Gram-stain procedure
Taxonomic classification of bacteria
Gram-positive and Gram-negative bacteria can be further subdivided according to their morphological and biochemical characteristics. The principal subdivisions include:
morphology (shape) on microscopy
cocci (spherical)
bacilli (rod-shaped)
Oxygen (O2) requirements
aerobes (grow in the presence of O2)
obligate aerobes require O2 to grow
facultative anaerobes can grow in the presence or absence of O2
anaerobes (require the exclusion of O2 to grow).
By combining the appearance on microscopy after Gram staining with the growth characteristics (aerobic/anaerobic), a rapid initial putative identification may be made as shown in Table 20.1.
| O2 requirements | Gram-positive bacteria | Gram-negative bacteria | ||
|---|---|---|---|---|
| Cocci | Bacilli (rods) | Cocci | Bacilli (rods) | |
| Obligate aerobes (require the presence of O2 to grow) | Micrococcus spp. | Nocardia spp. (Mycobacterium tuberculosis)a | Neisseria meningitidis N. gonorrhoeae | Pseudomonas aeruginosa Bordetella pertussis |
| Facultative anaerobes (can tolerate aerobic and anaerobic conditions) | Staphylococcus spp. (e.g. S. aureus) Streptococcus spp. Enterococcus spp. | Corynebacterium spp. (e.g. C. diphtheriae) Listeria monocytogenes Bacillus spp. | There are no common pathogens within the facultative anaerobic Gram-negative cocci group | Escherichia coli Klebsiella spp. Enterobacter spp. Citrobacter spp. Haemophilus influenzae (coccobacillus) |
| Obligate anaerobes (require the exclusion of O2 to grow) | Peptostreptococcus spp. | Clostridium spp. (e.g. C. tetani, C. difficile) Actinomyces israelii Lactobacillus spp. Propionibacterium spp. | Veillonella parvula | Bacteroides spp. Fusobacterium spp. |
a Weakly Gram-positive, stains better with Ziehl–Neelsen stain | spp. = two or more species
Biochemical properties and growth characteristics can also help to further subdivide related bacteria. For example, Gram-negative bacteria can be broadly divided upon their ability to ferment glucose:
Enterobacteriaceae are glucose fermenters
pseudomonads and related organisms are glucose non-fermenters.
Gram-negative organisms will grow well on the majority of laboratory media, but they are best identified on MacConkey agar. MacConkey agar contains bile salts and is selective for enteric bacteria. It is also an indicator medium, allowing differentiation between lactose fermenters (which produce a pink colour, e.g. Escherichia coli, Klebsiella spp.) and lactose nonfermenters (which produce a pale yellow colour, e.g. Pseudomonas aeroginosa, Serratia spp.).
New methods of identifying microorganisms
Molecular methods
Molecular techniques have had a direct influence on the clinical practice of medical microbiology. In many cases where traditional phenotypic methods (using enzyme reactions and characteristics) of microbial identification and typing are insufficient or time-consuming, molecular techniques can provide rapid and accurate data, potentially improving clinical outcomes.
Polymerase chain reaction (PCR) is used in microbiology to amplify (replicate many times) a single DNA sequence.
Gel electrophoresis is used routinely in microbiology to separate DNA, RNA or protein molecules using an electric field by virtue of their size, shape or electric charge.
Southern blotting, Northern blotting, Western blotting and Eastern blotting are molecular techniques for detecting the presence of microbial DNA sequences (Southern), RNA sequences (Northern), protein molecules (Western) or protein modifications (Eastern).
DNA sequencing and genomics have been used for many decades in molecular microbiology studies. Due to their relatively small size, viral genomes were the first to be completely analysed by DNA sequencing. A huge range of sequence and genomic data is now available for a number of species and strains of microorganisms. Increasingly, this is becoming the gold standard for identifying species, typing organisms and identifying antimicrobial resistance. It also allows the production of a genetic tree looking at relatedness of organisms to predict whether a transmission event could have occurred.
Specific examples of clinical use include identifying organisms through their molecular sequence when phenotypical methods have failed, rapid detection of organisms (e.g. influenza, methicillin-resistant Staphylococcus aureus [MRSA]), typing to review the relatedness of strains (e.g. Spa typing of Staphylococcus aureus), detection of resistance (e.g. rifampicin resistance in Mycobacterium tuberculosis).
Matrix-assisted laser desorption/ionisation/time of flight (MALDI/TOF)
MALDI/TOF is an ionisation technique used in mass spectrometry, allowing the analysis of biomolecules. The type of a mass spectrometer most widely used with MALDI is the TOF (time-of-flight mass spectrometer), mainly due to its large mass range.
MALDI/TOF spectra are used for the identification of microorganisms such as bacteria or fungi. A colony of the microbe in question is smeared directly on the sample target and overlayed with matrix. The mass spectra generated are analysed by dedicated software and compared with stored profiles. Species diagnosis by this procedure is much faster, more accurate and cheaper than other procedures based on immunological or biochemical tests. MALDI/TOF may become the standard method for species identification in medical microbiological laboratories over the next few years. It can also be used to determine resistance profiles of organisms where certain proteins are expressed.
Toxin-mediated effects of bacteria
The pathogenic potential of several bacteria is enhanced by the production of either exotoxins or endotoxins. Exotoxins are proteins secreted by bacteria. Some important exotoxins are listed in Table 20.2. The properties of exotoxins and endotoxins are compared in Table 20.3.
| Organism | Exotoxin | Action | Clinical significance |
|---|---|---|---|
| Staphylococcus aureus | Toxic shock syndrome toxin 1 | Polyclonal T cell activation Cytokine release Fever and shock | Toxic shock syndrome |
| Enterotoxins A–E | Vomiting | Food poisoning | |
| Epidermolytic toxin | Intraepidermal blisters and desquamation | Scalded skin syndrome | |
| Panton–Valentine leucocidin | Lysis of leucocytes | Invasive, pyogenic and necrotising infections | |
| Streptococcus pyogenes | Streptococcal pyrogenic exotoxins A and B | Endothelial damage Fever | Tissue oedema |
| Streptolysin O | Lysis of erythrocytes and leucocytes | Poststreptococcal rheumatic fever | |
| Clostridium spp. | C. tetani toxin | Sustained neuronal discharge | Motor spasms |
| C. botulinum toxin | Neuromuscular blockade | Botulism (food poisoning, wound botulism) | |
| C. difficile toxins A and B, binary toxin | Cytotoxicity | Pseudomembranous colitis (association with the use of broad-spectrum antibiotics) | |
| Corynebacterium diphtheriae | Diphtheria toxin | Inhibition of protein synthesis | Diphtheria |
| Vibrio cholera | Cholera enterotoxin | Activation of adenylate cyclase and gastrointestinal water loss | Cholera (torrential diarrhoea) |
| Shigella dysenteriae | Shiga toxin | Inhibition of protein synthesis and cell death | Bacterial dysentery |
| Verocytotoxigenic Escherichia coli | Verocytotoxin | Inhibition of protein synthesis and cell death | Bacterial dysentery Haemorrhagic colitis |
| Parameter of comparison | Exotoxins | Endotoxins |
|---|---|---|
| Producing organisms | Mostly Gram-positive organisms (and a few Gram-negative organisms) | Only Gram-negative organisms |
| Chemical composition | Proteins | Lipopolysaccharide |
| Site of production | Manufactured in the bacterial-cell cytoplasm | Bacterial outer-membrane component |
| Release | Secreted from cell | Released from membrane as vesicles (blebs) during bacterial cell death |
| Biochemical properties | Heat-labile Denatured by formaldehyde | Heat-stable Not denatured by formaldehyde |
| Antigenicity | Neutralised by specific antibodies; therefore immunity can be developed | Poorly antigenic; only partially neutralised by specific antibodies |
Important pathogenic bacteria and associated disease states
This section considers bacterial pathogens of the female urogenital tract and a variety of other bacterial infections that are of relevance to obstetrics or gynaecology.
Streptococcaceae
These are Gram-positive cocci that are facultative anaerobes. They typically grow in chains or pairs. Streptococci can be distinguished from other Gram-positive cocci biochemically by a negative catalase test (failure to hydrolyse hydrogen peroxide to O2 and H2O).
There are numerous genera within the Streptococcaceae. Most pathogens fall into two main genera:
Streptococcus
Enterococcus.
Classification of Streptococcaceae
The classification of streptococci is complicated by the presence of three overlapping methods of subdividing the group, namely on pattern of haemolysis, on serologic (Lancefield) group and on biochemical reactions (Table 20.4).
| Type of haemolysis | Species | Distinguishing microbial tests | Normal habitat | Disease |
|---|---|---|---|---|
| Alpha haemolysis | Viridans-type streptococci, e.g. Streptococcus mitis, S. mutans, S. anginosus, S. bovis | Optochin-resistant Not bile-soluble | Normal flora of oropharynx, gastrointestinal tract and genitourinary tract | S. mitis: endocarditis S. mutans: dental caries, endocarditis S. anginosus (also known as S. milleri): deep-tissue abscesses S. bovis: association with colonic carcinoma |
| Alpha haemolysis | Enterococcus spp., e.g. E. faecalis, E. faecium | Aesculin-positive | Part of bowel flora | Urinary tract infection Biliary/abdominal sepsis Endocarditis (rare) |
| Alpha haemolysis | S. pneumonia | ‘Draughtsman’ appearance of colonies Lancet-shaped diplococci on Gram stain Optochin-sensitive Bile-soluble | Commensal of the oropharynx | Pneumonia Meningitis Otitis media Sinusitis |
| Beta haemolysis | S. pyogenes | Lancefield group A | Asymptomatic carriage in the oropharynx | Pharyngitis/tonsillitis (‘strep throat’) Cellulitis/erysipelas Necrotising fasciitis Toxic shock syndrome Poststreptococcal phenomena (rheumatic fever, glomerulonephritis, scarlet fever) |
| Beta haemolysis | S. agalactiae | Lancefield group B | Normal flora of lower gastrointestinal tract and female genital tract; 30% of pregnant women are carriers | Early-onset neonatal infection (bacteraemia, meningitis, pneumonia) Late-onset neonatal infection (meningitis) Maternal infection (urinary tract infection, chorioamnionitis, septic abortion) |
| Beta haemolysis | S. equisimilis S. dysgalactiae | Lancefield groups C and G | May be mucosal commensals | As for group A streptococci but usually less virulent |
| Gamma haemolysis | Viridans-type streptococcia | Optochin-resistant Not bile-soluble | Normal flora of oropharynx, gastrointestinal tract and genitourinary tract | S. mitis: endocarditis S. mutans: dental caries, endocarditis S. anginosus (also known as S. milleri): deep-tissue abscesses S. bovis: association with colonic carcinoma |
| Gamma haemolysis | Enterococcus spp.a | Aesculin-positive | Part of bowel flora | Urinary tract infection Biliary/abdominal sepsis Endocarditis (rare) |
a Strains from this species may in fact be alpha- or gamma-haemolytic | any one strain will show only one type of haemolysis but there may be variation among different strains of the same species; other clinical characteristics are as for the alpha-haemolytic strains | spp. = two or more species
Haemolysis is visualised by culture of streptococci on blood agar:
clear zones of haemolysis surrounding the colonies indicate complete haemolysis (beta haemolysis)
green zones around the colonies indicate partial haemolysis (alpha haemolysis)
lack of zones around the colonies (also referred to as gamma haemolysis) indicates the presence of a nonhaemolytic strain.
Lancefield serotyping (developed by Rebecca Lancefield) is based on the fact that different streptococci have different cell-wall antigens (polysaccharides). This allows rapid differentiation of streptococci by agglutination. The method is very useful for subdividing beta-haemolytic streptococci. Although some alpha-haemolytic streptococci also possess Lancefield group antigens, this method is not recommended for these organisms.
Finally, streptococci can be subdivided on the basis of biochemical properties. For example, enterococci hydrolyse bile salts (as detected by use of the aesculin test), whereas most streptococci do not.
Streptococcus pyogenes (Lancefield group A)
This Streptococcus species possesses a variety of virulence factors, including exotoxins (see Table 20.2), that make it a significant pathogen. Some of the infections and clinical syndromes associated with S. pyogenes are described in Table 20.4.
Skin and soft-tissue infections
S. pyogenes is highly infectious and virulent and can be spread by contact with an infected person or with fomites. Persons with a microbiological diagnosis of S. pyogenes skin infection should be isolated at least until they have received 48 hours of intravenous antibiotic treatment.
Necrotising fasciitis
This is an important invasive streptococcal infection. It is characterised by necrosis spreading through the fascia and fat, with rapid progression to systemic toxicity, shock and death unless aggressively managed. Bacteria gain entry to the subcutaneous layer often through minor, and sometimes unnoticed, trauma but this may also occur as a postsurgical complication. Characteristically, the site of infection is erythematous or purple with marked pain, often progressing to bullae formation and skin necrosis. Affected persons may show features of an associated toxic shock syndrome.
Two types of necrotising fasciitis have been identified; they are classified according to the organisms involved:
necrotising fasciitis type 1 is polymicrobial; typical organisms include anaerobes, Gram-negative bacteria (Enterobacteriaceae), streptococci and staphylococci
necrotising fasciitis type 2 is caused by group A streptococci (with/without a concomitant staphylococci infection).
Treatment should be prompt and aggressive and requires a multidisciplinary approach between physicians, surgeons and microbiologists that involves:
resuscitation
immediate surgical debridement (pus and necrotic tissue should be sent for microscopy and culture)
broad-spectrum intravenous antibiotics (including streptococcal cover):
intravenous benzylpenicillin 1.2 g every 4 hours
intravenous clindamycin 600 mg four times a day
intravenous ciprofloxacin 400 mg twice a day.
Repeated surgical exploration and debridement may be required. Treatments such as hyperbaric O2 therapy and intravenous immunoglobulin may have a role, although convincing evidence of their effectiveness is currently lacking.
People with necrotising fasciitis should be isolated until cultures are negative for S. pyogenes.
Toxic shock syndrome
Persons with invasive streptococcal disease, including necrotising fasciitis, may also develop toxic shock syndrome. This is mediated by toxins produced by S. pyogenes, particularly streptococcal pyogenic exotoxins A and C. Affected people are unwell with evidence of a marked systemic inflammatory response and shock, often with few superficial signs of infection. Rapid identification and antibiotic treatment is required to avoid progression to organ failure and death.
Streptococcus agalactiae (Lancefield group B)
S. agalactiae (commonly referred to as group B streptococcus or GBS) is part of the normal flora of the lower gastrointestinal tract, the throat and, most importantly, the female genital tract. Carriage of GBS occurs in up to 30% of women during pregnancy but may be intermittent.
Neonatal group B streptococcal disease
About 60% of babies born to mothers colonised with GBS will themselves become colonised during passage through the vagina. Several factors increase the probability of the baby acquiring GBS:
premature birth (before week 37 of gestation)
prolonged rupture of membranes (lasting for more than 18 hours)
intrapartum fever (over 38 degrees C)
previous infant with GBS disease
heavy maternal carriage of GBS.
GBS infection in babies can be divided into early-onset and late-onset disease. Early-onset neonatal disease occurs in infants of women colonised with GBS, either during delivery or by retrograde spread of GBS from the vagina into the amniotic fluid. Onset of disease may be a few hours to a few days postpartum. It may present as bacteraemia, meningitis or pneumonia. The incidence of early-onset neonatal GBS disease in the UK is estimated to be 0.5 in 1000 births.
Prompt treatment of the infant is required, including antibiotics active against GBS, such as intravenous benzylpenicillin. However, there remains a significant morbidity amongst survivors, with neurological sequelae such as blindness and mental retardation reported in those with meningitis.
In disease occurring in older infants (late-onset neonatal disease), GBS is usually not acquired at delivery but through another source (for example contact with other children, relatives, hospital staff who are carriers of GBS). The most common presentation is meningitis with bacteraemia.
In pregnant women, ascending infection with GBS can result in urinary tract infection. Infection of the amniotic fluid may result in chorioamnionitis and abortion. Invasive GBS may also cause postpartum sepsis with bacteraemia.
Group B streptococcal disease in other groups of people
In healthy men and nonpregnant women, GBS rarely causes infection. However, it may cause invasive disease in elderly people with other underlying conditions or a degree of immunosuppression. Presentations include septicaemia, pneumonia, cellulitis, osteomyelitis and septic arthritis.
Maternal screening for group B streptococci and peripartum prophylaxis
Current UK guidelines on the peripartum prophylaxis of GBS disease, published by the Royal College of Obstetricians and Gynaecologists, differ from those in the USA in that they do not advocate the routine antenatal screening of all pregnant women for carriage of GBS. This is because the current evidence base is insufficient to determine with certainty its clinical and cost-effectiveness in the UK population. Therefore, a risk-based approach is used to determine which women should receive prophylactic antibiotics peripartum. Women with one or more of the following risk factors should receive antibiotics:
GBS disease in a previous baby or pregnancy
GBS found incidentally in the vagina or urine at any time during pregnancy
prolonged rupture of membranes at term (more than 18 hours)
preterm rupture of membranes in labour (before week 37 of gestation)
preterm rupture of membranes with known GBS (whether in labour or not)
intrapartum temperature (more than 38 degrees C).
The following antibiotic regimen are used for prophylaxis during labour:
Staphylococcaceae
These are facultatively anaerobic Gram-positive cocci that typically appear as clusters, like bunches of grapes, when viewed using a Gram-stain technique. In Table 20.5, the features of staphylococci are compared with those of streptococci.
| Parameter of comparison | Staphylococci | Streptococci |
|---|---|---|
| Gram stain and morphology | Gram-positive cocci in clusters | Gram-positive cocci in pairs or chains |
| Catalase test (hydrolysis of hydrogen peroxide) | Positive | Negative |
| Colonisation site | Skin and mucous membranes | Oropharynx, oral mucosa, gastrointestinal tract, genitourinary tract |
| Infections | S. aureus Skin and soft-tissue infections Bacteraemia Endocarditis Osteomyelitis and septic arthritis | Streptococci Skin and soft-tissue infections Bacteraemia Endocarditis Osteomyelitis and septic arthritis Pharyngitis (‘strep throat’) |
| Coagulase-negative staphylococci Device-associated infections | Pneumococci Pneumonia Meningitis Sinusitis | |
| Enterococci Urinary tract infections Abdominal/biliary sepsis | ||
| First-line antibiotics | Meticillin-sensitive strains Flucloxacillin | Streptococci/pneumococci Penicillin |
| Meticillin-resistant strains Vancomycin | Enterococci Amoxicillin, teicoplanin |
The genus Staphylococcus contains a number of species that are classified mainly upon their ability to clot plasma, which signifies the presence of the extracellular enzyme coagulase:
S. aureus is coagulase-positive
coagulase-negative staphylococci include S. epidermidis, S. haemolyticus, S. capitis and S. saprophyticus.
Staphylococcus aureus
The following features aid the differentiation of S. aureus from the other (coagulase-negative) staphylococci in the laboratory:
positive coagulase test
positive DNase test, indicating the production of a nuclease enzyme that can break down DNA
presence of surface protein A (detected by agglutination).
S. aureus may form part of the normal flora of the nose, skin and perineum. It may be spread from person to person by direct contact (for example on unwashed hands) or via fomites. S. aureus may, however, also cause disease. Its virulence may be enhanced by the production of exotoxin (see Table 20.2).
Meticillin-resistant S. aureus
S. aureus can gain resistance to all beta-lactam antibiotics by mutations in their cellular targets (the penicillin-binding proteins). This is referred to as meticillin-resistant S. aureus (MRSA); meticillin is a beta-lactam antibiotic used previously in laboratory testing but not in clinical practice. MRSA is a major problem in hospitals, where the pressure of antibiotic therapy helps select out meticillin-resistant strains. Like all strains of S. aureus, MRSA may be spread from person to person by direct contact or by fomites. People may be asymptomatic carriers of MRSA or develop the same range of infections caused by meticillin-sensitive S. aureus.
MRSA infections will not respond clinically to any of the beta-lactam antibiotics (flucloxacillin, other penicillins, cephalosporins, carbapenems). It may, however, still be sensitive to other antibiotics, such as glycopeptides (vancomycin, teicoplanin), tetracyclines, rifampicin and fusidic acid. However, the pharmacokinetic and pharmacodynamic properties of these antibiotics render them suboptimal when compared with flucloxacillin.
Measures for the prevention of MRSA carriage are therefore important to reduce the number of MRSA infections. Several measures can be adopted:
prevention of spread between persons
spacing hospital beds adequately
handwashing/use of alcohol hand gels between patients
cleaning of equipment (e.g. stethoscopes) between patients
identification of patients colonised with MRSA
screening of patients for MRSA carriage
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