The Finding Of Carbapenem-resistant Salmonella Stanley And Resistance Mechanisms

Antibiotics were among greatest discoveries of20th century which saved innumerable lives undoubtedly. As a result of extensive use of antimicrobials clinical treatments, animal husbandry and aquaculture, the incidence of multi-drug resistant bacteria has been increasing, leading to numerous treatment failure and even death. One of the main resistant mechanisms is that bacteria can produce antibiotics-hyrolyzing enzymes. The Enterobacteriaceae are competent enzyme producers and important clinical pathogens. By producing extended-spectrum-p-lactamases (ESBLs) and AmpC enzyme, they can be resistant to almost beta-lactams, which are the most important class of antibiotics. Carbapenems are advocated for the treatment of infections caused by ESBLs and or AmpC-producing Enterobacteriaceae owing to its stability for ESBLs and AmpC. Unfortunately, resistance to carbapenems quickly emerged in Enterobacteriaceae and became concerns of globle heathcare workers. Carbapenems-resistant Enterobacteriaceae usually remain susceptible in vitro only to colistin, tigecycline, fosfomycin. The novel metallo-β-lactamase NDM-1(New Delhi metallo-β-lactamase-1) was first found in2009. Bacteria producing NDM-1is typically resistant to almost all β-lactam antibiotics including carbapenems and has diseminated globally. Considering the potential for bacteria carrying NDM-1, this finding is worrisome and emphasizes the need for epidemiological studies and scrutiny of antimicrobial susceptibility.Salmonellosis is an important zoonosis. Salmonella is the most important foodborne pathogens, and it often leads to outbreaks worldwide. So the increasing of antibiotics-resistance of Salmonella has to be taken seriously. The research on Salmonella has been more than100years since Choleraesuis Salmonella was isolated by Salmon and Smith firstly. More than2000kinds of Salmonella serotypes have been reported now. Antibiotics have played an important role in controlling and prevention of salmonellosis. However, with the abuse and improper use of antibiotics, the antibiotic resistant rate of Salmonella is increasing gradually, exhibiting in multi-resistance or even carbapenems-resistance. The Salmonella can aquire carbapenem-resistant genes encoded carbapenemases. In2011, one blaNDM-1positive Salmonella strain was isolated in USA from rectal swab of one patient transferred from Indian. And NDM-1harboring Salmonella strain was found again in France from urine sample of another patient transferred from Indian in September2012. Both positive clinical Salmonella strains were discovered by resistance surveillance and the patients had no clear infection symptoms caused by Salmonella. In spite of rare reports of carbapenem-resistant Salmonella, the dissemination of antibiotic-resistant Salmonella and its resistance genes will be great threat to public health due to the high infectivity of Salmonella.We described here a NDM-1-prouducing Salmonella strain isolated from the stool of a child with acute diarrhea. The aim of this study was to analyze the clinical data, and carbapenem-resistance mechanism and disseminationPart I:The finding of carbapenem-resistant SalmonellaThe clinical Salmonella strain was isolated from an11-month-old girl at Lishui Central Hospital of Zhejiang province, China on July25,2012. Six days before admission, the patient developed a continued fever as high as40℃, accompanied with cough. Four days before admission, physical examination revealed rales in both lungs. The white blood count was8900/μl, with80%neutrophils. No obvious abnormalities were found in chest X-ray. She was diagnosed with acute bronchitis and given cefoxitin for3days and piperacillin-tazobactam for1day parenterally, but the fever persisted. Two days before admission, the patient developed diarrhea4to5times a day with loose stool containing mucus and blood. On the admission day, stool analysis revealed3to4white blood cells and1to3red blood cells per high power field. Salmonella was isolated from stool obtained on admission, identified with the Vitek-2Compact System, and serotyped by the local Centers for Disease Control.The patient was diagnosed as having bacterial enteritis and given intravenous azithromycin and latamoxef. Fever and diarrhea resolved over the next three days. On the fifth day of hospitalizaion, a stool culture was negative for Salmonella and the patient was discharged. At a follow-up visit3months later, no Salmonella or other carbapenem-resistant bacteria were isolated from stool samples from the patient or her grandmother and brother, who lived with her. The case confirmed to be sporadic after investigation, and epidemiologically it was not related the Indian subcontinent. The strain belongs to sequence type (ST)29by using multilocus sequence typing.Part II The study of carbapenem-resistance mechanism and disseminationThe Modified Hodge Test with Salmonella Stanley was weakly positive. Double-disk synergy test to detect metallo-β-lactamases was also positive. The strain was shown to possess blaNDM-1but was negative for other MBL genes. Carbapenem resistance could be transfered from Salmonella Stanley to E. coli C600RifR and K. pneumoniae13883RifR by conjugation at frequency about10-7～10-4over a period of15min. Electrophoresis showed that the donor and the transconjugant strains had the same plasmid profiles, which both contained a single plasmid named pHS36-NDM approximately140kb in size. PCR-based typing indicated that the plasmid belonged to IncA/C.The blaNDM-1stability experiments showed that only3clonies of clinical Salmonella Stanley strain lost the carbapenem resistance, which were collected at the3nd day and9th day of passages, respectively. The plasmids containing blaNDM-1were lost in these three colonies. No transconjugants lost carbapenem resistance during the14days of passages. The growth curve of the strain carrying pHS36-NDM was similar to the same species of bacteria without pHS36-NDM.Part III pHS36-NDM plasmid sequenced and bioinformatics analysis of NDM-1By using enzyme digestion, molecular cloning, PCR and the second generational high-throughput sequencing technology we have revealed the genetic information of pHS36-NDM plasmid. The results shows that the full length of plasmid is138001bp and the average G+C content is50.2%. Also, we analyze the open reading frame (ORF) by The ORF Finder software on the NCBI web site, and we found that the ORF number is763. However, comparing with public database, only170ORFs could be recognized. Interestingly, the pHS36-NDM plasmid encodes type IV conjugal transfer system protein gene such as traA, traB, traC, traD, traE, traF, traG, traH, tral, traK, traL, traW, traU, traN, traV etc and StbA gene which encode a plasmid stability protein, confirming the capability of horizontal transfer and passage stability of pHS36-NDM plasmid. Besides, the plasmid contains several mobile genetic elements including ISEcpl,1S4321, Tn1696, intll,ISKpnl4,ISAbl25and ISCR27, which increase the plasticity of plasmid. Moreover, the plasmid contained some drug resistance related gene, such as sull, blaCMY-6and blaNDM-1in accordance with antimicrobial sensitivity test. Moreover, the plasmid contains some repeat sequence related to the regulation of plasmid replication and transfer of and protein expression which raise the possibility that the plasmid had certain significance to regulating plasmid copies. The genetic environment of blaNDM-1gene shared very little identity with India strains, while some strains have been reported from other unrelated countries show a high degree of consistency. There was no conclusive evidence that the homology and relationship between these bacterias. We speculated that NDM-1may probably originate in environmental microbes, under the selection pressure of antibiotics and species evolution, bacteria can integrate the segments to adapt to changing environments.Also, we analyzed the physical and chemical properties of NDM-1protein by bio informatics analysis. Results showed that molecular formula of NDM-1was C1256H1973N353O377S14, and its molecular mass was28kDa contains269amino acids. The isoelectric point of NDM-1was5.88and the hydrophobicity GRAVY value was0.010. The half-life of NDM-1protein in Escherichia coli was more than10hours. The NDM-1belonged to the Lactamase_B (PF00753) protein family, its functional domain composition amino acid position:74to250. The crystal structure of NDM-1monomer alone or combined with some drug compounds such as captopril, meropenem and ampicillin have been resolved. NDM-1amino acid sequence alignment were performed and found NDM-1shows lower sequence identity with other MBLs, and the most closely related MBLs was VIM which show32%sequence identity with NDM-1Conclusions1. This is the first report of carbapenem-resistant Salmonella isolated from a child with acute diarrhea and resistant to all β-lactam antibiotics including compound preparations of P-Lactamase inhibitors and carbapenems. The resistance profile was attributed to blaNDM-1. That azithromycin is recommended for the treatment of invasive Salmonella infection containing NDM-1in children may have the good effect. We suggest that azithromycin be used for treatment based on antimicrobial susceptibility. The Salmonella Stanley isolate possessing NDM-1shows weakly positive result of Hodified Hodge Test, and it is epidemiologically not related to the Indian subcontinent, where they occur widely in hospital and community infections.2. The strain carries an approximately138001bp, and IncA/C type plasmid harboring blaNDM-1.The plsmid, which is named pHS36-NDM, could be transferred from Salmonella to Escherichia coli and Klebsiella pneumoniae at high frequency by conjugation and demonstrated high stability during14days’passages. IncA/C is important plasmid type carrying blaNDM-1.3. The full-length DNA of pHS36-NDM plasmid is138001bp. Multiple-alignments of complete sequences of plasmids and alignments of several sequences surrounding the blaNDM-1gene demonstrated that pHS36-NDM has high identity with sequences from Canada and Italy, but has low identity with sequences from Klebsiella pneumoniae05-506in India.