Gardnerella vaginalis: Introduction

• Gardnerella vaginalis is known by various names such as Haemophilus vaginalis and Corynebacterium vaginale.
• It was first described in 1953.
• It was placed formally in the new genus Gardnerella in 1980.
• Ultrastructural studies show the cell wall is seen as Gram-positive but it has got a thinner peptidoglycan layer.
• The layer is thinner than that of the Corynebacterium, Lactobacillus, or Staphylococcus
• Its peptidoglycan content constitutes about 20% of the total cell weight. It is similar to Enterobacteriaceae, such as Escherichia coli ( contain about 23%).
• It may be seen as Gram-positive, Gram-negative, or Gram-variable depending upon the various strains of vaginalis.
• The compounds which are present in the Gram-negative cell wall lipopolysaccharides are not present in the cell wall extracts of vaginalis.
• Example: meso-DAP, 2-keto-3-deoxy-D-manno-2-octonoic acid, hydroxy fatty acids.
• The cell wall of vaginalis is distinct from the cell wall of Gram-positive ( Corynebacterium type ) because of the absence of meso-DAP, arabinogalactans, and mycolic acids.
• G. vaginalis is found to be closely related to the bifidobacteria which is shown during its molecular studies.

Clinical Significance of Gardnerella vaginalis:

• G. vaginalis is a member of the normal vaginal microbiota.
• It was found to have an association with Bacterial vaginosis (BV)
• As this clinical syndrome is not caused by a single organism it is termed BV.
• In the Gram-stained smears of the vaginal discharge, inflammatory cells (seen with both Candida and Trichomonas vaginal infections) are not observed.
• Clinical characters of bacterial vaginosis caused by Gardnerella veginalis:
  • Malodorous vaginal discharge
  • Significant overgrowth in a number of G. vaginalis.
  • Growth in obligate anaerobes like Prevotella bivia, Prevotella disiens, Mycoplasma species, Peptostreptococci, and Mobiluncus species
  • Concomitant decrease in the numbers of normal vaginal lactobacilli.
  • BV is a risk factor for preterm birth and can cause an impact on adverse pregnancy outcomes.
  • BV is also a risk factor for obstetrical infections and pelvic inflammatory disease.
  • Initially, G. vaginalis was hypothesized as the etiological agent of this condition. Later, it was found that the other organisms were also involved in it.
  • G. vaginalis was present in the women with BV.
  • But, the presence of G. vaginalis was found in greater than 50% of women without BV.
  • In about 14% to 70 % of healthy women, vaginalis can be found in healthy women without BV.
  • Bacterial vaginosis is also indicated by the presence of a large number of G. vaginalis in the vagina.
  • It was found that the presence of these bacteria at concentrations of ≥2 × 107cfu/mL of vaginal fluid, vaginal pH greater than 4.5, had 95% sensitivity and 99% specificity.
  • In the male urethra also G. vaginalis was found.
  • For the diagnosis of BV, isolation of vaginalis by routine culture is not recommended because it is itself part of the vaginal microbiota.
  • Different studies had suggested as sexual transmission may not occur but from the intestinal tract, it may have colonized the vagina endogenously.
  • G. vaginalis was isolated from semen specimens of 50% of the men.
  • The organism was found to adhere to the cell membrane and it could penetrate the cytoplasm of both vaginal and male urethral epithelial cells.
  • Recolonization (reinfection) of the female vagina might occur due to a male partner. Bacteria might colonize the male lower genital tract asymptomatically.
  • G. vaginalis has also been associated with the complications of pregnancy.

Gardnerella vaginalis has also been isolated from the infants born from those mothers, particularly during and after delivery like:

• intrauterine infections
• intraamniotic infections
• Chorioamnionitis
• postabortal pelvic inflammatory disease
• postpartum endometritis after cesarean delivery.

Systemic and localized G. vaginalis may occur with the complications such as:

• Amnionitis
• episiotomy wound infection
• bacteremias
• Meningitis
• Cellulitis
• Conjunctivitis
• osteomyelitis

G. vaginalis is also isolated from the oropharyngeal cultures, gastric aspirates, and tracheal suction specimens of neonates.

• It is passed from the birth canal which is heavily colonized.
• It has also been isolated from:
• Bartholin’s gland abscesses
• postcesarean section
• postsurgical wound infections
• abdominal surgeries
• hysterectomies
• episiotomies

Rarely Gardnerella vaginalis has also caused infections in males.

• Occasionally, infections rather than the genitourinary tract are also caused by G. vaginalis.
• Bacteremia with G. vaginalis in men has been reported following transurethral prostatectomy, urogenital surgical procedures, and in association with renal calculi, and urinary retention secondary to obstruction.
• In both men and women, G. vaginalis also play a role in urinary tract infections.
• G. vaginalis has been isolated more from the urinary tract infection of females than the male because of its presence in females as the normal vaginal microbiota.
• From the symptomatic and asymptomatic patients, G. vaginals has been isolated from both the upper and lower urinary tracts.
• Upper urinary tracts include the ureters, renal pelvis, and calyx.
• The lower urinary tracts include the bladder.
• In a study, its isolation rate was found to be more in pregnant women than the non-pregnant women.
• Many of these patients are asymptomatic too.

Diagnosis of Bacterial Vaginosis: 

• Diagnosis of BV usually involves a patient with a malodorous vaginal discharge.
• After sexual contact, there may be minimal irritation.
• exposure to alkaline pH of vaginal secretions after sexual contact and during menstruation may cause fishy odors.
• The fishy odor is due to the volatilization of amines.
• Production of the fishy odor is the basis of the whiff test.
• In this test, for the production of a fishy odor, KOH is mixed with the discharge.
• Vaginal discharge of BV:
  • Homogeneous
  • White-gray
  • Maybe frothy
  • Vaginal pH is greater than 4.6
• The specificity of diagnosis is being even greater when the vaginal pH is ≥5
• Moderate to many vaginal epithelial cells can be seen by wet mounts.
• The large number of adherent bacteria having various morphologies can also be seen ( clue cells)
• The margins of the epithelial cells may be completely covered by adherent microorganisms.
• For the diagnosis of BV, its absence or possible presence, it is evaluated by various criteria.
• The relative amount of lactobacilli is evaluated.
• The morphotypes of Gardnerella and the Mobiluncus are also studied for it.
• These bacterial morphotypes cover the vaginal epithelial “clue” cells.
• The study of Gram reactions and morphologies also aids this diagnosis process.

Cultural Characteristics of  Gardnerella vaginalis

• The culture of vaginal specimens for the isolation of G. vaginalis in the case of diagnosis of BV should be discouraged.
• Because BV is not caused by a single species of bacteria and its isolation can be done even from more than 50% of the asymptomatic women.
• Wet preparation or the Gram-stained smears of vaginal discharge should be examined carefully and interpreted.
• After the prolonged incubation, on the Columbia NaladixicAcid Agar (CNA) medium, G. vaginalis can be recovered.
• Isolation of G. vaginalis can be done on routine SBA, CNA, and chocolate agar from the clinical specimens.
• Semi-selective media include HBT agar or V agar.
• After 48 hours of incubation on HBT agar, vaginalis forms small, clear zones of β-hemolytic colonies surrounding colonies with diffuse edges.
• For “routine media”, vaginalis grows better on Columbia agar-based media (i.e., CNA agar) than on blood agar made with a tryptic soy base.
• On CNA, vaginalis shows a subtle, “diffuse” hemolysis surrounding the colonies.
• This is noted initially in confluent areas of growth or after incubation for >72 hours.
• Growth is best at 35°C to 37°C in a 5% to 7% CO2 atmosphere.
• After 48 to 72 hours, most of the isolates are recovered.
• vaginalis can grow in most blood culture media but the anticoagulant additive sodium polyanethol sulfonate (SPS) is inhibitory to some G. vaginalis.
• By the additional testing, the suspected vaginalis should be confirmed from the systemic body sites such as blood, joint fluid, etc.

 

Presumptive identification of Gardnerella vaginalis is done by:

• typical cellular morphology on Gram-stained smears (small gram-positive, gram-negative, or gram-variable coccobacilli)
• characteristic growth on CNA agar with “diffuse” weak β-hemolysis
• negative oxidase and catalase tests.

Definitive identification of G. vaginalis is done by biochemical test:

Biochemical Characteristics for the Identification of Gardnerella vaginalis:

Characteristic	Reaction
Hemolysis on human blood bilayer Tween (HBT) agar	β haemolysis
Oxidase	Negative
Catalase	Negative
Hippurate hydrolysis	Positive
Acid production from:
Glucose	positive
Maltose	positive
Sucrose	positive
Mannitol	negative
Starch	positive
Zone of growth inhibition with:
Metronidazole (50 μg disk)	positive
Trimethoprim	positive
Sulfonamide	positive

 

Other characteristics for the confirmation of   Gardnerella vaginalis are:

• presence of α-glucosidase
• absence of β-glucosidase
• positive starch and hippurate hydrolysis reactions.
• Carbohydrate utilization tests are performed in medium containing Proteose Peptone no. 3, phenol red indicator, and 1% filter-sterilized carbohydrate.
• Production of acid from glucose, maltose, sucrose, and starch, but not from mannitol, sorbitol, raffinose, rhamnose, or salicin
• Hydrolysis of hippurate
• Absence of lysine or ornithine decarboxylases or arginine dihydrolase
• does not reduce nitrate
• does not produce indole, urease, or acetoin
• zones of inhibition around disks containing metronidazole (50 μg) and trimethoprim (5 μg)

Antimicrobial Susceptibility of  Gardnerella vaginalis:

• Susceptible to penicillin, ampicillin, erythromycin, clindamycin, trimethoprim, and vancomycin.
• Ciprofloxacin and imipenem show variable activity.
• Some strains may be resistant to tetracycline and minocycline.
• Most strains show marked resistance to amikacin, aztreonam, and sulfamethoxazole.