Analysis of distinct blaKPC-encoding plasmids in an Enterobacter kobei strain recovered from recreational coastal water.

In this study we present an in-depth characterization of two blaKPC-2 encoding plasmids found in the Enterobacter kobei FL23 strain recovered from recreational coastal water. The plasmids belong to distinct incompatibility groups and carry a diverse collection of resistance genes. Furthermore, the genetic context of the blaKPC-2 gene was different in each of them. While pEkFL23-IncX3 presents a new Tn4401k, a new isoform, similar to Tn4401b but with a truncated tnpA and a deleted tnpR; pEkFL23-IncU/P6 carries a new isoform of a non-Tn4401 element (NTEKPC), named NTEKPC-IIh. Its difference from NTEKPC-IId is the truncated Tn3 resolvase upstream blaKPC-2. Capacity of conjugation, maintenance rates and fitness cost of both replicons were also assessed. Both were transferred after mating assays, whereas only pEkFL23-IncX3 was transferred under the adverse conditions of Marine broth at 25 °C as a mating platform. A remarkable stability of both plasmids was observed in the parental and transconjugant strains. Finally, both replicons did not impose a significant fitness cost to their transformant hosts, with pEkFL23-IncU/P6 conferring a statistically significant (p < 0.05) advantage in head-to-head competitions. Our findings show that E. kobei FL23 is a disquieting case of a carbapenem-resistant bacteria identified in a community setting, being a possible silent disseminator of two seemingly stable and metabolic weightless multidrug resistance plasmids.

qnrVC occurs in different genetic contexts in Klebsiella and Enterobacter strains isolated from Brazilian coastal waters.

OBJECTIVES: In contrast to other qnr families, qnrVC has been reported mainly in Vibrio spp. and inserted in class 1 integrons. This study aimed to identify the variants of qnrVC genes detected in Klebsiella pneumoniae carbapenemase-2-producing Enterobacter and Klebsiella strains isolated from Brazilian coastal waters and the genetic contexts associated with their occurrence. METHODS: qnrVC variants were identified by Sanger sequencing. Stains were typified by pulsed-field gel electrophoresis. Antimicrobial susceptibility testing, conjugation assays, and whole genome sequencing (WGS) were applied to identify the strains' antimicrobial resistance profile, qnrVC and blaKPC-2 co-transference, and qnrVC genetic context. RESULTS: qnrVC1 was identified in 15 Enterobacter and 3 Klebsiella, and qnrVC4 in 2 Enterobacter strains. Pulsed-field gel electrophoresis revealed 12 clonal profiles of Enterobacter and one of Klebsiella. Strains were resistant to aminoglycosides, beta-lactams, fosfomycin, quinolones, and sulfamethoxazole-trimethoprim. Co-transference of qnrVC and blaKPC-2 were obtained from five representative Enterobacter strains, which showed resistance to ampicillin and amoxicillin-clavulanate, and reduced susceptibility to extended-spectrum cephalosporins, meropenem, and ciprofloxacin. WGS analysis from representative strains revealed one K. quasipneumoniae subsp. similipneumoniae, one E. soli, four E. kobei, and seven isolates belonging to Enterobacter Taxon 3. Long-read WGS showed qnrVC and blaKPC-2 were carried by the same replicon on Klebsiella and Enterobacter strains, and the qnrVC association with not previously described genetic environments composed of insertion sequences and truncated genes. These contexts occurred in small- and high-molecular-weight plasmids belonging to IncFII, IncP6, pKPC-CAV1321, and IncU groups. CONCLUSION: Our results suggest that the dissemination of qnrVC among Enterobacterales in Brazilian coastal waters is associated with several genetic recombination events.

Description of a new non-Tn4401 element (NTEKPC-IIe) harboured on IncQ plasmid in Citrobacter werkmanii from recreational coastal water.

OBJECTIVES: Here we describe an IncQ1-like plasmid carrying blaKPC-2 in a new non-Tn4401 element found in Citrobacter werkmanii recovered from coastal water. METHODS: In vitro and in silico approaches were used to assess antimicrobial resistance determinants, as well as blaKPC-2 vicinities. RESULTS: The LB-887 isolate showed a multidrug-resistant phenotype and was identified as C. werkmanii. Resistome analysis identified further acquired resistance determinants to ß-lactams, aminoglycosides, sulphonamides/trimethoprim, tetracyclines, chloramphenicol, macrolides, rifampicin and fluoroquinolones. Plasmidome included incompatibility groups IncA, IncC2, IncR, Col and IncQ families. The blaKPC-2 was inserted on a new variant of NTEKPC-II, called here NTEKPC-IIe, carried by an InQ1-like plasmid of 7930 kb (pKPC-LB887). NTEKPC-IIe differed from NTEKPC-IId by the complete absence of ISKpn6-tnpA. The InQ1-like backbone harbouring this element had been described in Enterobacterales recovered from clinical and environmental settings. CONCLUSION: Unravelling genetic structures related to blaKPC dissemination in different settings may provide clues on the main forces driving evolution of this important resistance determinant. Indeed, the occurrence of blaKPC in a new NTEKPC variant from an environmental source highlights the ongoing evolution of this mobile genetic element. In addition, blaKPC carriage on a small and highly mobilizable IncQ plasmid in C. freundii complex from recreational water, similar to others found in clinical isolates, may suggest its relevance for blaKPC-2 dissemination among different compartments.

shsA: A novel orthologous of sasX/sesI virulence genes is detected in Staphylococcus haemolyticus Brazilian strains.

The surface protein SasX, has a key role in methicillin-resistant Staphylococcus aureus (MRSA) colonization and pathogenesis, and has been associated with the epidemic success of some MRSA clones. To date, only one SasX homologous protein, named SesI, has been described in Staphylococcus epidermidis. In this work, we analyze the occurrence of the sasX gene and its genetic environment in Staphylococcus haemolyticus S. haemolyticus clinical strains (n = 62) were screened for the presence of the sasX gene and its carrier, the prophage Φ SPß-like. A deep characterization was done in one strain (MD43), through which we determined the complete nucleotide sequence for the S. haemolitycus sasX-like gene. Whole genome sequencing of strain MD43 was performed, and the gene, termed here because of its unique attributes, shsA, was mapped to the Φ SPß-like prophage sequence. The shsA gene was detected in 33 out of 62 strains showing an average identity of 92 and 96% with the sasX and sesI genes and at the amino acid level, 88% identity with SasX and 92% identity with SesI. The ~124Kb Φ SPß-like prophage sequence showed a largely intact prophage compared to its counterpart in S. epidermidis strain RP62A, including the sesI insertion site. In conclusion, we identified a new sasX ortholog in S. haemolyticus (shsA). Its horizontal spread from this reservoir could represent an emergent threat in healthcare facilities since so far, no S. aureus sasX+ strains have been reported in Brazil.

Colistin resistance emerges in pandrug-resistant Klebsiella pneumoniae epidemic clones in Rio de Janeiro, Brazil.

Klebsiella pneumoniae is an important human pathogen, able to accumulate and disseminate a variety of antimicrobial resistance genes. Resistance to colistin, one of the last therapeutic options for multi-drug-resistant bacteria, has been reported increasingly. Colistin-resistant K. pneumoniae (ColRKp) emerged in two hospitals in Rio de Janeiro state, Brazil in 2016. The aim of this study was to investigate if these ColRKp isolates were clonally related when compared between hospitals, to identify the molecular mechanisms of colistin resistance, and to describe other antimicrobial resistance genes carried by isolates. Twenty-three isolates were successively recovered, and the whole-genome sequence was analysed for 10, each of a different pulsed-field gel electrophoresis (PFGE) type. Although some PFGE clusters were found, none of them included isolates from both hospitals. Half of the isolates were assigned to CC258, three to ST152 and two to ST15. One isolate was pandrug resistant, one was extensively drug resistant, and the others were multi-drug resistant. Colistin resistance was related to mutations in mgrB, pmrB, phoQ and crrB. Eleven new mutations were found in these genes, including two nucleotide deletions in mgrB. All isolates were carbapenem resistant, and seven were associated with carbapenemase carriage (blaKPC-2 in six isolates and blaOXA-370 in one isolate). All isolates had a blaCTX-M, and two had a 16S ribosomal RNA methyltransferase encoding gene (armA and rmtB). ColRKp were composed of epidemic clones, but cross-dissemination between hospitals was not detected. Colistin resistance emerged with several novel mutations amid highly resistant strains, further restricting the number of drugs available and leading to pandrug resistance.