| BackgroundKlebsiella pneumoniae is widely considered as an opportunistic pathogen to be carried asymptomatically in the intestinal tract, skin, nose, and throat of healthy individuals. It is a frequent cause of urinary, respiratory tract infections and bloodstream infections in intensive care units and neonatal care facilities, which can spread readily between patients leading to nosocomial outbreaks. Over the past few decades, K.pneumoniae has been an important cause of community-acquired infections, such as septicemia, lower respiratory tract infection, urinary tract infection, cephalomeningitis and liver abscesses. With the characteristics of hypervirulent and serious drug resistance, community-acquired infections by K. pneumoniae got a high mortality. Similar to other members of the ESKAPE group(Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) of bacterial pathogens, K.pneumoniae has frequently been reported with severe antimicrobial drug resistance, especially in relation to third-generation cephalosporins, including resistance conferred by ESBLs, and to carbapenems. In the past three decades, a new virulent hypermucoviscous variant of Kpneumoniae defined as hypervirulent K. pneumoniae has emerged all over the world especially in Asian and South Africa. The clinical features of hypervirulent K.pneumoniae included the ability to cause severe community-acquired infections (e.g., liver abscesses, pneumonia and meningitis) in young healthy hosts and metastatic infections (e.g., endophthalmitis). Capsular types K1 and K2, to a lesser extent, K5, K20, K54 and K57 were thought to the major virulence determinants responsible for hypervirulent K. pneumoniae in nosocomial infections. In the previous studies, hypervirulent K. pneumoniae was isolated both from inpatients and heathy population. However, the source of hypervirulent K. pneumoniae infection was still unknown.The urban rodent-like animals include many kinds of mammalia animals in urban environment, which are similar to rat in shape and share a similar living habits. Most of urban rodent-like animals such as rodents and shrews play a role as reservoir hosts of causative agents for various bacterial, viral and parasitic zoonoses because of their pervasiveness, their propensity toward close contact with humans. Rodents and shrews have been reported previously to carry multidrug-resistant bacteria pathogen such as Escherichia coliy Staphylococcus aureus, Salmonella typhimurium and Yersinia enterocolitica in USA, UK, Vietnam, Canada. Several studies have supported the hypothesis of the dissemination of resistant bacteria and horizontal transfer of antimicrobial resistance genes among bacteria from wild animal to populations through direct and indirect interactions with humans, food, water or sewer systems. Nevertheless, there were no studies regardingpresence and microbiological characteristics of Kpneumoniae in urben rodent-like animals, particularly rodents. Wheather urben rodent-like animals play a role in dissemination of resistant K. pneumoniea remains unclear.According to the background above, some questions were raised in our study:in community-setting environment, ①prevalence of K. pneumoniae among healthy adults, diarrheal children and urban rodent-like animals;②antibioticresistance of K. pneumoniae among healthy adults, diarrheal children and urban rodent-like animals, and possibility for urban rodent-like animals to be source of drug-resistant K. pneumoniaeisolates and drug-resistant gene;③prevalence of hypervirulent K. pneumoniae among healthy adults, diarrheal children and urban rodent-like animals, and relationship of hypervirulent K. pneumoniae among them. To answer all questions above, we conducted a study to investigate the microbiological characteristics and antibiotic resistance K. pneumoniae among healthy adults, diarrheal children and urban rodent-like animals in Guangzhou city between May to September in 2015.ObjectiveTo investigate the microbiological characteristics and antibiotic resistance ofK. pneumoniae isolates from different sources of stool samples (healthy adults, diarrheal children and urban rodent-like animals), to explore relationships of resistant and hypervirulent K. pneumoniaeisoltes fromhealthy adults, diarrheal children and urban rodent-like animals, which would help to prevent and control the resistant and hypervirulent K. pneumoniaerelated community-acquired infections.Study contents1) To investigate the carriage rates of K. pneumoniae in healthy adults, diarrheal children and urban rodent-like animals;2) To characterize the epidemiology of K. pneumoniae isolates from urban rodent-like animals from a community-setting environment as well as data on distribution and demographic characteristics;3) To investigate antimicrobial susceptibility characteristics ofK. pneumoniae isolates using antimicrobial susceptibility testing and ESBLs-producing testing; to investigate prevalence of ESBL type including TEM, SHV and CTX-M among ESBLs-producing isolates;4) To investigate distribution of hypervirulent K. pneumoniae (including serotypesK1, K2, K5, K20, K54 and K57) and to study antimicrobial susceptibility characteristics, distribution of virulence genes and genotype of hypervirulent K. pneumoniae in Multi-locus sequence type (MLST) analysis among hypervirulent K. pneumoniae isolates.Methods1) Stool samples were consecutively collected from healthy adults and outpatient clinics of diarrheal children in Physical Examination Center the third affiliated hospital of Southern Medical University from June to September,2015. All stool samples were placed in transport medium in (2-8)℃ and transported to lab2) Urban rodent-like animals were captured around a hospital and residential area nearby in Baiyun District, Guangzhou city, Guangdong province in China from May to September,2015 (cover an area of 650,000 square meters). According to Industry standards for Entry-Exit Inspection and Quarantine of the PRC (SN/T 2788-2011), Sherman live traps were set 10 m apart in the evening and checked in the morning after a trapping night around human buildings including street corner, garbage, sewer (or rubbish bin), kitchen (or canteen in hospital and university) and storehouse every month. Trapped rats were anesthetized with diethyl to euthanasia in laboratory. Rats subsequently underwent a full necropsy in laboratory according to Bacteriological testing Standards of laboratory animal (GB/T 14926.42-2001).3) All stool samples were tested to isolate K. pneumoniae with microbiological cultivation meathods according to technical standard (SN/T 2788-2011). K. pneumoniae isolates were tested for their susceptibility by the Kirby-Bauer disk diffusion test on Mueller-Hinton agar for the following antibiotics:amikacin, amoxicillin/clavulanic acid, ampicillin, aztreonam, cefepime, cefotaxime, cefotetan. ceftazidime, cefuroxime sodium, cephazolin, chloramphenicol, ciprofloxacin, gentamicin, meropenem, nitrofurantoin, norfloxacin, piperacillin, tetracycline and trimethoprim/sulfamethoxazole. The eligible isolates were confirmed for the production of ESBLs by further phenotypic confirmatory test. Interpretations were done according to the guidelines of Clinical and Laboratory Standards Institute in 2014. Three types of ESBL gene including SHV, TEM and CTX-M were identified with polymerase chain reaction (PCR) among all ESBLs-producing isolates.4) To determine capsular genotypes of K. pneumoniae, we undertook extraction of nucleic acids using bacterial genome DNA extraction kit and identified hypervirulent K. pneumoniaeincluding serotypes K1, K2, K5, K20. K54 and K.57. Antimicrobial susceptibility test and virulence genes, including magA, wcaG, rmpA, uge, kfu and Aerobactin, were also detected by PCR among serotypes K1, K2, K5, K20, K54 and K57 isolates. MLST was performed to study the genotype of hypervirulent K. pneumoniae according to the protocol described on the MLST websiteof K. pneumoniae.5) All data were keyed in an Excel form,and Chi-square test or Fisher’s exact test was used in the comparison of categorical variables. Analysis was performed using SPSS version 20.0 with P< 0.05 considered statistically significant.Results1) In total, we consecutively collected 275 stool samples of healthy adults in Physical Examination Center and 105 stool samples of diarrheal children from outpatient clinics, and prevalence of K. pneumoniae in healthy adults and diarrheal children were 66.1%(182/275),65.7%(69/105), respectively. A total of 190 urban rodent-like animals (including 80 brown rats,3 mus musculus,2 Rattus flavipectus and 105 shrews) were trapped for the presence of K. pneumoniae in Jingxi community during the 5-month study period, and the capture rate was 26.8%. The capture rate of shrews was significant higher than brown rats, mus musculus and Rattus flavipectus(x2=189.834, P<0.001), while brown rats got the highest capture rate among urban rats (x2=147.225, P<0.001). However, there was no significant difference between the capture ratesbetween shrews and urban rats. Of the 190 urban rodent-like animals tested, K. pneumoniae was identified from 78.4%(149/190)urban rodent-like animals, and there was no significant difference for prevalence of K. pneumoniaebetween shrews and brown rats. None of mus musculus and Rattus flavipectus were positive for K. pneumoniae. Prevalence of K. pneumoniaefrom urban rodent-like animals was higher than those from healthy adults and diarrheal children (x2=9.266, P=0.010).2) All K. pneumoniae isolates were resistant to ampicillin. A total of 182 K. pneumoniae were isolated from healthy adults. K. pneumoniaeisolates got a susceptibility rate of 19.2% to piperacillin and a low susceptibility rate to the first-generation and third-generation cephalosporins (the susceptibility rates to cephazolin, cefotaxime and ceftazidime were 2.2%,35.2% and 65.4%, respectively). The susceptibility rate to the fourth-generation cephalosporins (eg.cefepime) was 54.4%. Isolates from healthy adults were susceptible to sulfonamides, quinolones and carbapenem antibiotics. Of the 69 K. pneumoniae isolated from diarrheal children, the susceptibility rate to piperacillin was only 19.2%. Isolates had high resistance to cephalosporins antibiotics. The susceptibility rates to cephazolin, cefotaxime, ceftazidime and cefepime were 1.4%,46.4%.82.6% and 69.6%. Isolates from diarrheal children were susceptible to sulfonamides, quinolones and carbapenem antibiotics. Of all 149 K. pneumoniae isolated from shrews and brown rats, the susceptibility rate to piperacillin, known as a common penicillin antibiotics, was 10.7%. Isolates from shrews andbrown rats had a high resistance to cephazolin and susceptibility rate was 3.4%. The susceptibility rates to cefotaxime and ceftazidime, known as third-generation cephalosporins, were 36.9% and 65.1% respectively. The susceptibility rates to fourth-generation cephalosporins (eg.cefepime) was 67.8%. Isolates were susceptible to sulfonamides, quinolones and carbapenem antibiotics. Amonghealthy adults, diarrheal children andurban rodent-like animals, multiple drug resistance rates were 47.8%,29.0% and 40.9% (x2=7.382, P=0.025) and the prevalences of ESBLs-producing isolates were17.0%, 14.5% and 10.7% (x2=2.661, P=0.264). Among all ESBLs-producing isolates, the positive rates of TEM and CTX-M types were38.6%(22/57)and28.1%(16/57)(x2=1.421, P=0.233). No SHV type isolates were identified. There was no significant difference for positive rates of TEM type among healthy adults, diarrheal children and urban rodent-like animals (x2=5.585, P=0.061). Though the positive rate for CTX-M type was higher from diarrlheal children than that from either healthy adults or urban rodent-like animals (x2=9.039, P=0.011), no difference was detected between healthy adults and urban rodent-like animals (x2=0.032, P=0.858).3) A total of 400 K. pneumoniaeisolates were tested for distribution of hypervirulent isolates by PCR, and prevalenceof serotypes K1, K2, K5, K20, K.54 and K57 were 1.0%,1.0%,1.5%,0.8%,0.5% and 1.3%, respectively. Among all isolates from humans, positive rates of K1, K2, K5, K20, K54 and K57 were 1.6%, 1.6%,0.4%,0.8%,0.8%,1.2%, respectively. Positive rates of K5, K20 and K57 for K. pneumoniae isolated from urban rodent-like animals were 3.4%,0.7% and 1.3%, and none of isolates were positive to K1, K2 and K54. All Kl and K.2 isolates were susceptible to most of antibiotics tested, while K5, K20 and K57 had a high resistance to penicillin and first-generation cephalosporins antibiotics. Some K5and K57 isolates were resistant to the third-generation cephalosporins. Among all hypervirulent isolates, positive rates of virulent genes uge, kfu, wcaG, aerobactin, rmpA and magA were 95.8%,33.3%,29.2%,29.2%,29.2% and 16.7%, respectively. Gene magA only existed in K1 isolates while uge were positive to all serotypes. All K1 isolates and some K20, K54 and K57 isolates carried wcaG gene. Gene aerobactin were identified among all rmpA-positive isolates. All K2 isolates were lack of kfu gene while other five serotypes were positive to it. Several new STs were identified among all hypervirulent isolates, ST 23, ST86, ST 37 and ST 65 were detected in our study. ST 268 was first identified in rats.Conclusions1) There is no significant difference of carriage rates between healthy adults and diarrheal children, while both of healthy adults and diarrheal children got a lower carriage cates than urban rodent-like animals.2) K. pneumoniae isolates from healthyadults and diarrheal children got a high drug resistance, which were similar to urban rodent-like animals. All isolates got a high level of resistance to penicillin and cephalosporins antibiotics, especially to first-generation and third-generation cephalosporins antibiotics. Isolates from both animals and human beings were susceptible to sulfonamides, quinolones and carbapenem antibiotics. The spectrums of drug resistance were parallel among isolates from urban rodent-like animals, healthy adults and diarrheal children, which suggested urban rodent-like animals can be the reservoir host and source of drug-resistant and hypervirulent K. pneumoniae.3) K1, K2, K5, K20, K54 and K57 serotypes were detected among healthyadults and diarrheal children, and K1, K2, K57 were the predominant serotypes. Serotypes K5, K20, K57 were predominant hypervirulent serotypes in brown rats and shrews, while K1, K2 and K54 has not been detected among animals. All K1 and K2 isolates were susceptible to most of antibiotics tested, while K5, K20 and K57 had a high resistance to penicillin and cephalosporins antibiotics. Gene magA played an important role in the virulence of K1 serotype and only existed in K1 isolates. Gene wcaG was detected in K1, K20, K54 and K57, which helped to evade the phagocytosis of macrophage. Gene rmpA may cooperate with aerobactin to help K. pneumoniae to get virulence.4) Urban rodent-like animals, healthy adults and outpatient clinics of diarrheal children carried some STs of hypervirulent isolates, which belonged to dominant clones in pyogenic live abscesses patients, indicating that shrews and rats can be reservoir host and source of hypervirulent K. pneumoniae. |
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