In the University Hospital of Trieste (northern Italy, at the border with Slovenia), clinical isolates of P. aeruginosa producing VIM-type MBLs were detected sporadically, for the first time, in 1999 (12). In 2001, a significant increase in the prevalence of imipenem-resistant P. aeruginosa isolates was observed at the Laboratory of Clinical Microbiology of that hospital (29%, vs. 19% in 2000 and 21% in 1999, respectively; p < 0.001 according to the χ² test; statistical analyses were conducted with Epi Info statistical software, version 6.03, Centers for Disease Control and Prevention, Atlanta, GA).

Of the 444 nonreplicate imipenem-resistant P. aeruginosa isolates collected in 2001, a total of 89 were randomly selected and analyzed for acquired MBL genes of the bla_IMP and bla_VIM types in dot-blot hybridization experiments carried out with purified genomic DNA spotted (0.5 μg per spot) on positively charged nylon membranes (ZetaProbe, Bio-Rad, Hercules, CA) with digoxygenin-labeled DNA probes. The probes were polymerase chain reaction amplicons containing internal fragments of the bla_IMP-1 (754–1,114 nt, EMBL/GenBank database entry S71932) or of the bla_VIM-1 gene (3,366–3,888 nt, EMBL/GenBank database entry Y18050), respectively obtained using primers IMP-DIA (forward, 5′-GGAATAGAGTGGCTTAATTCTC; reverse, 5′-GTGATGCGTCYCCAAYTTCACT) and VIM-DIA (forward, 5′-CAGATTGCCGATGGTGTTTGG; reverse, 5′-AGGTGGGCCATTCAGCCAGA) as described previously (13). Hybridization was carried out under conditions that allowed recognition, by each probe, of different allelic variants of the corresponding MBL determinant. None of the imipenem-resistant isolates were recognized by the bla_IMP probe, while 64 (72%) were recognized by the bla_VIM probe. In the 64 bla_VIM-positive isolates, the nature of the determinant was further investigated by analysis of the RsaI restriction fragment length polymorphism of the gene region amplified by the VIM-DIA primers as described previously. With this approach, the determinant was identified as bla_VIM-1-like in 54 isolates (84%), and as bla_VIM-2-like in the remaining 10 isolates (16%).

Figure

Figure. RAPD profiles of bla_VIM positive strains. Amplification products (8 μL) obtained with primer 208 (5′-ACGGCCGACC-3′) (14) were run on 2% agarose gel. Lanes A-E: RAPD-types as indicated in <>

The sources of the 64 bla_VIM-positive isolates were 52 inpatients from 15 different wards (including 10 medical wards, 4 surgical wards, and an intensive care unit), 5 patients from 4 different long-term care facilities for elderly persons, and 7 outpatients (Table 1). The degree of genomic relatedness of these isolates was investigated by Random Amplification of Polymorphic DNA (RAPD) (14) and by Amplified Fragment Length Polymorphism (AFLP) (15). Electrophoretic profiles generated by the techniques described earlier were compared by the GelComparII software (Applied Maths, Kortrijk, Belgium). Consistent results were obtained with both typing methods. Isolates sharing a Dice similarity coefficient >0.88 comparing their RAPD-profiles were assigned to the same cluster. Results of molecular typing indicated that most bla_VIM-positive isolates (61 [95%]) belonged to either of two clusters, indicated as cluster A and B respectively, while the remaining three isolates were unrelated with those clusters and also among each other (Figure). Cluster A included 53 isolates, all containing bla_VIM-1-like determinants. They were widely distributed in the hospital (15 wards), and were also found in three long-term care facilities and in six outpatients. Cluster B included eight isolates, all containing bla_VIM-2-like determinants. The isolates were from four wards where isolates of cluster A had also been detected. Of the three sporadic isolates, one (carrying a bla_VIM-2-like gene) was from a ward where isolates of clusters A and B had also been detected, the second (also carrying a bla_VIM-2-like gene) was from a long-term care facility different from those yielding isolates of cluster A, and the third (carrying a bla_VIM-1-like gene) was from an outpatient (Table 1). Genotyping of the 25 bla_VIM-negative isolates indicated that 5 belonged in cluster A, 1 in cluster B, while the remaining 19 were unrelated to the VIM producers and were overall distributed among 6 different genotypes (Table 1).

Imipenem MICs for the bla_VIM-positive isolates were always >64 μg/mL (range 64–512 μg/mL), while being always <64 μg/mL for the hybridization-negative isolates. Most of the bla_VIM-positive isolates (49 of 64 [76%]) exhibited a multidrug-resistant phenotype including all the tested drugs (imipenem, meropenem, ceftazidime, piperacillin, aztreonam, amikacin, gentamicin, tobramycin, and ciprofloxacin), except polymixin B. On the other hand, this virtually panresistant phenotype was observed in 7 (28%) of 25 bla_VIM-negative isolates (Table 2).

Our findings are of concern since they demonstrate that acquired MBLs can rapidly emerge and become a major cause of broad-spectrum β-lactam resistance among nosocomial pathogens. In our setting bla_VIM-positive P. aeruginosa isolates, which were sporadically detected for the first time in 1999 (12), represented approximately 20% of all P. aeruginosa isolates and 70% of the carbapenem-resistant P. aeruginosa isolates, respectively, during 2001. These figures exceed those reported for MBL producers from other settings (7,9,10). As an additional matter of concern, the bla_VIM-positive isolates were significantly more resistant than the bla_VIM-negative isolates to non-β-lactam antimicrobial agents as well.

In this survey, the bla_VIM-positive isolates were detected on a regular basis during the year and appeared to be widely distributed in the hospital and even outside of it. Molecular characterization showed the simultaneous circulation of different bla_VIM alleles (either bla_VIM1-like or bla_VIM-2-like) in multiple P. aeruginosa clones. Overall, these findings suggest that bla_VIM determinants have rapidly established a condition of high-level endemicity in this area. To the best of our knowledge, this study is the first in which a similar condition has been reported. Even the large outbreak reported in Greece was caused by a single clone and was apparently confined to the hospital wards (11). The finding of bla_VIM-negative P. aeruginosa isolates showing the same genotype as that of the two major clusters of bla_VIM-positive strains suggests a likely acquisition of the MBL determinants by strains already endemic in this area, followed by clonal expansion of the bla_VIM-positive strains.

The possibility that spreading transferable MBL genes among nosocomial gram-negative pathogens could emerge as a major problem in the clinical setting underscores the need for systematic surveillance of these resistance determinants. Considering that MBL producers were also isolated from outpatients and from long-term care facility patients, even if all of them showed at least one hospital treatment during the 6 months before, surveillance should not be restricted to nosocomial isolates but should also include isolates from community-acquired infections.