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Background: Bacillus cereus is a Gram-positive, spore-forming bacterium that produces a spectrum of effectors integral to bacterial niche adaptation and the development of various infections. Among those is EsxA, whose secretion depends on the EssC component of the type VII secretion system (T7SS). EsxA's roles within the bacterial cell are poorly understood, although postulations indicate that it may be involved in sporulation. However, the T7SS repertoire in B. cereus has not been reported, and its functions are unestablished. Methods: We used the type strain, B. cereus ATCC14579, to generate ΔessC mutant through homologous recombination using the homing endonuclease I-SceI mediated markerless gene replacement. Comparatively, we analyzed the culture supernatant of type strain and the ΔessC mutant through Liquid chromatography-tandem mass spectrometry (LC-MS/MS). We further generated T7SSb-specific gene mutations to explore the housekeeping roles of the T7SSb-dependent effectors. The sporulation process of B. cereus ATCC14579 and its mutants was observed microscopically through the classic Schaeffer-Fulton staining method. The spore viability of each strain in this study was established by enumerating the colony-forming units on LB agar. Results: Through LC-MS/MS, we identified a pair of nearly identical (94%) effector proteins named EsxA belonging to the sagEsxA-like subfamily of the WXG100 protein superfamily in the culture supernatant of the wild type and none in the ΔessC mutant. Homology analysis of the T7SSb gene cluster among B. cereus strains revealed diversity from the 3' end of essC, encoding additional substrates. Deletions in esxA1 and esxA2 neither altered cellular morphology nor growth rate, but the ΔesxA1ΔesxA2 deletion resulted in significantly fewer viable spores and an overall slower sporulation process. Within 24 h culture, more than 80% of wild-type cells formed endospores compared to less than 5% in the ΔesxA1ΔesxA2 mutant. The maximum spore ratios for the wild type and ΔesxA1ΔesxA2 were 0.96 and 0.72, respectively. Altogether, these results indicated that EsxA1 and EsxA2 work cooperatively and are required for sporulation in B. cereus ATCC14567. Conclusion: B. cereus ATCC14579 possesses two nearly identical T7SSb-dependent effectors belonging to the sagEsxA-like proteins. Simultaneous deletion of genes encoding these effectors significantly delayed and reduced sporulation, a novel finding for EsxA. [ABSTRACT FROM AUTHOR]

Background: Bacillus anthracis is a highly pathogenic bacterium that can cause lethal infection in animals and humans, making it a significant concern as a pathogen and biological agent. Consequently, accurate diagnosis of B. anthracis is critically important for public health. However, the identification of specific marker genes encoded in the B. anthracis chromosome is challenging due to the genetic similarity it shares with B. cereus and B. thuringiensis. Methods: The complete genomes of B. anthracis, B. cereus, B. thuringiensis, and B. weihenstephanensis were de novo annotated with Prokka, and these annotations were used by Roary to produce the pan-genome. B. anthracis exclusive genes were identified by Perl script, and their specificity was examined by nucleotide BLAST search. A local BLAST alignment was performed to confirm the presence of the identified genes across various B. anthracis strains. Multiplex polymerase chain reactions (PCR) were established based on the identified genes. Result: The distribution of genes among 151 whole-genome sequences exhibited three distinct major patterns, depending on the bacterial species and strains. Further comparative analysis between the three groups uncovered thirty chromosome-encoded genes exclusively present in B. anthracis strains. Of these, twenty were found in known lambda prophage regions, and ten were in previously undefined region of the chromosome. We established three distinct multiplex PCRs for the specific detection of B. anthracis by utilizing three of the identified genes, BA1698, BA5354, and BA5361. Conclusion: The study identified thirty chromosome-encoded genes specific to B.anthracis, encompassing previously described genes in known lambda prophage regions and nine newly discovered genes from an undefined gene region to the best of our knowledge. Three multiplex PCR assays offer an accurate and reliable alternative method for detecting B. anthracis. Furthermore, these genetic markers have value in anthrax vaccine development, and understanding the pathogenicity of B. anthracis. [ABSTRACT FROM AUTHOR]

Background: Tuberculosis medication nonadherence is a multi-dimensional public health problem with serious consequences worldwide. There is little information available for medication nonadherence in South Sudan. This study assessed the proportion, reasons, and associated factors for nonadherence among patients with TB in Wau Municipality, South Sudan. Methods: A health facility based cross-sectional study was conducted among 234 tuberculosis (TB) patients receiving first line anti-TB regimen in Wau Municipality. Urine isoniazid metabolite testing (IsoScreen®) was used to determine nonadherence (visualized by negative test results) and a questionnaire was used to describe the reasons for nonadherence. Modified poisson regression with robust standard errors was performed since the proportion of nonadherence was < 10%, to identify nonadherence associated factors using the WHO Multidimensional adherence model. Results: Out of 234 participants, 24.8% (95% CI, 19.2 − 30.3) were nonadherent to the TB treatment regimen. At multivariate analysis, nonadherence was significantly associated with: relief of symptoms (APR 1.93, 95% CI 1.12 − 3.34, p = 0.018), alcohol use (APR 2.12, 95% CI 1.33 − 3.96, p = 0.019) and waiting time to receive drugs (APR 1.77, 95% CI 1.11 − 2.83, p = 0.017). Conclusion: Tuberculosis medication nonadherence was high, and it's associated with patients' relived of symptoms, alcohol use, and prolonged waiting time at health facility. Hence, addressing these barriers and the use of multifaceted interventions e.g. counseling, health education and improve appointments are crucial to reduce nonadherence among patients with TB in South Sudan. [ABSTRACT FROM AUTHOR]

Introduction: Leptospirosis is a neglected emerging and zoonotic disease reported worldwide. This study sought to determine the molecular and serological prevalence of Leptospira spp. and the associated risk factors in slaughtered cattle from the Bahr El Ghazal region of South Sudan. Materials and methods: Between January 16th and February 25th, 2023, blood and urine samples were collected from 402 cattle at the Lokoloko Municipal Slaughterhouse in Western Bahr El-Ghazal State. Serum samples were tested using the microscopic agglutination test (MAT), with a panel of 12 serovars (sv) from 12 serogroups (sg) and 4 species (spp) of Leptospira spp. These serovars had been previously identified in Sudan and the East African region. Simultaneously, 400 corresponding urine samples were screened using qualitative real-time polymerase chain reaction (PCR) to detect the shedding of Leptospira spp. in urine. To identify the associated risk factors, the age, sex, breed and body condition score of each sampled cattle was noted at the time of sampling and subsequently analysed using logistic regression models. Results: Among the 402 serum samples screened, a substantial 81.8% (329/402, 95% CI 77.9–85.3) displayed seropositivity for Leptospira spp. with a MAT titre ≥ 100. The prevalence of urine shedding determined by PCR was 6% (23/400, 95% CI 3.8–8.4), while probable recent leptospirosis with a MAT ≥ 1:800 was observed in 33.1% (133/402, 95% CI 28.6–37.8) of the cattle. Multiple reactions were detected in 34.8% (140/402, 95% CI 30.6–39.5) serum samples. The seropositivity was against L. borgpetersenii sg. Tarassovi (78.6%; 316/402, 95% CI 74.4–82.3), followed by L. borgpetersenii sg. Ballum at 20.4% (82/402, 95% CI, 16.7–24.4%), L. kirschneri sg. Autumnalis At 8.7% (35/402, 95% CI 5.7–11.7), L. interrogans sg. of Pomona at 7.0% (28/402, 95% CI 4.5–9.5), and L. interrogans sg. Hebdomadis was 5.0% (20/402, 95% CI 2.8–7.2). Several risk factors are associated with seropositivity. Older animals (≥ 2 years) had 2.0 times greater odds (95% CI 1.14–3.5) of being seropositive than younger animals (< 2 years), P-value = 0.016. Female animals demonstrated 2.1 times greater odds (95% CI 1.2–3.6) of seropositivity than males did (P-value = 0.008). Additionally, Felata/Mbororo cattle exhibited 2.4 times greater odds (95% CI 1.3–4.5) of being seropositive than did local Nilotic cattle (P-value = 0.005). The agreement between the MAT and PCR results was poor, as indicated by a kappa statistic value of 0.001 and a P-value of 0.913. But there was a moderate agreement between MAT high titres ≥ 800 and PCR positivity with a kappa statistic value = 0.501 and a P-value < 0.001. Conclusion: In addition to the high seroprevalence, Leptospira spp. were found in the urine of slaughtered cattle, suggesting that leptospirosis is endemic to the study area. This finding underscores the significance of cattle as potential sources of infection for slaughterhouse workers, the general public, and other animal species. To address this issue effectively in the Bahr El Ghazal Region and South Sudan, a comprehensive strategy involving a multidisciplinary approach is essential to minimize disease among animals, hence reducing potential zoonotic risks to humans. [ABSTRACT FROM AUTHOR]

Antimicrobial resistance (AMR) among Escherichia coli from food animals is a rising problem, and heavy antimicrobial use in poultry is a contributing factor. In Zambia, studies linking poultry-associated AMR and antibiotic use (AMU) are rare. This study aimed to investigate commercial and medium-/small-scale poultry farmers' usage of antimicrobials based on a questionnaire survey in ten districts of Zambia. In addition, the study characterized extended-spectrum β-lactamase (ESBL)-producing E. coli isolates obtained from poultry in the same districts. Data regarding knowledge and usage of antimicrobials were collected from commercial and medium-/small-scale poultry farmers using a pre-tested structured questionnaire. At the same time, cloacal samples were collected and analyzed. One hundred and fifty E. coli isolates were tested for antimicrobial susceptibility using eight antibiotic classes. The isolates were further screened for ESBL production by streaking them on cefotaxime (CTX)-supplemented MacConkey agar, then subjecting them to sequencing on a NextSeq. The questionnaire survey showed that more medium-/small-scale than commercial poultry farmers used antimicrobials (OR = 7.70, 95% CI = 2.88–20.61) but less prescriptions (OR = 0.02, 95% CI = 0.00–0.08). Susceptibility testing revealed that resistance was highest to ampicillin (128/148, 86.5%) and tetracycline (101/136, 74.3%) and that the prevalence of multidrug resistance (MDR) (28/30, 93.3%) was high. Whole-genome sequencing (WGS) of eight (8/30, 26.7%) isolates with CTX Minimum Inhibitory Concentration (MIC) ≥ 4 µg/mL revealed the presence of ESBL-encoding genes blaCTX-M-14, blaCTX-M-55, and blaTEM. WGS also detected other AMR genes for quinolones, aminoglycosides, phenicols, tetracycline, macrolides, and folate-pathway antagonists. Altogether, the questionnaire survey results showed a higher proportion of AMU and lower prescription usage among medium-/small-scale farmers. In addition, our results emphasize the circulation of ESBL-producing E. coli strains with associated MDR. It is critical to educate farmers about AMR risks and to encourage responsible usage of antimicrobials. Furthermore, there is a need to strengthen regulations limiting access to antimicrobials. Finally, there is a need to establish a one health system to guide public health response. [ABSTRACT FROM AUTHOR]