Defect induced ferromagnetism in Mn3Ga.

Ni-substituted Mn3Ga displays a weak ferromagnetism embedded in an antiferromagnetic (AF) phase. Upon field cooling, the alloy exhibits exchange bias and an open hysteresis loop, signifying kinetic arrest at room temperature. For the first time, a kinetic arrest is seen in a compound due to the first order transition of an embedded defect phase. A systematic study of crystal structure, local structure, and magnetic properties of Mn3-xNixGa (x= 0, 0.25) alloys reveal the origin of ferromagnetism in Mn2.75Ni0.25Ga is due to the segregation of a Heusler-type environment around Ni in the cubic Mn3Ga matrix. Upon temper annealing at 400∘C, these local structural defects around the Ni phase separate into a modulated ferromagnetic (FM) Ni-Mn-Ga Heusler phase. A strong interaction between the AF host and the FM defect phase gives rise to exchange bias. The first-order transition of the defect phase seems to be responsible for the observed kinetic arrest in Mn2.75Ni0.25Ga.

Exploring Galleria mellonella larval model to evaluate antibacterial efficacy of Cecropin A (1-7)-Melittin against multi-drug resistant enteroaggregative Escherichia coli.

High throughput in vivo laboratory models is need for screening and identification of effective therapeutic agents to overcome microbial drug-resistance. This study was undertaken to evaluate in vivo antimicrobial efficacy of short-chain antimicrobial peptide- Cecropin A (1-7)-Melittin (CAMA) against three multi-drug resistant enteroaggregative Escherichia coli (MDR-EAEC) field isolates in a Galleria mellonella larval model. The minimum inhibitory concentration (MIC; 2.0 mg/L) and minimum bactericidal concentration (MBC; 4.0 mg/L) of CAMA were determined by microdilution assay. CAMA was found to be stable at high temperatures, physiological concentration of cationic salts and proteases; safe with sheep erythrocytes, secondary cell lines and commensal lactobacilli at lower MICs; and exhibited membrane permeabilization. In vitro time-kill assay revealed concentration- and time-dependent clearance of MDR-EAEC in CAMA-treated groups at 30 min. CAMA- treated G. mellonella larvae exhibited an increased survival rate, reduced MDR-EAEC counts, immunomodulatory effect and proved non-toxic which concurred with histopathological findings. CAMA exhibited either an equal or better efficacy than the tested antibiotic control, meropenem. This study highlights the possibility of G. mellonella larvae as an excellent in vivo model for investigating the host-pathogen interaction, including the efficacy of antimicrobials against MDR-EAEC strains.

Current approaches for the detection of Coxiella burnetii infection in humans and animals.

Q fever (coxiellosis), caused by Coxiella burnetii, is an emerging or re-emerging zoonotic disease of public health significance and with worldwide distribution. As a causal agent of the one among the 13 global priority zoonoses, having the infectious dose as low as one bacterium, C. burnetii has been regarded as an obligate intracellular bacterial pathogen. The agent has been classified as a Group B bioterrorism agent by the Centre for Disease Control and Prevention (CDC), and the disease is included in the World Organisation for Animal Health (OIE) list of notifiable diseases. It is mainly transmitted through airborne route in humans and animals. Isolation of C. burnetii, using standard routine laboratory culture techniques was impossible until formulation of axenic-based medium. However, it is still to be included among routinely isolated laboratory pathogen, accounting prolonged incubation period (~7 days) and requirement of specific oxygen concentration (2.5% O2). Therefore, indirect diagnostic tools have been mainly used for its diagnosis. So far serology has been mostly used for testing for C. burnetii infection. The detection of C. burnetii DNA by PCR in various clinical samples have also been widely used. The disease has remained largely under-reported, underdiagnosed and as a masked zoonosis; and therefore, needs to be explored through well-planned scientific studies for knowing its true status and likely it impact in humans and animals by employing state-of-the-art diagnostics, identifying its diverse and new host range, as well as risk factors involved in different geo-climatic, behavioural and social settings as well as risk groups. Here, we reviewed the current approaches used for the detection of C. burnetii infection in humans and animals at the population and individual level.

Antimicrobial efficacy of Cecropin A (1-7)- Melittin and Lactoferricin (17-30) against multi-drug resistant Salmonella Enteritidis.

The present study evaluated intracellular antibacterial efficacy of two short-chain cationic antimicrobial peptides (AMPs) namely, Cecropin A (1-7)-Melittin and lactoferricin (17-30) against three field strains of multi-drug resistant Salmonella Enteritidis. Initially, antimicrobial ability of both the AMPs was evaluated for their minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against multi-drug resistant S. Enteritidis strains. Besides, the AMPs were evaluated for its in vitro stability (high-end temperatures, proteases, physiological concentrations of cationic salts and pH) and safety (haemolytic assay in sheep erythrocytes; cytotoxicity assay in murine macrophage RAW 264.7 cell line and human epithelioma HEp-2 cell line and beneficial gut lactobacilli). Later, a time-kill assay was performed to assess the intracellular antibacterial activity of Cecropin A (1-7)-Melittin and lactoferricin (17-30) against multi-drug resistant S. Enteritidis in RAW 264.7 and HEp-2 cells. The observed MBC values of Cecropin A (1-7)-Melittin and lactoferricin (17-30) against multi-drug resistant S. Enteritidis (128 µM; 256 µM) were generally twice or four-fold greater than the MIC values (64 µM). Further, both the AMPs were found variably stable after exposure at high-end temperatures (70 °C and 90 °C), protease treatment (trypsin, proteinase K, lysozyme), higher concentration of physiological salts (150 mM NaCl and 2 mM MgCl2) and hydrogen ion concentrations (pH 4.0 to 8.0). Both the AMPs were found non-haemolytic on sheep erythrocytes, revealed minimal cytotoxicity in RAW 264.7 and HEp-2 cells, and was tested safe against beneficial gut lactobacilli (L. acidophilus and L. rhamnosus). Intracellular bacteriostatic effect with both cationic AMPs against multi-drug resistant S. Enteritidis was evident in RAW 264.7 cells; however, in both the cell lines, the significant bactericidal effect was not observed (P > 0.05) with both cationic AMPs understudy against multi-drug resistant S. Enteritidis. Based on the results of the present study, both the cationic AMPs understudy may not be useful for the intracellular elimination of multi-drug resistant S. Enteritidis; hence, further studies such as conjugation of these AMPs with either cell-penetrating peptides (CPP) and/or nanoparticles (NPs) are warranted.

Efficacy of Indolicidin, Cecropin A (1-7)-Melittin (CAMA) and Their Combination Against Biofilm-Forming Multidrug-Resistant Enteroaggregative Escherichia coli.

The present study examined the anti-biofilm efficacy of two short-chain antimicrobial peptides (AMPs), namely, indolicidin and cecropin A (1-7)-melittin (CAMA) against biofilm-forming multidrug-resistant enteroaggregative Escherichia coli (MDR-EAEC) isolates. The typical EAEC isolates re-validated by PCR and confirmed using HEp-2 cell adherence assay was subjected to antibiotic susceptibility testing to confirm its MDR status. The biofilm-forming ability of MDR-EAEC isolates was assessed by Congo red binding, microtitre plate assays and hydrophobicity index; broth microdilution technique was employed to determine minimum inhibitory concentrations (MICs) and minimum biofilm eradication concentrations (MBECs). The obtained MIC and MBEC values for both AMPs were evaluated alone and in combination against MDR-EAEC biofilms using crystal violet (CV) staining and confocal microscopy-based live/dead cell quantification methods. All the three MDR-EAEC strains revealed weak to strong biofilm-forming ability and were found to be electron-donating and weakly electron-accepting (hydrophobicity index). Also, highly significant (P < 0.001) time-dependent hydrodynamic growth of the three MDR-EAEC strains was observed at 48 h of incubation in Dulbecco's modified Eagle's medium (DMEM) containing 0.45% D-glucose. AMPs and their combination were able to inhibit the initial biofilm formation at 24 h and 48 h as evidenced by CV staining and confocal quantification. Further, the application of AMPs (individually and combination) against the preformed MDR-EAEC biofilms resulted in highly significant eradication (P < 0.001) at 24 h post treatment. However, significant differences were not observed between AMP treatments (individually or in combination). The AMPs seem to be an effective candidates for further investigations such as safety, stability and appropriate biofilm-forming MDR-EAEC animal models.

Apparent prevalence and risk factors associated with occurrence of Coxiella burnetii infection in goats and humans in Chhattisgarh and Odisha, India.

Coxiella burnetii is one of the most contagious pathogen associated with Q fever in humans, while, ruminants act as important source of infection for humans. In the present cross sectional study, a total of 464 samples were collected from 218 goats comprising of 218 sera, 218 blood and 28 milk from various parts of Chhattisgarh and Odisha region, India. Besides, environmental (33; soil- 4, faecal- 10, feed-6, drainage water- 6, drinking water- 7) and rodent (38) samples were also collected from the premises of the animals. Human sera samples (93) were collected from same sampling area comprised of workers at an organized dairy farm (43), and farmers (50). The samples were subjected to PCR targeting the trans and com1 genes and detection of antibodies using commercial ELISA kits. An overall 14.22% (95% CI: 10.2-19.47%) of the goat samples were positive using either PCR or ELISA. While, by using PCR and ELISA, 11.93% (26/218) and 9.63% (21/218) of the samples were positive for C. burnetii. A higher seropositivity (46.24%; 95% CI: 36.46-56.32%) was observed for antibodies against C. burnetii in samples collected from humans. None of the human, environmental and rodent samples were positive for C. burnetii using PCR. This seems to be the first cross sectional study to focus the hidden threat of coxiellosis among goat population and associated risk factors in India.

A comparative study for detection of extended spectrum ß-lactamase (ESBL) production by Enteroaggregative Escherichia coli (EAEC) strains using double disc, nitrocefin and PCR assays.

We explored and evaluated for the first time colorimetric nitrocefin assay in conjunction with the double disc test and PCR assay. We suggested the use of nitrocefin assay for rapid screening of ESBL-production by Enteroaggregative Escherichia coli.

[ЕFFICACY OF BIOMECHANICALLY SUBSTANTIATED METHODS OF MOBILIZATION OF ADIPOSO-CUTANEOUS FLAPS FROM THE ANTERIOR ABDOMINAL WALL].

There were studied the skin specimen from 40 dead persons, aged from 23 to 87 yrs, (52 ± 15.6) yrs at average, with the objective to investigate the biochemical properties and establishment of experimental deformation angles. Basing on the biomechanical tissues background of the anterior abdominal wall topographo-anatomic regions there were analyzed the results of treatment in the patients, оperated on for the anterior wall deformities and defects, concerning biomechanical possibilities of the flaps and classic methods of the tissues mobilization applied. There was established, that in borders of possible plastic deformity the tissues pressure distribution have persisted in normal range, and this have promoted the cicatricial tissue formation, which did not differ in characteristics from the adjacent connective tissues structures, what have had guaranteed the uncomplicated course of the wound process. Biomechanical properties of adiposo-cutaneous flaps must be taken into account while manipulating with them with the objective of obtaining an optimal distribution of pressure along the flaps and conditions for obtaining of the tissues adequate reaction on operative trauma, while performing interventions on the anterior abdominal wall for its defects and deformities, guaranteeing better esthetic result of treatment, reduction of postoperative morbidity and stationary stay.

A multiplex PCR for detection of Listeria monocytogenes and its lineages.

A novel multiplex PCR assay was developed to identify genus Listeria, and discriminate Listeria monocytogenes and its major lineages (LI, LII, LIII). This assay is a rapid and inexpensive subtyping method for screening and characterization of L. monocytogenes.

Antimicrobial effects of Lactobacillus plantarum and Lactobacillus acidophilus against multidrug-resistant enteroaggregative Escherichia coli.

The in vitro and in vivo antimicrobial effects of Lactobacillus plantarum and Lactobacillus acidophilus were evaluated individually and synergistically against multidrug-resistant enteroaggregative Escherichia coli (MDR-EAEC). In vitro evaluation of each probiotic strain when co-cultured with MDR-EAEC isolates revealed a reduction in MDR-EAEC counts (eosin-methylene blue agar) in a dose- and time-dependent manner: probiotics at a dose rate of 10(10) CFU inhibited MDR-EAEC isolates at 72 h post-inoculation (PI), whereas at lower concentrations (10(8) and 10(9) CFU) MDR-EAEC isolates were inhibited at 96 h PI. The synergistic antimicrobial effect of both probiotic strains (each at 10(10) CFU) was highly significant (P < 0.01) and inhibited the growth of MDR-EAEC isolates at 24 h PI. For in vivo evaluation, weaned mice were fed orally with 10(7) CFU of MDR-EAEC. At Day 3 post-infection, treated mice were fed orally with the probiotic strains (each at 10(10) CFU). Compared with the control, post-treatment a significant (P < 0.01) reduction in MDR-EAEC counts was observed in faeces by Day 2 and in intestinal tissues of treated mice by Days 3 and 4 as evidenced by plate count (mean 2.71 log and 2.27 log, respectively) and real-time PCR (mean 1.62 log and 1.57 log, respectively) methods. Histopathologically, comparatively mild changes were observed in the ileum and colon from Days 3 to 5 post-treatment with probiotics; however, from Day 6 the changes were regenerative or normal. These observations suggest that these probiotic strains can serve as alternative therapeutics against MDR-EAEC-associated infections in humans and animals.

Genetic diversity, virulence potential and antimicrobial susceptibility of Listeria monocytogenes recovered from different sources in India.

Listeria monocytogenes isolates (n = 36) recovered from human and animal clinical cases and foods from different geographical regions of India were characterized using multiplex PCR-based serotyping, pulsed field gel electrophoresis (PFGE), in vitro and in vivo pathogenicity tests and antibiogram profiling. Multiplex PCR-based serotyping distributed L. monocytogenes isolates into 3 serogroups, of which 91.67% belonged to 4b, 4d, 4e serogroup, followed by 5.56% to 1/2a, 3a and 2.78% to 1/2b, 3b serogroups. PFGE analysis using ApaI and AscI restriction enzymes revealed 17 pulsotypes among 36 L. monocytogenes isolates with 6 major clusters having similar fingerprint profile within their cluster and 11 unique fingerprint profiles. Interestingly, PFGE analysis inferred that foods of animal origin could be a significant source of infection for spread of listeriosis among human populations. Furthermore, on comparison of in vitro and in vivo pathogenicity tests, an overall good correlation was observed between hemolytic titer assay and chick embryo inoculation test as most of the isolates with a hemolytic titer of ≥ 16 were found to be lethal to chick embryo. All the isolates were found to be susceptible to tested antimicrobials except for one animal isolate which showed resistance towards co-trimoxazole.

Biofilm formation and genetic diversity of Salmonella isolates recovered from clinical, food, poultry and environmental sources.

In the present study, Salmonella isolates (n=40) recovered from clinical, food, poultry and environmental sources were characterized for serotype identification, genetic diversity and biofilm formation capability. Serotype identification using multiplex PCR assay revealed six isolates to be Salmonella Typhimurium, 14 as Salmonella Enteritidis, 11 as Salmonella Typhi, and the remaining nine isolates unidentified were considered as other Salmonella spp. Most of the Salmonella isolates (85%) produced biofilm on polystyrene surfaces as assessed by microtitre plate assay. About 67.5% isolates were weak biofilm producers and 17.5% were moderate biofilm producers. There was no significant difference in biofilm-forming ability among the Salmonella isolates recovered from different geographical regions or different sources. Among the genetic methods, Enterobacterial Repetitive Intergenic Consensus (ERIC) PCR revealed greater discriminatory power (DI, 0.943) followed by pulsed field gel electrophoresis (PFGE) (DI, 0.899) and random amplification of polymorphic DNA (RAPD) PCR (DI, 0.873). However, composite analysis revealed the highest discrimination index (0.957). Greater discrimination of S. Typhimurium and S. Typhi was achieved using PFGE, while ERIC PCR was better for S. Enteritidis and other Salmonella serotypes. A strong positive correlation (r=0.992) was observed between biofilm formation trait and clustered Salmonella isolates in composite genetic analysis.

Evaluation of a PCR targeting fimbrial subunit gene (fimA) for rapid and reliable detection of Enteroaggregative Escherichia coli recovered from human and animal diarrhoeal cases.

The present study describes the utility of a chromosomal associated fimbrial subunit gene (fimA) for screening 'typical' as well as 'atypical' Enteroaggregative Escherichia coli (EAEC) by PCR and its possible role as a promising molecular marker for rapid detection of 'typical' and 'atypical' EAEC pathotype.

Isolation and identification of Salmonella from diarrheagenic infants and young animals, sewage waste and fresh vegetables.

AIM: This study was carried out to determine the prevalence, distribution, and identification of Salmonella serotypes in diarrheagenic infants and young animals, including sewage waste and fresh vegetables. MATERIALS AND METHODS: A total of 550 samples were processed for the isolation of Salmonella spp., using standard microbiological and biochemical tests. Further polymerase chain reaction (PCR) detection of Salmonella genus was carried out using self-designed primers targeting invA gene and thereafter identification of important serotypes namely Salmonella Enterica serovar Typhimurium, Salmonella Enterica serovar Enteritidis, Salmonella Enterica serovar Typhi was performed using published standardized multiplex PCR. RESULTS: An overall low prevalence of 2.5% (14/550) was observed. The observed prevalence of Salmonella spp. in diarrheagenic infants was 1.2% (05/400), diarrheagenic young animals 4% (02/50), sewage waste 10% (05/50), and fresh vegetables 4% (02/50), respectively. In diarrheagenic infants, of the five Salmonella isolates identified, two were Salmonella Typhimurium, two Salmonella Enteritidis, and one was unidentified and hence designated as other Salmonella serovar. All the Salmonella isolates identified from diarrheagenic young animals and sewage waste belonged to other Salmonella serovar, whereas, of the two isolates recovered from fresh vegetables, one was identified as other Salmonella serovar, and one as Salmonella Typhimurium, respectively. CONCLUSION: Isolation of Salmonella spp. especially from sewage waste and fresh vegetable is a matter of great concern from public health point of view because these sources can accidentally serve as a potential vehicle for transmission of Salmonella spp. to animals and human beings.

Isolation of Coxiella burnetii from bovines with history of reproductive disorders in India and phylogenetic inference based on the partial sequencing of IS1111 element.

In the present study, a total of 158 blood samples from 148 bovines and 10 dogs having a history of reproductive disorders were screened for Coxiella burnetii by trans-PCR method. In case of bovines, 6.08% (9/148) blood samples comprised of 4.54% (4/88) cattle and 8.33% (5/60) buffaloes turned out to be positive for C. burnetii DNA while all the samples from dogs (10) were found negative. Of the 9 PCR-positive bovine blood samples, the organism could be isolated only from 3 cases of buffaloes by chick embryo inoculation method. Further, to predict the homology and genetic diversity, the recovered C. burnetii isolates designated as Y1, Y3 and Y7 were partially sequenced for IS1111 gene. On phylogenetic analysis, Y3 and Y7 isolates clustered to a common node away from Y1 isolate. This study may enlighten the nature of circulating C. burnetii isolates in different parts of the world. To the best of our knowledge, this appears to be the first report describing phylogenic analysis of C. burnetii isolates based on IS1111 gene sequence.

16S rRNA PCR followed by restriction endonuclease digestion: a rapid approach for genus level identification of important enteric bacterial pathogens.

The study describes a rapid approach for detection of common enteric bacterial pathogens, which involves partial amplification of the 16S rRNA gene by PCR using a colony from selective medium followed by restriction enzyme (RE) digestion using the EcoRI, HindIII and SalI enzymes. On the basis of RE digestion analysis different genera namely, Escherichia, Salmonella, Shigella, Vibrio, Campylobacter, Arcobacter, Yesinia and Listeria were differentiated.

Epidemiology and risk management of listeriosis in India.

Listeria monocytogenes is a foodborne pathogen that can cause serious invasive illness, mainly in certain well-defined high-risk groups, including elderly and immunocompromised patients, pregnant women, newborns and infants. In India, this pathogen has been isolated from humans, animals and foods. The incidence of Listeria is generally comparable to those reported elsewhere in the world. In humans, maternal/neonatal listeriosis is the most common clinical form reported. Among animal populations, spontaneous abortions, subclinical mastitis, meningoencephalitis and endometritis were the commonest forms reported. The disease largely remains undiagnosed and under reported. From reported analyses of a variety of foods for Listeria, milk and milk products, meat and meat products, seafood and vegetables have been reported to be contaminated in India. The legal framework for microbiological safety of foods against microbes including L. monocytogenes is summarised. The epidemiological studies would help in understanding of the sources of infection and persistence and their risk assessment, routes of transmission, clinical forms and allow for better management of the infection.

Genotypic characterization of Listeria monocytogenes isolated from humans in India.

Listeria monocytogenes is a foodborne pathogen associated with severe diseases in humans and animals. The genotypic analysis of 17 L. monocytogenes isolates recovered from humans in India during 2006-2009 using multiplex serotyping PCR allowing serovar predictions, conventional serology and by pulsed field gel electrophoresis (PFGE) is presented. The isolates were recovered from patients exhibiting various clinical conditions. A multiplex-PCR based serotyping assay revealed 88·24% (15/17) of the strains belonging to the serovar group 4b, 4d, 4e and 11·76% (2/17) to the serovar group 1/2b, 3b. Conventional serology indicated that 13 (76·47%) L. monocytogenes isolates to be of serotype 4b, 2 (11·76%) serotype 4d, and 2 (11·76%) serotype 1/2b. Ten ApaI and nine AscI pulsotypes were recognized among the 17 human isolates. PFGE analysis allowed discrimination among isolates of the same serotype and among isolates from the same sampling areas or those isolated from different areas. Thus, PFGE together with multiplex-PCR serotyping allows rapid discrimination of L. monocytogenes strains. In addition, the predominance of L. monocytogenes serotype 4b is of concern, as this serotype has been most frequently associated with human listeriosis outbreaks.

Prevalence of Q fever in domestic animals with reproductive disorders.

The occurrence of Coxiella burnetii in animals with reproductive disorders was studied. A total of 920 samples (genital and faecal swabs, milk, urine and serum) were collected from cows (88), buffaloes (33), sheep (43) and goats (53) with a history of reproductive disorders and screened for C. burnetii by a PCR assay targeting the repetitive transposon-like region of C. burnetii (trans-PCR), real-time PCR, indirect immunofluorescence assay (IFA), enzyme-linked immunosorbent assay (ELISA) and isolation method. The overall prevalence of Q fever in animals with the history of reproductive disorders turned out to be 13.82%. The species-wise prevalence of Q fever among animals was observed to be 12.78% in cattle, 16.66% in buffaloes, 11.04% in sheep and 6.13% in goats. In comparison to IFA, the highest sensitivity (85.18%) was shown by both PCR assays followed by ELISA (74.07%) and isolation method (14.81%) whereas, isolation method was the most specific (100%) followed by both PCR assays (99.47%) and ELISA (98.44%). The high excretion rate of pathogen particularly in milk observed in the study posses a potential public health threat from infected animals.