Molecular Typing of Staphylococcus epidermidis and Other CNS with Repetitive Element Sequence-Based PCR

The chromosomal distribution of the repetitive DNA sequence found in Mycoplasma pneumoniae (REP-MP2) provides an ideal target for detecting DNA fragment patterns specific to individual Staphylococcus epidermidis and S. haemolyticus strains. A REP-MP2 sequence-based PCR (rep-PCR) was developed and applied to CNS isolates. We identified a 450 bp genomic DNA fragment which was common and specific to S. epidermidis isolates and not found in other CNS. In addition, S. epidermidis isolates showed several bands that could be grouped into 14 different fragment patterns. Similarly, S. haemolyticus isolates were classified into 10 groups. Significant correlations between the typing patterns of S. epidermidis and resistance to oxacillin (P<0.05), gentamicin (P<0.01), erythromycin (P<0.02), and sulfamethoxazole-trimethoprim (P<0.001) were found. The rep-PCR method is a rapid and reproducible discriminatory means for molecular typing of S. epidermidis and other CNS.     

Analysis of pristinamycin-resistant Staphylococcus epidermidis isolates in the Tunisian Bone Marrow Transplant Center

Aims: We report the analysis of genetic determinants conferring resistance to pristinamycin in Staphylococcus epidermidis strains and epidemiology typing of these strains by pulsed‐field gel electrophoresis. Methods and Results: Staphylococcus epidermidis (346 isolates) were searched for strains with pristinamycin resistance. Pristinamycin‐resistant strains (seven isolates) were isolated in five patients with haematological cancer in the Bone Marrow Transplant Centre of Tunisia in 2002. Resistance to pristinamycin was observed in 2% of isolates. The seven pristinamycin‐resistant strains shared resistance to oxacillin (MIC = 8–512 μg ml^?1), gentamicin (MIC = 16–512 μg ml^?1), erythromycin (MIC > 1024 μg ml^?1), lincomycin (MIC > 1024 μg ml^?1), pristinamycin (MIC = 4–16 μg ml^?1) and rifampin (MIC = 128–256 μg ml^?1). erm genes were amplified: ermA from six strains and ermC from one. vga gene encoding streptogramins A resistance (pristinamycin résistance) was amplified from all strains and typed as vgaA by analysis after electrophoresis of restriction profiles of vga amplicons (two fragments with Sau3A of 164 and 378 bp; one fragment with EcoRI). Pulsed‐field gel electrophoresis (PFGE) of SmaI chromosomal DNA digests of the seven S. epidermidis isolates divided them into two distinct pattern types: pulsed‐field type A (classified from A1 to A6 subtypes) and type B. The six strains harbouring ermA genes belonged to the PFGE type A while the strain harbouring ermC genes belonged to the PFGE type B. We characterized an epidemic strain carrying the vgaA and ermA genes responsible for the outbreak. Conclusions: Two clones of pristinamycin‐resistant S. epidermidis were isolated in our patients. One of them, isolated in all patients, had expanded over six months suggesting acquisition by cross‐contamination. Significance and Impact of the study: Increasing isolation of pristinamycin resistant S. epidermidis strains is an alarming indicator of nosocomial dissemination. The vector will be determined to establish a system of epidemiological surveillance.     

Typing of Staphylococcus epidermidis Colonizing in Human Nares by Pulsed-Field Gel Electrophoresis

From the nares of 11 healthy adults, 253 strains of coagulase negative staphylococcus were isolated and 88% of them were identified as Staphylococcus epidermidis using the API STAPH system. Chromosomal DNA fingerprinting of the isolated strains revealed that each person carried multiple types of S. epidermidis in his or her nares. The colonization of the strains was not stable; the types of the isolates changed in the first and the second examinations 5 months apart. The results contrasted with previous findings in which only one strain of S. aureus colonized persistently in the nares of healthy adults.     

Restriction Fragment Length Polymorphism (RFLP) of Genomic DNA of Moraxella (Branhamella) catarrhalis Isolates in a Hospital

Epidemiological typing, based on restriction fragment length polymorphism (RFLP) by pulsed-field gel electrophoresis (PFGE), was attempted for the 38 clinical isolates of Moraxella catarrhalis obtained at Shinshu University Hospital during the years 1987 and 1993. Digestion with SmaI or NotI generated well separable, 12 to 5 genomic DNA fragments ranging from 1,000 kb to 30 kb and the strains could be classified into 14 or 13 types, respectively. The electrophoretic profile differed with the strain in most of them and was hence useful to distinguish the each strain. Investigation for their RFLP have, however, suggested that majority of them, including the type strain ATCC25238, may have derived from a common ancestor.     

Antibiotic resistance and molecular characterization of clinical isolates of methicillin-resistant coagulase-negative staphylococci isolated from bacteremic patients in oncohematology

Polymerase chain reaction (PCR) amplification of antibiotic resistance genes as well as staphylococcal cassette chromosome mec (SCCmec) typing and pulsed-field gel electrophoresis (PFGE) of SmaI macrorestriction fragments of genomic DNA were used to characterize 45 methicillin-resistant coagulase-negative staphylococci (MRCoNS) isolates responsible of bacteremia recovered in patients at the Bone Marrow Transplant Centre of Tunisia in 1998–2007. Among the 45 MRCoNS isolates, Staphylococcus epidermidis was the most prevalent species (75.6%) followed by Staphylococcus haemolyticus (22.2%) and Staphylococcus hominis (2.2%). Extended susceptibility profiles were generated for MRCoNS against 16 antimicrobial agents. Out of 45 mecA-positive strains, 43 (95.6%) were phenotypically methicillin-resistant and two (4.4%) were methicillin-susceptible. The msr(A) was the most prevalent gene (13 isolates; 48.1%) among erythromycin-resistant isolates. The erm(C) was found alone in seven (25.9%) or in combination with both erm(A) and erm(B) in two (7.4%) isolates. The aac(6′)-Ie-aph(2″)-Ia was the most prevalent gene among aminoglycoside-resistant isolates, detected alone in 14 isolates (33.3%) isolates, in combination with ant(4′)-Ia in 18 (42.8%) isolates, in combination with aph(3′)-IIIa in four (9.5%) or with both ant(4′)-Ia and aph(3′)-IIIa in two (4.7%) isolates. The ant(4′)-Ia was detected in three (7.1%) isolates and the aph(3′)-IIIa in one (2.4%) isolate. Among tetracycline-resistant isolates, six (85.7%) strains harbored the tet(K) gene and one (14.3%) strain carried tet(K) and tet(M) genes. SCCmec types IV (31%) and III (24.5%), the most prevalent types detected, were found to be more resistant to non-β-lactam antibiotics. A wide diversity of isolates was observed by PFGE among MRCoNS.     

Evaluation of DNA Fingerprinting by PFGE as an Epidemiologic Tool for Salmonella Infections

To evaluate DNA fingerprinting as an epidemiologic tool, pulsed-field gel electrophoresis (PFGE) was performed on isolates of Salmonella, including S. typhimurium, S. thompson, and S. enteritidis. Chromosomal DNA was digested with the restriction endonucleases Bln I and Xba I. The patterns of S. thompson and S. typhimurium isolates from various sources were different from one another. There was no correlation between the phage type and the digestion pattern of S. enteritidis isolates. Some strains belonging to one phage type were distinguished by their PFGE pattern in this study. These results suggest that the Bln I and Xba I digestion patterns of chromosomal DNA are useful for epidemiological analysis of an outbreak of Salmonella infection or food poisoning.     

Antibiotic resistance patterns of coagulase-negative staphylococcus strains isolated from blood cultures of septicemic patients in Turkey

The aim of this study is to determine antibiotic resistance patterns and slime production characteristics of coagulase-negative Staphylococci (CoNS) caused nosocomial bacteremia. A total of 200 CoNS strains were isolated from blood samples of patients with true bacteremia who were hospitalized in intensive care units and in other departments of Istanbul University Cerrahpasa Medical Hospital between 1999 and 2006. Among 200 CoNS isolates, Staphylococcus epidermidis was the most prevalent species (87) followed by Staphylococcus haemolyticus (23), Staphylococcus hominis (19), Staphylococcus lugdunensis (18), Staphylococcus capitis (15), Staphylococcus xylosus (10), Staphylococcus warneri (8), Staphylococcus saprophyticus (5), Staphylococcus lentus (5), Staphylococcus simulans (4), Staphylococcus chromogenes (3), Staphylococcus cohnii (1), Staphylococcus schleiferi (1), and Staphylococcus auricularis (1). Resistance to methicillin was detected in 67.5% of CoNS isolates. Methicillin-resistant CoNS strains were determined to be more resistant to antibiotics than methicillin-susceptible CoNS strains. Resistance rates of methicillin-resistant and methicillin-susceptible CoNS strains to the antibacterial agents, respectively, were as follows: gentamicin 90% and 17%, erythromycin 80% and 37%, clindamycin 72% and 18%, trimethoprim-sulfamethoxazole 68% and 38%, ciprofloxacin 67% and 23%, tetracycline 60% and 45%, chloramphenicol 56% and 13% and fusidic acid 25% and 15%. None of the strains were resistant to vancomycin and teicoplanin. Slime production was detected in 86 of 200 CoNS strains. Resistance to methicillin was found in 81% of slime-positive and in 57% of slime-negative strains. Our results indicated that there is a high level of resistance to widely used agents in causative methicillin-resistant CoNS strains. However fusidic acid has the smallest resistance ratio, with the exception of glycopeptides. Additionally, most S. epidermidis strains were slime-positive, with statistically significant (p<0.001) association between methicillin resistance and slime production.     

Molecular and Phenotypic Characterization of Staphylococcus epidermidis Isolates from Healthy Conjunctiva and a Comparative Analysis with Isolates from Ocular Infection

Staphylococcus epidermidis is a common commensal of healthy conjunctiva and it can cause endophthalmitis, however its presence in conjunctivitis, keratitis and blepharitis is unknown. Molecular genotyping of S. epidermidis from healthy conjunctiva could provide information about the origin of the strains that infect the eye. In this paper two collections of S. epidermidis were used: one from ocular infection (n = 62), and another from healthy conjunctiva (n = 45). All isolates were genotyped by pulsed field gel electrophoresis (PFGE), multilocus sequence typing (MLST), staphylococcal cassette chromosome mec (SCCmec), detection of the genes icaA, icaD, IS256 and polymorphism type of agr locus. The phenotypic data included biofilm production and antibiotic resistance. The results displayed 61 PFGE types from 107 isolates and they were highly discriminatory. MLST analysis generated a total of 25 STs, of which 11 STs were distributed among the ocular infection isolates and lineage ST2 was the most frequent (48.4%), while 14 STs were present in the healthy conjunctiva isolates and lineage ST5 was the most abundant (24.4%). By means of a principal coordinates analysis (PCoA) and a discriminant analysis (DA) it was found that ocular infection isolates had as discriminant markers agr III or agr II, SCCmec V or SCCmec I, mecA gene, resistance to tobramycin, positive biofilm, and IS256⁺. In contrast to the healthy conjunctiva isolates, the discriminating markers were agr I, and resistance to chloramphenicol, ciprofloxacin, gatifloxacin and oxacillin. The discriminant biomarkers of ocular infection were examined in healthy conjunctiva isolates, and it was found that 3 healthy conjunctiva isolates [two with ST2 and another with ST9] (3/45, 6.66%) had similar genotypic and phenotypic characteristics to ocular infection isolates, therefore a small population from healthy conjunctiva could cause an ocular infection. These data suggest that the healthy conjunctiva isolates do not, in almost all cases, infect the eye due to their large genotypic and phenotypic difference with the ocular infection isolates.     

Molecular detection of enterotoxins E, G, H and I in Staphylococcus aureus and coagulase-negative staphylococci isolated from clinical samples of newborns in Brazil

Aims: The objective of this study was to investigate the detection of SEE, SEG, SEH and SEI in strains of Staphylococcus aureus and coagulase‐negative staphylococci (CNS) using RT‐PCR. Methods and Results: In this study, 90 Staph. aureus strains and 90 CNS strains were analysed by PCR for the detection of genes encoding staphylococcal enterotoxins (SE) E, G, H and I. One or more genes were detected in 54 (60%) Staph. aureus isolates and in 29 (32·2%) CNS isolates. Staphylococcus epidermidis was the most frequently isolated CNS species (n = 64, 71·1%), followed by Staphylococcus warneri (n = 8, 8·9%) and other species (Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus lugdunensis, Staphylococcus simulans, Staphylococcus saprophyticus and Staphylococcus xylosus: n = 18, 20%). The genes studied were detected in Staph. epidermidis, Staph. warneri, Staph. haemolyticus, Staph. hominis, Staph. simulans and Staph. lugdunensis. The highest frequency of genes was observed in Staph. epidermidis and Staph. warneri, a finding indicating differences in the pathogenic potential between CNS species and highlighting the importance of the correct identification of these micro‐organisms. RT‐PCR used for the detection of mRNA revealed the expression of SEG, SEH and/or SEI in 32 (59·3%) of the 90 Staph. aureus isolates, whereas expression of some of these genes was observed in 10 (34·5%) of the 90 CNS isolates. Conclusions: Staphylococcus epidermidis was the most toxigenic CNS species. Among the other species, only Staph. warneri and Staph. lugdunensis presented a positive RT‐PCR result. PCR was efficient in confirming the toxigenic capacity of Staph. aureus and CNS. Significance and Impact of the Study: This study permitted to confirm the toxigenic capacity of CNS to better characterize the pathogenic potential of this group of micro‐organisms. In addition, it permitted the detection of SEG, SEH and SEI, enterotoxins that cannot be detected by commercially available immunological methods.     

A Simple and Practical Method for Typing and Strain Differentiatin of Herpes Simplex Virus Using Infected Cell DNAs

A simple and practical method for typing and strain differentiation of herpes simplex virus (HSV) isolates, based upon analysis of the restriction endonuclease cleavage patterns of viral DNAs, was established by using unlabeled infected cell DNAs. The preparation of infected cell DNA is technically easier than that of purified viral DNA or of radiolabeled viral DNA. The method provides a powerful and practical tool for epidemiological and clinical studies of HSV infection, which can be performed in most diagnostic laboratories. In order to select suitable restriction endonucleases for the study of HSV isolates, the cleavage patterns of viral DNAs (strains MacIntyre, HF, UW-268, and SAV) with 12 enzymes were analyzed. Several enzymes, Bam HI, Kpn I, Pst I, Sal I, Sst I, and Xho I, were found to be useful for both typing and strain differentiation. With this method, HSV isolates from different individuals and from the same individual were analyzed by digestion of their infected cell DNAs with Bam HI. Six isolates from epidemiologically unrelated individuals were readily typed and differentiated from each other. Three isolates from the same individual showed very similar patterns. However, there was a small degree of difference between the first two isolates and the third isolate.     

Molecular Epidemiology of Vibrio cholerae O1 Isolated from Sporadic Cholera Cases in Okinawa,Japan

In July 1994, 6 cholera cases due to Vibrio cholerae O1 El Tor Ogawa sporadically appeared in Okinawa. All 6 patients had no history of traveling abroad. In the period of this cholera outbreak, a strain of V. cholerae O1 El Tor Ogawa was detected from an imported fish at the Naha port quarantine station. The isolates were characterized to clarify whether or not, they belonged to a common clone. Phenotypes were identical except that one strain revealed cured Celebes and the others were original Celebes in kappa phage typing. The restriction fragment patterns of DNA of the isolates hybridized with an enzyme-labeled oligonucleotide probe for cholera toxin gene (ctx) were identical. Randomly amplified polymorphic DNA of the isolates were identical when a primer was used, but 2 patterns were seen when another primer was used. Pulsed-field gel electrophoresis of the chromosomal DNA digested with NotI restriction enzyme showed 3 patterns. The DNA fragment pattern of the strain isolated from the imported fish was different from the clinical isolates. These results suggested that there was no epidemiological relation among the strains of V. cholerae O1 isolated during this period.     

Bacteriocins Pep5 and Epidermin Inhibit Staphylococcus epidermidis Adhesion to Catheters

Pep5 and epidermin bacteriocins were tested on clinical strains of Staphylococcus epidermidis and S. aureus isolated from catheter-related infections. These bacteriocins were inhibitory to several isolates at a concentration of 640 activity units mL⁻¹. The ability of bacteriocins in inhibiting adhesion of S. epidermidis to silicone catheters was evaluated. When Pep5 and epidermin were added to in vitro catheter colonization experiments, there was a significant decrease in the cell number of S. epidermidis adhered to silicone catheters. Bacteriocins used to decrease bacterial attachment to medical devices may represent a novel strategy to control catheter-related infections.     

Molecular typing of Salmonella enterica serovar Sofia in Australia by pulsed‐field gel electrophoresis and repetitive element PCR typing

Aims: In this study, we used two molecular fingerprinting methods to investigate the genetic and clonal relationship shared by Australian Salmonella Sofia isolates. Methods and Results: A total of 84 Australian Salm. Sofia isolates from various states in Australia were typed using pulsed‐field gel electrophoresis (PFGE) (XbaI and SpeI) and repetitive element PCR (REP1R‐I primer). The previous problem of DNA degradation of Salm. Sofia strains was solved by modifying the lysis solution used to treat the bacterial plugs, allowing Salm. Sofia to be subtyped using PFGE. Molecular typing of isolates resulted in the generation of eight XbaI, six SpeI and five REP1 pattern profiles. Individual typing methods showed low discrimination index values (<0·5), indicating the poor discriminatory ability of the methods. However, the combination of the typing methods was able to improve the discrimination of isolates, further dividing them into 16 subtypes and raising the index value to 0·721. Conclusions: The combination of typing methods was shown to be the best approach to fingerprint Salm. Sofia. The Australian Salm. Sofia isolates only showed limited genetic diversity and probably share a clonal relationship. A majority of the Salm. Sofia isolates were not geographically restricted with the predominant pattern subtype observed amongst the isolates from various states. Significance and Impact of the Study: We have successfully devised a PFGE protocol that counteracts DNase activity of Salm. Sofia, enabling typing of this serovar.     

Safety-related properties of staphylococci isolated from food and food environments

Aims: To test some safety‐related properties within 321 staphylococci strains isolated from food and food environments. Methods and Results: The isolates were identified as Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Staphylococcus pasteuri, Staphylococcus sciuri, Staphylococcus warneri and Staphylococcus xylosus. Decarboxylase activity was quite common for the various Staphylococcus spp., and tyrosine was the most frequently decarboxylated amino acid. The frequency of antibiotic resistance was highest in Staph. pasteuri and Staph. xylosus. Several of the isolates were tolerant to QAC compounds, and in some cases, QAC tolerance was present in antibiotic‐resistant strains. Most of the strains displayed moderate to high adhesion rates to stainless steel and Teflon^®. The strains that readily formed biofilms belonged to the species Staph. aureus, Staph. epidermidis and Staph. pasteuri. Conclusions: An high incidence of some safety hazards was found within the staphylococcal strains of food origin tested in this study. In particular, amino acid decarboxylase activity and biofilm‐forming ability were common within strains, and antibiotic resistance and tolerance to QAC‐based compounds occurred frequently as well. These characteristics are an important safety concern for food industry. Significance and Impact of the Study: This work gives a first picture of safety hazards within staphylococcal species isolated from food environments. The presence of disinfectant‐resistant staphylococci is a concern because resistance can be genetically transferred between the various Staphylococcus species. This could lead an increase and spread of resistant enterotoxic staphylococci and/or pathogenic staphylococci.     

Rapid molecular identification and characteristics of <Emphasis Type="Italic">Lactobacillus</Emphasis> strains

Eleven type strains and 24 Lactobacillus isolates, preliminarily classified to the species due to phenotypic features, were investigated. Standard methods of identification with species-specific PCRs and typing with PFGE (with ApaI, NotI and SmaI restriction enzymes) allowed us to distinguish 16 unique strains belonging to 5 species (L. acidophilus, L. delbrueckii ssp. bulgaricus, L. plantarum, L. rhamnosus, L. salivarius). Alternative approach with 16S–23S rDNA ARDRA identification (with merely two restrictases, BsuRI and TaqI) and PCR-based typing (RAPD with two random- and rep-PCR with (GTG)₅ primers) showed to be more discriminative, i.e. 21 unique strains were classified in the same species as above. As a result, 7 out of 24 phenotypically species-assigned isolates were reclassified. The alternative procedure of rapid identification and typing of Lactobacillus isolates appeared to be equally effective and shortened from 1 week to 2–3 d (in comparison to the standard methods).     

Potential Utility of Random Amplified Polymorphic DNA (RAPD) and Restriction Endonuclease Assay (REA) as Typing Systems for Madurella mycetomatis

Two molecular methods were compared, random amplified polymorphic DNA (RAPD) and restriction endonuclease analysis (REA), in order to evaluate their ability to discriminate, and to characterize Madurella mycetomatis strains isolated from human mycetomas in different parts of the world. Both methods were able to cluster the Madurella mycetomatis isolates into the same number of distinct typing groups. However, RAPD, presenting several advantages over REA such as its rapidity, simplicity, and the accessibility for implementation in the laboratory, is a more sensitive and reproducible tool for the study of Madurella mycetomatis epidemiology than REA. Received: 24 March 1999 / Accepted: 24 June 1999     

Restriction Fragment Length Polymorphism Analysis of Epidemiologically Related Mycobacterium tuberculosis Isolates

Restriction fragment length polymorphism (RFLP) analysis of a large number of Japanese isolates of Mycobacterium tuberculosis, containing isolates from small outbreaks of M. tuberculosis infection, and clinical isolates of M. bovis BCG, was carried out using a DNA probe derived from the insertion sequence IS986. Clinical isolates of M. tuberculosis had a high degree of RFLP. The occurrences of the IS element varied from 1 to 19, the majority of isolates having 8 to 15 copies. Very similar fingerprints, however, were seen among strains isolated in the Kanto district. In particular, 3 strains were of the same pattern with or without an additional band. Similarity of the banding patterns of strains islated in the same district was observed in other areas. Six groups of strains, each group arising from a suspected common source of infection, were analyzed. Of these, 5 showed identical fingerprints within each group, but one showed different fingerprints. RFLP patterns of three strains isolated from individuals with lymphadenitis developed about two months after BCG vaccination, and one strain isolated from a bladder cancer patient with BCG instillation therapy were identical to those of BCG-Tokyo which had been used for the vaccination and therapy. These results confirm that RFLP analysis using IS986 is a suitable tool for epidemiology of tuberculosis.     

Antimicrobial resistance and species identification of staphylococci isolated from the meat of wild rabbits (<Emphasis Type="Italic">Oryctolagus cuniculus</Emphasis>) in Slovakia

In 2009, a total of 113 strains of staphylococci were isolated from the thigh muscles of ten hunted and 20 farmed wild rabbits (Oryctolagus cuniculus) in the Slovak Republic. Only two isolates (1.8%) possessed coagulase activity, the rest of 111 staphylococcal isolates were coagulase-negative. Among them, six isolates (5.4%) showed the production of DNase. In each isolate, resistance to eight antibiotics by means of agar dilution test was tested. Based on these results, 110 isolates were found to be resistant to at least one antibiotic. Only one isolate was susceptible to all eight antibiotics tested. Another two isolates were susceptible, however, they showed intermediate susceptibility to cefoxitin. Resistance to ampicillin (78.8%), erythromycin (58.4%), penicillin (51.3%) and oxacillin (46.0%) was found most frequently. Twenty-six isolates (23.0%) were resistant to novobiocin. On the other hand, resistance to cefoxitin (8.0%) and gentamicin (1.8%) were quite rare. Fifteen percent of isolates were resistant to one antibiotic, simultaneous resistance to two, three, four and five antibiotics was confirmed in 22.1%, 23.9%, 21.2% and 13.3% of isolates, respectively. Except for two coagulase-positive Staphylococcus aureus isolates (1.8%), seven species of coagulase-negative staphylococci were identified using the MALDI BioTyper (TM) sytem as follows: Staphylococcus warneri (45.1%), Staphylococcus epidermidis (21.2%), Staphylococcus pasteuri (13.3%), Staphylococcus xylosus (8.0%), Staphylococcus capitis (7.1%), Staphylococcus haemolyticus (1.8%) and Staphylococcus cohnii ssp cohnii (1.8%).     

Enzyme-Linked Lectinsorbent Assay Measures N-Acetyl-D-Glucosamine in Matrix of Biofilm Produced by Staphylococcus epidermidis

An enzyme-linked lectinsorbent assay (ELLA) was developed for quantification of in situ biofilm produced by Staphylococcus epidermidis in polystyrene 96-well tissue culture plates with phosphatase-labeled wheat germ agglutinin (WGA) as a specific probe for the GlcNAcβ-1,4 _n component of exocellular matrix material (EMM) that is responsible for intercellular adhesion and accumulation. The ELLA and the modified Christensen dye assay were used to test 13 laboratory strains of coagulase-negative staphylococci and 10 clinical isolates of S. epidermidis. Four biofilm-positive laboratory strains of S. epidermidis were positive by both tests, and six biofilm-negative strains were negative by both. One strain of S. hemolyticus was positive by the ELLA only. Two of the 10 clinical isolates of S. epidermidis were positive by both assays, two were negative by both, and the remaining were positive by the ELLA only. The ELLA was objective, reproducible, specific, sensitive, and useful for screening strains for their capacity to adhere to plastic, produce EMM, and form biofilm. Received: 15 February 1997 / Accepted: 16 April 1997     

Attachment and Biofilm Forming Capabilities of Staphylococcus epidermidis Strains Isolated from Preterm Infants

Staphylococcus epidermidis, a human commensal, is an important opportunistic, biofilm-forming pathogen and the main cause of late onset sepsis in preterm infants, worldwide. In this study we describe the characteristics of S. epidermidis strains causing late onset (>72 h) bloodstream infection in preterm infants and skin isolates from healthy newborns. Attachment and biofilm formation capability were analyzed in microtiter plates and with transmission electron microscopy (TEM). Clonal relationship among strains was studied with pulsed-field gel electrophoresis. Antimicrobial susceptibility testing was performed, as well as the detection of biofilm-associated genes and of the invasiveness marker IS256 with polymerase chain reaction. Blood and skin isolates had similar attachment and biofilm-forming capabilities and biofilm formation was not related to the presence of specific genes. Filament-like membrane structures were seen by TEM early in the attachment close to the device surface, both in blood and skin strains. Nine of the ten blood isolates contained the IS256 and were also resistant to methicillin and gentamicin in contrast to skin strains. S. epidermidis strains causing bloodstream infection in preterm infants exhibit higher antibiotic resistance and are provided with an invasive genetic equipment compared to skin commensal strains. Adhesion capability to a device surface seems to involve bacterial membrane filaments.