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Metallo-beta-lactamase VIM-2 in clinical isolates of Pseudomonas aeruginosa from Portugal

Resistance to carbapenems is emerging, and it is a great problem to therapeutics. Three isolates of Pseudomonas aeruginosa from a Portuguese hospital identified in urine and sputum, in 1995, presented a high-level resistance to imipenem (> 32 mg/L). Afterward, one isolate of P. aeruginosa recovered from urine of an ambulatory patient in 1998 showed high resistance to imipenem and meropenem. The resistance to carbapenems in these strains was associated with the production of a class B beta-lactamase, as was demonstrated by imipenem hydrolysis and inhibition by EDTA. Using primers described for bla(IMP) and bla(VIM), the amplification of the latter was observed in all isolates and a VIM-2 metallo-enzyme was identified. The pulsed-field gel electrophoresis (PFGE) patterns of these isolates were indistinguishable, suggesting dissemination to the community of this VIM-2 producer.

First description of CTX-M-15-producing Klebsiella pneumoniae in Portugal

The first CTX-M-15 β-lactamase was found in several enterobacterial isolates from India in 1999 (4). Worldwide spread of CTX-M-15 β-lactamases is now well documented (1-5, 7, 8, 12). CTX-M β-lactamases confer high-level resistance to cefotaxime, ceftriaxone, and aztreonam and are well inhibited by clavulanate and tazobactam (10, 11). n nKlebsiella pneumoniae 193KFFUL was isolated from a blood culture in September 2003 at the Hospital de Santa Maria. The clinical isolate was resistant to cefotaxime and cefepime (MIC, >256 μg/ml) and ceftazidime and aztreonam (MIC, 96 μg/ml) and susceptible to imipenem (MIC, 0.125 μg/ml). Tazobactam and clavulanate restored the activities of piperacillin, cefotaxime, ceftazidime, and cefepime as determined by E-test strips (Table u200b(Table1).1). Genomic DNA was prepared as described elsewhere (6), and PCR experiments were performed using specific primers in order to amplify bla genes coding for CTX-M β-lactamases. The set of primers CTX1 (5′-SCS ATG TGC AGY ACC AGT AA-3′) and CTX2 (5′-CCG CRA TAT GRT TGG TGG TG-3′), designed in accordance with consensus sequences from the blaCTX-M genes available at GenBank, produced an amplicon with 544 bp. In order to perform sequencing of the entire gene, PCR was performed with primers CTX-M-1F (5′-ATG GTT AAA AAA TCA CTG CGY C-3′) and CTX-M-1R (5′-TTA CAA ACC GTC GGT G-3′). The amplicon of 876 bp was cloned into the pCR2.1-TOPO vector, resulting in plasmid p193K1, and introduced into Escherichia coli TOP10 chemically competent cells. The sequenced gene shared 100% homology with blaCTX-M-15. E. coli 193K1 revealed MIC profiles similar to those of the parental strain, particularly for cefotaxime, whose MIC was >256 μg/ml. CTX-M-15 showed increased activity against ceftazidime because of a single nucleotide substitution (A-725→G) that has already been reported in CTX-M-16 (2, 4, 10). n n n nTABLE 1. n nMICs of β-lactam antibiotics alone or in association with β-lactam inhibitors for K. pneumoniae clinical isolate 193KFFUL and E. coli 193K1 harboring recombinant plasmid p193K1 n n n nTo explore the surrounding regions of blaCTX-M-15, PCR was performed with internal primers CTX1 and CTX2 and primers hybridizing to the ends of the insertion sequences ISEcp1 and IS903 (2) and to the conserved regions of class 1 integrons, 5′-CS and 3′-CS (6). Positive PCR products were obtained with primers ISEcp1F and CTX2 (911 bp) and primers CTX1 and IS903R (1,430 bp). Nucleotide sequence analysis indicated that blaCTX-M-15 was flanked upstream by an ISEcp1-like element and downstream by an IS903-like element. Insertion sequences such as ISEcp1 or IS903 have already been described as flanking regions of blaCTX-M-14, blaCTX-M-17, and blaCTX-M-19 (2, 4, 9). n nA class 1 integron was identified containing an aadA1 and an aadA2 gene not associated with the blaCTX-M-15 gene. n nThe −35 and −10 promoter sequences for blaCTX-M-15 expression are located at the end of an ISEcp1-like element upstream of its inverted repeat, which is 48 bp from the start codon ATG (data not shown), as already described for blaCTX-M-15 from India and Turkey and different from the blaCTX-M-15 gene described in Poland, in which the distance is 128 bp (4, 9). In addition, analysis of the downstream region of blaCTX-M-15 showed that 685 nucleotides had 97% similarity to IS903-C from blaCTX-M-17. n nThis is the first report identifying an IS903-like element downstream of the blaCTX-M-15 gene in a K. pneumoniae isolate from a Portuguese hospital.

Outbreak of GES-1 β-Lactamase-Producing Multidrug-Resistant Klebsiella pneumoniae in a University Hospital in Lisbon, Portugal

Plasmid-located extended-spectrum β-lactamase genes are mostly found in Klebsiella pneumoniae (4), which is an important cause of nosocomial infections (1). In this study, we report the existence of K. pneumoniae clinical isolates producing the Ambler class A enzyme GES-1. This enzyme has been reported in Europe from K. pneumoniae (4) and Pseudomonas aeruginosa (2). n nBetween February 1999 and 2001, 30 K. pneumoniae clinical isolates were collected from different patients, distributed among several wards (surgery and medical services and in different intensive care units) at the Hospital de Santa Maria, Lisbon, Portugal. n nTwenty-four isolates were identified from urine, four were identified from respiratory tract samples (three from sputum and one from bronchial exudate), and the remaining two isolates were found in blood and pus. n nAntibiotic susceptibility testing by disk diffusion (3) suggested the presence of an extended-spectrum β-lactamase. Synergies were observed among clavulanic acid-amoxicillin, cefotaxime, aztreonam, and cefepime. All isolates were resistant to clavulanic acid, ceftazidime, cefuroxime, gentamicin, kanamycin, netilmicin, nalidixic acid, and norfloxacin. They were susceptible to imipinem and cefepime and presented reduced susceptibilities to amikacin, cefotaxime, and aztreonam. n nThe analysis of genomic DNA, digested with XbaI and resolved by pulsed-field gel electrophoresis (1), revealed the same macrorestriction pattern among all isolates, classified as indistinguishable according to the work of Tenover et al. (6). n nOn the isoelectric focusing gel two β-lactamase activities with pIs of 5.9 and 7.6 were detected. The β-lactamase activity of pI 7.6 corresponds to chromosomal SHV penicil-linase, and the pI value of 5.9 represented the GES-1 β-lactamase. n nPlasmid extraction, performed according to the alkaline lysis method (5), revealed plasmids with molecular sizes ranging from 3 to 23 kb. From five selected K. pneumoniae strains we obtained Escherichia coli DH5α transformants more resistant to ceftazidime than to cefotaxime and aztreonam and harboring a plasmid with a molecular size of ca. 9 kb. Under standard PCR conditions, plasmid DNA preparations from K. pneumoniae and E. coli DH5α transformants were used as templates for amplification of the blaGES-1 gene with primers GES-1A and GES-1B (4). All isolates revealed an 864-bp PCR product. The resulting amplicon was cloned into the SmaI site of pBK-CMV. The E. coli TOP10 harboring pMFA-62 was selected for subsequent analysis and sequencing. MICs of β-lactam antibiotics were determined with E-test strips (AB Biodisk, Solna, Sweden). For the K. pneumoniae clinical isolates, E. coli DH5α transformants, and E. coli TOP10 harboring recombinant plasmid pMFA-62, the cefuroxime and ceftazidime MICs were >256 μg/ml, and the MIC ranges (in micrograms per milliliter) were 3 to 0.75 for aztreonam and 6 to 8 for cefotaxime (Table u200b(Table1).1). The nucleotide sequence of the cloned fragment revealed 100% identity with blaGES-1 from P. aeruginosa Pa695 (2) and differs by a single silent mutation at position 591 from blaGES-1 described elsewhere for K. pneumoniae ORI-1 (4). n n n nTABLE 1. n nMICs of β-lactams for K. pneumoniae clinical isolates, E. coli DH5α transformants, E. coli TOP10 harboring recombinant plasmid pMFA-62, and reference strains E. coli DH5α and E. coli TOP10 with pBK-CMV n n n nThe same macrorestriction pattern by pulsed-field gel electrophoresis indicated that an endemic K. pneumoniae strain producing GES-1 β-lactamase was presenting in different wards in the Hospital de Santa Maria. The persistence of these multiresistant microorganisms in the hospital may be associated with the existence of other resistance genes, inserted in multidrug-resistant integrons and/or plasmids.

First isolation of bla(VIM-2) in Klebsiella oxytoca clinical isolates from Portugal.

VIM-type carbapenemases were originally detected in Europe (2, 4, 7) and have been essentially found in Pseudomonas aeruginosa, as well as in Enterobacteriaceae (2, 6, 8). The blaVIM genes are often carried by mobile gene cassettes inserted into class 1 integrons (7, 11). The Klebsiella genus is responsible for the most frequent human nosocomial infections of the respiratory and urinary tracts. Four Klebsiella oxytoca clinical isolates were recovered by blood culture from neonatal patients at a pediatric hospital. The clinical isolates showed resistance to amoxicillin and ticarcillin, which was restored by clavulanate, aminoglycosides, and fluoroquinolones, and intermediate susceptibility to imipenem (MIC, 4 μg/ml) and broad-spectrum cephalosporins and aztreonam (MICs, 8 and 16 μg/ml) and were susceptible to meropenem (MIC, 0.075 μg/ml). A deformation of ellipses between the two gradient sections with E-test MBL (metallo-β-lactamase) strips was indicative of MBL production (10).

Diversity of chromosomal AmpC β-lactamases from Enterobacter cloacae isolates in a Portuguese hospital

Six clinical isolates of Enterobacter cloacae isolated in a Portuguese hospital, between April 1999 and November 2000, demonstrated resistance to almost all broad-spectrum cephalosporins, except to cefepime. These isolates were susceptible to quinolones and to aminoglycosides. Isoelectric focusing demonstrated production of beta-lactamases with pIs > 8.0 and by all six isolates, exhibiting a cephalosporinase phenotype. The results of pulsed field gel electrophoresis revealed that these isolates were genetically unrelated. The amino acid sequence of six AmpC beta-lactamases (Eclo1FF, Eclo6FF, Eclo9FF, Eclo10FF, Eclo11FF and Eclo15FF) shared 97-99% homology with the chromosomal AmpC beta-lactamase from E. cloacae P99 and 86-87% homology with those of two plasmid-mediated AmpC beta-lactamases, MIR-1 and ACT-1. This is the first report of chromosomal AmpC beta-lactamase production by E. cloacae isolates in a Portuguese hospital.

New Chromosomal AmpC β-Lactamase in Enterobacter cloacae

Several members of the Enterobacteriaceae, including Enterobacter spp., are naturally resistant to amoxicillin and cephalosporins. Enterobacter cloacae produces chromosomally encoded β-lactamases, also called cephalosporinases (1), and is a serious nosocomial pathogen, the third most prevalent bacterium isolated in intensive care settings (5, 8). We report here the study of a new chromosomal AmpC β-lactamase produced by E. cloacae FFUL2En isolated from the blood culture of a patient hospitalized in a medicine ward of Hospital de Santa Maria, Lisbon, Portugal. The antibiogram revealed resistance to aminopenicillins, aztreonam, and broad-spectrum cephalosporins, except imipenem, aminoglycosides, and quinolones. By isoelectrofocusing, the sonicate extracts expressed a pI of 8.68, suggesting the presence of a presumed AmpC enzyme. n nA total DNA preparation from E. cloacae FFUL2En was used in PCR experiments with two sets of primers, TN5 (5′-CGTTTGTCAGGCACAGTCAAATCCA) and TN4 (5′-TTACTGTAGCGCGTCGAGGATATGG) and the internal primers TN2 (5′-TTCCACTGCGGCTGCCAGT) and TN3 (5′-CGGATGAGGTCACGGATAACGCC), designed in accordance with consensus sequences from the ampC genes described for E. cloacae and available at GenBank. The amplicon with 1,234 bp was cloned into the SmaI site of the pBK-CMV vector (6) with a TOPO TA cloning kit, resulting in the plasmid p2En1. The β-lactam susceptibility pattern of Escherichia coli 2En1, harboring the recombinant plasmid p2En1, displayed cefoxitin, cefuroxime, ceftazidime, and piperacillin plus tazobactam MICs of >256 μg/ml and a cefepime MIC of 0.5 μg/ml. The MICs of cefotaxime and aztreonam were lower than those for the parental strain (Table u200b(Table1).1). The E. coli 2En1 transformant showed the same pI as the parental strain (pI 8.68), and the substrate profile of the enzyme EcloFFUL2En was determined with the transformant crude enzymatic extract (7). The Vmax values indicate that cephalothin, with a Vmax of 3,000.1 μM/min, is hydrolyzed more quickly than cefoxitin (Vmax = 3.7 μM/min). Ceftazidime and cefotaxime are not hydrolyzed at detectable levels (Vmax = <0.1 μM/min). n n n nTABLE 1. n nu2002 MICs of β-lactams for E. cloacae FFUL2En clinical isolate, E. coli 2En1 harboring recombinant plasmid p2En1, and reference strain E. coli TOP10 harboring the pBK-CMV plasmid n n n nIn order to perform the sequencing reactions, the amplicon of 1,234 bp was cloned in the pCR2.1-TOPO vector with a TOPO TA cloning kit, resulting in the plasmid p2En2. The sequence with 382 amino acids has an 86% identity with the AmpC of E. cloacae P99 and 98% identity with the plasmid-borne MIR-1 β-lactamase gene product (2). n nTo search for a possible chromosomal location of the blaAmpC gene, whole-cell DNA of E. cloacae FFUL2En was restricted with I-CeuI endonuclease (New England Biolabs), which recognizes a 26-bp sequence in rrn genes coding for the 23S large-subunit rRNA. After digestion, separation of the resulting fragments was performed on a contour-clamped homogeneous electric field-DRII apparatus, as described previously (3). n nThe restricted fragments of E. cloacae FFUL2En DNA were transferred to a nylon membrane by Southern blotting (9) and were hybridized by using a nonradioactive labeling and detection kit (Roche) with a PCR-obtained probe with primers TN5 and TN2 (see above), consisting of a 576-bp fragment of blaAmpC and a 16S rRNA gene probe amplified with universal primers described elsewhere (4). The blaAmpC probe hybridized only with the 630-kb fragment of E. cloacae FFUL2En. These data indicate the chromosomal location of the blaAmpC gene, coding for the AmpC β-lactamase EcloFFUL2En, in E. cloacae FFUL2En, which is closely related to the plasmid-borne MIR-1 from Klebsiella pneumoniae.