blaOXA Genes Research Articles

Investigation of antimicrobial resistance patterns and molecular typing of Pseudomonas aeruginosa isolates among Coronavirus disease-19 patients

BackgroundPseudomonas aeruginosa is a common co-infecting pathogen recognized among COVID-19 patients. We aimed to investigate the antimicrobial resistance patterns and molecular typing of Pseudomonas aeruginosa isolates among Coronavirus disease-19 patients.MethodsBetween December 2020 and July 2021, 15 Pseudomonas aeruginosa were isolated from COVID-19 patients in the intensive care unit at Sina Hospital in Hamadan, west of Iran. The antimicrobial resistance of the isolates was determined by disk diffusion and broth microdilution methods. The double-disk synergy method, Modified Hodge test, and polymerase chain reaction were utilized to detect Pseudomonas aeruginosa extended spectrum beta-lactamase and carbapenemase producers. Microtiter plate assay was performed to evaluate the biofilm formation ability of the isolates. The isolates phylogenetic relatedness was revealed using the multilocus variable-number tandem-repeat analysis method.ResultsThe results showed Pseudomonas aeruginosa isolates had the most elevated resistance to imipenem (93.3%), trimethoprim-sulfamethoxazole (93.3%), ceftriaxone (80%), ceftazidime (80%), gentamicin (60%), levofloxacin (60%), ciprofloxacin (60%), and cefepime (60%). In the broth microdilution method, 100%, 100%, 20%, and 13.3% of isolates showed resistance to imipenem, meropenem, polymyxin B, and colistin, respectively. Ten (66.6%) isolates were identified as multiple drug resistance. Carbapenemase enzymes and extended spectrum beta-lactamases were identified in 66.6% and 20% of the isolates, respectively and the biofilm formation was detected in 100% of the isolates. The blaOXA-48, blaTEM, blaIMP, blaSPM, blaPER, blaVEB, blaNDM, blaSHV, and blaCTX-M genes were detected in 100%, 86.6%, 86.6%, 40%, 20%, 20%, 13.3%, 6.6%, and 6.6% of the isolates, respectively. The blaVIM, blaGIM, blaGES, and blaMCR-1 genes were not identified in any of the isolates. The MLVA typing technique showed 11 types and seven main clusters and most isolates belong to cluster I, V and VII.ConclusionDue to the high rate of antimicrobial resistance, as well as the genetic diversity of Pseudomonas aeruginosa isolates from COVID-19 patients, it is indispensable to monitor the antimicrobial resistance pattern and epidemiology of the isolates on a regular basis.

Increased clonal dissemination of OXA-232-producing ST15 Klebsiella pneumoniae in Zhejiang, China from 2018 to 2021

BackgroundOXA-232-producing Klebsiella pneumoniae was first identified in China in 2016, and its clonal transmission was reported in 2019. However, there are no prevalence and genotypic surveillance data available for OXA-232 in China. Therefore, we investigated the trends and characteristics of OXA-232 type carbapenemase in Zhejiang Province, China from 2018 to 2021.MethodsA total of 3278 samples from 1666 patients in the intensive care units were collected from hospitals in Zhejiang Province from 2018 to 2021. Carbapenem-resistant isolates were initially selected by China Blue agar plates supplemented with 0.3 μg/ml meropenem, and further analyzed by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry identification, immune colloidal gold technique, conjugation experiment, antimicrobial susceptibility testing and whole genome sequencing.ResultsA total of 79 OXA-producing strains were recovered, with the prevalence increased from 1.8% [95% confidence interval (CI): 0.7–3.7%] in 2018 to 6.0% (95% CI: 4.4–7.9%) in 2021. Seventy-eight strains produced OXA-232 and one produced OXA-181. The blaOXA-232 gene in all strains was located in a 6141-bp ColKP3-type non-conjugative plasmid and the blaOXA-181 gene was located in a 51,391-bp ColKP3/IncX3-type non-conjugative plasmid. The blaOXA-232-producing K. pneumoniae was dominated (75/76) by isolates of sequence type 15 (ST15) that differed by less than 80 SNPs. All OXA-producing strains (100%, 95% CI: 95.4–100.0%) were multidrug-resistant.ConclusionsFrom 2018 to 2021, OXA-232 is the most prevalent OXA-48-like derivative in Zhejiang Province, and ST15 K. pneumoniae isolates belonging to the same clone are the major carriers. The transmission of ColKP3-type plasmid to E. coli highlighted that understanding the transmission mechanism is of great importance to delay or arrest the propagation of OXA-232 to other species.

Identification and Characterization of OXA-232-Producing Sequence Type 231 Multidrug Resistant Klebsiella pneumoniae Strains Causing Bloodstream Infections in China.

Klebsiella pneumoniae, a notorious pathogen for opportunistic health care-associated infections, represents increasing multidrug resistance, particularly to carbapenems. OXA-232 carbapenemase, as a variant of OXA-48, has been increasingly reported worldwide. ST231, an epidemic, multidrug resistant (MDR) K. pneumoniae clone in south and southeast Asia, has been found in other regions, including Europe. In the study, five OXA-232 carbapenemase-producing Klebsiella pneumoniae isolates, four of which belong to sequence type 231 (ST231) and one of which belongs to ST15, were isolated from two hospitals in China. All isolates displayed a MDR phenotype, being susceptible to only polymyxin B and colistin, and the blaOXA-232 gene was located on a ColKP3-type nonconjugative plasmid of 6.1 kb. A phylogenetic analysis of the global ST231 K. pneumoniae isolates (n = 231) suggested that the four ST231 isolates from this study gathered with strains from south Asia (especially India), indicating that the emerging Chinese ST231 clone was more closely related to south Asia isolates and might have spread from south Asia, where ST231 was a successful epidemic clone. Virulence assays suggested that the four ST231 strains were not highly virulent, as they displayed significantly lower virulence potential, compared with a ST23 K1 hypervirulent isolate in a G. mellonella infection and in mouse intraperitoneal infection models, although three ST231 strains harbored a plasmid-borne aerobactin-encoding iuc gene cluster. This is the first report of ST231 K. pneumoniae clinical strains bearing blaOXA-232 in China, and it highlights the emergence of the ST231 clone causing bloodstream infections in a health care setting as well as calls attention to the transmission of this emerging clone in China. IMPORTANCE OXA-232 carbapenemase, being a vital resistance mechanism against carbapenems, has recently been increasingly reported. In China, the identified OXA-232-producing K. pneumoniae isolates almost belonged to ST15 and were not hypervirulent, despite harboring a virulence plasmid. Here, we report the first occurrence in China of a MDR OXA-232-producing K. pneumoniae ST231 clone that is an epidemic ST type in south and southeast Asia. A phylogenetic analysis indicated that this emerging Chinese ST231 clone was more closely related to Indian isolates. The occurrence of this clone may have been driven through the transnational importation of Indian ST231 K. pneumoniae clones. Moreover, this study is the first to assess the virulence potential of ST231 clones that have never been estimated in previous studies. While the high burden of MDR K. pneumoniae is concerning, genomic surveillance can shed light on the transmission chains of novel MDR clones, and active surveillance should be enforced to restrict the spread of MDR isolates.

Healthcare-Associated Bloodstream Infections Due to Multidrug-Resistant Acinetobacter baumannii in COVID-19 Intensive Care Unit: A Single-Center Retrospective Study.

Healthcare-associated infections are an emerging cause of morbidity and mortality in COVID-19 intensive care units (ICUs) worldwide, especially those caused by multidrug-resistant (MDR) pathogens. The objectives of this study were to assess the incidence of bloodstream infections (BSIs) among critically ill COVID-19 patients and to analyze the characteristics of healthcare-associated BSIs due to MDR Acinetobacter baumannii in an COVID-19 ICU. A single-center retrospective study was conducted at a tertiary hospital during a 5-month period. The detection of carbapenemase genes was performed by PCR and genetic relatedness by pulsed-field gel electrophoresis (PFGE) and multilocus-sequence typing. A total of 193 episodes were registered in 176 COVID-19 ICU patients, with an incidence of 25/1000 patient-days at risk. A. baumannii was the most common etiological agent (40.3%), with a resistance to carbapenems of 100%. The blaOXA-23 gene was detected in ST2 isolates while the blaOXA-24 was ST636-specific. PFGE revealed a homogeneous genetic background of the isolates. The clonal spread of OXA-23-positive A. baumannii is responsible for the high prevalence of MDR A. baumannii BSIs in our COVID-19 ICU. Further surveillance of resistance trends and mechanisms is needed along with changes in behavior to improve the implementation of infection control and the rational use of antibiotics.

Dissemination of carbapenemase-producing Enterobacterales in Ireland from 2012 to 2017: a retrospective genomic surveillance study.

The spread of carbapenemase-producing Enterobacterales (CPE) is of major public health concern. The transmission dynamics of CPE in hospitals, particularly at the national level, are not well understood. Here, we describe a retrospective nationwide genomic surveillance study of CPE in Ireland between 2012 and 2017. We sequenced 746 national surveillance CPE samples obtained between 2012 and 2017. After clustering the sequences, we used thresholds based on pairwise SNPs, and reported within-host diversity along with epidemiological data to infer recent putative transmissions. All clusters in circulating clones, derived from high-resolution phylogenies, of a species (Klebsiella pneumoniae, Escherichia coli, Klebsiella oxytoca, Enterobacter cloacae, Enterobacter hormaechei and Citrobacter freundii) were individually examined for evidence of transmission. Antimicrobial resistance trends over time were also assessed. We identified 352 putative transmission events in six species including widespread and frequent transmissions in three species. We detected putative outbreaks in 4/6 species with three hospitals experiencing prolonged outbreaks. The bla OXA-48 gene was the main cause of carbapenem resistance in Ireland in almost all species. An expansion in the number of sequence types carrying bla OXA-48 was an additional cause of the increasing prevalence of carbapenemase-producing K. pneumoniae and E. coli.

Epidemic characteristics of the SXT/R391 integrated conjugative elements in multidrug-resistant Proteus mirabilis isolated from chicken farm

This study was designed to depict prevalence and antimicrobial resistance characteristics of Proteus mirabilis (P. mirabilis) strains in 4 chicken farms and to probe the transfer mechanism of resistance genes. A total of 187 P. mirabilis isolates were isolated from 4 chicken farms. The susceptibility testing of these isolates to 14 antimicrobials showed that the multidrug resistance (MDR) rate was as high as 100%. The β-lactamase resistance genes blaOXA-1, blaCTX-M-1G, blaCTX-M-9G and colistin resistance gene mcr-1 were highly carried in the P. mirabilis isolates. An MDR strain W47 was selected for whole genome sequencing (WGS) and conjugation experiment. The results showed that W47 carried 23 resistance genes and 64 virulence genes, and an SXT/R391 integrated conjugative elements (ICEs) named ICEPmiChn5 carrying 17 genes was identified in chromosome. ICEPmiChn5 was able to be excised from the chromosome of W47 forming a circular intermediate, but repeated conjugation experiments were unsuccessful. Among 187 P. mirabilis isolates, 144 (77.01%, 144/187) isolates carried ICEPmiChn5-like ICEs, suggesting that ICEs may be the major vector for the transmission of resistance genes among MDR chicken P. mirabilis strains in this study. The findings were conducive to insight into the resistance mechanism of chicken P. mirabilis strains and provide a theoretical basis for the use of antibiotics for the treatment of MDR P. mirabilis infections in veterinary clinic.

The Prevalence of Virulence Factor Genes among Carbapenem-Non-Susceptible Acinetobacter baumannii Clinical Strains and Their Usefulness as Potential Molecular Biomarkers of Infection

Healthcare-associated infections caused by multidrug-resistant Acinetobacter baumannii strains are a serious global threat. Therefore, it is important to expand the knowledge on the mechanisms of pathogenicity of these particular bacteria. The aim of this study was to assess the distribution of selected virulence factor genes (bap, surA1, omp33-36, bauA, bauS, and pld) among carbapenem-non-susceptible clinical A. baumannii isolates and to evaluate their potential usefulness as genetic markers for rapid diagnostics of A. baumannii infections. Moreover, we aimed to compare the virulence genes prevalence with the occurrence of carbapenemases genes. A total of 100 carbapenem-non-susceptible A. baumannii clinical isolates were included in the study. The presence of virulence factors and blaOXA genes was evaluated by real-time PCR. The occurrence of virulence factors genes was as follows: 100.0% for the bap and surA1 genes, 99.0% for the basD and pld genes. The bauA and omp33-36 genes were absent among the studied strains. The predominant genes (bap and surA1) are involved in biofilm formation and their presence among all clinical strains can be applied as a genetic marker to recognize A. baumannii infection. High frequencies of the basD gene-involved in siderophore biosynthesis and the gene encoding phospholipase D (pld)-were also noted among blaOXA-positive strains, showing their potential role in a pathogenicity of blaOXA-positive A. baumannii clinical strains.

Characterization of Third Generation Cephalosporin- and Carbapenem-Resistant Aeromonas Isolates from Municipal and Hospital Wastewater.

Antibiotic resistance (AR) remains one of the greatest threats to global health, and Aeromonas species have the potential to spread AR in the aquatic environment. The spread of resistance to antibiotics important to human health, such as third-generation cephalosporins (3GCs) and carbapenems, is of great concern. We isolated and identified 15 cefotaxime (3GC)- and 51 carbapenem-resistant Aeromonas spp. from untreated hospital and treated municipal wastewater in January 2020. The most common species were Aeromonas caviae (58%), A. hydrophila (17%), A. media (11%), and A. veronii (11%). Almost all isolates exhibited a multidrug-resistant phenotype and harboured a diverse plasmidome, with the plasmid replicons ColE, IncU, and IncR being the most frequently detected. The most prevalent carbapenemase gene was the plasmid-associated blaKPC-2 and, for the first time, the blaVIM-2, blaOXA-48, and blaIMP-13 genes were identified in Aeromonas spp. Among the 3GC-resistant isolates, the blaGES-5 and blaMOX genes were the most prevalent. Of the 10 isolates examined, three were capable of transferring carbapenem resistance to susceptible recipient E. coli. Our results suggest that conventionally treated municipal and untreated hospital wastewater is a reservoir for 3GC- and carbapenem-resistant, potentially harmful Aeromonas spp. that can be introduced into aquatic systems and pose a threat to both the environment and public health.

Multidrug-resistant Enterobacterales responsible for septicaemia in a neonatal intensive care unit in Morocco

Neonatal sepsis with multidrug resistant (MDR) bacteria is an important cause of morbidity and mortality, especially in low- and middle-income countries. Here, the molecular mechanisms of MDR in bacteria responsible of sepsis in neonates was determined. From July to December 2019, documented bacteraemia from 524 neonates hospitalized in a neonatal intensive care unit (NICU) in Morocco were collected. WGS was used to determine the resistome, MLST and phylogeny. Among the 199 documented bacteraemia, 40 (20%) and 20 (10%) were due to MDR K. pneumoniae (Kp) and E. hormaechei (Eh), respectively. Of these, 23 (38.5%) were early neonatal infections (≤ 3 days of life). Twelve different STs were observed among Kp isolates with ST1805 (n=10), and ST307 (n=8), being the most prevalent. Twenty-one Kp (53%) possessed blaCTX-M-15 gene, of which 6 co-produced an OXA-48, 2 an NDM-7, and 2 OXA-48 and NDM-7. BlaOXA-48 gene was present in 11 (27.5%), blaNDM-1 in 13 (32.5%), and blaNDM-7 in 4 isolates of Kp (10.0%). Eighteen Eh (90.0%) produced an ESBL. Three SHV-12 that co-produced CMY-4 and NDM-1, and 15 CTXM-15 of which 6 co-produced OXA-48. Twelve different STs belonging to 3 different Eh subspecies were observed with one to 4 isolates. Kp and Eh isolates belonging to the same ST had less than 20 SNP differences, and were found throughout the study period, highlighting their endemic presence in the NICU. Thirty percent of neonatal sepsis (23 early- and 37 late-neonatal sepsis) were due to highly-drug resistant carbapenemase- and/or ESBL producing-Enterobacterales.

The European Union Summary Report on Antimicrobial Resistance in zoonotic and indicator bacteria from humans, animals and food in 2020/2021.

Antimicrobial resistance (AMR) data on zoonotic and indicator bacteria from humans, animals and food are collected annually by the EU Member States (MSs) and reporting countries, jointly analysed by EFSA and ECDC and presented in a yearly EU Summary Report. This report provides an overview of the main findings of the 2020-2021 harmonised AMR monitoring in Salmonella spp., Campylobacter jejuni and C. coli in humans and food-producing animals (broilers, laying hens and turkeys, fattening pigs and bovines under 1year of age) and relevant meat thereof. For animals and meat thereof, indicator E. coli data on the occurrence of AMR and presumptive Extended spectrum β-lactamases (ESBL)-/AmpC β-lactamases (AmpC)-/carbapenemases (CP)-producers, as well as the occurrence of methicillin-resistant Staphylococcus aureus are also analysed. In 2021, MSs submitted for the first time AMR data on E. coli isolates from meat sampled at border control posts. Where available, monitoring data from humans, food-producing animals and meat thereof were combined and compared at the EU level, with emphasis on multidrug resistance, complete susceptibility and combined resistance patterns to selected and critically important antimicrobials, as well as Salmonella and E. coli isolates exhibiting ESBL-/AmpC-/carbapenemase phenotypes. Resistance was frequently found to commonly used antimicrobials in Salmonella spp. and Campylobacter isolates from humans and animals. Combined resistance to critically important antimicrobials was mainly observed at low levels except in some Salmonella serotypes and in C. coli in some countries. The reporting of a number of CP-producing E.coli isolates (harbouring bla OXA-48, bla OXA-181, and bla NDM-5 genes) in pigs, bovines and meat thereof by a limited number of MSs (4) in 2021, requests a thorough follow-up. The temporal trend analyses in both key outcome indicators (rate of complete susceptibility and prevalence of ESBL-/AmpC- producers) showed that encouraging progress have been registered in reducing AMR in food-producing animals in several EU MSs over the last years.

Comparative evaluation of phenotypic and genotypic methods for the rapid and cost-effective detection of carbapenemases in extensively drug resistant Klebsiella pneumoniae.

Carbapenemases are the enzymes that can hydrolyze carbapenems and other β-lactam antibiotics. These enzymes confer resistance to multiple antibiotics and act as a stumbling block in the treatment of infections caused by gram-negative bacteria. Therefore, rapid and specific detection of these enzymes is crucial for deciding the course of treatment and better clinical outcomes. This study was conducted to compare various phenotypic and PCR based methods for the detection of carbapenemases in carbapenem- and colistin-resistant Klebsiella pneumoniae. One hundred clinical isolates of extensively resistant Klebsiella pneumoniae were included in the study. Phenotypic detection for carbapenemases was performed by Rapidec® Carba NP (Biomerieux), modified carbapenem inactivation method (mCIM), imipenem-ethylenediaminetetraacetic acid disk synergy (EDS), double disk synergy test using mercaptopropionic acid (DDST-MPA), and combined disk method (CD) and for colistin by microbroth dilution method. Genotypic detection for carbapenemases and colistin resistance was performed by targeted PCR. The sensitivity of Carba NP test and mCIM were positive in 95% and 96% respectively and specificity was 100% for both methods. The sensitivity of EDS, DDST-MPA, and CD were 55.6%, 88.9% and 54.5% respectively. Among the carbapenem resistance genes, blaOXA-48 (82%) genes were the most prevalent. Among metallo-beta lactamases, blaVIM (56%) was most common followed by blaNDM (54%) and blaIMP (20%). The mcr-1 gene for colistin resistance was not detected in any isolate. Among the five phenotypic assays analyzed, the mCIM is the most simple, inexpensive, accurate and reproducible method for carbapenemase detection in Klebsiella pneumoniae. The DDST-MPA test provides the best sensitivity for the detection of carbapenemases, although specificity is low. These tests, when applied in a clinical laboratory and assessed by the microbiologist, can help in guiding the course of treatment.

β-Lactamase Genes (blaCTX-M, blaSHV, blaTEM, blaOXA1 and blaOXA2) and Phylogenetic Groups in ESBL Producing Commensal Escherichia coli Isolated from Faecal Samples from Dairy Farm in the Municipality of Debar

Abstract β-lactamases are a diverse class of enzymes produced by bacteria that present a major cause for resistance to β-lactams. In this study we analysed 159 fecal samples from dairy cows, for the presence of presumptive ESBL, AmpC, and carbapenemase-producing E. coli. Phylotyping was done using Clermont phylo-typing method, targeting arpA, ChuA, and YjaA genes, along with the DNA fragment TspE4.C2. Convetional PCR method was used to confirm the presence of bla genes among 39 phenotypically confirmed ESBL producing E. coli. The results showed presence of CTX-M, SHV, TEM and OXA1 bla genes in 28 (71.79%), 1 (2.56%), 29 (74.35%), 2 (5.12%) of isolates, respectively Twenty (51.28%) isolates showed presence of both blaCTX-M and TEM genes. The strain that carried the blaSHV gene was found to carry blaTEM gene as well, while one of the strains that carried blaOXA1 gene was also carrying blaCTX-M and TEM gene. The ration between isolates and phylo-groups was as follows: 9 (23.07%) strains were assigned to phyllo-group D; 14 (35.89%) to phyllo-group B; 16 (41.02%) to phyllo-group A. Out of the 39 strains where bla genes were identified, 29 (74.35%) were categorized as multi drug resistant.

Whole-genome sequence analysis of clinically isolated carbapenem resistant Escherichia coli from Iran

BackgroundThe emergence of carbapenem-resistant Enterobacterales (CRE) continues to threaten public health due to limited therapeutic options. In the current study the incidence of carbapenem resistance among the 104 clinical isolates of Escherichia coli and the genomic features of carbapenem resistant isolates were investigated.MethodsThe susceptibility to imipenem, tigecycline and colistin was tested by broth dilution method. Susceptibility to other classes of antimicrobials was examined by disk diffusion test. The presence of blaOXA-48, blaKPC, blaNDM, and blaVIM carbapenemase genes was examined by PCR. Molecular characteristics of carbapenem resistant isolates were further investigated by whole-genome sequencing (WGS) using Illumina and Nanopore platforms.ResultsFour isolates (3.8%) revealed imipenem MIC of ≥32 mg/L and positive results for modified carbapenem inactivation method and categorized as carbapenem resistant E. coli (CREC). Colistin, nitrofurantoin, fosfomycin, and tigecycline were the most active agents against all isolates (total susceptibility rate of 99, 99, 96 and 95.2% respectively) with the last three compounds being found as the most active antimicrobials for carbapenem resistant isolates (susceptibility rate of 100%). According to Multilocus Sequence Type (MLST) analysis the 4 CREC isolates belonged to ST167 (n = 2), ST361 (n = 1) and ST648 (n = 1). NDM was detected in all CREC isolates (NDM-1 (n = 1) and NMD-5 (n = 3)) among which one isolate co-harbored NDM-5 and OXA-181 carbapenemases. WGS further detected blaCTX-M-15, blaCMY-145, blaCMY-42 and blaTEM-1 (with different frequencies) among CREC isolates. Co-occurrence of NDM-type carbapenemase and 16S rRNA methyltransferase RmtB and RmtC was found in two isolates belonging to ST167 and ST648. A colistin-carbapenem resistant isolate which was mcr-negative, revealed various amino acid substitutions in PmrB, PmrD and PhoPQ proteins.ConclusionAbout 1.9% of E. coli isolates studied here were resistant to imipenem, colistin and/or amikacin which raises the concern about the outbreaks of difficult-to-treat infection by these emerging superbugs in the future.

Isolation, characterization, therapeutic potency, and genomic analysis of a novel bacteriophage vB_KshKPC-M against carbapenemase-producing Klebsiella pneumoniae strains (CRKP) isolated from Ventilator-associated pneumoniae (VAP) infection of COVID-19 patients

BackgroundCarbapenem-resistant Klebsiella pneumoniae (CRKP) is a significant clinical problem, given the lack of therapeutic options. The CRKP strains have emerged as an essential worldwide healthcare issue during the last 10 years. Global expansion of the CRKP has made it a significant public health hazard. We must consider to novel therapeutic techniques. Bacteriophages are potent restorative cases against infections with multiple drug-resistant bacteria. The Phages offer promising prospects for the treatment of CRKP infections.ObjectiveIn this study, a novel K. pneumoniae phage vB_KshKPC-M was isolated, characterized, and sequenced, which was able to infect and lyse Carbapenem-resistant K. pneumoniae host specifically.MethodsOne hundred clinical isolates of K. pneumoniae were collected from patients with COVID-19 associated with ventilator-associated acute pneumonia hospitalized at Shahid Beheshti Hospital, Kashan, Iran, from 2020 to 2021. Initially, all samples were cultured, and bacterial isolates identified by conventional biochemical tests, and then the ureD gene was used by PCR to confirm the isolates. The Antibiotic susceptibility test in the disc diffusion method and Minimum inhibitory concentrations for Colistin was done and interpreted according to guidelines. Phenotypic and molecular methods determined the Carbapenem resistance of isolates. The blaKPC, blaNDM, and blaOXA-23 genes were amplified for this detection. Biofilm determination of CRKP isolates was performed using a quantitative microtiter plate (MTP) method. The phage was isolated from wastewater during the summer season at a specific position from Beheshti Hospital (Kashan, Iran). The sample was processed and purified against the bacterial host, a CRKP strain isolated from a patient suffering from COVID-19 pneumoniae and resistance to Colistin with high potency for biofilm production. This isolate is called Kp100. The separated phages were diluted and titration by the double overlay agar plaque assay. The separate Phage is concentrated with 10% PEG and stored at −80 °C until use. The phage host range was identified by the spot test method. The purified phage morphology was determined using a transmission electron microscope. The phage stability tests (pH and temperature) were analyzed. The effect of cationic ions on phage adsorption was evaluated. The optimal titer of bacteriophage was determined to reduce the concentration of the CRKP strain. One-step growth assays were performed to identify the purified phage burst’s latent cycle and size. The SDS-PAGE was used for phage proteins analysis. Phage DNA was extracted by chloroform technique, and the whole genome of lytic phage was sequenced using Illumina HiSeq technology (Illumina, San Diego, CA). For quality assurance and preprocessing, such as trimming, Geneious Prime 2021.2.2 and Spades 3.9.0. The whole genome sequence of the lytic phage is linked to the GenBank database accession number. RASTtk—v1.073 was used to predict and annotate the ORFs. Prediction of ORF was performed using PHASTER software. ResFinder is used to assess the presence of antimicrobial resistance and virulence genes in the genome. The tRNAs can-SE v2.0.6 is used to determine the presence of tRNA in the genome. Linear genome comparisons of phages and visualization of coding regions were performed using Easyfig 2.2.3 and Mauve 2.4.0. Phage lifestyles were predicted using the program PHACTS. Phylogenetic analysis and amino acid sequences of phage core proteins, such as the major capsid protein. Phylogenies were reconstructed using the Neighbor-Joining method with 1000 bootstrap repeat. HHpred software was used to predict depolymerase. In this study, GraphPad Prism version 9.1 was used for the statistical analysis. Student’s t-test was used to compare the sets and the control sets, and the significance level was set at P ≤ 0.05.ResultsPhage vB_KshKPC-M is assigned to the Siphoviridae, order Caudovirales. It was identified as a linear double-stranded DNA phage of 54,378 bp with 50.08% G + C content, had a relatively broad host range (97.7%), a short latency of 20 min, and a high burst size of 260 PFU/cell, and was maintained stable at different pH (3–11) and temperature (45–65 °C). The vB_KshKPC-M genome contains 91 open-reading frames. No tRNA, antibiotic resistance, toxin, virulence-related genes, or lysogen-forming gene clusters were detected in the phage genome.Comparative genomic analysis revealed that phage vB_KshKPC-M has sequence similarity to the Klebsiella phages, phage 13 (NC_049844.1), phage Sushi (NC_028774.1), phage vB_KpnD_PeteCarol (OL539448.1) and phage PWKp14 (MZ634345.1).ConclusionThe broad host range and antibacterial activity make it a promising candidate for future phage therapy applications. The isolated phage was able to lyse most of the antibiotic-resistant clinical isolates. Therefore, this phage can be used alone or as a phage mixture in future studies to control and inhibit respiratory infections caused by these bacteria, especially in treating respiratory infections caused by resistant strains in sick patients.

Genomic Comparative Analysis of Two Multi-Drug Resistance (MDR) Acinetobacter baumannii Clinical Strains Assigned to International Clonal Lineage II Recovered Pre- and Post-COVID-19 Pandemic.

After the emergence of COVID-19, numerous cases of A. baumannii/SARS-CoV-2 co-infection were reported. Whether the co-infecting A. baumannii strains have distinctive characteristics remains unknown. A. baumannii AMA_NO was isolated in 2021 from a patient with COVID-19. AMA166 was isolated from a mini-BAL used on a patient with pneumonia in 2016. Both genomes were similar, but they possessed 337 (AMA_NO) and 93 (AMA166) unique genes that were associated with biofilm formation, flagellar assembly, antibiotic resistance, secretion systems, and other functions. The antibiotic resistance genes were found within mobile genetic elements. While both strains harbored the carbapenemase-coding gene blaOXA-23, only the strain AMA_NO carried blaNDM-1. Representative functions coded for by virulence genes are the synthesis of the outer core of lipooligosaccharide (OCL5), biosynthesis and export of the capsular polysaccharide (KL2 cluster), high-efficiency iron uptake systems (acinetobactin and baumannoferrin), adherence, and quorum sensing. A comparative phylogenetic analysis including 239 additional sequence type (ST) 2 representative genomes showed high similarity to A. baumannii ABBL141. Since the degree of similarity that was observed between A. baumannii AMA_NO and AMA166 is higher than that found among other ST2 strains, we propose that they derive from a unique background based on core-genome phylogeny and comparative genome analysis. Acquisition or shedding of specific genes could increase the ability of A. baumannii to infect patients with COVID-19.

Antimicrobial Resistance Among Gram-Negative Bacteria Isolated in the Newborn Intensive Care Unit at Abderrezak-Bouhara Hospital of Skikda, Algeria.

Background: This study aimed to determine the epidemiology of gram-negative bacteria (GNB) isolated in the newborn intensive care unit (NICU) population, to assess their antibiotic susceptibility patterns and possible associated risk factors. Methods: All neonates admitted to the NICU of Abderrezak-Bouhara hospital (Skikda, Algeria) with a clinical diagnosis of neonatal infections from March to May 2019 were included in the study. The extended-spectrum β-lactamase (ESBLs), plasmidic cephalosporinase (pAmpC), and carbapenemases genes were screened by polymerase chain reaction (PCR) and sequencing. PCR amplification of oprD among carbapenem-resistant Pseudomonas aeruginosa isolates was also performed. The clonal relatedness of the ESBLs isolates was studied using multilocus sequence typing (MLST). Results: Among 148 clinical specimens, 36 (24.3%) GNB strains were isolated from urine (n = 22), wound (n = 8), stool (n = 3), and blood (n = 3) samples. The bacterial species identified were Escherichia coli (n = 13), Klebsiella pneumoniae (n = 5), Enterobacter cloacae (n = 3), Serratia marcescens (n = 3), Salmonella spp. (n = 3), Proteus mirabilis (n = 1), P. aeruginosa (n = 5), and Acinetobacter baumannii (n = 3). PCR and sequencing showed that eleven Enterobacterales isolates harbored the blaCTX-M-15 gene, two E. coli isolates harbored the blaCMY-2 gene, and three A. baumannii isolates harbored both blaOXA-23 and blaOXA-51 genes. Also, five strains of P. aeruginosa were found to harbor mutations in the oprD gene. MLST showed that the K. pneumoniae strains belonged to ST13 and ST189, E. coli belonged to ST69, and E. cloacae belonged to ST214. Different risk factors that could predict positive GNB cultures were found, including female sex, Apgar score <8 at 5 min of life, enteral nutrition, antibiotic use, and extended length of hospitalization. Conclusion: Our study highlights the importance of determining the epidemiology of pathogens causing neonatal infections, their sequence types (ST), and their antibiotic susceptibility patterns to address rapidly a correct antibiotic treatment regimen.

Detection of virulence factor genes, antibiotic resistance genes and biofilm formation in clinical Gram-negative bacteria and first report from Türkiye of K.oxytoca carrying both blaOXA-23 and blaOXA-51 genes

Detection of virulence factor genes, antibiotic resistance genes and biofilm formation in clinical Gram-negative bacteria and first report from Türkiye of K.oxytoca carrying both blaOXA-23 and blaOXA-51 genes

Prevalence of Carbapenem-Resistant Enterobacteriaceae in Western Saudi Arabia and Increasing Trends in the Antimicrobial Resistance of Enterobacteriaceae.

The aim of the study is to estimate the prevalence rate of carbapenem-resistant Enterobacteriaceae (CRE) and to determine the types of carbapenemase genes present in patients admitted to King Abdulaziz Medical City (KAMC-J) and King Abdulaziz University Hospital (KAUH), both in Jeddah, Saudi Arabia. A total of 180 isolates were analyzed which were included on the basis of retrospective chart review of patients from KAMC-J and KAUH between 1st April 2017 to 30th March 2019. The prevalence of carbapenemase genes( blaIMP, blaVIM, blaKPC, blaNDM-1, and blaOXA-48)was evaluated by Xpert® Carba-R (Cepheid, Sunnyvale, CA, USA). We assessed the CRE prevalence and described their susceptibility to antimicrobial agents based on antibiogram reports. Results:Klebsiella pneumoniaeshowed a higher frequency of bla OXA-48 (79%) than bla NDM (11.7%) genes (p=0.007). The CRE prevalence in KAUH was 8% in 2017 and increased to 13% in 2018. In KAMC-J, the prevalence was 57% in 2018 and 61% in 2019. K. pneumoniae was found to be the most frequently isolated causative organism followed byEscherichia coli .The bla OXA-48 (76.1%) gene was predominantamong overall isolates followed by bla NDM (13.9%); both genes coexisted in 6.1% of the isolates. During the study period, the prevalence of CRE considerably rose in the two tertiary care institutions from western Saudi Arabia. In the CRE isolates, bla OXA-48was discovered to be the most common gene. We recommend an antimicrobial resistance surveillance system to detect the emergence of resistant genes through use of new rapid diagnostic tests and monitor antimicrobial use in order to improve clinical outcomes of CRE infections given the severity of infection associated with the CRE isolates as well as the limited treatment options available.

Development of a new multiplex real-time PCR assay for rapid screening of hospital-acquired infection agents

AimsA new multiplex real-time PCR (qPCR) assay was developed to detect antibiotic-resistant hospital-acquired infectious agents in nasal and rectal swab samples in 1.5 h without the need for nucleic acid extraction. MethodsSpiked negative clinical specimens were used for the analytical performance evaluation. Double-blind samples were collected from 1788 patients to assess the relative clinical performance of the qPCR assay to the conventional culture-based methods. Bio-Speedy® Fast Lysis Buffer (FLB) and 2× qPCR-Mix for hydrolysis probes (Bioeksen R&D Technologies, Istanbul, Turkey) and LightCycler® 96 Instrument (Roche Inc., Branchburg, NJ, USA) were used for all molecular analyses. The samples were transferred into 400 L FLB, homogenized and immediately used in qPCRs. The target DNA regions are vanA and vanB genes for vancomycin-resistant Enterococcus (VRE); blaKPC, blaNDM, blaVIM, blaIMP, blaOXA-23, blaOXA-48, blaOXA-58 genes for carbapenem-resistant Enterobacteriaceae (CRE); and mecA, mecC and spa for methicillin-resistant Staphylococcus aureus (MRSA). ResultsNo qPCR tests produced positive results for the samples spiked with the potential cross-reacting organisms. The limit of detection (LOD) of the assay for all targets was 100 colony-forming unit (cfu)/swab-sample. Results of the repeatability studies in two different centers were in 96%–100% (69/72–72/72) agreement. The relative specificity and sensitivity of the qPCR assay were respectively 96.8% and 98.8% for VRE; 94.9% and 95.1% for CRE; 99.9% and 97.1% for MRSA. ConclusionsThe developed qPCR assay can screen antibiotic-resistant hospital-acquired infectious agents in infected/colonized patients with an equal clinical performance to the culture-based methods.

Fusion plasmid enhanced the endemic extensively drug resistant Klebsiella pneumoniae clone ST147 harbored blaOXA-48 to acquire the hypervirulence and cause fatal infection

BackgroundKlebsiella Pneumoniae (Kp) sequence type (ST) 147 has emerged globally and spread rapidly, particularly the extensively drug resistant (XDR) isolates. However, the infections caused by this subtype is rare reported in China for now. The clinical, microbiological and genomic characteristics are unclear.MethodsA systemic retrospective study was conducted in a Chinese tertiary hospital. Clinical information of the infection cases was collected, and whole-genome sequencing and phenotypic experiments were performed on the ST147 isolates. The resistance and virulence genes were identified, and the plasmids harboring these genes were further studied.ResultsSix ST147 isolates from six patients among 720 available clincial Kp isolates were detected. Notably, two isolates, PEKP4035 and PEKP4265, represented both XDR and hypervirulence by acquiring blaOXA-48, blaCTX-M-15 and key virulence genes, iucA + rmpA2, representing no fitness cost and resulting fatal infection. Four of the six ST147 isolates presented with more nucleotide differences, whereas the PEKP4035 and PEKP4265 both isolated from the intensive care unit possessed 20 single nucleotide polymorphisms among one year, indicating the prolonged survive and transmission. Interestingly, the two isolates harbored the same fused plasmid composed of sul2 and iucA + rmpA2, which might be generated by recombination of a plasmid like KpvST101_OXA-48 with the pLVPK plasmid via IS26. Besides, two ~ 70 kb plasmids conferring multiple-drug resistance were also identified among the two isolates, which presented resistance genes including blaOXA-48, blaCTX-M-16, strA and strB. Interestingly, we reported that blaCTX-M-15, a common resistance gene within ST147, has successfully transferred into the chromosome by ISEcp1.ConclusionsXDR hypervirulent ST147 Kp is emerging, suggesting enhanced surveillance is essential.