Isolation of five Enterobacteriaceae species harbouring blaNDM-1 and mcr-1 plasmids from a single paediatric patient.

In Argentina, NDM metallo-ß-lactamase was first reported in 2013. By now, it has disseminated throughout the country in diverse Gram negative bacteria. Here, we report the case of a paediatric patient that underwent a 1-year hospitalisation due to erythrodermic psoriasis in 2014 and received multiple antimicrobial treatments. During his stay, five isolates were obtained from rectal swabs (rs) or blood culture (bc) suspicious of carbapenemase production: a K. quasipneumoniae subsp. quasipneumoniae (rs), Citrobacter freundii (rs), Escherichia coli (bc), Enterobacter cloacae (rs), and a Serratia marcescens (bc). The isolates were studied with broth microdilution, biparental conjugation and plasmid and whole genome sequencing (Illumina). All isolates harboured an 138,998-bp type 1 IncC plasmid that carried blaNDM-1, bleMBL, blaCMY-6, rmtC, aac(6')-Ib, and sul1 resistance genes. Additionally, the blaNDM-plasmids contained ISKpn8 an insertion sequence previously described as associated only to blaKPC. One isolate, a colistin-resistant E. coli, also carried a mcr-1-containing an IncI2 plasmid, which did not harbour additional resistance. The whole genome of K. quasipneumoniae subsp. quasipneumoniae isolate was fully sequenced. This isolate harboured, additionally to blaNDM, three plasmid-mediated quinolone resistance genes: qnrB4, qnrB52 and aac(6')-Ib-cr1. The E. cloacae isolate also harboured qnrA1. These findings alert to the underestimated horizontal dissemination of multidrug-resistant plasmids limiting treatment options with last resort antimicrobials.

Correction: Isolation of five Enterobacteriaceae species harbouring blaNDM-1 and mcr-1 plasmids from a single paediatric patient.

[This corrects the article DOI: 10.1371/journal.pone.0221960.].

Novel class 1 Integrons and sequence types in VIM-2 and VIM-11-producing clinical strains of Enterobacter cloacae.

All VIM-producing Enterobacteriaceae (six Enterobacter cloacae) submitted to the Argentinian Reference Laboratory in Antimicrobial Resistance in the period 2008-13 were characterized. The isolates were referred from 6 nosocomial institutions located in 5 different cities across the country. All isolates showed carbapenem disk diffusion inhibition zones ≤22mm and synergism between a carbapenem disk and EDTA/SMA. The six isolates were PCR positive for blaVIM. Imipenem MICs were ≤1 to 8µg/ml. Typing by PFGE and MLST distinguished six pulsotypes and sequence types with blaVIM located on novel class 1 integron arrays: ECL-1: ST182, In883; ECL-2, ST90, In885; ECL-3, ST88, In346 with blaVIM-11; ECL-4, ST184, In900; ECL-5, ST749-new, In900; ECL-6, ST91 and uncharacterized In. Only ECL-2 was able to transfer blaVIM-2 to E. coli J53 by biparental conjugation. blaVIM was located in plasmids of 53-82Kb and in the chromosome (ECL-1 and ECL-5). The diversity of clones, class 1 integrons, plasmids and location of blaVIM, reveals the plasticity of the genetic elements described and highlights the importance of surveillance programs as tools to identify the transmission of these highly resistant metallo-ß-lactamase-producing Enterobacteriaceae.

Performance of a PCR assay for the rapid identification of the Klebsiella pneumoniae ST258 epidemic clone in Latin American clinical isolates.

The worldwide dissemination of Klebsiella pneumoniae carbapenemase (KPC)-producing Klebsiella pneumoniae ST258 demands a rapid PCR-based typing method to detect unique genes of the ST258 clone. This study evaluates a PCR developed by Adler et al. (2014) for the detection of ST258 in K. pneumoniae clinical isolates centered on the identification of the pilv-I and prp genes. We tested 143 clinical isolates from Argentina (n=109), Chile (n=1), Colombia (n=1), Costa Rica (n=2), Ecuador (n=5), El Salvador (n=2), Nicaragua (n=5), Panamá (n=2), Paraguay (n=2), Perú (n=3) and Trinidad and Tobago (n=11) recovered from 2006 to 2015. blaKPC, pilv-l and prp genes were detected by PCR and sequenced by standard procedures. ST258 and non-ST258 were defined by PFGE and/or MLST. Isolates were grouped according to PFGE patterns: 58 were compatible with ST258 (group 1) and 85 with non-ST258 (group 2). MLST study was done on an arbitrary selection of isolates. The pilv-l gene was present only in ST258 isolates, regardless of the presence of the blaKPC gene. Results for the prp gene were variable. Its presence did not define ST258. The pilv-I PCR had a sensitivity and specificity of 100%, respectively, for the detection of ST258 in the isolates under investigation. Given our findings, the pilv-I PCR could replace more time and resource consuming methods, allowing for more rapid detection of the circulating high risk K. pneumoniae clone ST258 in Latin American (LA) countries.

In vivo horizontal dissemination of the blaKPC-2 gene carried on diverse genetic platforms among clinical isolates of Enterobacteriaceae.

This study investigated the molecular characteristics of six blaKPC-positive Enterobacteriaceae recovered from three patients in Argentina. Antimicrobial susceptibility testing was performed following Clinical and Laboratory Standards Institute (CLSI) 2014 recommendations. Molecular characterisation of the isolates was performed by biparental conjugation, PCR, sequencing, S1 nuclease restriction, and Southern blot hybridisation with a blaKPC probe using standard protocols and conditions. The isolates studied were as follows. Case 1: Escherichia coli (ECO-P1) and Klebsiella pneumoniae (KPN-P1) isolated from a rectal swab harboured blaKPC-2 in transposon Tn4401a on non-typeable and non-conjugative plasmids. Case 2: Enterobacter cloacae (ECL-P2) and K. pneumoniae (KPN-P2) were isolated from two blood cultures. blaKPC-2 was found in a novel genetic variant of ISKpn8-blaKPC-2-ISKpn6-like on conjugative plasmids of IncL/M type. Case 3, Citrobacter freundii (CFR-P3) and Klebsiella oxytoca (KOX-P3) were isolated from skin and skin-structure infection. The blaKPC gene was detected on ISKpn8-ΔblaTEM-blaKPC-2-ISKpn6-like located on an IncA/C conjugative plasmid. CFR-P3 and KOX-P3 harboured blaPER-2 in addition to the blaKPC gene. In conclusion, we document the horizontal dissemination of blaKPC-2 from diverse Enterobacteriaceae clinical isolates with different genetic backgrounds. This is the first report of E. coli harbouring blaKPC associated with Tn4401a in Argentina.

Intrapatient emergence of OXA-247: a novel carbapenemase found in a patient previously infected with OXA-163-producing Klebsiella pneumoniae.

Two genetically related Klebsiella pneumoniae strains carrying OXA-type carbapenemases were isolated from a single patient 1 month apart. Kpn163 harboured OXA-163 and Kpn247 a new variant named OXA-247 that showed susceptibility to carbapenems and expanded-spectrum cephalosporins similar to OXA-48. Our epidemiological, biochemical and molecular results suggest the intrapatient emergence of blaOXA -247 from blaOXA -163.