|Year : 2015 | Volume
| Issue : 5 | Page : 53-58
A method for early detection of antibiotic resistance in positive blood cultures: Experience from an oncology centre in eastern India
G Goel1, D Das1, S Mukherjee2, S Bose2, K Das2, R Mahato2, S Bhattacharya3
1 Department of Clinical Microbiology, Tata Medical Center, Kolkata, West Bengal, India
2 Laboratory Technologist in Microbiology, Tata Medical Center, Kolkata, West Bengal, India
3 Consultant Microbiologist, Tata Medical Center, Kolkata, West Bengal, India
|Date of Submission||05-Dec-2013|
|Date of Acceptance||05-Jun-2014|
|Date of Web Publication||6-Feb-2015|
Consultant Microbiologist, Tata Medical Center, Kolkata, West Bengal
Source of Support: None, Conflict of Interest: None
Purpose: For antibiotic susceptibility results, conventional culture and sensitivity methods takes 48 hours after a blood culture is flagged positive by automated systems. Early initiation of targeted antibiotic therapy is essential for effective management of sepsis to reduce morbidity, mortality, cost of treatment and prevent antibiotic resistance. Objective of this study was to evaluate Direct Sensitivity Test (DST) as a potential tool to get reliable antibiotic susceptibility results 24 hours earlier. Materials and Methods: Blood cultures flagged positive between May 2011 to December 2012 by BacT/ALERT were Gram stained. All uni-microbial gram-negative blood cultures were simultaneously cultured and processed for DST from broth using disk diffusion method using British Society of Antimicrobial Chemotherapy (BSAC) guidelines. DST results available next day were compared with conventional antibiotic susceptibility test (AST) performed by Vitek-2 on isolated colonies. Results of DST (test method) and AST (reference method) were compared for agreements or errors. Results: Of the 840 antibiotic gram-negative organism combinations tested, Categorical and essential agreements were 83.7% and 96.2% respectively. Minor, major and very major errors were 12.5%, 3.33% and 0.47%, respectively. Conclusions: DST using disk diffusion from positive blood culture broths helps to initiate early targeted antibiotic therapy. There is high concordance between DST and AST.
Keywords: Antibiotic resistance, blood culture, direct sensitivity, inoculum size, septicemia
|How to cite this article:|
Goel G, Das D, Mukherjee S, Bose S, Das K, Mahato R, Bhattacharya S. A method for early detection of antibiotic resistance in positive blood cultures: Experience from an oncology centre in eastern India. Indian J Med Microbiol 2015;33, Suppl S1:53-8
|How to cite this URL:|
Goel G, Das D, Mukherjee S, Bose S, Das K, Mahato R, Bhattacharya S. A method for early detection of antibiotic resistance in positive blood cultures: Experience from an oncology centre in eastern India. Indian J Med Microbiol [serial online] 2015 [cited 2019 Sep 15];33, Suppl S1:53-8. Available from: http://www.ijmm.org/text.asp?2015/33/5/53/150883
| ~ Introduction|| |
Sepsis is one of the major causes of mortality and morbidity in hospitals. Bloodstream infections affect approximately 2% of all hospitalised patients and up to 70% patients admitted in the Intensive Care Unit. ,. Mortality is high, ranging from 14% to 57%.  Early administration of adequate antibiotic therapy has been shown to reduce mortality , and has a positive impact on the outcome of bacteraemic patients. , It can reduce costs and may prevent development of bacterial resistance. ,
Kumar et al., demonstrated an increase in mortality of 7.6% for every hour by which antimicrobials were delayed in septic shock.  The Surviving Sepsis Campaign's 2008 "International guidelines for the management of severe sepsis and septic shock" also recommend that appropriate antimicrobial therapy be administered within 1 hour of recognition of severe sepsis or septic shock. 
The standard protocol for positive blood cultures involves Gram stain and subculture of blood culture broth onto solid media and overnight incubation to obtain isolated colonies, which are then used to make a standardized inoculum used for antibiotic susceptibility test conventional antimicrobial susceptibility testing (AST). Conventional methods of blood culture take 48 hours to get antibiotic susceptibility test results even after a blood culture is flagged positive by automated systems like BacT/ALERT or Bactec.
Direct susceptibility testing (DST) from positive blood culture broths is a well-established and recommended method in the diagnostic work-up of bloodstream infections. , Outcome-based studies assessing the effect of rapid reporting of susceptibility results have shown a decrease in the number of laboratory tests and procedures ordered,  decreased length of stay  and decreased health care costs and quicker modification of antimicrobial therapy. , Moreover, DST by disk diffusion method has added advantage of finding clones of bacteria identical in colony morphology and biochemical characteristics but different in antibiotic susceptibility.  Although not mentioned in Clinical and Laboratory Research Institute (CLSI), but inoculum standardisation from blood culture broth for direct sensitivity is mentioned in British society for Antimicrobial Chemotherapy (BSAC) methods. 
Previously DST of isolates from positive blood cultures has been evaluated with many automated testing systems, like the Phoenix, the VITEK system. , In all of these studies automated systems were directly inoculated with positive blood culture broths.
The current study was done in a newly built modern cancer care and bone marrow transplantation centre in eastern India. In this centre gram-negative bacteraemia clearly outnumbered that of gram positives. Mortality is also higher in gram-negative bacteraemic patients. Prevalence of infections caused by multi-drug resistant gram-negative bacilli, such as those caused by ESBL producers and Carbapenem-resistant organisms comprise the majority (ESBL: 70%, Carbapenem resistance: 25%).  In such high resistance settings, especially in view of immuno-compromised patients, the need to know reliable preliminary sensitivity pattern earlier becomes even more important. A DST algorithm from positive BacT ALERT blood culture broths by disk diffusion method was designed that circumvents the isolation process and expedites the reporting of preliminary sensitivity results.
A study comparing DST results for gram-negative isolates to those by AST with VITEK-2 was performed.
| ~ Materials and Methods|| |
The study was conducted from May 2011 to December 2012 at our tertiary-Cancer hospital and bone marrow transplantation centre. The automated blood culture system BacT ALERT with the culture bottles FA (adult) and PA (pediatric) is used in the hospital. All 275 blood cultures that were flagged as positive by the BacT ALERT system were followed. Positive blood culture bottles were first analyzed by Gram staining. Blood cultures with poly-microbial growth detected in the Gram stain or later, in the subcultures were excluded from the study. The study was conducted on both weekdays and weekends.
A total of 165 positive blood cultures showing uni-microbial gram-negative bacilli were included in study. All blood cultures showing gram-positive cocci (a total of 110) were also processed for DST followed by final identification and AST, but kept beyond the scope of this study. All broths were simultaneously cultured and processed for DST using disk diffusion method.
Direct susceptibility testing (DST) by disk diffusion method
For DST of gram-negative isolates, 1 drop (Dispovan needle size: 0.55x25 mm/24x1) of blood culture medium (BacT ALERT) was added to 5 ml 0.45% saline. This dilution was shown to result in semi-confluent growth on the Mueller-Hinton agar plates used for disk diffusion testing after incubation at 35-37°C in air for 18-20 hour as described by standards of the BSAC Methods for Antimicrobial Susceptibility Testing 2011 and 2012 [Figure 1]. 
|Figure 1: Direct sensitivity testing inoculums size as per British Society for Antimicrobial Chemotherapy (BSAC) criteria|
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The following panels of antimicrobial disks (Bio-Rad) were applied: Amoxicillin/clavulanate (20/10 μg), cefuroxime (30 μg), cefpodoxime (10 μg), ceftazidime (30 μg), ceftriaxone (30 μg), aztreonam (30 μg), amikacin (30 μg), gentamicin (10 μg), piperacillin-tazobactam (75/10 μg), imipenem (10 μg), meropenem (10 μg) and tigecycline (15 μg).
Zone inhibition diameters were interpreted as Sensitive (S), Intermediate (I) or Resistant (R), as per standards of the BSAC Methods for Antimicrobial Susceptibility Testing 2011 and 2012, as applicable.
Results of DST were communicated to clinical team on the same day to direct appropriate antibiotic treatment and evaluation note recorded in Hospital Information System. If the isolate was a presumptive multi-drug resistant organism (MDRO) based on DST, appropriate infection control measures were put in place on the same day to prevent spread of MDRO as per guidelines of Hospital Infection Control Committee.
Culture and antibiotic susceptibility testing (Reference method)
A drop of the positive blood culture broth was plated onto a MacConkey Agar and Columbia agar with 5% sheep blood (bioMerieux, France) and incubated at 37°C overnight to obtain isolated colonies. These colonies were added to sterile saline solution (bioMerieux, France) to make a suspension equivalent to a 0.5 McFarland standard, adjusted by using a DensiCHEK Plus (bioMerieux, Inc.) based on colorimetric principle.
AST of all isolates was performed with a pure overnight subculture with the VITEK-2 system as recommended by the manufacturer (bioMerieux, France). For identification and antibiotic susceptibility testing of gram-negative organisms GN ID card and N090 card, respectively were used (bioMerieux SA, France) as per manufacturer's instructions. Results are given as Sensitive (S), Intermediate (I) or Resistant (R) as per the database in instrument, which is regularly updated by the manufacturer.
Quality control strains, including Escherichia More Details coli ATCC 25922, Escherichia coli ATCC 35218, Pseudomonas aeruginosa ATCC 27853 and Klebsiella pneumoniae ATCC 700603 were tested weekly by the reference method (Vitek-2) for identification and AST.
Susceptibility results obtained by Direct Sensitivity, DST (test method) were compared to antibiotic susceptibility, AST by VITEK-2 (reference method). The tests were not repeated in the event of discordant AST results.
The following definitions were adopted: (1) Essential agreement or "Minor errors" (percentages of agreement obtained when minor discrepancies are ignored, i.e. "reference method" is S or R and "test method" is I; alternatively, "reference method" is I and "test method" is S or R); (2) Categorical agreement or "No Error" ("test method" and "reference method" susceptibility results agree using the respective criteria); (3) Major errors ("reference method" is S and "test method" is R; the percentage of major errors was calculated only for susceptible isolates); (4) Very major errors ("reference method" is R and "test method" is S; the percentage of very major errors was calculated only for resistant isolates).
| ~ Results|| |
A total of 162 gram-negative bacterial isolates were compared for DST and AST. One isolate each of Ralstonia mannitolilytica, Achromobacter xylosoxidans, Stenotrophomonas maltophilia, could not be compared as there is no BSAC criteria for DST interpretations.
DST results were typically available 18-24 hour after a blood culture was signaled positive by the BacT ALERT compared to 36-48 hour for AST results, secondary to overnight incubation required to produce isolated colonies. Thus, the DST results were available 18-24 h sooner than the AST results.
The absolute numbers and percentages of various isolates from blood cultures are given in [Table 1]. It is evident that majority of blood stream infections are caused by members of Enterobacteriaceae followed by Pseudomonas and Acinetobacter spp. and other rare gram-negative bacteria. [Table 2], [Table 3], [Table 4], [Table 5] show correlation of sensitivity patterns (DST) of relevant antibiotics as compared to that from reference method (VITEK-2) for Enterobacteriaceae, Pseudomonas, Acinetobacter and other gram-negative bacteria, respectively.
|Table 1: Species distribution among the positive blood cultures available for direct susceptibility test and antimicrobial susceptibility test|
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|Table 2: Correlation of direct susceptibility test and antimicrobial susceptibility test of enterobacteriaceae (n=113)|
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|Table 3: Correlation of direct susceptibility test and antimicrobial susceptibility test of Pseudomonas spp. (n=31)|
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|Table 4: Correlation of direct susceptibility test and antimicrobial susceptibility test of Acinetobacter spp (n=10)|
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|Table 5: Correlation of direct susceptibility test and antimicrobial susceptibility test of other gram‑negative bacilli (n=8)|
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For all 162 gram-negative isolates (840 antimicrobial- organism combinations), we observed 96.2% essential agreement between DST and AST. Thirty two antimicrobial-organism combinations did not show essential agreement. DST yielded 105 (12.5%) minor, 28 (5.3%) major, and 4 (1.4%) very major errors. The categorical agreement was observed in 703 antimicrobial-organism combinations (84.88%).
In Enterobacteriaceae (n = 113), no very major errors were found. Most of the major errors were detected in interpretation of amikacin (14.1%) and tigecycline (14.1%) followed by gentamicin (6%) and ceftazidime (3.3%). Maximum minor errors were found in tigecycline (45/105), amikacin (16/107) and piperacillin-tazobactam (16/107).
In non-Enterobacteriaceae (n = 49), one very major error, each was seen for ceftazidime in Pseudomonas (100%) and Acinetobacter (33%), one for meropenem in Pseudomonas (20%) and one for piperacillin-tazobactam in Aeromonas (100%).
There were five minor errors detected in Pseudomonas (n = 26) for ceftazidime, one in Acinetobacter (n = 8) for piperacillin-tazobactam and one in Aeromonas (n = 3) for imipenem.
| ~ Discussion|| |
DST of positive blood cultures can help clinicians to tailor antibiotic treatment about 24 hour earlier than final AST. Shortening the time to results of susceptibility testing of blood culture isolates can lead to significant reductions in patient morbidity, mortality, and costs. ,
DST inoculum as opposed to standard AST inoculum is better representative of bacterial isolates in the sample. DST by disk diffusion method selects some colony variants which may be potentially resistant to the particular antibiotic and are visible inside the sensitive zone or as double zones. These potentially resistant clones can be confirmed by Gram stain and identification tests to reassure purity of lawn culture of DST plates and then separately tested for antibiotic sensitivity. Alternately, the final report may advice against the use of that particular antibiotic, which although reported sensitive based on AST, but there are potential resistant clones which may get selected during treatment with that antibiotic and give rise to antibiotic resistance and treatment failure. This becomes even more important in dealing blood cultures of immuno-compromised neutropenic patients.
This study involved 162 gram-negative isolates and 840 organism-antibiotic combinations for comparison of direct and conventional AST methods. The number of gram-negative isolates for DST comparison included in this study was comparable to previously published studies of direct AST with positive blood cultures. ,,, The isolates spectrum was comparable to that found in other studies and countries. 
This study demonstrates that DST of gram-negative isolates from BacT ALERT bottles by using disk diffusion method performs well since 96.2% essential agreement, categorical agreement of 83.7%, and minor, major and very major errors of 12.5%, 5.3%, and 1.4% respectively were noted. Colistin was omitted from study comparison analysis as there are no disc diffusion interpretative guidelines available.
We observed that standardization of inoculum for DST is the critical step to achieve semi-confluent growth on the Mueller-Hinton agar plates. With our inoculum preparation method we obtained semi-confluent bacterial growth on the disk diffusion plates, as required by BSAC guidelines. DST plates need to be first examined for purity and proper semi-confluent lawn to enable it to be evaluated. In our experience we had to discard some DST plates sometime because of lesser or heavier inoculum size. These cultures had to be excluded from this comparison study. Fay and Oldfather (1979) also observed standardization of the inoculum in DST from blood cultures is important since different inoculum volumes may cause different inhibition zone diameters and recommended different inoculum volumes for gram-positive and gram-negative organisms.  The volumes used in our study were grossly comparable to those recommended by Fay and Oldfather (1979) although our preparation method differs from theirs in dilution of the blood culture medium in sterile saline instead of direct application of the blood culture medium onto the culture plate.  In our opinion, inhibitory effects of blood or culture medium components may be diminished by dilution of the positive blood culture medium in sterile saline. Results of quality control strains artificially spiked into blood culture bottles were within given limits.
Another important variable in DST is using antibiotic discs of correct potency. While performing DST, antibiotic discs are to be selected with caution as disc content of some antibiotics is different for CLSI and BSAC criteria, e.g. Ciprofloxacin. Disc quality is as important as disc potency. Need for regular quality control of antibiotic discs cannot be overemphasized. For some antibiotics there may not be disc diffusion criteria in BSAC guidelines. While selecting antibiotic panel for DST, hospital antibiotic formulary and clinician preferences need to be taken into account. Laboratories reporting DST as a preliminary sensitivity should prospectively correlate it with final AST to gain confidence in issuing preliminary reports. Moreover, this will monitor any consistent discordant result with an antibiotic, which may be linked to improper inoculum preparation or disc potency, storage or incubation conditions or any other systemic error. This will necessitate relooking into practices. Major and very major errors are of special concerns. Any particular antibiotic showing consistent discordance may be omitted from the DST panel. Frequent change of technical staff doing DST and vendor for antibiotic disc or media should be discouraged.
More extensive studies are required to gain confidence in reporting of DST results in India. Some laboratories even may wish to issue DST as a final blood culture report after proper standardization.
To conclude, DST using disk diffusion from positive blood culture broths helps to initiate early targeted antibiotic therapy. There is high concordance between DST and AST. Inoculum size and proper disc strengths (quality) are major variables for proper DST results. More studies are required to standardize inoculum size for DST.
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[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]