Introduction and aims : Carbapenem-resistant Acinetobacter baumannii (CRAB) poses a serious global threat, prioritized by the WHO for new antibiotic development. The ECDC proposed the integration of genomic sequencing into surveillance. This study characterizes phenotypic and genotypic features of nationwide-collected CRAB isolates from Spanish hospitals (2016-2020). Materials and methods : A total of 822 CRAB isolates sent to the Spanish reference laboratory were analyzed, and 108 representative isolates were selected from 48 hospitals in 25 Spanish provinces. Antibiotic susceptibility was determined according to EUCAST guidelines, and WGS was performed by Illumina. Resistome, virulome, phylogeny (cgMLST; 2390 genes), insertion sequences and plasmids were analyzed. Results : Of the 108 representative isolates, 68.5% produced OXA-23, 10.2% OXA-24, 7.4% OXA-58, 1.9% NDM-1 and 2.7% others. 3.7% co-produced two acquired carbapenemases, and six that did not have them showed the ISAba1 upstream of the blaOXA-51 like. The predominant STs were ST2pas (82.4%) and ST218Oxf (61.1%). blaOXA-66 chromosomal carbapenemase allele was present in 75% of the 108 isolates, armA 16s rRNA methyltransferase gene in 61.1% and the kL-7/OCL-18 was present in 59.3% of the isolates. The most active antibiotic was colistin. 65.4% of invasive cases were caused by isolates harboring all 38 virulence genes tested. In the 64.8% of isolates a plasmid type was detected, mainly pS32-1-like (54.6%). Conclusions : In Spain, CRAB dissemination is driven by interregional spread of isolates belonging to the ST218Oxf/ST2Pas clones, characterized by the blaOXA-23/blaOXA-66 genotype, the presence of armA, the KL7/OCL18 capsular profile and the presence of pS32-1 plasmid.

Circulating recombinant forms (CRFs) are important components of the HIV-1 pandemic. Previous studies have reported the propagation of diverse HIV-1 CRFs of South American origin in Europe. Here, through protease-reverse transcriptase (Pr-RT) and integrase sequence analyses, we identify a Spanish cluster (BC3) branching with a Brazilian virus (10BR_RJ009) previously classified as CRF146_BC. In Pr-RT, BC3 comprised 14 viruses and was nested within a larger cluster, comprising 22 Brazilian viruses and 1 Spanish virus branching outside of BC3. Near full-length genome analyses of five BC3 viruses revealed mosaic structures identical to 10BR_RJ009, with two breakpoints delimiting a ~0.3 kb subtype B fragment within an otherwise subtype C genome. Two other Brazilian viruses previously classified as CRF146_BC (10BR_RJ039 and 01_BR_RGS69) had one and two additional short subtype B fragments, respectively, and failed to group with the 10BR_RJ009/BC3 cluster in subtype C fragments. Based on these results, we contend that 10BR_RJ009 and BC3 viruses, but not 10BR_RJ039 and 01_BR_RGS69, should be classified as CRF146_BC. Bayesian analyses estimated the CRF146_BC emergence in Brazil to be around 1999 and its introduction in Europe around 2011. CRF146_BC is the 10th CRF of South American origin reported to circulate in Europe, reflecting the relationship between South American and European HIV-1 epidemics.

BACKGROUND:Antimicrobial resistance among Enterobacter cloacae complex and Klebsiella aerogenes is a growing clinical challenge, often driven by AmpC β-lactamase hyperproduction. This phenotype limits therapeutic options to cefepime or carbapenems. OBJECTIVES:To assess the in vitro activity of conventional β-lactams, such as cefepime, piperacillin/tazobactam or carbapenems, and new-generation agents, including ceftazidime/avibactam, cefepime/taniborbactam, imipenem/relebactam, meropenem/vaborbactam, and cefiderocol, against AmpC-hyperproducing E. cloacae complex and K. aerogenes. METHODS:A total of 314 clinical isolates (200 E. cloacae complex and 114 K. aerogenes) recovered between March and December 2024, from eight Spanish centres were tested by broth microdilution (EUCAST). AmpC hyperproduction was confirmed phenotypically and carbapenemase was screened phenotypically/molecularly. WGS was performed for (i) 84 E. cloacae complex isolates, and (ii) six isolates with reduced susceptibility to at least one novel agent. RESULTS:High resistance rates were observed for piperacillin/tazobactam (up to 82.4%) and cefepime (up to 20.5%), whereas carbapenems showed variable activity. New-generation agents exhibited excellent activity, with susceptibility rates ≥99% in both species. Resistance to at least one new agent was found in only six isolates. In the WGS-analysed E. cloacae complex subset, Enterobacter hormaechei predominated (75%) with high STs and blaACT allele diversity. In the six reduced-susceptibility isolates, no carbapenemase or explanatory acquired β-lactamase was found. CONCLUSIONS:Our findings underscore the need to take this phenotype into account in clinical practice and confirm the limited activity of conventional β-lactams against AmpC-hyperproducing E. cloacae complex and K. aerogenes, and support the use of newer agents as effective alternatives.

Antimicrobial resistance is a growing global healthcare crisis, driven by the rapid spread of resistant pathogens that compromise existing treatments. Carbapenem-resistant Klebsiella pneumoniae is a major public health threat, requiring novel therapeutic strategies. Phage therapy, which employs phages to target bacterial pathogens, is a promising approach, particularly when combined with antibiotics to enhance efficacy through synergistic interactions. In this study, time-kill curve assays were used to evaluate the synergy between the lytic phage vB_Kpn_2-P4 and meropenem against twelve K. pneumoniae clinical isolates from Spanish hospitals that carried diverse carbapenemases. Notably, in OXA-48-producing isolates, this combination prevented the emergence of resistant mutants, highlighting the therapeutic potential of phage-antibiotic synergy. The observed effect, linked to the presence of the pOXA-48 plasmid, suggests a promising strategy for combating multidrug-resistant bacteria.

Multi-Locus Sequence Typing (MLST) is a key method for allocation of Sequence Types (STs) for bacterial isolates. Traditionally, this is performed by the Sanger sequencing method, which can be highly time-consuming and laborious. In this study, we present NanoMLST, a high-throughput MLST workflow using multiplex PCR, Oxford Nanopore Technologies Next-Generation Sequencing, and the Krocus program for typing ESKAPE + E pathogens (Enterococcus faecium [E. faecium], Staphylococcus aureus, Klebsiella pneumoniae [K. pneumoniae], Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp., and Escherichia coli). Bacterial isolates were obtained from the Hospital Universitario La Paz's Microbiology Department and the Centro Nacional de Microbiología. Primers that can be multiplexed in a single PCR reaction were designed for the seven housekeeping genes for each species. DNA was extracted from single colonies by heating at 95°C for 10 min, mechanical lysis at 4.20 m/s for 2 min, and then by the MagCore extraction system. Multiplex PCRs were then performed with the respective primer mixes for each species, and libraries were prepared for sequencing by ONT Flongle cells. The Krocus program was then used to determine the STs from the raw FastQ reads. STs for 221 isolates were obtained through this workflow with an average time of 12 h per 24 isolates. In line with local data, the K. pneumoniae and E. faecium isolates were relatively oligoclonal, while the rest were polyclonal. STs from representative isolates showed 100% concordance between Sanger sequencing and the proposed workflow. NanoMLST offers a fast, cheaper, and less labor-intensive alternative for large-scale MLST applications targeting clinically important pathogens.

The global emergence of carbapenem-resistant Acinetobacter baumanii (CRAB) represents a significant public health threat. In the summer of 2022, a polyclonal CRAB outbreak occurred in our hospital, marking the first detection of an NDM-1 plus OXA-23 co-producing A. baumannii strain in Spain. The aim of this study was to phenotypically and genotypically characterize the clonal spread of NDM-1 and OXA-23 co-producing A. baumannii isolates and to describe the infection control measures implemented to contain the outbreak. Patients with multidrug-resistant A. baumannii isolates (July 2022–May 2023) were included in the study. Isolates were identified via MALDI-TOF, and antimicrobial susceptibility was tested using a broth microdilution method (DKMGN SensititreTM panels). Whole-genome sequencing was performed on 24 representative isolates. Phylogenetic analysis was performed using Ridom SeqSphere+ (cgMLST), while sequence typing was performed using ARIBA (Pasteur and Oxford schemes). A. baumannii isolates from the affected patients belonged to five different sequence types. The two main STs were ST1Pas/ST231Oxf (NDM-1- and OXA-23-co-producing), which accounted for 58%, and ST136Pas/ST406Oxf (OXA-23-producing), which accounted for 21%. All isolates were resistant to fluoroquinolones, trimethoprim/sulfamethoxazole, aminoglycosides, and carbapenems. In addition, 8% were resistant to colistin and 17% to cefiderocol. Finally, the affected patients were cohorted, and a thorough cleaning of the affected units was carried out. This study documents the clonal spread of an NDM-1- and OXA-23-co-producing A. baumannii strain in Spain, linked to a Libyan patient, highlighting the risk of cross-border spread. Although infection control measures successfully contained the outbreak, surveillance is essential as the incidence of CRAB infections is expected to increase.

OBJECTIVE:Citrobacter spp. are commonly found in the human intestine and can cause antibiotic-resistant infections. Inhibitors of LpxC, an enzyme involved in the synthesis of lipid A, have demonstrated potent activity against multiple Gram-negative species, but their activity in Citrobacter spp. has not been well-characterised. The objective of the present study was to evaluate the antimicrobial activity of LpxC inhibitors against carbapenemase-producing Citrobacter spp. clinical isolates. METHODS:The MICs of LpxC-2, LpxC-4, and CHIR-090 LpxC inhibitors were determined for 61 clinical isolates with high genetic diversity. Time-kill assays with the three LpxC inhibitors were performed with two Citrobacter spp. strains, with low (Cit 52) and high (Cit 110) LpxC inhibitor MIC values. RESULTS:LpxC-4 was shown to be the most potent inhibitor (MIC90 = 0.5 mg/L). In general, the three LpxC inhibitors demonstrated similar activity between Citrobacter spp. isolates harbouring type A (n = 11 isolates), B (n = 39 isolates), and D (n = 11 isolates) carbapenemases. All LpxC inhibitors showed bactericidal activity at concentrations 4× the MIC and no activity at ¼× the MIC at 24 h for the two Citrobacter spp. strains analysed. CONCLUSIONS:Although LpxC-4 had a lower MIC90 value compared with CHIR-090, CHIR-090 showed a bactericidal effect at short times and prevented the regrowth of both isolates in the time-kill assays. In terms of cross-resistance, all three structurally similar LpxC inhibitors showed a moderate positive correlation between their activity.

BACKGROUND:Community-acquired (CA) and healthcare-associated (HCA) infections caused by carbapenemase-producing Enterobacterales (CPE) are not well characterized. The objective was to provide detailed information about the clinical and molecular epidemiological features of nosocomial, HCA and CA infections caused by carbapenemase-producing Klebsiella pneumoniae (CP-Kp) and Escherichia coli (CP-Ec). METHODS:A prospective cohort study was performed in 59 Spanish hospitals from February to March 2019, including the first 10 consecutive patients from whom CP-Kp or CP-Ec were isolated. Patients were stratified according to acquisition type. A multivariate analysis was performed to identify the impact of acquisition type in 30-day mortality. RESULTS:Overall, 386 patients were included (363 [94%] with CP-Kp and 23 [6%] CP-Ec); in 296 patients (76.3%), the CPE was causing an infection. Acquisition was CA in 31 (8.0%) patients, HCA in 183 (47.4%) and nosocomial in 172 (48.3%). Among patients with a HCA acquisition, 100 (54.6%) had been previously admitted to hospital and 71 (38.8%) were nursing home residents. Urinary tract infections accounted for 19/23 (82.6%), 89/130 (68.5%) and 42/143 (29.4%) of CA, HCA and nosocomial infections, respectively. Overall, 68 infections (23%) were bacteremia (8.7%, 17.7% and 30.1% of CA, HCA and nosocomial, respectively). Mortality in infections was 28% (13%, 14.6% and 42.7% of CA, HCA and nosocomial, respectively). Nosocomial bloodstream infections were associated with increased odds for mortality (adjusted OR, 4.00; 95%CI 1.21-13.19). CONCLUSIONS:HCA and CA infections caused by CPE are frequent and clinically significant. This information may be useful for a better understanding of the epidemiology of CPE.

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.

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. IMPORTANCE The 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.

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.

[This corrects the article DOI: 10.3389/fmicb.2022.918362.].

Objectives To 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 (MLS B ) antibiotics. To assess molecular changes that could be involved in erythromycin resistance trends by whole genome analysis of representative isolates. Materials and methods We collected antibiotic susceptibility data for all first-blood S. aureus isolates in patients from 47 Spanish hospitals according to EARS-Net criteria. MLS B 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. Results For 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 < 0.001); however, it decreased from 68.7 to 61.8% ( p < 0.0001) in methicillin-resistant S. aureus (MRSA). Total consumption of MLS B antibiotics increased from 2.72 defined daily doses per 1,000 inhabitants per day (DID) in 2014 to 3.24 DID in 2016. By WGS, the macrolide resistance genes detected were erm (59.8%), msrA (46%), and mphC (45.2%). The erm genes were more prevalent in MSSA (44/57, 77.2%) than in MRSA (38/80, 47.5%). Most of the erm genes identified in MSSA after 2013 differed from the predominant ermC gene (17/22, 77.3%), largely because ermT was significantly associated with MSSA after 2013 (11/29, 37.9%). All 13 ermT isolates in this study, except one, belonged to ST398 and came from 10 hospitals and six Spanish provinces. Conclusion The significant increase in erythromycin resistance in blood MSSA correlated with the consumption of the MLS B antibiotics in Spain. These preliminary data seem support the hypothesis that the human ST398 MSSA clade with ermT -mediated resistance to erythromycin may be involved in this trend.

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 place the CRF47_BF sequences in a definitive phylogenetic context. The CRFx_BF sequences cluster into a single, not well supported, clade that includes their dominant parent subtypes (subtype B and subtype F). This clade also includes subtype D and excludes subsubtype F2. 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, coupled with 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 1993-1994 and spread to Spain from Brazil in approximately 1999-2000. The virus spread rapidly throughout Spain with increasing population sizes prior to 2010 and again between 2010 and 2017 with population declines to 2019 and a steady state through 2020. 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

Objectives:Little 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 methods:IMP-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.Results:Fifty 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.Conclusion:IMP-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.

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.

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.

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.