Trends, patterns and relationship of antimicrobial use and resistance in bacterial isolates tested between 2015-2020 in a national referral hospital of Zambia.

Increased antimicrobial resistance (AMR) among bacteria underscores the need to strengthen AMR surveillance and promote data-based prescribing. To evaluate trends and associations between antimicrobial usage (AMU) and AMR, we explored a dataset of 34,672 bacterial isolates collected between 2015 and 2020 from clinical samples at the University Teaching Hospital (UTH) in Lusaka, Zambia. The most frequently isolated species were Escherichia coli (4,986/34,672; 14.4%), Staphylococcus aureus (3,941/34,672; 11.4%), and Klebsiella pneumoniae (3,796/34,672; 10.9%). Of the 16 drugs (eight classes) tested, only amikacin and imipenem showed good (> 50%) antimicrobial activity against both E. coli and K. pneumoniae, while nitrofurantoin was effective only in E. coli. Furthermore, 38.8% (1,934/4,980) of E. coli and 52.4% (2,079/3,791) of K. pneumoniae isolates displayed multidrug resistance (MDR) patterns on antimicrobial susceptibility tests. Among S. aureus isolates, 44.6% (973/2,181) were classified as methicillin-resistant (MRSA). Notably, all the MRSA exhibited MDR patterns. The annual hospital AMR rates varied over time, while there was a weak positive relationship (r = 0.38, 95% CI = 0.11-0.60) between the monthly use of third-generation cephalosporins (3GCs) and 3GC resistance among Enterobacterales. Overall, the results revealed high AMR rates that fluctuated over time, with a weak positive relationship between 3GC use and resistance. To our knowledge, this is the first report to evaluate the association between AMU and AMR in Zambia. Our results highlight the need to strengthen antimicrobial stewardship programs and optimize AMU in hospital settings.

Detection of Extended-Spectrum Beta-Lactamase (ESBL)-Producing Enterobacteriaceae from Diseased Broiler Chickens in Lusaka District, Zambia.

Poultry products in Zambia form an integral part of the human diet in many households, as they are cheap and easy to produce. The burden of poultry diseases has, however, remained a major challenge. Growing consumer demand for poultry products in Zambia has resulted in non-prudent antimicrobial use on farms, intending to prevent and treat poultry diseases for growth optimisation and maximising profits. This cross-sectional study aimed to identify the different types of bacteria causing diseases in chickens in Lusaka and to detect the extended-spectrum lactamase (ESBL)-encoding genes. We collected 215 samples from 91 diseased chickens at three post-mortem facilities and screened them for Gram-negative bacteria. Of these samples, 103 tested positive for various clinically relevant Enterobacteriaceae, including Enterobacter (43/103, 41.7%), Escherichia coli (20/103, 19.4%), Salmonella (10/103, 9.7%), and Shigella (8/103, 7.8%). Other isolated bacteria included Yersinia, Morganella, Proteus, and Klebsiella, which accounted for 21.4%. E. coli, Enterobacter, Salmonella, and Shigella were subjected to antimicrobial susceptibility testing. The results revealed that E. coli, Enterobacter, and Shigella were highly resistant to tetracycline, ampicillin, amoxicillin, and trimethoprim-sulfamethoxazole, while Salmonella showed complete susceptibility to all tested antibiotics. The observed resistance patterns correlated with antimicrobial usage estimated from sales data from a large-scale wholesale and retail company. Six (6/14, 42.9%) E. coli isolates tested positive for blaCTX-M, whilst eight (8/14, 57.1%) Enterobacter samples tested positive for blaTEM. Interestingly, four (4/6, 66.7%) of the E. coli isolates carrying blaCTX-M-positive strains were also positive for blaTEM. Sanger sequencing of the PCR products revealed that five (5/6, 83.3%) of the abovementioned isolates possessed the blaCTX-M-15 allele. The results suggest the presence of potentially pathogenic ESBL-producing Enterobacteriaceae in poultry, threatening public health.

Effects of heat degradation of betanin in red beetroot (Beta vulgaris L.) on biological activity and antioxidant capacity.

Betanin is a red pigment of red beetroot (Beta vulgaris L.), providing the beneficial effects to maintain human health. Betanin is involved in the characteristic red color of red beetroot, and used as an edible dye. Betanin is known to be a highly unstable pigment, and water solutions of betanin are nearly fully degraded after heating at 99°C for 60 min in the experimental conditions of this study. The present study investigated the effects of red beetroot juice (RBJ) and betanin on immune cells, and found that stimulation with RBJ and betanin induces interleukin (IL)-1ß, IL-8, and IL-10 mRNA in a human monocyte derived cell line, THP-1 cells. This mRNA induction after stimulation with RBJ and betanin was not significantly changed after heat treatment when attempting to induce degradation of the betanin. Following these results, the effects of heat degradation of betanin on the inhibition of lipopolysaccharide (LPS) induced nitric oxide (NO) production in RAW264 cells and the antioxidant capacity were investigated. The results showed that the inhibition activity of RBJ and betanin with the LPS induced NO production is not altered after heat degradation of betanin. In addition, the results of FRAP (ferric reducing antioxidant power) and DPPH (1,1-Diphenyl-2-picrylhydrazyl) assays indicate that a not inconsiderable degree of the antioxidant capacity of RBJ and betanin remained after heat degradation of betanin. These results suggest that it is important to consider the effects of degradation products of betanin in the evaluation of the beneficial effects of red beetroot on health.

Effects of chondroitin sulfate oligosaccharides on osteoclast differentiation of RAW264 cells, and myotube differentiation of C2C12 cells.

Chondroitin sulfate (CS) is a glycosaminoglycan, and CS derived from various animal species is used in drugs and food supplements to alleviate arthralgia. The CS is a high molecular weight compound, and hydrolysis of CS by intestinal microbiota is thought to be required for absorption in mammalians. Chondroitin sulfate oligosaccharides (Oligo-CS) are produced by hydrolysis with subcritical water from CS isolated from a species of skate, Raja pulchra for the improvement of bioavailability. The present study conducted in vitro experiments using murine cell lines, to compare the biological activities of Oligo-CS and high molecular weight CS composed with the similar disaccharide isomer units of D-glucuronic acid and N-acetyl-D-glucosamine (CS-C). The results show that Oligo-CS inhibits osteoclast differentiation of RAW264 cells significantly at lower concentrations than in CS. The cell viability of a myoblast cell line, C2C12 cells, was increased when the cells were grown in a differentiated medium for myotubes with Oligo-CS, where there were no effects on the cell viability in CS. These results suggest that in vitro Oligo-CS exhibits stronger bioactivity than high-molecular weight CS.

Baseline and seasonal trends of Bacillus cereus and Bacillus subtilis from clinical samples in Japan.

Background: Outbreaks of Bacillus cereus bloodstream infections (BSIs) are a concern in Japanese medical settings. Aim: This study determined baseline values for B. cereus detection in clinical samples that are useful as reference values for hospitals when assessing the need for intervention. Method: A retrospective analysis of B. cereus detection in the Japan Nosocomial Infections Surveillance data from 2008 to 2014 was performed; it included 950 individual hospitals across the country. Findings: Bacillus spp. were detected in 0.54% of the clinical specimens submitted for bacteriological testing. Specimens positive for Bacillus spp. were mainly blood (24.6%), stool (26.5%), and respiratory specimens (23.3%). Identification of Bacillus spp. at the species level (i.e., B. cereus or B. subtilis) was reported in 55.3%, 14.7%, and 15.4% of cases, of which 88.9%, 48.3%, and 33.1% were B. cereus in blood, stool, and respiratory specimens, respectively. Of the 4105 hospital-years, 75.7% had blood specimens with Bacillus spp., with a median of 0.85 blood specimens/100 beds annually (interquartile range, 0.17-2.10). The B. cereus detection showed significant summer seasonality, regardless of specimen type or geographic distribution. The B. subtilis detection did not show seasonality, and its detection remained constant throughout the year. The seasonality of Bacillus spp. reflects the high proportion of B. cereus. Conclusions: The increased detection rate of Bacillus spp. during summer should be interpreted as a risk factor for B. cereus BSIs. A post-summer decrease in Bacillus spp. should not be interpreted as an effect of interventions.

Chlamydia trachomatis relies on the scavenger role of aryl hydrocarbon receptor with detyrosinated tubulin for its intracellular growth, but this is impaired by excess indole.

Although IFN-γ depletes tryptophan (Trp) as a defense against intracellular Chlamydia trachomatis (Ct) infected to hypoxic vagina, the presence of indole, a precursor of Trp, enables Ct to infect IFN-γ-exposed culture cells. Meanwhile, Trp-derived indole derivatives interact the aryl hydrocarbon receptor (AhR), which is a ligand-dependent transcription factor involved in the cellular homeostasis with tubulin dynamics. Here, the amounts of IFN-γ and indole in cervical swabs with known Ct infection status were measured, and Ct growth in the presence of indole was determined from the perspective of the AhR axis under hypoxia. A positive correlation between the amounts of IFN-γ and indole was found, and both of these amounts were lower in Ct-positive swabs than in Ct-negative ones. Indole as well as other AhR ligands inhibited Ct growth, especially under normoxia. Ct prompted the expression of detyrosinated tubulin (dTTub), but indole inhibited it. Indole did not stimulate the translocation of AhR to nucleus, and it blocked AhR activation in AhR-reporter cells. Ct growth was reduced more effectively under normoxia in AhR-knockdown cells, an effect that was enhanced by indole, which in turn diminished dTTub. Thus, Ct growth relies on the scavenger role of cytosolic AhR responsible for promoting dTTub expression.

Antimicrobial Susceptibility Profiles and Molecular Characterisation of Staphylococcus aureus from Pigs and Workers at Farms and Abattoirs in Zambia.

Pigs have been shown to be a reservoir for recently emerging livestock-associated Staphylococcus aureus (LA-SA), including methicillin resistant strains in many countries worldwide. However, there is sparse information about LA-SA strains circulating in Zambia. This study investigated the prevalence, phenotypic and genotypic characteristics of S. aureus from pigs and workers at farms and abattoirs handling pigs in Lusaka Province of Zambia. A total of 492 nasal pig swabs, 53 hand and 53 nasal human swabs were collected from farms and abattoirs in selected districts. Standard microbiological methods were used to isolate and determine antimicrobial susceptibility patterns of S. aureus. Polymerase Chain Reaction was used to confirm the species identity and detect antimicrobial resistance and virulence genes of isolates, whereas genetic diversity was evaluated using spa typing. Overall prevalence of S. aureus was 33.1%, 37.8% for pigs and 11.8% for humans. The isolates were resistant to several antibiotics with resistance ranging from 18% to 98% but were all susceptible to vancomycin. Typical LA-SA spa types were detected. The presence of plasmid mediated resistance genes such as tetM (12.8%), other resistance determinants and immune evasion cluster genes among the isolates is of great public health concern. Thus, continuous surveillance of S. aureus using a "One health" approach is warranted to monitor S.aureus infections and spread of antimicrobial resistance.

Chlamydia trachomatis L2/434/Bu Favors Hypoxia for its Growth in Human Lymphoid Jurkat Cells While Maintaining Production of Proinflammatory Cytokines.

The role of lymphocytes as a cornerstone of the inflammatory response in the invasive pathogenesis of Chlamydia trachomatis (Ct) LGV (L1-3) infection is unclear. Therefore, we assessed whether the adaptation of CtL2 to immortal lymphoid Jurkat cells under hypoxic conditions occurred through proinflammatory cytokine profile modification. The quantities of inclusion-forming units with chlamydial 16S rDNA confirmed that CtL2 grew well under hypoxic rather than normoxic conditions in the cells. Confocal microscopic imaging and transmission electron microscopy revealed the presence of bacterial progeny in the inclusions and showed that the inclusions were larger under hypoxic rather than normoxic conditions; this was supported by the results of 3D image construction. Furthermore, PCR-based analysis of proinflammatory cytokines revealed that the gene expression levels under hypoxic conditions were significantly higher than those under normoxic conditions. In particular, the expression of two genes (CXCL8 and CXCR3) was significantly diminished under normoxic conditions. Taken together, the results indicated that hypoxia promoted CtL2 growth in Jurkat cells while maintaining the levels of proinflammatory cytokines. Thus, Ct LGV infection in lymphocytes under hypoxic conditions might be crucial to a complete understanding of the invasive pathogenesis.

Chlamydia trachomatis Requires Functional Host-Cell Mitochondria and NADPH Oxidase 4/p38MAPK Signaling for Growth in Normoxia.

Chlamydia trachomatis (Ct) is an intracellular energy-parasitic bacterium that requires ATP derived from infected cells for its growth. Meanwhile, depending on the O2 concentration, the host cells change their mode of ATP production between oxidative phosphorylation in mitochondria (Mt) and glycolysis; this change depends on signaling via reactive oxygen species (ROS) produced by NADPH oxidases (NOXs) as well as Mt. It has been proposed that Ct correspondingly switches its source of acquisition of ATP between host-cell Mt and glycolysis, but this has not been verified experimentally. In the present study, we assessed the roles of host-cell NOXs and Mt in the intracellular growth of CtL2 (L2 434/Bu) under normoxia (21% O2) and hypoxia (2% O2) by using several inhibitors of NOXs (or the downstream molecule) and Mt-dysfunctional (Mtd) HEp-2 cells. Under normoxia, diphenyleneiodonium, an inhibitor of ROS diffusion, abolished the growth of CtL2 and other Chlamydiae (CtD and C. pneumoniae). Both ML171 (a pan-NOX inhibitor) and GLX351322 (a NOX4-specific inhibitor) impaired the growth of CtL2 under normoxia, but not hypoxia. NOX4-knockdown cells diminished the bacterial growth. SB203580, an inhibitor of the NOX4-downstream molecule p38MAPK, also inhibited the growth of CtL2 under normoxia but not hypoxia. Furthermore, CtL2 failed to grow in Mtd cells under normoxia, but no effect was observed under hypoxia. We conclude that under normoxia, Ct requires functional Mt in its host cells as an ATP source, and that this process requires NOX4/p38MAPK signaling in the host cells. In contrast to hypoxia, crosstalk between NOX4 and Mt via p38MAPK may be crucial for the growth of Ct under normoxia.

Cas9-guided haplotyping of three truncation variants in autosomal recessive disease.

An autosomal recessive disease is caused by biallelic loss-of-function mutations. However, when more than two disease-causing variants are found in a patient's gene, it is challenging to determine which two of the variants are responsible for the disease phenotype. Here, to decipher the pathogenic variants by precise haplotyping, we applied nanopore Cas9-targeted sequencing (nCATS) to three truncation COL7A1 variants detected in a patient with recessive dystrophic epidermolysis bullosa (EB). The distance between the most 5' and 3' variants was approximately 19 kb at the level of genomic DNA. nCATS successfully demonstrated that the most 5' and 3' variants were located in one allele while the variant in between was located in the other allele. Interestingly, the proband's mother, who was phenotypically intact, was heterozygous for the allele that harbored the two truncation variants, which could otherwise be misinterpreted as those of typical recessive dystrophic EB. Our study highlights the usefulness of nCATS as a tool to determine haplotypes of complicated genetic cases. Haplotyping of multiple variants in a gene can determine which variant should be therapeutically targeted when nucleotide-specific gene therapy is applied.

A GCDGC-specific DNA (cytosine-5) methyltransferase that methylates the GCWGC sequence on both strands and the GCSGC sequence on one strand.

5-Methylcytosine is one of the major epigenetic marks of DNA in living organisms. Some bacterial species possess DNA methyltransferases that modify cytosines on both strands to produce fully-methylated sites or on either strand to produce hemi-methylated sites. In this study, we characterized a DNA methyltransferase that produces two sequences with different methylation patterns: one methylated on both strands and another on one strand. M.BatI is the orphan DNA methyltransferase of Bacillus anthracis coded in one of the prophages on the chromosome. Analysis of M.BatI modified DNA by bisulfite sequencing revealed that the enzyme methylates the first cytosine in sequences of 5'-GCAGC-3', 5'-GCTGC-3', and 5'-GCGGC-3', but not of 5'-GCCGC-3'. This resulted in the production of fully-methylated 5'-GCWGC-3' and hemi-methylated 5'-GCSGC-3'. M.BatI also showed toxicity when expressed in E. coli, which was caused by a mechanism other than DNA modification activity. Homologs of M.BatI were found in other Bacillus species on different prophage like regions, suggesting the spread of the gene by several different phages. The discovery of the DNA methyltransferase with unique modification target specificity suggested unrevealed diversity of target sequences of bacterial cytosine DNA methyltransferase.

Complete Genome Sequences of 24 Strains of Bacillus cereus Isolated from Nosocomial Infection and Bacteremia Cases in Japan.

Bacillus cereus is mainly associated with foodborne illness but sometimes causes nosocomial infections. We previously reported that B. cereus strains of a specific sequence type, ST1420, were associated with nosocomial infection. Here, we determined the complete genome sequences of B. cereus strains isolated from nosocomial infection cases in Japanese hospitals.

Leptospira Is an Environmental Bacterium That Grows in Waterlogged Soil.

Leptospirosis is a zoonotic disease caused by infection with pathogenic leptospires. Consistent with recent studies by other groups, leptospires were isolated from 89 out of 110 (80.9%) soil or water samples from varied locations in the Philippines in our surveillance study, indicating that leptospires might have a life cycle that does not involve animal hosts. However, despite previous work, it has not been confirmed whether leptospires multiply in the soil environment under various experimental conditions. Given the fact that the case number of leptospirosis is increased after flood, we hypothesized that waterlogged soil, which mimics the postflooding environment, could be a suitable condition for growing leptospires. To verify this hypothesis, pathogenic and saprophytic leptospires were seeded in the bottles containing 2.5 times as much water as soil, and bacterial counts in the bottles were measured over time. Pathogenic and saprophytic leptospires were found to increase their number in waterlogged soil but not in water or soil alone. In addition, leptospires were reisolated from soil in closed tubes for as long as 379 days. These results indicate that leptospires are in a resting state in the soil and are able to proliferate with increased water content in the environment. This notion is strongly supported by observations that the case number of leptospirosis is significantly higher in rainy seasons and increased after flood. Therefore, we reached the following conclusion: environmental soil is a potential reservoir of leptospires. IMPORTANCE Since research on Leptospira has focused on pathogenic leptospires, which are supposed to multiply only in animal hosts, the life cycle of saprophytic leptospires has long been a mystery. This study demonstrates that both pathogenic and saprophytic leptospires multiply in the waterlogged soil, which mimics the postflooding environment. The present results potentially explain why leptospirosis frequently occurs after floods. Therefore, environmental soil is a potential reservoir of leptospires and leptospirosis is considered an environment-borne as well as a zoonotic disease. This is a significant report to reveal that leptospires multiply under environmental conditions, and this finding leads us to reconsider the ecology of leptospires.

Clonal relationship between multidrug-resistant Escherichia coli ST69 from poultry and humans in Lusaka, Zambia.

Multidrug-resistant (MDR) Escherichia coli in food animals such as chickens is an emerging public health concern in Zambia. Additionally, the country's high demand for poultry products necessitates further investigation into the link between poultry and human MDR E. coli. Twenty cefotaxime-resistant E. coli isolates collected from poultry in Lusaka, Zambia, were screened for multidrug resistance and sequenced on MiSeq and MinION platforms. Genomes were assembled de novo and compared with 36 previously reported cefotaxime-resistant E. coli isolates from inpatients at the University Teaching Hospital, Lusaka. All (20/20, 100%) poultry isolates exhibited resistance to ampicillin, chloramphenicol and doxycycline. Phylogenetic analysis and hierarchical clustering showed a high degree of genetic relatedness between E. coli O17:H18-ST69 from poultry and humans. The E. coli O17:H18-ST69 clone accounted for 4/20 (20%) poultry- and 9/36 (25%) human-associated isolates that shared two plasmids harboring 14 antimicrobial resistance (AMR) genes. However, comparison analysis showed that the isolates also had other AMR plasmids distinct for each niche. Our results suggested clonal transmission of MDR E. coli between poultry and humans, with the potential acquisition of niche-specific AMR plasmids. Thus, the control of MDR E. coli requires a One Health approach involving both human and animal health sectors.

Detection of revertant mosaicism in epidermolysis bullosa through Cas9-targeted long-read sequencing.

Revertant mosaicism (RM) is a phenomenon in which inherited mutations are spontaneously corrected in somatic cells. RM occurs in some congenital skin diseases, but genetic validation of RM in clinically revertant skin has been challenging, especially when homologous recombination (HR) is responsible for RM. Here, we introduce nanopore Cas9-targeted sequencing (nCATS) for identifying HR in clinically revertant skin. We took advantage of compound heterozygous COL7A1 mutations in a patient with recessive dystrophic epidermolysis bullosa who showed revertant skin spots. Cas9-mediated enrichment of genomic DNA (gDNA) covering the two mutation sites (>8 kb) in COL7A1 and subsequent MinION sequencing successfully detected intragenic crossover in the epidermis of the clinically revertant skin. This method enables the discernment of haplotypes of up to a few tens of kilobases of gDNA. Moreover, it is devoid of polymerase chain reaction amplification, which can technically induce recombination. We, therefore, propose that nCATS is a powerful tool for understanding complicated gene modifications, including RM.

Risk factors and spatio-temporal patterns of livestock anthrax in Khuvsgul Province, Mongolia.

Anthrax is a worldwide zoonotic disease. Anthrax has long been a public health and socio-economic issue in Mongolia. Presently, there is no spatial information on carcass burial sites as a potential hazard of future anthrax outbreaks and possible risk factors associated with anthrax occurrences in Mongolia. Here, we analyze retrospective data (1986-2015) on the disposal sites of livestock carcasses to describe historical spatio-temporal patterns of livestock anthrax in Khuvsgul Province, which showed the highest anthrax incidence rate in Mongolia. From the results of spatial mean and standard deviational ellipse analyses, we found that the anthrax spatial distribution in livestock did not change over the study period, indicating a localized source of exposure. The multi-distance spatial cluster analysis showed that carcass sites distributed in the study area are clustered. Using kernel density estimation analysis on carcass sites, we identified two anthrax hotspots in low-lying areas around the south and north regions. Notably, this study disclosed a new hotspot in the northern part that emerged in the last decade of the 30-year study period. The highest proportion of cases was recorded in cattle, whose prevalence per area was highest in six districts (i.e., Murun, Chandmani-Undur, Khatgal, Ikh-Uul, Tosontsengel, and Tsagaan-Uul), suggesting that vaccination should prioritize cattle in these districts. Furthermore, size of outbreaks was influenced by the annual summer mean air temperature of Khuvsgul Province, probably by affecting the permafrost freeze-thawing activity.

Development of ELISA based on Bacillus anthracis capsule biosynthesis protein CapA for naturally acquired antibodies against anthrax.

Anthrax is a zoonotic disease caused by the gram-positive spore-forming bacterium Bacillus anthracis. Detecting naturally acquired antibodies against anthrax sublethal exposure in animals is essential for anthrax surveillance and effective control measures. Serological assays based on protective antigen (PA) of B. anthracis are mainly used for anthrax surveillance and vaccine evaluation. Although the assay is reliable, it is challenging to distinguish the naturally acquired antibodies from vaccine-induced immunity in animals because PA is cross-reactive to both antibodies. Although additional data on the vaccination history of animals could bypass this problem, such data are not readily accessible in many cases. In this study, we established a new enzyme-linked immunosorbent assay (ELISA) specific to antibodies against capsule biosynthesis protein CapA antigen of B. anthracis, which is non-cross-reactive to vaccine-induced antibodies in horses. Using in silico analyses, we screened coding sequences encoded on pXO2 plasmid, which is absent in the veterinary vaccine strain Sterne 34F2 but present in virulent strains of B. anthracis. Among the 8 selected antigen candidates, capsule biosynthesis protein CapA (GBAA_RS28240) and peptide ABC transporter substrate-binding protein (GBAA_RS28340) were detected by antibodies in infected horse sera. Of these, CapA has not yet been identified as immunoreactive in other studies to the best of our knowledge. Considering the protein solubility and specificity of B. anthracis, we prepared the C-terminus region of CapA, named CapA322, and developed CapA322-ELISA based on a horse model. Comparative analysis of the CapA322-ELISA and PAD1-ELISA (ELISA uses domain one of the PA) showed that CapA322-ELISA could detect anti-CapA antibodies in sera from infected horses but was non-reactive to sera from vaccinated horses. The CapA322-ELISA could contribute to the anthrax surveillance in endemic areas, and two immunoreactive proteins identified in this study could be additives to the improvement of current or future vaccine development.

Silkworm model for Bacillus anthracis infection and virulence determination.

Bacillus anthracis is an obligate pathogen and a causative agent of anthrax. Its major virulence factors are plasmid-coded; however, recent studies have revealed chromosome-encoded virulence factors, indicating that the current understanding of its virulence mechanism is elusive and needs further investigation. In this study, we established a silkworm (Bombyx mori) infection model of B. anthracis. We showed that silkworms were killed by B. anthracis Sterne and cured of the infection when administered with antibiotics. We quantitatively determined the lethal dose of the bacteria that kills 50% larvae and effective doses of antibiotics that cure 50% infected larvae. Furthermore, we demonstrated that B. anthracis mutants with disruption in virulence genes such as pagA, lef, and atxA had attenuated silkworm-killing ability and reduced colonization in silkworm hemolymph. The silkworm infection model established in this study can be utilized in large-scale infection experiments to identify novel virulence determinants and develop novel therapeutic options against B. anthracis infections.

Immunization Coverage and Antibody Retention against Rabies in Domestic Dogs in Lusaka District, Zambia.

Rabies remains endemic in Zambia. Despite conducting canine vaccinations in Lusaka district, the vaccination coverage and actual seropositivity in the dog population in Lusaka district are rarely evaluated. This study estimated the seropositivity-based immunization coverage in the owned dog population in Lusaka district using the expanded program on immunization cluster survey method. The time-series trend of neutralizing antibodies against rabies in vaccinated dogs was also evaluated. Of 366 dogs in 200 dog-owning households in Lusaka district, blood samples were collected successfully from 251 dogs. In the sampled dogs, 42.2% (106/251) had an antibody titer ≥0.5 IU/mL. When the 115 dogs whose blood was not collected were assumed to be seronegative, the minimum immunization coverage in Lusaka district's owned dog population was estimated at 29.0% (95% confidence interval: 22.4-35.5). It was also found that a single vaccination with certified vaccines is capable of inducing protective levels of antibodies. In contrast, higher antibody titers were observed in multiple-vaccinated dogs than in single-vaccinated dogs, coupled with the observation of a decline in antibody titer over time. These results suggest the importance of continuous booster immunization to maintain herd immunity and provide useful information to plan mass vaccination against rabies in Zambia.

Direct Regulons of AtxA, the Master Virulence Regulator of Bacillus anthracis.

AtxA, the master virulence regulator of Bacillus anthracis, regulates the expression of three toxins and genes for capsule formation that are required for the pathogenicity of B. anthracis. Recent transcriptome analyses showed that AtxA affects a large number of genes on the chromosome and plasmids, suggesting a role as a global regulator. However, information on genes directly regulated by AtxA is scarce. In this work, we conducted genome-wide analyses and cataloged the binding sites of AtxA in vivo and transcription start sites on the B. anthracis genome. By integrating these results, we detected eight genes as direct regulons of AtxA. These consisted of five protein-coding genes, including two of the three toxin genes, and three genes encoding the small RNAs XrrA and XrrB and a newly discovered 95-nucleotide small RNA, XrrC. Transcriptomes from single-knockout mutants of these small RNAs revealed changes in the transcription levels of genes related to the aerobic electron transport chain, heme biosynthesis, and amino acid metabolism, suggesting their function for the control of cell physiology. These results reveal the first layer of the gene regulatory network for the pathogenicity of B. anthracis and provide a data set for the further study of the genomics and genetics of B. anthracis. IMPORTANCE Bacillus anthracis is the Gram-positive bacterial species that causes anthrax. Anthrax is still prevalent in countries mainly in Asia and Africa, where it causes economic damage and remains a public health issue. The mechanism of pathogenicity is mainly explained by the three toxin proteins expressed from the pXO1 plasmid and by proteins involved in capsule formation expressed from the pXO2 plasmid. AtxA is a protein expressed from the pXO1 plasmid that is known to upregulate genes involved in toxin production and capsule formation and is thus considered the master virulence regulator of B. anthracis. Therefore, understanding the detailed mechanism of gene regulation is important for the control of anthrax. The significance of this work lies in the identification of genes that are directly regulated by AtxA via genome-wide analyses. The results reveal the first layer of the gene regulatory network for the pathogenicity of B. anthracis and provide useful resources for a further understanding of B. anthracis.