Abstract
Staphylococcus aureus is known to be a predominant pathogen causing bloodstream infection (BSI) from burn units. Our study aimed to perform the clinical epidemiological analysis and virulence features of S. aureus strains isolated from the burn patients with BSI from a burn center in southeastern China during 2012–2016. A collection of 112 S. aureus isolates causing BSI from burn center of a tertiary care hospital in China was carried out. Antimicrobial susceptibility testing was conducted in accordance with the Clinical and Laboratory Standards Institute (CLSI) guidelines. Toxin gene profiles, multilocus sequence typing, staphylococcal protein A (spa) typing, accessory gene regulator (agr) locus typing, staphylococcal cassette chromosome mec (SCCmec) typing, and dendrographic analysis were used to characterize and analyze these isolates. Of 112 S. aureus isolates, 52 (46.4%) were methicillin-resistant S. aureus (MRSA) and 60 (53.6%) were methicillin-susceptible S. aureus (MSSA). ST239-SCCmec III-t030-agr I was the major prevalent clone (26 from MRSA and 6 from MSSA), which was followed by ST239-SCCmec III-t037-agr I (12, 10.0%) and ST5-SCCmec II-t002-agr I (11, 9.2%). The genotyping results showed high genetic diversity in molecular characterization and toxin gene profiles of the strains. Carriage of tsst-1 was mainly associated with ST239-SCCmec III-t030-agr I and ST30-SCCmec IV-t062-agr III, whereas lukS/F-PV was distributed in different clones. In conclusion, ST239-SCCmec III-t030-agr I is the commonest clone causing BSI among burn patients in eastern regions of China. In contrast to MRSA, polyclonality was statistically significantly higher among MSSA isolated from burn patients with BSI.
Introduction
I
The main task for successful clinical treatment of BSI depends on the information regarding the molecular characteristics and antimicrobial susceptibility patterns of S. aureus strains. 3 Molecular typing of S. aureus has significant value as a measure to control infection. 4 At present, there are several molecular typing methods, including multilocus sequence typing (MLST), staphylococcal protein A (spa) typing, staphylococcal cassette chromosome mec (SCCmec) typing, accessory gene regulator (agr) typing, and toxin gene profiling.5,6 As previously reported, ST239 and ST59 were prevalent in many South Asian countries, such as China, Japan, and Korea.7–9 Recently, several studies have focused on the MRSA BSI.10,11 However, there have been no published data about molecular characteristic and virulence factors of S. aureus isolates causing BSIs in patients with burn in China. The aim of our study was to characterize S. aureus bloodstream isolates from a regional burn center during 2012–2016 by a combination of different phenotypic and molecular typing method.
Materials and Methods
Bacterial isolates
This study was performed at the First Affiliated Hospital, Nanchang University, a 2,900-bed tertiary university hospital with ∼1,400,000 admissions annually in Southeastern China. This study included all S. aureus clinical isolates collected in a 5-year period (2012–2016) from positive blood cultures of patients admitted to the burn intensive care unit and burn ward of our hospital. S. aureus BSI patients were defined as patients with S. aureus in blood cultures that met the Centers for Disease Control and Prevention (CDC) criteria for primary BSI. 12 During this period, a total of 112 nonduplicate bloodstream S. aureus strains were collected in the study. The isolated strains were stored at temperature −80°C and subcultured on brain heart infusion medium before DNA extraction.
Identification and antimicrobial susceptibility test
S. aureus isolates were identified by traditional biochemical tests (catalase, DNase, and tube coagulase tests) and the VITEK-2 compact automated microbiology system (BioMérieux, France). Susceptibility of the isolates to penicillin, oxacillin, vancomycin, tetracycline, clindamycin, erythromycin, rifampin, linezolid, teicoplanin, moxifloxacin, quinupristin/dalfopristin, ciprofloxacin, gentamicin, trimethoprim/sulfamethoxazole, and levofloxacin was determined according to Clinical and Laboratory Standards Institute (CLSI) standards. 13 Resistance to oxacillin or cefoxitin was classified as methicillin resistant. The D-test was performed to detect inducible resistance to clindamycin. S. aureus ATCC25923 was used as a quality control for minimum inhibitory concentration detection.
Detection of toxin genes
DNA was extracted by the simplified alkaline-lysis method 14 and then was subjected to PCR amplification of several clinically significant toxin genes,15,16 including tsst-1, sea-see and seg-sej, lukS/F-PV, eta and etb, hla-hlg, and sasX. 17 Each PCR experiment was carried out with positive control and negative control (distilled water). The PCR products were detected by electrophoresis in a 1% agarose gel stained with ethidium bromide and visualized under UV light.
Molecular typing
All S. aureus isolates were performed with MLST, 8 spa typing, 5 and agr typing. 18 The sequence types (STs) were assigned by comparing the sequences with those of corresponding allelic profiles at MLST database (http://saureus.mlst.net), and clonal complexes (CCs) were determined using the program eBURST. The spa sequence was assigned using the spa database website (www.ridom.de/spaserver) to generate a unique spa type. MRSA strains were confirmed by PCR detecting mecA gene, and SCCmec types of MRSA isolates were determined as previously described. 19
Statistical analyses
Statistical analyses were performed using Statistical Package for the Social Sciences software version 17.0 (SPSS, Chicago, IL).The chi-square test with Yates correction or Fisher's exact test was used to compare the discrete variables. For continuous variables, Student's t test or the Mann–Whitney test was used to examine the difference. A p-value <0.05 was considered statistically significant.
Results
Clinical characteristics
During the 5-year period, 433 positive blood cultures were investigated for possible BSI, and the presence of S. aureus BSI was detected in 112 out of 433 cultures. Their demographic characteristics, clinical characteristics, and outcomes are summarized in Table 1. Of these patients, 60 were classified into the methicillin-susceptible S. aureus (MSSA) BSI group, and 52 were classified into the MRSA BSI group. The sex and age were not significantly different among the groups. However, the acute physiology and chronic health evaluation (APACHE) II score, the percentage total body surface area (TBSA) burned, the percentage TBSA with third-degree burn wounds, rates of central venous catheters, and presence of inhalation injury differed significantly between the groups.
APACHE, acute physiology and chronic health evaluation; BSI, bloodstream infection; ICU, intensive care unit; IQR, interquartile range; MSSA, methicillin-susceptible S. aureus; SD, standard deviation; TBSA, total body surface area.
Antimicrobial susceptibility testing
Among 112 S. aureus isolates, 52 (46.4%) were MRSA and 60 (53.6%) were MSSA. All isolates were susceptible to vancomycin, linezolid, quinupristin/dalfopristin, and teicoplanin. Five (4.5%) penicillin-susceptible isolates of the clinical isolates were β-lactamase producers. Moreover, inducible resistance to clindamycin was seen in 15 isolates (8 MRSA and 7 MSSA) by the D-test. The resistance rates for individual agents were significantly higher in MRSA isolates than in MRSA isolates, including oxacillin, gentamicin, ciprofloxacin, levofloxacin, moxifloxacin, rifampicin, erythromycin, clindamycin, and trimethoprim/sulfamethoxazole (Table 2).
Virulence factors
As shown in Table 3, the seg (59/112, 52.7%) was the most frequent enterotoxin gene, of which 17 isolated from MRSA and 42 isolates were from MSSA. Seventeen isolates carried lukS/F-PV gene and eight of which were MRSA. Fourteen (12.5%) isolates, including 2 from MSSA and 12 from MRSA, carried a tsst-1 gene. The lukS/F-PV-positive isolates were more commonly identified in CC8 and CC5, whereas tsst-1 was more frequent in CC30. The sasX gene was not detected among the S. aureus isolates. Three other toxin genes (sec, see, and seh) were not detected in the MRSA strains. Staphylococcal hla and hlg were the most frequently detected hemolysin genes. MSSA was more likely to contain seg gene (p < 0.0001), whereas those from MRSA were more likely to carry seb and sea (p = 0.0106 and p < 0.0001, respectively).
p-value and two-sided p-value were calculated by the chi-square or Fisher's exact test appropriately. Values in bold signify p < 0.05 compared with MSSA isolates.
lukS/F-PV, gene encoding Panton–Valentine leukocidin; sea-see and seg-sej, gene encoding staphylococcal enterotoxins; tsst, gene encoding toxic shock syndrome toxin 1; hla-hlg, gene encoding staphylococcal hemolysin.
Molecular epidemiological characteristics
Forty-two SCCmec type III and 17 SCCmec type IV were confirmed. Twenty-four spa types were identified. t030 (32/112, 28.6%) was the most common, followed by t002 (13/112, 11.6%), t062 (13/112, 11.6%), t037 (10/112, 8.9%), and t034 (7/112, 6.25%). t030 was detected in 26 MRSA isolates and 6 MSSA isolates, and t037 was detected in 4 MRSA isolates and 6 MSSA isolates.
Among all S. aureus isolates, 17 major CCs that correspond to 19 STs were identified by MLST. The most common ST was ST239 (42/112, 37.5%), followed by ST5 (13/112, 11.6%), ST188 (10/112, 8.9%), and ST30 (8/112, 7.1%). Seventeen CCs were identified according to eBURST analysis. CC8 (42/112, 37.5%) was the most prevalent clone, followed by CC5 (13/112, 11.6%), CC59 (11/112, 9.8%), and CC188 (10/112, 8.9%) (Table 4). In addition, the prevalence of the ST239 was significantly higher in lukS/F-PV-positive isolates, including eight SCCmec type III isolates. Other STs contain ST188 (6 MSSA), ST398 (2 MSSA), and ST6 (1 MSSA) in lukS/F-PV-positive isolates.
CC, clonal complex.
Four agr groups (I-IV) were discovered in 85, 15, 11, and 1 isolates, respectively. The results showed that agr I was the most frequent agr group, detected in 75.9% (85/112) of the strains. The groups of agr II, III, and IV were found less commonly in13.4%, 9.8%, and 0.9% of the isolates, respectively. There was no statistically significant difference in the prevalence of agr allele between MSSA and MRSA. ST239-SCCmec III-t030-agr I (26/52, 50%) was most commonly found in MRSA. However, ST188-t318-agr I (10/60, 16.7%) was the most prevalent clone in MSSA. More diversity was found in MSSA isolates (19 STs and 21 spa types) than in MRSA isolates (8 STs and 12 spa types).
Discussion
Despite radical advances in burn care and treatment, bacterial infection remains the major cause of death in modern burn units. 20 Extensive burn injuries render patients susceptible to S. aureus or MRSA infection. 21 Over the past two decades, the morbidity of BSI in burn patients caused by S. aureus, particularly MRSA, has been increased significantly in low- and middle-income countries. 22 In China, S. aureus was one of the most frequently isolated BSI pathogens in burn patients, and the average prevalence has continued to increase, reaching 55%. 23
In previous study, patients with S. aureus BSIs had a higher 30-day mortality rate (40.2%) and exhibited relatively great TBSA (34%), compared to the previous studies. 24 The overall MRSA prevalence rate was 46.4% in this study. The predominant resistance pattern indicates that vancomycin, linezolid, quinupristin/dalfopristin, and teicoplanin are efficacious drugs for treating S. aureus, including MRSA BSI. Consistent with the previous study, 25 ST239-t030/t037-SCCmec III-agr I clone was the most common among the MRSA strains, followed by ST5-SCCmec II-t002-agr I. Of note, the MSSA was more commonly detected ST188-t318-agr I. We found that ST239, ST30, ST338, ST51, ST88, and ST398 were distributed in both MSSA and MRSA, which suggested that these MRSA originated from MSSA clones through SCCmec transfer. 26 Among the MSSA isolates, ST239 and ST188 occurred at a relatively high frequency (36.7%; 22/60) in our patient population, indicating that some pandemic clones have arisen in Chinese hospitals and spread across the country.
Bien et al. 27 reported that the S. aureus pathogenicity is a complex multifactorial process. To gain insights into the potential pathogenic ability of S. aureus BSI in patients with burns, all the isolates in this study were screened for several virulence genes. Staphylococcal enterotoxins (SEs) are a family of structurally related exotoxin molecules produced by S. aureus. The presence of genes coding for SEs among S. aureus (52.7%, 59/112), isolated from burn patients, is fully consistent with that in the same region. 28 Enterotoxin positive strains in MRSA and MSSA were 42 (70.0%, 42/60) and 27 (38.5%, 20/52), respectively. MRSA is more likely to carry the seb and sea genes, while the seg and sei genes were detected exclusively in the MSSA isolates, which were somewhat similar to the findings of previous studies. 29 In addition, virulence gene profiling showed that the sec, see, and she genes were not found in MRSA strains.
Panton–Valentine leukocidin (PVL) is a bicomponent, pore-forming leukotoxin secreted by S. aureus strains epidemiologically linked to severe invasive infections, and it is the main virulence factor associated with increased virulence of community-associated MRSA isolates. 30 Previous studies demonstrate that the PVL-positive S. aureus population structure was polyclonal in China which contrasts starkly with results from previous studies.31,32 This difference may be affected by environmental, host, social, economic, and cultural factors.33,34 In our study, a relatively high prevalence of PVL-carrying isolates was observed to be associated with several MRSA clones.
The predominance of ST239-SCCmec III-t030/t037-agr I and ST5-SCCmec II-t003-agr II in the PVL-carrying MRSA isolates in our hospital is a cause for concern due to the propensity to spread worldwide and displace hospital strains.35,36 Notably, the tsst-1 gene encoding the toxic shock syndrome toxin TSST-1 was mainly found in ST30-SCCmec IV-t062-agr III and ST5-SCCmec II-t002-agr II strains.
In conclusion, our study illustrates the characteristics of the S. aureus isolates from burn patients in a hospital of Jiangxi from 2012 to 2016. Molecular typing revealed the presence of 30 different genotypes with ST239-t030/t37-SCCmec III-agr I in MRSA and ST188-t318-agr I in MSSA. MSSA isolates expressed greater diversity in molecular characteristics than MRSA. Toxin genes, in particular tsst-1 and lukS/F-PV, were associated with specific CCs. Our results reinforce the need for ongoing microbiological surveillance and appropriate selection of antibiotics.
Footnotes
Acknowledgments
Financial support was provided by the National Natural Science Foundation of China (81560323), Education Department of Jiangxi Province, China (GJJ160029), Jiangxi science and Technology Department in China (20171BBG70053 and 20161BAB205247), and Health and Family Planning Commission of Jiangxi Province (20155140).
Authors' Contributions
All authors equally participated in study design, data analysis and interpretation, and writing of the article.
Disclosure Statement
No competing financial interests exist.