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.].

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.

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.

Clonal dissemination of Klebsiella pneumoniae ST258 harbouring KPC-2 in Argentina.

The present work describes the abrupt emergence of Klebsiella pneumoniae carbapenemase (KPC) and characterizes the first 79 KPC-producing enterobacteria from Argentina (isolated from 2006 to 2010). The emergence of bla(KPC-2) was characterized by two patterns of dispersion: the first was the sporadic occurrence in diverse enterobacteria from distant geographical regions, harbouring plasmids of different incompatibility groups and bla(KPC-2) in an unusual genetic environment flanked by ISKpn8-Δbla(TEM-1) and ISKpn6-like. bla(KPC-2) was associated with IncL/M transferable plasmids; the second was the abrupt clonal spread of K. pneumoniae ST258 harbouring bla(KPC-2) in Tn4401a.

A simple test for the detection of KPC and metallo-ß-lactamase carbapenemase-producing Pseudomonas aeruginosa isolates with the use of meropenem disks supplemented with aminophenylboronic acid, dipicolinic acid and cloxacillin.

We evaluated the ability of the combination disk test (CDT) and the Modified Hodge Test (MHT) to discriminate between various carbapenemase-producing Pseudomonas aeruginosa isolates (KPC, n = 36; metallo-ß-lactamase (MBL), n = 38) and carbapenemase non-producers (n = 75). For the CDT, the optimal inhibitor concentrations and cut-off values were: 600 µg of 3-aminophenylboronic acid (APB) per disk (an increment of ≥4 mm), 1000 µg of dipicolinic acid (DPA) per disk (an increment of ≥5 mm) and 3000 µg of cloxacillin per disk (an increment of ≥3 mm). APB had excellent sensitivity (97%) and specificity (97%) for the detection of KPC enzymes. DPA detected MBL enzymes with a sensitivity and specificity of 97% and 81%, respectively. The MHT resulted in a low sensitivity (78%) and specificity (57%). The CDT could be very useful in daily practice to provide fast and reliable detection of KPC and MBL carbapenemases among P. aeruginosa isolates.

Fluoroquinolone-resistant Streptococcus agalactiae isolates from Argentina.

Fluoroquinolone resistance is a growing problem that has only recently emerged in S. agalactiae. Between 2005-2007, WHONET--Argentina network evaluated levofloxacin susceptibility in 1128 clinical S. agalactiae isolates, 10 (0.9%) of which proved to be resistant. Nine of them had come from 5 hospitals (in Buenos Aires City and 4 Argentinean provinces) and recovered from urine (n=7) and vaginal screening cultures (n=2). Three strains were also resistant to macrolides, lincosamides and B streptogramins due to the ermA gene. All nine fluoroquinolone-resistant isolates bore the same two mutations, Ser79Phe in ParC and Ser81Leu in GyrA proteins. Genetic relationships were analyzed by Apal-PFGE and two clones were determined, A (n=6) and B (n=3). To our knowledge, these are the first fluoroquinolone-resistant S. agalactiae isolates detected in Latin America.

Prevalence of mef and ermB genes in invasive pediatric erythromycin-resistant Streptococcus pneumoniae isolates from Argentina.

During the period 1993-2001, a total of 1,499 pneumococci isolates were recovered through the Argentinean surveillance of Streptococcus pneumoniae causing invasive disease in children under 6 years of age, 3.5% of which were erythromycin resistant. Among the 50 erythromycin-resistant strains available, 58% (n=29) harbored mefA/E genes (15 mefA, 30%; and 14 mefE, 28%), 34% (n=17) ermB, and 6% (n=3) both mefA/E plus ermB genes, while one isolate was negative for all the acquired genes studied. The England14-9 (42%), Poland6B-20 (20%) and Spain9V-3 (16%) clones were responsible for the emergence of pneumococcal macrolide resistance in pediatric population from Argentina.

Prevalence of mef and ermB genes in invasive pediatric erythromycin-resistant Streptococcus pneumoniae isolates from Argentina / Prevalencia de los genes mef y ermB en aislamientos invasivos de Streptococcus pneumoniae resistentes a eritromicina recuperados de pacientes pediátricos en Argentina

During the period 1993-2001, a total of 1,499 pneumococci isolates were recovered through the Argentinean surveillance of Streptococcus pneumoniae causing invasive disease in children under 6 years of age, 3.5% of which were erythromycin resistant. Among the 50 erythromycin-resistant strains available, 58% (n=29) harbored mefA/E genes (15 mefA, 30%; and 14 mefE, 28%), 34% (n=17) ermB, and 6% (n=3) both mefA/E plus ermB genes, while one isolate was negative for all the acquired genes studied. The England14-9 (42%), Poland6B-20 (20%) and Spain9v-3 (16%) clones were responsible for the emergence of pneumococcal macrolide resistance in pediatric population from Argentina.

En el marco del programa de vigilancia regional SIREVA, se analizaron 1499 aislamientos de Streptococcus pneumoniae causantes de enfermedad invasiva en menores de 6 años, recuperados entre 1993 y 2001. Se detectó un 3,5% de resistencia a eritromicina. De los 50 aislamientos resistentes a eritromicina que pudieron ser estudiados, el 58% (n=29) tenían los genes mefA/E (15 mefA, 30% y 14 mefE, 28%), el 34% (n=17) el gen ermB y el 6% (n=3) la combinación de genes mefA/E y ermB. Sólo un aislamiento fue negativo para todos los genes analizados. Los clones internacionales England14-9, Poland6B-20 y Spain9v-3 representaron el 78% del total de aislamientos resistentes (42, 20 y 16%, respectivamente) y se consideraron los responsables de la emergencia de la resistencia a macrólidos entre los neumococos que afectan a la población pediátrica de Argentina.

First report of VanA Enterococcus gallinarum dissemination within an intensive care unit in Argentina.

Enterococcusgallinarum is intrinsically resistant to low levels of vancomycin and has been described as a colonizing microorganism causing bacteraemia and infection among immunosupresed patients. Between August 2000 and February 2001, 15 highly glycopeptide-resistant E. gallinarum isolates, one from blood and the remaining from rectal swabs, were recovered in a general hospital of Buenos Aires Province, Argentina. All isolates were characterized by biochemical assays, and displayed MICs of vancomycin in the range 16-128 mg/l and MICs of teicoplanin in the range 16-32 mg/l. In all cases, PCR analysis yield positive results for both vanC1 and vanA genes. E. gallinarum isolates were classified as two clonal types by SmaI-PFGE: clone A (n = 8) and clone B (n = 7) and both harboured a transferable vanA element.

Improvement of a latex agglutination test for the evaluation of oxacillin resistance in coagulase-negative staphylococci.

The "Slidex MRSA Detection" test (Denka Seiken, Japan) is a latex agglutination assay able to detect PBP2a. We evaluated its ability to differentiate mecA-positive from mecA-negative coagulase-negative staphylococci. We included 100 coagulase-negative staphylococci clinical isolates belonging to 9 species, 54 mecA positive and 46 mecA negative, as characterized by PCR. The specificity achieved using the manufacturer's instructions was 100%, but the sensitivity was only 57%. To increase sensitivity, we introduced modifications into the standard protocol. Using either large inocula or oxacillin induction before test performance, we achieved 100% sensitivity.

[Methicillin resistance detection in Staphylococcus aureus: comparison between conventional methods and MRSA-Screen latex agglutination technique]. / Detección de meticilino-resistencia en Staphylococcus aureus: comparación de métodos convencionales y aglutinación con MRSA-Screen latex.

Methicillin-resistant Staphylococcus aureus (MRSA) is a significant pathogen that has emerged over the last four decades, causing both nosocomial and community-acquired infections. Rapid and accurate detection of methicillin resistance in S. aureus is important for the use of appropriate antimicrobial therapy and for the control of nosocomial spread of MRSA strains. We evaluated the efficiency of conventional methods for detection of methicillin resistance such as the disk diffusion, agar dilution, oxacillin agar screen test, and the latex agglutination test MRSA-Screen latex, in 100 isolates of S. aureus, 79 mecA positive and 21 mecA negative. The MRSA-Screen latex (Denka Seiken, Niigata, Japón), is a latex agglutination method that detects the presence of PLP-2a, product of mecA gene in S. aureus. The PCR of the mecA gene was used as the "gold standard" for the evaluation of the different methods tested. The percentages of sensitivity and specificity were as follows: disk difusión 97 and 100%, agar dilution 97 and 95%, oxacillin agar screen test 100 and 100%, and MRSA-Screen latex, 100 and 100 %. All methods presented high sensitivity and specificity, but MRSA-Screen latex had the advantage of giving a reliable result, equivalent to PCR, in only 15 minutes.

Detección de meticilino-resistencia en Staphylococcus aureus: Comparación de métodos convencionales y aglutinación con mrsa-Screen latex / Methicillin resistance detection in Staphylococcus aureus: Comparison between conventional methods and Mrsa-Screen latex agglutination technique

Staphylococcus aureus meticilino-resistente (MRSA) es un patógeno que ha emergido en las últimas cuatro décadas causando tanto infecciones nosocomiales como de la comunidad. La rápida y precisa detección de MRSA es relevante para guiar una apropiada terapia antibiótica y evitar la diseminación nosocomial de MRSA.En este trabajo se evaluó la eficiencia de métodos convencionales para la detección de meticilino-resistencia como difusión por discos, CIM en medio sólido, screening de oxacilina, y el nuevo test de aglutinación MRSA-Screen latex sobre 100 aislamientos de S. aureus, 79 mecA positivos y 21 mecA negativos. El test de aglutinación MRSA-Screen latex (Denka Seiken, Niigata, Japón) detecta la presencia de la PLP-2a, producto del gen mecA en cepas de S. aureus. La detección del gen mecA por PCR se utilizó como gold standard para comparar los resultados de los diferentes métodos. La sensibilidad y especificidad fueron 97 y 100 % para el método de difusión, 97 y 95 % para la CIM en medio sólido, 100 y 100 % para el screening de oxacilina y 100 y 100 % para MRSA-Screen latex. Todos los métodos presentaron alta sensibilidad y especificidad, pero el “MRSA-Screen latex” mostró la ventaja de poder brindar un resultado confiable, equivalente a la PCR, en sólo 15 minutos.

Methicillin-resistant Staphylococcus aureus (MRSA) is a significant pathogen that has emerged over the last four decades, causing both nosocomial and community-acquired infections. Rapid and accurate detection of methicillin resistance in S. aureus is important for the use of appropriate antimicrobial therapy and for the control of nosocomial spread of MRSA strains. We evaluated the efficiency of conventional methods for detection of methicillin resistance such as the disk diffusion, agar dilution, oxacillin agar screen test, and the latex agglutination test MRSA-Screen latex, in 100 isolates of S. aureus, 79 mecA positive and 21 mecA negative. The MRSA-Screen latex (Denka Seiken, Niigata, Japón), is a latex agglutination method that detects the presence of PLP-2a, product of mecA gene in S. aureus. The PCR of the mecA gene was used as the “gold standard” for the evaluation of the different methods tested. The percentages of sensitivity and specificity were as follows: disk difusión 97 and 100 %, agar dilution 97 and 95 %, oxacillin agar screen test 100 and 100 %, and MRSA-Screen latex, 100 and 100 %. All methods presented high sensitivity and specificity, but MRSA-Screen latex had the advantage of giving a reliable result, equivalent to PCR, in only 15 minutes.

Detección de meticilino-resistencia en Staphylococcus aureus: comparación de métodos convencionales y aglutinación con MRSA-Screen latex. / [Methicillin resistance detection in Staphylococcus aureus: comparison between conventional methods and MRSA-Screen latex agglutination technique]

Methicillin-resistant Staphylococcus aureus (MRSA) is a significant pathogen that has emerged over the last four decades, causing both nosocomial and community-acquired infections. Rapid and accurate detection of methicillin resistance in S. aureus is important for the use of appropriate antimicrobial therapy and for the control of nosocomial spread of MRSA strains. We evaluated the efficiency of conventional methods for detection of methicillin resistance such as the disk diffusion, agar dilution, oxacillin agar screen test, and the latex agglutination test MRSA-Screen latex, in 100 isolates of S. aureus, 79 mecA positive and 21 mecA negative. The MRSA-Screen latex (Denka Seiken, Niigata, Japón), is a latex agglutination method that detects the presence of PLP-2a, product of mecA gene in S. aureus. The PCR of the mecA gene was used as the [quot ]gold standard[quot ] for the evaluation of the different methods tested. The percentages of sensitivity and specificity were as follows: disk difusión 97 and 100

, agar dilution 97 and 95

, oxacillin agar screen test 100 and 100

, and MRSA-Screen latex, 100 and 100

. All methods presented high sensitivity and specificity, but MRSA-Screen latex had the advantage of giving a reliable result, equivalent to PCR, in only 15 minutes.