Marisa Almuzara

VIM-2-producing Pseudomonas putida, Buenos Aires.

To the Editor: Pseudomonas putida (0.03% of isolates from the culture collection of the Argentina Association of Microbiology, www.aam.org.ar) infections are mainly reported in immunocompromised patients, such as newborns, neutropenic patients, and cancer patients. They are usually susceptible to extended-spectrum cephalosporins, aminoglycosides, fluoroquinolones, and carbapenems. However, isolates have been identified that produce acquired metallo-β-lactamases (MBLs) and are resistant to most β-lactams, including carbapenems. Two multidrug-resistant P. putida isolates were obtained from clinical samples at the Sanatorio Mater Dei in Buenos Aires. One isolate was obtained in March 2005 from a urine specimen of a 76-year-old woman with a urinary tract infection who was using a urethral catheter. The second isolate was obtained in May 2005 from a tracheal aspirate of a 67-year-old man with nosocomial pneumonia. Bacteria were identified by using conventional biochemical tests and the API 20NE System (API, bioMerieux, Lyon, France). Susceptibility tests were performed according to standard procedures. Both isolates were resistant to imipenem and meropenem (MICs >32 μg/mL) but were susceptible to amikacin and colistin. Susceptibility data are shown in the Table. Table Antimicrobial drug susceptibility profiles of 2 blaVIM-2-carrying Pseudomonas putida isolates, Argentina Screening for MBLs was performed by using a double-disk diffusion method. Disks containing 1 μmol/L EDTA (metal chelator) were placed on Mueller-Hinton agar plates containing the 2 isolates. Disks containing carbapenem were placed 15 mm from disks containing EDTA. An increase in the inhibition zone of the disk containing drug near the disk containing EDTA was observed for both isolates, which suggested the presence of MBLs. PCR amplification of imp and vim genes was conducted by using primers based on conserved regions of the imp and vim genes (blaIMP-F: 5′-GAAGGCGTTTATGTTCATACTT-3′, blaIMP-R: 5′-GTTTGCCTTACCATATTTGGA-3′, blaVIMG-F: 5′-GGTGTTTGGTCGCATATC-3′, and blaVIMG-R 5′-TGGGCCATTCAGCCAGATC-3′) and heat-extracted DNA as template. Reactions were performed in a T-gradient instrument (Biometra, Gottingen, Germany) with the following reaction conditions: 1 cycle at 95°C for 5 min, 52°C for 15 min, and 72°C for 6 min, followed by 30 cycles at 95°C for 1 min, 52°C for 1 min, and 72°C for 1 min, and a final reaction at 72°C for 20 min. Amplified fragments were sequenced on both strands by using an ABI Prism DNA 3700 (Applied Biosystems, Foster City, CA, USA), and nucleotide sequences were compared by using BLAST (National Center for Biotechnology Information, Bethesda, MD, USA, http://www.ncbi.nlm.nih.gov/Tools/). Nucleotide sequences were completely homologous to the vim-2 coding gene. Two repetitive-element–based PCR (rep-PCR) assays (ERIC-PCR and REP-PCR) with primers REP-1 (5′-IGCGCCGICATCAGGC-3′), REP-2 (5′-CGTCTTATCAGGCCTAC-3′), ERIC-1 (5′-CACTTAGGGGTCCTCAATGTA-3′), and ERIC-2 (5′-AAGTAAGTGACTGGGGTGAGCG-3′) were used to characterize isolates. PCR conditions were 94°C for 2 min, 30 cycles at 94°C for 30 s, 50°C for 1 min, and 72°C for 4 min, and a final reaction at 72°C for 7 min. Banding patterns were visually analyzed after electrophoresis of samples. Variations in band intensity were not considered to indicate genetic differences. Banding patterns obtained by REP-PCR and ERIC-PCR assays were identical in both isolates (data not shown). Among the MBLs acquired by P. putida, IMP-1 was reported by Senda et al. in Japan in 1996 (1) and later reported in Taiwan and Japan (2). IMP-12 was the first IMP MBL described in P. putida in Europe (3). VIM-1 in P. putida was first reported in Europe (4), and VIM-2 in P. putida was first reported in Taiwan, Republic of Korea, Japan, and France (5,6). Our isolates were resistant to aztreonam (MIC 64 μg/mL). However, carbapenem-susceptible P. putida had low levels of susceptibility because the MIC50 was only 1 dilution below the current breakpoint (7,8). Aztreonam resistance could not be transferred by conjugation between IMP-1–producing (aztreonam-resistant) P. putida and P. aeruginosa (2) and is not associated with a transposon carrying blaVIM-2 (6). No evidence of extended-spectrum β-lactamases was detected in our isolates by classic synergy assays with clavulanate plus aztreonam, ceftazidime, or cefotaxime. VIM-6–producing P. putida isolates from Singapore (9) were more resistant to aztreonam (MIC >128 μg/mL), ceftazidime, and cefepime (MIC >256 μg/mL). Detection of blaVIM-2 in Pseudomonas in South America was initially reported by the SENTRY Antimicrobial Surveillance Program (10) and included 1 P. fluorescens isolate in Chile and 3 P. aeruginosa isolates in Venezuela. To the best of our knowledge, our report is the first of VIM-2 in P. putida in Latin America. VIM-2–producing P. putida, which were originally restricted to East Asia and only very recently found in France, may represent an emerging pathogen or function as reservoirs for resistance because of their widespread presence in the hospital environment.

Comparison of the Bruker MALDI-TOF Mass Spectrometry System and Conventional Phenotypic Methods for Identification of Gram-Positive Rods

In recent years, MALDI-TOF Mass Spectrometry (MS) method has emerged as a promising and a reliable tool for bacteria identification. In this study we compared Bruker MALDI-TOF MS and conventional phenotypic methods to identify a collection of 333 Gram-positive clinical isolates comprising 22 genera and 60 species. 16S rRNA sequencing was the reference molecular technique, and rpoB gene sequecing was used as a secondary gene target when 16Sr RNA did not allow species identification of Corynebacterium spp. We also investigate if score cut-offs values of ≥1,5 and ≥1,7 were accurate for genus and species-level identification using the Bruker system. Identification at species level was obtained for 92,49% of Gram-positive rods by MALDI-TOF MS compared to 85,89% by phenotypic method. Our data validates the score ≥1,5 for genus level and ≥1,7 for species-level identification in a large and diverse collection of Gram-positive rods. The present study has proved the accuracy of MALDI-TOF MS as an identification method in Gram-positive rods compared to currently used methods in routine laboratories.

First Case of Fulminant Sepsis Due to Wohlfahrtiimonas chitiniclastica

ABSTRACT We report the first case of fulminant sepsis due to Wohlfahrtiimonas chitiniclastica. This case is also the first one reported in South America. We emphasize the importance of recognizing bacteria that live in the larvae of a parasitic fly as the causative agent of severe infections in homeless patients.

In vitro susceptibility of Achromobacter spp. isolates: comparison of disk diffusion, Etest and agar dilution methods

In this study, we analysed the antimicrobial susceptibility of 92 strains of Achromobacter spp. isolated from clinical samples to 18 antimicrobial agents. The disk diffusion method and Etest were compared with the agar dilution method, and the breakpoints of susceptibility and resistance for the disk diffusion method for the antimicrobials tested were determined. The most active antibiotics were piperacillin, piperacillin/tazobactam and the carbapenems. By applying the linear least-squares regression method, breakpoints could be established for antibiotics active against this genus such as imipenem, meropenem, ertapenem and trimethoprim/sulfamethoxazole (SXT). Other active antibiotics, such as piperacillin and minocycline, could be tested by the Etest method. The less active antibiotics such as gentamicin, doxycycline and tetracycline could be tested by the disk diffusion method. For the rest of the antimicrobial agents tested, breakpoints could not be established owing to the high percentage of errors and/or the poor linear regression coefficient obtained. Therefore, these antimicrobial agents should be tested by minimal inhibitory concentration determination. In summary, we recommend the following zone diameter breakpoints for resistant and susceptible, respectively: < or = 11 mm and > or = 22 mm for imipenem; < or = 13 mm and > or = 24 mm for meropenem; < or = 17 mm and > or = 24 mm for ertapenem; < or = 15 mm and > or = 21 mm for gentamicin; < or = 27 mm and > or = 28 mm for SXT; < or = 20 mm and > or = 29 mm for tetracycline; and < or = 20 mm and > or = 24 mm for doxycycline.

Emergence and spread of plasmid-borne tet(B)::ISCR2 in minocycline-resistant Acinetobacter baumannii isolates.

ABSTRACT Resistance to minocycline has emerged in multidrug-resistant Acinetobacter baumannii isolates from Buenos Aires hospitals. Few reports about the description and dispersion of tet genes in this species have been published. We observed the presence of tet(B) in all minocycline-resistant isolates. This gene was found to be associated with the ISCR2 mobile element, which may, in part, explain its dispersion.

Evaluation of matrix-assisted laser desorption ionization-time-of-flight mass spectrometry for species identification of Nonfermenting Gram-Negative Bacilli

Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) to identify 396 Nonfermenting Gram-Negative Bacilli clinical isolates was evaluated in comparison with conventional phenotypic tests and/or molecular methods. MALDI-TOF MS identified to species level 256 isolates and to genus or complex level 112 isolates. It identified 29 genera including uncommon species.

First case of human infection due to Pseudomonas fulva, an environmental bacterium isolated from cerebrospinal fluid.

ABSTRACT We report the first case of human infection due to Pseudomonas fulva. P. fulva caused acute meningitis following the placement of a drainage system in a 2-year-old female. Additionally, the isolate displayed a VIM-2 carbapenemase in a class 1 integron context.

Novel gastric helicobacters and oral campylobacters are present in captive and wild cetaceans

The mammalian gastric and oral mucosa may be colonized by mixed Helicobacter and Campylobacter species, respectively, in individual animals. To better characterize the presence and distribution of Helicobacter and Campylobacter among marine mammals, we used PCR and 16S rDNA sequence analysis to examine gastric and oral samples from ten dolphins (Tursiops gephyreus), one killer whale (Orcinus orca), one false killer whale (Pseudorca crassidens), and three wild La Plata river dolphins (Pontoporia blainvillei). Helicobacter spp. DNA was widely distributed in gastric and oral samples from both captive and wild cetaceans. Phylogenetic analysis demonstrated two Helicobacter sequence clusters, one closely related to H. cetorum, a species isolated from dolphins and whales in North America. The second related cluster was to sequences obtained from dolphins in Australia and to gastric non-H. pylori helicobacters, and may represent a novel taxonomic group. Dental plaque sequences from four dolphins formed a third cluster within the Campylobacter genus that likely represents a novel species isolated from marine mammals. Identification of identical Helicobacter spp. DNA sequences from dental plaque, saliva and gastric fluids from the same hosts, suggests that the oral cavity may be involved in transmission. These results demonstrate that Helicobacter and Campylobacter species are commonly distributed in marine mammals, and identify taxonomic clusters that may represent novel species.

Full Resistance and Decreased Susceptibility to Carbapenems in IMP-13-Producing Pseudomonas aeruginosa Isolates from an Outbreak

Of 23 ceftazidime-resistant Pseudomonas aeruginosa isolates with simultaneous decreased susceptibility to carbapenems recovered from inpatients at Hospital Eva Peron, Provincia de Buenos Aires, Argentina, from December 2004 to December of 2005, 18 were positive by a double-disk phenotypic screening

Isolation of Kerstersia gyiorum from a Patient with Cholesteatomatous Chronic Otitis Media

ABSTRACT We describe the first case of a Kerstersia gyiorum strain isolated from a patient with cholesteatomatous chronic otitis media. We emphasize the isolation of members of the family Alcaligenaceae in serious infections and unusual sites and the importance of polyphasic identification addressing the definitive identification.