|Year : 2016 | Volume
| Issue : 1 | Page : 40-43
Spectrum of anaerobes encountered in surgical infections in a tertiary care hospital in Mangalore, coastal Karnataka: A prospective study
Beena Antony, Sherin Justin, Roshna Raveendran, Anup Kumar Shetty, Thomas S Kuruvilla, Rekha Boloor
Department of Microbiology, Father Muller Medical College, Mangalore, Karnataka, India
|Date of Web Publication||21-Jan-2016|
Department of Microbiology, Father Muller Medical College, Kankanady, District - Dakshina Kannada, Mangalore - 575 002, Karnataka
Source of Support: None, Conflict of Interest: None
Context: Anaerobes are significant pathogens as well as normal flora in a variety of body sites. As the conventional anaerobic culture techniques are laborious, time-consuming and relatively expensive, the interest in anaerobes started narrowing down in the recent past. Objective: This study is aimed at the isolation of bacteria encountered in the surgical infections with an emphasis on anaerobes and to create an awareness among the clinicians regarding the potential role of these silent pathogens Materials and Methods: The present prospective study that was conducted for a period of 1 year, employed 393 specimens collected from various surgical infections. All the samples were subjected for the isolation of both aerobes and anaerobes. Results: Out of 393 specimens, 193 anaerobes and 311 aerobes were obtained. Majority of the cases (226) exhibited polymicrobial etiology. Among the anaerobes, more frequently isolated organisms were nonsporing gram-negative bacilli, such as Bacteroides fragilis group, Prevotella-Porphyromonas group and Fusobacterium, which constitute 68.91% of the cases. Conclusions: This study emphasizes the need to employ anaerobic culture techniques routinely in microbiology laboratories and to create an awareness among the clinicians regarding the potential role of anaerobes.
Keywords: Anaerobes, Bacteroides fragilis group, Prevotella - Porphyromonas group, surgical infections
|How to cite this article:|
Antony B, Justin S, Raveendran R, Shetty AK, Kuruvilla TS, Boloor R. Spectrum of anaerobes encountered in surgical infections in a tertiary care hospital in Mangalore, coastal Karnataka: A prospective study. Muller J Med Sci Res 2016;7:40-3
|How to cite this URL:|
Antony B, Justin S, Raveendran R, Shetty AK, Kuruvilla TS, Boloor R. Spectrum of anaerobes encountered in surgical infections in a tertiary care hospital in Mangalore, coastal Karnataka: A prospective study. Muller J Med Sci Res [serial online] 2016 [cited 2021 Dec 2];7:40-3. Available from: https://www.mjmsr.net/text.asp?2016/7/1/40/174637
| Introduction|| |
Anaerobes are significant pathogens in diverse human clinical infections. They exist as the normal flora in a variety of body sites, especially gastrointestinal tract, urogenital tract and periodontal pockets in humans, which provide a suitable habitat for them due to the low O 2 concentration.  It is not surprising that anaerobes are more frequent than aerobes by 1,000 times in the gut and 5-10 times in the mouth, upper respiratory tract and female lower genital tract.  Though anaerobes live in complete harmony with the host and lead a symbiotic relationship with aerobic commensals, they can become pathogenic when the host's defense mechanisms impair. The review of literature has revealed a well-documented role of these organisms in case of various clinical infections such as intra-abdominal infections, female genital tract infections, orodental infections, brain abscess, and bone and soft tissue infections. ,,,, Generally, these infections are endogenous in origin, except some clostridial infections that are exogenous. Since the anaerobes occur as normal resident flora, these infections are polymicrobial in nature and they act synergistically to cause damage. The pathogenicity of the anaerobes has been attributed to the virulence factors such as capsule, fimbriae, enzymes and metabolic products. 
Conventional anaerobic culture techniques are laborious, time-consuming and relatively expensive. Newer techniques for the presumptive diagnosis of anaerobic infections are quite expensive and not readily available to most of the routine laboratories. The lack of reference centers for anaerobes in India is another hurdle to be crossed. In addition, many technical factors are likely to reduce the frequency of isolation rate of anaerobes. Due to these reasons as well as the recent developments in the field of molecular biology, most of the microbiologists retracted from this topic and hence the anaerobes lost their "charm."
However, increased clinical awareness and inclusion of selective media have given a new identity to these little known pathogens. Though scientific recognition of anaerobic bacteriology had been extended to India during the 1970s, the increased enthusiasm in the subject started narrowing down in the recent past. The present study is aimed at the isolation of anaerobes encountered in the surgical infections in Fr. Muller Medical College and Hospital, Mangalore, for a period of 1 year and to create awareness among the clinicians regarding the potential role of these "silent pathogens."
| Materials and Methods|| |
The present study was conducted on patients admitted in our institute, a tertiary care center of coastal Karnataka, for various surgical problems. A total of 393 samples were processed anaerobically that were collected during surgery. The study lasted for a period of 1 year starting from January 2012 to December 2012. The specimens comprising of wound swabs, necrotic tissue, aspirations, pus were subjected to gram stain prior to the culture. After performing gram stain, the specimen were inoculated directly onto anaerobic media such as Robertson's Cooked Meat Medium (RCM) [Figure 1], Laked Blood Agar (BA), Neomycin BA and Brain Heart Infusion Blood Agar (BHIBA) supplemented with yeast extract (0.5%), cysteine (0.05%), vitamin K (0.1 μg/mL), and hemin (1 μg/mL). A metronidazole disc (5 μg) was placed in the plates for the presumptive identification of anaerobes [Figure 2] and incubated in an anaerobic jar, Mark II Hi Gas Pak jar, a transparent, polycarbonate jar of 3.5 L capacity with sturdy aluminum lid, clamp, and sealing ring with built in safety features (Hi Media Pvt. Ltd, India) [Figure 3]. Anaerogas Pak, LE 002A Hi media was used for achieving anaerobiosis, which is a paper sachet, preserved in the foil bag that absorbs O 2 and generates carbon dioxide, immediately on contact with air. The jar was incubated at 37°C for 48-72 h. After incubation, the jar was opened and the colonies that showed a definite zone of inhibition to metronidazole were presumptively identified as anaerobes. Isolates were further characterized based on aerotolerance, gram stain, colony morphology and biochemical reactions according to the Anaerobic Laboratory Manuals. , RCM, that is a supplemental medium was incubated for 24 h, subcultured onto the anaerobic media as mentioned above and processed in the same way. Simultaneously, the samples were processed for the isolation of aerobes on BA and Mac Conkey's agar and the colonies were identified according to the standard procedures. 
|Figure 1: Robertson's Cooked Meat Medium (RCM) showing saccharolytic property (left), proteolytic property 9 (middle), uninoculated RCM (right)|
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| Results|| |
The specimen collected from various surgical samples that were included during the study is given in [Table 1]. Out of 393 specimen, 336 (85.5%) showed bacterial growth, 57 (14.5%) were sterile, 110 (28.0) cases with single bacterium either aerobe, anaerobe, or microaerophilic organisms. Two hundred and twenty-six specimens (57.5%) were of polymicrobial etiology. The results of the gram stain revealed that the morphological features were in accordance with the cultural characteristics in most of the cases. Polymicrobial nature of the specimen, a supportive evidence for anaerobes and the presence of pus cells were also observed A mixed growth of both aerobes and anaerobes was evident in 106 cases (27 %). The pattern of distribution of organisms is given in [Table 2].
[Table 3] gives the details of anaerobes isolated in the study. Out of 393 specimens, 193 anaerobes were obtained, among these more frequently isolated organisms were nonsporing gram-negative bacilli such as Bacteroides fragilis group, Prevotella-Porphyromonas group, and Fusobacterium, which constitutes 68.91% (i.e., 133 out of 193). Among these, B. fragilis group was the most common isolate [48 (24.87%)]. A total of 311 aerobic bacteria were isolated in the study - Staphylococcus aureus (71), including 16 methticillin resistant Stapylococcus aureus, Pseudomonas aeruginosa (41), Escherichia More Details coli (76), Klebsiella species (30), Citrobacter species (19), Proteus mirabilis (12), Enterococci (13), Microaerophilic Streptococci (15), Acinetobacter baumannii (23), and miscellaneous bacteria (11) constituting coagulase negative Staphylococci (6), diphtheroids (3) and Moraxella More Details species (2).
RCM having anaerobes had a very characteristic feature of foul smell. In many cases, the meat particles exhibited either a saccharolytic or proteolytic property.
| Discussion|| |
The role of anaerobic bacteria as a significant pathogen in human infections is well documented in the literature. Any condition that disrupts the anatomical barrier and permits the entry of normal flora or clinical conditions that produces a low redox potentials, such as surgery, trauma, necrosis, can pave the path to anaerobic infections  Even though anaerobes outnumber aerobes in many clinical settings, the general belief that metronidazole will provide an antibiotic coverage to them, made the clinicians not to give much attention to these troublesome pathogens. However, the emergence of metronidazole resistant anaerobes are reported by various investigators. , Though reports from India regarding metronidazole resistant anaerobes are scanty, all organisms grown anaerobically and resistant to metronidazole were subjected to aerotolerance test (growth at 37°C and in CO 2 ) to rule out the microaerophilic organisms and metronidazole resistant anaerobes.
Technical shortcomings in obtaining appropriate specimens, inadequate anaerobic conditions during transport, laborious culture procedures and resemblance to facultative anaerobes are some of the limiting factors that are likely to reduce the frequency of isolation rate of anaerobes. 
There are many clues for the presence of anaerobes in the specimen such as foul odor, location of infection in proximity to a mucosal surface, presence of gas in specimen, black coloration of the tissue and also the presence of "sulfur" granules. Failure of the previous therapeutic regimens that were not giving effective coverage of anaerobes is another indication for the presence of anaerobes. Samples that yield "No growth" reports in spite of the presence of organisms in the direct smear examination as well as unique morphology resembling anaerobes in the exudates are also valuable indications.  The cultural characteristics of the organisms also predict the presence of the anaerobic bacteria. Characteristic colonies on anaerobic selective media, growth in anaerobic zone of fluid media or agar deeps, zone of inhibition to metronidazole in the primary isolation media are some of them.
As anaerobes exist as normal flora, it is very difficult to assess the clinical significance. However, repeated isolation in some cases, correlation of culture with gram stain morphology, clinical suspicion and associated features will be able to solve this problem. Culture positivity of the anaerobes varies based on the specimen and the collection technique employed. Appropriate specimens are to be collected so as to exclude normal flora. Specimen such as throat swabs, expectorated sputum, feces, vaginal swabs are among those likely to be contaminated with normal flora and hence to be rejected. 
| Conclusion|| |
It was concluded in the present study, constituting 393 surgical infections, 193 anaerobes were isolated. Hence, the large volume of anaerobes that could have been isolated if these techniques were employed routinely can be estimated. The isolation rate might be improved by collecting appropriate specimen, avoiding the delay in transport, quick processing in the laboratory and also the inclusion of appropriate selective media. In brief, this short article strives to add a few more drops to the mighty ocean of anaerobic bacteriology and hopes to serve as a "food for the thought" to the clinicians and the microbiologists.
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Conflicts of Interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3]