Close amino acid sequence relationship between the new plasmid-mediated extended-spectrum beta-lactamase MEN-1 and chromosomally encoded enzymes of Klebsiella oxytoca.

Isolated from an Escherichia coli strain MEN-1 is a plasmid-mediated beta-lactamase that confers resistance to methoxy imino third-generation cephalosporins. The protein purified to homogeneity was digested by trypsin, chymotrypsin and endoproteinase Asp-N. Amino acid sequence determinations of the resulting peptides gave rise to the alignment of the 263 residues of the beta-lactamase. From amino acid sequence comparison MEN-1 was found to share more than 72% identity with the chromosomally mediated beta-lactamases of Klebsiella oxytoca. Therefore, MEN-1 is the first transferable extended-spectrum beta-lactamase which is not directly derived from the widespread TEMs or SHV-1 penicillinases with which it presents less than 39% identity.     

Identification of the 4-hydroxycinnamate decarboxylase (PAD) gene of Klebsiella oxytoca

A 7.1-kbp DNA fragment isolated from a wild strain of Klebsiella oxytoca was sequenced, leading to the identification of 10 open-reading frames (ORFs), including a 504-bp Pad gene. The Pad gene of the Gram-negative bacterium was subsequently expressed in Escherichia coli as a chimeric Pad. The deduced amino acid (AA) sequence of the Pad gene from wild-type K. oxytoca showed approximately 50% homology to those of other bacterial PADs from Gram-positive bacilli plus a coccus. These data and a genomic library search of some gamma-proteobacteria, including E. coli and Vibrio sp., indicated that PAD of K. oxytoca is a member of the bacterial PAD family characteristic of Gram-negative bacteria. Using Pad-specific PCR primers designed from the Gram-negative bacterial Pad of K. oxytoca, Pad genes of two further strains of K. oxytoca, another wild isolate and JCM 1665 and two PAD-positive Enterobacter spp. were successfully amplified for specific Pad detection.     

Single amino acid substitution between SHV-1 beta-lactamase and cefotaxime-hydrolyzing SHV-2 enzyme

SHV-2 beta-lactamase was purified from an overproducing variant of a clinical isolate of Escherichia coli resistant to cefotaxime. Pure protein was digested by trypsin and Lys-C endoproteinase. Proteolytic peptides, isolated by reverse-phase HPLC, were submitted to manual Edman degradation and aligned by homology with the sequence of SHV-1 beta-lactamase. A putative amino acid sequence was deduced. Structural comparison revealed that SHV-2 differed from SHV-1 by only one amino acid, Gly----Ser, at position 213 of the mature protein.     

Characterisation of an amylase from a psychrotrophic Vibrio isolated from a deep-sea mud sample

The gene coding for the class A beta-lactamase of Citrobacter diversus has been cloned and sequenced. It contains the information for a 294-amino-acid precursor protein, including a 27-residue N-terminal signal peptide. The deduced sequence of the N-terminal portion of the mature protein is in excellent agreement with that determined by microsequencing of the protein and readily explains the pI differences observed between the naturally occurring forms I and II of the enzyme. The sequence of the mature protein exhibits a very high degree of similarity with that of the Klebsiella oxytoca class A beta-lactamase.     

Comparison of beta-lactamase II from Bacillus cereus 569/H/9 with a beta-lactamase from Bacillus cereus 5/B/6.

A mutant of Bacillus cereus 5/B, strain 5/B/6, produces a beta-lactamase II-like enzyme but no beta-lactamase I. Beta-lactamases II and II 5/B/6 appear to show a high degree of homology, but there are significant differences in their enzymic properties.     

The beta-lactamase of Enterobacter cloacae P99. Chemical properties, N-terminal sequence and interaction with 6 beta-halogenopenicillanates.

The beta-lactamase of Enterobacter cloacae P99 consists of one polypeptide chain of Mr 39000 devoid of disulphide bridges and free thiol groups. It contains an unusually high proportion of tyrosine and tryptophan. The N-terminal sequence exhibits overlaps with the tryptic peptide obtained after labelling the active site with 6 beta-iodopenicillanate. The active-site serine residue is at position 64. The homology with the chromosomal beta-lactamase of Escherichia coli K 12 (ampC gene) is lower within the 25 residues of the N-terminal portion than around the active-site serine residue. The P99 beta-lactamase is inactivated by 6 beta-bromo- and 6 beta-iodo-penicillanate, with a second-order rate constant of 110-140M-1 X s-1 at 30 degrees C and pH 7.0, a value that is much lower than that observed with class-A beta-lactamases.     

Molecular characterization of a new plasmid-encoded SHV-type beta-lactamase (SHV-2 variant) conferring high-level cefotaxime resistance upon Klebsiella pneumoniae

Between 1986 and 1988, multiresistant Klebsiella pneumoniae strains exhibiting high-level cefotaxime resistance were isolated from patient specimens particularly of the intensive care units of the Aachen Technical University Hospital. The resistance gene responsible was shown to be encoded on a conjugative 66 kb plasmid designated pZMP1. The MIC values for cefotaxime of the original isolates and the transconjugants were greater than 128 mg l-1 and 64 mg l-1, respectively. Isoelectric focusing of protein preparations from the transconjugants showed a beta-lactamase with a pI of 7.6. A 3.6 kb BamHI fragment containing the beta-lactamase gene was cloned into pLG339 resulting in the recombinant plasmid pZMP1-1. A restriction map of the cloned insert was established and PstI subfragments of the insert were further subcloned into pBGS18. The nucleotide sequence of the complete 3.6 kb fragment was determined. Within 3663 bp an open reading frame of 858 kb was found to show 99% homology to the SHV-2 and -3 nucleotide sequences. The deduced amino acid sequence differed in one and two positions, respectively, from these established SHV enzymes. The 3' noncoding sequence exhibited nearly perfect homology to that of SHV-2, but the 5' upstream sequence showed homology of less than 50% to the corresponding SHV-2 sequence, indicating an altered promoter region of the variant SHV-enzyme. Kinetic analysis of the beta-lactamase revealed a 50-100% elevated hydrolytic effectivity on cefotaxime in comparison to other SHV enzymes. Cefoxitin, ceftazidime, aztreonam and imipenem were not hydrolysed by the enzyme. The variant enzyme was inhibited by commonly available beta-lactamase inhibitors. Clavulanic acid had the highest affinity for the enzyme and the greatest effectivity in blocking its action. Based on the genetic and kinetic data we propose to classify the enzyme as a new variant beta-lactamase of the SHV-type and name it SHV-2a.     

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.     

Evaluation of a recombinant Klebsiella oxytoca strain for ethanol production from cellulose by simultaneous saccharification and fermentation: comparison with native cellobiose-utilising yeast strains and performance in co-culture with thermotolerant yeast and Zymomonas mobilis

In the simultaneous saccharification and fermentation to ethanol of 100 g l(-1) microcrystalline cellulose, the cellobiose-fermenting recombinant Klebsiella oxytoca P2 outperformed a range of cellobiose-fermenting yeasts used in earlier work, despite producing less ethanol than reported earlier for this organism under similar conditions. The time taken by K. oxytoca P2 to produce up to about 33 g l(-1) ethanol was much less than for any other organism investigated, including ethanol-tolerant strains of Saccharomyces pastorianus, Kluyveromyces marxianus and Zymomonas mobilis. Ultimately, it produced slightly less ethanol (maximum 36 g l(-1)) than these organisms, reflecting its lower ethanol tolerance. Significant advantages were obtained by co-culturing K. oxytoca P2 with S. pastorianus, K. marxianus or Z. mobilis, either isothermally, or in conjunction with temperature-profiling to raise the cellulase activity. Co-cultures produced significantly more ethanol, more rapidly, than either of the constituent strains in pure culture at the same inoculum density. K. oxytoca P2 dominated the early stages of the co-cultures, with ethanol production in the later stages due principally to the more ethanol tolerant strain. The usefulness of K. oxytoca P2 in cellulose simultaneous saccharification and fermentation should be improved by mutation of the strain to increase its ethanol tolerance.     

Close evolutionary relationship between the chromosomally encoded beta-lactamase gene of Klebsiella pneumoniae and the TEM beta-lactamase gene mediated by R plasmids

Sixty-three percent homology of nucleotide sequence and 67% homology of deduced amino acid sequence were found between the chromosomally encoded beta-lactamase gene of Klebsiella pneumoniae and the TEM beta-lactamase of transposon Tn3. Moreover, 22 out of 24 amino acid residues are identical around the predicted active site. It is therefore suggested that these two kinds of beta-lactamases share a common evolutionary origin. The 0.5 kb DNA fragment of the cloned gene hybridized specifically with the chromosomal DNA of all the K. pneumoniae strains tested which had been isolated in Japan, USA and Europe.     

Group 1 beta-lactamases of Aeromonas caviae and their resistance to beta-lactam antibiotics

The contribution of beta-lactamase production to beta-lactam antibiotic resistance was examined in an Aeromonas caviae mutant strain, selected in vitro by cefotaxime and derived from a wild-type strain isolated in our laboratory from crude sewage. Both strains produced beta-lactamase. The mutant strain (AC7m) produced beta-lactamase constitutively, in contrast to the parental strain (AC7), which was inducible by cefoxitin. AC7m was regarded as a mutant from AC7, which over-expressed beta-lactamase. The mutant strain showed a remarkable reduction in sensitivity to most of the beta-lactam antibiotics tested, such as (i) aminopenicillins and their combinations with clavulanic acid and sulbactam, (ii) carboxypenicillins, (iii) ureidopenicillins, and (iv) cephalosporins. This strain remained susceptible to ceftazidime, imipenem, and aztreonam. Isoelectric focusing of sonic extracts revealed that both strains AC7 and AC7m shared a common major beta-lactamase band at pI 6.5. The plasmid DNA assays showed that the beta-lactamases expressed by each A. caviae strain were chromosomally encoded. Based on substrate and inhibitor profiles determined in sonic extracts for AC7 and AC7m, the enzymes displayed on isoelectric focusing at pI 6.5 were assigned to chromosomal Group 1 beta-lactamases. Imipenem would therefore be the appropriate choice for therapy of infections caused by A. caviae beta-lactamase over-expressing mutants.     

Antibiotic susceptibility and molecular mechanisms of cephalosporins resistance in Klebsiella isolates from patients with hospital-acquired infections]

Antibiotic susceptibility of nosocomial Klebsiella isolates from inpatients of 30 medical centres in 15 various regions of Russia was studied. In total 212 strains were tested. The Klebsiella genus was represented by the following species: Klebsiella pmeumoniae ss. pneumoniae (182 isolates, 85.8%), Klebsiella pneumonia ss. ozaenae (1 isolate, 0.5%), Klebsiella oxytoca (29 isolates, 13.7%). The susceptibility was determined by the broth microdilution method. Carbapenems (imipenem and meropenem) remained to be the most active antibacterial agents. However, 1 imipenem resistant strain and 2 meropenem resistant strains were isolated. As for the 3rd generation cephalosporins, the lowest MICs were observed with the use of the inhibitor-protected agents, such as ceftazidime/clavulanic acid (MIC50 0.25 mcg/ml, MIC90 64 mcg/ml). 48.8%, 16.9%, 29.7% and only 10.5% of the isolates was susceptible to cefepime, cefotaxime, ceftazidime and cefoperazone respectively. Detecting of the beta-lactamase genes (TEM, SHV and CTX) was performed by PCR in 42 strains of Klebsiella pneumoniae ss. pneumoniae. Alone or in various combination the TEM type beta-lactamases were detected in 16 (38.1%) isolates. SHV and CTX were detected in 29 (69%) and 27 (64.3%) isolates respectively. Combinations of 2 and 3 different determinants of resistance to beta-lactams were revealed in 23.8% and 26.2% of the isolates respectively. No isolates producing class B MBL among the carbapenem resistant nosocomial Klebsiella strains were detected.     

Possible Involvement of β-Lactamase in Sporulation in Bacillus cereus

Nonreverting beta-lactamase-negative strains were isolated from the beta-lactamase-constitutive strain, Bacillus cereus 569 H. These strains differed from both beta-lactamase-inducible and -constitutive strains not only in failure to produce beta-lactamase but also in failure to autolyze on aging, delayed sporulation, and failure to release free spores from sporangia when produced. The addition of B. cereus beta-lactamase of 15% purity to a final concentration of 10 IU/ml stimulates sporulation and particularly the release of free spores in culture from sporangia of strain 569 (inducible wild-type), 569/H (constitutive mutant of 569), and HPen(-), a nonreverting beta-lactamase strain isolated from 569/H in this laboratory. Cultures of HPen(-) did not release free spores without this treatment. Similar stimulation of sporulation and spore release by beta-lactamase from B. cereus were observed in another beta-lactamase-negative strain derived from 569/H as well as in certain sporogeny mutants of B. subtilis. The beta-lactamase preparation used in these experiments was free of peptidases, proteases, and autolysins capable of solubilizing wall from vegetative cells. These results, taken with our previous finding that a soluble peptidoglycan inducer becomes available in cultures of B. cereus only at sporulation and that normal derepression of beta-lactamase accompanies normal sporulation, suggest that beta-lactamase in B. cereus may be involved in peptidoglycan metabolism during sporulation and possibly the breakdown of sporangial wall with the concomitant release of mature spores.     

Involvement of a plasmid in exopolysaccharide production by Klebsiella oxytoca

A Klebsiella oxytoca isolate which can produce significant levels of an exopolysaccharide using whey as a growth substrate has been reported. The plasmid profile of this isolate was shown to be different from that of the non-exopolysaccharide-producing K. oxytoca ATCC 43863. Irreversible curing of the single plasmid in the K. oxytoca isolate was achieved using 30 g acriflavin/ml. The ability to produce the exopolysaccharide was lost with the curing of the plasmid (parent strain produced a medium viscosity of 1260 cP at 1 s^–1 compared to 1.6 cP at 200 s^–1 produced by the cured strain). However, the ability to metabolize lactose was not significantly affected by curing, and both the parent and the cured strain produced similar levels of viable cells (~10⁹ cfu/ml) after 62 h growth on lactose-rich medium. The exopolysaccharide-producing ability of the isolate was stable for at least 139 generations.     

Nucleotide sequence of the PSE-4 carbenicillinase gene and correlations with the Staphylococcus aureus PC1 beta-lactamase crystal structure

The nucleotide sequence of the PSE-4 beta-lactamase gene from Pseudomonas aeruginosa strain Dalgleish has been determined. The structural gene encodes a polypeptide product of 252 amino acids with an estimated molecular mass of 29,246 Da for the mature form of the protein. The PSE-4 gene has limited homology with other beta-lactamases at the DNA level. An alignment of all known class A beta-lactamases permitted as to identify specific residues important for enzyme structure and function. To confirm observations based on the linear sequences, we designed a new molecular model for PSE-4 beta-lactamase based on x-ray data from the Staphylococcus aureus PC1 beta-lactamase at 2.0-A resolution. The structural similarities between PSE-4 and class A beta-lactamases are more extensive than indicated by earlier biochemical studies. The combined structural and sequence information now available for a series of beta-lactamases identifies conserved residues in these molecules, giving insight of their divergence and ancestry. Analysis of the PSE-4 flanking DNA sequences revealed an integration site common to antibiotic resistance genes inserted into transposons of the Tn21 family with the target integration sequence AAGTT.     

Cytotoxic component(s) of Klebsiella oxytoca on HEp-2 cells

A cytotoxic component(s) was detected in culture filtrates of Klebsiella oxytoca isolated from patients with antibiotic-associated hemorrhagic colitis. Eleven of 12 isolates exhibited cytotoxicity on HEp-2 cells. The cytotoxic activity of K. oxytoca strain MH43-1 was stable for the treatment of 60 C for 30 min, but inactivated by the treatment of 100 C for 15 min. This cytotoxicity was not destroyed by the treatment with trypsin or pronase, and the component was filtrable through a membrane filter which cut off molecular weight 5,000.     

厚荚相思树根瘤菌HJ06菌株的16S rDNA全序列和nifA基因片段分析

对厚荚相思(Acaciacrassicarpa)根瘤菌HJ06菌株的16SrDNA全序列和nifA基因片段进行了测定。结果表明,HJ06菌株在以16SrDNA序列构建的系统发育树状图中位于根瘤菌属(Rhizobium)分支中,与根瘤菌属各个种的相似性达95%以上;从HJ06菌株克隆出的585bpnifA基因片段与Klebsiellapneumoniae的同源性达到99.3%,与Klebsiellaoxytoca的NifF,NifL,NifA,NifB蛋白基因的同源性为97.8%。     

Characterization and identification of virulent Klebsiella oxytoca isolated from abalone (Haliotis diversicolor supertexta) postlarvae with mass mortality in Fujian, China

An epidemic of mass mortality of abalone (Haliotis diversicolor supertexta) postlarvae aged 40 days or less has existed across south coast of China since the second half of 2002. Among 20 bacterial strains isolated from diseased abalone postlarvae on 2216E marine agar plates during an outbreak of postlarval disease in August 2005, a predominant strain (designated strain 20) was demonstrated to be virulent to postlarvae with an LD(50) value of 1.0x10(5) colony forming units (CFUml(-1)) on day 4, while the other 19 strains were either avirulent (16 strains) or weakly virulent (3 strains). The same bacterium could be re-isolated from postlarvae after bacterial challenge using 2216E marine agar plates. Preliminary toxicity tests of ECPs of strain 20 revealed that at 2.77mgproteinml(-1), crude ECPs completely liquefied postlarvae within 24h, leaving only shells. API 20E analysis identified strain 20 as Klebsiella oxytoca. 16S and ITS rDNA sequencing and phylogenetic analyses further confirmed this identification. Antibiotic susceptibility tests showed that strain 20 exhibited 94% of susceptibility to 16 various antibiotics tested and only showed resistance to streptomycin. Results of this work demonstrated that K. oxytoca is also linked to this epidemic in Fujian, China. This is considered to be the first report regarding K. oxytoca involved in the mass mortality of postlarval abalone in south China and the world.