Intestinal Colonization by Campylobacter jejuni, Clostridium difficile, and Clostridium perfringens among Commensal Rattus norvegicus in the Urban Areas of Tehran, Iran.

Background: Rattus norvegicus (R. norvegicus) population plays a significant role in the spread of numerous diseases in urban environments. The present study is aimed at investigating the presence of Campylobacter jejuni (C. jejuni), C. coli, Clostridium difficile (C. difficile), C. difficile toxigenic, and C. perfringens in R. norvegicus captured from urban areas of Tehran, Iran. Methods: From October 2021 to October 2022, 100 urban rats were trapped in 5 different districts of Tehran, Iran. The genomic DNA was extracted from fecal samples, and the presence of C. jejuni, C. coli, C. perfringens, and C. difficile species was evaluated using PCR assay. Moreover, PCR was used to assess the toxicity of C. difficile isolates. Results: Overall, 30% (n = 30/100) of fecal samples were positive for zoonotic pathogens. Based on the PCR on hippuricase (hipO), glycine (gly), CIDIF, and phospholipase C (plc) genes, C. perfringens and C. difficile were isolated from 18.2% (n = 14/77) and 5.2% (n = 4/77) of male rats. The highest frequency of C. perfringens and C. jejuni was 25% (n = 5/20) related to the south of Tehran. Toxigenic C. difficile was not detected in all regions. Conclusion: According to the findings, rats are the main reservoirs for diseases. Therefore, rodent control coupled with the implementation of surveillance systems should be prioritized for urban health.

Protective effects of different lyoprotectants on survival of clinical bacterial isolates in a hospital biobank.

Nowadays the significant role of biobanks in medical, diagnostic, industrial, and environmental research is well known. Bacterial biobanks could be used as a good resource for designing new treatments, biomedical and industrial researches, and laboratory diagnostics. To have a collection of bacteria from clinical samples and maintain their long-term viability, their preservation needs appropriate protective agents, like cryoprotectants and lyoprotectants. In this study, we collected and characterized Gram-negative and Gram-positive bacteria carrying important antibiotic resistance markers from different clinical samples of hospitalized children. Sucrose (10%), skimmed milk (10%), skimmed milk plus sodium glutamate (10% + 1%), and bovine serum albumin (BSA, 10%) were used as lyoprotectants during the freeze-drying procedure. The survival rate of the lyophilized samples was calculated by dilution plating and measuring the colony forming unit (CFU) after 3 months of storage. The culture analysis results indicated that 25 of the 27 studied bacterial genera (Dilutions 10-3 to 10-6), including Shigella, Methicillin-resistant S. aureus, Acinetobacter spp., Escherichia spp., Pseudomonas spp., Klebsiella spp., Enterococcus spp., were recovered in cultured fractions from all preservation conditions, while 2 genera were only detected in a single preservation condition (2/27, 7.4%). Based on the results, sucrose (10%) and skimmed milk (10%) presented the most protective features. The survival rates varied significantly according to types of the bacteria. Collectively, our results showed a diversity in the recovery of different bacterial genera after lyophilization. While statistically no significant difference was detected among the studied protective agents, sucrose (10%) and skimmed milk (10%) exhibited more effective lyoprotective properties for both Gram-positive and Gram-negative bacteria among the clinical isolates in our study.

Improved heat shock method for extracting total RNA from nasopharyngeal swab samples even with low viral load.

This study aimed to improve the heat shock method as a cost-effective and time-efficient for total RNA extraction. We compared the effectiveness of two total RNA extraction methods by using Real-Time PCR for nasopharynx swabs. Include: I; use of a commercial total RNA extraction kit as a standard. II; utilized a modified heat shock method (MHS). Time, centrifuge speed and duration, proteinase K, and RNA carrier were optimized. The optimized parameters included treating the sample with 5 µg/µL at 56°C for 5 minutes, heating at 95°C for 5 minutes followed by thermal shock in ice for 3 minutes, adding 4 µg/µL RNA carrier at room temperature for 3 minutes, and centrifuging at 7000 rpm for 10 minutes. This optimization demonstrated a sensitivity and specificity of 100% (CI: 95%) even in samples with low viral load. Our in-house method presents a rapid, and cost-effective alternative for total RNA extraction.

Detection of Yersinia enterocolitica, Shigella spp. and Salmonella spp. in Rattus norvegicus captured from Tehran, Iran.

Background: The present study aims to determine the presence of Yersinia spp., Yersinia pestis, Yersinia enterocolitica pathogen, Listeria monocytogenes, Salmonella spp., Shigella spp., Francisella tularensis and Borrelia spp. in brown rats of Tehran, Iran. Methods: PCR was used to detect various bacteria in 100 brown rats, Also, ELISA was used to detect antibodies against the F. tularensis and Borrelia spp. Results: A total of 16% and 13% of fecal samples were positive for Yersinia spp. and Y. enterocolitica pathogen. ELISA results were negative for F. tularensis and Borrelia. No specific antibodies (IgG) were against these bacteria. Conclusion: According to the results of our analysis, rats are significant transmitters and carriers of a variety of illnesses that can spread to both people and other animals.

Co-colonization of methicillin-resistant Staphylococcus aureus and Candida spp. in children with malignancies.

This study aimed to evaluate the interaction between methicillin-resistant Staphylococcus aureus(MRSA) and Candida spp. in the oral cavity of children with malignancies under chemotherapy. We evaluated the expression level of Als3p and mecA in Candida spp. and MRSA strains in both single colonization and co-colonization condition. Oral and nasal samples were collected by dry sponge swabs in 10 ml of sterile phosphate-buffered saline. The MRSA and Candida spp. was confirmed using the PCR method and mecA and Als3p genes, respectively. The SYBR Green-based quantitative real-time PCR was used to evaluate the relative expression levels of mecA and Als3p genes in MRSA and Candida spp., respectively. The frequency of S. aureus in oral-only and nasal-only swab samples were 14.1% (n = 24/170). 58.3% (n = 14/24) and 29.2% (n = 7/24) of S. aureus isolated from oral and nasal samples were MRSA, respectively. Among Candida species, C. albicans (n = 28/170; 16.5%) had the highest frequency. The oral co-colonization of MRSA and Candida spp. was detected in 4.7% (n = 8/170) patients. The overall average of gene expression levels among all Candida spp. and MRSA isolates indicated that the mecA and Als3p genes expression increased six and two times in co-colonization conditions compared to single colonization conditions, respectively. Our findings revealed the importance of polymicrobial infection in clinical settings and stated that it is possible that Candida spp. facilitates the infection of S. aureus and can lead to systemic infection in co-colonized patients.

Sequence-type diversity of invasive Streptococcus pneumoniae isolates in Iran among children under 15 years of age.

Background: Infection with viruses, bacteria, or other pathogens can lead to inflammation of the meninges. Finding the pathogen and identifying the most common type is necessary for each country. Using multi-locus sequence typing (MLST), the aim of this study was to determine the genetic relationship among S. pneumoniae isolated from CSF in children with bacterial meningitis. Materials and methods: : Fourteen isolates of S. pneumoniae from CSF in children with bacterial meningitis were included in this study. The seven housekeeping genes, primer, and analysis of the sequencing used in MLST were extracted from PubMLST. Results: The sequencing analysis showed four MLST types in the studied strains. The most frequent type is ST13649 and the least frequent are ST708 and ST285. Conclusion: Finding the bacterial sequence types (ST) enables comparing the ST in different, especially neighbouring, countries.

Evaluation of Covid-19 anti-spike IgG antibody five months after the second Covid-19 vaccination.

Background: Studies in different communities have shown significant differences in IgG antibody titers in the time period after the first and second doses of the vaccines. This study aimed to serologically evaluate the IgG anti-spike antibody titer five months after injection of the second COVID-19 vaccine in healthcare workers. Materials and method: This study was performed in healthcare personnel for whom five months had passed since their second anti-Covid-19 vaccination. The level of IgG antibody against SARS-CoV-2 spike protein was measured by ELISA. Healthcare workers in Mofid Children's hospital received three brands of vaccines: Sputnik V, Sinopharm, and AstraZeneca. Results: The mean titer of anti-spike IgG was 4.3±2.29 units. The percentage of positive cases of the antibody was estimated to be 96.4%. The titer of anti-spike IgG antibody was dependent on both the occupational area and a positive history of Covid-19 disease. Conclusion: About 96.4% of the staff vaccinated against Covid-19 had a high titer of anti-spike IgG antibody even five months after inoculation of the second dose. The field of occupational can affect the level of antibody present.

Endogenous Bacteremia Caused by Intestinal Colonization of Carbapenem-Resistant Enterobacteriaceae (CRE) in Immunocompromised Children.

OBJECTIVE: Carbapenem-resistant Enterobacteriaceae (CRE) infection is life-threatening, especially for immunocompromised children. The source tracking of CRE could prevent bacteremia during hospitalization. In this study, the intestinal colonization of CRE and their translocation to blood were investigated. METHODS: Stool samples from immunocompromised pediatric patients were collected after admission, and secondary stool and blood samples were collected in case of fever. After CRE phonotypic detection, the OXA-48, NDM-1, VIM, IMP, and KPC genes were detected by PCR. Enterobacterial Repetitive Intergenic Consensus Polymerase Chain Reaction (ERIC-PCR) was used to determine the phylogenic relatedness of the blood and fecal isolates. RESULTS: Bacteremia was recorded in 71.4% of the patients. Enterobacteriaceae spp. were recorded in 100% of the stool samples and 31% of the blood samples. The correlation between the length of stay (LOS), days of fever, chemotherapy regimens, and death rate was significant (p-value ≤ 0.05). OXA-48 was present in all CRE isolates in both the primary and the secondary stool samples and the blood samples. According to the phylogenetic data, 58.33% of the patients with bacteremia had identical blood and stool isolates. The death rate was 24.4% in children with CRE bacteremia. CONCLUSIONS: The primary intestinal colonization with CRE in immunocompromised pediatrics and their translocation to blood was established in this study. The implementation of infection control programs and the application of infection prevention strategies for immunocompromised children is necessary.

The intestinal carrier status of Enterococcus spp. in children: clonal diversity and alterations in resistance phenotypes before and after admission to a pediatric intensive care unit.

BACKGROUND: This study aimed to investigate the intestinal carrier status of Enterococcus spp. among children in a pediatric intensive care unit (PICU) and reveal the role of hospitalization in the alteration of resistance phenotypes and clonal diversity of the isolates during admission and discharge periods. METHODS: Two separate stool samples were collected from hospitalized patients in the pediatric intensive care unit at admission and discharge times. The culture was done, and Enterococcus species were tested for antimicrobial susceptibility and carriage of vanA-D gene subtypes. Random Amplified Polymorphic DNA (RAPD)-PCR was used for a phylogenetic study to check the homology of pairs of isolates. RESULTS: The results showed carriage of Enterococci at admission, discharge, and at both time points in 31%, 28.7%, and 40.1% of the cases, respectively. High frequencies of the fecal Enterococcus isolates with vancomycin-resistance (VR, 32.6% and 41.9%), high-level of gentamicin-resistance (HLGR, 25.6% and 27.9%), and multi-drug resistance phenotypes (MDR, 48.8% and 65.1%) were detected at admission and discharge times, respectively. Resistance to vancomycin, ampicillin, and rifampicin was higher among E. faecium, but resistance to ciprofloxacin was higher in E. faecalis isolates. The increased length of hospital stay was correlated with the carriage of resistant strains to vancomycin, ampicillin, and ciprofloxacin. While the homology of the isolates was low among different patients during hospitalization, identical (9%) and similar (21%) RAPD-PCR patterns were detected between pairs of isolates from each patient. CONCLUSIONS: The high rate of intestinal carriage of VR, HLGR-, and MDR-Enterococci at admission and during hospitalization in the PICU, and the impact of increased length of hospital stay on the fecal carriage of the resistant strains show the importance of antibiotic stewardship programs to control their transmission and spread in children.

Seroepidemiological and Molecular Survey for the Detection of SARS-CoV-2 Infection among Children in Iran, September 2020 to June 2021: 1-Year Cross-Sectional Study.

A population-based seroepidemiological and molecular survey on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was performed to detect induced antibodies to prior exposure and active infection of children aged 14 years or less in Tehran between 19 September 2020 and 21 June 2021. Moreover, correlations between the children's demographic data and coronavirus disease 2019 (COVID-19) symptoms with the infection status were investigated. Out of 1517 participants, cardinal symptoms of COVID-19 (fever > 38 °C and/or cough and/or diarrhea) were detected in 18%, and serological history of SARS-CoV-2 infection and polymerase chain reaction (PCR) positivity were confirmed in 33.2% and 10.7% of the weighted population, respectively. The prevalence of SARS-CoV-2 infection was significantly higher among 10-14-year-old children. Active infection was significantly higher in symptomatic children and during autumn 2020 and spring 2021. The quantitative reverse transcription real-time PCR (RT-qPCR) positivity was significantly higher among families with a lower socioeconomic status, whereas no association between RT-qPCR or seropositivity was determined with household size, underlying diseases, or gender. In conclusion, high SARS-CoV-2 infection prevalence and seroprevalence were detected in children in Tehran in different seasons. Infection prevalence was significantly higher in older children and in those with a positive history of close contact with infected cases and/or lower socioeconomic status.

Antibiotic Susceptibility Patterns for Carbapenem-Resistant Enterobacteriaceae.

Carbapenem is a broad-spectrum beta-lactam antibiotic considered the last choice for the treatment of antibiotic-resistant Gram-negative bacteria. Thus, the increasing rate of carbapenem resistance (CR) in Enterobacteriaceae is an urgent public health threat. This study aimed to evaluate the antibiotic susceptibility pattern of carbapenem-resistant Enterobacteriaceae (CRE) to new and old antibiotics. In this study, Klebsiella pneumoniae, E. coli, and Enterobacter spp. were collected from 10 hospitals in Iran for one year. CRE is recognized by resistance to meropenem and/or imipenem disk after identification of the collected bacteria. Antibiotic susceptibility of CRE against fosfomycin, rifampin, metronidazole, tigecycline, and aztreonam was detected by disk diffusion method and colistin by MIC. In this study, 1222 E. coli, 696 K. pneumoniae, and 621 Enterobacter spp. were collected from 10 hospitals in Iran in one year. Fifty-four E. coli (4.4%), 84 K. pneumoniae (12%), and 51 Enterobacter spp. (8.2%) were CRE. All CRE strains were resistant to metronidazole and rifampicin. Tigecycline has the highest sensitivity on CRE and levofloxacin for Enterobacter spp. Tigecycline showed an acceptable effectiveness rate of sensitivity on the CRE strain. Therefore, we suggest that clinicians consider this valuable antibiotic to treat CRE.

Assessment of early and post COVID-19 vaccination antibody response in healthcare workers: a multicentre cross-sectional study on inactivated, mRNA and vector-based vaccines.

In this multicentre study, we compared the status of antibody production in healthcare personnel (HCP) before and after vaccination using different brands of COVID-19 vaccines between March 2021 and September 2021. Out of a total of 962 HCP enrolled in our study, the antibody against the S1 domain of SARS-CoV-2 was detected in 48.3%, 95.5% and 96.2% of them before, after the first and the second doses of the vaccines, respectively. Our results showed post-vaccination infection in 3.7% and 5.9% of the individuals after the first and second doses of vaccines, respectively. The infection was significantly lower in HCP who presented higher antibody titres before the vaccination. Although types of vaccines did not show a significant difference in the infection rate, a lower infection rate was recorded for AstraZeneca after the second vaccination course. This rate was equal among individuals receiving a second dose of Sinopharm and Sputnik. Vaccine-related side effects were more frequent among AstraZeneca recipients after the first dose and among Sputnik recipients after the second dose. In conclusion, our results showed diversity among different brands of COVID-19 vaccines; however, it seems that two doses of the vaccines could induce an antibody response in most of HCP. The induced immunity could persist for 3-5 months after the second vaccination course.

Intestinal colonization of vancomycin-resistant Enterococcus in children admitted to Mofid children's hospital intensive care unit at admission and at discharge.

BACKGROUND: This study aimed to investigate the frequency of intestinal colonization by vancomycin-resistant Enterococcus (VRE) carrying vanA and vanB genes in patients at ICU admission and at discharge from ICU in Mofid children's Hospital, Tehran, Iran. METHOD: Sampling was performed using rectal swabs and vancomycin susceptibility testing for Enterococcus spp. was carried out using a minimum inhibitory concentration (MIC) assay on Muller Hinton Agar (MHA) medium using an E-test kit. The molecular detection of VRE isolates was performed by the PCR method using the vanA and vanB resistance genes. RESULTS: A total of 234 and 186 non-duplicate rectal swab samples were collected from patients at ICU admission and at discharge from ICU, respectively. Enterococcus spp. was detected in 34.6% (n = 81/234) of rectal swab samples collected from patients at ICU admission, of which 44.4% (n = 36/81) were VRE isolates. In contrast, the prevalence of Enterococcus spp. and VRE isolates among patients at discharge from ICU was 17.7% (n = 33/186) and 57.6% (n = 19/33), respectively. Out of 19 VRE isolated from patients at ICU admission, 4 (21%) and 1 (5.3%) contained vanA and vanB genes, respectively. In contrast, out of 36 VRE isolated from patients at discharge from ICU, 11 (30.5%) were positive for the vanA gene. CONCLUSION: Results revealed that the prevalence of Enterococcus spp. among patients at ICU admission was high. However, VRE was frequently isolated from patients who were hospitalized for several days in ICUs. The implementation of proper infection control strategies and the use of suitable protocols to guide the appropriate prescribing of antibiotics are necessary.

Tracing the Negative Results of Multiplex Real-Time PCR Assay for Diagnosis of Bacterial Pediatrics Meningitis.

The death because of meningitis remains high in some parts of the world. It is important to know the specific cause of meningitis because the treatment differs depending on the cause. This study aimed to trace the false-negative results of multiplex RT-PCR to detect Streptococcus pneumoniae, Haemophilus influenzae, and Neisseria meningitidis serogroup by two different molecular methods. In this study, the CSF of the suspicious pediatric for acute bacterial meningitis among children aged 1 month to 14 years who are admitted to the hospitals in four cities of a certain region of Iran was collected. S. pneumoniae, H. influenzae, and N. meningitidis in CSF samples were detected by single-tube multiplex RT-PCR and specific RT-PCR with a probe on the same specimens. In this cross-sectional study, 506 CSF samples were collected during one year. The multiplex RT-PCR can detect 3.3% and 2.2% of S. pneumoniae and H. influenzae, respectively. N. meningitidis was not detected. The CSF analysis was abnormal in 53% of 506 patients. On the other hand, 11.5%, 4.8%, and 4.1% of S. pneumoniae, H. influenzae, and N. meningitidis were identified, respectively, by specific RT-PCR assay, exactly on the same specimens. Various types of PCR can be used for pathogen identification. As we change the type of PCR in our study, we could approximately increase 15% our positive results and also consequently decrease our false-negative responses.

Evaluation of methicillin-resistant Staphylococcus virulence genes and antibiotics susceptibility in Iranian population.

Background: Methicillin resistance Staphylococcus aureus (MRSA) is one most important pathogens for human health. The ability of this organism for producing different kinds of disease is related to its virulence gene. The frequency of hemolysin alpha (hla), hemolysin beta (hlb), and exfoliative toxin A (eta) virulence genes of MRSA was evaluated, and the association of these genes with antibiotics susceptibility was investigated. Materials and Methods: In a cross-sectional study, a total of 695 Staphylococcus clinical samples from seven different provinces of Iran were evaluated. MRSA was detected by cefoxitin disk. Virulence genes were detected by polymerase chain reaction. Susceptibility to clindamycin and ciprofloxacin was evaluated according to the Clinical and Laboratory Standards Institute guideline. Results: From a total of 695 samples, 170 (24.46%) were found to be MRSA. 142, 82, and 132 samples of MRSA were hla, hlb, and eta positive, respectively. hla gene was significantly found more frequently in patients at least 18 years (P = 0.02). 105 (68.6%) and 93 (59.6%) of MRSA samples were resistance to ciprofloxacin and clindamycin, respectively. hlb gene was significantly more resistant to clindamycin (P = 0.04) and ciprofloxacin (P = 0.01). Logistic regression analysis displayed hlb-positive MRSA strains were significantly associated with ciprofloxacin (odds ratio [OR]: 3.6, 95% confidence interval [CI] = 1.637-8.00) and clindamycin (OR: 1.93, 95% CI 1.00-3.68). Conclusion: MRSA strains from Staphylococcus aureus which isolated from hospitalized Iranian patients are significantly resistant to clindamycin and ciprofloxacin and it is may be because of hlb virulence gene. These samples consist of both community-acquired MRS) and health-care associated MRSA, so we could not use this finding as a guide for local antibiotics usage.

Nanobiosensor Based on Sugar Code-AuNPs Aggregation: A Key to Opening New Gates in Rapid Diagnosis of Streptococcal Pharyngitis.

Streptococcal pharyngitis is mainly caused by Streptococcus pyogenes (GAS), which if left untreated can lead to rheumatic heart disease. The accurate diagnosis of streptococcal pharyngitis is a challenge for clinicians because several symptoms of streptococcal pharyngitis are similar to viral pharyngitis. There are some commercially available biosensors for the rapid diagnosis of streptococcal pharyngitis. Nevertheless, they are not widely used by physicians, mainly because of their high price and dependence on the instrument. Serotype M1 GAS is the most prevalent cause of streptococcal pharyngitis and binds to H-1 antigen, a sugar code found on oral epithelial cells. Here, we present a nanobiosensor based on aggregation of H-1 antigen-conjugated gold nanoparticles for the rapid, qualitative, and quantitative detection of M1 GAS, which is inspired by the sugar code-lectin interaction. It is noteworthy that M1 GAS was detected in a wide concentration range (1 × 103-1×106 CFU/ml) with a linear response and a short detection time of 20 min. Good reproducibility, easy-to-use, and relatively low production cost are among other attractive features of this nanobiosensor. This work provides a strategic roadmap for developing a new generation of biosensors via targeting the sugar code-lectin interaction in future studies.

Re-positive PCR of SARS-CoV-2 in health care persons during COVID-19 pandemic.

Reinfection rate with SARS-CoV-2 and degree of protection by the induced antibody after the first episode of the infection is not well known, so it makes a big dilemma for health care personnel (HCP) who work in the front line of combating SARS-CoV-2. In this study, we investigated the frequency of SARS-CoV-2 redetection among HCP after the initial onset of the infection in a children's hospital during one year. Out of 131 seropositive HCP, 13.7% of them were symptomatic and PCR positive during 74-360 days after first sampling. Analysis of demographic data of seropositive HCP showed a correlation between a higher number of family members, higher body mass index, and the existence of underlying diseases with SARS-CoV-2 redetection. In conclusion, reinfection is one of the important problems in the SARS-CoV-2 pandemic. Research on this topic can help us to find answers to questions for estimating the duration of human protection with produced immunity after the infection or vaccination.

Co-infection with dual Streptococcus pneumoniae serotypes as a cause of pediatric bacterial meningitis in Iran: a multi-center cross-sectional study.

BACKGROUND: Meningitis is considered a life-threatening infection with high mortality all over the world. Hemophilus influenzae (H. influenzae) and Streptococcus pneumoniae (S. pneumoniae) are regarded as the two most common infectious agents causing bacterial meningitis. This study aimed to identify H. influenzae and S. pneumoniae serotypes in blood and cerebrospinal fluid (CSF) of pediatric patients with meningitis, using polymerase chain reaction (PCR). METHODS: This multi-center cross-sectional study included 284 children with suspected meningitis referred to 4 target hospitals. Overall, 412 samples (128 blood and 284 CSF samples) were obtained from the patients from November 14, 2016 to November 15, 2017. The extracted DNA was examined using multiplex real time PCR to screen for S. pneumoniae and H. influenzae. S. pneumoniae serotyping was also done by multiplex PCR. RESULTS: Out of 284 CSF specimens, 22 were positive for ply S. pneumoniae. Of 20 DNA samples meeting the Quality Control (QC) standards for serotyping, 7 (35%), 6 (30%), 2 (10%), 2 (10%), 2 (10%), 1 (5%), 1 (5%), 1 (5%), 1 (5%) and 1 (5%) were positive for serotypes 3, 11A, 6A, 14, 7C, 23F, 23B, 19A, and 19F and 5, respectively. Overall, nine samples were positive for two serotypes, of whom 3 and 11A were the most common from Tehran province. Of note, one of these CSF samples showed a new co-infection with serotypes 7C and 14. Also, 6 samples (30%) were positive for H. influenzae detected by bexA primer. None of the blood samples were positive for S. pneumoniae or H. influenzae. CONCLUSION: Co-infection with S. pneumoniae serotypes can occur in bacterial meningitis and it might be missed if all serotypes are not evaluated in CSF specimens.

Evaluation of phenotypic and genotypic patterns of aminoglycoside resistance in the Gram-negative bacteria isolates collected from pediatric and general hospitals.

The purpose of the current study was to evaluate the phenotypic and genotypic patterns of aminoglycoside resistance among the Gram-negative bacteria (GNB) isolates collected from pediatric and general hospitals in Iran. A total of 836 clinical isolates of GNB were collected from pediatric and general hospitals from January 2018 to the end of December 2019. The identification of bacterial isolates was performed by conventional biochemical tests. Susceptibility to aminoglycosides was evaluated by the disk diffusion method (DDM). The frequency of genes encoding aminoglycoside-modifying enzymes (AMEs) was screened by the PCR method via specific primers. Among all pediatric and general hospitals, the predominant GNB isolates were Acinetobacter spp. (n = 327) and Escherichia coli (n = 144). However, E. coli (n = 20/144; 13.9%) had the highest frequency in clinical samples collected from pediatrics. The DDM results showed that 64.3% of all GNB were resistant to all of the tested aminoglycoside agents. Acinetobacter spp. and Klebsiella pneumoniae with 93.6%, Pseudomonas aeruginosa with 93.4%, and Enterobacter spp. with 86.5% exhibited very high levels of resistance to gentamicin. Amikacin was the most effective antibiotic against E. coli isolates. In total, the results showed that the aac (6')-Ib gene with 59% had the highest frequency among genes encoding AMEs in GNB. The frequency of the surveyed aminoglycoside-modifying enzyme genes among all GNB was found as follows: aph (3')-VIe (48.7%), aadA15 (38.6%), aph (3')-Ia (31.3%), aph (3')-II (14.4%), and aph (6) (2.6%). The obtained data demonstrated that the phenotypic and genotypic aminoglycoside resistance among GNB was quite high and it is possible that the resistance genes may frequently spread among clinical isolates of GNB.

Development of high-resolution melting (HRM) assay to differentiate the species of Shigella isolates from stool and food samples.

Shigella species, a group of intracellular foodborne pathogens, are the main causes of bacillary dysentery and shigellosis in humans worldwide. It is essential to determine the species of Shigella in outbreaks and food safety surveillance systems. The available immunological and molecular methods for identifying Shigella species are relatively complicated, expensive and time-consuming. High resolution melting (HRM) assay is a rapid, cost-effective, and easy to perform PCR-based method that has recently been used for the differentiation of bacterial species. In this study, we designed and developed a PCR-HRM assay targeting rrsA gene to distinguish four species of 49 Shigella isolates from clinical and food samples and evaluated the sensitivity and specificity of the assay. The assay demonstrated a good analytical sensitivity with 0.01-0.1 ng of input DNA template and an analytical specificity of 100% to differentiate the Shigella species. The PCR-HRM assay also was able to identify the species of all 49 Shigella isolates from clinical and food samples correctly. Consequently, this rapid and user-friendly method demonstrated good sensitivity and specificity to differentiate species of the Shigella isolates from naturally contaminated samples and has the potential to be implemented in public health and food safety surveillance systems.