Extended-Spectrum Beta-lactamase and Metallo-beta-lactamase Production among Escherichia coli and Klebsiella pneumoniae Strains from Urine of Pregnant Women in Afikpo, Ebonyi State, Nigeria

Aim: The aim of this study was to determine the occurrence of Extended-spectrum beta-lactamase (ESBL) and Metallo-beta-lactamase (MBL) among Escherichia coli and Klebsiella pneumoniae strains from pregnant women attending Mater Misericordia Hospital Afikpo, Ebonyi state, Nigeria. Study Design: This is a laboratory based prospective study carried out on pregnant women suspected of having urinary tract infection and was requested to undergo diagnosis at microbiology laboratory of the hospital. Place and Duration of Study: The study was conducted in the Department of Science Laboratory Technology, Akanu Ibiam Federal Polytechnic, Unwana, Afikpo, Ebonyi State, Nigeria from October, 2022 to January, 2023. Methodology: Clean-catch midstream urine samples were collected from 206 pregnant women suspected of having urinary tract infection and were requested to undergo medical diagnosis at microbiology laboratory of the hospital. The urine samples were processed following standard microbiological procedure. Antimicrobial susceptibility testing was determined using the disc diffusion method, while ESBL phenotypes were determine by the Double-Disc Synergy Test (DDST). Disc potentiation test was performed to check for MBL production. Results: Out of the 206 urine samples processed, 24 (11.7 %) E. coli and 12 (5.8 %) K. pneumoniae were isolated. The antimicrobial susceptibility of the isolates recorded a 100 % resistance with Amoxicillin/Clavulanic acid and Cotrimoxazole. The Gram-negative isolates showed a high sensitivity of 100 % to Netilmicin, Meropenem and Ofloxacin. Overall, 35 (97.2 %) multidrug resistance (MDR) was observed of the bacteria isolates. A total of 9 (37.5 %) E. coli and 4 (33.3 %) K. pneumoniae was found positive for ESBL production whereas, 5 (20.8 %) E. coli and 2 (16.7 %) K. pneumoniae were MBL positive. Conclusion: The level of drug resistance in this study underscores the need for regular surveillance for effective management of urinary tract infection in pregnancy.

Astragaloside IV alleviates lung inflammation in Klebsiella pneumonia rats by suppressing TGF-β1/Smad pathway

Astragaloside IV is a biologically active substance derived from the traditional Chinese medicine Astragalus mambranaceus Bunge, and has antioxidant, anti-inflammatory, and anti-apoptotic properties. In this study, we aimed to investigate the effects of astragaloside IV on Klebsiella pneumonia rats and the underlying mechanisms. Klebsiella pneumoniae (K. pneumoniae) rats were treated with different dosages of astragaloside IV (5, 10, and 20 mg/kg) by intragastric administration. The levels of pro-inflammatory cytokines interleukin (IL)-6, IL-1β, and tumor necrosis factor (TNF)-α in bronchoalveolar lavage fluid (BALF) were determined. Pathological changes of lung tissue were inspected by HE staining. The expression of transforming growth factor (TGF)-β1 in lung tissue was determined with immunohistochemistry, and the expression levels of TGF-β1, p-Smad2/Smad2, p-Smad3/Smad3, IκBα/p-IκBα, and p65/p-p65 in lung tissue were determined by western blot. The mechanism was further investigated with TGF-β1 inhibitor SB-431542. Astragaloside IV reduced the elevated levels of pro-inflammatory cytokines caused by K. pneumoniae and improved lung tissue damage in a dose-dependent manner. Astragaloside IV also decreased the expression of TGF-β1/Smad signaling pathway-related proteins and decreased the protein levels of inflammation-related p-IκBα and p65 in lung tissues induced by K. pneumoniae. Additionally, it was found that the effects of 20 mg/kg astragaloside IV were similar to SB-431542, which could improve pulmonary fibrosis induced by K. pneumoniae, decrease the levels of TGF-β1/Smad signaling pathway-related proteins in lung, and reduce inflammation at the same time. Astragaloside IV could alleviate the inflammation of rat pneumonia induced by K. pneumoniae through suppressing the TGF-β1/Smad pathway.

Escherichia coli y Klebsiella pneumoniae productoras de betalactamasas de espectro extendido en un hospital de La Habana

RESUMEN Introducción: En microorganismos gramnegativos la producción de enzimas betalactamasas es el mecanismo más común de resistencia. Las de espectro extendido constituyen un grupo importante por su capacidad de inactivar las cefalosporinas de tercera y cuarta generación y el aztreonam. Su detección es vital para indicar el tratamiento óptimo y las medidas de aislamiento que eviten la dispersión de los microorganismos que las portan. Objetivos: Determinar la incidencia y principales características de los aislados de Escherichiacoli y Klebsiellapneumoniae productores de betalactamasas de espectro extendido en muestras no urogenitales. Métodos: Estudio transversal realizado en el hospital "Salvador Allende" durante el año 2017. Se determinó la frecuencia de Escherichia coli y Klebsiella pneumoniae productoras de betalactamasas de espectro extendido, su procedencia según servicio del hospital, tipo de muestra clínica, y su sensibilidad antimicrobiana. La identificación de betalactamasas de espectro extendido se hizo por el método de doble disco de Jarlier. Resultados: Fueron productores de betalactamasas de espectro extendido 46 y 50 % de aislados de Escherichia coli y Klebsiella pneumoniae, respectivamente. La mayoría provenían de muestras de las salas del Instituto de Angiología, el antimicrobiano con mayor efectividad fue el meropenem, la sensibilidad al resto de los antimicrobianos estuvo por debajo de 80 % y no hubo aislados sensibles a las cefalosporinas de tercera generación. Conclusiones: Se demuestra una alta incidencia de aislados de Escherichia coli y Klebsiella pneumoniae productores de betalactamasas de espectro extendido en el Hospital "Salvador Allende" de La Habana, más marcada en las salas del Instituto de Angiología y en muestras de piel.

ABSTRACT Introduction: Beta-lactamase production is the most common resistance mechanism in gram-negative microorganisms. Extended-spectrum beta-lactamases are an important group of enzymes capable of inactivating third- and fourth-generation cephalosporins and aztreonam. Their detection is important to indicate the optimum treatment as well as isolation measures aimed at preventing the spread of carrier microorganisms. Objectives: Determine the incidence and main characteristics of isolates of extended-spectrum beta-lactamase-producing Escherichia coli and Klebsiella pneumoniae from non-urogenital samples. Methods: A cross-sectional study was conducted at Salvador Allende hospital during the year 2017. Determination was made of the frequency of extended-spectrum beta-lactamase-producing Escherichia coli and Klebsiella pneumoniae, their origin by hospital service, the type of clinical sample and their antimicrobial sensitivity. Identification of extended-spectrum beta-lactamases was based on the Jarlier double disc method. Results: Of the total Escherichia coli and Klebsiella pneumoniae isolates studied, 46% and 50%, respectively, were extended-spectrum beta-lactamase producers. Most had been obtained from samples taken in wards of the Institute of Angiology; the most effective antimicrobial was meropenem; sensitivity to the remaining antimicrobials was below 80%; no isolates were sensitive to third-generation cephalosporins. Conclusions: A high incidence was found of extended-spectrum beta-lactamase-producing Escherichia coli and Klebsiella pneumoniae isolates at Salvador Allende Hospital in Havana, more noticeably in Institute of Angiology wards and skin samples.

Biologically Synthesized Silver Nanoparticles for Enhancing Tetracycline Activity Against Staphylococcus aureus and Klebsiella pneumoniae

Abstract Phytochemical content of plant extracts can be used effectively to reduce the metal ions to nanoparticles in one-step green synthesis process. In this study, six plant extracts were used for the synthesis of silver nanoparticles (AgNPs). Biologically synthesized AgNPs was characterized using UV-Vis Spectrophotometer, Field Emission Scanning Electron Microscope (FE-SEM), X-ray diffraction (XRD), Energy Dispersive X-ray spectroscopy (EDX) and Fourier Transform Infrared (FTIR) spectroscopy. The individual and combined effects of AgNPs and tetracycline against S. aureus and K. pneumoniae were assessed. Ginger, onion and sidr extracts supported AgNPs formation while arak, garlic and mint extracts failed to convert the silver ions to AgNPs. The present findings revealed significant differences between the tested plant extracts in supporting AgNPs synthesis. AgNPs synthesized by ginger showed the highest individual and combined activity against tested strains followed by AgNPs prepared by sidr then that synthesized by onion. AgNPs significantly enhanced tetracycline activity (p≤0.05) against S. aureus and K. pneumoniae. The results of this study demonstrated that the combination of tetracycline and biologically synthesized AgNPs presented a useful therapeutically method for the treatment of bacterial infection and counterattacking bacterial resistance.

Aminoglycoside resistance genes in carbapenem-resistant Klebsiella pneumoniae / 中华临床感染病杂志

Objective To analyze the aminoglycoside ( AG ) antibiotics resistance rate of carbapenem-resistant Klebsiella pneumoniae ( CRKP ) and its molecular mechanisms.Methods One hundred and four strains of CRKP isolated from 4 hospitals in Zhejiang Province from January 2013 to June 2014 were collected, including 56 strains from Sir Run Run Shaw Hospital , Zhejiang University School of Medicine ( S hospital ), 22 from the First Affiliated Hospital, Zhejiang University School of Medicine ( Z hospital), 13 from Yiwu TCM Hospitals (Y Hospital) and 13 from Fuyang First People's Hospital (F Hospital).VITEK 2 Compact method and K-B disk method were used to detect the susceptibility of commonly used antibiotics including three kinds of AGs (kanamycin, gentamycin and amikacin ).PCR and sequencing techniques were used to screen the aminoglycoside resistance -related 16S rRNA methylation genes (rmtA, rmtB and armA) and the aminoglycoside modified enzyme resistance gene [aac(6′)Ⅰb].The relationship between drug resistance and carrier status of drug resistance genes was analyzed .Homologous analysis of rmtB-positive strains was performed using PFGE to examine the epidemic spread of strains in each hospital.Results All 104 CRKP strains were multi-drug resistant and had high resistance to cephalosporins, fluoroquinolones ( ciprofloxacin, levofloxacin ) and nitrofurantoin.The resistance rates to gentamicin, kanamycin and amikacin were 73.1%(76/104), 64.4%(67/104) and 56.7%(59/104), respectively.The carrying rates of aminoglycoside-resistance genes were: rmtB 56.7%( 59/104 ), aac (6′)Ⅰb 17.3%(18/104), armA 1.9%(2/104); while no rmtA was detected.Thirty-seven strains did not carry the screened genes.Amikacin-resistant strains were resistant to both kanamycin and gentamicin, and both were rmtB-positive strains.The PFGE classification results showed that 104 strains were divided into 11 clonal populations, and there were scattered non-population clones in each hospital. There were seven major clonal populations (Ⅰ-Ⅶ) carrying rmtB genes, of which typeⅠ, typeⅢand typeⅤwere prevalent in S hospital ; typeⅡ, typeⅥand TypeⅦwere popular in Z hospital ; the distribution of strains in Y hospital was scattered ; F hospital had one independent clone type Ⅳ(3 strains).Conclusion AGs still have certain sensitivity to CRKP strains.The main mechanism of strain resistance to AGs is the rmtB gene-mediated 16S rRNA methylase.

Targeting of Virulence Factors and Plasmid Profiling of Klebsiella pneumoniae Causing Urinary Tract Infection in Sylhet City of Bangladesh

ABSTRACT Studies were conducted to characterize Klebsiella pneumoniae isolates from urinary tract infection (UTI) patients in Sylhet city of Bangladesh. At the same time, all isolates were screened for some common virulence genes and four significant isolates were searched for plasmid number and sizes by mini alkaline-lysis method. Among five tested isolates from female UTI patients, gyrase subunit B2 (gyrb2) amplified in all isolates, lipase and nuclease detected in three isolates and serine protease amplifies in two isolates and gave the expected band of 1130 bp, 517 bp, 1055 bp and 211 bp respectively. Two of four isolates showed 9.82 kb plasmid band on agarose gel. Isolates bearing 9.82 kb plasmid were found to be resistant to multiple commercial antibiotics. At the same time all isolates were screened for in-vitro plate assay for proteolytic, lypolytic and hemolytic activity. Isolates with positive plasmid and more than one virulent gene with gyrB2 showed positive result in in-vitro culture plate with clear zone of proteolysis, hemolysis or lipolysis. This study will be helpful for further study in finding correlation or pattern of virulence properties for K. pneumoniae associated UTI in Bangladesh.

Characterisation of plasmid-mediated rmtB-1 in Enterobacteriaceae clinical isolates from São Paulo, Brazil

OBJECTIVES The emergence of 16S rRNA methyltranferases (16 RMTAses) has jeopardised the clinical use of aminoglycosides. RmtB is one of the most frequently reported in Gram-negatives worldwide. In this study, we aimed to estimate the frequency of 16S RMTAses encoding genes in Enterobacteriaceae isolated in a three-month period from a tertiary Brazilian hospital. METHODS All Gram-negatives classified as resistant to amikacin, gentamicin, and tobramycin by agar screening were selected for analysis. The presence of 16SRMTases encoding genes was verified by polymerase chain reaction (PCR). Antimicrobial susceptible profile was determined by broth microdilution. The genetic relationship among these isolates was accessed by pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Selected RmtB-producing isolates were characterised by whole genome sequencing (WGS) analysis. RESULTS Twenty-two of 1,052 (2.1%) Enterobacteriaceae were detected as producers of RmtB-1 [Klebsiella pneumoniae (n = 21) and Proteus mirabilis (n = 1)]. blaKPC-2 was identified among 20 RmtB-1-producing K. pneumoniae isolates that exhibited an identical PFGE and MLST (ST258) patterns. Two K. pneumoniae isolates, the A64216 (not harboring bla KPC-2), A64477 (harboring bla KPC-2) and one P. mirabilis isolate (A64421) were selected for WGS. rmtB-1 and bla KPC-2 genes were carried by distinct plasmids. While a plasmid belonging to the IncFIIk group harbored rmtB-1 in K. pneumoniae, this gene was carried by a non-typable plasmid in P. mirabilis. In the three analysed plasmids, rmtB-1 was inserted on a transposon, downstream a Tn2. CONCLUSION Our findings suggested that the rmtB-1 was harbored by plasmids distinct from those previously reported in Bolivia and China. It suggests that multiple mobilization events might have occurred in South America.

Molecular detection of ß-lactamase and integron genes in clinical strains of Klebsiella pneumoniae by multiplex polymerase chain reaction

Abstract INTRODUCTION: Infections caused by β-lactamase-producing gram-negative bacteria, such as Klebsiella pneumoniae, are increasing globally with high morbidity and mortality. The aim of the current study was to determine antimicrobial susceptibility patterns and the prevalence of antibiotic resistance genes (β-lactamase and integron genes) using multiplex PCR. METHODS One-hundred K. pneumoniae isolates were collected from different clinical samples. Antibiotic susceptibility testing was performed with thirteen different antibiotics. Multiplex-PCR was used to detect β-lactamase (bla TEM, bla CTX-M, bla SHV , bla VEB, bla PER, bla GES, bla VIM, bla IMP, bla OXA, and bla KPC) and integron genes (int I, int II, and int III). RESULTS: The highest and lowest rate of resistance was exhibited against amikacin (93%) and imipenem (8%), respectively. The frequency of β-lactamase-positive K. pneumoniae was 37%, and the prevalence of the bla TEM, bla CTX-M, bla SHV , bla VEB, bla PER, bla GES, bla VIM, bla IMP, bla OXA, and bla KPC genes was 38%, 24%, 19%, 12%, 6%, 11%, 33%, 0%, 28%, and 23%, respectively. Of the 100 isolates, eight (8%) were positive for class I integrons; however, class II and III integrons were not detected in any of the strains. CONCLUSIONS: These results indicate co-carriage of a number of β-lactamase genes and antibiotic resistance integrons on the same plasmids harboring multi-drug resistance genes. It seems that these properties help to decrease treatment complications due to resistant bacterial infections by rapid detection, infection-control programs and prevention of transmission of drug resistance.

High MDR and ESBL Producing Escherichia coli and Klesbiella pneumoniae from Urine, Pus and Sputum Samples.

Aims: This study was done to assess the prevalence of multidrug resistance and extended spectrum β-lactamase producing E. coli, K. pneumoniae in urine, pus and sputum. Place and Duration of Study: This study was done to assess the prevalence of MDR and ESBL producing E. coli and Klebsiella in urine, pus and sputum from March 2013 to April 2014 at KIST Medical College, Lalitpur, kathmandu, Nepal. Methodology: E. coli and K. pneumoniae were isolated from urine, pus and sputum samples in KIST Medical College, Lalitpur, Nepal. Antibiotic susceptibility test was performed by using disk diffusion method. MDR isolates which were suspected as ESBL producers were confirmed by using double disk synergy test and combined disk diffusion test for same isolates. Results: Out of 580 urine samples, (87/580) 15% showed significant growth of E. coli and K. pneumoniae while in 97 pus and 124 sputum (16/221) 7% showed significant growth of E. coli and K. pneumoniae. From the sputum among 9 isolates, 3 were E. coli and 6 were K. pneumoniae whereas in pus among 7 isolates, 6 were E. coli and one was K. pneumoniae. Out of E. coli (77) isolates from urine, (74/77) 96.10% were MDR and of K. pneumoniae (10) isolates from urine 90% were MDR. Among E. coli (74) MDR isolates 52/74 (70.27%) were ESBL producers whereas all MDR K. pneumoniae isolates from urine were ESBL producers. All the isolates of E. coli and K. pneumoniae from pus and sputum were MDR which were resistant to tested third generation cephalosporins. Among the isolates E. coli (55.55%) and K. pneumoniae (42.85%) isolates were ESBL producers. Conclusions: The high prevalence of MDR E. coli and K. pneumoniae was observed in urine, pus and sputum. The resistance pattern was alarmingly higher to all the antibiotics used except imipenem and amikacin. The prevalence of ESBL was higher so necessary step should be taken to prevent the spread and emergence of resistance.

Detection of virulent of Klebsiella isolates from ñlinical samples / Монголын Анагаах Ухаан

IntroductionKlebsiella spp is a well-known opportunistic pathogen associated with nosocomial infections such asurinary tract, septicaemia and pneumonia number of multi-drug resistant strains and infections causedby Klebsiella has progressively increased, causing treatment limitations.GoalIdentify of phenotype of Klebseilla isolates from ñlinical samplesMaterials and MethodsA total of 112 Klebsiella strains were isolated from clinical samples in State Central First Hospital and StateCentral Third Hospital from July 2015 through December 2015. The bacterial isolates were identifi edaccording to cultural characteristics, biochemical test and API20E. The serum resistance, capsule andhypermucoviscosity, cell surface protein (curly), a-hemolysin and ability to form biofi lm were sought byphenotypic assays. Antimicrobial susceptibility was tested by diffusion method.ResultA total of 112 Klebsiella samples were collected. The bacterial isolates were identifi ed according tocultural characteristics, biochemical test and API20E, the results revealed that 16.1 percent isolateswere identifi ed as K.oxytoca all of them 83.9 percent isolates were belong to K.pneumonia. Therewere observed for ampicillin (99 percent), nitrofurantoin (53.6 percent), cepalotin (50.6 percent) and51 percent of isolates were considered as a multiple drug resistant. Serum resistance properties ofK.pneumoniae was resistance 89.4 percent, intermediately susceptible 4.3 percent, sensitive 6.4percent and for K.oxytoca resistance 88.9 percent, intermediately susceptible 5.6 percent, sensitive 5.6percent. The hemolysin àalpha was detected in 32.2 percent, and gamma, beta in 66.96 percent, 0.9percent respectively. The capsule was observed in 46.5 percent and hypermucoviscosity in 27.7 percentof isolates. The cell surface protein (curly) and biofi lm were detected in 100 percent.Conclusion:Both K.pneumoniae and K.oxytoca isolates from clinical samples have similar virulent properties, andthe a-hemolysin and hypermucoviscosity positive isolates were more resistance to antibiotics.

Distribution of beta-Lactamase Genes Among Carbapenem-Resistant Klebsiella pneumoniae Strains Isolated From Patients in Turkey

BACKGROUND: The emergence of carbapenem-resistant Klebsiella pneumoniae poses a serious problem to antibiotic management. We investigated the beta-lactamases in a group of carbapenem-resistant K. pneumoniae clinical isolates from Turkey. METHODS: Thirty-seven strains of K. pneumoniae isolated from various clinical specimens were analyzed by antimicrobial susceptibility testing, PCR for the detection of beta-lactamase genes, DNA sequencing, and repetitive extragenic palindronic (REP)-PCR analysis. RESULTS: All 37 isolates were resistant to ampicillin, ampicillin/sulbactam, piperacillin, piperacillin/tazobactam, ceftazidime, cefoperazone/sulbactam, cefepime, imipenem, and meropenem. The lowest resistance rates were observed for colistin (2.7%), tigecycline (11%), and amikacin (19%). According to PCR and sequencing results, 98% (36/37) of strains carried at least one carbapenemase gene, with 32 (86%) carrying OXA-48 and 7 (19%) carrying NDM-1. No other carbapenemase genes were identified. All strains carried a CTX-M-2-like beta-lactamase, and some carried SHV- (97%), TEM- (9%), and CTX-M-1-like (62%) beta-lactamases. Sequence analysis of bla(TEM) genes identified a bla(TEM-166) with an amino acid change at position 53 (Arg53Gly) from bla(TEM-1b), the first report of a mutation in this region. REP-PCR analysis revealed that there were seven different clonal groups, and temporo-spatial links were identified within these groups. CONCLUSIONS: Combinations of beta-lactamases were found in all strains, with the most common being OXA-48, SHV, TEM, and CTX-M-type (76% of strains). We have reported, for the first time, a high prevalence of the NDM-1 (19%) carbapenemase in carbapenem-resistant K. pneumoniae from Turkey. These enzymes often co-exist with other beta-lactamases, such as TEM, SHV, and CTX-M beta-lactamases.

Comparison of culture and PCR assays for detection of bacteria in laboratory rats and mice / 中国比较医学杂志

Objective To compare the efficiency of bacteria culture and PCR assays for detection of Staphylococcus aureus ( S.aureus) , Pseudomonas aeruginosa ( P.aeruginosa) and Klebsiella pneumoniae ( K.pneumoniae) in laboratory rats and mice.Methods Bacteria culture combined with biochemical identification and PCR assay were used to detect 78 SPF rats and 422 SPF mice and the results of the two methods were compared .Results All the 78 rats were negative .Of the 422 mice, the positive rate by culture was 7.11%(30/422), of which, 10 were S.aureus, 22 were P.aeruginosa, and 2 were K.pneumoniae.The positive rate by PCR was 7.58%(32/422), of which, 10 were S.aureus, 25 were P. aeruginosa, and 2 were K.pneumoniae.Conclusions The high sensitivity , rapid procedure and easy to operate of PCR assay makes it valuable for rapid bacteria diagnosis and large-scale screening in laboratory animals .

Pathogenic determinants of clinical Klebsiella pneumoniae strains associated with their persistence in the hospital environment

Background: Klebsiella pneumoniae is considered an opportunistic pathogen associated with nosocomial infections occurring mainly in pediatric patients, such as premature infants placed in intensive care units. The aim of this study was to characterize K. pneumoniae strains isolated from different clinical sources based on their resistance to antibiotics and the presence of virulence factors associated with their persistence in the hospital environment. Methods: Fifty clinical strains of K. pneumoniae isolated from urine, blood, catheters, and cerebrospinal fluid sources were characterized. Susceptibility testing of antibiotics was performed by the Kirby-Bauer method (Clinical Laboratory Standards Institute, 2010). The ability to form a biofilm was determined by the 96-well microplate method. Capsule and fimbrial structures were visualized by transmission electron microscopy (TEM). Adherence was evaluated on A549 and HT29 cells. Assessment for the presence and expression of the ecpA, fimH, and mrkA genes was performed by PCR and RT-PCR. Results: Clinical strains of K. pneumoniae were isolated from 48% of urine, 24% of blood, 18% of catheters, and 10% of cerebrospinal fluid. Ninety-two percent of the strains showed resistance to cefpodoxime, whereas few strains showed resistance to imipenem and meropenem (4 and 2%, respectively). The extended spectrum-type beta-lactamase (ESBL) phenotype was identified in 97% of the strains positive for resistance to third-generation cephalosporins. In addition, 88% of the strains were multidrug resistant. All strains were able to form biofilms. Capsule and fimbirial structures were visualized by TEM. Based on our adhesion assays, the A549 cell line was more permissive to K. pneumoniae strains than the HT-29 cell line. K. pneumoniae strains amplified and expressed ecpA (100/70%), fimH (98/2%), and mrkA (84/48%) genes, respectively. Conclusion: The K. pneumoniae strains exhibited features that allowed them to survive in the hospital environment (formation of biofilm) and resist antimicrobial therapy (multidrug resistant MDR strains). These strains also possessed a capsule, adhesive properties, and expression of genes encoding colonization factors that favor the selection and persistence of these strains in hospitals.

Outbreak of carbapenem-resistant Klebsiella pneumoniae: two-year epidemiologic follow-up in a tertiary hospital

This study describes a carbapenem-resistant Klebsiella pneumoniae (CRKP) outbreak that occurred from October 2008-December 2010. Polymerase chain reaction assays were performed to detect the blaKPC gene and molecular typing was performed using pulsed-field gel electrophoresis (PFGE). There were 33 CRKP infections; PFGE revealed five genotypes: genotype A in five (15%), B in 18 (55%), C in eight (24%) and two unique profiles. Genotype B was disseminated in all hospital units and belonged to the same clone identified in 11 different hospitals in the state of São Paulo. Sixteen (48%) patients died. Seven isolates (21%) were resistant to polymyxin B and 45% were resistant to tigecycline and amikacin.

Study of qepA among clinical isolates of Klebsialla pneumoniae / 中华微生物学和免疫学杂志

Objective To investigate the prevalence of qepA, quinolone efflux protein, among 41 unique clinical strains of K. pneumoniae producing ESBLs and to study the qepA-bearing isolates using the Diversilab system. Methods Identification and antimicrobial susceptibility were performed by Vitek-2 Compact System. Screening of qepA was carried out by PCR amplification. The NCBI BLAST program was utilized for sequence comparisons. qnr-bearing strains was evaluated by the Repetitive-sequence-based PCR(Rep-PCR) employing the Diversilab system. And the existence of rmtB was detected among these qepA contained isolates. Results qepA were detected in 5 isolates( 12.2% ). The Rep-PCR profiles produced by the Diversilab system showed that 2/5 of isolates were indistinguishable. And 60% of qepA-positive isolates were detected to harbor rmtB gene. Conclusion The data suggest the emergence of qepA-borne K. pneumoniae.

Molecular Characteristics of Extended Spectrum beta-Lactamases in Escherichia coli and Klebsiella pneumoniae and the Prevalence of qnr in Extended Spectrum beta-Lactamase Isolates in a Tertiary Care Hospital in Korea

PURPOSE: Extended spectrum beta-lactamases (ESBLs) are cephalosporinases that confer resistance to a wide variety of oxyimino cephalosporins and create serious therapeutic problems. In addition, the quinolone resistance qnr genes are becoming increasingly prevalent in clinical isolates, some of which also produce ESBL. This study was designed to evaluate the occurrence and genotypic distribution of ESBL producing Escherichia coli (E. coli) and Klebsiella pneumoniae (K. pneumoniae) as well as the prevalence and distribution of qnr genes in ESBL-producing isolates in a tertiary care hospital in Korea. MATERIALS AND METHODS: We tested a total of 111 ESBL-producing isolates of E. coli and K. pneumoniae, which were collected at Kyung Hee Medical Center from November 2006 to June 2008. ESBL production was determined by the Clinical and Laboratory Standards Institute (CLSI) ESBL confirmatory test. The cefotaxime and ceftazidime resistance of the ESBL-producers were transferred to azide-resistant E. coli J53 by conjugation. The presence and identity of ESBL and qnr genes were determined by polymerase chain reaction (PCR) and nucleotide sequencing. RESULTS: The prevalence of ESBLs was 17.7% (297/1,680) of E. coli and 26.5% (240/904) of K. pneumoniae in our hospital during the study periods. Of the 111 collected isolates, 69 isolates were E. coli and 42 isolates were K. pneumoniae. The most prevalent ESBL genotype was CTX-M15. Among the ESBL-producing isolates, 4 E. coli (5.8%) and 17 K. pneumoniae (40.5%) contained qnr genes. qnrB4 was the most frequent type in both E. coli and K. pneumoniae. CONCLUSION: CTX-M15 was the most frequently encountered ESBL. In addition, a high prevalence of qnr genes among ESBL-producing K. pneumoniae was identified in this study.

Design of a molecular method for subspecies specific identification of Klebsiella pneumoniae by using the 16S ribosomal subunit gene / Diseño de un método molecular para la identificación específica de Klebsiella pneumoniae a nivel de subespecie, usando el gen que codifica para la subunidad ribosomal 16S

Introduction: Rhinoscleroma is caused by Klebsiella pneumoniae subsp. rhinoscleromatis and the ozena infections caused by K. pneumoniae subsp. ozaenae, both infections affect the upper respiratory tract. In the first clinical phases the symptoms are unspecific, and the disease can be misdiagnosed as a common cold, therefore antimicrobial therapy cannot reach effective results and patients must be following up for several years since the infection became chronic. Objective: To identify Klebsiella subspecies using a specific assay based on amplicons restriction of a gene which encodes 16S subunit ribosomal (rDNA16S). Methodology: Specific restriction patterns were generated; using reported sequences from rDNA16S gene and bioinformatics programs MACAW, PFE, GENEDOC and GENE RUNNER. Amplification and restriction assays were standardized. Results: Predictions in silico allowed us to propose an algorithm for Klebsiella species and subspecies identification. Two reference strains were included and two clinical isolates which were biotyped and identified by the proposed method. rDNA16S gene restriction patterns showed differences regarding the initially identified species for conventional methods. Additionally two patterns of bands were observed for K. pneumoniae subsp. rhinoscleromatis, indicating the polymorphisms presence in the rDNA16S gene. Conclusions: We confirmed the difficulty to identify K. pneumoniae subspecies by conventional methods. Implementation of this technique could allow accurate and rapid differentiation among K. pneumoniae subsp. ozaenae and K. pneumoniae subsp. rhinoscleromatis the aetiological agents of two frequently misdiagnosed infections. Antimicrobial therapy usually could be ineffective, especially in chronic patients. Finally we consider very important to enlarge the study by using more clinical and reference strains.

Introducción: El rinoescleroma es causado por Klebsiella pneumoniae subsp. rhinoscleromatis y la ocena por K. pneumoniae subsp. ozaenae, respectivamente. Estas infecciones se presentan sobre todo en el tracto respiratorio superior y originan una sintomatología inespecífica en sus fases iniciales por lo cual se pueden confundir con el catarro común. Las dificultades para establecer un diagnóstico oportuno tienen repercusiones negativas en la terapia antimicrobiana, que puede no ser efectiva y hacer que la enfermedad evolucione a una fase crónica cuyo seguimiento en el paciente puede necesitar muchos años. Objetivo: Diseñar un ensayo molecular para la identificación a nivel de subespecie de bacterias del género Klebsiella basado en restricción de amplicones del gen que codifica para la subunidad ribosomal 16S (ADNr 16S). Metodología: Se generaron patrones de restricción específicos, con secuencias informadas del gen ADNr 16S y los programas bioinformáticos MACAW, PFE, GENEDOC y GENE RUNNER. Se estandarizaron las condiciones para la amplificación y restricción del ensayo experimental.Resultados: Las predicciones in silico permitieron proponer un algoritmo para identificar a nivel de especie y subespecie los miembros del género Klebsiella. Se incluyeron dos cepas de referencia y dos aislamientos clínicos, que fueron biotipificados e identificados por el método propuesto; los patrones de restricción obtenidos del gen ADNr 16S evidenciaron diferencias respecto a la especie inicialmente identificada por métodos convencionales. Además, se encontraron dos patrones de bandas en K. pneumoniae. rhinoscleromatis, que indican la presencia de polimorfismos en el gen ADNr 16S para esta subespecie.

Design of a molecular method for subspecies specific identification of Klebsiella pneumoniae by using the 16S ribosomal subunit gene / Diseño de un método molecular para la identificación específica de Klebsiella pneumoniae a nivel de subespecie, usando el gen que codifica para la subunidad ribosomal 16S

Introduction: Rhinoscleroma is caused by Klebsiella pneumoniae rhinoscleromatis and the ozena infections caused by K. pneumoniae ozaenae, both infections affect the upper respiratory tract. In the first clinical phases the symptoms are unspecific, and the disease can be misdiagnosed as a common cold, therefore antimicrobial therapy cannot reach effective results and patients must be following up for several years since the infection became chronic. Objective: To identify Klebsiella subspecies using a specific assay based on amplicons restriction of a gene which encodes 16S subunit ribosomal (rDNA16S). Methodology: Specific restriction patterns were generated; using reported sequences from rDNA16S gene and bioinformatics programs MACAW, PFE, GENEDOC and GENE RUNNER. Amplification and restriction assays were standardized. Results: Predictions in silico allowed to propose an algorithm for Klebsiella species and subspecies identification. Two reference strains were included and two clinical isolates which were biotyped and identified by the proposed method. rDNA16S gene restriction patterns showed differences regarding the initially identified species for conventional methods. Additionally two patterns of bands were observed for K. pneumoniae rhinoscleromatis, indicating the polymorphisms presence in the rDNA16S gene. Conclusions: It was confirmed the difficulty to identify K. pneumoniae subspecies by conventional methods. Implementation of this technique could allow an accurate and rapid differentiation among K. pneumoniae ozaenae and K. pneumoniae rhinoscleromatis aetiological agents of two frequently misdiagnosed infections. Antimicrobial therapy usually could be ineffective, especially in chronic patients. Finally it is considered very important to enlarge the study by using more clinical and reference strains.

Introducción: El rinoescleroma es causado por Klebsiella pneumoniae rhinoscleromatis y la ocena por Klebsiella pneumoniae ozaenae respectivamente. Estas infecciones se presentan sobre todo en el tracto respiratorio superior y tienen una sintomatología inespecífica en sus fases iniciales por lo cual se pueden confundir con el catarro común. Las dificultades de establecer un diagnóstico oportuno tienen repercusiones negativas en la terapia antimicrobiana, porque puede no ser efectiva y hacer que la enfermedad evolucione a una fase crónica cuyo seguimiento puede implicar muchos años. Objetivo: Diseñar un ensayo molecular para la identificación a nivel de subespecie de bacterias del género Klebsiella basado en restricción de amplicones del gen que codifica para la subunidad ribosomal 16S (ADNr 16S).Metodología: Se generaron patrones de restricción específicos, utilizando secuencias informadas del gen ADNr 16S y los programas bioinformáticos MACAW, PFE, GENEDOC y GENE RUNNER. Se estandarizaron las condiciones para la amplificación y restricción para el ensayo experimental.Resultados: Las predicciones in silico permitieron proponer un algoritmo para la identificación a nivel de especie y subespecie de las especies del género Klebsiella. Se incluyeron dos cepas de referencia y dos aislados clínicos, que se biotipificaron e identificaron por el método propuesto; los patrones de restricción obtenidos del gen ADNr 16S evidenciaron diferencias con respecto a la especie inicialmente identificada por métodos convencionales. Además se encontraron dos patrones de bandas en Klebsiella pneumoniae rhinoscleromatis, indicando la presencia de polimorfismos en el gen ADNr 16S para esta subespecie. Conclusiones: Se confirmó la dificultad para identificar Klebsiella pneumoniae a nivel de subespecie por métodos convencionales.

Characteristics of aac(6')-Ib-cr Gene in Extended-Spectrum beta-Lactamase-Producing Escherichia coli and Klebsiella pneumoniae Isolated from Chungnam Area / 대한진단검사의학회지

BACKGROUND: Concomitant quinolone resistance in extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae is a crucial problem in the clinical management of infections. In foreign countries, the fluoroquinolone acetylating aminoglycoside-(6)-N-acetyltransferase (aac[6']-Ib-cr) gene, a novel plasmid-mediated quinolone resistance determinant has been reported to occur in conjunction with qnr. We aim to investigate the prevalence and characteristics of concomitant aac(6')-Ib-cr and qnr expression in ESBL-producing Escherichia coli and Klebsiella pneumoniae in Korea. METHODS: Between December 2007 and April 2008, we collected 60 and 69 clonally unrelated non-repetitive clinical isolates of ESBL-producing E. coli and K. pneumoniae, respectively. We studied the expressions of 11 types of ESBL-encoding genes, 4 types of 16s rRNA methylase genes; rmtA, rmtB, rmtC and armA, 3 types of qnr genes; qnrA, qnrB, qnrS and aac(6')-Ib. The presence of aac(6')-Ib-cr variants was detected by sequencing. The involvement of integrons was studied using multiplex PCR and sequencing of gene-cassette arrays. Conjugation experiments were performed to confirm plasmid-mediated resistance and the relationships among coharbored genes. RESULTS: We observed a high prevalence of the cr variant (61.1%) of aac(6')-Ib, and the prevalence of this variant in qnr and aac(6')-Ib-coharboring isolates (67.4%) was higher than in qnr-negative isolates (51.7%). The high prevalence of the cr variant was significantly related to the high minimum inhibitory concentrations (MICs) of ciprofloxacin, tobramycin, and amikacin and indicated the statistically significant roles of qnrB, qnrS, rmtA, and rmtB in quinolone and aminoglycoside resistance. CONCLUSIONS: The aac(6')-Ib-cr variants were widespread and showed significant relation to the high-level quinolone and aminoglycoside resistance in ESBL-producing E. coli and K. pneumoniae.

Phenotypic detection and occurrence of extended-spectrum beta-lactamases in clinical isolates of Klebsiella pneumoniae and Escherichia coli at a tertiary Hospital in Trinidad & Tobago

The incidence and distribution of ESBL producing microorganisms such as E. coli and K. pneumoniae have been demonstrated and varies in different health care facilities and as well as other countries This study was carried out to determine the frequency of occurrence and the antimicrobial susceptibility pattern of ESBL producing E. coli and K. pneumoniae species from clinical isolates at a tertiary hospital in Trinidad & Tobago. Standard microbiological procedures and automated MicroScan System was used to identify, screen for putative ESBL production and determine antimicrobial susceptibility of 1,118 clinical isolates of Enterobacteriaceae species at the microbiology laboratory of the Eric Williams Medical Science Complex, Trinidad & Tobago over a 36 months period. All ESBL producing isolates flagged by the automated system were further confirmed by E-test method. The E-test confirmed a 15.2 percent ESBL rate among the K. pneumoniae isolates and 9.3 percent among the E. coli isolates. There was also a 1.8 percent rate of ESBL production in K. pneumoniae and 0.2 percent in E. coli isolates from specimens received from community health facilities into the laboratory. Isolates recovered from the intensive care unit of the hospital had 2.1 percent E. coli and 8.2 percent K. pneumoniae ESBL producers. Although all ESBL positive isolates were completely susceptible to imipenem and meropenem; and all positive K. pneumoniae isolates were susceptible to amikacin, there was a low susceptibility of ESBL positive E. coli to the aminoglycosides. However, susceptibility of these ESBL producing isolates to the fluoroquinolones varied. There is a high rate of ESBL production among isolates of E. coli and K. pneumoniae at this hospital that is linked to the extensive inappropriate use of third generation cephalosporins in the country. Further molecular studies are needed to characterize the types of these ESBL prevailing in the country.