Original Articles
 

By Dr. Shobha KL , Mr. Gowrish G Rao , Mr. Anand M Kukkamalla
Corresponding Author Dr. Shobha KL
Department of Microbiology , Melaka Manipal Medical College (Manipal Campus ), Melaka Manipal Medical College , Manipal Campus , Manipal,Karnataka State - India 576104
Submitting Author Dr. Shobha KL
Other Authors Mr. Gowrish G Rao
MMMC(MC),Department of Microbiology, MMMC(MC)Manipal - India 576104

Mr. Anand M Kukkamalla
MMMC(MC),Department of Microbiology, MMMC(MC)nManipal - India 576104

MICROBIOLOGY

Urinary Tract Infection; Nonfermenters; Pseudomonas spp.

KL S, Rao GG, Kukkamalla AM. Prevalence Of Non-fermenters In Urinary Tract Infections In A Tertiary Care Hospital. WebmedCentral MICROBIOLOGY 2011;2(1):WMC001464
doi: 10.9754/journal.wmc.2011.001464
No
Submitted on: 10 Jan 2011 06:18:39 AM GMT
Published on: 10 Jan 2011 01:17:50 PM GMT

Abstract


Background and Objective: Aerobic nonfermenting gram-negative bacilli (non-fermenters) are now emerg­ing as important uropathogens. This study was done to know the prevalence of non-fermenters in Urinary Tract Infections (UTI) along with their antibiotic sensitivity.

Method: 5436 mid stream urine  specimens received in six months period from July to December 2007 were subjected to Gram,s staining ,semi-quantitative culture, biochemical tests and antibiotic sensitivity testing as per the standard procedures in the routine microbiology laboratory and the results were noted.

Results: Out of the 1271 culture positive cases with significant bactereuria, 120 (9.44%) were caused by non-fermenters. Among the non-fermenters, Pseudomonas spp. was the most common (80.84%) followed by Acinetobacter spp. (19.16%).  Majority of patients belonged to the age group of 15 to 50 years and most of them were females. Antibiotic sensitivity testing revealed that, among Pseudomonas spp., 75.25% isolates were sensitive to Piperacillin, 71.13% sensitive to Amikacin, 43.30% sensitive to Ciprofloxacin, 31.95% sensitive to Ceftazidime and 91.66% sensitive to Imipenem. In the case of Acinetobacter spp., 73.91% isolates were sensitive to Amikacin, 34.78% sensitive to Norfloxacin and all were sensitive to imipenem. Out of the 18 non-fermenter isolates which were resistant to commonly used antibiotics, 15 were from inpatients indicating the possibility of nosocomial infection.

Conclusion: The non-fermenters are emerging as important cause of Urinary Tract Infections. In this study, Pseudomonas spp. was the most common non-fermenter isolated in significant numbers in MSU specimens. But, Acinetobacter spp. appeared to be more resistant to invitro antibiotics in general.

Introduction


Urinary tract infections (UTI) are the second most common bacterial infections affecting humans throughout their lifetime. They are frequent cause of morbidity in outpa­tients as well as most frequent cause of nosocomial in­fection in many hospitals (Sussman M 1998) (9). Escherichia coli is the commonest urinary pathogen accounting for over 80% of commu­nity-acquired infection. Far less commonly Klebsiella spp, Proteus spp and Staphylococcus saprophyticus are responsible for community-acquired infection. The distri­bution of urinary pathogens in hospitalized patients is dif­ferent with Escherichia coli accounting for about 50% of infections. Enterococcus, Klebsiella, Enterobacter, Citrobacter, Serra­tia, Pseudomonas aeruginosa, Providencia, and Staphylo­coccus epidermidis account for most of the rest. (Bryan et al 1984) (1). Aerobic nonfermenting gram-negative bacilli (nonfer­menters) are a heterogeneous group of organisms that are either incapable of utilizing carbohydrates as a source of energy or degrade them via oxidative rather than fermentative pathway. (Koneman EWet al 1988) (4) This group includes organisms from diverse gen­era like Pseudomonas, Acinetobacter, Alcaligenes, Flavo­bacter, Oligella, Flavimonas, Agrobacter Weeksiella, etc. These organisms are common inhabitants of soil and water. They also exist as harmless parasites on the mucus membranes of humans and animals. Though primarily regarded as contaminants or incidental organisms, they are becoming increasingly important as opportunistic pathogens in immunocompromised patients. They can also cause infection by gaining access to normally sterile body sites through trauma. (Baron E.J et al 1990) (2). Though these organisms vary in their potential to cause infection and antibiotic sus­ceptibility, there are common disease manifestations and risk factors. These organisms have been implicated in causing septicemia, meningitis, osteomyelitis, wound infections, pneumonias and urinary tract infections. Risk factors include immunosuppression, trauma, foreign body, broad-spectrum antibiotic use, infused body fluids like saline irrigations (Hall G.S1995) (3) and also urinary catheterization (Katsumi Shigemura 2006) (13). Pseudomonas aeruginosa is the predominant and most well known organism out of this heterogeneous group (Katsumi Shigemura et al 2006) (13) (Jombo G.T.A et al 2008) (14). This is partly due to its easy recognition in the laboratory as it produces pyocyanin, a blue-green pigment. Other organisms usually get ignored as contaminants because identification up to species level is cumbersome and laborious and not possible in a busy routine microbiology laboratory (Stansfeld JM 1966) (8). It has been noted that the non-fermenter gram negative bacilli are being increasingly isolated in significant bacteriuria cases in routine urinary microbiology over the years indicating their potential in causing urinary tract infections. It is also well-known that most of these non-fermenter gram negative bacilli are resistant to many commonly used antibiotics for treatment (Gupta V et al 2002) (10)  (Jamal W et al 2009) (11)  (Nenek A et al 2008) (12)  (Takeyama K et al 2008) (15). Evaluation of significance of isolating these organisms is com­plicated by the fact that they are usually present in mixed culture and the clinicians are rather unfamiliar with their names. This retrospective study was done to know the prevalence of nonfermenters in UTI along with their antibiotic susceptibility.

Materials & Methods:


Mid stream urine (MSU) specimens collected from both inpatients and outpatients attending Kasturba Hospital, Manipal (1500 bed capacity) for routine culture and sensitivity test, from July to December 2007 were included in this study. These MSU specimens were studied for significant bactereuria by grams staining and culturing on blood agar and MacConkey agar according to Kass concept (Hall GS et al 1995) (3).  Those urine specimens  containing organisms and pus cells on grams staining and  yielding a pure culture of 10 5 or more CFU/ml are considered to be significant bactereuria cases. They were further proceeded for species identification by standard biochemical tests. (Rambaugh KP et al 1999) (7) In-vitro antibiotic sensitivity test was performed by Kirby Bauer’s disc diffusion method using Muller Hinton Agar as per Clinical Laboratory Standards Institute (CLSI) guidelines and susceptibility pattern was noted. (Quinn PJ 1988) (6) In this study, we have included only the non-fermenters causing UTI. First and second line antibiotics used for oxidase positive non-fermenters were gentamicin (10 μg), tobramycin (10 μg), netilmycin (30 μg), amikacin (30 μg), ciprofloxacin (5 μg) , ceftazidime & piperacillin (100 μg),  where as for oxidase negative non-fermenters gentamycin(10 μg), netilmycin(30 μg), amikacin(30 μg), norfloxacin (10μg), ampicillin (10 μg), amoxicillin-clavulanic acid(10 μg), cefuroxime(30 μg) & cefotaxime(30 μg). Isolates resistant for all first & second line antibiotics were tested against third line antibiotics which included aztreonam(30 μg) , cefpirome(30 μg) , ticarcillin-clavulanic acid(30 μg) , pipercillin-tazobactam(30 μg) , cefoperazone-sulbactum & imipenem(30 μg)  for both oxidase positive & negative non-fermenters. All culture media and antibiotic discs were from Hi­media Laboratories Pvt Ltd, Mumbai, India.

Results


Out of the five thousand three hundred and forty six MSU specimens, no significant growth was recorded in four thousand seventy five (76.22%) specimens where as one thousand two hundred seventy one (23.38%) specimens showed significant growth. This indicates that approximately ¼ of the total specimens are significant bactereuria cases.

Out of 1271 urinary isolates with significant bactereuria, number of fermenter gram negative bacilli was 1033 (81.27%), non-fermenters gram negative bacilli was 120 (9.44%), gram positive cocci was 81 (6.37%) and yeasts 37 (2.91%).

Thus fermenter GNBs caused more than ¾ cases of significant bactereuria where as the remaining ¼ cases were caused by non-fermenter GNBs, GPCs and yeasts. In case of non-fermenter GNBs, the number of Pseudomonas spp. was 97 (07.63 %) and Acinetobacter spp. 23 (01.80 %). Illustrations 1 and 2.

Discussion and conclusion


Urinary tract infections are found in all age groups. The incidence is higher in male infants due to presence of con­genital anomalies (Stansfeld J.M 1966) (8). From adulthood to the age group of 65 years, the incidence of UTI is very high in women as com­pared to men. In older patients there is a progressive decrease in the female to male ratio due to development of obstructive uropathy from the prostatic enlargement. The results of this study are in line with the general observation regarding the age groups (Stansfeld J.M 1966) (8).

In the present study, non-fermenter GNBs were found to be significant in 09.44% cases, second only to the fermenters (81.27%).  Pseudomonas aeruginosa is an established pathogen of urinary tract. (Rambaugh K.P et al 1999) (7). Pseudomonas spp. was the commonest non-fermenter isolate in the present study being significant in 07.63% of cases. Acinetobacters are ubiquitous in environment and in hospitals (Quinn P.J 1988) (6).  They account for 1 to 3 percent of nosocomial infections, being second only to P. aeruginosa as non-fermenters in their re­covery from clinical specimens. (Hall G.S 1995)(3)  Acinetobacters have been implicated in causation of a variety of illness including UTI. (Pederson M.B et al 1970) (5) They accounted for 01.80% cases in this study. A high level of drug resistance was observed in general for majority of strains of these bacterial species in different studies (Gupta V et al 2002) (10) (Jamal W et al 2009) (11) (Nenek A et al 2008) (12) (Jombo G.T.A et al 2008) (14) (Takeyama K et al 2008) (15) which is in line with the results of this study. Out of the 18 non-fermenter urinary isolates which were resistant to all first and second line antibiotics invitro, 15 were from inpatients.  This could be due to the fact that most of these isolates may be from UTI with nosocomial origin (Marcus N et al) (16).

Non-fermenters were found to be significant in 120 cases, in which 76 were in-patients and 44 were attending Outpatients Department. In this, 61 were males and 59 females.  Children below 15 years of age were ten in which six were males and four females. In the remaining 110 cases, 62 were in the age group between 15 to 50 years (20 males & 42 females) and 48 were above the age of 50 years (37 males & 11 females).

Out of 97 isolates of Pseudomonas spp., 85 were sensitive to at least one of the first and second line antibiotics. Further, 69 (71.13%) isolates were sensitive to Aminoglycosides, but Gentamicin was effective against only 36 (37.11%) isolates. 42 (43.30%) isolates were sensitive to Ciprofloxacin (fluoroquinolonee) whereas  73 (75.25%) isolates were sensitive to Piperacillin (Anti-Pseudomonas Penicillin) and 31 (31.95%) isolates were sensitive to Ceftazidime (Anti-Pseudomonas Cephalosporin).  12 isolates were tried with the susceptibility of imipenem,Piperacillin-Tazobactam ,Cefoperazone-sulbactam,Aztreonam,Cefpirome and Ticarcillin-Clavulanic acid ,11 (91.66%) were sensitive to Imipenem, 5 (41.66%) were sensitive to Piperacillin-Tazobactam and 6 (50%) were sensitive to Cefoperazone-Sulbactam. All 12 (100%) isolates were resistant to Aztreonam, Cefpirome and Ticarcillin-Clavulanic acid.Illustration 3 and 4.    

Out of 23 isolates of Acinetobacter spp, 17 (73.91%) were sensitive to at least one of the first and second line antibiotics. Further, 17 (73.91%) isolates were sensitive to Aminoglycosides, but Gentamycin was effective against only 10 (43.47%) isolates. None of the isolates (0%) were sensitive to Penicillins and Cephalosporins. Fluoroquinolone (Norfloxacin) sensitivity was seen with 8 (34.78%) isolates. Third line antibiotic sensitivity was performed for 6 isolates. All six (100%) were sensitive to Imipenem and Four (66.66%) isolates were sensitive to Cefoperazone-Sulbactam where as Piperacillin-Tazobactam was effective against 3 (50%) isolates and Cefpirome sensitivity was seen with only 2 (33.33%) isolates. None of the isolates in this category were sensitive to Aztreonam and Ticarcillin-Clavulanic acid. Illustration 5 and 6.   

Urinary tract infections are found in all age groups. The incidence is higher in male infants due to presence of con­genital anomalies (Stansfeld J.M 1966) (8). From adulthood to the age group of 65 years, the incidence of UTI is very high in women as com­pared to men. In older patients there is a progressive decrease in the female to male ratio due to development of obstructive uropathy from the prostatic enlargement. The results of this study are in line with the general observation regarding the age groups (Stansfeld J.M 1966) (8).

In the present study, non-fermenter GNBs were found to be significant in 09.44% cases, second only to the fermenters (81.27%).  Pseudomonas aeruginosa is an established pathogen of urinary tract. (Rambaugh K.P et al 1999) (7). Pseudomonas spp. was the commonest non-fermenter isolate in the present study being significant in 07.63% of cases. Acinetobacters are ubiquitous in environment and in hospitals (Quinn P.J 1988) (6).  They account for 1 to 3 percent of nosocomial infections, being second only to P. aeruginosa as non-fermenters in their re­covery from clinical specimens. (Hall G.S 1995)(3)  Acinetobacters have been implicated in causation of a variety of illness including UTI. (Pederson M.B et al 1970) (5) They accounted for 01.80% cases in this study. A high level of drug resistance was observed in general for majority of strains of these bacterial species in different studies (Gupta V et al 2002) (10) (Jamal W et al 2009) (11) (Nenek A et al 2008) (12) (Jombo G.T.A et al 2008) (14) (Takeyama K et al 2008) (15) which is in line with the results of this study. Out of the 18 non-fermenter urinary isolates which were resistant to all first and second line antibiotics invitro, 15 were from inpatients.  This could be due to the fact that most of these isolates may be from UTI with nosocomial origin (Marcus N et al) (16).

This study revealed that for Pseudomonas spp., Piperacillin and Amikacin are most effective (Sussman M 1998)(9) followed by Ciprofloxacin and Ceftazidime in the group of first and second line antibiotics and also Imipenem to be the most effective followed by Cefoperazone-Sulbactam and Piperacillin-Tazobactam in the group of third line reserved antibiotics. For Acinetobacter spp., Aminoglycosides in the group of first and second line antibiotics and Imipenem in the group of third line reserved antibiotics are found to be most effective.  

A previous study has reported that for Pseudomonas aeruginosa, amikacin, ceftazidime and piperacillin are the recommended antibiotics (Gupta V et al 2002) (10) which is in accordance with the results of our study. But other studies reported multiple drug resistance in Pseudomonas aeruginosa isolates (Takeyama K et al 2008) (15), (Jombo G.T.A et al 2008) (14), (Katsumi Shigemura 2006) (13). It has been also reported that Acinetobacter infection in hospitalised patients showed high degree of resistance to almost all the antibiotics used routinely necessitating its susceptibility testing for newer drugs (Gupta V et al 2002) (10)  which is supported by our study. Also, all strains of Acinetobacter spp. were found sensitive to imipenem in our study though emergence of resistance to this group of antibiotics has been reported in previous studies (Jamal W et al 2009) (11) (Nenek A et al 2008) (12).

From the present study it can be concluded that non-fermenter gram negative bacilli though regarded as contaminants are important bacteria causing hospital and community acquired UTI. If recovered in pure culture, a re­peat urine specimen should be taken to confirm their presence, and the strain should be identified up to the species level along with the antibiotic susceptibility testing for routinely used antibiotics.     

References:


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Source(s) of Funding


Manipal university,Manipal

Competing Interests


No competing interests

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