The role of NagC in Yersinia pestis and Yersinia pseudotuberculosis biofilm development and insect transmission

There are three species of human-pathogenic Yersiniae; two of which cause gastrointestinal disease (Yersinia pseudotuberculosis and Yersinia enterocolitica) and one which causes plague (Yersinia pestis), one of the deadliest diseases in human history. Whilst Y. pseudotuberculosis and Y. enterocolitica are transmitted faeco-orally, Y. pestis is transmitted by insects, classically the Oriental rat flea, Xenopsylla cheopis. All Yersiniae are capable of a method of bacterial messaging known as quorum sensing (QS), which allows individual cells to communicate in a density dependent manner using signalling molecules such as N-acyl-homoserine lactones (AHLs). QS regulates several virulence phenotypes, including the production of Yersinia virulence factors (YOPs), secreted by the type three secretion system (T3SS) into mammalian host cells, auto-aggregation and biofilm formation. Biofilms are aggregates of bacteria within a protective exopolysaccharide matrix and are especially important for Y. pestis, as transmission of plague relies on the biofilm-mediated blockage of the proventriculus within the digestive system of a flea. Understanding the mechanisms of biofilm development are therefore potentially important for developing methods to prevent or control plague transmission. Recent research has also suggested a more prominent role in the spread of plague for other insect vectors, such as body lice (Pediculus corporis). In the Yersiniae, the extracellular polymeric substance (EPS) matrix is composed primarily of the polysaccharide N-acetyl-D-glucosamine (poly-GlcNAc) and extracellular DNA. The production of GlcNAc but not poly-GlcNAc is known to be regulated by NagC, a DNA binding protein responsible for the repression of nagE-nagBACD (GlcNAc catabolic operon) and activation of glmUS (GlcNAc biosynthesis operon) in Escherichia coli, but there is little information on the NagC-dependent regulation of GlcNAc metabolism or on poly-GlcNAc biosynthesis via the hmsHFRS operon in the Yersiniae. This study set out to investigate the contribution of NagC in Y. pseudotuberculosis and Y. pestis to QS, poly-GlcNAc production and biofilm formation in vitro and in vivo on and within the established C. elegans and insect vectors. Artificially fed colonies of X. cheopis and P. corporis were investigated for use in experimental infection models. For both insect species, defibrinated human blood fed through a collagen membrane was the most successful combination for feeding, although problems with reproducible control of environmental conditions, such as humidity, prevented the insect colonies from becoming established. To investigate the contribution of NagC to QS, YOP production, auto-aggregation, poly-GlcNAc biosynthesis and biofilm formation, a ΔnagC mutant was constructed in Y. pestis and compared with a Y. pseudotuberculosis ΔnagC mutant as well as with the corresponding wild type strains. NagC did appear to influence AHL and YOP production in both Y. pseudotuberculosis and Y. pestis. NagC did not influence auto-aggregation in Y. pestis, although the rate of auto-aggregation was slower for the Y. pseudotuberculosis ΔnagC mutant. NagC was however required for the production of poly-GlcNAc (Y. pseudotuberculosis) and biofilm formation (both Y. pseudotuberculosis and Y. pestis). To determine whether NagC directly regulates poly-GlcNAc biosynthesis, the Y. pestis nagC was expressed in E. coli, the recombinant protein purified and subjected to electrophoretic mobility shift assays. The data obtained show that NagC binds to the promoter regions of the GlcNAc metabolic operons nagE-nagBACD and glmUS, as well as hmsHFRS in Y. pestis and hence directly regulates the production of poly-GlcNAc. Using in vitro (glass) and in vivo (nematode and insect) models, both Y. pestis and Y. pseudotuberculosis ΔnagC mutants were evaluated for biofilm formation. In addition, a ‘fake flea’ proventriculus model was developed to investigate attachment and biofilm formation on a chitin substrate. The Y. pseudotuberculosis ΔnagC mutant showed reduced biofilm formation on the chitin surface, compared with the parental strain. On the nematode C. elegans, the Y. pestis ΔnagC mutant produced substantially less biofilm formation than the parent. When Y. pseudotuberculosis was fed to P. corporis, the ΔnagC mutant showed an increased ability to clear the infection and increased survival rates compared with insects fed the parent strain. Taken together, these results indicate that NagC plays a central role in biofilm formation and in the ability of the Yersiniae to be transmitted by insect vectors

Does Africa have the toolkit to combat the next zoonotic pandemic?

The emergence of new zoonotic diseases remains virtually impossible to predict, but exposure to wildlife, abundant animal populations and the increasing destruction of natural habitats make them certain. Should a zoonotic outbreak emerge in Africa with pandemic potential, what are the continent’s strategies to prepare itself and the world? The first post in a new series explores the networks strengthening communication and trust between governments, local communities, health workers and scientists

Mapping the phylogeny and lineage history of geographically distinct BCG vaccine strains

The bacillus Calmette-Guérin (BCG) vaccine has been in use for prevention of tuberculosis for over a century. It remains the only widely available tuberculosis vaccine and its protective efficacy has varied across geographical regions. Since it was developed, the BCG vaccine strain has been shared across different laboratories around the world, where use of differing culture methods has resulted in genetically distinct strains over time. Whilst differing BCG vaccine efficacy around the world is well documented, and the reasons for this may be multifactorial, it has been hypothesized that genetic differences in BCG vaccine strains contribute to this variation. Isolates from an historic archive of lyophilized BCG strains were regrown, DNA was extracted and then whole-genome sequenced using Oxford Nanopore Technologies. The resulting whole-genome data were plotted on a phylogenetic tree and analysed to identify the presence or absence of regions of difference (RDs) and single-nucleotide polymorphisms (SNPs) relating to virulence, growth and cell wall structure. Of 50 strains available, 36 were revived in culture and 39 were sequenced. Morphology differed between the strains distributed before and after 1934. There was phylogenetic association amongst certain geographically classified strains, most notably BCG-Russia, BCG-Japan and BCG-Danish. RD2, RD171 and RD713 deletions were associated with late strains (seeded after 1927). When mapped to BCG-Pasteur 1172, the SNPs in sigK, plaA, mmaA3 and eccC5 were associated with early strains. Whilst BCG-Russia, BCG-Japan and BCG-Danish showed strong geographical isolate clustering, the late strains, including BCG-Pasteur, showed more variation. A wide range of SNPs were seen within geographically classified strains, and as much intra-strain variation as between-strain variation was seen. The date of distribution from the original Pasteur laboratory (early pre-1927 or late post-1927) gave the strongest association with genetic differences in regions of difference and virulence-related SNPs, which agrees with the previous literature

Phenotypic and Genotypic Characteristics of Antimicrobial Resistance in Citrobacter freundii Isolated from Domestic Ducks (Anas platyrhynchos domesticus) in Bangladesh

Antimicrobial resistance (AMR) in Citrobacter freundii poses a serious challenge as this species is one of the sources of nosocomial infection and causes diarrheal infections in humans. Ducks could be the potential source of multidrug-resistant (MDR) C. freundii; however, AMR profiles in C. freundii from non-human sources in Bangladesh have remained elusive. This study aimed to detect C. freundii in domestic ducks (Anas platyrhynchos domesticus) in Bangladesh and to determine their phenotypic and genotypic antibiotic susceptibility patterns. A total of 150 cloacal swabs of diseased domestic ducks were screened using culturing, staining, biochemical, polymerase chain reaction (PCR), and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) to detect C. freundii. Phenotypic and genotypic antibiotic susceptibility patterns were done by the disk diffusion method and PCR, respectively. In total, 16.67% (25/150) of the samples were positive for C. freundii. C. freundii isolates showed a range of 20% to 96% resistance to cefotaxime, gentamicin, levofloxacin, ciprofloxacin, cotrimoxazole, tetracycline, ampicillin, and cephalexin. More than 60% of the isolates were phenotypically MDR, and the index of multiple antibiotic resistance ranged from 0.07 to 0.79. Genes encoding resistance to beta-lactams [blaTEM-1-88% (22/25), blaCMY-2-56% (14/25), blaCMY-9-8% (2/25), and blaCTX-M-14-20% (5/25)], sulfonamides [sul1-52% (13/25), sul2-24% (6/25)], tetracyclines [tetA-32% (8/25) and tetB-4% (1/25)], aminoglycosides [aacC4-16% (4/25)], and fluoroquinolones [qnrA-4% (1/25), qnrB-12% (3/25), and qnrS-4% (1/25)] were detected in the isolated C. freundii. To the best of our knowledge, this is the first study in Bangladesh to detect MDR C. freundii with their associated resistance genes from duck samples. We suggest addressing the burden of diseases in ducks and humans and associated AMR issues using the One Health approach

Antimicrobial Resistance Patterns and Risk Factors Associated with <i>Salmonella</i> spp. Isolates from Poultry Farms in the East Coast of Peninsular Malaysia: A Cross-Sectional Study.

The burden of antimicrobial use in agricultural settings is one of the greatest challenges facing global health and food security in the modern era. Malaysian poultry operations are a relevant but understudied component of epidemiology of antimicrobial resistance. We aimed to identify the prevalence, resistance patterns, and risk factors associated with Salmonella isolates from poultry farms in three states of East Coast Peninsular Malaysia. Between 8 February 2019 and 23 February 2020, a total of 371 samples (cloacal swabs = 259; faecal = 84; Sewage = 14, Tap water = 14) was collected from poultry operations. Characteristics of the sampled farms and associated risk factors were obtained using semi-structured questionnaires. Presumptive Salmonella spp. isolates were identified based on colony morphology with subsequent biochemical and PCR confirmation. Susceptibility of isolates was tested against a panel of 12 antimicrobials using disk diffusion method. Our findings revealed that the proportion of Salmonella spp.-positive isolates across sample source were as following: cloacal swab (46.3%, 120/259); faecal (59.5%, 50/84); in tap water (14.3%, 2/14); and in sewage sample (35.7%, 5/14). Isolates from faecal (15.5%, 13/84), cloacal (1.2%, 3/259), and sewage (7.1%, 1/14) samples were significantly resistant to at least five classes of antimicrobials. Resistance to Sulfonamides class (52%, 92/177) was predominantly observed followed by tetracycline (39.5%, 70/177) and aminoglycosides (35.6%, 63/177). Multivariate regression analysis identified intensive management system (OR = 1.55, 95% CI = 1.00-2.40) as a leading driver of antimicrobial resistance (AMR) acquisition. A prevalence of resistance to common antimicrobials was recorded for sulfamethoxazole (33.9%), tetracycline (39.5%), and trimethoprim-sulphamethoxazole (37.9%). A close association between different risk factors and the prevalence of AMR of Salmonella strains suggests a concern over rising misuse of veterinary antimicrobials that may contribute to the emergence and evolution of multidrug-resistant pathogen isolates. One Health approach is recommended to achieve a positive health outcome for all species

Identification of Risk Factors Associated with Resistant Escherichia coli Isolates from Poultry Farms in the East Coast of Peninsular Malaysia: A Cross Sectional Study.

Antimicrobial resistance is of concern to global health security worldwide. We aimed to identify the prevalence, resistance patterns, and risk factors associated with Escherichia coli (E. coli) resistance from poultry farms in Kelantan, Terengganu, and Pahang states of east coast peninsular Malaysia. Between 8 February 2019 and 23 February 2020, a total of 371 samples (cloacal swabs = 259; faecal = 84; Sewage = 14, Tap water = 14) were collected. Characteristics of the sampled farms including management type, biosecurity, and history of disease were obtained using semi-structured questionnaire. Presumptive E. coli isolates were identified based on colony morphology with subsequent biochemical and PCR confirmation. Susceptibility of isolates was tested against a panel of 12 antimicrobials and interpreted alongside risk factor data obtained from the surveys. We isolated 717 E. coli samples from poultry and environmental samples. Our findings revealed that cloacal (17.8%, 46/259), faecal (22.6%, 19/84), sewage (14.3%, 2/14) and tap water (7.1%, 1/14) were significantly (p < 0.003) resistant to at least three classes of antimicrobials. Resistance to tetracycline class were predominantly observed in faecal samples (69%, 58/84), followed by cloacal (64.1%, 166/259), sewage (35.7%, 5/14), and tap water (7.1%, 1/84), respectively. Sewage water (OR = 7.22, 95% CI = 0.95-151.21) had significant association with antimicrobial resistance (AMR) acquisition. Multivariate regression analysis identified that the risk factors including sewage samples (OR = 7.43, 95% CI = 0.96-156.87) and farm size are leading drivers of E. coli antimicrobial resistance in the participating states of east coast peninsular Malaysia. We observed that the resistance patterns of E. coli isolates against 12 panel antimicrobials are generally similar in all selected states of east coast peninsular Malaysia. The highest prevalence of resistance was recorded in tetracycline (91.2%), oxytetracycline (89.1%), sulfamethoxazole/trimethoprim (73.1%), doxycycline (63%), and sulfamethoxazole (63%). A close association between different risk factors and the high prevalence of antimicrobial-resistant E. coli strains reflects increased exposure to resistant bacteria and suggests a concern over rising misuse of veterinary antimicrobials that may contribute to the future threat of emergence of multidrug-resistant pathogen isolates. Public health interventions to limit antimicrobial resistance need to be tailored to local poultry farm practices that affect bacterial transmission

A pandemic within a pandemic? Admission to COVID-19 wards in hospitals is associated with increased prevalence of antimicrobial resistance in two African settings

BACKGROUND: Patients who develop severe illness due to COVID-19 are more likely to be admitted to hospital and acquire bacterial co-infections, therefore the WHO recommends empiric treatment with antibiotics. Few reports have addressed the impact of COVID-19 management on emergence of nosocomial antimicrobial resistance (AMR) in resource constrained settings. This study aimed to ascertain whether being admitted to a COVID-19 ward (with COVID-19 infection) compared to a non-COVID-19 ward (as a COVID-19 negative patient) was associated with a change in the prevalence of bacterial hospital acquired infection (HAI) species or resistance patterns, and whether there were differences in antimicrobial stewardship (AMS) and infection prevention and control (IPC) guidelines between COVID-19 and non-COVID-19 wards. The study was conducted in Sudan and Zambia, two resource constrained settings with differing country-wide responses to COVID-19. METHODS: Patients suspected of having hospital acquired infections were recruited from COVID-19 wards and non-COVID-19 wards. Bacteria were isolated from clinical samples using culture and molecular methods and species identified. Phenotypic and genotypic resistance patterns were determined by antibiotic disc diffusion and whole genome sequencing. Infection prevention and control guidelines were analysed for COVID-19 and non-COVID-19 wards to identify potential differences. RESULTS: 109 and 66 isolates were collected from Sudan and Zambia respectively. Phenotypic testing revealed significantly more multi-drug resistant isolates on COVID-19 wards in both countries (Sudan p = 0.0087, Zambia p = 0.0154). The total number of patients with hospital acquired infections (both susceptible and resistant) increased significantly on COVID-19 wards in Sudan, but the opposite was observed in Zambia (both p = ≤ 0.0001). Genotypic analysis showed significantly more β-lactam genes per isolate on COVID-19 wards (Sudan p = 0.0192, Zambia p = ≤ 0.0001). CONCLUSIONS: Changes in hospital acquired infections and AMR patterns were seen in COVID-19 patients on COVID-19 wards compared to COVID-19 negative patients on non-COVID-19 wards in Sudan and Zambia. These are likely due to a potentially complex combination of causes, including patient factors, but differing emphases on infection prevention and control, and antimicrobial stewardship policies on COVID-19 wards were highlighted

Is Africa prepared for tackling the COVID-19 (SARS-CoV-2) epidemic. Lessons from past outbreaks, ongoing pan-African public health efforts, and implications for the future

Soon after the novel coronavirus, SARS-CoV-2 (2019-nCoV), was first identified in a cluster of patients with pneumonia (Li et al., 2020), in the Chinese city of Wuhan on 31 December 2019, rapid human to human transmission was anticipated (Hui et al., 2020). The fast pace of transmission is wreaking havoc and stirring media hype and public health concern (Ippolito et al., 2020) globally. When the World Health Organization (WHO) declared the disease, (now officially named COVID-19) a Public Health Emergency of International Concern (PHEIC) on 31st January 2020 (WHO, 2020a), the Director General Dr Tedros Ghebreyesus justified the decision by stating that WHOs greatest concern was the potential for the virus to spread to countries with weaker health systems. Repeated outbreaks of other preventable emerging and re-emerging infectious diseases with epidemic potential have taken their toll on the health systems of many African countries. The devastating 2014–2016 Ebola Virus Epidemic (WHO, 2020b) in West Africa, demonstrated how ill-prepared the affected countries were to rapidly identify the infection and halt transmission (WHO, 2020d, Largent, 2016, Hoffman and Silverberg, 2018, Omoleke et al., 2016). Similarly, the smoldering remnants of the 2018–19 Ebola Virus outbreak in the Democratic Republic of Congo, have demonstrated even for health services with considerable experience of dealing with a certain emerging pathogen, geography and sociopolitical instability, can hamper the response (Aruna et al., 2019)

The role of NagC in Yersinia pestis and Yersinia pseudotuberculosis biofilm development and insect transmission

There are three species of human-pathogenic Yersiniae; two of which cause gastrointestinal disease (Yersinia pseudotuberculosis and Yersinia enterocolitica) and one which causes plague (Yersinia pestis), one of the deadliest diseases in human history. Whilst Y. pseudotuberculosis and Y. enterocolitica are transmitted faeco-orally, Y. pestis is transmitted by insects, classically the Oriental rat flea, Xenopsylla cheopis. All Yersiniae are capable of a method of bacterial messaging known as quorum sensing (QS), which allows individual cells to communicate in a density dependent manner using signalling molecules such as N-acyl-homoserine lactones (AHLs). QS regulates several virulence phenotypes, including the production of Yersinia virulence factors (YOPs), secreted by the type three secretion system (T3SS) into mammalian host cells, auto-aggregation and biofilm formation. Biofilms are aggregates of bacteria within a protective exopolysaccharide matrix and are especially important for Y. pestis, as transmission of plague relies on the biofilm-mediated blockage of the proventriculus within the digestive system of a flea. Understanding the mechanisms of biofilm development are therefore potentially important for developing methods to prevent or control plague transmission. Recent research has also suggested a more prominent role in the spread of plague for other insect vectors, such as body lice (Pediculus corporis). In the Yersiniae, the extracellular polymeric substance (EPS) matrix is composed primarily of the polysaccharide N-acetyl-D-glucosamine (poly-GlcNAc) and extracellular DNA. The production of GlcNAc but not poly-GlcNAc is known to be regulated by NagC, a DNA binding protein responsible for the repression of nagE-nagBACD (GlcNAc catabolic operon) and activation of glmUS (GlcNAc biosynthesis operon) in Escherichia coli, but there is little information on the NagC-dependent regulation of GlcNAc metabolism or on poly-GlcNAc biosynthesis via the hmsHFRS operon in the Yersiniae. This study set out to investigate the contribution of NagC in Y. pseudotuberculosis and Y. pestis to QS, poly-GlcNAc production and biofilm formation in vitro and in vivo on and within the established C. elegans and insect vectors. Artificially fed colonies of X. cheopis and P. corporis were investigated for use in experimental infection models. For both insect species, defibrinated human blood fed through a collagen membrane was the most successful combination for feeding, although problems with reproducible control of environmental conditions, such as humidity, prevented the insect colonies from becoming established. To investigate the contribution of NagC to QS, YOP production, auto-aggregation, poly-GlcNAc biosynthesis and biofilm formation, a ΔnagC mutant was constructed in Y. pestis and compared with a Y. pseudotuberculosis ΔnagC mutant as well as with the corresponding wild type strains. NagC did appear to influence AHL and YOP production in both Y. pseudotuberculosis and Y. pestis. NagC did not influence auto-aggregation in Y. pestis, although the rate of auto-aggregation was slower for the Y. pseudotuberculosis ΔnagC mutant. NagC was however required for the production of poly-GlcNAc (Y. pseudotuberculosis) and biofilm formation (both Y. pseudotuberculosis and Y. pestis). To determine whether NagC directly regulates poly-GlcNAc biosynthesis, the Y. pestis nagC was expressed in E. coli, the recombinant protein purified and subjected to electrophoretic mobility shift assays. The data obtained show that NagC binds to the promoter regions of the GlcNAc metabolic operons nagE-nagBACD and glmUS, as well as hmsHFRS in Y. pestis and hence directly regulates the production of poly-GlcNAc. Using in vitro (glass) and in vivo (nematode and insect) models, both Y. pestis and Y. pseudotuberculosis ΔnagC mutants were evaluated for biofilm formation. In addition, a ‘fake flea’ proventriculus model was developed to investigate attachment and biofilm formation on a chitin substrate. The Y. pseudotuberculosis ΔnagC mutant showed reduced biofilm formation on the chitin surface, compared with the parental strain. On the nematode C. elegans, the Y. pestis ΔnagC mutant produced substantially less biofilm formation than the parent. When Y. pseudotuberculosis was fed to P. corporis, the ΔnagC mutant showed an increased ability to clear the infection and increased survival rates compared with insects fed the parent strain. Taken together, these results indicate that NagC plays a central role in biofilm formation and in the ability of the Yersiniae to be transmitted by insect vectors

Phenotypic and Genotypic Characteristics of Antimicrobial Resistance in Citrobacter freundii Isolated from Domestic Ducks (Anas platyrhynchos domesticus) in Bangladesh.

Antimicrobial resistance (AMR) in Citrobacter freundii poses a serious challenge as this species is one of the sources of nosocomial infection and causes diarrheal infections in humans. Ducks could be the potential source of multidrug-resistant (MDR) C. freundii; however, AMR profiles in C. freundii from non-human sources in Bangladesh have remained elusive. This study aimed to detect C. freundii in domestic ducks (Anas platyrhynchos domesticus) in Bangladesh and to determine their phenotypic and genotypic antibiotic susceptibility patterns. A total of 150 cloacal swabs of diseased domestic ducks were screened using culturing, staining, biochemical, polymerase chain reaction (PCR), and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) to detect C. freundii. Phenotypic and genotypic antibiotic susceptibility patterns were done by the disk diffusion method and PCR, respectively. In total, 16.67% (25/150) of the samples were positive for C. freundii. C. freundii isolates showed a range of 20% to 96% resistance to cefotaxime, gentamicin, levofloxacin, ciprofloxacin, cotrimoxazole, tetracycline, ampicillin, and cephalexin. More than 60% of the isolates were phenotypically MDR, and the index of multiple antibiotic resistance ranged from 0.07 to 0.79. Genes encoding resistance to beta-lactams [blaTEM-1-88% (22/25), blaCMY-2-56% (14/25), blaCMY-9-8% (2/25), and blaCTX-M-14-20% (5/25)], sulfonamides [sul1-52% (13/25), sul2-24% (6/25)], tetracyclines [tetA-32% (8/25) and tetB-4% (1/25)], aminoglycosides [aacC4-16% (4/25)], and fluoroquinolones [qnrA-4% (1/25), qnrB-12% (3/25), and qnrS-4% (1/25)] were detected in the isolated C. freundii. To the best of our knowledge, this is the first study in Bangladesh to detect MDR C. freundii with their associated resistance genes from duck samples. We suggest addressing the burden of diseases in ducks and humans and associated AMR issues using the One Health approach