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.     

Understanding the dynamics of imipenem-resistant Acinetobacter baumannii lineages within Portugal

A recent collection of 213 imipenem-resistant Acinetobacter baumannii (IRAB) clinical isolates was characterized for the presence of acquired carbapenem-hydrolysing class D β-lactamases (CHDLs) and clonality. A population structure analysis of IRAB was also conducted, with five molecular typing methods. Three main clusters, each one associated with a specific CHDL, were observed with multilocus sequence typing. Overall, our results suggest a switch in the dominant clone, with sequence type (ST) 92, carrying bla_OXA-23 (63.4%), replacing the closely related ST98, carrying bla_OXA-24/40 (22%). In addition, ST103, an independent lineage, was associated with bla_OXA-58-carrying isolates (14.6%).     

A PCR-based method to differentiate between Acinetobacter baumannii and Acinetobacter genomic species 13TU

A new PCR-based method that exploits differences in gyrB gene sequences was developed to distinguish between Acinetobacter baumannii and Acinetobacter genomic sp. 13TU. Among 118 clinical and reference Acinetobacter strains, 102 of which were previously speciated by amplified rDNA restriction analysis as belonging to the Acinetobacter calcoaceticus-A. baumannii complex, the method correctly identified 31 A. baumannii and 54 Acinetobacter genomic sp. 13TU isolates to the species level. The method was rapid, specific and easy to interpret.     

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.     

Clonal spread of carbapenem-resistant OXA-23-positive Acinetobacter baumannii in a Bulgarian university hospital

From October 1999 to September 2006, 29 carbapenem-resistant isolates of Acinetobacter baumannii were collected consecutively from patients hospitalized in different wards of the University Hospital in Pleven, Bulgaria. The bla _OXA-23 gene, associated with the upstream-located IS Aba1 , was identified as the mechanism responsible for carbapenem resistance in all isolates. The isolates belonged to two different clonal groups, indicating a sustained hospital outbreak. This study demonstrates both the epidemic potential of carbapenem-resistant A. baumannii and its longevity in the hospital environment.     

Molecular epidemiology of ampicillin-resistant clinical isolates of Salmonella enterica serovar Typhimurium

Thirty-nine multiresistant Salmonella enterica serovar Typhimurium (S. Typhimurium) isolates were obtained from 33 children and 6 adults hospitalized from 1996 to 1999 in the University Hospital of Amiens (France). S. Typhimurium was cultured from stools (n=36), blood samples (n=2) and peritoneal fluid (n=1). These isolates were characterized by biotyping, antibiotic susceptibility test, RAPD-PCR, and PFGE typing. Emergence of pentaresistant S. Typhimurium isolates (phenotype ACSSuTe) was observed, and five of them were resistant to nalidixic acid and of intermediate susceptibility to pefloxacin. Genotypic analysis of both RAPD and PFGE results showed that there were 7 different patterns. Thirty-three isolates gave an identical pattern (AI) and were considered as epidemic isolates; the six remaining patterns (each containing one isolate) corresponded to sporadic cases. Antibiotic susceptibility patterns, RAPD and PFGE patterns subdivided the 39 isolates into 9 clonally related groups. One of them (pattern AI and R-pattern a) was implicated in 74% of the cases.     

Identification of widespread, closely related Acinetobacter baumannii isolates in Portugal as a subgroup of European clone II

The aims of this study were to determine whether a multidrug-resistant Acinetobacter baumannii clone, known to be endemic in three tertiary-care Portuguese hospitals, had disseminated throughout Portugal, and whether this clone was related to one of the European clones I-III described previously. The isolates were first screened by pulsed-field gel electrophoresis and/or M13 random amplified polymorphic DNA fingerprint analysis. Ten representative isolates were compared by amplified fragment length polymorphism (AFLP) analysis, and were also compared with isolates contained in the AFLP library of the Leiden University Medical Centre. All of the Portuguese isolates clustered in European clone II (clone delineation level >80%). Following AFLP analysis, seven isolates clustered at >96%, indicating a striking degree of genetic relatedness and suggesting recent spread of a (sub)clone. Three isolates were slightly more separated from this main group, but all isolates clustered at 87.4%. Thus, the Portuguese multidrug-resistant isolates formed a sub-cluster of European clone II, suggesting that they belong to a recent lineage within clone II.     

Characterisation of Yersinia pestis isolates from natural foci of plague in the Republic of Georgia, and their relationship to Y. pestis isolates from other countries

Forty Yersinia pestis isolates from endemic foci of plague in the Republic of Georgia, and six Y. pestis isolates from neighbouring former Soviet Union countries, were analysed for their biochemical and phenotypic properties, and their genetic relatedness was compared with Y. pestis strains KIM and CO92 by pulsed-field gel electrophoresis (PFGE). In addition, 11 Y. pestis isolates from the USA, together with published nucleotide sequences from Y. pestis strains KIM, CO92 and 91001, were compared with the 46 isolates in the present collection using multilocus sequence typing (MLST), based on sequence data for the 16S rRNA, hsp60, glnA, gyrB, recA, manB, thrA and tmk loci. Four virulence gene loci (caf1, lcrV, psaA and pla) were also sequenced and analysed. Two sequence types (ST1 and ST2), which differed by a single nucleotide, were identified by MLST. With the exception of a single isolate (771G), all of the Georgian Y. pestis isolates belonged to ST2. PFGE also grouped the Georgian Y. pestis isolates separately from the non-Georgian isolates. Overall, PFGE discriminated the Y. pestis isolates more effectively than MLST. The sequences of three of the four virulence genes (lcrV, psaA and pla) were identical in all Georgian and non-Georgian isolates, but the caf1 locus was represented by two allele types, with caf1 NT1 being associated with the non-Georgian isolates and caf1 NT2 being associated with the Georgian isolates. These results suggest that Georgian Y. pestis isolates are of clonal origin.     

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.     

Genotypic diversity and antibiotic susceptibility of Acinetobacter baumannii isolates in a Bulgarian hospital

A set of 18 Acinetobacter baumannii isolates, collected prospectively in a Bulgarian hospital during episodes of increased A. baumannii occurrence during 2000-2002, was investigated for genotypic diversity and antibiotic susceptibility. Four genotypes were identified by amplified fragment length polymorphism genomic fingerprinting, one of which (type 1) accounted for 13 isolates, indicating that a specific strain was predominant. The single isolate allocated to type 2 was identified to European clone I. All isolates were resistant to multiple antibiotics, but most retained susceptibility to tobramycin and colistin, and all except one were susceptible to imipenem.     

Clonal spread of SCCmec type IV methicillin-resistant Staphylococcus aureus between community and hospital

The staphylococcal chromosome cassette (SCC)mec types of 382 hospital-acquired methicillin-resistant Staphylococcus aureus (HA-MRSA) isolates in Taiwan were analysed over a 7-year period (1999-2005). There was an abrupt increase in SCCmec type IV in HA-MRSA during 2005. The molecular epidemiology of a subset (n = 69) of HA-MRSA isolates with SCCmec types III, IV or V was characterised and compared with that of community-acquired MRSA (CA-MRSA) (n = 26, collected during 2005). Pulsed-field gel electrophoresis revealed three major pulsotypes (A, B and C) and 15 minor clones. Pulsotypes B and C, which contained isolates carrying SCCmec types IV and V, respectively, included both CA-MRSA and HA-MRSA isolates. Among 24 toxin genes analysed, five genes had significant differential distribution between CA-MRSA and SCCmec type III HA-MRSA. Furthermore, among SCCmec type IV isolates, the seb gene was detected more commonly in HA-MRSA. Analysis of representative members of the three major pulsotypes by multilocus sequence typing revealed two sequence types (STs), namely ST239 (SCCmecIII) and ST59 (SCCmecIV or SCCmecV). This suggests that ST59:SCCmecIV, which is usually community-acquired, has become an important nosocomial pathogen in the hospital studied.     

Molecular and phenotypic characterization of invasive group B streptococcus strains from infants in Norway 2006–2007

Multilocus sequence typing of an almost complete collection of invasive group B streptococcus (GBS) strains from infants in Norway, conducted in 2006–2007, revealed 27 sequence types (ST), of which 23 clustered into five clonal complexes. The case fatality rate of invasive GBS disease in infants was 16/98 (16.3%). Type V strains were predominant among strains resistant to erythromycin and clindamycin (11/18; 61.1%). All type V strains from fatal cases (5/16) were ST1, resistant to erythromycin and clindamycin, and belonged to three pulsed-field gel electrophoresis-clusters. Further analysis of virulence characteristics of these apparently highly virulent subtypes of type V, ST1 GBS strains is warranted.     

Conventional and molecular investigation of meningococcal isolates in relation to two outbreaks in the area of Athens, Greece

Two local outbreaks caused by serogroup B Neisseria meningitidis occurred in the Athens area of Greece during 2003. In total, 30 N. meningitidis isolates from patients and carriers, as well as sporadic cases, were investigated by conventional techniques (serogroup, serotype and serosubtype), multilocus sequence typing (MLST), analysis of variable number tandem repeats (VNTR) and random amplified polymorphic DNA (RAPD) analysis. Compared with the two other molecular techniques, VNTR analysis was a simple, reliable and highly discriminatory method for fine typing of meningococcal isolates, showing a good correlation with the epidemiological data for the two outbreaks analysed.     

Molecular typing of Clostridioides difficile isolates from clinical and non‐clinical samples in Iran

Clostridioides difficile is a major cause of nosocomial infectious diarrhea in hospitalized patients throughout the world. We aimed to characterize C. difficile isolates among hospitalized patients, hospital staffs, and hospital environment samples obtained in three tertiary care hospitals of Iran with regard to their molecular types between June 2016 and November 2017. The toxigenicity of C. difficile isolates was determined by toxigenic culture and multiplex‐PCR. Toxigenic C. difficile isolates collected were ribotyped using capillary gel electrophoresis‐based PCR and the database of WEBRIBO ( http://webribo.ages.at ). Of 500 clinical and non‐clinical samples, toxigenic C. difficile were identified in 35 of 250 stool samples (14%) and in 3 of 250 swabs (1.2%). The most frequently found ribotypes (RTs) were 039, AI‐12, and AI‐21 (15.8, 10.52, and 10.52% of all isolates, respectively). Further RTs were: 017, 001, AI‐3, AI‐15, AI‐18, AI‐10, AI‐4, and PR21195 (as new ribotype). The epidemic RTs (027 and 078) seen in the Europe, North America, and Asia were completely absent in this study.     

Molecular analysis of ampicillin-resistant sporadic Salmonella typhi and Salmonella paratyphi B clinical isolates

Objective: To study the mechanisms of antibiotic resistance in Salmonella typhi and Salmonella paratyphi B clinical isolates, and the clonality of resistant strains.
Method: Antibiotic susceptibility was tested by disk-agar diffusion. Conjugation experiments and plasmid analysis by agarose gel electrophoresis after Eco RI digestion were followed by hybridization to a digoxigenin-labeled TEM-type β-lactamase probe. DNA fingerprints were obtained by pulsed-field gel electrophoresis of Xba I-digested chromosomal DNA.
Results: Three S. typhi isolates (7% of the isolates studied), of which one was ampicillin resistant and the other two multiresistant (ampicillin, chloramphenicol, tetracycline, sulfamethoxazole/trimethoprim and streptomycin), and two ampicillin-resistant S. paratyphi B isolates (25% of the isolates studied) were further evaluated. A 34-MDa conjugative plasmid, previously isolated from Salmonella enteritidis , conferred ampicillin resistance. A 100-MDa conjugative plasmid encoded resistance to chloramphenicol, tetracycline and sulfamethoxazole/trimethoprim, as well as ampicillin. Chromosomal fingerprinting revealed two distinct resistant strains for each serovar which were different from a matched set of sensitive S. typhi strains.
Conclusions: Two conjugative, TEM-type β-lactamase-encoding plasmids conferred ampicillin resistance to S. typhi and S. paratyphi B. The 34-MDa plasmid was identical to that previously characterized from S. enteritidis , while the 100-MDa plasmid also encoded resistance to chloramphenicol, tetracycline and sulfamethoxazole/trimethoprim. Resistant isolates did not belong to a single clone but rather represented distinct strains.     

Outer membrane vesicles isolated from two clinical Acinetobacter baumannii strains exhibit different toxicity and proteome characteristics

Outer membrane vesicles (OMVs) are well-characterized virulence factors produced by Gram-negative bacteria. Here, we isolated two clinical Acinetobacter baumannii strains, the multidrug-resistant A. baumannii (MDRAb) A38 and non-MDRAb 5806. Strain A38 produced more abundant OMVs than strain 5806 when cultured to the early stationary phase. The results from cell proliferation assays and real-time PCR analyses indicated that A38 OMVs induced more powerful cytotoxicity and stronger innate immune responses compared with 5806 OMVs. Moreover, SDS-PAGE and LC-MS/MS analyses revealed that A38 OMVs contained more virulence factors, including Omp38, EpsA, Ptk, GroEL, hemagglutinin-like protein, and FilF. Taken together, the results of the present study suggest that MDRAb might produce abundant OMVs with more virulent factors facilitating the worse outcome, a finding that merits further study.     

Non-multiresistant methicillin-resistant Staphylococcus aureus bacteraemia in Sydney, Australia: emergence of EMRSA-15, Oceania, Queensland and Western Australian MRSA strains

AIMS: To describe clinical features and molecular epidemiology of non-multiresistant methicillin-resistant Staphylococcus aureus (MRSA) bacteraemia. METHODS: Patients with non-multiresistant MRSA isolated from blood at South Western Area Pathology Service from 1 January 1999 to 31 December 2001 were enrolled. Pulsed field gel electrophoresis, phage typing, and (selected instances) multilocus sequence and staphylococcal cassette chromosome typing was performed. PCR was used to detect Panton-Valentine leukocidin (PVL), toxic shock syndrome toxin-1 (TSST-1), and enterotoxin genes. RESULTS: Sixteen patients were detected: eight with UK EMRSA-15 (ST22-MRSA-IV), three with Oceania (South-West Pacific/Western Samoan phage pattern) (ST30-MRSA-IV), two with WA MRSA-5 (ST8-MRSA-IV), and one each with WA MRSA-1 (ST1-MRSA-IV), Queensland strain (ST93-MRSA-IV), and WA MRSA-15 (ST59-MRSA-IV). Prior hospital admissions occurred with six of the eight patients with UK EMRSA-15, none of the three with Oceania, and three of the five with other strains. Thirteen of 16 patients had underlying disease. Three of the three patients with Oceania strain bacteraemia were Polynesians; 11 of 13 of the others were Caucasians. PVL genes were detected in four of 16 isolates (all Oceania and Queensland strains). entC was detected in two EMRSA-15 strains; entA in one Oceania, two WA MRSA-5 and the WA MRSA-1 strain, with entA and entB in the WA MRSA-15 strain. tst was not detected. CONCLUSIONS: Multiple epidemic strains cause non-multiresistant MRSA bacteraemia. Most patients had risk factors. Oceania and Queensland strains possess the PVL gene.