Kegg Pathway: Clavulanic acid biosynthesis

BMC Infect Dis. 2008; 8: 55
Bauhofer A, Huttel M, Lorenz W, Sessler DI, Torossian A

BACKGROUND: In addition to their antimicrobial activity, antibiotics modulate cellular host defence. Granulocyte-colony stimulating factor (G-CSF) is also a well known immunomodulator; however little is known about the interactions of G-CSF with antibiotics. We investigated in septic rats the effects of two antibiotic combinations with G-CSF. METHODS: In two clinic modelling randomised trials (CMRTs), male Wistar rats were anesthetized, given antibiotic prophylaxis, had a laparotomy with peritoneal contamination and infection (PCI), and were randomly assigned (n = 18 rats/group) to: 1) PCI only; 2) PCI+antibiotic; and, 3) PCI+antibiotic+G-CSF prophylaxis (20 mug/kg, three times). This sequence was conducted first with 10 mg/kg coamoxiclav, and then with ceftriaxone/metronidazole (Cef/met, 10/3 mg/kg). In additional animals, the blood cell count, migration and superoxide production of PMNs, systemic TNF-alpha and liver cytokine mRNA expression levels were determined. RESULTS: Only the combination coamoxiclav plus G-CSF improved the survival rate (82 vs. 44%, p < 0.001). Improved survival with this combination was accompanied by normalised antimicrobial PMN migratory activity and superoxide production, along with normalised systemic TNF-alpha levels and a reduced expression of TNF-alpha and IL-1 in the liver. CONCLUSION: There are substantial differences in the interaction of antibiotics with G-CSF. Therefore, the selection of the antibiotic for combination with G-CSF in sepsis treatment should be guided not only by the bacteria to be eliminated, but also by the effects on antimicrobial functions of PMNs and the cytokine response.

Biochemistry. 2008 May 13; 47(19): 5312-6
Tremblay LW, Hugonnet JE, Blanchard JS

The intrinsic resistance of Mycobacterium tuberculosis to the beta-lactam class of antibiotics arises from a chromosomally encoded, extended spectrum, class A beta-lactamase, BlaC. Herein, we report the X-ray crystallographic structure of BlaC inhibited with clavulanate at a resolution of 1.7 A with an R-factor value of 0.180 and R-free value of 0.212 for the m/ z +154 clavulanate-derived fragment observed in the active site. Structural evidence reveals the presence of hydrogen bonds to the C1 carbonyl along with a coplanar arrangement of C1, C2, C3, and N4, which favors enolization to generate a trans-alpha,beta-eneamine, stabilizing the +154 adduct from hydrolysis. The irreversible inhibition of BlaC suggests that treatment of M. tuberculosis with a combination of a beta-lactam antibiotic and clavulanate may lead to rapid bactericidal activity.

APMIS. 2008 Apr; 116(4): 302-8
Lytsy B, Sandegren L, Tano E, Torell E, Andersson DI, Melhus A

Between May and December 2005, 64 multidrug-resistant isolates of Klebsiella pneumoniae were detected from patients admitted to Uppsala University Hospital. This represented a dramatic increase in ESBL-producing K. pneumoniae compared to previous years. To investigate the epidemiology and to characterize the resistance mechanisms of the isolates, a study was initiated. Antibiotic susceptibility was determined by means of the Etest and the disc diffusion method. Extended-spectrum beta-lactamase (ESBL) production was identified by Clavulanic acid synergy test and confirmed with PCR amplification followed by DNA sequencing. DNA profiles of the isolates were examined with pulsed-field gel electrophoresis (PFGE). All isolates were resistant or exhibited reduced susceptibility to cefadroxil, cefuroxime, cefotaxime, ceftazidime, aztreonam, piperacillin/tazobactam, ciprofloxacin, tobramycin, and trimethoprim-sulfamethoxazole. They produced ESBL of the CTX-M-15 type, and the involvement of a single K. pneumoniae clone was shown. This is the first major clonal outbreak of multiresistant ESBL-producing K. pneumoniae in Scandinavia. The outbreak demonstrates the epidemic potential of enterobacteria containing ESBLs of the CTX-M type, even in a country with a relatively low selective pressure and a low prevalence of multiresistant bacteria.

J Microbiol Biotechnol. 2008 Mar; 18(3): 417-26
Song JY, Kim ES, Kim DW, Jensen SE, Lee KJ

The effect of increasing levels of proclavaminate amidino hydrolase (Pah) on the rate of Clavulanic acid production in Streptomyces clavuligerus ATCC 27064 was evaluated by knock-in a gene (pah2) encoding Pah. A strain (SMF5703) harboring a multicopy plasmid containing the pah2 gene showed significantly retarded cell growth and reduced Clavulanic acid production, possibly attributable to the deleterious effects of the multicopy plasmid. In contrast, a strain (SMF5704) carrying a single additional copy of pah2 introduced into chromosome via an integrative plasmid showed enhanced production of Clavulanic acid and increased levels of pah2 transcripts. Analysis of transcripts of other genes involved in the Clavulanic acid biosynthetic pathway revealed a pattern similar to that seen in the parent. From these results, it appears that Clavulanic acid production can be enhanced by duplication of pah2 through integration of a second copy of the gene into chromosome. However, increasing the copy number of only one gene, such as pah2, does not affect the expression of other pathway genes, and so only modest improvements in Clavulanic acid production can be expected. Flux controlled by Pah did increase when the copy number of pah2 was doubled, suggesting that under these growth conditions, Pah levels may be a limiting factor regulating the rate of Clavulanic acid biosynthesis in S. clavuligerus.

Int J Antimicrob Agents. 2008 May; 31(5): 467-71
Jeong SH, Song W, Park MJ, Kim JS, Kim HS, Bae IK, Lee KM

A study using boronic acid (BA) was designed to detect the extended-spectrum beta-lactamases (ESBLs) in Enterobacteriaceae producing chromosomal AmpC beta-lactamases. A total of 197 clinical isolates of Enterobacter spp. (n=100), Serratia marcescens (n=62) and Citrobacter freundii (n=35) were analysed. Genes encoding ESBLs were detected by polymerase chain reaction (PCR) amplification followed by direct sequencing of PCR products. The Clinical and Laboratory Standards Institute confirmatory test detected only 72.1% of the ESBL-producing isolates. When a > or =5mm increase in the zone diameter of either the cefotaxime/Clavulanic acid and/or the ceftazidime/Clavulanic acid disks tested in combination with BA versus cefotaxime and/or ceftazidime containing BA was considered to be a positive for ESBL, the method detected 60 (98.4%) of the 61 isolates that harboured ESBLs and showed no false-positive results for ESBL-non-producing isolates. In conclusion, the BA disk test is a highly sensitive and specific method for the detection of ESBLs in Enterobacteriaceae producing chromosomal AmpC beta-lactamases.

Biotechnol Prog. 2008 Mar-Apr; 24(2): 432-5
Rodrigues LO, Cardoso JP, Menezes JC

The use of near-infrared spectroscopy (NIRS) is demonstrated in the first downstream processing (DSP) steps of an active pharmaceutical ingredient (API) manufacturing process. The first method developed was designed to assess the API content in the filtrate stream (aqueous) of a rotary drum vacuum filter. The PLS method, built after spectral preprocessing and variable selection, had an accuracy of 0.01% (w/w) for an API operational range between 0.20 and 0.45% (w/w). The robustness and extrapolation ability of the calibration was proved when samples from ultrafiltration and nanofiltration processes, ranging from 0 to 2% (w/w), were linearly predicted (R2=0.99). The development of a robust calibration model is generally a very time-consuming task, and once established it is imperative that it can be useful for a long period of time. This work demonstrates that NIR procedures, when carefully developed, can be used in different process conditions and even in different process steps of similar unit operations.

Indian J Med Res. 2008 Jan; 127(1): 85-8
Taneja N, Rao P, Arora J, Dogra A

BACKGROUND & OBJECTIVE: Production of extended spectrum beta-lactamases (ESBLs) and AmpC beta-lactamases are the most common mechanisms of antimicrobial resistance in Gram negative bacilli. A prospective study was undertaken to know the occurrence of ESBL and AmpC producing strains and their antibiotic susceptibilities to newer agents to guide empirical therapy for complicated urinary tract infections. METHODS: Over a period of five months (January to May 2003), organisms grown in pure culture and in significant numbers from urine sample were identified by standard biochemical tests and antibiotic susceptibility determined by disc diffusion method. Gram-negative bacilli that were resistant to third generation cephalosporins, ciprofloxacin and gentamicin/amikacin were defined as highly drug resistant uropathogens (HDRU). HDRU were further tested for ESBL and AmpC phenotypes. RESULTS: Uropathogens were isolated in significant numbers in 1979 (21.8%) of the total 9,072 samples, of which 438(22.1%) were HDRU. Two hundred and five consecutive HDRU isolates were tested for ESBL production and 36.5 per cent were found to be ESBL producers. The highest positivity was found to be in Klebsiella spp. (51.2%), followed by Escherichia coli (40.2%), Enterobacter aerogenes (33.4%) and Pseudomonas aeruginosa (27.9%). Both ESBL producers and non producers showed a high degree resistance to piperacillin (93.1 and 90.9%), amoxycillin-Clavulanic acid (93.4 and 90.9%), aztreonam (79.4 and 78%), cefepime (76.7 and 78%), and ampicillin-sulbactam (76.7 and 70.4%). The most effective antibiotics for ESBL producers were imipenem (8.2% resistance), piperacillin-tazobactam (9.5%) and ceftazidime-Clavulanic acid (23.2%). Among ESBL non-producers, piperacillin-tazobactam (31.06%), ceftazidime-Clavulanic acid (49.2%) and imipenem (11%) were less effective when compared to ESBL producers. Fifty three piperacillin and piperacillin-tazobactam positive and 20 negative isolates were further tested for AmpC production and found that all 53 positive isolates were also positive by for AmpC beta-lactamase. INTERPRETATION & CONCLUSION: Overall, 22.1 per cent of our isolates were highly drug resistant, and ESBL producers could explain only 36.5 per cent of HDRU in our study. Therefore, we assume that AmpC beta-lactamases are more important in our setting. Based on our finding a test using discs containing piperacillin and piperacillin-tazobactam ( PtPc) disc at a distance of 20 mm would act as a useful screening procedure for AmpC production as AmpC beta-lactamase producers are more susceptible to tazobactam as compared to Clavulanic acid.

Microbiology. 2008 Mar; 154(Pt 3): 744-55
Gomez-Escribano JP, Martín JF, Hesketh A, Bibb MJ, Liras P

The (p)ppGpp synthetase gene, relA, of Streptomyces clavuligerus was cloned, sequenced and shown to be located in a genomic region that is highly conserved in other Streptomyces species. relA-disrupted and relA-deleted mutants of S. clavuligerus were constructed, and both were unable to form aerial mycelium or to sporulate, but regained these abilities when complemented with wild-type relA. Neither ppGpp nor pppGpp was detected in the S. clavuligerus relA-deletion mutant. In contrast to another study, Clavulanic acid and cephamycin C production increased markedly in the mutants compared to the wild-type strain; Clavulanic acid production increased three- to fourfold, while that of cephamycin C increased about 2.5-fold. Complementation of the relA-null mutants with wild-type relA decreased antibiotic yields to approximately wild-type levels. Consistent with these observations, transcription of genes involved in Clavulanic acid (ceaS2) or cephamycin C (cefD) production increased dramatically in the relA-deleted mutant when compared to the wild-type strain. These results are entirely consistent with the growth-associated production of both cephamycin C and Clavulanic acid, and demonstrate, apparently for the first time, negative regulation of secondary metabolite biosynthesis by (p)ppGpp in a Streptomyces species of industrial interest.

J Antimicrob Chemother. 2008 Apr; 61(4): 792-7
Pérez-Llarena FJ, Cartelle M, Mallo S, Beceiro A, Pérez A, Villanueva R, Romero A, Bonnet R, Bou G

OBJECTIVES: In order to assess whether or not the Arg-276 of CTX-M-type enzymes is equivalent to the Arg-244 of IRT-TEM-derivative enzymes, we replaced the former with six different amino acids, some of them previously described as involved in resistance to beta-lactamase inhibitors in TEM-IRT derivatives. We also investigated the role of Arg276 in cefotaxime hydrolysis. METHODS: By site-directed mutagenesis and by use of the bla(CTX-M-1) gene as template, Arg-276 was replaced with six different amino acids (Trp, His, Cys, Asn, Gly and Ser). MICs of beta-lactams alone and in combination with beta-lactamase inhibitors were established. The seven enzymes (CTX-M-1 wild-type and six derived mutants) were purified by affinity chromatography, and kinetic parameters (k(cat), K(m), k(cat)/K(m)) towards cefalotin and cefotaxime were determined. Clavulanic acid IC(50) values were also assessed with all enzymes. RESULTS: No increase in MICs of beta-lactam/beta-lactamase inhibitor combination was detected with any of the six CTX-M-1-derived mutants, in agreement with the Clavulanic acid IC(50) values. The MICs of cefotaxime were clearly lower for the Escherichia coli harbouring the Trp, Cys, Ser and Gly CTX-M-1 mutant enzymes than for CTX-M-1, and these enzymes showed a clearly reduced catalytic efficiency towards cefotaxime. As regards cefalotin, there was a moderate reduction in catalytic efficiency for Cys and His. CONCLUSIONS: Arg-276 in CTX-M-type beta-lactamases is not equivalent to Arg-244 in IRT-type enzymes. Position Arg-276 appears to be important for cefotaxime hydrolysis in CTX-M-type enzymes, although different effects were obtained regarding the replaced amino acid.

Indian J Exp Biol. 2007 Dec; 45(12): 1068-72
Gayen JR, Majee SB, Das S, Samanta TB

Search for anti-beta-lactamase and synthesis of newer penicillin were suggested to overcome resistance to penicillin in chemotherapy. It was found that Clavulanic acid, an ant-beta-lactamase was ineffective due to its structural modification by bacteria. Thus, there is a need for the synthesis of newer pencillins. Retro-synthesis was inspired by the success of forward reaction i.e.conversion of penicillin G to 6-aminopenicillanic acid (6-APA) by biological process. In the present study a better enzymatic method of synthesis of newer pencillin by a beta-lactamase-free penicillin amidase produced by Alcaligenes sp. is attempted. Antibacterial and toxicological evaluation of the enzymatically synthesized beta-lactams are reported. Condensation of 6-APA with acyl donor was found to be effective when the reaction is run in dimethyl formamide (DMF 50% v/v) in acetate buffer (25 mM pH 5.0) at 37 degrees C. Periplasm entrapped in calcium alginate exihibited the highest yield (approximately 34%) in synthesis. The minimum inhibitory concentration of the synthetic products against Staphylococcus aureus and Salmonella typhi varied between 20-80 microg/ml. Some of the products exhibited antibacterial activity against enteric pathogens. It was interesting to note that product A was potent like penicillin G. LD50 value of three products (product A, B and C) was more than 12 mg/kg. Furthermore, these synthetic beta-lactams did not exihibit any adverse effect on house keeping enzymes viz., serum glutamate oxalacetate-trans-aminase, serum glutamate pyruvate -trans-aminase, acid phosphatase, alkaline phosphatase of the test animals. The hematological profile (RBC and WBC) of the test animals also remained unaffected.

Antimicrob Agents Chemother. 2008 Apr; 52(4): 1264-8
Meziane-Cherif D, Lambert T, Dupêchez M, Courvalin P, Galimand M

Brachyspira pilosicoli BM4442, isolated from the feces of a patient with diarrhea at the Hospital Saint-Michel in Paris, was resistant to oxacillin (MIC > 256 microg/ml) but remained susceptible to cephalosporins and to the combination of amoxicillin and Clavulanic acid. Cloning and sequencing of the corresponding resistance determinant revealed a coding sequence of 807 bp encoding a new class D beta-lactamase named OXA-63. The bla OXA-63 gene was chromosomally located and not part of a transposon or of an integron. OXA-63 shared 54% identity with FUS-1 (OXA-85), an oxacillinase from Fusobacterium nucleatum subsp. polymorphum, and 25 to 44% identity with other class D beta-lactamases (DBLs) and contained all the conserved structural motifs of DBLs. Escherichia coli carrying the bla OXA-63 gene exhibited resistance to benzylpenicillin and amoxicillin but remained susceptible to amoxicillin in combination with Clavulanic acid. Mature OXA-63 consisted of a 31.5-kDa polypeptide and appeared to be dimeric. Kinetic analysis revealed that OXA-63 exhibited a narrow substrate profile, hydrolyzing oxacillin, benzylpenicillin, and ampicillin with catalytic efficiencies of 980, 250, and 150 mM(-1) s(-1), respectively. The enzyme did not apparently interact with oxyimino-cephalosporins, imipenem, or aztreonam. Unlike FUS-1 and other DBLs, OXA-63 was strongly inhibited by Clavulanic acid (50% inhibitory concentration [IC50] of 2 microM) and tazobactam (IC50 of 0.16 microM) and exhibited low susceptibility to NaCl (IC50 of >2 M). OXA-63 is the first DBL described for the anaerobic spirochete B. pilosicoli.

Biotechnol Bioeng. 2008 Jun 15; 100(3): 439-47
Brethauer S, Held M, Panke S

Product inhibition of biological production systems is a widely observed phenomenon with prominent implications for the design and ultimately the success of biotechnological processes. In order to investigate whether such effects could limit the maximal concentrations in the production of the beta-lactamase inhibitor Clavulanic acid (CA) in Streptomyces clavuligerus cultivations under process-related conditions, we first validated the equivalence of a laboratory scale aerated stirred tank reactor and a medium scale (50 mL) cultivation device, which required optimization of gas transfer in the latter and finally allowed to conduct the required experiments in smaller volumes with correspondingly reduced consumption of compounds. With this, we investigated the effect of CA additions on two global performance parameters: consumption of the carbon source glycerol and oxygen consumption (measured as the oxygen uptake rate, OUR). Increased levels of CA severely interfered with the physiology of the producing strain at least at and above 1.6 g L(-1) CA, as indicated by a dose-dependent decrease in maximal OURs and glycerol consumption rates. As CA is rapidly degraded during cultivation, it was unclear whether CA itself or its decomposition products, a complex mixture containing amongst others several pyrazine derivatives, were responsible for the observed effects. We supplemented S. clavuligerus cultures with mixtures of the decomposition products and found altered OUR and glycerol consumption, but CA production was not influenced. Compared to the drastic effect of CA itself, it is clear that the products of CA degradation exert a much less severe effect on growing S. clavuligerus cultures.

J Antimicrob Chemother. 2008 Feb; 61(2): 309-14
Bhattacharjee A, Sen MR, Prakash P, Anupurba S

OBJECTIVES: To determine the in vitro activity of beta-lactamase inhibitors (Clavulanic acid and sulbactam) in combination with third-generation cephalosporins and monobactam against extended-spectrum beta-lactamase (ESBL)-producing members of the Enterobacteriaceae family. METHODS: A total of 361 ESBL-producing enterobacterial isolates obtained from patients of a university hospital were screened for the status of co-production of AmpC beta-lactamase. These strains were further subjected to an MIC study using third-generation cephalosporins and monobactam, and reductions were observed after combining with beta-lactamase inhibitors at a fixed concentration of 4 mg/L. RESULTS: Most of the isolates showed 8-fold reduction with sulbactam when combined with ceftriaxone, cefpodoxime and cefotaxime but not with ceftazidime and aztreonam, whereas Clavulanic acid showed the same result with all the cephalosporins tested. Further, both the inhibitors showed greater reduced MIC when combined with aztreonam. CONCLUSIONS: As the ability of Clavulanic acid to induce AmpC production may interfere with ESBL detection, sulbactam is likely to be preferred over Clavulanic acid after standardization of an appropriate concentration for ESBL detection in the scenario of increased prevalence of AmpC producers. Greater in vitro activity of these inhibitors when combined with aztreonam further indicates the need of studies to evaluate these combination antimicrobials in clinical settings as they can play a significant role for clinicians as viable alternatives to treat infections caused by such organisms.

J Agric Food Chem. 2008 Jan 23; 56(2): 448-54
Reyns T, De Boever S, De Baere S, De Backer P, Croubels S

A residue depletion study of amoxicillin (AMO) and its major metabolites, amoxicilloic acid (AMA) and amoxicillin diketopiperazine-2',5'-dione, was performed after a single oral (p.o.) and intravenous (i.v.) administration of amoxicillin (20 mg kg (-1)) and amoxicillin/Clavulanic acid (20 and 5 mg kg (-1)) to pigs. Animals were slaughtered 12, 36, 48, 60, 72, and 84 h after dosing. Tissue samples were analyzed using liquid chromatography-tandem mass spectrometry. Kidney samples contained high concentrations of amoxicilloic acid metabolite, which depleted much slower from tissues than amoxicillin, both after p.o. (t1/2AMO = 4.5 h vs t1/2AMA = 8 h) and i.v. (t1/2AMO = 4 h vs t1/2AMA = 8 h) administration. Moreover, after oral administration, significantly higher amoxicilloic acid concentrations were measured in liver and kidney than after i.v. administration. The coadministration of amoxicillin with Clavulanic acid provoked no significant differences in amoxicilloic acid tissue concentrations as compared to an amoxicillin dosing. The prolonged presence of residues of amoxicilloic acid in edible tissues can play an important role in food safety, because the compound could give rise to a possible health risk, although it is not included in the maximum residue limit legislation.

Indian J Med Res. 2007 Nov; 126(5): 417-27
Gupta V

The resistance to beta-lactam antibiotics is an increasing problem worldwide and beta lactamases production is the most common mechanism of drug resistance. Both global and Indian figures showed a marked increase in the number of beta-lactamases producing organisms. These enzymes extended spectrum beta-lactamases (ESBLs) are numerous and continuous mutation has led to the development of enzymes having expanded substrate profile. To date, there are more than 130 TEM type and more than 50 sulphydryl variable (SHV) type beta-lactamases found in Gram negative bacilli. ESBL producing Enterobacteriaceae are, as a rule, resistant to all cephalosporins and extended spectrum penicillins including the monobactam, aztreonam, while resistance to trimethoprim - sulphamethaxazole and aminoglycosides is frequently co-transferred on the same plasmid. Many ESBL producing organisms also express Amp C beta-lactamases. Amp C- beta-lactamases are clinically significant, as these confer resistance to cephalosporins in the oxyimino group, 7 alpha-methoxy cephalosporins, and are poorly inhibited by Clavulanic acid. Carbepenems are the drugs of choice for the treatment of infections caused by ESBL producing organisms but carbapenemases (MBLs) have emerged and have spread from Pseudomonas aeruginosa to Enterobacteriaceae. The routine clinical microbiology laboratories should employ simple methods to recognize these enzymes using various substrates and inhibitors. These organisms may lead to therapeutic dead ends. Presently, the therapy relies on beta-lactam/ beta-lactamases inhibitor combinations, carbepenems and piperacillin - tazobactam plus aminoglycoside combination. Proper infection control practices and barrier precautions are essential to contain the organisms producing beta-lactamases.

Clin Microbiol Infect. 2008 Jan; 14 Suppl 1: 189-93
Livermore DM, Hope R, Mushtaq S, Warner M

Clavulanate is a highly effective inhibitor of extended-spectrum beta-lactamases (ESBLs) in detection tests, but the commercial amoxycillin-clavulanate and ticarcillin-clavulanate combinations have borderline activity, at best, against most ESBL producers. Oxyimino-cephalosporin-clavulanate combinations are active in vitro against most ESBL-producing Escherichia coli and Klebsiella spp. isolates at < or =1-2 mg/L but are compromised against Enterobacter spp., whether ESBL-producing or not, where clavulanate-induced AmpC enzymes attack the cephalosporin. These problems can be overcome by combining clavulanate with cefepime or cefpirome, which are more stable to AmpC. The resulting combinations are active in vitro at < or =1 mg/L against virtually all ESBL-producing Enterobacteriaceae, including Enterobacter spp. AmpC-inducible organisms, such as Enterobacter, are less of a concern in the community, where ESBL-producing E. coli strains present growing problems, and where new oral treatments would be useful. Cefpodoxime-clavulanate is not ideal, in terms of pharmacological matching, but might be fit for purpose, certainly in comparison with fosfomycin and nitrofurantoin, which are used at present but which are suitable only for lower urinary tract infections. Clinical development of clavulanate with cefepime, cefpirome or cefpodoxime does not seem likely in the West, considering ownership and patent issues. Cefpisome-tazobactum is, however, being launched in India, where the licensing regime is more liberal. Combinations of clavulanate with modern anti-methicillin-resistant Staphylococcus aureus cephalosporins also deserve investigation, as these compounds remain labile to ESBLs.

Clin Microbiol Infect. 2008 Jan; 14 Suppl 1: 90-103
Drieux L, Brossier F, Sougakoff W, Jarlier V

Strains of Enterobacteriaceae producing an extended spectrum beta-lactamase have become a concern in medical bacteriology as regards both antimicrobial treatment and infection control in hospitals. Extended-spectrum beta-lactamase (ESBL) detection tests should accurately discriminate between bacteria producing these enzymes and those with other mechanisms of resistance to beta-lactams, e.g., broad-spectrum beta-lactamases, inhibitor-resistant beta-lactamases and cephalosporinase overproduction. Several phenotypic detection tests, based on the synergy between a third-generation cephalosporin and clavulanate, have been designed: the double-disk synergy test (DDST), ESBL Etests, and the combination disk method. These tests often need to be refined in order for them to detect an ESBL in some bacterial strains, such as those that also overproduce a cephalosporinase. The sensitivity of the DDST can be improved by reducing the distance between the disks of cephalosporins and clavulanate. The use of cefepime, a fourth-generation cephalosporin that is less rapidly inactivated by cephalosporinase than by ESBL, improves the detection of synergy with clavulanate when there is simultaneous stable hyperproduction of a cephalosporinase; alternatively, the cephalosporinase can be inactivated by performing phenotypic tests on a cloxacillin-containing agar. Some beta-lactamases can hydrolyse both third-generation cephalosporins and carbapenems, such as the metallo-beta-lactamases, which are not inhibited by clavulanate, but rather by EDTA. The production of an ESBL masked by a metallo-beta-lactamase can be detected by means of double inhibition by EDTA and clavulanate. Since extended-spectrum Ambler class D oxacillinases are weakly inhibited by clavulanate and not inhibited by EDTA, their detection is difficult in the routine laboratory.

Antimicrob Agents Chemother. 2008 Feb; 52(2): 551-6
Meziane-Cherif D, Decré D, Høiby EA, Courvalin P, Périchon B

Carnobacterium divergens clinical isolates BM4489 and BM4490 were resistant to penicillins but remained susceptible to combinations of amoxicillin-Clavulanic acid and piperacillin-tazobactam. Cloning and sequencing of the responsible determinant from BM4489 revealed a coding sequence of 912 bp encoding a class A beta-lactamase named CAD-1. The bla(CAD-1) gene was assigned to a chromosomal location in the two strains that had distinct pulsed-field gel electrophoresis patterns. CAD-1 shared 53% and 42% identity with beta-lactamases from Bacillus cereus and Staphylococcus aureus, respectively. Alignment of CAD-1 with other class A beta-lactamases indicated the presence of 25 out of the 26 isofunctional amino acids in class A beta-lactamases. Escherichia coli harboring bla(CAD-1) exhibited resistance to penams (benzylpenicillin and amoxicillin) and remained susceptible to amoxicillin in combination with Clavulanic acid. Mature CAD-1 consisted of a 34.4-kDa polypeptide. Kinetic analysis indicated that CAD-1 exhibited a narrow substrate profile, hydrolyzing benzylpenicillin, ampicillin, and piperacillin with catalytic efficiencies of 6,600, 3,200, and 2,900 mM(-1) s(-1), respectively. The enzyme did not interact with oxyiminocephalosporins, imipenem, or aztreonam. CAD-1 was inhibited by tazobactam (50% inhibitory concentration [IC(50)] = 0.27 microM), Clavulanic acid (IC(50) = 4.7 microM), and sulbactam (IC(50) = 43.5 microM). The bla(CAD-1) gene is likely to have been acquired by BM4489 and BM4490 as part of a mobile genetic element, since it was not found in the susceptible type strain CIP 101029 and was adjacent to a gene for a resolvase.

Chemotherapy. 2008; 54(1): 31-7
Zhao WH, Hu ZQ, Chen G, Ito R, Shimamura T

BACKGROUND: Some clinical isolates of Serratia marcescens showed high-level resistance to expanded-spectrum cephalosporins alone and in combination with beta-lactamase inhibitor, but the beta-lactamase extracted from these strains was sensitive to the inhibitors. This study examines the possible mechanisms responsible for the discrepancy. METHODS AND RESULTS: Three clinical isolates of S. marcescens bore the bla(TEM) gene coded on a plasmid. This was confirmed by detection of the bla gene using PCR analysis and by transferring resistance determinants to Escherichia coli via conjugation and transformation. All of the E. coli transconjugants and transformants acquired a similar level of resistance to penicillins and narrow-spectrum cephalosporins, and showed the reduced susceptibility to expanded-spectrum cephalosporins alone and in combination with clavulanate. As a result of the highly constitutive expression of the TEM gene, up to 247-690 U of beta-lactamase were produced by 10(10) cells of wild-type S. marcescens and the transconjugants and transformants. In the presence of 0.24 U/ml of TEM enzyme, the minimum inhibition concentrations of cefotaxime against E. coli ATCC 25922 increased from 0.125 to 512 microg/ml. The TEM-type beta-lactamase extracted from these strains was sensitive to clavulanate, and 62.2-92.1% of its activity was inhibited after preincubation with 0.1 mM clavulanate. CONCLUSION: The TEM-type beta-lactamase plays a critical role in the resistance of S. marcescens to beta-lactams, and the hyperproduction of inhibitor-susceptible TEM beta-lactamase is responsible for the resistance to beta-lactam-beta-lactamase inhibitor combinations.

J Microbiol Biotechnol. 2007 Sep; 17(9): 1538-45
Hung TV, Malla S, Park BC, Liou K, Lee HC, Sohng JK

Clavulanic acid (CA) is an inhibitor of beta-lactamase that is produced from Streptomyces clavuligerus NRRL3585 and is used in combination with other antibiotics in clinical treatments. In order to increase the production of CA, the replicative and integrative expressions of ccaR (encoding for a specific regulator of the CA biosynthetic operon) and cas2 (encoding for the rate-limiting enzyme in the CA biosynthetic pathway) were applied. Six recombinant plasmids were designed for this study. The pIBRHL1, pIBRHL3, and pIBRHL13 were constructed for overexpression, whereas pNQ3, pNQ2, and pNQ1 were constructed for chromosomal integration with ccaR, cas2, and ccaR-cas2, respectively. All of these plasmids were transformed into S. clavuligerus NRRL3585. CA production in transformants resulted in a significantly enhanced amount greater than that of the wild type, a 2.25-fold increase with pIBRHL1, a 9.28-fold increase with pNQ3, a 5.06-fold increase with pIBRHL3, a 2.93-fold increase with pNQ2 integration, a 5.79-fold increase with pIBRHL13, and a 23.8-fold increase with pNQ1. The integrative pNQ1 strain has been successfully applied to enhance production.