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Acquired antibiotic resistance in bacteria has become an important worldwide challenge. Currently, several bacteria, including Escherichia coli, have multidrug resistance profiles. Genes such as bla CTX-M-24 and bla KPC-2 (carbapenemase) are widespread. This research letter reports about a genomic surveillance study where multidrug-resistant E. coli containing CTX-M-24(IncF [F-:A1:B32]) and KPC-2(IncX3/IncU) plasmids were obtained from community- acquired urinary tract infection in Brazil.

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Chryseobacterium indologenes is a non-glucose-fermenting Gram-negative bacillus. This emerging multidrug resistant opportunistic nosocomial pathogen can cause severe infections in neonates and immunocompromised patients. This study aimed to present the first detailed draft genome sequence of a multidrug-resistant C. indologenes strain isolated from the cerebrospinal fluid of an infant hospitalized at the Neonatal Intensive Care Unit of Brazilian Tertiary Hospital. We first analyzed the susceptibility of C. indologenes strain to different antibiotics using the VITEK 2 system. The strain demonstrated an outstanding resistance to all the antibiotic classes tested, including ß-lactams, aminoglycosides, glycylcycline, and polymyxin. Next, C. indologenes was whole-genome-sequenced, annotated using Prokka and Rapid Annotation using Subsystems Technology (RAST), and screened for orthologous groups (EggNOG), gene ontology (GO), resistance genes, virulence genes, and mobile genetic elements using different software tools. The draft genome contained one circular chromosome of 4,836,765 bp with 37.32% GC content. The genomic features of the chromosome present numerous genes related to cellular processes that are essential to bacteria. The MDR C. indologenes revealed the presence of genes that corresponded to the resistance phenotypes, including genes to ß-lactamases (bla IND-13, bla CIA-3, bla TEM-116, bla OXA-209, bla VEB-15), quinolone (mcbG), tigecycline (tet(X6)), and genes encoding efflux pumps which confer resistance to aminoglycosides (RanA/RanB), and colistin (HlyD/TolC). Amino acid substitutions related to quinolone resistance were observed in GyrA (S83Y) and GyrB (L425I and K473R). A mutation that may play a role in the development of colistin resistance was detected in lpxA (G68D). Chryseobacterium indologenes isolate harbored 19 virulence factors, most of which were involved in infection pathways. We identified 13 Genomic Islands (GIs) and some elements associated with one integrative and conjugative element (ICEs). Other elements linked to mobile genetic elements (MGEs), such as insertion sequence (ISEIsp1), transposon (Tn5393), and integron (In31), were also present in the C. indologenes genome. Although plasmids were not detected, a ColRNAI replicon type and the most resistance genes detected in singletons were identified in unaligned scaffolds. We provided a wide range of information toward the understanding of the genomic diversity of C. indologenes, which can contribute to controlling the evolution and dissemination of this pathogen in healthcare settings.

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During a microbiological and genomic surveillance study conducted to investigate the molecular epidemiology of extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli from community-acquired urinary tract infections (UTI) and commercial meat samples, in a Brazilian city with a high occurrence of infections by ESBL-producing bacteria, we have identified the presence of CTX-M (-2, -14, -15, -24, -27 and -55)-producing E. coli of international clones ST38, ST117, ST131 and ST354. The ST131 was more prevalent in human samples, and worryingly the high-risk ST131-C1-M27 was identified in human infections for the first time. We also detected CTX-M-55-producing E. coli ST117 from meat samples (i.e., chicken and pork) and human infections. Moreover, the clinically relevant CTX-M-24-positive E. coli ST354 clone was detected for the first time in human samples. In summary, our results highlight a potential of commercialized meat as a reservoir of high-priority E. coli lineages in the community, whereas the identification of E. coli ST131-C1-M27 indicates that novel pandemic clones have emerged in Brazil, constituting a public health issue.

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The dissemination of carbapenem-resistant and third generation cephalosporin-resistant pathogens is a critical issue that is no longer restricted to hospital settings. The rapid spread of critical priority pathogens in Brazil is notably worrying, considering its continental dimension, the diversity of international trade, livestock production, and human travel. We conducted a nationwide genomic investigation under a One Health perspective that included Escherichia coli strains isolated from humans and nonhuman sources, over 45 years (1974-2019). One hundred sixty-seven genomes were analyzed extracting clinically relevant information (i.e., resistome, virulome, mobilome, sequence types [STs], and phylogenomic). The endemic status of extended-spectrum ß-lactamase (ESBL)-positive strains carrying a wide diversity of blaCTX-M variants, and the growing number of colistin-resistant isolates carrying mcr-type genes was associated with the successful expansion of international ST10, ST38, ST115, ST131, ST354, ST410, ST648, ST517, and ST711 clones; phylogenetically related and shared between human and nonhuman hosts, and polluted aquatic environments. Otherwise, carbapenem-resistant ST48, ST90, ST155, ST167, ST224, ST349, ST457, ST648, ST707, ST744, ST774, and ST2509 clones from human host harbored blaKPC-2 and blaNDM-1 genes. A broad resistome to other clinically relevant antibiotics, hazardous heavy metals, disinfectants, and pesticides was further predicted. Wide virulome associated with invasion/adherence, exotoxin and siderophore production was related to phylogroup B2. The convergence of wide resistome and virulome has contributed to the persistence and rapid spread of international high-risk clones of critical priority E. coli at the human-animal-environmental interface, which must be considered a One Health challenge for a post-pandemic scenario. IMPORTANCE A One Health approach for antimicrobial resistance must integrate whole-genome sequencing surveillance data of critical priority pathogens from human, animal and environmental sources to track hot spots and routes of transmission and developing effective prevention and control strategies. As part of the Grand Challenges Explorations: New Approaches to Characterize the Global Burden of Antimicrobial Resistance Program, we present genomic data of WHO critical priority carbapenemase-resistant, ESBL-producing, and/or colistin-resistant Escherichia coli strains isolated from humans and nonhuman sources in Brazil, a country with continental proportions and high levels of antimicrobial resistance. The present study provided evidence of epidemiological and clinical interest, highlighting that the convergence of wide virulome and resistome has contributed to the persistence and rapid spread of international high-risk clones of E. coli at the human-animal-environmental interface, which must be considered a One Health threat that requires coordinated actions to reduce its incidence in humans and nonhuman hosts.

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Klebsiella pneumoniae is an opportunistic pathogen that can cause several infections, mainly in hospitalised or immunocompromised individuals. The spread of K. pneumoniae emerging virulent and multidrug-resistant clones is a worldwide concern and its identification is crucial to control these strains especially in hospitals. This article reports data related to multi-resistant K. pneumoniae strains, isolated from inpatients in the city of Manaus, Brazil, harbouring virulence and antimicrobial-resistance genes, including high-risk international clones belonging to clonal group (CG) 258. Twenty-one strains isolated from different patients admitted to four hospitals in the city of Manaus, located in the state of Amazonas, Northern Brazil (Amazon Rainforest region) were evaluated. The majority of strains (61.9% n = 13) were classified as multidrug-resistant (MDR), and five strains (23.8%) as extensively drug-resistant (XDR). Several virulence and antimicrobial-resistance genes were found among the strains and eight strains (38.1%) presented the hyper-mucoviscous phenotype. MLST analysis demonstrated a great diversity of STs among the strains, totaling 12 different STs (ST11, ST23, ST198, ST277, ST307, ST340, ST378, ST462, ST502, ST3991, ST3993 and ST5209). Three of these (ST11, ST23 and ST340) belong to CG258.

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Hemodialysis patients are at high risk for bloodstream infections associated with highest morbidity and mortality rates. Bacterial species not commonly related to such infections has been hardly identified by traditional methods. Pseudocitrobacter is a novel genus of the order Enterobacterales that is associated with carbapenemase genes and nosocomial infection. In this context, we have investigated nine cases of bloodstream infections by carbapenem-resistant Gram-negative bacilli in patients assisted at a hemodialysis unit in Brazil. The infections were caused by a metallo-ß-lactamase (IMP-1)-producing clone (> 90% XbaI-PFGE similarity) of Pseudocitrobacter vendiensis, displaying a multidrug-resistant profile to broad-spectrum cephalosporins, carbapenems, chloramphenicol, and trimethoprim-sulfamethoxazole. S1-PFGE and Southern blot hybridization revealed that blaIMP-1 was carried by a 200-kb IncC/ST3 plasmid. Patients were successfully treated with amikacin, and strict disinfection procedures and hand washing protocols were reinforced. We report the emergence of P. vendiensis, a recently described species of the genus, in bloodstream infections of patients undergoing hemodialysis. Considering the epidemic potential of carbapenemase-producing Enterobacterales in hospital settings, surveillance of this emerging pathogen is of utmost importance.

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The increasing prevalence and dissemination of carbapenemase-producing Enterobacterales represent a serious concern for public health. We studied the genetic features of a multidrug-resistant isolate of high-risk clone ST147 Klebsiella pneumoniae coharboring mcr-1 and blaNDM-1 recovered from a human clinical urine sample in 2017 in Peru. Whole-genome sequencing and conjugation assays identified mcr-1 and blaNDM-1 genes on two different conjugative plasmids, which belong to IncI2 and IncFIB/HI1B incompatibility groups, respectively. The presence of blaCTX-M-15 (in the studied isolate, located on the chromosome) and mutations in GyrA S83I and ParC S80I were detected, as expected for ST147. In addition, other ß-lactamases (blaTEM-26 and blaOXA-1) and PMQR (qnrE2 and aac(6')-Ib-cr) among several resistance determinants were identified. The coexistence not previously described of these genes in the same high-risk clone is a cause for serious concern that supports the need for implementation of genomic surveillance studies.

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WGS-based surveillance has significantly improved the ability to track global spread and emergence of multidrug-resistant clones of clinically relevant pathogens. In this study, we performed the genomic characterization and comparative analysis of an Acinetobacter baumannii (strain Ac56) belonging to the sequence type ST374, which was isolated for the first time in Brazil, in 1996. Genomic analysis of Ac56 predicted a total of 5373 genes, with 3012 being identical across nine genomes of A. baumannii isolates of ST374 from European, Asian, North and South American countries. GoeBURST analysis grouped ST374 lineages into clonal complex CC3 (international clone IC-III). Resistome analysis of ST374 clone predicted genes associated with resistance to heavy metals and clinically relevant beta-lactams and aminoglycosides antibiotics. In this regard, in two closely related A. baumannii strains, the intrinsic blaADC gene was linked to the insertion sequence ISAba1; including the Ac56 strain, where it has been possibly associated with intermediate susceptibility to meropenem. Other four carbapenem-resistant A. baumannii strains carried the ISAba1/blaOXA-23 gene array, which was associated with the transposon Tn2008 or with Tn2006 in an AbaR4-type resistance island. While most virulence genes were shared for A. baumannii strains of ST374, three isolates from Thailand harbored KL49 capsular loci, previously identified in the hypervirulent A. baumannii LAC-4 strain. Analysis of thirty-four predicted plasmids showed eight major groups, of which GR-6 (LN-1) and GR-2 (LN-2) were prevalent. All strains, including the earliest isolate Ac56 harbored at least one complete prophage, whereas none CRISPR-associated (cas) gene was detected. In summary, genomic data of A. baumannii ST374 reveal a potential of this lineage to become a successful clone.

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ABSTRACT Acquired antibiotic resistance in bacteria has become an important worldwide challenge. Currently, several bacteria, including Escherichia coli, have multidrug resistance profiles. Genes such as bla CTX-M-24 and bla KPC-2 (carbapenemase) are widespread. This research letter reports about a genomic surveillance study where multidrug-resistant E. coli containing CTX-M-24(IncF [F-:A1:B32]) and KPC-2(IncX3/IncU) plasmids were obtained from community- acquired urinary tract infection in Brazil.

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Klebsiella variicola is mainly associated with opportunistic infections and frequently identified as Klebsiella pneumoniae. This misidentification implies a wrong epidemiology result as well as incorrect attribution to K. pneumoniae as the etiology of some severe infections. Recently, huge efforts have been made to study K. variicola, however, the biological aspects of this species are still unclear. Here we characterized five K. variicola strains initially identified as K. pneumoniae, with a Vitek-2 System and 16S rRNA sequencing. One-step multiplex polymerase chain reaction and Whole Genome Sequencing (WGS) identified them as K. variicola. Additionally, WGS analysis showed that all the strains are closely related with K. variicola genomes, forming a clustered group, apart from K. pneumoniae and K. quasipneumoniae. Multilocus sequence typing analysis showed four different sequence types (STs) among the strains and for two of them (Kv97 and Kv104) the same ST was assigned. All strains were multidrug-resistant (MDR) and three showed virulence phenotypes including invasion capacity to epithelial cells, and survival in human blood and serum. These results showed the emergence of new K. variicola clones with pathogenic potential to colonize and cause infection in different tissues. These characteristics associated with MDR strains raise great concern for human health.

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The global spread of antibiotic-resistant bacteria and their resistance genes is a critical issue that is no longer restricted to hospital settings, but also represents a growing problem involving environmental and food safety. In this study, we have performed a microbiological and genomic investigation of critical priority pathogens resistant to broad-spectrum cephalosporins and showing endophytic lifestyles in fresh vegetables sold in a country with high endemicity of extended-spectrum ß-lactamases (ESBLs). We report the isolation of international high-risk clones of CTX-M-15-producing Escherichia coli, belonging to clonal complexes CC38 and CC648, and Klebsiella pneumoniae of complex CC307 from macerated tissue of surface-sterilized leaves of spinach, cabbage, arugula, and lettuce. Regardless of species, all ESBL-positive isolates were able to endophytically colonize common bean (Phaseolus vulgaris) seedlings, showed resistance to acid pH, and had a multidrug-resistant (MDR) profile to clinically relevant antibiotics (i.e., broad-spectrum cephalosporins, aminoglycosides, and fluoroquinolones). Genomic analysis of CTX-M-producing endophytic Enterobacterales revealed a wide resistome (antibiotics, biocides, disinfectants, and pesticides) and virulome, and genes for endophytic fitness and for withstanding acidic conditions. Transferable IncFIB and IncHI2A plasmids carried bla CTX-M-15 genes and, additionally, an IncFIB plasmid (named pKP301cro) also harbored genes encoding resistance to heavy metals. These data support the hypothesis that fresh vegetables marketed for consumption can act as a figurative Trojan horse for the hidden spread of international clones of critical WHO priority pathogens producing ESBLs, and/or their resistance genes, to humans and other animals, which is a critical issue within a food safety and broader public and environmental health perspective.IMPORTANCE Extended-spectrum ß-lactamases (ESBL)-producing Enterobacterales are a leading cause of human and animal infections, being classified as critical priority pathogens by the World Health Organization. Epidemiological studies have shown that spread of ESBL-producing bacteria is not a problem restricted to hospitals, but also represents a growing problem involving environmental and food safety. In this regard, CTX-M-type ß-lactamases have become the most widely distributed and clinically relevant ESBLs worldwide. Here, we have investigated the occurrence and genomic features of ESBL-producing Enterobacterales in surface-sterilized fresh vegetables. We have uncovered that international high-risk clones of CTX-M-15-producing Escherichia coli and Klebsiella pneumoniae harboring a wide resistome and virulome, carry additional genes for endophytic fitness and resistance to acidic conditions. Furthermore, we have demonstrated that these CTX-M-15-positive isolates are able to endophytically colonize plant tissues. Therefore, we believe that fresh vegetables can act as a figurative Trojan horse for the hidden spread of critical priority pathogens exhibiting endophytic lifestyles.

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The emergence and rapid spread of carbapenemase-producing Enterobacterales represents a serious public health concern. Critically, these global priority bacteria have begun to be reported in companion animals, implying a potential risk of cross-transmission between humans and pets. Using long-read (MinION) and short-read (Illumina) sequencing technologies, we have identified and characterized a hypermucoviscous KPC-2-producing Klebsiella pneumoniae strain belonging to the high-risk international clone ST11/CG258, in a dog with urinary tract infection. Strikingly, the blaKPC-2 gene was carried by a 54-kb IncN plasmid assignated to ST15, which shared 99.8 and 96.8% pairwise identity with IncN-pST15 plasmids from human and environmental K. pneumoniae strains, respectively; all come from an area with high endemicity of KPC-2. Our findings suggest that IncN-pST15 plasmids conferring carbapenem resistance can play as important a role as clonal transmission of K. pneumoniae, representing another major challenge for One Health.

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Serratia fonticola is a human pathogen widely found in the environment, with birds being reported as possible natural hosts. During an epidemiological and genomic surveillance study conducted to monitor the occurrence of extended-spectrum ß-lactamase (ESBL)-producing Enterobacterales in South American wild birds, we identified an ESBL-positive S. fonticola in a fecal sample collected from a Hudsonian Whimbrel, during its non-breeding range on the Pacific Coast of Chile. Whole genome sequencing analysis and "in silico" modeling revealed a novel variant of the class A ESBLs FONA family, designated FONA-7, which shows 96.28% amino acid identity with FONA-6; with amino acid substitutions occurring in the signal peptide sequence (Thr22→Ser), and in the mature protein (Ser39→Asn and Thr227→Ile). This finding denotes that migratory birds can be potential vectors for the transboundary spread of ESBL-producing bacteria, creating a further theoretical risk for the origin of novel plasmid-encoded ß-lactamases.

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This study used whole-genome sequencing to analyze the first case of NDM-1-producing Acinetobacter baumannii belonging to the novel sequence type 1465/CC216 recovered in Brazil. The study identified an unusual plasmid carrying blaNDM-1 gene, in which some genes of the Tn125 transposon were lost. Besides, on the chromosome, the strain reported here presented blaOXA-106 gene, a variant of blaOXA-51 gene, and blaADC-25 with ISAba1 upstream. The isolation of new STs of A. baumannii carrying blaNDM-1 genes elicits our concerns about the possible spread of these genes among clinically relevant bacteria.

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The emergence of invasive Haemophilus influenzae infections in vaccinated patient is a public health concern. We have investigated the genomic basis of invasiveness and possible vaccine failure in H. influenzae causing invasive disease in vaccinated and unvaccinated children in Brazil. Three H. influenzae strains isolated from blood cultures of pediatric patients were sequenced. Serotype, MLST, resistome and virulome were predicted using bioinformatic tools, whereas single nucleotide polymorphisms (SNPs) analysis of cap loci and the presence of the putative virulence-enhancing IS1016-bexA partial deletion were predicted in silico. Infections were caused by H. influenzae type a (Hia), type b (Hib) and nontypeable (NTHi), belonging to international high-risk clones of sequence types ST23, ST6 and ST368, respectively, which have been identified in North American, European and Asian countries. Convergence of ampicillin resistance and virulence in Hib-ST6 was supported by blaTEM-1B and deletion in the bexA gene, whereas presence of SNPs in the cap-b locus was associated with antigenic modifications of the capsule structure. Hia-ST23 and NTHi-ST368 strains carried galU, lpsA, opsX, rfaF, iga1, lgtC and lic1/lic2 virulence genes, associated with colonization, adaptation and damage to the lung, or invasiveness. In summary, deletion in the bexA gene and presence of SNPs in the cap locus of Hib could be contributing to invasive disease and possible vaccine failure in pediatric patients, whereas serotype replacement of Hib with type "a" and NTHi strains denotes the ability of non-vaccine serotypes to re-colonize vaccinated patients. Finally, the dissemination of international high-risk clones of H. influenzae emphasizes the importance of monitoring changes in the molecular epidemiology of invasive H. influenzae disease.

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Integrative conjugative elements (ICEs) are widespread in many bacterial species, often carrying antibiotic resistance determinants. In the present work, we screened a collection of Proteus mirabilis clinical isolates for the presence of type 1 SXT/R391 ICEs. Among the 76 isolates analyzed, 5 of them carry such elements. The complete sequences of these elements were obtained. One of the isolates carried the CMY-2 beta-lactamase gene in a transposon and is nearly identical to the element ICEPmiJpn1 previously described in Japan, and later shown to be present in other parts of the world, indicating global spread of this element. Nevertheless, the Brazilian isolate carrying ICEPmiJpn1 is not clonally related to the other lineages carrying the same element around the world. The other ICEs identified in this work do not carry known antibiotic resistance markers and are diverse in variable gene content and size, suggesting that these elements may be responsible for the acquisition of other advantageous traits by bacteria. Some sequences carried by these elements in Brazilian strains were not previously found in other SXT/R391 variants.

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A multidrug-resistant CTX-M-15-producing Klebsiella pneumoniae (KpP1 strain) was isolated from a native Amazonian fish (Brachyplatystoma filamentosum) at the Brazilian Amazon. The strain was identified by MALDI-TOF. The genome was extracted, purified and a Nextera DNA Flex library was prepared and sequenced by Illumina platform. The sequenced genome was de novo assembled using Unicycler and in silico prediction accomplished by curated bioinformatics tools. The size of the genome is 5.6 Mb with 5715 genes. Whole-genome sequencing analysis revealed the presence of wide resistome, with genes conferring resistance to clinically relevant antibiotics, heavy metals and disinfectants. The KpP1 strain was assigned to the sequence type ST3827, KL111 (wzi113) and O3b locus. Native freshwater fish sold in wet markets of the Amazonian region could be an important vehicle for transmission of multidrug-resistant bacteria to humans. This study may give genomic insights on the spread of critical-priority WHO pathogens in a One Health context.

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OBJECTIVE: Carbapenemase-producing Acinetobacter baumannii has been recognized as a critical priority pathogen by the World Health Organization. We hereby report the identification and the draft genome sequence of a carbapenem-resistant A. baumannii isolated from a patient with community-onset urinary tract infection, in a Chilean Patagonian city. METHODS: The whole genome was sequenced on an Illumina NextSeq platform and de novo assembled using Unicycler v.0.4. Resistome analysis and epidemiological investigation (based on MLST data and Pasteur scheme) were performed using bioinformatics tools available from the Center for Genomic Epidemiology. RESULTS: The genome size was calculated at 3890824 bp, with a GC content of 39.1%, comprising 3864 total genes, 30 tRNAs, 3 rRNAs, 4 ncRNAs, and 109 pseudogenes. Carbapenem-resistant A. baumannii Ab3_Ch strain belonged to the international sequence type ST15 (clonal complex, CC15), and harboured the ISAba-1-blaOXA-219 gene array, along to blaTEM-1B and blaADC-6 ß-lactamase genes, and aac(3)-IIa and aph(3')-VIa aminoglycoside resistance genes. Additionally, efflux pump encoding genes (abaF, abaQ, abeS, adeI, adeK, adeL, adeN, adeR, adeS, and amvA) were identified, and mutations in the quinolone resistance-determining region of gyrA (Ser81Leu) and parC (Ser84Leu) were considered responsible for fluoroquinolone resistance. CONCLUSION: This genome sequence data could be used for comparative genomic studies of critical priority A. baumannii strains, as well as to understand the specific features of hospital-associated A. baumannii lineages of international clonal complexes emerging in community settings.

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