Unraveling the genomic characteristics of a Klebsiella quasipneumoniae clinical isolate carrying blaNDM-1.

OBJECTIVE: Despite the increasing reports of blaNDM in Enterobacterales in Brazil, comprehensive whole genome sequencing (WGS) data remains scarce. To address this knowledge gap, our study focuses on the characterization of the genome of an NDM-1-producing Klebsiella quasipneumoniae subsp. quasipneumoniae (KQPN) clinical strain isolated in Brazil. METHODS: The antimicrobial susceptibility profile of the A-73.113 strain was performed by agar dilution or broth microdilution following the BrCAST/EUCAST recommendations. WGS was performed using the Illumina® NextSeq platform and the generated reads were assembled using the SPAdes software. The sequences obtained were submitted to the bioinformatics pipelines to determine the sequence type, resistome, plasmidome, and virulome. RESULTS: The A-73.113 strain was identified as KQPN and was susceptible to polymyxins (MICs, ≤0.25 µg/mL), tigecycline (MIC, 0.5 µg/mL), ciprofloxacin (MIC, 0.5 µg/mL), and levofloxacin (MIC, 1 µg/mL). WGS analysis revealed the presence of genes conferring resistance to ß-lactams (blaNDM-1, blaCTX-M-15, blaOXA-9, blaOKP-A-5, blaTEM-1), aminoglycosides [aph(3')-VI, aadA1, aac(6')-Ib], and fluoroquinolones (oqxAB, qnrS1, aac(6')-Ib-cr]. Additionally, it was verified the presence of the plasmid replicons Col(pHAD28), IncFIA(HI1), IncFIB(K) (pCAV1099-114), IncFIB(pQil), and IncFII(K), as well as virulence-encoding genes: fimABCDEFGHIK (type 1 fimbria), pilW (type IV pili), iutA (aerobactin), entABCDEFS/fepABCDG/fes (Ent siderophores), iroE (salmochelin), and allABCDRS (allantoin utilization). Furthermore, we found that A-73.113 strain belongs to ST1040. CONCLUSION: Here we report the genomic characteristics of an NDM-1-producing KQPN ST1040 strain isolated from blood culture in Brazil. These data will enhance our comprehension of how this species contributes to the acquisition and dissemination of blaNDM-1 in Brazilian nosocomial settings.

Unveiling novel threats: Urban river isolation of Aeromonas veronii with unusual VEB-28 extended-spectrum ß-lactamase and distinct mcr variants.

Aeromonas spp. are frequently encountered in aquatic environments, with Aeromonas veronii emerging as an opportunistic pathogen causing a range of diseases in both humans and animals. Recent reports have raised public health concerns due to the emergence of multidrug-resistant Aeromonas spp. This is particularly noteworthy as these species have demonstrated the ability to acquire and transmit antimicrobial resistance genes (ARGs). In this study, we report the genomic and phenotypic characteristics of the A. veronii TR112 strain, which harbors a novel variant of the Vietnamese Extended-spectrum ß-lactamase-encoding gene, blaVEB-28, and two mcr variants recovered from an urban river located in the Metropolitan Region of São Paulo, Brazil. A. veronii TR112 strain exhibited high minimum inhibitory concentrations (MICs) for ceftazidime (64 µg/mL), polymyxin (8 µg/mL), and ciprofloxacin (64 µg/mL). Furthermore, the TR112 strain demonstrated adherence to HeLa and Caco-2 cells within 3 h, cytotoxicity to HeLa cells after 24 h of interaction, and high mortality rates to the Galleria mellonella model. Genomic analysis showed that the TR112 strain belongs to ST257 and presented a range of ARGs conferring resistance to ß-lactams (blaVEB-28, blaCphA3, blaOXA-912) and polymyxins (mcr-3 and mcr-3.6). Additionally, we identified a diversity of virulence factor-encoding genes, including those encoding mannose-sensitive hemagglutinin (Msh) pilus, polar flagella, type IV pili, type II secretion system (T2SS), aerolysin (AerA), cytotoxic enterotoxin (Act), hemolysin (HlyA), hemolysin III (HlyIII), thermostable hemolysin (TH), and capsular polysaccharide (CPS). In conclusion, our findings suggest that A. veronii may serve as an environmental reservoir for ARGs and virulence factors, highlighting its importance as a potential pathogen in public health.

Capture ELISA for KPC Detection in Gram-Negative Bacilli: Development and Standardisation.

The detection of KPC-type carbapenemases is necessary for guiding appropriate antibiotic therapy and the implementation of antimicrobial stewardship and infection control measures. Currently, few tests are capable of differentiating carbapenemase types, restricting the lab reports to their presence or not. The aim of this work was to raise antibodies and develop an ELISA test to detect KPC-2 and its D179 mutants. The ELISA-KPC test was designed using rabbit and mouse polyclonal antibodies. Four different protocols were tested to select the bacterial inoculum with the highest sensitivity and specificity rates. The standardisation procedure was performed using 109 previously characterised clinical isolates, showing 100% of sensitivity and 89% of specificity. The ELISA-KPC detected all isolates producing carbapenemases, including KPC variants displaying the ESBL phenotype such as KPC-33 and -66.

In Vitro and In Vivo Synergism of Fosfomycin in Combination with Meropenem or Polymyxin B against KPC-2-Producing Klebsiella pneumoniae Clinical Isolates.

Fosfomycin disodium is a potential therapeutic option to manage difficult-to-treat infections, especially when combined with other antimicrobials. In this study, we evaluated the activity of fosfomycin in combination with meropenem or polymyxin B against contemporaneous KPC-2-producing K. pneumoniae clinical isolates (KPC-KPN). Synergistic activity was assessed by checkerboard (CKA) and time-kill (TKA) assays. TKA was performed using serum peak and trough concentrations. The activity of these combinations was also assessed in the Galleria mellonella model. Biofilm disruption was assessed by the microtiter plate technique. CKA resulted in an 8- to 2048-fold decrease in meropenem MIC, restoring meropenem activity for 82.4% of the isolates when combined with fosfomycin. For the fosfomycin + polymyxin B combination, a 2- to 128-fold reduction in polymyxin B MIC was achieved, restoring polymyxin B activity for 47% of the isolates. TKA resulted in the synergism of fosfomycin + meropenem (3.0-6.7 log10 CFU/mL decrease) and fosfomycin + polymyxin B (6.0-6.2 log10 CFU/mL decrease) at peak concentrations. All larvae treated with fosfomycin + meropenem survived. Larvae survival rate was higher with fosfomycin monotherapy (95%) than that observed for fosfomycin + polymyxin B (75%) (p-value < 0.0001). Finally, a higher biofilm disruption was observed under exposure to fosfomycin + polymyxin B (2.4-3.4-fold reduction). In summary, we observed a synergistic effect of fosfomycin + meropenem and fosfomycin + polymyxin B combinations, in vitro and in vivo, against KPC-KPN, as well as biofilm disruption.

Capture ELISA for KPC detection in gram-negative bacilli: development and standardisation

The detection of KPC-type carbapenemases is necessary for guiding appropriate antibiotic therapy and the implementation of antimicrobial stewardship and infection control measures. Currently, few tests are capable of differentiating carbapenemase types, restricting the lab reports to their presence or not. The aim of this work was to raise antibodies and develop an ELISA test to detect KPC-2 and its D179 mutants. The ELISA-KPC test was designed using rabbit and mouse polyclonal antibodies. Four different protocols were tested to select the bacterial inoculum with the highest sensitivity and specificity rates. The standardisation procedure was performed using 109 previously characterised clinical isolates, showing 100% of sensitivity and 89% of specificity. The ELISA-KPC detected all isolates producing carbapenemases, including KPC variants displaying the ESBL phenotype such as KPC-33 and -66.

Genome sequencing of an XDR Klebsiella pneumoniae ST101 strain isolated from a Brazilian Amazon river.

OBJECTIVE: Herein, this study aimed to perform the genomic characterization of a blaKPC-2 positive Klebsiella pneumoniae (KP1.1JP) strain isolated from the surface water of river located the Brazilian Amazon region. METHODS: Antimicrobial susceptibility testing was performed following BrCAST/EUCAST recommendations. Genomic DNA was extracted and sequenced using the Illumina® NextSeq platform and the assembly of the generated reads was performed using the SPAdes software. Research on the sequence type, resistance and virulence encoding genes, and plasmid replicon typing was carried out. RESULTS: The KP1.1JP strain was resistant to all ß-lactams, aminoglycosides, and fluoroquinolones tested. The genome size was 5 626 346 bp, distributed in 203 contigs and a guanine and cytosine content of 57.02%. The values of N50 and N75 were 285 583 bp and 173 927 bp, respectively. We verified that KP1.1JP belongs to ST101 and carries genes encoding resistance to ß-lactams (blaCTX-M-15, blaTEM-1B, blaOXA-1, blaSVH-182, and blaKPC-2), aminoglycosides [aac(3')-IIa, aph(3')-Vla], fluoroquinolones [aac(6')-Ib-cr], phenicol (catA1, catA2, catB3), tetracycline [tet(D)], trimethoprim (dfrA14), and fosfomycin (fosA). Additionally, the following virulence encoding genes were also detected: mrkABCDFHIJ (Fimbria type 3); fimABCDRFGHIK (Fimbria type 1); entABCDEFS and fepABCDG (siderophores); iroN, irp1, and irp2 (salmochelins); fyuA and ybtAEPQSTUX (yersiniabactin); and iutA (aerobactin). CONCLUSIONS: We report the occurrence of a K. pneumoniae ST101 strain carrying blaKPC-2 gene in an Amazon river in Brazil. The genomic characteristics of this strain will contribute to a better understanding of the spread of pathogens of clinical importance in the environment based on a One Health perspective.