Diffusion
Dilution
Diffusion and dilution
Stokes method
MIC method
1. Broth dilution
2. Agar dilution
E test method
Kirby-Bauer method
Selection of the appropriate method will depend on the intended degree of accuracy, convenience, urgency, availability of resources, availability of technical expertise, and cost.
Diffusion Methods
The Kirby-Bauer and Stokes methods are usually used for antimicrobial susceptibility testing; the Kirby-Bauer method is recommended by the Clinical and Laboratory Standards Institute (CLSI, formerly, NCCLS). CLSI is an international, interdisciplinary, nonprofit, nongovernmental organization composed of medical professionals, government, industry, healthcare providers, educators, etc. which promotes accurate AST and appropriate reporting by developing standard reference methods, by indicating interpretative criteria and quality control parameters for standard test methods. Interpretative criteria of CLSI are developed based on the international collaborative studies; they are well correlated with MICs and the clinical data. Based on study results, CLSI interpretative criteria are revised frequently. CLSI is approved by FDA-USA and recommended by the World Health Organization (WHO) [3].
The Kirby-Bauer test is a qualitative assay, whereby disks of filter paper pre-impregnated with a standard concentration of a particular antibiotic are lightly pressed onto the agar surface. The test antibiotic immediately begins to diffuse outward from the disks, creating a gradient of antibiotic concentration in the agar such that the highest concentration is found close to the disk with decreasing concentrations further away from the disk. After an overnight incubation, the bacterial growth around each disk is observed. If the test isolate is susceptible to a particular antibiotic, a clear area of “no growth” is observed around that particular disk [4]. The zone around an antibiotic disk that has no growth is referred to as the “zone of inhibition” (Fig. 27.1), since this approximates the minimum antibiotic concentration sufficient to prevent growth of the test isolate. This zone is then measured in mm and compared to a standard interpretation chart of Tables 2A through I (Zone Diameter Interpretative Standards and Equivalent Minimum Inhibitory Concentration Breakpoints) of the NCCLS M100-S12, Performance Standards for AST, and the organisms are reported as either susceptible, intermediate, or resistant to the agents that have been tested [5].
Fig. 27.1
Mueller-Hinton agar plate for susceptibility testing of Staphylococcus species to various antibiotics after an incubation period of 24 h by disk diffusion method. The diameter of the clear zone around each drug indicates whether the test organism is susceptible or not
Dilution Methods
The Broth dilution method involves subjecting the isolate to a series of concentrations of antimicrobial agents in a broth environment. Microdilution testing uses about 0.05–0.1 mL total broth volume and can be conveniently performed in a microtiter plates. Macrodilution testing uses broth volumes at about 1.0 mL in standard test tubes. For both of these broth dilution methods, the lowest concentration at which the isolate is completely inhibited (as evidenced by the absence of visible bacterial growth) is recorded as the minimal inhibitory concentration or MIC [3].
A procedure similar to broth dilution is agar dilution. Agar dilution method follows the principle of establishing the lowest concentration of the serially diluted antibiotic concentration at which bacterial growth is still inhibited [3].
Dilution and Diffusion Methods
E test, also known as the Epsilometer test, is an “exponential gradient” testing methodology where the “E” refers to the Greek symbol “epsilon” (ε). The E test which is a quantitative method for antimicrobial susceptibility testing applies to both the dilution of antibiotic and diffusion of antibiotic into the medium. It utilizes a plastic test strip impregnated with a gradually decreasing concentration of a particular antibiotic. The strip also displays a numerical scale that corresponds to the antibiotic concentration contained therein. Following the incubation, a symmetrical inhibition ellipse is produced (Fig. 27.2). The intersection of the inhibitory zone edge and the calibrated carrier strip indicates the MIC value over a wide concentration range (>10 dilutions) with inherent precision and accuracy [3]. This method provides for a convenient quantitative test of antibiotic resistance of a clinical isolate. However, a separate strip is needed for each antibiotic, and therefore the cost of this method could be high.
Fig. 27.2
Mueller-Hinton agar plate for susceptibility testing of Staphylococcus species to various antibiotics after an incubation period of 24 h by E test method. (Left) The inhibition ellipse is produced and the intersection of the inhibitory zone edge and the calibrated carrier strip indicates the MIC against each drug. (Right) No inhibitory zone around the strip signifies complete resistance of the isolate to the test drug
Changing Trends in Antibacterial Susceptibility Profile
The earliest reports of susceptibility testing in endophthalmitis included mainly case reports and the panel of drugs tested. Mark and Gaynon reported a case of Acinetobacter anitratus endophthalmitis in 1983 [6]; this patient was treated with penicillin, but the infection did not respond. It was then found that the organism was sensitive in vitro to gentamicin, tobramycin, kanamycin, and amikacin; it was intermediately sensitive to carbenicillin and resistant to penicillin. These early reports paved the way for a larger series, thus establishing the evidence for adequate treatment of both gram-positive and gram-negative organisms in confirmed cases of endophthalmitis.
Gram-Positive Bacteria
Global Perspective
Davis et al. suspected a change in the expected sensitivity of coagulase-negative staphylococci when three cases of endophthalmitis due to multiple resistant organisms failed to respond to intravitreal cefazolin and gentamicin [7]. On reviewing their records from 1973 to 1986, they found increased resistance to gentamicin and methicillin. No isolates were resistant to vancomycin. The Endophthalmitis Vitrectomy Study (EVS, February 1990 to January 1994) that recruited patients with acute bacterial endophthalmitis corroborated this observation [8]. In the EVS, all gram-positive organisms were susceptible to vancomycin (100%). Susceptibilities of coagulase-negative micrococci in decreasing order were as follows: amikacin, 86.1%; ciprofloxacin, 77.9%; oxacillin, 62.7%; and ceftazidime, 62.1%. Corresponding susceptibilities of “other” gram-positive organisms in decreasing order were: ceftazidime, 74.3%; ciprofloxacin, 67.6%; oxacillin, 60.0%; and amikacin, 49.3%. In addition, all of five isolates of Streptococcus pneumoniae tested were susceptible to ceftazidime.
Benz et al. reviewed the 5-year (1996–2001) microbiology records of patients with culture-proven endophthalmitis at the Bascom Palmer Eye Institute [9] and found that among the 246 gram-positive organisms identified, the sensitivities were the following: vancomycin 100%, gentamicin 78.4%, ciprofloxacin 68.3%, cefazolin 66.8%, and ceftazidime 63.6%. During the period of the study, 1996–2001, there was a significant decrease in the sensitivity of gram-positive organisms to ciprofloxacin (from 72% in 1996, to 36% in 2001). Although levofloxacin showed better activity in 2001 against gram-positive isolates than ciprofloxacin (62% vs. 36%), it still had incomplete gram-positive coverage. In comparison, during the same period sensitivities of gram-positive isolates to gentamicin remained relatively stable. The widespread and routine use of third-generation fluoroquinolones as therapeutic and prophylactic medications in North America may have led to an increase in microbial resistance against them. The difference from the EVS study could be due to geographic differences [8], as well as the inclusion criteria in EVS acute-onset endophthalmitis associated with cataract surgery or secondary intraocular lens placement versus all categories of endophthalmitis. Similarly, Recchia et al. reviewed post-cataract surgery endophthalmitis data in the Wills Eye Hospital for 11 years, 1989–2000, and reported statistically significant resistance of gram-positive bacteria to ciprofloxacin and resistance of coagulase-negative staphylococci to ciprofloxacin (20–38%) and cefazolin (19–40%) [10]. Resistance to bacitracin, trimethoprim-sulfamethoxazole, and vancomycin remained statistically unchanged; 30% of all isolates (and 35% of coagulase-negative staphylococci) were resistant to ofloxacin [8].
A 25-year review of culture-positive endophthalmitis collected from 1987 to 2011 at the New York Eye and Ear Infirmary by Gentile and co-workers have documented a statistically significant decrease in microbial susceptibility over time for ampicillin, cefazolin, cefotetan, cephalothin, ceftriaxone, clindamycin, erythromycin, and methicillin/oxacillin [11].They also observed an increase in susceptibility to gentamicin, imipenem, and tobramycin. Susceptibility to fluoroquinolones for all isolates ranged from a low 67% for levofloxacin to a high 81% for gatifloxacin during the time period from 2000 to 2011. Only levofloxacin showed a decrease in microbial susceptibility that approached significance. Vancomycin displayed 99.7% susceptibility against 727 gram-positive isolates, and 99.3% (143/144) susceptibility was observed for linezolid (approved by the Food and Drug Administration only in 2000).
Similar studies outside the USA, conducted at the Federal University of São Paulo, Brazil from 2006 to 2009, showed that 79.5% and 89.5% of coagulase-negative staphylococci (CoNS) were sensitive to gatifloxacin and moxifloxacin, respectively [12]. Additionally, most fourth-generation quinolone-resistant samples were also methicillin resistant. In a previous report from the same institute, 2000 to 2005, all CoNS (100%) were susceptible to both moxifloxacin and gatifloxacin [13]. Falavarjani et al. at Tehran studied the antibiotic resistance in 21 culture-proven endophthalmitis cases; resistance to penicillin G (7 isolates), oxacillin (5 isolates), clindamycin (4 isolates), cefazolin (2 isolates), ceftazidime (5 isolates), ciprofloxacin (2 isolates), ceftriaxone (2 isolates), and imipenem (1 isolate) [14]. There was no resistance to vancomycin.
A retrospective analysis on 912 cases of post-traumatic endophthalmitis at Zhongshan Ophthalmic Center, Guangzhou, China, from 1990 to 2009 showed that S. epidermidis had the greatest susceptibility to ceftazidime (90.7%), followed by cefuroxime (88.9%), but showed at least some resistance to all other antibiotics tested [15]. S. saprophyticus was highly susceptible to ceftazidime (100%) and cefuroxime (100%), followed by ciprofloxacin (from 93.3% to 96.4%, p > 0.05). B. subtilis showed susceptibility (100%) to ciprofloxacin, gentamicin, ofloxacin, cefuroxime, and ceftazidime. However, there was a variation of antibiotic susceptibility analysis among the isolates between the different time periods. During the first decade (1990–1999), ciprofloxacin was the most effective antibiotic against bacterial isolates, followed by cefoperazone. For the second decade (2000–2009), ceftazidime showed the greatest level of activity against most bacterial isolates, followed by cefuroxime. Neomycin showed little activity against most bacterial isolates, except B. subtilis, which was highly sensitive to all the tested antibiotics except erythromycin and ampicillin. All cases with culture-proven endophthalmitis from the Eye, Ear, Nose, and Throat Hospital, Shanghai Medical College, between April 2004 and April 2014 were examined [16]. The authors found that 97.6% of 369 gram-positive isolates were sensitive to vancomycin. Three B. cereus isolates and six isolates of Streptococcus species were resistant to vancomycin. One hundred percent of the isolated staphylococcal species were susceptible to vancomycin. The other antibiotic susceptibilities were as follows: levofloxacin, 85.1%; gentamicin, 78.7%; rifampin, 77.2%; ofloxacin, 77.2%; chloramphenicol, 76.4%; and ciprofloxacin, 73.7%.
Indian Perspective
In the Indian subcontinent, the earliest reports were from the Endophthalmitis Research Group at the L.V. Prasad Eye Institute in Hyderabad, India. They reported microbiological profile of post-traumatic and postoperative endophthalmitis in the period 1991–1997 [17, 18]. In traumatic endophthalmitis, the gram-positive cocci were most susceptible to cefazolin (93.4%) and ciprofloxacin (93.2%), and gram-positive bacilli were most susceptible to ciprofloxacin (100%). In postoperative endophthalmitis, the gram-positive isolates were most susceptible to cefazolin (93.1%) followed by ciprofloxacin (86%); the gram-positive bacilli were 100% susceptible to several antibiotics, including vancomycin.
A study from Chennai, between 1995 and 1998, showed that among the gram-positive bacteria tested, 41/53 (77.3%) were sensitive to gentamicin, and 47/53 (88.6%) to cefotaxime, 88.4% (46/52) to ciprofloxacin, 92.6% (38/41) to cefazolin, and 72.9% (27/37) to ceftazidime [19]. All the gram-positive bacteria (100%) were sensitive to vancomycin. The resistance of gram-positive bacteria to ceftazidime and ciprofloxacin was comparatively lower than the EVS results. Contradicting to these reports is a study by Vedantham et al. from Madurai [20]. In a series of 42 post-traumatic endophthalmitis managed at the Aravind Eye Hospital (January 2000–December 2001), majority of the organisms were susceptible to chloramphenicol and ciprofloxacin, and the susceptibility to vancomycin and amikacin was poor. Resistance of this magnitude to vancomycin and amikacin has not previously been reported in the literature. Another retrospective analysis of culture-proven endophthalmitis treated at the Aravind Eye Hospital, Tirunelveli, South India, over a 10-year period, 1997–2006, showed that the highest percentage of gram-positive cocci were susceptible to both cefazolin (100%) and moxifloxacin (100%) followed by chloramphenicol (98.3%), vancomycin (96.6%), and gatifloxacin (95.3%) [21]. The gram-positive bacilli were completely susceptible to amikacin and to all four tested fluoroquinolones, ciprofloxacin, ofloxacin, gatifloxacin, and moxifloxacin. Amikacin, gatifloxacin, and moxifloxacin also showed 100% sensitivity against Nocardia spp. of endophthalmitis isolates.
The susceptibility patterns of isolates from patients with exogenous endophthalmitis, January 2003–December 2013, at a tertiary eye care referral hospital of the Northeast India were slightly different [22]. While all the gram-positive bacteria were sensitive to vancomycin, only 54.5% showed sensitivity to amikacin and 45.5% to cefotaxime (33.3%). Sensitivity to ceftazidime and ciprofloxacin was observed in 55.3% and 48.7%, respectively.
More recently, Jindal et al. evaluated the antimicrobial susceptibility of isolates on 581 patients with culture-proven post-traumatic endophthalmitis at L.V. Prasad Eye Institute, Hyderabad, from January 2006 to March 2013 [23]. Comparing with the earlier published report from the same institute [17], they found that the susceptibility of gram-positive organisms continues to be highest to vancomycin; the susceptibility of CoNS to ciprofloxacin had reduced from 100% in 1999 to 77.3% in 2013.
A comparative analysis of all these studies is shown in Table 27.2.
Table 27.2
Antibiotic susceptibility profile of gram- positive bacteria isolated from endophthalmitis as reported in literature
Sl | Author et al. (year) | Study period | Location | Overall antibiotic susceptibility (%) | ||||||
|---|---|---|---|---|---|---|---|---|---|---|
Vancomycin | Gentamicin | Ciprofloxacin | Gatifloxacin | Moxifloxacin | Amikacin | Cefazolin | ||||
1 | Davis et al. [7] | 1973–1986 | USA | 100 | 59 | NA | NA | NA | NA | NA |
2 | Han et al. [8] | 1990–1994 | USA | 100 | NA | 75.3 | NA | NA | 77 | NA |
3 | Benz et al. [9] | 1996–2001 | USA | 100 | 78.4 | 68.3 | NA | NA | NA | NA |
4 | Recchia et al. [10] | 1989–2000 | USA | 95 | NA | 67.2 | NA | NA | NA | 67.2 |
5 | Gentile et al. [11] | 1987–2011 | USA | 99.7 | 74.8 | 71 | 78 | 75.3 | NA | 48 |
6 | Melo et al. [12] | 2006–2009 | Brazil | 100 | 78.3 | 54 | 86 | 92 | 90.2 | NA |
7 | Bispo et al. [13] | 2000–2005 | Brazil | 100 | NA | 89.6 | 100 | 100 | NA | NA |
8 | Falvarjani et al. [14] | 2005–2015 | Iran | 100 | NA | 90 | NA | NA | NA | NA |
9 | Long et al. [15] | 1990–2009 | China | NA | 100 | 100 | NA | NA | NA | NA |
10 | Liu et al. [16] | 2004–2014 | China | 97.6 | 78.7 | 73.7 | NA | NA | NA | 12.7 |
11 | Kunimoto et al. [17] | 1991–1997 | India | 100 | NA | 50 | NA | NA | 100 | 75 |
12 | Kunimoto et al. [18] | 1989–2000 | India | 86 | 84.5 | 95 | NA | NA | 89 | 87 |
13 | Anand et al. [19] | 1995–1998 | India | 100 | 77.3 | 88.4 | NA | NA | NA | 92.6 |
14 | Ramakrishnan et al. [21] | 1997–2006 | India | 92 | 61 | 95.5 | 97.2 | 100 | 65.5 | 91.3 |
15 | Bhatacharjee et al. [22] | 2003–2013 | India | 100 | NA | 48.7 | NA | NA | NA | 54.5 |
16 | Jindal et al. [23] | 2006–2013 | India | 94.4 | 78.3 | 78.1 | 91 | 85 | 66 | 88 |
The rates of resistance of gram-positives to antimicrobials (L.V. Prasad Eye Institute from India) over a 10-year period (January 2005 to December 2015) are shown in Fig. 27.3.
Fig. 27.3
Trends of antibiotic resistance by gram-positive organisms, 2005–2015 of all culture-proven endophthalmitis seen at the L.V. Prasad Eye Institute, Hyderabad (unpublished data)
Gram-Negative Bacteria
Global Perspective
In the EVS, of the 19 gram-negative isolates tested for antibiotic susceptibility, the frequencies of susceptible isolates were amikacin, 89.5%; ceftazidime, 89.5%; and ciprofloxacin, 94.7% [8]. While 17/19 isolates were susceptible to both amikacin and ceftazidime, 2/19 were resistant to both. Additionally, one gram-negative isolate was resistant to only ceftazidime, and another isolate was resistant to only ciprofloxacin, but the later isolate was susceptible to both amikacin and ceftazidime. Benz et al. have reported that among the gram-negative organisms, the sensitivities were the following: ciprofloxacin 94.2%, ceftazidime 80%, amikacin 81%, and gentamicin 75% [9]. Recchia et al. showed that all 15 tested gram-negative bacteria were completely sensitive to ceftazidime; but it could not be said for the aminoglycoside antibiotics (gentamicin, tobramycin, and amikacin) [10]. A higher percentage of gram-negative isolates in this study was additionally susceptible to gatifloxacin (95%, 19/20) and moxifloxacin (100%).
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