In vitro activity of cefepime/zidebactam (WCK 5222) against recent Gram-negative isolates collected from high resistance settings of Greek hospitals.

Cefepime/zidebactam is in clinical development for the treatment of carbapenem-resistant Gram-negative infections. MICs of cefepime/zidebactam (1:1) and comparators against Enterobacterales (n = 563), Pseudomonas (n = 172) and Acinetobacter baumannii (n =181) collected from 15 Greek hospitals (2014-2018) were determined by reference broth microdilution method. The isolates exhibited high carbapenem resistance rates [(Enterobacterales (75%), Pseudomonas (75%) and A. baumannii (98.3%)]. Cefepime/zidebactam showed MIC50/90 of 0.5/2 mg/L, against Enterobacterales including metallo-ß-lactamases (MBL)-producers. Reduced susceptibility rates to tigecycline (16.8%), colistin (47.4%), ceftazidime/avibactam (59.8%), and imipenem/relebactam (61%) indicated high prevalence of multi-drug resistance among Greek Enterobacterales. Cefepime/zidebactam exhibited MIC50/90 of 8/16 mg/L against Pseudomonas including MBL-producers. The MIC50/90 of ceftazidime/avibactam and imipenem/relebactam were high (≥32 mg/L). Cefepime/zidebactam showed MIC90 of 64 mg/L against A. baumannii which is within its therapeutic scope. Other antibiotics including colistin showed limited activity against A. baumannii. The activity of cefepime/zidebactam against multi-drug-resistant isolates is attributable to zidebactam mediated novel ß-lactam-enhancer mechanism.

Abundance of Colistin-Resistant, OXA-23- and ArmA-Producing Acinetobacter baumannii Belonging to International Clone 2 in Greece.

Carbapenem resistant Acinetobacter baumannii (CRAB) represents one of the most challenging pathogens in clinical settings. Colistin is routinely used for treatment of infections by this pathogen, but increasing colistin resistance has been reported. We obtained 122 CRAB isolates from nine Greek hospitals between 2015 and 2017, and those colistin resistant (ColR; N = 40, 32.8%) were whole genome sequenced, also by including two colistin susceptible (ColS) isolates for comparison. All ColR isolates were characterized by a previously described mutation, PmrBA226V, which was associated with low-level colistin resistance. Some isolates were characterized by additional mutations in PmrB (E140V or L178F) or PmrA (K172I or D10N), first described here, and higher colistin minimum inhibitory concentrations (MICs), up to 64 mg/L. Mass spectrometry analysis of lipid A showed the presence of a phosphoethanolamine (pEtN) moiety on lipid A, likely resulting from the PmrA/B-induced pmrC overexpression. Interestingly, also the two ColS isolates had the same lipid A modification, suggesting that not all lipid A modifications lead to colistin resistance or that other factors could contribute to the resistance phenotype. Most of the isolates (N = 37, 92.5%) belonged to the globally distributed international clone (IC) 2 and comprised four different sequence types (STs) as defined by using the Oxford scheme (ST 425, 208, 451, and 436). Three isolates belonged to IC1 and ST1567. All the genomes harbored an intrinsic bla OXA-51 group carbapenemase gene, where bla OXA-66 and bla OXA-69 were associated with IC2 and IC1, respectively. Carbapenem resistance was due to the most commonly reported acquired carbapenemase gene bla OXA-23, with ISAba1 located upstream of the gene and likely increasing its expression. The armA gene, associated with high-level resistance to aminoglycosides, was detected in 87.5% of isolates. Collectively, these results revealed a convergent evolution of different clonal lineages toward the same colistin resistance mechanism, thus limiting the effective therapeutic options for the treatment of CRAB infections.

Activity of TP-6076 against carbapenem-resistant Acinetobacter baumannii isolates collected from inpatients in Greek hospitals.

TP-6076 is a synthetic fluorocycline antibiotic that inhibits bacterial protein synthesis. In this study, carbapenem-resistant Acinetobacter baumannii clinical isolates from 13 Greek hospitals were tested for susceptibility to TP-6076 and comparator antibiotics. Broth microdilution plates were used to determine minimum inhibitory concentrations (MICs). A total of 121 non-duplicate A. baumannii isolates were tested. The MIC50 and MIC90 values of TP-6076 were 0.03 mg/L and 0.06 mg/L, respectively. Tigecycline was the second most active antibiotic (MIC90, 2 mg/L), followed by minocycline (MIC90, 8 mg/L). TP-6076 exhibited MIC90 values that were one dilution lower against tigecycline- and minocycline-susceptible isolates than against resistant isolates. There was no difference in the MIC90 value for colistin-susceptible or -resistant isolates. In conclusion, TP-6076 exhibited greater antimicrobial activity in vitro against carbapenem-resistant A. baumannii than comparator antibiotics.

Activity of cefiderocol (S-649266) against carbapenem-resistant Gram-negative bacteria collected from inpatients in Greek hospitals.

Background: Cefiderocol (S-649266), a siderophore cephalosporin, utilizes a novel mechanism of entry into the periplasmic space of Gram-negative bacteria and is broadly stable to ESBLs and carbapenemases. Methods: A collection of carbapenem-resistant Gram-negative bacteria isolated from clinical specimens in 18 Greek hospitals was tested for susceptibility to cefiderocol, meropenem, ceftazidime, cefepime, ceftazidime/avibactam, ceftolozane/tazobactam, aztreonam, amikacin, ciprofloxacin, colistin and tigecycline. Broth microdilution plates were used to determine MICs. Results: In total 189 non-fermentative Gram-negative bacteria (107 Acinetobacter baumannii and 82 Pseudomonas aeruginosa ) and 282 Enterobacteriaceae (including 244 Klebsiella pneumoniae , 14 Enterobacter cloacae and 11 Providencia stuartii ) were studied. For both A. baumannii and P. aeruginosa the MIC 90 of cefiderocol was 0.5 mg/L. For K. pneumoniae , E. cloacae and P. stuartii the MIC 90 of cefiderocol was 1, 1 and 0.5 mg/L, respectively. Tigecycline was the second most active antibiotic, followed by colistin. Conclusions: Cefiderocol exhibited greater antimicrobial activity in vitro against carbapenem-resistant Gram-negative bacteria than comparator antibiotics.

Aerococcus urinae, a cause of cystitis with malodorous urine in a child: clinical and microbiological challenges.

Introduction. An infection of the lower urinary tract associated with an extremely unpleasant odour due to Aerococcus urinae in an otherwise healthy 5-year-old boy is described herein. Case presentation. Interestingly, imaging examination revealed the presence of a bladder diverticulum. Routine microbiological examination based on Gram staining, colony morphology and catalase reactivity suggested that the responsible pathogen could belong either to staphylococci, α-haemolytic streptococci or enterococci, which are more common urine isolates. Of note is that the VITEK 2 automated system could not identify the micro-organism. Susceptibility testing showed full sensitivity to ß-lactam antibiotics and resistance to trimethoprim/sulfamethoxazole. The isolate was subjected to 16S rRNA gene sequence analysis because of its unusual characteristics. It was identified as A. urinae and the sequence was deposited in GenBank under the accession number KU207150. Conclusion.A. urinae should be considered as a causative agent of urinary-tract infection associated with malodorous urine.

Epidemiological characteristics of infections caused by Bacteroides, Prevotella and Fusobacterium species: a prospective observational study.

In order to investigate differences among infections due to Gram-negative anaerobic bacteria (Bacteroides, Prevotella and Fusobacterium spp.), clinical, epidemiological, and microbiological data were collected and evaluated from 206 anaerobic infections. The most frequently isolated species was Bacteroides fragilis. The majority of the cases were intra-abdominal infections (49%) followed by skin and soft tissue infections (24.7%). Logistic regression analysis showed that Bacteroides spp. strains were more often isolated from intra-abdominal infections (p = 0.002), whereas Prevotella spp. were isolated more frequently from cases with shorter duration of hospitalization (p = 0.026), and less frequently from bloodstream infections (p = 0.049). In addition, Bacteroides spp. were associated with coinfection due to Enterobacteriaceae species (p = 0.007), whereas Prevotella spp. were associated with coinfection due to Staphylococcus spp. (p = 0.002). Patients with an infection due to B. fragilis, were more frequently admitted in a general surgical ward (p = 0.017), or have been treated with a 2nd generation cephalosporin before anaerobic infection onset (p = 0.05). Total mortality was 10.9% and was associated with bacteremia (p = 0.026), and hematological (p = 0.028), or solid organ malignancy (p = 0.007). Metronidazole resistance was detected only among Prevotella spp. (16.2%) and B. fragilis group (0.8%) isolates. In conclusion, this study indicated differences between infections due to the most frequently isolated Gram-negative anaerobic species, differences that may affect the design and implementation of empirical antimicrobial chemotherapy guidelines.

In vitro activity of tigecycline against 2423 clinical isolates and comparison of the available interpretation breakpoints.

MICs to tigecycline and 12 antimicrobials were performed by microdilution method, against 2423 nonduplicate pathogens recently isolated in 17 Greek hospitals. The Food and Drug Administration (FDA) and European Committee on Antimicrobial Susceptibility Testing (EUCAST) criteria were used comparatively for interpretation of tigecycline MICs. Tigecycline exhibited potent in vitro activity against the majority of the isolates tested. (MIC(90) values of 0.5, 1, 2, 0.125, 1, 0.25, 0.125, and 1 mg/L were observed for Escherichia coli, Klebsiella pneumoniae, Enterobacter spp., Moraxella catarrhalis, Acinetobacter spp., Staphylococcus aureus, Enterococcus spp., and Streptococcus pneumoniae isolates, respectively.) Tigecycline activity was the same, irrespective of the resistance profile to other antimicrobials (Gram-negative pathogens susceptible or resistant to imipenem, Enterococcus spp., S. aureus, or S. pneumoniae isolates, susceptible or resistant to vancomycin, methicillin or penicillin, respectively). Interpretation using EUCAST and FDA breakpoints differed among isolates of K. pneumoniae and Enterobacter spp. having tigecycline MICs of 2 to 4 mg/L. In conclusion, tigecycline exhibited potent activity against pathogens recently isolated in a region that experiences high antimicrobial resistance rates. Indications that the available criteria might categorize differently tigecycline susceptibility status in K. pneumoniae and Enterobacter spp. isolates were also detected.

Moxifloxacin resistance is prevalent among Bacteroides and Prevotella species in Greece.

OBJECTIVES: Moxifloxacin is recommended in the empirical treatment of infections involving Gram-negative anaerobes. However, current European data regarding its activity against anaerobic pathogens are limited. In order to evaluate its potency, we comparatively studied the activity of moxifloxacin against recently isolated Gram-negative anaerobes. METHODS: Four hundred and ninety-five Gram-negative anaerobic clinical isolates (296 Bacteroides fragilis group, 58 non-fragilis Bacteroides spp. and 141 Prevotella spp.) were prospectively recovered in six Greek hospitals. Moxifloxacin MICs were determined in comparison with those of penicillin, piperacillin/tazobactam, cefoxitin, imipenem, metronidazole and clindamycin. RESULTS: Overall moxifloxacin MIC(50) and MIC(90) were 2 and 32 mg/L, respectively. Based on the current CLSI breakpoints (susceptible, < or =2 mg/L; resistant, > or =8 mg/L), almost half of the total isolates (49%) were non-susceptible to moxifloxacin (32% resistant; 17% intermediate). This was more evident among the non-fragilis Bacteroides species, where 47% of the isolates were resistant and 14% intermediate to moxifloxacin. Species variation was noticed, with the highest non-susceptible rates detected among Prevotella oralis (90%), Prevotella bivia (80%), Bacteroides thetaiotaomicron (75%), Bacteroides uniformis (70%) and Bacteroides capillosus (67%) species. Among the 19 (4%) isolates that were metronidazole non-susceptible (MIC > or = 16 mg/L), only 4 (21%) were additionally non-susceptible to moxifloxacin. CONCLUSIONS: High resistance rates to moxifloxacin among Bacteroides and Prevotella spp. were recorded, exceeding those previously reported in Europe and contraindicating its use as monotherapy for infections involving Gram-negative anaerobes without prior microbiological confirmation. For empirical usage, moxifloxacin should be combined with metronidazole in order to cover for these pathogens.

Genotyping of human Campylobacter jejuni isolates in Greece by pulsed-field gel electrophoresis.

BACKGROUND: Pulsed-field gel electrophoresis (PFGE) typing has been recognized by several groups as a relatively simple and quick method for genotyping of Campylobacter jejuni (C. jejuni). The present study was carried out to determine the genetic variations among clinical isolates of C. jejuni from Greece and to establish a database, which could be used for future epidemiological and clinical studies. METHODS: A total of 93 C. jejuni clinical isolates of known flagellin subunit A (flaA) genotype, serotype, and antimicrobial susceptibility pattern, were collected from a general hospital in the Attica region of Greece, between the years 2000 and 2003. The PFGE profiles of SmaI DNA digests of each strain were compared using a bin analysis based on 44 molecular size intervals. RESULTS: Forty-three different PFGE types, designated as C. jejuni (C. j.) 1 Greece (GR) to C. j. 43 GR, were identified. There was no statistically significant association of PFGE type with flaA genotype, serotype, or antimicrobial susceptibility pattern. However, PFGE typing did show a remarkable discriminatory ability within the non-serotypable group. CONCLUSION: Evaluating our results, we observed that (i) there was no statistically significant clonality of a certain PFGE type among the strains examined, and (ii) the discriminatory ability of PFGE typing was much better than that of the other typing methods. This is the first report of the use of bin patterns to compare the PFGE genotypes identified.

The first database comprised of flagellin gene (flaA) types of Campylobacter jejuni human clinical isolates from Greece.

BACKGROUND: Flagellin subunit A gene (flaA) typing of Campylobacter has been recognized by several groups as a relatively simple and quick genotyping method. The present study aimed to create, for the first time in Greece, a database with flaA restriction patterns, which could be used for future epidemiological and clinical studies. A total of 207 C. jejuni clinical isolates of known serotype were collected from 5 general hospitals of the area of Attica, during the period 2000-2003. RESULTS: The RFLP profiles of each strain were matched in 44 bins of 0 or 1. Thirty nine different flaA types, designated as flaA 1 GR to flaA 39 GR (GR: Greece) were found. There was no significant association of certain genotypes with certain serotypes. However flaA typing showed a remarkable discriminatory ability inside the non-typable (NT) group. CONCLUSIONS: Evaluating our results we observed (i) that there was no clonality of a certain flaA type among the strains and the serotypes examined and (ii) that the discriminatory ability of flaA typing was much better than that of serotyping. Giving a simple and detailed description of the data analysis, we are the first who publish the bin patterns for the flaA genotypes found.

Frequency and predictors of colonization of the respiratory tract by VIM-2-producing Pseudomonas aeruginosa in patients of a newly established intensive care unit.

The aim of this study was to examine the frequency and predictors of colonization of the respiratory tract by metallo-beta-lactamase (MBL)-producing Gram-negative bacteria in patients admitted to a newly established intensive care unit (ICU) of a tertiary care hospital. Specimens of tracheobronchial aspirates for microbiological studies were obtained every day for the first 3 days of the ICU stay and subsequently every third day for the rest of the ICU stay. PCR analysis and nucleotide sequencing were performed to identify bacteria that had MBL genes. Thirty-five patients (20 male, 15 female) were hospitalized during the initial 3 month period of functioning of the ICU. Colonization of the lower respiratory tract by Gram-negative bacteria was found in 29 of 35 patients (83 %) during the first 6-20 days (median 13 days) following admission to the ICU (13 patients with Acinetobacter baumannii, ten with Pseudomonas aeruginosa, three with Enterobacter aerogenes, two with Klebsiella pneumoniae and one with Stenotrophomonas maltophilia). Six of 29 patients (21 %) colonized with Gram-negative bacteria had bla(VIM-2)-positive P. aeruginosa isolates; one of these patients developed clinical infection due to this micro-organism. Previous use of carbapenems (P=0.01) or other beta-lactams (P=0.03), as well as a stay in the ICU of >20 days (P<0.001), were associated with colonization with bla(VIM-2)-producing P. aeruginosa. In conclusion, colonization by Gram-negative bacteria of the respiratory tract of patients in this newly established ICU was common (83 %). Use of beta-lactams, including carbapenems, was associated with subsequent colonization of the respiratory tract with MBL-positive P. aeruginosa.

Frequent detection of cytomegalovirus in the intestine of patients with inflammatory bowel disease.

BACKGROUND: Although a growing number of reports have described inflammatory bowel disease (IBD) complicated with cytomegalovirus (CMV) infection, there are limited molecular studies that investigate CMV genome in intestinal sections of patients with IBD. METHODS: A cross-sectional prospective study was conducted between September 2000 and June 2003 in a cohort of 85 patients diagnosed with IBD (58 with ulcerative colitis and 27 with Crohn's disease) in two adult gastrointestinal referral centers in Athens, Greece. Prevalence of CMV infection was estimated by pathologic studies in intestinal sections and by molecular assays in blood and intestinal tissue samples and compared with a control group of 42 individuals with noninflammatory disease. RESULTS: Immunohistochemical staining showed CMV antigen in 10 IBD patients (7 with ulcerative colitis; 9 with severe disease), whereas CMV antigen was not detected in any of the controls. CMV genome in both the intestinal tissue and blood was found by polymerase chain reaction in 23 (27.1%) of the total IBD patients, in 18 (31.0%) of those with ulcerative colitis, and in 5 (18.5%) of those with Crohn's disease. In addition, five (5.9%) IBD patients (2 with ulcerative colitis and 3 with Crohn's disease) had detectable CMV genome in their intestinal samples but not in their blood. In the control group, five (11.9%) individuals had detectable CMV genome in their blood, but only one (2.2%) in his intestine. CONCLUSION: Patients with ulcerative colitis had more often detectable CMV genome in their blood as well as in their intestinal tissue samples as compared with controls (P = 0.022 and P < 0.0001, respectively). However, patients with Crohn's disease had more often detectable CMV genome only in their intestinal tissue samples as compared with controls (P = 0.001). Detection of CMV genome in blood or intestinal tissue was significantly associated with short duration of IBD (P = 0.0088 and 0.04, respectively) but not with age, sex, severity of the disease, activity at colonoscopy, pancolitis, administration of a specific treatment, and surgery. In this cross-sectional prospective study, detection of CMV genome or antigen in the intestine was commonly associated with IBD.

Prevalence of infection by HTLV-I/II among pregnant women and high-risk groups in the Peloponnese peninsula, Greece.

Although screening for human T-cell lymphotropic virus types I and II (HTLV-I/II) antibodies in volunteer blood donors has been systematic in Greece since 1995, the epidemiology and the determinants of HTLV-I/II infection are not well defined among population groups. During 1997-2005, the prevalence of HTLV-I/II infection was investigated in a sample of 2016 pregnant women, 102 multitransfused haematologic and oncologic patients, 93 thalassaemic patients and 57 intravenous drug users originating from four geographic areas of Pelopennese peninsula, Greece. One recipient of HTLV-I infected blood and the relatives of a woman died from adult T-cell leukaemia/lymphoma (ATTL) related to HTLV-I have also been tested. The subjects were initially screened by an enzyme immunoassay whereas Western blot, INNO-LIA HTLV, polymerase chain reaction and nucleotide sequencing confirmed the infection. One thalassaemic patient had proved HTLV-I infection giving an overall prevalence of 11 per 1000. In the recipient of the infected blood and in two of the five relatives of the woman died from ATTL, HTLV-I infection was also detected. In none of the pregnant women, multitransfused patients and intravenous drug users HTLV-I/II infection was confirmed. These data suggest that HTLV-I is present in Greece among populations at high-risk. However, they would not support the need for HTLV-I/II antenatal screening in Greece.

VIM-1 metallo-beta-lactamase in Acinetobacter baumannii.

In 2004 and 2005, 5 metallo-beta-lactamase (MBL)-positive Acinetobacter baumannii isolates were found in 2 Greek hospitals. Isolates were unrelated and carried blaVIM-1 in a class 1 integron; bla(OXA-51-) and bla(OXA-58-like) carbapenemase genes were also detected. VIM-1 MBL in Acinetobacter spp. causes concern, given the increasing resistance of this species.

In vitro activities of tigecycline against recently isolated Gram-negative anaerobic bacteria in Greece, including metronidazole-resistant strains.

The in vitro activity of tigecycline was compared with those of benzylpenicillin, piperacillin + tazobactam, cefoxitin, imipenem, metronidazole, clindamycin, and tetracycline against 249 Gram-negative anaerobic bacteria (158 Bacteroides fragilis group, 27 non-fragilis Bacteroides spp., 44 Prevotella spp., and 20 miscellaneous), recently isolated from 8 general hospitals in Athens, Greece. Overall tigecycline MIC(50) and MIC(90) were 0.25 and 2 mg/L, respectively, whereas B. fragilis group MIC(50) and MIC(90) were 0.5 and 4 mg/L, respectively. In total, 93% of the isolates were susceptible to tigecycline (MIC /= 32 mg/L) was detected. In addition, tigecycline exhibited good activity against metronidazole- and tetracycline-resistant isolates (MIC(90), 0.5 and 8 mg/L, respectively). In summary, tigecycline exhibits good in vitro activity against Gram-negative anaerobic bacteria isolated in Greece, as well as stability to the most common occurring resistance mechanisms, attributes that make this parenteral agent an attractive alternative for use against infections involving these microorganisms.

VIM-12, a novel plasmid-mediated metallo-beta-lactamase from Klebsiella pneumoniae that resembles a VIM-1/VIM-2 hybrid.

A transferable plasmid from Klebsiella pneumoniae carried a class 1 integron containing bla(VIM-12), a novel bla(VIM)-type gene, flanked by two copies of aacA7. bla(VIM-12) was clustered between bla(VIM-1) and bla(VIM-2) and differed from bla(VIM-1) by 18 nucleotides that were all located at the 3' end and matched the corresponding nucleotides in bla(VIM-2). The bla(VIM-12)-associated 59-base element was identical to that described in bla(VIM-2) alleles.

Ruling out Bacillus anthracis.

Optimization of methods for ruling out Bacillus anthracis leads to increased yields, faster turnaround times, and a lighter workload. We used 72 environmental non-B. anthracis bacilli to validate methods for ruling out B. anthracis. Most effective were the use of horse blood agar, motility testing after isolates had a 2-h incubation in trypticase soy broth, and screening isolates with a B. anthracis-selective agar.

Prevalence and characterization of the mechanisms of macrolide, lincosamide and streptogramin resistance in viridans group streptococci.

The presence of erm genes conferring constitutive and inducible resistance, as well as that of the mefA gene conferring only constitutive resistance, was investigated using PCR in 70 erythromycin resistant (MIC>or=1 mg/l) strains of viridans group streptococci (VGS) (18 Streptococcus mitis biotype 1, 16 S. mitis biotype 2, 15 S. oralis, 12 S. salivarius and nine S. sanguis) isolated from the oropharynx of healthy Greek children. All of the 56 isolates belonging to resistance phenotype M harbored the mefA gene. All of the 14 isolates constitutively resistant to macrolides and lincosamides (phenotype CR) harbored the ermB gene. Co-presence of both genes was not observed, whereas class A erm gene (previously known as ermTR) was not detected. Our results are consistent with a possible role of VGS as a reservoir of resistance genes now prevalent in pathogenic species of streptococci.