Necrotising soft tissue infections, otherwise known as NSTI’s are brutal infections with the possibility of being fatal (Weese. et al, 2009) and affects a plethora of the living tissues, including all layers of the dermis, adipose tissue and muscle (Campos. et al, 2017). One bacterial species that is able to cause an NSTI is Staphylococcus pseudintermedius, a Gram positive, cocci bacterium, part of the Staphylococcus family, of which there are at least 22 species (Dubois. et al, 2009).
Figure 1. Staphylococcus pseudintermedius stained in crystal violet via Gram Staining (Willows, 2015)
S. pseudintermedius is a single celled bacterium, measuring 1 umhickness and are predominantly found in clusters shown in Figure 1 (Devriese. et al, 2005, Foster, 1996). Many species of Staphylococcus are found on the surface of the animals’ skin but a symptomatic infection is only caused when the bacteria enter the body, this could be from a cut allowing the bacteria enter the blood stream or if the bacteria are ingested from personal grooming or from breastfeeding young. S. pseudintermedius is a leading cause of skin, ear and post-operative infections in dogs and cats (Beale. et al, 2009).
Figure 2. S. pseudintermedius taxonomy (Devriese. et al, 2005)
Staphylococcus bacteria are either coagulase positive postie or negative, with S. pseudintermedius being coagulase positive. DNA-ribosomal RNA hybridisation show that the bacteria have several strains with varying number of alleles (Bannoehr. et al, 2007)
One case study (Campos. et al, 2017) shows a NSTI infection caused by S. pseudintermedius in a six – year – old female beagle. The dog was presented to the veterinary clinic after being found in boarding kennels in lateral decubitus with pale mucous membranes and apathetic. The patients’ clinical signs included intense signs of pain, oedema, erythema, loss of sensation and crepitations stretching from the dorso-lumbar region of the right flank to the mammary glands. On physical examination a small perforated wound was found, 1cm in diameter, possibly obtained during a fight in the kennels. The area surrounding the lesion, however, was inflamed and erythematous. One section, measuring 4cm in diameter, was found to be black in colour and had lost sensation completely. The areas showing oedema, towards the right mammary glands had changed colour and had decreased sensation after being palpated.
Figure 3. Irregularities in skin colour, especially near the right mammary gland indicated by the black arrow (Campos. et al, 2017).
Both normocytic and normochromic anaemia were displayed in the blood samples, and cell cultured biopsies showed necrotising S. pseudintermedius and S. agalacitate, leading to the diagnosis of a Type II NSTI, which is more commonly seen in younger animals. Type I is predominantly seen in older animals with a weaker immune system, the type of infection roughly dictates the treatment protocol or guidelines followed (Surgical Critical Care, 2014). Antibiotic treatment was administered using ceftriaxone, metronidazole and tramadol clorhidrate as an analgesic. Early diagnosis and regular wound cleaning allowed the case to have a good prognosis.
Pathogenicity and diagnosis
Replication and infection
S. pseudintermedius reproduces asexually by binary fission, the way that the majority of bacteria do. In binary fission, one bacterium separates into two new genetically identical cells – the genetic information is duplicated, and each new cell gains one copy (CALS, 2020). Generally, bacteria species have a FtsZ protein that during binary fission, allows the two cells to cleave from one another, but S. pseudintermedius produces a biofilm, a complex extracellular layer of polysaccharide biopolymers, that causes the cells to partially penetrate and stick to one another. This biofilm aids in creating clusters and has a degree of pathogenicity (Lopez. et al, 2017).
Infection can be brought on by the entrance of the bacteria into the bloodstream, in this case was likely to have been through the perforated wound, as S. pseudintermedius has little virulence on the skin surface. Once in the blood stream and tissues and replicates therefore increasing its pathogenicity. As seen in this case, the dog obtained a perforates wound, where the bacteria were most likely to have entered.
Cutaneous fragments of 0.8 cm were collected from the patient, immediately afterwards, purulent and fetid material came from the openings and pus is more often than not indicative of a bacterial infection (Mayo Clinic, 2017). DNA sequencing via PCR is the most reliable method of distinguishing S. pseudintermedius due to its different phenotypic markers (Bannoehr. et al, 2008). The histopathology of the subcutaneous tissue cuts showed areas of prevalent neutrophilic or fibrinous infiltration, diagnosing the patient with necrotizing fibrino-purulent cellulitis. Additionally, PCR sequencing can identify S. pseudintermedius by the presence of the sodA gene (Hiramatsu. et al, 2007).
As the case study suggests, the most recommended diagnostic method is exploratory surgery, with a positive diagnosis being given with the presence of greyish, necrotic tissue, pus and lack of resistance to digital pressure. In some cases, such as this, surgical debridement is deemed unnecessary.
S. pseudintermedius has several virulence factors. The most predominant pathogenic factors in canines is Protein A (Garbacz. et al, 2013), a surface protein with 4 or 5 domains that bind to immunoglobulin G, when the two are bound together, the affected IgG molecules cannot be recognized by neutrophil receptors, allowing the infection to progress and not be targeted by phagocytes.
The bacterium also possesses a gene that produce an exfoliative toxin, known as S. pseudintermedius exfoliative toxin (SIET), this toxin is able to bind to MHC class II, making it less able to initiate an immune response, perpetuating the infection (Langley. et al, 2017). Moreover, the toxin is able to degrade cell walls – causing erythema, exfoliation and crusting of skin. This aids in explaining the patient’s clinical presentation with the discolouration seen on the affected areas.
The production of Enterotoxins produced by S. pseudintermedius can be linked to the erythema and loss of sensation seen in the patient. One of these toxins is. Luk-I, which destroys leukocytes and necrosis of tissues (Garbacz. et al, 2013). These toxins also cause the non-specific activation of polyclonal T cell proliferation and exponential cytokine release, which can cause systemic toxicity- increasing the rates of mortality. The regulation of this toxin is controlled by an expression gene that is upregulated in the initial infection (Futagawa-Saito. et al, 2006)
An additional pathogenic factor of the bacteria is the production of beta-hemolysin. Hemolysins are enzyme that act on lipids within the erythrocyte membrane (Foster, 1996). This causes the tissue to become necrotic as the oxygen supply has been cut off, and the lack resistance in the tissue due to burst cells, in addition to the grey/purple appearance of affected areas displayed in Figure 3.
Another pathogenic enzyme the bacteria produces is DNase, and it is quite potent (Beale, M. et al, 2005). DNase cleaves to DNA in a non-specific manner and breaks down 5’-phosphodiester bonds releasing dinucleotides trinucleotides and oligonucleotides (ThermoFisher, 2001).
One of the factors that causes inflammation in a S. pseudintermedius NSTI is its ability to produce a biofilm. The extracellular protein and polysaccharide matrices that exist in the colonies contribute to inflammatory responses which positively confers with the inflammation seen around the wound site and the surrounding tissue. Interleukin-1 beta and interleukin-6 mRNA are expressed in the grouped colonies and an active TLR signalling pathway is observed on animal derived agars and the Inflammatory response is increased if the relevant coding is upregulated (Arima. et al, 2018).
Figure 4. Biofilm production from cocci bacteria (Singh. et al, 2013).
In the case, once the diagnosis was established, the treatment was as follows. 50 mg/kg IV 12/12h of Ceftriaxone was given alongside 25 mg/kg IV 12/12h of Metronidazole. This treatment was determined by doing an antibiotic susceptibility test by disc diffusion assays. The S. pseudintermedius isolates were found to be susceptible to the following antibiotics: Ceftriaxone, Enrofloxacin, Amoxicillin and Clavulanic Acid, Penicillin, Gentamicin, Sulfamethoxazole and Trimethoprim, and Azithromycin. Metronidazole also used due to the possibility of anaerobic microbes being present, despite these cultures not being collected. The importance of disc diffusion assays is that bacteria are becoming ever more antibiotic resistant and as a common infection, S. pseudintermedius already has a resistant strain, MRSP – methicillin resistant Staphylococcus pseudintermedius, including strains that test positive for Luk-I and SEIT (Ruscher. et al, 2010). The MRSP strains are resistant to beta-lactam antibiotics (The Bella Moss Foundation, 2020). Disc diffusion assays allow clinicians to choose the most effective antibiotic and reduce the risk of increasing resistance.
An additional reason antibiotic disc diffusion assays would have important before deciding on a drug therapy course is that Gram positive bacteria have different susceptibilities to antibiotics due to their cell structure, in conjunction to their ability to produce biofilms.
Gram positive bacteria have a thick outer membrane made up of layers of peptidoglycans, lipoproteins and other molecules (Microbeonlince.com, 2015), including multidrug efflux pumps that lie throughout the outer, as well as inner, membranes of the outer membrane of Gram-positive bacteria. These can pump out antibiotics using a proton-motive force and transport proteins (Kaatsz. & Shcindler. 2016), removing beta-lactams before they are able to take effect. This raises the minimum inhibitory concentrations needed for a drug to be effective (Nikaido. 1998). Beta-lactamases in antibiotics can also be degraded by the bacteria’s enzymes. In addition to having a role in the pathogenicity of S. pseudintermedius, the biofilm also plays a role in the microbes’ susceptibility to treatment: the biofilm produced by the bacteria also contributes to protecting the colonies from antibiotics due to the lipopolysaccharides.
Disc diffusion assays showed susceptibility to Ceftriaxone, so it was used in this case to control and inhibit further infection in the infection by inhibiting cell wall synthesis. Looking at the pharmacodynamics, the active substance in the drug binding to PBP’s, thus inhibiting the final transpeptidation of peptidoglycan in cell walls. This cases the cells to eventually lyse due to arrested cell wall assembly (Ravizzola. et al, 1985)
As well as receiving tramadol as an analgesic, the patient also received wound management, which included thorough cleaning every 12 hours with diluted iodopovidone (0.01%) as well as a silver sulfadiazine cream applied subcutaneously and to lesions, all in combination with compression bandages.
After 15 days, the biochemical test values were at normal levels however there was a high presence of immature and abnormally sized blood cells. After a month of continued treatment, there was considerable clinical improvement, with successful skin recovery and growth and a small area of scar tissue where the necrotized tissue had been and where the biopsy sample was collected. The patient persisted with some signs of anaemia but overall was cleared to discontinue treatment.
Other cases of necrotising bacteria, such as described by Abma et al (2013), proposed that surgery and then the use antibiotics and regular wound cleaning as post-operative care, is the best treatment option as it more effectively eradicates the bacteria from the body and mortality rates are increased without aggressive surgical debridement, However, in this case the attending clinicians decided it would be too much skin and muscle to remove and opted for a non-surgical route. Silver sulfadiazine also possesses immediate bactericidal effects and residual bacteriostatic effects (Atiyeh. et al, 2009). Bactericidal action as well as antibiotic treatment in this instance seemed to prove effective without causing the animal unnecessary pain and discomfort, as well as not compromising their quality of life by prolonged recovery.
It can be assumed that the approach taken in this case study was different to similar cases due to the rapid diagnosis, and the choice not to debride a large area of tissue. Intuitive understanding of the clinical signs prompted the appropriate diagnostic methods, aiding the prognosis of the patient
As well as necrotising tissue, S. pseudintermedius releases toxins that could cause the patient to suffer a systemic toxic shock, if not diagnosed and treated rapidly. As discussed, delayed immune responses from Protein A increase chances of STS occurring. Other methods of diagnosis, such as a urine analysis would be an adequate way to see if the bacteria have become systemic, making it easier to avoid or control a toxic shock occurring.
Conducting diffusion disc assays was a pinnacle part of treating bacterial infections as it reduces the possibilities of bacteria becoming resistant and ensures that the best possible medication is given on the first attempt. By using a bactericidal in conjunction with the antibiotic increases the chances of eradicating the bacteria. Awareness of appropriate antibiotic use was also shown in this case, as one case saw the appearance of MSRP in a site that was previously eradicated of S. pseudintermedius (Mayer. & Rubin, 2012).
As seen in this case study, the patient was subject to prompt diagnostic techniques and appropriate treatment protocols, showing the clinicians understanding of the importance of early and efficient diagnosis. The severity of S. pseudintermedius caused was able to be reduced, resulting in successful eradication of the bacteria and leaving the patient with a good quality of life.
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