Characterization of Genus Proteus Isolated from Various Clinical Specimens and Detection of Extended-Spectrum β-Lactamase Production

Abstract

Background: 

Among Gram-negative bacteria, the presence of beta-lactamases is a defined modality of resistance to antibiotics of the beta-lactam group. Proteus species also has been showing various degrees of resistance to these groups of antibiotics and thereby appearing multidrug resistant. The aim of our study was to know the occurrence of Proteus species from different clinical samples their antibiotic susceptibility, extended-spectrum β-lactamase (ESBL) production, and the associated patient risk factors.

Materials and Methods: 

This observational descriptive study was done from December 2020 to December 2021. All Proteus isolates obtained from various clinical samples, were biochemically identified, antibiogram, and screening for ESBL production was done using a combination of cefotaxime and cefotaxime clavulanic acid antibiotic discs.

Results: 

Seventy-nine Proteus isolates were obtained from hospitalized patients. In that, Proteus mirabilis was the most common isolate followed by Proteus vulgaris. Proteus infections were common in the age groups of 41–60 years and males were affected more than females. Out of the 79 Proteus isolates 12 were ESBL producers. Among these, 25% of ESBL producers were P. mirabilis and 75% of them were P. vulgaris.

Conclusion: 

The most common isolate was P. mirabilis among hospitalized patients and P. vulgaris was the main ESBL producers when compared to P. mirabilis. These pathogens mainly caused wound and urinary tract infections. The patient age groups ranged mainly from 49–60 years. Diabetes mellitus was the most significant risk factor in these Proteus infections.

Introduction

Multidrug-resistant bacteria pose a serious danger to human health globally and cause a global epidemic in every health-care environment. Antibiotics that are widely prescribed to cure bacterial infections are β-lactam antibiotics. Most Gram-negative bacteria, on the other hand, contain β-lactamases, which attack β-lactam antibiotics. Extended-spectrum β-lactamases (ESBL’s) are a kind of β-lactamase enzyme formed by Gram-negative bacteria belonging to the Enterobacteriaceae family. The predominant ESBL producers are Escherichia coli, Klebsiella pneumoniae, and Proteus spp.^[1] Speciating Proteus and analyzing their tendency to develop resistance in various samples is of utmost importance to a treating physician. The intestine of human beings contains different types of aerobic and mainly anaerobic microbial flora. Most of these organisms are normal commensals and symbionts. Outside the intestine, they can cause wound, urinary tract, nosocomial, and various other types of infections. Proteus species are differentiated from the majority of other lineages of Enterobacteriaceae because of the property of swarming on culture media.^[2] This organism is a motile, nonsporing, chemoheterotrophic bacterium with a diversity of transmission modes.^[3]

The other members of the Protea family are Proteus, Providencia, and Morganella. P. mirabilis, P. penneri, P. vulgaris, P. myxofaciens, and P. hauseri and three unknown genome species, i.e., Proteus genome species 4, 5, and 6 make up the genus Proteus.^[4]Proteus is a participant in a majority of diseases and infections obtained in healthcare. Proteus is a part of the normal flora of the gastrointestinal tract and it is the most common species in the environment and in hospital settings, and have been found in humans. Proteus mirabilis and Proteus vulgaris are common species that infect humans. However, P. mirabilis is said to typically associated with kidney stone and chronic bacteriuria which are the most common reasons of bacteriuria.^[5] Since Proteus species have several modes of transmission, they can lead to infections in a variety of body areas as well as population and health-care facility infections. Proteus infections are the third most prevalent source of healthcare-associated infections, with risk factors ranging from 9.8% to 14.6%.^[6]

Antibiotic resistance is very common among Proteus strains, and in the β-lactamases group, the synthesis of ESBL and AmpC type of β-lactamase is mostly seen. ESBL’s are plasmid-mediated and hydrolyze penicillins, cephalosporins, and monobactams, but are never hydrolyzed by cephamycin or carbapenems. They are also prevented by β-lactamase inhibitors such as clavulanic acid, sulbactam, and tazobactam. Carbapenems are the drug of choice for ESBL producers. The genes that code for multiple antibiotic resistance and β-lactamases are communicable, it is imperative that testing of β-lactamases in any hospital is followed.^[7]

Beta-lactam antibiotics are preferred against different Gram-negative bacteria. Beta-lactam class of antibiotics are lysed by AmpC β lactamases an enzyme produced by these bacteria^[8] as well as a few different isolates are sensitive to latter generation Cephalosporins but Extended-spectrum bacteria may be detoxified by new β-lactamases Penicillins and Cephalosporins because of spontaneous mutation. Extended-spectrum β-lactamases (ESBL’s) are also seen in Salmonella spp, Enterobacter spp, and fungi.^[9] Cephalosporins are antibiotics that have a wide spectrum of activity toward Gram-positive and Gram-negative orgaisms.^[10] The body’s reaction to cephalosporins with a broad range of action for Enterobacteriaceae family members that do not induce β-lactamases is the secretion of mutant variants of old β-lactams called extended-spectrum β-lactamases.^[11] This transformation focuses on various genetic pathways in the acquisition of resistant genes to several antibiotic groups, which has invariably turned P. mirabilis into a pandrug-resistant bacteria with difficult-to-treat infections.^[12]

Materials and Methods

This observational, descriptive study was done after obtaining ethical committee clearance in the microbiology laboratory of a tertiary care institute for 1 year. The clinical samples from patients with infection were screened for Proteus species. Samples from blood/serum collected for serological testing, and patients from whom strains other than Proteus have been isolated from other samples were excluded from the study. Seventy-nine isolates were analyzed.

Clinical samples such as urine, wound swab, tracheal aspirate, sputum, ear swab, body fluids, and blood were collected and inoculated aseptically onto blood agar and MacConkey’s agar and incubated for 18–24 h at 37°C. The morphology of the colonies studied was its size, shape, color, pigmentation, and hemolytic properties. Proteus like colonies was identified by Gram stain and relevant biochemical tests. The antibiogram of the strains were tested by the Kirby–Bauer disk diffusion method. The following antimicrobial agents tested were: Ampicillin, Amoxyclav, Gentamicin, Cotrimoxazole, Amikacin, Ciprofloxacin, Aztreonam, Piperacillin-tazobactam, Imipenem, and Meropenem. The diameter of the zone size of growth-inhibition observed was measured and then compared to the zone size interpretative chart provided by Clinical & Laboratory Standards Institute (CLSI).

Any Proteus strain found to be a suspected ESBL producer was screened for ESBL by the methodology outlined in the CLSI 2012 guidelines. The following antibiotics discs were used as indicators, i.e., ceftriaxone (30 μg), ceftazidime (30 μg), and cefotaxime (30 μg). Any strain showing an inhibitory zone size ≤25 mm with ceftriaxone, ≤22 mm for ceftazidime, and ≤27mm for cefotaxime were presumably considered to be an ESBL strain. 0.5 McFarland turbidity standard suspension was made from the colonies of Proteus spp. isolates and using this inoculum, a lawn culture was made on Muller Hinton Agar (MHA) plate. Antibiotic disks of Ceftazidime and a combination of Ceftazidime + Clavulanic acid (30 mcg/10 mcg) were kept on the surface of MHA at a distance of 15 mm between the discs. The plate was then incubated overnight at 37°C. An increase of ≥5 mm in zone diameter of Ceftazidime + Clavulanic acid with its comparison to the zone diameter of Ceftazidime alone concluded that the organism was an ESBL producer as shown in Figure 1.^[13]

Figure 1: A Proteus strain showing ESBL production by the double disk synergy test (DDST)

All the available data were analyzed. Nominal data were expressed by number and percentage differences and any association between categorical variables was tested by Chi-square test and was considered statistically significant at P < 0.05 in addition to the odds ratio and 95% confidence interval calculations.

Results

Among the total of 79 Proteus strains obtained during the period of study, 50.6% were isolated as P. mirabilis and 49.4% isolated as P. vulgaris. The age group of the patients from whom the Proteus species were isolated ranged from 4 to 90 years, with a high percentage of cases in the age group of 41–60 years. Of the 79 Proteus isolates, 64.6% were male patients, 35.4% were female. Of the 79 isolates, 39.2% of Proteus species were isolated from wound swabs, 30.4% from urine, 27% from pus swabs, 1.3% from pleural fluid, and 1.3% from ascitic fluid. Among the Proteus isolates 15.2% were ESBL producers and 84.8% were non-ESBL producers. In this, 25% of ESBL producers were P. mirabilis and 75% of ESBL producers were P. vulgaris. 74.7% of patients were free from any type of risk factors that can predispose to a Proteus infection. 1.3% of patients were found to be anemic and also another 1.3% of patients had a diagnosed carcinoma. 12.7% of patients were diabetic, 7.6% of them had hypertension and 2.5% of them had hypothyroidism.

Discussion

Proteus species are often culprits in hospital and community-acquired infections. Since this bacteria has a range of methods of dissemination, it can infect a series of anatomical locations in the body.^[14] β lactams are vulnerable to wide-type isolates of this genus. The majority of acquired tolerance is due to enzymes.^[15] The problem is more aggravated in developing countries where infection control practices and antibiotic policies are almost nonexistent. Methicillin-resistant Staphylococcus aureus, ESBL producers, and Vancomycin-resistant Enterococci are increasingly reported all over the world. Mortality, morbidity, and treatment cost have increased due to infections caused by these pathogens.^[16]

Our study revealed that of the 79 Proteus isolates, 40 isolates were P. mirabilis (50.6%) and 39 isolates were P. vulgaris (49.4%). This was similar to an article by Mahima et al., in which she said that P. mirabilis (80.90%) and P. vulgaris (70.90%) were the two most common Proteus species found in 110 specimens.^[17] A majority of the patients belonged to the 41–60 years age group (46.8%) followed by the above 60 (36.7%) years age group. The Proteus species affected more males (64.6%) than females (35.4%). These findings corroborate a study by Bahashwan and Shafey et al.^[18]

ESBL detection method in our study was using the double disk diffusion method that disclosed that 15% of our Proteus strains were ESBL producers and strains of P. vulgaris produced more ESBL than P. mirabilis. A similar finding was evident in a study by De Champs et al. in 2002 who concluded that beta-lactamases were found in 147 (52%) of 282 P. mirabilis isolates.^[19] He also showed that 172 isolates with ESBL phenotype, of which 95 carried ESBL genes and 54 carried the Amp C gene.^[19]

In our study, the genus Proteus were predominately isolated from wound swabs (39.2%) followed by samples of urine (30.4%), and from pus samples (27.8%). Cyprien Ntirenganya et al. showed that the Proteus species was the most predominant urinary tract pathogen.^[12] Other bacteria such as E. coli, Pseudomonas aeruginosa, and Acinetobacter species are more frequent in causing urinary tract infections (UTI’s). P. mirabilis is most commonly seen as pathogens in human urinary tract than P. vulgaris.^[20] A similar study by Yah et al. detected 148 species of Proteus strains of which 97 were P. mirabilis and 51 were P. vulgaris strains from wounds of diabetic patients.^[21]

In our study too, we have seen that, 12.7% of the patients with Proteus infections had diabetes which was seen as a major risk factor. 7.6% of the patients had hypertension and 2.5% of patients had hypothyroidism. A study conducted by Chen et al., showed hydronephrosis, hyperthermia or hypothermia and a serum C-reactive protein concentration >100 mg/L were risk factors for P. mirabilis bacteremic UTI.^[22] Diabetes mellitus was linked to a variety of infections, particularly targeting the skin, mucous membranes, soft tissue, urinary tract, respiratory tract, surgical and/or hospital-associated infections triggered by Enterobacteriaceae species as seen by Akash MS et al.^[23]

A comparable activity of cefoxitin and cephalothin against select Enterobacteriaceae and its correlation with enzymatic resistance mechanisms showed cefoxitin to be totally resistant to most Gram-negative β-lactamases, which was also shown by other researchers.^[10] The current literature texts by Vuye A et al. showed that in-patients with P. mirabilis bacteremia, the ESBL development was linked to a higher risk of death, although in our study, we did not isolate any Proteus species from blood culture samples. To reduce mortality by P. mirabilis bacteremia, a rapid identification of ESBL expression and prompt antimicrobial therapy were found very crucial.^[10] Comparing the two studies, our study further concluded that drug resistance due to ESBL production was more common in the case of P. mirabilis with some risk factors, and these bacteria were found resistant to cefazolin, cefotaxime and ceftriaxone.

A study by Senthamari S et al. showed that it is important to be mindful of certain species tolerance as they are a possible source of specific strains that might be passed around to several microbes. As a result, increasing drug resistance is a widespread issue, and its control is a cause of worry.^[6]

Feglo PK et al. showed that P. mirabilis, P. vulgaris, and P. penneri were the species commonly implicated among Proteus infections. These species were found susceptible to Amikacin, Gentamicin, and third-generation Cephalosporins, even to those that were immune to ampicillin, tetracycline, chloramphenicol, and cotrimoxazole, since these antibiotics never could be used for the diagnosis of the Proteus pathogen.^[6]

Another research project by Alhaj et al. revealed that the high rate of ESBL secreting Proteus spp was found in samples in an Yemeni community clinical setting, necessitating long-term disease prevention strategies including antibacterial control and systematic detection of ESBL secreting isolates.^[1]P. mirabilis has a predilection for urological complications such as UTI’s, cystitis, pyelonephritis, and prostatitis. Apart from this wound infections, and infection in burns patients, both community-acquired and catheter-associated UTI are common. They have also been linked to infections of the respiratory tract, chronic suppurative otitis media, endophthalmitis, and infections of the central nervous system, such as meningitis and meningoencephalitis.^[2]

Another survey published by Bahashwan and Shafey stated that, in the treatment of Proteus spp infections, the antibiotic imipenem (IMP) was indicated. If there is an allergic reaction to imipenem, users can still use amikacin (AK) along with (IMP). Proteus infections can necessitate the use of a range of therapies in extreme cases like a combination of Imipenem-Amikacin.^[23] The occurrence of P. mirabilis tolerance to beta-lactams has increased significantly. In 60% of cases of infection, P. mirabilis has been the only organism implicated. These aspects should be considered when selecting an infection as unveiled by a two-way survey by Syed Suhail Ahmed.^[13]

In another similar study de Champs et al. revealed that, out of 172 isolates, 24% of the Proteus isolates produced ESBL, which was confirmed by double disk diffusion method using cefotaxime (30 μg) and a combination of cefotaxime/clavulanic acid (30/10 μg).^[15] A comparison with our current study showed that 15.2% of ESBL producers were detected with this same procedure. This same method was followed by Pandey et al., that yielded, 48.86% of the total clinical samples as ESBL producers.^[14] In another study Bourgeois TA et al. showed 3.7% of ESBL producers among P. mirabilis which were identified using Allele-specific PCR.^[24] The double-disk diffusion procedure is a convenient and cost-effective method to confirm ESBL production on a day-to-day routine laboratory workflow.

Magliano et al. revealed that among nosocomial pathogens causing UTI, 5.2% were caused by P. mirabilis and were prevalent in ≤14 years’ age group.^[25] Infection among wounds with P. mirabilis was higher in the 16-30 year old age group, followed by the 5–15 and 30-50 year old age groups in our study. This was a similar conclusion to a study by Zafar et al.^[26] We too find that Proteus species were more frequently isolated in wound swabs than from urine samples among those in the age groups of 40–60 years, which was followed by those above 60 years. Thus P. mirabilis was the most common significant species isolated mainly from wound swabs among the age groups of 41–60 male patients. 15.2% of them were found to be ESBL producers and patients presented with a variety of risk factors.

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Conflicts of interest

There are no conflicts of interest.