Isolation and characterization of a broad-spectrum phage of multiple drug resistant Salmonella and its therapeutic utility in mice.

Salmonella are causes of livestock, poultry, and other animal diseases but they also have the potential to infect people. Currently, antibiotics are the first choice for treatment of Salmonella infections. Thus, the utility of phage has become the research focus for scientists for several reasons. There are efficient, non-toxic, ubiquitous, easy to prepare and can result in the lysis of host bacteria. In this study, a broad spectrum bacteriophage of Salmonella was isolated from the fecal samples of a poultryfarm and we studied the morphological aspects, thermal stability, pH stability, optimal multiplicity of infection (MOI), and one-step growth curve of this phage. This phage was named Salmonella phage SaFB14 and lysed 54.12%(105/194)Salmonella spp. SaFB14 belongs to the Siphoviridae and has a polyhedron head with a diameter approximating 60 × 60 nm and a tail approximating 140 nm. The optimum growth temperature was 37 °C and maintained high activity over a widepH range(pH3-10) with an optimum of pH 7.0. The optimal MOI was 0.1. A one-step growth curve showed that its latency time was 10 min, burst time was 70 min, and burst was 23 particles. In order to study the therapeutic effect of phage SaFB14 in infected mice, mice were challenged with 2 × 109 CFU/mouse Salmonella (cs20130523-001-1). Each mouse was injected to 2 × 1010 PFU SaFB14 1 h later. SaFB14 protected 40% of mice from infection. Then, the same dose of phage was given to mice for 3 days continuously. After 3 days treatment, the survival rate increased to 60%.In conclusion, phage SaFB14 showed wide host range and good activity in vivo, it is promising against diseases caused by Salmonella.

A Phage-Like IncY Plasmid Carrying the mcr-1 Gene in Escherichia coli from a Pig Farm in China.

We report here a new type of plasmid that carries the mcr-1 gene, the pMCR-1-P3 plasmid, harbored in an Escherichia coli strain isolated from a pig farm in China. pMCR-1-P3 belongs to the IncY incompatibility group and is a phage-like plasmid that contains a large portion of phage-related sequences. The backbone of this plasmid is different from that of other mcr-1-carrying plasmids reported previously.

Isolation and characterization of a multidrug-resistant Staphylococcus aureus infecting phage and its therapeutic use in mice.

In recent years, the emergence of multidrug-resistant bacteria has limited the selection of drugs for treating bacterial infections, reduced clinical efficacy, and increased treatment costs and mortality. It is urgent to find alternative antibiotics. In order to explore a new method for controlling methicillin-resistant Staphylococcus aureus (S. aureus), this study isolated and purified a multidrug-resistant S. aureus broad-spectrum phage JPL-50 from wastewater. JPL-50 belongs to the Siphoviridae family after morphological observation, biological characterization, and transmission electron microscopy (TEM) fragmentation spectrum analysis. It can cleave 84% of tested S. aureus (168/200), in which 100% of tested mastitis-associated strains (48/48) and 72.04% of MRSA strains (67/93) were lysed. In addition, it has an optimal growth temperature of about 30°C, a high activity within a wide pH range (pH 3-10), and an optimal multiplicity of infection of 0.01. The one-step growth curve shows a latent time of 20 min, an explosive time of 80 min. JPL-50 was 16 927 bp in length and was encoded by double-stranded DNA, with no genes associated with bacterial resistance or virulence factors detected. In a therapeutic study, injection of the phage JPL-50 once and for 7 times in 7 days protected 40% and 60% of the mice from fatal S. aureus infection, respectively. More importantly, JPL-50-doxycycline combination could effectively inhibit host S. aureus in vitro and reduce the use of doxycycline within 8 h. In conclusion, the bacteriophage JPL-50 has a wide lysis spectrum, high lysis rate, high tolerance to extreme environments, and moderate in vivo activity, providing ideas for developing multidrug-resistant S. aureus infections.

Molecular epidemiological characteristics and genetic evolutionary relationships of methicillin-resistant Staphylococcus aureus of different avian origins in Qingdao, China, using whole-genome sequencing.

Introduction: To understand the prevalence of avian methicillin-resistant Staphylococcus aureus (MRSA) and the current status of drug resistance in Qingdao, a comprehensive molecular epidemiological investigation and analysis of evolutionary relationships of MRSA isolates from broiler and layer chickens and waterfowl was conducted. Material and Methods: One hundred and two avian MRSA strains were identified by multi-locus sequence typing, staphylococcal protein A (spa) and staphylococcal cassette chromosome mec (SCCmec) typing, and whole-genome sequencing. Results: The sequence type (ST) 9-t899-SCCmec IVb type represented the highest proportion of avian-derived MRSA strains (71.57%), with ST398 type strains occasionally observed in broilers and waterfowl. The poultry-derived MRSA strains were all resistant to eight or more antimicrobials. Avian-derived MRSA strains carried 20 resistance genes, 109 virulence genes and 10 plasmids. Strains carrying the cfr oxazolidinone resistance gene were occasionally seen in broiler- and layer-derived MRSA. Single nucleotide polymorphism (SNP) core genome evolution and locus difference analysis showed that the closest strains were all of ST9-t899 type (to which also affiliated the highest number of strains) and this type occurred on all three kinds of poultry farm, but the SNP difference loci between strains of the same type ranged from 0 to 1472. Conclusion: The dominant type of MRSA from different poultry sources in Qingdao is ST9-t899-SCCmec IVb, which is commonly resistant to a variety of antimicrobial drugs and carries a variety of resistance genes and a large number of virulence genes. Sequence type 9-t899 type is widely spread among the three kinds of poultry investigated, but there are differences in affiliations.

Changes in antibiotic resistance of Escherichia coli during the broiler feeding cycle.

The purpose of this study was to investigate the drug-resistant phenotypes and genes of Escherichia coli in animal, environmental, and human samples before and after antibiotic use at a large-scale broiler farm to understand the respective effects on E. coli resistance during the broiler feeding cycle. The antibiotic use per broiler house was 143.04 to 183.50 mg/kg, and included tilmicosin, florfenicol, apramycin, and neomycin. All strains isolated on the first day the broilers arrived (T1; day 1) were antibiotic-resistant bacteria. E. coli strains isolated from animal samples were resistant to ampicillin, tetracycline, and sulfamethoxazole (100%), and those isolated from environmental samples were resistant to 5 different drugs (74.07%, 20 of 27). E. coli strains isolated on the last day before the broilers left (T2; day 47) had a higher resistance rate to florfenicol (100%, 36 of 36) than at T1 (P < 0.05). Multidrug resistance increased from T1 (84.21%, 32 of 38) to T2 (97.22%, 35 of 36). Most strains were resistant to 5 classes of antibiotics, and 2 strains were resistant to 6 classes of antibiotics. Among 13 identified drug resistance genes, 11 and 13 were detected at T1 and T2, respectively. NDM-1 was detected in 4 environmental samples and 1 animal sample. In conclusion, the use of antibiotics during breeding increases E. coli resistance to antibacterial drugs. Drug-resistant bacteria in animals and the environment proliferate during the feeding cycle, leading to the widespread distribution of drug resistance genes and an increase in the overall resistance of bacteria.

Risk Prevention and Control Points Through Quantitative Evaluation of Campylobacter in a Large Broiler Slaughterhouse.

Chickens contaminated with Campylobacter are a major risk factor for human Campylobacter disease. As a result of the slaughter process, infections should be strictly controlled due to complete exposure of the chickens and the cross-contamination of pathogens. Using @RISK software, quantitative evaluation models of Campylobacter contamination during slaughtering in a large broiler slaughterhouse were constructed. Broiler scalding was set as the starting point of evaluation and four major processes including defeathering, eviscerating, pre-cool rinsing, and splitting-transmission were included. Through the simulation of the constructed model, 90% probability of Campylobacter in 100 g chickens after slaughtering were distributed between 0.3 and 50.2 MPN, which was consistent with simulated actual monitoring data 0-16.6 MPN, indicating that the model shows high credibility. In addition, growth curves of Campylobacter during whole slaughtering showed that contamination significantly increased after defeathering, and increased again after pre-cool rinsing. Using correlation coefficients to analyze the sensitivity of each parameter in the model, it was determined that the concentration of Campylobacter in the pre-cool pond water (correlation coefficient: 0.95) was the most critical risk point of sanitary control in this slaughterhouse. In conclusion, this study is the first to incorporate environmental factors during broiler slaughtering into the risk evaluation of Campylobacter contamination, which provides guidance for the sanitary control and risk management of Campylobacter contamination during broiler slaughtering.

Surveillance of antimicrobial resistance among Escherichia coli from chicken and swine, China, 2008-2015.

The objective of this study was to investigate the antimicrobial resistance trend in Escherichia coli from food animals in China. During 2008-2015, a total of 15,130 E. coli were isolated from chicken and swine from seven provinces. The susceptibilities of these isolates to nine classes of antimicrobial agents were determined using broth microdilution susceptibility method. The findings of this study include: (1) multi-drug resistance was highly prevalent in E. coli; (2) these E. coli isolates showed high resistant rate (>80%) to several old drugs, including ampicillin, tetracycline and sulfisoxazole; (3) increasing resistance to colistin, florfenicol and ceftiofur was observed; (4) the E. coli isolates from different provinces had different resistance patterns. All these data highlight the rising problem of antimicrobial resistance. It is urgent to improve the management of animal production and enhance the proper use of antimicrobials in China as well as the other countries.

Molecular classification and drug resistance analysis of Escherichia coli isolated from poultry in China.

We aimed to understand the distribution of Escherichia coli in poultry and to reveal the virulence factors, the drug resistance and molecular epidemic regularity and characteristics of isolate strains from 6 provinces in China and to complete the characteristics of E. coli for the risk assessment. A total of 87 E. coli isolates were analyzed with 7 virulence genes by PCR drug sensitivity test in 13 kinds of antimicrobial agents and analyzed with PFGE and MLST genotyping. The PFGE genotyping of 87 isolates yielded 75 PFGE type. MLST analysis of isolates identified the 39 STs, the 7 housekeeping genes had the different variation. The most prevalent virulence genes were iucD (74.7%), followed by iss (55.2%), Irp2 (43.7%), tsh (28.7%), cva (19.5%), papC (9.2%) and vat (8.1%). All isolates were resistant to two or three antimicrobial agents highly resistant to SXT, TE (85.06%), SF (83.91%), AM (66. 67%), to fluoroquinolones (ENR, 63.22%, NOR, 50.57%) and to GM (57.47%). E. coli strains resistant spectrum was wide gene was polymorphism the distribution had a certain timeliness and regional in part region of China. These were a solid foundation for the epidemiological investigation and traceability laid.

Developing and optimizing an immunoaffinity cleanup technique for determination of quinolones from chicken muscle.

An immunoaffinity chromatographic method was developed using an antibody mediated immunosorbent to selectively extract and purify 10 quinolones (marbofloxacin, norfloxacin, ciprofloxacin, lomefloxacin, danofloxacin, enrofloxacin, difloxacin, sarafloxacin, oxolinic acid, and flumequine) in chicken muscle followed by HPLC. The operating conditions of the immunoaffinity chromatography (IAC) column were optimized, and the IAC has been successfully used for the isolation and purification of 10 quinolones from chicken muscle tissue. The optimized immunoaffinity column sample cleanup procedure combined with HPLC coupling to fluorescence detection afforded low limits of detection (0.1 ng g(-1) for danfloxacin and 0.15 ng g(-1) for all other quinolones tested). The method was also applied to determine quinolone residues in commercial muscle samples.