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BACKGROUND: The biocontrol potential of soil microbes can reduce the extensive use of hazardous synthetic fungicides. This study was designed to find a strain of rhizobacteria indigenous to Pakistan with potential biocontrol against early blight of tomato caused by Alternaria solani and to characterize its biocontrol mechanisms. RESULTS: Among 88 strains tested for antagonism against A. solani on agar media, S27, Dt10 and 423, identified by 16S rRNA sequencing as strains of Bacillus amyloliquefaciens, B. cereus and Stenotrophomonas rhizophila, respectively, were the most inhibitory. When applied to detached tomato leaflets in Petri dish assays, the strains reduced lesion development by over 30% compared to the control. In greenhouse pot trials, the bacterial strains reduced early blight severity by over 50%. In three field trials, all three strains applied to tomato foliage slowed early blight disease progress and reduced disease severity, with B. amyloliquefaciens S27 reducing the area under the disease progress curve by up to 70%. All three strains showed protease, catalase and oxidase activities in vitro, but none produced β‐1,3‐glucanase and only B. cereus Dt10 showed slight chitinase activity. In a greenhouse experiment in which the bacteria were applied to tomato foliage prior to pathogen inoculation, bacteria‐treated leaves had higher β‐1,3‐glucanase and chitinase levels than leaves inoculated only with the pathogen, indicating priming induction of response. CONCLUSION: Three rhizobacteria strains have the potential to control early blight of tomato under Pakistan's growing conditions, with B. amyloliquefaciens S27 being the most promising candidate for commercial development. Antagonism and induction of the priming response may be mechanisms of biocontrol by the bacterial strains. © 2023 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

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During a survey of mushrooms of Pakistan, Macrocybe gigantea was collected from University of the Punjab, Lahore, Pakistan under the Morus species. For the domestication of this wild species, its culturability and cultivation potential was assessed by using different synthetic culture media and substrates. Among these different media used, maximum cultural growth was observed on Potato Dextrose Agar (PDA) medium at 30 °C followed by Malt Extract Agar (MEA), Compost Extract Agar (CEA), Glucose Peptone Agar (GPA), and Saboraud Dextrose Agar (SDA). Strains on PDA medium were used for production of spawning material on wheat, sorghum and barley grains. Sorghum grains at 30 °C were the best combination for spawn production. A mixed substrate of wheat straw and Tea waste at 30 °C produced the highest yield. Mineral analysis of the wild and cultivated strain revealed that both forms enrich Potassium and Calcium. These findings show that this giant edible mushroom species could be domesticated on the number of media and substrates. Its domestication can provide nutritional, economical, medicinal and tasty food to the growing population that would otherwise be restricted to natural production at a specific time of the year. [ABSTRACT FROM AUTHOR]

Objective: To determine the diagnostic accuracy of Colistin agar for detection of Colistin resistance in clinical isolates of Multi- Drug Resistant Gram-Negative Bacilli. Study Design: Cross-sectional validation study. Place and Duration of Study: Department of Microbiology, Armed Forces Institute of Pathology, Rawalpindi Pakistan, from Feb to Aug 2019. Methodology: A total of 100 Multi-Drug Resistant Gram-Negative Bacilli in clinical isolates were included. Isolates were identified using Gram stain, Catalase, Oxidase, API 20E, and API 20NE. After approval from the institutional ethical review committee, Colistin susceptibility was determined simultaneously by Colistin agar and Broth Micro Dilution Minimum Inhibitory Concentration method as per CLSI. For susceptibility criteria, EUCAST guidelines were followed. Results were validated with the gold standard test, i.e., Broth Micro Dilution. Results: Out of 100 Multi-Drug Resistant clinical isolates, the distribution was K. pneumoniae n=60, E.coli n=16, A. baumannii n=11, C. freundii n=8, and E. cloacae n=5. The sensitivity, specificity, positive predictive value, negative predictive value, and diagnostic accuracy of Colistin agar for detection of Colistin resistance, keeping Broth Micro Dilution Minimum Inhibitory Concentration method as the gold standard was 96.67%, 97.14%, 93.55%, 98.55%, and 97%, respectively. Conclusion: Colistin agar has excellent diagnostic accuracy for the detection of colistin resistance with standardized inoculum density. Due to its ease of use, cost-effectiveness, and accurate results, it can be used in lab setups deficient in manpower and advanced equipment for Broth Micro Dilution or genetic sequencing. [ABSTRACT FROM AUTHOR]

Pseudomonas aeruginosa and Aspergillus fumigatus infections frequently co-localize in lungs of immunocompromised patients and individuals with cystic fibrosis (CF). The antifungal activity of P. aeruginosa has been described for its filtrates. Pyoverdine and pyocyanin are the principal antifungal P. aeruginosa molecules active against A. fumigatus biofilm metabolism present in iron-limited or iron-replete planktonic P. aeruginosa culture filtrates, respectively. Using various P. aeruginosa laboratory wild-type strains (PA14, PAO1, PAK), we found antifungal activity against Aspergillus colonies on agar. Comparing 36 PA14 and 7 PAO1 mutants, we found that mutants lacking both major siderophores, pyoverdine and pyochelin, display higher antifungal activity on agar than their wild types, while quorum sensing mutants lost antifungal activity. Addition of ferric iron, but not calcium or magnesium, reduced the antifungal effects of P. aeruginosa on agar, whereas iron-poor agar enhanced antifungal effects. Antifungal activity on agar was mediated by PQS and HHQ, via MvfR. Among the MvfR downstream factors, rhamnolipids and elastase were produced in larger quantities by pyoverdine–pyochelin double mutants and showed antifungal activity on agar. In summary, antifungal factors produced by P. aeruginosa on agar differ from those produced by bacteria grown in liquid cultures, are dependent on quorum sensing, and are downregulated by the availability of ferric iron. Rhamnolipids and elastase seem to be major mediators of Pseudomonas ' antifungal activity on a solid surface. [ABSTRACT FROM AUTHOR]

This study was carried out to evaluate the prevalence of mycobiota and aflatoxin (AF) B1 contamination in sesame seeds from rainfed and irrigated zones of the Punjab, Pakistan. For this purpose, 100 sesame seed samples were collected directly from the fields of major sesame-producing areas in rainfed and irrigated zones. The agar plate method was used for isolation of mycobiota, and thin-layer chromatography was used to determine AFB1 concentrations. Seed samples were then stored for 12 months. After 12 months, the seeds were again analyzed for mycobiota and AFB1 for comparison. All samples were positive for fungal growth under fresh and stored conditions. Twenty-one fungal species of 10 genera were isolated. Aspergillus flavus was the most prevalent contaminant found in fresh and stored sesame seeds from rainfed and irrigated zones, followed by Aspergillus niger, Alternaria alternata, and Fusarium oxysporum. The least prevalent fungi were Aspergillus ochraceus and Cladosporium oxysporum. Analysis revealed that 92% of fresh and 99% of stored seed samples were contaminated with AFB1. In the rainfed zone, 88% of fresh and 100% of stored seed samples were contaminated with AFB1, with mean concentrations of 15.74 and 33.8 ppb, respectively. In the irrigated zone, 96% of fresh and 98% of stored seed samples were contaminated with AFB1, with mean concentrations of 20.5 and 27.56 ppb, respectively. AFB1 concentrations >20 ppb were found in 20% of fresh and 100% of stored seeds samples from the rainfed zone and in 28% of fresh and 60% of stored samples from the irrigated zone and thus were not fit for human consumption as per the maximum limit (20 ppb) assigned by the U.S. Food and Drug Administration and the Food and Agriculture Organization of the United Nations. This report is the first on the mycobiota and AFB1 contamination in sesame seeds from rainfed and irrigated zones of the Punjab, Pakistan. These baseline data are an initial step in the effort to deal with this significant food safety issue. Fresh and stored sesame seeds were positive for fungal growth. The leading contaminant in fresh and stored sesame seeds was Aspergillus flavus. Overall, 92% of fresh and 99% of stored seed samples were contaminated with AFB1. In the rainfed zone, 88% of fresh and 100% of stored samples were contaminated with AFB1. In the irrigated zone, 96% of fresh and 98% of stored samples were contaminated with AFB1. [ABSTRACT FROM AUTHOR]

Traditional polyherbal formulations are essential part of health system in various populations in the subcontinent region including Pakistan, however scientific evaluation of such claims are not significant. In this investigation, the antibiofilm and antimicrobial activities of Glycyrrhiza glabra and Viola odorata, two important ingredients of a traditional herbal formulation were determined in synergism. Various fractions of plants extract were prepared in the order of polarity. All fractions were tested against Gram negative (Streptococcus pneumoniae, Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa) and Gram positive (Staphylococcus aureus and Salmonella typhi) bacteria and antimicrobial activity was assessed using agar diffusion method. The MIC (Minimum Inhibitory concentration), was assessed. Finally antibiofilm activities were determined using 96 micro wells plate by calculating percent inhibition. In case of Viola oldorata, the inhibition zone ranged between (6 mm to 18 mm±1.24) against the test strains. The highest inhibition was recorded against gram positive bacteria Strep: Pneumoniae (20 mm±0.99) followed by S. aureus (18 mm±1.02) using methanolic extract. A moderate level of inhibition was seen in case of Glycyrrhiza glabra against the microbial strains. It was noticed that Glycyrrhiza Glabra was more active against gram negative bacteria. The methanolic fraction presented highest activity against e coli (18 mm±0.96) followed by K. pneumoniae and P. aureginosa (12 mm±1.22).The MIC ranged between 4–8 mg/mL. During antibiofilm assay, a moderate level of inhibition occurs using the Viola odorata (40%inhibition) and Glycyrrhiza glabra (45%inhibition), that increased by using combination of both plants in 50:50 ratio. It was concluded that the effective properties of indigenous poly herbal formulation are due to synergism causing greater antibiofilm characteristics against the biofilm producer strains as well as other pathogenic microorganisms. [ABSTRACT FROM AUTHOR]

Clostridium perfringens (C. perfringens) is widely distributed in broiler chickens causing clinical and subclinical enteritis and is especially known for causing necrotic enteritis (NE). There are numerous reports of NE outbreaks in Pakistan as well as China but there is a lack of information related to PFGE profile from both the countries. To close this gap, we designed this study and obtained samples from broiler chicken farms located in 3 different regions of Pakistan and 4 different regions of China. A total of 79 fecal swabs (Pakistan=29; China=50) were collected and grown on FTA media. Further, isolates were grown on TSE agar and black colonies were selected for DNA extraction. All 79 isolates were tested for toxin profiles by PCR (a-gene; beta-2; netB gene) and PFGE profiling (pulsotypes analysis). Toxinotyping results revealed that all the isolates (n=50) from China were type A (a-toxin positive) while 23 and 6 isolates (n=29) from Pakistan were type A (a-toxin positive) and type G (a-toxin, NetB positive), respectively. Toxinotyping revealed a-toxin is highly prevalent in both the countries while from Pakistani isolates, NetB toxin was also detected. PFGE discriminated 79 isolates into 45 different PFGE patterns (pulsotypes). The analysis further showed different pulsotypes originating from China and Pakistan and isolates were subtyped by SmaI. The results showed high genetic polymorphism in C. perfringens even within the same strain. These preliminary findings of genetic variations will further help to design control strategies. [ABSTRACT FROM AUTHOR]

Objective: The objective of the present study was to investigate the association of altered serum resistin levels to RETN gene (420C>G) polymorphism in women with polycystic ovarian syndrome (PCOS) and in healthy controls. Material and Methods: Eighty (40 PCOS cases and 40 healthy controls) individuals were included. Whole blood and serum samples were taken from all participants. Enzyme linked immunosorbent (ELISA) was performed for measuring the levels of serum resistin. Whole blood was used for extracting total genomic DNA by the phenol-chloroform method. Polymerase chain reaction with fragment length polymorphism was performed for detecting single nucleotide polymorphism (SNP) in the promoter region (-420 C>G) of the resistin (RETN) gene by amplifying the oligonucleotide sequence of the SNP. The amplified products were first confirmed on 2.0% agarose gel for product size, and then restriction digestion of these products was performed by using the Bpil restriction enzyme. After completion of digestion, the products were resolved on 2.5% agarose gel with a 100 bp DNA ladder, and the bands were inspected to infer genotype. Data analysis was done using SPSS software and the association between serum resistin levels and RETN genotypes was analyzed. Results: There was no significant difference (p=0.125) observed in serum resistin levels between PCOS cases (mean±SD=19.33±3.50) and healthy controls (mean±SD=13.48±1.31). The frequency of the G allele was high in PCOS cases (65%) than in controls (53.7%). The GG genotype frequency of SNP (-420 C>G) was high in PCOS cases (40%) than in controls (20%), but no association was found (p=0.148). The high serum resistin levels were significantly associated with the GG genotype in PCOS cases (p=0.027). Conclusion: High serum resistin levels are not associated with the genotypes of RETN (-420 C>G) polymorphism in PCOS women and controls, although women with PCOS had high GG genotype levels of serum resistin. Further studies with large sample size should be conducted to explore the mechanism of genetic factors in complex diseases like PCOS. [ABSTRACT FROM AUTHOR]

Objectives: MRSA isolates with vancomycin MIC of 1-2 ìg/ml have been linked with treatment failure and heteroresistant VISA phenotype. This study was aimed at comparing two screening methods i.e. GRD Etest and Vancomycin Screen agar in detection of heteroresistance. Study Design: Comparative Study. Setting: Pathology Department of Post Graduate Medical Institute, Lahore. Period: May 2014 to May 2015. Material & Methods: The present study was carried out on 41 Methicillin Resistant Staphylococcus aureus strains isolated from different clinical specimens collected from Lahore General Hospital, Lahore. After screening for methicillin resistance, vancomycin MIC was determined by standard E test. Isolates with a vancomycin MIC of 1-2 ìg/ml were screened for heteroresistance by Glycopeptide Resistance Detection (GRD) E-test and Vancomycin screen agar. Data was entered and analyzed by using SPSS version 20.0. Results: When compared with E test GRD, Vancomycin screen agar (V3) showed 100% sensitivity with a 95% CI 39.76% to 100% and the specificity was 65 % with a 95 % CI 47.46% to 79.79%. Its PPV was 23% and NPV was 100% with an overall diagnostic accuracy of 68%. When compared with E test GRD, Vancomycin screen agar (V4) showed a sensitivity of 75% with a 95% CI 19.41% to 99.37% and a specificity of 86.47% with a 95% CI 71.91 to 95.59%. Its PPV was 37.5% and NPV predictive value was 96.96% with an overall diagnostic accuracy of 85.36%. Conclusion: In developing countries like Pakistan, where E tests are costly and difficult to use in routine laboratories, a screening test, which does not miss heteroresistant VISA may be of clinical use. [ABSTRACT FROM AUTHOR]

Rice bacterial leaf blight is an injurious disease among all disquieting problems of rice in Pakistan. The key and main procedure for its control is identification and characterization of its causal agent. To achieve the goals, a comprehensive surveillance was made to collect infected samples. Associated bacteria were isolated from these diseased specimens by direct plating method. Then cultures were subjected for purification and morphological characterization. Their colonies were small, medium and large. The shapes of colonies were filamentous, circular and irregular on Nutrient Agar (N.A) media. Their elevation was convex, raised and their edges were entire and undulate. The color of colonies was yellow, pale yellow, light orange, off white, creamy and reddish. Most of the colonies were smooth. [ABSTRACT FROM AUTHOR]

Emergence of plasmid mediated colistin and extended spectrum β-lactamases (ESBL) resistant genes has been impacted the efficacy of colistin and β-lactams drugs like 3rd, 4th generation cephalosporin. Current study was aimed to investigate antimicrobial resistance genes (ARGs) among Escherichia coli isolates from meat producing commercial broilers in Pakistan. Two hundred (n=200) fecal samples were collected during January-2018 to August-2019. For isolation of E. coli, pink colonies on MacConkey agar were transferred to EMB agar. Metallic sheen color colonies were tested biochemically using API-20E kit. The molecular identification of E. coli (n=153) was targeted by amplification of uid gene through polymerase chain reaction (PCR) and different ARGs i.e. gentamicin, streptomycin, tetracycline, colistin, β-lactams drugs, quinolone and ampicillin followed by sequence analysis. Genotypically, followed by phenotypically of resistant ARGs of isolated PCR-confirmed E. coli (153) shoed resistant against gentamicin (aac(3)-IV), streptomycin (aadA1), tetracycline (tetA), colistine (mcr-1), ampicillin (bla-TEM) and bla-CTX-M were 86%, 88%, 86%, 88%, 83% & 77% respectively. 33/38 (86%) of the isolate was positive for quinolone resistance. Colistine (mcr-1), ESBLs (bla-TEM) and (bla-CTX-M) resistance genes were 88%, 83% and 77% respectively. About 33 isolated E. coli harbored the both mcr-1 and ESBLs genes. All of E. coli isolates were found sensitive to ceftriaxone (CTX-30) and imipenem (IMP-10). The Isolated E. coli showed single or multi clade decadency. The E. coli and ARGs sequences showed single or multi clade decadency. This is first comprehensive study from Pakistan that described the molecular evidences of ARGs and their co-existence in single isolates originated from commercial poultry. Commercial chicken (Broilers) can act as melting pot of antibiotic resistance genes for human being. It is alarming situation for surveillance of antibiotic resistance program because of more regulated prescription of antimicrobial agents in Pakistan.

The mycelial growth and primordial initiation of the strains of Agaricus bitorquis and other species isolated from different areas of Pakistan, and their comparison was investigated with cultivated strains including AS60, AS61, AS65, AS51, W20, W2F, K26, and K32. In contrast to the most important and widely cultivated mushroom, Agaricus bisporus, which grows best at 25°C and produces fruiting bodies below 20°C, the tropical Agaricus species show optimum mycelial growth around 30°C. Comparison of the growth rates of different isolates showed that most of the strains grew within the range of 20-30°C with the optimum at 30°C. Most of the strains formed primordia at both 25° and 30°C whereas; W2-F and K32 produced areas of dense hyphal aggregates at 30°C. A. bisporus (AS51) failed to form pin heads at either. temperature. Strain AS60 and W20 initiated most of the pin heads at 30°C. Strain AS60 formed pin heads at both the temperatures with the maximum number at 30°C after full colonization of the agar surface. [ABSTRACT FROM AUTHOR]

Clostridium perfringens is a causative agent of enteric infections in animals including poultry by producing twenty different types of toxins. A single strain produces only a subset of these toxins, which form the basis of its classification into seven toxinotypes (A-G). C. perfringens toxinotype A is a widespread cause of necrotic enteritis (NE) in poultry. The current study was conducted to determine the prevalence of different toxins and antimicrobial susceptibility of C. perfringens isolated from Pakistan NE affected poultry. A total of 134 intestinal samples of the diseased birds were collected postmortem and processed for isolation of C. perfringens using tryptose sulphite cycloserine (TSC) agar supplemented with d -cycloserine. Isolates were confirmed by Gram's staining, biochemical and molecular analyses. Toxinotyping was performed by multiplex PCR. Antimicrobial susceptibility profile of isolates was performed by Kirby Bauer disc diffusion method. A total of 34 strains of C. perfringens were isolated from 134 samples with prevalence rate of 25.37%. All the isolated strains were toxinotype A, as they were positive for alpha toxin (CPA) and negative for other tested toxins such as beta (CPB), epsilon (ETX), iota (ITX), enterotoxin (CPE), toxin perfringens large (TpeL) and necrotic B-like toxin (NetB). Interestingly, all the isolated strains of C. perfringens were multidrug resistant. The highest resistance was observed against Neomycin, Trimethoprim, Tetracycline and Lincomycin which are routinely used at Pakistan poultry production. C. perfringens toxinotype A is prevalent in Pakistan poultry. Incidence of C. perfringens with prevalence rate of 25.37% can pose serious threat to Pakistan's poultry industry given that all the isolated strains were multidrug resistant. Our findings highlight the need for new antibiotics and antibiotic alternatives to overcome multidrug resistance. • Clostridium perfringens is 25.37% prevalent in Pakistan poultry. • All the C. perfringens strains isolated from poultry were toxinotype A. • All the C. perfringens strains were multidrug resistant. • The highest antibiotic resistance was observed against Neomycin, Trimethoprim, Tetracycline and Lincomycin. [ABSTRACT FROM AUTHOR]

Lentinus squarrosulus (Mont.) Singer, a wild edible macrofungus, possesses confirmed organoleptic, nutritional, and medicinal attributes. During the exploration of fungal biota of Punjab, Pakistan, L. squarrosulus was collected under the Morus alba from University of the Punjab, Lahore. For the domestication of this wild significant species, its culturability and cultivation potential was assessed by using different synthetic culture media and substrates. Among these different media used, maximum cultural growth was observed on the malt-extract agar (MEA) medium at 35°C followed by the potato-dextrose agar (PDA), compost extract agar (CEA), glucose-peptone agar (GPA), and Sabouraud-dextrose agar (SDA). Cultured strains on MEA medium were used to produce spawning material on wheat, sorghum, and barley grains. Sorghum grains at 35°C were found to be the best combination for spawn production of this fungus. Yield and biological efficiency were investigated on a variety of substrates. A mixed substrate of wheat straw and tea waste at 30°C showed the optimum yield. Tea-waste medium was used as the casing material and proved very effective. Mineral analysis of the wild and cultivated strain was carried out and revealed that both forms were enriched with macronutrients. These results showed that the L. squarrosulus strain from Pakistan possesses good nutrient and growth potential and its domestication can compete with nutritional and medicinal properties of commonly growing mushrooms.

Abstract: This study was carried out to evaluate the prevalence of mycobiota and aflatoxin (AF) B1 contamination in sesame seeds from rainfed and irrigated zones of the Punjab, Pakistan. For this purpose, 100 sesame seed samples were collected directly from the fields of major sesame-producing areas in rainfed and irrigated zones. The agar plate method was used for isolation of mycobiota, and thin-layer chromatography was used to determine AFB1 concentrations. Seed samples were then stored for 12 months. After 12 months, the seeds were again analyzed for mycobiota and AFB1 for comparison. All samples were positive for fungal growth under fresh and stored conditions. Twenty-one fungal species of 10 genera were isolated. Aspergillus flavus was the most prevalent contaminant found in fresh and stored sesame seeds from rainfed and irrigated zones, followed by Aspergillus niger, Alternaria alternata, and Fusarium oxysporum. The least prevalent fungi were Aspergillus ochraceus and Cladosporium oxysporum. Analysis revealed that 92% of fresh and 99% of stored seed samples were contaminated with AFB1. In the rainfed zone, 88% of fresh and 100% of stored seed samples were contaminated with AFB1, with mean concentrations of 15.74 and 33.8 ppb, respectively. In the irrigated zone, 96% of fresh and 98% of stored seed samples were contaminated with AFB1, with mean concentrations of 20.5 and 27.56 ppb, respectively. AFB1 concentrations >20 ppb were found in 20% of fresh and 100% of stored seeds samples from the rainfed zone and in 28% of fresh and 60% of stored samples from the irrigated zone and thus were not fit for human consumption as per the maximum limit (20 ppb) assigned by the U.S. Food and Drug Administration and the Food and Agriculture Organization of the United Nations. This report is the first on the mycobiota and AFB1 contamination in sesame seeds from rainfed and irrigated zones of the Punjab, Pakistan. These baseline data are an initial step in the effort to deal with this significant food safety issue., (Copyright ©, International Association for Food Protection.)

Frequency of extensively drug-resistant tuberculosis in Pakistan increased from 1.5% in 2006 to 4.5% in 2009 (p<0.01). To understand the epidemiology, we genotyped selected strains by using spoligotyping, mycobacterial interspersed repetitive units-variable number of tandem repeats, and IS6110 restriction fragment length polymorphism analysis. [ABSTRACT FROM AUTHOR]