Exploring the resistome, virulome, and mobilome of multidrug-resistant Klebsiella pneumoniae isolates: deciphering the molecular basis of carbapenem resistance.

BACKGROUND: Klebsiella pneumoniae, a notorious pathogen for causing nosocomial infections has become a major cause of neonatal septicemia, leading to high morbidity and mortality worldwide. This opportunistic bacterium has become highly resistant to antibiotics due to the widespread acquisition of genes encoding a variety of enzymes such as extended-spectrum beta-lactamases (ESBLs) and carbapenemases. We collected Klebsiella pneumoniae isolates from a local tertiary care hospital from February 2019-February 2021. To gain molecular insight into the resistome, virulome, and genetic environment of significant genes of multidrug-resistant K. pneumoniae isolates, we performed the short-read whole-genome sequencing of 10 K. pneumoniae isolates recovered from adult patients, neonates, and hospital tap water samples. RESULTS: The draft genomes of the isolates varied in size, ranging from 5.48 to 5.96 Mbp suggesting the genome plasticity of this pathogen. Various genes conferring resistance to different classes of antibiotics e.g., aminoglycosides, quinolones, sulfonamides, tetracycline, and trimethoprim were identified in all sequenced isolates. The highest resistance was observed towards carbapenems, which has been putatively linked to the presence of both class B and class D carbapenemases, blaNDM, and blaOXA, respectively. Moreover, the biocide resistance gene qacEdelta1 was found in 6/10 of the sequenced strains. The sequenced isolates exhibited a broad range of sequence types and capsular types. The significant antibiotic resistance genes (ARGs) were bracketed by a variety of mobile genetic elements (MGEs). Various spontaneous mutations in genes other than the acquired antibiotic-resistance genes were observed, which play an indirect role in making these bugs resistant to antibiotics. Loss or deficiency of outer membrane porins, combined with ESBL production, played a significant role in carbapenem resistance in our sequenced isolates. Phylogenetic analysis revealed that the study isolates exhibited evolutionary relationships with strains from China, India, and the USA suggesting a shared evolutionary history and potential dissemination of similar genes amongst the isolates of different origins. CONCLUSIONS: This study provides valuable insight into the presence of multiple mechanisms of carbapenem resistance in K. pneumoniae strains including the acquisition of multiple antibiotic-resistance genes through mobile genetic elements. Identification of rich mobilome yielded insightful information regarding the crucial role of insertion sequences, transposons, and integrons in shaping the genome of bacteria for the transmission of various resistance-associated genes. Multi-drug resistant isolates that had the fewest resistance genes exhibited a significant number of mutations. K. pneumoniae isolate from water source displayed comparable antibiotic resistance determinants to clinical isolates and the highest number of virulence-associated genes suggesting the possible interplay of ARGs amongst bacteria from different sources.

Comprehensive insights into Klebsiella pneumoniae: unravelling clinical impact, epidemiological trends and antibiotic-resistance challenges.

Klebsiella pneumoniae, a challenging opportunistic bacterium, became a notable global health concern owing to its clinical impact, widespread epidemiology and escalating antibiotic resistance. This comprehensive review delves into the multifaceted dimensions of K. pneumoniae, with a focus on its clinical implications, epidemiological patterns and the critical issue of antibiotic resistance. The review also emphasizes the implications of K. pneumoniae in the context of antimicrobial stewardship and infection control. Epidemiological aspects are scrutinized, shedding light on the global distribution and prevalence of K. pneumoniae. Factors influencing its transmission and persistence in healthcare facilities and communities are examined, with patient demographics, healthcare practices and geographical variations. The review centres on antibiotic resistance, a critical issue in the era of bacteria displaying resistance to multiple drugs. The mechanisms of resistance used by K. pneumoniae against various classes of antibiotics are elucidated, along with the alarming rise of carbapenem-resistant strains. It also highlights ongoing research efforts and innovative strategies aimed at addressing this critical public health issue. This comprehensive review offers a holistic understanding of K. pneumoniae, emphasizing its clinical significance, global epidemiology and the immediate necessity for effective strategies to combat antibiotic resistance. It serves as a valuable resource for healthcare practitioners, researchers and policymakers seeking to manage better and mitigate the impact of this pathogen on public health.

Epidemiology of cutaneous leishmaniasis in children of Khyber Pakhtunkhwa, Pakistan.

OBJECTIVES: In Pakistan, cutaneous leishmaniasis is an emerging tropical disease and a very high number (>70%) of children are afflicted by this marring infection. This study aimed to scrutinise the prevalence, spatial distribution and socio-demographic and behavioural risk factors associated with cutaneous leishmaniasis in children aged <5-15 years in Khyber Pakhtunkhwa. METHODS: A total of 1, 559 clinically confirmed records of children diagnosed with cutaneous leishmaniasis (January-December) from 2020 and 2022 were obtained from selected district hospitals. In addition, a risk factors-related questionnaire was administered to 1, 011 households (400 in 2020 and 611 in 2022) in nine districts during a household survey. RESULTS: The maximum number of cutaneous leishmaniasis cases was recorded in 2022 (n = 877, 56.25%) as compared to 2020 (n = 682, 43.75%). The hospital records showed a greater number of male patients in the 2022 cohort (n = 603, 68.76%). The highest number of cases were observed in children aged 5-9 years in 2022 (n = 282, 32.16%) and 2020 (n = 255, 37.39%). In 2020 and 2022, cutaneous leishmaniasis cases showed peak aggregation in March (n = 118, 17.3%) and January (n = 322, 36.72%). From a spatial analysis, the maximum number of cutaneous leishmaniasis cases was recorded at 59-1700 m elevation in various land-use/land-cover and climatic regions with quaternary alluvium rock formations. A multivariate logistic regression model analysis of risk factors from the households survey suggested that age group, socio-economic status, construction materials of the house, use of insect repellents, Afghan refugee camps in the village/district, knowledge and biting times of sand flies, frequent use of mosquito bed nets, presence of domestic animals in the house, knowledge of the transmission period and peak month of leishmaniasis infection increased the risk of acquiring cutaneous leishmaniasis (p value < 0.05). CONCLUSION: Our analysis demonstrated that cutaneous leishmaniasis in children is influenced by a variety of environmental, socio-demographic and behavioural risk factors in Khyber Pakhtunkhwa. The increase in recorded cases of cutaneous leishmaniasis in children in 2022 compared to 2020 suggests that the infection likely extended to new foci in the province.

Association of AFF3 Gene Polymorphism rs10865035 with Rheumatoid Arthritis: A Population-Based Case-Control Study on a Pakistani Cohort.

Rheumatoid arthritis (RA) is one of the complex diseases with the involvement of the genetic as well as environmental factors in its onset and severity. Different genome-wide association and candidate gene studies have shown the role of several genetic variants in multiple loci/genes with ethnical and geographical variations. This study was designed to detect the association of a single-nucleotide polymorphism (SNP) rs10865035 in the AFF3 gene with the genetic background of rheumatoid arthritis (RA) in the Pakistani cohort. A total of 703 individuals, including 409 RA patients and 294 healthy controls, were genotyped using TaqMan assay and Tri primer ARMS-PCR (amplification-refractory mutation system-polymerase chain reaction) methods. The association of rs10865035 with the RA was statistically determined using different models. Interestingly, besides the homozygous recessive model (G/G vs. A/G + A/A) (OR = 1.693(1.06-2.648); P = 0.025), all other models, which included the codominant (χ 2 = 5.169; P = 0.075), homozygous dominant (A/A vs. G/G + A/G) (OR = 0.867 (0.636-1.187); P = 0.41), heterozygous (A/G vs. A/A + GG) (OR = 0.491 (0.667-1.215); P = 0.49), and additive model (OR = 0.826 (0.665-1.027); P = 0.08) showed insignificant distribution of the genotypes among the cases and controls. These findings suggest that the AFF3 gene (rs10865035) has no significant role in the onset of RA in the Pakistani population.

Isolation and characterization of bacteriophage to control multidrug-resistant Pseudomonas aeruginosa planktonic cells and biofilm.

Pseudomonas aeruginosa is Gram-negative bacterium, one of the leading cause of drug-resistant nosocomial infections in developing countries. This bacterium possesses chromosomally encoded efflux pumps, poor permeability of outer-membrane and high tendency for biofilm formation which are tools to confer resistance. Bacteriophages are regarded as feasible treatment option for control of resistant P. aeruginosa. The aim of the current study was isolate and characterized a bacteriophage against P. aeruginosa with MDR and biofilm ability. A bacteriophage MA-1 with moderate host range was isolated from waste water. The phage was considerable heat and pH stable. Electron microscopy revealed that phage MA-1 belongs to Myoviridae family. Its genome was dsDNA (≈50 kb), coding for eighteen different proteins (ranging from 12 to 250 KDa). P. aeruginosa-2949 log growth phase was significantly reduced by phage MA-1 (2.5 × 103 CFU/ml) as compared to control (without phage). Phage MA-1 also showed significant reductions of 2.0, 2.5 and 3.2 folds in 24, 48, and 74 h old biofilms after 6 h treatment with phage respectively as compared to control. It was concluded from this study that phage MA-1 has capability of killing P. aeruginosa planktonic cells and biofilm, but for complete eradication cocktail will more effective to avoid resistance.

Identification of most damaging nsSNPs in human CCR6 gene: In silico analyses.

Single nucleotide polymorphisms in CCR6 (C-C chemokine receptor type 6) gene have been found to be the possible cause of many diseases like rheumatoid arthritis, psoriasis, lupus nephritis and systemic sclerosis and other autoimmune diseases. Therefore, identification of structurally and functionally important polymorphisms in CCR6 is important in order to study its potential malfunctioning and discovering therapeutic targets. Several bioinformatics tools were used to identify most damaging nsSNPs that might be vital for CCR6 structure and function. The in silico tools included PROVEAN, SIFT, SNP&GO and PolyPhen2 followed by I-Mutant MutPred and ConSurf. Phyre2 and I-TASSER were used for protein 3-D Modelling while gene-gene interaction was predicted by STRING and GeneMANIA. Our study suggested that three nsSNPs rs1376162684, rs751102128 and rs1185426631 are the most damaging in CCR6 gene while 7 missense SNPs rs1438637216, rs139697820, rs768420505, rs1282264186, rs1394647982, rs769360638 and rs1263402382 are found to revert into stop codons. Prediction of post-transcriptional modifications highlighted the significance of rs1376162684 because it effected potential phosphorylation site. Gene-gene interactions showed relation of CCR6 with other genes depicting its importance in several pathways and co-expressions. In future, studying diseases related to CCR6 should include investigation of these 10 nsSNPs. Being the first of its type, this study also proposes future perspectives that will help in precision medicines. For such purposes, CCR6 proteins from patients of autoimmune diseases should be explored. Animal models can also be of significance find out the effects of CCR6 in diseases.

Bacteriophages: an overview of the control strategies against multiple bacterial infections in different fields.

Bacteriophages (phages/viruses) need host bacteria to replicate and propagate. Primarily, a bacteriophage contains a head/capsid to encapsidate the genetic material. Some phages contain tails. Phages encode endolysins to hydrolyze bacterial cell wall. The two main classes of phages are lytic or virulent and lysogenic or temperate. In comparison with antibiotics, to deal with bacterial infections, phage therapy is thought to be more effective. In 1921, the use of phages against bacterial infections was first demonstrated. Later on, in humans, phage therapy was used to treat skin infections caused by Pseudomonas species. Furthermore, phages were successfully employed against infections in animals - calves, lambs, and pigs infected with Escherichia coli. In agriculture, for instance, phages have successfully been used e.g., Apple blossom infection, caused by Erwinia amylovora, was effectively catered with the use of bacteriophages. Bacteriophages were also used to control E. coli, Salmonella, Listeria, and Campylobacter contamination in food. Comparatively, phage display is a recently discovered technology, whereby, bacteriophages play a significant role. This review is an effort to collect almost recent and relevant information regarding applications and complications associated with the use of bacteriophages.

Progression pattern of rheumatoid arthritis: A study of 500 Pakistani patients.

To estimate the most prevalent age of patients and disease status and progression in terms of severity at different age groups in the Pakistani Rheumatoid Arthritis (RA) patients. A total of five hundred (500) RA patients were enrolled during October, 2009 to October, 2013. A screening questionnaire was filled for each patient satisfying America College of Rheumatology (ACR) criteria under the supervision of certified rheumatologists. Epidemiological and demographic variables were statistically analyzed for correlation with progression of the disease using SPSS ver 17.0.1 software. In general, rheumatoid arthritis preferentially affects women with female to male ratio of about 3:1; however, patients with above 60 years of age have equal female to male ratio. The most prevalent age is 45-60 years. The disease severity increases with increase in the age and reaches to its peak in above 60 years of age (p=0.001). The pattern of progression of RA in the Pakistani patients is almost consistent with other relevant studies conducted on European and European derived populations.

Characterization of new Myoviridae bacteriophage WZ1 against multi-drug resistant (MDR) Shigella dysenteriae.

Shigella dysenteriae is a normal inhabitant of the human gastrointestinal tract, but sometimes it causes severe infection known as shigellosis (bacillary dysentery). Bacteriophages are considered very safe and effective agents for controlling bacterial infections and contaminations. In this study, we describe the isolation and characterization of bacteriophage WZ1, isolated from waste water which inhibits the growth of S. dysenteriae. Phage WZ1 showed maximum stability at 37 °C and was stable up to 65 °C but was totally inactive at 70 °C. The pH stability increased from low to high and was totally inactive at pH 3 while maximum stability was observed at optimal pH 7. Phage WZ1 adsorption rate to the host bacterium was significantly enhanced by the addition of CaCl2 . It has a latent time and burst time of 24 min and about 430 virions/cell, respectively. Transmission electron microscopy of phage WZ1 revealed a head width of 10 ± 0.5 nm and length of 10 ± 0.2 nm with a contractile tail of 128 ± 25 nm long and 21 ± 0.5 nm wide and belongs to family Myoviridae of order Caudovirales. Twelve structural proteins ranging from 22 to 150 kDa were detected by Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE). The genome was found to be double stranded DNA with an approximate size of 38 kb. It has a very good reduction potential for S. dysenteriae by lowering abruptly the optical density of the planktonic S. dysenteriae culture. Phage WZ1 is a very promising candidate for phage therapy and other applications such as phage typing.

Genomic diversity and nutritional analysis of multi-drug resistant extended spectrum ß-lactamase Producing-Klebsiella pneumoniae genes isolated from mastitic cattle milk in district peshawar, Pakistan.

The increasing incidence of resistance extended spectrum-beta lactamase (ESBL) producing Klebsiella pneumonia become worldwide issue. The current study aimed to determine the genomic diversity of ESBL-producing K. pneumoniae in milk samples collected from cows with mastitis as well as their antibiotic sensitivity profiles and genetic identification in Peshawar, Pakistan. The california mastitis test (CMT) was initially used to verify the presence for mastitis in 700 collected milk samples. The molecular identification of the 16SrRNA gene confirmed 120/700 (17.14 %) propagation of K. pneumonia. Out of these isolates MDR ESBL-producing isolates were 60/120 (50 %). The lactose were found (M = 3.96 ± 0.28, SD = 2.19), followed by fats (M = 3.12 ± 0.11, SD = 0.90), protein (M = 5.97 ± 0.24, SD = 1.84), sodium (M = 55.74 ± 2.07, SD = 15.81), potassium (M = 138.5 ± 1.53, SD = 11.71), chloride (M = 0.74 ± 0.03, SD = 0.24), calcium (M = 10.27 ± 0.31, SD = 2.42), and chlorine (M = 2.80 ± 0.22, SD = 1.70), respectively. Amikacin (80 %), ceftazidime (71 %), and tetracycline (71 %) were shown to be the most effective antimicrobials against all of the isolates. The occurrence of the blaSHV gene was observed at 56.00 % whereas the blaTEM gene and blaCTX-M gene were 36.00 %, and 30.00 %. The distribution of blaCTX-M subgroup genes was followed by blaCTX-M-1 (38.00 %), blaCTX-M-9 (22.20 %), and blaCTX-M-15 (61.10 %). Co-occurrence of blaCTX-M+ blaSHV was (15.00 %), blaCTX-M+ blaTEM were (6.60 %), and blaSHV + blaTEM were (10.00 %), respectively. The inappropriate, prolonged and common use of antibiotics may apply selective pressure for propagation and the occurrence of resistant isolates.

Environmental and socio-demographic factors associated with cutaneous leishmaniasis in district Khyber, Pakistan; alarming spread of the disease to new foci.

Cutaneous leishmaniasis (CL) is a sand fly-borne infection of significant public health concern in Pakistan (endemic for CL). This study aimed to scrutinize the environmental and socio-economic risk factors for CL in district Khyber (located on Pak-Afghan border), Pakistan. Clinically confirmed 2881 CL case records for (January-December) 2017 and 2020 were obtained from district hospital. In addition, a questionnaire for CL risk factors assessment was administered to 525 households (175 in 2017 and 350 in 2020) in 40 villages throughout the district in a household survey. Higher number of CL cases were recorded in 2020 (N = 1824 belonging to 90 villages) compared to 2017 (N = 1057 from 42 villages). Highest number of CL patient cases were recorded in tehsil Jamrud (N = 2248, 39.01 %), followed by Landi Kotal (N = 398, 6.91 %) and Bara (N = 235, 4.08 %). Records showed higher number of CL cases in males (N = 1,659, 57.58 %) compared to females (N = 1,222, 42.41 %). In 2017 and 2020 the disease burden peaked in January. GIS-based spatial analyses of hospital records revealed that CL cases were abundant at 294-1,916 m elevation, in agriculture and range lands. Univariate logistic regression model analysis of risk factors suggested that higher education, family size >15, knowledge of CL, having family member/s that suffered from CL in the past, knowledge about biting time of sand flies, use of mosquito spray, presence of Afghan refugees in the village/s and living in mud-made houses increased the risk of acquiring CL. The multivariable logistic regression model showed none of the risk factors to be statistically significant. Findings of the study are crucial towards effective and targeted control of CL in district Khyber.

Correction: Nutritional analysis and characterization of carbapenemase producing-Klebsiella pneumoniae resistant genes associated with bovine mastitis infected cow's milk.

[This corrects the article DOI: 10.1371/journal.pone.0293477.].

Synthesis and analysis of silica nanocarriers for pectinase immobilization: Enhancing enzymatic stability for continuous industrial applications.

Pectinolytic enzymes are among the important group of industrial enzymes that have wide applications in different food industries. In this study, pectinase-based silica nanocarriers were synthesized using co-precipitation and cross-linking techniques. The resulting silica nanoparticles were investigated using scanning electron microscopy (SEM), energy-dispersive electron microscopy (EDEX), and X-ray diffraction (XRD) for determination of its morphology, elemental composition, and crystalline pattern. Under the optimal immobilization conditions like 1.5 % glutaraldehyde, 3000 IU/mg pectinase concentration, 90 min immobilization time and 40 °C immobilization temperature, pectinase showed maximum immobilization yield. The immobilization of pectinase onto the silica nanocarriers led to enhanced catalytic characteristics, displaying higher enzymatic activity across various temperature and pH levels compared to soluble pectinase. Moreover, the immobilization substantially improved the temperature stability of pectinase, exhibiting 100 % of its initial activity even after 120 h of pre-incubation at 50 °C. Additionally, the silica nanocarrier pectinase retained 100 % of its original activity even after being reused 10 times in a single batch of reactions. These findings indicate that the immobilization of silica nanocarriers effectively enhances pectinase's industrial capabilities, making it economically feasible for industrial use and an efficient system for various biotechnological applications.

Investigation of Three Morchella Species for Anticancer Activity Against Colon Cancer Cell Lines by UPLC-MS-Based Chemical Analysis.

In search of new anticancer agents, natural products including fungal compounds had been used as potential anticancer agents. The aim of this study was to investigate the anticancer activity of Morchella extracts against colon cancer cell line and UPLC-DAD-MS/MS analysis for the identification of compounds. The cytotoxic activity of the three Morchella species was examined for their anti-carcinogenic properties against the colon cancer cell lines. Phytochemical analyses were performed to screen Morchella for the presence of anti-cancerous compounds. All the fungal extracts inhibited the viability of colon cancer cells in a dose-dependent manner. Major compounds identified in Morchella included amino acid, fatty acid, sterol, flavonoid, peptide, glutamic acid, alkaloid, terpenoid, cyclopyrrolones, and coumarin. Several new compounds were detected among all the three Morchella extracts. In conclusion, all the fungal extracts showed potential inhibition of colon cancer cells and actively arrested the cell viability. It was concluded that the identified bioactive compounds might be the main constituents contributing to the anticancer activity of Morchella against human colon cancer cell lines. Thus, Morchella extracts are a potential source of bioactive compounds with cytotoxicity and could potentially be used as functional food supplements. Due to the nature of impressive findings, this investigation should be undertaken further to allow the studies to explore and develop a potential cytotoxic agents against colon cancer.

Multidrug resistant Klebsiella Pneumoniae reservoir and their capsular resistance genes in cow farms of district Peshawar, Pakistan.

Klebsiella pneumoniae is a major zoonotic pathogen that causes a variety of severe illnesses as well as mastitis. The distribution of mastitis-causing K. Pneumoniae and its virulence factors vary by country and geographical location. The present study aimed to find out the occurrence of Multidrug-resistant (MDR) K. Pneumoniae and their capsular resistance genes which were undocumented previously in cow farms of district Peshawar, Pakistan. A total of 700 milk samples from symptomatic mastitic cows were screened for MDR K. Pneumoniae. Furthermore, the characterization of capsular resistance genes was done by molecular techniques. Among these samples, K. pneumoniae was found 180/700 (25.7%), while MDR K. pneumoniae was found 80/180 (44.4%). The antibiogram analysis revealed high resistance to Vancomycin (95%) while highly sensitive to Ceftazidime (80%). The distribution of capsular genes shows the most common serotype K2 gene 39/80 (48.7%), followed by serotype K1 gene 34/80 (42.5%), serotype K5 17/80 (21.2%), and serotype K54 13/80 (16.2), respectively. Moreover, the co-occurrence of serotypes K1+K2 was found at 11.25%, KI+K5 was 05%, K1+K54 was 3.75%, and K2+K5 was 7.5%, respectively. A statistically significant association (p ≤ 0.05) was found between predicted and discovered K. pneumoniae values. In conclusion, the presence of MDR K. pneumoniae in combination with capsular genes may be a possible threat to dairy farm animals and humans in Peshawar, Pakistan. It may give us special attention to follow up on hygienic practices in livestock management.

Nutritional analysis and characterization of carbapenemase producing-Klebsiella pneumoniae resistant genes associated with bovine mastitis infected cow's milk.

The current study was designed to analyze nutritional parameters and to characterize carbapenemase producing-Klebsiella pneumoniae isolates from bovine mastitic cow's milk. Out of 700 milk samples K. pneumoniae was identified by phenotypic and molecular techniques along with their antibiogram analysis and nutritional analysis was performed using the procedure of Association of Official Analytical Chemists. Carbapenemase-producing K. pneumoniae was detected by phenotypic CarbaNP test followed by molecular characterization of their associated resistant genes blaVIM, blaKPC, blaOXA-48, blaNDM, and blaIMP along with insertion sequence common region 1 (ISCR1) and integrons (Int1, Int2, and Int3) genes. Among nutritional parameters, fat content was observed (2.99%) followed by protein (2.78%), lactose (4.32%), and total solid (11.34%), respectively. The prevalence of K. pneumoniae among bovine mastitis was found 25.71%. Antibiogram analysis revealed that more effective antibiotics was ceftazidime (80%) followed by amikacin (72%), while highly resistant antibiotics was Fusidic acid (100%). Distribution of carbapenemase producer K. pneumoniae was found 44.4%. Among carbapenem resistant genes blaKPC was found 11.25%, blaVIM 2.75%, blaNDM 17.5%, and blaOXA-48 7.5%, while blaIMP gene was not detected. Furthermore, distribution of ISCR1 was found 40%, while integron 1 was found 61.2% followed by integron 2 (20%), and integron 3 (5%). In conclusion, the recent scenario of carbapenemase resistant K. pneumoniae isolates responsible for mastitis may affect not only the current treatment regime but also possess a serious threat to public health due to its food borne transmission and zoonotic potential.

Challenges and Approaches of Culturing the Unculturable Archaea.

Since Carl Woese's discovery of archaea as a third domain of life, numerous archaeal species have been discovered, yet archaeal diversity is poorly characterized. Culturing archaea is complicated, but several queries about archaeal cell biology, evolution, physiology, and diversity need to be solved by culturing and culture-dependent techniques. Increasing interest in demand for innovative culturing methods has led to various technological and methodological advances. The current review explains frequent hurdles hindering uncultured archaea isolation and discusses features for more archaeal cultivation. This review also discusses successful strategies and available media for archaeal culturing, which might be helpful for future culturing practices.

Co-occurrence of antibiotic resistance and virulence Genes in Methicillin Resistant Staphylococcus aureus (MRSA) Isolates from Pakistan.

Introduction: Methicillin resistant Staphylococcus aureus (MRSA) is one of the major human pathogen that is associated with hospital as well as community acquired infections and is responsible for huge amount of life-threatening diseases. Objective: Objective of the study was to determine MRSA prevalence, their antibiotic sensitivity patterns, frequency of virulence genes (sea, seb, sed, tst, hla, hld) and their co-occurrence with resistance marker mecA among Rawalpindi and its nearby regions of Pakistani clinical isolates. Methodology: The present study was carried out to identify the virulence and antibiotic resistance genes that co-occur in MRSA through polymerase chain reaction. Antibiotic sensitivity, presence of virulence genes and their co-occurrence with resistance marker mecA were analyzed. Results: These isolates were found resistant to number of antibiotics i.e. Amoxicillin (16.1%), Cefixime (48.38%), Doxycycline (27.415), Trimethoprim/sulfamethoxazole (37.09%), Clindamycin (30.64%), Erythromycin (83.87%), Penicillin (100%), Vancomycin (4.83%), Ciprofloxacin (70.96%), Tetracycline (20%), Linezolid (3.22%) and Fusidic acid (11.295). The frequency of antibiotic resistant gene (mecA) was 69.35% and that of virulence genes hla, hld, sea, seb, sed and tst was 100, 100, 53.2, 30.6, 3.2 and 24.2% respectively. Amongst all examined genes, hla and hld genes had the highest and sed gene had the lowest frequency. The maximum coexistence of genes was observed for hla+hld+mecA gene combination (42 out of 62 isolates). Conclusion: This study reports the presence of multidrug resistant, vancomycin-resistant and mecA negative MRSA isolates in infected patients of Rawalpindi and nearby regions of Pakistan that may have attributed to treatment failures, adaptability of new virulence characteristics and spread of antibiotic resistance.

Spatial Distribution, Seasonal Abundance and Physio-Chemical Assessment of Mosquito Larval Breeding Sites in Mardan District, Khyber Pakhtunkhwa, Pakistan.

Background: Mosquitoes (Diptera: Culicidae) are haemotophagus insects and are vectors of many arthropod-borne diseases. Present study aimed to explore species composition, seasonal abundance, spatial distribution and physio-chemical properties of larval breeding sites of mosquitoes in District Mardan, Khyber Pakhtunkhwa, Pakistan. Methods: Both adults and larvae of mosquitoes were collected through light traps, insecticide spray, mouth aspirator and larval standard dipping method in District Mardan from May to November 2017. Water samples from larval sites were physio-chemically analysed. Results: 5078 (3704 adults and 1374 larvae) mosquito specimens were collected in Mardan, Katlang and Takhtbhai tehsils. Six species in four genera were reported. Culex pipiens (89.80%) and Armigeres subalbatus (9.20%) were the most abundant species. Diversity was high in Takhtbhai (0.29) followed by Katlang (0.28) and Mardan (0.25). Greater number of specimens were recorded in peridomestic sites (93.97%) as compared to domestic habitats (6.03%). Culex pipiens larval abundance had negative correlation with pH whereas it correlated positively with electric conductivity, salinity, and TDS (total dissolved sulphur). Mosquito abundance peaked in August and July while the lowest was in May. Their monthly abundance had positive correlation with rainfall (r= 0.5069), relative humidity (r= 0.4439) and mean minimum temperature (r= 0.2866). Number of mosquitoes was highest at low elevation < 347m asl (above sea level) in agriculture land and near to water bodies (streams). Conclusion: Culex pipiens being the most abundant species, was susceptible to high pH. Mosquitoes preferred habitats were at low elevation in agriculture land.

Knowledge, attitude, and practices associated with avian influenza among undergraduate university students of East Java Indonesia: A cross-sectional survey.

Background: Several public health strategic actions are required for effective avian influenza (AI) prevention and control, as well as the development of a communication plan to keep undergraduate students sufficiently informed on how to avoid or reduce exposure. The aim of the survey was to measure the level of knowledge, attitudes and practices (KAPs) toward AI among undergraduate university students in East Java, Indonesia, and observe the correlation between KAPs and the factors associated with the control and prevention of AI.  Methods: A cross-sectional survey was conducted among undergraduate students to collect information about AI-related KAPs. Students were selected from three faculties of Universitas Airlangga Surabaya Indonesia (Faculty of Veterinary Medicine, Faculty of Fisheries and Marine, and Faculty of Science and Technology). Students voluntarily responded to a pre-designed questionnaire. Results: A total of 425 students (222 female; and 203 male), of ages ranging from 18 years (n=240) to 20-30 years (n=185), responded to the survey. This cohort consisted of 157 students from the Faculty of Fisheries and Marine, 149 from the Faculty of Veterinary Medicine, and 119 from the Faculty of Science and Technology. The results indicated that appropriate knowledge was obtained by 76.94% of students; significantly higher levels were seen in Faculty of Veterinary Medicine students as compared to the other two faculties (p<0.05). 72.89% of students documented positive attitudes; veterinary medicine students had significantly more positive attitudes than other faculties (p<0.05). Proactive behaviors were observed in 56.90% of students. The aggregate scores for KAPs were 6.93 ± 0.77 (range: 0-9) for knowledge, 7.6 ± 1.25 (range: 0-10) for attitude, and 9.1 ± 1.5 (range: 0-12) for practice.