The Gram-negative cocci of clinical importance Neisseriaceae The Gram-negative cocci of clinical importance

Neisseriaceae Classification - Family Neisseriaceae- are all  Gram negative aerobic rods and cocci. Contains 4 genera: Neisseria- cocci Moraxella, subgenus Branhamella- cocci; other species within the genus are short rods Acinetobacter- rods Kingella- rods

Neisseriaceae We  will only be discussing Neisseria and Branhamella which  morphologically and biochemically resembles Neisseria, but shows DNA homology  to  Moraxella. Some are found as part  of  the  normal flora of the naso and oropharynx and are usually considered to be non-pathogenic. Two species- N. meningitidis (MGC) and N. gonorrhoeae (GC) are considered to be pathogenic although N.  meningitidis may be found as part of the nasopharynx normal flora in 10-15% of the population.

Neisseria and Branhamella Morphology and General Characteristics Gram negative diplococci that are coffee- bean  shaped  with  adjacent flattened edges. In a direct smear GC may be found intracellularly in PMNs or extracellularly.

Neisseria species Gram stain

Intracellular GC

Morphology and General Characteristics B. catarrhalis and Neisseria species other than MGC and  GC will grow on nutrient agar without blood. MGC and GC are more  fastidious and  require serum or blood in addition to other growth  factors. GC  is more fastidious and requires chocolate agar, whereas  MGC  may grow  on  CBA.  Colonies of both are small  and  translucent  to grayish-white.   Colonies  of non-pathogenic species may be  dry  and wrinkled, opaque or yellow, and they tend to be self-adherent.

MGC colonies

N. sicca colonies

N. subflava colonies

Morphology and General Characteristics A selective media is usually used to recover MGC and GC when the specimen is likely to be contaminated with other bacterial normal normal  flora. Modified Thayer Martin (MTM) is the most common of these. It is chocolate agar containing Vancomycin  (inhibits  G  +), Colistin (inhibits  G -),  Nystatin  (inhibits fungi), and Trimethoprim (inhibits swarming Proteus). Occasional strains of GC are sensitive to Vancomycin, therefore, both  chocolate agar and MTM should always be used. The colonies may require 48-72 hours for growth. Lewis Martin agar is similar to MTM, but it has anisomycin substituted for nystatin for improved inhibition of Candida albicans.

GC growth on MTM

Morphology and General Characteristics Members of the genus Neisseria and B.  catarrhalis  are aerobic with little or no growth under anaerobic conditions. MGC and GC require a CO2 concentration of 3-10% for growth,  particularly for primary isolation. In the lab we will be growing the Neisseria in candle jars.

Growth in candle jars

Morphology and General Characteristics GC  and MGC are particularly sensitive  to  temperature extremes  and drying, therefore successful cultivation depends upon careful collection and careful processing of specimens. Ideally, specimens should be plated directly onto culture media and incubated immediately. The reason for this is that MGC and GC may produce autolytic enzymes at 250 C and an alkaline pH leading to lysis of the cells.  Most MGC and GC produce capsules.

Neisseria and Branhamella Biochemical identification - need to distinguish between  B. catarrhalis and both pathogenic and non-pathogenic Neisseria. To distinguish from other organisms – use colony  morphology Gram stain (g-dc), and the fact that it is catalase and oxidase positive.

Oxidase test on plate

Oxidase test on paper

Biochemical identification To differentiate between  the different Neisseria species and B.  catarrhalis. Carbohydrate utilization tests -  These are not fermentation tests because the organisms  use  carbohy­ drates  oxidatively. Cystine-tryptic-agar (CTA) sugars  are  the reference tests, but require a heavy inoculation, incubation in a non-CO2 incubator and observation for 24-72 hours. Some fastidious GC won't  grow  or grow very poorly on CTA and the small  amount  of acid  produced  is neutralized by alkaline products  formed  from peptone utilization. The  sugars  glucose,  maltose,  lactose, sucrose and fructose are commonly used. Pertinent results: GC  + for glucose only; MGC + for glucose and maltose; B. catarrhalis - for all.

CTA sugars

Biochemical identification We now have non growth dependent tests which depend  on preformed enzymes. A heavy inoculation of organism is used with a small volume of carbohydrate in a buffered salt solution. Results are read in 4 hours. Kits are now available that can identify the different organisms by  using enzyme profiles obtained using chromogenic  substrates. Results are in 4 hours and are non growth dependent. This is what we will do in the lab. DNAse test - B. catarrhalis is + for DNAse , all Neisseria are -.   Nitrate reduction - GC and MGC are - for nitrate reduction,  B.  catarrhalis is +. Some nonpathogenic Neisseria species are + and others are - .

Neisseria and Branhamella Serologic and Other Identification methods – Many kits are available for serologic identification of MGC and GC Auxotyping distinguishes GC on the basis of  nutritional requirements.   Gonococcal strains that are associated with disseminated disease have  a multiple  requirement  for  arginine, hypoxanthine and uracil. Nucleic acid based tests – both probe based and PCR based tests have been developed for the identification of GC directly from clinical samples.

Neisseria and Branhamella Mechanisms of Pathogenicity – Neisseria gonorrhoeae – Pili- important in attachment to human columnar  epithelial cells- may inhibit phagocytosis.  Protein I (Por) - the principal protein found on  the  outer membrane- functions as a porin to allow small molecules to diffuse thru the membrane and is important for intracellular survival. Is protective against host’s inflammatory response and serum complement mediated killing Protein  II (Opa)- a minor protein found on the  outer  membrane. It is involved in attachment to PMNs and other  leukocytes where it induces the endocytic uptake of GC After endocytosis, GC is  transported in vesicles to the base of the cells and released into subepithelial  tissue. Both pili  and protein II are subject to antigenic variation;even if antibodies are formed against them they  are  not effective.

Induced uptake

Neisseria and Branhamella Protein III (rmp) – an outer membrane protein that may associate with Protein I to form pores. Blocks host serum bacteriocidal (IgG) action against the organism by eliciting formation of blocking antibodies Lipopooligosaccharide- (LOS) - comparable to LPS of Gram negative bacilli (endotoxin). May undergo antigenic variation and causes mucous membrane damage, fever and toxicity. Specific activities include: Adherence and invasion Molecular mimicry of host structures Release in membrane blebs Suppression of leukotreine B4 synthesis→ inhibits PMN activation Activates alternative complement pathway → inflammation Activates TNF → inflammation

LOS and Membrane blebs

Neisseria and Branhamella Capsule – helps prevent phagocytosis and protects organism from antimicrobial peptides IgA1 protease- cleaves IgA to allow mucosal  attachment of GC.   Ability  to acquire iron from the  host  by  producing outer membrane proteins that capture it under conditions of  iron limitation. Enhanced capacity occurs in those that cause disseminated gonococcal infection (DGI).

Iron acquisition

GC cell wall structure

Neisseria and Branhamella Strains of GC that cause DGI  possess  certain  types of Protein I,  are  resistant  to phagocytosis  and  serum bactericidal activity, and are  able  to multiply unchecked in the presence of PMNs. Neisseria meningitidis – Pili- allow binding to specific nasopharyngeal receptors. Type IV pili are essential for the interaction of MGC with brain endothelial cells

Neisseria and Branhamella Capsule (polysaccharide) – antibodies, if made, are protective. Only certain serotype antigens are found during  epidemics.   Ability to acquire iron from human transferrin IgA1 Protease Class 1,2, and 3 outer membrane proteins ( Por) that function as porins and are important for intracellular survival Class 5 outer membrane protein (Opa) involved in attachment, induced uptake (invasion). Class 4 outer membrane protein (rmp) important in blocking host serum bacteriocidal (IgG) action against the organism by eliciting formation of blocking antibodies Opc protein – important for attachment and invasion LOS with same functions as GC LOS Pili, Opa, LOS, and to a lesser extent, Opc, undergo antigenic variation  

Neisseria and Branhamella Clinical Significance – Neisseria gonorrhoeae - usually transmitted by direct sexual contact. Used to be the most common reportable disease  in the  U.S. Those infected commonly have a concomitant Chlamydia trachomatis infection (most common reportable disease  in the  U.S.  today). Asymptomatic infections- Males= 10%, Females= 20-75%. The problem with asymptomatic individuals is that they are carriers and can transmit the disease to others.  

Clinical significance of GC Uncomplicated infections - in males incubation= 1-4 days followed  by  acute urethritis with profuse purulent discharge filled  with GC. If untreated may spread to cause  prostitis  and epididymitis - with proper antibiotic treatment this is rare. With oropharyngeal infection mild pharyngitis. With rectal infection - proctitis; Uncomplicated infections - in females following the incubation may have increased vaginal discharge, burning or frequency of urination and menstrual  abnormalities.   Infected mothers can transmit  GC  to  their babies  at birth causing gonococcal ophthalmia neonatorum.  This can lead to blindness, therefore all babies are given  erythromycin eye drops at birth to prevent this.  

Uncomplicated GC infection Pus containing discharge

Neonatal ophthalmia neonatorum

Clinical significance of GC Complicated infections- Occur more frequently in women - spread  from  the cervix  into the fallopian tubes resulting in endometritis,  salpingitis,  and  peritonitis= pelvic inflammatory  disease  (PID). Occurs in 10-20% of infected women. Symptoms include lower abdominal  pain, abnormal vaginal and cervical discharge, and  uterine tenderness. This can result in ectopic pregnancy and infertility.

Salpingitis

Complicated infections of GC Spread to the bloodstream may result in DGI in both men  and  women (1%). Can result in endocarditis, arthritis, maculopapular rash, and meningitis.  

Clinical significance Neisseria meningitis- causes endemic and epidemic epidemic cerebral  meningitis. Most common in children under 1 year,  adolescents, and young adults. Can be part of the normal flora of  the nasopharynx  and  cause disease when there is a failure  in  host resistance  or when a particularly virulent strain colonizes  the mucous membranes. Asymptomatic carriers are the primary  source of spread thru aerosols. Colonization of the nasopharynx usually results  in a subclinical infection or a mild upper respiratory tract infection  like the common cold. Invasion of the bloodstream occurs only in individuals lacking bactericidal antibodies or deficient in certain complement components (C5-C8). Chronic irritation or damage to the respiratory mucosa may be predisposing factors.  Invasion of the bloodstream may result in septicemia (meningococcemia)  or meningitis.

MGC infections Meningococcemia- symptoms include high fever,  shaking, chills, muscle pain, and petechial rash (hallmark of MGC infections). Disease may  be chronic, moderate or fulminant.   The  fulminant type is called the Waterhouse-Friderichsen syndrome and it is characterized by disseminated intravascular coagulation (DIC),  with hemorrhaging into  the skin, adrenal glands and  other  internal organs.   This  results in rapid death due to acute generalized toxemia and shock. Most of these symptoms are due to the activity of the endotoxin (LOS).   Patients that survive may lose their limbs from tissue necrosis (gangrene of the skin and soft tissues)

Petechial rash

Waterhouse-Friderichsen syndrome

Waterhouse-Friderichsen syndrome

MGC infections Meningitis- symptoms include fever, headache, stiffness of  back and neck, and petechial rash. In severe cases there  is severe cerebral hyperemia (accumulation of blood) and  tissue swelling. May progress to convulsions and coma This is invariably fatal without rigorous antimicrobial therapy. 10%-14% of cases are fatal even with treatment For patients who recover 11%-19% have permanent hearing loss or mental retardation  

Meningitis

Clinical significance Other Neisseria species - rarely associated with endocarditis, septicemia, and meningitis. B.  catarrhalis - may be a significant cause of otitis media  and maxillary sinusitis in children May  cause  pneumonia and bronchitis in immunocompromised individuals. May also  occasionally cause endocarditis, meningitis and septicemia.

Neisseriaceae Treatment and antimicrobial susceptibility – GC -  used to use only penicillin. Resistant strains are emerging  so -lactamase testing should be done. For penicillin resistant strains or penicillin sensitive individuals substitute ceftriaxone or spectinomycin for penicillin (1 dose to treat GC) plus 7 days of doxycycline to treat possible coexisting C. trachomatis infection. Amoxicillin or ampicillin or Penicillin  G  plus probenecid can be used for uncomplicated cases   MGC- I.V. penicillin or chloramphenicol and erythromycin or ceftriaxone. Prophylaxis with rifampin, ciprofloxacin, or ceftriaxone for household and other close contacts

Treatment Vaccines (tetravalent to polysaccharide capsular antigens A, C, Y, W135) are available for the virulent types  of  MGC. Most human infections are due to types A, B, C, Y, and W135. They are given to those at risk, including college students living in dorms. A group B vaccine consisting of OM antigens has been developed, but is not available in the United States.