https://orcid.org/0000-0001-6545-7616
Figures
Abstract
Klebsiella pneumoniae is a major zoonotic pathogen that causes a variety of severe illnesses as well as mastitis. The distribution of mastitis-causing K. Pneumoniae and its virulence factors vary by country and geographical location. The present study aimed to find out the occurrence of Multidrug-resistant (MDR) K. Pneumoniae and their capsular resistance genes which were undocumented previously in cow farms of district Peshawar, Pakistan. A total of 700 milk samples from symptomatic mastitic cows were screened for MDR K. Pneumoniae. Furthermore, the characterization of capsular resistance genes was done by molecular techniques. Among these samples, K. pneumoniae was found 180/700 (25.7%), while MDR K. pneumoniae was found 80/180 (44.4%). The antibiogram analysis revealed high resistance to Vancomycin (95%) while highly sensitive to Ceftazidime (80%). The distribution of capsular genes shows the most common serotype K2 gene 39/80 (48.7%), followed by serotype K1 gene 34/80 (42.5%), serotype K5 17/80 (21.2%), and serotype K54 13/80 (16.2), respectively. Moreover, the co-occurrence of serotypes K1+K2 was found at 11.25%, KI+K5 was 05%, K1+K54 was 3.75%, and K2+K5 was 7.5%, respectively. A statistically significant association (p ≤ 0.05) was found between predicted and discovered K. pneumoniae values. In conclusion, the presence of MDR K. pneumoniae in combination with capsular genes may be a possible threat to dairy farm animals and humans in Peshawar, Pakistan. It may give us special attention to follow up on hygienic practices in livestock management.
Citation: Saddam S, Khan M, Jamal M, Rehman SU, Slama P, Horky P (2023) Multidrug resistant Klebsiella Pneumoniae reservoir and their capsular resistance genes in cow farms of district Peshawar, Pakistan. PLoS ONE 18(2): e0282245. https://doi.org/10.1371/journal.pone.0282245
Editor: Sherin Reda Rouby, Beni Suef University Faculty of Veterinary Medicine, EGYPT
Received: December 17, 2022; Accepted: February 10, 2023; Published: February 27, 2023
Copyright: © 2023 Saddam et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper and its Supporting Information files.
Funding: This research was funded by the Higher Education Commission of Pakistan under the NRPU project (Sr. Number: 8633). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Cow mastitis is one of the most common infections in dairy cows. It exclusively affects the dairy cow’s reproductive cycle and maternity, resulting in decreased milk output and quality. It also raises treatment costs and creates significant economic losses in dairy sectors worldwide [1]. Mastitis causes $35 billion in global economic losses each year, with the US dairy industry losing $2 billion [2, 3]. Furthermore, mastitis can endanger human and animal health by transferring antibiotic-resistant bacteria and causing food poisoning [2]. Klebsiella pneumoniae is a major environmental pathogen that causes mastitis, as well as a zoonotic pathogen that may cause a variety of severe illnesses [4, 5]. Numerous properties involved in K. pneumoniae pathogenesis have been identified, like virulence factors, but their capability in toxicity and drug resistance remains unknown [6].
The pathogenic mechanism of K. pneumoniae is also determined by virulence factors. Capsular, iron carriers, pili, and lipopolysaccharide (LPS) have all been associated in K. pneumoniae adhesion, invasion, and proliferation [7, 8]. The capsular can stop K. pneumoniae from being recognized by the host immune system through immune escape mechanisms like inhibition of early inflammatory response, anti-phagocytosis, inhibition of dendritic cell maturation, and neutralization of antimicrobial peptides to lessen the body’s immune response [8]. These bacteria may take iron from the host via four siderophores, including salmochelin, aerobactin, yersiniabactin, and enterobactin for metabolism and infection [9]. As the researches are still in its early stages, K. pneumoniae pathogenesis has long been linked to four key components K. pneumoniae capsule antigens, adherence factors, lipopolysaccharides, and Siderophores. The virulence factor for K. pneumoniae was capsular polysaccharide (K antigen). The sparkling and mucoid presence of K. pneumoniae settlements on agar plates due to this antigen shapes a thick hydrophilic case. At least 77 K antigen serotypes have been recognized to date, referred to as K1, K2, etc. The K antigens assume a significant part in shielding cells from opsonophagocytosis and serum killing [10].
Mastitis incidence varies by country and geographical location because of variances in legislation, veterinarian and laboratory facilities, and farmer management techniques. Therefore, the present study was designed to find out the occurrence of multi-drug resistant (MDR) K. Pneumoniae in cow farms and their comparative analysis with capsular resistance genes in the district of Peshawar, Pakistan.
Materials and methods
Sample collection and isolation of K. pneumoniae
The animal study protocol was approved by the Institutional Review Board of Abdul Wali Khan University Mardan, Pakistan. The study was performed in various areas of Peshawar, Pakistan, located at 34.0151°N, 71.5249°E (S1 Table). The samples were collected from symptomatic mastitis cows in sterile falcon tubes after discarding a few drops of milk on verbal permission from the dairy farm management. All samples were sealed with air-tight closing to prevent contamination and were processed in the microbiology Laboratory, Abdul Wali Khan University Mardan, Pakistan. The milk samples were inoculated with Luria Bertani broth medium for 24 hours at 37°C. After incubation, the medium was subcultured on K. pneumonia specific media Simmons citrate agar (SCA) followed by Rodrigues et al. [11] method. Further confirmation was done by gram staining and string test. Molecular confirmation of K. pneumoniae isolates was done by Polymerase chain reaction (PCR) using their specific 16s RNA primer (Table 1) [12].
Detection of MDR K. pneumoniae
Mueller-Hinton agar was used for antibiotic susceptibility of K. pneumoniae isolates for commonly used 14 different antibiotics discs (Table 2), using the standard Kirby-Bauer disk diffusion technique [16]. Multi antibiotic resistance (MAR) index was calculated for MDR K. pneumoniae determination, as described by Ayandele et al. [17]. The following formula used for the MAR index:
MAR Index = a/b (a = number of antibiotics to which isolate resistant, b = total antibiotics tested)
Detection of capsular resistance genes
Total nucleic acid from the MDR K. pneumoniae isolates was extracted through WizPrepTM gDNA Mini Kit (Seongnam-si, Gyeonggi-do, 13209, Republic of Korea). Molecular characterization of four (04) capsular encoding genes; serotype K1, serotype K2, serotype K5, and serotype K54 was performed through PCR by using gene-specific primers, as given in Table 2. PCR Primer-specific fragments were visualized in 01% agarose gel electrophoresis using Gel DocTMEZ (BIO-RAD).
Results
A total of 700 samples were collected from symptomatic mastitis cows. All samples were cultured on specific media SCA for the isolation of K. pneumoniae. After the appearance of a yellowish colony, gram-negative and string test positive, the bacterium was assumed as K. pneumoniae. They were confirmed by molecular analysis, the 16s RNA gene size 260bp was conformed as K. pneumoniae (S1 Fig in S1 Raw images). Among the total collected samples, K. pneumoniae growth was observed in 180 (25.7%).
MDR K. pneumoniae
All isolated K. pneumoniae colonies were subjected for the screening of their sensitivity pattern. The MDR K. pneumoniae occurrence was observed n = 80 (44.4%) with observed MAR ranges from 0.21–0.92. The antibiogram analysis revealed high resistance to Vancomycin (95%) while highly sensitive to Ceftazidime (80%). The other antibiotic’s antibiogram results were presented in Fig 1 and S2 Table.
Occurrence of capsular genes
MDR K. pneumoniae was further screened for the occurrence of capsular genes. The obtained electrophoretogram of capsular resistance genes was presented in S2-S5 Figs in S1 Raw images. The high occurrence was found of serotype K2 gene n = 39 (48.7%) followed by serotype K1 gene n = 34 (42.5%), serotype K5 n = 17 (21.2%), and serotype K54 gene n = 13 (16.2), respectively. Moreover, the co-occurrence of serotypes K1+K2 was detected n = 09 (11.25%), KI+K5 was n = 04 (05%), K1+K54 was n = 03 (3.75%), and K2+K5 was n = 06 (7.5%), respectively (Fig 2). Further area wise distribution of MDR K. pneumoniae with their capsular genes was presented in Table 3.
Discussion
One of the dairy cow illnesses that pose the greatest economic challenges is mastitis, particularly for small-scale producers across the world. Mastitis is typically more prevalent in nations with underdeveloped dairy industries and lax udder cleanliness [18]. In the present study the symptomatic mastitic milk samples from cows in Peshawar, Pakistan were collected. The samples were cultured for the estimation of occurrence frequency and isolation of MDR K. pneumoniae. Furthermore, the capsular genes K1, K2, K5, and K54 occurrence incidence were also determined among the isolated MDR K. pneumoniae.
Klebsiella mastitis is a significant problem in the USA [18]. In Bangladesh, Klebsiella spp. was found in 62.5% of mastitic milk [19] while in India found 20% to 24% in different studies [20, 21]. In the present study, among the total 700 collected milk samples of cows, K. pneumoniae incidence was found 25.7%. In contrast to our study, a recent study in Pakistan indicated 7.9% of in Peshawar, Khyber Pakhtunkhwa among cows [22], 8% incidence due to K. pneumoniae in Punjab buffaloes [23], and 11% in Sindh province among buffaloes [24]. The current study variation may be due to specie, veterinarian facilities, and farmer management techniques.
Antibiotic overuse contributes to the development of multi-drug resistant (MDR) organisms. The pace of MDR Klebsiella spp., growth is accelerating daily. Globally, concerns including the rise of MDR bacteria, the use of antibiotics to treat them, and zoonotic diseases are considered critical [19]. The MAR index is determined by dividing the number of antibiotic isolates that are resistant by the total number of antibiotics the organism has been to expose [17]. In the current study, MDR K. pneumoniae was found at 44.4% with a MAR range from 0.21–0.92. These results are in agreement with previous studies of Salauddin et al. [19], who reported a 62.5% incidence of MDR K. pneumoniae and Osman et al. [18] reported 40%, while in contrast to our study, the Yang et al. [25] reported 9.78% MDR K. pneumoniae. The present study antibiogram analysis of cow milk isolated MDR K. pneumoniae showed high resistance to Vancomycin, Fusidic acid, Amoxicillin, Sulfamethazine, and Chloramphenicol, while highly sensitive to Ceftazidime, Ciprofloxacin, Levofloxacin, Amikacin, Gentamycin, Tetracycline, and Imipenem. These results are mostly similar to the previously reported study of Ali et al. [22]. The variation in the resistance profile of the present study is due to the overuse of these antibiotics for the treatment of mastitis as well as infected milk consumers.
Capsular and other virulence components are crucial to K. pneumoniae’s pathogenic process. Through immune escape mechanisms like anti-phagocytosis, inhibition of early inflammatory response, neutralization of antimicrobial peptides to lessen the body’s immune response, and inhibition of dendritic cell maturation, capsular can stop K. pneumoniae from being recognized by the host immune system [8]. Herein this study, the most common capsular gene was found K2 gene (48.7%), while serotype K1 gene was found at 42.5%, followed by serotype K5 (21.2%), and serotype K54 (16.2%). In agreement with these findings, Osman et al. [18] also reported the distribution of the K1 gene (66.7%) and K2 gene (55.6%), while Gao et al. [26] reported the K5 gene (10%) and K54 gene (8%).
Conclusions
In conclusion, the current study results showed the high occurrence of MDR K. pneumoniae in cow farms of district Peshawar Pakistan. We must pay particular care since K. pneumoniae present in mastitic milk has capsular resistance genes, which might lead to clinical infections in cows and those who consume milk. The development of veterinary diagnostic labs for routine animal screening and antibiogram analysis is currently necessary to combat empirical antibiotic usage and resistance to certain antibiotic groups. It must be ensured follow up the hygiene practices in dairy farms.
Supporting information
S1 Table. Study area of district Peshawar, Pakistan with its geographical coordinates from where cow farms located.
https://doi.org/10.1371/journal.pone.0282245.s001
(DOCX)
S2 Table. Antibiotics sensitivity pattern of MDR K. pneumoniae.
https://doi.org/10.1371/journal.pone.0282245.s002
(DOCX)
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