Carbapenem-resistant Serratia Marcescens Research Articles

A preliminary exploration on the mechanism of the carbapenem-resistance transformation of Serratia marcescens in vivo

BackgroundThe infection of carbapenem-resistant organisms was a huge threat to human health due to their global spread. Dealing with a carbapenem-resistant Serratia marcescens (CRSM) infection poses a significant challenge in clinical settings. This study aims to provide insights into strategies for controlling CRSM infection by exploring the transformation mechanism of carbapenem-resistance.MethodsWe used whole genome sequencing (WGS) to investigate the mechanism of carbapenem resistance in 14 S. marcescens isolates in vivo. The expression level of related genes and the minimum inhibitory concentration of meropenem (MICMEM) were also evaluated to confirm the mechanism of carbapenem resistance.ResultsSeven groups of S. marcescens, each consisting of two strains, were collected from a hospital and displayed a shift in MICMEM from low to high levels. Homology analysis revealed that the isolates in five groups were significantly different from the remaining two. WGS and experimental evidence indicated that four groups of strains developed carbapenem resistance by acquiring the blaKPC (obtaining group), while two groups (persisting group) increased the expression level of the blaKPC. In contrast, isolates in the last group (missing group) did not carry the blaKPC. All strains possessed multiple β-lactamase genes, including blaCTX−M−14, blaSRT−1, and blaSRT−2. However, only in the missing group, the carbapenem-resistant strain lost an outer membrane protein-encoding gene, leading to increased blaCTX−M−14 expression compared to the carbapenem-susceptible strain.ConclusionThe study findings suggest that S. marcescens strains developed diverse carbapenem resistance in vivo through the evolution of drug resistance, rather than through clone replacement. We hypothesize that carbapenem resistance in S. marcescens was due to certain clonal types with a distinct mechanism.

The genomic characterization of carbapenem-resistant Serratia marcescens at a tertiary hospital in South Africa.

Serratia marcescens is an opportunistic nosocomial pathogen, and recent reports have highlighted the rapid increase in multidrug resistance in this organism. There is a paucity in genomic data for carbapenem-resistant S. marcescens (CRSM). A retrospective cohort study describing laboratory-confirmed CRSM from a tertiary academic hospital in Cape Town, South Africa, for the period 2015-20, was performed. Stored CRSM and contemporary isolates were submitted for WGS using Illumina MiSeq, with the Nextera DNA Flex Library Preparation Kit. Sequence data were analysed in-house using srst2 and Tychus, and CRSM and contemporary isolates were compared. Twenty-one CRSM and four contemporary isolates were sequenced and analysed. Twenty-four different resistance genes were identified, with all isolates having at least two resistance genes, and seventeen isolates harbouring three or more genes. This correlated well with phenotypic results. The blaOXA-48-like carbapenemase was the most common carbapenemase identified, in 86% (18/21) of CRSM. A core SNP difference tree indicated that the CRSM could be grouped into three clusters. Eleven isolates had shared plasmids. Several genes and SNPs were identified in the CRSM, which may putatively augment virulence, but this requires further functional characterization. A diverse resistome was observed in CRSM, which was also reflected phenotypically, with blaOXA-48-like the most commonly carbapenemase. Though distinct clusters were observed, no clonality was noted, and a limited number of isolates shared plasmids. This study provides genomic data for emerging CRSM and highlights the importance of ongoing genomic surveillance to inform infection prevention control and antimicrobial stewardship initiatives.

Genomic characterization of two carbapenem-resistant Serratia marcescens isolates causing bacteremia: Emergence of KPC-2-encoding IncR plasmids.

The occurrence and transmission of carbapenemase-producing-Enterobacterales (CPE) on a global scale has become a major issue. Clinical reports are rarely providing information on the genomic and plasmid features of carbapenem-resistant Serratia marcescens. Our objective was to investigate the resistance and transmission dynamics of two carbapenem-resistant S. marcescens that are resistant to carbapenem and have caused bacteremia in China. Blood specimens were taken from two individuals with bacteremia. Multiplex PCR was employed to identify genes that code for carbapenemase. Antimicrobial susceptibility tests and plasmid analysis were conducted on S. marcescens isolates SM768 and SM4145. The genome of SM768 and SM4145 were completely sequenced using NovaSeq 6000-PE150 and PacBio RS II platforms. Antimicrobial resistance genes (ARGs) were predicted using the ResFinder tool. S1 nuclease pulsed-field gel electrophoresis (S1-PFGE) and southern blotting were employed to analyze plasmids. Two S. marcescens that produced KPC-2 were identified from bloodstream infections. The antimicrobial susceptibility testing demonstrated that both of the isolates had a resistance to various antibiotics. The whole-genome sequence (WGS) and plasmid analysis revealed the presence of bla KPC-2-bearing IncR plasmids and multiple plasmid-borne antimicrobial resistance genes in the isolates. Our comparative plasmid analysis suggested that the two IncR plasmids identified in this study could be derived from a common ancestor. Our findings revealed the emergence of bla KPC-2-bearing IncR plasmid in China, which could be a hindrance to the transmission of KPC-2-producing S. marcescens in clinical settings.

Exploring the antibacterial, antibiofilm, and antivirulence activities of tea tree oil-containing nanoemulsion against carbapenem-resistant Serratia marcescens associated infections

Carbapenem-resistant Serratia marcescens (CRE-S. marcescens) has recently emerged as an opportunistic human pathogen that causes various nosocomial and respiratory tract infections. The prognosis for CRE-S. marcescens-related infections is very poor and these infections are difficult to treat. This study investigated the synthesis of tea tree oil nanoemulsion (TTO-NE) and its impact on CRE-S. marcescens both in vitro and in vivo. TTO-NE was characterized by dynamic light scattering (DLS) and effectively eradicated bacterial planktonic and sessile forms, reduced bacterial virulence factors, and generated reactive oxygen species (ROS) in the bacterial cell. Notably, TTO-NE was efficient in reducing the colonization of CRE-S. marcescens in a C. elegans in vivo model. The data suggest that TTO-NE might be an excellent tool to combat infections associated with CRE-S. marcescens.

Phenotypic and genotypic characteristics of a carbapenem-resistant Serratia marcescens cohort and outbreak: describing an opportunistic pathogen

Serratia marcescens is an emerging opportunistic pathogen with high genetic diversity. This article describes the microbiological characteristics of isolates and the risk factors for infections caused by carbapenem-resistant S. marcescens. A retrospective study of patients colonized (n=43) and infected (n=20) with carbapenem-resistant S. marcescens over a 3-year period was conducted. Polymerase chain reaction for carbapenemase genes and molecular typing of all available strains was performed. Forty-two isolates were analysed, including three environmental samples identified during an outbreak. Thirty-five carbapenem-resistant S. marcescens carried blaKPC-2, one isolate was blaNDM-positive and four isolates carried blaOXA-101. The genomes were grouped into three clusters with 100% bootstrap; three patterns of mutations on ompC and ompF were found. The strains carried virulence genes related to invasion and haemolysis, and the environmental strains presented fewer mutations on the virulence genes than the clinical strains. Multi-variate analysis showed that previous use of polymyxin (P=0.008) was an independent risk factor for carbapenem-resistant S. marcescens infection. This study highlighted that blaKPC-2 in association with ompC or ompF mutation was the most common mechanism of resistance in the study hospital, and that previous use of polymyxin was an independent risk factor for carbapenem-resistant S. marcescens. There was a predominant clone, including the environmental isolates, suggesting that cross-transmission was involved in the dissemination of this pathogen.

Analysis of the molecular characteristics of a blaKPC-2-harbouring untypeable plasmid in Serratia marcescens.

Serratia marcescens has attracted increasing attention worldwide as a neglected opportunistic pathogen of public health concern, especially due to its antimicrobial resistance features, which usually cause nosocomial infections in immunocompromised or critically ill patients. In our study, four carbapenem-resistant Serratia marcescens (CRSM) clinical isolates were characterized in our hospital from February 2018 to May 2018. The conjugation experiment confirmed the transferability of the carbapenem resistance gene. The types of carbapenem resistance genes were detected by PCR. The homology of the strains was analysed by pulsed field gel electrophoresis (PFGE). The characteristics of the plasmid and environment of carbapenem resistance genes were analysed after whole genome sequencing was performed. Then, we compared the amino acid sequence of the replication initiation protein and constructed a dendrogram by the neighbour-joining method. All four isolates showed carbapenem resistance conferred by a blaKPC-2-harbouring plasmid. They had exactly the same bands confirmed by PFGE and were defined as the homologous strains. The blaKPC-2 genes in all of the isolates were located in a 42,742 bp plasmid, which was located in the core region of antibiotic resistance and was composed of Tn3 family transposons, recombinant enzyme genes, ISKpn6 and ISKpn27. The core region of antibiotic resistance formed a 'Tn3-ISKpn6-blaKPC-ISKpn27-Tn3' structure, which was an independent region as a movable element belonging to transposon Tn6296 and its derivatives. The plasmid had a similar skeleton to incX6 plasmids and a similar amino acid sequence to the replication initiation protein. The plasmid was defined as an untypeable blaKPC-2-harbouring plasmid named the 'IncX6-like' plasmid. The four CRSM isolates were mainly clonally disseminated with a blaKPC-2-harbouring plasmid in our hospital. The pKPC-2-HENAN1602 plasmid (CP047392) in our study was first reported in Serratia marcescens, which belongs to an untypeable group named the 'IncX6-like' plasmid. The carbapenem-resistant gene structure surrounding blaKPC-2 as a sole accessory module can be acquired by horizontal gene transfer and might lead to serious nosocomial infection.

In vitro anti-biofilm efficacy of sanguinarine against carbapenem-resistant Serratia marcescens

Sanguinarine, a plant-derived benzophenanthridine alkaloid, was studied in terms of its anti-biofilm effects against carbapenem-resistant Serratia marcescens (CRSM). Minimum inhibitory concentrations (MICs) and cell membrane integrity were measured to investigate the antimicrobial mechanism of sanguinarine. Additionally, the extent of biofilm formation by CRSM exposed to sanguinarine was measured by crystal violet staining and visualized via field emission scanning electron microscopy and confocal laser scanning microscopy. Sanguinarine displayed moderate activity against CRSM, with a MIC90 of 32 μg ml−1. Moreover, cell membrane integrity was severely disrupted by sanguinarine at 64 μg ml−1, and biofilm formation was sharply inhibited at 32 μg ml−1. The minimum biofilm eradication concentration was 512 μg ml−1 against mature CRSM biofilms. The overall results suggest that sanguinarine is a potential anti-biofilm agent that can be explored to treat CRSM infections.

Antimicrobial and Antibiofilm Activities and Mechanism of Action of Chelerythrine Against Carbapenem-Resistant Serratia marcescens In Vitro.

Aim: To evaluate the antimicrobial and antibiofilm effects of chelerythrine (CHE) against carbapenem-resistant Serratia marcescens (CRSM). Materials and Methods: The minimum inhibitory concentration (MIC) of CHE against CRSM was determined using the agar dilution method. Changes in intracellular adenosine triphosphate (ATP) concentration, intracellular pH, cell membrane potential, and cell membrane integrity were investigated to assess the influence of CHE on the cell membrane. The effects of CHE on cell morphology were observed by field emission scanning electron microscopy (FESEM) and transmission electron microscopy. The antibiofilm formation of CHE was measured by crystal violet staining and visualized with confocal laser scanning microscopy (CLSM) and FESEM. The influence of CHE on biofilm components was further investigated using CLSM specific combined with double-dyeing methods. Results: Our results showed that CHE had an MIC at 125 μg/mL against CRSM was capable of inhibiting the growth of CRSM and destroying its cell membrane integrity, as well as obviously changing the cell morphology. Sub-MIC CHE displayed robust inhibitory effects against CRSM biofilm formation by mediating the production of biofilm components. In addition, CLSM- and FESEM-mediated evaluation of the damage of biofilm cells and biofilm persistence revealed that at high concentrations, CHE could compromise the cells within biofilms and remove preformed biofilms. Conclusion: CHE shows promise as a natural antimicrobial substance against biofilm-positive CRSM, with the potential to serve as an alternative therapeutic agent.

Molecular Characterization of Carbapenem-Resistant Serratia marcescens Clinical Isolates in a Tertiary Hospital in Hangzhou, China.

IntroductionAlthough carbapenem-resistant Enterobacteriaceae (CRE) have been thoroughly investigated as the pathogens most commonly associated with clinical infections, data on Serratia marcescens are inadequate and superficial.MethodsIn this study, we characterized 36 carbapenem-resistant Serratia marcescens (CRSM) isolates in our hospital from April 2018 to March 2019 by analysing whole-genome sequencing (WGS) data. The molecular typing of the isolates was performed using both pulsed-field gel electrophoresis (PFGE) and core genome multilocus sequence typing (cgMLST).ResultsThirty-three of the 36 isolates showed carbapenem resistance conferred by a blaKPC-2-harbouring plasmid, while the remaining three isolates were characterized by overexpression of beta-lactamase combined with porin loss. The blaKPC-2 genes in all the isolates were located on a plasmid of ~103 kb, except one, which was on a plasmid of ~94 kb. The gene structure surrounding blaKPC-2 in the plasmids was confirmed by integration of a partial Tn4401 structure and an intact IS26 as previously reported. Most of the plasmids also contained a mobile genetic element (MGE) comprising qnr and ISKpn19, which provided evidence of horizontal transfer of antibiotic resistance genes.ConclusionThe thirty-six CRSM isolates were mainly clonally disseminated with a blaKPC-2-harbouring plasmid in our hospital. The gene structure surrounding blaKPC-2 as an MGE, as well as the qnr segment, might be acquired by horizontal gene transfer, and it could aggravate the infection and increase the difficulty of clinical treatment.

Microextraction by Packed Sorbent (MEPS) for the determination of Polybrominated Diphenyl Ether (PBDE) in eggs by Gas Chromatography-Mass Spectrometry (GC-MS)

We evaluated the genetic environment of blaGES-16 found in 2 carbapenem-resistant Serratia marcescens clinical isolates recovered from patients hospitalized at a tertiary hospital located in Rio de Janeiro, Brazil. We also compared the kinetics constants for GES-16 and GES-5 against several β-lactams. Both S. marcescens isolates showed identical PFGE pattern and carried the carbapenemase-encoding gene blaGES-16 and the extended-spectrum β-lactamase encoding gene blaOXA-10. The blaGES-16 was inserted at the first position of a defective class 1 integron, composed by a fragmented integrase gene that lacked its attI1 recombination site, followed by dfr22, aac(6′)-IIc, and aadA1 genes. This integron was located on a 30-kb nonconjugative plasmid. The GES-16 showed 2 amino acid substitutions (Gln38Glu and Gly170Ser) compared to GES-1. Kinetic analysis showed that GES-16 presented hydrolytic activity against all β-lactams tested, except for aztreonam. Imipenem was the carbapenem more efficiently hydrolyzed (highest kcat/Km) by GES-16. The kinetic parameters of GES-16 were similar to those of GES-5. In conclusion, we identified a new GES-type enzyme with carbapenemase activity in S. marcescens. The increasing diversity of such resistance determinants confirms the ongoing evolution of these β-lactamases towards a broader spectrum of activity.

Genetic and biochemical characterization of GES-16, a new GES-type β-lactamase with carbapenemase activity in Serratia marcescens

We evaluated the genetic environment of blaGES-16 found in 2 carbapenem-resistant Serratia marcescens clinical isolates recovered from patients hospitalized at a tertiary hospital located in Rio de Janeiro, Brazil. We also compared the kinetics constants for GES-16 and GES-5 against several β-lactams. Both S. marcescens isolates showed identical PFGE pattern and carried the carbapenemase-encoding gene blaGES-16 and the extended-spectrum β-lactamase encoding gene blaOXA-10. The blaGES-16 was inserted at the first position of a defective class 1 integron, composed by a fragmented integrase gene that lacked its attI1 recombination site, followed by dfr22, aac(6′)-IIc, and aadA1 genes. This integron was located on a 30-kb nonconjugative plasmid. The GES-16 showed 2 amino acid substitutions (Gln38Glu and Gly170Ser) compared to GES-1. Kinetic analysis showed that GES-16 presented hydrolytic activity against all β-lactams tested, except for aztreonam. Imipenem was the carbapenem more efficiently hydrolyzed (highest kcat/Km) by GES-16. The kinetic parameters of GES-16 were similar to those of GES-5. In conclusion, we identified a new GES-type enzyme with carbapenemase activity in S. marcescens. The increasing diversity of such resistance determinants confirms the ongoing evolution of these β-lactamases towards a broader spectrum of activity.

Emergence of Serratia marcescens isolates possessing carbapenem-hydrolysing β-lactamase KPC-2 from China

Eighty-three carbapenem-resistant Serratia marcescens isolates were recovered from Zhejiang Provincial People's Hospital, China. The minimum inhibitory concentrations of imipenem, meropenem, and ertapenem for all isolates were 2 to >128μg/mL. Polymerase chain reaction indicated that 63 S.marcescens isolates produced Klebsiella pneumoniae carbapenemase (KPC)-2. Clone A (15 isolates) and clone B (41 isolates) were the two dominant clones and clone A strains were gradually replaced by clone B strains between 2011 and 2014. The results indicate that blaKPC-2-positive S.marcescens emerged in our hospital as the major mechanism of carbapenem resistance.

Coproduction of KPC-2 and IMP-10 in Carbapenem-Resistant Serratia marcescens Isolates from an Outbreak in a Brazilian Teaching Hospital

We describe an outbreak caused by KPC-2- and IMP-10-producing Serratia marcescens isolates in a Brazilian teaching hospital. Tigecycline was the only active antimicrobial agent tested. The blaIMP-10 gene was located in a new class 1 integron, named In990, carried by a nonconjugative plasmid, in contrast to blaKPC-2.

In Memoriam: John P. Quinn, MD

In Memoriam: John P. Quinn, MD

A Fitness Cost Associated With the Antibiotic Resistance Enzyme SME-1 β-Lactamase

The bla(TEM-1) beta-lactamase gene has become widespread due to the selective pressure of beta-lactam use and its stable maintenance on transferable DNA elements. In contrast, bla(SME-1) is rarely isolated and is confined to the chromosome of carbapenem-resistant Serratia marcescens strains. Dissemination of bla(SME-1) via transfer to a mobile DNA element could hinder the use of carbapenems. In this study, bla(SME-1) was determined to impart a fitness cost upon Escherichia coli in multiple genetic contexts and assays. Genetic screens and designed SME-1 mutants were utilized to identify the source of this fitness cost. These experiments established that the SME-1 protein was required for the fitness cost but also that the enzyme activity of SME-1 was not associated with the fitness cost. The genetic screens suggested that the SME-1 signal sequence was involved in the fitness cost. Consistent with these findings, exchange of the SME-1 signal sequence for the TEM-1 signal sequence alleviated the fitness cost while replacing the TEM-1 signal sequence with the SME-1 signal sequence imparted a fitness cost to TEM-1 beta-lactamase. Taken together, these results suggest that fitness costs associated with some beta-lactamases may limit their dissemination.

Plasmid-mediated carbapenem-hydrolysing β-lactamase KPC-2 in carbapenem-resistant Serratia marcescens isolates from Hangzhou, China

Sir, Carbapenems are considered to be one of the few drugs that are useful for the treatment of infections caused by multidrugresistant isolates with high levels of AmpC or extended-spectrum b-lactamase (ESBL) production. While carbapenem resistance has become a serious problem for non-fermenting bacteria, it has remained uncommon in Enterobacteriaceae. However, identification of carbapenem-resistant Enterobacteriaceae is increasingly common. Especially, reports of nosocomial infection due to carbapenem-resistant Serratia spp. have become significantly more common, which has mainly been attributed to production of b-lactamases that are capable of hydrolysing carbapenems such as IMP-1, IMP-6, VIM-2, and SME-1, 2 and 3. The KPC class A carbapenemases are capable of hydrolysing carbapenems, penicillins, cephalosporins and aztreonam, and are inhibited by clavulanic acid and tazobactam. The first report of this b-lactamase, KPC-1, was from a carbapenemresistant Klebsiella pneumoniae. KPC-2 was then found in isolates of K. pneumoniae, Salmonella enterica, Klebsiella oxytoca and Enterobacter spp. Soon afterward, KPC-3 was found in K. pneumoniae and Enterobacter cloacae from New York. Recently, KPCs were found in France, South America and Israel. We describe three clinical isolates of carbapenem-resistant Serratia marcescens that produce KPC-2. This is the first report of detection of a plasmid-encoded carbapenem-hydrolysing enzyme KPC-2 in S. marcescens. We collected three clinical isolates of carbapenem-resistant S. marcescens from our hospital in February 2006. The three isolates exhibited either resistance or reduced susceptibility to carbapenems with imipenem MICs of 8, 64 and 8 mg/L. The isolates were also resistant to penicillins, cephalosporins and aztreonam, but were susceptible to quinolones and aminoglycosides (Table 1). Following mating of the three S. marcescens isolates with an Escherichia coli (EC600) by conjugation, all of the resulting transconjugants exhibited a phenotype of resistance to b-lactams similar to that of the S. marcescens isolate used. The imipenem and meropenem MICs of transconjugants were 1 mg/L. They were also resistant to penicillins and aztreonam, and were resistant or intermediately resistant to cephalosporins, but were susceptible to quinolones and aminoglycosides (Table 1). The three S. marcescens isolates and three transconjugants, one derived from a mating with each parent strain, had identical plasmid profiles and restriction patterns (data not shown). Following the elimination of plasmids by repeated SDS treatment, the S. marcescens isolates became susceptible to all antibiotics except ampicillin. Isoelectric focusing analysis of crude enzyme extracts from the isolates (data not shown) demonstrated the production of two b-lactamases with apparent pIs of 6.5 and 6.7. The E. coli transconjugants produced the pI 6.7 b-lactamase only. Neither pI 6.7 nor pI 6.5 b-lactamase was detected in the three plasmid-eliminated S. marcescens strains. Given the pI of 6.7, we suspected the production of a KPC b-lactamase. To confirm the presence of a blaKPC gene, we conducted PCR amplification using the primers KPC-F and KPC-R. The reaction gave a positive result and DNA sequencing identified the gene as blaKPC-2. The data show that the carbapenem-hydrolysing enzyme in the three test S. marcescens isolates is KPC-2. The enzyme is encoded on a conjugative plasmid and mainly contributed to resistance to carbapenems. We presume that the unidentified pI 6.5 b-lactamase was either the degradation product or the precursor of KPC-2. Unidentified carbapenemase activity bands around pI 6.3–6.5 have also been detected from K. pneumoniae, which were presumed to be the degradation product of KPCs. The resistance against ampicillin remaining when the blaKPC-containing plasmid had been cured is probably due to the low activity of chromosomal AmpC in S. marcescens, which was not detected in IEF but confirmed through enzyme assay. The three S. marcescens isolates had identical plasmid profiles and restriction patterns, indicating that the same plasmid spread among these isolates. Most blaKPC-encoding plasmids could be conjugated into E. coli, except the blaKPC-1-encoding plasmid described by Yigit et al. Many blaKPC-encoding plasmids also encode blaTEM-1 and various ESBLs. However, only a blaKPC gene was identified from the plasmid in S. marcescens in this study. KPC family enzymes have been detected in a variety of Enterobacteriaceae but mainly in North America. The emergence of a KPC-2 type carbapenem-hydrolysing enzyme in China is alarming. It indicates that the spread of blaKPC genes is no longer a regional problem.