A fatal outbreak of ST11 carbapenem-resistant hypervirulent Klebsiella pneumoniae in a Chinese hospital: a molecular epidemiological study

Summary

Background

Hypervirulent Klebsiella pneumoniae strains often cause life-threatening community-acquired infections in young and healthy hosts, but are usually sensitive to antibiotics. In this study, we investigated a fatal outbreak of ventilator-associated pneumonia caused by a new emerging hypervirulent K pneumoniae strain.

Methods

The outbreak occurred in the integrated intensive care unit of a new branch of the Second Affiliated Hospital of Zhejiang University (Hangzhou, China). We collected 21 carbapenem-resistant K pneumoniae strains from five patients and characterised these strains for their antimicrobial susceptibility, multilocus sequence types, and genetic relatedness using VITEK-2 compact system, multilocus sequence typing, and whole genome sequencing. We selected one representative isolate from each patient to establish the virulence potential using a human neutrophil assay and Galleria mellonella model and to establish the genetic basis of their hypervirulence phenotype.

Findings

All five patients had undergone surgery for multiple trauma and subsequently received mechanical ventilation. The patients were aged 53–73 years and were admitted to the intensive care unit between late February and April, 2016. They all had severe pneumonia, carbapenem-resistant K pneumoniae infections, and poor responses to antibiotic treatment and died due to severe lung infection, multiorgan failure, or septic shock. All five representative carbapenem-resistant K pneumoniae strains belonged to the ST11 type, which is the most prevalent carbapenem-resistant K pneumoniae type in China, and originated from the same clone. The strains were positive on the string test, had survival of about 80% after 1 h incubation in human neutrophils, and killed 100% of wax moth larvae (G mellonella) inoculated with 1 × 10⁶ colony-forming units of the specimens within 24 h, suggesting that they were hypervirulent K pneumoniae. Genomic analyses showed that the emergence of these ST11 carbapenem-resistant hypervirulent K pneumoniae strains was due to the acquisition of a roughly 170 kbp pLVPK-like virulence plasmid by classic ST11 carbapenem-resistant K pneumoniae strains. We also detected these strains in specimens collected in other regions of China.

Interpretation

The ST11 carbapenem-resistant hypervirulent K pneumoniae strains pose a substantial threat to human health because they are simultaneously hypervirulent, multidrug resistant, and highly transmissible. Control measures should be implemented to prevent further dissemination of such organisms in the hospital setting and the community.

Funding

Chinese National Key Basic Research and Development Program and Collaborative Research Fund of Hong Kong Research Grant Council.

Introduction

Klebsiella pneumoniae is a major Gram-negative bacterial pathogen that can cause invasive hospital-acquired infections among immunocompromised patients.¹ Pyogenic liver abscesses caused by K pneumoniae have become a serious clinical challenge in Asia. In Taiwan, more than 3000 new cases of pyogenic liver abscesses occur each year.² Compared with the classic K pneumoniae strains that cause other types of opportunistic infections, pyogenic liver abscess-associated K pneumoniae strains often have substantially higher virulence and are therefore designated hypervirulent K pneumoniae. Hypervirulent K pneumoniae has the ability to cause life-threatening, community-acquired infections such as liver abscesses, pneumonia, meningitis, and endophthalmitis in young and healthy individuals and is therefore associated with high morbidity and mortality.³ These strains can efficiently acquire iron and produce an increased amount of capsular substance compared with classic K pneumoniae, which confers a hypermucoviscous phenotype that is detectable as a positive result on the string test (a viscous string of >5 mm in length is produced when touched with an inoculation loop).³ The hypervirulent phenotype of K pneumoniae is thought to be attributable to the carriage of a virulence plasmid harbouring two capsular polysaccharide (CPS) regulator genes (rmpA and rmpA2) and several siderophore gene clusters that contribute to the hypermucoviscous phenotype.4, 5 Correlation between carriage of the virulence plasmid and the hypervirulence phenotype has also been reported.⁶ To date, most hypervirulent K pneumoniae strains have been K1 or K2 types and have remained antibiotic-sensitive.⁷

Research in context

Evidence before this study

We searched PubMed with no language restrictions for reports that were published from Jan 1, 2000, to May 5, 2017, and contained the terms “Hypervirulent and Klebsiella pneumoniae”, “Hypervirulent and Klebsiella pneumoniae and antimicrobial resistance”, “Klebsiella pneumoniae and ST11 and antimicrobial resistance”, “China and Hypervirulent Klebsiella pneumoniae”, “Hypervirulent Klebsiella pneumoniae and virulence plasmid”, “ST11 and Klebsiella pneumoniae and virulence plasmid ”, “Hypervirulent Klebsiella pneumoniae and virulence ”, and “Klebsiella pneumoniae and ventilator-associated pneumonia”. We found only one publication that described the emergence of carbapenem-resistant K2/ST65 and K2/ST25 hypervirulent Klebsiella pneumoniae strains, as well as the first case of ST11 hypervirulent K pneumoniae infection in Beijing, China, in 2015, which was defined on the basis of string test result. However, no virulence assays, such as human neutrophil survival assay or those involving animal models, were used to confirm the hypervirulence phenotype of this strain, in which no virulence plasmid or virulence-encoding genes were detectable. Additionally, the study found no significant difference between the clinical symptoms of patients infected by this carbapenem-resistant hypervirulent K pneumoniae strain and those infected with classic K pneumoniae strains. To date, carbapenem resistance and hypervirulence phenotypes are associated predominantly with K1 and K2 type organisms carrying virulence plasmids, with only carbapenem-resistant K1 hypervirulent K pneumoniae strains having been reported to cause lethal infections. No report has described the phenotypic features and underlying resistance mechanisms of the hypervirulent ST11 K pneumoniae strains or clinical manifestations of diseases caused by such strains.

Added value of this study

Our results showed that carbapenem-resistant ST11 hypervirulent K pneumoniae (ST11 carbapenem-resistant hypervirulent K pneumoniae) strains have emerged and can cause fatal ventilator-associated pneumonia in patients in hospital. Such strains have disseminated across various regions of China, accounting for as much as 3% of clinical ST11 carbapenem-resistant K pneumoniae infections in the country. ST11 carbapenem-resistant hypervirulent K pneumoniae has a hypervirulence phenotype characterised by a positive string test result, extremely high survival on exposure to human neutrophils, and high virulence in a wax moth (Galleria mellonella) larva infection model. The emergence of ST11 carbapenem-resistant hypervirulent K pneumoniae strains was due to the acquisition of a roughly 170 kbp virulence plasmid carrying the rmpA2 and aerobactin biosynthesis genes by classic ST11 carbapenem-resistant K pneumoniae strains.

Implications of all the available evidence

ST11 carbapenem-resistant hypervirulent K pneumoniae strains are expected to pose a substantial threat to human health because these strains are simultaneously hypervirulent, multidrug resistant, and highly transmissible. Because of the genetic similarity between ST11 and ST258 types of K pneumoniae, ST258 hypervirulent K pneumoniae strains might have emerged and become transmissible in the USA and in European countries. Future research should focus on the development of intervention measures to prevent further dissemination of such organisms in hospital settings.

In reports from 2013, the US Centers for Diseases Control and Prevention (CDC) described the emergence of carbapenem-resistant Enterobacteriaceae strains, which commonly cause untreatable or hard-to-treat infections among patients in hospitals, as an urgent public health threat. Carbapenem-resistant K pneumoniae strains account for roughly 70–90% of clinical carbapenem-resistant Enterobacteriaceae infections in the European Union and China.8, 9 The most common clinical carbapenem-resistant K pneumoniae strains are those of the clonal group (CG) 258, with ST258 and ST11 being the most prevalent multilocus sequence types in different parts of the world.¹⁰ ST258 is a hybrid clone composed of 80% of ST11 genome and 20% of ST442 genome.10, 11 ST258 has disseminated worldwide since its emergence in the early 2000s and has become particularly prevalent in North America, Latin America, and several European countries.¹⁰ However, in Asia, the dominant clone is ST11 carbapenem-resistant K pneumoniae, which accounts for up to 60% of carbapenem-resistant K pneumoniae in China.⁹ Consequently, in recent years, ST11 has been regarded as the most transmissible clone contributing to the increasing prevalence of carbapenem-resistant K pneumoniae in China. In this study, we investigate a fatal ventilator-associated pneumonia outbreak in an intensive care unit (ICU) in a Chinese hospital with the aim of identifying the molecular basis for the hypervirulence of the emerging ST11 carbapenem-resistant K pneumoniae strains responsible for this outbreak.