Marcela Nastro

First nosocomial outbreak of VIM-16-producing Serratia marcescens in Argentina.

Seven metallo-β-lactamase-positive isolates of Serratia marcescens were recovered from three patients hospitalized in a neonatal ward in an Argentinean hospital during the period July-September 2011. All the isolates were multidrug-resistant, they belonged to a single clone, and carried a blaVIM-16 -containing class I integron structure. This represents the first nosocomial outbreak of metallo-β-lactamase in Enterobacteriaceae in Argentina.

In vitro activity of minocycline alone or in combination in multidrug-resistant Acinetobacter baumannii isolates.

Minocycline (MIN) usually shows good activity against Acinetobacter baumannii strains. The reintroduction to the market of intravenous MIN provides an additional agent to the limited options for the treatment of A. baumannii infections. The activity of MIN as a single agent and in combination with rifampicin (RIF), colistin (COL) or imipenem (IMI) was evaluated by means of killing curves and 24  h-time-kill curves in five A. baumannii isolates which were selected on the basis of different antimicrobial resistance profiles. MIN showed bacteriostatic activity in three isolates (2 ×  or 16 ×  MIC) and bactericidal activity in the other isolates (64 ×  MIC). In isolates harbouring the tetB gene, the associations studied were always indifferent. However, in isolates not harbouring tetB, the use of MIN in combination showed a rapid synergistic effect (at 4  h) in four out of nine combinations (two with RIF and one each with IMI and COL). At 24  h, this effect was observed in six out of nine combinations (two in each association). MIN in combination with RIF, IMI and COL showed bactericidal synergy in most of the isolates which did not harbour the tetB gene, but the combinations were not synergistic in tetB-positive isolates.

Activity of the colistin–rifampicin combination against colistin-resistant, carbapenemase-producing Gram-negative bacteria

Abstract Colistin (COL) is one of the few antimicrobials that retain activity against carbapenemase-producing Gram-negative bacteria (GNB). However, the emergence of COL resistance has renewed the use of combination therapy. The aim of this study was to determine the activity of COL plus rifampicin (RIF) against clinical isolates of COL-resistant, carbapenemase-producing GNB. We employed 36 COL-resistant carbapenemase-producing isolates (27 Klebsiella pneumoniae, 5 Serratia marcescens, and 4 Acinetobacter baumannii) belonging to 36 patients. E-test/agar dilution of all strains was performed with E-test strips of COL placed on agar plates with and without RIF. In 11 patients, the synergy was confirmed by time-kill studies. Synergy was detected in 34 isolates, whereas indifference was detected in two S. marcescens. The E-test/agar dilution method showed comparable results to the time-kill studies. Seven patients infected with these isolates (two meningitis, four sepsis, and one urinary tract infection) were treated with the combination successfully.

Hyperendemic clone of KPC producing Klebsiella pneumoniae ST 258 in Buenos Aires hospitals

Since KPC-type carbapenemases were first reported in Klebsiella pneumoniae in 2001 in the USA (Yigit et al., 2001), they have become a frequent resistant marker encountered also in other Enterobacteriaceae and Pseudomonads from the Americas, Europe, Asia and Middle East (Villegas et al., 2007; Nordmann et al., 2009; Walsh, 2010). Their wide spread across multiple continents and species has been associated with a mobile genetic element, Tn4401 (Naas et al., 2008). More recently, the molecular epidemiology of KPCproducing K. pneumoniae isolates has revealed the successful dissemination of a single sequence type 258 clone (Kitchel et al., 2009). Although in Argentina KPC-2 producing K. pneumoniae were first detected in 2006 (Pasterán et al., 2008), a substantial increase was observed in 2010, in Buenos Aires (http://www.ine. gov.ar/publi_pdfs/Carbapenemasas.pdf). To characterize this occurrence, single, non-repetitive K. pneumoniae isolates obtained from 57 patients from May 2009 through April 2010 in six hospitals in Buenos Aires were included (Hosp 1:3, Hosp 2:5, Hosp 3:30, Hosp 4:3, Hosp 5: 2, Hosp 6:14). Antimicrobial susceptibility tests were conducted by disk diffusion according to CLSI recommendations (CLSI, 2010). Taking into account the local resistance prevalence in hospital-acquired K. pneumoniae infections, the selected antibiotic disks were placed on two primary 90 mm Mueller Hinton agar plates as follows: plate 1: ampicillin, cephalotin, gentamicin, amikacin, ciprofloxacin and trimethoprime/sulfamethoxazole; plate 2: amoxicillin/clavulanic acid, piperacillin/tazobactam, cefotaxime, ceftazidime, imipenem, and meropenem. As it has been previously reported, phenyl boronic acid is a good inhibitor of both AmpC and KPC b-lactamases (Yagi et al., 2005; Pasteran et al., 2009), we included a disk containing 300 lg phenyl boronic acid in the center of the second plate (distance between disks was 25 mm center to center). An enhancement in the inhibition zone of the carbapenemcontaining disks adjacent to the one containing the inhibitor was considered a positive screening for KPC. All the isolates displayed inhibition zones for imipenem and/or meropenem 621 mm and a positive synergy with phenyl boronic acid. Minimal inhibitory concentrations (MICs) were determined according to CLSI. The isolates showed a multidrug-resistant phenotype with varying levels of carbapenem resistance (Table 1). MIC50 and MIC90 values were as follows (lg/ml) ampicillin: >256 and >256, cephalotin: >256 and >256, ceftazidime: 256 and >256, cefotaxime: 128 and >256, imipenem: 2 and 16, meropenem: 2 and 32, colistin: 4 and 8, amikacin: >128 and >128 and gentamicin 2 and 4, respectively. Three isolates displayed MICs of colistin of 128 lg/ml. The presence of blaKPC was confirmed by PCR amplification using the following primers: KPC-F: 50ATGTCAC TGTATCGCCGTCT 30 and KPC-R: 50TTTT CAGAGCCTTACTGCCC 30 on heat-extracted DNA as template

Outbreak of blaOXA-72-producing Acinetobacter baumannii in South America.

Thirty-five Acinetobacter baumannii isolates were recovered from two medical centres in Guayaquil City, Ecuador, from November 2012 to October 2013. Isolates were identified using MALDI-TOF and confirmed by rpoB. PCR methods were employed for epidemiological analysis.Thirty-three A. baumannii isolates were resistant to all β-lactams. The blaOXA-24/40-like gene was detected in 30 isolates. DNA sequencing identified the blaOXA-24/40-like amplicon as blaOXA-72. The 30 isolates harbouring blaOXA-72 strains showed the same PCR pattern. We report the first outbreak of blaOXA-72-producing A. baumannii in South America. This is the first study carried out in the Republic of Ecuador.

Impact of heteroresistance to colistin in meningitis caused by Acinetobacter baumannii

We read with interest the paper by Li and colleagues concerning colistin hetero-resistance in Acinetobacter baumannii clinical isolates. The incidence of colistin-heteroresistance is still not well known due to its difficult detection. Moreover, its clinical impact has only been discussed in our previous report and in a communication of David. In a recent review of patients with meningitis due to A. baumannii treated with colistin, heteroresistant isolates have not been described. The aim of the present work is to report our experience regarding the clinical impact of colistin-heteroresistance in post neurosurgical meningitis caused by A. baumannii. Seven isolates were recovered from two cases (patients 1 and 2) of A. baumannii colistin heteroresistant in post neurosurgical meningitis were detected in the intensive care unit of Hospital de Clinicas Jose de San Martin of Buenos Aires city during the period 2004e2009. The selection intra-treatment with colistin of A. baumannii colistinresistant was analyzed. Strains were identified by phenotypic tests and genomospecies was determined by ARDRA (Amplified ribosomal DNA restriction analysis). Minimal inhibitory concentrations (MICs) to colistin (against the resistant subpopulations and the original strains) and the other antimicrobials (ampicillinesulbactam, ceftriaxone, ceftazidima, imipenem, meropenem, amikacin, gentamicin and levofloxacin) were performed by dilution method in cation-adjusted MuellerHinton agar and interpreted according to the Clinical and Laboratory Standards Institute. Pulsed-field electrophoresis (PFGE) was performed with ApaI as described previously. Population analysis profiles (PAPs) were determined according to a previous report. Timeekill studies were performed twice and thus, results were analyzed by using mean colony count values from the duplicate plates for each isolate, with the following concentrations: colistin sulfate: 4,8,12 and 80 MIC and rifampicin 4 mg/ ml. This study was performed with the colistin susceptible heteroresistant isolates from each patient (Ab86 and

Molecular epidemiology of carbapenem-resistant Acinetobacter baumannii in South America.

One hundred and twenty-six epidemiologically sequential, unrelated, carbapenem-resistant Acinetobacter baumannii isolates from nine hospitals in six countries of South America were collected between July 2013 and June 2014. Genes coding for Ambler class D and B carbapenemases were sought by PCR. All isolates were typed using the 3-locus sequence typing and blaOXA-51-like sequence-based typing techniques. The blaOXA-23 gene was recovered in all the participating hospitals and in all the isolates of seven of nine medical centres. The blaOXA-72 gene was only recovered in the two medical centres from Guayaquil city, Ecuador. Trilocus sequence typing revealed the presence of sequence groups SG2, SG4 and SG5. blaOXA-51-like sequence-based typing revealed the presence of blaOXA-132, blaOXA-65, blaOXA-69 and blaOXA-64. Our results showed that the population of carbapenem-resistant A. baumannii in South America was principally associated with ST79, ST25 and ST15 (92 %) and harboured the blaOXA-23 gene mainly. CC2 was not detected.

Rapid Blue-Carba test: reduction in the detection time of carbapenemases performed from a 4-hour bacterial lawn

The increase in carbapenem-resistant gram-negative bacteria is a matter of concern due to the limited therapeutic options available. In severe infections caused by these isolates, the rapid detection of the mechanisms of resistance is vital. We described a slightly modified version of the Blue-Carba test, rapid Blue-Carba test, which allows the detection of carbapenemases at 4 h of incubation from a haze of bacterial growth obtained from a positive blood culture. It was able to detect carbapenemase-producing isolates (Enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter baumannii) with a sensitivity and specificity of 98.1 and 100%, respectively. It is a rapid, easy-to-perform and an inexpensive technique that can be applied to routine laboratories, together with the simultaneous identification by mass spectrometry which would help to screen non-enzymatic carbapenem resistance; this method allows the detection of clinically relevant multidrug-resistant bacteria and the early implementation of accurate therapeutic interventions.

Emergencia de la resistencia a colistina en Klebsiella pneumoniae: caracterización microbiológica y epidemiológica de aislamientos productores y no productores de carbapenemasa de tipo KPC

Resumen Se estudiaron 64 aislamientos de K. pneumoniae resistentes a colistina recuperados de materiales clinicos de 57 pacientes atendidos entre los anos 2010 y 2012 en el Hospital de Clinicas Jose de San Martin, Universidad de Buenos Aires, con el objetivo de describir las caracteristicas microbiologicas y epidemiologicas y los factores relacionados con la emergencia de estos aislamientos. Se incluyeron en el estudio 54 aislamientos de K. pneumoniae sensibles a colistina contemporaneos a los aislamientos resistentes. La relacion de similitud genetica entre los aislamientos se investigo mediante la tecnica de PCR. El 50 % de los aislamientos resistentes presentaron KPC-2, el 45,3 % BLEE y el 4,7 % restante solo presento resistencia a las aminopenicilinas. Todos los aislamientos portadores de KPC (resistentes o sensibles a colistina), a excepcion de uno, fueron indistinguibles geneticamente, mientras que los portadores de BLEE se agruparon en 7 clones distintos, y se distinguieron de los clones recuperados en los aislamientos sensibles a colistina. El uso previo de colistina se asocio como el principal factor vinculado con la adquisicion de esta resistencia y con la internacion en UCI en los aislamientos sin KPC. A partir del ano 2010 la resistencia a colistina comenzo a emerger, alcanzo el 3 % en los aislados nosocomiales y se mantuvo estable en los anos subsiguientes, debido a la seleccion de las subpoblaciones resistentes en el clon epidemico en los aislamientos productores de KPC, y en los no productores de KPC por dispersion de clones resistentes a colistina.

Trends in the resistance profiles of Acinetobacter baumannii endemic clones in a university hospital of Argentina

A total of 925 Acinetobacter spp. isolates were collected from routine clinical samples of patients admitted to the university hospital of Buenos Aires city during the period 2004–2012. From this collection, 129 isolates identified as Acinetobacter baumannii were selected for molecular studies. Minimal inhibitory concentrations (MICs) of antimicrobials were determined by agar dilution method. Colistin (COL) heteroresistance was investigated by means of population analysis studies. PCR-based methods were used for epidemiological analysis and for the screening of carbapenemases and the blatetB gene. We have observed a steady rise in the MIC50 of imipenem (IMI)-resistant isolates and an increment in the presence of blaOXA-23-like gene (74–100%) as well. A rapid increasing rate of minocycline (MIN) resistance and a rise of the MIC50 of the resistant isolates have been detected since the year 2008. All isolates harboured the tet (B) gene. An increase in the value of the tigecycline (TIG) MIC was seen from the year 2007 onwards. This loss of activity was observed among different clones. A rise of COL heteroresistance from 46.4% in 2004 to 95% in 2012 was detected. During this period, COL consumption also increased (11.1-fold). However, COL resistance remained sporadic.