The impact of antibiotics on the gut microbiota of children recovering from watery diarrhoea.

Infectious diarrhoeal diseases remain a substantial health burden in young children in low- and middle-income countries. The disease and its variable treatment options significantly alter the gut microbiome, which may affect clinical outcomes and overall gut health. Antibiotics are often prescribed, but their impact on the gut microbiome during recovery is unclear. Here, we used 16S rRNA sequencing to investigate changes in the gut microbiota in Vietnamese children with acute watery diarrhoea, and highlight the impact of antibiotic treatment on these changes. Our analyses identified that, regardless of treatment, recovery was characterised by reductions in Streptococcus and Rothia species and expansion of Bacteroides/Phocaeicola, Lachnospiraceae and Ruminococcacae taxa. Antibiotic treatment significantly delayed the temporal increases in alpha- and beta-diversity within patients, resulting in distinctive patterns of taxonomic change. These changes included a pronounced, transient overabundance of Enterococcus species and depletion of Bifidobacterium pseudocatenulatum. Our findings demonstrate that antibiotic treatment slows gut microbiota recovery in children following watery diarrhoea.

Multidrug resistance plasmids underlie clonal expansions and international spread of Salmonella enterica serotype 1,4,[5],12:i:- ST34 in Southeast Asia.

Salmonella enterica serotype 1,4,[5],12:i:- (Typhimurium monophasic variant) of sequence type (ST) 34 has emerged as the predominant pandemic genotype in recent decades. Despite increasing reports of resistance to antimicrobials in Southeast Asia, Salmonella ST34 population structure and evolution remained understudied in the region. Here we performed detailed genomic investigations on 454 ST34 genomes collected from Vietnam and diverse geographical sources to elucidate the pathogen's epidemiology, evolution and antimicrobial resistance. We showed that ST34 has been introduced into Vietnam in at least nine occasions since 2000, forming five co-circulating major clones responsible for paediatric diarrhoea and bloodstream infection. Most expansion events were associated with acquisitions of large multidrug resistance plasmids of IncHI2 or IncA/C2. Particularly, the self-conjugative IncA/C2 pST34VN2 (co-transferring blaCTX-M-55, mcr-3.1, and qnrS1) underlies local expansion and intercontinental spread in two separate ST34 clones. At the global scale, Southeast Asia was identified as a potential hub for the emergence and dissemination of multidrug resistant Salmonella ST34, and mutation analysis suggests of selection in antimicrobial responses and key virulence factors.

Tumour microbiomes and Fusobacterium genomics in Vietnamese colorectal cancer patients.

Perturbations in the gut microbiome have been associated with colorectal cancer (CRC), with the colonic overabundance of Fusobacterium nucleatum shown as the most consistent marker. Despite its significance in the promotion of CRC, genomic studies of Fusobacterium is limited. We enrolled 43 Vietnamese CRC patients and 25 participants with non-cancerous colorectal polyps to study the colonic microbiomes and genomic diversity of Fusobacterium in this population, using a combination of 16S rRNA gene profiling, anaerobic microbiology, and whole genome analysis. Oral bacteria, including F. nucleatum and Leptotrichia, were significantly more abundant in the tumour microbiomes. We obtained 53 Fusobacterium genomes, representing 26 strains, from the saliva, tumour and non-tumour tissues of six CRC patients. Isolates from the gut belonged to diverse F. nucleatum subspecies (nucleatum, animalis, vincentii, polymorphum) and a potential new subspecies of Fusobacterium periodonticum. The Fusobacterium population within each individual was distinct and in some cases diverse, with minimal intra-clonal variation. Phylogenetic analyses showed that within four individuals, tumour-associated Fusobacterium were clonal to those isolated from non-tumour tissues. Genes encoding major virulence factors (Fap2 and RadD) showed evidence of horizontal gene transfer. Our work provides a framework to understand the genomic diversity of Fusobacterium within the CRC patients, which can be exploited for the development of CRC diagnostic and therapeutic options targeting this oncobacterium.

Dynamic of the human gut microbiome under infectious diarrhea.

Despite the widespread implementation of sanitation, immunization and appropriate treatment, infectious diarrheal diseases still inflict a great health burden to children living in low resource settings. Conventional microbiology research in diarrhea have focused on the pathogen's biology and pathogenesis, but initial enteric infections could trigger subsequent perturbations in the gut microbiome, leading to short-term or long-term health effects. Conversely, such pre-existing perturbations could render children more vulnerable to enteropathogen colonization and diarrhea. Recent advances in DNA sequencing and bioinformatic analyses have been integrated in well-designed clinical and epidemiological studies, which allow us to track how the gut microbiome changes from disease onset to recovery. Here, we aim to summarize the current understanding on the diarrheal gut microbiome, stratified into different disease stages. Furthermore, we discuss how such perturbations could have impacts beyond an acute diarrhea episode, specifically on the child's nutritional status and the facilitation of antimicrobial resistance.

The evolutionary history of Shigella flexneri serotype 6 in Asia.

Shigella flexneri serotype 6 is an understudied cause of diarrhoeal diseases in developing countries, and has been proposed as one of the major targets for vaccine development against shigellosis. Despite being named as S. flexneri, Shigella flexneri serotype 6 is phylogenetically distinct from other S. flexneri serotypes and more closely related to S. boydii. This unique phylogenetic relationship and its low sampling frequency have hampered genomic research on this pathogen. Herein, by utilizing whole genome sequencing (WGS) and analyses of Shigella flexneri serotype 6 collected from epidemiological studies (1987-2013) in four Asian countries, we revealed its population structure and evolutionary history in the region. Phylogenetic analyses supported the delineation of Asian Shigella flexneri serotype 6 into two phylogenetic groups (PG-1 and -2). Notably, temporal phylogenetic approaches showed that extant Asian S. flexneri serotype 6 could be traced back to an inferred common ancestor arising in the 18th century. The dominant lineage PG-1 likely emerged in the 1970s, which coincided with the times to most recent common ancestors (tMRCAs) inferred from other major Southeast Asian S. flexneri serotypes. Similar to other S. flexneri serotypes in the same period in Asia, genomic analyses showed that resistance to first-generation antimicrobials was widespread, while resistance to more recent first-line antimicrobials was rare. These data also showed a number of gene inactivation and gene loss events, particularly on genes related to metabolism and synthesis of cellular appendages, emphasizing the continuing role of reductive evolution in the adaptation of the pathogen to an intracellular lifestyle. Together, our findings reveal insights into the genomic evolution of the understudied Shigella flexneri serotype 6, providing a new piece in the puzzle of Shigella epidemiology and evolution.

Exploring the Genomic Diversity and Antimicrobial Susceptibility of Bifidobacterium pseudocatenulatum in a Vietnamese Population.

Bifidobacterium pseudocatenulatum is a member of the human gut microbiota, and specific variants of B. pseudocatenulatum have been associated with health benefits such as improving gut integrity and reducing inflammatory responses. Here, we aimed to assess the genomic diversity and predicted metabolic profiles of B. pseudocatenulatum cells found colonizing the gut of healthy Vietnamese adults and children. We found that the population of B. pseudocatenulatum from each individual was distinct and highly diverse, with intraclonal variation attributed largely to a gain or loss of carbohydrate-utilizing enzymes. The B. pseudocatenulatum genomes were enriched with glycosyl hydrolases predicted to target plant-based nondigestible carbohydrates (GH13, GH43) but not host-derived glycans. Notably, the exopolysaccharide biosynthesis region from organisms isolated from healthy children showed extensive genetic diversity and was subject to a high degree of genetic modification. Antimicrobial susceptibility profiling revealed that the Vietnamese B. pseudocatenulatum cells were uniformly susceptible to beta-lactams but exhibited variable resistance to azithromycin, tetracycline, ciprofloxacin, and metronidazole. The genomic presence of ermX and tet variants conferred resistance against azithromycin and tetracycline, respectively; ciprofloxacin resistance was associated with a mutation(s) in the quinolone resistance-determining region (GyrA, S115, and/or D119). Our work provides the first detailed genomic and antimicrobial resistance characterization of B. pseudocatenulatum found in the Vietnamese population, which can be exploited for the rational design of probiotics. IMPORTANCE Bifidobacterium pseudocatenulatum is a beneficial member of the human gut microbiota. The organism can modulate inflammation and has probiotic potential, but its characteristics are largely strain dependent and associated with distinct genomic and biochemical features. Population-specific beneficial microbes represent a promising avenue for the development of potential probiotics, as they may exhibit a more suitable profile in the target population. This study investigates the underexplored diversity of B. pseudocatenulatum in Vietnam and provides more understanding of its genomic diversity, metabolic potential, and antimicrobial susceptibility. Such data from indigenous populations are essential for selecting probiotic candidates that can be accelerated into further preclinical and clinical investigations.

The Gut Microbiome of Healthy Vietnamese Adults and Children Is a Major Reservoir for Resistance Genes Against Critical Antimicrobials.

Antimicrobials are a key group of therapeutic agents. Given the animal/human population density and high antimicrobial consumption rate in Southeast Asia, the region is a focal area for monitoring antimicrobial resistance (AMR). Hypothesizing that the gastrointestinal tract of healthy individuals in Vietnam is a major source of AMR genes that may be transferred to pathogens, we performed shotgun metagenomic sequencing on fecal samples from 42 healthy Vietnamese people (21 children and 21 adults). We compared their microbiome profiles by age group and determined the composition of AMR genes. An analysis of the taxonomic profiles in the gut microbiome showed a clear differentiation by age, with young children (age <2 years) exhibiting a unique structure in comparison to adults and older children. We identified a total of 132 unique AMR genes, with macrolide, lincosamide, and streptogramin class resistance genes (ermB and lnuC) and tetracycline resistance genes being almost ubiquitous across the study population. Notably, samples from younger children were significantly associated with a greater number of AMR genes than other age groups, including key signature genes associated with AMR pathogens (eg, blaCTX-M, mphA). Our data suggest that the gut microbiome of those living in Vietnam, particularly young children, is a substantial reservoir of AMR genes, which can be transferred to circulating enteric pathogens. Our data support the generation of longitudinal cohort studies of those living in urban and rural areas of developing countries to understand the behavior of these AMR reservoirs and their role in generating multidrug-resistant and extensively drug-resistant pathogens.

Evolutionary histories and antimicrobial resistance in Shigella flexneri and Shigella sonnei in Southeast Asia.

Conventional disease surveillance for shigellosis in developing country settings relies on serotyping and low-resolution molecular typing, which fails to contextualise the evolutionary history of the genus. Here, we interrogated a collection of 1,804 Shigella whole genome sequences from organisms isolated in four continental Southeast Asian countries (Thailand, Vietnam, Laos, and Cambodia) over three decades to characterise the evolution of both S. flexneri and S. sonnei. We show that S. sonnei and each major S. flexneri serotype are comprised of genetically diverse populations, the majority of which were likely introduced into Southeast Asia in the 1970s-1990s. Intranational and regional dissemination allowed widespread propagation of both species across the region. Our data indicate that the epidemiology of S. sonnei and the major S. flexneri serotypes were characterised by frequent clonal replacement events, coinciding with changing susceptibility patterns against contemporaneous antimicrobials. We conclude that adaptation to antimicrobial pressure was pivotal to the recent evolutionary trajectory of Shigella in Southeast Asia.

Colonization with Staphylococcus aureus and Klebsiella pneumoniae causes infections in a Vietnamese intensive care unit.

Pre-existing colonization with Staphylococcus aureus or Klebsiella pneumoniae has been found to increase the risk of infection in intensive care patients. We previously conducted a longitudinal study to characterize colonization of these two organisms in patients admitted to intensive care in a hospital in southern Vietnam. Here, using genomic and phylogenetic analyses, we aimed to assess the contribution these colonizing organisms made to infections. We found that in the majority of patients infected with S. aureus or K. pneumoniae, the sequence type of the disease-causing (infecting) isolate was identical to that of corresponding colonizing organisms in the respective patient. Further in-depth analysis revealed that in patients infected by S. aureus ST188 and by K. pneumoniae ST17, ST23, ST25 and ST86, the infecting isolate was closely related to and exhibited limited genetic variation relative to pre-infection colonizing isolates. Multidrug-resistant S. aureus ST188 was identified as the predominant agent of colonization and infection. Colonization and infection by K. pneumoniae were characterized by organisms with limited antimicrobial resistance profiles but extensive repertoires of virulence genes. Our findings augment the understanding of the link between bacterial colonization and infection in a low-resource setting, and could facilitate the development of novel evidence-based approaches to prevent and treat infections in high-risk patients in intensive care.

Commensal Escherichia coli are a reservoir for the transfer of XDR plasmids into epidemic fluoroquinolone-resistant Shigella sonnei.

Despite the sporadic detection of fluoroquinolone-resistant Shigella in Asia in the early 2000s and the subsequent global spread of ciprofloxacin-resistant (cipR) Shigella sonnei from 2010, fluoroquinolones remain the recommended therapy for shigellosis1-7. The potential for cipR S. sonnei to develop resistance to alternative second-line drugs may further limit future treatment options8. Here, we aim to understand the evolution of novel antimicrobial resistant (AMR) S. sonnei variants after introduction into Vietnam. We found that cipR S. sonnei displaced the resident ciprofloxacin-susceptible (cipS) lineage while rapidly acquiring additional resistance to multiple alternative antimicrobial classes. We identified several independent acquisitions of extensively drug-resistant/multidrug-resistant-inducing plasmids, probably facilitated by horizontal transfer from commensals in the human gut. By characterizing commensal Escherichia coli from Shigella-infected and healthy children, we identified an extensive array of AMR genes and plasmids, including an identical multidrug-resistant plasmid isolated from both S. sonnei and E. coli in the gut of a single child. We additionally found that antimicrobial usage may impact plasmid transfer between commensal E. coli and S. sonnei. These results suggest that, in a setting with high antimicrobial use and a high prevalence of AMR commensals, cipR S. sonnei may be propelled towards pan-resistance by adherence to outdated international treatment guidelines.

Dissecting the molecular evolution of fluoroquinolone-resistant Shigella sonnei.

Shigella sonnei increasingly dominates the international epidemiological landscape of shigellosis. Treatment options for S. sonnei are dwindling due to resistance to several key antimicrobials, including the fluoroquinolones. Here we analyse nearly 400 S. sonnei whole genome sequences from both endemic and non-endemic regions to delineate the evolutionary history of the recently emergent fluoroquinolone-resistant S. sonnei. We reaffirm that extant resistant organisms belong to a single clonal expansion event. Our results indicate that sequential accumulation of defining mutations (gyrA-S83L, parC-S80I, and gyrA-D87G) led to the emergence of the fluoroquinolone-resistant S. sonnei population around 2007 in South Asia. This clone was then transmitted globally, resulting in establishments in Southeast Asia and Europe. Mutation analysis suggests that the clone became dominant through enhanced adaptation to oxidative stress. Experimental evolution reveals that under fluoroquinolone exposure in vitro, resistant S. sonnei develops further intolerance to the antimicrobial while the susceptible counterpart fails to attain complete resistance.

Out of Asia: the independent rise and global spread of fluoroquinolone-resistant Shigella.

Shigella are ranked among the most prevalent aetiologies of diarrhoeal disease worldwide, disproportionately affecting young children in developing countries and high-risk communities in developed settings. Antimicrobial treatment, most commonly with fluoroquinolones, is currently recommended for Shigella infections to alleviate symptoms and control disease transmission. Resistance to fluoroquinolones has emerged in differing Shigella species (S. dysenteriae, flexneri and sonnei) since the turn of the 21st century, originating in endemic areas, and latterly spreading into non-endemic regions. Despite occurring independently, the emergence of fluoroquinolone resistance in these different Shigella species shares striking similarities regarding their epidemiology and resistance mechanisms. Here, we review and discuss the current epidemiology of fluoroquinolone-resistant Shigella species, particularly in the light of recent genomic insights.

Rapid environmental effects on gut nematode susceptibility in rewilded mice.

Genetic and environmental factors shape host susceptibility to infection, but how and how rapidly environmental variation might alter the susceptibility of mammalian genotypes remains unknown. Here, we investigate the impacts of seminatural environments upon the nematode susceptibility profiles of inbred C57BL/6 mice. We hypothesized that natural exposure to microbes might directly (e.g., via trophic interactions) or indirectly (e.g., via microbe-induced immune responses) alter the hatching, growth, and survival of nematodes in mice housed outdoors. We found that while C57BL/6 mice are resistant to high doses of nematode (Trichuris muris) eggs under clean laboratory conditions, exposure to outdoor environments significantly increased their susceptibility to infection, as evidenced by increased worm burdens and worm biomass. Indeed, mice kept outdoors harbored as many worms as signal transducer and activator of transcription 6 (STAT6) knockout mice, which are genetically deficient in the type 2 immune response essential for clearing nematodes. Using 16S ribosomal RNA sequencing of fecal samples, we discovered enhanced microbial diversity and specific bacterial taxa predictive of nematode burden in outdoor mice. We also observed decreased type 2 and increased type 1 immune responses in lamina propria and mesenteric lymph node (MLN) cells from infected mice residing outdoors. Importantly, in our experimental design, different groups of mice received nematode eggs either before or after moving outdoors. This contrasting timing of rewilding revealed that enhanced hatching of worms was not sufficient to explain the increased worm burdens; instead, microbial enhancement and type 1 immune facilitation of worm growth and survival, as hypothesized, were also necessary to explain our results. These findings demonstrate that environment can rapidly and significantly shape gut microbial communities and mucosal responses to nematode infections, leading to variation in parasite expulsion rates among genetically similar hosts.

A Double-blind, Randomized, Placebo-controlled Trial of Lactobacillus acidophilus for the Treatment of Acute Watery Diarrhea in Vietnamese Children.

BACKGROUND: Probiotics are the most frequently prescribed treatment for children hospitalized with diarrhea in Vietnam. We were uncertain of the benefits of probiotics for the treatment of acute watery diarrhea in Vietnamese children. METHODS: We conducted a double-blind, placebo-controlled, randomized trial of children hospitalized with acute watery diarrhea in Vietnam. Children meeting the inclusion criteria (acute watery diarrhea) were randomized to receive either 2 daily oral doses of 2 × 10 CFUs of a local probiotic containing Lactobacillus acidophilus or placebo for 5 days as an adjunct to standard of care. The primary end point was time from the first dose of study medication to the start of the first 24-hour period without diarrhea. Secondary outcomes included the total duration of diarrhea and hospitalization, daily stool frequency, treatment failure, daily fecal concentrations of rotavirus and norovirus, and Lactobacillus colonization. RESULTS: One hundred and fifty children were randomized into each study group. The median time from the first dose of study medication to the start of the first 24-hour diarrhea-free period was 43 hours (interquartile range, 15-66 hours) in the placebo group and 35 hours (interquartile range, 20-68 hours) in the probiotic group (acceleration factor 1.09 [95% confidence interval, 0.78-1.51]; P = 0.62). There was also no evidence that probiotic treatment was efficacious in any of the predefined subgroups nor significantly associated with any secondary end point. CONCLUSIONS: This was a large double-blind, placebo-controlled trial in which the probiotic underwent longitudinal quality control. We found under these conditions that L. acidophilus was not beneficial in treating children with acute watery diarrhea.

South Asia as a Reservoir for the Global Spread of Ciprofloxacin-Resistant Shigella sonnei: A Cross-Sectional Study.

BACKGROUND: Antimicrobial resistance is a major issue in the Shigellae, particularly as a specific multidrug-resistant (MDR) lineage of Shigella sonnei (lineage III) is becoming globally dominant. Ciprofloxacin is a recommended treatment for Shigella infections. However, ciprofloxacin-resistant S. sonnei are being increasingly isolated in Asia and sporadically reported on other continents. We hypothesized that Asia is a primary hub for the recent international spread of ciprofloxacin-resistant S. sonnei. METHODS AND FINDINGS: We performed whole-genome sequencing on a collection of 60 contemporaneous ciprofloxacin-resistant S. sonnei isolated in four countries within Asia (Vietnam, n = 11; Bhutan, n = 12; Thailand, n = 1; Cambodia, n = 1) and two outside of Asia (Australia, n = 19; Ireland, n = 16). We reconstructed the recent evolutionary history of these organisms and combined these data with their geographical location of isolation. Placing these sequences into a global phylogeny, we found that all ciprofloxacin-resistant S. sonnei formed a single clade within a Central Asian expansion of lineage III. Furthermore, our data show that resistance to ciprofloxacin within S. sonnei may be globally attributed to a single clonal emergence event, encompassing sequential gyrA-S83L, parC-S80I, and gyrA-D87G mutations. Geographical data predict that South Asia is the likely primary source of these organisms, which are being regularly exported across Asia and intercontinentally into Australia, the United States and Europe. Our analysis was limited by the number of S. sonnei sequences available from diverse geographical areas and time periods, and we cannot discount the potential existence of other unsampled reservoir populations of antimicrobial-resistant S. sonnei. CONCLUSIONS: This study suggests that a single clone, which is widespread in South Asia, is likely driving the current intercontinental surge of ciprofloxacin-resistant S. sonnei and is capable of establishing endemic transmission in new locations. Despite being limited in geographical scope, our work has major implications for understanding the international transfer of antimicrobial-resistant pathogens, with S. sonnei acting as a tractable model for studying how antimicrobial-resistant Gram-negative bacteria spread globally.

Inducible colistin resistance via a disrupted plasmid-borne mcr-1 gene in a 2008 Vietnamese Shigella sonnei isolate.

OBJECTIVES: The objective of this study was to assess the presence of mcr-1 in Shigella sonnei isolated in Vietnam. METHODS: WGS data were analysed for the presence of the mcr-1 gene sequence. The association of mcr-1 with a plasmid was assessed by PCR and by conjugation. RESULTS: Through genome sequencing we identified a plasmid-associated inactive form of mcr-1 in a 2008 Vietnamese isolate of Shigella sonnei. The plasmid was conjugated into Escherichia coli and mcr-1 was activated upon exposure to colistin, resulting in highly colistin-resistant transconjugants. CONCLUSIONS: This is the first description of the mcr-1 gene in Shigella, which is atypical given that colistin is not ordinarily used to treat diarrhoea. Our data suggest the mcr-1 gene has been circulating in human-restricted pathogens for some time but likely carries a selective fitness cost.

A high-resolution genomic analysis of multidrug-resistant hospital outbreaks of Klebsiella pneumoniae.

Multidrug-resistant (MDR) Klebsiella pneumoniae has become a leading cause of nosocomial infections worldwide. Despite its prominence, little is known about the genetic diversity of K. pneumoniae in resource-poor hospital settings. Through whole-genome sequencing (WGS), we reconstructed an outbreak of MDR K. pneumoniae occurring on high-dependency wards in a hospital in Kathmandu during 2012 with a case-fatality rate of 75%. The WGS analysis permitted the identification of two MDR K. pneumoniae lineages causing distinct outbreaks within the complex endemic K. pneumoniae. Using phylogenetic reconstruction and lineage-specific PCR, our data predicted a scenario in which K. pneumoniae, circulating for 6 months before the outbreak, underwent a series of ward-specific clonal expansions after the acquisition of genes facilitating virulence and MDR. We suggest that the early detection of a specific NDM-1 containing lineage in 2011 would have alerted the high-dependency ward staff to intervene. We argue that some form of real-time genetic characterisation, alongside clade-specific PCR during an outbreak, should be factored into future healthcare infection control practices in both high- and low-income settings.

Introduction and establishment of fluoroquinolone-resistant Shigella sonnei into Bhutan.

Shigella sonnei is a major contributor to the global burden of diarrhoeal disease, generally associated with dysenteric diarrhoea in developed countries but also emerging in developing countries. The reason for the recent success of S. sonnei is unknown, but is likely catalysed by its ability to acquire resistance against multiple antimicrobials. Between 2011 and 2013, S. sonnei exhibiting resistance to fluoroquinolones, the first-line treatment recommended for shigellosis, emerged in Bhutan. Aiming to reconstruct the introduction and establishment of fluoroquinolone-resistant S. sonnei populations in Bhutan, we performed whole-genome sequencing on 71 S. sonnei samples isolated in Bhutan between 2011 and 2013.We found that these strains represented an expansion of a clade within the previously described lineage III, found specifically in Central Asia. Temporal phylogenetic reconstruction demonstrated that all of the sequenced Bhutanese S. sonnei diverged from a single ancestor that was introduced into Bhutan around 2006. Our data additionally predicted that fluoroquinolone resistance, conferred by mutations in gyrA and parC, arose prior to the introduction of the founder strain into Bhutan. Once established in Bhutan, these S. sonnei had access to a broad gene pool, as indicated by the acquisition of extended-spectrum ß-lactamase-encoding plasmids and genes encoding type IV pili. The data presented here outline a model for the introduction and maintenance of fluoroquinolone-resistant S. sonnei in a new setting. Given the current circulation of fluoroquinolone-resistant S. sonnei in Asia, we speculate that this pattern of introduction is being recapitulated across the region and beyond.