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Objectives To explore the in vitro antimicrobial susceptibility among clinically important Gram-negative bacteria (GNB) in Taiwan. Methods From 2016 through 2018, a total of 5458 GNB isolates, including Escherichia coli (n = 1545), Klebsiella pneumoniae (n = 1255), Enterobacter species (n = 259), Pseudomonas aeruginosa (n = 1127), Acinetobacter baumannii complex (n = 368), and Stenotrophomonas maltophilia (n = 179), were collected. The susceptibility results were summarized by the breakpoints of minimum inhibitory concentration (MIC) of CLSI 2020, EUCAST 2020 (for colistin), or published articles (for ceftolozane/tazobactam). The resistance genes among multidrug-resistant (MDR) or extensively drug-resistant (XDR)-GNB were investigated by multiplex PCR. Results Significantly higher rates of non-susceptibility (NS) to ertapenem and carbapenemase production, predominantly KPC and OXA-48-like beta-lactamase, were observed in Enterobacterales isolates causing respiratory tract infection than those causing complicated urinary tract or intra-abdominal infection (12.7%/3.44% vs. 5.7%/0.76% or 7.7%/0.97%, respectively). Isolates of Enterobacter species showed higher rates of phenotypic extended-spectrum β-lactamase and NS to ertapenem than E. coli or K. pneumoniae isolates. Although moderate activity (54–83%) was observed against most potential AmpC-producing Enterobacterales isolates, ceftolozane/tazobactam exhibited poor in vitro (44.7–47.4%) activity against phenotypic AmpC Enterobacter cloacae isolates. Additionally, 251 (22.3%) P. aeruginosa isolates exhibited the carbapenem-NS phenotype, and their MDR and XDR rate was 63.3% and 33.5%, respectively. Fifteen (75%) of twenty Burkholderia cenocepacia complex isolates were inhibited by ceftolozane/tazobactam at MICs of ≤4 μg/mL. Conclusions With the increase in antibiotic resistance in Taiwan, it is imperative to periodically monitor the susceptibility profiles of clinically important GNB.

Objectives IMP-type carbapenemases are rarely detected in Europe and limited information is available to guide the treatment of infections caused by carbapenemase-producing Enterobacterales (CPE) producing these carbapenemases. Accurate antimicrobial susceptibility testing (AST) results are essential for optimal antibiotic management. Here we report discrepancies in AST of IMP-producing Enterobacterales (IMP-CPE) complicating the management of severe sepsis. Methods Antimicrobial susceptibilities were analysed by in-house VITEK® 2, Etest and broth microdilution (BMD). Carbapenemase-encoding genes were detected by PCR. Whole-genome sequencing (WGS) was performed using an Illumina MiSeq platform. Results Minimum inhibitory concentrations (MICs) determined by VITEK® 2 for Enterobacter hormaechei and Klebsiella oxytoca blood culture isolates were ≥16 mg/L for meropenem and ≤0.5 mg/L for ertapenem. In contrast, Etest analysis and BMD returned MICs of 2 mg/L and 1 mg/L, respectively. Both isolates tested positive for IMP carbapenemase-encoding genes by PCR. WGS revealed that both isolates carried the same blaIMP-4 gene. Based on VITEK® 2 susceptibilities, initial treatment was with tigecycline and amikacin. After subsequent deterioration, the patient was successfully treated with ertapenem and amikacin. Conclusion This case highlights that automated AST by VITEK® 2 can over-report meropenem resistance for IMP carbapenemase-producers compared with Etest and BMD. Clinicians need to be cautious deciding against carbapenem treatment based on VITEK® 2 susceptibility testing results for IMP-positive Enterobacterales. Tigecycline was inferior to carbapenem treatment for pyelonephritis caused by isolates expressing IMP carbapenemases, however specific evidence guiding the treatment of these infections is lacking.

Funding: This research was funded by the University of St Andrews. The aim of the study was to determine the efficacy of carbapenem-only combination treatments derived from four approved drugs (meropenem, doripenem, ertapenem and imipenem) against a MDR strain of P. aeruginosa in a Galleria mellonella larvae infection model. G. mellonella larvae were infected with P. aeruginosa NCTC 13437 (carrying the VIM 10 carbapenamase) and the efficacy of the six possible dual, four triple, and one quadruple carbapenem combination(s) were compared to their constituent monotherapies. Four of these combinations showed significantly enhanced survival compared to monotherapies and reduced the bacterial burden inside infected larvae but without complete elimination. Bacteria that survived combination therapy were slower growing, less virulent but with unchanged carbapenem MICs—observations that are consistent with a persister phenotype. In vitro time-kill assays confirmed that the combinations were bactericidal and confirmed that a low number of bacteria survived exposure. Mass spectrometry was used to quantify changes in the concentration of carbapenems in the presence of carbapenemase-carrying P. aeruginosa. The rate of degradation of individual carbapenems was altered, and often significantly reduced, when the drugs were in combinations compared with the drugs alone. These differences may account for the enhanced inhibitory effects of the combinations against carbapenem-resistant P. aeruginosa and are consistent with a ‘shielding’ hypothesis. In conclusion, carbapenem combinations show promise in combating MDR P. aeruginosa and are worthy of additional study and development. Publisher PDF

Extended-Spectrum Beta-Lactamase (ESBL) producing bacteria is a type of resistance that leads to complex management of patients in intensive care due to their resistance to first, second, and third-generation Cephalosporin and monobactam antibiotics. The most ESBL-producing bacteria are found in the family Enterobacteriaceae, especially Klebsiella pneumoniae and Escherichia coli. The purpose of this research was to determine the sensitivity pattern of ESBLproducing bacteria in Intensive Care Units (ICUs) of Ulin Hospital, Banjarmasin, in the period of 2016-2018. This research was a descriptive study with a cross-sectional approach using data from the laboratory medical records of patients with positive ESBL in the ICUs of Ulin Hospital, Banjarmasin, between 2016 and 2018. The research sample was taken by the total sampling method. This research obtained 216 isolates of ESBL-producing bacteria consisting of 155 (71.8%) isolates of Klebsiella pneumoniae and 61 (28.2%) Escherichia coli. It was found that the Cephalosporin antibiotics (Cefazolin, Ceftazidime, Ceftriaxone, and Cefepime) and monobactam antibiotic (Aztreonam) had the lowest sensitivity. Aminoglycoside antibiotics (Amikacin), Carbapenem (Ertapenem and Meropenem), and Tetracycline (Tigesycline) were the most sensitive antibiotics. It was concluded that both Klebsiella pneumoniae and Escherichia coli were the most dominant ESBL-producing bacteria and showed good sensitivity to the Amikacin, Ertapenem, Meropenem, and Tigecycline.

Objective. To evaluate in vitro activity of biapenem and other clinically available carbapenems against Russian clinical isolates of Enterobacterales, Pseudomonas aeruginosa и Acinetobacter spp., including isolates with acquired fermentative mechanisms of resistance to β-lactams. Materials and Methods. A total of 3139 Enterobacterales isolates, 793 P. aeruginosa isolates and 634 Acinetobacter spp. isolates from hospitalized patients in 63 hospitals from 35 Russian cities were included in the study during 2018-2019. Minimal inhibitory concentrations (MIC) for biapenem and other antimicrobials were determined in accordance with ISO 20776-1:2006. Carbapenemases genes were detected by commercially available real-time PCR kits AmpliSens® MDR KPC/OXA-48-FL and AmpliSens® MDR MBL-FL (Central Research Institute of Epidemiology, Russia). Data analysis and reporting was performed using AMRcloud online platform (www.amrcloud.net). Results. For all tested Escherichia coli isolates MIC50/90 were 0.06/0.125 mg/l for biapenem, 0.125⁄0.25 mg/l for imipenem, and 0.06/0.06 mg/l for meropenem. When MIC50/90 for ertapenem (0.015/0.125 mg/l for all isolates tested) were comparable to those of biapenem, a greater number of nosocomial E. coli isolates had MIC >4 mg/l for ertapenem (3.6%) than for biapenem (2.6%). MIC50/90 of Klebsiella pneumoniae for biapenem were 0.5⁄16 mg/l, for both imipenem and meropenem – 0.5⁄32 mg/l, for ertapenem – 2⁄32 mg/l. Resistance to oxyimino-β-lactams had no significant influence on activity of biapenem against Enterobacterales isolates. For 321 carbapenemase-producing K. pneumoniae isolates (ОХА-48 – 63.9%, NDM – 27.7%) biapenem has shown no advantages over imipenem and meropenem. МПК50/90 for nosocomial and community-acquired P. aeruginosa isolates were 8⁄64 mg/l and 0,5⁄16 mg/l for biapenem, 8⁄128 mg/l and 1⁄16 mg/l – for imipenem, 16⁄64 mg/l and 0,5⁄32 mg/l – for meropenem. All carbapenems, including biapenem, had very low in vitro activity against carbapenemaseproducing P. aeruginosa isolates. МПК50/90 of Acinetobacter spp. were 64⁄128 mg/l for biapenem, 64⁄128 mg/l – for imipenem, and 128⁄128 mg/l – for meropenem. Conclusions. According to the MIC distributions and MICs50/90 values independently of the presence of fermentative mechanisms of resistance to β-lactams, in vitro activity of biapenem against Russian clinical isolates of Enterobacterales, P. aeruginosa and Acinetobacter spp. was comparable to those of imipenem and meropenem.

Sandip Patil,1– 3 Hongyu Chen,4 Chunna Guo,4 Xiaoli Zhang,3 Pei-Gen Ren,1 Ngiambudulu M Francisco,5 Feiqiu Wen2,3 1Shenzhen Institute of Advanced Technology, Shenzhen University Town, Shenzhen, Guangdong Province, People’s Republic of China; 2Department of Haematology and Oncology, Shenzhen Children’s Hospital, Shenzhen, Guangdong Province, People’s Republic of China; 3Paediatric Research Institute, Shenzhen Children’s Hospital, Shenzhen, Guangdong Province, People’s Republic of China; 4Department of Laboratory Medicine, Shenzhen Children’s Hospital, Shenzhen, Guangdong Province, People’s Republic of China; 5Grupo de Investigação Microbiana e Imunológica, Instituto Nacional de Investigação em Saúde (National Institute for Health Research), Luanda, AngolaCorrespondence: Feiqiu WenDepartment of Haematology and Oncology, Shenzhen Children’s Hospital, Shenzhen, Guangdong Province, People’s Republic of ChinaTel +86 18938690333Fax +86-755 83009888Email fwen62@163.comAim: We investigated the clonal diversity of carbapenemase-producing Klebsiella pneumoniae isolates from the Shenzhen Children’s Hospital, China, and drew conclusions on the clinical and public health impact of these isolates as multidrug-resistant.Methods: From January 2014 to December 2018, a total number of 36 unique carbapenemase-producing clinical isolates of Klebsiella pneumoniae were collected out of 900 clinical isolates in paediatric patients from the Shenzhen Children’s Hospital, China. After carbapenemase production confirmation, antimicrobial susceptibility, resistance determinants and phylogenetic relationship were determined.Results: The isolates showed resistance to ceftazidime, ertapenem, ampicillin, cefazolin, ceftriaxone, cefotetan, ticarcillin, cefaclor, cefpodoxime, azlocillin, cefcapene, mezlocillin and ampicillin-sulbactam. Of the 36 Klebsiella pneumoniae carbapenemase genes coding isolates, blaNDM was the mostly detected 50% (n=18) followed by blaKPC and blaIMP 19% (n=7), blaVIM 17% (n=6), blaOXA-48-like 8% (n=3) and blaSME 5% (n=2), whereas extended-spectrum β-lactamase (blaSHV) was predominantly detected 92% (n=33) followed by blaCTX-M 53% (n=19) and blaCMY 28% (n=10). Pulsed-field gel electrophoresis typing showed eight different patterns, and twenty-five distinct sequences types were observed with ST307 being predominantly identified 11% (n=4), followed by ST2407 8% (n=3). Plasmid replicon typing results indicated that IncFIS, IncHI2, IncFIC and IncFIA plasmids carry blaCTX-M, blaSHV and blaNDM genes.Conclusion: This study reports on the occurrence and spread of carbapenemase and extended-spectrum β-lactamase encoding genes co-existence in sporadic Klebsiella pneumoniae ST307 in paediatric patients from the Shenzhen Children’s Hospital, China.Keywords: Klebsiella pneumoniae, carbapenemase, ESBLs, antimicrobial susceptibility, molecular characterization

Background We describe antibiotic resistance trends of Klebsiella pneumoniae pathogens, responsible for urinary tract infections (UTIs) and intra-abdominal infections (IAIs), isolated from different organs and tissues, hospital departments and Chinese regions between 2014 and 2017. Methods Resistances of UTIs and IAIs derived K. pneumoniae isolates from 17 hospitals in 7 Chinese regions to amikacin, imipenem, piperacillin-tazobactam, ertapenem, and cefepime were unequivocally established. Results Overall resistance rates of K. pneumoniae IAI isolates obtained from gallbladder and abscesses increased to amikacin (14.29–30.95%) and for liver, gallbladder, and abscesses to imipenem (14.29–38.10%), piperacillin-tazobactam (23.81–38.10%), and ertapenem (23.81–38.10%) in 2017, but were constant (20–30%) for K. pneumoniae isolates from UTIs from 2014 to 2017. In medical and surgical ICUs, resistance rates to all tested antibiotics rose to ∼60% for IAIs, which was also reflected in higher resistance rates of hospital acquired (HA) compared to community acquired (CA) infections. In medical ICUs resistance rates increased to 50–60% for amikacin, imipenem, and ertapenem for UTI-derived K. pneumoniae isolates in 2017. Resistance rates to all tested antibiotics were highest in the east Jiangzhe region of China, being ∼60% for K. pneumoniae isolates from IAIs and 40% for K. pneumoniae isolates from UTIs to ertapenem and imipenem, as well as > 40% for piperacillin-tazobactam in 2017. Conclusion In China, ICUs resistance rates to K. pneumoniae IAIs and UTIs isolates was increased in 2017 for all tested antimicrobials including carbapenems, which makes them no longer suitable for empiric treatment. In the east Jiangzhe region this was a general trend that was independent of the type of hospital department.

Background We describe the molecular epidemiology and resistance patterns of blaOXA-48 Klebsiella pneumoniae and Escherichia coli in Taiwan. Methods In this multicenter surveillance study from January 2012 to August 2015, the identified blaOXA-48 Enterobacteriaceae isolates were subjected to antibiotics susceptibility testing. PCR method was used for detecting concomitant other beta-lactamases. Outer membrane porins were analyzed. Genetic relatedness and molecular epidemiology of the isolates were determined through pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Plasmid incompatibility was determined using PCR-based replicon typing. Results Forty-three blaOXA-48 K. pneumoniae and two E. coli isolates were analyzed. The annual incidence of blaOXA-48 K. pneumoniae isolates from 2012 to 2015 were 0%, 1.1%, 2.4%, and 7.6%, respectively. Forty-three (95.5%) of 45 isolates were non-susceptible to broad-spectrum beta-lactams (ceftriaxone, ceftazidime, cefepime, piperacillin/tazobactam), Forty-two (93.3%) of the 45 isolates showed resistance against all tested carbapenems (imipenem, meropenem, doripenem, and ertapenem). Molecular characterization revealed that they co-produced at least one extended-spectrum beta-lactamases or AmpC beta-lactamases, with at least one outer membrane porin loss. Thirty-eight (88.3%) of the 43 K. pneumoniae isolates belonged to ST11. PFGE analysis of 43 K. pneumoniae isolates revealed dissemination of multiple clones. Six of the 12 tested K. pneumoniae representatives of different pulso-types belonged to IncA/C. Conclusion Concomitant loss of porins and production of other beta-lactamases renders the blaOXA-48-producing isolates in Taiwan a high-level carbapenem resistance and broad resistance against many beta-lactam antibiotics. Following dissemination of multiple clones of blaOXA-48 K pneumoniae ST 11, a trend of increased blaOXA-48 prevalence was noted.

Our aim was to assess whether newer carbapenems with a better administration profile than meropenem (ertapenem, faropenem and tebipenem) were more effective against Mycobacterium tuberculosis including M/XDRTB and determine if there was a synergistic/antagonistic effect with amoxicillin or clavulanate (inhibitor of beta-lactamases that MTB possesses) in vitro. Whilst meropenem is given three times a day intravenously, ertapenem, though given parenterally, is given once a day, faropenem and tebipenem are given orally. Eighty-two clinical drug-sensitive and -resistant MTB strains and a laboratory strain, H37Rv, were assessed by a microdilution methodology against ertapenem, faropenem, tebipenem and meropenem with and without amoxicillin or clavulanic acid. Ertapenem showed a limited activity. The addition of amoxicillin and clavulanate did not translate into significant improvements in susceptibility. Sixty-two isolates (75.6%) exhibited susceptibility to faropenem; the addition of amoxicillin and clavulanate further reduced the MIC in some isolates. Faropenem showed a limited activity (MIC of 8 mg/L or lower) in 21 strains completely resistant to meropenem (MIC of 16 mg/L or higher). Fifteen of the meropenem-resistant strains were susceptible to tebipenem. Carbapenems’ activity has been reported extensively. However, there remains uncertainty as to which of them is most active against TB and what the testing methodology should be.

Introduction: Carbapenem-resistant Klebsiella pneumoniae is an emerging healthcare-associated pathogen with dynamic molecular epidemiology. This study presents a retrospective analysis of the distribution and antibiotic resistance patterns of ertapenem-resistant ESBL-producing K. pneumoniae strains recovered during an outbreak from 2012 to 2014 in a Croatian University hospital. Methods We aimed to estimate genetic relatedness of clinical isolates and underlying mechanisms that conferred the ertapenem- resistant phenotype. Results: Expression analysis of genes involved in the antibiotic resistance showed reduced expression of major non-selective porin channel OmpK35. Reduced expression of OmpK36 porin channel in isolates resistant to at least one more carbapenem, apart from the ertapenem, was found to a lesser degree. Pulsed-field gel electrophoresis analysis of genomic DNA revealed that almost all isolates belonged to the same genetic clone. Conclusions: Caution regarding ertapenem- resistant, carbapenemase-negative porin- deficient mutants of K. pneumoniae is required as they are widespread, and under selective pressure this could result in a local clonal outbreak.

Combined antibiotic therapy is widely used for infections caused by carbapenem-resistant K. pneumoniae. The objective of this work was to identify the synergistic activity of combinations of two carbapenems against multidrug- and extensively drug-resistant K. pneumoniae strains producing various types of carbapenemases. For 60 antibiotic-resistant K. pneumoniae strains isolated in 8 cities of Belarus, the minimum inhibitory concentrations (MIC) of colistin and carbapenems were determined by subsequent broth microdilution method, and the genes of carbapenemases and phosphoethanolamine transferases were detected. The checkerboard method was used to determine the sensitivity to the combination of ertapenem and doripenem. High MIC values of carbapenems were revealed for NDM carbapenemase-producing strains (MIC50 of meropenem 64 mg/L, MIC50 of doripenem 64 mg/L). Doripenem was more active; MIC of doripenem ≤ 16 mg/L (low level of resistance) was determined in 28 (46.7%) strains, MIC of meropenem ≤ 16 mg/L - in 8 (13.3% of strains). The effect of potentiating the activity of doripenem with ertapenem at a fixed pharmacokinetic / pharmacodynamic concentration was observed for 20.0% of the strains producing KPC carbapenemase and 29.0% of the strains producing OXA-48 carbapenemase. The potentiating effect was independent of the presence of colistin resistance. Thus, the ability of ertapenem to potentiate the antimicrobial activity of doripenem and meropenem against some of the strains producing serine carbapenemases (KPC and OXA-48) was confirmed. The necessity of routine determination of the true MIC values of carbapenems was shown to optimize their dosage regimens and select the combination antibiotic therapy regimens.

Background: The ever-increasing magnitude of antimicrobial resistance encountered in human pathogens has led to limited treatment options for bacterial infections, consequently reducing antimicrobial efficacy while increasing treatment costs, morbidity, and mortality. In clinical setup, laboratory-based in vitro antimicrobial susceptibility testing is the cornerstone for guiding therapy and enables the monitoring of antimicrobial resistance trends. Aim: To characterize the distribution of bacteria isolated from various specimens and their antibiotic susceptibility profiles in Mary Begg Health facilities. Material & Methods: This was a retrospective, cross-sectional, quantitative, descriptive study that involved the review of 569 laboratory files from three Mary Begg Health facilities from the period of January 2019 to June 2020. A systematic random sampling method was used and SPSS version 21.0 was used for data analysis. Results: The distribution of bacteria based on Gram stain reaction found that most bacteria that were isolated were Gram negative bacilli, 79.5% (171/215). The most common bacterium isolated was Escherichia coli, 46.5% (100/215) followed by Staphylococcus aureus, 12.1% (26/215) and Klebsiella pneumoniae, 17 7.9% (17/215). The study found that E. coli was highly resistant to amoxicillin (95.0%), Ampicillin (90.0%) and Cotrimoxazole (77.0%), respectively. In contrast, E. coli was highly sensitive to Amikacin (96.0%), Ertapenem (91.0%) and Ceftriaxone (80.0%) S. aureus species isolated were sensitive to Gentamicin (65.4%) and Clindamycin (46.2%) but highly resistant to Cotrimoxazole (80.8%). Conclusion: The most frequent isolates were Escherichia coli followed by Staphylococcus aureus and majority of them were from urine specimens. Key words: Antimicrobial, Resistant, Antimicrobial Resistance, Antimicrobial susceptibility testing, Mary Begg Health services.

Extended-spectrum cephalosporin-resistant Escherichia coli (ESCREC) are a growing threat. Leading ESCREC lineages include sequence type ST131, especially its (blaCTX-M-15-associated) H30Rx subclone and (blaCTX-M-27-associated) C1-M27 subset within the H30R1 subclone. The comparative activity against such strains of alternative antimicrobial agents, including the recently developed aminoglycoside plazomicin, is undefined, so was investigated here. We assessed plazomicin and 11 comparators for activity against 216 well-characterized ESCREC isolates (Minnesota, 2012-2017) and then compared broth microdilution MICs with phylogenetic and clonal background, beta-lactamase genotype (blaCTX-M; group 1 and 9 variants), and co-resistance. Percent susceptible was > 99% for plazomicin, meropenem, imipenem, and tigecycline; 96-98% for amikacin and ertapenem; and ≤ 75% for the remaining comparators. For most comparators, MICs varied significantly in relation to multiple bacterial characteristics, in agent-specific patterns. By contrast, for plazomicin, the only bacterial characteristic significantly associated with MICs was ST131 subclone: plazomicin MICs were lowest among O16 ST131 isolates and highest among ST131-H30R1 C1-M27 subclone isolates. Additionally, plazomicin MICs varied significantly in relation to resistance vs. susceptibility to comparator agents only for amikacin and levofloxacin. For most study agents, antimicrobial activity against ESCREC varied extensively in relation to multiple bacterial characteristics, including clonal background, whereas for plazomicin, it varied only by ST131 subclone (C1-M27 isolates least susceptible, O16 isolates most susceptible). These findings support plazomicin as a reliable alternative for treating ESCREC infections and urge continued attention to the C1-M27 ST131 subclone.

According to the WHO global report, ESBL (Extended-Spectrum Beta-Lactamase)-producing microorganisms is the most common cause of urinary tract infection; hospital-acquired infection as well as a foodborne infection. These include Escherichia coli, Salmonella, Shigella and Vibrio cholera. The carbapenems which include doripenem, ertapenem, imipenem and meropenem are the known choice of medication for life-threatening infections which is caused by ESBL-producing microorganisms. Recently there is the emergence of carbapenem-resistant Enterobacteriaceae and Pseudomonas which makes treatment an obstacle for the medical personnel for various infections. Natural products have been reported to be emerged as a potential possibility to be explored as antimicrobial drugs. The plant extracts could serve as an alternate source of resistance modifying agents owing to the wide variety of secondary metabolites (eg. flavonoids, phenols, tannins, alkaloids, etc). Mushroom are have been considered as a source that attracts the researchers to explore its properties as it has been established with various bioactive components that possess many health beneficial effects such as antimicrobial and antifungal compounds. In this paper, we summarizes the information available in the literature on the ESBL, mushroom and current treatment, with a special focus on the three different types of mushrooms (Fistulina hepatica, Leucopaxillus giganteus and Pleurotus Ostreatus) against spectrum beta-lactamase (ESBL) producing microorganisms. Electronic databases including SciFinder, Web of Science, Science Direct, PubMed, and Google Scholar were screened using the following keywords: “antibacterial, antimicrobial, mushroom, ESBL, Enterobacter, Fistulina hepatica, Leucopaxillus giganteus and Pleurotus Ostreatus”. We intend to enhance understanding in the field and promote further work on the development of phytocomponent based antimicrobial compounds.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) is an important bacterium of nosocomial infections. In this study, CRKP strains, which were mainly isolated from fecal samples of 14 patients in three wards of the hospital in the Silesia Voivodship, rapidly increased from February to August 2018. Therefore, we conducted microbiological and molecular studies of the CRKP isolates analyzed. Colonized patients had critical underlying diseases and comorbidities; one developed bloodstream infection, and five died (33.3%). Antibiotic susceptibilities were determined by the E-test method. A disc synergy test confirmed carbapenemase production. CTX-Mplex PCR evaluated the presence of resistance genes bla CTX-M-type, bla CTX-M-1, bla CTX-M-9, and the genes bla SHV, bla TEM, bla KPC-2, bla NDM-1, bla OXA-48, bla IMP, and bla VIM-1 was detected with the PCR method. Clonality was evaluated by Multi Locus Sequence Typing (MLST) and Pulsed Field Gel Electrophoresis (PFGE). Six (40%) strains were of XDR (Extensively Drug-Resistant) phenotype, and nine (60%) of the isolates exhibited MDR (Multidrug-Resistant) phenotype. The range of carbapenem minimal inhibitory concentrations (MICs, μg/mL) was as follows doripenem (16 to >32), ertapenem (> 32), imipenem (4 to > 32), and meropenem (> 32). PCR and sequencing confirmed the bla CTX-M-15, bla KPC-2, bla OXA-48, and bla VIM-1 genes in all strains. The isolates formed one large PFGE cluster (clone A). MLST assigned them to the emerging high-risk clone of ST147 (CC147) pandemic lineage harboring the bla OXA-48 gene. This study showed that the K. pneumoniae isolates detected in the multi-profile medical centre in Katowice represented a single strain of the microorganism spreading in the hospital environment.

The emergent bacterial resistance to antibiotics, most especially Carbapenems, has become a common phenomenon. The aim of the recent study was the observation and evaluation of the antibacterial susceptibility of Klebsiella pneumoniae in clinical specimens to different Carbapenems. One hundred isolates of K. pneumoniae isolated from different clinical sites, such as leg, caesarean section (CS), head, buttock, breast, thigh, and arm were tested. Using disc diffusion method, the isolates were tested for susceptibility to different antibiotics including Tobramycin, Ciprofloxacin, Aztreonam, Colistin sulphate, Ceftriaxone, Cefepime, Cefoxitin, Ceftazidime, Ertapenem, Meropenem, and Imipenem. The results were interpreted according to the Clinical and Laboratory Standard Institute disk diffusion standard. All K. pneumoniae isolates were highly susceptible to all classes of Carbapenems: Imipenem (99%), Meropenem (96%) and Ertapenem (91%). However, they were highly resistant to Ciprofloxacin (97%), Ceftriaxone (91%) and Tobramycin (73%). Despite the recent emergence of multi-drug resistant bacteria to Carbapenems, this study showed that Carbapenems could still be used in treating different infections caused by multi-drug resistant K. pneumoniae.

Faecal carriage of Carbapenem-resistant Enterobacteriaceae (CRE) is being observed as an important risk factor for bacteremia among patients with hematological malignancies. A prospective surveillance study was conducted among these patients to determine the gut colonization of CRE. Rectal/perianal swabs were collected to isolate CRE. Carbapenem resistance was detected by disk diffusion, modified-Hodge, Carba-NP test, and PCR for blaNDM-1, blaKPC, blaOXA-48, blaVIM, blaIMP genes. A total of 209 CRE isolates were identified from 151 patients. E. coli was the most common (83.2%) CRE identified, followed by Klebsiella spp. (9.6%). The majority of CRE were observed resistant to ertapenem (86%). blaNDM-1 was the most common gene (57.3%), followed by blaOXA-48 (37.8%). 26.8% isolates found to carry both blaNDM-1 and blaOXA-48 genes. CRE is increasingly observed to cause bacteremia among hematological malignancy patients due to increased colonization. Screening for gut CRE colonization is necessary to guide empirical therapy and apply infection control measures among these patients.

Yann Mouanga Ndzime,1,2 Richard Onanga,1 Roland Fabrice Kassa Kassa,1 Michelle Bignoumba,1 Pierre Philippe Mbehang Nguema,1,3 Amahani Gafou,1 Roméo Wenceslas Lendamba,1 Kelly Mbombe Moghoa,1 Cyrille Bisseye2 1Unité de Recherche et d’Analyses Médicales, Laboratoire de Bactériologie, Centre Interdisciplinaire de Recherches Médicales de Franceville, Franceville, BP 769, Gabon; 2Laboratoire de Biologie Moléculaire et Cellulaire (LABMC), Université des Sciences et Techniques de Masuku, Franceville, BP 943, Gabon; 3Institut de Recherche en Ecologie Tropicale, Centre National de la Recherche Scientifique et Technologique (CENAREST), Libreville, BP 13354, GabonCorrespondence: Yann Mouanga Ndzime Tel +24177206217 Email yann_mouangandzime@yahoo.frIntroduction: Urinary tract infection is one of the major causes of consultation, microbiologic exploration, intensive use of antibiotics worldwide, and the second leading cause of clinical consultation in community practice. Many bacteria play a role in the urinary tract infections etiology, including Enterobacteriaceae such as Escherichia coli (E. coli) and Klebsiella spp.Objective: The study’s main objective was to examine the epidemiology of E. coli and Klebsiella pneumoniae (K. pneumoniae) uropathogenic strains resistant to antibiotics in Franceville.Methodology: The study was carried out between January 2018 and June 2019 in Franceville South-East Gabon. We examined a total of 1086 cytobacteriological urine samples. The identification of E. coli and K. pneumoniae strains was carried out using the Vitek-2 compact automated system and the antibiogram with the disk diffusion method according to the European Committee on Antimicrobial Susceptibility Testing recommendations.Results: The prevalence of urinary tract infections was 29.2% (317/1086), of which 25.1% and 4.1% were mono-infections and co-infections, respectively. The prevalence of UTIs with E. coli was 28.7% (91/317) with a predominance of isolation in women. K. pneumoniae was responsible for 16.2% (61/317) of UTIs. E. coli and K. pneumoniae Uropathogenic strains showed resistance to beta-lactams, quinolones and cotrimoxazole, whereas Nitrofurantoin, Amikacin, Imipenem and Ertapenem were the most active antibiotics against E. coli and K. pneumoniae uropathogenic strains.Conclusion: This study showed a high prevalence of urinary tract infections with a major implication of E.coli and K. pneumoniae strains. E. coli and K. pneumoniae presented high frequency of resistance to antibiotics, highlighting the need to adapt their use accordingly at the local level.Keywords: urinary tract infection, antibiotic resistance, E. coli, K. pneumoniae

Antibiotic resistance is one of the most important public health problem and one of the most critical steps in preventing resistance is the monitorization of the resistance. Local, regional and global monitoring enables the spread of antibiotic resistance to be understood more clearly. In this study, it was aimed to evaluate the results of the pilot study for the establishment of molecular-based carbapenem surveillance system in Escherichia coli and Klebsiella pneumoniae isolates and to investigate the carbapenemase epidemiology in Turkey. Hospitals (n= 28) from 26 different statistical level II regions from Turkey were included in the study. The hospitals participated in the study submitted ten carbapenem susceptible and ten carbapenem resistant E.coli and K.pneumoniae isolates to our laboratory that were isolated in two different periiods of six-month either between 1 March-31 August or 1 April-30 September 2019. A total of 509 isolates were collected from 26 of the 28 participating hospitals in the study. Isolates were identified by matrix assisted laser desorptionization-time of flight mass spectrophotometry (MALDI TOF MS) (Bruker Daltonics, Germany) method and antibiotic susceptibility tests for imipenem, meropenem and colistin were studied by broth microdilution. Moreover, susceptibilities to amikacin, amoxicillin-clavulanic acid, ampicillin, aztreonam, cefepime, cefotaxime, ceftazidime, ciprofloxacin, ertapenem, gentamicin, piperacillin-tazobactam, tobramycin and trimethoprim-sulfamethoxazole were determined by disc diffusion method. The resistance genes were investigated in isolates which were found to be phenotypically resistant to carbapenem and colistin, in house method was used to investigate carbapenemase genes and a commercial colistin resistant real-time PCR kit (Biospeedy, Turkey) was used for colistin resistance genes. In total, 493 of the 509 isolates collected from hospitals were identified as E.coli (25.7%, n= 127) and K.pneumoniae (74.3%, n= 366) and included in the study. It was determined that 31% of the isolates evaluated were from community-acquired infections and 69% were either from healthcare-associated infections or from colonization sites. Among the tested isolates, 248 (50.3%) were susceptible to carbapenems and 245 (49.7%) were resistant. The types of carbapenemases in carbapenemase-producing isolates were OXA-48 (52.2%), KPC (16.1%), NDM-1 (15%), OXA-48 + NDM-1 (12.6%), KPC + NDM-1 (2.8%) and VIM (0.5%) and OXA-48+VIM (0.5%). Resistance to colistin was detected in 23.3% of the isolates but mcr1-8 genes were not detected. It was found that all colistin resistant isolates are resistant to at least one of the carbapenems. The importance of a molecular-based antimicrobial resistance surveillance system in our country was demonstrated with this pilot study. It is thought that continuous monitoring of these epidemiological features will contribute to the management of infections due to carbapenemase-producing organisms.

Background: Raoultella terrigena is a Gram-negative bacterium mainly reported as aquatic and soil organism. It is rarely involved in human infections. This study investigated the epidemiology of R. terrigena infections in the National Bone Marrow Transplant Center of Tunis (NBMTC) between January 2010 and March 2018, their associated antibiotics resistance patterns, and the molecular features of extended-spectrum β-lactamase (ESBL) producing strains. Materials and Methods: Our retrospective study concerned hematopoietic stem cell transplant (HSCT) adult recipients hospitalized at the NBMTC and infected with R. terrigena. The search of the ESBL and carbapenemases genes for multidrug-resistant strains was performed by PCR amplification. Results: Twelve strains of R. terrigena were responsible for infections in 10 hematopoietic stem cell transplant recipients (1.2% of total HSCT recipients). They were responsible for skin (n = 4) and urinary tract infections (n = 4). The first-line antibiotherapy was based on a monotherapy in one case and a dual therapy in eleven cases. Imipenem was the most prescribed antibiotic (n = 7/12). Mortality was attributable to R. terrigena infection in two over ten patients. Nine strains were producing ESBL. Five strains were resistant to ertapenem, two to imipenem, ten to ciprofloxacin, and five to amikacin. BlaCTX-M1, blaOXA-48, and blaKPC were found in seven, four, and one strains, respectively. Conclusion: Low prevalence of R. terrigena infections in HSCT recipients but high rates of attributable mortality and multidrug-resistant strains.

Background: Klebsiella pneumoniae is one of the leading causes of nosocomial infections. Carbapenems are used as the last resort for the treatment of multidrug resistant Gram-negative bacterial infections. In recent years, resistance to these lifesaving drugs has been increasingly reported due to the production of carbapenemase. The main objective of this study was to detect the carbapenem-resistant genes blaNDM-1 and blaVIM in K. pneumoniae isolated from different clinical specimens. Methods: A total of 585 clinical specimens (urine, pus, sputum, blood, catheter tips, and others) from human subjects attended at Annapurna Neurological Institute and Allied Sciences, Kathmandu were obtained in the period between July 2018 and January 2019. The specimens were isolated and identified for K. pneumoniae. All K. pneumoniae isolates were processed for antimicrobial susceptibility testing (AST) using the disk diffusion method. The isolates were further phenotypically confirmed for carbapenemase production by the modified Hodge test (MHT) using imipenem (10 μg) and meropenem (10 μg) discs. Thus, confirmed carbapenemase-producing isolates were further screened for the production of blaNDM-1 and blaVIM using conventional polymerase chain reaction (PCR). Results: Among the clinical isolates tested, culture positivity was 38.29% (224/585), and the prevalence of K. pneumoniae was 25.89% (58/224). On AST, K. pneumoniae exhibited resistance toward carbapenems including ertapenem, meropenem, and imipenem, while it showed the highest susceptibility rate against to tigecycline (93.1%; 54/58). Overall, AST detected 60.34% (35/58) carbapenem-resistant isolates, while the MHT phenotypically confirmed 51.72% (30/58) isolates as carbapenemase-producers and 48.28% (28/58) as carbapenemase nonproducers. On subsequent screening for resistant genes among carbapenemase-producers by PCR assay, 80% (24/30) and 3.33% (1/30) isolates were found to be positive for blaNDM-1 and blaVIM, respectively. In the same assay among 28 carbapenem nonproducing isolates, 9 (32.14%) isolates were positive for blaNDM-1 gene while none of them were tested positive for blaVIM gene. Conclusions: Molecular detection of resistant genes provides greater specificity and sensitivity than those with conventional techniques, thus aiding in accurate identification of antimicrobial resistance and clinical management of the disease.

Background and Aim: Selection and dissemination of plasmid-encoded extended-spectrum β-lactamase (ESBL) among Enterobacteriaceae confers resistance to beta-lactam antibiotics. The purpose of this study was to determine the prevalence and molecular characteristics of ESBL-producing organisms isolated from dairy cattle with a uterine infection. Materials and Methods: Bacterial isolates (n=62) were characterized by biochemical test for genus and species determination. Antimicrobial susceptibility tests were performed by Kirby–Bauer disk diffusion method using panel of antibiotics for initial screening of ESBL organism. Phenotypic confirmation of ESBL-suspected strains was done by combination disk method and double-disk method. Multiplex polymerase chain reaction (PCR) was carried out for phylogrouping of Escherichia coli isolates as well as for genotyping ESBL genes. Enterobacterial repetitive intergenic consensus-PCR method was used for genotypic characterization of isolates. Results: Antibiotic susceptibility profile of E. coli (n=40) isolates showed high rates of resistance for ampicillin (95.0%), cefpodoxime (97.5%), cefotaxime (87.5%), and ceftriaxone (70%). However, low rates of resistance were observed for cefoxitin (25%), amoxicillin/clavulanic acid (20%), ceftazidime (17.5%), gentamicin (10%), and ertapenem (7.5%). A total of 39/40 E. coli isolates were confirmed as ESBL with Epsilometer test as well as the genotypic method and 28 (70%) of them were multidrug-resistant. Genotype blaCTX-M was observed as a predominant beta-lactamase type with the preponderance of CTX-M Group 1. The following combinations were observed: blaTEM + blaCTX-M in 15 (36.2%) isolates, blaTEM / blaSHV in 8 (5.2%) isolates, and blaCTX-M / blaSHV in 6 (5.2%) isolates. The phylogenetic grouping of E. coli strains revealed the highest prevalence for B1 (22.0%) followed by A (20%). Conclusion: This report shows a high frequency of ESBL E. coli from cattle with postpartum uterine infections. These isolates showed reduced susceptibility to common antibiotics used for the treatment of uterine infections greater affecting the therapeutic outcome.

Resistance of representatives of the order Enterobacterales to ertapenem 12.1%. The highest frequency of insensitivity to this antimicrobial drug was noted among isolates of K. pneumoniae 29.4%. Among all enterobacterial isolates, resistance to imipenem and meropenem was 17.2% and 20%. The proportion of P. aeruginosa strains is 50.9% resistant to meropenem and imipenem, respectively, and 45% to doripenem. In turn, A. baumannii is resistant to meropenem - 66.6%, imipenem - 63.6%, doripenem - 83.3%. The following resistance genes were found in K. pneumoniae: NDM (n=2), KPC (n=10), OXA (n=1); in P. aeruginosa: VIM (n=8), NDM (n=1), OXA (n=1); A. baumannii OXA (n=1). At present, it is optimal to use molecular methods, in particular real-time PCR, to effectively monitor the distribution of carbapenemase producers, which tend to be widely distributed in a hospital setting. Molecular methods allow you to quickly get the result (during the working day) and give an adequate decision on antibiotic therapy.

Introduction A rapid increase in multidrug-resistant (MDR) strains is being seen across the globe especially in the Southeast Asian region, including India. Carbapenems and colistin form the mainstay of treatment against gram-negative pathogens, especially extended-spectrum beta-lactamase (ESBL)- and metallo-beta-lactamse (MBL)-producing isolates. However, due to increased resistance to carbapenems and toxicity of colistin, especially in intensive care units (ICUs), carbapenem-sparing antibiotics like ceftriaxone-sulbactam-EDTA (CSE) combination needs to be evaluated. Materials and methods Bacterial isolates cultured from various clinical samples from all ICUs for a period of 9 months were evaluated. Bacterial identification was performed by matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) and antibiotic susceptibility testing were performed by disk diffusion and E test method. Antibiogram of various antibiotics was noted. Extended-spectrum beta-lactamase- and MBL-producing bacteria were identified by phenotypic methods. Antibiotic sensitivity results of CSE were compared with the comparator drugs like colistin, carbapenems, and tigecycline in Enterobacteriaceae, Acinetobacter spp., and Pseudomonas spp. along with ESBL and MBL producers. Results A total of 2,760 samples of blood, cerebrospinal fluid (CSF), respiratory samples, tissue, and pus were collected from ICUs with maximum isolates from pus (37%) followed by respiratory samples (31%) and blood (27%). Escherichia coli and Klebsiella pneumoniae were the predominant gram-negative pathogens accounting for 56% of the isolates followed by Acinetobacter spp. (23%) and Pseudomonas spp. (15%). Extended-spectrum beta-lactamase screening was positive for 57% (1,069/1,877) isolates; whereas 43% (732/1,877) were MBL producers. According to the antibiotic susceptibility results, CSE was the most effective antibiotic showing 94% sensitivity for carbapenem-sensitive Enterobacteriaceae and 97% for carbapenem-resistant Acinetobacter and Pseudomonas spp. Among the other drugs, colistin was found to be the most effective showing almost 95% sensitivity in both the Enterobacteriaceae and non-Enterobacteriaceae group (both ESBL + OXA/NDM). Ceftriaxone-sulbactam-EDTA was also found much more effective (95%) as compared to Colistin (89%) toward ESBL- and MBL-producing strains of Enterobacteriaceae and non-Enterobacteriaceae group. Among the carbapenems, imipenem was the most effective drug against Enterobacteriaceae showing 34% sensitivity and ertapenem proved to be least effective. Conclusion In our present study, CSE emerged as a potent antibacterial agent against MDR gram-negative infections; both for ESBL as well as MBL producers. Hence, in light of present study, we strongly recommend inclusion of CSE in routine sensitivity panel and may be used as a carbapenem- and colistin-sparing drug and a promising option against ESBL and MBL producers especially in ICU. How to cite this article Singh S, Sahu C, Patel SS, Singh A, Yaduvanshi N. A Comparative In Vitro Sensitivity Study of "Ceftriaxone-Sulbactam-EDTA" and Various Antibiotics against Gram-negative Bacterial Isolates from Intensive Care Unit. Indian J Crit Care Med 2020;24(12):1213-1217.

Objetivo. Comunicar el primer reporte de Klebsiella pneumoniae productora de carbapenemasas. El estudio: Diagnóstico microbiológico empleando métodos fenotípicos: sinergia del doble disco, Test de Hodge modificado, inmunocromatografia y la inactivación del carbapenémico. Hallazgos: Se confirmaron cuatro aislamientos (orinas 3 y sangre 1) de K. pneumoniae productoras de carbapenemasas que además presentaron una CMI de 4 y > = 8 para el ertapenem y > = 16 µg/mL a imipenem y meropenem. Conclusiones: los métodos fenotípicos estandarizados son de utilidad en la confirmación de carbapenemasas y con ello una alternativa para la mejor toma de opciones terapéuticas y vigilancia epidemiológica.

The spread of antibiotic resistance has been associated with consumption of food contaminated with pathogenic and non-pathogenic resistant bacteria. The objective of this study was to determine the prevalence of antibiotic resistant Escherichia coli isolates ( E. coli ) from local and imported retail raw chicken meat in Qatar. A total of 270 whole chicken carcasses locally-produced (chilled) and imported (chilled and frozen) were obtained from three different hypermarket stores in Qatar. A total of 216 E. coli isolates from the chicken samples were subjected to antibiotic susceptibility testing using disc diffusion method. Furthermore, extended-spectrum β-lactamase (ESBL) production was determined via double disc synergetic test. In addition, isolates harboring colistin resistant was determined using multiplex-PCR and DNA sequencing. Nearly 89% (192/216) of the isolates were resistant to at least one of the 18 antibiotics. In general, isolates from local and imported chicken carcasses showed relatively higher resistance to sulfamethoxazole (62%), tetracycline (59.7%), ampicillin and trimethoprim (52.3%), ciprofloxacin (47.7%), cephalothin, and colistin (31.9%). On the other hand, less resistance was recorded against amoxicillin/clavulanic acid (6%), ceftriaxone (5.1%), nitrofurantoin (4.2%) and piperacillin/tazobactam (4.2%), cefepime (2.3%), meropenem (1.4%), ertapenem (0.9%), and amikacin (0.9%). Nine isolates (4.2%) were ESBL producers. Furthermore, 63.4% (137/216) were multidrug resistant (MDR). The percentage of MDR, ESBL producers, and colistin-resistant isolates were significantly higher among local chilled isolates compared to imported chilled and frozen chicken samples. Our findings indicate the high antibiotic resistant E. coli prevalence in chicken meat sold at retail in Qatar.

In recent years, the prevalence of carbapenem-resistant Enterobacteriaceae (CRE) has risen substantially, and the study of CRE resistance mechanisms has become increasingly important for antibiotic development. Although much research has focused on genomic resistance factors, relatively few studies have examined CRE pathogens through changes in gene expression. In this study, we examined the gene expression profile of a CRE Escherichia coli clinical isolate that is sensitive to meropenem but resistant to ertapenem to explore transcriptomic contributions to resistance and to identify gene knockdown targets for carbapenem potentiation. We sequenced total and short RNA to analyze the gene expression response to ertapenem or meropenem treatment and found significant expression changes in genes related to motility, maltodextrin metabolism, the formate hydrogenlyase complex, and the general stress response. To validate these findings, we used our laboratory's Facile Accelerated Specific Therapeutic (FAST) platform to create antisense peptide nucleic acids (PNAs), gene-specific molecules designed to inhibit protein translation. PNAs were designed to inhibit the pathways identified in our transcriptomic analysis, and each PNA was then tested in combination with each carbapenem to assess its effect on the antibiotics' minimum inhibitory concentrations. We observed significant PNA-antibiotic interaction with five different PNAs across six combinations. Inhibition of the genes hycA, dsrB, and bolA potentiated carbapenem efficacy in CRE E. coli, whereas inhibition of the genes flhC and ygaC conferred added resistance. Our results identify resistance factors and demonstrate that transcriptomic analysis is a potent tool for designing antibiotic PNA.

Introduction. Group B streptococci (GBS) are globally recognized as one of the leading causes of neonatal sepsis and meningitis and is also known to cause adverse pregnancy outcomes such as stillbirths and miscarriages. Thus, detailed investigation of GBS in pregnant women has special significance in public health related researches. Objectives. The present study is aimed at evaluating the prevalence of GBS colonization among pregnant women in Kathmandu city. Methods. The study was carried out among 125 pregnant women at their trimester (35–37 weeks) (during the time period between January and June in 2018). The prevalence was determined by the culture method in HiCrome Strep B Selective Agar Base and then by using the PCR technique. The serotypes were evaluated by multiplex PCR analysis, while the antibiotics susceptibility tests were performed using the disk diffusion method. Results. Among 125 samples studied, GBS were recorded in 24 samples (implying a prevalence of 19.2%). Furthermore, using the multiplex PCR, among 24 GBS-positive samples, 13 (54.17%) were found to be typeable while 11 (45.83%) were nontypeable. The most abundant serotype recorded in this study was type III (33.33) while the serotypes IV, V, VI, VII, and VIII were not found. Conclusion. The isolates were sensitive towards some antibiotics such as linezolid and ceftriaxone 100%, whereas penicillin 50% and vancomycin 75% but were resistant to tetracycline and ertapenem. Serotype III was found to be predominant in the samples collected during the study period. The observed prevalence was significantly associated with the gestational period, whereas no relationship was found for other risk factors.

Introduction. Acinetobacter baumannii is a critical priority pathogen listed by the World Health Organization due to increasing levels of resistance to carbapenem classes of antibiotics. It causes wound and other nosocomial infections, which can be life-threatening. Hence, there is an urgent need for the development of new classes of antibiotics. Aim. To study the interaction of carabapenems with class D beta-lactamases (oxacillinases) and analyse drug resistance by studying enzyme–substrate complexes using modelling approaches as a means of establishing correlations with the phenotypic data. Methodology. The three-dimensional structures of carbapenems (doripenem, ertapenem, imipenem and meropenem) were obtained from DrugBank and screened against class D beta-lactamases. Further, the study was extended with their variants. The variants’ structure was homology-modelled using the Schrödinger Prime module (Schrödinger LLC, NY, USA). Results. The first discovered intrinsic beta-lactamase of Acinetobacter baumannii , OXA-51, had a binding energy value of −40.984 kcal mol−1, whereas other OXA-51 variants, such as OXA-64, OXA-110 and OXA-111, have values of −60.638, –66.756 and −67.751 kcal mol−1, respectively. The free energy values of OXA-51 variants produced better results than those of other groups. Conclusions. Imipenem and meropenem showed MIC values of 2 and 8 µg ml−1, respectively against OXA-51 in earlier studies, indicating that these are the most effective drugs for treatment of A. baumannii infection. According to our results, OXA-51 is an active enzyme that shows better interactions and is capable of hydrolyzing carbapenems. When correlating the hydrogen-bonding interaction with MIC values, the predicted results are in good agreement and might provide initial insights into performing similar studies related to OXA variants or other antibiotic–enzyme-based studies.

This study investigated the occurrence of Extended Spectrum Beta-Lactamase (ESBL) genes in ESBL- producing Escherichia coli isolated from wastewater and sludge of a tertiary hospital treatment plant. Wastewater (treated and untreated) and sludge were collected over a period of four months for the isolation of E. coli on CHROMagar E. coli amended with 6 μg/mL of cefotaxime. The identity of the bacteria was confirmed by detection of uidA gene. Screening for ESBL production was done using the Double Disc Synergy Method (DDST). Detection of ESBL genes was done using quantitative PCR. Antibiotic susceptibility testing was carried out using the disc diffusion method. A total of thirty-three distinct E. coli were obtained with twelve being ESBL producers. blaTEM, blaCTX₋M₋₂, blaCTX₋M₋₈ and blaCTX₋M₋₂₅ were detected in eight isolates. All the ESBL- producing E. coli harboured blaCTX ₋M₋₁ and blaCTX ₋M₋₉. blaCMY ₋₁ was detected in one of the twelve ESBL- producers. There was 100% resistance to tetracycline, sulphonamide, ertapenem, cefpodoxime and cefotaxime, and 75% showing resistance to ciprofloxacin, ceftazidime and amoxycillin-clavulanate respectively in the ESBL positive isolates.

Purpose The Chinese Meropenem Surveillance Study (CMSS) was conducted every 2 years from 2010 to 2018 to monitor the antimicrobial activity of commonly used antimicrobial agents against nosocomial gram-negative bacilli in China. Methods From 2010 to 2018, 6,537 gram-negative bacilli were collected from 14 teaching hospitals. The minimum inhibitory concentrations (MICs) of meropenem and other antimicrobial agents were determined using the agar dilution and broth microdilution methods. Results Continuous surveillance indicated that, except for Klebsiella pneumoniae, the susceptibility of Enterobacterales to carbapenems was relatively stable over time. Carbapenems had the highest activity against the tested isolates, with MIC90 values (MIC for 90% of organisms) ranging from 0.032 mg/L to 8 mg/L. More than 90% of bacteria were susceptible to either meropenem or imipenem; more than 80% were susceptible to ertapenem. The prevalence of extended-spectrum beta-lactamase (ESBL)-producing E. coli, K. pneumoniae, and P. mirabilis each year was 50.4-64.3%, 18-41.2%, and 1.9-33.8%, respectively. The prevalence of carbapenem-resistant K. pneumoniae (CRKP) and carbapenem-resistant Acinetobacter baumannii (CRAB) continued to increase significantly over time, from 7.6% to 21.2% and 64.6% to 69.3%, respectively. The prevalence of CRKP was higher from urinary tract infections (25.4%) than from bloodstream infections (14.2%), intra-abdominal infections (14.5%), and respiratory infections (14.4%). In total, 129 CRKP isolates were evaluated by PCR; of these, 92 (71.3%) carried the bla KPC-2 gene. Colistin maintained very high in vitro antimicrobial activity against P. aeruginosa and A. baumannii (more than 95% of isolates exhibited susceptibility at all timepoints). Conclusion The results indicate an increase in K. pneumoniae resistance to carbapenems over time, mainly owing to KPC-type carbapenemase production. A. baumannii was severely resistant to carbapenems in China. Ongoing MIC-based resistance surveillance, like CMSS, provides additional data for clinical anti-infective treatment.

Purpose. The objective of this study was to evaluate the microbiological spectrum and antibiotic susceptibilities of isolates in posttraumatic endophthalmitis over a 15-year period. Methods. A retrospective study of 3,163 posttraumatic endophthalmitis cases was conducted between July 2004 and July 2019. The outcome measures included the microbiological spectrum and antibiotic susceptibilities. Chi-squared tests were conducted to detect trends in changes in antibiotic sensitivity over the 15-year period. P values of Results. Of the 3,163 cases of posttraumatic endophthalmitis, 1,003 culture-positive isolates were identified. Among these, there were 848 (84.5%) Gram-positive isolates, 109 (10.9%) Gram-negative isolates, and 46 (4.6%) fungal isolates. The most common isolates were Staphylococcal species. There was a significant increase in the percentage of fungal isolates over the 15-year period (P=0.02). Gram-positive organisms showed the greatest level of susceptibility to vancomycin (99.6%). The susceptibilities of the 109 Gram-negative isolated organisms were as follows: levofloxacin (95.8%), meropenem (95.7%), ciprofloxacin (93.5%), tobramycin (90.8%), imipenem (88.9%), trimethoprim-sulfamethoxazole (TMP-SMX) (87.7%), ertapenem (80%), and ceftazidime (79.1%). The susceptibility of Gram-positive organisms to several antibiotics, including levofloxacin (P=0.004), ciprofloxacin (P<0.001), and chloramphenicol (P=0.001) decreased over time, whereas the susceptibility to TMP-SMX increased over time (P<0.001). The susceptibility of Gram-negative bacilli to ceftazidime decreased over time (P=0.03). Conclusions. Over the 15-year study period, most isolates were Gram-positive cocci, especially coagulase-negative staphylococci (CNS). Vancomycin seemed to be the most effective antibiotic for Gram-positive bacteria. Gram-negative bacteria appeared to be most susceptible to fluoroquinolones. A number of antibiotics showed an increasing trend of microbial resistance.

Introduction: Klebsiella pneumoniae, a common hospital- and community-acquired pathogen, is notorious for multidrug resistance. This study aimed to better understand the correlation of clinical presentation and microbiological characteristics of the isolates causing bloodstream infections (BSIs) in Taiwan. Methodology: We retrospectively collected 150 isolates derived from K. pneumoniae bacteremia patients in Taiwan in both 2014 and 2016. Clinical data, bacterial serotyping and drug susceptibility tests were comparatively analyzed. Results: Demographic data showed that diabetes mellitus (DM) was the most common underlying disease (44.0%). The overall 30-day mortality rate was 19.3%, and higher mortality was found in patients with malignancy than others (P = 0.023). Serotype distribution was diverse. The major isolates belonged to non-PCR-typeable serotypes (58.7%) associated with hospital-acquired infections (P = 0.007) and in non-DM patients (P < 0.001), while K2 and K20 significantly caused infections and in DM patients (P = 0.046 and P = 0.006, respectively); however, only K2 showed more community-acquired infection (P = 0.022) than other typeable serotypes. Resistance to antibiotics in clinical isolates in the year 2016 was > 24%, including cefazolin (54%), ampicillin-sulbactam (25%) and cefuroxime (25%). Susceptibility to gentamicin, flomoxef, and tigecycline reduced between the two time periods (2014 and 2016). However, the isolates remained highly susceptible to amikacin and ertapenem (> 95%). Conclusions: Patients with cancer had a higher 30-day mortality rate than others. Amikacin and ertapenem are the drugs of choice for the treatment of multidrug-resistant K. pneumoniae BSIs in Taiwan.

In this study, we report a clinical isolate of a carbapenem-, colistin-, and fosfomycin-resistant Escherichia coli isolate DC-3737 co-harboring blaNDM-1, mcr-1, and fosA3 from an inpatient in China. Antimicrobial susceptibility testing, polymerase chain reaction, multi-locus sequence typing, conjugation experiment, and Southern blot hybridization were performed on E. coli DC-3737 isolated from the wound. Plasmid analysis is presented and the locations of blaNDM-1, mcr-1, fosA3, and other resistance genes were identified as well. E. coli DC-3737 was resistant to ampicillin, ceftriaxone, ceftazidime, ciprofloxacin, levofloxacin, gentamicin, tobramycin, trimethoprim-sulfamethoxazole, imipenem, meropenem, ertapenem, fosfomycin and colistin, and with intermediate susceptibility to amikacin. It was typed as sequence type 27. The isolate possessed blaNDM-1, mcr-1, fosA3, blaCTX-M-9, blaTEM-1, aac (6')-Ib-cr and sul1 simultaneously. In addition, the mutations in quinolone resistance-determinant regions (QRDRs) such as Ser83Leu and Asp87Asn in gyrA, and Ser80Ile in parC were detected. Conjugation assays revealed that blaNDM-1, fosA3, sul1, mcr-1, and blaCTX-M-9 genes could successfully transfer their resistance phenotype to E. coli strain J53. Plasmid analysis and Southern hybridization showed that DC-3737 possessed Z-type self-transmissible plasmid bearing blaNDM-1, fosA3, and sul1. Moreover, mcr-1, blaCTX-M-9, and blaTEM-1 were located on a ~60-kb IncFIB type self-transmissible plasmid. This is the first report of blaNDM-1, mcr-1 and fosA3 co-harboring in E. coli in China. Moreover, it is also the first description of the co-harboring of blaNDM and fosA3 in a single Z plasmid in Enterobacteriaceae species. The identification of E. coli DC-3737 co-harboring blaNDM-1, mcr-1, and fosA3 in this study highlights the need to increase epidemiologic surveillance and the need for new classes of antibiotics to address multidrug-resistant bacteria.

Introduction: The emergence and global spread of carbapenem-resistant E. coli is of great concern to the health care facilities due to high rates of morbidity and mortality associated with them. It is necessary to know its epidemiology and the resistant pattern in a geographical area to formulate a antibiotic stewardship policy. Objective: To study the occurrence and mechanisms of carbapenem resistance in Escherichia coli. Materials and Methods: A total of 1172 clinical strains of E. coli obtained from various clinical specimens were screened for carbapenem resistance during the study period. Strains showing reduced susceptibility to imipenem &/or ertapenem &/or meropenem were included in the study. The resistance mechanisms were identified using various phenotypic methods. Results: Total of 53/1172 were found to be carbapenem resistant E. coli (CRE). The most common sample in which CRE were isolated was urine (n=26, 49.1%). A total of 50 isolates were confirmed as Metallobeta- lactamase (MBL) producers using Ezy MICTM strip. One of the three non carbapenemase producing isolate was positive for ESBL with porin loss and the other two isolates were positive for AmpC with porin loss. Conclusion: MBL production being the most common mechanism of carbapenem resistance, the study indicates the importance of regular monitoring of drug resistance in the hospital for an urgent action to be taken for antibiotics stewardship in the institute. Keywords: Escherichia coli, Carbapenem resistance, Super bug, Carba NP, MBL, Carbapenem resistance mechanisms.

Objective To investigate the molecular characteristics of carbapenem-resistant Enterobacteriaceae (CRE) from county hospitals in China. Materials and methods Forty-three sequential non-duplicate CRE strains (including 33 Klebsiella pneumoniae isolates, 4 Enterobacter cloacae isolates, 3 Escherichia coli isolates, 1 Serratia marcescens, 1 Morganella morganii and 1 Citrobacter freundii) were collected from 4 county hospitals and 2 municipal hospitals. Antimicrobial susceptibility testing was conducted by broth microdilution method, using 3-aminophenylboronic acid and EDTA and the modified carbapenem inactivation method (mCIM) to screen phenotype of carbapenemase. β-Lactamases were characterized by polymerase chain reaction (PCR) and DNA sequencing. The transferability of bla NDM-5 was investigated by transformation experiment. Clonal relatedness was evaluated by pulsed-field gel electrophoresis and multilocus sequence typing . Results The results of antimicrobial susceptibility testing indicated that 43 CRE strains were resistant to most of the antimicrobial agents, except tigecycline and colistin. Overall, 93%, 93%, and 97.7% of these strains were resistant to imipenem, meropenem, and ertapenem, respectively. PCR and DNA sequencing indicated that 67.4% (29/43) were bla KPC-2 positive isolates, in which 3.4% (1/29) was coproduced with bla NDM-1. In addition, 7.0% (3/43), 4.7% (2/43), 4.7% (2/43), 2.3% (1/43), 2.3% (1/43) were bla NDM-1, bla NDM-16, bla NDM-4, bla NDM-5, bla IMP-4 positive isolates, respectively. The 29 bla KPC-2-positive isolates belonged to 12 different PFGE type and designated as ST11 (n=20) and ST15, ST39, ST116, ST667, ST2245, ST2338. The plasmid bearing bla NDM-5 could be transferred into recipient E. coli J53 through transformation. Conclusion Our study indicated the dissemination of CRE between the tertiary hospitals and secondary hospitals.

Alteration in two-component systems (TCSs), which are signal transduction pathways in prokaryotes, can result in antibiotic resistance. Recently, it has been shown that the overexpression, using a multicopy cloning vector, of the dcuR, rcsB, and yehT genes, which code for the response regulator (RR) part of TCSs, enhanced the minimal inhibitory concentrations (MICs) of carbapenems in Escherichia coli K-12 derivative KAM3. Herein, the contribution to carbapenem resistance of the DcuS/DcuR, RcsC/RcsB, and YehU/YehT TCSs was assessed in E. coli K-12 derivative BW25113 (A phylogroup) and 536 (B2 phylogroup) recipient strains in combination with extended-spectrum β-lactamase that exhibit a weak carbapenemase activity. The genes encoding both the sensor kinase (SK) and the RR, on the one hand, and the genes encoding the SK only, on the other hand, of these regulating pathways were disrupted. Subsequently, the mutants and their parental strains were transformed by a recombinant plasmid encoding the CTX-M-15 gene, before testing their susceptibility to carbapenems and their fitness. Results showed a trade-off between enhanced MICs for ertapenem, which remained above the clinical resistance breakpoint, and decreased growth rate, specifically for the 536 strain SK mutants. In conclusion, mutations in dcuS/dcuR, rcsC/rcsB, and yehU/yehT genes may be a pivotal first-step event in the development of carbapenem resistance.

Background The emergence of antibiotic resistance is increasing and there are few effective antibiotics to treat infections caused by resistant and multidrug resistant bacterial pathogens. This study aimed to evaluate the in vitro activity of ceftolozane–tazobactam against clinical bacterial isolates from Brazil. Methods A total of 673 Gram-negative bacterial isolates including Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and other Enterobacterales collected from 2016 to 2017 were tested, most of them isolated from patients in intensive care units. Minimum inhibitory concentrations (MIC50/90) were determined by broth microdilution for amikacin, aztreonam, cefepime, cefotaxime, cefoxitin, ceftolozane–tazobactam, ceftazidime, ceftriaxone, ciprofloxacin, colistin, ertapenem, imipenem, levofloxacin, meropenem, and piperacillin-tazobactam using dried panels. Antimicrobial susceptibility results were interpreted according to Clinical and Laboratory Standards Institute criteria. Results Susceptibility rates to ceftolozane–tazobactam ranged from 40.4% to 94.9%. P. aeruginosa susceptibility rate to ceftolozane–tazobactam was 84.9% (MIC50/90, 1/16 μg/mL) and 99.2% to colistin. For E. coli, ceftolozane–tazobactam inhibited 94.9% (MIC50/90, 0.25/1 μg/mL) of the microorganisms. The susceptibility rate of K. pneumoniae to ceftolozane–tazobactam was 40.4% (MIC50/90, 16/>32 μg/mL). Other Enterobacterales have shown susceptibility rates of 81.1% (MIC50/90, 0.5/16 μg/mL) to ceftolozane–tazobactam, 93.9% to meropenem, 90.9% to amikacin (90.9%), and 88.6% to ertapenem. In non-carbapenemase producing isolates, AmpC mutations were found three isolates. Conclusions Ceftolozane–tazobactam has shown relevant activity against a large variety of the analyzed microorganisms collected from multiple centers in Brazil, showing promising results even in multidrug resistant strains.

Objectives To investigate the carbapenem resistance mechanism of a carbapenem-resistant clinical Pseudomonas aeruginosa isolate. Methods A carbapenem-resistant P. aeruginosa isolate was recovered from a tracheal swab from a patient of a general ward in central Germany. Various phenotypic tests confirmed production of a carbapenemase that could not be identified further by PCR. A novel bla gene was identified by WGS and its carbapenemase activity was verified by heterologous expression in an Escherichia coli cloning strain. Kinetic parameters of the novel β-lactamase were determined by spectrophotometric measurements using purified enzyme. Results WGS confirmed the presence of a novel class A carbapenemase. The novel bla gene was named GPC-1 (GPC standing for German Pseudomonas Carbapenemase) and exhibited 77% amino acid identity to BKC-1. WGS also showed that blaGPC-1 was located on the chromosome surrounded by multiple ISs as part of a 26 kb genetic island. Heterologous expression of GPC-1 in E. coli TOP10 led to increased MICs of penicillins, oxyimino-cephalosporins, aztreonam and imipenem, but not of meropenem or ertapenem. Spectrophotometric measurements supported the MIC studies, but detected a slight hydrolysis of ertapenem and meropenem when using high concentrations of purified enzyme. Conclusions The biochemical characterization of GPC-1 emphasizes the ongoing emergence of novel carbapenemases. Strains expressing a weak carbapenemase like GPC-1 might go unrecognized by routine diagnostics due to low MICs for the bacterial strains producing such enzymes.

Objective: The spread of carbapenemase-producing Enterobacteriaceae is a global challenge that leads to an increase in health-care cost, treatment failures, high morbidity, and mortality. This study was aimed at determining the prevalence of New-Delhi Metallo Beta-Lactamase (NDM), and oxacillinase-48 (OXA-48) genes in clinical isolates of Escherichia coli obtained from a tertiary hospital in Nnewi, South-eastern, Nigeria. Materials and Methods: E. coli isolated from several clinical specimens including blood, urine, and wound swabs from patients receiving care at the hospital, were screened for resistance to meropenem and ertapenem antibiotics (Oxoid, UK) by the Kirby–Bauer disk-diffusion method. All isolates, which showed reduced sensitivity to the tested antibiotics, were then subjected to phenotypic confirmation of carbapenemase production using the Modified Hodge test. The NDM and OXA-48 genes were then detected using the polymerase chain reaction techniques. Results: Of the 187 E. coli isolates, 41 (21.9%) screened positive as suspected carbapenemase producers, while the prevalence of carbapenemase-producing E. coli in this study was 21/187 (11.23%). The prevalence of NDM and OXA-48 genes in the entire sample population was 3/187 (1.6%) and 12/187 (6.4%), respectively. Conclusion: The results obtained showed that NDM and OXA-48 carbapenemase-mediated resistance occurred in the study location. Hence, a reinforcement of infection prevention and control practices in the hospital will be required to curb the propagation of these resistant organisms.

Antimicrobial stewardship programs (ASP) promote appropriate antimicrobial use. We present a 4-year retrospective study that evaluated the clinical impact of the acceptance of the recommendations made by a meropenem-focused ASP. A total of 318 meropenem audits were performed. The ASP team (comprising infectious disease physicians, pharmacists and microbiologists) considered meropenem use in 96 audits (30.2%) to be inappropriate. The reasons to consider these uses inappropriate were the possibility of de-escalating to a narrower-spectrum antibiotic, in 66 (68.7%) audits, and unnecessary meropenem use, in 30 (31.3%) audits. The ASP team recommended de-escalation in 66 audits (68.7%) and discontinuation of meropenem in 30 audits (31.3%). ASP interventions were stratified according to whether or not recommendations were followed. The group in which recommendations were accepted and followed (i.e., accepted audit, AA) included 66 audits (68.7%) and the group in which recommendations were not followed (i.e., rejected audit, RA) included 30 (31.3%) audits. The comorbidity of the AA group (Charlson score) was higher than in the RA group (7.0 (5.0–9.0) vs. 6.0 (4.0–7.0), p = 0.02). Discontinuation of meropenem was recommended in 83.3% of audits in the AA group vs. 62.2% in the RA group (OR 3.05 (1.03–8.99), p = 0.04). Ertapenem de-escalation resulted in a 100% greater rate of follow-up compared with the non-carbapenem option (100% vs. 51.9%, OR 1.50 (1.21–1.860), p = 0.001). Significant differences were observed in the AA group when cultures were taken before antibiotic prescription—98.5% vs. 83.3% (p = 0.01, OR 13.0 (1.45–116.86))—or when screening cultures were taken—45.5% vs. 19.2% (p = 0.03, OR 3.5 (1.06–11.52)). There were no differences between the groups in terms of overall mortality and 30-day mortality, length of stay, Clostridiodes difficile infection, 30-day readmission or hospitalization costs. In conclusion, meropenem ASP recommendations contributed to a decrease in meropenem prescription without worsening clinical and economic outcomes.