Enterobacter hormaechei Bacteria from Paper Napkins Confirmed by 16S rRNA Gene and Experimental Infection in Rats

Only a small portion of the microorganisms present in the environment of paper-processing technologies (those that create toxins and bad tastes) are detrimental to the operation or the security of the finished goods. Microbes typically enter machinery through the raw ingredients and chemicals used to produce paper. They usually produce biofilms on a variety of surfaces and flourish in the conditions of the paper mill. Circulating process water is a tried-and-true technique for preserving and distributing microbial contamination in mills, claim (Suihko and Skytta, 1997). The bacteria that produce enzymes and toxins could cause issues if they diffuse from the paper into food and skin scrapes (Swartz, 2004; Suominen et al., 1997).
       
Microbiological quality is an important consideration in risk assessment due to disposal fibers providing a favorable environment for bacteria development when materials are collected, storedand processed (Blanco et al., 1996; Sorelle and Belgard, 1991). Although even though the vast majority of food sellers were aware of safe food handling procedure, they were not properly followed when preparing food, which lead to increased levels of microbial contamination in their produce (Krishnasree et al., 2018).
       
The species of the Enterobacter cloacae complex includes a large variety of bacteria that have been widely seen in numerous circumstances and have the potential to act as human pathogens (Mezzatesta et al., 2012). These microorganisms are members of the Enterobacteriaceae family, which has been linked to the colonization of the human respiratory system and has the potential to promote pathological progression leading to recurrent lung infections (Abbas et al., 2020). They are also regarded as frequent nosocomial infections (Gaston, 1988) capable of causing various diseases, such as bacteremia, septicemia, pneumonia, cystitis, hospital-acquired bacteremiaand injury contagions (Xiong et al., 2008). These creatures, which today belong to the species Ent. hormaechei, Ent. dissolvens, Ent. cloacae, Ent. kobei and Ent. ludwigii were discovered to contain highly heterogeneous genomes. In clinical instances, E. hormaechei emerged as the most frequently recognized species (Hoffmann and Roggenkamp, 2003; Morand et al., 2009; Boone and Castenholz, 2001).
       
For reproducible molecular typing approaches for identification, open, trustworthy and cumulative datasets must be available (Paauw et al., 2009; Abhyankar et al., 2022). This information suggests that both animal and human health may be at risk from the possible spread of this strain. High-quality identification data bases used to be rare, but they were sometimes available for gene sequences encoding particular housekeeping genes.
       
Numerous environmental niches contain E. hormaechei (Davin-Regli et al., 1997). It is frequently regarded as a pathogen that causes nosocomial infections (Paauw et al., 2009) and it seldom affects animals outside humans. Animals with respiratory illness complex (Khalifa et al., 2021) also a dead fox with an infection in its uterus (Shan-Shan et al., 2017), diarrheal piglets Lu-Yao et al., (2017), respiratory disease in calves, goats 0 and sheep (Wang et al., 2020), fish pathogen (Nallaiah et al., 2022), combination of respiratory diseases in fauna (Khalifa et al., 2020) have all tested positive for E. hormaechei infections. E. hormaechei was just recently identified as the pathogen responsible for pathological alterations in sheep in 2022 (Hongfei et al., 2022).
       
The goal of this investigation was to detect isolates coming from regular paper napkins and to build a database for personal identification. The later commercial database is more reliable, but it is still restricted to a small number of species that are important for medical and food microbiology and environmental isolates like those from the primary material of the paper industry are not present. We first isolated E. hormaechei to experimentally infected and determine the pathogenicity of E. hormaechei in rat newborns and adult rats by two ways of infection.

Ethical approval
 
The ethical approval for the current experiment was obtained from the ethics committee of Veterinary Medicine at Al-Qasim Green University and approved to guide the care and use of laboratory animals (ESCVM, No. 1192022).
 
Samples and isolation
 
The experiment was conducted during the period from of September 2022 to February 2023 at the AL-Qasim green university, Bacteriology department laboratory led to the isolation of 50 aerobic bacterial isolates. Samples from recycled paper napkin (Dima)®,  were taken from a factories in Hillah , Babil province, Iraq. A 0.2 mg random sample of each napkin was homogenized in a Stomacher for 1 minute after being combined with a Peptone water tube solution for the culture of the bacteria for 12 hours. For 24 hours, MacConkey agar and Nutrient agar were cultured and incubated at 30°C after receiving a trans-loopful of the solution. By serially diluting 10-fold, you can determine the quantity of colony-forming units and their corresponding concentrations. For the molecular lab, the isolates were kept at 20°C in 5% glycerol.
 
Physiological and morphological characteristics
 
The 50 E. hormaechei isolates came from paper napkins. Biochemical testing was performed using the Vitek 2 Compactand morphological (cell size and shape, pigment production and Gram reaction) studies were conducted through culture.
 
PCR reactions and analysis of the 16S rDNA sequence
 
For three isolates of E. hormaechei, Primer sets 143 are for general use (5-AGAGTTTGATCATGGCTCAG-3) and DG 74 is for specific use (5-ACGGTTACCTTGTTACGACTT-3) (Weisburg et al., 1991). “30 cycles of initial denaturation at 94°C for two minutes, annealing at 56°C for thirty seconds, extension at 72°C for four minutes and a final extension at 72°C for ten minutes were performed after the initial denaturation at 94°C for five minutes”. The amplified products were treated in a 1X TBE buffer before being electrophoretically stained on a 1.5% agarose gel with RedSafe Nucleic Acid Stain.
       
According to the primer synthesizer company’s instructions, the primers (which were initially lyophilized) and the amplified fragment were directly sequenced using the Sanger sequencing method (Macrogen, Korea).
       
Using the G-spin Genomic DNA Kit, total DNA was extracted from the strain that had been isolated after 20 hours at 37°C in LB broth for three isolates. Following the established methods (Martínez et al., 2014), an Opti-DNA marker gene was used in order to run the previously released polymerase chain reaction (PCR) primers (Poirel et al., 2011).
       
The partial region of 16S rRNA of E. cloaca is 353 bpand the consensus sequence was used to search for homologous sequences at Gen Bank.
 
Experimental infection
 
In group one, there are eight male baby rats (2 weeks to 28 days old, weighing 21-35 g). Using the intranasal grip, uniformly distribute 10-20 microliters of E. hormaechei suspension (2×10⁷ mg/ml CFU) into the nose. Allow the inoculum to flow gradually, allowing the rat enough time to breathe in the inoculum (Southam et al., 2002). Eight adult female rats (2 months post-partum, weighing 63-95 g) are given a volume dose of 0.5 ml of bacterial suspension intra vaginally in group two, while the third group is the control group. Following infection, check the rat every 24-72 hours for medical conditions that cause signs including fatigue, frizzy hairand weight loss.
       
According to (Abbas et al., 2012) the lungs and uterus of the experimental animals were dissected into histopathological sections using the H and E stain.

The group from which all 50 isolates were cultivated for 24 hours at 30°C on MacConkey agar was identified as Enterobacter hormaechei. With a high growth rate, the resulting colonies were round, smooth, pinkand had regular edges that were 1-2 mm in diameter, as shown in Fig 1.
 

       
The number of colony-forming units is 2 × 10⁷ mg/ml CFU concentrations. A Gram stain was used to identify these colonies after they had been cleansed and it revealed red bacilli. The name N.L. gen. Nov. hormaechei honors Estenio Hormaeche, a scientist from Uruguay who co-founded and characterized the genus Enterobacter with P.R. Edwards and Hormaeche (1960). Nimbalkar et al., (2022) reported that E. hormaechei is also known to produce siderophores.

The target 16S rRNA fragment was found in the gDNA of the three E. hormaechei isolates that demonstrated Safe-Green (353 bp) amplification, as shown in Fig 2. The isolated strain utilized in this study is listed in NCBI-Gen Bank submissions as E. hormaechei strains IE1, 2and 3and the 16S rRNA gene sequence is designated by the accession number MN032348.1.
 

       
Sequences of the type strain’s 16S rRNA and strains of E. hormaechei and E. cloacae are 100–99% similar. To determine the links between the detected Enterobacter sp. strain and the published sequences with the highest homologies, a phylogenetic analysis was performed. The linked taxa clustered together in the bootstrap test with 101 repetitions. The strain of Enterobacter sp. can be categorized as E. hormaechei based on this phylogenetic study (Table 2).
      
E. hormaechei strains are described as fermentative, non-pigmented, gram-negativeand oxidase-negative rods that share traits with members of the genus Enterobacter and the family Enterobacteriaceae (Table 1). Table 1 provides a summary of the isolate’s biochemical characteristics. The microscopic appearance and morphology of colonies on growth media, as well as biochemical studies, were used to determine that the isolated bacterial strain belonged to the family Enterobacteriaceae, which displayed a pattern similar to E. hormaechei as O’Hara et al., (1989) reported and Hoffmann et al., (2005) indicated motility in SIM-agar at 37°C. The results of the biochemical assays supported the diagnosis of E. hormaechei. Even still, the urease result was uncommon because 99% of the strains were reported to have positive outcomes.
       




The development of molecular techniques enables the study of a sizable portion of the bacterial genome. Because the PCR technique is faster at identifying the type of bacteria present in milk samples, (Vatalia et al., 2020) utilized it to identify Enterobacter spp. as one of the leading mastitis pathogens. The most often used molecular technique in taxonomic investigations is the analysis of variable sections of bacterial 16S rRNA gene sequences according to Ibal et al., (2019). Reclassification of bacteria may be feasible by using this molecular sequence to create phylogenetic trees based on changes in nitrogenous bases between species. Similar finding was reported Sabaa et al., (2023) and Jenkins et al., (2012).
       
Three to five days after colonization, the pathogenesis causes rats to exhibit disease signs, which are preceded by weight loss. The rats will adopt slumped positions and exhibit less activity and reactivity to stimulation, including handling, as their condition worsens. Since blood was used to isolate the germs, sepsis and/or pneumonia are usually indicators of sickness at this stage.
       
Histopathological examination of infected rat lung revealed thickening of interalveolar septa with edema and infiltration of mononuclear cells, as well as diffuse edema filling the alveolar lumen with thickening of interstitial tissue and proliferation of bronchial-associated lymphoid tissue (Fig 3 (b, c and d), respectively, compared with Fig 3 (a) This demonstrates the lung’s typical anatomy.
 

 

       
Moreover, the intestinal histopathological section exhibits destruction and necrosis of intestinal villi with infiltration of lymphoplasmacytic cells in lamina properia (Fig 4).
 

       
While uterine histopathological sections show widespread hemosiderin pigment accumulation in the endometrial layer, diffuse infiltration of inflammatory cells with vacuolation of epithelial cells in the endometrium and myometrium layers, as well as focal necrosis of endometrium epithelium cells with degeneration of endometrium glands, respectively [Fig 5 (a, b and c)] and additionally the presence of atresia of oviduct epithelia (Fig 6).
 

 

       
Histological features of infected rats’ lungs observed alveolar septum thickening and mononuclear infiltration (Fig 3) were also recorded in Hongfei et al., (2022) and Zhicheng et al., (2020) the lungs of dead sheep, alveolar septal thickening and bronchial epithelial cell destruction, the release of mucus and the presence of neutrophils were seen. Similar to this, in Carvalho-Assef et al., (2014), Lung histopathology samples that contained the Enterobacter hormaechei strain showed thickened alveolar septa, erythrocyte displacement and inflammatory cell invasion along the pulmonary alveolar septa.
       
Fig 4 the intestine of an infected rat gives destructions and necrosis like in a piglet’s intestinal illness has been documented, Additionally, only one uterine infection led to the isolation of E. hormaechei from the uterine secretions of a dead fox, despite the fact that few investigations of E. hormaechei are connected to infection in animals also reported by Shan-Shan et al., (2017); Paauw et al., (2009) and Lu-Yao et al., (2017). E. hormaechei was also isolated from blood after infected rat. According to Daurel et al., (2009); Giammanco et al., (2011); Pereira et al., (2015); Rafferty et al., (2011); Sampaio et al., (2014) and Yang et al., (2018) Injuries, tissues, organsand body fluids of patients can all contain E. hormaechei.

The study’s findings imply that the presence of Enterobacter hormaechei in recycled napkins is a sign of contamination and that the organism can infect an organ and cause sickness.