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 species in clinical samples, antibiotic sensitivity pattern and ESBL production

Jitendra Kumar Pandey1*, Akanksha Narayan2 and Shikhar Tyagi3

Department of , 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): (10 µg), (30 µg), Different clinical samples such as urine, (30 µg), (30 purulent material from wounds or µg), ceftriaxone (30 µg), cefotaxime (30 abscesses, ear swabs, sputum, blood or µg), (10 µg), (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 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 mediated beta fermentation and ornithine lactamases capable of hydrolyzing decarboxylase production. P. vulgaris and monobactams (NCTC 4175) and P. mirabilis (NCTC () 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 (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 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 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 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. Figure.2 Showing antibiotic sensitivity pattern of Proteus vulgaris.

SENSITIVITY %

70 60 50 40 30 SENSITIVITY % 20 10 0

257

Int.J.Curr.Microbiol.App.Sci (2013) 2(10): 253-261

Figure.3 Shows antibiotic sensitivity pattern wise distribution of Proteus penneri in clinical samples

SENSITIVITY %

80 80 60 60 60 60 60 60 60 40 40 40 20 20 20 20 20 20 SENSITIVITY % 20 0 L F Z S Z T K F T P X D N I M M C P T A A A F G A O N E C X O C C C G C L

Figure.4 Showing Antibiotic sensitivity pattern of Proteus mirabilis

SENSITIVITY %

100

50 SENSITIVITY %

0 CIP LOM CPZ CTX AMC PF CXM OF CAZ GEN NT AK

Figure.5 Multi-Drug Resistant (MDR) of Proteus isolates

80% 100.00% 28.13% 36.84% 50.00% MDR PROTEUS SPECIES 0.00% PROTEUS PROTEUS PROTEUS MIRABILIS VULGARIS PENNERI

258

Int.J.Curr.Microbiol.App.Sci (2013) 2(10): 253-261

Figure.6 Species wise distribution of ESBL producing Proteus strain

SPECIES WISE DISTRIBUTION

8.33, 8%

PROTEUS MIRABILIS PROTEUS VULGARIS 33.33, 33% PROTEUS PENNERI

58.33, 59%

three Proteus species were found to be Navi Mumbai and other parts of multi-drug resistant. Proteus penneri Maharashtra, India. Our findings thus showed the maximum MDR with 80% partially supports the findings of followed by Proteus vulgaris with 36.84% those from Europe and Asia; [Reslinski, and then Proteus mirabilis with 28.13%. 2005; Chung, 1999] which showed Proteus species to be more commonly Species identification and surveillance of encountered in urine than in other clinical is essential in specimens. management and control of infections. These practices are usually absent in most According to our study maximum of our hospitals mainly due to the high infection in urine sample was of costs involved. In this study we P.vulgaris which encountered 5 4 . 5 4 % investigated the presence of Proteus which is in contrast with the finding species in 4995 clinical samples which supports that P. vulgaris and P. collected between January 2012 and penneri infections of the urinary tract are December 2012 at MGM Hospital. Three rare (Chung, 1999; Foxman, 2000; Nawal, Proteus species (P. mirabilis, P. vulgaris 1994) whereas P. mirabilis has a higher and P. penneri) were identified to be propensity for colonizing the urinary responsible for causing infections in tract due to difference in its various anatomical sites. P. mirabilis was pathogenicity (Mobley, 1994). Proteus the most common species isolated, infections were also common among the accounting for 57.14 % of all the in-patients (71.42 %) as compared to infections and hence responsible for the out-patients (28.57 %). Out of the 56 majority of Proteus infections. This result clinical specimens from which Proteus agrees with similar studies conducted in was recovered, 38 (67.85 %) were England, Wales and Northern Ireland collected from males and 18 (32.14 %) [Chow, 1979; Jones, 2003]. Wounds from females. The study showed a recorded the highest percentage of significant difference between the males Proteus isolates (67.85 %) followed by and females infected with Proteus. The urine (19.64%). Proteus is therefore a Proteus infections were detected in all common cause of wound infections in age groups from <1 to 99 years where

259 Int.J.Curr.Microbiol.App.Sci (2013) 2(10): 253-261

60-69 years age group registering as the highest group infected (23.21 %). The Proteus species isolated were found to antibiotics for the treatment of Proteus have high antimicrobial resistance against infections at MGM Hospital. -lactamase third generation of Cephalosporin production and multi-drug resistance have antibiotics. All the Proteus species all been exhibited by the isolates. This showed sensitivity to Cefotaxime, study is therefore a step towards the Ciprofloxacin, Lomefloxacin, generation of national data on the , Cefuroxime, Ofloxacin, prevalence of antimicrobial resistant Ceftazidime, Gentamycin, Netilline and pathogens in . Amikacin.. All the three Proteus species were found to be multi drug resistant. Acknowledgement 48.86% of the total clinical samples were ESBL producers. Highest being the We will like to acknowledge the Medical Proteus mirabilis with 58.33% followed Microbiology Laboratory technicians of by Proteus vulgaris with 33.33% and MGM HOSPITAL for their assistance in Proteus mirabilis with 8.33%. The high this study. antibiotic resistance of Proteus may be an indication of the resistance levels References among the enterobacteriaceae and perhaps salmonellae since indiscriminate ingestion Barrow, G.I., and Felthan, R.K.A. 2003. of antibiotics provides selective pressure, Cowan and Steel s Manual for the leading to a higher prevalence of resistant Identification of Medical . 3rd bacteria (Levy, 1999) which is very Ed. Cambridge University Press. common in developing countries like Cambridge UK. 351-353. India. Not only are these species Cheesebrough, M., 2000. District potential causes of infections but also Laboratory Practice in Tropical potential reservoirs of resistance genes Countries. Part 2, Cambridge that could be transferred to other bacterial University Press, UK. 132 143. pathogens. The high levels of -lactamase Chow, A.W., et al. 1979. A nosocomial production and multi-drug resistance of outbreak of infection due to multiple the isolates are indications of an increase strains of resistant Proteus mirabilis: in the resistance menace reported by Role of intestinal colonization as a earlier studies (Newman, 2006) major reservoir. J. Infect. Dis. 139:621 627 P. mirabilis, P. vulgaris and P. penneri Chung, H.I., et al. 1999. Prevalence of are the species implicated in Proteus Proteus species in urinary tract infections; wounds recorded the highest infections in a regional hospital in incidence of Proteus infections at MGM Trinidad. 62:438-442. Hospital, Navi Mumbai. The species Douglas, G., et al. 2000. Bennett's were susceptible to Cefotaxime, Principles and Practice of Infectious Ofloxacin, Gentamycin, Amikacin, Diseases. 5th ed. Philadelphia, Pa: Lomefloxacin, Ciprofloxacin and Churchill Livingstone. 9:121-126 Cefaperazone. They were, however Emori, G.T., et al. 1993. An overview of resistant to ampicillin, Netilline and nosocomial infections, including the Cefuroxime and Pefloxacin and hence role of the microbiology laboratory, these must not form part of the empirical Clin. Microbiol. Rev. 6: 428.

260

Int.J.Curr.Microbiol.App.Sci (2013) 2(10): 253-261

Foxman, B., 2000. Urinary tract infections: Ohene, A., 1997. Bacterial pathogens and Incidence and risk factors. Am. J. Pub. their antimicrobial susceptibility in Health, 80:331-333. Kumasi, Ghana, East Afri. Med. J. Heinzelmann, M., et al. 2002. Factors 74:450 455. predisposing to bacterial invasion and Patterson, J.E., et al. 1999. Association infection. Am J Surg; 183(2): 179-90 of antibiotic utilization . Jones, R., et al. 2003 Bacteraemia, Measurement and control of multiple England, Wales and Northern Ireland: drug resistance.19(8):120-128. Commun Dis Rep CDR. Wkly [serial Reslinski, A., et al. 2005. Prevalence of online] multi-drug resistant Proteus species Levy. S.B., 1999. Antibiotic resistance: an in clinical specimens and their ecological inbalance. Ciba Found. susceptibility to antibiotics, Med. Symp. 207:1-14. Dosw. Micribial, 57(2):175-184. Mobley, H.L.T., 1994. Virulence of Tenssaie, Z.W., 2001. Multiple Proteus mirabilis in urinary tract antimicrobial resistance in gram infections. Molecular pathogenesis negative bacilli isolated from and clinical management. ASM Press, clinical specimens, Jimma Hospital, Washington, D.C. 245 269. Southwest Ethiopia, Ethiopia Med. J. Nawal, V.I., et al. 1994. In vitro 39: 305 312. antimicrobial activities against Proteus Yah, S.C., et al. 2001. Widespread plasmid species isolated from patients in a resistance genes among Proteus Tertiary care Hospital in Jordan: species in diabetic wounds of patients Frequency of isolation and in Ahmadu Bello University Teaching . Scienti.Res.Essay. Hospital (ABUTH) Zaria, Afr. J. 3(4):122-126. Biotechnol. 6(15):1757-1762. Newman, M.J., et al. 2006. Resistance to Yao, J.D.C, et al., 1999. Antibacterial Antimicrbial Drugs in Ghana. The agents. Manual of clinical Ghanaian Dutch collaboration for microbiology. 7th ed. Washington, Health Research and Development, 1- D.C. American Society for 6. Microbiology. 1474 1504.

261