Study of Cryptic and Non Cryptic Bacterial

Urethritis and Prostatitis

Thewaini,Q.N.O , Zainab,M.J
.University of Babylon , Collage of Science , Department of Biology ,Hilla

Abstract
Sixty samples of urethral discharge and Prostatic exudates were collected and cultured in three
techniques namely;1-Serial culture contain: whole urethral and prostatic exudates, sediment and
supernate after centrifugation, sediment and supernate after lysing 2-Dilution technique, 3-Biphasic
media. The results by using these techniques CWDBs were found outside of the inflammatory cells,
adhere on the surfaces of pus cells and cryptic inside the pus cell of urethra and prostate.
The biphasic media and lysing sediment were the best techniques comparative with routine method by
using Z test . The more common bacterial causes were Neisseria gonorrhoeae with a percentage of
25.3% , followed by Staphylococcus saprophyticus, (20.4%), and Escherichia coli(18%).

‫الخالصة‬
‫زراعة‬: ‫) عينة من طرح األحليل وسائل البروستات وزرعت بثالث تقنيات هي تقنية الزرع المتسلسل التي تضمنت‬60(‫جمعت‬
‫تقنية التخافيف وتقنية‬، ‫زراعة الرائق والراسب بعد النبذ وزراعة الرائق والراسب بعد التحلل‬,‫سائل البروستات وطرح االحليل الكلي‬
‫ملتصقَا بسطوح األغشية الخلوية أو‬، ‫أن المسبب المرضي أما أن يكون خارج الخاليا الملتهبة‬.‫المزرعة ثنائية الطور و بينت النتائج‬
‫ واوضحت النتائج إن تقنية المزرعة ثنائية الطور والراسب المكسر هي أفضل الطرق في العزل مقارنة مع‬.‫متخفيًا داخل الخاليا‬
‫ تلتها‬%25.3 ‫ و تبين أن بكتريا نيسيريا السيالن هي المسبب األكثر شيوعًا وبنسبة‬.Z ‫الطريقة الروتينية المستعملة كما بينها اختبار‬
.%18 ‫ ثم بكتريا اشريكيا القولون بنسبة‬%20.4 ‫بكتريا المكورات العنقودية بنسبة‬

Introduction
Sexually transmitted disease (STD) subjects were classified as symptomatic if
symptoms appear such as discharge, dysuria, and pelvic pain were reported by the
subject (Betts,2003). While asymptomatic case if the subject dose not report
symptoms. Asymptomatic is transmitted by human-to-human contact and is highly
adapted to the genital tract, surviving poorly outside the human
body(Mimis,2004).The antimicrobials and in the antigenic variability by which it
evades host defenses. Most notably Chlamydia trachomatis, N. gonorrhoeae cause
infections principally of the urethra in men and the endocervix in women, although it
may also infect extra genital mucosal sites, including the pharynx and rectum. Ocular
infections also occur, and in neonates can cause blindness(Workowiski,2006).
Genital infection in men usually presents with a urethral discharge, but silent
infections are common in women and in the case of extra genital infections.
Urethritis is the most frequent STD syndrome seen in men. Customarily, clinicians
categorize, urethritis into gonococcal and non gonococcal etiologies. The relative
frequency of non gonococcal Urethritis (NGU) and gonococcal Urethritis varies by
population studied, but overall, more cases of NGU than gonococcal Urethritis are
now being seen all around the world (Betts,2003). Up to one-fourth of heterosexual
men with gonorrhea also have simultaneous Chlamydia trachomatis (CT) infections.
C.trachomatis causes 20 to 40% of cases of NGU, and some studies indicated that
Mycoplasma genitalium and Ureaplasma urealyticum may cause an additional 10 to
20%. The remaining cases probably result from sexually transmitted pathogens, but
their precise etiology remains unclear. (Kriger,1999) .Both gonococcal and non
gonococcal Urethritis typically cause urethral disease on the other hand Prostatitis is a
condition that involves inflammation of the prostate and sometimes the area around it
(Nickel,2004). There are several types of prostatitis, each with a range of symptoms.

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Some men with prostatitis have a great deal of pain, while others are not really
bothered by their prostatitis; the rest have symptoms that fall somewhere between
those two extremes. Even mild symptoms can have a negative impact on quality of
life, especially if they persist or recur Prostatitis is fairly common, especially among
men younger than 50. Studies suggest that as many as 10 percent of adult males suffer
from prostatitis. This study was aimed to isolate and identify the intact cell wall and
cell wall defective bacteria from urethitis and prostatitis cases then study their
antibiotic sensitivity .
Material and methods
A- Collection and transport of specimens
For the collection of urethral specimens, a swab with a narrow diameter or a sterile
bacteriological loop should be inserted 3–4 cm into the urethra and gently rotated
before withdrawal. Purulent discharge can be collected directly on a swab also
prostatic fluid specimens were collected and transferred into their respective specimen
transport swabs from 30 patients with Urethritis and 30 prostatic patients men with
simple prostatitis , for culture on blood ,MacConky ,chocolate and variant agar in
seven modified methods (Brown,2007)

B-Direct examination
In most cases of gonorrhoeae in the male, the discharge is purulent, and numerous
polymorph nuclear leukocytes (>10 per oil-immersion field) can be seen in the
urethral smear directly making and after drying and fixation the smears were stained
with Leishman's stain . However, this is not always with the case in NGU, which
yields a less severe inflammatory reaction. Smears with more than 4 PMN leukocytes
per oil-immersion field, and without intracellular Gram-negative diplococci, are
highly suggestive of NGU.( Collins etal.,2004)

C-Routine methods:
The inoculation of specimens should be made directly onto the culture medium
(blood, MacConky ,variant and .chocolate agar) by streaking method. Blood&
MacConky plates inoculated in ordinary condition while variant and chocolate agar
plates should be placed in a candle jar into an atmosphere containing 5–10%carbon
dioxide "Co2",then the detection of isolated colonies by using some of biochemical
tests (MacFaddin,2000).

D-Modified culture techniques:(AL-Nassery ,2003)

Swabs of urethra and prostate were cultured by using seven modifiable methods
in three techniques :serial cutler, dilution and biphasic media to detect the cell wall
&cell wall defective bacteria as fallow:
1-Routine culturing of the sample by streaking on the culture media
2-Cultuering of the supernatant after centrifuging of the transport media which is
brain heart infusion broth.
3- Culturing of unwashed sediment.
4- Culturing of washed sediment with normal saline.
5- Culturing of lyses sediment with distal water after centrifugation.
6- Culturing of diluted supernatant
7-Biphasic media(consist of variant broth and variant agar in universal bottles)

415
 Result &Discussion
The results being reported as culture study (Table 1-6) ,male and female
distribution (Table 7),Identification (Table 8) and Sensitivity test (Table 9,10).The
distribution of infection according to sex appear that the total percentage of patients
with Urethritis and prostatitis for both sexes was (78.3%) while the total percentage
of the patients of urethritis for females was (21.6%).This result refer to present of
relative variation between the two types of diseases
( Bradshow,2006 and Betts,2003). The direct examination for the colonies that grew
on the cultured media and using of biochemical testes appear that the cell wall
microorganism involved in Urethritis and prostatitis was
N.gonorrhoae ,S.saprophyticus .K.pneumoniae.S.pnumoniae.E.coli and S.pyogenes
while the cell wall defective bacteria involved in these disease were
E.coli ,N.gonorrhoae and S.saprophyticu(Denham etal.,2005 and Mimis
etal.,2004)The presence of N.gonorrhoeae in the genital tract due to its ability to
adhere with the mucosal layer of this area because it has Pilli which also protect the
bacteria from phagoctosis(Brooks,2007) .Infected of the genital tract with
S.saprophyticus is happened by the association of virulence factors found in this
bacteria such as vironectin ,fibronectin and collagen proteins(Pulsson etal.,1999).The
antibiotic sensitivity test for the cell wall bacteria (gram positive and negative)showed
that cell wall N.gonorrhoeae was sensitive to tetracycline and Nalidixic acid while it
was resistamt to penicillin and ciprofloxacin. Penicillin G Resistant N. gonorrhoeae is
widespread in most developed countries, which has necessitated a change to newer
drugs for treatment of gonococcal infections. Recent reports indicated that resistance
to these newer drugs is increasing, highlighting the need for accurate therapeutic
recommendations. In some countries or communities, however, N. gonorrhoeae
isolates are still susceptible to penicillin, so the use of this antibiotic for single-dose
treatments of medically under-resourced patients is beneficial. (Gain etal.,2004).
Antimicrobial resistance mechanisms include the production of antibiotic-degrading
enzymes, modification of the target site, and decreased influx or increased efflux of
the antibiotic(Workowiski etal.,2006). Penicillin G was used to treat gonococcal
infections onto the mid-1980s, until the emergence and spread of resistance rendered
the antibiotic ineffective. These strains utilized either plasmid-mediated or
chromosomally mediated mechanisms to become resistant to penicillin G
(Gootz,2006). The spread of strains harboring a plasmid-encoded TEM-like _-
lactamase has been very rapid has forced the withdrawal of penicillin for treating
gonococcal infections in many areas. Chromosomally mediated resistance to
Penicillin, Tetracycline, and Macrolides .The resistance of gonococci to ciprofloxacin
is rapidly increasing and has reached high levels(40% of the strains in France in
2007). As a consequence ciprofloxacin might no longer be compatible with empirical
treatment in so far that its resort to ciprofloxacin would require prior microbiological
documentation and evidence of in vitro susceptibility to ciprofloxacin. Due to cross-
resistance, other fluoroquinolones (ofloxacin, norfloxacin and pefloxacin) cannot also
be recommended. This is all the more important that these molecules are known as
beingess active than ciprofloxacin with respect to gonococci, the activity of
norfloxacin is the worst, and ofloxacin has the additional disadvantage of a poor
pharyngeal diffusion. .In general cell wall defective bacteria was resist to penicillin
and all antibiotics act on the cell wall(Proctor etal.,1998 and Von-Eiff 2008). By
using different culture techniques the results were showing that the causative agent
may found in different shapes (cell wall, cell wall defective or transmissible) and can
grow on different media so the causative agent either be outside the cells ,adhere on

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Journal of Babylon University/Pure and Applied Sciences/ No.(2)/ Vol.(21): 2013

the cell surface or inside the pus cell. The appearance of the microorganism in the
biphasic media and dilution techniques refer to present of bacteria inside the pus cell
as cell wall defective and can pass through the Millipore filter with diameter 0.22m
(Domingue etal.,1997). In some time these techniques gave negatives results in all
steps this results indicate that the causative agent may be virus or fungi which can not
be isolated by these techniques or it is cryptic in protective area of the
body .Thus ,pathogen and stealth pathogens are associated with gonococcal and non
.gonococcal urethritis and prostatitis in this are (Denham etal.,2005)
Specimen B.A M.A V.A Ch.A
+ - + - + - + -
Whole blood 14 0 10 4 14 0 3 11
Supernatant 10 4 3 11 10 4 10 4
Sediment 10 4 3 11 10 4 10 4
Washed sediment 14 0 6 8 14 0 14 0
Lyses sediment 14 0 6 9 14 0 14 0
Table 1 The culture positive in all culture methods
A: Serial culture methods
B-Dilution
Dilution + -
Lysing cell supernate 1:10 14 0
1:100 9 4
1:500 6 8

C-Biphasic medium
Specimen B.A M.A V.A Ch.A
+ - + - + - + -
Lysing cell sediment 14 0 10 4 14 0 3 11

Table 2 culture is negative in the direct culture& washed sediment& positive to

other technique methods
A-Serial culture
Specimen B.A M.A V.A Ch.A
+ - + - + - + -
Whole blood 0 24 7 17 0 24 3 21
Supernatant 0 6 2 22 0 6 4 20

Sediment 0 6 2 22 0 6 4 20
Washed sediment 0 0 3 21 0 0 3 21
Lysed sediment 0 24 3 21 0 24 3 21

B:Dilution methd
Dilution + -
Lysing cell supernate 1:10 24 0
1:100 7 17
1:500 3 21
C:Biphaic media technique
Specimen B.A M.A V.A Ch.A
+ - + - + - +

417
 Lysing cell sediment 18 6 18 6 18 6 18 6

Table 3 The culture is negative for all techniques & positive in dilution method
& first step of serial culture
A: Serial method

Specimen B.A M.A V.A Ch.A

+ - + - + -
Whole blood 4 4 2 6 4 4 2 6
Supernatant 0 8 0 8 0 8 0 8
Sediment 0 8 0 8 0 8 0 8
Washed sediment 0 8 0 8 0 8 0 8
Lyses sediment 0 8 0 8 0 8 0 8

B: Dilution
Dilution + -
Lysing cell supernate 1:10 4 4
1:100 4 4
1:500 4 4

C:Biphasic media technique

Specimen B.A M.A V.A Ch.A
+ - + - + - + -
Lysing cell sediment 0 8 0 8 0 8 0 8

Table 4: Table 3 The culture is positive for serial culture methods(except the
first step) & positive in dilution method
A: Serial culture
Specimen B.A M.A V.A Ch.A
+ - + - + - + -
Whole blood 0 3 0 3 0 3 0 3
Supernatant 3 0 0 3 3 0 3 0
Sediment 3 0 0 3 3 0 3 0
Washed sediment 3 0 0 3 3 0 3 0
Lyses sediment 3 0 0 3 3 0 3 0
B: Dilution
Specimen + - + -
Lysing cell supernate 1:10 3 0
1:100 3 0
1:500 2 1

C:Biphasic media technique

Specimen B.A M.A V.A Ch.A
+ - + - + - + -
Lysing cell sediment 3 0 0 3 3 0 3 0

Table 5 The culture positive in serial culture &dilution methods

&negative in Biphasic media technique

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A: Serial culture method

Specimen B.A M.A V.A Ch.A
+ - + - + - + -
Whole blood 4 4 5 3 4 4 0 3
Supernatant 4 4 4 4 4 4 0 3
Sediment 8 0 8 0 8 0 0 3
Washed sediment 8 0 8 0 8 0 0 3
Lysed sediment 8 0 8 0 8 0 0 3
Dilution
Dilution + -
Lysing cell supernate 1:10 5 3
1:100 0 8
1:500 0 8

C:Biphasic media technique

Specimen B.A M.A V.A Ch.A
+ - + - + - + -
Lysing cell sediment 0 8 0 8 0 8 0 8
Table 6 The culture negative in all culture methods
A: Serial culture methods
Specimen B.A M.A V.A Ch.A
+ - + - + - + -
Whole blood 0 3 0 3 0 3 0 3
Supernatant 0 3 0 3 0 3 0 3
Sediment 0 3 0 3 0 3 0 3
Washed sediment 0 3 0 3 0 3 0 3
Lysed sediment 0 3 0 3 0 3 0 3

B : Dilution method
Dilution + -
Lysing cell supernate 1:10 0 3
1:100 0 3
1:500 0 3

C:Biphasic media technique

Specimen B.A M.A V.A Ch.A
+ - + - + - + -
Lysing cell sediment 0 3 0 3 0 3 0 3
)-(: Growth No growth:)+(

Table7: Distribution of Prostatitis &Urethritis according to sex

Number of patients )%(Male )%(Female

Number of patient with 17)56.7( 13)43.3(

Urethritis
Number of patients with 30)100( -
prostatitis 419
Total Number 47)78.3( 13)21.7(
 Table 8:Cusetive bacteria of Urethritis& Prostatitis
Causative agent Urethritis Prostatitis Total Number
Bacteria CWB CWDB CWB CWDB
No )%(. No )%(. No )%(. No )%(. No )%(
G-ve 10 )12.0( 2 )2.4( 8 )6.9( 1 )1.2( 21 )25.3(
N.gonorrhoae
5 )6.0( 1 )1.1( 8 )6.9( 1 )1.2( 15 )18(
E.coli
4 )4.8( 0 )0( 5 )6( 0 )0( 9 )10.8(
K.pneumoniae
P.aeruginosa 5 )6.0( 0 )0( 3 )3.6( 0 )0( 8 )9.6(

G+ve 5 )6.0( 5 )6.0( 5 )6( 2 )1.2( 17 )20.5(

S.saprophyticus
5 )6.0( 0 )0( 3 )3.6( 0 )0( 8 )9.6(
S.pyogenes
K.pneumoniae 2 )2.4( 0 )0( 3 )3.6( 0 )0( 5 )6(
Total 36 )43.4( 8 )9.6(( 35 42.2( 4 )4.8( 83 )100(
)

CWB: Cell Wall Bacteria CWDB :Cell Wall Defective Bacteria

Antibiotic sensitivity of cell wall bacteria(gram positive& negative)

Table(9)

Antibiotic S.saprophyticus N.gonorrhoeae E.coli

S R S R S R
.No )%( .No )%( .No )%( .No )%( .No )%( .No )%(
Ampicillin 4 )23.5( 13 )76.5( 5 )23.8( 16 )76.2( - - - -
Penicillin 6 )35.3( 11 )64.7( 3 )14.2( 18 )85.8( - - - -
Chloramphynicol 13 )76.5( 4 )23.5( 0 )0( 21 )100( 8 )53.3( 7 )46.7(
Tetracycline 11 )64.7( 6 )35.3( 13 )61.6( 8 )38.4( 8 )53.3( 7 )46.7(
Streptomycin 9 )52.9( 8 )47.0( 3 )14.2( 18 )85.8( 5 )33.3( 10 )66.7(
Impineme 17 )100( 0 )0( 5 )23.8( 16 )76.2( 9 )60( 6 )40(
Nalidixic acid 9 )52.9( 8 )47.1( 15 )71.4( 6 )28.6( 10 )66.7( 5 )33.3(
Timethoprime 6 )35.3( 11 )64.7( 0 )0( 21 )100( 6 )40( 9 )60(
Meropinem 15 )88.2( 2 )11.8( 7 )33.3( 16 )76.1( 9 )60( 6 )40(
Ciprofloxacillin 11 )64.7( 6 )3.3( 5 )23.8( 16 )76.2( 8 )53.3( 7 )46.7(

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 Journal of Babylon University/Pure and Applied Sciences/ No.(2)/ Vol.(21): 2013

Table 10:Antibiotic sensitivity of cell wall defective bacteria

(gram positive and negative)

Antibiotic S.saprophyticus N.gonorrhoeae E.coli

S R S R S R
.No )%( .No )%( .No )%( .No )%( .No )%( .No )%(
Chloramphynicol 3 )42.8( 4 )57.1( 2 )66.7( 1 )33.3( 2 )100( 0 )0(
Tetracycline 5 )71.4( 2 )28.5( 0 )0( 3 )100( 0 )0( 2 )100(
Streptomycin 5 )71.4( 2 )28.5( 0 )0( 3 )100( 0 )0( 2 )100(
Nalidixic acid 5 )71.4( 2 )28.5( 0 )0( 3 )100( 0 )0( 2 )100(
Timethoprime 0 )0( 7 )100( 0 )0( 3 )100( 0 )0( 2 )100(
Ciprofloxacillin 6 )85.7( 1 )14.3( 2 )66.7( 1 )33.3( 2 )100( 0 )0(

S: sensitive R:Resistant

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