Sonia Alejandra Gomez

Accumulation of resistance elements makes infections with Klebsiella pneumoniae (Kp) difficult-to-treat. Reported discrepancies between in vitro susceptibility testing and clinical effectiveness of cell wall-targeting antibiotics in treatment of Kp infections are indicative of additional complexity. Here, we found that non-bactericidal antibiotic effects contribute to killing of carbapenem-resistant (CR) Kp isolates in the presence of polymorphonuclear neutrophils (PMN). PMN alone had no effect on Kp survival, but they increased in vitro killing of CR-Kp in the presence of 2nd and 3rd generation cephalosporines (3GC), fosfomycin and carbapenems. Notably, subinhibitory concentrations of meropenem and cefotaxime were sufficient and synergy was not affected by carbapenemases. Permeabilization and changes in cell morphology correlated with in vitro susceptibility to antibiotics but were not altered by PMN. Subsequently, phagocytosis was found to be required for synergy, and the induction of PMN-derived reactive oxygen species (ROS) was identified as the most sensitive parameter predicting synergism. In accordance, synergy was not observed with ROS-defective ST258 Kp. K1/K2+ hypervirulent Kp also failed to display synergy. Importantly, the synergistic effect could not be restored by opsonophagocytosis of K1+ Kp with anti-K1 antibodies. Taken together, this study demonstrates that betalactam resistance in CR-Kp can be overcome by PMN. This offers new therapeutic options for CR-Kp and triggers the need for more refined immune and bacteriological diagnostics.

Accumulation of resistance elements makes infections with Klebsiella pneumoniae (Kp) difficult-to-treat. Reported discrepancies between in vitro susceptibility testing and clinical effectiveness of cell wall-targeting antibiotics in treatment of Kp infections are indicative of additional complexity. Here, we found that non-bactericidal antibiotic effects contribute to killing of carbapenem-resistant (CR) Kp isolates in the presence of polymorphonuclear neutrophils (PMN). PMN alone had no effect on Kp survival, but they increased in vitro killing of CR-Kp in the presence of 2nd and 3rd generation cephalosporines (3GC), fosfomycin and carbapenems. Notably, subinhibitory concentrations of meropenem and cefotaxime were sufficient and synergy was not affected by carbapenemases. Permeabilization and changes in cell morphology correlated with in vitro susceptibility to antibiotics but were not altered by PMN. Subsequently, phagocytosis was found to be required for synergy, and the induction of PMN-derived reactive oxygen species (ROS) was identified as the most sensitive parameter predicting synergism. In accordance, synergy was not observed with ROS-defective ST258 Kp. K1/K2+ hypervirulent Kp also failed to display synergy. Importantly, the synergistic effect could not be restored by opsonophagocytosis of K1+ Kp with anti-K1 antibodies. Taken together, this study demonstrates that betalactam resistance in CR-Kp can be overcome by PMN. This offers new therapeutic options for CR-Kp and triggers the need for more refined immune and bacteriological diagnostics.

Enterobacter spp., after Klebsiella pneumoniae , is among the most common hospital-acquired enterobacteria associated with antimicrobial resistance (AMR) worldwide, yet it remains underexplored. We determined the taxonomic and epidemiological distribution of 131 representative carbapenemase-producing Enterobacter spp. recovered from infections across Argentina between 2016 and 2022. The sample included one isolate per patient from 17 jurisdictions and 68 hospitals. Outbreak-related and surveillance isolates were excluded. Sequencing was performed using Illumina, and bioinformatic analyses were used to determine species (average nucleotide identity [ANI] ≥94%) and subspecies (ANI ≥98%) using BactInspector/fastANI. Sequence types (STs) were assigned by multilocus sequence typing (MLST) via PubMLST, and resistance genes were identified using AMRfinderPlus/Resfinder. Samples were obtained from ( n ; %) urine (43; 33%), blood (41; 31%), and other sites (47; 36%). Seven species were identified, with Enterobacter hormaechei (EH) predominating ( 120; 92%) and comprising five subspecies. E. hormaechei subsp. steigerwaltii was the most abundant (60; 45%), followed by E. hormaechei subsp. xiangfangensis (29; 22%). Enterobacter cloacae subsp. cloacae was underrepresented (3; 2.3%). KPC-2 was the predominant carbapenemase, followed by NDM-1 and OXA-163. A total of 58 STs were identified, 38 represented by a single isolate. EH included 50 STs, with the most abundant being ST88 (12; 9%), ST78 (12; 9%), and ST66 (8; 6%). No clear associations were observed between species/subspecies, clones, and carbapenemase types. Overall, the population structure of carbapenemase-producing Enterobacter spp. in Argentina is complex and diverse, characterized by multiple species, subspecies, clonal diversity, and varied carbapenemase types and combinations. Notably, 50% of the isolates belonged to E. hormaechei subsp . steigerwaltii , suggesting a major role of this subspecies in the dissemination of AMR. IMPORTANCE: Carbapenemase-producing Enterobacter spp. rank as the most common Enterobacterales associated with carbapenemase production. Despite their clinical relevance, the taxonomy and population structure of this group remain poorly characterized. This study provides a comprehensive genomic overview of clinical carbapenemase-producing Enterobacter spp. circulating in Argentina, revealing a highly diverse population composed of species, subspecies, and clonal types, harboring mainly KPC-2, followed by NDM-1 and combinations. We identified international clones within E. hormaechei , such as ST88 and ST78, which may play important roles in the dissemination of antimicrobial resistance. Notably, 50% of the isolates belonged to E. hormaechei subsp. steigerwaltii , suggesting a major role of this subspecies in the dissemination of antimicrobial resistance (AMR). By improving our understanding of this complex bacterial group, this work supports the strengthening of antimicrobial resistance surveillance networks and the development of targeted strategies for infection control and antibiotic stewardship in healthcare settings.

Introduction: The spread of carbapenemase-producing bacteria (CPB) is exacerbated in hospital settings, making the surveillance of rectal carriage of CPB crucial to halt their spread. However, the processing time until detection with traditional methods and the cost of new techniques limit their implementation. We aimed to evaluate the effectiveness of infection prevention and control (IPC) measures guided by a novel algorithm (NA) for rectal swab processing, which incorporated a modified Blue-Carba test (mBCT), in reducing carbapenemase-producing bacteria (CPB) rectal carriage prevalence in general wards of a tertiary-care hospital from Argentina. Additionally, we assessed the impact of this algorithm on microbiological turnaround time (mTAT) and time to positive results (TPR).Materials and methods: An experimental and quasi-experimental designs were combined into a prospective interrupted time series study structured in three phases: P1 (February 2022-July 2022), P2 (August 2022-January 2023; intervention) and P3 (February 2023-July 2023). Briefly, the NA included as key steps a 6-hour pre-incubation at 37 °C in nutrient broth, followed by a 15-minute centrifugation at 3,200 rpm. The mBCT was set at pH 10.7 using 9 mg of imipenem in a final volume of 150 μL and was validated against conventional methods testing 1,120 samples. It was subsequently implemented to assess its impact on hospital CPB prevalence and the effectiveness of IPC measures. Patients were randomly selected for CPB rectal screening during Phases 1 and 3 and provided informed consent for inclusion.Results: The mBCT significantly shortened the mTAT and TPR compared to standard approaches (<24 h vs. 4d, p < 0.001), showing moderate sensitivity [54.6% (IC95% 45.2–63.7)] and high specificity [99.8% (IC 95% 99.3–100)]. The IPC intervention guided by the mBCT reduced CPB prevalence in general wards (8.1% vs. 13.8%, p = 0.006).Conclusion: The implementation of the NA reduced mTAT with high sensitivity, while the mBCT also contributed to reducing TPR with high specificity. Integrating the NA and mBCT into IPC protocols led to a decrease in CPB rectal carriage prevalence in general wards, underscoring their diagnostic, epidemiological and thus IPC benefits.

Ceftazidime-avibactam (CZA) therapy has significantly improved survival rates for patients infected by carbapenem-resistant bacteria, including KPC producers. However, resistance to CZA is a growing concern, attributed to multiple mechanisms. In this study, we characterized four clinical CZA-resistant Klebsiella pneumoniae isolates obtained between July 2019 and December 2020. These isolates expressed novel allelic variants of bla _KPC-2 resulting from changes in hotspots of the mature protein, particularly in loops surrounding the active site of KPC. Notably, KPC-80 had an K269_D270insPNK mutation near the Lys270-loop, KPC-81 had a del_I173 mutation within the Ω-loop, KPC-96 showed a Y241N substitution within the Val240-loop and KPC-97 had an V277_I278insNSEAV mutation within the Lys270-loop. Three of the four isolates exhibited low-level resistance to imipenem (4 µg/mL), while all remained susceptible to meropenem. Avibactam and relebactam effectively restored carbapenem susceptibility in resistant isolates. Cloning mutant bla _KPC genes into pMBLe increased imipenem MICs in recipient Escherichia coli TOP10 for bla _KPC-80 , bla _KPC-96 , and bla _KPC-97 by two dilutions; again, these MICs were restored by avibactam and relebactam. Frameshift mutations disrupted omp K35 in three isolates. Additional resistance genes, including bla _TEM-1 , bla _OXA-18 and bla _OXA-1 , were also identified. Interestingly, three isolates belonged to clonal complex 11 (ST258 and ST11) and one to ST629. This study highlights the emergence of CZA resistance including unique allelic variants of bla _KPC-2 and impermeability. Comprehensive epidemiological surveillance and in-depth molecular studies are imperative for understanding and monitoring these complex resistance mechanisms, crucial for effective antimicrobial treatment strategies. IMPORTANCE: The emergence of ceftazidime-avibactam (CZA) resistance poses a significant threat to the efficacy of this life-saving therapy against carbapenem-resistant bacteria, particularly Klebsiella pneumoniae -producing KPC enzymes. This study investigates four clinical isolates exhibiting resistance to CZA, revealing novel allelic variants of the key resistance gene, bla _KPC-2 . The mutations identified in hotspots surrounding the active site of KPC, such as K269_D270insPNK, del_I173, Y241N and V277_I278insNSEAV, prove the adaptability of these pathogens. Intriguingly, low-level resistance to imipenem and disruptions in porin genes were observed, emphasizing the complexity of the resistance mechanisms. Interestingly, three of four isolates belonged to clonal complex 11. This research not only sheds light on the clinical significance of CZA resistance but also shows the urgency for comprehensive surveillance and molecular studies to inform effective antimicrobial treatment strategies in the face of evolving bacterial resistance.

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 -Tn As2 ), 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, bla _NDM-1 was embedded in a partially conserved structure flanked by two IS Kox2 . In addition, all plasmids harbored aph(3')-Ia , aph(3')-VI , and qnr D1 genes and aac(6´)Ib-cr , bla _OXA-1 , cat B3, 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 bla _NDM-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.

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, bla_CVI-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-β-lactamase was confirmed. Genome analysis revealed bla_CVI-1, a subclass B2 metallo-β-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, bla_CVI-1, a metallo-β-lactamase that can be detected by routine methods. Prompt suspicion of C. violaceum infection is crucial to treating this rare pathogen rapidly and properly.

Background. The global spread of carbapenemase-producing Enterobacterales has become an epidemiological risk for healthcare systems by limiting available antimicrobial treatments. The COVID-19 pandemic worsened this scenario, prompting the emergence of extremely resistant microorganisms. Methods. Between March 2020 and September 2021, the NRL confirmed 82 clinical Enterobacterales isolates harboring a combination of bla_KPC and MBL genes. Molecular typing was analyzed by PFGE and MLST. Modified double-disk synergy (MDDS) tests were used for phenotypic studies. Results. Isolates were submitted from 28 hospitals located in seven provinces and Buenos Aires City, including 77 K. pneumoniae, 2 K. oxytoca, 2 C. freundii, and 1 E. coli. Almost half of K. pneumoniae isolates (n = 38; 49.4%), detected in 15 hospitals, belong to the CC307 clone. CC11 was the second clone, including 29 (37.7%) isolates (22, ST11 and 7, ST258) from five cities and 12 hospitals. Three isolates belonging to CC45 were also detected. The carbapenemase combinations observed were as follows: 55% bla_KPC-2 plus bla_NDM-5; 32.5% bla_KPC-2 plus bla_NDM-1; 5% bla_KPC-3 plus bla_NDM-1; 5% bla_KPC-2 plus bla_IMP-8; and 2.5% strain with bla_KPC-2 plus bla_NDM-5 plus bla_OXA-163. Aztreonam/avibactam and aztreonam/relebactam were the most active combinations (100% and 91% susceptible, respectively), followed by fosfomycin (89%) and tigecycline (84%). Conclusions. The MDDS tests using ceftazidime-avibactam/EDTA and aztreonam/boronic acid disks improved phenotypic classification as dual producers. The successful high-risk clones of K. pneumoniae, such as hyper-epidemic CC307 and CC11 clones, drove the dissemination of double carbapenemase-producing isolates during the COVID-19 pandemic.