Safety issues in semen banks during the COVID-19 pandemic: data from a European survey.

PURPOSE: We performed a survey among European semen banks enquiring safety protocols during the COVID-19 pandemic. We report the experience from a center searching SARS-CoV-2 mRNA in semen of patients undergoing cryopreservation from May 2020 to January 2021. METHODS: A questionnaire was submitted to accredited semen banks of the European Academy of Andrology (EAA) and the Italian Society of Andrology and Sexual Medicine (SIAMS). A total of 22 centers answered to the survey. SARS-CoV-2 mRNA in semen was evaluated by RT-PCR in 111 subjects banking in the Semen Bank of Careggi University Hospital (Florence, Italy). RESULTS: No particularly drastic safety measures were adopted by the majority of the centers to prevent the risk of contamination or transmission of the virus. The most common strategy (77.3%) was the administration of an anamnestic questionnaire. About half of the centers request a negative nasopharyngeal swab (NPS) before cryopreservation. Few centers use a quarantine tank, in case of late response of NPS, and only 4 store in a dedicated tank in case of infection. SARS-CoV-2 mRNA was not found in 111 semen samples cryopreserved in the Florentine bank. CONCLUSIONS: European semen banks use different measures to handle semen samples for cryopreservation during COVID-19 pandemic. The request of NPS is advised to better manage couples undergoing ART and to protect the personnel operating in the bank/ART center. Finally, due to the areas of uncertainties of an almost unknown virus, it is absolutely recommended the use of safe devices for sample handling and storage.

Semen impairment and occurrence of SARS-CoV-2 virus in semen after recovery from COVID-19.

STUDY QUESTION: How is the semen quality of sexually active men following recovery from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection? SUMMARY ANSWER: Twenty-five percent of the men with recent SARS-Cov-2 infections and proven healing were oligo-crypto-azoospermic, despite the absence of virus RNA in semen. WHAT IS KNOWN ALREADY: The presence of SARS-CoV-2 in human semen and its role in virus contagion and semen quality after recovery from coronavirus disease 2019 (COVID-19) is still unclear. So far, studies evaluating semen quality and the occurrence of SARS-CoV-2 in semen of infected or proven recovered men are scarce and included a limited number of participants. STUDY DESIGN, SIZE, DURATION: A prospective cross-sectional study on 43 sexually active men who were known to have recovered from SARS-CoV2 was performed. Four biological fluid samples, namely saliva, pre-ejaculation urine, semen, and post-ejaculation urine, were tested for the SARS-CoV-2 genome. Female partners were retested if any specimen was found to be SARS-CoV-2 positive. Routine semen analysis and quantification of semen leukocytes and interleukin-8 (IL-8) levels were performed. PARTICIPANTS/MATERIALS, SETTING, METHODS: Questionnaires including International Index of Erectile Function and Male Sexual Health Questionnaire Short Form were administered to all subjects. The occurrence of virus RNA was evaluated in all the biological fluids collected by RT-PCR. Semen parameters were evaluated according to the World Health Organization manual edition V. Semen IL-8 levels were evaluated by a two-step ELISA method. MAIN RESULTS AND THE ROLE OF CHANCE: After recovery from COVID-19, 25% of the men studied were oligo-crypto-azoospermic. Of the 11 men with semen impairment, 8 were azoospermic and 3 were oligospermic. A total of 33 patients (76.7%) showed pathological levels of IL-8 in semen. Oligo-crypto-azoospermia was significantly related to COVID-19 severity (P < 0.001). Three patients (7%) tested positive for at least one sample (one saliva; one pre-ejaculation urine; one semen and one post-ejaculation urine), so the next day new nasopharyngeal swabs were collected. The results from these three patients and their partners were all negative for SARS-CoV-2. LIMITATIONS, REASONS FOR CAUTION: Although crypto-azoospermia was found in a high percentage of men who had recovered from COVID-19, clearly exceeding the percentage found in the general population, the previous semen quality of these men was unknown nor is it known whether a recovery of testicular function was occurring. The low number of enrolled patients may limit the statistical power of study. WIDER IMPLICATIONS OF THE FINDINGS: SARS-CoV-2 can be detected in saliva, urine, and semen in a small percentage of men who recovered from COVID-19. One-quarter of men who recovered from COVID-19 demonstrated oligo-crypto-azoospermia indicating that an assessment of semen quality should be recommended for men of reproductive age who are affected by COVID-19. STUDY FUNDING/COMPETING INTEREST(S): None. TRIAL REGISTRATION NUMBER: N/A.

Synergistic activity of synthetic N-terminal peptide of human lactoferrin in combination with various antibiotics against carbapenem-resistant Klebsiella pneumoniae strains.

The spread of multi-drug resistant (MDR) Klebsiella pneumoniae strains producing carbapenemases points to a pressing need for new antibacterial agents. To this end, the in-vitro antibacterial activity of a synthetic N-terminal peptide of human lactoferrin, further referred to as hLF1-11, was evaluated against K. pneumoniae strains harboring different carbapenemase genes (i.e. OXA-48, KPC-2, KPC-3, VIM-1), with different susceptibility to colistin and other antibiotics, alone or in combination with conventional antibiotics (gentamicin, tigecycline, rifampicin, clindamycin, and clarithromycin). An antimicrobial peptide susceptibility assay was used to assess the bactericidal activity of hLF1-11 against the different K. pneumoniae strains tested. The synergistic activity was evaluated by a checkerboard titration method, and the fractional inhibitory concentration (FIC) index was calculated for the various combinations. hLF1-11 was more efficient in killing a K. pneumoniae strain susceptible to most antimicrobials (including colistin) than a colistin-susceptible strain and a colistin-resistant MDR K. pneumoniae strain. In addition, hLF1-11 exhibited a synergistic effect with the tested antibiotics against MDR K. pneumoniae strains. The results of this study indicate that resistance to hLF1-11 and colistin are not strictly associated, and suggest an hLF1-11-induced sensitizing effect of K. pneumoniae to antibiotics, especially to hydrophobic antibiotics, which are normally not effective on Gram-negative bacteria. Altogether, these data indicate that hLF1-11 in combination with antibiotics is a promising candidate to treat infections caused by MDR-K. pneumoniae strains.

Non-tuberculous mycobacteria: epidemiological pattern in a reference laboratory and risk factors associated with pulmonary disease.

The diseases caused by non-tuberculous mycobacteria (NTM), in both AIDS and non-AIDS populations, are increasingly recognized worldwide. Although the American Thoracic Society published the guidelines for diagnosis of NTM pulmonary disease (NTM-PD), the diagnosis is still difficult. In the first part of the study, we collected data on NTM isolates in the Mycobacteriology Laboratory of Careggi Hospital (Florence, Italy) and analysed the epidemiological data of NTM isolates. Then, to analyse the risk factors associated to NTM-PD, we studied the presence of ATS/IDSA criteria for NTM-PD in patients who had at least one positive respiratory sample for NTM and were admitted to the Infectious Disease Unit and the Section of Respiratory Medicine. We selected 88 patients with available full clinical data and, according to ATS/IDSA criteria, classified 15 patients (17%) as NTM-PD cases and 73 as colonized patients (83%). When comparing colonized and NTM-PD patients we did not find significant differences of age, gender and comorbidity. We observed that Mycobacterium avium and M. intracellulare were statistically associated with NTM-PD (P = 0·001) whereas M. xenopi was statistically associated with colonization. Although the number of studied patients is limited, our study did not identify risk factors for NTM-PD that could help clinicians to discriminate between colonization and disease. We underline the need of close monitoring of NTM-infected patients until the diagnosis is reasonably excluded. Further larger prospective studies and new biological markers are needed to identify new useful tools for the diagnosis of NTM-PD.

Colistin resistance superimposed to endemic carbapenem-resistant Klebsiella pneumoniae: a rapidly evolving problem in Italy, November 2013 to April 2014.

Consecutive non-replicate clinical isolates (n=191) of carbapenem non-susceptible Enterobacteriaceae were collected from 21 hospital laboratories across Italy from November 2013 to April 2014 as part of the European Survey on Carbapenemase-producing Enterobacteriaceae (EuSCAPE) project. Klebsiella pneumonia carbapenemase-producing K. pneumoniae (KPC-KP) represented 178 (93%) isolates with 76 (43%) respectively resistant to colistin, a key drug for treating carbapenamase-producing Enterobacteriaceae. KPC-KP colistin-resistant isolates were detected in all participating laboratories. This underscores a concerning evolution of colistin resistance in a setting of high KPC-KP endemicity.

Effects of saline nebulization on SARS-CoV-2 RNA spreading and exhaled bio-aerosol particles in COVID-19 patients.

BACKGROUND: Nebulized therapy is the mainstay for treating obstructive airway diseases, but there is heightened concern about the potential risk for SARS-CoV-2 transmission during nebulization in COVID-19 patients. AIM: To investigate the effects of 0.9% saline nebulization on SARS-CoV-2 RNA spreading in 11 COVID-19 patients (five females, mean age 62.45 ± 9.31 years); also to ascertain whether saline nebulization changed the number of exhaled bio-aerosol particles in six out of the 11 patients. METHODS: Air samples were collected using suction pumps equipped with 0.45 µm PTFE filters and positioned around the patient's bed. Exhaled particles were quantified by using an optical particle counter. FINDINGS: At baseline (i.e. before nebulization) SARS-CoV-2 was detected more frequently in the pumps close to the patient than in those far away. After saline nebulization, the detection of SARS-CoV-2 in the pumps close to the patient was comparable to that observed at baseline. In the pumps far from the patient, saline nebulization slightly, but not significantly, increased SARS-CoV-2 RNA detection compared to baseline. Overall, no significant changes in the SARS-CoV-2 RNA detection were observed after saline nebulization. At baseline, exhaled particle emission varied among patients, with two of them showing higher emission of particles than the remaining patients. Saline nebulization induced a marked decrease in exhaled particles in the two patients who displayed high emission at baseline, whereas no changes were observed in the remaining patients. Saline nebulization did not significantly change SARS-CoV-2 RNA spreading. CONCLUSION: Saline nebulization does not significantly increase SARS-CoV-2 spreading.

Epidemic diffusion of KPC carbapenemase-producing Klebsiella pneumoniae in Italy: results of the first countrywide survey, 15 May to 30 June 2011.

Carbapenem-resistant Enterobacteriaceae (CRE) are emerging as a public health problem in various settings. In Italy, a rapid and remarkable increase of carbapenem-non-susceptible Klebsiella pneumoniae has been reported since 2010. Here we report on the results of a countrywide cross-sectional survey, carried out from 15 May to 30 June 2011 to investigate the diffusion of CRE in Italy and to characterise the most prevalent resistance mechanisms and their dissemination patterns. CRE were reported from most (23 of 25) participating laboratories, with an overall proportion of 3.5% and 0.3% among consecutive non-duplicate clinical isolates of Enterobacteriaceae from inpatients (n=7,154) and outpatients (n=6,595), respectively. K. pneumoniae was the most frequent species (proportion of carbapenem-non-susceptible isolates: 11.9%), while a minority of CRE of other species were detected. Carbapenemase production was detected in the majority (85%) of CRE. KPC-type enzymes were by far the most common (89.5% of carbapenemase producers), followed by VIM-1 (9.2%) and OXA-48 (1.3%). KPC-producing K. pneumoniae (KPC-KP) were detected in most centres and contributed majorly to the epidemic dissemination of CRE recently observed in our country. Dissemination of KPC-KP was mostly sustained by strains of clonal complex 258 (ST-258 producing KPC-2 or KPC-3, and ST-512 producing KPC-3), while a minority belonged to ST-101.

Carbapenemase-producing Enterobacteriaceae in Europe: a survey among national experts from 39 countries, February 2013.

The spread of carbapenemase-producing Enterobacteriaceae (CPE) is a threat to healthcare delivery, although its extent differs substantially from country to country. In February 2013, national experts from 39 European countries were invited to self-assess the current epidemiological situation of CPE in their country. Information about national management of CPE was also reported. The results highlight the urgent need for a coordinated European effort on early diagnosis, active surveillance, and guidance on infection control measures.

Rationale and evidence for the adjunctive use of N-acetylcysteine in multidrug-resistant infections.

Bacterial multidrug resistance has been a serious issue for healthcare systems in recent decades, responsible for many infections and deaths. Due to the increasing incidence of antimicrobial resistance and scarce treatment options, research is focused on finding possible therapeutic adjuvants able to increase the efficacy of antibiotics. The aim of this article is a review of available evidence on the use of N-acetylcysteine (NAC). MEDLINE/PubMed was searched for appropriate keywords. In vitro and in vivo preclinical studies, clinical studies, reviews, and meta-analyses were retrieved and selected based on relevance. A narrative review article was written, reporting published evidence and the expert opinion of the authors. Among possible adjunctive treatments, NAC has attracted the interest of researchers as a candidate for re-purposing. It is a widely used drug with a good tolerability profile, mainly used as a mucolytic agent, with antioxidant, anti-inflammatory properties and antibacterial activity. NAC acts on different mechanisms and stages of infections, resulting in inhibition of biofilm formation, disruption of preformed biofilms, and reduction of bacterial viability. NAC may be administered as an aerosol in many types of infections, including cystic fibrosis, bronchiectasis and infective flare of chronic obstructive pulmonary disease (COPD), and by the intravenous route in severe systemic infections (including septic shock) such as those caused by carbapenemase (KPC)-producing Klebsiella pneumoniae (Kp) and Carbapenem-Resistant Acinetobacter baumannii (CR-Ab). A rationale exists for using NAC as an adjunctive treatment in multidrug-resistant (MDR) infections, based on in vitro, in vivo and clinical evidence, and future research is needed to identify candidate patients and optimal schedules for specific clinical conditions.

Evaluation of VITEK® 2 AST cards (AST-N376 and AST-N397) for susceptibility testing of challenging Gram negatives.

Due to the increasing diffusion of MDR/XDR Gram-negatives it is necessary to offer reliable antibiotic susceptibility testing (AST), which also include new drugs. Here we evaluated the performances of the VITEK®2 AST-N376 and the AST-N397 cards. A collection of 180 clinical Gram-negative bacteria, producing relevant resistance mechanisms, were tested using VITEK 2 and MERLIN, in parallel. Discrepancies between the 2 systems were solved by the reference broth microdilution method. The workflow timing of the VITEK®2 system was also assessed. Overall, the VITEK®2 cards proved to be reliable in determining AST for the molecules evaluated, even if compliance with ISO acceptance criteria for accuracy assessment was not reached for some combinations and showed a short hands-on time for panels preparation. In conclusion, VITEK®2 is a valid system that ensures accurate results for AST of the molecules evaluated in this study and speeds up the workflow in the laboratory of diagnostic microbiology.

Changing epidemiology of extended-spectrum ß-lactamases in Argentina: emergence of CTX-M-15.

A multicenter survey, carried out in 2010 in Argentina, showed an increased prevalence of extended-spectrum ß-lactamase (ESBL)-producing enterobacteria, with some changes in the molecular epidemiology of circulating ESBLs. While enzymes of the CTX-M-2 group remain endemic, the emergence of CTX-M-15 and of enzymes of the CTX-M-8 and CTX-M-9 groups was observed. The CTX-M-15-positive isolates represented 40% of CTX-M producers and included representatives of Escherichia coli ST131 and Klebsiella pneumoniae ST11.

Carbapenem non-susceptible Klebsiella pneumoniae from Micronet network hospitals, Italy, 2009 to 2012.

Carbapenem-resistant Klebsiella pneumoniae has recently been reported as a new, multidrug-resistant nosocomial pathogen in several hospitals from various Italian regions. Through Micronet, a new Italian sentinel laboratory-based surveillance network, we studied the trend of non-susceptibility of K. pneumoniae to selected carbapenems (imipenem and/or meropenem) in 14 of the 15 hospitals participating in the network. Analysis of data from 1 January 2009 to 30 April 2012 revealed a statistically significant increasing trend (p<0.01) in the proportion of carbapenem non-susceptible K. pneumoniae isolates from clinical specimens (from 2.2 % in 2009 to 19.4% in 2012). The increase in the proportion of non-susceptibility was very large for isolates from the respiratory tract (from 5.3% in 2009 to 38.5% in 2012) and blood (from 5.4% in 2009 to 29.2% in 2012). The results demonstrate the urgent need in Italy for infection control, guidelines, antibiotic stewardship programmes and utilisation of surveillance systems, such as Micronet, which are capable of receiving data from hospitals in real time for many pathogens and types of clinical specimens.

Evolution and spread of a multidrug-resistant Proteus mirabilis clone with chromosomal AmpC-type cephalosporinases in Europe.

Proteus mirabilis isolates obtained in 1999 to 2008 from three European countries were analyzed; all carried chromosomal AmpC-type cephalosporinase bla(CMY) genes from a Citrobacter freundii origin (bla(CMY-2)-like genes). Isolates from Poland harbored several bla(CMY) genes (bla(CMY-4), bla(CMY-12), bla(CMY-14), bla(CMY-15), and bla(CMY-38) and the new gene bla(CMY-45)), while isolates from Italy and Greece harbored bla(CMY-16) only. Earlier isolates with bla(CMY-4) or bla(CMY-12), recovered in France from Greek and Algerian patients, were also studied. All isolates showed striking similarities. Their bla(CMY) genes resided within ISEcp1 transposition modules, named Tn6093, characterized by a 110-bp distance between ISEcp1 and bla(CMY), and identical fragments of both C. freundii DNA and a ColE1-type plasmid backbone. Moreover, these modules were inserted into the same chromosomal site, within the pepQ gene. Since ColE1 plasmids carrying ISEcp1 with similar C. freundii DNA fragments (Tn6114) had been identified earlier, it is likely that a similar molecule had mediated at some stage this DNA transfer between C. freundii and P. mirabilis. In addition, isolates with bla(CMY-12), bla(CMY-15), and bla(CMY-38) genes harbored a second bla(CMY) copy within a shorter ISEcp1 module (Tn6113), always inserted downstream of the ppiD gene. Sequence analysis of all mobile bla(CMY-2)-like genes indicated that those integrated in the P. mirabilis chromosome form a distinct cluster that may have evolved by the stepwise accumulation of mutations. All of these observations, coupled to strain typing data, suggest that the bla(CMY) genes studied here may have originated from a single ISEcp1-mediated mobilization-transfer-integration process, followed by the spread and evolution of a P. mirabilis clone over time and a large geographic area.

Escherichia coli and Staphylococcus aureus: bad news and good news from the European Antimicrobial Resistance Surveillance Network (EARS-Net, formerly EARSS), 2002 to 2009.

Based on data collected by the European Antimicrobial Resistance Surveillance Network (EARS-Net) and the former EARSS, the present study describes the trends in antimicrobial susceptibility patterns and occurrence of invasive infections caused by Escherichia coli and Staphylococcus aureus in the period from 2002 to 2009. Antimicrobial susceptibility results from 198 laboratories in 22 European countries reporting continuously on these two microorganisms during the entire study period were included in the analysis. The number of bloodstream infections caused by E. coli increased remarkably by 71% during the study period, while bloodstream infections caused by S. aureus increased by 34%. At the same time, an alarming increase of antimicrobial resistance in E. coli was observed, whereas for S. aureus the proportion of meticillin resistant isolates decreased. The observed trend suggests an increasing burden of disease caused by E. coli. The reduction in the proportion of meticillin-resistant S. aureus and the lesser increase in S. aureus infections, compared with E. coli, may reflect the success of infection control measures at hospital level in several European countries.

Carbapenem-non-susceptible Enterobacteriaceae in Europe: conclusions from a meeting of national experts.

The emergence and global spread of carbapenemase-producing Enterobacteriaceae is of great concern to health services worldwide. These bacteria are often resistant to all beta-lactam antibiotics and frequently co-resistant to most other antibiotics, leaving very few treatment options. The epidemiology is compounded by the diversity of carbapenem-hydrolysing enzymes and the ability of their genes to spread between different bacterial species. Difficulties are also encountered by laboratories when trying to detect carbapenemase production during routine diagnostic procedures due to an often heterogeneous expression of resistance. Some of the resistance genes are associated with successful clonal lineages which have a selective advantage in those hospitals where antimicrobial use is high and opportunities for transmission exist; others are more often associated with transmissible plasmids. A genetically distinct strain of Klebsiella pneumoniae sequence type (ST) 258 harbouring the K. pneumoniae carbapenemases (KPC) has been causing epidemics of national and international proportions. It follows the pathways of patient referrals, causing hospital outbreaks along the way. Simultaneously, diverse strains harbouring New Delhi metallo-beta-lactamase (NDM-1) are repeatedly being imported into Europe, commonly via patients with prior medical exposure in the Indian subcontinent. Since the nature and scale of carbapenem-non-susceptible Entrobacteriaceae as a threat to hospital patients in Europe remains unclear, a consultation of experts from 31 countries set out to identify the gaps in diagnostic and response capacity, to index the magnitude of carbapenem-non-susceptibility across Europe using a novel five-level staging system, and to provide elements of a strategy to combat this public health issue in a concerted manner

Rapid microbiological tests for bloodstream infections due to multidrug resistant Gram-negative bacteria: therapeutic implications.

BACKGROUND: Treating severe infections due to multidrug-resistant Gram-negative bacteria (MDR-GNB) is one of the most important challenges for clinicians worldwide, partly because resistance may remain unrecognized until identification of the causative agent and/or antimicrobial susceptibility testing (AST). Recently, some novel rapid test for identification and/or AST of MDR-GNB from positive blood cultures or the blood of patients with bloodstream infections (BSIs) have become available. OBJECTIVES: The objective of this narrative review is to discuss the advantages and limitations of different rapid tests for identification and/or AST of MDR-GNB from positive blood cultures or the blood of patients with BSI, as well as the available evidence on their possible role to improve therapeutic decisions and antimicrobial stewardship. SOURCES: Inductive PubMed search for publications relevant to the topic. CONTENT: The present review is structured in the following way: (a) rapid tests on positive blood cultures; (b) rapid tests directly on whole blood; (c) therapeutic implications. IMPLICATIONS: Novel molecular and phenotypic rapid tests for identification and AST show the potential for favourably influencing patients' outcomes and results of antimicrobial stewardship interventions by reducing both the time to effective treatment and the misuse of antibiotics, although the interpretation about their impact on actual therapeutic decisions and patients' outcomes is still complex. Factors such as feasibility and personnel availability, as well as the detailed knowledge of the local microbiological epidemiology, need to be considered very carefully when implementing novel rapid tests in laboratory workflows and algorithms. Providing high-level, comparable evidence on the clinical impact of rapid identification and AST is becoming of paramount importance for MDR-GNB infections, since in the near future rapid identification of specific resistance mechanisms could be crucial for guiding rapid, effective, and targeted therapy against specific resistance mechanisms.

Isothermal microcalorimetry minimal inhibitory concentration testing in extensively drug resistant Gram-negative bacilli: a multicentre study.

OBJECTIVES: To evaluate the performance of an isothermal microcalorimetry (IMC) method for determining the MICs among extensively drug-resistant Gram-negative bacilli. METHODS: A collection of 320 clinical isolates (n = 80 of each) of Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter baumannii from Sweden, Spain, Italy and the Netherlands were tested. The MICs were determined using the IMC device calScreener (Symcel, Stockholm, Sweden) and ISO-broth microdilution as the reference method. Essential agreement, categorical agreement, very major errors (VME), major errors (ME) and minor (mE) errors for each antibiotic were determined. RESULTS: Data from 316 isolates were evaluated. Four errors (two ME, one VME, one mE) among 80 K. pneumoniae, six errors (four ME, one VME, one mE) among 79 E. coli, 15 errors (seven VME, three ME, five mE) among 77 P. aeruginosa and 18 errors (12 VME, two ME, four mE) among 80 A. baumannii were observed. Average essential agreement and categorical agreement of the IMC method were 96.6% (95% confidence interval, 94.2-99) and 97.1% (95% confidence interval, 95.4-98.5) respectively when the MICs were determined at the end of 18 hours. Categorical agreement of the IMC method for prediction of MIC by the end of 8 hours for colistin, meropenem, amikacin, ciprofloxacin and piperacillin/tazobactam were 95%, 91.4%, 94%, 95.2% and 93.7% respectively. CONCLUSIONS: The IMC method could accurately determine the MICs among extensively drug-resistant clinical isolates of E. coli, K. pneumoniae, P. aeruginosa and A. baumannii isolates.

Variable performance of different commercial systems for testing carbapenem susceptibility of KPC carbapenemase-producing Escherichia coli.

OBJECTIVES: The aim was to evaluate different methods for testing carbapenem susceptibility of Escherichia coli producing KPC-type carbapenemase. METHODS: Susceptibility to imipenem, meropenem and ertapenem was assayed using the reference broth microdilution method and several commercial methods (Vitek2, MicroScan, Etest, MIC Test Strip) starting from the same bacterial suspension. Susceptibility to imipenem and meropenem was also tested by Sensititre and disc diffusion (Bio-Rad). Results were interpreted according to EUCAST clinical breakpoints. Essential agreement (EA), category agreement (CA) and error rates were calculated as described by the International Organization for Standardization (ISO) guidelines and also considering the new EUCAST definitions. Genotypic diversity of isolates was evaluated with a RAPD profiling protocol. RESULTS: Of 54 KPC-positive E. coli isolates, 5.6%, 7.4% and 0% were susceptible standard dosing regimen (S), 55.6%, 72.2% and 0% susceptible increased exposure (I), and 38.9%, 20.4% and 100.0% resistant (R) to imipenem, meropenem and ertapenem, respectively, using the reference broth microdilution method. CA lower than 90% were observed with all systems for imipenem and meropenem using both the ISO and the modified EUCAST criteria. With ertapenem, CA >90% was observed with all methods except Vitek2. RAPD profiling revealed a remarkable genotypic diversity of the isolates, supporting that results were not biased by an oligoclonal nature of the collection. CONCLUSIONS: Commercial methods can be unreliable for testing susceptibility to carbapenems of KPC-producing E. coli. Susceptibility should be confirmed by reference broth microdilution.

The spread of CTX-M-type extended-spectrum beta-lactamases.

CTX-M-type enzymes are a group of class A extended-spectrum beta-lactamases (ESBLs) that are rapidly spreading among Enterobacteriaceae worldwide. More that 50 allotypes are known, clustered into six sub-lineages. The CTX-M-encoding genes have been captured from the chromosome of Kluyvera spp. on conjugative plasmids that mediate their dissemination among pathogenic enterobacteria. CTX-M-type ESBLs exhibit powerful activity against cefotaxime and ceftriaxone but generally not against ceftazidime, which has important implications for laboratory detection. However, several CTX-M variants with enhanced ceftazidimase activity have been detected. The rapid and massive spread of CTX-M-type ESBLs is rapidly changing the ESBL epidemiology and, in some geographical areas, these enzymes are now the most prevalent ESBLs in Enterobacteriaceae.