Hongyu Ren

Population structure and minimum core genome typing of Legionella pneumophila.

Legionella pneumophila is an important human pathogen causing Legionnaires’ disease. In this study, whole genome sequencing (WGS) was used to study the characteristics and population structure of L. pneumophila strains. We sequenced and compared 53 isolates of L. pneumophila covering different serogroups and sequence-based typing (SBT) types (STs). We found that 1,896 single-copy orthologous genes were shared by all isolates and were defined as the minimum core genome (MCG) of L. pneumophila. A total of 323,224 single-nucleotide polymorphisms (SNPs) were identified among the 53 strains. After excluding 314,059 SNPs which were likely to be results of recombination, the remaining 9,165 SNPs were referred to as MCG SNPs. Population Structure analysis based on MCG divided the 53 L. pneumophila into nine MCG groups. The within-group distances were much smaller than the between-group distances, indicating considerable divergence between MCG groups. MCG groups were also supplied by phylogenetic analysis and may be considered as robust taxonomic units within L. pneumophila. Among the nine MCG groups, eight showed high intracellular growth ability while one showed low intracellular growth ability. Furthermore, MCG typing also showed high resolution in subtyping ST1 strains. The results obtained in this study provided significant insights into the evolution, population structure and pathogenicity of L. pneumophila.

Frequency, Antimicrobial Resistance and Genetic Diversity of Klebsiella pneumoniae in Food Samples

This study aimed to assess the frequency of Klebsiella pneumoniae in food samples and to detect antibiotic resistance phenotypes, antimicrobial resistance genes and the molecular subtypes of the recovered isolates. A total of 998 food samples were collected, and 99 (9.9%) K. pneumoniae strains were isolated; the frequencies were 8.2% (4/49) in fresh raw seafood, 13.8% (26/188) in fresh raw chicken, 11.4% (34/297) in frozen raw food and 7.5% (35/464) in cooked food samples. Antimicrobial resistance was observed against 16 antimicrobials. The highest resistance rate was observed for ampicillin (92.3%), followed by tetracycline (31.3%), trimethoprim-sulfamethoxazole (18.2%), and chloramphenicol (10.1%). Two K. pneumoniae strains were identified as extended-spectrum β-lactamase (ESBL)–one strain had three beta-lactamases genes (blaSHV, blaCTX-M-1, and blaCTX-M-10) and one had only the blaSHV gene. Nineteen multidrug-resistant (MDR) strains were detected; the percentage of MDR strains in fresh raw chicken samples was significantly higher than in other sample types (P<0.05). Six of the 18 trimethoprim-sulfamethoxazole-resistant strains carried the folate pathway inhibitor gene (dhfr). Four isolates were screened by PCR for quinolone resistance genes; aac(6’)-Ib-cr, qnrB, qnrA and qnrS were detected. In addition, gyrA gene mutations such as T247A (Ser83Ile), C248T (Ser83Phe), and A260C (Asp87Ala) and a parC C240T (Ser80Ile) mutation were identified. Five isolates were screened for aminoglycosides resistance genes; aacA4, aacC2, and aadA1 were detected. Pulsed-field gel electrophoresis-based subtyping identified 91 different patterns. Our results indicate that food, especially fresh raw chicken, is a reservoir of antimicrobial-resistant K. pneumoniae, and the potential health risks posed by such strains should not be underestimated. Our results demonstrated high prevalence, antibiotic resistance rate and genetic diversity of K. pneumoniae in food in China. Improved control and prevention strategies are urgently needed.

Legionnaires' disease caused by Legionella pneumophila serogroups 5 and 10, China.

To the Editor: Legionnaires’ disease is a systemic infection caused by gram-negative bacteria belonging to the genus Legionella. The primary clinical manifestation is pneumonia. Legionella spp. are typically found in natural and artificially hydrated environments. Legionella pneumophila is the species responsible for ≈90% of human cases of infection. L. pneumophila is divided into 15 serogroups, among which serogroup 1 is the most prevalent disease-causing variant (1). In contrast, rare cases are caused by other serogroups. We describe a case of Legionnaires’ disease caused by co-infection with L. pneumophila serogroups 5 and 10 and the genotype characteristics of these strains. The case-patient was a 77-year-old man who had chronic hepatitis B for 50 years, ankylosing spondylitis for 40 years, and chronic cholecystitis for 5 years. On September 17, 2012, he was admitted to Wuxi People’s Hospital (Wuxi, China) for treatment after a continuous cough for 15 days and a high fever for 2 days. At admission, the patient had a blood pressure of 130/65 mm Hg, a pulse rate of 102 beats/minute, and a body temperature of 37.4°C, which increased to 38.4°C four hours later. Laboratory tests showed a leukocyte count of 9,200 cells/μL (88.7% neutrophils) and a C-reactive protein level of 31 mg/L in serum. Lung inflammation was identified by computed tomography. The result of a urinary antigen test for L. pneumophila serogroup 1 (Binax, Portland, ME, USA) was negative. Bronchoalveolar lavage was performed, and fluid was collected for bacterial culture and molecular analysis. Real-time PCRs were performed with primers specific for the 5S rRNA gene of the genus Legionella (2) and the L. pneumophila-specific mip gene (3). Legionella colonies isolated from bronchoalveolar lavage fluid grew on buffered-charcoal yeast extract agar. Nine Legionella-like colonies were isolated, and all showed positive results by PCRs. The colonies were identified as L. pneumophila serogroups 2–14 by using the Legionella latex test (Oxoid, Basingstoke, UK). Among these colonies, 5 were identified as L. pneumophila serogroup 5, and 4 were identified as serogroup 10 by using a monoclonal antibody (Denka Seiken, Tokyo, Japan). Environmental investigations were conducted in the patient’s house and hospital room, but L. pneumophila serogroup 5 and 10 were not detected in any of the locations tested. Pulsed-field gel electrophoresis (PFGE) (4) was used to investigate the 9 L. pneumophila strains. Two PFGE patterns that were 94% similar were observed; each pattern represented 1 serogroup. The PFGE patterns were compared with those of a reference database of L. pneumophila for China. All L. pneumophila in the database, including 41 strains isolated from the city in which the patient resided in 2012, had patterns different from those of the 9 strains. Two clinical L. pneumophila strains of different serogroups were further analyzed by sequence typing (5,6). Sequence type (ST) indicated that allele numbers for flaA, pilE, asd, mip, mompS, proA, and neuA genes were 6, 10, 15, 28, 21, 7, and 207 for serogroup 5 strains and 6, 10, 15, 10, 21, 40, and 207 for serogroup 10 strains. By querying the ST database for L. pneumophila (http://www.ewgli.org), we found that both profiles were new and assigned these 2 strains the numbers ST1440 (serogroup 5) and ST1439 (serogroup 10). STs of these 2 isolates differed from each other by only 2 alleles (3 nt in the mip gene and 1 nt in the proA gene), which suggested that the isolates might be more closely related to each other than suggested by serologic analysis. Human infections with L. pneumophila serogroups 5 and 10 have been rarely reported (1,7). Our study confirms human infection with 2 L. pneumophila serogroups that did not involve serogroup 1. Results for this case-patient also indicated that a negative urinary antigen test result should not be a reason for ruling out Legionnaires’ disease because the urinary antigen kit used detects only L. pneumophila serogroup 1 antigen. L. pneumophila serogroups 5 and 10 are probably underrecognized pathogenic serogroups. Culture and molecular analysis should be performed to obtain an accurate diagnosis. Rare co-infections with L. pneumophila serogroup strains have been identified by culture methods (8,9). The cases reported previously and in this study indicate that co-infections with different serogroups are more common than currently recognized and that multiple colonies should be tested for accurate epidemiologic investigations. Qin et al. reported that pathogenic Legionella strains of different species, serogroups, and genotypes were isolated from the same hot spring water samples (10). This finding suggests that co-infections with different Legionella strains may occur under certain conditions. In China, Legionnaires’ disease is usually ignored in the differential diagnosis of pneumonia because most clinicians lack experience with this disease. This case highlights the need to familiarize physicians with diagnostic methods for identifying Legionella pneumonia in clinics in China and for further epidemiologic surveillance to monitor this disease and improve public health disease control strategies.

Defining and Evaluating a Core Genome Multilocus Sequence Typing Scheme for Whole-Genome Sequence-Based Typing of Klebsiella pneumoniae

At present, the most used methods for Klebsiella pneumoniae subtyping are multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). However, the discriminatory power of MLST could not meet the need for distinguishing outbreak and non-outbreak isolates and the PFGE is time-consuming and labor-intensive. A core genome multilocus sequence typing (cgMLST) scheme for whole-genome sequence-based typing of K. pneumoniae was developed for solving the disadvantages of these traditional molecular subtyping methods. Firstly, we used the complete genome of K. pneumoniae strain HKUOPLC as the reference genome and 907 genomes of K. pneumoniae download from NCBI database as original genome dataset to determine cgMLST target genes. A total of 1,143 genes were retained as cgMLST target genes. Secondly, we used 26 K. pneumoniae strains from a nosocomial infection outbreak to evaluate the cgMLST scheme. cgMLST enabled clustering of outbreak strains with <10 alleles difference and unambiguous separation from unrelated outgroup strains. Moreover, cgMLST revealed that there may be several sub-clones of epidemic ST11 clone. In conclusion, the novel cgMLST scheme not only showed higher discriminatory power compared with PFGE and MLST in outbreak investigations but also showed ability to reveal more population structure characteristics than MLST.

Distribution of Secretion Systems in the Genus Legionella and Its Correlation with Pathogenicity

The genus Legionella comprises over 60 species, which are important human pathogens. Secretion systems in Legionella pneumophila have been studied extensively because of the essential role of protein secretion in bacterial infection. However, there are few reports describing the secretion systems in non-L. pneumophila species. In this study, we analyzed the distribution of secretion systems in L. pneumophila and 18 species of non-L. pneumophila based on whole genome sequences. A total of 74 whole genome sequences from 19 species of Legionella were analyzed. Type II and IVB secretion systems were detected in all Legionella strains, but the type I secretion systems was restricted to L. pneumophila. The type IVA secretion system was randomly distributed among different species. Furthermore, we found the type VI secretion system in three non-L. pneumophila strains (Legionella cherrii DSM 19213, Legionella dumoffii Tex-KL, and Legionella gormanii ATCC 33297). In population structure analysis, L. pneumophila formed a conservative cluster and was located at the terminal of the evolutionary tree. At the same time, L. pneumophila, especially eight clone groups (named MCGG1–MCGG8), showed higher intracellular growth ability than non-L. pneumophila species. These results suggest that L. pneumophila has acquired additional secretion systems during evolution, resulting in increased pathogenicity.

Whole genome sequence revealed the fine transmission map of carbapenem-resistant Klebsiella pneumonia isolates within a nosocomial outbreak

BackgroundCarbapenem-resistant Klebsiella pneumoniae (CRKP) is a major cause of nosocomial infections worldwide. The transmission route of CRKP isolates within an outbreak is rarely described. This study aimed to reveal the molecular characteristics and transmission route of CRKP isolates within an outbreak of nosocomial infection.MethodsCollecting case information, active screening and targeted environmental monitoring were carried out. The antibiotic susceptibility, drug-resistant genes, molecular subtype and whole genome sequence of CRKP strains were analyzed.ResultsBetween October and December 2011, 26 CRKP isolates were collected from eight patients in a surgical intensive care unit and subsequent transfer wards of Beijing Tongren hospital, China. All 26 isolates harbored blaKPC-2, blaSHV-1, and blaCTX-M-15 genes, had the same or similar pulsed-field gel electrophoresis patterns, and belonged to the sequence type 11 (ST11) clone. By comprehensive consideration of genomic and epidemiological information, a putative transmission map was constructed, including identifying one case as an independent event distinct from the other seven cases, and revealing two transmissions starting from the same case.ConclusionsThis study provided the first report confirming an outbreak caused by K. pneumoniae ST11 clone co-harboring the blaKPC-2, blaCTX-M-15, and blaSHV-1 genes, and suggested that comprehensive consideration of genomic and epidemiological data can yield a fine transmission map of an outbreak and facilitate the control of nosocomial transmission.

Serotype and MLST-based inference of population structure of clinical Streptococcus pneumoniae from invasive and noninvasive pneumococcal disease

Streptococcus pneumoniae is an important human pathogen causing various diseases. In this study, S. pneumoniae from invasive (IPD) and noninvasive pneumococcal disease (NIPD) were studied by serotype and multilocus sequence typing (MLST) for population structure characteristics. A total of 169 clinical S. pneumoniae, containing 63 IPD and 106 NIPD strains, were analyzed. 19F, 19A, 6A, 6B, 1, 14 and 23F were the dominant serotypes in both IPD and NIPD strains. By MLST, 169 strains were divided into 85 sequence types (STs) with an index of discrimination (IOD) value of 0.9606. The seven predominant STs were ST271, ST320, ST876, ST3173, ST236, ST81 and ST342, which were mainly associated with serotypes 19F, 19A, 14, 6A, 19F, 1, and 1/23F, respectively. The 63 IPD strains were divided into 20 serotypes (IOD=0.9135) and 44 STs (IOD=0.9795); the 106 NIPD strains were divided into 16 serotypes (IOD=0.8334) and 49 STs (IOD=0.9430). In conclusion, the serotypes and ST distribution of IPD and NIPD strains analyzed in this study are similar to the profiles observed in other cities of China, suggesting that the clinical S. pneumoniae isolates were derived from clones generally circulating in China. The strains showed a variety of serotypes and STs, and the IPD strains showed higher serotype and genetic diversity than NIPD strains.