emm Types and clusters and macrolide resistance of pediatric group A streptococcal isolates in Central Greece during 2011-2017.

BACKGROUND: The surveillance of emm types and macrolide susceptibility of group A streptococcus (GAS) in various areas and time periods enhances the understanding of the epidemiology of GAS infections and may guide treatment strategies and the formulation of type-specific vaccines. Greece has emerged as a country with high macrolide use. However, studies suggest a gradual reduction in macrolide consumption after 2007. METHODS: During a 7-year period (2011-2017), 604 GAS isolates were recovered from consecutive children presenting with pharyngeal or nonpharyngeal infections in Central Greece; 517 viable isolates underwent molecular analysis, including emm typing. RESULTS: Isolates belonged to 20 different emm types (in decreasing order of prevalence: 1, 89, 4, 12, 28, 3, 75 and 6, accounting for 88.2% of total isolates). The emm types comprised 10 emm clusters (five most common clusters: E4, A-C3, E1, A-C4 and A-C5). The emm89 isolates were acapsular ('new clade'). Overall macrolide resistance rate was 15.4%, and cMLSB emerged as the predominant resistance phenotype (56.4%). The lowest annual resistance rates occurred in 2014 (13.1%), 2016 (5.5%) and 2017(8.0%) (P for trend = 0.002). Consumption of macrolide/lincosamide/streptogramin B declined by 22.6% during 2011-2017. Macrolide resistance and emm28 and emm77 types were associated (both P<0.001). The most frequently identified genetic lineages of macrolide-resistant GAS included emm28/ST52, emm77/ST63, emm12/ST36, emm89/ST101 and emm4/ST39. We estimated that 98.8% of the isolates belonged to emm types incorporated into a novel 30-valent M protein vaccine. CONCLUSIONS: In Central Greece during 2011-2017, the acapsular emm89 isolates comprised the second most prevalent type. Susceptibility testing and molecular analyses revealed decreasing GAS macrolide resistance rates, which may be attributed to the reduction in the consumption of macrolides and/or the reduced circulation of macrolide-resistant clones in recent years. Such data may provide valuable baseline information in targeting therapeutic intervention and the formulation of type-specific GAS vaccines.

Ceftazidime-Avibactam To Treat Life-Threatening Infections by Carbapenem-Resistant Pathogens in Critically Ill Mechanically Ventilated Patients.

Data on the effectiveness of ceftazidime-avibactam (CAZ-AVI) in critically ill, mechanically ventilated patients are limited. The present retrospective observational cohort study, which was conducted in two general intensive care units (ICUs) in central Greece, compared critically ill, mechanically ventilated patients suffering from carbapenem-resistant Enterobacteriaceae (CRE) infections receiving CAZ-AVI to patients who received appropriate available antibiotic therapy. Clinical and microbiological outcomes and safety issues were evaluated. A secondary analysis in patients with bloodstream infections (BSIs) was conducted. Forty-one patients that received CAZ-AVI (the CAZ-AVI group) were compared to 36 patients that received antibiotics other than CAZ-AVI (the control group). There was a significant improvement in the Sequential Organ Failure Assessment (SOFA) score on days 4 and 10 in the CAZ-AVI group compared to that in the control group (P = 0.006, and P = 0.003, respectively). Microbiological eradication was accomplished in 33/35 (94.3%) patients in the CAZ-AVI group and 21/31 (67.7%) patients in the control group (P = 0.021), and clinical cure was observed in 33/41 (80.5%) versus 19/36 (52.8%) patients (P = 0.010), respectively. The results were similar in the BSI subgroups for both outcomes (P = 0.038 and P = 0.014, respectively). The 28-day survival was 85.4% in the CAZ-AVI group and 61.1% in the control group (log-rank test = 0.035), while there were 2 and 12 relapses in the CAZ-AVI and control groups, respectively (P = 0.042). A CAZ-AVI-containing regime was an independent predictor of survival and clinical cure (odds ratio [OR] = 5.575 and P = 0.012 and OR = 5.125 and P = 0.004, respectively), as was illness severity. No significant side effects were recorded. In conclusion, a CAZ-AVI-containing regime was more effective than other available antibiotic agents for the treatment of CRE infections in the high-risk, mechanically ventilated ICU population evaluated.

Implementation of the Rapid Polymyxin™ NP test directly to positive blood cultures bottles.

The present study assessed the performance of Rapid Polymyxin™ NP test to detect colistin resistance directly from 132 blood cultures found positive for Enterobacterales by FilmArray. Additionally, colistin MICs of isolated microorganisms were determined by the commercial broth microdilution method ComASP™ Colistin, used as the gold standard comparator. The Rapid Polymyxin™ NP test correctly detected all colistin-resistant isolates. However, the test has misidentified as resistant 4 colistin-susceptible isolates (1 Escherichia coli and 3 Klebsiella pneumoniae). Molecular characterization of colistin-resistant isolates showed that K. pneumoniae, which belonged to ST15 and ST147, carried alterations in the mgrB. Moreover, the first Greek mcr-1-positive colistin-resistant E. coli was detected. The rapidity (2-3 h) of the results, combined with its excellent negative predictive value (100%), allows implementation of the test for routine testing of blood cultures mainly in the clinical settings that are endemic for carbapenem-resistant bacteria, avoiding misuse of colistin and preventing the spread of colistin-resistant bacteria.

IncC blaKPC-2-positive plasmid characterised from ST648 Escherichia coli.

OBJECTIVES: This study describes the characterisation of type 2 IncC plasmids pC-Ec20-KPC and pC-Ec2-KPC, carrying theblaKPC-2 gene, from two multiresistant Escherichia coli recovered in University Hospital of Larissa (Greece) in 2018. METHODS: E. coli strains Ec-2Lar and Ec-20Lar were recovered from rectal swabs of two patients during monthly surveillance cultures. Transfer experiments by conjugation were carried out using rifampicin-resistant E. coli A15 laboratory strain as recipient. blaKPC-carrying plasmids were characterised by S1 profiling. Isolates were typed by MLST. Whole-genome sequencing was performed using the Sequel platform. RESULTS: Both E. coli isolates, belonging to ST648, transferred blaKPC-2 to E. coli A15 by conjugation. Plasmid analysis revealed that the transconjugants harboured blaKPC-positive plasmids of different sizes. Analysis of plasmid sequences showed that in both isolates the blaKPC-2 gene was carried on a type 2 IncC plasmid (pC-Ec20-KPC and pC-Ec2-KPC, respectively). Both plasmids carried the ARI-B resistance island consisting of several resistance genes, intact and truncated copies of several mobile elements, and a 25 571-bp segment harbouring coding sequences for an iron transporter. The blaKPC-2 gene was part of transposon Tn4401a, which was bounded by 5-bp direct repeats (TCCTT) suggesting its transposition into the IncC plasmids. CONCLUSION: To our knowledge, this is the first report on complete nucleotide sequences of type 2 IncC plasmids. These findings, which hypothesise the acquisition of KPC-2-encoding transposon Tn4401a by an IncC replicon, indicate the ongoing need for molecular surveillance studies of multidrug-resistant pathogens. In addition, they underline the increasing clinical importance of the IncC plasmid family.

Antimicrobial Agent Susceptibility and Typing of Staphylococcal Isolates from Subclinical Mastitis in Ewes.

Objective was to study susceptibility to antimicrobial agents of 142 staphylococcal isolates from subclinical mastitis in ewes. In total, 41.5% of these were resistant and 5.6% multidrug resistant. More coagulase-negative staphylococci (47.0%) were resistant than Staphylococcus aureus (18.5%) isolates. Resistance was greater to penicillin (22.5%), tetracycline, or ampicillin (18.3%). More biofilm-forming (20.6%) isolates were resistant to tetracycline than nonbiofilm-forming (0.0%) ones. Presence of tetK was associated with presence of icaA in the same strains. Further, 76.6% of resistant isolates versus 57.7% of susceptible ones were recovered immediately postpartum and 23.4% of resistant isolates versus 9.9% of susceptible ones were recovered in farms that practiced routine administration of antimicrobial agents at the end of a lactation period. Most S. aureus (59.3%) were classified in ST133 and most Staphylococcus epidermidis were classified in ST100, ST142, or ST152 (19.0% each). There was no association of sequence types with resistance. Whole genome sequencing showed that, in a Staphylococcus lentus strain, the ermB gene was part of transposon Tn917 integrated into the chromosome; also, a small plasmid was observed in an ermC-carrying Staphylococcus hominis strain and, finally, in an S. aureus and an S. epidermidis strains, small tetK-carrying plasmids (pSau-2716Lar, pSau-3893Lar) of 4.439 kb were found.

The clinical significance of carbapenem-resistant Klebsiella pneumoniae rectal colonization in critically ill patients: from colonization to bloodstream infection.

PURPOSE: To highlight the clinical significance of carbapenem-resistant Klebsiella pneumoniae (CRKP) rectal colonization by examining the risk factors for CRKP rectal colonization and subsequent bloodstream infection (BSI) in critically ill patients. METHODOLOGY: Prospective study of CRKP rectal colonization in an intensive care unit (ICU) during a 39-month period. CRKP strains isolated from both the blood cultures and corresponding rectal specimens (n=96) of patients were screened by PCR for the presence of antibiotic resistance-associated genes. Molecular analyses were conducted to investigate the clonal relatedness of CRKP strains from the rectal and blood specimens. RESULTS: Among the 498 patients, 226 were rectally colonized by CRKP, 48 of whom developed a CRKP BSI. The median time from hospital admission to the detection of CRKP rectal colonization was 8 days, while the median time from colonization to BSI was 4 days. The duration of ICU stay, patient/nurse ratio and prior use of antianaerobic antimicrobials were associated with CRKP rectal colonization. No specific factor was associated with BSIs in the colonized patients. The blaKPC-2 gene was detected in all 96 strains, which were all classified as sequence type ST-258. Representative pairs (n=48) of CRKP strains colonizing and infecting the same patient shared the same pulsotype. CONCLUSION: Our results indicate that hospitalized patients become infected with their colonizing strains, supporting the strong association between colonization and BSI. Limiting antianaerobic antimicrobial administration, reducing the duration of ICU stay and maintaining a low patient/nurse ratio are possible strategies to restrict rectal CRKP colonization in ICUs.

MLSB-Resistant Staphylococcus aureus in Central Greece: Rate of Resistance and Molecular Characterization.

The aim of the present study was to determine the rate and mechanisms of resistance to macrolides, lincosamides, and streptogramin B (MLSB) antibiotics of Staphylococcus aureus collected in Central Greece. Of the 2,893 S. aureus collected during 2012-2017, 1,161 isolates (40.2%) exhibited resistance to at least one of the MLSB agents. The rate of erythromycin resistance was statistically significantly higher in methicillin-resistant S. aureus (MRSA) (58.6%) than in methicillin-sensitive S. aureus (MSSA) isolates (20.7%) (p = 0.002). Two hundred seventy-five representative MLSB-resistant S. aureus, including 81 MSSA and 194 MRSA isolates, were further studied. Thirty-eight MSSA isolates carried ermC, 26 MSSA were positive for ermA, whereas 17 isolates carried msrA gene. Among MRSA, the ermA gene was identified in the majority of the isolates (n = 153). Thirty-seven MRSA isolates carried ermC; three isolates carried msrA, whereas the remaining MRSA was positive for two genes (ermA and ermC). Phylogenetic analysis showed that ST225, which belongs to CC5, was the most prevalent, accounting for 137 MRSA isolates. Higher genetic diversity was found in the group of MSSA isolates, which comprised of 13 sequence types. Whole-genome sequencing data showed that all ermA-positive S. aureus, with the exception of one ST398 isolate, harbored the ermA-carrying Tn554 transposon integrated into their chromosomes. Furthermore, Illumina sequencing followed by polymerase chain reaction screening identified that ermC, which was identified in a polyclonal population of MSSA and MRSA isolates, was carried by small plasmids, like pNE131. These findings highlighted the important role of high-risk clones and of mobile elements carrying resistance genes in the successful dissemination of MLSB-resistant staphylococci.

Evaluation of rapid polymyxin NP test to detect colistin-resistant Klebsiella pneumoniae isolated in a tertiary Greek hospital.

The efficiency of Rapid Polymyxin NP test for detection of colistin-resistant isolates was tested against a collection of 131 non-repetitive Klebsiella pneumoniae, including 98 colistin-resistant and 33 colistin-susceptible isolates. In addition, the performance of this test was compared with those of the automated systems, BD Phoenix™ and VITEK®2, and the Etest. Determination of imipenem and meropenem MICs showed that 95 of colistin-resistant (Col-R) isolates were also resistant to at least one carbapenem. Characterization of colistin resistance mechanisms showed that 75 out of 98 Col-R isolates were associated with the presence of alterations in the mgrB gene, while no mcr genes were detected among our isolates. Rapid Polymyxin NP correctly detected 97 out of 98 colistin-resistant isolates (Geometric mean MIC value 9.89 mg/L), except one ST147 K. pneumoniae harboring a wild-type mgrB gene (MIC: 8 mg/L), yielding a sensitivity 99%. The other methods gave more false-negative results with colistin-resistant strains; BD Phoenix™,VITEK®2, and the gradient Etest missed five, two and three colistin-resistant, respectively (95%, 98% and 97%). The Rapid Polymyxin NP test gave false positive results with six isolates, for which colistin MICs were 1-2 mg/L (specificity 82%). Despite the fact that Rapid Polymyxin exhibited lower specificity than other methods (82% versus 94%, 88% and 85%), it is easy-to-perform and rapid. Thus, these findings indicate that the Rapid Polymyxin NP test can be an initial tool for the detection of colistin-resistant isolates.

First Description in Greece of mphC-Positive Staphylococci Causing Subclinical Mastitis in Ewes.

The aim of the present study was to describe the first mphC-positive staphylococci, including two Staphylococcus lentus (Sle-087lar and Sle-091lar) and one Staphylococcus xylosus (Sxy-228lar), isolated from samples of animal origin, in Greece. Isolates Sle-087lar and Sxy-228lar were resistant to erythromycin, whereas Sle-091lar was resistant to erythromycin and lincomycin. All three isolates were susceptible to the remaining antibiotics. PCR screening showed that isolate Sle-091lar carried also ermB. For Sxy-228lar, whole-genome sequencing (WGS) and de novo assembly obtained an mphC-positive contig of 57.3-kb exhibiting high similarity with the genome of mphC-negative S. xylosus S170. However, mphC of Sxy-228lar was 91% similar to that found in plasmid pJW2311 from S. xylosus JW2311. Additionally, WGS data showed that Sle-087lar and Sle-091lar harbored mphC-carrying sequences being highly similar to the recently announced genome of the mphC-carrying S. lentus isolate 050AP from Tanzania. However, differences were observed in the mphC environment, suggesting the independent acquisition of the gene by each isolate. Sle-091lar also harbored transposon Tn917, which carries ermB resistance gene, integrated into S. lentus chromosome. These findings indicated that acquisition of resistance genes can lead to the emergence of multiresistant staphylococci, causing animal infections with economic burden.

Emergence of Escherichia coli sequence type 410 (ST410) with KPC-2 ß-lactamase.

Fifteen carbapenem-non-susceptible Escherichia coli isolates obtained during the period May 2010 to April 2011 in a hospital and a long-term care facility (LTCF) in Larissa (Central Greece) were investigated. Minimum inhibitory concentrations (MICs) to various antimicrobial agents were determined by Etest. Carriage of bla genes, including bla(KPC-2) and bla(CTX-M), was documented by polymerase chain reaction (PCR) and sequencing. Production of ß-lactamases was confirmed by isoelectric focusing. Transfer of resistance was carried out by conjugation. Plasmid incompatibility groups were determined by PCR-based replicon typing and replicon sequence typing. Isolates were genotyped by multilocus sequence typing. Ten E. coli isolates with KPC-2 were derived from seven patients in the University Hospital of Larissa. Six patients had previously been treated for prolonged time periods in a LTCF located in the same city. The remaining isolate was from a patient previously treated in an Athens hospital. Screening of faecal samples from 20 randomly selected LTCF patients yielded eight enterobacteria with KPC-2, of which five were E. coli, showing the wide spread of KPC-2-producers in this institution and confirming that it was the focus of the outbreak. Fourteen of the isolates were classified as sequence type 410 (ST410); the remaining isolate belonged to a novel ST (ST2281). All 15 isolates carried a KPC-2-encoding plasmid of the Inc group FIIK. Additional plasmids encoding enzymes of the CTX-M-1 family were identified in 11 isolates. The bla(KPC-2)-carrying plasmid IncFIIK, widespread amongst Klebsiella pneumoniae in Greece, has probably been acquired by E. coli ST410 known to be associated with CTX-M production. Diffusion of bla(KPC-2) in common pathogens such as E. coli is of concern.