Relationship between adhesion to intestinal Caco-2 cells and multidrug resistance in Klebsiella pneumoniae clinical isolates.

Klebsiella pneumoniae is an opportunistic gram-negative pathogen involved in outbreaks of nosocomial infections in intensive care units. Strains are resistant to multiple antibiotics, and 15 to 30% of them are also resistant to the broad-spectrum cephalosporins by the production of R plasmid-encoded extended-spectrum beta-lactamases. Because the gastrointestinal tracts of patients have been shown to be the reservoir for nosocomial strains of K. pneumoniae, we looked for a correlation between antibiotic resistance and adhesion of K. pneumoniae strains to intestinal cells. We investigated adhesion to the human intestinal epithelial Caco-2 cell line of 61 clinical K. pneumoniae strains isolated in hospitals in Clermont-Ferrand, France. None of the strains tested expressed the previously described adhesive factors CF29K and KPF-28. Adhesive properties were found for 42.6% of the strains tested (26 strains). Just 7.7% (2 strains) of the 26 strains producing only the chromosomally encoded SHV-1 beta-lactamase adhered to the Caco-2 cell line, whereas 68.5% (24 strains) of the 35 strains producing a plasmid-encoded beta-lactamase were adherent. All the adherent strains, and even the two strains producing only the SHV-1 enzyme, harbored at least one self-transmissible R plasmid. At variance for CAZ-1/TEM-5 or CAZ-5/SHV-4 beta-lactamase-producing K. pneumoniae strains, curing and mating experiments demonstrated that the self-transmissible R plasmids encoding the TEM-1, CTX-1/TEM-3, CAZ-2/TEM-8, CAZ-6/TEM-24, or CAZ-7/TEM-16 beta-lactamase were not involved in the adhesion of K. pneumoniae strains to intestinal epithelial cells. Nevertheless, there was an association of multiple antibiotic resistance, including resistance to extended-spectrum cephalosporins, and adhesive properties in K. pneumoniae clinical isolates.     

Identification of SHV-type extended spectrum beta-lactamase genes in Pseudomonas aeruginosa by PCR-restriction fragment length polymorphism and insertion site restriction-PCR

We propose a simple and rapid method to discriminate SHV-type extended spectrum beta-lactamase (ESBL) genes in P. aeruginosa based on PCR techniques (PCR-RFLP and RSI-PCR). We studied 22 producing ESBL P. aeruginosa strains isolated from seven immunocompromised patients (19 isolates) and from environmental swabs (three isolates) at the Bone Marrow Transplantation Center of Tunis. Screening PCR with primer pairs designed to detect gene encoding TEM, SHV, OXA group I, OXA group II, OXA-18 and PER-1 ESBL was positive for bla(OXA18) and bla(SHV) genes in all isolates. Pulsed field gel electrophoresis using SpeI endonuclease defined five genotypic groups. For at least one isolate corresponding to each genotype observed, restriction of PCR products by DdeI and BsrI revealed the same restriction pattern that the bla(SHV-1) negative control; in the same way, RSI-PCR products digestion by NruI, thus excluding 35, 238 and 240 mutations characterizing reported ESBL in P. aeruginosa (SHV-2a, SHV5 et SHV12), and suggesting that studied bla(SHV) genes were not ESBL ones. Genomic DNA hybridization by southern blot with probe consisting in bla(SHV-1) gene was positive in these isolates. Sequencing the full-length open reading frame revealed nucleotide sequence of the bla(SHV-1). PCR-RFLP and RSI-PCR results were then confirmed. This approach is effective for screening P. aeruginosa for ESBL genes carriage in epidemiological studies and for detecting new variants.     

Molecular epidemiology of Klebsiella pneumoniae strains that produce SHV-4 beta-lactamase and which were isolated in 14 French hospitals.

Preliminary results suggested that the diffusion in France of the SHV-4 extended-spectrum beta-lactamase was probably due to the spread of one single epidemic strain of Klebsiella pneumoniae. In this study, we tested various phenotypic and genotypic markers to compare K. pneumoniae strains producing this enzyme isolated in 14 French hospitals between 1987 and 1989. All of the strains were of the same capsule serotype, K25. Twelve of them were of the same biotype: weak urease activity and no sucrose fermentation. Among the six plasmid profiles observed, one accounted for eight strains. Large plasmids of 170 kb encoding SHV-4 beta-lactamase were present in all strains of K. pneumoniae and could be transferred by conjugation with high frequency to Escherichia coli J53-2 or HB101 from all except one strain. Plasmid EcoRI restriction patterns suggested that these plasmids were closely related and similar to pUD18 encoding SHV-3 beta-lactamase, originally described in France and differing from SHV-4 by one amino acid substitution. Ribotyping with EcoRI and HindIII and genomic fingerprinting with XbaI by pulsed-field gel electrophoresis were concordant and suggested that 12 of the isolates recovered from the 14 hospitals were probably the same strain. Dissemination in France of the SHV-4 extended-spectrum beta-lactamase was thus essentially due to the diffusion of a single K. pneumoniae clone.     

Effects of inoculum and beta-lactamase activity in AmpC- and extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli and Klebsiella pneumoniae clinical isolates tested by using NCCLS ESBL methodology

Escherichia coli and Klebsiella pneumoniae isolates with extended-spectrum beta-lactamases (ESBLs) or AmpC cephalosporinases generally respond as predicted to NCCLS tests for ESBL production. However, inoculum size may affect MICs. The effect of inoculum level in clinical isolates expressing beta-lactamases were studied at inocula within 0.5 log unit of the standard inoculum, using broth microdilution methodology with ceftazidime, cefotaxime, cefepime, cefpodoxime, and aztreonam. Strains with TEM-1 or no beta-lactamases gave consistent MIC results with inocula of 10(5) and 10(6) CFU/ml. When the bacteria were screened for ESBL production and the lower inoculum was used, several strains with ESBLs, including CTX-M-10, TEM-3, TEM-10, TEM-12, TEM-6, SHV-18, and K1, gave false-negative results for one or more antimicrobial agents (MICs below the NCCLS screening concentration for detecting suspected ESBLs). When the higher inoculum was used, MICs of at least one antimicrobial agent increased at least fourfold in strains producing TEM-3, TEM-10, TEM-28, TEM-43, SHV-5, SHV-18, and K1. All antimicrobial agents showed an inoculum effect with at least one ESBL producer. Confirmatory clavulanate effects were seen for both inocula for all ESBL-producing strains with all antimicrobial agents tested, except for the CTX-M-10-producing E. coli with ceftazidime and the SHV-18-producing K. pneumoniae with cefotaxime. In kinetic studies, cefpodoxime and cefepime were hydrolyzed by ESBLs in a manner similar to that of cefotaxime. When total beta-lactamase activity and hydrolysis parameters were evaluated, however, no single factor was predictive of inoculum effects. These results indicate that the NCCLS screening and confirmation tests are generally predictive of ESBL production, but false-negative results can arise when a lower inoculum is used in testing.     

Coexistence of SHV-4- and TEM-24-producing Enterobacter aerogenes strains before a large outbreak of TEM-24-producing strains in a French hospital

In 1996, a monitoring program was initiated at the teaching hospital of Amiens, France, and carried out for 3 years. All extended-spectrum beta-lactamase (ESBL)-producing Enterobacter aerogenes isolates recovered from clinical specimens were collected for investigation of their epidemiological relatedness by pulsed-field gel electrophoresis and enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) and determination of the type of ESBL harbored by isoelectric focusing and DNA sequencing. Molecular typing revealed the endemic coexistence, during the first 2 years, of two clones expressing, respectively, SHV-4 and TEM-24 ESBLs, while an outbreak of the TEM-24-producing strain raged in the hospital during the third year, causing the infection or colonization of 165 patients. Furthermore, this strain was identified as the prevalent clone responsible for outbreaks in many French hospitals since 1996. This study shows that TEM-24-producing E. aerogenes is an epidemic clone that is well established in the hospital's ecology and able to spread throughout wards. The management of the outbreak at the teaching hospital of Amiens, which included the reinforcement of infection control measures, failed to obtain complete eradication of the clone, which has become an endemic pathogen.     

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.     

Comparative activities of 15 beta-lactam antibiotics against 590 strains of Klebsiella pneumoniae according to the production of beta-lactamase

In October 1988, all non repetitive strains of K. pneumoniae isolated in 17 hospitals have been studied. Among these 590 strains: 451 (76%) only produce the specific beta-lactamase of the species SHV-1 (pI 7,7) or SHV-1 type (pI 7,1), while 74 (12.5%) produce a TEM-1 or TEM-2 type beta-lactamase, and 65 (11%) an extended broad spectrum beta-lactamase: 22 CTX-1, 5 SHV-2, 4 SHV-3, 26 SHV-4, 8 SHV-5. The minimum inhibitory concentrations of the following antibiotics were performed by a liquid micro dilution technic: amoxicillin (AMX), amoxicillin + clavulanic acid (CL), 5 mg/l, ticarcillin (TIC), piperacillin (PIP), cefazolin (CEZ), cefamandole (CFM), cefoperazone (CFP), cefotaxime (CTX), cefotaxime + clavulanic acid 5 mg/l, cefotaxime + sulbactam (SUL) 5 mg/l, cefpirome (CPI), ceftazidime (CAZ), azthreonam (AZT), latamoxef (MOX), cefoxitin (FOX), cefotetan (CTT), temocillin (TMO), imipenem (IMI). The "wild" strains with SHV-1 beta-lactamase are resistant to AMX and have a decreased susceptibility to TIC and PIP, but are susceptible to other antibiotics. The TEM producing strains are more resistant to PIP and TIC, have a decreased susceptibility to CEZ and CFM but are susceptible to other antibiotics. For the extended broad-spectrum beta-lactamase producing strains, the MIC of penicillin antibiotics (AMX, TIC, PIP) are very high and also the MIC of CEZ, CFM and CFP. The MIC of CTX are higher for CTX-1 or SHV-4 producing strains, than for SHV-2, SHV-3, or SHV-5 producing strains. The combination with CL is more efficacious than the one with SUL to reduce the MIC of CTX in susceptibility area.(ABSTRACT TRUNCATED AT 250 WORDS)     

Extended-spectrum beta-lactamase-producing Enterobacteriaceae in Bulgarian hospitals

The aim of the study was to describe the emergence, the spread, and the prevalence of extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae in Bulgaria. Over eight years (1996-2003), 442 ESBL-screen-positive isolates were collected in nine medical institutions in four Bulgarian towns. Class A ESBLs of the SHV, TEM, and CTX-M groups were identified in seven species. SHV-type enzymes persisted during the whole study period, TEM-ESBLs appeared first in 1999, and CTX-M-types appeared first in 2001. The rate of CTX-M enzyme producers increased rapidly between 2001 and 2003, while the rate of SHV producers decreased. Six different ESBL-types were identified, namely, SHV-2, -5, and -12, CTX-M-3 and -15, and a new TEM-3-like variant (TEM-139). The most widespread enzymes were SHV-12, CTX-M-15, and CTX-M-3 found in seven centers. TEM-139 was identified mainly in one center. A trend for strains harboring more than one ESBL gene, for example, CTX-M + SHV, was observed since 2002. Plasmid fingerprinting and random amplified polymorphic DNA analysis typing revealed wide dissemination of identical plasmids among different bacterial species and hospitals, as well as clonal spread of ESBL producers. Our data contribute to clarify the dynamics in the prevalence of ESBLs in Bulgaria and demonstrate the importance of molecular procedures for their analysis.     

Molecular epidemiology of extended-spectrum beta-lactamases produced by clinical isolates in a university hospital in Greece: detection of SHV-5 in Pseudomonas aeruginosa and prevalence of SHV-12

To assess the nature and diversity of various types of SHV and TEM derivatives in our hospital a survey was conducted. Sixty-seven extended-spectrum beta-lactamases (ESBL)-producing nosocomial pathogens, isolated over a 12-month period, were analyzed by means of PCR and direct sequencing. SHV-5 was the predominant ESBL found in our region (38 strains). Other less frequent variants included SHV-2 and SHV-12 with two and three isolates, respectively. For the first time, an outbreak of 11 Pseudomonas aeruginosa producing SHV-5 was encountered. All blaTEM-positive strains carried the non-ESBL TEM-1. The incidence of non-SHV non-TEM ESBLs was remarkably high as almost one out of three isolates harbored such an ESBL. The epidemiological and clinical impact of these findings must be carefully investigated and interpreted.     

Clinical and molecular analysis of extended-spectrum {beta}-lactamase-producing enterobacteria in the community setting

During a previous survey, five extended-spectrum beta-lactamase (ESBL)-producing enterobacteria (ESBLE) (two Enterobacter aerogenes isolates expressing TEM-24 b, two Escherichia coli isolates expressing TEM-21 or TEM-24 b, and one Klebsiella pneumoniae isolate expressing SHV-4/TEM-15) responsible for urinary tract infections (UTIs) were found among 1,584 strains collected from community patients. The aim of the present study was to elucidate the route of emergence of these typically nosocomial organisms in the community. Thus, the files of the five patients were analyzed over at least a decade, and potentially related ESBLE from hospitals or clinics were examined. Their enzymes were characterized at a molecular level, and the strains were typed by amplified-primed PCR, enterobacterial repetitive intergenic consensus PCR, and restriction plasmid profile. All patients (C1 to C5) had risk factors for ESBLE acquisition, including past history of hospitalization (2.5 to 23 months before). Four (C1 and C3 to C5) had previously received antibiotics (concomitantly to 35 months earlier), two (C1 and C3) had indwelling urinary catheters and recurrent UTIs, and three (C2, C3, and C5) formerly experienced ESBLE-induced UTIs (2 to 11 months before). The same ESBLE and/or an identical or similar ESBL-encoding plasmid was identified in the hospital ward (C1 to C4) or in a clinic (C5) where the patients had previously resided. Patients C1 and C4, infected with different ESBLE carrying a closely related plasmid, were hospitalized in the same unit. Persistence of ESBLE over at least a 5-year period was demonstrated for patient C3. Thus, community-acquired UTIs in these patients likely resulted from nosocomially acquired ESBLE or an ESBL-encoding plasmid followed by a prolonged digestive carriage.     

SHV-5 extended-spectrum beta-lactamases in clinical isolates of Escherichia coli in Malaysia

Escherichia coli isolates resistant to ceftazidime isolated in the University Malaya Medical Center (UMMC) Kuala Lumpur, Malaysia, between the years 1998 and 2000 were studied for extended-spectrum beta-lactamase (ESBL) production. All strains were analysed phenotypically and genotypically and found to be ESBL-producing organisms harbouring SHV-5 beta-lactamase. This was confirmed by PCR-SSCP and nucleotide sequencing of the blaSHV amplified gene. As there was no evidence of ESBL activity in E. coli prior to this, coupled with the fact that there was a predominance of SHV-5 beta-lactamases in Klebsiella pneumoniae isolates in UMMC, we postulate that the E. coli obtained the SHV-5 beta-lactamase genes by plasmid transfer from the ESBL-producing K. pneumoniae.     

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.     

Investigation of the new QNR-based mechanism of quinolone resistance among enterobacterial strains isolated in Henri-Mondor hospital 2002-2005

AIM OF THE STUDY: To assess the prevalence of the novel plasmid-mediated resistance to quinolones in enterobacteria isolated in our hospital. MATERIALS AND METHODS: We have screened 737 enterobacterial strains isolated in Henri-Mondor hospital between 2002 and 2005 for the presence of the qnr gene by PCR using specific primers. Among them, 282 had a phenotype in concordance with extended spectrum betalactamase (ESBL). Qnr-positive strains were phenotypically and genetically characterized, and epidemiological link between the cases was investigated. RESULTS: Five qnr+ strains were described. The global prevalence was 0.7% but 5/282 among ESBL producing strains and 0/437 among quinolone-resistant enterobacteria non producing ESBL. The sequences of the PCR products were identical to qnrA in the environment of the integron In36. All the strains harboured also the ESBL SHV-12 gene. Transfer of qnr by conjugation raised quinolone MICs from 2 to 24 times. However clinical strains harboured a higher level of quinolone resistance and harboured also DNA gyrase and topoisomerase IV mutations. Two strains were epidemiologically related by molecular typing and contact tracing revealed that the patients have been previously hospitalized in the same tertiary care center. CONCLUSION: We described the first investigation of qnr-positive strains in one hospital in France over 4 years. Although the qnr gene prevalence is low, nosocomial transmission is already shown and the transfer of the qnr containing integron among ESBL producing strains may predict future epidemic. Surveillance will be necessary to confirm this low prevalence rate of qnr in France.     

Evaluation of plasmid-encoded beta-lactamase resistance inEscherichia coli blood culture isolates

The frequency of beta-lactam resistance was determined among 313 strains ofEscherichia coli, 119 ofEnterobacter/Klebsiella/Proteus spp., and 48 ofPseudomonas spp. isolated from blood cultures (at Turku University Central Hospital and Turku City Hospital) in 1983–1987. During this period the MIC50 of ampicillin forEscherichia coli increased from 8 to 32 µg/ml, the MIC90 of piperacillin from 16 to > 32 µg/ml and the MIC90 of cefuroxime from 4–8 to 16 µg/ml. Among 172 ampicillin-resistant isolates beta-lactamase-mediated resistance was characterized by DNA hybridization with TEM-1, SHV-1, OXA-1, OXA-2, PSE-1, PSE-2 and PSE-4 beta-lactamase probes and by isoelectric focusing. Beta-lactamase types found were TEM-1, TEM-2, SHV-1 and OXA-1. Isoelectric focusing did not show any other plasmid-mediated beta-lactamase varieties. Piperacillin-resistant strains showed mostly TEM-1 activity, but also produced OXA-1 and chromosomal beta-lactamase. Interestingly, a decrease in cefuroxime susceptibility inEscherichia coli occurred in a few OXA-1 producing strains as well as in strains that produced only chromosomal beta-lactamase. TwoEscherichia coli strains that overproduced chromosomal beta-lactamase had increased ceftazidime MIC values (8–16 µg/ml).     

Comparative antibacterial activity of ceftibuten (SH 39 720) against 150 K. pneumoniae strains producing different beta-lactamases

Comparative antibacterial activity of ceftibuten (SH 39 720) on 150 K. pneumoniae strains producing different beta-lactamases. MICs of ceftibuten, cefotaxime + clavulanic acid (10 mg/l), ceftazidime and azthreonam were determined for 150 clinical isolates of K. pneumoniae: 15 "wild" type producing only the SHV-1 type beta-lactamase, 15 TEM-1 and 120 producing new "extended spectrum" beta-lactamases (48 CTX-1, 9 SHV-2, 4 SHV-3, 35 SHV-4, 24 SHV-5). Against SHV-1 and TEM-1 strains the bacteriostatic activity of ceftibuten was close to that of the other beta-lactams tested. This activity was preserved against strains producing CTX-1, SHV-2, SHV-3 beta-lactamases and MICs were comparable to those of cefotaxime + clavulanic acid. The MICs of ceftibuten against strains producing SHV-4 and SHV-5 beta-lactamases were higher (1 to 8 mg/l) but this level of antibacterial activity was superior to that of the other beta-lactam antibiotics. The bactericidal activity of ceftibuten was evaluated by kill kinetic studies: this activity was preserved against the CTX-A and SHV-2 strains--with concentrations equal to 2 or 4 x CMI a 4 log10 reduction of the bacterial inoculum was obtained at 24 h. A such reduction was not obtained with cefotaxime. This preserved antibacterial activity of ceftibuten against K. pneumoniae producing some new "extended spectrum" beta-lactamases need further studies to state the structure-activity relation-ships of this antibiotic.     

Survey of Extended-Spectrum β-Lactamases in Clinical Isolates of Escherichia coli and Klebsiella pneumoniae: Prevalence of TEM-52 in Korea

Two hundred ninety isolates of Escherichia coli were investigated for the production of extended-spectrum beta-lactamases (ESBLs). Fourteen (4.8%) of the 290 strains were found to produce ESBLs. Each of the 14 strains produced one or two ESBLs, as follows: 10 strains produced TEM-52, 1 strain produced SHV-2a, 1 strain produced SHV-12, 1 strain produced a CMY-1-like enzyme, and 1 strain expressed SHV-2a and a CMY-1-like enzyme. Another two strains for which the MICs of ceftazidime and cefoxitin were high, were probable AmpC enzyme hyperproducers. Because of the high prevalence of TEM-52 in E. coli isolates, we further investigated the TEM-type ESBLs produced by Klebsiella pneumoniae in order to observe the distribution of TEM-52 enzymes among Enterobacteriaceae in Korea. All TEM enzymes produced by 12 strains of K. pneumoniae were identified as TEM-52. To evaluate the genetic relatedness among the organisms, ribotyping of TEM-52-producing E. coli and K. pneumoniae was performed. The ribotyping profiles of the organisms showed similar but clearly different patterns. In conclusion, TEM-52 is the most prevalent TEM-type ESBL in Korea.     

Frequency and distribution of beta-lactamases in 1792 strains of Klebsiella pneumoniae in France between 1985 and 1988

In october 1985, 1987 and 1988, all the clinical isolates of K. pneumoniae (respectively 530, 654, 590 strains) were collected in 20 hospitals. The beta-lactamases were identified by analytical isoelectrofocusing and by substrate and inhibition profiles. 76 to 81% of the strains produced only one beta-lactamase: SHV-1 type, pI 7.7 (61 to 65%) or PI 7.1 (14%). The TEM-1 betalactamase (pI 5.4) was produced in 1985 by 21% of the strains, 9% in 1987, and 11% in 1988: TEM-2, pI 5.6 by 2% in 1985-87-88. The extended broad spectrum beta-lactamases, able to hydrolyse amino-thiazol-oximino-beta-lactam antibiotics, TEM or SHV type enzymes (SHV-2, pI 7.7, SHV-3, pI 7.1; SHV-4/CAZ-5, pI 7.8; SHV-5/CAZ-4 pI 8.2; CTX-1/TEM-3, pI 6.3) were also detected: 0.75% of the strain (3 strains) in 1985, 8.4% (55 strains) in 1987, 11% (65 strains) in 1988. These extended broad spectrum beta-lactamases were found in 2 hospitals in 1985, 10 in 1987 and 9 in 1988.     

Molecular characterization of extended-spectrum beta-lactamases produced by nosocomial isolates of Enterobacteriaceae from an Italian nationwide survey

Extended-spectrum beta-lactamases (ESBLs) are widespread in hospital settings worldwide. The present investigation was undertaken to assess the distribution and prevalence of ESBLs belonging to the TEM and SHV families in 448 ESBL-producing clinical isolates of Enterobacteriaceae collected from 10 different Italian hospitals. The natures of TEM and SHV determinants were identified by direct sequencing of PCR-amplified genes. TEM-52 and SHV-12 were the most common variants, and they were found in most hospitals and in several different species. Other less frequent variants included TEM-5, TEM-12, TEM-15, TEM-19, TEM-20, TEM-24, TEM-26, TEM-43, TEM-60, TEM-72, TEM-87, SHV-2a, SHV-5, and SHV-11. Proteus mirabilis was the most common producer of TEM-type ESBLs, while Klebsiella pneumoniae was the most common producer of SHV-type ESBLs. The distribution of TEM- and SHV-type ESBL variants in Enterobacteriaceae from Italian hospitals exhibited notable differences from those from other geographical settings.