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.

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.

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.

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.

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.

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.

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.

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.

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.

Enhanced modulation of gut microbial dynamics affecting body weight in birds triggered by natural growth promoters administered in conventional feed.

This study explored the effects of natural growth promoters (phytogenic feed additives and organic acids) on animal performance, carcass characteristics, blood parameters, gut microflora composition, and microbe-host interactions in broiler chickens over a 42-day feeding period. Two-hundred-fifty-day-old chicks were randomly assigned to one of five treatments: (i) control diets (CON); (ii) control diets + 40 g/tons antibiotic growth promoter (AB); (iii) control diets + 3 kg/tons organic acids (ORG); (iv) control diets + 3 kg/tons phytogenic feed additives (PHY); (v) control diets + 3 kg/tons organic acids + phytogenic feed additive combination (COM). A non-significant differences (p > 0.05) were observed in broiler performance among treatments at 21 days of age; however, a gradually increasing body weight gain and reduced feed conversion ratio were observed at 42 days in treatments versus control group. Biochemical indices were non-significant (p > 0.05) except for decreased cholesterol (p < 0.05) and increased A/G ratio (p < 0.05) recorded in the treatment groups. The addition of PHY and ORG improved total counts of Enterococcus spp. and Lactobacillus spp. (p < 0.05) as well as reduced caecal and ileal Campylobacter spp. and Escherichia coli (p < 0.05). Correlation analysis elucidated beneficial bacteria (Enterococcus spp. and Lactobacillus spp.) were positively and pathogenic bacteria (Campylobacter spp. and E. coli) were negatively correlated (p < 0.05) with host weight gain. The findings indicated that dietary supplementation of PHY and ORG sustained balanced gut microflora, which in turn improved body weight. This study broadens the significance of using PHY and ORG as safe alternatives to antibiotic growth promoters for achieving healthier and economical broiler production.

Isolation of selenium-resistant bacteria and advancement under enrichment conditions for selected probiotic Bacillus subtilis (BSN313).

The main aim of this work was to screen, isolate, and identify a probiotic selenium (Se)-resistant strain of Bacillus subtilis, using the 16S rDNA sequencing approach and subsequently optimize conditions. Initially, conditions were enhanced in two univariate optimization environments: shakings flask and a bioreactor. After solving optimization for selected variables, conditions were further optimized using orthogonal array testing. The results were further evaluated by the analysis of variance, in support of Se enrichment. In a bioreactor, based on R and F values, the order of effect of selected conditions on Se enrichment was stirring speed > initial pH > temperature > Se addition time. The stirring speed of the bioreactor was most significant, due to the suspension of reduced Se, as it formed. After absolute optimization, strain BSN313 was able to enrich Se up to 2,123 µg/g of dry weight, which is 7.58 times greater than the baseline Se-resistance. PRACTICAL APPLICATIONS: Systematic studies of selenium enrichment conditions will facilitate the successful development of an organic selenium source and the safe use of Bacillus subtilis strain (BSN313) as a food supplement. Selenium-enriched probiotic bacteria are reported to provide many health benefits to the host, due to antipathogenic, antioxidative, anticarcinogenic, antimutagenic, and anti-inflammatory activities.

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.

Determination of antimicrobial effect of protamine by transmission electron microscopy and SDS PAGE on Pseudomonas aeruginosa isolates from diabetic foot infection.

OBJECTIVES: Diabetic foot infection is one of the major complications of diabetes leading to lower limb amputations. Isolation and identification of bacteria causing diabetic foot infection, determination of antibiotic resistance, antimicrobial potential of protamine by electron microscopy and SDS-PAGE analysis, arethe aims of this study. MATERIALS AND METHODS: 285 pus samples from diabetic foot infection patients were collected from different hospitals of Karachi and Capital Health Hospital, Halifax, Canada. Clinical history of each patient was recorded. Bacterial isolates were cultured on appropriate media; identification was done by morphology, cultural and biochemical tests. Effect of protamine against multi drug resistant strains of Pseudomona aeruginosa was checked by minimum inhibitory concentration in 96 well micro-titer plates. The isolates were grown in bactericidal concentration of protamine on plates to isolate mutants. Effect of protamine on protein expression was checked by SDS- PAGE and ultra-structural morphological changes by transmission electron microscopy. RESULTS: Results indicated prevalence of foot infection as 92% in diabetic patients. Major bacterial isolates were Staphylococcus aureus 65 (23%), P. aeruginosa 80 (28.1%), Klebsiella spp. 37 (13%), Proteus mirabilis 79 (27.7%), and Escherichia coli 24 (12%). These isolates were highly resistant to different antibiotics. MIC value of protamine was 500 µg/ml against P. aeruginosa. SDS-PAGE analysis revealed that protamine can suppress expression of various virulence proteins and electron micrographs indicated condensation of cytoplasm and accumulation of protamine in cytoplasm without damaging the cell membrane. CONCLUSION: P. aeruginosa and S. aureus were the major isolates expressing multi-drug resistance and protamine sulfate represented good antimicrobial potential.

Study of class 1 integrons in multidrug-resistant uropathogenic Escherichia coli isolated from different hospitals in Karachi.

OBJECTIVES: Escherichia coli is the key pathogen in the family producing ESBL (extended spectrum ß-lactamase) and associated with community-acquired infections. Therefore, this study was planned to determine the antibiotic susceptibility pattern of uropathogenic E. coli, prevalence of the ESBL gene group and class 1 integrons. MATERIALS AND METHODS: Clinical isolates of uropathogenic E. coli were isolated from different hospitals of Karachi. Antibiotic susceptibility test was performed by Kirby-Bauer Methods. Presence of ß- lactamases genes (CTX, TEM, and SHV) and integron 1 were identified by polymerase chain reaction (PCR). RESULTS: Out of 500, 105 isolates were identified as multi-drug resistant (MDR) uropathogenic E. coli. The subject MDR isolates showed the highest resistance to aztreonam, amoxil/ clavulanic acid, ampicillin, cotrimoxazole, ceftriaxone, cefipime, and cefuroxime. Genetic analysis showed that the majority of the MDR E. coli carry CTX M1 (57.1%) followed by TEM (33.3%) and SHV (9.5%). Moreover, 79% of MDR E. coli harbored class 1 integrons, whereas all three conserved genes for class 1 integrons were present in 58% of MDR E. coli. CONCLUSION: This study is helpful to provide information regarding the antibiotic susceptibility pattern, distribution ESBLs and class 1 integrons among uropathogenic E. coli.

Relationship of cell surface hydrophobicity with biofilm formation and growth rate: A study on Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli.

OBJECTIVES: This study was designed to determine the relationship of Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli isolates in multispecies biofilms and their individual phenotypic characters in biofilm consortia. MATERIALS AND METHODS: The subject isolates were recovered from different food samples and identified on the basis of growth on differential and selective media. Tube methods, Congo-red agar method, and scanning electron microscopy (SEM) were used to study biofilms phenotypes. The hydrophobicity of the strains was evaluated by the adhesion to apolar solvent. RESULTS: The results showed that E. coli dominated the pre-biofilm stage. It has been observed that E. coli adopted biofilm life much before S. aureus and P. aeruginosa. However, after adopting biofilm lifestyle, slowly and gradually, P. aeruginosa dominated the consortia and dispersed other stakeholders. The subject isolates of P. aeruginosa produce cis-2-decanoic acid to disperse or inhibit S. aureus and E. coli biofilms. Gas-chromatography and mass spectrometry results showed that cis-2-decanoic was higher in the co-culture condition and increased at late log-phase or at stationary phase. Although majority of S. aureus were unable to compete with P. aeruginosa, however, a minor population competed, survived, and persisted in biofilm consortia as small colony variants. The survivors showed higher expression of sigB and sarA genes. P. aeruginosa showed comparatively higher hydrophobic surface properties. CONCLUSION: Comparative analysis showed that cell surface hydrophobicity, growth rate, and small colony variants (SCVs) are correlated in biofilm consortia of the P. aeruginosa, S. aureus, and E. coli.

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.

Antibacterial fatty acids destabilize hydrophobic and multicellular aggregates of biofilm in S. aureus.

Present study is based on 20 methicillin-resistant Staphylococcus aureus (MRSA) isolates recovered from different food items. These isolates were identified on the basis of colony morphology, Gram staining and growth on different selective and differential media. Studies on 16S RNA and positive reactions on DNase agar and Prolex Latex Agglutination system confirm it as Staphylococcus aureus. Oxacillin susceptibility testing and PCR with mecA gene-specific primer results showed that these isolates are MRSA-carrying mecA gene that belongs to SCCmecA type IV and also harbor agr type II. Phenotypic study revealed that these isolates adopt biofilm mode of growth after exposure to subinhibitory doses of oxacillin. The biofilm and cell surface hydrophobicity have a strong correlation. It was noticed that affinity to hexadecane (apolar-solvent) of planktonic cells was low, suggesting its hydrophilic character. However, as the cells are exposed to oxacillin, they adopt biofilm mode of life and the affinity to apolar solvent increases, indicating a hydrophobic character. In biofilm consortia, the cells with more hydrophobic surfaces show incomplete septation and produce multicellular aggregates. This is due to reduced expression of atl gene. This was confirmed by real-time PCR studies. Moreover, the planktonic or wild-type phenotypes of these isolates were more tolerant to antibacterial effect of the fatty acids used; that is, cis-2-decanoic acid and cis-9-octadectanoic acid. These fatty acids were more effective against biofilms. After exposure to these fatty acids, established biofilms were dispersed and surviving cells were unable to readopt biofilm mode of life. The planktonic or wild-type phenotypes produce fatty acid-modifying enzyme (FAME) to inactivate the bactericidal activity of fatty acids by esterification to cholesterol. The biofilm indwellers are metabolically inactive and unable to produce FAME; hence, they are vulnerable to antibiofilm effect of cis-2-decanoic acid and cis-9-octadectanoic acid.