In vitro activity of tigecycline and occurrence of tetracycline resistance determinants in isolates from patients enrolled in phase 3 clinical trials for community-acquired pneumonia.

The in vitro activity of tigecycline was evaluated against baseline pathogens isolated from patients enrolled in phase 3 clinical trials for community-acquired pneumonia conducted in 29 countries worldwide. Tigecycline was active against the most prevalent pathogens, including Streptococcus pneumoniae (MIC(90) 0.06 mg/L), Staphylococcus aureus (MIC(90) 0.25 mg/L), Haemophilus influenzae (MIC(90) 0.5 mg/L) and Klebsiella pneumoniae (MIC(90) 1 mg/L). Twelve isolates of S. pneumoniae expressing tet(M) and two isolates of K. pneumoniae producing extended-spectrum beta-lactamases isolated during the study were susceptible to tigecycline. The excellent in vitro activity of tigecycline against these clinical isolates confirmed its potential utility against pathogens associated with community-acquired pneumonia.

Uses of Staphylococcus aureus GeneChips in genotyping and genetic composition analysis.

Understanding the relatedness of strains within a bacterial species is essential for monitoring reservoirs of antimicrobial resistance and for epidemiological studies. Pulsed-field gel electrophoresis (PFGE), ribotyping, and multilocus sequence typing are commonly used for this purpose. However, these techniques are either nonquantitative or provide only a limited estimation of strain relatedness. Moreover, they cannot extensively define the genes that constitute an organism. In the present study, 21 oxacillin-resistant Staphylococcus aureus (ORSA) isolates, representing eight major ORSA lineages, and each of the seven strains for which the complete genomic sequence is publicly available were genotyped using a novel GeneChip-based approach. Strains were also subjected to PFGE and ribotyping analysis. GeneChip results provided a higher level of discrimination among isolates than either ribotyping or PFGE, although strain clustering was similar among the three techniques. In addition, GeneChip signal intensity cutoff values were empirically determined to provide extensive data on the genetic composition of each isolate analyzed. Using this technology it was shown that strains could be examined for each element represented on the GeneChip, including virulence factors, antimicrobial resistance determinants, and agr type. These results were validated by PCR, growth on selective media, and detailed in silico analysis of each of the sequenced genomes. Collectively, this work demonstrates that GeneChips provide extensive genotyping information for S. aureus strains and may play a major role in epidemiological studies in the future where correlating genes with particular disease phenotypes is critical.

Determining incidence of extended spectrum beta-lactamase producing Enterobacteriaceae, vancomycin-resistant Enterococcus faecium and methicillin-resistant Staphylococcus aureus in 38 centres from 17 countries: the PEARLS study 2001-2002.

The PEARLS study prospectively monitored selected nosocomial pathogens from 38 centres in 13 European, three Middle Eastern countries and South Africa during 2001-2002. Extended spectrum beta-lactamase (ESBL) production rates among Escherichia coli, Klebsiella pneumoniae, and Enterobacter spp. were 5.4% (142/2609), 18.2% (401/2,206) and 8.8% (204/2,328), respectively, for all study sites. The overall ESBL production rate for the combined Enterobacteriaceae was 10.5% (747/7,143), highest in Egypt, 38.5%, and Greece, 27.4%, and lowest in The Netherlands, 2.0%, and Germany, 2.6%. IEF, PCR and DNA sequencing determined 10.7% false positives among Enterobacter spp. when using NCCLS guidelines to screen for ESBL production. The prevalence of nosocomial methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecium was 32.4% (294/908) and 8.7% (83/949), respectively. PEARLS provides baseline data against which prospective changes in resistant determinants and outcomes can be measured in this ongoing study.

Mannopeptimycins, new cyclic glycopeptide antibiotics produced by Streptomyces hygroscopicus LL-AC98: antibacterial and mechanistic activities.

Mannopeptimycins alpha, beta, gamma, delta, and epsilon are new cyclic glycopeptide antibiotics produced by Streptomyces hygroscopicus LL-AC98. Mannopeptimycins gamma, delta, and epsilon, which have an isovaleryl substitution at various positions on the terminal mannose of the disaccharide moiety, demonstrated moderate to good antibacterial activities. Mannopeptimycin epsilon was the most active component against methicillin-resistant staphylococci and vancomycin-resistant enterococci (MICs, 2 to 4 micro g/ml for staphylococci and streptococci and 4 to 32 micro g/ml for enterococci), while mannopeptimycins gamma and delta were two- to fourfold less active. Mannopeptimycins alpha and beta, which lack the isovaleryl substitution and the disaccharide moiety, respectively, had poor antibacterial activities. The in vivo efficacies of the mannopeptimycins in Staphylococcus aureus mouse protection studies paralleled their in vitro activities. The median effective doses of mannopeptimycins gamma, delta, and epsilon were 3.8, 2.6, and 0.59 mg/kg of body weight, respectively. The mannopeptimycins were inactive against cell wall-deficient S. aureus and caused spheroplasting of Escherichia coli imp similar to that observed with penicillin G in an osmotically protective medium. Mannopeptimycin delta rapidly inhibited [(3)H]N-acetylglucosamine incorporation into peptidoglycan in Bacillus subtilis and had no effect on DNA, RNA, or protein biosynthesis. On the basis of the observations presented above, an effect on cell wall biosynthesis was suggested as the primary mode of action for mannopeptimycin delta. The mannopeptimycins were inactive against Candida albicans, did not initiate hemolysis of human erythrocytes, and did not promote potassium ion leakage from E. coli imp, suggesting a lack of membrane damage to prokaryotic or eukaryotic cells.

Extended-spectrum beta-lactamases in the 21st century: characterization, epidemiology, and detection of this important resistance threat.

Beta-lactamases continue to be the leading cause of resistance to beta-lactam antibiotics among gram-negative bacteria. In recent years there has been an increased incidence and prevalence of extended-spectrum beta-lactamases (ESBLs), enzymes that hydrolyze and cause resistance to oxyimino-cephalosporins and aztreonam. The majority of ESBLs are derived from the widespread broad-spectrum beta-lactamases TEM-1 and SHV-1. There are also new families of ESBLs, including the CTX-M and OXA-type enzymes as well as novel, unrelated beta-lactamases. Several different methods for the detection of ESBLs in clinical isolates have been suggested. While each of the tests has merit, none of the tests is able to detect all of the ESBLs encountered. ESBLs have become widespread throughout the world and are now found in a significant percentage of Escherichia coli and Klebsiella pneumoniae strains in certain countries. They have also been found in other Enterobacteriaceae strains and Pseudomonas aeruginosa. Strains expressing these beta-lactamases will present a host of therapeutic challenges as we head into the 21st century.

Ceftriaxone-resistant salmonella infection acquired by a child from cattle.

BACKGROUND: The emergence of resistance to antimicrobial agents within the salmonellae is a worldwide problem that has been associated with the use of antibiotics in livestock. Resistance to ceftriaxone and the fluoroquinolones, which are used to treat invasive salmonella infections, is rare in the United States. We analyzed the molecular characteristics of a ceftriaxone-resistant strain of Salmonella enterica serotype typhimurium isolated from a 12-year-old boy with fever, abdominal pain, and diarrhea. METHODS: We used pulsed-field gel electrophoresis and analysis of plasmids and beta-lactamases to compare the ceftriaxone-resistant S. enterica serotype typhimurium from the child with four isolates of this strain obtained from cattle during a local outbreak of salmonellosis. RESULTS: The ceftriaxone-resistant isolate from the child was indistinguishable from one of the isolates from cattle, which was also resistant to ceftriaxone. Both ceftriaxone-resistant isolates were resistant to 13 antimicrobial agents; all but one of the resistance determinants were on a conjugative plasmid of 160 kb that encoded the functional group 1 beta-lactamase CMY-2. Both ceftriaxone-resistant isolates were closely related to the three other salmonella isolates obtained from cattle, all of which were susceptible to ceftriaxone. CONCLUSIONS: This study provides additional evidence that antibiotic-resistant strains of salmonella in the United States evolve primarily in livestock. Resistance to ceftriaxone, the drug of choice for invasive salmonella disease, is a public health concern, especially with respect to children, since fluoroquinolones, which can also be used to treat this disease, are not approved for use in children.

Characterization of expanded-spectrum cephalosporin resistance in E. coli isolates associated with bovine calf diarrhoeal disease.

Antibiotic resistance among Escherichia coli isolates from diarrhoeal disease in cattle was studied. Many of the isolates were multiply resistant to beta-lactams, including expanded-spectrum cephalosporins, aminoglycosides, sulphonamides, tetracycline and fluoroquinolones. In many of the isolates, IEF revealed a strong beta-lactamase band compatible with overexpression of the AmpC beta-lactamase, either alone or in addition to TEM-type enzymes. Several of the isolates also possessed genes encoding virulence factors associated with animal and human diarrhoeal diseases. These results suggest that the use of antibiotics in animals could lead to a reservoir of antibiotic-resistant bacteria that could potentially infect humans.

Automated thermal cycling is superior to traditional methods for nucleotide sequencing of bla(SHV) genes.

Genes encoding SHV-1 and SHV-2 were sequenced by different methods. Nucleotide sequencing of the coding strand by standard dideoxy-chain termination methods resulted in errors in the interpretation of the nucleotide sequence and the derived amino acid sequence in two main regions which corresponded to nucleotide and amino acid changes that had been reported previously. The automated thermal cycling method was clearly superior and consistently resulted in the correct sequences for these genes.

Clinical characteristics and molecular epidemiology associated with imipenem-resistant Klebsiella pneumoniae.

Eight patients were infected or colonized with imipenem-resistant Klebsiella pneumoniae (IRKP) from December 1994 to November 1995. Initial Klebsiella isolates were susceptible to imipenem but resistant to all cephalosporins, aminoglycosides, and beta-lactam inhibitor combinations. All patients had been in the surgical intensive care unit and had undergone abdominal surgery or tracheostomy during hospitalization. The average age of the patients was 71 years (range, 41-81 years). All patients were treated with imipenem for 5 to 36 days, and IRKP was recovered from each during or after therapy. Pulsed-field gel electrophoresis (PFGE) of the IRKP isolates revealed three distinct clonal patterns. Paired sequential isolates of imipenem-susceptible K. pneumoniae and IRKP from two patients had identical PFGE patterns, suggesting the development of clonal stepwise resistance to imipenem during therapy. Thus, imipenem resistance in Klebsiella may occur when this agent is used for treatment of infection due to ceftazidine- and aminoglycoside-resistant strains.

Multiple antibiotic-resistant Klebsiella and Escherichia coli in nursing homes.

CONTEXT: Infections caused by ceftazidime sodium-resistant gram-negative bacteria that harbor extended-spectrum beta-lactamases (ESBLs) are increasing in frequency in hospitals in the United States. OBJECTIVES: To report a citywide nursing home-centered outbreak of infections caused by ESBL-producing gram-negative bacilli and to describe the clinical and molecular epidemiology of the outbreak. DESIGN: Hospital-based case-control study and a nursing home point-prevalence survey. Molecular epidemiological techniques were applied to resistant strains. SETTINGS: A 400-bed tertiary care hospital and a community nursing home. PATIENTS: Patients who were infected and/or colonized with ceftazidime-resistant Escherichia coli, Klebsiella pneumoniae, or both and controls who were admitted from nursing homes between November 1990 and July 1992. MAIN OUTCOME MEASURES: Clinical and epidemiological factors associated with colonization or infection by ceftazidime-resistant E coli or K pneumoniae; molecular genetic characteristics of plasmid-mediated ceftazidime resistance. RESULTS: Between November 1990 and October 1992, 55 hospital patients infected or colonized with ceftazidime-resistant E coli, K pneumoniae, or both were identified. Of the 35 admitted from 8 nursing homes, 31 harbored the resistant strain on admission. All strains were resistant to ceftazidime, gentamicin, and tobramycin; 96% were resistant to trimethoprim-sulfamethoxazole and 41% to ciprofloxacin hydrochloride. In a case-control study, 24 nursing home patients colonized with resistant strains on hospital admission were compared with 16 nursing home patients who were not colonized on hospital admission; independent risk factors for colonization included poor functional level, presence of a gastrostomy tube or decubitus ulcers, and prior receipt of ciprofloxacin and/or trimethoprim-sulfamethoxazole. In a nursing home point-prevalence survey, 18 of 39 patients were colonized with ceftazidime-resistant E coli; prior receipt of ciprofloxacin or trimethoprim-sulfamethoxazole and presence of a gastrostomy tube were independent predictors of resistance. Plasmid studies on isolates from 20 hospital and nursing home patients revealed that 17 had a common 54-kilobase plasmid, which conferred ceftazidime resistance via the ESBL TEM-10, and mediated resistance to trimethoprim-sulfamethoxazole, gentamicin, and tobramycin; all 20 isolates harbored this ESBL. Molecular fingerprinting showed 7 different strain types of resistant K pneumoniae and E coli distributed among the nursing homes. CONCLUSIONS: Nursing home patients may be an important reservoir of ESBL-containing multiple antibiotic-resistant E coli and K pneumoniae. Widespread dissemination of a predominant antibiotic resistance plasmid has occurred. Use of broad-spectrum oral antibiotics and probably poor infection control practices may facilitate spread of this plasmid-mediated resistance. Nursing homes should monitor and control antibiotic use and regularly survey antibiotic resistance patterns among pathogens.

CTX-M-5, a novel cefotaxime-hydrolyzing beta-lactamase from an outbreak of Salmonella typhimurium in Latvia.

At a children's hospital in Riga, Latvia, isolates identified as Salmonella typhimurium were found to be resistant to expanded-spectrum cephalosporins. Two of the resistant strains were analyzed for the mechanism of cephalosporin resistance. Isoelectric focusing revealed a common beta-lactamase with a pI of 8.8. In addition, one of the strains produced a pI 7.6 beta-lactamase. A transconjugant producing only the pI 7.6 enzyme was susceptible to expanded-spectrum cephalosporins; therefore, this enzyme was most likely SHV-1. Transformants producing only the pI 8.8 beta-lactamase were resistant to cefotaxime and aztreonam but were susceptible or intermediate to ceftazidime. A substrate profile determined spectrophotometrically with purified enzyme revealed potent activity against cefotaxime, with a relative kcat value of 95 (benzylpenicillin equal to 100). The enzyme showed lower relative kcat values for ceftazidime (3.3) and aztreonam (9.3). In addition, the enzyme was inhibited by clavulanate, sulbactam and tazobactam, with 50% inhibitory concentrations of 19, 100, and 3.4 nM, respectively. These results indicated the presence of an unusual extended-spectrum beta-lactamase. The gene expressing the pI 8.8 beta-lactamase was cloned. Nucleotide sequencing revealed a beta-lactamase gene that differs from the gene encoding CTX-M-2, which also originated from S. typhimurium, by 11 nucleotides, 4 of which result in amino acid substitutions: Ala27Thr, Val230Gly, Glu254Ala, and Ile278Val. These results indicated the presence of a novel extended-spectrum beta-lactamase, designated CTX-M-5, that specifically confers resistance to cefotaxime.

Ceftazidime-resistant Klebsiella pneumoniae and Escherichia coli isolates producing TEM-10 and TEM-43 beta-lactamases from St. Louis, Missouri.

Ceftazidime-resistant Escherichia coli and Klebsiella pneumoniae (49 and 102 isolates, respectively) were collected from Barnes-Jewish Hospital, St. Louis, Mo., from 1992 to 1996. They were uniformly resistant to ceftazidime, generally resistant to aztreonam, and variably susceptible to cefotaxime. Four representative E. coli strains and 15 Klebsiella strains were examined. From one to four beta-lactamases were produced per strain, with three possible enzymes related to ceftazidime resistance: enzymes with pI values of 5.6, 6.1, or 7.6. By pulsed-field gel electrophoresis there were at least 13 different Klebsiella strain types and 3 different E. coli strain types, indicating that the outbreak was not clonal. After cloning and sequencing of the beta-lactamase-encoding genes, the enzyme with a pI of 5.6 was identified as TEM-10. The enzyme with a pI of 6.1 was a novel TEM variant (TEM-43) with Lys at 104, His at 164, and Thr at 182. TEM-43 showed broad-spectrum hydrolytic activity against all penicillins, with the highest hydrolysis rate for ceftazidime compared to those for the other expanded-spectrum cephalosporins. Aztreonam was also a good substrate for TEM-43, with hydrolytic activity similar to that of ceftazidime and affinity higher than that of ceftazidime. The TEM-43 beta-lactamase was well inhibited by clavulanic acid and tazobactam at concentrations of < 10 nM. Sulbactam was less effective than the other inhibitors. The Thr182 mutation previously reported in an inhibitor-resistant beta-lactamase did not cause the TEM-43 enzyme to become resistant to any of the inhibitors.

Penicillin-binding proteins and induction of AmpC beta-lactamase.

In competition assays for radiolabeled penicillin, penicillin-binding proteins (PBPs) 4, 7a, and 7b showed very high affinities for strong inducers of AmpC beta-lactamase. Loss of PBP 4 resulted in diminished inducibility. This suggests that if PBPs are involved in induction of AmpC beta-lactamase, there is probably a redundancy in function among the different PBPs.

Imipenem resistance in Klebsiella pneumoniae is associated with the combination of ACT-1, a plasmid-mediated AmpC beta-lactamase, and the foss of an outer membrane protein.

Six Escherichia coli and 12 Klebsiella pneumoniae isolates from a single hospital expressed a common beta-lactamase with a pI of approximately 9.0 and were resistant to cefoxitin and cefotetan (MIC ranges, 64 to > 128 and 16 to > 128 micrograms/ml, respectively). Seventeen of the 18 strains produced multiple beta-lactamases. Most significantly, three K. pneumoniae strains were also resistant to imipenem (MICs, 8 to 32 micrograms/ml). Spectrophotometric beta-lactamase assays with purified enzyme indicated hydrolysis of cephamycins, in addition to cephaloridine and benzylpenicillin. The 4ene encoding the pI 9.0 beta-lactamase (designated ACT-1 for AmpC type) was cloned and sequenced, which revealed an ampC-type beta-lactamase gene that originated from Enterobacter cloacae and that had 86% sequence homology to the P99 beta-lactamase and 94% homology to the partial sequence of MIR-1. Southern blotting revealed that the gene encoding ACT-1 was on a large plasmid in some of the K. pneumoniae strains as well as on the chromosomes of all of the strains, suggesting that the gene is located on an easily mobilized element. Outer membrane protein profiles of the K. pneumoniae strains revealed that the three imipenem-resistant strains were lacking a major outer membrane protein of approximately 42 kDa which was present in the imipenem-susceptible strains. ACT-1 is the first plasmid-mediated AmpC-type beta-lactamase derived from Enterobacter which has been completely sequenced. This work demonstrates that in addition to resistance to cephamycins, imipenem resistance can occur in K. pneumoniae when a high level of the ACT-1 beta-lactamase is produced in combination with the loss of a major outer membrane protein.

TEM-28 from an Escherichia coli clinical isolate is a member of the His-164 family of TEM-1 extended-spectrum beta-lactamases.

TEM-28 (pI 6.1), expressed by an Escherichia coli clinical isolate, is a novel beta-lactamase which hydrolyzed ceftazidime, cefotaxime, and aztreonam with rates of 25, 1.1, and 5.6, respectively, relative to that for benzylpenicillin (100). The nucleotide sequence of blaTEM-28 differed from that of blaTEM-1 by two base changes, resulting in amino acid substitutions of Arg-164 to His and Glu-240 to Lys.

SHV-7, a novel cefotaxime-hydrolyzing beta-lactamase, identified in Escherichia coli isolates from hospitalized nursing home patients.

Four ceftazidime-resistant Escherichia coli strains were isolated from elderly nursing home patients in a New York hospital during 1993. Strains MCQ-2, MCQ-3, and MCQ-4 were determined to be identical by pulsed-field gel electrophoresis and plasmid profiles, whereas strain MCQ-1 was unique. Strain MCQ-1 was determined to produce a TEM-10 beta-lactamase. Strains MCQ-2, MCQ-3, and MCQ-4 were also noted to be resistant to cefotaxime. These three strains produced two beta-lactamases with pIs of 5.4 (TEM-1) and 7.6. beta-Lactamase assays revealed that the pI 7.6 enzyme hydrolyzed cefotaxime faster (at a relative hydrolysis rate of 30% compared with that of benzylpenicillin) than either ceftazidime or aztreonam (relative hydrolysis rates of 13 and 3.3%, respectively). Nucleotide sequencing of the gene encoding the pI 7.6 beta-lactamase from strain MCQ-3 revealed a blaSHV-type gene differing from the gene encoding SHV-1 at four nucleotides which resulted in amino acid substitutions: phenylalanine for isoleucine at position 8, serine for arginine at position 43, serine for glycine at position 238, and lysine for glutamate at position 240. This novel SHV-type extended-spectrum beta-lactamase is designated SHV-7.

Development of test panel of beta-lactamases expressed in a common Escherichia coli host background for evaluation of new beta-lactam antibiotics.

A test panel of 35 different beta-lactamases expressed in a common Escherichia coli host was created to compare the effect that each beta-lactamase had on susceptibility to various beta-lactam antibiotics. A comparison of the MICs obtained with this panel generally reflected differences in the substrate profiles of the various beta-lactamases examined. In addition, several strains of the panel were subjected to selection with porin-specific bacteriophages to obtain mutants lacking either the OmpC or OmpF porin protein. A mutation in either OmpC or OmpF did change the susceptibilities of certain strains expressing beta-lactamase to certain beta-lactam antibiotics. However, the loss of a single porin did not predictably alter susceptibility to any given beta-lactam drug. This panel of strains producing various beta-lactamases was found to be a useful tool for comparing the effects of different beta-lactamases and outer membrane permeability upon susceptibility to beta-lactam drugs.

Multiply resistant Klebsiella pneumoniae strains from two Chicago hospitals: identification of the extended-spectrum TEM-12 and TEM-10 ceftazidime-hydrolyzing beta-lactamases in a single isolate.

Ceftazidime-resistant Klebsiella pneumoniae strains began to appear when ceftazidime usage was increased in two unrelated Chicago hospitals. These strains produced a beta-lactamase with an isoelectric point of 5.6 (RP-5.6) and strong hydrolyzing activity against ceftazidime. Two different restriction digest profiles were associated with the ceftazidime resistance plasmids. A second beta-lactamase with a pI of 5.2 (RP-5.2) was coproduced in two representative strains. The second beta-lactamase hydrolyzed ceftazidime, cefotaxime, and aztreonam with relative hydrolysis rates of < 8% of that observed for benzylpenicillin. Both enzymes were inhibited by clavulanic acid and tazobactam. Nucleotide sequencing of the genes coding for RP-5.2 and RP-5.6 revealed sequences identical to those of the TEM-12 and TEM-10 beta-lactamase genes, respectively. Both genes were derived from a TEM-1 sequence related to that of the gene encoded on the Tn2 transposon. Single point mutations are required to progress from TEM-1 to TEM-12 and from TEM-12 to TEM-10. Extracts from broths grown from single cell isolates of the strain producing TEM-12 and TEM-10 were shown to contain both enzymes. Transconjugants producing either the TEM-12 or the TEM-10 beta-lactamase were obtained. A significant finding was that both enzymes were encoded by plasmids with identical restriction digest patterns. These studies show that mutations leading to extended-spectrum beta-lactamases can occur sequentially in the same organism, with the genes encoding both enzymes maintained stably.

Use of a predictor panel to evaluate susceptibility test methods proposed for piperacillin-tazobactam.

A predictor panel of clinical isolates that produce a variety of types and amounts of beta-lactamases was used to assess the accuracy of dilution and disk diffusion susceptibility tests for piperacillin-tazobactam. Combinations of piperacillin-tazobactam with a fixed ratio of 8:1 and with tazobactam held constant at 4 micrograms/ml were examined in dilution tests performed in agar. In addition, disks containing 100 and 10 micrograms of piperacillin and tazobactam, respectively, were examined in diffusion tests. Three very major discrepancies between MICs determined with an 8:1 ratio and MICs determined with tazobactam held constant at 4 micrograms/ml were noted. These involved strains that appeared to be susceptible in tests with the 8:1 ratio but resistant when tazobactam was held constant at 4 micrograms/ml. However, the differences were only twofold. Error rate-bounded analysis with the disk containing 100 and 10 micrograms of piperacillin and tazobactam, respectively, revealed low error rates, regardless of whether MICs were determined with an 8:1 ratio or tazobactam held constant at 4 micrograms/ml. Thus, a predictor panel was useful in the identification of accurate susceptibility test for piperacillin-tazobactam.

Genetically diverse ceftazidime-resistant isolates from a single center: biochemical and genetic characterization of TEM-10 beta-lactamases encoded by different nucleotide sequences.

Ceftazidime-resistant isolates of Escherichia coli and Klebsiella pneumoniae produced a plasmid-mediated beta-lactamase with a pI of 5.6 with biochemical characteristics comparable to those of the TEM-10 beta-lactamase. Plasmids from the two strains were nonidentical. Both TEM-10 sequences differed from TEM-1 by substitutions of Ser-162 and Lys-237. The nucleotide sequences of the two genes were identical except for three silent nucleotide substitutions corresponding to the nucleotide differences in the Tn2 TEM-1 or Tn3 TEM-1 genes. The original TEM-10 plasmid was identical to that found in the E. coli isolate and coded for a gene that corresponded to the TEM-10 beta-lactamase from Tn2.