ampC Gene Research Articles

Characterization of Pseudomonas aeruginosa resistance to ceftolozane-tazobactam due to ampC and/or ampD mutations observed during treatment using semi-mechanistic PKPD modeling.

A double ampC (AmpCG183D) and ampD (AmpDH157Y) genes mutations have been identified by whole genome sequencing in a Pseudomonas aeruginosa (PaS) that became resistant (PaR) in a patient treated by ceftolozane/tazobactam (C/T). To precisely characterize the respective contributions of these mutations on the decreased susceptibility to C/T and on the parallel increased susceptibility to imipenem (IMI), mutants were generated by homologous recombination in PAO1 reference strain (PAO1- AmpCG183D, PAO1-AmpDH157Y, PAO1-AmpCG183D/AmpDH157Y) and in PaR (PaR-AmpCPaS/AmpDPaS). Sequential time-kill curve experiments were conducted on all strains and analyzed by semi-mechanistic PKPD modeling. A PKPD model with adaptation successfully described the data, allowing discrimination between initial and time-related (adaptive resistance) effects of mutations. With PAO1 and mutant-derived strains, initial EC50 values increased by 1.4, 4.1, and 29-fold after AmpCG183D , AmpDH157Y and AmpCG183D/AmpDH157Y mutations, respectively. EC50 values were increased by 320, 12.4, and 55-fold at the end of the 2 nd experiment. EC50 of PAO1-AmpCG183D/AmpDH157Y was higher than that of single mutants at any time of the experiments. Within the PaR clinical background, reversal of AmpCG183D, and AmpDH157Y mutations led to an important decrease of EC50 value, from 80.5 mg/L to 6.77 mg/L for PaR and PaR-AmpCPaS/AmpDPaS, respectively. The effect of mutations on IMI susceptibility mainly showed that the AmpCG183D mutation prevented the emergence of adaptive resistance. The model successfully described the separate and combined effect of AmpCG183D and AmpDH157Y mutations against C/T and IMI, allowing discrimination and quantification of the initial and time-related effects of mutations. This method could be reproduced in clinical strains to decipher complex resistance mechanisms.

Characterization of Carbapenemase- and ESBL-Producing Gram-Negative Bacilli Isolated from Patients with Urinary Tract and Bloodstream Infections.

A total of 199 Gram-negative bacterial isolates from urinary tract infections and 162 from bloodstream infections were collected from 12 healthcare systems throughout the United States between May 2021 and August 2022. The isolates, phenotypically non-susceptible to 2nd or 3rd generation cephalosporins or carbapenems, were characterized through antimicrobial susceptibility testing and whole genome sequence analysis to obtain a broad snapshot of beta-lactamase-mediated resistance among these two sample types. Overall, 23 different carbapenemase genes were detected among 13 species (20.5% of isolates). The blaKPC-3 and blaKPC-2 subtypes were the most common carbapenemase genes identified, followed by blaNDM and the co-carriage of two different blaOXA carbapenemases by Acinetobacter baumannii isolates. All carbapenemase-producing A. baumannii isolates were mCIM negative. Extended-spectrum beta-lactamase genes were identified in 66.2% of isolates; blaCTX-M-15 was the most common. AmpC genes, both plasmid and chromosomal, were detected in 33.2% of isolates. Importantly, 2.8%, 8.3%, and 22.2% of blaKPC-positive organisms were susceptible to ertapenem, imipenem, and meropenem, respectively. The correlation between broth microdilution and disk diffusion results was high for most drugs except cefepime, where the detection of resistance was statistically lower by disk diffusion. Thus, there were gaps in the accuracy of susceptibility testing for some mechanisms of resistance.

The functional repertoire of AmpR in the AmpC β-lactamase high expression and decreasing β-lactam and aminoglycosides resistance in ESBL Citrobacter freundii

Citrobacter freundii is characterized by AmpC β-lactamases that develop resistance to β-lactam antibiotics. The production of extended-spectrum β-lactamase (ESBL) is substantially high in Escherichia coli, C. freundii, Enterobacter cloacae, and Serratia marcescens, but infrequently explored in C. freundii. The present investigation characterized the ESBL C. freundii and delineated the genes involved in decrease in antibiotics resistance. We used the VITEK-2 system and Analytical Profile Index (API) kit to characterize and identify the Citrobacter isolates. The mRNA level of AmpC and AmpR was determined by RT-qPCR, and gel-shift assay was performed to evaluate protein-DNA binding. Here, a total of 26 Citrobacter strains were isolated from COVID-19 patients that showed varying degrees of antibiotic resistance. We examined and characterized the multidrug resistant C. freundii that showed ESBL production. The RT-qPCR analysis revealed that the AmpC mRNA expression is significantly high followed by a high level of AmpR. We sequenced the AmpC and AmpR genes that revealed the AmpR has four novel mutations in comparison to the reference genome namely; Thr64Ile, Arg86Ser, Asp135Val, and Ile183Leu while AmpC remained intact. The ΔAmpR mutant analysis revealed that the AmpR positively regulates oxidative stress response and decreases β-lactam and aminoglycosides resistance. The AmpC and AmpR high expression was associated with resistance to tazobactam, ampicillin, gentamicin, nitrofurantoin, and cephalosporins whereas AmpR deletion reduced β-lactam and aminoglycosides resistance. We conclude that AmpR is a positive regulator of AmpC that stimulates β-lactamases which inactivate multiple antibiotics.

Prevalence of AmpC, ESBL, and colistin resistance genes in Enterobacterales isolated from ready-to-eat food in Algeria.

Antimicrobial resistance among bacteria present in ready-to-eat foods is an emerging concern. Hence, this study investigated the presence of extended-spectrum and AmpC β-lactamases (ESBL/AmpC)-producing Enterobacterales (ESBL-E) and the dissemination of mcr-1 in ESBL-E from ready-to-eat food samples (RTE) in Algeria. RTE food samples (n = 204) were aseptically collected and selectively cultured using MacConkey agar. The isolates were screened for ESBL production using the DDST test, confirmed ESBL-E isolates were identified using different conventional methods and MALDI-TOF MS, antibiotic susceptibility was determined using the disc diffusion and broth microdilution assay, ESBL-E isolates were analyzed for colistin and ESBL/AmpC encoding genes by PCR, and food samples were analyzed by univariate and multiple logistic regression. Overall, 48 (17.4%) of the 276 Enterobacterales were confirmed as ESBL producers, with a high prevalence in soups (40%), salads (25%), and cream-filled pastries (23.8%). Antibiotic susceptibility testing revealed that all the ESBL-E isolates were found multi-drug resistant. PCR revealed that blaTEM, blaCTX-M, blaCMY-2, blaOXA-1, and blaSHV were the most frequently detected. blaCTX-M-9 and blaCTX-M-1 were the predominant CTX-M types. Furthermore, four isolates were positive for mcr-1; three of them harbored the colistin resistance gene and ESBL/AmpC genes (2 E. cloacae and 1 S. enterica). To the best of our knowledge, this is the first report that detects the presence of the mcr-1 gene in ESBL-E strains isolated from RTE foods in Algeria. These findings suggest an urgent need for strict policies that prevent the spread and transmission of ESBL-E in food.

Study on the Role of ampG in the Regulation of Plasmid-Mediated ampC -Induced Expression in Klebsiella pneumoniae.

In this study, we constructed ampG knock-out and knock-in strains from a clinically isolated Kp1strain carrying ampR-ampC in its plasmid and compared them with the Kp NTUH-K2044 strain to investigate the relationship between ampG and ampR-ampC-induced expression. We created the ampG gene deletion mutant strains Kp1-ΔampG and Kp NTUH-K2044-ΔampG with pKO3-km plasmid using homologous recombination technology. We constructed the Kp NTUH-K2044-RC and Kp NTUH-K2044-ΔampG-RC drug resistance model strains with plasmid pACYC184. We constructed the ampG knock-in strains by introducing the ampG genes of Kp1, Enterobacter cloacae 029M, Pseudomonas aeruginosa PAO1, Escherichia coli ATCC25922, and Salmonella typhimurium LT2 into the ampG gene-deleted strains with carrier pet-30a. Real-time polymerase chain reaction (real-time PCR) was used to detect the relative expressions of ampC and ampG mRNAs. Compared with Kp1, the induction phenotype of the ampC of Kp1-ΔampG strain disappeared, the ampC expression was reduced, and the minimal inhibitory concentration (MIC) values of cefoxitin and ceftazidime significant decrease from 128 μg/mL to 1 μg/mL. Based on Kp1, five strain were successfully constructed to complement the ampG genes from five knock-in strain, and all of the above complemented strains showed inducible expression of ampC and restored the expression of ampG to varying degrees, as well as restored resistance to the antimicrobial drugs cefoxitin and ceftazidime (P < 0.05). The ampC and ampG genes were barely expressed in Kp NTUH-K2044-ΔampG-RC when compared with Kp NTUH-K2044-RC. The expressions of ampG and ampC in each knock-in strain were recovered, the induction phenotype of ampC was restored, and the MIC values of cefoxitin and ceftazidime were increased. (P < 0.05). In this study, we found that ampG was an essential regulator for the plasmid-mediated ampC-induced expression in K. pneumoniae.

BlaPSZ-1, a novel AmpC gene identified from a Pantoea isolate.

Pantoea species of the family Erwiniaceae are well-known plant pathogens and animal and human conditional pathogens. Due to the widespread and continuous use of antimicrobials, multidrug-resistant strains continue to emerge, making clinical treatment difficult; therefore, there is an increasing need to clarify the mechanisms of drug resistance. A rabbit anal fecal sample was collected by a swab and the streak plate method was used to isolate single colonies. The standard agar dilution method was used to determine the minimum inhibitory concentrations (MICs) against antimicrobials. The complete genome sequence of the bacterium was obtained using Next-Generation Sequencing platforms. The potential resistance gene was annotated based on the Comprehensive Antibiotic Resistance Database (CARD) and verified by molecular cloning. The β-lactamase PSZ-1 was expressed via the pCold I expression vector and its enzyme kinetic parameters were analyzed. The genetic environment and evolutionary process of the novel resistance gene-related sequences were analyzed by bioinformatic methods. The isolate Pantoea endophytica X85 showed some degree of resistance to penicillins as well as cephalosporins. A novel AmpC resistance gene, designated blaPSZ-1 in this research, was identified to be encoded in the plasmid (pPEX85) of P. endophytica X85. BlaPSZ-1 showed resistance to penicillins and several first-, second-and third-generation cephalosporins as well as aztreonam, but it did not show resistance to the fourth-generation cephalosporins or carbapenems tested. Enzyme kinetic assays revealed that it could hydrolyze amoxicillin, penicillin G, cephalothin, and cefazolin, and its hydrolytic activity could be strongly inhibited by the inhibitor avibactam, which was generally consistent with antimicrobial susceptibility testing results. No hydrolytic activity was observed for third-generation cephalosporins or aztreonam. In this study, a novel AmpC β-lactamase gene, designated blaPSZ-1, was characterized and it was encoded in the plasmid of the bacterium P. endophytica X85. It shows resistance to penicillins and several cephalosporins. The discovery of novel drug resistance mechanisms can help guide the scientific use of drugs in animal husbandry and clinical practice, effectively avoiding the abuse of antimicrobials and thus preventing the further development and spread of bacterial resistance.

Colonization of Residents and Staff of an Italian Long-Term Care Facility and an Adjacent Acute Care Hospital Geriatrics Unit by Multidrug-Resistant Bacteria.

In 2022, we undertook a point prevalence screening study for Enterobacterales with extended-spectrum β-lactamases (ESBLs), high-level AmpC cephalosporinases and carbapenemases, and also methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE) in a long-term care facility (LTCF) and the associated acute-care hospital Geriatrics unit in Bolzano, Northern Italy. Urine samples and rectal, inguinal, oropharyngeal, and nasal swabs were plated on selective agar plates. Metadata of the patients, including demographic data, were collected, and risk factors for colonization were determined. ESBL, AmpC, carbapenemase, and quinolone resistance genes were investigated by the HybriSpot 12 PCR AUTO System. The following colonization percentages by multidrug-resistant (MDR) bacteria have been found in LTCF residents: all MDR organisms, 59.5%; ESBL producers, 46.0% (mainly CTX-M-type enzymes); carbapenemase producers, 1.1% (one Klebsiella pneumoniae with KPC-type); MRSA, 4.5%; VRE, 6.7%. Colonization by MDR bacteria was 18.9% for LTCF staff and 45.0% for Geriatrics unit patients. Peripheral vascular disease, the presence of any medical device, cancer, and a Katz Index of 0 were significant risk factors for colonization of LTCF residents by MDR bacteria in univariate and/or multivariate regression analysis. To conclude, the ongoing widespread diffusion of MDR bacteria in the LTCF suggests that efforts should be strengthened on MDR screening, implementation of infection control strategies, and antibiotic stewardship programs targeting the unique aspects of LTCFs. ClinicalTrials.gov ID: 0530250-BZ Reg01 30/08/2022.

Roles of intrinsic and acquired resistance determinants in multidrug-resistant clinical Pseudomonas aeruginosa in Bangladesh

Introduction: Pseudomonas aeruginosa is an ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, P. aeruginosa, and Enterobacter spp.) pathogen and one of the leading etiologies in multiple nosocomial infections. Treatment of P. aeruginosa is becoming increasingly difficult due to its ever-increasing antibiotic resistance trends. This study investigated clinical multidrug resistance (MDR) P. aeruginosa (MDR-PA), their intrinsic resistance determinants, including the presence of chromosomal AmpC β-lactamase (Ampicillinase), decreased expression of outer membrane porin protein OprD and selected acquired β-lactamase resistance genes.&#x0D; Methods: Out of 238 clinical specimens, including urines from urinary tract-infected patients, wound swabs, burn swabs, and catheter aspirates, were collected from two major hospitals in Savar, Dhaka, Bangladesh. Samples were inoculated with Cetrimide agar to isolate presumptive P. aeruginosa. Bacteria were identified by cultural, biochemical characterization, 16S rDNA sequencing, and phylogenetic analysis. Virulence-associated genes of P. aeruginosa, namely, toxA, lasB, and plcH, were identified by polymerase chain reaction (PCR). Antibiotic susceptibilities of the isolates were investigated against ten antibiotics belonging to seven groups by disc-diffusion method followed by a selected minimum inhibitory concentration (MIC) assay. Phenotypic expression of Metallo-β-lactamases (MBLs) production was checked by the double disc synergistic test selectively among the imipenem-resistant isolates. Acquisition of β-lactam resistance trait was examined by PCR detection of bla-genes variants. Mutational loss of the OprD was analyzed by PCR to investigate intrinsic resistance determinants. Phenotypic overexpression of chromosomal AmpC was assayed with the identification of the AmpC gene by PCR. The expression level of OprD was assessed by real-time quantitative PCR (RT-qPCR).&#x0D; Results: Fifty-three P. aeruginosa was identified, with an overall isolation of 22.3% (53/238), where urine remains the most prevalent source. Virulence genes toxA, lasB, and plcH were identified in the isolates of 92.4%, 96.2%, and 94.3%. The highest phenotypic antimicrobial resistance was observed against ampicillin and ceftriaxone (100%), followed by cefotaxime (96%), tetracycline (89%), azithromycin (72%), imipenem (31%), ciprofloxacin (29%), levofloxacin (29%), gentamycin (27%) and ceftazidime (14%). The antibiogram pattern revealed 85% of isolates as multidrug-resistant, while 12% were considered extensively drug-resistant (XDR)-P. aeruginosa. The carriage of β-lactamase genes blaTEM, blaSHV, and blaOXA was detected in 4%, 2%, and 2% cephalosporin-resistant isolates, respectively. Double disc synergistic test revealed 87% of imipenem-resistant isolates expressing MBL-mediated resistance phenomenon. All seven ceftazidime-resistant isolates showed the presence of the AmpC gene with phenotypic overproduction of the AmpC enzyme, indicating AmpC-mediated ceftazidime resistance. Mutational loss of OprD was observed in 12% of phenotypically multidrug-resistant isolates, and RT-qPCR analysis revealed reduced expression of OprD porin protein at various levels in the outer membrane of multidrug-resistant isolates.&#x0D; Conclusions: This study depicts the high prevalence of MDR-PA in clinical specimens in Bangladesh. The identified intrinsic and acquired antimicrobial resistance determinants play synergistic roles in emerging MDR-PA.&#x0D; Bangladesh Journal of Medical Science Vol. 22 No. 03 July’23 Page : 489-507

Plasmid-mediated AmpC in Klebsiella pneumoniae and Escherichia coli from septicaemic neonates: diversity, transmission and phenotypic detection

Presence and dissemination of plasmid-mediated AmpC genes (pAmpCs) have made bacteria cephalosporin-resistant and assessment of their prevalence and diversity is essential. Coexistence of pAmpCs with New Delhi metallo-β-lactamase (blaNDM) has facilitated their spread and NDM interferes with correct pAmpC phenotypic identification. Assessment of pAmpCs in different species and sequence types (STs), co-transmission with blaNDM and phenotypic detection were analysed among Klebsiella pneumoniae (n=256) and Escherichia coli (n=92) isolated from septicaemic neonates over 13 years. pAmpCs were present in 9% (30/348) of strains, 5% in K. pneumoniae and 18% in E. coli. pAmpC genes (blaCMY and blaDHA) were detected, blaCMY-42 and blaDHA-1 variants being predominant. Strains were resistant to most antimicrobials tested. blaCMY and blaDHA were dominant among E. coli (14/17) and K. pneumoniae (9/13), respectively. pAmpC-bearing strains belonged to diverse STs, including epidemic K. pneumoniae ST11 and ST147. Some strains co-harboured carbapenemase genes, blaNDM (17/30) and blaOXA-48 (5/30). In 40% (12/30) of strains, pAmpC genes were transferred by conjugation, of which 8/12 exhibited co-transfer with blaNDM. pAmpCs were frequently found in replicons as follows: blaDHA-1 with IncHIB-M, blaCMY-4 with IncA/C, blaCMY-6 with IncA/C, and blaCMY-42 with IncFII. The combination disk-diffusion test correctly detected pAmpC in 77% (23/30) of pAmpC-bearing strains. However, correct detection of pAmpC was higher in strains that did not harbour blaNDM vs. those with blaNDM (85% vs. 71%). Presence of pAmpCs along with carbapenemases, linkage with multiple STs, and replicon types indicated their potential for spread. pAmpCs can go undetected in the presence of blaNDM; hence, regular surveillance is required.

Occurrence of Carbapenemases, Extended-Spectrum Beta-Lactamases and AmpCs among Beta-Lactamase-Producing Gram-Negative Bacteria from Clinical Sources in Accra, Ghana

Beta-lactamase (β-lactamase)-producing Gram-negative bacteria (GNB) are of public health concern due to their resistance to routine antimicrobials. We investigated the antimicrobial resistance and occurrence of carbapenemases, extended-spectrum β-lactamases (ESBLs) and AmpCs among GNB from clinical sources. GNB were identified using matrix-assisted laser desorption/ionization time of flight–mass spectrometry (MALDITOF-MS). Antimicrobial susceptibility testing was performed via Kirby–Bauer disk diffusion and a microscan autoSCAN system. β-lactamase genes were determined via multiplex polymerase chain reactions. Of the 181 archived GNB analyzed, Escherichia coli and Klebsiella pneumoniae constituted 46% (n = 83) and 17% (n = 30), respectively. Resistance to ampicillin (51%), third-generation cephalosporins (21%), and ertapenem (21%) was observed among the isolates, with 44% being multi-drug resistant (MDR). β-lactamase genes such as AmpCs ((blaFOX-M (64%) and blaDHA-M and blaEDC-M (27%)), ESBLs ((blaCTX-M (81%), other β-lactamase genes blaTEM (73%) and blaSHV (27%)) and carbapenemase ((blaOXA-48 (60%) and blaNDM and blaKPC (40%)) were also detected. One K. pneumoniae co-harbored AmpC (blaFOX-M and blaEBC-M) and carbapenemase (blaKPC and blaOXA-48) genes. blaOXA-48 gene was detected in one carbapenem-resistant Acinetobacter baumannii. Overall, isolates were resistant to a wide range of antimicrobials including last-line treatment options. This underpins the need for continuous surveillance for effective management of infections caused by these pathogens in our settings.

Extended-spectrum beta-lactamases found in Escherichia coli isolates obtained from blood cultures and corresponding stool specimen

With extended-spectrum β-lactamases (ESBLs) and CTX-M enzymes being on the rise, antimicrobial treatment of enterobacterial infections is becoming more and more challenging. Our study aimed at a molecular characterization of phenotypically ESBL-positive E. coli strains obtained from blood cultures of patients of the University Hospital of Leipzig (UKL), Germany. The presence of CMY-2, CTX-M-14 and CTX-M-15 was investigated using Streck ARM-D Kit (Streck, USA). Real-time amplifications were performed by QIAGEN Rotor-Gene Q MDx Thermocycler (QIAGEN, Thermo Fisher Scientific, USA). Antibiograms as well as epidemiological data were evaluated. Among 117 cases, 74.4% of the isolates showed a resistance to ciprofloxacin, piperacillin and ceftazidime or cefotaxime while being susceptible to imipenem/meropenem. The proportion of ciprofloxacin resistance was significantly higher than the proportion of ciprofloxacin susceptibility. At least one of the investigated genes was detected in 93.1% of the blood culture E. coli isolates: CTX-M-15 (66.7%), CTX-M-14 (25.6%) or the plasmid-mediated ampC gene CMY-2 (3.4%). 2.6% were tested positive for two resistance genes. 94 of the corresponding stool specimens tested positive for ESBL producing E. coli (94/112, 83.9%). 79 (79/94, 84%) E. coli strains found in the stool samples matched with the respective patient’s blood culture isolate phenotypically (MALDI-TOF, antibiogram). The distribution of resistance genes was in accordance with recent studies in Germany as well as worldwide. This study provides indications of an endogenous focus of infection and emphasize the importance of screening programs for high-risk patients.

In-vitro activity of oral third-generation cephalosporins plus clavulanate against ESBL-producing Enterobacterales isolates from the MERINO trial

Extended-spectrum-beta-lactamase (ESBL)-producing Enterobacterales as a cause of community-acquired uncomplicated urinary tract infection (UTI) is on the rise. Currently, there are minimal oral treatment options. New combinations of existing oral third-generation cephalosporins paired with clavulanate may overcome resistance mechanisms seen in these emerging uropathogens. Ceftriaxone-resistant Escherichia coli and Klebsiella pneumoniae containing CTX-M-type ESBLs or AmpC, in addition to narrow-spectrum OXA and SHV enzymes, were selected from blood culture isolates obtained from the MERINO trial. Minimum inhibitory concentration (MIC) values of third-generation cephalosporins (cefpodoxime, ceftibuten, cefixime, cefdinir), both with and without clavulanate, were determined. One hundred and one isolates were used with ESBL, AmpC and narrow-spectrum OXA genes (e.g. OXA-1, OXA-10) present in 84, 15 and 35 isolates, respectively. Susceptibility to oral third-generation cephalosporins alone was very poor. Addition of 2 mg/L clavulanate reduced the MIC50 values (cefpodoxime MIC50 2 mg/L, ceftibuten MIC50 2 mg/L, cefixime MIC50 2 mg/L, cefdinir MIC50 4 mg/L) and restored susceptibility (33%, 49%, 40% and 21% susceptible, respectively) in a substantial number of isolates. This finding was less pronounced in isolates co-harbouring AmpC. In-vitro activity of these new combinations may be limited in real-world Enterobacterales isolates co-harbouring multiple antimicrobial resistance genes. Pharmacokinetic/pharmacodynamic data would be useful for further evaluation of their activity.

Phenotypic and genetic screening of Klebsiella pneumoniae isolates from human UTI patients for beta-lactamases and their genetic diversity analysis by ERIC and REP PCRs.

Klebsiella pneumoniae is one of the major nosocomial pathogens responsible for pneumoniae, septicaemia, liver abscesses, and urinary tract infections. Coordinated efforts by antibiotic stewardship and clinicians are underway to curtail the emergence of antibiotic-resistant strains. The objective of the present study is to characterize K. pneumoniae strains through antibiotic resistance screening for production of beta-lactamases (β-lactamases) such as extended spectrum beta lactamases (ESBLs), AmpC β-lactamases, and carbapenemases by phenotypic and genotypic methods and genetic fingerprinting by enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) and repetitive element palindromic PCR (REP-PCR). A total of 85 K. pneumoniae strains isolated from 504 human urinary tract infections (UTI) were used in this study. Only 76 isolates showed positive in phenotypic screening test (PST), while combination disc method (CDM) as phenotypic confirmatory test (PCT) confirmed 72 isolates as ESBL producers. One or more β-lactamase genes were detected by PCR in 66 isolates (91.66%, 66/72) with blaTEM gene being the most predominant (75.75%, 50/66). AmpC genes could be detected in 21 isolates (31.8%, 21/66) with FOX gene being the predominant (24.24%, 16/66), whereas NDM-I was detected in a single strain (1.51%, 1/66). Genetic fingerprinting using ERIC-PCR and REP-PCR revealed wide heterogeneity among β-lactamase producing isolates with discriminatory power of 0.9995 and 1, respectively.

Characteristics of antibiotic resistance mechanisms and genes of Klebsiella pneumoniae.

Klebsiella pneumoniae is an important multidrug-resistant (MDR) pathogen that can cause a range of infections in hospitalized patients. With the growing use of antibiotics, MDR K. pneumoniae is more prevalent, posing additional difficulties and obstacles in clinical therapy. To provide a valuable reference to deeply understand K. pneumoniae, and also to provide the theoretical basis for clinical prevention of such bacteria infections, the antibiotic resistance and mechanism of K. pneumoniae are discussed in this article. We conducted a literature review on antibiotic resistance of K. pneumoniae. We ran a thorough literature search of PubMed, Web of Science, and Scopus, among other databases. We also thoroughly searched the literature listed in the papers. We searched all antibiotic resistance mechanisms and genes of seven important antibiotics used to treat K. pneumoniae infections. Antibiotics such as β-lactams, aminoglycosides, and quinolones are used in the treatment of K. pneumoniae infection. With both chromosomal and plasmid-encoded ARGs, this pathogen has diverse resistance genes. Carbapenem resistance genes, enlarged-spectrum β-lactamase genes, and AmpC genes are the most often β-lactamase resistance genes. K. pneumoniae is a major contributor to antibiotic resistance worldwide. Understanding K. pneumoniae antibiotic resistance mechanisms and molecular characteristics will be important for the design of targeted prevention and novel control strategies against this pathogen.

Phenotypic and genotypic characteristics of beta-lactamase dominant with CARBA, AmpC, and ESBL-producing bacteria in municipal wastewater influent in Helsinki, Finland

Analysing samples of municipal wastewater influent (before treatment) can help to map the status of antibiotic-resistant bacteria (ARB) at the population level in sewershed communities and may also help in predicting the public health risks of ARB in surface water because of the outfall of wastewater. In this study, we investigated the bacterial isolates carrying beta-lactamase genes in wastewater and compared their genotypic and phenotypic characteristics. A total of 399 bacterial isolates grown on CHROMagarESBL (n=207) and CHROMagarKPC (n=192) from composite wastewater influent samples (n=7) from the Viikinmäki wastewater treatment plant (Helsinki) were subcultured, nucleic acid was extracted, and the prevalence of different beta-lactamase genes was screened with multiplex polymerase chain reaction (PCR). All PCR-positive isolates were identified with MALDI-TOF. A total of 32.6% of isolates (130 of 399) were PCR positive for at least one resistance gene, and 13% of these positive isolates out of 130 had at least three resistance genes. Among the 22 detected genes, blaGES group was the most prevalent, at 25.8% (n=198; many isolates carried multiple genes), followed by blaMOX (13.1%) and blaTEM (10.1%) as most frequently detected. Furthermore, out of 18 different bacterial species/genera detected as carrying beta-lactamase genes, A. hydrophila/caviae (28.5%), Enterobacter spp. (16.9%), and E. coli (14.6%) were the most prevalent. Enterobacter spp., Aeromonas spp., and K. cryocescens potentially carried AmpC genes, and E. coli carried ESBL genes. We recorded a huge variety of beta-lactamases (blaAmpC, blaESBL, and blaCARBA) genes in many potential pathogens that probably originated from both enteric and environmental sources.

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

BackgroundCarbapenem-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.MethodsOne 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.ResultsAll 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.ConclusionThe 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.

High prevalence of clonally related ST182 NDM-1-producing Enterobacter cloacae complex clinical isolates in Greece

NDM-type metallo-β-lactamase (MBL)-producing Enterobacterales remain uncommon in the European region, especially among species other than Klebsiella pneumoniae and Escherichia coli. The aim of this study was to describe epidemiological and molecular characteristics of a widespread NDM-1-producing Enterobacter cloacae complex outbreak in Greece. Over a 6-year period (March 2016-March 2022), a retrospective study was conducted in a tertiary care Greek hospital. Ninety single-patient carbapenem-non-susceptible E. cloacae complex clinical isolates were recovered consecutively. The isolates were subjected to further investigation, including antimicrobial susceptibility testing and combined disc tests for carbapenemase production, polymerase chain reaction and sequencing for resistance genes, molecular fingerprinting by pulsed-field gel electrophoresis (PFGE), plasmid profiling, replicon typing, conjugation experiments, genotyping by multi-locus sequence typing (MLST), whole-genome sequencing and phylogenetic analysis. Phenotypic and molecular testing confirmed the presence of blaNDM-1 in 47 (52.2%) of the E. cloacae complex isolates. MLST analysis clustered all but four of the NDM-1 producers into a single MLST sequence type (ST182), whereas single isolates belonged to different sequence types (ST190, ST269, ST443 and ST743). PFGE analysis revealed that ST182 isolates were clustered into a single clonal type, with three subtypes, which differed from the clonal types detected among the remaining carbapenem non-susceptible E. cloacae complex isolates identified during the study period. All ST182 blaNDM-1-carrying isolates also harboured the blaACT-16 AmpC gene, while the blaESBL, blaOXA-1 and blaTEM-1 genes were detected in most cases. In all clonal isolates, the blaNDM-1 gene was located on an IncA/C-type plasmid, and flanked upstream by an ISAba125 element and downstream by bleMBL. Conjugation experiments failed to produce carbapenem-resistant transconjugants, indicating a low dynamic for horizontal gene transfer. Application of enforced infection control measures led to the absence of new NDM-positive cases for periods of time during the survey. This study represents the largest clonal outbreak of NDM-producing E. cloacae complex in Europe.

Phenotypic and genotypic characterization of pathogenic Escherichia coli identified in resistance mapping of β-lactam drug-resistant isolates from seafood along Tuticorin coast.

The ubiquity of pathogenic E. coli isolate possessing antimicrobial resistance was investigated in seafood samples procured from major seafood supply chain markets established for export and domestic consumption along Tuticorin coast. Out of 63 seafood samples examined, 29 (46%) were found to be contaminated by pathogenic E. coli harbouring one or more genes of virulent potential. Based on virulome profiling, 9.55% of isolates belonged to enterotoxigenic E. coli (ETEC), 8.08% to enteroaggregative E. coli (EAEC), 7.35% to enterohemorrhagic E. coli (EHEC), 2.20% to enteropathogenic E. coli (EPEC), and 2.20% to uropathogenic E. coli (UPEC). All the 34 virulome positive and haemolytic pathogenic E. coli have been serogrouped as O119, O76, O18, O134, O149, O120, O114, O25, O55, O127, O6, O78, O83, O17 and clinically significant O111, O121, O84, O26, O103, and O104 (non-O157 STEC) serotypes in this study. Multi-drug resistance (MDR) (≥ 3 antibiotic classes/sub-classes) was exhibited in 38.23% of the pathogenic E. coli, and 17.64% were extensive drug resistant (XDR). Extended spectrum of β-lactamase (ESBL) genotypes were confirmed in 32.35% isolates and 20.63% isolates harboured ampC gene. One sample (Penaeus semisulcatus) collected from landing centre (L1) harboured all ESBL genotypes blaCTX-M, blaSHV, blaTEM, and ampC genes. Hierarchical clustering of isolates revealed the separation of ESBL isolates into three clusters and non-ESBL isolates into three clusters based on phenotypic and genotypic variations. Based on dendrogram analysis on antibiotic efficacy pattern, carbapenems and β-lactam inhibitor drugs are the best available treatment for ESBL and non-ESBL infections. This study emphasizes the significance of comprehensive surveillance of pathogenic E. coli serogroups that pose serious threat to public health and compliance of AMR antimicrobial resistant genes in seafood that hinder seafood supply chain.

Molecular characterization and detection of antibiotic resistant genes of bacteria isolated from yoghurt in Port Harcourt Metropolis, Nigeria

Currently, molecular identification is replacing the conventional method because of its precision and reliability whereas the effectiveness of antibacterial treatments has continuously declined due to antimicrobial resistance (AMR). This study aimed to identify the bacterial isolates; phenotypically and molecularly as well as detect the resistant genes after susceptibility testing of the isolates obtained from yoghurt samples. Standard microbiological techniques and molecular analysis were applied on both samples (commercial and home-made yoghurt) for species validation. Forty-four (44) bacterial species were identified, phenotypically belonging to three (3) genera; Bacillus, Staphylococcus and Lactobacillus and an additional genus Bifidobacterium emerged from molecular analysis. The microbial load of the yoghurt samples was not statistically significant at (p≥0.05). A sensitivity test on the species was carried out using Kirby-Bauer disc diffusion method with some standard antibiotics. The results revealed that Bacillus and Staphylococcus species were resistant to ampicillin and augmentin (100%) but susceptible to ofloxacin and gentamicin respectively. Lactobacillus spp. were susceptible to ofloxacin and ceftazidime (100%), and resistant to ampicillin, augmentin, and ciprofloxacin (100%). The six most resistant species were molecularly identified as S. aureus CP019117, S. epidermidis AB68833, B. megaterium KC246043, B. cereus NC004722, Lactobacillus casei NC008526 and Bifidobacterium lactis CP003941. Resistant bacteria with mecA gene are S. aureus and S. epidermidis and those with ampC gene are Bifidobacterium lactis and Lactobacillus casei. However, neither gene was found in the genome of any Bacillus species. However, the data also revealed that the bacterial species in home-made yoghurt samples were negative for mecA and ampC resistant genes but positive in the commercial samples. These genes contributed to the bacterial isolates' high levels of multidrug resistance (MDR). The presence of resistant genes in bacterial species from commercial yoghurt samples remains a challenge for food safety. Therefore, good manufacturing practice, proper hygiene and sanitation is hereby advocated to avoid serious emerging foodborne illnesses.

Prevalence and molecular analysis of antibiotic resistance of Pseudomonas aeruginosa isolated from clinical and environmental specimens in Basra, Iraq.

The steady increase in the spread of multidrug-resistant Pseudomonas aeruginosa (MDR) has become a major threat to the global health systems, including Iraq. This study aimed to investigate the prevalence and the molecular basis of antibiotic resistance in Pseudomonas aeruginosa isolated from clinical and environmental samples. Pseudomonas aeruginosa strains were identified by standard microbiological procedures followed by PCR confirmation. Antibiotic susceptibility testing, for 16 antimicrobials, was conducted according to the Clinical and Laboratory Standard Institute (CLSI) standardized by disk diffusion and VITEK 2 methods. Detection of beta-lactamases (ESBLs, AmpC and carbapenemase) activities and related encoding genes was performed by using phenotypic methods and PCR technique respectively. A total of 81 clinical specimens and 14 environmental samples were positive for P. aeruginosa. Antimicrobial susceptibility test showed high rates of resistance to antipseudomonal cephalosporines (74.74 to 98.95%), aztreonam (82.11%), antipseudomonal carbapenems (68.4%), piperacillin/tazobactam (69.5%) ciprofloxacin (71.6%), and aminoglycosides (69%), with emergence of resistance to colistin (7.4%) among tested P. aeruginosa. Among the tested isolates, 69 (72.63%) strains were MDR, of which 63 (91.3%) strains were extremely drug resistance (XDR). Most of the isolated strains harbored one or more of ESBL genes (bla SHV-2a, bla CTX-M-28, bla VEB-2, bla OXA-677, bla PER) with predominant bla OXA-677, but none of the MBLs (GIM, SIM, SPM, IMP) and AmpC (FOX) genes were detected. The results highlighted a high prevalence rate of MDR and XDR and emergence of colistin resistance P. aeruginosa at Basra hospitals, Iraq.