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Bacteriological profile of both aerobic and anaerobic organisms from deep seated abscess.

INTRODUCTION: Abscess is an accumulation of pus in tissue and it is caused by suppuration deep within a tissue, an organ or confined space. [1] Abscess can occur in any part of body as a superficial infection or deep seated infection associated with any internal organs.

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. [2]

Culture of Anaerobic Bacteria [3]: 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 [5]. In Staphylococcus aureus methicillin resistance was detected by using cefoxitin disc (30mcg). [5] 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, [6] where 31% samples were from same region.

In a review study conducted by Brook et al [7] 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 [6] and Saini et al [8] 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 [8] and Pramodini et al [6] 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 [6] 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 [9] study have recovered predominantly anaerobic organisms. In Brook et al [9] 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 [5] 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 [5] 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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DOI: 10.14260/jemds/2015/1431

REFERENCES:

[1.] Tucker C. Acute and chronic inflammation. In: Cotran RS, Kumar V, Collins T, Robbins SL (editors). Robbins Pathologic Basis of Disease. 6th ed. Philadelphia: WB Saunders Company; 1999, 50-89.

[2.] Forbes BA, Sahm DF, Weissfeld AS. Bailey and Scott's Diagnostic microbiology. Mosby Elsevier. 12th ed. 2007; 842-55.

[3.] Patrick R Murray, Hellen Jo Baron, James H Jorgensen, Marie Louise Landry, Michael A Pfaller. Manual of Clinical Microbiology, 9th ed. Vol I; 2007: 862-932.

[4.] Dianem. Titron, Ian R, Poxton, Hellen Jo Baron. Bacteriodes, Porphyromonas, Prevotella, Fusobacterium and other gram negative rods. Manual of Clinical Microbiology, 9th ed. Vol. I; 2007: Ch. 58, 911-932.

[5.] Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing: Twenty-third Informational Supplment M100-S23. 2013; 72-86.

[6.] Pramodhini S, Thenmozhivalli PR, Selvi R, Dillirani V, Vasumathi A, Agatha D. Bacteriological profile of superficial and deep seated abscesses and their antibiogram in a tertiary care hospital, South India. Indian Journal of Medical Microbiololgy. June 2012, 123-129.

[7.] Brook I. Microbiology of polymicrobial abscesses and implications for therapy. Journal of Antimicrobial Chemotherapy (2002) 50, 805-810.

[8.] Saini S, Gupta N; Aparna, Lokveer, Griwan M.S. Surgical Infection: A Microbiological study.Brazilian Journal of Infectious diseases. 2004; 8(2)118-125.

[9.] Brook I, Frazier E H; Microbiology of liver and spleen abscesses. Journal of Medical Microbiology. 01/1999; 47(12): 1075-80.

[10.] Brook I, Frazier E H. Aerobic and anaerobic bacteriology of perirectal abscesses. Journal Clinical Microbiology.1997; 35: 2974-2997.

[11.] Brook I, Finegold S M and Cherry J D. Bacteriology of aspiration pneumonia in children. Pediatric Research. 1977; 11: 568-5.

Sreekanth Basireddy (1), Ribekha Zachariah (2), Manisha Singh (3), Vasanti Kabra (4)

AUTHORS:

(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

E-mail: drsinghmanisha@gmail.com

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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