Phenotypic Characterisation of Proteus Species Isolated from Different Clinical Samples with Special Reference to Antibiotic Resistance Pattern in a Tertiary Care Centre

1. Masters in Science, Department of Microbiology, SRM Medical College Hospital and Research Centre, SRM Institute of Science and Technology, SRM Nagar, Chennai, Tamil Nadu, India. 2. Associate Professor, Department of Microbiology, SRM Medical College Hospital and Research Centre, SRM Institute of Science and Technology, SRM Nagar, Chennai, Tamil Nadu, India. 3. Professor and Head, Department of Microbiology, SRM Medical College Hospital and Research Centre, SRM Institute of Science and Technology, SRM Nagar, Chennai, Tamil Nadu, India.

Abstract

Introduction: The Proteus species are vulnerable to cause community-acquired and Healthcare Acquired Infections (HCAI). This organism is grouped under opportunistic pathogen and implicates a wide range of infection in humans. The emergence of antimicrobial resistance in this species is alarming and life threatening.

Aim: To evaluate the prevalence and the resistance pattern of Proteus species isolated from various clinical samples by conventional culture methods.

Materials and Methods: This was a cross-sectional study conducted from May 2020 to April 2021. The study was conducted at SRM Medical College Hospital and Research Centre, Chennai, Tamil Nadu, India, after approval from the Institutional Ethical Committee. Proteus isolates from various clinical samples like wound swab, pus, urine, Cerebrospinal Fluid (CSF), tracheal swabs, endotracheal aspirate, vaginal swabs, blood, body fluids, ear swab, tissue were collected as per standard protocols. The phenotypic characterisation and resistance pattern of Proteus isolates were done by conventional culture methods. The analysis was done using Statistical Package for the Social Sciences (SPSS) software version 24.0.

Results: Out of 100 isolates, the higher number of Proteus species was isolated from pus sample (35%), wound swab (22%) and urine sample (25%). Males were found to have a higher prevalence (65%) of infections by Proteus species than females. Proteus mirabilis was the most common isolated species (89%) in this study. The Extended Spectrum Beta Lactamase (ESBL) producers among Proteus species were found to be 39%, AmpC producers to be 20% and carbapenemase producer 1%. Proteus species had the highest sensitivity to piperacillin tazobactam (100%), tigecycline (100%), meropenem and imipenem (98%) in this study.

Conclusion: It was concluded that there was an increasing occurrence of drug resistance Proteus species. Their resistance is contributed by production of ESBL, AmpC and carbapenemase enzymes. The screening test alone is sufficient for detection of antibiotic resistance. Hence, there is a need for reliable phenotypic confirmatory test to identify the resistance among Proteus species.

Antimicrobial resistance, Non lactose fermenter, Proteus mirabilis, Proteus vulgaris, Swarming

The Proteus genus is included in tribe Proteeae comes under the family of Enterobacteriaceae which are pleomorphic gram negative bacilli. The Proteus species are mostly saprophytic which can be isolated commonly from sewage and decomposing animal matter (1). In concordance to humans, this species is a commensal of skin and intestine. They are grouped under opportunistic pathogen that causes infections mainly in urinary tract, skin and soft tissues (2). The main pathogenic organism in this family includes Proteus mirabilis (P. mirabilis), P. vulgaris and P. penneri.

Among these species, the most commonly isolated species is P. mirabilis which causes Urinary Tract Infection (UTI) and wound infection. In addition, P. mirabilis is also found to be the cause of empyema, bacteraemia, neonatal meningoencephalitis, renal calculi, diarrhoea and osteomyelitis. P. vulgaris is mostly isolated from patients who are immunocompromised like people who are on long term antibiotics (1),(2). Proteus species corresponds to 10-15% of complicated UTI in catheterised patients. P. mirabilis is considered as the most commonly encountered pathogen causing UTI. The prevalence of UTI in chronically catheterised patients by Proteus is about 20-45% (3).

P. penneri is capable of outbreaks of nosocomial infections (4). Few articles have documented that P. penneri can be isolated from the patients suffering from UTI, wound and epidural ulcers and samples such as pus, conjunctiva, bronchoalveolar lavage (5). It is isolated from the urine at a prevalence of 50%, Skin and Soft tissue Infection (SSI) with 25% and blood samples at 15% (6). The Proteus species is also found to cause UTI in patients suffering from urinary tract abnormalities and catheterised patients. The Proteus infection is found to be the reason for the development of renal stones and pyelonephritis (6). The virulence factors of Proteus species is varied and stated to be the production of urease, swarming, formation of biofilm, formation of calculi, proteolytic enzymes, fimbriae and haemolysin production (7),(8).

The Proteus species are also found to be infecting diabetic patients at a higher rate. They can cause tissue destruction in these patients (9). They are opportunistic pathogens and the main source of transmission includes equipment’s used in hospital, open wounds, trauma and patient to patient spread through contaminated surfaces (4). The Multidrug-Resistant (MDR) Proteus species is increasing in health sectors. The increase in resistance to antibiotics accounts to challenge in the treatment of patient which increases the death rate. The mechanism by which Proteus species shows resistance to antibiotics includes the production of ESBL, carbapenamase and AmpC (10).

The majority of resistance is caused by ESBLs which is a major group of beta-lactamases (11). The ESBL shows resistance to ceftazidime but sensitivity to ceftazidime in combination with clavulanic acid. The ESBL production shows a high risk for the community and accounts for many outbreaks. The major threats posed by ESBLs include ineffectiveness of antibiotics, high cost, increased hospital stay and increased complications. The treatments are becoming limited due to the rapid increase in the ESBL. The screening of ESBLs is required to control the infection worldwide (12),(14). Another major class, AmpC beta-lactamases cause resistance to the penicillin group of drugs, cephalosporins and monobactams (15). The next important resistance mechanism is resistance to carbapenems. Carbapenemase resistance poses a major difficulty in treating the severely ill patients. The evolvement of MDR implies major threat to therapeutics and has become the major source of HCAI (16). The antimicrobial resistance is becoming the main burden to healthcare system. The current study assesses the prevalence of infections caused by Proteus species including the resistance pattern which is helpful in characterisation of the Proteus species as there is less data available regarding the resistance in Proteus species.

Material and Methods

The present study was a descriptive, cross-sectional study done between May 2020 to April 2021. The present study was approved by IEC of SRM Medical College Hospital and Research Centre, Chennai, Tamil Nadu, India (Ethical clearance number: 1934/IEC/2020).

Inclusion criteria: All non duplicate Proteus isolates from various clinical samples like pus, sputum, urine, CSF, tracheal swab, endotracheal aspirate, vaginal swab, blood, body fluids, ear swab, and tissue were included. Samples collected before initiation of antibiotic therapy were included in the study.

Exclusion criteria: Isolates other than Proteus species from various clinical samples were excluded from the study.

Sample size calculation: Study sample size approx. 100, n=4pq/d²

(Calculated and approved by Statistician).

p=Prevalence rate=5.82% (17)

p=5.8/100=0.582

q=(100-p)

n=4×0.0582×0.9418/0.0025

=0.2193/0.0025

=87.7

=88- final sample size of 100 was taken

Study Procedure

Identification: Clinical samples were obtained in an aseptic manner and processed using standard microbiological techniques (1). Conventional culture techniques were used to isolate the Proteus species. It produces swarming on blood agar, non lactose fermenting colonies on MacConkey agar. The biochemical tests were used to speciate Proteus. It shows positive for phenylalanine deaminase test (PPA), abundant H₂S production with gas in Triple Sugar Iron (TSI) agar, and is a rapid urease producer. Indole is positive in P. vulgaris that distinguishes it from P. mirabilis which is indole negative. P. penneri is distinguished from P. mirabilis by maltose fermentation and the absence of ornithine decarboxylase (1),(18).

Antibiotic Susceptibility Testing (AST): The assessment of antibiotic susceptibility is carried on Muller Hinton Agar (MHA) by the Kirby Bauer disc diffusion method. The Clinical and Laboratory Standards Institute (CLSI) guidelines for 2020 were used to interpret the zone sizes (19). The following antibiotic disc were used ampicillin (10 (μg), amoxicillin-clavulanate (20μg/10μg), ceftazidime (30μg) cetazidime with clavulanuic acid (30μg/20 μg), cefipime (30 μg), ceftriaxone (30 μg), cefuroxime (30 μg), ertapenem (10 μg), meropenem (10 μg), piperacillin tazobactam (100 μg/10 μg), amikacin (30 μg), chloramphenicol (30 μg), ciprofloxacin (5 μg), cefotaxime (30 μg), cefoxitin (30 μg), cefazolin (30 μg), gentamicin (10 μg), imipenem (10 mg), tetracycline (30 μg), tigecycline (15 μg), nitrofurantoin (300 μg) (purchased from Himedia, Mumbai, India). The zone size was interpreted after 18-24 hours of incubation at 37°C.

Phenotypic Confirmatory Tests for ESBL, AmpC and Carbapenamase Producers

Double disc diffusion test for detecting ESBL: Two antibiotic discs were used namely ceftazidime (30 μg) and cetazidime with clavulanic acid (CAC) (30 μg/20 μg). The CAC is placed in centre of MHA plate and ceftazidime is placed with a distance about 1.5 cm from each other. The inhibition zone development towards CAC disc after incubating at 37°C for 24 hours indicated the presence of an ESBL positive organism (11),(12).

Modified three-dimensional test for AmpC producers: On MHA, lawn culture is put, and a cefoxitin disc of 30 μg is placed in the plate. A sterile surgical blade is used to cut a 3 cm long slit 2-3 mm away from cefoxitin drug disc. A well is done at the other end using a pipette tip. The AmpC beta-lactamase enzyme is obtained by alternate freeze and thawing for 7-8 times before centrifuging at 2500 rpm (revolution per minute) for 15 mins. The AmpC beta-lactamase enzymes were released into the suspending fluid as a result of this method. The prepared well is then filled with a total of 20-30 μL of the supernatant containing the extract. After allowing the enzyme to fill and disperse into the slit for 5-10 minutes, the plates were kept at 37°C up to 24 hours for incubation. A slight heart shaped zone of lysis towards cefoxitin drug suggested a positive result. In this test, the American Type Culture Collection (ATCC) Escherichia coli 25922 was used as the indicator strain (20).

Modified Hodge Test (MHT): In peptone broth ATCC E. coli 25922 is grown and adjusted to McFarland standards (0.5). The MHA plate is streaked with a 1/10 dilution. In the centre of the test region, a meropenem 10 μg disc is mounted. The test organism is streaked from edge of discs and kept at 37°C for 16-24 hours. The carbapenem discs were subjected to quality control in compliance with CLSI guidelines. Klebsiella pneumoniae ATCC 1705 was used as the control organism (21),(22).

Statistical Analysis

The information thus obtained is entered in MS-excel (Microsoft office professional 13) spread sheet for each isolate and the analysis was done using SPSS software version 24.0.

Results

Out of 100 Proteus isolates, 39 were isolated between the age group of 61-75 years, 34 were between the age group of 46-60 years and around 15 between the age group of 31-45 year (Table/Fig 1). Proteus isolates were collected, subjected to identification by conventional culture, biochemical and AST. Culture was done on MacConkey agar, Blood agar, and Phenylalanine deaminase test (PPA) were all used to validate the Proteus species. In MacConkey agar Proteus isolates produced non lactose fermenting colonies. In blood agar Proteus species produce a swarming growth which is a unique characteristic feature of this organism (Table/Fig 2). The PPA test was positive for all Proteus species due to the fact that this species is able to produce an enzyme called phenylalanine deaminase that converts phenylalanine to phenylpyruvic acid. The prevalence of Proteus mirabilis was found to be 89% whereas Proteus vulgaris was found to be 11%. The specimen wise distribution of the Proteus species; maximum number of Proteus species was isolated from the pus (35%) followed by the urine samples (25%) and wound swab (22%) and other samples as shown in (Table/Fig 3). The maximum number of Proteus species were isolated from wound infection (60%) followed by UTI (20%) (Table/Fig 4).

Antibiotic susceptibility testing: The AST was performed on the isolates, screening ESBL, AmpC β-lactamase and carbapenamase producers. The zone sizes were interpreted according to CLSI 2020 guidelines. The ESBL producer shows ≥5 differences in zone size between ceftazidime and ceftazidime with clavulanic acid. The resistance to cefoxitin is considered as AmpC producer. The resistance to imipenem, meropenem and ertapenem is considered to be carbapenamase producer. Proteus species had the highest sensitivity to piperacillin tazobactam (100%), tigecycline (100%) and meropenem and imipenem (98%) in this study (Table/Fig 5). The present study demonstrates ampicillin resistance to be 64% and 43% resistance to cefazolin. From the AST report it was found that there were 41 ESBL producer, 25 AmpC producer and two carbapenamase producer.

Confirmatory tests for ESBL, AmpC and Carbapenamase producers:

a. Double disc diffusion test for detecting ESBL: The 41 test isolates which were found to be ESBL producer by standard AST was subjected to phenotypic confirmatory test. The confirmatory test was positive in 39 isolates (Table/Fig 6).

b. Modified three-dimensional test for AmpC producers: The 25 test isolates which were found to be AmpC producer by standard AST was subjected to phenotypic confirmatory test. The confirmatory test was positive in 20 isolates (Table/Fig 6).

c. Modified Hodge Test (MHT): The two test isolates which were found to be carbapenamase producer by standard AST was subjected to phenotypic confirmatory test. The confirmatory test was positive in one isolate (Table/Fig 6).

Discussion

Proteus species is found throughout the environment and is a normal part of the human gastrointestinal tract's flora. Escherichia coli is considered the most common cause of uncomplicated cystitis, pyelonephritis, and prostatitis. Proteus is considered the third most common cause, especially in hospital acquired infections (23). The maximum numbers of Proteus species was between the age group of 61-75 years (39%), 46-60 years (34%) and 31-45 years (15%). Maheswary D and Chitralekha S and Prasad RR et al., found similar results (17),(24). The 89% of the Proteus species was found to be P. mirabilis in this analysis and 11% was P. vulgaris. The variation in prevalence rate between the previous studies may be due to difference in demography and also the Proteus is getting highly virulent and spreads at higher rate (Table/Fig 7) (17),(25),(26).

Since, the antibiotic resistance patterns of Proteus species vary with different species hence species reporting is essential. In comparison to P. vulgaris that has inherent resistance to variety of antimicrobial agents P. mirabilis was found to be the most susceptible species. The highest number of Proteus species is collected from the pus sample (35%), followed by the urine samples (25%). Proteus species was also isolated from blood (1%), ear swab (5%), tissue (10%) and from tracheal aspirate (2%) sample. Bahashwan SA and Shafey HME, Feglo PK et al., Leulmi Z et al., and Shenoy SM et al., reported maximum isolates from pus whereas some studies have reported isolates more commonly from urine than other clinical specimens (23),(27),(28),(29),(30),(31),(32).

When there are predisposing conditions such as surgery or catheterisation, Proteus plays a major role in UTI. Adhesion factors, flagella (swarming), Immunoglobulin A (IgA) protease, and the urease enzyme are all virulence factors that assist the organism to establish an infection. The formation of bladder or kidney stones as a result of the activity of the urease enzyme, which causes polyvalent cations such as Mg²⁺ and Ca²⁺ to precipitate out of the urine and form struvite stones, which obstruct the urinary tract or catheters, making treatment difficult and allowing the bacteria to persist and multiply and causing the complications. Males are more likely to have a UTI from Proteus (29). In present study, the majority of isolates (65%) came from male UTI patients with old age group. Various research have shown a similar gender distribution (27),(33).

Proteus species had the highest sensitivity to piperacillin tazobactam (100%), tigecycline (100%) and meropenem and imipenem (98%). Kengne M et al., recorded 100% sensitivity for piperacillin tazobactam and meropenem and Preethishree P et al., reported 100% sensitivity for piperacillin tazobactam and meropenem (34),(35). In the present study, amikacin is found to be 88% sensitive whereas gentamicin was found to be 72% sensitive. Alexis A and Sakthivennila M recorded 60% gentamicin resistance (36). The present study demonstrates ampicillin resistance to be 64%. In comparison to the above analysis, Kargar M et al., reported a high sensitivity percentage of 84% to gentamicin (37). In contrast to this analysis Kengne M et al., recorded an increase in ampicillin resistance of about 91% (34). The present study demonstrated the screening test to be 41% ESBL producers and the confirmatory test was positive for 39%. In this present study, AmpC beta lactamases production was detected by screening test in 25% of cases and by confirmatory test of 20% of cases. The carbapenemase producers were found to be 2% in the screening test and 1% in the confirmatory test. Similar results were found in Maheswary D and Chitralekha S (17). Hence, this study emphasises the data of prevalence along with antimicrobial resistance pattern of Proteus species which will provide a good understanding of the resistance pattern at species level and aids in rational use of antibiotics ultimately helping to reduce the increasing antimicrobial resistance threat.

Limitation(s)

The detection of gene responsible for the antimicrobial resistance by molecular methods was not included in the study.

Conclusion

There was an increasing occurrence of drug resistance in Proteus species. Only screening test will not be sufficient for detection of antibiotic resistance. Hence, reliable phenotypic confirmatory test to identify the resistance among Proteus species is required. The confirmatory test such as combined disc diffusion test (ESBL detection), 3-dimensional AmpC assay (AmpC detection) and MHT (Carbapenemase detection) is found to be more significant, reliable and can be carried out easily in a routine microbiology testing laboratories aiding to identify the resistant species and significantly helps in the rationale use of antibiotics.

DOI and Others

DOI: 10.7860/JCDR/2022/51928.15901

Date of Submission: Aug 14, 2021
Date of Peer Review: Sep 15, 2021
Date of Acceptance: Nov 06, 2021
Date of Publishing: Jan 01, 2022

Author declarati on:
• Financial or Other Competing Interests: None
• Was Ethics Committee Approval obtained for this study? Yes
• Was informed consent obtained from the subjects involved in the study? Yes
• For any images presented appropriate consent has been obtained from the subjects. Yes

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Etymology: Author Origin