Non-radioactive PCR-SSCP with a single PCR step for detection of inhibitor resistant beta-lactamases in Escherichia coli

A method based on PCR-SSCP has been developed to detect presumptive Inhibitor-Resistant TEM (IRT) beta-lactamases in Escherichia coli. The capacity of this technique to differentiate genes from 11 control strains encoding IRT beta-lactamases was evaluated with PCR products digested with RsaI. All the bla(TEM) genes studied could be distinguished by their electrophoretic mobilities. Applied to 29 epidemiologically unrelated clinical isolates of E. coli resistant to amoxicillin-clavulanate (MIC, > or =32 microg/ml), the electrophoretic mobilities of the digested bla(TEM) PCR products were identical to those of the reference bla(TEM-1A) (6 strains) and bla(TEM-1B) (18 strains) genes. The remaining five bla(TEM) PCR products displayed SSCP profiles different from those of the reference bla(TEM) genes and their nucleotide sequence identified them as bla(TEM-1C) in one strain, bla(TEM-30/IRT-2) in two strains, bla(TEM-37/IRT-8) in one strain, and bla(TEM-40/IRT-11) in one isolate. Overexpression of the wild-type bla(TEM-1) gene, as detected by high-level resistance to beta-lactams and enzyme assay, accounted for resistance in the 24 E. coli containing bla(TEM-1). We report a simple one PCR step SSCP that can be used in epidemiological studies for rapid preliminary detection of IRT beta-lactamases; identification should be confirmed by sequence data.     

An IS1-like element is responsible for high-level synthesis of extended-spectrum beta-lactamase TEM-6 in Enterobacteriaceae.

Resistance of Escherichia coli strain HB251 to the newer beta-lactam antibiotics, in particular ceftazidime and aztreonam, results from production of the extended-spectrum beta-lactamase TEM-6. The corresponding structural gene, bla(T)-6, and its promoter region were amplified by the polymerase chain reaction. Analysis of the sequence of the amplification product showed that bla(T)-6 differed by two nucleotide substitutions from bla(T)-1, the gene encoding TEM-1 penicillinase in plasmid pBR322. The mutations led to the substitution of a lysine for a glutamic acid at position 102 and of a histidine for an arginine at position 162 of the unprocessed TEM-1 protein. The presence of a 116 bp DNA insert upstream from bla(T)-6 resulted in the creation of hybrid promoter P6 in which the -10 region was that of TEM-1 promoter P3 whereas the -35 canonical sequence TTGACA was provided by the right end of the insert. P6 was found to be 10 times more active than P3 and to confer higher levels of antibiotic resistance upon the host. Analysis of the sequence of the insert indicated that the 116 bp fragment is related to insertion sequence IS1 but differs from it by three internal deletions that removed regions encoding the transposase. The distribution of the IS1-like element in clinical isolates of Enterobacteriaceae was studied by the polymerase chain reaction and by DNA-DNA hybridization. The element appeared to be widespread and was detected in strains producing TEM-6 or other TEM variants.     

Simple and reliable multiplex PCR assay for detection of blaTEM,bla(SHV) and blaOXA-1 genes in Enterobacteriaceae

Third-generation cephalosporin resistance is often mediated by TEM- and SHV-type beta-lactamases in Enterobacteriaceae. TEM-type and OXA-1 enzymes are the major plasmid-borne beta-lactamases implicated in amoxicillin-clavulanic acid resistance in Escherichia coli isolates. We have developed a rapid and simple multiplex polymerase chain reaction (PCR) which discriminates bla(TEM), bla(SHV) and bla(OXA-1) genes by generating fragments of 516, 392 and 619 bp respectively. Multiplex PCR analysis of 51 amoxicillin-clavulanate resistant E. coli isolates detected bla(TEM) and bla(SHV) genes in 45 and two strains, respectively, and only one strain harboured a bla(OXA-1) gene. Twenty-three of the 40 cefotaxime-resistant Enterobacteriaceae isolates produced amplicons with a size compatible with the presence of bla(TEM) (13 strains), bla(SHV) (six strains) genes or the association of both genes (four strains). These results were verified by colony hybridisation. Therefore, multiplex PCR is a suitable tool for initial rapid screening of bla genes in Enterobacteriaceae.     

Biochemical and genetic characteristics of TEM-29B, a novel extended spectrum beta-lactamase

A clinical strain of Escherichia coli (strain Ec 41553) that was resistant to ceftazidime produced a TEM-type beta-lactamase with a pI of 5.4. Clavulanic acid restored the ceftazidime activity, thus suggesting an extended spectrum beta-lactamase (ESBL). The gene encoding ESBL was located in a plasmid of 57 kb. After cloning and sequencing, the ESBL (TEM-29B) showed one amino acid replacement with respect to the TEM-1 sequence, Arg-164 to His. This change increased mainly the rate of hydrolysis of ceftazidime but not of cefotaxime and aztreonam. The relevance of this substitution in the increase of ceftazidime MIC is thus stressed.     

Oligonucleotide probes for the detection of TEM-1 and TEM-2 beta-lactamase genes and their transposons

We describe the use of molecular probes to detect the TEM-type beta-lactamase genes. As a general probe, we prepared a 656 base pair restriction fragment, entirely within the TEM structural gene. This probe was specific for the TEM family, hybridizing only with TEM-1 and TEM-2. The TEM-1 and TEM-2 beta-lactamases differ by only one amino acid. We synthesized two oligonucleotides whose central bases correspond to this difference. The use of these oligonucleotides enables us to discriminate between TEM-1 and TEM-2 genes. Using oligonucleotides homologous to parts of Tn3, we also monitored the presence of TnA-like transposons in bacteria harboring different beta-lactamase genes. Only the TEM-1 and TEM-2 genes were found to be on transposons with terminal sequences identical to those of Tn3. All hybridization experiments were performed with both dot-blot and colony-hybridization techniques, and the suitability of these two methods for epidemiological studies is compared.     

Nucleotide sequence of the thermostable direct hemolysin gene (tdh gene) of Vibrio mimicus and its evolutionary relationship with the tdh genes of Vibrio parahaemolyticus

During an outbreak of infection with ampicillin-resistant, TEM-1 beta-lactamase-producing Escherichia coli serotype O15, some strains were noted to differ from the majority in that they showed reduced susceptibility to amoxycillin/clavulanic acid (Augmentin), ureidopenicillins and first generation cephalosporins and produced increased amounts of beta-lactamase. The plasmid from one such isolate was compared with that from an isolate that produced normal amounts of beta-lactamase. Restriction analysis with EcoRI revealed extra fragments in the plasmid from the beta-lactamase hyperproducer and use of DNA-DNA hybridisation with a biotinylated TEM-1 probe showed genetic rearrangement in the beta-lactamase hyperproducer so that the TEM gene appeared to be present in larger amounts and was located on a smaller fragment than for the plasmid from the strain that produced normal amounts of beta-lactamase.     

Antibiotic-resistant Escherichia coli bacteria, including strains with genes encoding the extended-spectrum beta-lactamase and QnrS, in waterbirds on the Baltic Sea Coast of Poland

Individual cloacal swabs of mallards (Anas platyrhynchos) and of herring gulls (Larus argentatus), as well as samples of waterbird feces obtained in 2008 and 2009, were cultivated for Escherichia coli. Isolates of E. coli were tested for susceptibilities to 12 antimicrobial agents by the disk diffusion method. Moreover, the samples were subcultivated on MacConkey agar (MCA) containing cefotaxime (2 mg liter(-1)) to detect E. coli with extended-spectrum beta-lactamase (ESBL) and subsequently on MCA supplemented with ciprofloxacin (0.05 mg liter(-1)) and MCA with nalidixic acid (20 mg liter(-1)) to isolate fluoroquinolone-resistant E. coli. PCR was used to detect specific antibiotic resistance genes. We found 9 E. coli isolates producing ESBL with bla genes: bla(CTX-M-1) (6 isolates), bla(CTX-M-9) plus bla(TEM-1b) (1 isolate), bla(CTX-M-15) plus bla(OXA-1) (1 isolate), and bla(SHV-12) (1 isolate). In the isolate with bla(CTX-M-15), the gene aac(6)-Ib-cr was also detected. The bla genes were harbored by transferable plasmids of the IncN and IncI1 groups. Nine quinolone-resistant E. coli isolates with qnrS genes were found and characterized. The gene qnrS was associated with a Tn3-like transposon on the IncX1 plasmid together with bla(TEM-1) in two isolates. The gene qnrS was also harbored by conjugative plasmids of the IncN and IncX2 groups. Even if populations of wild birds are not directly influenced by antibiotic practice, we have demonstrated that antibiotic-resistant E. coli strains, including strains with various ESBL and qnrS genes, are found in the feces of wild birds on the coast of the Baltic Sea in Poland.     

Nucleotide sequences of the genes coding for the TEM-like β-lactamases IRT-1 and IRT-2 (formerly called TRI-1 and TRI-2)

Abstract Two bla _TEM-like genes were characterized that encoded IRT β-lactamases (previously called TRI) in clinical isolates of Escherichia coli resistant to amoxycillin alone and to combinations of amoxycillin with β-lactamase inhibitors. Plasmids carrying this resistance were isolated from E. coli K 12 transconjugants and the genes were sequenced after amplification of defined fragments, using TEM-1-specific primers. The gene for IRT-1 β-lactamase resembled the bla _TEM-1B gene, and that for IRT-2 resembled bla _TEM-2. However, both IRT enzymes have a glutamine residue at position 37, which is characteristic of TEM-1. The unique nucleotide difference with parental genes corresponding to amino acid variation was observed at nucleotide position 929. The consequence of C to T transition in the bla _IRT-1 gene and C to A transversion in the bla _IRT-2 gene was the substitution of arginine 241 in the native protein by cysteine and serine, respectively, in the mutants. Thus, the nature of amino acid 241 is critical in conferring resistance or susceptibility to β-lactamase inhibitors. Furthermore, these basic to neutral amino acid replacements explain the more acidic p I (p I =5.2) of these IRT enzymes compared to that of TEM-1 (p I =5.4). The presence of cysteine-241 in IRT-1 also explains the selective sensitivity of this β-lactamase to inhibition by p -chloromercuribenzoate.     

Phenotypic and molecular characterization of TEM-116 extended-spectrum beta-lactamase produced by a Shigella flexneri clinical isolate from chickens.

Extended-spectrum beta-lactamases (ESBLs) produced by a clinical isolate of Shigella flexneri from chickens were detected with confirmatory phenotypic tests of the Clinical and Laboratory Standards Institute, and minimum inhibitory concentrations of several antibacterial drugs against the isolate were determined by the twofold dilution method. The genotype and subtype of the ESBL-producing S. flexneri isolate were identified by PCR amplifying of ESBL genes and DNA sequencing analysis. The results revealed that the isolate was able to produce ESBLs. They were resistant to third-generation cephalosporins such as ceftiofur and ceftriaxone and showed characteristics of multidrug resistance. The ESBL gene from the S. flexneri isolate was of the TEM type. Sequence analysis indicated that the TEM-type gene had 99.1% and 99.2% identity to TEM-1D ESBL and TEM-1 beta-lactamase, respectively, at the nucleotide level. The amino acid sequence inferred from the TEM-type gene revealed three substitutions compared with the TEM-1 and TEM-1D enzymes: Ser51Gly, Val82Ila and Ala182Val. When it was compared with TEM-116 (99.8% identity), there were only two mutations (A(151)G and T(403)C) in the TEM-type gene, resulting in the substitution of Ser to Gly at position 51 in the amino acid sequence. The TEM type was a TEM-116 derivative.     

Crystal structure and kinetic analysis of beta-lactamase inhibitor protein-II in complex with TEM-1 beta-lactamase

The structure of the 28 kDa beta-lactamase inhibitor protein-II (BLIP-II) in complex with the TEM-1 beta-lactamase has been determined to 2.3 A resolution. BLIP-II is a secreted protein produced by the soil bacterium Streptomyces exfoliatus SMF19 and is able to bind and inhibit TEM-1 with subnanomolar affinity. BLIP-II is a seven-bladed beta-propeller with a unique blade motif consisting of only three antiparallel beta-strands. The overall fold is highly similar to the core structure of the human regulator of chromosome condensation (RCC1). Although BLIP-II does not share the same fold with BLIP, the first beta-lactamase inhibitor protein for which structural data was available, a comparison of the two complexes reveals a number of similarities and provides further insights into key components of the TEM-1-BLIP and TEM-1-BLIP-II interfaces. Our preliminary results from gene knock-out studies and scanning electron microscopy also reveal a critical role of BLIP-II in sporulation.     

Substitution of Thr for Ala-237 in TEM-17, TEM-12 and TEM-26: alterations in β-lactam resistance conferred on Escherichia coli

Non-naturally occurring mutants of TEM-17 (E104K), TEM-12 (R164S) and TEM-26 (E104K:R164S) extended-spectrum (ES) beta-lactamases bearing threonine at position 237 were constructed by site-specific mutagenesis and expressed under isogenic conditions in Escherichia coli. Quantification of beta-lactamase activities and immunoblotting indicated that Ala-237-->Thr did not significantly affect expression levels of these ES enzymes. Minimum inhibitory concentrations of beta-lactam antibiotics showed that the presence of threonine at position 237 exerted a dominant effect increasing the enzymes' preference for various early generation cephalosporins over penicillins. Activity against broad-spectrum oxyimino-beta-lactams was also changed. The effect of Ala-237-->Thr on the activity against ceftazidime, aztreonam, cefepime and cefpirome of all three ES TEM enzymes was detrimental. Introduction of Thr-237 improved activity against cefotaxime and ceftriaxone in TEM-12 and TEM-26, but not in TEM-17.     

Triplet nucleotide removal at random positions in a target gene: the tolerance of TEM-1 beta-lactamase to an amino acid deletion

The deletion of amino acids is one of the evolutionary mechanisms by which nature adapts the function of proteins. A simple method has been developed that mimics this event in vitro by introducing a deletion of exactly three nucleotides at random positions in a target gene. The method involved the engineering of the mini-Mu transposon to introduce a recognition sequence for the restriction enzyme MlyI. The new transposon, MuDel, was capable of efficient insertion into a target DNA sequence. To determine the efficacy of the method, the bla gene that encodes the TEM-1 beta-lactamase was used as the target and a small library containing 22 different sequence variants was created. Of these 22 variants, 8 were identified that conferred resistance to ampicillin on Escherichia coli. Each of the TEM-1 variants possessed a distinct ampicillin minimum inhibitory concentration, ranging from 500 to >10,000 microg/ml. Sequence analysis revealed that active TEM-1 variants contained deletions not just in loops but also helices, and included regions known to be involved in catalysis, antibiotic resistance and inhibitor binding. This new technology is transferable to most genes, permitting an extensive analysis of deletion mutations on protein function.     

Overexpression and biosynthetic deuterium enrichment of TEM-1 beta-lactamase for structural characterization by magnetic resonance methods

An expression system has been developed that allows high levels of production of TEM-1 beta-lactamase with ease of biosynthetic incorporation of nuclear isotopes. The gene for mature TEM-1 beta-lactamase fused to the leader sequence of the ompA protein was subcloned into the pET-24a(+) vector by introduction of an NdeI restriction site at the first codon of the fused genes and transformed into Escherichia coli BL21 (DE3) cells. With protein induction at 25 degrees C supported by LB medium supplemented with osmolytes (300 mM sucrose and 2.5 mM betaine), the extracellular, mature form of wild-type TEM-1 beta-lactamase was recovered at a level of 140 mg/L. The production level of E166N, E240C, E104C, and M272C mutants depended on the mutation but was invariably higher than reported by others for expression systems of the wild-type enzyme. Comparison of different carbon sources on the efficiency of biosynthetic incorporation of covalent deuterium showed maximal (90%) incorporation with minimal medium containing 99% (2)H(2)O and sodium d(3)-acetate (99 atom% (2)H). The yield of deuterium-enriched wild-type enzyme was 80 mg/L with yields for mutants proportionally reduced. The high level of protein deuteration achieved with this system allowed detection of the hyperfine coupling between the paramagnetic nitroxyl group of a spin-labeled penicillin substrate and hydrogens on the penicillin moiety in a cryokinetically isolated acylenzyme reaction intermediate because of the decrease in overlapping resonances of active site residues. The overexpression system is readily adaptable for other target proteins and facilitates studies requiring large quantities of protein in isotopically enriched forms.     

Antibiotic susceptibility of bacterial strains isolated from patients with various infections

AIMS: Isolates from various samples obtained during 1998 and 1999 were identified and their susceptibility to third-generation cephalosporins, monobactams and/or cephamycins studied along with any production of ESBLs. METHODS AND RESULTS: Of these samples, bacteria most frequently isolated by the conventional techniques and Vitek GNI card were Escherichia coli (37%), Klebsiella pneumoniae (27%) and Enterobacter cloacae (16%). Using disk diffusion and double-disk synergy tests, we found that 71% strains produced ESBLs and 18% strains produced ESBLs and cephamycinases. Banding patterns of PCR amplification with the designed primers showed that 57% strains were capable of harbouring bla(SHV) genes. The bla(TEM), bla(CMY) and bla(AmpC) genes were harboured by 55%, 31% and 12% strains, respectively. Forty-five percent of strains contained more than two types of beta-lactamase genes. In particular, one strain contained bla(TEM), bla(SHV), bla(CMY) and bla(AmpC) genes. CONCLUSIONS: The percentage of ESBL-producing strains was high. The most prevalent beta-lactamase gene was bla(SHV) gene. The bla(CMY) genes have been prevalent in cephamycin-resistant strains. The multidrug-resistant strains resistant to third-generation cephalosporins and cephamycins were detected in high percentage. SIGNIFICANCE AND IMPACT OF THE STUDY: Resistance mechanisms to beta-lactams, comprising mostly extended-spectrum beta-lactamase (ESBL) production, lead to the resistance against even recently developed beta-lactams in enterobacteria, which is now a serious threat to antibiotic therapy. The high prevalence of bla(CMY) genes and multidrug-resistant genes may also cause therapeutic failure and lack of eradication of these strains by third-generation cephalosporins or cephamycins.     

A fitness cost associated with the antibiotic resistance enzyme SME-1 beta-lactamase

The bla(TEM-1) beta-lactamase gene has become widespread due to the selective pressure of beta-lactam use and its stable maintenance on transferable DNA elements. In contrast, bla(SME-1) is rarely isolated and is confined to the chromosome of carbapenem-resistant Serratia marcescens strains. Dissemination of bla(SME-1) via transfer to a mobile DNA element could hinder the use of carbapenems. In this study, bla(SME-1) was determined to impart a fitness cost upon Escherichia coli in multiple genetic contexts and assays. Genetic screens and designed SME-1 mutants were utilized to identify the source of this fitness cost. These experiments established that the SME-1 protein was required for the fitness cost but also that the enzyme activity of SME-1 was not associated with the fitness cost. The genetic screens suggested that the SME-1 signal sequence was involved in the fitness cost. Consistent with these findings, exchange of the SME-1 signal sequence for the TEM-1 signal sequence alleviated the fitness cost while replacing the TEM-1 signal sequence with the SME-1 signal sequence imparted a fitness cost to TEM-1 beta-lactamase. Taken together, these results suggest that fitness costs associated with some beta-lactamases may limit their dissemination.     

Dissemination of transferable CTX-M-type extended-spectrum beta-lactamase-producing Escherichia coli in Korea

AIMS: Among 365 Escherichia coli isolated in 2003, 31 cefotaxime-resistant isolates were obtained from clinical specimens taken from adults hospitalized in Busan, Korea. Six extended-spectrum beta-lactamase (ESBL)-producing isolates were investigated further to determine the mechanism of resistance. METHODS AND RESULTS: These isolates were analysed by antibiotic susceptibility testing, pI determination, plasmid profiles, transconjugation test, PCR-restriction fragment length polymorphism (RFLP), enterobacterial repetitive consensus (ERIC)-PCR and DNA sequencing. All six of these isolates were found to contain the CTX-M-type ESBL genes. Five clinical isolates and their transconjugants produced CTX-M-3. One clinical isolate (K17391) and its transconjugant (trcK17391) produced CTX-M-15. Five clinical isolates also produced another TEM-1. One clinical isolate (K12776) also contained another TEM-52. CTX-M-3 ESBL gene was responsible for the resistance to piperacillin, cephalothin, cefotaxime, cefepime and aztreonam. CTX-M-15 or TEM-52 was especially responsible for the resistance to ceftazidime. CONCLUSIONS: These results appear to represent the in vivo evolution of CTX-M-type beta-lactamase genes (bla(CTX-M-3) --> bla(CTX-M-15)) under the selective pressure of antimicrobial therapy (especially ceftazidime). PCR-RFLP is a reliable method to discriminate CTX-M-15 gene from CTX-M-3 gene. ERIC-PCR analysis revealed that dissemination of CTX-M-3 was not due to a clonal outbreak of a resistant strain but to the intra-species spread of resistance to piperacillin, cephalothin, cefotaxime, cefepime and aztreonam in Korea. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of the occurrence of CTX-M-1 cluster ESBLs in Korea. A more comprehensive survey of these ESBL types from Korea is urgently needed because of the in vivo evolution of CTX-M-15 from CTX-M-3. The emergence of these CTX-M-type ESBLs suggests that diagnostic laboratories should screen for ESBLs with ceftazidime as well as cefotaxime; they should still perform clavulanate synergy tests on resistant isolates.     

Construction by polymerase chain reaction and intragenic DNA probes for three main types of transferable β-lactamases (TEM, SHV, CARB)

Intragenic DNA probes were synthesized by polymerase chain reaction using fragments of the genes of three major types of beta-lactamases (TEM, SHV, CARB) as templates. The TEM probe hybridized with the genes encoding TEM-1, TEM-2 and six extended-spectrum related enzymes (TEM-3 to TEM-7, TEM-2O) in colony hybridizations and Southern-blot analysis. The SHV probe hybridized with the genes for SHV-1, OHIO-1 and four derived extended-spectrum beta-lactamases (SHV-2, SHV-3, SHV-4 and SHV-5). The CARB probe hybridized with the genes for PSE-1 (CARB-2), PSE-4 (CARB-1), CARB-3 and CARB-4. None of the probes hybridized with genes for any of eight oxacillin-hydrolysing enzymes, PSE-2, OXA-1 to OXA-7, ROB-1 and chromosomal beta-lactamases of various Enterobacteriaceae (except Klebsiella pneumoniae) and Pseudomonas aeruginosa. Investigations of Escherichia coli clinical isolates using these probes indicate the presence of a novel type of extended-spectrum, transferable beta-lactamase.     

Complete sequencing of the bla(NDM-1)-positive IncA/C plasmid from Escherichia coli ST38 isolate suggests a possible origin from plant pathogens

The complete sequence of the plasmid pNDM-1_Dok01 carrying New Delhi metallo-β-lactamase (NDM-1) was determined by whole genome shotgun sequencing using Escherichia coli strain NDM-1_Dok01 (multilocus sequence typing type: ST38) and the transconjugant E. coli DH10B. The plasmid is an IncA/C incompatibility type composed of 225 predicted coding sequences in 195.5 kb and partially shares a sequence with bla(CMY-2)-positive IncA/C plasmids such as E. coli AR060302 pAR060302 (166.5 kb) and Salmonella enterica serovar Newport pSN254 (176.4 kb). The bla(NDM-1) gene in pNDM-1_Dok01 is terminally flanked by two IS903 elements that are distinct from those of the other characterized NDM-1 plasmids, suggesting that the bla(NDM-1) gene has been broadly transposed, together with various mobile elements, as a cassette gene. The chaperonin groES and groEL genes were identified in the bla(NDM-1)-related composite transposon, and phylogenetic analysis and guanine-cytosine content (GC) percentage showed similarities to the homologs of plant pathogens such as Pseudoxanthomonas and Xanthomonas spp., implying that plant pathogens are the potential source of the bla(NDM-1) gene. The complete sequence of pNDM-1_Dok01 suggests that the bla(NDM-1) gene was acquired by a novel composite transposon on an extensively disseminated IncA/C plasmid and transferred to the E. coli ST38 isolate.