blaPER Genes Research Articles

Exploring Biofilm-Related Traits and Bile Salt Efficacy as Anti-Biofilm Agents in MDR Acinetobacter baumannii.

Acinetobacter baumannii has been designated as a critical priority pathogen by the World Health Organization for the development of novel antimicrobial agents. This study aimed to investigate both the phenotypic and genotypic traits of multidrug-resistant (MDR) A. baumannii strains, along with the effects of natural bile salts on biofilm formation. The research analyzed phenotypic traits, including autoaggregation, hydrophobicity, twitching motility, lectin production, and biofilm formation, as well as genotypic traits such as the presence of bap and blaPER-1 genes in twenty wound and eight environmental MDR A. baumannii isolates. While all strains were identified as good biofilm producers, no statistically significant correlation was detected between the examined traits and biofilm formation. However, differences in biofilm production were observed between environmental and wound isolates. The natural bile salts Na-cholate, Na-deoxycholate, and Na-chenodeoxycholate demonstrated effective anti-A. baumannii activity (MIC = 0.25-10 mg mL-1), with significant anti-biofilm effects. Na-deoxycholate and Na-chenodeoxycholate inhibited 94-100% of biofilm formation at super-MIC concentrations (8-32 mg mL-1). This study underscores the urgent need for innovative strategies to combat antibiotic resistance and biofilm formation in A. baumannii, highlighting the potential of natural bile salts as promising biofilm inhibitors and encouraging further research into their modification and combination with other antimicrobials.

Ceftazidime/avibactam resistance is associated with PER-3-producing ST309 lineage in Chilean clinical isolates of non-carbapenemase producing Pseudomonas aeruginosa.

Ceftazidime/avibactam (CZA) is indicated against multidrug-resistant Pseudomonas aeruginosa, particularly those that are carbapenem resistant. CZA resistance in P. aeruginosa producing PER, a class A extended-spectrum β-lactamase, has been well documented in vitro. However, data regarding clinical isolates are scarce. Our aim was to analyze the contribution of PER to CZA resistance in non-carbapenemase-producing P. aeruginosa clinical isolates that were ceftazidime and/or carbapenem non-susceptible. Antimicrobial susceptibility was determined through agar dilution and broth microdilution, while bla PER gene was screened through PCR. All PER-positive isolates and five PER-negative isolates were analyzed through Whole Genome Sequencing. The mutational resistome associated to CZA resistance was determined through sequence analysis of genes coding for PBPs 1b, 3 and 4, MexAB-OprM regulators MexZ, MexR, NalC and NalD, AmpC regulators AmpD and AmpR, and OprD porin. Loss of bla PER-3 gene was induced in a PER-positive isolate by successive passages at 43°C without antibiotics. Twenty-six of 287 isolates studied (9.1%) were CZA-resistant. Thirteen of 26 CZA-resistant isolates (50%) carried bla PER. One isolate carried bla PER but was CZA-susceptible. PER-producing isolates had significantly higher MICs for CZA, amikacin, gentamicin, ceftazidime, meropenem and ciprofloxacin than non-PER-producing isolates. All PER-producing isolates were ST309 and their bla PER-3 gene was associated to ISCR1, an insertion sequence known to mobilize adjacent DNA. PER-negative isolates were classified as ST41, ST235 (two isolates), ST395 and ST253. PER-negative isolates carried genes for narrow-spectrum β-lactamases and the mutational resistome showed that all isolates had one major alteration in at least one of the genes analyzed. Loss of bla PER-3 gene restored susceptibility to CZA, ceftolozane/tazobactam and other β-lactamsin the in vitro evolved isolate. PER-3-producing ST309 P. aeruginosa is a successful multidrug-resistant clone with blaPER-3 gene implicated in resistance to CZA and other β-lactams.

Distribution of blaOXA-10, blaPER-1, and blaSHV genes in ESBL-producing Pseudomonas aeruginosa strains isolated from burn patients

Pseudomonas aeruginosa is resistant to a wide range of extended spectrum-lactamases (ESBLs) antibiotics because it produces several kinds of ESBLs. The goal of the current investigation was to identify the bacteria that produce extended spectrum -lactamases and the genes that encode three different ESBLs, such as blaOXA-10, blaPER-1 and blaSHV genes in Pseudomonas aeruginosa isolated from burn patients. In this investigation, 71 Pseudomonas aeruginosa isolates were isolated from burn wounds in Burn and Plastic Surgery Hospital, Duhok City between July 2021 to June 2022. For the purpose of finding the blaOXA-10, blaPER-1, and blaSHV ESBL expressing genes, Polymerase Chain Reaction (PCR) was used. Among 71 Pseudomonas aeruginosa isolates, 26.36% (29/71) were isolated from males and 38.18% (42/71) from females, and 76.06% (54/71) of the isolates were multidrug resistant. They exhibited higher resistance against Piperacillin with resistance rates of 98.59%. Among the ESBL-producing isolates tested, blaOXA-10 was found in 59.26% (32), blaPER-1 was found in 44.44% (24), and blaSHV was found in 11.11% (6). All isolates must undergo antimicrobial susceptibility testing because only a few numbers of the available antibiotics are effective for the treatment of this bacterium. This will prevent the development of resistance in burn units and aids in the management of the treatment plan.

Comparison of loop-mediated isothermal amplification, multiplex PCR, and REP- PCR techniques for identification of carbapenem-resistant Acinetobacter baumannii clinical isolates

Background and Objectives: 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.
 Materials and Methods: 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.
 Results: 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.
 Conclusion: 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.

Complete genetic characterization of carbapenem-resistant Acinetobacter johnsonii, co-producing NDM-1, OXA-58, and PER-1 in a patient source.

The emergence of carbapenemase-producing Acinetobacter spp. has been widely reported and become a global threat. However, carbapenem-resistant A. johnsonii strains are relatively rare and without comprehensive genetic structure analysis, especially for isolates collected from human specimen. Here, one A. johnsonii AYTCM strain, co-producing NDM-1, OXA-58, and PER-1 enzymes, was isolated from sputum in China in 2018. Antimicrobial susceptibility testing showed that it was resistant to meropenem, imipenem, ceftazidime, ciprofloxacin, and cefoperazone/sulbactam. Whole-genome sequencing and bioinformatic analysis revealed that it possessed 11 plasmids. bla OXA-58 and bla PER-1 genes were located in the pAYTCM-1 plasmid. Especially, a complex class 1 integron consisted of a 5' conserved segment (5' CS) and 3' CS, which was found to carry sul1, arr-3, qnrVC6, and bla PER-1 cassettes. Moreover, the bla NDM-1 gene was located in 41,087 conjugative plasmids and was quite stable even after 70 passages under antibiotics-free conditions. In addition, six prophage regions were identified. Tracking of closely related plasmids in the public database showed that pAYTCM-1 was similar to pXBB1-9, pOXA23_010062, pOXA58_010030, and pAcsw19-2 plasmids, which were collected from the strains of sewage in China. Concerning the pAYTCM-3 plasmids, results showed that strains were collected from different sources and their hosts were isolated from various countries, such as China, USA, Japan, Brazil, and Mexico, suggesting that a wide spread occurred all over the world. In conclusion, early surveillance is warranted to avoid the extensive spread of this high-risk clone in the healthcare setting.

Evaluation of the Ability to Form Biofilms in KPC-Producing and ESBL-Producing Klebsiella pneumoniae Isolated from Clinical Samples.

The appearance of Klebsiella pneumoniae strains producing extended-spectrum β-lactamase (ESBL), and carbapenemase (KPC) has turned into a significant public health issue. ESBL- and KPC-producing K. pneumoniae's ability to form biofilms is a significant concern as it can promote the spread of antibiotic resistance and prolong infections in healthcare facilities. A total of 45 K. pneumoniae strains were isolated from human infections. Antibiograms were performed for 17 antibiotics, ESBL production was tested by Etest ESBL PM/PML, a rapid test was used to detect KPC carbapenemases, and resistance genes were detected by PCR. Biofilm production was detected by the microtiter plate method. A total of 73% of multidrug resistance was found, with the highest resistance rates to ampicillin, trimethoprim-sulfamethoxazole, cefotaxime, amoxicillin-clavulanic acid, and aztreonam. Simultaneously, the most effective antibiotics were tetracycline and amikacin. blaCTX-M, blaTEM, blaSHV, aac(3)-II, aadA1, tetA, cmlA, catA, gyrA, gyrB, parC, sul1, sul2, sul3, blaKPC, blaOXA, and blaPER genes were detected. Biofilm production showed that 80% of K. pneumoniae strains were biofilm producers. Most ESBL- and KPC-producing isolates were weak biofilm producers (40.0% and 60.0%, respectively). There was no correlation between the ability to form stronger biofilms and the presence of ESBL and KPC enzymes in K. pneumoniae isolates.

Expression of Biofilm-Related Genes in Extensively Drug-Resistant Acinetobacter baumannii

Background: Acinetobacter baumannii is an important cause of nosocomial pneumonia in patients requiring long-term mechanical ventilation. Besides, extensively drug-resistant (XDR) strains cause infection in intensive care unit (ICU) patients. Chronic infections of A. baumannii and antimicrobial resistance are associated with biofilm formation. Several virulence genes, such as blaPER-1, pgaA, and bap, are involved in biofilm formation. Objectives: The current study examines the expression levels of biofilm formation-related genes in pneumonia patients. Methods: The sputum samples were collected from patients hospitalized in the ICU, and A. baumannii ATCC 19606, the reference strain, was isolated and cultured on blood agar, eosin methylene blue agar, and chocolate agar medium. The media were then incubated at 37°C for 18 - 24 hours. Next, Gram-Thirty XDR A. baumannii isolates were collected from the sputum samples of ICU patients at Besat Hospital in Tehran, Iran. Bacterial isolates were characterized for antibiotic resistance patterns and biofilm-forming ability. Subsequently, RNA was extracted from the biofilm-forming isolates. A real-time polymerase chain reaction (PCR) assay was performed to evaluate the expression levels of the blaPER-1, pgaA, and bap genes. Transcripts were normalized to 16S rRNA as an internal control, and gene expression fold changes were calculated. Statistical analysis was performed using an unpaired two-tailed t-test (P < 0.05) with SPSS (V. 16). Results: The disk diffusion susceptibility test revealed that all 30 (100%) isolates were resistant to piperacillin-tazobactam, cefepime, ceftriaxone, ceftazidime, gentamicin, imipenem, meropenem, levofloxacin, and ciprofloxacin. All 30 isolates from ICU-admitted patients (100%) were classified as XDR, and 27 (90%) isolates demonstrated the ability to form biofilms. The obtained results indicated a significant difference in gene expression levels. The fold change in expression for blaPER-1, bap, and pgaA was 7.473, 11.964, and 5.277, respectively. Conclusions: In our study, XDR A. baumannii primarily caused ventilator-associated pneumonia, and an observed increase in the expression of biofilm-related genes was noted in these strains. Healthcare centers should implement appropriate infection control programs to manage nosocomial infections, particularly in the ICU.

Diversity of genetic platforms harboring the blaPER-2 gene in Enterobacterales and insights into the role of ISPa12 in its mobilization and dissemination

The production of PER-like extended-spectrum β-lactamases has recently been associated with reduced susceptibility to the last resort drugs aztreonam/avibactam and cefiderocol. PER-2 has been mainly confined to Argentina and neighboring countries. Until now, only three plasmids harboring blaPER-2 genes have been characterized but very little is known about the involvement of different plasmid groups in its dissemination. The diversity of genetic platforms associated with blaPER-2 genes from a collection of PER-producing Enterobacterales was analysed by describing the close environment and the plasmid backbones. Full sequences of 11 plasmids were obtained by short read (Illumina) and long read (Oxford Nanopore or PacBio) sequencing technologies. De novo assemblies, annotation and sequence analysis were performed by Unicycler, Prokka and BLAST. Plasmid analysis revealed that the blaPER-2 gene is encoded on plasmids of different incompatibility groups (A, C, FIB, HI1B, N2), indicating that this gene may have been disseminated through a variety of plasmids. Comparison with the few publicly available nucleotide sequences describing the blaPER-2 genetic environment, including those from the environmental species Pararheinheimera spp. (considered as the progenitor of blaPER genes), indicates a role of ISPa12 in blaPER-2 gene mobilization from the chromosome of Pararheinheimera spp. Also, the blaPER-2 gene was carried by a novel ISPa12-composite transposon, Tn7390. In addition, its association with ISKox2-like elements in the close genetic environment in all plasmids analysed suggests a role of these insertion sequence elements in further dissemination of blaPER-2 genes.

Antimicrobial susceptibility profile and detection of extended spectrum β-lactamase resistance genes in pseudomonas aeruginosa isolated from post-operative wounds

The spread of Pseudomonas aeruginosa is difficult to control with disinfectants and antibiotics due to the resistance found in P. aeruginosa isolates. The study investigated the susceptibility profile and detect extended spectrum β-lactamase (ESBL) resistance genes in the Pseudomonas aeruginosa isolated from post-operative wound infection. Wound swabs were cultured on MacConkey and sub-cultured on Cetrimide agar. The isolates were identified base on their macroscopic and microscopic characteristics as well as 16SrRNA. Disc diffusion technique was used to check the susceptibility profile of the isolates and the extended spectrum beta-lactam (ESBL) producers were determined by growing the isolates on Mueller Hinton agar plate in the presence of amoxyclav. 40 positive isolates were recovered in the study with 21 isolates been multi-drug resistance (MDR). Before plasmid curing, Streptomycin, ceftazidime, Augmentin, and Gentamicin has the highest resistance rate of 80% (n=32), 60% (n=24), 60% (n=24) and 60% (n=24) respectively. However, after plasmid curing, the result showed reduction in resistance rates. Streptomycin and ceftazidime showed high reduction in resistance from 80% to 25% and from 60 to 12.7% respectively indicating that resistance was plasmid-mediated. Pseudomonas aeruginosa F23 and Pseudomonas aeruginosa S15 isolates shown to possess both the plcH and bla PER genes with bands amplicons size of 307bp and 520bp respectively. Antibiotic susceptibility pattern after plasmid curing showed that 45% of the isolates produces beta-lactamase enzymes i.e., 18 out of 40 isolates were inactive to all beta-lactam group of antibiotics.

Resistance to Cefiderocol Involved Expression of PER-1 β-Lactamase and Downregulation of Iron Transporter System in Carbapenem-Resistant Acinetobacter baumannii.

Cefiderocol (CFDC) is a promising antimicrobial agent against multidrug resistant Gram-negative bacteria. However, CFDC resistance has emerged in carbapenem-resistant Acinetobacter baumannii (CR-AB) but the underlying mechanisms remain unclear. Whole-genome sequencing and transcriptome sequencing were performed on CFDC-non-susceptible and CFDC-susceptible isolates. Two different recombinant plasmids was electro-transformed into the E. coli BL21 strain to determine the impact of blaPER and the combined impact of blaPER-1 and blaOXA-23 on CFDC resistance. Fifty-five CR-AB isolates with minimum inhibitory concentrations (MICs) ranged from 0.06 mg/L to >256 mg/L were sequenced, including 47 CFDC-non-susceptible and eight CFDC-susceptible isolates. Two CFDC-non-susceptible isolates belonged to ST104 whereas the remaining isolates belonged to ST2, and blaPER-1 was present only in CFDC-non-susceptible isolates. Amino acid substitutions were noted in penicillin-binding proteins (PBPs) in four CFDC-susceptible isolates, with slightly elevated MICs. The MICs of recombinant E. coli BL21 carrying the blaPER-1 gene increased 64-fold and recombinant E. coli BL21 carrying both the blaPER-1 and blaOXA-23 genes increased 8-fold but both remained within the susceptibility range. Transcriptome sequencing of 17 CFDC-non-susceptible isolates and eight CFDC-susceptible isolates revealed that transcriptional levels of various iron transport proteins, such as fiu, feoA, and feoB, and the energy transduction system, TonB-ExbB-ExbD, were relatively downregulated in CFDC-non-susceptible isolates. GO enrichment analysis revealed that the upregulated genes in CFDC-non-susceptible isolates were mainly associated with redox homeostasis and stress response. Besides, the expression levels of the blaOXA-23 and exbD genes were negatively correlated with the MICs. PER-1 production, iron transport system downregulation, and mutations in PBPs may synergistically impart high-level resistance to CFDC in CR-AB.

Susceptibility of cefiderocol and other antibiotics against carbapenem-resistant, Gram-negative bacteria.

BackgroundCefiderocol is a promising antimicrobial agent against carbapenem-resistant, Gram-negative bacteria, but susceptibility data from the Chinese mainland are lacking. The aim of the present study was to test the susceptibility of cefiderocol against carbapenem-resistant, Gram-negative bacteria collected from Beijing, China.MethodsCarbapenem-resistant Klebsiella pneumoniae (CR-KP; n=105), carbapenem-resistant Acinetobacter baumannii (CR-AB; n=126), carbapenem-resistant Pseudomonas aeruginosa (CR-PA; n=74), and Stenotrophomonas maltophilia (SM; n=72) isolates were collected from inpatients at 4 tertiary hospitals in Beijing, China. Minimum inhibitory concentrations (MICs) for cefiderocol were determined using iron-depleted cation-adjusted Mueller Hinton broth (CAMHB), and for comparators using CAMHB, according to the recommended Clinical and Laboratory Standards Institute (CLSI) methodology. Carbapenemase and other β-lactamase gene profiles were determined using polymerase chain reaction (PCR).ResultsCefiderocol inhibited 100% of CR-KP and CR-PA, and 98.6% of the SM isolates at the susceptibility breakpoint concentration of 4 mg/L. However, the susceptibility rate for cefiderocol against CR-AB was only 62.7%, with MIC90 values as high as 128 mg/L. Nearly all the cefiderocol-susceptible CR-AB isolates were found to be positive for blaOXA-23 and blaTEM, whereas all the cefiderocol-resistant CR-AB isolates were found to be positive for the blaPER genes, in addition to blaOXA-23 and blaTEM.ConclusionsCefiderocol showed potent in vitro activity against CR-KP, CR-PA, and SM isolates collected from Beijing, China. However, the resistance rate for cefiderocol against CR-AB was higher than that reported by other research centers, and the presence of blaPER might contribute to resistance in non-susceptible CR-AB isolates.

Plasmid Carrying blaCTX-M-15, blaPER-1, and blaTEM-1 Genes in Citrobacter spp. From Regional Hospital in Mexico

Introduction:Citrobacter spp. is an opportunistic bacteria that have been recognized as significant pathogens in patients with underlying diseases or immunocompromised status. The aim of this study was to identify extended-spectrum β-lactamases in clinical isolates of Citrobacter spp.Methods:This cross-sectional study was conducted at Hospital Central “Dr. Ignacio Morones Prieto” in San Luis Potosi, Mexico. Nineteen isolates of Citrobacter spp. were obtained from clinical specimens between April to December 2015. Four isolates were resistant to third-generation cephalosporins. The presence of genes encoding ESBL (blaCTX-M-15, blaTEM-1, blaVEB-1, blaSHV, and blaPER-1) was analyzed by PCR. For this purpose, plasmid DNA was extracted and horizontally transferred to recipient E. coli Top 10.Results:blaCTX-M-15 and blaVEB-1 genes were detected in Citrobacter freundii and Citrobacter sedlakii, whereas blaPER-1 gene was identified in 1 isolate of Citrobacter freundii. In contrast, blaSHV gene was not detected in any isolate. One strain carried blaCTX-M-15, blaTEM-1, blaVEB-1, and blaPER-1 genes, most in a 275-kb plasmid.Conclusion:This study shows the presence of different types of ESBL in clinical isolates of Citrobacter freundii and Citrobacter sedlakii, which confer resistance to broad-spectrum β-lactams. The plasmid identified in this study harboring ESBL genes could play an important role in the dissemination of antibiotic resistance.

1287. Double Disk Diffusion Study to Evaluate the Synergistic Effect Between Cefiderocol and Ceftazidime-Avibactam Against Cefiderocol-Non-susceptible Acinetobacter baumannii

Abstract Background Cefiderocol (CFDC), a siderophore cephalosporin, has broad coverage of Gram-negative bacteria and has been approved for clinical use in USA and Europe. The SIDERO-WT surveillance studies showed that CFDC shows >95% susceptibility against Acinetobacter baumannii. Against many of CFDC non-susceptible isolates, most of which had blaPER gene, the combination use of avibactam significantly decreased the MIC by broth microdilution (BMD). In this study, we evaluated the appropriate methodology to evaluate the synergistic effect by disk diffusion studies. Methods The susceptibility testing was conducted as recommended by the CLSI using CFDC non-susceptible isolates (MIC of >4 µg/mL based on CLSI breakpoint). The MIC by BMD was determined using iron-depleted cation-adjusted Mueller-Hinton broth, in the presence or absence of 4 µg/mL of avibactam. The disk diffusion was evaluated using Mueller-Hinton agar, and the synergy was evaluated by using disk stacking methods. For disk stacking methods, CFDC disk was placed on agar on which bacterial suspension of 0.5 McFarland units was spread, then the ceftazidime-avibactam (CZA) disks was stacked on the top, followed by adding a drop (30 µL) of saline on the stacked disks. As an alternative method, CZA was immersed in saline for 1 second instead of adding a drop of saline, followed by the stacking on the top. The disk zone size was determined after 24-hour incubation at 37°C. Results Against blaPER-positive A. baumannii which showed >64 µg/mL MIC of CFDC and CZA, CFDC MIC decreased to 0.25 µg/mL in the presence of avibactam. The disk diffusion methods also showed isolates resistant to CFDC and CZA and showed susceptiblilty disk zone to CFDC by stacking both disks. On the other hand, against blaNDM-positive A. baumannii which showed 64 µg/mL MIC of CFDC and CZA, the disk diffusion methods showed resistance even when stacking both disks. Against multiple isolates, the MIC of CFDC without or with avibactam was correlated well with the disk zone produced by CFDC disk alone or stacked with CZA disks, respectively (Figure). Conclusion The synergistic effect between CFDC and avibactam by BMD methods could be detected by disk stacking methodology using CFDC and CZA disks. Disclosures Yoshinori Yamano, PhD, Shionogi (Employee) Miki Takemura, MS, SHIONOGI & CO., LTD. (Employee) Roger Echols, MD, Shionogi (Consultant) Christopher Longshaw, PhD, Shionogi (Employee)

Virulence Characteristics of Biofilm-Forming Acinetobacter baumannii in Clinical Isolates Using a Galleria mellonella Model.

Acinetobacter baumannii is a Gram-negative coccobacillus responsible for severe hospital-acquired infections, particularly in intensive care units (ICUs). The current study was designed to characterize the virulence traits of biofilm-forming carbapenem-resistant A. baumannii causing pneumonia in ICU patients using a Galleria mellonella model. Two hundred and thirty patients with hospital-acquired or ventilator-associated pneumonia were included in our study. Among the total isolates, A. baumannii was the most frequently isolated etiological agent in ICU patients with pneumonia (54/165, 32.7%). All A. baumannii isolates were subjected to antimicrobial susceptibility testing by the Kirby–Bauer disk diffusion method, while the minimum inhibitory concentrations of imipenem and colistin were estimated using the broth microdilution technique. The biofilm formation activity of the isolates was tested using the microtiter plate technique. Biofilm quantification showed that 61.1% (33/54) of the isolates were strong biofilm producers, while 27.7% (15/54) and 11.1% (6/54) showed moderate or weak biofilm production. By studying the prevalence of carbapenemases-encoding genes among isolates, blaOXA-23-like was positive in 88.9% of the isolates (48/54). The BlaNDM gene was found in 27.7% of the isolates (15/54 isolates). BlaOXA-23-like and blaNDM genes coexisted in 25.9% (14/54 isolates). Bap and blaPER-1 genes, the biofilm-associated genes, coexisted in 5.6% (3/54) of the isolates. For in vivo assessment of A. baumannii pathogenicity, a Galleria mellonella survival assay was used. G. mellonella survival was statistically different between moderate and poor biofilm producers (p < 0.0001). The killing effect of the strong biofilm-producing group was significantly higher than that of the moderate and poor biofilm producers (p < 0.0001 for each comparison). These findings highlight the role of biofilm formation as a powerful virulence factor for carbapenem-resistant A. baumannii that causes pneumonia in the ICU.

Detection of tetM, armA, blaPER-1 and blaIMP genes in E. coli isolates among the gram negative bacteria that cause urinary tract infections

The purpose of this study was to define the prevalence of gram negative bacteria that cause urinary tract infections (UTIs) and to look for the genes tetM, blaIMP, and ND M-1 in uropathogenic Escherichia coli isolates. This study was conducted on 205 urine samples collected from outpatients having a urinary tract infection for Baquba teaching hospital. The VITEK 2 compact device, which is fully automated, was used to identify the causative microorganisms and also used in antimicrobials susceptibility test. Combined-disk test was used for phenotypic determination of beta lactamases producing isolates and after DNA extraction, PCR was used to determine the genotypic status of the 16S rRNA, tetM, blaIMP, and NDM-1 genes..Out of 205 urine samples, only 72(35.12%) were found to have gram negative bacteria, isolated microorganisms were Escherichia coli 44 (35.7%), K. Pneumonia 18(14.6%), P. aeruginosa 8(6.5 %) were 2 (1.6 %) diagnosed as Burkholderia cepacia. Overall highest susceptibility was observed for imipenem, meropenem, amikacin, momocycline, colistin and to tazobactam and 32 isolates from E. coli were confirmed to be ESBL producers. 16S rRNA gene sequencing attributed the isolates to Escherichia coli. Among the 10 E. coli isolates recovered from the UTI patients, isolates which positive for tetM ( 90%) and which negative for armA, blaPER-1 and blaIMP. The most common gram negative bacteria found to cause UTIs were E. coli and K. pneumoniae. This study found that a few pathogens of E. coli are resistant to IPM and MEM. The frequency of tetM genes that are responsible for this resistance among pathogenic E. coli isolates in diyala, was high.

High frequency of blaPER-1 gene in clinical strains of Acinetobacter baumannii and its association with quorum sensing and virulence factors

Acinetobacter baumannii is known as the main pathogenic organisms, especially for the nosocomial infections. Multi-drug resistance and the presence of different virulence factors are recognized as causes of the organism's survival and the infection's risk. In this study, 70 Acinetobacter baumannii isolates were collected from three hospitals located in the Northwest of Iran. Antimicrobial susceptibility of these isolates was determined by agar dilution and disk diffusion methods. Production of gelatinase, proteolytic, and hemolytic activities of the isolates was also assessed. The frequencies of epsA, pld, ptk, luxR, luxI, and blaPER-1 genes were investigated by polymerase chain reaction (PCR). Furthermore, molecular typing of the isolates was performed using random amplified polymorphic DNA (RAPD-PCR). All the obtained isolates were multi drug resistant and 58.5% of them were extensively drug resistance. Six of the 70 study isolates were found to be resistant to all the tested antibiotics. Nine (12.8%) of the isolates were found to be resistant to colistin (MIC = 4 μg/mL). 38.5%, 45.7%, and 15.5% of the isolates were positive for gelatinase production, protease, and hemolysis, respectively. The frequency of the genes was as follows: pld 100%, epsA 91.9%, luxI 85.7%, ptk 80%, luxR 75.7% and blaPER-1 64.2%. Based on the RAPD patterns obtained, the 70 isolates in this study clustered into twelve major genotypes. In this study, the prevalence of blaPER-1 was worryingly high. On the other hand, a significant relationship was found between the presence of this gene and the production of some virulence factors in the strains.

Resistance to nitrofurantoin is an indicator of extensive drug-resistant (XDR) Enterobacteriaceae.

Introduction. Nitrofurantoin is one of the preferred antibiotics in the treatment of uropathogenic multidrug-resistant (MDR) infections. However, resistance to nitrofurantoin in extensively drug-resistant (XDR) bacteria has severely limited the treatment options.Gap statement. Information related to co-resistance or collateral sensitivity (CS) with reference to nitrofurantoin resistant bacteria is limited.Aim. To study the potential of nitrofurantoin resistance as an indicator of the XDR phenotype in Enterobacteriaceae.Methods. One hundred (45 nitrofurantoin-resistant, 21 intermediately resistant and 34 nitrofurantoin-susceptible) Enterobacteriaceae were analysed in this study. Antibiotic susceptibility testing (AST) against nitrofurantoin and 17 other antimicrobial agents across eight different classes was performed by using the Vitek 2.0 system. The isolates were screened for the prevalence of acquired antimicrobial resistance (AMR) and efflux pump genes by PCR.Results. In total, 51 % of nitrofurantoin-resistant and 28 % of intermediately nitrofurantoin resistant isolates exhibited XDR characteristics, while only 3 % of nitrofurantoin-sensitive isolates were XDR (P=0.0001). Significant co-resistance was observed between nitrofurantoin and other tested antibiotics (β-lactam, cephalosporin, carbapenem, aminoglycoside and tetracycline). Further, the prevalence of AMR and efflux pump genes was higher in the nitrofurantoin-resistant strains compared to the susceptible isolates. A strong association was observed between nitrofurantoin resistance and the presence of bla PER-1, bla NDM-1, bla OXA-48, ant(2) and oqxA-oqxB genes. Tigecycline (84 %) and colistin (95 %) were the only antibiotics to which the majority of the isolates were susceptible.Conclusion. Nitrofurantoin resistance could be an indicator of the XDR phenotype among Enterobacteriaceae, harbouring multiple AMR and efflux pump genes. Tigecycline and colistin are the only antibiotics that could be used in the treatment of such XDR infections. A deeper understanding of the co-resistance mechanisms in XDR pathogens and prescription of AST-based appropriate combination therapy may help mitigate this problem.

Screening of Antimicrobial Resistance Genes and Epidemiological Features in Hospital and Community-Associated Carbapenem-Resistant Pseudomonas aeruginosa Infections.

IntroductionResearching carbapenem-resistant isolates enables the identification of carbapenemase-producing bacteria and prevents their spread.MethodsP. aeruginosa isolates were recovered from Medicine Faculty of Recep Tayyip Erdoğan University and identified by conventional methods and the automated Vitek 2 Compact system. Antimicrobial susceptibility experiments were performed in accordance with CLSI criteria and the automated Vitek 2 Compact system. The PCR method was investigated for the presence of β-lactamase resistance genes. PFGE typing was performed to show clonal relation among samples.ResultsSeventy P. aeruginosa isolates were isolated from seventy patients. Of the patients, 67.1% had contact with the health service in the last 90 days and 75.7% of the patients had received antimicrobial therapy in the previous 90 days. Twenty-four isolates were carbapenem resistant, 2 isolates were multidrug-resistant except colistin, and none of the samples had colistin resistance. The gene encoding β-lactamase or metallo-β-lactamase was found in a total of 36 isolates. The blaVEB and blaPER genes were identified in 1 and 5 isolates alone or 17 and 13 isolates in combination with other resistance genes, respectively. The blaNDM was the most detected metallo-β-lactamase encoding gene (n=18), followed by blaKPC (n=12). blaIMP and blaVIM were detected in 5 and 1 isolates, respectively. Also, the association of blaVEB-blaPER and blaVEB-blaKPC-blaNDM was found to be very high. Much more resistance genes and co-occurrence were detected in hospital-acquired samples than community-acquired samples. No difference was found between the community and hospital-associated isolates according to PFGE results. Simultaneously from 6 patients, other microorganisms were also isolated and 5 of them died.ConclusionThe average length of stay (days) was found to be significantly higher in HAI group than CAI group. The death of 5 patients with fewer or no resistance genes showed that the co-existence of other microorganisms in addition to resistance genes was important on death.

Characterization of antibiotic resistance profiles in Pseudomonas aeruginosa isolates from burn patients

ObjectiveTo investigate the prevalence of multidrug-resistant (MDR) Pseudomonas aeruginosa (PA) producing extended-spectrum beta-lactamases (ESBLs) and metallo-beta-lactamases (MBLs) in burn patients in Algeria. MethodsBetween April 2016 and October 2019, 47 non-redundant isolates of PA were collected from 47 burn patients admitted to the Department of Burns at the Military Hospital of Algiers in Algeria. Antibiotic susceptibility testing was performed by agar diffusion and the Phoenix automated method. Resistance genes were identified by PCR, and molecular typing of isolates was carried out by enterobacterial repetitive intergenic consensus (ERIC) sequences-polymerase chain reaction (PCR). ResultsAmong the 47 non-redundant MDR PA strains isolated, 59.57% were phenotypically ESBLs-positive, and 100% were phenotypically MBL-positive. The ESBL-positive isolates were subsequently screened for six groups of bla genes encoding ESBL-type enzymes, namely blaCTX-M2, blaPER, blaTEM, blaSHV, blaVEB, and blaGES. Out of the 28 ESBL-producing strains, 23 (82.14%) were blaCTX-M2 positive; 18 (38.29%) were blaPER positive, and 16 (34.04%) were blaTEM positive, while 5 (17.9%) were co-harboring blaCTX-M2, blaTEM, and blaPER genes. The blaSHV, blaVEB, and blaGES genes were not detected in any of the ESBL positive isolates. Since all isolates were MBL-positive, all 47 strains were screened for the blaNDM-1, blaIMP, blaVIM genes that produce MBLs; however, none of these genes were detected. Additional screening for the oprD gene demonstrated that 45 (95.74%) of the isolates were positive for this gene. Finally, ERIC PCR revealed 11 distinct PA clones among the blaCTX-M2 positive strains. ConclusionThis is the first study to report the presence of CTX-M2-producing PA in the North Africa region and the first to detect blaCTX-M2-positive and blaPER-positive PA clinical isolates in Algeria, therefore demonstrating the spread of such MDR strains to this part of the world. Identification of bacterial genotypic alterations that confer antibiotic resistance is critical in determining the most effective antimicrobial strategies to be employed. Therefore, our findings could potentially facilitate clinical decision making regarding the antibiotics of choice for the treatment of burn patients that suffer from PA infections in Algeria.

Microbiological and molecular characterization of antimicrobial resistance in uropathogenic Escherichia coli from peruvian public hospitals.

We characterized the antimicrobial resistance of 70 Escherichia coli isolates obtained from patients with a urinary tract infection (UTI) from 8 public hospitals in Peru. Resistance profiles were identified using the automated MicroScan® system. A standard polymerase chain reaction was used for the detection of the bla TEM, bla CTX-M, bla SHV and bla PER genes. The 65.7% (46/70) of the isolates presented a multidrug-resistant phenotype and 55.7% (39/70) were extended-spectrum beta-lactamases producers. High levels of resistance were detected for ampicillin (77,1%), ciprofloxacin (74,3%), trimethoprim/sulfamethoxazole (62,9%), cefepime (57,1%), and cefuroxime (57,1%). The bla TEM gene was the most frequent (31,4%), followed by bla CTX-M (18,6%) and bla SHV (2,9%) genes. These results show high resistance levels to antimicrobials of clinical use in E. coli isolates from hospital UTI patients in Peru.