DUF916 and DUF3324 in the WxL protein cluster bind to WxL and link bacterial and host surfaces.

Bacterial WxL proteins contain peptidoglycan-binding WxL domains, which have a dual Trp-x-Leu motif and are involved in virulence. It was recently shown that WxL proteins occur in gene clusters, containing typically a small WxL protein (which in the mature protein consists only of a WxL domain), a large WxL protein (which contains a C-terminal WxL domain with N-terminal host-binding domains), and a conserved protein annotated as a Domain of Unknown Function (DUF). Here we analyze this DUF and show that it contains two tandem domains-DUF916 and DUF3324-which both have an IgG-like fold and together form a single functional unit, connected to a C-terminal transmembrane helix. DUF3324 is a stable domain, while DUF916 is less stable and is likely to require a stabilizing interaction with WxL. The protein is suggested to have an important role to bind and stabilize WxL on the peptidoglycan surface, via the DUF916 domain, and to bind to host cells via the DUF3324 domain. AlphaFold2 predicts that a ß-hairpin strand from DUF916 inserts into WxL adjacent to its N-terminus. We therefore propose to rename the DUF916-DUF3324 pair as WxL Interacting Protein (WxLIP), with DUF916, DUF3324 and the transmembrane helix forming the first, second and third domains of WxLIP, which we characterize as peptidoglycan binding domain (PGBD), host binding domain (HBD), and transmembrane helix (TMH) respectively.

Bioinformatic analysis of WxL domain proteins.

The WxL domain is found on the cell surface of many bacteria, most of which are commensal gut bacteria. Its functions are generally identified as being related to virulence and/or peptidoglycan attachment, but there is so far no clear function or structure for this domain. Here, a range of bioinformatics tools were used to clarify the structure and function. These indicate that WxL domains occur in cell surface-associated gene clusters that always contain a small WxL, large WxL and DUF916 domain; and that the small and large WxL proteins have distinct structure despite sharing two conserved WxL motifs. The two WxL motifs form a hydrophobic surface buried inside the protein. The likely function of the WxL domain is to attach to bacterial peptidoglycan, forming a platform to allow associated domains in the cluster to interact with host proteins.

Kashmiri refugees at the verge of hepatitis B and C epidemic in the State of Azad Jammu and Kashmir, Pakistan.

OBJECTIVE: To determine the seroprevalence of hepatitis B and C among immigrants residing refugee camps in Muzaffarabad, Azad Kashmir, Pakistan, and to identify possible risk factors for hepatitis B virus (HBV) and hepatitis C virus (HCV) transmission. METHODS: Around 1,225 individuals inhabiting Muzaffarabad refugee camps, participated in the study. A qualitative Immuno-Chromatographic Technique was used for initial screening and PCR test was used for detection of HBV and HCV in participants. The major risk factors for HBV and HCV transmission were assessed using a questionnaire approach. RESULTS: Around 86 (7.0%) individuals were observed for hepatitis B surface antigen (HBsAg) presence, and 215 (17.5%) individuals were found positive for Anti-HCV. Only 32 (2.6%) individuals were confirmed for HBV DNA and 126 (10.3%) individuals were positive for HCV RNA after PCR. Demographically, both HBsAg and Anti-HCV were found more prevalent in female (4.4% HBsAg and 10.8% Anti-HCV) population as compared to male (2.6% HBsAg and 6.7% Anti-HCV) population. Surprisingly, the HBsAg (23.5%) and Anti-HCV (41.1%) appeared to be more frequent in the age group 62-75 years. Previous history of hepatitis in the family (p < 0.0001), blood transfusion (p = 0.0197) dental treatment (p < 0.0001) and tattooing or piercing on any part of the body (p = 0.0028) were assessed as significant risk factors in HBV and HCV transmission. CONCLUSIONS: Presence of 7.0% HBsAg and 17.5% Anti-HCV in a small fragment of the migrant population cannot be overlooked. Lack of awareness among people and negligence of health department could escalate the situation.

Kashmiri refugees at the verge of hepatitis B and C epidemic in the State of Azad Jammu and Kashmir, Pakistan

ABSTRACT OBJECTIVE To determine the seroprevalence of hepatitis B and C among immigrants residing refugee camps in Muzaffarabad, Azad Kashmir, Pakistan, and to identify possible risk factors for hepatitis B virus (HBV) and hepatitis C virus (HCV) transmission. METHODS Around 1,225 individuals inhabiting Muzaffarabad refugee camps, participated in the study. A qualitative Immuno-Chromatographic Technique was used for initial screening and PCR test was used for detection of HBV and HCV in participants. The major risk factors for HBV and HCV transmission were assessed using a questionnaire approach. RESULTS Around 86 (7.0%) individuals were observed for hepatitis B surface antigen (HBsAg) presence, and 215 (17.5%) individuals were found positive for Anti-HCV. Only 32 (2.6%) individuals were confirmed for HBV DNA and 126 (10.3%) individuals were positive for HCV RNA after PCR. Demographically, both HBsAg and Anti-HCV were found more prevalent in female (4.4% HBsAg and 10.8% Anti-HCV) population as compared to male (2.6% HBsAg and 6.7% Anti-HCV) population. Surprisingly, the HBsAg (23.5%) and Anti-HCV (41.1%) appeared to be more frequent in the age group 62-75 years. Previous history of hepatitis in the family (p < 0.0001), blood transfusion (p = 0.0197) dental treatment (p < 0.0001) and tattooing or piercing on any part of the body (p = 0.0028) were assessed as significant risk factors in HBV and HCV transmission. CONCLUSIONS Presence of 7.0% HBsAg and 17.5% Anti-HCV in a small fragment of the migrant population cannot be overlooked. Lack of awareness among people and negligence of health department could escalate the situation.

Attitude and perception towards vaccination against poliomyelitis in Peshawar, Pakistan.

OBJECTIVE: This research aimed to quantitatively assess the general public's awareness, attitude and perception of polio and its vaccination in Peshawar KPK, Pakistan. METHODS: We conducted a survey-based study to understand the surge in polio cases from 2015 to 2019 in the Peshawar city of the Khyber Pakhtunkhwa (KPK), Pakistan. A pre-tested questionnaire-based study was conducted in 2019 to assess the attitude and general perception of residents of Peshawar KPK towards polio vaccination. RESULTS: Out of 241 country-wide polio cases, 63 (26.1%) polio cases were reported in Peshawar city from 2015-2019. The questionnaire revealed that individuals between 18-30 years of age had sufficient knowledge (65.1%) about polio. Male and female participants had equal awareness (~ 43%). Participants with higher education (45.9%), those with better financial status (49.5%), individuals with children < 5 years of age (46.4%), and those who had experience of a polio patient (63.1%) had better knowledge. Participants inhabiting the central city were better aware (50.5%) of polio than individuals living in the outskirts. CONCLUSION: The data indicated that poor knowledge and negative attitudes of people towards polio vaccination are the main causes of the polio eradication program's failure. Moreover, religious beliefs, unchecked migration between the Pak-Afghan border, and lack of knowledge about polio vaccination are identified as critical barriers to polio eradication.

Characterizations of novel pesticide-degrading bacterial strains from industrial wastes found in the industrial cities of Pakistan and their biodegradation potential.

BACKGROUND: Lack of infrastructure for disposal of effluents in industries leads to severe pollution of natural resources in developing countries. These pollutants accompanied by solid waste are equally hazardous to biological growth. Natural attenuation of these pollutants was evidenced that involved degradation by native microbial communities. The current study encompasses the isolation of pesticide-degrading bacteria from the vicinity of pesticide manufacturing industries. METHODS: The isolation and identification of biodegrading microbes was done. An enrichment culture technique was used to isolate the selected pesticide-degrading bacteria from industrial waste. RESULTS: Around 20 different strains were isolated, among which six isolates showed significant pesticide biodegrading activity. After 16S rRNA analysis, two isolated bacteria were identified as Acinetobacter baumannii (5B) and Acidothiobacillus ferroxidans, and the remaining four were identified as different strains of Pseudomonas aeruginosa (1A, 2B, 3C, 4D). Phylogenetic analysis confirmed their evolution from a common ancestor. All strains showed distinctive degradation ability up to 36 hours. The Pseudomonas aeruginosa strains 1A and 4D showed highest degradation percentage of about 80% for DDT, and P. aeruginosa strain 3C showed highest degradation percentage, i.e., 78% for aldrin whilst in the case of malathion, A. baumannii and A. ferroxidans have shown considerable degradation percentages of 53% and 54%, respectively. Overall, the degradation trend showed that all the selected strains can utilize the given pesticides as sole carbon energy sources even at a concentration of 50 mg/mL. CONCLUSION: This study provided strong evidence for utilizing these strains to remove persistent residual pesticide; thus, it gives potential for soil treatment and restoration.

Attitude and perception towards vaccination against poliomyelitis in Peshawar, Pakistan

ABSTRACT OBJECTIVE: This research aimed to quantitatively assess the general public's awareness, attitude and perception of polio and its vaccination in Peshawar KPK, Pakistan. METHODS: We conducted a survey-based study to understand the surge in polio cases from 2015 to 2019 in the Peshawar city of the Khyber Pakhtunkhwa (KPK), Pakistan. A pre-tested questionnaire-based study was conducted in 2019 to assess the attitude and general perception of residents of Peshawar KPK towards polio vaccination. RESULTS: Out of 241 country-wide polio cases, 63 (26.1%) polio cases were reported in Peshawar city from 2015-2019. The questionnaire revealed that individuals between 18-30 years of age had sufficient knowledge (65.1%) about polio. Male and female participants had equal awareness (~ 43%). Participants with higher education (45.9%), those with better financial status (49.5%), individuals with children < 5 years of age (46.4%), and those who had experience of a polio patient (63.1%) had better knowledge. Participants inhabiting the central city were better aware (50.5%) of polio than individuals living in the outskirts. CONCLUSION: The data indicated that poor knowledge and negative attitudes of people towards polio vaccination are the main causes of the polio eradication program's failure. Moreover, religious beliefs, unchecked migration between the Pak-Afghan border, and lack of knowledge about polio vaccination are identified as critical barriers to polio eradication.

Characterisation of Bacteriocins Produced by Lactobacillus spp. Isolated from the Traditional Pakistani Yoghurt and Their Antimicrobial Activity against Common Foodborne Pathogens.

Lactic acid bacteria (LAB) are widely known for their probiotic activities for centuries. These bacteria synthesise some secretory proteinaceous toxins, bacteriocins, which help destroy similar or interrelated bacterial strains. This study was aimed at characterising bacteriocins extracted from Lactobacillus spp. found in yoghurt and assessing their bactericidal effect on foodborne bacteria. Twelve isolated Lactobacillus spp. were examined to produce bacteriocins by the organic solvent extraction method. Bacteriocins produced by two of these strains, Lactobacillus helveticus (BLh) and Lactobacillus plantarum (BLp), showed the most significant antimicrobial activity, especially against Staphylococcus aureus and Acinetobacter baumannii. Analysis of SDS-PAGE showed that L. plantarum and L. helveticus bacteriocins have a molecular weight of ~10 kDa and ~15 kDa, respectively. L. plantarum (BLp) bacteriocin was heat stable while L. helveticus (BLh) bacteriocin was heat labile. Both bacteriocins have shown activity at acidic pH. Exposure to a UV light enhances the activity of the BLh; however, it had negligible effects on the BLp. Different proteolytic enzymes confirmed the proteinaceous nature of both the bacteriocins. From this study, it was concluded that bacteriocin extracts from L. helveticus (BLh) can be considered a preferable candidate against foodborne pathogens as compared to L. plantarum (BLp). These partially purified bacteriocins should be further processed to attain purified product that could be useful for food spoilage and preservation purposes.

Association of Virulence Genes with Antibiotic Resistance in Pakistani Uropathogenic E. coli Isolates.

BACKGROUND: Escherichia coli various strains can cause alarmingly serious infections. Countries like Pakistan harbour the class of bacteria with one of the highest rates of resistance, but very little has been done to explore their genetic pool. OBJECTIVES: This study was designed to find out the frequency of virulence genes of Uropathogenic E. coli and their association with antibiotic resistance along with the evolutionary adaptation of the selected gene through the phylogenetic tree. METHODS: Isolates from 120 urinary tract infected patients were collected. Antibiotic sensitivity was detected by the disk diffusion method and DNA extraction was done by the boiling lysis method followed by PCR-based detection of virulence genes. The final results were analysed using the chi-square test. RESULTS: The isolates were found to be least susceptible to nalidixic acid, followed by ampicillin, cotrimoxazole, cefotaxime, ciprofloxacin, aztreonam, amoxicillin, gentamycin, nitrofurantoin and imipenem. The iucC was the most common virulence gene among the resistant isolates. About 86% of the collected samples were found to be multi-drug resistant. Statistical analysis revealed a significant association between the iucC gene and resistance to ampicillin (P=0.03) and amoxicillin (P=0.04), and also between fimH and resistance to aztreonam (P=0.03). CONCLUSION: This study unravels the uncharted virulence genes of UPEC in our community for the very first time. We report a high frequency of the iucC and fimH virulence genes. This, along with their positive association with resistance to beta-lactam antibiotics in the studied community, indicates their important role in the development of complicated UTIs.

Probiotic Properties of Lactobacillus helveticus and Lactobacillus plantarum Isolated from Traditional Pakistani Yoghurt.

Probiotic bacteria are of utmost importance owing to their extensive utilisation in dairy products and in the prevention of various intestinal diseases. The objective of this study was to assess the probiotic properties of bacteriocin-producing isolates of Lactobacillus helveticus and Lactobacillus plantarum isolated from traditional Pakistani yoghurt. In this study, ten bacteriocin-producing isolates were selected to screen for the probiotic property. The isolates showed resistance to acidic pH (6-6.5), bile salt (0.01-1%), and 1-7% NaCl salt and showed good growth at acidic pH and antibacterial activity against ten different foodborne pathogens. Interestingly, these isolates were proved to be effective against Actinobacter baumannii but least effective against Klebsiella pneumoniae and Pseudomonas aeruginosa. A few isolates were found to be resistant to some antibiotics like vancomycim, gentamycin, erythromycin, streptomycin, and clindamycin. Our results provide strong evidence in favour of traditional Pakistani yoghurts as a potential source of bacteriocin-producing bacteria with an added benefit of the probiotic property. Specifically, LBh5 was considered a good probiotic isolate as compared to other isolates used in the study. Further extensive research should be done on isolation and characterisation of probiotic isolates from local fermented foods, and then, these isolates should be used in the development of probiotic enriched food supplements in Pakistan.