Molecular epidemiology of extended-spectrum beta-lactamase-producing Klebsiella pneumoniae isolates in a district hospital in Taiwan

Thirty-one of 104 clinical isolates of Klebsiella pneumoniae collected over a period of 8 months were found to be putative extended-spectrum beta-lactamase (ESBL) producers. Isoelectric focusing and an iodine overlay agar method were used for preliminary identification of the ESBLs. They were further identified by DNA sequencing. Seventy-one percent of the isolates were found to produce SHV-5. The variation in the ESBL patterns of these isolates was slight, with only five patterns being identified. The strains were typed by pulsed-field gel electrophoresis (PFGE), and 16 different genotypes were identified. When the PFGE patterns were analyzed by the algorithmic clustering method called the unweighted-pair group method using arithmetic averages, five clusters were found. However, significant genetic variations were found among 11 isolates and between each cluster. A plasmid of 36 kb was found in all clinical isolates and in the transconjugants. Our results indicate that the increase in the number of ESBL-producing K. pneumoniae isolates in this hospital is due mainly to the dissemination of a resistance plasmid rather than to the clonal spread of a few epidemic strains.     

Cloning and sequencing of the gene encoding Toho-2, a class A beta-lactamase preferentially inhibited by tazobactam

Escherichia coli TUM1083, which is resistant to ampicillin, carbenicillin, cephaloridine, cephalothin, piperacillin, cefuzonam, and aztreonam while being sensitive to cefoxitin, moxalactam, cefmetazole, ceftazidime, and imipenem, was isolated from the urine of a patient treated with beta-lactam antibiotics. The beta-lactamase (Toho-2) purified from the bacteria hydrolyzed beta-lactam antibiotics such as penicillin G, carbenicillin, cephaloridine, cefoxitin, cefotaxime, ceftazidime, and aztreonam and especially had increased relative hydrolysis rates for cephalothin, cephaloridine, cefotaxime, and ceftizoxime. Different from other extended-spectrum beta-lactamases, Toho-2 was inhibited 16-fold better by the beta-lactamase inhibitor tazobactam than by clavulanic acid. Resistance to beta-lactams was transferred by conjugation from E. coli TUM1083 to E. coli ML4909, and the transferred plasmid was about 54.4 kbp, belonging to the incompatibility group IncFII. The cefotaxime resistance gene for Toho-2 was subcloned from the 54.4-kbp plasmid. The sequence of the gene was determined, and the open reading frame of the gene was found to consist of 981 bases. The nucleotide sequence of the gene (DDBJ accession no. D89862) designated as bla(toho) was found to have 76.3% identity to class A beta-lactamase CTX-M-2 and 76.2% identity to Toho-1. It has 55.9% identity to SHV-1 beta-lactamase and 47.5% identity to TEM-1 beta-lactamase. Therefore, the newly isolated beta-lactamase designated as Toho-2 produced by E. coli TUM1083 is categorized as an enzyme similar to Toho-1 group beta-lactamases rather than to mutants of TEM or SHV enzymes. According to the amino acid sequence deduced from the DNA sequence, the precursor consisted of 327 amino acid residues. Comparison of Toho-2 with other beta-lactamase (non-Toho-1 group) suggests that the substitutions of threonine for Arg-244 and arginine for Asn-276 are important for the extension of the substrate specificity.     

The role of residue 238 of TEM-1 beta-lactamase in the hydrolysis of extended-spectrum antibiotics

beta-Lactamases inactivate beta-lactam antibiotics by catalyzing the hydrolysis of the amide bond in the beta-lactam ring. The plasmid-encoded class A TEM-1 beta-lactamase is a commonly encountered beta-lactamase. It is able to inactivate penicillins and cephalosporins but not extended-spectrum antibiotics. However, TEM-1-derived natural variants containing the G238S amino acid substitution display increased hydrolysis of extended-spectrum antibiotics. Two models have been proposed to explain the role of the G238S substitution in hydrolysis of extended-spectrum antibiotics. The first proposes a direct hydrogen bond of the Ser238 side chain to the oxime group of extended-spectrum antibiotics. The second proposes that steric conflict with surrounding residues, due to increased side chain volume, leads to a more accessible active site pocket. To assess the validity of each model, TEM-1 mutants with amino acids substitutions of Ala, Ser, Cys, Thr, Asn, and Val have been constructed. Kinetic analysis of these enzymes with penicillins and cephalosporins suggests that a hydrogen bond is necessary but not sufficient to achieve the hydrolytic activity of the G238S enzyme for the extended-spectrum antibiotics cefotaxime and ceftazidime. In addition, it appears that the new hydrogen bond interaction is to a site on the enzyme rather than directly to the extended-spectrum antibiotic. The data indicate that, for the G238S substitution, a combination of an optimal side chain volume and hydrogen bonding potential results in the most versatile and advantageous antibiotic hydrolytic spectrum for bacterial resistance to extended-spectrum antibiotics.     

Penectomy: a technique to reduce blood loss

Twelve SHV-type extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli lac mutant isolates were recovered in October 1997 from 11 patients of the neonatal ward in a Warsaw hospital. The outbreak was clonal; however, some of the isolates expressed a much higher level of resistance to several beta-lactam antibiotics, including expanded-spectrum cephalosporins. This phenotype has been attributed to beta-lactamase hyperproduction correlating with the multiplication of ESBL gene copies, as was demonstrated for representative isolates.     

Outbreak of ceftazidime-resistant Klebsiella pneumoniae in a pediatric hospital in Warsaw, Poland: clonal spread of the TEM-47 extended-spectrum beta-lactamase (ESBL)-producing strain and transfer of a plasmid carrying the SHV-5-like ESBL-encoding gene

In 1996 a large, 300-bed pediatric hospital in Warsaw, Poland, started a program of monitoring infections caused by extended-spectrum beta-lactamase (ESBL)-producing microorganisms. Over the first 3-month period eight Klebsiella pneumoniae isolates were identified as being resistant to ceftazidime. Six of these were found to produce the TEM-47 ESBL, which we first described in a K. pneumoniae strain recovered a year before in a pediatric hospital in Lód?, Poland, which is 140 km from Warsaw. Typing results revealed a very close relatedness among all these isolates, which suggested that the clonal outbreak in Warsaw was caused by a strain possibly imported from Lód?. The remaining two isolates expressed the SHV-5-like ESBL, which resulted from the horizontal transfer of a plasmid carrying the blaSHV gene between nonrelated strains. The data presented here exemplify the complexity of the epidemiological situation concerning ESBL producers typical for large Polish hospitals, in which no ESBL-monitoring programs were in place prior to 1995.     

Extended-spectrum beta-lactamases in clinical isolates of Klebsiella pneumoniae from Zagreb, Croatia

Forty clinical isolates of Klebsiella pneumoniae, from various clinical specimens, with reduced susceptibility to ceftazidime, were tested for extended-spectrum beta-lactamase (ESBL) production. ESBL production was demonstrated by an 8-fold reduction in the minimum inhibitory concentration (MIC) of ceftazidime combined with clavulanate (2 mg/L) compared to ceftazidime alone in all strains. The aim of this investigation was the biochemical and molecular characterization of the ESBL produced by K. pneumoniae strains and their Escherichia coli transconjugants. Transfer of ceftazidime resistance was demonstrated in 23 of 40 strains. Thirteen strains produced an ESBL with the isoelectric point of 8.2 which was encoded by a self-transferable multiresistance plasmid of 150 kb. The substrate profile was similar to that of the SHV-5 isolated initially in Chile. Seven of these 12 strains had an additional TEM beta-lactamase. Six isolates and their transconjugants produced a plasmid-encoded ESBL with an isoelectric point close to 5.4. The remaining 21 strains produced an ESBL with an isoelectric point of 7.6 (thus probably SHV-2) which was encoded on a plasmid transferable to E. coli in 4 strains only. Four of those strains possessed an additional plasmid encoded TEM beta-lactamase with an isoelectric point close to 5.4. The transconjugants harbored a multiresistance plasmid of 150 kb. Thus SHV-2 and SHV-5 enzymes appear to have been the most common ESBLs in K. pneumoniae from Zagreb during 1994-1995.     

beta-Lactamases responsible for resistance to expanded-spectrum cephalosporins in Klebsiella pneumoniae, Escherichia coli, and Proteus mirabilis isolates recovered in South Africa

Although resistance to the expanded-spectrum cephalosporins among members of the family Enterobacteriaceae lacking inducible beta-lactamases occurs virtually worldwide, little is known about this problem among isolates recovered in South Africa. Isolates of Klebsiella pneumoniae, Escherichia coli, and Proteus mirabilis resistant to expanded-spectrum cephalosporins recovered from patients in various parts of South Africa over a 3-month period were investigated for extended-spectrum beta-lactamase production. Antibiotic susceptibility was determined by standard disk diffusion and agar dilution procedures. Production of extended-spectrum beta-lactamases was evaluated by using the double-disk test, and the beta-lactamases were characterized by spectrophotometric hydrolysis assays and an isoelectric focusing overlay technique which simultaneously determined isoelectric points and general substrate or inhibitor characteristics. DNA amplification and sequencing were performed to confirm the identities of these enzymes. The P. mirabilis and E. coli isolates were found to produce TEM-26-type, SHV-2, and SHV-5 extended-spectrum beta-lactamases. An AmpC-related enzyme which had a pI of 8.0 and which conferred resistance to cefoxitin as well as the expanded-spectrum cephalosporins was found in a strain of K. pneumoniae. This is the first study which has identified organisms producing different extended-spectrum beta-lactamases from South Africa and the first report describing strains of P. mirabilis producing a TEM-26-type enzyme. The variety of extended-spectrum beta-lactamases found among members of the family Enterobacteriaceae isolated from major medical centers in South Africa is troubling and adds to the growing list of countries where these enzymes pose a serious problem for antimicrobial therapy.     

Topographic variation in biglycan and decorin synthesis by articular cartilage in the early stages of osteoarthritis: an experimental study in sheep

From October 1988 to January 1992, nine isolates of Pseudomonas aeruginosa carrying transferable plasmids encoding imipenem-hydrolyzing beta-lactamase (pI = c. 9.5) were recovered from nine different patients in a neurosurgical ward of a hospital in Japan. The beta-lactamase activities of the sonicated extracts from the transconjugants were inhibited by EDTA and this was partially reversible by the addition of zinc cation. The substrate specificity and pI of the beta-lactamase were similar to those of the metallo beta-lactamases from P. aeruginosa and Serratia marcescens TN9106. All strains were resistant to imipenem, carbenicillin and antipseudomonal cephems including ceftazidime, cefsulodin, cefpirome, while four and five strains were susceptible to piperacillin and aztreonam, respectively. Both low level imipenem resistance and high level cephem resistance were co-transferred with the production of metallo beta-lactamase, while resistance to piperacillin, aztreonam, and high level imipenem-resistance were not selected. Production of chromosomal cephalosporinase in piperacillin resistant strains was derepressed, and production of outer membrane protein of D2 was diminished in highly imipenem resistant strains. Six strains were isolated in 1991, and the amounts of antipseudomonal agents, especially imipenem, used in the neurosurgical ward increased markedly in this year. Only three of the nine isolates had the same serotype, pyocin type and phage type. Our results suggest that the repeated isolation of imipenem and cephem-resistant P. aeruginosa producing metallo beta-lactamase was related to the high usage of antipseudomonal beta-lactam antibiotics such as imipenem, and was exacerbated by the dissemination of a plasmid.     

Extended-spectrum beta-lactamases from Klebsiella pneumoniae strains isolated at an Italian hospital

Eighteen strains of Klebsiella pneumoniae recently isolated from hospitalized patients were resistant or moderately resistant to oxyimino-cephalosporins (ceftazidime and/or cefotaxime), aztreonam, cefoxitin and all but one were susceptible to imipenem. Analysis of enzymes produced by these clinical isolates revealed a wide pattern of extended-spectrum beta-lactamases. All isolates produced one or more beta-lactamases that were characterized preliminarily by their isoelectric point. Strains isolated early were from patients in the Intensive Care Unit and produced an ES beta-lactamase with an apparent pI of 7.6, whereas the later isolates were from surgical and medical wards of the same hospital and produced ES beta-lactamases with apparent pI of 8.2 and 8.4, respectively. This suggests the emergence of SHV-5 and MIR-1 beta-lactamases in our hospital. Agarose gel electrophoresis of plasmid DNA revealed the presence of a similar plasmid of approximate size 60 Kb in all isolates.     

A TEM-derived extended-spectrum beta-lactamase in Pseudomonas aeruginosa

A clinical strain of Pseudomonas aeruginosa, PAe1100, was found to be resistant to all antipseudomonal beta-lactam antibiotics and to aminoglycosides, including gentamicin, amikacin, and isepamicin. PAe1100 produced two beta-lactamases, TEM-2 (pI 5.6) and a novel, TEM-derived extended-spectrum beta-lactamase called TEM-42 (pI 5.8), susceptible to inhibition by clavulanate, sulbactam, and tazobactam. Both enzymes, as well as the aminoglycoside resistance which resulted from AAC(3)-IIa and AAC(6')-I production, were encoded by an 18-kb nonconjugative plasmid, pLRM1, that could be transferred to Escherichia coli by transformation. The gene coding for TEM-42 had four mutations that led to as many amino acid substitutions with respect to TEM-2: Val for Ala at position 42 (Ala42), Ser for Gly238, Lys for Glu240, and Met for Thr265 (Ambler numbering). The double mutation Ser for Gly238 and Lys for Glu240, which has so far only been described in SHV-type but not TEM-type enzymes, conferred concomitant high-level resistance to cefotaxime and ceftazidime. The novel, TEM-derived extended-spectrum beta-lactamase appears to be the first of its class to be described in P. aeruginosa.     

Selection strategy for site-directed mutagenesis based on altered beta-lactamase specificity

Conventional approaches to oligonucleotide-directed mutagenesis rely upon the application of a selection strategy to maximize mutagenesis efficiencies. We have developed a mutagenesis procedure that incorporates a novel antibiotic resistance for selection. The selection involves altering the substrate specificity of TEM-1 beta-lactamase, the enzyme responsible for bacterial resistance to beta-lactam antibiotics such as ampicillin. The gene encoding beta-lactamase is commonly found on cloning and shuttle vectors used in molecular biology. Amino acid substitutions in several active site residues of beta-lactamase result in increased hydrolytic activity against extended-spectrum penicillins and cephalosporins. This increased activity confers a novel resistance specific to the mutant and thus provides the basis of the selection strategy. We describe a simple and efficient mutagenesis procedure and its application to creating a range of oligonucleotide-directed mutants.     

Pseudomonas aeruginosa isolates from patients with cystic fibrosis have different beta-lactamase expression phenotypes but are homogeneous in the ampC-ampR genetic region

Pseudomonas aeruginosa isolates from 1 of 17 cystic fibrosis patients produced secondary beta-lactamase in addition to the ampC beta-lactamase. Isolates were grouped into three beta-lactamase expression phenotypes: (i) beta-lactam sensitive, low basal levels and inducible beta-lactamase production; (ii) beta-lactam resistant, moderate basal levels and hyperinducible beta-lactamase production; (iii) beta-lactam resistant, high basal levels and constitutive beta-lactamase production. Apart from a base substitution in the ampR-ampC intergenic region of an isolate with moderate-basal-level and hyperinducible beta-lactamase production, sensitive and resistant strains were identical in their ampC-ampR genetic regions. Thus, enhanced beta-lactamase expression is due to mutations in regulatory proteins other than AmpR.     

Epidemiological study of an outbreak due to multidrug-resistant Enterobacter aerogenes in a medical intensive care unit

In 1993, 63 isolates of Enterobacter aerogenes were collected from 41 patients in a medical intensive care unit (ICU). During the same period, only 46 isolates from 32 patients were collected in the rest of the hospital. All isolates were analyzed by antibiotic resistance phenotype, and 77 representative isolates were differentiated by plasmid restriction analysis, ribotyping, and arbitrarily primed (AP)-PCR. The extended-spectrum beta-lactamases produced by 22 strains were characterized by determination of their isoelectric points and by hybridization of plasmid DNA with specific probes. The isolates were divided into 25 antibiotic resistance phenotypes, either susceptible (group I) or resistant (group II) to aminoglycosides, and exhibited three phenotypes of resistance to beta-lactams: chromosomally derepressed cephalosporinase alone or associated with either extended-spectrum beta-lactamases (mainly of the SHV-4 type) or imipenem resistance. The results of the tests divided the 77 representative isolates (group I, n = 21; group II, n = 56) into 15 plasmid profiles, 14 ribotypes, and 15 AP-PCR patterns. Although the resistant isolates (group II) exhibited different plasmid profiles, ribotyping and AP-PCR analysis demonstrated an identical chromosomal pattern, indicating an epidemiological relatedness. They were mainly found in the medical ICU and occasionally in other units. The susceptible strains (group I) had various and distinct markers and were mainly isolated in units other than the medical ICU. In conclusion, the presence of a nosocomial outbreak in an ICU and the spread of a multidrug-resistant epidemic strain throughout the hospital was confirmed. Ribotyping and AP-PCR represent discriminatory tools for the investigation of nosocomial outbreaks caused by E. aerogenes.     

Inhibitor-resistant OXY-2-derived beta-lactamase produced by Klebsiella oxytoca

Klebsiella oxytoca strains are generally moderately resistant to amoxicillin and ticarcillin due to the activities of the chromosomally encoded OXY-1 and OXY-2 class A beta-lactamase families. These enzymes have the ability to hydrolyze not only penicillins but also cephalosporins, including cefuroxime, ceftriaxone, and aztreonam, and are inhibited by clavulanic acid. A Klebsiella oxytoca strain was isolated from a culture of blood from a patient who had been treated with amoxicillin-clavulanate (3 g/day) for 10 days 1 month earlier. This strain harbored an unusual phenotype characterized by resistance to amoxicillin-clavulanate. It produced an OXY-2-type beta-lactamase (pI 6.3), as confirmed by PCR amplification with primers specific for the OXY-2-encoding gene. Gene sequencing revealed a point mutation (A-->G) corresponding to the amino acid substitution Ser-->Gly at position 130. This mutant enzyme was poorly inhibited by inhibitors, and its kinetic constants compared to those of the parent enzyme were characterized by an increased Km value for ticarcillin, with a drastically reduced activity against cephalosporins, as is observed with inhibitor-resistant TEM enzymes. The substitution Ser-->Gly-130 was previously described in the inhibitor-resistant beta-lactamase SHV-10 derived from an SHV-5 variant, but this is the first report of such a mutant in OXY enzymes from K. oxytoca.     

Extrachromosomal resistance in gram-negative organisms: the evolution of beta-lactamase

beta-Lactamases are the major defense used by bacteria to overcome the effects of penicillins, cephalosporins and related beta-lactam antibiotics. In the antibiotic era, the enzymes have evolved to become more prevalent, to appear in new hosts, to be expressed at higher levels, to be acquired by plasmids and to change catalytic properties to increase affinity for what were meant to be nonhydrolysable substrates or to reduce affinity for beta-lactamase inhibitors.     

Characterization of SFO-1, a plasmid-mediated inducible class A beta-lactamase from Enterobacter cloacae

Enterobacter cloacae 8009 produced an inducible class A beta-lactamase which hydrolyzed cefotaxime efficiently. It also hydrolyzed other beta-lactams except cephamycins and carbapenems. The activity was inhibited by clavulanic acid and imipenem. The bla gene was transferable to Escherichia coli by electroporation of plasmid DNA. The molecular mass of the beta-lactamase was 29 kDa and its pI was 7.3. All of these phenotypic characteristics of the enzyme except for inducible production resemble those of some extended-spectrum class A beta-lactamases like FEC-1. The gene encoding this beta-lactamase was cloned and sequenced. The deduced amino acid sequence of the beta-lactamase was homologous to the AmpA sequences of the Serratia fonticola chromosomal enzyme (96%), MEN-1 (78%), Klebsiella oxytoca chromosomal enzymes (77%), TOHO-1 (75%), and FEC-1 (72%). The conserved sequences of class A beta-lactamases, including the S-X(T)-X(S)-K motif, in the active site were all conserved in this enzyme. On the basis of the high degree of homology to the beta-lactamase of S. fonticola, the enzyme was named SFO-1. The ampR gene was located upstream of the ampA gene, and the AmpR sequence of SFO-1 had homology with the AmpR sequences of the chromosomal beta-lactamases from Citrobacter diversus (80%), Proteus vulgaris (68%), and Pseudomonas aeruginosa (60%). SFO-1 was also inducible in E. coli. However, a transformant harboring plasmid without intact ampR produced a small amount of beta-lactamase constitutively, suggesting that AmpR works as an activator of ampA of SFO-1. This is the first report from Japan describing an inducible plasmid-mediated class A beta-lactamase in gram-negative bacteria.     

Extended-spectrum beta-lactamase-producing Klebsiella pneumoniae strains causing nosocomial outbreaks of infection in the United Kingdom

Representative isolates from 10 distinct extended-spectrum beta-lactamase-producing strains of Klebsiella pneumoniae that caused hospital outbreaks in the United Kingdom from 1991 to 1994 were examined for relationships between their enzymes and plasmids. The beta-lactamases were identified by a combination of isoelectric focusing and gene sequencing. SHV-2 beta-lactamase was produced by isolates from four outbreaks, SHV-5 was involved in three, and SHV-4, TEM-15, and TEM-26 were involved in one outbreak each. All of the extended-spectrum beta-lactamases were encoded by self-transmissible plasmids, with sizes ranging from about 70 to 160 kb. No similarities between the restriction digest patterns of the extended-spectrum beta-lactamase-encoding plasmids were detected, except to some extent between those that produced TEM-15 and TEM-26. Thus, outbreaks of hospital infection with these organisms in the United Kingdom from 1991 to 1994 involved distinct organisms and resistance plasmids and appeared to be unrelated.     

Characterization of ceftriaxone-resistant Enterobacteriaceae: a multicentre study in 26 French hospitals. Vigil'Roc Study Group

During a multicentre study performed in 26 French hospitals, 287 (3.2%) of 9038 Enterobacteriaceae isolated, mainly Enterobacter spp., Serratia spp., Citrobacter spp. and Klebsiella spp. were classified as ceftriaxone resistant on the basis of an MIC > 4 mg/L or the presence of an extended-spectrum beta-lactamase. Extended-spectrum beta-lactamase was present mainly in Klebsiella pneumoniae (65 strains, 10.2%) and very rarely in Escherichia coli, Proteus mirabilis, Klebsiella oxytoca, Citrobacter spp. and Enterobacter spp. The extended-spectrum beta-lactamases conferred low-level resistance to ceftriaxone in nearly 60% of the strains harbouring them, emphasizing the need for routine testing for the presence of these enzymes. Among transconjugants three types of extended-spectrum beta-lactamase were identified. Those resembling TEM-3 were the most common, but TEM-21, and SHV-4 were also found. Clavulanate and to a lesser extent sulbactam inhibited all the extended-spectrum beta-lactamases encountered in this study.     

Chromosomally encoded ampC-type beta-lactamase in a clinical isolate of Proteus mirabilis

A clinical strain of Proteus mirabilis (CF09) isolated from urine specimens of a patient displayed resistance to amoxicillin (MIC >4,096 microg/ml), ticarcillin (4,096 microg/ml), cefoxitin (64 microg/ml), cefotaxime (256 microg/ml), and ceftazidime (128 microg/ml) and required an elevated MIC of aztreonam (4 microg/ml). Clavulanic acid did not act synergistically with cephalosporins. Two beta-lactamases with apparent pIs of 5.6 and 9.0 were identified by isoelectric focusing on a gel. Substrate and inhibition profiles were characteristic of an AmpC-type beta-lactamase with a pI of 9.0. Amplification by PCR with primers for ampC genes (Escherichia coli, Enterobacter cloacae, and Citrobacter freundii) of a 756-bp DNA fragment from strain CF09 was obtained only with C. freundii-specific primers. Hybridization results showed that the ampC gene is only chromosomally located while the TEM gene is plasmid located. After cloning of the gene, analysis of the complete nucleotide sequence (1,146 bp) showed that this ampC gene is close to blaCMY-2, from which it differs by three point mutations leading to amino acid substitutions Glu --> Gly at position 22, Trp --> Arg at position 201, and Ser --> Asn at position 343. AmpC beta-lactamases derived from that of C. freundii (LAT-1, LAT-2, BIL-1, and CMY-2) have been found in Klebsiella pneumoniae, E. coli, and Enterobacter aerogenes and have been reported to be plasmid borne. This is the first example of a chromosomally encoded AmpC-type beta-lactamase observed in P. mirabilis. We suggest that it be designated CMY-3.     

Characterization of multiple-antibiotic-resistant Salmonella typhimurium stains: molecular epidemiology of PER-1-producing isolates and evidence for nosocomial plasmid exchange by a clone

We characterized epidemiologic and genetic features of nosocomially originated multiple-antibiotic-resistant Salmonella typhimurium isolates from two hospitals. A total of 32 multiply resistant strains, isolated during a 28-month period, were studied. Four resistance phenotypes were distinguished on the basis of the results of disc diffusion tests. Group 1 was resistant to chloramphenicol, gentamicin, tobramycin, amikacin, and the newer cephalosporins because of the production of an extended-spectrum beta-lactamase (PER-1). Group 2 exhibited the same pattern plus resistance to sulfamethoxazole-trimethoprim (Sxt). Except for Sxt resistance, dominant phenotypes of both groups were transferred on an identical plasmid, pSTI1 (81 MDa). Group 3 was resistant to ampicillin, chloramphenicol, gentamicin, tobramycin, and Sxt. This pattern was also transferred on an 81-MDa plasmid (pSTI2) which differed from pSTI1 on the basis of EcoRI and HindIII restriction fragments. Group 4 was resistant to ampicillin, chloramphenicol, and tetracycline, and a 74-MDa nonconjugative plasmid was detected. Restriction fragment length polymorphism of RNA-encoding DNA and arbitrarily primed PCR tests revealed that bacteria from groups 1, 2, and 3 were clonally related. Epidemiologic data also supported the clonal-dissemination hypothesis. We concluded that S. typhimurium isolates acquire and exchange multiple-resistance plasmids in hospital microflora.