Biofilm colonization of Mycobacterium abscessus: New threat in hospital-acquired surgical site infection.

INTRODUCTION: Rapidly growing non-tuberculous mycobacteria (NTM) are hazardous cause of post-operative soft tissue infection leading to nosocomial outbreaks following various surgical procedures, especially laparoscopic surgeries using heat sensitive, non-autoclavable surgical instruments. METHODOLOGY: Surgery department of our hospital noticed increase in rate of post-laparoscopic abdominal port site infection (PSI) and informed the Microbiology Department. A prospective investigational study of defined cases with the aim of source tracing and formulation of infection control measures was initiated. Pus or wound scrapings were collected and processed for aerobic, anaerobic bacteria and Mycobacterium, both by staining and culture. Environmental samples were collected from laparoscopic instruments, and different parts of operation theatre (OT). Mycobacterial isolates were speciated by line probe assay. All the cases were treated with clarithromycin and ofloxacin±amikacin. RESULTS: Among 15 cases of PSI, 11 patients had undergone laparoscopic cholecystectomy, 3 had laparoscopic mesh hernioplasty and one had laparoscopic orchidopexy. Of the 13 pus/discharge specimens examined, 11 revealed growth of NTM. All the isolates were identified as Mycobacterium abscessus by line probe assay. Scraping of biofilm from the disinfectant tray also produced growth of the same organism. Plastic trays used for disinfectants were replaced with metal trays and instructed to do mechanical scrubbing before autoclaving at regular interval. No similar PSI cases were notified after those measures were taken, till date. CONCLUSIONS: This study has shown the need of culture and identification of pathogens causing persistent post surgical wound infections and illuminated importance of rapid source tracing in resource constraint situation which could control outbreak.

Isolation of bacteria from diabetic foot ulcers with special reference to anaerobe isolation by simple two-step combustion technique in candle jar.

BACKGROUND & OBJECTIVES: Although polymicrobial infections involving both aerobic and anaerobic bacteria are very common in diabetic foot ulcers, in many centres of developing countries, anaerobes are rarely isolated due to technical difficulties. This can be overcome by using a new simple, innovative technique of a combination of candle combustion and use of acidified copper-coated steel wool, as reported here. METHODS: In-house developed method was used in a prospective clinico-microbiological study for anaerobes from randomly selected 43 patients with diabetic foot ulcers along with conventional method of anaerobic culture in GasPak system and aerobic culture by standard laboratory procedures. For primary isolation of anaerobes, Brucella blood agar supplemented with hemin (5 µg/ml) and menadione (1 µg/ml) was used. Antibiotic sensitivity tests were performed by the standard disc diffusion method for aerobes and E-test method for anaerobes. RESULTS: All the 43 samples were culture positive, of which aerobic Gram-negative bacteria (GNB) predominated, followed by Staphylococcus aureus, Enterococcus and diphtheroids. Anaerobes isolated from 21 samples were Peptostreptococcus, Bacteroides, Porphyromonas, Veillonella spp. and Clostridium perfringens by both GasPak and in-house developed and modified candle jar techniques. Imipenem and metronidazole were most sensitive while clindamycin, penicillin and cefoxitin were least sensitive drugs for anaerobes. Aerobic GNB were found to be multidrug resistant, especially to penicillin and cephalosporins. The most sensitive drug was piperacillin-tazobactam. INTERPRETATION & CONCLUSIONS: For isolation of anaerobes from clinical specimens such as diabetic foot ulcers, modified candle jar technique was found to be as reliable as GasPak system. This modified technique needs to be tested for many other clinical materials which are not yet evaluated.

Biofilm colonization in chronic treatment refractory infections presenting with discharging sinuses: A study in a tertiary care hospital of Eastern India.

INTRODUCTION: Treatment refractory chronic recurrent infections mean those chronic infections which recur by same causal agents with similar drug responsiveness after apparent relief following full course of recommended antimicrobial management. 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. (1.5%). 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. 88.88%, S. aureus 81.25%, M. abscessus 75%, Citrobacter spp. 83.33%, Proteus spp. 66.66%, E. coli spp. 33.33%, and Enterobacter spp. 0%. 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. S. 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.