Anthrax, a severe zoonotic disease, is infrequently reported in anthrax-endemic regions of Pakistan. Despite clinical reports indicating its presence, particularly cutaneous anthrax, there is insufficient laboratory evidence regarding disease occurrence and environmental persistence. The present study aimed to confirm Bacillus anthracis presence, accountable for animal mortality and human infection, while exploring environmental transmission factors. Between March 2019 and July 2021, a total of 19 outbreaks were documented. Of these, 11 affected sheep/goats in Zhob district and 8 affected cattle/sheep in Bajour Agency. Clinical signs suggestive of Bacillus anthracis outbreak were observed in 11 animals. Blood and swab samples were collected for confirmation. The study followed a One Health approach, analyzing animal, environmental (soil/plant), and human samples. Of the 19 outbreaks, 11 were confirmed positive for anthrax based on growth characteristics, colony morphology, and PCR. Soil and plant root samples from the outbreak areas were collected and analyzed microscopically and molecularly. Cutaneous anthrax was observed in six humans, and swab samples were taken from the lesions. Human serum samples (n = 156) were tested for IgG antibodies against PA toxin and quantitative analysis of anthrax toxin receptor 1 (ANTXR1). Bacillus anthracis was detected in 65 out of 570 (11.40%) soil samples and 19 out of 190 (10%) plant root samples from the outbreak areas. Four out of six human samples from cutaneous anthrax lesions tested positive for Bacillus anthracis. Human anthrax seroprevalence was found to be 11% and 9% in two districts, with the highest rates among butchers and meat consumers. The highest ANTXR1 levels were observed in butchers, followed by meat consumers, farm employees, meat vendors, veterinarians, and farm owners. These findings highlight the persistence of anthrax in the region and emphasize the potential public health risks.

Bacillus anthracis, the causative agent of anthrax, is a highly virulent zoonotic pathogen primarily affecting domesticated and wild herbivores. Human exposure to B. anthracis is primarily through contact with infected animals or contaminated animal products. In Pakistan, where livestock vaccines are largely unavailable and infected carcasses are often disposed of improperly, the risk to humans, wildlife and livestock is significant. Currently, the diagnosis of anthrax infections and outbreak tracing necessitates the isolation and culturing of B. anthracis, a process that requires BSL-3 facilities. In this study, we show that positive identification, genome reconstruction and lineage assignment can be accomplished using bioinformatic analysis of DNA extracted directly from environmental samples that would otherwise provide the starting material for isolation and culturing. This approach does not require laboratory target enrichment as is necessary for other pathogens, due in part to the extremely high bacterial load in the bloodstream in the deceased animals. Using these methods, we greatly expand the knowledge of endemic B. anthracis in Pakistan. We provide the first reference B. anthracis genomes from Pakistan since the 1970s and identify A.Br.014 Aust94 as a minor circulating sublineage alongside the dominant A.Br.047 Vollum. Future work will focus on the limits of detection and will determine if this bioinformatic method can be expanded more broadly for B. anthracis or other pathogens to replace typical culture-based methods.

Bacterial biofilm formation involves multigenic signaling and regulatory pathways that control the transition from motile to sessile lifestyle, production of extracellular polymeric matrix, and maturation of the biofilm complex 3D structure. Biofilms are extensively studied because of their importance in biomedical, ecological and industrial settings. Genetic approaches based on gene inactivation are powerful for mechanistic studies but often are labor intensive, limiting systematic gene surveys to the most tractable bacterial hosts. Here, we adapted the CRISPR interference (CRISPRi) system for use in P. fluorescens . We found that CRISPRi is applicable to three genetically and physiologically diverse species, SBW25, WH6 and Pf0-1 and affords extended periods of time to study complex phenotypes such as cell morphology, motility and biofilm formation. In SBW25, CRISPRi-mediated silencing of the GacA/S two-component system and genes regulated by cylic-di-GMP produced phenotypes similar to those previously described after gene inactivation in various Pseudomonas . Combined with detailed confocal microscopy of biofilms, our study also revealed novel phenotypes associated with biofilm architecture and extracellular matrix biosynthesis as well as the potent inhibition of SBW25 biofilm formation mediated by the PFLU1114 protein. Thus, CRISPRi is a reliable and scalable approach to interrogate gene networks in the diverse P. fluorescens group.

Bacillus anthracis , the causative agent of anthrax, is a highly virulent zoonotic pathogen primarily affecting domesticated and wild herbivores. Human exposure to B. anthracis is primarily through contact with infected animals or contaminated animal products. In Pakistan, where livestock vaccines are largely unavailable and infected carcasses are often disposed of improperly, the risk to humans, wildlife and livestock is significant. Currently, diagnosis of anthrax infections and outbreak tracing necessitates the isolation and culturing of B. anthracis , a process that requires BSL-3 facilities. In this study, we show that positive identification, genome reconstruction and lineage assignment can be accomplished using bioinformatic analysis of DNA extracted directly from environmental samples that would otherwise provide the starting material for isolation and culturing. This approach does not require laboratory target enrichment as is necessary for other pathogens, due in part to the extremely high bacterial load in the bloodstream in the deceased animals. Using these methods, we greatly expand the knowledge of endemic B. anthracis in Pakistan. We provide the first reference B. anthracis genomes from Pakistan since the 1970s and identify A.Br.014 Aust94 as a minor circulating sublineage alongside dominant A.Br.047 Vollum. Future work will focus on limits of detection and will determine if this bioinformatic method can be expanded more broadly for B. anthracis or other pathogens to replace typical culture-based methods.

Anthrax is an endemic and undercharacterized zoonotic disease in the Middle East, including Jordan. Between 2018 and 2020, we conducted a comprehensive investigation of 13 confirmed anthrax outbreaks in Jordan, analyzing 822 samples from animal farm environments, including carcasses, asymptomatic livestock, and abiotic environmental surfaces. All samples were tested by qPCR targeting the chromosomal marker (ba177) and the plasmid marker pXO1 (pag). Among carcass samples, 75/195 (38.5%) were ba177-positive, of which 81% harbored the pXO1. Of specimens from live-animals and from environmental surfaces, 218/627 (35%) were positive by qPCR, likely reflecting environmental contamination during active outbreak periods. Serological analysis using anti-protective antigen (PA) ELISA revealed a high seroprevalence of 53% (75/141) among asymptomatic animals, indicating widespread sub-clinical exposure to B. anthracis antigens and previously undocumented endemicity. An integrated approach combining qPCR with ELISA demonstrated that 11% of seropositive animals with paired swab testing also yielded swab samples that were positive by qPCR, suggesting environment-to-host transition. Molecular strain typing using canonical SNP (canSNP) analysis identified the rare sublineage C.USA.A1055 within lineage C.Br.A1005 across two distinct outbreaks, suggesting the environmental persistence of this endemic lineage. Overall, our findings provide the first systematic molecular and serological surveillance baseline data for Jordan, demonstrating a complex genomic persistence and subclinical exposure landscape. This study suggests the need for enhanced surveillance strategies under the One Health framework to mitigate the risk of anthrax endemicity.