The emergence of carbapenem-resistant Gram-negative pathogens poses a serious threat to public health worldwide. In particular, the increasing prevalence of carbapenem-resistant Klebsiella pneumoniae is a major source of concern. Klebsiella pneumoniae carbapenemases (KPC) and carbapenemases of the oxacillinase-48 (OXA-48) type have been reported worldwide. New Delhi metallo--lactamase (NDM) carbapenemases were originally identified in Sweden in 2008 and have spread worldwide rapidly. In this review, we summarize the epidemiology of K. pneumoniae producing three carbapenemases (KPCs, NDMs, and OXA-48-like). Although the prevalence of each resistant strain varies geographically, K. pneumoniae producing KPCs, NDMs, and OXA-48-like carbapenemases have become rapidly disseminated. In addition, we used recently published molecular and genetic studies to analyze the mechanisms by which these three carbapenemases, and major K. pneumoniae clones, such as ST258 and ST11, have become globally prevalent. Because carbapenemase-producing K. pneumoniae are often resistant to most -lactam antibiotics and many other non--lactam molecules, the therapeutic options available to treat infection with these strains are limited to colistin, polymyxin B, fosfomycin, tigecycline, and selected aminoglycosides. Although combination therapy has been recommended for the treatment of severe carbapenemase-producing K. pneumoniae infections, the clinical evidence for this strategy is currently limited, and more accurate randomized controlled trials will be required to establish the most effective treatment regimen. Moreover, because rapid and accurate identification of the carbapenemase type found in K. pneumoniae may be difficult to achieve through phenotypic antibiotic susceptibility tests, novel molecular detection techniques are currently being developed.

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Global Dissemination of Carbapenemase-Producing Klebsiella pneumoniae: Epidemiology, Genetic Context, Treatment Options, and Detection Methods

Carbapenems, once considered the last line of defense against of serious infections with Enterobacteriaceae, are threatened with extinction. The increasing isolation of carbapenem-resistant Gram-negative pathogens is forcing practitioners to rely on uncertain alternatives. As little as five years ago, reports of carbapenem resistance in Enterobacteriaceae, common causes of both community and healthcare-associated infections, were sporadic and primarily limited to case reports, tertiary care centers, intensive care units, and outbreak settings. Carbapenem resistance mediated by -lactamases, or carbapenemases, has become widespread and with the paucity of reliable antimicrobials available or in development, international focus has shifted to early detection and infection control. However, as reports of Klebsiella pneumoniae carbapenemases (KPCs), New Delhi metallo--lactamase (NDM-1), and more recently OXA-48 become more common and with the conveniences of travel, the assumption that infections with highly resistant Gram-negative pathogens are limited to the infirmed and the heavily antibiotic and healthcare exposed are quickly being dispelled. Herein, we provide a status report describing the increasing challenges clinicians are facing and forecast the "stormy waters" ahead.

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“Stormy waters ahead”: global emergence of carbapenemases

In 2012, the European Centre for Disease Prevention and Control (ECDC) launched the 'European survey of carbapenemase-producing Enterobacteriaceae (EuSCAPE)' project to gain insights into the occurrence and epidemiology of carbapenemase-producing Enterobacteriaceae (CPE), to increase the awareness of the spread of CPE, and to build and enhance the laboratory capacity for diagnosis and surveillance of CPE in Europe. Data collected through a post-EuSCAPE feedback questionnaire in May 2015 documented improvement compared with 2013 in capacity and ability to detect CPE and identify the different carbapenemases genes in the 38 participating countries, thus contributing to their awareness of and knowledge about the spread of CPE. Over the last two years, the epidemiological situation of CPE worsened, in particular with the rapid spread of carbapenem-hydrolysing oxacillinase-48 (OXA-48)- and New Delhi metallo-beta-lactamase (NDM)-producing Enterobacteriaceae. In 2015, 13/38 countries reported inter-regional spread of or an endemic situation for CPE, compared with 6/38 in 2013. Only three countries replied that they had not identified one single case of CPE. The ongoing spread of CPE represents an increasing threat to patient safety in European hospitals, and a majority of countries reacted by establishing national CPE surveillances systems and issuing guidance on control measures for health professionals. However, 14 countries still lacked specific national guidelines for prevention and control of CPE in mid-2015.

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Carbapenemase-producing Enterobacteriaceae in Europe: assessment by national experts from 38 countries, May 2015

OXA-48 beta-lactamase producers are emerging as an important threat mostly in the Mediterranean area. We report here the molecular epidemiology of a collection of OXA-48 beta-lactamase-positive enterobacterial isolates (n=107) recovered from European and north-African countries between January 2001 and December 2011. This collection included 67 Klebsiella pneumoniae, 24 Escherichia coli and 10 Enterobacter cloacae. Using the EUCAST breakpoints, ninety-eight isolates (91.6%) were of intermediate susceptibility or resistant to ertapenem, whereas 66% remained susceptible to imipenem. Seventy-five per cent of the isolates co-produced an extended-spectrum beta-lactamase, most frequently CTX-M-15 (77.5%). Susceptibility testing to non-beta-lactam antibiotics showed that colistin, tigecycline, amikacin, and fosfomycin remain active against most of the isolates. Multilocus sequence typing indicated that the most common sequence types (ST) were ST101 and ST38 for K. pneumoniae and E. coli, respectively. The bla(OXA-48) gene was located on a 62 kb IncL/M plasmid in 92.5% of the isolates, indicating that a single plasmid was mainly responsible for the spread of that gene. In addition, this study identified multiple cases of importation of OXA-48 beta-lactamase producers at least in Europe, and spread of OXA-48 beta-lactamase producers giving rise to an endemic situation, at least in France.

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Intercontinental spread of OXA-48 beta-lactamase-producing Enterobacteriaceae over a 11-year period, 2001 to 2011.

SUMMARY Surveillance studies have shown that OXA-48-like carbapenemases are the most common carbapenemases in Enterobacterales in certain regions of the world and are being introduced on a regular basis into regions of nonendemicity, where they are responsible for nosocomial outbreaks. OXA-48, OXA-181, OXA-232, OXA-204, OXA-162, and OXA-244, in that order, are the most common enzymes identified among the OXA-48-like carbapenemase group. OXA-48 is associated with different Tn1999 variants on IncL plasmids and is endemic in North Africa and the Middle East. OXA-162 and OXA-244 are derivatives of OXA-48 and are present in Europe. OXA-181 and OXA-232 are associated with ISEcp1, Tn2013 on ColE2, and IncX3 types of plasmids and are endemic in the Indian subcontinent (e.g., India, Bangladesh, Pakistan, and Sri Lanka) and certain sub-Saharan African countries. Overall, clonal dissemination plays a minor role in the spread of OXA-48-like carbapenemases, but certain high-risk clones (e.g., Klebsiella pneumoniae sequence type 147 [ST147], ST307, ST15, and ST14 and Escherichia coli ST38 and ST410) have been associated with the global dispersion of OXA-48, OXA-181, OXA-232, and OXA-204. Chromosomal integration of blaOXA-48 within Tn6237 occurred among E. coli ST38 isolates, especially in the United Kingdom. The detection of Enterobacterales with OXA-48-like enzymes using phenotypic methods has improved recently but remains challenging for clinical laboratories in regions of nonendemicity. Identification of the specific type of OXA-48-like enzyme requires sequencing of the corresponding genes. Bacteria (especially K. pneumoniae and E. coli) with blaOXA-48, blaOXA-181, and blaOXA-232 are emerging in different parts of the world and are most likely underreported due to problems with the laboratory detection of these enzymes. The medical community should be aware of the looming threat that is posed by bacteria with OXA-48-like carbapenemases.

The Global Ascendency of OXA-48-Type Carbapenemases.

Objectives First detected in Enterobacteriaceae isolates in Turkey, the OXA-48 carbapenemase has gradually disseminated in the wider Mediterranean area and Europe. Despite reports from other European regions, until now no such isolates have been detected in Greece. We describe the characteristics of the first outbreak caused by OXA-48-producing Klebsiella pneumoniae in Greece. Methods From December 2011 to March 2012, 13 ertapenem-resistant K. pneumoniae isolates, which were positive by the modified Hodge test while remaining negative by phenotypic screening for metallo-β-lactamase (MBL) and KPC production, were recovered from nine patients. Patient records were retrieved to access patterns of acquisition. Resistance genes were identified by PCR and sequencing. ompK35, ompK36 and the genetic environment of the bla(OXA-48) gene were investigated. Plasmid profiling, conjugation experiments, PFGE and multilocus sequence typing (MLST) were performed. Results All isolates harboured the bla(OXA-48) gene along with the bla(CTX-M-15) and bla(OXA-1) genes. The bla(OXA-48) gene was located on a self-transferable IncL/M-type plasmid of ~62 kb, which harboured no other resistance genes. IS1999 was located upstream of the bla(OXA-48) gene. Genetic disruptions of the ompK35 and ompK36 genes were not detected. The isolates belonged to a unique PFGE clone and MLST assigned them to sequence type ST11. All cases were characterized as hospital acquired and none of them was linked to immigration or history of travel in endemic areas. Conclusions Carbapenem resistance due to MBL and KPC carbapenemases is currently on an endemic scale in Greece and this report highlights the wider undetected dissemination of yet another carbapenemase in this region.

Outbreak of OXA-48 carbapenemase-producing Klebsiella pneumoniae in Greece involving an ST11 clone

Globally, candidemia displays geographical variety in terms of epidemiology and incidence. In that respect, a nationwide Greek study was conducted, reporting the epidemiology of Candida bloodstream infections and susceptibility of isolates to antifungal agents providing evidence for empirical treatment. All microbiologically confirmed candidemia cases in patients hospitalized in 28 Greek centres during the period 2009–2018 were recorded. The study evaluated the incidence of infection/100,000 inhabitants, species distribution, and antifungal susceptibilities of isolated strains. Overall, 6057 candidemic episodes occurred during the study period, with 3% of them being mixed candidemias. The average annual incidence was 5.56/100,000 inhabitants, with significant increase over the years (p = 0.0002). C. parapsilosis species complex (SC) was the predominant causative agent (41%), followed by C. albicans (37%), C. glabrata SC (10%), C. tropicalis (7%), C. krusei (1%), and other rare Candida spp. (4%). C. albicans rates decreased from 2009 to 2018 (48% to 31%) in parallel with a doubling incidence of C. parapsilosis SC rates (28% to 49%, p < 0.0001). Resistance to amphotericin B and flucytosine was not observed. Resistance to fluconazole was detected in 20% of C. parapsilosis SC isolates, with a 4% of them being pan-azole-resistant. A considerable rising rate of resistance to this agent was observed over the study period (p < 0.0001). Echinocandin resistance was found in 3% of C. glabrata SC isolates, with 70% of them being pan-echinocandin-resistant. Resistance rate to this agent was stable over the study period. This is the first multicentre nationwide study demonstrating an increasing incidence of candidemia in Greece with a species shift toward C. parapsilosis SC. Although the overall antifungal resistance rates remain relatively low, fluconazole-resistant C. parapsilosis SC raises concern.

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Increasing Incidence and Shifting Epidemiology of Candidemia in Greece: Results from the First Nationwide 10-Year Survey

Background: Bloodstream infections (BSI) caused by highly resistant pathogens in non-ICU COVID-19 departments pose important challenges. Methods: We performed a comparative analysis of incidence and microbial epidemiology of BSI in COVID-19 vs. non-COVID-19, non-ICU departments between 1 September 2020-31 October 2021. Risk factors for BSI and its impact on outcome were evaluated by a case-control study which included COVID-19 patients with/without BSI. Results: Forty out of 1985 COVID-19 patients developed BSI. The mean monthly incidence/100 admissions was 2.015 in COVID-19 and 1.742 in non-COVID-19 departments. Enterococcus and Candida isolates predominated in the COVID-19 group (p < 0.001 and p = 0.018, respectively). All Acinetobacter baumannii isolates were carbapenem-resistant (CR). In the COVID-19 group, 33.3% of Klebsiella pneumoniae was CR, 50% of Escherichia coli produced ESBL and 19% of Enterococcus spp. were VRE vs. 74.5%, 26.1% and 8.8% in the non-COVID-19 group, respectively. BSI was associated with prior hospitalization (p = 0.003), >2 comorbidities (p < 0.001), central venous catheter (p = 0.015), severe SARS-CoV-2 pneumonia and lack of COVID-19 vaccination (p < 0.001). In the multivariate regression model also including age and multiple comorbidities, only BSI was significantly associated with adverse in-hospital outcome [OR (CI95%): 21.47 (3.86–119.21), p < 0.001]. Conclusions: BSI complicates unvaccinated patients with severe SARS-CoV-2 pneumonia and increases mortality. BSI pathogens and resistance profiles differ among COVID-19/non-COVID-19 departments, suggesting various routes of pathogen acquisition.

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Bloodstream Infections in a COVID-19 Non-ICU Department: Microbial Epidemiology, Resistance Profiles and Comparative Analysis of Risk Factors and Patients’ Outcome

Objectives The aim of this study was to examine the performance of the chromogenic β LACTA™ test for the rapid detection of expanded-spectrum cephalosporin (ESC) non-susceptibility among Enterobacteriaceae in a region endemic for potent β-lactamases. Methods The β LACTA™ test was applied prospectively on 235 consecutive Enterobacteriaceae clinical isolates and 163 previously characterised ESC-non-susceptible Enterobacteriaceae producing a range of β-lactamases. Results The β LACTA™ test exhibited excellent sensitivity (96.1%) and specificity (98.5%) for the detection of ESC non-susceptibility in the 235 clinical isolates, which harboured mainly extended-spectrum β-lactamase (ESBL) and KPC- and NDM-type enzymes. Among the 163 challenged archived isolates, some false-negative or uninterpretable results were detected, mostly among ESC-non-susceptible isolates with AmpC/VIM/OXA-48-like enzymes. Conclusion β LACTA™ may be effectively applied in regions where ESC non-susceptibility among Enterobacteriaceae is mainly due to ESBL, KPC or NDM β-lactamases.

Performance of the β LACTA™ test for rapid detection of expanded-spectrum cephalosporin-non-susceptible Enterobacteriaceae

Isolated post-traumatic cerebral mucormycosis represents an extremely rare and severe disease. A case of isolated cerebral mucormycosis infection caused by Lichtheimia spp. in a 21-year-old multi-trauma patient is presented. The patient was hospitalized in the intensive care unit and underwent craniotomy due to brain injuries. Two weeks following the initial procedure, pus drained from the surgical wound was microscopically examined and cultured, yielding Lichtheimia spp. Imaging showed parietal, temporal and frontal abscesses at the right side. The patient was commenced on amphotericin B and underwent surgical debridement, while histopathological examination of the affected tissue demonstrated broad, aseptate hyphae, findings typical for mucormycetes. The patient passed away due to heavy traumatic injuries after 2 months. It is speculated that direct inoculation was the portal of entry for infection, and that high steroid use for 2 weeks following inoculation contributed to the severity of infection that developed. Isolated cerebral mucormycosis in immunocompetent hosts is an extremely rare, but severe disease. Diagnosis is established through direct microscopy, histopathology and/or cultures. PCR-based techniques are useful either to detect mucormycetes in tissues, especially when cultures are negative, or to accurately identify the fungi grown in cultures at the species level. A high suspicion index, especially in the necrotic lesions of traumas, is of the utmost importance for early diagnosis. Appropriate surgical debridement, as well as antifungal therapy, including amphotericin B, represents the treatment of choice.

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Vasiliki Mamali

Papers

Outbreak of OXA-48 carbapenemase-producing Klebsiella pneumoniae in Greece involving an ST11 clone

Increasing Incidence and Shifting Epidemiology of Candidemia in Greece: Results from the First Nationwide 10-Year Survey

Bloodstream Infections in a COVID-19 Non-ICU Department: Microbial Epidemiology, Resistance Profiles and Comparative Analysis of Risk Factors and Patients’ Outcome

Performance of the β LACTA™ test for rapid detection of expanded-spectrum cephalosporin-non-susceptible Enterobacteriaceae

Isolated Cerebral Mucormycosis Caused by Lichtheimia Species in a Polytrauma Patient