Identification and Antibiogram of Klebsiella pneumoniae Isolated from Camels with Molecular Recognition of Some Virulence and Antibiotic Resistance Encoding Genes

Camelids are unparalleled group of mammals with distinguished physiological and immunological statuses. The members of this family have developed features to accommodate diverse and challenging environments. In Africa and Middle East, camels are nursed, and contribute significantly to the economy of certain communities which rely on camels for meat, milk, sport and transportation (FAOSTAT, 2016). In spite of resilience to the environment, camels are susceptible to numerous bacterial pathogens. A group of the most common infectious diseases affect camels are the respiratory illnesses which are usually attributed to multifactorial etiology (Zhu et al., 2019).

Respiratory sickness in camels is a growing concern and is leading cause for significant production losses, higher mortalities, and costly preventive and curative efforts (Kane et al., 2005). One of the most prevalent bacterial pathogens impact respiratory illnesses is Klebsiella pneumoniae which is a Gram’s negative, facultative anaerobic, nonmotile and opportunistic pathogen belonging to the family Enterobacteriacae. The K. pneumoniae cause a wide range of symptoms in both humans and animals including pneumonia, septicemia, diarrhea, liver abscesses, meningitis and urinary tract infections (Guo et al., 2017; Navon-Venezia et al., 2017).

Recently, mucoid-hypervirulent K. pneumonia (hvKP) strains have emerged worldwide and found to harbor genes that coding several virulence features that help in the microbial pathogenicity (Shon et al., 2013). Two important genes found to be implemented in the virulence of these hvKP include rmpA gene (regulator of mucoid phenotypes) and magA gene which is associated with K1 capsular serotype. Both genes have been reported to enhance the mucoviscosity and pathogenicity, resulting in severe septicemia and death (Yeh et al., 2006; Hsu et al., 2011).

Furthermore, the global concern of increasing antimicrobial resistance among microbes was extended to K. pneumonia strains. Antibiotics of the β-lactam and aminoglycosides groups are broadly prescribed worldwide. The β-lactams which include cephalosporins, carbapenems, penicillin, amoxicillin and ampicillin are mainly neutralized by production of beta-lactamase. K. pneumonia is considered as one of extended-spectrum beta-lactamase (ESBL) producing bacteria (WHO, 2014; Rahman et al., 2018). On other side, aminoglycosides are potent natural or semisynthetic broad-spectrum antibiotics derived from actinomycetes, which act via prohibition of protein synthesis and widely used for treatment of diseases caused by Enterobacteriaceae.

The K. pneumonia strains may harbor genes responsible to establish resistance against many antibiotics. These include blaTEM and aadB which are responsible for amoxicillin and gentamicin/kanamycin/tobramycin resistence, respectivley (Cameron et al., 1986).

In the current study, the living and slaughtered camels in Gharbia Governorate were screened for the prescence of K. pneumoniae by traditional and advanced molecular techniques. The chracterized isolates were analysed for the prescence of virulence associated (rmpA and magA) and antibiotic resistant genes.

A total of 205 samples (116 nasal swabs and 89 lung tissues) were obtained from apparently healthy (n≈33) as well as respiratory manifested camels (n≈83) from established farms and abattoirs in Gharbia Governorate during 2018. Nasal samples were incubated in nutrient broth while lung tissues were processed for the bacteriological examination.

Bacterial isolation was carried out following standard techniques described by Gundogan and Avci (2013). Nasal swabs and tissue samples were primarily inoculated in to blood agar plates (HiMedia, India) and incubated for 24-48 h at 37oC. Thereafter, the suspected colonies were streaked on MacConkey bile salt lactose agar (Oxoid Basingstoke, UK), XLD agar media (Oxoid Basingstoke, UK) and Eosin methylene blue agar (HiMedia, India) and incubated aerobically at 37oC for 24-48 h. After incubation, the plates were examined for the nature of growth and appearance characters of bacterial colonies. The preliminary morphological identification was performed based on Gram’s staining.

The pure colonies were used for biochemical identification through classical biochemical tests, including catalase, oxidase, indole, methyl-red, Voges-Proskauer, urease production and citrate utilization, string test for mucoviscosity, gelatin liquefaction, hydrogen sulphide production on TSI agar and carbohydrates fermentation. Furthermore, the purified colonies were confirmed by application of API 20E system (BioMerieux, Marcy l’Etoile, France) following manufacturer’s guidelines.

The sensitivity of the obtained isolates to ten commonly used antibiotics was performed using disk diffusion tech­nique on Mueller-Hinton agar (Oxoid Basingstoke, UK) and interpreted accordingly CLSI (2018). Antibiotic discs (Oxoid Basingstoke, UK) listed in the Table III were used to assess antibiotic sensitivity.

DNA was extracted from the K. pneumoniae isolates using QIAamp DNA mini kit following the manufacturer’s guidelines. A total of five isolates from pneumonic lung tissues were used for genotyping. The extracted DNA was subjected to uniplex PCR assay using oligonucleotide specific primers for tested genes as mentioned in Table I. PCR master mix and cycling protocol was adapted according to EmeraldAmp GT PCR Master Mix (Takara, Japan). Amplification was carried out using Biometra thermal cycler.

The reaction was performed under different temperature and duration conditions as described in Table II. Aliquots of amplified PCR products were electrophorised on agarose gel of 1.5% (AB gene) in 1x TBE buffer at 25ºC. For gel analysis, 15 µl of PCR products were loaded in each gel slot. A 100 bp DNA ladder (QIAGEN Inc, CA, USA) was used to define the fragment sizes. The gel was photographed using a gel documentation system (Alpha Innotech, Biometra) and the data were analyzed via different softwares.

A total of 8 isolates of K. pneumoniae from pneumonic camels’ nasal swabs, and 5 isolates obtained from pneumonic lung tissues. The overall incidence of

Klebsiella was 8.96%. As regards to apparently healthy camel, one isolate was recovered from nasal swabs and also one isolate from lung tissues. The overall resulting incidence of isolates was 3.33%.

The results were based on the cultural appearance of colonies, Gram’s staining, classical and confirmatory biochemical tests. On blood agar, Klebsiella species appeared as greyish white non hemolytic colonies, whereas on MacConkey’s agar the colonies appeared mucoid, slimy, rose-pink colonies due to lactose fermenter. Furthermore, Klebsiella spp. appeared purple colonies on EMB agar while bright yellow colonies on XLD agar.

All Klebsiella isolates were identified as Klebsiella pneumoniae according to their classical and API 20 E biochemical reactions. The K. pneumoniae shows yellow slants, yellow butt with gas production in triple sugar test. The isolates were negative for oxidase activity, and showed positivity with indole, methyl red and hydrogen sulfide test. The bacterial isolates showed positive results for catalase activity, Voges Proskauer test, Simmons citrate tests, and were found to be positive for urease, lactose, sucrose, maltose, raffinose, trehalose, D-mannitol, D-sorbitol, L-arabinose and L-rhamnose

The in vitro susceptibility of obtained 15 isolates of K. pneumoniae exhibited all the isolates as sensitive to levofloxacin, imipenem (100%) followed by norfloxacin (93.3%) and ceftriaxon (73%). On the other side, all isolates were resistant to amoxicillin, ampicillin and gentamycin (100%) as outlined in Table III.

Uniplex PCR was applied on the selected five K. pneumoniae isolates to detect the virulence genes (magA, rmpA). Analysis indicated a detection of two and zero isolates by a percentage of 40% and 0%, respectively. On the other hand, the both genes responsible for antibiotic resistance (blaTAM, aadB) were found in all five isolates by a percentage rate of 100%.

The role of K. pneumoniae as a significant etiological pathogen for causing pneumonia especially interstitial and chonic pleuropneumonia with mortalities in neonatal dromedary camels was reported (Al-Tarazi, 2001; Narnaware et al., 2020).

Our data revealed eight isolates of K. pneumoniae from pneumonic camels’ nasal swabs, and 5 isolates obtained from pneumonic lung tissues. Therefore, the overall incidence of isolates was 8.96%. As regards to apparently healthy camel, one isolate was recovered from nasal swabs and also one isolate from lung tissues, reulting an overall incidence of 3.33%.

Lower similar incidences (6.3%) have also been mentioned by Abubakar et al. (2008) from Nigerian normal and diseased camel lungs and Ismail et al. (2014) from Egypt (0.5%) in apparently healthy camels. Ahmed and Musa (2015) have recorded a lower rate (0.1%) among pneumonic camels in Sudan. Also, the low prevalence of K. pneumonia in camel was noticed as 16 out of 232 camels (6.9%) in Tunisia (Saidani et al., 2019).

On the other side, higher isolation rates (25.0%) were reported by Azizollah et al. (2009) among healthy dromedaries in central Iran. Sharma et al. (2013) have identified K. pneumoniae isolates as high as 26.9% and 49% in nasal swabs obtained from apparent healthy as well as acute respiratory infected camels, respectively. A total of 70 pneumonic lung tissues obtained from abattoirs in Cairo, Egypt revealed isolation of K. pneumoniae in 26.7% of samples (Wareth et al., 2014). A moderate incidence was reported in cross-sectional bacteriological study performed in Afar Region, Ethiopia among 74 examined camels. The K. pneumoniae was isolated from pneumonic tracheas and lungs by 17.3% while the isolation was noticed in 13.5% of samples collected from other normal animals (Gebru et al., 2018).

Antibiotics are being used extensively to inhibit or to treat microbial infections in human and veterinary practices. Therefore, a great concern has been growing pertaining to antimicrobial resistance in recent years included in what is termed as One Health concept (Matar et al., 2020).

Our data revealed that all tested K. pneumoniae isolates were sensitive to both imipenem and levofloxacin (100%), and have also showed considerably high susceptibility against ciprofloxacin (93.3%) and ceftriaxon (73%). On the other hands, it was observed that all recovered isolates were resistant to amoxicillin, ampicillin and gentamycin.

The choice of quinolone group of antibiotics in effective treatment of diseases caused by K. pneumoniae has been observed earlier (Coskun et al., 2020). The resistance of K. pneumoniae to amoxicillin, ampicillin or gentamycin has been reported in several previous reports (El-Mahmood et al., 2009; Moawad et al., 2011; Sharma et al., 2013; Ahmed and Musa, 2015; Borgio et al., 2021; Fouad et al., 2022).

In our study, all the five tested isolates showed presence of blaTEM and aadB genes and interestingly this existence was expressed in all isolates clinically by resistance to amoxicillin, ampicillin or gentamycin in susceptibility test as mentioned before. The high presence of these resistance genes which are responsible for antibiotic resistance among clinical K. pneumoniae isolates have been documented earlier (Stolle et al., 2013; Lev et al., 2018; Sivaraman et al., 2020; Ejikeugwu et al., 2021; Wareth and Neubauer, 2021).

K. pneumonia is characterized by numerous virulence factors which are playing important roles in the microbial pathogenesis. The magA is considered virulence factor encoded for an outer membrane protein, and help in the resistance of phagocytosis. The rmpA is another virulence factor that facilitates the expression of the hypermucoviscous phenotypes (Fang et al., 2004).

In our study, we have found two out of five tested K. pneumoniae harbor magA gene (40%) while no isolate carry rmpA gene. The variation of these two genes’ existence among K. pneumonia clinical isolates has been reported and the absence incidence are documented (Pinsky et al. (2009), Sharma et al. (2013), Wen-Liang et al. (2015)). On the other side, high detection of magA and rmpA (77.8%) was reported by Osman et al. (2014) rmpA (45.7%) and Tan et al. (2019). Hartman et al. (2009), Turton et al (2010) and Lev et al. (2018) have demonstrated a moderate (25%) ratio of rmpA gene detection.

In conclusion, K. pneumoniae may become a significant pathogen among farmed animals such as camels with the emergence of multidrug resistance that may constitute public health concern. A special vigilance is required to avoid camels becoming a possible reservoir for K. pneumonia.

This research received no external funding.

As per CPCSEA guidelines, ethical approval was not needed as the study included clinical and postmortem samples.

The authors have declared no conflict of interest.