Antibiotic Resistance Characteristics And Prevalence Regularity Of Carbapenem-resistant Klebsiella Pneumoniae In A Wastewater Treatment Plant

Background and objectivesCarbapenem-resistant Klebsiella pneumoniae（CRKP）is a common pathogenic bacterium of hospital-acquired infection,which often manifests as multi-drug resistance or even pan-drug resistance.Clinically,the antibiotic treatment options are severely limited,leading to increased high morbidity and mortality.The widespread use and abuse of carbapenem antibiotics has led to an increase in the detection rate of CRKP.year by year.At present,CRKP has spread globally.The reports of infections and outbreaks of CRKP in hospitals are also gradually increasing.The widespread spread of CRKP has become an important public health issue of worldwide concern,and its prevalence and spread in the environment warrants more and tremendous attention.Wastewater treatment plants gather sewage from communities,hospitals,enterprises,etc.,and contain a large number of antibiotic resistant bacteria（ARB）and antibiotic resistance genes（ARGs）.While wastewater treatment plants are very effective at removing conventional contaminants（ammonia,phosphate,etc.）,their ability to remove various drugs and other compounds（including antibiotics）is very limited,and there still have a certain bacterial load in the effluent.Sewage discharge and sludge land use of wastewater treatment plants are important channels for releasing and diffusing ARB and ARGs into the environment.Therefore,the existence of ARB and ARGs in wastewater treatment plants is closely related to human public health,and is a research hotspot at home and abroad.Previous studies have shown that different treatment processes of wastewater treatment plants can affect the distribution and concentration of ARB in wastewater treatment plants.However,only a few studies have investigated the distribution and changes of ARBs in various wastewater treatment processes.Most studies only focus on the comparative analysis of the ARB concentration in the inlet and outlet water,and the results are still controversial.In addition,seasonal variation can affect the source,growth and reproduction of ARB in wastewater treatment plants,and then affect the distribution and concentration of ARB.Therefore,the dynamic study of various functional areas and seasonal variation factors is helpful to clarify the occurrence of ARB in wastewater treatment plants.At present,ARB species such as heterotrophic bacteria,coliforms,E.coli often targeted in studies on the distribution of ARB in wastewater treatment plants.Studies on the distribution of CRKP in different functional areas and seasons of wastewater treatment plants are scare.In addition,hospitals and wastewater treatment plants are important media of ARB transmission to the environment,and the characteristics and prevalence of CRKP in hospitals and wastewater treatment plants are closely related to environmental CRKP.Among them,the characteristics and prevalence of CRKP are closely related to environmental CRKP.Therefore,in order to effectively prevent and control the spread of CRKP in the environment,we need to clarify the characteristic of CRKP in wastewater treatment plants and its relationship with clinical strains.Therefore,the purposes of this study are to master the antibiotic resistance,virulence and molecular epidemic characteristics of CRKP in a large-scale wastewater treatment plant in Jinan,China;to confirm the occurrence characteristics of CRKP in different treatment processes and seasons;to explore the relationship between clinically important CRKP in wastewater treatment plant and nosocomial infection-related CRKP.MethodsSamples（sewage,sludge and mud cake）were collected from different functional areas of a wastewater treatment plant in Jinan in 2018 winter（January to February）,spring（May）,summer（August）and autumn（November）,respectively.An additional sampling was carried out in January 2019.Samples were screened by MacConkey medium supplemented with 2μg/mL meropenem for carbapenem-resistant gramnegative bacilli.Carbapenem resistance genes including blaNDM,blaKPC,blaVIM,blaIMP and blaoXA-48 were detected using PCR and Sanger sequencing,and CRKP strains were determined based on the results of bacterial identification.Antimicrobial susceptibility testing（AST）of CRKP was performed to confirm the antibiotic-resistant phenotype.S1-PFGE and Southern blot were conducted to determine whether carbapenem ARGs were located on the plasmid and the size of the plasmid.SPSS was used to analyze the difference of CRKP detection rate among different functional units,different sampling seasons and different sampling time points within the day,as well as the relationship between ambient temperature and CRKP detection rate.Illumina Novaseq 6000-PE150 platform were used to perform the whole genome sequencing of isolated CRKP,and antibiotic-resistant genotype,MLST,plasmids,capsule serotype（K type）,virulence genes of CRKP and genetic relationships among CRKP strains were analyzed.Results1.49 CRKP isolates were isolated from wastewater treatment plant.Different ST types were found in these CRKP isolates,among which ST 11 is the dominant MLST type（n=33）,followed by ST766（n=4）,ST15（n=2）,ST220（n=2）,ST22,ST45,ST364,ST414,ST1999,ST2158,ST4584.All CRKP isolates carried carbapenem ARGs blaKPC-2 and/or blaNDM,one or more β-lactam ARGs（blaCTX-M,blaTEM-1B,blaSHV,blaCMY-6,blaOXA-1,blaLAP-2,blaOKP）and fosfomycin resistance gene fosA,and showed high levels of resistance to multiple antibiotics.The results of drug-resistant plasmid analysis showed that ST11 CRKP mainly carried blaKPC-2-positive IncFII-IncR plasmid,with the size ranging from～90kb to～23 8kb.Some of the plasmids had higher genome coverage and nucleotide identity with the multiple drug-resistant plasmid pKPC-CRHVKP-C789 detected in hospital.ST766 CRKP all carried blaNDM-positive IncX3 plasmid with a size of～46kb,which had high genomic coverage and nucleotide identity with IncX3 plasmids widely reported at home and abroad.2.A total of 10 capsule serotypes（K type）were identified,ST11-KL64-wzi64（n=25）was dominate type,followed by ST11-KL47-wzi209（n=3）,KL19-wzi19（n=2）,KL15-wzi50,KL24-wzi101,KL9-wzi9.KL28-wzi84,KL114-wzi354,KL116-wzi180 and KL123-wzi44.All CRKP isolates carried one or more genes encoding virulence factors,including virulence genes encoding siderophores（aerobactin,enterobactin,yersinomycin）,fimbriae（type Ⅰ and type Ⅲ fimbriae）,and capsule.The IncHI1B/IncFIB（K）virulence plasmid carrying the virulence gene cluster rmpA2-iucABCD-iutA was detected from some KPC-2 producing ST11 CRKP isolates.This type of virulence plasmid had high genomic coverage and nucleotide identity with hypervirulent plasmid pVir-CR-hvKP-C789 and pVir-CR-HvKP4.3.In 2018,the detection rates of CRKP in different functional areas were water inlet（41.7%,10/24）,anaerobic tank（16.7%,4/24）,biological aerobic tank（8.3%,2/24）,activated sludge tank（12.5%,3/24）,sludge thickening tank（20.8%,5/24）,mud cake storage area（0.0%,0/24）,water outlet（25.0%,6/24）,and the difference was statistically significant（P<0.05）.The detection rate of CRKP in water inlet was higher than that in biological aerobic tank,activated sludge tank and mud cake storage area.The detection rate of CRKP in anaerobic tank,sludge thickening tank and water outlet was higher than that in mud cake storage area（P<0.05）.In 2018,the detection rates of CRKP in different sampling seasons were 28.6%（12/42）in summer,16.7%（7/42）in spring and autumn and 9.5%（4/42）in winter,with no statistical significance（P>0.05）.The results of Chi-square test for linear trends showed that the detection rate of CRKP increased linearly with the increase of temperature in the range of-10℃ to 40℃（P<0.05）.There is no statistically significant difference in the detection rate of CRKP among different sampling time points within the day（P>0.05）.The detection rate of CRKP in winter of 2019 was 52.4%（11/21）.4.The minimum spanning tree（MST）results generated based on cgMLST analysis showed that ST11-KL64 and ST11-KL47 CRKP isolated from wastewater treatment plant and hospital patients constituted clonal clusters.The detection of ST11-KL64 and ST11-KL47 CRKP isolates in hospital was time-correlated with the detection of cloned isolates in wastewater treatment plant.In addition,ST766,ST220 and ST 15 clonal clusters were also detected.Single nucleotide polymorphism（SNP）analysis showed that there were few SNP differences among ST11 CRKP clones,and most ST11-KL64 strains had few SNP differences with the reported carapenem-resistant hypervirulent K.pneumoniae strain C789.BLASTN comparison showed that these strains had high genomic coverage and nucleotide consistency with C789.Conclusions1.The CRKP detected in WWT has the coexistence of multiple drug resistance genes and has multiple drug resistance as well as potential of high virulence.2.The wastewater treatment plant in this study had a certain effect on CRKP removal,however,multi-drug resistant and hypervirulent CRKP strains were still contained in the final effluent.Wastewater treatment plant urgently needs to strengthen the disinfection of wastewater before discharge and improve the disinfection technology of wastewater.Ambient temperature has a certain influence on the detection of CRKP,and the relationship between the detection of CRKP and season needs to be further studied.The highest detection rate of CRKP in the winter of 2019 may be related to the influenza outbreak.This study demonstrated the prevalence and dissemination of ’high risk’CRKP clone ST11-KL64 and ST11-KL47 in wastewater treatment plant,hospital and between them.The hospital wastewater are likely to be an important source of CRKP contamination in wastewater treatment plant.The hospital urgently needs to take measures to curb the infection and spread of such "superbacteria",and strengthen the disinfection of hospital sewage.