L-Arabinose-ornithine-Irgasan medium for differentiating Serratia species.

A semisolid medium (designated Serratia differentiation medium) containing L-arabinose, ornithine, and selective inhibitor was used to differentiate three clinically encountered Serratia species. The inhibitor, Irgasan DP-300, was incorporated to eliminate false-positive reactions from most remaining Enterobacteriaceae. The suspected Serratia colony was inoculated as a stab into the medium. Serratia marcescens was indicated by a change in color from olive to purple following 18 h of incubation, whereas S. rubidaea (not listed in Bergey's Manual of Determinative Bacteriology) was indicated by a change to bright yellow. S. liquefaciens (described in Bergey's Manual of Determinative Bacteriology [8th ed., 1974] as Enterobacter liquefaciens) produced a small purple band at the top of the medium and a yellow or yellow-green butt. Absence of growth and color change following incubation indicates that the suspected colony is a non-Serratia. Thirty-six Serratia strains and 97 other Enterobacteriaceae and Pseudomonadaceae strains were tested. Two strains of the non-Serratia Enterobacteriaceae (one each of Citrobacter freundii and Proteus morganii) and two strains of Pseudomonas aeruginosa produced a color change in the medium. All of the Serratia strains tested were correctly identified using this medium, while 96% of the other species tested were inhibited.     

Evaluation of RapID onE system for identification of 379 strains in the family Enterobacteriaceae and oxidase-negative, gram-negative nonfermenters.

The ability of the RapID onE system (Innovative Diagnostic Systems, Inc., Norcross, Ga.) to identify 364 strains in the family Enterobacteriaceae and 15 oxidase-negative, gram-negative, nonfermentative rods was evaluated. Kits were inoculated with no. 2 McFarland standard suspensions, and reactions were interpreted after 4 h of incubation at 35 degrees C. Overall, the method correctly identified (to the species level or to the genus level for salmonellas and non-Shigella sonnei Shigella species) 363 strains (95.8%) without additional tests. For four strains (1.0%), additional tests were required to delineate the correct identification from a range of two or more possibilities; these included one Serratia liquefaciens (Serratia marcescens or Serratia liquefaciens), one Serratia rubidaea (Serratia rubidaea or Serratia odorifera), one Salmonella typhi (Leminorella richardii or Salmonella sp.) and one Yersinia enterocolitica (Yersinia frederiksenii, Yersinia intermedia, or Yersinia enterocolitica). Twelve strains (3.2%) were misidentified or yielded codes with no identification; these comprised one Citrobacter amalonaticus (no identification), three Enterobacter hormaechei (not in the RapID onE database; two Enterobacter amnigenus, one Enterobacter sp.), one Serratia liquefaciens (Enterobacter cloacae), one Serratia rubidaea (no identification), four Serratia fonticola (not in RapID onE database; two Enterobacter aerogenes, one Serratia marcescens, one not identified), one Proteus mirabilis (Proteus penneri), and one Proteus vulgaris (Providencia rustigianii). If the seven strains not included in the database had been excluded, correct identification rates would have risen to 97.6% without additional tests and 98.7% with additional tests, with misidentification rates dropping to 1.3%. The RapID onE system is easy to set up and the results are easy to read, and the system provides an accurate, nonautomated commercially available method for the same-day identification of members of the family Enterobacteriaceae and oxidase-negative, gram-negative nonfermenters.     

L-prolineaminopeptidase activity as a tool for identification and differentiation of Serratia marcescens, Serratia liquefaciens and Hafnia alvei strains

S. marcescens (316 strains), S. liquefaciens (10 strains) and Hafnia alvei (20 strains), in contrast to 18 other Enterobacteriaceae species, hydrolyzed L-proline-4-nitroanilide within 30 min at 37 degrees C. In this way, rapid identification of these species is possible. The substrate is applied in solution or in a paper disc. The substrate optimum of Hafnia alvei strains proved to be 16 times lower than that of the Serratia spp. Application of a tenth of the substrate concentration necessary for identification of Serratia spp. allows a rapid differentiation between the two species.     

Antigenic analysis of Serratia marcescens fimbriae with monoclonal antibodies.

Monoclonal antibodies (MAbs) were raised against the purified fimbriae of Serratia marcescens US46, a strain expressing three morphologically distinct fimbriae. The widths of these fimbriae were 7, 4.5, and 3 nm, respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified fimbriae showed three bands with molecular weights of 21,000, 20,000, and 19,000, respectively. This strain had mannose-resistant (MR) hemagglutinating activity and was agglutinated by yeast cells. Therefore, strain US46 appeared to have both MR and mannose-sensitive fimbriae. In the immunoblot analysis, all MAbs reacted with the 20,000-molecular-weight subunit when given a choice of three differently sized subunits. Immunoelectron microscopy showed these MAbs attached to the MR fimbriae with the largest width (7 nm). The antigenic cross-reactivity of fimbriae was examined by an MAb-mediated agglutination test. All MR strains of S. marcescens and some mannose-sensitive strains were agglutinated by the MAbs. The serological homogeneity of MR fimbriae was confirmed by a spot test, using the crude purified fimbriae from several MR strains of S. marcescens. In other gram-negative rods, clinical isolates of Klebsiella spp. with hemagglutinating activity were agglutinated, but clinical isolates of Escherichia coli and Enterobacter spp. were not.     

Serratia infections: from military experiments to current practice

Serratia species, in particular Serratia marcescens, are significant human pathogens. S. marcescens has a long and interesting taxonomic, medical experimentation, military experimentation, and human clinical infection history. The organisms in this genus, particularly S. marcescens, were long thought to be nonpathogenic. Because S. marcescens was thought to be a nonpathogen and is usually red pigmented, the U.S. military conducted experiments that attempted to ascertain the spread of this organism released over large areas. In the process, members of both the public and the military were exposed to S. marcescens, and this was uncovered by the press in the 1970s, leading to U.S. congressional hearings. S. marcescens was found to be a certain human pathogen by the mid-1960s. S. marcescens and S. liquefaciens have been isolated as causative agents of numerous outbreaks and opportunistic infections, and the association of these organisms with point sources such as medical devices and various solutions given to hospitalized patients is striking. Serratia species appear to be common environmental organisms, and this helps to explain the large number of nosocomial infections due to these bacteria. Since many nosocomial infections are caused by multiply antibiotic-resistant strains of S. marcescens, this increases the danger to hospitalized patients, and hospital personnel should be vigilant in preventing nosocomial outbreaks due to this organism. S. marcescens, and probably other species in the genus, carries several antibiotic resistance determinants and is also capable of acquiring resistance genes. S. marcescens and S. liquefaciens are usually identified well in the clinical laboratory, but the other species are rare enough that laboratory technologists may not recognize them. 16S rRNA gene sequencing may enable better identification of some of the less common Serratia species.     

New fimbrial hemagglutinin in Serratia species.

Strains of Serratia marcescens, Serratia liquefaciens, Serratia marinorubra, and Serratia plymuthica produced one or more of the following hemagglutinins (HAs): mannose-sensitive HA and mannose-resistant K-HA (MR/K-HA) and P-HA (MR/P-HA) (J. P. Duguid and D. C. Old, in E. H. Beachey (ed.), Bacterial adherence, vol. 6., p. 185-217, 1980). Most strains (82%) were multiply hemagglutinating. The properties of the three HAs are described. Each HA was associated with a distinct type of fimbria: mannose-sensitive HA with type 1 fimbriae. MR/K-HA with type 3 fimbriae, and MR/P-HA with a new type of thin fimbriae provisionally called MR/P fimbriae. This is the first report of the production of MR/P-HA and MR/P fimbriae by Serratia species. The range of Serratia HAs, which may reflect in vivo colonization potential, is more complex than previously reported.     

Type 3 fimbriae among enterobacteria and the ability of spermidine to inhibit MR/K hemagglutination.

The distribution of the gene cluster encoding type 3 fimbriae among various isolates of the family Enterobacteriaceae was investigated by using 112 clinical and nonclinical isolates. Closely related DNA sequences were detected in all Klebsiella strains, in most Enterobacter isolates, in a smaller number of Escherichia coli and Salmonella spp., and in a single isolate each of Yersinia enterocolitica and Serratia liquefaciens but not in isolates of Morganella or Providencia species or Serratia marcescens. Except for E. coli and Salmonella strains, the presence of gene sequences was correlated with the phenotypic expression of either the MR/K hemagglutinin or fimbriae that reacted with specific antibodies. In one isolate of Y. enterocolitica the expression of type 3 fimbriae was plasmid determined. The polyamine spermidine was identified as an inhibitor of MR/K hemagglutinating activity, exhibiting an MIC of 1.2 mM. Spermidine inhibited the hemagglutination of 37 MR/K-positive clinical isolates from various genera. However, one clinical isolate of Enterobacter cloacae and most (four of five) nonclinical Klebsiella isolates were not completely inhibited.     

Role of cell-bound hemolysin as a pathogenicity factor for Serratia infections.

The hemolytic activities of clinical isolates of Serratia marcescens, of Serratia liquefaciens, and of Escherichia coli strains containing a cloned hemolysin gene of S. marcescens were determined. Hemolysis was induced only by cells and not by spent media. The hemolytically active bacteria induced the release of the leukotriene C4 and of much less leukotriene B4 from polymorphonuclear leukocytes, the release of histamine from rat mast cells, and chemoluminescence of neutrophils. The hemolytic activity was correlated with the response of the leukocytes, but quantitative differences were recorded with regard to the release of the inflammatory mediators. Therefore, other factors in addition to the hemolysin contribute to the stimulation of leukotriene generation and histamine release. It is concluded that the hemolysin via these inflammatory mediators can increase vascular permeability, edema formation, and granulocyte accumulation and thus contributes to the pathogenicity of Serratia species.     

Bacteriocin typing of Serratia marcescens. A simplified system.

The authors describe a simplified system for the detection of bacteriocin production by Serratia marcescens with the use of six indicator strains, which include Escherichia coli, Klebsiella pneumoniae, Citrobacter diversus, Enterobacter aerogenes (two strains), and Serratia rubidaea grown on arabinose minimal medium plates. Of the 64 possible bacteriocin types, 11 were observed; 66% of the isolates tested were found to be one of three types. Occasionally more than one bacteriocin type was observed in an individual specimen; however, serotyping or antibiograms, or both, also indicated this was a different strain. The marcescin types were stable markers. With the use of this technic, different endemic strains of Serratia were shown to predominate in various areas of the hospital. In addition, when urinary tract isolates were compared with respiratory tract isolates, significant differences were found in the predominate types. The typing of these isolates by bacteriocin production was supported by serotype and antibiotype findings. The results suggest that this simple system may be a useful tool in a general hospital.     

In-vitro activities of cefamandole and cephalothin against 1,881 clinical isolates. A multi-center study

By use of an agardilution technic, 1,881 clinical isolates were tested against cefamandole and cephalothin. The isolates represented 18 genera, recovered in five geographically separate centers within the United States. The majority of strains were susceptible (MICs less than or equal to 8 micrograms/ml) to both drugs. Cefamandole showed greater activity against most of the bacterial pathogens. Enterococci, Serratia spp., and Acinetobacter spp. were resistant to both drugs. Cephalothin was more active against Staphylococcus aureus, and both cephalosporins were relatively inactive against methicillin-resistant strains of S. aureus. Enterobacter spp. and indole-positive Proteus spp. were susceptible to cefamandole but resistant to cephalothin.     

In vitro activity of aztreonam--a new monocyclic beta-lactam antibiotic

The in vitro antibacterial activity of aztreonam (SQ 26776), a new beta-lactam antibiotic, was measured by the agar dilution technique. Aztreonam is known to have a narrow spectrum with activity only against Gram negative bacteria. The strains tested were 223 clinical isolates from blood cultures obtained at Rigshospitalet, Copenhagen, Denmark. Its activity against E. coli, Klebsiella spp., Proteus spp., Enterobacter spp., Citrobacter, Salmonella typhimurium and Serratia marcescens was satisfactory with MIC values normally below 0.5 mg/l. However, six out of 135 strains of E. coli showed surprisingly high MIC values of eight and 16 mg/l. The activity against Pseudomonas spp. and Acinetobacter spp. were limited, but the majority were inhibited by concentrations of aztreonam between 2.0 and 8.0 mg/l. The MIC values for the tested anaeobic bacteria were high, ranging from 8.0 to above 512 mg/l. With its narrow spectrum of activity, aztreonam seems to be a valuable addition to the antibiotic arsenal. Clinical studies will determine its real value.     

In vitro antimicrobial activity of diethyldithiocarbamate and dimethyldithiocarbamate against methicillin-resistant Staphylococcus

Staphylococcus aureus has appeared which is highly resistant to both methicillin and aminoglycosides. Current therapy involves long-term intravenous therapy of vancomycin. Since vancomycin is currently the only drug used to treat these patients, there is a need to develop additional antimicrobial therapy. The in vitro antimicrobial effect of the metal chelator, diethyldithiocarbamate (DDTC) and its structural analog dimethyldithiocarbamate (DMTC) were investigated. Both DDTC and DMTC were effective against S. aureus including methicillin-resistant S. aureus (MRSA). By agar diffusion, DDTC at 10 micrograms per disk produced zone sizes of 12 to 21 mm and at 100 micrograms per disk produced zone sizes of 26 to 34 mm against MRSA. The DMTC produced slightly greater zone sizes against MRSA of 16 to 24 mm and 24 to 37 mm for 10 micrograms per disk and 100 micrograms per disk, respectively. The minimum inhibitory concentration (MIC) for DMTC against MRSA was 6 micrograms per ml. Both DDTC and DMTC were also effective against enterococci, Proteus mirabilis, Klebsiella pneumoniae, Klebsiella oxytoca, Escherichia coli, Enterobacter cloacae, Enterobacter aerogenes, Salmonella species, Serratia marcescens and Citrobacter freundii at 100 micrograms per disk. The MICs of DMTC for Klebsiella pneumoniae, Klebsiella oxytoca, Escherichia coli, Salmonella and Citrobacter freundii were approximately 128 micrograms per ml while the MICs for Proteus vulgaris, Proteus mirabilis, Pseudomonas aeruginosa and Serratia marcescens was greater than or equal to 256 micrograms per ml. In addition, DMTC was synergistic with gentamicin against MRSA and coagulase-negative staphylococcus species, Enterobacter cloacae, Klebsiella pneumoniae and Pseudomonas aeruginosa. Additive and synergistic effects of DMTC were displayed with gentamicin against S. aureus including methicillin-resistant S. aureus.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thin-layer chromatographic technique for rapid detection of bacterial phospholipases.

Silica gel thin-layer chromatography was employed to detect lecithinase activity induced from bacterial resting cell preparations induced from bacterial resting cell preparations incubated at 37 C for 4 h in the presence of purified egg yolk lecithin. Bacillus subtilis, Bacillus cereus, Serratia marcescens, and Pseudomonas aeruginosa hydrolyzed lecithin with the formation of free fatty acids as the sole lipid-soluble product. In none of the Escherichia coli and Citrobacter freundii strains tested could lecithinase activity be detected. Four among eight strains of Enterobacter aerogenes and one among 12 strains of Proteus tested produced negligible amounts of free fatty acid.     

Improved O-serotyping method for Serratia marcescens.

In a previous study, we found that some O serotypes of Serratia marcescens, as defined by agglutination tests, were not based on lipopolysaccharide (LPS) O antigens. We developed a dot enzyme immunoassay with a high degree of LPS specificity and tested 104 distinct clinical strains. Only 7 of the 24 existing O antigens were found in more than one strain: O12/O14 (30.8% of strains examined), O21 (12.5%), O8 (8.7%), O6/O7 (5.8%), O4 (3.8%), O18 (2.9%), and O9 (2.9%). Two new antigens, S1254 (13.5%) and S3255 (3.8%), were also found. Agglutination tests with O antisera identified the LPS antigen in only 36 strains. Prodigiosin production was restricted to serotypes O8, O6, and S3255 and strains with a rough or semirough LPS phenotype. Dot immunoassay appears to offer greater accuracy than agglutination tests for serotype identification in S. marcescens.     

Effects of human and rabbit serum on viability, permeability, and envelope lipids of Serratia marcescens.

The major action of serum on gram-negative organisms is thought to be on the microbial envelope. We compared the effects of normal human and rabbit serum on the envelope lipids of two strains of Serratia marcescens, one sensitive and one resistant to the bactericidal effects of serum. During killing by either serum, the sensitive strain underwent rapid permeability changes coincident with degradation of microbial phospholipids. The resistant strain exhibited none of these effects. The phospholipid degradation that accompanies killing of the sensitive strain by serum could be caused by phospholipases present in serum or by Serratia's own phospholipid-splitting enzymes. The results indicate that phospholipid breakdown is caused by activation of bacterial of bacterial phospholipases and not by serum phospholipases. This conclusion is based upon the following findings.(i1 Although rabbit serum phospholipase A was at least 10 times more active than human serum phospholipase A, phospholipid degradation in the sensitive Serratia strain was comparable during (equally rapid) killing by human or rabbit serum. (ii) Heat treatment (56 C) of both sera eliminated bactericidal activity as well as microbial lipid degradation but abolished phospholipase activity of human serum only. (iii) Virtually complete removal of phospholipase A activity from human serum by adsorption onto autoclaved Micrococcus lysodeikticus had no effect on the extent of phospholipid hydrolysis or on bactericidal activity. Activation by serum of endogenous phospholipase activity in S. marcescens was accompanied by enhanced incorporation of lipid precursors into bacterial lipids. No evidence was found for increased turnover of protein or ribonucleic acid during killing by serum.     

Evidence of in vivo transfer of a plasmid encoding the extended-spectrum beta-lactamase TEM-24 and other resistance factors among different members of the family Enterobacteriaceae

The epidemiological study of several multidrug-resistant Enterobacteriaceae isolated from five patients demonstrated in vivo dissemination of a 100-kb plasmid encoding the extended-spectrum beta-lactamase TEM-24 from a clonal strain of Enterobacter aerogenes to different strains of Klebsiella pneumoniae, Escherichia coli, Proteus vulgaris, Proteus mirabilis, and Serratia marcescens.     

Prevalence of derepressed ampC mutants and extended-spectrum beta-lactamase producers among clinical isolates of Citrobacter freundii, Enterobacter spp., and Serratia marcescens in Korea: dissemination of CTX-M-3, TEM-52, and SHV-12

The resistance mechanism of extended-spectrum cephalosporins in clinical isolates of Citrobacter freundii, Enterobacter spp., and Serratia marcescens was studied. Of 152 isolates, 45 isolates (29.6%) were derepressed AmpC mutants and 39 isolates (25.7%) produced extended-spectrum beta-lactamase (ESBLs). The most prevalent ESBLs were CTX-M enzymes, followed by TEM-52 and SHV-12.     

Carbenicillin resistance of gram-negative bacteria: incidence, biochemical and genetic determinism

Of nine hundred ampicillin resistant (Amp-R) enterobacteria strains, isolated in hospital between July and December 1981, 73,7% are also carbenicillin-resistant (Carb-R). This particular double resistance varies depending upon the species considered: indole positive Proteus (23%), Enterobacter cloacae (64%), Citrobacter freundii (67%), Acinetobacter calcoaceticus (73%), Proteus mirabilis (75%), Serratia marcescens (90%), Escherichia coli (91%), Providencia stuartii (96%) and Klebsiella pneumoniae (100%). The biochemical and genetic basis of resistance to beta-lactamines was studied in 27 strains belonging to these 9 species. A constitutive beta-lactamase was found in all the strains. These enzymes were identified by determination of the isoelectric point on crude sonic extracts, the enzymic activity profile, the inhibition by clavulanic acid and cloxacillin of enzyme activity. Two types of enzymes were predominant: TEM-1 (20 strains) and TEM-2 (7 strains); two strains of Klebsiella pneumoniae produced both SHV-1 and TEM-1. The transfer by conjugation to E. coli K12 of ampicillin and carbenicillin resistance was obtained with 14 strains: (E. coli: 9, C. freundii: 1, K. pneumoniae: 1, E. cloacae: 2, P. stuartii: 1). In all strains but one E. coli we noted the co-transfer of other antibiotic resistance markers.     

Occurrence and characterization of carbapenemase-producing Enterobacteriaceae: report from the SENTRY Antimicrobial Surveillance Program (2000-2004)

Emergence and dissemination of Enterobacteriaceae isolates harboring carbapenemases in various geographic regions represents a significant threat to the management of nosocomial infections. Enterobacteriaceae isolates from the SENTRY Antimicrobial Surveillance Program (2000-2004) demonstrating decreased susceptibility to imipenem and meropenem (minimum inhibitory concentration [MIC], > or =2 mg/L) were evaluated for the production of metallo-beta-lactamases and serine carbapenemases using disk approximation and polymerase chain reaction (PCR) tests. Carbapenemase-producing strains were epidemiologically typed by automated riboprinting and pulsed-field gel electrophoresis (PFGE) to establish clonality. Among 37,557 Enterobacteriaceae (5 genus groups) evaluated, 119 (0.32%) had increased carbapenem MIC values, and a carbapenemase was identified in 51 (42.9%) of these strains. KPC-2 and KPC-3 were the most frequently occurring carbapenemases (24 isolates, 20.2%) in the United States and were detected in Klebsiella spp, Citrobacter spp., Enterobacter spp., and Serratia marcescens strains isolated in New York, Arkansas, and Virginia. SME-2-producing S. marcescens were isolated in the New York City area, Texas, and Ohio, while NMC-A was found in one E. cloacae strain from New York. In contrast, metallo-beta-lactamases were prevalent in Europe. IMP-1-producing E. cloacae (11 isolates) were detected in Turkey, while VIM-1-producing strains were found in Italy (Enterobacter spp.) and Greece (Klebsiella pneumoniae). Clonal dissemination of carbapenemase-producing strains was observed in several medical centers on both continents. The occurrence of carbapenemases in various Enterobacteriaceae remains rare but appears to be spreading geographically (not in Latin America), mainly with metallo-beta-lactamases being found in Mediterranean Europe and KPC enzymes in the New York City area.     

N-acylhomoserine lactone-dependent cell-to-cell communication and social behavior in the genus Serratia

Members of the genus Serratia are increasingly responsible for nosocomial infections, the treatment of which may be complicated by the appearance of multi-antibiotic-resistant strains. Some but not all Serratia strains and species produce N-acylhomoserine lactones (AHLs), and possess luxR and luxI homologous genes. Phylogenetic comparisons have provided evidence for the lateral transfer of these quorum-sensing systems, and in at least one strain of S. marcescens, transfer via a complex transposon has been experimentally demonstrated. AHL-dependent quorum sensing in Serratia controls population surface migration, biofilm development, the biosynthesis of a carbapenem antibiotic and production of the red pigment, prodigiosin. Serratia also possesses LuxS and produces autoinducer-2 (AI-2) which appears to function as a second quorum-sensing system controlling many of the same phenotypes as the LuxR/AHL systems.