A novel type of AmpC beta-lactamase, ACC-1, produced by a Klebsiella pneumoniae strain causing nosocomial pneumonia.

A Klebsiella pneumoniae strain resistant to oxyimino cephalosporins was cultured from respiratory secretions of a patient suffering from nosocomial pneumonia in Kiel, Germany, in 1997. The isolate harbors a bla resistance gene located on a transmissible plasmid. An Escherichia coli transconjugant produces a beta-lactamase with an isoelectric point of 7.7 and a resistance phenotype characteristic of an AmpC (class 1) beta-lactamase except for low MICs of cephamycins. The bla gene was cloned and sequenced. It encodes a protein of 386 amino acids with the active site serine of the S-X-X-K motif at position 64, as is characteristic for class C beta-lactamases. Multiple alignment of the deduced amino acid sequence with 21 other AmpC beta-lactamases demonstrates only very distant homology, reaching at maximum 52.3% identity for the chromosomal AmpC beta-lactamase of Serratia marcescens SR50. The beta-lactamase of K. pneumoniae KUS represents a new type of AmpC-class enzyme, for which we propose the designation ACC-1 (Ambler class C-1).

Differential autocrine regulation of intestine epithelial cell proliferation and differentiation by insulin-like growth factor (IGF) system components.

The mechanisms which regulate cell turnover in the intestinal epithelium are incompletely understood. The present study was performed to characterize the role of autocrine IGF system components in intestine epithelial cell proliferation and differentiation comparing rapidly growing crypt cells (IEC-6) with differentiating enterocytes (CaCo-2). The autocrine release of IGF-I, IGF-II and IGFBP-1 through -3 was determined by specific RIAs and western ligand blotting. In addition, binding and growth-promoting activity of insulin, IGF-I and IGF-II was investigated. Enterocytic differentiation was assessed by measuring the brush-border enzymes alkaline phosphatase and sucrase. During IEC-6 growth, the autocrine release of IGF-I and -II increased, whereas IGFBP-2 levels decreased. Specific receptors for IGF-I and IGF-II but not insulin could be detected. IGF-I was 100-fold more potent than insulin to stimulate IEC-6 cell proliferation. In contrast, CaCo-2 cells revealed higher binding of insulin than IGF-I/-II and no release of IGF-I. At switch from CaCo-2 cell proliferation to differentiation a marked increase in the secretion of IGF-II (10-fold), IGFBP-1 (2.5-fold), IGFBP-2 (3-fold), and IGFBP-3 (6-fold) was measured. Our data indicate that IGF system components differentially modulate enterocytic cell proliferation and differentiation.

Discrimination of Burkholderia multivorans and Burkholderia vietnamiensis from Burkholderia cepacia genomovars I, III, and IV by PCR.

We present a PCR procedure for identification of Burkholderia cepacia, Burkholderia multivorans, and Burkholderia vietnamiensis. 16S and 23S ribosomal DNAs (rDNAs) of B. multivorans and B. vietnamiensis were sequenced and aligned with published sequences for definition of species-specific 18-mer oligonucleotide primers. Specific antisense 16S rDNA primers (for B. cepacia, 5'-AGC ACT CCC RCC TCT CAG-3'; for B. multivorans, 5'-AGC ACT CCC GAA TCT CTT-3') and 23S rDNA primers (for B. vietnamiensis, 5'-TCC TAC CAT GCG TGC AA-3') were paired with a general sense primer of 16S rDNAs (5'-AGR GTT YGA TYM TGG CTC AG-3') or with a sense primer of 23S rDNA (5'-CCT TTG GGT CAT CCT GGA-3'). PCR with these primers under optimized conditions is appropriate to specifically and rapidly identify B. multivorans, B. vietnamiensis, and B. cepacia (genomovars I, III, and IV are not discriminated). In comparison with the polyphasic taxonomic analyses presently necessary for species and genomovar identification within the B. cepacia complex, our procedure is more rapid and easier to perform and may contribute to clarifying the clinical significance of individual members of the complex in cystic fibrosis.

Proinsulin stimulates growth of small intestinal crypt-like cells acting via specific receptors.

The mechanisms that regulate cell turnover in the intestinal epithelium are incompletely understood. Here we tested the hypothesis that proinsulin, present in serum and pancreatic juice in picomolar concentrations, stimulates growth of the rat small intestinal crypt-like cell line IEC-6 under serum-free conditions. Proinsulin binding was assessed by competitive ligand binding studies. Proinsulin and insulin-like growth factor I (IGF-I) stimulated cell proliferation up to threefold above controls, with half-maximal action already in the picomolar range and with additive effects. In early confluent cell monolayers, proinsulin bound with higher affinity (IC50 1.3 +/- 0.05 nM) and capacity (87,200 +/- 2,500 receptors/cell) than IGF-I (4.0 +/- 0.6; 23,700 +/- 2,200, P < 0. 05). C-peptide competed with 10-fold lower affinity for binding of 125I-proinsulin but not for 125I-IGF-I or 125I-insulin, suggesting a specific binding epitope of the proinsulin molecule within or close to the C-peptide region. In contrast, insulin showed approximately 100-fold lower binding affinity and growth-promoting potency than proinsulin or IGF-I. We conclude that proinsulin stimulates growth of small intestinal crypt cells through specific binding and may play a physiological role in the regulation of intestinal epithelial cell proliferation.

[Imipenem resistance in Pseudomonas aeruginosa]. / Imipenem-Resistenz bei Pseudomonas aeruginosa.

In 1997 in western Austria, 9.9% of Pseudomonas aeruginosa strains from patients of general practitioners were resistant to imipenem as well as 18.2% of the isolates from hospitals and 20.2% of the strains at a university teaching hospital. Within the hospital the imipenem resistance varied from 9.9% among out-patients to 28.7% in isolates from intensive care units. In medical/surgical words, up to 15.1% of P. aeruginosa strains were resistant to imipenem. The incidence of imipenem-resistant P. aeruginosa strains correlates to the use of carbapenems. In June 1997, 10 consecutive isolates from 8 patients were obtained and typed using restriction fragment length polymorphism analysis (RFLP) and Pyocin typing. All 10 isolates were resistant to meropenem as well as to imipenem. The finding (by RFLP and Pyocin typing) of individual bacterial types in each isolate strongly contradicts the spread of infection by cross infection. However, all patients were proven to have been treated with imipenem during the 3 months prior to testing. In 1997, 13,880 g of imipenem were used at the university hospital in Innsbruck. The use of carbapenems appears to be the main cause for the increased incidence of imipenem-resistant P. aeruginosa strains.

Molecular procedure for rapid detection of Burkholderia mallei and Burkholderia pseudomallei.

A PCR procedure for the discrimination of Burkholderia mallei and Burkholderia pseudomallei was developed. It is based on the nucleotide difference T 2143 C (T versus C at position 2143) between B. mallei and B. pseudomallei detected in the 23S rDNA sequences. In comparison with conventional methods the procedure allows more rapid identification at reduced risk for infection of laboratory personnel.

Discrimination of Burkholderia gladioli from other Burkholderia species detectable in cystic fibrosis patients by PCR.

A procedure for molecular identification of Burkholderia gladioli is described. Specific 16S and 23S rRNA gene signature sequences were defined as primers for PCR. The method allows rapid and specific discrimination of B. gladioli from related species (B. cepacia, B. multivorans, B. vietnamiensis, B. mallei, B. pseudomallei, Ralstonia pickettii, and R. eutropha) and should contribute to the clarification of its role as a human pathogen, e.g., in cystic fibrosis.

Cefotaxime-resistant Enterobacteriaceae isolates from a hospital in Warsaw, Poland: identification of a new CTX-M-3 cefotaxime-hydrolyzing beta-lactamase that is closely related to the CTX-M-1/MEN-1 enzyme.

A group of cefotaxime-resistant Citrobacter freundii and Escherichia coli isolates were collected by a clinical laboratory in a hospital in Warsaw, Poland, in July 1996. Detailed analysis has shown that all of these produced a beta-lactamase (pI, 8.4) belonging to the CTX-M family, one of the minor extended-spectrum beta-lactamase families with a strong cefotaxime-hydrolyzing activity. Sequencing has revealed that C. freundii isolates produced a new CTX-M-3 enzyme which is very closely related to the CTX-M-1/MEN-1 beta-lactamase, sporadically identified in Europe over a period of 6 years. Amino acid sequences of these two beta-lactamases differ at four positions: Val77Ala, Asp114Asn, Ser140Ala, and Asn288Asp (the first amino acid of each pair refers to CTX-M-1/MEN-1 and second refers to CTX-M-3). The partial sequence of the E. coli CTX-M gene was identical to the corresponding region of bla(CTX-M-3), but a transconjugant of the E. coli isolate expressed higher levels of resistance to beta-lactams than did C. freundii transconjugants. These resistance differences correlated with differences in plasmid DNA restriction patterns. Our results suggest that CTX-M genes have been spread among different species of the family Enterobacteriaceae in the hospital and that the CTX-M-3-expressing C. freundii strain causing routine urinary tract infections has been maintained for a relatively long time in the hospital environment.

Ceftazidime-resistant Enterobacteriaceae isolates from three Polish hospitals: identification of three novel TEM- and SHV-5-type extended-spectrum beta-lactamases.

Twelve ceftazidime-resistant isolates of the family Enterobacteriaceae (11 Klebsiella pneumoniae isolates and 1 Escherichia coli isolate) were collected in 1995 from three Polish hospitals located in different cities. All were identified as producers of extended-spectrum beta-lactamases (ESBLs). Detailed analysis of their beta-lactamase contents revealed that six of them expressed SHV-5-like ESBLs. The remaining six were found to produce three different TEM enzymes, each characterized by a pI value of 6.0 and specified by new combinations of amino acid substitutions. The amino acid substitutions compared to the TEM-1 beta-lactamase sequence were Gly238Ser, Glu240Lys, and Thr265Met for TEM-47; Leu21Phe, Gly238Ser, Glu240Lys, and Thr265Met for TEM-48; and Leu21Phe, Gly238Ser, Glu240Lys, Thr265Met, and Ser268Gly for TEM-49. The new TEM beta-lactamases, TEM-47, TEM-48, and TEM-49, belong to a subfamily of TEM-2-related enzymes. Genes coding for TEM-47 and TEM-49 could have originated from the TEM-48-encoding sequence by various single genetic events. The new TEM derivatives probably document the already advanced microevolution of ESBLs ongoing in Polish hospitals, in a majority of which no monitoring of ESBL producers was performed before 1996.

A novel class C beta-lactamase (FOX-2) in Escherichia coli conferring resistance to cephamycins.

An Escherichia coli strain resistant to a broad spectrum of beta-lactams, including cephamycins, was isolated from a patient suffering from urinary tract infection. A resistance plasmid (pMVP-7) was transferred from the clinical isolate to an Escherichia coli recipient. Both strains produce a cefoxitin-hydrolyzing beta-lactamase focusing at pI 6.7. The phenotype was similar to that of a Klebsiella pneumoniae strain producing cephamycinase FOX-1, so primers were selected from the FOX-1 sequence to amplify the bla gene of the transconjugant. The PCR product obtained was sequenced. The percentage of identity of the deduced amino acid sequence with sequences of other AmpC-type beta-lactamases was 96.9% with FOX-1, 74.9% with CMY-1, and 67.7% with MOX-1. This new plasmid-mediated enzyme is most closely related to FOX-1 (11 amino acid exchanges). We therefore propose the designation FOX-2.

Comparative characterization of the cephamycinase blaCMY-1 gene and its relationship with other beta-lactamase genes.

A plasmidic beta-lactamase which hydrolyzed cephamycins was first detected and reported in 1989. At that time its description was restricted to phenotypic characteristics. We analyzed nucleotide sequence of its gene and explored it genetic relationship with other bla genes. The deduced amino acid sequence of the blaCMY-1 product was compared with those of other known plasmidic cephamycinases and of chromosomal AmpC beta-lactamases. The results indicate that the relationship of CMY-1 is closest to MOX-1 among the plasmidic cephamycinases and to AmpC of Pseudomonas aeruginosa among the chromosomal cephalosporinases. We conclude that the plasmidic cephamycinases described up to now may be classified into three families, as follows: CMY-1, MOX-1, and FOX-1 with AmpC of P. aeruginosa; CMY-2, BIL-1 and LAT-1 with AmpC of Citrobacter freundii; and MIR-1 with AmpC of Enterobacter cloacae. Plasmidic cephamycinases are now recognized as clinically relevant class C beta-lactamases.

Characterization of beta-lactamase gene blaPER-2, which encodes an extended-spectrum class A beta-lactamase.

Plasmidic extended-spectrum beta-lactamases of Ambler class A are mostly inactive against ceftibuten. Salmonella typhimurium JMC isolated in Argentina harbors a bla gene located on a plasmid (pMVP-5) which confers transferable resistance to oxyiminocephalosporins, aztreonam, and ceftibuten. The beta-lactamase PER-2 (formerly ceftibutenase-1; CTI-1) is highly susceptible to inhibition by clavulanate and is located at a pI of 5.4 after isoelectric focusing. The blaPER-2 gene was cloned and sequenced. The nucleotide sequence of a 2.2-kb insert in vector pBluescript includes an open reading frame of 927 bp. Comparison of the deduced amino acid sequence of PER-2 with those of other beta-lactamases indicates that PER-2 is not closely related to TEM or SHV enzymes (25 to 26% homology). PER-2 is most closely related to PER-1 (86.4% homology), an Ambler class A enzyme first detected in Pseudomonas aeruginosa. An enzyme with an amino acid sequence identical to that of PER-1, meanwhile, was found in various members of the family Enterobacteriaceae isolated from patients in Turkey. Our data indicate that PER-2 and PER-1 represent a new group of Ambler class A extended-spectrum beta-lactamases. PER-2 so far has been detected only in pathogens (S. typhimurium, Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis) isolated from patients in South America, while the incidence of PER-1-producing strains so far has been restricted to Turkey, where it occurs both in members of the family Enterobacteriaceae and in P. aeruginosa.

Sequences of beta-lactamase genes encoding CTX-M-1 (MEN-1) and CTX-M-2 and relationship of their amino acid sequences with those of other beta-lactamases.

Amino acid sequences determined either by protein sequencing or by DNA sequencing are identical for cefotaximases CTX-M-1 and MEN-1, whereas CTX-M-2 is 84% identical to CTX-M-1/MEN-1. Both beta-lactamases are distantly related to other plasmidic class A enzymes (homology to TEM-1 is 38.1% for CTX-M-1/MEN-1 and 36.5% for CTX-M-2); the closest relationship was with the chromosomal beta-lactamase of Klebsiella oxytoca E23004 (homologies of 74.5% for CTX-M-1/MEN-1 and 77.9% for CTX-M-2). The cefotaximases CTX-M-1/MEN-1 and CTX-M-2 represent two members of a new subgroup of plasmidic class A beta-lactamases.

Characterization of the plasmidic beta-lactamase CMY-2, which is responsible for cephamycin resistance.

The phenotype of Klebsiella pneumoniae HEL-1 indicates a plasmidic cephamycinase gene (blaCMY-2). Its sequence shows one open reading frame coding for a protein of 381 amino acids. CMY-2 is classified as class C beta-lactamase that is closely related to the plasmidic enzymes BIL-1 and LAT-1 and the chromosomal AmpC of Citrobacter freundii. The blaCMY-2 gene possibly was translocated onto a plasmid of C. freundii which spread to K. pneumoniae.

Comparative pharmacodynamics of clarithromycin and azithromycin against respiratory pathogens.

The differences between the antibacterial activities of new macrolides such as clarithromycin (CLA) and azithromycin (AZI) against common respiratory tract pathogens are only minor. However, CLA and AZI constitute macrolides with extremely different pharmacokinetic profiles. This constellation presents an opportunity to evaluate the effect of the pharmacokinetic profile on antibacterial kinetics comparatively. In a pharmacodynamic model simulating the dynamics of serum concentrations in bacterial cultures, both CLA and AZI demonstrate bactericidal activity at concentrations reached in human blood at recommended dosages (CLA 250 mg b.i.d., AZI 500 mg o.i.d.). Bactericidal activity of CLA against the variety of pathogens included is superior to that of AZI in the rate and the extent of killing in this model. These results are considered to correlate with the antibacterial effect of macrolides in vivo in cases where pathogens enter the blood stream. Furthermore, mutants with susceptibility reduced between 8 and 16 times in relation to the initial strain of all strains having an initial minimal inhibitory concentration (MIC) < or = 0.25 mg/l, are selected during exposure to AZI, but not to CLA. The pharmacokinetic profiles of CLA and AZI thus strongly influence their antibacterial effect in the pharmacodynamic model, allowing both higher bactericidal activity and greater reduction of the risk of selection of resistant mutants with CLA than with AZI. As a whole, the pharmacodynamics of these macrolides are determined more by the proportion of the MICs to the maximum serum concentration than by the relation of the MICs to the area under the curve.

Influence of cholesterol supply on cell growth and differentiation in cultured enterocytes (CaCo-2).

When used as treatment for hypercholesterolemia HMG-CoA reductase inhibitors will first pass through and act upon the gut mucosa. Although cholesterol availability is essential for cell growth of the intestinal mucosa adverse intestinal events are rare which is possibly due to hitherto undefined compensatory mechanisms. In the present work we therefore studied the long-term influence of mevinolin on proliferation and differentiation of CaCo-2 cells as an enterocyte model and their response upon the cholesterol supply of different origin. Mevinolin caused a marked and dose-dependent inhibition of cell proliferation, microvilli length and alkaline phosphatase. This parallel suppression was reversed by the addition of either exogenous free cholesterol, endogenous cholesterol from mevalonolactone or LDL but not HDL3. Surprisingly, sucrase activity reacted in an inverse fashion to alkaline phosphatase activity. Mevinolin induced enzyme activity and this was further enhanced by mevalonolactone supply, while cholesterol and LDL normalized sucrase to controls. In conclusion, the presence of luminal cholesterol as well as plasma LDL as the cholesterol source for the enterocyte may prevent mevinolin toxicity.

A new plasmidic cefotaximase from patients infected with Salmonella typhimurium.

Salmonella typhimurium strains resistant to most beta-lactams, co-trimoxazole, tobramycin and gentamicin were isolated from patients in two hospitals in Buenos Aires, Argentina, beginning in August 1990. The patients were suffering from meningitis, septicaemia or enteritis. Therapy including ampicillin, ceftriaxone and gentamicin failed. The strains produced a plasmidic (pMVP-4) extended broad-spectrum beta-lactamase which is more active against cefotaxime than against ceftazidime (Vmax for cefotaxime 350 times higher than for ceftazidime). This cefotaximase demonstrates similarity to the previously described CTX-ase-M-1 from Escherichia coli, but it is distinctly different from CTX-ase-M-1 by its isoelectric point (7.9 for CTX-ase-M-2 in comparison with 8.9 for CTX-ase-M-1) as well as in its lower susceptibility to the beta-lactamase inhibitors sulbactam, clavulanic acid, tazobactam and BRL 42715. Thus, the beta-lactamase produced by S. typhimurium strains from Argentina appears to represent a new member (CTX-ase-M-2) of a novel group of plasmidic extended broad-spectrum beta-lactamases designated as cefotaximases.

RU 29 246, the active compound of the cephalosporin-prodrug-ester HR 916. I. Antibacterial activity in vitro.

The aminothiazolyl-cephalosporin RU 29 246 is the active metabolite of the prodrug-pivaloyl-oxyethyl-ester HR 916. RU 29 246 in vitro activity includes a wide range of clinically relevant bacterial pathogens. Against methicillin-sensitive Staphylococci RU 29 246 (MIC90 of 0.25 approximately 2 micrograms/ml) was clearly more active than cefaclor, cefuroxime, cefpodoxime, cefixime and ceftibuten, but slightly less active than cefdinir. RU 29 246 inhibited hemolytic Streptococci of the serogroups A, B, C and G as well as penicillin-sensitive Streptococcus pneumoniae at concentrations similar to cefdinir, cefpodoxime and cefuroxime (MIC90 less than or equal to 0.13 micrograms/ml), but less than the other oral cephalosporins investigated (cefixime, cefaclor and ceftibuten). MIC90s of RU 29 246 against Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Salmonella spp., Shigella spp., Proteus mirabilis and Haemophilus influenzae were less than or equal to 0.5 micrograms/ml. Only RU 29 246 and cefdinir demonstrated moderate activity against Acinetobacter baumannii (MIC90 greater than or equal to 4 micrograms/ml). Most strains of Pseudomonas spp., Serratia marcescens, Enterobacter spp., Hafnia alvei and Bacteroides spp. were resistant to RU 29 246. RU 29 246 killed Escherichia coli and Staphylococcus aureus at a rate of 99% to 99.9% at concentrations of two times MIC. The pH value of the medium (range 5.5 to 8.5) and the inoculum size (range 10(5) to 10(7) cfu/ml) had no or only low influence on the antibacterial activity of RU 29 246. RU 29 246 is a broad spectrum cephalosporin including in its activity both Gram-positive and Gram-negative pathogens and therefore--depending on the bioavailability of its prodrug--looks promising as to its therapeutic perspective.

RU 29 246, the active compound of the cephalosporin-prodrug-ester HR 916. II. Stability to beta-lactamases and affinity for penicillin-binding proteins.

The aminothiazolyl-cephalosporin RU 29 246, the active metabolite of the prodrug-ester HR 916, is active against strains producing the widespread plasmid-encoded TEM-1, TEM-2 and SHV-1 beta-lactamases. Except for TEM-7 the activity of RU 29 246 against strains producing extended broad spectrum beta-lactamases (TEM-3, TEM-5, TEM-6, SHV-2, SHV-4, SHV-5, CMY-1, CTX-M), however, is low. Relative hydrolysis rates of RU 29 246 are comparable with those of cefpodoxime, the active metabolite of CS-807, and are extremely low for the TEM-1 and SHV-1 beta-lactamases. The compound demonstrates remarkable inhibitory activity against the chromosomal beta-lactamase of Enterobacter cloacae P99. In the presence of 1.7 microM this enzyme loses 50% of its activity. At concentrations of 0.43, 0.003 and 0.01 micrograms/ml the compound binds preferentially to the penicillin-binding protein (PBP) 3 of Escherichia coli K12, to the PBPs 2x and 3 of Streptococcus pneumoniae R6 and to PBP 1 of Staphylococcus aureus SG 511, respectively.