Update on clinical significance of coagulase-negative staphylococci.

The clinical significance of coagulase-negative Staphylococcus species (CNS) continues to increase as strategies in medical practice lead to more invasive procedures. Hospitalized patients that are immunocompromised and/or suffering from chronic diseases are the most vulnerable to infection. Since CNS are widespread on the human body and are capable of producing very large populations, distinguishing the etiologic agent(s) from contaminating flora is a serious challenge. For this reason, culture identification should proceed to the species and strain levels. A much stronger case can be made for the identification of a CNS etiologic agent if the same strain is repeatedly isolated from a series of specimens as opposed to the isolation of different strains of one or more species. Strain identity initially can be based on colony morphology, and then one or more molecular approaches can be used to gain information on the genotype. Many of the CNS species are commonly resistant to antibiotics that are being indicated for staphylococcal infections, with the exception of vancomycin. The widespread use of antibiotics in hospitals has provided a reservoir of antibiotic-resistant genes. The main focus on mechanisms of pathogenesis has been with foreign body infections and the role of specific adhesins and slime produced by Staphylococcus epidermidis. Slime can reduce the immune response and opsonophagocytosis, thereby interfering with host defense mechanisms. As we become more aware of the various strategies used by CNS, we will be in a better position to compromise their defense mechanisms and improve treatment.     

An epidemiological assessment of coagulase-negative staphylococci from an intensive care unit.

Detection of an unusual combination of four resistance markers among coagulase-negative staphylococci (CNS) isolated in the same intensive care unit led to the undertaking of an epidemiological assessment. Seventeen CNS isolates from the same unit and 38 epidemiologically unrelated Staphylococcus epidermidis isolates were typed by eight methods, including analysis of immunoblot patterns and hybridisation patterns (HP) obtained with three probes. The probes comprised plasmids carrying the genes encoding 16S rRNA (pBA2), aacA-aphD (pSF815A), and aacA-aphD with part of IS256 (pIP1307). Immunoblot patterns and HP with pIP1307 indicated that 14 of the 17 CNS isolates from the same unit resulted from the spread of an epidemic strain.     

Biotyping coagulase-negative staphylococci.

The biochemical profiles obtained with Staph-Ident (Analytab Products, Plainview, N.Y.) panels were combined with the results of adherence and synergistic hemolysis tests to define biotypes among 1,064 clinical isolates representing eight species of coagulase-negative staphylococci. The 672 isolates of Staphylococcus epidermidis were aligned in 69 of 144 potential biotypes in our scheme because of 18 different biochemical profiles and the eight physiologic subtypes. Isolates of most other species were in fewer biotypes because of more uniform adherence and synergistic hemolysis data, as well as fewer biochemical profiles. Since adherence and synergistic hemolysis may prove to be related to virulence and pathogenicity, biotyping with these test results would help evaluate the reliability of adherence and synergistic hemolysis as possible indices of the clinical significance of some of these organisms. When the antimicrobial susceptibility and plasmid profiles obtained on two clusters of S. epidermidis isolates were compared with the biotyping results, one cluster was not further differentiated by plasmid profiles, but was by antimicrobial profiles; the other cluster with only two biotypes was further divided into five distinct types by plasmid profiles but was not separated at all by antimicrobial profiles.     

Ribotyping of coagulase-negative staphylococci.

The discriminative capacity of ribotyping was initially assessed without knowledge of results obtained for the same isolates by use of more established typing methods. Forty-eight isolates of coagulase-negative staphylococci (CNS) from peritoneal fluids were studied. They were collected prospectively during 31 consecutive episodes of infection associated with peritoneal dialysis in 17 patients. DNA was digested by the restriction endonucleases EcoRI or HindIII and ribotyped by means of a biotinylated cDNA probe to 16S + 23S staphylococcal ribosomal RNA gene sequences. These methods in combination produced a total of 27 types which compared well with numbers of groups distinguished by other typing methods: limited biotype-antibiotic resistogram (ARB; 28), antibiotic resistogram alone (25), API-Staph (12), phage typing (9) and plasmid analysis (22). Ribotyping was highly reproducible and typed all isolates, including those that were not phage-typable (35) or did not contain plasmids (4). When used in a hierarchical manner with ARB, ribotyping results produced 13 additional types in comparison with the other three methods. When used hierarchically with all other typing systems, a further five types were found among isolates from two patients. However, some of the differences observed as a result of ribotyping could have been due to subtle changes produced by mutation, lysogenisation or gene transposition. Since the method requires additional time, expense and technical expertise, it is likely to be useful only when answers to specific epidemiological problems are required or as an initial screen before using other methods of genetic analysis.     

An epidemiological study of blood culture isolates of coagulase-negative staphylococci demonstrating hospital-acquired infection.

We applied pulsed-field gel electrophoresis (PFGE) after SmaI digestion and random amplification of polymorphic DNA (RAPD) analysis with nine oligonucleotide primers to 146 blood culture isolates of Staphylococcus epidermidis and 25 blood culture isolates of Staphylococcus haemolyticus. These were obtained over a 12-month period from patients on the neonatal and hematology units of the Central Manchester Health Care Trust. PFGE demonstrated two clusters of isolates of S. epidermidis (type A and type B) on the neonatal ward and a single cluster (type C) on the hematology unit. Type A was represented by 10 indistinguishable isolates from nine patients, type B was represented by 20 isolates from 14 patients, and type C was represented by 26 isolates from 10 patients. Type A isolates were resistant to chloramphenicol and type C isolates were resistant to ciprofloxacin, mirroring current antibiotic usage. There was no evidence of cross infection due to S. haemolyticus. RAPD analysis, on the basis of a single band difference, produced 58 types of S. epidermidis and 12 types of S. haemolyticus with primer 8 (ATG TAA GCT CCT GGG GAT TCA C; 5' to 3') and 54 types of S. epidermidis and 10 types of S. haemolyticus with primer 9 (AAG TAA GTG ACT GGG GTG AGC G; 5' to 3'). Combining the results confirmed cross infection. Types A, B, and C were concurrently isolated from the hands of the staff of the appropriate unit. Partial control was achieved by withdrawing ciprofloxacin use in the case of the hematology unit and improving hand hygiene in both units.     

Clinical significance of coagulase-negative staphylococci.

Although coagulase-negative staphylococci (C-NS) have been implicated in certain human infections, they are generally regarded as contaminants, and their clinical significance is questioned. To assess their role as pathogens, we studied 205 isolates of C-NS from wounds and body fluids (blood, urine, pleural and peritoneal fluids, etc.). Patient's charts were reviewed, and, by using strict criteria, a determination was made regarding the clinical significance of these isolates. The organisms were then identified to determine whether certain species of C-NS were associated with specific infections. S epidermidis sensu stricto accounted for 81% of the C-NS isolated. The frequencies of other species were: S. haemolyticus (6%), S. hominis (5%), S. capitis (4%), S. warneri (3%), and others (1%). Only two isolates were novobiocin resistant; neither was identified as S. saprophyticus. By using our criteria, 22% of the C-NS were considered to be clinically significant, and the majority of these (93%) was S. epidermidis. The most common source of the clinically relevant C-NS isolates was wounds. These data suggest that identification of C-NS species other than S. epidermidis may be of limited value in predicting clinical significance.     

Bacteriophage typing of coagulase-negative staphylococci.

Cultures comprising the 10 species of coagulase-negative staphylococci proposed by Kloos and Schleifer (J. Clin. Microbiol. 1:82--88, 1975) were typed with bacteriophages isolated from Staphylococcus epidermidis. Although only 10.5% were typable, 50% of those identified as S. epidermidis were typed. Cultures from patients with middle ear infections were also classified by this system and phage typed.     

Speciation of coagulase-negative staphylococci in the clinical laboratory

The purpose of this study was to evaluate the efficacy of the API Staph System for the speciation of coagulase-negative staphylococci. Three hundred and seventy-one coagulase-negative clinical isolates were studied; 50 % of these could be speciated using the code profiles of the API System. By reference to the Kloos and Schleifer schema, 93 % of the isolates could be speciated. The distribution of the various staphylococcal species in clinical specimens was determined. It was concluded that the API Staph System would be a satisfactory method of speciation if the data base could be expanded. Such speciation may at times be helpful in interpreting the significance of coagulase-negative staphylococcal isolates in the clinical laboratory.     

Emergence of teicoplanin-resistant coagulase-negative staphylococci.

Over a period of 5 years we have recovered 32 clinical isolates of coagulase-negative staphylococci (CoNS) exhibiting either decreased levels of susceptibility or true resistance to teicoplanin (MICs, 16 to 128 micrograms/ml); these isolates make up 0.55% of the total CoNS isolated by us. Twenty-nine of the strains were also methicillin resistant, and all were susceptible to vancomycin. Fourteen of the strains were Staphylococcus epidermidis, fourteen were Staphylococcus haemolyticus, and four were Staphylococcus hominis. In one case, a strain of S. haemolyticus was isolated with a vancomycin-resistant, teicoplanin-resistant Enterococcus faecalis strain. All strains were nosocomially acquired and were isolated from 17 different wards. Teicoplanin resistance occurred as a sporadic phenomenon, and none of the isolates were epidemiologically related. The isolates were from 30 patients, 13 of whom presented with true infections (43%). Five (38%) of the 13 patients with true infections had been previously treated with vancomycin. None of the infected patients were previously treated with teicoplanin. The in vivo development of resistance to teicoplanin among CoNS strains limits the therapy of infections by these microorganisms. There is a need for surveillance of nosocomial isolates of CoNS to determine resistance to glycopeptides.     

Rapid, automated identification of novobiocin-resistant, coagulase-negative staphylococci.

A modified automated method that uses the MS-2 system (Abbott Laboratories, Diagnostics Div., Irving, Tex.) to verify the reaction of coagulase-negative staphylococci to novobiocin is described. This technique permits the testing of a great number of specimens in an average time of 99 min and results in a 100% match with the traditional method of culturing.     

Encapsulation of coagulase-negative staphylococci

It is becoming clear that encapsulation is frequent among coagulase negative staphylococci and is unrelated to the formation of extracellular polysaccharide slime by many strains. Crude slime may contain capsular polysaccharides or proteins, as well as cell wall components, but this is probably the result of cell wall turnover in growing bacteria. As in coagulase-positive staphylococci the capsules confer resistance to phagocytosis and can be regarded as important virulence factors. The observation that within the species S. epidermidis several different capsular types can be distinguished serologically suggests the possibility of using the presence and serotype of capsule for biotyping. There is a great need for detailed structural studies of capsular polysaccharides and for the investigation of the role of proteins in the capsules. Several published analyses fail to account for substantial proportions of the weight of isolated capsular material, indicating the presence of components yet to be recognised. Preliminary studies have revealed interesting biological activities of capsular components in humans and experimental animals and further work is likely to provide important new information about the pathogenesis of opportunistic infections by this group of bacteria.     

The evaluation of a typing scheme for coagulase-negative staphylococci suitable for epidemiological studies

A typing scheme was devised for an epidemiological study of infection with coagulase-negative staphylococci in patients undergoing continuous ambulatory peritoneal dialysis (CAPD). The scheme was constructed in four stages suitable for the screening of large numbers of isolates: antibiogram, biotype, phage type, and plasmid profile. The discrimination and reproducibility of the scheme was established by the examination of 50 isolates from 33 consecutive episodes of peritonitis affecting 18 patients. The discrimination of the scheme was 76%, with a reproducibility of 86%. Indistinguishable strains occurred in individual patients only, demonstrating that no cross-infection between patients occurred during the 10-month period of collection of strains, and suggesting that the discriminating power of the scheme was, in fact, much higher. The antibiogram, selected as the first stage of the scheme because it was the simplest and cheapest test, proved to be the most discriminatory stage, providing 60% of the final discriminatory power.     

Isolation of methicillin-resistant coagulase-negative staphylococci from chickens.

Methicillin-resistant coagulase-negative staphylococci were isolated from the nares and skin of 1- to 8-week-old healthy chickens in three flocks from a farm. Isolation of methicillin-resistant coagulase-negative staphylococci was positive for 72 (25.7%) of the 280 chickens tested, with the frequency varying from 2.2 to 100% according to flock. A total of 45 appropriate isolates were selected and subjected to identification. Of the 45 methicillin-resistant coagulase-negative staphylococcal isolates selected, 37 were identified as Staphylococcus sciuri, 5 were identified as Staphylococcus epidermidis, and 3 were identified as Staphylococcus saprophyticus. The distribution of the species was different among the flocks. Comparative analysis of the SmaI-digested chromosomal DNA by pulsed-field gel electrophoresis revealed that the isolates could have originated from a single clone of each of S. sciuri and S. saprophyticus and three clones of S. epidermidis. By two methods based on the PCR technique, the mecA gene was detected in all five representative isolates of each methicillin-resistant coagulase-negative staphylococcal clone. The nucleotide sequence of a PCR fragment obtained from an isolate of S. sciuri was completely identical to the corresponding region of mecA genes reported in human methicillin-resistant Staphylococcus aureus isolates and Staphylococcus epidermidis isolates. The representative methicillin-resistant coagulase-negative staphylococcal isolates were resistant to many beta-lactam antibiotics, and some isolates were also resistant to macrolide and aminoglycoside antibiotics. This is the first evidence of the existence of methicillin-resistant coagulase-negative staphylococci from animals possessing the mecA gene.     

Evaluation of methods for typing coagulase-negative staphylococci.

One hundred and forty-two coagulase-negative staphylococci (CNS) isolated from dialysate effluent or skin of patients receiving continuous ambulatory peritoneal dialysis (CAPD) were typed by extended antibiogram (16 antibiotics) and biotype (26 reactions). These isolates were then typed by supplementary methods to determine the most suitable typing method for an epidemiological study of antibiotic resistance. These included phage typing, reverse phage typing, plasmid typing, whole-cell protein typing by SDS-PAGE with analysis by densitometry, and immunoblotting. The percentage of isolates typed successfully by the supplementary methods were: phage typing 20%, reverse phage typing 0%, plasmid typing 66%, SDS-PAGE 100%, immunoblotting 100%. The discrimination of each method was: phage typing 20%, plasmid typing 37%, SDS-PAGE 69%, immunoblotting 57%. Reproducibility was 88% for phage typing and 97% for plasmid typing. The reproducibility of the whole-cell protein typing was 83% if the same extracts were used but only 43% when separate protein extracts were analysed on separate occasions. However, strain relatedness was highly reproducible. The determination of an antibiogram-biotype profile was not a sufficiently accurate typing method for an epidemiological study of antibiotic resistance. Whole-cell protein typing by SDS-PAGE or immunoblotting was technically demanding but was the most effective of the supplementary methods for detecting erroneous discrimination and false matching produced by antibiogram-biotype combinations.     

Micromethod for biochemical identification of coagulase-negative staphylococci.

We have endeavored to elaborate a suitable method for easy and rapid identification in clinical microbiology laboratories of the different species of infection-inducing, coagulase-negative staphylococci. Ten type strains described by Kloos and Schleifer and 269 strains isolated from 95 patients were tested; the classical tests were used for determination of Staphylococcus species. Strains were identified by using the Kloos-Schleifer reference method and the micromethod simultaneously. After preliminary tests on 77 substrates, 19 were retained, 15 for determination of species and 4 to reveal biotypes. The substrates were placed in wells in a rigid strip of inert plastic. Inoculation of wells was carried out with rich microbial suspensions in a special medium; reading of substrate reactions was done after incubation for 48 h at 35 degree C. The intrasystem reproducibility was excellent, from 91 to 100% for the 19 substrates. It was in excellent agreement with the reference method, 100% for type strains and 97.9% for hospital-isolated strains. Because it is simple and easy to reproduce, the micromethod will be most useful in clinical and ecological microbiology laboratories.     

Phage-typing of coagulase-negative staphylococci. Factors influencing typability

Factors influencing the phage-typability of coagulase-negative Micrococcaceae have been studied in 2,778 clinical isolates comprising A) 209 consecutive isolates from one laboratory, B) 2,107 clinical strains submitted for phage-typing for epidemiological reasons, and C) 462 strains representing all isolates of presumed clinical significance found in two laboratories during one month. The reproducibility was acceptable at duplicate repeated typing of the same strains as well as by typing epidemiologically-related pairs of strains from the same patient. Strains of Staphylococcus haemolyticus were seldom typable, whereas strains of S. epidermidis and S. hominis had a higher typability. Methicillin-resistant strains and other multiple-resistant strains were rarely typable (11-13%). The typability was higher among susceptible strains (36%) and strains resistant to penicillin only (43-50%). The typability of strains of the same species and antibiotic-resistance pattern differed between hospitals compared and decreased markedly over the years for multiple-resistant S. epidermidis isolates.