Antibiotic resistance through the lens of One Health: A study from an urban and a rural area in Sri Lanka.

This study aimed to investigate and compare the proportion of AMR Escherichia coli (E. coli) between urban (Dompe in the Western province) and rural (Dambana in the Sabaragamuwa province) areas in Sri Lanka. The overall hypothesis of the study is that there is a difference in the proportion of AMR E. coli between the urban and the rural areas. Faecal samples were collected from healthy humans (n = 109), dairy animals (n = 103), poultry (n = 35), wild mammals (n = 81), wild birds (n = 76), soil (n = 80) and water (n = 80) from both areas. A total of 908 E. coli isolates were tested for susceptibility to 12 antimicrobials. Overall, E. coli isolated from urban area was significantly more likely to be resistant than those isolated from rural area. The human domain of the area had a significantly higher prevalence of AMR E. coli, but it was not significantly different in urban (98%) and rural (97%) areas. AMR E. coli isolated from dairy animals, wild animals and water was significantly higher in the urban area compared with the rural area. There was no significant difference in the proportion of multidrug resistance (MDR) E. coli isolated from humans, wild animals and water between the two study sites. Resistant isolates found from water and wild animals suggest contamination of the environment. A multi-sectorial One Health approach is urgently needed to control the spread of AMR and prevent the occurrences of AMR in Sri Lanka.

Exploring Barriers to One Health Antimicrobial Stewardship in Sri Lanka: A Qualitative Study among Healthcare Professionals.

Antimicrobial resistance (AMR) is a global health threat, but little is known about the perceptions regarding antimicrobials and AMR among healthcare professionals in Sri Lanka. This research aimed to take a One Health approach to explore the knowledge, attitudes and perceptions of antibiotic stewardship and AMR among healthcare professionals in Sri Lanka. A qualitative study, using telephone interviews, allowing for an in-depth exploration of attitudes, beliefs and perspectives was conducted. Healthcare professionals from both the medical and veterinary sectors were included (n = 29). Interviews were conducted by an independent interviewer and were audio-recorded and transcribed. Conventional qualitative content analysis was undertaken. Four main categories were identified: (1) understanding of AMR and observing AMR, (2) barriers to antimicrobial stewardship, (3) personal factors in, and as a result of, inappropriate antibiotic usage and (4) how to tackle AMR. Healthcare professionals showed poor awareness regarding the spread of AMR and identified inappropriate prescribing behaviours by their inter- and intra-professional colleagues. Patient demands and the influence of pharmaceutical companies were factors contributing to poor prescribing behaviour. Suggestions for the future are stricter regulation of AMR control policy, effective government involvement, and awareness campaigns for healthcare professionals and the public.

Misconceptions of Antibiotics as a Potential Explanation for Their Misuse. A Survey of the General Public in a Rural and Urban Community in Sri Lanka.

Reducing the growth of antimicrobial resistance (AMR) through public understanding is a goal of the World Health Organization. It is especially important in countries where antibiotics are widely available for common ailments without prescription. This study assessed understanding of antibiotics and AMR alongside perception of antibiotic usage among the general public in two diverse Sri Lankan communities: ordinary urban and indigenous rural. A cross-sectional questionnaire survey was conducted, gaining 182 urban and 147 rural responses. The majority of urban respondents (69.2%) believed that they had very good or good knowledge about antibiotics compared to 40.1% of rural respondents. Belief about knowledge and actual knowledge (measured via a test question) were correlated (r = 0.49, p = 0.001) for rural respondents, but not for urban respondents. Several misconceptions about antibiotics were highlighted, including that Paracetamol, a painkiller, was thought to be an antibiotic by more than 50% of both urban and rural respondents. In addition, 18.5% of urban and 35.4% of rural participants would keep and re-use what they perceived as leftover antibiotics. It is urgent that we pay attention to educating the general public regarding the identified misconceptions of these powerful drugs and their appropriate use.

Antimicrobial Resistance Patterns of Fecal Escherichia coli in Wildlife, Urban Wildlife, and Livestock in the Eastern Region of Sri Lanka, and Differences between Carnivores, Omnivores, and Herbivores.

Antimicrobial resistance (AMR) is a major public health concern. We identified AMR in fecal Escherichia coli of wildlife (WL), urban wildlife (UWL), and livestock in the eastern region in Sri Lanka and compared the prevalence of AMR bacteria from carnivores, omnivores, and herbivores. Fecal samples were collected from 165 animals: WL (n=47), UWL (n=54), and livestock (n=64). Esherichia coli was cultured from 129 samples, with isolation rates of 76% from WL (36/47), 70% from UWL (45/54), and 75% from livestock (48/64). Testing E. coli isolates against 12 antimicrobials using the disk diffusion method revealed that the proportions of E. coli isolates resistant to at least one antimicrobial were WL 52.7%, UWL 20%, and livestock 52%. Multidrug-resistant E. coli isolates were detected in WL, UWL, and livestock. Overall, the prevalence of E. coli isolates with AMR was significantly lower in UWL compared with WL and livestock. The number of isolates showed AMR was significantly higher in E. coli from carnivores than in isolates from omnivores and in herbivores. We conclude that AMR E. coli in Sri Lanka is widespread in WL, UWL, and livestock. The higher incidence of AMR bacteria in carnivores compared with herbivores and omnivores suggest that the mechanisms of spread of AMR may vary among wild animals, which requires further investigation.

Characterization and Antimicrobial Resistance of Environmental and Clinical Aeromonas Species Isolated from Fresh Water Ornamental Fish and Associated Farming Environment in Sri Lanka.

The aims of this study were to characterize and investigate antimicrobial susceptibility and presence of integrons in 161 Aeromonas spp. isolated from ornamental freshwater fish farming environment, apparently healthy and diseased fish. Phylogenetic analyses of the gyrB gene sequences identified Aeromonas veronii as the most abundant species (75.8%) followed by Aeromonashydrophila (9.3%), Aeromonas caviae (5%), Aeromonas jandaei (4.3%), Aeromonas dhakensis (3.7%), Aeromonas sobria (0.6%), Aeromonas media (0.6%), and Aeromonas popoffii (0.6%). Susceptibility to thirteen antimicrobials was determined and antimicrobial resistance frequencies were: amoxicillin (92.5%), enrofloxacin (67.1%), nalidixic acid (63.4%), erythromycin (26.1%), tetracycline (23.6%), imipenem (18%), trimethoprim-sulfamethoxazole (16.8%), and gentamicin (16.8%). Multi-drug resistance (MDR) was widespread among the isolates (51.6%, 83/161) with 51.6% (63/122) A. veronii isolates being MDR. In addition, 68.3% of isolates had multiple antibiotic resistance (MAR) indexes higher than 0.2, suggesting that they originated from a high-risk source of contamination where antimicrobials are often used. In all, 21.7% isolates carried class 1 integrons, with 97.1% having gene cassettes, while there were 12 isolates carrying class 2 integron gene cassettes. Our findings highlight that the aquatic environment and ornamental fish act as reservoirs of multidrug resistant Aeromonas spp. and underline the need for a judicious use of antimicrobials and timely surveillance of antimicrobial resistance (AMR) in aquaculture.

Colonization with selected antibiotic resistant bacteria among a cohort of Sri Lankan university students.

BACKGROUND: Antibiotic Resistance is an imminent global public health threat. Antibiotic resistance emerged in healthcare settings and has now moved on to the community settings. This study was conducted to identify the rates of asymptomatic colonization with selected antibiotic resistant organisms, (Methicillin Resistant Staphylococcus aureus (MRSA), Extended Spectrum Beta Lactamase (ESBL) producing Escherichia coli and Klebsiella spp and carbapenem resistant E.coli and Klebsiella spp) - among a group of university students in Sri Lanka. Identification of genetic determinants of MRSA and ESBL was an additional objective of the study. METHODS: A self - collected nasal swab and a peri-rectal swab collected after passing stools were obtained. Routine microbiological methods were used for the isolation S.aureus from the nasal swab and E.coli and Klebsiella species from the peri-rectal swab. Antibiotic sensitivity testing was performed as recommended by clinical and laboratory standard institute (CLSI). Three (3) genes that are responsible for ESBL production; blaCTX-M, blaSHV, and blaTEM were tested using previously described primers and PCR procedures. Identification of MecA and PVL genes attributed to MRSA was also done with PCR. RESULTS: A total of 322 participants between 21 and 28 years were recruited representing 5 different faculties of study. Seventy one (22.0%) were colonized with S.aureus and 14 among them with MRSA, making the MRSA colonization rate of 4.3%. Forty five (15%) of the participants were colonized with an ESBL producing E.coli or Klebsiella spp. No one was colonized with carbapenem resistant E.coli or Klebsiella species. Of the 45 ESBL producers the commonest genetic determinant identified was blaCTX-M (n = 36), while 16 isolates had blaTEM and 7 had blaSHV. Similarly, of the 14 isolates identified as MRSA, 3 (21.4%) were found to be PVL positive while 11 (78.6%) were MecA positive. CONCLUSIONS: A high rate of colonization with ESBL producing E.coli and Klebsiella species was noted in our study group.

Methicillin-resistant Staphylococcus pseudintermedius among dogs in the description of novel SCCmec variants.

The presence and genetic characteristics of methicillin-resistant Staphylococcus pseudintermedius (MRSP) in Sri Lanka was investigated to add additional insight into global spread, emergence and evolution of MRSP. A total of 234 samples from dogs visiting veterinary clinics were cultured for staphylococci and the genomes of the MRSP isolates were sequenced, to identify resistance genes, the multilocus sequence types (MLST) and spa types. From a questionnaire the history of antimicrobial treatment and patient information was obtained. S. pseudintermedius was isolated from 116/229 samples, eight of these were MRSP. Six MRSP CC45 isolates contained a pseudo-SCC element ΨSCCmec57395. Two isolates belonging to ST429 (CC761) and ST121 (CC121) contained novel variants of the SCCmec Type V(T) element. The elements were designated SCCmecV(T)SL/066, that carried additional transposon-related genes, and SCCmecV(T)SL/154 that carried a type III restriction-modification system, a type 7 ccr gene complex, and a cadA coding sequence. Thirty-seven percent of the dogs received antimicrobial treatment at the time of sampling of which four dogs were MRSP-positive. The proportion of MRSP among S. pseudintermedius is low compared to other countries, despite the fact that in Sri Lanka antimicrobials for treatment of dogs are available over the counter. Important is the finding of novel type V(T) SCCmec elements, which further underlines the high recombination frequency of SCC elements. The ΨSCCmec57395 was found in isolates of CC45, which is the only sequence type of MRSP known to contain this pseudo-cassette.

Campylobacter in Broiler Chicken and Broiler Meat in Sri Lanka: Influence of Semi-Automated vs. Wet Market Processing on Campylobacter Contamination of Broiler Neck Skin Samples.

Broiler meat can become contaminated with Campylobacter of intestinal origin during processing. The present study aimed to identify the prevalence of Campylobacter in broiler flocks and meat contamination at retail shops, and determine the influence of semi-automated and wet market processing on Campylobacter contamination of neck skin samples. Samples were collected from semi-automated plants (n = 102) and wet markets (n = 25). From each batch of broilers, pooled caecal samples and neck skin samples were tested for Campylobacter. Broiler meat purchased from retail outlets (n = 37) was also tested. The prevalence of Campylobacter colonized broiler flocks was 67%. The contamination of meat at retail was 59%. Both semi-automated and wet market processing resulted to contaminate the broiler neck skins to the levels of 27.4% and 48%, respectively. When Campylobacter-free broiler flocks were processed in semi-automated facilities 15% (5/33) of neck skin samples became contaminated by the end of processing whereas 25% (2/8) became contaminated after wet market processing. Characterization of isolates revealed a higher proportion of C. coli compared to C. jejuni. Higher proportions of isolates were resistant to important antimicrobials. This study shows the importance of Campylobacter in poultry industry in Sri Lanka and the need for controlling antimicrobial resistance.

Activation of murine dendritic cells and macrophages induced by Salmonella enterica serovar Typhimurium.

Macrophages and dendritic cells (DCs) are antigen-presenting cells (APCs), and the direct involvement of both cell types in the immune response to Salmonella has been identified. In this study we analysed the phenotypic and functional changes that take place in murine macrophages and DCs in response to live and heat-killed Salmonella enterica serovar Typhimurium. Both types of cell secreted proinflammatory cytokines and nitric oxide (NO) in response to live and heat-killed salmonellae. Bacterial stimulation also resulted in up-regulation of costimulatory molecules on macrophages and DCs. The expression of major histocompatibility complex (MHC) class II molecules by macrophages and DCs was differentially regulated by interferon (IFN)-gamma and salmonellae. Live and heat-killed salmonellae as well as lipopolysaccharide (LPS) inhibited the up-regulation of MHC class II expression induced by IFN-gamma on macrophages but not on DCs. Macrophages as well as DCs presented Salmonella-derived antigen to CD4 T cells, although DCs were much more efficient than macrophages at stimulating CD4 T-cell cytokine release. Macrophages are effective in the uptake and killing of bacteria whilst DCs specialize in antigen presentation. This study showed that the viability of salmonellae was not essential for activation of APCs but, unlike live bacteria, prolonged contact with heat-killed bacteria was necessary to obtain maximal expression of the activation markers studied.

Salmonella enterica serovar Typhimurium expressing mutant lipid A with decreased endotoxicity causes maturation of murine dendritic cells.

A major Salmonella component involved in cellular activation is the lipopolysaccharide (LPS) molecule which can act as a dendritic cell (DC) stimulator. The structure of the lipid A domain of the LPS molecule dictates its immunostimulatory capacity with various cell types. In this study, the role of lipid A as an integral component of Salmonella in stimulating murine DCs was studied by using a Salmonella enterica serovar Typhimurium lpxM mutant with defective lipid A. This study revealed that a mutation in lpxM did not significantly affect the ability of bacteria to activate DCs. Although the lpxM mutant less tumor necrosis factor alpha, interleukin-1beta, and inducible nitric oxide synthase than the parental strain, this was only seen at lower multiplicities of infection (MOIs). Both strains upregulated surface molecule expression on DCs and augmented the T-cell-stimulating capacity of these cells in an MOI-independent manner. Thus, the lpxM mutation did not appear to affect the stimulatory capacity of the Salmonella mutant.