Athanasios Tsakris

Comparative Evaluation of Colistin Susceptibility Testing Methods among Carbapenem-Nonsusceptible Klebsiella pneumoniae and Acinetobacter baumannii Clinical Isolates

ABSTRACT We compared six colistin susceptibility testing (ST) methods on 61 carbapenem-nonsusceptible Klebsiella pneumoniae (n = 41) and Acinetobacter baumannii (n = 20) clinical isolates with provisionally elevated colistin MICs by routine ST. Colistin MICs were determined by broth microdilution (BMD), BMD with 0.002% polysorbate 80 (P80) (BMD-P80), agar dilution (AD), Etest, Vitek2, and MIC test strip (MTS). BMD was used as the reference method for comparison. The EUCAST-recommended susceptible and resistant breakpoints of ≤2 and >2 μg/ml, respectively, were applied for both K. pneumoniae and A. baumannii. The proportions of colistin-resistant strains were 95.1, 77, 96.7, 57.4, 65.6, and 98.4% by BMD, BMD-P80, AD, Etest, MTS, and Vitek2, respectively. The Etest and MTS methods produced excessive rates of very major errors (VMEs) (39.3 and 31.1%, respectively), while BMD-P80 produced 18% VMEs, AD produced 3.3% VMEs, and Vitek2 produced no VMEs. Major errors (MEs) were rather limited by all tested methods. These data show that gradient diffusion methods may lead to inappropriate colistin therapy. Clinical laboratories should consider the use of automated systems, such as Vitek2, or dilution methods for colistin ST.

Single-Locus-Sequence-Based Typing of blaOXA-51-like Genes for Rapid Assignment of Acinetobacter baumannii Clinical Isolates to International Clonal Lineages

ABSTRACT Single-locus bla OXA-51-like sequence-based typing (SBT) was evaluated for its ability to determine correctly sequence types (STs) in Acinetobacter baumannii clinical isolates, in comparison with the Pasteurs multilocus sequence typing (MLST) reference method and 3-locus sequence typing (3-LST). The comparative study was performed in 585 multidrug-resistant (MDR) A. baumannii clinical isolates recovered from 21 hospitals located throughout Greece, Italy, Lebanon, and Turkey. The isolates belonged to nine clonal complexes (CCs) that correspond to 12 distinct sequence types (STs) and to one singleton ST. These clonal lineages predominate worldwide among nosocomial MDR A. baumannii strains. The most common clone was CC2 (ST2 and ST45; n = 278 isolates) followed by CC1 (ST1 and ST20; n = 155), CC25 (n = 65), ST78 (n = 62), CC15 (ST15 and ST84; n = 9), CC10 (n = 4), CC3 (n = 4), CC6 (n = 3), CC54 (n = 3), and CC83 (n = 2). Using the bla OXA-51-like SBT method, all 585 isolates of the study were typed and assigned correctly to the nine CCs and the singleton ST78. The 3-LST method was not able to classify isolates belonging to CC6, CC10, CC54, and CC83, which are not yet characterized in its database. The low-cost and convenient bla OXA-51-like SBT method, compared with 3-LST and MLST, discriminated all epidemic and sporadic lineages of our collection and could be effectively applied to type rapidly A. baumannii strains.

Wide dissemination of linezolid-resistant Staphylococcus epidermidis in Greece is associated with a linezolid-dependent ST22 clone

OBJECTIVES Dependence on linezolid was recently described as significant growth acceleration of linezolid-resistant Staphylococcus epidermidis (LRSE) isolates upon linezolid exposure. We investigated the possible contribution of linezolid dependence to LRSE dissemination in Greece. METHODS Linezolid resistance rates were estimated in six tertiary hospitals located throughout Greece between 2011 and 2013. Sixty-three randomly selected LRSE recovered in these hospitals during this period were studied. Growth curve analysis was conducted with and without linezolid. Clonality of the isolates was investigated by PFGE and MLST. RESULTS During the study period, the LRSE rate in the participating hospitals rose significantly from 6.9% to 9% (P = 0.006); the increase was more prominent in ICUs (from 15.1% to 20.9%; P = 0.005). Forty-seven (74.6%) of the 63 LRSE, derived from all study hospitals, clearly exhibited linezolid dependence, growing significantly faster in the presence of 16 and 32 mg/L linezolid. Of note, 61 (96.8%) LRSE exhibited a single macrorestriction pattern and belonged to ST22, which included all linezolid-dependent LRSE. The remaining two LRSE belonged to unique STs. Five of six linezolid-dependent isolates tested also exhibited linezolid dependence upon exposure to 8 mg/L linezolid. Interestingly, five of six ST22 linezolid-non-dependent isolates tested developed linezolid dependence when linezolid exposure preceded growth analysis. CONCLUSIONS The rapid LRSE dissemination in Greek hospitals threatens linezolid activity. The observation that most LRSE belonged to ST22 and expressed dependence on linezolid clearly implies that the spread of linezolid resistance should have been driven by this trait, which provided the LRSE with a selective advantage under linezolid pressure.

Prevention of Malaria Resurgence in Greece through the Association of Mass Drug Administration (MDA) to Immigrants from Malaria-Endemic Regions and Standard Control Measures.

Greece was declared malaria-free in 1974 after a long antimalarial fight. In 2011–2012, an outbreak of P. vivax malaria was reported in Evrotas, an agricultural area in Southern Greece, where a large number of immigrants from endemic countries live and work. A total of 46 locally acquired and 38 imported malaria cases were detected. Despite a significant decrease of the number of malaria cases in 2012, a mass drug administration (MDA) program was considered as an additional measure to prevent reestablishment of the disease in the area. During 2013 and 2014, a combination of 3-day chloroquine and 14-day primaquine treatment was administered under direct observation to immigrants living in the epicenter of the 2011 outbreak in Evrotas. Adverse events were managed and recorded on a daily basis. The control measures implemented since 2011 continued during the period of 2013–2014 as a part of a national integrated malaria control program that included active case detection (ACD), vector control measures and community education. The MDA program was started prior to the transmission periods (from May to December). One thousand ninety four (1094) immigrants successfully completed the treatment, corresponding to 87.3% coverage of the target population. A total of 688 adverse events were recorded in 397 (36.2%, 95% C.I.: 33.4–39.1) persons, the vast majority minor, predominantly dizziness and headache for chloroquine (284 events) and abdominal pain (85 events) for primaquine. A single case of primaquine-induced hemolysis was recorded in a person whose initial G6PD test proved incorrect. No malaria cases were recorded in Evrotas, Laconia, in 2013 and 2014, though three locally acquired malaria cases were recorded in other regions of Greece in 2013. Preventive antimalarial MDA to a high-risk population in a low transmission setting appears to have synergized with the usual antimalarial activities to achieve malaria elimination. This study suggests that judicious use of MDA can be a useful addition to the antimalarial armamentarium in areas threatened with the reintroduction of the disease.

Predominance of international clone 2 OXA-23-producing-Acinetobacter baumannii clinical isolates in Greece, 2015: results of a nationwide study ★

In a previous nationwide study in Greece, OXA-58 was the sole carbapenemase present among carbapenem-resistant Acinetobacter baumannii (CRAB) isolated between 2000 and 2009. In this study, the antibiotic resistances, carbapenemase gene content and clonal relatedness of 194 single-patient CRAB clinical isolates collected randomly during 2015 from 11 tertiary hospitals located throughout Greece were investigated. Antimicrobial susceptibility was determined using commercial and dilution methods. PCR assays for carbapenemase genes were performed. Clonality was tested by a scheme based on two multiplex PCRs and single-locus blaOXA-51-like sequence-based typing. Furthermore, Pasteurs multilocus sequence typing (MLST) scheme and pulsed-field gel electrophoresis (PFGE) were applied to 31 selected representative isolates. The most active antibiotics were trimethoprim/sulfamethoxazole (SXT) (34.6% of isolates susceptible), minocycline (71.6%), colistin (72.7%) and tigecycline (MIC50/90 values, 1/2 mg/L). The blaOXA-23-like gene was identified in 188 isolates (96.9%), blaOXA-23-like together with blaOXA-58-like in 3 isolates (1.5%), blaOXA-58-like in 2 isolates (1.0%) and blaOXA-40-like in 1 isolate (0.5%). ISAba1 was found upstream of the blaOXA-23-like gene in all isolates. International clone (IC) 2 comprised 157 isolates (80.9%), IC1 comprised 36 isolates (18.6%) and ST78 comprised 1 isolate (0.5%). All IC2 and IC1 isolates tested by MLST were ST2 and ST1, respectively. Seven PFGE types were detected. IC2 isolates were resistant to more antibiotics than IC1, except for SXT. This nationwide study showed that CRAB isolates in Greek hospitals currently produce almost uniformly the OXA-23 carbapenemase and belong mainly to IC2 and, to a lesser extent, IC1. Of particular concern, colistin susceptibility is recently severely reduced.

Colistin-Resistant Acinetobacter baumannii Clinical Strains with Deficient Biofilm Formation

ABSTRACT In two pairs of clinical colistin-susceptible/colistin-resistant (Csts/Cstr) Acinetobacter baumannii strains, the Cstr strains showed significantly decreased biofilm formation in static and dynamic assays (P < 0.001) and lower relative fitness (P < 0.05) compared with those of the Csts counterparts. The whole-genome sequencing comparison of strain pairs identified a mutation converting a stop codon to lysine (*241K) in LpsB (involved in lipopolysaccharide [LPS] synthesis) in one Cstr strain and a frameshift mutation in CarO and the loss of a 47,969-bp element containing multiple genes associated with biofilm production in the other.

Pneumococcal Mastoiditis in Children Before and After the Introduction of Conjugate Pneumococcal Vaccines.

Objectives: To determine whether serotype distribution and antibiotic resistance of Streptococcus pneumoniae acute mastoiditis (AM) in children have changed in the post pneumococcal conjugate vaccines (PCVs) era. Methods: Medical records of pneumococcal AM cases, in a tertiary pediatric hospital were reviewed from January 1999 to December 2014. S. pneumoniae isolates were serotyped using the quellung reaction and tested for antibiotic susceptibility by E-test and for macrolide resistance genes by polymerase chain reaction. Results: Among 334 children with AM, S. pneumoniae was isolated from 89 (26.6%) with median age 22 months (interquartile range: 12–30 months). S. pneumoniae was recovered from ear fluid (58%), mastoid specimens (35.2%) and blood (6.8%). Resistance to penicillin, erythromycin and clindamycin was 12.4%, 49.4% and 18%, respectively. Distribution of pneumococcal serotypes before (1999–2005), after the introduction of PCV7 (2006–2010) and after PCV13 (2011–2014) was found: for the PCV7 serotypes 81%, 25% and 0% (P < 0.0001), for PCV13 additional serotypes 16.3%, 70.8% and 63.6% (P < 0.0001) and for non-PCV serotypes 2.3%, 4.1% and 36.3% (P = 0.0002), respectively. Significant increase was detected for the serotype 19A after PCV7, and this trend was not changed after PCV13 (2.3%, 50% and 50%, respectively; P < 0.0001). A significant proportion of resistant isolates to penicillin (54.5%) and erythromycin (34.8%) was identified as 19A. Conclusions: After the introduction of PCV7, a significant increase of serotype 19A and replacement of PCVs serotypes was identified. After PCV13, the overall proportion of pneumococcal mastoiditis and the incidence of serotype 19A were not significantly declined. A significant proportion of resistant isolates to penicillin and erythromycin is attributed to serotype 19A.

The Impact of Antibiotic Stewardship Programs in Combating Quinolone Resistance: A Systematic Review and Recommendations for More Efficient Interventions

Quinolones are among the most commonly prescribed antibiotics worldwide. A clear relationship has been demonstrated between excessive quinolone use and the steady increase in the incidence of quinolone-resistant bacterial pathogens, both in hospital and community sites. In addition, exposure to quinolones has been associated with colonization and infection with healthcare-associated pathogens such as methicillin-resistant Staphylococcus aureus and Clostridium difficile in hospitalized patients. Therefore, the management of quinolone prescribing in hospitals through antibiotic stewardship programs is considered crucial. Although suggestions have been made by previous studies on the positive impact of stewardship programs concerning the emergence and spread of multidrug-resistant bacteria at hospital level, the association of quinolone-targeted interventions with reduction of quinolone resistance is vague. The purpose of this article was to evaluate the impact of stewardship interventions on quinolone resistance rates and healthcare-associated infections, through a literature review using systematic methods to identify and select the appropriate studies. Recommendations for improvements in quinolone-targeted stewardship programs are also proposed. Efforts in battling quinolone resistance should combine various interventions such as restriction formulary policies, prospective audits with feedback to prescribers, infection prevention and control measures, prompt detection of low-level resistance, educational programs, and guidelines for optimal quinolone usage. However, the effectiveness of such strategies should be assessed by properly designed and conducted clinical trials. Finally, novel approaches in diagnostic stewardship for rapidly detecting bacterial resistance, including PCR-based techniques, mass spectrometry, microarrays, and whole-genome sequencing as well as the prompt investigation on the clonality of quinolone-resistant strains, will strengthen our ability to personalize quinolone prescribing to individual patients.

West Nile Virus Seroprevalence in the Greek Population in 2013: A Nationwide Cross-Sectional Survey

Cases of West Nile Virus (WNV) disease were recorded for three consecutive years in Greece following the year 2010 outbreak. A cross-sectional serologic survey was conducted to estimate the WNV seroprevalence and assess the ratio of infection to neuroinvasive disease. A stratified left-over sampling methodology was used including age and residence strata. A total of 3,962 serum samples was collected and tested for WNV Immunoglobulin G (IgG) antibodies by Enzyme–Linked Immunosorbent Assay (ELISA). All positive samples were further tested by Plaque Reduction Neutralization Test (PRNT) and WNV Immunoglobulin M (IgM) antibodies. WNV IgG antibodies were detected in 82 samples and 61 were also positive in PRNT representing a weighted seroprevalence of 2.1% (95% C.I.: 1.7–2.6) and 1.5% (95% C.I.: 1.2–2.0), respectively. Multivariable analysis showed that seroprevalence was associated with age and residence. The overall ratio of neuroinvasive disease to infected persons was estimated at 1:376 (95% C.I.: 1:421–1:338), while the elderly people had the highest ratio. This nationwide study provided valuable data regarding the epidemiology of WNV in Greece based on the fact that elderly people have higher risk of being both infected and having severe disease.

Molecular analysis of Streptococcus pyogenes macrolide resistance of paediatric isolates during a 7 year period (2007–13)

OBJECTIVES The molecular characterization of paediatric group A Streptococcus (GAS) isolates regarding macrolide resistance and relevant emm types in Athens, Greece. METHODS Pharyngeal and non-pharyngeal GAS isolates were collected during a 7 year period (2007-13) and examined for antibiotic susceptibility, macrolide resistance genes [mef(A), erm(A) and erm(B)] and relevant emm types. RESULTS Overall, 20.4% (270/1324) of GAS isolates were resistant to macrolides. The macrolide resistance rate varied during the study period with a maximum rate observed in 2008 (29.57%) and a minimum rate observed in 2013 (10.95%) (P value for trend = 0.007). During the same period, consumption of macrolides was gradually reduced by 56.6%. No difference was observed in macrolide resistance between pharyngeal and non-pharyngeal isolates (P = 0.7). Among macrolide-resistant isolates, mef(A) was detected in 87 (32.2%), erm(A) in 136 (50.4%), erm(B) in 44 (16.3%) and both mef(A) and erm(A) in 3 (1.1%) isolates. The most prevalent emm types among macrolide-resistant isolates were emm77 (31.5%), emm4 (18.1%) and emm12 (10.7%). Ten emm types (77, 4, 12, 28, 1, 22, 11, 2, 44 and 89) accounted for 90.3% of macrolide-resistant isolates. emm types 4, 22, 44 and 77 were more prevalent in macrolide-resistant compared with macrolide-susceptible isolates, whereas emm types 1, 3, 5, 6, 75 and 89 were more prevalent in macrolide-susceptible compared with macrolide-resistant isolates. CONCLUSIONS GAS macrolide resistance remained significant in our area during the study period. A substantial decline in the resistance rate was observed in the last year of the study, which may be related to reduced consumption of macrolides.