Single-Locus-Sequence-Based Typing of blaOXA-51-like Genes for Rapid Assignment of Acinetobacter baumannii Clinical Isolates to International Clonal Lineages

Single-locus bla_OXA-51-like sequence-based typing (SBT) was evaluated for its ability to determine correctly sequence types (STs) in Acinetobacter baumannii clinical isolates, in comparison with the Pasteur''s multilocus sequence typing (MLST) reference method and 3-locus sequence typing (3-LST). The comparative study was performed in 585 multidrug-resistant (MDR) A. baumannii clinical isolates recovered from 21 hospitals located throughout Greece, Italy, Lebanon, and Turkey. The isolates belonged to nine clonal complexes (CCs) that correspond to 12 distinct sequence types (STs) and to one singleton ST. These clonal lineages predominate worldwide among nosocomial MDR A. baumannii strains. The most common clone was CC2 (ST2 and ST45; n = 278 isolates) followed by CC1 (ST1 and ST20; n = 155), CC25 (n = 65), ST78 (n = 62), CC15 (ST15 and ST84; n = 9), CC10 (n = 4), CC3 (n = 4), CC6 (n = 3), CC54 (n = 3), and CC83 (n = 2). Using the bla_OXA-51-like SBT method, all 585 isolates of the study were typed and assigned correctly to the nine CCs and the singleton ST78. The 3-LST method was not able to classify isolates belonging to CC6, CC10, CC54, and CC83, which are not yet characterized in its database. The low-cost and convenient bla_OXA-51-like SBT method, compared with 3-LST and MLST, discriminated all epidemic and sporadic lineages of our collection and could be effectively applied to type rapidly A. baumannii strains.     

Wide dispersion of ST175 clone despite high genetic diversity of carbapenem-nonsusceptible Pseudomonas aeruginosa clinical strains in 16 Spanish hospitals

During the COMParative Activity of Carbapenems Testing (COMPACT) surveillance study, 448 Pseudomonas aeruginosa clinical isolates were obtained from 16 Spanish hospitals. Nonsusceptibility (EUCAST breakpoints) to imipenem (35%), meropenem (33%), and/or doripenem (33%) was observed with 175 isolates (39%). Simultaneous resistance to these three drugs was observed with 126 of the 175 isolates (72%). Except for colistin, high resistance rates were observed among noncarbapenem antibiotics. Clonal relatedness was investigated by pulsed-field gel electrophoresis (PFGE) with SpeI, discriminating 68 patterns. Multilocus sequence typing (MLST) was performed on 84 isolates representing different PFGE types and all participating hospitals. Thirty-nine sequence types (STs) could be distinguished, and of these, ST175 (48 isolates, 10 hospitals), ST646 (16 isolates, 4 hospitals), ST532 (13 isolates, 3 hospitals), and ST111 (13 isolates, 7 hospitals) were the most frequently encountered. Minimum-spanning tree analysis confirmed a wide dissemination of different clones among participant hospitals, particularly ST175. PFGE pattern comparison within the four most frequent STs revealed that ST175 isolates were relatively uniform, while ST646, ST532, and ST111 isolates were highly diverse, with almost every isolate belonging to a unique pulsotype, even when originating from the same center. The population of carbapenem-nonsusceptible P. aeruginosa isolates from 16 hospitals is highly diverse, with one ST (ST175) representing a highly conserved clone disseminated in 10 of the 16 participant hospitals. This ST175 clone should be added to the list of P. aeruginosa clones at high risk for epidemic spread, such as the Liverpool, Manchester, and Melbourne clones previously found in cystic fibrosis patients and ST235 in the nosocomial setting.     

Epidemiological characterization and distribution of carbapenem-resistant Acinetobacter baumannii clinical isolates in Italy

This study was aimed at tracing the molecular characteristics of carbapenem-resistant Acinetobacter baumannii (CRAB) clinical isolates in Italy with both pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Two hundred and two CRAB isolates were collected during 2004–2009, in two different surveillance periods, from 22 Italian hospitals that were representative for both distribution and infection. PFGE was performed, and the MLST scheme used was based on the gene sequence as published on the MLST Pasteur website http://www.pasteur.fr/mlst. Representatives of the major European clones I (RUH 875) and II (RUH 134) were used as controls. The two groups of isolates were characterized for their carbapenem resistance genes: 154 of 202 carried bla_OXA-58 alone, 21 of 202 also carried bla_OXA-23, and 27 of 202 carried bla_OXA-23 alone. No isolates were positive for bla_OXA-24. Genotype analysis of all isolates identified four distinct patterns by PFGE, which correlated with four distinct sequence types (STs) by MLST. The distribution of these four clusters in Italy confirmed the propensity of A. baumannii for nosocomial cross-transmission in a vast geographical area. We observed that clones A and B had similarities with European clone II and I respectively. By MLST, clone A was ST2, like European clone II, and clone B was ST1, like European clone I. PFGE and MLST showed the same discriminatory power and reproducibility. In addition, the two methods were concordant in defining CRAB Italian clones and in correlating them with the two pan-European clones.     

Molecular epidemiology of a clonal outbreak of multidrug-resistant Acinetobacter baumannii in a university hospital in Italy

This study investigated the molecular epidemiology of a clonal outbreak of multidrug-resistant Acinetobacter baumannii that occurred between June 2003 and June 2004 in a tertiary-care hospital in Naples, Italy. A. baumannii was isolated from 74 patients, of whom 38 were infected and 36 were colonised. Thirty-three patients had ventilator-associated pneumonia, three had hospital-acquired pneumonia, and two had sepsis. Genotypic analysis of 45 available A. baumannii isolates revealed two distinct pulsed-field gel electrophoresis (PFGE) patterns. Of these, PFGE pattern 1 was represented by isolates from 44 patients and was identical to that of an epidemic A. baumannii clone isolated in another hospital of Naples during 2002. All A. baumannii isolates of PFGE type 1 showed identical multiresistant antibiotypes, characterised by resistance to all antimicrobial agents tested, including carbapenems, with the exception of colistin. In these isolates, inhibition of OXA enzymes by 200 mM NaCl reduced the imipenem MIC by up to four-fold. Molecular analysis of antimicrobial resistance genes showed that all A. baumannii isolates of PFGE type 1 harboured a class 1 integron containing the aacA4, orfX and bla(OXA-20) gene cassettes, an ampC gene and a bla(OXA-51)-like allele. Moreover, a bla(OXA-58)-like gene surrounded by the regulatory elements ISAba2 and ISAba3 was identified in a 30-kb plasmid from A. baumannii isolates of PFGE type 1, but not PFGE type 2. Thus, selection of a single A. baumannii clone producing an OXA-58-type carbapenem-hydrolysing oxacillinase was responsible for the increase in the number of A. baumannii infections that occurred in this hospital.     

Outbreak of Pseudomonas aeruginosa infections with PER-1 extended-spectrum beta-lactamase in Warsaw, Poland: further evidence for an international clonal complex

Forty-one Pseudomonas aeruginosa isolates with extended-spectrum beta-lactamases (ESBLs) from a hospital in Warsaw, Poland, were analyzed. Thirty-seven isolates from several wards were collected over 9 months in 2003 and 2004. The isolates were recovered from patients with multiple types of infections, mostly respiratory tract and postoperative wound infections. All 41 isolates produced the PER-1 ESBL, originally observed in Turkey but recently also identified in several countries in Europe and the Far East. The bla(PER-1) gene resided within the Tn1213 composite transposon, which was chromosomally located. Pulsed-field gel electrophoresis and multilocus sequence typing (MLST) revealed the presence of three separate clones among the isolates. Two of these, corresponding to sequence types (STs) ST244 and ST235, were responsible for parallel outbreaks. Apart from PER-1, all the isolates produced OXA-2 oxacillinase. ST235 isolates additionally expressed a novel enzyme, OXA-74, differing by one amino acid from the OXA-17 ESBL identified originally in PER-1- and OXA-2-positive P. aeruginosa isolates from Ankara, Turkey, in 1992. These earlier Ankara isolates with PER-1, OXA-2, and OXA-17 were also classified into ST235, which is a single-locus variant of two other STs, ST227 and ST230. ST227, ST230, and ST235 all correspond to the recently described clonal complex BG11, which seems to be internationally distributed, having spread in Turkey, Greece, Italy, Hungary, Poland, Sweden, and much of Russia. It is associated with various beta-lactamases, including PER-1 and VIM metalloenzymes. This work further demonstrates the value of MLST of P. aeruginosa.     

Characteristics of Haemophilus influenzae type b responsible for meningitis in Poland from 1997 to 2004

Two hundred forty-five H. influenzae isolates responsible for meningitis in Poland from 1997 to 2004 were studied. Among these, 233 (95.1%) belonged to serotype b (Hib), 2 belonged to serotype f, and 10 were noncapsulated. The relatedness of all isolates was evaluated by pulsed-field gel electrophoresis (PFGE), and selected representatives were evaluated by multilocus sequence typing. Resistance to ampicillin was identified in 34 (14.6%) of the Hib isolates and was associated with the production of beta-lactamase only. Except for four isolates nonsusceptible to chloramphenicol, all isolates were susceptible to cefotaxime, ciprofloxacin, and rifampin. The PFGE analysis divided the Hib isolates into five PFGE types; however, all of them were possibly related. The most common PFGE type, with 25 subtypes, was characteristic for 97.4% of the isolates. The most prevalent PFGE subtype found in our study was also the most common among the Hib isolates responsible for invasive disease in Italy and the Czech Republic and was found among isolates causing lower respiratory tract infections in Poland. The most prevalent sequence types (STs) in the studied group were ST6 and ST92. Four new STs were found: ST188, ST189, ST190, and ST268. Results of this study support the evidence that the genetic structure of encapsulated H. influenzae is clonal. The continuing high number of meningitis cases due to Hib in Poland underlines the need for mass vaccination against Hib in Poland.     

Association between β-lactamase-encoding bla(OXA-51) variants and DiversiLab rep-PCR-based typing of Acinetobacter baumannii isolates

This study investigated the correlation between bla(OXA-51) variants and Acinetobacter baumannii worldwide clonal lineages 1 to 8 (WW1 to -8). The bla(OXA-51-like) genes of 102 A. baumannii isolates were sequenced. Using DiversiLab repetitive-sequence-based PCR (rep-PCR) typing, 92 of these isolates had previously been assigned to WW1 to -8 and 10 were unclustered. Clustering of DNA sequences was performed using the neighbor-joining method and the Jukes-Cantor phylogenetic correction. bla(OXA-51) variants were in good correlation with DiversiLab-defined clonal lineages. Sequence-based typing of bla(OXA-51) variants has the potential to be applied for epidemiologic characterization of A. baumannii and to identify worldwide clonal lineages 1 to 8.     

Molecular characterization of vancomycin-resistant Enterococcus faecium isolates from Korea

A total of 98 vancomycin-resistant Enterococcus faecium (VREF) isolates from four tertiary-care hospitals in Korea during the period between 1998 and 2004 were analyzed for genotypic characteristics using the multiplex PCR, multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), and esp gene analysis. Ninety-two isolates of VREF with VanA phenotype and five of six isolates with VanB phenotype possessed the vanA gene. MLST analysis revealed 9 sequence types (STs), which belonged to a single clonal complex (CC78, clonal lineage C1). Five strains showing incongruence between phenotype and genotype (VanB-vanA) did not belong to the same genotypic clone. The esp gene was detected in all VREF strains, showing 12 different esp repeat profiles. Data suggest that an epidemic clonal group of VREF, CC78 with esp gene, is also present in Asia and has differentiated into multiple diverse genotypic clones during the evolutionary process.     

Molecular epidemiology of OXA-48-producing Klebsiella pneumoniae in France

We characterized 53 OXA-48-producing Klebsiella pneumoniae (OXA-48-Kp) isolated between 2011 and 2013 in 21 French hospitals. All the isolates were genotyped using MLST and PFGE and the population structure of the species was determined by a nucleotide-based analysis of the entire K. pneumoniae MLST database. Most of the OXA-48-Kp isolates also produced CTX-M-15 and remained susceptible to imipenem and meropenem. The isolates were distributed into 20 STs, of which five were dominant (ST15, ST101, ST147, ST395 and ST405). All the OXA-48-Kp clustered in the major clade of K. pneumoniae KpI.     

Contemporary methicillin-resistant Staphylococcus aureus clones in Hong Kong

Two hundred non-duplicate methicillin-resistant Staphylococcus aureus (MRSA) isolates causing bacteremia in patients in four major Hong Kong hospitals during the period 2000 to 2001 were characterized by antibiogram, pulsed-field gel electrophoresis (PFGE) using SmaI restriction enzymes, and determination of staphylococcal cassette chromosome mec (SCCmec) types. Nine PFGE types, A to I, were obtained. PFGE type A constituted 50% (99/200) of all isolates and was present in isolates from all four hospitals. PFGE types A to E, had previously been identified as the major types at one of the hospitals from 1988 to 2000. The majority had a resistance profile to tetracycline (T), erythromycin (E), clindamycin (D), gentamicin (G), tobramycin (To), and ciprofloxacin (Ci), and belonged to SCCmec type III; and representatives belonged to clonal complex 239 (CC 239) (MRSA with SCCmec type III and sequence type 239, designated ST 239-MRSA-III). PFGE types F to I were new patterns that had not been previously identified in isolates from Hong Kong. PFGE type F constituted 18% (35/200) of MRSAs, had resistance profile TEGToCi, and belonged to CC 5 (ST 5-MRSA-II). PFGE type G included 13% (26/200) of MRSAs, had resistance profile TECi, and belonged to CC 45 with SCCmec type I or II. PFGE type H had characteristics similar to those of CC 239, while PFGE type I included three isolates, two of which expressed resistance to oxacillin and fusidic acid only. Two of these strains had SCCmec IVa and carried sequence type 389, with a multilocus sequence typing allelic profile of 3-35-19-2-20-26-39. Contemporary MRSAs causing bacteremia in Hong Kong hospitals belong to three clonal complexes (CC 5, CC 45, and CC 239). The most prevalent MRSA clone in Hong Kong belongs to CC 239, with PFGE types A to E and H, SCCmec type III, ST 239, and a resistance profile of TEDGToCi.     

Emergence in Brazil of methicillin-resistant Staphylococcus aureus isolates carrying SCCmecIV that are related genetically to the USA800 clone

An increasing incidence of nosocomial infections caused by non-multiresistant methicillin-resistant Staphylococcus aureus (nMMRSA) has been reported worldwide. The present study genotyped nMMRSA isolates obtained from hospitals in two cities in Brazil. The hospital isolates displayed pulsed-field gel electrophoresis (PFGE) patterns that were similar to those of the USA100 (ST5-SCCmecII) and USA 800 (ST5-SCCmecIV) strains, which are related to the New York/Japan and paediatric clones, respectively. Carriage of SCCmecIV and the classification by multilocus sequence typing (MLST) of a representative of this PFGE pattern in clonal complex 5 (CC5) confirmed the genetic relationship of the Brazilian isolates with USA800. The USA800-related Brazilian isolates were responsible for severe nosocomial infections in compromised adults and elderly patients in Brazil. A higher growth rate, an ability to form biofilm on inert polystyrene surfaces and the presence of the egc locus may have contributed, at least in part, to the fitness of these organisms as global nosocomial pathogens.     

Molecular Epidemiology of Carbapenem-Resistant Acinetobacter baumannii Isolates in the Gulf Cooperation Council States: Dominance of OXA-23-Type Producers

The molecular epidemiology and mechanisms of resistance of carbapenem-resistant Acinetobacter baumannii (CRAB) were determined in hospitals in the states of the Cooperation Council for the Arab States of the Gulf (Gulf Cooperation Council [GCC]), namely, Saudi Arabia, United Arab Emirates, Oman, Qatar, Bahrain, and Kuwait. Isolates were subjected to PCR-based detection of antibiotic resistance genes and repetitive sequence-based PCR (rep-PCR) assessments of clonality. Selected isolates were subjected to multilocus sequence typing (MLST). We investigated 117 isolates resistant to carbapenem antibiotics (either imipenem or meropenem). All isolates were positive for OXA-51. The most common carbapenemases were the OXA-23-type, found in 107 isolates, followed by OXA-40-type (OXA-24-type), found in 5 isolates; 3 isolates carried the ISAba1 element upstream of bla_OXA-51-type. No OXA-58-type, NDM-type, VIM-type, or IMP-type producers were detected. Multiple clones were detected with 16 clusters of clonally related CRAB. Some clusters involved hospitals in different states. MLST analysis of 15 representative isolates from different clusters identified seven different sequence types (ST195, ST208, ST229, ST436, ST450, ST452, and ST499), as well as three novel STs. The vast majority (84%) of the isolates in this study were associated with health care exposure. Awareness of multidrug-resistant organisms in GCC states has important implications for optimizing infection control practices; establishing antimicrobial stewardship programs within hospital, community, and agricultural settings; and emphasizing the need for establishing regional active surveillance systems. This will help to control the spread of CRAB in the Middle East and in hospitals accommodating transferred patients from this region.     

Characterization of Acinetobacter baumannii from intensive care units and home care patients in Palermo, Italy

In this study 45 isolates of Acinetobacter baumannii identified from patients in intensive care units of three different hospitals and from pressure ulcers in home care patients in Palermo, Italy, during a 3-month period in 2010, were characterized. All isolates were resistant to at least three classes of antibiotics, but susceptible to colistin and tygecycline. Forty isolates were non-susceptible to carbapenems. Eighteen and two isolates, respectively, carried the bla(OXA-23-like) and the bla(OXA-58-like) genes. One strain carried the VIM-4 gene. Six major rep-PCR subtype clusters were defined, including isolates from different hospitals or home care patients. The sequence type/pulsed field gel electrophoresis group ST2/A included 33 isolates, and ST78/B the remaining 12. ST2 clone proved to be predominant, but a frequent involvement of the ST78 clone was evident.     

Clonal spread of a vancomycin-resistant Enterococcus faecium strain among bloodstream-infecting isolates in Italy

Recent data indicated that the rate of vancomycin resistance in bloodstream-infecting enterococcal isolates in Italy is one of the highest in Europe. The aims of this study were to characterize bloodstream-infecting vancomycin-resistant enterococci (VRE) obtained from various Italian hospitals and to establish whether the isolates were clonally related. During the years 2001 to 2003, a total of 39 VRE isolates were obtained from 19 hospital laboratories in various areas of Italy. Species identification and resistance genotypes of the isolates were obtained by multiplex PCR. Further characterization included antibiotic susceptibility testing, pulsed-field gel electrophoresis (PFGE) of SmaI-digested genomic DNA, detection of virulence genes (esp and hyl), and multilocus sequence typing (MLST) of selected isolates. VRE were identified as 31 Enterococcus faecium (VREfm) isolates and 8 E. faecalis isolates. All but one isolate carried the vanA gene; one VREfm isolate carried the vanB gene. Analysis of the PFGE profiles showed that 28 VREfm isolates shared a similar electrophoretic profile, designed type 1, and were clonally related. All type 1 isolates were resistant to ampicillin, streptomycin, gentamicin, and rifampin and were positive for the esp gene. MLST identified an allelic profile (ST78) comprising purK allele 1, belonging to the C1 clonal lineage, characteristic of human infection and hospital outbreak isolates. The vanB-carrying VREfm isolate, of PFGE type 2, was shown to be a single-locus variant of ST78. Our data indicate that the recent increase in the number of bloodstream infections caused by VRE in Italy is due to the spread of a hospital-adapted, multidrug-resistant VREfm clone belonging to an internationally disseminated lineage.     

Molecular epidemiologic analysis and antimicrobial resistance of Helicobacter cinaedi isolated from seven hospitals in Japan

Helicobacter cinaedi colonizes the colons of human and animals and can cause colitis, cellulitis, and sepsis in humans, with infections in immunocompromised patients being increasingly recognized. However, methods for analyzing the molecular epidemiology of H. cinaedi are not yet established. A genotyping method involving multilocus sequence typing (MLST) was developed and used to analyze 50 H. cinaedi isolates from Japanese hospitals in addition to 6 reference strains. Pulsed-field gel electrophoresis (PFGE) results were also compared with the MLST results. Based on the genomic information from strain CCUG18818, 21 housekeeping genes were selected as candidates for MLST and were observed to have high homology (96.5 to 100%) between isolates. Following a comparison of the 21 housekeeping genes from 8 H. cinaedi isolates, 7 genes were chosen for MLST, revealing 14 sequence types (STs). The isolates from 3 hospitals belonged to the same STs, but the isolates from the other 4 hospitals belonged to different STs. Isolates belonging to ST6 were analyzed by PFGE and showed similar, but not identical, patterns between isolates. Isolates belonging to ST9, ST10, and ST11, which belonged to the same clonal complex, had the same pattern. All isolates were found to contain mutations in GyrA and the 23S rRNA gene that confer ciprofloxacin and clarithromycin resistance, respectively, in H. cinaedi. These results raise concerns about the increase in H. cinaedi isolates resistant to clarithromycin and ciprofloxacin in Japan.     

Genetic diversity of carbapenem-resistant isolates of Acinetobacter baumannii in Europe.

In total, 96 carbapenem-resistant isolates of Acinetobacter baumannii were obtained from 25 hospitals in 17 European countries. Imipenem MICs ranged from <4 to 128 mg/L on retesting by Etest, with MICs > or =16 mg/L being associated with the carriage of genes encoding at least one other class D carbapenemase in addition to the intrinsic OXA-51-like enzyme. Molecular typing results obtained by random amplified polymorphic DNA analysis, followed by pulsed-field gel electrophoresis (PFGE) of ApaI-digested chromosomal DNA, were highly congruent, with 17 different PFGE types being delineated at a cut-off similarity level of 85%. With few exceptions, multiple isolates from a single hospital belonged to the same PFGE type. Seven sequence groups were identified among the 96 A. baumannii isolates, with the majority of isolates (n = 81) belonging to the previously defined sequence groups 1 and 2, which each included eight PFGE types. These two multinational lineages included the previously defined European clones II and I, respectively, but the problem of resistant A. baumannii in Europe appeared not to be confined solely to these two European clones. Rather, two broader lineages of carbapenem-resistant A. baumannii now seem to be spreading throughout Europe.     

OXA-51-like beta-lactamases and their association with particular epidemic lineages of Acinetobacter baumannii.

Sixty diverse clinical Acinetobacter baumannii isolates of worldwide origin were assigned to sequence groups, based on a multiplex PCR for the ompA, csuE and bla(OXA-51-like) genes. The majority (77%) of isolates belonged to sequence groups 1 and 2 (SG1 and SG2), with sequence group 3 (SG3) and non-grouped isolates accounting for the remainder. The isolates were not closely related according to pulsed-field gel electrophoresis (PFGE), and the majority were sensitive to imipenem and meropenem. The construction of a linkage map of OXA-51-like beta-lactamase sequence relationships revealed two closely related clusters of enzymes, one focused around OXA-66 and the other around OXA-69. Isolates belonging to SG1 encoded an enzyme from the OXA-66 cluster, while those belonging to SG2 encoded an enzyme from the OXA-69 cluster. All SG3 isolates encoded OXA-71, which does not form part of a close enzyme grouping. Major multinational lineages accounted for a significant proportion of A. baumannii clinical isolates, and the evolution of the OXA-51-like enzymes appears to be an ongoing process.     

Molecular epidemiology of clinical Acinetobacter baumannii and Acinetobacter genomic species 13TU isolates using a multilocus sequencing typing scheme

To further expand the limited multilocus sequence typing (MLST) database for Acinetobacter baumannii , 53 clinical isolates from various outbreaks in Europe and the USA, collected between 1991 and 2004, plus the A. baumannii reference strain ATCC 19606^T and 20 clinical Acinetobacter genomic species 13TU isolates from the same period, were analyzed using a new MLST scheme based on fragments of the gltA , gyrB , gdhB , recA , cpn60 , gpi and rpoD genes. Data were compared with typing results generated using pulsed-field gel electrophoresis (PFGE) and randomly amplified polymorphic DNA (RAPD)-PCR. In total, 50 sequence types (STs) were distinguished among the A. baumannii isolates investigated, and the MLST data were in high concordance with the PFGE and RAPD-PCR results. Only five clonal complexes were identified by eBURST analysis, including the 21 STs listed in a previous study, suggesting high diversity among the A. baumannii isolates. With one exception, there was no relatedness among isolates from outbreaks in different countries (Europe) or regions (USA). No intercontinental spread was revealed. Acinetobacter genomic species 13TU isolates could also be analyzed using the A. baumannii MLST scheme (18 different STs) and could be distinguished from A. baumannii isolates according to characteristic sequences. It was concluded that the MLST scheme provides a high level of resolution and is a promising tool for studying the epidemiology of A. baumannii and Acinetobacter genomic species 13TU.     

Molecular evolution of methicillin-resistant Staphylococcus aureus in the metropolitan area of Cologne, Germany, from 1984 to 1998

To investigate the molecular evolution of methicillin-resistant Staphylococcus aureus (MRSA) in a large metropolitan area in Germany, 398 nonrepetitive MRSA isolates recovered from patients from various teaching and nonteaching hospitals in Cologne between 1984 and 1998 were characterized by pulsed-field gel electrophoresis (PFGE). On this basis, 95 representative isolates were selected and further investigated by multilocus sequence typing (MLST), spa typing, and staphylococcal cassette chromosome mec (SCCmec) typing. Overall, there were 9 MLST types and 16 spa types. The most prevalent sequence types (STs) were ST239 (38% of isolates), ST247 (29%), and ST228 (18%); the most prevalent spa types were 37 (32%) and 51 (29%). ST239 comprised five major PFGE types and various unique PFGE patterns, and ST5 comprised two PFGE types. While the same PFGE pattern was not observed among strains with different STs, spa type 37 was observed among strains representing two different STs (ST239 and ST241), and these belonged to the same clonal complex as single-locus variants. ST239 was the earliest predominant ST, with the highest prevalence from 1984 to 1988 (96%), followed by ST247 from 1989 to 1993 (83%) and ST228 from 1994 to 1998 (40%). Spa type 37 was the most prevalent from 1984 to 1988 (96%), spa type 51 was the most prevalent from 1989 to 1993 (83%), and spa types 1 and 458 were the most prevalent from 1994 to 1998 (26% and 14%, respectively). The prevalence of SCCmec type III decreased from 96% from 1984 to 1988 to 8% from 1989 to 1993, the prevalence of SCCmec type I increased from 4% from 1984 to 1988 to 97% from 1989 to 1993 and decreased to 62% from 1994 to 1998. While the genetic diversity of MRSA increased from 1984 to 1998, one prevalent ST usually accounted for most of the isolates in a given time period.     

Similar Frequencies of Pseudomonas aeruginosa Isolates Producing KPC and VIM Carbapenemases in Diverse Genetic Clones at Tertiary-Care Hospitals in Medellín,Colombia

Carbapenem-resistant Pseudomonas aeruginosa has become a serious health threat worldwide due to the limited options available for its treatment. Understanding its epidemiology contributes to the control of antibiotic resistance. The aim of this study was to describe the clinical and molecular characteristics of infections caused by carbapenem-resistant P. aeruginosa isolates in five tertiary-care hospitals in Medellín, Colombia. A cross-sectional study was conducted in five tertiary-care hospitals from June 2012 to March 2014. All hospitalized patients infected by carbapenem-resistant P. aeruginosa were included. Clinical information was obtained from medical records. Molecular analyses included PCR for detection of bla_VIM, bla_IMP, bla_NDM, bla_OXA-48, and bla_KPC genes plus pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) for molecular typing. A total of 235 patients were enrolled: 91.1% of them were adults (n = 214), 88.1% (n = 207) had prior antibiotic use, and 14.9% (n = 35) had urinary tract infections. The bla_VIM-2 and bla_KPC-2 genes were detected in 13.6% (n = 32) and 11.5% (n = 27), respectively, of all isolates. Two isolates harbored both genes simultaneously. For KPC-producing isolates, PFGE revealed closely related strains within each hospital, and sequence types (STs) ST362 and ST235 and two new STs were found by MLST. With PFGE, VIM-producing isolates appeared highly diverse, and MLST revealed ST111 in four hospitals and five new STs. These results show that KPC-producing P. aeruginosa is currently disseminating rapidly and occurring at a frequency similar to that of VIM-producing P. aeruginosa isolates (approximately 1:1 ratio) in Medellín, Colombia. Diverse genetic backgrounds among resistant strains suggest an excessive antibiotic pressure resulting in the selection of resistant strains.