Genomic Epidemiology of Azithromycin-Nonsusceptible Neisseria gonorrhoeae, Argentina, 2005-2019.

Azithromycin-nonsusceptible Neisseria gonorrhoeae strains are an emerging global public health threat. During 2015-2018, the prevalence of azithromycin-nonsusceptible gonococcal infection increased significantly in Argentina. To investigate the genomic epidemiology and resistance mechanisms of these strains, we sequenced 96 nonsusceptible isolates collected in Argentina during 2005-2019. Phylogenomic analysis revealed 2 main clades, which were characterized by a limited geographic distribution, circulating during January 2015-November 2019. These clades included the internationally spreading multilocus sequence types (STs) 1580 and 9363. The ST1580 isolates, which had MICs of 2-4 µg/mL, had mutations in the 23S rRNA. The ST9363 isolates, which had MICs of 2-4 or >256 µg/mL, had mutations in the 23S rRNA, a mosaic mtr locus, or both. Identifying the geographic dissemination and characteristics of these predominant clones will guide public health policies to control the spread of azithromycin-nonsusceptible N. gonorrhoeae in Argentina.

[Evolution of the performance of Latin America Reference Laboratories in the detection of mechanisms of antimicrobial resistanceEvolução do desempenho dos Laboratórios de Referência na América Latina na detecção de mecanismos de resistência antimicrobiana]. / Evolución del desempeño de Laboratorios de Referencia de América Latina en la detección de mecanismos de resistencia a los antimicrobianos.

OBJECTIVE: The objective is to present the results of the Latin American Program for Quality Assurance in Bacteriology and Antimicrobial Resistance (LA-EQAS) between 2000 and 2018 and the evolution of the detection of resistance mechanisms with clinical impact. METHODS: The participating National Reference Laboratories (NRLs) received 25 surveys with 10 strains in each one, representing a total of 86 bacterial species and 40 resistance mechanisms. To evaluate the performance of the NRLs, five indicators were analyzed: bacterial identification, interpretation of susceptibility testing, acceptable ranges for zones of inhibition, inferred resistance mechanism, and delay time for the response. RESULTS: The average concordance was 82.6% (range: 74-95%) for bacterial identification, 93.3% (85-98%) for the interpretation of susceptibility testing, 84.6% (70-94%) for the zones of inhibition, and 82.5% (73-96%) for the inferred resistance mechanisms. The average delay time for the response was 34 days. Improvements in the detection of mechanisms of clinical importance, such as resistance to methicillin, macrolides and glycopeptides in Gram-positive cocci, and extended-spectrum, AmpC plasmid and carbapenemase beta-lactamases in Gram-negative bacilli, were observed. CONCLUSIONS: The LA-EQAS is an excellent tool for continuous quality improvement in the diagnosis of infections due to multiresistant microorganisms in NRLs in Latin America.

OBJETIVO: O objetivo deste trabalho é apresentar os resultados do Programa Latino-Americano de Garantia da Qualidade em Bacteriologia e Resistência Antimicrobiana (LA-EQAS, na sigla em inglês) entre 2000 e 2018 e a evolução na detecção de mecanismos de resistência com impacto clínico. MÉTODOS: Os Laboratórios Nacionais de Referência (LNRs) participantes receberam 25 inquéritos com 10 cepas bacterianas cada, representando um total de 86 espécies bacterianas e 40 mecanismos de resistência. Para avaliar o desempenho dos LNRs, foram analisados cinco indicadores: identificação bacteriana, interpretação dos testes de sensibilidade, faixas das zonas de inibição aceitáveis, mecanismo de resistência inferido e tempo de demora na resposta. RESULTADOS: A concordância média foi de 82,6% (intervalo: 74-95%) na identificação bacteriana, 93,3% (85-98%) na interpretação dos testes de sensibilidade, 84,6% (70-94%) nas zonas de inibição, 82,5% (73-96%) no mecanismo de resistência inferido e 34 dias na demora na resposta. Observou-se uma melhoria na detecção de mecanismos clinicamente relevantes, como a resistência a meticilina, macrolídeos e glicopeptídeos em cocos Gram-positivos, beta-lactamases de espectro ampliado, AmpC plasmídica e carbapenemases em bacilos Gram-negativos. CONCLUSÕES: O LA-EQAS é uma excelente ferramenta para a melhoria contínua da qualidade no diagnóstico de infecções por microrganismos multirresistentes nos LNRs da América Latina.

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.

Carbapenemase-Producing Extraintestinal Pathogenic Escherichia coli From Argentina: Clonal Diversity and Predominance of Hyperepidemic Clones CC10 and CC131.

Extraintestinal pathogenic Escherichia coli (ExPEC) causes infections outside the intestine. Particular ExPEC clones, such as clonal complex (CC)/sequence type (ST)131, have been known to sequentially accumulate antimicrobial resistance that starts with chromosomal mutations against fluoroquinolones, followed with the acquisition of bla CTX-M-15 and, more recently, carbapenemases. Here we aimed to investigate the distribution of global epidemic clones of carbapenemase-producing ExPEC from Argentina in representative clinical isolates recovered between July 2008 and March 2017. Carbapenemase-producing ExPEC (n = 160) were referred to the Argentinean reference laboratory. Of these, 71 were selected for genome sequencing. Phenotypic and microbiological studies confirmed the presence of carbapenemases confirmed as KPC-2 (n = 52), NDM-1 (n = 16), IMP-8 (n = 2), and VIM-1 (n = 1) producers. The isolates had been recovered mainly from urine, blood, and abdominal fluids among others, and some were from screening samples. After analyzing the virulence gene content, 76% of the isolates were considered ExPEC, although non-ExPEC isolates were also obtained from extraintestinal sites. Pan-genome phylogeny and clonal analysis showed great clonal diversity, although the first phylogroup in abundance was phylogroup A, harboring CC10 isolates, followed by phylogroup B2 with CC/ST131, mostly H30Rx, the subclone co-producing CTX-M-15. Phylogroups D, B1, C, F, and E were also detected with fewer strains. CC10 and CC/ST131 were found throughout the country. In addition, CC10 nucleated most metalloenzymes, such as NDM-1. Other relevant international clones were identified, such as CC/ST38, CC155, CC14/ST1193, and CC23. Two isolates co-produced KPC-2 and OXA-163 or OXA-439, a point mutation variant of OXA-163, and three isolates co-produced MCR-1 among other resistance genes. To conclude, in this work, we described the molecular epidemiology of carbapenemase-producing ExPEC in Argentina. Further studies are necessary to determine the plasmid families disseminating carbapenemases in ExPEC in this region.

[Capability of national reference laboratories in Latin America to detect emerging resistance mechanisms]. / Capacidad de los laboratorios nacionales de referencia en Latinoamérica para detectar mecanismos de resistencia emergentes.

OBJECTIVE: To evaluate the capability of 17 national reference laboratories participating in the Latin American Quality Control Program in Bacteriology and Antibiotic Resistance (LA-EQAS) to detect emerging resistance mechanisms- namely: resistance of enterobacteria to carbapenems due to the presence of Klebsiella pneumoniae carbapenemase (KPC) and metallo-beta-lactamase (MBL) type IMP, and intermediate resistance of Staphylococcus aureus isolates to vancomycin (vancomycin-intermediate resistant S. aureus-VISA). METHODS: The following three isolates were sent to the 17 participating LA-EQAS laboratories: KPC -producing Klebsiella pneumoniae PAHO-161, IMP-producing Enterobacter cloacae PAHO-166, and S. aureus PAHO-165 with intermediate resistance to vancomycin. Performance of each of the following operations was evaluated: interpretation of sensitivity tests, detection of the resistance mechanism, and assessment of either inhibition halo size (disk diffusion method) or minimum inhibitory concentration (MIC). RESULTS: Concordance in the detection of resistance mechanisms was 76.4%, 73.3%, and 66.7% for the K. pneumoniae PAHO-161, E. cloacae PAHO-166, and S. aureus PAHO-165 strains, respectively. Concordance between the inhibition areas observed by the participating laboratories and the ranges established by the coordinating laboratory was acceptable for all three isolates, at 90.8%, 92.8%, and 88.9%, respectively. CONCLUSIONS: Overall concordance in on the detection of KPC, MBL, and VISA resistance mechanisms was 72.1%. We consider the national reference laboratories in Latin America capable of recognizing these emerging resistance mechanisms and expect that maximum levels of concordance will be reached in the future.

Evolución del desempeño de Laboratorios de Referencia de América Latina en la detección de mecanismos de resistencia a los antimicrobianos

[RESUMEN]. Objetivo. El objetivo es presentar los resultados del Programa Latinoamericano de Aseguramiento de la Calidad en Bacteriología y Resistencia a los Antimicrobianos (LA-EQAS) entre 2000 y 2018 y la evolución en la detección de mecanismos de resistencia de impacto clínico. Métodos. Los Laboratorios Nacionales de Referencia (LNR) participantes recibieron 25 encuestas con 10 cepas cada una, representando un total de 86 especies bacterianas y 40 mecanismos de resistencia. Para evaluar el desempeño de los LNR, se analizaron cinco indicadores: identificación bacteriana, interpretación de las pruebas de sensibilidad, rangos de las zonas de inhibición aceptables, mecanismo de resistencia inferido, y tiempo de demora en la respuesta. Resultados. La concordancia media fue 82,6% (rango: 74-95%) en la identificación bacteriana, 93,3% (85-98%) en la interpretación de las pruebas de sensibilidad, 84,6% (70-94%) en las zonas de inhibición, 82,5% (73-96%) en el mecanismo de resistencia inferido, y la demora en la respuesta, 34 días. Se observó una mejora en la detección de mecanismos de relevancia clínica como resistencia a meticilina, macrólidos y glucopéptidos en cocos gram positivos, y betalactamasas de espectro extendido, AmpC plasmídico y carbapenemasas en bacilos gram negativos. Conclusiones. El LA-EQAS es una excelente herramienta para la mejora continua de la calidad en el diagnóstico de las infecciones por microorganismos multirresistentes en los LNR de América Latina.

[RESUMEN]. Objetivo. El objetivo es presentar los resultados del Programa Latinoamericano de Aseguramiento de la Calidad en Bacteriología y Resistencia a los Antimicrobianos (LA-EQAS) entre 2000 y 2018 y la evolución en la detección de mecanismos de resistencia de impacto clínico. Métodos. Los Laboratorios Nacionales de Referencia (LNR) participantes recibieron 25 encuestas con 10 cepas cada una, representando un total de 86 especies bacterianas y 40 mecanismos de resistencia. Para evaluar el desempeño de los LNR, se analizaron cinco indicadores: identificación bacteriana, interpretación de las pruebas de sensibilidad, rangos de las zonas de inhibición aceptables, mecanismo de resistencia inferido, y tiempo de demora en la respuesta. Resultados. La concordancia media fue 82,6% (rango: 74-95%) en la identificación bacteriana, 93,3% (85-98%) en la interpretación de las pruebas de sensibilidad, 84,6% (70-94%) en las zonas de inhibición, 82,5% (73-96%) en el mecanismo de resistencia inferido, y la demora en la respuesta, 34 días. Se observó una mejora en la detección de mecanismos de relevancia clínica como resistencia a meticilina, macrólidos y glucopéptidos en cocos gram positivos, y betalactamasas de espectro extendido, AmpC plasmídico y carbapenemasas en bacilos gram negativos. Conclusiones. El LA-EQAS es una excelente herramienta para la mejora continua de la calidad en el diagnóstico de las infecciones por microorganismos multirresistentes en los LNR de América Latina.

[RESUMO]. Objetivo. O objetivo deste trabalho é apresentar os resultados do Programa Latino-Americano de Garantia da Qualidade em Bacteriologia e Resistência Antimicrobiana (LA-EQAS, na sigla em inglês) entre 2000 e 2018 e a evolução na detecção de mecanismos de resistência com impacto clínico. Métodos. Os Laboratórios Nacionais de Referência (LNRs) participantes receberam 25 inquéritos com 10 cepas bacterianas cada, representando um total de 86 espécies bacterianas e 40 mecanismos de resistência. Para avaliar o desempenho dos LNRs, foram analisados cinco indicadores: identificação bacteriana, interpretação dos testes de sensibilidade, faixas das zonas de inibição aceitáveis, mecanismo de resistência inferido e tempo de demora na resposta. Resultados. A concordância média foi de 82,6% (intervalo: 74-95%) na identificação bacteriana, 93,3% (85-98%) na interpretação dos testes de sensibilidade, 84,6% (70-94%) nas zonas de inibição, 82,5% (73-96%) no mecanismo de resistência inferido e 34 dias na demora na resposta. Observou-se uma melhoria na detecção de mecanismos clinicamente relevantes, como a resistência a meticilina, macrolídeos e glicopeptídeos em cocos Gram-positivos, beta-lactamases de espectro ampliado, AmpC plasmídica e carbapenemases em bacilos Gram-negativos. Conclusões. O LA-EQAS é uma excelente ferramenta para a melhoria contínua da qualidade no diagnóstico de infecções por microrganismos multirresistentes nos LNRs da América Latina.

Laboratory-based prospective surveillance for community outbreaks of Shigella spp. in Argentina.

BACKGROUND: To implement effective control measures, timely outbreak detection is essential. Shigella is the most common cause of bacterial diarrhea in Argentina. Highly resistant clones of Shigella have emerged, and outbreaks have been recognized in closed settings and in whole communities. We hereby report our experience with an evolving, integrated, laboratory-based, near real-time surveillance system operating in six contiguous provinces of Argentina during April 2009 to March 2012. METHODOLOGY: To detect localized shigellosis outbreaks timely, we used the prospective space-time permutation scan statistic algorithm of SaTScan, embedded in WHONET software. Twenty three laboratories sent updated Shigella data on a weekly basis to the National Reference Laboratory. Cluster detection analysis was performed at several taxonomic levels: for all Shigella spp., for serotypes within species and for antimicrobial resistance phenotypes within species. Shigella isolates associated with statistically significant signals (clusters in time/space with recurrence interval ≥365 days) were subtyped by pulsed field gel electrophoresis (PFGE) using PulseNet protocols. PRINCIPAL FINDINGS: In three years of active surveillance, our system detected 32 statistically significant events, 26 of them identified before hospital staff was aware of any unexpected increase in the number of Shigella isolates. Twenty-six signals were investigated by PFGE, which confirmed a close relationship among the isolates for 22 events (84.6%). Seven events were investigated epidemiologically, which revealed links among the patients. Seventeen events were found at the resistance profile level. The system detected events of public health importance: infrequent resistance profiles, long-lasting and/or re-emergent clusters and events important for their duration or size, which were reported to local public health authorities. CONCLUSIONS/SIGNIFICANCE: The WHONET-SaTScan system may serve as a model for surveillance and can be applied to other pathogens, implemented by other networks, and scaled up to national and international levels for early detection and control of outbreaks.

Capacidad de los laboratorios nacionales de referencia en Latinoamérica para detectar mecanismos de resistencia emergentes / Capability of national reference laboratories in Latin America to detect emerging resistance mechanisms

Objetivo. Evaluar la capacidad de 17 laboratorios nacionales de referencia que participan en el Programa Latinoamericano de Control de Calidad en Bacteriología y Resistencia a los Antimicrobianos (LA-EQAS) para detectar mecanismos de resistencia emergentes, a saber: resistencia de enterobacterias a carbapenemes por presencia de Klebsiella pneumoniae carbapenemasa (KPC); resistencia de enterobacterias a carbapenemes por presencia de metalobetalactamasas (MBL) tipo IMP, y resistencia intermedia a vancomicina de aislamientos de Staphylococcus aureus (VISA). Métodos. Se enviaron los siguientes tres aislamientos a los 17 laboratorios participantes del LA-EQAS: Klebsiella pneumoniae OPS-161 productor de KPC, Enterobacter cloacae OPS-166 productor de IMP y S. aureus OPS-165 con resistencia intermedia a vancomicina. Se evaluó la interpretación de las pruebas de sensibilidad y detección del mecanismo de resistencia y el tamaño de los halos de inhibición (método de difusión por discos) o valor de la concentración inhibitoria mínima (CIM). Resultados. La concordancia en la detección de los mecanismos de resistencia fue de 76,4%, 73,3% y 66,7% con respecto a la cepas K. pneumoniae OPS-161, E. cloacae OPS-166 y S. aureus OPS-165, respectivamente. La concordancia entre las zonas de inhibición obtenidas por los laboratorios participantes y los rangos establecidos por el laboratorio coordinador fue aceptable en los tres aislamientos, ya que alcanzó 90,8%, 92,8% y 88,9%, respectivamente, para cada cepa. Conclusiones. La concordancia global en la detección de los mecanismos de resistencia KPC, MBL y VISA fue de 72,1%. Consideramos que los laboratorios nacionales de referencia de América Latina son capaces de reconocer estos mecanismos de resistencia emergentes y se espera que en el futuro la concordancia alcance su nivel máximo.

Objective. To evaluate the capability of 17 national reference laboratories participating in the Latin American Quality Control Program in Bacteriology and Antibiotic Resistance (LA-EQAS) to detect emerging resistance mechanisms— namely: resistance of enterobacteria to carbapenems due to the presence of Klebsiella pneumoniae carbapenemase (KPC) and metallo-beta-lactamase (MBL) type IMP, and intermediate resistance of Staphylococcus aureus isolates to vancomycin (vancomycinintermediate resistant S. aureus—VISA). Methods. The following three isolates were sent to the 17 participating LA-EQAS laboratories: KPC-producing Klebsiella pneumoniae PAHO-161, IMP-producing Enterobacter cloacae PAHO-166, and S. aureus PAHO-165 with intermediate resistance to vancomycin. Performance of each of the following operations was evaluated: interpretation of sensitivity tests, detection of the resistance mechanism, and assessment of either inhibition halo size (disk diffusion method) or minimum inhibitory concentration (MIC). Results. Concordance in the detection of resistance mechanisms was 76.4%, 73.3%, and 66.7% for the K. pneumoniae PAHO-161, E. cloacae PAHO-166, and S. aureus PAHO- 165 strains, respectively. Concordance between the inhibition areas observed by the participating laboratories and the ranges established by the coordinating laboratory was acceptable for all three isolates, at 90.8%, 92.8%, and 88.9%, respectively. Conclusions. Overall concordance in on the detection of KPC, MBL, and VISA resistance mechanisms was 72.1%. We consider the national reference laboratories in Latin America capable of recognizing these emerging resistance mechanisms and expect that maximum levels of concordance will be reached in the future.