Molecular characterization of Klebsiella pneumoniae isolates from stool specimens of outpatients in sentinel hospitals Beijing, China, 2010–2015

Background Klebsiella pneumoniae (K. pneumoniae) is an opportunistic pathogen associated with community-acquired infections and nosocomial infections. From 2010 to 2015, K. pneumoniae testing was included into the exiting diarrhea-syndrome surveillance with objective to estimate the prevalence of K. pneumoniae in diarrhea-syndrome patients, test antibiotics susceptibility and investigate molecular characteristics. Methods Stool specimens from diarrhea-syndrome outpatients were cultured and identified the pathogens by the Vitek2 Compact instrument. The isolated K. pneumoniae strains were tested for antibiotics susceptibility by minimal inhibitory concentration (MIC) method, and subtyped by pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Results 22 K. pneumoniae strains were identified from 4340 stool specimens of outpatients who visited sentinel hospitals in Beijing during 2010–2015. All strains were sensitive to gentamicin, nalidixic acid, ciprofloxacin, ceftriaxone, cefotaxime, cefepime, imipenem. The highest resistance rate of K. pneumoniae strains was 100% to amoxicillin–clavulanate, followed by 72.7% to ampicillin. These 22 K. pneumoniae strains were characterized into 21 different PFGE types and 20 MLST types with less similarity. Conclusions The detection rate of K. pneumoniae in stool specimens from outpatients with diarrhea syndromes was about 0.5% in Beijing. Less similarity of the isolated strains indicated the unlikely long-term circulating of K. pneumoniae in the community. ST23 was the most common genotype. Drug resistance of the community-acquired K. pneumoniae was not a serious problem in comparing with hospital-acquired infections. High vigilance in the community-acquired K. pneumoniae strains and investigation of pathogens’ microbiological characteristics are valuable for signals detection for drug resistance in population and strains variation.

increasingly nosocomial infections caused by K. pneumoniae might impose an increasing risk of infections in communities, from 2010 to 2015, K. pneumoniae testing was included into the exiting diarrhea-syndrome surveillance with objective to estimate the prevalence of K. pneumoniae in diarrheasyndrome outpatients, test antibiotics susceptibility and investigate microbiological characteristics.

Identification of bacterial strains
Stool specimens from diarrhea-syndrome outpatients were collected through Beijing diarrhea-syndrome surveillance from 2010 to 2015. Diarrheagenic bacteria strains were cultured and isolated from stool specimens firstly. Any isolated positive bacteria strains were further identified the pathogens (e.g., Salmonella, Shigella spp., diarrheagenic Escherichia coli, Vibrio parahemolyticus, and K. pneumoniae) by the Vitek2 Compact instrument (bioMérieux. Marcy, France). The isolated K. pneumoniae strains were proceeding for antibiotics susceptibility test and molecular characterization.

Antibiotics susceptibility testing
Antibiotics susceptibility testing for the K. pneumoniae strains was measured by minimal inhibitory concentration (MIC) method. The MICs were interpreted by the standards of Clinical and Laboratory Standards Institute (CLSI) document M100-S26:2016 [6]. The following 14 antibiotics sourced from Shanghai Xingbai Co. (AST panel for aerobic gram negative bacilli) were used for antimicrobial susceptibility test: ciprofloxacin, ampicillin, amoxicillinclavulanate, chloramphenicol, sulfisoxazole, trimethoprimsulfamethoxazole, nalidixic acid, ceftriaxone, gentamicin, tetracycline, cefotaxime, cefoxitin, cefepime, imipenem. Escherichia coli strain ATCC 25922 was in use as qualitycontrol strains for the antibiotics susceptibility testing. MIC level at or above 2 μg/mL for cefotaxime and ceftriaxone indicates the strains possibly produced extended-spectrum beta-lactamases (ESBL), which needs further confirmation. MIC for ceftazidime combination with clavulanate decrease at least three twofold concentration in contrast with the MIC value for ceftazidime alone (e.g., ceftazidime MIC = 8 μg/mL; ceftazidime-clavulanate MIC = 1 μg/mL) confirms the existing ESBL production strains [6].

Pulse field gel electrophoresis
K. pneumoniae strains were subtyped by pulse field gel electrophoresis (PFGE) method using restriction endonucleases XbaI [7]. The restriction endonuclease XbaI (Fermentas) was used to digest the prepared genomic DNA and resultant DNA fragments were separated in a PFGE CHEF-DR III system (Bio-Rad Laboratories) in 0.5× Tris-borate-EDTA buffer at 120 V for 18.5 h, with pulse times ranging from 6 to 36 s. PFGE dendrogram was generated by BioNumerics version 7.1 (Applied Math, Belgium) with the unweighted pairgroup method (UPGMA) and Dice coefficient. Isolates that exhibited a PFGE profile with more than 80% similarity were considered as closely related strains [8,9].

Multilocus sequence typing
Multilocus sequence typing (MLST) was performed to subtype K. pneumoniae strains using seven housekeeping genes (gapA, infB, mdh, pgi, phoE, rpoB, and tonB) [10]. Seven gene fragments of the K. pneumoniae strains were amplified by PCR and sequenced. The allele sequences and sequence types (STs) were determined by the Institute Pasteur Klebsiella MLST database (http://bigsdb.web. pasteur.fr/klebsiella/klebsiella.html). BioNumerics version 7.1 software was used to create the minimum spanning tree. In the minimum spanning tree, the founder ST was defined as the greatest number of single-locus variants. Types were represented by circles and the size of a circle indicated the number of strains with this particular type.

Antibiotics susceptibility results
Antibiotics susceptibility is shown in Table 1. The result showed that 22 K. pneumoniae strains were all sensitive to gentamicin, nalidixic acid, ciprofloxacin, ceftriaxone, cefotaxime, cefepime, imipenem, followed by 95.5% to trimethoprim-sulfamethoxazole, cefoxitin, and 63.6% to sulfisoxazole. The highest resistance rate of K. pneumoniae strains was 100% to amoxicillin-clavulanate, and followed by 72.7% to ampicillin. Moreover, all 22 K. pneumoniae strains were sensitive to ceftriaxone, cefotaxime, the MIC was less than 1 μg/mL, and no ESBL production strain was identified.

PFGE typing
22 isolates showed 21 different PFGE types. The dendrogram of the PFGE image showed that four K. pneumoniae strains isolated during 2011-2014 had over 80% similarity, which categorized into one PFGE cluster (cluster A), while other strains had less similarity (Fig. 1).

MLST typing
MLST was performed for all 22 isolates to investigate the genetic relationships. MLST analysis revealed 20 different sequence types (STs), including three ST23 strains and one ST65. In addition to identifying ST23 and ST65 which are prevail in Asia countries, ST2362, ST2363, ST2364, ST2365, ST2366, ST2367, ST2368, ST2369, and ST2370 were discerned for the first time. In the minimum spanning tree, there were two clone groups: one clone group contained ST23 and ST1660, and the other clone group contained ST17 and ST20 (Fig. 2).

Discussion
K. pneumoniae is one of the most common Gram-negative pathogen found in human's nasopharynx and in the intestinal tract [1]. It existed with 60-70% the carrier rate in the hospital environment, and was notified as a common pathogen caused nosocomial infection [1]. However, the community-acquired K. pneumoniae infection was rare reported. This study, through inclusion of K. pneumoniae into the existing diarrhea-syndrome surveillance, was able to detect K. pneumoniae infection in community and its risk for possible persistent transmission in population in Beijing.
The detection rate of K. pneumoniae in stool specimens from outpatients with diarrhea syndromes was about 0.5% (22/4340), which further demonstrated the existence of the community-acquired K. pneumoniae infection [11]. The prevalence in this study was much lower than the previous detection rate of K. pneumoniae in stool samples ranges from 5 to 38% in hospital patients [11], which emphasized the importance of nosocomial infection control as well necessary vigilance for detecting community-acquired K. pneumoniae.
Less similarity of the strains typed by PFGE or MLST indicated the unlikely long-term transmission of K. pneumoniae in the community. This study identified ST23 as the most common genotype, which were reported in many previous hospital-acquired infection studies in Asia [12,13]. Noticeable this study isolated one ST65 strain, a mucoid phenotype and harbored rmpA gene for aerobactin, which more likely caused community-acquired infection [14,15] and indicated as one of the independent risk factors for bacteremia in patients with pneumonia [13]. Moreover, the microbiological character of newly detected ST2362, ST2363, ST2364, ST2365, ST2366, ST2367, ST2368, ST2369, and ST2370, these strains presented clinical character are not thoroughly explicit, which needs further studies.
No ESBL production strains existed from stool specimens of diarrhea-syndrome outpatients in recent years illustrated that the community-acquired K. pneumoniae was not a serious public health problem. All the strains were sensitive to some antibiotics (e.g., cephalosporins, quinolones, and fluoroquinolones) universally in use for clinical treatment. However, hospital-acquired K. pneumoniae infection with resistance to multiple antibiotics agents has been increasing [16]. The Klebsiella species strains (e.g., TEM-type and SHV-type ESBLs, CTX-M type ESBLs) caused several predominant nosocomial infections [17]. This study did not identify the drug resistance for K. pneumoniae strains, but the diarrhea-syndrome surveillance identified serious drug resistance for Shigella spp. [4]. Given the consideration of horizontal transfer of drug resistance related genes as one of the most important mechanisms for the dramatically quickly dissemination of multi drug resistance among bacteria [18], monitoring the drug resistance of the community-acquired K. pneumoniae strains can provide a significant signal of drug resistance in population.

Conclusions
The detection rate of K. pneumoniae in stool specimens from outpatients with diarrhea syndromes was about 0.5% in Beijing. Less similarity of the isolated strains indicated the unlikely long-term transmission of K. pneumoniae in the community. ST23 was the most common genotype. Drug resistance of the community-acquired K. pneumoniae was not a serious problem in comparing with hospital-acquired infections. High vigilance in the drug resistance of the community-acquired K. pneumoniae strains can provide a significant signal of drug resistance in population.   3 3 3 3 3 3 3 3  3  3 3 3 3 3 3 3 3  3   3 3 3 3 3 3 3 3  3   3 3 3 3 3 3 3 3  3   3 3 3 3 3 3 3 3  3   3 3 3 3 3 3 3