Fast microbiological diagnostics (MDx) are needed to ensure early targeted antimicrobial treatment in sepsis. This systematic review focuses on the impact on antimicrobial management and patient outcomes of MDx for pathogen and resistance gene identification compared with blood cultures. PubMed was searched for clinical studies using either whole blood directly or after short-term incubation. Twenty-five articles were retrieved describing the outcomes of 8 different MDx. Three interventional studies showed a significant increase in appropriateness of antimicrobial therapy and a nonsignificant change in time to appropriate therapy. Impact on mortality was conflicting. Length of stay was significantly lower in 2 studies. A significant decrease in antimicrobial cost was demonstrated in 6 studies. The limitations of this systematic review include the low number and observed heterogeneity of clinical studies. In conclusion, potential benefits of MDx regarding antimicrobial management and some patient outcomes were reported. More rigorous intervention studies are needed focusing on the direct benefits for patients.

Urinary tract infections (UTIs) are the most frequent hospital infections and among the most commonly observed community acquired infections. Alongside their clinical importance, they are notorious because the pathogens that cause them are prone to acquiring various resistance determinants, including extended-spectrum beta-lactamases (ESBL); plasmid-encoded AmpC β-lactamases (p-AmpC); carbapenemases belonging to class A, B, and D; qnr genes encoding reduced susceptibility to fluoroquinolones; as well as genes encoding enzymes that hydrolyse aminoglycosides. In Escherichia coli and Klebsiella pneumoniae, the dominant resistance mechanisms are ESBLs belonging to the CTX-M, TEM, and SHV families; p-AmpC; and (more recently) carbapenemases belonging to classes A, B, and D. Urinary Pseudomonas aeruginosa isolates harbour metallo-beta-lactamases (MBLs) and ESBLs belonging to PER and GES families, while carbapenemases of class D are found in urinary Acinetobacter baumannii isolates. The identification of resistance mechanisms in routine diagnostic practice is primarily based on phenotypic tests for the detection of beta-lactamases, such as the double-disk synergy test or Hodge test, while polymerase chain reaction (PCR) for the detection of resistance genes is mostly pursued in reference laboratories for research purposes. As the emergence of drug-resistant bacterial strains poses serious challenges in the management of UTIs, this review aimed to appraise mechanisms of resistance in relevant Gram-negative urinary pathogens, to provide a detailed map of resistance determinants in Croatia and the world, and to discuss the implications of these resistance traits on diagnostic approaches. We summarized a sundry of different resistance mechanisms among urinary isolates and showed how their prevalence highly depends on the local epidemiological context, highlighting the need for tailored interventions in the field of antimicrobial stewardship.

The management of infectious diseases has proven to be a daunting task for clinicians worldwide, and the rapid development of antibiotic resistance among Gram-negative bacteria is making it even more challenging. The first-line therapy is empirical, and it most often comprises β-lactam antibiotics. Among Gram-negative bacteria, Proteus mirabilis, an important community and hospital pathogen associated primarily with urinary tract and wound infection, holds a special place. This review’s aim was to collate and examine recent studies investigating β-lactam resistance phenotypes and mechanisms of Proteus species and the global significance of its β-lactam resistance evolution. Moreover, the genetic background of resistance traits and the role of mobile genetic elements in the dissemination of resistance genes were evaluated. P. mirabilis as the dominant pathogen develops resistance to expanded-spectrum cephalosporins (ESC) by producing extended-spectrum β-lactamases (ESBL) and plasmid-mediated AmpC β-lactamases (p-AmpC). β-lactamase-mediated resistance to carbapenems in Enterobacterales, including Proteus spp., is mostly due to expression of carbapenemases of class A (KPC); class B (metallo-β-lactamases or MBLs of IMP, VIM, or NDM series); or class D or carbapenem-hydrolyzing oxacillinases (CHDL). Previously, a dominant ESBL type in P. mirabilis was TEM-52; yet, lately, it has been replaced by CTX-M variants, particularly CTX-M-14. ESC resistance can also be mediated by p-AmpC, with CMY-16 as the dominant variant. Carbapenem resistance in Proteus spp. is a challenge due to its intrinsic resistance to colistin and tigecyclin. The first carbapenemases reported belonged to class B, most frequently VIM-1 and NDM-5. In Europe, predominantly France and Belgium, a clonal lineage positive for OXA-23 CHDL spreads rapidly undetected, due to its low-level resistance to carbapenems. The amazing capacity of Proteus spp. to accumulate a plethora of various resistance traits is leading to multidrug or extensively drug-resistant phenotypes.

Resistance to carbapenems in Enterobacterales has become a matter of the highest concern in the last decade. Recently, Enterobacterales harboring multiple carbapenemases were detected in three hospital centers in Croatia and in the outpatient setting, posing a serious therapeutic challenge for clinicians. In this study, we analyzed eight Klebsiella pneumoniae and two Enterobacter cloacae complex isolates with multiple carbapenemases, with regard to antibiotic susceptibility, β-lactamase production and plasmid content. The isolates demonstrated uniform resistance to amoxicillin/clavulanate, piperacillin/tazobactam, cefuroxime, ceftazidime, cefotaxime, ceftriaxone and ertapenem. Among novel β-lactam/inhibitor combinations, ceftazidime/avibactam exhibited moderate activity, with 50% of isolates susceptible. All isolates demonstrated resistance to imipenem/cilastatin/relebactam, and all but one to ceftolozane/tazobactam. Four isolates exhibited a multidrug-resistant phenotype (MDR), whereas six were allocated to an extensively drug-resistant phenotype (XDR). OKNV detected three combinations of carbapenemases: OXA-48+NDM (five isolates), OXA-48+VIM (three isolates) and OXA-48+KPC (two isolates). Inter-array testing identified a wide variety of resistance genes for β-lactam antibiotics: bla_CTX-M-15, bla_TEM, bla_SHV, bla_OXA-1, bla_OXA-2, bla_OXA-9, aminoglycosides: aac6, aad, rmt, arm and aph, fluoroquinolones: qnrA, qnrB and qnrS, sulphonamides: sul1 and sul2 and trimethoprim: dfrA5, dfrA7, dfrA14, dfrA17 and dfrA19. mcr genes were reported for the first time in Croatia. This study demonstrated the ability of K. pneumoniae and E. cloacae to acquire various resistance determinants under the selection pressure of antibiotics widely used during the COVID-19 pandemic. The novel inter-array method showed good correlation with OKNV and PCR, although some discrepancies were found.

Ventilator-associated pneumonia (VAP) is a serious and common complication in patients admitted to intensive care unit (ICU) and contributes to mortality. Multidrug Gram-negative bacteria such as Acinetobacter baumannii, Pseudomonas aeruginosa, and Klebsiella pneumoniae are frequently associated with VAP in ICU. A prospective study was set up in three ICUs of the University Hospital Center Zagreb and one ICU in General Hospital Pula from September 2017 to March 2018. Antibiotic susceptibility was determined by broth microdilution method. Production of extended-spectrum β-lactamases (ESBLs) was determined by double-disk synergy test and carbapenemases by Hodge and carbapenem inactivation method (CIM). The genes encoding ESBLs, carbapenemases of class A, B and D and qnr genes were determined by PCR. In total 97 Gram-negative bacteria isolates were analyzed. P. aeruginosa demonstrated high resistance rates for imipenem and meropenem with 74% and 68% of resistant strains, respectively. Moderate resistance rates were observed for ceftazidime andpiperacillin/tazobactam, ciprofloxacin and gentamicin (44%). All except three A. baumannii isolates, were resistant to carbapenems and to all other antibiotics apart from colistin and amikacin. Eight A. baumannii isolates were positive for bla _OXA-23 and 12 for bla _OXA-24 genes. Four K. pneumoniae and two E. cloacae strains were ESBL positive and harboured group 1 of CTX-M β-lactamases. Three P. mirabilis strains were positive for plasmid-mediated ampC β-lactamase of CMY family. Two carbapenem-resistant K. pneumoniae harboured OXA-48 and one carbapenem-resistant E. cloacae VIM-1. A high proportion of multidrug-resistant P. aeruginosa, K. pneumoniae and extensively resistant A. baumannii was reported. Acquired resistance mechanisms, mainly production of carbapenemases and ESBLs were dominant in A. baumannii and K. pneumoniae, respectively. Resistance of P. aeruginosa isolates was more likely due to upregulation of efflux pumps or porin loss. A marked diversity of β-lactamases was identified in Enterobacteriaceae.