Β-lactamases In Clinical Isolates Research Articles

Evaluation of six commercial and in-house phenotypic tests for detection of AmpC β-lactamases: is routine detection possible?

Phenotypic characterization of the prevalent AmpC β-lactamases in clinical isolates is essential for making informed empirical decisions and critical for strengthening antimicrobial stewardship programmes. This study focused on assessing six assays, two in-house and four commercial phenotypic tests for detection of AmpC, to study the feasibility of making its detection a routine diagnostic microbiology laboratory activity. A total of 116 non-duplicate Gram-negative bacteria that were resistant to third-generation cephalosporins and amoxicillin/clavulanic acid, and resistant or susceptible to piperacillin/tazobactam and carbapenems, were screened by cefoxitin discs for AmpC. These isolates were subjected to two in-house (AmpC Tris-EDTA and disc approximation) methods and four commercial tests: D69C AmpC Detection Set; D72C ESBL, AmpC & Carbapenemase Detection Set; combination disc test: ESBL + AmpC Screen Disc Kit; and AmpC MIC Test Strip for confirmation of AmpC production. Ten whole-genome-sequenced AmpC-confirmed Gram-negative isolates were used as positive controls and one as a negative control. The prevalence of AmpC β-lactamases was 16%. Escherichia coli was a major carrier of plasmid-mediated AmpC (26.5%), followed by Klebsiella pneumoniae (23.4%). Phenotypically, 61% of AmpCs were detected by Tris-EDTA (accuracy: 73.8%), 76% by disc approximation (accuracy: 89.2%), 75% by the D69C AmpC Detection Set (accuracy: 95.4%), 74% by the D72C AmpC, ESBL & Carbapenemase Detection Set (accuracy: 95.4%), 76% by the combination disc test (accuracy: 95.4%) and 63% by AmpC MIC Test Strip (accuracy: 87.7%). The sensitivity and specificity of D69C were 97.9% and 88.2%, respectively, and 95.9% and 93.8% for the combination disc test, while for the disc approximation test and D72C they were 93.9% and 75%, and 93.9% and 100%, respectively. Screening by cefoxitin screening was less sensitive (75%) and specific (25%). Disc approximation and the combination disc test detect AmpC in Enterobacterales and also Pseudomonas aeruginosa and Acinetobacter species. We recommend the in-house disc approximation test and the commercial D69C, as well as the combination disc test, as excellent tools for detection of AmpC. The cefoxitin test overcalls AmpC and cannot be considered a good stand-alone test for AmpC detection.

Plasmid Carrying blaCTX-M-15, blaPER-1, and blaTEM-1 Genes in Citrobacter spp. From Regional Hospital in Mexico

Introduction:Citrobacter spp. is an opportunistic bacteria that have been recognized as significant pathogens in patients with underlying diseases or immunocompromised status. The aim of this study was to identify extended-spectrum β-lactamases in clinical isolates of Citrobacter spp.Methods:This cross-sectional study was conducted at Hospital Central “Dr. Ignacio Morones Prieto” in San Luis Potosi, Mexico. Nineteen isolates of Citrobacter spp. were obtained from clinical specimens between April to December 2015. Four isolates were resistant to third-generation cephalosporins. The presence of genes encoding ESBL (blaCTX-M-15, blaTEM-1, blaVEB-1, blaSHV, and blaPER-1) was analyzed by PCR. For this purpose, plasmid DNA was extracted and horizontally transferred to recipient E. coli Top 10.Results:blaCTX-M-15 and blaVEB-1 genes were detected in Citrobacter freundii and Citrobacter sedlakii, whereas blaPER-1 gene was identified in 1 isolate of Citrobacter freundii. In contrast, blaSHV gene was not detected in any isolate. One strain carried blaCTX-M-15, blaTEM-1, blaVEB-1, and blaPER-1 genes, most in a 275-kb plasmid.Conclusion:This study shows the presence of different types of ESBL in clinical isolates of Citrobacter freundii and Citrobacter sedlakii, which confer resistance to broad-spectrum β-lactams. The plasmid identified in this study harboring ESBL genes could play an important role in the dissemination of antibiotic resistance.

MOLECULAR CHARACTERIZATION OF FECAL ESCHERICHIA COLI ISOLATED FROM ZOO ANIMALS.

Accredited zoos and animal parks play an important role in animal health research and conservation, providing important insights on matters of public health including zoonotic infectious diseases and antimicrobial resistance (AMR). The emergence and spread of AMR is a complex phenomenon that jeopardizes human and animal health and also threatens the long-term survival of endangered species. The presence of β-lactamases in clinical isolates is particularly significant as they can jeopardize the efficacy of critically important antimicrobials. Although the presence of β-lactamases and extended-spectrum β-lactamases (ESBLs) producing Enterobacteriaceae in zoo animals has been reported, data are not available for northern European countries. In addition, few data are available on phylogenetic grouping of Escherichia coli isolated from zoo animals that can provide additional information on the host-bacterium relationship and on the pathogenicity of isolates. This study aimed to characterize fecal E. coli isolated from 33 healthy zoo animals from 22 species in Ireland, using conventional and molecular microbiological methods. All E. coli isolates were ampicillin resistant, but combined resistance to amoxicillin and clavulanic acid was not detected. Three E. coli isolates sampled from one Amur tiger, one Bornean orangutan, and one Southern white rhino were multidrug resistant, and blaTEM was detected in E. coli recovered from the Amur tiger and the Bornean orangutan. Other β-lactamases, including ESBLs and AmpCs and plasmid-mediated mcr-1 and mcr-2, were not detected. Overall, E. coli isolates investigated were susceptible to the majority of the antimicrobials tested, and only two animals shed E. coli carrying β-lactamase-encoding genes. The majority of isolates belonged to phylogenetic group B1. The screening of the AMR phenotype and genotype of zoo animal E. coli provides useful data that is relevant to antimicrobial stewardship in the zoo veterinary services and relevant to the bank of knowledge needed for tackling AMR.

Rapid detection of beta-lactamase production including carbapenemase by thin layer chromatography

ObjectivesTo develop a rapid and simple method that can identify the presence of β-lactamases in clinical isolates and samples, and determine their activity on different types of β-lactam antibiotics, including carbapenems, within one hour. MethodsIn this study, we describe a thin layer chromatography-based method for rapid detection of β-lactamases including carbapenemases. The method relies on the examination of changes in the migration rate of β-lactams in chromatography, due to degradation by β-lactamase enzymes. A total of 44 isolates, 29 carbapenemase-producers and 15 non-carbapenemase-producers, were screened by this method. ResultsThe method has proven to be able to distinguish β-lactamases as carbapenemase or non-carbapenemase producing strains with high sensitivity in one hour. ConclusionsThe method developed, provides information about the production of β-lactamases by bacteria and β-lactam drugs inactivated by these enzymes, including carbapenems. This new method may play an important role in guiding antimicrobial treatment, especially in critically ill patients infected bacteria producing β-lactamases.

Extended Spectrum β-lactamases in Clinical Isolates of Escherichia coli and Enterobacter cloacae Collected from Nablus District - Palestine

Extended Spectrum β-lactamases in Clinical Isolates of Escherichia coli and Enterobacter cloacae Collected from Nablus District - Palestine

High Prevalence of AmpC β-Lactamases in Clinical Isolates of Escherichia coli in Ilam, Iran

ObjectivesWidespread use of β-lactam antibiotics could cause resistance to this group of antibiotics in pathogenic bacteria through the production of the enzyme β-lactamases. The aim of this study is to determine the molecular detection of AmpC β-lactamases among clinical Escherichia coli isolated from Ilam hospitals in Ilam, Iran. MethodsOne hundred and twelve clinical isolates of E. coli were collected from hospitalized patients and were identified by biochemical tests. They were evaluated for extended spectrum beta-lactamases (ESBLs) production, and the positive strains were subjected to AmpC enzymes; for detection of AmpC cluster genes, multiplex polymerase chain reaction was applied. ResultsThe analysis showed 62.5% of isolates were ESBLs positive and that five strains revealed the AmpC cluster genes. This is the first report of FOXM cluster genes in E. coli in Iran. ConclusionBased on our results, the prevalence of AmpC β-lactamases is increasing in Iran, which caused failure in antibiotic therapy. So, the current study recommended the revision of antibiotic policy in Iranian hospitals.

Characterization of AmpC, CTX-M and MBLs types of β-lactamases in clinical isolates of Klebsiella pneumoniae and Escherichia coli producing Extended Spectrum β-lactamases in Kerman, Iran.

Background:Extended spectrum β-lactamases (ESBLs) and AmpC β-lactamases enzyme are major sources of resistance to β-lactam antibiotics especially in Enterobacteriaceae such as Escherichia coli and Klebsiella pneumoniae. Increasing frequency of the co-existence of ESBLs with AmpC-β-lactamases in bacteria is a serious threat for treating bacterial infections.Objectives:The aim of this study was to determine the presence of AmpC and CTX-M types of β-lactamases in clinical isolates of E. coli and K. pneumoniae producing ESBLs.Materials and Methods:Resistance to different antibiotics was determined using the standard disk diffusion method. ESBLs, MBLs and AmpC-β-lactamases were detected by the combination double disk test (CDDT) method and polymerase chain reaction (PCR) was used to determine blaCTX-M genes in the ESBLs and AmpC positive isolates.Results:The prevalence of ESBLs and AmpC-β-lactamase producer isolates was 181 (43.8%) and 133 (37.2%), respectively. The prevalence of blaCTX-M among isolates was 61 (14.7%).Conclusions:Outbreak of isolates co-expressing AmpC-β-lactamases and ESBLs can cause serious problems in the future, regarding the treatment of infections caused by these common enteric pathogens.

Co-production of ESBL and AmpC β-Lactamases in Clinical Isolates of A. baumannii and A. lwoffii in a Tertiary Care Hospital From Northern India.

Acinetobacter baumannii is an important cause of health care associated infections which are difficult to control and treat, because of widespread antimicrobial resistance which is possessed by this organism. The aim of the present study was to know the prevalence of ESBLs and AmpC β-lactamases in clinical isolates of Acinetobacter spp. which were cultured from various clinical specimens by using different phenotypic methods. Study was conducted over a period of one year at the Microbiology Department of a tertiary care teaching hospital. A total of 100 consecutive, non-duplicate strains of Acinetobacter species which were isolated from various clinical samples were included. All the isolates were identified by standard microbiological procedures and antimicrobial susceptibility testing was done by Kirby-Bauer disc diffusion technique. Isolates which showed reduced susceptibilities to third generation cephalosporins were tested for ESBL production by CLSI double disc synergy method and also by using sulbactam as an inhibitory agent. Isolates which showed reduced susceptibilities to cefoxitin were tested for AmpC detection by doing AmpC disc test. SPSS, version 17 was used to calculate p-value. If the p-value was <0.05, it was considered to be significant. Out of 100 isolates, 82 were Acinetobacter baumannii and 18 were Acinetobacter lwoffii. ESBL were mentioned in 4% of the Acinetobacter isolates and in 77% of the isolates by using clavulanic acid and sulbactam as inhibitory agents respectively. AmpC β-lactamase production was detected in 60% isolates of Acinetobacter spp. Co-production of both ESBL and AmpC enzymes were seen in 29% of the Acinetobacter strains. Failure in detecting β-lactamases contributes to their uncontrolled spread and therapeutic failures. Hence, these β-lactamases should be detected routinely and they should be reported to clinicians in time, so that inappropriate use of antibiotics can be stopped in time.

Molecular Characteristics of Extended-Spectrum β-Lactamases in Clinical Isolates from Escherichia coli at a Japanese Tertiary Hospital

The prevalence of ESBL has been increasing worldwide. In this study, we investigated the molecular characteristics of ESBL among clinical isolates of Escherichia coli from a Japanese tertiary hospital. A total of 71 consecutive and nonduplicate clinical isolates of ESBL-positive E. coli collected at Tohoku University Hospital between January 2008 and March 2011 were studied. The antimicrobial susceptibility profile of these strains was determined. PCR and sequencing were performed to identify genes for β-lactamase (bla TEM, bla SHV, bla OXA-1-like, and bla CTX-M) and plasmid-mediated quinolone resistance determinants (PMQR). The isolates were also analyzed by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Of the 71 strains, 68 were positive for CTX-M, 28 were positive for TEM, four were positive for OXA-1, and one was positive for SHV. Sequencing revealed that CTX-M-14 was the most prevalent (31/71), followed by CTX-M-27 (21/71) and then CTX-M-15 (9/71). Of the 28 TEM-positive strains, one was TEM-10 and the rest were TEM-1. One SHV-positive strain was SHV-12. The 21 CTX-M-27-producing isolates were divided into 14 unique PFGE types, while the 9 CTX-M-15 producers were divided into 8 types. Based on MLST, 9 CTX-M-14 procedures, 19 CTX-M-27 procedures, and 8 CTX-M-15 producers belonged to ST131. Thirty-five (94.6%) of the 37 ST131 E. coli strains showed resistance to levofloxacin, which was a higher rate than among non-ST131 strains (63.6%). Among ESBL-producing isolates, one, two, and six possessed qnrB, qnrS, qepA, and aac(6′)-Ib-cr, respectively. Of the 6 isolates with aac(6′)-Ib-cr, 4 carried the CTX-M-15 gene. Our data suggest that CTX-M-15-producing E. coli ST131 has emerged as a worldwide pandemic clone, while CTX-M-27 (a variant of CTX-M-14) is also spreading among E. coli ST131 in Japan.

Evaluation of four phenotypic methods to detect plasmid-mediated AmpC β-lactamases in clinical isolates

Four phenotypic methods (three dimensional test, AmpC test, cloxacillin synergy test and cefotetan/cefotetan-cloxacillin E-test) to detect plasmid-mediated AmpC β-lactamases (pAmpC) were compared in 125 clinical Enterobacteriaceae isolates with AmpC profile: 74 E. coli (bla (CMY-2): 70; bla (DHA-1): 4), five K. pneumoniae (bla (CMY-2): 2; bla (DHA-1): 3), six P. mirabilis (bla (CMY-2): 6) and 40 negative isolates for pAmpC β-lactamases. All evaluated methods showed a good sensitivity (>95%) but low values of specificity (<60%) in E. coli, explained by an increase of AmpC expression caused by chromosomal ampC promoter/attenuator mutations (-42, -18, -1, +58, predominantly). The cefotetan/cefotetan-cloxacillin or cloxacillin synergy test may be advocated as phenotypic screening test, and the AmpC test as confirmatory test for detection of pAmpC in isolates that lack or minimally express chromosomally encoded AmpC β-lactamases. In the case of E. coli, the phenotypic evaluated tests were not able to differentiate between chromosomal ampC overexpression or acquisition of plasmid-encoded ampC genes.

Molecular characteristics of extended-spectrum β-lactamases in clinical isolates of Enterobacteriaceae from the Philippines

β-Lactamases, including extended-spectrum β-lactamases (ESBLs) and AmpC β-lactamases, are major resistance mechanisms of Enterobacteriaceae. Emergence of plasmid-mediated quinolone resistance (PMQR) determinants in ESBL-producing isolates poses a global threat. The molecular characterisitcs of ESBL and PMQR determinants in the Philippines are not well characterized. In this study, we investigated ESBLs and AmpC β-lactamases in clinical isolates of Enterobacteriaceae from the Philippines, and analyzed the association between ESBL and PMQR genes. A total of 91 amoxicilin non-susceptible Enterobacteriaceae were collected at the Research Institute for Tropical Medicine of the Philippines from 2006 to 2008. AmpC- or ESBL-producing isolates were screened by detecting a zone diameter for cefoxitin≤14mm or cefpodoxime≤20mm, respectively. Possible ESBL-producing strains were assessed by the ESBL confirmation test of the Clinical and Laboratory Standards Institute. PCR and sequencing were performed to detect the ESBL and PMQR genes. The number of ESBL-producers and AmpC-producers confirmed phenotypically was 17 (18.7%) and 61 (67.0%), respectively. Of 17 phenotypic ESBL-producers, 14 isolates had ESBL genes, including 6 of Escherichia coli, 3 of Enterobacter cloacae, 2 of Enterobacter aerogenes, 2 of Klebsiella pneumoniae, and 1 of Klebsiella ozaenae. Among these isolates, there were 13, 4, and 12 with blaCTX-M, blaSHV, and blaTEM, respectively. Of the blaCTX-M-positive isolates, blaCTX-M-15 shows the highest prevalence, followed by blaCTX-M-3 and blaCTX-M-14. Of 14 ESBL-producers identified by PCR, 4, 6, and 7 isolates were positive for qnrB, qnrS, and aac(6′)-Ib-cr, respectively. The frequency of aac(6′)-Ib-cr positivity was significantly higher among CTX-M-15-producing isolates. Thus, we identified blaCTX-M, aac(6′)-Ib-cr, and qnr in ESBL-producing Enterobacteriaceae from the Philippines, and revealed a significant association between blaCTX-M-15 and aac(6′)-Ib-cr. Local epidemiological data are important for implementing appropriate antimicrobial therapy and effective infection control measures. Continuous monitoring of antimicrobial resistance genes in the Philippines will be required.

P24.02 Regional variation in the prevalence of extended-spectrum β-lactamases in clinical isolates of Enterobacter and Citrobacter species in Japan

P24.02 Regional variation in the prevalence of extended-spectrum β-lactamases in clinical isolates of Enterobacter and Citrobacter species in Japan

Emergence of class 1 integron-associated GES-5 and GES-5-like extended-spectrum β-lactamases in clinical isolates of Pseudomonas aeruginosa in South Africa

Several different Guiana extended-spectrum (GES) enzymes have been described occurring in Enterobacteriaceae and Pseudomonas aeruginosa worldwide. Polymerase chain reaction and gene sequencing analysis confirmed bla GES genes identified in three P. aeruginosa clinical isolates from South Africa as bla GES-5 and bla GES-5-like, respectively. Compared with GES-1, the GES-5-like protein exhibited an A21E amino acid change, a novel mutation not previously described in this family. Integron structures identified upstream from the bla GES-5 and bla GES-5-like genes were found to be identical to bla GES-2-carrying integrons described previously from the same geographical region. These findings confirm the establishment and persistence of integron-associated GES-type extended-spectrum β-lactamases (ESBLs) in the South African nosocomial environment. This study describes the first isolation of class 1 integron-associated bla GES-5 and the emergence of a novel GES-5-like ESBL in South Africa.

Use of boronic acid disk methods to detect the combined expression of plasmid-mediated AmpC β-lactamases and extended-spectrum β-lactamases in clinical isolates of Klebsiella spp., Salmonella spp., and Proteus mirabilis

A study using boronic acid (BA), an AmpC enzyme inhibitor, was designed to detect the combined expression of plasmid-mediated AmpC β-lactamases (pAmpCs) and extended-spectrum β-lactamases (ESBLs) in bacterial isolates naturally lacking chromosomal ampC genes. A total of 122 Klebsiella spp., Salmonella spp., and Proteus mirabilis isolates producing or nonproducing pAmpCs and/or ESBLs were analyzed. Detection of genes encoding ESBLs and AmpCs was confirmed by polymerase chain reaction (PCR) followed by sequencing of PCR products. A ≥5-mm increase in zone diameter for i) cefoxitin (FOX) and/or cefotetan (CTT) containing BA versus FOX and/or CTT alone was considered positive for AmpC; ii) ceftazidime (CAZ)-clavulanate (CA) and/or cefotaxime (CTX)–CA tested in combination with BA versus CAZ and/or CTX containing BA was considered positive for ESBL. The disk tests of FOX and/or CTT alone and with BA detected 98.4% of organisms producing pAmpCs. All of the 21 pAmpC and ESBL coproducers were accurately detected ESBL by the disk tests of CTX–CA and/or CAZ–CA containing BA and CTX and/or CAZ containing BA. In conclusion, The BA disk test using Clinical and Laboratory Standards Institute methodology is simple and very efficient method to detect pAmpC and ESBL in organisms naturally lacking chromosomal AmpC enzymes. In particular, the method accurately detects the isolates that harbor both AmpCs and ESBLs.

Accelerated detection of extended-spectrum β-lactamases in clinical isolates of Enterobacteriaceae

A prospective study is carried out to evaluate the performance of a protocol for the accelerated detection of extended-spectrum β-lactamases (ESBLs) in clinical isolates of Escherichia coli, Klebsiella pneumoniae and other Gram-negative bacteria. A modified double-disc test (MDDT) is incorporated in a Gram-negative template for routine susceptibility testing. The MDDT identified accurately ESBLs in all isolates subsequently confirmed as ESBLproducers by the standard Clinical Laboratory Standards Institute (CLSI) combined disc method. Of 1213 isolates tested, 98 (8%) were positive for ESBLs by MDDT and 95 (7.8%) were positive by the CLSI method. ESBLs were detected in 48 (7.8%) E. coli, 21 (8%) K. pneumoniae, 12 (5.8%) Proteus mirabilis, 13 (18.8%) Providencia stuartii and four (6.8%) Enterobacter cloacae isolates. Time required for ESBL detection by the MDDT method was one day. The protocol described provides a simple, rapid and low-cost method for early detection of ESBLs in Gram-negative bacteria.

Acceptable performance of VITEK 2 system to detect extended-spectrum β-lactamases in clinical isolates of Escherichia coli: a comparative study of phenotypic commercial methods and NCCLS guidelines

The disk approximation method, Etest (AB Biodisk, Solna, Sweden), and VITEK 2 system (bioMérieux, Marcy l'Etoile, France) were used to study 399 extended-spectrum β-lactamase (ESBL)-producing (115 strains) and non-ESBL-producing (284 strains) clinical isolates of Escherichia coli after recommended procedures. Comparative study of the phenotypic findings yielded data on the sensitivity, specificity, and positive and negative predictive values and performance of each method. The sensitivity (100% using 2 substrates), specificity (99.3–100%), and predictive values of the disk approximation, Etest, and VITEK 2 methods were similar.

Relationship between penicillin-binding protein patterns and beta-lactamases in clinical isolates of Bacteroides fragilis with different susceptibility to beta-lactam antibiotics.

This study examines the role of the penicillin-binding proteins (PBPs) of Bacteroides fragilis in the mechanism of resistance to different beta-lactam antibiotics. Six of the eight strains used were beta-lactamase-positive by the nitrocefin assay. These strains displayed reduced susceptibility to imipenem (MIC, 2-16 mg l(-1)) and some of them were resistant to the actions of ampicillin, cefuroxime, cephalexin, cefoxitin and piperacillin. When studying specific enzymic activity, the capacity to degrade cefuroxime was only detected in strains AK-4, R212 and 0423 and the capacity to degrade cephalexin was only detected in strains R212 and 2013E; no specific activity was detected on imipenem. Metallo-beta-lactamase activity was only detected in strains AK-2 and 119, despite the fact that the cfiA gene was identified in four strains (AK-2, 2013E, 119 and 7160). The cepA gene was detected in six of the eight strains studied. Three high-molecular-mass PBPs were detected in all strains; however, in some cases, PBP2Bfr and/or PBP3Bfr appeared as a faint band. PBP4Bfr and PBP5Bfr were detected in six strains. PBP6Bfr only was detected in B. fragilis strains AK-2, 0423, 119 and 7160. By analysis of the sequence of B. fragilis chromosomal DNA and comparison with genes that are known to encode PBPs in Escherichia coli, six genes that encode PBP-like proteins were detected in the former organism. The gene that encodes the PBP2 orthologue of E. coli (pbpABfr, PBP3Bfr) was sequenced in six of the eight strains and its implications for resistance were examined. Differences in the PBP3Bfr amino acid sequences of strains AK-2 and 119 and their production of beta-lactamases indicate that these differences are not involved in the mechanism of resistance to imipenem and/or cephalexin.

Detection and Typing of Extended-Spectrum β-Lactamases in Clinical Isolates of the Family Enterobacteriaceae in a Medical Center in Turkey

To determine and type the extended-spectrum beta-lactamases (ESBLs) among the family Enterobacteriaceae in a medical center, a total of 668 clinical isolates were screened. Of the 668 isolates, the 80 strains were presumptively defined as ESBL producers according to the result of disk method using ESBL marker antibiotics (aztreonam, ceftazidime, and cefoxitin). These 80 strains were retested with the double-disk synergy test (DDST), the E-test ESBL strip, a 5-microg ceftazidime disk, and agar dilution MICs of ceftazidime with and without clavulonic acid. Isoelectric focusing was performed to confirm ESBL production and type the beta-lactamases. By evaluation of the results of all tests used for ESBL detection together with isoelectric focusing, 33 (4.9%) of the 668 isolates were described as ESBL producer. The positive results of the agar dilution test, DDST, the E-test strip, and 5-microg ceftazidime disk were 32, 26, 27, and 26 of the 33 strains, respectively. ESBL positivity was 48.8% in Klebsiella species, 15.4% in Citrobacter species, 4.9% in Enterobacter species and 1.1% in Escherichia coli strains. The ESBL enzymes frequently determined were SHV-2/6-like (pI 7.6), SHV-5-like (pI 8.2), SHV-4-like (pI 7.8), and SHV-3-like (pI 7). SHV-derived enzymes were commonly observed in Klebsiella spp whereas TEM-related enzymes were seen in E. coli strains. The results of this study indicated that SHV-2/6-derived (pI 7.6) ESBL expression among the isolates of the family Enterobacteriaceae is an important problem in our medical center.

Outer-membrane proteins pattern and detection of β-lactamases in clinical isolates of imipenem-resistant Acinetobacter baumannii from Brazil

In order to compare imipenem-sensitive and -resistant Acinetobacter baumannii strains isolated from three patients, ribotyping, plasmid, β-lactamase detection and outer-membrane analysis were performed. Ribotyping and the use of a β-lactam during the period when the strains were isolated suggested that they had a common origin and that resistance occurred in vivo. Outer membrane analysis showed no difference between susceptible and resistant strains with the exception of an A2 imipenem-resistant strain that lost a protein band of 31–36 kDa. Beta-lactamases were detected using isoelectric focusing in all strains ( pI of 7.4). In addition, two β-lactamases ( pI of 5.9 and 6.7) were found in imipenem-resistant isolates. The double-disc technique demonstrated the presence of a β-lactamase capable of imipenem inactivation in resistant strains. Plasmid analysis showed that all susceptible strains had the same pattern, one resistant strain did not have any plasmid, one had the same plasmid pattern of its susceptible pair and only one had a different pattern when compared with its susceptible pair.

Biotinylated oligonucleotide probes for the detection and the characterization of TEM-type extended broad spectrum β-lactamases in enterobacteriaceae

Point mutations in the nucleotide sequence of the structural genes for the TEM-type penicillinases can broaded their substrate spectrum towards all β-lactams except cephamycins and imipenem. The presence of such variants on self-transferable plasmids accounts for the dissemination of this new type of resistance to numerous species of Enterobacteriaceae in various countries. We have synthetized biotinylated oligonucleotide probes for the detection and the discrimination of parental and mutated nucleotide sequences of TEM enzymes. Seven clinical isolates belonging to four species and harbouring TEM-1, TEM-3 or TEM-6 were studied. The results obtained indicate that detection of TEM-derived broad spectrum β-lactamases in clinical isolates of Enterobacteriaceae is possible with biotinylated oligonucleotide probes.