Saira Bashir

Molecular profiling of antimicrobial resistance and integron association of multidrug-resistant clinical isolates of Shigella species from Faisalabad, Pakistan

Bacillary dysentery, common in developing countries, is usually caused by Shigella species. A major problem in shigellosis is the rapid emergence of multidrug-resistant strains. This is the first detailed molecular study on drug resistance of Shigella isolates from the Faisalabad region of Pakistan. Ninety-five Shigella isolates obtained after screening of 2500 stool samples were evaluated for in vitro resistance to commonly used antimicrobial agents; the presence or absence of 20 of the most relevant drug resistance genes; and the prevalence of integrons 1, 2, and 3. Shigella flexneri was found to be the most prevalent and most resistant species. Collectively, high resistance was found towards ampicillin (96.84%), tetracycline (93.68%), streptomycin (77.89%), and chloramphenicol (72.63%). Significant emerging resistance was detected towards the modern frontline drugs ciprofloxacin (12.63%), cefradine (17.89%), ceftriaxone (20.00%), cefoperazone (22.10%), and cefixime (28.42%). Prevalence rates for bla(TEM), bla(CTX-M), gyrA, gyrB, qnrS, aadA1, strAB, tetA, tetB, catA, and catP were 78.94%, 12.63%, 20.00%, 21.05%, 21.05%, 67.36%, 42.10%, 12.63%, 53.68%, 33.68%, and 25.26%, respectively. Class 2 integrons (42.10%) were more common in the local isolates. Simultaneous detection of class 1 and 2 integrons in some isolates and a rapidly emerging resistance to modern frontline drugs are the major findings of this study.

Effects of ampicillin, gentamicin, and cefotaxime on the release of Shiga toxins from Shiga toxin-producing Escherichia coli isolated during a diarrhea episode in Faisalabad, Pakistan.

The Shiga toxin-producing Escherichia coli (STEC) is an emerging foodborne pathogen. The proportion of cases attributed to STEC in an episode of diarrhea in the Faisalabad region of Pakistan was investigated. In addition, as increase in Shiga toxin (Stx) release after exposure to various antimicrobial agents is widely reported, we also elucidated the in vitro effects of three commonly used antibiotics (ampicillin, gentamicin, and cefotaxime) on Stx release. Isolation and detection of STEC was done using enzyme-linked immunosorbent assay and polymerase chain reaction, followed by phenotypic characterization. In vitro Stx release from isolated STEC was determined using enzyme-linked immunosorbent assay, and Stx-induced verocytotoxicity was quantified using cytotoxicity detection assay. STEC was detected in 5 (21.7%) of 23 patients. Exposure to minimum inhibitory concentration (MIC) of ampicillin, gentamicin, and cefotaxime resulted in a considerable decrease in toxin release and level of cytotoxicity in most of the STEC isolates when compared with control (without antibiotic exposure). Exposure to sub-MIC of ampicillin resulted in a relative increase in Stx release and cytotoxicity (p <or= 0.05) in three of the four isolates tested, whereas a decreasing trend was observed in isolates exposed to sub-MICs of gentamicin and cefotaxime. Sub-MIC of gentamicin resulted in largest decrease in Stx release and a similar trend was observed with cefotaxime to a lesser extent. In conclusion, these in vitro observations suggested that sub-MIC of ampicillin may stimulate Stx release and level of cytotoxicity and therefore should be avoided. Gentamicin did not show such effects and therefore may be considered for STEC antimicrobial therapy.

Virulence profile of different phylogenetic groups of locally isolated community acquired uropathogenic E. coli from Faisalabad region of Pakistan.

BackgroundUropathogenic E.coli (UPEC) are among major pathogens causing urinary tract infections. Virulence factors are mainly responsible for the severity of these emerging infections. This study was planned to investigate the distribution of virulence genes and cytotoxic effects of UPEC isolates with reference to phylogenetic groups (B2, B1, D and A) to understand the presence and impact of virulence factors in the severity of infection in Faisalabad region of Pakistan.MethodsIn this study phylogenetic analysis, virulence gene identification and cytotoxicity of 59 uropathogenic E.coli isolates obtained from non-hospitalized patients was studied.ResultsAmong 59 isolates, phylogenetic group B2 (50%) was most dominant followed by groups A, B1 (19% each) and D (12%). Isolates present in group D showed highest presence of virulence genes. The prevalence hlyA (37%) was highest followed by sfaDE (27%), papC (24%), cnf1 (20%), eaeA (19%) and afaBC3 (14%). Highly hemolytic and highly verotoxic isolates mainly belonged to group D and B2. We also found two isolates with simultaneous presence of three fimbrial adhesin genes present on pap, afa, and sfa operons. This has not been reported before and underlines the dynamic nature of these UPEC isolates.ConclusionsIt was concluded that in local UPEC isolates from non-hospitalized patients, group B2 was more prevalent. However, group D isolates were most versatile as all were equipped with virulence genes and showed highest level of cytotoxicity.

Field evaluation of Eimeria tenella (local isolates) gametocytes vaccine and its comparative efficacy with imported live vaccine, LivaCox®

The present paper describes the field evaluation of local gametocyte vaccine and its comparative efficacy with commercial anticoccidial vaccine, LivaCox®, used in breeder and broiler flocks in Pakistan. Humoral immune response in vaccinated and control chickens was monitored by enzyme-linked immunosorbent assay. Results demonstrated significantly elevated antibody titres in vaccinated groups as compared to control groups conducted both in Laboratory and field experiments. Significantly (P < 0.01) higher antibody titres in local gametocyte-vaccinated group as compared to LivaCox®-vaccinated chickens were recorded. Splenic cell migration inhibition assay was used to detect the cell-mediated immune (CMI) response, and results were expressed in terms of per cent migration index. Lower per cent migration index in LivaCox®-vaccinated chickens indicated the higher CMI response, as compared to local gametocyte-vaccinated chickens, although the difference was statistically non-significant (P > 0.05). Results of the challenge studies in laboratory experiments revealed significantly higher (P < 0.05) oocyst count in LivaCox®-vaccinated group as compared to local gametocyte-vaccinated chickens.Maximum protection (75%) against mixed species of genus Eimeria was recorded in chickens vaccinated with gametocyte vaccines as compared to LivaCox®-vaccinated group. The mean body weight gains in chickens vaccinated with local gametocyte vaccine were significantly better (P < 0.05) than in chickens vaccinated with LivaCox® vaccine, both in laboratory and field experiments. Majority of the chickens (70–72%) in control group demonstrated severe lesions (3.0–4.0), while 20–26% chickens showed moderate lesions (2.0). On the other hand, local gametocyte- and LivaCox®-immunized chickens developed 78% and 85% mild to moderated lesions (1.0–2.0), respectively. Results of the present study provide a probable explanation for cross-protection induced by Eimeria tenella gametocyte vaccines against other species of genus Eimeria.

Multiple drug resistance patterns in various phylogenetic groups of uropathogenic E.coli isolated from Faisalabad region of Pakistan

The objective of this work was the phylogenetic characterization of local clinical isolates of uropathogenic E. coli with respect to drug resistance. A total of 59 uropathogenic E. coli responsible for community acquired urinary tract infections were included in this study. A triplex PCR was employed to segregate each isolate into four different phylogenetic groups (A, B1, B2 and D). Drug resistance was evaluated by disc diffusion method. The drugs used were ampicillin, aztreonam, cefixime, cefoperazone, ceftriaxone, cephradine among β-lactam group; amikacin, gentamicin, and streptomycin among aminoglycosides; nalidixic acid and ciprofloxacin from quinolones; trimethoprim-sulfomethoxazole, and tetracycline. Among 59 uropathogenic E. coli isolates majority belonged to phylogenetic group B2 (50%) where as 19% each belonged to groups A and B1, and 12% to group D. All the isolates were multiple drug resistant (MDR). Most effective drugs against Group A, B1, and B2 were gentamicin, amikacin and cefixime; ceftriaxone and quinolones; and ceftriaxone and amikacin, respectively. Group D isolates were found to be highly resistant to all drugs. Our results have shown emergence of MDR isolates among uropathogenic E. coli with dominance of phylogenetic group B2. However, it was found that group D isolates were though less frequent, more drug resistant as compared with group B2. Groups A and B1 were relatively uncommon. Amikacin, ceftriaxone and gentamicin were the most effective drugs in general.

A profile of drug resistance genes and integrons in E. coli causing surgical wound infections in the Faisalabad region of Pakistan

Escherichia coli are one of the leading causes of infection in wounds. Emerging multiple drug resistance among E. coli poses a serious challenge to antimicrobial therapy for wounds. This study was conducted to ascertain a baseline profile of antimicrobial resistance in E. coli isolates infecting surgical wounds. A total of 64 pus samples from hospitalized patients were screened and 29 (45.3%) were found to have E. coli, which were identified biochemically and confirmed by molecular methods. Using the disc diffusion method, antimicrobial resistance was observed toward tetracycline (100%), cefradine (100%), nalidixic acid (93.1%), ampicillin (86.2%), gentamicin (86.2%), cefixime (82.8%), ceftriaxone (82.8%), aztreonam (82.8%), ciprofloxacin (75.9%), streptomycin (72.4%), cefoperazone (65.5%), chloramphenicol (58.6%) and amikacin (58.6%). In an effort to find relevant genes, 11 different genes were targeted by PCR. Among these, the mutated gyrA gene was found to be the most prevalent (82.8%), followed by the TEM (72.4%), catP (68.9%), catA1 (68.9%), tetB (62.1%), blt (58.6%), blaCTX−M−15 (27.6%), blaTEM (20.7%), blaOXA (17.2%), tetA (17.2%) and aadA1 (13.8%) genes. The presence of integrons was also studied among these isolates. The prevalence of class 1 integrons was the highest (44.8%), followed by class 2 (27.6%). Three (10.3%) isolates carried both class 1 and class 2 integrons (first report from E. coli infecting wounds). The high incidence of integrons points toward their facilitation for carriage of antimicrobial resistance genes; however, in nearly 37% isolates, no integrons were detected, indicating the significance of alternative mechanisms of gene transfer. Another salient finding was that all isolates were multidrug-resistant E. coli.

In vitro and in vivo toxicological evaluations of methyl ferulate, methyl p-coumarate, and pulegone 1,2-epoxide.

Abstract Context: Toxicological screening of natural compounds for medicinal purposes. Objectives: The objective of this study is to evaluate the toxicity of methyl ferulate (MF), methyl p-coumarate (MpC), and pulegone 1,2-epoxide (PE) with in vitro and in vivo assays. Materials and methods: The in vitro toxicity of MF, MpC, and PE was assessed at a concentration of 10 mg/ml with the Ames assay using two strains of Salmonella typhimurium TA98 and TA100. Human red blood cells (RBC) were used to determine the hemolytic activity of these compounds. The cytotoxicity of above compounds was determined with brine shrimp lethality bioassay (BSLB) at the concentrations of 0.1–20 mg/ml. While dermal and ocular irritation studies were conducted on healthy rabbits (n = 8) for 96 and 12 h post-topical application of test compounds, respectively. Results: PE produced 6–8% hemolysis of RBCs at all the tested concentrations while MF and MpC produced 10–5% hemolysis up to 20 mg/ml, and 50–85% hemolysis at concentrations of 40 and 80 mg/ml, respectively. The Ames assay indicated that MF, MpC, and PE were non-mutagenic as the test values were not significantly higher as compared with background values of the assay. BSLB suggested the lethal concentration (LC50) values of MF, MpC, and PE as 4.38, 6.74, and 25.91 mg/ml, respectively. In vivo ocular and dermal irritation scores of MF, MpC, and PE were comparable with ethanol (control) in rabbits indicating the non-irritant nature of these natural compounds. Conclusion: The present studies suggest that these compounds are non-toxic/non-irritant and might be used for medicinal purposes.

Estimation of Atrazine in the Stratum Corneum and its Toxic Effects in Skin Following Topical Application to Rabbits

ABSTRACT For risk assessment, a workable non-invasive method for the estimation of atrazine in skin was needed. Moreover, the dermato-toxic effects of different concentrations of atrazine have not been studied so far. Fifteen milligrams of 35% solution of atrazine in ethanol was topically applied to each of six different clipped sites on the back of rabbits (n = 6). Each site was tape-stripped with 10 consecutive adhesive tapes at 0.5, 1, 2, 3, 4, and 6 h of topical dosing to remove the stratum corneum (SC). Atrazine in tapes was extracted with methanol and analyzed with HPLC. The amount of atrazine detected in the SC at 6 h (1955.79 ± 47.22 μg) is equivalent to 13.03% of the total applied dose. In dermal toxicity studies, 500 μl of 35%, 70% atrazine in ethanol and a commercial preparation of atrazine (Balance®) was dosed on clipped backs of rabbits (n = 4) for 4 days under occluded vs non-occluded conditions. Ethanol alone served as control. On the 5th day, rabbits were euthanized and skin was scored for erythema and then examined microscopically. Significant differences (p < 0.05) in erythema scores were observed with 70% atrazine and Balance® as compared to the control under occluded conditions. Significant differences in epidermal thickness and cell layers were observed with Balance® and 74% atrazine as compared to control in both dosing conditions. There were non-significant differences in erythema, epidermal thickness, or cell layers in occluded vs non-occluded applications of atrazine in ethanol, indicating that atrazine is equally toxic regardless of its application procedures. Moreover, under non-occluded application, very slight erythema was observed but microscopically significant epidermal hyperplasia was noticed. This indicates that even if there are no significant gross skin manifestations of atrazine this can still produce significant damage to the epidermal barrier and hence can result in increase in penetration of self or other toxic substances.

In-vitro and in-vivo anthelmintic potential of different medicinal plants against Ascaridia galli infection in poultry birds

Infectious diseases are major constraint that hinders the poultry industry. Among them parasitic diseases are very common and Ascaridia galli is one of the most common parasitic roundworms found in poultry. Haemorrhages, diarrhoea and listlessness are signs of infection. Parasitic infections such as A. galli are treated with chemical anthelmintics (piperazine, albendazole, levamisole, Ivermectin, benzimidazoles and fenbendazole). These synthetic chemicals can promote resistance, so there is need for alternative ways to treat the disease. Medicinal plants have the potential to combat such parasitism and the development of anthelmintic resistance appears to be very slow against such treatment. This review covers the studies related to the screening of plant materials having in vitro and in vivo anthelmintic activities against A. galli throughout the world. Medicinal plants showing in vitro anthelmintic activity include Anacardium occidentale, Allium sativum, Tribulus terrestris, Bassia latifolia, Piper betle, Morinda citrifolia L.I, Cassia occidentalis L. and Aloe secundiflora while in vivo studies include the use of Psorelia corylifolia, Piper betle, Pilostigma thonningi, Caesalpinia crista, Ocimum gratissimum and Anacardium occidentale. In conclusion, medicinal plants appear to have good anthelmintic activities in poultry and may substitute conventionally used synthetic drugs, and their use may moderate drug resistance in endemic pathogen populations and drug residues in poultry meat.

Antiviral and immune boosting activities of different medicinal plants against Newcastle disease virus in poultry

The poultry industry is affected by economically important problems such as Newcastle disease (ND). Newcastle disease virus (NDV) belongs to the Paramyxoviridae family and manifests as different strains; lentogenic, mesogenic and velogenic. ND affects the quality of eggs and impairs growth performance of birds. Various efforts have been made to control economic losses due to this disease, including using live and killed vaccines, which do not confer 100% immunity in all cases. Due to mutations within viral strains, NDV can become resistant and difficult to control, and there is a need to search for alternative measures. Medicinal plants are considered as a complementary means to control this virus, especially in developing countries, because they have been implicated in treatments for a variety of infectious and non-infectious diseases. Alkaloids, flavonoids, saponins and tannins found in plants have been regarded as novel antiviral agents. The following paper encompasses the studies related to plants having in vitro antiviral activity against ND such as Momordica balamina, Adansonia digitata, Artemisia annua, Azadirachta indica, Psidium guajava, Moringa oleifera and plants having good in vivo antiviral activity such as Aloe secundiflora, Cucumis metuliferus and Anthocleista nobilis as well. It discusses plants with the potential to improve the immunity of infected birds, such as Mangrove halophytes, Nigella sativa, Polysavone, Melissa officinalis, Momordica cochinchinensis, Echinacea purpurea, Withania somnifera and Aspargus Racemosus.