Genomic characterization of New Delhi metallo-beta-lactamase-producing species of Morganellaceae, Yersiniaceae, and Enterobacteriaceae (other than Klebsiella) from Brazil over 2013-2022.

Over the last decade, New Delhi metallo-beta-lactamase (NDM) carbapenemase has silently spread in Brazil. In this study, we analyzed a large collection of Enterobacterales other than Klebsiella spp. received in our reference laboratory between 2013 and 2022. A total of 32 clinical isolates displaying different pulsed-field gel electrophoresis profiles, and represented by 11 species in the families Enterobacteriaceae (Citrobacter freundii, Citrobacter portucalensis, Enterobacter hormaechei, and Escherichia coli), Morganellaceae (Morganella morganii, Proteus mirabilis, Proteus vulgaris, Providencia rettgeri, Providencia stuartii, and Raoultella ornithinolytica), and Yersiniaceae (Serratia marcescens) had their whole genomes sequenced and further analyzed. Antimicrobial susceptibility was determined by disk diffusion, except for polymyxin B, assessed by broth microdilution. The blaNDM-1 allele was predominant (n = 29), but blaNDM-5 was identified in an E. coli specimen with a novel ST, and the blaNDM-7 allele was found in E. hormaechei ST45 and E. coli ST1049. Polymyxin was active against all but one Enterobacteriaceae isolate: an mcr-1-producing E. coli presenting minimal inhibitory concentration (4 mg/L). Isolates producing extended-spectrum ß-lactamases were common: cefotaximase from Munich (CTX-M)-15 (n = 10), CTX-M-2 (n = 4), and CTX-M-8 (n = 3) were detected, and the mcr-1-producing E. coli was found to co-produce both CTX-M-8 and CTX-M-55 ß-lactamases. The mcr-9 gene was found in 5/8 E. hormaechei isolates, distributed in four different sequence types, all of them presenting susceptibility to polymyxin. This study showed that NDM-producing Enterobacterales other than Klebsiella are already spread in Brazil, in diversified species, and cocarrying important resistance genes. Prompt detection and effective implementation of measures to prevent further spread are mandatory for mitigating the dissemination of NDM carbapenemase in hospital settings and preserving the already limited antimicrobial therapy options.

Novel Metallo-ß-Lactamase blaCVI-1 Isolated from a Chromobaterium violaceum Clinical Strain Resistant to Colistin.

OBJECTIVE: We aimed to describe a colistin (COL)-resistant (R) Chromobacterium violaceum (Cvi) isolate from a septic patient in Argentina expressing a previously unknown gene, blaCVI-1. METHODS: In 2019, a 12 year old child was injured with a thorn in a lagoon. The child was hospitalized due to sepsis and multiple abscesses. Cvi was isolated from skin and soft tissue and tracheal aspirate. The patient was successfully treated with imipenem (IMI) plus amikacin. Antimicrobial susceptibility was assessed by disk diffusion, broth microdilution, and the E-test. Carbapenemase activity was assayed by double-disk synergy and microbiological tests. Resistance, virulence, and additional gene searches were performed by in silico analysis of sequences obtained by whole-genome sequencing (WGS). A maximum likelihood phylogenetic tree was built with public Cvi genomes. RESULTS: R was seen for IMI and COL. Expression of a metallo-ß-lactamase was confirmed. Genome analysis revealed blaCVI-1, a subclass B2 metallo-ß-lactamase with 62.66% ID with CphA from A. hydrophila (WP081086394). R to COL could be attributed to the arnC and arnT genes. Virulence factors required for invasion and toxicity were also found. No plasmids were detected. The phylogeny tree showed two main clades with geographical distinction, and the isolate studied here stands alone in a branch closely related to two clinical isolates from the USA. CONCLUSIONS: This is the second report of infection by Cvi in Argentina. This pathogen carried a new gene, blaCVI-1, a metallo-ß-lactamase that can be detected by routine methods. Prompt suspicion of C. violaceum infection is crucial to treating this rare pathogen rapidly and properly.

Genomic characterization of new Delhi metallo-beta-lactamase-producing species of Morganellaceae, Yersiniaceae, and Enterobacteriaceae (other than Klebsiella) from Brazil over 2013-2022

Over the last decade, New Delhi metallo-beta-lactamase (NDM) carbapenemase has silently spread in Brazil. In this study, we analyzed a large collection of Enterobacterales other than Klebsiella spp. received in our reference laboratory between 2013 and 2022. A total of 32 clinical isolates displaying different pulsed-field gel electrophoresis profiles, and represented by 11 species in the families Enterobacteriaceae (Citrobacter freundii, Citrobacter portucalensis, Enterobacter hormaechei, and Escherichia coli), Morganellaceae (Morganella morganii, Proteus mirabilis, Proteus vulgaris, Providencia rettgeri, Providencia stuartii, and Raoultella ornithinolytica), and Yersiniaceae (Serratia marcescens) had their whole genomes sequenced and further analyzed. Antimicrobial susceptibility was determined by disk diffusion, except for polymyxin B, assessed by broth microdilution. The blaNDM-1 allele was predominant (n = 29), but blaNDM-5 was identified in an E. coli specimen with a novel ST, and the blaNDM-7 allele was found in E. hormaechei ST45 and E. coli ST1049. Polymyxin was active against all but one Enterobacteriaceae isolate: an mcr-1–producing E. coli presenting minimal inhibitory concentration (4 mg/L). Isolates producing extended-spectrum β-lactamases were common: cefotaximase from Munich (CTX-M)-15 (n = 10), CTX-M-2 (n = 4), and CTX-M-8 (n = 3) were detected, and the mcr-1–producing E. coli was found to co-produce both CTX-M-8 and CTX-M-55 β-lactamases. The mcr-9 gene was found in 5/8 E. hormaechei isolates, distributed in four different sequence types, all of them presenting susceptibility to polymyxin. This study showed that NDM-producing Enterobacterales other than Klebsiella are already spread in Brazil, in diversified species, and cocarrying important resistance genes. Prompt detection and effective implementation of measures to prevent further spread are mandatory for mitigating the dissemination of NDM carbapenemase in hospital settings and preserving the already limited antimicrobial therapy options.

Increased frequency of blaNDM in a tertiary care hospital in southern Brazil.

Resistance to carbapenems due to metallo-beta-lactamase NDM-1 was first described in Brazil in 2013. To date, only a few scattered reports of the prevalence of NDM-1 in the country have been reported, and most of them indicated a very low prevalence of this metalloenzyme. In the present study, we report a steady increase in the frequency of NDM among Enterobacterales resistant to carbapenems in a tertiary care hospital in southern Brazil. Carbapenemase genes were evaluated by multiplex real-time polymerase chain using high-resolution melting analysis among 3501 isolates of 8 different species of Enterobacterales recovered from January 2015 to May 2020. The blaKPC-like was identified in 3003 isolates (85.8%) and the blaNDM-like was the second most common gene (351 isolates-10%). There was a steady increase in frequency of blaNDM-like, from 4.2% in 2015 to 24% in 2020. The increase of blaNDM frequency raises an important matter as novel therapeutic options are currently very limited for the treatment of patients infected by bacteria carrying the blaNDM.

LMB-1 producing Citrobacter freundii from Argentina, a novel player in the field of MBLs.

Carbapenemase-producing Enterobacterales expressing OXA-48, KPC, NDM, VIM or IMP enzymes are increasingly reported worldwide. We have characterized LMB-1, a novel metallo-ß-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 ß-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 ß-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.

Occurrence of IMP-1 in non-baumannii Acinetobacter clinical isolates from Brazil.

The aim of this study was to characterize the presence of carbapenemase-encoding genes in distinct species of Acinetobacter spp. isolated from Brazilian hospitals. Five carbapenem-resistant Acinetobacter spp. isolates (two Acinetobacter pittii, two Acinetobacter bereziniae and one Acinetobacter junii) recovered from two distinct hospitals between 2000 and 2016 were included in this study. All of the isolates harboured blaIMP-1, which was inserted into In86, a class 1 integron. Pulsed field gel eletrophoresis analysis showed that both A. pittii were identical, while the two A. berezinae isolates were considered to be clonally related. In this study, we demonstrated that mobile elements carrying carbapenemase-encoding genes such as In86 may persist for a long period, allowing their mobilization from A. baumannii to other Acinetobacter spp. that are usually susceptible to multiple antimicrobials.

Spread of Clonally Related Escherichia coli Strains Harboring an IncA/C1 Plasmid Encoding IMP-8 and Its Recruitment into an Unrelated MCR-1-Containing Isolate.

Ten IMP-8-producing Escherichia coli isolates were recovered from surveillance cultures of a neonatal intensive care unit; eight of the isolates were clonally related. A 168.2-kb blaIMP-8 plasmid was fully sequenced, and it corresponded to the recently described IncA/C1-ST13 plasmid. This plasmid was detected in all isolates, even in those that were not clonally related. One unrelated isolate was also resistant to colistin and positive for mcr-1 This marker was located in a 62.7-kb IncI2 plasmid, which was also fully sequenced.

Shaping Substrate Selectivity in a Broad-Spectrum Metallo-ß-Lactamase.

Metallo-ß-lactamases (MBLs) are the major group of carbapenemases produced by bacterial pathogens. The design of MBL inhibitors has been limited by, among other issues, incomplete knowledge about how these enzymes modulate substrate recognition. While most MBLs are broad-spectrum enzymes, B2 MBLs are exclusive carbapenemases. This narrower substrate profile has been attributed to a sequence insertion present in B2 enzymes that limits accessibility to the active site. In this work, we evaluate the role of sequence insertions naturally occurring in the B2 enzyme Sfh-I and in the broad-spectrum B1 enzyme SPM-1. We engineered a chimeric protein in which the sequence insertion of SPM-1 was replaced by the one present in Sfh-I. The chimeric variant is a selective cephalosporinase, revealing that the substrate profile of MBLs can be further tuned depending on the protein context. These results also show that the stable scaffold of MBLs allows a modular engineering much richer than the one observed in nature.

Genomic Epidemiology of NDM-1-Encoding Plasmids in Latin American Clinical Isolates Reveals Insights into the Evolution of Multidrug Resistance.

Bacteria that produce the broad-spectrum Carbapenem antibiotic New Delhi Metallo-ß-lactamase (NDM) place a burden on health care systems worldwide, due to the limited treatment options for infections caused by them and the rapid global spread of this antibiotic resistance mechanism. Although it is believed that the associated resistance gene blaNDM-1 originated in Acinetobacter spp., the role of Enterobacteriaceae in its dissemination remains unclear. In this study, we used whole genome sequencing to investigate the dissemination dynamics of blaNDM-1-positive plasmids in a set of 21 clinical NDM-1-positive isolates from Colombia and Mexico (Providencia rettgeri, Klebsiella pneumoniae, and Acinetobacter baumannii) as well as six representative NDM-1-positive Escherichia coli transconjugants. Additionally, the plasmids from three representative P. rettgeri isolates were sequenced by PacBio sequencing and finished. Our results demonstrate the presence of previously reported plasmids from K. pneumoniae and A. baumannii in different genetic backgrounds and geographically distant locations in Colombia. Three new previously unclassified plasmids were also identified in P. rettgeri from Colombia and Mexico, plus an interesting genetic link between NDM-1-positive P. rettgeri from distant geographic locations (Canada, Mexico, Colombia, and Israel) without any reported epidemiological links was discovered. Finally, we detected a relationship between plasmids present in P. rettgeri and plasmids from A. baumannii and K. pneumoniae. Overall, our findings suggest a Russian doll model for the dissemination of blaNDM-1 in Latin America, with P. rettgeri playing a central role in this process, and reveal new insights into the evolution and dissemination of plasmids carrying such antibiotic resistance genes.

Evolutionary Genetic Analysis Uncovers Multiple Species with Distinct Habitat Preferences and Antibiotic Resistance Phenotypes in the Stenotrophomonas maltophilia Complex.

The genus Stenotrophomonas (Gammaproteobacteria) has a broad environmental distribution. Stenotrophomonas maltophilia is its best known species because it is a globally emerging, multidrug-resistant (MDR), opportunistic pathogen. Members of this species are known to display high genetic, ecological and phenotypic diversity, forming the so-called S. maltophilia complex (Smc). Heterogeneous resistance and virulence phenotypes have been reported for environmental Smc isolates of diverse ecological origin. We hypothesized that this heterogeneity could be in part due to the potential lumping of several cryptic species in the Smc. Here we used state-of-the-art phylogenetic and population genetics methods to test this hypothesis based on the multilocus dataset available for the genus at pubmlst.org. It was extended with sequences from complete and draft genome sequences to assemble a comprehensive set of reference sequences. This framework was used to analyze 108 environmental isolates obtained in this study from the sediment and water column of four rivers and streams in Central Mexico, affected by contrasting levels of anthropogenic pollution. The aim of the study was to identify species in this collection, defined as genetically cohesive sequence clusters, and to determine the extent of their genetic, ecological and phenotypic differentiation. The multispecies coalescent, coupled with Bayes factor analysis was used to delimit species borders, together with population genetic structure analyses, recombination and gene flow estimates between sequence clusters. These analyses consistently revealed that the Smc contains at least 5 significantly differentiated lineages: S. maltophilia and Smc1 to Smc4. Only S. maltophilia was found to be intrinsically MDR, all its members expressing metallo-ß-lactamases (MBLs). The other Smc lineages were not MDR and did not express MBLs. We also obtained isolates related to S. acidaminiphila, S. humi and S. terrae. They were significantly more susceptible to antibiotics than S. maltophilia. We demonstrate that the sympatric lineages recovered display significantly differentiated habitat preferences, antibiotic resistance profiles and ß-lactamase expression phenotypes, as shown by diverse multivariate analyses and robust univariate statistical tests. We discuss our data in light of current models of bacterial speciation, which fit these data well, stressing the implications of species delimitation in ecological, evolutionary and clinical research.

Detection of blaVIM-7 in an extensively drug-resistant Pseudomonas aeruginosa isolate belonging to ST1284 in Brazil.

We described for the first time an extensively drug-resistant Pseudomonas aeruginosa isolate belonging to ST1284 carrying a plasmid-mediated blaVIM-7 in Brazil. The blaVIM-7 was harbored by an integron that also carried aacA4 and blaOXA-46. Multiple virulence factors were also detected.

Prevalence and antimicrobial susceptibility of non-fermenting Gram-negative bacilli isolated from clinical samples at a tertiary care hospital

Abstract INTRODUCTION: We compared the prevalence and antimicrobial susceptibility of non-fermenting gram-negative bacilli (NFGNB) isolated from clinical samples at a Brazilian tertiary care hospital in 2008 and 2013. METHODS: Collected data included patient's name, age, sex, inpatient unit, laboratory record number, type of biological material, culture test result, and antimicrobial susceptibility of isolated strains. RESULTS: Out of 19,112 culture tests analyzed, 926 (4.8%) were positive for NFGNB. Among these, 45.2% were metallo-beta-lactamase (MBL) producing strains. CONCLUSION: Between 2008 and 2013, the number of MBL-producing NFGNB isolates increased by 21.5%, which was accompanied by a consequent reduction in susceptibility to antimicrobials.

Identification of São Paulo metallo-beta-lactamase-1-producing Pseudomonas aeruginosa in the Central-West region of Brazil: a case study

Abstract: Metallo-beta-lactamase production is an important mechanism for carbapenem resistance of Pseudomonas aeruginosa , which represents an emerging public health challenge. We report the case of a patient admitted to an intensive care unit, with sepsis caused by multidrug-resistant São Paulo Metallo-beta-lactamase-1-producing P. aeruginosa . This is the first case of infection by this pathogenic strain in the State of Mato Grosso do Sul, Brazil. Thus, infection control measures are required for preventing future spread and outbreaks.

Molecular epidemiology of SPM-1-producing Pseudomonas aeruginosa by rep-PCR in hospitals in Parana, Brazil.

BACKGROUND: Infections caused by multidrug resistant microorganisms are a global health problem, and Pseudomonas aeruginosa is an important nosocomial pathogen, easily disseminated in the hospital environment. The aim of this study was to determine SPM-1 in P. aeruginosa strains in 30 Brazilian hospitals and the genetic similarity of isolates. METHODS: We analyzed 161 isolates of carbapenem-resistant P. aeruginosa. Imipenem/EDTA and imipenem strip were used for phenotypic detection of MBL production; and real-time polymerase chain reaction (PCR) for genetic detection. Genetic similarity was determined by rep-PCR. RESULTS: We obtained 136/161 (84.5%) isolates with positive phenotypic result for metallo-ß-lactamase (MBL) and the blaSPM-1 gene was identified in 41 isolates. There was a predominant profile (>95% of genetic similarity) in 92.7% of isolates. This predominant profile was widely disseminated in Paraná state. CONCLUSION: SPM-1 is the main MBL identified in carbapenem-resistant P. aeruginosa in Southern Brazil. The genetic similarity among some isolates suggests a clonal expansion.

Beta-lactamase Enzymes of Clinical Pseudomonas aeruginosa Strains.

OBJECTIVES: In this study, the production of extended spectrum beta-lactamase (ESBL), metallo-betalacatamase (MBL) and AmpC beta-lactamase enzymes of Pseudomonas aeruginosa (P aeruginosa) strains which were isolated from clinical samples were investigated. AmpC gene was also detected by the polymerase chain reaction (PCR) analysis. METHODS: A hundred strains of P aeruginosa were included in the study. The presence of ESBL was investigated with combined disk confirmation test, MBL was investigated with E-test method and AmpC beta-lactamase was investigated with disk induction test. In order to detect the production of AmpC betalactamase genotypically, the PCR method was used. RESULTS: Only one strain was found to be MBL positive. Four per cent of strains were found to be ESBL positive. AmpC beta-lactamase production was positive in 73% of the strains with disk induction test. AmpC gene was detected in 96% of the studied strains with the PCR method. CONCLUSION: While ESBL and MBL rates in this study were significantly lower than those found in other studies, the rate of AmpC beta-lactamase was higher. Although AmpC gene was detected in some strains (23%), they were not found to produce AmpC beta-lactamase with disk induction test.

Comparison of phenotypic tests for the detection of metallo-beta-lactamases in clinical isolates of Pseudomonas aeruginosa.

Metallo-ß-lactamase (MBL)-producing gram-negative bacteria are an increasing public health concern worldwide. Screening tests for the rapid and specific identification of these pathogens are essential, and should be included among routine diagnostics in laboratories. This study aimed to determine the MBL frequency among carbapenem-resistant Pseudomonas aeruginosa isolates, and to evaluate the accuracy of different tests in screening for MBL production. From January 2001 to December 2008, a total of 142 imipenem-non-susceptible P. aeruginosa strains were isolated from distinct clinical samples from hospitalized patients. These isolates were examined by PCR, MBL E-test, double-disk synergy test (DDST), and combined disk (CD) test. The minimal inhibitory concentration (MIC; µg/mL) was determined by agar dilution, and pulsed field gel electrophoresis (PFGE) was performed on all samples. Sequencing was performed to confirm and define the MBL variant and subtype. Using PCR and DNA sequence analysis, 93 strains were confirmed positive for MBLs, 91 strains for the blaSPM-1 gene, 1 strain for the blaIMP-1 gene, and 1 strain for the blaIMP-16 gene. PFGE displayed a clonal pattern. The sensitivities, specificities, positive and negative predictive values were evaluated for all tests. The DDST assay (CAZ-MPA) was the optimal method for screening MBL production in P. aeruginosa strains. However, the results of the CD assay (IMP/EDTA) showed close agreement with those of the DDST. In addition, the CD assay allowed a more objective interpretation and did not require the use of a toxic substance.

Metalo beta lactamasas: el fin de los beta lactámicos? / Metallo beta lactamases: the end of beta lactams?

Uno de los grupos de antibióticos más importantes, es el grupo de los beta-lactámicos. Para ejercer su acción antimicrobiana los beta-lactámicos requieren penetrar la pared celular y atacar las Proteínas Ligadoras de Penicilinas (Penicilin Binding Proteins: PBP). El mecanismo más utilizado por los bacilos Gram negativos para adquirir resistencia a beta-lactámicos, es la inactivación de las drogas por las enzimas beta-lactamasas. La producción de beta-lactamasas tipo carbapenemasas es un mecanismo de resistencia de gran importancia. Estas enzimas, codificadas por genes que en su mayoría están localizados en elementos genéticos tales como los integrones o insertados en elementos móviles como transposones y plásmidos, se han extendido rápidamente entre los agentes patógenos de importancia clínica, como Enterobacterias, P. aeruginosa y A. baumanii. Las metalo-beta-lactamasas (MbetaL) pertenecientes al grupo B de Ambler y el grupo 3 de Bush, poseen cuatro características principales: (i) Poseen actividad contra los carbapenemes, (ii) No hidrolizan los monobáctamicos como el aztreonam, (iii) Son inhibidas por quelantes como el EDTA o el Mercapto acetato de Sodio; y (iv) Requieren cationes +2 divalentes, generalmente Zn como cofactor para su actividad catalítica. La aproximación de un disco conteniendo un agente quelante a uno que contiene un carbapeneme podría resultar una herramienta útil para la detección de MbetaLs. El efecto sinérgico entre los carbapenemes [imipenem (IPM) y/o meropenem (MEN)] y el agente quelante sería indicativo de la presencia de MbetaL. La aparición de metalo-beta-lactamasas como una amenaza sustancial a la salud debe impulsar a las autoridades de salud para formular un plan de contención, para su implementación a nivel nacional. Este plan debe asegurar la detección temprana de casos, la vigilancia permanente de brotes y una estrategia en entornos con presencia espor dica o ausencia completa de productores metalo-beta-lactamasa.

One of the most important groups of antibiotics, is the group of beta-lactams. To exert its antimicrobial action the beta-lactam require penetrate and attack the cell wall Penicillin Binding Proteins (PBP). The mechanism used by Gram negative bacilli to acquire resistance tobeta-lactams, is drug inactivation by beta-lactamase enzymes. The production of beta-lactamase carbapenemases is a resistance mechanism of great importance. These enzymes encoded by genes that are located mostly in genetic elements such as integrons or inserted in mobile elements such as transposons and plasmids, have spread rapidly among clinically important pathogens such as Enterobacteriaceae, P. aeruginosa and A. baumannii. The metallo-beta-lactamases (MbetaL) in group B and group 3 Ambler Bush, have four main characteristics: (i) possess activity against carbapenems, (ii) not hydrolyze monobactams like aztreonam, (iii) are inhibited by chelating agents such as EDTA or sodium acetate Mercapto and (iv) require divalent cations, usually Zn+2 cofactor for its catalytic activity. The approximation of a disc containing a chelating agent containing one carbapeneme could be a useful tool for detecting MbetaLs. The synergistic effect between carbapenems [imipenem (IPM) and/or meropenem (MEN)] and the chelating agent would be indicative of the presence of MbetaL. The emergence of metallo-beta-lactamase as a substantial threat to health should prompt health officials to develop a plan of containment, for implementation at the national level. This plan should ensure early case detection, continuous surveillance of outbreaks and strategy in environments with sporadic presence or complete absence of metallo-beta-lactamase.