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Background. The etiological structure of implant-associated infection and antibiotic resistance of pathogens are important when choosing empirical antibiotic therapy. COVID-19 pandemic and increased consumption of antibiotics by the population could provoke an increase in antibiotic resistance.The aim of the work. To compare the spectrum of leading pathogens of implantassociated infection in the pre- and post-Covid period and to assess antibiotic resistance.Materials and methods. A continuous retrospective study of biomaterial samples from traumatology and orthopedic patients with implant-associated infection was carried out for 2018–2019 and 2021–2022. The sample consisted of 548 microorganism strains (n = 237 and n = 317, respectively) in 442 cases of infectious complications. The antibiotic resistance of all isolated microorganisms, including those from microbial associations, was assessed.Results. The leading pathogen of monomicrobial implant-associated infection in both study periods was Staphylococcus epidermidis (33–37 %). In 2021–2022, the proportion of microbial associations increased (from 12.5 to 17.5 %; p = 0.147) with the appearance of fungi in the microbial landscape. In the post-Covid period, the increase in Staphylococcus aureus resistance to tetracycline and doxycycline was revealed; the isolation of methicillin-resistant strains among Staphylococcus aureus decreased from 4 cases (out of 187) to 3 (out of 232); 100 % sensitivity to rifampicin and co-trimoxazole was maintained. An increase in Staphylococcus epidermidis resistance to all tested antibiotics was detected (statistically significant increase in resistance to fluoroquinolones; p = 0.002–0.003) with the isolation of methicillin-resistant strains in 80.5% and 80.9% of cases, respectively. All staphylococcal isolates were susceptible to vancomycin and linezolid. Enterobacteriaceae representatives showed a decrease in resistance to carbapenems and an increase in resistance to co-trimoxazole; in Pseudomonas aeruginosa and Acinetobacter baumannii, there is an increase in resistance to carbapenems and fluoroquinolones. All gram-negative microorganisms were sensitive to colistin.Conclusion. The high frequency of isolation of methicillin-resistant staphylococci determines the choice of vancomycin for empirical therapy. Increasing resistance of staphylococci to fluoroquinolones may limit their use. Increasing resistance of gram-negative bacteria and a narrow spectrum of antibiotics acting on carbapenemase producers may reduce the effectiveness of therapy.

Carbapenem resistance (CR) is a major global health concern. CR is a growing challenge in clinical settings due to its rapid dissemination and low treatment options. The characterization of its molecular mechanisms and epidemiology are highly studied. Nevertheless, little is known about the spread of CR in food-producing animals, seafood, aquaculture, wildlife, their environment, or the health risks associated with CR in humans. In this review, we discuss the detection of carbapenem-resistant organisms and their mechanisms of action in pigs, cattle, poultry, seafood products, companion animals, and wildlife. We also pointed out the One Health approach as a strategy to attempt the emergency and dispersion of carbapenem-resistance in this sector and to determine the role of carbapenem-producing bacteria in animals among human public health risk. A higher occurrence of carbapenem enzymes in poultry and swine has been previously reported. Studies related to poultry have highlighted P. mirabilis, E. coli, and K. pneumoniae as NDM-5- and NDM-1-producing bacteria, which lead to carbapenem resistance. OXA-181, IMP-27, and VIM-1 have also been detected in pigs. Carbapenem resistance is rare in cattle. However, OXA- and NDM-producing bacteria, mainly E. coli and A. baumannii, are cattle's leading causes of carbapenem resistance. A high prevalence of carbapenem enzymes has been reported in wildlife and companion animals, suggesting their role in the cross-species transmission of carbapenem-resistant genes. Antibiotic-resistant organisms in aquatic environments should be considered because they may act as reservoirs for carbapenem-resistant genes. It is urgent to implement the One Health approach worldwide to make an effort to contain the dissemination of carbapenem resistance.

ABSTRACT Fosfomycin (FOS) has been recently reintroduced into clinical practice, but its effectiveness against multidrug-resistant (MDR) Enterobacterales is reduced due to the emergence of FOS resistance. The copresence of carbapenemases and FOS resistance could drastically limit antibiotic treatment. The aims of this study were (i) to investigate fosfomycin susceptibility profiles among carbapenem-resistant Enterobacterales (CRE) in the Czech Republic, (ii) to characterize the genetic environment of fosA genes among the collection, and (iii) to evaluate the presence of amino acid mutations in proteins involved in FOS resistance mechanisms. During the period from December 2018 to February 2022, 293 CRE isolates were collected from different hospitals in the Czech Republic. FOS MICs were assessed by the agar dilution method (ADM), FosA and FosC2 production was detected by the sodium phosphonoformate (PPF) test, and the presence of fosA-like genes was confirmed by PCR. Whole-genome sequencing was conducted with an Illumina NovaSeq 6000 system on selected strains, and the effect of point mutations in the FOS pathway was predicted using PROVEAN. Of these strains, 29% showed low susceptibility to fosfomycin (MIC, ≥16 μg/mL) by ADM. An NDM-producing Escherichia coli sequence type 648 (ST648) strain harbored a fosA10 gene on an IncK plasmid, while a VIM-producing Citrobacter freundii ST673 strain harbored a new fosA7 variant, designated fosA7.9. Analysis of mutations in the FOS pathway revealed several deleterious mutations occurring in GlpT, UhpT, UhpC, CyaA, and GlpR. Results regarding single substitutions in amino acid sequences highlighted a relationship between ST and specific mutations and an enhanced predisposition for certain STs to develop resistance. This study highlights the occurrence of several FOS resistance mechanisms in different clones spreading in the Czech Republic. IMPORTANCE Antimicrobial resistance (AMR) currently represents a concern for human health, and the reintroduction of antibiotics such as fosfomycin into clinical practice can provide further option in treatment of multidrug-resistant (MDR) bacterial infections. However, there is a global increase of fosfomycin-resistant bacteria, reducing its effectiveness. Considering this increase, it is crucial to monitor the spread of fosfomycin resistance in MDR bacteria in clinical settings and to investigate the resistance mechanism at the molecular level. Our study reports a large variety of fosfomycin resistance mechanisms among carbapenemase-producing Enterobacterales (CRE) in the Czech Republic. Our study summarizes the main achievements of our research on the use of molecular technologies, such as next-generation sequencing (NGS), to describe the heterogeneous mechanisms that reduce fosfomycin effectiveness in CRE. The results suggest that a program for widespread monitoring of fosfomycin resistance and epidemiology fosfomycin-resistant organisms can aide timely implementation of countermeasures to maintain the effectiveness of fosfomycin.

This study was undertaken to detect the presence of blaNDM-1 gene among the carbapenem resistant Escherichia coli and Klebsiella pneumoniae isolates, confirmed through using Modified Hodge Test (MHT). During the six months of this study (November, 2016 – April, 2017), a total of 1503 clinical samples including urine, blood, wound swabs and Cerebrospinal fluid (CSF) were collected and processed at Microbiology Laboratory, International Friendship Children’s Hospital, Kathmandu, Nepal. Among the E. coli and K. pneumoniae isolates, 14.1%, 17.1% were resistant to meropenem, whereas 11.28%, 17.1% were resistant to imipenem, respectively. From the pool of 94 E. coli and 35 K. pneumoniae isolates, 34 E. coli and 18 K. pneumoniae were screened as possible carbapenemase producers. For screening of carbapenemase enzyme production among these isolates, resistances to third generation cephalosporins and carbapenems were taken into consideration. Two isolates of E. coli and 3 isolates of K. pneumoniae were confirmed as carbapenemase producers by MHT. Furthermore, Polymerase chain reaction (PCR) was carried out among the MHT positive bacteria for detection of blaNDM-1 gene. Genetic analysis of MHT positive isolates showed that 1\2 E. coli and 2\3 K. pneumoniae isolates were New Delhi metallo-β-lactamase producers (NDM-1). Results of this study demonstrated the presence of blaNDM-1 gene among the carbapenemase producing E. coli and K. pneumoniae isolates, and were recorded to be 50% and 66.6%, respectively.

Objective: To gauge the recent breadth of MDR E. coli along with antibiogram of carbapenemase producing (CP) E. coli among children from an institute which receives patients from all over Punjab. Methods: The bacterial strains of E. coli isolated from various specimens of patients were collected from April 2017 to August 2018 and processed using standard biochemical tests and API 20E system (bioMerieux). Phenotypic screening for CP E. coli was done by the modified Hodge test, whereas antibiotic susceptibility testing was done with Kirby-Bauer disc diffusion technique. Results: Total of 6,468 bacterial strains were isolated, out of which 1,552 (24%) were E. coli. Carbapenem resistance was observed in 245 (16%) strains, amongst which 113 (46%) were confirmed to be CP. E. coli isolated from males were higher as compared to females (p<0.05). Majority of the organisms were isolated from blood (37.2%) samples. The hospital discharged about 65% of patients, while 23% left against medical advice. Overall MDR amongst E. coli was 93.26%. Colistin sulphate (15.9%) and nitrofurantoin (16.8%) showed the most efficacy followed by amikacin (15%) and fosfomycin (10.6%). Conclusion: The isolation of high number of MDR E. coli amongst the paediatric patients is worrisome, which could serve as a potential source of horizontal genes transfer to other genera. [ABSTRACT FROM AUTHOR]

Enterobacteriaceae cause different types of community- and hospital-acquired infections. Moreover, the spread of multidrug-resistant Enterobacteriaceae is a public health problem and the World Health Organization pointed them among the pathogens in which the search of new antibiotics is critical. The objective of this study was to analyze the in vitro activity of pentamidine alone and in combination with gentamicin, tobramycin, amikacin, tigecycline, rifampicin, or doripenem against eight clinical strains of carbapenemase-producing and/or colistin-resistant Enterobacteriaceae: five carbapenemase-producing Klebsiella pneumoniae, one carbapenemase-producing Escherichia coli, and two colistin-resistant Enterobacter cloacae. MIC and MBC were determined following standard protocols. MIC results were interpreted for all the antibiotics according to the EUCAST breakpoints but for rifampicin in which the French FSM breakpoint was used. Bactericidal and synergistic activity of pentamidine alone and in combination with antibiotics at concentrations of 1xMIC was measured by time-kill curves. For one selected strain, K. pneumoniae OXA-48/CTX-M-15 time-kill curves were performed also at 1/2xMIC of pentamidine. All studies were performed in triplicate. Pentamidine MIC range was 200–800 μg/mL. The 50, 12.5, 62.5, 87.5, and 62.5% of the strains were susceptible to gentamicin, tobramycin, amikacin, tigecycline, and doripenem, respectively. Only the two E. cloacae strains were susceptible to rifampicin. Pentamidine alone at 1xMIC showed bactericidal activity against all strains, except for the E. cloacae 32 strain. The bactericidal activity of pentamidine alone was also observed in combination. The combinations of pentamidine were synergistic against E. cloacae 32 with amikacin and tobramycin at 24 h and with tigecycline at 8 h. Pentamidine plus rifampicin was the combination that showed synergistic activity against more strains (five out of eight). Pentamidine plus doripenem did not show synergy against any strain. At 1/2xMIC, pentamidine was synergistic with all the studied combinations against the K. pneumoniae OXA-48/CTX-M-15 strain. In summary, pentamidine alone and in combination shows in vitro activity against carbapenemase-producing and/or colistin-resistant Enterobacteriaceae. Pentamidine appears to be a promising option to treat infections caused by these pathogens.

Despite the relevance of carbapenemase-producing Klebsiella pneumoniae (CP-Kp) infections there are a scarce number of studies to evaluate in vivo the efficacy of combinations therapies. The bactericidal activity of colistin, rifampin, and its combination was studied (time–kill curves) against four clonally unrelated clinical isolates of CP-Kp, producing VIM-1, VIM-1 plus DHA-1(acquired AmpC β-lactamase), OXA-48 plus CTX-M-15 (extended spectrum β-lactamase) and KPC-3, respectively, with colistin MICs of 0.5, 64, 0.5, and 32 mg/L, respectively. The efficacies of antimicrobials in monotherapy and in combination were tested in a murine peritoneal sepsis model, against all the CP-Kp. Their efficacies were tested in the pneumonia model against the OXA-48 plus CTX-M-15 producers. The development of colistin-resistance was analyzed for the colistin-susceptible strains in vitro and in vivo. In vitro, colistin plus rifampin was synergistic against all the strains at 24 h. In vivo, compared to the controls, rifampin alone reduced tissue bacterial concentrations against VIM-1 and OXA-48 plus CTX-M-15 strains; CMS plus rifampin reduced tissue bacterial concentrations of these two CP-Kp and of the KPC-3 strain. Rifampin and the combination increased the survival against the KPC-3 strain; in the pneumonia model, the combination also improved the survival. No resistant mutants appeared with the combination. In conclusion, CMS plus rifampin had a low and heterogeneous efficacy in the treatment of severe peritoneal sepsis model due to CP-Kp producing different carbapenemases, increasing survival only against the KPC-3 strain. The combination showed efficacy in the less severe pneumonia model. The combination prevented in vitro and in vivo the development of colistin resistant mutants.

Preventing transmission of carbapenemase-producing, carbapenem-resistant Enterobacteriaceae (CP-CRE) is a public health priority. A phenotype-based definition that reliably identifies CP-CRE while minimizing misclassification of non–CP-CRE could help prevention efforts. To assess possible definitions, we evaluated enterobacterial isolates that had been tested and deemed nonsusceptible to >1 carbapenem at US Emerging Infections Program sites. We determined the number of non-CP isolates that met (false positives) and CP isolates that did not meet (false negatives) the Centers for Disease Control and Prevention CRE definition in use during our study: 30% (94/312) of CRE had carbapenemase genes, and 21% (14/67) of Klebsiella pneumoniae carbapenemase–producing Klebsiella isolates had been misclassified as non-CP. A new definition requiring resistance to 1 carbapenem rarely missed CP strains, but 55% of results were false positive; adding the modified Hodge test to the definition decreased false positives to 12%. This definition should be considered for use in carbapenemase-producing CRE surveillance and prevention.

Despite the relevance of carbapenemase-producing Klebsiella pneumoniae (CP-Kp) infections there are a scarce number of studies to evaluate in vivo the efficacy of combinations therapies. The bactericidal activity of colistin, rifampin, and its combination was studied (time-kill curves) against four clonally unrelated clinical isolates of CP-Kp, producing VIM-1, VIM-1 plus DHA-1(acquired AmpC β-lactamase), OXA-48 plus CTXM-15 (extended spectrum β-lactamase) and KPC-3, respectively, with colistin MICs of 0.5, 64, 0.5, and 32 mg/L, respectively. The efficacies of antimicrobials in monotherapy and in combination were tested in a murine peritoneal sepsis model, against all the CP-Kp. Their efficacies were tested in the pneumonia model against the OXA-48 plus CTX-M-15 producers. The development of colistin-resistance was analyzed for the colistin-susceptible strains in vitro and in vivo. In vitro, colistin plus rifampin was synergistic against all the strains at 24 h. In vivo, compared to the controls, rifampin alone reduced tissue bacterial concentrations against VIM-1 and OXA-48 plus CTXM-15 strains; CMS plus rifampin reduced tissue bacterial concentrations of these two CP-Kp and of the KPC-3 strain. Rifampin and the combination increased the survival against the KPC-3 strain; in the pneumonia model, the combination also improved the survival. No resistant mutants appeared with the combination. In conclusion, CMS plus rifampin had a low and heterogeneous efficacy in the treatment of severe peritoneal sepsis model due to CP-Kp producing different carbapenemases, increasing survival only against the KPC-3 strain. The combination showed efficacy in the less severe pneumonia model. The combination prevented in vitro and in vivo the development of colistin resistant mutants. [ABSTRACT FROM AUTHOR]

During the last decade, antimicrobial resistance in bacteria from food-producing animals has become a major research topic. In this review, different emerging resistance properties related to bacteria of food-producing animals are highlighted. These include: extended-spectrum β-lactamase-producing Enterobacteriaceae; carbapenemase-producing bacteria; bovine respiratory tract pathogens, such as Pasteurella multocida and Mannheimia haemolytica, which harbor the multiresistance mediating integrative and conjugative element ICE Pmu1; Gram-positive and Gram-negative bacteria that carry the multiresistance gene cfr; and the occurrence of numerous novel antimicrobial resistance genes in livestock-associated methicillin-resistant Staphylococcus aureus. The emergence of the aforementioned resistance properties is mainly based on the exchange of mobile genetic elements that carry the respective resistance genes. [ABSTRACT FROM AUTHOR]