Ongoing epidemic of blaVIM-1-positive Klebsiella pneumoniae in Athens, Greece: a prospective survey.

OBJECTIVES: To determine the current frequency and study the characteristics of VIM-1-producing Klebsiella pneumoniae isolates from bloodstream infections in Greek hospitals. METHODS: All blood isolates of K. pneumoniae were prospectively collected during 2004-06 in three teaching hospitals located in Athens. MICs of antibiotics were determined by the Etest. Extended-spectrum- (ESBL) and metallo-beta-lactamase (MBL) production was examined by clavulanate- and EDTA-based techniques, respectively. Isolates were typed by PFGE of XbaI-digested genomic DNA. Detection of bla(VIM-1) and mapping of the VIM-1-encoding integrons were performed by PCR and sequencing. Beta-lactamase activities were analysed by IEF and imipenem hydrolysis was assessed by spectrophotometry. VIM-1-encoding plasmids were transferred to Escherichia coli by conjugation and transformation and characterized by Inc/rep typing and RFLP. RESULTS: Sixty-seven (37.6%) of 178 K. pneumoniae blood isolates were bla(VIM-1)-positive (VPKP); 77.8% of these were from ICUs. All VPKP isolates were multidrug-resistant. The MICs of carbapenems for VPKP varied from the susceptible range to high-level resistance overlapping with those of MBL-negative isolates. The EDTA-imipenem synergy methods had reduced sensitivity in detecting VPKP isolates when the MICs were in the susceptible range. ESBL production was common among VPKP isolates (n = 45, 67.2%) as indicated by resistance to aztreonam and confirmed by a clavulanate-based double-disc synergy test. The responsible ESBL was always an SHV-5-type enzyme as indicated by IEF. PFGE identified eight clusters (A-H) of VPKP isolates with related (>80%) patterns, as well as four unique types. Both inter-hospital spread of several clones and genotypic similarities among susceptible, ESBL-positive and VPKP isolates were also observed. Location of bla(VIM-1) and expression of VIM-1 were studied in 12 isolates representing the eight PFGE clusters. In all isolates, bla(VIM-1) was part of a class 1 integron that also carried aacA4, dhfrI, aadA and sulI. In eight isolates (clusters C, D, G and H), the bla(VIM-1) integron was located in transferable IncN plasmids. A cluster F isolate carried a VIM-1-encoding, self-transferable plasmid that was not typeable by Inc/rep typing. VIM-1-encodingreplicons were not identified in three isolates (PFGE clusters A, B and E). VPKP isolates exhibited differences in imipenem-hydrolysing activities which, however, were not correlated with the respective carbapenem MICs. CONCLUSIONS: A multiclonal epidemic of bla(VIM-1)-carrying K. pneumoniae is under way in the majorhospitals in Greece. Microorganisms producing both VIM-1 and SHV-5 constitute the prevalent multidrug-resistant population of K. pneumoniae in this setting.

Multicentre survey of the in-vitro activity of seven antimicrobial agents, including ertapenem, against recently isolated Gram-negative anaerobic bacteria in Greece.

The in-vitro activities of penicillin, ticarcillin-clavulanic acid, cefoxitin, imipenem, ertapenem, metronidazole and clindamycin were evaluated against 138 Gram-negative anaerobic isolates (82 Bacteroides fragilis group, 17 non-fragilis Bacteroides spp., 31 Prevotella spp., four Fusobacterium spp., two Veillonella spp., one Porphyromonas sp. and one Tissierella praeacuta) collected from six general hospitals in Athens, Greece. Overall rates of non-susceptibility (both resistant and intermediately-resistant) to penicillin and ticarcillin-clavulanic acid were 81.8% and 2.3%, respectively. The rates of non-susceptibility to cefoxitin and clindamycin were 30.3% and 31.1%, respectively, and that for metronidazole was 4.3% (four Prevotella spp. isolates, one Porphyromonas sp. isolate and one B. fragilis isolate). Only the single B. fragilis isolate was nim-positive by PCR. Only one B. fragilis isolate was resistant to both carbapenems tested, while six more Bacteroides spp. isolates were imipenem-susceptible and ertapenem-non-susceptible. The MIC range, MIC(50) and MIC(90) values were comparable for imipenem and ertapenem, although ertapenem MIC(90)s were one or two two-fold dilutions higher.

Characterisation of VanA and VanB elements from glycopeptide-resistant Enterococcus faecium from Greece.

Ten glycopeptide-resistant Enterococcus faecium isolates from separate patients in Laikon General Hospital, Athens were studied. Eight isolates had the VanA phenotype and represented variants of three strains based on SmaI macrorestriction banding patterns. Their VanA elements were compared with the prototype element, Tn1546, by an overlapping PCR method. Three related isolates contained resistance elements indistinguishable from Tn1546 (designated Greek type I). The other five isolates all contained identical elements that differed from Tn1546 by the presence of IS1251 between vanS and vanH, by a point mutation (G --> T) at nucleotide position 8234 within vanX and by a partial loss of transposition gene orf1 (designated Greek type II). Two distinct strains of E. faecium with the VanB phenotype were obtained. HhaI digestion of an amplified fragment of the vanB gene indicated that both strains contained the vanB2 allele, and further PCR assays confirmed that the vanB2 gene cluster was located within a Tn5382-like element.

In vitro activity of tigemonam against multiresistant nosocomial Enterobacteriaceae.

Tigemonam, an oral monobactam that exhibits beta-lactamase stability similar to that of aztreonam, was tested in vitro against 240 species of Enterobacteriaceae (50 Escherichia coli, 48 Klebsiella pneumoniae, 52 Enterobacter cloacae, 32 Proteus mirabilis, 22 Proteus indole-positive [Providencia sp.], 24 Serratia sp., and 12 Citrobacter sp. All strains were resistant to ampicillin and first-generation cephalosporins. In addition, 77.4% were resistant to amoxicillin plus clavulanic acid, 46.8% to cefuroxime, 23.3% to ceftriaxone, 22.2% to aztreonam, 46.9% to cotrimoxazole, and 0.9% to norfloxacin. Tigemonam at a concentration of 4 micrograms/mL or less inhibited 72.7% of the strains with minimum inhibitory concentrations ranging from 0.03 or less to more than 512 micrograms/mL. The highest intrinsic activity was observed against Proteus sp. Tigemonam proved to be a bactericidal antibiotic. Cross-resistance was chiefly observed with aztreonam and ceftriaxone. It is concluded that tigemonam should play an important role in the treatment of nosocomial infections that do not require parenteral therapy and in the treatment of multiresistant community-acquired infections.

Risk factors for coexistence of fluoroquinolone resistance and ESBL production among Enterobacteriaceae in a Greek university hospital.

The purpose of this study was to identify risk factors for fluoroquinolone resistance (QR) among ESBL- producing Enterobacteriaceae causing nosocomial infections. The study was conducted in Laikon General Hospital in Athens, Greece, during the period January 2004 - January 2005. Epidemiological and clinical data were collected from the medical charts of the patients diagnosed with nosocomial infections due to an ESBL-producing Enterobacteriaceae. QR was 60% among the 84 ESBL-producing Enterobacteriaceae isolates. Infection from QR-ESBL bacteria was associated with increased hospital stay (p=0.028); QRESBL bacteria were isolated later during hospitalization than fluoroquinolone susceptible (QS)-ESBL (p=0.089); factors associated with QR were immune-deficiency (p=0.047), previous use of carbapenems (p=0.08) and fluoroquinolones (p=0.067), and admission to the Transplantation Unit (p=0.047). In addition, QR-ESBL bacteria were more likely to be resistant to co-trimoxazole (p<0.001), gentamicin (p=0.054) and tobramycin (p=0.004). Logistic regression analysis indicated that admission to the transplantation unit was an independent risk factor for infection due to a QR-ESBL isolate. Results of this study question ciprofloxacin's usefulness as a valid alternative to carbapenems in our hospital for the treatment of infections due to ESBL-producing bacteria. In addition strategies for addressing the QR-ESBL situation should focus on limiting fluoroquinolone and carbapenem consumption and emphasize on barrier precautions in patients with longer hospitalization, immunosuppression, or admission to the transplantation unit.

Surveillance study of resistance in Haemophilus species in Greece.

The results are presented of the first surveillance study in Greece on resistance in strains of Haemophilus influenzae (n = 61) and Haemophilus parainfluenzae (n = 96) to six antibiotics. Strains were isolated in a three-month period in 1987 in most cases from sputum specimens from adult patients with lung infections. The overall rate of resistance to ampicillin was 28.3%, to chloramphenicol 2.7%, to cefaclor 2.7%, to tetracycline 10.6%, to erythromycin 38.1% and to cotrimoxazole 5.3%. It is evident that the resistance rate of Haemophilus spp. is steadily increasing in Greece as in other European countries. Regular surveys of resistance patterns for both older and newer antimicrobial agents are therefore necessary.

Comparative in vitro activity of cefepime (BMY 28142) against multiresistant nosocomial isolates of Pseudomonas aeruginosa.

The in vitro activity of a new parenteral cephalosporin cefepime (BMY 28142) was compared with that of ceftazidime, cefotaxime, piperacillin, imipenem, gentamicin, amikacin and ciprofloxacin against 173 recent multiresistant Pseudomonas aeruginosa isolates of nosocomial origin using an agar dilution technique with an inoculum of 10(4) CFU per spot. The activity of cefepime was comparable to that of ceftazidime, superior to that of cefotaxime, piperacillin, gentamicin and amikacin, but inferior to that of imipenem and ciprofloxacin. Cross-resistance of Pseudomonas aeruginosa to ceftazidime and cefepime occurred in nearly 50% of the cefepime resistant strains and 61.5% of the ceftazidime resistant strains respectively.

In-vitro activity of meropenem, a new carbapenem, against multiresistant Pseudomonas aeruginosa compared with that of other antipseudomonal antimicrobials.

The novel carbapenem meropenem, which is stable to dehydropeptadase action, was studied in vitro against 173 nosocomial isolates of Pseudomonas aeruginosa derived from pus, expectorated sputum, bronchial secretions, urine or blood of infected patients. Susceptibilities to meropenem, imipenem, ceftazidime, cefotaxime, piperacillin, gentamicin, amikacin and ciprofloxacin were determined by the agar dilution technique with an inoculum of 10(4) cfu/spot. The proportion of sensitive isolates with MICs below the breakpoint was highest for meropenem (91% less than or equal to 8 mg/l); the proportion of sensitive isolates for the remaining antibacterials was as follows: ciprofloxacin 87% less than or equal to 2 mg/l; imipenem 85% less than or equal to 8 mg/l; ceftazidime 77.5% less than or equal to 16 mg/l; piperacillin 60% less than or equal to 64 mg/l; amikacin 44% less than or equal to 8 mg/l; gentamicin 34% less than or equal to 4 mg/l and cefotaxime 15% less than or equal to 16 mg/l. Strains moderately resistant to imipenem (MIC 16-32 mg/l) were mostly sensitive to meropenem, but highly resistant strains (MIC 128-256 mg/l) were invariably resistant (MIC greater than 128 mg/l) to the new carbapenem.

In Vitro Activity of Tigemonam Against Multiresistant Nosocomial Enterobacteriaceae.

Tigemonam, an oral monobactam that exhibits beta-lactamase stability similar to that of aztreonam, was tested in vitro against 240 species of Enterobacteriaceae (50 Escherichia coli, 48 Klebsiella pneumoniae, 52 Enterobacter cloacae, 32 Proteus mirabilis, 22 Proteus indole-positive [Providencia sp.], 24 Serrada sp., and 12 Citrobacter sp. All strains were resistant to ampicillin and first-generation cephalosporins. In addition, 77.4% were resistant to amoxicillin plus clavulanic acid, 46.8% to cefuroxime, 23.3% to ceftriaxone, 22.2% to aztreonam, 46.9% to cotrimoxazole, and 0.9% to norfloxacin. Tigemonam at a concentration of 4 µg/mL or less inhibited 72.7% of the strains with minimum inhibitory concentrations ranging from 0.03 or less to more than 512 µg/mL. The highest intrinsic activity was observed against Proteus sp. Tigemonam proved to be a bactericidal antibiotic. Cross-resistance was chiefly observed with aztreonam and ceftriaxone. It is concluded that tigemonam should play an important role in the treatment of nosocomial infections that do not require parenteral therapy and in the treatment of multiresistant community-acquired infections.