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Aim: To determine the prevalence of multidrug (MDR) and extensively drug-resistant (XDR) pathogens from pediatric blood samples Methods: In total, 4543 children's blood samples were processed in the BacT/ALERT system. Confirmation of the isolates and MIC was determined in VITEK® 2 system. Molecular identification of blaIMP, blaVIM and blaOXA-48 was done by PCR. Results: Of 4543 blood cultures, 458 (10%) were positive for bacterial growth and Salmonella Typhi (415; 90%) remained the primary pathogens. Antibiogram revealed 208 (50.1%) and 137 (33%) were MDR and XDR S. Typhi, respectively. Klebsiella pneumoniae displayed 46% resistance to imipenem. One hundred twelve (81.7%) XDR Typhi were positive for blaCTXM, whereas 14 (66.6%) blaVIM were found in carbapenem-resistant bacteria. Conclusion: A high prevalence of MDR and XDR pathogens was found in peads blood culture.

Sana Ilyas,1 Muhammad Hidayat Rasool,1 Muhammad Javed Arshed,2 Muhammad Usman Qamar,1 Bilal Aslam,1 Ahmad Almatroudi,3 Mohsin Khurshid1 1Department of Microbiology, Government College University, Faisalabad, Pakistan; 2National Veterinary Laboratory, National Agriculture Research Council, Islamabad, Pakistan; 3Department of Medical Laboratories, College of Applied Medical Sciences, Qassim University, Buraydah, Saudi ArabiaCorrespondence: Muhammad Hidayat RasoolDepartment of Microbiology, Government College University, Faisalabad 38000, PakistanTel +92 41-9203023Email drmhrasool@gcuf.edu.pkBackground and Aim: The extended-spectrum beta-lactamases (ESBLs), as well as carbapenemases, are considered as the foremost resistance determinants throughout the world. However, the relevant data especially related to the sequence types of ESBL and carbapenemases producing Escherichia coli from the poultry is limited from Pakistan. Here, we present the data on the genetic diversity of E. coli strains isolated from the poultry birds from the poultry farms located in Islamabad, Pakistan, and the underlying resistance mechanisms to beta-lactam agents.Methods: Of 250 broilers from 25 different farms (10 birds from each farm), the cecal samples were obtained and analyzed for the presence of ESBLs producing E. coli (ESBL-Ec) as well as carbapenemases producing E. coli (CPEc) strains using selective agar for ESBL and carbapenemases screening. The susceptibility profiling of the ESBL-Ec and CPEc isolates was evaluated followed by multi-locus sequence typing.Results: A total of 119 strains were positive for ESBL production whereas 37 strains were found positive to produce carbapenemases in addition to ESBLs. The MLST analysis has shown a diversity of isolates as the E. coli isolates from poultry birds correspond to a total of 16 sequence types (STs). The ST131 (22/48, 46%) followed by ST8051 (10/48, 21%) were the main STs in this study. The blaCTX-M gene was detected in all the poultry E. coli strains whereas the blaTEM was found in 45.5% of strains. The blaVIM was found in all 37 CPEc isolates whereas the blaNDM and blaIMP were found in 31/37 (83.8%) and 16/37 (43.2%) CPEc isolates respectively.Conclusion: The overall results have shown the prevalence of diverse genotypes among the ESBL-Ec and carbapenemase-producing E. coli (CPEC) from poultry. Furthermore, the study documents poultry birds as a persisting reservoir of extensively antimicrobial-resistant E. coli ST131 in Pakistan, suggesting a potential threat to public health.Keywords: MLST, E. coli, poultry, Pakistan, AMR, ESBL

Probiotic potential of Enterococcus spp. is widely investigated around the globe. The biochemically and molecular characterized E. faecium strains isolated from Dahi (continental yogurt) were evaluated to tolerate simulated gastric environment, bile, sodium chloride, temperature, and pH. The safety was assessed by disc diffusion, broth microdilution, antibiotic resistance genes screening, and hemolytic ability. Enterococci survived simulated gastrointestinal conditions and depicted growth at temperature (15 to ≥42°C), pH (≤2.5 to ≥9.5), 0.3% bile salt and 3% NaCl. All strains were sensitive to ampicillin, vancomycin, kanamycin, gentamicin, streptomycin, tetracycline and ciprofloxacin and harbored vanR, vanX, qnrB2, qnrS, tetK, and tetW resistance genes. E. faecium strains inhibited the E. coli (85%) and S. Typhi (50%) whereas the 10% cell-free culture supernatant (CFCS) of E. faecium halted the growth of E. coli while 15% CFCS completely suppressed S. Typhi. The cell-free culture supernatant retained antibacterial nature after pH and proteinase K treatment, however, it lost activity after heat treatment (≥95°C). The genetic screening revealed that all isolates are capable to produce putrescine biogenic amine. Further assessment of strains for lack of infectivity, cytotoxicity in animals, adhesion to Caco-2 cells and characterization of enterocins is essential to conclude the probiotic potential of these strains.

Newcastle disease (ND) is a highly fatal, infectious, viral disease, and despite immunization with live and inactivated vaccines, the disease is still endemic, causing heavy morbidity and mortality leading to huge economic losses to the poultry industry in Pakistan. Therefore, the present study was aimed for the first time in the country at using novel virosomal technology to develop the ND vaccine using an indigenous highly virulent strain of the virus. ND virosome was prepared using Triton X-100, and SM2 Bio-Beads were used to remove the detergent and reconstitute the viral membrane into virosome. Confirmation was done by transmission electron microscopy and protein analysis by SDS-PAGE. In vitro cell adhesion property was observed by incorporating green fluorescent protein (GFP), producing plasmid into virosome and in vitro cell culture assay. Sterility, safety, and stability of the vaccine were tested before in vivo evaluation of immunogenicity and challenge protection study in commercial broiler. The virosome vaccine was administered (30 μg/bird) at days 7 and 14 through the intranasal route in comparison with commercially available live and inactivated ND vaccines. Results revealed significantly high ( p < 0.05 ) and clinically protective hemagglutination inhibition (HI) antibody titers at 7, 14, 21, and 28 days postimmunization with the virosome vaccine in comparison to the negative control. The GMTs were comparable to live and inactivated vaccines with nonsignificant ( p > 0.05 ) differences throughout the experiment. Antibody levels increased in all vaccinated groups gradually from the 7th day and were maximum at 28th-day postvaccination. In the virosome-administered group, GMT was 83.18 and 77.62 at 21st and 28th-days postvaccination, respectively. Challenge revealed 100%, 90%, and 80% protection in virosome, live, and inactivated vaccinated groups, respectively. Under given experimental conditions, we can conclude that ND virosome vaccine prepared from the indigenous virus was found to be safe and immunogenic.

Introduction Intense livestock farming practices enforcing the farmers to use antibiotics as food supplements on a routine basis. Aberrant use of antibiotics is associated with the emergence of antibiotics resistance and resistant superbugs. Keeping in view the current scenario, the present study was designed for the first time from Pakistan with a specific aim to estimate the prevalence of the carbapenem-resistant Klebsiella pneumoniae in veterinary settings and the waste in Pakistan. Methods A total of 138 samples from various veterinary sources were collected by employing a nonprobability sampling technique. Isolation and phenotypic identification of carbapenem-resistant K. pneumoniae were performed according to the CLSI standard. Molecular detection of various antibiotic resistance genes (ARGs) was done through PCR by using specific primers against each ARG. According to the pasture scheme, the multilocus sequence typing (MLST) was performed to characterize the K. pneumoniae sequence types (STs). Results According to the results of the study, overall 9.4% (13/138) isolates were confirmed carbapenem-resistant K. pneumoniae. Among various carbapenem ARGs particularly, the bla NDM-1 was found in 92.3% (12/13) isolates followed by bla OXA-48 84.6% (11/13). MLST results revealed that overall 3 STs were found in the study which includes ST29, ST11, and ST258. Taking together, this is the first study to our best knowledge which demonstrated the prevalence of carbapenem-resistant K. pneumoniae and its various STs prevalent in veterinary settings and the waste of Pakistan. Conclusion Based on the above-mentioned facts, we suggested that veterinary settings and waste are the potential source and reservoir of carbapenem-resistant K. pneumoniae, which may be disseminated to the environment and ultimately can affect the public and companion livestock health.

Introduction The aminoglycosides are widely used for the therapeutic management of infections caused by gram-negative bacteria, including the Acinetobacter baumannii strains. However, the resistance to the members of the aminoglycoside family, such as amikacin, gentamicin, and tobramycin, is increasingly being common among the clinical isolates. Purpose This study aimed to investigate the presence of 16SrRNA methylases and aminoglycoside modifying enzymes (AMEs) genes among aminoglycoside resistant A. baumannii isolates and to study the genetic diversity of the clinical population of A. baumannii in local hospitals. Material and methods The 143 A. baumannii clinical strains were analyzed for antimicrobial susceptibility, genetic screening for enzymes conferring aminoglycosides resistance followed by the multilocus sequence typing. Results The 133/143 (93%) isolates were non-susceptible to at least one of the tested aminoglycosides, including amikacin, gentamicin, and tobramycin. The MIC distribution has shown that 87.486.7% strains were resistant to amikacin and gentamicin, respectively. The aphA6, aadB, aacC1, and aphA1 were found in 74.1%, 59.4%, 16.1%, and 11.2% isolates, respectively, whereas the armA was found in 28% of the strains having a higher MIC value (MIC; ≥256µg/mL). The MLST data have shown that the ST589 and ST2 were the most common STs and corresponded to 51 (35.7%) and 38 (26.6%) isolates, respectively, and few of the isolates corresponding to these STs were found to harbor the armA gene with a variable genotypic profile for AMEs. Discussion The study has reported the incidence of various enzymes conferring aminoglycoside resistance among the A. baumannii clones for the first time from Pakistan. The findings suggest the possibility of transmission of aminoglycoside resistance determinants through the lateral gene transfer as well as clonal dissemination.

Klebsiella pneumoniae is an important pathogen that causes pneumonia and bloodstream infections, especially in neonates and intensive care patients. The carbapenems remain an important therapeutic option for clinicians, particularly against cephalosporin-resistant Gram-negative pathogens. This increased use of carbapenems at clinics has resulted in the evolution and spread of carbapenem-resistant K. pneumoniae. In this study, we isolated six bla K. pneumoniae carbapenemase (KPC)-producing strains belonging to sequence type 258 (ST258) from clinical, environmental, and veterinary sources. Antibiotic susceptibility was performed on these isolates and the genes responsible for extended-spectrum beta-lactamases and metallo-beta-lactamase production were screened. The molecular typing was done using multilocus sequence typing. Isolated strains were resistant to various antibiotic classes, including carbapenems, and carried the carbapenem-resistant gene, blaKPC. All strains were susceptible to tigecycline and colistin. This is the first report detecting K. pneumoniae ST258 strains in Pakistan.

Objective The rise of carbapenem resistance among Acinetobacter baumannii represents a challenge for the therapeutic management of infections. The present study aimed to investigate the sequence types and carbapenem resistance in A. baumannii strains collected from various clinical specimens from the patients admitted to tertiary care hospitals in Pakistan. Methods A total of 156 A. baumannii clinical strains were analyzed for antimicrobial susceptibility, followed by genetic screening for the carbapenem-resistant determinants. All the A. baumannii strains were typed using multilocus sequence typing by the Pasteur scheme. Results One thirty-nine of the 156 isolates (89.1%) were carbapenem-resistant and out of these 136 carried the blaOXA-23-like genes. Interestingly the sequence type (ST) 589 was the most common sequence type that was classified as clonal complex 1 (CC1). The ST2 was the second most common sequence type that corresponds to the clonal complex 2/92 (Pasteur scheme/oxford scheme), however, it was distributed in all the hospitals. Conclusion The diverse clones of carbapenem-resistant A. baumannii including the already reported STs as well as new STs carrying OXA-23 are mainly distributed in Pakistan. This is the first study that described the molecular epidemiology of widely disseminated A. baumannii in Pakistan. The findings will help to improve the knowledge of predominant sequence types and will be valuable for the deeper understanding of resistance mechanisms among various MLST types.

Introduction The mounting incidence of multidrug-resistant bacterial strains and the dearth of novel antibiotics demand alternate therapies to manage the infections caused by resistant superbugs. Bacteriophages and phage=derived proteins are considered as potential alternates to treat such infections, and have several applications in health care systems. The aim of this review is to explore the hidden potential of bacteriophage proteins which may be a practical alternative approach to manage the threat of antibiotic resistance. Results Clinical trials are in progress for the use of phage therapy as a tool for routine medical use; however, the existing regulations may hamper their development of routine antimicrobial agents. The advancement of molecular techniques and the advent of sequencing have opened new potentials for the design of engineered bacteriophages as well as recombinant bacteriophage proteins. The phage enzymes and proteins encoded by the lysis cassette genes, especially endolysins, holins, and spanins, have shown plausible potentials as therapeutic candidates. Conclusion This review offers an integrated viewpoint that aims to decipher the insights and abilities of bacteriophages and their derived proteins as potential alternatives to antibiotics.

Background: K. pneumoniae is ubiquitous and known to be a notorious pathogen of human, animal, and plant-based food. K. pneumoniae is a recognized trafficker of antibiotic resistance genes (ARGs) between and from different ecological niches. Current study was designed to decipher the transmission dynamics and genetic relatedness of MDR K. pneumoniae at the human-animal-environment interface in Pakistan. Methods: A total (n=775) samples were collected from September 2017 to August 2019 from the human, animal, and environmental sources by applying the convenient random sampling technique. HiChrome Klebsiella Selective agar (Himedia®) was used for the isolation of K. pneumoniae, VITEK™ identification system (bioMerieux, France) was used isolates identification. CLSI guidelines 2018 were followed for antibiotic susceptibility testing. The range of antibiotic resistance genes (ARGs) was detected through PCR by using specific primers. Multi Locus Sequence Typing (MLST) was performed according to the Pasteur scheme and sequence types (STs) were allotted to the isolates. Findings: A total of 120 (15.7 %) samples were confirmed K. pneumoniae. The distribution of K. pneumoniae among humans, animals, and the environment were 17.4 %, 15.17%, and 7.9 % respectively. Isolates have shown significant resistance against all the subjected antibiotics except colistin. In acquired resistance genes, the highest percentage prevalence (67.5 %) of blaCTX-M was estimated in the isolates, various carbapenem resistance genes that were found in the study were blaNDM-1 (43.3%), blaOXA-48 (38 %), and (1.67 %) blaKPC-2. Conclusion: Overall, 21 distinct sequence types (STs) and 13 clonal complexes (CCs) were found through the MLST analysis. Taking together, the dissemination of genetically related K. pneumoniae clones among the community is alarming for the health care system of the country. Health policymakers should consider the role of all the integral parts of human, animal, and associated environment intently to cope with this serious public and animal health concern. Funding Statement: This study was supported by grants from the major science and technology special project of Yunnan Province, No. 2019ZF004. Declaration of Interests: All author declare No conflict of interests. Ethics Approval Statement: The study was conducted in a span of two years duration from September 2017 to August 2019; proper permission was granted by the Ethical Review Board (ERB) of Government College University, Faisalabad, Pakistan.

Background. The term “persisters” refers to a small bacterial population that persists during treatment with high antibiotic concentration or dose in the absence of genetic resistance. The present study was designed to investigate the transcriptional response in indigenous Klebsiella pneumoniae under the ciprofloxacin stress. Methods. Isolation and identification of K. pneumoniae were carried out through standard microbiological protocols. The characterization of quinolone resistance was performed by estimating the quinolone susceptibility testing, MIC estimation, and detecting the QRDR and PMQR. Transcriptional response of the isolates to ciprofloxacin was determined using qPCR. Results. Among 34 isolates, 23 (67%) were resistant to ciprofloxacin. Both QRDR (gyrA and gyrB) and PMQR (qnrA, qnrB, and qnrS) were detected in the isolates, and all were found resistant to ciprofloxacin. The mRNA levels of both mutS and euTu under the influence of ciprofloxacin were significantly increased. On ciprofloxacin exposure, the mRNA levels of the DNA damage response element (mutS) were raised in a time-dependent fashion. K. pneumoniae showed high-level resistance to ciprofloxacin in the presence of mutations in QRDR and PMQR genes. Conclusion. The transcriptional response revealed the upregulation of DNA repair and protein folding elements (mutS and euTu) in ciprofloxacin stress and delayed cell division. The ciprofloxacin was found to trigger various stress responses in a time- and concentration-dependent manner.

The bacterial species employ various types of molecular communication systems recognized as quorum sensing for the synchronization of differential gene expression to regulate virulence traits and biofilm formation. A variety of quorum sensing inhibitors; molecules that interfere with quorum sensing among bacteria have been examined which can block the action of autoinducers. Moreover, the studies have scrutinized various enzymes for their quorum quenching activity resulting in the degradation of signaling molecules or blocking of gene expression. So far, the studies have found that these approaches are not only capable to reduce the pathogenicity and biofilm formation but also resulted in increased bacterial susceptibility to antibiotics and bacteriophages. The effectiveness of these strategies has been validated in different animal models and it seems that these practices will be transformed in near future to develop the medical devices including catheters, implants, and dressings for the prevention of bacterial infections. Although many of these approaches are still in the research stage, the increasing library of quorum quenching molecules and enzymes will open innovative perspectives for the development of antibacterial approaches which will extend the therapeutic arsenal against the pathogenic bacterial species.

The role of the human microbiome in the brain and behavioral development is an area of increasing attention. Recent investigations have found that diverse mechanisms and signals including the immune, endocrine and neural associations are responsible for the communication between gut microbiota and the brain. The studies have suggested that alteration of intestinal microbiota using probiotic formulations may offer a significant role in the maturation and organization of the brain and can shape the brain and behavior as well as mood and cognition in human subjects. The understanding of the possible impact of gut microflora on neurological function is a promising phenomenon that can surely transform the neurosciences and may decipher the novel etiologies for neurodegenerative and psychiatric disorders.

Human gut microbiota consist of numerous microorganisms, but the most abundant species are Bacteroides and Firmicutes. Each human possesses a specific gut microbiota, which can be altered by diet, antibiotics, lifestyle, and genetic background. Gut microbiota perform vital functions, but in this article, we aimed to elaborate the effects of modified composition of microbiota on host metabolism. Ligands for G protein coupled receptors (GPCRs) are short-chain fatty acids (SCFAs) located on endocrine glands, epithelial cells, and adipocytes. SCFAs are produced in the distal gut by bacterial fermentation of nondigestible polysaccharides; they induce the various beneficial effects including decrease serum glucose level, insulin resistance, as well as inflammation; and they increase glucagon-like peptide-1 (GLP-1) secretion. Fasting-induced adipose factor (FIAF) is suppressed by gut microbiota and results in the increased storage of fatty acids in the adipose tissues and liver. An increased lipopolysaccharide level due to altered gut microflora cause the initiation of inflammation associated with type 2 diabetes mellitus (T2DM). Intestinal dysbiosis and metabolic endotoxemia are considered key mechanisms that seem to be associated with the development of T2DM and obesity. Therapeutic interventions that can be used for the treatment of diabetes include metformin, dietary modulation, probiotics, prebiotics, fecal microbiota transplantation and bariatric surgery.

The aim of the current investigation was to determine the antibacterial and antibiofilm potential of MgO nanoparticles (NPs) against antibiotic-resistant clinical strains of bacteria. MgO NPs were synthesized by a wet chemical method and further characterized by scanning electron microscopy and energy dispersive X-ray. Antibacterial activity was determined by broth microdilution and agar diffusion methods. The Bradford method was used to assess cellular protein leakage as a result of loss of membrane integrity. Microtiter plate assay following crystal violet staining was employed to determine the effect of MgO NPs on biofilm formation and removal of established biofilms. MIC values ranged between 125 and 500 μg/mL. Moreover, treatment with MgO NPs accelerated rate of membrane disruption, measured as a function of leakage of cellular proteins. Leakage of cellular protein content was greater among gram-negative bacteria. Cell adherence assay indicated 25.3-49.8% inhibition of bacterial attachment to plastic surfaces. According to a static biofilm method, MgO NPs reduced biofilm formation potential from 31% to 82.9% in a time-dependent manner. Moreover, NPs also significantly reduced the biomass of 48, 72, 96 and 120 hr old biofilms (P < 0.05). Cytotoxicity experiments using a neutral red assay revealed that MgO NPs are non-toxic to HeLa cells at concentrations of 15-120 μg/mL. These data provide in vitro scientific evidence that MgO NPs are effective and safe antibiofilm agents that inhibit adhesion, biofilm formation and removal of established biofilms of multidrug-resistant bacteria.

Aim: The ISAba1 insertion sequence supports the overexpression of various oxacillinases genes by providing promoter and therefore, confers carbapenem resistance. The study aimed to determine the clonal diversity, genetic attributes of carbapenem resistance and the role of ISAba1 in the expression of oxacillinases genes among Acinetobacter baumannii clinical isolates. Materials & methods: PCR was performed to amplify various antimicrobial resistance determinants and ISAba1 insertion elements in 137 A. baumannii clinical isolates. Results: The bla OXA51 and bla OXA23-like genes and ISAba1 upstream to these genes were found in almost all multidrug-resistant isolates belonging to multiple clones. Conclusion: This is the first comprehensive study from Pakistan that highlighted the emergence of ISAba1 harboring carbapenems resistant A. baumannii stains in tertiary care hospitals.

Background: Giardia intestinalis is the most common protozoan infecting the small intestine of human beings and a major cause of enteric infection, especially in children throughout the world. It is a highly diverse protozoan, which comprises a complex of eight genetic assemblages that are further differentiated into sub-assemblages. Objectives: A cross-sectional study was conducted to detect the frequency, molecular detection and assemblage identification of G. intestinalis in children of Punjab, Pakistan. Methods: A total of 800 stool samples were collected from children ranging 0 - 10 years of age with gastrointestinal disturbances and subjected to direct microscopy, enzyme-linked immunosorbent assay and polymerase chain reaction targeting small-subunit ribosomal RNA (SSU rRNA) and triosephosphate isomerase (tpi) genes. A predesigned questionnaire was filled prior to sampling from the guardian of each child to collect information. Results: The results indicated that the prevalence of 9.5% (76/800) was achieved by microscopy, ELISA and PCR targeting SSU-rRNA gene. The genetic DNA from 69 out of 76 (90.80%) was successfully amplified by tpi gene. Among these tpi gene-positive samples, 38 were successfully typed in assemblage B (55.07%) followed by 20 (28.98%) in assemblage A and 11 (15.94%) in mixed type assemblages (A & B). Residency and socioeconomic status were statistically associated with giardiasis. Among the clinical presentations, abdominal pain is prominent in assemblage B (57.89%) and vomiting in assemblage A (40%) type infections. Conclusions: Advanced molecular tools for giardiasis are well-adapted to get true prevalence, better discrimination of assemblages and their correlation with clinical signs.

The carbapenem-resistant Acinetobacter baumannii (CRAB) has got global attention as a notorious nosocomial pathogen. This study describes a case of urinary tract infection in a 2-years old pet female cat infected with A. baumannii. The susceptibility profiling, screening for the resistance determinants, and the multilocus sequence typing was performed. The A. baumannii isolate was found to harbor the blaOXA23-like gene and corresponded to International clone II that has been widely reported to be involved in human infections. The study proposes that the pets may contribute towards the spread of clinically relevant antimicrobial-resistant pathogens. The carbapenem-resistant Acinetobacter baumannii (CRAB) has got global attention as a notorious nosocomial pathogen. This study describes a case of urinary tract infection in a 2-years old pet female cat infected with A. baumannii. The susceptibility profiling, screening for the resistance determinants, and the multilocus sequence typing was performed. The A. baumannii isolate was found to harbor the blaOXA23-like gene and corresponded to International clone II that has been widely reported to be involved in human infections. The study proposes that the pets may contribute towards the spread of clinically relevant antimicrobial-resistant pathogens.

Background Urbanization and industrialization are the main anthropogenic activities that are adding toxic heavy metals to the environment. Among these, chromium (in hexavalent: Cr+6 and/or trivalent Cr+3) is being released abundantly in wastewater due to its uses in different industrial processes. It becomes highly mutagenic and carcinogenic once it enters the cell through sulfate uptake pathways after interacting with cellular proteins and nucleic acids. However, Cr+6 can be bio-converted into more stable, less toxic and insoluble trivalent chromium using microbes. Hence in this study, we have made efforts to utilize chromium tolerant bacteria for bio-reduction of Cr+6 to Cr+3. Methods Bacterial isolate, K1, from metal contaminated industrial effluent from Kala Shah Kaku-Lahore Pakistan, which tolerated up to 22 mM of Cr6+ was evaluated for chromate reduction. It was further characterized biochemically and molecularly by VITEK®2 system and 16S rRNA gene sequencing respectively. Other factors affecting the reduction of chromium such as initial chromate ion concentration, pH, temperature, contact-time were also investigated. The role of cellular surface in sorption of Cr6+ ion was analyzed by FTIR spectroscopy. Results Both biochemical and phylogenetic analyses confirmed that strain K1 was Staphylococcusaureus that could reduce 99% of Cr6+ in 24 hours at 35 °C (pH = 8.0; initial Cr6+ concentration = 100 mg/L). FTIR results assumed that carboxyl, amino and phosphate groups of cell wall were involved in complexation with chromium. Our results suggested that Staphylococcusaureus K1 could be a promising gram-positive bacterium that might be utilized to remove chromium from metal polluted environments.

Introduction: The multidrug-resistant (MDR) Salmonella enterica serovar Typhi isolates have been increasingly reported from the Asian and African countries. The emergence of isolates with decreased susceptibility to fluoroquinolones and cephalosporins has worsened the situation. Recently, an outbreak from Sindh, Pakistan was reported caused by extensively drug-resistant (XDR) S. Typhi strains. Methodology: In the present study, a total of 82 cases of typhoid have been investigated during 2018 from the febrile children referred to a tertiary care hospital in the population-wise largest province (Punjab) of Pakistan. S. Typhi was identified by standard microbiological techniques and isolates were characterized for antimicrobial resistance profiling and minimum inhibitory concentrations were determined. The presence of various ESBL genes in S. Typhi was confirmed by the PCR. Results: Out of the 82 isolates tested, 35 (43%) were found to be XDR; resistant to the first-line drugs. The resistance to third-generation cephalosporins was mainly mediated by extended-spectrum beta-lactamases i.e. blaTEM and blaCTX-M genes. Conclusions: The higher prevalence of ESBL producing Salmonella typhi clinical strains raises the concern about transmission prevention and infection management in the community as well as clinical settings. Moreover, the study highlights the problem concerning the declining antibiotic arsenal for the therapeutic management of typhoid fever and the emergence and spread of XDR strains in Pakistan.

Background: Zinc is an essential micronutrient required for optimum plant growth. Zinc-solubilizing bacteria convert applied inorganic zinc to available forms that could be used by plants. Research design: In present study, experiments were conducted to isolate, characterize, and evaluate Zn solubilization potential of different bacteria. Results: Among 10 isolated strains, Pseudomonas protegens (RY2, MF351762) was found to be the most promising strain having zinc-solubilizing potential on 4 different insoluble zinc sources. In quantitative assay, Zn solubilization by RY2 was significantly higher than other strains at different incubation time. P. protegens RY2 was selected (based on zinc solubilizing and plant growth promoting activities like P solubilization and ACC deaminase) for plant experiments. Meanwhile, available Zn release rate in soil was determined at day 10 of incubation. Chickpea seeds were inoculated with RY2 strain and ZnO is used as zinc source. Growth parameters and quantifying zinc content of shoot and root using atomic absorption spectrophotometer were determined. Enhanced shoot and root dry weights and lengths were observed in chickpea plants compared to control. Maximum increase of 44%, 67%, and 75% in T2 (Soil + RY2), T5 (Soil + ZnO + RY2), and T7 (Soil + manure + ZnO + RY2), respectively, was found in shoot length compared to control (T1). Conclusion: The study indicated that zinc-solubilizing RY2 strain possesses potential for enhanced Zn in soil so it would allow reduced inorganic Zn application.

Purpose – The purpose of this paper is to evaluate the frequency and antimicrobial susceptibility pattern of pathogens present in ready-to-eat salads available at a local market. Design/methodology/approach – A 100 salad samples were collected aseptically. Each sample (25 g) was homogenized in 225 ml of sterile peptone water and was serially diluted up to 1×106. Dilutions were inoculated on nutrient agar by surface spread plate technique. Aerobic colony count (ACC) was estimated by counting the colonies. Bacterial isolates were cultured on blood and MacConkey agar and identified on the basis of their morphology, culture characteristics and confirmed by API 20E and 20NE. Antimicrobial susceptibility was determined as per CLSI 2014. Findings – ACC range was 1.1×103 cfu/g to 5.8×109 cfu/g. Among these the highest ACC was found in channa chat (4.9×104 to 5.8×109 cfu/g). A total of 127 microorganisms were identified; 73 were gram negative rods (GNRs) and 24 were gram positive cocci (GPC). Among GNRs; Klebsiella spp. (n=18) was the predominant whereas among GPC, Staphylococcus aureus (n=6) were the chief pathogen. Klebsiella spp. showed 100 percent resistance to ampicillin, 89-78 percent to amoxicillin/clavulanic acid and 33 percent to imipenem, however, Enterobacter spp. were resistant to ampicillin (100 percent) amoxicillin/clavulanic acid (77 percent) and imipenem (23 percent). Staphylococcus aureus showed resistance to co-amoxiclav (83 percent) and penicillin (75 percent). Practical implications – This study revealed that effective control measures must been implemented and respected by throughout the entire food chain and better surveillance studies should be performed at national level to reduce the spread of bacteria by fresh salads. Originality/value – This paper explore the high prevalence of multidrug-resistant pathogens in different salads and most of the salads were found to be unhygienic for consumption.

BackgroundExtensively drug-resistant (XDR) Salmonella Typhi has been reported in Sindh province of Pakistan since 2016. The potential for further spread is of serious concern as remaining treatment options are severely limited. We report the phenotypic and genotypic characterisation of 27 XDR S. Typhi isolated from patients attending Jinnah Hospital, Lahore, Pakistan.MethodsIsolates were identified by biochemical profiling; antimicrobial susceptibility was determined by a modified Kirby-Bauer method. These findings were confirmed using Illumina whole genome nucleotide sequence data. All sequences were compared to the outbreak strain from southern Pakistan and typed using the S. Typhi genotyping scheme.FindingsTwenty-seven XDR S. Typhi isolates were identified from patients at Jinnah Hospital in Lahore between January and April 2019. All isolates were confirmed by sequence analysis to harbour an IncY plasmid and the CTX-M-15 ceftriaxone resistance determinant. All isolates were of the same genotypic background as the outbreak strain from Sindh province.SummaryWe report the first emergence of XDR S. Typhi in Punjab province of Pakistan confirmed by whole genome sequencing.FundingThis work was supported by the BBSRC-funded QIB Institute Strategic Programme “Microbes in the Food Chain” BB/R012504/1 and its constituent projects Microbial Survival in the Food Chain (BBS/E/F/000PR10349) and Research Infrastructure (BBS/E/F/000PR10352), and the BBSRC funded Core Capability Grant (project number BB/CCG1860/1).Research in contextEvidence before this studyTyphoid fever is endemic in Pakistan, with widespread resistance to first line drugs: ampicillin, co-trimoxazole, and chloramphenicol, and to fluoroquinolones. In 2017 the first report of additional resistance to ceftriaxone was published as XDR typhoid fever from southern Pakistan. Treatment of XDR typhoid fever has been a clinical challenge; options are scarce, and the level of chronic carriage is unknown. Current treatment is dependent upon azithromycin and this represents a major threat for the community as resistance has been reported from Bangladesh.Added value of this studyThis study is the first genomic report of XDR typhoid fever in a central part of Pakistan, describing the same genetic background as strains responsible for XDR typhoid in Sindh province. We have confirmed that the outbreak strain of XDR S. Typhi is now present in the most populated province and second largest city of Pakistan.Implications of all the available evidenceThe present study highlights the gravity of the situation - the spread of this strain is of serious concern. The clinical situation remains unchanged since this study was performed; we believe it is imperative that future research focuses upon the biology, transmission and control of this strain.

Cotton ( Gossypium hirsutum) wilt is one of the destructive disease caused by Fusarium oxysporum f. sp. vasinfectum and lead to 100% yield loss under favorable conditions. This study aims to estimate the potential of biological control agents Saccharothrix algeriensis NRRL B-24137 (SA) and chemical fungicides against cotton wilt pathogen under in-vitro and in-vivo conditions. The in-vitro study revealed that carbendazim showed maximum mycelia growth inhibition with a mean of 91% over control, which was further validated in glasshouse assay. In-vitro dual culture test of biocontrol agents with F. oxysporum determined that SA had a potential to inhibit mycelia growth by 68% compared to control. Further in glasshouse assay, the combination of the SA and carbendazim (10 µg/mL) showed a significant ( p < 0.05) disease control. Moreover, results demonstrated that carbendazim and SA remarkably decreased the disease development up to 83% and subsequently, significant improvement was observed in the plant growth parameters (plant length, root length, and plant weight) compared to untreated plants. Conclusively, exploration and utilization of bioagent for fungal diseases in cotton may provide a better line with maximum efficacy and with lesser adverse effects, which will pave a way toward better consequences in fungal treatments.

Introduction: The spread of carbapenem-resistant Acinetobacter baumannii (CRAB) is difficult to control especially in the hospitals due to the successful mobilization and evolution of the genetic elements harboring the resistant determinants. The study was conducted to examine the distribution of aminoglycosides, tetracycline, and sulfonamide-resistant determinants among CRAB isolates that carry the blaOXA-23 gene. Methodology: For a total of 160 CRAB strains isolated at tertiary care hospitals of Pakistan that mainly carried blaOXA-23 gene were included in the study to evaluate the assortment of antibiotic resistance genes. Results: The susceptibility rates of CRAB for other than beta-lactam drugs were 2.5% for both ciprofloxacin and aminoglycosides and 18% and 25% for sulfonamides and tetracyclines, respectively. Polymyxin B (MIC90, 1 g/mL) Colistin (MIC90, 1 g/mL) and Tigecycline (MIC90, 2 g/mL) were most active against these extensively drug-resistant CRAB isolates. The isolates were found to possess various genes mainly the tetB and sul2 for tetracycline and sulfonamide but the genes conferring resistance to aminoglycosides were varied with various combinations. Conclusion: Despite the CRAB clones containing blaOXA-23 have been previously reported in Pakistani hospitals, the screening of genetic determinants responsible for other antimicrobial agents is crucial for developing an effective surveillance and mitigation system for infection management.

Objective: To determine the occurrence of bacterial pathogens responsible for diarrhea and to engender information regarding the effectiveness of commonly used antibiotic against diarrhea. Methods: This cross-sectional study was conducted between April and July 2014. Samples were collected from the Divisional Headquarter and Allied Hospital, Faisalabad, Pakistan. The differential and selective media were used to isolate bacterial pathogens, which were identified through cultural characteristics, microscopy, and biochemical tests. Disc diffusion assay was carried out using Muller Hinton agar medium, and minimum inhibitory concentration was determined using broth dilution method against isolated pathogens. Results: One hundred and forty-one (100%) samples were positive for some bacteria. Frequency of occurrence was Bacillus cereus ( B. cereus ) (66%), Escherichia coli (E. coli) (48.5%), Salmonella typhi (S. Typhi) (27.7%), Pseudomonas aeruginosa (P. aeruginosa) (8.5%), and Staphylococcus aureus (S. aureus) (4.3%). Single pathogen was detected in 20 (14.2%) samples whereas combinations were found in 121 (85.8%) samples. Bacillus cereus and E. coli were the most frequently detected pathogens followed by the S. Typhi , P. aeruginosa , and Staph. aureus . The percentage occurrence of isolated pathogens was 31% in B. cereus , 31% in E. coli , 18% in S. Typhi , 5% in P. aeruginosa , and 3% in Staph. aureus . Conclusion: Pseudomonas aeruginosa showed resistance against Amoxicillin and Cefotaxime, whereas S. aureus was found resistant against Cefotaxime. Statistical analysis using one way Analysis of Variance revealed that Ofloxacin and Gentamicin had significant ( p less than 0.05) differences against all isolates as compared with other antibiotics used in this study. Saudi Med J 2016; Vol. 37 (3): 274-279 doi: 10.15537/smj.2016.3.14449 How to cite this article Rasool MH, Siddique AB, Saqalein M, Asghar MJ, Zahoor MA, Aslam B, et al. Occurrence and antibacterial susceptibility pattern of bacterial pathogens isolated from diarrheal patients in Pakistan. Saudi Med J 2016; 37: 274-279.

Heavy metal contamination now a day is one of the major global environmental concerns. Textile effluents of Faisalabad Pakistan are heavily contaminated with heavy metals and demands to explore native microorganisms as effective bioremediation tool. Study aimed to isolate heavy metal tolerant bacteria from textile effluents of Faisalabad Pakistan and to evaluate their biosorptive potential. Out of 30 collected samples 13 isolates having metal tolerance potential against Ni and Co were screened out. Maximum tolerable concentration and multi metal resistance was determined. A native bacterial strain showing maximum tolerance to Ni and Co and multi metal resistance against Ni, Co and Cr at different levels was selected and named as Abuzar Microbiology 1 (AMIC1). Molecular characterization confirmed it as Klebsiella variicola which was submitted in First fungal culture bank of Pakistan (FCBP-WB-0688). ICP-OES revealed that it reduced Ni (50, 49%) and Co (71, 68.6%) after 24 and 48 h, respectively. FT-IR was used to analyze functional groups and overall nature of chemical bonds. Changes in spectra of biomass were observed after absorption of Ni and Co by K. variicola. SEM revealed morphological changes in bacteria in response to metal stress. Both metals affected bacterial cell wall and created pores in it. However effect of Ni was more pronounced than Co. It was concluded that K. variicola, a native novel strain possessed significant heavy metal tolerance and bioremediation potential against Ni and Co. It may be used in future for development of bioremediation agents to detoxify textile effluents at industrial surroundings.

The present study aimed at measuring the antibody titres against H9N2 influenza viruses and its molecular detections in asymptomatic commercial layers from Faisalabad district of Punjab, Pakistan. Overall 120 blood samples, 24 tissue samples from each organ (trachea, lung and intestine) were collected from the 12 commercial layer flocks selected randomly at the age of 35-50 weeks without any direct clinical manifestation of avian influenza. Serum collected from 120 birds was tested for antibodies against H9N2 by using Haemagglutination Inhibition assay. Calculated geometric mean titers of 5.37 revealed the infectivity of the flock with H9N2 Influenza virus. To investigate the presence of virus in the study population, trachea, lung and intestine tissue samples were processed for RNA isolation and subjected to molecular detection of H9 gene of H9N2 subtypes using one step Reverse Transcriptase Polymerase Chain Reaction (RT-PCR). Out of 24 samples taken from each organ, 22, 21, and 18 samples from trachea, lungs, and intestine showed viral RNA, respectively. Taken together, results showed that the H9N2 is endemic and widely distributed in asymptotic layers. Furthermore results indicated that H9N2 subtype may survive in layers without showing any symptoms.

Background In recent years, antibiotic resistance has been indicated as a paramount threat to public health. The use of bacteriophages appears to be a safer alternative for the control of bacterial infections. Objectives The present study aims to explore sewage water for the presence of indigenous bacteriophages, and to investigate their antibacterial potential against Methicillin-resistant Staphylococcus aureus (MRSA). Methods Bacterial isolates were first collected and identified from pus samples taken from the surgical and burn units using standard microbiological procedures. A cefoxitin disk screen test was then used and interpreted according to the clinical laboratory standards institute (CLSI) guidelines for the detection of MRSA. The sewage samples were processed and the phages enriched using S. aureus as a host organism. Turbid and clear plaques of different sizes were isolated using an overlay method, purified, and then enumerated by means of a dilution method. Results The phages exhibited good lytic activity against MRSA when tested in-vitro, and the highest activity was attained within three to six hours of phage infection. The isolated phage pq/48 was also found efficient in decreasing the bacterial count during an in-vivo trial in rabbits. A protein analysis using SDS-PAGE revealed 10 proteins of between 20 kDa and 155 kDa in size. Conclusions The overall results indicated that bacteriophages isolated from sewage exhibited excellent lytic activity against MRSA strains. In conclusion, bacteriophages can be further characterized and appear to be a promising candidate for phage therapy against MRSA in the future.

Combating the problem of microbial resistance is one of the major challenges the medical sciences facing today. Inhibition of resistance mechanism in bacteria seems much better approach than developing new antibiotics only. The present study was aimed to investigate the effect of different concentrations of prochlorperazine in increasing the effectiveness of fluoroquinolones against Escherichia coli, Staphylococcus aureus and Streptococcus pyogenes. The bacteria were isolated from indigenous sources and identified through cultural characteristics, Grams’ staining and biochemical tests. The master suspensions were subjected to viable count and inoculated at the rate of 106 CFU/ml of the media for antimicrobial sensitivity testing. The four fluoroquinolones i.e. ciprofloxacin, levofloxacin, pefloxacin and norfloxacin were first applied to bacterial cultures alone and then in combination with four different concentrations (16μg/ml, 32μg/ml, 64μg/ml and 128μg/ml) of prochlorperazine and inhibition of growth was recorded in terms of diameter of zones of inhibition. A linear relationship was found between the increase in concentration of prochlorperazine and diameter of zones of inhibition against all the fluoroquinolones. The diameter of zones of inhibition were significantly (p

The present study was conducted for the isolation and characterization of gymnemic acid from indigenous Gymnema sylvestre leaves. Four different methods of extraction were employed to obtain the maximum yield. The method where the defatted leaves were extracted under continuous hot extraction in Soxhlet apparatus with 95% ethanol gave the maximum yield of gymnemic acid (6.15% m.f.b.). The yield was minimum in aqueous extraction method (1.66% m.f.b.) Gymnemic acid was purified by preparative chromatographic method in two solvent systems. Its circular Thin Layer Chromatography (TLC) also resulted in to a single concentric ring. Gymnemic acid was hydrolyzed by two methods to confirm its glycosidic nature. The glycon portion thus obtained gave a positive test with Fehling’s solution showing the presence of reducing sugars and indicating that gymnemic acid was a glycoside. The aglycone portion on thin layer chromatographic examination in two systems showed the presence of four genins. Therefore the gymnemic acid was a glycoside and was triterpenoid saponins in character. The resinous extract obtained on defatting the leaves with the petroleum ether in first method of extraction separated out in to four spots on TLC. The presence of stigmasterol, -amyrin, -amyrin acetate and lupeol was indicated when the reference samples of these compounds were run on the same chromatoplate.

Peste des petits ruminants virus (PPRV) is causing infectious disease with high morbidity and mortality rate in domestic and wild small ruminants of Pakistan with valuable economical losses. The present study was carried out to investigate risk factors of PPRV in domestic small ruminants which were present in the vicinity of wildlife parks. A total of 265 sera samples (27 wild ruminants and 238 domesticated small ruminants) from apparently healthy animals from two different wildlife parks were collected and analysed for PPRV antibodies. Also, 20 nasal swabs from domestic small ruminants showing respiratory signs were collected to check for presence of PPRV antigen. Competitive ELISA revealed highest proportions of anti-PPRV antibodies in domestic small ruminants around the Wildlife Park at Lahore (35%) as compared to Faisalabad (13%), with no existence of PPRV antibodies in tested serum of wild ruminants at these parks. Higher seropositivity was observed in females (25.6%) than in males (5.1%) and in goats (34.5%) compared to sheep (11.2%). The results of N-gene based RT-PCR highlight the absence of PPRV due to lack of current PPR outbreak in the region during study period. Even though grazing was not a significant risk factor, there is still a possibility of wildlife-livestock interactions for feed and water reservoirs, resulting in spillover of PPR to wildlife. Keeping in view the high seropositivity and risk of PPR, vaccination should be adopted to avoid circulation of PPRV among wild and domestic small ruminants (sheep and goats).

Probiotics are viable microorganisms with the capacity to alter the gastrointestinal microbiota of the host. The recent scientific advancements and development of probiotic formulations have rekindled the importance of these clinical interpretations, underlining the starring role of the gut flora in host metabolism, defense and immune regulation. Despite encouraging preliminary results from randomized clinical trials of probiotics for various clinical conditions including irritable bowel syndrome, necrotizing enterocolitis, gastroenteritis, antibiotic-associated diarrhea, infantile colic, and improvement of digestion and immune function, further evidence is needed to determine the reproducibility of the findings and elucidate the underlying mechanisms. In this review, we have considered the postnatal development of gut flora and appraised the role of probiotics in health and disease condition among infants.

The present work was carried out to study the seroprevalence of Mycoplasma mycoides ssp. capri in small and large ruminants and their possible role in its transmission among goat population. A total of 1288 serum samples were collected from randomly selected small and large animals including goat, sheep, cattle, buffalo and camel slaughtered in Faisalabad abattoir. The percentages of positive sera were recorded after preliminary screening of all samples through counter immuno-electrophoresis (CIE) test. To rule out the possibility of false positive results and to determine the geometric mean antibody titer (GMT), the positive samples through CIE were subjected to indirect haemagglutination (IHA) test. Seroprevalence was recorded in each species of animal. Overall results showed that goat sera were highly seropositive through CIE (7.3) and IHA (1.6), followed by sheep and cattle. In sheep, positive percentage was 2.6 and 1.5 through CIE and IHA tests, respectively. In cattle, 1.9% was positive through CIE and 0.7% through IHA test. Camel and buffalo population were positive for CIE as 1.3 and 0.8%, respectively. There was no evidence of IHA antibody response in their serum. The GMT through IHA test was 3.5, 3.0 and 2.3 in goat, sheep and cattle, respectively. The overall prevalence was 3.0 ± 0.9 and 0.8 ± 0.5% through CIE and IHA tests, respectively. The CIE was found an efficient, sensitive and economical test for initial screening whereas IHA test was found more specific and used for antibody titration. The percentage of positive samples was higher in sheep and cattle after goat which indicates their possible role in the transmission of M. mycoides ssp. capri among goat population.

Pathogenic E. coli was isolated from the birds suffering from colibacillosis showing characteristic lesions. Two types of coliphages T and T were isolated from the sewage water collected from different 1 4 areas of Faisalabad, Pakistan. These phages were separately tested for in vitro lysis of their host cell on tryptone agar plates, with equal (1:1)) concentration (10 CFU and 10 PFU) of E. coli and phages, which 7 7 1 2 deprived of phages and E. coli respectively. The 13 birds of group D that were given only the phages did 2 show neither the signs of disease nor mortality. The results indicated a good protection level using coliphages when compared with the control group. It was concluded that phages may be used effectively as good alternatives of those antibiotics to which bacteria have become resistant.

The present study was aimed at exploring the native wild isolates of Penicillium chrysogenum series in terms of their penicillin production potential. Apart from the standard medium, the efforts were made to utilize suitable agro-industrial wastes for the maximum yield of penicillin. Two series of P. chrysogenum were isolated from local sources and named as P. chrysogenum series UAF R1 and P. chrysogenum series UAF R2. The native series were found to possess better penicillin production potential than the already reported series of P. chrysogenum. However, P. chrysogenum series UAF R1 was found to be the best candidate for high yield of penicillin starting at 100 hour as compared to P. chrysogenum series UAF R2 which produced the highest yield of penicillin at 150 hours for a shorter period of time. Addition of Corn Steep Liquor (CSL) to the fermentation medium resulted in the production of 1.20g/L penicillin by P. chrysogenum series UAF R1 and P. chrysogenum series UAF R2. The fermentation medium in which Sugar Cane Bagasse (SCB) was replaced with CSL resulted in the highest yield of penicillin (1.92g/L) by both native series of P. chrysogenum. The penicillin production was increased by 62.5% in medium with SCB as compared to that with CSL. The penicillin yield of medium containing lactose and phenyl acetate was higher than that of control medium. Overall results revealed that P. chrysogenum series UAF R1 and P. chrysogenum series UAF R2 may be recommended for better yield of natural penicillin and this efficiency may be further enhanced by utilizing SCB as substrate in the growth medium.

The present study was conducted to find out the ethambutol resistance pattern of indigenous isolates of Mycobacterium tuberculosis from Tuberculosis diagnosed human patients. A total of 172 specimens were collected from six different sources and comprised of 84.9% sputum, 10.5% pus and 4.7% bronchial washings. There were 70.9% males and 29.1% females with 84.30% pulmonary and 15.69% extra-pulmonary tuberculosis. The Mycobacterium tuberculosis isolates collected from primary culture were further studied to determine their pattern and level of resistance. The inoculums were prepared using 0.5 Mac Farland turbidity standards. Five different concentration of ethambutol were used in Lowenstein Jensen (LJ) medium i.e. 2μg/ml, 4μg/ml, 6μg/ml, 8μg/ml and 10μg/ml for sensitivity testing. Data showed 10 (5.8%) resistant and 162 (94.2%) sensitive Mycobacterium tuberculosis out of total 172 clinical isolates. The growth was not inhibited at 1st (2μg/ml) and 2nd (4μg/ml) drug levels, while growth of 50% isolates inhibited at 3rd level (6μg/ml), 30% inhibited at 4th level (8μg/ml) and 20% at 5th level (10μg/ml). The last three levels are above the therapeutic index and not recommended in actual clinical practice. It is thus conceivable to explore some other more effective chemotherapeutic agents, modify combinations or find more effective procedures to stop morbidity and mortality due to ethambutol resistant Mycobacterium tuberculosis.

The present work was carried out to develop an effective vaccine to control infectious bursal disease (IBD). The very virulent infectious bursal disease virus (vvIBDV) was adapted to grow in Vero-cell line after three serial passages. The virus was then attenuated by further serial passages and pathogenicity of different passages was determined in broiler chicks free from antibodies against IBDV. The ninth passage virus was completely attenuated, as it was non-pathogenic to chicks. The birds inoculated with ninth passage virus had bursa to body weight ratio comparable to un-inoculated control and zero mean lesion score. This ninth passage virus was evaluated as live attenuated Vero-cell adapted vaccine. A commercial live cell culture origin vaccine (IBD-VAC, Korea) was used for comparison. Experimental trials were conducted in 3-week-old broiler chicks. Clinical signs, gross and histopathological lesions and bursa to body weight ratio were all comparable to un-vaccinated control group in case of live attenuated Vero-cell vaccine (group A). High virus-neutralizing antibody titres were also found at 7, 14, and 21 days post-vaccination in group A. On challenge, 100% protection was achieved in group A and 65% in case of commercial vaccine (group B) in terms of bursal lesions. All the birds in control group showed sever bursal lesions post-challenge. The study indicated newly developed infectious bursal disease vaccine was found to be safe and immunogenic.