Bacteriology

Regis P. Kowalski

Jules L. Baum

Bacteria are single-celled prokaryotes and one of the earliest life forms. Although bacteria naturally inhabit many exterior and interior areas of the human body, the normal human cornea harbors no colonizing bacteria, but bacteria from the eyelids constantly contaminate the ocular surface. Host defense mechanisms of the eye and adnexa provide excellent corneal protection for the elimination of microbes through (a) mechanical, (b) immunologic, (c) chemical, and (d) innate mechanisms (1).

Bacteria colonize the cornea in infection, or some other pathologic process that compromises basic defense mechanisms, thus allowing growth. Bacteria that cause disease are labeled as pathogens because they possess virulence factors that enable the survival of these organisms in the cornea. These virulence factors include specific antigens, proteolytic enzymes, hemolysins, and toxins (2). In general, nonpathogens do not possess significant virulence factors, but under compromised conditions, may gain access to the cornea. Bacteria become established in the cornea after a breakdown of the corneal epithelium due to mechanical or nutritional stress, and proliferate under continued favorable conditions for bacterial growth (3).

The cornea may become infected from a pathogen in a wide range of bacteria, classified in part by the staining characteristics of the cell wall. The classic differential staining system is based on the Gram stain, which separates bacteria into gram-positive and gram-negative bacteria (Fig. 12-1). The staining procedure starts with placement of a corneal tissue specimen or a suspension of bacteria isolated from culture media on a glass microscope slide. The specimen is air-dried and fixed with methanol or a comparable fixative. In consecutive steps, it is stained with crystal violet, rinsed with tap water, stained with iodine, decolorized with alcohol (alcohol/acetone mixture), and counterstained with safranin. Gram-positive bacteria have a high content of peptidoglycan and a low content of lipid compared with gram-negative bacteria. When the lipid layer is dissolved, crystal violet and iodine form a complex in the cell wall that appears blue under microscopic examination, denoting gram-positive bacteria. Gram-negative bacteria appear red because the lipid layer is not removed by the decolorizing step. A blue crystal violet complex can not form in the cell wall and safranin counterstains the bacteria. Other stains, such as an acid-fast stain, are also used to differentiate Mycobacterium species from other bacteria (4). Gram-positive bacteria are more frequently isolated from patients with keratitis than are gram-negative bacteria. In our laboratory (The Charles T. Campbell Ophthalmic Microbiology Laboratory, Pittsburgh, PA, 1993 to 2002), 56% of the bacterial keratitis isolates were gram-positive and 44% were gram-negative (Fig. 12-2). This ratio is consistent with other reports from China (60.2% vs. 39.6%) (5), England (54.7% vs. 45.3%) (6), and Boston (60% vs. 40%) (7).

BACTERIAL IDENTIFICATION METHODS

The vast majority of bacteria are easily cultivated in the basic microbiology laboratory using culture media. A broth-based agar medium supplemented with 5% to 10% sheep blood (blood agar), a chocolate agar (red cells partially lysed by heat) plate, and an optional mannitol salt agar plate will isolate almost all corneal bacterial pathogens. Anaerobic media such as enriched thioglycollate broth medium and broth-based agar media incubated under anaerobic conditions suffice for anaerobic bacterial isolation. Most bacteria appear on culture media with distinct characteristics that allow for immediate identification. Colony shape, size, color, and edge appearance, medium color changes due to production of end products (e.g., mannitol salt agar), and hemolysis of blood in supplemented media (i.e., beta-, alpha-Streptococci) due to production of lysins are examples of identification characteristics. As a rule, most bacteria can be identified to a bacterial genus or as gram-positive or gram-negative by appearance, but further identification to species requires the use of biochemicals (e.g., indole, glucose fermentation), antisera (e.g., coagulase), and changing growth conditions (e.g., Haemophilus growth factors). Bacteria are also characterized by specific oxygen requirements. Strict aerobic
bacteria (aerobes) require oxygen for growth, whereas oxygen is toxic to other bacteria. These bacteria are classified as anaerobes. Many bacteria are facultative anaerobes in that growth occurs under either aerobic or anaerobic conditions. Bacteria also grow better at certain temperature and atmospheric conditions. In general, bacteria are incubated for isolation at body temperature (37°C or 98.6°F) and under a 6% CO₂ atmospheric condition. Mycobacteria require different incubating temperatures, different light conditions, and extended incubation periods for isolation and identification.

FIGURE 12-1. Microphotographs of gram-positive (blue) and gram-negative (red) bacteria that infect the cornea. (Original magnification × 100, oil immersion; photographed by Lisa M. Karenchak.) Row 1, left: diplococci (Streptococcus pneumoniae); row 1, middle: pleomorphic rods (diphtheroids); row 1, right: diplobacilli (Moraxella). Row 2, left: chain of cocci (Streptococcus); row 2, middle: rods with endospores (Bacillus); row 2, right: rods (Pseudomonas aeruginosa). Row 3, left: grapelike clusters of cocci (Staphylococcus aureus); row 3, middle: thin filaments (Actinomyces); row 3, right: nonstaining ghost rods (Mycobacterium chelonae). (See color figure.)

FIGURE 12-2. Distribution of bacteria isolated from cases of keratitis (1993 to 2002). Gram-positive bacteria comprised 56% of isolates, and gram-negative bacteria 44%. (Courtesy of the Charles T. Campbell Ophthalmic Microbiology Laboratory, The University of Pittsburgh Medical Center, Pittsburgh, PA.)

Antibiotic susceptibility patterns are often used to identify and characterize bacteria. Susceptibility to vancomycin usually denotes a gram-positive bacteria, whereas bacitracin resistance is a characteristic of Haemophilus species. Staphylococcus aureus that are resistant to oxacillin are designated as methicillin resistant. Tables 12-1 and 12-2 include the susceptibility patterns of bacterial keratitis pathogens isolated from the Charles T. Campbell Ophthalmic Microbiology Laboratory 1993 to 2002. Although this chapter does not deal with keratitis therapy, susceptibility patterns are important in epidemiology and strain characterization. Minimum inhibitory concentrations or disk diffusion patterns using standard methods are determined and compared with established standards that interpret susceptibility (8,9). In the United States, bacterial susceptibility is determined using the serum standards established by the National Committee of Clinical Laboratory Standards (NCCLS). Most general laboratories do not test all the antibiotics presented in Tables 12-1 and 12-2 because some of these antibiotics are not used for treatment of systemic disease. There are no standards for interpretation
of antibacterial susceptibility for bacterial keratitis isolates because therapy is topical instead of systemic. NCCLS susceptibility interpretations can be used for ocular isolates if the assumption is made that the antibiotic concentration in the ocular tissue is at least as great as the antibiotic concentrations achieved in the blood serum.

TABLE 12-1. SUSCEPTIBILITY OF GRAM-POSITIVE BACTERIAL ISOLATES FROM KERATITIS TO COMMON ANTIBIOTICS (PERCENTAGE SUSCEPTIBLE, 1993-2002)

Bacteria	No.	BAC	CHL	VAN	GEN	CIP	OFX	TRI	PB	CEF	TOB	SULF	OXA
Staphylococcus aureus	264	97	98	100	89	71	74	89	1	92	72	95	63
Coagulase-negative staphylococci	95	94	90	100	66	45	45	55	25	88	64	78	26
Streptococcus pneumoniae	52	100	94	100	4	54	79	22	2	100	0	94
Streptococcus viridans	73	100	97	100	42	33	75	43	4	99	22	100
Beta-hemolytic streptococci	8	100	100	100	25	63	88	25	0	100	0	38
Nutritionally variant streptococci	6	50	100	100	17	33	100	25	0	83	0	17
Enterococcus species	10	80	40	90	10	40	50	60	0	20	10	30
Nonhemolytic streptococci	2	100	100	100	0	0	0	0	0	50	0	0
Diphtheroids	32	91	84	100	81	58	65	6	91	91	78	75
Bacillus species	5	40	80	100	100	100	100	100	60	80	100	100
BAC, bacitracin; CHL, chloramphenicol; VAN, vancomycin; GEN, gentamicin; CIP, ciprofloxacin; OFX, ofloxacin; TRI, trimethoprim; PB, polymyxin B; CEF, cefazolin; TOB, tobramycin; SULF; sulfasoxazole; OXA, oxacillin. (Data courtesy of the Charles T. Campbell Ophthalmic Microbiology Laboratory, The University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania.)

TABLE 12-2. SUSCEPTIBILITY OF GRAM-NEGATIVE BACTERIAL ISOLATES FROM KERATITIS TO COMMON ANTIBIOTICS (PERCENTAGE SUSCEPTIBLE; 1993-2002)

Bacteria	No.	BAC	CHL	VAN	GEN	CIP	OFX	TRI	PB	CEF	TOB	SULF
Pseudomonas aeruginosa	122	0	0	0	96	97	92	0	100	0	98	2
Serratia marcescens	122	0	89	0	99	100	100	84	3	0	88	84
Serratia liquefaciens	4	0	100	0	100	100	100	100	100	0	100	100
Serratia species	7	0	100	0	100	100	100	83	43	0	100	67
Moraxella species	40	100	100	98	100	100	100	5	100	98	100	100
Haemophilus species	26	4	100	4	77	100	100	85	96	56	81	65
Acinetobacter species	20	10	25	15	100	95	95	0	95	10	95	100
Enterobacter species	18	0	94	6	100	100	100	86	100	50	100	100
Hafnia alvei	1	0	100	0	100	100	100	—	0	0	100	100
Klebsiella species	5	0	80	0	100	100	100	50	100	100	100	80
Escherichia coli	10	10	90	0	100	100	100	100	100	90	100	90
Citrobacter species	2	0	100	0	100	100	100	100	100	50	100	100
Shigella sonnei	1	0	100	0	100	100	100	100	100	100	100	100
Proteus mirabilis	14	0	92	0	100	100	100	57	0	86	100	100
Proteus vulgaris	2	0	50	0	100	100	100	—	0	50	100	100
Morganella morganii	4	0	100	0	100	100	100	50	0	0	100	100
Providencia stuartii	1	0	0	0	0	0	0	0	0	0	0	—
Branhamella catarrhalis	8	100	100	13	100	100	88	0	100	75	100	88
Neisseria subflava	1	100	100	100	100	100	100	—	100	100	100	100
Pasteurella haemolytica	1	0	0	100	100	100	100	—	100	100	100	100
Capnocytophaga species	1	100	100	50	100	100	100	100	100	50	100	100
Alcaligenes species	7	0	43	0	29	71	86	0	100	0	29	100
Flavobacterium species	3	66	66	100	33	100	100	—	33	33	0	100
Stenotrophomonas maltophilia	4	0	67	25	25	75	75	0	75	0	25	100
Burkholderia cepacia	1	0	0	0	0	100	100	0	100	0	0	100
Brevundimonas vesicularis	1	0	—	100	0	0	100	—	100	0	0	100
Pseudomonas stutzeri	1	0	—	0	100	100	100	—	100	0	100	0
Pseudomonas species	1	0	0	0	100	0	0	0	0	0	100	100
BAC, bacitracin; CHL, chloramphenicol; VAN, vancomycin; GEN, gentamicin; CIP, ciprofloxacin; OFX, ofloxacin; TRI, trimethoprim; PB, polymyxin B; CEF, cefazolin; TOB, tobramycin; SULF, sulfasoxazole. (Data courtesy of the Charles T. Campbell Ophthalmic Microbiology Laboratory, The University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania.)

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Sep 18, 2016 | Posted by drzezo in OPHTHALMOLOGY | Comments Off

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