Epidemiological Investigation of Legionella pneumophila Serogroup 2 to 14 Isolates from Water Samples by Amplified Fragment Length Polymorphism and Sequence-Based Typing and Detection of Virulence Traits.

The aim of this study is to explore the dispersion, clonality, and virulence of Legionella pneumophila serogroups 2 to 14 in the Greek environment. Eighty L. pneumophila serogroup 2 to 14 strains isolated from water distribution systems of hotels, hospitals, athletic venues, and ferries in Greece were tested by monoclonal antibodies (MAbs) for serogroup discrimination and molecularly by amplified fragment length polymorphism (AFLP) for genetic diversity. Fifty-six of 80 strains were also typed by the sequence-based typing (SBT) method. Αll strains were further analyzed for detection of two pathogenicity loci: Legionella vir homologue (lvh) and repeats in structural toxin (rtxA). Thirty-seven strains (46.2%) belonged to serogroup 6, 26 strains (32.5%) to serogroup 3, and 7 (8.8%) to other serogroups (4, 5, 8, and 10). Ten strains (12.5%) were nontypeable (NT) into the known serogroups. Thirty-nine different AFLP types were found among the 80 L. pneumophila serogroup 2 to 14 strains, and 24 different SBT types were found among the 56 strains tested. Among the 80 strains, the lvh locus was present in 75 (93.8%), the rtxA locus was found in 76 (95%), and both loci were found in 73 (91.3%) strains. This study showed that there is genetic variability of L. pneumophila serogroups 2 to 14 in the Greek environment as well as a high percentage of the pathogenicity loci. Ιntroducing an effective diagnostic test for L. pneumophila serogroups 2 to 14 in urine and promoting the examination of respiratory specimens from patients hospitalized for pneumonia in Greek hospitals are essential. IMPORTANCE: In this study, the dispersion, clonality, and virulence of environmental isolates of Legionella pneumophila serogroups 2 to 14 (Lp2-14) in Greece were investigated. Genetic variability of Lp2-14 in the Greek environment was identified together with the presence of the pathogenicity loci in a high percentage of the isolates. Despite the high prevalence of Lp2-14 in the Greek environment, no clinical cases were reported, which may be due to underdiagnosis of the disease. Almost all the legionellosis cases are diagnosed in Greece by using the urine antigen test, which is specific for Lp1. There is an urgent need to improve the clinical diagnosis of legionellosis by introducing an effective diagnostic test for Lp2-14 in urine and by promoting the PCR examination of respiratory specimens from patients with compatible clinical symptoms.

Characterization of KPC-encoding plasmids from two endemic settings, Greece and Italy.

OBJECTIVES: Global dissemination of KPC-type carbapenemases is mainly associated with the spread of high-risk clones of Klebsiella pneumoniae and of KPC-encoding plasmids. In this study, we explored the population structure of KPC-encoding plasmids from the recent epidemics of KPC-producing K. pneumoniae (KPC-Kp) in Greece and Italy, the two major European endemic settings. METHODS: Thirty-four non-replicate clinical strains of KPC-Kp representative of the early phases (2008-11) of the Greek (n = 22) and Italian (n = 12) epidemics were studied. Isolates were typed by MLST, and blaKPC-carrying plasmids were characterized by S1 profiling, PCR-based replicon typing and RFLP. Transfer experiments by conjugation or transformation were carried out with Escherichia coli recipients. Eleven plasmids, representative of all different restriction profiles, were completely sequenced. RESULTS: The representative Greek strains belonged to 14 sequence types (STs), with a predominance of ST258. The representative Italian strains belonged to three STs, with a predominance of clonal complex 258 (ST258, ST512). The 34 strains carried plasmids of variable size (78-166 kb), either with blaKPC-2 or blaKPC-3 gene embedded in a Tn4401a transposon. Plasmids from Greek strains were mostly of a single RFLP type (A) and resembled the archetypal pKpQIL KPC-encoding plasmid, while plasmids from Italian strains belonged to a more heterogeneous population, showing five RFLP profiles (A, C-F). Types A and C resembled pKpQIL or deletion derivatives thereof, while types D-F included plasmids with hybrid structures between pKpQIL, pKPN3 and pKPN101-IT. CONCLUSIONS: pKpQIL-like plasmids played a major role in the dissemination of blaKPC in Greece and Italy, but evolved with different dynamics in these endemic settings.

Characterisation of IncA/C2 plasmids carrying an In416-like integron with the blaVIM-19 gene from Klebsiella pneumoniae ST383 of Greek origin.

The complete nucleotide sequences of three multidrug resistance (MDR) IncA/C-like plasmids from Enterobacteriaceae isolates carrying the VIM-type carbapenemase-encoding integrons In4863 (blaVIM-19-aacA7-dfrA1-ΔaadA1-smr2) or In4873 (blaVIM-1-aacA7-dfrA1-ΔaadA1-smr2) were determined, which are the first In416-like elements identified in Greece. Plasmids pKP-Gr642 and pKP-Gr8143 were from Klebsiella pneumoniae ST383 isolates, whereas plasmid pEcl-Gr4873 was from an Enterobacter cloacae ST88 isolate. Sequencing showed that pKP-Gr642 (162787bp) and pKP-Gr8143 (154395bp) consisted of the type 1 IncA/C2 conserved backbone, the blaCMY-2-like gene-containing region, and the ARI-B (with the sul2 gene) and ARI-A (with a class 1 integron) resistance islands, like the plasmid pUMNK88_161 from the USA. The third plasmid, pEcl-Gr4873 (153958bp), exhibited extensive similarity with the type 2 IncA/C2 plasmid pR55 from France. pEcl-Gr4873 carried only one resistance island of a hybrid transposon structure inserted in a different location to ARI-A in type 1 A/C2 plasmids. In all three plasmids, the In416-like integrons In4863 or In4873 were identified within non-identical class II transposon structures. All three In416-like-carrying regions presented significant similarities with the MDR region of the IncA/C2 plasmid pCC416 from Italy, carrying the prototype In416 integron (blaVIM-4-aacA7-dfrA1-ΔaadA1-smr2). These findings provided the basis for speculations regarding the evolution of IncA/C2 plasmids with In416-like integrons, and confirmed the rapid evolution of some IncA/C2 plasmid lineages. Considering the broad host range of IncA/C2 molecules, it seems that pKP-Gr642, pKP-Gr8143 and pEcl-Gr4873 plasmids might support the diffusion of In416-like integrons among Enterobacteriaceae.

An update of the evolving epidemic of blaKPC-2-carrying Klebsiella pneumoniae in Greece (2009-10).

OBJECTIVES: To follow the epidemic of KPC-2-producing Klebsiella pneumoniae in Greece. METHODS: KPC-2-producing isolates (n = 378) were collected during January 2009-April 2010 in 40 Greek hospitals. bla(KPC) and bla(VIM) were detected by PCR. Carbapenemase production was confirmed by spectrophotometry. Sequences flanking bla(KPC-2) and their plasmid carriers were studied. Isolates were typed by PFGE and multilocus sequence typing (MLST). RESULTS: All 378 isolates were bla(KPC-2) positive; 18 also carried bla(VIM-1/VIM-4). Higher isolation frequencies were observed in Athens and Crete. Isolates were classified into 13 PFGE types and 11 sequence types (STs). ST258 was predominant (n = 322), followed by ST147 (n = 20), ST383 (n = 9), ST133 (n = 6), ST274 (n = 4) and ST323 (n = 3). Of the remaining isolates, seven were distributed into five STs (11, 17, 340 and the novel 494 and 495) and seven were not typed. bla(KPC-2) could not be transferred from ST258 isolates, in contrast to isolates of ST17, ST133, ST147, ST274, ST494 and ST495. All bla(KPC-2)-encoding plasmids were of similar size (∼100 kb) and showed indistinguishable restriction fragment length polymorphism (RFLP) patterns except those from the ST340 isolates. Sequences flanking bla(KPC-2) revealed that the Tn4401a isoform was present in plasmids from all STs except ST340 containing Tn4401b. Co-production of VIM enzymes was observed in isolates of ST147, ST323 and ST383. CONCLUSIONS: Apart from the epidemic of KPC-2-producing K. pneumoniae belonging to ST258 in Greece, diffusion of bla(KPC-2) to at least 10 additional STs has taken place. Notably, strains from three of the latter STs (147, 323 and 383) were found to carry both bla(KPC-2) and bla(VIM).

Antimicrobial resistance of Esherichia coli urinary isolates from primary care patients in Greece.

BACKGROUND: Most of antimicrobial susceptibility surveillance studies focus on isolates from hospitalized patients. A retrospective analysis of microbiological data of the antimicrobial susceptibility of Escherichia coli urinary isolates from primary care patients in Greece was performed here. MATERIAL/METHODS: The in vitro susceptibility to ampicillin, amoxicillin/clavulanate, cefaclor, cefprozil, trimethoprim-sulfamethoxazole (cotrimoxazole), amikacin, and norfloxacin of 2460 E. coli isolates (01/2005-06/2005) from the urine specimens of patients tested at the laboratories of three Greek primary care diagnostic centers were analyzed. Only the first isolate per patient (2074 females and 386 males) were included in the analysis. RESULTS: The proportion of E. coli urinary isolates that were resistant to cotrimoxazole was 20.8% and 26.4% for females and males, respectively. There were noteworthy differences between age groups; 37.8% isolates from females <15 years old were resistant to cotrimoxazole compared with 18.9%, 17%, and 23.3% for the 15-35, 35-45, and >55-year-old females, respectively (P<0.001). The proportion of isolates resistant to ampicillin was very high (from 32.1% to 45.3% and 38% to 63% for the urinary isolates from females and males, respectively, in the different age groups examined), while it was relatively low for amikacin (up to 4.1%); 17.8% and 5.5% of the isolates from males and females, respectively, were resistant to norfloxacin (18.2% for males >55 years old). CONCLUSIONS: These findings offer help to clinicians in deciding the appropriate empirical treatment for primary care patients with urinary tract infection and emphasize the increasing problem of antimicrobial resistance even in the primary care setting in Greece.

Correlation between bacterial indicators and bacteriophages in sewage and sludge.

The use of bacteriophages as potential indicators of faecal pollution has recently been studied. The correlation of the number of bacterial indicators and the presence of three groups of bacteriophages, namely somatic coliphages (SOMCPH), F-RNA-specific phages (FRNAPH) and phages of Bacteroides fragilis (BFRPH), in raw and treated wastewater and sludge is presented in this study. Raw and treated wastewater and sewage sludge samples from two wastewater treatment plants in Athens were collected on a monthly basis, over a 2-year period, and analysed for total coliforms, Escherichia coli, intestinal enterococci and the three groups of bacteriophages. A clear correlation between the number of bacterial indicators and the presence of bacteriophages was observed. SOMCPH may be used as additional indicators, because of their high densities and resistance to various treatment steps.

Novel GES/IBC extended-spectrum beta-lactamase variants with carbapenemase activity in clinical enterobacteria.

Two clinical isolates, an Escherichia coli and a Klebsiella pneumoniae, with decreased susceptibility to carbapenems were studied. This phenotype was associated with production of novel GES/IBC variant beta-lactamases, designated GES-3 (from E. coli) and GES-4 (from K. pneumoniae), exhibiting carbapenemase activity. Both enzymes possessed Ser at Ambler's position 170 instead of Gly found in the beta-lactamases GES-1 and IBC-1 that lack carbapenemase activity. Additionally, position 104 in GES-4 was occupied by a Lys as in IBC-1. bla(GES-3) and bla(GES-4) occurred as gene cassettes in the variable regions of class 1 integrons carried by plasmids. The structure of the GES-4-encoding integron was similar to that of previously described IBC-1 integrons. The GES-3-encoding integron was, most likely, truncated at the 3' conserved segment.

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.

Epidemiology of multiresistant Enterococcus avium isolates in a Greek tertiary care hospital.

A retrospective survey of the isolation rate of Enterococcus avium during the period March 1994-February 2000 conducted in Laikon General Hospital using the WHONET software, revealed a peak in the isolation rates of this species during March 1995-February 1996. The ten strains isolated during this time were studied further. No glycopeptide resistance was detected but resistance to ampicillin, ciprofloxacin, erythromycin, gentamicin (high-level) and streptomycin (high-level) was present in nine, ten, nine, three and seven of the isolates, respectively. The genes aac(6')-Ie+aph(2")-Ia and ant(6)-I, encoding for high-level gentamicin and streptomycin resistance, respectively, were detected only in the isolates with the corresponding phenotypes. Beta-lactamase production and haemolysis were not detected. There was evidence of ward-, floor- and building-specific distribution among the different aminoglycoside resistance phenotypes. DNA fingerprinting by PFGE grouped six of the ten isolates in a single cluster with 83% similarity, even though they expressed various resistance phenotypes. These results suggest dissemination of resistance genes among both genetically related and unrelated strains.

Molecular analysis of ampicillin-resistant sporadic Salmonella typhi and Salmonella paratyphi B clinical isolates.

OBJECTIVE: To study the mechanisms of antibiotic resistance in Salmonella typhi and Salmonella paratyphi B clinical isolates, and the clonality of resistant strains. METHODS: Antibiotic susceptibility was tested by disk-agar diffusion. Conjugation experiments and plasmid analysis by agarose gel electrophoresis after EcoRI digestion were followed by hybridization to a digoxigenin-labeled TEM-type beta-lactamase probe. DNA fingerprints were obtained by pulsed-field gel electrophoresis of Xbal-digested chromosomal DNA. RESULTS: Three S. typhi isolates (7% of the isolates studied), of which one was ampicillin resistant and the other two multiresistant (ampicillin, chloramphenicol, tetracycline, sulfamethoxazole/trimethoprim and streptomycin), and two ampicillin-resistant S. paratyphi B isolates (25% of the isolates studied) were further evaluated. A 34-MDa conjugative plasmid, previously isolated from Salmonella enteritidis, conferred ampicillin resistance. A 100-MDa conjugative plasmid encoded resistance to chloramphenicol, tetracycline and sulfamethoxazole/trimethoprim, as well as ampicillin. Chromosomal fingerprinting revealed two distinct resistant strains for each serovar which were different from a matched set of sensitive S. typhi strains. CONCLUSIONS: Two conjugative, TEM-type beta-lactamase-encoding plasmids conferred ampicillin resistance to S. typhi and S. paratyphi B. The 34-MDa plasmid was identical to that previously characterized from S. enteritidis, while the 100-MDa plasmid also encoded resistance to chloramphenicol, tetracycline and sulfamethoxazole/trimethoprim. Resistant isolates did not belong to a single clone but rather represented distinct strains.

Multiresistant Pseudomonas aeruginosa serogroup O:11 outbreak in an intensive care unit.

OBJECTIVE: To determine whether 15 multiresistant Pseudomonas aeruginosa isolates from an intensive care unit (ICU) outbreak were related, were endemic, and belonged to the O:12 European clone. METHODS: Forty-six P. aeruginosa isolates from a large hospital were investigated with respect to their antibiotic resistance profiles, serogroups, bacteriocin types and DNA fingerprints obtained by pulsed-field gel electrophoresis (PFGE) of genomic DNA digested with Xbal. RESULTS: Fourteen of the ICU outbreak isolates were indeed identical with respect to their serogroup, O:11, pyocin type, 10/a, and PFGE type, A. Clone A was endemic and dominant throughout the hospital, even though, within the ICU, it underwent phenotypic alterations, such as loss of cell wall lipopolysaccharide side-chains, or acquisition of ceftazidime and imipenem resistance. Bacteriocin typing was more discriminatory than serotyping, but PFGE could differentiate further among phenotypically identical strains. It also allowed the tracking of an O:6 strain, as it was becoming gradually more resistant and undergoing a bacteriocin-type conversion while remaining genotypically unaltered. CONCLUSIONS: Using three typing methods, a nosocomial multiresistant strain distinct from the previously described dominant European O:12 clone was characterized, and the ability of PFGE to identify clonal isolates even when these appear phenotypically distinct was demonstrated.