43038 CH14 0135

The Effect ofAntibiotics on Bacteria

t is valuable for the physician to have laboratory data indicatingwhether a bacterium is susceptible or resistant to various anti-biotics. This data, together with knowledge of possible side

effects, patient allergies, and other information, will be used in prescribingthe antibiotic. The laboratory test should be rapid, accurate, and relativelyinexpensive to perform.

In most laboratories, the effect of antibiotics on bacteria is determinedby the Standardized Disk Susceptibility Test as outlined in the FederalRegister. The procedure also is referred to as the Kirby-Bauer method,after its developers. Bacteria are swabbed on Mueller-Hinton agar, whichgives reproducible results and does not inhibit sulfonamides. Paper diskscontaining known amounts of the antibiotic are added, and after incubation,the plates are observed for clear zones of inhibition surrounding the disks.By referring to a standardized table, the effect of the antimicrobial drugon the organism may be ascertained. The physician may be reasonablycertain that if the stated dose of antibiotic exists in the tissues, then bacterialinhibition will take place.

pecial Materials

• Broth cultures of two selected bacterial species• Plates of Mueller-Hinton agar, or materials for their preparation• Sterile swabs and beakers of disinfectant• Antibiotic disks• Forceps in beakers of 70% ethyl alcohol• Millimeter rulers

rocedure

1. Obtain or prepare two plates of Mueller-Hinton agar. Label them on thebottom side with your name, the date, and the name of the procedure, anddesignate one plate for each of two organisms to be tested. The depth of theagar should be about 4 millimeters. Obtain broth cultures of the two organismsand two sterile swabs.

2. Dip a swab into a broth culture of bacteria, press it to the inside of the tubeto express the excess fluid, and inoculate one of the Mueller-Hinton platesby making a lawn of bacteria. The swabbing should be performed at three

P

S

T H E E F F E C T O F A N T I B I O T I C S O N B A C T E R I A 14 135

14

I PURPOSE: to evaluate thesensitivity of bacterial speciesto antibiotics.

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different angles, and a final sweep should be made around the rim of theplate as shown in Figure 8.2 in Exercise 8. Discard the swab in the beakerof disinfectant after use. Inoculate the second plate with the second or-ganism in like manner.

3. Allow the plates to dry at room temperature for about 10 minutes so that theexcess moisture is absorbed.

4. Using a sterile flamed forceps or an automatic dispenser, apply a series ofantibiotic disks to the surfaces of the plates as directed by the instructor. Thedisks should be about 3 cm from each other to prevent overlapping of anti-biotic, and at least 2 cm from the edge of the plate. If the forceps is used, itshould be flamed lightly after applying each disk. Light pressure may be applied to the disks using a forceps or inoculating loop to ensure firmcontact.

5. Incubate the plates in the inverted position at 37° C for 16 to 18 hours, andthen refrigerate them until observed. The short incubation time is neces-sary because bacteria may overcome the inhibition, and bacterial over-growth may occur during extended periods.

6. Examine the plates for the presence of clear zones of inhibition sur-rounding the disks, as shown in Figure 14.1. These zones represent areaswhere bacterial growth was inhibited by the antibiotic. With a millimeterruler, measure the diameter of the zone, including the disk. Measure-ments should be made to the nearest millimeter. Use the bottom of theplate for your measurements. The determinations should be entered inTable 14.2 in the Results section.

7. Referring to Table 14.1, determine whether the organism was susceptibleto the antibiotic (S), showed intermediate susceptibility (I), or was resistant(R). Enter these determinations in Table 14.2 next to the diameters. Also,note whether tiny colonies appear within the zone of inhibition. Diagramsof the plates may be entered in the spaces provided.

136 14 T H E E F F E C T O F A N T I B I O T I C S O N B A C T E R I A

!After swabbing plates,always place the swabs intodisinfectant to avoidcontaminating nearbymaterials.

Quick ProcedureAntibiotic Susceptibility Test

1. Inoculate a plate of testmedium with bacteria.

2. Place antibiotic disks onthe surface.

3. Incubate.

4. Observe and measurezones of inhibition.

5. Determine S, I, or Rfrom table.

1 2 3 4 5 6 7 8 9 10

Disk

Zone of inhibition

Bacterial lawn

F I G U R E 1 4 . 1Measurement of zones of inhibition on antibiotic susceptibilityplates.

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continued*Courtesy BD Biosciences.

TABLE

Diameter of Zone of Inhibition and Interpretation*

RESISTANT INTERMEDIATE SUSCEPTIBLEDISK CODE ANTIMICROBIAL AGENT TESTED (≤ mm) (mm range) (≥ mm)

�-LACTAMS - PENICILLINS

AM 10 Ampicillin 10 �g

Gram-negative enteric organisms 13 14–16 17Staphylococci 28 — 29Enterococci 16 — 17Streptococci (not S. pneumoniae) 21 22–29 30Haemophilus spp. 18 19–21 22Listeria monocytogenes 19 — 20

AZ 75 Azlocillin 75 �g

Pseudomonas aeruginosa 17 — 18

CB 100 Carbenicillin 100 �g

P. aeruginosa 13 14–16 17Other gram-negative organisms 19 20–22 23

ME 5 Methicillin 5 �g

Staphylococci 9 10–13 14

MZ 75 Mezlocillin 75 �g

P. aeruginosa 15 — 16Other gram-negative organisms 17 18–20 21

NF 1 Nafcillin 1 �g


OX 1 Oxacillin 1 �g


P 10 Penicillin G 10 units

Staphylococci 28 — 29Enterococci 14 — 15Streptococci (not S. pneumoniae) 19 20–27 28Neisseria gonorrhoeae 26 27–46 47L. monocytogenes 19 — 20

PIP 100 Piperacillin 100 �g


TIC 75 Ticarcillin 75 �g


�-LACTAM/�-LACTAMASE INHIBITOR COMBINATIONS

AMC 30 Amoxicillin 20 �g/Clavulanic Acid 10 �g

Staphylococci 19 — 20Other organisms 13 14–17 18

SAM 20 Ampicillin 10 �g/Sulbactam 10 �g

Gram-negative enterics and staphylococci 11 12–14 15

PTZ 110 Piperacillin 100 �g/Tazobactam 10 �g

P. aeruginosa 17 — 18Other gram-negative organisms 17 18–20 21Staphylococci 17 — 18

14.1

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TABLE

Zone Diameters continued

RESISTANT INTERMEDIATE SUSCEPTIBLEDISK CODE ANTIMICROBIAL AGENT TESTED (� mm) (mm range) (� mm)

TIM 85 Ticarcillin 75 �g/Clavulanic Acid 10 �g

P. aeruginosa 14 — 15Other gram-negative organisms 14 15–19 20Staphylococci 22 — 23

CEPHALOSPORINS AND OTHER CEPHEMS

CEC 30 Cefaclor 30 �g 14 15–17 18

Haemophilus spp. 16 17–19 20

MA 30 Celamandole 30 �g 14 15–17 18

CZ 30 Cefazolin 30 �g 14 15–17 18

FEP 30 Cefepime 30 �g 14 15–17 18

Haemophilus spp. — — 26N. gonorrhoeae — — 31

CAT 10 Cefetamet 10 �g 14 15–17 18

Haemophilus spp. 14 15–17 18N. gonorrhoeae — — 29

CFM 5 Cefixime 5 �g 15 16–18 19


CMZ 30 Cetmetazole 30 �g 12 13–15 16

N. gonorrhoeae 27 28–32 33

CID 30 Cefonicid 30 �g 14 15–17 18


CFP 75 Cefoperazone 75 �g 15 16–20 21

CTX 30 Cefotaxime 30 �g 14 15–22 23Haemophilus spp. — — 26N. gonorrhoeae — — 31

CTT 30 Cefotetan 30 �g 12 13–15 16


FOX 30 Cefoxitin 30 �g 14 15–17 18


CPD 10 Cefpodoxime 10 �g 17 18–20 21


CPR 30 Cefprozil 30 �g 14 15–17 18


CAZ 30 Ceftazidime 30 �g 14 15–17 18


ZOX 30 Ceftizoxime 30 �g 14 15–19 20


CRO 30 Ceftriaxone 30 �g 13 14–20 21


14.1

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TABLE



CXM 30 Cefuroxime 30 �g (oral) 14 15–22 23

Haemophilus spp. 16 17–19 20N. gonorrhoeae 25 26–30 31

CXM 30 Cefuroxime 30 �g (parenteral) 14 15–17 18

CR 30 Cephalothin 30 �g 14 15–17 18

LOR 30 Loracarbef 30 �g 14 15–17 18


MOX 30 Moxalactam 30 �g 14 15–22 23

CARBAPENEMS

IPM 10 Imipenem 10 �g 13 14–15 16

Haemophilus spp. – – 16

MONOBACTAMS

ATM 30 Aztreonam 30 �g 15 16–21 22

Haemophilus spp. – – 26

GLYCOPEPTIDES

TEC 30 Telcoplanin 30 �g 10 11–13 14

VA 30 Vancomycin 30 �gEnterococci 14 15–16 17Other gram-positive organisms 9 10–11 12S. pneumoniae — — 17

AMINOGLYCOSIDES

AN 30 Amikacin 30 �g 14 15–16 17

GM 10 Gentamicin 10 �g 12 13–14 15

K 30 Kanamycin 30 �g 13 14–17 18

NET 30 Netilmicin 30 �g 12 13–14 15

S 10 Streptomycin 10 �g 11 12–14 15

TM 10 Tobramycin 10 �g 12 13–14 15

MACROLIDES

AZM 15 Azithromycin 15 �g 13 14–17 18

Haemophilus spp. – – 12S. pneumoniae 13 14–17 18

CLR 15 Clarithromycin 15 �g 13 14–17 18

Haemophilus spp. 10 11–12 13S. pneumoniae 16 17–20 21

E 15 Erythromycin 15 �g 13 14–22 23

S. pneumoniae 15 16–20 21

TETRACYCLINES

DO 30 Doxycycline 30 �g 12 13–15 16

MIN 30 Minocycline 30 �g 14 15–18 19

14.1

continued

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TABLE



TE 30 Tetracycline 30 �g 14 15–18 19

Haemophilus spp. 25 26–28 29N. gonorrhoeae 30 31–37 38S. pneumoniae 17 18–21 22

QUINOLONES

CIN 100 Cinoxacin 100 �g 14 15–18 19

CIP 5 Ciprofloxacin 5 �g 15 16–20 21

Haemophilus spp. – – 21N. gonorrhoeae – – 36

ENX 10 Enoxacin 10 �g 14 15–17 18

N. gonorrhoeae 32

FLX 5 Fleroxacin 5 �g 15 16–18 19

Haemophilus spp. – – 19N. gonorrhoeae 27 28–32 33

LOM 10 Lomefloxacin 10 �g 18 19–21 22


NA 30 Nalidixic Acid 30 �g 13 14–18 19

NOR 10 Norfloxacin 10 �g 12 13–16 17

OFX 5 Ofloxacin 5 �g 12 13–15 16

Haemophilus spp. — — 16N. gonorrhoeae — — 31S. pneumoniae 12 13–15 16

OTHERS

C 30 Chloramphenicol 30 �g 12 13–17 18

Haemophilus spp. 25 26–28 29S. pneumoniae 20 — 21

CC 2 Clindamycin 2 �g 14 15–20 21

S. pneumoniae 15 16–18 19

FD 300 Nitrofurantoin 300 �g 14 15–16 17

RA 5 Rifampin 5 �g 16 17–19 20Haemophilus spp. 16 17–19 20S. pneumoniae 16 17–18 19

G300 Sulfisoxazole 300 �g 12 13–16 17

TMP 5 Trimethoprim 5 �g 10 11–15 16

SxT 25 Trimethoprim 1.25 �g/ 10 11–15 16Sulfamethoxazole 23.75 �g

Haemophilus spp. 10 11–15 16S. pneumoniae 15 16–18 19

14.1


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uestions

1. List several factors that must be standardized to enhance the usefulness ofthe antibiotic susceptibility test by the Kirby-Bauer method.

2. Which antibiotics may be broad-spectrum antibiotics and which may benarrow-spectrum antibiotics, as determined from the results in this exercise?

3. How might one explain the appearance of colonies of bacteria within thezone of inhibition?

4. Why is it more advisable to use a swab instead of a loop for preparing alawn of bacteria on an agar plate?

5. Explain why a shorter incubation time of 16 to 18 hours is preferred in thistest to the usual 24 to 48 hours.

Q

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Table 14.2. Susceptibility or Resistance of Bacteria

Organisms Tested

1. 2.Zone Diameter S, I, or R Zone Diameter S, I, or R

Name

Date Section

Exercise Results

The Effect of Antibiotics on Bacteria

14

Antibiotic


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Observations and Conclusions:


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43038 CH14 0135