A retrospective study on molecular epidemiology trends of carbapenem resistant Enterobacteriaceae in a teaching hospital in Malaysia

Introduction

Carbapenem resistance is a recognised worldwide threat by the World Health Organisation (WHO). One of the major causes for the emergence of carbapenem resistant pathogens is the overuse of carbapenems in the management of infections caused by extended-spectrum beta-lactamases (ESBLs)-expressing Enterobacteriaceae (Nordmann, Dortet & Poirel, 2012; Slama, 2008; Rahal, 2008). Indeed, given that carbapenem is recommended for the treatment of ESBL infections (Paterson et al., 2004), a steady rise in carbapenem resistance had been recorded in Malaysia with the increasing use of the antibiotics (Institute for Medical Research, 2020).

Carbapenem-resistant Klebsiella pneumoniae (CRKp) has persisted and has been gradually rising since its first discovery. For instance, in southeastern United States, a five-fold increase in CRKp infections were recorded from 2008 to 2012 (Thaden et al., 2014). Although carbapenem resistance data is limited, particularly in Southeast Asia, a meta-analysis revealed high estimated carbapenem resistance among Klebsiella spp. in Indonesia, the Philipines, Thailand and Vietnam and among E. coli in Indonesia and Myanmar (Malchione et al., 2019). Among the recorded carbapenem resistant Enterobacteriaceae (CRE), K. pneumoniae is the most prevalent species in Malaysia (Institute for Medical Research, 2020; Zaidah et al., 2017).

The widespread distribution of CRKp over different geographical regions is commonly associated with high-risk clones and the acquisition of carbapenemase genes encoded in mobile genetic elements (David et al., 2019; Pitout, Nordmann & Poirel, 2015). In a parallel survey by the European survey of carbapenemase-producing Enterobacteriaceae (EuSCAPE), a 1.3% increase in population-weighted mean percentage in carbapenem-resistant K. pneumoniae and Escherichia coli (E. coli) was recorded in EU member states from 2011–2014 (Xu et al., 2015; Thaden et al., 2014; European Centre for Disease Prevention and Control, 2020). In Malaysia, the reported carbapenemases that produced by Enterobacteriaceae are blaNDM, blaIMP, blaOXA-48, blaOXA-181 and blaKPC (Hamzan et al., 2015; Wang et al., 2016; Lau et al., 2020).

CRE is commonly associated with nosocomial infection. A previous study had identified several factors associated with CRE acquisition such as previous overseas hospitalisation, ICU admission and comorbidities (cardiovascular disease and hematology condition) (Ling et al., 2015). Furthermore, studies had also revealed that usage of mechanical ventilation, presence of central venous catheter and receiving immunosuppressants were associated with mortality (Low et al., 2017; Wang et al., 2016). Understanding the transmission of CRE as well as their resistance mechanism is essential to inform the infection control strategy for containing the spread of CRE. In this study, we investigated the epidemiology of CRE (from 2014 to 2015) in a teaching hospital in Klang Valley, Malaysia. The risk factors associated with mortality among the patients infected or colonised with CRE was also examined.

Materials and Methods

Results

Molecular characterization of CRKp by PFGE

Based on dendrogram (S1–S3), the 140 CRKp strains were separated into seven major clusters (Cluster I, II, III, IV, V, VI, VII). More than half of the CRKp strains (n = 81, 57.6%) were clustered into Cluster I (11 pulsotypes) followed by Cluster IV (n = 8, 5.7%) and V (n = 7, 5.0%). A total of 48 pulsotypes (XbaI1–XbaI48) have been generated from the PFGE analysis. Among the detected pulsotypes, pulsotype XbaI4. which belonged to Cluster I is the predominant pulsotype that consists of 62 strains (44.3%) which is 100% clonally similar to each other. This pulsotype has been sporadically isolated in January (n = 1), February (n = 1) and April (n = 1) of 2014. However, a steep increment of this pulsotype was observed since December 2014 (n = 5) until the first quarter of 2015 (n = 28). The isolation of this pulsotype was later decreased from 2^nd quarter of 2015 (Fig. 1).

Discussion

K. pneumoniae is a human commensal that colonises the mucosal surfaces such as gastrointestinal tract and respiratory tract (Podschun & Ullmann, 1998). Gastrointestinal tract has been reported as the important reservoir for the transmission and infection of resistant pathogens (Dorman & Short, 2017). The injudicious use of antibiotics has accelerated the development of antibiotic resistance. Over the years, there has been an increase of cases caused by multidrug-resistant K. pneumoniae and the trends of K. pneumoniae strains harbouring resistance genes such as extended spectrum β-lactamase (ESBL) and carbapenemase genes have also been observed (Müller-Schulte et al., 2020; Sirot et al., 1988; Nordmann, Cuzon & Naas, 2009). The propensity for colonisation, transmission, and survival under selection pressure of antibiotics could have enhanced their survival rate, thus contributing to the high infection rate in the hospital setting.

The surveillance of the major carbapenemase genes such as blaKPC, blaNDM, blaVIM, blaIMP and blaOXA has been carried out in many countries (Hamzan et al., 2015; Rimrang et al., 2012; Hoang et al., 2013; Karuniawati, Saharman & Lestari, 2013). The reported dominant carbapenemase gene in Singapore and China were blaNDM and blaKPC respectively (Teo et al., 2014; Zhang et al., 2017). However, in our study, blaOXA-48 and blaNDM were the two main carbapenemase genes detected. blaOXA-48 and blaNDM carried on IncL group plasmid and IncFIIK plasmid have been reported. These plasmids have been associated with low fitness burden, high plasmid stability and high transconjugation frequency (Chaalal et al., 2021). In addition, close genetic association between the bleMBL and blaNDM-1 genes has been reported. These two genes are controlled by the same promoter, and this will lead to constitutive expression of blaNDM and bleMBL that encodes a bleomycin resistant protein (BRP). BRF is a functional protein that prevent DNA damage, thus stabilise the plasmid-borne blaNDM. Studies have shown that blaBRF present in both non-clinical and clinical environment. In clinical environment, the medical use of bleomycin as an anticancer agent, contribute to selective pressure that led to further spread of blaNDM in the environment (Dortet, Nordmann & Poirel, 2012). Finally, the dissemination of carbapenemase genes is possibly affected by characteristics of the primary reservoir (which depends on population density, hygiene level, antibiotic selection pressure in that specific area) and population exchange (imported cases from endemic areas) (Nordmann & Poirel, 2014).

Several carbapenem resistance mechanisms could be used among the strains without carbapenemase genes. For this group of strains, the resistance could be attributed by permeability defect (porin loss or mutation) coupled with combination of AmpC expression; or ESBL expression; or non-targeted carbapenemase genes in this study (Crowley, Benedí & Doménech-Sánchez, 2002; Shin et al., 2012; García-Fernández et al., 2010). On the other hand, ertapenem mono resistant non-carbapenemase producing K. pneumoniae (EMRNCP-K.p) is also believed to acquire ESBL genes and/or alteration of porins (Chung, Yong & Lee, 2016). In our present study, only a small number of strains showed the loss of porins that could possibly changes the outer membrane permeability. These group of strains were believed to survive poorly compared to other strains. Previous studies had shown that carbapenem resistant bacteria without porins are usually unstable, thus conferred lower fitness, growth and transmission ability in the hospital environment (Nordmann, Dortet & Poirel, 2012). However, it is noteworthy that the presence of porin associated genes may not guarantee the expression. Expression of porin can also be disrupted by insertional inactivation that alters translation resulting in early termination of translation (Chung, Yong & Lee, 2016; Kaczmarek et al., 2006).

Majority of the CRKp strains co-harbouring blaNDM and blaOXA-48 belonged to Cluster I. On the other hand, 1 strain that did not harbour any targeted carbapenemase genes also grouped in Cluster I. Hence, it is suggested that strains that shared the same genetic backbone might not utilising the same resistance mechanisms.

Although carbapenemase plays an important role in carbapenem resistance, but carbapenemase did not affect the mortality of patients with CRE infection based on statistical analysis. This agrees with previous studies (Daikos et al., 2009). However, there are studies reported the correlations between mortality and severity of underlying diseases as well as the degree of carbapenem resistance (based on MIC value) (Daikos et al., 2009; Daikos et al., 2014). In our study, patients who received definitive treatment at more than 3 days from sample isolation date was significantly associated with mortality compared to patients that received definitive treatment at less or equal than 3 days from sample isolation date. The rapid initiation of appropriate therapy (definitive treatment) has previously been shown to affect in-hospital mortality, but highly dependent on rapid diagnostic that produce accurate and reliable results (Deresinski, 2007; Kumar et al., 2006). In addition, previous studies also suggested the importance of active screening of patients colonised with carbapenemase-producing CRE for implementation of infection control measures, such as isolation, precautions during invasive procedures, patient transport and environment disinfection (Banerjee & Humphries, 2017).

Based on the comparison of the finding reported by Low et al. (2017) with our current study, the trend of carbapenem-resistance mechanisms has been changed from year 2013 to 2015. Overall, more resistance mechanisms have been observed in year 2013 compared to 2014 and 2015. For instance, blaKPC-2 and blaIMP have been observed in 2013 but not detected in the latter years. The previous study found that CRKp strains with blaKPC in 2013 were associated with Tn4401b transposon (Low et al., 2017). This isoform has been reported to exhibit lower resistance towards carbapenems when compared to other isoforms (Kitchel et al., 2010). Further, these strains did not belong to the high-risk clones that are responsible for the spread of KPC-harbouring K. pneumoniae such as ST258 and ST11 (Lee et al., 2016), hence it only appeared sporadically. On the other hand, in 2013, only 1 strain co-harbouring blaNDM and blaIMP-8 were found but blaNDM has been the main resistance mechanisms among the strains in 2014 although superseded by blaOXA-48 in 2015. Moreover, the increase of strains that did not harbour the five targeted carbapenemase genes has been observed since 2014. In addition, only blaOXA-48 variant was detected among the strains in 2013 and 2014 but in 2015, 3 blaOXA-232 variants were observed. To date, only 2 blaOXA variants (blaOXA-48, blaOXA-181) have been reported in Malaysia (Lau et al., 2021; Lau et al., 2020). This study shed some lights on the changes of main carbapenem-resistance mechanisms in the hospital within the 3-year period; however, further study is required with more recent patient data set to track the genetic shift of these clinical bacterial strains.

The in-hospital mortality rate of CRKp in 2013, 2014 and 2015 were 35.3%, 27.3% and 42.6% respectively. When comparing the CRKp strains among the predominant pulsotype, the in-hospital mortality is increasing from 2013 to 2015 in which the mortality rate of 2013, 2014 and 2015 were 11.1%, 16.7% and 69.6% respectively. The reason behind of the increment could not be concluded based on the collected details. A further study which includes host factors such as white blood cells and nutritional status as well as bacterial virulence factors are needed.

Conclusions

Emergence of CRE was observed in our study setting from 2014 to 2015. The predominant species was K. pneumoniae. The genotypic characterisation of CRKp revealed that the predominant pulsotype had circulated in our setting since 2013. The circulation of this pulsotype with more than one carbapenemase gene could have a negative impact on the management of patients. Hence, this study is important to provide better understanding on the spread of CRE and carbapenemase gene.

Supplemental Information