Department of Microbiology Bacteraemia Guideline

Gram negative bacillus bacteraemia

• Aim
• Background
• Bacteriological differential diagnosis (Gram guideline only)
• Clinical differential diagnosis
• Technical issues
• Antimicrobial treatment
• Supplementary investigations

Quick reference guide to the management of Gram negative bacillus bacteraemia

This document provides guidelines for doctors on the management of patients with confirmed bacteraemias (blood cultures). This document is supplementary to, and should be used in conjunction with, the antimicrobial guidelines.

Gram stain: Gram negative bacillus

Aim

The aim of this guideline is to:

• Provide education to junior microbiology registrars
• Support communication of Gram stain results from microbiologists to ward doctors
• Support ward doctors in treating and investigating bacteraemic patients

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Background

The blood culture process: Timings of culture, identification, susceptibility tests and clinical liaison.

How to use this guideline: This guideline should be used to help in the management of patients with a confirmed bacteraemia. The guideline should be used to support interaction with specialist advice e.g. Microbiology.

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Bacteriological differential diagnosis (Gram guideline only)

Gram negative bacilli are a common cause of bacteraemia. It should always be taken seriously, with the vast majority of bacteraemias being the result of a significant infection. Contamination very rarely accounts for the finding of Gram negative bacilli in the blood culture.
As the name suggests, this is a descriptive term of Gram stain morphology and culture, or an alternative identification method, is needed to fully identify the organism. Gram negative bacilli can cause a wide range of possible infections. Background information on Gram negative bacteria is provided below. This information is broadly applicable to most patients with Gram negative bacteraemia, but should be used in conjunction with disease specific guidelines and discussion with an infection specialist.

Clinical and microbiological characteristics of Gram negative bacteria

Enterobacteriaceae
Clinical

• Escherichia species i.e. E. coli: E. coli are a normal part of the bacteria growing in the human bowel. As such they cause infections when the integrity of the colon is breached e.g. colonic perforations. They will also cause infections in areas of the body close to the gastrointestinal tract e.g. urinary tract infections and biliary tract infections. In some patients e.g. hospitalised patients, E. coli commonly colonise the skin and mucous membranes e.g. oral cavity. And so there is potential for it to be detected in clinical samples at these sites as well as cause infections. E. coli is often identified in clinical samples e.g. urine and sputum, when it is not associated with a clinical infection. In this situation it is described as colonising. Clinical assessment is required to differentiate colonisation from infection, although bacteraemias are almost universally related to an infection. Some E. coli strains are associated with specific diseases e.g. Enterotoxigenic E. coli causes diarrhoeal disease, though these are not commonly a cause of bacteraemia.
• Non E. coli Enterobacteriaceae (Klebsiella, Enterobacter, Citrobacter, Morganella, Proteus, Providencia, Serratia): These bacteria are able to survive for long periods (years) within the human gastrointestinal tract, but they are principally environmental bacteria. They are less common causes of infection than E. coli, but still do cause infections. The spectrum of infections these bacteria cause can be considered the same as E. coli.
• Salmonella: Non typhi salmonella have become more common as a cause of bacteraemia when previously they caused only diarrhoeal disease. Both typhi and non typhi salmonella bacteraemia should result in a patient being tested for HIV. A travel and food history may suggest Salmonella infection is a possibility.

Microbiological
The Enterobacteriaceae are a large family of Gram negative bacilli, primarily found in the gastrointestinal tract of humans and other animals. They are facultative anaerobes, are oxidase negative and catalase positive. Three species; Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis account for 80-95% of all Enterobacteriaceae from clinical specimens.

• Escherichia species.
  There are six species, of which four are known to cause human disease. By far the most common of these is Escherichia coli. Its ability to ferment lactose helps in its identification in the laboratory (however a very small percentage can be non-lactose fermenting). There are several strains of E. coli associated with human disease; some have enterotoxins or other virulence factors. Some strains are encapsulated with a K antigen.

• Klebsiella spp.
  Klebsiella pneumoniae is the most frequently isolated species. They are lactose fermenting, non-motile bacilli with a very large polysaccharide capsule, which gives the colonies a distinctive mucoid appearance on the agar plate. This capsule can also be seen on the Gram stain.

• Enterobacter species are lactose fermenters. They also ferment glucose, producing acid and gas. Unlike Klebsiella species, they are usually motile and are less often capsulated. Enterobacter organisms can readily develop resistance to beta-lactam antibiotics, due to the presence of inducible beta-lactamase enzymes such as AmpC.
  
  
• Citrobacter species take their name from their ability to grow on Simmons citrate media. They are usually non-motile non-lactose fermenters, but can sometimes be slow/late lactose fermenters. C. freundii can be mistaken for Salmonella spp. as it produced hydrogen sulphide. Their ability to agglutinate with Salmonella polyvalent antisera, can also lead to misidentification of Citrobacter spp. as Salmonella spp.

• Morganella morganii. The genus Morganella contains the single species; Morganella morganii. This organism is mobile and a non-lactose fermenter. Like Citrobacter spp, its ability to agglutinate with Salmonella polyvalent antisera can lead to misidentification.

• Proteus spp. are very motile organisms, this can be seen as “swarming” on the agar plate. They can rapidly hydrolyse urea and are non-lactose fermenters. Some organisms carry an AmpC beta-lactamase enzyme which can induce resistance in the presence of beta-lactam antibiotics.

• Providencia spp. These organisms are similar to Proteus sp., but do not hydrolyse urea. Another distinguishing feature from Proteus spp. is that they are motile, but do not swarm.

• Serratia spp. The most commonly isolated species are Serratia liquefaciens and Serratia marcesens. S. marcesens produces distinctive red pigmented colonies. Like Citrobacter spp., they are slow or non-lactose fermenters. Unlike Citrobacter spp., Serratia spp. are usually motile.

• Salmonella species. There are seven subspecies and over 240 serovars. They are motile, oxidase negative and non-lactose fermenters. Most produce hydrogen sulphide, with the exception of S. Paratyphi A which doesn’t. Also of note, S. Typhi is a weak producer of hydrogen sulphide. Salmonellae possess lipopolysaccharide somatic (O) heat-stable antigens, and flagella (H) heat-labile antigens. S. Typhi and S. Paratyphi C produce a Vi polysaccharide capsule, which can mask the somatic antigens. Polyvalent antisera and biochemical tests are important identification methods. All isolates are submitted to the reference laboratory for serotype confirmation. S Typhi and S Paratyphi are biohazard group 3 organisms.

• Hafnia alvei is the only species in the genus Hafnia. Hafnia alvei produces grey colonies on blood agar, is motile and can resemble Salmonella spp. biochemically and can agglutinate with Salmonella polyvalent antisera.

Aerobes

Clinical

• Pseudomonas’s normal habitat is water based. The human body is able to provide moist environments which it can survive in. It is therefore commonly cultured from a number of human body sites e.g. urine, wounds, sputum. As for E. coli it is frequently a coloniser of these sites and clinical assessment is needed to differentiate infection from colonisation. Pseudomonas causing a bacteraemia is almost always representative of a clinical infection (cf. contamination).
• Stenotrophomonas maltophilia is an environmental organism which is resistant to multiple classes of antibiotics. It is only normally cultured when a patient has been on broad spectrum antibiotics eradicating all other Enterobacteriaceae.

Microbiological

• Pseudomonas species are non-spore forming, motile, oxidase positive aerobes. Diffusible pigments pyocyanin and pyoverdin give Pseudomonas aeruginosa its green-blue colouration.

• Stenotrophomonas maltophilia are non-lactose fermenting, motile, aerobic Gram negative bacilli. Their colonies are a pale yellow on blood agar and they are often oxidase negative and catalase positive. They are resistant to imipenem, a feature which can help in the identification process.

Anaerobes

Clinical

• Anaerobes are a normal part of the gastrointestinal tract and it is within the abdomen they cause most infections. They are commonly involved in polymicrobial intra-abdominal infections and are often required to from abscesses. They can be involved with significant skin and soft tissue infections e.g. Fusobacterium necrophorum from Lemièrre’s disease.

Microbiological

• Bacteroides species are bacillus/ rod shaped organisms that vary in size; are often pleomorphic and demonstrate terminal or central swellings, vacuoles or filaments. They are non-spore forming. The most commonly isolated species from clinical samples is Bacteroides fragilis.
• Fusobacterium spp. Fusobacteria are long and thin bacilli which may be spindle-shaped, e.g., Fusobacterium nucleatum, or pleomorphic Fusobacterium necrophorum. They are non-spore forming and non-motile. F. necrophorum can be associated with serious infections such as Lemièrre’s disease.
• Prevotella species. The genus Prevotellais composed of mainly saccharolytic, pigmented or non- pigmented species that were previously classified as Bacteroides, and these are usually
  pleomorphic. They are non-spore forming.

Facultative anaerobe (non- enterobacteriaceae)

• Capnocytophaga species are long and thin Gram negative rods. They are facultative anaerobes. They will grow on blood or chocolate agar, but not on MacConkey agar. Capnocytophaga canimorsus is oxidase positive and catalase positive. Most commonly they are associated with animal bite wounds.

Microaerophilic:

Campylobacter spp.
Characteristic comma/ seagull shaped, oxidase positive, motile Gram negative bacilli. Bacteraemia is more common with C. intestinalis than it is with C. jejuni. The latter will grow at 42ºC, whereas C. intestinalis grows at 25ºC and is also resistant to naladixic acid. It is an uncommon cause of bacteraemia, but can cause bacteraemia in patients who are pregnant.

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Clinical differential diagnosis

There is a wide differential diagnosis for Gram negative bacillus infections; this reflects the varied site and numerous species that may be cultured. A full systems review is recommended to identify the source of Gram negative bacillus bacteraemia. The differential diagnosis includes:

• Intra-abdominal infection
• Hepato-biliary infection
• Urinary tract infection
• Intravascular access device infection
• Pneumonia
• Meningitis
• Peritonitis
• Skin and soft tissue infection e.g. cellulitis/necrotising fasciitis
• Bone & joint infections
• Endocarditis and cardiac device infections
• Other: E.g. Obstetric, Lemièrre’s disease

• Contamination: Environmental Gram negative bacillus (always assume a Gram negative bacteraemia is not a contaminant until proven otherwise).

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Technical issues

Specificity/Common bacteriological misdiagnoses:

Gram stains give a provisional idea of the identification of an organism and require growth of the organism for full identification. Gram negative bacilli (unlike Gram positive organisms) do not retain methyl/crystal violet dye in the staining process and are stained red by a counterstain. Many exist in more than one form and/or stain irregularly and therefore may be mis-identified as each other. Culture growth allows for further biochemical and molecular testing in order to confirm identification.

Gram stain interpretation requires skill and experience. The reading of a Gram stain is subjective and open to descriptive variation depending on the reader of the Gram stain. Some organisms are more difficult to identify than others. As such, short and chunky Gram-negative bacilli can sometimes be misidentified as cocco-bacilli and vice-versa.

Limitations of the test:
Gram stains of positive blood culture bottles provide provisional guidance only. They do not give an accurate identification of organisms nor do they provide susceptibilities. Culture is required for further identification and susceptibilities. Some Gram negative organisms can resist decolourisation in the staining process and as such can be mistaken for Gram positive organisms, whilst Gram positive organisms can sometimes be over decolourised and be mistaken for Gram negative organisms.

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Antimicrobial treatment

• Antimicrobial therapy can often be improved with knowledge of the Gram stain result. An antibiotic may be started, stopped, or have its dose changed.
• Antibiotic therapy should always be reviewed the day after the Gram result as bacterial growth will have allowed further speciation of the bacteria, and sensitivity tests to be completed.
  History of the following are important to determine appropriate antimicrobial therapy

•  
  • Previous GNB (and it’s treatment)
  • History of multidrug-resistant Gram negative bacteria (such as but not limited to: AmpC beta-lactamases, Extended-spectrum beta-lactamases and Carbapenemase producing organisms). If there is a history of resistance please discuss with Microbiology for treatment advice.
  • Foreign travel
  • Source of infection
  • Allergy status and nature of allergy

The table below outlines some of the common organisms associated with each of the clinical syndromes. Please be aware that infections caused by Gram Negative Bacilli can present in unusual ways, and that this list is by no means exhaustive

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Supplementary Investigations

Consider further investigations as appropriate to source of infection, please see relevant guidelines.

E. coli bacteraemia in a neonate is consistent with a diagnosis of meningitis, urgent
lumbar puncture should be considered.

Further Action:

Notification to the local health protection unit is required for Salmonella Typhi & Salmonella Paratyphi.
Source isolation and appropriate infection prevention and control measure are required for multidrug-resistant Gram negative bacteria (such as but not limited to: AmpC beta-lactamases, Extended-spectrum beta-lactamases and Carbapenemase producing organisms).

References