Background: Multidrug-resistant pathogens are a significant cause of morbidity globally. The recent rise in resistant bacterial strains is largely attributed to the extensive use of antibiotics in human and animal treatment. In recent years, there has been a notable increase in drug-resistant Klebsiella pneumoniae strains, posing a major challenge for clinicians worldwide. 
 
Methods: In the current study, 121 samples were obtained from bovine mastitis milk and faeces, dairy cattle farm premises, chicken cloacal swabs, poultry farm litter and water. The samples were processed for the isolation and identification of Klebsiella species. Klebsiella isolates were identified by cultural, morphological and biochemical characteristics and were subsequently confirmed by PCR assays that targeted species-specific genes. The isolates were subjected to in vitro antibiotic sensitivity test. The DNA from all the isolates was extracted and subjected to PCR to identify the presence of 10 different Antibiotic Resistance Genes (ARG) using published primers. The ARGs targeted were blaTEM, blaSHV, blaOXA, blaIMP, DHAM, MOXM, tetA, tetB, sul1 and aadA. 
 
Result: Out of 121 samples, twenty-six (21.4%) samples were identified as Klebsiella pnuemoniae isolates. The occurence of Klebsiella pnuemoniae in samples from different sources was- 3/33 (09%) from mastitis milk, 8/27 (29.6%) from faecal samples and 04/14 (28.5%) from environmental samples collected from dairy farms. Similarly, Klebsiella pnuemoniae prevalence was recorded to be 6/33 (18.1%) in cloacal swabs and 5/14 (35.7%) from environmental samples collected from poultry farms. On in vitro antibiotic sensitivity test, all the Klebsiella pneumoniae isolates were found to be resistant to penicillin, amoxicillin, cefuroxime and ampicillin/ sulbactam (100% each), followed by cefalexin (65.3%), streptomycin (53.8%), tetracycline (38.4%), imipenum and co-trimoxazole (34.6% each), ofloxacin (30.7%), norfloxacin (27%), ceftriaxone (23%), amikacin (15.38%) and gentamicin (11.5%). All the K. pneumoniae isolates were found to be Multi Drug Resistant (MDR). Out of 26 isolates, 25 (96%) were positive for Sul1, 22 (84.6%) for blaTEM, 19 (73%) for blaSHV, 19 (3%) for tetA, 16 (61.5%) for aadA, 10 (38.4%) for OXA, 9 (34.6%) for tetB and 2 (7.6%) for DHAM and MOXM each. None of the isolates harboured blaIMP gene.

Multi Drug Resistant (MDR) Klebsiella pnuemoniae Isolated from Different Sources

<div>Background: The emergence of a multidrug resistance phenotype is most likely outcome of co-existing multiple mechanisms. The underlying molecular mechanism of carbapenem resistance is poorly understood in bacterial isolates of animal origin. The present study investigates the resistance mechanism for reduced susceptibility to carbapenem in E. coli. 
&nbsp;&nbsp; 
Methods: The study includes 12 multidrug resistant E.coli isolates showing decreased susceptibility to imipenem. Carbapenemase production was evaluated by the mCarba-NP test. The presence of the carbapenem resistance gene was confirmed by multiplex PCR. Efflux pump activity was accessed by broth microdilution method with or without selective efflux pump inhibitor. We analyzed differential expression of the outer membrane porin genes (ompF, ompC) and efflux pump genes (acrA, acrB) by quantitative real-time PCR analysis.&nbsp; 
 
Result: We observed increased expression of acrA and acrB genes in all the E. coli isolates under imipenem stress. A positive correlation (p&lt;0.001) was evident between imipenem MIC and acrA and acrB over-expression in these isolates. A significant decrease in ompF and ompC expression was observed in these isolates under imipenem stress. The change in MIC was not correlated with the corresponding OmpC and OmpF gene expression. The results suggest the importance of efflux pump and porins proteins in the emergence of multidrug resistance besides carbapenemase production.</div>

Imipenem Induced Transcriptional Modulation of Multidrug Efflux Pumps and Porin Genes in Carbapenem Resistant Escherichia coli Isolates of Animal Origin

Background: Nowadays, the emergence and dissemination of Extended-spectrum &beta;-lactamase (ESBL) and Carbapenemase producing Enterobacteriaceae (CPE) constitutes a major public health concern. The extensive and inappropriate use of antibiotics may lead to development of multidrug resistant (MDR) organisms. The antibiotic resistant genes are mostly situated conjugative plasmids, which are horizontally transmitted from one species to another in highly populous country like India.

Methods: Total 240 samples were collected from Ayodhya and Sultanpur districts of Awadh region of Uttar Pradesh (India). Confirmation of isolates was done by PCR analysis using species specific Uid A gene. ESBL and Carbapenemase producing isolates were confirmed using DDST, ESBL-E-strip and MBL E-strip test. Plasmid profile of these isolates was performed using GeneJet Plasmid Miniprep Kit and gel-electrophoresis.

Result: In this study total 186 (77.5%) isolates of E. coli were confirmed by PCR analysis. Total 130(54.16%) isolates were confirmed as ESBL and 10(4.16%) as Carbapenemase producer using E-ESBL strip and MBL-E strip test. All ESBL and Carbapenemase positive isolates were found 99-100% sensitive against chloramphenicol, polypeptides and aminoglycosides group of antibiotics and 80-100% resistance against 3rd, 4th group of cephalosporins, monobactam and penicillin group of antibiotics. Total 76.18% ESBL positive and 100% Carbapenemase positive isolates were found MDR. Plasmid profiling revealed total 63.84% ESBL positive isolates&nbsp;carried plasmid of molecular weight in range of 1.5 to &gt; 10kb with 1-3 number of bands and 80.0% Carbapenemase positive isolates carried plasmid with molecular weight 3 to &gt;10 kb with 1-3 number of bands.

Multidrug Resistance Pattern and Plasmid Profiling of ESBL and Carbapenemase Producing E. coli Isolates of Bovine Origin in Awadh Region of Uttar Pradesh

Background: Antimicrobial resistance being the major emerging problem for management of mastitis in cow hence, the research work was undertaken in a geographical area around Shirwal town in Satara district of Western Maharashtra, India to investigate the occurrence of multidrug resistance in Staphylococcus aureus (MDRSA) isolated from mastitic cow milk samples. Further assessment was carried out for antimicrobial efficacy of formulation of Green Silver Nanoparticles (GAgNPs) synthesized from Azadirechta indica against MDRSA of mastitis origin. 
 
Methods: A total of 250 milk samples from cows suspected of mastitis were screened by the California Mastitis Test (CMT). The CMT-positive samples were subjected to bacteriological investigation for the presence of S. aureus as an etiological agent. The isolates identified as S. aureus were further authenticated by the molecular method targeting 16s-rRNA and nuc gene. All isolates of S. aureus were subjected to an antimicrobial susceptibility test. The randomly selected&nbsp; MDRSA isolates were subjected to antimicrobial susceptibility testing with GAgNPs formulation were prepared from silver nitrate by reducing with Azadirachta indica leaf extract.&nbsp; 
 
Result: Out of 250 cow milk samples suspected for having mastitis were screened with CMT, 170 tested positive. From these milk samples 145 isolates of S. aureus were isolated and bacteriologically identified. When processed for antibiotic sensitivity 60 were detected having multidrug resistance. Maximum resistance was shown against beta-lactam antibiotics. GAgNP synthesized and characterized with visual appearance, spectroscopy and TEM (Transmission electron microscopy) and tested for in vitro antimicrobial efficacy against MDRSA showed significant antimicrobial property.&nbsp;

Efficacy of Green Silver Nanoparticles (GAbNPs) against Multidrug Resistance in Staphylococcus aureus from Mastitis-infected Cows

<div style="text-align: justify;">Background: Postweaning multisystemic wasting syndrome (PMWS) is caused by porcine circovirus type 2 (PCV2). It is a globally emerging clinical condition which affects nursing piglets and growing pigs. PCV2 requires various cofactors for fully development of PMWS. The present report describes the pathology of PMWS associated with classical swine fever virus (CSFV), porcine parvo virus (PPV) and multidrug resistant enteric Escherichia coli infections in an organized swine farm, Rajasthan. In India, this is the first report on pathology of PMWS associated with all the three viruses and multidrug resistant enteric colibacillosis.</div>

<div style="text-align: justify;">Methods: Detailed necropsy was conducted on pig carcass submitted to Post-mortem Facility, Division of Pathology, ICAR-Indian Veterinary Research Institute (IVRI), Izatnagar in the year 2017 from an organized swine farm, Rajasthan, India. Heart blood and diarrhoeic intestinal contents were collected aseptically and stored separately at 4&deg;C for bacteriological examination and antibiotic sensitivity test. Tissue samples were collected in sterile container and stored at -40&deg;C for DNA and RNA extraction and polymerase chain reaction (PCR) and also collected in 10% neutral buffered saline for histopathological processing.</div>

<div style="text-align: justify;">Result: The organized farm was having history of 33% (200/600) of mortality among 2 months to 6 months old piglets. Gross lesions were wasting, lymphadenopathy, non-collapsed lungs, hydropericardium, hepatomegaly, icterus, enteritis and pale kidneys, button shaped ulcers in the caecum and colon etc. Histopathologically, lymphoid depletion, reticuloendothelial cell proliferation in lymphoid tissue, fibrin thrombi in spleen, severe hepatitis, interstitial pneumonia, interstitial nephritis, severe necrosis in large intestine, meningitis, perivascular cuffing (PVC), gliosis, neuronal degeneration in cerebrum etc were observed. Molecular diagnosis and bacteriological examination confirmed presence of PCV2, PPV, CSFV and multidrug resistant Escherichia coli.</div>

Pathology of PMWS Associated with Classical Swine Fever Virus, Porcine Parvo Virus and Multidrug Resistant Enteric Escherichia coli Infections in an Organized Swine Farm, Rajasthan, India

Background: Rural poultry farmers are gradually changing from the backyard poultry production to intensive production, which made poultry more vulnerable to diseases and lead to indiscriminate use of antimicrobials in poultry production. As per OIE, FAO and WHO, multiple spread antimicrobial resistant pathogenic bacteria are a serious global human and animal health problem. Hence the study has been carried out to know the antimicrobial resistance status of poultry in Tirunelveli, Tamil Nadu, India. 
 
Methods: The study was aimed to find the occurrence of extended spectrum beta lactamases (ESBLs) producing E. coli from poultry origin. Caecum samples from the broiler (n=180), desi chicken (n=180) and Japanese quail (n=180) were processed for the isolation and phenotypic identification of ESBL&nbsp;E. coli. The isolates were tested for antimicrobial susceptibility test and characterization of ESBL and virulence genes done by PCR. 
 
Result: On phenotypic confirmation of ESBL producing E. coli by combined disk diffusion test (CDDT), a total of 9.63% samples were positive for extended spectrum beta lactamase producer (ESBL). Multi drug resistance (MDR) was observed in 38.23% of the broiler isolates, 27.27% and 57.14% in desi chicken and Japanese quail isolates, respectively. All the phenotypically confirmed ESBL producing E. coli isolates (n=52) were positive for uspA gene by PCR confirming the isolates as E. coli. The overall presence of the blaTEM gene was 57.69%, with broiler isolates having the highest prevalence of 67.47%, followed by desi chicken 36.36% and Japanese quail 42.85%. In contrast, the blaSHV gene was found in only 17.64% of the broiler isolates, but none were positive for the blaCTXM and blaOXA-1 gene.

Prevalence and molecular characterization of Multidrug-Resistant ESBL-Producing E. coli in commercial poultry

<div>Background: Mitigation process to curb the ever increasing problem of antimicrobial resistance through development of new class of antimicrobials is slow and costly affairs. Research on alternative to conventional antimicrobials using plant based products as good source of numerous phytochemicals have potential to cope up the antimicrobial resistance. The present study was formulated on detection of in vitro antimicrobial and antibiofilm properties of methanol leaf extract of Citrus maxima against clinical isolates of Staphylococcus aureus. 
 
Methods: Leaves of Citrus maxima plants were collected from the campus of College of Veterinary Sciences and Animal Husbandry, Central Agricultural University, Aizawl, Mizoram and processed for preparation of methanol crude extract. The plant extracts were evaluated for their phytochemical and antioxidant properties using DPPH (2, 2-Diphenyl-1-picrylhydrazyl) method. Twenty well characterized biofilm producing and multidrug resistant Staphylococcus aureus strains recovered from milk of mastitic cows from Mizoram were received from the cultural repository of the department. The plant extracts were subjected to determine their antimicrobial and antibiofilm activities against all the bacterial isolates including&nbsp;S. aureus (ATCC 29213) by in vitro agar well diffusion method and 96 well microtiter plate methods, respectively. The MIC value of the plant extracts were determined by microdilution method. 
 
Result: In the methanol leaf extract of C. maxima alkaloids, glycosides, terpenoids, tannin and phenol and flavonoids were detected by qualitative analysis. Saponin, protein, free amino acids, steroids and carbohydrates were not detected. The free radical scavenging potential of the extract was found to be 10.66&plusmn;1.84% to 36.10&plusmn;1.98%, which was comparatively lower than ascorbic acid (83.39&plusmn;0.13% to 89.76&plusmn;0.24%). A total of 8 (40.0%), 5 (25.0%) and 7 (35.0%) strains were recorded as weak, moderate and strong biofilm producer. Maximum antibacterial activity against standard culture was observed with the zone of inhibition of 18 mm at 200 mg/mL concentration and MIC value at 25 mg/mL. Maximum antimicrobial activities against clinical isolates were recorded with 11.8&plusmn;1.13 mm zone of inhibition at 200 mg/mL and MIC value at 25 mg/mL. The clinical isolates exhibited highest (85.94&plusmn;1.00%) biofilm inhibition at 6.25 mg/mL. To the best of our knowledge, this is the first-ever report on antibiofilm and antioxidative activities of C. maxima leaf extracts against any bacteria.</div>

Antimicrobial and Antibiofilm Activities of Methanol Leaf Extract of Citrus maxima against Clinical Isolates of Multidrug Resistant Staphylococcus aureus

<div>Background: In the present study two hundred milk samples were collected from cows and buffaloes with no history of clinical mastitis in the ongoing lactation, from the Sirohi district of Southern Rajasthan. 
 
Methods: The pooled sample (collected from each quarter) and examined for the status of subclinical mastitis by Modified California mastitis test and Somatic cell count respectively. Positive samples were further investigated for isolation and identification of the major mastitis-causing pathogens: S. aureus, predominant Streptococcal species and E. coli for assessing antimicrobial resistance models in southern Rajasthan. 
 
Result: The results of the current study indicate high levels of multi-drug antibiotic resistance among bacteria that commonly cause mastitis, particularly ampicillin, penicillin, tetracycline, erythromycin and methicillin. However, the highest sensitivity was conferred to ceftriaxone, gentamicin, and co-trimoxazole, suggestive of judicious use of these antibiotics in the treatment of bovine mastitis. Concurrent implementation of gradient PCR indicated the presence of mecA and blaZ genes in 51.9% and 81.4% of S. aureus isolates, respectively. Meanwhile, 56.6% of the streptococcal isolate contained the tetracycline-conferring tetM gene and none of the streptococci contained the ermB gene. The 92.3% E. coli isolates contained the tetA gene and the tetB gene for tetracycline resistance.</div>

Multidrug-resistant Bacterial Isolates in Bovine Subclinical Mastitis from Southern Rajasthan

<div>Background: Mastitis, the inflammation of parenchyma of mammary gland is frequently considered to be costliest and complex disease prevalent in India. Mastitis is caused by pathogens like Staphylococcus spp., Streptococcus spp., Mycoplasma bovis, E. coli, Klebsiella spp., Citrobacter spp., Enterobacter spp. and Entercoccus. The treatment of mastitis in animals is carried out using antibiotics. Treatment failure in mastitis is due to increased antibiotic resistance of mastitis pathogens and also due to indiscriminate use of antibiotics without testing in vitro antibiotic sensitivity test against causal organisms. In comparison to cultural method, PCR assays takes less time for detection of bacteria from the mastitis milk samples. Present research work was carried out regarding isolation, identification and multiple drug resistance profile of clinical bovine mastitis associated pathogens using conventional as well as molecular approach.&nbsp; 
 
Methods: In the present study, 73 mastitis milk samples were collected from Anand and Panchmahal district of Gujarat. The milk samples were subjected for cultural isolation and DNA extraction for identification of bacteria by cultural and PCR method. Antimicrobial sensitivity pattern of the isolates were carried by disc diffusion method and isolates were categorized in multiple drug resistant. &nbsp; &nbsp;&nbsp; 
 
Result: In the present study, Out of 73 mastitis milk samples collected from cows 48 (65.75%) cows were positive for bacterial isolation and S. aureus was the most predominant bacterial species. PCR from the mastitis milk additionally detected bacteria in culturally negative milk samples. Most sensitive drug was gentamicin and most of the isolates (90.19%) showed the multiple drug resistance for the two to nine drugs with 0.1 to 0.6 multiple antibiotic resistance index.</div>

<div>Isolation, Molecular Identification and Multidrug Resistance Profiling of Bacteria Causing Clinical Mastitis in Cows</div>

<div>Background: We investigated the occurrence of blaCTX-M carrying extended spectrum beta lactamase (ESBL) producing Escherichia coli in pigs from 8 North-eastern states of India with special emphasis on the transferability of ESBL gene from resistant E. coli strains to the susceptible Salmonella strains by in vitro and in vivo. 
 
Methods: Fecal samples (n=790) were collected from pigs reared under organized and unorganized farming set up of entire North-eastern region of India. All the samples were processed for isolation and identification of E. coli. All the isolates were subjected to antimicrobial sensitivity assay by disc diffusion method followed by determination of ESBLs producing ability by double disc synergy test (DDST). All the ESBLs producing isolates were screened for blaCTX-M gene by PCR using specific primers. The representative blaCTX-M gene positive isolates were used as donor to determine the ability to transfer of resistance gene in Salmonella by in vitro and in vivo assays with and without antibiotic selection pressure. &nbsp; 
 
Result: A total of 2,291 E. coli was isolated, of which 1113 and 1178 were from organized and unorganized farms, respectively. Majority of the isolates were multi-drug resistant with highest resistance against amoxicillin (84.81%) followed by cefalexin (77.17%), sulphafurazole (56.79%), piperacillin (46.40%), tetracycline (38.29%) and cefexime (35.66%). Isolates from unorganized farms showed higher resistance than the isolates recovered from organized farms. A total of 654 (28.55%) isolates were confirmed as ESBL producers by double disc synergy test (DDST) method, of which 65 (2.84%) isolates were positive for blaCTX-M gene. Genotypically, isolates with specific amino acids substitution revealed variation in their antibiotic susceptibility by phenotypic method. blaCTX-M gene could be successfully transferred horizontally from E. coli (donor) to Salmonella (recipient) by in vitro (3.6&plusmn;2.07x10-8 to 4.4&plusmn;2.88x10-8 transconjugate per donor) and in vivo method. By in vivo method in pig model, the frequency of transfer was higher under the antibiotic selection pressure (6.6&plusmn;3.05x10-5 to 7.2&plusmn;1.92x10-5 trans-conjugants per donor) than without antibiotic pressure (5.6&plusmn;2.3x10-4 to 6.8&plusmn;3.35x10-4 trans-conjugants per donor).</div>