. PER-1 production rates were 32.3, 33.9, 14.9 and 37.9% in the 1998-2000 period, 2001, 2002 and 2003, respectively. bla(PER-1) was detected in 46.2 and 35.9% of ceftazidime-resistant Pseudomonas aeruginosa and Acinetobacter baumannii isolates, respectively. ERIC-PCR results revealed that dissemination of two endemic clones for both P. aeruginosa (X and Y) and A. baumannii (A and B) was responsible for the high prevalence. Results of the conjugation tests and plasmid curing experiments suggested that bla(PER-1) was located on the chromosome in the representative strains. It was also shown for the first time that bla(PER-1) in a clinical isolate was associated with class-1 integron which could facilitate dissemination of bla(PER-1) among bacteria.     

Emergence and spread of carbapenem-resistant Acinetobacter baumannii in a tertiary care hospital in Turkey

An intensive care unit (ICU)-based OXA-23-producing multiple-drug resistant Acinetobacter baumannii (MDRAB) outbreak was detected between October 2005 and October 2006. A total of 47 patients were infected/colonized with the outbreak strain. Clinical data were available from 37 patients. The all-cause mortality rate among the patients exposed to the epidemic strain was 35% (13/37). The outbreak strain and the resistance determinants were characterized both by microbiological methods and by molecular techniques. Cloning and sequencing experiments identified ISAbaI-associated bla(oxa-23) on the chromosome. Screening of imipenem-resistant Acinetobacter isolated from the ICU during the outbreak period with PCR identified 97 isolates as positive for the ISAbaI-bla(oxa-23) structure. Pulsed-field gel electrophoresis and plasmid analyses with selected nonrepetitive isolates revealed the clonality. Disk diffusion on cloxacillin-supplemented agar media and the real-time PCR experiments showed that outbreak isolates are overexpressing the ampC enzyme. This study highlights the occurrence of OXA-23-producing and ampC-overexpressing MDRAB in ICUs.     

Outbreaks of imipenem-resistant Acinetobacter baumannii producing carbapenemases in Korea

Among 53 Acinetobacter baumannii isolates collected in 2004, nine imipenem-resistant isolates were obtained from clinical specimens taken from patients hospitalized in Busan, Korea. Nine carbapenemase-producing isolates were further investigated in order to determine the mechanisms underlying resistance. These isolates were then analyzed via antibiotic susceptibility testing, microbiological tests of carbapenemase activity, pI determination, transconjugation test, enterobacterial repetitive consensus (ERIC)-PCR, and DNA sequencing. One outbreak involved seven cases of infection by A. baumannii producing OXA-23 beta-lactamase, and was found to have been caused by a single ERIC-PCR clone. During the study period, the other outbreak involved two cases of infection by A. baumannii producing IMP-1 beta-lactamase. The two clones, one from each of the outbreaks, were characterized via a modified cloverleaf synergy test and an EDTA-disk synergy test. The isoelectric focusing of the crude bacterial extracts detected nitrocefin-positive bands with pI values of 6.65 (OXA-23) and 9.0 (IMP-1). The PCR amplification and characterization of the amplicons via direct sequencing showed that the clonal isolates harbored blaIMP-1 or blaOXA-23 determinants. The two clones were characterized by a multidrug resistance phenotype that remained unaltered throughout the outbreak. This resistance encompassed penicillins, extended-spectrum cephalosporins, carbapenems, monobactams, and aminoglycosides. These results appear to show that the imipenem resistance observed among nine Korean A. baumannii isolates could be attributed to the spread of an IMP-1- or OXA-23-producing clone. Our microbiological test of carbapenemase activity is a simple method for the screening of clinical isolates producing class D carbapenemase and/or class B metallo-beta-lactamase, in order both to determine their clinical impact and to prevent further spread.     

Modification and evaluation of the Carba NP test by use of paper strip for simple and rapid detection of carbapenemase-producing <Emphasis Type="Italic">Enterobacteriaceae</Emphasis>

Carbapenemase-producing Enterobacteriaceae (CPE) isolates have now emerged worldwide. We therefore modified the phenotypic Carba NP test by use of a filter paper strip for easily and rapidly identifying CPE in routine laboratory. A collection of 56 CPE and carbapenemase-producing Pseudomonas spp. isolates (including 28 NDM-1, 11 IMP-14a, 1 IMP-1, 1 IMP-4, 1 IMP-9, 1 IMP-15, 4 VIM-2, 1 VIM-1, 1 IMP-14a with VIM-2, 3 OXA-48, 3 OXA-181 and 1 KPC-2 producers) and 41 non-CPE isolates (including 19 ESBL, 7 pAmpC, 3 AmpC, 9 ESBL with pAmpC and 3 non-ESBL & non-AmpC producers) as confirmed by the PCR methods were tested by the paper strip method using pharmaceutical imipenem/cilastatin as a substrate. Bacterial colonies of each isolate were applied directly on filter paper strips dropped with either imipenem-phenol red (test strip) or phenol red solution alone (control strip). The reaction was read within 5 min. This test failed to detect 3 OXA-181, 2 OXA-48 and 3 IMP-14a producers (85.7 % sensitivity), whereas no false positives were seen (100 % specificity). Further evaluation of the paper strip test in 267 CPE screening-positive isolates from three hospitals by their medical technologists showed 92.0 % sensitivity (100 % for NDM producers) and 100 % specificity compared with the PCR methods. Because of its ease, rapidness and cost effective, the paper strip test has a potential for routine CPE testing in low-resource laboratories particularly in areas with high prevalence of NDM enzymes, leading to appropriate antimicrobial therapy and infection control strategy.     

First report of the bla(OXA-58) gene in a clinical isolate of Acinetobacter baumannii in Rio de Janeiro, Brazil

Carbapenemase production is an important mechanism of carbapenem resistance among nonfermentative Gram-negative isolates. This study aimed to report the detection of bla(OXA-58) gene in multiresistant clinical isolates of Acinetobacter baumannii recovered from inpatients in a public hospital. Polymerase chain reaction tests were performed to detect the bla(OXA-23-like), bla(OXA-24-like), bla(OXA-58-like) and bla(OXA-51-like) genes. The bla(OXA-58) and bla(OXA-23) genes were detected in one and three isolates, respectively. Sequencing of the bla(OXA-58-like) amplicon revealed 100% identity with the A. baumannii bla(OXA-58) gene listed in the GenBank database. This is the first report of an OXA-58-producing A. baumannii isolate in Rio de Janeiro, Brazil.     

PER-1 is still widespread in Turkish hospitals among Pseudomonas aeruginosa and Acinetobacter spp

PER-1 type beta-lactamases were screened among ceftazidime-resistant clinical isolates of Acinetobacter spp. and Pseudomonas aeruginosa. A total of 176 non-repetitive isolates (84 Acinetobacter spp. and 92 P. aeruginosa) were collected during a three month surveillance period. Isolates were obtained from seven intensive care units of seven university hospitals. All strains were screened for bla(PER-1) alleles by PCR. Of the strains, 31% and 55.4% of Acinetobacter spp. and P. aeruginosa were positive for bla(PER-1) type genes, respectively.     

Distribution of blaOXA genes among carbapenem-resistant Acinetobacter baumannii nosocomial strains in Poland

Acinetobacter baumannii is an important nosocomial pathogen occurring particularly in intensive care (ICU) as well as burn therapy units (BTU). A. baumannii strains have emerged as resistant to almost all antimicrobial agents, including carbapenems. b-lactamase-mediated resistance is the most common mechanism for carbapenem resistance in this species. Carbapenem-hydrolysing class D b-lactamases - OXA are widespread among A. baumannii strains. It is suggested that ISAba1 plays an important role in drug resistance. The aims of the study were detection of OXA encoding genes and presence of ISAba1. The study included the total of 104 isolates of carbapenem-resistant A. baumannii, obtained from patients hospitalized in ICU and BTU of Specialized Hospital in Krakow. Multiplex PCR was applied for detection of selected OXA carbapenemases encoding genes. PCR analysis showed the presence of bla OXA-51-like gene and ISAba1 in all isolates. 46 strains carried bla OXA-51-like and bla OXA-23-like genes while 48 bla OXA-51-like and bla OXA-40-like genes. 3 isolates carried: bla OXA-51-like , bla OXA-23-like and bla OXA-40-like genes. 7 strains encoded an OXA-51-like carbapenemase but were negative for enzymes belonging to the other families tested. Comparative analysis of ICU and BTU isolates revealed the dominance of: bla OXA-51-like and bla OXA-40-like among ICU while bla OXA-51-like and bla OXA-23-like in BTU.     

Inhibitors of the FEZ-1 metallo-beta-lactamase

Metallo-beta-lactamases (MBLs) catalyze the hydrolysis of beta-lactams including penicillins, cephalosporins and carbapenems. Starting from benzohydroxamic acid (1) structure-activity studies led to the identification of selective inhibitors of the FEZ-1 MBL, e.g., 2,5-substituted benzophenone hydroxamic acid 17 has a K(i) of 6.1+/-0.7microM against the FEZ-1 MBL but does not significantly inhibit the IMP-1, BcII, CphA or L1 MBLs.     

The role of bla(OXA-like carbapenemase) and their insertion sequences (ISS) in the induction of resistance against carbapenem antibiotics among Acinetobacter baumannii isolates in Tehran hospitals

This study aimed to evaluate the occurrence and dissemination of bla(OXA-like) carbapenemase genes and their insertion sequences among Acinetobacter baumannii isolates, taken from different hospitals in Tehran city and also their roles in the induction of resistance to carbapenem drugs. A total number of 100 non duplicate Acinetobacter baumannii with different origins, were isolated from patients with proved nosocomial infections at eight university hospital in Tehran city. Antimicrobial susceptibility of these strains was done by E-test against 7 antimicrobial agents according to CLSI guideline. PCR of bla(OXA-51-like), bla(OXA-23-like), bla(OXA-24-like), bla(OXA-58-like), IS(ABA-1), IS(1133) was carried out by specialized primers and then these strains were typed by REP-fingerprinting. Colistin, imipenem and meropenem were the most sensitive antibiotics against Acinetobacter baumannii isolates with 96%, 51% and 51% sensitivity respectively. All the isolates had a bla(OXA-51-like) intrinsic to these species. The rates of bla(OXA-23), 23 and 58-like were 38%, 32% and 1% respectively. Coexistence of bla(OXA-51/23/24-like) was observed among 16% of these isolates. All bla(OXA-23-like) carbapenemase genes had only one IS(ABA1). REP fingerprinting showed 5 genotypes among carbapenem resistant isolates, 16 of them being genotype A. This study emphasized on the major role of bla(OXA-like) carbapenemase, particularly bla(OXA-23-like) carbapenemase and their IS(ABA1), in the dissemination of carbapenem resistant Acinetobacter baumannii. This study confirmed a presumptive role of IS element neighboring the carbapenemase gene in the elevation of resistance to carbapenem drug among Acinetobacter baumannii isolates for the first time in Iran.     

Mechanism of imipenem resistance in metallo-β-lactamases expressing pathogenic bacterial spp. and identification of potential inhibitors: An in silico approach

The World Health Organization reports that millions of people around the world are infected with antibiotic-resistant bacteria. Such resistance is more common in Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae strains because of the expression of the metallo-β-lactamases (MBLs) namely Imipenemase (IMP)-1, IMP-2, New Delhi metallo-β-lactamases-, Verona imipenemase (VIM)-4, VIM-5, and VIM-7. We did an in silico analysis to understand the resistance mechanism of imipenem at the structural level. Our modeling studies reveal that the VIM-4-imipenem complex has highest binding energy and forms a stable complex as indicated by a consensus score (C-score) value of 5.44. The intense interaction between the substrate and the β-lactamases leads to the increased hydrolysis of the substrate resulting in rapid hydrolysis of the antibiotic imipenem by VIM-4. Virtual screening of compounds from the ZINC database targeting VIM-4 was done, and we found compound ZINC44608383 as the high binding energy compound with the C-score value of 5.58. This compound could be exploited for inhibitor design and development. The current study helps us to understand the resistance mechanism of imipenem in MBL-expressing strains. Also, we have identified a probable inhibitor for VIM-4. We believe that our results will be useful for researchers in designing potent inhibitors for VIM-4.     

多重耐药铜绿假单胞菌的耐药性及耐药基因检测分析

目的探讨呼吸内科病房分离的多重耐药铜绿假单胞菌相关耐药基因。方法采用纸片扩散法进行体外药敏试验,聚合酶链反应（PCR）对其中32株多重耐药铜绿假单胞菌进行相关基因TEM、SHV、CTX-M-9、DHA、VIM、PER、OXA、IMP和OprD2检测,并对耐药基因进行测序。结果 32株多重耐药铜绿假单胞菌对多黏菌素B耐药率是3.1%,对其余抗菌药物耐药率为53%～100%,β-内酰胺酶基因OXA-10、TEM-1、DHA-1、PER-1和IMP-1基因阳性率分别为46.9%、21.9%、15.6%、12.5%和31.3%,未检出SHV基因、CTX-M-9基因和VIM基因;另外OprD2基因缺失率达68.8%。结论呼吸内科病房多重耐药的铜绿假单胞菌携带多种β-内酰胺酶基因,应引起高度重视。