Detección de los genes de ß-lactamasas blaTEM, blaSHV y blaCTX-M en aislamientos de Escherichia coli comunitarios / Detection of ß-lactamase genes bla TEM, bla SHV and bla CTX-M in community isolates of Escherichia coli

A nivel mundial la resistencia a los antibióticos es un problema de salud pública, tanto en el ámbito hospitalario como en el comunitario. La producción de ß-lactamasas es el principal mecanismo de resistencia en enterobacterias y la mayoría de enzimas responsables pertenecen a las familias TEM, SHV y CTX-M. El objetivo de este estudio fue detectar los genes de ß-lactamasas blaTEM, blaSHV y blaCTX-M en cepas comunitarias de Escherichia coli productoras de BLEE aisladas de urocultivos de pacientes que acudieron al Laboratorio Clínico Popular de la Universidad de San Carlos de Guatemala en el año 2016. Se detectó la presencia de al menos uno de los genes en el 90% de los 79 aislamientos y un 53.2% presentó los tres genes. La frecuencia fue de 57% para blaCTX-M, 84% para bla SHV y 85% para blaTEM. La detección de los genes codificadores de las enzimas TEM-1, SHV-11, CTX-M15 y CTX-M55 corresponde a la primera caracterización molecular de aislamientos de E. coli productoras de BLEE en Guatemala y son importantes para entender su propagación en el ámbito comunitario. Los aislamientos de E. coli productoras de BLEE mostraron alta resistencia a ciprofloxacina y trimetoprim sulfametoxazol (78%) y bajos niveles de resistencia para fosfomicina (2.5%) y nitrofurantoina (7.6%). El 11.39% de las cepas presentó resistencia a un grupo de antibióticos no betalactámicos. Es importante establecer una vigilancia activa para la resistencia de estos antibióticos en cepas comunitarias ya que son la primera opción de tratamiento para cepas productoras de BLEE

Globally, resistance to antibiotics is a public health problem, both in the hospital and in the community environment. e production of ß-lactamases is the main mechanism of resistance in enterobacteria and usually the cause of resistance are the enzymes to the families TEM, SHV and CTX-M. e principal aim of this study was to detect - ß-lactamase genes blaTEM, blaSHV and blaCTX-M in community strains of ESBL-producing Escherichia coli isolated from urine cultures of patients attended in the Laboratorio Clínico Popular of the Universidad de San Carlos de Guatemala in 2016. At least, one of the genes was detected in 90% of the 79 isolates and in 53.2% of the isolates the three genes were detected. e frequency was 57% for blaCTX-M, 84% for blaSHV and 85% for blaTEM. e detection of the genes coding for TEM-1, SHV-11, CTX-M15 and CTX-M55 represent the first molecular characterization of ESBL-producing E. coli isolates in Guatemala and it is important to understand the spread of these strains at the community environment. e ESBL-producing E. coli isolates showed high resistance to ciprofloxacin and trimethoprim sulfamethoxazole (78%) and low resistance levels for fosfomycin (2.5%) and nitrofurantoin (7.6%). e 11.39% of the strains showed resistance to a group of non-beta-lactam antibiotics. It is important to establish an active surveillance for these antibiotics in community strains because they are the first line treatment for strains producing ESBL

New Delhi metallo-β-lactamase and extended spectrum β-lactamases co-producing isolates are high in community-acquired urinary infections in Assam as detected by a novel multiplex polymerase chain reaction assay.

Background: The ability of microorganisms to evade antibiotic pressure is challenging in healthcare as patients have little or no drug treatment options. Detection of the prevalence of antibacterial resistance pattern helps towards improved antibiotic policy and empirical treatment. Objectives: We carried out antibiogram profiling and documented the prevalence and co-prevalence of New Delhi metallo-β-lactamase (NDM) and extended spectrum β-lactamases (ESBL) encoding genes in urinary Escherichia coli and Klebsiella pneumonia isolates. Materials and Methods: Antibiotic susceptibilities were tested for 241 isolates of E. coli and K. pneumoniae from urine samples collected from out- and hospitalised patients. Polymerase chain reaction (PCR) was carried out on isolates tested positive for phenotypic production of metallo-β-lactamase and ESBL. A multiplex PCR assay was designed to detect the genes. Results: Multiplex PCR assay designed had a limit of detection of 103 CFU/mL in vitro. NDM detected was significantly higher among K. pneumoniae compared to E. coli (69.2% vs. 18.2%; P = 0.001). Of 17, 14 NDM positive isolates also harboured ESBL genes. The co-production of CTX-M + TEM + NDM (3/9; 33.3% and 5/8; 62.5%) was most common in K. pneumoniae and E. coli, respectively while CTX-M + TEM + SHV + NDM was found in one isolate. Of the 156 phenotypically ESBL producing isolates, CTX-M, TEM and SHV was detected by PCR in 85, 53 and 24 isolates, respectively. Conclusion: NDM and ESBL co-producing isolates were both community (64.7%) and hospital (35.29%) acquired among E. coli. Antibiotic resistance can be effectively evaluated by a cost and time effective molecular method, such as the multiplex PCR used in this study, which complement culture and sensitivity tests.

Prevalence and identification of extended spectrum β-lactamases (ESBL) in Escherichia coli isolated from a tertiary care hospital in North-East India.

Extended-spectrum β-lactamases (ESBLs) are rapidly evolving group of β-lactamase enzymes produced by the Gram negative bacteria. In this study, we determined the antimicrobial sensitivity pattern of Escherichia coli isolates and prevalence of TEM, SHV and CTX-M genes in ESBL positive E. coli isolated from the patients admitted to a tertiary care hospital in North-East India. A total of 85 multidrug-resistant isolates of E. coli obtained from clinical samples; urine (n=80), sputum (n=3), body fluid (n=1), vaginal discharge (n=1) were screened for resistance to third generation cephalosporins. ESBL production in resistant isolates was determined by double disk synergy test (DDST) and phenotypic confirmatory test (PCT). ESBL positive isolates were subjected to PCR for detection of TEM, SHV and CTX-M genes. Imipenem was found to be most effective against E. coli (susceptible isolates 96.47%) while ciprofloxacin was the least effective antibiotic (resistant isolates 60%). Among 33 ESBL positive isolates confirmed via PCT, preponderance in female population (60.6%) was noted. The most prevalent gene was blaSHV (63.04%) followed by blaTEM and blaCTX-M (60.86 and 54.34%, respectively) in ESBL positive E. coli. Most of the extensively used antibiotics, appear to be ineffective against the ever-mutating bacteria. This resistance urges cautious antimicrobial management on priority. Further, it helps in effectively designing the chemotherapeutic regimen for patients of a particular geographic area.

First description of SHV‑148 mediated extended‑spectrum cephalosporin resistance among clinical isolates of Escherichia coli from India.

Purpose: The present study was aimed to investigate the genetic context, association with IS26 and horizontal transmission of SHV‑148 among Escherichia coli in Tertiary Referral Hospital of India. Methodology: Phenotypic characterisation of extended‑spectrum beta‑lactamases (ESBLs) was carried out as per CLSI criteria. Molecular characterisation of blaSHV and integron was carried out by polymerase chain reaction (PCR) assay and confirmed by sequencing. Linkage of IS26 with blaSHV‑148 was achieved by PCR. Purified products were cloned on pGEM‑T vector and sequenced. Strain typing was performed by pulsed field gel electrophoresis with XbaI digestion. Transferability experiment and antimicrobial susceptibility was performed. Results: A total of 33 isolates showed the presence of SHV‑148 variant by sequencing and all were Class 1 integron borne. PCR and sequencing results suggested that all blaSHV‑148 showed linkage with IS26 and were present in the upstream portion of the gene cassette and were also horizontally transferable through F type of Inc group. Susceptibility results suggest that tigecycline was most effective. Conclusion: The present study reports for the first time of SHV‑148 mediated extended spectrum cephalosporin resistance from India. Association of their resistance gene with IS26 and Class 1 integron and carriage within IncF plasmid signifies the potential mobilising unit for the horizontal transfer.

Establishment and application of multiplex touchdown PCR for detection of extended-spectrum β-lactamases-producing Enterobacteriaceae and me-thicillin-resistant Staphylococcus aureus in bloodstream infection / 中国感染控制杂志

Objective To develop a multiplex touchdown PCR for simultaneous detection of extended-spectrum β-lactamases (ESBLs )-producing Enterobacteriaceae and methicillin-resistant Staphylococcus aureus (MRSA ). Methods Blood culture positive specimens from 102 hospitalized patients were collected between March 2013 and September 2014,four pairs of specific primers were designed based on SHV,TEM,and OXA genes of ESBLs-pro-ducing Enterobacteriaceae and MecA gene of MRSA,drug-resistant genes were amplified with single touchdown PCR and multiplex touchdown PCR, the results were compared with Kirby-Bauer disk diffusion method. Results Each single PCR amplified a specific band,four drug-resistant genes were also detected by multiplex touchdown PCR;the lower detection limits of multiplex touchdown PCR for DNA of MecA,SHV,TEM,and OXA were 4.37 ng,2.19 ng,4.53 ng,and 3.59 ng,respectively.Compared with Kirby-Bauer disk diffusion method, the overall sensitivity and specificity of multiplex touchdown PCR were 100.00% and 88.24% respectively,for ES-BLs were 100.00% and 87.23% respectively,for MRSA were both 100.00%.Conclusion A higher sensitivity and specificity multiple touchdown PCR assay has been developed,and it can be used in the rapid diagnosis and epidemi-ology investigation of bloodstream infection caused by ESBLs-producing Enterobacteriaceae and MRSA,and is help-ful for guiding antimicrobial use in clinic.

Epidemiology of Diabetes Mellitus and Molecular Characterization of Bacterial Isolates Recovered from Cases with Asymptomatic Bacteriauria in Trinidad and Tobago.

Aims: To highlight the observed features including socio-demographic, economic and biochemical characteristics seen among uncontrolled diabetic adults that should be areas of concern or focus by healthcare providers during the management of diabetes in the country. Also to perform molecular characterization of bacterial organisms prevalent among a cross section of diabetic patients with asymptomatic bacteriauria. Study Design: This was a cross sectional prospective and descriptive study. Place and Duration of Study: Study was carried out among patients attending two noncommunicable chronic diseases health centers in Trinidad & Tobago over a 6 months period in 2012. Methodology: Following informed consent, diabetic volunteers were recruited to participate in the study. Participants fulfilled study criteria that included absence of urinary symptoms, not catheterized, no history of UTI or any form of uropathy. Blood samples were screened for Hb1Ac, serum electrolytes and urea values; urine for microscopy, culture and sensitivity. Enterobacteriaceae isolates from urine culture were subjected to screening for CTX-M, TEM, and SHV by amplification of gene fragments by conventional PCR and for KPC, and NDM and OXA48 targets by real-time PCR using Sybergreen melting curve analysis. Results: Four hundred and fourteen diabetics were surveyed. Significant (15.7%; 65/414) bacteriauria was noted in sixty five subjects. Majority, 81.5% (53/65) with positive urine cultures had high HBA1c values. Escherichia coli 48.57% (34/70) and Klebsiella pneumonia 25.7% (18/70) were the most recovered organisms, with 87.1% (61/70) from urine samples and 75.4% (49/65) from female subjects. Urine samples from males 24.6% (16/65) yielded mostly Staphylococcus epidermidis 14.3% (10/7) and Staphylococcus aureus 5.7% (4/70) respectively. All Enterobacteriaceae isolates were negative for KPC, NDM and OXA-48. Although the blaTEM and bla SHV were detected in both the E. coli and K. pneumoniae isolates as expected. Conclusion: Escherichia coli was the prevalent Enterobacteriaceae among the patients with asymptomatic bacteriauria. Poor diabetic control is significantly and strongly associated with bacteriauria that was more prevalent among female diabetics. Although none of the antimicrobial resistant targets were encountered among the Enterobacteriaceae, there is still the need to keep an eye on these targets and diabetic subjects in the country.

Caracterização de beta-lactamases de espectro ampliado (ESBLs), genes de resistência aos antimicrobianos e conteúdo plasmidial em cepas de Escherichia coli e Salmonella spp. não tifóides isoladas do ambiente hospitalar e da comunidade / Characterization of extended spectrum Beta-lactamase (ESBLs), antimicrobial resistance genes, and plasmid content in Escherichia coli and Salmonella spp. isolates recovered from hospital and community

Enterobactérias produtoras de ESBLs são descritas tanto no ambiente hospitalar quanto na comunidade em todo o mundo. No Brasil, esses microrganismos também têm emergido como uma causa importante de infecções, sendo as enzimas CTX-M as prevalentes. O objetivo deste estudo foi analisar diferentes aspectos genotípicos relacionados à expressão da resistência aos antimicrobianos em cepas Escherichia coli e de Salmonella spp, tais como: a diversidade de ESBLs, os genes de resistência aos antimicrobianos e o conteúdo plasmidial. Os aspectos epidemiológicos das cepas produtoras de ESBLs também foram investigados. Foram estudadas 88 cepas de enterobactérias, sendo 43 E. coli e 45 cepas de Salmonella spp., de origem hospitalar e da comunidade (principalmente alimentos), isoladas na cidade do Rio de Janeiro. A expressão de ESBL foi observada em sete cepas de E. coli (7/43, 16,3%) e em uma cepa de Salmonella Typhimurium (1/45, 2,3%) e as enzimas foram identificadas como variantes de CTX-M e SHV-5, respectivamente. Entre as cepas de E. coli, a enzima CTX-M-2 foi a mais frequente (n = 4), sendo detectada em cepas isoladas de swab retal de pacientes hospitalizados, enquanto as enzimas CTX-M-59 (uma variante de CTX-M) (n = 1) e CTX-M-9 (n = 2) foram identificadas em cepas isoladas a partir de espécimes clínicos. Salmonella Typhimurium produtora de SHV-5 foi isolada do ambiente hospitalar (fórmula infantil). As cepas de E. coli produtoras das enzimas CTX-M pertenceram a grupos filogenéticos (A, B1, D) e STs (ST34, ST69, ST101) diferentes, sendo os genes blaCTX-M identificados em plasmídeos com tipo de replicon IncA/C de cerca de 150 kb (blaCTX-M-2, blaCTX-M-9, blaCTX-M-59) ou 80 kb (blaCTX-M-2)...

ESBL-producing Enterobacteriaceae have been described in hospitals and in the community worldwide. In Brazil, ESBL-producing Enterobacteriaceae have also emerged as an important cause of infections, being CTX-M enzymes the most prevalent ESBLs. The objective of this study was to analyze different genotypic aspects related to expression of antimicrobial resistance in isolates of Escherichia coli and Salmonella spp., such as: diversity of ESBLs, antibiotic resistance genes and plasmid content. Epidemiological features of ESBL-producing isolates were also investigated. We studied 88 isolates of enterobacteria, 43 E. coli and 45 Salmonella serotypes of hospital and community (mainly food) origin, isolated in the city of Rio de Janeiro. ESBL expression was observed in seven E. coli isolates (7/43; 16,3%) and in one Salmonella Typhimurium (1/45; 2,3%) and the enzymes identified as CTX-M variants and SHV-5, respectively. Among the E. coli isolates, CTX-M-2 was the most frequent (n=4), being detected in isolates recovered from rectal swabs of hospitalized patients, whereas CTX-M-59 (a CTX-M-2-variant) (n=1) and CTX-M-9 (n=2) were identified in E. coli isolated from clinical specimens. SHV-5-producing S. Typhimurium was isolated from the hospital environment (infant formula). CTX-M-producing E. coli belonged to different phylogenetic groups (A, B1, D) and STs (ST34, ST69, ST101), being blaCTX-M genes were identified in IncA/C plasmids of approximately 150 kb (blaCTX-M-2, blaCTX-M-9, blaCTX-M-59) or 80 kb (blaCTX-M-2)...

Extended-spectrum beta-lactamase-producing bacteria isolated from hematologic patients in Manaus, State of Amazonas, Brazil

Antibiotic therapy in hematologic patients, often weak and susceptible to a wide range of infections, particularly nosocomial infections derived from long hospitalization periods, is a challenging issue. This paper presents ESBL-producing strains isolated from such hematologic patients treated at the Amazon Hematology and Hemotherapy Foundation (HEMOAM) in the Brazilian Amazon Region to identify the ESBL genes carried by them as well as the susceptibility to 11 antimicrobial agents using the E-test method. A total of 146 clinical samples were obtained from July 2007 to August 2008, when 17 gram-negative strains were isolated in our institution. The most frequent isolates confirmed by biochemical tests and 16S rRNA sequencing were E. coli (8/17), Serratia spp. (3/17) and B.cepacia (2/17). All gram-negative strains were tested for extended-spectrum-beta-lactamases (ESBLs), where: (12/17) strains carried ESBL; among these, (8/12) isolates carried blaTEM, blaCTX-M, blaOXA, blaSHV genes, (1/12) blaTEM gene and (3/12) blaTEM, blaCTX-M, blaOXA genes. Antibiotic resistance was found in (15/17) of the isolates for tetracycline, (12/17) for ciprofloxacin, (1/17) resistance for cefoxitin and chloramphenicol, (1/17) for amikacin and (3/17) cefepime. This research showed the presence of gram-negative ESBL-producing bacteria infecting hematologic patients in HEMOAM. These strains carried the blaTEM, blaSHV, blaCTX-M and blaOXA genes and were resistant to different antibiotics used in the treatment. This finding was based on a period of 13 months, during which clinical samples from specific populations were obtained. Therefore, caution is required when generalizing the results that must be based on posological orientations and new breakpoints for disk diffusion and microdilution published by CLSI 2010.

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 bla SHV 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 bla SHV-28 genotype and three had the bla SHV-1 genotype. With our new real-time PCR assay, we detected 29 out of 29 bla SHV 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.

Detection of TEM & SHV genes in Escherichia coli & Klebsiella pneumoniae isolates in a tertiary care hospital from India.

Background & objectives: Extended spectrum beta lactamases (ESBLs) have been observed in virtually all the species of family Enterobacteriaceae. The enzymes are predominantly plasmid mediated and are derived from broad-spectrum beta lactamase TEM-1, TEM-2 or SHV-1 by a limited number of mutations. This study was undertaken to characterize ESBL producers among Escherichia coli and Klebsiella pneumoniae by PCR-RFLP, which were initially screened by phenotypic method. Methods: A total of 100 isolates of each species (E. coli and K. pneumoniae) were screened for ESBL production. PCR analysis for b-lactamase genes of the family TEM and SHV was also carried out. PCR products of TEM and SHV genes were subjected to digest with three different restriction enzymes. The digested products were run on 1.5 per cent agarose gel, stained and examined for DNA bands. Results: PCR carried out on plasmid DNA alone detected 30 per cent ESBL positive isolates using TEM primer and 38 per cent using SHV primer, whereas PCR for both plasmid and chromosomal DNA showed 56 per cent positivity for TEM and 60 per cent positivity for SHV. Interpretation & conclusion: RFLP yielded homogeneous band pattern, suggesting that there may be a point source or a common evolutionary origin for all the ESBL isolates.

Some etiological and clinical aspects of pyelonephritis and antibiotic resistance of pathogens causing children’s pyelonephritis in Mongolia / Монголын Анагаах Ухаан

IntroductionPyelonephritis is generally the result of an ascending infection of the urinary tract (reflux is seen in 30- 50% of affected children) most common organism is Escherichia coli (Heiberger R.E., 2006). The bacteria that are most likely to cause pyelonephritis are those that normally occur in the feces. Pathogens isolated from patients with pyelonephritis include both gram positive and gram negative bacteria. R.E.Neiberger reported that of gram negative bacteria E.coli, Klebsielle, Enterobacter, Proteus, Pseudomonas and of gram positive bacteria Enterococcus, Staphylococcus saphrophiticus, group B.Streptococcus were prevalent in children with pyelonephritis. In another study conducted at the Pennsylvania University, USA, 70-95% of causes were related with E.coli, Proteus, Klebsiella, Pseudomonas, Staphylococcus saphrophiticus and Еnterobacter (Sammuel Baron, 2004). In the Indian study E.coli accounted for 88.1%, Pseudomonas aeruginosa for 7.1% and Klebsiella was in 4.8% of cases (Namalwar B.R., Vijayakumar M., Janani Sankar., Ramnath B and Prahlad N., 2004). Serotypes 01; 02; 04; 06; 07 were considered as most prevalent among E.coli causes of UTI (Sammuel Baron, 2004). Escherichia coli causes about 85% of acute bladder and kidney infections in patient with no obstruction or history of surgical procedures. Proteus, Klebsiella, Enterobacter or Pseudomonas are other common causes of infection(Namalwar B.R., Vijayakumar M., Janani Sankar., Ramnath B and Prahlad N., 2004). Once these organisms enter the urinary tract, they cling to the tissues that line the tract and multiply in them (Parveen J Kumar Michael L Clark Clinical medicine. London, 2003). Pyelonephritis is characterized by tubulo-interstitial inflammation, hyperaemia and oedema. Affected patients present with the abrupt onset of fever and chills, constant dull flank pain, and symptoms of cystitis (dysuria, frequency, urgency). Urinalysis usually procedures microabscess. Urine cultures will frequency be negative in the setting of haematogenousinfection (Namalwar B.R., Vijayakumar M., Janani Sankar., Ramnath B and Prahlad N., 2004). Asymptomatic infections of the urinary tract or Asymptomatic bactreuria are common. In childgood, about 1 percent of girls have asymptomatic bacteruria (Namalwar B.R., Vijayakumar M., Janani Sankar., Ramnath B and Prahlad N., 2004). In Mongolia females aged between 20-40 years old accounted for 60.3% of all patients with chronic pyelonephritis. In cases of bacteriuria E.Coli was 74.05%, with highest sensitivity to claforan (91.25%) and ciprofloxacin (90.65%) (B.Selengee, 2003). Last 5 years (2004-2008) 3709 children were treated in Nephrological department of Mathernal and Children’s Scientific Center, but between them 741(19.97%) children were illned by pyelonephritis. However no study was conducted on bacterial causes of pyelonephritis in children in Mongolia. Purpose:The main goal of study was to indicate the etiological and clinical specialaty of children’s pyelonephritis. Especially, to indicate the prevalently occurred type and the etiological bacterial groups also the clinical specialaty of children’s pyelonephritis. To study microbiological spectrum of pathogens causing pyelonephritis in Mongolian children, sensitivity of them to antimicrobial agent, mechanisms of their resistance and to identify genes related with microbial pathogenity.Materials and Мethods:My study covered 254 children wich illned by pyelonephritis, these were treated in Nephrological department of Mathernal and Children’s Scientific Center in last 5 years (2004-2008). From patient’s history we also used the clinical laboratorial analysis results such as biochemical, blood count and urine analysis. We used retrospective and prospective statistical methods for study on pathient’s history of children wich sickned by pyelonephritis and bacteriological method for indicating groups of bacteria. We collected information about patients by questionary chart. During study we also used electro-sonography(echo), Xray, radio-isotope, computer-tomography and reno-vascular angiography methods. Antibiotic susceptibility testing covered total 212 bacterial species to 19 kind of antibiotics by disk diffusion(DD) and minimium inhibitory concentration(MIC) methods. Strains were identified by API tests strip (BioMerieux, France) and antibiotic susceptibility was determined by disk diffusion method according to the ELSI recommendations. Types of the beta lactamase were determined by PCR using the blaTEM, blaSHV and blaCTX-M specific primer. 1080 bp, 400 bp and 570 bp PCR product were interpreted as positive for blaTEM, blaSHV and blaCTX-M genes. WHONET5.1 programm was used for the analysis of susceptibility testing according to the WHO recommendation. Pathogens from 1 month consequent urine culture of patients treated at the pediatric nephrology, urology units and outpatient clinic at the MCHRC and patients from nephrology and urology departments at the CCH were isolated using disk diffusion test and minimum inhibitory concentration. Bacterial resistance and pathogenity related genes were identified using PCR method.Results:There were the girls prevalently sicked by pyelonephritis (n=181 or 71.25%) than boys (n=73 or 28.74%) and the primary (n=155 or 61.02%) and secondary chronic pyelonephritis (n=99 or 38.97%) also was prevalently occurred. During pyelonephritis are occurring following clinical symptoms these are high temperature, edema eyelid, loss of appetite, sludged urinate, skin dryness, womiting and nicthuria. By our study the main clinical symptoms of children’s pyelonephritis are high temperature (58.55%), back pain (51.0%), edema of eyelid (34.42%) and polyuria (25.79%). Also the main influencing factors for inducing children’s pyelonephritis are hyphoplasia (33.3%), hydronephrosis (19.23%), single kidney (15.38%) and vesico-uretheral reflux (6.41%). Study was conducted on 110 positive cultures (E.coli-52, Citrobacter spp.-8, E.cloacae-10, Proteus spp.-7, K.pneumoniae-7, P.aeruginosae-22 ба A.baumennii-2, S.aureus-1, Enterococci-1). The bacterial sensitivity and resistance to antimicrobial agents differed between various pathogens. From Gram negative oxydase negative Enterobacteriaceae by PCR testing, 19 isolates contained blaTEM types, 11 strains contained blaSHV types and 16 strains contained blaCTX-M type genes. On the study of PCR the virulence gene “aer” were determined 50% of E.coli strains. blaTEM type resistance can divided to 5 groups, from them 2 groups had the resistance to single beta-lactamic antibiotics, but 3 groups had to both beta-lactamic and aminoglycoside antibiotics.blaSHV type resistance had 2 groups antibiotics, these were beta-lactamic and aminoglycoside antibiotics. blaCTX-M type resistance had 2 groups, these were beta-lactamic and aminoglycoside antibiotics. Both the blaTEM and blaSHV type co-resistance had 2 groups, these were CEP and CXA antibiotics. blaTEM and CTX-M type resistance can divided to 4 groups, from them 1 group had the resistance to single beta-lactamic antibiotics, other 3 groups had to aminoglycoside antibiotics. Both the blaSHV and CTX-M type co-resistance had 2 groups, these were lactamic and aminoglycoside antibiotics. blaTEM, SHV and CTX-M type co-resistance can divided to 3 groups, these were also beta-lactamic and aminoglycoside antibiotics. On the study of PCR the virulence “aer (aerobacterin) gene” expression was identified in 50% of E.coli strains.Conclusion:There were the girls (181 cases or 71.25%) prevalently sick by pyelonephritis than boys(73 cases or 28.74%) was prevalently occurred.In Mongllia the primary pyelonephritis(155 cases or 61.02%) was occurred prevalently than secondary chronic pyelonephritis( 99 cases or 38.97%). The gasteroenterological and toxic symptoms prevalently occurred in clinical symptoms, but later kidney and urinary tract symptoms noticed.In Mongolia the main influencing factors for inducing children’s pyelonephritis are hyphoplasia (33.3%), hydronephrosis (19.23%), single kidney (15.38%) and vesico-uretheral reflux (6.41%).The prevalent pathogen causing pyelonephritis in Mongolia is E.coli. Other causes include Enterobacter spp., Klebsilla spp., Proteus spp., P.aeruginosa, Aceinobacter baumenii and S.aureus. The virulence gene “aer” were determined 50% of E.coli strains. The most prevalent resistance was resistance to beta-lactam, aminoglycoside, tetracycline and trimethoprim sulfometohoxazole. Resistance to beta-lactamic antibiotics was related with blaSHV, blaTEM, blaCTX-M gene experessions in 25-40% of cases. blaTEM, blaSHV and bla CTX-M type of the beta-lactamase were determined 25-40% of Gram negative oxidase negativebacilli. Pathogenic “aer” gene was identified in 50% of E.coli in Mongolia.

Solexa sequencing and resistant analysis of the metagenome of plasmid in Klebsiella pneumoniae / 中华微生物学和免疫学杂志

Objective High-throughout sequencing of all plasmid of 206 strains of Klebsiella pneu-moniae using Solexa/Illumina sequencing technology in order to investigate the resistance for plasmids in Klebsiella pneumoniae. Methods Bacterial isolates were obtained over the years 2002-2008. Solexa/Illumi-na sequencing technology was used to sequence both samples (S1 and S2) to a depth of between 10-560 fold coverage. We used SOAP provided by BGI to find SNPS and use velvet package to assemble these sequences and gained some long sequences, and MAQ programs developed in the laboratory were used to annotate SNPs and compare lineage-specific mutations in SHV-ESBLs. Results The Metagenome of plasmid encodes a 13 variety of resistance-related genes with exceptionally high copy numbers, including ABC-type efflux pumps and 4 variety of β-lactamases, SHV-ESBLs is abroad presence. We systematically investigated single nucleo-tide substitutions in plasmids metagenome, and found an amount of nonsynonymous mutations in the SHV-ESBLs genes. Conclusion Probabily in press of positive selection, we can clearly see these nonsynonymous changes predominantly occurred in plasmid SHV-ESBLs genes. And our findings indicate a unspecial low-level resistance contribute to antimicrobial efflux in the metagenome of plasmid in Klebsiella pneumoniae.

First Outbreak of Multidrug-Resistant Klebsiella pneumoniae Producing both SHV-12-Type Extended-Spectrum beta-Lactamase and DHA-1-Type AmpC beta-Lactamase at a Korean Hospital

PURPOSE: Coexistence of different classes of beta-lactamases in a single bacterial isolate may pose diagnostic and therapeutic challenges. We investigated a spread of Klebsiella pneumoniae isolates co-producing an AmpC beta-lactamase and an extended-spectrum beta-lactamase (ESBL) in a university hospital. MATERIALS AND METHODS: Over a three-month period, a total of 11 K. pneumoniae isolates, which exhibited resistance to cefotaxime, aztreonam, and cefoxitin, were isolated. These isolates showed positive to ESBLs by double disk tests. Minimal inhibitory concentrations (MICs) were determined by broth microdilution testing. All isolates were examined by isoelectric focusing, PCR and sequence analysis to identify bla(SHV) and bla(DHA), and molecular typing by pulsed-field gel electrophoresis (PFGE). RESULTS: All 11 isolates were highly resistant (MIC, > or = 128microngram/ml) to ceftazidime, aztreonam, and cefoxitin, while they were susceptible (MIC, < or = 2microngram/ml) to imipenem. The bla(SHV-12) and bla(DHA-1) genes were detected by PCR and sequence analysis. PFGE revealed a similar pattern in 10 of the 11 strains tested. CONCLUSION: This is the first outbreak report of K. pneumoniae in Korea which co-produced SHV-12 and DHA-1 beta-lactamase, and we suggest a clonal spread of multidrug-resistant K. pneumoniae at a hospital.

SHV-type extended-spectrum beta-lactamase (ESBL) are encoded in related plasmids from enterobacteria clinical isolates from Mexico

OBJECTIVE: In this work we report the molecular characterization of beta-lactam antibiotics resistance conferred by genes contained in plasmids from enterobacteria producing extended-spectrum beta-lactamases (ESBL). MATERIAL AND METHODS: Fourteen enterobacterial clinical isolates selected from a group of strains obtained from seven different hospitals in Mexico during 1990-1992 and 1996-1998 were analyzed at the Bacterial Resistance Laboratory (National Institute Public Health, Cuernavaca). Molecular characterization included PFGE, IEF of beta-lactamases, bacterial conjugation, PCR amplification and DNA sequencing, plasmid extraction and restriction. RESULTS: Isolates were genetically unrelated. ESBL identified were SHV-2 (5/14) and SHV-5 (9/14) type. Cephalosporin-resistance was transferable in 9 of 14 (64 percent) clinical isolates with only one conjugative plasmid, DNA finger printing showed a similar band pattern in plasmids. CONCLUSIONS: The dissemination of cephalosporin resistance was due to related plasmids carrying the ESBL genes.

OBJETIVO: En este trabajo se reporta la caracterización molecular de la resistencia a antibiótico beta-lactámicos conferida por genes contenidos en plásmidos de enterobacterias productoras de beta-lactamasas de espectro extendido (BLEEs). MATERIAL Y MÉTODOS: Catorce aislamientos clínicos de enterobacterias fueron seleccionados por conveniencia de un banco de cepas obtenidas de siete diferentes hospitales de México durante los periodos 1990-1992 y 1996-1998 y fueron procesados en el Laboratorio de Resistencia Bacteriana (Instituto Nacional de Salud Pública, Cuernavaca). En la caracterización se empleó PFGE, IEF para beta-lactamasas, conjugación bacteriana, amplificación por PCR y secuenciación de DNA, extracción y restricción de plásmidos. RESULTADOS: Las 14 cepas fueron no relacionadas genéticamente. Se identificaron BLEEs tipo SHV-2 (5/14) y SHV-5 (9/14). La resistencia a cefalosporinas fue transferida por conjugación en 9 de 14 (64 por ciento) aislamientos clínicos mediante un plásmido que mostró un patrón de restricción similar entre ellos. CONCLUSIÓN: Se sugiere que la diseminación de la resistencia a cefalosporinas fue debida a plásmidos relacionados que contienen los genes que codifican BLEEs.

Detection of common extended \ufffd?spectrum \xdf \ufffd?Lactamase genes in EAEC strains

Background:blaTEM, bla SHV, blaCTX \ufffd?M, blaOXA genes encode for extended spectrum \u03b2 \ufffd?lactamases resistance to broad \ufffd?spectrumcephalosporins. Many species belonging the family Enterobacteriaceae possess these genes. Objectives: To determine the distribution of blaTEM, bla SHV, blaCTX \ufffd?M and blaOXA genes in enteroaggregation E.coli (EAEC) strains. Subjects and method: 67 EAEC strains causing diarrhea and 18 strains isolated from healthy children were screened by PCR with primers specific to blaTEM, bla SHV, blaCTX \ufffd?M \ufffd?1and blaOXA genes. Results: The prevalence of ESBL genes in diarrheagenic EAEC strains and those isolated from healthy children were 83.6 and 72.2 %, respectively. The highest prevalence blaTEM gene (82% in diarrheagenic EAEC strains and 72.2% in isolated from healthy children) was followed by that of blaOXA gene (11.9 and 11.1% in two EAEC groups). Only 2 strains possess blaSHV gene. The blaCTX \ufffd?M \ufffd?1 was not detected in any EAEC strain. Conclusion: our findings have not only provided additional understanding of the distribution of blaTEM, bla SHV, blaCTX \ufffd?M - 1 and blaOXA genes in EAEC strains but also have a given significance in selecting antibiotics for treatment.

Extended \ufffd?spectrum \xdf \ufffd?Lactamase in E.Coli, K.Pneumoniae and Enterobacter spp

Background:Resistance to antibiotics due to extended \ufffd?spectrum \ufffd?Lactamase (ESBL) which increased quickly, made treatment much more difficult. However, this matter was not enough to be concerned in our country. Objectives: To investigate the prevalence of ESBL producing among clinical isolates of E.coli, K.pneumoniae and Enterobacter spp and the classification of ESBLs gene by PCR. Subjects and method: 663 strains, including 248 E.coli, 393 K.pneumoniae, 22 Enterobacter spp, isolated from patients in Viet Tiep hospital (Hai Phong), Bach Mai and Pediatric hospital (Ha Noi). ESBLs were detected using modified double \ufffd?disc method. The classification of ESBLs producing strains was implemented by PCR. Results:the percentage of ESBL producing in E.coli, K.pneumoniae and Enterobacter spp is 20.2; 18.3 and 36.4%, respectively. The ESBLs producing strains were co \ufffd?resistant to most of the tested antibiotics. These strains were prevalent in intensive care units (sputum or respiratory fluid samples). TEM, SHV, CTX \ufffd?M, OXA were 87.7; 62.3; 24.6 and 12.3%, respectively. They were detected alone or in combination in the same strain. Conclusion: The rate of ESBLs producing strains is high. ESBLs were marker for multi \ufffd?drug resistance. TEM and SHV type ESBLs are most prevalent in the tested strains.

Rapid Detection of Antibiotic Resistance of Klebsiella pneumoniae by Denaturing High-performance Liquid Chromatography (DHPLc) / 中华医院感染学杂志

OBJECTIVE To type the genes of plasmid DNA in 54 clinical Klebsiella pneumoniae isolates producing extended -spectrum beta-lactamases (SHV) by denaturing high-performance liquid chromatography (DHPLc) and evaluate their sensitivity and specificity, and explore a rapid and convenient method for detecting the antibiotic resistance of K. pneumoniae. METHODS Plasmid DNA from each extended-spectrum beta-lactamase (SHV) producing strain was subjected to PCR amplification. After we performed DNA sequencing of these amplicons and identification of mutation and their genotype, DHPLc was undertaken to investigate whether its results correlate the distinctive chromatogram with each genotype. RESULTS All the strains were found abnormal elution peaks (two or three peaks) which were different from each other. The result of DNA sequencing demonstrated that all the strains had DNA mutation in comparison with SHV-1. Moreover, DHPLc could produce specific peak patterns that correlate with genotype. CONCLUSIONS The sensitivity of DHPLc is 100% in this study. And each genotype is corresponded to specific peak pattern. So we can use DHPLc technique to type the genes of plasmid DNA in K. pneumoniae and detect mutations rapidly. DHPLc not only has high accuracy , but also is a convenient and rapid technique for the detection of mutation in the bacterial genome. It has a great potential clinical value.

Genotype analysis of ESBLs-producing Klebsiella pneumoniae isolates / 中国感染与化疗杂志

Objective To identify the genotypes of ESBLs-producing Klebsiella pneumoniae isolates from the First Affiliated Hospital,Shantou University Medical College.Methods The MICs of 10 antibiotics were determined by agar-dilution against the clinical isolates of ESBLs-producing K.pneumoniae.PCR were performed with specific primers for blaTEM,blaSHV, blaCTX-M and blaOXA respectively.PCR products were cloned and sequenced.Results The results of PCR showed that a- mong the 83 strains of ESBLs-producing K.pneumoniae,75 were positive for blaTEM,41 positive for blaSHV,25 poitive for blaCTX-M,9 positive for hlaOXA.Three genotypes were found in 13 strains(15.7%),2 genotypes in 59 strains (71.1%) and single genotype in only 11 strains(13.2%).The genes of CTX-M-3,TEM-1 and SHV were found co-existent in 9 strains. The strains carrying 2 or 3 ESBL genes were more resistant to antibiotics than those carrying only 1 ESBL gene.Conclusions The genotypes of ESBLs-producing Klebsiella pneumoniae in this hospital are blaTEM,blaSHV,blaCTX-M and blaOXA. Most strains carry 2 or 3 ESBL genes.

β-Lactamasas de Espectro Expandido en Enterobacterias aisladas en Centros de Salud de Caracas

Las BLEEs son β-lactamasas producidas por una variedad de bacterias gramnegativas, que confieren resistencia a cefalosporinas de tercera y cuarta generación y aztreonam. Son principalmente producidas por K. pneumoniae y E. coli, aunque la naturaleza plasmídica de los genes que las codifican ha permitido una amplia diseminación a otros géneros bacterianos. Se realizó la detección fenotípica y molecular de BLEE a 224 aislados de Enterobacterias provenientes de ocho Centros de Salud de Caracas. El 91,1% de las cepas analizadas mediante el método de doble disco y el NCCLS 2004, fueron productoras de BLEE. El análisis de CIM para ceftazidime, cefotaxime, cefepime y aztreonam mostró una mayor proporción de cepas BLEE con actividad ceftazidimasa, compatibles con las familias SHV y/o TEM, y en menor proporción pertenecientes a la familia CTX-M. Los resultados del PCR basados en la amplificación de los genes blaSHV, blaTEM y blaCTXM, confirmaron el predominio de SHV-BLEE (72%) y CTX-M-BLEE (21,1%). Los carbapenemos con un 100% de sensibilidad. constituyen la mejor opción terapéutica. Esta investigación permitió detectar el tipo de BLEE circulante en ocho centros hospitalarios y es el primer reporte de la determinación de BLEE del tipo CTX-M, en nuestro país.

The extended-spectrum ß-lactamases (ESBLs) are ß-lactamases produced by a variety of gram-negative bacteria that confers resistance to cephalosporins of third and quarter generation and aztreonam. They are most commonly produced by isolates of K. pneumoniae and E. coli, although the plasmidic nature of the genes that encode them has allowed a wide dissemination to other bacterial genera. A phenotypical and molecular detection of ESBLs were carried out to 224 Enterobacteria isolates from eight Health Centers of Caracas . A 91.1% of stumps analyzed by double disk method and the NCCLS 2004, were ESBLs' producers. The MIC analysis for ceftazidime, cefotaxime, cefepime and aztreonam showed a bigger proportion of stumps ESBL with ceftazidimase activity compatible to the SHV families and/or TEM, and in a smaller proportion to the CTX-M family. The PCR results based on genes blaSHV, blaTEM and blaCTX-M, amplification confirmed a predominance of SHV-ESBL (72%) and CTX-M-ESBL (21.1%). Therefore, carbapenems with 100% of sensibility constitute the best therapeutic option. This investigation has allowed the detection of circulating ESBLs type in eight hospitals, and is also the first report that determines the CTX-M type ESBLs in our country.

Emergence of CTX-M-9 Extended-Spectrum beta-Lactamase-Producing Enterobacter cloacae Isolates / 대한임상미생물학회지

BACKGROUND: The aim of this study is to assess the prevalence and to investigate the molecular epidemiology of Ambler class A extended-spectrum beta-lactamase (ESBL)-producing Enterobacter cloacae isolates in a university hospital in Busan, Korea. METHODS: Non-duplicated clinical isolates of E.cloacae from patients admitted in Kosin University Gospel Hospital were collected during the period from January through September, 2003. ESBL-production was examined by the double-disk synergy test (DDST) and the transferability of cefotaxime-resistance by conjugation. MICs of beta-lactam antibiotics were determined by the agar dilution method and Ambler class A ESBL genes were searched by PCR amplification. Enterobacterial repetitive intergenic consensus (ERIC) PCR was performed to investigate epidemiological relationships among bla CTX-M-9 gene-carrying E.cloacae isolates. RESULTS: Antimicrobial resistance rates of E.cloacae isolates (n=148) to ceftazidime, cefotaxime, and aztreonam were 50.0%, 29.6%, and 48.0%, respectively. Among 50 E.cloacae isolates intermediate or resistant to more than one expanded-spectrum beta-lactam agent, 41 (27.7%) showed positive results in DDST; of these 41 isolates, 1 was found to carry bla TEM-52 gene, 16 carried bla SHV-12 gene, 4 bla CTX-M-9 gene, and 19 both bla SHV-12 and bla CTX-M-9 genes. The 23 E.cloacae isolates carrying bla CTX-M-9 gene showed 9 different profiles by ERIC PCR. CONCLUSION: ESBL-producing E.cloacae was not uncommon in a university hospital in Busan, Korea. The commonest types of ESBLs produced by E.cloacae isolates were SHV-12 and CTX-M-9. CTX-M-9 ESBL-producing E.cloacae isolates showed diverse ERIC-PCR profiles, indicating that they were not originated from a common source.