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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.

AIM To investigate the occurrence of ESBL and colistin-resistant E. coli and its genotypic characterization and identification of virulence determinants in cases of bovine mastitis in three provinces of China. MATERIALS AND METHODS Five hundred and thirty-five milk samples presenting mastitis symptoms were screened for the presence of ESBL, colistin-resistant and different virulence genes. Susceptibility testing was identified by the micro-dilution method. Escherichia coli isolates were used to detect ESBL genes (blaCTX-M , blaSHV and blaTEM ) and colistin-resistant genes mcr-(1-9). Multiplex PCR approach was used for the detection of major blaCTX-M groups, different phylogroups and virulence genes. The clonal relationship was then evaluated with MLST, MLSA and PFGE. RESULTS Multi-drug resistance (MDR) was found in more than 85% of ESBL and colistin-resistant E. coli isolates. Genotypic characterization showed a dominance of the blaCTX-M-1 group, and the most prevalent alleles observed were blaCTX-M-28 (38·37%), blaCTX-M-14 (17·44%), blaCTX-M-66 (13·95%) and blaCTX-M-55 (10·46%). The targeted virulence genes were detected in 97·89% of isolates. Sequence types ST58 and ST410 were the most predominant (2/20 = 20%). The majority of the E. coli isolates carrying ESBL and mcr-1 were clonally unrelated. CONCLUSION High level of association was observed between ESBL-producing and COL-resistance in E. coli of bovine mastitis. SIGNIFICANCE AND IMPACT OF THE STUDY To the best of our knowledge, this is the first report which shows the genetic diversity of ESBL and mcr-1, and various virulent features of E. coli strains isolated from bovine clinical mastitis in three different provinces of China. The major carriers of the blaCTX-M-1 and blaCTX-M-9 were blaCTX-M-28 and blaCTX-M-14 alleles respectively. The association of ESBL-producing E. coli with mcr-1 is of particular concern.

The current study aimed to evaluate the efficacy of selenium nanoparticles (SeNPs), combined or loaded with chitosan (COS), in broiler chickens reared under standard management protocols. The parameters under investigation were production performance, organ development, components of the intestinal barrier, and ileal microbial count. Two hundred and forty day-old chicks were raised in five groups, with each group containing eight replicates (

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

Both mcr-1 phosphoethanolamine transferase enzymes and extended-spectrum β-lactamases (ESBLs) are the main plasmid-mediated mechanisms of resistance to colistin and third-generation cephalosporins,...

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.

Purpose The coexistence of mobile colistin (COL)-resistant gene mcr-1 with extended-spectrum beta-lactamase (ESBL) gene in Escherichia coli has become a serious threat globally. The aim of this study was to investigate the increasing resistance to COL and in particular its coexistence with ESBL-producing E. coli recovered from pig farms in China. Materials and methods E. coli were isolated from 14 pig farms in Jiangsu China. Susceptibility testing was identified by micro-dilution method. PCR assay and nucleotide sequencing were used to detect COL-resistant genes, mcr-1 to -5, as well as ESBL genes, bla CTX-M, bla SHV and bla TEM. Conjugation experiment, plasmid replicon typing of the multidrug resistance (MDR), S1-PFGE and DNA southern hybridization were performed to study the transferability of these genes. Results Overall, 275 E. coli isolates were recovered from a total of 432 cloacal and nasal swabs. More than 90% of the isolates were MDR, of which 70.18% were resistant to COL. Of these 275 isolates, mcr-1 was identified as the most predominant gene carried by 71.63% (197/275) of isolates, 39.59% (78/197) of the isolates were harboring both mcr-1 and ESBL genes (bla CTX-M, bla SHV and bla TEM). ESBL genotyping showed that bla CTX-M was the most predominant ESBL (68.49%) followed by bla SHV (16.4%) and bla TEM (15%). Sequencing revealed that the most common variants of bla CTX-M identified were, bla CTX-M-15 (69%), bla CTX-M-55 (29%) and bla CTX-M-1 (1.8%). IncHI2, IncFIB, IncFIC, IncN and IncX4 were found to be the most common Inc-types found both in donors and in transconjugants and were associated with the transfer of the mcr-1 and ESBL encoding genes. Six strains carried a total of five different plasmids: approximately 97-, 130-, 160-, 227- and 242-kb plasmids. Conclusion The coexistence of the mcr-1- and bla CTX-M-15-carrying isolates displaying high MDR, recovered from E. coli of pig origin, is a major concern for both humans and veterinary medicine.

Bacteriophages have been recently revisited as an alternative biocontrol tool due to the limitations of antibiotic treatment. In this study, we reported on the biological characteristics and genomic information of vB_KpnS_GH-K3 (abbreviated as GH-K3), a Klebsiella phage of the Siphoviridae family, which was previously isolated from a hospital sewage system. One-step growth curve analysis indicated that the burst size of GH-K3 was 291 PFU/cell. GH-K3 maintained a stable titer in a broad range of pH values (6-10) and temperature (up to 50 °C). Based on bioinformatics analysis, GH-K3 comprises of 49,427 bp containing a total of 77 open reading frames (ORFs), which share high degree of nucleotide similarity and close evolutionary relationships with at least 12 other Klebsiella phages. Of note, GH-K3 gp32 was identified as a unique ORF. The major segment of gp32 sequence at the C-terminus (residues 351-907) was found highly variable as determined by its mismatch with the nucleotide and protein sequences available at NCBI database. Furthermore, HHpred analysis indicated that GH-K3 gp32 contains three domains (PDB ID: 5W6S_A, 3GQ8_A and 1BHE_A) similar to depolymerase (depoKP36) of Klebsiella phage KP36 suggestive of a potential depolymerase activity during host receptor-binding in the processes of phage infection. Altogether, the current data revealed a novel putative depolymerase-like protein which is most likely to play an important role in phage-host interaction.

Staphylococcus aureus is one of the most important pathogens causing rabbit necrotizing pneumonia and brings huge economic losses to rabbit production. This study investigated the preventive effect of a phage on rabbit necrotizing pneumonia caused by S. aureus. S. aureus S6 was isolated from the lungs of rabbits suffering necrotizing pneumonia and identified. A novel phage named VB-SavM-JYL01 was isolated by using S. aureus S6 as a host and showed a broader host range than the phages GH15 and K. The genome of VB-SavM-JYL01 lacked bacterial virulence-, antibiotic resistance- and lysogenesis-related genes. A single intranasal administration of VB-SavM-JYL01 (3 × 109 PFU) could effectively improve the survival rate at 48 h to 90% (9/10) compared with the survival rate of 10% and 80% observed with the PBS or linezolid treatment, respectively. The bacterial count in the lungs of rabbits treated with the phage VB-SavM-JYL01 was 4.18 × 104 CFU/g at 24 h, which was significantly decreased compared to that of rabbits treated with PBS (7.38 × 107 CFU/g) or linezolid (3.12 × 105 CFU/g). The phage treatment significantly alleviated lung tissue damage. The levels of total proteins, Panton-Valentine leukocidin (PVL), alpha-toxin (Hla) and cytokines in the lungs of the rabbits treated with the phage were significantly lower than those of the rabbits treated with PBS and similar to those of the rabbits treated with linezolid. These data demonstrate the potential utility of phage as an alternative for preventing rabbit necrotizing pneumonia caused by S. aureus.

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 β-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.

Salmonella enterica subsp. enterica serovar Abortusequi is a frequently reported pathogen causing abortion in mares. In this study, the preventive and therapeutic effects of phage PIZ SAE-01E2 against S. Abortusequi in a mouse model of abortion were investigated. Phage PIZ SAE-01E2 was stable at different temperatures (4 to 70°C) and pH values (pH 4 to 10) and could lyse the majority of the Salmonella serogroup O:4 and O:9 strains tested (25/28). There was no lysogeny-related, toxin, or antibiotic resistance-related gene in the genome of PIZ SAE-01E2. All of these characteristics indicate that PIZ SAE-01E2 has the potential for use in phage therapy. In in vivo experiments, 2 × 103 CFU/mouse of S. Abortusequi ATCC 9842 was sufficient to lead to murine abortion (gestational day 14.5) within 48 h. A single intraperitoneal inoculation of PIZ SAE-01E2 (108 PFU/mouse, multiplicity of infection = 105) 1 h before or after S. Abortusequi challenge provided effective protection to all pregnant mice (10/10). After 24 h of treatment with phage PIZ SAE-01E2, the bacterial loads in both the placenta and the uterus of the infected mice were significantly decreased ( 106 CFU/g). In addition, the levels of inflammatory cytokines in the placenta and blood of the mice in the phage administration groups were significantly reduced (P IMPORTANCES. Abortusequi is an important pathogen that can induce abortions in mares. Although S. Abortusequi has been well controlled in Europe and the United States due to strict breeding and health policies, it is still widespread in African and Asian countries and has proven difficult to control. In China, abortions caused by S. Abortusequi have also been reported in donkeys. So far, there is no commercial vaccine. Thus, exploiting alternative efficient and safe strategies to control S. Abortusequi infection is essential. In this study, a new lytic phage, PIZ SAE-01E2, infecting S. Abortusequi was isolated, and the characteristics of PIZ SAE-01E2 indicated that it has the potential for use in phage therapy. A single intraperitoneal inoculation of PIZ SAE-01E2 before or after S. Abortusequi challenge provided effective protection to all pregnant mice. Thus, PIZ SAE-01E2 showed the potential to block abortions induced by S. Abortusequi in vivo.

Inflammation is the hallmark of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli-induced bovine mastitis. Organic selenium can activate pivotal proteins in immune responses and regulate the immune system. The present study aimed to investigate whether selenomethionine (SeMet) attenuates ESBL E. coli-induced inflammation in bovine mammary epithelial cells (bMECs) and macrophages. Cells were treated with 0, 5/10, 10/20, 20/40, or 40/60 μM SeMet for 12 h and/or inoculated with ESBL-E. coli [multiplicity of infection (MOI) = 5] for 4/6 h, respectively. We assessed inflammatory responses, including selenoprotein S (SeS), Toll-like receptor 4 (TLR4), Ikappa-B (IκB), phospho-NF-κB p65 (Ser536), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and lactate dehydrogenase (LDH) activities. Treatment with 40/60 μM SeMet promoted cell viability and inhibited LDH activities in both bMECs and macrophages. Inoculation with ESBL-E. coli reduced cell viability, which was attenuated by SeMet treatment in bMECs and macrophages. SeMet increased ESBL E. coli-induced downregulation of SeS and decreased LDH activities, TLR4, IκB, phospho-NF-κB p65 (Ser536), IL-1β, and TNF-α protein expressions in bMECs and macrophages. In addition, knockdown of SeS promoted protein expression of TLR4-mediated nuclear factor-kappa (NF-κB) pathway and BAY 11-708 inhibited TNF-α and IL-1β protein levels in bMECs and macrophages after ESBL-E. coli treatment. Moreover, ESBL-E. coli inoculation increased monocyte chemoattractant protein 1 (MCP-1), C–C motif ligand 3 (CCL-3), and CCL-5 mRNA expressions in bMECs. In conclusion, ESBL-E. coli induced expression of MCP-1, CCL-3, and CCL-5 in bMECs and then recruited and activated macrophages, whereas SeMet attenuated ESBL E. coli-induced inflammation through activated SeS-mediated TLR4/NF-κB signaling pathway in bMECs and macrophages.

Salmonella enterica subsp. enterica serovar Abortusequi is a frequently reported pathogen causing abortion in mares. In this study, the preventive and therapeutic effects of phage P(IZ) SAE-01E2 against S. Abortusequi in a mouse model of abortion were investigated. Phage P(IZ) SAE-01E2 was stable at different temperatures (4 to 70°C) and pH values (pH 4 to 10) and could lyse the majority of the Salmonella serogroup O:4 and O:9 strains tested (25/28). There was no lysogeny-related, toxin, or antibiotic resistance-related gene in the genome of P(IZ) SAE-01E2. All of these characteristics indicate that P(IZ) SAE-01E2 has the potential for use in phage therapy. In in vivo experiments, 2 × 10(3) CFU/mouse of S. Abortusequi ATCC 9842 was sufficient to lead to murine abortion (gestational day 14.5) within 48 h. A single intraperitoneal inoculation of P(IZ) SAE-01E2 (10(8) PFU/mouse, multiplicity of infection = 10(5)) 1 h before or after S. Abortusequi challenge provided effective protection to all pregnant mice (10/10). After 24 h of treatment with phage P(IZ) SAE-01E2, the bacterial loads in both the placenta and the uterus of the infected mice were significantly decreased (10(6) CFU/g). In addition, the levels of inflammatory cytokines in the placenta and blood of the mice in the phage administration groups were significantly reduced (P

Bovine mastitis is an inflammatory condition of mammary glands causing huge economic losses for dairy industries. Infection with extended-spectrum β-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-1β (IL-1β), 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.

Biofilm is involved in a variety of infections, playing a critical role in the chronicity of infections. Enterobacter cloacae is a biofilm-forming and multi-drug-resistant (MDR) nosocomial pathogen leading to significant morbidity and mortality. This study aimed at isolation of a bacteriophage against MDR clinical strain of E. cloacae and its efficacy against bacterial planktonic cells and biofilm. A bacteriophage MJ2 was successfully isolated from wastewater and was characterized. The phage exhibited a wide range of thermal and pH stability and demonstrated considerable adsorption to host bacteria in the presence of CaCl2 or MgCl2. Transmission electron microscopy (TEM) showed MJ2 head as approximately 62 and 54 nm width and length, respectively. It had a short non-contractile tail and was characterized as a member of the family Podoviridae [order Caudovirales]. The phage MJ2 was found to possess 11 structural proteins (12–150 kDa) and a double-stranded DNA genome with an approximate size of 40 kb. The log-phase growth of E. cloacae both in biofilm and suspension was significantly reduced by the phage. The E. cloacae biofilm was formed under different conditions to evaluate the efficacy of MJ2 phage. Variable reduction pattern of E. cloacae biofilm was observed while treating it for 4 h with MJ2, i.e., biofilm under static conditions. The renewed media with intervals of 24, 72, and 120 h showed biomass decline of 2.8-, 3-, and 3.5-log, respectively. Whereas, the bacterial biofilm formed with dynamic conditions with refreshing media after 24, 72, and 120 h demonstrated decline in growth at 2.5-, 2.6-, and 3.3-log, respectively. It was, therefore, concluded that phage MJ2 possessed considerable inhibitory effects on MDR E. cloacae both in planktonic and biofilm forms.

Both

A thorough characterisation of the genetics, physiology and metabolism of Escherichia coli has led to the availability of a large number of strains and vectors suitable for recombinant protein expression. Despite the relative ease in using E. coli for achieving amplified expression of many recombinant proteins, for some proteins this can be a frustrating and time-consuming process leading to very low expression or no expression at all. This is especially true for membrane proteins, which introduce additional challenges. A number of factors can be considered and optimised for achieving required levels of amplified expression of recombinant proteins in E. coli that are broadly classified as host strain, expression vector and growth conditions. In this paper we summarise these factors and consolidate the common challenges encountered and approaches to overcome them, focusing in particular on cases where there is low amplified expression or no expression at all of the desired recombinant protein, due to various reasons.

// Tariq Ali 1, * , Sadeeq ur Rahman 2, * , Limei Zhang 1 , Muhammad Shahid 1 , Dandan Han 1 , Jian Gao 1 , Shiyao Zhang 1 , Pamela L. Ruegg 3 , Umer Saddique 4 and Bo Han 1 1 Department of Clinical Veterinary Medicine, College of Veterinary Medicine, China Agricultural University, Beijing, P.R. China 2 College of Veterinary Sciences and Animal Husbandry, Abdul Wali Khan University, Garden Campus, Mardan, Pakistan 3 Department of Dairy Science, University of Wisconsin, Madison, WI, USA 4 Department of Animal Health, The University of Agriculture, Peshawar, Pakistan * These authors have contributed equally and co-first authors Correspondence to: Bo Han, email: hanbo@cau.edu.cn Keywords: E. coli , ESBL, multilocus sequence typing, PCR-based replicon typing, split network analysis Received: June 15, 2017 Accepted: August 23, 2017 Published: October 04, 2017 ABSTRACT 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.

Mycoplasmosis, the respiratory infection, is a well-established cause of heavy economic losses in sheep and goats farming. The occurrence and antibiotics susceptibility profiles of clinical isolates of Mycoplasma mycoides subsp. capri ( Mmc ) from suspected cases of contagious caprine pleuropneumonia (CCPP) were investigated in small ruminants of three climatic- southern, central and northern- regions of Khyber Pakhtunkhwa province of Pakistan. Specie specific-PCR for Mmc confirmed 24% (198/825) isolates with significantly ( P ˂ 0.001) higher number (83/275; 30.18%) recovered from the northern-, followed by southern- (68/275; 24.73%) and central-regions (47/275; 17.09%), respectively. Subsequently, a total of 54 PCR-confirmed isolates (18 from each zone) were subjected to antibiogram assay. Five different common antimicrobial agents including ceftiofur, enrofloxacin, gentamycin, oxytetracycline and tylosin were tested by disc diffusion and broth microdilution methods The antibiotic susceptibility profiles of Mmc isolates indicated that enrofloxacin exhibited the highest ( P ˂ 0.001) in vitro antibacterial activity with a maximum zone of inhibition (19 ± 0.71 mm) and the lowest MIC (0.001 ± 0.0002 mg/mL) amongst the tested antimicrobial agents, followed by gentamycin (11 ± 0.45 mm and 0.01 ± 0.001 mg/mL zone of inhibition and MIC, respectively). All these isolates were found resistant against tylosin, oxytetracycline and ceftiofur sodium: antibiotics that are favored in clinical cases of CCPP. This study concludes a high occurrence of Mmc in small ruminants during infection of CCPP, and increased resistance rate against commonly used antibiotics.

Klebsiella pneumoniae (K. pneumoniae) spp. are important nosocomial and community-acquired opportunistic pathogens, which cause various infections. We observed that K. pneumoniae strain K7 abruptly mutates to rough-type phage-resistant phenotype upon treatment with phage GH-K3. In the present study, the rough-type phage-resistant mutant named K7RR showed much lower virulence than K7. Liquid chromatography-tandem mass spectrometry (LC-MS-MS) analysis indicated that WcaJ and two undefined glycosyltransferases (GTs)- named GT-1, GT-2- were found to be down-regulated drastically in K7RR as compared to K7 strain. GT-1, GT-2, and wcaJ are all located in the gene cluster of capsular polysaccharide (CPS). Upon deletion, even of single component, of GT-1, GT-2, and wcaJ resulted clearly in significant decline of CPS synthesis with concomitant development of GH-K3 resistance and decline of virulence of K. pneumoniae, indicating that all these three GTs are more likely involved in maintenance of phage sensitivity and bacterial virulence. Additionally, K7RR and GT-deficient strains were found sensitive to endocytosis of macrophages. Mitogen-activated protein kinase (MAPK) signaling pathway of macrophages was significantly activated by K7RR and GT-deficient strains comparing with that of K7. Interestingly, in the presence of macromolecular CPS residues (>250 KD), K7(ΔGT-1) and K7(ΔwcaJ) could still be bounded by GH-K3, though with a modest adsorption efficiency, and showed minor virulence, suggesting that the CPS residues accumulated upon deletion of GT-1 or wcaJ did retain phage binding sites as well maintain mild virulence. In brief, our study defines, for the first time, the potential roles of GT-1, GT-2, and WcaJ in K. pneumoniae in bacterial virulence and generation of rough-type mutation under the pressure of bacteriophage.

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 (&lt, 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 (&le, 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.

We report on occurrence of ESBL producing E. coli recovered from poultry meat samples collected from live bird market of district Mardan, Khyber Pakhtunkhwa province, Pakistan. We characterized these isolates for ESBL-genotypes, integron typing, insertion sequence and susceptibility against a panel of 22 different classes of drugs. Our results showed that of 100 samples, 28 E. coliisolates were multidrug resistant showing resistance to more than two antibiotics. The predominant gene wasbla VIM bla NDM-1 4/28 (14.2%). Class-1 integron was detected in 21 (75%), class-2 and class-3 integrons were detected in 25% and 7.1, respectively. ISCR1 element was identified among11isolates (39.28%). Interestingly, ISCR1 was investigated for its link with carbapenemase encoding genes and results showed that it was found linked with two bla VIM genes suggesting involvement in its mobilization.

This study investigated batch-to-batch variations in carbohydrates 1) chemical profile; 2) Cornell Net Carbohydrate and Protein System (CNCPS) carbohydrates subfractions; 3) in situ ruminal degradation kinetics of neutral detergent fiber (NDF); and 4) molecular structure spectral characteristics of carbohydrates (structural carbohydrates, SCHO; cellulosic compounds, CELC; total carbohydrates, TCHO and non-structural carbohydrates, NSCHO) in corn byproducts. The second aim was to correlate the variations in carbohydrates molecular structures with carbohydrates nutritional traits. The corn byproducts selected in this study were corn gluten feed, corn germ, corn germ cake and corn gluten meal. The preprocessed corn kernels were used as control. The results revealed large differences in carbohydrate chemical composition (P

The objectives of this study were to investigate batch-to-batch variation in 1) crude protein (CP) chemical profile; 2) the Cornell Net Carbohydrate and Protein System (CNCPS) CP subfraction profiles 3) ruminal degradation kinetics of CP; 4) the DVE/OEB2010 model estimated metabolizable protein supply to dairy cows; 5) molecular structure spectral characteristics of protein primary (amide I and II) and secondary (α-helix and β-sheet) structures among different types of coproducts and among three batches of corn coproducts using parent corn kernels of each batch as reference; and 6) to correlate the batch-to-batch variation in protein molecular structures to the corresponding variation in CP chemical composition, digestibility and metabolism in dairy cows. Corn coproducts, namely, corn gluten feed, corn germ, corn germ cake and corn gluten meal from the same wet milling manufacturer were evaluated. The parent corn kernel was used as control. The results showed large differences in the CP chemical profile (P

Bovine anaplasmosis, caused by Anaplasma marginale , is a non-contagious tick borne disease. The main objective of the current study was to investigate comparative frequency of anaplasmosis in three different cattle breeds (European breed/ Holstein Friesian and Jersey breed, indigenous breed and cross breed (1st and 2nd pedigree) and transplacental transmission using real time polymerase chain reaction for detection. Of the total 96 blood samples analyzed, our results indicated an overall incidence 45.83% (44/96) of A. marginale with highest incidence 62.5% (20/32) in European breed, followed by 42.4% (14/33) in cross breed and 35.4% (11/31) in indigenous breed. Most importantly, our results indicated that 13.7% (4/29) calves were found positive for the presence of A. marginale indicating transplacental transmission. Furthermore, indirect ELISA revealed an overall incidence rate of 34.3% (33/96) more likely indicating current or previous exposure. Finally, Giemsa staining determined that 15% (15/96) animals were found positive by examining red blood cells. Statistical analysis showed significantly higher (P 0.05) difference was found among the crossbred and indigenous breed of cattle. Moreover, non significant (P> 0.05) effect of age group was observed on the incidence of A. marginale . White blood cell count and mean corpuscular volume were significantly (P

Bacteriophages (phages/viruses) need host bacteria to replicate and propagate. Primarily, a bacteriophage contains a head/capsid to encapsidate the genetic material. Some phages contain tails. Phages encode endolysins to hydrolyze bacterial cell wall. The two main classes of phages are lytic or virulent and lysogenic or temperate. In comparison with antibiotics, to deal with bacterial infections, phage therapy is thought to be more effective. In 1921, the use of phages against bacterial infections was first demonstrated. Later on, in humans, phage therapy was used to treat skin infections caused by Pseudomonas species. Furthermore, phages were successfully employed against infections in animals - calves, lambs, and pigs infected with Escherichia coli. In agriculture, for instance, phages have successfully been used e.g., Apple blossom infection, caused by Erwinia amylovora, was effectively catered with the use of bacteriophages. Bacteriophages were also used to control E. coli, Salmonella, Listeria, and Campylobacter contamination in food. Comparatively, phage display is a recently discovered technology, whereby, bacteriophages play a significant role. This review is an effort to collect almost recent and relevant information regarding applications and complications associated with the use of bacteriophages.

Theβ-lactams—a large class of diverse compounds—due to their excellent safety profile and broad antimicrobial spectrum are considered to be the most widely used therapeutic class of antibacterials prescribed in human and veterinary clinical practices. This, unfortunately, has also given rise to a continuous increased resistance globally in health care settings as well as in the community due to their permanent selective force driving diversification of the resistance mechanism. Resistance againstβ-lactams is increasing rapidly as novelβ-lactamases, enzymes that degradeβ-lactams, are being discovered each day such as recent emergence of extended spectrumβ-lactamases (ESBL) that have the ability to inactivate most of the cephalosporins. The complexity and diversity of ESBL are increasing so rapidly that more than 170 variants have thus far been described for only a single genotype, theblaCTX-M-encoding ESBL. This review is to organize all the current updated literature describing genomic features, organization, and mechanism of resistance and mode of dissemination of all known ESBLs.

Prevalence of zoonotic Mycobacterium bovis (bTB) disease in human population is underreported from the North of Pakistan. Here, we report on the proportion of human bTB disease among the overall TB patients, drug resistance pattern of bTB isolates, and knowledge, attitude, and practices (KAP)-based analysis of bTB disease. For this purpose, sputum samples from a total of 300 clinically diagnosed TB patients and 100 randomly selected school children suspected of pulmonary TB were processed by culture as well as polymerase chain reaction (PCR) for isolation, identification, and confirmation of Mycobacterium tuberculosis (mTB) and bTB species. Isolates of bTB were processed for drug susceptibility tests. Data on KAP regarding TB were obtained on a pretested questionnaire. Sputum-based PCR results indicated that 288/300 (96%) were confirmed as mTB, while 12/300 (4%) were found as bTB diseases. Interestingly, none of the school child was declared positive for either mTB or bTB. Notably, 274/300 (91.3%) positively cultured samples were identified as mTB, 13/300 (4.3%) as bTB, while 5/300 (1.7%) as mixed containing both. Importantly, except one, all of the bTB isolates were found resistant to pyrazinamide. Surprisingly, most of the bTB isolates (~70%) were found resistant to a broad range of first- and second-line anti-TB drugs. SplitsTree and recombination analysis indicated no evidence of intergenic recombination. Finally, residence, occupation, presence of animals at home, and sleeping alongside animals were found significantly associated with occurrence of bTB disease. To the best of our knowledge, we report for the first time on the high (4%) burden of bTB disease in human TB patients in Peshawar, Pakistan.