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148 Postgrad Med J 2001;77:148–156

REVIEWS Postgrad Med J: first published as 10.1136/pmj.77.905.148 on 1 March 2001. Downloaded from

Use of the diagnostic bacteriology : a practical review for the clinician

W J Steinbach, A K Shetty

Lucile Salter Packard Children’s Hospital at EVective utilisation and understanding of the Stanford, Stanford Box 1: technique University School of clinical bacteriology laboratory can greatly aid Medicine, 725 Welch in the diagnosis of infectious diseases. Al- (1) Air dry specimen and fix with Road, Palo Alto, though described more than a century ago, the methanol or heat. California, USA 94304, Gram stain remains the most frequently used (2) Add stain. USA rapid diagnostic test, and in conjunction with W J Steinbach various biochemical tests is the cornerstone of (3) Rinse with water to wash unbound A K Shetty the clinical laboratory. First described by Dan- dye, add mordant (for example, iodine: 12 potassium iodide). Correspondence to: ish pathologist Christian Gram in 1884 and Dr Steinbach later slightly modified, the Gram stain easily (4) After waiting 30–60 seconds, rinse with [email protected] divides into two groups, Gram positive water. Submitted 27 March 2000 and Gram negative, on the basis of their (5) Add decolorising solvent ( or Accepted 5 June 2000 wall and permeability to ) to remove unbound dye.

Growth on artificial medium Obligate intracellular (6) with . Chlamydia Legionella Gram positive bacteria stain blue Coxiella Ehrlichia Rickettsia (retained crystal violet). Gram negative bacteria stain red (decolorised and then counterstained). No Spirochetes Ureaplasma Leptospira Spirillum organic solvents (box 1).3–5 Information derived Treponema from a Gram stain and several simple bio- Aerobic chemical tests can be enormous, often provid- Cocci Coccobacilli ing a presumptive diagnosis and significantly Gram positive Gram negative Gram positive Gram negative Gram negative : influencing patient care. Unfortunately the

Enterococcus Arcanobacterium training for correctly interpreting the Gram http://pmj.bmj.com/ 6 Edwardsiella Bordetella stain is disappearing. The goal of this article is to review the utility of the clinical bacteriology Erysipelothrix Escherichia Campylobacter Gardnerella Hafnia Eikenella laboratory and discuss its role in the diagnosis Klebsiella Franciscella of common clinical . Morganella Proteus Helicobacter Gram stain basics Nocardia Providencia The Gram stain classifies bacteria (fig 1)

Serratia phenotypically based on diVerences in cell wall on October 1, 2021 by guest. Protected copyright. thickness with diVering glycosaminopeptide Yersinia and compositions: Gram positive bacteria have a layer 10–15 Non-enterobacteriaceae: (Fermentative) times thicker than Gram negative bacteria. The Aeromonas cell wall, synonymous with the peptidoglycan layer, is a rigid framework of cross linked pep- Plesiomonas tidoglycan forming the outermost component of the cell. The more complex Gram negative (Non-fermentative) bacteria also have an outer membrane beyond Acinetobacter the peptidoglycan layer that consists of li- Alcaligenes popolysaccharide (endotoxin), lipoprotein, and Burkholderia Flavobacterium phospholipids. In some Gram negative there also exists a periplasmic space between Stenotrophomonas Anaerobic the outer membrane and the inner cytoplasmic

Cocci Bacilli membrane with â-lactamases that degrade Gram positive Gram negative Gram positive Gram negative â-lactam . The present hypothesis for the mechanism of Bacteroides the Gram stain states the cell wall acts as a Bifidobacterium Fusobacterium Porphyromonas physical permeability barrier restricting diVu- 7 Eubacterium Prevotella sion of the stain complex, and any micro- Propionibacterium organism with a cell wall able to retard the Figure 1 Classification of clinically significant bacteria by . eZux of the crystal violet-iodine complex

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should be Gram positive. The mechanism fur- cells to stain Gram negatively. The age of the Postgrad Med J: first published as 10.1136/pmj.77.905.148 on 1 March 2001. Downloaded from ther implies that solvent decolorisation causes culture also influences the degree of Gram significant damage to the cell surfaces of Gram positivity, with cells 48 hours old sometimes negative bacteria, and only limited damage to more Gram positive than younger cells.8 Gram positive bacteria. This suggests Gram Several bacteria are unable to be Gram negative bacteria are more “leaky”, causing stained for a variety of reasons. Mycobacteria these thin walled -rich cells to lose their and nocardia have a high concentration of lip- crystal violet stain and appear red from the ids called mycolic acids in their cell walls and counterstain. Gram positive cells, thick walled are “acid-fast” because they resist decolorisa- and lipid-poor, appear blue from retaining the tion with an organic solvent. The spirochetes original crystal violet. (treponema, borrelia, leptospira, spirillum) are too thin and are best seen with darkfield Gram stain utility microscopy. Legionella, rickettsia, coxiella, Gram stain interpretation gives immediate ehrlichia, and are primarily intra- information regarding the presence or absence cellular and although possess outer and inner of bacterial disease and can guide initial membranes similar to Gram negative bacteria, treatment. Additionally, epithelial lack a peptidoglycan layer to take up a Gram and inflammatory cells are stained in a Gram stain adequately. Mycoplasma and ureaplasma stain, thus providing information about the do not have a cell wall to absorb the stain. immune response and quality of the specimen. A well prepared sample can show- Specific bacteria case the organism’s colour, size, shape, and GRAM POSITIVE COCCI arrangement, allowing cellular morphology to The two principal medically important genera further separate bacteria into four major are staphylococcus and streptococcus, ar- groups. Cocci are spherical or oval, bacilli are ranged in irregular grapelike clusters and rod-like or cylindrical, are comma-like chains, respectively (fig 2). The orientation and degree of attachment at the time of cell division or curved, and spirochetes are flexible (spirilla determines the type of arrangement: staphylo- if rigid) and helical. Additionally, coccobacilli cocci divide in three planes while streptococci are unusually short bacilli, and fusiform bacilli divide in only one plane. Enterococci are are bacilli with tapered ends. closely related to the streptococci yet are now known to be phylogenetically distinct and Limitations therefore comprise their own genus. The Several substances have been shown to convert , which degrades hydrogen Gram results. Ultraviolet light, antibi- peroxide to and water, diVerentiates otics, prolonged heat fixation, crushing of catalase positive staphylococci from catalase unprotected cells on a slide, or autolysis by negative streptococci and enterococci. such as ribonucleases or have all been shown to cause Gram positive STAPHYLOCOCCI

Catalase Staphylococci are a major component of the http://pmj.bmj.com/ normal human flora and the presence of Positive Negative , which accelerates the formation of a

Staphylococcus fibrin clot from fibrinogen, diVerentiates the species. Although there are 29 species of Coagulase coagulase negative staphylococci, most clinical Positive Negative isolates are either Staphylococcus epidermidis or S aureus Novobicin Staphylococcus saprophyticus. Staphylococcus epi- dermidis is part of the normal skin flora. on October 1, 2021 by guest. Protected copyright. Sensitive Resistant Although often occurring as a contaminant in specimens, S epidermidis may S epidermidis S saprophyticus cause in neonates, the immunocom- promised, and in patients with an indwelling central line, shunt placement, or prosthetic implant. Staphylococcus saprophyticus occurs Streptococcus chiefly in the periurethral and urethral flora Haemolysis where it shows a tropism for urinary tract epi- Beta Alpha (or gamma) thelium and causes urinary tract in

Bacitracin Bile esculin sexually active adolescent girls, second only to in this age group.9 SensitiveResistant Sensitive Resistant Positive Negative is an important patho- S pyogenes S agalactiae S pneumoniae Viridans Nutrionally variant gen, causing skin infections, , (most group C, G) group streptococcus , and septicaemia. It is dis- 6.5% NaCl growth tinguished on the positive results of coagulase, Positive Negative mannitol fermentation, and deoxyribonuclease S bovis (group D) tests.10 Selective media, such as mannitol salt Growth in tellurite , may be used for isolating S aureus when screening for carriage in infection control Positive Negative investigations. In the last decade the prevalence E faecalis E faecium of resistance to G among isolates of S Figure 2 DiVerentiating aerobic Gram positive cocci. aureus and S epidermidis has consistently

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exceeded 90%.11 Resistance to penicillin G is Capsular can be identified Postgrad Med J: first published as 10.1136/pmj.77.905.148 on 1 March 2001. Downloaded from due to the production of â-lactamases under using an latex particle agglutination the control of transmissible plasmids and can test for the cerebrospinal fluid, serum, or urine. be overcome with â-lactamase resistant (sec- Group B streptococci as well as Listeria ond generation) , such as nafcillin or monocytogenes, both major neonatal pathogens, methicillin. are treated with a penicillin while is Since the first case reports of methicillin added in the nursery for Gram negative cover- resistant S aureus (MRSA) in the United States age, namely E coli. in 1968, MRSA has become an increasing Group C streptococci species (chiefly Strep- problem.12 Several hospitals have reported tococcus equisimilis) are â-haemolytic and have methicillin resistance prevalence of 39% been identified as a cause of pharyngitis but are among S aureus and 75% among S epidermidis not associated with non-supporative complica- isolates,11 an example where Gram stain and tions like due to decreased biochemical diVerentiation can greatly influ- of the group specific ence antibiotic choice. Methicillin resistance to compared to the M . Group G strepto- S aureus is mediated by the chromosomal mecA cocci also produce a wide zone of â-haemolysis gene, which encodes a novel altered penicillin and occasionally cause cellulitis and bone and binding protein (PBP-2A) that causes resist- infections, often requiring the addition of ance to all â-lactam antibiotics, including an aminoglycoside with a penicillin for therapy. .13 The recent identification of derive their name from strains of S aureus with intermediate level the Latin word viridis, a reference to the green resistance to has caused great con- colour seen in the á-haemolysis, however some cern because vancomycin is the drug of choice species are â or ã-haemolytic. Viridans strepto- for MRSA.14 cocci, the preferred term since “Streptococcus viridans” implies a single species and not a STREPTOCOCCI group of species, lack classic virulence factors Streptococci may be classified according to the possessed by other streptococci and therefore type of haemolysis when cultured on blood have a low pathogenic potential in the normal agar, namely â, á,orã-haemolysis. host. Viridans streptococci are ubiquitous â-Haemolysis refers to complete haemolysis of inhabitants of the mouth and produce an the red blood cells in agar and therefore extracellular which may have a role in shows a clear zone around colonies due to the mediating bacterial adherence to heart valves production of enzymes called haemolysins. in .20 Viridans streptococci account á-Haemolysis is an incomplete haemolysis for 40.3% of bacterial endocarditis cases while producing a greenish discoloration around the other bacteria account for a minority of cases: colonies, while ã-haemolysis refers to non- S aureus (23.8%), S epidermidis (4.7%), and haemolysis. â-Haemolytic streptococci are fur- enterococci (4.0%).21 ther classified into Lancefield groups (A-H, Streptococci that grow in the intestine are K-V), based on the antigenic “C” carbohydrate now designated enterococci. Before recent

in the cell wall and reactions to pools of antis- reclassification raised enterococci to genus http://pmj.bmj.com/ era as originally described by Rebecca Lance- level, group D streptococci were divided into field. Groups A, B, C, D, and G are the groups enterococcal species (chiefly Enterococcus faeca- most commonly associated with human infec- lis, ) and non-enterococcal tions. species () based on the diVer- Group A () strepto- ential ability of the enterococci to grow in cocci, a responsible for a wide range hypertonic 6.5% saline solution. Most entero- of superficial and deep infections, are further cocci produce ã or á-haemolysis on blood agar

classified into certain types according to the M and all are able to grow on MacConkey on October 1, 2021 by guest. Protected copyright. protein, an antiphagocytic fibrillar molecule medium that contains bile salts. Most human that interferes with deposition of complement clinical isolates are either E faecalis (74–90%) C3b on the streptococcal cell wall surface.15 or E faecium (5%–16%)22 and biochemical tests Group A â-haemolytic streptococci antigen can further diVerentiate these two, important detection tests have been designed based on in planning therapy since E faecium is more extraction of the group specific carbohydrate antibiotic resistant than E faecalis. Enterococci antigen followed by detection with an are resistant to multiple drugs, including tagged reagent to produce a colour change. uniform resistance to cephalosporins, and Sensitivity ranges from 79% to 87%; specificity empiric treatment requires a penicillin plus an ranges from 90% to 96%.16 No penicillin aminoglycoside for synergy. resistant group A â-haemolytic streptococcal shows a characteris- strains have been identified. Although penicil- tic diplococci on Gram staining and is consist- lin tolerance has been described, with de- ently á-haemolytic and optochin sensitive. S creased bacterial killing by growth inhibiting pneumoniae possess a capsule antibiotic concentrations, its clinical signifi- which interferes with ; this capsule cance has not been defined.17 can be made to swell for rapid identification Group B () strepto- (quellung reaction) and diVerentiation into one cocci produce a narrow zone of â-haemolysis, of more than 90 . Penicillin and and may be identified presumptively by a posi- resistant S pneumoniae are tive CAMP test.18 The organism is a major emerging as a result of alterations in penicillin pathogen in neonatal , with increasing binding . Because resistance is not a attack rates inversely related to birth weight.19 result of the production of â-lactamases,

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major cause of and sepsis, Fermentation N gonor- Postgrad Med J: first published as 10.1136/pmj.77.905.148 on 1 March 2001. Downloaded from rhoeae, the cause of gonorrhoea and pelvic inflammatory disease, and None Glucose Maltose and glucose (formerly catarrhalis), which can cause respiratory infections including otitis M catarrhalis N gonorrhoeae N meningitidis media, , and pneumonia. These cocci, all diplococci, possess the enzyme cytochrome c and consequently are oxidase positive. Capsule Nitrate reduction Because the trace metals and fatty acids found in blood agar inhibit both neisseria spe- Yes No Yes No cies they are cultured on “chocolate” agar, a ° N meningitidis N gonorrhoeae M catarrhalis N gonorrhoeae blood agar heated to 80 C to inactivate the M catarrhalis N meningitidis inhibitors. Non-selective is used for usually sterile sites such as cerebrospinal V Figure 3 Di erentiating aerobic Gram negative cocci. fluid, blood, or synovial fluid. Thayer-Martin antibiotics with â-lactamase inhibitors such as or Martin-Lewis selective media is used for clavulonic acid are not helpful. sites where contamination of other bacterial In recent years the proportion of penicillin flora is suspected, such as urethral cultures. non-susceptible pneumococcal invasive iso- The two major Neisseria spp can be diVerenti- lates has varied from 0%–41%.23 Of these ated from each other by carbohydrate utilisa- isolates, 5%–21% exhibit penicillin resistance. tion tests. The incidence of cefotaxime and Moraxella catarrhalis is one of the three main non-susceptible pneumococcal isolates has agents in acute and sinusitis increased to 20% in some areas. The Streptococ- besides S pneumoniae and non-typable Haemo- cus pneumoniae Therapeutic Working Group philus influenzae. Nearly 100% of strains of M recently advocated using higher dose amoxicil- catarrhalis produce â-lactamases. Although lin in less invasive infections, such as acute oti- remains an eVective empiric tis media, to overcome penicillin binding therapy for acute otitis media, suspicion or iso- protein resistance in high risk patients.24 The lation of M catarrhalis warrants the addition of concern for resistance has also brought about a â-lactamase inhibitor (that is, clavulonic the practice of simultaneously using vancomy- acid). cin and a third generation cephalosporin, ceftriaxone or cefotaxime, as empiric coverage GRAM POSITIVE BACILLI for meningitis due to suspected S pneumoniae Bacillus, clostridium, listeria, and corynebacte- in patients beyond 3 months of age. Vancomy- rium are the four medically important genera cin addresses the possibility of cephalosporin of Gram positive rods (fig 4), with anaerobic resistance while a well absorbed cephalosporin growth diVerentiating the forming compensates for the poor cerebrospinal fluid clostridium and bacillus while mobility diVer- penetrability of vancomycin.25 entiates the two non-spore forming Gram

positive bacilli. Most Bacillus spp are non- http://pmj.bmj.com/ pathogenic, but is the cause of GRAM NEGATIVE COCCI the disease while is a There are three medically important Gram cause of food poisoning. Clostridium spp include negative cocci (fig 3): ,a the causative agents of , food poi- soning, , , and antibiotic asso- Spore forming ciated . Yes No can be diagnosed by

Gram stain alone with the appearance of Gram on October 1, 2021 by guest. Protected copyright. Bacillus A haemolyticum positive rods in small, grey colonies with a nar- Corynebacterium row zone of â-haemolysis resembling diph- Capsule, penicillin E rhusiopathiae theroids; it may be assumed to be a contami- sensitive G vaginalis nant. Listeria monocytogenes is a common cause Lactobacillus of infection in neonates and the immunocom- Yes No L monocytogenes B anthacis B cereus Mycobacterium promised and infection in pregnancy accounts Nocardia for 27% of all cases of listerosis, usually occur- ring in the third trimester due to a decline in cell mediated immunity seen at 26–30 weeks’ Motile gestation.26 Because L monocytogenes are uni- Yes No formly not susceptible to cephalosporins, the practice of beginning and cefo- L monocytogenes Corynebacterium taxime as empiric coverage is B cereus Erysipelothrix questionable. Additionally, routine use of these antibiotics may contribute to cephalosporin Haemolysis Catalase resistance among strains of , , and in the nursery.27 Gamma Beta Positive Negative Klebsiella spp spp Corynebacterium diphtheriae, the cause of B anthracisA haemolyticum Bacillus A haemolyticum diphtheria, are non-motile Gram positive rods B cereus C diphtheriae Erysipelothrix with metachromatic granules, often arranged L monocytogenes L monocytogenes Lactobacillus as “Chinese lettering” on Gram stain. A throat Figure 4 DiVerentiating aerobic Gram positive cocci. swab should be cultured on LöZer’s medium

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to inhibit normal flora and enhance the ment glucose (fermentation of other Postgrad Med J: first published as 10.1136/pmj.77.905.148 on 1 March 2001. Downloaded from metachromasia and a tellurite plate to highlight varies), reduce nitrates to nitrites, and are oxi- the reduction of tellurium salt in the organ- dase negative (figs 5–7). Suspected enteric ism.28 In patients with the clinical picture of bacteria are inoculated on a blood as tonsillopharygneal diphtheria characterised by well as a selective medium such as MacCon- a thick, grey, adherent membrane over the ton- key’s agar or eosin- agar to sup- sils and throat the Gram stain can make the press unwanted Gram positive organisms by diagnosis as the methylene blue stain reveals bile salts and bacteriostatic dyes. Lactose the typical metachromatic granules. fermenters form coloured colonies while triple agar, composed of ferrous sulfate GRAM NEGATIVE BACILLI and three sugars (glucose, fructose, and Enteric tract sucrose), determine fermentation as well as The family enterobacteriaceae, often called hydrogen sulphide production. Urea agar is “enterics” due to their normal habitat in the used to determine production, which colon of humans and animals, are diVerenti- hydrolyses urea to ammonia and carbon ated by a range of biochemical tests but all fer- dioxide and turns the pH alkaline.

Enterobacteriaceae Non-enterobacteriaceae (catalase positive, oxidase negative, nitrates reduced to nitrites, glucose fermented) Fermentative Non-fermentative

Citrobacter Proteus Aeromonas Acinetobacter Edwardsiella Providencia Pasteurella Alcaligenes Enterobacter Salmonella Plesiomonas Burkholderia Escherichia Serratia Vibrio Flavobacterium Hafnia Shigella Pseudomonas Klebsiella Yersinia Stenotrophomonas Morganella Lactose fermenter

Positive Negative

Escherichia coli Edwardsiella tarda Enterobacter aerogenes, cloacae Morganella morganii Citrobacter (50%) Providencia Pseudomonas Salmonella typhi , parahaemolyticus

Yersinia pestis, enterocolitica, pseudotuberculosis http://pmj.bmj.com/

Urease H2S production

Positive Negative Positive Negative

Citrobacter Alcaligenes Citrobacter diversus Klebsiella Edwardsiella tarda Escherichia coli Morganella Pasteurella P vulgaris, mirabelis Klebsiella on October 1, 2021 by guest. Protected copyright. Proteus Salmonella Salmonella Morganella Providencia Shigella Providencia Y enterocolitica Y pestis Serratia marcescens Y pseudotuberculosis Shigella Y pseudotuberculosis, enterocolitica

Oxidase Motile

Positive Negative Yes No

Aeromonas hydrophilia Acinetobacter Enterobacter Flavobacterium Alcaligenes Klebsiella pneumoniae Escherichia Klebsiella Burkholderia cepacia Stenotrophomonas maltophilia Proteus Shigella , C fetus Pseudomonas Y pestis Flavobacterium Salmonella Serratia Y pseudotuberculosis, enterocolitica Pasteurella multocida V cholerae, V parahaemolyticus Figure 5 DiVerentiating aerobic Gram negative bacilli.

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the ; the “H” antigen is the Aerobic Gram negative coccobacilli Postgrad Med J: first published as 10.1136/pmj.77.905.148 on 1 March 2001. Downloaded from Bartonella flagellar antigen. In haemolytic uraemic syn- Brucella melitensis drome E coli O157:H7 produces a shiga-like Campylobacter verotoxin, named because it is toxic to Vero Chlamydiae (African green monkey) cell culture. Es- Ehrlichia Eikenella corrodens cherichia coli O157:H7 is easily separated as it Franciscella tularensis does not ferment sorbitol and forms pale colo- Helicobacter nies on sorbitol MacConkey agar. Kingella Salmonella spp include the causes of typhoid Bartonella and paratyphoid fevers, , sepsis, Legionella pneumonphila Rickettsia and osteomyelitis, especially in patients with sickle cell disease. Unlike salmonella, shigella Motile Haemolysis does not produce hydrogen sulphide gas (neither ferment lactose) and is immobile. Yes No Gamma Beta Shigella produces bloody diarrhoea by invasion C jejuni Bordetella pertussis H influenzae Bordetella pertussis of the mucosa of the distal ileum and colon and H pylori Brucella melitensis Kingella kingae is much more virulent than salmonella: as few Legionella Franciscella Haemophilus influenzae as 100 organisms are necessary for disease as Kingella kingae opposed to the 10 000 organisms required with 29 Figure 6 DiVerentiating aerobic Gram negative coccobacilli (including obligate salmonella or V cholerae. More selective intracellular). media such as xylose-lysine deoxycholate may be used to isolate shigella or salmonella from A methylene blue stain of a fecal sample will faecal specimens. determine whether polymorphonuclear cells Five major non-enterobacteriacae also in- (PMNs) are present. The presence of PMNs habit the enteric tract. Vibrio causes indicates the involvement of an invasive organ- cholera and is a comma shaped, oxidase ism, such as shigella, salmonella, campylo- positive Gram negative bacillus and its charac- bacter, rather than a -producing organism teristic appearance can help make a presump- such as Vibrio cholerae, E coli, or Clostridium tive diagnosis. Campylobacter are also comma perfringens. Escherichia coli and salmonella pro- or S shaped, oxidase positive, and often duce disease both within and outside the interpreted as coccobacilli on Gram stain. enteric tract; in contrast, shigella, vibrio, Campylobacter jejuni causes enterocolitis while campylobacter, and helicobacter produce dis- Campylobacter intestinalis causes bacteraemia; ease primarily within the enteric tract. the two are diVerentiated by nalidixic acid sen- Escherichia coli is the most abundant faculta- sitivity. Helicobacter pylori, the cause of gastritis tive anaerobe in the colon and faeces, although and , is urease positive and vastly outnumbered by the may be demonstrated by Giemsa staining of Bacteriodes fragilis, and the five major subdivi- gastric . Anaerobic Gram negative sions each cause diVerent clinical pictures. bacilli such as Bacteroides fragilis are abundant

in the human colon whereas http://pmj.bmj.com/ Escherichia coli O157:H7, famous in the public Fusobacterium spp media for outbreaks of food poisoning, is so and others are normal flora in the human oral cavity. named by its . The “O” or somatic antigen, is the outer polysaccharide portion of Cocci Bacilli Of the six serotypes of H influenzae (a-f), type b Gram positive Gram negative Gram positive Gram negative (Hib) causes the majority of invasive disease Non-spore forming such as meningitis and epiglotitis. The H influ- Peptococcus niger Actinomyces israelii Bacteroides fragilis Peptostreptococcus Bifidobacterium Fusobacterium enzae species involved in acute otitis media and on October 1, 2021 by guest. Protected copyright. Eubacterium Porphyromonus sinusitis are largely unencapsulated and, there- Propionibacterium Prevotella fore, non-typable strains. The incidence of Spore forming invasive Hib disease has declined dramatically Clostridium since the introduction of the polyribosylribose phosphate in April 1985. Depending Haemolysis on local patterns, 10% to 40% of H influenzae 30 Yes No isolates produce â-lactamases. Latex particle agglutination for detection of capsular antigen C perfringens C difficile C septicum Eubacterium in the cerebrospinal fluid is available, but anti- Propionibacterium acnes Fusobacterium gen detection in the serum and urine can be Peptostreptococcus unreliable due to asymptomatic nasopharyn- Prevotella Veillonella geal carriage. Cultures of H influenzae require the growth factors haemin (X) and/or nicotina- mide adenine diphosphate (V) provided by Beta lactamase production heated blood agar. Yes No Legionella spp are bacilli that stain faintly Gram negative with the standard Gram stain Bacteroides fragilis Clostridium and specimens do not stain with haema- Fusobacterium Eubacterium Prevotella melaninogenica Peptostreptococcus toxylin and eosin, requiring the use of the Diet- Prevotella oralis Propionibacterium acnes erle silver impregnation stain. Because these Veillonella organisms require high concentrations of iron Figure 7 DiVerentiating anaerobic bacteria. and cysteine to grow, Legionella pneumophilia

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fails to grow on ordinary media and is cultured genera are urease positive and a common cause Postgrad Med J: first published as 10.1136/pmj.77.905.148 on 1 March 2001. Downloaded from on buVered charcoal extract medium or of urinary tract infections. Proteus spp are char- investigated directly by immunofluorescence. acterised by their ability to “swarm” on blood The majority of human disease is caused by L agar plates. pneumophilia serogroup 1, which can be detected in the urine by radioimmunoassay, enzyme Zoonotic immunoassay, or serologically.31 Most species Because of the risks that the major zoonotic produce some â-lactamases. bacteria such as Brucella spp, tularen- Bordetella pertussis, the cause of whooping sis, , and Pasteurella multocida pose cough, occurs as Gram negative coccobacilli to laboratory personnel they are seldom singly or in pairs. Bordetella pertussis can best be cultured; consequently, diagnosis is made sero- isolated from nasopharyngeal swabs (calcium logically. Brucella melitensis, the agent in what alginate) obtained during the catarrhal stage was originally known as Malta fever, is when the organisms attach to the ciliated localised in the reticuloendothelial system of the upper respiratory tract and where it survives within as a fac- cause decreased cilia activity and epithelial cell ultative intracellular parasite. Tularaemia (rab- death.32 The special medium used for culture bit fever or deer fly fever) is an infection caused in the past, Bordet-Gengou medium, by . Humans often serve as has now been replaced with Regan-Lowe agar, accidental hosts who acquire infection after a half strength charcoal agar with horse blood bitten by a dermacentor tick or removing the and cephalexin.33 Direct fluorescent antibody hide of an infected animal. Yersinia pestis is the staining is also used, but is less sensitive than encapsulated organism responsible for the culture. No single serological test is diagnostic “,” and mimics of pertussis. A profound leukocytosis, with up appendicitis. Laboratory identification of yers- to 70% lymphoctes, can be seen. These are inia in stool can be made by characteristic generally “typical” lymphocytes, as opposed to “safety-pin” bipolar appearance in Wayson’s the classic “atypical” lymphocytes seen in stain, the use of fluorescent antibody testing, or Epstein-Barr virus infections. serological testing with passive haemagglutina- Pseudomonas and related species include tion or enzyme immunoassay.35 Pasteurella mul- bacteria that are ubiquitous, some of which are tocida is part of the normal oral flora of domes- important pathogens. Pseudomonas aeruginosa tic cats and dogs and is may cause infection in causes a wide variety of infections, including bite wounds. wound infections, urinary tract infections, and septicaemia. Pseudomonas aeruginosa is a non- SPIROCHETES lactose fermenter, oxidase positive, and isolates Spirochetes are spiral, motile organisms that can be classified as smooth, rough, or mucoid are not easily cultivated in the routine labora- based on their appearance on agar. The tory. The three genera of importance are mucoid strains isolated from patients with borrelia, treponema, and leptospira. Borrelia cystic fibrosis produce alginate, a polysaccha- burgdorferi causes Lyme disease, while Borrelia 34

ride polymer with antiphagocytic activity. All recurrentis and Borrelia hermsii cause relapsing http://pmj.bmj.com/ psuedomonads have chormosomally encoded fever, so named for its anitgenic variation dur- â-lactamases (not plasmid mediated) which are ing relapses of the illness. Diagnosis of Lyme inducible and therefore may not be detectable disease is made with serological tests, most in vitro until exposed to â-lactam antibiotics in commonly enzyme immunoassay, and due to vivo. the concern for cross reactivity with other spi- Recently there have been numerous taxo- rochetal a second step using western nomic changes in the pseudomonas genus, cre- immunoblot is now advocated for verification 36 ating the separate genera stenotrophomonas of enzyme immunoassay results. Cultures for on October 1, 2021 by guest. Protected copyright. and burkholderia. Burkholderia cepacia (for- B burgdoferi are rarely positive, but culture of merly Pseudomonas cepacia) is an increasingly the organism from the tick vector is usually important pathogen in patients with cystic positive. Borrelia recurrentis can be seen in fibrosis. It requires a unique agar for isolation Giemsa stains of blood films from infected and can be detected by its resistance to . patients. Stenotrophomonas maltophilia (formerly Xan- , the cause of , thomonas maltophilia)isdiVerentiated as oxi- may be identified as tightly wound spirochetes dase negative. Virtually all isolates of S using dark field microscopy since only non- maltophilia are resistant to penicillins, cepha- pathogenic treponemes have ever been grown losporins, and aminoglycosides and all are in culture. Generally serological tests are used highly resistant to imipenem; - in the diagnosis of syphilis with non- is the drug of choice. treponemal antigens such as cardiolipin from The encapsulated Klebsiella pneumoniae his- beef heart reacting with serum antibodies torically has been recognised as the causative (called reagins). Flocculation tests like the agent in pneumonia characterised by thick, Venereal Disease Research Laboratory and bloody “currant-jelly” . Serratia marces- detect these antibodies. cens produces a striking red pigment, and it and Treponema pallidum in treponemal specific tests Enterobacter cloacae are often nosocomial infec- react with immunofluorescence in the fluores- tions related to invasive procedures. Previously cent treponemal antibody absorbed test or four species were classified as proteus, however haemagglutination in the microtitre haemag- two of these have been renamed Providencia glutination assay T pallidum. Whereas a non- rettgeri and Morganella morganii. All three treponemal test usually becomes non-reactive

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after successful therapy, treponemal tests 2 Gram C. Uber die isolirte Farung der Schizomycetin in Schnitt-und Trockenpraparaten. Fortschr Med 1884;2:185– Postgrad Med J: first published as 10.1136/pmj.77.905.148 on 1 March 2001. Downloaded from remain reactive for life despite successful 9. therapy. 3 Bartholomew JW. Variables influencing results and the pre- cise definition of steps in gram staining as a means of stand- Leptospira interrogans, the cause of lepto- ardizing the results obtained. Stain Technol 1962;37:139–55. spirosis, is occasionally isolated from blood and 4 Bartholomew JW, Mittwer T. The Gram stain. Bacteriol Rev urine in special cultures, but diagnosis is made 1952;16:1–29. 5 Bottone EJ. The Gram stain: the century-old quintessential through a marked rise in enzyme immunoassay rapid diagnostic test. Lab Med 1988;19:288–91. or agglutination antibodies. 6 Mandel LP, Schaad DC, Cookson BT, et al.Evaluation of an interactive computer program to teach Gram-stain interpretation. Acad Med 1996;71(10 suppl):S100–2. OBLIGATE INTRACELLULAR ORGANISMS 7 Popescu A, Doyle RJ. The Gram stain after more than a century. Biotech Histochem 1996;71:145–51. These bacteria lack some of the mechanism for 8 Biswas BB, Basu PS, Pal MK. Gram staining and its production of energy and therefore grow only molecular mechanism. Int Rev Cytol 1970;29:1–27. 9 Joklik WK, Willett HP, Amos DB, et al. Staphylococcus. In: inside host cells. Their cell walls resemble Joklik WK, Willet HP, Amos DB, et al,eds.Zinsser microbiol- Gram negative bacteria, but lack muramic ogy. 20th Ed. East Norwalk, CT: Appleton & Lange, 1992: 401–16. acid. Chlamydia trachomatis is the most com- 10 Wilkinson BJ. Biology. In: Crossley KB, Archer GL, eds. The mon chlamydiae pathogen and new nucleic staphylococci in human disease. New York: Churchill- acid amplification using ligase chain reactions Livingstone, 1997: 1–38. 11 Maranan MC, Moreira B, Boyle-Vavra S, et al. Antimicro- is more sensitive than cell culture and detects bial resistance in staphylococci. Epidemiology, molecular antigen in the urine.37 Diagnosis of rickettsiae is mechanisms, and clinical relevance. Infect Dis Clin North Am 1997;11:813–49. usually made serologically and Rocky Moun- 12 Barrett FF, McGehee RF, Finland M. Methicillin-resistant tain () is best Staphylococcus aureus at Boston City Hopsital. N Engl J Med 1968;279:441–8. detected through indirect fluorescent antibody 13 Archer G, Niemeyer DM. Origin and evolution of DNA and indirect haemagglutination, but antibodies associated with resistance to methicillin in staphylococci. Trends Microbiol 1994;2:343–7. are detected 7–10 days after illness. No micro- 14 Centers for Disease Control and Prevention. Staphylococ- biological test is readily available for rapid cus aureus with reduced susecptibility to vancomycin— diagnosis early in the illness; the polymerase United States, 1997. MMWR Morb Mortal Wkly Rep1997; 46:813–15. chain reaction has been used during the acute 15 Joklik WK, Willett HP, Amos DB, et al. Streptococcus. In: phase. This test, while specific, is insensitive Joklik WK, Willet HP, Amos DB, et al,eds.Zinsser microbiol- ogy. 20th Ed. East Norwalk, CT: Appleton & Lange, 1992: and performs only slightly better on skin biop- 417–31. sies than blood specimens.38 16 Pichichero, ME. Group A beta-hemolytic streptococcal infections. Pediatr Rev 1998;19:291–302. 17 Kim KS. Clinical perspectives on penicillin tolerance. J ORGANISMS WITH NO CELL WALL Pediatr 1988;112:509–14. 18 Christie R, Atkins NE, Munch-Peterson E. A note of a lytic are small, non-motile, freeliving phenomenon shown by group B streptococci. Aust J Exp organisms that lack a cell wall, which means Biol Med Sci 1944;22:197–200. 19 Baker CJ, Edwards MS. Streptococcus agalactiae (group B there are no Gram stain results and antibiotics streptococcus). In: Long SS, Pickering LK, Prober CG, eds. that inhibit cell wall synthesis (for example, Principles and practice of pediatric infectious diseases.New York: Churchill-Livingstone, 1997: 812–18. penicillins and cephalosporins) are ineVective. 20 Haslam DB, St Geme III JW. Viridans streptococci, The majority of infections caused by Myco- nutritionally variant streptococci, and Streptococcus bovis. include pneumonia and tra- In: Long SS, Pickering LK, Prober CG, eds. Principles and plasma pneumoniae practice of pediatric infectious diseases. New York: Churchill- cheobronchitis, while can Livingstone, 1997: 821–3. http://pmj.bmj.com/ cause , postpartum infection, and pel- 21 Starke JR. . In: Feigin RD, Cherry JD, eds. Textbook of pediatric infectious diseases. 4th Ed. vic inflammatory disease. Mycoplasmas are Philadelphia: WB Saunders, 1998: 315–38. slow growing so diagnosis is made serologi- 22 English BK, Shenep JL. Enterococcal and viridans strepto- coccal infections. In: Feigin RD, Cherry JD, eds. Textbook of cally. In children cold agglutinins, immu- pediatric infectious diseases. 4th Ed. Philadelphia: WB noglobulin M autoantibodies against type O Saunders, 1998: 1106–20. ° 23 American Academy of Pediatrics Committee on Infectious red blood cells that agglutinate at 4 C but not Diseases. Therapy for children with invasive pneumococcal at 37°C, are not as reliable as in adults. infections. Pediatrics 1997;99:289–99.

24 Dowell SF, Butler JC, Giebink S, et al. Acute otitis media: on October 1, 2021 by guest. Protected copyright. Ureaplasma can be distinguished from myco- management and surveillance in an era of pneumococcal plasma by its ability to produce urease. resistance—a report from the drug-resistant Streptococcus pneumoniae Therapeutic Working Group. Pediatr Infect Dis J 1999;18:1–9. Conclusion 25 Willoughby R, Polack F. Meningitis: what’s new in diagno- The clinical bacteriology laboratory can be sis and management. Contemp Pediatr 1998;15:49–70. 26 Lorber B. Listeria monocytogenes. In: Long SS, Pickering pivotal in guiding clinicians to make a rapid LK, Prober CG, eds. Principles and practice of pediatric infec- diagnosis and initiate appropriate treatment. tious diseases. New York: Churchill-Livingstone, 1997: 873–9. The Gram stain is the microbiologists’ century 27 American Academy of Pediatrics. Escherichia coli and other old quintessential first line diagnostic tool Gram-negative bacilli. In: Peter G, ed. 1997 Red book: report of the Committee on Infectious Diseases. 24th Ed. Elk Grove allowing preliminary identification of bacteria. Village, IL: American Academy of Pediatrics, 1997: 202–4. HousestaV physicians should receive formal 28 Levinson WE, Jawetz E. Corynebacterium diphtheriae. In: training in the interpretation of the Gram stain Levinson WE, Jawetz E, eds. Medical & immunology. 3rd Ed. Norwalk, CT: Appleton & Lange, and other basic clinical bacteriological tests. A 1994: 84–6. more rigorous and confident use of clinical 29 Levinson WE, Jawetz E. Shigella. In: Levinson WE, Jawetz E, eds. & immunology. 3rd Ed. microbiological knowledge may allow greater Norwalk, CT: Appleton & Lange, 1994: 96. precision in diagnosis and focused narrow 30 American Academy of Pediatrics. Haemophilus influenzae infections. In: Peter G, ed. 1997 Red book: report of the Com- spectrum antibiotic treatment, thus curbing mittee on Infectious Diseases. 24th Ed. Elk Grove Village, IL: the growing trend of inappropriate antibiotic American Academy of Pediatrics, 1997: 220–31. use in the current era of increased antimicro- 31 American Academy of Pediatrics. Legionella pneumophilia infections. In: Peter G, ed. 1997 Red book: report of the Com- bial resistance. mittee on Infectious Diseases. 24th Ed. Elk Grove Village, IL: American Academy of Pediatrics, 1997: 319–21. 32 Levinson WE, Jawetz E. Bordetella. In: Levinson WE, 1 Friedlander C. Die Mikrokokken der Pneumonie. Fortschr Jawetz E, eds. Medical microbiology & immunology. 3rd Ed. Med 1883;1:715–33. Norwalk, CT: Appleton & Lange, 1994: 108–9.

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33 Joklik WK, Willett HP, Amos DB, et al. Bordetella. In: Joklik 36 American Academy of Pediatrics. Lyme disease. In: Peter G, WK, Willet HP, Amos DB, et al,eds.Zinsser microbiology. ed. 1997 Red book: report of the Committee on Infectious Postgrad Med J: first published as 10.1136/pmj.77.905.148 on 1 March 2001. Downloaded from 20th Ed. East Norwalk, CT: Appleton & Lange, 1992: 473– Diseases. 24th Ed. Elk Grove Village, IL: American Academy 80. of Pediatrics, 1997: 329–33. 34 Brady MT, Feigin RD. Pseudomonas and related species. 37 American Academy of Pediatrics. Chlamydia trachomatis In: Feigin RD, Cherry JD, eds. Textbook of pediatric infectious In: Peter G, ed. 1997 Red book: report of the Committee on diseases. 4th Ed. Philadelphia: WB Saunders, 1998: 1401– Infectious Diseases. 24th Ed. Elk Grove Village, IL: American 13. Academy of Pediatrics, 1997: 170–4. 35 American Academy of Pediatrics. Plague. In: Peter G, ed. 38 American Academy of Pediatrics. Rocky Mountain spotted 1997 Red book: report of the Committee on Infectious Diseases. fever. In: Peter G, ed. 1997 Red book: report of the Committee 24th Ed. Elk Grove Village, IL: American Academy of Pedi- on Infectious Diseases. 24th Ed. Elk Grove Village, IL: Ameri- atrics, 1997: 408–10. can Academy of Pediatrics, 1997: 452–6. http://pmj.bmj.com/ on October 1, 2021 by guest. Protected copyright.

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