In vitro susceptibility of urinary pothogens to trimethoprim, nitrofurantoin and trimethoprim-nitrofurantoin combinationPervin Anklesaria1, Swati Paranjpe1, Kusum Gupta1, Vidya Acharya1, Teresa Paul1, UK Sheth2
1 Department of Pharmacology, Clinical Drug trial Unit, Council of Scientific and Industrial Research (CSIR) and The Department of Medicine, Seth G. S. Medical College, Parel, Bombay-400012., India
2 Department of Pharmacology, Seth G. S. Medical College, Parel, Bombay-400012, India
Trimethoprim and Nitrofurantoin are both effective against urinary tract infections. Trimethoprim till now has been used in combination with sulphamethoxazole. We have investigated the combination of Trimethoprim and Nitrofurantoin on 182 pathogenic cultures isolated from 165 cases of urinary tract infection which had a significant colony count of >10 5 orgs/ml. 65% of the strains exhibited synergy to the combination. Clinical results of this combination are very promising and are reported in another communication.
Infection of the urinary tract is one of the most common bacteriological diseases encountered. This condition produces a favourable environment for the continuous multiplication of bacteria in the urinary system. Urine culture is the only satisfactory method of diagnosis of active infection.
Infection of the urinary tract leads to the administration of antimicrobial drugs. The objective of antimicrobial therapy is to permanently eliminate bacteria from the urinary tract, which is determined by quantitative bacterial counts decreasing within 48-72 hours after initiation of therapy.  The urinary level of an antimicrobial agent generally determines the outcome of therapy when the infection is unaccompanied by sepsis.  Good urinary concentrations are achieved after oral administration of most of the commonly used antimicrobial agents namely Nitrofurantoin (NF), Trimethoprim (TMP) , ampicillin, cephalexin etc. It is well known that NF produces untoward side effects such as nausea and vomiting in a considerable number of patients.
In recent years, as many urinary tract infections show high degree of resistance to sulfonamides and antibiotics, interest has centered on either new antibiotics which have not been much used or combination of two drugs acting at different sites on the organism. Successful introduction of cotrimoxazole which is a combination of an antifolic agent Trimethoprim (TMP) and sulfamethoxazole is an example of such strategy. Trimethoprim and sulfonamides both act at different stages in the synthesis of folic acid for the formation of DNA. TMP alone in adequate doses also has been reported to be effective in the treatment of urinary tract infection.
In our hospital a number of bacteria isolated from urinary tract infection have been found to be sensitive to Nitrofurantoin. The practical difficulty has been intolerance to oral administration of Nitrofurantoin. The mechanism of action of NF is not still well defined. We decided to check the effectiveness of a combination of TMP and NF in decreased doses on the isolated organisms from urinary tract infections. Both these drugs are excreted well in urine and if one could find any additive or synergistic effects of the combination, clinically, in cases sensitive to these drugs, smaller doses of NF could be used with better tolerance and adequate effectiveness.
182 bacterial strains were isolated from 165 cases of urinary tract infection which had a significant colony count of >10 5 orgs/ml. The bacteria were identified according to Cowan and Steel. 
The minimal inhibitory concentrations (MIC) of TMP, NF and the combination of the two drugs in varying ratios were determined for all the strains. The medium used was nutrient agar pH 7.4, plus 5% lysed horse blood. An overnight broth culture of the organisms appropriately diluted with nutrient broth was used as the inoculum to streak the agar plates containing the antibacterial agent.
A dilution of 1: 1000 of the overnight broth culture was used for E. coli, Staphylococci, A. anitratus and A. faecalis. For Proteus, Pseudomonas, Morganella, Klebsiella and Enterobacter strains a dilution of 1:10,000 and for Streptococci a dilution of 1:10 was used.
The organisms isolated from 165 cases of urinary tract infection were 175 Gram negative bacteria and 7 Gram positive bacteria, namely Escherichia More Details coli (98), Enterobacter aerogenes , Acinetobacter anitratus (7), Alcaligenes faecalis (6), Klebsiella pneumoniae (4), Alkalescens-dispar group (1), Pseudomonas aeruginosa ), Morganella morganii (1), Proteus mirabilis (7), Proteus rettgeri (1), Proteus inconstans s (3), mixed culture of Enterobacter + Proteus (3), unidentified Gram negative bacilli (1), Staphylococcus aureus (3), Staphylococcus epidermidis (2) and Streptococcus pyogenes r>
The MICs of TMP and NF were determined for the 182 urinary pathogens of which 114 strains (62.6%) had an MIC of < 1µg/ml of TMP and 121 strains (66%) had an MIC of < 100 µg/ml of NF [Figure 1] and [Figure 2].
Out of the 182 bacterial species, 120 strains (65%) demonstrated synergy when tested with varying ratios of the combination of TMP and NF. The results of these 120 susceptible strains are presented in [Table 1]. The Fractional Inhibitory Concentration (FIC) of each drug, which is defined as the MIC of the drug in combination divided by the MIC of the drug alone, teas calculated for the individual strains. The sum of the FICs of TMP and NF for each organism gave the FIC index. Potentiation by the combination was demonstrated for the 120 strains by the FIC index being < 1.0, the lower the index, the greater the synergy. 
The FIC indices varied from 0.13 (1 strain of E. coli) to 0.9 (2 strains of A. faecalis). 26 strains had FIC indices < 0.5; 41 strains between 0.5 to 0.7 and 53 strains had an FIC index of 0.7 to 0.9 [Table 2].
Of the 62 strains that did not exhibit synergy, 20 were E. coli, 23 -E. aerogenes, 5-A. anitratus, 1 -A. faecalis, 4 -K. pneunaoniae, 3 -P. aeruginosa, 1 -Morganella morganii, 2 -P. mirabilis, 1 -Enterobacter + Proteus, and 2 -Staphylococcus aureus.
The above 182 strains were also tested by the paper disc method  against all the common antibiotics and chemotherapeutic agents as well as the combination of 1.25 µg TMP plus 25 µg NF per disc.
Enhancement of antibacterial activity of chemotherapeutic agents is desired, especially in the treatment of urinary tract infection. The use of a combination of antimicrobial agents for the management of severe infections is common in modern practice.
In our in vitro study, we have found that a combination of TMP and NF is useful in inhibiting bacterial strains at low concentrations as compared to the individual drugs when acting alone. A significant reduction in the MIC of NF when combined with TMP is evident [Table 1].
The MIC of TMP of the various urinary pathogens is comparable with those reported by Acar et al  in their study of urinary tract infections.
Bushby  stated that synergy is maximal when the organisms are susceptible to both drugs. This was not the case in our study where we found that, though the bacteria were susceptible to both TMP and NF, the FIC indices varied from 0.13 to 0.8 showing a wide range synergy. Our findings show that strains resistant to TMP and sensitive to NF, exhibit maximum synergy with the exception of 2 strains of E. coli and those resistant to both drugs, demonstrate moderate degree of potentiation except 3 strains of A. f aecalis, 1 strain of A-D group and 2 strains of P. aeruginosa which showed minimal synergy.
TMP acts by inhibiting the enzyme dihydrofolate reductase of the bacterial cell.  Since the action of NF is not known, the synergistic action of the combination cannot be explained.
As the drugs were not combined in the optimal ratio of their MICs, the excess of one drug reduced the MIC of the other drug considerably below that of the optimal ratio. This finding is supported by Bushby. 
Clinical trials with this combination have shown promising results and are reported in another communication.
[Figure 1], [Figure 2]
[Table 1], [Table 2]