Millennium Initiative for Collaborative Research on Bacterial Resistance

In 2024, Chile experienced an outbreak of invasive group A Streptococcus (iGAS) infections. Although increasing reports of GAS outbreaks have been described worldwide, it remains unclear whether these events are driven by increased virulence of circulating lineages or by lineage replacement. In parallel, the burden of hospitalized noninvasive GAS infections (h-niGAS), which may present with severe disease, remains poorly characterized. This study aimed to compare epidemiological, clinical, antimicrobial resistance, and genomic features of iGAS and h-niGAS infections in Chile over a six-year period. We analyzed 2,307 GAS isolates collected between 2018 and 2024, including 2,249 noninvasive isolates and 58 iGAS isolates. Noninvasive cases were classified as outpatient niGAS or hospitalized niGAS (h-niGAS). Antimicrobial susceptibility testing was performed for all isolates. emm typing was performed on isolates from hospitalized patients (28 iGAS and 94 h-niGAS). Whole-genome sequencing was performed on outbreak-related isolates, followed by virulence gene profiling and phylogenetic analysis. Clinical characteristics were compared between iGAS and non-iGAS cases and across time periods. Among niGAS isolates, 2,119 were obtained from outpatients and 130 from hospitalized patients. h-niGAS isolates showed the highest proportion of clindamycin resistance (42%), whereas iGAS isolates showed the lowest (16%). Notably, clindamycin resistance among iGAS increased from 0% during 2020-2023 to nearly 20% in 2024. The most frequent emm types were emm1, emm12, and emm4. The frequency of emm1 increased from 0% in 2020-2023 to 10% in 2024, while overall emm diversity declined markedly (Simpson's reciprocal index from 17.3 to 6.63). Clinically, iGAS infections were associated with immunosuppression, chronic liver disease, viral coinfection, and trauma. During the 2024 outbreak, iGAS cases did not differ clinically from those reported in 2018-2023; however, h-niGAS cases required more surgical procedures (p = 0.005) and medical evaluations (p = 0.025). Whole-genome sequencing revealed predominance of emm1, including three M1UK strains harboring all 27 defining single-nucleotide polymorphisms. emm1 isolates carried a higher number of virulence-associated genes compared with non-emm1 isolates (p < 0.05). Phylogenetic analysis showed close relatedness to strains from the United Kingdom, Argentina, and the United States. These findings suggest that the Chilean outbreak was driven by the expansion of the emm1 lineage rather than an increase in strain virulence. The identification of M1UK strains and the reduced emm diversity support a model of lineage introduction and clonal expansion. The significant clinical burden observed among h-niGAS cases underscores the importance of including hospitalized noninvasive infections in surveillance efforts. Ongoing integrated clinical and genomic surveillance of both iGAS and h-niGAS is essential to monitor emerging lineages and antimicrobial resistance trends.

Ceftazidime/avibactam (CZA) is indicated against multidrug-resistant Pseudomonas aeruginosa, particularly those that are carbapenem resistant. CZA resistance in P. aeruginosa producing PER, a class A extended-spectrum β-lactamase, has been well documented in vitro. However, data regarding clinical isolates are scarce. Our aim was to analyze the contribution of PER to CZA resistance in non-carbapenemase-producing P. aeruginosa clinical isolates that were ceftazidime and/or carbapenem non-susceptible. Antimicrobial susceptibility was determined through agar dilution and broth microdilution, while bla PER gene was screened through PCR. All PER-positive isolates and five PER-negative isolates were analyzed through Whole Genome Sequencing. The mutational resistome associated to CZA resistance was determined through sequence analysis of genes coding for PBPs 1b, 3 and 4, MexAB-OprM regulators MexZ, MexR, NalC and NalD, AmpC regulators AmpD and AmpR, and OprD porin. Loss of bla PER-3 gene was induced in a PER-positive isolate by successive passages at 43°C without antibiotics. Twenty-six of 287 isolates studied (9.1%) were CZA-resistant. Thirteen of 26 CZA-resistant isolates (50%) carried bla PER. One isolate carried bla PER but was CZA-susceptible. PER-producing isolates had significantly higher MICs for CZA, amikacin, gentamicin, ceftazidime, meropenem and ciprofloxacin than non-PER-producing isolates. All PER-producing isolates were ST309 and their bla PER-3 gene was associated to ISCR1, an insertion sequence known to mobilize adjacent DNA. PER-negative isolates were classified as ST41, ST235 (two isolates), ST395 and ST253. PER-negative isolates carried genes for narrow-spectrum β-lactamases and the mutational resistome showed that all isolates had one major alteration in at least one of the genes analyzed. Loss of bla PER-3 gene restored susceptibility to CZA, ceftolozane/tazobactam and other β-lactamsin the in vitro evolved isolate. PER-3-producing ST309 P. aeruginosa is a successful multidrug-resistant clone with blaPER-3 gene implicated in resistance to CZA and other β-lactams.

The siderophore-cephalosporin cefiderocol(FDC) presents a promising treatment option for carbapenem-resistant (CR) P. aeruginosa (PA). FDC circumvents traditional porin and efflux mediated resistance by utilizing TonB-dependent receptors (TBDRs) to access the periplasmic space. Emerging FDC resistance has been associated with loss of function mutations within TBDR genes or the regulatory genes controlling TBDR expression. Further, difficulties with antimicrobial susceptibility testing (AST) and unexpected negative clinical treatment outcomes have prompted concerns for heteroresistance, where a single lineage isolate contains resistant subpopulations not detectable by standard AST. This study aimed to evaluate the prevalence of TBDR mutations among clinical isolates of P. aeruginosa and the phenotypic effect on FDC susceptibility and heteroresistance. We evaluated the sequence of pirR , pirS , pirA , piuA or piuD from 498 unique isolates collected before the introduction of FDC from 4 clinical sites in Portland, OR (1), Houston, TX (2), and Santiago, Chile (1). At some clinical sites, TBDR mutations were seen in up to 25% of isolates, and insertion, deletion, or frameshift mutations were predicted to impair protein function were seen in 3% of all isolates (n=15). Using population analysis profile testing, we found that P. aeruginosa with major TBDR mutations were enriched for a heteroresistant phenotype and undergo a shift in the susceptibility distribution of the population as compared to susceptible strains with wild type TBDR genes. Our results indicate that mutations in TBDR genes predate the clinical introduction of FDC, and these mutations may predispose to the emergence of FDC resistance.

The breast microbiome presents a diverse microbial community that could affectshealth and disease states, in the context of breast cancer. Sequencingtechnologies have allowed describing the diversity and abundance of microbialcommunities among individuals. The complex tumoral microenvironment thatincludes the microbial composition could influence tumor growth. The imbalanceof diversity and abundance inside the microbial community, known as dysbiosisplays a crucial role in this context. One the most prevalent bacterial generadescribed in breast invasive carcinoma are Bacillus, Pseudomonas,Brevibacillus, Mycobacterium, Thermoviga, Acinetobacter, Corynebacterium,Paenibacillus, Ensifer, and Bacteroides.Paenibacills genus shows a relation with patient survival.When the Paenibacills genus increases its abundance in patientswith breast cancer, the survival probability decreases. Within this dysbioticenvironment, various bacterial metabolites could play a pivotal role in theprogression and modulation of breast cancer. Key bacterial metabolites, such ascadaverine, lipopolysaccharides (LPS), and trimethylamine N-oxide (TMAO), havebeen found to exhibit potential interactions within breast tissuemicroenvironments. Understanding the intricate relationships between dysbiosisand these metabolites in breast cancer may open new avenues for diagnosticbiomarkers and therapeutic targets. Further research is essential to unravel thespecific roles and mechanisms of these microbial metabolites in breast cancerprogression.

and a meta-analysis of the prevalence of antibiotic-resistant bacteria (ARB) in marine bivalves. These studies employed modern methodologies such as high-throughput qPCR and whole-40 genome sequencing. 41 with ARGs at both sites. In addition to their relevance in their specific geographical areas, 59these results provide valuable insights for potentially standardizing tracers for ARGs 60 dissemination in the environment. 61 In conclusion, these studies provide an excellent update on the role of aquatic 94 ecosystems in the evolution and dissemination of AMR globally. The editors encourage the 95 scientific community to take note of the results and challenges presented in this special issue. 96

Carbapenem-resistant Pseudomonas aeruginosa (CRPA) is one of the pathogens that urgently needs new drugs and new alternatives for its control. The primary strategy to combat this bacterium is combining treatments of beta-lactam with a beta-lactamase inhibitor. The most used combinations against P. aeruginosa are ceftazidime/avibactam (CZA) and ceftolozane/tazobactam (C/T). Although mechanisms leading to CZA and C/T resistance have already been described, among which are the resistance-nodulation-division (RND) efflux pumps, the role that these extrusion systems may play in CZA, and C/T baseline susceptibility of clinical isolates remains unknown. For this purpose, 161 isolates of non-carbapenemase-producing (Non-CP) CRPA were selected, and susceptibility tests to CZA and C/T were performed in the presence and absence of the RND efflux pumps inhibitor, Phenylalanine-arginine β-naphthylamide (PAβN). In the absence of PAβN, C/T showed markedly higher activity against Non-CP-CRPA isolates than observed for CZA. These results were even more evident in isolates classified as extremely-drug resistant (XDR) or with difficult-to-treat resistance (DTR), where CZA decreased its activity up to 55.2% and 20.0%, respectively, whereas C/T did it up to 82.8% (XDR), and 73.3% (DTR). The presence of PAβN showed an increase in both CZA (37.6%) and C/T (44.6%) activity, and 25.5% of Non-CP-CRPA isolates increased their susceptibility to these two combined antibiotics. However, statistical analysis showed that only the C/T susceptibility of Non-CP-CRPA isolates was significantly increased. Although the contribution of RND activity to CZA and C/T baseline susceptibility was generally low (two-fold decrease of minimal inhibitory concentrations [MIC]), a more evident contribution was observed in a non-minor proportion of the Non-CP-CRPA isolates affected by PAβN [CZA: 25.4% (15/59); C/T: 30% (21/70)]. These isolates presented significantly higher MIC values for C/T. Therefore, we conclude that RND efflux pumps are participating in the phenomenon of baseline susceptibility to CZA and, even more, to C/T. However, the genomic diversity of clinical isolates is so great that deeper analyzes are necessary to determine which elements are directly involved in this phenomenon.

ABSTRACTCarbapenem-resistant Acinetobacter baumannii (CRAb) is a public health threat accounting for a significant number of hospital-acquired infections. Despite the importance of this pathogen, there is scarce literature on A. baumannii molecular epidemiology and evolutionary pathways relevant to resistance emergence in South American strains. We analyzed the genomic context of 34 CRAb isolates recovered from clinical samples between 2010 and 2013 from two hospitals in Santiago, Chile, using whole-genome sequencing. Several Institut Pasteur scheme sequence types (STs) were identified among the 34 genomes studied here, including ST1, ST15, ST79, ST162, and ST109. No ST2 (the most widespread sequence type) strain was detected. Chilean isolates were phylogenetically closely related, forming lineages specific to South America (e.g., ST1, ST79, and ST15). The genomic contexts of the resistance genes were diverse: while genes were present in a plasmid in ST15 strains, all genes were chromosomal in ST79 strains. Different variants of a small Rep_3 plasmid played a central role in the acquisition of the oxa58 carbapenem and aacC2 aminoglycoside resistance genes in ST1, ST15, and ST79 strains. The aacC2 gene along with blaTEM were found in a novel transposon named Tn6925 here. Variants of Tn7 were also found to play an important role in the acquisition of the aadA1 and dfrA1 genes. This work draws a detailed picture of the genetic context of antibiotic resistance genes in a set of carbapenem-resistant A. baumannii strains recovered from two Chilean hospitals and reveals a complex evolutionary picture of antibiotic resistance gene acquisition events via multiple routes involving several mobile genetic elements.IMPORTANCE Treating infections caused by carbapenem-resistant A. baumannii (CRAb) has become a global challenge given that CRAb strains are also often resistant to a wide range of antibiotics. Analysis of whole-genome sequence data is now a standard approach for studying the genomic context of antibiotic resistance genes; however, genome sequence data from South American countries are scarce. Here, phylogenetic and genomic analyses of 34 CRAb strains recovered from 2010 to 2013 from two Chilean hospitals revealed a complex picture leading to the generation of resistant lineages specific to South America. From these isolates, we characterized several mobile genetic elements, some of which are described for the first time. The genome sequences and analyses presented here further our understanding of the mechanisms leading to multiple-drug resistance, extensive drug resistance, and pandrug resistance phenotypes in South America. Therefore, this is a significant contribution to elucidating the global molecular epidemiology of CRAb.