SHV ESBL Research Articles

Genetic Characterization of Extended-Spectrum Beta-Lactamase (ESBL) and Metallo-Beta-Lactamase (MBL) Producing Klebsiella pneumoniae from Diabetic Foot Ulcer (DFU)

ABSTRACT Background: Antibiotic resistance in common pathogenic bacteria is linked with the genetic makeup. The genetic basis of antibiotic resistance may vary in different species or pathophysiological conditions. Objectives: We studied the antibiotic resistance in Klebsiella pneumonia isolates from DFU in the western Indian population. We also studied the presence of ESBL and MBL mechanisms of antibiotic resistance along with the prevalence of the genes involved in ESBL (TEM ESBL , SHV ESBL , and CTX-M ESBL ) and MBL (NDM-1 bla , KPC bla , OXA-48 bla , and VIM bla ) production. Results: A total of 161 K. pneumoniae isolates were analyzed; among which 50.93% were positive for ESBL and 45.96% were positive for MBL production. Most of the isolates were resistant to antibiotics used in the present study and partially resistant to Imipenem and Amikacin. There was no relation between the antibiotic resistance of the isolates and the production of ESBL or MBL mechanism of antibiotic resistance. Further, TEM ESBL was the most prevalent gene in K. pneumoniae isolates followed by CTX-M ESBL , NDM-1 bla , SHV ESBL , and KPC bla . VIM bla was the least prevalent gene found in K. pneumoniae isolates. There was no difference in the prevalence of the genes with respect to the presence or absence of ESBL and MBL mechanism of resistance. Further, there was no relation between the prevalence of the genes and antibiotic resistance in K. pneumoniae isolates. Conclusion: These results along with the literature review suggest that the prevalence of the genes involved in antibiotic resistance mechanisms are widespread in India and their distribution varies in different studies.

The role of the plasmid-mediated fluoroquinolone resistance genes as resistance mechanisms in pediatric infections due to Enterobacterales.

Fluoroquinolones (FQs) are not commonly prescribed in children, yet the increasing incidence of multidrug-resistant (MDR) Enterobacterales (Ent) infections in this population often reveals FQ resistance. We sought to define the role of FQ resistance in the epidemiology of MDR Ent in children, with an overall goal to devise treatment and prevention strategies. A case-control study of children (0-18 years) at three Chicago hospitals was performed. Cases had infections by FQ-susceptible, β-lactamase-producing (bla) Ent harboring a non- or low-level expression of PMFQR genes (PMFQS Ent). Controls had FQR infections due to bla Ent with expressed PMFQR genes (PMFQR Ent). We sought bla genes by PCR or DNA (BD Max Check-Points assay®) and PMFQR genes by PCR. We performed rep-PCR, MLST, and E. coli phylogenetic grouping. Whole genome sequencing was additionally performed on PMFQS Ent positive isolates. Demographics, comorbidities, and device, antibiotic, and healthcare exposures were evaluated. Predictors of infection were assessed. Of 170 β-lactamase-producing Ent isolates, 85 (50%) were FQS; 23 (27%) had PMFQR genes (PMFQS cases). Eighty-five (50%) were FQR; 53 (62%) had PMFQR genes (PMFQR controls). The median age for children with PMFQS Ent and PMFQR Ent was 4.3 and 6.2 years, respectively (p = NS). Of 23 PMFQS Ent, 56% were Klebsiella spp., and of 53 PMFQR Ent, 76% were E. coli. The most common bla and PMFQR genes detected in PMFQS Ent were bla SHV ESBL (44%) and oqxAB (57%), and the corresponding genes detected in PMFQR Ent were bla CTX-M-1-group ESBL (79%) and aac(6')-Ib-cr (83%). Whole genome sequencing of PMFQS Ent revealed the additional presence of mcr-9, a transferable polymyxin resistance gene, in 47% of isolates, along with multiple plasmids and mobile genetic elements propagating drug resistance. Multivariable regression analysis showed that children with PMFQS Ent infections were more likely to have hospital onset infection (OR 5.7, 95% CI 1.6-22) and isolates containing multiple bla genes (OR 3.8, 95% CI 1.1-14.5). The presence of invasive devices mediated the effects of healthcare setting in the final model. Differences in demographics, comorbidities, or antibiotic use were not found. Paradoxically, PMFQS Ent infections were often hospital onset and PMFQR Ent infections were community onset. PMFQS Ent commonly co-harbored multiple bla and PMFQR genes, and additional silent, yet transferrable antibiotic resistance genes such as mcr-9, affecting therapeutic options and suggesting the need to address infection prevention strategies to control spread. Control of PMFQS Ent infections will require validating community and healthcare-based sources and risk factors associated with acquisition.

Antimicrobial Resistance Profiling and Molecular Epidemiological Analysis of Extended Spectrum β-Lactamases Produced by Extraintestinal Invasive Escherichia coli Isolates From Ethiopia: The Presence of International High-Risk Clones ST131 and ST410 Revealed.

The treatment of invasive Escherichia coli infections is a challenge because of the emergence and rapid spread of multidrug resistant strains. Particular problems are those strains that produce extended spectrum β-lactamases (ESBL’s). Although the global characterization of these enzymes is advanced, knowledge of their molecular basis among clinical E. coli isolates in Ethiopia is extremely limited. This study intends to address this knowledge gap. The study combines antimicrobial resistance profiling and molecular epidemiology of ESBL genes among 204 E. coli clinical isolates collected from patient urine, blood, and pus at four geographically distinct health facilities in Ethiopia. All isolates exhibited multidrug resistance, with extensive resistance to ampicillin and first to fourth line generation cephalosporins and sulfamethoxazole-trimethoprim and ciprofloxacin. Extended spectrum β-lactamase genes were detected in 189 strains, and all but one were positive for CTX-Ms β-lactamases. Genes encoding for the group-1 CTX-Ms enzymes were most prolific, and CTX-M-15 was the most common ESBL identified. Group-9 CTX-Ms including CTX-M-14 and CTX-27 were detected only in 12 isolates and SHV ESBL types were identified in just 8 isolates. Bacterial typing revealed a high amount of strains associated with the B2 phylogenetic group. Crucially, the international high risk clones ST131 and ST410 were among the sequence types identified. This first time study revealed a high prevalence of CTX-M type ESBL’s circulating among E. coli clinical isolates in Ethiopia. Critically, they are associated with multidrug resistance phenotypes and high-risk clones first characterized in other parts of the world.

2558. Predicting β-Lactam Resistance Using Whole Genome Sequencing (WGS) in Klebsiella pneumoniae: The Challenge of β-Lactam Inhibitors

BackgroundAntimicrobial susceptibility testing (AST) is the major driver in designing effective therapy. As multiple resistance determinants can demonstrate the same phenotype (e.g., inhibitor resistant [IR], extended spectrum [ES], and carbapenem hydrolyzing [CH] β-lactamases), critical information provided from AST for therapy, stewardship, and infection control is currently lacking. WGS provides more comprehensive genetic data, explaining phenotype, and provides insight into clonality. Efforts are in development that apply novel statistical methods (e.g., PRIMERS I-IV) and machine learning (Sci Reports, 2108, 8, 421) to interpret results accurately and anticipate AST. Using a collection of clinical strains that spanned a 3.5-year period, we tested how well the detection of problematic IR, ES, and CH bla resistance genes predicted phenotype.MethodsFourty-one isolates were chosen for AST from a collection of 1,777 WGS K. pneumoniae. Isolates chosen possessed the following β-lactamases: (9 isolates) NDM; (3) NDM and OXA-48; (5) KPC-8 or KPC-14; (24) with a very complex β-lactamase background (all possessed an inhibitor resistant TEM (IRT), SHV ESBL, +/− CTX-M, and/or +/− KPC). AST was performed using CLSI methods for piperacillin/tazobactam (PIP/TAZO), ceftazidime (CAZ), aztreonam (ATM), ceftazidime/avibactam (CAZ/AVI), CAZ/AVI/ATM, and ceftolozane/tazobactam (TOL/TAZO) by disk diffusion assay.ResultsPresented below. ConclusionIn all cases, blaNDM-1 and blaNDM-1/OXA-48 containing isolates were resistant to CAZ/AVI; the addition of ATM fully restored susceptibility to CAZ/AVI. Surprisingly, clinical K. pneumoniae isolates bearing KPC-8 (V240G) and KPC-14 did not test fully resistant to CAZ/AVI, suggesting a more complex mechanism than the D179Y variant of KPC-3. Lastly, despite the complexity of the β-lactamase background, CAZ/AVI retained potency. Interestingly, TOL/TAZO maintained efficacy in these same complex backgrounds in the absence of NDM, KPC, and SHV-12. As previously shown in PRIMERS I-II, PIP/TAZO resistance was not observed in the majority of isolates as was predicted by the genotype. WGS in K. pneumoniae to predict AST results and potentially guide clinical decisions is improved for novel combinations like CAZ/AVI.Disclosures All authors: No reported disclosures.

Ceftazidime-avibactam activity tested against Enterobacteriaceae isolates from U.S. hospitals (2011 to 2013) and characterization of β-lactamase-producing strains.

Ceftazidime-avibactam (MIC50/90, 0.12/0.25 μg/ml) inhibited 99.9% (20,698/20,709) of Enterobacteriaceae isolates at ≤8 μg/ml. This compound was active against resistant subsets, including ceftazidime-nonsusceptible Enterobacter cloacae (MIC50/90, 0.25/0.5 μg/ml) and extended-spectrum β-lactamase (ESBL) phenotype isolates. An ESBL phenotype was noted among 12.4% (1,696/13,692 isolates from targeted species) of the isolates, including 776 Escherichia coli (12.0% for this species; MIC50/90, 0.12/0.25 μg/ml), 721 Klebsiella pneumoniae (16.3%; MIC50/90, 0.12/0.25 μg/ml), 119 Klebsiella oxytoca (10.3%; MIC50/90, 0.06/0.25 μg/ml), and 80 Proteus mirabilis (4.9%; MIC50/90, 0.06/0.12 μg/ml) isolates. The most common enzymes detected among ESBL phenotype isolates from 2013 (n = 743) screened using a microarray-based assay were CTX-M-15-like (n = 307), KPC (n = 120), SHV ESBLs (n = 118), and CTX-M-14-like (n = 110). KPC producers were highly resistant to comparators, and ceftazidime-avibactam (MIC50/90, 0.5/2 μg/ml) and tigecycline (MIC50/90, 0.5/1 μg/ml; 98.3% susceptible) were the most active agents against these strains. Meropenem (MIC50/90, ≤0.06/≤0.06 μg/ml) and ceftazidime-avibactam (MIC50/90, 0.12/0.25 μg/ml) were active against CTX-M-producing isolates. Other enzymes were also observed, and ceftazidime-avibactam displayed good activity against the isolates producing less common enzymes. Among 11 isolates displaying ceftazidime-avibactam MIC values of >8 μg/ml, three were K. pneumoniae strains producing metallo-β-lactamases (all ceftazidime-avibactam MICs, >32 μg/ml), with two NDM-1 producers and one K. pneumoniae strain carrying the bla(KPC-2) and bla(VIM-4) genes. Therapeutic options for isolates producing β-lactamases may be limited, and ceftazidime-avibactam, which displayed good activity against strains, including those producing KPC enzymes, merits further study in infections where such organisms occur.

Application of Molecular Diagnostics in Primary Detection of ESBL Directly from Clinical Specimens.

The infections caused by extended-spectrum β-lactamase (ESBL)-producing organisms are associated with increased mortality. The real-time polymerase chain reaction (PCR) method, which enables detection of ESBLs directly from patients' clinical material, was developed. This study focused on blaCTX-M and blaSHV determination in endotracheal aspirates. Each sample was identified with standard microbiological procedures and simultaneously analyzed for the presence of nucleic acids, which encode CTX-M and SHV ESBL enzymes using real-time PCR. A total of 341 samples were investigated. In the set, 27 ESBL-positive samples were identified by phenotypic methods, while 60 positive samples were identified by the PCR method. Of the 60 PCR-positive samples, 58 were positive for the blaCTX-M. In two samples, the ESBL blaSHV-ESBL gene was detected. One phenotypically positive sample was PCR negative. The real-time PCR assay does not require a cultivation step and therefore enables detection of ESBL in 6 hours. The rapid method is necessary for early and adequate antimicrobial treatment.

CTX-M-producing Escherichia coli and Klebsiella pneumoniae isolated from community-acquired urinary tract infections in Valledupar, Colombia

ObjectiveDescribe the presence of CTX-M-1 phylogenetic subgroup extended-spectrum β-lactamases (ESBL), associated with TEM and SHV genes, and the gene encoding cephalosporinase, CMY-2 in Escherichia coli and Klebsiella pneumoniae isolates from community-acquired urinary tract infections. Methods102 E. coli and 21K. pneumoniae were collected from patients with culture-proven urinary tract infection (UTI), during February and March, 2011. Antimicrobial susceptibility test was performed by disk diffusion according to the standards of the Clinical Laboratory Standard Institute. Screening for cephalosporins-resistant E. coli and K. pneumoniae was performed by PCR assay for blaTEM, blaSHV, blaCTX-M-1,-2,-8,-9, blaPER-2 and blaCMY-2 genes. Statistical analysis was performed by chi-squared test and multivariate logistic regression analysis. ResultsESBL production was detected in 12 (11.7%) E. coli and four (19%) K. pneumoniae isolates. TEM ESBLs were detected in seven E. coli and three K. pneumoniae isolates. SHV ESBLs were found in four K. pneumoniae isolates. CTX-M-1 phylogenetic subgroup was positive in seven E. coli and three K. pneumoniae isolates. CMY-2 β-lactamase gene was detected in nine E. coli and one K. pneumoniae isolates. A significant association of ESBL expression in E. coli was observed with resistance to tobramycin (p≤0.001), tetracycline (p=0.043), and ciprofloxacin (p≤0.001). In K. pneumoniae isolates, significant association was found with resistance to tobramycin and ciprofloxacin (p=0.006), and trimethoprim-sulfamethoxazole (p=0.043). Multivariate analyses did not show association between ESBL production in E. coli and K. pneumoniae, and resistance to non-β-lactams drugs. ConclusionsCTX-M ESBL in uropathogens isolated from the community is cause for concern due to the enormous potential for multidrug resistance from strains that produce these enzymes, which could lead to failure of empirically-administered therapies and development of complicated UTIs.

Susceptibility of Klebsiella pneumoniae Isolates from Intra-Abdominal Infections and Molecular Characterization of Ertapenem-Resistant Isolates

A total of 2,841 clinical isolates of Klebsiella pneumoniae from intra-abdominal infections worldwide were collected in the Study for Monitoring Antimicrobial Resistance Trends (SMART) during 2008 and 2009. Overall, 22.4% of isolates had extended-spectrum β-lactamases (ESBLs). The most active antibiotics among the 11 tested were imipenem, amikacin, and ertapenem, though even these, like all other comparators, were less consistently active against ESBL-positive isolates than against ESBL-negative isolates. Globally, 6.5% of isolates were ertapenem resistant based on the June 2010 clinical breakpoints published by the Clinical and Laboratory Standards Institute, with MICs of ≥1 μg/ml. Molecular characterization of 43 isolates with ertapenem MICs of ≥4 μg/ml showed that they variously produced CTX-M or SHV ESBLs combined with altered impermeability and/or had KPC (n = 28), OXA-48 (n = 3), or VIM (n = 1) carbapenemases. Further monitoring of ertapenem susceptibility and molecular characterization of ertapenem-resistant isolates are needed.

Ligand-Dependent Disorder of the Ω Loop Observed in Extended-Spectrum SHV-Type β-Lactamase

Among Gram-negative bacteria, resistance to β-lactams is mediated primarily by β-lactamases (EC 3.2.6.5), periplasmic enzymes that inactivate β-lactam antibiotics. Substitutions at critical amino acid positions in the class A β-lactamase families result in enzymes that can hydrolyze extended-spectrum cephalosporins, thus demonstrating an "extended-spectrum" β-lactamase (ESBL) phenotype. Using SHV ESBLs with substitutions in the Ω loop (R164H and R164S) as target enzymes to understand this enhanced biochemical capability and to serve as a basis for novel β-lactamase inhibitor development, we determined the spectra of activity and crystal structures of these variants. We also studied the inactivation of the R164H and R164S mutants with tazobactam and SA2-13, a unique β-lactamase inhibitor that undergoes a distinctive reaction chemistry in the active site. We noted that the reduced Ki values for the R164H and R164S mutants with SA2-13 are comparable to those with tazobactam (submicromolar). The apo enzyme crystal structures of the R164H and R164S SHV variants revealed an ordered Ω loop architecture that became disordered when SA2-13 was bound. Important structural alterations that result from the binding of SA2-13 explain the enhanced susceptibility of these ESBL enzymes to this inhibitor and highlight ligand-dependent Ω loop flexibility as a mechanism for accommodating and hydrolyzing β-lactam substrates.

Rapid Identification of Gram-Negative Bacteria with and without CTX-M Extended-Spectrum β-Lactamase from Positive Blood Culture Bottles by PCR Followed by Microchip Gel Electrophoresis

We evaluated the usefulness of PCR analysis of the 16S-23S rRNA gene internal transcribed spacer (ITS) and the CTX-M extended-spectrum β-lactamase (ESBL) followed by microchip gel electrophoresis (MGE) for direct identification and CTX-M detection of Gram-negative bacteria (GNB) from positive blood culture bottles. Of 251 GNB isolated from blood cultures containing a single bacterium, 225 (90%) were correctly identified at the species level directly from positive blood culture bottles by comparing the ITS-PCR patterns of the sample strain with those of the control strains. There were no cases of incorrect identification. Limitations encountered included the inability to detect mixed cultures (four bottles) as well as some species (Enterobacter species and Klebsiella oxytoca) demonstrating identical ITS-PCR patterns. A total of 109 ESBL-producing isolates from various clinical materials obtained between January 2005 and December 2008 were examined for bla(CTX-M), bla(SHV), and bla(TEM) genes by PCR and sequences of PCR products. CTX-M ESBL was detected in 105 isolates, and SHV ESBL was detected in two isolates. The remaining two isolates (K. oxytoca) were shown to harbor bla(OXY.) Twenty (19%) of 104 Escherichia coli isolates from blood cultures were suspected to produce ESBL by the combination disk method, and these isolates were shown to harbor CTX-M ESBL by PCR-MGE. The results were obtained within 1.5 h at a calculated cost of $6.50 per specimen. In conclusion, simultaneous detection of ITS length polymorphisms and bla(CTX)-(M) by single PCR followed by MGE is useful for rapid, cost-effective, and reliable species-level identification of CTX-M ESBL-producing GNB responsible for bloodstream infections.

Real-time polymerase chain reaction for rapid detection of genes encoding SHV extended-spectrum β-lactamases

Purpose: This study aimed to develop an improved method for the detection of bacterial SHV-type extended-spectrum β-lactamases (ESBLs). Materials and Methods: Our method was based on real-time polymerase chain reaction (PCR) in which the amplification of the product was monitored with a fluorescent probe. This method enabled the detection of blaSHV genes with high degrees of sensitivity and specificity. Results: Based on ESBL phenotyping methods and bla gene DNA sequencing, we identified 240 bla genes from 662 Enterobacteriaceae isolated from clinical culture specimens. Of these 240 isolates, 26 had the blaSHV-28 genotype and three had the blaSHV-1 genotype. With our new real-time PCR assay, we detected 29 out of 29 blaSHV genes in ESBL-producing isolates. Conclusion: This method represents a powerful tool for epidemiological studies of SHV ESBLs. Furthermore, it has potential for use in diagnostic microbiology.

Identification of Clonal Clusters of Klebsiella pneumoniae Isolates from Chennai by Extended Spectrum Beta Lactamase Genotyping and Antibiotic Resistance Phenotyping Analysis

Problem statement: An increased resistance to antibiotics has been reported in Klebsiella pneumoniae, an opportunistic gram negative pathogen belonging to Entrobacteraceae due to the evolution and spread of plasmid encoded extended spectrum beta lactamases and other genes conferring cross-resistance to other antibiotics. This is of concern due to the increasing cost of antibiotic treatment and the spread of multidrug resistance to more pathogenic microorganisms. This study was undertaken to analyze the extent of the problem, to identify the most prevalent MDR isolates of Klebsiella pneumoniae in Chennai and to identify new plant based drugs. Approach: About 188 clinical isolates of Klebsiella pneumoniae were collected from Chennai during the period Nov. 2007-Oct. 2008 and their resistance to different groups of antibiotics were analyzed. These isolates were further characterized by molecular studies to identify the ESBL genes conferring the resistance phenotype. Plant extracts were tested against the MDR isolates to identify new treatment methods. Results: Our results showed that the combination therapy of clavulanic acid with cephalosporins and fluroroquinolones-norfloxacin and ciprofloxacin were the most effective antibiotics for treatment of Klebsiella pneumoniae infections. However resistance to clavulanic acid was increasing among the isolates (17%). All the isolates harbored a plasmid containing one or more of the genes for ESBL resistance. Plasmid isolation from the Isolates followed by ESBL PCR genotyping showed that CTX-M was present in 75 % isolates and TEM in 73% isolates either alone or in combination with the other ESBL genes. SHV ESBL type was present only in 42% of isolates. We identified 4 presumptive clonal spreads which were likely to be prevalent in India by clustering based on ESBL genotypic and antibiotic resistance. The isolates with both SHV and CTX were correlated with the most pathogenic and clavulanic acid resistant isolates. The extracts of Hemidesmus indicus and Terminalia arjuna had a significant antimicrobial activity against the MDR isolates. Conclusion: There was an increase in the spread of the ESBL genes and clavulanic acid resistance in the clinical isolates of Klebsiella pneumoniae in Chennai. Our data also supported a higher incidence of CTX-M genotype in India though the SHV genotype was associated with the most resistant forms of ESBL. Future research with separation of plant extracts to identify bioactive principles will pave the way for newer plant based antibiotics.

First Report of the Emergence of CTX-M-Type Extended-Spectrum β-Lactamases (ESBLs) as the Predominant ESBL Isolated in a U.S. Health Care System

CTX-M-type extended-spectrum beta-lactamases (ESBLs) have become increasingly common worldwide, with the notable exception of the United States, where TEM- and SHV-type ESBLs have appeared to predominate. We have noted the emergence of ESBLs in our health care system (the University Health System in San Antonio, TX), especially in Escherichia coli isolates, that preferentially hydrolyze cefotaxime rather than ceftazidime, suggesting the possibility of CTX-M-type enzymes. Microbiology laboratory records were reviewed to identify ESBL-producing isolates and to compare the diameters of ceftazidime disk diffusion zones of inhibition to cefotaxime zone diameters. All isolates had been initially detected and confirmed using the procedures recommended by the Clinical and Laboratory Standards Institute. A total of 94 stored ESBL-producing isolates recovered between January 2000 and June 2006 (predominately from blood and normally sterile fluids) were retrieved for further study and screened using PCR primers specific for the presence of CTX-M, TEM, and SHV ESBLs. Only small numbers of retained ESBL-producing isolates were available for study in 2000 and 2002. The percentages of available ESBL-producing organisms in the following years were found to produce CTX-M enzymes: 2000, 25%; 2001, 10%; 2002, 0%; 2003, 60%; 2004, 69%; 2005, 89%; and 2006, 70%. The most common CTX-M-type ESBL was CTX-M-15, followed by CTX-M-16, CTX-M-8, and CTX-M-14. Comparing the disk diffusion zone diameters of cefotaxime and ceftazidime was helpful with the initial recognition of CTX-M-producing E. coli, which had an average cefotaxime zone diameter 7 mm smaller than the ceftazidime zone. However, comparing ceftazidime and cefotaxime zones for CTX-M-producing Klebsiella spp. was not helpful with initial recognition. CTX-M enzymes were also identified in Proteus mirabilis, Enterobacter spp., and Morganella morganii. Based on pulsed-field gel electrophoresis typing of the E. coli isolates, the CTX-M-producing isolates did not represent the spread of a single clone in the institution or in the community. In conclusion, CTX-M-type ESBLs are now the most common ESBL type isolated from patients in our health care system and may also be present but unrecognized in other U.S. locales.

Klebsiella pneumoniae Isolate Producing at Least Eight Different β-Lactamases, Including AmpC and KPC β-Lactamases

Reports of the production of multiple β-lactamases in a single gram-negative pathogen are increasing (1, 2, 6). Isolates of Klebsiella species producing KPC-2, SHV extended-spectrum β-lactamases (ESBLs), and inhibitor-resistant TEM-30 β-lactamases have been reported as endemic in New York City (1). The present report identifies a Klebsiella pneumoniae isolate from New York City which produced up to 10 different β-lactamases, including a FOX-like plasmid-mediated AmpC, in addition to the previously reported KPC, SHV ESBL, and IRT β-lactamases (1). The K. pneumoniae isolate was obtained from the sputum of a patient with a mesothelioma. No carbapenem antibiotics were given to the patient prior to the isolation of the K. pneumoniae isolate. CLSI (formerly NCCLS) disk diffusion and broth microdilution assays (4) revealed that the K. pneumoniae isolate lacked susceptibility to levofloxacin (>16 μg/ml), amikacin (32 μg/ml), piperacillin-tazobactam (>128 μg/ml), ceftazidime (128 μg/ml), cefotaxime (64 μg/ml), aztreonam (>128 μg/ml), and cefoxitin (>64 μg/ml) but was susceptible by microbroth tests to tigecycline (0.5 μg/ml), minocycline (4 μg/ml), polymyxin B (1 μg/ml), cefepime, and imipenem. However, when broth microdilution tests were performed with an inoculum of 107 CFU/ml instead of 105 CFU/ml, the MICs of both imipenem and cefepime increased (4 μg/ml to 32 μg/ml and 8 μg/ml to >128 μg/ml, respectively). The MICs of tigecycline, minocycline, and polymyxin B were not affected when a higher inoculum was used. The presence of an ESBL was indicated by the CLSI ESBL confirmatory disk tests but not by the CLSI ESBL broth microdilution confirmatory tests (4). Isoelectric focusing (IEF) indicated that this isolate produced up to 10 different β-lactamases. Characterization of these enzymes was performed, as previously described, using IEF, inhibitor profiles, cefotaxime gel hydrolysis assays, and PCR (5-7, 8, 9). Using these techniques, 8 of 10 β-lactamases were identified. Three of these enzymes hydrolyzed cefotaxime. The pI and inhibitor profiles of these enzymes suggested the production of an SHV-12-like ESBL, a KPC-like carbapenem-hydrolyzing enzyme, a FOX-like AmpC, a PSE-1-like β-lactamase, and an OXA β-lactamase. In addition, TEM-1-like, TEM-30-like, and SHV-1-like enzymes were also identified (Table ​(Table1).1). PCR amplification using family-specific primers substantiated the presence of these genes within the K. pneumoniae isolate and identified the OXA β-lactamase as an OXA-9-like β-lactamase. Sequence data generated using the same primers that amplified the blaOXA product also suggested that the gene was blaOXA-9. TABLE 1. Identification of the β-lactamases produced by the K. pneumoniae isolate Accurate β-lactam susceptibility testing can be expected to become increasingly difficult over the next few years due to an increase in isolates producing multiple β-lactamases. The numbers and types of β-lactamases produced by the K. pneumoniae isolate from New York described in this report are a cause for concern, especially with respect to detecting ESBL, AmpC, and KPC-type enzymes. The CLSI microbroth ESBL confirmatory tests were unable to detect the presence of the SHV ESBL in this isolate, and these tests also suggested imipenem susceptibility, even though the isolate produced a KPC-type enzyme. As the number of pathogens producing multiple β-lactamases continues to rise, the difficulties in identifying the mechanisms responsible for β-lactam MICs will increase (1, 2, 6, 10). Clinical laboratories need the option of molecular testing in addition to phenotypic testing for the identification of resistance mechanisms that may be masked by the production of multiple enzymes. Additional testing options for these highly resistant pathogens may help avert future outbreaks like the ones reported for KPC-producing K. pneumoniae isolates in New York (1-3).

Real-time PCR and melting curve analysis for reliable and rapid detection of SHV extended-spectrum beta-lactamases.

Extended-spectrum beta-lactamases (ESBLs), e.g., ESBLs of the TEM or SHV type, compromise the efficacies of expanded-spectrum cephalosporins. An SHV non-ESBL that hydrolyzes only narrow-spectrum cephalosporins can be converted into an SHV ESBL through substitutions at three amino acid positions, 179, 238, or 238--240. In order to improve detection of SHV ESBLs, a novel method, based on real-time PCR monitored with fluorescently labeled hybridization probes and followed by melting curve analysis, was developed. It is able to (i) detect bla(SHV) genes with high degrees of sensitivity and specificity, (ii) discriminate between bla(SHV non-ESBL) and bla(SHV ESBL), and (iii) categorize the SHV ESBL producers into three phenotypically relevant subgroups. This method, termed the SHV melting curve mutation detection method, represents a powerful tool for epidemiological studies with SHV ESBLs. It even has the potential to be used in the diagnostic microbiology laboratory, because up to 32 clinical isolates can be processed in less than 1 h by starting with just a few bacterial colonies.

Comparison of Five Different Methods for Detection of SHV Extended-Spectrum β-Lactamases

Five different methods for detection of different types of SHV extended-spectrum β-lactamases (ESBL) were compared: minimum inhibitory concentration (MIC) determination of β-lactam with and without clavulanic acid, double-disk synergy test (DDST), inhibitor potentiated disk diffusion test (IPDDT), three-dimensional test (TDT) and PCR/Nhe I test. MIC determination of β-lactam with and without clavulanic acid was the most sensitive method regardless of the type of β-lactamase. However the specificity of this method was a little above 90%. IPDDT turned out to be a very sensitive method too but it lacks specificity because 26.9% of ceftazidime sensitive strains (putative ESBL negative), gave a positive result. It is important to put all four disks on the plate because ceftazidime and aztreonam were more sensitive indicators for SHV-5 and SHV-12 β-lactamase producers while cefotaxime and ceftriaxone were more reliable in detecting SHV-2 β-lactamase producers. The DDST detected all SHV-5 and SHV-12 β-lactamase producers and 95.2% of SHV-2, so it was less sensitive than MIC determination but was highly specific, since there were no false negative results observed. The sensitivity of DDST can be improved by using all four disks and placing them at the smaller distance from the central disk (2.5 cm). The TDT was the least sensitive method, particularly for SHV-5 and SHV-12 β-lactamase producers. The PCR/Nhe I test for detection of ESBL blaSHV genes is a highly sensitive and specific method but it is rather laborious and thus not very practical for use in routine clinical laboratories. Nevertheless it has potential to serve as the gold standard in epidemiological investigations on ESBLs. According to the results of this investigation MIC determination of β-lactam with and without clavulanic acid, even if only one antibiotic is used and the PCR/Nhe I tests are the most reliable methods for detection of SHV ESBLs.

Contribution of natural amino acid substitutions in SHV extended-spectrum beta-lactamases to resistance against various beta-lactams.

SHV extended-spectrum beta-lactamases (ESBLs) arise through single amino acid substitutions in the parental enzyme, SHV-1. In order to evaluate the effect of genetic dissimilarities around the structural gene on MICs, we had previously devised an isogenic system of strains. Here, we present an extended version of the system that now allows assessment of all major types of SHV beta-lactamases as well as of two types of promoters of various strengths. Moreover, we devised a novel vector, pCCR9, to eliminate interference of the selection marker. A substitution within the signal sequence, I8F found in SHV-7, slightly increased MICs, suggesting more efficient transfer of enzyme precursor into the periplasmic space. We also noted that combination of G238S and E240K yielded higher resistance than G238S alone. However, the influence of the additional E240K change was more pronounced with ceftazidime and aztreonam than with cefotaxime and ceftriaxone. The SHV enzymes characterized by the single change, D179N, such as SHV-8, turned out to be the weakest SHV ESBLs. Only resistance to ceftazidime was moderately increased compared to SHV-1.

Prevalence of SHV-12 among clinical isolates of Klebsiella pneumoniae producing extended-spectrum beta-lactamases and identification of a novel AmpC enzyme (CMY-8) in Southern Taiwan.

Twenty (8.5%) of 234 nonrepetitive clinical isolates of Klebsiella pneumoniae from southern Taiwan were found to produce extended-spectrum beta-lactamases (ESBLs): 10 strains produced SHV-12, 4 produced SHV-5, 2 produced a non-TEM non-SHV ESBL with a pI of 8.3, 3 produced a novel AmpC beta-lactamase designated CMY-8 with a pI of 8.25, and 1 produced SHV-12 and an unidentified AmpC enzyme with a pI of 8.2. The CMY-8 enzyme confers a resistance phenotype similar to CMY-1 and MOX-1, and sequence comparisons showed high homologies (>95%) of nucleotide and amino acid sequences among these three enzymes. Plasmid and pulse-field gel electrophoresis analyses revealed that all isolates harboring an SHV-derived ESBL were genetically unrelated, indicating that dissemination of resistance plasmids is responsible for the spread of SHV ESBLs among K. pneumoniae in this area. All three isolates carrying CMY-8 had identical genotypic patterns, suggesting the presence of an epidemic strain.