Effect of florfenicol on nirS-type denitrifying communities structure of water in an aquatic microcosm model.

Florfenicol is used worldwide for its low side effects and strong bactericidal effect. Florfenicol is physicochemically stable and can persist in natural water bodies and affect water denitrification. Indoor aquatic microcosm models were constructed and water samples were collected at different florfenicol concentrations (0.1, 1, 10, and 100 mg/L) on days 0, 7, 30, and 60 to extract the microbial genome DNA and determine the water properties. qPCR and amplicon sequencing were used to study the dynamic changes of nirS gene and nirS-type denitrifying communities structure, diversity and abundance, respectively. The results showed that higher florfenicol concentrations caused accumulation of nitrate and ammonium nitrogen in water. Florfenicol stress caused orders of magnitude changes in nirS gene abundance, showing a trend of increasing first and then decreasing. 100 mg/L florfenicol addition led to a sustained increase of nirS gene abundance in water bodies. The florfenicol addition altered denitrifying community structure and suppressed the richness and diversity index of denitrifying bacteria in water body. Over time, the richness and diversity index gradually recovered. Proteobacteria was always the dominant denitrifying phylum in water. The relative abundance of Pseudomonas and beta proteobacterium showed obvious positive correlation with nirS gene abundance and were the dominant genera under florfenicol stress. Our study provided a scientific basis for the rational use of florfenicol in aquaculture to maintain a healthy and stable microecological environment, and also provided a preliminary understanding of the response characteristics of water denitrifying microorganisms to florfenicol exposure.

Effects of Florfenicol on nirS-Type Denitrification Community Structure of Sediments in an Aquatic Microcosm Model.

Florfenicol is one of the most widely used antibiotics in aquaculture and veterinary clinics because of its low side effects and strong bactericidal effect. A total of 45~60% of florfenicol is not absorbed by the animal body and accumulates in the aquatic environment through a variety of pathways, which affects denitrification. Indoor aquatic microcosm models were constructed and sediment samples were collected at different florfenicol concentrations (0.1, 1, 10, and 100 mg/L) on days 0, 7, 30, and 60 to extract the microbial genome DNA and determine the water properties. qPCR and amplicon sequencing were used to study the dynamic changes in the nirS gene and nirS-type denitrification community structure, diversity, and abundance, respectively. The results showed that high florfenicol stress influenced the sediment's physicochemical properties, reducing conductivity, alkaline dissolved nitrogen, and organic matter content. In addition, the abundance of nirS, a functional denitrification gene, increased obviously with increased florfenicol concentrations but decreased the diversity of nirS-type denitrification microorganisms. Proteobacteria was the dominant denitrifying phylum in the sediment. Our study provides a scientific basis for the rational use of florfenicol in aquaculture to maintain a healthy and stable microecological environment and also provides a preliminary understanding of the response characteristics of water denitrifying microorganisms to florfenicol exposure.

Resistance and virulence gene analysis and molecular typing of Escherichia coli from duck farms in Zhanjiang, China.

Introduction: The widespread use of antibiotics in animal agriculture has increased the resistance of Escherichia coli, and pathogenic E. coli often harbor complex virulence factors. Antimicrobial resistance in pathogenic bacteria can cause public health problems. Correlation analyses of the resistance, virulence, and serotype data from the pathogenic bacteria found on farms and in the surrounding environment can thus provide extremely valuable data to help improve public health management. Methods: In this investigation, we have assessed the drug resistance and virulence genes as well as the molecular typing characteristics of 30 E. coli strains isolated from duck farms in the Zhanjiang area of China. Polymerase chain reaction was used to detect the drug resistance and virulence genes as well as serotypes, and whole-genome sequencing was used to analyze the multilocus sequence typing. Results: The detection rates for the oqxA resistance gene and fimC virulence gene were highest (93.3%, respectively). There were no correlations between the drug resistance and virulence gene numbers in the same strain. The epidemic serotype was O81 (5/24), ST3856 was an epidemic sequence type, and strains I-9 and III-6 carried 11 virulence genes. The E. coli strains from the duck farms in the Zhanjiang area were thus found to have a broad drug resistance spectrum, various virulence genes, complex serotypes, and certain pathogenicity and genetic relationship. Discussion: Monitoring the spread of pathogenic bacteria and the provision of guidance regarding the use of antibiotics in the livestock and poultry industries will be required in the future in the Zhanjiang area.

Effects of antibiotics (enrofloxacin) on microbial community of water and sediment in an aquatic ecological model.

In order to explore the impact of antibiotics (enrofloxacin) on microbial community in aquatic environment, an indoor aquatic ecological model was built, and different concentrations of enrofloxacin (0.05, 0.5, 5, and 50 mg/L) were added in the aquatic ecological model. In addition, the water and sediment samples were collected on the 0, 7, 30, and 60 days, and the changes in microbial community were studied through 16S rDNA high-throughput sequencing. The results showed that when the concentration of enrofloxacin was 50 mg/L, the relative abundance of Actinomycetes was increased. In the water, the bacterial richness and diversity communities first decreased and then gradually recovered with the passage of time; On the 7th day, the diversity and richness index of species in the treatment groups with enrofloxacin at 5 and 50 mg/L decreased to the lowest; On the 30th day, the diversity and richness index of species began to rise; On the 60th day, the diversity index and richness index of water species began to increase, while the diversity index and richness index of sediment species decreased. In conclusion, the addition of enrofloxacin negatively affected the microbial community structure in an indoor aquatic ecological model, 50 mg/L enrofloxacin could increase the relative abundance of Actinomycetes, and decrease the diversity and richness index of water and sediment.

Effects of Broad-Spectrum Antibiotic (Florfenicol) on Resistance Genes and Bacterial Community Structure of Water and Sediments in an Aquatic Microcosm Model.

This study evaluates the effects of a broad-spectrum antibiotic (florfenicol) on antibiotic resistance genes (ARGs) and bacterial community structure in aquatic environments. We constructed an indoor aquatic microcosm model, adding different concentrations of florfenicol (0.1, 1, 10, 100 mg L-1), and water and sediment samples were collected after 0, 7, 30, and 60 days. qPCR and 16S rDNA amplicon sequencing were used to study the changes in the ARGs and bacterial community structure of the collected samples. The results show that the inclusion of florfenicol resulted in an increased abundance of the floR and optrA genes. Adding 100 mg L-1 florfenicol to the water increased the abundance of optrA gene copies with the maximum on the Day 7, and increased the abundance of floR gene copies with the maximum on Day 30. Adding 100 mg L-1 florfenicol to the sediment increased the abundance of floR and optrA genes by one order of magnitude on Day 60. Meanwhile, the average number of operational taxonomic units (OTUs) in the water samples was 257, and the average number of OTUs in sediment samples was 823. The bacterial community diversity and richness in sediments were higher than those in water. The difference between the maximal and minimal values of the Shannon diversity index in the water and sediment samples was 4.36 and 1.95, respectively. The effect of florfenicol on the bacterial community structure in water was much higher than that in sediment. At 30 days, the diversity index and richness index of the florfenicol treatment groups with 1 and 10 mg L-1 concentrations began to increase; at 60 days, the diversity and richness indices of the 100 mg L-1 florfenicol treatment group began to increase. The samples at the same sampling time in the sediments clustered closer together. The results of this study provide a scientific basis for guiding the rational use of florfenicol in aquaculture, maintaining a healthy and stable microecological environment in aquaculture, and provide theoretical data for environmental ecological risk assessment and safety management caused by microbial resistance under the abuse of florfenicol.

Untangling an AGS Outbreak Caused by the Recombinant GII.12[P16] Norovirus With Nanopore Sequencing.

For a rapidly spreading virus such as NoV (norovirus), pathogen identification, genotype classification, and transmission tracing are urgent for epidemic control. Here, we applied the Nanopore metatranscriptomic sequencing to determine the causative pathogen of a community AGS (Acute gastroenteritis) outbreak. The results were also confirmed by RT-PCR. The NGS (Next Generation Sequencing) library was constructed within 8 hours and sequence analyses were carried out in real-time. NoV positive reads were detected in 13 of 17 collected samples, including two water samples from sewage treatment tank and cistern. A nearly complete viral genome and other genome fragments could be generated from metatranscriptomic sequencing of 13 samples. The NoV sequences from water samples and cases are identical suggesting the potential source of the outbreak. The sequencing results also indicated the outbreak was likely caused by an emerging recombinant GII.12[P16] virus, which was only identified in the United States and Canada in 2017-2018. This is the first report of this emerging variant in mainland China, following the large outbreaks caused by the recombinant GII.17[P17] and GII.2[P16] in 2014 and 2016, respectively. Closely monitoring of the prevalence of this recombinant strain is required. Our data also highlighted the importance of real-time sequencing in emerging pathogens' surveillance.

Viral infection and transmission in a large, well-traced outbreak caused by the SARS-CoV-2 Delta variant.

The SARS-CoV-2 Delta variant has spread rapidly worldwide. To provide data on its virological profile, we here report the first local transmission of Delta in mainland China. All 167 infections could be traced back to the first index case. Daily sequential PCR testing of quarantined individuals indicated that the viral loads of Delta infections, when they first become PCR-positive, were on average ~1000 times greater compared to lineage A/B infections during the first epidemic wave in China in early 2020, suggesting potentially faster viral replication and greater infectiousness of Delta during early infection. The estimated transmission bottleneck size of the Delta variant was generally narrow, with 1-3 virions in 29 donor-recipient transmission pairs. However, the transmission of minor iSNVs resulted in at least 3 of the 34 substitutions that were identified in the outbreak, highlighting the contribution of intra-host variants to population-level viral diversity during rapid spread.

Identification of antimicrobial resistance genes and drug resistance analysis of Escherichia coli in the animal farm environment.

BACKGROUND: Antibiotics are widely used to prevent and control diseases and infection for reducing the morbidity and mortality of animals, because of the high-density stocking in modern food-source animal production. However, the overuse of antibiotics in animal farms results in antimicrobial resistance (AMR), and causes public health issues through the food chain. Therefore, the AMR analysis of the farms and their surrounding environments is great significance to public health. METHODS: To investigate the distribution of AMR genes and analyze the antimicrobial drug resistance of Escherichia coli in feces and surrounding soil of animal farm in Zhanjiang, China. E. coli was isolated and identified through PCR, and the distribution of 21 common antimicrobial drug resistance genes were also detected by using PCR. The minimum inhibitory concentration (MIC) of the isolated E. coli strains against 22 drugs was detected using the broth double dilution method. RESULTS: The results showed that the different AMR genes were detected in both feces and soil, and the detection rate of each AMR gene was higher than 50%. The detection rate of most AMR genes in feces was higher than those in soil. Besides, the isolated 88 strains of E. coli were resistant to 22 kinds of antimicrobial drugs. The highest drug resistance rate (100%) was observed for amoxicillin, colistin, doxycycline and oxytetracycline, and the drug resistance rate of cephalosporins was less than 10%. The drug resistance rate of the isolated strains of E. coli from feces was higher than those from soil, however, in both of feces and soil, most of the isolated strains of E. coli from (77.55% of isolates from feces, 79.49% of isolates from soil and total 78.41%) showed multi-drug resistance (resistant to 15-22 drugs). CONCLUSION: Overall, the detection rate of AMR genes in feces and soil from hog farms was high, and the isolated strains of E. coli from both feces and soil showed multi-drug resistance. Also, the results showed that the AMR genes and drug resistance in the feces and soil from the hog farms are similar. These findings suggested that the AMR genes could be transmitted horizontally from the animal feces to surrounding environments of farms. Therefore, it is urgent need to strengthen the monitoring and guide the rational use of antimicrobial drugs in the hog industry of Zhanjiang, China.

Molecular surveillance of coxsackievirus A16 in southern China, 2008-2019.

A national surveillance system on hand, foot, and mouth disease (HFMD) was launched in 2008 in China. Since then, millions of HFMD cases have been reported each year, with enterovirus A71 (EV-A71), coxsackievirus A16 (CV-A16), and coxsackievirus A6 (CV-A6) as the major causative pathogens. Long-term surveillance of viral infection rates and genetic changes is essential for understanding the disease epidemiology pattern. Here, we analyzed molecular surveillance data on CV-A16 covering a period of 12 years (2008-2019) in Guangdong, China, one of the regions reporting the largest number of HFMD cases. Full VP1 sequences of 456 strains were determined to examine the genetic diversity and changes in the distribution of CV-A16 variants. Our study revealed an irregular pattern of CV-A16 infections in Guangdong. Different from the cyclic epidemics observed in some Asia-Pacific regions, there was a continuously high CV-A16 infection rate from 2008 to 2014, and after a period of lower epidemic activity in 2015-2017, an upsurge of CV-A16 infection was observed in 2018-2019. Cocirculation of subgenotypes B1a and B1b was observed, but while subgenotype B1a was predominant from 2008 to 2012, it appears to have been replaced by B1b, which has circulated as the predominant subgenotype since 2013. Phylogenetic analysis showed that most of the circulating CV-A16 strains are endemic, with occasional transmission between neighboring regions. The re-emergence of B1a in 2016-2019 in Guangdong was likely the result of introduction(s) from Southeast Asia. These results highlight the importance of continuous molecular surveillance from different areas, which will improve our understanding of the origin of the epidemic and facilitate the development of strategies for HFMD disease control.

Capturing noroviruses circulating in the population: sewage surveillance in Guangdong, China (2013-2018).

Noroviruses (NoVs) are the leading cause of acute gastroenteritis (AGE) outbreaks. Since 2014, novel genetic variants of NoV have been continuously identified and have caused a sharp increase in the number of AGE outbreaks. The specific geographical distribution and expanding genetic diversity of NoV has posed a challenge to conventional surveillance. Here, we describe the long-term dynamic correlation between NoV distribution in sewage and in the local population through the molecular surveillance of NoV in Guangdong, 2013-2018. The relative viral loads of the GI and GII genotypes in sewage were calculated through RT-PCR. A high-throughput sequencing method and operational taxonomic unit (OTU) clustering pipeline were developed to illustrate the abundances of different genotypes and genetic variants in sewage. Our results showed that the NoV viral loads and the emergence of new variants in sewage were closely associated with NoV outbreak risks in the population. Compared with the outbreaks surveillance, the dominance of the newly emerged variants, GII.P17-GII.17 and GII.P16-GII.2, could be detected one or two months ahead in sewage of a hub city. In addition, the dynamics of pre-epidemic variants, which were rarely detected in clinics, could be captured through sewage surveillance, thus improving our understanding of the origin and evolution of these novel epidemic variants. Our data highlight that sewage surveillance could provide nearly real-time and high-throughput data on NoV circulation in the community. With the advances in sequencing techniques, the sewage surveillance system could also be extended to other related infectious diseases.

Antimicrobial Resistance of Escherichia coli From Aquaculture Farms and Their Environment in Zhanjiang, China.

Antimicrobial resistance (AMR) has become a major concern worldwide. To evaluate the AMR of Escherichia coli in aquaculture farms of Zhanjiang, China, a total of 90 samples from the water, soil, and sediment of three aquaculture farms (farms I, II, and III) in Zhanjiang were collected, and 90 strains of E. coli were isolated for drug resistance analysis and AMR gene detection. The results indicated that the isolated 90 strains of E. coli have high resistance rates to penicillin, amoxicillin, ampicillin, tetracycline, compound sulfamethoxazole, sulfisoxazole, chloramphenicol, florfenicol, and rifampin (≥70%). Among these antimicrobial drugs, the resistance rate to rifampicin is as high as 100%. Among the isolated 90 strains of E. coli, all of them were resistant to more than two kinds of antimicrobial drugs, the number of strains resistant to nine kinds of drugs was the largest (19 strains), and the most resistant strain showed resistance to 16 kinds of antibacterial drugs. Regarding the AMR genes, among the three aquaculture farms, the most resistance genes were detected in farm II (28 species). The detection rate of bla TEM , bla CIT , bla NDM , floR, OptrA, cmlA, aphA1, Sul2, oqxA, and qnrS in 90 isolates of E. coli was high (≥50%). The detection rate of carbapenem-resistant genes, such as bla KPC , bla IMP , and cfr, was relatively lower ( ≤ 30%), and the detection rate of mcr2 was the lowest (0). At least four AMR genes were detected for each strain, and 15 AMR genes were detected at most. Among them, the number of strains that carried 10 AMR genes was the largest (15 strains). Finally, a correlation analysis found that the AMR genes including bla TEM , bla CIT , floR, OptrA, cmlA, aac(3)-II, Sul2, ereA, ermB, oqxB, qnrA, mcr1, and mcr2 had a high correlation rate with drug resistance (≥50%). To summarize, the 90 strains of E. coli isolated from water, surrounding soil, and sediment samples showed resistance to multi-antimicrobial drugs and carried various antimicrobial resistance genes. Thus, it is essential to strengthen the rational use of antimicrobial drugs, especially the amide alcohol drugs, and control the AMR in the aquaculture industry of Zhanjiang, China.

The kinetics of viral load and antibodies to SARS-CoV-2.

OBJECTIVES: The aim was to understand persistence of the virus in body fluids the and immune response of an infected host to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), an agent of coronavirus disease 2019 (COVID-19). METHODS: We determined the kinetics of viral load in several body fluids through real time reverse transcription polymerase chain reaction, serum antibodies of IgA, IgG and IgM by enzyme-linked immunosorbent assay and neutralizing antibodies by microneutralization assay in 35 COVID-19 cases from two hospitals in Guangdong, China. RESULTS: We found higher viral loads and prolonged shedding of virus RNA in severe cases of COVID-19 in nasopharyngeal (1.3 × 106 vs 6.4 × 104, p < 0.05; 7∼8 weeks) and throat (6.9 × 106 vs 2.9 × 105, p < 0.05; 4∼5 weeks), but similar in sputum samples (5.5 × 106 vs 0.9 × 106, p < 0.05; 4∼5 weeks). Viraemia was rarely detected (2.8%, n = 1/35). We detected early seroconversion of IgA and IgG at the first week after illness onset (day 5, 5.7%, n = 2/35). Neutralizing antibodies were produced in the second week, and observed in all 35 included cases after the third week illness onset. The levels of neutralizing antibodies correlated with IgG (rs = 0.85, p < 0.05; kappa = 0.85) and IgA (rs = 0.64, p < 0.05; kappa = 0.61) in severe, but not mild cases (IgG, rs = 0.42, kappa = 0.33; IgA, rs = 0.32, kappa = 0.22). No correlation with IgM in either severe (rs = 0.17, kappa = 0.06) or mild cases (rs = 0.27, kappa = 0.15) was found. DISCUSSION: We revealed a prolonged shedding of virus RNA in the upper respiratory tract, and evaluated the consistency of production of IgG, IgA, IgM and neutralizing antibodies in COVID-19 cases.

Clinical, immunological and virological characterization of COVID-19 patients that test re-positive for SARS-CoV-2 by RT-PCR.

BACKGROUND: Some COVID-19 cases test positive again for SARS-CoV-2 RNA following negative test results and discharge, raising questions about the meaning of virus detection. Better characterization of re-positive cases is urgently needed. METHODS: Clinical data were obtained through Guangdong's COVID-19 surveillance network. Neutralization antibody titre was determined using microneutralization assays. Potential infectivity of clinical samples was evaluated by cell inoculation. SARS-CoV-2 RNA was detected using three different RT-PCR kits and multiplex PCR with nanopore sequencing. FINDINGS: Among 619 discharged COVID-19 cases, 87 re-tested as SARS-CoV-2 positive in circumstances of social isolation. All re-positive cases had mild or moderate symptoms at initial diagnosis and were younger on average (median, 28). Re-positive cases (n = 59) exhibited similar neutralization antibodies (NAbs) titre distributions to other COVID-19 cases (n = 218) tested here. No infectious strain could be obtained by culture and no full-length viral genomes could be sequenced from re-positive cases. INTERPRETATION: Re-positive SARS-CoV-2 cases do not appear to be caused by active reinfection and were identified in ~14% of discharged cases. A robust NAb response and potential virus genome degradation were detected in almost all re-positive cases, suggesting a substantially lower transmission risk, especially through respiratory routes.

Identification of Common Deletions in the Spike Protein of Severe Acute Respiratory Syndrome Coronavirus 2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel coronavirus first identified in December 2019. Notable features that make SARS-CoV-2 distinct from most other previously identified betacoronaviruses include a receptor binding domain and a unique insertion of 12 nucleotides or 4 amino acids (PRRA) at the S1/S2 boundary. In this study, we identified two deletion variants of SARS-CoV-2 that either directly affect the polybasic cleavage site itself (NSPRRAR) or a flanking sequence (QTQTN). These deletions were verified by multiple sequencing methods. In vitro results showed that the deletion of NSPRRAR likely does not affect virus replication in Vero and Vero-E6 cells; however, the deletion of QTQTN may restrict late-phase viral replication. The deletion of QTQTN was detected in 3 of 68 clinical samples and 12 of 24 in vitro-isolated viruses, while the deletion of NSPRRAR was identified in 3 in vitro-isolated viruses. Our data indicate that (i) there may be distinct selection pressures on SARS-CoV-2 replication or infection in vitro and in vivo; (ii) an efficient mechanism for deleting this region from the viral genome may exist, given that the deletion variant is commonly detected after two rounds of cell passage; and (iii) the PRRA insertion, which is unique to SARS-CoV-2, is not fixed during virus replication in vitro These findings provide information to aid further investigation of SARS-CoV-2 infection mechanisms and a better understanding of the NSPRRAR deletion variant observed here.IMPORTANCE The spike protein determines the infectivity and host range of coronaviruses. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has two unique features in its spike protein, the receptor binding domain and an insertion of 12 nucleotides at the S1/S2 boundary resulting in a furin-like cleavage site. Here, we identified two deletion variants of SARS-CoV-2 that either directly affect the furin-like cleavage site itself (NSPRRAR) or a flanking sequence (QTQTN), and we investigated these deletions in cell isolates and clinical samples. The absence of the polybasic cleavage site in SARS-CoV-2 did not affect virus replication in Vero or Vero-E6 cells. Our data indicate the PRRAR sequence and the flanking QTQTN sequence are not fixed in vitro; thus, there appears to be distinct selection pressures on SARS-CoV-2 sequences in vitro and in vivo Further investigation of the mechanism of generating these deletion variants and their infectivity in different animal models would improve our understanding of the origin and evolution of this virus.

Prolonged Persistence of SARS-CoV-2 RNA in Body Fluids.

We prospectively assessed 49 coronavirus disease cases in Guangdong, China, to estimate the frequency and duration of detectable severe acute respiratory syndrome coronavirus 2 RNA in human body fluids. The prolonged persistence of virus RNA in various body fluids may guide the clinical diagnosis and prevention of onward virus transmission.

Genomic Epidemiology of SARS-CoV-2 in Guangdong Province, China.

Coronavirus disease 2019 (COVID-19) is caused by SARS-CoV-2 infection and was first reported in central China in December 2019. Extensive molecular surveillance in Guangdong, China's most populous province, during early 2020 resulted in 1,388 reported RNA-positive cases from 1.6 million tests. In order to understand the molecular epidemiology and genetic diversity of SARS-CoV-2 in China, we generated 53 genomes from infected individuals in Guangdong using a combination of metagenomic sequencing and tiling amplicon approaches. Combined epidemiological and phylogenetic analyses indicate multiple independent introductions to Guangdong, although phylogenetic clustering is uncertain because of low virus genetic variation early in the pandemic. Our results illustrate how the timing, size, and duration of putative local transmission chains were constrained by national travel restrictions and by the province's large-scale intensive surveillance and intervention measures. Despite these successes, COVID-19 surveillance in Guangdong is still required, because the number of cases imported from other countries has increased.

Identification of drug resistance genes and drug resistance analysis of Salmonella in the duck farm environment of Zhanjiang, China.

This study investigated and identified the distribution of drug resistance genes in feces, soil, and water of duck farms in Zhanjiang, China, and analyzed the drug resistance of Salmonella in the duck farm environment. PCR was used to assess the distribution of 25 resistance genes that are common in the duck farm environment. The isolation, biochemical identification, PCR identification of Salmonella, and the minimum inhibitory concentration (MIC) of 22 drugs were measured by micro-broth double dilution. In water, 25 drug resistance genes were detected, 24 in soil, and 23 in feces. Among them, the detection rate of the aadA1 gene in soil reached 100%, 13 drug resistance genes had a detection rate above 80%, and five species had a detection rate below 50%. In water, the detection rate of the floR and aadA1 genes was 100%, 12 drug resistance genes had a detection rate above 80%, and eight genes had a detection rate below 50%. In feces, nine drug resistance genes had a detection rate of 100%, nine genes had a detection rate above 80%, and one gene had a detection rate below 50%. In addition, 92 strains of Salmonella were isolated and identified, and their resistance rate to nine drugs was as high as 100%. All isolated Salmonella can tolerate at least nine drugs, 55.43% (51/92) of the strains can tolerate more than 16 drugs, and 4.35% (4/92) of the strains were resistant to up to 21 drugs. In conclusion, the present experiment suggested that drug resistance genes were ubiquitous in the duck farm environment in Zhanjiang and that these drug resistance genes may spread horizontally between feces, soil, and water. Moreover, drug resistance and multi-drug resistance were found for 92 isolated Salmonella strains from the duck farm environment. The government should consequently strengthen the regulation of antimicrobial drug use in duck farms.

Combination of amplified rDNA restriction analysis and high-throughput sequencing revealed the negative effect of colistin sulfate on the diversity of soil microorganisms.

Colistin sulfate is widely used in both human and veterinary medicine. However, its effect on the microbial ecologyis unknown. In this study, we determined the effect of colistin sulfate on the diversity of soil microorganisms by amplified rDNA restriction analysis (ARDRA) and high-throughput sequencing.ARDRAshowed that the diversity of DNA from soil microorganisms was reduced after soil was treated with colistin sulfate, with the most dramatic reductionobserved after 35days of treatment. High-throughput sequencing showed that the Chao1 and abundance-based coverage estimators (ACE) were reduced in the soils treated with colistin sulfate for 35 dayscompared to those treated with colistin sulfate for 7days. Furthermore, Chao1 and ACE tended to be lower when higher concentration of colistin sulfate was used, suggesting that the microbial abundance is reduced by colistin sulfate in a dose-dependent manner. Shannon index showed that the diversity of soil microorganism was reduced upon treatment with colistin sulfate compared to the untreated control group. Following 7days of treatment, Bacillus, Clostridiumand Sphingomonas were sensitive to all the concentration of colistin sulfate used in this study. Following 35days of treatment, the abundance of Choroplast, Haliangium, Pseudomonas, Lactococcus, and Clostridium was significantly decreased. Our results demonstrated that colistin sulfate especially at high concentration (≥5mg/kg) could alter the population structure of microorganisms and consequently the microbial community function in soil.

[Effects of colistin sulfate residue on soil microbial community structure].

By using fumigation extraction and phospholipid fatty acid (PLFA) methods, the change of characteristics of soil microbial community structure caused by residue of colistin sulfate (CS) was studied. The results showed that the CS (w(cs) > or = 5 mg x kg(-1)) had a significant effect on the microbial biomass carbon (MBC) and it was dose-dependent where MBC decreased with the increase of CS concentration in soil. The MBC in soil decreased by 52. 1% when the CS concentration reached 50 mg x kg(-1). The total PLFA of soil in each CS treatment was significantly decreased during the sampling period compared with the control group and showed a dose-dependent relationship. The soil microbial community structure and diversity in the low CS group (w(cs) = 0.5 mg x kg(-1)) were not significantly different from the control group on 7th and 49th day. However, they were significantly different on 21st and 35th day especially in the high CS group (w(cs) = 50 mg x kg(-1)). It was concluded that CS could change the structure of soil microorganisms and varied with time which might be caused by the chemical conversion and degradation of CS in soil.