Surviving under pollution stress: Antibacterial and antifungal activities of the Oyster species (Magallana bilineata and Magallana cuttackensis).

Antibiotic effectively controls the bacterial and fungal infections. Pathogens developing multi-drug resistance are a global health concern, which necessitate developing new molecules to overcome the resistance dilemma. This study explores the intrinsic ability of marine oysters synthesizing bioactive compounds. The tissue extracts prepared in n-hexane from two oysters, Magallana bilineata and Magallana cuttackensis compared for broad spectral antimicrobial activates against the fungal, Gram positive and negative pathogens. Regardless, both the species tolerated the same pollution indices; the M. bilineata exhibits stronger antimicrobial activities as compared to M. cuttackensis. M. bilineata potentially inhibited the bacterial growth with minimal inhibitory concentration (0.75-20 µg/ml) and fungal pathogens (0.75-5 µg/ml) as compared to ciprofloxacin and miconazole. Inhibitory potential complimented with reduce bactericidal and fungicidal concentrations required to observed susceptible zone of inhibition (ZOI). The inhibition augmented with increased antimicrobial index (AMI) and total activity index (TAI) against the human pathogen than those of M. cuttackensis. It is widely acknowledged that there is a need to develop novel antimicrobial agents to minimize the threat of emerging multiple antimicrobial resistant pathogens. Therefore, the oysters surviving in the pollution stress modulate the physiological and immune response may exploit to develop potential antibiotics.

Biosynthesis of Selenium Nanoparticles (via Bacillus subtilis BSN313), and Their Isolation, Characterization, and Bioactivities.

Among the trace elements, selenium (Se) has great demand as a health supplement. Compared to its other forms, selenium nanoparticles have minor toxicity, superior reactivity, and excellent bioavailability. The present study was conducted to produce selenium nanoparticles (SeNPs) via a biosynthetic approach using probiotic Bacillus subtilis BSN313 in an economical and easy manner. The BSN313 exhibited a gradual increase in Se reduction and production of SeNPs up to 5-200 µg/mL of its environmental Se. However, the capability was decreased beyond that concentration. The capacity for extracellular SeNP production was evidenced by the emergence of red color, then confirmed by a microscopic approach. Produced SeNPs were purified, freeze-dried, and subsequently characterized systematically using UV-Vis spectroscopy, FTIR, Zetasizer, SEM-EDS, and TEM techniques. SEM-EDS analysis proved the presence of selenium as the foremost constituent of SeNPs. With an average particle size of 530 nm, SeNPs were shown to have a -26.9 (mV) zeta potential and -2.11 µm cm/Vs electrophoretic mobility in water. SeNPs produced during both the 24 and 48 h incubation periods showed good antioxidant activity in terms of DPPH and ABST scavenging action at a concentration of 150 µg/mL with no significant differences (p > 0.05). Moreover, 200 µg/mL of SeNPs showed antibacterial reactivity against Escherichia coli ATCC 8739, Staphylococcus aureus ATCC 9027, and Pseudomonas aeruginosa ATCC 25923. In the future, this work will be helpful to produce biogenic SeNPs using probiotic Bacillus subtilis BSN313 as biofactories, with the potential for safe use in biomedical and nutritional applications.

Study of class 1 integrons and plasmid profile among multiple drug resistant uropathogenic Escherichia coli.

The emergence of multidrug resistance in uropathogenic Escherichia coli (UPEC) is associated with the presence of drug resistant plasmids and integrons which facilitate horizontal gene transfer which impose serious challenges in patients with urinary tract infections (UTIs). The proposed research study is designed to determine emerging antibiotic resistance trends and the presence of plasmids and class 1 integron in UPEC. A total 74 strains of urinary pathogens were procured among them 50 UPEC isolates were selected and their antibiotic resistance pattern was performed by CLSI guidelines. Plasmid DNA of UPEC strains was extracted by kit method and profiling was done using gel electrophoresis. Class 1 integron genes intI1, sul1 and qacEΔ1 were detected by multiplex PCR in UPEC. Among gram negative urinary isolates, 50 (68%) isolates were E. coli, while the rest were Klebsiella, Pseudomonas, Enterobacter etc. All the tested UPEC were totally resistant to quinolones while sensitive to fosfomycin, imipenem and colistin antibiotics. Majority of multidrug resistant UPEC showed common resistant phenotype of fluoroquinolones, cephalosporins, sulfamethoxazole/trimethoprim and aminoglycosides. Out of the 50 UPEC isolates 46 (92%) were multi-drug resistant having one to three plasmids of more than 1kb and 41 (82%) possessed class 1 integron genes. Over all association between antibiotic resistance and presence of class 1 integron genes showed statistically significant results (p<0.05). Our results also depict a strong correlation between multidrug resistance and presence of class 1 integron in UPEC isolates (p<0.05). The presence of multiple plasmid bands in MDR E. coli strains and high prevalence of class 1 integrons indicate the role of plasmids and integrons in the horizontal transmission of antibiotic resistance genes in UPEC.

Identification and growth optimization of a Marine Bacillus DK1-SA11 having potential of producing broad spectrum antimicrobial compounds.

Control of harmful bacteria in food, aquaculture, pharmaceuticals, agriculture, hospitals and recreation water pools are of great global concern. Marine bacteria are an enormous source of bio-controlling agents. The aim of this study was to identify and optimize the growth conditions including effect of different biotic and abiotic factors on antimicrobial activity of strain DK1-SA11 isolated from Qingdao Bay of China Yellow Sea. Microscopic characterization, API® 20E and 50 CHB kit base carbohydrates utilization, 16S rDNA and DNA gyrB gene sequencing studies identified the bacterium as Bacillus subtilis subsp. spizizenii DK1-SA11. Antimicrobial spectrum of cell free supernatant (CFS) has shown antimicrobial activities against all test strains including methicillin-resistant Staphylococcus aureus, E. coli O157:H7, Candida albicans, Klebsiella pneumoniae, Listeria monocytogenes, Vibrio parahaemolyticus, E. coli, Pseudomonas fluorescens, Salmonella typhimurium and Vibrio cholerae. Among all the media tested, Marine Broth 2216 was found to be the best medium for bacterial growth and production of antibacterial compounds. The other optimum conditions for growth were pH:7 and incubation temperature: 25°C with < 180 rpm for 60-72 h. Out of 49 different carbohydrates tested, D-mannose increases the antibacterial activity by 33.3% while D-arabitol decreases it by 44.4%. Crude CFS showed activity even after three months of storage below -20°C and boiling for 10 min, whereas it loses 100% of its antimicrobial activity after enzymatic treatments of lipase, trypsin and papain. The production of antimicrobial compounds and broad spectrum of antimicrobial activity against all tested pathogens suggested that the strain DK1-SA11 can be used as a source for probiotics, synbiotics and antibiotics.

Broad spectrum anti-microbial compounds producing bacteria from coast of Qingdao bays.

Anti-microbial resistance burden and hazard associated with chemical treatment of infections demanded for new anti-microbial natural products. Marine associated microorganisms are the enormous source of bioactive compounds. In this study we have isolated 272 marine bacteria among them 136 (50%) were antagonistic to at least one of the four pathogenic strains Listeria monocytogenes, Vibrio cholerae, E. coli and S. aureus. Only two strains exhibited antibacterial activity against all four test strains, which were identified by 16S rDNA sequencing as Bacillus sp. DK1-SA11 and Vibrio sp. DK6-SH8. Marine isolate DK1-SA11 has potential to resist boiling temperature and pH 2-12. Furthermore cell free extract (CFE) inhibited all test organisms including superbug MRSA and pathogenic yeast Candida albicans. Marine isolate Bacillus sp. DK1-SA11 could be a potential combatant for the battle of drugs and bugs.

Source tracking of extensively drug resistant Salmonella Typhi in food and raw vegetables using molecular approaches.

INTRODUCTION: Extensively drug resistant (XDR) strains of the Salmonella lineages have been reported to spread from Africa to South Asia. XDR strains are resistant to fluoroquinolones, chloramphenicol, co-trimoxazole, and ampicillin, resulting in treatment failure. The objectives of this study included the investigation of transmission of S. Typhi lineages and the identification of the potentially contaminated sources of the XDR typhoid outbreak from different urban areas by using molecular techniques. METHODOLOGY: Environmental samples, including food samples, were collected from different towns and the susceptibility of each isolate to the antimicrobial agents was examined. Molecular identification of different Salmonella lineages including S. Typhi, S. Paratyphi A, H58, and XDR was carried out through multiplex PCR. RESULTS AND CONCLUSIONS: A total of 328 environmental samples including raw vegetables, water, and bakery items were collected. More than half of the tested samples (64%) found harboring Salmonella spp. The Salmonella was confirmed through PCR amplification of species-specific markers that showed the presence of S. Typhi (40%), S. Paratyphi A (8%), H58 (7%), and XDR S. Typhi (6%). Raw vegetables had the highest number of Salmonella spp., indicating consumption of raw vegetables as a possible source of salmonellosis. XDR status was also affirmed through phenotypic antimicrobial susceptibility testing.

Biofilm formation and dispersal of Staphylococcus aureus under the influence of oxacillin.

A total of 180 food borne isolates of methicillin resistant Staphylococcus aureus (MRSA) (oxacillin MICs 864 µg/ml) were used in the present study to investigate the effect of oxacillin on biofilm formation and its detachment process. Majority (98.3%) of these isolates were found to carry icaA gene. Out of 180 isolates 35.5% were identified as MRSA and 64.4% were methicillin sensitive S. aureus (MSSA). Biofilm studies by con-red agar and tube methods revealed that 57% of the MRSA isolates were biofilm producers. Polymerase chain reaction studies suggested that all of the biofilm positive MRSA isolates belong to SCCmec type IV and carry agr type II. This showed the strong association of SCCmec IV agr type II and biofilm formation in food borne MRSA. Conversely, only 13.7% of the MSSA isolates were biofilm positive and majority was found to carry agr type II. It has been noticed that oxacillin has regulatory effect on icaA expression and induce the icaA dependent polysaccharide intracellular adhesin (PIA) production and biofilm formation. This was confirmed by Real Time PCR studies of MRSA and MSSA isolates. Quantitative analysis showed that most of the MRSA isolates started biofilm formation after 24 h of incubation in the presence of sub-inhibitory concentration of oxacillin and achieved highest adhesion on glass slide after 48 h. The control in the absence of oxacillin showed slow conversion from planktonic to biofilm mode of growth (Table 1). Another novel feature of most of these biofilm producing isolates is the reduction in (Optical Density) OD, which is noticed after 48 h of incubation. Possibly, after 48 h oxacillin loses its toxicity or consumed the cells re-adapt to the planktonic state, possibly, by the activation of accessory gene regulator A (agrA) which has an important role in biofilm dispersal.

Effect of vancomycin on the cytoplasmic membrane fatty acid profile of vancomycin-resistant and -susceptible isolates of Staphylococcus aureus.

This study was designed to analyze the effect of vancomycin on the cytoplasmic membrane fatty acid (FA) composition of vancomycin-resistant Staphylococcus aureus (VRSA), vancomycin-intermediate resistant S. aureus (VISA), and vancomycin-susceptible S. aureus. One low-level vancomycin-resistant isolate (LLR-VRSA) termed CP2, along with two vancomycin intermediate-resistant S. aureus isolates (VISA-CP1) and Mu50 (ATCC #700699), were studied. The LLR-VRSA isolate CP2, recovered from the blood sample of a postoperative cardiac patient, exhibited vanA type vancomycin resistance [minimum inhibitory concentration (MIC) 16 µg/ml], and the vanA cassette was located on a plasmid. CP1, isolated from the pus sample of the same patient, exhibited vancomycin intermediate resistance (MIC 8 µg/ml) in the absence of the vanA, vanB, or vanC gene. As susceptible controls, we used PSA (vancomycin MIC 2 µg/ml), which was isolated from the pus sample of a neonate, and S. aureus (ATCC# 29213). Membrane FA analysis was carried out using gas chromatography coupled with mass spectrometry. For this purpose, CP1, CP2, Mu50, and the susceptible control isolates were grown in the presence and absence of vancomycin. Comparative analysis showed an increase in the relative proportion of unsaturated FAs during growth under vancomycin stress. The isolate CP2 (LLR-VRSA) exhibited a higher MIC to vancomycin than the other isolates used in present study (16 µg/ml) and under vancomycin stress conditions, quantitatively, it showed a high rate of conversion of saturated to unsaturated membrane FAs than CP1, Mu50 (VISA isolate) and the susceptible control PSA. The rate of saturated-to-unsaturated FA conversion increased as the concentration of vancomycin in the growth media was increased. Therefore, it is concluded that S. aureus tend to modify their membrane lipid chemistry from saturated to unsaturated in order to survive in a vancomycin stress environment.

Role of extra-cellular fatty acids in vancomycin induced biofilm formation by vancomycin resistant Staphylococcus aureus.

In the present study a vancomycin resistant Staphylococcus aureus (S. aureus) (VRSA) (Labeled as CP2) was isolated from the blood of a post-operative cardiac patient is described. It harbors a plasmid which carry vanA gene and exhibited low-level vancomycin resistance (MIC 16µg/mL), was sensitive to teicoplanin. It has been observed that sub-lethal dose of vancomycin induced biofilm formation by CP2 on nylon and silicon indwelling. The results divulge new insights into associations between vancomycin induced biofilms and extra-cellular fatty acids. Gas chromatography coupled with mass spectrometry (GC-MS) revealed that biofilm matrix of CP2 contains a variety of saturated and un-saturated fatty acids, especially, diverse species of octadecanoic (C18:0) and octadecenoic acids (C18:1). A large difference in fatty acids composition was noticed in biofilms, isolated from hydrophobic and hydrophilic surfaces. CP2 produced thicker layer of biofilms on hydrophobic silicon and nylon surfaces which contains variety of saturated, un-saturated and cyclic fatty acids. Contrary to this on hydrophilic glass surfaces it produced thinner layer of biofilm which contains only straight chain saturated fatty acids. These fatty acid components seem to play a crucial role in cell-cell communication and in the establishment of biofilms, consequently, advantageous for pathogens to survive in hospital environment under enormous antibiotics pressure.

Design and in silico analysis of mRNA vaccine construct against Salmonella.

Salmonella infections are continuously growing. Causative serovars have gained enhanced drug resistance and virulence. Current vaccines have fallen short of providing sufficient protection. mRNA vaccines have come up with huge success against SARS-CoV-2; Pfizer-BioNTech and Moderna vaccines have resulted in >90% efficacy with efficient translocation, expression, and presentation of antigen to the host immune system. Herein, based on the same approach a mRNA vaccine construct has been designed and analyzed against Salmonella by joining regions of genes of outer membrane proteins C and F of S. Typhi through a flexible linker. Construct was flanked by regulatory regions that have previously shown better expression and translocation of encoded protein. GC content of the construct was improved to attain structural and thermodynamic stability and smooth translation. Sites of strong binding miRNAs were removed through codon optimization. Protein encoded by this construct is structurally plausible, highly antigenic, non-allergen to humans, and does not cross-react to the human proteome. It is enriched in potent, highly antigenic, and conserved linear and conformational epitopes. Most conserved conformational epitopes of core protein lie on extended beta hairpins exposed to the cellular exterior. Stability and thermodynamic attributes of the final construct were found highly comparable to the Pfizer-BioNTech vaccine construct. Both contain a stable stem-loop structure downstream of the start codon and do not offer destabilizing secondary structures upstream of the start codon. Given structural and thermodynamic stability, effective immune response, and epitope composition the construct is expected to provide broad-spectrum protection against clinically important Salmonella serovars.Communicated by Ramaswamy H. Sarma.

Banned Sudan dyes in spices available at markets in Karachi, Pakistan.

Sudan dyes were investigated in branded and non-branded spices, commonly available in the markets of Karachi, Pakistan. High performance liquid chromatography (HPLC) with a variable wavelength detector (VWD) was applied to determine Sudan dyes I-IV. The non-branded samples had higher concentrations of Sudan dyes than the maximum limits of 0.1 mg/kg. The highest concentration of Sudan dye (I) was found in turmeric powder (8460 mg/kg) and the lowest concentration (1.50 mg/kg) of Sudan (IV) in Chaat Masala. This indicates that the use of non-branded spices is not safe, whereas no Sudan dye was found in the branded spice samples. Further studies regarding the higher carcinogenic risk posed by Sudan dye adulterated spices in Pakistan is strongly advised.

Nanotubes Formation in P. aeruginosa.

The present study discusses a biofilm-positive P. aeruginosa isolate that survives at pH levels ranging from 4.0 to 9.0. The biofilm consortia were colonized with different phenotypes i.e., planktonic, slow-growing and metabolically inactive small colony variants (SCVs). The lower base of the consortia was occupied by SCVs. These cells were strongly attached to solid surfaces and interconnected through a network of nanotubes. Nanotubes were observed at the stationary phase of biofilm indwellers and were more prominent after applying weight to the consortia. The scanning electron micrographs indicated that the nanotubes are polar appendages with intraspecies connectivity. The micrographs indicated variations in physical dimensions (length, width, and height) and a considerable reduction in volume due to weight pressure. A total of 35 cells were randomly selected. The mean volume of cells before the application of weight was 0.288 µm3, which was reduced to 0.144 µm3 after the application of weight. It was observed that a single cell may produce as many as six nanotubes, connected simultaneously to six neighbouring cells in different directions. The in-depth analysis confirmed that these structures were the intra-species connecting tools as no free nanotubes were found. Furthermore, after the application of weight, cells incapable of producing nanotubes were wiped out and the surface was covered by nanotube producers. This suggests that the nanotubes give a selective advantage to the cells to resist harsh environmental conditions and weight pressure. After the removal of weight and proper supply of nutrients, these phenotypes reverted to normal planktonic lifestyles. It is concluded that the nanotubes are not merely the phenomenon of dying cells; rather they are a connectivity tool which helps connected cells to tolerate and resist environmental stress.

Biogenic Selenium Nanoparticles and Their Anticancer Effects Pertaining to Probiotic Bacteria-A Review.

Selenium nanoparticles (SeNPs) can be produced by biogenic, physical, and chemical processes. The physical and chemical processes have hazardous effects. However, biogenic synthesis (by microorganisms) is an eco-friendly and economical technique that is non-toxic to human and animal health. The mechanism for biogenic SeNPs from microorganisms is still not well understood. Over the past two decades, extensive research has been conducted on the nutritional and therapeutic applications of biogenic SeNPs. The research revealed that biogenic SeNPs are considered novel competitors in the pharmaceutical and food industries, as they have been shown to be virtually non-toxic when used in medical practice and as dietary supplements and release only trace amounts of Se ions when ingested. Various pathogenic and probiotic/nonpathogenic bacteria are used for the biogenic synthesis of SeNPs. However, in the case of biosynthesis by pathogenic bacteria, extraction and purification techniques are required for further useful applications of these biogenic SeNPs. This review focuses on the applications of SeNPs (derived from probiotic/nonpathogenic organisms) as promising anticancer agents. This review describes that SeNPs derived from probiotic/nonpathogenic organisms are considered safe for human consumption. These biogenic SeNPs reduce oxidative stress in the human body and have also been shown to be effective against breast, prostate, lung, liver, and colon cancers. This review provides helpful information on the safe use of biogenic SeNPs and their economic importance for dietary and therapeutic purposes, especially as anticancer agents.

Effect of sub-lethal doses of vancomycin and oxacillin on biofilm formation by vancomycin intermediate resistant Staphylococcus aureus.

Biofilms are means of protection to bacteria against antibiotics and antibodies. Catheters and others tube devices used by patients are prone to accumulation of thick layers of biofilms as hiding place for etiologic agents, resulting in substantial morbidity and mortality. Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of hospital-acquired infections. Vancomycin remains the only treatment of choice for MRSA infections. In the present study a vancomycin resistant S. aureus (VRSA) (Labeled as CP2) was isolated from the blood of a post-operative cardiac patient. It harbors a plasmid which carry vanA gene and exhibited low-level vancomycin resistance (MIC 16 µg/ml), high level of oxacillin/methicillin resistance (MIC 500 µg/ml) and was sensitive to teicoplanin. CP2 also found to carry icaA gene on its chromosome. This strain exhibited resistance to triton-X100 induced autolysis under sub-inhibitory concentration of vancomycin and produced some extracellular matrix material that surrounding the cells. These characteristic features have warranted us to study the biofilm formation by CP2 on biomedical indwellings in presence of vancomycin and oxacillin. Our findings suggest that sub-lethal dose of vancomycin induced the biofilm formation by CP2 on nylon and silicon indwellings whereas oxacillin facilitated the biofilm formation on glass surfaces exclusively. This implicates that not only the antibiotics but also the indwelling material influences biofilm formation. Therefore, these implants serve as potential surfaces for bacterial adhesion that lead to biofilm formation, thus provide hiding places for pathogens from the actions of antimicrobials.

Phylogenetic Group B2 Expressed Significant Biofilm Formation among Drug Resistant Uropathogenic Escherichia coli.

Biofilm is an important virulent marker attributed to the development of urinary tract infections (UTIs) by uropathogenic E. coli (UPEC). Drug-resistant and biofilm-producing UPEC are highly problematic causing catheter-associated or recurrent UTIs with significant morbidity and mortality. The aim of the current study was to investigate the prevalence of biofilm formation and phylogenetic groups in drug-resistant UPEC to predict their ability to cause disease. This prospective study was conducted at the Department of Microbiology, University of Karachi from January to June 2019. A total of 50 highly drug-resistant UPEC were selected for this study. UPEC isolates were screened to form biofilm by Congo-red agar (CRA) and microtiter plate (MTP) technique. The representative biofilm-producing isolates were analysed by scanning electron microscopy (SEM) monitoring. Phylogenetic analysis was done by PCR method based on two preserved genes; chuA, yjaA and TspE4-C2 DNA fragment. On CRA 34 (68%) UPEC were slime producers, while on MTP 20 (40%) were strong biofilm producers, 19 (38%) moderate and 11 (22%) were low to negligible biofilm producers. Molecular typing confirmed that phylogenetic group B2 was prevalent in drug resistant UPEC strains. Pathogenic strains belonged to phylogenetic group B2 and D were found to have greater biofilm forming ability as compare to non-pathogenic commensal strains that belonged to phylogenetic group A. Our results indicate that biofilm formation vary in drug resistant UPEC belonged to different phylogenetic groups. This study indicates possible link between in vitro biofilm formation and phylogenetic groups of UPEC, therefore this knowledge might be helpful to predict the pathogenic potential of UPEC and help design strategies for controlling UTIs.

A Size-dependent Bioaccumulation of Metal Pollutants, Antibacterial and Antifungal Activities of Telescopium telescopium, Nerita albicilla and Lunella coronata.

We assessed metal/metalloid pollutants (through multi-indices) in seawater, sediments, tissues and shells of gastropods using various indices such as contamination degree (modified/unmodified; Cd/mCd; 1875/187.5). From sediment indices; e.g. the potential ecological risk index/enrichment factor (Eri/EF; 3396.8/105.5) indicated the area to be highly contaminated with metal/metalloid pollutants. Indeed, bioaccumulation with these materials was gastropod size dependent. Antimicrobial and percentage activity indices (AMI/PAI) for; T. telescopium was (AMI/PAI; 1.59/159), N. albicilla (1.14/114) and L. coronata (0.95/95) against E. coli. Similarly T. telescopium (1.33/133), N. albicilla (1.19/119) and L. coronata (1.14/114) have AMI/PAI against A. terreus. The total activity index (TAI), for T. telescopium was the highest, while L. coronata has lowest for all pathogens. This study indicates, T. telescopium, N. albicilla and L. coronata, surviving under metal/metalloid stress exhibited altered natural defense to pathogens which was related to the degree of toxin bioaccumulation.

Evaluation of heavy metal pollutants in salt and seawater under the influence of the Lyari River and potential health risk assessment.

Heavy metals intoxication through edible salt poses serious health hazards. The conducted research assessed the levels of heavy metals, health risks of salt samples, and the pollution index of seawater obtained from saltpans located at Sandpit, Karachi, Pakistan, which receive untreated effluents through the Lyari River. Seawater (n = 27) and salt samples (n = 27) were prepared for the detection of Al, Cd, Pb, Cr, Fe, Cu, Hg, Ni, As and Zn through atomic absorption spectroscopy, mean concentrations ± S.D. (mg/mL) were compared with the National Environmental Quality Standards, Pakistan. The levels of Cr (40.06 ± 0.21) were the highest followed by Fe (39.77 ± 0.08) > As (25.12 ± 0.21) > Ni > Pb > Al > Hg > Zn > Cd > Cu. In sea salt (Mean ± S·D mg/Kg), the Cr (47.79 ± 0.19), Fe (47.5 ± 0.15), As (30.62 ± 0.22), and Ni were redundant elements followed by Al > Pb > Hg > Zn > Cu > Cd. The water comprehensive pollution index was 1000 times greater than the critical values. The single factor pollution index was highest for Hg (1321), followed by Cr (40), Ni (36), Pb (32), Al (31.4), Cd (31.3), and As (25). Health risk assessment indices (from salt samples), including target hazard quotient (THQ) for As, Pb, Al, Hg, and Cr were two to six times higher than the noncarcinogenic THQ reference range. Similarly, the hazardous index indicated that salt was 20 times hazardous (HI = 20.29), and the carcinogenic rate index for the heavy metals i.e., Cd, As, Cr, and Ni was above the reference CR value i.e., 1 × 10-4. In conclusion, the experimental and theoretical approaches recommend that the use of contaminated salt may impart health hazards.

Correlation between antibiotic resistance and phylogenetic types among multidrug-resistant Escherichia coli isolated from urinary tract infections.

OBJECTIVES: Emergence of multidrug resistance has reduced the choice of antimicrobial regimens for UTIs. To understand the association of phenotype and genotype among uropathogens. MATERIALS AND METHODS: Six hundred and twenty-eight (628) urine samples were collected and analyzed. Antibiotic sensitivity pattern was determined by the Kirby-Bauer Disc Diffusion Method and minimum inhibitory concentration (MIC) was tested by the E test. Fluoroquinolone resistant mutations in QRDR of gyrA and ParC, phylogenetic groups, and PAIusp subtype were detected by PCR. RESULTS: Most prevalent uropathogens were Escherichia coli (53.2%) followed by Klebsiella pneumoniae (21%). Multidrug- resistance was observed in > 50% cases for third-generation cephalosporins and ciprofloxacin and lowest in meropenem. E. coli (66.2%) and K. pneumonia (64.4%) were extended-spectrum ß-lactamases (ESBLs) producers. MIC to trimethoprim-sulfamethoxazole was highest in E. coli (>1024 µg/ml). In 80 (24%) of the 334 E. coli isolates analyzed in detail, 54 fluoroquinolones (FQ) resistant isolates carried mutations (S83L, D87N, S80I, E84V) in QRDR of gyrA and ParC. Out of 54 FQ-resistant isolates, 43 (79.6%) isolates belonged to the phylogenetic group B2, and 11(20.4%) belonged to group D. Isolates belonged to group B2, 38 (88.4%) of the 43 isolates carried PAIusp subtype IIa and high frequency of mutation E84V in ParC was detected in 37 (97.4%). Other mutations, such as S80I, S83L in gyrA and D87N in ParC were found in all resistant isolates. CONCLUSION: Correlations between phenotype and genotype provided a basis to understand the resistance development in uropathogens, and PAIusp subtyping indicated that E. coli belonged to the B2 group.

Characterization of a vancomycin intermediate-resistant Staphylococcus aureus isolated from a hospital.

A Vancomycin intermediate resistant strain of Staphylococcus aureus (S. aureus) labeled as CP2 (MIC 16 microg/ml) was isolated from an in-patient of local Cardiac Hospital of Karachi. CP2 showed typical characters of Vancomycin intermediate resistant S. aureus (VISA) i.e. high level of oxacillin resistance, thickened cell wall with rough surface and reduced autolytic activities associated with murein hydrolase (MH) enzyme. This strain may have acquired vancomycin resistance due to long term exposure to antibiotic during the treatment of the patient. Therefore, it implicates the importance of monitoring the usage and also to control of the abuse of antibiotics for prevention of any further proliferation of this type of notorious bugs.

An effective weapon against biofilm consortia and small colony variants of MRSA.

OBJECTIVES: This study was designed to investigate the effect of AgNPs (10 nm and 30 nm) on different phenotypes of Staphylococcus aureus biofilm consortia. MATERIALS AND METHODS: A total of eighteen biofilm-producing isolates of Methicillin-Resistant S. aureus (MRSA) were used in the present study. Tube methods, Congo-red agar method, and scanning electron microscopy (SEM) were used to study biofilm phenotypes. Population analysis assay on a tryptone soya agar (TSA) plate was applied to study the different phenotypes of biofilm consortia. The effect of AgNPs was evaluated by broth dilution assay. RESULTS: Results showed that biofilm consortia harbour different phenotypes, i.e., planktonic, metabolically inactive cells, and small colony variants (SCVs) or persister cells. The focus of the present study is the effect of AgNPs on biofilm consortia of MRSA, particularly on the SCVs population. Large size AgNPs (30 nm) were unable to diffuse through extracellular matrix material coverings of the biofilm consortia; they were only active against the planktonic population that occupies the outer surface of consortia. The smaller AgNPs (10 nm), on the other hand, were found to diffuse through the matrix material and hence were effective against planktonic as well as metabolically inactive population of consortia. Moreover, 30 nm AgNPs take 6 hr to disperse off and kill planktonic and upper surface indwellers. The 10 nm AgNPs disperse and kill the majority of biofilm indwellers within 20 min. CONCLUSION: The present study showed that 10 nm AgNPs can easily penetrate inside the biofilm and are active against all of the indwellers of consortia.