Emergence of NDM-producing Enterobacterales infections in companion animals from Argentina.

Antimicrobial resistance is considered one of the most critical threat for both human and animal health. Recently, reports of infection or colonization by carbapenemase-producing Enterobacterales in companion animals had been described. This study report the first molecular characterization of NDM-producing Enterobacterales causing infections in companion animals from Argentina. Nineteen out of 3662 Enterobacterales isolates analyzed between October 2021 and July 2022 were resistant to carbapenemes by VITEK2C and disk diffusion method, and suspected to be carbapenemase-producers. Ten isolates were recovered from canine and nine from feline animals. Isolates were identified as K. pneumoniae (n = 9), E. coli (n = 6) and E. cloacae complex (n = 4), and all of them presented positive synergy among EDTA and carbapenems disks, mCIM/eCIM indicative of metallo-carbapenemase production and were also positive by PCR for blaNDM gene. NDM variants were determined by Sanger sequencing method. All 19 isolates were resistant to ß-lactams and aminoglycosides but remained susceptible to colistin (100%), tigecycline (95%), fosfomycin (84%), nitrofurantoin (63%), minocycline (58%), chloramphenicol (42%), doxycycline (21%), enrofloxacin (5%), ciprofloxacin (5%) and trimethoprim/sulfamethoxazole (5%). Almost all isolates (17/19) co-harbored blaCTX-M plus blaCMY, one harbored blaCTX-M alone and the remaining blaCMY. E. coli and E. cloacae complex isolates harbored blaCTX-M-1/15 or blaCTX-M-2 groups, while all K. pneumoniae harbored only blaCTX-M-1/15 genes. All E. coli and E. cloacae complex isolates harbored blaNDM-1, while in K. pneumoniae blaNDM-1 (n = 6), blaNDM-5 (n = 2), and blaNDM-1 plus blaNDM-5 (n = 1) were confirmed. MLST analysis revealed the following sequence types by species, K. pneumoniae: ST15 (n = 5), ST273 (n = 2), ST11, and ST29; E. coli: ST162 (n = 3), ST457, ST224, and ST1196; E. cloacae complex: ST171, ST286, ST544 and ST61. To the best of our knowledge, this is the first description of NDM-producing E. cloacae complex isolates recovered from cats. Even though different species and clones were observed, it is remarkable the finding of some major clones among K. pneumoniae and E. coli, as well as the circulation of NDM as the main carbapenemase. Surveillance in companion pets is needed to detect the spread of carbapenem-resistant Enterobacterales and to alert about the dissemination of these pathogens among pets and humans.

KPC-2 allelic variants in Klebsiella pneumoniae isolates resistant to ceftazidime-avibactam from Argentina: blaKPC-80, blaKPC-81, blaKPC-96 and blaKPC-97.

Ceftazidime-avibactam (CZA) therapy has significantly improved survival rates for patients infected by carbapenem-resistant bacteria, including KPC producers. However, resistance to CZA is a growing concern, attributed to multiple mechanisms. In this study, we characterized four clinical CZA-resistant Klebsiella pneumoniae isolates obtained between July 2019 and December 2020. These isolates expressed novel allelic variants of blaKPC-2 resulting from changes in hotspots of the mature protein, particularly in loops surrounding the active site of KPC. Notably, KPC-80 had an K269_D270insPNK mutation near the Lys270-loop, KPC-81 had a del_I173 mutation within the Ω-loop, KPC-96 showed a Y241N substitution within the Val240-loop and KPC-97 had an V277_I278insNSEAV mutation within the Lys270-loop. Three of the four isolates exhibited low-level resistance to imipenem (4 µg/mL), while all remained susceptible to meropenem. Avibactam and relebactam effectively restored carbapenem susceptibility in resistant isolates. Cloning mutant blaKPC genes into pMBLe increased imipenem MICs in recipient Escherichia coli TOP10 for blaKPC-80, blaKPC-96, and blaKPC-97 by two dilutions; again, these MICs were restored by avibactam and relebactam. Frameshift mutations disrupted ompK35 in three isolates. Additional resistance genes, including blaTEM-1, blaOXA-18 and blaOXA-1, were also identified. Interestingly, three isolates belonged to clonal complex 11 (ST258 and ST11) and one to ST629. This study highlights the emergence of CZA resistance including unique allelic variants of blaKPC-2 and impermeability. Comprehensive epidemiological surveillance and in-depth molecular studies are imperative for understanding and monitoring these complex resistance mechanisms, crucial for effective antimicrobial treatment strategies. IMPORTANCE: The emergence of ceftazidime-avibactam (CZA) resistance poses a significant threat to the efficacy of this life-saving therapy against carbapenem-resistant bacteria, particularly Klebsiella pneumoniae-producing KPC enzymes. This study investigates four clinical isolates exhibiting resistance to CZA, revealing novel allelic variants of the key resistance gene, blaKPC-2. The mutations identified in hotspots surrounding the active site of KPC, such as K269_D270insPNK, del_I173, Y241N and V277_I278insNSEAV, prove the adaptability of these pathogens. Intriguingly, low-level resistance to imipenem and disruptions in porin genes were observed, emphasizing the complexity of the resistance mechanisms. Interestingly, three of four isolates belonged to clonal complex 11. This research not only sheds light on the clinical significance of CZA resistance but also shows the urgency for comprehensive surveillance and molecular studies to inform effective antimicrobial treatment strategies in the face of evolving bacterial resistance.

Co-integrate Col3m bla NDM-1-harboring plasmids in clinical Providencia rettgeri isolates from Argentina.

The first cases of bla NDM in Argentina were detected in three Providencia rettgeri (Pre) recovered from two hospitals in Buenos Aires city in 2013. The isolates were genetically related, but the plasmid profile was different. Here, we characterized the bla NDM-1-harboring plasmids of the first three cases detected in Argentina. Hybrid assembly obtained from short- and long-read sequencing rendered bla NDM-1 in Col3M plasmids of ca. 320 kb (p15268A_320) in isolate PreM15268, 210 kb (p15758B_210) in PreM15758, and 225 kb (p15973A_225) in PreM15973. In addition, PreM15758 harbored a 98-kb circular plasmid (p15758C_98) flanked by a putative recombination site (hin-TnAs2), with 100% nucleotide ID and coverage with p15628A_320. Analysis of PFGE/S1-nuclease gel, Southern hybridization with bla NDM-1 probe, hybrid assembly of short and long reads suggests that pM15758C_98 can integrate by homologous recombination. The three bla NDM-1-plasmids were non-conjugative in vitro. Moreover, tra genes were incomplete, and oriT was not found in the three bla NDM-1-plasmids. In two isolates, blaNDM-1 was embedded in a partially conserved structure flanked by two ISKox2. In addition, all plasmids harbored aph(3')-Ia, aph(3')-VI, and qnrD1 genes and aac(6´)Ib-cr, bla OXA-1, catB3, and arr3 as part of a class 1 integron. Also, p15268A_320 and p15973A_225 harbored bla PER-2. To the best of our knowledge, this is the first report of clinical P. rettgeri harboring blaNDM-1 in an atypical genetic environment and located in unusual chimeric Col3M plasmids. The study and continuous surveillance of these pathogens are crucial to tracking the evolution of these resistant plasmids and finding solutions to tackle their dissemination. IMPORTANCE Infections caused by carbapenem hydrolyzing enzymes like NDM (New Delhi metallo-beta-lactamase) represent a serious problem worldwide because they restrict available treatment options and increase morbidity and mortality, and treatment failure prolongs hospital stays. The first three cases of NDM in Argentina were caused by genetically related P. rettgeri recovered in two hospitals. In this work, we studied the genetic structure of the plasmids encoding bla NDM in those index cases and revealed the enormous plasticity of these genetic elements. In particular, we found a small plasmid that was also found inserted in the larger plasmids by homologous recombination as a co-integrate element. We also found that the bla NDM plasmids were not able to transfer or move to other hosts, suggesting their role as reservoir elements for the acquisition of resistance genes. It is necessary to unravel the dissemination strategies and the evolution of these resistant plasmids to find solutions to tackle their spread.

Emergence of Hyper-Epidemic Clones of Enterobacterales Clinical Isolates Co-Producing KPC and Metallo-Beta-Lactamases during the COVID-19 Pandemic.

BACKGROUND: The global spread of carbapenemase-producing Enterobacterales has become an epidemiological risk for healthcare systems by limiting available antimicrobial treatments. The COVID-19 pandemic worsened this scenario, prompting the emergence of extremely resistant microorganisms. METHODS: Between March 2020 and September 2021, the NRL confirmed 82 clinical Enterobacterales isolates harboring a combination of blaKPC and MBL genes. Molecular typing was analyzed by PFGE and MLST. Modified double-disk synergy (MDDS) tests were used for phenotypic studies. RESULTS: Isolates were submitted from 28 hospitals located in seven provinces and Buenos Aires City, including 77 K. pneumoniae, 2 K. oxytoca, 2 C. freundii, and 1 E. coli. Almost half of K. pneumoniae isolates (n = 38; 49.4%), detected in 15 hospitals, belong to the CC307 clone. CC11 was the second clone, including 29 (37.7%) isolates (22, ST11 and 7, ST258) from five cities and 12 hospitals. Three isolates belonging to CC45 were also detected. The carbapenemase combinations observed were as follows: 55% blaKPC-2 plus blaNDM-5; 32.5% blaKPC-2 plus blaNDM-1; 5% blaKPC-3 plus blaNDM-1; 5% blaKPC-2 plus blaIMP-8; and 2.5% strain with blaKPC-2 plus blaNDM-5 plus blaOXA-163. Aztreonam/avibactam and aztreonam/relebactam were the most active combinations (100% and 91% susceptible, respectively), followed by fosfomycin (89%) and tigecycline (84%). CONCLUSIONS: The MDDS tests using ceftazidime-avibactam/EDTA and aztreonam/boronic acid disks improved phenotypic classification as dual producers. The successful high-risk clones of K. pneumoniae, such as hyper-epidemic CC307 and CC11 clones, drove the dissemination of double carbapenemase-producing isolates during the COVID-19 pandemic.

Characterization of third generation cephalosporin-resistant Escherichia coli clinical isolates from Ushuaia, Argentina / Caracterización de aislamientos clínicos de Escherichia coli resistente a cefalosporinas de tercera generación de Ushuaia, Argentina

Abstract Escherichia coli is one of the main human pathogens causing different hospital- and community-acquired infections. During the period from January 2013 to March 2015, 1.96% (32/1632) of E. coli isolates recovered at the Hospital Regional de Ushuaia, Tierra del Fuego province, were resistant to third-generation cephalosporins (TGCs). These isolates were resistant to cefotaxime (91%) and/or ceftazidime (28%). No resistance to carbapenems was detected. Twenty-six isolates were positive for blaCTX-M gene, grouped as CTX-M-1/15 (54%); CTX-M-9/14 (25%); CTX-M-2 (17%); and CTX-M-1/15 plus CTX-M-9/14 (4%). Five TGC-resistant strains were positive for blaCMY gene, while one strain harbored TEM-19 ESBL. Twelve isolates were identified as ST131 E. coli hyperepidemic clone, and one as ST69. Genome sequence analysis of seven blaCTX-M-15 E. coli selected isolates confirm the circulation of ST131, ST617 and ST405 international high-risk clones in the city of Ushuaia.

Resumen Escherichia coli es uno de los principales patógenos humanos causantes de diferentes infecciones de inicio hospitalario y comunitario. Se determinó que el 1,96% (32/1.632) de los aislamientos de E. coli recuperados entre enero de 2013 y marzo de 2015 en el Hospital Regional de Ushuaia, provincia de Tierra del Fuego, fueron resistentes a cefalosporinas de tercera generación (CTG). Estos aislamientos fueron resistentes a cefotaxima (91%) y/o a ceftazidima (28%). No se detectó resistencia a los carbapenemes. Veintiséis aislamientos fueron positivos para el gen blaCTX-M, agrupados como CTX-M-1/15 (54%), CTX-M-9/14 (25%), CTX-M-2 (17%) y CTX-M-1/15 más CTX-M-9/14 (4%). Cinco cepas resistentes a CTG dieron positivo para el gen blaCMY, mientras que un aislamiento presentó la BLEE TEM-19. Doce aislamientos se identificaron como clon hiperepidémico E. coli ST131 y uno como ST69. El análisis de las secuencias del genoma de siete aislamientos seleccionados de E. coli blaCTX-M-15 confirmó la circulación de los clones internacionales de alto riesgo ST131, ST617 y ST405 en la ciudad de Ushuaia.

Characterization of third generation cephalosporin-resistant Escherichia coli clinical isolates from Ushuaia, Argentina.

Escherichia coli is one of the main human pathogens causing different hospital- and community-acquired infections. During the period from January 2013 to March 2015, 1.96% (32/1632) of E. coli isolates recovered at the Hospital Regional de Ushuaia, Tierra del Fuego province, were resistant to third-generation cephalosporins (TGCs). These isolates were resistant to cefotaxime (91%) and/or ceftazidime (28%). No resistance to carbapenems was detected. Twenty-six isolates were positive for blaCTX-M gene, grouped as CTX-M-1/15 (54%); CTX-M-9/14 (25%); CTX-M-2 (17%); and CTX-M-1/15 plus CTX-M-9/14 (4%). Five TGC-resistant strains were positive for blaCMY gene, while one strain harbored TEM-19 ESBL. Twelve isolates were identified as ST131 E. coli hyperepidemic clone, and one as ST69. Genome sequence analysis of seven blaCTX-M-15E. coli selected isolates confirm the circulation of ST131, ST617 and ST405 international high-risk clones in the city of Ushuaia.

Emergence of KPC-31, a KPC-3 Variant Associated with Ceftazidime-Avibactam Resistance, in an Extensively Drug-Resistant ST235 Pseudomonas aeruginosa Clinical Isolate.

A ceftazidime-avibactam-resistant KPC-producing Pseudomonas aeruginosa strain was isolated in Argentina from a tracheal aspirate. The patient was treated with ceftazidime-avibactam in combination with other agents for 130 days. Whole-genome sequencing of P. aeruginosa identified a D179Y substitution in the Ω loop of KPC-3, corresponding to KPC-31, integrated at the chromosome. The strain belonged to the sequence type 235/O11 (ST235/O11) high-risk clone. Evaluation of carbapenemase detection assays most used by clinical laboratories failed to identify the isolate as a KPC producer.

A New Twist: The Combination of Sulbactam/Avibactam Enhances Sulbactam Activity against Carbapenem-Resistant Acinetobacter baumannii (CRAB) Isolates.

An increasing number of untreatable infections are recorded every year. Many studies have focused their efforts on developing new ß-lactamase inhibitors to treat multi-drug resistant (MDR) isolates. In the present study, sulbactam/avibactam and sulbactam/relebactam combination were tested against 187 multi-drug resistant (MDR) Acinetobacter clinical isolates; both sulbactam/avibactam and sulbactam/relebactam restored sulbactam activity. A decrease ≥2 dilutions in sulbactam MICs was observed in 89% of the isolates when tested in combination with avibactam. Sulbactam/relebactam was able to restore sulbactam susceptibility in 40% of the isolates. In addition, the susceptibility testing using twenty-three A. baumannii AB5075 knockout strains revealed potential sulbactam and/or sulbactam/avibactam target genes. We observed that diazabicyclooctanes (DBOs) ß-lactamase inhibitors combined with sulbactam restore sulbactam susceptibility against carbapenem-resistant Acinetobacter clinical isolates. However, relebactam was not as effective as avibactam when combined with sulbactam. Exploring novel combinations may offer new options to treat Acinetobacter spp. infections, especially for widespread oxacillinases and metallo-ß-lactamases (MBLs) producers.

Simple Phenotypic Tests To Improve Accuracy in Screening Chromosomal and Plasmid-Mediated Colistin Resistance in Gram-Negative Bacilli.

CLSI and EUCAST recommend that only broth microdilution (BMD) should be used for routine colistin susceptibility testing; however, this technique can be difficult to perform in resource-poor settings. The purpose of this study was to evaluate the accuracy of a colistin agar spot test (COL-AS) and a colistin drop test (COL-DT) compared to BMD. COL-AS and COL-DT were assessed with a collection of 271 Gram-negative bacilli clinical isolates: 195 Enterobacterales (including 63 mcr-1 positive strains), 37 Acinetobacter spp., and 39 Pseudomonas aeruginosa For COL-AS, 3.0 µg/ml (final concentration) of colistin was added to a Mueller-Hinton agar plate and subsequently swabbed with a 0.5 McFarland standard suspension of the tested strain within a 1 cm2 spot. For COL-DT, 10 µl of a 16 µg/ml colistin solution was dripped on the surface of a Mueller-Hinton agar plate, previously inoculated with a lawn of the tested strain (0.5 McFarland standard). Colistin solution was made either by dissolving powder or by disk elution in cation-adjusted Mueller-Hinton broth (CA-MHB). Overall, 141/271 (52%) isolates were categorized as colistin resistant by reference BMD. COL-AS yielded a categorical agreement (CA) of 95.5% compared to BMD, with 0.7% very major errors and 3.8% major errors. COL-DT yielded a CA of 96.2% compared to BMD, with 0.7% and 0% very major errors and 3.1% and 3.8% major errors, for colistin powder and disk elution solutions, respectively. Most major errors occurred for mcr-1 strains with MICs that fluctuated from 2 to 4 µg/ml according to the method used. In conclusion, we developed and validated methods suited to the systematic screening of resistance to colistin in Gram-negative bacilli.

Plasmidic resistance to colistin mediated by mcr-1 gene in Escherichia coli clinical isolates in Argentina: A retrospective study, 2012-2018.

OBJECTIVE: To describe the resistance profile and the genetic characteristics of Escherichia coli isolates that harbor the mobilizable colistin resistance gene mcr-1 in Argentina. METHODS: This was a retrospective study of 192 E. coli isolates positive for mcr-1 obtained from 69 hospitals of Buenos Aires City and 14 Argentinean provinces in 2012 - 2018. The antimicrobial susceptibility was performed by agar diffusion, broth macrodilution, and/or agar dilution. Standard polymerase chain reaction (PCR) was performed to detect resistance genes and incompatibility groups; specific PCR was applied to discriminate between blaCTX-M allelic groups and mcr-1.5 variant. The genetic relatedness among isolates was evaluated by XbaI-pulsed field gel electrophoresis and multilocus sequence typing in a subset of isolates. RESULTS: All E. coli isolates showed minimal inhibitory concentrations to colistin ≥ 4µg/mL; nearly 50% were resistant to third-generation cephalosporins, with CTX-M-2 being the main extended-spectrum ß-lactamase detected. Five E. coli were carbapenemase-producers (3 NDM, 2 KPC). The mcr-1.5 variant was detected in 13.5% of the isolates. No genetic relationship was observed among the mcr-1-positive E. coli clinical isolates, but a high proportion (164/192; 85.4%) of IncI2 plasmids was detected. CONCLUSIONS: The presence of IncI2 plasmids among highly diverse E. coli clones suggests that the mcr-1 gene's wide distribution in Argentina may be driven by the horizontal transmission of IncI2 plasmids.

Plasmid carrying mcr-9 from an extensively drug-resistant NDM-1-producing Klebsiella quasipneumoniae subsp. quasipneumoniae clinical isolate.

The extensively drug-resistant NDM-1-producing Klebsiella quasipneumoniae subsp. quasipneumoniae M17277 (Kqsq-M17277) was only susceptible to colistin and fosfomycin. Whole genome sequencing of Kqsq-M17277 was performed through Illumina (short reads) and Oxford Nanopore Technologies (long reads). Hybrid assembly of short and long reads rendered circular contigs of the Kqsq-M17277 chromosome and three plasmids of 477,340 (p17277A_477), 138,998 (p17277C_138, which harbored blaNDM-1) and 123,307 bp (p17277B_123). In silico analysis showed that p17277A_477 was not previously described, belonged to the IncHI2 incompatibility group and contained 51 complete or partial (≥300 bp) insertion sequences and Tn3 family transposons, which encompassed, as a whole, 14% of the p17277A_477 sequence. In silico search for antimicrobial resistance genes revealed that p17277A_477 harbored the recently described plasmid-encoded mcr-9 gene (colistin resistance), besides other resistance genes. This is the first worldwide report of a K. quasipneumoniae harboring mcr-9 and blaNDM-1 and the first report of mcr-9 in Latin America. This gene was embedded in a genetic structure, i.e., IS903B-like/mcr-9/wbuC/IS26, that was found in 71% of the mcr-9-harboring sequences of the NCBI Nucleotide Collection database. The qseC and qseB genes involved in mcr-9 expression were not found in Kqsq-M17277, which could explain its colistin susceptibility. Fifteen additional resistance genes were detected in p17277A_477. Ten of them [aac(6')-IIc, aadA2, aph(3')-Ia, blaDHA-1, dfrA12, ereA2, mphA, qnrB4, sul1 and tet(D)] were found clustered within a 44,907-bp-long array of IS26-bounded resistance genes. The plasmid-mediated quinolone resistance gene qnrB4 was located in a complex class 1 integron within this IS26-bounded resistance gene cluster. p17277A_477 was self-transferable by conjugation to Escherichia coli J53, azide-resistant. The Tra1 and Tra2 conjugative transfer regions of the IncHI2 plasmid R478 were found by in silico search. p17277A_477 is a complex plasmid that provides a large baggage of both resistance genes and genetic resources for plasmid rearrangements and additional resistance gene acquisition.

Distinct Mechanisms of Dissemination of NDM-1 Metallo-ß-Lactamase in Acinetobacter Species in Argentina.

A 4-year surveillance of carbapenem-resistant Acinetobacter spp. isolates in Argentina identified 40 strains carrying blaNDM-1 Genome sequencing revealed that most were Acinetobacter baumannii, whereas seven represented other Acinetobacter spp. The A. baumannii genomes were closely related, suggesting recent spread. blaNDM-1 was located in the chromosome of A. baumannii strains and on a plasmid in non-A. baumannii strains. A resistance gene island carrying blaPER-7 and other resistance determinants was found on a plasmid in some A. baumannii strains.

Multidrug-resistant Escherichia coli harbouring mcr-1 and blaCTX-M genes isolated from swine in Argentina.

OBJECTIVES: Multidrug-resistant Escherichia coli isolates recovered from food-producing animals are a global public-health concern, especially those with transferable mechanisms of antimicrobial resistance such as extended-spectrum ß-lactamase (ESBL) and mcr-1 genes. Here we report for the first time E. coli recovered from diarrhoeic and healthy pigs harbouring blaCTX-M and/or mcr-1 from Argentinean farms. METHODS: During 2017, a total of 34 E. coli were recovered from 31 faecal samples from diarrhoeic piglets and healthy fattening pigs from five pig farms in three Argentinean provinces. Antimicrobial susceptibility was evaluated by agar diffusion and resistance genes were identified by PCR. Multiplex PCR was applied to screen for ST69, ST73, ST95 and ST131 clones. Genetic relationships were evaluated by XbaI-PFGE. RESULTS: A high diversity of resistance profiles was observed (20 profiles among 34 isolates), and 71% of isolates were multidrug-resistant. Resistance to third-generation cephalosporins (3GCs) was observed in 28 isolates and was associated with blaCTX-M (24), blaCMY (3) and blaPER-2 (1) genes. blaCTX-M alleles were grouped by specific PCR as follow: 17 blaCTX-M-8/25; 4 blaCTX-M-1/15; 2 blaCTX-M-2; and 1 blaCTX-M-9/14. Twelve isolates were positive for mcr-1, of which six were also resistant to 3GCs and were positive for blaCTX-M-8/25 (4), blaCTX-M-1/15 (1) or blaCMY (1). High genetic diversity was observed, discriminating 29 profiles. One ST131 and two ST95 human-associated clones were detected. CONCLUSION: Here we describe E. coli isolates recovered from diarrhoeic piglets and healthy fattening pigs harbouring ESBL and/or mcr-1 genes. 3GC resistance was mainly associated with CTX-M, in particular with blaCTX-M-8/25 alleles.

Characterization of a multidrug resistant Citrobacter amalonaticus clinical isolate harboring blaNDM-1 and mcr-1.5 genes.

A multidrug resistant isolate, identified as Citrobacter amalonaticus using MALDI-TOF MS and confirmed by genomic analysis, was recovered from a pediatric patient in a hospital from Buenos Aires, Argentina. By whole-genome sequencing a total of 16 resistance genes were detected, including blaNDM-1 and mcr-1.5. To the best of our knowledge this is the first description of these two genes together in a clinical isolate of the Citrobacter genus.

Multiple clones of metallo-ß-lactamase-producing Acinetobacter ursingii in a children hospital from Argentina.

Acinetobacter spp. are opportunistic pathogens being A. baumannii the most frequently identified in nosocomial settings. A. ursingii was mainly described as causing bacteremia and outbreaks in neonatal intensive care units. Ten A. ursingii isolates were recovered from rectal swab screening for carbapenemase-producing bacteria between June 2013 and December 2015 from a children hospital in Argentina. All ten isolates were metallo-ß-lactamase-producing, nine were positive for blaIMP-1 and one for blaNDM-1. IMP-positive isolates were also positive for blaOXA-58 gene. All isolates were susceptible to ciprofloxacin, colistin and minocycline, and nine were susceptible to ampicillin-sulbactam and gentamicin. Two A. ursingii displayed high level of resistance to aztreonam associated with blaCTX-M-15 in one isolate, and blaVEB-1 in the other. Eight SmaI-PFGE patterns were recognized. We evaluated the usefulness of Acinetobacter MLST-Pasteur scheme, to analyse A. ursingii isolates, however the rpoB gene was not amplified. A new set of primers were designed for specific amplification and sequencing, allowing the analysis of rpoB gene for this species. New alleles and the sequence types 748, 749, 750, 751, 993, 1186, 1187, and 1189 were included at the Acinetobacter MLST-Pasteur database. Those isolates showing related PFGE patterns were assigned to the same ST. To the best of our knowledge, this is the first report of MBL-producing A. ursingii in Argentina. The inclusion of A. ursingii species to the Acinetobacter MLST-Pasteur scheme allows deeper molecular characterization and a better understanding about the epidemiology of this germen.

Resistencia a colistín mediado por el gen mcr-1 identificado en cepas de Escherichia coli y Klebsiella pneumoniae: primeros reportes en el Perú / Resistance to colistin mediated by the mcr-1 gene identified in strains of Escherichia coli and Klebsiella pneumoniae: first reports in Peru

Introducción. Ante la aparición de reportes de la presencia del gen mcr-1 y su posible diseminación por plásmidos en los países de la región y dado que este gen confiere resistencia a colistín, fármaco que es la última línea de tratamiento contra bacterias multirresistentes, es importante conocer su presencia en nuestro país en microorganismos que lo expresen. Métodos. Se realizó un estudio descriptivo y transversal. Se incluyeron microorganismos aislados de urocultivos de pacientes ambulatorios de un centro de salud privado en Lima, Perú, en agosto del año 2017. De 326 urocultivos positivos se seleccionaron 10 aislamientos entre cepas de Escherichia coli y Klebsiella pneumoniae que presentaron concentración mínima inhibitoria ≥ 4µg/ mL (interpretado como resistente para colistín) por el sistema automatizado Microscan Walkaway 96 plus. Se utilizaron los siguientes métodos: colistín agar spot, predifusión con tabletas de colistín, microdilución en caldo colistin y PCR para el gen mcr-1. Resultados. Se determinó que 7 aislamientos, todas Escherichia coli, expresaron la presencia del gen mcr-1 por PCR, el cual confiere resistencia plasmídica a polipéptidos. De las cepas restantes, dos Escherichia coli y una Klebsiella pneumoniae, resultaron positivos para resistencia a colistín en las pruebas fenotípicas pero no en la PCR para gen mcr-1 lo cual sugiere un mecanismo de resistencia a colistín no asociado a gen mcr-1. Conclusiones. Se obtuvieron 7 aislamientos de Escherichia coli resistentes a colistín y con expresión del gen mcr-1.

Introduction. Given the appearance of reports of the presence of the mcr-1 gene and its possible dissemination by plasmids in the countries of the region and given that this gene confers resistance to colistin, the drug that is the last line of treatment against multiresistant bacteria, it is important to know its presence in our country in microorganisms that express it. Methods. Descriptive and cross-sectional study was carried out. Microorganisms isolated from urine culture of outpatients from a private health center in Lima, Peru, were included in august 2017. Out of 326 positive urine cultures, 10 isolates were selected between strains of Escherichia coli and Klebsiella pneumoniae that had a minimum inhibitory concentration ≥4µg/mL (interpreted as resistant for colistín) by the automated system Microscan Walkaway 96 plus. The following methods were used: colistin agar spot, prediffusion with colistin tablets, microdilution in colistin broth and PCR for the mcr-1 gene. Results. It was determined that 7 isolates, all Escherichia coli, expressed the presence of the mcr-1 gene by PCR, which confers plasmid resistance to polypeptides. Of the remaining strains, two Escherichia coli and one Klebsiella pneumoniae, were positive for resistance to colistin in the phenotypic tests but not in the PCR for mcr-1 gene, which suggests a mechanism of colistin resistance not associated with the mcr-1 gene. Conclusions. Seven isolates of Escherichia coli resistant to colistin and with expression of the mcr-1 gene were obtained.

Genetic Diversity of KPC-Producing Escherichia coli, Klebsiella oxytoca, Serratia marcescens, and Citrobacter freundii Isolates from Argentina.

The predominance of Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae was caused by the spread of ST258 clone. In Latin America, KPC was reported in 2006, with the isolation of genetically unrelated K. pneumoniae in Colombia. Since then, the expansion of blaKPC in either K. pneumoniae ST258 or other Enterobacteriaceae (ETB) species was increasingly reported. In this study, we characterized 89 KPC-producing Escherichia coli, Klebsiella oxytoca, Serratia marcescens, and Citrobacter freundii that were received between 2010 and 2014. The results revealed that all isolates harbored blaKPC-2. Moreover, the dissemination of KPC by non-K. pneumoniae was mainly caused by the dispersion of ETB mostly genetically unrelated. E. coli is a community pathogen that may serve as the vehicle for the spread of KPC into community settings. Recently, KPC was detected in E. coli ST131, an international epidemic and multidrug-resistant clone. We found that 5/29 KPC-producing E. coli belonged to ST131 and four were blaCTXM-15 producers. The detection of blaKPC in ST131 should be closely monitored to prevent further dissemination. The blaKPC is generally located within Tn4401 transposon capable of mobilization through transposition found in plasmids in ST258. Less is known about the diversity of blaKPC genetic elements that disseminate horizontally among other species of ETB. We found that 16/29 E. coli and 2/18 S. marcescens harbored blaKPC-2 in Tn4401a. In 71 isolates, blaKPC-2 was located amidst diverse Tn3-derived genetic elements bearing non-Tn4401 structure. Further studies on the plasmids that encode blaKPC-2 in these clinical isolates may provide additional insight into its transmission mechanisms.

Molecular characteristics of mcr-1-carrying plasmids and new mcr-1 variant recovered from polyclonal clinical Escherichia coli from Argentina and Canada.

We have characterized nine mcr-1-harboring plasmids from clinical Escherichia coli isolates previously described in Argentina and Canada. Three of these plasmids carried a mcr-1-variant called here mcr-1.5. All these E. coli isolates were not clonally related and were recovered in different years and locations. However, their mcr-1-harboring plasmids showed high identity among them and to others characterized in other countries, which strongly suggests that this plasmid-type is playing an important role in spreading this mechanism of resistance to polymyxins.