Gabriel Gutkind

Chromosome-Encoded CTX-M-3 from Kluyvera ascorbata: a Possible Origin of Plasmid-Borne CTX-M-1-Derived Cefotaximases

ABSTRACT A gene identical to plasmid-borne blaCTX-M-3 is present in the chromosome of one Kluyvera ascorbata strain. It is associated with a structure including an inverted repeat right and an open reading frame 477-like gene probably involved in the mobilization of blaCTX-M-3. Two other K. ascorbata strains rendered the previously described blaKLUA-9 gene.

Novel Class 1 Integron (InS21) Carrying blaCTX-M-2 in Salmonella enterica Serovar Infantis

ABSTRACT The genetic organization of the region coding for CTX-M-2 in Salmonella enterica serovar Infantis was determined by PCR mapping. This gene seems to have been mobilized from the Kluyvera ascorbata chromosome to a complex sulI-type integron, similar to In6 and In7.

CTX-M-12 β-Lactamase in a Klebsiella pneumoniae Clinical Isolate in Colombia

ABSTRACT We describe the detection of the CTX-M-12 β-lactamase from a clinical isolate of Klebsiella pneumoniae in Colombia. Screening of nosocomial Klebsiella spp. and Escherichia coli isolates from a network of teaching hospitals revealed the presence of CTX-M enzymes in multiple cities. This is the first description of CTX-M in Colombia.

Enteropathogenic Escherichia coli Strains Carrying Genes Encoding the PER-2 and TEM-116 Extended-Spectrum β-Lactamases Isolated from Children with Diarrhea in Uruguay

ABSTRACT We studied 13 extended-spectrum β-lactamase (ESBL)-producing enteropathogenic Escherichia coli isolates from children suffering acute diarrhea in Uruguay. ESBL characterization in crude extracts showed a single band at pI 5.4. PCR amplification and sequencing data allowed identification of blaPER-2 and blaTEM-116. Retrospective analysis suggests that these strains were disseminated in the community, even if unnoticed, prior to their access to the hospital environment more than a decade ago.

β-Cyclodextrin hydrogels for the ocular release of antibacterial thiosemicarbazones

Two types of hydrophilic networks with conjugated beta-cyclodextrin (β-CD) were developed with the aim of engineering useful platforms for the localized release of an antimicrobial 5,6-dimethoxy-1-indanone N4-allyl thiosemicarbazone (TSC) in the eye and its potential application in ophthalmic diseases. Poly(2-hydroxyethyl methacrylate) soft contact lenses (SCLs) displaying β-CD, namely pHEMA-co-β-CD, and super-hydrophilic hydrogels (SHHs) of directly cross-linked hydroxypropyl-β-CD were synthesized and characterized regarding their structure (ATR/FT-IR), drug loading capacity, swelling and in vitro release in artificial lacrimal fluid. Incorporation of TSC to the networks was carried out both during polymerization (DP method) and after synthesis (PP method). The first method led to similar drug loads in all the hydrogels, with minor drug loss during the washing steps to remove unreacted monomers, while the second method evidenced the influence of structural parameters on the loading efficiency (proportion of CD units, mesh size, swelling degree). Both systems provided a controlled TSC release for at least two weeks, TSC concentrations (up to 4000μg/g dry hydrogel) being within an optimal therapeutic window for the antimicrobial ocular treatment. Microbiological tests against P. aeruginosa and S. aureus confirmed the ability of TSC-loaded pHEMA-co-β-CD network to inhibit bacterial growth.

Changing epidemiology of extended-spectrum β-lactamases in Argentina: emergence of CTX-M-15

ABSTRACT A multicenter survey, carried out in 2010 in Argentina, showed an increased prevalence of extended-spectrum β-lactamase (ESBL)-producing enterobacteria, with some changes in the molecular epidemiology of circulating ESBLs. While enzymes of the CTX-M-2 group remain endemic, the emergence of CTX-M-15 and of enzymes of the CTX-M-8 and CTX-M-9 groups was observed. The CTX-M-15-positive isolates represented 40% of CTX-M producers and included representatives of Escherichia coli ST131 and Klebsiella pneumoniae ST11.

Detection of class 1 and 2 integrons, extended-spectrum β-lactamases and qnr alleles in enterobacterial isolates from the digestive tract of Intensive Care Unit inpatients

In this study, we searched for extended-spectrum β-lactamases (ESBLs), class 1 and 2 integrons, and qnrA, qnrB and qnrS genes in 56 oxyimino-cephalosporin and/or ciprofloxacin-resistant enterobacterial isolates obtained from the gastrointestinal tract of patients admitted in an Intensive Care Unit in Uruguay. ESBLs were detected in 11 isolates (6 CTX-M-2, 3 CTX-M-9, 1 CTX-M-15 and 1 PER-2). qnr genes and integrons were detected in 5 and 24 isolates, respectively. Eight different antibiotic resistance gene cassettes were found within six different genetic arrangements. Two types of complex class 1 integrons carrying insertion sequence ISCR1 were found, one showing bla(CTX-M-2)-orf3 and the other qnrA1-ampR. Ten of the thirteen isolates carrying class 2 integrons presented the element IS5 inserted between intI2 and dfrA1, whereas another class 2 integron lacked the internal stop codon usually present in intI2. This is the first report of the occurrence of qnrA, qnrB and bla(CTX-M-9) in Uruguay. Dissemination of the different groups of CTX-M enzymes (i.e. groups 1, 2 and 9) appears to be a recent phenomenon in South America.

Community-associated methicillin-resistant Staphylococcus aureus, eastern Argentina

Sixty-nine community-associated methicillin-resistant Staphylococcus aureus recovered in 6 healthcare centers from northeastern and eastern Argentina were genotyped by pulsed-field gel electrophoresis. The predominant pulsotype was widely distributed harbored SCCmec type IV and Panton-Valentine leukocidin genes. Representative isolates were characterized by multilocus sequence typing and spa typing, demonstrating that this clone belonged to ST5 and spa type 311.

Ciprofloxacin-Resistant Enterobacteria Harboring the aac(6′)-Ib-cr Variant Isolated from Feces of Inpatients in an Intensive Care Unit in Uruguay

The presence of aac(6′)-Ib-cr is associated with decreased susceptibility to aminoglycosides (kanamycin, amikacin, and tobramycin) and to norfloxacin and ciprofloxacin (9). This allelic variant of aac(6′)-Ib was found to be linked to the extended-spectrum β-lactamase (ESBL) gene blaCTX-M-15 in isolates from many countries (4, 6, 7), while association of aac(6′)-Ib with the blaCTX-M-2 ESBL gene has been widely reported in Uruguay and Argentina (3, 11). In this work we looked for the presence of aac(6′)-Ib and the aac(6′)-Ib-cr variant and their putative ESBL coresistance markers in fecal isolates of enterobacteria resistant to ciprofloxacin and/or ceftazidime from inpatients in an intensive care unit (ICU) in Montevideo, Uruguay. From 1 March to 31 October 2006, 106 patients were admitted to this ICU and followed daily until discharge. Rectal swabs obtained at 1, 4, 7, 10, 13, and 16 days after admission were plated on MacConkey agar plus ceftazidime (4 mg/liter) or ciprofloxacin (2 mg/liter). Enterobacterial isolates were identified by classical methods, including only the first isolate of each bacterial species per patient in this study. Antibiotic resistance profiling, screening, and confirmatory testing for ESBL detection were performed by disk diffusion assay, and results were interpreted following the CLSI guidelines (2). A total of 58/106 patients (55.2%) were colonized with ciprofloxacin- and/or ceftazidime-resistant enterobacteria, and 68 isolates were included in this study. Of these, 48 were resistant to gentamicin and 24 to amikacin (Table ​(Table11). TABLE 1. Main characteristics of the 68 studied isolatesa All aminoglycoside-resistant isolates were screened for aac(6′)-Ib by PCR; amplicons were analyzed by restriction with BstF5I, as described by Park et al. (8). PCR products that were not digested by the enzyme [tentatively assigned to aac(6′)-Ib-cr] were confirmed to contain aac(6′)-Ib-cr by double-strand sequencing. Only two Escherichia coli isolates were positive for aac(6′)-Ib-cr detection. Recalling the observed links between blaCTX-M-15 and aac(6′)Ib-cr (4, 6, 7) and between aac(6′)Ib and blaCTX-M-2, the two aac(6′)-Ib-cr-positive isolates were further analyzed by PCR to detect CTX-M-1 and CTX-M-2 group ESBL genes using previously described primers (3, 5). Both isolates were positive only for CTX-M-1 group genes, identified as blaCTX-M-15 after sequencing. Both isolates were obtained at the time of patient admission into the ICU and showed identical pulsed-field gel electrophoresis patterns (10). Both patients were previously hospitalized before ICU admission, suggesting that this strain could be endemic in the hospital, where it could be horizontally transferred. All the other E. coli isolates yielded different pulsotypes (data not shown) compared with these. PCR assays for the detection of class 1 integrons and ISCR1 elements were performed according to the method of Di Conza et al. (3). Both isolates carried a class 1 integron containing the dfr17 and aadA5 gene cassettes, while ISCR1 elements were not detected. So far we have not been able to transfer these resistance genes, either by transformation or by conjugation. This is the first report of aac(6′)-Ib-cr in Uruguay. In accordance with a previous report (6), blaCTX-M-15 and aac(6′)-Ib-cr do not seem to be associated with class 1 integrons. Demonstration of a link to IS26 as previously reported (1) is pending.

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.