Outbreak of colistin and carbapenem-resistant Klebsiella pneumoniae ST16 co-producing NDM-1 and OXA-48 isolates in an Iranian hospital.

BACKGROUND: Colistin and carbapenem-resistant Klebsiella pneumoniae (Col-CRKP) represent a significant and constantly growing threat to global public health. We report here an outbreak of Col-CRKP infections during the fifth wave of COVID-19 pandemic. METHODS: The outbreak occurred in an intensive care unit with 22 beds at a teaching university hospital, Isfahan, Iran. We collected eight Col-CRKP strains from seven patients and characterized these strains for their antimicrobial susceptibility, determination of hypermucoviscous phenotype, capsular serotyping, molecular detection of virulence and resistance genes. Clonal relatedness of the isolates was performed using MLST. RESULTS: The COVID-19 patients were aged 24-75 years with at least 50% pulmonary involvement and were admitted to the intensive care unit. They all had superinfection caused by Col-CRKP, and poor responses to antibiotic treatment and died. With the exception of one isolate that belonged to the ST11, all seven representative Col-CRKP strains belonged to the ST16. Of these eight isolates, one ST16 isolate carried the iucA and ybtS genes was identified as serotype K20 hypervirulent Col-CRKP. The blaSHV and blaNDM-1 genes were the most prevalent resistance genes, followed by blaOXA-48 and blaCTX-M-15 and blaTEM genes. Mobilized colistin-resistance genes were not detected in the isolates. CONCLUSIONS: The continual emergence of ST16 Col-CRKP strains is a major threat to public health worldwide due to multidrug-resistant and highly transmissible characteristics. It seems that the potential dissemination of these clones highlights the importance of appropriate monitoring and strict infection control measures to prevent the spread of resistant bacteria in hospitals.

Immunization with recombinant DcaP-like protein and AbOmpA revealed protections against sepsis infection of multi-drug resistant Acinetobacter baumannii ST2Pas in a C57BL/6 mouse model.

BACKGROUNDS: The prevalence of infections associated with multi-drug resistant (MDR) Acinetobacter baumannii is increasing worldwide. Therefore, the introduction of effective vaccines against this bacterium seems necessary. METHODS: AbOmpA and DcaP-like protein were selected as promising and putative immunogenic candidates based on previous in silico studies. Three formulations including AbOmpA, DcaP-like protein, and AbOmpA + DcaP-like protein were injected into C57BL/6 mice three times with Alum adjuvant. The specific production of IgG antibodies (e.g. total IgG, IgG1 and IgG2c) and cytokines (e.g. IL-4, IL-6, and IL-17A), were evaluated. LD50% of MDR A. baumannii ST2Pas was measured using Probit's method. After the challenge with bacteria, a decrease in bacterial loads (DLs) in the lung and spleen of mice was measured. Then serum bactericidal assay was performed to determine the function of antibodies on day 42. In addition, histopathological examinations of the spleen and lung, the number of macrophage and neutrophil, as well as the rate of lymphocyte infiltration were assessed. RESULTS: The highest level of total IgG was reported in the group immunized with DcaP-like protein on day 42. The survival rate of mice was 80% in the AbOmpA immunized group and 100% for the rest of two groups. DLs in the spleen of mice immunized with AbOmpA, DcaP-like protein, and combination form were 3.5, 3, and 3.4 Log10 (CFU/g), respectively. While in the lung, the DLs were 7.5 Log10 (CFU/g) for the AbOmpA group and 5 for the rest of two groups. The levels of IL-6, IL-4, and IL-17A were significantly decreased in all immunized groups after the bacterial challenge (except for IL-17A in the group of AbOmpA). The bactericidal effect of antibodies against DcaP-like protein was more effective. No histopathological damage was observed in the combination immunized group. The DcaP-like protein was more effective in neutrophil and macrophage deployment and decreased lymphocyte infiltration. CONCLUSION: The results of immunization with AbOmpA + DcaP-like protein induced a protective reaction against the sepsis infection of MDR A. baumannii. It seems that in the future, these proteins can be considered as promising components in the development of the A. baumannii vaccine.

Various arrangements of mobile genetic elements among CC147 subpopulations of Klebsiella pneumoniae harboring blaNDM-1: a comparative genomic analysis of carbapenem resistant strains.

BACKGROUND: Certain clonal complexes (CCs) of Klebsiella pneumoniae such as CC147 (ST147 and ST392) are major drivers of blaNDM dissemination across the world. ST147 has repeatedly reported from our geographical region, but its population dynamics and evolutionary trajectories need to be further studied. METHODS: Comparative genomic analysis of 51 carbapenem-nonsusceptible strains as well as three hypervirulent K. pneumoniae (hvKp) recovered during 16-months of surveillance was performed using various bioinformatics tools. We investigated the genetic proximity of our ST147 strains with publicly available corresponding genomes deposited globally and from neighbor countries in our geographic region. RESULTS: While IncL/M plasmid harboring blaOXA-48 was distributed among divergent clones, blaNDM-1 was circulated by twenty of the 25 CC147 dominant clone and were mostly recovered from the ICU. The NDM-1 core structure was bracketed by a single isoform of mobile genetic elements (MGEs) [ΔISKpn26-NDM-TnAs3-ΔIS3000-Tn5403] and was located on Col440I plasmid in 68.7% of ST392. However, various arrangements of MGEs including MITESen1/MITESen1 composite transposon or combination of MITESen1/ISSen4/IS903B/IS5/ISEhe3 on IncFIb (pB171) were identified in ST147. It seems that ST392 circulated blaNDM-1 in 2018 before being gradually replaced by ST147 from the middle to the end of sample collection in 2019. ST147 strains possessed the highest number of resistance markers and showed high genetic similarity with four public genomes that harbored blaNDM-1 on the same replicon type. Mainly, there was a convergence between clusters and isolated neighboring countries in the minimum-spanning tree. A conserved arrangement of resistance markers/MGEs was linked to methyltransferase armA which was embedded in class 1 integron in 8 isolates of ST147/ST48 high-risk clones. CONCLUSION: Our findings highlight the dynamic nature of blaNDM-1 transmission among K. pneumoniae in Iran that occurs both clonally and horizontally via various combinations of MGEs. This is the first analysis of Iranian ST147/NDM + clone in the global context.

Clonal relatedness of carbapenem-resistant Acinetobacter baumannii: high prevalence of ST136pas in a burn center.

BACKGROUND: Carbapenem-resistant Acinetobacter baumannii (CRAB) is a global health crisis. This study aimed to determine the clonal relatedness of antibiotic-resistant A. baumannii isolates in hospitalized patients who suffered from burn wound infection. METHODS: One hundred and six A. baumannii isolates from 562 patients with burn wound infections, were identified and examined for antimicrobial susceptibility. Detection and characterization of carbapenem-hydrolyzing class D OXA-type beta-lactamases (CHDLs) were performed by PCR assays. The clonal relatedness of A. baumannii isolates was determined by multilocus sequence typing (MLST) according to the Pasteur scheme, dual-sequence typing of blaOXA-51-like and ampC genes, and RAPD-PCR method. RESULTS: All isolates were carbapenem-resistant while susceptible to colistin, minocycline, doxycycline, and ampicillin-sulbactam. The intrinsic blaOXA-51-like was detected in all isolates, and blaOXA-23-like was identified in 92.5% of isolates. However, blaOXA-143-like and blaOXA-58-like genes were not detected among isolates. Four distinct blaOXA-51-like alleles were determined as follows: blaOXA-317 (67.0%), blaOXA-90 (9.4%), blaOXA-69 (17.0%), and blaOXA-64 (6.6%) and four ampC (blaADC) allele types including ampC-25 (6.6%), ampC-39 (9.4%), ampC-1 (17.0%), and blaADC-88 (67.0%) were identified. MLST (Pasteur scheme) analysis revealed four ST types including ST136 (singleton), ST1 (CC1), ST25 (CC25), and ST78 (singleton) in 71, 18, 7, and 10 of A. baumannii strains, respectively. Five RAPD clusters including A (1.9%), B (26.4%), C (57.5%), D (7.5%), and E (1.9%) were characterized and 5 (4.7%) strains were found to be singletons. CONCLUSION: The present study demonstrated that there was a high prevalence of blaOXA-23-like producing CRAB in the clinical setting. The majority of isolates belonged to ST136 (singleton). However, blaOXA-23-like producing multi-drug resistant international clones including ST1, and emerging lineages (e.g. ST25 and ST78) were also identified. Interestingly, in this study ST2 was not detected.

Antibiotic Susceptibility, Biofilm-Forming Ability, and Prevalence of Extended-Spectrum Beta-Lactamase (ESBL)- and Biofilm-Associated Genes Among Klebsiella pneumoniae Isolates from Hospitalized Patients in Northwest of Iran.

Klebsiella pneumoniae is an opportunistic bacterium, which is globally recognized for its high prevalence and antimicrobial resistance (AMR). Biofilm-forming capability, susceptibility testing, and phenotypic confirmatory test for extended-spectrum beta-lactamase (ESBL)-producing isolate recognition of 104 K. pneumoniae isolates were performed according to the Clinical Laboratory Standard Institute (CLSI) guidelines. The prevalence of ESBL-associated genes bla-VIM, bla-NDM, and bla-OXA-48, as well as biofilm-associated genes luxS, fimH1, wza, and mrkD, was determined by multiplex PCR. The highest resistance rate was against ampicillin (100.0%). Among the 104 K. pneumoniae isolates, 52 (50.0%) and 31 (29.8%) isolates were determined as multi- and extensively drug resistant (MDR, XDR), respectively. Moreover, 21 (40.4%) isolates were determined as ESBL producing. Among 50 biofilm-producing K. pneumoniae isolates, 7 (14.0%), 15 (30.0%), and 28 (56.0%) isolates exhibited high, moderate, and weak levels of biofilm formation, respectively. A number of 41 (78.8%) isolates were susceptible to colistin, and 10 (19.2%) were resistant. AMR was significantly higher (P < 0.05) in the biofilm-forming isolates compared with non-biofilm formers.

Detection of multidrug-resistant Acinetobacter baumannii from burn patients and healthcare workers in Iran.

Multidrug-resistant (MDR) Acinetobacter baumannii is a serious global health threat. Burn patients are at high risk to acquire A. baumannii infections from endogenous sources. This study evaluated carbapenem resistance and clonal relatedness of A. baumannii isolated from burn patients and healthcare workers (HCWs).The study was performed in 100 non-duplicated A. baumannii isolates from nasal and hand samples of hospitalized burn patients and HCWs in two hospitals of Iran from June 2020 to August 2021. Antimicrobial susceptibility testing was performed and carbapenemase genes were detected by PCR. Clonal relatedness of A. baumannii isolates was determined by two single-locus sequence-based typing of blaOXA-51-like and ampC and by multilocus sequence typing (MLST).All A. baumannii isolates were found to be MDR while susceptible to colistin. The intI1, conserved segments of class 1 integron (intI1 CS), blaIMP, blaVIM, blaOXA-51-like, and blaOXA-23-like, genes were detected in 32.5%, 29.1%, 36%, 95.3%, 100%, 100%; and 14.3%, 14.3%, 21.4%, 92.9%, 100%, and 85.7% of isolates from patients and from healthcare workers, respectively. The blaOXA-58, and blaOXA-143 were not detected among the isolates. Using dual-locus blaOXA-51-like and ampC sequence-based typing (SBT), the isolates obtained from nasal samples of burn patients were grouped into 3 clusters including blaOXA-317, blaADC-88 (72.1%); blaOXA-64, ampC-25 (18.6%); and blaOXA-69, ampC-1 (9.3%). While only allele type blaOXA-317, blaADC-88 was determined among isolates from HCWs. MLST results showed A. baumannii ST136, ST25, and ST1 from burn patients. However, A. baumannii strains from HCWs belonged to ST136. Our findings indicate high prevalence of globally spreading of MDR A. baumannii ST136 carrying blaOXA-23-like from nasal and hand samples of burn patients and HCWs.

Subtractive genomic approach toward introduction of novel immunogenic targets against Clostridioides difficile: Thinking out of the box.

Clostridioides difficile is one of the major causatives of nosocomial infections worldwide. Antibiotic-associated diarrhea, pseudomembranous colitis, and toxic megacolon are the most common forms of C. difficile infection (CDI). Considering the high antibiotic resistance of C. difficile isolates and the low efficacy of immunization with toxin-related vaccines, we suggested that surface-exposed and secreted proteins could be considered as potential immunogenic targets against CDI. Various immuninformatics databases were used to predict antigenicity, allergenicity, B-cell epitopes, MHC-II binding sites, conserved domains, prevalence and conservation of proteins among the most common sequence types, molecular docking, and immunosimulation of immunogenic targets. Finally, 16 proteins belonging to three functional groups were identified, including proteins involved in the cell wall and peptidoglycan layer (nine proteins), flagellar assembly (five proteins), spore germination (one protein), and a protein with unknown function. Molecular docking results showed that among all the mentioned proteins, WP_009892971.1 (Acd) and WP_009890599.1 (a C40 family peptidase) had the strongest interactions with human Toll-like receptor 2 (TLR-2) and TLR-4. This study proposes a combination of C. difficile toxoid (Tcd) and surface-exposed proteins such as Acd as a promising vaccine formulation for protection against circulating clinical strains of C. difficile.

Identification of novel putative immunogenic targets and construction of a multi-epitope vaccine against multidrug-resistant Corynebacterium jeikeium using reverse vaccinology approach.

The emergence of multidrug-resistant Corynebacterium jeikeium has limited treatment options and resulted in the inability to treat C. jeikeium infections, especially in immunocompromised patients. To our knowledge, no studies have been conducted to evaluate C. jeikeium antigens for vaccine development. Given the lack of effective treatments against C. jeikeium, this study aimed to identify potential immunogenic targets against C. jeikeium as a nosocomial pathogen using a reverse vaccinology approach. To achieve this goal, we performed several immuninformatics analyses, including antigenicity, allergenicity, PSI-BLAST to the human proteome, physiochemical properties, B-cell and T-cell epitopes, molecular docking, and immunosimulation. In addition, quartile scoring and prevalence assessment were used to select the most abundant immunogenic targets in different C. jeikeium strains. Finally, protein-protein interactions were performed and the multi-epitope vaccine was developed. Five putative immunogenic targets were presented as short-listed proteins in this study, including three enzymatic proteins (WP_011273969.1, WP_041626322.1, and WP_005292204.1), one protein with DUF3235 domain (WP_011273103.1), and one hypothetical protein (WP_005293648.1). Four linear B-cell epitopes of putative immunogenic targets, including WP_011273103.1 (LNSKPTPRNAAAKPKAK), WP_011273969.1 (GEGAQGSAAPADAQATANE), WP_005292204.1 (ASVSAAQKADGIAP), and WP_041626322.1 (YSKKVAEEMGVG) were selected and inserted into the mutant TbpB C-lobe protein. This platform can effectively present multiple epitopes to the immune system. However, experimental in vitro and in vivo analysis is required to confirm the safety, immunoreactivity, and efficacy of these putative immunogenic targets.

High heterogeneity of fecal carriage extended-spectrum beta-lactamase-producing E. coli isolated from iranian community and clinical settings.

BACKGROUND: Extended-spectrum beta-lactamase-producing enterobacteria (ESBL-PE) in carriers have become a global health problem. Using molecular typing techniques, including PFGE, could be useful to determine the source of bacterial dissemination. The current study aimed to investigate the intestinal carriage of ESBL-producing E. coli (ESBL-EC) and clonal relatedness among ESBL-EC isolated from hospitalized and outpatient fecal carriers in Iran. METHODS: A total of 120 rectal swabs were collected; 50.8% (61/120) from intensive care unit (ICU) inpatients and 49.2% (59/120) from outpatients. MacConkey agar enriched with cefotaxime was used to screen the ESBL-EC. PCR assays were performed to detect ESBL and carbapenemase genes. Pulse-fields gel electrophoresis (PFGE) was performed to assess clonal relatedness. RESULTS: Totally, 60.0% (72/120) were carrier for ESBL-EC. The rates of resistance against ceftazidime and cefepime were 90.2% (65/72) and 93.0% (67/72), respectively. The rates of blaCTX-M-15, blaTEM, blaSHV, blaNDM-1, blaOXA-48 and blaIMP was 90.2% (65/72), 50.0% (36/72), 5.5% (4/72), 4.1% (3/72), 4.1% (3/72) and 1.3% (1/72), respectively. Based on a cut-off 80%, 69 ESBL-EC isolates could be categorized in 10 mini-cluster and 47 isolates were considered as singletons. DISCUSSION: High heterogeneity among isolates from ESBL-EC suggests that this bacterium probably has a different source of dissemination. Screening of carriers in hospitals and communities could help the infection control program in public health.

Nanosized paclitaxel-loaded niosomes: formulation, in vitro cytotoxicity, and apoptosis gene expression in breast cancer cell lines.

BACKGROUND: In this study, the optimized niosomal formulation containing paclitaxel using non-ionic surfactants and cholesterol was designed and its cytotoxic effects against different breast cancer cell lines and apoptosis gene expression analysis were also investigated. METHODS AND RESULTS: Due to enhancing equation variables, the Box-Behnken method has been applied. Lipid/drug molar ratio, the amounts of Span 60, and cholesterol were selected as the target for optimization. The particle size of niosome loaded paclitaxel and entrapment efficiency proportion have been considered in the role of dependent variables. Then the cytotoxic activity of the optimized formulation was evaluated using an MTT assay against different breast cancer cell lines including MCF-7, T-47D, SkBr3, and MDA-MB-231. The expression level of Bax and Bcl-2 apoptosis genes was determined by Real-Time PCR. In this study, the optimized niosomal formulation revealed that the synthesized niosomes had a spherical appearance and had an average size of 192.73 ± 5.50 nm so that the percentage of drug loading was 94.71 ± 1.56%. Moreover, this formulation showed a controlled and slowed release of paclitaxel at different pH (7.4, 6.5, and 5.4). The cytotoxicity results demonstrated that cell viability in all concentrations of niosome loaded paclitaxel had profound cytotoxic effects on all studied breast cancer cell lines compared to the free paclitaxel (p < 0.05). In addition, the expression of apoptosis genes was much higher than that of free paclitaxel indicating the susceptibility of cells to apoptosis. CONCLUSIONS: As a result, niosomal formulations containing paclitaxel can be used as a new drug delivery system to increase cytotoxicity and treatment of breast cancer in the upcoming future.

Comparative in silico characterization of Klebsiella pneumoniae hypervirulent plasmids and their antimicrobial resistance genes.

BACKGROUND: The hypervirulent pathotype of Klebsiella pneumoniae (hvKp) is mainly mediated by large virulent plasmids. It seems that these hypervirulent plasmids (HVPs) are accumulating antimicrobial resistance genes (ARGs) and are turning quickly into drug-resistant hypervirulent hybrids. Therefore, molecular mechanisms involved in this convergence needs to be investigated to control their global spread. METHODS: In this study, the complete sequence of 79 non-redundant hypervirulent plasmids were retrieved from GenBank and their genetic features, hypervirulence and antimicrobial resistance patterns (AMR) as well as their putative transmission capability were compared using bioinformatics tools. RESULTS: The majority of HVPs belonged to clonal complex (CC)23, and sequence type (ST)11. IncFIB and IncHI1B were the most prevalent plasmid replicon types. Out of 79 plasmids, 78 were positive for iutA and iucA. The iucC, iucB and iucD genes were found in 77 plasmids. Almost 26% of the HVPs were potentially conjugative of which 71% carried AGRs. ARGs against beta-lactams, carbapenems, quinolones, aminoglycosides, chloramphenicols, tetracyclines and macrolides were detected in 30% of HVPs. Class 1 integron and prophage structures harboring multiple ARGs were found in eight plasmids. Insertion sequences (IS)6, IS110 and IS1380 appeared to be important genetic elements in transmission of ARGs. CONCLUSIONS: The high prevalence of iucA and iutA suggests their strong capability for rapid and accurate genetic markers for discrimination of hvKp in the laboratory. This study indicated the important role of mobile genetic elements (MGEs) in the emergence of drug-resistance in hypervirulent strains. The high prevalence of putative conjugative hybrids implies higher incidence of multidrug-resistant (MDR)-hvKp strains in near future.

Detection of ESBL and AmpC producing Klebsiella pneumoniae ST11 and ST147 from urinary tract infections in Iran.

In the present study a total of 200 Klebsiella pneumoniae isolates were collected from patients with urinary tract infections (UTIs) in Tehran, Iran. Antibiotic resistance was determined by disk diffusion and broth dilution methods. Detection of extended-spectrum ß-lactamases (ESBLs) and AmpCs was performed using phenotypic tests. Polymerase chain reaction (PCR) was applied to detect the ESBL, AmpC, and integron genes. Analysis of AmpC and cassette arrays of integron genes was performed using DNA sequencing. Plasmids were analyzed by PCR-based replicon typing and conjugation. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) were applied to explore the genomic relatedness among the isolates. The highest levels of resistance were observed against ampicillin (100%), followed by piperacillin (57.5%), ceftazidime (46%), trimethoprim/sulfamethoxazole (44%), ciprofloxacin (32.5%), and imipenem (19%). Approximately, 66.5% of isolates harbored at least one of the beta-lactamase genes (blaTEM, blaSHV, blaCTX-M, and blaOXA-1). In addition, 22.5% of isolates carried at least one of the AmpC genes including blaDHA and blaCIT. Integron class I was the most prevalent integron among resistant isolates. According to the results of replicon typing, IncFII, IncL/M, and IncA/C were the most frequent replicons, respectively. All selected isolates were able to transfer blaCTX-M, also two isolates transferred the blaDHA-1 gene to Escherichia coli K12 through conjugation. Finally, 21 isolates were categorized into 4 pulsotypes and 11 unique clusters in PFGE. MLST identified ST147 and ST11 sequence types but ST147 was the most prevalent in the current study.

Fabrication and Characterization of Thymol-Loaded Chitosan Nanogels: Improved Antibacterial and Anti-Biofilm Activities with Negligible Cytotoxicity.

Thymol is a monoterpene phenolic derivative extracted from the Thymus vulgaris which has antimicrobial effects. In the present study, thymol-loaded chitosan nanogels were prepared and their physicochemical properties were characterized. The encapsulation efficiency of thymol into chitosan and its stability were determined. The in vitro antimicrobial and anti-biofilm activities of thymol-loaded chitosan nanogel (Ty-CsNG), free thymol (Ty), and free chitosan nanogel (CsNG) were evaluated against both Gram-negative and Gram-positive multidrug-resistant (MDR) bacteria including Staphylococcus aureus, Acinetobacter baumanii, and Pseudomonas aeruginosa strains using the broth microdilution and crystal violet assay, respectively. After treatment of MDR strains with sub-minimum inhibitory concentration (Sub-MIC) of Ty-CsNG, free Ty and CsNG, biofilm gene expression analysis was studied. Moreover, cytotoxicity of Ty-CsNG, free Ty, and CsNG against HEK-293 normal cell line was determined using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) method. The average size of Ty-CsNG was 82.71±9.6 nm, encapsulation efficiency was 76.54±0.62 % with stability up to 60 days at 4 °C. Antibacterial activity test revealed that Ty-CsNG reduced the MIC by 4-6 times in comparison to free thymol. In addition, the expression of biofilm-related genes including ompA, and pgaB were significantly down-regulated after treatment of strains with Ty-CsNG (P<0.05). In addition, free CsNG displayed negligible cytotoxicity against HEK-293 normal cell lines and presented a biocompatible nanoscale delivery system. Based on the results, it can be concluded that Ty-CsNG can be considered a promising candidate for enhancing antimicrobial and anti-biofilm activities.

Emergence of carbapenem resistant Acinetobacter baumannii clonal complexes CC2 and CC10 among fecal carriages in an educational hospital.

Carbapenem-resistant Acinetobacter baumannii strains are increasing worldwide. In this study, samples were collected from hospital environments, extra hospital environments, and fecal carriages. 76% (89/117) of bacterial isolates were detected as A. baumannii strains. The imipenem resistance in the hospital environment, fecal carriages, extra hospital environments, and clinical isolates was 37.7% (17/45), 100% (9/9), 0% (0/45), and 92.9% (92/99), respectively. The blaVIM and blaOXA-23 were detected in 6.6% (3/45) and 2.2% (1/45) of strains isolated from hospital environments. Interestingly, strains isolated from fecal carriages had blaVIM, blaOXA-23, and blaIMP genes which resembled carbapenem resistance genes in clinical strains. The structure of clonal relatedness among all non-clinical isolates was as follows: CC2, 37% (33/89); CC1, 22.4% (20/89); CC3, 12.3% (11/89); CC25, 7.8% (7/89); CC10, 4.4% (4/89) and CC15, 2.2% (2/89). Comparison of clonal relatedness among clinical and non-clinical isolates indicated that widespread clones including CC2, CC3, and CC10 were common clonal complexes between two categories.

The combination of CipA and PBP-7/8 proteins contribute to the survival of C57BL/6 mice from sepsis of Acinetobacter baumannii.

Due to the emergence of multi-drug resistant Acinetobacter baumannii strains, there is an urgent need to develop several new strategies to control this bacterium. In this context, vaccination may be the best approach to reduce the morbidity and mortality associated with MDR isolates in vulnerable groups. Serum resistance factors have a key role in the pathogenesis of A. baumannii and can be considered as potential vaccine candidates. This project aimed to evaluate the immunological reactivity of CipA and PBP-7/8 as two serum resistance factors in a combination form against sepsis infections of A. baumannii. Recombinant proteins were obtained and immunological evaluations were performed against sepsis infection in the C57BL/6 mouse model. The data showed a statistically significant increase in total IgG levels in all three immunization regimens (CipA, PBP-7/8, and CipA + PBP-7/8) compared to the control group. The ratios of IgG2c/IgG1 in the CipA, PBP-7/8, and CipA + PBP-7/8 schedules were 8.7, 46.50, and 33.29, respectively. It appears that the immunization schedules developed a strong polarized Th1 response. The cytokine profiles of the three plans showed that IFN-γ was highly concentrated in the combination plan. However, the highest concentration of IL-17 belonged to the PBP-7/8 plan. In conclusion, the data of total IgG, survival rates and splenic bacterial loads showed that the CipA + PBP-7/8 plan was more effective than each protein individually.

Two new rapid PCR-based methods for identification of Acinetobacter baumannii isolated from clinical samples.

The accurate identification of Acinetobacter spp. is challenging due to their high phenotypic and biochemical similarities. Because clinical relevance and antibiotic susceptibility are significantly different among different genomic species of Acinetobacter, the exact identification of A. baumannii is necessary and it can help us prevent inappropriate antibiotic use and inferior clinical care. This project employed a sequence-specific PCR assay for the rpoB region in A. baumannii to distinguish it from non-Acinetobacter baumannii Acinetobacter species. Moreover, a duplex PCR assay was used to detect blaOXA-51-like and gluconolactonase genes as a second identification method. In this study, 210 isolates of Acinetobacter spp. were considered and identified by PCR-sequencing of rpoB gene as a reference test. PCR-sequencing of rpoB revealed that 179 isolates were A. baumannii and 31 were non- A. baumannii Acinetobacter strains. PCR amplification targeting the rpoB gene as the first method, detected 182 isolates of A. baumannii, while duplex PCR assay confirmed 163 isolates as A. baumannii. Data analysis indicated that the sensitivities of sequence-specific PCR of the rpoB gene and duplex PCR assay were 100% and 91.06%, respectively, while specificities were 91.18% and 100%, respectively. Given the data, it was revealed that these two methods showed a reasonable potential for the accurate identification of A. baumannnii from non- A. baumannii species. Sequence-specific PCR assay for the rpoB gene and duplex PCR assay for blaOXA-51-like and gluconolactonase genes are rapid, reliable and cost-effective methods which can be used in clinical laboratories for the accurate identification of A. baumannii.

Molecular study of metallo-ß-lactamases and integrons in Acinetobacter baumannii isolates from burn patients.

BACKGROUND: Productions of metallo-ß-lactamases enzymes are the most common mechanism of antibiotic resistance to all beta-lactam classes (except monobactams) in Acinetobacter baumannii. MBLs are usually associated with gene cassettes of integrons and spread easily among bacteria. The current study was performed to detect the genes encoding MBLs and integron structures in A. baumannii isolates from burn patients. METHODS: This study was performed on 106 non-duplicate A. baumannii isolates from burn patients referred to Shahid Motahari Hospital in Tehran. Antibiotic susceptibility of A. baumannii isolates was performed using disk diffusion and broth microdilution method in accordance with the CLSI guidelines. The presence of class 1 integron and associated gene cassettes as well as MBLs-encoding genes including blaVIM, and blaIMP were investigated using PCR and sequencing techniques. RESULTS: In this cross-sectional study all (100%) of the A. baumannii isolates examined were multidrug resistant. All isolates were sensitive to colistin and simultaneously all were resistant to imipenem. PCR assays showed the presence of blaVIM and blaIMP genes in 102 (96.2%) and 62 (58.5%) isolates of A. baumannii respectively. In addition, 62 (58.5%) of the A. baumannii isolates carried integron class 1, of which 49 (79.0%) were identified with at least one gene cassette. Three types of integron class 1 gene cassettes were identified including: arr2, cmlA5, qacE1 (2300 bp); arr-2, ereC, aadA1, cmlA7, qacE1 (4800 bp); and aac(3)-Ic, cmlA5 (2250 bp). CONCLUSION: A high prevalence of MBLs genes, especially blaVIM, was identified in the studied MDR A. baumannii isolates. In addition, most of the strains carried class 1 integrons. Furthermore, the gene cassettes arrays of integrons including cmlA5 and cmlA7 were detected, for the first time, in A. baumannii strains in Iran.

The face of hypervirulent Klebsiella pneumoniae isolated from clinical samples of two Iranian teaching hospitals.

Hypervirulent Klebsiella pneumoniae (hvKp) has emerged as a pathogen of global concern. In this study, both phenotypic and genotypic tests were used to detect hvKp. Antimicrobial resistance profiles and clonal relatedness of clinical isolates were also determined. We found that 34.2% (163/477) of the isolates were tellurite resistant, and among them 102 hvKp isolates detected with iucA or iutA or peg-344 as molecular markers. The blaSHV (80.4%), followed by blaCTX-M-15 (76.5%) and blaTEM (67.6%), blaOXA-48 (53.9%), and blaNDM-1 (32.3%) were detected, while blaKPC-1 was not present in any hvKp isolates. It was found that the majority of hvKp isolates belonged to capsular serotype K20 and ompK36 group C, which is related to clonal group (CG) 23 (e.g. ST23). A high percentage of multidrug-resistant hvKp (76.6%) and high resistance to imipenem (67%) indicated a serious problem that should be addressed in the clinical setting.

Comparative genome analysis of colistin-resistant OXA-48-producing Klebsiella pneumoniae clinical strains isolated from two Iranian hospitals.

BACKGROUND: Carbapenemase-producing Klebsiella pneumoniae (CP-KP) is becoming extensively disseminated in Iranian medical centers. Colistin is among the few agents that retains its activity against CP-KP. However, the administration of colistin for treatment of carbapenem-resistant infections has increased resistance against this antibiotic. Therefore, the identification of genetic background of co-carbapenem, colistin-resistance K. pneumoniae (Co-CCRKp) is urgent for implementation of serious infection control strategies. METHODS: Fourteen Co-CCRKp strains obtained from routine microbiological examinations were subjected to molecular analysis of antimicrobial resistance (AMR) using whole genome sequencing (WGS). RESULTS: Nine of 14 K. pneumoniae strains belonged to sequence type (ST)-11 and 50% of the isolates had K-locus type 15. All strains carried blaOXA-48 except for P26. blaNDM-1 was detected in only two plasmids associated with P6 and P26 strains belonging to incompatibility (Inc) groups; IncFIB, IncHI1B and IncFII. No blaKPC, blaVIM and blaIMP were identified. Multi-drug resistant (MDR) conjugative plasmids were identified in strains P6, P31, P35, P38 and P40. MICcolistin of K. pneumoniae strains ranged from 4 to 32 µg/ml. Modification of PmrA, PmrB, PhoQ, RamA and CrrB regulators as well as MgrB was identified as the mechanism of colistin resistance in our isolates. Single amino acid polymorphysims (SAPs) in PhoQ (D150G) and PmrB (R256G) were identified in all strains except for P35 and P38. CrrB was absent in P37 and modified in P7 (A200E). Insertion of ISKpn72 (P32), establishment of stop codon (Q30*) (P35 and P38), nucleotides deletion (P37), and amino acid substitution at position 28 were identified in MgrB (P33 and P42). None of the isolates were positive for plasmid-mediated colistin resistance (mcr) genes. P35 and P38 strains carried iutA, iucD, iucC, iucB and iucA genes and are considered as MDR-hypervirulent strains. P6, P7 and P43 had ICEKp4 variant and ICEKp3 was identified in 78% of the strains with specific carriage in ST11. CONCLUSION: In our study, different genetic modifications in chromosomal coding regions of some regulator genes resulted in phenotypic resistance to colistin. However, the extra-chromosomal colistin resistance through mcr genes was not detected. Continuous genomic investigations need to be conducted to accurately depict the status of colistin resistance in clinical settings.

New putative vaccine candidates against Acinetobacter baumannii using the reverse vaccinology method.

Infections caused by multi-drug resistance Acinetobacter baumannii are increasing worldwide. Discovery of the vaccine against this bacterium as a cost-effective and preventive strategy seems necessary. This study has introduced 11 new putative vaccine candidates against A. baumannii using the reverse vaccinology method. We considered 33 genomes of A. baumannii strains and selected the outer membrane and secreted proteins as putative vaccine candidates using Vaxign web tool. Finally, 11 proteins were confirmed as promising vaccine candidates. These targets belonged to proteins involved in cell division (NlpD), fimbria or pili assembly (FimA, PapC, and PapC associated with usher system), iron acquisition (FhuA, BfnH, FatA-like protein, and IutA), DcaP-like protein and two novel hypothetical proteins (HP-1 and HP-2). The analysis of linear and conformational B-cell epitopes showed that the outer membrane proteins including DcaP-like protein and HP-2 had high conserved surface-exposed epitopes that they can consider as excellent putative vaccine targets in the upcoming immunological assays.