Molecular Characterization of Bacterial Isolates from Soil Samples and Evaluation of their Antibacterial Potential against MDRS.

Some soil microbes, with their diverse inhabitance, biologically active metabolites, and endospore formation, gave them characteristic predominance and recognition among other microbial communities. The present study collected ten soil samples from green land, agricultural and marshy soil sites of Khyber Pakhtunkhwa, Pakistan. After culturing on described media, the bacterial isolates were identified through phenotypic, biochemical and phylogenetic analysis. Our phylogenetic analysis revealed three bacterial isolates, A6S7, A1S6, and A1S10, showing 99% nucleotides sequence similarity with Brevibacillus formosus, Bacillus Subtilis and Paenibacillus dendritiformis. The crude extract was prepared from bacterial isolates to assess the anti-bacterial potential against various targeted multidrug-resistant strains (MDRS), including Acinetobacter baumannii (ATCC 19606), Methicillin-resistant Staphylococcus aureus (MRSA) (BAA-1683), Klebsiella pneumoniae (ATCC 13883), Pseudomonas aeruginosa (BAA-2108), Staphylococcus aureus (ATCC 292013), Escherichia coli (ATCC25922) and Salmonella typhi (ATCC 14028). Our analysis revealed that all bacterial extracts possess activity against Gram-negative and Gram-positive bacteria at a concentration of 5 mg/mL, efficiently restricting the growth of E. coli compared with positive control ciprofloxacin. The study concluded that the identified species have the potential to produce antimicrobial compounds which can be used to control different microbial infections, especially MDRS. Moreover, the analysis of the bacterial extracts through GC-MS indicated the presence of different antimicrobial compounds such as propanoic acid, oxalic acid, phenol and hexadecanoic acid.

Comparative in vitro activity of selected antibacterial agents against Escherichia coli isolated from hospitalized patients suffering UTI.

Escherichia coli is the most studied among those bacteria causing urinary tract infections. This study was aimed to find out antibacterial activity and minimum inhibitory concentration of selected antibacterial agents against E. coli isolates of hospitalized UTI patients. The specimens were inoculated on Eosin Methylene Blue medium. E. coli isolates were identified via colonial morphology, biochemical testing and API-20 kit. The susceptibility pattern of antibacterial agents was determined applying disc diffusion method (Kirby-Bauer) and dilution tube method. Among all, 38.82% (n=158/407) specimens were positive for E. coli, while the rest showed either no growth or exhibited colonies other than E. coli. while observing the susceptibility pattern, Imipenem was found the most effective (73.42%) antibacterial agent, followed by nitrofurantoin (52.53%), cefpirome (44.94%) and tazobactam/ piperacillin (44.94%), whereas the E. coli isolates were highly resistant to sulfamethoxazole/trimethoprim (71.52%), followed by Amoxicillin-clavulanic acid (67.72%), nalidixic acid (66.46%) and Tobramycin (62.03%), when tested by disc diffusion method. The isolates were susceptible to cefpirome (39.87%) and tobramycin (39.87%) and resistant to sulfamethoxazole/trimethoprim (75.32%), followed by levofloxacin (61.39%), when tested by tube dilution method. The study concluded high degree of resistance against Sulfamethoxazole/trimethoprim, in contrast, cephalosporin and Imipenem exhibited good potency which can be recommended for UTI.

The evaluation of antibiotic susceptibility pattern and associated risk factors of UTI in tertiary care hospital of Peshawar.

UTIs are majorly caused by species of bacteria in patients of almost all ages. The study was aimed to determine the prevalence rate of uropathogens, its antibiotic susceptibility pattern and associated risk factors. Urine samples were collected from n=470 participants using sterilized containers and were inoculated on culture media. The isolates were identified via gram-staining and biochemical characterization. A total of 43.20% samples were positive. Female contributed the highest prevalence rate, 78.82% as compared to male, 21.18%. The highest prevalence 40.90% was observed in the age-group 31-45, followed by 16-30 with 36.90%. Escherichia coli (47.80%) was the most prevalent, followed by Klebsiella pneumoniae (18.2%), Enterococcus faecalis (12.80%), Pseudomonas aeruginosa (10.30%) and Proteus mirabilis (7.40%). Staphylococcus aureus showed high sensitivity (100%) to amikacin, meropenem, imipenem, fosfomycin, vancomycin, clindamycin and linezolid while in case of E. faecalis, vancomycin and linezolid were highly potent. Amikacin and meropenem showed the highest (100%) potency followed by imipenem While Fosfomycin was highly potent to E. coli, K. pneumoniae, P. mirabilis and P. aeruginosa with potency rate 89.97%, 92.31%, 100% and 100% respectively. In the current study, the positivity rate was highly observed in female. E. coli and K. pneumoniae were found the most ubiquitous for UTI.

Molecular detection of Shiga toxin-producing Escherichia coli (STEC) O157 in sheep, goats, cows and buffaloes.

BACKGROUND: Shiga toxin-producing E. coli (STEC) are important foodborne pathogens that causing serious public health consequences worldwide. The present study aimed to estimate the prevalence ratio and to identify the zoonotic potential of E. coli O157 isolates in slaughtered adult sheep, goats, cows and buffaloes. MATERIALS AND METHODS: A total of 400 Recto-anal samples were collected from two targeted sites Rawalpindi and Islamabad. Among them, 200 samples were collected from the slaughterhouse of Rawalpindi included sheep (n = 75) and goats (n = 125). While, 200 samples were collected from the slaughterhouse of Islamabad included cows (n = 120) and buffalos (n = 80). All samples were initially processed in buffered peptone water and then amplified by conventional PCR. Samples positive for E. coli O157 were then streaked onto SMAC media plates. From each positive sample, six different Sorbitol fermented pink-colored colonies were isolated and analyzed again via conventional PCR to confirm the presence of rfbE O157 gene. Isolates positive for rfbE O157 gene were then further analyzed by multiplex PCR for the presence of STEC other virulent genes (sxt1, stx2, eae and ehlyA) simultaneously. RESULTS: Of 400 RAJ samples only 2 (0.5%) showed positive results for E. coli O157 gene, included sheep 1/75 (1.33%) and buffalo 1/80 (1.25%). However, goats (n = 125) and cows (n = 120) found negative for E. coli O157. Only 2 isolates from each positive sample of sheep (1/6) and buffalo (1/6) harbored rfbE O157 genes, while five isolates could not. The rfbE O157 isolate (01) of sheep sample did not carry any of STEC genes, while the rfbE O157 isolate (01) of buffalo sample carried sxt1, stx2, eae and ehlyA genes simultaneously. CONCLUSION: It was concluded that healthy adult sheep and buffalo are possibly essential carriers of STEC O157. However, rfbE O157 isolate of buffalo RAJ sample carried 4 STEC virulent genes, hence considered an important source of STEC infection to humans and environment which should need to devise proper control systems.

Comparative Effect of Fertilization Practices on Soil Microbial Diversity and Activity: An Overview.

Continuously increasing human population demands increased food production, which needs greater fertilizer's input in agricultural lands to enhance crop yield. In this respect, different fertilization practices gained acceptance among farmers. We reviewed effect of three main fertilization practices (Conventional-, Organic-, and Bio-fertilization) on soil microbial diversity, activity, and community composition. Studies reported that over application of inorganic fertilizers decline soil pH, change soil osmolarity, cause soil degradation, disturb taxonomic diversity and metabolism of soil microbes and cause accumulation of extra nutrients into the soil such as phosphorous (P) accumulation. On the contrary, organic fertilizers increase organic carbon (OC) input in the soil, which strongly encourage growth of heterotrophic microbes. Organic fertilizer vermicompost application provides readily available nutrients to both plants as well as microbes and encourage overall microbial number in the soil. Most recently, role of beneficial bacteria in long-term sustainable agriculture attracted attention of scientists towards their use as biofertilizer in the soil. Studies documented favorable effect of biofertilization on microbial Shannon, Chao and ACE diversity indices in the soil. It is concluded from intensive review of literature that all the three fertilization practices have their own way to benefit the soil with nutrients, but biofertilization provides long-term sustainability to crop lands. When it is used in integration with organic fertilizers, it makes the soil best for microbial growth and activity and increase microbial diversity, providing nutrients to soil for a longer time, thus improving crop productivity.

Antibiogram of ESBL and MBL producing Pseudomonas aeruginosa among the population of Hazara division, KPK, Pakistan.

OBJECTIVE: To investigate the frequency rate and sensitivity pattern of extended-spectrum beta-lactamase and metallobeta- lactamase producing Pseudomonas aeruginosa isolated from major hospitals. METHODS: The cross-sectional study was conducted in the Microbiology section of the Pathology Department of Ayub Medical College, Abbottabad, Pakistan, from September 2017 to April 2018, and comprised clinical samples collected from different medical wards of major hospitals in the study area. For the selective growth of Pseudomonas aeruginosa, Cetrimide agar was used, and different antibiotics were evaluated for the sensitivity pattern following Kirby-Bauer diffusion method. Pseudomonas aeruginosa producing extended-spectrum beta lactamase and metallo-beta-lactamase were identified through double disk synergy test and imipenem ethylenediaminetetraacetic acid tests respectively. Patient's demographic and medical history was noted on a proforma. Data was analysed using SPSS 22.0. RESULTS: Of the 242 samples screened, 46 (19%) were positive for Pseudomonas aeruginosa. These samples were highly sensitive to levofloxacin, amikacin, imipenem, meropenem and ciprofloxacin (p<0.05). Of the positive cases, 11 (23.91%) were detected for extended-spectrum beta-lactamase production, while 3 (6.52%) samples were detected for metallo-beta-lactamase production. CONCLUSIONS: Pseudomonas aeruginosa samples were widely resistant to most antibiotics, but were sensitive for some antibiotics which may be recommended by physicians when treating Pseudomonas aeruginosa infection.

Ichthyenterobacterium magnum gen. nov., sp. nov., a member of the family Flavobacteriaceae isolated from olive flounder (Paralichthys olivaceus).

A novel marine bacterium isolated from the intestine of cultured flounder (Paralichthys olivaceus) was studied by using a polyphasic taxonomic approach. The isolate was Gram-stain-negative, pleomorphic, aerobic, yellow and oxidase- and catalase-negative. Phylogenetic analysis of 16S rRNA gene sequences indicated that isolate Th6(T) formed a distinct branch within the family Flavobacteriaceae and showed 96.6% similarity to its closest relative, Bizionia hallyeonensis T-y7(T). The DNA G+C content was 29 mol%. The major respiratory quinone was MK-6. The predominant fatty acids were iso-C(15 : 1) G, iso-C(15 : 0), iso-C(15 : 0) 3-OH, iso-C(17 : 0) 3-OH and summed feature 3 (C(15 : 1)ω6c and/or C(16 : 1)ω7c). On the basis of the phenotypic, chemotaxonomic and phylogenetic characteristics, the novel bacterium has been assigned to a novel species of a new genus for which the name Ichthyenterobacterium magnum gen. nov., sp. nov. is proposed. The type strain is Th6(T) ( = JCM 18636(T) = KCTC 32140(T)).

Flavirhabdus iliipiscaria gen. nov., sp. nov., isolated from intestine of flounder (Paralichthys olivaceus) and emended descriptions of the genera Flavivirga, Algibacter, Bizionia and Formosa.

A Gram-stain-negative, orange-coloured, rod-shaped bacterium, designated strain Th68(T), was isolated from the intestine of flounder (Paralichthys olivaceus). The isolate required sea salts for growth. Gliding motility was not observed. Flexirubin-type pigments were present. 16S rRNA gene sequence analysis indicated that strain Th68(T) represented a distinct phyletic line within the family Flavobacteriaceae with less than 96.1% similarity to members of the recognized genera of the family. The DNA G+C content was 33.0 mol%. The major fatty acids were iso-C(15 : 0), iso-C(15 : 1) G, iso-C(17 : 0) 3-OH and iso-C(15 : 0) 3-OH. The major polar lipids were phosphatidylethanolamine, two unidentified aminolipids and two unidentified polar lipids. Menaquinone 6 (MK-6) was the only respiratory quinone. On the basis of the phenotypic, chemotaxonomic and phylogenetic data, strain Th68(T) represents a novel species of a new genus in the family Flavobacteriaceae , for which the name Flavirhabdus iliipiscaria gen. nov., sp. nov. is proposed. The type strain of Flavirhabdus iliipiscaria is Th68(T) ( = JCM 18637(T) = KCTC 32141(T)).

Biological Transformation of Zearalenone by Some Bacterial Isolates Associated with Ruminant and Food Samples.

Zearalenone (ZEA) is a secondary metabolite produced by Fusarium spp., the filamentous fungi. Food and feed contamination with zearalenone has adverse effects on health and economy. ZEA degradation through microorganisms is providing a promising preventive measure. The current study includes isolation of 47 bacterial strains from 100 different food and rumen samples. Seventeen isolates showed maximum activity of ZEA reduction. A bacterial isolate, RS-5, reduced ZEA concentration up to 78.3% through ELISA analysis and 74.3% as determined through HPLC. Ten of the most efficient strains were further selected for comparison of their biodegradation activity in different conditions such as incubation period, and different growth media. The samples were analyzed after 24 h, 48 h, and 72 h of incubation. De Man Rogosa Sharp (MRS) broth, Tryptic soy broth, and nutrient broth were used as different carbon sources for comparison of activity through ELISA. The mean degradation % ± SD through ELISA and HPLC were 70.77% ± 3.935 and 69.11% ± 2.768, respectively. Optimum reducing activity was detected at 72 h of incubation, and MRS broth is a suitable medium. Phylogenetic analysis based on 16S rRNA gene nucleotide sequences confirmed that one of the bacterial isolate RS-5 bacterial isolates with higher mycotoxin degradation is identified as Bacillus subtilis isolated from rumen sample. B05 (FSL-8) bacterial isolate of yogurt belongs to the genus Lactobacillus with 99.66% similarity with Lactobacillus delbrukii. Similarly, three other bacterial isolates, D05, H05 and F04 (FS-17, FSL-2 and FS-20), were found to be the sub-species/strains Pseudomonas gessardii of genus Pseudomonas based on their similarity level of (99.2%, 96% and 96.88%) and positioning in the phylogenetic tree. Promising detoxification results were revealed through GC-MS analysis of RS-5 and FSL-8 activity.

Distribution of Toxoplasma gondii IgM and IgG antibody seropositivity among age groups and gestational periods in pregnant women.

Background: Toxoplasmosis is a globally distributed parasitic disease. The present study aimed to estimate the prevalence and geographic distribution of toxoplasmosis, as well assess the risk of animal contact in disease development and determine the percentage of toxoplasmois-associated IgM and IgG seropositivity among different age groups. In addition, it aimed to estimate the proportion of toxoplasma IgM seropositivity among pregnancy trimesters. Methods: A total of 500 pregnant women were included in this study. From each participant, a 5-ml venous blood sample was collected and centrifuged to obtain serum that was tested for Toxoplasma gondii IgM and IgG antibodies using immunochromatographic testing and ELISA. Results: The overall seroprevalence of toxoplasmosis was 24.8%, with rates of acute infection of 8%. Among positive cases in every trimester, 54.34% of first trimester positive cases had a serologic marker of acute toxoplasmosis. Out of the 40 pregnant women with previous history of cow/buffalo contact and toxoplasmosis, 40% were seropositive for toxoplasma IgM; and out of 30 women with prior history of dog contact, 16.66% had serological marker of acute toxoplasmosis. Conclusions: In this study, there is a high prevalence of toxoplasmosis and contact with domestic animals is a risk factor for this illness. Therefore, it is necessary to test every pregnant women for toxoplasmosis and distinguish the type of infection, as well as the conduction of public health education programs to generate the awareness.

Study on awareness, knowledge, and practices towards antibiotic use among the educated and uneducated people of Khyber Pakhtunkhwa Province, Pakistan

INTRODUCTION: Antimicrobial resistance developed through the inadequate use of antibiotics; is an overriding task for global public health. OBJECTIVE: To explore awareness, knowledge, and practices, and compare the elements associated with antibiotic misuse in different University students and uneducated people of Khyber Pakhtunkhwa Province, Pakistan. METHODS: Cross-sectional study was conducted from July to December 2020 using a validated questionnaire. Data were collected from eleven different university students and uneducated people of Khyber Pakhtunkhwa, Pakistan. RESULTS: 3,600 questionnaires were completed, consisting of 56.9% Male and 43.0% Female. 1,999 (55.5%) of the antibiotic users reported through the survey used non-prescription antibiotics within a one-month study period. Out of the participants, 230 (6.3%) were uneducated or their education level was below matric rest were university students. 1999 (55.5%) reported buying Antibiotics with Medical Prescription. Most self-medicated participants (56.9%) stop taking antibiotics when they feel better. More than 90% of the respondents answered that doctors and pharmacist staff do not guide them well that how to use antibiotics. 2,171 (60.03%) respondents mistakenly believed that antibiotics improve restoration from coughs and colds. Only 720 (20%) respondents knew that antibiotics also disturb normal flora and 547 participants (15.9%) agree that unnecessary use of antibiotics causes bacterial resistance. CONCLUSION: Finding from this study may have important implications for public health policy in Khyber Pakhtunkhwa, Pakistan given the growing global resistance to antibiotics and the reported health issues related to their improper use.