Interactions among beneficial mutations (that is, epistasis) are often strong enough to direct adaptation through alternative mutational paths. Although alternative solutions should display similar fitness under the primary selective conditions, their properties across secondary environments may differ widely. The extent to which these cryptic differences are to be expected is largely unknown, despite their importance-for example, in identifying exploitable collateral sensitivities among mutations conferring antibiotic resistance. Here we use directed evolution to characterize the diversity of mutational paths through which the prevalent carbapenemase Klebsiella pneumoniae carbapenemase-2 can evolve high activity against the clinically relevant antibiotic ceftazidime, an initially poor substrate. We identified 40 different substitutions-including many that are common in clinical settings-spread among 18 different mutational trajectories. Initial mutations determined four major groups into which the trajectories can be classified, a signature of strong epistasis. Despite similar final ceftazidime resistance, groups diverged markedly across multiple phenotypic dimensions, from molecular traits, such as in-cell stability and catalytic efficiency, to macroscopic traits, such as growth rate and activity against other β-lactam antibiotics. Our results indicate that cryptic yet consequential phenotypic differences can accumulate rapidly under strong selection, unpredictably shaping the long-term success of resistance enzymes in their journey across hosts and environments.

BACKGROUND:A drastic increase in carbapenem resistance among Klebsiella pneumoniae isolates occurred during the period 2019-22. Three epidemiological changes could be evidenced: (i) NDM became the predominant carbapenemase; (ii) NDM-5 replaced NDM-1; and (iii) the emergence of NDM-producing K. pneumoniae ST258 (NDM-KpST258). MATERIALS AND METHODS:Carbapenem-resistant K. pneumoniae isolates from patients on the ICU of a university hospital of Buenos Aires were studied during the period 2019-22. Identification was performed by MS and susceptibility by the Phoenix system (broth microdilution for colistin). Carbapenemase production was detected phenotypically. Molecular studies included PCR with specific primers and WGS (in some isolates). RESULTS:NDM-producing K. pneumoniae was statistically associated with the use of ceftazidime/avibactam between 2019 and April 2021, whereas in the period from May 2021 to December 2022, it seemed to be related to the presence of NDM-5-KpST258. A gradual increase in the number of urease-negative NDM-Kp-ST258 during 2019-22 was observed. The plasmid origin of NDM-5 was supported by its presence on the IncFII incompatibility group plasmid. CONCLUSIONS:Our study describes the first outbreak of NDM5-KpST258 at an ICU in Argentina, remarkably associated with considerable changes in the carbapenemase epidemiology. The intrinsic characteristics of ST258 may contribute to increased spread of NDM in hospital settings, resembling KPC-2 dissemination.

The production of PER-like extended-spectrum β-lactamases has recently been associated with reduced susceptibility to the last resort drugs aztreonam/avibactam and cefiderocol. PER-2 has been mainly confined to Argentina and neighboring countries. Until now, only three plasmids harboring blaPER-2 genes have been characterized but very little is known about the involvement of different plasmid groups in its dissemination. The diversity of genetic platforms associated with blaPER-2 genes from a collection of PER-producing Enterobacterales was analysed by describing the close environment and the plasmid backbones. Full sequences of 11 plasmids were obtained by short read (Illumina) and long read (Oxford Nanopore or PacBio) sequencing technologies. De novo assemblies, annotation and sequence analysis were performed by Unicycler, Prokka and BLAST. Plasmid analysis revealed that the blaPER-2 gene is encoded on plasmids of different incompatibility groups (A, C, FIB, HI1B, N2), indicating that this gene may have been disseminated through a variety of plasmids. Comparison with the few publicly available nucleotide sequences describing the blaPER-2 genetic environment, including those from the environmental species Pararheinheimera spp. (considered as the progenitor of blaPER genes), indicates a role of ISPa12 in blaPER-2 gene mobilization from the chromosome of Pararheinheimera spp. Also, the blaPER-2 gene was carried by a novel ISPa12-composite transposon, Tn7390. In addition, its association with ISKox2-like elements in the close genetic environment in all plasmids analysed suggests a role of these insertion sequence elements in further dissemination of blaPER-2 genes.

β-Lactams are among the most prescribed antibiotics worldwide, mainly due to their weak toxicity and good efficacy [...].

OXA-48-producing Enterobacterales have now widely disseminated throughout the world. Several variants have now been reported, differing by just a few amino-acid substitutions or deletions, mostly in the region of the loop β5-β6.

The spread of carbapenem-resistant Enterobacterales has raised concern in clinical settings due to the limited therapeutic options available. OXA-48-like enzymes are still sporadic in South America. The aim of this study was to characterize a multidrug-resistant Escherichia coli isolate from a hospitalized patient in Buenos Aires city. The isolate was characterized phenotypically by determination of its susceptibility pattern, synergistic and colorimetric tests, and molecularly, by PCR, whole genome sequencing, and plasmid analysis. It belonged to ST-744, phylogroup A, and serotype O162/O89: H9. It remained susceptible to ceftazidime, meropenem, aminoglycosides, trimethoprim/sulfamethoxazole, and tigecycline. The presence of blaOXA-232 harbored by a nonconjugative plasmid ColKp3, and blaCTX-M-14, mcr-1.1, and fosL1 in 2 conjugative plasmids, together with their genetic environment, was revealed. To the best of our knowledge, this is the first report of the coproduction of the enzyme OXA-232 and the mcr-1.1 gene in an E. coli clinical isolate in South America in a patient who had not received colistin therapy.

Since the first description of OXA-48, more than forty variants have been recovered from Enterobacterales isolates. Whereas some OXA-48-related enzymes have been reported as conferring similar resistance patterns, namely, the hydrolysis of carbapenems and penicillins with very weak or almost no activity against expanded-spectrum cephalosporins, some have reduced carbapenem and temocillin hydrolysis, and others hydrolyze expanded-spectrum cephalosporins and carbapenems only marginally. With such drastic differences in the hydrolytic profile, especially of carbapenems, it becomes urgent to establish hydrolytic cutoffs in order to determine when an OXA-48-like enzyme may be considered as a carbapenemase or not. With this aim, the coefficient of activity for imipenem (kcat/Km) was determined for a total of 30 enzymes, including OXA-48, OXA-48-like natural variants, and OXA-48 synthetic mutants. In addition, six different methods for the detection of carbapenemase-producers were performed. The coefficients of activity for imipenem for all the different enzymes went from 550 mM−1·s−1 to 0.02 mM−1·s−1. In order to match the coefficient of activity results with the biochemical confirmatory tests, we suggest the value of 0.27 mM−1·s−1 as the cutoff above which an OXA-48 variant may be considered a carbapenem-hydrolyzing enzyme.

Carbapenem resistance (CR) is an emerging health issue. Epidemiological surveys on carbapenem-resistant Gram-negative bacilli (CR-GNB) in Lebanon remain scarce. In this study, we determined the prevalence of CR-GNB isolated between 2015 to 2019 in three hospitals in northern Lebanon: 311 CR-Enterobacterales (out of 11210; 2.8%), 155 CR-Pseudomonas (out of 1034; 15%) and 106 CR- Acinetobacter (out of 184; 57.6%) were identified. CR mechanisms were determined for 146 randomly chosen isolates: the Carba NP test revealed an enzymatic resistance to carbapenems in 109 isolates (out of 146, 74.7%). Produced carbapenemases were evaluated by the NG-Test Carba5, NG-Test OXA-23 immunochromatographic assays and PCR. Carbapenemase-producing (CP) Enterobacterales expressed blaOXA-48-like, blaNDM-like and blaVIM-like genes and CP-Pseudomonas expressed blaIMP-like and blaVIM-like genes, whereas CP-Acinetobacter expressed blaOXA-23-like genes. The NG-Test Carba5 results were confirmed by PCR sequencing and revealed several variants, such as NDM-19, VIM-62 and OXA-162, never described so far in Lebanon. Isolates with discordant results were sequenced by WGS and highlighted novel variants of the natural oxacillinases of Pseudomonas aeruginosa: blaOXA-50-like genes. Their role in carbapenem resistance should be further studied. Overall, our findings highlight an alarming situation and encourage health care centers to establish performant registration systems that could help in limiting resistance spread.

Objetivo: describir brote causado por acinetobacter baumannii recuperado en dos centros asistenciales de la ciudad de Guayaquil, mediante técnicas de epidemiología molecular. Materiales y métodos: treinta y tres aislamientos de A. baumannii fueron recuperados de dos centros médicos de la ciudad de Guayaquil, Ecuador, entre noviembre de 2012 y octubre de 2013. Los aislamientos fueron identificados mediante MALDI-TOF y por la presencia de blaOXA-51. El análisis epidemiológico se realizó mediante PCR. Resultados: 33 aislamientos fueron sensibles solo a colistina. En 29 se detectó OXA-24/40. La secuenciación del ADN identificó a blaOXA-24/40 como blaOXA-72. Todos presentaron el mismo patrón de PCR. Conclusión: se presenta el primer brote de blaOXA-72 en aislados de A. baumannii en América del sur. Este es el primer estudio llevado a cabo en la República de Ecuador.

OXA-48 carbapenemase has rapidly spread in many countries worldwide with several OXA-48-variants being described, differing by a few amino acid (AA) substitutions or deletions, mostly in the β5-β6 loop. While single AA substitutions have only a minor impact on OXA-48 hydrolytic profiles, others with 4 AA deletions result in loss of carbapenem hydrolysis and gain of expanded-spectrum cephalosporin (ESC) hydrolysis. We have replaced the β5-β6 loop of OXA-48 with that of OXA-18, a clavulanic-acid inhibited oxacillinase capable of hydrolyzing ESCs but not carbapenems. The hybrid enzyme OXA-48Loop18 was able to hydrolyze ESCs and carbapenems (although with a lower kcat), even though the β5-β6 loop was longer and its sequence quite different from that of OXA-48. The kinetic parameters of OXA-48Loop18 were in agreement with the MIC values. X-ray crystallography and molecular modeling suggest that the conformation of the grafted loop allows the binding of bulkier substrates, unlike that of the native loop, expanding the hydrolytic profile. This seems to be due not only to differences in AA sequence, but also to the backbone conformation the loop can adopt. Finally, our results provide further experimental evidence for the role of the β5-β6 loop in substrate selectivity of OXA-48-like enzymes and additional details on the structure-function relationship of β-lactamases, demonstrating how localized changes in these proteins can alter or expand their function, highlighting their plasticity.

Klebsiella pneumoniae is one of the so-called ESKAPE pathogens. These organisms are the main cause of nosocomial infections worldwide, causing life-threatening infections amongst critically ill and immunocompromised individuals. They are characterized by drug resistance mechanisms. Klebsiella pneumoniae carbapenemase (KPC)-producing isolates display resistance to multiple antimicrobial agents, usually including last-resort alternative options, leading to an urgent need to develop new drugs or combinations. In Argentina sequence type (ST) 258 harbouring blaKPC-2 emerged in 2010 and remained prevalent until the last few years, when the emergence of different STs such as ST25, ST11 and ST307 appeared likely to change the local epidemiology [[1]Cejas D. Elena A. Guevara Nuñez D. Sevillano Platero P. De Paulis A. Magariños F. et al.Changing epidemiology of KPC-producing Klebsiella pneumoniae in Argentina: emergence of hypermucoviscous ST25 and high-risk clone ST307.Glob Antimicrob Resist. 2019; 18: 238-242Crossref PubMed Scopus (45) Google Scholar]. Ceftazidime/avibactam (CZA) is a novel β-lactam/β-lactamase inhibitor combination that inactivates KPC and OXA-48 carbapenemases, AmpC and extended-spectrum β-lactamases (ESBL), but it is not active against metallo-β-lactamases. In the last 2 years, CZA was introduced in some nosocomial centres in Argentina with promising expectations. The combination of enhanced expression of KPC, porin mutations and the increase of ceftazidime (CAZ) hydrolysis in some variants has been reported as the main resistance mechanisms to this antibiotic combination [[2]Shields R.K. Nguyen M.H. Press E.G. Chen L. Kreiswirth B.N. Clancy C.J. Emergence of ceftazidime–avibactam resistance and restoration of carbapenem susceptibility in carbapenemase-producing K. pneumoniae: a case report and review of literature.Open Forum Infect Dis. 2017; 4ofx101Crossref PubMed Scopus (84) Google Scholar,[3]Oueslati S. Tlili L. Exilie C. Bernabeu S. Iorga B. Bonnin R.A. et al.Different phenotypic expression of KPC β-lactamase variants and challenges in their detection.J Antimicrob Chemother. 2020; 75: 769-771Crossref PubMed Scopus (13) Google Scholar]. Three CZA-resistant K. pneumoniae isolates harbouring KPC-8 were obtained from urine samples of patients who had previously been treated with multiple antimicrobials but had not received CZA therapy in a hospital in Buenos Aires during the period June to November 2019. Identification was performed by MALDI-TOF MS and susceptibility testing by Phoenix System (NMIC-406 panel) and broth microdilution for CZA and colistin (Med Chem Express). The isolates were characterized phenotypically and genotypically by PCR using specific primers for the detection of blaCTX-M, blaKPC, blaOXA, blaNDM, blaVIM and blaIMP and were confirmed by sequencing (Macrogen). Repetitive-element PCR and multilocus sequence typing were also performed. Plasmid conjugation assay was performed in lysogeny broth with Escherichia coli J53 AzR as the recipient strain and KpB1 as the donor strain. KpB1, KpB2 and KpB3 were resistant to all β-lactams, including CZA (MIC: 16 mg/L) and to amikacin, gentamicin, trimethoprim/sulfamethoxazole and fluoroquinolones. They remained susceptible to colistin (MIC: 1 mg/L), fosfomycin and tigecycline. The three isolates generated indistinguishable repetitive-element PCR fingerprints, and KpB1 belonged to ST11 according to multilocus sequence typing. The phenotypic synergy test was positive for ESBL detection ceftriaxone, amoxicillin clavulanic acid and ceftazidime (CRO-AMC-CAZ). Double-disc synergy tests for carbapenemases using imipenem, phenylboronic acid and meropenem (IMI-PBA-MER), and imipenem, EDTA and meropenem (IMI-EDTA-MER) were performed. KPC production was confirmed, whereas the test was negative for the presence of metallo-β-lactamases. Genotypic analysis confirmed that the isolates harboured blaKPC-8 and blaCTX-M-15. The conjugation experiment performed on KpB1 revealed the plasmid location of the KPC-8 coding gene, a result confirmed by the colourimetric Blue-Carba Test, by the phenylboronic acid synergy test and by PCR, on the transconjugant (KpB1TC). The antimicrobial susceptibility data of KpB1 and KpB1TC are listed in Table 1. Both KpB1 and KpB1TC displayed resistance to extended-spectrum cephalosporins, including CAZ, the resistance of which was inhibited by adding avibactam). This effect was observed in KpB1TC with avibactam at ≥4 mg/L, compared to KpB1 with avibactam ≥10 mg/L (data not shown). The KpB1TC MIC to CZA was eightfold higher (1 mg/L) than E. coli J53 AzR.Table 1Antimicrobial drug susceptibility for KpB1, Escherichia coli J53 AzR and KpB1TCAntimicrobial agentMIC (mg/L)KpB1E. coli J53 AzRKpB1TCAmpicillin>16≤4>16Ampicillin/sulbactam>16/8≤4/2>16/8Piperacillin/tazobactam>64/4≤4/4>64/4Cefazolin>8≤2>8Cefoxitin16≤4≤4Ceftriaxone>4≤1>4Ceftazidime128≤164Cefepime>16≤116Ertapenem8≤0.250,5Imipenem4≤0.251Meropenem4≤0.51Amikacin>32≤8>32Gentamicin>8≤2>8Trimethoprim/sulfamethoxazole>2/38≤0.5/9.5>2/38Colistin≤1≤1≤1Ciprofloxacin>2≤0.125≤0.125Levofloxacin>4≤1≤1Fosfomycin≤16≤16≤16Tigecycline2≤1≤1Ceftazidime/avibactam160.1251KpB1, KpB2 and KpB3 showed the same susceptibility profile. Open table in a new tab KpB1, KpB2 and KpB3 showed the same susceptibility profile. Among acquired carbapenemases reported in Argentina, KPC-2 is absolutely prevalent and to a lesser extent KPC-3, while KPC-8 was not previously detected [[1]Cejas D. Elena A. Guevara Nuñez D. Sevillano Platero P. De Paulis A. Magariños F. et al.Changing epidemiology of KPC-producing Klebsiella pneumoniae in Argentina: emergence of hypermucoviscous ST25 and high-risk clone ST307.Glob Antimicrob Resist. 2019; 18: 238-242Crossref PubMed Scopus (45) Google Scholar]. KPC-8 differs from KPC-2 by two amino acid substitutions (V240G and H274Y) and from KPC-3 by one amino acid substitution (V240G). Mutations within the KPC Ω loop (R164 to D179) have been described in KPC-2 and KPC-3. These substitutions have been related to enhanced affinity towards CAZ, prevention of binding to avibactam and reversion of carbapenem resistance [[2]Shields R.K. Nguyen M.H. Press E.G. Chen L. Kreiswirth B.N. Clancy C.J. Emergence of ceftazidime–avibactam resistance and restoration of carbapenem susceptibility in carbapenemase-producing K. pneumoniae: a case report and review of literature.Open Forum Infect Dis. 2017; 4ofx101Crossref PubMed Scopus (84) Google Scholar,[4]Compain F. Arthur M. Impaired inhibition by avibactam and resistance to the ceftazidime-avibactam combination due to the D179Y substitution in the KPC-2 β-lactamase.Antimicrob Agents Chemother. 2017; 61e00451-17Crossref PubMed Scopus (57) Google Scholar]. Mutations distantly located from the Ω loop in the regions covering the amino acids 240–243 (close to the hinge loop) and 263–277 (in the vicinity of the X loop and the hinge loop) have been also described [[5]Mueller L. Masseron A. Prod'Hom G. Galperine T. Greub G. Poirel L. et al Phenotypic, biochemical and genetic analysis of KPC-41, a KPC-3 variant conferring resistance to ceftazidime-avibactam and exhibiting reduced carbapenemase activity.Antimicrob Agents Chemother. 2019; 63: e01111-e01119Crossref Scopus (40) Google Scholar,[6]Göttig S. Frank D. Mungo E. Nolte A. Hogardt M. Besier S. et al.Emergence of ceftazidime/avibactam resistance in KPC-3–producing Klebsiella pneumoniae in vivo.J Antimicrob Chemother. 2019; 74: 3211-3216Crossref PubMed Scopus (47) Google Scholar]. Substitution V240G, as observed in KPC-8, may enhance backbone flexibility, so that larger substrates might be accommodated in the active site. KPC-8–producing isolates presented an 80-fold increase in CAZ MICs compared to KPC-2 due as a result of the enhanced CAZ catalytic efficiency of KPC-8, in addition to the protein expression level and its stability and solubility [[7]Mehta S.C. Rice K. Palzkill T. Natural variants of the KPC-2 carbapenemase have evolved increased catalytic efficiency for ceftazidime hydrolysis at the cost of enzyme stability.PLoS Pathog. 2015; 11e1004949Crossref PubMed Scopus (68) Google Scholar]. An increase in CZA MICs in KPC-8–producing K. pneumoniae isolates was reported in 2017 by Shields et al. [[2]Shields R.K. Nguyen M.H. Press E.G. Chen L. Kreiswirth B.N. Clancy C.J. Emergence of ceftazidime–avibactam resistance and restoration of carbapenem susceptibility in carbapenemase-producing K. pneumoniae: a case report and review of literature.Open Forum Infect Dis. 2017; 4ofx101Crossref PubMed Scopus (84) Google Scholar]. In our study the fact that the addition of avibactam restored CAZ activity suggests, as was observed by Shields et al., that the resistance to CZA in these KPC variants may be related to the high CAZ MIC value. Shields et al. described the emergence of mutations in plasmid-borne blaKPC-3 in isolates recovered after 10 to 19 days' CZA treatment with respect to baseline isolates. All these isolates belonged to ST258, while KPC-8–producing isolates recovered in the present study corresponded to ST11. The presence of this ST was reported in our region to be initially associated with the production of CTX-M-15 and more recently with KPC-2 [[1]Cejas D. Elena A. Guevara Nuñez D. Sevillano Platero P. De Paulis A. Magariños F. et al.Changing epidemiology of KPC-producing Klebsiella pneumoniae in Argentina: emergence of hypermucoviscous ST25 and high-risk clone ST307.Glob Antimicrob Resist. 2019; 18: 238-242Crossref PubMed Scopus (45) Google Scholar]. Moreover, decreased CZA susceptibility was reported in 2017 by Shen et al. [[8]Shen Z. Ding B. Ye M. Wang P. Bi Y. Wu S. et al.High ceftazidime hydrolysis activity and porin OmpK35 deficiency contribute to the decreased susceptibility to ceftazidime/avibactam in KPC-producing Klebsiella pneumoniae.J Antimicrob Chemother. 2017; 72: 1930-1936Crossref PubMed Scopus (54) Google Scholar] in KPC-2–producing clinical isolates corresponding to ST11. These isolates displayed an increased gene expression of blaKPC-2 and the coproduction of ESBLs, which confers higher CAZ hydrolysis activity, added to the loss of OmpK35. KPC-8 is a KPC variant with low prevalence worldwide and in our region, but there is a potential risk that the increasing use of CZA may exert selective pressure in favour of its dissemination. In conclusion, we reported three CZA-resistant K. pneumoniae harbouring KPC-8 clinical isolates. At the time of this writing, there were no previous reports of CZA resistance in South America in carbapenem-resistant Enterobacteriaceae. Funding was received by UBACyT 20020150100174BA (to Marcela Radice) and UBACyT 20020130100167BA (to Angela Famiglietti), Argentina. All authors report no conflicts of interest relevant to this letter.

Carbapenemase-producing Enterobacterales expressing OXA-48, KPC, NDM, VIM or IMP enzymes are increasingly reported worldwide. We have characterized LMB-1, a novel metallo-β-lactamase (MBL) of Ambler class B3 from Citrobacter freundii 164 (Cf164) clinical isolate from Buenos Aires, Argentina. Cf164 displayed reduced susceptibility to carbapenems but gave inconsistent results with carbapenemase confirmatory tests, indicating the presence of a weak carbapenemase. Analysis of whole-genome sequencing (WGS) of Cf164 using Resfinder revealed four β-lactamase genes coding for CTX-M-8, PER-2, TEM-1 and CMY-150, a novel chromosomally-encoded CMY variant. Kinetic parameters of purified CMY-150 did not reveal any carbapenemase activity. However, CMY-150 conferred higher minimum inhibitory concentrations (MICs) to E. coli for ceftazidime and aztreonam compared with CMY-2. The in-house-developed β-lactamase search software (ResMiner) in WGS data revealed a novel subclass B3 MBL named LMB-1. LMB-1 conferred resistance to penicillins and expanded-spectrum cephalosporins and reduced susceptibility to carbapenems in E. coli. The blaLMB-1 gene was located on a 176-kb IncA/C2 plasmid. LMB-1 shared 99% amino acid sequence identity with the MBL encoded in the chromosome of Rheinheimera pacifica, it's likely progenitor. Despite repeated attempts, LMB-1 could not be purified, thus only specific activities could indicate hydrolysis of carbapenems. Here we report on CMY-150, a novel CMY-2 variant that confers increased ceftazidime and aztreonam MICs to E. coli and the first description of LMB-1 in Argentina. This work underlines the need for several carbapenemase-producing Enterobacteriaceae (CPE) confirmatory tests, as this novel enzyme might have been missed using only one.

OXA-48 carbapenemase has rapidly spread in many countries worldwide with several OXA-48-variants being described, differing by a few amino acid (AA) substitutions or deletions, mostly in the beta 5-beta 6 loop. While single AA substitutions have only a minor impact on OXA-48 hydrolytic profiles, others with 4 AA deletions result in loss of carbapenem hydrolysis and gain of expanded-spectrum cephalosporin (ESC) hydrolysis. We have replaced the beta 5-beta 6 loop of OXA-48 with that of OXA-18, a clavulanic-acid inhibited oxacillinase capable of hydrolyzing ESCs but not carbapenems. The hybrid enzyme OXA-48Loopl8 was able to hydrolyze ESCs and carbapenems (although with a lower k(cat)), even though the beta 5-beta 6 loop was longer and its sequence quite different from that of OXA-48. The kinetic parameters of OXA-48Loop18 were in agreement with the MIC values. X-ray crystallography and molecular modeling suggest that the conformation of the grafted loop allows the binding of bulkier substrates, unlike that of the native loop, expanding the hydrolytic profile. This seems to be due not only to differences in AA sequence, but also to the backbone conformation the loop can adopt. Finally, our results provide further experimental evidence for the role of the beta 5-beta 6 loop in substrate selectivity of OXA-48-like enzymes and additional details on the structure-function relationship of beta-lactamases, demonstrating how localized changes in these proteins can alter or expand their function, highlighting their plasticity.

We investigated the presence of carbapenemases in carbapenem-resistant Pseudomonas aeruginosa isolates, which were collected over a 14-month period in a Turkish hospital, with in-depth molecular characterization of carbapenemase-producing isolates. Among 45 study isolates, 2 isolates were identified as carbapenemase producers by both Carba NP and Carbapenem Inactivation Method tests, and only 1 of them gave a positive result in polymerase chain reaction tests for a carbapenemase gene (blaVIM). Whole genome sequencing of the 2 isolates revealed the presence of blaVIM-5 gene in an ST308 isolate, while the other one expressed IMP-7 in an ST357 isolate; both STs are considered high-risk clones. The 2 carbapenemase-producing isolates were multidrug resistant, as they harbored other resistance determinants, including a variant of the recently described plasmid-encoded fluoroquinolone resistance determinant crpP gene, crpP-2. We report for the first time P. aeruginosa high-risk clones carrying VIM-5– and IMP-7–type carbapenemases with multiple resistance determinants in Turkey.

BACKGROUND SME carbapenemases are increasingly reported, especially from North and South America. Here, we describe an SME-4-producing Serratia marcescens (SME-Sm) clinical isolate from Argentina and compare its genome with other SME-Sm and Sm isolates recovered from public databases. METHODS Sm isolates were characterized by WGS using Illumina technology, susceptibility testing and MIC determination. Carbapenemase activity was revealed by biochemical tests based on imipenem hydrolysis. A whole-genome phylogeny was estimated for all the Sm isolates retrieved from public databases with kSNP3 and a whole-genome phylogenetic analysis based on non-recombinant core SNPs was inferred for Sm complete genomes and for those encoding any blaSME variants. RESULTS Sm163 was resistant to amoxicillin, temocillin, aztreonam and carbapenems, remaining susceptible to extended-spectrum cephalosporins. WGS analysis of Sm163 revealed a genome of 5139329 bp and a chromosomally encoded blaSME-4 carbapenemase gene located on a genomic island closely related to SmarGI1-1 of Sm N11-02820. Comparison of the Sm genomes revealed that the 14 SME-Sm isolates possess this genomic island inserted at the same loci, that 13/14 belong to clade 1 and that 11/14 form a well-defined subcluster of cluster I of Sm clade 1, while Sm163 belongs to clade 2, suggesting that an SME-encoding genomic island may have been transferred between isolates from different clades. CONCLUSIONS To the best of our knowledge this is the first report of an SME-4-encoding Sm from Argentina. The blaSME-4 gene is located on a SmarGI1-1-like genomic island. The genome of Sm163 belongs to clade 2, unlike all the other SME-Sm isolates, which belong to clade 1.

Due to the paucity of remaining antibiotics for treating infections caused by carbapenem-resistant Enterobacteriaceae, polymyxins have become the last resort antibiotics.As a consequence, colistin resistance is increasingly reported worldwide.The aim of this study was to analyze colistin-resistant E. coli clinical isolates, recovered between 2014 and 2016 at the University Hospital of Buenos Aires, Argentina.Nine clinical colistin resistant E. coli isolates were studied.These isolates were recovered from urine samples of 5 inpatients and 4 outpatients.Whole genome sequencing was performed using Illumina technology.Plasmid characterization and mating-out assay was done using E. coli J53 as receptor strain.Antibiotic susceptibility (MIC) of clinical isolates and their transconjugants was determined using broth microdilution method.WGS analysis revealed the presence of mcr-1 gene in six out of the 9 isolates: 4 isolates carried mcr-1 and 2 carried mcr-1.5 alleles.All the clinical isolates had MIC values for colistin in the range of 4-16 mg/L.The three isolates lacking any mcr variant, presented point mutations in the chromosomal pmrA or pmrB genes.The mcr-1 gene were located on plasmids similar to the prototypical Incl2-type (KY471308, pMCR-M15049) differing only by little deletions.Until this date mcr-1.5 allele was reported once in Argentina and in Japan, suggested a transcontinental dissemination of this variant.

ABSTRACT The bla PER-2 -harboring plasmid pCf587 (191,541 bp) belongs to lineage IncA/C 1 and is closely related to pRA1. It contains a large resistance island including the bla PER-2 gene between two copies of IS Kox2 -like elements, the toxin-antitoxin module pemK-pemI , several other resistance genes inserted within a Tn 2 transposon, a Tn 21 -like structure, and a class 1 integron. pCf587 belongs to sequence type 13 (ST13), a new plasmid multilocus sequence typing (pMLST) ST.