Bacteriological profile of both aerobic and anaerobic organisms from deep seated abscess.
Deep seated abscess occur at various sites like lung, brain, intra-abdominal, retroperitoneal. Abscess is associated not only with significant morbidity but also mortality. Abscesses are often polymicrobial in nature involving both aerobic and anaerobic infection.
The microbial pathogens, as well as, their antibiotic sensitivity pattern, may change from time to time and place-to-place and with the inadvertent use of antibiotics, the resistant pattern of these organisms has become unpredictable. Therefore, knowledge of the aetiology and current drug resistance pattern of the common pathogenic bacteria in a particular region is useful in clinical practise.
The present study was taken up with an objective to find the prevalence of aerobes and anaerobes in abscesses and to determine their antimicrobial susceptibility pattern.
MATERIAL AND METHODS: A prospective study was conducted in the Department of Microbiology, SVS Medical College & Hospital, Mahabubnagar for a period of 18 months from January 2013 to June 2014.
All the clinically diagnosed patients having deep seated abscesses from various sites with suspected bacterial aetiology, attending the out patient department and those admitted in wards were included. Patients other than deep seated abscesses, causes other than bacterial organisms and patients on antibiotic treatment were excluded.
Collection of Specimen: The specimens were collected aseptically by experienced personnel and were sent to the microbiology department immediately. In brief, the surrounding skin and the area were cleaned with spirit, povidone iodine solution and normal saline with a sterile cotton swab. Specimens were collected in a sterile syringe, following which the tip of the needle sealed with rubber cork and transported to the lab and processed immediately. The aspirated samples taken from each patient was subjected for smear preparation, aerobic and anaerobic culture.
Culture of Aerobic Bacteria: Pus was directly cultured on blood agar and Mac Conkey agar and incubated aerobically at 37[degrees]C for overnight and observed for growth. The organisms were identified by using standard biochemical tests. 
Culture of Anaerobic Bacteria : Samples were inoculated directly on blood agar and Mac Conkey agar and kept in an anaerobic jar (Anaerobic System Mark II LE 002) with Gaspak (Anaerogas Pack 3.5 L LE 002A)(Hi media, Mumbai) at 37[degrees]C for 48 hrs for anaerobic culture.
Samples were also inoculated into Robertson cooked meat broth (RCM) and the inoculated RCM broth was incubated till it was turbid, not earlier than 48 hours. Smears from RCM broth was made and stained with Gram stain. It was then subcultured onto blood agar, Mac Conkey agar plates and incubated anaerobically for 48 hrs at 37[degrees]C.
Identification of Anaerobic Bacteria [3,4]: The anaerobic bacteria are exceptionally difficult to identify up to species level in a routine diagnostic laboratory. In the present study the organisms were identified only to the genus level by using simple and reliable laboratory methods. These include gram staining, colony morphology, pigment production, swarming growth, haemolysis, motility, esculin hydrolysis and spore formation. Antibiotic discs Penicillin (2U), Vancomycin (5[micro]g), Kanamycin (1000[micro]g), Colistin (10[micro]g), Rifampicin (15[micro]g) were also used wherever necessary for identification.
Aerotolerent test was done for all the anaerobically isolated organisms in the anaerobic jar by sub culturing the colony on to an agar plate and incubating aerobically. Only the organisms with no growth aerobically were considered as obligate anaerobes and others were considered as facultative anaerobe.
Antibiotic susceptibility testing was done by Kirby Bauer disc diffusion method for all the aerobic organisms . In Staphylococcus aureus methicillin resistance was detected by using cefoxitin disc (30mcg).  Susceptibility testing for the anaerobic organisms is always a difficult task as there are no disc diffusion guidelines by either CLSI or EUCAST. Routine susceptibility testing for all clinical isolates of anaerobes was not recommended by the current Clinical and Laboratory Standards Institute (CLSI) guidelines and was not done in the present study.
RESULTS: A total of 103 samples of deep seated abscesses obtained during the study period were processed. Out of this 72 (70%) samples have shown growth of either aerobic or anaerobic or mixed growth. The remaining 31(30%) samples have not shown any observable growth and were considered sterile.
The predominant age group from which majority of the samples obtained were in between 16 to 30 years followed by 31-45 yrs, constituting 38 (37%) and 36 (35%) out of 103 respectively. This is followed by the age group 46 - 60yrs 13 (12%) and above 60 yrs 11 (11%).
Out of 72 culture positive samples, in 54 (75%) samples aerobic organisms were isolated, out of these 54, 47 (87%) samples showed growth of only one aerobic organism in pure form and the remaining 7(13%) samples two different aerobic organisms were isolated in culture.
Obligate anaerobic organisms were isolated in 14 (19%) samples out of these, 10 (71%) samples showed growth of single anaerobic organism and the remaining 4 (29%) isolates showed the growth of two anaerobic microorganisms.
In the remaining 4(6%) samples mixed growth containing both aerobic and anaerobic organism was observed.
In these 87 isolates obtained from 72 culture positive samples, 65 were aerobic organisms (75%) and 22 were obligate anaerobes (25%). Among these 65 aerobic organisms gram positive isolates constituted 27 (42%) and gram negative organisms constituted 38 (58%).
The most commonly isolated gram negative organism was E.coli constituting 16 (42%) out of 38 gram negative organisms isolated followed by Klebsiella spp 8 (21%) and Pseudomonas spp 6 (16%). Together, Enterobacteriaceae members were the predominant group isolated constituting 30 (79%) out of 38 gram negative bacteria isolated.
Among gram positive aerobes, Staphylococcus aureus was the predominant organism isolated constituting 19 (70%) out of 27 gram positive isolates. Other gram positive aerobic organisms were of streptococci & enterococci species accounting to 8 (30%) of the gram positive isolates.
A total of 22 obligate anaerobic organisms were isolated in the present study. Among them the gram positive Peptostreptococci spp were the most common organisms isolated from culture constituting 9 (41%) out of 22 isolates. The only other gram positive organism isolated was Clostridium spp with only accounting for 2 (9%) of the total anaerobic isolates.
Among the gram negative anaerobic isolates Bacteriodes spp were the predominant organisms isolated accounting to 5 (23%) of the total anaerobes followed by Fusobacterium spp 4 (18%) and Prevotella spp 2 (9%).
Total of 65 aerobic gram negative bacilli isolated from culture. All these organisms were highly resistant to Amoxyclav (100%), followed by Ceftriaxone (73%) and ciprofloxacin (66%). But all these isolates were sensitive to Imipenem (100%). Resistance to Aminoglycoside antibiotic Amikacin was also considerably low (35%).
Regarding the antimicrobial susceptibilities of the Gram positive aerobic organisms, S. aureus was highly resistant to Ampicillin and Amoxyclav with 100% isolates showing resistance to it. In the present study the MRSA accounted for 9(47%) out of 19 isolates. Resistance to Fluoroquinolones and Macrolides was also very high with 12(63%) and 11(58%) out of 19 being resistant. All the organisms were uniformly sensitive to Vancomycin (with MIC 90 1mcg/ml) and Linezolid. Streptococcus species were also highly sensitive to Vancomycin (100%), Linezolid (100%).
DISCUSSION: Majority of the deep-seated abscesses in present study are obtained from the abdominal region accounting for 28% of the total samples, which correlates with Pramodini et al,  where 31% samples were from same region.
In a review study conducted by Brook et al  on deep abscesses, more than two thirds of the total abscesses are obtained from the abdominal region accounting 585(75%) out of total 778 samples. Out of these 585 abdominal abscesses majority were from retroperitoneal (27%) region.
Isolation of the organisms varies from site to site and also on the methodologies adopted during the study. A total of 103 samples from various deep-seated abscesses were processed. Out of which 72 were culture positive with an isolation rate of 1.2 organisms per sample. This is similar to studies conducted by Pramodini et al  and Saini et al  where average numbers of organisms were 1.45 organisms per sample, which correlate well with our study.
Out of the total 72 culture positives, 57 were in pure culture while the remaining 15 were mixed isolates. These findings are similar to the findings of Saini et al  and Pramodini et al  where single organism and polymicrobial infections accounted for 65% and 35%, 88% and 12% respectively.
In our study, aerobes were isolated to an account of 65 out of 87(74.7%) followed by 22 out of 87(25.2%) anaerobes, similar to Pramodini et al  study where 50 out of 67(74.6%) organisms isolated were aerobes and the remaining 17 were anaerobes (25.4%). In contrast to present study Brook et al  study have recovered predominantly anaerobic organisms. In Brook et al  study, of liver and splenic abscess a total of 116 isolates were obtained out of which only 43 (37%) were aerobic and 73(63%) were anaerobes.
Among aerobic gram positive organisms Staphylococcus aureus was the most common species isolated (29%) in the present study followed by Enterococcus spp (6%).The most common gram negative organism was E.coli (24%) followed by Klebsiella spp (12%). Among anaerobes, Peptostreptococci spp was the most common isolate in present study (41%), followed by Bacteriodes spp which constituted 23%.
In the present study, MRSA accounted for 47% of the Staphylococcus aureus isolates similar to Pramodini et al  where MRSA accounted for 33%. Among gram-negative bacilli resistance to Ceftriaxone was 63% in E. coli and Klebsiella spp, 66% in Citrobacter spp and 100% in Proteus spp. None of the isolates were resistant to Imipenem, making this drug as the most effective drug in the treatment of infections. Other antibiotics that are effective include Amikacin (34%) and Cefoperazone sulbactam (21%) respectively.
Present study findings correlate with many other studies conducted by different authors. In Pramodini et al  study the occurrence of ESBL producing Enterobacteriaceae was about 32.6% with majority of the (47%) Klebsiella spp being resistant to third generation Cephalosporins like Cefotaxime and Ceftazidime. Resistance to Fluoroquinolones like Ciprofloxacin was also quite high in their study accounting for 54% in Klebsiella spp and 46% resistance in E. coli isolates.
The environment of an abscess is detrimental to many antimicrobials. The abscess capsule, the low pH level and the presence of binding proteins or inactivating enzymes such as [beta]-lactamase may impair the activity of many antimicrobial agents. Management of mixed aerobic and anaerobic infections requires surgical correction and drainage of pus and the administration of antimicrobial agents effective against both aerobic and anaerobic bacteria. Without adequate therapy infection will persist.
A careful attempt should be made to identify the causative microorganisms, as many of the isolates are resistance to multiple drugs.
CONCLUSION: Deep-seated abscess are caused by a variety of etiological agents and are often difficult to treat. Identifying the etiological agents and knowing its susceptibility pattern will help in the better management of the patients.
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Sreekanth Basireddy (1), Ribekha Zachariah (2), Manisha Singh (3), Vasanti Kabra (4)
(1.) Sreekanth Basireddy
(2.) Ribekha Zachariah
(3.) Manisha Singh
(4.) Vasanti Kabra
PARTICULARS OF CONTRIBUTORS:
(1.) Assistant Professor, Department of Microbiology, SVS Medical College, Yenugonda, Mahabubnagar, Telangana.
(2.) Tutor, Department of Microbiology, SVS Medical College, Yenugonda, Mahabubnagar, Telangana.
(3.) Professor, Department of Microbiology, SVS Medical College, Yenugonda, Mahabubnagar, Telangana
(4.) Professor & HOD, Department of Microbiology, SVS Medical College, Yenugonda, Mahabubnagar, Telangana.
FINANCIAL OR OTHER COMPETING INTERESTS: None
NAME ADDRESS EMAIL ID OF THE CORRESPONDING AUTHOR:
Dr. Manisha Singh, Professor, Department of Microbiology, SVS Medical College, Yenugonda, Mahabubnagar, Telangana
Date of Submission: 19/06/2015. Date of Peer Review: 20/06/2015. Date of Acceptance: 09/07/2015. Date of Publishing: 14/07/2015.
Table 1: Identification of Anaerobic Gram Negative Bacilli based on Antibiotics RIF PEN (15 KAN (1000 VAN (5 Organism (2U) [micro]g) [micro]g) [micro]g) B. fragilis group R S R R Pigmented group a) Porphyromonas sp V(S/R) S R S b) Prevotella spp V(S/R) S R R Fusobacterium spp S S S R CL (10 Organism [micro]g) B. fragilis group R Pigmented group a) Porphyromonas sp R b) Prevotella spp V(S/R) Fusobacterium spp S Table 2: Antibiotics zone sizes for Anaerobic Gram Negative Bacilli Antibiotics Sensitive Resistance Penicillin [greater than or < 12mm (2 U) equal to] 12mm Vancomycin [greater than or < 10mm (5 [micro]g) equal to] 10mm Kanamycin [greater than or < 12mm (1000 [micro]g) equal to] 12mm Colistin [greater than or < 10mm (10 [micro]g) equal to] 10mm Rifampicin [greater than or < 12mm (15 [micro]g) equal to] 15mm Table 3: Antibiotic resistant pattern of GNB in percentage SI. No. Organisms AMC AK CAZ IMP CIP PIT 1 E.coli 100 25 62.5 0 68.7 34 2 Klebsiella spp 100 37.5 62.5 0 62.5 50 3 Citrobacter spp 100 66.6 66.6 0 66.6 0 4 Proteus spp 100 33.3 100 0 66.6 33.3 5 Pseudomonas spp NA 50 50 0 33.3 0 6 Acinetobacter spp NA 0 100 0 100 50 SI. No. Organisms CTR COT CFS 1 E.coli 62.5 50 31 2 Klebsiella spp 62.5 50 25 3 Citrobacter spp 66.6 100 0 4 Proteus spp 100 100 33.3 5 Pseudomonas spp NA NA 0 6 Acinetobacter spp NA 100 0 Table 4: Antibiotic resistant pattern of GPC in percentage SI. Organism AMP AMC CIP DO E GEN No. 1 S. aureus 100 100 63 52.6 58 47 2 Enterococci spp. 25 25 50 25 75 50 3 Streptococci spp. 0 0 50 50 0 0 SI. CX VA CD LE LZ No. 1 47 0 31.5 63 0 2 -- 0 0 50 0 3 -- 0 0 50 0 Table 5: Comparison of our study with other studies-aerobic (6,8,9,10,11) Itzhak S. Santosh brook Pramodhini Saini et al et al et al (1997) (2012) (2004) Staphylococcus 34 (26%) 19(38%) 16(30%) aureus Enterococci 9 (7%) Streptococci 3(16%) 3(6%) Escherichia coli 19 (15%) 14(28%) 7(13%) Klebsiella spp 3 (2%) 10(20%) 5(9%) Proteus spp 12 (9%) 4(18%) 2(4%) Citrobacter spp -- -- -- Pseudomonas spp 4 (3%) -- 6(11%) Brook and Itzhak Present Finegold brook Study et al et al (2014) (1977) (1999) Staphylococcus 1(4%) 6(10%) 19(29%) aureus Enterococci 4(6%) Streptococci 10 (40%) 3(4%) 4(6%) Escherichia coli 4 (16%) 19(33%) 16(24%) Klebsiella spp 4 (16%) 3(5%) 8(12%) Proteus spp 2 (8%) 2(3%) 3(4%) Citrobacter spp -- 6(10%) 3(4%) Pseudomonas spp 2 (8%) 2(3%) 6(9%) Table 6: Comparison of our study with other studies-anaerobic (6,8,9,10,11) Santosh Itzhakbrook Pramodhini Saini et al (1997) et al (2012) et al (2004) Peptostreptococci 35% (41%) (60%) Clostridium spp 10% -- -- Bacteriodes spp. 16% (59%) -- Fusobacterium spp 10% -- (20%) Prevotella spp 10% -- Brook and Present Finegold Itzhak brook Study et al (1977) et al (1999) (2014) Peptostreptococci (29%) (27%) (41%) Clostridium spp (12%) (12%) (9%) Bacteriodes spp. (26%) (33%) (23%) Fusobacterium spp (6%) (6%) (18%) Prevotella spp (18%) (7%) (9%) Fig 1: Distribution according to sites in percentage Oto facial 22 Respiratory System 19 Abdominal 27 CNS 6 Anal & Perianal 20 Breast 5 Bone 1 Note: Table made from bar graph. Fig 2: Percentage of organism isolated Aerobic 75 Anaerobic 6 Mixed Aerobe and Anaerobe 19 Note: Table made from pie chart.
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|Title Annotation:||ORIGINAL ARTICLE|
|Author:||Basireddy, Sreekanth; Zachariah, Ribekha; Singh, Manisha; Kabra, Vasanti|
|Publication:||Journal of Evolution of Medical and Dental Sciences|
|Date:||Jul 16, 2015|
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