Clinical Isolates Of Serratia Marcescens Research Articles

Effect of cultivation conditions on hemolysin production from clinical isolates of Serratia marcescens

Sixty-four isolates were collected from clinical sources in Baghdad and Mosul city. The identification of S. marcescens confirmed by using API 20E and VITEK-2 systems. Twelve isolates of S. marcescens produced hemolysin, which were detected by two ways, qualitative hemolytic assay and quantitative hemolytic assay. The optical density of hemolysin producing isolates at standard condition (37oC, pH=7, 24h) showed that Serratia marcescens (SmU9) isolate gave the highest absorbance of hemolysin production (0.7) but at (25°C, pH=7,24h) the absorbance of hemolysin production turn into (3.0), compared with the absorbance at 571nm for Control without hemolysin (0.060) and completely hemolysis by Triton x-100 (6.0). However, the optimum optical density of the best hemolysin producing isolate (SmU9) at the optimum cultivation conditions (25°C, pH=9, 24h, 150 rpm, 1% inoculum size in Nutrient broth) was (6.0).

SlpE is a calcium-dependent cytotoxic metalloprotease associated with clinical isolates of Serratia marcescens

Serralysin-like proteases are found in a wide variety of bacteria. These metalloproteases are frequently implicated in virulence and are members of the widely conserved RTX-toxin family. We identified a serralysin-like protease in the genome of a clinical isolate of Serratia marcescens that is highly similar to the canonical serralysin protein, PrtS. This gene was named serralysin-like protease E, SlpE, and was found in the majority (67%) of tested clinical isolates, but was absent from most tested non-clinical isolates including the insect pathogen and reference S. marcescens strain Db11. Purified recombinant SlpE exhibited calcium-dependent protease activity similar to metalloproteases PrtS and SlpB. Induction of slpE in the low-protease-producing S. marcescens strain PIC3611 highly elevated extracellular protease activity, and extracellular secretion required the lipD type 1 secretion system gene. Transcription of slpE was highly reduced in an eepR transcription factor mutant. Mutation of the slpE gene in a highly proteolytic clinical isolate reduced its protease activity, and evidence suggests that SlpE confers cytotoxicity of S. marcescens to the A549 airway carcinoma cell line. Together, these data reveal SlpE to be an EepR-regulated cytotoxic metalloprotease associated with clinical isolates of an important opportunistic pathogen.

Correlation between Prodigiocin , Biofilm formation and Drug Resistant in Virulent Serratia marcescens

One hundred and fifty samples were collected from urine and blood, with a male to female ratio 1.45:1, culture positivity seen in the urinary tract infection was the most frequent (77%), followed by blood infection (18%). A total of 86 (57.33%) bacterial isolates were obtained. The study focused on 9 clinical isolates of Serratia marcescens (15.2%), differentiated into Prodigiosin producer (11.6%) and non- Prodigiosin producer (3.6%). Almost all isolates (6/9) showed visible growth at 37°C. All S. marcescens strains of different sources were hemolytic, whereas the formation of a pellicle was noticed among 7 isolates (77.77%) from 9 isolates. The biofilm formation performed was assessed through the crystal violet assay noticed that out of the 9 S. marcescens isolates 5 (55.5%) were non-adherent, 4 (44.4%) were weakly adherent, No isolate showed strongly adherent. The tube method for assay biofilm formation showed good correlation with the Tissue culture plate (TCP) method assay for biofilm forming isolates and total 2 (22.2%) isolate were picked up as strong and 5 (55.5%) were moderat biofilm producers. The tube test correlates well with the TCP test for biofilm producing isolates but it was difficult to discriminate between weak and biofilm negative isolates due to the variability in observed results by different observers. All 9 isolates were sensitive to the IPM and AZM (100%). The biofilm MICs were found to be much higher than the planktonic MICs. The medium with antibiotic used not inhibited the prodigiosin production at the MIC for planktonic cells. Therefore prodigiosin production in media with antibiotics used not marker of the growth and activity of bacterial cells. Biofilms are considerably less susceptible to antibiotics than their planktonic counterparts; biofilm production may vary largely among different strains of the same species isolated from different sites.

Chromosome and plasmid-mediated quinolone resistance mechanism in clinical isolates of Serratia marcescens

Objective To investigate the prevalence of plasmid-mediated quinolone resistance (PMQR) determinants ［qnr, aac(6′)-Ib-cr and qepA］ and mutations in quinolone resistance-determining regions (QRDRs) of gyrA and parC and their association with fluoroquinolone susceptibility in clinical isolates of Serratia marcescens in Anhui. Methods The minimum inhibition concentration (MIC) of 104 strains of S.marcescens collected from various clinical specimens from 34 hospitals during 2005 to 2010 were determined by agar dilution method.The qnr, aac(6′)-Ib, qepA, gyrA and parC genes were screened by polymerase chain reaction (PCR) in 31 strains resistant to ciprofloxacin, and positive results were subsequently confirmed by sequencing.The conjugation experiments were performed for qnr and aac(6′)-Ib-cr positive strains.The MIC of S.marcescens isolates, recipient strains and conjugants were tested by agar dilution method for quinolones and other antimicrobial agents. Results Six strains of the 31 S.marcescens isolates harboured qnr and/or aac(6′)-Ib-cr genes.Among those 6 strains, 2 strains harboured a qnrB6 gene, 1 harboured a qnrS2 gene, and 4 harboured aac(6′)-Ib-cr, whereas no qnrA-, qnrC- or qnrD-positive isolate was detected.None of the 31 isolates carried the qepA gene.Mutations in the QRDR of gyrA and parC genes were detected in 9 and 7 isolates, respectively.The conjugation experiments were successfully carried out in 5 isolates of 6 PMQR determinants-postive strains.The MIC of conjugants for quinolones were increased evidently compared to recipient strains. Conclusions Chromosome and plasmid-mediated resistance determinants play an important role in quinolone resistance in clinical isolates of S.marcescens.And more important is that the PMQR determinants can be horizontal transmitted.It is necessary to continuously survey and watch for the spread of PMQR in S.marcescens in public health control program.（Chin J Lab Med,2012,35:706-710） Key words: Serratia marcescens; Quinolones; Chromosomes; Plasmids; Drug resistance，bacterial

Detection of the plasmid-mediated quinolone resistance determinants in clinical isolates of Serratia marcescens in China

Detection of the plasmid-mediated quinolone resistance determinants in clinical isolates of Serratia marcescens in China

Identification of plasmid- and integron-borne bla IMP-1 and bla IMP-10 in clinical isolates of Serratia marcescens

The emergence of carbapenem-hydrolysing metallo-beta-lactamases (MBLs) is a serious threat to the clinical utility of carbapenems. This study identified plasmid- and integron-borne bla(IMP-1) and bla(IMP-10) in clinical isolates of Serratia marcescens. The bla(IMP-1) and bla(IMP-10) gene cassettes were carried by a class 1 integron and followed by the aac(6')-IIc gene cassette. The bla(IMP-1) and bla(IMP-10) gene cassettes were preceded by a weak P(ant) promoter, TGGACA(N)(17)TAAGCT, and an inactive P2 promoter, TTGTTA(N)(14)TACAGT. These genes were easily transferred to Escherichia coli by conjugation and transformation, indicating that they are located on transferable plasmids. Due to the acquisition of bla(IMP-1), the susceptibility of E. coli transconjugants to imipenem, meropenem, panipenem and biapenem decreased by 32-, 256-, 64- and 128-fold, respectively. In comparison, after gaining bla(IMP-10), the susceptibility of E. coli transconjugants to the four carbapenems decreased by 64-, 2048-, 256- and 64-fold, respectively. Strains harbouring bla(IMP-10) showed higher-level resistance to imipenem, meropenem and panipenem than the strains harbouring bla(IMP-1), although the nucleotide sequences of the class 1 integrons carrying bla(IMP-10) and bla(IMP-1) were identical except for a single point mutation.

First detection of a class 2 integron among clinical isolates of Serratia marcescens

Serratia marcescens is a frequent nosocomial isolate and is associated with a variety of clinical sources, including blood, urine and sputum, and can cause significant infection. Infections can be difficult to treat due to its resistance to a variety of antimicrobial agents. An investigation of a population of 30 clinical strains revealed the presence of a class 2 integron among nine of the isolates, which represents the first isolation of this integron in Serratia species. This integron contained the gene cassettes dfrA1, sat1 and aadA1, conferring resistance to trimethoprim, streptothricin and streptomycin/ spectinomycin, respectively. One of these isolates also carried a class 1 integron identified by sequence analysis as containing the open reading frames aacC1 (encoding gentamicin resistance), ORFX, ORFY and aadA1. Polymerase chain reaction analysis confirmed the presence of the qacE∆1 and sul1 markers, which are common among class 1 integrons.

Hyperproduction of Inhibitor-Susceptible TEM β-Lactamase Is Responsible for Resistance of Serratia marcescens to β-Lactam-β-Lactamase Inhibitor Combinations

Background: Some clinical isolates of Serratia marcescens showed high-level resistance to expanded-spectrum cephalosporins alone and in combination with β-lactamase inhibitor, but the β-lactamase extracted from these strains was sensitive to the inhibitors. This study examines the possible mechanisms responsible for the discrepancy. Methods and Results: Three clinical isolates of S. marcescens bore the bla_TEM gene coded on a plasmid. This was confirmed by detection of the bla gene using PCR analysis and by transferring resistance determinants to Escherichia coli via conjugation and transformation. All of the E. coli transconjugants and transformants acquired a similar level of resistance to penicillins and narrow-spectrum cephalosporins, and showed the reduced susceptibility to expanded-spectrum cephalosporins alone and in combination with clavulanate. As a result of the highly constitutive expression of the TEM gene, up to 247–690 U of β-lactamase were produced by 10¹⁰ cells of wild-type S. marcescens and the transconjugants and transformants. In the presence of 0.24 U/ml of TEM enzyme, the minimum inhibition concentrations of cefotaxime against E. coli ATCC 25922 increased from 0.125 to 512 µg/ml. The TEM-type β-lactamase extracted from these strains was sensitive to clavulanate, and 62.2–92.1% of its activity was inhibited after preincubation with 0.1 mM clavulanate. Conclusion:The TEM-type β-lactamase plays a critical role in the resistance of S. marcescens to β-lactams, and the hyperproduction of inhibitor-susceptible TEM β-lactamase is responsible for the resistance to β-lactam-β-lactamase inhibitor combinations.

Efficacy of Contact Lens Multipurpose Solutions Against Serratia Marcescens

To compare the susceptibilities of clinical isolates of Serratia marcescens and the standard ISO ATCC 13880 strain to five contact lens multipurpose disinfection solutions (MPDSs). Five commercially available MPDSs, containing either a polymeric biguanide or polyquaternium, were tested using ISO/CD 14729 stand-alone test for contact lens care products against four ocular isolates of S. marcescens and the strain ATCC 13880. An average log reduction in bacterial numbers at the manufacturer's minimum recommended disinfection time was determined and compared with the criteria for stand-alone disinfection products for each MPDS against each bacterial strain. All the MPDSs tested met the stand-alone criteria of 3-log reduction of viable bacteria against the ATCC strain of S. marcescens. However, there was more variability in their ability to meet disinfection criteria when tested against the clinical isolates. Two of the clinical isolates were significantly more resistant to disinfection than was the recommended ISO strain (p < or = 0.034). Two of the polyquaternium-1-based disinfection solutions (solutions D and E, p < or = 0.005) were less effective overall than the other MPDSs against S. marcescens. The importance of strain selection for the testing of MPDSs is indicated, and the use of a single laboratory strain may be insufficient to provide assurance that the disinfection solution will be effective against clinical isolates. Furthermore, clinical isolates of S. marcescens may show increased resistance to disinfection with polyquaternium.

Prevalence of decreased susceptibility to carbapenems among Serratia marcescens, Enterobacter cloacae, and Citrobacter freundii and investigation of carbapenemases

Between March and July 2002, total of 612 clinical isolates of Serratia marcescens, Enterobacter cloacae, and Citrobacter freundii (201 S. marcescens, 228 E. cloacae, and 183 C. freundii) were collected from 13 clinical laboratories in a nationwide distribution. Imipenem and meropenem minimum inhibitory concentrations (MICs) were determined using the agar dilution method according to the National Committee for Clinical Laboratory Standards guidelines. For the isolates with a decreased susceptibility to carbapenems (MICs of ≥2 μg/mL), isoelectric focusing, polymerase chain reaction (PCR) amplification of the carbapenemase genes ( bla IMP-1, bla VIM-2, bla SME-1, bla OXA-23, bla OXA-25, bla KPC-1), and sequencing were performed. The prevalence of S. marcescens, E. cloacae, and C. freundii with a decreased susceptibility to imipenem was 17.9% (36/201), 0.4% (1/228), and 0.5% (1/183), respectively, and to meropenem, it was 11.4% (23/201), 0% (0/228), and 0.5% (1/183), respectively. The bla VIM-2 was the only carbapenemase detected, and was found in 0.5% (1/201) of S. marcescens and 0.5% (1/183) of C. freundii isolate.

Probing the Role of Asp-120(81) of Metallo-β-lactamase (IMP-1) by Site-directed Mutagenesis, Kinetic Studies, and X-ray Crystallography

Metallo-beta-lactamase IMP-1 is a di-Zn(II) metalloenzyme that efficiently hydrolyzes beta-lactam antibiotics. Wild-type (WT) IMP-1 has a conserved Asp-120(81) in the active site, which plays an important role in catalysis. To probe the catalytic role of Asp-120(81) in IMP-1, the IMP-1 mutants, D120(81)A and D120(81)E, were prepared by site-directed mutagenesis, and various kinetics studies were conducted. The IMP-1 mutants exhibited 10(2)-10(4)-fold drops in k(cat) values compared with WT despite the fact that they contained two Zn(II) ions in the active site. To evaluate the acid-base characteristics of Asp-120(81), the pH dependence for hydrolysis was examined by stopped-flow studies. No observable pK(a) values between pH 5 and 9 were found for WT and D120(81)A. The rapid mixing of equimolar amounts of nitrocefin and all enzymes failed to result in the detection of an anion intermediate of nitrocefin at 650 nm. These results suggest that Asp-120(81) of IMP-1 is not a factor in decreasing the pK(a) for the water bridging two Zn(II) ions and is not a proton donor to the anionic intermediate. In the case of D120(81)E, the nitrocefin hydrolysis product, which shows a maximum absorption at 460 nm, was bound to D120(81)E in the protonated form. The three-dimensional structures of D120(81)A and D120(81)E were also determined at 2.0 and 3.0 A resolutions, respectively. In the case of D120(81)E, the Zn-Zn distance was increased by 0.3 A compared with WT, due to the change in the coordination mode of Glu-120(81)OE1 and the positional shift in the conserved His-263(197) at the active site.

Studies on a cytotoxin produced by clinical isolates of Serratia marcescens

Studies on a cytotoxin produced by clinical isolates of Serratia marcescens

Sequences of homologous beta-lactamases from clinical isolates of Serratia marcescens with different substrate specificities.

Genes for two group 1 beta-lactamases, SRT-1 and SST-1, were sequenced. These beta-lactamases were produced by clinical isolates of Serratia marcescens, isolates GN16694 and GN19450, respectively. The resulting enzymes were 96% identical. SRT-1 hydrolyzed oxyimino cephalosporins, but SST-1 hardly hydrolyzed them. At residue 213 in the third motif, which is conserved among group 1 beta-lactamases, SRT-1 and SST-1 had Lys and Glu, respectively. By site-directed mutagenesis, the substitution of Glu by Lys at residue 213 in SST-1 resulted in an enzyme that hydrolyzed oxyimino cephalosporins.

Preventive Effect of Dapsone on Renal Scarring following Mannose-Sensitive Piliated Bacterial Infection

Renal scarring has been thought to occur in the later stages of chronic pyelonephritis. We previously reported that mannose-sensitive (MS) piliated bacteria promoted renal scarring, which was prevented by antioxidants. The preventive effect of diaphenylsulfone (dapsone), which has a scavenging activity on active oxygen species, on renal scarring was examined. Female Sprague-Dawley rats were inoculated with clinical isolates of Serratia marcescens which had both MS and mannose-resistant pili or with recombinant strains which had MS pili on their surface; they were then administered 20 mg/kg of dapsone or not. Dapsone significantly suppressed scarring following infection of the kidney. The bacterial counts in the kidneys were not different in dapsone-treated and nontreated rats. We conclude that dapsone is effective in preventing renal scarring, and it is suggested that the clinical use of this drug may prevent renal scar formation following pyelonephritis.

Detection of cytotoxic activity on Vero cells in clinical isolates of Serratia marcescens.

Cytotoxin production was studied in 60 Serratia marcescens strains isolated from hospitalized patients. Association of cytotoxic activity with serotype, source of isolation and presence of plasmids was also evaluated. Thirteen of the 60 S. marcescens strains produced a cytotoxic effect on Vero cells. These strains were isolated from distinct clinical sources and classified into seven different serotypes (O1:H7; O4:NM; O10:NT; O19:NM; O6,14:H4; O6,14:NM and O6,14:H1). No relationship was observed between cytotoxic activity and clinical source or serotypes of the strains. Plasmids from five cytotoxin-producing S. marcescens strains were transferred to E. coli K12/711. The transconjugants did not exhibit cytotoxicity, indicating that the cytotoxic effect is not plasmid-mediated among these strains. Although a cytotoxic activity was demonstrated in filtrates of some S. marcescens strains, further studies should be performed to assess the role of this toxin in pathogenesis.

Typing of nosocomial Strains of Serratia marcescens: Comparison of restriction enzyme cleaved genomic DNA fragment (PFGE) analysis with bacteriocin typing, biochemical profiles and serotyping

Eighty-eight selected clinical isolates of Serratia marcescens, representing 27 putative outbreaks of nosocomial cross-infection encountered during 1980-1995, were tested comparatively by bacteriocin typing, carbon source assimilation tests, serotyping (O and H antigens), and restriction pattern (RFLP) analysis of restriction cleaved (SpeI, XbaI) genomic DNA fragments after pulsed-field gel electrophoresis (PFGE). Serotyping served as the "gold standard" of the phenotypic methods. One pseudo-outbreak (bacteriocin typing incriminated type 26) was uncovered through serotyping as well as the biochemical profile and confirmed by PFGE analysis of genomic DNA. Bacteriocin typing and determination of biochemical profiles disclosed several instances of phenotypic variation; serotyping revealed two episodes of shifts from motility (H12) to nonmotility. Resolution of restricted genomic DNA fragments with the PFGE procedure permitted detection of 27 PFGE patterns (A-M, N-1-N-3, O-1, O-2, P-1-P-3, Q-1-Q-3, R-1-R-3, S-1, and T). Based on the analysis of PFGE patterns against the background of epidemiological data, the number of nosocomically significant strains of S. marcescens could be reduced to 16 (PFGE patterns A-M, N-2, O-1, P-2, and T). It was concluded that PFGE analysis of restricted genomic DNA of S. marcescens was superior to the three phenotypic methods.

Immunochemical characterization of two new O serotypes of Serratia marcescens (O27 and O28)

In an earlier study of the distribution of O-serotypes among clinical isolates of Serratia marcescens, two apparently new serotypes were identified, represented by strains S1254 and S3255. Studies using ELISA, immunoblotting and the Quellung reaction have shown that they qualify for inclusion in the O-antigenic typing scheme on three counts: (1) they possess chemically distinct O-antigenic repeating units, (2) the O-antigens are serologically distinguishable from all others, and (3) they are found in a significant proportion of clinical S. marcescens strains (13% and 6% respectively). S1254, the type strain for serotype O27, is an acapsular strain which expressed a glucorhamnan with a disaccharide repeating unit as its lipopolysaccharide side chain. It cross-reacts with serotype O4, the O antigen of which is an O-acetylated form of the O27 glucorhamnan, but this cross-reaction can be eliminated by reciprocal cross-absorption. S3255, the type strain for serotype O28, has a mannose homopolymer as its O-antigen and is the only S. marcescens serotype with a trimeric repeating-unit structure. However, it cross-reacts with the O5 serotype strain due to similarities in their acidic capsular polysaccharides. Cross-absorption and the production of serum to an acapsular variant of serotype strain O28 produced typing reagents which could differentiate serotypes O5 and O28.

Association of colony variation in Serratia marcescens with the differential expression of protease and type 1 fimbriae

Several clinical isolates of Serratia marcescens were found to dissociate on peptone glycerol agar into colonies with red and pink or white and gray phenotypes that differ in the expression of proteolytic activity and mannose-sensitive type of hemagglutination. Colonies of red and white type were proteolytically active but did not express hemagglutination, whereas pink and gray colonies were protease-deficient but agglutinated guinea pig erythrocytes. Site-directed mutagenesis of a red laboratory strain S. marcescens SM6 resulted in selection of protease negative derivative prt::G7 which expressed the pink phenotype with hemagglutinating activity. It is suggested that a DNA-regulatory element may be involved in this type of colony variation.

Association of colony variation in Serratia marcescens with the differential expression of protease and type 1 fimbriae.

Several clinical isolates of Serratia marcescens were found to dissociate on peptone glycerol agar into colonies with red and pink or white and gray phenotypes that differ in the expression of proteolytic activity and mannose-sensitive type of hemagglutination. Colonies of red and white type were proteolytically active but did not express hemagglutination, whereas pink and gray colonies were protease-deficient but agglutinated guinea pig erythrocytes. Site-directed mutagenesis of a red laboratory strain S. marcescens SM6 resulted in selection of protease negative derivative prt::G7 which expressed the pink phenotype with hemagglutinating activity. It is suggested that a DNA-regulatory element may be involved in this type of colony variation.

Nucleotide Sequence and Expression of a Gentamicin Resistance Gene Isolated from the R Plasmid in Serratia marcescens

The 1.0 kb fragment carrying a gentamicin resistance determinant was subcloned from the R plasmid in clinical isolates of Serratia marcescens and its nucleotide sequence was determined. It contains an open reading frame composed of 858 nucleotides, which shows 97% homology to the aacC2 gene in nucleotide sequence. Minicells carrying the 1.0 kb insert DNA on the plasmids produced a 30 kd protein that was consistent with the expected size of the deduced amino acid sequence from the open reading frame. A search for the promoter revealed a putative promoter of which the −35 region was provided by the right end of Tn3, suggesting that the expression of the gentamicin resistance gene is dependent on the Tn3 sequence.