Prevalence, genomic characteristics, and transmission dynamics of mcr-1-positive Salmonella enterica Typhimurium from patients with infectious diarrhea.

BACKGROUND: Previous studies reported the prevalence of mcr-1 among clinical infected Salmonella isolates in China. However, the transmission dynamics of mcr-1 in different ecological niches were not well investigated. Our objective is to exhibit the transmission dynamics of mcr-1 in Salmonella. METHODS: 598 Salmonella isolates were recovered from ten hospitals; besides 936 pig faces and 167 pork samples were collected from January 2015 to December 2017 in Guangzhou, China. PCR and sequencing were used to identify mcr-1-positive Salmonella. Antimicrobial susceptibility testing was performed with 16 antimicrobials. Conjugation, S1-PFGE, and Southern blot were used to determine the transferability and location of mcr-1. Whole-genome sequencing was used to investigate pangenome, phylogeny, plasmid, and transposon. RESULTS: Eleven mcr-1-positive Salmonella isolates were identified from patients with infectious diarrhea. Five pig fecal samples and three pork samples contained mcr-1-positive Salmonella isolates. All isolates were multi-drug resistant. The mcr-1 genes were located on ∼210-250 kb IncHI2-pST3 plasmids, and 12 mcr-1 genes were transferable. All isolates were assigned to ST34 or its genetically closed STs. The distribution of the core-genome network was significantly correlated with source distributions. The accessory genes-based network demonstrated that the diverse clonal complexes could share highly similar accessory genomes. CONCLUSIONS: The prevalence of mcr-1-positive Salmonella among different sources was low. Clonal transmission could not be the main reason for the expansion of mcr-1-positive Salmonella, but be attributed to the horizontal transfer of IncHI2-pST3 plasmid. Continuous surveillance on Salmonella should be performed to investigate the response of colistin banning in food-producing animals by mcr-1-positive Salmonella populations.

Genomic patterns and characterizations of chromosomally-encoded mcr-1 in Escherichia coli populations.

The emergence and transmission of the mobile colistin resistance gene (mcr-1) threatened the extensive use of polymyxin antimicrobials. Accumulated evidence showed that the banning of colistin additive in livestock feed efficiently reduce mcr-1 prevalence, not only in animals but also in humans and environments. However, our previous study has revealed that a small proportion of Escherichia coli could continually carry chromosomally-encoded mcr-1. The chromosomally-encoded events, indicated the existence of stabilized heritage of mcr-1 and revealed a potential threat in the antimicrobial stewardship interventions, are yet to be investigated. In this study, we systematically investigated the genetic basis of chromosomally-encoded mcr-1 in prevalence and potential mechanisms of lineage, plasmid, insertion sequence, and phage. Our results demonstrated that the emergence of chromosomally-encoded mcr-1 could originate from multiple mechanisms, but mainly derived through the recombination of ISApl1/Tn6330. We reported a specific transmission mechanism, which is a phage-like region without lysogenic components, could associate with the emergence and stabilization of chromosomally-encoded mcr-1. These results highlighted the potential origin and risks of chromosomally-encoded mcr-1, which could be a heritable repository and thrive again when confronted with new selective pressures. To the best of our knowledge, this is the first study to systematically reveal the genomic basis of chromosomally-encoded mcr-1, and report a specific transmission pattern involved in phage-like region. Overall, we demonstrate the origin mechanisms and risks of chromosomally-encoded mcr-1. It highlights the need of public attention on chromosome-encoded mcr-1 to prevent from its reemergence.

Rapid Fulminant Progression and Mortality Secondary to Aeromonas dhakensis Septicemia with Hepatitis B Virus Infection Following the Ingestion of Snakehead Fish in Mainland China: A Case Report.

Aeromonas dhakensis is an important ubiquitous Gram-negative and freshwater bacterium detected in different reservoirs. It can cause invasive diseases in humans. Herein, we report the first case in Mainland China of a fulminant death of a 29-year-old man as a result of a new, unexpected association between septicemic A. dhakensis and hepatitis B viral infection (HBV). Herein, the patient died from multiple organ failure 5 d postadmission after the ingestion of Snakehead Fish meal. The isolated bacterium was initially misidentified as Aeromonas hydrophila using VITEK-2, while whole-genome sequencing (WGS) revealed that the isolate is A. dhakensis. WGS revealed the occurrence of three antimicrobial genes of resistance: imiH, cphA2, and blaOXA-12; besides, major virulence factors were detected. In silico, multilocus sequence typing (MLST) showed that our A. dhakensis 17FW001 belonged to a novel sequence type (ST557). A comparative genomic analysis of our isolate with nine selected Aeromonas species was done, which elucidated the pathogenicity of our A. dhakensis. In conclusion, we reported for the first time the association between A. dhakensis and HBV in Mainland China. We revealed that septicemic A. dhakensis could result in severe adverse clinical outcomes that end up with unexpected fulminant death especially when it is accompanied with HBV and sheds light on the virulence of A. dhakensis and the high rate of its misdiagnosis that requires to urgently consider screening of all cases of A. dhakensis for HBV in the future. Besides, caution should be taken while dealing with snakeheads which act as a vector for A. dhakensis.

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.

Colistin and its role in the Era of antibiotic resistance: an extended review (2000-2019).

Increasing antibiotic resistance in multidrug-resistant (MDR) Gram-negative bacteria (MDR-GNB) presents significant health problems worldwide, since the vital available and effective antibiotics, including; broad-spectrum penicillins, fluoroquinolones, aminoglycosides, and ß-lactams, such as; carbapenems, monobactam, and cephalosporins; often fail to fight MDR Gram-negative pathogens as well as the absence of new antibiotics that can defeat these "superbugs". All of these has prompted the reconsideration of old drugs such as polymyxins that were reckoned too toxic for clinical use. Only two polymyxins, polymyxin E (colistin) and polymyxin B, are currently commercially available. Colistin has re-emerged as a last-hope treatment in the mid-1990s against MDR Gram-negative pathogens due to the development of extensively drug-resistant GNB. Unfortunately, rapid global resistance towards colistin has emerged following its resurgence. Different mechanisms of colistin resistance have been characterized, including intrinsic, mutational, and transferable mechanisms.In this review, we intend to discuss the progress over the last two decades in understanding the alternative colistin mechanisms of action and different strategies used by bacteria to develop resistance against colistin, besides providing an update about what is previously recognized and what is novel concerning colistin resistance.

Involvement of Transcription Elongation Factor GreA in Mycobacterium Viability, Antibiotic Susceptibility, and Intracellular Fitness.

There is growing evidence that GreA aids adaptation to stressful environments in various bacteria. However, the functions of GreA among mycobacteria remain obscure. Here, we report on cellular consequences following deletion of greA gene in Mycobacterium spp. The greA mutant strain (ΔgreA) was generated in Mycobacterium smegmatis, Mycobacterium tuberculosis (MTB) H37Ra, and M. tuberculosis H37Rv. Deletion of greA results in growth retardation and poor survival in response to adverse stress, besides rendering M. tuberculosis more susceptible to vancomycin and rifampicin. By using RNA-seq, we observe that disrupting greA results in the differential regulation of 195 genes in M. smegmatis with 167 being negatively regulated. Among these, KEGG pathways significantly enriched for differentially regulated genes included tryptophan metabolism, starch and sucrose metabolism, and carotenoid biosynthesis, supporting a role of GreA in the metabolic regulation of mycobacteria. Moreover, like Escherichia coli GreA, M. smegmatis GreA exhibits a series of conservative features, and the anti-backtracking activity of C-terminal domain is indispensable for the expression of glgX, a gene was down-regulated in the RNA-seq data. Interestingly, the decrease in the expression of glgX by CRISPR interference, resulted in reduced growth. Finally, intracellular fitness significantly declines due to loss of greA. Our data indicates that GreA is an important factor for the survival and resistance establishment in Mycobacterium spp. This study provides new insight into GreA as a potential target in multi-drug resistant TB treatment.

Dynamics of mcr-1 prevalence and mcr-1-positive Escherichia coli after the cessation of colistin use as a feed additive for animals in China: a prospective cross-sectional and whole genome sequencing-based molecular epidemiological study.

BACKGROUND: The global dissemination of colistin resistance encoded by mcr-1 has been attributed to extensive use of colistin in livestock, threatening colistin efficacy in medicine. The emergence of mcr-1 in common pathogens, such as Escherichia coli, is of particular concern. China banned the use of colistin in animal feed from May 1, 2017. We investigated subsequent changes in mcr-1 prevalence in animals, humans, food, and the environment, and the genomic epidemiology of mcr-1-positive E coli (MCRPEC). METHODS: Sampling was done before (October to December, 2016) and after (October to December, 2017, and 2018, respectively) the colistin ban. 3675 non-duplicate pig faecal samples were collected from 14 provinces (66 farms) in China to measure intervention-related changes in mcr-1 prevalence. 15 193 samples were collected from pigs, healthy human volunteers, patients colonised or infected with Enterobacteriaceae who were admitted to hospital, food and the environment in Guangzhou, to characterise source-specific mcr-1 prevalence and the wider ecological effect of the ban. From these samples, 688 MCRPEC were analysed with whole genome sequencing, plasmid conjugation, and S1 pulsed-field gel electrophoresis with Southern blots to characterise associated genomic changes. FINDINGS: After the ban, mcr-1 prevalence decreased significantly in national pig farms, from 308 (45%) of 684 samples in 2016 to 274 (19%) of 1416 samples in 2018 (p<0·0001). A similar decrease occurred in samples from most sources in Guangzhou (959 [19%] of 5003 samples in 2016; 238 [5%] of 4489 samples in 2018; p<0·0001). The population structure of MCRPEC was diverse (23 sequence clusters); sequence type 10 clonal complex isolates were predominant (247 [36%] of 688). MCRPEC causing infection in patients admitted to hospital were genetically more distinct and appeared less affected by the ban. mcr-1 was predominantly found on plasmids (632 [92%] of 688). Common mcr-1 plasmid types included IncX4, IncI2, and IncHI2 (502 [76%] of 656); significant increases in IncI2-associated mcr-1 and a distinct lineage of mcr-1-associated IncHI2 were observed post ban. Changes in the frequency of mcr-1-associated flanking sequences (ISApl1-negative MCRPEC), 63 core genome single nucleotide polymorphisms, and 30 accessory genes were also significantly different after the ban (Benjamini-Hochberg-adjusted p<0·05), consistent with rapid genetic adaptation in response to changing selection pressures. INTERPRETATION: A rapid, ecosystem-wide, decline in mcr-1 was observed after the use of colistin in animal feed was banned, with associated genetic changes in MCRPEC. Withdrawal of antimicrobials from animal feed should be an important One Health measure contributing to the wider control of antimicrobial resistance globally. FUNDING: National Natural Science Foundation of China.

Formulation and characterization of chlorhexidine HCl nanoemulsion as a promising antibacterial root canal irrigant: in-vitro and ex-vivo studies.

Introduction and aim: Chlorhexidine Hydrochloride [Chx.HCl] has a broad-spectrum antibacterial effect, sustained action and low toxicity so it has been recommended as a potential root canal irrigant. The aim of this study was to improve the penetration ability, cleansing and antibacterial effect of Chx.HCl using a newly formulated Chx.HCl nanoemulsion and use it as root canal irrigant. Methods: Chx.HCl nanoemulsions were prepared using two different oils; Oleic acid and Labrafil M1944CS, two surfactants; Tween 20 and Tween 80 and co-surfactant; Propylene Glycol. Pseudoternary phase diagrams were constructed to designate the optimum systems. The prepared nanoemulsion formulae were evaluated for their drug content, emulsification time, dispersibility, droplet size, in-vitro drug release, thermodynamic stability, In-vitro antibacterial activity and ex-vivo study for the selected formula. Comparisons were made of Chx.HCl nanoemulsion with two different concentrations 0.75% and 1.6% vs Chx.HCl normal particle size as root canal irrigant for their penetration ability, cleansing effect and antibacterial effect. Results: The selected formula was F6 with composition of 2% Labrafil, 12% Tween 80 and 6% Propylene glycol. It has small particle size (12.18 nm), short emulsification time (1.67 seconds), and fast dissolution rate after 2 minutes. It was found to be a thermodynamically/physically stable system. The higher concentration of Chx.HClnanoemulsion1.6% shows the best penetration ability compared to Chx.HCl normal particle size due to the smaller particle size. Chx.HCl nanoemulsion 1.6% has the lowest mean value of the remaining debris surface area (2001.47 µm2) when compared to normal particle size material (2609.56 µm2). Conclusion: Chx.HCl nanoemulsion preparation has better cleansing ability and antibacterial effect with high efficacy on Enterococcus faecalis, where high reduction rate or complete eradication of bacterial cells has been achieved.

Multiplex loop-mediated isothermal amplification (multi-LAMP) assay for rapid detection of mcr-1 to mcr-5 in colistin-resistant bacteria.

Purpose: The discovery of the plasmid-mediated colistin resistance genes, mcr, revealed a mechanism of transmission of colistin resistance, which is a major, global public health concern especially among individuals infected with carbapenem-resistant Gram-negative bacteria. To monitor the spread and epidemiology of mcr genes, a convenient and reliable method to detect mcr genes in clinical isolates is needed, especially in the primary care institutions. This study aimed to establish a restriction endonuclease-based multiplex loop-mediated isothermal amplification (multi-LAMP) assay to detect mcr genes (mcr-1 to mcr-5) harbored by colistin-resistant bacteria. Methods: A triple-LAMP assay for mcr-1, mcr-3, and mcr-4 and a double-LAMP assay for mcr-2 and mcr-5 were established. The sensitivity and specificity of the LAMP reactions were determined via electrophoresis and visual detection. Results: The sensitivity of the LAMP assay was 10-fold greater than that of PCR, with high specificity among the screened primers. Specific mcr genes were distinguished in accordance with band numbers and the fragment length of the digested LAMP amplification products. Furthermore, the LAMP assay was confirmed as a rapid and reliable diagnostic technique upon application for clinical samples, and the results were consistent with those of conventional PCR assay. Conclusion: The multi-LAMP assay is a potentially promising method to detect mcr genes and will, if implemented, help prevent infections by drug-resistant bacteria in primary-care hospitals due to rapid and reliable surveillance. To our knowledge, this is the first study to report the application of LAMP to detect mcr-2 to mcr-5 genes and the first time that multi-LAMP has been applied to detect mcr genes.

Antimicrobial resistance, virulence genes profiling and molecular relatedness of methicillin-resistant Staphylococcus aureus strains isolated from hospitalized patients in Guangdong Province, China.

PURPOSE: The main objective of this study was to decipher the prevalence, antimicrobial resistance, major virulence genes and the molecular characteristics of methicillin-resistant Staphylococcus aureus (MRSA) isolated from different clinical sources in southern China. MATERIALS AND METHODS: The present study was performed on 187 non-duplicate S. aureus clinical isolates collected from three tertiary hospitals in Guangdong Province, China, 2010-2016. Antimicrobial susceptibility testing was performed by the disk diffusion method and by measuring the minimum inhibitory concentration. Screening for resistance and virulence genes was performed. Clonal relatedness was determined using various molecular typing methods such as multilocus sequence typing, spa and staphylococcal chromosomal cassette mec (SCCmec) typing. Whole genome sequencing was performed for three selected isolates. RESULTS: Out of 187 isolates, 103 (55%) were identified as MRSA. The highest prevalence rate was found among the skin and soft tissue infection (SSTI) samples (58/103), followed by sputum samples (25/103), blood stream infection samples (15/103) and others (5/103). Antimicrobial susceptibility results revealed high resistance rates for erythromycin (64.1%), clindamycin (48.5%), gentamicin (36.9%) and ciprofloxacin (33.98%). All isolates were susceptible to vancomycin. Resistance genes and mutation detected were as follows: aac(6')-aph(2") (24.3%), dfrG (10.7%), rpoB (21.4%), cfr (0%), fexA (1.94%), gyrA (35.92%), gyrB (0.97%), grlA (20.4%), grlB (10.68%), ermA (21.4%), ermB (18.44%), ermC (21.4%) and lnuA (18.44%). Profiling of virulence genes revealed the following: sea (11.7%), seb (21.4%), sec (0.97%), sed (0.97%), hla (86.41%), hlb (17.48%), hlg (10.68%), hld (53.4%), Tsst-1 (3.9%) and pvl (27.2%). Clonal relatedness showed that ST239-SCCmecA III-t37 clone was the most prevalent clone. CONCLUSION: Our study elucidated the prevalence, antibiotic resistance, pathogenicity and molecular characteristics of MRSA isolated from various clinical sources in Guangdong, China. We found that the infectious rate of MRSA was higher among SSTI than other sources. The most predominant genotype was ST239-SCCmecA III-t37 clone, indicating that ST239-t30 clone which was previously predominant had been replaced by a new clone.

Plasmid-mediated colistin resistance gene mcr-1 in Escherichia coli and Klebsiella pneumoniae isolated from market retail fruits in Guangzhou, China.

BACKGROUND: As a result of the growing prevalence of the plasmid-mediated mobile colistin resistance gene mcr-1 among Gram-negative bacteria, the surveillance of mcr-1 has been globally applied. In our study, we aimed to shed light on the possibility of transmission of mcr-1-resistant isolates through market retail fruits. METHODS AND RESULTS: Herein, 133 different fruit surface samples were collected and screened for the different MCR variants (mcr-1 to mcr-8) using PCR and confirmed with sequencing. We identify for the first time mcr-1-carrying Escherichia coli and Klebsiella pneumoniae from market retail fruits in Guangzhou, China. Minimum inhibitory concentrations were detected by the broth microdilution method. Liquid mating was performed to check the transferability of the mcr-1 gene. Pulsed field gel electrophoresis analysis of S1 nuclease-digested DNA and Southern blotting were performed to check the location of the mcr-1 gene. Then, whole-genome sequencing and in silico multilocus sequence typing analysis were performed. CONCLUSION: We showed that E. coli GB110 can mediate the spreading of antibiotic resistance genes through the food chain, while K. pneumoniae GB015 was considered to be the progenitor of the most successful multidrug-resistant clone. Since fruits are usually consumed fresh, this may serve as a direct source of mcr-1-producing bacteria in humans that requires prompt surveillance and intervention to limit the spread of resistance.

High prevalence of bla(NDM-1) carbapenemase-encoding gene and 16S rRNA armA methyltransferase gene among Acinetobacter baumannii clinical Isolates in Egypt.

The main objective of this study was to decipher the molecular mechanism of resistance to carbapenems and aminoglycosides in a large series of 150 Acinetobacter baumannii clinical isolates collected from July 2012 to September 2013 in Egypt. We report for the first time the emergence of bla(NDM-1) and the cooccurrence of 16S rRNA methylase armA with bla(NDM-1) and bla(OXA-23) in Egyptian hospitals. Multilocus sequence typing identified 27 distinct sequence types, 11 of which were novel.