Correction: Nutritional analysis and characterization of carbapenemase producing-Klebsiella pneumoniae resistant genes associated with bovine mastitis infected cow's milk.

[This corrects the article DOI: 10.1371/journal.pone.0293477.].

Nutritional analysis and characterization of carbapenemase producing-Klebsiella pneumoniae resistant genes associated with bovine mastitis infected cow's milk.

The current study was designed to analyze nutritional parameters and to characterize carbapenemase producing-Klebsiella pneumoniae isolates from bovine mastitic cow's milk. Out of 700 milk samples K. pneumoniae was identified by phenotypic and molecular techniques along with their antibiogram analysis and nutritional analysis was performed using the procedure of Association of Official Analytical Chemists. Carbapenemase-producing K. pneumoniae was detected by phenotypic CarbaNP test followed by molecular characterization of their associated resistant genes blaVIM, blaKPC, blaOXA-48, blaNDM, and blaIMP along with insertion sequence common region 1 (ISCR1) and integrons (Int1, Int2, and Int3) genes. Among nutritional parameters, fat content was observed (2.99%) followed by protein (2.78%), lactose (4.32%), and total solid (11.34%), respectively. The prevalence of K. pneumoniae among bovine mastitis was found 25.71%. Antibiogram analysis revealed that more effective antibiotics was ceftazidime (80%) followed by amikacin (72%), while highly resistant antibiotics was Fusidic acid (100%). Distribution of carbapenemase producer K. pneumoniae was found 44.4%. Among carbapenem resistant genes blaKPC was found 11.25%, blaVIM 2.75%, blaNDM 17.5%, and blaOXA-48 7.5%, while blaIMP gene was not detected. Furthermore, distribution of ISCR1 was found 40%, while integron 1 was found 61.2% followed by integron 2 (20%), and integron 3 (5%). In conclusion, the recent scenario of carbapenemase resistant K. pneumoniae isolates responsible for mastitis may affect not only the current treatment regime but also possess a serious threat to public health due to its food borne transmission and zoonotic potential.

Co-existence of blaIMP, blaNDM-1, and blaSHV, genes of Pseudomonas aeruginosa isolated from Quetta: Antimicrobial resistance and clinical significance.

Objective: Molecular detection and co-presence of carbapenem-resistant genes in the isolates of Pseudomonas aeruginosa are less commonly reported from Quetta. In the present study, we determined to highlight the antibiotic sensitivity profile and genetic mechanism of carbapenem resistance. Methods: The cross-sectional study was conducted from May to September 2018 at the Hi-tech laboratory, Centre for Advance Studies in Vaccinology and Biotechnology, University of Baluchistan, Quetta. Biochemical and molecular methods were ascertained for the recognition of the isolates and minimum inhibitory concentration was performed using E-test and broth microdilution methods. The molecular basis of carbapenemase activity was determined by identifying carbapenemase genes in the isolates. Results: Of the (n=23) P. aeruginosa isolated from pus aspirates obtained from surgical/burn units, we have detected blaIMP (n=7/8) 87.5%, blaNDM-1 (n=5/8) 62.5%, and blaSHV (n=4/8) 50%. The co-existence of multiple antibiotic-resistant genes, blaIMP, blaNDM-1 and blaSHV was found in (n=2/8) 25% isolates. These isolates displayed resistance against a range of antimicrobials from ß-lactams, tetracyclines, cephalosporins, quinolones, monobactams, aminoglycosides, sulphonamides, phosphoric acid, macrolides, and polypeptide groups, suggesting extensive-drug resistance. Conclusion: The emergence of MBL and ESBL producers is an alarming threat in the region. It is of great importance to determine the resistance mechanism of bacterial bugs. The lack of new antimicrobials particularly against gram-negative bacteria is quite alarming worldwide.

Bioinformatics and immunoinformatics approach to develop potent multi-peptide vaccine for coxsackievirus B3 capable of eliciting cellular and humoral immune response.

Coxsackievirus B3 (CVB3) is a viral pathogen of various human disorders with no effective preventative interventions. Herein, we aimed to design a chimeric vaccine construct for CVB3 using reverse vaccinology and immunoinformatics approaches by screening the whole viral polyprotein sequence. Firstly, screening and mapping of viral polyprotein to predict 21 immunodominant epitopes (B-cell, CD8+ and CD4+ T-cell epitopes), fused with an adjuvant (Resuscitation-promoting factor), appropriate linkers, HIV-TAT peptide, Pan DR epitope, and 6His-tag to assemble a multi-epitope vaccine construct. The chimeric construct is predicted as probable antigen, non-allergen, stable, possess encouraging physicochemical features, and indicates a broader population coverage (98 %). The tertiary structure of the constructed vaccine was predicted and refined, and its interaction with the Toll-like receptor 4 (TLR4) was investigated through molecular docking and dynamics simulation. Computational cloning of the construct was carried out in pET28a (+) plasmid to guarantee the higher expression of the vaccine protein. Lastly, in silico immune simulation foreseen that humoral and cellular immune responses would be elicited in response to the administration of such a potent chimeric construct. Thus, the design constructed could vaccinate against CVB3 infection and various CVB serotypes. However, further in vitro/in vivo research must assess its safety and effectiveness.

Synergistic Activity of Tetrandrine and Colistin against mcr-1-Harboring Escherichia coli.

Before the emergence of plasmid-mediated colistin resistance, colistin was once considered the last drug of choice for infections caused by carbapenem-resistant bacteria. Currently, researchers are relentlessly exploring possible alternative therapies that could efficiently curb the spread of drug resistance. In this study, we aim to investigate the synergistic antibacterial activity of tetrandrine in combination with colistin against mcr-1-harboring Escherichia coli. We examined the antibacterial activity of tetrandrine in combination with colistin in vivo and in vitro and examined the bacterial cells by fluorescence, scanning, and transmission electron microscopy (TEM) to explore their underlying mechanism of action. We further performed a computational analysis of MCR-1 protein and tetrandrine to determine the interaction interface of these two molecules. We confirmed that neither colistin nor tetrandrine could, on their own, inhibit the growth of mcr-1-positive E. coli. However, in combination, tetrandrine synergistically enhanced colistin activity to inhibit the growth of E. coli both in vivo and in vitro. Similarly, molecular docking showed that tetrandrine interacted with the three crucial amino acids of the MCR-1 protein in the active site, which might inhibit MCR-1 from binding to its substrates, cause MCR-1 to lose its ability to confer resistance. This study confirmed that tetrandrine and colistin have the ability to synergistically overcome the issue of colistin resistance in mcr-1-harboring E. coli.

Incidence and molecular characterization of ESBL-producing and colistin-resistant Escherichia coli isolates recovered from healthy food-producing animals in Pakistan.

OBJECTIVES: To investigate the occurrence and molecular features of ESBL-producing and colistin-resistant Escherichia coli isolates recovered from healthy food-producing animals in Pakistan. METHODS: A total of 153 E. coli isolates were recovered from 250 faecal samples collected from livestock and poultry. The antibiotic susceptibility, resistant determinants and mobile genetic elements were determined for all the isolates. The clonal relatedness was analysed by MLST. Plasmids harbouring, localization and transferability of mcr-1 gene were carried out by Southern hybridization, S1-PFGE and transconjugation. RESULTS: Out of 153 E. coli strains, 49.01% isolates were ESBLs producers, whereas 18.95% were resistant to colistin and 84.31% of the isolates. Multidrug resistance was found in 84% of the isolates. The ESBL-producing E. coli in buffaloes, cattle, sheep, goat and broilers faecal samples were 60%, 74%, 54%, 50% and 68%, respectively. Among the ESBLs genes, blaCTX-M was the most prevalent group detected in 98.66%, while only mcr-1 of the colistin-resistant genes could be PCR amplified in 29 isolates. The common MGEs found were ISECP1 (35.13%), ISCR1 (33.78%), ISApl1 (20.27%) and Inti1 (58.10%). The most predominant Inc. types found were IncFIB 46.66%, followed by IncFIA 30.66%, IncFIC 26.66%, IncFrepB 26.66%, IncHI2 26.66%, IncP 22.66% and IncX4 21.33%. The most frequent sequence type detected was ST58. Southern blot and S1-PFGE confirmed the plasmid harbouring of mcr-1 gene. CONCLUSION: The co-occurrence of mcr-1 and ESBLs-encoding genes, along with MGEs in E. coli from healthy food animals in Pakistan, is a major concern. SIGNIFICANCE AND IMPACT OF STUDY: Antimicrobial resistance can be transferred from animals to humans by direct contact or via the food chain and environment. The prevalence and co-occurrence of ESBL and colistin resistance genes from food-producing animals is rare in Pakistan. To our knowledge, this is the first report to find ESBLs and mcr-1-harbouring E. coli from the faecal samples of the healthy food-producing animals in Pakistan. The presence of ARGs in association with MGEs, co-harbouring the virulence factors, as determined in the current study, is a severe threat to livestock and the human community as it has horizontally and food web transferability.

Prevalence and Molecular Characterization of Cystic Echinococcosis in Livestock Population of the Malakand Division, Khyber Pakhtunkhwa, Pakistan.

Cystic echinococcosis (CE) is a neglected zoonotic disease prevalent in Pakistan, but the genetic diversity of the cestode is largely unexplored in the country. This study investigated the molecular epidemiology of CE infecting the livestock population of the Malakand division, Khyber Pakhtunkhwa, Pakistan. A total of 1,200 livestock, including buffaloes, cattle, goats, and sheep, were examined for echinococcosis from November 2017-2018 at different slaughterhouses in the Malakand division. Hydatid cysts were collected from different organs, and hydatid cyst fluid (HCF) was examined microscopically and used for DNA extraction. The LSU (rrnl) and NAD1 genes were amplified and sequenced. The overall prevalence of CE was 17% (204/1,200), including cows (21.7%), buffaloes (17.4%), goats (10%), and sheep (9.6%). The infection was relatively more prevalent among males (17%) than females (16.9%) and animals of older age (>5 years) (p = 0.710). Liver (63.2%) and lungs (25%) were more affected as compared to kidneys (6.8%) and heart (4.9%). HCF analysis indicated that 52.0% of the cysts were sterile and (48.0%) were fertile. Sequencing and phylogenetic analyses confirmed 80.0% of the isolates as Echinococcus granulosus sensu stricto (G1-G3) in all animal species, while Echinococcus equinus (G4) and Echinococcus ortleppi (G5) were present in buffaloes. The present study concluded that CE is prevalent in the livestock population of Malakand. Besides E. granulosus s. s. (G1-G3), E. ortleppi genotype (G5) and E. equinus (G4) in livestock were also reported.

Characterization and resistant determinants linked to mobile elements of ESBL-producing and mcr-1-positive Escherichia coli recovered from the chicken origin.

The spread of antimicrobial resistance (AMR) in Escherichia coli is a complex process linked with various mobile genetic elements (MGEs) like plasmids, transposons, and integrons. This study aimed to determine the co-occurrence of ESBL and mcr-1 and their physical linkage with MGEs in E. coli. E. coli strains of chicken origin were obtained from different commercial farms of eastern China from 2010 to 2011. Antimicrobial sensitivity testing, identification of different antibiotic-resistant genes (ARGs), and prevalence and evidence involvement of integrons, ISEcp1, ISCR1, and ISApl1, were determined. A multiplex PCR was used to detect virulence genes and the phylogenetic clustering of isolates. Conjugation experiments, plasmid replicon typing were performed to know the transferability of ARGs and MGEs. A total of 83.33% of isolates were found to be multidrug-resistant (MDR). The incidence rate of blaCTX-M, blaSHV,blaTEM, and mcr-1 was found to be 30%, 10.95%, 8.09%, and 36.66%, respectively. The most prevalent combination was noticed for mcr-1 and blaCTX-M 73%, whereas the most prominent blaCTX-M alleles found, were blaCTX-M-55 46%, followed by blaCTX-M-14 31%, and blaCTX-M-15 13%. The frequency of ISEcp1, ISCR1, ISApl1, and int1 was 27.77%, 53.70%, 51.85%, and 70.37% respectively. Most ß-lactamases, especially blaCTX-M, blaSHV, and blaTEM, were associated with ISEcp1, ISCR1, and Integron 1, whereas the ISAPl1-mcr-1 segment was observed in mcr-1-positive E. coli isolates. Phylogrouping revealed that group A was the most predominant phylotype, whereas the common virulence genes detected in these isolates were EHEC, EAEC, and EPEC. Conjugation assay also indicated that multiple genetic elements were involved; common plasmids identified were FIB 61.11%, followed by IncHI2 48.14%, and FrepB 33.33%. Propagation of such MDR strains carrying multiple resistance elements among the bacterial population is a threat of worry.

Chemical composition, ruminal degradation kinetics, and methane production (in vitro) of winter grass species.

BACKGROUND: Information about the nutritive value, dry matter (DM) digestibility, and methane (CH4 ) emission potential of grass species is required for their optimal utilization in ruminant rations. The present study was designed: (i) to quantify the nutrient profile, mineral composition and in vitro dry matter digestibility (IVDMD) of winter grass species commonly available in northern Pakistan; and (ii) to measure the in vitro gas production (IVGP) and CH4 emission of the grass species during 72 h in vitro ruminal fermentation. Seven grass species, namely, Cenchrus ciliaris, Setaria anceps, Panicum antidotale, P. maximum, Pennisetum purpureum, Pennisetum orientale, and Atriplex lentiformis were assessed. RESULTS: A high level of variability (P < 0.001) was observed among grass species for the content of all measured nutrients, IVDMD, IVGP, and CH4 -production. Notably, the content (g kg-1 DM) of crude protein varied from 59.8 to 143.3, neutral detergent fiber from 560.3 to 717.9, IVDMD from 375.1 to 576.2, and 72 h cumulative IVGP from 97.6 to 227.4 mL g-1 organic matter (OM) and CH4 from 48 to 67 mL g-1 OM. Among the grasses, P. antidotale had greater content (g kg-1 DM) of crude protein (CP) (143.3), IVDMD (576.2), and 72 h cumulative IVGP (227.4 mL g-1 OM), and produced the smallest amount of total CH4 (48 mL g-1 OM) during 72 h fermentation. In contrast, A. lentiformis had the lowest content (g kg-1 DM) of CP (59.8), IVDMD (375.1), 72 h cumulative IVGP (97.6 mL g-1 OM), and produced a greater amount of total CH4 (67 mL g-1 OM) during 72 h fermentation. CONCLUSION: The findings of the current study highlight that it is possible to select and further develop grass species with high nutritional value and lower CH4 -production, which can improve livestock productivity, farm profitability, and long-term environment sustainability. © 2020 Society of Chemical Industry.

In vitro immune responses of bovine mammary epithelial cells induced by Escherichia coli, with multidrug resistant extended-spectrum ß-lactamase, isolated from mastitic milk.

Bovine mastitis is an inflammatory condition of mammary glands causing huge economic losses for dairy industries. Infection with extended-spectrum ß-lactamase (ESBL)-producing sequence types (ST) 410-Escherichia coli (ESBL-ST410 E. coli) is considered a leading cause of bovine mastitis in China. However, pathogenic effects of these strains in an in vitro model, e.g. bovine mammary epithelial cells (bMECs), are unknown. Therefore, our objectives were to explore pathogenesis (adhesion and invasion, inflammation, oxidative stress and apoptosis) of ESBL-E. coli (highly prevalent in bovine mastitis) in bMECs. Non-pathogenic E. coli DH5α and a prototypical E. coli P4 were included as negative and positive controls, respectively. The bMECs were infected with our isolated ST410 strains, plus DH5α and P4, with assessment of the following end points: adhesive and invasive capabilities; lactate dehydrogenase (LDH) activities; inflammatory responses, including concentrations of interleukin-1ß (IL-1ß), IL-6, IL-10 and tumor necrosis factor-α; oxidative stress including intracellular reactive oxygen species production, malondialdehyde concentrations, activities of glutathione peroxidase and superoxide dismutase; and apoptosis. All ST410 strains had greater adhesive and invasive capabilities and increased LDH release, with varying degrees of inflammatory responses, oxidative stress and apoptosis compared to blank and DH5α groups, similar to P4-infected bMECs. In particular, ST410(4) was more likely than the other 3 isolates to adhere to and invade bMECs and increase LDH activities, cytokine release, oxidative stress and apoptosis. Thus, ST410 isolates had pathogenic manifestations of adhesive and invasive capabilities; furthermore, they induced inflammation, oxidative stress and apoptosis in bMECs. Finally, ST410(4) was the most pathogenic strain.

The Characteristics and Genome Analysis of vB_AviM_AVP, the First Phage Infecting Aerococcus viridans.

Aerococcus viridans is an opportunistic pathogen that is clinically associated with various human and animal diseases. In this study, the first identified A. viridans phage, vB_AviM_AVP (abbreviated as AVP), was isolated and studied. AVP belongs to the family Myoviridae. AVP harbors a double-stranded DNA genome with a length of 133,806 bp and a G + C content of 34.51%. The genome sequence of AVP showed low similarity (<1% identity) to those of other phages, bacteria, or other organisms in the database. Among 165 predicted open reading frames (ORFs), there were only 69 gene products exhibiting similarity (≤65% identity) to proteins of known functions in the database. In addition, the other 36 gene products did not match any viral or prokaryotic sequences in any publicly available database. On the basis of the putative functions of the ORFs, the genome of AVP was divided into three modules: nucleotide metabolism and replication, structural components, and lysis. A phylogenetic analysis of the terminase large subunits and capsid proteins indicated that AVP represents a novel branch of phages. The observed characteristics of AVP indicate that it represents a new class of phages.

Characteristics and genetic diversity of multi-drug resistant extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli isolated from bovine mastitis.

A characterization of the drug resistance profiles, identification of PCR-based replicon typing, and multilocus sequence typing (MLST) and analysis of 46 ESBL-producing Escherichia coli from cows with mastitis are described. All multidrug-resistant isolates of various phylogenetic groups (A = 31, B1= 3, B2 = 2, D = 10) were ESBL-producers of genotypes CTX-M-15 (29), CTX-M-55 (4), CTX-M-14 (4), CTX-M-3 (1), CTX-M-1 (1), TEM (22) and SHV (8) that were found on conjugative plasmids of diverse incompatibility groups (primarily IncF). Transconjugation experiments indicated successful (100%) trans-conjugation, which was verified phenotypically and genotypically. A total of 28 sequence types (ST) were identified, with 10% of isolates being ST410, and 9 other ST that were assigned arbitrary numbers, reflecting the degree of diversity. Multilocus sequence analysis revealed two lineages, a dominant and a small lineage. Split-decomposition showed intraspecies recombination clearly contributed in genetic recombination generating genotypic diversity among the isolates, and a lack of interspecies recombination. This coherent analysis on genetic structure of multidrug-resistant pathogenic E. coli population isolated from mastitic-milk weaponized with resistance elements from a large, rapidly developing country will be a helpful contribution for epidemiology and surveillance of drug resistance patterns, and understanding their global diversity.

Type V secretion: from biogenesis to biotechnology.

The two membranes of Gram-negative bacteria contain protein machines that have a general function in their assembly. To interact with the extra-cellular milieu, Gram-negatives target proteins to their cell surface and beyond. Many specialized secretion systems have evolved with dedicated translocation machines that either span the entire cell envelope or localize to the outer membrane. The latter act in concert with inner-membrane transport systems (i.e. Sec or Tat). Secretion via the Type V secretion system follows a two-step mechanism that appears relatively simple. Proteins secreted via this pathway are important for the Gram-negative life-style, either as virulence factors for pathogens or by contributing to the survival of non-invasive environmental species. Furthermore, this system appears well suited for the secretion of biotechnologically relevant proteins. In this review we focus on the biogenesis and application of two Type V subtypes, the autotransporters and two-partner secretion (TPS) systems. For translocation across the outer membrane the autotransporters require the assistance of the Bam complex that also plays a generic role in the assembly of outer membrane proteins. The TPS systems do use a dedicated translocator, but this protein shows resemblance to BamA, the major component of the Bam complex. Interestingly, both the mechanistic and more applied studies on these systems have provided a better understanding of the secretion mechanism and the biogenesis of outer membrane proteins. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.