Occurrence of bla DHA-1 mediated cephalosporin resistance in Escherichia coli and their transcriptional response against cephalosporin stress: a report from India.

BACKGROUND: Treatment alternatives for DHA-1 harboring strains are challenging as it confers resistance to broad spectrum cephalosporins and may further limit treatment option when expressed at higher levels. Therefore, this study was designed to know the prevalence of DHA genes and analyse the transcription level of DHA-1 against different ß-lactam stress. METHODS: Screening of AmpC ß-lactamase phenotypically by modified three dimensional extract method followed by Antimicrobial Susceptibility and MIC determination. Genotyping screening of ß-lactamase genes was performed by PCR assay followed by their sequencing. The bla DHA-1 transcriptional response was evaluated under different cephalosporin stress by RT PCR. Transferability of bla DHA gene was performed by transformation and conjugation and plasmid incompatibility typing, DNA fingerprinting by enterobacterial repetitive intergenic consensus sequences PCR. RESULTS: 16 DHA-1 genes were screened positive from 176 Escherichia coli isolates and primer extension analysis showed a significant increase in DHA-1 mRNA transcription in response to cefotaxime at 8 µg/ml (6.99 × 102 fold), ceftriaxone at 2 µg/ml (2.63 × 103 fold), ceftazidime at 8 µg/ml (7.06 × 103 fold) and cefoxitin at 4 µg/ml (3.60 × 104 fold) when compared with untreated strain. These transcription data were found significant when analyzed statistically using one way ANOVA. Four different ESBL genes were detected in 10 isolates which include CTX-M (n = 6), SHV (n = 4), TEM (n = 3) and OXA-10 (n = 1), whereas, carbapenemase gene (NDM) was detected only in one isolate. Other plasmid mediated AmpC ß-lactamases CIT (n = 9), EBC (n = 2) were detected in nine isolates. All DHA-1 genes detected were encoded in plasmid and incompatibility typing from the transformants indicated that the plasmid encoding bla DHA-1 was carried mostly by the FIA and L/M Inc group. CONCLUSION: This study demonstrates the prevalence of DHA-1 gene in this region and highlights high transcription of DHA-1 when induced with different ß-lactam antibiotics. Therefore, cephalosporin treatment must be restricted for the patients infected with pathogen expressing this resistance determinant.

Characterization of a DHA-1-producing Klebsiella pneumoniae strain involved in an outbreak and role of the AmpR regulator in virulence.

A clonal strain of Klebsiella pneumoniae producing the plasmid-encoded cephalosporinase DHA-1 was isolated from four patients admitted to the teaching hospital of Clermont-Ferrand, France, in 2006. It was responsible for severe infections in three of the patients; the fourth was colonized only in the gastrointestinal tract. The strain had at least two plasmids encoding resistance to antibiotics (quinolones, aminoglycosides, chloramphenicol, sulfonamides, and trimethoprim), as shown by disk diffusion assay, and harbored only a few genes for virulence factors (wabG and mrkD), as shown by PCRs. DHA-1 synthesis is regulated by an upstream, divergently transcribed gene, ampR, which is also involved in the expression of virulence factors in Pseudomonas aeruginosa. To investigate the role of AmpR in K. pneumoniae, we cloned the wild-type ampR gene from the DHA-1 clonal isolate into a previously characterized K. pneumoniae background plasmid-cured strain, CH608. ampR was also introduced into a CH608 isogenic mutant deleted of ampD, in which AmpR is present only in its activator form, resulting in constitutive hyperproduction of the ß-lactamase. We showed that ampR was involved in the upregulation of capsule synthesis and therefore in resistance to killing by serum. AmpR also modulated biofilm formation and type 3 fimbrial gene expression, as well as colonization of the murine gastrointestinal tract and adhesion to HT-29 intestinal epithelial cells. These results show the pleiotropic role of ampR in the pathogenesis process of K. pneumoniae.

Variable within- and between-herd diversity of CTX-M cephalosporinase-bearing Escherichia coli isolates from dairy cattle.

bla(CTX-M) beta-lactamases confer resistance to critically important cephalosporin drugs. Recovered from both hospital- and community-acquired infections, bla(CTX-M) was first reported in U.S. livestock in 2010. It has been hypothesized that veterinary use of cephalosporins in livestock populations may lead to the dissemination of beta-lactamase-encoding genes. Therefore, our objectives were to estimate the frequency and distribution of coliform bacteria harboring bla(CTX-M) in the fecal flora of Ohio dairy cattle populations. In addition, we characterized the CTX-M alleles carried by the isolates, their plasmidic contexts, and the genetic diversity of the bacterial isolates themselves. We also evaluated the association between ceftiofur use and the likelihood of recovering cephalosporinase-producing bacteria. Thirty fresh fecal samples and owner-reported ceftiofur use data were collected from each of 25 Ohio dairy farms. Fecal samples (n = 747) yielded 70 bla(CTX-M)-positive Escherichia coli isolates from 5/25 herds, 715 bla(CMY-2) E. coli isolates from 25/25 herds, and 274 Salmonella spp. from 20/25 herds. The within-herd prevalence among bla(CTX-M)-positive herds ranged from 3.3 to 100% of samples. Multiple pulsed-field gel electrophoresis (PFGE) patterns, plasmid replicon types, and CTX-M genes were detected. Plasmids with CTX-M-1, -15, and -14 alleles were clonal by restriction fragment length polymorphism (RFLP) within herds, and specific plasmid incompatibility group markers were consistently associated with each bla(CTX-M) allele. PFGE of total bacterial DNA showed similar within-herd clustering, with the exception of one herd, which revealed at least 6 different PFGE signatures. We were unable to detect an association between owner-reported ceftiofur use and the probability of recovering E. coli carrying bla(CTX-M) or bla(CMY-2).

Molecular characterization of extended-spectrum cephalosporinase-producing Salmonella enterica serovar Choleraesuis isolates from patients in Thailand and Denmark.

The objective of this study was to characterize extended-spectrum cephalosporinase (ESC)-producing isolates of Salmonella enterica serovar Choleraesuis recovered from patients in Thailand and Denmark. Twenty-four blood culture isolates from 22 patients were included in the study, of which 23 isolates were recovered from 21 Thai patients during 2003, 2007, or 2008 and one isolate was recovered from a Danish traveler to Thailand. ESC production was confirmed in 13 out of the 24 isolates by MIC testing. Microarray and plasmid profiling (replicon typing and restriction fragment length polymorphism [RFLP]) were used to characterize the genetic mechanisms of antimicrobial resistance in the 13 ESC-producing isolates. Pulsed-field gel electrophoresis (PFGE) and MIC testing were used to compare the clonality between the 13 ESC-producing isolates and the 11 non-ESC-producing isolates. Based on susceptibility patterns, the ESC-producing isolates were more closely related than non-ESC-producing isolates. Microarray, PCR, plasmid profiling, and replicon typing revealed that the 13 ESC-producing isolates harbored either bla(CMY-2) containing incA/C or bla(CTX-M-14) containing incFIIA, incFrepB, and an unknown replicon located on plasmids ranging in size from 75 to 200 kb. The RFLP and replicon typing clustered the isolates into four distinct groups. PFGE revealed 16 unique patterns and five clusters; each cluster contained two or three of the 24 isolates. The isolate from the Danish patient was indistinguishable from two Thai clinical isolates by PFGE. This study revealed the emergence of the bla(CTX-M-14) gene among several clones of Salmonella serovar Choleraesuis. Numerous plasmids were identified containing up to two different ESC genes and four distinct replicons. A "travel-associated" spread was confirmed. Overall, a high degree of clonal diversity between isolates resistant and susceptible to cephalosporins was observed. The findings represent a serious threat to public health for the Thai people and tourists.

First Report of DHA-1 Producing Enterobacter cloacae Complex Isolate in Bulgaria.

THE AIM: of the present study was to reveal the characteristics of an. MATERIALS AND METHODS: Susceptibility testing, conjugation experiments, isoelectric focusing, PCR and sequencing were carrying out. RESULTS: Of 176. CONCLUSIONS: To the best of our knowledge this is the first report of DHA-1 producing isolate in Bulgaria. The emergence of DHA-1 producing.

Analysis of the beta-lactam resistance phenotypes of Escherichia coli. An 8-year survey conducted in Greece.

There have been few studies, outside of France, on the resistance of Escherichi coli to beta-lactams by means of resistance phenotypes. In the present 8-year study from a tertiary Greek hospital, a statistically significant decrease in wild-type strains was noted, with a parallel increase in strains producing penicillinase. Of the total 6,089 isolates analyzed, 62.47% had no acquired resistance mechanisms while 35.70% produced penicillinase, 0.61% cefalosporinase and 0.94% extended-spectrum beta-lactamase. No overexpression of chromosomal cephalosporinase or synthesis of inhibitor-resistant enzymes was found. A shift in the pattern of penicillinase production was noted as, in the early years of the study, intermediate- and high-level penicillinase predominated whereas, in later years, low-level penicillinase prevailed.

Acquisition of plasmid-mediated cephalosporinase producing Enterobacteriaceae after a travel to the tropics.

Travelers are at high risk of acquiring multi-drug resistant Enterobacteriaceae (MRE) while traveling abroad. Acquisition of extended spectrum beta-lactamase producing Enterobacteriaceae (ESBL-E) while traveling has been extensively described, but not that of plasmid-mediated cephalosporinase producing Enterobacteriaceae (pAmpC-E). Here, we characterized the pAmpC-E acquired in 574 French travelers to tropical areas enrolled in the VOYAG-R study. Among the 526 MRE isolated at return, 57 (10.8%) from 49 travelers were pAmpC-E. The acquisition rate of pAmpC-E was 8.5% (49/574) ranging from 12.8% (25/195) in Asia, 7.6% (14/184) in Latin America to 5.1% (10/195) in Africa. The highest acquisition rates were observed in Peru (21.9%), India (21.4%) and Vietnam (20%). The carriage of pAmpC-E decreased quickly after return with 92.5% of colonized travelers being negative at one month. Most enzymes were CMY types (96.5%, n = 55, only met in Escherichia coli), including 40 CMY-2 (70.2%), 12 CMY-42 (21.1%), 1 CMY-6 and two new CMY-2 variants. The remaining were two DHA observed in Klebsiella pneumoniae. CMY-2 producing strains were acquired worldwide whereas CMY-42, except for one, were all acquired in Asia. BlaCMY-2 genes were associated with different plasmid types, including IncI1 (45. 2%), IncF (10%), IncF-IncI (7.5%), IncA/C (5%) and IncR (2.5%) whereas blaCMY-42 were all associated with IncI1 plasmids. Even though the pAmpC-E acquisition rate was much lower than that of ESBL-E, it was significant, especially in Asia, showing that pAmpC-E, especially CMY-type producing E. coli have spread in the community settings of tropical regions.

[Clinically important beta-lactamases of gram-negative bacteria: AmpC]. / Klinicky významné beta-laktamázy gramnegativních bakterii: AmpC.

Beta-lactamases are the most common cause of beta-lactam resistance in Gram-negative bacteria. With third-generation and fourth-generation cephalosporins being introduced into practice, new beta-lactamases have evolved, able to hydrolyze these antibiotics. AmpC-type beta lactamases (cephalosporinases) are serine enzymes with the ability to hydrolyze penicillins, monobactams and cephalosporins of all generations, including cephamycins. Over the last two decades, transferable plasmid-mediated class C beta-lactamases have been reported with increasing frequency. The genes for resistance to other groups of antibiotics are usually carried on the same mobile element as the AmpC genes. A reliable method for AmpC detection in routine diagnosis has not been available yet. The issue of AmpC-type beta lactamases is summarized, including their identification, interpretation of susceptibility test results and recommended treatment of infection caused by AmpC producers.

Cephalosporinase over-expression resulting from insertion of ISAba1 in Acinetobacter baumannii.

ISAba1-like sequences were identified immediately upstream of the bla(ampC) gene in ceftazidime-resistant Acinetobacter baumannii isolates, but were absent in ceftazidime-susceptible A. baumannii isolates. AmpC over-expression resulted from insertion of ISAba1-like sequences upstream of bla(ampC). ISAba1 provided strong promoter sequences, and it was demonstrated that the change in the ribosome binding site sequence resulting from insertion of ISAba1 did not influence expression of the bla(ampC) gene. Sequence analysis revealed that AmpC sequences of A. baumannii isolates were almost identical and that ISAba1 elements had a high percentage of identity.

[Inducible cephalosporinases from Proteus morganii]. / Céphalosporinases inductibles de Proteus morganii

It has been shown that two Proteus morganii strains produce an inducible cephalosporinase. No significative difference was shown between them: they present the same isoelectric poit: 8,3 and very similar kinetic constants. The parameter tau, proportional to the half life of antibiotic at low concentration, in presence of beta lactamase, shows that cefamandole is the most stable cephalosporin studied.

Beta-lactamase-mediated resistance to extended spectrum cephalosporins among clinical isolates of Pseudomonas aeruginosa.

The role of chromosomal cephalosporinases and secondary beta-lactamases in resistance to extended spectrum cephalosporins in clinical isolates of Pseudomonas aeruginosa was investigated. Strains 687, 59, and 58 expressed an inducible chromosomal cephalosporinase, efficiently enhanced with cefoxitin and imipenem. The inducible activity in strain 802 was produced at a moderately elevated basal level and may be involved in resistance to extended spectrum cephalosporins and aztreonam. All strains produced secondary beta-lactamases inhibited by clavulanate: strains 687, 59, and 58 had carbenicillinases with pIs of 5.7 and 5.3. Strain 802 expressed a secondary beta-lactamase of pI 7.6 which may be a novel extended spectrum beta-lactamase different from known enzymes of P. aeruginosa.

Sensitivities of strains of enteropathogenic Escherichia coli to cephalexin and other antibiotics.

The sensitivity patterns of strains of enteropathogenic Escherichia coli to nine antibiotics were determined. Most strains were sensitive to gentamicin, kanamycin, neomycin, and colistin. Sensitivity to cephalexin was generally greater than sensitivity to ampicillin. Compared with sensitivity patterns of strains isolated in previous years, no significant change in sensitivity patterns of recently isolated strains was detected. All ampicillin-resistant strains destroyed the drug by producing beta-lactamase. The activity of this enzyme against cephalexin was significantly lower than its activity against ampicillin. The role of beta-lactamase, the correlation between its production and resistance to beta-lactamase antibiotics, and the similarity between beta-lactamase produced by EEC and the classified beta-lactamases produced by other enteric bacteria and Escherichia coli, are discussed.

Selective digestive decontamination by erythromycin-base in a polyvalent intensive care unit.

OBJECTIVE: To study the effect of selective digestive tract decontamination by erythromycin-base on the incidence of carriage and infection with MR Enterobacteriaceae producing an extended spectrum beta-lactamase (ESB). DESIGN: After a 10-week prospective survey to ascertain the baseline incidence in two bays (1 and 3) of the same ICU, bay 1 was compared with bay 3 during a further survey of 6 months. The patients in bay 1 received erythromycin-base. SETTING: Two non-contiguous bays, 1 and 3, of 4 beds, in the same polyvalent ICU of a university hospital. PATIENT: Consecutive patients with unit stay longer than 2 days; 34 patients were included during the control period, 43 in bay 1 (decontamination) and 46 in bay 3 (control) during the trial period. INTERVENTION: Erythromycin-base, 1 g t.i.d. in powder form administered by gastric tube to patients in bay 1 from admission to discharge. MEASUREMENTS AND RESULTS: Digestive tract carriage was monitored by cultures of gastric and rectal swab specimens, sampled twice a week. Enterobacteriaceae were isolated on Drigalski agar with incorporated ceftazidime (4 mg/l). In bay 1 there was a decrease in ESB producing Enterobacteriaceae (23% vs 10%, p = 0.0004) from rectal swab, especially in K. pneumoniae (15% vs 2%, p = 10(-5)), during the decontamination period in comparison to the control period. During the trial period the only differences observed between bays 1 and 3 were in the gastric samples: K. pneumoniae were less often isolated in bay 1 than in bay 3 (0% vs 3%, p = 0.03). Intestinal carriage with multiresistant Enterobacteriaceae occurred in 28% patients in bay 1 and 30% patients in bay 3 during the trial period (p = 0.79). Erythromycin-base did not delay the carriage by patients in bay 1 (log rank test p = 0.42). CONCLUSION: Erythromycin-base was not effective in preventing digestive tract carriage due to Enterobacteriaceae resistant to third generation cephalosporin by production of chromosomal cephalosporinase. The decrease in isolates containing K. pneumoniae in bay 1 cannot be definitively attributed to erythromycin-base, since the number of this species in bay 3 was low.

Multicenter evaluation of the antimicrobial activity for six broad-spectrum beta-lactams in Venezuela: comparison of data from 1997 and 1998 using the Etest method. Venezuelan Antimicrobial Resistance Study Group.

The minimum inhibitory concentrations of six broad-spectrum beta-lactam antimicrobial agents were determined in 1998 by use of the Etest versus a total of 502 bacteria in seven Venezuelan hospital laboratories. These data were compared with results of a similar study performed in 1997. The organisms tested included 309 recent clinical isolates of Enterobacteriaceae, 70 Pseudomonas aeruginosa, 54 Acinetobacter species, and 69 oxacillin-susceptible Staphylococcus aureus. Extended spectrum beta-lactamase production was noted among 30% of Klebsiella pneumoniae isolates. Hyperproduction of Amp C cephalosporinase producing resistance to ceftazidime and cefotaxime was observed with 10 to 37% of isolates of Enterobacter spp., Serratia spp., and Citrobacter freundii. The overall rank order of activity of the six beta-lactams tested in this study against all clinical isolates was imipenem (96.6% susceptible) > cefepime (90.4%) > piperacillin/tazobactam (85.7%) > ceftazidime (73.5%) > cefotaxime (70.5%) > piperacillin (55.0%). These findings were very similar to those reported for 1997.

In vitro evaluation of cefepime and other broad-spectrum beta-lactams in 22 medical centers in Japan: a phase II trial comparing two annual organism samples. The Japan Antimicrobial Resistance Study Group.

An antimicrobial resistance surveillance study in Japan is presented representing the second year (Phase II) results from 22 medical centers. Each participant laboratory tested (Etest, AB BIODISK, Solna, Sweden) 100 organisms, 10 strains each from 10 species groups including Escherichia coli, Klebsiella spp., Enterobacter spp., Citrobacter spp., indole-positive Proteae, Serratia spp., Acinetobacter spp., Pseudomonas aeruginosa, and oxacillin-susceptible Staphylococcus aureus and coagulase-negative staphylococci. Generally only modest variations in the activity of the studied broad-spectrum beta-lactams was observed compared to the study a year before. Specifically, extended spectrum beta-lactamase (ESBL) rates in E. coli increased (2.9 to 8.1%), but the ESBL rate in Klebsiella spp. fell (8.6 to 5.0%). Overall the resistance to the beta-lactams varied from a 4.7% decrease (ceftazidime as a consequence of a modified staphylococcal breakpoint criteria) to a 1.0% increase (cefepime, not significant). The rank order of spectrums in 1998 only changed for cefoperazone-sulbactam (6.1% resistance) that was active against more strains than cefpirome (6.8% resistance). The overall spectrum rank order for the 1998 Japan sample (% resistance) was: cefepime (3.2%) > imipenem (4.1%) > cefoperazone-sulbactam (6.1%) > cefpirome (6.8%) > ceftazidime (8.4%) > piperacillin (19.9%). As with a similar study in 1997, imipenem-resistant isolates of P. aeruginosa and Serratia spp. were discovered with metalloenzymes, usually found in the same medical centers. These results demonstrate the continued in vitro activity and potential sustained clinical efficacy of several broad-spectrum beta-lactams in Japan. Rapid emergence of new or novel resistance were not wide spread using a precise quantitative MIC system. Continued surveillance in this nation would be prudent to document the activity of this clinically valuable class of safe, antimicrobial agents.

Multicenter evaluation of antimicrobial resistance to six broad-spectrum beta-lactams in Colombia using the Etest method. The Colombian Antimicrobial Resistance Study Group.

The need for comprehensive and quantitative accurate antimicrobial resistance surveillance systems has become acute as a guide to problem recognition and to focus local interventions. A multilaboratory (10 medical centers) Colombia surveillance project was initiated in early 1997 to monitor the potency and spectrum of six (cefepime, cefotaxime, ceftazidime, cefoperazone/sulbactam, aztreonam, and imipenem) broad-spectrum antimicrobial agents tested against 100 organisms per participant center (802 strains). Ten groups of organisms were tested by a reference-quality method (Etest; AB BIODISK, Solna, Sweden) with results validated by concurrent quality control and additional challenge strain analysis. Results from nine qualifying medical centers were tabulated, and 95.7 to 96.8% of quality assurance tests were within expected ranges. Only cefepime (90.1-100.0% susceptible) and imipenem (96.3-100.0%) were active against all Enterobacteriaceae at > 90% of susceptible isolates using the breakpoint concentrations recommended by the National Committee for Clinical Laboratory Standards. Among ceftazidime- (or cefotaxime- or aztreonam-) resistant Enterobacter spp. and Citrobacter freundii, cefepime remained active, but not cefoperazone with sulbactam. Escherichia coli and Klebsiella spp. strains having resistance phenotypes consistent with extended spectrum beta-lactamase production were discovered in approximately 5 to 10% of isolates. All tested drugs except ceftazidime (31.8-57.7% susceptible) were active against > 94% of oxacillin-susceptible staphylococci. Similar rates of resistance (9.1-14.8%) were observed in Pseudomonas aeruginosa for five of six drugs (not cefotaxime; 15.9% of strains were susceptible). Acinetobacter spp. isolates were most susceptible to imipenem (95.8%), cefepime (86.1%), and cefoperazone/sulbactam (83.3%). Overall for the 1997 order of antimicrobial spectrums for these tested compounds was: imipenem (96.6%) > cefepime (93.6%) > cefoperazone/sulbactam (90.5%) > cefotaxime (74.9%) > aztreonam (74.3% for Gram-negative bacilli only) > ceftazidime (73.2%). These data should be used to guide empiric regimens in Colombia, and additionally will provide a resistance statistical baseline to which future studies in this nation can be compared.

R-factor mediated beta-lactamase production by Haemophilus influenzae.

Production of beta-lactamase by 15 strains of Haemophilus influenzae has been investigated. All the strains produce a constitutive beta-lactamase, which readily hydrolyses penicillin G, ampicillin, and cephaloridine. The beta-lactamase produced by these strains is indistinguishable from the type-IIIa enzyme commonly found in strains of Escherichia coli. The beta-lactamase gene has been transferred from the enzyme-producing strains of Haemophilus to strains of H. parainfluenzae and a strain of E. coli.

beta-Lactamase and beta-lactam antibiotics resistance in acinetobacter anitratum (syn: A. calcoaceticus).

The cephalosporin beta-lactamase (cephalosporinase) produced by Acinetobacter was studied. The enzyme was partially purified by means of column chromatography and its properties were investigated. The enzyme was induced by benzylpenicillin, 6-aminopenicillanic acid and cephaloridine. Its molecular weight is 30,000 its optimal temperature 40C, and its optimal pH 7.25 similar to 7.50. Substrate specificity studies using various cephalosporins and penicillins, showed that the enzyme functioned as a cephalosporinase rather than penicillinase.

Localization of cephalosporinase in Enterobacter cloacae by immunocytochemical examination.

Enterobacter cloacae NUH10 was isolated at Nagasaki University Hospital in 1987. E. cloacae NUH10 is a mutant strain which produces high levels of cephalosporinase. E. cloacae ATCC 23355 is known to be sensitive to so-called third generation cephems and produces an inducible cephalosporinase. The polyclonal antibody to cephalosporinase extracted from E. cloacae NUH10 was utilized in post-embedding immunogold labeling in order to localize this protein in E. cloacae ATCC 23355 and E. cloacae NUH10. Immunocytochemical localization of the cephalosporinase in both strains was observed with and without incubation with an inducer. Cephalosporinase was detected in both the cytoplasm and periplasmic space of E. cloacae ATCC 23355 and E. cloacae NUH10 incubated in medium including cefoxitin as an inducer. In the case of incubation without the inducer, a small quantity of cephalosporinase was located in the periplasmic space in either strain of bacteria. Western blot analysis showed that cephalosporinase was predominantly localized in the periplasmic space rather than in the cytoplasmic space.

Characteristics of aztreonam as a substrate, inhibitor and inducer for beta-lactamases.

Aztreonam was investigated as to its characteristics as a substrate, inhibitor and inducer for the well-defined beta-lactamases of Gram-negative bacteria, and its antibacterial efficacy as to bacterial cells producing eight types of beta-lactamases was also evaluated. Aztreonam was hydrolyzed at measurable rates by class A beta-lactamases, a TEM-2 type penicillinase and the Proteus vulgaris cephalosporinase with a broad substrate range. However, the affinity of aztreonam for the class A enzymes was low, this property being well reflected by its high antibacterial activity toward producers of class A beta-lactamases. Aztreonam was extremely stable as to the typical class C cephalosporinase of Citrobacter freundii, and acted as a competitive and progressive inhibitor for the beta-lactamase. While the MICs of aztreonam in the cases of the constitutive producers of class C beta-lactamases were evidently affected by enzyme production. An experiment involving aztreonam as a inhibitor in combination with a hydrolyzable beta-lactam gave ambiguous results, however, a strong synergistic effect was found in combination with mecillinam. Using Pseudomonas aeruginosa, aztreonam was confirmed to be a poor inducer of beta-lactamases.