Authentication of traditional meat products (Haleem) sold in India: a first report confirming species mislabelling.

Traditional meat products like Haleem play a pivotal role in the culinary landscapes of Indian consumers, along with high economic value and business potential. Due to anticipated gains associated with adulterating 'Haleem' and constant evasion from regulatory oversight, the susceptibility to adulteration has substantially increased. Furthermore, no reports/surveillance regarding their labelling compliance has been reported. Hence, we conducted a 2-year surveillance using 100 samples collected from Hyderabad, India, using the Chipron™ DNA macroarray analysis technique. The method was validated for sensitivity (1%), specificity, and with proficiency test samples. Following this, the surveillance samples (beef, chicken, and mutton Haleem) were tested. The surveillance revealed an alarming adulteration of 46% of the samples, with different proportions of adulterant species. Adulteration of unconventional meat like camel meat was also found. These concerning results necessitate the requirement of stricter and constant regulatory surveillance to safeguard consumer trust and preserve the authenticity of traditional meat products. Supplementary Information: The online version contains supplementary material available at 10.1007/s13197-024-05947-9.

Duplex real-time PCR assay with high-resolution melt analysis for the detection and quantification of Listeria species and Listeria monocytogenes in meat products.

Listeria contamination in foods of animal origin is one of the most concerning food safety issues. A duplex, SYBR green-based, real-time PCR assay was developed with high-resolution melting analysis-based differentiation of the genus Listeria and Listeria monocytogenes. The primers were designed and tested against other related foodborne pathogens. The assay was optimized for standard parameters in a non-orthogonal fashion and validated following international standards. The LODabs and LOQ of the assay were calculated to be 0.78 and 1.56 ng of the target DNA. The LODrel of the assay was found to be 1% Listeria DNA in background DNA. The assay was evaluated for applicability in artificially spiked samples, providing a 120 CFU/ml detection. The assay was validated with proficiency test samples and also with samples collected for surveillance analysis. This well-established and validated assay can be utilized as a qualitative and quantitative tool for addressing the Listeria contamination in the food safety contexts. Supplementary Information: The online version contains supplementary material available at 10.1007/s13197-023-05695-2.

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.

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.

Association of Myroides odoratimimus in immunocompromized piglets with post weaning multisystemic wasting syndrome.

AIM: To study the association of opportunistic infection due to Myroides odoratimimus in piglets immunocompromised by porcine circovirus type 2 (PCV2) infection. METHODS AND RESULTS: The clinical samples (n = 101) were analysed bacteriologically. The isolates were identified by their phenotypes and MALDI TOF-MS analysis as Myroides species. The phylogram constructed based on nucleotide sequences of the 16S rRNA gene showed identity (~99%) with the M. odoratimimus isolates. The minimum inhibitory concentration values for antibiotics revealed M. odoratimimus to be resistant against carbapenem, cephalosporins, aminoglycosides and fluoroquinolones. The presence of PCV2 in affected tissue samples was confirmed by amplification of the 565 bp region of ORF2 of the PCV2 genome. The topology of the phylogenetic tree grouped the PCV2 with cluster-2d. CONCLUSIONS: PCV2 being immunosuppressive in nature might have impaired the immunity thereby increasing the susceptibility of immunocompromised piglets to opportunistic pathogens such as M. odoratimimus leading to disease severity and high mortality. The M. odoratimimus isolates were found to be multidrug resistant and evidenced for uncertain clinical relevance and hence could act as hidden source of public health hazard. SIGNIFICANCE AND IMPACT OF THE STUDY: Myroides odoratimimus is a rarely reported human pathogen. We reported the incidence of infection due to seemingly rare isolates of M. odoratimimus causing an outbreak of pneumonia in piglets. This appears, to the best of authors' knowledge, to be the first outbreak due to Myroides recorded in animal clinical cases described in the literature.

Distribution of Orientia tsutsugamushi in rodents and mites collected from Central India.

Orientia tsutsugamushi, the causative agent of scrub typhus, is an obligate intracytosolic bacterium transmitted among humans and small mammals by some species of larval trombiculid mites (chiggers). It has been recognized as a pathogen of major public health concern in the Asia-Pacific region. As disease is considered as a neglected, there exists a gap in our knowledge of the disease with regard to the sporadic epidemiologic data in endemic areas. The purpose of the study was to find out the vector as well as pathogen distribution in rodents present in the scrub typhus-reported areas in central India. We studied the seasonal variations of occurrence in O. tsutsugamushi in rodents and mites by molecular detection targeting the 56-kDa and 47-kDa genes. Rodent and mite samples were collected during December 2015 to July 2017. A total of 127 samples from rodents, seven pools of mites, and four pools of fleas were collected and processed for DNA isolation. Nested PCRs targeting the 56-kDa and 47-kDa surface antigen genes were performed. In addition, quantification of bacterial load was done by qPCR targeting the 47-kDa gene. During the pre-monsoon season, O. tsutsugamushi was detected in 12% and 10% samples employing the 56-kDa and 47-kDa nested PCRs, respectively, whereas, during post-monsoon season, the respective detection rates were 13.33% and 26.66%. This study predicted a bimodal pattern during the months of pre-monsoon and post-monsoon season with a peak in post-monsoon. Thus, the impact of season on the perpetuation of O. tsutsugamushi in the host was observed.

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.

Isolation and characterization of multidrug-resistant Leclercia species from animal clinical case.

Leclercia adecarboxylata, a Gram-negative bacillus of family Enterobacteriaceae, is an uncommonly identified pathogen isolated from environmental and clinical specimens. Most of the human infections are polymicrobial and commonly occur in immunocompromised hosts, although nosocomial infections in immunocompetent hosts have been documented. Here, we describe the case of isolation of Leclercia species as polymicrobial infection from bovine suffering from respiratory distress in Chhattisgarh state of India. The isolates were identified by their phenotypes, 16S rDNA sequencing and MALDI-TOF-MS. The isolate was found to be resistant to aminoglycosides and fluoroquinolone antibiotics and intermediate resistant to cephalosporins and evidenced for uncertain clinical relevance and could act as hidden source of public health hazard. SIGNIFICANCE AND IMPACT OF THE STUDY: Leclercia adecarboxylata is a rarely reported human pathogen. We report here the case from bovine suffering from respiratory distress; the sample yielded Leclercia species as polymicrobial culture. The isolate was found to be multidrug resistant and evidenced for uncertain clinical relevance and could act as hidden source of public health hazard. The limited literature available on this organism is reviewed, and the potential implications of findings are discussed. To the best of our knowledge, this is the first report of isolation and characterization of multidrug-resistant Leclercia species from animal clinical case from India.

Mangrove Ecosystems: An Adopted Habitat for Pathogenic Salmonella spp.

Mangroves are affected by industrial and anthropogenic factors. Although mangroves have been widely studied, investigations of pathogens that may affect public health significance are largely lacking even while incidences of diseases linked with the consumption of mangrove-associated food have increased. A total of 150 samples of water, sediment, and biota were collected from ten mangrove ecosystems in Goa, India. Total viable counts of pathogens such as E. coli, Listeria, Salmonella, and Vibrio spp. ranged from 1.25 to 3.9 × 10(3) cfu/ mL, which were above the relevant standards. Salmonella counts were the highest at 3.1 to 3.9 × 10(3)cfu/mL, with a prevalence of 40%. Considering its high prevalence, the virulence of Salmonella spp. was studied. The invA gene was detected in 35% of the Salmonella isolates by polymerase chain reaction (PCR). The findings suggested that pathogens adapt to this habitat, resulting in contamination of the indigenous fauna.

Next generation sequencing technologies: tool to study avian virus diversity.

Increased globalisation, climatic changes and wildlife-livestock interface led to emergence of novel viral pathogens or zoonoses that have become serious concern to avian, animal and human health. High biodiversity and bird migration facilitate spread of the pathogen and provide reservoirs for emerging infectious diseases. Current classical diagnostic methods designed to be virus-specific or aim to be limited to group of viral agents, hinder identifying of novel viruses or viral variants. Recently developed approaches of next-generation sequencing (NGS) provide culture-independent methods that are useful for understanding viral diversity and discovery of novel virus, thereby enabling a better diagnosis and disease control. This review discusses the different possible steps of a NGS study utilizing sequence-independent amplification, high-throughput sequencing and bioinformatics approaches to identify novel avian viruses and their diversity. NGS lead to the identification of a wide range of new viruses such as picobirnavirus, picornavirus, orthoreovirus and avian gamma coronavirus associated with fulminating disease in guinea fowl and is also used in describing viral diversity among avian species. The review also briefly discusses areas of viral-host interaction and disease associated causalities with newly identified avian viruses.

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.

Prevalence and genetic profiles of Escherichia coli from mangroves and mangrove associated foods off Goa, India.

A total of 120 samples comprising of water (45), sediment (45) and mangrove originated food (30) collected from mangrove ecosystems of Goa were screened for Escherichia coli employing ISO-16654 method. Seventy-one (59.16%) samples were positive for E. coli. The E. coli isolates were further characterized by serotyping, virulence gene profiling and pulsed field gel electrophoresis (PFGE). Water and sediment samples were analyzed for physico-chemical parameters. The serotypes reported were O1, O10, O13, O17, O36, O41, O50, O68, O105, O116, O141, O148, O159, O162 and rough types while, 23 strains could not be typed. The stx1 and stx2 genes were detected in 33(46.47%) and 16(22.53%) isolates, respectively. The XbaI restriction digestion patterns of the stx positive strains were diverse. Interestingly, few strains isolated from diarrheal patients and from water, sediment and food from mangrove sources were genetically similar. The study showed that the mangrove ecosystem could be a potential reservoir for pathogenic E. coli.

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.

Differentiation of Staphylococcus aureus and Staphylococcus epidermidis by PCR for the fibrinogen binding protein gene.

Mastitis is one of the most common and burdensome diseases afflicting dairy animals. Among other causes of mastitis, staphylococci are frequently associated with clinical and subclinical mastitis. Although Staphylococcus aureus is the predominant species involved, Staphylococcus epidermidis and other coagulase-negative staphylococci are increasingly being isolated from cases of bovine mastitis. Although Staph. aureus and Staph. epidermidis can be easily differentiated based on their biochemical properties, such phenotypic identification is time consuming and laborious. This study aimed to rapidly identify Staph. aureus and Staph. epidermidis. Accordingly, a multiplex PCR was developed and we found that a single gene encoding the adhesin fibrinogen binding protein could be used to identify and differentiate the two species. Consequently, a multiplex reaction combining a triplex PCR for Staph. aureus and a duplex PCR for Staph. epidermidis was standardized, first using bacterial cultures and then with pasteurized milk spiked with live organisms or DNA extracted from the organisms. The test could specifically detect Staph. aureus and Staph. epidermidis even in the presence of a dozen other organisms. The limit of detection for detecting Staph. aureus and Staph. epidermidis separately was 10 to 100 cfu/mL for simplex PCR and 10(4)cfu/mL for multiplex PCR. Conversely, the limit was 10(6)cfu/mL by multiplex PCR for simultaneous detection of both the organisms when spiked into culture medium or pasteurized milk. Overnight enrichment enhanced the assay sensitivity 100-fold. The assay had a high diagnostic sensitivity and specificity. The application of the test was verified on 602 field isolates of staphylococci that had been characterized earlier by phenotypic methods. Importantly, 25 coagulase-negative isolates were identified as Staph. aureus by the multiplex PCR. The test could be adapted for use in clinical diagnostic laboratories.

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.

Multiplex PCR assay for species identification of bovine mastitis pathogens.

AIM: To develop and evaluate a multiplex PCR (mPCR) assay for simultaneous detection of 10 bacterial species causing bovine mastitis namely, Staphylococcus aureus, Staphylococcus chromogenes, Staphylococcus epidermidis, Staphylococcus sciuri, Staphylococcus haemolyticus, Staphylococcus simulans, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis and Escherichia coli in milk. METHODS AND RESULTS: A two-tube mPCR assay was developed. The accuracy of the mPCR was evaluated using 56 standard reference strains and 705 strains comprising of E. coli (n = 99), staphylococci (n = 522) and streptococci (n = 84). The threshold of detection of the mPCR assay was 10 fg of genomic DNA and <10(3) CFU ml(-1). A comparative evaluation of mPCR with culture method using 115 milk samples from subclinical mastitis showed mPCR to be more efficacious. Subsequently, the mPCR showed successful detection of target bacteria, when applied directly for the assessment of 36 bulk milk samples. CONCLUSION: The developed mPCR assay was found to be simple, rapid, reliable and specific in species identification of 10 bacteria at a time. SIGNIFICANCE AND IMPACT OF THE STUDY: The assay will be useful for the detection of mastitis, testing bacteriological safety of milk and for species level differentiation. The assay will be of value in the dairy sector for diagnosis and research. The early and accurate identification of pathogens will enable timely interventions for the treatment and control of bovine mastitis.

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.

Isolation and Confirmation of Listeria Species from Seafood off Goa Region by Polymerase Chain Reaction.

Several food borne outbreaks have highlighted the importance of Listeria monocytogenes to the public health and have been recognized as an emerging, important food borne pathogen, and a causative agent of listerioses. A number of genes are involved in the manifestation of Listeria virulence, hlyA is one among them. In the present study, 111 marine fish samples including prawns, finfishes and bivalves were screened for the presence of Listeria species. The isolates were characterized biochemically and further L. monocytogenes were confirmed by polymerase chain reaction (PCR) technique using the hlyA gene as a tool to differentiate between L. monocytogenes and other non-pathogenic Listeria species. Out of 111 samples 5 (4.5%) samples were positive for L. monocytogenes. Among the three different types of samples bivalves were found to have maximum percent (12.5) of L. monocytogenes followed by prawns (3.84) and finfishes (2.9). Among all the 111 samples, 15 (13.51%) samples were positive for other Listeria species. It was observed that Listeria occurrence is more in shellfishes than in fin fishes. All the isolates were sensitive towards five different antibiotics in sequence ciprofloxacin > sulphafurazole > norfloxacin > ampicillin and gentamicin.