Isolation, characterization, therapeutic and prophylactic applications of a lytic bacteriophage to combat multi-drug resistance Shigella flexneri: an animal study model

dgkh000543 10.3205/dgkh000543 urn:nbn:de:0183-dgkh0005436 Research Article Isolation, characterization, therapeutic and prophylactic applications of a lytic bacteriophage to combat multi-drug resistance Shigella flexneri: an animal study model Isolierung, Charakterisierung, therapeutische und prophylaktische Anwendung eines lytischen Bakteriophagen bei der Bekämpfung der multiresistenten Shigella flexneri: Ein Tierversuchsmodell Abbasi Fashami Abbasi Fashami Parisa P

Infectious Diseases and Tropical Medicine Research Center, Babol University of Medical Sciences, Babol, Iran

author Pournajaf Pournajaf Abazar A

Infectious Diseases and Tropical Medicine Research Center, Babol University of Medical Sciences, Babol, Iran

author Amirmozafari Amirmozafari Nour N

Department of Microbiology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran

author Hallajzadeh Hallajzadeh Masoume M

Department of Microbiology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran

author Mahabadi Mahabadi Vahid Pirhajati VP

Neuroscience Research Center, Iran University of Medical Sciences, Tehran, Iran

author Saghiri Saghiri Reza R

Department of Biochemistry, Pasteur Institute of Iran, Tehran, Iran

author Khafri Khafri Soraya S

Department of Social Medicine, Babol University of Medical Sciences, Babol, Iran

author Golmoradi Zadeh Golmoradi Zadeh Rezvan R

Department of Microbiology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran

author Akrami Akrami Sousan S

Department of Microbiology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran

author Asgharzadeh Asgharzadeh Sajjad S

Department of Microbiology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran

author Rajabnia Rajabnia Mehdi M PhD

Infectious Diseases and Tropical Medicine Research Center, Babol University of Medical Sciences, Babol, Iran, Phone: +98 11 32190624Infectious Diseases and Tropical Medicine Research Center, Babol University of Medical Sciences, Babol, Iran

rajabniamehdi549@gmail.com author German Medical Science GMS Publishing House

Düsseldorf

610 S. flexneri lytic bacteriophage dysentery therapeutic option S. flexneri lytische Bacteriophagen Dysenterie therapeutische Option 20250430 engl This is an Open Access article distributed under the terms of the Creative Commons Attribution 4.0 License. Dieser Artikel ist ein Open-Access-Artikel und steht unter den Lizenzbedingungen der Creative Commons Attribution 4.0 License (Namensnennung). 2196-5226 20 GMS Hygiene and Infection Control GMS Hyg Infect Control 14 Babol University of Medical Sciences Hintergrund: Shigella (S.) flexneri ist eine der wichtigsten Krankheitsursachen bei Kindern mit Dysenterie im Iran. Bakteriophagen sind eine aussichtsreiche Option, um das Problem der Arzneimittelresistenz bei Krankheitserregern zu lösen. Dementsprechend zielt die vorliegende Studie darauf ab, lytische Bakteriophagen von S. flexneri zu isolieren und ihre Auswirkungen auf die Verringerung der Ausscheidung von Shigella in Mausmodellen mit S. flexneri Diarrhoe zu untersuchen.Methode: Es wurde S. flexneri ATCC 12022 verwendet. Die Identifizierung der aus Krankenhausabwässern isolierten Phagen erfolgte mit einem TEM-Elektronenmikroskop. Es wurden Stabilitätstests durchgeführt, um die Empfindlichkeit der isolierten Phagen gegenüber verschiedenen Faktoren wie Temperatur, pH-Wert und Gallensalzen zu bestimmen. Um die Sicherheit und Wirksamkeit der Bakteriophagen bei der Reduzierung von Shigellen im Stuhl zu gewährleisten, wurden männliche syrische Mäuse (C57) im Alter von 6 Wochen und einem Gewicht von 22–25 g verwendet. Die Behandlung mit Phagen erfolgte: (I) 1 h vor, (II) 1 h nach, (III) 5 h nach und (IV) 1 h vor +1 h nach der bakteriellen Infektion.Ergebnisse: Die in der Studie verwendeten Bakteriophagen gehören zur Familie der Myoviridae. Die Verabreichung einer Dosis des Bakteriophagen vor der Infektion kann eine Verbesserung des Verlaufs nach der Transfektion bewirken. Die Verabreichung von Bakteriophagen nach der Infektion hat einen therapeutischen Einfluss.Schlussfolgerung: Die Untersuchung des Bakteriophagen unter In-vivo- und In-vitro-Bedingungen zeigt, dass er eine vollständige Lyse von S. flexneri bewirkt. Dementsprechend kann der Phage eine potenzielle therapeutische Option für die Behandlung der durch multiresistente S. flexneri verursachten Diarrhoe sein. Background: Shigella (S.) flexneri is one of the most important causes of</PlainText></TextGroup> disease in children with diarrhea in Iran. Today, bacteriophages are an attractive option to resolve the drug resistance problem among pathogenic agents. Accordingly, the present study aimed to isolate a lytic bacteriophage of <Mark2>S. flexneri</Mark2> and investigate its impact on reducing excretion of <Mark2>Shigella</Mark2> in mouse models suffering from bacillary dysentery.</Pgraph><Pgraph><Mark1>Method:</Mark1> <Mark2>S. flexneri</Mark2> ATCC12022 was used. Identification of the phage isolated from hospital wastewater was performed using Transmission Electron Microscopy (TEM). Stability tests were performed to determine the sensitivity of isolated phages to various factors such as temperature, pH and bile salts. A male Syrian mouse model (C57), with mice 6 weeks of age weighing 22–25 g, was used to ensure safety and efficacy of the bacteriophage in reducing <Mark2>Shigella</Mark2> in stool. Treatment with the phage was performed (I) 1 h before, (II) 1 h after, (III) 5 h after, and (IV) 1 h before +1 h after bacterial challenge. </Pgraph><Pgraph><Mark1>Results:</Mark1> TEM indicated that the bacteriophage used in this study belongs to the Myoviridae family. Administration of one dose of bacteriophage before the infection can accelerate improvement post-transfection, and administration of bacteriophage post-infection has a therapeutic influence. </Pgraph><Pgraph><Mark1>Conclusion:</Mark1> <Mark2>In vivo</Mark2> and <Mark2>in vitro</Mark2> results indicate that our bacteriophage causes complete lysis of <Mark2>S. flexneri</Mark2>. Thus, this phage could be a therapeutic option for treating bacillary dysentery resulting from multidrug-resistant <Mark2>S. flexneri</Mark2>.</Pgraph></Abstract> <TextBlock name="Introduction" linked="yes"> <MainHeadline>Introduction</MainHeadline><Pgraph>Diarrhea resulting from bacteria, viruses, and parasit<TextGroup><PlainText>es i</PlainText></TextGroup>s a public health concern. Among diarrhea-causing pathogens, <Mark2>Shigella</Mark2> spp. play a significant role in developing bloody and inflammatory diarrhea <TextLink reference="1"></TextLink>. For instance, <Mark2>Shigella flexneri</Mark2> is one of the most important causes of this disease among children suffering from diarrhea in Iran <TextLink reference="2"></TextLink>. <Mark2>Shigella</Mark2> pathogenesis is based on the bacterial ability to attack and proliferate in the colon’s epithelium, causing severe inflammation and epithelial degeneration. More than 164 million cases are reported annually, with 1.<TextGroup><PlainText>1 m</PlainText></TextGroup>illion mortalities, in response to shigellosis in developing countries; most patients are children under five years of age <TextLink reference="3"></TextLink>. Most <Mark2>Shigella</Mark2> infections respond to antibiotic treatment, but increasing consumption of antibiotics has led to the emergence and spread of antibiotic-resistant strains, which has led to the development of multi-drug resistance, making it a serious concern of the World Health Organization <TextLink reference="4"></TextLink>. Thus, finding novel alternatives for infection control is essential. Bacteriophages (or phages) specifically attack bacterial cells, causing their death; however, they do not affect human or animal cells <TextLink reference="5"></TextLink>. Phage therapy involves the targeted usag<TextGroup><PlainText>e of b</PlainText></TextGroup>acteriophages for treating infections resulting from bacteria, which in case of interacting with the target bacteria, can infect and kill them <TextLink reference="5"></TextLink>. Unlike antibiotics, the approach of phages is limited and does not disrupt the normal equilibrium of the intestinal microflora <TextLink reference="6"></TextLink>. That is, they only cause minor damage to the beneficial bacteria of the body, such as intestinal and lactic-acid bacteria <TextLink reference="7"></TextLink>. Meanwhile, phages function in a constrained way, and after killing the harmful bacteria, they enter the inactive phase of their lifecycle and show virtually no activity against non-host bacteria. They are not allergens and have only minor side effects <TextLink reference="8"></TextLink>, <TextLink reference="9"></TextLink>. Considering the therapeutic properties of phages, the present study aims to isolate specific lytic bacteriophages of <Mark2>S. flexneri</Mark2> and explore their impact on reducing <Mark2>Shigella</Mark2> excretion in a mouse model suffering from bacillary dysentery.</Pgraph></TextBlock> <TextBlock name="Materials and methods" linked="yes"> <MainHeadline>Materials and methods</MainHeadline><Pgraph>This cross-sectional study was performed within a 1<TextGroup><PlainText>0-m</PlainText></TextGroup>onth period from May 2020 to February 2021 and was approved by the research ethics committee of Babol University of Medical Sciences (code IR.MUBABOL.REC.1399.101).</Pgraph><SubHeadline>Source of S. flexneri and isolation and purification of the phage</SubHeadline><Pgraph><Mark2>S. flexneri</Mark2> ATCC 12022 was obtained from the Microbiology Department of IUMS (Iran University of Medical Sciences), Tehran, Iran. To isolate the specific lytic phage of <Mark2>S. flexneri</Mark2>, the typical isolation protocol was used with some modifications <TextLink reference="6"></TextLink>. A wastewater sample was collected from the first basin of the Motahari hospital wastew<TextGroup><PlainText>a</PlainText></TextGroup>ter treatment plant and utilized as the source of the phage. All samples were collected after centrifuging at 2,500×g for 20 min, and then filtered through a syringe filter (45.0 µm). Then, 10 ml of the bacteria in the logarithmic phase (4-h fresh culture) was added to 10 ml of treated wastewater as well as 10 ml Brain Heart Infusion (BHI) culture medium with double concentration (2×). The mixture was incubated overnight in a shaker incubator at 37°C and 90 rpm. Then, the suspension was centrifuged and the supernatant was filtered (45.0 µm). The presence of the specific lytic phage in the suspensi<TextGroup><PlainText>on w</PlainText></TextGroup>as explored using the double-layer agar (DLA) method. Em<TextGroup><PlainText>e</PlainText></TextGroup>rgent transparent plaques were considered to demonstrate the presence of the lytic phage of <Mark2>S. flexneri</Mark2>. A plaque was chosen, and the purification method was performed three times consecutively. The phage was typically replicated according to the protocol of Sambrook and Russell <TextLink reference="10"></TextLink>.</Pgraph><Pgraph>To determine the titer of phages, pure and filtered lysates of phages were used to prepare serial dilutions, after which DLA was used. The obtained plaques were counted, whereby the titer of phages was calculated according to the following equation:</Pgraph><Pgraph>pfu/ml=number of plaques/dilution factor × phage.</Pgraph><Pgraph>To eliminate the residuals of bacteria and to obtain a more concentrated phage sample, the phage suspension (43.3×10<Superscript>11</Superscript> pfu/ml) was centrifuged (Beckman optima XL-90 Ultracentrifuge) at 4,000×g for 120 min; thereafter, the residue was dissolved in SM buffer. Then, the phage suspension was placed in a copper grid with carbon coating (Ted Pella Inc, USA), after which negative staining was performed with phosphotungstic acid (PTA) 2%. Finally, the sample was examined using transmission electron microscopy (TEM; Zeiss LEO 906 (Germany) <TextLink reference="11"></TextLink>.</Pgraph><SubHeadline>Stability studies</SubHeadline><Pgraph>To determine the sensitivity of the isolated phages, stability tests were performed with some modifications. To determine the stability at different temperatures, lysates (diluted at a 1:100 ratio) were exposed to 40–80°C for 60 min, and to –20°C as well as –80°C for 48 hours. To evaluate their stability, the different titers were deplaqued using the DLA method and the phage changes were compared against the stock sample <TextLink reference="12"></TextLink>.</Pgraph><Pgraph>To evaluate the stability at different pHs, different amounts of 1 n NaOH and 1 n HCl were added to the BHI medium to obtain the desired pH (1–3, 5–7, 9–11). Next, 100 µL of the phage suspension diluted at a 1:100 ratio was added to 900 µL of each BHI medium with a different pH. The samples were incubated for 1 hour or 16 hours in the incubator at 37°C. Thereafter, to evaluate their stability by DLA method, plaques were obtained from their different titers, and phage changes were compared with the original titer <TextLink reference="11"></TextLink>, <TextLink reference="13"></TextLink>.</Pgraph><Pgraph>To evaluate the stability of the phage in bile salt, BHI medium containing 1% and 2% concentrations of oxgall powder (bovine bile, Sigma-Aldrich, Germany) was used. 100 µL of the phage suspension diluted at a 1:100 ratio was added to 9.9 mL of medium containing 1% or 2% bile, and then incubated at 37°C for 1 or 3 hours. After the incubation, the phage suspension was prepared as a serial dilution and phage survival was examined using the double-layer agar technique <TextLink reference="14"></TextLink>.</Pgraph><SubHeadline>Determination of phage adsorption extent</SubHeadline><Pgraph>The bacteria were cultured in two flasks and incubated at 37°C until reaching OD<Subscript>600nm</Subscript>=0.2–0.5 nm. Phage diluted at a 1:100 ratio was added to one of the flasks with the same amount of bacterial culture. The second flask with no phages was used as the control. Both flasks were placed inside a shaker-equipped incubator at 37°C and 130 rpm. Using a spectrophotometer (Beckman Coulter), OD<Subscript>600nm</Subscript> was measured for each flask once every 10 min for 2 hours <TextLink reference="11"></TextLink>, <TextLink reference="15"></TextLink>.</Pgraph><SubHeadline>One-step growth curve</SubHeadline><Pgraph>The host bacteria were incubated in the BHI culture m<TextGroup><PlainText>edi</PlainText></TextGroup>um until reaching the logarithmic phase (OD<Subscript>600</Subscript><TextGroup><Subscript>nm</Subscript><PlainText>=0.4–0.5 nm</PlainText></TextGroup>). 6 mL of 4-hour bacterial culture plus 6 mL phage suspension diluted at a 1:100 ratio was incubated for 15 min inside a shaker incubator at 37°C. Thereafter, the suspension was centrifuged for 10 min at 10,000×g to precipitate the bacteria. The resulting sediment was washed three times with normal saline and transferred to a falcon tube. All falcon tube were placed inside the shaker incubator at 37°C and 130 rpm. 1,00<TextGroup><PlainText>0 µ</PlainText></TextGroup>L was withdrawn from each falcon tube and centrifuged every 10 min for 2 hours, then passed across a 45.0-µm filter. Ultimately, after preparing the serial dilutions, each sample was used to investigate the plaque formation using the DLA method.</Pgraph><SubHeadline>Effect of the isolated phage on S. flexneri in vivo</SubHeadline><Pgraph>To prepare the bacterial strain required for the formation of bacillary dysentery disease in the mice, <Mark2>S. flexneri</Mark2> spp. ATCC 12022 was incubated in BHI medium until reaching OD<Subscript>600nm</Subscript>=0.8–1 nm in a shaker incubator at 37°C at 15<TextGroup><PlainText>0 r</PlainText></TextGroup>pm. To prepare the bacteriophage for treatment, phage lysates were centrifuged at 4,000 rpm for 30 min and then filtered (22.0 µm).</Pgraph><Pgraph>As the model host, male Syrian mice C57 (6 weeks old, weight 22 g±3.2 g), acquired from the IUMS experimental study center, were used. The mice were fed freely and routinely, and had access to fresh water around the clock. The animal studies were conducted bas<TextGroup><PlainText>ed on the gu</PlainText></TextGroup>idelines of the Ethics Committee for Animal Experimentsat IUMS. </Pgraph><Pgraph>To evaluate the effectiveness of phage therapy, the mice were examined in five groups: </Pgraph><Pgraph><OrderedList><ListItem level="1" levelPosition="1" numString="1.">Group I: The prophylaxis group received phage 1 h before bacterial force-feeding; </ListItem><ListItem level="1" levelPosition="2" numString="2.">group II: treatment group in which the phage was administered after transfection with <Mark2>Shigella</Mark2>; </ListItem><ListItem level="1" levelPosition="3" numString="3.">group III: treatment group which received phage <TextGroup><PlainText>5 h</PlainText></TextGroup>ours after bacterial force-feeding; </ListItem><ListItem level="1" levelPosition="4" numString="4.">group IV: treatment group which received phage <TextGroup><PlainText>1 h</PlainText></TextGroup>our before and 1 hour after bacterial force-feeding; </ListItem><ListItem level="1" levelPosition="5" numString="5.">group V: control group (received bacteria only) <TextLink reference="16"></TextLink>. </ListItem></OrderedList></Pgraph><Pgraph>200 µL bacteria and an equal amount (200 µL) of phage were force-fed to the mice. In group IV, 100 µL <TextGroup><PlainText>phage was</PlainText></TextGroup> force-fed before and 100 µL after bacterial force-feeding. Separate sterile needles were used for bacterial and phage force-feeding in order to prevent confounding results. </Pgraph><SubHeadline>Colony count</SubHeadline><Pgraph>Fresh stool was collected from all groups on days 1 and 3. The stools were investigated in terms of appearance and weight, and then colony count was done for them according to the study by Mai et al. <TextLink reference="17"></TextLink> . Biochemical tests were used to confirm that the strains were <Mark2>Shigella</Mark2>. </Pgraph><SubHeadline>Statistical analysis</SubHeadline><Pgraph>SPSS software version 25 (SPSS, Chicago, IL, USA) was used to analyze the data. Kruskal-Wallis and adjusted Bonferroni ad-hoc tests were applied to determi<TextGroup><PlainText>ne c</PlainText></TextGroup>orrelation between variables. p≤0.05 was considered statistically significant.</Pgraph></TextBlock> <TextBlock name="Results" linked="yes"> <MainHeadline>Results</MainHeadline><Pgraph>Plaque formation indicated the presence of lytic phage against <Mark2>S. flexneri</Mark2>. Fully transparent plaques formed, having diameters of 0.3–0.8 mm. One of the plaq<TextGroup><PlainText>ues w</PlainText></TextGroup>as chosen, enriched, and used for subsequent tests. According to TEM results, the isolated phage had an icosahedral head with 80 nm diameter, attached directly to the 13<TextGroup><PlainText>1-n</PlainText></TextGroup>m-long non-contracting tail. Based on the morphological features, this phage was classified as a member of the <Mark2>Myoviridae</Mark2> family, order <Mark2>Caudovirales</Mark2>. </Pgraph><Pgraph>Also, according to the equation mentioned in the methods section, the phage titer was determined to be 43.3×10<Superscript>11</Superscript>. </Pgraph><SubHeadline>Effect of temperature on phage survival</SubHeadline><Pgraph>To analyze the stability of phages at high temperatures, freephages were exposed to 40–80°C. The results showed that they are resistant to heat up to 60°C, but their number approaches zero at 70°C. Most of these phages appear to be inactive at 80°C (Figure 1A <ImgLink imgNo="1" imgType="figure" />). One method of long-term storage involves freezing. Examination of the stability of phages at –20 and –70°C showed that keeping phages at –20°C does not significantly alter their biology, but at –70°C, the number of phages is reduced. Phages begin to proliferate again by removing them from –20 and –70°C and adding fresh culture of the host bacterium (<Mark2>S. flexneri</Mark2>) plus incubation at 37°C for 24 hours (Figure 1B <ImgLink imgNo="1" imgType="figure" />).</Pgraph><SubHeadline>Effect of pH on phage survival</SubHeadline><Pgraph>The pH resistance test showed that during incubation of phages for 16 hours, plaque is formed only at pH=7 (2×10<Superscript>7</Superscript> pfu/ml). However, in 1 h incubation, the phage had good stability at acidic pHs, while the number of phages was undetectable at pHs 9 and 11. The results showed that high pH may present a barrier to the phage (Figure 2 <ImgLink imgNo="2" imgType="figure" />).</Pgraph><SubHeadline>Stability of the phages in bile salts</SubHeadline><Pgraph>Bacteriophages showed better stability in 1% bile within 1 hour and 3 hours compared to 2% bile (Figure 3 <ImgLink imgNo="3" imgType="figure" />).</Pgraph><SubHeadline>Bacterial reduction assay for the phage</SubHeadline><Pgraph>To determine the bacterial reduction assay of bacteriophages, OD was measured for both falcons (one of them as control) once every 10 min for 2 hours (to observe bacterial lysis). The results of this investigation showed that with an increase in the control OD (bacterial increase), the phages also revealed an ascending trend (Figure 4 <ImgLink imgNo="4" imgType="figure" />).</Pgraph><SubHeadline>The results of one-step growth curve</SubHeadline><Pgraph>This test was done to determine the phage’s incubation period and burst size. Based on the results, the incubation period was 35 min, and phage burst size was 377 pfu/cell per infected cell. The burst size was calculated based on the mean ratio of the phage performance of the bacterial cells to the average released particles of the infected phage (Figure 5 <ImgLink imgNo="5" imgType="figure" />) <TextLink reference="13"></TextLink>.</Pgraph><SubHeadline>Effect of the phage on S. flexneri in vivo</SubHeadline><Pgraph>The groups of mice that received phage showed greater improvement within 24 hours compared to control group. Among phage-receiving groups, group I followed by group IV had the most significant improvement (Figure 6 <ImgLink imgNo="6" imgType="figure" />). After 72 hours, fresh stool samples were collected again from the mice groups. Before homogenizing into normal saline, the samples were visually examined and weighed. Serial dilution was prepared from this suspension for colony counting. It was dispersed uniformly on a MacConk<TextGroup><PlainText>ey ag</PlainText></TextGroup>ar plate using a Pasteur pipette, and incubated overnig<TextGroup><PlainText>ht a</PlainText></TextGroup>t 37°C.</Pgraph><Pgraph>Colonies were counted the next day and recorded as colony forming units (cfu)/g. Compared to the control group, a decrease in the number of cfu’s was observed in all groups of mice. The greatest reduction was observed in group I, followed by group IV (Figure 7 <ImgLink imgNo="7" imgType="figure" />). </Pgraph><Pgraph>The number of cfu’s of <Mark2>S. flexneri</Mark2> across different mouse groups on days 1 and 3 indicates that the number of cfu’s decreased on the third day, showing which had considerable improvement over time compared to the control group. Group I, followed by group IV, showed the greatest improvement (Figure 8 <ImgLink imgNo="8" imgType="figure" />).</Pgraph></TextBlock> <TextBlock name="Discussion" linked="yes"> <MainHeadline>Discussion</MainHeadline><Pgraph>Diarrhea resulting from the <Mark2>Shigella</Mark2> bacterium is generally called shigellosis <TextLink reference="18"></TextLink>. Clinical and epidemiological studies suggest that <Mark2>Shigella</Mark2> is one of the major causes of diarrhea, especially in children under five, with the dominant <Mark2>Shigella</Mark2> species in developing countries being <Mark2>S. flexneri</Mark2> <TextLink reference="19"></TextLink>. The disease is usually transferred from person to person or through food and water <TextLink reference="20"></TextLink>. In industrial countries, milder cases are more common, while more severe cases and often fatal dysentery occur in developing countries. Considering the high transmissivity of the disease due to low infective dose (200 cfu’s), and since humans are considered the only reservoir of <Mark2>Shigella</Mark2>, a control treatment-based program is requir<TextGroup><PlainText>ed alo</PlainText></TextGroup>ngside observing hygienic principles <TextLink reference="21"></TextLink>. The emergence of antibiotic-resistant strains has led to the failure of effective treatments against shigellosis as well as other bacterial infections, potentially increasing health-care costs as well as mortality <TextLink reference="16"></TextLink>. Despite efforts of more than a century, a certified vaccine for <Mark2>Shigella</Mark2> is not yet available <TextLink reference="18"></TextLink>. Therefore, finding an alternative strategy for controlling infection is essential. Indeed, phages come to the forefront because of the emergence of antibiotic-resistant bacteria <TextLink reference="22"></TextLink>, and shigellosis is one of the first human diseases for which treatment outcome has improved using phage therapy <TextLink reference="23"></TextLink>. Mikeladze et al. <TextLink reference="24"></TextLink> reported 50% reduction in mortality of bloody diarrhea patients through phage therapy. Anpilov et al. <TextLink reference="25"></TextLink> also reported a tenfold decrease in incidence of bloody diarrhea in patients treated with phages.</Pgraph><Pgraph>The studied phage was isolated from the wastewater of Shahid Motahari burn accident hospital (the first <TextGroup><PlainText>basin of</PlainText></TextGroup> the treatment plant). According to some studies, wastewater per se and hospital wastewater in particular generally contains a large diversity of microorganisms due to contaminated excreted materials <TextLink reference="26"></TextLink>. The isolated phage was highly lytic and produced transparent plaques ranging from 0.3 to 0.8 mm in diameter. The morphologic<TextGroup><PlainText>al ch</PlainText></TextGroup>aracteristics indicated that the isolated phage belonged to the <Mark2>Myoviridae</Mark2> family, <Mark2>Caudovirales</Mark2> order. </Pgraph><Pgraph>Several studies have documented that phages differ from each other in terms of pH and heat stability. Investigating the stability of the studied phage across a wide range of temperatures and pH is important. This study showed that the phage could tolerate temperatures up to 65°C, while at 70°C, their numbers dropped to almost zero. It seems that most of these phages are inactive at 80°C, which is due to the protein coating of the phage. Furthermore, the stability of the present phage at –20°C offers a perfect condition under which phages may be kept easily. Numerous studies have reported that most phages can survive within a wide pH range of 5 to 9, maintaining the native viral structure and stability <TextLink reference="27"></TextLink>. However, the <Mark2>Myoviridae</Mark2> phage isolated in this study had good stability for 1 hour at low pH values. This indicates that the mentioned phage can tolerate the acidic pH of the stomach, and oral administration of the phage has no significant effect on its lytic activity. Therefore, assessment of phage survival through the digestive system is essential. Accordingly, stability measurements in 1% and 3% bile were performed for the mentioned phage for periods of 1 and 3 hours. It was found that the studied phage has relatively good stability in the bile salts, although when exposed to the simulated intestinal fluid, slight negative effects were observed on the survival of the free phages.</Pgraph><Pgraph>The longer the incubation period of the phage, the larger the burst size will be. The burst size of the phage is the average number of phages produced after infection in a bacterial cell <TextLink reference="28"></TextLink>. In 2015, Zhang et al. <TextLink reference="29"></TextLink> isolated a high-risk phage from <Mark2>Lacticaseibacillus (L.)</Mark2> casei ATCC 393 and reported an incubation period of about 75 min, which followed by a relatively short burst period of 4<TextGroup><PlainText>5 m</PlainText></TextGroup>in. The burst size was approximately 16/pfu per each infected cell at 30°C. In 2017, Sunthornthummas et al. <TextLink reference="30"></TextLink> reported a 55-min incubation period for <Mark2>L. paracasei</Mark2> phage ΦT25 at 37°C, which decreased with a 55 min incremental period and average burst size of around 38 phage particles in each infected cell. For the studied phage, a single-stage growth curve was drawn, showing a biphasic curve that identified the incubation period and burst size of the phage. According to the single-stage growth curve, the phase incubation period was 35 min, and its burst size was 377 pfu/cell phage particles in each infected cell. The large burst size of the phage in this study indicates its suitability for use in treatment. After 40 min, a new generation of phages starts to generate and eventually a large number of new phages are produced. These features suggest the therapeutic potential of the studied phages. These phages seem to produce a large number of new generation phages within 40 min, and fulfill the need for high concentration of phages and high doses for treatment.</Pgraph><Pgraph>The interaction of phages and host cells plays a significant role in practical applications. The effectiveness of phage activity is associated with the host cells, which causes an increased probability of binding of phages to the host cells. If the phage reaches the site of bacterial infection, it will be effective in eliminating the infection. When the bacterial infection is systemic and the bacteria are scattered throughout the animal’s body, it seems likely that ensuring a sufficient number of phages would enhance the therapeutic effect, as it increases the probability of targeted binding of the phage to the bacteria <TextLink reference="31"></TextLink>. The first stage in the phage infection process is binding and absorption, which determines the specificity <TextLink reference="32"></TextLink>. In our study, in which OD was measured for the phage and control samples for two hours, this phage considerably reduced the bacterial number under <Mark2>in vitro</Mark2> conditions (Figure 5 <ImgLink imgNo="5" imgType="figure" />). Concurrent with an increase in the number of host bacteria, the phage also begins to grow. Thus, we used this phage for<Mark2> in vivo</Mark2> experiments. </Pgraph><Pgraph>To observe the effectiveness of the isolated phages <Mark2>in vivo</Mark2>, a mouse model was employed for prophylaxis and treatment of bacillary dysentery. Our observations indicated that phage therapy was highly effective for prophylaxis. Meanwhile, the comparison of the five groups using the Kruskal-Wallis test (p<0.05) indicated that all five groups were significant together, while for pair-wise comparison of the groups, the adjusted Bonferroni ad-hoc test was utilized, which showed pair-wise significance. Groups I and IV, representing the prophylaxis and prophylaxis plus treatment, respectively, showed the greatest statistically significant differences (p<0.001) compar<TextGroup><PlainText>ed t</PlainText></TextGroup>o other groups. </Pgraph><Pgraph>Phage therapy is developing as a promising treatment of infections resulting from multi-drug resistant bacteria. There are numerous reports about phages that have been isolated and therapeutically implemented against antibiotic-resistant <Mark2>Shigella</Mark2> pathogens <TextLink reference="13"></TextLink>, <TextLink reference="22"></TextLink>. The results of prophylaxis and treatment with the phage studied here showed that administration of the phage either before or after infection yields quite similar effects. Furthermore, <Mark2>in vivo</Mark2> methods demonstrated that the free phages can be recovered from the stool in very small amounts following oral administration to the mice. These results confirm studies showing that phages survive passage through the gastrointestinal tract and can be excreted from animal feces in a dose-dependent manner. Some researchers have proposed that phages enter the bloodstream of the laboratory animals within 2 to 4 hours (following one oral dose), and are found in internal organs (liver, spleen, kidneys, etc.) within 10 hours. Also, the data related to the persistence of the administered phages indicate that the phages can remain in the body for a relatively long time, i.e., several days <TextLink reference="33"></TextLink>. Although phages can circulate well in the bloodstream and in different organs, some researchers have shown that phages may be neutralized by antibodies, thereby inhibiting the targeted lysis of bacteria <TextLink reference="34"></TextLink>. According to Sulakvelidze et al. <TextLink reference="35"></TextLink>, the production of neutralizing antibodies should not be a major obstacle during the primary treatment of acute infections, as the phage kinetic of action is far faster than the production of the host’s neutralizing antibodies. In addition, if the phage neutralizing antibodies still exist during implementation of the second course of treatment or if a rapid immune response occurs before phages exert their action, by re-administering the phage or elevating its concentration, this problem can be overcome <TextLink reference="35"></TextLink>. </Pgraph><Pgraph>Overall, our results present definite proof of effective treatment of bacillary dysentery in mouse models using phages, which is in line with several other reports. It clearly indicates that phages can function as a therapeutic agent against bacterial infections. Previous studies have shown that compared to antibiotic therapy, phage therapy presents more effective results, and it seems to be a safe agent to replace antibiotics <TextLink reference="36"></TextLink>. Although phage therapy is a promising substitute for antibiotics against special and treatment-resistant infections, precise assessment of its therapeutic potential is an absolute prerequisite before clinical application <TextLink reference="37"></TextLink>.</Pgraph></TextBlock> <TextBlock name="Notes" linked="yes"> <MainHeadline>Notes</MainHeadline><SubHeadline>Acknowledgements</SubHeadline><Pgraph>We would like to thank Babol University of Medical Sciences for funding this study. </Pgraph><SubHeadline>Authors’ ORCIDs </SubHeadline><Pgraph><UnorderedList><ListItem level="1">Mehdi Rajabnia: <Hyperlink href="https://orcid.org/0000-0002-5343-0878">0000-0002-5343-0878</Hyperlink></ListItem><ListItem level="1">Sousan Akrami: <Hyperlink href="https://orcid.org/0000-0001-6643-140X">0000-0001-6643-140X</Hyperlink></ListItem></UnorderedList></Pgraph><SubHeadline>Ethical approval</SubHeadline><Pgraph>The study was approved by the research ethics committee of Babol University of Medical Sciences; Babol, Iran ( code IR.MUBABOL.REC.1399.101).</Pgraph><SubHeadline>Funding</SubHeadline><Pgraph>The study was financially supported by Babol University of Medical Sciences (grant number: 744132621).</Pgraph><SubHeadline>Competing interests</SubHeadline><Pgraph>The authors declare that they have no competing interests.</Pgraph></TextBlock> <References linked="yes"> <Reference refNo="1"> <RefAuthor>Zhu Z</RefAuthor> <RefAuthor>Wang W</RefAuthor> <RefAuthor>Cao M</RefAuthor> <RefAuthor>Zhu Q</RefAuthor> <RefAuthor>Ma T</RefAuthor> <RefAuthor>Zhang Y</RefAuthor> <RefAuthor>Liu G</RefAuthor> <RefAuthor>Zhou X</RefAuthor> <RefAuthor>Li B</RefAuthor> <RefAuthor>Shi Y</RefAuthor> <RefAuthor>Zhang J</RefAuthor> <RefTitle>Virulence factors and molecular characteristics of Shigella flexneri isolated from calves with diarrhea</RefTitle> <RefYear>2021</RefYear> <RefJournal>BMC Microbiol</RefJournal> <RefPage>214</RefPage> <RefTotal>Zhu Z, Wang W, Cao M, Zhu Q, Ma T, Zhang Y, Liu G, Zhou X, Li B, Shi Y, Zhang J. 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DOI: 10.1186/s12866-018-1234-4</RefTotal> <RefLink>https://doi.org/10.1186/s12866-018-1234-4</RefLink> </Reference> </References> <Media> <Tables> <NoOfTables>0</NoOfTables> </Tables> <Figures> <Figure width="472" height="690" format="png"> <MediaNo>1</MediaNo> <MediaID>1</MediaID> <Caption><Pgraph><Mark1>Figure 1: A: Measurement of the bacteriophage resistance at 40–80°C; B: Comparison of phages at –20 and –70°C against the stoke sample</Mark1></Pgraph></Caption> </Figure> <Figure width="472" height="353" format="png"> <MediaNo>2</MediaNo> <MediaID>2</MediaID> <Caption><Pgraph><Mark1>Figure 2: Survival rate of the bacteriophages at various pHs within 1 hour</Mark1></Pgraph></Caption> </Figure> <Figure width="565" height="326" format="png"> <MediaNo>3</MediaNo> <MediaID>3</MediaID> <Caption><Pgraph><Mark1>Figure 3: Comparison of bacteriophage resistance in 1% and 2% bile within 1 and 3 h</Mark1></Pgraph></Caption> </Figure> <Figure width="568" height="307" format="png"> <MediaNo>4</MediaNo> <MediaID>4</MediaID> <Caption><Pgraph><Mark1>Figure 4: Extent of absorption of bacteriophages over time</Mark1></Pgraph></Caption> </Figure> <Figure width="564" height="288" format="png"> <MediaNo>5</MediaNo> <MediaID>5</MediaID> <Caption><Pgraph><Mark1>Figure 5: Single-stage growth of the </Mark1><Mark1><Mark2>S. flexneri</Mark2></Mark1><Mark1> phage</Mark1></Pgraph></Caption> </Figure> <Figure width="698" height="402" format="png"> <MediaNo>6</MediaNo> <MediaID>6</MediaID> <Caption><Pgraph><Mark1>Figure 6: Assessment of the colony forming number of </Mark1><Mark1><Mark2>S. flexneri</Mark2></Mark1><Mark1> in five different groups after 24 hours</Mark1></Pgraph></Caption> </Figure> <Figure width="728" height="386" format="png"> <MediaNo>7</MediaNo> <MediaID>7</MediaID> <Caption><Pgraph><Mark1>Figure 7: Assessment of colony forming number of </Mark1><Mark1><Mark2>S. flexneri</Mark2></Mark1><Mark1> in five different groups after 72 hours</Mark1></Pgraph></Caption> </Figure> <Figure width="727" height="372" format="png"> <MediaNo>8</MediaNo> <MediaID>8</MediaID> <Caption><Pgraph><Mark1>Figure 8: Comparison of the number of colony forming unit of </Mark1><Mark1><Mark2>S. flexneri</Mark2></Mark1><Mark1> in different mouse groups on days 1 and 3</Mark1></Pgraph></Caption> </Figure> <NoOfPictures>8</NoOfPictures> </Figures> <InlineFigures> <NoOfPictures>0</NoOfPictures> </InlineFigures> <Attachments> <NoOfAttachments>0</NoOfAttachments> </Attachments> </Media> </OrigData> </GmsArticle>