Chiara Crestani

Background: The global health threat of antimicrobial resistance involves the human, animal and environmental sectors. Data from Cameroon are scarce. Objectives: This study aimed to define extended-spectrum beta-lactamase-producing Escherichia coli (ESBL-Ec) rates and associated risk factors across the three sectors in Douala, Cameroon, and to define molecular characteristics of isolates. Methods: From June 2022 to May 2023, we collected blood cultures from hospitalized patients, rectal swabs from healthy pregnant women, caeca from broiler chickens and environmental wastewater. Samples were screened for ESBL-Ec using CHROMAgar™ ESBL and cefotaxime-supplemented Tryptone Bile X-glucuronide agar. Antimicrobial susceptibility testing was performed by disk diffusion following EUCAST guidelines. Whole-genome sequencing was carried out using Illumina technology. Results: Of 628 samples, 374 yielded ESBL-Ec. Prevalence was 54.6% (131/240) in pregnant women, 70.4% (169/240) in chickens and 93.1% (67/72) in wastewater. The proportion of ESBL-Ec among E. coli-positive-blood cultures was 9.2% (7/76). Multi-family household living was independently associated with ESBL-Ec carriage among pregnant women (adjusted odds ratio = 1.7, 95% CI 1.0–3.1, P = 0.03). High co-resistance (>70%) was observed for tetracycline, ciprofloxacin and trimethoprim/sulfamethoxazole. Sequencing of 32 isolates revealed 45 distinct resistance genes, including blaCTX-M-15 (n = 13, 40.6%), blaCTX-M-55 (n = 11, 34.4%) and last-resort antibiotic resistance genes mcr-1 and blaOXA-181. High-risk sequence types included ST131 (pregnant women) and ST10 (chickens). Notably, ST48 was shared between pregnant women and chickens, and ST155 between pregnant women and wastewater. Conclusion: Cross-sectoral ESBL-Ec in Douala exhibits high genomic diversity and alarming resistance. The occurrence of last-resort genes requires immediate One Health surveillance and coordinated interventions.

DNA methylation is a universal mechanism of epigenetic control that in bacteria can regulate genetic flux and gene expression, contributing to the emergence and success of discrete lineages. The CC17 lineage of the important multi-host pathogen group B Streptococcus has emerged as a hypervirulent clone in human neonates, the molecular basis for which remains elusive. In this study we identify a novel type II restriction modification system that is uniquely associated with the CC17 clade. Using in vitro and in vivo techniques, we show that this system acts as a barrier to genetic exchange, which drives the genetic recalcitrance and low levels of homologous recombination associated with the CC17 clade. Strikingly, the restriction modification system also directly regulates expression of the transcriptional activator NanR and promotes murine vaginal colonization and ascension to the uterus, implicating a role for DNA methylation in promoting persistence at polymicrobial mucosal surfaces. Together, these findings reveal for the first time the importance of restriction modification system activity in defining the lineage structure and virulence potential of group B Streptococcus, uncovering a novel mechanism that underpins the global success of the CC17 clade as a major neonatal pathogen.

Purpose: Diphtheria is caused by toxigenic strains of the Corynebacterium diphtheriae complex, mainly Corynebacterium diphtheriae and C. ulcerans . The diagnosis of diphtheria relies on detecting the diphtheria toxin (DT), for which Engler’s method of Elek’s immunoprecipitation test is the gold standard. A recent optimization of Engler’s method was proposed by Melnikov and colleagues, showing higher sensitivity for C. ulcerans . The goal of our study was to test and adapt this optimized method, and to re-analyze apparent non-toxigenic tox gene bearing (NTTB) isolates from our collection. Methods: We included 48 C. ulcerans, C. ramonii and C. diphtheriae isolates previously categorized as NTTB but for which no genetic explanation was found for the lack of DT expression. DT production was tested using Melnikov’s method with further modifications made by us: i ) increasing the antitoxin concentration; ii ) using 5°C as the incubation temperature after 24h; and iii ) modifying the layout of control and test strains on agar plates. Results: 35 of 38 C. ulcerans , 3 C. ramonii and 8 of 10 C. diphtheriae were found to be toxigenic. No genetic explanation was found regarding two non-toxigenic isolates (1 C. diphtheriae and 1 C. ulcerans ), whereas for one C. diphtheriae, IS1132 was detected upstream of the tox gene. Conclusion: Our modified implementation of Melnikov’s Elek test improved our ability to detect diphtheria toxin production. Most isolates previously considered as NTTB but with no genetic explanation, were shown to be toxigenic using the novel method.

Group B Streptococcus (GBS) is a pathogen of global relevance in neonatal and maternal disease as well as bovine mastitis. Two closely related clonal groups, denoted 103 and 314 (CG103/314) have been detected in humans and cattle on multiple continents in recent decades but are poorly characterised compared to other host-generalist clades. We examined their potential origins, host-switching events and presence of a suite of genetic markers for antimicrobial resistance, virulence and host association using a newly assembled dataset of 248 CG103/314 genomes from humans, cattle, and food originating from five continents. We detected multiple host switches between humans and cattle, and significant regional differences in AMR gene distribution, possibly reflecting local differences in antimicrobial use across countries and hosts and indicating a capacity for regional adaptation to selective pressures. Across the evolutionary history of CG103/314 from both host species, the prevalence of the Lac.2 operon, a genetic marker associated with bovine host adaptation, was high, whereas the prevalence of the scpB-lmb gene pair, a genetic marker of human host adaptation in other GBS clonal groups, was very low. All isolates with scpB-lmb were associated with human disease rather than carriage. Our dataset displayed biases typical of research into multi-host pathogens, when sampling is often focused on a specific host species or setting. Consistent, balanced, contemporaneous and sympatric sampling efforts across host species and sources are needed for a full understanding of the distribution and emergence of CG103/314 and similar multi-host pathogens impacting food safety and public health.

CRISPR-Cas9 immune systems protect bacteria from foreign DNA. However, immune efficiency is constrained by Cas9 off-target cleavages and toxicity. How bacteria regulate Cas9 to maximize protection while preventing autoimmunity is not understood. Here, we show that the master regulator of virulence, CovR, regulates CRISPR-Cas9 immunity against mobile genetic elements in Streptococcus agalactiae, a pathobiont responsible for invasive neonatal infections. We show that CovR binds to and represses a distal promoter of the cas operon, integrating immunity within the virulence regulatory network. The CovR-regulated promoter provides a controlled increase in off-target cleavages to counteract mutations in the target DNA, restores the potency of old immune memory, and stimulates the acquisition of new memory in response to recent infections. Regulation of Cas9 by CovR is conserved at the species level, with lineage specificities suggesting different adaptive trajectories. Altogether, we describe the coordinated regulation of immunity and virulence that enhances the bacterial immune repertoire during host-pathogen interaction.

High-throughput massive parallel sequencing has significantly improved bacterial pathogen genomics, diagnostics, and epidemiology. Despite its high accuracy, short-read sequencing struggles with the complete genome reconstruction and assembly of extrachromosomal elements such as plasmids. Long-read sequencing with Oxford Nanopore Technologies (ONT) presents an alternative that offers benefits including real-time sequencing and cost efficiency, particularly useful in resource-limited settings. However, the historically higher error rates of ONT data have so far limited its application in high-precision genomic typing. The recent release of ONT's R10.4.1 chemistry, with significantly improved raw read accuracy (Q20+), offers a potential solution to this problem. The aim of this study is to evaluate the performance of ONT's latest chemistry for bacterial genomic typing against the gold-standard Illumina technology, focusing on three respiratory pathogens of public health importance, Klebsiella pneumoniae, Bordetella pertussis, and Corynebacterium diphtheriae, and their related species. Using the Rapid Barcoding Kit V14, we generate and analyze genome assemblies with different basecalling models, at different simulated depths of coverage. ONT assemblies are compared to the Illumina reference for completeness and core genome multilocus sequence typing (cgMLST) accuracy (number of allelic mismatches). Our results show that genomes obtained from raw ONT data basecalled with Dorado SUP v0.9.0, assembled with Flye, and with a minimum coverage depth of 35×, optimized accuracy for all bacterial species tested. Error rates are consistently <0.5% for each cgMLST scheme, indicating that ONT R10.4.1 data are suitable for high-resolution genomic typing applied to outbreak investigations and public health surveillance.

Corynebacterium ulcerans is an emerging zoonotic pathogen which causes diphtheria-like infections. Although C. ulcerans is found in multiple domestic and wild animal species, most human cases are linked with pets. Our ability to decipher cross-host species transmission dynamics and to understand the emergence of clinically relevant clones (e.g., diphtheria toxin-positive) is currently hampered by a limited knowledge of C. ulcerans strain diversity and genome evolution. Here, we explore the genomic population structure and evolution of C. ulcerans with 582 isolates from diverse hosts and geographical locations. A newly developed core genome genotyping scheme captures the population structure of C. ulcerans both at deep and shallow phylogenetic levels, uncovering its main sublineages and offering high strain subtyping resolution for epidemiological surveillance. Additionally, we reveal the diversity and distribution of the diphtheria toxin gene (tox), and those of its associated mobile elements. Considering the entire Corynebacterium diphtheriae Species Complex, we find four diphtheria toxin families, five tox-prophage families, and a novel tox-carrying genetic element. We show that some toxin families are shared across Corynebacterium species, revealing tox-prophage cross-species transfer. Our work enhances knowledge on the ecology and evolution of C. ulcerans and provides a genomic framework for tracking the dissemination of emerging sublineages.

Group B Streptococcus (GBS) is a commensal bacterium that can cause severe infections in infants and adults with comorbidities. Resistance and reduced susceptibility to antibiotics are continually on the rise, and vaccines remain in development. Prophages have been reported to contribute to GBS evolution and pathogenicity. However, no studies are available to date on prophage contribution to the epidemiology of GBS isolates from humans in South America. In the context of an Argentinian multicentric study, we had previously phenotypically characterized 365 human GBS isolates from invasive disease, urinary infections and maternal colonization. These isolates had been whole-genome sequenced, and their prophage presence was bioinformatically determined. In this study, we genomically characterized the isolates and analysed the prophage content in the context of the epidemiological data. The phylogenetic analysis of the 365 genomes with 103 GBS from public databases revealed that Argentinian GBS were related to isolates from around the world. The most prevalent lineages, independent of the isolated source, were CC23/Ia and CC12/Ib. Genes encoding virulence factors involved in immune response evasion, tissue damage and adherence to host tissues, and invasion were found in all of the genomes in accordance with previously described lineage distribution. According to the prevalent capsular types and the distribution of specific virulence factors in Argentinian GBS, over 95% coverage would be expected from the vaccines currently under development. Antibiotic resistance determinants (ARDs) to at least one antibiotic class were found in 90% of the genomes, including novel mutations in pbp2x, while more than 15% carried ARDs to three or more classes. GBS collected from urinary infections carried a significantly higher proportion of ARDs to multiple antibiotic classes than the rest of the isolates. A total of 454 prophages were found among the 468 genomes analysed, which were classified into 23 prophage types. Prophage presence exhibited variations based on GBS clonal complex and capsular type. A possible association between an increased GBS pathogenicity and the carriage of prophages with integrase type GBSInt8 and/or the presence of genes that encode the Phox Homology domain has been observed. The highest prevalence of prophages per genome was found in lineages CC17/III and CC19/III, while the lowest amount was observed in CC12/Ib. Overall, the highest density of prophages, virulence factors and ARDs determinants was found in CC19 isolates, mostly of capsular type III, independent of the isolates’ source. This is the first analysis of the human-associated GBS population in South America based on whole-genome sequencing data, which will make a significant contribution to future studies on the global GBS population structure.

Group B Streptococcus (GBS) is a commensal bacterium that can cause severe infections in infants and adults with comorbidities. Resistance and reduced susceptibility to antibiotics is continually on the rise, and vaccines remain in-development. Prophages have been reported to contribute to GBS evolution and pathogenicity. However, no studies are available to date on prophage contribution to the epidemiology of GBS isolates from humans in South America. In the context of an Argentinian multicentric study, we had previously phenotypically characterised 365 human GBS isolates from invasive disease, urinary infections and maternal colonisation. These isolates had been whole genome sequenced and their prophage presence bioinformatically determined. In this study, we genomically characterised the isolates and analysed the prophage content in the context of the epidemiological data. The phylogenetic analysis of the 365 genomes with 103 GBS from public databases revealed that Argentinian GBS were related to isolates from around the world. The most prevalent lineages, independently of the isolate source, were CC23/Ia and CC12/Ib. Genes encoding virulence factors involved in immune response evasion, tissue damage and adherence to host tissues and invasion were found in all of the genomes in accordance with previously described lineage distribution. According to the prevalent capsular types and the distribution of specific virulence factors in Argentinian GBS, over 95% coverage would be expected from the vaccines currently under development. Antibiotic resistance determinants (ARDs) to at least one antibiotic class were found in 90% of the genomes, including novel mutations in pbp 2x, while more than 15% carried ARDs to 3 or more classes. GBS collected from urinary infections carried a significantly higher proportion of ARDs to multiple antibiotic classes than the rest of the isolates. A total of 454 prophages were found among the 468 genomes analysed, which were classified into 23 prophage types. Prophage presence exhibited variations based on GBS clonal complex and capsular type. A possible association between an increased GBS pathogenicity and the carriage of prophages with integrase type GBS Int 8 and/or the presence of genes that encode the Phox Homology domain has been observed. The highest prevalence of prophages per genome was found in lineages CC17/III and CC19/III, while the lowest amount was observed in CC12/Ib. Overall, the highest density of prophages, virulence factors and ARDs determinants was found in CC19 isolates, mostly of capsular type III, independently of the isolates source. This is the first analysis of the human-associated GBS population in South America based on whole genome sequencing data, which will make a significant contribution to future studies on the global GBS population structure. Data summary: Supplementary results and figures can be found in Supplementary Material 1. Supplementary tables can be found in Supplementary Material 2. All datasets analysed in this study are detailed in the Supplementary Materials. Metadata about the genomes analysed can be found in the microreact project created for this study: https://microreact.org/project/gbs-pangenomic-analysis . Impact statement: In Latin America studies on the epidemiology of Group B Streptococcus (GBS) are scarce, especially those describing clonal complex and serotype distribution, and the role of prophages in GBS epidemiology has not been studied. This article addresses the first genomic characterisation of the human-isolated GBS population in Latin America based on whole genome sequencing data, with special focus on the analysis of prophage content. We determine the clonal complex and serotype distribution of 365 GBS isolates collected from clinical samples in an Argentinian multicenter study and analyse the presence of prophages and virulence and antibiotic resistance determinants in the context of the epidemiological data. Through these analyses, we were able to determine how GBS population structure in Argentina differs from other parts of the world and to predict the potential coverage of the in-development GBS vaccines. We also found a possible association between the carriage of certain types of prophages and an increased GBS pathogenicity. In the context of increased global efforts to develop new strategies to prevent GBS infections through vaccine development, this study makes a significant contribution to our understanding of the global GBS population structure.