CYSTEINE-DEPENDENT UROPATHOGENS: Isolation, Identification and Susceptibility to Antimicrobial Agents

Tarek M. Gibreel1 and Khalifa Sifaw Ghenghesh2

Central Hospital Laboratories1 and Dept. of Medical Microbiology2, Faculty of Medicine, Al-Fateh University, Tripoli-Libya

*Correspondence: Tarek M. Gibreel, BPh, MSc Microbiology Unit, Central Hospital Laboratories, Tripoli – Libya e-mail:tarekgibreel@hotmail.com Or: Prof. Khalifa Sifaw Ghenghesh e-mail: ghengesh_micro@yahoo.com To cite this paper: Gibreel TM., and Ghenghesh KS. 2002. Cysteine-Dependent Uropathogens: Isolation, Identification and Susceptibility to Antimicrobial Agents. Jamahiriya Med J; 2 (2): 52-54.

ABSTRACT: Members of the family Enterobacteriacea that are auxotrophic for

cysteine because of defects in the sulfur assimilatory pathway account for up

to 2 % of significant isolates from urinary tract infections. In the present work

bacterial isolates from Libyan patients with urinary tract infections were

examined for cysteine-dependence and susceptibility to antibiotics. Methods

of isolation, identification and antimicrobial susceptibility testing were used in

presence and absence of cysteine supplementation. Of 675 enterobacterial

isolates examined, nearly 1.5% were cysteine-dependent uropathogens

(CDUP). A significant association of CDUP with abnormal urological

conditions was observed. The means by which laboratory procedures can be

adapted to fully identify such organisms are simple and should be used

whenever auxotrophy is expected.

INTRODUCTION: Strains of the family Enterobacteriaceae auxotrophic for a variety of

nutrients are not infrequently isolated from clinical specimens. Such

organisms grow poorly on isolation media and form so called "dwarf colonies"

(1). These organisms are known to cause difficulty in identification and in

antimicrobial susceptibility testing as a result of their dependence on various

growth factors to achieve normal colony morphology and growth rate. They

include cysteine-dependent Escherichia coli (1) and Klebsiella sp. (2) and

thymine-dependent E. coli (3), Proteus mirabilis (4), and Salmonella

typhimurium (5).

Strains of cysteine-dependent E. coli represent about 2% of all isolates

of this species from urinary tract infections and are more frequently recovered

from elderly patients with chronic urinary tract infections and/or with long

standing urinary tract abnormalities (1,6). The deviation from prototrophs to

auxotrophs is enhanced under conditions of cysteine and other sulfur

compound excess that is seen in the urine of patients with decreased renal

function, or patient on prolonged co-trimoxazole (combination of trimethoprim

and sulfamethoxazole) (3). Cysteine requiring isolates have been shown to

lack the ability to assimilate sulfate, as a functional sulfur assimilatory pathway

resulting in the formation of cysteine. Laboratory media commonly incorporate

peptone in their constituents. These peptones are not fully characterized with

regard to their amino acid contents or concentration. Cysteine is normally

denatured during the production of peptones, so cysteine-supplemented

media have been developed to facilitate the isolation of cysteine auxotrophs

(7). The amounts of cysteine that allow the maximum growth yields of

auxotrophs but without toxic effects in the growth of auxotrophic and

prototrophic strains have been determined in such media (8).

The presence of cysteine-dependent E. coli in urine or blood usually is

an indication of underlying renal condition and should prompt a search for

such a condition (9,10). However, reports published on these organisms are

few and in Libya are lacking. The present study was undertaken to determine

the prevalence of cysteine-dependent uropathogens among isolates from

Libyan patients with urinary tract infections, describe the methods used to

isolate and identify them, and determine their antibiotic susceptibilities. MATERIALS AND METHODS: Patients and specimens:

Included in the present study 2053 patients (1032 adults and 1021

children) aged between <1 month and >65 years old. All were suspected

cases of urinary tract infection attending Al-Jala Pediatric Hospital and Tripoli

Medical Center during the period from January to July 1999. A total of 2053

urine specimens, were collected in sterile universal containers containing 2%

boric acid or adhesive urine bags and transported to the laboratory and

processed within 2 hours from collection.

Culture of Urine and Determination of Significant Bacteriuria:

Urine specimens were cultured onto Blood agar, MacConeky agar and

CLED agar, by the semiquantitative method using standard loops. A cut–off

point of 100.000 CFU/ml was used for this study, the colonies were counted

and if more than 100 colonies were present, the urine specimen was

considered to have greater than 105 CFU/ml indicating significant bacteriuria.

Identification of the isolated uropathogens: Standard diagnostic microbiological methods for identification of

isolates were used (11,12).

Detection of the cysteine-dependent uropathogens (CDUP): Each Gram-negative isolate that grew as lactose-fermenting pinpoint

colonies (dwarf colonies) on MacConkey agar (MA) plates, and was oxidase

negative and catalase positive, was inoculated onto CLED agar plate, MA

plate and Davis Minimal medium (DMM) (13). Then filter paper discs

impregnated with solution containing 0.63 mM (0.1mg/ml) L-cysteine HCL

were placed on both MA and DMM plates (14). A blood agar plate and

another MA plate were inoculated as controls. All plates were incubated at

37ºC for 24 hours.

Identification of CDUP:

API 20E Sysytem (bio-Merieux, France) was used for the identification

of the auxotrophs. The inocula were derived from suspensions of organisms

in 5 ml of sterile normal saline alone and sterile saline supplemented with

cysteine HCL 0.63mM (0.1mg/ml). Suspensions were prepared using three

dwarf colonies from growth on MA, and for inoculum prepared from CLED

agar one normal sized colony was used and it provided an inoculum of similar

cell density. The test reaction were initially recorded after 24 hours and then

after 48 hours for detection of weak reactions. The profile numbers thus

generated were used to identify these isolates by using the Analytical Profile

Index catalog for the Enterobacteriaceae (bio-Merieux).

Antibiotic susceptibility testing:

The method of Kirby and Bauer was used (15). The isolates were

tested against ampicillin, amoxicillin-clavulanic acid combination (augmentin),

gentamicin, nalidixic acid, nitrofurantoin, co-trimoxazole, cephalexin,

ciprofloxacin and norfloxacin. The inocula suspensions were adjusted to

McFarland 0.5 turbidity standard. Mueller-Hinton agar plates supplemented

with 0.63mML-cysteine HCL were used. The zones of inhibition were

compared against standard zone diameter scales.

RESULTS:

Of 2053 urine specimens examined, 625 were from inpatients, of which

130 were patients with different urological abnormalities, and 1428 were from

outpatients. Significant bacteriuria was detected in 41% (255/625) of

inpatients and in 29% (420/1428) of outpatients. In total significant bacteruria

was found in 33% (675/2053) of patients examined. Also, significant

uropathogens were detected in 86% (112/130) of inpatients with urological

abnormalities and in 29% (143/495) of inpatients with non-urological

abnormalities.

Isolation and identification of cysteine-dependent uropathogens (CDUP):

After 24 hours of incubation, CLED agar plates showed growth of

lactose fermenting colonies that reached a diameter of 4 mm. There were

satellitism and lactose fermenting activity around the cysteine discs on both

MA and DMM. However, pinpoint colonies were noted on MA control plate.

Ten CDUP were detected representing around 1.5% of the total isolates. The

prevalence of CDUP among uropathogens from patients and inpatients with

urinary tract infections is shown in Table 1.

CDUP were identified using API 20E system. Using inocula not

supplemented with cysteine it was not possible to identify CDUP after 24

hours incubation of the API 20E system, however, incubating the system for

further 24h (48h incubation) permitted the identification of the CDUP. On the

other hand using inocula supplemented with cysteine resulted in identification

of CDUP within 24h incubation. Of the 10 isolated CDUP 7 were identified as

E. coli and 3 as Klebsiella pneumoniae.

Reviewing the clinical history of patients from which the CDUP were

detected resulted in 7 of these patients being on co-trimoxazole either for

treatment or prophylaxis. Although the supplementation with cysteine did not

change the susceptibility results to most of antibiotics tested, 6 isolates were

resistant to co-trimoxazole and 2 to co-trimoxazole and augmentin. One

isolate was resistant to cephalexin after supplementation of the sensitivity

medium with cysteine. All 10 CDUP strains were susceptible to gentamicin,

nalidixic acid, nitrofurantoin, ciprofloxacin and norfloxacin.

Table 1. Prevalence of cysteine-dependent uropathogens (CDUP) among bacterial isolates from inpatients and outpatients with urinary tract infections. -------------------------------------------------------------------------------------------------------- UP from Inpatients with UP from outpatient Total

urological non-urological abnormalities abnormalities [n=112] [n=143] [n=420] [n=675]

--------------------------------------------------------------------------------------------------------CDUP 9(8)* 1(0.7) 0(0.0) (1.5) -------------------------------------------------------------------------------------------------------- (%), *statistically significant (P< 0.05, Chi-squares test).

DISCUSSION: Cysteine-dependent uropathogens (CDUP) in 1.5% of the significant

bacteriuria detected in the present study. This finding is comparable to the

rate of 1.5-2% reported by others (1). The mechanism for development of

most autotrophic bacteria is largely unknown (16). However, the obvious

occurrence of cysteine auxotrophs in patients with long standing urinary tract

abnormalities probably correlates to excess cysteine and sulfur compounds in

the urine of those patients or prolonged co-trimoxazole therapy used for those

patients (3). Our findings support this hypothesis. In the present work, CDUP

were significantly detected in inpatients with urinary tract abnormalities (8%, 9

out of 112) when compared with inpatients with no such abnormalities (0.7%,

1 out of 420) or with outpatients (0%). Furthermore, 7 (70%) of the 10 CDUP

were detected in patients who were on co-trimoxazole therapy.

The difficulties that were encountered with the identification and

antimicrobial susceptibilities of cysteine requiring isolates were overcome

using both extended incubation times and supplementation with cysteine.

These problems can be attributed to the poor growth of the auxotrophs in

growth-dependent tests. Such tests are the carbohydrate fermentation tests

and cysteine-deficient culture media. This is being a consequence of the lack

of appropriate nutrients for these organisms in peptones, which are a basic

component of the culture media (14,16). The growth of the auxotrophs on the

Mueller-Hinton agar markedly enhanced by the cysteine supplementation but

it rarely changes the antimicrobial susceptibility pattern except that for co-

trimoxazole. This antimicrobial agent is commonly used in Libya for the

treatment of urinary tract infections and for prophylaxis in patients with chronic

urinary tract abnormalities.

It is difficult to estimate the extent of the problems posed by

auxotrophic bacteria to the bacteriology laboratories, as there had been no

previous reports of such organisms in Libya. The means by which laboratory

procedures can be adapted to fully identify these organisms are simple and

should be used when auxotrophy is suggested on the basis of unusual

colonial morphology or when inadequate growth occurs in identification or

susceptibility testing systems. Urologists and bacteriology laboratory

technicians should pay more attention to CDUP, to have a better idea in the

future on their role in disease processes in Libyan patients.

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