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.

Synergistic effects of Cinnamomum cassia L. essential oil in combination with polymyxin B against carbapenemase-producing Klebsiella pneumoniae and Serratia marcescens.

Multidrug resistance prompts the search for new sources of antibiotics with new targets at bacteria cell. To investigate the antibacterial activity of Cinnamomum cassia L. essential oil (CCeo) alone and in combination with antibiotics against carbapenemase-producing Klebsiella pneumoniae and Serratia marcescens. The antimicrobial susceptibility of the strains was determined by Vitek® 2 and confirmed by MALDI-TOF/TOF. The antibacterial activity of CCeo and its synergism with antibiotics was determined using agar disk diffusion, broth microdilution, time-kill, and checkboard methods. The integrity of the bacterial cell membrane in S. marcescens was monitored by protein leakage assay. CCeo exhibited inhibitory effects with MIC = 281.25 µg.mL-1. The association between CCeo and polymyxin B showed a decrease in terms of viable cell counts on survival curves over time after a 4 hour-treatment with a FIC index value of 0.006. Protein leakage was observed with increasing concentrations for CCeo and CCeo + polymyxin B treatments. CCeo showed antibacterial activity against the studied strains. When associated with polymyxin B, a synergistic effect was able to inhibit bacterial growth rapidly and consistently, making it a potential candidate for the development of an alternative treatment and drug delivery system for carbapenemase-producing strains.

Blowing epithelial cell bubbles with GumB: ShlA-family pore-forming toxins induce blebbing and rapid cellular death in corneal epithelial cells.

Medical devices, such as contact lenses, bring bacteria in direct contact with human cells. Consequences of these host-pathogen interactions include the alteration of mammalian cell surface architecture and induction of cellular death that renders tissues more susceptible to infection. Gram-negative bacteria known to induce cellular blebbing by mammalian cells, Pseudomonas and Vibrio species, do so through a type III secretion system-dependent mechanism. This study demonstrates that a subset of bacteria from the Enterobacteriaceae bacterial family induce cellular death and membrane blebs in a variety of cell types via a type V secretion-system dependent mechanism. Here, we report that ShlA-family cytolysins from Proteus mirabilis and Serratia marcescens were required to induce membrane blebbling and cell death. Blebbing and cellular death were blocked by an antioxidant and RIP-1 and MLKL inhibitors, implicating necroptosis in the observed phenotypes. Additional genetic studies determined that an IgaA family stress-response protein, GumB, was necessary to induce blebs. Data supported a model where GumB and shlBA are in a regulatory circuit through the Rcs stress response phosphorelay system required for bleb formation and pathogenesis in an invertebrate model of infection and proliferation in a phagocytic cell line. This study introduces GumB as a regulator of S. marcescens host-pathogen interactions and demonstrates a common type V secretion system-dependent mechanism by which bacteria elicit surface morphological changes on mammalian cells. This type V secretion-system mechanism likely contributes bacterial damage to the corneal epithelial layer, and enables access to deeper parts of the tissue that are more susceptible to infection.

Comprehensive analysis of resistance-nodulation-cell division superfamily (RND) efflux pumps from Serratia marcescens, Db10.

We investigated the role of the resistance-nodulation-cell division superfamily (RND) efflux system on intrinsic multidrug resistance in Serratia marcescens. We identified eight putative RND efflux system genes in the S. marcescens Db10 genome that included the previously characterized systems, sdeXY, sdeAB, and sdeCDE. Six out of the eight genes conferred multidrug resistance on KAM32, a drug hypersensitive strain of Escherichia coli. Five out of the eight genes conferred resistance to benzalkonium, suggesting the importance of RND efflux systems in biocide resistance in S. marcescens. The energy-dependent efflux activities of five of the pumps were examined using a rhodamine 6 G efflux assay. When expressed in the tolC-deficient strain of E. coli, KAM43, none of the genes conferred resistance on E. coli. When hasF, encoding the S. marcescens TolC ortholog, was expressed in KAM43, all of the genes conferred resistance on E. coli, suggesting that HasF is a major outer membrane protein that is used by all RND efflux systems in this organism. We constructed a sdeXY deletion mutant from a derivative strain of the clinically isolated multidrug-resistant S. marcescens strain and found that the sdeXY deletion mutant was sensitive to a broad spectrum of antimicrobial agents.

Differential genomic damage in different tumor lines induced by prodigiosin.

Prodigiosin is a secondary metabolite produced by Serratia marcercens. As this pigment is suggested to be a cancer drug, genotoxicity studies are necessary. The aim of the present investigation was to evaluate the genotoxic effects of prodigiosin on tumoral and normal cell lines, NCIH-292, MCF-7 and HL-60. A normal line BGMK was used as control. Genomic damage induced by prodigiosin was observed in all tumor lines as well as the control line. The pigment induced the formation of micronuclei in tumor cells. The present data confirm the antitumor potential of prodigiosin. However, these findings also raise concerns regarding its target-specific action, as genotoxic effects on normal cells also occurred.

Genetic dissection of Anopheles gambiae gut epithelial responses to Serratia marcescens.

Genetic variation in the mosquito Anopheles gambiae profoundly influences its ability to transmit malaria. Mosquito gut bacteria are shown to influence the outcome of infections with Plasmodium parasites and are also thought to exert a strong drive on genetic variation through natural selection; however, a link between antibacterial effects and genetic variation is yet to emerge. Here, we combined SNP genotyping and expression profiling with phenotypic analyses of candidate genes by RNAi-mediated silencing and 454 pyrosequencing to investigate this intricate biological system. We identified 138 An. gambiae genes to be genetically associated with the outcome of Serratia marcescens infection, including the peptidoglycan recognition receptor PGRPLC that triggers activation of the antibacterial IMD/REL2 pathway and the epidermal growth factor receptor EGFR. Silencing of three genes encoding type III fibronectin domain proteins (FN3Ds) increased the Serratia load and altered the gut microbiota composition in favor of Enterobacteriaceae. These data suggest that natural genetic variation in immune-related genes can shape the bacterial population structure of the mosquito gut with high specificity. Importantly, FN3D2 encodes a homolog of the hypervariable pattern recognition receptor Dscam, suggesting that pathogen-specific recognition may involve a broader family of immune factors. Additionally, we showed that silencing the gene encoding the gustatory receptor Gr9 that is also associated with the Serratia infection phenotype drastically increased Serratia levels. The Gr9 antibacterial activity appears to be related to mosquito feeding behavior and to mostly rely on changes of neuropeptide F expression, together suggesting a behavioral immune response following Serratia infection. Our findings reveal that the mosquito response to oral Serratia infection comprises both an epithelial and a behavioral immune component.

Serratia marcescens suppresses host cellular immunity via the production of an adhesion-inhibitory factor against immunosurveillance cells.

Injection of a culture supernatant of Serratia marcescens into the bloodstream of the silkworm Bombyx mori increased the number of freely circulating immunosurveillance cells (hemocytes). Using a bioassay with live silkworms, serralysin metalloprotease was purified from the culture supernatant and identified as the factor responsible for this activity. Serralysin inhibited the in vitro attachment of both silkworm hemocytes and murine peritoneal macrophages. Incubation of silkworm hemocytes or murine macrophages with serralysin resulted in degradation of the cellular immune factor BmSPH-1 or calreticulin, respectively. Furthermore, serralysin suppressed in vitro phagocytosis of bacteria by hemocytes and in vivo bacterial clearance in silkworms. Disruption of the ser gene in S. marcescens attenuated its host killing ability in silkworms and mice. These findings suggest that serralysin metalloprotease secreted by S. marcescens suppresses cellular immunity by decreasing the adhesive properties of immunosurveillance cells, thereby contributing to bacterial pathogenesis.

The integration of molecular tools into veterinary and spatial epidemiology.

At the interface of molecular biology and epidemiology, the emerging discipline of molecular epidemiology offers unique opportunities to advance the study of diseases through the investigation of infectious agents at the molecular level. Molecular tools can increase our understanding of the factors that shape the spatial and temporal distribution of pathogens and disease. Both spatial and molecular aspects have always been important to the field of infectious disease epidemiology, but recently news tools have been developed which increase our ability to consider both elements within a common framework. This enables the epidemiologist to make inferences about disease patterns in space and time. This paper introduces some basic concepts of molecular epidemiology in a veterinary context and illustrates the application of molecular tools at a range of spatio-temporal scales. Case studies - a multi-state outbreak of Serratia mastitis, a national control program for campylobacteriosis, and evolution of foot-and-mouth-disease viruses - are used to demonstrate the importance of considering molecular aspects in modern epidemiological studies. The discipline of molecular epidemiology is in its infancy and our contribution aims to promote awareness, understanding and uptake of molecular epidemiology in veterinary science.

CTX-M and TEM as predominant types of extended spectrum beta-lactamases among Serratia marcescens isolated from solid organ recipients.

BACKGROUND: Serratia marcescens is an important pathogen in hospital infections since organisms resistant to multiple antimicrobials pose a special threat particularly among transplant patients. The aim of this work was to assess the number of strains producing beta-lactamases with extended spectrum (ESBL) among S. marcescens isolated from our patients. MATERIALS AND METHODS: We investigated S. marcescens isolated from 2005 to 2008 for ESBL. The phenotype methods were applied and additionally we chose strains for polymerase chain reactions using primers for the most popular types of ESBL. RESULTS: Over the investigated time, 257 patients were infected with S. marcescens with 188 (73%) displaying an ESBL-positive phenotype. A Molecular analysis showed that most of them produced both CTX-M and TEM beta-lactamases. In the last year, the percentage of ESBL-producing strains decreased, but also in the last year, we isolated S. marcescens resistant to carbapenems from three patients. CONCLUSIONS: The CTX-M type of ESBL predominated among ESBLs produced by strains of S. marcescens. The appearance of strains resistant to carbapenems is alarming.

Genome-wide RNAi screen identifies genes involved in intestinal pathogenic bacterial infection.

Innate immunity represents the first line of defense in animals. We report a genome-wide in vivo Drosophila RNA interference screen to uncover genes involved in susceptibility or resistance to intestinal infection with the bacterium Serratia marcescens. We first employed whole-organism gene suppression, followed by tissue-specific silencing in gut epithelium or hemocytes to identify several hundred genes involved in intestinal antibacterial immunity. Among the pathways identified, we showed that the JAK-STAT signaling pathway controls host defense in the gut by regulating stem cell proliferation and thus epithelial cell homeostasis. Therefore, we revealed multiple genes involved in antibacterial defense and the regulation of innate immunity.

Outbreak of infection by extended-spectrum beta-lactamase SHV-5-producing Serratia marcescens in a Mexican hospital.

Previous outbreaks caused by Serratia marcescens have been associated with contaminated medical equipment, intravenous fluids and inadequate hygiene. We carried out the molecular characterization of an outbreak produced by a cephalosporin-resistant S. marscescens that occurred in a Mexican hospital in August 1999. The lethality of this outbreak was 26%. Positive isolates were collected from 20 patients, one medical staff and three chlorhexidine disinfectant solutions. Results of PFGE, beta-lactamase patterns, sequencing of PCR amplifications, plasmid profiles, and mating experiments showed that the outbreak occurred by the dissemination of a S. marcescens SHV-5 producing strain. The adequate enforcement of procedures under the supervision of an infection control resulted in the abrupt end of the outbreak.

Serratia marcescens osteomyelitis in an infant.

Neutrophil dysfunction can result from oxidative burst defect or from glucose-6-phosphate dehydrogenase (G6PD) deficiency; we noted both in the same patient. A 4-month-old male infant with G6PD deficiency presented with swelling of the left middle finger, left leg, and right big toe. At 5 weeks of age he was hospitalized for fever for 2 days. A maternal uncle died at 5 years of age and a male maternal cousin died at the age of 21 months, both reportedly diagnosed with chronic granulomatous disease (CGD). On physical examination, he had a swollen erythematous left third finger, left distal leg swelling, and right big toe abscess. None of these areas was significantly tender. WBC was 18.7 x 10(3)/mm(3) with 37% PMN and 5% bands. The x-ray films showed osteomyelitis in the left third proximal phalanx and the distal right first metatarsal. Culture from the toe abscess grew Serratia marcescens. His neutrophil oxidative burst was tested by the dihydrorhodamine-123 assay and was markedly suppressed, typical of CGD. The mother and maternal grandmother were found to be CGD carriers. He was treated with i.v. antibiotics for 4 weeks and was discharged on prophylactic trimethoprim, itraconazole and interferon gamma, with substantial reduction in infections. Infection in this infant was unusual in its nature, in affecting multiple sites, and in its causative organism. Immune deficiency was suspected, particularly of the phagocytic component, but could not be attributed to his moderate degree of primary G6PD deficiency. Additional immunologic evaluation and the family history led to the diagnosis of X-linked CGD.

Molecular epidemiology of Serratia marcescens outbreaks in two neonatal intensive care units.

OBJECTIVE: Serratia marcescens can cause serious infections in patients in neonatal intensive care units (NICUs), including sepsis, pneumonia, urinary tract infection, and conjunctivitis. We report the utility of genetic fingerprinting to identify, investigate, and control two distinct outbreaks of S. marcescens. DESIGN: An epidemiologic investigation was performed to control two clusters of S. marcescens infections and to determine possible routes of transmission. Molecular typing by pulsed-field gel electrophoresis determined the relatedness of S. marcescens strains recovered from neonates, the environment, and the hands of healthcare workers (HCWs). SETTING: Two geographically distinct level III-IV NICUs (NICU A and NICU B) in two university-affiliated teaching hospitals in New York City. RESULTS: In NICU A, one major clone, "F," was detected among isolates recovered from four neonates and the hands of one HCW. A second predominant clone, "A," was recovered from four sink drains and one rectal surveillance culture from an asymptomatic neonate. In NICU B, four neonates were infected with clone "D," and three sink drains harbored clone "H." The attributable mortality rate from bloodstream infections was 60% (3 of 5 infants). The antimicrobial susceptibilities of clone F strains varied for amikacin, cefepime, and piperacillin/tazobactam. CONCLUSIONS: S. marcescens causes significant morbidity and mortality in preterm neonates. Cross-transmission via transient hand carriage of a HCW appeared to be the probable route of transmission in NICU A. Sinks did not harbor the outbreak strains. Antimicrobial susceptibility patterns did not prove to be an accurate predictor of strain relatedness for S. marcescens.

Clustering of Serratia marcescens infections in a neonatal intensive care unit.

OBJECTIVES: To study clusters of infections caused by Serratia marcescens in a neonatal intensive care unit (NICU) and to determine risk factors for S. marcescens infection or colonization. DESIGN: Genotyping of S. marcescens isolates was performed by pulsed-field gel electrophoresis (PFGE). A retrospective case-control study was conducted. SETTING: A tertiary-care pediatric hospital with a 16-bed NICU. PATIENTS: All neonates with at least one culture positive for S. marcescens in the NICU during December 1999 to July 2002. Case-patients (n = 11) treated in the NICU during December 1999 to February 2000 were included in the case-control study. Neonates treated in the NICU for at least 72 hours during the same period with cultures negative for S. marcescens were used as control-patients (n = 27). RESULTS: S. marcescens was cultured from 19 neonates; 9 were infected and 10 were colonized. PFGE analysis identified three epidemic strains; each cluster consisted of identical isolates, except one isolate in the first cluster that was different. The risk factors identified were low birth weight, prematurity, prolonged respiratory therapy, prolonged use of antibiotics, and maternal infection prior to delivery. Overcrowding and understaffing were recorded simultaneously with the clusters. CONCLUSIONS: PFGE analysis showed three independent clusters. Several factors contributed to spread of the epidemic strains: (1) there were many severely premature and susceptible neonates, (2) the NICU was overcrowded during the clusters, and (3) transmission was likely to occur via the hands of staff. Cohorting and improvement of routine infection control measures led to the cessation of each cluster.

Rapid eradication of a cluster of Serratia marcescens in a neonatal intensive care unit: use of epidemiologic chromosome profiling by pulsed-field gel electrophoresis.

OBJECTIVE: To investigate a cluster of patients infected and colonized with Serratia marcescens in a neonatal intensive care unit (NICU). METHODS: In June 2001, two neonates in the NICU had clinical infections with S. marcescens and one died. Infection control surveillance data for the NICU revealed that S. marcescens was rarely isolated from clinical specimens. Surveillance and environmental cultures were performed and isolates were typed using pulsed-field gel electrophoresis. Staff and neonates were cohorted and a waterless, alcohol-based handwashing agent was introduced. A case-control study was performed. RESULTS: From June 2 through August 20, 2001, 11 neonates with S. marcescens infection and colonization were identified. The incidence of S. marcescens infections increased from 0.19 per 1,000 patient-days in 2000 to 0.52 per 1,000 patient-days in 2001 (P < .0001). In the first 3 weeks of the investigation, there were 2 sets of patients and sinks with indistinguishable strains; however, in subsequent weeks, all isolates were of unique strains, signifying no further transmission of the two initial predominant strains. Neonates with S. marcescens were more likely to have a lower gestational age and birth weight. There was no association between cases and healthcare workers (HCWs). CONCLUSIONS: A cluster of S. marcescens was quickly terminated after the introduction of preventive measures including cohorting of infected and colonized neonates and HCWs, contact precautions, surveillance cultures, and a waterless, alcohol-based hand antiseptic. Chromosomal typing determined that strains with an indistinguishable pattern were no longer present in the unit after control measures were implemented.

A nosocomial outbreak of Serratia marcescens producing inducible Amp C-type beta-lactamase enzyme and carrying antimicrobial resistance genes within a class 1 integron.

We investigated an outbreak of Serratia marcescens in the adult intensive care unit of the University Hospital of Napoli. The outbreak involved 13 cases of infection by S. marcescens over a nine-month period and was caused by a single pulsed-field gel electrophoresis clone. The epidemic strain was multiply antibiotic resistant, producing an inducible Amp C-type beta-lactamase enzyme and carrying the trimethoprim-resistance gene and the adenyltransferase gene, which confers resistance to streptomycin and spectinomycin, within a class 1 integron. Antimicrobial therapy with beta-lactams was associated with S. marcescens acquisition in the intensive care unit.

Cloning and nucleotide sequence of the DNA gyrase gyrA gene from Serratia marcescens and characterization of mutations in gyrA of quinolone-resistant clinical isolates.

The sequence of the DNA gyrase gyrA gene of Serratia marcescens ATCC 14756 was determined. An open reading frame of 2,640 nucleotides coding for a polypeptide with a calculated molecular mass of 97,460 was found, and its sequence complemented the sequence of an Escherichia coli gyrA temperature-sensitive mutation. Analysis of the PCR products of the quinolone resistance-determining regions of gyrA genes from six quinolone-resistant clinical isolates revealed a single amino acid substitution, Ser-83 to Arg or Asp-87 to Tyr, in all six mutants, suggesting that a mutational alteration in gyrA is a common mechanism of quinolone resistance in S. marcescens.

Genetic and molecular R-plasmid analysis of Enterobacteriaceae hospital strains at Children's Hospitals of the former USSR.

R-plasmids from Enterobacteriaceae clinical strains, mainly Klebsiella and Serratia, isolated at different neonatal and children's hospitals of different cities of the former USSR for 10 years, were studied for their possible influence on the bacterial host phenotype. Hospital R-plasmids of stable inheritance persisted in hospitals from 2 to 7 years and were disseminated among strains of different genera (Klebsiella, Serratia, Enterobacter) and among different units. The data showed a possibility of long-term molecular rearrangements of R-plasmids in the hospital settings and an acquisition of genetic determinants encoding enterotoxin production. A novel R-plasmid encoding cytotoxicity to HEp-2 cells involved in two nosocomial outbreaks due to K. pneumoniae strains was reported. K. pneumoniae population heterogeneity was evaluated by using the plasmid parameters of strains. Their heterogeneity of a bacterial population was significantly lower during nosocomial outbreaks than in interepidemic periods.

[An epidemic of primary bacteremia due to an endemic strain of Serratia marcescens in an intensive care unit]. / Epidemia de bacteremias primarias por una cepa endémica de Serratia marcescens en una unidad de terapia intensiva.

An outbreak of Serratia marcescens bacteremia detected in the intensive care unit (ICU) of a tertiary care center on the last days of October, 1985, is described. The rate of primary S. marcescens nosocomial bacteremia during the pre-epidemic period (January-September 1985) was 6.25 per cent; and for the post-epidemic period compared with the epidemic were significantly different (p < 0.0001). The outbreak strains belonged to the biotype A8b, which has been endemic in our hospital. The responsible organism exhibited an unusual antimicrobial resistance pattern associated to the presence of a specific plasmid (greater than 50 kilobases), which showed similar fragments after restriction endonuclease digestion. No specific risk factors were identified in the case-control study. The outbreak was probably related to a greater influx of infected patients, resulting in less careful infection control measures, due to the emergency situation which suffered the hospital after the earthquakes in 1985. The unusual high rate of blood isolation of S. marcescens at the ICU was the first sign of the outbreak. The prompt reinforcement of infection control policies facilitated its resolution.