Profile of Cutaneous Bacterial Flora in Pemphigus Patients

Introduction

Pemphigus (derived from the Greek word “pemphix” meaning bubble) is a heterogeneous group of bullous diseases, autoimmune in nature, which affect the skin and mucous membranes.^[1] The condition is characterized by acantholysis (cell adhesion loss) and development of blisters within the epidermis; and is mediated by immunoglobulin G autoantibodies formed against adhesion molecules.^[1,2] Incidence has been estimated to vary from 0.09 to 1.8% among the dermatology outpatient attendees and 0.76 to 16.1 cases per million in the general population.^[3,4] Systemic glucocorticoids and immunosuppressants are the mainstay of therapy of this chronic disorder, the advent of which has largely led to the improvement of the overall prognosis of patients suffering from pemphigus. However, long-term use of immunosuppressants in high doses and the resultant immunocompromised state of patients predispose to high risk of infections which account for 34.3 to 55.5% of all deaths.^[4] Moreover, infectious complications are also liable to occur due to the disease process itself because of disruption of the epidermal barrier. Thus, if not treated, pemphigus can be fatal, usually from overwhelming opportunistic infection such as septicemia and pneumonia, secondary to cutaneous bacterial infections.^[4,5] Therefore, timely identification of infection, its causative bacteriological agent, and appropriate treatment can play a major role in reducing the mortality. In this study, we aim to look into the cutaneous bacterial infection profile in pemphigus patients presenting to a tertiary-level hospital and examine their respective pattern of susceptibility to various antimicrobial agents.

Materials and Methods

The retrospective study, exempted from review by the Institutional Ethics Committee, was performed from July 2018 to June 2020 over a 2-year time frame in the department of microbiology of a tertiary-level health care setting in the Eastern region of India. Pus samples or swabs received from patients with pemphigus after swabbing the lesions with sterile normal saline were subjected to culture using standard microbiological techniques.^[6] Identification and antimicrobial susceptibility testing of the bacterial isolates was performed per manufacturer's instructions by VITEK-2 Compact automated system (bioMérieux, Marcy l'Etoile, France). The resultant minimum inhibitory concentrations were interpreted as per the Clinical and Laboratory Standards Institute's (CLSI) breakpoints as susceptible, intermediate, or resistant.^[7] Tigecycline results were interpreted as per the European Committee on Antimicrobial Susceptibility Testing guidelines.^[8] Susceptibility testing for antimicrobial agents for which CLSI breakpoints were available, but not included in the VITEK panel, were supplemented by the use of disc-diffusion or EzyMIC testing (HiMedia, Mumbai, Maharashtra, India). Strains used for quality control purposes were Staphylococcus aureus ATCC 29213, S. aureus ATCC 25923, Escherichia coli ATCC 25922, and Pseudomonas aeruginosa ATCC 27853. Results of strains having intermediate resistance were included in the percentage of resistant isolates.

Results and Discussion

During the period of study, samples from 315 patients were received, which were comprised of 203 (64.4%) males and 112 (35.5%) females. The lowest age was that of a 1-year-old male child and the highest a 87-year-old female patient (age range, 1–87 years). One hundred six (33.6%) patients were admitted, while 209 (66.3%) were from the outpatient department. A total of 252 (80.0%) samples were culture-positive, with growth of diphtheroid, other commensal skin flora, environmental bacillus species, and more than three morphotypes in 41 samples which were not processed further. The remaining 211 samples yielded 245 bacterial isolates comprising of 158 Gram-positive cocci and 87 Gram-negative bacilli. Culture from 34 (13.5%) patients grew two organisms each, while that from the remaining 177 (70.2%) grew a single organism. S. aureus (138, 56.3%) was found to be isolated most frequently, followed by P. aeruginosa (41, 16.7%), E. coli (16, 6.5%), and Klebsiella pneumoniae (14, 5.7%) (►Table 1). Apart from S. aureus, other Gram-positive organisms included Streptococcus pyogenes (9, 3.7%), Enterococcus spp. (7, 2.9%), and coagulase-negative staphylococci (4, 1.6%). Total number of Enterobacterales isolated was 42 (►Table 1).

Analysis of susceptibility profile among the predominant Gram-positive cocci revealed that methicillin resistance occurred in 24.6% (34/138) of S. aureus isolates (►Table 2). The resistance pattern of S. aureus to other antibiotics was as follows: penicillin (116, 84.1%), erythromycin (97, 70.3%), clindamycin (76, 55.1%), ciprofloxacin (84, 60.9%), levofloxacin (69, 50.0%), trimethoprim-sulfamethoxazole (30, 21.7%), chloramphenicol (29, 21.0%), gentamicin (14, 10.1%), and tetracycline (3, 2.2%). S. pyogenes were the next most common Gram-positive bacteria from the pemphigus lesions and all (n = 9, 100%) were susceptible to penicillin, ceftriaxone, vancomycin, linezolid, and tigecycline. Resistance was observed to erythromycin, clindamycin, chloramphenicol, levofloxacin, and tetracycline in 4 (44.4%), 3 (33.3%), 2 (22.2%), 1 (11.1%), and 1 (11.1%) isolates of S. pyogenes. The D-test (indicating inducible clindamycin resistance) was positive in 43 (31.1%) S. aureus and 1 (11.1%) S. pyogenes isolates, respectively. As regards enterococci, only one (14.3%) exhibited simultaneous penicillin and high-level gentamicin resistance; rest being susceptible to all the tested antimicrobials. As regards Gram-negative bacilli, among the 42 Enterobacterales isolates, 23 (54.8%), 6 (14.3%), and 4 (9.5%) were resistant to the third-generation cephalosporins, piperacillin-tazobactam, and carbapenems, respectively (►Table 2). Resistance to ciprofloxacin, trimethoprim-sulfamethoxazole, gentamicin, and amikacin was observed in 21 (50.0%), 13 (30.9%), 4 (9.5%), and 2 (4.8%) isolates, while none were resistant to colistin and tigecycline (except Proteus which is intrinsically resistant to both). Isolates of P. aeruginosa (n = 41) exhibited the following resistance pattern: ceftazidime (10, 24.4%), cefepime (10, 24.4%), ciprofloxacin (8, 19.5%), aztreonam (8, 19.5%), piperacillin-tazobactam (6, 14.6%), carbapenem (6, 14.6%), gentamicin (4, 9.8%), and amikacin (3, 7.3%) (►Table 2). The lone isolate of Burkholderia cepacia complex was susceptible to its tested panel of antibiotics, namely, ceftazidime, meropenem, ticarcillin-clavulanate, levofloxacin, and chloramphenicol.

The most common comorbidity in pemphigus patients is infection, which may lead to increased morbidity and mortality.^[9] In the current study, 211 of 315 (66.9%) patients had clinically and microbiologically proven infected pemphigus lesions substantiating the large burden of cutaneous infection in these patients. Furthermore, 33.6% of patients were hospitalized, which emphasizes the severity of the infection leading to hospital admission. Infections have been observed in 60.6 to 68% of pemphigus patients in various studies from the world, with cutaneous infections observed in 10.32 to 71.4% of them.^[5,10–12]

In the current study, S. aureus (56.3%) was the most common organism isolated from pemphigus patients followed by P. aeruginosa (16.7%) which is consistent with many other studies.^[5,11–15]

In the study by Esmaili et al,^[11] Qadim et al,^[5] and Solanki et al,^[13]S. aureus accounted for 93.7, 82.9, and 72% of the isolates, which is quite high than that observed in the current study. However, in the study by Kiran et al,^[12]S. aureus accounted for a lower frequency of 40.8% of the isolates.^[12] This difference in the frequency isolation may be due to local regional variation in the bacterial population related to specific patient groups or the prevalent pattern of antimicrobial prescription practices or therapy leading to selection of isolates.

Frequency of methicillin resistance (24.6%) observed in the present study, is similar to a very recent study on skin and soft tissue infections from North India reporting 24.18% methicillin resistance among S. aureus isolates.^[16] However, an evaluation of S. aureus infections in pemphigus patients by Motallebi et al revealed a higher methicillin resistance of 43.2%.^[17] In the current study, maximum resistance in S. aureus isolates was observed for penicillin (84.1%) followed by erythromycin (70.3%) and ciprofloxacin (60.9%). A study, by Kiran et al, also found similar resistance pattern of penicillin (90%), erythromycin (55%), and ciprofloxacin (55%) in pemphigus patients.^[12] Li et al found resistance rates of penicillin G, erythromycin, and clindamycin to be 91.9, 75.8, and 45.2%, respectively, among 62 S. aureus isolated from hospitalized patients with pemphigus.^[4]S. pyogenes, the second most common Gram-positive bacteria found in the current study displayed maximum resistance to erythromycin and clindamycin (44.4 and 33.3%, respectively), which is a persisting problem as observed by other researchers.^[18,19] Susceptibility to penicillin, has fortunately been retained in these studies^[18,19] as well as in our study which is the antimicrobial agent of choice.

Among Gram-negative bacteria, a high resistance was observed for third-generation cephalosporins (24.4–54.8%), ciprofloxacin (19.5–50.0%), and trimethoprim-sulfamethoxazole (30.9%) as that observed by Esmaili et al.^[11] In contrast, 100% of the P. aeruginosa isolates were sensitive to ceftazidime and ciprofloxacin as well as to amikacin and gentamicin in the study by Kiran et al.^[12] Colistin and tigecycline resistance was not exhibited by any of the isolates in the current study (except Proteus species which are intrinsically resistant to both). Carbapenem resistance was low (9.5–14.6%), which was also found in the study by Kiran et al.^[12] However, at other places, the burden of carbapenem-resistant Gram-negative bacteria in various types of skin and soft tissue infections is increasing.^[20] Carbapenem-resistant Enterobacteriaceae with distinct carbapenemase-encoding genes exhibit variation in their geographic spread, as well as display differing susceptibility to the newer therapeutic agents such as the newer beta-lactam combination agents and cefiderocol.^{[20,21,22,23]} Hence, there is a need for molecular characterization of carbapenemase genes in carbapenem-resistant bacteria in routine clinical practice. It is known that infection by multidrug-resistant organisms like methicillin-resistant S. aureus and carbapenem-resistant organisms may be associated with serious systemic complications and multiorgan failure leading to prolonged hospitalization, higher economic burden, and increased mortality.^[20,21,22] So outlining the profile of antimicrobial susceptibility of the isolates and administration of appropriate antibiotics are recommended for better patient outcome.

There are certain limitations of the study in view of its retrospective design. It would have been better if any relationship could have been delineated between the number and type of isolates and the duration of steroid treatment in the patients. Molecular characterization of the resistant isolates or their resistance mechanisms would also have contributed to crucial information. To conclude, cutaneous pemphigus lesions are infected by a large variety of bacterial species that demonstrate varying antimicrobial susceptibility pattern, emphasizing the need for continuous monitoring to help in the appropriate choice of empiric or definitive therapy.