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ABSTRACT Klebsiella pneumoniae is one of the most threatening multi-drug-resistant pathogens today, with phage therapy being a promising alternative for personalized treatments. However, the intrinsic capsule diversity in Klebsiella spp. poses a substantial barrier to the phage host range, complicating the development of broad-spectrum phage-based treatments. Here, we have isolated and genomically characterized phages capable of infecting each of the acquired 77 reference serotypes of Klebsiella spp., including capsular types widespread among high-risk K. pneumoniae clones causing nosocomial infections. We demonstrated the possibility of isolating phages for all capsular types in the collection, revealing high capsular specificity among taxonomically related phages, in contrast to a few phages that exhibited broad-spectrum infection capabilities. To decipher the determinants of the specificity of these phages, we focused on their receptor-binding proteins, with particular attention to depolymerases. We also explored the possibility of designing a broad-spectrum phage cocktail based on phages isolated in reference capsular-type strains and determining the ability to lyse relevant clinical isolates. A combination of 12 phages capable of infecting 55% of the reference Klebsiella spp. serotypes was tested on a panel of carbapenem-resistant K. pneumoniae clinical isolates. Thirty-one percent of isolates were susceptible to the phage cocktail. However, our results suggest that in a highly variable encapsulated bacterial host, phage hunting must be directed to the specific Klebsiella isolates. This work is a step forward in the understanding of the complexity of phage–host interactions and highlights the importance of implementing precise and phage-specific strategies to treat K. pneumoniae infections worldwide.IMPORTANCEThe emergence of resistant bacteria is a serious global health problem. In the absence of effective treatments, phages are a personalized and effective therapeutic alternative. However, little is still known about phage–host interactions, which are key to implementing effective strategies. Here, we focus on the study of Klebsiella pneumoniae, a highly pathogenic encapsulated bacterium. The complexity and variability of the capsule, where in most cases phage receptors are found, make it difficult for phage-based treatments. Here, we isolated a large collection of Klebsiella phages against all the reference strains and in a cohort of clinical isolates. Our results suggest that clinical isolates represent a challenge, especially high-risk clones. Thus, we propose targeted phage hunting as an effective strategy to implement phage-derived therapies. Our results are a step forward for new phage-based strategies to control K. pneumoniae infections, highlighting the importance of understanding phage–host interactions to design personalized treatments against Klebsiella spp.

IntroductionCarbapenemase-Producing Escherichia coli (CP-Eco) isolates, though less prevalent than other CP-Enterobacterales, have the capacity to rapidly disseminate antibiotic resistance genes (ARGs) and cause serious difficult-to-treat infections. The aim of this study is phenotypically and genotypically characterizing CP-Eco isolates collected from Spain to better understand their resistance mechanisms and population structure.MethodsNinety representative isolates received from 2015 to 2020 from 25 provinces and 59 hospitals Spanish hospitals were included. Antibiotic susceptibility was determined according to EUCAST guidelines and whole-genome sequencing was performed. Antibiotic resistance and virulence-associated genes, phylogeny and population structure, and carbapenemase genes-carrying plasmids were analyzed.Results and discussionThe 90 CP-Eco isolates were highly polyclonal, where the most prevalent was ST131, detected in 14 (15.6%) of the isolates. The carbapenemase genes detected were blaOXA-48 (45.6%), blaVIM-1 (23.3%), blaNDM-1 (7.8%), blaKPC-3 (6.7%), and blaNDM-5 (6.7%). Forty (44.4%) were resistant to 6 or more antibiotic groups and the most active antibiotics were colistin (98.9%), plazomicin (92.2%) and cefiderocol (92.2%). Four of the seven cefiderocol-resistant isolates belonged to ST167 and six harbored blaNDM. Five of the plazomicin-resistant isolates harbored rmt. IncL plasmids were the most frequent (45.7%) and eight of these harbored blaVIM-1. blaOXA-48 was found in IncF plasmids in eight isolates. Metallo-β-lactamases were more frequent in isolates with resistance to six or more antibiotic groups, with their genes often present on the same plasmid/integron. ST131 isolates were associated with sat and pap virulence genes. This study highlights the genetic versatility of CP-Eco and its potential to disseminate ARGs and cause community and nosocomial infections.

ObjectivesTo describe and analyse erythromycin resistance trends in blood isolates of Staphylococcus aureus (EARS-Net Spain, 2004–2020) and the association of these trends with the consumption of macrolide, lincosamide, and streptogramin B (MLSB) antibiotics. To assess molecular changes that could be involved in erythromycin resistance trends by whole genome analysis of representative isolates.Materials and methodsWe collected antibiotic susceptibility data for all first-blood S. aureus isolates in patients from 47 Spanish hospitals according to EARS-Net criteria. MLSB antibiotic consumption was obtained from the Spanish Agency for Medicines and Medical Devices (2008–2020). We sequenced 137 representative isolates for core genome multilocus sequence typing, resistome and virulome analysis.ResultsFor the 36,612 invasive S. aureus isolates, methicillin resistance decreased from 26.4% in 2004 to 22.4% in 2020. Erythromycin resistance in methicillin-susceptible S. aureus (MSSA) increased from 13.6% in 2004 to 28.9% in 2020 (p

ABSTRACT In this study, we determined the presence of virulence factors in nonoutbreak, high-risk clones and other isolates belonging to less common sequence types associated with the spread of OXA-48-producing Klebsiella pneumoniae clinical isolates from The Netherlands (n = 61) and Spain (n = 53). Most isolates shared a chromosomally encoded core of virulence factors, including the enterobactin gene cluster, fimbrial fim and mrk gene clusters, and urea metabolism genes (ureAD). We observed a high diversity of K-Locus and K/O loci combinations, KL17 and KL24 (both 16%), and the O1/O2v1 locus (51%) being the most prevalent in our study. The most prevalent accessory virulence factor was the yersiniabactin gene cluster (66.7%). We found seven yersiniabactin lineages—ybt 9, ybt 10, ybt 13, ybt 14, ybt 16, ybt 17, and ybt 27—which were chromosomally embedded in seven integrative conjugative elements (ICEKp): ICEKp3, ICEKp4, ICEKp2, ICEKp5, ICEKp12, ICEKp10, and ICEKp22, respectively. Multidrug-resistant lineages—ST11, ST101, and ST405—were associated with ybt 10/ICEKp4, ybt 9/ICEKp3, and ybt 27/ICEKp22, respectively. The fimbrial adhesin kpi operon (kpiABCDEFG) was predominant among ST14, ST15, and ST405 isolates, as well as the ferric uptake system kfuABC, which was also predominant among ST101 isolates. No convergence of hypervirulence and resistance was observed in this collection of OXA-48-producing K. pneumoniae clinical isolates. Nevertheless, two isolates, ST133 and ST792, were positive for the genotoxin colibactin gene cluster (ICEKp10). In this study, the integrative conjugative element, ICEKp, was the major vehicle for yersiniabactin and colibactin gene clusters spreading. IMPORTANCE Convergence of multidrug resistance and hypervirulence in Klebsiella pneumoniae isolates has been reported mostly related to sporadic cases or small outbreaks. Nevertheless, little is known about the real prevalence of carbapenem-resistant hypervirulent K. pneumoniae since these two phenomena are often separately studied. In this study, we gathered information on the virulent content of nonoutbreak, high-risk clones (i.e., ST11, ST15, and ST405) and other less common STs associated with the spread of OXA-48-producing K. pneumoniae clinical isolates. The study of virulence content in nonoutbreak isolates can help us to expand information on the genomic landscape of virulence factors in K. pneumoniae population by identifying virulence markers and their mechanisms of spread. Surveillance should focus not only on antimicrobial resistance but also on virulence characteristics to avoid the spread of multidrug and (hyper)virulent K. pneumoniae that may cause untreatable and more severe infections.

ObjectivesLittle is known about IMP-producing Enterobacterales (IMP-Ent) in Europe. We analyzed at genomic and phenotypic level IMP-Ent isolates circulating in Spain in a 9-year period.Materials and methodsIMP-Ent isolates submitted to our reference laboratory were included. Antibiotic susceptibility was performed using microdilution method (EUCAST), and IMP-carbapenemase activity was measured with carbapenemase inhibitors, the β-CARBA method, the modified Hodge test (MHT), and the modified carbapenemase inhibition method (mCIM). All isolates collected were sequenced for high-resolution single-nucleotide polymorphism (SNP) typing, core genome multilocus sequence typing (cgMLST), and resistome analysis.ResultsFifty IMP-Ent isolates, collected from 19 hospitals in 13 Spanish provinces, were detected: Klebsiella pneumoniae (IMP-Kpn) (24; 48%), Enterobacter roggenkampii (13; 26%), Enterobacter hormaechei (8, 16%), Klebsiella oxytoca (two; 4%), Enterobacter asburiae (one, 2%), Serratia marcescens (one; 2%) and Escherichia coli (one; 2%). All isolates were positive by the MHT and β-CARBA tests; 48 (96%) were mCIM positive; 12 (24%) and 26 (52%) displayed positive inhibition with dipicolinic (meropenem) and EDTA (ertapenem), respectively. Five IMP-carbapenemase types were identified: IMP-8 (22; 44%), IMP-22 (17; 34%), IMP-13 (7; 14%), IMP-28 (two; 4%), and IMP-15 (two; 4%), predominating IMP-8 in K. pneumoniae and IMP-22 in E. roggenkampii. IMP-28 was exclusively identified in K. oxytoca and IMP-15 in E. hormaechei. Predominant STs were ST405 (29.2%), ST15 (25%) and ST464 (20.8%) in IMP-Kpn; ST96 (100%) in E. roggenkampii and ST182 (62.5%) in E. hormachei. Colistin and amikacin were the most active non-carbapenem antibiotics against IMP-Ent.ConclusionIMP-Ent isolates remain infrequent in Spain, although in recent years have been circulating causing nosocomial outbreaks, being IMP-8-producing K. pneumoniae and IMP-22-producing E. roggenkampii the most frequently detected in this study. Inhibition with EDTA or dipicolinic acid presented false negative results in some IMP-producing strains. Active microbiological and molecular surveillance is essential for a better comprehension and control of IMP-Ent dissemination.

ObjectivesCARB-ES-19 is a comprehensive, multicenter, nationwide study integrating whole-genome sequencing (WGS) in the surveillance of carbapenemase-producing K. pneumoniae (CP-Kpn) and E. coli (CP-Eco) to determine their incidence, geographical distribution, phylogeny, and resistance mechanisms in Spain.MethodsIn total, 71 hospitals, representing all 50 Spanish provinces, collected the first 10 isolates per hospital (February to May 2019); CPE isolates were first identified according to EUCAST (meropenem MIC > 0.12 mg/L with immunochromatography, colorimetric tests, carbapenem inactivation, or carbapenem hydrolysis with MALDI-TOF). Prevalence and incidence were calculated according to population denominators. Antibiotic susceptibility testing was performed using the microdilution method (EUCAST). All 403 isolates collected were sequenced for high-resolution single-nucleotide polymorphism (SNP) typing, core genome multilocus sequence typing (cgMLST), and resistome analysis.ResultsIn total, 377 (93.5%) CP-Kpn and 26 (6.5%) CP-Eco isolates were collected from 62 (87.3%) hospitals in 46 (92%) provinces. CP-Kpn was more prevalent in the blood (5.8%, 50/853) than in the urine (1.4%, 201/14,464). The cumulative incidence for both CP-Kpn and CP-Eco was 0.05 per 100 admitted patients. The main carbapenemase genes identified in CP-Kpn were blaOXA–48 (263/377), blaKPC–3 (62/377), blaVIM–1 (28/377), and blaNDM–1 (12/377). All isolates were susceptible to at least two antibiotics. Interregional dissemination of eight high-risk CP-Kpn clones was detected, mainly ST307/OXA-48 (16.4%), ST11/OXA-48 (16.4%), and ST512-ST258/KPC (13.8%). ST512/KPC and ST15/OXA-48 were the most frequent bacteremia-causative clones. The average number of acquired resistance genes was higher in CP-Kpn (7.9) than in CP-Eco (5.5).ConclusionThis study serves as a first step toward WGS integration in the surveillance of carbapenemase-producing Enterobacterales in Spain. We detected important epidemiological changes, including increased CP-Kpn and CP-Eco prevalence and incidence compared to previous studies, wide interregional dissemination, and increased dissemination of high-risk clones, such as ST307/OXA-48 and ST512/KPC-3.

Circulating recombinant forms (CRFs) are important components of the HIV-1 pandemic. Those derived from recombination between subtype B and subsubtype F1, with 18 reported, most of them of South American origin, are among the most diverse. In this study, we identified a HIV-1 BF1 recombinant cluster that is expanding in Spain, transmitted mainly via heterosexual contact, which, analyzed in near full-length genomes in four viruses, exhibited a coincident BF1 mosaic structure, with 12 breakpoints, that fully coincided with that of two viruses (10BR_MG003 and 10BR_MG005) from Brazil, previously classified as CRF72_BF1. The three remaining Brazilian viruses (10BR_MG002, 10BR_MG004, and 10BR_MG008) previously identified as CRF72_BF1 exhibited mosaic structures highly similar, but not identical, to that of the Spanish viruses and to 10BR_MG003 and 10BR_MG005, with discrepant subtypes in two short genome segments, located in pol and gp120env. Based on these results, we propose that the five viruses from Brazil previously identified as CRF72_BF1 actually belong to two closely related CRFs, one comprising 10BR_MG002, 10BR_MG004, and 10BR_MG008, which keep their CRF72_BF1 designation, and the other, designated CRF122_BF1, comprising 10BR_MG003, 10BR_MG005, and the viruses of the identified Spanish cluster. Three other BF1 recombinant genomes, two from Brazil and one from Italy, previously identified as unique recombinant forms, were classified as CRF72_BF1. CRF122_BF1, but not CRF72_BF1, was associated with protease L89M substitution, which was reported to contribute to antiretroviral drug resistance. Phylodynamic analyses estimate the emergence of CRF122_BF1 in Brazil around 1987. Given their close phylogenetic relationship and similar structures, the grouping of CRF72_BF1 and CRF122_BF1 in a CRF family is proposed.

CRF47_BF is a circulating recombinant form (CRF) of the human immunodeficiency virus type 1 (HIV-1), the etiological agent of AIDS. CRF47_BF represents one of 19 CRFx_BFs and has a geographic focus in Spain, where it was first identified in 2010. Since its discovery, CRF47_BF has expanded considerably in Spain, predominantly through heterosexual contact (∼56% of the infections). Little is known, however, about the origin and diversity of this CRF or its epidemiological correlates, as very few samples have been available so far. This study conducts a phylogenetic analysis with representatives of all CRFx_BF sequence types along with HIV-1 M Group subtypes to validate that the CRF47_BF sequences share a unique evolutionary history. The CRFx_BF sequences cluster into a single, not well supported, clade that includes their dominant parent subtypes (B and F). This clade also includes subtype D and excludes sub-subtype F2. However, the CRF47_BF sequences all share a most recent common ancestor. Further analysis of this clade couples CRF47_BF protease-reverse transcriptase sequences and epidemiological data from an additional 87 samples collected throughout Spain, as well as additional CRF47_BF database sequences from Brazil and Spain to investigate the origin and phylodynamics of CRF47_BF. The Spanish region with the highest proportion of CRF47_BF samples in the data set was the Basque Country (43.7%) with Navarre next highest at 19.5%. We include in our analysis epidemiological data on host sex, mode of transmission, time of collection, and geographic region. The phylodynamic analysis indicates that CRF47_BF originated in Brazil around 1999–2000 and spread to Spain from Brazil in 2002–2003. The virus spread rapidly throughout Spain with an increase in population size from 2011 to 2015 and leveling off more recently. Three strongly supported clusters associated with Spanish regions (Basque Country, Navarre, and Aragon), together comprising 60.8% of the Spanish samples, were identified, one of which was also associated with transmission among men who have sex with men. The expansion in Spain of CRF47_BF, together with that of other CRFs and subtype variants of South American origin, previously reported, reflects the increasing relationship between the South American and European HIV-1 epidemics.

Viruses of HIV-1-infected individuals whose transmission is related group phylogenetically in transmission clusters (TCs). The study of the phylogenetic relations of these viruses and the factors associated with these individuals is essential to analyze the HIV-1 epidemic. In this study, we examine the role of TCs in the epidemiology of HIV-1 infection in Galicia and the Basque County, two regions of northern Spain. A total of 1,158 HIV-1-infected patients from both regions with new diagnoses (NDs) in 2013–2018 were included in the study. Partial HIV-1 pol sequences were analyzed phylogenetically by approximately maximum-likelihood with FastTree 2. In this analysis, 10,687 additional sequences from samples from HIV-1-infected individuals collected in Spain in 1999–2019 were also included to assign TC membership and to determine TCs’ sizes. TCs were defined as those which included viruses from ≥4 individuals, at least 50% of them Spaniards, and with ≥0.95 Shimodaira-Hasegawa-like node support in the phylogenetic tree. Factors associated to TCs were evaluated using odds ratios (OR) and their 95% CI. Fifty-one percent of NDs grouped in 162 TCs. Male patients (OR: 2.6; 95% CI: 1.5–4.7) and men having sex with men (MSM; OR: 2.1; 95% CI: 1.4–3.2) had higher odds of belonging to a TC compared to female and heterosexual patients, respectively. Individuals from Latin America (OR: 0.3; 95% CI: 0.2–0.4), North Africa (OR: 0.4; 95% CI: 0.2–1.0), and especially Sub-Saharan Africa (OR: 0.02; 95% CI: 0.003–0.2) were inversely associated to belonging to TCs compared to native Spaniards. Our results show that TCs are important components of the HIV-1 epidemics in the two Spanish regions studied, where transmission between MSM is predominant. The majority of migrants were infected with viruses not belonging to TCs that expand in Spain. Molecular epidemiology is essential to identify local peculiarities of HIV-1 propagation. The early detection of TCs and prevention of their expansion, implementing effective control measures, could reduce HIV-1 infections.

During the COVID-19 pandemic, intensive care units (ICUs) operated at or above capacity, and the number of ICU patients coinfected by nosocomial microorganisms increased. Here, we characterize the population structure and resistance mechanisms of carbapenemase-producing Klebsiella pneumoniae (CP-Kpn) from COVID-19 ICU patients and compare them to pre-pandemic populations of CP-Kpn. We analyzed 84 CP-Kpn isolates obtained during the pandemic and 74 CP-Kpn isolates obtained during the pre-pandemic period (2019) by whole genome sequencing, core genome multilocus sequence typing, plasmid reconstruction, and antibiotic susceptibility tests. More CP-Kpn COVID-19 isolates produced OXA-48 (60/84, 71.4%) and VIM-1 (18/84, 21.4%) than KPC (8/84, 9.5%). Fewer pre-pandemic CP-Kpn isolates produced VIM-1 (7/74, 9.5%). Cefiderocol (97.3–100%) and plazomicin (97.5–100%) had the highest antibiotic activity against pandemic and pre-pandemic isolates. Sequence type 307 (ST307) was the most widely distributed ST in both groups. VIM-1-producing isolates belonging to ST307, ST17, ST321 and ST485, (STs infrequently associated to VIM-1) were detected during the COVID-19 period. Class 1 integron Int1-blaVIM-1-aac(6′)-1b-dfrB1-aadAI-catB2-qacEΔ1/sul1, found on an IncL plasmid of approximately 70,000 bp, carried blaVIM-1 in ST307, ST17, ST485, and ST321 isolates. Thus, CP-Kpn populations from pandemic and pre-pandemic periods have similarities. However, VIM-1 isolates associated with atypical STs increased during the pandemic, which warrants additional monitoring and surveillance.