Differences in the epidemiology between paediatric and adult invasive Streptococcus pyogenes infections.

In order to investigate for possible differences between paediatric and adult invasive Streptococcus pyogenes (iGAS) infections, a total of 142 cases were identified in 17 Greek hospitals during 2003-2007, of which 96 were children and 46 adults. Bacteraemia, soft tissue infections, streptococcal toxic shock syndrome (STSS), and necrotizing fasciitis were the main clinical presentations (67·6%, 45·1%, 13·4%, and 12·0% of cases, respectively). Bacteraemia and lymphadenitis were significantly more frequent in children (P=0·019 and 0·021, respectively), whereas STSS was more frequent in adults (P=0·017). The main predisposing factors in children were varicella and streptococcal pharyngotonsillitis (25% and 19·8%, respectively), as opposed to malignancy, intravenous drug abuse and diabetes mellitus in adults (19·6%, 15·2% and 10·9%, respectively). Of the two dominant emm-types, 1 and 12 (28·2% and 8·5%, respectively), the proportion of emm-type 12 remained stable during the study period, whereas emm-type 1 rates fluctuated considerably. Strains of emm-type 1 from children were associated with erythromycin susceptibility, STSS and intensive-care-unit admission, whereas emm-type 12 isolates from adults were associated with erythromycin and clindamycin resistance. Finally, specific emm-types were detected exclusively in adults or in children. In conclusion, several clinical and epidemiological differences were detected, that could prove useful in designing age-focused strategies for prevention and treatment of iGAS infections.

Carbapenemases in Klebsiella pneumoniae and other Enterobacteriaceae: an evolving crisis of global dimensions.

SUMMARY: The spread of Enterobacteriaceae, primarily Klebsiella pneumoniae, producing KPC, VIM, IMP, and NDM carbapenemases, is causing an unprecedented public health crisis. Carbapenemase-producing enterobacteria (CPE) infect mainly hospitalized patients but also have been spreading in long-term care facilities. Given their multidrug resistance, therapeutic options are limited and, as discussed here, should be reevaluated and optimized. Based on susceptibility data, colistin and tigecycline are commonly used to treat CPE infections. Nevertheless, a review of the literature revealed high failure rates in cases of monotherapy with these drugs, whilst monotherapy with either a carbapenem or an aminoglycoside appeared to be more effective. Combination therapies not including carbapenems were comparable to aminoglycoside and carbapenem monotherapies. Higher success rates have been achieved with carbapenem-containing combinations. Pharmacodynamic simulations and experimental infections indicate that modification of the current patterns of carbapenem use against CPE warrants further attention. Epidemiological data, though fragmentary in many countries, indicate CPE foci and transmission routes, to some extent, whilst also underlining the lack of international collaborative systems that could react promptly and effectively. Fortunately, there are sound studies showing successful containment of CPE by bundles of measures, among which the most important are active surveillance cultures, separation of carriers, and assignment of dedicated nursing staff.

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.

Prevalence of emm types 1 and 12 from invasive Streptococcus pyogenes disease in Greece--results of enhanced surveillance.

Among a total of 101 isolates from the first systematic multicentre surveillance effort concerning invasive Streptococcus pyogenes disease in Greece, conducted between 2003 and 2005 and covering 38% of the population, emm types 1 and 12 were prevalent, being responsible for 27 and nine cases, respectively. The isolates from the remaining 65 cases were assigned to 26 other emm types. Erythromycin resistance (12 isolates) was primarily mef(A)-mediated, although all emm type 1 strains were susceptible. Tetracycline resistance, due mostly to tet(M), was detected in 26 isolates. Subtyping by pulsed-field gel electrophoresis yielded 50 chromosomal fingerprints, thus discriminating further among ten of the 28 observed emm types.

Ongoing epidemic of blaVIM-1-positive Klebsiella pneumoniae in Athens, Greece: a prospective survey.

OBJECTIVES: To determine the current frequency and study the characteristics of VIM-1-producing Klebsiella pneumoniae isolates from bloodstream infections in Greek hospitals. METHODS: All blood isolates of K. pneumoniae were prospectively collected during 2004-06 in three teaching hospitals located in Athens. MICs of antibiotics were determined by the Etest. Extended-spectrum- (ESBL) and metallo-beta-lactamase (MBL) production was examined by clavulanate- and EDTA-based techniques, respectively. Isolates were typed by PFGE of XbaI-digested genomic DNA. Detection of bla(VIM-1) and mapping of the VIM-1-encoding integrons were performed by PCR and sequencing. Beta-lactamase activities were analysed by IEF and imipenem hydrolysis was assessed by spectrophotometry. VIM-1-encoding plasmids were transferred to Escherichia coli by conjugation and transformation and characterized by Inc/rep typing and RFLP. RESULTS: Sixty-seven (37.6%) of 178 K. pneumoniae blood isolates were bla(VIM-1)-positive (VPKP); 77.8% of these were from ICUs. All VPKP isolates were multidrug-resistant. The MICs of carbapenems for VPKP varied from the susceptible range to high-level resistance overlapping with those of MBL-negative isolates. The EDTA-imipenem synergy methods had reduced sensitivity in detecting VPKP isolates when the MICs were in the susceptible range. ESBL production was common among VPKP isolates (n = 45, 67.2%) as indicated by resistance to aztreonam and confirmed by a clavulanate-based double-disc synergy test. The responsible ESBL was always an SHV-5-type enzyme as indicated by IEF. PFGE identified eight clusters (A-H) of VPKP isolates with related (>80%) patterns, as well as four unique types. Both inter-hospital spread of several clones and genotypic similarities among susceptible, ESBL-positive and VPKP isolates were also observed. Location of bla(VIM-1) and expression of VIM-1 were studied in 12 isolates representing the eight PFGE clusters. In all isolates, bla(VIM-1) was part of a class 1 integron that also carried aacA4, dhfrI, aadA and sulI. In eight isolates (clusters C, D, G and H), the bla(VIM-1) integron was located in transferable IncN plasmids. A cluster F isolate carried a VIM-1-encoding, self-transferable plasmid that was not typeable by Inc/rep typing. VIM-1-encodingreplicons were not identified in three isolates (PFGE clusters A, B and E). VPKP isolates exhibited differences in imipenem-hydrolysing activities which, however, were not correlated with the respective carbapenem MICs. CONCLUSIONS: A multiclonal epidemic of bla(VIM-1)-carrying K. pneumoniae is under way in the majorhospitals in Greece. Microorganisms producing both VIM-1 and SHV-5 constitute the prevalent multidrug-resistant population of K. pneumoniae in this setting.

Guidelines for the validation and application of typing methods for use in bacterial epidemiology.

For bacterial typing to be useful, the development, validation and appropriate application of typing methods must follow unified criteria. Over a decade ago, ESGEM, the ESCMID (Europen Society for Clinical Microbiology and Infectious Diseases) Study Group on Epidemiological Markers, produced guidelines for optimal use and quality assessment of the then most frequently used typing procedures. We present here an update of these guidelines, taking into account the spectacular increase in the number and quality of typing methods made available over the past decade. Newer and older, phenotypic and genotypic methods for typing of all clinically relevant bacterial species are described according to their principles, advantages and disadvantages. Criteria for their evaluation and application and the interpretation of their results are proposed. Finally, the issues of reporting, standardisation, quality assessment and international networks are discussed. It must be emphasised that typing results can never stand alone and need to be interpreted in the context of all available epidemiological, clinical and demographical data relating to the infectious disease under investigation. A strategic effort on the part of all workers in the field is thus mandatory to combat emerging infectious diseases, as is financial support from national and international granting bodies and health authorities.

Spread of the Streptococcus pneumoniae Taiwan19F-14 clone among children in Greece.

Serotype 19F pneumococci were a leading cause of infections among children in Athens, Greece during 2001-2006. In total, 143 19F isolates were typed by pulsed-field gel electrophoresis (PFGE), and 38 isolates representing the main PFGE types were also characterised by multilocus sequence typing. A diversity of distinct strains belonging to sequence types 236, 1035, 274, 172 and 319 were identified, but multidrug-resistant isolates related to the Taiwan(19F)-14 clone (ST236) constituted 76.9% of the isolates. Spread of the Taiwan(19F)-14 clone explains, in part, the high incidence of antibiotic resistance observed among pneumococci reported recently from Athens.

Sources of diversity of carbapenem resistance levels in Klebsiella pneumoniae carrying blaVIM-1.

OBJECTIVES: To elucidate the mechanisms responsible for the diversity of beta-lactam resistance phenotypes among isolates of a VIM-1-producing Klebsiella pneumoniae (VPKP) strain that is endemic in Greek hospitals. METHODS: Five VPKP clinical isolates were studied. MICs of beta-lactams were determined by agar dilution. PFGE of XbaI-digested genomic DNA was used for typing. Profiles of outer membrane proteins (OMPs) were determined by SDS-PAGE. Selected isolates were transformed with a plasmid encoding the Omp36K porin. beta-Lactamase activities were analysed by IEF and imipenem hydrolysis was assessed by spectrophotometry. VIM-1-encoding, self-transmissible plasmids were characterized by replicon typing, RFLP and hybridization with bla(VIM)- and IS26-specific probes. Characterization of integrons was performed by PCR, cloning and sequencing. RESULTS: Isolates exhibited highly similar PFGE patterns. Imipenem MICs were 2, 4, 16, 32 and 64 mg/L. The isolate with the highest imipenem MIC (Vipm-64) lacked a 36 kDa OMP. Expression of a cloned OmpK36 in this isolate reduced the imipenem MIC to susceptibility levels. Imipenem-hydrolysing activity was significantly higher in Vipm-16 as compared with the other isolates that expressed similar amounts of VIM-1. All isolates transferred beta-lactam resistance to Escherichia coli through conjugative, IncN plasmids that exhibited differences in the RFLP and hybridization patterns with bla(VIM)- and IS26-specific probes. The Vipm-16 plasmid, mediating the higher imipenem MICs among transconjugants, carried two copies of bla(VIM-1). Cloning and sequencing showed In-e541-like integrons truncated at the 5'CS by insertion of IS26 elements at two different positions. CONCLUSIONS: A VIM-1-producing strain of K. pneumoniae has evolved through OMP alterations and rearrangements in the bla(VIM-1)-carrying plasmid probably mediated by IS26, generating isolates with imipenem MICs ranging from susceptibility to resistance.

Spread of efflux pump-overexpressing, non-metallo-beta-lactamase-producing, meropenem-resistant but ceftazidime-susceptible Pseudomonas aeruginosa in a region with blaVIM endemicity.

OBJECTIVES: To investigate the resistance mechanisms of meropenem-resistant, ceftazidime-susceptible Pseudomonas aeruginosa isolates, in a clinical setting where VIM-2 or VIM-4 metallo-beta-lactamase (MBL)-producing pseudomonads are common. METHODS: During May to December 2003, 13 consecutive meropenem-resistant, ceftazidime-susceptible P. aeruginosa isolates were recovered from separate patients at the University Hospital of Larissa, Thessaly, Greece. The isolates were studied by Etest MBL, PCR for blaVIM, blaIMP and blaSPM genes and PFGE. Experiments were performed to detect synergy between meropenem or other antimicrobials and the efflux pump inhibitor carbonyl cyanide-m-chlorophenylhydrazone (CCCP). The isolates were also tested by PCR and RT-PCR for the expression of the genes mexB and mexY, which encode the efflux pumps MexAB-OprM and MexXY-OprM. RESULTS: Twelve of the isolates, belonging to six distinct PFGE types, gave negative results in the MBL Etest and lacked genes encoding MBLs but exhibited synergy between meropenem and CCCP, indicating that efflux pump activity contributed to the meropenem resistance. All 12 isolates were positive for mexB and 11 were also positive for mexY genes. RT-PCR showed that 10 and five isolates over-expressed mexB and mexY, respectively. One isolate was blaVIM-2-positive and did not show synergy with CCCP, or harbour mexB or mexY. CONCLUSIONS: In our hospital, where MBL-producing P. aeruginosa were previously prevalent, meropenem resistance due to the overexpression of efflux pumps has also now emerged. Early recognition of this resistance mechanism should allow the use of alternative beta-lactams, such as ceftazidime, which would be inactive even against phenotypically susceptible MBL producers.

Repeated occurrence of diverse extended-spectrum beta-lactamases in minor serotypes of food-borne Salmonella enterica subsp. enterica.

Screening of Greek nontyphoid salmonellae from 2000 to 2002 yielded three extended-spectrum beta-lactamase (ESBL)-producing human isolates. Salmonella enterica serotype Brandenburg harbored a multiresistant SHV-5 gene-carrying plasmid. S. enterica serotype Blockley and S. enterica serotype Hadar harbored a TEM-52 gene-carrying plasmid. An S. enterica serotype Virchow strain producing plasmid-mediated CTX-M-32 was isolated twice from poultry end products. All ESBL plasmids were self-transferable and carried by clones currently common in Greece.

Validation of a PCR-based method for detection of food-borne thermotolerant campylobacters in a multicenter collaborative trial.

A PCR-based method for rapid detection of food-borne thermotolerant campylobacters was evaluated through a collaborative trial with 12 laboratories testing spiked carcass rinse samples. The method showed an interlaboratory diagnostic sensitivity of 96.7% and a diagnostic specificity of 100% for chicken samples, while these values were 94.2 and 83.3%, respectively, for pig samples.

Expanded-spectrum cephalosporin resistance in non-typhoid Salmonella.

Expanded-spectrum cephalosporins (ESCs) such as ceftriaxone, together with fluorinated quinolones, are the choice antibiotics in the treatment of invasive salmonella infections. Resistance to ESCs among non-typhoid salmonella has been recognised since the late 1980s. Currently, ESC-resistant salmonella strains are reported world-wide and in some areas their incidence is significant. Resistance is mainly due to acquisition of multi-resistant plasmids encoding a variety of extended-spectrum and AmpC-type beta-lactamases. The origins of ESC-resistant salmonellae are diverse. Exchange of resistance determinants between salmonellae and nosocomial enterobacteria seems to be frequent, at least in developing countries. Also, the use of newer beta-lactams in animal husbandry and veterinary medicine may have facilitated the spread of ESC-resistant salmonella strains in livestock.

A common clone of erythromycin-resistant Streptococcus pneumoniae in Greece and the UK.

OBJECTIVE: To investigate the possible genetic relationship among erythromycin-resistant Streptococcus pneumoniae strains isolated in Greece and the UK. METHODS: During 1995-97, 140 S. pneumoniae strains were isolated from clinical specimens submitted to the microbiology departments of the two main children's hospital in Athens. All erythromycin-resistant strains were further studied with respect to the presence of genes encoding for the two major mechanisms of macrolide resistance, their serotypes, and pulsed-field gel electrophoresis (PFGE) types, in comparison to a previously characterized UK erythromycin-resistant clone. RESULTS: Eleven of the 140 isolates (7.9%) were resistant to erythromycin; nine of these were susceptible to penicillin. Serotyping allocated seven, three and one isolates to serotypes 14, 19F and serogroup 6, respectively. The mefA gene was detected in seven isolates (five serotype 14 and two serotype 19F), ermB in two (one serotype 19F and the serogroup 6 isolate), whilst in the remaining two isolates no resistance gene could be detected by polymerase chain reaction (PCR). Pulsed-field gel electrophoresis of genomic DNA showed that five Greek serotype 14 isolates belonged to the same chromosomal type as the serotype 14 erythromycin-resistant UK clone. CONCLUSIONS: The present study showed that erythromycin resistance among the S. pneumoniae isolates was mostly owing to the efflux mechanism and suggested a possible clonal spread of serotype 14 erythromycin-resistant S. pneumoniae strains between Greece and the UK.

Infrequent detection of acquired metallo-beta-lactamases among carbapenem-resistant Pseudomonas isolates in a Greek hospital.

OBJECTIVE: To study the possible distribution of metallo-beta-lactamases among nosocomial Pseudomonas isolates in a Greek hospital with a recent high prevalence of carbapenem-resistant Pseudomonas isolates. METHODS: All carbapenem-resistant (imipenem- and/or meropenem-resistant) (MICs > 8 mg/L) Pseudomonas non-replicate isolates recovered from clinical infections in the Microbiology Laboratory of Saint Demetrios Hospital, Thessaloniki, Greece, from April 1998 to November 2000 were studied for the presence of metallo-beta-lactamases. They were tested by a disk diffusion test, PCR analysis, and nucleotide sequencing. DNA fingerprints were obtained by pulsed-field gel electrophoresis (PFGE) of XbaI-digested chromosomal DNA. RESULTS: In total, 24 carbapenem-resistant isolates (23 P. aeruginosa and one P. putida) were recovered. The serotypes observed among the P. aeruginosa isolates were, in order of decreasing frequency, O:11 (52%), O:3 and O:12 (17% each), and O:6 (13%). PFGE grouped 17 of the P. aeruginosa isolates into four clusters, each containing from two to seven isolates, while the remaining isolates exhibited unique genotypes. blaVIM-2 was detected in the P. putida isolate and a P. aeruginosa serotype O:3 isolate. The latter strain was genotypically distinct from other contemporaneous or older carbapenem-resistant P. aeruginosa Greek isolates. CONCLUSION: These findings suggest that, although the prevalence of metallo-beta-lactamases is low, the integron-associated blaVIM genes can spread to P. aeruginosa serotypes that have not been previously associated with carbapenem resistance in our region, as well as to other pseudomonal species.