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ABSTRACTAn outbreak of multidrug-resistant Pseudomonas aeruginosastrains producing VIM-type metallo-ß-lactamases (MBLs) has occurred in an Italian hospital since 2000 (C. Lagatolla, E. A. Tonin, C. Monti-Bragadin, L. Dolzani, F. Gombac, C. Bearzi, E. Edalucci, F. Gionechetti, and G. M. Rossolini, Emerg. Infect. Dis. 10:535-538, 2004). In this work, using molecular methods, we characterized 128 carbapenem-resistant isolates (including 98 VIM-positive isolates) collected from that hospital from 2000 to 2002 to investigate the dynamics of the dissemination of MBL producers in the clinical setting. Genotyping by random amplification of polymorphic DNA and pulsed-field gel electrophoresis showed that most VIM-positive isolates belonged to two different clonal lineages, producing either a VIM-1- or a VIM-2-like MBL, whose ancestors were detected for the first time in the hospital in 1999, suggesting that clonal expansion played a predominant role in the dissemination of these isolates. The 86 clonally related isolates carrying a blaVIM-1-like gene on an In70-like integron were clearly related to a VIM-1-positive P. aeruginosaclone circulating in various Italian hospitals since the late 1990s. VIM-negative P. aeruginosastrains related to the VIM-1-positive clone were detected during the same period, suggesting that the latter strain was derived from a clonal lineage already circulating in the hospital. In the VIM-2-like positive clone, the MBL gene was carried by an unusual class 1 integron, named In71, lacking the 3' conserved sequence region typical of sul1-associated integrons. A different class 1 integron with an original structure carrying a blaVIM-2determinant, named In74, was detected in a sporadic isolate. A retrospective investigation did not reveal the presence of strains related to any of the VIM-producing isolates earlier than 1997.

A novel OXA-type enzyme, named OXA-46, was found to be encoded by a gene cassette inserted into a class 1 integron from a multidrug-resistant Pseudomonas aeruginosa clinical isolate. The variable region of the integron also contained a blaVIM-1 metallo-{szligbeta}-lactamase cassette and a duplicated aacA4 aminoglycoside acetyltransferase cassette. OXA-46 belongs to the OXA-2 lineage of class D {szligbeta}-lactamases. It exhibits 78% sequence identity with OXA-2 and the highest similarity (around 92% identity) with another OXA-type enzyme detected in clinical isolates of Burkholderia cepacia and in unidentified bacteria from a wastewater plant. Expression of blaOXA-46 in Escherichia coli decreased susceptibility to penicillins and narrow-spectrum cephalosporins but not to extended-spectrum cephalosporins, cefsulodin, aztreonam, or carbapenems. The enzyme was overproduced in E. coli and purified by two anion-exchange chromatography steps (approximate yield, 6 mg/liter). OXA-46 was made of a 28.5-kDa polypeptide and exhibited an alkaline pI (7.8). In its native form OXA-46 appeared to be dimeric, and the oligomerization state was not affected by EDTA. Kinetic analysis of OXA-46 revealed a specificity for narrow-spectrum substrates, including oxacillin, other penicillins (but not temocillin), and narrow-spectrum cephalosporins. The enzyme apparently did not interact with temocillin, oxyimino-cephalosporins, or aztreonam. OXA-46 was inactivated by tazobactam and carbapenems and, although less efficiently, also by clavulanic acid. Enzyme activity was not affected either by EDTA or by divalent cations and exhibited low susceptibility to NaCl. These findings underscore the functional and structural diversity that can be encountered among class D {szligbeta}-lactamases.

ABSTRACTA novel OXA-type enzyme, named OXA-46, was found to be encoded by a gene cassette inserted into a class 1 integron from a multidrug-resistant Pseudomonas aeruginosaclinical isolate. The variable region of the integron also contained a blaVIM-1metallo-β-lactamase cassette and a duplicated aacA4aminoglycoside acetyltransferase cassette. OXA-46 belongs to the OXA-2 lineage of class D β-lactamases. It exhibits 78% sequence identity with OXA-2 and the highest similarity (around 92% identity) with another OXA-type enzyme detected in clinical isolates of Burkholderia cepaciaand in unidentified bacteria from a wastewater plant. Expression of blaOXA-46in Escherichia colidecreased susceptibility to penicillins and narrow-spectrum cephalosporins but not to extended-spectrum cephalosporins, cefsulodin, aztreonam, or carbapenems. The enzyme was overproduced in E. coliand purified by two anion-exchange chromatography steps (approximate yield, 6 mg/liter). OXA-46 was made of a 28.5-kDa polypeptide and exhibited an alkaline pI (7.8). In its native form OXA-46 appeared to be dimeric, and the oligomerization state was not affected by EDTA. Kinetic analysis of OXA-46 revealed a specificity for narrow-spectrum substrates, including oxacillin, other penicillins (but not temocillin), and narrow-spectrum cephalosporins. The enzyme apparently did not interact with temocillin, oxyimino-cephalosporins, or aztreonam. OXA-46 was inactivated by tazobactam and carbapenems and, although less efficiently, also by clavulanic acid. Enzyme activity was not affected either by EDTA or by divalent cations and exhibited low susceptibility to NaCl. These findings underscore the functional and structural diversity that can be encountered among class D β-lactamases.

ABSTRACTThree epidemiologically independent Pseudomonas aeruginosaisolates, representative of the first VIM-1 metallo-β-lactamase producers detected at three different hospitals in northern Italy, were investigated to determine their genomic relatedness and to compare the structures of the genetic supports for the VIM-1 determinants. The three isolates, all of serotype O11, appeared to be clonally related according to the results of genotyping by macrorestriction analysis of genomic DNA by pulsed-field gel electrophoresis and random amplification of polymorphic DNA. Investigation of the genetic support for the blaVIM-1determinant revealed that it was carried on identical or almost identical integrons (named In70.2 and In70.3) located within a conserved genomic context. The integrons were structurally related to In70 and In110, two plasmid-borne blaVIM-1-containing integrons from Achromobacter xylosoxidansand Pseudomonas putidaisolates, respectively, from the same geographic area (northern Italy) and were found to be inserted close to the ressite of a Tn5051-like transposon, different from any of those described previously, that was apparently carried on the bacterial chromosome. The present findings suggest that the three VIM-1-producing isolates are members of the same clonal complex which have been spreading in hospitals in northern Italy since the late 1990s and point to a common ancestry of their blaVIM-1-containing integrons.

ABSTRACTA Pseudomonas putidastrain showing broad-spectrum resistance to β-lactams, including expanded-spectrum cephalosporins and carbapenems, was isolated from a patient with a urinary tract infection at the University Hospital of Varese in northern Italy. The isolate was found to produce metallo-β-lactamase activity and to harbor a 50-kb plasmid, named pVA758, carrying a new blaIMPdeterminant, named blaIMP-12. Plasmid pVA758 was not self-transferable by conjugation to either Escherichia colior Pseudomonas aeruginosabut could be introduced by electroporation and maintained in the latter host, where it conferred resistance or decreased susceptibility to various β-lactams. The IMP-12 enzyme is quite divergent from other IMP variants: its closest relatives are IMP-8 and IMP-2 (89 and 88% sequence identity, respectively), and IMP-1 is 85% identical to IMP-12. The blaIMP-12determinant is carried on an integron-borne gene cassette whose attCrecombination site is related to those present in cassettes containing blaIMP-1, blaIMP-6, blaIMP-7, blaIMP-10, and blaIMP-11and unrelated to that present in cassettes containing blaIMP-2and blaIMP-8. IMP-12 was overproduced in E. coliby using a T7-based expression system and was purified by cation-exchange chromatography followed by gel filtration. Kinetic analysis revealed that, like other IMP variants, IMP-12 exhibits an overall preference for cephalosporins and carbapenems rather than for penicillins and does not hydrolyze temocillin and aztreonam. However, IMP-12 also exhibits some notable functional differences from other IMP variants, including uniformly poor activity toward penicillins (kcat/Kmvalues, around 104M−1· s−1) and a remarkably high Km(around 900 μM) for imipenem.

ABSTRACTSuccessful carbapenem-based chemotherapy for the treatment of Pseudomonasinfections has been seriously hindered by the recent appearance of IMP- and VIM-type metallo-ß-lactamases, which confer high-level resistance to carbapenems and most other ß-lactams. Recently, multidrug-resistant Pseudomonas putidaisolates for which carbapenem MICs were =32 µg/ml were recovered from cultures of urine from three inpatients in the general intensive care unit of the Ospedale di Circolo, Varese, Italy. Enzyme assays revealed production of a metallo-ß-lactamase activity, while molecular analysis detected in each isolate a blaVIM-1determinant carried by an apparently identical medium-sized plasmid. Conjugation experiments were unsuccessful in transferring the ß-lactamase determinant to Escherichia colior Pseudomonas aeruginosa. Macrorestriction analysis by pulsed-field gel electrophoresis demonstrated that the isolates were of clonal origin. PCR mapping and sequencing of the variable region of the plasmid-borne class 1 integron carrying the blaVIM-1determinant (named In110) showed that the blaVIM-1-containing cassette was identical to that previously found in strains of different species from other Italian hospitals and that the cassette array of In110 was not identical but clearly related to that of In70 (a blaVIM-1-containing plasmid-borne integron from an Achromobacter xylosoxidansisolate), pointing to a common origin of this cassette and to a related evolutionary history of their cognate integrons.

ABSTRACTIntegron carriage by 36 epidemiologically unrelated Acinetobacter baumanniiisolates collected over an 11-year period from patients in six different Italian hospitals was investigated. Sixteen type 1 integron-positive isolates (44%) were found, 13 of which carried the same array of cassettes, i.e., aacC1, orfX, orfX′, and aadA1a.As ribotype analysis of the isolates demonstrated a notable genetic diversity, horizontal transfer of the entire integron structure or ancient acquisition was hypothesized.

ABSTRACTAn Achromobacter xylosoxydansstrain showing broad-spectrum resistance to β-lactams (including carbapenems) and aminoglycosides was isolated at the University Hospital of Verona (Verona, Italy). This strain was found to produce metallo-β-lactamase activity and to harbor a 30-kb nonconjugative plasmid, named pAX22, carrying ablaVIM-1determinant inserted into a class 1 integron. Characterization of this integron, named In70, revealed an original array of four gene cassettes containing, respectively, theblaVIM-1gene and three different aminoglycoside resistance determinants, including an aacA4allele, a new aph-like gene named aphA15, and an aadA1allele. The aphA15gene is the first example of an aph-like gene carried on a mobile gene cassette, and its product exhibits close similarity to the APH(3′)-IIa aminoglycoside phosphotransferase encoded by Tn5(36% amino acid identity) and to an APH(3′)-IIb enzyme fromPseudomonas aeruginosa(38% amino acid identity). Expression of the cloned aphA15gene in Escherichia colireduced the susceptibility to kanamycin and neomycin as well as (slightly) to amikacin, netilmicin, and streptomycin. Characterization of the 5′ and 3′ conserved segments of In70 and of their flanking regions showed that In70 belongs to the group of class 1 integrons associated with defective transposon derivatives originating from Tn402-like elements. The structure of the 3′ conserved segment indicates the closest ancestry with members of the In0-In2 lineage. In70, with its array of cassette-borne resistance genes, can mediate broad-spectrum resistance to most β-lactams and aminoglycosides.

A blaVIM‐2metallo‐β‐lactamase determinant, identical to that previously identified in Pseudomonas aeruginosaCOL‐1 isolate from a French hospital, was detected on a 28‐kb plasmid carried by a nosocomial isolate of P. aeruginosafrom Verona, Italy. In this plasmid the blaVIM‐2determinant was inserted into a class 1 integron of original structure, named In72, that contains a partially deleted intI1integrase gene and two gene cassettes. The first cassette carries an aacA4aminoglycoside acetyl transferase determinant. The second cassette carries a blaVIM‐2determinant followed by a partially deleted attCsite. The structure of In72 was notably different from that of In56, the blaVIM‐2‐containing integron found in the COL‐1 isolate, revealing the existence of molecular heterogeneity among blaVIM‐2‐containing integrons in clinical isolates of P. aeruginosafrom Europe.

ABSTRACTA metallo-β-lactamase determinant was cloned from a genomic library of Legionella(Fluoribacter)gormaniiATCC 33297Tconstructed in the plasmid vector pACYC184 and transformed into Escherichia coliDH5α, by screening for clones showing a reduced susceptibility to imipenem. The product of the cloned determinant, named FEZ-1, contains a 30-kDa polypeptide and exhibits an isoelectric pH of 7.6. Sequencing revealed that FEZ-1 is a molecular-class B β-lactamase which shares the closest structural similarity (29.7% of identical residues) with the L1 enzyme of Stenotrophomonas maltophilia, being a new member of the highly divergent subclass B3 lineage. All the residues that in L1 are known to be directly or indirectly involved in coordination of the zinc ions were found to be conserved also in FEZ-1, suggesting that the geometry of zinc coordination in the active site of the latter enzyme is identical to that of L1. Unlike L1, however, FEZ-1 appeared to be monomeric in gel permeation chromatography experiments and exhibited a distinctive substrate specificity with a marked preference for cephalosporins and meropenem. The properties of FEZ-1 overall resembled those of a β-lactamase previously purified from the same strain of L. gormanii(T. Fujii, K. Sato, K. Miyata, M. Inoue, and S. Mitsuhashi, Antimicrob. Agents Chemother. 29:925–926, 1986) and are as yet unique among class B enzymes, reinforcing the notion that considerable functional heterogeneity can be encountered among members of this class. A system for overexpression of theblaFEZ-1gene in E. coli, based on the T7 phage promoter, was also developed.

ABSTRACTThe metallo-β-lactamase determinant of Acinetobacter baumanniiAC-54/97, a clinical isolate from Italy that was previously shown to produce an enzyme related to IMP-1, was isolated by means of a PCR methodology which targets amplification of gene cassette arrays inserted into class 1 integrons. Sequencing revealed that this determinant was an allelic variant (namedblaIMP-2) of blaIMPfound in Japanese isolates and that it was divergent from the latter by 12% of its nucleotide sequence, which evidently had been acquired independently. Similar to blaIMP,blaIMP-2was also carried by an integron-borne gene cassette. However, the 59-base element of theblaIMP-2cassette was unrelated to those of theblaIMPcassettes found in Japanese isolates, indicating a different phylogeny for the gene cassettes carrying the two allelic variants. Expression of the integron-borneblaIMP-2gene in Escherichia coliresulted in a significant decrease in susceptibility to a broad array of β-lactams (ampicillin, carbenicillin, cephalothin, cefoxitin, ceftazidime, cefepime, and carbapenems). The IMP-2 enzyme was purified from an Escherichia colistrain carrying the cloned determinant, and kinetic parameters were determined with several β-lactam substrates. Compared to IMP-1, the kinetic parameters of IMP-2 were similar overall with some β-lactam substrates (cefoxitin, ceftazidime, cefepime, and imipenem) but remarkably different with others (ampicillin, carbenicillin, cephaloridine, and meropenem), revealing a functional significance of at least some of the mutations that differentiate the two IMP variants. Present findings suggest that the environmental reservoir of blaIMPalleles could be widespread and raise a question about the global risk of their transfer to clinically relevant species.

ABSTRACTThe location and environment of the acquiredblaIMPgene, which encodes the IMP-1 metallo-β-lactamase, were investigated in a JapanesePseudomonas aeruginosaclinical isolate (isolate 101/1477) that produced the enzyme. In this isolate,blaIMPwas carried on a 36-kb plasmid, and similar to the identical alleles found in Serratia marcescensand Klebsiella pneumoniaeclinical isolates, it was located on a mobile gene cassette inserted into an integron. The entire structure of this integron, named In31, was determined. In31 is a class 1 element belonging to the same group of defective transposon derivatives that originated from Tn402-like ancestors such as In0, In2, and In5. The general structure of In31 appeared to be most closely related to that of In5 from pSCH884, suggesting a recent common phylogeny for these two elements. In In31, the blaIMPcassette is the first of an array of five gene cassettes that also includes anaacA4cassette and three original cassettes that have never been described in other integrons. The novel cassettes carry, respectively, (i) a new chloramphenicol acetyltransferase-encoding allele of the catBfamily, (ii) a qacallele encoding a new member of the small multidrug resistance family of proteins, and (iii) an open reading frame encoding a protein of unknown function. All the resistance genes carried on cassettes inserted in In31 were found to be functional in decreasing the in vitro susceptibilities of host strains to the corresponding antimicrobial agents.

ABSTRACTIn addition to the BlaB metallo-β-lactamase,Chryseobacterium(Flavobacterium)meningosepticumCCUG 4310 (NCTC 10585) constitutively produces a 31-kDa active-site serine β-lactamase, named CME-1, with an alkaline isoelectric pH. The blaACMEgene that encodes the latter enzyme was isolated from a genomic library constructed in the Escherichia coliplasmid vector pACYC184 by screening for cefuroxime-resistant clones. Sequence analysis revealed that the CME-1 enzyme is a new class A β-lactamase structurally divergent from the other members of this class, being most closely related to the VEB-1 (also named CEF-1) and PER β-lactamases and the Bacteroideschromosomal cephalosporinases. TheblaACMEdeterminant is located on the chromosome and exhibits features typical of those of C. meningosepticumresident genes. The CME-1 protein was purified from an E. colistrain that overexpresses the cloned gene via a T7-based expression system by means of an anion-exchange chromatography step followed by a gel permeation chromatography step. Kinetic parameters for several substrates were determined. CME-1 is a clavulanic acid-susceptible extended-spectrum β-lactamase that hydrolyzes most cephalosporins, penicillins, and monobactams but that does not hydrolyze cephamycins and carbapenems. The enzyme exhibits strikingly different kinetic parameters for different classes of β-lactams, with both Kmandkcatvalues much higher for cephalosporins than for penicillins and monobactams. However, the variability of both kinetic parameters resulted in overall similar acylation rates (kcat/Kmratios) for all types of β-lactam substrates.

ABSTRACTProduction of a metallo-β-lactamase activity was detected in a carbapenem-resistant Pseudomonas aeruginosaclinical isolate (isolate VR-143/97) from an Italian inpatient at the Verona University Hospital (northern Italy). The metallo-β-lactamase determinant was isolated from a genomic library of VR-143/97, constructed in an Escherichia coliplasmid vector, by screening for clones with reduced susceptibility to imipenem. Sequencing of the cloned gene revealed that it encoded a new class B β-lactamase that was named VIM-1. At the sequence level VIM-1 was rather divergent from the other class B enzymes (16.4 to 38.7% identity), overall being more similar to members of subclass B1 including the β-lactamase II of Bacillus cereus(Bc-II), the Bacteroides fragilisCcrA, the Chryseobacterium meningosepticumBlaB, and the cassette-encoded IMP-1 enzymes. Among these, VIM-1 showed the highest degree of similarity to Bc-II. Similarly to blaIMP,blaVIMwas also found to be carried on a gene cassette inserted into a class 1 integron. TheblaVIM-containing integron was located on the chromosome of P. aeruginosaVR-143/97, and the metallo-β-lactamase-encoding determinant was not transferable toE. coliby conjugation. Expression of the integron-borneblaVIMgene in E. coliresulted in a significant decrease in susceptibility to a broad array of β-lactams (ampicillin, carbenicillin, piperacillin, mezlocillin, cefotaxime, cefoxitin, ceftazidime, cefoperazone, cefepime, and carbapenems), revealing a very broad substrate specificity of the VIM-1 enzyme.

ABSTRACTA novel gene, aac(3)-Ic, encoding an AAC(3)-I aminoglycoside 3-N-acetyltransferase, was identified on a gene cassette inserted into a Pseudomonas aeruginosaintegron that also carries a blaVIM-2and a cmlA7gene cassette. The aac(3)-Icgene product is 59 and 57% identical to AAC(3)-Ia and AAC(3)-Ib, respectively, and confers resistance to gentamicin and sisomicin.

ABSTRACTA new natural TEM-2 derivative, named TEM-72, was identified in aProteus mirabilisstrain and in a Morganella morganiistrain 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 ofblaTEM-72in Escherichia coliwas capable of decreasing the host susceptibility to the above drugs.

A blaVIM-2 metallo-β-lactamase determinant, identical to that previously identified in Pseudomonas aeruginosa COL-1 isolate from a French hospital, was detected on a 28-kb plasmid carried by a nosocomial isolate of P. aeruginosa from Verona, Italy. In this plasmid the blaVIM-2 determinant was inserted into a class 1 integron of original structure, named In72, that contains a partially deleted intI1 integrase gene and two gene cassettes. The first cassette carries an aacA4 aminoglycoside acetyl transferase determinant. The second cassette carries a blaVIM-2 determinant followed by a partially deleted attC site. The structure of In72 was notably different from that of In56, the blaVIM-2-containing integron found in the COL-1 isolate, revealing the existence of molecular heterogeneity among blaVIM-2-containing integrons in clinical isolates of P. aeruginosa from Europe.