Daniela Cejas

First isolate of KPC-2-producing Klebsiella pneumonaie sequence type 23 from the Americas.

ABSTRACT KPC-2-producing Klebsiella pneumoniae isolates mainly correspond to clonal complex 258 (CC258); however, we describe KPC-2-producing K. pneumoniae isolates belonging to invasive sequence type 23 (ST23). KPC-2 has scarcely been reported to occur in ST23, and this report describes the first isolation of this pathogen in the Americas. Acquisition of resistant markers in virulent clones could mark an evolutionary step toward the establishment of these clones as major nosocomial pathogens.

First National Survey of Antibiotic Susceptibility of the Bacteroides fragilis Group: Emerging Resistance to Carbapenems in Argentina

ABSTRACT The antibiotic susceptibility rates of 363 clinical Bacteroides fragilis group isolates collected from 17 centers in Argentina during the period from 2006 to 2009 were as follows: piperacillin-tazobactam, 99%; ampicillin-sulbactam, 92%; cefoxitin, 72%; tigecycline, 100%; moxifloxacin, 91%; and clindamycin, 52%. No metronidazole resistance was detected in these isolates during this time period. Resistance to imipenem, doripenem, and ertapenem was observed in 1.1%, 1.6%, and 2.3% of B. fragilis group strains, respectively. B. fragilis species showed a resistance profile of 1.5% to imipenem, 1.9% to doripenem, and 2.4% to ertapenem. This is the first report of carbapenem resistance in Argentina. The cfiA gene was present in 8 out of 23 isolates, all of them belonging to the B. fragilis species and displaying reduced susceptibility or resistance to carbapenems (MICs ≥ 4 μg/ml). Three out of eight cfiA-positive isolates were fully resistant to carbapenems, while 5 out of 8 isolates showed low-level resistance (MICs, 4 to 8 μg/ml). The inhibition by EDTA was a good predictor of the presence of metallo-β-lactamases in the fully resistant B. fragilis strains, but discrepant results were observed for low-level resistant isolates. B. fragilis was more susceptible to antimicrobial agents than other Bacteroides species. Bacteroides vulgatus species was the most resistant to ampicillin-sulbactam and piperacillin-tazobactam, and B. thetaiotaomicron/ovatus strains showed the highest level of resistance to carbapenems, with an unknown resistance mechanism. B. vulgatus and the uncommon non-Bacteroides fragilis species were the most resistant to moxifloxacin, showing an overall resistance rate of 15.1%.

Identification of the first blaCMY-2 gene in Salmonella enterica serovar Typhimurium isolates obtained from cases of paediatric diarrhoea illness detected in South America

The objectives of this study were to investigate clinical isolates of Salmonella enterica serovar Typhimurium resistant to β-lactam antibiotics, to characterise their mechanisms of antibiotic resistance and to evaluate the possible biological cost of expressing resistance genes. Two oxyimino-cephalosporin-resistant Salmonella isolates obtained from children with diarrhoea were characterised. The occurrence of plasmid-encoded blaCMY-2 genes was confirmed by molecular methods and conjugation assays; transcription levels were determined by quantitative real-time PCR (qRT-PCR). The genomic context of the β-lactamases, replicon type and addiction systems were analysed by PCR. Genomic relatedness of both isolates was studied by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) assays. Growth curves, motility and invasiveness assays in Caco-2 cells were performed to analyse the bacterial fitness of both isolates. Both isolates carried a blaCMY-2-like allele in an IncI plasmid and belonged to the same MLST sequence type (ST19); nevertheless, they showed extensive differences in their PFGE profiles and virulotypes. Isolate STM709 appeared to lack the Salmonella virulence plasmid and displayed less motility and invasiveness in cultured cells than isolate STM910. qRT-PCR showed that isolate STM709 had higher blaCMY-2 mRNA levels compared with STM910. Altogether, the results suggest that a plasmid carrying blaCMY-2 could be disseminating among different clones of S. Typhimurium. Different levels of blaCMY-2 mRNA could have an effect on the fitness of this micro-organism, resulting in lower invasiveness and motility.

OXA-258 from Achromobacter ruhlandii: a Species Specific Marker

ABSTRACT A new bla OXA-258 gene is described as a species-specific taxonomic marker for Achromobacter ruhlandii isolates (all recovered from cystic fibrosis patients). Even though OXA-258 differs from OXA-114 variants, isolates could be misidentified as A. xiloxosidans by the amplification of an inner fragment from the OXA-coding gene. A robust identification of A. ruhlandii can be achieved by sequencing this single OXA gene, as well as by a more laborious recently proposed multilocus sequence-typing (MLST) scheme.

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

Plasmid-mediated colistin resistance in Escherichia coli recovered from healthy poultry

Fil: Dominguez, Johana Elizabeth. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Houssay; Argentina. Instituto Nacional de Tecnologia Agropecuaria. Centro de Investigacion en Ciencias Veterinarias y Agronomicas. Instituto de Patobiologia; Argentina

First detection of CMY-2 plasmid mediated β-lactamase in Salmonella Heidelberg in South America

Salmonella enterica serovar Heidelberg ranks among the most prevalent causes of human salmonellosis in the United States and Canada, although it has been infrequently reported in South American and European countries. Most Salmonella infections are self-limiting; however, some invasive infections require antimicrobial therapy. In this work we characterized an oxyimino-cephalosporin resistant S. Heidelberg isolate recovered from an inpatient in a Buenos Aires hospital. CMY-2 was responsible for the β-lactam resistance profile. S. Heidelberg contained a 97kb plasmid belonging to the Inc N group harboring blaCMY-2. ISEcp1 was located upstream blaCMY-2 driving its expression and mobilization. The isolate belonged to sequence type 15 and virotyping revealed the presence of sopE gene. In this study we identified the first CMY-2 producing isolate of S. Heidelberg in Argentina and even in South America.

Spread of Clonally Related Escherichia coli Strains Harboring an IncA/C1 Plasmid Encoding IMP-8 and Its Recruitment into an Unrelated MCR-1-Containing Isolate.

ABSTRACT Ten IMP-8-producing Escherichia coli isolates were recovered from surveillance cultures of a neonatal intensive care unit; eight of the isolates were clonally related. A 168.2-kb blaIMP-8 plasmid was fully sequenced, and it corresponded to the recently described IncA/C1-ST13 plasmid. This plasmid was detected in all isolates, even in those that were not clonally related. One unrelated isolate was also resistant to colistin and positive for mcr-1. This marker was located in a 62.7-kb IncI2 plasmid, which was also fully sequenced.

Fast and easy detection of CMY-2 in Escherichia coli by direct MALDI-TOF mass spectrometry

Fast typing methods for third generation cephalosporin resistance mechanisms are needed to guide appropriate treatment and prevent potential dissemination events. In this study we used a novel short and fast methodology for the identification of CMY-2 in 50 well characterized clinical isolates of E. coli by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry- MALDI-TOF MS. Samples were prepared using the double layer sinapinic acid technique for detection of intact proteins Comparison among mass spectral profile of different strains between m/z 35,000-45,000 Da showed that two groups of isolates could be differentiated after peak analysis. A single distinctive peak with different intensities, at approximately m/z 39,800 Da was found in all CMY-2 producing strains (transconjugant, transformant and wild type) and consistently absent in the control groups (ESBL producers and susceptible strains). Statistical results showed 100% values for sensitivity and specificity, indicating a perfect test and a high discriminative power. In this study, we demonstrated that MALDI-TOF MS has the potential to detect directly the most clinically relevant acquired AmpC β-lactamase, the CMY-2-enzyme, in E. coli with a less time-consuming process as compared to conventional methods. Our results may constitute the basis for further research to detect other β-lactamases, or even other resistance markers.

Simultaneous Carriage of mcr-1 and Other Antimicrobial Resistance Determinants in Escherichia coli From Poultry

The use of antimicrobial growth promoters (AGPs) in sub-therapeutic doses for long periods promotes the selection of resistant microorganisms and the subsequent risk of spreading this resistance to the human population and the environment. Global concern about antimicrobial resistance development and transference of resistance genes from animal to human has been rising. The goal of our research was to evaluate the susceptibility pattern to different classes of antimicrobials of colistin-resistant Escherichia coli from poultry production systems that use AGPs, and characterize the resistance determinants associated to transferable platforms. E. coli strains (n = 41) were obtained from fecal samples collected from typical Argentine commercial broiler farms and susceptibility for 23 antimicrobials, relevant for human or veterinary medicine, was determined. Isolates were tested by PCR for the presence of mcr-1, extended spectrum β-lactamase encoding genes and plasmid-mediated quinolone resistance (PMQR) coding genes. Conjugation and susceptibility patterns of the transconjugant studies were performed. ERIC-PCR and REP-PCR analysis showed a high diversity of the isolates. Resistance to several antimicrobials was determined and all colistin-resistant isolates harbored the mcr-1 gene. CTX-M-2 cefotaximase was the main mechanism responsible for third generation cephalosporins resistance, and PMQR determinants were also identified. In addition, co-transference of the qnrB determinant on the mcr-1-positive transconjugants was corroborated, which suggests that these resistance genes are likely to be located in the same plasmid. In this work a wide range of antimicrobial resistance mechanisms were identified in E. coli strains isolated from the environment of healthy chickens highlighting the risk of antimicrobial abuse/misuse in animals under intensive production systems and its consequences for public health.