W. B. van Leeuwen

Comparison of Staphylococcus aureus Isolates from Bovine and Human Skin, Milking Equipment, and Bovine Milk by Phage Typing, Pulsed-Field Gel Electrophoresis, and Binary Typing

ABSTRACT Staphylococcus aureus isolates (n = 225) from bovine teat skin, human skin, milking equipment, and bovine milk were fingerprinted by pulsed-field gel electrophoresis (PFGE). Strains were compared to assess the role of skin and milking equipment as sources of S. aureus mastitis. PFGE of SmaI-digested genomic DNA identified 24 main types and 17 subtypes among isolates from 43 herds and discriminated between isolates from bovine teat skin and milk. Earlier, phage typing (L. K. Fox, M. Gershmann, D. D. Hancock, and C. T. Hutton, Cornell Vet. 81:183-193, 1991) had failed to discriminate between isolates from skin and milk. Skin isolates from humans belonged to the same pulsotypes as skin isolates from cows. Milking equipment harbored strains from skin as well as strains from milk. We conclude that S. aureus strains from skin and from milk can both be transmitted via the milking machine, but that skin strains are not an important source of intramammary S. aureus infections in dairy cows. A subset of 142 isolates was characterized by binary typing with DNA probes developed for typing of human S. aureus. Typeability and overall concordance with epidemiological data were lower for binary typing than for PFGE while discriminatory powers were similar. Within several PFGE types, binary typing discriminated between main types and subtypes and between isolates from different herds or sources. Thus, binary typing is not suitable as replacement for PFGE but may be useful in combination with PFGE to refine strain differentiation.

Coagulase and protein A polymorphisms do not contribute to persistence of nasal colonisation by Staphylococcus aureus

The nasal carriage rate of Staphylococcus aureus was examined in a longitudinal study of 31 healthy Danish volunteers. Each person was classified as persistent (>8 positive cultures from 10 examinations), an intermittent carrier (50-80% positive cultures) or an ocassional carrier (positive cultures on 10-40% of ocassions only). One hundred and twenty strains from these persons were subjected to phage typing and random amplification of polymorphic DNA (RAPD) analysis. Phage and RAPD typing were in close agreement. RAPD confirmed the spread of a particular S. aureus clone (phage type 95) throughout Denmark. However, no common genotype or phenotype characteristics of S. aureus that could separate persistent from intermittent or incidental colonisers were identified. The immunoglobulin binding protein A and the prothrombin binding coagulase protein are both putative S. aureus virulence or defence factors. Analysis of polymorphisms in the variable repeat regions in the genes for these proteins showed no correlation between the number of repeat units and, consequently, the protein structure with the ability of strains to persist in the human nasal mucosa. The amount of protein A, detectable by its IgG binding activity, appeared not to be correlated to persistence of carriage. Thus protein A and coagulase gene polymorphisms do not seem to play a significant role in the propensity of S. aureus to colonise human nasal epithelium. Furthermore, based on the genetic heterogeneity encountered among the S. aureus strains it is suggested that within the current study population, no single clonal lineage of S. aureus has increased capability to colonise the human nasal epithelium.

Distribution, antibiotic susceptibility and tolerance of bacterial isolates in culture-positive cases of endocarditis in The Netherlands

During a two-year period data were collected nationwide in The Netherlands on 438 episodes of bacterial endocarditis (BE) in 432 patients. Of the strains isolated in these patients 419 were available for analysis. Of these, 326 were isolated in native valve endocarditis (NVE) and 93 in prosthetic valve endocarditis (PVE). Viridans streptococci, staphylococci and enterococci together constituted 87 % of the isolates. More than 46 % of the viridans streptococci consisted ofStreptococcus sanguis. Enterococus faecalis andStaphylococcus aureus were the predominant species in the late form of PVE. The majority of the viridans streptococci and haemolytic streptococci were highly susceptible to penicillin. Five of 35 strains of coagulase negative staphylococci were resistant to methicillin. Eleven percent of a random sample of the streptococci collected were tolerant to penicillin. After repeated exposure to a concentration gradient of an appropriate β-lactam antibiotic, this figure increased to 49 %. Of the staphylococci, 5–6 % of the strains were tolerant before induction and 16–20 % after induction. Of theEnterococcus strains (n=40), 12.5 % showed high-level resistance to one or more aminoglycoside.

Genetic diversification of methicillin-resistant Staphylococcus aureus as a function of prolonged geographic dissemination and as measured by binary typing and other genotyping methods.

The aim of the present study was to determine the extent of genome evolution among methicillin-resistant Staghylococcus aureus (MRSA) strains. Three different collections of strains were analysed, comprising locally, nationally and internationally disseminated genotypes. Various genotyping assays displaying different levels of resolution were used. Geographically and temporally diverse MRSA strains comprised the international group. MRSA strains recovered during an outbreak in a New York City hospital and Portuguese MRSA isolates, all resembling the so-called Iberian clone, were included in the local and national collections, respectively. Genotypes were determined by genome scanning typing techniques and procedures which analyse specific DNA elements only. The outbreak strains showed subclonal variation, whereas the Portuguese isolates displayed an increased number of genotypes. Among the epidemiologically unrelated MRSA strains, the different genotyping techniques revealed a wide heterogeneity of types. Different typing techniques appeared to show different levels of resolution, which could be correlated with the extent of geographic spread; the more pronounced the spread, the higher the degree of genome evolution. Binary typing and randomly amplified polymorphic DNA analysis are the typing methods of choice for determining (non)identity among strains that have a recent common ancestor and have undergone yet limited dissemination.

Simultaneous persistence of methicillin-resistant and methicillin-susceptible clones of Staphylococcus aureus in a neonatal ward of a Warsaw Hospital

Fifty-seven methicillin-resistant Staphylococcus aureus (MRSA) isolates from babies (N = 31), carriers amongst health care workers (N = 16; 10% of all staff members) and the environment (N = 10); 39 MSSA isolates, from babies (N = 18), health care workers (N = 5) and environment (N = 16) were analysed. The strains were from the neonatal ward of a teaching hospital in Warsaw and were collected over a period of 16 months (1993/1994). The isolates were characterized by phage-typing, arbitrary-primed polymerase chain reaction (AP PCR), DNA repeat polymorphism within the protein A gene and the resistance pattern to antimicrobial agents. The presence of the mecA gene was determined by PCR. MRSA were classified as heterogeneously resistant to methicillin, susceptible to other antimicrobial agents and, except for three isolates, appeared to be genotypically almost identical. The first example of mupirocin resistant MRSA in Poland was documented. Amongst MSSA isolates, increased variability was seen, however, the persistence of one predominate clone of MSSA was shown. In this particular hospital environment, several different strains of both MRSA and MSSA were capable of maintaining persistent colonization.

Typing of Staphylococcus aureus isolated from nasal carriers

Eighteen Staphylococcus aureus strains (9 pairs) were isolated from 9 apparently healthy persons (including 5 persons from medical staff) regarded as persistent nasal carriers in a 5-7-day or 3-5-month period, respectively. The 18 S. aureus strains were characterized phenotypically and by genotypic methods. Biochemical properties determined with a commercial identification system, production of haemolysins as well as most of the antibiotic resistance date revealed an identity between both strains of each pair. This putative identity was confirmed for most of the paired strains by phage typing and plasmid profiling for all 9 pairs by determination of the number of repeats in the X region of the spa gene and by macrorestriction analysis of the chromosomal DNA of the isolates. The present results indicate that a single clone of S. aureus might colonize continuously persistent nasal carriers over a 5-7 day period, even for a period of 3-5 months.

External quality assessment of the molecular diagnostics and genotyping of meticillin-resistant Staphylococcus aureus

Two multicentre external quality assessments (EQA) for the molecular detection and genotyping of meticillin-resistant Staphylococcus aureus (MRSA) were arranged. Firstly, 11 samples containing various amounts of inactivated MRSA strains, meticillin-susceptible S. aureus (MSSA), meticillin-resistant coagulase-negative staphylococci (MRCoNS) or Escherichia coli were distributed to 82 laboratories. Samples containing 102 or 103 MRSA cells were correctly scored in only 16 and 46% of the datasets returned, respectively. Two of the used MSSA strains contained an SCCmec cassette lacking the mecA gene. There was a marked difference in the percentage of correct results for these two MSSA strains (37 and 39%) compared to the MSSA strain lacking the SCCmec cassette (88%). Secondly, a panel for MRSA genotyping, consisting of ten samples (two identical, three genetically related and five unique strains) was distributed to 19 laboratories. Seventy-three percent of the datasets recorded all samples correctly. Most pulsed-field gel electrophoresis (PFGE) protocols proved to be suboptimal, resulting in inferior resolution in the higher or lower fragment regions. The performance of molecular diagnostics for MRSA shows no significant changes since our first EQA in 2006. The first molecular typing results are encouraging. Both assessments indicate that programme expansion is required and that major performance discrepancies continue to exist.

Efficacy of ampicillin therapy in experimental listeriosis in mice with impaired T-cell-mediated immune response.

The importance of intact host defense mechanisms for successful antimicrobial therapy was investigated via a comparison of the activities of ampicillin against experimental Listeria monocytogenes infections in normal mice and congenitally athymic (nude) mice. Nude mice were used for these experiments because recovery from infection with this organism depends on development of cellular immunity induced specifically by a T-cell-mediated reaction. When infections ampicillin per mouse (32 doses of 25 mg each), which is twenty times the dose required for a cure of infections in normal mice (8 doses of 5 mg each), would not cure infections in nude mice. With a reduction in inoculum to 10(5) colony-forming units, cures were obtained with a total ampicillin dose of 800 mg (32 doses of 25 mg each), but not with 400 mg (16 doses of 25 mg each). These studies show clearly that the efficacy of ampicillin against infections with L. monocytogenes is dependent upon intact host defense mechanisms.

Degree and stability of tolerance to penicillin inStreptococcus pyogenes

The degree of antibiotic tolerance may be assessed by determining the tolerance percentage of a bacterial strain, defined as the surviving fraction of an inoculum that has been exposed for 24 hours to a high concentration of a β-lactam antibiotic. In 61 clinical isolates ofStreptococcus pyogenes, tolerance percentages ranged from 0 to 0.43. From the slopes of the killing curves it can be deduced that killing starts to be delayed at a tolerance percentage of 0.1. Although a tolerance percentage exceeding 0.1 was observed in 41.4 % of the strains, the incidence of clinically relevant forms of tolerance is expected to occur in a smaller fraction of the strains. Tolerance percentages of two strains stored at 20 °C, 4 °C or −70 °C (tolerance percentages 0.43 and 0.36) decreased to 0.03 or less in six weeks. Tolerance percentages could be completely restored in these strains, but not in sensitive strains, by successive selection for this property on penicillin gradients of increasing concentration. In four strains isolated from a family outbreak, identical levels of tolerance percentage could be selected for with the same technique.

The Role of New Technologies in Medical Microbiological Research and Diagnosis

Microbial culture is exemplified by the Petri dish, a tool that (one century after its invention) still remains one of the “gold standards” for microbiological analysis. However, current trends towards automation, massively paralleled assays, and miniaturization (as well as the observation that we still cannot culture most microorganisms), suggest that new ideas in microbial culture are required. In the Petri dish, nutrient containing agar is typically used as the matrix on which microorganisms are cultured. However, new materials such as nanofibres and nanoporous materials may be better choices as supporting matrixes. Further, emerging techniques in microengineering and the fabrication of low cost materials are helping to create new porous disposables that are of sufficiently low cost that they may be used in the routine microbiology laboratory. These disposables are in turn allowing the development of novel miniature culture methods to take place, methods such as microchemostats, cages for growing microorganisms, and “habitats on a chip”. One particularly useful porous ceramic is Porous Aluminium Oxide (PAO), which can be utilized to generate highly subdivided culture chips that possess up to one million separate, miniaturized, growth areas. Indeed, this material has applications in microbiological diagnostics, microbiological research and industrial microbiology. In this chapter, the applications, advantages, and limitations of porous matrixes and accompanying culture chips will be examined. It is expected that these advances will yield significant improvements in microbial culture when compared to the classical Petri dish.