Extended-spectrum beta-lactamases among Klebsiella pneumoniae from Iraqi patients with community-acquired pneumonia

SUMMARY OBJECTIVE: Beta-lactams resistance is a major clinical problem in treating pneumonia. This study aimed to detect the extended-spectrum beta-lactamases (ESBL) genes in Klebsiella pneumoniae among patients with community-acquired pneumonia (CAP) in Al-Najaf City, Iraq. METHODS: A total of 511 sputum samples were obtained from all suspected patients with CAP in Al-Najaf City, Iraq, from March 2020 to September 2020. Sputum samples were subjected to microbiological tests. The disk diffusion method was used to test antibiotic sensitivity. Production of ESBLs was identified using phenotypic and genotypic methods. RESULTS: The total prevalence of K. pneumoniae was 31.9% (163/511). Using CHROM agar, 41 (25.2%) isolates were ESBL producers. The imipenem 0.0% (n=0/41) and norfloxacin 0.0% (n=0/41) were the most effective antibiotics. The multiplex polymerase chain reaction showed that 46.3% (n=19/41) of isolates harbored ESBL genes. Out of 19 ESBL producers, 47.4% and 15.8% harbored blaCTX-M and blaSHV, respectively. While blaCTX-M and blaSHV genes were detected in 7 (36.8%) isolates, simultaneously. CONCLUSIONS: The imipenem and norfloxacin can be used in empirical treatment of K. pneumoniae isolates in Iraq. The emergence of K. pneumoniae strains harboring ESBL resistance genes necessitates the development of a regular surveillance program to prevent the spreading of these isolates more in Iraqi health care systems.

Detection of Extended-Spectrum Beta-Lactamase-Producing Enterobacteriaceae in Desi Chickens in Andhra Pradesh

A total of 150 cloacal swabs were collected from desi chickens, 217 Enterobacteriaceae isolates were identified. The phenotypic antimicrobial resistance among Enterobacteriaceae was studied for 14 selected antibiotics by disc diffusion method. The selection of antibiotics was based on usage of antibiotics in commercial poultry farms and also based on priority of critically important antibiotics in humans. All Enterobacteriaceae isolates were subjected to multiplex PCR - I and II for detection of blaTEM, blaSHV and blaOXA genes and blaCTX-M, group 1 and 2 genes. Predominant β- Lactamase genes in gut microbiota of desi chicken include blaTEM (90.55%) followed by blaCTX-M group I (25.86%) and blaSHV (9.44%) genes. All the samples were found to be negative for blaOXA and blaCTX-M group 2 genes

Prevalence of Extended Spectrum Beta Lactamase Genotypes in Klebsiella pneumonia from Respiratory Tract Infections at Tertiary Care Hospital.

Background: Extended Spectrum Beta Lactamases(ESBLs) are rapidly evolving group of β-lactamaseenzymes that are of particular concern to clinicians andepidemiologists. Most ESBLs have been evolved bygenetic mutation from blaTEM and blaSHV genes, andare well described in Klebsiella pneumoniae. Aim andObjective: To investigate the ESBL genotypes in K.pneumoniae isolates from Respiratory Tract Infections(RTIs). Material and Methods: Clinical isolates of K.pneumoniae were obtained from RTI -sputum samples.Antimicrobial susceptibility was determined by KirbyBauer disc diffusion method. ESBL was detectedphenotypically and multiplex Polymerase ChainReaction (PCR) specific for blaTEM, blaSHV andblaCTX-M genes was performed to identify genotypes.Results: During the 19 months period, a total of 212 ofK. pneumoniae were found from RTIs. Of these 212isolates, 60 isolates (28.3%) were ESBL producers byphenotypic method. Of these 212 isolates, 96 wererandomly selected for multiplex PCR for blaTEM,blaSHV and blaCTX-M genes. The findings ofmultiplex PCR showed that 24 isolates (25%)possessed blaTEM gene and only 4 isolates (4.1%)possessed each blaSHV and blaCTX-M gene alone.Isolates having both blaTEM+blaSHV genes were 20(20.8%), and both blaTEM+blaCTX-M genes were 12(12.5%); and isolate possessing all threeblaTEM+blaSHV+blaCTX-M genes were 20 (20.8%).The overall prevalence of blaTEM, blaSHV andblaCTX-M genes in this study was 79.1%, 45.8% and37.5% respectively. Imipenem was most effectiveantibiotic. Conclusion: Spread of ESBL producing K.pneumoniae is a major concern, as it causes limitationsto optimal treatment. Multiplex PCR can be used as arapid method to identify ESBL genotypes in K.pneumoniae. It will prove valuable for surveillance andestablishing the treatment line against drug resistantorganisms, thus saving precious time and resources. Inour study blaTEM genotype was most prevalent.

An emerging threat of ceftriaxone-resistant non-typhoidal salmonella in South India: Incidence and molecular profile

Background: Non-typhoidal Salmonella (NTS) infection is a serious public health problem globally. Although NTS infections are self-limited, antimicrobial therapy is recommended for severe infections and immunocompromised patients. Antimicrobial resistance (AMR) in these pathogens further limits its therapeutic options. Here, we report an incidence of ceftriaxone resistance in NTS over the past 9 years in a southern Indian region. Materials and Methods: Molecular mechanisms of resistance in ceftriaxone-resistant NTS have been tested by both phenotypic and molecular methods. Minimum inhibitory concentration was determined by the E-test and broth microdilution method. AMR gene markers of ?-lactamases such as AmpCs (blaMOX, blaCMY, blaDHA, blaFOX, blaACC and blaACT) and extended-spectrum ?-lactamases (ESBLs) (blaSHV, blaTEM, blaVEB, blaPER, blaCTXM-1like,blaCTXM-2like, blaCTXM-8like, blaCTXM-9like and blaCTXM-25like) were screened. The presence of IncH12 and IncI1 plasmid was also analysed. Results: The study reports a 5% prevalence of ceftriaxone resistance in NTS. The most common serogroup was Salmonella Group B followed by Salmonella Group E and Salmonella group C1/C2. The occurrence of blaCTX-M-1, blaTEM, blaCMY and blaSHV genes was observed in 54%, 54%, 48% and 3% of the isolates, respectively. Interestingly, few isolates carried dual resistance genes (ESBLs and AmpCs). IncH12 and IncI1 plasmid was identified in isolates carrying ESBL and AmpC genes, respectively. Conclusion: This study shows that ceftriaxone resistance is mainly mediated by ?-lactamases such as ESBL and AmpC. As the incidence of ceftriaxone resistance is rising gradually over the years, it is imperative to monitor the AMR in this species.

Caracterización molecular y detección de genes blaCTX-M grupos 1 y 9 en Klebsiella pneumoniae resistentes a ceftazidima, en un hospital de San José de Cúcuta, Colombia / Molecular characterization and detection of genes blaCTX-M groups 1 and 9 in Klebsiella pneumoniae resistant to ceftazidime, in a hospital in San José de Cúcuta, Colombia

Resumen Introducción: La expresión de β-lactamasas CTX-M pertenecientes a los grupos 1 y 9 en Klebsiella pneumoniae produce grados altos de resistencia a ceftazidima, y presentan una amplia distribución mundial. Objetivo: Identificar y caracterizar los genes blaCTX-M-Grupo1 y blaCTX-M-Grupo9 en aislados de K. pneumoniae resistentes a ceftazidima en un hospital de San José de Cúcuta, Colombia. Material y Método: Se diseñaron partidores para la identificación de K. pneumoniae y los genes blaCTX-M mediante reacción de polimerasa en cadena (RPC). Posteriormente se realizó el análisis de la relación genética de estos aislados por medio de la RPC basada en secuencias repetitivas (RPC-REP). Resultados: Treinta y ocho por ciento de los 24 aislados identificados por RPC como K. pneumoniae presentaron los genes blaCTX-M-3, blaCTX-M-15 y blaCTX-M-32 (Grupo CTX-M-1) y 42% los genes blaCTX-M14, blaCTX-M-24 y blaCTX-M-27 (Grupo CTX-M-9). El análisis filogenético agrupó los aislados de K. pneumoniae en cuatro clusters, mostrando correlación en los clusters I, II y IV, al comparar los perfiles genéticos con el tipo de muestra y grupo de genes. Discusión: Se encontró una frecuencia similar de los genes blaCTX-M-Grupo1 y blaCTX-M-Grupo 9 en aislados de K. pneumoniae resistentes a ceftazidima. La correlación entre la RPC-REP con los grupos de CTX-M y el tipo de muestra reveló la presencia de tres patrones clonales.

Background: The expression of CTX-M β-lactamases belonging to groups 1 and 9 in Klebsiella pneumoniae produces high levels of resistance to ceftazidime, and they have a wide distribution worldwide. Aim: To identify and characterize the blaCTX-M-Group1 and blaCTX-M-Group9 genes in K. pneumoniae isolates resistant to ceftazidime in a hospital in San José de Cúcuta, Colombia. Material and Methods: Primers were designed for the identification of K. pneumoniae and blaCTX-M genes by PCR. Subsequently, the genetic relationship of these isolates was analyzed by REP-PCR. Results: A 38% of the 24 isolates identified by PCR as K. pneumoniae showed blaCTX-M-3. blaCTX-M-15 y blaCTX-M-32 genes (Group CTX-M-1) and 42% blaCTX-M14. blaCTX-M-24 y blaCTX-M-27 genes (Group CTX-M-9). The phylogenetic analysis grouped the K. pneumoniae isolates into 4 clusters, showing correlation in clusters I, II and IV, when comparing the genetic profiles with the type of sample and group of genes. Discussion: We found a similar frequency of blaCTX-M-Group 1 and blaCTX-M-Group 9 genes in isolates of K. pneumoniae resistant to ceftazidime. The correlation between the REP-PCR with the CTX-M groups and the type of sample revealed the presence of three clonal patterns.

Antimicrobial resistance, virulence & plasmid profiles among clinical isolates of Shigella serogroups

Background & objectives: Bacillary dysentery caused by Shigella spp. remains an important cause of the crisis in low-income countries. It has been observed that Shigella species have become increasingly resistant to most widely used antimicrobials. In this study, the antimicrobial resistance, virulence and plasmid profile of clinical isolates of Shigella species were determined. Methods: Sixty clinical Shigella isolates were subjected to whole-genome sequencing using Ion Torrent platform and the genome sequences were analyzed for the presence of acquired resistance genes, virulence genes and plasmids using web-based software tools. Results: Genome analysis revealed more resistance genes in Shigella flexneri than in other serogroups. Among ?-lactamases, blaOXA-1was predominantly seen followed by the blaTEM-1B and blaEC genes. For quinolone resistance, the qnr S gene was widely seen. Novel mutations in gyr B, par C and par E genes were observed. Cephalosporins resistance gene, blaCTX-M-15 was identified and plasmid-mediated AmpC ?-lactamases genes were found among the isolates. Further, a co-trimoxazole resistance gene was identified in most of the isolates studied. Virulence genes such as ipaD, ipaH, virF, senB, iha, capU, lpfA, sigA, pic, sepA, celb and gad were identified. Plasmid analysis revealed that the IncFII was the most commonly seen plasmid type in the isolates. Interpretation & conclusions: The presence of quinolone and cephalosporin resistance genes in Shigella serogroups has serious implications for the further spread of this resistance to other enteric pathogens or commensal organisms. This suggests the need for continuous surveillance to understand the epidemiology of the resistance.

Beta-lactamase and integron-associated antibiotic resistance genes of Klebsiella pneumoniae isolated from Tilapia fishes (Oreochromis niloticus)

Tilapia fishes (Oreochromis niloticus) are commonly consumed and exported in Thailand. Bacterial isolation anddrug resistance from farmed tilapia fished in Thailand were previously reported. This study was purposed to studyon the distribution of human pathogenic bacteria in tilapia fishes, which were collected from Thai farms (n = 180)and fresh markets (n = 160) by identification, antibiotic susceptibility test; and conduct to identify virulence genesby molecular technique. Pathogen isolations were collected from internal organs of fish samples for identificationand test of antibiotic susceptibility according to Clinical and Laboratory Standards Institute (CLSI) criteria. blaCTX-Mand Int1 genes detection of antibiotic resistance bacteria was performed by molecular based techniques. Klebsiellapneumoniae, Edwardsiella tarda, and coagulase-negative Staphylococci were most frequent bacteria isolated fromfarming tilapia fishes, respectively. However, Escherichia coli, coagulase-negative Staphylococci, and K. pneumoniawere frequently distributed from tilapia fishes in markets of Bangkok area. Klebsiella pneumoniae, E. coli, and Proteusmirabilis were resisted to penicillin and ampicillin. Klebsiella pneumoniae is the most important isolated bacteria dueto the distribution in tilapia fishes and positive for blaCTX-M and Int1 gene detection. However, E. coli and P. mirabiliswere lack of blaCTX-M and Int1 genes, possibly there may reserve other antibiotic resistance genes

pMEX01, a 70kb Plasmid Isolated From Escherichia Coli That Confers Resistance to Multiple ß-Lactam Antibiotics

Abstract Multidrug-resistant microorganisms are of great concern to public health. Genetic mobile elements, such as plasmids, are among the most relevant mechanisms by which bacteria achieve this resistance. We obtained an Escherichia coli strain CM6, isolated from cattle presenting severe diarrheic symptoms in the State of Querétaro, Mexico. It was found to contain a 70 kb plasmid (pMEX01) with a high similarity to the pHK01-like plasmids that were previously identified and described in Hong Kong. Analysis of the pMEX01 sequence revealed the presence of a blaCTX-M-14 gene, which is responsible for conferring resistance to multiple β-lactam antibiotics. Several genes putatively involved in the conjugative transfer were also identified on the plasmid. The strain CM6 is of high epidemiological concern because it not only displays resistance to multiple β-lactam antibiotics but also to other kinds of antibiotics.

Phylogeny and Detection of blaTEM, blaSHV, blaCTX-M Genes in Escherichia coli Isolates from Patients with Urinary Tract Infections in Taif Hospitals, Saudi Arabia

Background: Urinary tract infections (UTIs) represent one of the most common diseases that are encountered in clinical practice and are caused mainly by Escherichia coli (E. coli). Aims: The objectives of this study were to identify and compare the blaTEM, blaSHV and blaCTX-M as marker of beta-lactamase genes in E. coli strains isolated from patients with UTIs collecting from King Abdul-Aziz hospital in Taif region, Saudi Arabia. Study Design: In vitro experimental and molecular study. Place and Duration of Study: Genetic engineering and biotechnology unit, Taif University, from September, 2016 to November, 2017. Methodology: Beta-lactame antibiotics are prescribed in most infectious disease including UTIs. Twenty one isolates identified as E. coli using microbial identification and confirmed by 16S rDNA. Results: These isolates were susceptible to Imipenem (100%), Ampicillin (90%) and Cefoxitin, but resistant to Cefepime (38%). Existance of selected bla-genes (blaTEM, blaSHV and blaCTX-M) were detected in the 21 isolates by PCR. Moreover, phylogeny tree was drawn based on 16S rDNA sequence. The results of this study show significant differences in susceptibility to different beta-lactam antibiotics among the bla-genes in E. coli isolates. Conclusion: Therefore, our findings instead of our data provide some new epidemiological information about the clonal nature of E. coli isolated from patients with UTIs in Taif region, KSA.

First Report of blaCTX-M-15 Extended Spectrum Beta-Lactamase (ESBL) Producing E. Coli Isolated from Cloacal Swabs of Birds in South Eastern Nigeria

Introduction: In this study, we investigated the presence of some beta-lactamases namely SHV, TEM and the most widely spread extended spectrum beta-lactamase (blaCTX-M-15) genes in E. coli isolated from four different bird species including ducks, pigeons, weaverbirds and bats in Ebonyi State, Nigeria. Methodology: Genes for ESBL production was determined based on PCR amplification of the genes encoding the enzymes including TEM, SHV and CTX-M-15 using specific primers. One hundred and fifty cloacal swabs each from ducks and pigeons and 100 each from weaverbirds and bats were respectively collected using sterile swab sticks. Antibiotic susceptibility test on the isolates was conducted using disc diffusion method while the phenotypic determination of ESBL was carried out using Double Disc Synergy Tests (DDST). Observations: Results from this study showed that E. coli was present in all the 4 bird species investigated. Of the 117 isolates screened for ESBL production, only 3(5.56 %) and 9(16.67 %), respectively were positive from ducks and pigeons while none was positive from bats and weaverbirds. Results of the molecular studies showed that the ESBL producing E. coli from pigeons were negative for SHV genes, positive for TEM and CTX-M-15 while those from ducks did not harbour any of the beta-lactamase and ESBL genes investigated.  Conclusion: The detection of similar types of beta-lactamase and ESBL genes (TEM and CTX-M-15) in pigeon samples indicates the possible involvement of some bird species in the spread of multidrug resistant genes across human population. This is the first report of CTX-M-15 ESBL in bird species from Southeast Nigeria.

Resistance mechanisms of blaCTX-M-55 in a clinical Shigella sonnei strain / 军事医学

Objective To characterize the resistance mechanisms of a clinical Shigella sonnei strain harboring blaCTX-M-55 .Methods A double-disk synergy test was conducted to detect ESBL.Antibiotic resistance genes were determined by PCR followed by amplicon sequencing.Conjugation experiments were performed to verify the transferability of the plasmids carrying ESBL genes.The minimum inhibitory concentration values were tested using VITEK 2.The transposition unit was confirmed by DNA sequencer,and the transcriptional start site was identified using primer extension assay.Results Strain #1083 produced CTX-M-55,which was encoded by plasmid p1083-CTXM that could be transferred into E.coli through conjugation experiments to confer corresponding antibiotic resistance to the transconjugant #1083-EC600.The transposition unit mediating the transfer of blaCTX-M-55 was ISEcp1-blaCTX-M-55 -Δorf477.ISEcp1 offered strong promoter regions for the resistance genes,facilitating their expressions.Besides,the expressions were constant,not induced by antibiotics.Conclusion BlaCTX-M-55 on plasmids is the major resistance genes for strain #1083.Their expressions and spread are mediated by the insertion sequence ISEcp1.

Emergence of CTX-M-15 producing E. coli O25b-ST131 clone in a tertiary care hospital of Bangladesh

Background: Extended-spectrum β-lactamase (ESBL) producing uropathogens has become prevalent worldwide. E. coli O25b-ST131 clone, associated with blaCTX-M-15, has been reported from many parts of the world and is frequently associated with multidrug resistance. Thus far, there are no reports about this clone in Bangladesh. The objective of this study was to investigate ESBL producing uropathogens and to survey the prevalence of E. coli O25b-ST131 clone among ESBL positive E. coli isolates. Methods: From symptomatic urinary tract infection cases, a total of 800 urine samples were collected. Bacterial identification and antimicrobial susceptibility testing was performed using established methods. Screening of ESBL producers was done using the disk diffusion method. Screening positive isolates were phenotypically confirmed by double disk synergy (DDS) test. Genes encoding ESBLs (blaCTX-M-15, blaOXA-1) were identified both by PCR and DNA sequencing. Phenotypic positive ESBL producers were also studied by PCR for existence of class 1 integron. Subsequently, O25b-ST131 clone was identified by allele specific PCR. Results: Of 138 gram-negative uropathogens, 45 (32.6%) were positive for ESBLs. ESBL producers showed high frequency of antimicrobial resistance except imipenem. Among 45 ESBL producers, 36 (80%) produced blaCTX-M-15, 18 (40%) produced blaOXA-1. Fifteen (33.3%) strains simultaneously produced both blaOXA-1 and blaCTX-M-15. Class 1 integron was present in 30 (66.7%) isolates. Of the 31 blaCTX-M-15 positive E. coli, 22 (71%) were positive for E. coli O25b-ST131 clone and all (100%) belonged to B2 phylogenetic group. Conclusion: Rising antimicrobial resistance among uropathogens, and especially the emergence of blaCTX-M-15 positive E. coli O25b-ST131 clone in Bangladesh has provided urgency to the development of novel preventive and therapeutic strategies.

Carriage of ESBL and AmpC-Positive Enterobacteriaceae in Gastrointestinal Tract of Healthy Community Subjects and Hospitalized Patients and Detection of blaCTX-M Gene in ESBL Positive Isolates.

Aim: The study aimed at analyzing ESBL and AmpC positive Enterobacteriaceae in the gastrointestinal tracts of healthy community subjects and hospitalized patients and detection of blaCTX-M type gene in ESBL positive isolates. Methods: Bacteria were isolated from stool samples of the study population. Production of ESBL-type beta-lactamases was screened by double-disk synergy test as well as automated system, and AmpC enzyme production was detected by the AmpC disk test. ESBL positive isolates were subjected to detection of the blaCTX-M gene. Results: A total of 792 stool samples (50% from healthy subjects and 50% from hospitalized patients) were studied. The prevalence rates of ESBL-positive Enterobacteriaceae were 9.3% in hospitalized patients and 4.4% in healthy community subjects. Production of the AmpC enzyme was detected in

Prevalence of blaTEM , blaSHV and blaCTX-M genes in clinical isolates of Escherichia coli and Klebsiella pneumoniae from Northeast India.

Aim: This study was carried out to determine the presence of blaTEM, blaSHV and blaCTX-M genes in extended-spectrum β-lactamase (ESBL) producing Escherichia coli (E. coli) and Klebsiella pneumoniae (K. pneumoniae) at a tertiary care referral hospital in Northeast India. Materials and Methods: A total of 270 E. coli and 219 K. pneumoniae isolates were recovered during the period between August 2009 and July 2010. Kirby-Bauer disk diffusion method was performed to determine the antibiotic resistance pattern. Screening and phenotypic confi rmatory test for ESBL production were performed using standard disc diffusion methods. Each of the initial ESBL screening test isolate was investigated for the presence of blaTEM, blaSHV and blaCTX-M genes via polymerase chain reaction (PCR) using gene-specifi c primers. Results: P henotypic confi rmatory test able to detect ESBL production in 73.58% of E. coli and 67.24% of K. pneumoniae. However, PCR amplifi cation showed the presence of one or more ESBL genes in each of the initial ESBL screening positive isolate. Among three ESBL genotypes, the most prevalent genotype was found to be blaCTX-M in E. coli (88.67%) and blaTEM in K. pneumoniae (77.58%) ESBL producing isolates. Majority of ESBL producing isolates possess more than one ESBL genes. Conclusion: T his study constituted a primer report on high prevalence of blaTEM and blaCTX-M genes in ESBL producing isolates of E. coli and K. pneumoniae and denotes the need of more extensive studies on these antibiotic genes to determine the magnitude of the problem of antibiotic resistance exiting in this locality.

Genes blaTEM, blaSHV y blaCTX-M en enterobacterias productoras de b-lactamasas de espectro extendido aisladas de pacientes con infección intrahospitalaria / blaTEM, blaSHV y blaCTX-M genes in extended-spectrum b-lactamases producing enterobacterias isolated from patients with hospital-acquired infections

El objetivo de este estudio fue identificar los genes blaTEM, blaSHV y blaCTX-M en aislados clínicos de enterobacterias productoras de b-lactamasas de espectro extendido (BLEE), recolectadas entre septiembre y noviembre de 2005. Además de la resistencia a las cefalosporinas de tercera generación, los aislados también mostraron resistencia a cloranfenicol (59,2%) amikacina (37,0%) y gentamicina (40,7%) y se mostraron sensibles a imipenem y meropenem. Nueve cepas lograron transferir la resistencia a las cefalosporinas de tercera generación, así como la producción de BLEE. En los aislados clínicos se detectaron los genes blaSHV, blaTEM y blaCTX-M, donde los tipos blaTEM-1, blaSHV-1, blaSHV-5 blaSHV-5-2a y blaCTX-M-1 fueron los prevalentes; mientras que en las transconjugantes sólo se detectaron blaTEM-1, blaSHV-5 y blaSHV-5-2a. Se identificaron en total siete tipos de genes, de los cuales cinco eran codificantes de enzimas tipo BLEE, lo que demuestra que en el centro hospitalario la resistencia a las cefalosporinas de tercera generación es debida a diversas enzimas.

The objective of the present investigation was to identify the blaTEM, blaSHV and blaCTX-M genes on extended-spectrum b-lactamases (ESBL) producing Enterobacteriaceae from clinical isolates, collected between September and November 2005. In addition to third-generation cephalosporin resistance, the isolates also showed resistance to chloramphenicol (59.2%), amikacin (37.0%) and gentamicin (40.7%), and demonstrated sensitivity to imipenem and meropenem. Nine strains were capable of transferring third-generation cephalosporin resistance, as well as the production of ESBL. In the clinical isolates, the genes blaSHV, blaTEM and blaCTX-M were detected, being more prevalent the types blaTEM-1, blaSHV-1, blaSHV-5 blaSHV-5-2a and blaCTX-M-1; while in the trans-conjugated only blaTEM-1, blaSHV-5 y blaSHV-5-2a were found. In total, seven types of genes were identified, five of which were codifying genes for ESBL-type enzymes. This demonstrates that in the hospital center, resistance to third-generation cephalosporin is mediated by several enzymes.

Multidrug resistance genes, including blaKPC and blaCTX-M-2, among Klebsiella pneumoniae isolated in Recife, Brazil / Genes de multirresistência, incluindo blaKPC e blaCTX-M-2, em Klebsiella pneumoniae isoladas em Recife, Brasil

INTRODUCTION: The prevalence of cephalosporins and carbapenem-resistant Klebsiella pneumoniae strains is rising in Brazil, with potential serious consequences in terms of patients' outcomes and general care. METHODS: This study characterized 24 clinical isolates of K. pneumoniae from two hospitals in Recife, Brazil, through the antimicrobial susceptibility profile, analyses of β-lactamase genes (blaTEM, blaSHV,blaCTX-MblaKPC, blaVIM, blaIMP, and blaSPM), plasmidial profile and ERIC-PCR (Enterobacterial repetitive intergenic consensus-polymerase chain reaction). RESULTS: ERIC-PCR and plasmidial analysis grouped the isolates in 17 and 19 patterns, respectively. Six isolates from one hospital presented the same pattern by ERIC-PCR, indicating clonal dissemination. All isolates presented blaSHV, 62.5% presented blaCTX-M-2, 29% blaTEM, and 41.7% blaKPC. Metallo-β-lactamase genes blaand blawere not detected. Eleven isolates were identified carrying at least 3 β-lactamase studied genes, and 2 isolates carried blaSHVblaTEM, blaCTX-M-2 and blaKPC simultaneously. CONCLUSIONS: The accumulation of resistance genes in some strains, observed in this study, imposes limitations in the therapeutic options available for the treatment of infections caused by K. pneumoniae in Recife, Brazil. These results should alert the Brazilian medical authorities to establish rigorous methods for more efficiently control the dissemination of antimicrobial resistance genes in the hospital environment.

INTRODUÇÃO: A prevalência de cepas de Klebsiella pneumoniae resistentes a cefalosporinas e carbapenêmicos está aumentando no Brasil, com sérias consequências em termos de desfechos dos pacientes e cuidados gerais. MÉTODOS: Este estudo caracterizou 24 isolados clínicos de K. pneumoniae provenientes de dois hospitais de Recife, Brasil, através do perfil de susceptibilidade a antimicrobianos, análise de genes de β-lactamase (blaTEM,blaSHV,blaCTX-MblaKPC,blaVIM, blaIMP,and blaSPM), perfil plasmidial e ERIC-PCR (Enterobacterial repetitive intergenic consensus-polymerase chain reaction). RESULTADOS: A análise da ERIC-PCR e do perfil plasmidial agrupou os isolados em 17 e 19 perfis, respectivamente. Seis isolados de um hospital apresentaram o mesmo padrão de ERIC-PCR, indicando disseminação clonal. Todos os isolados apresentaram blaSHV, 62,5% apresentaram blaCTX-M-2, 29% blaTEM e 41,7% blaKPC. Genes de metalo-β-lactamase blaVIM, blaIMP e blaSPM não foram detectados. Onze isolados foram identificados carreando, pelo menos, três dos genes de β-lactamase estudados, dentre estes, dois isolados continham blaSHV,blaTEM, blaCTX-M-2 e blaKPC simultaneamente. CONCLUSÕES: O acúmulo de genes de resistência em algumas cepas, observado nesse estudo, impõem limitações nas opções terapêuticas disponíveis para o tratamento de infecções causadas por K. pneumoniae em Recife, Brasil. Estes resultados devem alertar as autoridades médicas brasileiras para estabelecer rigorosos métodos para controlar eficientemente a disseminação de genes de resistência a antimicrobianos no ambiente hospitalar.

blaCTX-M-2 and blaCTX-M-28 extended-spectrum â-lactamase genes and class 1 integrons in clinical isolates of Klebsiella pneumoniae from Brazil

Twenty-eight Klebsiella pneumoniae clinical isolates that exhibited an extended-spectrum cephalosporin-resistance profile from a city in the Northeast of Brazil were analysed by PCR and DNA sequencing in order to determine the occurrence of blaCTX-M genes and class 1 integrons. We determined the occurrence of the blaCTX-M-2 gene in six K. pneumoniae isolates and describe the first detection of the blaCTX-M-28 gene in South America. Seven isolates carried class 1 integrons. Partial sequencing analysis of the 5'-3'CS variable region in the class 1 integrons of three isolates revealed the presence of aadA1, blaOXA-2 and dfr22 gene cassettes.

Ambiente genético del gen blaCTX-M-12 en aislamientos hospitalarios de klebsiella pneumoniae / The blaCTX-M-12 gene's genetic environmnt in klebsiella pneumoniae hospital isolates

En Colombia se han detectado genes del grupo CTX-M-1 con alta frecuencia en aislamientos de Klebsiella pneumoniae causantes de infección intrahospitalaria. El conocimiento de los factores genéticos que pueden favorecer la diseminación de estos genes entre especies bacterianas es un aspecto importante para el control de la resistencia. En este estudio se identificaron los plásmidos portadores del gen blaCTX-M-12 en 21 aislamientos clínicos de K. pneumoniae. Se evaluó por conjugación la transferencia de resistencia a antibióticos. Integrones, secuencias de inserción y otros elementos genéticos fueron detectados por amplificación del ADN plasmídico con la reacción en cadena de la polimerasa (PCR). Mediante análisis por PCR se determinó la relación entre el gen blaCTX-M-12 y los elementos genéticos detectados. En todos los aislamientos, el gen blaCTX-M-12 se encontró en plásmidos conjugativos de tamaños entre 65 y 106 kpb. La transferencia por conjugación de estos elementos móviles puede explicar la amplia diseminación de este gen entre enterobacterias causantes de infección nosocomial en hospitales de Bogotá, Colombia. El gen blaCTX-M-12 se encontró corriente abajo de ISEcp1, secuencia de inserción que se ha asociado con la movilización de determinantes genéticos de resistencia. Los promotores de ISEcp1, detectados por análisis de secuencia, pueden facilitar la expresión de la cefotaximasa codificada por este gen.

Genes from CTX-M-1 group have been detected with great frequency in Colombia in intrahospital infection-causing Klebsiella pneumoniae isolates. Knowledge regarding the genetic factors favouring such genes’ dissemination amongst bacterial species is an important issue for resistance control blaCTX-M-12 gene-carrying plasmids were identified in this study in 21 clinical K. pneumoniae isolates. Antibiotic resistance transfer was evaluated by mating. Integrons, insertion sequences and other genetic elements were detected by plasmid DNA amplification using polymerase chain reaction (PCR). The relationship between the blaCTX-M-12 gene and other genetic elements was determined by PCR analysis. The blaCTX-M-12 gene was disemifound on 52 to 106 Kpb conjugative plasmids in all isolates. These mobile elements’ transfer by mating may explain their wide dissemination amongst nosocomial infection-causing enterobacteria in hospitals in Bogota, Colombia. The blaCTX-M-12 gene was found downstream from ISEcp1, this being an insertion sequence which has been associated with resistance genetic determinants’ mobilisation. ISEcp1 promoters (detected by sequence analysis) may increase the expression of cefotaximase encoded by this gene.