Three challenging cases of infections by multidrug-resistant Serratia marcescens in patients admitted to intensive care units

Abstract

The occurrence of multidrug-resistant Serratia marcescens strains represents a serious public health threat. The purpose here is to report three cases of carbapenem-resistant S. marcescens infections with unfavorable clinical outcomes and provide a molecular description of the antibiotic resistance determinants at a genomic level. We performed bacterial identification by VITEK 2 and MALDI-TOF. The minimal inhibitory concentrations of antimicrobials were determined according to the Clinical and Laboratory Standards Institute guidelines, except for tigecycline, for which they were determined using Etest strips. Preliminary screening for the presence of carbapenemases was performed by ertapenem hydrolysis using MALDI-TOF MS. Whole-genome sequencing was provided to identify genes responsible for virulence and antimicrobial resistance. Here we report three challenging cases of S. marcescens that were resistant to the most commonly used antibiotics. Otherwise, we performed a genome description, which includes several genes involved in the resistance and virulence. These cases illustrate serious infection due to multidrug-resistant organisms and the complexity of treatment. Our results highlight the need to evaluate isolates regularly during long-term hospital stay to achieve optimal quality of clinical care and thus improve patient outcomes.

Supplementary Information

The online version contains supplementary material available at 10.1007/s42770-021-00477-4.

Introduction

Serratia marcescens is a common pathogen involved in nosocomial infections affecting several body sites such as meninges, blood, and lungs, significantly impacting morbidity and mortality rates [1]. In general, carbapenems are the main therapeutic options to treat these infections, since S. marcescens frequently exhibit resistance to broad-spectrum cephalosporins due to the production of chromosomally encoded AmpC enzyme and/or acquisition of plasmid-borne extended-spectrum β-lactamases [2]. However, the emergence of carbapenem-resistant S. marcescens strains in several countries has jeopardized the clinical use of carbapenems [3]. The mechanisms of carbapenem resistance in S. marcescens are related to the loss of porins associated with AmpC overexpression, production of Ambler class A (KPC and SME), or class B metallo-β-lactamases (MβLs; IMP and VIM) [4]. The spread of carbapenem-resistant S. marcescens strains in the nosocomial environment is a matter of concern. Since this pathogen is intrinsically resistant to polymyxins, carbapenems are often the last resort drugs for the treatment of infections caused by multidrug-resistant gram-negative bacilli [5].

Methods

Clinical cases description

All clinical cases described here were obtained between February and April 2012 from patients hospitalized at a tertiary hospital in Brazil’s Midwestern region, serving as a referral center for 32 cities, with an average of 9800 annual admissions per year. The institution has 237 beds, distributed between infirmaries and adult, pediatric, and neonatal intensive care units. This study was conducted with the approval of the research ethics committee from the Universidade Federal da Grande Dourados (no. 039439/2012).

Case 1

A 67-year-old male with a medical history of tabagism and alcoholism was admitted to the hospital, presenting anemia and leukocytosis. After clinical worsening, the patient was referred to the intensive care unit (ICU) and required mechanical ventilation and vasoactive drugs. After 23 days of hospitalization, he was transferred to the ICU of our hospital, and ceftriaxone antibiotic therapy was implemented. Chest x-ray was performed evidencing bilateral pleural effusion, not punctured due to thrombocytopenia, but tracheal aspirate samples were collected on April 16, 2012. A diuretic drug was prescribed, and ceftriaxone was substituted by imipenem. Six days after hospitalization at our institution, chronic lymphocytic leukemia was diagnosed, and broad-spectrum antibiotic therapy started, with the replacement of imipenem by piperacillin/tazobactam. Pseudomonas spp. resistant only to gentamicin were isolated from tracheal aspirates, and the current antibiotic therapy was replaced by the combination of vancomycin and meropenem. Due to severe respiratory effort, a tracheostomy was performed on day eight, and carbapenemase-producing S. marcescens was isolated from tracheal aspirates, and vancomycin was substituted by amikacin. After 22 days, the patient developed septicemia and cardiorespiratory arrest with non-successful cardiopulmonary resuscitation.

Case 2

A 57-year-old male patient was referred to the ICU with a medical history of acute respiratory failure to aspiration pneumonia. At the time of admission, empiric antibiotic therapy with imipenem/cilastatin was instituted, on February 08, 2012, tracheal aspirates culture was performed, and carbapenemase-producing S. marcescens was isolated (colony counts more than 100,000 CFU/mL), but negative uroculture and blood culture. Antibiotic therapy with ciprofloxacin started remaining for 10 days. After 9 days of hospitalization, the exchange of the central access and a tracheostomy was performed. The patient was discharged from ICU and moved to the infirmary remaining without antibiotic therapy. After 20 days, stable and waiting to perform a gastrostomy, the patient presented a febrile peak, abundant secretion in the tracheostomy region, and turbid urine. Cultures were requested, and carbapenemase-producing S. marcescens was isolated from uroculture. Presenting hemodynamic instability, the patient developed septicemia and died after 2 months of hospitalization.

Case 3

A 52-year-old male patient was admitted to the ICU presenting fever and dyspnea with a history of productive cough for more than a week. On the admission day, mechanical ventilation use was initiated. The patient had a previous history of AIDS, was diagnosed a few months ago, and, without treatment, also made abusive use of tobacco and alcohol. On the admission day, uroculture and blood culture were requested, both negative; empirical antibiotic therapy with piperacillin/tazobactam and trimethoprim/sulfamethoxazole was implemented. After 12 days of hospitalization, on February 13, 2012, multidrug-resistant S. marcescens was isolated from uroculture. The previous treatment with meropenem and vancomycin was replaced by ciprofloxacin after 10 days. However, therapy was not successful, and the patient continued to show clinical signs of persisting infection. The patient developed septicemia and died after 2 months of hospitalization.

Microbiological investigation

Bacterial species were identified using the VITEK® 2 automated system (bioMérieux, Hazelwood, MO) and confirmed by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) using the Microflex LT spectrometer (BrukerDaltonics, MA, USA) [6]. The minimal inhibitory concentrations (MICs) of antimicrobials were determined by broth microdilution according to the Clinical and Laboratory Standards Institute (CLSI) guidelines [7], except for tigecycline, for which they were determined using Etest strips (bioMérieux, Marcy l’Étoile, France). Preliminary screening for the presence of carbapenemases was performed by ertapenem hydrolysis using MALDI-TOF MS [6]. All isolates were resistant to ertapenem (MIC₅₀, >32 mg/L), imipenem (MIC₅₀, >16 mg/L), and meropenem (MIC₅₀, >8 mg/L). Tigecycline (MIC, 0.5 mg/L) was the only antimicrobial agent tested with activity.

Genomic DNA was extracted from fresh cultures using Illustra Bacterium Genomic Prep Mini Spin kit (GE Healthcare, Sao Paulo, SP, Brazil). The strains had its genome sequenced through the Ion Torrent Personal Genome Machine (PGM) (Life Technologies, Saint Aubin, France) sequencing platform, utilizing a 318 chip (Life Technologies) and single-end libraries, to generate readings of 400 bp on the BAM format, which were then converted to the FASTQ format with the bamTofastq tool from the BEDTools package [8]. Reading quality was measured through the FASTX-Tool kit (http://hannonlab.cshl.edu/), and we omitted sequences with a Phred score lower than 20. De novo assembly was performed by three distinct assemblers, SPAdes [9], MIRA (https://sourceforge.net/p/mira-assembler/), and Newbler (http://www.454.com/), and the contigs generated by the last one were utilized in the following steps in light of the higher contiguity. Genome annotation was performed using Genix [10] and manually revised with Artemis.

Read sets were screened for known alleles of genes using a read mapping approach with SRST2. For acquired resistance genes, it was used the ARG-ANNOT database [11]. Plasmid replicon sequences were identified using ARIBA to screen reads for replicons in the PlasmidFinder database (http://cge.cbs.dtu.dk/services/PlasmidFinder/). Virulence genes were identified by comparison to the genes downloaded from the Virulence Factor Database (VFDB) (http://www.mgc.ac.cn/VFs/). To verify potential clonality among the three isolates, a whole-genome multi-locus sequence typing (wgMLST) analysis was performed using PGAdb-builder (https://www.nature.com/articles/srep36213) and based on a core genome alignment of the pangenome computed from a total of 84 complete genomes of S. marcescens obtained from NCBI (Online resource 1-Supplementary Data). Only genes present in at least 95% of the strains were included in the core genome. The isolates’ final assembly was obtained using WGS ranging from 160 to 230 contigs covering ~5.2 Mb of the S. marcescens genome. All isolates presented a similar number of protein-coding sequences (CDSs) and tRNA copies (Online Resource 2-Supplementary Data). The detailed distribution of genes in SEED subsystems for all strains is shown in Online Resource 3-Supplementary Data. Whole-genome sequence analysis showed that all S. marcescens strains were highly related with ANI values >99.0. In addition, pangenome analysis showed that isolates shared 4625 genes in their core genome. All three strains harbored several antimicrobial-resistant genes encoding resistance against beta-lactams (bla_IMP-10, bla_KPC-2, bla_SRT-2), aminoglycosides (aac(6´)-Ib-cr, aac(6´)-Ib3, aac(6´)-Ic, aadA1), fluoroquinolones (oqxB), and sulfonamides (sul1). Plasmid replicon sequences were identified using PlasmidFinder. The isolates were found to harbor IncL/M plasmids, commonly associated with the dissemination of beta-lactamases and aminoglycosides resistance genes. The identified virulence genes included aerobactin, fimbrial adherence, iron uptake, magnesium uptake, phospholipase, protease, stress adaptation, toxins, and yersiniabactin determinants (Table 1). On wgMLST analysis, a total of 3004 genes were included on the core genome do S. marcescens, which represent only 15% of genes identified by the tool, while the pangenome has 20,714 genes that occurred in at least one strain, and these values are close to those observed by Abreo et al. [12]. Based on these results, an analysis of the strains ICU-2, ICU-3, and ICU-4 revealed a computed genetic distance of zero, indicating that these three isolates may belong to the same clonal group.

Table 1.

Virulence and resistance genes of carbapenem-resistant Serratia marcescens strains

	Genes
Virulence factors
Aerobactin	iucD, iutA
Fimbrial adherence	ecpA, ecpB, ecpC, ecpD, ecpE, fimB, fimD, lpfC, matC, matE, papC, papD, stfC, stfD, stiB
Iron uptake	chuA, chuS, chuU, fptA, iroN, sitA, sitB, sitC, sitD
Magnesium uptake	mgtB, mgtC
Non-hemolytic phospholipase C	plcN
Protease	aprA
Stress adaptation	katA, sodCI
Toxins	hlyB
Yersiniabactin	yplA
Resistance
Aminoglycosides	aac(6´)-Ib-cr, aac(6´)-Ib3, aac(6´)-Ic, aadA1
Beta-lactams	blaIMP-10, blaKPC-2, blaSRT-2
Quinolones	oqxB
Sulfonamides	sul1

Discussion

Here we described three cases of sepsis caused by multidrug-resistant S. marcescens recovered from 3 patients in the course of 2 months. In Brazil, resistance to carbapenems in S. marcescens has rarely been reported and is associated mainly with KPC-2 production [13–15]. Production of IMP-10 in Brazil was previously reported in S. marcescens and Acinetobacter baumannii [14, 16]. Production of both carbapenemases is a matter of great concern since effective treatment of infections caused by these pathogens is a considerable challenge for clinicians since S. marcescens exhibits intrinsic resistance to polymyxins, drugs of last resort for treatment [17]. With whole-genome sequencing (WGS), we have identified genes responsible for the virulence and antimicrobial resistance of these strains.

The genotypic virulence profile was consistent with this lineage [12], with many virulence factors identified. Our analysis revealed that all S. marcescens strains were carrying virulence genes with homology to the E. coli common pilus (ecpA, ecpB, ecpC, ecpD, ecpE), which are significantly associated with virulent strain [18]. The co-existence of antibiotic resistance and virulence factors favor colonization, which often lead to the emergence of untreatable infections [19]. Moreover, the successful evolution of the infection also depends on several host-dependent factors [1, 20]. The patients were ≥50 years old or displayed comorbidities such as diabetes, heart failure, decubitus ulcers, chronic obstructive pulmonary disease, systemic hypertension, cancer, and AIDS. Additionally, during hospitalization, they were subjected to central venous catheterization, mechanical ventilation, or surgical procedures. All these factors may have contributed to the mortality of patients evaluated in this study.

The origin of the index isolate is unclear; however, patient 1 was previously hospitalized in another hospital, suggesting that strain was introduced into our hospital by colonized patients who previously had been hospitalized elsewhere. The three patients shared the same ICU for an overlapping period. The potential spread of these strains may be due to occupational transmission, such as cross-transmission from colonized or infected [1]. WgMLST analysis showed that the three isolated strains belong to the same clonal group, representing a probable clonal spreading. Consequently, our analysis hypothesizes possible patient-to-patient transmission events only based on the high genetic relatedness among strains. Frequent screening of patients, especially those receiving long-term healthcare and/or extensive antibiotic treatment, is required to control and prevent transmissions [21]. Patients had failed antibiotic treatments, and based on susceptibility testing, we hypothesized that the patients would have improved with continued tigecycline/polymyxin therapy. However, this antibiotic was not available in the hospital at the time of patient’s presentation. Thus they were treated with a combination therapy of polymyxin B/aminoglycoside and ciprofloxacin. Tigecycline in combination with polymyxin may have a possible synergistic interaction, resulting in an enhanced antibacterial effect during therapy with these agents [22].

The use of WGS plays an important role in clinical microbiology. In our study, WGS findings reflect the considerable antimicrobial resistance displayed by these isolates to multiple antibiotics and are consistent with the clinical features observed. Both carbapenemase-encoding genes were found in isolates. The obtained data showed the high genetic relatedness among the three isolates, which can be exploited to better understand the cross-transmission of such pathogens in hospital settings and prevent dissemination. In conclusion, genomic surveillance of these key phenotypic may improve our understanding of the pathogenicity of multidrug-resistant organisms and control their evolution. In addition, tigecycline might be a safe and effective therapy for a serious infection caused by a carbapenem-resistant S. marcescens strain. This case report could constitute an argument for initiating the therapy at an early time point in the course of a complicated infection.