Dissemination of Klebsiella pneumoniae ST147 NDM-1 in Poland, 2015-19.

OBJECTIVES: To assess the spread of New Delhi metallo-ß-lactamase-1 (NDM-1)-producing Klebsiella pneumoniae ST147 organisms in Poland since an introduction from Tunisia in March 2015, including their phylogenetic position in the global population of the high-risk clone. METHODS: Out of 8925 unique NDM-positive K. pneumoniae isolates identified in Poland from April 2015 till December 2019, 126 isolates, including the Tunisian imports, were related by PFGE and blaNDM gene-carrying Tn125 transposon derivatives. Forty-seven representative isolates were sequenced by Illumina MiSeq. The phylogeny, resistome, virulome and plasmid replicons were analysed and compared with the international ST147 strains. Plasmids of six isolates were studied by the MinION sequencing. RESULTS: A high homogeneity of the 47 isolates was observed, with minor variations in their resistomes and plasmid replicon profiles. However, the detailed SNP comparison discerned a strict outbreak cluster of 40 isolates. All of the organisms were grouped within the ST147 phylogenetic international lineage, and four NDM-1 producers from Tunisia, Egypt and France were the closest relatives of the Polish isolates. Yersiniabactin genes (YbST280 type) were located within the ICEKpn12-like element in most of the outbreak isolates, characterized by O2v1 and KL64 antigen loci. The blaNDM-1 genes were located in double-replicon IncFIIK2+IncFIBK plasmids. CONCLUSIONS: The continuous spread of K. pneumoniae ST147 NDM-1 in Poland since 2015, largely in the Warsaw area, is demonstrated by this genomic analysis. The isolates showed a high degree of homogeneity, and close relatedness to organisms spreading in the Mediterranean region.

Cutaneous ulcers revealing diphtheria: A re-emerging disease imported from Indian Ocean countries?

INTRODUCTION: Diphtheria due to Corynebacteriumdiphtheriae (C. diphtheriae) has become rare in developed countries. In France only 10 cases of toxigenic diphtheria have been reported since 1989, in all cases causing pharyngitis and all emanating from endemic countries with exception of one contact case. We report herein 13 cases with cutaneous diphtheria, in 5 of which diphtheria toxin was produced, and all imported into France between 2015 and 2018. OBSERVATIONS: Thirteen patients aged 4 to 77 years presented painful and rapidly progressive round ulcerations of the legs, that were superficial and in some cases purulent, with an erythematous-purple border covered with greyish membrane. Bacteriological sampling of ulcers revealed the presence of C. diphtheriae. Only 6 patients had been properly immunized over the preceding 5 years. DISCUSSION: These cases underline the resurgence of cutaneous diphtheria and the circulation of toxigenic strains in France following importation from Indian Ocean countries. This may constitute an important reservoir for ongoing transmission of the disease. Re-emergence of this pathogen stems from the current migratory flow and decreased adult booster coverage. CONCLUSION: Cutaneous diphtheria should be considered in cases of rapidly developing painful skin ulcers with greyish membrane, especially among patients returning from endemic areas, regardless of their vaccination status. The clinician should order specific screening for C. diphtheriae from the bacteriologist, since with routine swabbing Corynebacteriaceae may be reported simply as normal skin flora. Vaccination protects against toxigenic manifestations but not against actual bacterial infection. Early recognition and treatment of cutaneous diphtheria and up-to-date vaccination are mandatory to avoid further transmission and spread of both cutaneous and pharyngeal diphtheria.

Genomic background of the Klebsiella pneumoniae NDM-1 outbreak in Poland, 2012-18.

OBJECTIVES: To characterize genomes of Klebsiella pneumoniae ST11 NDM-1 responsible for a countrywide outbreak in Poland and compare them phylogenetically with other Polish and international ST11 strains. METHODS: Seventy-one carbapenemase-producing K. pneumoniae ST11 isolates from Poland, including 66 representatives of the NDM-1 epidemic from 2012-18, were sequenced using Illumina MiSeq. Additionally, three outbreak isolates were also sequenced using MinION. The clonality and phylogenetic analysis was done by core-genome MLST and SNP approaches. Resistomes, virulomes, K/O antigens and plasmid replicons were screened for. The detailed plasmid analysis was based on full assemblies using Oxford Nanopore Technologies data. RESULTS: Chromosomes of the outbreak isolates formed an essentially homogeneous cluster (though accumulating SNPs gradually with time), differing remarkably from other Polish NDM-1/-5-, KPC-2- or OXA-48-producing K. pneumoniae ST11 strains. The cluster belonged to a clade with 72 additional isolates identified worldwide, including closely related NDM-1 producers from several countries, including organisms from Bulgaria and Greece. All these had KL24 and O2v1 antigens and the chromosomal yersiniabactin locus YbST230 residing in the ICEKp11 element. The specific blaNDM-1-carrying Tn125 transposon derivative, named Tn125A, was located in IncFII/pKPX-1- and/or IncR-like plasmids; however, the IncRs rearranged extensively during the outbreak, contributing to highly dynamic plasmid profiles and resistomes. CONCLUSIONS: The K. pneumoniae ST11 NDM-1 genotype that has been expanding in Poland since 2012 is largely monoclonal and represents a novel international high-risk lineage that is also spreading in other countries.

Re-emergence of Corynebacterium diphtheriae.

BACKGROUND: Diphtheria is re-emerging in Europe. A total of 36 cases were reported in Europe in 2015 versus 53 cases between 2000 and 2009. PATIENTS: We report two cases of Corynebacterium diphtheriae infection in a French hospital in 2016: a cutaneous infection with negative toxin testing in a French traveller, and a respiratory diphtheria carriage with positive toxin testing in an Afghan refugee diagnosed with pulmonary tuberculosis. The vaccination history of the Afghan patient could not be retrieved.

Phylogenetic lineages, clones and ß-lactamases in an international collection of Klebsiella oxytoca isolates non-susceptible to expanded-spectrum cephalosporins.

OBJECTIVES: The objective of this study was to examine Klebsiella oxytoca clonal and phylogenetic diversity, based on an international collection of carriage isolates non-susceptible to expanded-spectrum cephalosporins (ESCs). METHODS: The study material comprised 68 rectal carriage K. oxytoca isolates non-susceptible to ESCs recovered in 2008-11 from patients in 14 hospitals across Europe and Israel. ESC resistance was tested phenotypically; genes encoding ESBLs, AmpC cephalosporinases and carbapenemases were amplified and sequenced. The isolates were typed by PFGE and MLST, followed by sequencing of blaOXY genes. RESULTS: MLST and PFGE distinguished 34 STs and 47 pulsotypes among the isolates, respectively. Six STs were split into several pulsotypes each. Five STs were more prevalent (n = 2-9) and occurred in several countries each, including ST2, ST9 and ST141, which belong to a growing international clonal complex (CC), CC2. Four phylogenetic lineages were distinguished, each with another type of chromosomal OXY-type ß-lactamase. Three of these, with OXY-1/-5, OXY-2 types and OXY-4, corresponded to previously described phylogroups KoI, KoII and KoIV, respectively. A single isolate from Israel represented a distinct lineage with a newly defined OXY-7 type. The phylogroups showed interesting differences in mechanisms of ESC resistance; KoI strains rarely overexpressed the OXY enzymes but commonly produced ESBLs, whereas KoII strains often were OXY hyperproducers and carried ESBLs much less frequently. AmpCs (DHA-1) and carbapenemases (VIM-1) occurred sporadically. CONCLUSIONS: The study confirmed the high genetic diversity of the collection of K. oxytoca ESC-non-susceptible isolates, composed of phylogroups with distinct types of OXY-type ß-lactamases, and revealed some STs of broad geographical distribution.

Campylobacter coli cultured from the stools of a patient with immunoproliferative small intestinal disease.

Campylobacter has been associated with immunoproliferative small intestinal disease (IPSID), on the basis of 16S rDNA sequencing, in situ hybridization, and immunohistochemistry. Here, for the first time, we have cultured Campylobacter from the stools of a patient with IPSID. Phenotypic analysis and whole genome sequencing identified Campylobacter coli. PCR on a IPSID tissue biopsy sample was positive for Campylobacter coli and negative for Campylobacter jejuni. These findings further support a causative role for Campylobacter in the development of IPSID.

Klebsiella pneumoniae resistant to third-generation cephalosporins in five African and two Vietnamese major towns: multiclonal population structure with two major international clonal groups, CG15 and CG258.

The molecular epidemiology of third-generation cephalosporin-resistant (3GC-R) Klebsiella pneumoniae in developing countries is poorly documented. From February 2007 to March 2008, we collected 135 3GC-R K. pneumoniae isolates from seven major towns in Maghreb (Morocco), West Africa (Senegal, Côte d'Ivoire), Central Africa (Cameroon), East Africa (Madagascar) and Southeast Asia (Vietnam). Their genetic diversity, assessed by multilocus sequence typing, was high (60 sequence types), reflecting multiclonality. However, two major clonal groups, CG15 (n = 23, 17% of isolates) and CG258 (n = 18, 13%), were detected in almost all participating centres. The two major clonal groups have previously been described in other parts of the world, indicating their global spread. The high diversity of enterobacterial repetitive intergenic consensus sequence-PCR banding patterns at the local level indicates that most isolates were epidemiologically unrelated. The isolates were characterized by the presence of multiple resistance determinants, most notably the concomitant presence of the aac(6')-Ib-cr, qnr and blaCTX-M-15 genes in 61 isolates (45%) belonging to 31 sequence types. These isolates were detected across a large geographical area including Cameroon (n = 1), Vietnam (n = 4), Madagascar (n = 10), Côte d'Ivoire (n = 12), Morocco (n = 13) and Senegal (n = 21). These results have major implications for patient management and highlight a potential reservoir for resistance determinants.

Severity of Escherichia coli bacteraemia is independent of the intrinsic virulence of the strains assessed in a mouse model.

Extraintestinal pathogenic Escherichia coli (ExPEC) strains, a major cause of bacteraemia, typically belong to phylogenetic group B2 and express diverse accessory traits that contribute to virulence in mouse infection models. However, their high genomic diversity obscures the relationship between virulence factors and severity of infection in patients. In this study, we analysed concomitantly the strain's expression of virulence in a mouse model, genomic determinants and the clinical severity of infection in 60 bacteraemic patients. We show that bacterial virulence based on an animal model study and virulence factor determination is not correlated with pejorative outcome of E. coli human blood infections.

The emergence of multidrug-resistant Klebsiella pneumoniae of international clones ST13, ST16, ST35, ST48 and ST101 in a teaching hospital in the Paris region.

Despite infection control measures, an important increase in the extended-spectrum ß-lactamase (ESBL)-producing Klebsiella pneumoniae incidence density occurred in our hospital from 2006 onwards. This study, focusing on the 2005-2007 period, was performed in an attempt to explain this increase. ESBLs were characterized, isolates were typed by ERIC2-PCR, and sequence type (ST) of clustered isolates was determined. Temporal-spatial relationships of patients were analysed to assess possible cross-contamination. Of the 74 ESBL-producing isolates, 30 (40%) were detected at admission, 53 (71∙5%) produced CTX-M enzymes, 40 displayed unique ERIC2-PCR profiles and 34 were assigned into six clusters: ST16 (n=21), ST101, ST48, ST35, ST13, and ST436. Relationships were identified in 22 of the 34 patients harbouring clustered isolates. This study highlights the complex epidemiology of ESBL-producing K. pneumoniae in the mid-2000s with potential cross-contamination for only 30% of the 74 patients in our hospital, and the emergence of clones that are currently spreading worldwide.

Primary liver abscess due to CC23-K1 virulent clone of Klebsiella pneumoniae in France.

Since the mid-1980s, Klebsiella pneumoniae hypermucoviscous isolates have emerged in Taiwan and other Asian countries. We reported the first autochthonous European liver abscess due to an ST57 isolate, which belongs to virulent clonal complex CC23-K1. This case highlights the emergence in France and Europe of hypermucoviscous virulent K. pneumoniae isolates.

Clinical relevance of cagA and vacA gene polymorphisms in Helicobacter pylori isolates from Senegalese patients.

The molecular epidemiology of Helicobacter pylori in Africa is poorly documented. From January 2007 to December 2008, we investigated 187 patients with gastric symptoms in one of the main tertiary hospitals in Dakar, Senegal. One hundred and seventeen patients were culture-positive for H. pylori. Polymorphisms in vacA and cagA status were investigated by PCR; the 3'-region of cagA was sequenced, and EPIYA motifs were identified. Bacterial heterogeneity within individuals was extensively assessed by using an approach based on vacA and cagA heterogeneity. Fourteen per cent of H. pylori-positive patients displayed evidence of mixed infection, which may affect disease outcome. Patients with multiple vacA alleles were excluded from subsequent analyses. Among the final study population of 105 patients, 29 had gastritis only, 61 had ulcerated lesions, and 15 had suspicion of neoplasia based on endoscopic findings. All cases of suspected neoplasia were histologically confirmed as gastric cancer (GC). The cagA gene was present in 73.3% of isolates. CagA proteins contained zero (3.7%), one (93.9%) or two (2.4%) EPIYA-C segments, and all were western CagA. Most of the isolates possessed presumed high-vacuolization isotypes (s1i1m1 (57.1%) or s1i1m2 (21.9%)). Despite the small number of cases, GC was associated with cagA (p 0.03), two EPIYA-C segments in the C-terminal region of CagA (p 0.03), and the s1 vacA allele (p 0.002). Multiple EPIYA-C segments were less frequent than reported in other countries, possibly contributing to the low incidence of GC in Senegal.

Human leptospira isolates circulating in Mayotte (Indian Ocean) have unique serological and molecular features.

Leptospirosis is one of the most widespread zoonoses in the world. However, there is a lack of information on circulating Leptospira strains in remote parts of the world. We describe the serological and molecular features of leptospires isolated from 94 leptospirosis patients in Mayotte, a French department located in the Comoros archipelago, between 2007 and 2010. Multilocus sequence typing identified these isolates as Leptospira interrogans, L. kirschneri, L. borgpetersenii, and members of a previously undefined phylogenetic group. This group, consisting of 15 strains, could represent a novel species. Serological typing revealed that 70% of the isolates belonged to the serogroup complex Mini/Sejroe/Hebdomadis, followed by the serogroups Pyrogenes, Grippotyphosa, and Pomona. However, unambiguous typing at the serovar level was not possible for most of the strains because the isolate could belong to more than one serovar or because serovar and species did not match the original classification. Our results indicate that the serovar and genotype distribution in Mayotte differs from what is observed in other regions, thus suggesting a high degree of diversity of circulating isolates worldwide. These results are essential for the improvement of current diagnostic tools and provide a starting point for a better understanding of the epidemiology of leptospirosis in this area of endemicity.

Widespread carbapenem resistant Acinetobacter baumannii clones in Italian hospitals revealed by a multicenter study.

Population diversity, susceptibility to antibiotics including carbapenems of 277 Acinetobacter baumannii strains collected in 17 Italian hospitals over a 6-months' period was assessed. Semi-automated rep-PCR was used for screening strains for genotypic relatedness. AFLP analysis and MLST were used as definitive methods for strain, species and/or clone identification. Among the 277 strains, 49 rep-PCR types were distinguished with four types (1-4) predominant, indicating both intra- and interhospital spread. AFLP analysis allowed to distinguish 51 types and largely confirmed rep-typing results. Isolates with predominant rep-types 1 and 2 (in 3 and 9 hospitals) were allocated to EU clones I and II, respectively. Rep-type 3 (8 hospitals) belonged to a new clone ("Italian clone"). Rep-type 4 was found in 2 neighbouring hospitals. Two isolates from 2 locations belonged to EU clone III. Twenty-five isolates were identified by AFLP-analysis to A. pittii, emphasizing misidentification by phenotypic methods. MLST confirmed clone identification by AFLP; demonstrating also that the "Italian clone" was ST78, recently detected in different Mediterranean countries. Multidrug resistance, defined as resistance to 9 out of the 11 drugs tested, was common in 10 out of 17 hospitals. The high prevalence of carbapenem resistance was associated with OXA-58 found in 9 out of the 10 hospitals. A high percentage of noted very major errors in susceptibility testing, especially for amikacin and meropenem, was probably due to heteroresistant strains. The occurrence of carbapenem and multidrug resistance in A. baumannii was mainly confined to a limited number of clonal lineages of A. baumannii.

Epidemiology of methicillin-resistant Staphylococcus aureus lineages in five major African towns: emergence and spread of atypical clones.

The epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) in Africa is poorly documented. From January 2007 to March 2008, we collected 86 MRSA isolates from five African towns, one each in Cameroon, Madagascar, Morocco, Niger and Senegal. Although one or two major clones, defined by the sequence type and staphylococcal cassette chromosome mec type, predominated at each site, genetic diversity (ten clones) was relatively limited in view of the large geographical area studied. Most of the isolates (n = 76, 88%) belonged to three major clones, namely ST239/241-III, a well-known pandemic clone (n = 34, 40%), ST88-IV (n = 24, 28%) and ST5-IV (n = 18, 21%). The latter two clones have only been sporadically described in other parts of the world. The spread of community-associated MRSA carrying the Panton-Valentine leukocidin genes is a cause for concern, especially in Dakar and possibly throughout Africa.

Epidemiology of methicillin-susceptible Staphylococcus aureus lineages in five major African towns: high prevalence of Panton-Valentine leukocidin genes.

The epidemiology of methicillin-susceptible Staphylococcus aureus (MSSA) in Africa is poorly documented. From January 2007 to March 2008, 555 S. aureus isolates were collected from five African towns in Cameroon, Madagascar, Morocco, Niger, and Senegal; among these, 456 unique isolates were susceptible to methicillin. Approximately 50% of the MSSA isolates from each different participating centre were randomly selected for further molecular analysis. Of the 228 isolates investigated, 132 (58%) belonged to five major multilocus sequence typing (MLST) clonal complexes (CCs) (CC1, CC15, CC30, CC121 and CC152) that were not related to any successful methicillin-resistant S. aureus (MRSA) clones previously identified in the same study population. The luk-PV genes encoding Panton-Valentine leukocidin (PVL), present in 130 isolates overall (57%), were highly prevalent in isolates from Cameroon, Niger, and Senegal (West and Central Africa). This finding is of major concern, with regard to both a source of severe infections and a potential reservoir for PVL genes. This overrepresentation of PVL in MSSA could lead to the emergence and spread of successful, highly virulent PVL-positive MRSA clones, a phenomenon that has already started in Africa.

Molecular epidemiological investigation of multidrug-resistant Acinetobacter baumannii strains in four Mediterranean countries with a multilocus sequence typing scheme.

Thirty-five multidrug-resistant Acinetobacter baumannii strains, representative of 28 outbreaks involving 484 patients from 20 hospitals in Greece, Italy, Lebanon and Turkey from 1999 to 2009, were analysed by multilocus sequence typing. Sequence type (ST)2, ST1, ST25, ST78 and ST20 caused 12, four, three, three and two outbreaks involving 227, 93, 62, 62 and 31 patients, respectively. The genes bla(oxa-58), bla(oxa-23) and bla(oxa-72) were found in 27, two and one carbapenem-resistant strain, respectively. In conclusion, A. baumannii outbreaks were caused by the spread of a few strains.

Multilocus microsatellite markers for molecular typing of Candida glabrata: application to analysis of genetic relationships between bloodstream and digestive system isolates.

Candida glabrata has emerged as the second most common etiologic agent, after Candida albicans, of superficial and invasive candidiasis in adults. Strain typing is essential for epidemiological investigation, but easy-to-use and reliable typing methods are still lacking. We report the use of a multilocus microsatellite typing method with a set of eight markers on a panel of 180 strains, including 136 blood isolates from hospitalized patients and 34 digestive tract isolates from nonhospitalized patients. A total of 44 different alleles were observed, generating 87 distinct genotypes. In addition to perfect reproducibility, typing ability, and stability, the method had a discriminatory power calculated at 0.97 when all 8 markers were associated, making it suitable for tracing strains. In addition, it is shown that digestive tract isolates differed from blood culture isolates by exhibiting a higher genotypic diversity associated with different allelic frequencies and preferentially did not group in clonal complexes (CCs). The demonstration of the occurrence of microevolution in digestive strains supports the idea that C. glabrata can be a persistent commensal of the human gut.

Molecular epidemiology of extended-spectrum beta-lactamase-producing Klebsiella pneumoniae strains in a university hospital in Tunis, Tunisia, 1999-2005.

During a period of 6 years and 5 months (January 1999 to May 2005), 103 extended-spectrum beta-lactamase (ESBL)-producing Klebsiella pneumoniae isolates, each from an individual patient or site, were collected at Mongi Slim University Hospital Centre, Tunis, Tunisia. The objectives of our work were the characterization of the bla genes encoding ESBLs, the investigation of clonal diversity of strains, and identification of the transmission modes of the resistance genes. We carried out detection by PCR and sequencing of the bla(SHV), bla(CTX-M) and bla(TEM) genes, transferability studies, plasmid replicon typing, and analysis by multilocus sequence typing (MLST) on selected isolates. Forty-seven isolates were found to be producers of CTX-M-type ESBLs, of which 43 were CTX-M-15, two CTX-M-14 and two CTX-M-27. Fifty-eight isolates were producers of SHV-12, and three were producers of SHV-2a. More than one ESBL was detected in seven isolates, as five produced both CTX-M-15 and SHV-12, and two produced both CTX-M-27 and SHV-12. By a PCR-based replicon typing method, the plasmids carrying the bla(SHV-2a) or bla(CTX-M-15) genes were assigned to IncFII or, more rarely, to IncL/M types. Of 12 plasmids carrying the bla(SHV-12) gene, only one could be typed: it was positive for the HI2 replicon. The MLST results showed large genetic background diversity in the SHV-12-producing isolates and dissemination of specific clones of the CTX-M-15-producing isolates within the same ward and among wards, and suggested endemicity with horizontal dissemination of the bla(CTX-M-15) and the bla(SHV-12) genes.

Polyphasic characterization of xanthomonads pathogenic to members of the Anacardiaceae and their relatedness to species of Xanthomonas.

We have used amplified fragment length polymorphism (AFLP), multilocus sequence analysis (MLSA) and DNA-DNA hybridization for genotypic classification of Xanthomonas pathovars associated with the plant family Anacardiaceae. AFLP and MLSA results showed congruent phylogenetic relationships of the pathovar mangiferaeindicae (responsible for mango bacterial canker) with strains of Xanthomonas axonopodis subgroup 9.5. This subgroup includes X. axonopodis pv. citri (synonym Xanthomonas citri). Similarly, the pathovar anacardii, which causes cashew bacterial spot in Brazil, was included in X. axonopodis subgroup 9.6 (synonym Xanthomonas fuscans). Based on the thermal stability of DNA reassociation, consistent with the AFLP and MLSA data, the two pathovars share a level of similarity consistent with their being members of the same species. The recent proposal to elevate X. axonopodis pv. citri to species level as X. citri is supported by our data. Therefore, the causal agents of mango bacterial canker and cashew bacterial spot should be classified as pathovars of X. citri, namely X. citri pv. mangiferaeindicae (pathotype strain CFBP 1716) and X. citri pv. anacardii (pathotype strain CFBP 2913), respectively. Xanthomonas fuscans should be considered to be a later heterotypic synonym of Xanthomonas citri.