Four years of monitoring antibiotic resistance in microorganisms from bacteremic patients.

From the second semester of 2002 to the end of the first semester of 2005, a total of 2544 bacterial strains were isolated from the blood stream of patients with clinical sepsis and bacteremia hospitalized in six University Hospitals in the Slovak Republic. Almost 30% of strains were coagulase-negative staphylococci (CONS), about 14% were Staphylococcus aureus and, of the Gram-negative bacteria, up to 9% were Klebsiella pneumoniae. All CONS, S. aureus and Enterococcus spp. strains were found to be still susceptible to vancomycin, but the resistance of CONS and/or S. aureus to macrolides and fluoroquinolones dramatically increased during the period of this study. Among Gram-negative bacteria, increasing levels of resistance to higher generation cephalosporins, to fluoroquinolones resistance in Pseudomonas aeruginosa and Acinetobacter spp. to meropenem was recorded, which is alarming. The results were periodically submitted to cooperating hospitals with proposals for rationalizing the prophylactic and general use of indicated antibiotics as well as for improving hospital hygiene measures and anti-epidemic practices.

Transferable antibiotic resistance in nosocomial Stenotrophomonas maltophilia strain.

Stenotrophomonas maltophilia (298/85) was isolated from the extensively inflamed conjunctiva of a neonate in a regional hospital in Ostrava, Czech Republic. It was resistant to all available antibiotics except cefepime and trimethoprim. The donor S. maltophilia strain 298/85 transferred carbenicillin and cephaloridine resistance determinants to recipient strains of Escherichia coli K-12 3110 rif+ and Proteus mirabilis P-38 rif+. All transconjugant colonies were co-resistant also to kanamycin, cefotaxime, and aztreonam. Active hydrolysis of imipenem in the original strain was inhibited by ethylene diamine tetra-acetic acid, and hydrolysis of cefotaxime and aztreonam in the original strain and in the E. coli K-12 transconjugant was inhibited by clavulanate. In contrast, ceftazidime was hydrolyzed by the original strain and was not inhibited by clavulanate, indicating a different character of the resistance to cefotaxime or aztreonam and ceftazidime.

Origin of transferable cefotaxime resistance in a clinical isolate of Escherichia coli by mutation.

A clinical isolate of Escherichia coli susceptible to cefotaxime and ceftazidime from a polytraumatic pediatric patient, transferred a series of determinants of resistance to antibiotics, including cephalotin-cefazolin, to a recipient strain of E. coli K-12. When cephalotin-resistant clones of K-12 were tested for their resistance to cefotaxime on solid media with this antibiotic, nine clones could be isolated which were resistant to high concentrations of cefotaxime. All nine clones were able to transfer this resistance, associated with resistance to cephalotin-cefazolin, to the K-12 recipient. Mutations to ceftazidime resistance could be obtained by a similar procedure from cephalotin-cefotaxime-resistant transconjugants of the first cycle of transfers. It is concluded that transferable resistance to cefotaxime, and probably also to ceftazidime, could originate by selection of spontaneous mutants of cephalotin-resistant E. coli, transferring this resistance.

Extended-spectrum beta-lactamase producing strains of Enterobacter cloacae transferring resistance to cefotaxime and ceftazidime.

Transferability of ceftazidime resistance and of extended-spectrum beta-lactamase (ESBL) production was recorded in three strains of Enterobacter cloacae from Frankfurt University Clinics. All three strains were resistant to rifampicin, carbenicillin, cephaloridine, cefotaxime, ceftazidime, aztreonam, imipenem and meropenem. They transferred directly determinants of resistance to carbenicillin and cephaloridine to Escherichia coli K-12 No. 185 nal+ recipient strain. All transconjugant colonies also contained determinants of resistance to cefotaxime, ceftazidime and aztreonam. In the second cycle of transfers, determinants of resistance to carbenicillin, cephaloridine, cefotaxime, ceftazidime and aztreonam were transferred en bloc to the recipient Escherichia coli K-12 No. 3110 rif+ indicating that the "transfer factor" (tra+) mediating the process of conjugational transfer had also been co-transferred. Double-disk diffusion test showed the same pattern of production of ESBLs both in original strains and in transconjugants. Transfer of cefotaxime, ceftazidime and aztreonam was accompanied by the transfer of ESBL production.

Sudden increase in Pseudomonas aeruginosa nosocomial strains with broad host range transfer of antibiotic resistance.

We investigated transfer of antibiotic resistance from 51 multiply resistant strains of Pseudomonas aeruginosa isolated from seriously ill patients in the Frankfurt University Clinics. Nine isolates directly transferred resistance to three recipient strains used. Ticarcillin and cephalothin resistance determinants were accepted from eight isolates, and in one case a kanamycin resistance determinant was transferred. The total spectrum of resistance transferred demonstrated that several donor strains transferred a different set of resistance determinants to all three recipient strains. Two P. aeruginosa isolates transferred spectrum of seven resistance determinants including ceftazidime, cefepime and aztreonam, three isolates transferred five determinants and four isolates transferred four resistance determinants. The fact that identical spectra of multiple drug resistance were transferred to recipient strains belonging to three different species (Escherichia coli, Proteus mirabilis and P. aeruginosa), indicates a broad host range in all three transferable genetic elements not observed in previous transfers from P. aeruginosa strains.

Low-Frequency transduction of imipenem resistance and high-frequency transduction of ceftazidime and aztreonam resistance by the bacteriophage AP-151 isolated from a Pseudomonas aeruginosa strain.

Bacteriophage AP-151, isolated from a multidrug resistant Pseudomonas aeruginosa strain, was found to transduce antibiotic resistance determinants to recipient strains of P. aeruginosa. Resistance to cefotaxime, ceftazidime, aztreonam, imipenem and meropenem was transduced as a block, at different frequencies, to two P. aeruginosa strains. Resistance was two logarithms higher (in the range 10(-5)) for cefotaxime, ceftazidime or aztreonam than for imipenem in recipient strain PAO-1670. The frequency of transduced imipenem resistance was also lower in recipient strain ML-1008. This phenomenon reflects the difference in the lytic activity of AP-151 in both strains, as the titer of the AP-151 phage in the PAO strain was found to be restricted to 10(-4)-10(-5) in contrast to the titer of the same phage in the ML strain which was 10(-10). The limited lytic activity in the PAO recipient strain was correlated with higher transducing activity. It can be concluded that some wild-type bacteriophages of P. aeruginosa might have highly individual relations between lytic and transducing activity in various potential recipient nosocomial strains of P. aeruginosa. The nature of resistance to ceftazidime and imipenem was studied using clavulanate and EDTA as inhibitors of individual class of beta-lactamases, indicating the presence of extended-spectrum beta-lactamase and a metallo-beta-lactamase in this isolate.

Host range variability in the transfer of antibiotic resistance determinants from Pseudomonas aeruginosa strains.

Twelve multiply drug-resistant strains of Pseudomonas aeruginosa, isolated in Frankfurt University Clinics during the year 1998, transferred determinants of antibiotic resistance with remarkable variability in the host range of transferred plasmids. As a new phenomenon, two of them transferred resistance genes to both groups of recipients, i.e. to Enterobacteriaceae as well as to a strain of P. aeruginosa. Six strains had more restrictive activity and transferred resistance determinants to Enterobacteriaceae only, while four additional strains demonstrated transferability only to P. aeruginosa. Two isolates transferred imipenem resistance and one cefepime resistance. Differences in selection pressure occurring during therapy of individual patients seems to be one of the possible factors influencing the spectrum of antibiotic resistance genes present in P. aeruginosa and their transfer to susceptible strains.

Transferable resistance to cefotaxime in nosocomial Klebsiella pneumoniae and Escherichia coli strains due to their production of extended-spectrum beta-lactamase in Slovakia.

Transferable resistance to cefotaxime was demonstrated in 21 nosocomial strains of Klebsiella pneumoniae and Escherichia coli subsequently isolated from patients in two large University clinics. Using the double-disk diffusion test, we could detect, in each such strain, as well as in E. coli 3110 K-12 transconjugants after the transfer, the production of an Extended Spectrum Beta-Lactamase (ESBL). Ceftibuten was demonstrated to be effective against the majority of strains studied.

First occurrence of transferable extended-spectrum beta-lactamase hydrolyzing cefoperazone in multiresistant nosocomial strains of Klebsiella pneumoniae from two hospitals in Czech and Slovak Republics.

The occurrence of positive synergy between antibiotic discs of amoxicillin/clavulanate and cefoperazone was registered in two Klebsiella pneumoniae strains, isolated from hospitals in Czech and Slovak Republic, indicating the presence of genes coding for an extended-spectrum beta-lactamase active also against cefoperazone, a broad-spectrum cephalosporin. Sulbactam inhibited the hydrolysis of cefoperazone by cell-free lysates of these strains which substantiates its use in combination with cefoperazone. Resistance to cephalothin, cefotaxime, ceftazidime, cefoperazone, cefepime and aztreonam was transferred from K. pneumoniae isolates to Escherichia coli K-12 3110 and to Proteus mirabilis P-38 recipient strains.

Two nosocomial strains of Stenotrophomonas maltophilia transferring antibiotic resistance to Proteus mirabilis P-38 recipient strain.

In this report we describe a specific transfer of carbenicillin and cephaloridine resistance determinants from two different strains of Stenotrophomonas maltophilia: No. 215 and 221 isolated from two critically ill patients treated in different Intensive Care Units of a large University Hospital in Ostrava, Czech Republic. These strains were resistant to flouroquinolones and the following beta-lactam drugs: carbenicillin, cephaloridine, cefotaxime, ceftazidime, cefepime, imipenem, meropenem and aztreonam. Both strains transferred carbenicillin and cephaloridine resistance determinants, with rather different frequency, to Proteus mirabilis P-38. All carbenicillin-selected transconjugants were found by an indirect selection method to be co-resistant to cephaloridine only. In a second cycle of transfers Proteus mirabilis R+ strains directly transferred carbenicillin and cephalothin determinants to Escherichia coli K-12 No. 185 nal+ lac+ recipient strain.

Transferable resistance to specific antibiotics in nosocomial strains of Pseudomonas aeruginosa from two teaching hospitals.

We describe the transfer of resistance to kanamycin, carbenicillin and cephaloridine to a recipient strain of Escherichia coli K-12 No. 3110 from three strains of Pseudomonas aeruginosa out of 146 strains tested in 1995. The P.aeruginosa No. 201, 203, 208 donor strains were isolated from patients in the University Clinics in Frankfurt, Germany. They were resistant to most beta-lactam antibiotics including cephalosporins of the 1st, 2nd and 3rd generation, imipenem, meropenem and aztreonam. They transferred kanamycin, carbenicillin and cephaloridine resistance determinants to recipient strain E.coli K-12 3110. These determinants were accompanied in strain P. aeruginosa No. 203, with a transfer of ceftazidime resistance determinants and in P.aeruginosa No. 208 with transfer of cefotaxime, ceftazidime and aztreonam resistance determinants. Transfer of antibiotic resistance was also studied in 13 nosocomial strains of P.aeruginosa collected for their ceftazidime and/or imipenem resistance in a large Teaching Hospital in Ostrava, Czech Republic. Six of these strains transferred carbenicillin and/or cephaloridine resistance to the E.coli K-12 3110 recipient strain. Resistance to kanamycin and cefotaxime was also co-transferred with carbenicillin and cephaloridine determinants. Ceftazidime, imipenem or ofloxacin resistance was not transferred and is thus, most probably, of chromosomal origin.

Mobilization of antibiotic resistance for transfer in Pseudomonas aeruginosa.

We describe a phenomenon of mobilization of antibiotic resistance from non-transferring strains of Pseudomonas aeruginosa by cultivation with strains of P.aeruginosa capable of transferring determinants of antibiotic resistance to a susceptible recipient strain by triparental cross. In this report we described two strains of P.aeruginosa capable to mobilize for transfer the resistance determinants in strains of P.aeruginosa with multiply antibiotic resistance which were not themselves transferable to Pseudomonas recipient strains. Two strains of P.aeruginosa No. 282 and 283 from Bata's Hospital in Zlín, Czech Republic, were used as donor strains for experiments in triparental crosses. They were resistant to carbenicillin (CAR), kanamycin (KAN), cefalotin-cefazolin (CFR), cefotaxime (CTX), ceftazidime (CAZ) and aztreonam (AZA). Both strains transferred CFR resistance to Escherichia coli K-12 rif+ recipient, but not to P.aeruginosa PAO recipients. In a second system, a strain of P.aeruginosa No. 76 from Frankfurt University Clinics was used as a donor strain. It transfers CAR resistance to PAO-1670 rif+, but not to E. coli K-12 rif+. Two strains of P.aeruginosa from Frankfurt University Clinics No. 76 and 229 were used as intermediary recipient strains. They were resistant to CAR, KAN, CTX, CAZ, AZA, imipenem (IMP) and ofloxacin (OFL). Strain No. 229 did not transfer any antibiotic resistance to any of both final recipient strains. Strain No. 76 transfers, as indicated, CAR resistance determinant to PAO 1670 rif+ recipient strain. Strains of E. coli K-12 No. 3110 rif+ and P.aeruginosa PAO 1670 rif+ were used as final recipient strains.

The origin, by mutation, of high level resistance to ceftazidime and cefotaxime in a clinical isolate of Enterobacter cloacae.

Although strains of Enterobacter sp. produce a chromosomal AmpC beta-lactamase, they have been, in general, susceptible to cefotaxime or ceftazidime. But now resistance to ceftazidime has increased in nosocomial Enterobacter strains, reaching the level of 40%. A chromosomal mutation in the amp operon coding the production of AmpC type beta-lactamase may cause a change in the conversion of a susceptible strain of Enterobacter to a highly resistant mutant. The production of AmpC enzyme is inducible and third-generation cephalosporins are weak inducers of AmpC production. Spontaneous mutations (or insertions of transposons) in the regulatory region of amp operon might create constitutive (derepressed) overproducers of large amounts of AmpC molecules. As a consequence, such cells acquire a stable resistance to beta-lactam antibiotics including cefotaxime or ceftazidime. We describe the origin of mutants of a clinical isolate of Enterobacter cloacae that acquired high-level resistance to ceftazidime followed by the resistance to cefotaxime and/or aztreonam due to a second mutation.

Further occurrence of extended-spectrum beta-lactamase-producing Salmonella enteritidis.

We describe the further occurrence of Salmonella enteritidis producing an extended spectrum beta-lactamase (ESBL) and transferring cefotaxime, ceftazidime and aztreonam resistance. In a previous communication we describe the isolation of a strain of S. enteritidis (No. 35) from a septic neonate with high-level resistance to cefuroxime, cefotaxime, ceftazidime and aztreonam. Six weeks later, a second neonate was found, in the same Pediatric University Hospital, to be infected with apparently the same strain of S. enteritidis (No. 100) which was isolated from blood with similar properties as the strain No. 35. In this strain we were able to study, in more detail, the transferability of its resistance to 3rd generation cephalosporins and its ESBL production. In this report we describe results of transfer experiments, relative rates of hydrolysis (Vmax) and ESBL tests which indicate that the capacity to produce ESBL was transferred from the multiply-resistant strain of S. enteritidis No. 100 to S. typhimurium LT-2 rif+str+.

Monitoring of antibiotic resistance in bacterial isolates from bacteremic patients.

The aim of the study was to monitor the prevalence of pathogens and development of resistance in bacteria isolated from bacteremic patients. Five University Clinics and/or Regional Hospitals in the Slovak Republic participated in the study and a total of 421 isolates were collected in the second half of the year 2002. The most prevalent organisms were coagulase-negative staphylococci (CONS) (19%), Staphylococcus aureus (18.3%), among Gram-negative bacteria Escherichia coli (13.3%), Klebsiella pneumoniae (11.4%) and Pseudomonas aeruginosa (7.8%) followed by enterococci, Acinetobacter baumannii and Enterobacter sp. All CONS and S. aureus were susceptible to vancomycin; resistance to oxacillin was observed for 55% of the CONS and only for 4% of S. aureus isolates. A higher prevalence of resistance to erythromycin, clindamycin, gentamicin and ofloxacin was found in CONS in comparison to S. aureus. Enterococcus sp. isolates were fully susceptible to vancomycin and teicoplanin. Gentamicin, amoxicillin/clavulanate, third generation cephalosporins and ciprofloxacin showed good activity against E. coli. Although 17% of K. pneumoniae isolates were resistant to ciprofloxacin, it was the most effective drug against K. pneumoniae; the prevalence of resistance to other antibiotics was rather higher. Gentamicin and ciprofloxacin were the most active against Enterobacter sp. isolates and ceftazidime and meropenem against P. aeruginosa.

Transferable antibiotic resistance in multiresistant nosocomial Acinetobacter baumannii strains from seven clinics in the Slovak and Czech Republics.

Sixty-seven multiresistant nosocomial Acinetobacter baumannii isolates from patients hospitalized mostly in intensive care units of seven clinics in Slovak and Czech Republic were tested to determine their ability to transfer antibiotic resistance. All isolates were resistant to kanamycin, ticarcillin, cephalothin, cefotaxime, ceftazidime, aztreonam and susceptible to carbapenems, sulbactam and ampicillin/sulbactam. Sixty-five out of 67 strains transferred resistance determinants to Escherichia coli K-12 and Proteus mirabilis P-38 recipients. Analysis of selected transconjugants by an indirect selection method showed a more variable pattern of transferred resistance determinants. The clonal spread of strains transferring resistance seems to be an additional risk for occurrence of strains resistant to ceftazidime and aztreonam.

Further studies of transferable antibiotic resistance in strains of Pseudomonas aeruginosa from four clinical settings in three countries.

This paper describes transferability of antibiotic resistance determinants in clinical isolates of Pseudomonas aeruginosa resistant to imipenem, cefotaxime and ceftazidime obtained from different clinical settings in three different countries. Two strains of Enterobacteriaceae (Escherichia coli K-12 and Proteus mirabilis P-38) and two strains of P. aeruginosa (PAO and ML) were used as recipient strains. The conjugative transfer of resistance was very specific, i.e. donor strains of P. aeruginosa transferred individual resistance determinants either to recipient strains E. coli K-12 and P. mirabilis P-38, or to P. aeruginosa PAO or ML. In a case when three different species (P. aeruginosa, Acinetobacter calcoaceticus, Stenotrophomonas maltophilia) were isolated from a single patient, a block of resistance determinants was transferred both to Enterobacteriaceae and P. aeruginosa recipients. A single plasmid with rather specific properties was suspected of being exchanged among strains of these different species. It was recommended to study the overall situation of transferability of individual resistance determinants in bacterial strains belonging to various species of nosocomial bacteria, especially of P. aeruginosa, by using a rather specific methodology.

Transduction of imipenem resistance by the phage F-116 from a nosocomial strain of Pseudomonas aeruginosa isolated in Slovakia.

Generalized transducting Pseudomonas aeruginosa phage F-116 was propagated in an Imipenem (IMI)-resistant clinical isolate of P. aeruginosa No. 191 and the phage was then used to transduce the IMI-resistance to a susceptible auxotrophic mutant strain of the same species. Transduction seems to be a suitable method for study of resistance determinant(s) and mechanism of resistance in this important species. It was found that the IMI-resistance of the clinical strain No. 191 is caused by production of a betalactamase specifically hydrolyzing this antibiotic.

Transduction of antibiotic resistance in Pseudomomas aeruginosa: relationship between lytic and transducing activity of phage isolate AP-423.

Isolation and propagation of a wild type phage, isolate AP-423, from an apparently lysogenic strain of Pseudomonas aeruginosa, resistant to a series of anti-pseudomonadal antibiotics, and its use for transduction of resistance determinants is described. The phage isolate AP-423 showed a phenomenon of host restriction, i.e. it was lysogenic only for some of the recipient strains tested. Its transduction capacity, both in sets of genes transduced and frequency of transduction, was different in two recipient strains of P. aeruginosa. This phage showed also some restriction in titers, to which it could be propagated, only in certain recipient strains.

Two high-frequency-transduction phage isolates from lysogenic strains of Pseudomonas aeruginosa transducing antibiotic resistance.

Two high frequency transduction (HFT) phage isolates, obtained from seriously ill patients, transducing individual determinants of antibiotic resistance with a frequency of 10(-5) (phage isolate AP-103) and 10(-6) (phage isolate AP-343), are described. The frequency of transduction depended on the transduced determinant(s) of resistance used for the detection of transductants and on the individual recipient antibiotic-susceptible strain of Pseudomonas aeruginosa (PAO and/or ML series). A multiple-antibiotic resistance was transduced by the phage isolate AP-343 to all tested recipient strains. The appearance of such phages in clinical conditions with an unusually high frequency of transduction might contribute to the dissemination of antibiotic resistance genes among nosocomial strains of P. aeruginosa. The existence of HFT phages might reflect an increased efficiency of transduction of antibiotic resistance among P. aeruginosa strains, and thus an increased risk of spread of antibiotic resistance even to recently introduced anti-pseudomonadal antibiotics among pseudomonads with unfavourable and unwanted epidemiological consequences in hospital conditions.