Evaluations of the Effects of Extremely Low-Frequency Electromagnetic Fields on Growth and Antibiotic Susceptibility of Escherichia coli and Pseudomonas aeruginosa.

We aimed to investigate the effects of exposure to extremely low-frequency electromagnetic fields (2 mT; 50 Hz) on the growth rate and antibiotic sensitivity of E. coli ATCC 25922 and P. aeruginosa ATCC 27853. The electromagnetic field treatment significantly influenced the growth rate of both strains when incubated in the presence of subinhibitory concentrations of kanamycin (1 µg/mL) and amikacin (0.5 µg/mL), respectively. In particular, at 4, 6, and 8 h of incubation the number of cells was significantly decreased in bacteria exposed to electromagnetic field when compared with the control. Additionally, at 24 h of incubation, the percentage of cells increased (P. aeruginosa∼42%; E. coli∼5%) in treated groups with respect to control groups suggesting a progressive adaptive response. By contrast, no remarkable differences were found in the antibiotic susceptibility and on the growth rate of both bacteria comparing exposed groups with control groups.

QnrB9 in association with TEM-116 extended-spectrum beta-lactamase in Citrobacter freundii isolated from sewage effluent: first report from Italy.

The aim of the present study was to investigate the mechanism of quinolone and beta-lactam resistance in an isolate of Citrobacter freundii PC2/08 collected from sewage effluent from l'Aquila, Italy. QnrB-9 and bla(TEM-116 )were co-expressed in a large plasmid identified in C. freundi PC2/08 strain. Compared to TEM-1, TEM-116 showed two single mutations: V84I and A184V. The plPC2/08 plasmid conferred resistance to several beta-lactams and fluoroquinolones. Tazobactam could be considered a good inhibitor whereas clavulanic acid was unable to restore susceptibility to amoxicillin. The QnrB-9 element seems to confer the same level of resistance to levofloxacin and ciprofloxacin with minimum inhibitory concentration (MIC) values of 4 mg/l for either. In this study, we confirm the common association of plasmid-mediated quinolone resistance with extended-spectrum beta-lactamase (ESbetaL) production. This is the first finding in Italy of qnrB9 and TEM-116 in a non-clinical or animal strain.

In vitro selection and characterization of mutants in TEM-1-producing Escherichia coli by ceftazidime and ceftibuten.

The present work was undertaken to study the ability of ceftazidime and ceftibuten to selectin vitro Escherichia coli HB101 harboring bla(TEM-1) beta-lactamase gene. Minimum inhibitory concentrations (MICs) of ceftazidime and ceftibuten were increased by a factor of 32, overcoming in the case of ceftazidime the breakpoint for clinical resistance. Outer membrane protein analysis and PCR for bla(TEM )alleles revealed that ceftazidime and ceftibuten select for different resistance mechanisms. Ceftazidime created mutants that encode an extended-spectrum beta-lactamase (TEM-12) and exhibit decreased expression of OmpF. Ceftibuten was unable to select for extended-spectrum beta-lactamase expressing mutants but reduced the expression of two porins, OmpC and OmpF. The stability of ceftibuten to hydrolysis and the difference in the structure of these beta-lactam antibiotics could be responsible for the selection of different mechanisms of resistance.

Meropenem stability to beta-lactamase hydrolysis and comparative in vitro activity against several beta-lactamase-producing Gram-negative strains.

The interaction between meropenem and class A, B, C and D beta-lactamases was studied by a spectrophotometric method. Class A, C and D beta-lactamases were unable to confer in vitro resistance to carbapenems. Surprisingly, several class B metallo-beta-lactamases expressed in Escherichia coli failed to confer resistance when a conventional inoculum (105 cfu/mL) was used.

Ceftibuten stability to active-site serine and metallo-beta-lactamases.

Ceftibuten is an oral third-generation cephalosporin active against a wide range of bacteria and shows an improved stability to hydrolysis by several beta-lactamases because of the carboxyethilidine moiety at position 7 of the ss-acyl side chain. The kinetic interactions between ceftibuten and active-site serine and metallo-ss-lactamases were investigated. The activity of several TEM-derived extended spectrum beta-lactamases (ESbetaLs) against ceftibuten, cefotaxime and ceftazidime was compared using K(m), K(cat) and K(cat)/K(m). Ceftibuten behaved as a poor substrate for class A and B beta-lactamases compared with cefotaxime. The chromosomal class C beta-lactamase from Enterobacter cloacae 908R gave a high K(cat) value (21 s(-1)), whereas there was poor activity with enzymes from Acinetobacter baumannii and Morganella morganii and ceftibuten. Ceftibuten resists hydrolysis in the presence of typical respiratory or urogenital-tract pathogens producing beta-lactamases.

TEM-72, a new extended-spectrum beta-lactamase detected in Proteus mirabilis and Morganella morganii in Italy.

A new natural TEM-2 derivative, named TEM-72, was identified in a Proteus mirabilis strain and in a Morganella morganii strain isolated in Italy in 1999. Compared to TEM-1, TEM-72 contains the following amino acid substitutions: Q39K, M182T, G238S, and E240K. Kinetic analysis showed that TEM-72 exhibits an extended-spectrum activity, including activity against oxyimino-cephalosporins and aztreonam. Expression of bla(TEM-72) in Escherichia coli was capable of decreasing the host susceptibility to the above drugs.

A kinetic study on the interaction between tazobactam (a penicillanic acid sulphone derivative) and active-site serine beta-lactamases.

The interaction between tazobactam and several chromosome- and plasmid-encoded (TEM, SHV, PSE types) class A and C beta-lactamases was studied by spectrophotometry. Tazobactam behaved as a competitive inhibitor or inactivator able to restore in several cases the efficiency of piperacillin as a partner beta-lactam. A detailed kinetic analysis permitted measurement of the acylation efficiency for some cephalosporinases and broad-spectrum beta-lactamases; the presence of a turn-over of acyl-enzyme complex was also evaluated.

Bactericidal activity of levofloxacin and ciprofloxacin on clinical isolates of different phenotypes of Pseudomonas aeruginosa.

Levofloxacin has been reported to have in vitro activity against both gram-positive and gram-negative bacteria. A recent survey carried out at our Institution showed clinical isolates of Pseudomonas aeruginosa to be more susceptible to levofloxacin than to ciprofloxacin. The in vitro activity of the two fluoroquinolones was evaluated further by looking at their bactericidal activity against two strains of each of the following antibio-phenotypes of P. aeruginosa: levofloxacin- and ciprofloxacin-susceptible, levofloxacin-susceptible/ciprofloxacin-resistant, levofloxacin-susceptible/ciprofloxacin-susceptible and ceftazidime-resistant, (National Committee for Clinical Laboratory Standards susceptibility breakpoints were used). MIC and MBC values were measured and time-kill experiments were carried out. Drugs were used at susceptibility or resistance breakpoint concentrations in the time-kill experiments and results were recorded over 12 h in an attempt to link in vitro results with the clinical situation The polypeptide profiles of outer membrane preparations of the six strains were examined by gel electrophoresis. Levofloxacin was shown to be more bactericidal than ciprofloxacin in the time-kill experiments. No differences were observed between the outer membrane proteins of the six strains. Levofloxacin showed greater bactericidal activity against P. aeruginosa clinical isolates than ciprofloxacin.

Italian survey on comparative levofloxacin susceptibility in 334 clinical isolates of Pseudomonas aeruginosa.

A national survey on susceptibility patterns of 334 Pseudomonas aeruginosa isolates from intensive care units and hematology and oncology wards from 13 Italian hospitals compared the in vitro activity of levofloxacin, an injectable oral fluoroquinolone, to those of ciprofloxacin, ofloxacin, ceftazidime, imipenem, amikacin, and gentamicin. Amikacin and imipenem had the best susceptibility profiles. The activity of levofloxacin was superior to those of the other quinolones and was comparable to that of ceftazidime. The effect of levofloxacin in vitro on P. aeruginosa clinical isolates suggests that further clinical investigations are warranted.

A Kinetic Study on the Interaction between Tazobactam (A Penicillanic Acid Sulphone Derivative) and Active-Site Serine beta-Lactamases.

The interaction between tazobactam and several chromosome- and plasmid-encoded (TEM, SHV, PSE types) class A and C beta-lactamases was studied by spectrophotometry. Tazobactam behaved as a competitive inhibitor or inactivator able to restore in several cases the efficiency of piperacillin as a partner beta-lactam. A detailed kinetic analysis permitted measurement of the acylation efficiency for some cephalosporinases and broad-spectrum beta-lactamases; the presence of a turn-over of acyl-enzyme complex was also evaluated.

In vitro activity of piperacillin/tazobactam against 615 Pseudomonas aeruginosa strains isolated in intensive care units.

From May 1996 to September 1997, 615 Pseudomonas aeruginosa strains isolated from patients in intensive care units collected from different Italian laboratories were studied. The susceptibility of piperacillin/tazobactam, in comparison with other antipseudomonal antibiotics, to their NCCLS breakpoints was evaluated: amikacin 79. 6%, carbenicillin 67.0%, ceftazidime 73.4%, ciprofloxacin 55.8%, imipenem 64.1%, piperacillin 88.1%, piperacillin/tazobactam 92.4% and ticarcillin/clavulanic acid 69.0%. Seventy-three strains were selected because of their resistance to piperacillin and the mechanisms underlying such a resistance were investigated. Isoelectric focusing and hydrolysis assays revealed the presence of 15 plasmid-mediated beta-lactamases. Chromosomal beta-lactamase derepression was demonstrated in 34 isolates. The remaining 24 piperacillin-resistant strains did not produce beta-lactamases and an 'intrinsic mechanism' of resistance was inferred. The piperacillin/tazobactam combination restored resistance in 25 piperacillin strains. Nine of these were derepressed for chromosomal beta-lactamase, 8 showed impermeability and 8 showed plasmid enzymes.

Ceftazidime and aztreonam resistance in Providencia stuartii: characterization of a natural TEM-derived extended-spectrum beta-lactamase, TEM-60.

A plasmid-encoded beta-lactamase produced from a clinical strain of Providencia stuartii has been purified and characterized. The gene coding for the beta-lactamase was cloned and sequenced. It appears to be a new natural TEM-derived enzyme, named TEM-60. Point mutations (Q39K, L51P, E104K, and R164S) are present with respect to the TEM-1 enzyme; the mutation L51P has never been previously reported, with the exception of the chromosomally encoded extended-spectrum beta-lactamase PER-1. Kinetic parameters relative to penicillins, cephalosporins, and monobactams other than mechanism-based inactivators were related to the in vitro susceptibility phenotype.

Interactions of biapenem with active-site serine and metallo-beta-lactamases.

Biapenem, formerly LJC 10,627 or L-627, a carbapenem antibiotic, was studied in its interactions with 12 beta-lactamases belonging to the four molecular classes proposed by R. P. Ambler (Philos. Trans. R. Soc. Lond. Biol. Sci. 289:321-331, 1980). Kinetic parameters were determined. Biapenem was readily inactivated by metallo-beta-lactamases but behaved as a transient inhibitor of the active-site serine enzymes tested, although with different acylation efficiency values. Class A and class D beta-lactamases were unable to confer in vitro resistance toward this carbapenem antibiotic. Surprisingly, the same situation was found in the case of class B enzymes from Aeromonas hydrophila AE036 and Bacillus cereus 5/B/6 when expressed in Escherichia coli strains.

In vitro activity of cefodizime (HR-221) in combination with beta-lactamase inhibitors.

Cefodizime (formerly HR221) was tested either for in vitro microbiological activity or for its stability to beta-lactamases in the presence of two beta-lactamase inhibitors (clavulanic acid, tazobactam). Cefodizime was a poor substrate of class C enzymes but hyperproducer strains were generally resistant with or without a beta-lactamase inhibitor used in combination. On the contrary, class A enzymes were able to hydrolyze cefodizime. However, strains expressing class A beta-lactamase were susceptible to cefodizime in combination with clavulanic acid.

High specificity of cphA-encoded metallo-beta-lactamase from Aeromonas hydrophila AE036 for carbapenems and its contribution to beta-lactam resistance.

The Aeromonas hydrophila AE036 chromosome contains a cphA gene encoding a metallo-beta-lactamase highly active against carbapenem antibiotics. This enzyme was induced in strain AE036 to the same extent by both benzylpenicillin and imipenem. When the cphA gene was inserted into plasmid pACYC184, used to transform Escherichia coli DH5 alpha, the MICs of imipenem, meropenem, and penem HRE664 for recombinant clone DH5 alpha(pAA20R), expressing the Aeromonas metallo-beta-lactamase, were significantly increased, but those of penicillins and cephalosporins were not. When the metallo-beta-lactamase purified from E. coli DH5 alpha(pAA20R) was assayed with several beta-lactam substrates, it hydrolyzed carbapenems but not penicillins or cephalosporins efficiently. These results demonstrate that this metallo-beta-lactamase possesses an unusual spectrum of activity compared with all the other class B enzymes identified so far, being active on penems and carbapenems only. This enzyme may thus contribute to the development of resistance to penems and carbapenems but not other beta-lactams.

Cloning and nucleotide sequencing of the gene encoding the beta-lactamase from Citrobacter diversus.

The gene coding for the class A beta-lactamase of Citrobacter diversus has been cloned and sequenced. It contains the information for a 294-amino-acid precursor protein, including a 27-residue N-terminal signal peptide. The deduced sequence of the N-terminal portion of the mature protein is in excellent agreement with that determined by microsequencing of the protein and readily explains the pI differences observed between the naturally occurring forms I and II of the enzyme. The sequence of the mature protein exhibits a very high degree of similarity with that of the Klebsiella oxytoca class A beta-lactamase.

Characterization of a beta-lactamase produced in Mycobacterium fortuitum D316.

A beta-lactamase from Mycobacterium fortuitum D316 was purified and some physico-chemical properties and substrate profile determined. On the basis of its N-terminal sequence and of its sensitivity to beta-iodopenicillanate inactivation, the enzyme appeared to be a class A beta-lactamase, but its substrate profile was quite unexpected, since nine cephalosporins were among the eleven best substrates. The enzyme also hydrolysed ureidopenicillins and some so-called 'beta-lactamase-stable' cephalosporins.

Broad spectrum beta-lactamases of Citrobacter diversus.

Citrobacter diversus NF85 produced a chromosomal beta-lactamase that was induced by a variety of beta-lactam antibiotics. Two major forms of the enzyme, with isoelectric points (pI's) of 5.7 and 6.2, were found in crude cell extracts. Derepressed mutants of NF85, generated by nitrosoguanidine treatment, displayed different levels of beta-lactamase expression to the parent strain and had different patterns of resistance to a range of beta-lactam antibiotics. Those mutants of NF85 that were totally derepressed, expressing high, constitutive levels of enzyme, were found to have an additional beta-lactamase activity with a pI of 6.8.

CTX and its desacetyl derivative (des-CTX): interaction with some representative beta-lactamases and their related pattern of resistance to newer selected clinical isolates.

In this study the kinetic features of cefotaxime (CTX) and desacetyl-cefotaxime towards several representative beta-lactamases were investigated. Desacetyl-CTX was more stable to hydrolysis in comparison with cefotaxime for all the investigated enzymes. However, a cephalosporinase produced in Acinetobacter was progressively inactivated by both CTX and des-CTX. After prolonged incubation, dialysis partially restored the enzyme activity. Finally, both compounds were tested against selected resistant strains. It is concluded that des-CTX, because of either poor hydrolysis or prolonged half-life in body fluids, could contribute in vivo to the good antimicrobial properties of cefotaxime.