Whole-Genome Sequencing and Molecular Analysis of Ceftazidime-Avibactam-Resistant KPC-Producing Klebsiella pneumoniae from Intestinal Colonization in Elderly Patients.

Ceftazidime-avibactam (CAZ-AVI) is an active antibiotic combination of a ß-lactam-ß-lactamase inhibitor against carbapenemase-producing Enterobacterales. Reports of resistance to CAZ-AVI other than metallo-ß-lactamases have increased in recent years. The aim of this study was to analyze KPC-Klebsiella pneumoniae (KP) isolates resistant to CAZ-AVI from the intestinal carriage of hospitalized elderly patients in Italy, in February 2018-January 2020. Characterization of CAZ-AVI-resistant KP isolates, including MLST, resistome, virulome and plasmid content, was performed by WGS analysis. Out of six CAZ-AVI-resistant KP isolates, three belonged to ST101 and three to ST512; two isolates produced KPC-3 (both ST512), four had mutated KPC-3 (KPC-31, in ST101 and ST512, and KPC-46, both ST101). All CAZ-AVI-resistant KP isolates were multidrug-resistant and carried several resistance genes. The yersiniabactin ybt9 gene cluster was present in all ST101 isolates, while, in ST512 isolates, no virulence genes were detected. Several plasmids were detected: IncF was present in all isolates, as well as IncR and Col440 in ST101 and IncX3 in ST512 isolates. In conclusion, it is important to monitor the circulation of K. pneumoniae resistant to CAZ-AVI to prevent the spread of clones causing difficult-to-treat infections. The presence of mutated KPC-3 in high-risk K. pneumoniae clones resistant to CAZ-AVI in hospitalized patients deserves attention.

Whole Genome Sequencing and Molecular Analysis of Carbapenemase-Producing Escherichia coli from Intestinal Carriage in Elderly Inpatients.

The spread of carbapenemase-producing (CP) Enterobacterales is currently a worldwide concern, especially in the elderly. Twelve CP-E. coli isolated from rectal swabs of colonized inpatients aged ≥65 years from four hospitals in two Italian cities (Milan and Rome) were analyzed by whole genome sequencing (WGS) to obtain multi-locus sequence typing (MLST), identification of carbapenemase-encoding genes, resistome, plasmid content, and virulence genes. MLST analysis showed the presence of 10 unrelated lineages: ST410 (three isolates from three different hospitals in two cities) and ST12, ST38, ST69, ST95, ST131, ST189, ST648, ST1288, and ST1598 (one isolate each). Most isolates (9/12, 75%) contained a serine-ß-lactamase gene (5 blaKPC-3, 2 blaKPC-2, and 2 blaOXA-181), while three isolates harbored a metallo-ß-lactamase gene (two blaNDM-5 and one blaVIM-1). In most CP-E. coli, the presence of more than one plasmid was observed, with the predominance of IncF. Several virulence genes were detected. All isolates contained genes enhancing the bacterial fitness, such as gad and terC, and all isolates but one, fimH, encoding type 1 fimbriae. In conclusion, CP-E. coli clones colonizing elderly patients showed heterogeneous genetic backgrounds. We recommend strict surveillance to monitor and prevent the spread of successful, high-risk clones in healthcare settings.

Virulence Determinants in Staphylococcus aureus Clones Causing Osteomyelitis in Italy.

Staphylococcus aureus is the most common pathogen causing osteomyelitis (OM). The aim of this study was to explore the clonal complex (CC) distribution and the pattern of virulence determinants of S. aureus isolates from OM in Italy. Whole-genome sequencing was performed on 83 S. aureus isolates from OM cases in six hospitals. Antibiotic susceptibility tests showed that 30.1% of the isolates were methicillin-resistant S. aureus (MRSA). The most frequent CCs detected were CC22, CC5, CC8, CC30, and CC15, which represent the most common lineages circulating in Italian hospitals. MRSA were limited in the number of lineages (CC22, CC5, CC8, and CC1). Phylogenetic analysis followed the sequence type-CC groupings and revealed a non-uniform distribution of the isolates from the different hospitals. No significant difference in the mean number of virulence genes carried by MRSA or MSSA isolates was observed. Some virulence genes, namely cna, fib, fnbA, coa, lukD, lukE, sak, and tst, were correlated with the CC. However, different categories of virulence factors, such as adhesins, exoenzymes, and toxins, were frequently detected and unevenly distributed among all lineages. Indeed, each lineage carried a variable combination of virulence genes, likely reflecting functional redundancy, and arguing for the importance of those traits for the pathogenicity in OM. In conclusion, no specific genetic trait in the most frequent lineages could explain their high prevalence among OM isolates. Our findings highlight that CCs detected in OM isolates follow the epidemiology of S. aureus infections in the country. It is conceivable that any of the most common S. aureus CC can cause a variety of infections, including OM.

Staphylococcus aureus clones causing osteomyelitis: a literature review (2000-2020).

OBJECTIVES: Staphylococcus aureus is the most common causative organism of osteomyelitis (OM). Nevertheless, the molecular epidemiology of S. aureus causing OM remains ill-defined. This study aimed to address the global epidemiology of S. aureus clones from OM patients. METHODS: Literature databases were searched for studies reporting the molecular typing of S. aureus involved in OM published between 1 January 2000 and 29 July 2020. Data from 32 articles that fulfilled the inclusion criteria were analysed for year of publication, country of patients, methicillin susceptibility and genotypic characteristics of S. aureus isolates. RESULTS: Pandemic clones CC5, CC8, CC22, CC30 and CC45 were the most common in OM. The distribution of clones differed greatly among studies owing to the local epidemiology of S. aureus and the MSSA heterogeneity. PVL-positive MRSA clones belonging to ST80/CC80 and ST8/CC8/USA300 were the most common among paediatric patients in Europe and the USA; greater variability was observed in the adult population. In Europe, MRSA belonged to PVL-negative CC5, CC8 and CC22 indicating a nosocomial origin of infections; in Asia PVL-positive ST59/CC59 MRSA was the most frequent. PVL-positive clones were often detected in haematogenous OM in children and adults. Although MSSA were polyclonal, PVL-negative ST398/CC398 MSSA was the most prevalent clone in diabetic foot OM. CONCLUSION: All major S. aureus clones circulating both in hospital and community settings appear to be capable of causing OM. Future studies reporting molecular typing and genomic data will provide more insights into the epidemiology and pathobiology of S. aureus clones causing OM.

The changing epidemiology of carbapenemase-producing Klebsiella pneumoniae in Italy: toward polyclonal evolution with emergence of high-risk lineages.

BACKGROUND: Previous studies showed that the epidemic of carbapenem-resistant Klebsiella pneumoniae (CR-KP) observed in Italy since 2010 was sustained mostly by strains of clonal group (CG) 258 producing KPC-type carbapenemases. In the framework of the National Antibiotic-Resistance Surveillance (AR-ISS), a countrywide survey was conducted in 2016 to explore the evolution of the phenotypic and genotypic characteristics of CR-KP isolates. METHODS: From March to July 2016, hospital laboratories participating in AR-ISS were requested to provide consecutive, non-duplicated CR-KP (meropenem and/or imipenem MIC >1 mg/L) from invasive infections. Antibiotic susceptibility was determined according to EUCAST recommendations. A WGS approach was adopted to characterize the isolates by investigating phylogeny, resistome and virulome. RESULTS: Twenty-four laboratories provided 157 CR-KP isolates, of which 156 were confirmed as K. pneumoniae sensu stricto by WGS and found to carry at least one carbapenemase-encoding gene, corresponding in most cases (96.1%) to blaKPC. MLST- and SNP-based phylogeny revealed that 87.8% of the isolates clustered in four major lineages: CG258 (47.4%), with ST512 as the most common clone, CG307 (19.9%), ST101 (15.4%) and ST395 (5.1%). A close association was identified between lineages and antibiotic resistance phenotypes and genotypes, virulence traits and capsular types. Colistin resistance, mainly associated with mgrB mutations, was common in all major lineages except ST395. CONCLUSIONS: This WGS-based survey showed that, although CG258 remained the most common CR-KP lineage in Italy, a polyclonal population has emerged with the spread of the new high-risk lineages CG307, ST101 and ST395, while KPC remained the most common carbapenemase.

Whole genome sequencing of macrolide resistant Streptococcus pneumoniae serotype 19A sequence type 416.

BACKGROUND: The resistance of Streptococcus pneumoniae to macrolides is becoming an increasingly important issue and thus it is important to understand the genetics related to adaptation of this species to the widespread use of antibiotics in Europe. The 58 isolates of S. pneumoniae belonging to sequence type (ST) 416 and serotype 19A and to several different phenotypes originated from Italy, Portugal and Czech Republic were thus sequenced on Illumina MiSeq. The aim of the study was to describe genetical origine of isolates, investigate their macrolide resistance and suggest reasons for spread of ST416 in the Czech Republic. RESULTS: Investigation of genes associated with serotype determined serotype switch between 15B and 19A serotypes and core genome multilocus sequence typing (cgMLST) confirmed the origine of concerned isolates in Netherlands15B-37 clone. Inspected genomes proved variability of genes associated with the macrolide resistance even within closely genetically relative isolates. CONCLUSIONS: Participation of 19A/ST416 on the spread of Netherlands15B-37 is accompanied by serotype switch between 19A and 15B serotypes and with acquisition of genes involved in macrolide resistance to the clone that was originally macrolide susceptible. There is evident tendency to interchanging and modifications of these and surrounding genes, that could lead to accelerate spreading of this sequence type in regions with high macrolide consumption.

Cross-border spread of blaNDM-1- and blaOXA-48-positive Klebsiella pneumoniae: a European collaborative analysis of whole genome sequencing and epidemiological data, 2014 to 2019.

Analysis of sequencing data for 143 blaNDM-1- and blaOXA-48-positive Klebsiella pneumoniae isolates from 13 European national collections and the public domain resulted in the identification of 15 previously undetected multi-country transmission clusters. For 10 clusters, cases had prior travel/hospitalisation history in countries outside of the European Union including Egypt, Iran, Morocco, Russia, Serbia, Tunisia and Turkey. These findings highlight the benefit of European whole genome sequencing-based surveillance and data sharing for control of antimicrobial resistance.

First detection of autochthonous extensively drug-resistant NDM-1 Pseudomonas aeruginosa ST235 from a patient with bloodstream infection in Italy, October 2019.

BACKGROUND: Pseudomonas aeruginosa (PA) is one of the most common and serious causes of healthcare-associated bacteremia. The emergence and dissemination of multidrug-resistant (MDR) and extensively drug-resistant (XDR) PA strains pose a major clinical concern. ST235-PA is a high-risk clone which shows a high capacity to acquire antibiotic resistance. Here we describe the first autochthonous New Delhi metallo-ß-lactamase (NDM)-producing Pseudomonas aeruginosa ST235 identified in Italy. CASE PRESENTATION: In October 2019, a patient residing in an elderly health care and rehabilitation facility, was hospitalized and died from sepsis caused by an XDR-PA. The strain belonged to the high-risk clone sequence type ST235. Whole genome sequencing (WGS) revealed the presence of genes encoding NDM-1 and multiple ß-lactamases, many clinically significant multidrug efflux pump complexes and also the virulence gene ExoU, which is associated with a high cytotoxic phenotype. CONCLUSIONS: Few strains of NDM-1-PA have been identified worldwide, all belonging to ST235. The combination of ST235 and ExoU is a predictor of highly unfavorable prognosis. The potential spread of these high-risk clones in healthcare settings is worrisome because treatment options are limited. Early identification of high-risk clones could help in outbreaks investigation and infections control.

Two multi-fragment recombination events resulted in the ß-lactam-resistant serotype 11A-ST6521 related to Spain9V-ST156 pneumococcal clone spreading in south-western Europe, 2008 to 2016.

BackgroundThe successful pneumococcal clone Spain9V-ST156 (PMEN3) is usually associated with vaccine serotypes 9V and 14.AimOur objective was to analyse the increase of a serotype 11A variant of PMEN3 as cause of invasive pneumococcal disease (IPD) in Spain and its spread in south-western Europe.MethodsWe conducted a prospective multicentre study of adult IPD in Spain (2008-16). Furthermore, a subset of 61 penicillin-resistant serotype 11A isolates from France, Italy, Portugal and Spain were subjected to whole genome sequencing (WGS) and compared with 238 genomes from the European Nucleotide Archive (ENA).ResultsAlthough the incidence of serotype 11A in IPD was stable, a clonal shift was detected from CC62 (penicillin-susceptible) to CC156 (penicillin-resistant). By WGS, three major 11A-CC156 lineages were identified, linked to ST156 (n = 5 isolates; France, Italy and Portugal), ST166 (n = 4 isolates; France and Portugal) and ST838/6521 (n = 52 isolates; France, Portugal and Spain). Acquisition of the 11A capsule allowed to escape vaccine effect. AP200 (11A-ST62) was the donor for ST156 and ST838/6521 but not for ST166. In-depth analysis of ST838/6521 lineage showed two multi-fragment recombination events including four and seven fragments from an 11A-ST62 and an NT-ST344 representative, respectively.ConclusionThe increase in penicillin-resistant serotype 11A IPD in Spain was linked to the spread of a vaccine escape PMEN3 recombinant clone. Several recombination events were observed in PMEN3 acquiring an 11A capsule. The most successful 11A-PMEN3 lineage spreading in south-western Europe appeared after two multi-fragment recombination events with representatives of two major pneumococcal clones (11A-ST62 and NT-ST344).

Daptomycin Resistant Staphylococcus aureus Clinical Strain With Novel Non-synonymous Mutations in the mprF and vraS Genes: A New Insight Into Daptomycin Resistance.

Objectives: Daptomycin (DAP) resistance in Staphylococcus aureus is uncommon but there are increasing reports of the emergence of resistance during DAP therapy. Most clinical DAP-resistant S. aureus isolates investigated carried mutations in the mprF gene. The aim of this study was to identify mutations between a clinical pair of methicillin-susceptible S. aureus (MSSA) isolates (DAP-susceptible and DAP-resistant). Additionally, the activity of genes previously associated with DAP resistance was assessed. Materials and Methods: Two MSSA isolates from patient with left-sided endocarditis were analyzed by whole genome sequencing (WGS) and reverse transcription-quantitative real-time PCR (RT-qPCR). The first isolate, DAP-susceptible, was obtained before initiation of treatment and the second isolate, DAP-resistant, was recovered after 4 weeks of DAP therapy. Results: Comparison of complete genomes of DAP-susceptible and its DAP-resistant variant identified two non-synonymous and one synonymous mutations. The non-synonymous mutations consisted of a S829L substitution in mprF and a T331I substitution in vraS. The RT-qPCR experiments revealed an increased expression of vraS, dltA, mprF, and sceD genes in DAP-resistant variant. Strikingly, the expression of dltA and mprF genes was significantly downregulated by DAP. Conclusion: The mprF and vraS genes were previously associated with DAP resistance, however, none of the mutations described in this study had been previously identified and linked to DAP resistance. Moreover, we provide a new insight into the DAP action on S. aureus, in which the expression of key genes in DAP resistance is decreased by the antibiotic.

Update on prevalence and mechanisms of resistance to linezolid, tigecycline and daptomycin in enterococci in Europe: Towards a common nomenclature.

Vancomycin-resistant enterococci (VRE) are important nosocomial pathogens. Invasive VRE infections are difficult to treat since common therapeutic options including ampicillin and glycopeptides often fail. In vitro, most VRE remain susceptible to last-resort antibiotics such as linezolid, tigecycline and daptomycin. However, neither tigecycline nor linezolid act in a bactericidal manner, and daptomycin has proven activity only at high dosages licensed for treating enterococcal endocarditis. Despite these pharmacological and therapeutic limitations, reports on resistance to these last-resort drugs in VRE, and enterococci in general, have increased in recent years. In this review, we briefly recapitulate the current knowledge on the mode of action as well as the known and novel mechanisms of resistance and describe surveillance data on resistance to linezolid, tigecycline and daptomycin in enterococci. In addition, we also suggest a common nomenclature for designating enterococci and VRE with resistances to these important last-resort antibiotics.

Impact of pneumococcal conjugate vaccine (PCV7 and PCV13) on pneumococcal invasive diseases in Italian children and insight into evolution of pneumococcal population structure.

BACKGROUND: The use of PCV7 for children immunization was gradually implemented in the Italian regions starting from 2006 and was replaced by PCV13 in 2010-2011. In this study we aimed to assess the PCV impact on invasive pneumococcal diseases (IPD) incidence, serotype distribution and antibiotic resistance in Italian children under 5years old. METHODS: All IPD cases in children from 5 Italian regions (Emilia-Romagna, Lombardia, A. P. Bolzano, A. P. Trento, and Piemonte) reported through the nationwide surveillance system during 2008-2014 were included in this study. Pneumococcal isolates were subjected to serotyping, antibiotic susceptibility testing, and clonal analysis according to standard methods. RESULTS: During the study period overall IPD incidence decreased from 7.8 cases/100,000 inhabitants in 2008 to 3.0 cases/100,000 in 2014 (61% decrease, P<0.001). In particular, from 2008 to 2014, PCV7-type IPD decreased from 2.92 to 0.13 cases/100,000 inhabitants (95% decrease, P<0.001) while PCV13-non-PCV7 type IPD decreased from 3.2 to 0.89 cases/100,000 inhabitants (72% decrease, P=0.008). Conversely, non-vaccine serotype (NVS) IPD increased overtime, becoming more common than PCV13 serotype IPD in 2013-2014. Emergent NVS 24F and 12F were the most prevalent in 2014. Antibiotic resistance testing revealed an overall increasing trend in penicillin resistance, from 14% in 2008 to 23% in 2014. Erythromycin resistance showed a downward trend, from 38% in 2008 to 27% in 2014. While in 2008 PCV13 serotypes were the major responsible for antibiotic resistance, during the following years antimicrobial resistance due to NVS increased, mainly as a result of expansion of pre-existing clones. CONCLUSIONS: Both PCVs led to a substantial decrease in vaccine-related IPD incidence in the children population. However NVS-related IPD increased, becoming the most prevalent in the last two-years period. Continuous surveillance is an essential tool to monitor evolution of pneumococcal population causing IPD in children.

ICESpy009, a Conjugative Genetic Element Carrying mef(E) in Streptococcus pyogenes.

Efflux-mediated macrolide resistance due to mef(E) and mel, carried by the mega element, is common in Streptococcus pneumoniae, for which it was originally characterized, but it is rare in Streptococcus pyogenes In S. pyogenes, mega was previously found to be enclosed in Tn2009, a composite genetic element of the Tn916 family containing tet(M) and conferring erythromycin and tetracycline resistance. In this study, S. pyogenes isolates containing mef(E), apparently not associated with other resistance determinants, were examined to characterize the genetic context of mega. By whole-genome sequencing of one isolate, MB56Spyo009, we identified a novel composite integrative and conjugative element (ICE) carrying mega, designated ICESpy009, belonging to the ICESa2603 family. ICESpy009 was 55 kb long, contained 61 putative open reading frames (ORFs), and was found to be integrated into hylA, a novel integration site for the ICESa2603 family. The modular organization of the ICE was similar to that of members of the ICESa2603 family carried by different streptococcal species. In addition, a novel cluster of accessory resistance genes was found inside a region that encloses mega. PCR mapping targeting ICESpy009 revealed the presence of a similar ICE in five other isolates under study. While in three isolates the integration site was the same as that of ICESpy009, in two isolates the ICE was integrated into rplL, the typical integration site of the ICESa2603 family. ICESpy009 was able to transfer macrolide resistance by conjugation to both S. pyogenes and S. pneumoniae, showing the first evidence of the transferability of mega from S. pyogenes.

Invasive pneumococcal disease in children and adults in seven Italian regions after the introduction of the conjugate vaccine, 2008-2014.

OBJECTIVE: To describe the trend of invasive pneumococcal disease in the years 2008-2014; to verify the impact of the conjugate vaccine and monitor the occurrence of serotype replacement. DESIGN: Prospective observational study based on data from the national surveillance for invasive bacterial diseases coordinated by the Istituto superiore di sanità. SETTING AND PARTICIPANTS: Seven Italian regions (A.P. Bolzano, A.P. Trento, Emilia-Romagna, Friuli-Venezia Giulia, Lombardia, Piemonte, Veneto), accounting for 43% of the national population. MAIN OUTCOME MEASURES: Number of cases and incidence of invasive pneumococcal diseases: global, stratified by age groups and by serotypes included or not in the PCV13. RESULTS: In 2008-2014, in the 0-4 age group IPD incidence for all serotypes decreased from 7.1 to 2.9/100,000; incidence for vaccine serotypes (VT) decreased from 5.5 to 1.1/100,000, while incidence for non-vaccine serotypes (NVT) increased from 1.6 to 2.0/100,000 (2.5 in 2013). In the >64 age group, IPD incidence increased from 5.3 to 7.5/100,000; VT incidence decreased from 3.9 to 3.2 (4.9 in 2010 and 4.3 in 2013), whereas NVT incidence increased from 1.4 to 4.4/100,000. CONCLUSION: Use of the conjugate vaccine has reduced the number of cases of IPD by VT in children; the increase in IPD by NVT, above all in older age groups, suggests a serotype replacement.

Tn5253 family integrative and conjugative elements carrying mef(I) and catQ determinants in Streptococcus pneumoniae and Streptococcus pyogenes.

The linkage between the macrolide efflux gene mef(I) and the chloramphenicol inactivation gene catQ was first described in Streptococcus pneumoniae (strain Spn529), where the two genes are located in a module designated IQ element. Subsequently, two different defective IQ elements were detected in Streptococcus pyogenes (strains Spy029 and Spy005). The genetic elements carrying the three IQ elements were characterized, and all were found to be Tn5253 family integrative and conjugative elements (ICEs). The ICE from S. pneumoniae (ICESpn529IQ) was sequenced, whereas the ICEs from S. pyogenes (ICESpy029IQ and ICESpy005IQ, the first Tn5253-like ICEs reported in this species) were characterized by PCR mapping, partial sequencing, and restriction analysis. ICESpn529IQ and ICESpy029IQ were found to share the intSp 23FST81 integrase gene and an identical Tn916 fragment, whereas ICESpy005IQ has int5252 and lacks Tn916. All three ICEs were found to lack the linearized pC194 plasmid that is usually associated with Tn5253-like ICEs, and all displayed a single copy of a toxin-antitoxin operon that is typically contained in the direct repeats flanking the excisable pC194 region when this region is present. Two different insertion sites of the IQ elements were detected, one in ICESpn529IQ and ICESpy029IQ, and another in ICESpy005IQ. The chromosomal integration of the three ICEs was site specific, depending on the integrase (intSp 23FST81 or int5252). Only ICESpy005IQ was excised in circular form and transferred by conjugation. By transformation, mef(I) and catQ were cotransferred at a high frequency from S. pyogenes Spy005 and at very low frequencies from S. pneumoniae Spn529 and S. pyogenes Spy029.

Pneumococcal carriage in young children one year after introduction of the 13-valent conjugate vaccine in Italy.

BACKGROUND: In mid 2010, the 7-valent pneumococcal conjugate vaccine (PCV7) was replaced by the 13-valent conjugate vaccine (PCV13) for childhood immunization in Italy. Our objective in this study was to obtain a snapshot of pneumococcal carriage frequency, colonizing serotypes, and antibiotic resistance in healthy children in two Italian cities one year after PCV13 was introduced. METHODS: Nasopharyngeal swabs were obtained from 571 children aged 0-5 years from November 2011-April 2012. Pneumococcal isolates were serotyped and tested for antimicrobial susceptibility. Penicillin and/or erythromycin non-susceptible isolates were analyzed by Multi Locus Sequence Typing (MLST). RESULTS: Among the children examined, 81.2% had received at least one dose of PCV7 or PCV13 and 74.9% had completed the recommended vaccination schedule for their age. Among the latter, 57.3% of children had received PCV7, 27.1% PCV13, and 15.6% a combination of the two vaccines. The overall carriage rate was 32.9%, with children aged 6-35 months the most prone to pneumococcal colonization (6-23 months OR: 3.75; 95% CI: 2.19-6.43 and 24-35 months OR: 3.15, 95%CI: 2.36-4.22). A total of 184 pneumococcal isolates were serotyped and divided into PCV7 (5.4%), PCV13 (18.0%), and non-PCV13 (82.0%) serotypes. Serotypes 6C, 24F, and 19A were the most prevalent (10.3%, 8.6%, and 8.1%, respectively). The proportion of penicillin non-susceptible (MIC >0.6 mg/L) isolates was 30.9%, while 42.3% were erythromycin resistant. Non-PCV13 serotypes accounted for 75.4% and 70.8% of the penicillin and erythromycin non-susceptible isolates, respectively. CONCLUSIONS: Our results revealed low rates of PCV7 and PCV13 serotypes in Italian children, potentially due to the effects of vaccination. As the use of PCV13 continues, its potential impact on vaccine serotypes such as 19A and cross-reactive serotypes such as 6C will be assessed, with this study providing a baseline for further analysis of surveillance isolates.

Increase of pneumococcal serotype 19A in Italy is due to expansion of the piliated clone ST416/CC199.

The emergence of Streptococcus pneumoniae serotype 19A, following use of the heptavalent pneumococcal conjugate vaccine (PCV7), has been favoured by multiple antibiotic resistance of this serotype and by other unknown factors. The aim of this study was to examine 19A isolates from invasive pneumococcal diseases (IPD) obtained before and after PCV7 implementation to ascertain which characteristics, including the presence of pili, might have favoured the emergence of this serotype in Italy. All S. pneumoniae isolates from IPD collected at the Italian National Institute of Health in the years 2001-2003 and 2006-2009 were serotyped. The 19A isolates were submitted to antimicrobial susceptibility testing by Etest and were genotyped by a combination of pulsed field gel electrophoresis (PFGE) and Multi Locus Sequence Typing (MLST). The presence of the pilus islets PI-1 and PI-2 was detected by PCR assays targeting a marker gene in each islet. The proportion of 19A isolates from IPD significantly increased from 4 % in 2001-2003 to 12 % in 2006-2009. This was largely due to the expansion of a clone characterized by sequence type (ST) 416, clonal complex (CC) 199, already present in Italy before PCV7 implementation. This clone included isolates susceptible to penicillin and containing PI-1 genes. Other CCs contributed to the emergence of serotype 19A: CC63 and CC193, already present in 2001-2003, and new-emerging CCs or clones such as CC230, CC320 and ST5204, that include drug-resistant and/or pilus-positive isolates. The expansion of serotype 19A in Italy might have been favoured not only by antibiotic resistance, but also by other bacterial factors such as the presence of pili.

Serotype and clonal evolution of penicillin-nonsusceptible invasive Streptococcus pneumoniae in the 7-valent pneumococcal conjugate vaccine era in Italy.

The percentage of invasive penicillin-nonsusceptible pneumococci (PNSSP) isolated in Italy in the seven-valent pneumococcal conjugate vaccine (PCV7) era moderately increased in comparison to the pre-PCV7 era. Increase of nonvaccine serotypes was observed among PNSSP. The most frequent PNSSP clones were the same as those identified in the pre-PCV7 era, although they were present in different proportions. Clonal expansion, emergence of new clones, and acquisition of penicillin resistance by established clones contributed to the maintenance of penicillin resistance.

Point mutations in wchA are responsible for the non-typability of two invasive Streptococcus pneumoniae isolates.

Non-typable Streptococcus pneumoniae (NTPn) strains are typically isolated from nasopharyngeal carriage or from conjunctivitis. Since the isolation of NTPn from invasive disease is rare, we characterized the genetic basis of the non-typability of two isolates obtained in Italy from two cases of bacteraemic pneumonia. MLST revealed that both NTPn belonged to ST191, which, according to the MLST database, is associated with serotype 7F. Sequencing of the capsular locus (cps) confirmed the presence of a 7F cps in both strains and revealed the existence of distinct single point mutations in the wchA gene (a glycosyltransferase), both leading to the translation of proteins truncated at the C terminus. To verify that these mutations were responsible for the non-typability of the isolates, a functional 7F WchA was overexpressed in both NTPn. The two NTPn along with their WchA-overexpressing derivatives were analysed by transmission electron microscopy and by high-resolution magic angle spinning NMR spectroscopy. Both NTPn were devoid of a polysaccharide capsule, and WchA overexpression was sufficient to restore the assembly of a serotype 7F capsule on the surface of the two NTPn. In conclusion, we identified two new naturally occurring point mutations that lead to non-typability in the pneumococcus, and demonstrated that WchA is essential for the biosynthesis of the serotype 7F capsule.