Development and validation of a quick, automated, and reproducible ATR FT-IR spectroscopy machine-learning model for Klebsiella pneumoniae typing.

The reliability of Fourier-transform infrared (FT-IR) spectroscopy for Klebsiella pneumoniae typing and outbreak control has been previously assessed, but issues remain in standardization and reproducibility. We developed and validated a reproducible FT-IR with attenuated total reflectance (ATR) workflow for the identification of K. pneumoniae lineages. We used 293 isolates representing multidrug-resistant K. pneumoniae lineages causing outbreaks worldwide (2002-2021) to train a random forest classification (RF) model based on capsular (KL)-type discrimination. This model was validated with 280 contemporaneous isolates (2021-2022), using wzi sequencing and whole-genome sequencing as references. Repeatability and reproducibility were tested in different culture media and instruments throughout time. Our RF model allowed the classification of 33 capsular (KL)-types and up to 36 clinically relevant K. pneumoniae lineages based on the discrimination of specific KL- and O-type combinations. We obtained high rates of accuracy (89%), sensitivity (88%), and specificity (92%), including from cultures obtained directly from the clinical sample, allowing to obtain typing information the same day bacteria are identified. The workflow was reproducible in different instruments throughout time (>98% correct predictions). Direct colony application, spectral acquisition, and automated KL prediction through Clover MS Data analysis software allow a short time-to-result (5 min/isolate). We demonstrated that FT-IR ATR spectroscopy provides meaningful, reproducible, and accurate information at a very early stage (as soon as bacterial identification) to support infection control and public health surveillance. The high robustness together with automated and flexible workflows for data analysis provide opportunities to consolidate real-time applications at a global level. IMPORTANCE We created and validated an automated and simple workflow for the identification of clinically relevant Klebsiella pneumoniae lineages by FT-IR spectroscopy and machine-learning, a method that can be extremely useful to provide quick and reliable typing information to support real-time decisions of outbreak management and infection control. This method and workflow is of interest to support clinical microbiology diagnostics and to aid public health surveillance.

Emergence of NDM-1 and KPC-3 carbapenemases in Kluyvera cryocrescens: Investigating genetic heterogeneity and acquisition routes of blaNDM-1 in Enterobacterales species in Portugal.

OBJECTIVES: A higher diversity of species, clones and genes have been increasingly implicated in carbapenemases spread, though the mobile genetic elements responsible for their acquisition and dispersion at local and global levels are less explored, particularly in species other than Klebsiella pneumoniae or Escherichia coli. We aim to explain the emergence of NDM-1 and KPC-3 carbapenemases in a Kluyvera cryocrescens isolate, and to shed light on the heterogeneity of genetic platforms and acquisition routes of blaNDM-1 in diverse Enterobacterales species in Portugal. METHODS: A KPC-3 and NDM-1-producing K. cryocrescens colonizing a hospitalized patient in 2019 was characterized by whole-genome sequencing and antibiotic resistance profiling following standard methods. Conjugative transfer of carbapenemases genes was assessed by filter mating. Plasmids were reconstructed with in silico and in vitro approaches. blaNDM-1 genetic context was compared with that of diverse NDM-1-producing Enterobacterales species, previously described in Portugal. RESULTS: K. cryocrescens K629 showed a multidrug resistance profile. Resistance gene blaKPC-3 was harboured by a Tn4401d transposon within a worldwide-spread IncN-ST15 plasmid (pKLU-KPC3), whereas blaNDM-1 was located in a Tn3000 within a non-typeable mosaic plasmid (pKLU-NDM1). The heterogeneous blaNDM-1 genetic platforms and variable plasmid backbones identified in various Enterobacterales species suggested multiple introductions of blaNDM-1 in Portugal, mediated by variable insertion sequences. CONCLUSIONS: We report the convergence of KPC-3 and NDM-1 in K. cryocrescens and the variable dissemination modes of these carbapenemases in different Enterobacterales species, underlining the need to track down genetic platforms responsible for carbapenemases diffusion.

From Farm to Fork: Persistence of Clinically Relevant Multidrug-Resistant and Copper-Tolerant Klebsiella pneumoniae Long after Colistin Withdrawal in Poultry Production.

Concerns about colistin-resistant bacteria in animal food-environmental-human ecosystems prompted the poultry sector to implement colistin restrictions and explore alternative trace metals/copper feed supplementation. The impact of these strategies on the selection and persistence of colistin-resistant Klebsiella pneumoniae in the whole poultry production chain needs clarification. We assessed colistin-resistant and copper-tolerant K. pneumoniae occurrence in chickens raised with inorganic and organic copper formulas from 1-day-old chicks to meat (7 farms from 2019 to 2020), after long-term colistin withdrawal (>2 years). Clonal diversity and K. pneumoniae adaptive features were characterized by cultural, molecular, and whole-genome-sequencing (WGS) approaches. Most chicken flocks (75%) carried K. pneumoniae at early and preslaughter stages, with a significant decrease (P < 0.05) in meat batches (17%) and sporadic water/feed contamination. High rates (>50%) of colistin-resistant/mcr-negative K. pneumoniae were observed among fecal samples, independently of feed. Most samples carried multidrug-resistant (90%) and copper-tolerant (81%; silA and pcoD positive and with a MICCuSO4 of ≥16 mM) isolates. WGS revealed accumulation of colistin resistance-associated mutations and F type multireplicon plasmids carrying antibiotic resistance and metal/copper tolerance genes. The K. pneumoniae population was polyclonal, with various lineages dispersed throughout poultry production. ST15-KL19, ST15-KL146, and ST392-KL27 and IncF plasmids were similar to those from global human clinical isolates, suggesting chicken production as a reservoir/source of clinically relevant K. pneumoniae lineages and genes with potential risk to humans through food and/or environmental exposure. Despite the limited mcr spread due to the long-term colistin ban, this action was ineffective in controlling colistin-resistant/mcr-negative K. pneumoniae, regardless of feed. This study provides crucial insights into the persistence of clinically relevant K. pneumoniae in the poultry production chain and highlights the need for continued surveillance and proactive food safety actions within a One Health perspective. IMPORTANCE The spread of bacteria resistant to last-resort antibiotics such as colistin throughout the food chain is a serious concern for public health. The poultry sector has responded by restricting colistin use and exploring alternative trace metals/copper feed supplements. However, it is unclear how and to which extent these changes impact the selection and persistence of clinically relevant Klebsiella pneumoniae throughout the poultry chain. We found a high occurrence of copper-tolerant and colistin-resistant/mcr-negative K. pneumoniae in chicken flocks, regardless of inorganic and organic copper formulas use and a long-term colistin ban. Despite the high K. pneumoniae isolate diversity, the occurrence of identical lineages and plasmids across samples and/or clinical isolates suggests poultry as a potential source of human K. pneumoniae exposure. This study highlights the need for continued surveillance and proactive farm-to-fork actions to mitigate the risks to public health, relevant for stakeholders involved in the food industry and policymakers tasked with regulating food safety.

Phylogenomics of Globally Spread Clonal Groups 14 and 15 of Klebsiella pneumoniae.

Klebsiella pneumoniae sequence type 14 (ST14) and ST15 caused outbreaks of CTX-M-15 and/or carbapenemase producers worldwide, but their phylogeny and global dynamics remain unclear. We clarified the evolution of K. pneumoniae clonal group 14 (CG14) and CG15 by analyzing the capsular locus (KL), resistome, virulome, and plasmidome of public genomes (n = 481) and de novo sequences (n = 9) representing main sublineages circulating in Portugal. CG14 and CG15 evolved independently within 6 main subclades defined according to the KL and the accessory genome. The CG14 (n = 65) clade was structured in two large monophyletic subclades, CG14-I (KL2, 86%) and CG14-II (KL16, 14%), whose emergences were dated to 1932 and 1911, respectively. Genes encoding extended-spectrum ß-lactamase (ESBL), AmpC, and/or carbapenemases were mostly observed in CG14-I (71% versus 22%). CG15 clade (n = 170) was segregated into subclades CG15-IA (KL19/KL106, 9%), CG15-IB (variable KL types, 6%), CG15-IIA (KL24, 43%) and CG15-IIB (KL112, 37%). Most CG15 genomes carried specific GyrA and ParC mutations and emerged from a common ancestor in 1989. CTX-M-15 was especially prevalent in CG15 (68% CG15 versus 38% CG14) and in CG15-IIB (92%). Plasmidome analysis revealed 27 predominant plasmid groups (PG), including particularly pervasive and recombinant F-type (n = 10), Col (n = 10), and new plasmid types. While blaCTX-M-15 was acquired multiple times by a high diversity of F-type mosaic plasmids, other antibiotic resistance genes (ARGs) were dispersed by IncL (blaOXA-48) or IncC (blaCMY/TEM-24) plasmids. We first demonstrate an independent evolutionary trajectory for CG15 and CG14 and how the acquisition of specific KL, quinolone-resistance determining region (QRDR) mutations (CG15), and ARGs in highly recombinant plasmids could have shaped the expansion and diversification of particular subclades (CG14-I and CG15-IIA/IIB). IMPORTANCE Klebsiella pneumoniae represents a major threat in the burden of antibiotic resistance (ABR). Available studies to explain the origin, the diversity, and the evolution of certain ABR K. pneumoniae populations have mainly been focused on a few clonal groups (CGs) using phylogenetic analysis of the core genome, the accessory genome being overlooked. Here, we provide unique insights into the phylogenetic evolution of CG14 and CG15, two poorly characterized CGs which have contributed to the global dissemination of genes responsible for resistance to first-line antibiotics such as ß-lactams. Our results point out an independent evolution of these two CGs and highlight the existence of different subclades structured by the capsular type and the accessory genome. Moreover, the contribution of a turbulent flux of plasmids (especially multireplicon F type and Col) and adaptive traits (antibiotic resistance and metal tolerance genes) to the pangenome reflect the exposure and adaptation of K. pneumoniae under different selective pressures.

Taking hospital pathogen surveillance to the next level.

High-throughput bacterial genomic sequencing and subsequent analyses can produce large volumes of high-quality data rapidly. Advances in sequencing technology, with commensurate developments in bioinformatics, have increased the speed and efficiency with which it is possible to apply genomics to outbreak analysis and broader public health surveillance. This approach has been focused on targeted pathogenic taxa, such as Mycobacteria, and diseases corresponding to different modes of transmission, including food-and-water-borne diseases (FWDs) and sexually transmitted infections (STIs). In addition, major healthcare-associated pathogens such as methicillin-resistant Staphylococcus aureus, vancomycin-resistant enterococci and carbapenemase-producing Klebsiella pneumoniae are the focus of research projects and initiatives to understand transmission dynamics and temporal trends on both local and global scales. Here, we discuss current and future public health priorities relating to genome-based surveillance of major healthcare-associated pathogens. We highlight the specific challenges for the surveillance of healthcare-associated infections (HAIs), and how recent technical advances might be deployed most effectively to mitigate the increasing public health burden they cause.

Citrobacter meridianamericanus sp. nov., isolated from a soil sample.

A Gram-stain-negative strain, designated BR102T, isolated from a soil sample in Brazil was characterized by a polyphasic approach. Comparative 16S rRNA gene sequences indicated that strain BR102T belonged to the genus Citrobacter. The recN- and whole-genome-based phylogeny, and multilocus sequence analysis based on concatenated partial fusA, leuS, pyrG and rpoB sequences strongly supported a clade encompassing strain BR102T and a strain from public database that was distinct from currently recognized species of the genus Citrobacter. Average nucleotide identity and digital DNA-DNA hybridization values between strain BR102T and the closest relative Citrobacter freundii ATCC 8090T were 91.8 and 48.8 %, respectively. The ability to metabolize different compounds further discriminated strain BR102T from other closely related species of the genus Citrobacter. The novel variants bla CMY-179 and qnrB97, which encoded a CMY-2-like ß-lactamase and a QnrB-type protein, respectively, were identified in strain BR102T. BR102T was resistant to ampicillin, amoxicillin/clavulanate and cefoxitin. The DNA G+C content of strain BR102T is 51.3 mol%. Based on these results, strain BR102T represents a novel species of the genus Citrobacter, for which the name Citrobacter meridianamericanus sp. nov. is proposed. The type strain is BR102T (=MUM 22.55T=IMI 507229T).

Silver Nanostar-Based SERS for the Discrimination of Clinically Relevant Acinetobacter baumannii and Klebsiella pneumoniae Species and Clones.

The development of rapid, reliable, and low-cost methods that enable discrimination among clinically relevant bacteria is crucial, with emphasis on those listed as WHO Global Priority 1 Critical Pathogens, such as carbapenem-resistant Acinetobacter baumannii and carbapenem-resistant or ESBL-producing Klebsiella pneumoniae. To address this problem, we developed and validated a protocol of surface-enhanced Raman spectroscopy (SERS) with silver nanostars for the discrimination of A. baumannii and K. pneumoniae species, and their globally disseminated and clinically relevant antibiotic resistant clones. Isolates were characterized by mixing bacterial colonies with silver nanostars, followed by deposition on filter paper for SERS spectrum acquisition. Spectral data were processed with unsupervised and supervised multivariate data analysis methods, including principal component analysis (PCA) and partial least-squares discriminant analysis (PLSDA), respectively. Our proposed SERS procedure using silver nanostars adsorbed to the bacteria, followed by multivariate data analysis, enabled differentiation between and within species. This pilot study demonstrates the potential of SERS for the rapid discrimination of clinically relevant A. baumannii and K. pneumoniae species and clones, displaying several advantages such as the ease of silver nanostars synthesis and the possible use of a handheld spectrometer, which makes this approach ideal for point-of-care applications.

Rapid Detection of Plasmid AmpC Beta-Lactamases by a Flow Cytometry Assay.

Plasmidic AmpC (pAmpC) enzymes are responsible for the hydrolysis of extended-spectrum cephalosporins but they are not routinely investigated in many clinical laboratories. Phenotypic assays, currently the reference methods, are cumbersome and culture dependent. These methods compare the activity of cephalosporins with and without class C inhibitors and the results are provided in 24-48 h. Detection by molecular methods is quicker, but several genes should be investigated. A new assay for the rapid phenotypic detection of pAmpC enzymes of the Enterobacterales group-I (not usually AmpC producers) based on flow cytometry technology was developed and validated. The technology was evaluated in two sites: FASTinov, a spin-off of Porto University (Portugal) where the technology was developed, and the Microbiology Department of Ramón y Cajal University Hospital in Madrid (Spain). A total of 100 strains were phenotypically screened by disk diffusion for the pAmpC with the new 2 h assay. Molecular detection of the pAmpC genes was also performed on discrepant results. Forty-two percent of the strains were phenotypically classified as pAmpC producers using disk diffusion. The percentage of agreement of the flow cytometric assay was 93.0%, with 95.5% sensitivity and 91.1% specificity. Our proposed rapid assay based on flow cytometry technology can, in two hours, accurately detect pAmpC enzymes.

High diversity of pathogenic Escherichia coli clones carrying mcr-1 among gulls underlines the need for strategies at the environment-livestock-human interface.

The expansion of mcr-carrying bacteria is a well-recognized public health problem. Measures to contain mcr spread have mainly been focused on the food-animal production sector. Nevertheless, the spread of MCR producers at the environmental interface particularly driven by the increasing population of gulls in coastal cities has been less explored. Occurrence of mcr-carrying Escherichia coli in gull's colonies faeces on a Portuguese beach was screened over 7 months. Cultural, molecular and genomic approaches were used to characterize their diversity, mcr plasmids and adaptive features. Multidrug-resistant mcr-1-carrying E. coli were detected for 3 consecutive months. Over time, multiple strains were recovered, including zoonotic-related pathogenic E. coli clones (e.g. B2-ST131-H22, A-ST10 and B1-ST162). Diverse mcr-1 genetic environments were mainly associated with ST2/ST4-HI2 (ST10, ST131, ST162, ST354 and ST4204) but also IncI2 (ST12990) plasmids or in the chromosome (ST656). Whole-genome sequencing revealed enrichment of these strains on antibiotic resistance, virulence and metal tolerance genes. Our results underscore gulls as important spreaders of high-priority bacteria and genes that may affect the environment, food-animals and/or humans, potentially undermining One-Health strategies to reduce colistin resistance.

NDM-1 Introduction in Portugal through a ST11 KL105 Klebsiella pneumoniae Widespread in Europe.

The changing epidemiology of carbapenem-resistant Klebsiella pneumoniae in Southern European countries is challenging for infection control, and it is critical to identify and track new genetic entities (genes, carbapenemases, clones) quickly and with high precision. We aimed to characterize the strain responsible for the first recognized outbreak by an NDM-1-producing K. pneumoniae in Portugal, and to elucidate its diffusion in an international context. NDM-1-producing multidrug-resistant K. pneumoniae isolates from hospitalized patients (2018-2019) were characterized using FTIR spectroscopy, molecular typing, whole-genome sequencing, and comparative genomics with available K. pneumoniae ST11 KL105 genomes. FT-IR spectroscopy allowed the rapid (ca. 4 h after incubation) identification of the outbreak strains as ST11 KL105, supporting outbreak control. Epidemiological information supports a community source but without linkage to endemic regions of NDM-1 producers. Whole-genome comparison with previous DHA-1-producing ST11 KL105 strains revealed the presence of different plasmid types and antibiotic resistance traits, suggesting the entry of a new strain. In fact, this ST11 KL105 clade has successfully disseminated in Europe with variable beta-lactamases, but essentially as ESBL or DHA-1 producers. We expand the distribution map of NDM-1-producing K. pneumoniae in Europe, at the expense of a successfully established ST11 KL105 K. pneumoniae clade circulating with variable plasmid backgrounds and beta-lactamases. Our work further supports the use of FT-IR as an asset to support quick infection control.

Allogenous Selection of Mutational Collateral Resistance: Old Drugs Select for New Resistance Within Antibiotic Families.

Allogeneous selection occurs when an antibiotic selects for resistance to more advanced members of the same family. The mechanisms of allogenous selection are (a) collateral expansion, when the antibiotic expands the gene and gene-containing bacterial populations favoring the emergence of other mutations, inactivating the more advanced antibiotics; (b) collateral selection, when the old antibiotic selects its own resistance but also resistance to more modern drugs; (c) collateral hyper-resistance, when resistance to the old antibiotic selects in higher degree for populations resistant to other antibiotics of the family than to itself; and (d) collateral evolution, when the simultaneous or sequential use of antibiotics of the same family selects for new mutational combinations with novel phenotypes in this family, generally with higher activity (higher inactivation of the antibiotic substrates) or broader spectrum (more antibiotics of the family are inactivated). Note that in some cases, collateral selection derives from collateral evolution. In this article, examples of allogenous selection are provided for the major families of antibiotics. Improvements in minimal inhibitory concentrations with the newest drugs do not necessarily exclude "old" antibiotics of the same family of retaining some selective power for resistance to the newest agents. If this were true, the use of older members of the same drug family would facilitate the emergence of mutational resistance to the younger drugs of the family, which is frequently based on previously established resistance traits. The extensive use of old drugs (particularly in low-income countries and in farming) might be significant for the emergence and selection of resistance to the novel members of the family, becoming a growing source of variation and selection of resistance to the whole family. In terms of future research, it could be advisable to focus antimicrobial drug discovery more on the identification of new targets and new (unique) classes of antimicrobial agents, than on the perpetual chemical exploitation of classic existing ones.

From farm to fork: Colistin voluntary withdrawal in Portuguese farms reflected in decreasing occurrence of mcr-1-carrying Enterobacteriaceae from chicken meat.

Expansion of mcr-carrying Enterobacteriaceae (MCR-E) is a well-recognized problem affecting animals, humans and the environment. Ongoing global control actions involve colistin restrictions among food-animal production, but their impact on poultry-derived products is largely unknown, justifying comprehensive farm-to-fork studies. Occurrence of MCR-E among 53 chicken-meat batches supplied from 29 Portuguese farms shortly after colistin withdrawal was evaluated. Strains (FT-IR/MLST/WGS), mcr plasmids and their adaptive features were characterized by cultural, molecular and genomic approaches. We found high rates of chicken-meat batches (80%-100% - 4 months; 12% - the last month) with multiple MDR + mcr-1-carrying Escherichia coli (Ec-including ST117 and ST648-Cplx) and Klebsiella pneumoniae (Kp-ST147-O5:K35) clones, some of them persisting over time. The mcr-1 was located in the chromosome (Ec-ST297/16-farms) or dispersed IncX4 (Ec-ST602/ST6469/5-farms), IncHI2-ST2/ST4 (Ec-ST533/ST6469/5 farms and Kp-ST147/6-farms) or IncI2 (Ec-ST117/1-farm) plasmids. WGS revealed high load and diversity in virulence, antibiotic resistance and metal tolerance genes. This study supports colistin withdrawal potential efficacy in poultry production and highlights both poultry-production chain as a source of mcr-1 and the risk of foodborne transmission to poultry-meat consumers. Finally, in the antibiotic reduction/replacement context, other potential co-selective pressures (e.g., metals-Cu as feed additives) need to be further understood to guide concerted, effective and durable actions under 'One Health' perspective.

Phylogenomic analysis of a highly virulent Escherichia coli ST83 lineage with potential animal-human transmission.

The presence of specific virulence features conditions severe forms of urinary tract disease, but the frequency and distribution of these highly virulent extraintestinal pathogenic Escherichia coli strains in animals and humans is unclear. We used whole genome sequencing, comparative genomics, histological and clinical data to characterize the genetic basis for pathogenesis and origin of E. coli Ec_151217, a strain (B2, ST83, O83:H5:K5) that caused an extremely aggressive upper urinary tract infection (UTI) in a cat. We show that Ec_151217 and 52% of other highly related ST83 genomes (O6 and O83) identified from different animal species and human infections carry two copies of the hemolysin A operon, though this duplication is infrequent (~1%) among closed ExPEC genomes from multiple sources. Our data enlarges the list of E. coli genetic backgrounds carrying hlyA operon duplication which is potentially involved in severity of UTI, and demonstrates that it seems to occur infrequently amongst ExPEC. Its identification in E. coli lineages (diverse ST83 serotypes) of potential animal-human transmission is of concern and anticipates the need to screen larger collections.

MicroMundo@UPorto: an experimental microbiology project fostering student's antimicrobial resistance awareness and personal and social development.

Antimicrobial resistance (AMR) is a global societal challenge requiring the contribution of professionals along with general community citizens for their containment. Portugal is one of the European countries where a lack of knowledge on the correct use of antimicrobials and AMR problematic is preeminent. Moreover, youth demotivation to pursue science careers is emerging. To address these problems an innovative experimental service-learning pedagogical strategy, MicroMundo@UPorto, was implemented in Portugal during 2018 through University of Porto as a partner of the global Citizen Science project 'Tiny Earth' responding to the AMR crisis. In this first edition of MicroMundo@UPorto, university students (n = 41; Pharmaceutical Sciences and Nutrition Sciences) organized in eight teams tutored by university professors/researchers (n = 13) on Microbiology and AMR theoretical and practical aspects as well on communication skills to enable their guidance of younger school students (n = 140/3 schools) in experiments to discover antimicrobial-producing microorganisms while exploring the soil microbial diversity. Post-survey-based evaluation revealed that this project allowed university students to acquire diverse personal, social and scientific skills while increasing AMR awareness, in the One-Health perspective, and interest for science in school students. This University to Society approach can be successfully extended across Portugal and for education in Microbiology in general, with benefits for the future generations contributing to socially responsible and scientifically-literate citizens.

The Darkest Place Is under the Candlestick-Healthy Urogenital Tract as a Source of Worldwide Disseminated Extraintestinal Pathogenic Escherichia coli Lineages.

Since the discovery of the urinary microbiome, including the identification of Escherichia coli in healthy hosts, its involvement in UTI development has been a subject of high interest. We explored the population diversity and antimicrobial resistance of E. coli (n = 22) in the urogenital microbiome of ten asymptomatic women (representing 50% of the sample tested). We evaluated their genomic relationship with extraintestinal pathogenic E. coli (ExPEC) strains from healthy and diseased hosts, including the ST131 lineage. E. coli prevalence was higher in vaginal samples than in urine samples, and occasionally different lineages were observed in the same individual. Furthermore, B2 was the most frequent phylogenetic group, with the most strains classified as ExPEC. Resistance to antibiotics of therapeutic relevance (e.g., amoxicillin-clavulanate conferred by blaTEM-30) was observed in ExPEC widespread lineages sequence types (ST) 127, ST131, and ST73 and ST95 clonal complexes. Phylogenomics of ST131 and other ExPEC lineages revealed close relatedness with strains from gastrointestinal tract and diseased host. These findings demonstrate that healthy urogenital microbiome is a source of potentially pathogenic and antibiotic resistant E. coli strains, including those causing UTI, e.g., ST131. Importantly, diverse E. coli lineages can be observed per individual and urogenital sample type which is relevant for future studies screening for this uropathogen.

Multiplicity of Carbapenemase-Producers Three Years after a KPC-3-Producing K. pneumoniae ST147-K64 Hospital Outbreak.

Carbapenem resistance rates increased exponentially between 2014 and 2017 in Portugal (~80%), especially in Klebsiella pneumoniae. We characterized the population of carbapanemase-producing Enterobacterales (CPE) infecting or colonizing hospitalized patients (2017-2018) in a central hospital from northern Portugal, where KPC-3-producing K. pneumoniae capsular type K64 has caused an initial outbreak. We gathered phenotypic (susceptibility data), molecular (population structure, carbapenemase, capsular type) and biochemical (FT-IR) data, together with patients' clinical and epidemiological information. A high diversity of Enterobacterales species, clones (including E. coli ST131) and carbapenemases (mainly KPC-3 but also OXA-48 and VIM) was identified three years after the onset of carbapenemases spread in the hospital studied. ST147-K64 K. pneumoniae, the initial outbreak clone, is still predominant though other high-risk clones have emerged (e.g., ST307, ST392, ST22), some of them with pandrug resistance profiles. Rectal carriage, previous hospitalization or antibiotherapy were presumptively identified as risk factors for subsequent infection. In addition, our previously described Fourier Transform infrared (FT-IR) spectroscopy method typed 94% of K. pneumoniae isolates with high accuracy (98%), and allowed to identify previously circulating clones. This work highlights an increasing diversity of CPE infecting or colonizing patients in Portugal, despite the infection control measures applied, and the need to improve the accuracy and speed of bacterial strain typing, a goal that can be met by simple and cost-effective FT-IR based typing.

Fourier transform infrared (FT-IR) spectroscopy typing: a real-time analysis of an outbreak by carbapenem-resistant Klebsiella pneumoniae.

The spread of multidrug-resistant (MDR) Klebsiella pneumoniae in the nosocomial setting represents a big challenge to infection control teams. We have recently developed a simple spectroscopic-based method with excellent accuracy, turnaround time and cost-effectiveness (Rodrigues et al. mSystems 2020) for bacterial typing. Here, we applied our method in a real clinical context to support early identification of an outbreak involving KPC-3-producing K. pneumoniae ST147 isolates. Our results further support that attenuated total reflectance Fourier transform infrared (FT-IR) spectroscopy can provide enough information to support early and adequate infection control measures and therapeutic choices in the context of nosocomial outbreaks and hospital surveillance.

Acquired AmpC ß-Lactamases among Enterobacteriaceae from Healthy Humans and Animals, Food, Aquatic and Trout Aquaculture Environments in Portugal.

We aimed to investigate the occurrence of acquired AmpC ß-lactamases (qAmpC), and characterize qAmpC-producing Enterobacteriaceae from different non-clinical environments in Portugal. We analysed 880 Enterobacteriaceae resistant to third-generation cephalosporins recovered from 632 non-clinical samples [healthy human and healthy animal (swine, chickens) faeces; uncooked chicken carcasses; aquatic and trout aquaculture samples]. Bacterial and qAmpC identification, antibiotic susceptibility, clonal (PFGE, MLST) and plasmid (S1-/I-CeuI-PFGE, replicon typing, hybridization) analysis were performed using standard methods. The occurrence of qAmpC among Enterobacteriaceae from non-clinical origins was low (0.6%; n = 4/628 samples), corresponding to CMY-2-producing Escherichia coli from three healthy humans (HH) and one uncooked chicken carcass (UCC). We highlight a slight increase in CMY-2 human faecal carriage in the two periods sampled [1.0% in 2013-2014 versus 0% in 2001-2004], which is in accordance with the trend observed in other European countries. CMY-2-producing E. coli belonged to B22-ST4953 (n = 2, HH), A0-ST665 (n = 1, HH) or A1-ST48 (n = 1, UCC) clones. blaCMY-2 was identified in non-typeable and IncA/C2 plasmids. This study is one of the few providing an integrated evaluation of the qAmpC-producing Enterobacteriaceae occurrence, which was low, from a very large collection of different non-clinical origins. Further surveillance in contemporary collections can provide an integrated epidemiological information of potential shifts in reservoirs, transmission routes and mechanisms of dissemination of blaqAmpC in non-clinical settings.