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Oxacillin Resistance in Staphylococcus Epidermidis Essay (Critical Writing)

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Background and contents of the study

“Staphylococcus epidermidis is a bacterium that is often located on the human skin and is sometimes associated with infections” (5). In most cases, the infection is usually associated with “medical devices and is common in people with a weakened immune system” (7). Morphologically, Staphylococcus epidermidis are tiny bacteria (approximately one micrometer in diameter), gram positive cocci. The cocci are visualized individually under the microscope (5). S. epidermidis grow fast on blood agar and other media that are non selective. As in the case of “S. aureus, S. epidermidis are not hemolytic on blood agar” (4). S. epidermidis colonies are often white or cream colored both on slants and solid cultures. The bacterium does not “synthesize the enzyme coagulase and hence is said to be coagulase negative” (6). The “appropriate nomenclature for the pigmented colonies of Staphylococcus aureus and Staphylococcus albus (now epidermidis) was coined by Rosenbach” (5). “Staphylococcus epidermidis has been since established as a leading cause of bacteremia” (3).

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Coagulase-negative staphylococci (CoNS) have been identified to be significantly associated with bloodstream infections following hematopoietic stem cell transplantation (HSCT). Studies conducted recently have revealed the existence of clonal lineages for CoNS which persist in healthcare settings and can spread between different hospitals. An epidemic staphylococcus epidermidis clone has been identified by multilocus sequence typing studies (MLST). DNA branding patterns can be used as indicators of strain dissimilarities (6). The clone is identified as sequence type ST27 (ST2 according to the new MLST scheme) and is found to be the main cause of the greater part of nosocomial S. epidermidis infections wthin hospital setups. The biofilm generated by S. epidermidis has been identified as the principle factor that facilitates colonization of medical devices by the bacteria. The generation of biofilm can take place via different mechanisms; however, the most common method is via polysaccharide intercellular adhesion/poly-N-acetylglucosamine (PIA/PNAG). A four-gene operon icaADBC, found in various disease causing S. epidermidis strains has been identified as the gene that codes the enzyme facilitating PIA/PNAG synthesis. This study is based on a patient who contracted a staphylococcal infection that persisted for a long period, eventually leading to death. The study sought to find out the association of the epidemic ST27/ST2 clone and examine the genomic instability and the variations in biofilm expression and oxacillin resistance that was observed in the course of the infection.

The samples strains of S. epidermidis were plated on blood agar, different colonies representing different morphotypes selected, analyzed, 16S rRNA sequenced then PCR used to detect the icA and icaA. Biofilm testing was perfomed using the 96-well, polystyrene tissue culture plates. The TSB growth medium was used and S. epidermidis RP62A and staphylococs camosus TM300 used as positive and negative controls, respectively. Treatments with sodium periodate and proteinase K, were used to evaluate if the biofilm was facilitated by PIA production or by proteinase K respectively. In vitro generation of the biofilm-negative variants was done using the Congo red Agar and the frequencies calculated. The genomic DNA for pulsed-field gel electrophoresis (PFGE) was prepared and resolved in a CHEF-DR III apparatus. The MLST was conducted using the revised scheme. SCC mec typing and mecA PCR were carried out according to the description on the manual. For IS256 detection, the chromosomal DNA was isolation was done then EcoRI digested, blotted, and hybridized using IS256-specific gene probes. “Inter –IS256 PCR analysis is porposed as an efficient molecular typing assay for epidemiological studies on S. epidermidis” (2).

Primary culture conducted on the CSF and blood samples showed colonies of different sizes and color. On 16S rNA sequencing, the colonies were identified to be of Staphylococcus epidermidis. PCR detection and antibiotic analysis revealed the presence of both oxacillin-resistant/mecA-positive and oxacillin-susecptible/mecA-negative colonies in blood culture samples of between 13 to 40 days. Later stage isolates were all oxacillin resistant. Typing was conducted for SCCmec and this revealed VI cassette in all isolates, there was however no mecA gene in the oxacillin-sensitive variants. In addition the SCC mec type VI-specific PCR fragments could still be detected in the mecA-negative/oxacillin-sensitive variants. Thus the SCCmec cassette may have been lost partly and the lost part must have contained the mecA gene. The differences in oxacillin resistance were seen to be linked to biolfilm formation. The in vitro biofilm forming tests showed that mecA negative isolates were also biofilm negative and vice versa. This substantiated the observation of ica genes in biofilm positive isolates. However, in the samples obtained early during the infection, ica and mecA-positive colonies were detected together with ica and mecA-negative colonies. Samples collected later in the infection produced much more biofilm( ica positive isolates).

The treatment of the biofilms with proteinase K and metaperiodate revealed that weak biofilms were mediated by proteins while strong biofilms were mediated by PIA production later in the infection. IS256 was detected in all the isolates using the southern hybridization but with aberrations among the different groups. The results seemed to suggest infection by different strains of S epidermidis. However, molecular typing showed similar restriction patterns indicating that the isolates may be having a clonal origin. The absence of a 100-kb DNA fragment in the mecA– and ica-negative isolates was the only difference observed. The clonal origin was shown by both MLST schemes. Analysis by Congo red agar revealed the presence of biofilm positive and negative isolates. Furthermore, PCRs demonstrated a biofilm negative phenotype in the red colonies and lack of ica and mecA genes. The PFGE restriction patterns of the variants were found to be identical to those of the ica– and mecA-negative deletion mutants detected in the patient. Thus the study revealed the instability of the ica and mecA genes as a molecular basis for the observed heterogeneity that is confused with mixed infection by different strains. “Strains with different PFGE patterns are shown to have different susceptibility patterns” (1).

Staphylococcus epidermidis is chiefly a commensal bacterium that is rarely pathogenic and in most cases it is hard to tell whether a particular isolate is the cause of the infection or it is due to contamination. In the above study, the visualization of different colony morphologies indicated a mixed culture. But molecular typing showed that the isolates had a common clonal origin. ST27/ST2 strain that is emerging as an epidemic nosocomial pathogen in medical facilities was identified. According to the findings it can be suggested that the viability and expansive distribution is due to its genetic makeup, whereby most of the strains poses the ica operon and are able to synthesize biofilms. In addition the strains seem to have the ability to acquire and readily exchange mobile genetic elements, such as different SCCmec cassettes and are elements. The S. epidermidis that was detected in the current patient gave rise to different genetic and phenotypic variations. The biofilm formation, which is thought to be S. epidermidis way of coping with environmental variations, is responsible for the difficulty seen in curing the infection. In this study biofilm formation and oxacillin susceptibility were shown as variable characteristics. They were linked to the retention or deletion of the ica and mecA genes.

Both biofilm and resistant variants were present in the beginning of the infection but later, the ica and mecA positive strong biofilm producing variants seemed to outnumber the negative variants. Surprisingly, the ica-positive variants isolated in the beginning of the synthesized protein mediated biofilm and not PIA mediated biofilms, despite the fact that the isolates had icaADBC operon. The icaADBC operons obtained early and later stages of the infection had no significant differences. The molecular explanation for these phenomena is yet to be known and more studies need to be conducted. However, studies conducted in the recent past show that S. epidermidis is able to switch its mode of biofilm formation. The results obtained in this study indicate that this could be of biological importance during the infection process. It is generally believed that PIA-mediated biofilm production offers a selective advantage in established, device mediated infection and is shown to represent a loss of fitness when the isolate colonizes the skin. Studies conducted reveal that PIA producing variants are not required when S. epidermidis colonizes the skin where the bacterium is normally present (4). In this study it was seen that PIA production was absent early in the infection but present later in the infection, thus indicating that PIA production may be dependent on the environmental conditions since all the isolates in both cases had the same genetic makeup.

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This may indicate that PIA production is essential for withstanding the innate immune or effects of the antibiotics. The variants in this study lacked a large DNA fragment that comprises the Ica genes and parts of the SCCmec type VI cassette. The deletions occurred in vitro at a frequency of 10 -2 per cell generation. Normally, S. epidermidis biofilm variations occur at a magnitude of between 10 -5 to 10 -6 per cell generation. Deletion of such large portions of Genomic DNA at this frequency is a new phenomenon whose underlying mechanism is yet to be explained. In this study the deletion was associated with reduced number of IS256 copies. This was also shown in another study with a related strain. In the study, the deletion of the ica genes was associated with IS256 activity. This study shows that specific S. epidemidis clonal lineages may present a great risk to vulnerable patients. Special attention needs to be utilized in the identification of these clonal lineages as they are easily regarded as contaminants. Usually, positive blood culture with CoNS presents a challenge for clinicians and normally, the initiation of antibiotic treatment is usually done when more than one bottle is positive and the patient has a central venous catheter. As revealed by this study, this practice puts the patient at a greater risk. Occurrence of ST27/ST2 strains in hospital settings around the world suggest that the pathogenic potential of these strains should be taken seriously. If the strains are detected early, then patient management can be greatly improved.

Critical Review

The above study does not seek to establish a completely novel idea. Other studies contacted earlier have pointed towards the difficult associated with determining the specific isolates associated with the disease and those that may be just contaminants. This study however, reveals some previously unkown information about Staphylococcus epidermidis. For instance, the switching between protein mediated and PIA mediated biofilm production and the deletion of a large portion of the Genomic DNA even at a high frequency and the possibility that biofilm production is for adaption to the changing environmental conditions. The study is important to medical microbiology as it seems to confirm the various observations that are often labeled on the bacterium. For instance, the difficulty associated with identifying the particular variants associated with the patient’s illness. The study also calls for increased surveillance by microbiologists to identify infections caused by S. epidermidis early enough so that proper treatment intervention can be planned and thus reduce risk to the patient. The righting is clear but the results section is long and includes information that could have been in the discussion or materials and methods parts. The Abstract does not outline the aims and main methods of the study; it seems to jump to the results and conclusions.

Furthermore, the mention of PIA mediated biofilm production in the abstract has not received the same emphasis and weight seen in the main paper. The introduction states the research question but there is no sufficient background for the reader to understand the research question. The methods utilized in the study are appropriate for the research question. However, they are not sufficient for replication of the study by a different microbiologist. There are several instances when the authors are failing to give the details of a method and then followed by a statement “as described earlier”. Some known methods are described in detail for instance, the different colors observed when after staining with Congo red is common knowledge that is repeated severally by the authors. The authors have referenced all the methods used except the new ones developed by them. In the results section, the authors have presented their findings with proper explanation for what was observed.

The results are well represented. However, the ones captured in the text, particularly about biofilm formation seem to be an interpretation of what is in the table. Thus results section, to some extend contains what should have been captured in the discussion section. For instance, the mention that “Proteinase K and metaperiodate treatment demonstrated that weaker biofilms were protein mediated and strong bifilms were PIA mediated” In the discussion section, the authors have put in a gret deal of effort to explain there findings and have related their findings to other studies conducted in the same area. However, there are quite a number of issues the author is concluding that more studies should be carried out to confirm. For instance, the authors have not been able to explain why a large portion of genomic DNA appears to be deleted and yet this is an important factor in reaching to their conclusion. Most of the conclusions drawn by the authors have not been empirically verified and thus more studies need to be conducted before the conclusions can be accepted. Referencing has been done properly and all the tables have all the legends required for clear understanding.

References

Boigiel T., Agnieszka M., Deptuka A., Gospodarek E, and Zaklad K. 2009. Relatedness of methicillin-resistant coagulase-negative staphylococci. Med Dosw Mikrobiol. 61:111-8.

Deplano A., Vaneechoutte M., Verschraegen G., and Struelens MJ. 1997. Typing of staphylococcus aureus and staphylococcus epidermidis strains by PCR analysis of inter-IS256 spacer length polymorphisms. J Clin Microbiol. 35:2580-7.

Fidalgo S., Vazquez F., Mendoza MC, and Perez F. 2009. Bacteremia due to staphylococcus epidermidis: microbiologic, epidemiologic, clinical, and prognostic features. Rev Infect Dis. 12:520-8.

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Fitzpatrick F., Humphery H, and H O’Gara JP. 2005. The genetics of staphylococcal biofilm formation- Will greater understanding of pathogenesis lead to better management of device related infection. Clin Microbiol Infect. 12:967-73.

Goldman, D, and Pier, G. 1993. Pathogenesis of infections related to intravascular catheterization. Clin Microbial Rev. 1993 2: 176-92.

Parisi JT. Coagulase-negative staphylococci and the epidemological typing of staphylococcus epidermidis. Microbiol Rev. 1985. 49: 126-39.

Petters G, and Von Eiff C. 2002. Pathogenesis of infections due to coagulase-negative staphylococci. Lancet infect Dis. 11:677-85.

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