Genomic characterization of carbapenemase-producing Klebsiella pneumoniae ST307 revealed multiple introductions in Buenos Aires, Argentina.

OBJECTIVES: To describe at genomic level nine carbapenemase-producing Klebsiella pneumoniae ST307 (Kp-ST307) clinical isolates recovered in Buenos Aires during 2017 to 2021, investigating their resistome, virulome, and phylogeny. METHODS: Antimicrobial susceptibility was determined according to Clinical and Laboratory Standards Intitute (CLSI). Genomic DNA was sequenced by Illumina MiSeq and analysed using SPAdes, PROKKA, and Kleborate. Phylogeny of 355 randomly selected Kp-ST307 genomes and those from nine local isolates was inferred by a maximum-likelihood approach. The tree was visualized using Microreact. RESULTS: Besides resistance to ß-lactams and fluoroquinolones, six out of nine Kp-ST307 were also resistant to ceftazidime/avibactam (CZA). This difficult-to-treat resvistance phenotype was mediated by blaSHV-28 and GyrA-83I/ParC-80I mutations in addition to carbapenemase coding genes. Among CZA susceptible isolates, two of them harboured blaKPC-3 while the other harboured blaKPC-2+blaCTX-M-15. Regarding CZA-resistant isolates, three harboured blaKPC-3+blaNDM-1+blaCMY-6, two carried blaKPC-2+blaNDM-5+blaCTX-M-15, and blaNDM-5+blaCTX-M-15 were detected in the remaining isolate. Furthermore, five colistin-resistant isolates presented a nonsense mutation in mgrB. Global Kp-ST307 isolates were distributed in two deep-branching lineages while local isolates were set in the main clade of the phylogenetic tree. The five isolates from the same hospital, harbouring blaKPC-3 or blaKPC-3+blaNDM-1+blaCMY-6, clustered in a monophyletic subclade with Italian isolates. Also, an isolate harbouring blaKPC-2+blaNDM-5+blaCTX-M-15 recovered in another hospital was closed to this group. The remaining local Kp-ST307 were grouped in other subclades containing isolates of diverse geographical origin. CONCLUSION: The inferred resistome was consistent with the resistant phenotype. Phylogeny suggested multiple introduction events in our region and a single major introduction in one hospital followed by local spread.

Genetic and Biochemical Characterization of AXC-2 from Achromobacter ruhlandii.

Achromobacter spp. are intrinsically resistant to multiple antibiotics and can also acquire resistance to those commonly used for the treatment of respiratory infections, especially in patients with cystic fibrosis. The aim of this study was to perform the genetic and biochemical characterization of AXC-2 from A. ruhlandii and to analyze all available AXC variants. Steady-state kinetic parameters were determined on a purified AXC-2 enzyme. It exhibited higher catalytic efficiencies towards amino-penicillins and older cephalosporins, while carbapenems behaved as poor substrates. Phylogenetic analysis of all blaAXC variants available in the NCBI was conducted. AXC was encoded in almost all A. ruhlandii genomes, whereas it was only found in 30% of A. xylosoxidans. AXC-1 was prevalent among A. xylosoxidans. AXC variants were clustered in two main groups, correlating with the Achromobacter species. No association could be established between the presence of blaAXC variants and a specific lineage of A. xylosoxidans; however, a proportion of AXC-1-producing isolates corresponded to ST 182 and ST 447. In conclusion, this study provides valuable insights into the genetic context and kinetic properties of AXC-2, identified in A. ruhlandii. It also provides a thorough description of all AXC variants and their association with Achromobacter species and various lineages.

Genomic characterization of blaVIM-11-harbouring plasmids recovered from Pseudomonas aeruginosa.

OBJECTIVES: To the best of our knowledge, no genomic descriptions of blaVIM-11-harbouring plasmids are available in literature so far. The aim of this study was to describe the genomic features of three blaVIM-11-harbouring plasmids recovered from Pseudomonas aeruginosa isolated in Argentina in different periods. METHODS: blaVIM-11-harbouring plasmids from three clinical P. aeruginosa isolates were transferred by transformation into P. aeruginosa PAO-1. Then, genomic DNA of these transformants was extracted and sequenced using NovaSeq 6000 System-Illumina. De novo assemblies were generated using Unicycler program and reads were mapped against a reference genome of P. aeruginosa PAO-1. Plasmids sequences were predicted identifying the reads that did not map the reference sequence of PAO-1. These reads were recovered and assembled de novo. In silico predictions were carried out using bioinformatics tools. RESULTS: One Plasmid (pP6VIM-11) was distributed in 2 contigs, a second plasmid (pPOta2VIM-11) was found in a single contig, and the last one (pP936401VIM-11) was fragmented into 4 contigs. pP6VIM-11 and pPOta2VIM-11 belonged to the IncP-1ß group, displaying 64% of coverage and 83.9% of identity among them. pP936401VIM-1 plasmid corresponded to the IncN group. The bioinformatic analysis revealed that blaVIM-11 was located in a class 1 integron, flanked by insertion sequences, exhibiting potential for its dissemination. However, none of the plasmids were conjugative. CONCLUSION: This study corresponded to the first description and deposit of blaVIM-11-harbouring plasmids in P. aeruginosa, which expands the limited knowledge about their molecular epidemiology.

Molecular Mechanisms of Resistance to Ceftazidime/Avibactam in Clinical Isolates of Enterobacterales and Pseudomonas aeruginosa in Latin American Hospitals.

Ceftazidime-avibactam (CZA) is the combination of a third-generation cephalosporin and a new non-ß-lactam ß-lactamase inhibitor capable of inactivating class A, C, and some D ß-lactamases. From a collection of 2,727 clinical isolates of Enterobacterales (n = 2,235) and P. aeruginosa (n = 492) that were collected between 2016 and 2017 from five Latin American countries, we investigated the molecular resistance mechanisms to CZA of 127 (18/2,235 [0.8%] Enterobacterales and 109/492 [22.1%] P. aeruginosa). First, by qPCR for the presence of genes encoding KPC, NDM, VIM, IMP, OXA-48-like, and SPM-1 carbapenemases, and second, by whole-genome sequencing (WGS). From the CZA-resistant isolates, MBL-encoding genes were detected in all 18 Enterobacterales and 42/109 P. aeruginosa isolates, explaining their resistant phenotype. Resistant isolates that yielded a negative qPCR result for any of the MBL encoding genes were subjected to WGS. The WGS analysis of the 67 remaining P. aeruginosa isolates showed mutations in genes previously associated with reduced susceptibility to CZA, such as those involved in the MexAB-OprM efflux pump and AmpC (PDC) hyperproduction, PoxB (blaOXA-50-like), FtsI (PBP3), DacB (PBP4), and OprD. The results presented here offer a snapshot of the molecular epidemiological landscape for CZA resistance before the introduction of this antibiotic into the Latin American market. Therefore, these results serve as a valuable comparison tool to trace the evolution of the resistance to CZA in this carbapenemase-endemic geographical region. IMPORTANCE In this manuscript, we determine the molecular mechanisms of ceftazidime-avibactam resistance in Enterobacterales and P. aeruginosa isolates from five Latin American countries. Our results reveal a low rate of resistance to ceftazidime-avibactam among Enterobacterales; in contrast, resistance in P. aeruginosa has proven to be more complex, as it might involve multiple known and possibly unknown resistance mechanisms.

Emergence and clonal expansión of Klebsiella pneumoniae ST307, simultaneously producing KPC-3 and NDM-1

Abstract MDR Klebsiella pneumoniae ST307 is a high-risk clone, whose genetic features contribute to its adaptation to hospital environments and the human host. This study describesthe emergence and clonal dissemination of K. pneumoniae ST307, recovered during November2018 to February 2019 in a hospital in Buenos Aires city, which concurrently harbored KPC-3and NDM-1. These isolates were resistant to all -lactams and to the ceftazidime/avibactamcombination. Molecular studies showed that blaKPC-3was located in Tn4401a platform, whileblaNDM-1was surrounded upstream by ISKpn14 followed by a partial sequence of ISAba125 anddownstream by bleMBL-trpF, located in a 145.5 kb conjugative plasmid belonging to the Inc A/Cgroup. The dissemination of K. pneumoniae ST307 isolates co-producing KPC-3 and NDM-1 couldlead to a worrisome scenario due to the remarkable features of this clone and its resistanceprofile.

Resumen Klebsiella pneumoniae ST307 es un clon de alto riesgo, cuyas características genéticas contribuyen a su adaptación al entorno hospitalario y al huésped humano. Este estudio describe la emergencia y diseminación clonal de aislamientos de K. pneumoniae ST307 productores de KPC-3 y NDM-1, recuperados en un hospital de Buenos Aires. Estos aislamientos fueron resistentes a todos los p-lactámicos y a la combinación ceftacidima/avibactam. Los estudios moleculares evidenciaron que el contexto genético de blaKPC-3 se correspondió con el Tn4401a, mientras que blaNDM-1 estuvo flanqueado corriente arriba por ISKpn14 y una secuencia parcial de ISAba125 y corriente abajo por bleMBL - trpF, localizado a su vez en un plásmido conjugativo de 145.5 kb perteneciente al grupo Inc A/C. La emergencia de aislamientos de K. pneumoniae ST307 coproductores de KPC-3 y NDM-1 pone de manifiesto una situación altamente preocupante debido a las características de este clon y a su perfil de multirresistencia.

Molecular mechanisms leading to ceftolozane/tazobactam resistance in clinical isolates of Pseudomonas aeruginosa from five Latin American countries.

Objectives: Identify molecular mechanisms responsible for the in vitro non-susceptibility to ceftolozane/tazobactam (TOL) in a group of 158 clinical isolates of Pseudomonas aeruginosa from five Latin American countries collected before the introduction of TOL into the clinical practice. Methods: Clinical isolates of P. aeruginosa (n = 504) were collected between January 2016 and October 2017 from 20 hospitals located in Argentina, Brazil, Chile, Colombia, and Mexico. Minimum inhibitory concentrations (MICs) to TOL were determined by standard broth microdilution and interpreted according to CLSI breakpoints. Initially, production of carbapenemases in TOL non-susceptible isolates was assessed by Rapidec® followed by qPCR to detect bla KPC, bla NDM-1, bla VIM, and bla IMP. Illumina® WGS was performed for isolates in which non-susceptibility to TOL was not mediated by carbapenemases. Results: A total of 158 (31.3%) isolates were non-susceptible to TOL. In 74 (46.8%) of these isolates, non-susceptibility to TOL was explained by the production of at least one carbapenemase. WGS revealed that some isolates carried ESBLs, mutated bla PDC and ampD, associated with decreased susceptibility to TOL. Conclusion: Substitutions found in PDC and carbapenemase production were the most common presumed mechanisms of resistance to TOL detected in this study. This study shows that epidemiological surveillance is warranted to monitor the emergence of novel mechanisms of resistance to TOL that might compromise its clinical utility.

Outbreak of Klebsiella pneumoniae ST11 Resistant To Ceftazidime-Avibactam Producing KPC-31 and the Novel Variant KPC-115 during COVID-19 Pandemic in Argentina.

We describe an outbreak of Klebsiella pneumoniae sequence type 11 (ST11) producing KPC variants resistant to ceftazidime-avibactam. Six patients hospitalized in the intensive care unit (mostly due to critical COVID pneumonia) presented infection or colonization by this bacterium. They had several comorbidities and required mechanical ventilation, central venous catheters, and urinary catheters. All 6 patients had a history of fecal colonization with KPC-producing Enterobacterales (KPC-E). Three of them had previous episodes of infection with ceftazidime-avibactam-susceptible KPC-producing K. pneumoniae, which were treated with ceftazidime-avibactam. Several phenotypic methods failed to detect carbapenemase production in these 6 ceftazidime-avibactam-resistant isolates, and they showed in vitro susceptibility to imipenem and meropenem. All of them rendered positive results for blaKPC by PCR, and amplicon sequencing identified blaKPC-31 variant in 5 isolates and a novel variant, named blaKPC-115, in the other. Moreover, matrix-assisted laser desorption ionization-time of flight mass spectrometry was able to detect KPC in all isolates. Ceftazidime-avibactam-resistant isolates, as well as those recovered from previous infection episodes (KPC-3-producing K. pneumoniae, ceftazidime-avibactam susceptible), displayed a unique pulse type and belonged to ST11. Based on whole-genome sequencing results of selected isolates, less than 7 single-nucleotide polymorphisms were identified among them, which was indicative of the presence of a unique clone. Both in vivo selection and horizontal transmission seemed to have occurred in our hospital. Detection of these strains is challenging for the laboratory. History of previous KPC-E infections or colonization and systematic testing for resistance to ceftazidime-avibactam might help raise awareness of this possibility. IMPORTANCE Klebsiella pneumoniae is one of the main bacteria that cause infections in health care settings. This pathogen has developed a high level of resistance to many antibiotics. Some K. pneumoniae isolates can produce an enzyme known as carbapenemase KPC, making carbapenems (considered the last line for therapy) not effective to treat their infections. The combination ceftazidime-avibactam, approved by FDA in 2015, is useful to treat infections caused by KPC-producing K. pneumoniae. This study describes the emergence, in one hospital in Argentina, of K. pneumoniae isolates that produce KPC variants (KPC-31 and KPC-115) resistant to ceftazidime-avibactam. The ceftazidime-avibactam-resistant bacteria were isolated in inpatients, including some that previously received this combination as treatment. Transmission of this strain to other patients also occurred in the studied period. Detection of these bacteria is challenging for the laboratory. The knowledge and awareness of the emergence of this pathogen in our region are highly valuable.

Report of two events of nosocomial outbreak and pseudo-outbreak due to contamination with Achromobacter spp / Comunicación de 2 eventos de brote y seudobrote nosocomial debido a contaminación con Achromobacter spp

Abstract Achromobacter spp. are increasingly recognized as emerging pathogens in immunocompromised patients or suffering cystic fibrosis, but unusual in immunocompetent hosts or individuals that underwent surgery. In this study we describe two simultaneous events attributable to two different Achromobacter spp. contaminated sources. One event was related to an episode of pseudo-bacteremia due to sodium citrate blood collection tubes contaminated with Achromobacter insuavis and the other to Achromobacter genogroup 20 infection and colonization caused by an intrinsically contaminated chlorhexidine soap solution. Both threatened the appropriate use of antimicrobials. Molecular approaches were critical to achieving the accurate species identification and to assess the clonal relationship, strengthening the need for dedicated, multidisciplinary and collaborative work of microbiologists, specialists in infectious diseases, epidemiologists and nurses in the control of infections to clarify these epidemiological situations.

Resumen Achromobacter spp. son reconocidas con mayor frecuencia como patógenos emergentes en pacientes con fibrosis quística e inmunodeprimidos, pero son inusuales en hospedadores inmunocompetentes o quirúrgicos. En este estudio describimos 2 eventos simultáneos atribuibles a 2 fuentes contaminadas con Achromobacter spp. Uno correspondió a un episodio de seudobacteriemia por tubos de citrato de sodio contaminados con Achromobacter insuavis y el otro a infecciones y colonizaciones debidas al uso de solución jabonosa de clorhexidina intrínsecamente contaminada con Achromobacter genogrupo 20. Ambos episodios pusieron en peligro el uso apropiado de antimicrobianos. Los enfoques moleculares fueron fundamentales para lograr la identificación precisa de las especies y evaluar la relación clonal de los aislamientos, lo que refuerza la necesidad del trabajo perseverante y multidisciplinario de microbiólogos, especialistas en enfermedades infecciosas, epidemiólogos y enfermeras en el control de infecciones para el esclarecimiento de estas situaciones epidemiológicas.

Comparative In Vitro Activity of Ceftolozane/Tazobactam against Clinical Isolates of Pseudomonas aeruginosa and Enterobacterales from Five Latin American Countries.

BACKGROUND: Ceftolozane/tazobactam (C/T) is a combination of an antipseudomonal oxyiminoaminothiazolyl cephalosporin with potent in vitro activity against Pseudomonas aeruginosa and tazobactam, a known ß-lactamase inhibitor. The aim of this study was to evaluate the activity of C/T against clinical isolates of P. aeruginosa and Enterobacterales collected from five Latin American countries between 2016 and 2017, before its clinical use in Latin America, and to compare it with the activity of other available broad-spectrum antimicrobial agents. METHODS: a total of 2760 clinical isolates (508 P. aeruginosa and 2252 Enterobacterales) were consecutively collected from 20 hospitals and susceptibility to C/T and comparator agents was tested and interpreted following the current guidelines. RESULTS: according to the CLSI breakpoints, 68.1% (346/508) of P. aeruginosa and 83.9% (1889/2252) of Enterobacterales isolates were susceptible to C/T. Overall, C/T demonstrated higher in vitro activity than currently available cephalosporins, piperacillin/tazobactam and carbapenems when tested against P. aeruginosa, and its performance in vitro was comparable to fosfomycin. When tested against Enterobacterales, it showed higher activity than cephalosporins and piperacillin/tazobactam, and similar activity to ertapenem. CONCLUSIONS: these results show that C/T is an active ß-lactam agent against clinical isolates of P. aeruginosa and Enterobacterales.

Emergence and clonal expansion of Klebsiella pneumoniae ST307, simultaneously producing KPC-3 and NDM-1.

MDR Klebsiella pneumoniae ST307 is a high-risk clone, whose genetic features contribute to its adaptation to hospital environments and the human host. This study describes the emergence and clonal dissemination of K. pneumoniae ST307, recovered during November 2018 to February 2019 in a hospital in Buenos Aires city, which concurrently harbored KPC-3 and NDM-1. These isolates were resistant to all ß-lactams and to the ceftazidime/avibactam combination. Molecular studies showed that blaKPC-3 was located in Tn4401a platform, while blaNDM-1 was surrounded upstream by ISKpn14 followed by a partial sequence of ISAba125 and downstream by bleMBL-trpF, located in a 145.5kb conjugative plasmid belonging to the Inc A/C group. The dissemination of K. pneumoniae ST307 isolates co-producing KPC-3 and NDM-1 could lead to a worrisome scenario due to the remarkable features of this clone and its resistance profile.

Characterisation of blaKPC-2-harbouring plasmids recovered from Pseudomonas aeruginosa ST654 and ST235 high-risk clones.

OBJECTIVE: The main objectives were to describe two blaKPC-2 plasmids recovered from Pseudomonas aeruginosa isolates belonging to the ST654 and ST235 high-risk clones, and to compare with complete sequences of blaKPC-2 harbouring plasmids available in public databases. METHODS: Antimicrobial susceptibility was determined according to CLSI (Clinical and Laboratory Standards Institute) guidelines. Genomes were sequenced using an Illumina MiSeq platform, and blaKPC-2 plasmid sequences were achieved using MinION platform. Sequences were analysed using Unicycler and RAST. In silico predictions of the isolates sequence type (ST), antimicrobial resistance genes, plasmid replicon typing and MOB relaxases were fulfilled using bioinformatics tools. RESULTS: PA_2047 and PA_HdC isolates corresponded to the high-risk clones ST654 and ST235, respectively. The carbapenem resistance was mediated by KPC-2. Both blaKPC-2 harbouring plasmids, pPA_2047 and pPA_HdC, were different among them, non-conjugative and untypable by PlasmidFinder. pPA_2047 presented high identity with a Pae-13 plasmid, and these both located blaKPC-2 in Tn4401b isoform. pPA_HdC displayed a novel architecture, and the genetic context of blaKPC-2 was original. Besides the blaKPC-2 gene, resistance genes to aminoglycosides and quinolones were detected, including the novel phosphotransferase CrpP in PA_HdC. CONCLUSION: This study expands the limited knowledge about the molecular epidemiology of blaKPC-2 in P. aeruginosa from Latin America. Two novel plasmids harbouring blaKPC-2 were described that were untypable by their incompatibility group. The plasmid recovered from P. aeruginosa PA_HdC (ST235) displayed a novel architecture and an original context for blaKPC-2. On the other hand, the genetic platform carrying blaKPC-2 in P. aeruginosa PA_2047 (ST654) seems to a be a classical one.

Full characterization of plasmids from Achromobacter ruhlandii isolates recovered from a single patient with cystic fibrosis (CF) / Caracterización completa de plásmidos provenientes de aislamientos de Achromobacter ruhlandii recuperados de un único paciente con fibrosis quística

Abstract In the last decade Achromobacter spp. has been associated with chronic colonizationin patients with cystic fibrosis (CF). Although Achromobacter xylosoxidans is the most frequentspecies recovered within this genus, other species such as A. ruhlandii have also been reportedin these patients. Descriptions of mobile elements are scarce in Achromobacter and none ofthem have been originated in A. ruhlandii. The aim of this study was to report the full char-acterization of a plasmid which was maintained in four clonally related A. ruhlandii isolates.Between 2013 and 2015, nine A. ruhlandii isolates were recovered from a pediatric patientwith CF at a hospital in Buenos Aires. Four selected clonally related isolates were sequencedby Illumina MiSeq, annotated using RAST and manually curated. The presence of a unique plas-mid of 34096-bp and 50 CDS was observed in the four isolates, displaying only 1 nucleotidesubstitution translated into one amino acid change among them. These plasmids have a class 1integron containing the aac-(6)-Ib gene, a mercury resistance operon region and the relE/stbEtoxin/antitoxin system. Plasmids showed 79% similarity and 99% identity with pmatvim-7 fromPseudomonas aeruginosa. This is the first full description and characterization of a plasmid fromA. ruhlandii which was maintained over time.

Resumen Durante la última década, Achromobacter spp. han sido asociadas con la colonización crónica en pacientes con fibrosis quística. Si bien Achromobacter xylosoxidans es la especie más frecuentemente recuperada, otras especies como Achromobacter ruhlandii también fueron reportadas en nuestra región. Sin embargo, pocos reportes se han centrado en la descripción de elementos móviles, y ninguno de ellos los documenta en A. ruhlandii. El objetivo de este estudio fue reportar la caracterización completa de un plásmido conservado en 4 aislamientos clonalmente relacionados de A. ruhlandii. Se recuperaron 9 aislamientos de A. ruhlandii entre 2013 y 2015 de un único paciente con fibrosis quística proveniente de un hospital pediátrico de Buenos Aires, Argentina. Se realizó la secuenciación completa del genoma de los 4 aislamientos seleccionados según el perfil de resistencia antibiótica en un equipo Illumina MiSeq. Estos fueron anotados mediante RAST y curados manualmente. Se detectó la presencia de un solo plásmido de 34.096 pb y 50CDS en los 4 aislamientos, observándose únicamente un cambio nucleotídico traducido en un cambio aminoacídico en un aislamiento. Los plásmidos ensamblados se caracterizaron por presentar un integrón de clase 1 que contenía el gen aac-(6')-Ib, un operón de resistencia a mercurio y el sistema de toxina-antitoxina relE/stbE. Cabe destacar que estos plásmidos poseen un 79% de similitud y un 99% de identidad con el plásmido pmatvim-7 de Pseudomonas aeruginosa. Esta es la primera descripción y caracterización completa de un plásmido proveniente de A. ruhlandii.

Report of two events of nosocomial outbreak and pseudo-outbreak due to contamination with Achromobacter spp.

Achromobacter spp. are increasingly recognized as emerging pathogens in immunocompromised patients or suffering cystic fibrosis, but unusual in immunocompetent hosts or individuals that underwent surgery. In this study we describe two simultaneous events attributable to two different Achromobacter spp. contaminated sources. One event was related to an episode of pseudo-bacteremia due to sodium citrate blood collection tubes contaminated with Achromobacter insuavis and the other to Achromobacter genogroup 20 infection and colonization caused by an intrinsically contaminated chlorhexidine soap solution. Both threatened the appropriate use of antimicrobials. Molecular approaches were critical to achieving the accurate species identification and to assess the clonal relationship, strengthening the need for dedicated, multidisciplinary and collaborative work of microbiologists, specialists in infectious diseases, epidemiologists and nurses in the control of infections to clarify these epidemiological situations.

Full characterization of plasmids from Achromobacter ruhlandii isolates recovered from a single patient with cystic fibrosis (CF).

In the last decade Achromobacter spp. has been associated with chronic colonization in patients with cystic fibrosis (CF). Although Achromobacter xylosoxidans is the most frequent species recovered within this genus, other species such as A. ruhlandii have also been reported in these patients. Descriptions of mobile elements are scarce in Achromobacter and none of them have been originated in A. ruhlandii. The aim of this study was to report the full characterization of a plasmid which was maintained in four clonally related A. ruhlandii isolates. Between 2013 and 2015, nine A. ruhlandii isolates were recovered from a pediatric patient with CF at a hospital in Buenos Aires. Four selected clonally related isolates were sequenced by Illumina MiSeq, annotated using RAST and manually curated. The presence of a unique plasmid of 34096-bp and 50 CDS was observed in the four isolates, displaying only 1 nucleotide substitution translated into one amino acid change among them. These plasmids have a class 1 integron containing the aac-(6')-Ib gene, a mercury resistance operon region and the relE/stbE toxin/antitoxin system. Plasmids showed 79% similarity and 99% identity with pmatvim-7 from Pseudomonas aeruginosa. This is the first full description and characterization of a plasmid from A. ruhlandii which was maintained over time.

Full characterization of an IncR plasmid harboring qnrSI recovered from a VIM-11-producing Pseudomonas aeruginosa

Abstract Metallo-p-lactamases (MBL) producing Pseudomonas aeruginosa isolates have been well characterized. Quinolones are commonly used in the treatment of carbapenem-resistant P. aeruginosa infections; however, data about PMQR in this species are scarce. The objective of this study was to report the simultaneous presence of qnrS and blaV-M-n in P. aeruginosa, and to characterize the qnrS-harboring plasmid. Thirty-eight carbapenem-resistant P. aeruginosa isolates were recovered from a hospital in Buenos Aires during 2012. Screening forMBL was assessed by the double disk synergy test using EDTA and carbapenem discs. Plasmid DNA extraction was performed by a method using phenol-chloroform. PCR followed by sequencing was carried out to determine each MBL and PMQR allele. PCR-BseGI-RFLP was performed to detect aac-(6')-Ib-cr. The gyrA-QRDR was sequenced in those PMQR-harboring isolates. Plasmid incompatibility groups and addiction systems were characterized by PCR. The PMQR-carrying plasmid was sequenced using Illumina technology, annotated using RAST and manually curated. Eleven/38 isolates were VIM producers (blaVIM-2 and blaVIM-11) while 1/38 harbored blaIMP-13. One isolate harbored blaVIM-11 and the PMQR qnrSI; however, both markers were located in different plasmids. The qnrSí-harboring plasmid (pP6qnrS1) was 117 945 bp in size, presented 154 CDS and corresponded to the IncR group. In addition to qnrSI, it harbored several aminoglycoside resis-tance markers. Although pP6qnrS1 was non-conjugative, it presented an oriT which made it possible for this plasmid to be transferable. This is the first report on P. aeruginosa carrying both blaVIM-11 and qnrSI, plus the first detection of an IncR plasmid in Argentina.

Resumen Las quinolonas son comúnmente utilizadas en el tratamiento de las infecciones producidas por Pseudomonas aeruginosa resistentes a carbapenems (PARC); aun así, la información sobre la resistencia a quinolonas mediada por plásmidos (PMQR) en esta especie es escasa. El objetivo de este trabajo fue reportar la presencia simultánea de los genes qnrS y blaVIM-11 en PARC y caracterizar el plásmido portador de qnrS. Durante 2012 se recuperaron 38 PARC en un hospital de Buenos Aires. El tamizaje para detectar producción de metalo-beta-lactamasas (MBL) se llevó a cabo mediante sinergia de doble disco utilizando EDTA y carbapenems. El ADN plasmídico fue extraído utilizando fenolcloroformo. Para determinar los alelos de los genes implicados en la síntesis de MBL y de PMQR, se llevó a cabo PCR-secuenciación. Para la detección de aac-(6')-Ib-cr se realizó PCR-BseGI-RFLP. En aquellos aislamientos portadores de PMQR se secuenció el gen gyrA. Los grupos de incompatibilidad y sistemas de adicción fueron caracterizados por PCR. El plásmido portador de PMQR fue secuenciado completamente y curado manualmente. De 38 aislamientos, 11 fueron productores de VIM (blaVIM-2 y blaVIM-11), mientras que uno contenía blaIMP-13. Si bien un aislamiento fue portador de blaVIM-11 y de qnrSI, dichos marcadores se encontraban en distintos plásmidos. El plásmido portador de qnrSI (pP6qnrS1) presentó un tamaño de 117.945 pby 154 secuencias codificantes (CDS); este correspondió al grupo de incompatibilidad IncR. Además de qnrSI, el plásmido portaba diversos marcadores de resistencia a aminoglucósidos. Aun cuando pP6qnrS1 no resultó conjugativo, presentó un oriT, de modo que posiblemente sea transferible. Este es el primer informe acerca de PARC portadora de blaVIM-11 y de qnrSI en simultáneo, además, es la primera descripción de un plásmido IncR en Argentina.

Expansion and improvement of MALDI-TOF MS databases for accurate identification of Achromobacter species.

Different MALDI-TOF MS databases were evaluated for the identification of Achromobacter species. The in-house and extended database generated in this study rendered more accurate identification (58/64 and 57/64 isolates, respectively) in comparison with the Bruker commercial database (42/64 isolates), especially in those infrequent species that are not available or poorly represented.

Diversity of Achromobacter species recovered from patients with cystic fibrosis, in Argentina / Diversidad de las especies de Achromobacter recuperadas de pacientes con fibrosis quística en Argentina

Abstract Different phenotype-based techniques and molecular tools were used to describe the distribution of different Achromobacter species in patients with cystic fibrosis (CF) in Argentina, and to evaluate their antibiotic resistance profile. Phenotypic identification was performed by conventional biochemical tests, commercial galleries and MALDI-TOF MS. Genetic approaches included the detection of A. xylosoxidans specific marker blaoxa-114, the amplificaron and sequencing of the 16S rRNA gene, nrdA and blaOXA complete sequence, and MLST analysis. Phenotypic approaches, even MALDI-TOF, rendered inconclusive or misleading results. On the contrary, concordant results were achieved with the nrdA sequencing or sequence type (ST) analysis, and the complete blaOXA sequencing, allowing a reliable discrimination of different Achromobacter species. A. xylosoxidans accounted for 63% of Achromobacter infections and A. ruhlandii accounted for 17%. The remaining species corresponded to A. insuavis, A. dolens, A. marplatensis and A. pulmonis. Antimicrobial susceptibilities were determined by the agar dilution method according to CLSI guidelines. Piperacillin, piperacillin/tazobactam and car-bapenems were the most active antibiotics. However, the emergence of carbapenem-resistant isolates was detected. In conclusion, prompt and accurate identification tools were necessary to determine that different Achromobacter species may colonize/infect the airways of patients with CF. Moreover, antimicrobial therapy should be administered based on the susceptibility profile of individual Achromobacter sp. isolates. © 2019 Asociación Argentina de Microbiología. Published by Elsevier España, S.L.U. This is an open access article under the CC BY-NC-ND license (https://creativecommons.org/licenses/by-nc-nd/4.0/).

Resumen Se emplearon diversas técnicas fenotípicas y moleculares para describir la distribución de diferentes especies del género Achromobacter en pacientes con fibrosis quística (FQ) en Argentina, y se evaluó el perfil de resistencia a los antibióticos. Se realizó la identificación fenotípica por pruebas bioquímicas convencionales, galerías comerciales y MALDI-TOF MS. El enfoque genético incluyó la detección del marcador especie-específico de A. xylosoxidans bla[PRESERVECIRC]tu, la amplificación y la secuenciación de los genes ARNr 16S, nrdA y secuencia completa de blaOXA, y el análisis por MLST. Los enfoques fenotípicos, incluso la técnica de MALDI-TOF, proporcionaron resultados no concluyentes o erróneos. Por el contrario, se obtuvieron resultados concordantes entre la secuenciación del gen nrdA o el análisis de secuenciotipos (ST) y la secuenciación completa de blaOXA, lo que permitió una discriminación confiable de las diferentes especies de Achromobacter. A. xylosoxidans representó el 63% de las infecciones por Achromobacter y A. ruhlandii representó el 17%. Las especies restantes correspondieron a A. insuavis, A. dolens, A. marplatensis y A. pulmonis. Se determinó la sensibilidad a antimicrobianos por el método de dilución en agar de acuerdo al CLSI. Los antibióticos más activos fueron piperacilina, piperacilina/tazobactam y carbapenemes. Sin embargo, se detectó la emergencia de aislamientos resistentes a carbapenemes. En conclusión, resultaron necesarias herramientas de identificación rápida y precisas para determinar las diferentes especies del género Achro-mobacter capaces de colonizar/infectar las vías respiratorias de los pacientes con FQ. Asimismo, la terapia antimicrobiana debería llevarse a cabo en función del perfil de sensibilidad de los aislamientos individuales de Achromobacter spp. © 2019 Asociacion Argentina de Microbiología. Publicado por Elsevier Espana, S.L.U. Este es un artículo Open Access bajo la licencia CC BY-NC-ND (https://creativecommons.org/licenses/by-nc-nd/4.0/).

In Vitro Susceptibility to Ceftazidime/Avibactam and Comparators in Clinical Isolates of Enterobacterales from Five Latin American Countries.

Background: High rates of resistance to third-generation cephalosporins and carbapenems in Enterobacterales have been reported in Latin America. Ceftazidime/avibactam (CZA) is the combination of a third-generation cephalosporin and a non-ß-lactam ß-lactamase inhibitor, which has shown activity against isolates producing class A, C and D ß-lactamases. Herein, we evaluated the activity of CZA and comparators against clinical isolates of Enterobacterales in Latin America. Methods: The activity of CZA and comparators was evaluated against clinical isolates of Enterobacterales from Argentina, Brazil, Chile, Colombia and Mexico that were collected between January 2016 and October 2017. One specific phenotypic subset was evaluated. A carbapenem non-susceptible (CNS) phenotype was defined as any isolate displaying a minimum inhibitory concentration (MIC) ≥1 mg/L for ertapenem. Results: CZA was active against 95.8% of all isolates and 77.5% of CNS isolates. Fosfomycin (FOS) and tigecycline (TGC) were the second most active antibiotics with 93.4% of Enterobacterales being susceptible. Conclusions: The results of this study underline the potential therapeutic role of CZA in Latin America.

Full characterization of an IncR plasmid harboring qnrS1 recovered from a VIM-11-producing Pseudomonas aeruginosa.

Metallo-ß-lactamases (MBL) producing Pseudomonas aeruginosa isolates have been well characterized. Quinolones are commonly used in the treatment of carbapenem-resistant P. aeruginosa infections; however, data about PMQR in this species are scarce. The objective of this study was to report the simultaneous presence of qnrS and blaVIM-11 in P. aeruginosa, and to characterize the qnrS-harboring plasmid. Thirty-eight carbapenem-resistant P. aeruginosa isolates were recovered from a hospital in Buenos Aires during 2012. Screening for MBL was assessed by the double disk synergy test using EDTA and carbapenem discs. Plasmid DNA extraction was performed by a method using phenol-chloroform. PCR followed by sequencing was carried out to determine each MBL and PMQR allele. PCR-BseGI-RFLP was performed to detect aac-(6')-Ib-cr. The gyrA-QRDR was sequenced in those PMQR-harboring isolates. Plasmid incompatibility groups and addiction systems were characterized by PCR. The PMQR-carrying plasmid was sequenced using Illumina technology, annotated using RAST and manually curated. Eleven/38 isolates were VIM producers (blaVIM-2 and blaVIM-11) while 1/38 harbored blaIMP-13. One isolate harbored blaVIM-11 and the PMQR qnrS1; however, both markers were located in different plasmids. The qnrS1-harboring plasmid (pP6qnrS1) was 117945bp in size, presented 154 CDS and corresponded to the IncR group. In addition to qnrS1, it harbored several aminoglycoside resistance markers. Although pP6qnrS1 was non-conjugative, it presented an oriT which made it possible for this plasmid to be transferable. This is the first report on P. aeruginosa carrying both blaVIM-11 and qnrS1, plus the first detection of an IncR plasmid in Argentina.

Diversity of Achromobacter species recovered from patients with cystic fibrosis, in Argentina.

Different phenotype-based techniques and molecular tools were used to describe the distribution of different Achromobacter species in patients with cystic fibrosis (CF) in Argentina, and to evaluate their antibiotic resistance profile. Phenotypic identification was performed by conventional biochemical tests, commercial galleries and MALDI-TOF MS. Genetic approaches included the detection of A. xylosoxidans specific marker blaoxa-114, the amplification and sequencing of the 16S rRNA gene, nrdA and blaOXA complete sequence, and MLST analysis. Phenotypic approaches, even MALDI-TOF, rendered inconclusive or misleading results. On the contrary, concordant results were achieved with the nrdA sequencing or sequence type (ST) analysis, and the complete blaOXA sequencing, allowing a reliable discrimination of different Achromobacter species. A. xylosoxidans accounted for 63% of Achromobacter infections and A. ruhlandii accounted for 17%. The remaining species corresponded to A. insuavis, A. dolens, A. marplatensis and A. pulmonis. Antimicrobial susceptibilities were determined by the agar dilution method according to CLSI guidelines. Piperacillin, piperacillin/tazobactam and carbapenems were the most active antibiotics. However, the emergence of carbapenem-resistant isolates was detected. In conclusion, prompt and accurate identification tools were necessary to determine that different Achromobacter species may colonize/infect the airways of patients with CF. Moreover, antimicrobial therapy should be administered based on the susceptibility profile of individual Achromobacter sp. isolates.