SHV-1 Beta-lactamase Research Articles

Tazobactam Inactivation of SHV-1 and the Inhibitor-resistant Ser130 → Gly SHV-1 β-Lactamase: INSIGHTS INTO THE MECHANISM OF INHIBITION

The increasing number of bacteria resistant to combinations of beta-lactam and beta-lactamase inhibitors is creating great difficulties in the treatment of serious hospital-acquired infections. Understanding the mechanisms and structural basis for the inactivation of these inhibitor-resistant beta-lactamases provides a rationale for the design of novel compounds. In the present work, SHV-1 and the Ser(130) --> Gly inhibitor-resistant variant of SHV-1 beta-lactamase were inactivated with tazobactam, a potent class A beta-lactamase inhibitor. Apoenzymes and inhibited beta-lactamases were analyzed by liquid chromatography-electrospray ionization mass spectrometry (LC-ESI/MS), digested with trypsin, and the products resolved using LC-ESI/MS and matrix-assisted laser desorption ionization-time of flight mass spectrometry. The mass increases observed for SHV-1 and Ser(130) --> Gly (+ Delta 88 Da and + Delta 70 Da, respectively) suggest that fragmentation of tazobactam readily occurs in the inhibitor-resistant variant to yield an inactive beta-lactamase. These two mass increments are consistent with the formation of an aldehyde (+ Delta 70 Da) and a hydrated aldehyde (+ Delta 88 Da) as stable products of inhibition. Our results reveal that the Ser --> Gly substitution at amino acid position 130 is not essential for enzyme inactivation. By examining the inhibitor-resistant Ser(130) --> Gly beta-lactamase, our data are the first to show that tazobactam undergoes fragmentation while still attached to the active site Ser(70) in this enzyme. After acylation of tazobactam by Ser(130) --> Gly, inactivation proceeds independent of any additional covalent interactions.

Mechanisms of reduced susceptibility to amoxycillin-clavulanic acid in Escherichia coli strains from the health region of Tortosa (Catalonia, Spain)

This study investigated the mechanisms involved in reduced susceptibility to amoxycillin-clavulanic acid and the prevalence of enzymes compatible with inhibitor-resistant TEM (IRT) beta-lactamases produced by Escherichia coli isolates from patients in north-eastern Spain. The resistance mechanisms of 158 strains showing resistance or intermediate resistance to amoxycillin-clavulanic acid among 1122 ampicillin-resistant clinical isolates of E. coli were assessed on the basis of their beta-lactam resistance phenotypes. beta-Lactamases produced by strains showing resistant phenotypes suggestive of inhibitor-resistant penicillinase production were characterised by their isoelectric point. Specific activity and the concentration of clavulanic acid required to inhibit beta-lactamase activity by 50% (IC50) were determined in strains harbouring enzymes that focused at pI 5.2 or 5.4 in order to achieve presumptive identification of IRT beta-lactamases. Resistance phenotypes were consistent with overproduction of TEM-1, TEM-2 or SHV-1 beta-lactamases in 56 strains, with AmpC cephalosporinase hyperproduction in 46 strains, and with production of inhibitor-resistant penicillinases in 49 strains. Of the latter isolates, 17 produced moderately high or high levels of enzymes co-focusing with TEM-1, 17 produced enzymes co-focusing with OXA-1 (n = 12) or with PSE-1 (n = 5), either alone or in association with TEM-1, while only 15 produced enzymes with a phenotype characteristic of IRT beta-lactamases. It was concluded that resistance to amoxycillin-clavulanic acid in E. coli isolates from this area was mainly associated with presumptive overproduction of TEM-1, TEM-2 or SHV-1 beta-lactamases (46%) or of AmpC cephalosporinase (29%), while the occurrence of enzymes categorised as IRT beta-lactamases was unusual (9.5%).

Tazobactam forms a stoichiometric trans-enamine intermediate in the E166A variant of SHV-1 beta-lactamase: 1.63 A crystal structure.

Many pathogenic bacteria develop antibiotic resistance by utilizing beta-lactamases to degrade penicillin-like antibiotics. A commonly prescribed mechanism-based inhibitor of beta-lactamases is tazobactam, which can function either irreversibly or in a transient manner. We have demonstrated previously that the reaction between tazobactam and a deacylation deficient variant of SHV-1 beta-lactamase, E166A, could be followed in single crystals using Raman microscopy [Helfand, M. S., et al. (2003) Biochemistry 42, 13386-13392]. The Raman data show that maximal populations of an enamine-like intermediate occur 20-30 min after "soaking in" has commenced. By flash-freezing crystals in this time frame, we were able to trap the enamine species. The resulting 1.63 A resolution crystal structure revealed tazobactam covalently bound in the trans-enamine intermediate state with close to 100% occupancy in the active site. The Raman data also indicated that tazobactam forms a larger population of enamine than sulbactam or clavulanic acid does and that tazobactam's intermediate is also the most long-lived. The crystal structure provides a rationale for this finding since only tazobactam is able to form favorable intra- and intermolecular interactions in the active site that stabilize this trans-enamine intermediate. These interactions involve both the sulfone and triazolyl groups that distinguish tazobactam from clavulanic acid and sulbactam, respectively. The observed stabilization of the transient intermediate of tazobactam is thought to contribute to tazobactam's superior in vitro and in vivo clinical efficacy. Understanding the structural details of differing inhibitor effectiveness can aid the design of improved mechanism-based beta-lactamase inhibitors.

Clonal and horizontal dissemination of Klebsiella pneumoniae expressing SHV-5 extended-spectrum beta-lactamase in a Mexican pediatric hospital.

One hundred eighty-four clinical isolates of Klebsiella pneumoniae were recovered from August 1996 to October 1997 at the Pediatric Hospital of the Instituto Mexicano del Seguro Social in Mexico City, Mexico. Most of the isolates were collected from the neonatal intensive care unit and infant wards, which are located on the same floor of the hospital. Isolates were genotypically compared by pulsed-field gel electrophoresis with XbaI restriction of chromosomal DNA. Of 184 clinical isolates, 91 belonged to cluster A and comprised three subtypes (A1, A2, and A3), while 93 isolates, comprising two minor clones, B (10 isolates) and C (7 isolates), and 76 unique patterns, were considered unrelated isolates (URI). Susceptibility patterns were indistinguishable in both groups. Fifty extended-spectrum beta-lactamase-producing isolates, including 34 from clone A and 16 from URI, were examined for further studies. Molecular and genetic analysis showed that 47 of 50 clinical isolates expressed the SHV-5 beta-lactamase. This enzyme, in combination with TEM-1, was encoded in a >or=170-kb conjugative plasmid. Results indicate that dissemination of this resistance was due to clonal and horizontal spread.

Understanding Resistance to β-Lactams and β-Lactamase Inhibitors in the SHV β-Lactamase: LESSONS FROM THE MUTAGENESIS OF SER-130

Bacterial resistance to beta-lactam/beta-lactamase inhibitor combinations by single amino acid mutations in class A beta-lactamases threatens our most potent clinical antibiotics. In TEM-1 and SHV-1, the common class A beta-lactamases, alterations at Ser-130 confer resistance to inactivation by the beta-lactamase inhibitors, clavulanic acid, and tazobactam. By using site-saturation mutagenesis, we sought to determine the amino acid substitutions at Ser-130 in SHV-1 beta-lactamase that result in resistance to these inhibitors. Antibiotic susceptibility testing revealed that ampicillin and ampicillin/clavulanic acid resistance was observed only for the S130G beta-lactamase expressed in Escherichia coli. Kinetic analysis of the S130G beta-lactamase demonstrated a significant elevation in apparent Km and a reduction in kcat/Km for ampicillin. Marked increases in the dissociation constant for the preacylation complex, KI, of clavulanic acid (SHV-1, 0.14 microm; S130G, 46.5 microm) and tazobactam (SHV-1, 0.07 microm; S130G, 4.2 microm) were observed. In contrast, the k(inact)s of S130G and SHV-1 differed by only 17% for clavulanic acid and 40% for tazobactam. Progressive inactivation studies showed that the inhibitor to enzyme ratios required to inactivate SHV-1 and S130G were similar. Our observations demonstrate that enzymatic activity is preserved despite amino acid substitutions that significantly alter the apparent affinity of the active site for beta-lactams and beta-lactamase inhibitors. These results underscore the mechanistic versatility of class A beta-lactamases and have implications for the design of novel beta-lactamase inhibitors.

Following the reactions of mechanism-based inhibitors with beta-lactamase by Raman crystallography.

The reactions between three clinically relevant inhibitors, tazobactam, sulbactam, and clavulanic acid, and SHV beta-lactamase (EC 3.5.2.6) have been followed in single crystals using a Raman microscope. The data are far superior to those obtained for the enzyme in aqueous solution and allow us to identify species on the reaction pathway and to measure the rates of the accumulation and decay of these species. A key intermediate on the reaction pathway is an acyl enzyme formed between Ser70 and the lactam ring's C=O group. By using the E166A deacylation deficient variant of the enzyme, we were able to focus on the process of acyl enzyme formation. The Raman data show that all three inhibitors form an enamine-type acyl enzyme reaching maximal populations at 10, 22, and 29 min for sulbactam, clavulanic acid, and tazobactam, respectively. The enamine intermediate exhibits a characteristic and relatively intense band near 1595 cm(-1) due to a stretching motion of the O=C-C=C-NH moiety that shifts to lower frequency upon NH <--> ND exchange. This feature was used to follow the kinetics of enamine buildup and decay in the crystal. Quantum mechanical calculations support the assignment of the 1595 cm(-1) band, as well as several other bands, to a trans-enamine species. The Raman data also demonstrate that the lactam ring opens prior to enamine formation since the lactam ring carbonyl (C=O) peak disappears prior to the appearance of the enamine 1595 cm(-1) band. Tazobactam appears to form approximately twice as much enamine intermediate as sulbactam and clavulanic acid, which correlates with its superior performance in the clinic, a finding that may bear on future drug design.

Multiple antibiotic-resistance mechanisms including a novel combination of extended-spectrum beta-lactamases in a Klebsiella pneumoniae clinical strain isolated in Argentina.

Klebsiella pneumoniae M1803, isolated from a paediatric patient with chronic urinary infection, presented nine antimicrobial resistance mechanisms harboured on two conjugative megaplasmids, in addition to the chromosomally mediated SHV-1 beta-lactamase. These nine antimicrobial resistance mechanisms comprised two extended-spectrum beta-lactamases (ESBLs) (PER-2 and CTX-M-2), TEM-1-like, OXA-9-like, AAC(3)-IIa, AAC(6')-Ib, ANT(3")-Ia and resistance determinants to tetracycline and chloramphenicol. During fluoroquinolone treatment, a variant derived from M1803 (named M1826) was selected, with an overall increase of MICs, in particular of cefoxitin and carbapenems. No enzymic activity against these latter drugs was found. Mutations in the region analogous to the quinolone resistance-determining region were not found. Strain M1826 was deficient in OmpK35/36 expression, which produced the decrease in the susceptibility to cefoxitin, carbapenems and fluoroquinolones. The blaCTX-M-2 gene was located in an unusual class 1 integron, which includes Orf513, as occurred in the recently described In35. In addition, Tn3 and Tn1331 were detected in both K. pneumoniae isolates. This is the first report of in vivo selection of an OmpK35/36 deficiency in a K. pneumoniae strain that produced a novel combination of two ESBLs (CTX-M-2 and PER-2) during fluoroquinolone treatment in a paediatric patient with chronic urinary infection.

Beta-lactamase characterization in Escherichia coli isolates with diminished susceptibility or resistance to extended-spectrum cephalosporins recovered from sick animals in Spain.

A total of 1439 Escherichia coli isolates from sick animals were received from the Spanish Network of Veterinary Antimicrobial Resistance Surveillance (VAV) from 1997 to 2001. Antimicrobial susceptibility tests were performed and diminished susceptibility to cefotaxime and ceftazidime was identified in 2.5% and 2.8% of the isolates, respectively. Beta-lactamase characterization was carried out in the group of 20 E. coli isolates with both characteristics. The MIC ranges of different beta-lactams showed by these 20 isolates were as follows (in microg/ml): ampicillin (64-->256), amoxicillin-clavulanic acid (4-64), ticarcillin (8-->128), cefazolin (32-->256), cefoxitin (4-->128), cefotaxime (1-64), ceftazidime (2-->64), ceftriaxone (0.5-64), imipenem (< or = 0.06-0.25), and aztreonam (2-->32). TEM, SHV, CMY, and FOX beta-lactamase genes were analyzed by PCR and sequencing. The beta-lactamase genes detected were the following ones (number of isolates): bla(TEM-1b) (3), bla(TEM-1a) (1), bla(TEM-30f) (2), bla(TEM-1b) + bla(CMY-2) (2), and bla(SHV-12) (1). Sequences of the promoter and/or attenuator region of the chromosomal ampC gene were studied in all the 20 isolates. Mutations at position -42 or -32 were detected in 16 isolates and these mutations were associated with the presence of a TEM type beta-lactamase in 6 isolates. Besides, a high variety of plasmidic beta-lactamases was detected including TEM-30 and CMY-2. To our knowledge, this is the first time that TEM-30 beta-lactamase has been detected in E. coli isolates of animal origin.

Unexpected Advanced Generation Cephalosporinase Activity of the M69F Variant of SHV β-Lactamase

Infections with bacteria that contain hydrolytic beta-lactamase enzymes are becoming a serious problem in the United States. Mutations at Met-69, an amino acid proximal to the active site Ser-70 in the TEM-1 and SHV-1 beta-lactamases, have emerged as a puzzling cause of bacterial resistance to inhibitors of beta-lactamases. Site-saturation mutagenesis of the 69 position in SHV beta-lactamase was performed to determine how mutations of this non-catalytic residue play a role in increasing 50% inhibitory concentrations (IC(50) concentrations) for clinically important beta-lactamase enzyme inhibitors. Two distinct phenotypes are evident in the variant beta-lactamases studied: significantly increased minimum inhibitory concentrations (microg/ml) and IC(50) concentrations to clavulanic acid for the Met69Ile, Leu, and Val substitutions, and unanticipated increased minimum inhibitory concentrations and hydrolytic activity toward ceftazidime, an advanced generation cephalosporin antibiotic, for the Met69Lys, Tyr- and Phe-substituted enzymes. Molecular modeling studies emphasize the conserved structure of these substitutions despite great variation in substrate specificity. This study demonstrates the key role of Met-69 in defining substrate specificity of SHV beta-lactamases and alerts us to new phenotypes that may emerge clinically.

SHV-12, SHV-5, SHV-2a and VEB-1 extended-spectrum beta-lactamases in Gram-negative bacteria isolated in a university hospital in Thailand.

Sixty-one extended-spectrum beta-lactamase (ESBL)-producing isolates were collected from Srinagarind Hospital, Thailand. These included 43 Enterobacteriaceae and 18 Pseudomonadaceae. The 43 Enterobacteriaceae were found to produce the following ESBLs: 26 (60.5%) SHV-12, 13 (30.2%) SHV-5, two (4.7%) SHV-2a, one (2.3%) VEB-1 and one (2.3%) unidentified. Twenty-four isolates (55.8%) also carried bla(TEM-1B), as well as bla(SHV) or bla(VEB-1). Plasmid DNA from transconjugants carrying the bla(SHV-12) gene showed various restriction patterns, indicating the distribution of the bla(SHV-12) gene among different antibiotic resistance plasmids. In contrast, bla(SHV-5) in 13 isolates was found on a single plasmid of c. 130 kb. Pulsed-field gel electrophoresis (PFGE) analysis of genomic DNA from these isolates revealed that nine of 11 Klebsiella pneumoniae gave the same pattern, indicating clonal spread of the strain within the hospital, together with the occasional spread of the plasmid to other strains. Among the pseudomonad isolates, 16 Pseudomonas aeruginosa and one Pseudomonas putida had bla(VEB-like) and one P. aeruginosa had bla(SHV-12). Nine of the 16 isolates carrying bla(VEB-like) (56.3%) had identical PFGE patterns, suggesting the dissemination of this gene, also by clonal spread. At least six different bla(VEB-like-)containing integrons were found among the 18 isolates. This is the first report of bacteria producing SHV-12 and SHV-2a in Thailand and the first report of SHV-12 in P. aeruginosa, of VEB-1 in Citrobacter freundii and a VEB-1-like beta-lactamase in P. putida. These findings indicate that ESBL genes in the Far East are part of a gene pool capable of broad horizontal gene transfer, in that these genes can transfer between different families of Gram-negative bacilli.

Effect of inoculum size on the antibacterial activity of cefpirome and cefepime against Klebsiella pneumoniae strains producing SHV extended-spectrum β-lactamases

To determine the effects of varying inoculum size on in vitro susceptibility of SHV extended-spectrum beta-lactamase (ESBL)-producing Klebsiella pneumoniae isolates to cefepime and cefpirome compared to previously established cephalosporins and aztreonam. Antibiotic susceptibilities were determined by disk diffusion test, the MIC broth microdilution method, and time-kill studies with two different inocula of 10(5) and 10(7) CFU/mL. The strains were classified into four groups according to the type of beta-lactamase they produce: SHV-2, SHV-5, SHV-12, and ESBL-negative klebsiellae. The antibacterial activities of cefpirome and cefepime were comparable to that of cefotaxime, but were significantly greater than those of ceftazidime and aztreonam. An inoculum effect was detected for all broad-spectrum cephalosporins, but it was more pronounced with cefpirome and cefepime compared to older cephalosporins. The disk diffusion test proved to be not sensitive enough for the detection of an inoculum effect, particularly for cefepime. The present study found that most SHV-producing klebsiellae have MICs of cefpirome that imply susceptibility at the moderate inoculum size, in spite of the fact that, according to the NCCLS, all ESBL producers should be considered resistant to all cephalosporins, independent of MIC values. With a high inoculum, most of the strains seemed to be resistant to both antibiotics. Furthermore, the bactericidal activities of cefpirome and cefepime against isogenic Escherichia coli strains producing SHV-2, SHV-4 and SHV-5 beta-lactamases, respectively, were also inoculum dependent. Bactericidal activity against SHV-4 and SHV-5 beta-lactamase producers was obtained only at the moderate inoculum, whereas the SHV-2 beta-lactamase producer was efficiently killed with both antibiotics, regardless of the inoculum size.

SHV-1 beta-lactamase is mainly a chromosomally encoded species-specific enzyme in Klebsiella pneumoniae.

The nature of the SHV-1 beta-lactamase gene was analyzed in 97 epidemiologically unrelated Klebsiella pneumoniae strains isolated from clinical samples. beta-Lactamase bands that focused at a pI of 7.6 (SHV-1-type) in 74 strains, at a pI of 7.1 (LEN-1-type) in 13 strains, and at a pI of 5.4 (TEM-1-type) in 10 strains were detected by analytical isoelectric focusing (IEF). Among the 74 SHV-1-producing strains, 40 had, in addition to the pI 7.6 band, an additional band on IEF: 20 had a band with a pI of 7.1 and 20 had a band with a pI of 5.4. Most of the 74 SHV-1-producing strains (76.7%) carried plasmids. Transfer of beta-lactam resistance by conjugation was possible in only 9.3% of the strains tested. SHV-1 gene-specific PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of the chromosomal DNA was positive for 93 of the 97 strains and negative for only 4 of the 10 samples with K. pneumoniae TEM-1 producers. In an attempt to approximate the location of the SHV gene locus by endonuclease restriction analysis, RFLP analysis with Southern blotting of chromosomal DNA with a labeled SHV-1 fragment as a probe was used to study the 97 strains. A trial with EcoRI showed at least one positive hybridization band for 96 strains; two bands were detected for 8 strains. The hybridization was negative for only one TEM-1 beta-lactamase-producing strain. DNA sequence analysis showed no differences in promoter regions or extra stop-triplet sequences; only point mutations determined different allelic variants. The novel SHV-type variants are designated SHV-32 and SHV-33. As a result of the RFLP and sequencing analyses, it can be postulated that the loci for SHV-1 and LEN-1 genes are arranged in tandem. Our results strongly support the hypothesis that the ancestor of the SHV-1 beta-lactamase originated from the K. pneumoniae chromosome.

Diversity of SHV and TEM beta-lactamases in Klebsiella pneumoniae: gene evolution in Northern Taiwan and two novel beta-lactamases, SHV-25 and SHV-26.

A total of 113 blood culture isolates of Klebsiella pneumoniae from 10 hospitals in northern Taiwan were studied for SHV and TEM beta-lactamase production. bla(SHV) was amplified from all isolates by PCR. TEM-type resistance, was found in 32 of the isolates and was of the TEM-1 type in all isolates. SHV-1, -2, -5, -11, and -12 and two novel enzymes were identified. These novel enzymes were designated SHV-25 and SHV-26 and had pIs of 7.5 and 7.6, respectively. Amino acid differences in comparison to the amino acid sequence of bla(SHV-1) were found at positions T18A (ThrACC-->AlaGCC), L35Q (LeuCTA-->GluCAA), and M129V (MetATG-->ValGTG) for SHV-25 and at position A187T (AlaGCC-->ThrACC) for SHV-26. The results of substrate profiles and MIC determinations showed that the novel enzymes did not hydrolyze extended-spectrum cephalosporins, rendering the isolates susceptible to these agents. Inhibition profiles revealed that the 50% inhibitory concentration for SHV-26 was higher than those for SHV-1 and SHV-25, resulting in an intermediate resistance to amoxicillin-clavulanic acid. Forty-nine ribotypes were identified, suggesting that major clonal spread had not occurred in any of the hospitals. According to the amino acid sequence, SHV beta-lactamases in Taiwan may basically be derived through stepwise mutation from SHV-1 or SHV-11 and further subdivided by four routes. The stepwise mutations initiated from SHV-1 or SHV-11 to SHV-2, SHV-5, and SHV-12 comprise the evolutionary change responsible for extended-spectrum beta-lactamase (ESBL) production in Taiwan. The stepwise mutations that lead to a non-ESBL (SHV-25) and the beta-lactamase (SHV-26) with reduced susceptibility to clavulanic acid are possibly derived from SHV-11 and SHV-1, respectively. The results suggest a stepwise evolution of SHV beta-lactamases in Taiwan.

Outer membrane protein change combined with co-existing TEM-1 and SHV-1 beta-lactamases lead to false identification of ESBL-producing Klebsiella pneumoniae.

Nine isolates of Klebsiella pneumoniae, obtained from one colonized and eight bacteraemic patients on a paediatric ward, were shown to be identical by PFGE, indicating an outbreak. Screening for extended-spectrum beta-lactamase (ESBL) production using the double-disc synergy test, Etest for ESBLs and agar diffusion tests indicated ESBL production. The isolates showed reduced susceptibility to cefotaxime but not to other third-generation cephalosporins. Molecular studies revealed production of TEM-1 and SHV-1 but no ESBLs were identified. Deficiency in expression of an outer membrane protein (OmpK35) was also observed. These observations led us to postulate that the extremely low level of OmpK35 expression and the co-existence of TEM-1 and SHV-1 resulted in an increased MIC of cefotaxime and the false designation of the isolates as ESBL producers. All the infected infants were treated with either third-generation cephalosporins alone or multiple antibiotics including a third-generation cephalosporin, and recovered and were discharged without sequelae.

Amino acid substitutions causing inhibitor resistance in TEM beta-lactamases compromise the extended-spectrum phenotype in SHV extended-spectrum beta-lactamases.

Three amino acid substitutions, Met-69-->Ile, Arg-244-->Ser and/or Asn-276-->Asp, mediate inhibitor resistance (IR) in TEM beta-lactamases. They were introduced in all possible combinations at homologous positions into either SHV-1 or the respective extended-spectrum beta-lactamases (ESBLs), SHV-2 or SHV-5. Susceptibility testing of the resulting set of seven variants of each parental strain, all in an isogenic background, was performed. The phenotypes of the constructions revealed that most substitutions resulted in reduced resistance to most tested single beta-lactam formulations. This decrease over-compensated for the expected increase in inhibitor resistance, so that most mutants showed no rise in resistance to inhibitor/beta-lactam combinations, although increases of MICs from one- to 43-fold compared with the respective parental strains were also measured. Combination of several IR-determining substitutions impaired both phenotypes in the carrier strains even more. None of the 14 mutants derived from the ESBLs, SHV-2 and SHV-5, showed a clinically relevant combined ESBL-IR phenotype. These findings indicate that the SHV beta-lactamase does not benefit proportionally from simultaneous substitution of residues relevant for the ESBL and the IR phenotype.

Extended-spectrum TEM- and SHV-type beta-lactamase-producing Klebsiella pneumoniae strains causing outbreaks in intensive care units in Italy.

The aim of the present study was to investigate the production of extended-spectrum beta-lactamases (ESbetaLs) and the epidemiological correlations in a total of 107 Klebsiella pneumoniae strains resistant to third- and fourth-generation cephalosporins. The strains were collected from patients in four intensive care units (3 neonatal and 1 general) in three hospitals in Italy between March 1996 and July 1997. All strains were found to produce ESbetaLs. Phenotypic (antibiotyping and ESbetaL patterns) and genotypic (plasmid profile and pulsed-field gel electrophoresis) analyses showed that a single strain had been responsible for each outbreak in each of the four intensive care units. Isoelectric focusing, activity on substrates and gene sequencing showed that the strains produced SHV-5, SHV-2a, SHV-12 and TEM-52 beta-lactamases. This is not only the first time that ESbetaL-producing Klebsiella pneumoniae strains have been reported as causing epidemics in Italian hospitals, it is also, to the best of our knowledge, the first time that an outbreak caused by a TEM-52 ESbetaL-producing Klebsiella pneumoniae strain has been reported. The data presented here illustrate the complexity of determining the epidemiological pattern of ESbetaL producers in large hospitals that do not have an ESbetaL-monitoring program.

In vitro activities of parenteral beta-lactam antimicrobials against TEM-10-, TEM-26- and SHV-5-derived extended-spectrum beta-lactamases expressed in an isogenic Escherichia coli host.

The in vitro activities were determined and time-kill studies of cefepime, imipenem-cilastatin, meropenem and piperacillin-tazobactam were performed against SHVand TEM-derived extended-spectrum beta-lactamases (ESBLs). Sequence-confirmed SHV-5, TEM-10 and TEM-26 beta-lactamases were transferred into Escherichia coli C600N by conjugation. Imipenem and meropenem were more active (MIC range 0. 0625-0.25 mg/L) than cefepime (MIC range 2-8 mg/L) and piperacillin-tazobactam (MIC range 8-32 mg/L). Regrowth of strains expressing TEM-10 and TEM-26 was noted at all cefepime and piperacillin-tazobactam concentrations studied. Imipenem-cilastatin and meropenem demonstrated rapid, sustained bactericidal activity uninfluenced by the type of ESBL expressed.

Characterisation of extended-spectrum beta-lactamases of the SHV family using a combination of PCR-single strand conformational polymorphism (PCR-SSCP) and PCR-restriction fragment length polymorphism (PCR-RFLP).

Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) has been developed to extend the identification of SHV beta-lactamases previously characterised by PCR-single strand conformational polymorphism (PCR-SSCP) analysis alone. Eight bacteria, each producing a different SHV beta-lactamase, were used in this study. These bacteria harbour bla(SHV-1), bla(SHV-2a), bla(SHV-3), bla(SHV-4), bla(SHV-5) (two strains), bla(SHV-11) and bla(SHV-12). All isolates were characterised by PCR-SSCP and PCR-RFLP with DdeI and NheI digestion. By a combination of these techniques, the genes encoding these beta-lactamases could be differentiated from each other. In addition, the PCR-RFLP technique theoretically can be applied to distinguish the genes encoding SHV-7, SHV-9, SHV-10, SHV-15, SHV-17 and SHV-24 from those encoding other SHV variants. We report a simple PCR-RFLP technique that can be used in epidemiological studies to enable the rapid characterisation of known SHV beta-lactamases in a combination with the previously published PCR-SSCP analysis.

Evolution and spread of SHV extended-spectrum beta-lactamases in gram-negative bacteria.

Resistance to beta-lactam antibiotics has been a problem for as long as these drugs have been used in clinical practice. In clinically significant bacteria the most important mechanism of resistance is the production of one or more beta-lactamases, enzymes that hydrolyse the beta-lactam bond characteristic of this family of antibiotics. Prominent among the beta-lactamases produced by the Enterobacteriaceae is the SHV family. The first reported SHV beta-lactamase had a narrow spectrum of activity. By the accumulation of point mutations at sites that affect the active site of the enzyme, a family of derivatives of SHV-1 has evolved. Derivatives of SHV-1 either have an extended spectrum of activity, capable of inactivating third-generation cephalosporins, or are resistant to beta-lactamase inhibitors. This review describes the evolution and spread of the SHV family of beta-lactamases, introducing the structure-function analysis made possible by DNA sequence analysis. It also reviews the methods used to characterize members of this family of beta-lactamases, indicating some of the difficulties involved.

Aspartic acid for asparagine substitution at position 276 reduces susceptibility to mechanism-based inhibitors in SHV-1 and SHV-5 beta-lactamases.

In SHV-type beta-actamases, position 276 (in Ambler's numbering scheme) is occupied by an asparagine (Asn) residue. The effect on SHV-1 beta-lactamase and its extended-spectrum derivative SHV-5 of substituting an aspartic acid (Asp) residue for Asn276 was studied. Mutations were introduced by a PCR-based site-directed mutagenesis procedure. Wild-type SHV-1 and -5 beta-lactamases and their respective Asn276-->Asp mutants were expressed under isogenic conditions by cloning the respective bla genes into the pBCSK(+) plasmid and transforming Escherichia coli DH5alpha. Determination of IC50 showed that SHV-1(Asn276-->Asp), compared with SHV-1, was inhibited by 8- and 8.8-fold higher concentrations of clavulanate and tazobactam respectively. Replacement of Asn276 by Asp in SHV-5 beta-lactamase caused a ten-fold increase in the IC50 of clavulanate; the increases in the IC50s of tazobactam and sulbactam were 10- and 5.5-fold, respectively. Beta-lactam susceptibility testing showed that both Asn276-->Asp mutant enzymes, compared with the parental beta-lactamases, conferred slightly lower levels of resistance to penicillins (amoxycillin, ticarcillin and piperacillin), cephalosporins (cephalothin and cefprozil) and some of the expanded-spectrum oxyimino beta-lactams tested (cefotaxime, ceftriaxone and aztreonam). The MICs of ceftazidime remained unaltered, while those of cefepime and cefpirome were slightly elevated in the clones producing the mutant beta-lactamases. The latter clones were also less susceptible to penicillin-inhibitor combinations. Asn276-->Asp mutation was associated with changes in the substrate profiles of SHV-1 and SHV-5 enzymes. Based on the structure of TEM-1 beta-lactamase, the potential effects of the introduced mutation on SHV-1 and SHV-5 are discussed.