Carriage of distinct blaKPC-2 and blaOXA-48 plasmids in a single ST11 hypervirulent Klebsiella pneumoniae isolate in Egypt.

BACKGROUND: Carbapenem-resistant hypervirulent K. pneumoniae (CR-hvKP) causes serious infections with significant morbidity and mortality. However, the epidemiology and transmission mechanisms of CR-hvKP and the corresponding carbapenem-resistant plasmids require further investigation. Herein, we have characterized an ST11 K. pneumoniae strain EBSI041 from the blood sample encoding both hypervirulence and carbapenem resistance phenotypes from a patient in Egypt. RESULTS: K. pneumoniae strain EBSI041 showed multidrug-resistance phenotypes, where it was highly resistant to almost all tested antibiotics including carbapenems. And hypervirulence phenotypes of EBSI041 was confirmed by the model of Galleria mellonella infection. Whole-genome sequencing analysis showed that the hybrid plasmid pEBSI041-1 carried a set of virulence factors rmpA, rmpA2, iucABCD and iutA, and six resistance genes aph(3')-VI, armA, msr(E), mph(E), qnrS, and sul2. Besides, blaOXA-48 and blaSHV-12 were harboured in a novel conjugative IncL-type plasmid pEBSI041-2. The blaKPC-2-carrying plasmid pEBSI041-3, a non-conjugative plasmid lacking the conjugative transfer genes, could be transferred with the help of pEBSI041-2, and the two plasmids could fuse into a new plasmid during co-transfer. Moreover, the emergence of the p16HN-263_KPC-like plasmids is likely due to the integration of pEBSI041-3 and pEBSI041-4 via IS26-mediated rearrangement. CONCLUSION: To the best of our knowledge, this is the first report on the complete genome sequence of KPC-2- and OXA-48-coproducing hypervirulent K. pneumoniae from Egypt. These results give new insights into the adaptation and evolution of K. pneumoniae during nosocomial infections.

Characterization of a Plasmid-Encoded Resistance-Nodulation-Division Efflux Pump in Klebsiella pneumoniae and Klebsiella quasipneumoniae from Patients in China.

Two multidrug-resistant (MDR) mcr-1-harboring Klebsiella pneumoniae isolates from patients with urinary tract infections and one MDR Klebsiella quasipneumoniae isolate from a patient with bloodstream infection were identified to carry tmexCD1-toprJ1 The addition of the efflux pump inhibitor reduced the tigecycline MIC against all three isolates by 8- to 16-fold. pKQBSI104-1 was transferred from K. quasipneumoniae to Escherichia coli J53 via conjugation. The tmexCD1-toprJ1-carrying plasmids pKP15ZE495-1 (102,569 bp) and pKQBSI104-1 (121,996 bp) were completely sequenced and analyzed.

Colocation of the Polymyxin Resistance Gene mcr-1 and a Variant of mcr-3 on a Plasmid in an Escherichia coli Isolate from a Chicken Farm.

A colistin-resistant Escherichia coli isolate from a commercial poultry farm in China carried two colistin resistance genes, mcr-1 and variant of mcr-3, in an IncP plasmid. The variant of the mcr-3 gene, named mcr-3.11, encoded two amino acid substitutions compared with the mcr-3 gene. A novel genetic structure, ISKpn40-mcr-3-dgkA-ISKpn40, might be the key element mediating the translocation of mcr-3 through the formation of a circular form. The mcr-1 and mcr-3 genes, which are colocated on a plasmid, might pose a huge threat to public health.

Colocation of the Multiresistance Gene cfr and the Fosfomycin Resistance Gene fosD on a Novel Plasmid in Staphylococcus arlettae from a Chicken Farm.

The novel 63,558-bp plasmid pSA-01, which harbors nine antibiotic resistance genes, including cfr, erm(C), tet(L), erm(T), aadD, fosD, fexB, aacA-aphD, and erm(B), was characterized in Staphylococcus arlettae strain SA-01, isolated from a chicken farm in China. The colocation of cfr and fosD genes was detected for the first time in an S. arlettae plasmid. The detection of two IS431-mediated circular forms containing resistance genes in SA-01 suggested that IS431 may facilitate dissemination of antibiotic resistance genes.

Colistin Resistance Gene mcr-1 and Its Variant in Escherichia coli Isolates from Chickens in China.

The mcr-1 gene was detected in 5.11% (58/1136) of Escherichia coli isolates of chicken origin from 13 provinces in China. A novel mcr-1 variant, named mcr-1.3, encoding an Ile-to-Val functional variant of MCR-1 was identified in a sequence type 155 (ST155) strain. An mcr-1.3-containing IncI2 plasmid, pHeN867 (60,757 bp), was identified. The transfer of pHeN867 led to a 32-fold increase in the MIC of colistin in the recipient, exhibiting an effect on colistin resistance that was similar to that of mcr-1.

Characterization of pHeBE7, an IncFII-type virulence-resistance plasmid carrying blaCTX-M-98b, blaTEM-1, and rmtB genes, detected in Escherichia coli from a chicken isolate in China.

Recently, a novel variant of the CTX-M enzyme, CTX-M-98, was detected in Escherichia coli isolates from food animals. However, few plasmids carrying blaCTX-M-98 have been fully characterized. In this study, we sequenced the complete pHeBE7 plasmid, an 86,015-bp plasmid that contains the blaCTX-M-98b, blaTEM-1, rmtB, and traT genes, using whole-genome sequencing. The backbone of pHeBE7 shows a high similarity (>99%) to pMC-NDM, which carries the blaNDM-1 gene, however its mosaic regions remain relatively unique among sequenced plasmids. We discovered that a typical ISEcp1-blaCTX-M-IS903 element in the mosaic region harbors the blaCTX-M-98b gene. Conjugation and growth competition assays indicate that pHeBE7 can be easily transmitted and that it confers a limited fitness cost to the recipient cell. The genetic characterization of pHeBE7 may improve our knowledge of how antibiotic resistance disseminates in enterobacteria.

Prevalence, Antimicrobial Resistance, and Relatedness of Salmonella Isolated from Chickens and Pigs on Farms, Abattoirs, and Markets in Sichuan Province, China.

This study aims at investigating the distribution, antimicrobial resistance, and genetic relationship of Salmonella isolated from 18 farms, their downstream abattoirs, and markets of chickens and pigs in Sichuan province, China. A total of 193 Salmonella isolates were identified from 693 samples with an isolation rate of 26.27% (88/335) in chickens and 29.33% (105/358) in pigs. Salmonella was isolated more frequently in abattoirs and markets than from farms. Serotypes were determined according to the White-Kauffmann-Le Minor scheme and 16 different serotypes were identified, with Derby being the most common, followed by Typhimurium and Meleagridis. Antimicrobial resistance phenotypes and genotypes were studied by using the disk diffusion method and polymerase chain reaction (PCR) amplification, respectively. Overall, 44.04% (n = 85) of all isolates were multidrug resistant (MDR) and resistance to nalidixic acid (51.30%) was the most frequently observed. blaCTX-M-55 was the most prevalent extended-spectrum ß-lactamases gene, and polymyxin resistance gene mcr-1 was present in strains with various serotypes. Multilocus sequence typing indicated that sequence type (ST) had a close relationship with serotype, and 34.20% of all strains were ST40, which was the most prevalent. The unweighted pair group method with arithmetic means (UPGMA) dendrogram of pulsed-field gel electrophoresis showed that Salmonella isolates belonging to the same serovar from different parts of the production chain were highly genetic related, indicating that Salmonella as well as resistance genes could potentially be transmitted from farms to markets. Our study highlights the fact that Salmonella isolates from chicken and pig production chain were frequently exhibiting MDR profiles, and the dissemination of MDR Salmonella from farm to market could pose significant threats to food safety and public health.

Suppression of reproductive characteristics of the invasive plant Mikania micrantha by sweet potato competition.

BACKGROUND: As a means of biologically controlling Mikania micrantha H.B.K. in Yunnan, China, the influence of sweet potato [Ipomoea batatas (L.) Lam.] on its reproductive characteristics was studied. The trial utilized a de Wit replacement series incorporating six ratios of sweet potato and M. micrantha plants in 25 m(2) plots over 2 years. RESULTS: Budding of M. micrantha occurred at the end of September; flowering and fruiting occurred from October to February. Flowering phenology of M. micrantha was delayed (P < 0.05), duration of flowering and fruiting was reduced (P < 0.05) and duration of bud formation was increased (P < 0.05) with increasing proportions of sweet potato. Reproductive allocation, reproductive investment and reproductive index of M. micrantha were significantly reduced (P < 0.05) with increasing sweet potato densities. Apidae bees, and Calliphoridae or Syrphidae flies were the most abundant visitors to M. micrantha flowers. Overall flower visits decreased (P < 0.05) as sweet potato increased. Thus the mechanism by which sweet potato suppressed sexual reproduction in M. micrantha was essentially two-fold: causing a delay in flowering phenology and reducing pollinator visits. The number, biomass, length, set rate, germination rate, and 1000-grain dry weight of M. micrantha seeds were suppressed (P < 0.05) by sweet potato competition. With proportional increases in sweet potato, sexual and asexual seedling populations of M. micrantha were significantly reduced (P < 0.05). The mortality of both seedling types increased (P < 0.05) with proportional increases in sweet potato. CONCLUSIONS: These results suggest that sweet potato significantly suppresses the reproductive ability of the invasive species M. micrantha, and is a promising alternative to traditional biological control and other methods of control. Planting sweet potato in conjunction with other control methods could provide a comprehensive strategy for managing M. micrantha. The scenario of controlling M. micrantha by utilizing a crop with a similar growth form may provide a useful model for similar management strategies in other systems.

Genomic characterization revealing the high rate of tet(X4)-positive Escherichia coli in animals associated with successful genetic elements.

Introduction: The rapid spread of plasmid-mediated tet(X4) conferring high tigecycline resistance poses a significant threat to public health. Escherichia coli as the most common pathogen which carries tet(X4) has been widely disseminated in China. Thus, comprehensive investigations are required to understand the mechanism of transmission of tet(X4)-positive E. coli. Methods: In this study, a total of 775 nonduplicate samples were collected in Guangdong, China from 2019 to 2020. We screened for tet(X4)-positive E. coli by PCR amplification and species identification. Furthermore, we analyzed the phylogenetics and genetic context of tet(X4)-positive E. coli through whole-genome sequencing and long-reads sequencing. Results: Overall, 146 (18.84%) tet(X4)-positive E. coli were isolated, comprising 2 isolates from humans and 144 isolates from pigs. The majority of tet(X4)-positive E. coli exhibited resistance to multiple antibiotics but all of them were susceptible to amikacin and colistin. Phylogenetic analysis showed that ST877, ST871, and ST195 emerged as the predominant sequence types in tet(X4)-positive E. coli. Further analysis revealed various genetic environments associated with the horizontal transfer of tet(X4). Notably, a 100-kbp large fragment insertion was discovered downstream of tet(X4), containing a replicon and a 40-kbp gene cluster for the bacterial type IV secretion system. Discussion: The high colonization rate of tet(X4)-positive E. coli in animals suggests that colonization as a key factor in its dissemination to humans. Diverse genetic context may contribute to the transfer of tet(X4). Our findings underline the urgent need for controlling the spread of plasmid-mediated tigecycline resistance.

Characterization of two multidrug-resistant Klebsiella pneumoniae harboring tigecycline-resistant gene tet(X4) in China.

Objectives: Tigecycline is recognized as one of the last-line antibiotics to treat serious bacterial infection caused by carbapenem-resistant Klebsiella pneumoniae (CRKP). The plasmid-borne gene tet(X4) mediates high resistance to tigecycline. However, the prevalence and genetic context of tet(X4) in K. pneumoniae from various sources are not fully understood. Here, we investigated the prevalence of tet(X4)-positive K. pneumoniae and characterized the genetic context of tet(X4)-bearing plasmids in K. pneumoniae isolates. Methods: Polymerase chain reaction (PCR) was used to detect the tet(X4) gene. The transferability of the tet(X4)-carrying plasmids was tested by conjugation assays. The Galleria mellonella infection model was used to test virulence of tet(X4)-positive strains. Whole-genome sequencing and genome-wide analysis were performed to identify the antimicrobial resistance and the virulence genes, and to clarify the genetic characteristics of the tet(X4)-positive isolates. Results: Among 921 samples, we identified two tet(X4)-positive K. pneumoniae strains collected from nasal swabs of two pigs (0.22%, 2/921). The two tet(X4)-positive isolates exhibited high minimum inhibitory concentrations to tigecycline (32-256 mg/L) and tetracycline (256 mg/L). The plasmids carrying the tet(X4) gene can transfer from the donor strain K. pneumoniae to the recipient strain Escherichia coli J53. Genetic analysis of the complete sequence of two tet(X4)-carrying plasmids pTKPN_3-186k-tetX4 and pTKPN_8-216k-tetX4 disclosed that the tet(X4) gene was flanked by delta ISCR2 and IS1R, which may mediate the transmission of the tet(X4) gene. Conclusion: The prevalence of tet(X4)-positive K. pneumoniae among different sources was low. ISCR2 and IS1R may contribute to the horizontal transfer of tet(X4) gene. Effective measures should be taken to prevent the transmission of tet(X4)-producing K. pneumoniae in humans or animals.

Identification of Two CDK5R1-Related Subtypes and Characterization of Immune Infiltrates in Alzheimer's Disease Based on an Integrated Bioinformatics Analysis.

Background: Alzheimer's disease (AD) is a neurodegenerative disorder and the major cause of senile dementia. The Reelin pathway has been involved in both learning and AD pathogenesis. However, the specific Reelin-related gene signature during the pathological process remains unknown. Methods: Reelin-related gene (CDK5R1) expression was analyzed using the GEO datasets. The relevant genes of CDK5R1 were identified using differential expression analysis and weighted gene correlation network analysis (WGCNA) based on the GSE43850 dataset. ConsensusClusterPlus analysis was applied to identify subtypes (C1 and C2) of AD. The CIBERSORT algorithm was used to assess the immune cell infiltration between the two AD subtypes. Results: CDK5R1 was downregulated in AD. 244 differentially expressed CDK5R1-related genes (DECRGs) between the two subgroups were mainly enriched in GABAergic synapse, neuroactive ligand-receptor interaction, synapse organization, neurotransmitter transport, etc. Furthermore, the GSVA results indicated that immune-related pathways were significantly enriched in the C1 subgroup. Interestingly, 10 Reelin pathway-related genes (CRK, DAB2IP, LRP8, RELN, STAT5A, CDK5, CDK5R1, DAB1, FYN, and SH3KBP1) were abnormally expressed between the two subgroups. The proportion of T cell gamma delta, monocytes, macrophage M2, and dendritic cells activated decreased from C1 to C2, while the proportion of plasma cells, T cell follicular helper, and NK cells activated increased. Conclusion: Two CDK5R1-related subtypes of AD were identified, helping us to better understand the role of CDK5R1 in the pathological process of AD.

The Emergence of a Multidrug-Resistant and Pathogenic ST42 Lineage of Staphylococcus haemolyticus from a Hospital in China.

Staphylococcus haemolyticus is an opportunistic pathogen associated with hospital-acquired infections. However, the genetic diversity of S. haemolyticus among the patients and the hospital environment is largely unknown. Here, we isolated 311 S. haemolyticus strains from different sampling sites of patients and hospital environment. Genomic analysis showed that ST42 is an emerging clone widely disseminated in the hospital. S. haemolyticus ST42 strains exhibited decreased susceptibilities for multiple antibiotics compared with other STs and carried significantly more antibiotic resistance genes (ARGs). Furthermore, ST42 strains harbored more virulence genes per isolate than in other STs, and the capsular biosynthesis genes capDEFG were more prevalent in ST42 strains. Using the Galleria mellonella infection model, we demonstrated that ST42 strains are highly virulent compared with non-ST42 strains. Taken together, our data identified an emerging ST42 clone of S. haemolyticus with aggregated ARGs and virulence determinants in the hospital, representing a significant health threat in terms of both disease and treatment. IMPORTANCES. haemolyticus is an emerging opportunistic pathogen with a high burden of antimicrobial resistance. We performed molecular epidemiological analysis of S. haemolyticus that was isolated from a hospital, and found that the phylogenetic lineages are diverse accompanied by a dominant epidemic clonal lineage ST42. We demonstrated that S. haemolyticus ST42 strains have been disseminated among patients and the hospital environment. The data provide mechanistic insight and indicate that S. haemolyticus ST42 strains are multidrug-resistance and virulent clones via accumulating more ARGs and virulence genes.

Outcomes and Risk Factors of Bloodstream Infections Caused by Carbapenem-Resistant and Non-Carbapenem-Resistant Klebsiella pneumoniae in China.

Purpose: To compare antimicrobial resistance, virulence, clinical characteristics, and risk factors between carbapenem-resistant K. pneumoniae (CRKP) and carbapenem-susceptible K. pneumoniae (CSKP) isolates from patients with bloodstream infections (BSIs) in China. Patients and Methods: The clinical data of 103 patients with K. pneumoniae BSI from 10 hospitals were retrospectively analyzed. The minimum inhibitory concentrations of 15 antibiotics against the bacteria were determined. A Galleria mellonella infection model was used to evaluate virulence of the isolates. Kaplan-Meier curves were calculated to evaluate the 28-day and in-hospital survival rates of the isolates. The risk factors for CRKP and CSKP infection and respective mortality rate were evaluated by univariate analysis, and independent risk factors were evaluated using the multivariate logistic regression model. Results: Our results indicated that CRKP isolates were more resistant to most tested antibiotics than CSKP isolates. The G. mellonella infection model was used to demonstrate that CRKP isolates were more virulent than CSKP isolates. We found that in-hospital deaths occurred in 39.3% (22/56) of patients with CRKP BSIs and were significantly higher than those in patients with CSKP infections (19.1%, 9/47). Patients infected with CRKP isolates had poorer outcomes than those infected with the CSKP strains. For in-hospital mortality of CRKP BSIs, the independent risk factors included carbapenem-resistant Enterobacterales bacteremia and length of hospitalization after the onset of BSI. Conclusion: Our findings confirm that CRKP isolates are more drug-resistant than CSKP isolates and are associated with poorer outcomes. To prevent CRKP infection, strict infection control strategies and active surveillance should be implemented in hospitals.

Identification of Plasmid-Mediated Tigecycline-Resistant Gene tet(X4) in Enterobacter cloacae from Pigs in China.

Two tet(X4)-positive Enterobacter cloacae isolates TECL_1 and TECL_2 were isolated from pigs in China. S1-PFGE and Southern blotting showed that tet(X4) located on plasmids in the size of ∼290 kb and ∼190 kb in TECL_1 and TECL_2, respectively. Conjugation experiment demonstrated that the tet(X4)-harboring plasmid can transfer from the donor strain TECL_1 and TECL_2 to the recipient strain Escherichia coli J53, and the tigecycline resistance of transconjugants was increased by 128-fold and 64-fold compared with E. coli J53, respectively. We obtained the complete plasmid sequence of pTECL_2-190k-tetX4 (190,185 bp) from E. cloacae TECL_2 and found that the plasmid was a hybrid plasmid with replicon types of IncFIA, IncHI1A and IncHI1B. We further analyzed 85 tet(X4)-carrying plasmids in the public database and clarified that pTECL_2-190k-tetX4-like plasmid was widespread in multiple species of Enterobacteriaceae. IMPORTANCE We identified two tet(X4)-positive E. cloacae isolates, which has not been previously reported. We obtained the complete sequence of pTECL_2-190k-tetX4 and found that it was a hybrid plasmid with multiple replicon types, including IncFIA, IncHI1A and IncHI1B. By comparing all the known tet(X4)-carrying plasmids, we found that pTECL_2-190k-tetX4-like plasmid has been disseminated across various species in China. Our study expanded the identification of tet(X4)-positive species and emphasized that pTECL_2-190k-tetX4-like plasmid has spread widely in various species.

Emergence of Hypervirulent Carbapenem-Resistant Klebsiella pneumoniae Coharboring a blaNDM-1-Carrying Virulent Plasmid and a blaKPC-2-Carrying Plasmid in an Egyptian Hospital.

The emergence of carbapenem-resistant Klebsiella pneumoniae (CRKP) isolates in Egyptian hospitals has been reported. However, the genetic basis and analysis of the plasmids associated with carbapenem-resistant hypervirulent K. pneumoniae (CR-HvKP) in Egypt have not been presented. Therefore, we attempted to decipher the plasmid sequences that are responsible for transferring the determinants of carbapenem resistance, particularly blaNDM-1 and blaKPC-2 Out of 34 K. pneumoniae isolates collected from two tertiary hospitals in Egypt, 31 were CRKP. Whole-genome sequencing revealed that our isolates were related to 13 different sequence types (STs). The most prevalent ST was ST101, followed by ST383 and ST11. Among the CRKP isolates, one isolate named EBSI036 has been reassessed by Nanopore sequencing. Genetic environment analysis showed that EBSI036 carried 20 antibiotic resistance genes and was identified as a CR-HvKP strain: it harbored four plasmids, namely, pEBSI036-1-NDM-VIR, pEBSI036-2-KPC, pEBSI036-3, and pEBSI036-4. The two carbapenemase genes blaNDM-1 and blaKPC-2 were located on plasmids pEBSI036-1-NDM-VIR and pEBSI036-2-KPC, respectively. The IncFIB:IncHI1B hybrid plasmid pEBSI036-1-NDM-VIR also carried some virulence factors, including the regulator of the mucoid phenotype (rmpA), the regulator of mucoid phenotype 2 (rmpA2), and aerobactin (iucABCD and iutA). Thus, we set out in this study to analyze in depth the genetic basis of the pEBSI036-1-NDM-VIR and pEBSI036-2-KPC plasmids. We report a high-risk clone ST11 KL47 serotype of a CR-HvKP strain isolated from the blood of a 60-year-old hospitalized female patient from the intensive care unit (ICU) in a tertiary care hospital in Egypt, which showed the cohabitation of a novel hybrid plasmid coharboring the blaNDM-1 and virulence genes and a blaKPC-2-carrying plasmid.IMPORTANCE CRKP has been registered in the critical priority tier by the World Health Organization and has become a significant menace to public health. The emergence of CR-HvKP is of great concern in terms of both disease and treatment. In-depth analysis of the carbapenemase-encoding and virulence plasmids may provide insight into ongoing recombination and evolution of virulence and multidrug resistance in K. pneumoniae Thus, this study serves to alert contagious disease clinicians to the presence of hypervirulence in CRKP isolates in Egyptian hospitals.

Molecular characterization of carbapenem-resistant and virulent plasmids in Klebsiella pneumoniae from patients with bloodstream infections in China.

Bloodstream infections (BSIs) caused by carbapenem-resistant Klebsiella pneumoniae (CRKP) are potentially life-threatening and an urgent threat to public health. The present study aims to clarify the characteristics of carbapenemase-encoding and virulent plasmids, and their interactions with the host bacterium. A total of 425 Kp isolates were collected from the blood of BSI patients from nine Chinese hospitals, between 2005 and 2019. Integrated epidemiological and genomic data showed that ST11 and ST307 Kp isolates were associated with nosocomial outbreak and transmission. Comparative analysis of 147 Kp genomes and 39 completely assembled chromosomes revealed extensive interruption of acrR by ISKpn26 in all Kp carbapenemase-2 (KPC-2)-producing ST11 Kp isolates, leading to activation of the AcrAB-Tolc multidrug efflux pump and a subsequent reduction in susceptibility to the last-resort antibiotic tigecycline and six other antibiotics. We described 29 KPC-2 plasmids showing diverse structures, two virulence plasmids in two KPC-2-producing Kp, and two novel multidrug-resistant (MDR)-virulent plasmids. This study revealed a multifactorial impact of KPC-2 plasmid on Kp, which may be associated with nosocomial dissemination of MDR isolates.

Exploring the profile of antimicrobial resistance genes harboring by bacteriophage in chicken feces.

Bacteriophage may play an important role in antimicrobial resistance genes (ARGs) transmission. However, the contribution of bacteriophage to the spread of ARGs in environment, especially in poultry farm environment, is rarely known. In this study, the prevalence of ARGs in bacteriophage DNA was investigated in chicken feces from 30 different poultry farms in China. Then the abundance of the aac(6')-Ib-cr, blaCTX-M, ermB, floR, mcr-1, sul1, tetM and intI1 genes was determined by qPCR in bacteriophage and compared with certain representative plasmid DNA samples. The results showed that 12 ARGs (aac(6')-Ib-cr, aph(3')-IIIa, blaCTX-M, ermB, ermF, floR, mcr-1, qnrS, sul1, sul2, vanA, tetM genes) and class 1 integron gene intI1 were detected in bacteriophage DNA fraction. The sul1, tetM and aac(6')-Ib-cr genes were most prevalent with high detection rates of 77%, 61% and 55%, respectively. To our best knowledge, this study firstly reported the presence of the mcr-1 gene in bacteriophage DNA derived from farms environments. We found that the gene copy (GC) numbers of the aac(6')-Ib-cr, ermB and sul1 genes were as high as 5.47, 5.22 and 5.54 log10 GC/g, respectively. Both the prevalence and abundance of ARGs in broiler fecal wastes were also generally higher than in laying hens. In addition, although the GC numbers of the aac(6')-Ib-cr, floR and tetM genes in plasmid DNA was higher than that in phage DNA fraction by 4.68, 3.59 and 3.9 orders of magnitude, respectively, the absolute abundances of the blaCTX-M and mcr-1 genes in phage DNA were close to or even higher than that in plasmid DNA at farm SIL2, SIL4 and SIB1. As potential vessels for ARGs, bacteriophage could not be ignored due to their unique extracellular persistence in environments. Overall, this is the first comprehensive survey about bacteriophage carried ARGs from farms in different regions in China.

Co-Occurrence of mcr-9 and bla NDM-1 in Enterobacter cloacae Isolated from a Patient with Bloodstream Infection.

BACKGROUND: Bloodstream infection (BSI) caused by carbapenem-resistant Enterobacteriaceae are potentially life-threatening related to poorer outcomes. Colistin is considered one of the last-resort treatments against human infections caused by multidrug-resistant (MDR) Gram-negative bacteria. Therefore, emergence of strains from the blood that co-harboring mcr and carbapenem resistance genes were considered as a serious problem. PURPOSE: In this study, two mcr-9-harboring MDR Enterobacter cloacae isolates BSI034 and BSI072 recovered from BSI patients were identified, one of which co-harbored mcr-9 and bla NDM-1. The genetic characteristics of the MDR plasmid needed to be clarified. METHODS: S1-PFGE and Southern blotting were conducted to determine the location of mcr-9. Whole-genome sequencing was performed to obtain the complete genome and plasmid sequences. The resistome and virulence genes of the strains, accompanied by the genetic characteristics of mcr-9- and bla NDM-1-harboring plasmids, were analyzed. RESULTS: Whole-genome sequencing showed that BSI034 harbored mcr-9-carrying IncHI2-type pBSI034-MCR9 and bla NDM-1-carrying IncX3-type pBSI034-NDM1. The 278,517 bp pBSI034-MCR9 carried mcr-9 along with the other 19 resistance genes. mcr-9 was flanked by IS903B (1057 bp) and IS26 (820 bp) in the same orientation. In addition to resistance genes, strain BSI034 also carried a chromosome-located Yersinia high-pathogenicity island, which harbored genes of yersiniabactin biosynthesis operon ybtSXQPAUTE, irp1/2, and fyuA. CONCLUSION: We described the complete genome and mcr-9/bla NDM-1-co-harboring plasmid of E. cloacae from a BSI patient. Notable differences were observed within mosaic modules between pBSI034-MCR9 and other mcr-9-harboring plasmids due to extensive recombination via horizontal gene transfer.

Characterization of Resistance Patterns and Detection of Apramycin Resistance Genes in Escherichia coli Isolated from Chicken Feces and Houseflies after Apramycin Administration.

The aim of this study was to evaluate the influence of apramycin administration on the development of antibiotic resistance in Escherichia coli (E. coli) strains isolated from chicken feces and houseflies under field conditions. Chickens in the medicated group (n = 25,000) were given successive prophylactic doses (0.5 mg/l) of apramycin in their drinking water from Days 1 to 5, while no antibiotics were added to the un-medicated groups drinking water (n = 25,000). Over 40 days, a total of 1170 E. coli strains were isolated from fecal samples obtained from medicated and un-medicated chickens and houseflies from the same chicken farm. Apramycin MIC90 values for E. coli strains obtained from the medicated group increased 32-128 times from Days 2 to 6 (256-1024 µg/ml) when compared to those on Day 0 (8 µg/ml). Strains isolated from un-medicated chickens and houseflies had consistently low MIC90 values (8-16 µg/ml) during the first week, but showed a dramatic increase from Days 8 to 10 (128-1024 µg/ml). The apramycin resistance gene aac(3)-IV was detected in E. coli strains from medicated (n = 71), un-medicated (n = 32), and housefly groups (n = 42). All strains positive for aac(3)-IV were classified into 12 pulsed-field gel electrophoresis (PFGE) types. PFGE types A, E, and G were the predominant types in both the medicated and housefly groups, suggesting houseflies play an important role in spreading E. coli-resistant strains. Taken together, our study revealed that apramycin administration could facilitate the occurrence of apramycin-resistant E. coli and the apramycin resistance gene acc(3)-IV. In turn, these strains could be transmitted by houseflies, thus increasing the potential risk of spreading multi-drug-resistant E. coli to the public.