Genetic insights of antibiotic resistance, pathogenicity (virulence) and phylogenetic relationship of Escherichia coli strains isolated from livestock, poultry and their handlers - a one health snapshot.

BACKGROUND: Pathogenic and non-pathogenic strains of Escherichia coli harbouring antibiotic resistance genes (ARGs) from any source (clinical samples, animal settings, or environment) might be transmitted and contribute to the spread and increase of antibiotic resistance in the biosphere. The goal of this study was to investigate the genome to decipher the repertoire of ARGs, virulence genes carried by E. coli strains isolated from livestock, poultry, and their handlers (humans), and then unveil the genetic relatedness between the strains. METHODS: Whole genome sequencing was done to investigate the genetic makeup of E. coli isolates (n = 20) [swine (n = 2), cattle (n = 2), sheep (n = 4), poultry (n = 7), and animal handlers (n = 5)] from southern India. The detection of resistome, virulome, biofilm forming genes, mobile genetic elements (MGE), followed by multilocus sequence typing (MLST) and phylogenetic analyses, were performed. RESULTS: E. coli strains were found to be multi drug resistant, with a resistome encompassing > 20 ARGs, the virulome-17-22 genes, and > 20 key biofilm genes. MGE analysis showed four E. coli isolates (host: poultry, swine and cattle) harbouring composite transposons with ARGs/virulence genes (blaTEM, dfr, qnr/nleB, tir, eae,and esp) with the potential for horizontal transfer. MLST analyses revealed the presence of ST937 and ST3107 in both livestock/poultry and their handlers. Phylogenomic analyses with global E. coli isolates (human/livestock/poultry hosts) showed close relatedness with strains originating from different parts of the world (the United States, China, etc.). CONCLUSION: The current study emphasizes the circulation of strains of pathogenic sequence types of clinical importance, carrying a diverse repertoire of genes associated with antibiotic resistance, biofilm formation and virulence properties in animal settings, necessitating immediate mitigation measures to reduce the risk of spread across the biosphere.

Mapping Heterogeneous Population Structure of Mannheimia haemolytica Associated with Pneumonic Infection of Sheep in Southern State Karnataka, India.

Mannheimia haemolytica is recognized as principal pathogen associated with pneumonic pasteurellosis leading to huge economic losses to small ruminant farmers. Even though the disease causes huge economic losses, epidemiology of M. haemolytica is less studied, hindering the formulation of effective control strategies. Current study aimed to highlight molecular characterisation of M. haemolytica strains isolated from ovine pneumonic infection. M. haemolytica 27 isolates with two reference strains were characterised using capsular and virulence gene typing, multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) methods. M. haemolytica serotype A2 recognized as predominant serotype (74%) followed by A6 (11%) and A1 (5%) serotypes. Virulence gene profiling by PCRs showed dominance of all five virulent genes [such as adh and gcp (100% each)] followed by gs60 (88.8%), lktC (85.2%), tbpB (51.9%) and least nmaA gene (14.8%). MLST profiling delineated M. haemolytic isolates into 11 sequence types (STs) with most prevalent being ST37 (27.9%) and ST16 (23%) and nine new STs (ST37, 38, 39, 40, 41, 42, 47, 48, and 49). These new STs did not belong to any of the three clonal complexes (CC4, CC8 and CC28). ST16 was exclusively noted in A1 and A6 serotypes. Amongst 25 isolates, 22 pulsotypes (GD 0.88) recorded indicated variability of the M. haemolytica isolates in PFGE analysis. In conclusion, the study suggested dominance of M. haemolytica serotype A2 harbouring different virulent genes, diverse STs and pulsotypes responsible for pneumonic pasteurellosis frequently encountered in sheep.

Seroprevalence and associated risk factors of leptospirosis in bovine dairy farms in Telangana state, India.

The current cross-sectional study aimed to determine the seroprevalence of Leptospira infection in bovine dairy farms in the Telangana state of India, as well as the associated risk factors, in order to implement effective preventive measures for disease control. A total of 469 blood samples were collected from 67 herds/farms in different areas, covering 20 administrative districts in the state. These samples consisted of 253 from cattle and 216 from buffaloes. Questionnaires were used to collect data on host and epidemiological factors. The collected sera were tested using the gold standard serological test, the Microscopic Agglutination Test (MAT), which employed a panel of 18 reference serovars for Leptospira exposure. The statistical analysis of epidemiological data was carried out to identify the risk factors associated with Leptospira exposure. The overall observed seroprevalence at the animal and farm levels was 41.4% and 77.6%, respectively. The most prevalent anti-leptospiral antibodies were observed against the serogroups Icterohaemorrhagiae (32.4%), Pomona (22.2%), Javanica (19.1%), Australis (17.0%), Bataviae (15.5%), Autumnalis (12.9%), Hebdomadis (12.9%), and others, in the total reacting samples. At the animal level, the significant risk factors associated with exposure to Leptospira species were breed (p = 0.03) and health status (p = 0.03). Furthermore, the multivariate statistical analysis of farm factors revealed that farm size (p = 0.05), presence of dogs (p = 0.04) and rodents (p = 0.01) on the farm, use of fodder from wet soils (p = 0.04), and proximity to water bodies (p = 0.04) were significantly associated with exposure to Leptospira in the studied region. This study provides the first report from India highlighting the important risk factors at the herd/farm and animal level associated with Leptospira infections in cattle and buffaloes. The findings contribute to strengthening the one-health strategy by facilitating the design and planning of appropriate control measures to alleviate the burden of leptospirosis in bovines.

Genomic insights of beta-lactamase producing Klebsiella quasipneumoniae subsp. similipneumoniae belonging to sequence type 1699 from retail market fish, India.

Klebsiella quasipneumoniae is a recently described species and often misidentified as Klebsiella pneumoniae. Here, we report the genomic characterization of Klebsiella quasipneumoniae subsp. similipneumoniae (India238 strain) isolated from fish. The annotated genome acknowledged the presence of blaCTX-M-15, blaOKP-B-1, fosA5, oqxAB and virulence genes. The strain with ST1699 and serotypes KL52 and OL103 also harboured insertion sequences (ISs): ISKpn26 and ISEc9. Three complete phage genomes were identified in contigs 1 and 6 of the bacterial genome, enhancing the prospects of genome manipulation. The study highlights the pitfall of conventional microbiological identification methods to distinguish K. pneumoniae and K. quasipneumoniae. This is the first Indian study documenting the incidence of extended-spectrum beta-lactamase (ESBL)-producing K. quasipneumoniae subsp. similipneumoniae from a non-clinical environment, equipped with virulomes and associated mobile genetic elements. Given that fish can act as a potential vector for transmission of antimicrobial resistance genes, our findings have paramount importance on human health.

Countrywide cross-sectional study of swine brucellosis sero-prevalence in Indian subcontinent during 2018-2019.

Brucellosis in swine is a contagious disease with greater zoonotic potential caused by Brucella suis. The study describes PAN India swine brucellosis sero-prevalence in 5431 stratified random serum samples collected during 2018-2019 from 26 out of 29 states and two out of seven union territories. The serum samples were tested for anti-Brucella antibodies by indirect ELISA and overall, 4.33% apparent prevalence (AP) was recorded. The AP is ≥ 10% in five states among 26 states, P ≥ 50% in four districts out of 117 districts screened and cent percent prevalence in two epi units out of 264 sampled. Significantly high seropositivity (p < 0.05) in male (6.08%) than female pigs (3.46%) and in ≥ 24-month-old pigs indicated older and male pigs as potential carriers of the disease. The study recorded endemicity of the swine brucellosis in few regions of India requiring periodical surveillance for control of the disease. Brucella testing of boars before breeding and awareness among farmers and veterinarians will aid in reduction of disease burden in the absence of vaccination policy.

Whole genome global insight of antibiotic resistance gene repertoire and virulome of high - risk multidrug-resistant Uropathogenic Escherichiacoli.

Elucidation of genetic determinants via whole genome sequence (WGS) analyses can help understand the high risk multidrug-resistant (MDR) Uropathogenic Escherichia coli (UPEC) associated with urinary tract infections (UTI) and its evasion strategies from treatment. We investigated the WGS of 30 UPEC strains from UTI samples across the world (2016-2019) and found 25 UPEC strains carrying 2-23 antibiotic resistance genes (ARGs) scattered across 1-3 plasmids per strain. Different ARGs (blaTEM, blaCTXM, blaNDM, blaOXA, blaCMY) encoding extended-spectrum beta-lactamases (TEM, CTXM, CMY) and carbapenemases (NDM, OXA) were found in 24/30, ARGs encoding aminoglycoside modifying enzymes (AAC, APH, AAD) variants in 23/30, trimethoprim ARGs (dfrA17, dfrA12, dfrA5, dfrB4 variants) encoding dihydrofolate reductase in 19/30 and sulfonamide ARGs (sul1, sul2, sul3) encoding dihydropteroate synthase and macrolide ARGs (mph1) encoding macrolide 2' phosphotransferase in 15/30 UPEC strains. Collectively the ARGs were distributed in different combinations in 40 plasmids across UPEC strains with 20 plasmids displaying co-occurrence of multiple ARGs conferring resistance to beta lactam, aminoglycoside, sulfonamide, trimethoprim and macrolide antibiotics. These resistance plasmids belonged to seven incompatibility groups (IncF, IncI, IncC, IncH, IncN, IncB and Col), with IncFI and IncFII being the predominant resistance plasmids. Additionally, we observed co-occurrence of specific mutation pattern in quinolone resistance determining region (QRDR) viz., DNA gyrase (gyrA: S83L, D87N), and topoisomerase IV (parC: S80I, E84V; parE: I529L) in 18/30 strains. The strains also harbored diverse virulence genes, such as fimH, gad, iss, iha, ireA, iroN, cnf1 and san. Multilocus sequence typing (MLST) reconfirmed ST131(n = 10) as the predominant global high-risk clonal strain causing UTI. In summary, our findings contribute to better understand the plasmid mediated ARGs and its encoded enzymes that may contribute in antibiotic inactivation/modification or alteration in the antibiotic target site in high risk MDR hypervirulent UPEC strains causing UTI. The study reinforces the need to characterize and design appropriate inhibitors to counterattack different enzymes and devise strategies to curtail resistance plasmid.

Virulence and intermediate resistance to high-end antibiotic (teicoplanin) among coagulase-negative staphylococci sourced from retail market fish.

This study reports the distribution of enterotoxigenic determinants among staphylococci and the susceptibility of staphylococci to various classes of antibiotics. We observed all the isolates as resistant to beta-lactam antibiotics and a few as resistant to non-beta-lactam antibiotics such as clindamycin (47.4%), erythromycin (44.7%), gentamicin (23.7%), norfloxacin (34.2%), tetracycline (26.3%), trimethoprim-sulfamethoxazole (15.8%) etc. The resistance of S. sciuri (n = 1) and S. haemolyticus (n = 1) to rifampicin and intermediate resistance of S. gallinarum (n = 2) to teicoplanin, a high-end antibiotic, are also observed in this study. The multidrug-resistance (≥ 3 classes of antibiotics) was recorded in 23 (60.5%) isolates. The virulomes such as sea, seb, seg and sei were identified predominantly in S. haemolyticus. Surprisingly, certain isolates which were phenotypically confirmed as biofilm-producers by Congo red agar (CRA) test did not harbor biofilm-associated loci. This implies the protein-mediated mechanism of biofilm formation as an alternative to polysaccharide intercellular adhesin (PIA) in staphylococci. However, icaAD locus which encodes PIA was identified in 10 (26.3%) isolates and the eno locus, encoding elastin-binding protein which can accelerate the biofilm production, is identified in all the isolates. The possession of type V SCCmec elements by the S. haemolyticus (15.8%) raised the concern about the rapid dissemination of mecA gene to other species of staphylococci including the virulent S. aureus. In short, this study acknowledges the toxigenicity of coagulase-negative staphylococci (CoNS). Through this study, surveillance of antimicrobial resistance and transference of virulomes in staphylococci is warranted.

Animal disease surveillance: Its importance & present status in India.

Animal disease surveillance encompasses systematic collection of long-term data on disease events, risk factors and other relevant parameters followed by analyzing the same with reference to temporal and spatial characteristics to arrive at a conclusion so that necessary preventive measures can be taken. In India, the animal disease surveillance is done through National Animal Disease Reporting System, which is a web-based information technology system for disease reporting from States and Union Territories with the aim to record, monitor livestock disease situation and to initiate the preventive and curative action in a swift manner during disease emergencies. National Animal Disease Referral Expert System is a dynamic geographic information system and remote sensing-enabled expert system that captures an incidence of 13 economically important livestock diseases from all over the country and also provides livestock disease forecasting. The laboratories under State and Central governments, several research institutes under the Indian Council of Agricultural Research and veterinary colleges are involved in livestock disease diagnosis including zoonotic diseases. An integrated surveillance system is necessary for early detection of emerging/zoonotic diseases in humans. This review provides information on disease reporting and surveillance systems in animal health sector and the need for One Health approach to improve and strengthen the zoonotic disease surveillance system in India.

Molecular assessment of antimicrobial resistance and virulence in multi drug resistant ESBL-producing Escherichia coli and Klebsiella pneumoniae from food fishes, Assam, India.

The present study investigated the prevalence of Extended-Spectrum Beta Lactamase (ESBL) -producing E. coli and K. pneumoniae from the food fishes in retail markets in Assam, India. A total of 54 ESBL-producing E. coli and 12 K. pneumoniae isolates were recovered from 79 fish samples and were analyzed for antimicrobial resistance genes (ARGs) and virulence genes. E. coli isolates were categorized as multi drug resistant with resistance up to 12 different antibiotics with multiple antibiotic resistances (MAR) index ranging from 0.26 to 0.63. In E. coli, 100% resistance to cefotaxime along with 6% resistance to ceftazidime (third-generation cephalosporins) was observed. Moreover, 85% of the E. coli isolates were resistant to cefepime, a fourth-generation cephalosporin. K. pneumoniae showed resistance to 11 different antibiotics with MAR index value ranging from 0.21 to 0.57. All K. pneumoniae isolates showed 100% resistance to cefotaxime, 67% resistance to ceftazidime and 75% resistance to cefepime. Molecular characterization of ARGs revealed the presence of CTX-M group 1(CTX-M-15) in almost all E. coli isolates (98%, n = 53) and 100% in K. pneumoniae. A combination of uniplex and multiplex PCRs revealed fewer ARGs in E. coli isolates, with each isolate carrying 3 to 5 genes (tetA, dfrA1, sul1, sul2, qnrB, qnrS, aac(6')-Ib-cr). Majority of the E. coli were assigned to low-virulence phylogroup B1 and A while 8% of them belonged to pathogenic phylogroup D. 31 unique genetic profiles were identified for E. coli isolates by Pulsed-Field Gel Electrophoresis (PFGE) typing. K. pneumoniae isolates were highly diverse with 11 unique genetic profiles and a substantial ARG profile (blaTEM, blaSHV, blaOXA-1-like, tetA, strA, strB, dfrA1, sul1, sul2, qnrB, qnrS, aac(6')-Ib-cr, oqxA, oqxB). The frequency of ARGs ranged between 4 and 11. All K. pneumoniae isolates belonged to capsular serotype with wzi gene. Virulence gene iutA was prominent in all isolates while ybtS and kfu were confirmed in two isolates. Our findings raise concerns that fishes bought for consumption may serve as potential reservoirs of AMR genes and pose serious threat to public health. The study emphasizes the need for extensive surveillance of resistant strains in aquaculture and related settings, their in-depth analysis of population structure and transmission dynamics.

Understanding policy dilemmas around antibiotic use in food animals & offering potential solutions.

The looming concern of antimicrobial resistance (AMR) has prompted the government of many countries of the world to act upon and come up with the guidelines, comprehensive recommendations and policies concerning prudent use of antibiotics and containment of AMR. However, such initiatives from countries with high incidence of antibiotic-resistant bacteria in food animals are still in infancy. This review highlights the existing global policies on antibiotics use in food animals along with details of the various Indian policies and guidelines. In India, in spite of availability of integrated policies for livestock, poultry and aquaculture sector, uniform regulations with coordinated initiative are needed to formulate strict policies regarding antimicrobial use both in humans and animals. In an attempt to create effective framework to tackle the AMR, the Indian Council of Medical Research initiated a series of dialogues with various stakeholders and suggested various action points for urgent implementation. This review summarizes the recommendations made during the various consultations. The overarching aim of this review is to clearly delineate the action points which need to be carried out urgently to regulate the antibiotic use in animals.

Assessment of fluorescence polarization assay: a candid diagnostic tool in Brucella abortus strain 19 vaccinated areas.

Brucellosis caused by the bacteria of the genus Brucella is an important zoonosis and constitutes a serious public health hazard. Brucellosis is diagnosed mainly by the Rose Bengal plate test and indirect ELISA, both of which have poor specificity because false positive serological reactions occur when screening animals that have been vaccinated with B. abortus S19. Fluorescence polarization assay (FPA) was evaluated for screening samples from cattle and buffalo calves with persistent antibody titers induced by vaccination. The standardized FPA exhibited relative sensitivity and specificity of 0.94 and 0.95, respectively, and the area under the curve, kappa and accuracy were 0.98, 0.87 and 0.95, respectively. Comparison of competitive ELISA and FPA revealed that, FPA is more specific than competitive ELISA. The high specificity, sensitivity and 95% accuracy of FPA indicate that, it is suitable for testing vaccinated animals because it can distinguish between infected from vaccinated animals.

Prevalence of Leptospira serogroup-specific antibodies in cattle associated with reproductive problems in endemic states of India.

In this study, the seroprevalence and distribution of Leptospira in dairy cattle in endemic states of India were investigated in association with reproductive problems of the cattle. A total of 373 cattle serum samples from 45 farms in Maharashtra, Gujarat, Andhra Pradesh, Telangana, Karnataka, Tamil Nadu, Punjab, Haryana, Chhattisgarh, Sikkim and Uttarakhand states were collected from animals with a history of reproductive disorders like abortion, repeat breeding, anoestrus and endometritis, and also from apparently healthy animals. These samples were screened for Leptospira serogroup-specific antibodies by microscopic agglutination test (MAT) using a panel of 18 live reference serovar antigens. The seropositivity of 70.51% (263/373, 95% CI 0.65 to 0.75) was associated with reproductive problems (χ2 = 55.71, p < 0.01) and sampled states (χ2 = 32.99, p < 0.01) and independent of apparently healthy animals (χ2 = 15.6, p > 0.10) and age groups of cattle (χ2 = 0.91, p > 0.10). Further, the odds (risk-relation) of reproductive disorders was 5.29 compared to apparently healthy animals (0.25 odds). The frequency distribution of predominant serogroup-specific Leptospira antibodies were determined against the serovars: Hardjo (27.76%), Pyrogenes (18.63%), Canicola and Javanica (17.49%), Hebdomadis (17.11%), Shermani and Panama (16.73%), Djasiman (16.35%), Tarassovi, Grippotyphosa and Pomona (15.97%), Icterohaemorrhagiae (15.59%), Copenhageni (14.83%), Australis (13.69%), Kaup and Hurstbridge (10.65%), Bankinang (10.27%) and Bataviae (9.51%). In conclusion, dairy cattle have a role in maintaining important several serovars besides well-known Hardjo serovar in endemic states of India and warrant mitigating measures to reduce the incidence of cattle leptospirosis including need for an intensive surveillance programme, preventive vaccination and control strategies.

Biocompatible Injectable Hydrogel with Potent Wound Healing and Antibacterial Properties.

Two component injectable hydrogels that cross-link in situ have been used as noninvasive wound-filling devices, i.e., sealants. These materials carry a variety of functions at the wound sites, such as sealing leaks, ceasing unwanted bleeding, binding tissues together, and assisting in wound healing processes. However, commonly used sealants typically lack antibacterial properties. Since bacterial infection at the wound site is very common, bioadhesive materials with intrinsic antibacterial properties are urgently required. Herein, we report a biocompatible injectable hydrogel with inherent bioadhesive, antibacterial, and hemostatic capabilities suitable for wound sealing applications. The hydrogels were developed in situ from an antibacterial polymer, N-(2-hydroxypropyl)-3-trimethylammonium chitosan chloride (HTCC), and a bioadhesive polymer, polydextran aldehyde. The gels were shown to be active against both Gram-positive and Gram-negative bacteria, including drug-resistant ones such as methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus faecium (VRE), and ß-lactam-resistant Klebsiela pneumoniae. Mechanistic studies revealed that the gels killed bacteria upon contact by disrupting the membrane integrity of the pathogen. Importantly, the gels were shown to be efficacious in preventing sepsis in a cecum ligation and puncture (CLP) model in mice. While only 12.5% of animals survived in the case of mice with punctured cecam but with no gel on the punctured area (control), 62.5% mice survived when the adhesive gel was applied to the punctured area. Furthermore, the gels were also shown to be effective in facilitating wound healing in rats and ceasing bleeding from a damaged liver in mice. Notably, the gel showed negligible toxicity toward human red blood cells (only 2-3% hemolysis) and no inflammation to the surrounding tissue upon subcutaneous implantation in mice, thus proving it as a safe and effective antibacterial sealant.

Chitosan Derivatives Active against Multidrug-Resistant Bacteria and Pathogenic Fungi: In Vivo Evaluation as Topical Antimicrobials.

The continuous rise of antimicrobial resistance and the dearth of new antibiotics in the clinical pipeline raise an urgent call for the development of potent antimicrobial agents. Cationic chitosan derivatives, N-(2-hydroxypropyl)-3-trimethylammonium chitosan chlorides (HTCC), have been widely studied as potent antibacterial agents. However, their systemic structure-activity relationship, activity toward drug-resistant bacteria and fungi, and mode of action are very rare. Moreover, toxicity and efficacy of these polymers under in vivo conditions are yet to be established. Herein, we investigated antibacterial and antifungal efficacies of the HTCC polymers against multidrug resistant bacteria including clinical isolates and pathogenic fungi, studied their mechanism of action, and evaluated cytotoxic and antimicrobial activities in vitro and in vivo. The polymers were found to be active against both bacteria and fungi (MIC = 125-250 µg/mL) and displayed rapid microbicidal kinetics, killing pathogens within 60-120 min. Moreover, the polymers were shown to target both bacterial and fungal cell membrane leading to membrane disruption and found to be effective in hindering bacterial resistance development. Importantly, very low toxicity toward human erythrocytes (HC50 = >10000 µg/mL) and embryo kidney cells were observed for the cationic polymers in vitro. Further, no inflammation toward skin tissue was observed in vivo for the most active polymer even at 200 mg/kg when applied on the mice skin. In a murine model of superficial skin infection, the polymer showed significant reduction of methicillin-resistant Staphylococcus aureus (MRSA) burden (3.2 log MRSA reduction at 100 mg/kg) with no to minimal inflammation. Taken together, these selectively active polymers show promise to be used as potent antimicrobial agents in topical and other infections.

Side Chain Degradable Cationic-Amphiphilic Polymers with Tunable Hydrophobicity Show in Vivo Activity.

Cationic-amphiphilic antibacterial polymers with optimal amphiphilicity generally target the bacterial membranes instead of mammalian membranes. To date, this balance has been achieved by varying the cationic charge or side chain hydrophobicity in a variety of cationic-amphiphilic polymers. Optimal hydrophobicity of cationic-amphiphilic polymers has been considered as the governing factor for potent antibacterial activity yet minimal mammalian cell toxicity. However, the concomitant role of hydrogen bonding and hydrophobicity with constant cationic charge in the interactions of antibacterial polymers with bacterial membranes is not understood. Also, degradable polymers that result in nontoxic degradation byproducts offer promise as safe antibacterial agents. Here we show that amide- and ester (degradable)-bearing cationic-amphiphilic polymers with tunable side chain hydrophobicity can modulate antibacterial activity and cytotoxicity. Our results suggest that an amide polymer can be a potent antibacterial agent with lower hydrophobicity whereas the corresponding ester polymer needs a relatively higher hydrophobicity to be as effective as its amide counterpart. Our studies reveal that at higher hydrophobicities both amide and ester polymers have similar profiles of membrane-active antibacterial activity and mammalian cell toxicity. On the contrary, at lower hydrophobicities, amide and ester polymers are less cytotoxic, but the former have potent antibacterial and membrane activity compared to the latter. Incorporation of amide and ester moieties made these polymers side chain degradable, with amide polymers being more stable than the ester polymers. Further, the polymers are less toxic, and their degradation byproducts are nontoxic to mice. More importantly, the optimized amide polymer reduces the bacterial burden of burn wound infections in mice models. Our design introduces a new strategy of interplay between the hydrophobic and hydrogen bonding interactions keeping constant cationic charge density for developing potent membrane-active antibacterial polymers with minimal toxicity to mammalian cells.

Comparative evaluation of recombinant LigB protein and heat-killed antigen-based latex agglutination test with microscopic agglutination test for diagnosis of bovine leptospirosis.

This study aimed to develop latex agglutination test (LAT) using recombinant leptospiral immunoglobulin-like protein (LigB) (rLigB) antigen and compare its diagnostic efficacy with LAT using conventional heat-killed leptospiral antigen and microscopic agglutination test (MAT) in diagnosing bovine leptospirosis. The PCR-amplified 1053-bp ligB gene sequences from Leptospira borgpetersenii Hardjo serovar were cloned in pET 32 (a) vector at EcoRI and NotI sites and expressed in BL21 E. coli cells as fusion protein with thioredoxin (-57 kDa) and characterized by SDS-PAGE and immunoblot. Out of 390 serum samples [cattle (n = 214), buffaloes (n = 176)] subjected to MAT, 115 samples showed reciprocal titre≥100 up to 1600 against one or more serovars. For recombinant LigB protein/antigen-based LAT, agglutination was observed in the positive sample, while no agglutination was observed in the negative sample. Similarly, heat-killed leptospiral antigen was prepared from and used in LAT for comparison with MAT. A two-sided contingency table was used for analysis of LAT using both the antigens separately against MAT for 390 serum samples. The sensitivity, specificity and positive and negative predictive values of recombinant LigB LAT were found to be 75.65, 91.27, 78.38 and 89.96 %, respectively, and that of heat-killed antigen-based LAT were 72.17, 89.82, 74.77 and 88.53 %, respectively, in comparison with MAT. This developed test will be an alternative/complementary to the existing battery of diagnostic assays/tests for specific detection of pathogenic Leptospira infection in bovine population.

Membrane Disruption and Enhanced Inhibition of Cell-Wall Biosynthesis: A Synergistic Approach to Tackle Vancomycin-Resistant Bacteria.

Resistance to glycopeptide antibiotics, the drugs of choice for life-threatening bacterial infections, is on the rise. In order to counter the threat of glycopeptide-resistant bacteria, we report development of a new class of semi-synthetic glycopeptide antibiotics, which not only target the bacterial membrane but also display enhanced inhibition of cell-wall biosynthesis through increased binding affinity to their target peptides. The combined effect of these two mechanisms resulted in improved in vitro activity of two to three orders of magnitude over vancomycin and no propensity to trigger drug resistance in bacteria. In murine model of kidney infection, the optimized compound was able to bring bacterial burden down by about 6 logs at 12 mg kg(-1) with no observed toxicity. The results furnished in this report emphasize the potential of this class of compounds as future antibiotics for drug-resistant Gram-positive infections.

Knowledge and practices related to antibiotics among poultry producers and veterinarians in two Indian states.

Background: Antibiotics are frequently utilized in livestock, particularly poultry, for therapy and growth promotion, resulting in antimicrobial resistance. Multidrug-resistant bacteria are frequent in poultry samples from India. The purpose of this study was to better understand main antibiotic consumption patterns in poultry value chains, as well as antibiotic knowledge and practices among the stakeholders. Methods: A cross-sectional survey was conducted in Assam and Karnataka, India. The poultry farmers were interviewed on antibiotic usage, antibiotic knowledge, feeding practices, and preventive measures on the farm. Poultry farmers reported their veterinarians, and we also interviewed them on knowledge and practices related to antimicrobial use in poultry and antimicrobial resistance. Item response theory (IRT) was used to assess the association between the answers and demographic factors. Results: This survey interviewed 62 poultry farmers and 11 veterinarians. Small poultry farms with fewer than 4000 birds were owned by 51.6% of farmers. Most poultry farmers had heard about antibiotics, and 62.9% thought they cured all diseases. If one chicken is sick, 72.6% said others should be given antibiotics to prevent the disease. All veterinarians utilized tetracyclines, aminoglycosides, and cephalexin on the poultry farms. Over half (54.5%) stated antibiotics prevent diseases, and 72.7% said they treat and prevent diseases. Some (45.5%) said antibiotics boost growth. IRT analysis showed that 8 questions assessed a knowledge scale well. Univariable analysis showed that Assam farmers and women were likely to have have more knowledge. Conclusion: The poultry farmers were mostly unaware of the relation between antibiotic use and antimicrobial resistance. Despite being aware, the veterinarians agreed with use antibiotics as a prophylactic measure. It is vital that these stakeholders understand the repercussions of such widespread antibiotic use. In order to increase knowledge, frequent trainings and antimicrobial stewardship programmes with effective communication and incentives for behaviour change should be conducted.

Peste des Petits Ruminants (PPR) vaccine R&D investment: financial assessment of vaccine development and administration in India.

The present study assessed the financial viability of Peste des Petits Ruminants (PPR) vaccine Research & Development (R&D) investment in India and the Gross Technology Revenue (GTR) accrual to the different stakeholders. The Net Present Value (NPV), Internal Rate of Return (IRR) and Benefit Cost Ratio (BCR) of PPR vaccine development and administration were USD 16,326.6 million (INR 130,612 crore), USD 184,54.2 million (INR 147,633 crore) and USD 21,645.6 million (INR 173,164 crore); 162.2%, 167.6% and 169.7% and 43.3:1, 48.8:1 and 57.1:1, respectively under low, medium and high disease incidence scenarios. The estimated cumulative GTR accrued during 2001-02 to 2017-18 by the innovating public research institutions (Indian Council of Agricultural Research-Indian Veterinary Research Institute (ICAR-IVRI) and Tamil Nadu Veterinary and Animal Sciences University (TANUVAS)), private vaccine producers, public sector biologicals and government revenues in terms of taxes was USD 0.696 million (INR 5.568 crore) for ICAR-IVRI and USD 0.033 million (INR 0.26 crore) for TANUVAS; USD 5.00 million (INR 40 crore); USD 7.141 million (INR 57.1 crore) and USD 0.671 million (INR 5.36 crore), respectively. Overall, financial benefits of PPR vaccine development and administration to control PPR in India outweighs the investment in manifolds.

Prevalence and epidemiological characteristics of methicillin-resistant Staphylococcus aureus isolated from cattle in Bangalore India as a part of the One Health approach.

In India, limited studies are available on the epidemiological aspects of methicillin-resistant Staphylococcus aureus (MRSA) infections in both animal and human settings. Herein, we investigated the prevalence, antimicrobial resistance profile and molecular characteristics of MRSA isolates recovered from cattle using the One Health approach. Out of 66 mecA-positive staphylococci, species-specific multiplex PCR detected 24 % (n=16) of isolates as MRSA. Maximum antibiotic resistance was seen against cloxacillin (94 %, n=15) and least for enrofloxacin and cephalothin (each 13 %, n=2). Overall, 13 % (n=2) of MRSA isolates were multidrug-resistant. Molecular characterization by SCCmec typing identified 88 % (n=14) of MRSA isolates as type V. Twelve isolates (75 %) belonged to novel spa-type t17242, of which 67 % (n=8) belonged to agr type I. MLST analysis revealed ST 1687 (50 %, n=8) as the most predominant sequence type. Circulation of different MRSA clones among the cattle populace offers a risk of transmission to humans through direct contact, food chain or environmental contamination. Thus, continuous monitoring of MRSA strains is imperative for early diagnosis and for establishing effective treatment strategies to restrain the disease burden caused by MRSA infections.