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: Mammaliicoccus sciuri is a multidrug resistant human pathogen. Literature on its role causing bovine mastitis is scarce. Detection of M. sciuri from a cow with clinical mastitis in Malkangiri district of Odisha stimulated to ascertain its status in a larger population and to study its characteristics in genotypic and phenotypic levels.&nbsp; 
 
Methods:&nbsp;520 lactating cows from various herds of Malkangiri district of Odisha, India were screened for presence of clinical mastitis. Milk samples were collected aseptically from 15 cows with clinical mastitis. 16S rRNA amplification was performed to know presence of bacterial pathogen (s). Conventional PCR was carried out to detect resistance genes against &beta;-lactam (blaZ), aminoglycoside (aacA-aphD) and tetracycline (tetK, tetM). In vitro sensitivity test was studied against M. sciuri isolates using nine antibiotic discs. Phylogenetic analysis was performed to compare its origin.&nbsp; 
 
Result: M. sciuri was isolated in 6 (40.0%) mastitis positive milk samples. In vitro antibiotic sensitivity test against M. sciuri isolates showed highest degree of resistant to penicillin (100%) followed by, cefoperazone (33.3%), streptomycin (33.3%), tetracycline (16.0%). One isolate of M. sciuri had tetK, tetM resistance gene. Phylogenetic analysis showed (75-100%) similarity with other isolates obtained from GeneBank. Present study highlights tetK gene in M. sciuri.&nbsp;</div>

Genotypic and Phenotypic Characterization of Mammaliicoccus sciuri - A MDR Strain causing Clinical Mastitis in Cows of Odisha, India

<div>Background: Antimicrobial resistance (AMR) is a global concern for human and animal health. As the development and commercialization of a new class of antibiotics are difficult, time-consuming and extremely costly, exploration of alternatives like bacteriophages is considered one of the most suitable sources. There is a paucity of databases on the isolation of bacteriophages and exploration of their antibacterial properties against pathogenic MDR E. coli. The current study was undertaken to isolate and identify the bacteriophages from sewages to evaluate their lytic activity against MDR pathogenic Escherichia coli isolated from domestic animals in Mizoram. 
 
Methods: Total 50 samples were collected to process for isolation, identification and characterization of bacteriophages. Simultaneously, a total of 192 rectal swabs from pigs were collected, followed by isolation and identification of Escherichia coli. All the isolates were subjected to detection of selected virulence genes by PCR and determination of their antimicrobial sensitivity profile using commercial disks. All the bacteriophage isolates were assessed for their lytic effect against the MDR pathogenic E. coli isolates. 
 
Result: A total of six bacteriophages were isolated. Transmission electron microscopy revealed that bacteriophages belong to the Myoviridae family. Total 163 E. coli was isolated, out of which 59 isolates were pathogenic, which could be classified under STEC, EPEC, EHEC and ETEC pathotypes. By disk diffusion assay, majority of the E. coli showed resistance against commonly used antibacterial agents, viz., ampicillin, ciprofloxacin, cefpodoxime, ceftriaxone, nalidixic acid, tetracycline, doxycycline, gentamicin and streptomycin. Except one, all the bacteriophages exhibited lytic activity against pathogenic and MDR E. coli field isolates. Bacteriophages isolated and identified from sewages of Mizoram exhibited antimicrobial activities against field isolates of multidrug-resistant pathogenic E. coli isolated from pigs. Bacteriophages can be considered as a potential alternative to antibiotics to control the menace of AMR.</div>

Isolation, Identification and Characterization of Bacteriophages as a Potential Antibacterial Agent Against Multidrug-Resistant Pathogenic Escherichia coli Isolated from Swine in North Eastern Region of India

<div>Background: Higher prevalence of ESBL producers is alarming in dairy sector. So limiting antimicrobial use may curtail the selection and persistence of predominant ESBL and conjugative plasmids among strains. The study was aimed to determine the occurrence of extended-spectrum beta-lactamase (ESBL) producing E. coli as well as their genetic diversity, antimicrobial resistance and integrons in bovines.&nbsp; 
 
Methods:&nbsp;A total of 180 faecal samples were screened for the presumptive ESBL producing E. coli isolates. All ESBL producing isolates were subjected to the screening test and were confirmed as ESBL producers by double disc. Further, these ESBL producers were tested for the presence of bla genes and those found positive were tested for multidrug resistance (MDR) by disk diffsion. MDR positive isolates were further tested for the presence of intI 1, intI 2 and intI 3 genes. 
 
Result: A total of 360 presumptive ESBL producing E. coli isolates were obtained. Out of which, 154 (42.77%) isolates were found to be resistant and were confirmed as ESBL producers. Of 154 ESBL isolates, 120 (77.92%) isolates carried the gene/s screened for blaTEM, blaCTX-M and blaSHV genes are were declared as multi drug resistant. Out of 120 MDR isolates, 59 tested positive for the integron gene.</div>

Molecular Characterization and Antimicrobial Resistance Profiling of Extended Spectrum Beta-lactamase (ESBL) Producing Escherichia coli in Bovines from J and K, India

<div style="text-align: justify;">Colibacillosis is a disease of severe economic significance to all poultry producers worldwide, characterized by a diverse array of lesions. Due to enormous exploitation of antibiotics in broilers, an increased number of resistant bacterial strains have developed in recent years. 106 E. coli strains were collected from broilers suffering from colibacillosis. The resistance to different classes of antimicrobials was determined. The presence of integrons was determined by Polymerase Chain Reaction (PCR). All the statistical analyses were carried out using STATA&trade;. All the isolates were multidrug resistance (MDR), i.e. resistant to at least one agent in three or more antimicrobial categories. 37 (34.90%) isolates were found positive for Class 1 integrons. There was significant difference in carriage of integrons in different phylogentic groups, p=0.001, however, there was no difference among different serotypes for carriage of integrons. Significant difference was observed for resistance towards co-trimoxazole (p=0.002), piperacillin (p=0.034) and ciprofloxacin (p=0.046) in Class 1 integron carrying isolates and Class 1 integron negative isolates. Significant difference was observed for resistance towards ceftriaxone (p=0.004) and cefotaxime (p=0.027) in relation to phylogenetic groups. Also, significant difference was observed among different serotypes for resistance towards co-trimoxazole (p=0.001), gentamicin (p=0.005) and kanamycin (p=0.041).</div>

<div style="text-align: justify;">Risk factors for antimicrobial resistance in Escherichia coli isolates from poultry in Haryana</div>

Background: Escherichia coli is a Gram-negative commensal bacterium that normally resides in the digestive tract of humans and animals which is used as an indicator of the extended-spectrum beta-lactamase (ESBL) gene in an individual and can carry out gene transfer. Transmission of ESBL E. coli from wild animals has not been given much attention even though wild animals can act as vectors that spread ESBL in the environment so that it can pose a risk to public health. This research aims to identify the blaTEM gene which codes for ESBL in E. coli from bats that live in caves in Lombok, Indonesia.&nbsp; 
 
Methods: A total of 135 samples from bat rectal swabs were cultured on Eosin Methylene Blue Agar media and biochemical suspects were identified using the Indole, Methyl red, Voges-Proskauer, Citrate + and H2S (IMVIC) test. The E. coli bacteria obtained were tested for sensitivity using 7 classes of antibiotics, namely amoxicillin, ciprofloxacine, sulfamethoxazole/trimethoprim, tetracycline, gentamicin, cefotaxime and azithromycin. Bacteria that show multidrug resistance are subjected to PCR testing to detect the blaTEM gene. 
 
Result: Of the 135 test samples, it was found that 97 (71.85%) samples were positive for E. coli, 12 (12.37%) samples were Multidrug Resistance (MDR) and 2 (2.06%) samples had the blaTEM gene as the ESBL coding gene in bats that live in caves on the island of Lombok, Indonesia. The presence of the blaTEM gene in E. coli from bats can be indicated as a reservoir for MDR and ESBL transmission so that it can have an impact on public health.

Cave Bats as Carriers of Extended Spectrum Beta-lactamase Produced by Escherichia coli from the Island of Lombok, Indonesia

Background: Escherichia coli infection from inside the chicken&rsquo;s body may be present if the previously slaughtered chicken was contaminated with pathogens or the cage&rsquo;s hygiene is inadequate. One of the resistance mechanisms against antibiotics used by Gram-negative bacteria of the Enterobacteriaceae family is the production of extended-spectrum &beta;-lactamases (ESBL). Research is required to determine which E. coli genotypes in broiler chicken samples collected from Surabaya&rsquo;s traditional marketplaces produce ESBL.&nbsp; 
 
Methods: A total of 100 small intestine samples collected from broiler chickens were used in this study. After initial processing all the samples were separately injected into Eosin Methylene Blue Agar (EMBA) and Indole, Methyl Red, Voges Proskauer and Citrate (IMViC) tests were used to confirm the results. A test for antibiotic sensitivity based on the Kirby-Bauer method called the Double Disk Synergy Test (DDST) was used to analyze isolates that showed resistance to aztreonam and multidrug resistance (MDR). PCR testing was performed on ESBL-verified isolates to detect the TEM and CTX-M genes. 
 
Result: The results of the test for E. coli antibiotic resistance revealed that 14 isolates were proven to be multidrug-resistant (MDR) and six of them were found to be E. coli that produced ESBL. Four isolates were found to carry the TEM and CTX-M genes after genotyping analysis. Understanding the dangers of using antibiotics as feed additives and growth promoters is crucial for farmers to address the problem of ESBL-producing E. coli bacteria in chickens. Furthermore, the government needs to take action to monitor and enforce stronger controls on the use of antibiotics, which are even readily available on the market.

Identification of the Genotypes of Extended-spectrum &beta;-lactamase Producing Escherichia coli in the Small Intestine of Broiler Chickens at a Traditional Market in Surabaya, Indonesia

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: Escherichia coli cause mammary gland inflammation in dairy cows with striking local and sometimes severe systemic clinical symptoms. This disease affects many high producing cows in dairy herds and may cause several cases of death per year in the most severe cases. The present study was planned to evaluate the antibiogram and virulence factors of the isolated bacteria responsible for mastitis.&nbsp; 
 
Methods: In this study, 300 milk samples were collected and subjected to identify clinical mastitis (based on clinical symptoms) and sub clinical mastitis (using California mastitis test). E. coli was isolated and identified from mastitis positive samples and further tested for antibiogram and virulence factors such as haemolysis, biofilm formation and fimH and pap genes presence. 
 
Result: Out of 300 milk samples collected, 121 (40.33%) were positive for mastitis. E. coli was isolated from only 30 (24.79%) out of 121 mastitis positive samples. On Serotyping for somatic antigen, 13 E. coli isolates belonged to somatic serogroup O83, 2 to O157 and one each to O8, O20, O49, O119, O128 and O145. The highest sensitivity of E. coli isolates was recorded for ciprofloxacin, followed by gentamicin, tetracycline, nitrofurantoin, chloramphenicol, amikacin, cefixime, trimethoprim, ampicillin/sulbactam, ceftriaxone, cefoperazone, cefotaxime, kanamycin, cefotaxime/clavulanic acid and amoxycillin in the decreasing order. Multidrug resistance (MDR) was recorded in 96.66% of isolates.&nbsp;Out of 30 E. coli isolates, 46.66% were haemolytic and 40.0% positive for Congo red dye binding. Biofilm production was shown by 76.66% isolates. Molecular characterization revealed presence of fimH gene in 9 isolates, but pap gene was not detected in any of the strain. It may be concluded that E. coli is an emerging environmental mastitogen in cows and isolation of MDR strains with virulence factors is of serious concern.

Antibiogram, Haemolysis and Biofilm Properties of Escherichia coli from Bovine Mastitis

<div>Background: Antibiotic resistance is an emerging concern in the therapy of clinical infections worldwide. Previous studies conducted in our laboratory have confirmed an increase in the prevalence of extended spectrum beta-lactamase (ESBL) among the Gram-negative bacterial pathogens associated with dogs, which could act as a potential source for the transfer of these resistant pathogens or their genetic determinants to human. Since carbapenems are the last resort drugs against these resistant pathogens, the study was aimed to isolate and characterise carbapenem resistance among Escherichia coli (E. coli), Klebsiella pneumoniae (K. pneumoniae) and Pseudomonas aeruginosa (P. aeruginosa) associated with common clinical infections in dogs. 
 
Methods: A total of 100 samples were collected from lesional skin, urine and anterior vagina of dogs presented to the Veterinary Hospitals of Kerala Veterinary and Animal Sciences University at Mannuthy and Thrissur. The samples were cultured onto Brain Heart Infusion Agar (BHIA), Eosin Methylene Blue (EMB) and Mac Conkey (MAC) for isolation of bacteria. Identification of the isolates was performed based on cultural, morphological and biochemical characteristics. The isolates were subjected to antimicrobial susceptibility test (ABST) against the 12 commonly used beta-lactam and non&ndash;beta-lactam group of antibiotics by disc diffusion method and further subjected to screening for ESBL double disc diffusion method. Carbapenem-resistant isolates were subjected to phenotypic confirmatory test for carbapenemase production employing Imipenem-EDTA and Ertapenem-boronic acid minimum inhibitory concentration (MIC) strip method. 
 
Result: Forty four Gram-negative bacterial isolates obtained were viz., E. coli (30), K. pneumonia (11) and P. aeruginosa (3) from the 100 samples. Apart from these, other isolates obtained were Staphylococcus spp. (53) and Bacillus spp. (2). All the Gram-negative isolates were subjected to ABST employing 12 common antibiotics belonging to beta-lactam and non-beta-lactam groups. Multidrug resistance (MDR) could be observed in 28 E. coli, 11 K. pneumoniae and three P. aeruginosa isolates. All the 42 MDR isolates showed positive results for ESBL production. A total of 14 isolates out of the 44 Gram-negative bacilli were found to be resistant to carbapenem either to imipenem, meropenem or ertapenem. Among the 14 Gram-negative isolates, nine turned out to be positive for metallo-beta-lactamase (MBL) and none for K. penumoniae carbapenemase (KPC) on phenotypic confirmatory test for detecting major carbapenemase enzymes. The present study documented that Gram- negative bacteria like E. coli, K. pneumoniae and P. aeruginosa isolated from dogs are showing an increase rate of resistance against carbapenems which are the last resort drugs against ESBL producers. Hence, there is an urgent need to curb the irrational and excessive use of antibiotics in veterinary sector.</div>

<div>Phenotypic Characterisation of Klebseilla pneumoniae Carbapenemase and Metallo Beta-lactamase in Carbapenem Resistant Gram-negative Bacteria</div>