Nuclease overexpression mutants of Serratia marcescens

A family of mutants overexpressing the Serratia marcescens extracellular nuclease has been known for decades. A number of these alleles are characterized here at the molecular level, and the mutant genes are identified, yielding a likely model for their phenotype. The known mutations exert their effect indirectly on nucA expression by elevating the basal SOS response of the cell. Mutations have been found in xerC and uvrD, both of which result in partial SOS induction. A classic nucsu allele, that of strain W1050, is also likely to be in xerC.     

Pathology of Serratia marcescens mastitis in cattle

Microbiological, cytological, histopathological, and immunohistochemical investigations were carried out on four dairy cows affected by Serratia marcescens mastitis. The animals under study were from a herd of 120 lactating cows bred in the province of Rome. In the above herd, S. marcescens mastitis showed a prevalence of 20.8%. S. marcescens was the only bacterial agent isolated, prior to and after slaughter, from the teat milk, the mammary gland and the supramammary lymph nodes of the four cows under study. Cytologically, the four subjects exhibited high cell counts in their milk, with an average of up to 5,570,000 cells/ml in S.marcescens-infected quarters. Macroscopically, nodular lesions were apparent scattered throughout the mammary parenchyma, with enlargement of the regional lymph nodes. Histologically, a chronic, non-purulent mastitis, characterized by a marked fibrous tissue proliferation and the coexistence of corpora amylacea within the glandular alveoli, was observed in association with chronic hyperplastic lymphadenitis involving the supramammary lymph nodes of the four cows. Immunohistochemically, S. marcescens was demonstrated, by means of monoclonal antibodies, both in the mammary gland and in the supramammary lymph nodes from these four animals.     

L-Norvaline and L-homoisoleucine formation by Serratia marcescens,

Two unnatural amino acids were found as by-products in isoleucine fermentation from threonine by Serratia marcescens. These amino acids were identified as L-norvaline (2-aminopentanoic acid) and L-homoisoleucine (2-amino-4-methylhexanoic acid). Formation of L-norvaline and L-homoisoleucine was not observed when L-leucine was added to the medium. The leucine auxotroph derived from the isoleucine accumlator did not produce L-norvaline or L-homoisoleucine even during the accumulation of large amounts of isoleucine. It is suggested that L-norvaline and L-homoisoleucine formation is closely related to leucine biosynthesis.     

The biological properties of Serratia marcescens bacteriophages]

Sixteen phages active against bacteria of the genus Serratia have been divided into 4 groups on the basis of the study of their biological properties. As a result, 5 typing phages have been selected, which permitted the typing of 77.3% of S. marcescens cultures under study, divided into 13 phage types.     

Specific phosphatidylethanolamine dependence of Serratia marcescens cytotoxin activity

A region of dorsomedial frontal cortex (DMFC) has been implicated in planning and executing saccadic eye movements; hence it has been referred to as a supplementary eye field (SEF). Recently, activity related to executing smooth-pursuit eye movements has been recorded from the DMFC, and microstimulation here has been shown to evoke smooth eye movements. This report documents neuronal activity present in smooth-pursuit tasks where the predictability of target motion was manipulated. The activity of many neurons in the DMFC reached a peak when a predictable change in target motion occurred. Furthermore, the peak activity of some cells was systematically shifted by manipulating the duration of the target event, indicating that the network these neurons were in could learn the temporal characteristics of new target motion. Finally, the activity of most neurons tested was greater when target motion was predictable than when it was unpredictable. The results suggest that the DMFC participates in planning smooth-pursuit eye movements based on past stimulus history.     

T-cell specific immunosuppression by prodigiosin isolated from Serratia marcescens

One of the characteristics of obesity-associated diabetes is an elevated fasting plasma insulin concentration with a weak insulin secretory response to subsequent glucose stimulation. Evidence suggests that hyperglycemia and hyperlipidemia may contribute to the initiation and progression of this disordered islet glucose sensing. It has been proposed that reducing hyperglycemia and hyperlipidemia per se may improve islet glucose sensing. Here we studied glucose-dependent insulin release in islets isolated from ob/ob mice treated with dopamine agonists (bromocriptine and SKF38393, BC/SKF) which significantly reduced circulating glucose and lipid levels of ob/ob mice. Islets from BC/SKF-treated mice showed a marked decrease of the elevated basal insulin release to levels similar to lean mice. Such treatment also induced a higher secretory response to glucose stimulation compared with that in ob/ob mice with sustained hyperglycemia and hyperlipidemia. Similarly, when islets from untreated ob/ob mice were cultured for 7 days in 11 mM glucose in the absence of free fatty acid, the basal insulin release was significantly decreased and high glucose stimulated insulin release increased compared with that from islets cultured in medium containing 30 mM glucose and 2 mM oleate. The BC/SKF-induced reduction of elevated basal insulin release was associated with decreased hexokinase activity and basal cyclic AMP content in islet tissue. Our results demonstrate that dopamine agonist treatment improves basal insulin release in ob/ob mice and this effect may be mediated, in part, by a reduction of hyperglycemia and hyperlipidemia.     

Aminoglycoside resistance patterns of Serratia marcescens strains of clinical origin

Aminoglycoside resistance patterns of 147 Serratia marcescens strains of clinical origin were studied. All strains analysed belonged to three different bacterial populations. The periods of study and the institutions the strains were isolated from correlated significantly with the resistance patterns shown by the strains. The most frequent resistance patterns found were the following: ACC (6')-I at the Hospital Infantil de México (Children's Hospital of México), and ANT (2') + AAC(6')-I at the Instituto Nacional de Pediatría (INPed or National Institute of Pediatrics) in Mexico City. Furthermore, the isolation frequency of aminoglycoside-sensitive strains decreased remarkably at the INPed over a 12-year period. These results suggest that there has been a selection of Serratia marcescens strains that are very resistant to aminoglycosides.     

Environmental reservoirs for Serratia marcescens intramammary infections in dairy cows

Via special media, Serratia marcescens isolates were found in 3 bedding pack samples and in 2 milking parlor floor samples, and in milk samples from 19 cows during an episode of mastitis in a dairy cow herd. Chromosomal digest patterns of isolated S marcescens were indistinguishable for 18 of the milk samples and all bedding pack samples. Our findings provide strong evidence that the bedding pack was the reservoir of S marcescens associated with the outbreak of intramammary infections. Additionally, our ability to match digest patterns of isolates in the bedding pack and milk confirms the theory that S marcescens is an environmental pathogen capable of causing mastitis.     

Genetic analysis of the chitinase system of Serratia marcescens 2170

To carry out a genetic analysis of the degradation and utilization of chitin by Serratia marcescens 2170, various Tn5 insertion mutants with characteristic defects in chitinase production were isolated and partially characterized. Prior to the isolation of the mutants, proteins secreted into culture medium in the presence of chitin were analyzed. Four chitinases, A, B, C1, and C2, among other proteins, were detected in the culture supernatant of S. marcescens 2170. All four chitinases and a 21-kDa protein (CBP21) lacking chitinase activity showed chitin binding activity. Cloning and sequencing analysis of the genes encoding chitinases A and B of strain 2170 revealed extensive similarities to those of other strains of S. marcescens described previously. Tn5 insertion mutagenesis of strain 2170 was carried out, and mutants which formed altered clearing zones of colloidal chitin were selected. The obtained mutants were divided into five classes as follows: mutants with (i) no clearing zones, (ii) fuzzy clearing zones, (iii) large clearing zones, (iv) delayed clearing zones, and (v) small clearing zones. Preliminary characterization suggested that some of these mutants have defects in chitinase excretion, a negatively regulating mechanism of chitinase gene expression, an essential factor for chitinase gene expression, and a structural gene for a particular chitinase. These mutants could allow researchers to identify the genes involved in the degradation and utilization of chitin by S. marcescens 2170.     

Epidemiological survey of an outbreak of multiresistant Serratia marcescens by PCR-fingerprinting

During an outbreak of Serratia marcescens from May to November 1993 43 strains obtained from 27 ICU patients infected or colonized with multiresistant S. marcescens were genotypically characterized with random amplified polymerase chain reaction (RAPD-PCR)-fingerprinting. In addition, 43 epidemiologically unrelated control isolates were selected. PCR-fingerprinting identified ten different genotypes of S. marcescens among the outbreak related strains. One predominant genotype was demonstrated in 21/43 isolates of 11/27 patients. A cluster of this genotype was found in seven/eight patients on the cardiosurgical ICU. The epidemiologically unrelated strains all showed different genotypes as compared to the predominant type. This survey proved RAPD-PCR to be a highly discriminatory and reproducible method for epidemiological studies of S. marcescens strains in nosocomial outbreaks.     

Epidemiology of Serratia marcescens between 1987 and 1995 at Vall d'Hebron Hospital

BACKGROUND: Nosocomial infection have a relative high prevalence, which is necessary to reduce in order to improve the quality of patient care. The aims of this work is to study the behaviour of Serratia marcescens in our hospital as between 1987 and 1995. METHODS: Between February 1987 and March 1995 we detected 679 isolates of Serratia marcescens in 504 patients. We used serotype as first marker and phagotype as second, and evaluation of PGFE as a discriminator of doubtful strains was done. RESULTS: 35.8% of the strains were from the respiratory tract, 37.2% from urinary tract; and 11.7% were isolated in blood culture, among them 23.3% came form children younger than 7 years. We noticed a tendency to decrease the number of isolates along the studied period. The most frequent serotypes were O:6, O:3 and O:2; representing the 36.0% of the total. Serotypes O:6;14, O:4, O:5, O:11 and polyagglutinables accounted for 37% of the total. The frequency was variable from year to year, and the predominant serotypes were different in every one of the hospitals in which the center is divided. A 60% of the patients were hospitalized in the General Hospital Vall d'Hebron building, in ICU (20%) and in chirurgical services (25%). Ninety-six patients had more than one isolate, 91 of them (94.8%) can be classified by phenotypic test. The PFGE is discriminatory in three of the five unclassificable isolates. In more than 35% of the patients the strains isolated along the time are different. The 66.7% of the patients acquired Serratia strains in the same admission, and in some cases with few days of difference. We detected 17 cross infections, predominantly in ICUs. With PFGE we could discriminate isolates which produced cross infections between patient who are not in the same hospital. CONCLUSIONS: Although the prevalence of Serratia marcescens is diminishing, it is able to produce crossed infections that, in general, affected few patients. The serotype and phagotype discriminate 94.8% of isolates. The PFGE is high discriminatory and reproducible, only 6.8% of the 44 strains tested by this method can not be typed.     

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.     

An epidemiological study of Serratia marcescens isolates from nosocomial infections by enzyme electrophoresis

Serratia marcescens isolates from 164 patients with suspected nosocomial infection in several hospitals in the greater Paris region were investigated by analysis of the electrophoretically demonstrable allelic variations of gene loci coding for five esterases and five other enzymes. All the loci were polymorphic and the mean number of alleles per locus was 6.1. A total of 72 distinctive electrophoretic types (ETs) representing multilocus genotypes was distinguished. The isolates were divided into two groups according to their resistance to antibiotics: 82 multiresistant isolates (MRI) and 82 relatively susceptible isolates (RSI). Seventy-two MRI (88%) were in four genetically related ETs: ET1, ET2, ET8 and ET9; ET1 was found in 48 isolates, whereas the remaining MRI were in 10 ETs, and all RSI in 61 ETs. Three ETs contained both MRI and RSI. The mean coefficients of genetic diversity for the 10 enzyme loci among ETs and isolates were smaller for MRI than for RSI, while the modal ET of MRI resembled that of RSI. The epidemiological significance of isolates varied according to their ET. Thus, isolates belonging to ET1, ET2 and ET8 were responsible for outbreaks or for sporadic infections, whereas isolates of other ETs were responsible for only sporadic infections. The temporal distribution of ET1 isolates among hospitals identified seven outbreaks in seven clinical departments.     

Production, purification and characterization of a 50-kDa extracellular metalloprotease from Serratia marcescens

The extracellular metalloprotease (SMP 6.1) produced by a soil isolate of Serratia marcescens NRRL B-23112 was purified and characterized. SMP 6.1 was purified from the culture supernatant by ammonium sulfate precipitation, acetone fractional precipitation, and preparative isoelectric focusing. SMP 6.1 has a molecular mass of approximately 50,900 Da by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE). The following substrates were hydrolyzed: casein, bovine serum albumin, and hide powder. SMP 6.1 has the characteristics of a metalloprotease, a pH optimum of 10.0, and a temperature optimum of 42 degrees C. The isoelectric point of the protease is 6.1. Restoration of proteolytic activity by in-gel renaturation after SDS-PAGE indicates a single polypeptide chain. SMP 6.1 is inhibited by EDTA (9 micrograms/ml) and not inhibited by antipain dihydrochloride (120 micrograms/ml), aprotinin (4 micrograms/ml), bestatin (80 micrograms/ml), chymostatin (50 micrograms/ml), E-64 (20 micrograms/ml), leupeptin (4 micrograms/ml), Pefabloc SC (2000 micrograms/ml), pepstatin (4 micrograms/ml), phosphoramidon (660 micrograms/ml), or phenylmethylsulfonyl fluoride (400 micrograms/ml). SMP 6.1 retains full activity in the presence of SDS (1% w/v), Tween-20 (1% w/v), Triton X-100 (1% w/v), ethanol (5% v/v), and 2-mercaptoethanol (0.5% v/v). The extracellular metalloprotease SMP 6.1 differs from the serratiopeptidase (Sigma) produced by S. marcescens ATCC 27117 in the following characteristics: isoelectric point, peptide mapping and nematolytic properties.     

Serratia marcescens S-layer protein is secreted extracellularly via an ATP-binding cassette exporter, the Lip system

The Serratia marcescens Lip exporter belonging to the ATP-binding cassette (ABC) exporter is known to be involved in signal peptide-independent extracellular secretion of a lipase and a metalloprotease. Although the genes of secretory proteins and their ABC exporters are usually all reported to be linked in several gram-negative bacteria, neither the lipase nor the protease gene is located close to the Lip exporter genes, lipBCD. A gene (slaA) located upstream of the lipBCD genes was cloned, revealing that it encodes a polypeptide of 100 kDa and is partially similar to the Caulobacter crescentus paracrystalline cell surface layer (S-layer) protein. The Lip exporter-deficient mutants of S. marcescens failed to secrete the SlaA protein. Electron micrography demonstrated the cell surface layer of S. marcescens. The S-layer protein was secreted to the cultured media in Escherichia coli cells carrying the Lip exporter. Three ABC exporters, Prt, Has and Hly systems, could not allow the S-layer secretion, indicating that the S. marcescens S-layer protein is strictly recognized by the Lip system. This is the first report concerning secretion of an S-layer protein via its own secretion system.     

Cloning and DNA sequence analysis of an aac(3)-Vb gene from Serratia marcescens

The AAC(3)-V resistance mechanism is characterized by high-level resistance to the aminoglycosides gentamicin, netilmicin, 2'-N-ethylnetilmicin, and 6'-N-ethylnetilmicin and moderate resistance levels to tobramycin. Serratia marcescens 82041944 contains an AA(3)-V resistance mechanism as determined from aminoglycoside resistance profiles. This strain, however, does not exhibit hybridization with a probe derived from the previously cloned aac(3)-Va gene, (R. Allmansberger, B. Br?u, and W. Piepersberg, Mol. Gen. Genet. 198:514-520, 1985). High-pressure liquid chromatography analysis of the acetylation products of sisomicin carried out by extracts of S. marcescens 82041944 have demonstrated the presence of an AAC(3) enzyme. We have cloned the gene encoding this acetyltransferase and have designated it aac(3)-Vb. Nucleotide sequence comparisons show that the aac(3)-Va and aac(3)-Vb genes are 72% identical. The predicted AAC(3)-Vb protein is 28,782 Da. Comparisons of the deduced amino acid sequences show 75% identity and 84% similarity between the AAC(3)-Va and AAC(3)-Vb proteins. The use of a DNA fragment internal to the aac(3)-Vb as a hybridization probe demonstrated that the aac(3)-Vb gene is very rare in clinical isolates possessing an AAC(3)-V mechanism.     

Antibiotic resistance and putative virulence factors of Serratia marcescens with respect to O and K serotypes

Recent revision of the O serotyping scheme for Serratia marcescens has allowed the definitive serological identification of a collection of 511 epidemiologically distinct strains in terms of both lipopolysaccharide (O) antigens and capsular (K) antigens. High levels of typability were achieved, 88% and 91% respectively, with only 2% failing to type with either method. In most cases, non-typability was due to a lack of antigen, i.e., the strains produced only rough LPS or were acapsular, suggesting that typability would be little improved by the discovery of additional serotypes. The distribution of the 58 O:K serotypes was very uneven, with O14:K14 accounting for 30% of the 423 clinical strains in the collection, but only 5% of the 88 non-clinical, environmental strains. Thus, the prevalence of O14:K14 strains in hospitals is not reflected in the environment. Similar conclusions were valid for O27:K14, O21:K3 and O21:K14 strains, as well as those with rough lipopolysaccharide. Conversely, the proportions of O6:K3, O6:K14, O8:K14 or O28:K28 strains were significantly lower among the clinical collection than among their environmental counterparts (12% in total rather than 65%). This suggests that O14:K14 may have a selective advantage in colonising or infecting hospitalised patients and, therefore, that the O14 and K14 polysaccharides themselves may contribute towards the apparent pathogenicity of these serotypes.