The pathogenesis of Klebsiella pneumoniae in respiratory tract infections has been widely investigated in several studies [12, 13, 17,18,19]. It is an opportunistic pathogen causing both community-acquired and nosocomial infections [20], ranging from mild urinary tract infections to severe pneumonia with high morbidity and mortality rates [21,22,23]. Various studies have investigated the role of gender on the infectivity of K. pneumoniae or other pneumonic pathogens on animal models of pneumonia, such as mice (Swiss albino, C57BL/6, BALB/c, NMRI, DBA/2, Swiss Webster, etc.) and Sprague Dawley rats [2, 8, 14, 24, 25]. However, previous studies show that these investigations have not been carried out on the Wistar rat model, the experimental model most used for drug discovery studies in tropical areas and, particularly in sub-Saharan Africa due to the ease of this rodent species to acclimating and adapting to environmental conditions.

Especially, the K. pneumoniae strains have been used to induce pneumonia in the sus-mentioned animal models with determinate pathogenic characteristics [2, 12, 26]. However, despite the progress in understanding the pathogenesis of this germ, the preliminary research hasn’t focused on understanding the gender impact on the standardisation of the physiopathogenesis of K. pneumoniae ATCC 43816 in Wistar rats’ model when used for lobar pneumonia induction. Therefore, this study aimed at the sex-dependent vulnerability of Wistar rat’s respiratory tract infection model using intranasal instillation of K. pneumoniae ATCC 43816. Thus, the evaluated parameters in pulmonary tissues and whole blood of K. pneumoniae ATCC 43816-infected groups were significantly pathological when compared to the control group, consistent with findings from previous studies [2, 12,13,14, 27].

Different inoculum doses were evaluated to determine the optimal K. pneumoniae ATCC 43816 dose for inducing pneumonia in Wistar rats without causing death. Among the doses tested (1.5 × 105, 1.5 × 106, 1.5 × 107 and 1.5 × 108 CFU/mL), only the animals inoculated with 1.5 × 108 bacteria/mL showed significant lethality in the female group starting from day 12 of inoculation. The optimal dose for inducing stable lobar pneumonia was found to be 1.5 × 106 CFU/mL for female Wistar rats and 1.5 × 108 CFU/mL for male Wistar rats. Animals receiving these doses exhibited a loss of 10% to 12.5% of their initial body weight, indicating the development of lobar pneumonia. In comparison, Vivek-Kumar et al. reported an optimal infectious dose of 1 × 106 CFU/mL K. pneumoniae MTCC 109 inducing stable pneumonia in a non-specified male rat model [2]. It is interesting to note that while this result approximately equals the optimal dose that we report in this study for female Wistar rats, it’s a hundred lower than the optimal dose we found for male rats.

Determination of the LD50 of K. pneumoniae ATCC 43816 inoculum revealed its infective role in pneumonia induction in both male and female Wistar rats, with a lower LD50 observed in females. It thus appears that K. pneumoniae ATCC 43816 could be more pathogenic and virulent in female rats compared to males. These results align with reports in other previous studies using female C57BL/6 mice inoculated with the pneumonic pathogen W. chondrophila [28, 29]. Further similar results were found in studies using various strains of pneumonic pathogens and experimental animal models [2, 9, 29, 30].

It is worth noting that the intranasal instillation technique used in this study has limitations, such as variability in the volume of lung deposition among different animals testing, as some bacteria may remain in the upper respiratory tract, get exhaled, or end up in the digestive tract. Hence, residual bacteria could be either stay in the upper respiratory tract, exhaled, or deviated into the digestive tract [31]. Interestingly, the same evolution of K. pneumoniae ATCC 43816 load in lung tissues, as attested by Fig. 1C in the male rats depending on the inoculated doses and inoculation time, was similar to that of the females (Fig. 1D), demonstrating the model’s robustness and the reproducibility of this study’s, and highlighting a low variability of K. pneumoniae ATCC 43816 lung deposition. No bacteria were detected in the blood samples of both genders on day 5 post-infection for the lowest doses of 1.5 × 105 and 1.5 × 106 CFU/mL. This could be attributed to bacterial exhalation or the phagocyte action of polymorphonuclear cells like neutrophils and alveolar macrophages recruited to the inflamed lung tissues. In contrast, other studies have reported a significant increase in blood bacterial loads with the same range of doses within 6 h to 1 day post-infection with Gram-negative bacteria, reaching 4.05 log10 to 10 log10 bacteria/mL in blood samples [2, 12, 25].

In response to this infection, we observed that the K. pneumoniae ATCC 43816 proliferation triggers intense inflammation characterized by a disruption of endothelial permeability [30, 32], resulting in purulent fluid release, parenchymal oedema, and a massive influx of polymorphonuclear cells recruited by diapedesis towards the conjunctive inflamed tissues [32]. The gender dependence on K. pneumoniae ATCC 43816-induced pulmonary infection is further elicited by histomorphometry images that we used to obtain a spatiotemporal evaluation of inflammatory granulomas and pulmonary oedema caused by the pathogen’s virulence. Indeed, the appearance, amplification and/or regression of the inflammatory manifestations could be observed through the evolution of granuloma areas and histomorphometry of inflammatory granulomas in pulmonary parenchymas during the inoculation period. The quantification of this pathogenic evolution through the graphic representations of granuloma areas (Figs. 2 and 4), indicated the regression of inflammatory granulomas in male rats receiving the inoculum doses of 1.5 × 105 and 1.5 × 106 CFU/mL on days 15, and 1.5 × 107 CFU/mL on days 10 post-inoculation. However, male rats receiving the highest dose of 1.5 × 108 CFU/mL and females across all doses exhibited more regular, stable temporal and spatial evolution in inflammatory granuloma appearance during K. pneumoniae ATCC 43816 pneumonia, reflecting an aggravation of the pneumonic infection through the inoculation time. These results could be interpreted as an indication of a greater susceptibility of Wistar female rat vis a vis the pathogenicity of K. pneumoniae for the tested infecting doses.

Gender differences in immune function have been well-established in humans and animals [8, 9]. In animal models of respiratory infections, sex has been shown to influence susceptibility and severity of the pneumonia disease, however in inconsistent results [8]. This inconsistency may vary depending on the family or specie of the pneumonic microorganisms used. The results obtained in this study suggest that the examined parameters promoting the pathogenesis of K. pneumoniae ATCC 43816 pneumonia in Wistar rats confirm the gender/sex-hormones duality rule. It’s been shown that the impact of sex on K. pneumoniae pneumonia pathogenesis always aligns with the influence of sex hormones [9, 10]. Furthermore, males generally exhibit weaker humoral and cell-mediated immune responses compared to females as corroborated by a similar report to this study, showing that female mice tend to be more susceptible to lung infection than males in strains of Pseudomonas aeruginosa, a Gram-negative bacteria [33]. Female animals have also been shown to display greater weight loss and significant bacterial load, showing a more vigorous inflammatory response in the lungs than males [8, 33]. However, contrasting results exist showing that male animals could develop more severe inflammatory manifestations than females following pulmonary pathogenic infection. For instance, Carey et al. have shown that male mice develop more severe granulomatous lung lesions than females following infection with Mycobacteria as Mycobacterium marinum or Mycobacteria intracellulare [8].

These contrasting results give an indication of what could guide the relationship between the animal’s gender and the type and character of specific pro-inflammatory responses. Indeed, based on a general tendency, it has been proposed that males generally exhibit weaker humoral and cells mediated immune responses compared to females [9, 34]. During these pro-inflammatory processes, males would produce an acute alveolar inflammatory response, characterised by predominantly neutrophilic infiltration, oedema and haemorrhage. Females, on the other hand, initiate a chronic peribronchial inflammatory response characterised by mononuclear cell and neutrophil infiltration [10, 34]. It has also been shown that sex hormones and their metabolites (e.g. oestrogen, estradiol, estrone, estriol, progesterone) seem to directly or indirectly influence pro-inflammatory processes and host resistance to the infectious agent [9, 10, 34]. Similarly, Fuseini and Newcomb also showed that oestrogens and their metabolites can trigger lung pro-inflammatory, while male hormones, such as testosterone, usually plays the opposite role [35]. In the case of lower respiratory tract infections, specifically by K. pneumoniae pneumonia, these sex hormones induce the overproduction of pro-inflammatory mediators and secretory hyperactivity, which consequently cause aggravation and greater severity of the pathology in female Wistar rats compared to males [9, 33, 34].

Nevertheless, our results show that high infectious doses of K. pneumoniae ATCC 43816 (≥ 1.5 × 108 CFU/mL) would over-rule gender and, humoral and cells-mediated influences, over-activating the immune system by the excessive and uncontrolled recruitment of pro-inflammatory mediators in both males and females and leading to the installation of severe pneumonia.

Finally, in relation to the animals’ body weight changes, this study has found no relationship with the animals’ gender. However, a regular increase in bacteraemia, along with a regular increase in the surface of inflammatory granulomas seemed to be accompanied by an inoculum dose-dependent decrease in the animal’s body weight. These results are corroborated by previous studies by Pilloux et al., showing that female C57BL/6 mice infected with W. chondrophila, showed a significant body weight loss (p < 0.05) along with the appearance and aggravation of the pneumonia disease [14]. Thus, the installation of severe pneumonia induced by K. pneumoniae ATCC 43816 seems to be accompanied by a body weight loss, notwithstanding the animals’ gender.