Iranian Journal of Microbiology
Volume:15 Issue: 5, Oct 2023

The type VI secretion system (T6SS) was identified as a novel virulence factor in many Gram-negative bacteria. This study aimed to investigate the frequency of the T6SS genes in Klebsiella pneumoniae-causing different nosocomial infections, and to study the association between T6SS, antibiotic resistance, and biofilm formation in the isolated bacteria.

A total of fifty-six non-repetitive K. pneumoniae isolates were collected from different inpatients admitted at Sohag University Hospital from September 2022 to March 2023. Samples were cultured, colonies were identi- fied, and antimicrobial sensitivity was done by VITEK® 2 Compact. Biofilm formation was checked using Congo red agar method. T6SS genes, and capsular serotypes were detected by PCR.

Fifty-six K. pneumoniae isolates were obtained in culture. 38 isolates (67.86%) produced biofilm and 44 (78.57%) were positive for T6SS in PCR. There was a significant association between the presence of T6SS and resistance to the following antibiotics: meropenem, ciprofloxacin, and levofloxacin. All biofilm-forming bacteria had T6SS, with significant differences towards T6SS -positive bacteria. There was no significant association between T6SS, and the presence of certain capsular types.

The T6SS-positive K. pneumoniae has greater antibiotic resistance, and biofilm-forming ability which is con- sidered a potential pathogenicity of this emerging gene cluster.

Klebsiella pneumoniae is increasingly developing resistance to last-resort antibiotics such as carbapenems. This study aimed to investigate the dissemination of common carbapenemase encoding genes among 48 clinical isolates of carbapenem-resistant Klebsiella pneumoniae (CRKP).

Antimicrobial susceptibility testing was performed by broth dilution and disc diffusion methods. The phenotypic evaluation of carbapenemase production was performed by using Modified Carbapenem Inactivation Meth- od. Presence of carbapenemase encoding genes bla KPC , bla , bla OXA-48-like , bla , and bla VIM was screened by PCR.

Overall, carbapenemases were produced in all CRKP isolates. The bla OXA-48-like and bla NDM were the most prevalent genes detected among all and 66.6% (n=32) of CRKP isolates respectively. The bla VIM was detected in only one isolate co-harboring NDM and OXA-48-like carbapenemases. The bla KPC and bla IMP genes were not identified in any of the isolates. While tigecycline was the most active agent against CRKP isolates with low resistance rate (4.1%), high rate of resistance was observed to colistin (66.6%), amikacin (79%) and most of other tested antimicrobials.

Our results revealed predominant prevalence of OXA-48-like and NDM carbapenemases among CRKP clinical isolates. High rate of resistance to last-resort agents such as colistin among CRKP isolates is a source of great concern.

The study aimed to investigate the distribution of genes encoding integrons, extended spec- trum beta-lactamase (ESBL) in E. coli isolated from UTIs, as well as the genetic diversity among the isolates.

E. coli isolates were recovered from the patients with UTI in Kerman Iran. Antibiotic susceptibility was done according to CLSI guidelines. The presence of ESBL genes and integrons was evaluated using PCR. PCR and sequencing were applied for the evaluation of cassette content of integrons. Genotyping of the isolates was performed by multiple-locus variable-number tandem repeat analysis (MLVA).

Imipenem was the most effective antibiotic, while the highest resistance was observed to streptomycin. In total 40.2% of isolates were ESBL producers. Of 69 integron-positive isolates, 59 only had class I integrons, 4 only had class II integrons and 6 had both types. The most common gene cassette found within class I integrons was dfrA17-aadA5 (n=27). The E. coli isolates were divided into 16 MLVA clusters.

The current study demonstrated the simultaneous presence of class I integrons and ESBLs involved in the resis- tance of UPEC isolates to antibacterial agents. Our finding also revealed that the E. coli isolates belonged to diverse clones.

An increase in the antibiotic resistance of Shigella isolates has caused major global challenges in antimicrobial therapy. Knowledge of local antibiotic resistance trends is essential for selecting appropriate antibiotic treat- ment regimens. This study aimed to evaluate the frequency of efflux-mediated tetracycline resistance (tet) and plasmid-me- diated quinolone resistance (qnr) genes among Shigella isolates.

This survey investigated 91 Shigella isolates, obtained from children with acute diarrhea. The iso- lates were identified using standard biochemical tests and confirmed by polymerase chain reaction (PCR) assay. Besides, the susceptibility of isolates to six selected antibiotics was assessed by the disk diffusion method. All tetracycline-resistant and nalidixic acid and ciprofloxacin resistant strains were screened for tet and qnr genes by a multiplex PCR assay.

According to the results of antibiotic susceptibility tests, the highest level of antibiotic resistance was related to tet- racycline (80.2%) and doxycycline (78.1%), respectively. All isolates were sensitive to tigecycline. The PCR results showed that 40.6%, 3.1%, 21.8%, 61.6% and 28.7% of the isolates carried qnrA, qnrB, qnrS, tetA, and tetB genes, respectively. None of the isolates contained tetC and tetD genes.

The current findings revealed that tetA and qnrA genes might play a key role in conferring tetracycline and quinolone resistance.

Antibiotic resistance is an indicator of the passively acquired and circulating resistance genes. Salmonella Gallinarum significantly affects the poultry food industry. The present study is the first study of the S. Gallinarum biofilm in Iran, which is focused on the characterization of the S. Gallinarum serovars and their acquired antibiotic resistance genes circulating in poultry fields in central and northwestern Iran.

Sixty isolates of S. Gallinarum serovar were collected from feces of live poultry. The bacteria were isolated using biochemical tests and confirmed by Multiplex PCR. Biofilm formation ability and the antibacterial resistance were evaluated using both phenotypic and genotypic methods. The data were analyzed using SPSS software.

According to Multiplex PCR for ratA, SteB, and rhs genes, all 60 S. Gallinarum serovars were Gallinarum biovars. In our study, the antibiotic resistance rate among isolated strains was as follows: Penicillin (100%), nitrofurantoin (80%), nalidixic acid (45%), cefoxitin (35%), neomycin sulfate (30%), chloramphenicol (20%), and ciprofloxacin (5%). All isolates were susceptible to imipenem, ertapenem, ceftriaxone, ceftazidime, and ceftazidime+clavulanic acid. All sixty isolates did not express the resistance genes IMP, VIM, NDM, DHA, bla , and qnrA. On the other hand, they expressed GES (85%), OXA48 qnrB (75%), Fox M (70%), SHV (60%), CITM (20%), KPC (15%), FOX (10%), MOXM (5%), and qnrS (5%). All S. Galli- narum isolates formed biofilm and expressed sdiA gene.

Considering that the presence of this bacteria is equal to the death penalty to the herd, the distribution of resis- tance genes could be a critical alarm for pathogen monitoring programs in the region. This study showed a positive correla- tion between biofilm formation and 50% of tested resistance genes. Also, it was found that the most common circulating S. gallinarum biovars are multidrug-resistant.

 Detecting the source of a potential outbreak of multidrug resistant (MDR) Acinetobacter baumannii is necessary to be investigated. This study aimed to detect the possibility of A. baumannii outbreak in a hospital setting using a combination of random amplified polymorphism DNA- polymerase chain reaction (RAPD-PCR), antibiograms, and the presence of oxacillinase genes.

 The antibiogram of 31 clinical isolates and six environmental isolates of A. baumannii were determined by Vitek® 2 Compact. Oxacillinase genes (OXA-23, -24, -51, and -58) were detected by PCR, and RAPD-PCR was conducted using DAF-4 and ERIC-2 primers. The Similarity Index and dendrogram were generated using GelJ v2.3 software.

 The antibiograms showed that all MDR A. baumannii isolates has very limited susceptibility to cephalosporins, but mostly susceptible to tigecycline. All isolates were positive for blaOXA-51-like gene, thirty-two of 37 total isolates (86.5%) were positive for blaOXA-23-like gene, and none were positive for blaOXA-24-like and blaOXA-58-like genes. RAPD-PCR showed that the DAF-4 primer on average had more band visualization and lower Similarity Index’s variation compared to the ERIC-2. The discriminatory power of DAF-4 was 0.906. There was a significant correlation between the DAF-4 dendrogram pattern with the antibiogram (r=0.494, p<0.001) and the presence of blaOXA-23-like gene (r=0.634, p<0.001) from all ICU A isolates. Six out of fourteen ICU A isolates belonged to the same cluster with >95% Similarity Index, while one clinical isolate having an identical dendrogram and antibiogram pattern with an environmental isolate within this cluster.

 There is a high probability of MDR A. baumannii outbreak within ICU A detected by multiple analysis of RAPD-PCR, antibiogram and the blaOXA-23-like gene profiles. This combinatorial approach is conceivable to mitigate possible outbreak situations of A. baumannii in the local hospital without sophisticated microbiology laboratory.

Acinetobacter baumannii, an opportunistic pathogen, is related to hospital-acquired infections and increased mortality. This study aimed to develop the loop-mediated isothermal amplification (LAMP) test for the fast-de- tecting of A. baumannii isolates as well as determining genetic relatedness for these isolates via the REP-PCR technique.

LAMP primers and multiplex PCR primers were designed for recognizing A. baumannii isolates harboring the bla SHV-1 , bla PER-1 , bla , AMPC, qnr, and aac (6)-1 genes, were collected (October 2020 to February 2021) from Shahid Motahari Hospital, Tehran, Iran. Combination disc test (CDT) results were used to assess the phenotypic iden- tification of isolates from ESBL producers. The sensitivity of the LAMP method was evaluated using a range of serial dilu- tions of genomic DNA. Results were compared between the LAMP technique, and multiplex PCR. The genetic diversity of clinical isolates was determined by REP-PCR.

Among one hundred A. baumannii samples and based on the combined disc test, 56% of isolates were ESBL pro- ducers. The sensitivity of the LAMP technique for the identification of A. baumannii was 4.06 ng/μl whilst the multiplex PCR was (16.2 ng/μl). Regarding multiplex PCR, (68%) of the isolates were bla SHV-1 positive, (40%) bla , (85%) aac (6́)-1, AMPC (67%), bla TEM-1 (63%), and (15%) qnr respectively. While in LAMP, (69%) of isolates were bla positive, (86%) aac (6')-1, and (20%) qnr. The results of AMPC, bla TEM-1 , and bla PER-1 genes showed 100% compatibility between multiplex PCR and LAMP assays. The results of REP-PCR indicated there were 17 clones, clone A at 14% was the most prevalent of the isolates.

Wherever equipment and financial constraints are crucial, the LAMP test offers a better and more potent detec- tion rate for the identification of A. baumannii isolates than multiplex PCR. Furthermore, the genetic diversity of A. bauman- nii in these clinical isolates showed frequent commonality of genotypes.

The role of microRNAs (miRNAs) in tuberculosis infection is well established. As microR- NAs are able to change expression profiles according to different conditions, they can be useful biomarkers. Iranians and Afghans with tuberculosis were studied for three immune-related miRNAs (miR-let-7f, miR-125a, and miR-125b).

A total of 60 Iranian and Afghan patients with active pulmonary TB were enrolled in the Pulmo- nary Department of the Pasteur Institute of Iran. Serum and sputum samples were collected simultaneously from all partici- pants. A Real-time PCR was conducted to detect differentially expressed miRNAs.

Iranian (P<0.0001) and Afghan (P<0.0001) serum samples and Afghan (P<0.0001) sputum samples overexpressed miR-125a, whereas Iranian sputum samples showed downregulation (P=0.0039). In both Iranian (P<0.0001; P=0.0007) and Afghan (P<0.0001; P<0.0001) serum and sputum samples, miR-125b was overexpressed. Furthermore, miR-let-7f down- regulation was observed in serum and sputum samples (P<0.0001), whereas Iranian sputum samples had no statistically significant differences (P=0.348).

Overexpression of miR-125a and miR-125b has been detected in Iranian and Afghan samples. In both races, miR-let-7f downregulation has been confirmed. Identification of miRNA profiles under different conditions opens the door to evaluating potential new biomarkers for diagnosis, disease monitoring, and therapeutic markers in TB infection.

One of the highly conserved outer membrane proteins expressed only by pathogenic Lepto- spires is Loa22. The study aims is to achieve the optimum conditions for high expression of recombinant Loa22 (rLoa22) protein.

Complete coding sequence of loa22 gene sub-cloned into a pET32a (+) expression vector. BL21 competent E. coli (pLysS) used as expression host for transformation. The recombinant clones selected on ampicillin plates and subjected to PCR by using pET T7 primers. Then expression conditions optimized by adjusting parameters such as cul- ture media, induction time, temperature, and IPTG concentration.

SDS-PAGE analysis showed that high production of rLoa22 protein obtained when post induction incubation, IPTG concentration, and duration of induction were 37ºC, 0.1 M and 5 h in 2×TY medium respectively. The purification of rLoa22 protein under native conditions using Ni-NTA pull-down was optimum in one hour binding at 37°C, five times washing process and elution buffer with a pH 7.4 and a 0.3 M imidazole concentration.

The findings of the study led to high production of pure Loa22 protein, which can form the basis for future investigation on the design of rapid diagnostic tests and more effective vaccine candidates for leptospirosis.

Plant growth-promoting bacteria (PGPB) may reduce the negative effects of salinity stress. The aim of this study was to optimize Bacillus megaterium RTS1 and characterize the effect of the PGPB on the physiolog- ical characteristics of tomato (Lycopersicon esculentum).

The Central composite design (CCD) of response surface methodology (RSM) was used to opti- mize Bacillus megaterium RTS1 to produce maximum cell biomass and spores. Then the effect of the PGPB on the physi- ological characteristics of tomato (Lycopersicon esculentum), including membrane stability, leaf relative water content per- centage, anthocyanin and carotenoids content, chlorophyll photosynthetic parameters, sugar and starch level, superoxide anion and antioxidant activity under salt stress conditions. The NFB medium was inoculated with 5% bacterial culture and the fermentation was carried out in a 10-lit fermenter.

After optimization, the amount of cell biomass by the model was 9.45 log10 CFUs/mL, which showed a 1.2-fold increase compared to the non-optimized medium. Usage of bacteria under the optimal conditions of the culture medium may increase the stability of the membrane and improve the relative water content. Bacteria were able to prevent the excessive increase of anthocyanins. Oxidative stress led to an increase in the content of chlorophyll a, while causing the degradation of chlorophyll b. Bacterial inoculation led to an increase in the level of sugar and starch compared to the control. PGPB showed an increasing effect on the amount of superoxide anion production and caused a significant increase in the antioxidant activity under salinity stress conditions.

The PGPB can be a promising way to boost physiological characteristics of tomato plant under salinity stress. Also, sporulation capacity of Bacillus megaterium with high bacterial cell density in fermenter produce a sustainable product for tomato plants.

With entering the “post-antibiotic era”, antibiotic resistance is one of the most important prob- lems in food security, health and medicine. Invention of nanoparticles with intrinsic antimicrobial activity has been provided a new tool to combat the problem, including some metal nanoparticles. But protein nanoparticles have been often used as nano-carrier for antibiotic drugs, not for their own antibiotic activity. In this article we have fabricated a very small BSA-NP without any chemical modification on BSA molecules showing antibacterial activity.

Bovine serum albumin nanoparticle (BSA-NP) was synthesized using botton-up approach, by dissolution of BSA in urea-containing Tris buffer for 60 min at 60°C. Then, the BSA solution was dialyzed against distilled water in order to nanoparticle formation. The resulted BSA-NP has been characterized by dynamic light scattering (DLS), field emission surface electron microscopy (FESEM), SDS-PAGE, Fourier transform infrared spectroscopy (FTIR) and UV-spectrophotometery. Minimum inhibitory concentration (MIC) method was used for evaluation of antibacterial activity of BSA-NP against Staphylococcus aureus and Pseudomonas aeruginosa.

The results obtained by DLS technique indicated that BSA molecules were self-assembled into small aggregates with a hydrodynamic diameter of 23.23 ± 2.1 nm. With a small polydispersity index (PDI=0.522), the nanoparticles had good spherical uniformity. The nanoparticles made from a single type of protein molecule (BSA) and have a relatively transparent appearance. The BSA-NPs caused a decrease in cell growth of both P. aeruginosa and S. aureus. Moreover, they had a bac- teriostatic effect on P. aeruginosa (MIC=112×10-5 μM).

In this study, using a green synthesis method, we succeeded in synthesizing a very small uniform BSA nanopar- ticles without any chemical modification on BSA molecules. It also has bacteriostatic properties against P. aeruginosa. Therefore, it is hypothesized that our BSA-NPs may be effective as a new approach to combat antibiotic resistance.

Hepatitis B is a common chronic viral infection in humans. Universal use of hepatitis B vaccine is crucial for controlling the infection, but the duration of vaccine-induced immunity remains uncertain. This study aimed to assess hepatitis B antibody levels (anti-HBs) after vaccination in infancy and adolescence, and explore the relation- ship between immunity levels and variables such as age, sex, BMI, place of birth, and duration since last vaccination among students at Hormozgan University of Medical Sciences from 2019 to 2021.

The study included 1134 students who completed a questionnaire and provided blood samples for ELISA-based measurement of antibody titers.

The findings revealed that 727 students (64.1%) had no protective antibody level (anti-HBs <10 mIU/ml), 299 (26.4%) had partial immunity (anti-HBs 10-100 mIU/ml), and 108 (9.5%) had complete immunity (anti-HBs >100 mIU/ml). No statistically significant relationships were observed between anti-HBs titer and age, sex, or BMI. However, antibody titer decreased with increasing time since last vaccination (P<0.001).

This study highlights the decline in antibody titer over time following primary vaccination. Sustained immunity against hepatitis B virus relies on antibody durability or robust immunological memory, suggesting the importance of timing booster vaccinations.

Viral clearance studies are an essential part of a manufacturer's plan to ensure the safety of an injectable biologic product. In this way, viral safety is a critical quality attribute for biologics such as monoclonal antibodies (Mabs). Evaluation of virus purification by downstream processes is a key component of risk mitigation. In this study, the capability of continuous monoclonal antibody purification steps was evaluated in the process of instant monoclonal antibody purification in different stages of purification, and the amount of reduction or inactivation of each step was determined.

Four enveloped and non-enveloped viral models VSV, Reovirus, EMCV, and HSV1 were used for spiking in selected samples in the designated tests, to have a comprehensive examination of the ability to clear the virus such as the type of genetic material, chemical resistance, and particle size. A TCID and qPCR methods were used to measure viral reduction. Two cell lines, Vero (African green monkey kidney) and L929 (Mouse fibroblast) were used for 4 model viruses propagation. The steps that were evaluated included 4 steps monoclonal antibody purification; cation exchange chro- matography, acidic pH treatment, affinity chromatography, and nanofiltration.

The nano-filter stage showed the highest viral reduction and cation exchange chromatography showed the lowest reduction. The cumulative decrease using TCID is equal to 19.27 [log10] for all steps and for the qPCR method is equal to 12.47 [log10] in three steps of nano-filter, affinity chromatography, and ion exchange chromatography.

The overall average reduction coefficient for all four model viruses is significantly high, which indicates the high capacity of the monoclonal antibody production process in inactivating and removing viruses leads to reducing the load of all four model viruses.