Ipgmer Kolkata Biochemistry Manual
Institute of Post Graduate Medical Education & Research (S.S.K.M. Hospital) Kolkata West Bengal India. Ipgmer kolkata biochemistry manual. La chase au tresor pour bebe kangourou comment des parents homosexuels forment une famille french edition. Digital voice recorder instructions read online or download pdf olympus vn 4100pc user manual. Aha pals pretest 2013 manual code. View online or download olympus vn 4100pc instructions.
MATERIALS AND METHODS: Fifty different samples were collected from patients with chronic surgical site infections, laparoscopic port site infections, anal fistula, mesh hernioplasty, chronic dacryocystitis, chronic osteomyelitis, and chronic burn wounds. Samples were processed for culture, identification, antibiotic sensitivity testing using standard microbiological techniques. Biofilm (BF) forming capacity for aerobic organisms were tested by tissue culture plate method.
Those for anaerobes and atypical mycobacteria were studied by a novel method using atomic force microscopy (AFM). In vivo BF colonization in lacrimal mucosae of chronic dacryocystitis, patients were studied from histopathological sections by Gram staining, H and E, and fluorescent in situ hybridization (FISH). RESULTS: Out of fifty different samples, sixty-three isolates were obtained in pure culture as follows: Staphylococcus aureus (25.39%), Escherichia coli (14.28%), Klebsiella pneumonia (14.28%), Mycobacterium abscessus (12.69%), Citrobacter spp. (9.52%), Bacteroides fragilis (6.3%), Pseudomonas aeruginosa (4.7%), Proteus spp. (4.7%), Staphylococcus epidermidis (3.1%), Enterobacter spp.
(1.5%), Morganella morganii (1.5%), and Peptostreptococcus spp. Among the isolates, 74% were found to be BF producers in the following frequency: P. Aeruginosa 100%, S. Epidermidis 100%, B. Fragilis 100%, Klebsiella spp.
Aureus 81.25%, M. Abscessus 75%, Citrobacter spp. 83.33%, Proteus spp.
33.33%, and Enterobacter spp. CONCLUSION: AFM has been proven to be a useful method for detection of in vitro grown BF including those for anaerobes and atypical Mycobacteria. In vivo BF detection becomes possible by FISH.
Aureus was the most common isolate. Among the aerobic isolates, P.
Aeruginosa and S. Epidermidis were found to be the most common BF producers. Atypical mycobacteria were also found to be BF producers. Diagnosis of BF s in chronic infections significantly changes the management strategy as these infections can no longer be dealt simply with antibiotics alone but require mechanical removal of the foci along with antibiotic coverage for complete cure.
Introduction About two-third of bacterial infection is caused by biofilm (BF) producer strains. BF-mediated diseases show typical clinical characteristics.
Ipgmer Kolkata Biochemistry Manuals
Materials and Methods A study of 12 months duration was conducted in a Tertiary Care Hospital of Eastern India. During this period, samples were collected from the cases of treatment refractory chronic discharging sinuses which were persistent or recurrent even after full course of sensitive antimicrobials or those cases which underwent good remission but were not cured even after 1 month of empirical antimicrobial therapy. Samples included discharge material or resected tissues from cases of anal fistulae, chronic osteomyelitis, chronic dacryocystitis, chronic SSIs, including laparoscopic port site infections , mesh hernioplasty , etc. According to the Center for Disease Control and Prevention, Atlanta, definition, SSIs are those that develop within 30 days after an operation or within 1 year of placing an implant in situ and the infection appears related to the surgery.
Chronic infections without presentation of discharging sinus, or history of antimicrobial therapy and those received treatment with proved resistant drugs, were not included in the study. Infections other than bacterial origin or by slow growing Mycobacteria or reinfection caused by unrelated/other organisms were also excluded from the study while sampling. Samples collected were processed by direct Gram staining, ZN staining followed by aerobic culture in MacConkey's agar and 5% sheep blood agar (SBA) for 48 h. Bedside inoculation was done on prereduced Brucella blood agar enriched with hemin and menadione for anaerobic samples, and they were incubated in modified candle jar system. Anaerobic indicator Modified methylene blue indicator was used. Clinical specimens showing acid-fast Bacilli on ZN staining and those obtained from SSIs were inoculated on LJ medium besides MacConkey agar and SBA. Fluorescent in situ hybridization protocol for biofilm study (in brief) The slide was fixed in 4% paraformaldehyde solution for 3 h.
Lysozyme enzymatic buffer was added and incubated for 4 h at 45°C. The slide was washed in wash buffer and air dried, followed by incubation in FISH buffer (contains EUB 338 Probe) in a humidity chamber at 55°C for 4 h. Next to it, the slide was washed with wash buffer and air dried and exposed to 10 microliter/ml working concentration of ConA: Alexa fluor488 for 1 h at room temperature followed by Hoechst 33252 stain (2 µg/ml) stain for 1 h at room temperature. All staining were done in dark. Results Out of fifty different samples, sixty-three isolates were obtained in pure culture (15 samples showed growth of two organisms, thirty-three samples showed growth of single organism, and two samples of lacrimal mucosae showed no growth which were processed by FISH) as follows: Staphylococcus aureus (25.39%), Escherichia coli (14.28%), Klebsiella pneumonia (14.28%), M. Abscessus (12.69%), Citrobacter spp.(9.52%), Bacteroides fragilis (6.3%), Pseudomonas aeruginosa (4.7%), Proteus spp (4.7%), Staphylococcus epidermidis (3.1%), Enterobacter spp.
(1.5%), Morganella morganii (1.5%), and Peptostreptococcus spp. Among the isolates, 74% were found to be BF producers in the following frequency: P. Aeruginosa 100%, S. Epidermidis 100%, B. Fragilis 100%, Klebsiella spp. Aureus 81.25%, M.
Abscessus 75%, Citrobacter spp. 83.33%, Proteus spp. 33.33%, Enterobacter spp. Discussion It is a very common clinical experience that in spite of treatment with recommended antibiotics, some of the chronic infections such as anal fistula, chronic dacryocystitis, mesh hernioplasty infections, SSIs, laparoscopic port site infections, osteomyelitis, and chronic nonhealing wounds are known to recur.
This is a common feature of BF due to high resistance persisters within exopolysaccharide substances. Hence, we have reviewed such clinical conditions in the light of BF by in vitro studies.
In the present study, many of the isolates were found to be BF producers by microtiter tissue culture plate method, in vitro grown colony BF study by AFM, and in vivo by H and E, FISH, and Gram-staining. From anal fistulae cases, five anaerobic isolates were obtained by an innovative modified candle jar technique and their BF status were demonstrated for the first time by AFM after colony BF formation applying modified candle jar method. Four among the five isolates were BF producers.
Strong BF producer S. Aureus was isolated in some cases.
Viewing anal fistula in the light of BF will help in effective management. In mesh hernioplasty cases, S. Epidermidis, and M. Abscessus were isolated. Most isolates were strong BF producers. Clinical history of repeated antibiotic treatment was given by patients not resulting in cure. We advocated removal of mesh under antimicrobial coverage and it was found that cure occurred only after removal of the mesh.
Reslinski et al. Reports a case of mesh hernioplasty done for an incisional hernia (postlaparotomy scar) patient wherein the presence of a BF was confirmed. Laparoscopic port site infections showed growth of M. Abscessus and S. Aureus isolates and three out of five M. Abscessus isolates were BF producers. For the first time, we detected BF production by atypical mycobacteria by a novel approach of growing in vitro colony BF on polycarbonate membrane followed by detection of BF status by visualization of characteristic surface topography under atomic force microscope.
Atypical mycobacteria are well known to form BF as survival strategy as found in different studies. From chronic osteomyelitis cases S.
Morganii, and E. Coli were isolated, and all of them were BF producers. Morganii has been a rare inclusion in the list, both as a pathogen for chronic osteomyelitis and a BF producer, as found in this study., Chronic osteomyelitis cases are known to harbor BF. A study group from the United States have reported BF colonization in osteomyelitis of jaw demonstrated by SEM and histopathology. The main principles of osteomyelitis treatment are debridement of the necrotic tissues in a radical manner, filling up of the dead space, and effective long-term antibiotic therapy which is ideal for BF infection.
Frequent investigations and change of antimicrobial therapy should be discouraged. Many cases of chronic nonhealing ulcer with even no history of immunocompromised state and history of treatment with culture sensitive antibiotics did not result in complete cure.
Specimens from these cases showed growth of BF producer isolates of S. Aureus and P. The wounds showed improvement after surgical debridement under prolonged antibiotic treatment which is ideal for BF wound management. In a study by James et al., 60% chronic wounds were found to be BF positive in comparison to 6% acute wounds. Five chronic dacryocystitis cases were studied.
Three of them showed growth of S. Aureus and S.
Asnt level 3 exam questions. Epidermidis which were BF producer (by microtiter plate method). No organism was grown in vitro in two cases but showed lakes of polysaccharide on H and E staining. BF persisters may often not grow in cultures.
However, fluorescent microscopy was done after staining the histological cross section with Alexa (green), hoechst 33252 (blue tissue stain), and eubacterial stain EUB 338 probe (red) for FISH. Red indicated eubacteria inside the BF matrix which was indicated by green alexa under fluorescent microscope.
Gram staining of the histological sections showed colonization by Gram-positive cocci. In a similar study, Oates et al. Demonstrated that putative BF production by colonizing microorganisms in cases of chronic diabetic foot ulcers by FISH. In a study reported by Kosarsoy et al., BF was detected by SEM in 12 out of 14 (85.7%) specimens from lacrimal mucosa of cases of chronic dacryocystitis. Culture-directed optimal antibiotic treatment during acute stages may prevent BF colonization.
Once colonization has occurred, lacrimal syringing, and dacryocystorhinostomy/dacryocystectomy (mechanical removal of focus) remain the main modality of treatment. SSIs (such as postopen cholecystectomy, postlaparotomy, and postmastectomy) were often polymicrobial, and the isolates were BF producers. The polymicrobial nature and potential involvement of BFs in SSIs may be analogous to periodontal disease, where a diverse community of microorganisms acting in consort over time results in a chronic infection. Surgical debridement and removal of leftover suture materials (in two cases only) under antibiotic coverage showed improvement in the cases studied followed by complete cure. In a study report, Kanthju et al.
Showed the presence of Bacilli and cocci within BFs on explanted sutures from a case of chronic SSI by confocal microscopy. As sessile form is not amenable to therapeutic dose of most sensitive drugs and often require mechanical/chemical curing, rational policy for management of such treatment refractory cases should be planned with destabilizing the colonized surface under cover of antibiotic. However, it is preferred to prevent BF like colonization by early intervention while infective agents are still in planktonic form. Summary and Conclusion About 74% of the isolates from chronic discharging sinuses were found to be BF producers. Therefore, chronic discharging sinuses should be viewed in the light of BF, besides other risk factors for chronic infections as the management algorithm would change according to the presence or absence of BF. Antimicrobials tested to be active in vitro, may not be so in vivo, in the face of BF colonization.
Mechanical removal under antimicrobial coverage is still the most reliable treatment option for BF infections.