Sequence of the Citrobacter freundii OS60 chromosomal ampC beta-lactamase gene

The Citrobacter freundii OS60 ampC beta-lactamase gene was sequenced and found to encode a 380-amino-acid-long precursor with a 19-residue signal peptide. The mature protein has a predicted molecular mass of 39781 Da. The first 60 residues of the purified enzyme, as determined by sequential Edman degradation, are identical to the amino acid sequence inferred from the gene sequence. Also, the amino acid composition determined for the purified beta-lactamase and that given by the gene sequence are in good agreement. 77% of the amino acid positions hold identical residues in the C. freundii and Escherichia coli K12 chromosomal AmpC beta-lactamases. This clearly puts the C. freundii enzyme into the class C of beta-lactamases. Of the 68 amino-terminal residues determined for the Enterobacter cloacae P99 beta-lactamase, 44 are identical to the corresponding residues of the C. freundii enzyme. All three enzymes, as well as that of Pseudomonas aeruginosa 18S/H are highly similar around the active-site serine at position 64 of the mature protein.     

Inactivation of the ampD gene causes semiconstitutive overproduction of the inducible Citrobacter freundii beta-lactamase.

In Citrobacter freundii and Enterobacter cloacae, synthesis of AmpC beta-lactamase is inducible by the addition of beta-lactams to the growth medium. Spontaneous mutants that constitutively overproduce the enzyme occur at a high frequency. When the C. freundii ampC beta-lactamase gene is cloned into Escherichia coli together with the regulatory gene ampR, beta-lactamase expression from the clone is inducible. Spontaneous cefotaxime-resistant mutants were selected from an E. coli strain carrying the cloned C. freundii ampC and ampR genes on a plasmid. Virtually all isolates had chromosomal mutations leading to semiconstitutive overproduction of beta-lactamase. The mutation ampD2 in one such mutant was caused by an IS1 insertion into the hitherto unknown ampD gene, located between nadC and aroP at minute 2.4 on the E. coli chromosome. The wild-type ampD allele cloned on a plasmid could fully trans-complement beta-lactamase-overproducing mutants of both E. coli and C. freundii, restoring the wild-type phenotype of highly inducible enzyme synthesis. This indicates that these E. coli and C. freundii mutants have their lesions in ampD. We hypothesize that induction of beta-lactamase synthesis is caused by blocking of the AmpD function by the beta-lactam inducer and that this leads directly or indirectly to an AmpR-mediated stimulation of ampC expression.     

Identification of the active site of Citrobacter freundii beta-lactamase using dansyl-penicillin

The active site sequence of a beta-lactamase encoded by chromosomal gene(s) in Citrobacter freundii GN346 was determined using dansyl-penicillin as a fluorescent probe. The tryptic digest of the labelled enzyme gave a fluorescent peptide containing 22 amino acids. The sequence of this peptide was identical to the consensus sequence of class C beta-lactamases, Gly-Ser-X-Ser-Lys. The residue labelled was the serine adjacent to the glycine. The active site sequence corresponded to positions 46-67 of the entire sequence of the Citrobacter freundii beta-lactamase determined on the basis of the DNA sequence of the structural gene [(1986) Eur. J. Biochem. 156, 441-445]. The labelled serine corresponded to Ser-64.     

The<Emphasis Type="Italic">Escherichia fergusonii iucABCD iutA</Emphasis> genes are located within a larger chromosomal region similar to pathogenicity islands

Three strains of Escherichia fergusonii (EF873, EF1496, EF939) of 50 strains tested produced the hydroxamate siderophore aerobactin. Screening of a cosmid library of the strain EF873 chromosomal DNA (in aerobactin nonproducing Escherichia coli VCS257) for aerobactin production identified iucABCD and iutA gene orthologues. The predicted IucABCD and IutA proteins showed 59-65% identity to the corresponding proteins of Shigella flexneri and E. coli. Aerobactin molecules synthesized by E. fergusonii and E. coli strains stimulated growth of aerobactin indicator strains harboring either E. coli or E. fergusonii iutA genes. In the 12 kb upstream and 17 kb downstream regions of the iuc and iut genes, 20 additional ORFs were identified. Their gene products showed homology to proteins from E. coli, S. flexneri, Klebsiella aerogenes, Pseudomonas aeruginosa and Vibrio cholerae. Probes recognizing DNA sequences from a region of more than 25 kb, which included the iucABCD and iutA genes, hybridized with chromosomal DNA of two aerobactin-producing strains (EF873 and EF939), but not with other nonproducing E. fergusonii strains tested. These data, together with the genetic organization of this region, suggest that E. fergusonii iucABCD iutA genes are a portion of a larger segment of DNA similar to pathogenicity islands of other bacteria.     

Characterization of an extended-spectrum class C beta-lactamase of Citrobacter freundii

Citrobacter freundii GC3 is a clinical isolate which showed moderate resistance to oxyimino beta-lactams such as ceftazidime and aztreonam. This drug resistance was due to an extended-spectrum class C beta-lactamase encoded by chromosomal gene(s). The GC3 beta-lactamase showed high amino acid sequence homology to a known C. freundii beta-lactamase, i.e., 346 of 361 amino acids were identical with those of C. freundii GN346 beta-lactamase (Tsukamoto, K. et al, Eur. J. Biochem. 188, 15-22, 1990). Asp198 was the only dissimilar amino acid found in the omega loop region, known as the hot spot for extended-spectrum resistance in class C beta-lactamases (Haruta, S. et al, Microbiol. Immunol. 42, 165-169, 1998). However, Asp198 was eliminated as a cause of the extended-spectrum resistance by the substitution of Asn for Asp198. Subsequent investigation suggested that the moderate resistance to oxyimino beta-lactams is attributable to the replacement of amino acids on the enzyme's surface area, far from the active-site. Some or all of the replacements are assumed to delicately modify the active-site configuration. The GC3 beta-lactamase is the first example of an extended-spectrum class C beta-lactamase in which mutations are independent of the omega loop.     

CMY-39型AmpC酶新基因亚型的序列分析与原核表达

目的对弗劳地枸橼酸杆菌所产CMY-39型AmpC酶新基因亚型进行基因克隆和重组表达。方法以产CMY-39型AmpC酶新基因亚型的弗劳地枸橼酸杆菌总基因组DNA为模板,PCR扩增CMY-39,将其克隆入pGEM-T载体后测定该核苷酸序列,再将CMY-39基因克隆到pET-32 a（＋）系统进行重组,重组菌在大肠埃希菌BL21中表达,SDS-PAGE电泳鉴定酶蛋白的表达。结果 PCR扩增出大小为1 146 bp的基因片段,与GenBank上CMY-39的基因序列同源性为99%。大肠埃希菌BL21转化pET-32 a（＋）/CMY-39重组质粒后,AmpC酶三维试验为阳性。此基因能在大肠埃希菌中大量表达,SDS-PAGE电泳显示,蛋白分子质量大约为60 kD。结论此基因为CMY-39新基因亚型,登陆号为HM565135;成功表达重组的CMY-39型酶,为进一步做酶动力学及酶的其他分子生物学特性研究奠定了基础。     

Characterization of the gene encoding the beta-lactamase of the psychrophilic marine bacterium Moritella marina strain MP-1

The beta-lactamase gene (mbla) of the psychrophilic marine bacterium Moritella marina strain MP-1 was identified in a previously isolated genomic DNA fragment and it was expressed in Escherichia coli cells. The mbla gene encoded a protein consisting of 287 amino acid residues. Its predicted amino acid sequence showed approximately 50% identity with that of a number of class A beta-lactamases, especially with that of CARB/PSE type of beta-lactamases (carbenicillinases). E. coli transformed with the plasmid containing mbla grew on an ampicillin-containing plate at 37 degrees C but not at 42 degrees C, suggesting that the beta-lactamase of this bacterium is heat-labile.     

Characterization of a new beta-lactamase gene from isolates of Vibrio spp. in Korea

PCR was performed to analyze the beta-lactamase genes carried by ampicillin-resistant Vibrio spp. strains isolated from marine environments in Korea between 2006 and 2009. All 36 strains tested showed negative results in PCR with the primers designed from the nucleotide sequences of various known beta-lactamase genes. This prompted us to screen new beta-lactamase genes. A novel beta-lactamase gene was cloned from Vibrio alginolyticus KV3 isolated from the aquaculture water of Geoje Island of Korea. The determined nucleotide sequence (VAK-3 beta-lactamase) revealed an open reading frame (ORF) of 852 bp, encoding a protein of 283 amino acids (aa), which displayed low homology to any other beta-lactamase genes reported in public databases. The deduced 283 aa sequence of VAK-3, consisting of a 19 aa signal peptide and a 264 aa mature protein, contained highly conserved peptide segments specific to class A beta-lactamases including the specific amino acid residues STFK (62-65), SDN (122-124), E (158), and RTG (226-228). Results from PCR performed with primers specific to the VAK-3 beta-lactamase gene identified 3 of the 36 isolated strains as V. alginolyticus, Vibrio cholerae, and Photobacterium damselae subsp. damselae, indicating the utilization of various beta-lactamase genes including unidentified ones in ampicillin-resistant Vibrio spp. strains from the marine environment. In a mating experiment, none of the isolates transfered the VAK-3 beta-lactamase gene to the Escherichia coli recipient. This lack of mobility, and the presence of a chromosomal acyl-CoA flanking sequence upstream of the VAK-3 beta- lactamase gene, led to the assumption that the location of this new beta-lactamase gene was in the chromosome, rather than the mobile plasmid. Antibiotic susceptibility of VAK-3 beta-lactamase was indicated by elevated levels of resistance to penicillins, but not to cephalosporins in the wild type and E. coli harboring recombinant plasmid pKV-3, compared with those of the host strain alone. Phylogenetic analysis showed that VAK-3 beta-lactamase is a new and separate member of class A beta-lactamases.     

Differentiation of Citrobacter strains using chromosomal DNA restriction patterns

The aim of this study was checking of the usefulness of chromosomal DNA restriction patterns in differentiation of Citrobacter strains. Molecular characterization of total 56 isolates of Citrobacter from Poland and Czech Republic, was performed by pulsed-field gel electrophoresis after digestion of chromosomal DNA with restriction endonuclease Xba I (5'-TCTAGA-3'). Chromosomal DNA of all tested Citrobacter strains gave after electrophoresis 12 to 21 bands and patterns consisting of 12 to 21 fragments ranging in size from 790 kb to 48.5 kb and smaller, which where not distinguishable. Pulsed-field gel electrophoresis patterns were useful for comparing Citrobacter strains. Identical restriction patterns generated by PFGE were observed in the case of selected strains e.g. strains C. sedlakii studied in this study, coming from an outbreak, having the some phenotype. In addition, PFGE patterns can be used to evaluate the clonal relatedness among bacterial isolates. PFGE can be helpful for assessing genetic relatedness among strains epidemiologicaly unrelated e.g. C. werkmanii strains tested in this study. The sum of DNA fragments after Xba I digestion indicates the genome size of Citrobacter strains. This suggests that PFGE should be useful for epidemiological investigations of Citrobacter strains.     

Detection and characterization of beta-lactamase genes in subgingival bacteria from patients with refractory periodontitis

Fifty-three beta-lactamase-producing strains of oral bacteria isolated from patients with refractory periodontitis in Norway and USA were screened for the presence of the bla(TEM), bla(SHV), bla(OXA), bla(ampC), bla(cfxA), and bla(cepA/cblA) genes by the polymerase chain reaction (PCR). The PCR products were characterized by direct sequencing of the amplified DNA. Thirty-four of the 53 enzyme-producing strains (64%) were positive in one of the PCR assays. All beta-lactamase-producing Prevotella and Capnocytophaga spp. were CfxA positive. TEM-type beta-lactamases were identified in one strain each of Escherichia coli and Neisseria sp., and one strain of Citrobacter freundii possessed an AmpC-type beta-lactamase. Screening for gene cassettes and genes known to be associated with integrons did not reveal the presence of integrons in these oral bacteria. Sequence analyses showed that most CfxA positive Prevotella and Capnocytophaga isolates from patients with refractory periodontitis harboured variants of the CfxA2 and CfxA3 enzyme. The present study also showed that many different genetic determinants of beta-lactamase production are found in bacteria isolated from refractory periodontitis, many of which remain to be characterized.     

Characteristics of selected virulence factors of Enterobacter cloacae strains isolated from clinical specimens

We demonstrated that Enterobacter cloacae possesses a selective haemolytic activity on sheep erythrocytes. All the screened strains showed a haemolytic activity on sheep erythrocytes when cultures were preincubated with beta-mercaptoethanol. The investigation circulation of the genes encoding extended spectrum beta-lactamases (ESBL) shows that beta-lactamase producers can be ascribed to specific patterns of plasmids. We also demonstrated that genetic material from E. coli can be transferred and established in selected Enterobacter cloacae strains. In a survival tests we demonstrated that similarly to Salmonella or Vibrio clinical isolates Enterobacter cloacae doesn't demonstrate acid tolerance.     

Isolation and characterization of a beta-lactamase-inhibitory protein from Streptomyces clavuligerus and cloning and analysis of the corresponding gene.

Culture filtrates of Streptomyces clavuligerus contain a proteinaceous beta-lactamase inhibitor (BLIP) in addition to a variety of beta-lactam compounds. BLIP was first detected by its ability to inhibit Bactopenase, a penicillinase derived from Bacillus cereus, but it has also been shown to inhibit the plasmid pUC- and chromosomally mediated beta-lactamases of Escherichia coli. BLIP showed no inhibitory effect against Enterobacter cloacae beta-lactamase, and it also showed no activity against an alternative source of B. cereus penicillinase. BLIP was purified to homogeneity, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave a size estimate for BLIP of 16,900 to 18,000. The interaction between purified BLIP and the E. coli(pUC) beta-lactamase was investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and determined to be noncovalent, with an estimated 1:1 molar stoichiometry. The BLIP gene was isolated on a 13.5-kilobase fragment of S. clavuligerus chromosomal DNA which did not overlap a 40-kilobase region of DNA known to contain genes for beta-lactam antibiotic biosynthesis. The gene encoded a mature protein with a deduced amino acid sequence of 165 residues (calculated molecular weight of 17,523) and also encoded a 36-amino-acid signal sequence. No significant sequence similarity to BLIP was found by pairwise comparisons using various protein and nucleotide sequence data banks or by hybridization experiments, and no BLIP activity was detected in the culture supernatants of other Streptomyces spp.     

Population genetics of the nomenspecies Enterobacter cloacae

The genetic heterogeneity of the nomenspecies Enterobacter cloacae is well known. Enterobacter asburiae, Enterobacter cancerogenus, Enterobacter dissolvens, Enterobacter hormaechei, Enterobacter kobei, and Enterobacter nimipressuralis are closely related to it and are subsumed in the so-called E. cloacae complex. DNA-DNA hybridization studies performed previously identified at least five DNA-relatedness groups of this complex. In order to analyze the genetic structure and the phylogenetic relationships between the clusters of the nomenspecies E. cloacae, 206 strains collected from 22 hospitals, a veterinarian, and an agricultural center in 11 countries plus all 13 type strains of the genus and reference strain CDC 1347-71(R) were examined with a combination of sequence and PCR-restriction fragment length polymorphism (PCR-RFLP) analyses of the three housekeeping genes hsp60, rpoB, and hemB as well as ampC, the gene of a class C beta-lactamase. Based on the neighbor-joining tree of the hsp60 sequences, 12 genetic clusters (I to XII) and an unstable sequence crowd (xiii) were identified. The robustness of the genetic clusters was confirmed by analyses of rpoB and hemB sequences and ampC PCR-RFLPs. Sequence crowd xiii split into two groups after rpoB analysis. Only three strains formed a cluster with the type strain of E. cloacae, indicating that the minority of isolates identified as E. cloacae truly belong to the species; 13% of strains grouped with other type strains of the genus, suggesting that the phenotypes of these species seem to be more heterogeneous than so far believed. Three clusters represented 70% of strains, but none of them included a type or reference strain. The genetic clustering presented in this study might serve as a framework for future studies dealing with taxonomic, evolutionary, epidemiological, or pathogenetic characteristics of bacteria belonging to the E. cloacae complex.     

Exploring the effectiveness of tazobactam against ceftazidime resistant Escherichia coli: insights from the comparison between susceptibility testing and beta-lactamase inhibition

Thirteen clinical isolates of Escherichia coli resistant to ceftazidime that possessed an AmpC and other (beta-lactamases were identified. The effectiveness of different formulations of piperacillin/tazobactam to other beta-lactams was compared. Antibiotic susceptibility testing, polymerase chain reaction, amplification of blaTEM, blaSHV and blaAmpC, and enzyme-linked immunosorbent assays to identify AmpC beta-lactamases were performed. Hydrolysis rates were obtained and residual enzymatic activity was determined. Cefepime and ertapenem were more active than piperacillin/tazobactam. In contrast, increasing the relative proportion of tazobactam improved susceptibility testing. Twenty micromolar tazobactam inhibited total beta-lactamase activity (as measured by nitrocefin hydrolysis rates) by greater than 75% against all isolates tested: in 11 of 13 E. coli isolates, total beta-lactamase activity was inhibited by 90%. The observed differences between MIC determinations and susceptibility to enzymatic inactivation by tazobactam against E. coli containing AmpC and other -lactamases may be due to the final tazobactam concentration achieved in the periplasmic space. Factors determining this are critical considerations in assessing beta-lactamase inhibitor potency.     

Prediction of two- and three-amino-acid sequences of Citrobacter Freundii beta-lactamase from its amino acid composition

The repeated amino-acid sequences in Citrobacter Freundii beta-lactamase may be indispensable for its function, because such repetitions cannot be simply attributed to a chance. In order to fully explore the functional units in Citrobacter Freundii beta-lactamase, it may need to analyse all the amino acid pairs, triplets, etc. along Citrobacter Freundii beta-lactamase from one terminal to the other terminal, to count their frequencies and calculate their probabilities. The amino-acid sequence of Citrobacter Freundii beta-lactamase was counted according to two-, three- and four-amino-acid sequences. The counted frequency and probability were compared with the predicted frequency and probability. The amino acid sequences, which appear in Citrobacter Freundii beta-lactamase and can be predicted from its amino acid composition according to a purely random mechanism, should not be deliberately evolved and conserved. By contrast, the amino acid sequences, which appear in Citrobacter Freundii beta-lactamase but cannot be predicted from its amino acid composition according to a purely random mechanism, should be deliberately evolved and conversed. Accordingly 99 (26.053%) and 33 (8.684%) of 380 two-amino-acid sequences can be predicted by the frequency and probability according to a purely random mechanism. Some kinds of amino acid sequences, which absent in Citrobacter Freundii beta-lactamase and can be predicted from its amino acid composition according to a purely random mechanism, should not be deliberately excluded from Citrobacter Freundii beta-lactamase. By contrast, some kinds of amino acid sequences, which absent in Citrobacter Freundii beta-lactamase and cannot be predicted from its amino acid composition according to a purely random mechanism, should be deliberately excluded from Citrobacter Freundii beta-lactamase. Accordingly 89 (48.370%) and 41 (22.283%) of 184 kinds of absent two-amino-acid sequences can be predicted by the frequency and probability according to a purely random mechanism, and 7236 (99.848%) of 7247 kinds of absent three-amino-acid sequences can be predicted by the frequency according to a purely random mechanism. The amino acids, whose probabilities in following certain preceding amino acids can be predicted from Citrobacter Freundii beta-lactamase amino acid composition according to a purely random mechanism, should not be deliberately evolved and conversed, accordingly 2 (0.526%) of 380 counted first order Markov transition probabilities for the second amino acid in two-amino-acid sequences match the predicted conditional probabilities.     

Yersiniabactin and other siderophores produced by clinical isolates of Enterobacter spp. and Citrobacter spp

We analyzed the ability of extraintestinal strains of Enterobacter spp. and Citrobacter spp. to employ different siderophore-mediated strategies of iron acquisition. All strains produced iron-chelating compounds. Cross-feeding assays indicated that most isolates of both Enterobacter spp. and Citrobacter spp. excreted catecholate siderophore enterobactin, less produced aerobactin, and single strains excreted hydroxamates different from aerobactin. Besides, we analyzed if the strains had the ability to produce the siderophore yersiniabactin coded by the Yersinia high-pathogenicity island (HPI). The presence of HPI genes was observed in single isolates of three species: E. cloaceae, E. aerogenes and C. koseri. A detailed polymerase chain reaction analysis revealed differences in the genetic organization of the HPIs; however, in a cross-feeding test we proved that yersiniabactin was produced and the island was functional.     

弗劳地枸橼酸盐杆菌基因种与小儿腹泻关系、耐药性的研究

从１４５７例腹泻患儿大便中分离出１６４株弗劳地枸橼酸盐杆菌复合群的菌株,而对照组中分离率仅４．２６％,具统计学差异。为探讨其致病性,采用不耐热性肠毒素,耐热性肠毒素基因探针,菌液直接ＬＴ－ＰＣＲ,家兔肠袢结扎和刚果红结合试验检测细胞的腹泻毒力因子,证实４７．１％的菌株产生ＬＴ,系腹泻重要毒力因子,不具侵袭性因子。