Abstract: This study investigates the prevalence of Proteus spp. bacterial isolates from uropathogenic infections as well as their susceptibility and antibiogram patterns to some selected antibiotics. A total of 1927 patients presenting at University of Benin Teaching Hospital were screened for Urinary Tract Infection (UTI). Of the total 1927 sample studied, 14.58% were positive for UTI. About 281 samples positive for UTI, 14.59% were positive for Proteus spp. distributed in to Proteus mirabilis (68.29%) and Proteus morganii (31.71%). The highest distribution was observed in the month of September, followed by August and November with July, October and December presenting the least. Proteus mirabilis was not susceptible to Cloxacilin, Erythromycin and Cephalexine but was highly sensitive to Peflacine, Ciproxin, Cefuroxine and Ofloxacin. Proteus morganii was not susceptible to Ampicllin, Amoxicillin, Ofloxacin, Ciproxin, Cephalexin and Cefotaxime but was highly sensitive to Cloxacillin, Genticin and Cefuroxime. The Ajumali’s mnemonic coding showed that no two strains of any of Proteus mirabilis and Proteus morganii were same. Judging by these results, multidrug-resistant Proteus mirabilis and Proteus morganii is becoming a growing public health problem and hence, the need for antimicrobial sensitivity screening for accurate antibiotic prescription is becoming high.
INTRODUCTION
Urinary Tract Infection (UTI) is one of the most common bacterial infection in humans both in the community and hospital settings and are the most common bacterial infections encountered by clinicians in developing countries (Dalela et al., 2012; Kashef et al., 2010). Worldwide, approximately 150 million people are diagnosed with UTIs resulting in over USD 6 billion health care expenditures (Weichhart et al., 2008).
Worrisome is the fact that drug resistance among bacteria causing UTI has increased since the introduction to UTI chemotherapy (Nerurkar et al., 2012; Sood and Gupta, 2012; Bahadin et al., 2011; Haider et al., 2010; Tseng et al., 2008). Besides, the etiological agents and their susceptibility patterns of UTI vary in regions and geographical locations. According to De Francesco et al. (2007), the etiologyand drug resistance change through time. Hence, knowledge of the local bacterial etiology and susceptibility patterns is required to trace any change that might have occurred in time so that updated recommendation for optimal empirical therapy of UTI can be made as reported by Leegaard et al. (2000).
In this study, the Proteus species are of interest which according to De Champs et al. (2000) are a major cause of diseases acquired outside the hospital and in cases eventually require hospitalization. Proteus is a genus of gram-negative bacteria belonging to the family of Enterobactericeae. They are distinguishable from other genera by their ability to swarm across an agar surface (Jacobsen et al., 2008). Proteus ranks third as the cause of hospital-acquired infections (Stamm, 1999) and they are widespread in the environment and makes up part of the normal flora of the human gastrointestinal tract.
Significantly, there has been a report in Europe on the evolution and spread of multidrug-resistant Proteus mirabilis clone with chromosomal AmpC-type beta-lactamase (D’Andrea et al., 2011; Luzzaro et al., 2009). In addition, a report exists on a wide diversity between institutions in the prevalence of pathogens and in their antimicrobial susceptibility (Fridkin, 2001) and it is said to be particularly worse in resource-poor countries where sale of antibiotics is poorly controlled (Onile, 1997).
Considering the literatures above, spread of multidrug-resistant pathogen is representing a growing public health problem in the world. In fact, with the increasing phenomenal evolution and multidrug-resistance of many bacterial pathogens (with special interest on multidrug-resistance Proteus spp.), there is the need for regular review of antimicrobial sensitivity pattern among clinically isolated Proteus spp. for accurate decision on antibiotic prescription. This study was therefore, designed to investigate the prevalence of Proteus spp. bacterial isolates from uropathogenic infections as well as their susceptibility and antibiogram patterns to some selected antibiotics.
MATERIALS AND METHODS
Specimen: A total of 1,927 clinical specimen comprising of Mid-Stream Urine (MSU), Super-Public Urine (SPU) and catheter specimen were collected from in-and-out patients in UBTH, between July, 2009 and December, 2009 for this study (Orhue, 2004). These samples were taken to the laboratory for standard microbiological analysis with 30 min of collection.
Isolation and identification: The specimen was inoculated onto nutrient agar, blood agar and MacConkey agar plates by streaking. Inoculated plates were then incubated aerobically at 37°C for 24 h. After 24 h of incubation, discrete colonies were picked up and gram stained and further subculturing was done to obtain pure cultures and biochemical tests carried out.
Antibiotics under study are as follows (Momoh et al., 2011):
| • | Ciprofloxacin:This drug is a fluoroquinolone and acts by inhibiting DNA topoisomerases (gyrases); thereby, inhibiting bacterial DNA synthesis |
| • | Erythromycin: This drug belongs to the class of macrolide, it is bacteriostatic, binding to the 23-RNA of the 50s ribosomal subunit to inhibit peptide chain elongation during protein synthesis |
| • | Augmentin: This is a combination of amoxycillin and clavulinc acid. The clavulinic acid helps protect the amoxycillin from being inactivated by the enzyme beta-lactamsae, an enzyme produced by pathogenic bacteria |
| • | Gentamycin: This drug is an aminoglycoside which binds to small ribosome subunits and interfere with protein synthesis by directly inhibiting protein synthesis |
| • | Cefuroxime: This is a broad spectum antibiotic of the cephalosporin class. It is an alternate drug of choice when patients are allergic to the penicillins or when there is a need to overcome beta-lactamase inactivation |
Antibiotics susceptibility testing (antibiogram): This was done by the multi-discs diffusion using 21 different antibiotics. The multi discs were placed on the plates which were previously inoculated, few minutes earlier, then the plates were incubated at 37°C for 24 h, thereafter, the plates were examined for zones of inhibition around the different antibiotic disc. Staphylococcus aureus Oxford stain NTC 6751 was used as control for gram positive organisms.
Mnemonic coding: The Ajumali’s mnemonic coding method as earlier described by Joghi et al. (1984), was adopted as a typing scheme to re-arrange the nominal antibiotics into arbitrary numeric values, making it easy for the differentiation of strains. Using this pneumonic coding scheme, a sensitive result was scored as (+) while a resistance was scored as (-). Also, the 21 different antibiotics were divided into a group of 3 antibiotics each, following their mechanisms of action as well as, their clinical applications and these 3 antibiotics were given numerical values of 1, 2 and 4.
Thus, a perfect sensitivity to the 3 antibiotics will give a summation of 1+2+4 = 7. While complete resistance to the 3 antibiotics will give a summation of 0+0+0 = 0. The other values as obtained by adding up these numerical values thus, an isolate can receive a score of 0-7 in each triplet segment which, when the seven triplet segments are combined together, gives a seven (7) digit numerical value as the antibiogram types (Orhue, 2004).
Data analysis: All data was analyzed using simple descriptive statistic.
RESULTS
Of the total 1927 sample, 14.58% (281 samples) were positive for UTI among whom female represents 15.37% and male 13.46% (Table 1). Among the 281 samples positive for UTI, 14.59% were positive for Proteus spp. Proteus mirabilis (68.29%) and Proteus morganii (31.71%) were the Proteus organisms that were the only Proteus spp. presented (Table 2).
Table 3 shows the distribution of the Proteus spp. isolated during the studied period. Although, the total number in each month did not differ, however, the month of September presented the highest isolates (9 isolates) followed by the months of August and November (7 isolates each) with July, October and December producing the least isolates (6 isolates each).
| Table 1: | Distribution of the sampled population and prevalence of UTI |
| Table 2: | Prevalence of Proteus spp. isolated from the sampled population |
| Table 3: | Distribution of the Proteus spp. isolated from the sampled population |
| Table 4: | Cumulative frequency of susceptibility of bacterial isolates to antibiotics |
Table 4 shows the susceptibility of the Proteus spp. isolates to 21 different antibiotics. Proteus mirabilis was not susceptible to Cloxacilin, Erythromycin and Cephalexine. On the other hand, Proteus morganii was not susceptible to Ampicllin, Amoxicillin, Ofloxacin, Ciproxin, Cephalexin and Cefotaxime. Higher susceptibility was observed with Peflacine, Ciproxin, Cefuroxine and Ofloxacin for the case of Proteus mirabilis while for the case of Proteus morganii high susceptibility was observed with Cloxacillin, Genticin and Cefuroxime (Table 4). Table 5 and 6 show the sensitivity and antibiogram types of Proteus mirabilis and Proteus morganii, respectively. The Ajumali’s mnemonic coding showed that no two strains of any of the Proteus spp. were the same for the Proteus mirabilis and Proteus morganii.
DISCUSSION
In the current study, the prevalence of UTI observed to be 14.58% with Proteus spp. infection accounting for 14.59% of cases. Although, two strains of Proteus spp. were observed, the most prevalence was Proteus mirabilis accounting for 68.29% and Proteus morganii accounting for 31.71%. This high prevalence of Proteus spp. in UTI is not in line to fact that Proteus is seems to be a common cause of wound infections in West Africa (Feglo et al., 2010; Newman et al., 2006; Yah et al., 2007) but agrees with the report in Europe and Asia that Proteus is commonly encountered in urine than in other clinical specimens (Reslinski et al., 2005; Orrett, 1999).
According to the finding of this study, Proteus spp. infection was highest in the month of September.
| Table 5: | Sensitivity and antibiogram of Proteus mirabilis |
| Table 6: | Sensitivity and antibiogram types of Proteus morganii |
This finding is similar to the study by Bahashwan and El Shafey (2013) who reported Proteus spp. infections to be highest during summer season from 22 June to 22 September. Indeed, studies have reported relationship between bacterial infections and seasonal variation (Bryan, 2011; Smith and Hogan, 2008; O’Hara et al., 2000) and it has long been known that bacterial infections peak during summer season.
Interestingly, Proteus spp. acted differently in terms of sensitivity and resistivity to the studied antibiotics and similar report has been documented by Bahashwan and El Shafey (2013). In this study, the isolated Proteus spp. showed diversity in antibiotic susceptibility and sensitivity. In fact, the most effective antibiotics for Proteus mirabilis were Peflacine, Ciproxin, Cefuroxine and Ofloxacin while for Proteus morganii were Cloxacillin, Genticin and Cefuroxime. Specifically, Proteus mirabilis was resistance to Cloxacilin, Erythromycin and Cephalexine while Proteus morganii was resistance to Ampicllin, Amoxicillin, Ofloxacin, Ciproxin, Cephalexin and Cefotaxime. In line with the finding of this study, resistance of Proteus spp. against ampicillin, Doxycycline, Amoxycillin, Cephalothin, Erythromycin and some other antibiotics have been reported (Kibret and Abera, 2014; Feglo et al., 2010; Newman et al., 2006; Dance et al., 1987; Chow et al., 1979). Increasing drug resistance to these and other antimicrobials has been documented from previous studies (Tseng et al., 2008), hence, increase in immergence of antimicrobial drug resistance. This study is therefore, a step towards the generation of national data on the prevalence of antimicrobial resistance patterns of Proteus spp.
The Ajumali’s coding of Proteus mirabilis and Proteus morganii (Table 5 and 6) presented different antibiogram type, making them phenotypically different from one another, even though they are of the same species. This indicates a higher resolving strain differentiation effect of the Proteus mirabilis and Proteus morganii. This typing method is so specific that it can easily pass off as a phenotypic DNA antibiogram typing method (Momoh et al., 2011). This therefore, indicates that an appropriate pneumonic coding can be able to resolve strains of the same microorganisms into their different and specific antibiogram types. By implication this information is very important for laboratory physician as those with knowledge of the various strain distribution and differentiation can tackle Multi-Drug Resistant (MDR) strains effectively (Momoh et al., 2011).
CONCLUSION
This study has demonstrated that there is high prevalence of Proteus mirabilis and Proteus morganii in UTI in the community under study and these strains of Proteus spp. are with higher antimicrobial resistance. Hence, multidrug-resistant Proteus mirabilis and Proteus morganii is becoming a growing public health problem. It is therefore, recommended that there is need to fund research to regularly review antimicrobial sensitivity pattern as this will be beneficial for clinician and laboratory physician when selecting antibiotic for prescription. In addition, the need for Antibiotics susceptibility testing for patients before prescribing antibiotics cannot be overemphasized as this may reduce the growing multidrug-resistance.
ACKNOWLEDGMENT
Author wishes to acknowledge the assistance provided by Akpamu U and others that made this study possible and publishable.