Prevalence and diversity of extended-spectrum β-lactamases in faecal Escherichia coli isolates from healthy humans in Spain

Extended-spectrum β-lactamase (ESBL)-producing Escherichia coli isolates were detected in seven of 105 faecal samples from healthy humans, from two Spanish cities, during 2007. In these isolates, five ESBLs were detected, CTX-M-14 ( n  = 2), CTX-M-1 ( n  = 2), CTX-M-32 ( n  = 1), CTX-M-8 ( n  = 1) and TEM-52 ( n  = 1). Both bla _CTX-M-14a (surrounded by IS Ecp1 -IS 903 ) and bla _CTX-M-14b variants (included in an integron structure) were identified in this study. This is the first time that the bla _CTX-M-8 gene and ESBLs of the CTX-M-8 group have been found in Europe and Spain, respectively. Faecal E. coli of healthy humans therefore constitute a reservoir of bla _CTX-M genes with different surrounding genetic elements.     

Molecular characterisation of extended-spectrum β-lactamase-producing Escherichia coli and Klebsiella spp. isolates at a tertiary-care centre in Lebanon

The prevalence of bla _CTX-M, bla _TEM and bla _SHV genes among extended-spectrum β-lactamase (ESBL)-producing clinical isolates of Escherichia coli ( n  = 50) and Klebsiella spp. ( n  = 50) from Lebanon was 96%, 57% and 67%, and 40%, 82% and 84%, respectively. Genotyping revealed that the clonal diversity was unrelated to the presence of bla genes. Sequence analysis of 16 selected isolates identified the bla _CTX-M-15, bla _TEM-1, bla _OXA-1 and six bla _SHV genes, as well as the gene encoding the quinolone-modifying enzyme AAC(6')-Ib-cr. The genes encoding CTX-M-15 and AAC(6')-Ib-cr were carried on a 90-kb plasmid of the pC15-1a or pCTX-15 type, which transferred both ESBL production and quinolone resistance from donors to transconjugants.     

Prevalence and characterization of extended-spectrum beta-lactamases-producing Salmonella enterica isolates in Saragossa, Spain (2001-2008)

We analyzed the prevalence of resistance to extended-spectrum cephalosporins (ESCs) among clinical strains of Salmonella enterica collected by the Laboratory of Clinical Microbiology in the University Clinical Hospital Lozano Blesa in the region of Aragón (Spain), for which very few epidemiological information exists. A total of 2,092 strains of S. enterica were identified in stool samples from patients with gastroenteritis. Five isolates showed an extended-spectrum beta-lactamase (ESBL) phenotype: four isolates of S. enterica serotype Virchow harbored the ESBL-encoding bla(CTX-M-9) gene and an isolate of serotype Enteritidis carried a bla(CTX-M-1) gene, which, to the best of our knowledge, is described here for the first time in this serotype of S. enterica. The five ESC-resistant isolates were also resistant to spectinomycin, streptomycin, kanamycin, sulfonamides, tetracycline, and trimethoprim as well as to nalidixic acid. The ESBL isolate of serotype Enteritidis, however, remained susceptible to kanamycin and nalidixic acid. A class 1 integron of 1.5?kb was detected for the four serotype Virchow isolates with the gene cassette dfrA16-aadA2. The bla(CTX-M-9) gene was carried by an ～300-kb IncHI2 conjugative plasmid in the case of the S. enterica serotype Virchow isolates. The bla(CTX-M-1) gene was carried by an ～100-kb IncI1-N conjugative plasmid for the serotype Enteritidis ESC-resistant isolate. All the four ESC-resistant strains of S. enterica serotype Virchow clustered together in a XbaI pulsed-field gel electrophoresis, which also revealed a strong similarity between them and some pulsotypes of S. enterica serotype Virchow from France.     

Characterization of Acinetobacter baumannii carrying blaOXA-23, blaPER-1 and armA in a Korean hospital

Forty-two multidrug-resistant (MDR) Acinetobacter baumannii isolates were obtained during outbreaks in a Korean hospital. The co-carriage of bla _OXA-23, bla _OXA-51, bla _PER-1 and armA was observed in 23 isolates, and they were susceptible only to colistin and minocycline. The MDR A. baumannii isolates were found to belong to sequence group 1 using sequence-based typing.     

Emergence of extended-spectrum-beta-lactamase (CTX-M-9)-producing multiresistant strains of Salmonella enterica serotype Virchow in poultry and humans in France

During 2002 to 2003, eight Salmonella enterica serotype Virchow poultry and poultry product isolates from various sources (chicken farms, poultry slaughterhouse, or retail store) and one S. enterica rough strain isolated from human feces were found to produce extended-spectrum beta-lactamase CTX-M-9. Poultry and poultry product isolates were recovered from different locations in the southwest of France. The human rough isolate had sequences of flagellin genes (fliC and fljB) typical of serotype Virchow and ribotyping and pulsed-field gel electrophoresis (PFGE) patterns closely similar to those of serotype Virchow strains. PFGE confirmed the clonal relationship between the poultry isolates, while the human isolate displayed a pattern with 94% homology. The bla(CTX-M-9) gene was located on a conjugative plasmid and was shown to be linked to orf513. Plasmid profiling found a very similar EcoRI restriction pattern in six transconjugants studied, including transconjugants obtained from the human isolate. A single hatchery, supplying chicks to the six farms, was identified. Emergence of extended-spectrum beta-lactamase-producing S. enterica strains in food animals is a major concern, as such strains could disseminate on a large scale and lead to antibiotic therapy difficulties.     

Carbapenem-resistant and OXA-23-producing Acinetobacter baumannii isolates in the United Arab Emirates

Five carbapenem-resistant Acinetobacter baumannii isolates, collected from the United Arab Emirates in 2006, were investigated to identify the mechanism(s) responsible for carbapenem resistance. Genotyping was performed by pulsed-field gel electrophoresis, and the location of the bla _OXA-23 gene was determined by using the endonuclease I Ceu I technique and mating-out assays. The four isolates in which the bla _OXA-23 gene was located on the chromosome within a Tn 2006 composite transposon were clonally related. The single non-clonally related isolate harboured the bla _OXA-23 gene on a 70-kb transferable plasmid. This study reports on the dissemination of OXA-23-producing A. baumannii isolates in the Middle East.     

Repeated occurrence of diverse extended-spectrum beta-lactamases in minor serotypes of food-borne Salmonella enterica subsp. enterica

Screening of Greek nontyphoid salmonellae from 2000 to 2002 yielded three extended-spectrum beta-lactamase (ESBL)-producing human isolates. Salmonella enterica serotype Brandenburg harbored a multiresistant SHV-5 gene-carrying plasmid. S. enterica serotype Blockley and S. enterica serotype Hadar harbored a TEM-52 gene-carrying plasmid. An S. enterica serotype Virchow strain producing plasmid-mediated CTX-M-32 was isolated twice from poultry end products. All ESBL plasmids were self-transferable and carried by clones currently common in Greece.     

Occurrence of qnrA-positive clinical isolates in French teaching hospitals during 2002–2005

Bacteria harbouring the novel qnrA plasmid-mediated mechanism of quinolone resistance have been described in different countries, but the frequency of their occurrence has not been investigated. In total, 1,468 clinical isolates of Enterobacteriaceae with quinolone resistance or extended-spectrum beta-lactamase (ESBL) phenotypes were collected from eight teaching hospitals in France during 2002-2005 and screened for qnrA. Overall, 28 isolates (22 Enterobacter cloacae, three Klebsiella pneumoniae, one Citrobacter freundii, one Klebsiella oxytoca and one Proteus mirabilis) were positive for qnrA, representing 1.9% of all isolates, 3.3% of ESBL-producing isolates (22% of the E. cloacae isolates) and 0% of non-ESBL-producing isolates. The prevalence of qnrA among consecutive ESBL-producing isolates in 2004 from the eight hospitals was 2.8% (18/639). Of the qnrA-positive isolates, 100% were intermediately-resistant or resistant to nalidixic acid, and 75% to ciprofloxacin. Twenty-one of the 22 qnrA-positive E. cloacae isolates were obtained from two hospitals in the Paris area, and molecular typing and plasmid content analysis showed clonal relationships for five, three and two isolates, respectively. The qnrA genetic environment was similar to that of the In36 integron. The remaining two isolates had qnrA variants (30 and 29 nucleotide differences, respectively, compared with the original sequence) and an unknown genetic environment. The ESBL gene associated with qnrA was bla(SHV-12) in most of the isolates, but bla(PER-1) and bla(SHV-2a) were found in two isolates. In France, it appears that qnrA-positive isolates are predominantly E. cloacae isolates producing SHV-12, and may be associated with the dissemination of an In36-like integron.     

Ceftriaxone resistance of nontyphoidal Salmonella enterica isolates in Northern Taiwan attributable to production of CTX-M-14 and CMY-2 beta-lactamases

Among 3,027 nontyphoidal Salmonella enterica isolates identified between January 1999 and December 2002 in a medical center in northern Taiwan, 31 were resistant to the extended-spectrum cephalosporin ceftriaxone (1.02% [31/3,027]), including 2 in 1999 (0.36% [2/549]), 13 in 2000 (1.49% [13/870]), 7 in 2001 (0.78% [7/893]), and 9 in 2002 (1.26% [9/715]). Sixteen of these isolates belonged to Salmonella serogroup B, nine belonged to serogroup C, four belonged to serogroup D, and two belonged to serogroup E. The majority were from stool cultures. The mechanism of resistance was investigated for eight isolates, including three S. enterica serovar Typhimurium, one S. enterica serovar Wagenia, one S. enterica serovar Senftenberg, one S. enterica serovar Derby, one S. enterica serovar Panama, and one S. enterica serovar Duesseldorf isolate. All eight patients from whom these isolates were recovered had community-acquired infections. All eight isolates were resistant to ampicillin, ceftriaxone, and cefotaxime but susceptible to imipenem and ciprofloxacin. Ceftriaxone resistance was due to the production of the CMY-2 AmpC beta-lactamase by seven isolates and the CTX-M-14 beta-lactamase by the remaining isolate. Both beta-lactamase genes were carried on conjugative plasmids. In a 2.5-kb region encompassing the bla(CMY-2) gene, at nucleotide 49 upstream of the start codon of bla(CMY-2), three of the seven bla(CMY-2)-positive isolates had an A nucleotide and four had a G nucleotide. In conclusion, the ceftriaxone resistance of nontyphoidal Salmonella isolates in our hospital was attributed to the CTX-M-14 and CMY-2 beta-lactamases.     

Epidemic multidrug-resistant Acinetobacter baumannii related to European clonal types I and II in Rome (Italy)

The molecular epidemiology and the genetic basis of antibiotic resistance in 88 multidrug-resistant (MDR) Acinetobacter baumannii strains isolated during 18 months from infected patients in seven intensive care units (ICUs) in Rome were investigated. Random amplified polymorphic DNA and macrorestriction analysis identified two predominant clonal types, genetically related to the European epidemic clones I (type 2) and II (type 1), accounting for 98.9% of A. baumannii ICU isolates. Type 1 was isolated from all ICUs under survey. Class 1 integrons of 2.2 and 2.5 kb were detected in type 1 and type 2 isolates, respectively. The integron structures were similar to those previously determined for epidemic A. baumannii strains from various European countries, and suggestive of integron rearrangement/exchange among isolates related to the European epidemic clones I and II. Carbapenem resistance was associated with the presence of the bla _OXA-58 gene in type 1 isolates. The results indicate that the A. baumannii type 1 clone has a high potential of spreading among hospitals.     

Two different extended-spectrum beta-lactamases (ESBLs) in one of the first ESBL-producing salmonella isolates in Poland

Two extended-spectrum beta-lactamase (ESBL)-producing salmonella isolates, Salmonella enterica serovar Enteritidis and Salmonella enterica serovar Typhimurium, were analyzed. Both isolates produced the CTX-M-3 ESBL; however, their bla(CTX-M-3) genes were located on different plasmids. The serovar Typhimurium isolate also expressed another ESBL, SHV-2a, and probably the two ESBL genes had been acquired independently by the strain.     

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.     

Nosocomial outbreak caused by Salmonella enterica serotype Livingstone producing CTX-M-27 extended-spectrum beta-lactamase in a neonatal unit in Sousse, Tunisia

In this study, we report an outbreak of Salmonella enterica serotype Livingstone resistant to extended-spectrum cephalosporins that occurred in a neonatal ward of the maternity department of Farhat Hached Hospital, Sousse, Tunisia, in 2002. A total of 16 isolates were recovered from 16 babies hospitalized in the ward during the period 1 to 16 July. All these babies developed diarrhea, and three of them developed septicemia. All the isolates demonstrated resistance to ceftriaxone and ceftazidime due to the production of an extended-spectrum beta-lactamase (ESBL). The isolates were also resistant to aminoglycosides (kanamycin, tobramycin, netilmicin, gentamicin, and amikacin) and sulfamethoxazole-trimethoprim. DNA profiles were determined by pulsed-field gel electrophoresis using the XbaI and SpeI endonucleases and by ribotyping with PstI digestion. They yielded the same patterns, showing that the outbreak was caused by a single clone. The ESBL was identified as CTX-M-27 by sequencing of PCR products and by isoelectric focusing. The ESBL resistance was transferred by a 40-kb conjugative plasmid. The mobile insertion sequence ISEcp1 was found to be located upstream of bla(CTX-M-27) in the same position as that known for a bla(CTX-M-14) sequence. A new gene named dfrA21, encoding resistance to trimethoprim and carried by a 90-kb plasmid, was characterized. The dfrA21 gene was inserted as a single resistance cassette in a class I integron. The babies were treated with colistin, and all except two recovered. The outbreak came to an end when appropriate actions were taken: patient isolation, hand washing, and disinfection of the ward.     

Using nucleic acid microarrays to perform molecular epidemiology and detect novel β-lactamases: a snapshot of extended-spectrum β-lactamases throughout the world

The worldwide dissemination of extended-spectrum-β-lactamase (ESBL)- and carbapenemase-producing Enterobacteriaceae is a major concern in both hospital and community settings. Rapid identification of these resistant pathogens and the genetic determinants they possess is needed to assist in clinical practice and epidemiological studies. A collection of Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, and Proteus mirabilis isolates, including phenotypically ESBL-positive (n = 1,093) and ESBL-negative isolates (n = 59), obtained in 2008-2009 from a longitudinal surveillance study (SMART) was examined using an in vitro nucleic acid-based microarray. This approach was used to detect and identify bla(ESBL) (bla(SHV), bla(TEM), and bla(CTX-M) genes of groups 1, 2, 9, and 8/25) and bla(KPC) genes and was combined with selective PCR amplification and DNA sequencing for complete characterization of the bla(ESBL) and bla(KPC) genes. Of the 1,093 phenotypically ESBL-positive isolates, 1,041 were identified as possessing at least one bla(ESBL) gene (95.2% concordance), and 59 phenotypically ESBL-negative isolates, used as negative controls, were negative. Several ESBL variants of bla(TEM) (n = 5), bla(SHV) (n = 11), bla(CTX-M) (n = 19), and bla(KPC) (n = 3) were detected. A new bla(SHV) variant, bla(SHV-129), and a new bla(KPC) variant, bla(KPC-11), were also identified. The most common bla genes found in this study were bla(CTX-M-15), bla(CTX-M-14), and bla(SHV-12). Using nucleic acid microarrays, we obtained a "molecular snapshot" of bla(ESBL) genes in a current global population; we report that CTX-M-15 is still the dominant ESBL and provide the first report of the new β-lactamase variants bla(SHV-129) and bla(KPC-11).     

Susceptibility of strains of Streptococcus agalactiae to macrolides and lincosamides, phenotype patterns and resistance genes

The Group B streptococcus ( Streptococcus agalactiae ) is a pathogen of increasing importance in human disease. We therefore studied the susceptibility of clinical isolates of S. agalactiae to penicillin G, erythromycin, azithromycin and clindamycin using National Committee for Clinical Laboratory Standards methodology, and we also determined the phenotypes of macrolide-lincosamide susceptibility and the resistance genes implicated in a group of selected isolates of the different phenotypes. We used 221 isolates collected between 1997 and 1999 in two Health Authority Areas in Móstoles and Granada, Spain. The minimal concentration for 90% inhibition (MIC₉₀) for penicillin G was 0.12 mg/L and all the isolates tested were susceptible. One hundred and eighty-five (83.7%) were susceptible to erythromycin and azithromycin and 191 (86.4%) were susceptible to miocamycin and clindamycin. Twenty-three isolates (10.4%) had a constitutive MLS_B phenotype, seven (3.2%) an inducible phenotype, and six (2.7%) an M phenotype. All except one of the MLS_B phenotype isolates tested ( n  = 23) carried erm genes; in two strains with the mef (A) gene, all the M phenotype ( n  = 6) isolates tested carried mef genes, while erm and mef (A) genes were absent in all the macrolide-lincosamide-susceptible ( n  = 12) isolates tested. In our environment, resistance to macrolide and lincosamide in S. agalactiae was present in 10–16% of the isolates. The majority of resistant strains had the MLS_B phenotype.     

Characterization of the first extended-spectrum beta-lactamase-producing Salmonella isolate identified in Canada

A single Salmonella enterica serovar Typhimurium isolate with an UT2 phage type producing an extended-spectrum beta-lactamase (ESBL) was identified in Canada in 2000. The isolate harbored two plasmids, one containing a bla(TEM-1) gene and the other containing a bla(SHV-2a) gene. The ESBL gene was located on a 70-kb transferable plasmid which also carried tetracycline and trimethoprim resistance elements.     

Prevalence, antimicrobial resistance, and extended-spectrum beta-lactamases characterization of Salmonella isolates in Apulia, southern Italy (2001-2005)

The prevalence, antimicrobial susceptibility, and production of extended-spectrum beta-lactamases (ESBLs) of Salmonella collected from several hospitals in Apulia (southern Italy) were evaluated. The most common Salmonella isolates were Salmonella enterica serovar Typhimurium (44.6%), S. enterica serovar Enteritidis (33.4 %), S. enterica serovar Infantis (3.2 %), S. enterica serovar Typhi (1.5%), and S. enterica serovar Bovismorbificans (1.5%). The other serovars accounted for less than 1% each. Our data show a high resistance to ampicillin, tetracycline, and chloramphenicol. The isolates were pansensitive (53.5%), resistant to one antimicrobial agent (10.5%), resistant to two antimicrobial agents (22.1%), resistant to three antimicrobial agents (10.8%), and to four antimicrobial agents (2.7%). Resistance to more than four antibiotics was observed in 0.5% of strains. The presence of ESBL was found in only one strain of S. enterica serovar Bovismorbificans. The CTX-M-1 type-producing strain was identified by isoelectric focusing and molecular analysis. Results were consistent with the presence of a pI 8.6 ESBL active on cefotaxime, ceftazidime, cefepime, and aztreonam. Mating experiments showed that the CTX-M-1 determinant was transferable. To the best of our knowledge, this is the first report of CTX-M-1 type ESBL in Salmonella serovar Bovismorbificans.     

Characterisation and molecular epidemiology of extended-spectrum β-lactamase-producing Enterobacter cloacae isolated from a district teaching hospital in Taiwan

Enterobacter cloacae (n = 110) isolates from a district hospital in Taiwan were screened for extended-spectrum beta-lactamases (ESBLs). In total, 17 ESBL-producers were identified, based on the combination-disk synergy test using cefotaxime and ceftazidime +/- clavulanic acid. Investigation of ESBL genes in 33 ceftazidime-resistant isolates revealed the SHV-12 gene in the same 17 ESBL-producers. In addition, one isolate also carried the CTX-M-3 gene, and two isolates also carried the CTX-M-9 gene. No major epidemic clone of ESBL-producers was identified by pulsed-field gel electrophoresis. Routine screening for the ESBL phenotype, focusing on ceftazidime-resistant E. cloacae, should be undertaken in this area.     

Infrequent detection of acquired metallo-β-lactamases among carbapenem-resistant Pseudomonas isolates in a Greek hospital

Objective  To study the possible distribution of metallo-β-lactamases among nosocomial Pseudomonas isolates in a Greek hospital with a recent high prevalence of carbapenem-resistant Pseudomonas isolates.
Methods  All carbapenem-resistant (imipenem- and/or meropenem-resistant) (MICs > 8 mg/L) Pseudomonas non-replicate isolates recovered from clinical infections in the Microbiology Laboratory of Saint Demetrios Hospital, Thessaloniki, Greece, from April 1998 to November 2000 were studied for the presence of metallo-β-lactamases. They were tested by a disk diffusion test, PCR analysis, and nucleotide sequencing. DNA fingerprints were obtained by pulsed-field gel electrophoresis (PFGE) of Xba I-digested chromosomal DNA.
Results  In total, 24 carbapenem-resistant isolates (23 P. aeruginosa and one P. putida ) were recovered. The serotypes observed among the P. aeruginosa isolates were, in order of decreasing frequency, O:11 (52%), O:3 and O:12 (17% each), and O:6 (13%). PFGE grouped 17 of the P. aeruginosa isolates into four clusters, each containing from two to seven isolates, while the remaining isolates exhibited unique genotypes. bla _VIM-2 was detected in the P. putida isolate and a P. aeruginosa serotype O:3 isolate. The latter strain was genotypically distinct from other contemporaneous or older carbapenem-resistant P. aeruginosa Greek isolates.
Conclusion  These findings suggest that, although the prevalence of metallo-β-lactamases is low, the integron-associated bla _VIM genes can spread to P. aeruginosa serotypes that have not been previously associated with carbapenem resistance in our region, as well as to other pseudomonal species.