Int.J.Curr.Microbiol.App.Sci (2013) 2(10): 253-261 ISSN: 2319-7706 Volume 2 Number 10 (2013) pp. 253-261 http://www.ijcmas.com Original Research Article Prevalence of Proteus species in clinical samples, antibiotic sensitivity pattern and ESBL production Jitendra Kumar Pandey1*, Akanksha Narayan2 and Shikhar Tyagi3 Department of Microbiology, MGM Medical College and Hospital, Sector-18, Kamothe, Navi Mumbai- 410209, Maharashtra, India 2Department of Microbiology, Doon (PG) Paramedical College & Hospital, Chakrata Road, Dehradun, Uttarakhand, India 3Department of clinical Research, Jamia Hamdard University, New Delhi, India *Corresponding author A B S T R A C T K e y w o r d s Different Proteus species may vary with the type of infections they cause in both the community and hospital environments. However, in many laboratories in Antimicrobial developing countries, differentiation of the genus Proteus into species is not susceptibility; generally done during bacteriological diagnosis due to high cost and special skills antibiotic involved. This study aimed at determining the prevalence of different Proteus resistance; species in MGM Hospital, Navi Mumbai , their antibiotic resistance pattern and -lactamase. how they relate to patients demographic data. Introduction Proteus species are among the commonly however, bacteriological diagnosis up to implicated pathogens in hospital as well as the identification of species is rare in community acquired infections (Douglas, many laboratories in Ghana due to the cost 2000; Emori, 1993). This pathogen has and special skills involved. There is a diverse mode of transmission, and therefore limited documented information hence can cause infection in different relating to patients demographics and anatomical sites of the body. Some of the antibiotic susceptibility levels for incriminating sources of transmission are infections caused by the various species of soil, contaminated water, food, Proteus (Yao, 1999; Tenssaie, 2001; equipments, intravenous solutions, the Patterson, 1999). This study seeks to hands of patients and healthcare personnel determine the prevalence of the various (Emori, 1993; Heinzelmann, 2002). There Proteus infections in relation to patients are reports of 9.8 to 14.6% prevalence demographics and the response of the rates of Proteus infections in MGM different species to commonly prescribed (Newman, 2006; Ohene, 1997). antibiotics at the MGM Medical College, Different species of Proteus are Navi Mumbai encountered in human infections; 253 Int.J.Curr.Microbiol.App.Sci (2013) 2(10): 253-261 Materials and Methods test the susceptibility of the Proteus isolates to different antimicrobial agents Isolation site (obtained from BDH London, UK): ampicillin (10 µg), tetracycline (30 µg), Different clinical samples such as urine, chloramphenicol(30 µg), cefuroxime (30 purulent material from wounds or µg), ceftriaxone (30 µg), cefotaxime (30 abscesses, ear swabs, sputum, blood or µg), gentamicin (10 µg), amikacin (10 µg) aspirates (of joint fluid, pleural fluid, and co-trimoxazole (25 µg). The inocula ascitic fluid and pus) collected from 4995 were prepared by growing the various patients suspected of bacterial infection at Proteus species on separate agar plates MGM were cultured to isolate the and colonies from the plate were organisms. Demographic data (such as transferred with inoculating loop into 3 ml age, sex, in-patient and out- patient status) of normal saline in a test tube. The of the patients was recorded prior to density of these suspensions was sample collection. adjusted to 0.5 McFarland standards. The surface of Muller-Hinton agar Cultivation and Identification (Oxoid Cambridge, UK) plate was evenly inoculated with the organisms using a The clinical samples collected were sterile swab. The swab was dipped into the aseptically inoculated on plates of Blood suspension and pressed against the side of agar, Cystine-Lactose-Electrolyte- the test tube to remove excess fluid. The Deficient (CLED) agar and MacConkey wet swab was then used to inoculate the agar (Oxoid Cambridge, UK) and Muller-Hinton agar by evenly streaking incubated at 37 oC for 24 h. The across the surface. By means of Disc morphological characteristics of the Dispenser (Oxoid Cambridge, UK), the colonies including size, shape, colour, antibiotic discs were applied to the surface pigmentation and haemolytic nature were of the inoculated agar and the plates were recorded. Suspected Proteus colonies incubated overnight at 37 oC. The were isolated and identified through diameter of zone of growth-inhibition biochemical tests according to Barrow observed was measured and compared to and Felthan: [Barrow, 2003] based on the chart provided by National Committee whether they were positive for nitrate for Clinical Laboratory Standards reduction; H2S gas production; methyl-red (NCCLS). and urease reactions; and negative for lactose fermentation. Indole production -Lactamase Production Test differentiated P. vulgaris isolates from the other species; P.mirabilis and P.penneri Extended spectrum Beta lactamases were identified by maltose (ESBLs) are plasmid mediated beta fermentation and ornithine lactamases capable of hydrolyzing decarboxylase production. P. vulgaris cephalosporins and monobactams (NCTC 4175) and P. mirabilis (NCTC (aztreonam) but are inhibited by beta 8309) were the reference strains employed. lactamase inhibitors. Antimicrobial susceptibility test Screening Tests Modified Kirby-Bauer disk diffusion The isolate is a potential ESBL producer if method (Cheesebrough, 2000) was used to the Zone of inhibition around the 254 Int.J.Curr.Microbiol.App.Sci (2013) 2(10): 253-261 following is : Result and Discussion Ceftazidime (30µg) 22mm Proteus species isolated Ceftriaxone (30µg) 25 mm Cefotaxime (30µg) 27mm Three Proteus species were recovered Cefpodoxime (10µg) 17mm from 56 of the 4995 clinical samples collected (Table 1) and this gave a Disc Approximation Test prevalence rate of 1.12%. 38 of these samples (67.85 %) were taken from male Using Amoxyclav (Ac), Ceftriaxone and patients and 18 (32.14 %) from females. Ceftazidime All the age groups except 90-99 years age group had at least one species present. P. Procedure mirabilis being the highest with 57.14% (Figure 1) that could be detected among Muller Hinton Agar was prepared and all the age groups (Table 2) except <1 Lawn culture of test organism was years old and 90-99years old age groups. prepared and inoculated antibiotic disc P. vulgaris accounted for 33.92 % of the were placed in the centre and both the Proteus isolates and was present in all the cephalosporin discs at a distance of 25mm age groups except 1-9 years, 80-89 years on either side of Antibiotic. and 90 - 99 years age group. P. penneri (8.92 %) was absent in samples obtained Interpretation from < 1years, 1-9 years, 10-19 years, 30-39 years, 50 - 59 and 90 - 99 years Extension of edge of inhibition zone of age groups. Wound samples contributed cephalosporin towards Ac indicates the highest percentage of Proteus potential ESBL Producer (67.85%) followed by urine. Confirmatory Tests Antimicrobial susceptibility of the Proteus isolates ESBL double disc synergy test. The Proteus isolates recovered were Procedure highly susceptible to Cefotaxime, Ofloxacin, Gentamycin, Amikacin, Muller Hinton Agar was prepared and Lomefloxacin, Ciprofloxacin and Lawn culture of test organism was Cefaperazone . However, 44.64% of prepared and inoculated antibiotic disc Proteus isolates exhibited resistance to were placed in the centre (Caz disk and ampicillin, 25% to Netilline and 21.42% Caz + Cac disc) and incubated overnight each Cefuroxime and Pefloxacin(Figure at 37o C. 2). 4 out of 5 P. penneri isolates were resistant to at least 3 antibiotics while Interpretation 28.12 % of P. mirablis and 36.84 % of P. vulgaris were found to be multiple drug The test organism is considered an ESBL resistant. producer if the zone size around the ceftazidime plus clavulanic acid disk is Multi-drug resistance was defined as increased >5 mm vs the third generation resistance to at least 3 antibiotics. All the cephalosporin (Ca) disk alone. 255 Int.J.Curr.Microbiol.App.Sci (2013) 2(10): 253-261 Table.1 Prevalence of Proteus in clinical samples. No. of Samples No. of Proteus Types of Specimens % isolated URINE 1698 11 0.647 PUS 899 38 4.22 BLOOD 1423 2 0.14 ET 180 1 0.55 SPUTUM 772 1 0.12 EAR SWAB 23 3 13.04 BODY FLUIDS 385 0 0 TOTAL 5380 56 1.05 Table.2 Age wise distribution of isolated Proteus species out of 56 Age Groups Proteus mirabilis Proteus vulgaris Proteus penneri Total out of 56 % <1 Year 0 1 0 1 1.78 1-9 Year 1 0 0 1 1.78 10-19 Year 3 2 0 5 8.92 20-29 Year 2 2 1 5 8.92 30-39 Year 5 2 0 7 12.5 40-49 Year 6 2 1 9 16.07 50-59 Year 4 4 0 8 14.28 60-69 Year 7 5 1 13 23.21 70-79 Year 3 2 1 5 8.92 80-89 Year 1 0 1 2 3.57 90-99 Year 0 0 0 0 0 TOTAL % 32/56(57.14%) 20/50(33.92%) 5/56(8.92%) Figure.1 Species distribution 8.93% Proteus mirabilis 33.93% 57.14% Proteus vulgaris Proteus penneri 256 Int.J.Curr.Microbiol.App.Sci (2013) 2(10): 253-261 Table.3 Distribution of Proteus species among In-patient and Out-patient in relation to specimen type Samples&No Proteus Out- Total No. of Total No. of In- Patients Studied Species Patients Species Isolates Pus Pm 19 5 24 (n=899) Pv 8 3 11 38 Pp 2 1 3 Urine Pm 3 1 4 (n=1698) Pv 4 2 6 11 Pp 0 1 1 Sputum Pm 0 1 1 (n=772) Pv 0 0 0 1 Pp 0 0 0 ET Pm 0 0 0 (n=180) Pv 0 0 0 1 Pp 1 0 1 Ear Swab Pm 1 1 2 (n=23) Pv 1 0 1 3 Pp 0 0 0 Blood Pm 0 1 1 (n=1423) Pv 1 0 1 2 Pp 0 0 0 40/56 (71.42%) 16/56 56 56 (28.57%) Pm=Proteus mirabilis; Pv=Proteus vulgaris; Pp=Proteus penneri; n=number of clinical specimens tested.